U.S. patent application number 15/501784 was filed with the patent office on 2017-08-10 for antifungal paenibacillus strains, fusaricidin-type compounds, and their use.
The applicant listed for this patent is BASF SE. Invention is credited to Luis ANTELO, Birgit BLANZ, Heike BRUSER, Kerstin HAGE, Kristin KLAPPACH, Till OPATZ, Louis Pergaud SANDJO, Karl-Heinrich SCHNEIDER, Isabella SIEPE, Petra SPROTE, Eckhard THINES.
Application Number | 20170223968 15/501784 |
Document ID | / |
Family ID | 51260746 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170223968 |
Kind Code |
A1 |
SIEPE; Isabella ; et
al. |
August 10, 2017 |
ANTIFUNGAL PAENIBACILLUS STRAINS, FUSARICIDIN-TYPE COMPOUNDS, AND
THEIR USE
Abstract
The present invention relates to novel isolated bacterial
strains, which are members of the genus Paenibacillus, originally
isolated from soil and showing antagonistic activity against a
broad range of pathogens and being capable of producing
antimicrobial metabolites. It was found that the strains Lu16774
and Lu17007 belong to a novel subspecies named Paenibacillus
polymyxa ssp. plantarum while the strain Lu17015 belongs to a novel
species which is proposed to be Paenibacillus epiphyticus. The
present invention also relates to microbial pesticide compositions
comprising at least one of such novel bacterial strains, whole
culture broth or a cell-free extract or a fraction thereof or at
least one metabolite thereof, and/or a mutant of at least one of
said novel bacterial strains having all the identifying
characteristics of the respective bacterial strain or whole culture
broth, cell-free extract, fraction and/or metabolite of the mutant
thereof showing antagonistic activity against plant pathogens. The
present invention also relates to a method of controlling or
suppressing plant pathogens or preventing plant pathogen infections
by applying such composition. The present invention also relates to
novel fusaricidin-type compounds which are metabolites produced by
the strains of the present invention.
Inventors: |
SIEPE; Isabella;
(Dossenheim, DE) ; BRUSER; Heike; (Speyer, DE)
; KLAPPACH; Kristin; (Neustadt, DE) ; SCHNEIDER;
Karl-Heinrich; (Kleinkarlbach, DE) ; SPROTE;
Petra; (Mannheim, DE) ; HAGE; Kerstin;
(Speyer, DE) ; BLANZ; Birgit; (Limburgerhof,
DE) ; THINES; Eckhard; (Mehlingen, DE) ;
ANTELO; Luis; (Hochspeyer, DE) ; SANDJO; Louis
Pergaud; (Longkak-Yaounde, CM) ; OPATZ; Till;
(Oberursel, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BASF SE |
Ludwigshafen |
|
DE |
|
|
Family ID: |
51260746 |
Appl. No.: |
15/501784 |
Filed: |
August 4, 2015 |
PCT Filed: |
August 4, 2015 |
PCT NO: |
PCT/EP2015/067925 |
371 Date: |
February 3, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 1/20 20130101; A01N
63/10 20200101; C07K 14/195 20130101 |
International
Class: |
A01N 63/02 20060101
A01N063/02; C07K 14/195 20060101 C07K014/195 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 4, 2014 |
EP |
14179620.1 |
Claims
1.-19. (canceled)
20. A Paenibacillus strain, which is selected from the group
consisting of: a) strain Lu16774 deposited with DSMZ under
Accession No. DSM 26969; b) strain Lu17007 deposited with DSMZ
under Accession No. DSM 26970; c) strain Lu17015 deposited with
DSMZ under Accession No. DSM 26971; and d) a strain which comprises
a DNA sequence exhibiting d1) at least 99.6% nucleotide sequence
identity to the DNA sequences SEQ ID NO:4 or SEQ ID NO:9; or d2) at
least 99.8% nucleotide sequence identity to the DNA sequence SEQ ID
NO:14; or d3) at least 99.9% nucleotide sequence identity to the
DNA sequences SEQ ID NO:5 or SEQ ID NO:10; or d4) at least 99.2%
nucleotide sequence identity to the DNA sequence SEQ ID NO:15; or
d5) at least 99.2% nucleotide sequence identity to the DNA
sequences SEQ ID NO:6 or SEQ ID NO:11; or d6) at least 99.8%
nucleotide sequence identity to the DNA sequence SEQ ID NO:16; or
d7) at least 99.8% nucleotide sequence identity to the DNA
sequences SEQ ID NO:7 or SEQ ID NO:12; or d8) at least 99.3%
nucleotide sequence identity to the DNA sequence SEQ ID NO:17; or
d9) 100.0% nucleotide sequence identity to the DNA sequences SEQ ID
NO:8 or SEQ ID NO:13; or d10) 100% nucleotide sequence identity to
the DNA sequence SEQ ID NO:18.
21. The Paenibacillus strain according to claim 20, which is
selected from the group consisting of: a) strain Lu16774 deposited
with DSMZ under Accession No. DSM 26969; b) strain Lu17007
deposited with DSMZ under Accession No. DSM 26970; c) strain
Lu17015 deposited with DSMZ under Accession No. DSM 26971; and d)
strains which comprise a DNA sequence exhibiting d1) at least 99.8%
nucleotide sequence identity to any one of the DNA sequences SEQ ID
NO:4, SEQ ID NO:9 and SEQ ID NO:14; or d2) 100.0% nucleotide
sequence identity to any one of the DNA sequences SEQ ID NO:5, SEQ
ID NO:10 and SEQ ID NO:15; or d3) exhibiting at least 99.8%
nucleotide sequence identity to any one of the DNA sequences SEQ ID
NO:6, SEQ ID NO:11 and SEQ ID NO:16; or d4) at least 99.8%
nucleotide sequence identity to any one of the DNA sequences SEQ ID
NO:7, SEQ ID NO:12 and SEQ ID NO:17; or d5) 100.0% nucleotide
sequence identity to any one of the DNA sequences SEQ ID NO:8, SEQ
ID NO:13 and SEQ ID NO:18.
22. The Paenibacillus strain according to claim 20, wherein said
Paenibacillus strain has antifungal activity against at least two
of the plant pathogen selected from the group consisting of
Alternaria spp., Botrytis cinerea, Phytophthora infestans and
Sclerotinia sclerotiorum.
23. The Paenibacillus strain according to claim 20, wherein said
Paenibacillus strain produces at least one of the following
compounds: ##STR00007## in a growth medium comprising at least one
source of carbon and one source of nitrogen.
24. The Paenibacillus strain according to claim 23, wherein said
Paenibacillus strain is further capable of producing at least three
compounds selected from the group consisting of fusaricidin A,
fusaricidin B, fusaricidin C, fusaricidin D, LI-F06a, LI-F06b and
LI-F08b.
25. The Paenibacillus strain according to claim 20, wherein said
Paenibacillus strain comprises at least one of the following
compounds: ##STR00008##
26. The Paenibacillus strain according to claim 25, wherein said
Paenibacillus strain further comprises at least three compounds
selected from the group consisting of fusaricidin A, fusaricidin B,
fusaricidin C, fusaricidin D, LI-F06a, LI-F06b and LI-F08b.
27. A substantially purified culture of the Paenibacillus strain
according to claim 20.
28. A whole culture broth or a cell-free extract of the
Paenibacillus strain according to claim 20.
29. A compound of formula I ##STR00009## wherein R is selected from
15-guanidino-3-hydroxypentadecanoic acid and 12-guanidinododecanoic
acid; X.sup.1 is threonine; X.sup.2 is isoleucine; X.sup.3 is
tyrosine; X.sup.4 is threonine; X.sup.5 is selected from glutamine
and asparagine; X.sup.6 is alanine; and wherein an arrow defines a
single amide bond either between the carbonyl moiety of R and the
amino group of the amino acid X.sup.1 or between the carbonyl group
of one amino acid and the amino group of a neighboring amino acid,
wherein the tip of the arrow indicates the attachment to the amino
group of said amino acid X.sup.1 or of said neighboring amino acid;
and wherein the single line without an arrow head defines a single
ester bond between the carbonyl group of X.sup.6 and the hydroxyl
group of X.sup.1; or an agriculturally acceptable salt thereof.
30. The compound according to claim 29 selected from compounds 1A
and 1B: ##STR00010## or an agriculturally acceptable salt
thereof.
31. A method of preparing a compound of formula I ##STR00011##
wherein R is selected from 15-guanidino-3-hydroxypentadecanoic acid
and 12-guanidinododecanoic acid; X.sup.1 is threonine; X.sup.2 is
isoleucine; X.sup.3 is tyrosine; X.sup.4 is threonine; X.sup.5 is
selected from glutamine and asparagine; X.sup.6 is alanine; and
wherein an arrow defines a single amide bond either between the
carbonyl moiety of R and the amino group of the amino acid X.sup.1
or between the carbonyl group of one amino acid and the amino group
of a neighboring amino acid, wherein the tip of the arrow indicates
the attachment to the amino group of said amino acid X.sup.1 or of
said neighboring amino acid; and wherein the single line without an
arrow head defines a single ester bond between the carbonyl group
of X.sup.6 and the hydroxyl group of X.sup.1; or an agriculturally
acceptable salt thereof, which method comprises culturing the
Paenibacillus strain of claim 20 and recovering said compound or
salt thereof from the whole culture broth.
32. A composition comprising a) a Paenibacillus strain according to
claim 20; or b) a substantially purified culture of a); or c) a
whole culture broth or a cell-free extract of a); or d) a compound
of formula I ##STR00012## wherein R is selected from
15-guanidino-3-hydroxypentadecanoic acid and 12-guanidinododecanoic
acid; X.sup.1 is threonine; X.sup.2 is isoleucine; X.sup.3 is
tyrosine; X.sup.4 is threonine; X.sup.5 is selected from glutamine
and asparagine; X.sup.6 is alanine; and wherein an arrow defines a
single amide bond either between the carbonyl moiety of R and the
amino group of the amino acid X.sup.1 or between the carbonyl group
of one amino acid and the amino group of a neighboring amino acid,
wherein the tip of the arrow indicates the attachment to the amino
group of said amino acid X.sup.1 or of said neighboring amino acid;
and wherein the single line without an arrow head defines a single
ester bond between the carbonyl group of X.sup.6 and the hydroxyl
group of X.sup.1; or an agriculturally acceptable salt thereof; and
an auxiliary.
33. The composition of claim 32, further comprising a
pesticide.
34. The composition of claim 33, wherein the pesticide is a further
biopesticide.
35. A plant propagation material having a coating comprising the
composition according to claim 32.
36. (canceled)
37. A method of controlling, suppressing plant pathogens or
preventing plant pathogen infection, wherein the plant pathogens,
their habitat or plants to be protected against plant pathogen
attack, or the soil or propagation material are treated with an
effective amount of the composition according to claim 32.
38. The method of claim 37, wherein the plant pathogens and/or
harmful microorganisms are selected from harmful fungi.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel isolated bacterial
strains, which are members of the genus Paenibacillus, originally
isolated from soil and showing antagonistic activity against a
broad range of pathogens and being capable of producing
antimicrobial metabolites. The present invention also relates to
microbial pesticide compositions comprising at least one of such
novel bacterial strains, whole culture broth or a cell-free extract
or a fraction thereof or at least one metabolite thereof, and/or a
mutant of at least one of said novel bacterial strains having all
the identifying characteristics of the respective bacterial strain
or whole culture broth, cell-free extract, fraction and/or
metabolite of the mutant thereof showing antagonistic activity
against plant pathogens. The present invention also relates to a
method of controlling or suppressing plant pathogens or preventing
plant pathogen infections by applying such composition. The present
invention also relates to novel fusaricidin-type compounds which
are metabolites produced by the strains of the present
invention.
BACKGROUND OF THE INVENTION
[0002] In the technical field of controlling phytopathogenic fungi
affecting plants or crops it is well known to apply active compound
compositions comprising biopesticides, for example selected from
bacteria, like spore-forming bacteria, or fungi which are not
detrimental to the plant or crop to be treated and which biological
control agents may be further combined with classical organic
chemical antagonists of plant pathogens.
[0003] Biopesticides have been defined as a form of pesticides
based on micro-organisms (bacteria, fungi, viruses, nematodes,
etc.) or natural products (compounds or extracts from biological
sources) (U.S. Environmental Protection Agency:
http://www.epa.gov/pesticides/biopesticides/).
[0004] Biopesticides are typically created by growing and
concentrating naturally occurring organisms and/or their
metabolites including bacteria and other microbes, fungi, viruses,
nematodes, proteins, etc. They are often considered to be important
components of integrated pest management (IPM) programmes, and have
received much practical attention as substitutes to synthetic
chemical plant protection products (PPPs).
[0005] Biopesticides fall into two major classes, microbial and
biochemical pesticides: [0006] (1) Microbial pesticides consist of
bacteria, fungi or viruses (and often include the metabolites that
bacteria and fungi produce). Entomopathogenic nematodes are also
classed as microbial pesticides, even though they are
multi-cellular. [0007] (2) Biochemical pesticides are naturally
occurring substances that control pests or provide other crop
protection uses as defined below, but are relatively non-toxic to
mammals.
[0008] For controlling phytopathogenic fungi several microbial
pesticides comprising spore-forming bacteria such as Bacillus
subtilis have been described earlier, see e.g. WO 1998/050422; WO
2000/029426; WO 1998/50422 and WO 2000/58442.
[0009] WO 2009/0126473 discloses agriculturally acceptable aqueous
compositions comprising bacterial or fungal spores contained in an
aqueous/organic solvent and which may further comprise insect
control agents, pesticides, fungicides or combinations thereof.
Spores of bacteria of the genus Bacillus are a preferred
species.
[0010] WO 2006/017361 discloses compositions for controlling plant
pathogens and comprising at least one beneficial bacterium, at
least one beneficial fungus, at least on nutrient and at least one
compound which extends the effective lifetime of such a
composition. The group of beneficial bacteria e.a. comprises
bacteria of Paenibacillus polymyxa and Paenibacillus durum.
[0011] EP-A-1 168 922 relates to compositions for affecting plant
growth and/or imparting disease resistance comprising at least two
plant-growth promoting Rhizobacteria strains and a chitinous
compound, wherein said strains are selected from the genera
Bacillus, Paenibacillus, Brevibacillus, Virgibacillus,
Alicyclobacillus, and Aneurinibacilus. No particular Paenibacillus
strains are, however, exemplified in support of the claimed
combinations.
[0012] WO 1999/059412 discloses a Paenibacillus polymyxa strain
PKB1 (bearing ATCC accession no. 202127) active against several
phytopathogenic fungi.
[0013] WO 2006/016558 discloses Paenibacillus sp. strains BS-0048,
BS-0074, BS-0277 and P. polymyxa strain BS-0105 as well as
fusaricidin A and fusaricidin B for protection of plants from
infections with fungi. A further antifungal Paenibacillus strain
BRF-1 has been isolated from soybean rhizosphere (African J.
Microbiol. Res. 4(24), 2692-2698, 2010).
[0014] WO 2011/069227 discloses a P. polymyxa strain JB05-01-1
(bearing ATCC accession no. PTA-10436) having a highly inhibitory
effect against pathogenic bacteria, pre-dominantly food-borne human
pathogenic bacteria.
[0015] Budi et al. (Appl Environ Microbiol, 1999, 65, 5148-5150)
have isolated Paenibacillus sp. strain B2 from mycorrhizosphere of
Sorghum bicolor having antagonistic activity towards soil borne
fungal pathogens like Phytophthora parasitica.
[0016] A Paenibacillus peoriae strain 11.D.3 isolated by Delaporte,
B. (Lab Cytol Veg, Paris, France) and deposited in the open
collection of Agricultural Research Service, USDA, U.S.A. under the
NRRL Accession No. BD-62 (Int. J. Syst Bacteriol. 46(4), 988-1003,
1996, hereinafter also referred to as strain BD-62) from soil in
Cote d'Ivoire showed antifungal activity against several
phytopathogenic bacteria and fungi (J. Appl. Microbiol. 95,
1143-1151, 2003). NRRL is the abbreviation for the Agricultural
Research Service Culture Collection, an international depositary
authority for the purposes of deposing microorganism strains under
the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT
OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE, having the
address National Center for Agricultural Utilization Research,
Agricultural Research Service, U.S. Department of Agriculture, 1815
North University Street, Peoria, Ill. 61604, USA.
[0017] The antimicrobial activity of numerous Paenibacillus
strains, i. a. a P. peorirae strain, against numerous bacterial,
fungal and yeast pathogens has been reported elsewhere (Lett. Appl.
Microbiol. 43, 541-547, 2006).
[0018] Raza et al. (Brazilian Arch. Biol. Techol. 53, 1145-1154,
2010; Eur. J. Plant Pathol. 125: 471-483, 2009) described a
fusaricidin-type compound-producing Paenibacillus polymyxa strain
SQR-21 effective against Fusarium oxysporum.
[0019] Fusaricidins are a group of antibiotics isolated from
Paenibacillus spp., which belong to the class of cyclic
lipodepsipeptides. Their common structural features which are
conserved throughout the family are as follows: a macrocyclic ring
consisting of 6 amino acid residues, three of which are L-Thr,
D-allo-Thr and D-Ala, as well as the
15-guanidino-3-hydroxypentadecanoic acid tail attached to the
N-terminal L-Thr residue by an amide bond (ChemMedChem 7, 871-882,
2012; J. Microbiol. Meth. 85, 175-182, 2011, Table 1 herein). These
compounds are cyclized by a lactone bridge between the N-terminal
L-Thr hydroxyl group and the C-terminal D-Ala carbonyl group. The
position of the amino acid residues within the depsipeptide cycle
are usually numbered starting with the abovementioned L-Thr which
itself also carries the GHPD chain and ending with the C-terminal
D-Ala. Non-limiting examples of fusaricidins isolated from
Paenibacillus are designated LI-F03, LI-F04, LI-F05, LI-F07 and
LI-F08 (J. Antibiotics 40(11), 1506-1514, 1987; Heterocycles 53(7),
1533-1549, 2000; Peptides 32, 1917-1923, 2011) and fusaricidins A
(also called LI-F04a), B (also called LI-F04b), C (also called
LI-F03a) and D (also called LI-F03b) (J. Antibiotics 49(2),
129-135, 1996; J. Antibiotics 50(3), 220-228, 1997). The amino acid
chain of a fusaricidin is not ribosomally generated but is
generated by a non-ribosomal peptide synthetase. Structural
formulae of known fusaricidins are shown in Table 1 (Biotechnol
Lett. 34, 1327-1334, 2012; FIG. 1 therein). The compounds
designated as LI-F03a, LI-F03b up to LI-F08a and LI-F08b are herein
also referred to as fusaricidins LI-F03a, LI-F03b up to LI-F08a and
LI-F08b due to their structure within the fusaricidin family (see
Table 1).
TABLE-US-00001 TABLE 1 Structures of the fusaricidin family.
Fusaricidin X.sup.2 X.sup.3 X.sup.5 A (Ll-F04a) D-Val L-Val D-Asn B
(Ll-F04b) D-Val L-Val D-Gln C (Ll-F03a) D-Val L-Tyr D-Asn D
(Ll-F03b) D-Val L-Tyr D-Gln Ll-F05a D-Val L-Ile D-Asn Ll-F05b D-Val
L-Ile D-Gln Ll-F06a D-allo-Ile L-Val D-Asn Ll-F06b D-allo-Ile L-Val
D-Gln Ll-F07a D-Val L-Phe D-Asn Ll-F07b D-Val L-Phe D-Gln Ll-F08a
D-Ile L-allo-Ile D-Asn Ll-F08b D-Ile L-allo-Ile D-Gln
##STR00001## [0020] wherein an arrow defines a single (amide) bond
either between the carbonyl moiety of GHPD and the amino group of
L-Thr (L-threonine) or between the carbonyl group of one amino acid
and the amino group of a neighboring amino acid, wherein the tip of
the arrow indicates the attachment to the amino group of said amino
acid L-Thr or of said neighboring amino acid; and [0021] wherein
the single line (without an arrow head) defines a single (ester)
bond between the carbonyl group of D-Ala (D-alanine) and the
hydroxyl group of L-Thr; and wherein GHPD is
15-guanidino-3-hydroxypentadecanoic acid.
[0022] Among isolated fusaricidin antibiotics, fusaricidin A has
shown the most promising antimicrobial activity against a variety
of clinically relevant fungi and gram-positive bacteria such a
Staphylococcus aureus (MIC value range: 0.78-3.12 .mu.g/ml)
(ChemMedChem 7, 871-882, 2012). The synthesis of fusaricidin
analogues that contain 12-guanidino-dodecanoic acid (12-GDA) or
12-amino-dodecanoic acid (12-ADA) instead of naturally occurring
GHPD has been established but the replacement of GHPD by 12-ADA
resulted in complete loss of the antimicrobial activity while the
replacement of GHPD by 12-GDA retained antimicrobial activity
(Tetrahedron Lett. 47, 8587-8590, 2006; ChemMedChem 7, 871-882,
2012).
[0023] Fusaricidins A, B, C and D are also reported to inhibit
plant pathogenic fungi such as Fusarium oxysporum, Aspergillus
niger, Aspergillus oryzae, and Penicillum thomii (J. Antibiotics
49(2), 129-135, 1996; J. Antibiotics 50(3), 220-228, 1997).
Fusaricidins such as LI-F05, LI-F07 and LI-F08 have been found to
have certain antifungal activity against various plant pathogenic
fungi such as Fusarium moniliforme, F. oxysporum, F. roseum,
Giberella fujkuroi, Helminthosporium sesamum and Penicillium
expansum (J. Antibiotics 40(11), 1506-1514, 1987). Fusaricidins
also have antibacterial activity to Gram-positive bacteria
including Staphylococcus aureus (J. Antibiotics 49, 129-135, 1996;
J. Antibiotics 50, 220-228, 1997). In addition, fusaricidins have
antifungal activity against Leptosphaena maculans which causes
black root rot of canola (Can. J. Microbiol. 48, 159-169, 2002).
Moreover, fusaricidins A and B and two related compounds thereof,
wherein D-allo-Thr is bound via its hydroxyl group to an additional
alanine using an ester bridge, produced by certain Paenibacillus
strains were found to induce resistance reactions in cultured
parsley cells and to inhibit growth of Fusarium oxysporum (WO
2006/016558; EP 1 788 074 A1).
[0024] WO 2007/086645 describes the fusaricidin synthetase enzyme
and its encoding gene as isolated from Paenibacillus polymyxa
strain E681 which enzyme is involved in the synthesis of
fusaricidins A, B, C, D, LI-F03, LI-F04, LI-F05, LI-F07 and
LI-F08.
[0025] The genome of several Paenibacillus polymyxa strains has
been published so far: inter alia for strain M-1 (NCBI acc. no.
NC_017542; J. Bacteriol. 193 (29), 5862-63, 2011; BMC Microbiol.
13, 137, 2013), strain CR1 (GenBank acc. no. CP006941; Genome
Announcements 2 (1), 1, 2014) and strain SC2 (GenBank acc. nos.
CP002213 and CP002214; NCBI acc. no. NC_014622; J. Bacteriol. 193
(1), 311-312, 2011), for further strains see legend of FIG. 12
herein. The P. polymyxa strain M-1 has been deposited in China
General Microbiological Culture Collection Center (CGMCC) under
acc. no. CGMCC 7581.
[0026] Montefusco et al. describe in Int. J. Systematic Bacteriol.
(43, 388-390, 1993) a novel bacterial species of the genus Bacillus
and suggest the name Bacillus peoriae which may be distinguished
from other Bacillus strains as for example Bacillus badius, B.
coagulans, B. polymyxa and others. Said novel Bacillus strain is
reported to produce spores, to be gram-positive and to produce
catalase, without producing oxidase. Further biochemical
characteristics are summarized therein. The strain, which may be
isolated from soil or rotting vegetable materials, was designated
BD-57 and was deposited at the Agricultural Research Service, USDA,
U.S.A. as NRRL B-14750 and also at the DSMZ (see below) as strain
DSM 8320. Based on further biochemical and genetic analysis said
strain later has been renamed as Paenibacillus peoriae (see Int. J.
Systematic Bacteriol. 46, 988-1003, 1996). A more recent assessment
of the diversity of Paenibacillus spp. in the maize rhizosphere
using PCR-DGGE method was described in J. Microbiol. Methods 54,
213-231, 2003.
[0027] Biopesticides for use against crop diseases have already
established themselves on a variety of crops. For example,
biopesticides already play an important role in controlling downy
mildew diseases. Their benefits include: a 0-Day Pre-Harvest
Interval and the ability to use under moderate to severe disease
pressure.
[0028] A major growth area for biopesticides is in the area of seed
treatments and soil amendments. Biopesticidal seed treatments are
e.g. used to control soil borne fungal pathogens that cause seed
rots, damping-off, root rot and seedling blights. They can also be
used to control internal seed borne fungal pathogens as well as
fungal pathogens that are on the surface of the seed. Many
biopesticidal products also show capacities to stimulate plant host
defenses and other physiological processes that can make treated
crops more resistant to a variety of biotic and abiotic
stresses.
[0029] However, biopesticides under certain conditions can also
have disadvantages, such as high specificity (requiring an exact
identification of the pest/pathogen and the use of multiple
products), slow speed of action (thus making them unsuitable if a
pest outbreak is an immediate threat to a crop), variable efficacy
due to the influences of various biotic and abiotic factors (since
biopesticides are usually living organisms, which bring about
pest/pathogen control by multiplying within the target insect
pest/pathogen), and resistance development.
[0030] Therefore there is a need for further bacterial strains and
for further antimicrobial metabolites which antagonize
phytopathogenic microorganisms, in particular fungi, which are
characterized by a broad spectrum of activity against all classes
of phytopathogenic fungi.
DESCRIPTION OF THE INVENTION
[0031] Said problem was, surprisingly solved by providing novel
strains of bacteria of the genus Paenibacillus which are
characterized by a unique profile of antagonistic activity against
phytopathogenic fungi, also extending to plant leaf pathogens, as
for example selected from Alternaria spp., Botrytis cinerea,
Phytophthora infestans, and Sclerotinia sclerotiorum. Said
bacterial strains have been deposited with the International
Depositary Authority: Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH, Inhoffenstra.beta.e 7 B, 38124 Braunschweig,
Germany (hereinafter DSMZ).
[0032] Furthermore, the whole culture broth, the culture medium and
cell-free extracts of these bacterial strains showed inhibitory
activity at least against Alternaria spp., Botrytis cinerea and
Phytophthora infestans. Bioactivity guided fractionation of organic
extracts led to the isolation of two novel fusaricidin-type
compounds (compounds 1A and 1B), the structure of which were
elucidated by 1D- and 2D-NMR spectroscopy as well as mass
spectrometry.
[0033] Thus, the present invention relates to an isolated
microorganism, being a member of the family Paenibacillus, having
at least one of the identifying characteristics of one of the
following strains: [0034] 1) Paenibacillus sp. strain Lu16774
deposited with DSMZ under Accession No. DSM 26969,
[0035] 2) Paenibacillus sp. strain Lu17007 deposited with DSMZ
under Accession No. DSM 26970, and [0036] 3) Paenibacillus sp.
strain Lu17015 deposited with DSMZ under Accession No. DSM
26971.
[0037] As used herein, the term Paenibacillus sp. strain is
identical to the term Paenibacillus strain.
[0038] As used herein, "isolate" refers to a pure microbial culture
separated from its natural origin, such an isolate obtained by
culturing a single microbial colony. An isolate is a pure culture
derived from a heterogeneous, wild population of
microorganisms.
[0039] As used herein, "strain" refers to isolate or a group of
isolates exhibiting phenotypic, physiological, metabolic and/or
genotypic traits belonging to the same lineage, distinct from those
of other isolates or strains of the same species.
[0040] A further embodiment relates to a whole culture broth, a
supernatant or a cell-free extract or a fraction or at least one
metabolite of at least one of the microorganisms as defined above
which preferably exhibit antagonistic activity against at least one
plant pathogen.
[0041] As used herein, "whole culture broth" refers to a liquid
culture of a microorganism containing vegetative cells and/or
spores suspended in the culture medium and optionally metabolites
produced by the respective microorganism.
[0042] As used herein, "culture medium", refers to a medium
obtainable by culturing the microorganism in said medium,
preferably a liquid broth, and remaining when cells grown in the
medium are removed, e.g., the supernatant remaining when cells
grown in a liquid broth are removed by centrifugation, filtration,
sedimentation, or other means well known in the art; comprising
e.g. metabolites produced by the respective microorganism and
secreted into the culture medium. The "culture medium" sometimes
also referred to as "supernatant" can be obtained e.g. by
centrifugation at temperatures of about 2 to 30.degree. C. (more
preferably at temperatures of 4 to 20.degree. C.) for about 10 to
60 min (more preferably about 15 to 30 min) at about 5,000 to
20,000.times.g (more preferably at about 15,000.times.g).
[0043] As used herein, "cell-free extract" refers to an extract of
the vegetative cells, spores and/or the whole culture broth of a
microorganism comprising cellular metabolites produced by the
respective microorganism obtainable by cell disruption methods
known in the art such as solvent-based (e.g. organic solvents such
as alcohols sometimes in combination with suitable salts),
temperature-based, application of shear forces, cell disruption
with an ultrasonicator. The desired extract may be concentrated by
conventional concentration techniques such as drying, evaporation,
centrifugation or alike. Certain washing steps using organic
solvents and/or water-based media may also be applied to the crude
extract preferably prior to use.
[0044] As used herein, the term "metabolite" refers to any
component, compound, substance or byproduct (including but not
limited to small molecule secondary metabolites, polyketides, fatty
acid synthase products, non-ribosomal peptides, ribosomal peptides,
proteins and enzymes) produced by a microorganism (such as fungi
and bacteria, in particular the strains of the invention) that has
any beneficial effect as described herein such as pesticidal
activity or improvement of plant growth, water use efficiency of
the plant, plant health, plant appearance, or the population of
beneficial microorganisms in the soil around the plant activity
herein.
[0045] As used herein, "isolate" refers to a pure microbial culture
separated from its natural origin, such an isolate obtained by
culturing a single microbial colony. An isolate is a pure culture
derived from a heterogeneous, wild population of
microorganisms.
[0046] As used herein, "strain" refers to isolate or a group of
isolates exhibiting phenotypic and/or genotypic traits belonging to
the same lineage, distinct from those of other isolates or strains
of the same species.
[0047] A further embodiment relates to novel compounds of formula
I
##STR00002##
wherein [0048] R is selected from
15-guanidino-3-hydroxypentadecanoic acid (GHPD) and
12-guanidinododecanoic acid (12-GDA); [0049] X.sup.1 is threonine;
[0050] X.sup.2 is isoleucine; [0051] X.sup.3 is tyrosine; [0052]
X.sup.4 is threonine; [0053] X.sup.5 is selected from glutamine and
asparagine; [0054] X.sup.6 is alanine; and wherein an arrow defines
a single (amide) bond either between the carbonyl moiety of R and
the amino group of the amino acid X.sup.1 or between the carbonyl
group of one amino acid and the amino group of a neighboring amino
acid, wherein the tip of the arrow indicates the attachment to the
amino group of said amino acid X.sup.1 or of said neighboring amino
acid; and wherein the single line (without an arrow head) defines a
single (ester) bond between the carbonyl group of X.sup.6 and the
hydroxyl group of X.sup.1; and the agriculturally acceptable salts
thereof, and to methods of preparing compounds of formula I of the
invention which method comprises culturing the strains of the
invention and isolating said compounds of formula I from the whole
culture broth.
[0055] According to a further embodiment, the invention further
relates to compounds 1A and 1B, which are of formula I, wherein R
is GHPD and wherein X.sup.5 is asparagine in case of compound 1A
and X.sup.5 is glutamine in case of compound 1B:
##STR00003##
[0056] The present invention further relates to compositions
comprising the strains, whole culture broth, cell-free extracts,
culture media, or compounds of formula I and their salts of the
invention, as well as to their use for controlling or suppressing
plant pathogens or preventing plant pathogen infection or for
protection of materials against infestation destruction by harmful
microorganisms, and to corresponding methods which comprise
treating the pathogens, their habitat or the materials or plants to
be protected against pathogen attack, or the soil or propagation
material with an effective amount of the compositions, strains,
whole culture broth, cell-free extracts, culture media, or
compounds of formula I and their salts of the invention.
[0057] Further embodiments of the invention are disclosed in the
following detailed description of the invention, the claims and the
figures.
[0058] The invention relates to the microorganism strains [0059] 1)
Paenibacillus sp. strain Lu16774 deposited with DSMZ under
Accession No. DSM 26969, 2) Paenibacillus sp. strain Lu17007
deposited with DSMZ under Accession No. DSM 26970, and [0060] 3)
Paenibacillus sp. strain Lu17015 deposited with DSMZ under
Accession No. DSM 26971.
[0061] The strains Lu16774, Lu17007 and Lu17015 have been isolated
from soil samples from a variety of European locations including
Germany and deposited under the Budapest Treaty with the Deutsche
Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under the
abovementioned Accession numbers on Feb. 20, 2013 by BASF SE,
Germany.
[0062] The genus Paenibacillus (formerly rRNA group 3 bacilli) has
been characterized phenotypically and physiologically (Antonie van
Leeuwenhoek 64, 253-260 (1993)) by: [0063] rod-shaped cells of
Gram-positive structure, [0064] weak reaction with Gram's stain,
often even stain negatively, [0065] differentiation into
ellipsoidal endospores which distinctly swell the sporangium
(mother cell), [0066] facultative anaerobic growth with strong
growth in absence of air irrespective of whether nitrate is present
or not, [0067] fermentation of a variety of sugars, [0068] acid and
gas formation from various sugars including glucose, [0069] no acid
production from adonitol and sorbitol, [0070] Urease-negative (with
exception of P. validus), [0071] arginine dihydrolase negative,
[0072] no utilization of citrate, [0073] no growth in presence of
10% sodium chloride, [0074] secretion of numerous extracellular
hydrolytic enzymes degrading DNA, protein, starch; and/or [0075]
G+C content of DNA from 40% to 54%.
[0076] The genus Paenibacillus (formerly rRNA group 3 bacilli) has
also been characterized by 16S rDNA analysis (Antonie van
Leeuwenhoek 64, 253-260 (1993)): [0077] having a specific 22-base
sequence in a variable region V5 of the 16S rDNA (5' to 3'):
TCGATACCCTTGGTGCCGAAGT (Antonie van Leeuwenhoek 64, 253-260 (1993),
see Table 3 therein); and/or [0078] by hybridization of isolated or
PCR-amplified chromosomal DNA with BG3 probe
(5'-TCGATACCCTTGGTGCCGAAGT-3') (see Antonie van Leeuwenhoek 64,
253-260 (1993)).
[0079] The deposited strains Lu16774, Lu17007 and Lu17015 of the
invention were determined to belong to the genus Paenibacillus on
the following morphological and physiological observations (see
Example 2.3 herein): [0080] rod-shaped cells [0081] ellipsoidal
spores [0082] swollen sporangium [0083] anaerobic growth [0084]
fermentation of a variety of sugars including glucose, arabinose,
xylose, mannit, fructose, raffinose, trehalose and glycerol with
acid formation [0085] gas production from glucose [0086] arginine
dihydrolase negative [0087] no utilization of citrate [0088] no
growth in presence of 5% or more sodium chloride [0089] production
of extracellular hydrolytic enzymes degrading starch, gelatine,
casein and esculin.
[0090] Further, the deposited strains Lu16774, Lu17007 and Lu17015
of the invention were also determined to belong to the genus
Paenibacillus by 16S rDNA analysis by having the
Paenibacillus-specific 22-base sequence in 16S rDNA (5' to 3'):
TABLE-US-00002 5'-TCGATACCCTTGGTGCCGAAGT-3'
(see SEQ ID NO:1 (nucleotides 840-861), SEQ ID NO:2 (840-861), SEQ
ID NO:3 (844-865) and SEQ ID NO:4 (840-861) in sequence listings
herein).
[0091] Further, sequencing of the complete 16S rDNA in comparison
to 24 different Paenibacillus strains resulted in clustering of the
deposited strains Lu16774, Lu17007 and Lu17015 with the type
strains of Paenibacillus brasiliensis, P. knbbensis, P. jamilae, P.
peoriae, and P. polymyxa, more preferably to P. peoriae, in
particular Paenibacillus peoriae strain BD-62 (see FIGS. 1 and 2
herein). It is known that P. polymyxa and P. peoriae have 16S rDNA
sequence identity values of 99.6 to 99.7% (J. Gen. Appl. Microbiol.
48, 281-285 (2002)).
[0092] "Percent Identity" or "percent similarity" between two
nucleotide sequences means percent identity of the residues over
the complete length of the aligned sequences and is determined by
comparing two optimally locally aligned sequences over a comparison
window defined by the length of the local alignment between the two
sequences, such as, for example, the identity calculated (for
rather similar sequences) after manual alignment with the aid of
the program AE2 (Alignment Editor 2). Local alignment between two
sequences only includes segments of each sequence that are deemed
to be sufficiently similar according to the criterion that depends
on the algorithm used to perform the alignment (e.g. AE2, BLAST,
secondary structure of the rRNA molecule or alike). The percentage
identity is calculated by determining the number of positions at
which the identical nucleic acid occurs in both sequences to yield
the number of matched positions, dividing the number of matched
positions by the total number of positions in the window of
comparison and multiplying the result by 100.
[0093] To determine the percent sequence identity of two nucleic
acid sequences (e.g., one of the nucleotide sequences of Table 1
and a homolog thereof), the sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in the sequence
of one nucleic acid for optimal alignment with the other nucleic
acid). The bases at corresponding positions are then compared. When
a position in one sequence is occupied by the base as the
corresponding position in the other sequence then the molecules are
identical at that position. It is to be understood that for the
purposes of determining sequence identity when comparing a DNA
sequence to an RNA sequence, a thymidine nucleotide is equivalent
to a uracil nucleotide.
[0094] For alignment, the sequence data was put into the program
AE2 (http://iubio.bio.indiana.edu/soft/molbio/unix/ae2.readme),
aligned manually according to the 30 secondary structure of the
resulting rRNA molecule and compared with representative 16S rRNA
gene sequences of organisms belonging to the Firmcutes (Nucl. Acids
Res. 27, 171-173, 1999). To obtain % identity values for multiple
sequences, all sequences of were aligned with each other (multiple
sequence alignment). Further, to obtain % identity values between
two sequences over a longer stretch of aligned sequences in
comparison to multiple alignment, a manual pairwise sequence
alignment was done as described above using AE2 (pairwise sequence
alignment).
[0095] Further, standardized, automated ribotyping is performed
using the Qualicon RiboPrintersystem with the Paenibacillus strains
Lu16774, Lu17007 and Lu17015 in comparison to the P. peoriae BD-62
using the restriction enzyme EcoRI resulted in similarity of all
three novel strains to P. peoriae BD-62 of between 0.24 and 0.5
(Example 2.2 and FIG. 12).
[0096] In sum, the strains have been designated to the following
taxonomic groups.
[0097] The Paenibacillus strains Lu16774 and Lu17007 both belong to
the species Paenibacillus polymyxa.
[0098] Thus, the invention relates to the microorganism strains
[0099] 1) Paenibacillus polymyxa strain Lu16774 deposited with DSMZ
under Accession No. DSM 26969, [0100] 2) Paenibacillus polymyxa
strain Lu17007 deposited with DSMZ under Accession No.
[0101] DSM 26970, and [0102] 3) Paenibacillus sp. strain Lu17015
deposited with DSMZ under Accession No. DSM 26971.
[0103] According to the results of the phylogenetic analysis
presented herein (FIGS. 12 to 22) and unpublished results of
Professor Borriss, Germany, it is proposed that the heterogenous
species Paenibacillus polymyxa requires a new taxonomic
classification into two subspecies:
1) Paenibacillus polymyxa ssp. polymyxa and 2) Paenibacillus
polymyxa ssp. plantarum; and 3) a novel species Paenibacillus nov.
spec. epiphyticus.
[0104] The type strain P. polymyxa DSM 36 together with the P.
polymyxa strains SQR-21, CF05, CICC 10580, NRRL B-30509 and A18
form in each of the maximum likelihood dendrograms analysed for
five conserved house keeping genes (dnaN, gyrB, recA, recN and
rpoA) a separate cluster (FIGS. 17-21).
[0105] Very similar results have been obtained by determination of
the Average Amino acid Identity (AAI) which is frequently used for
determination of phylogenetic relationship amongst bacterial
species. This method is based on the calculation of the average
identity of a core genome on amino acid level (Proc. Natl. Acad.
USA 102, 2567-2572, 2005). According to the resulting AAI-matrix in
FIG. 22, P. polymyxa DSM 36 forms together with the P. polymyxa
SQR-21 strain a sub cluster that is different from the two other
sub clusters shown therein.
[0106] The strains Lu16674 and Lu17007 together with strain P.
polymyxa M-1, 1-43, SC2 and Sb3-1 form the second sub cluster in
each of the maximum likelihood dendrograms analysed for five
conserved house keeping genes (dnaN, gyrB, recA, recN and rpoA)
(FIGS. 17-21). According to AAI-matrix in FIG. 22 based on the
analysis of the core genome, this second sub cluster is confirmed
by its representative strains Lu16674 and Lu17007 together with the
P. polymyxa M-1 and SC2 strains.
[0107] The difference between the two sub clusters is not so
significant to justify a new species, but the AAI identity levels
between the representatives of both clusters is of about 97.5%
justifying the classification into two separate subspecies
[0108] Thus, it is proposed to nominate the first sub cluster
according to the type P. polymyxa strain DSM 36.sup.T Paenibacillus
polymyxa ssp. polymyxa. Besides strain DSM 36, the P. polymyxa
strains SQR-21, CF05, CICC 10580, NRRL B-30509 and A18 shall belong
to the subspecies Paenibacillus polymyxa ssp. polymyxa.
[0109] Further, it is proposed to nominate the second sub cluster
as novel subspecies Paenibacillus polymyxa ssp. plantarum. Besides
the strains Lu16674 and Lu17007, the P. polymyxa strains M-1, 1-43,
SC2 and Sb3-1 shall belong to Paenibacillus polymyxa ssp.
plantarum.
[0110] The strain Lu17015 has only 94.9% identity (AAI) amongst the
genes of the core genome with the type strain Paenibacillus
polymyxa DSM36=ATCC 842 (FIG. 22). Thus, the strain Lu17015 could
not have been designated to the species Paenibacillus polymyxa nor
to any other known Paenibacillus species. Similar values are found
for the strains E681 (94.7%) and CR2 (94.9%). Amongst each other,
these three strains have at least 98.1% identity (AAI). According
to the species definition of Konstantinides and Tiedje (Proc Natl.
Acad. Sci. USA. 102, 2567-2572, 2005), the strain Lu17015 and also
the strains E681 and CR2 can be designated to a novel species.
Thus, a new species Paenibacillus spec. nov. epiphyticus is
proposed herewith. Consequently, the Paenibacillus strain Lu17015
belongs to Paenibacillus epiphyticus. It is proposed that said
strain shall be the type strain. Likewise, the dendrograms based on
the sequence comparisons of the five house keeping genes (FIGS.
17-21) show that this cluster of distant from all other P. polymyxa
strains. Besides Lu17015, it is proposed that the P. polymyxa
strains E681, CR2 TD94, DSM 365 and WLY78 shall belong to
Paenibacillus spec. nov. epiphyticus.
[0111] Thus, the invention relates to the microorganism strains
[0112] 4) Paenibacillus polymyxa ssp. plantarum strain Lu16774
deposited with DSMZ under Accession No. DSM 26969, [0113] 5)
Paenibacillus polymyxa ssp. plantarum strain Lu17007 deposited with
DSMZ under Accession No. DSM 26970, and [0114] 6) Paenibacillus
epiphyticus strain Lu17015 deposited with DSMZ under Accession No.
DSM 26971.
[0115] In addition to the strains Lu16774, Lu17007 and Lu17015, the
invention relates to any Paenibacillus strain, whether physically
derived from the original deposit of any of the strains Lu16774,
Lu17007 and Lu17015 or independently isolated, so long as they
retain at least one of the identifying characteristics of the
deposited Paenibacillus strains Lu16774, Lu17007 and Lu17015. Such
Paenibacillus strains of the invention include any progeny of any
of the strains Lu16774, Lu17007 and Lu17015, including mutants of
said strains.
[0116] The term "mutant" refers a microorganism obtained by direct
mutant selection but also includes microorganisms that have been
further mutagenized or otherwise manipulated (e.g., via the
introduction of a plasmid). Accordingly, embodiments include
mutants, variants, and or derivatives of the respective
microorganism, both naturally occurring and artificially induced
mutants. For example, mutants may be induced by subjecting the
microorganism to known mutagens, such as X-ray, UV radiation or
N-methyl-nitrosoguanidine, using conventional methods. Subsequent
to said treatments a screening for mutant strains showing the
desired characteristics may be performed.
[0117] Mutant strains may be obtained by any methods known in the
art such as direct mutant selection, chemical mutagenesis or
genetic manipulation (e.g., via the introduction of a plasmid). For
example, such mutants are obtainable by applying a known mutagen,
such as X-ray, UV radiation or N-methyl-nitrosoguanidine.
Subsequent to said treatments a screening for mutant strains
showing the desired characteristics may be performed.
[0118] A Paenibacillus strain of the invention is in particular one
which comprises a DNA sequence exhibiting at least at least 99.6%,
preferably at least 99.8%, even more preferably at least 99.9%, and
in particular 100.0% nucleotide sequence identity to any one of the
16S rDNA sequences of the strains Lu16774, Lu17007 and Lu17015,
i.e. to any one of those nucleotide sequences set forth in the
Sequence listing being SEQ ID NO:1, SEQ ID NO:2 and SEQ ID
NO:3.
[0119] According to a further embodiment, a Paenibacillus strain of
the invention is in particular one which comprises a DNA sequence
exhibiting 100% nucleotide sequence identity to any one of the 16S
rDNA sequences of the strains Lu16774, Lu17007 and Lu17015, i.e. to
any one of those nucleotide sequences set forth in the Sequence
listing being SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
[0120] According to a further embodiment, a Paenibacillus strain of
the invention is one whose complete 16S rDNA sequence has after
optimal alignment within the aligned sequence window at least 99.6%
identity to at least one of the sequences SEQ ID NO:1 and SEQ ID
NO:2 or at least 99.8% identity to SEQ ID NO:3; preferably at least
99.8% identity to at least one of the sequences SEQ ID NO:1, SEQ
ID:2 and SEQ ID NO:3; more preferably at least 99.9% identity to at
least one of the sequences SEQ ID NO:1, SEQ ID NO:2 and SEQ ID
NO:3; even more preferably greater than 99.9% identity to at least
one of the sequences SEQ ID NO:1, SEQ ID:2 and SEQ ID NO:3; in
particular 100% identity to at least one of the sequences SEQ ID
NO:1, SEQ ID:2 and SEQ ID NO:3.
[0121] According to a further embodiment, a Paenibacillus strain of
the invention is selected from the group consisting of: [0122] a)
strain Lu16774 deposited with DSMZ under Accession No. DSM 26969;
[0123] b) strain Lu17007 deposited with DSMZ under Accession No.
DSM 26970; [0124] c) strain Lu17015 deposited with DSMZ under
Accession No. DSM 26971; and [0125] d) a strain which comprises a
DNA sequence exhibiting [0126] d1) at least 99.6% nucleotide
sequence identity to the DNA sequences SEQ ID NO:4 or SEQ ID NO:9;
or [0127] d2) at least 99.8% nucleotide sequence identity to the
DNA sequence SEQ ID NO:14; or [0128] d3) at least 99.9% nucleotide
sequence identity to the DNA sequences SEQ ID NO:5 or SEQ ID NO:10;
or [0129] d4) at least 99.2% nucleotide sequence identity to the
DNA sequence SEQ ID NO:15; or [0130] d5) at least 99.2% nucleotide
sequence identity to the DNA sequences SEQ ID NO:6 or SEQ ID NO:11;
or [0131] d6) at least 99.8% nucleotide sequence identity to the
DNA sequence SEQ ID NO:16; or [0132] d7) at least 99.8% nucleotide
sequence identity to the DNA sequences SEQ ID NO:7 or SEQ ID NO:12;
or [0133] d8) at least 99.3% nucleotide sequence identity to the
DNA sequence SEQ ID NO:17; or [0134] d9) 100.0% nucleotide sequence
identity to the DNA sequences SEQ ID NO:8 or SEQ ID NO:13; or
[0135] d10) at least 99.8% nucleotide sequence identity to the DNA
sequence SEQ ID NO:18.
[0136] A Paenibacillus strain of the invention is in particular one
which comprises a dnaN DNA sequence exhibiting at least 99.6%
nucleotide sequence identity to the DNA sequences SEQ ID NO:4 or
SEQ ID NO:9 or which comprises a DNA sequence exhibiting at least
99.8% nucleotide sequence identity to the DNA sequence SEQ ID
NO:14.
[0137] According to a further embodiment, a Paenibacillus strain of
the invention is one whose complete dnaN DNA sequence has after
optimal alignment within the aligned sequence window at least 99.6%
identity to at least one of the DNA sequences SEQ ID NO:4 and SEQ
ID NO:9 or at least 99.8% identity to SEQ ID NO:14; preferably at
least 99.9% identity to SEQ ID NO:14; in particular 100% identity
to SEQ ID NO:14.
[0138] A Paenibacillus strain of the invention is in particular one
which comprises a DNA sequence exhibiting at least 99.8%, in
particular 100.0% nucleotide sequence identity to any one of the
dnaN DNA sequences of the strains Lu16774, Lu17007 and Lu17015,
i.e. to any one of those DNA sequences SEQ ID NO:4, SEQ ID NO:9 and
SEQ ID NO:14.
[0139] A Paenibacillus strain of the invention is in particular one
which comprises a gyrB DNA sequence exhibiting at least 99.9%
nucleotide sequence identity to the DNA sequences SEQ ID NO:5 or
SEQ ID NO:10 or which comprises a DNA sequence exhibiting at least
99.2% nucleotide sequence identity to the DNA sequence SEQ ID
NO:15.
[0140] According to a further embodiment, a Paenibacillus strain of
the invention is one whose complete gyrB DNA sequence has after
optimal alignment within the aligned sequence window at least 99.9%
identity to at least one of the DNA sequences SEQ ID NO:5 and SEQ
ID NO:10 or at least 99.9% identity to SEQ ID NO:15; preferably at
least 99.9% identity to SEQ ID NO:15; in particular 100% identity
to SEQ ID NO:15.
[0141] A Paenibacillus strain of the invention is in particular one
which comprises a DNA sequence exhibiting 100.0% nucleotide
sequence identity to any one of the gyrB DNA sequences of the
strains Lu16774, Lu17007 and Lu17015, i.e. to any one of those DNA
sequences SEQ ID NO:5, SEQ ID NO:10 and SEQ ID NO:15.
[0142] A Paenibacillus strain of the invention is in particular one
which comprises a recF DNA sequence exhibiting at least 99.2%
nucleotide sequence identity to the DNA sequences SEQ ID NO:6 or
SEQ ID NO: 11 or which comprises a DNA sequence exhibiting at least
99.8% nucleotide sequence identity to the DNA sequence SEQ ID
NO:16.
[0143] According to a further embodiment, a Paenibacillus strain of
the invention is one whose complete recF DNA sequence has after
optimal alignment within the aligned sequence window at least 99.2%
identity to at least one of the DNA sequences SEQ ID NO:6 and SEQ
ID NO: 11 or at least 99.8% identity to SEQ ID NO:16; preferably at
least 99.9% identity to SEQ ID NO:16; in particular 100% identity
to SEQ ID NO:16.
[0144] A Paenibacillus strain of the invention is in particular one
which comprises a DNA sequence exhibiting at least 99.8%, in
particular 100.0% nucleotide sequence identity to any one of the
recF DNA sequences of the strains Lu16774, Lu17007 and Lu17015,
i.e. to any one of those DNA sequences SEQ ID NO:6, SEQ ID NO:11
and SEQ ID NO:16.
[0145] A Paenibacillus strain of the invention is in particular one
which comprises a recN DNA sequence exhibiting at least 99.8%
nucleotide sequence identity to the DNA sequences SEQ ID NO:7 or
SEQ ID NO:12 or which comprises a DNA sequence exhibiting at least
99.3% nucleotide sequence identity to the DNA sequence SEQ ID
NO:17.
[0146] According to a further embodiment, a Paenibacillus strain of
the invention is one whose complete recN DNA sequence has after
optimal alignment within the aligned sequence window at least 99.8%
identity to at least one of the DNA sequences SEQ ID NO:7 and SEQ
ID NO:12 or at least 99.3% identity to SEQ ID NO:17; preferably at
least 99.6% identity to SEQ ID NO:17; in particular 100% identity
to SEQ ID NO:17.
[0147] A Paenibacillus strain of the invention is in particular one
which comprises a DNA sequence exhibiting at least 99.8%, in
particular 100.0% nucleotide sequence identity to any one of the
recN DNA sequences of the strains Lu16774, Lu17007 and Lu17015,
i.e. to any one of those DNA sequences SEQ ID NO:7, SEQ ID NO:12
and SEQ ID NO:17.
[0148] A Paenibacillus strain of the invention is in particular one
which comprises a rpoA DNA sequence exhibiting 100.0% nucleotide
sequence identity to the DNA sequences SEQ ID NO:8 or SEQ ID NO:13
or which comprises a DNA sequence exhibiting at least 99.8%
nucleotide sequence identity to the DNA sequence SEQ ID NO:18.
[0149] According to a further embodiment, a Paenibacillus strain of
the invention is one whose complete rpoA DNA sequence has after
optimal alignment within the aligned sequence window 100.0%
identity to at least one of the DNA sequences SEQ ID NO:8 and SEQ
ID NO:13 or at least 99.8% identity to SEQ ID NO:18; preferably at
least 99.9% identity to SEQ ID NO:17; in particular 100% identity
to SEQ ID NO:18.
[0150] A Paenibacillus strain of the invention is in particular one
which comprises a DNA sequence exhibiting 100.0% nucleotide
sequence identity to any one of the rpoA DNA sequences of the
strains Lu16774, Lu17007 and Lu17015, i.e. to any one of those DNA
sequences SEQ ID NO:8, SEQ ID NO:13 and SEQ ID NO:18.
[0151] A further embodiment relates to an isolated microorganism,
being a member of the family Paenibacillus, having at least one of
the identifying characteristics of one of the following strains:
[0152] 1) Paenibacillus strain Lu16774 deposited with DSMZ under
Accession No. DSM 26969, [0153] 2) Paenibacillus strain Lu17007
deposited with DSMZ under Accession No. DSM 26970, or [0154] 3)
Paenibacillus strain Lu17015 deposited with DSMZ under Accession
No. DSM 26971.
[0155] A further embodiment relates to a Paenibacillus strain,
which is selected from the group consisting of: [0156] 1) strain
Lu16774 deposited with DSMZ under Accession No. DSM 26969, [0157]
2) strain Lu17007 deposited with DSMZ under Accession No. DSM
26970, and [0158] 3) strain Lu17015 deposited with DSMZ under
Accession No. DSM 26971, [0159] 4) strains having at least one of
the identifying characteristics of one of said strains Lu16774,
Lu17007 and Lu17015.
[0160] Another embodiment of the invention relates to an isolated
microorganism selected from strains: [0161] 1) Paenibacillus strain
Lu16774 deposited with DSMZ under Accession No. DSM 26969, [0162]
2) Paenibacillus strain Lu17007 deposited with DSMZ under Accession
No. DSM 26970, and [0163] 3) Paenibacillus strain Lu17015 deposited
with DSMZ under Accession No. DSM 26971; showing antagonistic
activity against at least one plant pathogen, and being capable of
producing at least one fusaricidin-type compound; or a mutant
strain thereof retaining said capability, i.e. retaining said
antagonistic activity against at least one plant pathogen, and
retaining said capability of producing at least one
fusaricidin-type compound.
[0164] A further embodiment relates to a microorganism selected
from: [0165] 1) Paenibacillus strain Lu16774 deposited with DSMZ
under Accession No. DSM 26969, [0166] 2) Paenibacillus strain
Lu17007 deposited with DSMZ under Accession No. DSM 26970, and
[0167] 3) Paenibacillus strain Lu17015 deposited with DSMZ under
Accession No. DSM 26971; or a mutant strain thereof having all the
identifying characteristics of one of said strains.
[0168] An identifying characteristic of the deposited Paenibacillus
strains Lu16774, Lu17007 and Lu17015 is that they are capable of
producing at least one compound of formula I, preferably selected
from compounds 1A and 1B, in particular producing compounds 1A and
1B, which are metabolites of the respective strains; and the
agriculturally acceptable salts thereof.
[0169] Thus, according to one aspect of the invention,
Paenibacillus strains of the invention are capable of producing at
least one compound of formula I, more preferably producing
compounds 1A or 1B, in particular producing compounds 1A and 1B;
and the agriculturally acceptable salts thereof.
[0170] Thus, according to one aspect of the invention,
Paenibacillus strains of the invention are capable of producing at
least one compound of formula I, more preferably producing
compounds 1A or 1B, in particular producing compounds 1A and 1B;
and the agriculturally acceptable salts thereof, in a growth medium
comprising at least one source of carbon and one source of nitrogen
as defined herein.
[0171] Thus, according to one aspect of the invention,
Paenibacillus strains of the invention in a growth medium
comprising at least one source of carbon and one source of nitrogen
as defined herein produce at least one compound of formula I, more
preferably produce compounds 1A or 1B, in particular produce
compounds 1A and 1B; and the agriculturally acceptable salts
thereof.
[0172] Another embodiment of the invention relates to an isolated
microorganism selected from [0173] 1) Paenibacillus strain Lu16774
deposited with DSMZ under Accession No. DSM 26969, [0174] 2)
Paenibacillus strain Lu17007 deposited with DSMZ under Accession
No. DSM 26970, and [0175] 3) Paenibacillus strain Lu17015 deposited
with DSMZ under Accession No. DSM 26971; showing antagonistic
activity against at least one plant pathogen, and being capable of
producing at least one fusaricidin-type compound of formula I,
preferably selected from compounds 1A and 1B, in particular
producing compounds 1A and 1B; or a mutant strain thereof retaining
said capability, i.e. retaining said antagonistic activity against
at least one plant pathogen, and retaining said capability of
producing at least one fusaricidin-type compound of formula I,
preferably selected from compounds 1A and 1B, in particular
producing compounds 1A and 1B.
[0176] Another embodiment of the invention relates to an isolated
microorganism selected from [0177] 1) Paenibacillus strain Lu16774
deposited with DSMZ under Accession No. DSM 26969, [0178] 2)
Paenibacillus strain Lu17007 deposited with DSMZ under Accession
No. DSM 26970, and [0179] 3) Paenibacillus strain Lu17015 deposited
with DSMZ under Accession No. DSM 26971; showing antagonistic
activity against at least one plant pathogen, and being in a growth
medium comprising at least one source of carbon and one source of
nitrogen as defined herein capable of producing at least one
fusaricidin-type compound of formula I, preferably selected from
compounds 1A and 1B, in particular producing compounds 1A and 1B;
or a mutant strain thereof retaining said capability, i.e.
retaining said antagonistic activity against at least one plant
pathogen, and retaining said capability of producing at least one
fusaricidin-type compound of formula I, preferably selected from
compounds 1A and 1B, in particular producing compounds 1A and
1B.
[0180] Another embodiment of the invention relates to an isolated
microorganism selected from [0181] 1) Paenibacillus strain Lu16774
deposited with DSMZ under Accession No. DSM 26969, [0182] 2)
Paenibacillus strain Lu17007 deposited with DSMZ under Accession
No. DSM 26970, and [0183] 3) Paenibacillus strain Lu17015 deposited
with DSMZ under Accession No. DSM 26971; showing antagonistic
activity against at least one plant pathogen, and producing at
least one fusaricidin-type compound of formula I, preferably
selected from compounds 1A and 1B, in particular producing
compounds 1A and 1B; or a mutant strain thereof retaining said
capability, i.e. retaining said antagonistic activity against at
least one plant pathogen, and retaining said capability of
producing at least one fusaricidin-type compound of formula I,
preferably selected from compounds 1A and 1B, in particular
producing compounds 1A and 1B.
[0184] A further identifying characteristic of the deposited
Paenibacillus strains Lu16774, Lu17007 and Lu17015 or a mutant
strain thereof is that they are capable of producing at least one
compound selected from the group consisting of fusaricidin A,
fusaricidin B, fusaricidin C, fusaricidin D, LI-F06a, LI-F06b and
LI-F08b in addition to their capability of producing at least one
compound of formula I, preferably selected from compounds 1A and
1B, in particular producing compounds 1A and 1B.
[0185] Thus, according to a further aspect of the invention,
Paenibacillus strains of the invention are capable of producing at
least one fusaricidin of formula I, preferably selected from
compounds 1A and 1B, in particular producing compounds 1A and 1B,
as disclosed herein, and are capable of producing at least one
compound selected from the group consisting of fusaricidin A,
fusaricidin B, fusaricidin C, fusaricidin D, LI-F06a, LI-F06b and
LI-F08b.
[0186] According to a further aspect of the invention,
Paenibacillus strains of the invention are capable of producing at
least one fusaricidin of formula I, preferably selected from
compounds 1A and 1B, in particular producing compounds 1A and 1B,
as disclosed herein, and are capable of producing at least three
compounds selected from the group consisting of fusaricidin A,
fusaricidin B, fusaricidin C, fusaricidin D, LI-F06a, LI-F06b and
LI-F08b.
[0187] According to a further aspect of the invention,
Paenibacillus strains of the invention are capable of producing at
least one fusaricidin of formula I, preferably selected from
compounds 1A and 1B, in particular producing compounds 1A and 1B,
as disclosed herein, and are capable of producing at least five
compounds selected from the group consisting of fusaricidin A,
fusaricidin B, fusaricidin C, fusaricidin D, LI-F06a, LI-F06b and
LI-F08b.
[0188] According to a further aspect of the invention,
Paenibacillus strains of the invention are capable of producing at
least one fusaricidin of formula I, preferably selected from
compounds 1A and 1B, in particular producing compounds 1A and 1B,
as disclosed herein, and are capable of producing fusaricidin A,
fusaricidin B, fusaricidin C, fusaricidin D and LI-F08b.
[0189] A further identifying characteristic of the deposited
Paenibacillus strains are their antifungal activity. In particular,
these strains were found to be effective against infestion with
plant pathogens including Alternaria spp., Botrytis cinerea,
Phytophthora infestans, and Sclerotinia sclerotiorum wherein
Alternaria spp. is preferably selected from A. solani and A.
alternata, in particular A. solani.
[0190] Thus, according to a further aspect of the invention,
Paenibacillus strains of the invention have antifungal activity,
particularly against a plant pathogen selected from the group
consisting of Alternaria spp., Botrytis cinema, Phytophthora
infestans, and Sclerotinia sclerotiorum, wherein Alternaria spp. is
preferably selected from A. solani and A. alternata, in particular
A. solani More particularly, Paenibacillus strains of the invention
have antifungal activity against at least two or against all four
of said pathogens.
[0191] According to a further aspect of the invention,
Paenibacillus strains of the invention have antifungal activity
against the plant pathogens Alternaria solani, Botrytis cinema,
Phytophthora infestans, and Sclerotinia sclerotiorum.
[0192] Antagonistic activity of the Paenibacillus strains against
plant pathogens can be shown in an in-vitro confrontation assays
using the desired phytopathogenic fungi such as Alternaria spp.,
Botrytis cinema, Phytophthora infestans, and Sclerotinia
sclerotiorum wherein Alternaria spp. is preferably selected from A.
solani and A. alternata, in particular A. solani
[0193] As growth medium for these phytopathogenic fungi, ISP2
medium is used comprising per litre: 10 g malt extract (Sigma
Aldrich, 70167); 4 g Bacto yeast extract (Becton Dickinson,
212750); 4 g glucose monohydrate (Sigma Aldrich, 16301); 20 g Agar
(Becton Dickinson, 214510), pH about 7, Aq. bidest. As growth
medium for PHYTIN, V8 medium is used comprising per litre: 200 ml
of vegetable juice, 3 g calcium carbonate (Merck Millipore,
1020660250); 30 g Agar (Becton Dickinson, 214510), pH 6.8, Aq.
bidest.
[0194] The Paenibacillus strains are point-inoculated on one side
of an agar plate. An agar block (approx. 0.3 cm.sup.2) containing
one actively growing plant pathogen was put in the center of the
plate. After incubating for 7-14 days at about 25.degree. C., the
growth of the plant pathogen is examined, especially for inhibition
zones. The following antagonistic effects can be evaluated:
Antibiosis is scored by evaluation of the diameter of the
fungi-free zone (zone of inhibition). Competition is scored by
comparing the diameter of the growth of the fungal pathogen on
plates with bacterial strains in comparison to control plates.
Mycoparasitism can be documented in case the bacteria overgrows the
fungal pathogen and also mycoparasite the pathogens. This can be
visualized by microscopy.
[0195] Another identifying characteristic of the deposited
Paenibacillus strains Lu16774, Lu17007 and Lu17015 is that they are
capable of producing and secreting at least one lytic enzyme
preferably selected from chitinase, cellulase and amylase (see
Example 6), even more preferably at least chitinase and cellulose;
in particular in a growth medium comprising at least one source of
carbon and one source of nitrogen as defined herein.
[0196] Thus, according to a further aspect of the invention,
Paenibacillus strains of the invention are capable of producing and
secreting at least one lytic enzyme preferably selected from
chitinase, cellulase and amylase, even more preferably at least
chitinase and cellulose; in particular in a growth medium
comprising at least one source of carbon and one source of nitrogen
as defined herein.
[0197] More specifically, the present invention relates to the
deposited strains Lu16774, Lu17007 and Lu17015 and any
Paenibacillus strain having one or more of the identifying
characteristics of the deposited strain, wherein the identifying
characteristics are selected from the group consisting of: [0198]
(a) antifungal activity against a plant pathogen selected from the
group consisting of Alternaria spp., Botrytis cinerea, Phytophthora
infestans, and Sclerotinia sclerotiorum, wherein Alternaria spp. is
preferably selected from A. solani and A. alternata, in particular
A. solani, as disclosed herein; [0199] (b) the capability of
producing at least one fusaricidin-type compound of formula I, in
particular compounds 1A and/or 1B, as disclosed herein; [0200] (c)
the capability of producing at least one compound selected from the
group consisting of fusaricidins A, B, C, D, LI-F06a, LI-F06b and
LI-F08b, as disclosed herein; and [0201] (d) the capability of
producing and secreting at least one lytic enzyme selected from the
group consisting of chitinase, cellulose and amylase, as disclosed
herein. [0202] More preferably, said Paenibacillus strain has the
capabilities referred to as (b), (c) and (d) in a growth medium
comprising at least one source of carbon and one source of nitrogen
as defined herein.
[0203] In particular, Paenibacillus strains of the invention have
two or more of the identifying characteristics of the deposited
strain, with strains having at least the characteristics (a) and
(b) being particularly preferred. For instance, according to a
preferred embodiment, the strains of the invention (a) have an
antifungal activity against a plant pathogen selected from the
group consisting of Alternaria spp., Botrytis cinerea, Phytophthora
infestans, and Sclerotinia sclerotiorum, wherein Alternaria spp. is
preferably selected from A. solani and A. alternata, in particular
A. solani and (b) are capable of producing at least one compound of
formula I, and particularly compound 1B. According to a further
preferred embodiment, the strains of the invention (a) have an
antifungal activity against three or against all of the plant
pathogens selected from the group consisting of Alternaria spp.,
Botrytis cinerea, Phytophthora infestans, and Sclerotinia
sclerotiorum, wherein Alternaria spp. is preferably selected from
A. solani and A. alternata, in particular A. solani and (b) are
capable of producing at least one compound of formula I, more
preferably producing compounds 1A or 1B, in particular of producing
compounds 1A and 1B.
[0204] According to an embodiment of the invention, the strains of
the invention are provided in isolated or substantially purified
form.
[0205] The terms "isolated" or "substantially purified" are meant
to denote that the strains of the invention have been removed from
a natural environment and have been isolated or separated, and are
at least 60% free, preferably at least 75% free, and more
preferably at least 90% free, even more preferably at least 95%
free, and most preferably at least 99% free from other components
with which they were naturally associated. An isolate obtained by
culturing a single microbial colony is an example of an isolated
strain of the invention.
[0206] The strains of the invention may be provided in any
physiological state such as active or dormant. Dormant strains may
be provided for example frozen, dried, or lyophilized or partly
desiccated (procedures to produce partly desiccated organisms are
given in WO 2008/002371) or in form of spores.
[0207] According to an embodiment of the invention, the strains of
the invention are provided in the form of spores.
[0208] According to a further embodiment of the invention, the
strains of the invention are provided as a whole culture broth
comprising a strain of the invention.
[0209] The culture is preferably an isolated or substantially
purified culture.
[0210] An "isolated culture" or "substantially purified culture"
refers to a culture of the strains of the invention that does not
include significant amounts of other materials which normally are
found in natural habitat in which the strain grows and/or from
which the strain normally may be obtained. Consequently, such
"isolated culture" or "substantially purified culture" is at least
60% free, preferably at least 75% free, and more preferably at
least 90% free, even more preferably at least 95% free, and most
preferably at least 99% free from other materials which normally
are found in natural habitat in which the strain grows and/or from
which the strain normally may be obtained. Such an "isolated
culture" or "substantially purified culture" does normally not
include any other microorganism in quantities sufficient to
interfere with the replication of the strain of the invention.
Isolated cultures of the invention may, however, be combined to
prepare a mixed culture of the strains of the invention and a
further biopesticide, preferably a microbial pesticide.
[0211] The invention relates to methods for the fermentative
production of antipathogenic biopesticides as described herein.
[0212] The strains as used according to the invention can be
cultivated continuously or discontinuously in the batch process or
in the fed batch or repeated fed batch process. A review of known
methods of cultivation will be found in the textbook by Chmiel
(Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik
(Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by
Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag,
Braunschweig/Wiesbaden, 1994)).
[0213] The medium that is to be used for cultivation of the
microorganism must satisfy the requirements of the particular
strains in an appropriate manner. Descriptions of culture media for
various microorganisms are given in the handbook "Manual of Methods
for General Bacteriology" of the American Society for Bacteriology
(Washington D. C., USA, 1981).
[0214] These media that can be used according to the invention
generally comprise one or more sources of carbon, sources of
nitrogen, inorganic salts, vitamins and/or trace elements.
Preferred sources of carbon are sugars, such as mono-, di- or
polysaccharides. Very good sources of carbon are for example
glucose, fructose, mannose, galactose, ribose, sorbose, ribulose,
lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars
can also be added to the media via complex compounds, such as
molasses, or other by-products from sugar refining. It may also be
advantageous to add mixtures of various sources of carbon. Other
possible sources of carbon are oils and fats such as soybean oil,
sunflower oil, peanut oil and coconut oil, fatty acids such as
palmitic acid, stearic acid or linoleic acid, alcohols such as
glycerol, methanol or ethanol and organic acids such as acetic acid
or lactic acid. Sources of nitrogen are usually organic or
inorganic nitrogen compounds or materials containing these
compounds. Examples of sources of nitrogen include ammonia gas or
ammonium salts, such as ammonium sulfate, ammonium chloride,
ammonium phosphate, ammonium carbonate or ammonium nitrate,
nitrates, urea, amino acids or complex sources of nitrogen, such as
corn-steep liquor, soybean flour, soybean protein, yeast extract,
meat extract and others. The sources of nitrogen can be used
separately or as a mixture. Inorganic salt compounds that may be
present in the media comprise the chloride, phosphate or sulfate
salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium,
manganese, zinc, copper and iron. Inorganic sulfur-containing
compounds, for example sulfates, sulfites, dithionites,
tetrathionates, thiosulfates, sulfides, but also organic sulfur
compounds, such as mercaptans and thiols, can be used as sources of
sulfur. Phosphoric acid, potassium dihydrogenphosphate or
dipotassium hydrogenphosphate or the corresponding
sodium-containing salts can be used as sources of phosphorus.
Chelating agents can be added to the medium, in order to keep the
metal ions in solution. Especially suitable chelating agents
comprise dihydroxyphenols, such as catechol or protocatechuate, or
organic acids, such as citric acid. The fermentation media used
according to the invention may also contain other growth factors,
such as vitamins or growth promoters, which include for example
biotin, riboflavin, thiamine, folic acid, nicotinic acid,
pantothenate and pyridoxine. Growth factors and salts often come
from complex components of the media, such as yeast extract,
molasses, com-steep liquor and the like. In addition, suitable
precursors can be added to the medium. The precise composition of
the compounds in the medium is strongly dependent on the particular
experiment and must be decided individually for each specific case.
Information on media optimization can be found in the textbook
"Applied Microbiol. Physiology, A Practical Approach" (Publ. P. M.
Rhodes, P. F. Stanbury, IRL Press (1997) p. 53-73, ISBN 0 19 963577
3). Growing media can also be obtained from commercial suppliers,
such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO)
etc.
[0215] Preferred growth media that can be used according to the
invention comprise one or more sources of carbon selected from
L-arabinose, N-acetyl-D-glucosamine, D-galactose, L-aspartaic acid,
D-trehalose, D-mannose, glycerol, D-gluconic acid, D-xylose,
D-mannitol, D-ribose, D-fructose, .alpha.-D-glucose, maltose,
D-melibiose, thymidine, .alpha.-methyl-D-Galactoside,
.alpha.-D-lactose, lactulose, sucrose, uridine, .alpha.-hydroxy
glutaric acid-.gamma.-lactone, .beta.-methyl-D-glucoside, adonitol,
maltotriose, 2-deoxyadenosine, adenosine, citric acid, mucic acid,
D-cellobiose, inosine, L-serine, L-alanyl-glycine, D-galacturonic
acid, .alpha.-cyclodextrin, .beta.-cyclodextrin, dextrin, inulin,
pectin, amygdalin, gentiobiose, lactitol, D-melezitose,
.alpha.-methyl-D-glucoside, .beta.-methyl-D-galactoside,
.beta.-methyl-D-xyloside, palatinose, D-raffinose, stachyose,
turanose, .gamma.-amino butyric acid, D-gluosamine, D-lactic acid,
L-lysine, 3-hydroxy 2-butanone; and one or more sources of nitrogen
selected from ammonia, nitrite, nitrate, L-alanine, L-asparagine,
L-aspartic acid, L-glutamic acid, L-glutamie, glycine, aminoacid
dimes: Ala-Asp, AlaGln, Ala-Glu, Ala-His, Gly-Gln, Gly-Glu,
Gly-Met, and Met-Ala; in particular nitrate. These media can be
supplemented with inorganic salts and vitamins and/or trace
elements. The strains are capable to produce compounds 1A and 1B in
these growth media.
[0216] All components of the medium are sterilized, either by
heating (20 min at 2.0 bar and 121.degree. C.) or by sterile
filtration. The components can be sterilized either together, or if
necessary separately. All the components of the medium can be
present at the start of growing, or optionally can be added
continuously or by batch feed.
[0217] The temperature of the culture is normally between
15.degree. C. and 36.degree. C., preferably 25.degree. C. to
33.degree. C. and can be kept constant or can be varied during the
experiment. The pH value of the medium should be in the range from
5 to 8.5, preferably around 7.0. The pH value for growing can be
controlled during growing by adding basic compounds such as sodium
hydroxide, potassium hydroxide, ammonia or ammonia water or acid
compounds such as phosphoric acid or sulfuric acid. Antifoaming
agents, e.g. fatty acid polyglycol esters, can be used for
controlling foaming. To maintain the stability of plasmids,
suitable substances with selective action, e.g. antibiotics, can be
added to the medium. Oxygen or oxygen-containing gas mixtures, e.g.
the ambient air, are fed into the culture in order to maintain
aerobic conditions. The temperature of the culture is normally from
20.degree. C. to 45.degree. C. Culture is continued until a maximum
of the desired product has formed. This is normally achieved within
10 hours to 160 hours.
[0218] In particular, the strains of the invention may be
cultivated in a medium a variety of standard microbiology media
such as Luria-Bertani Broth (LB), trypticase-soy broth (TSB), yeast
extract/malt extract/glucose broth (YMG, ISP2) at 15.degree. C. to
36.degree. C. for 18 to 360 h in liquid media or in agar-solidified
media on a petri dish. Aeration may be necessary. The bacterial
cells (vegetative cells and spores) can be washed and concentrated
(e.g. by centrifugation at temperatures of about 15 to 30.degree.
C. for about 15 min at 7,000.times.g).
[0219] The invention also relates to culture medium obtainable by
culturing the strains of the invention in a medium and separating
the medium from the culture broth (thus, remaining when cells grown
in the medium are removed from the whole culture broth), e.g., the
supernatant of a whole culture broth, i.e., the liquid broth
remaining when cells grown in broth and other debris are removed by
centrifugation, filtration, sedimentation, or other means well
known in the art.
[0220] The supernatant can be obtained e.g. by centrifugation at
temperatures of about 2 to 30.degree. C. (more preferably at
temperatures of 4 to 20.degree. C.) for about 10 to 60 min (more
preferably about 15 to 30 min) at about 5,000 to 20,000.times.g
(more preferably at about 15,000.times.g).
[0221] Such culture medium contains pesticidal metabolites which
are produced by the cultured strain.
[0222] The invention also relates to cell-free extracts of the
strains of the invention. To produce a cell-free extract, the
strains of the invention may be cultivated as described above. The
cells can be disrupted also by high-frequency ultrasound, by high
pressure, e.g. in a French pressure cell, by osmolysis, by the
action of detergents, lytic enzymes or organic solvents, by means
of homogenizers or by a combination of several of the methods
listed. The extraction can be carried out preferably with an
organic solvent or solvent mixture, more preferably an alcohol
(e.g. methanol, ethanol, n-propanol, 2-propanol or alike), even
more preferably with 2-propanol (e.g. in a 1:1 ratio to the culture
volume). Phase separation may be enhanced by addition of salts such
as NaCl. The organic phase can be collected and the solvent or
solvent mixture may be removed by conventional distillation and/or
drying followed by resuspension in methanol and filtration.
[0223] Such extract contains pesticidal metabolites which are
produced by the cultured strain.
[0224] Pesticidal metabolites that are specific to the strains of
the invention may be recovered from such medium or extract
according to conventional methods in particular when the strains of
the invention have been cultivated as described above.
[0225] The methodology of the present invention can further include
a step of recovering individual pesticidal metabolites.
[0226] The term "recovering" includes extracting, harvesting,
isolating or purifying the compound from culture media or cell-free
extracts. Recovering the compound can be performed according to any
conventional isolation or purification methodology known in the art
including, but not limited to, treatment with a conventional resin
(e.g., anion or cation exchange resin, non-ionic adsorption resin,
etc.), treatment with a conventional adsorbent (e.g., activated
charcoal, silicic acid, silica gel, cellulose, alumina, etc.),
alteration of pH, solvent extraction (e.g., with a conventional
solvent such as an alcohol, ethyl acetate, hexane and the like),
distillation, dialysis, filtration, concentration, crystallization,
recrystallization, pH adjustment, lyophilization and the like. For
example the metabolites can be recovered from culture media by
first removing the microorganisms. The remaining broth is then
passed through or over a cation exchange resin to remove unwanted
cations and then through or over an anion exchange resin to remove
unwanted inorganic anions and organic acids.
[0227] Several metabolites have been found in whole culture broth
of the novel Paenibacillus strains. Nine metabolites have been
studied in detail and identified (see Example 7, FIG. 1). Two of
them were found to be novel (compound 1A and compound 1B).
Compounds 1A and 1B have been found to be produced by all three
Paenibacillus strains of the invention (see Table 17) but none of
them was found in the whole culture broth of the related
Paenibacillus peoriae strain NRRL BD-62.
[0228] Thus the present invention also relates to compounds of
formula I
##STR00004##
wherein [0229] R is selected from
15-guanidino-3-hydroxypentadecanoic acid (GHPD) and
12-guanidinododecanoic acid (12-GDA); [0230] X.sup.1 is threonine;
[0231] X.sup.2 is isoleucine; [0232] X.sup.3 is tyrosine; [0233]
X.sup.4 is threonine; [0234] X.sup.5 is selected from glutamine and
asparagine; [0235] X.sup.6 is alanine; and wherein an arrow defines
a single (amide) bond either between the carbonyl moiety of R and
the amino group of the amino acid X.sup.1 or between the carbonyl
group of one amino acid and the amino group of a neighboring amino
acid wherein the tip of the arrow indicates the attachment to the
amino group of said neighboring amino acid; and wherein the single
line (without an arrow head) defines a single (ester) bond between
the carbonyl group of X.sup.6 and the hydroxyl group of X.sup.1;
and the agriculturally acceptable salts thereof.
[0236] According to a further embodiment, X.sup.1 in formula I is
preferably L-threonine.
[0237] According to a further embodiment, X.sup.2 in formula I is
preferably D-isoleucine or D-allo-isoleucine.
[0238] According to a further embodiment, X.sup.3 in formula I is
preferably L-tyrosine.
[0239] According to a further embodiment, X.sup.4 in formula I is
preferably D-allo-threonine.
[0240] According to a further embodiment, X.sup.5 in formula I is
preferably D-glutamine or D-asparagine.
[0241] According to a further embodiment, R in formula I is
preferably GHPD.
[0242] The sketch of formula I for compounds of formula I may also
be depicted as follows:
##STR00005##
wherein [0243] X is selected from
--NH--(C.dbd.O)--CH.sub.2--CH(OH)--(CH.sub.2).sub.12--NH--C(.dbd.NH)NH.su-
b.2 and --NH--(C.dbd.O)--(CH.sub.2).sub.11--NH--C(.dbd.NH)NH.sub.2;
[0244] R.sup.1 is 1-hydroxyethyl; [0245] R.sup.2 is 1-methylpropyl
(sec-butyl); [0246] R.sup.3 is 4-hydroxybenzyl; [0247] R.sup.4 is
1-hydroxyethyl; [0248] R.sup.5 is selected from carbamoylethyl and
carbamoylmethyl; [0249] R.sup.6 is methyl.
[0250] Likewise, the preferred embodiments based on this
alternative sketch of formula I
[0251] are as follows:
[0252] R.sup.1 in this formula I is preferably
(1S,2R)-1-hydroxyethyl.
[0253] R.sup.2 in this formula I is preferably
(1R,2R)-1-methylpropyl or (1R,2S)-1-methylpropy.
[0254] R.sup.3 in this formula I is preferably
(S)-4-hydroxy-benzyl.
[0255] R.sup.4 in this formula I is preferably
(1S,2R)-1-hydroxyethyl.
[0256] R.sup.5 in this formula I is preferably (R)-carbamoylethyl
and (R)-carbamoylmethyl.
[0257] X in this formula I is preferably
--NH--(C.dbd.O)--CH.sub.2--CH(OH)--(CH.sub.2).sub.12--NH--C(.dbd.NH)NH.su-
b.2.
[0258] According to a further embodiment, the invention further
relates to compounds 1A and 1B, which are of formula I, wherein R
is GHPD and wherein X.sup.4 is asparagine in case of compound 1A
and X.sup.4 is glutamine in case of compound 1B:
##STR00006##
[0259] The pesticidal metabolites from the strains of the invention
are preferably selected from compounds of formula I wherein R is
GHPD, in particular selected from compounds 1A and 1B, which can be
obtained by extraction and isolation from cultures, i.e. whole
culture broths, of the strains of the invention.
[0260] Further, the fusaricidin-type compounds of formula I
including those wherein R is GHTD can be synthesized in analogy to
methods known in the art (Biopolymers 80(4), 541, 2005; J. Peptide
Sci. 12S, 219, 2006; Tetrahedron Lett. 47(48), 8587-90, 2006;
Biopolymers 88(4), 568, 2007; ChemMedChem 7, 871-882, 2012).
[0261] The invention also relates to the agriculturally acceptable
salts, particularly acid addition salts of said fusaricidin-type
compounds of formula I. Said salts can be obtained by conventional
methods well known in the art, e.g. by reacting the compounds of
the invention with a suitable acid to form an acid addition salt.
Anions of useful acid addition salts are primarily chloride,
bromide, fluoride, iodide, hydrogensulfate, methylsulfate, sulfate,
dihydrogenphosphate, hydrogen-phosphate, nitrate, bicarbonate,
carbonate, hexafluorosilicate, hexafluorophosphate, benzoate and
also the anions of C.sub.1-C.sub.4-alkanoic acids, preferably
formate, acetate, propionate and butyrate.
[0262] Consequently, the invention also relates to a whole culture
broth of a microorganism comprising at least one compound of
formula I or an agriculturally acceptable salt thereof, preferably
selected from compounds 1A and 1B or an agriculturally acceptable
salt thereof, in particular said whole culture broth comprises
compounds 1A and 1B or an agriculturally acceptable salt
thereof.
[0263] According to a further embodiment, the invention also
relates to a whole culture broth of a microorganism of the genus
Paenibacillus comprising at least one compound of formula I or an
agriculturally acceptable salt thereof, preferably selected from
compounds 1A and 1B or an agriculturally acceptable salt thereof,
in particular said whole culture broth comprises compounds 1A and
1B or an agriculturally acceptable salt thereof.
[0264] According to a further embodiment, the invention also
relates to a whole culture broth of at least one Paenibacillus
strain of the invention as identified and defined above comprising
at least one compound of formula I or an agriculturally acceptable
salt thereof, preferably selected from compounds 1A and 1B or an
agriculturally acceptable salt thereof, in particular said whole
culture broth comprises compounds 1A and 1B or an agriculturally
acceptable salt thereof.
[0265] Said fusaricidin-type compounds are secreted into the
culture medium of the respective microorganism capable of producing
it.
[0266] Consequently, the invention also relates to a culture medium
and/or a cell-free extract of a microorganism comprising at least
one compound of formula I or an agriculturally acceptable salt
thereof, preferably selected from compounds 1A and 1B or an
agriculturally acceptable salt thereof, in particular said culture
medium and/or a cell-free extract comprises compounds 1A and 1B or
an agriculturally acceptable salt thereof.
[0267] According to a further embodiment, the invention also
relates to a culture medium and/or a cell-free extract of a
microorganism of the genus Paenibacillus comprising at least one
compound of formula I or an agriculturally acceptable salt thereof,
preferably selected from compounds 1A and 1B or an agriculturally
acceptable salt thereof, in particular said culture medium and/or a
cell-free extract comprises compounds 1A and 1B or an
agriculturally acceptable salt thereof.
[0268] According to a further embodiment, the invention also
relates to culture medium and/or a cell-free extract of at least
one Paenibacillus strain of the invention as identified and defined
above comprising at least one compound of formula I or an
agriculturally acceptable salt thereof, preferably selected from
compounds 1A and 1B or an agriculturally acceptable salt thereof,
in particular said culture medium and/or a cell-free extract
comprises compounds 1A and 1B or an agriculturally acceptable salt
thereof.
[0269] The invention further relates to agrochemical compositions
comprising an auxiliary as defined below and at least one or more
of the strains, whole culture broths, cell-free extracts, culture
media and compounds of formula I, of the invention,
respectively.
[0270] As used herein, "composition" in reference to a product
(microbial strain, agent or formulation) of the present invention
refers to a combination of ingredients, wherein "formulating" is
the process of using a formula, such as a recipe, for a combination
of ingredients, to be added to form the formulation. Such
composition is also referred herein to as formulation.
[0271] The strains, whole culture broths, cell-free extracts,
culture media, compounds of formula I, and compositions of the
invention, respectively, are suitable as antifungal agents or
fungicides. They are distinguished by an outstanding effectiveness
against a broad spectrum of phytopathogenic fungi, including
soil-borne fungi, which derive especially from the classes of the
Plasmodiophoromycetes, Peronosporomycetes (syn. Oomycetes),
Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and
Deuteromycetes (syn. Fungi imperfecti). Some are systemically
effective and they can be used in crop protection as foliar
fungicides, fungicides for seed dressing and soil fungicides.
Moreover, they are suitable for controlling harmful fungi, which
inter alia occur in wood or roots of plants.
[0272] The strains, whole culture broths, cell-free extracts,
culture media, compounds of formula I, and compositions of the
invention, respectively, are particularly important in the control
of a multitude of phytopathogenic fungi on various cultivated
plants, such as cereals, e.g. wheat, rye, barley, triticale, oats
or rice; beet, e.g. sugar beet or fodder beet; fruits, such as
pomes, stone fruits or soft fruits, e.g. apples, pears, plums,
peaches, almonds, cherries, strawberries, raspberries, blackberries
or gooseberries; leguminous plants, such as lentils, peas, alfalfa
or soybeans; oil plants, such as rape, mustard, olives, sunflowers,
coconut, cocoa beans, castor oil plants, oil palms, ground nuts or
soybeans; cucurbits, such as squashes, cucumber or melons; fiber
plants, such as cotton, flax, hemp or jute; citrus fruit, such as
oranges, lemons, grapefruits or mandarins; vegetables, such as
spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes,
potatoes, cucurbits or paprika; lauraceous plants, such as
avocados, cinnamon or camphor; energy and raw material plants, such
as corn, soybean, rape, sugar cane or oil palm; corn; tobacco;
nuts; coffee; tea; bananas; vines (table grapes and grape juice
grape vines); hop; turf; sweet leaf (also called Stevia); natural
rubber plants or ornamental and forestry plants, such as flowers,
shrubs, broad-leaved trees or evergreens, e.g. conifers; and on the
plant propagation material, such as seeds, and the crop material of
these plants.
[0273] Preferably, the strains, whole culture broths, cell-free
extracts culture media, compounds of formula I; and compositions of
the invention, respectively, are used for controlling a multitude
of fungi on field crops, such as potatoes sugar beets, tobacco,
wheat, rye, barley, oats, rice, corn, cotton, soybeans, rape,
legumes, sunflowers, coffee or sugar cane; fruits; vines;
ornamentals; or vegetables, such as cucumbers, tomatoes, beans or
squashes.
[0274] The term "plant propagation material" is to be understood to
denote all the generative parts of the plant such as seeds and
vegetative plant material such as cuttings and tubers (e.g.
potatoes), which can be used for the multiplication of the plant.
This includes seeds, roots, fruits, tubers, bulbs, rhizomes,
shoots, sprouts and other parts of plants, including seedlings and
young plants, which are to be transplanted after germination or
after emergence from soil. These young plants may also be protected
before transplantation by a total or partial treatment by immersion
or pouring.
[0275] Preferably, treatment of plant propagation materials with
the strains, whole culture broths, cell-free extracts culture
media, compounds of formula I; and compositions of the invention,
respectively, is used for controlling a multitude of fungi on
cereals, such as wheat, rye, barley and oats; rice, corn, cotton
and soybeans.
[0276] The term "cultivated plants" is to be understood as
including plants which have been modified by breeding, mutagenesis
or genetic engineering including but not limiting to agricultural
biotech products on the market or in development (cf.
http://cera-gmc.org/, see GM crop database therein). Genetically
modified plants are plants, which genetic material has been so
modified by the use of recombinant DNA techniques that under
natural circumstances cannot readily be obtained by cross breeding,
mutations or natural recombination. Typically, one or more genes
have been integrated into the genetic material of a genetically
modified plant in order to improve certain properties of the plant.
Such genetic modifications also include but are not limited to
targeted post-translational modification of protein(s), oligo- or
polypeptides e.g. by glycosylation or polymer additions such as
prenylated, acetylated or famesylated moieties or PEG moieties.
[0277] Plants that have been modified by breeding, mutagenesis or
genetic engineering, e.g. have been rendered tolerant to
applications of specific classes of herbicides, such as auxin
herbicides such as dicamba or 2,4-D; bleacher herbicides such as
hydroxylphenylpyruvate dioxygenase (HPPD) inhibitors or phytoene
desaturase (PDS) inhibitors; acetolactate synthase (ALS) inhibitors
such as sulfonyl ureas or imidazolinones;
enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors, such
as glyphosate; glutamine synthetase (GS) inhibitors such as
glufosinate; protoporphyrinogen-IX oxidase inhibitors; lipid
biosynthesis inhibitors such as acetyl CoA carboxylase (ACCase)
inhibitors; or oxynil (i. e. bromoxynil or ioxynil) herbicides as a
result of conventional methods of breeding or genetic engineering.
Furthermore, plants have been made resistant to multiple classes of
herbicides through multiple genetic modifications, such as
resistance to both glyphosate and glufosinate or to both glyphosate
and a herbicide from another class such as ALS inhibitors, HPPD
inhibitors, auxin herbicides, or ACCase inhibitors. These herbicide
resistance technologies are e.g. described in Pest Managem. Sci.
61, 2005, 246; 61, 2005, 258; 61, 2005, 277; 61, 2005, 269; 61,
2005, 286; 64, 2008, 326; 64, 2008, 332; Weed Sci. 57, 2009, 108;
Austral. J. Agricult. Res. 58, 2007, 708; Science 316, 2007, 1185;
and references quoted therein. Several cultivated plants have been
rendered tolerant to herbicides by conventional methods of breeding
(mutagenesis), e.g. Clearfield.RTM. summer rape (Canola, BASF SE,
Germany) being tolerant to imidazolinones, e.g. imazamox, or
ExpressSun.RTM. sunflowers (DuPont, USA) being tolerant to sulfonyl
ureas, e.g. tribenuron. Genetic engineering methods have been used
to render cultivated plants such as soybean, cotton, corn, beets
and rape, tolerant to herbicides such as glyphosate and
glufosinate, some of which are commercially available under the
trade names RoundupReady.RTM. (glyphosate-tolerant, Monsanto,
U.S.A.), Cultivance.RTM. (imidazolinone tolerant, BASF SE, Germany)
and LibertyLink.RTM. (glufosinate-tolerant, Bayer CropScience,
Germany).
[0278] Furthermore, plants are also covered that are by the use of
recombinant DNA techniques capable to synthesize one or more
insecticidal proteins, especially those known from the bacterial
genus Bacillus, particularly from Bacillus thuringiensis, such as
.delta.-endotoxins, e.g. CryIA(b), CryIA(c), CryIF, CryIF(a2),
CryIIA(b), CryIIIA, CryIIIB(b1) or Cry9c; vegetative insecticidal
proteins (VIP), e.g. VIP1, VIP2, VIP3 or VIP3A; insecticidal
proteins of bacteria colonizing nematodes, e.g. Photorhabdus spp.
or Xenorhabdus spp.; toxins produced by animals, such as scorpion
toxins, arachnid toxins, wasp toxins, or other insect-specific
neurotoxins; toxins produced by fungi, such Streptomycetes toxins,
plant lectins, such as pea or barley lectins; agglutinins;
proteinase inhibitors, such as trypsin inhibitors, serine protease
inhibitors, patatin, cystatin or papain inhibitors;
ribosome-inactivating proteins (RIP), such as ricin, maize-RIP,
abrin, luffin, saporin or bryodin; steroid metabolism enzymes, such
as 3-hydroxysteroid oxidase, ecdysteroid-IDP-glycosyl-transferase,
cholesterol oxidases, ecdysone inhibitors or HMG-CoA-reductase; ion
channel blockers, such as blockers of sodium or calcium channels;
juvenile hormone esterase; diuretic hormone receptors (helicokinin
receptors); stilbene synthase, bibenzyl synthase, chitinases or
glucanases. In the context of the present invention these
insecticidal proteins or toxins are to be understood expressly also
as pre-toxins, hybrid proteins, truncated or otherwise modified
proteins. Hybrid proteins are characterized by a new combination of
protein domains, (see, e.g. WO 02/015701). Further examples of such
toxins or genetically modified plants capable of synthesizing such
toxins are disclosed, e.g., in EP-A 374 753, WO 93/007278, WO
95/34656, EP-A 427 529, EP-A 451 878, WO 03/18810 und WO 03/52073.
The methods for producing such genetically modified plants are
generally known to the person skilled in the art and are described,
e.g. in the publications mentioned above. These insecticidal
proteins contained in the genetically modified plants impart to the
plants producing these proteins tolerance to harmful pests from all
taxonomic groups of arthropods, especially to beetles (Coeloptera),
two-winged insects (Diptera), and moths (Lepidoptera) and to
nematodes (Nematoda). Genetically modified plants capable to
synthesize one or more insecticidal proteins are, e.g., described
in the publications mentioned above, and some of which are
commercially available such as YieldGard.RTM. (corn cultivars
producing the Cry1Ab toxin), YieldGard.RTM. Plus (corn cultivars
producing Cry1Ab and Cry3Bb1 toxins), Starlink.RTM. (corn cultivars
producing the Cry9c toxin), Herculex.RTM. RW (corn cultivars
producing Cry34Ab1, Cry35Ab1 and the enzyme
Phosphinothricin-N-Acetyttransferase [PAT]); NuCOTN.RTM. 33B
(cotton cultivars producing the Cry1Ac toxin), Bollgard.RTM. I
(cotton cultivars producing the Cry1Ac toxin), Bollgard.RTM. II
(cotton cultivars producing Cry1Ac and Cry2Ab2 toxins); VIPCOT.RTM.
(cotton cultivars producing a VIP-toxin); NewLeaf.RTM. (potato
cultivars producing the Cry3A toxin); Bt-Xtra.RTM.,
NatureGard.RTM., KnockOut.RTM., BiteGard.RTM., Protecta.RTM., Bt11
(e.g. Agrisure.RTM. CB) and Bt176 from Syngenta Seeds SAS, France,
(corn cultivars producing the Cry1Ab toxin and PAT enzyme), MIR604
from Syngenta Seeds SAS, France (corn cultivars producing a
modified version of the Cry3A toxin, c.f. WO 03/018810), MON 863
from Monsanto Europe S.A., Belgium (corn cultivars producing the
Cry3Bb1 toxin), IPC 531 from Monsanto Europe S.A., Belgium (cotton
cultivars producing a modified version of the Cry1Ac toxin) and
1507 from Pioneer Overseas Corporation, Belgium (corn cultivars
producing the Cry1F toxin and PAT enzyme).
[0279] Furthermore, plants are also covered that are by the use of
recombinant DNA techniques capable to synthesize one or more
proteins to increase the resistance or tolerance of those plants to
bacterial, viral or fungal pathogens. Examples of such proteins are
the so-called "pathogenesis-related proteins" (PR proteins, see,
e.g. EP-A 392 225), plant disease resistance genes (e.g. potato
cultivars, which express resistance genes acting against
Phytophthora infestans derived from the mexican wild potato Solanum
bulbocastanum) or T4-lysozym (e.g. potato cultivars capable of
synthesizing these proteins with increased resistance against
bacteria such as Erwinia amylvora). The methods for producing such
genetically modified plants are generally known to the person
skilled in the art and are described, e.g. in the publications
mentioned above.
[0280] Furthermore, plants are also covered that are by the use of
recombinant DNA techniques capable to synthesize one or more
proteins to increase the productivity (e.g. bio mass production,
grain yield, starch content, oil content or protein content),
tolerance to drought, salinity or other growth-limiting
environmental factors or tolerance to pests and fungal, bacterial
or viral pathogens of those plants.
[0281] Furthermore, plants are also covered that contain by the use
of recombinant DNA techniques a modified amount of substances of
content or new substances of content, specifically to improve human
or animal nutrition, e.g. oil crops that produce health-promoting
long-chain omega-3 fatty acids or unsaturated omega-9 fatty acids
(e.g. Nexera.RTM. rape, DOW Agro Sciences, Canada).
[0282] Furthermore, plants are also covered that contain by the use
of recombinant DNA techniques a modified amount of substances of
content or new substances of content, specifically to improve raw
material production, e.g. potatoes that produce increased amounts
of amylopectin (e.g. Amflora.RTM. potato, BASF SE, Germany).
[0283] The strains, whole culture broths, cell-free extracts,
culture media, compounds of formula I, and compositions of the
invention, respectively, are particularly suitable for controlling
the following plant diseases:
Albugo spp. (white rust) on ornamentals, vegetables (e.g. A.
candida) and sunflowers (e.g. A. tragopogonis); Alternaria spp.
(Alternaria leaf spot) on vegetables, rape (A. brassicola or
brassicae), sugar beets (A. tenuis), fruits, rice, soybeans,
potatoes (e.g. A. solani or A. alternata), tomatoes (e.g. A. solani
or A. alternata) and wheat; Aphanomyces spp. on sugar beets and
vegetables; Ascochyta spp. on cereals and vegetables, e.g. A.
tritici (anthracnose) on wheat and A. hordei on barley; Bipolaris
and Drechslera spp. (teleomorph: Cochliobolus spp.), e.g. Southern
leaf blight (D. maydis) or Northern leaf blight (B. zeicola) on
corn, e.g. spot blotch (B. sorokiniana) on cereals and e.g. B.
oryzae on rice and turfs; Blumeria (formerly Eysiphe) graminis
(powdery mildew) on cereals (e.g. on wheat or barley); Botrytis
cinerea (teleomorph: Botryotinia fuckeliana grey mold) on fruits
and berries (e.g. strawberries), vegetables (e.g. lettuce, carrots,
celery and cabbages), rape, flowers, vines, forestry plants and
wheat; Bremmia lactucae (downy mildew) on lettuce; Ceratocystis
(syn. Ophiostoma) spp. (rot or wilt) on broad-leaved trees and
evergreens, e.g. C. ulmi (Dutch elm disease) on elms; Cercospora
spp. (Cercospora leaf spots) on corn (e.g. Gray leaf spot: C.
zeae-maydis), rice, sugar beets (e.g. C. beticola), sugar cane,
vegetables, coffee, soybeans (e.g. C. sojina or C. kikuchi) and
rice; Cladosporium spp. on tomatoes (e.g. C. fulvum: leaf mold) and
cereals, e.g. C. herbarum (black ear) on wheat; Claviceps purpurea
(ergot) on cereals; Cochliobolus (anamorph: Helminthosporium of
Bipolaris) spp. (leaf spots) on corn (C. carbonum), cereals (e.g.
C. sativus, anamorph: B. sorokiniana) and rice (e.g. C. miyabeanus,
anamorph: H. oryzae); Colletotrichum (teleomorph: Glomerella) spp.
(anthracnose) on cotton (e.g. C. gossypii), corn (e.g. C.
graminicola: Anthracnose stalk rot), soft fruits, potatoes (e.g. C.
coccodes black dot), beans (e.g. C. lindemuthianum) and soybeans
(e.g. C. truncatum or C. gloeosponoides); Corticium spp., e.g. C.
sasakii (sheath blight) on rice; Corynespora cassiicola (leaf
spots) on soybeans and ornamentals; Cycloconium spp., e.g. C.
oleaginum on olive trees; Cylindrocarpon spp. (e.g. fruit tree
canker or young vine decline, teleomorph: Nectria or Neonectria
spp.) on fruit trees, vines (e.g. C. liriodendri teleomorph:
Neonectria liriodendri Black Foot Disease) and ornamentals;
Dematophora (teleomorph: Roselinia) necatrix (root and stem rot) on
soybeans; Diaporthe spp., e.g. D. phaseolorum (damping off) on
soybeans; Drechslera (syn. Helminthosporium, teleomorph:
Pyrenophora) spp. on corn, cereals, such as barley (e.g. D. teres,
net blotch) and wheat (e.g. D. tritici-repentis: tan spot), rice
and turf; Esca (dieback, apoplexy) on vines, caused by Formitiporia
(syn. Phellinus) punctata, F. mediterranea, Phaeomoniella
chlamydospora (earlier Phaeoacremonium chlamydosporum),
Phaeoacremonium aleophilum and/or Botryosphaea obtusa; Elsinoe spp.
on pome fruits (E. pyri), soft fruits (E. veneta anthracnose) and
vines (E. ampelina anthracnose); Entyloma oryzae (leaf smut) on
rice; Epicoccum spp. (black mold) on wheat; Erysiphe spp. (powdery
mildew) on sugar beets (E. betae), vegetables (e.g. E. pisi), such
as cucurbits (e.g. E. cichoracearum), cabbages, rape (e.g. E.
cruciferarum); Eutypa lata (Eutypa canker or dieback, anamorph:
Cytosporina lata, syn. Libertella blepharis) on fruit trees, vines
and ornamental woods; Exserohilum (syn. Helminthosporium) spp. on
corn (e.g. E. turcicum); Fusarium (teleomorph: Gibberella) spp.
(wilt, root or stem rot) on various plants, such as F. graminearum
or F. culmorum (root rot, scab or head blight) on cereals (e.g.
wheat or barley), F. oxysporum on tomatoes, F. solani(f. sp.
glycines now syn. F. virguliforme) and F. tucumaniae and F.
brasiliense each causing sudden death syndrome on soybeans, and F.
verticillioides on corn; Gaeumannomyces graminis (take-all) on
cereals (e.g. wheat or barley) and corn; Gibberella spp. on cereals
(e.g. G. zeae) and rice (e.g. G. fujikuroi Bakanae disease);
Glomerella cingulata on vines, pome fruits and other plants and G.
gossypii on cotton; Grainstaining complex on rice; Guignardia
bidwellii (black rot) on vines; Gymnosporangium spp. on rosaceous
plants and junipers, e.g. G. sabinae (rust) on pears;
Helminthosporium spp. (syn. Drechslera, teleomorph: Cochliobolus)
on corn, cereals and rice; Hemileia spp., e.g. H. vastatrix (coffee
leaf rust) on coffee; Isariopsis clavispora (syn. Cladosporium
vitis) on vines; Macrophomina phaseolina (syn. phaseoli) (root and
stem rot) on soybeans and cotton; Microdochium (syn. Fusarium)
nivale (pink snow mold) on cereals (e.g. wheat or barley);
Microsphaera diffusa (powdery mildew) on soybeans; Monilinia spp.,
e.g. M. laxa, M. fructicola and M. fructigena (bloom and twig
blight, brown rot) on stone fruits and other rosaceous plants;
Mycosphaerella spp. on cereals, bananas, soft fruits and ground
nuts, such as e.g. M. graminicola (anamorph: Septoria tritici,
Septoria blotch) on wheat or M. fijiensis (black Sigatoka disease)
on bananas; Peronospora spp. (downy mildew) on cabbage (e.g. P.
brassicae), rape (e.g. P. parasitica), onions (e.g. P. destructor),
tobacco (P. tabacina) and soybeans (e.g. P. manshurica); Phakopsora
pachyrhizi and P. melbomiae (soybean rust) on soybeans; Phialophora
spp. e.g. on vines (e.g. P. tracheiphila and P. tetraspora) and
soybeans (e.g. P. gregata stem rot); Phoma lingam (root and stem
rot) on rape and cabbage and P. betae (root rot, leaf spot and
damping-off) on sugar beets; Phomopsis spp. on sunflowers, vines
(e.g. P. viticola can and leaf spot) and soybeans (e.g. stem rot:
P. phaseoli, teleomorph: Diaporthe phaseolorum); Physoderma maydis
(brown spots) on corn; Phytophthora spp. (wilt, root, leaf, fruit
and stem root) on various plants, such as paprika and cucurbits
(e.g. P. capsici), soybeans (e.g. P. megasperma, syn. P. sojae),
potatoes and tomatoes (e.g. P. infestans late blight) and
broad-leaved trees (e.g. P. ramorum: sudden oak death);
Plasmodiophora brassicae (club root) on cabbage, rape, radish and
other plants; Plasmopara spp., e.g. P. viticola (grapevine downy
mildew) on vines and P. halstedii on sunflowers; Podosphaera spp.
(powdery mildew) on rosaceous plants, hop, pome and soft fruits,
e.g. P. leucotricha on apples; Polymyxa spp., e.g. on cereals, such
as barley and wheat (P. graminis) and sugar beets (P. betae) and
thereby transmitted viral diseases; Pseudocercosporella
herpoinchoides (eyespot, teleomorph: Tapesia yallundae) on cereals,
e.g. wheat or barley; Pseudoperonospora (downy mildew) on various
plants, e.g. P. cubensis on cucurbits or P. humili on hop;
Pseudopezicula tracheiphila (red fire disease or `rotbrenner`,
anamorph: Phialophora) on vines; Puccinia spp. (rusts) on various
plants, e.g. P. triticina (brown or leaf rust), P. striiformis
(stripe or yellow rust), P. hordei(dwarf rust), P. graminis (stem
or black rust) or P. recondita (brown or leaf rust) on cereals,
such as e.g. wheat, barley or rye, P. kuehnii(orange rust) on sugar
cane and P. asparagi on asparagus; Pyrenophora (anamorph:
Drechslera) tritici-repentis (tan spot) on wheat or P. teres (net
blotch) on barley; Pyriculania spp., e.g. P. oryzae (teleomorph:
Magnaporthe grisea, rice blast) on rice and P. grisea on turf and
cereals; Pythium spp. (damping-off) on turf, rice, corn, wheat,
cotton, rape, sunflowers, soybeans, sugar beets, vegetables and
various other plants (e.g. P. ultimum or P. aphanidermatum);
Ramularia spp., e.g. R. collo-cygni (Ramularia leaf spots,
Physiological leaf spots) on barley and R. beticola on sugar beets;
Rhizoctonia spp. on cotton, rice, potatoes, turf, corn, rape,
potatoes, sugar beets, vegetables and various other plants, e.g. R.
solani (root and stem rot) on soybeans, R. solani(sheath blight) on
rice or R. cerealis (Rhizoctonia spring blight) on wheat or barley;
Rhizopus stolonifer(black mold, soft rot) on strawberries, carrots,
cabbage, vines and tomatoes; Rhynchosporium secalis (scald) on
barley, rye and triticale; Sarocladium oryzae and S. attenuatum
(sheath rot) on rice; Sclerotinia spp. (stem rot or white mold) on
vegetables and field crops, such as rape, sunflowers (e.g. S.
sclerotiorum) and soybeans (e.g. S. rolfsii or S. sclerotiorum);
Septoria spp. on various plants, e.g. S. glycines (brown spot) on
soybeans, S. tritici (Septoria blotch) on wheat and S. (syn.
Stagonospora) nodorum (Stagonospora blotch) on cereals; Uncinula
(syn. Erysiphe) necator (powdery mildew, anamorph: Oidium tucken)
on vines; Setospaeria spp. (leaf blight) on corn (e.g. S. turcicum,
syn. Helminthosporium turcicum) and turf; Sphacelotheca spp. (smut)
on corn, (e.g. S. reiliana. head smut), sorghum und sugar cane;
Sphaerotheca fuliginea (powdery mildew) on cucurbits; Spongospora
subterranea (powdery scab) on potatoes and thereby transmitted
viral diseases; Stagonospora spp. on cereals, e.g. S. nodorum
(Stagonospora blotch, teleomorph: Leptosphaeria [syn.
Phaeosphaeria] nodorum) on wheat; Synchytrium endobioticum on
potatoes (potato wart disease); Taphrina spp., e.g. T. deformans
(leaf curl disease) on peaches and T. pruni (plum pocket) on plums;
Thielaviopsis spp. (black root rot) on tobacco, pome fruits,
vegetables, soybeans and cotton, e.g. T. basicola (syn. Chalara
elegans); Tilletia spp. (common bunt or stinking smut) on cereals,
such as e.g. T. tritici (syn. T. caries, wheat bunt) and T.
controversa (dwarf bunt) on wheat; Typhula incarnata (grey snow
mold) on barley or wheat; Urocystis spp., e.g. U. occulta (stem
smut) on rye; Uromyces spp. (rust) on vegetables, such as beans
(e.g. U. appendiculatus, syn. U. phaseoli) and sugar beets (e.g. U.
betae); Ustilago spp. (loose smut) on cereals (e.g. U. nuda and U.
avaenae), corn (e.g. U. maydis. corn smut) and sugar cane; Venturia
spp. (scab) on apples (e.g. V. inaequalis) and pears; and
Verticilium spp. (wilt) on various plants, such as fruits and
ornamentals, vines, soft fruits, vegetables and field crops, e.g.
V. dahliae on strawberries, rape, potatoes and tomatoes.
[0284] The strains, whole culture broths, cell-free extracts,
culture media, compounds of formula I, and compositions of the
invention, respectively, are also suitable for controlling harmful
pathogens, especially fungi, in the protection of stored products
or harvest and in the protection of materials.
[0285] The term "protection of materials" is to be understood to
denote the protection of technical and non-living materials, such
as adhesives, glues, wood, paper and paperboard, textiles, leather,
paint dispersions, plastics, cooling lubricants, fiber or fabrics,
against the infestation and destruction by harmful microorganisms,
such as fungi and bacteria. As to the protection of wood and other
materials, the particular attention is paid to the following
harmful fungi: Ascomycetes such as Ophiostoma spp., Ceratocystis
spp., Aureobasidium pullulans, Sclerophoma spp., Chaetomium spp.,
Humicola spp., Petriella spp., Trichurus spp.; Basidiomycetes such
as Coniophora spp., Coriolus spp., Gloeophyllum spp., Lentinus
spp., Pleurotus spp., Poria spp., Serpula spp. and Tyromyces spp.,
Deuteromycetes such as Aspergillus spp., Cladosporium spp.,
Penicillium spp., Trichorma spp., Alternaria spp., Paecilomyces
spp. and Zygomycetes such as Mucor spp., and in addition in the
protection of stored products and harvest the following yeast fungi
are worthy of note: Candida spp. and Saccharomyces cerevisiae.
[0286] The method of treatment according to the invention can also
be used in the field of protecting stored products or harvest
against attack of fungi and microorganisms. According to the
present invention, the term "stored products" is understood to
denote natural substances of plant or animal origin and their
processed forms, which have been taken from the natural life cycle
and for which long-term protection is desired. Stored products of
crop plant origin, such as plants or parts thereof, for example
stalks, leafs, tubers, seeds, fruits or grains, can be protected in
the freshly harvested state or in processed form, such as
pre-dried, moistened, comminuted, ground, pressed or roasted, which
process is also known as post-harvest treatment. Also falling under
the definition of stored products is timber, whether in the form of
crude timber, such as construction timber, electricity pylons and
barriers, or in the form of finished articles, such as furniture or
objects made from wood. Stored products of animal origin are hides,
leather, furs, hairs and the like. The combinations according the
present invention can prevent disadvantageous effects such as
decay, discoloration or mold. Preferably "stored products" is
understood to denote natural substances of plant origin and their
processed forms, more preferably fruits and their processed forms,
such as pomes, stone fruits, soft fruits and citrus fruits and
their processed forms.
[0287] The strains, whole culture broths, cell-free extracts,
culture media, compounds of formula I, and compositions of the
invention, respectively, may be used for improving the health of a
plant.
[0288] The invention also relates to a method for improving plant
health by treating a plant, its propagation material and/or the
locus where the plant is growing or is to grow with an effective
amount of the strains, whole culture broths, cell-free extracts,
culture media, compounds of formula I, and compositions.
[0289] The term "plant health" is to be understood to denote a
condition of the plant and/or its products which is determined by
several indicators alone or in combination with each other such as
yield (e.g. increased biomass and/or increased content of valuable
ingredients), plant vigor (e.g. improved plant growth and/or
greener leaves ("greening effect")), quality (e.g. improved content
or composition of certain ingredients) and tolerance to abiotic
and/or biotic stress. The above identified indicators for the
health condition of a plant may be interdependent or may result
from each other.
[0290] Healthier plants are desirable since they result among
others in better yields and/or a better quality of the plants or
crops, specifically better quality of the harvested plant parts.
Healthier plants also better resist to biotic and/or abiotic
stress. A high resistance against biotic stresses in turn allows
the person skilled in the art to reduce the quantity of pesticides
applied and consequently to slow down the development of
resistances against the respective pesticides.
[0291] It has to be emphasized that the above mentioned effects of
the strains, whole culture broths, cell-free extracts, culture
media, compounds of formula I, and compositions of the invention,
respectively, i.e. enhanced health of the plant, are also present
when the plant is not under biotic stress and in particular when
the plant is not under pest pressure.
[0292] For example, for seed treatment and soil applications, it is
evident that a plant suffering from fungal or insecticidal attack
shows reduced germination and emergence leading to poorer plant or
crop establishment and vigor, and consequently, to a reduced yield
as compared to a plant propagation material which has been
subjected to curative or preventive treatment against the relevant
pest and which can grow without the damage caused by the biotic
stress factor. However, the methods according to the invention lead
to an enhanced plant health even in the absence of any biotic
stress. This means that the positive effects of the strains, whole
culture broths, cell-free extracts, culture media, compounds of
formula I, and compositions of the invention, respectively, cannot
be explained just by the pesticidal activities of strains, whole
culture broths, cell-free extracts, culture media, compounds of
formula I, and compositions of the invention, respectively, but are
based on further activity profiles. Accordingly, the application of
the strains, whole culture broths, cell-free extracts, culture
media, compounds of formula I, and compositions of the invention,
respectively, can also be carried out in the absence of pest
pressure.
[0293] In an equally preferred embodiment, the present invention
relates to a method for improving the health of plants grown from
said plant propagation material, wherein the plant propagation
material is treated with an effective amount of at least one
strains, whole culture broths, cell-free extract, culture medium,
compound of formula I, or a composition of the invention.
[0294] Each plant health indicator listed below, which is selected
from the groups consisting of yield, plant vigor, quality and
tolerance of the plant to abiotic and/or biotic stress, is to be
understood as a preferred embodiment of the present invention
either each on its own or preferably in combination with each
other.
[0295] According to the present invention, "increased yield" of a
plant means that the yield of a product of the respective plant is
increased by a measurable amount over the yield of the same product
of the plant produced under the same conditions, but without the
application of the strains, whole culture broths, cell-free
extracts, culture media, compounds of formula I, and compositions
of the invention, respectively.
[0296] For seed treatment e.g. as inoculant and/or foliar
application forms, increased yield can be characterized, among
others, by the following improved properties of the plant:
increased plant weight; and/or increased plant height; and/or
increased biomass such as higher overall fresh weight (FW); and/or
increased number of flowers per plant; and/or higher grain and/or
fruit yield; and/or more tillers or side shoots (branches); and/or
larger leaves; and/or increased shoot growth; and/or increased
protein content; and/or increased oil content; and/or increased
starch content; and/or increased pigment content; and/or increased
chlorophyll content (chlorophyll content has a positive correlation
with the plant's photosynthesis rate and accordingly, the higher
the chlorophyll content the higher the yield of a plant) and/or
increased quality of a plant.
[0297] "Grain" and "fruit" are to be understood as any plant
product which is further utilized after harvesting, e.g. fruits in
the proper sense, vegetables, nuts, grains, seeds, wood (e.g. in
the case of silviculture plants), flowers (e.g. in the case of
gardening plants, ornamentals) etc., that is anything of economic
value that is produced by the plant.
[0298] According to the present invention, the yield is increased
by at least 4%. In general, the yield increase may even be higher,
for example 5 to 10%, more preferable by 10 to 20%, or even 20 to
30%
[0299] According to the present invention, the yield--if measured
in the absence of pest pressure--is increased by at least 2% In
general, the yield increase may even be higher, for example until
4% to 5% or even more.
[0300] Another indicator for the condition of the plant is the
plant vigor. The plant vigor becomes manifest in several aspects
such as the general visual appearance.
[0301] For foliar applications, improved plant vigor can be
characterized, among others, by the following improved properties
of the plant: improved vitality of the plant; and/or improved plant
growth; and/or improved plant development; and/or improved visual
appearance; and/or improved plant stand (less plant verse/lodging
and/or bigger leaf blade; and/or bigger size; and/or increased
plant height; and/or increased tiller number; and/or increased
number of side shoots; and/or increased number of flowers per
plant; and/or increased shoot growth; and/or enhanced
photosynthetic activity (e.g. based on increased stomatal
conductance and/or increased CO.sub.2 assimilation rate)); and/or
earlier flowering; and/or earlier fruiting; and/or earlier grain
maturity; and/or less non-productive tillers; and/or less dead
basal leaves; and/or less input needed (such as fertilizers or
water); and/or greener leaves; and/or complete maturation under
shortened vegetation periods; and/or easier harvesting; and/or
faster and more uniform ripening; and/or longer shelf-life; and/or
longer panicles; and/or delay of senescence; and/or stronger and/or
more productive tillers; and/or better extractability of
ingredients; and/or improved quality of seeds (for being seeded in
the following seasons for seed production); and/or reduced
production of ethylene and/or the inhibition of its reception by
the plant.
[0302] Another indicator for the condition of the plant is the
"quality" of a plant and/or its products.
[0303] According to the present invention, enhanced quality means
that certain plant characteristics such as the content or
composition of certain ingredients are increased or improved by a
measurable or noticeable amount over the same factor of the plant
produced under the same conditions, but without the application of
the strains, whole culture broths, cell-free extracts, culture
media, compounds of formula I, and compositions of the invention,
respectively.
[0304] Enhanced quality can be characterized, among others, by
following improved properties of the plant or its product:
increased nutrient content; and/or increased protein content;
and/or increased oil content; and/or increased starch content;
and/or increased content of fatty acids; and/or increased
metabolite content; and/or increased carotenoid content; and/or
increased sugar content; and/or increased amount of essential amino
acids; and/or improved nutrient composition; and/or improved
protein composition; and/or improved composition of fatty acids;
and/or improved metabolite composition; and/or improved carotenoid
composition; and/or improved sugar composition; and/or improved
amino acids composition; and/or improved or optimal fruit color;
and/or improved leaf color; and/or higher storage capacity; and/or
better processability of the harvested products.
[0305] Another indicator for the condition of the plant is the
plant's tolerance or resistance to biotic and/or abiotic stress
factors. Biotic and abiotic stress, especially over longer terms,
can have harmful effects on plants.
[0306] Biotic stress is caused by living organisms while abiotic
stress is caused for example by environmental extremes. According
to the present invention, "enhanced tolerance or resistance to
biotic and/or abiotic stress factors" means (1.) that certain
negative factors caused by biotic and/or abiotic stress are
diminished in a measurable or noticeable amount as compared to
plants exposed to the same conditions, but without being treated
with the strains, whole culture broths, cell-free extracts, culture
media, compounds of formula I, and compositions of the invention,
respectively, and (2.) that the negative effects are not diminished
by a direct action of the strains, whole culture broths, cell-free
extracts, culture media, compounds of formula I, and compositions
of the invention, respectively, on the stress factors, e.g. by its
fungicidal or insecticidal action which directly destroys the
microorganisms or pests, but rather by a stimulation of the plants'
own defensive reactions against said stress factors.
[0307] Negative factors caused by biotic stress such as pathogens
and pests are widely known and are caused by living organisms, such
as competing plants (for example weeds), microorganisms (such as
phytopathogenic fungi and/or bacteria) and/or viruses.
[0308] Negative factors caused by abiotic stress are also
well-known and can often be observed as reduced plant vigor (see
above), for example:
[0309] less yield and/or less vigor, for both effects examples can
be burned leaves, less flowers, pre-mature ripening, later crop
maturity, reduced nutritional value amongst others.
[0310] Abiotic stress can be caused for example by: extremes in
temperature such as heat or cold (heat stress/cold stress); and/or
strong variations in temperature; and/or temperatures unusual for
the specific season; and/or drought (drought stress); and/or
extreme wetness; and/or high salinity (salt stress); and/or
radiation (for example by increased UV radiation due to the
decreasing ozone layer); and/or increased ozone levels (ozone
stress); and/or organic pollution (for example by phytotoxic
amounts of pesticides); and/or inorganic pollution (for example by
heavy metal contaminants).
[0311] As a result of biotic and/or abiotic stress factors, the
quantity and the quality of the stressed plants decrease. As far as
quality (as defined above) is concerned, reproductive development
is usually severely affected with consequences on the crops which
are important for fruits or seeds. Synthesis, accumulation and
storage of proteins are mostly affected by temperature; growth is
slowed by almost all types of stress; polysaccharide synthesis,
both structural and storage is reduced or modified: these effects
result in a decrease in biomass (yield) and in changes in the
nutritional value of the product.
[0312] As pointed out above, the above identified indicators for
the health condition of a plant may be interdependent and may
result from each other. For example, an increased resistance to
biotic and/or abiotic stress may lead to a better plant vigor, e.g.
to better and bigger crops, and thus to an increased yield.
Inversely, a more developed root system may result in an increased
resistance to biotic and/or abiotic stress. However, these
interdependencies and interactions are neither all known nor fully
understood and therefore the different indicators are described
separately.
[0313] In one embodiment the strains, whole culture broths,
cell-free extracts, culture media, compounds of formula I, and
compositions of the invention, respectively, effectuate an
increased yield of a plant or its product. In another embodiment
the strains, whole culture broths, cell-free extracts, culture
media, compounds of formula I, and compositions of the invention,
respectively, effectuate an increased vigor of a plant or its
product. In another embodiment the strains, whole culture broths,
cell-free extracts, culture media, compounds of formula I, and
compositions of the invention, respectively, effectuate in an
increased quality of a plant or its product. In yet another
embodiment the strains, whole culture broths, cell-free extracts,
culture media, compounds of formula I, and compositions of the
invention, respectively, effectuate an increased tolerance and/or
resistance of a plant or its product against biotic stress. In yet
another embodiment the strains, whole culture broths, cell-free
extracts, culture media, compounds of formula I, and compositions
of the invention, respectively, effectuate an increased tolerance
and/or resistance of a plant or its product against abiotic
stress.
[0314] The strains, whole culture broths, cell-free extracts,
culture media and compounds of formula I, respectively, are
employed as such or in form of compositions by treating the fungi
or the plants, plant propagation materials, such as seeds, soil,
surfaces, materials or rooms to be protected from fungal attack
with a fungicidally effective amount of the active substances. The
application can be carried out both before and after the infection
of the plants, plant propagation materials, such as seeds, soil,
surfaces, materials or rooms by the fungi.
[0315] The term "effective amount" denotes an amount which is
sufficient for controlling harmful fungi on cultivated plants or in
the protection of materials and which does not result in a
substantial damage to the treated plants. Such an amount can vary
in a broad range and is dependent on various factors, such as the
fungal species to be controlled, the treated cultivated plant or
material, the climatic conditions and the strains, whole culture
broths, cell-free extracts, culture media and compounds of formula
I or salt thereof, of the invention, respectively, used.
[0316] Plant propagation materials may be treated with the strains,
whole culture broths, cell-free extracts, culture media, compounds
of formula I, and compositions of the invention, respectively,
prophylactically either at or before planting or transplanting.
[0317] The strains of the invention can be formulated as an
inoculant for a plant. The term "inoculant" means a composition
that includes an isolated strain of the invention and optionally a
carrier, which may include a biologically acceptable medium.
[0318] Such inoculants and other suitable compositions can be
prepared as compositions comprising besides the active ingredients
at least one auxiliary (inert ingredient) by usual means (see e.g.
H. D. Burges: Formulation of Microbial Biopesticides, Springer,
1998).
[0319] To produce a dry formulation, bacterial cells, preferably
spores can be suspended in a suitable dry carrier (e.g. clay). To
produce a liquid formulation, cells, preferably spores, can be
re-suspended in a suitable liquid carrier (e.g. water-based) to the
desired spore density. The spore density number of spores per ml
can be determined by identifying the number of colony-forming units
(CFU) on agar medium e.g. potato dextrose agar after incubation for
several days at temperatures of about 20 to about 30.degree. C.
[0320] According to one embodiment, individual components of the
composition according to the invention such as parts of a kit or
parts of a binary or ternary mixture may be mixed by the user
himself in a spray tank or any other kind of vessel used for
applications (e.g seed treater drums, seed pelleting machinery,
knapsack sprayer) and further auxiliaries may be added, if
appropriate. When living microorganisms, such as the Paenibacillus
strains of the invention, form part of such kit, it must be taken
care that choice and amounts of the other parts of the kit (e.g.
chemical pesticidal agents) and of the further auxiliaries should
not influence the viability of the microbial pesticides in the
composition mixed by the user. Especially for bactericides and
solvents, compatibility with the respective microbial pesticide has
to be taken into account.
[0321] The strains, whole culture broths, cell-free extracts,
culture media and/or compounds of formula I of the invention can be
converted into customary types of agrochemical compositions, e.g.
solutions, emulsions, suspensions, dusts, powders, pastes,
granules, pressings, capsules, and mixtures thereof. Examples for
composition types are suspensions (e.g. SC, OD, FS), emulsifiable
concentrates (e.g. EC), emulsions (e.g. EW, EO, ES, ME), capsules
(e.g. CS, ZC), pastes, pastilles, wettable powders or dusts (e.g.
WP, SP, WS, DP, DS), pressings (e.g. BR, TB, DT), granules (e.g.
WG, SG, GR, FG, GG, MG), insecticidal articles (e.g. LN), as well
as gel formulations for the treatment of plant propagation
materials such as seeds (e.g. GF). These and further compositions
types are defined in the "Catalogue of pesticide formulation types
and international coding system", Technical Monograph No. 2,
6.sup.th Ed. May 2008, CropLife International.
[0322] The compositions are prepared in a known manner, such as
described by Mollet and Grubemann, Formulation technology, Wiley
VCH, Weinheim, 2001; or Knowles, New developments in crop
protection product formulation, Agrow Reports DS243, T&F
Informa, London, 2005.
[0323] Suitable auxiliaries are solvents, liquid carriers, solid
carriers or fillers, surfactants, dispersants, emulsifiers,
wetters, adjuvants, solubilizers, penetration enhancers, protective
colloids, adhesion agents, thickeners, humectants, repellents,
attractants, feeding stimulants, compatibilizers, bactericides,
anti-freezing agents, anti-foaming agents, colorants, tackifiers
and binders.
[0324] Suitable solvents and liquid carriers are water and organic
solvents, such as mineral oil fractions of medium to high boiling
point, e.g. kerosene, diesel oil; oils of vegetable or animal
origin; aliphatic, cyclic and aromatic hydrocarbons, e.g. toluene,
paraffin, tetrahydronaphthalene, alkylated naphthalenes; alcohols,
e.g. ethanol, propanol, butanol, benzylalcohol, cyclohexanol;
glycols; DMSO; ketones, e.g. cyclohexanone; esters, e.g. lactates,
carbonates, fatty acid esters, gamma-butyrolactone; fatty acids;
phosphonates; amines; amides, e.g. N-methylpyrrolidone, fatty acid
dimethylamides; and mixtures thereof.
[0325] Suitable solid carriers or fillers are mineral earths, e.g.
silicates, silica gels, talc, kaolins, limestone, lime, chalk,
clays, dolomite, diatomaceous earth, bentonite, calcium sulfate,
magnesium sulfate, magnesium oxide; polysaccharides, e.g.
cellulose, starch; fertilizers, e.g. ammonium sulfate, ammonium
phosphate, ammonium nitrate, ureas; products of vegetable origin,
e.g. cereal meal, tree bark meal, wood meal, nutshell meal, and
mixtures thereof.
[0326] Suitable surfactants are surface-active compounds, such as
anionic, cationic, nonionic and amphoteric surfactants, block
polymers, polyelectrolytes, and mixtures thereof. Such surfactants
can be used as emulsifier, dispersant, solubilizer, wetter,
penetration enhancer, protective colloid, or adjuvant. Examples of
surfactants are listed in McCutcheon's, Vol. 1: Emulsifiers &
Detergents, McCutcheon's Directories, Glen Rock, USA, 2008
(International Ed. or North American Ed.).
[0327] Suitable anionic surfactants are alkali, alkaline earth or
ammonium salts of sulfonates, sulfates, phosphates, carboxylates,
and mixtures thereof. Examples of sulfonates are
alkylarylsulfonates, diphenylsulfonates, alpha-olefin sulfonates,
lignine sulfonates, sulfonates of fatty acids and oils, sulfonates
of ethoxylated alkylphenols, sulfonates of alkoxylated arylphenols,
sulfonates of condensed naphthalenes, sulfonates of dodecyl- and
tridecylbenzenes, sulfonates of naphthalenes and alkylnaphthalenes,
sulfosuccinates or sulfosuccinamates. Examples of sulfates are
sulfates of fatty acids and oils, of ethoxylated alkylphenols, of
alcohols, of ethoxylated alcohols, or of fatty acid esters.
Examples of phosphates are phosphate esters. Examples of
carboxylates are alkyl carboxylates, and carboxylated alcohol or
alkylphenol ethoxylates.
[0328] Suitable nonionic surfactants are alkoxylates, N-substituted
fatty acid amides, amine oxides, esters, sugar-based surfactants,
polymeric surfactants, and mixtures thereof. Examples of
alkoxylates are compounds such as alcohols, alkylphenols, amines,
amides, arylphenols, fatty acids or fatty acid esters which have
been alkoxylated with 1 to 50 equivalents. Ethylene oxide and/or
propylene oxide may be employed for the alkoxylation, preferably
ethylene oxide. Examples of N-substituted fatty acid amides are
fatty acid glucamides or fatty acid alkanolamides. Examples of
esters are fatty acid esters, glycerol esters or monoglycerides.
Examples of sugar-based surfactants are sorbitans, ethoxylated
sorbitans, sucrose and glucose esters or alkylpolyglucosides.
Examples of polymeric surfactants are home- or copolymers of
vinylpyrrolidone, vinyl alcohols, or vinyl acetate.
[0329] Suitable cationic surfactants are quaternary surfactants,
for example quaternary ammonium compounds with one or two
hydrophobic groups, or salts of long-chain primary amines. Suitable
amphoteric surfactants are alkylbetains and imidazolines. Suitable
block polymers are block polymers of the A-B or A-B-A type
comprising blocks of polyethylene oxide and polypropylene oxide, or
of the A-B-C type comprising alkanol, polyethylene oxide and
polypropylene oxide. Suitable polyelectrolytes are polyacids or
polybases. Examples of polyacids are alkali salts of polyacrylic
acid or polyacid comb polymers. Examples of polybases are polyvinyl
amines or polyethyleneamines.
[0330] Suitable adjuvants are compounds, which have a negligible or
even no pesticidal activity themselves, and which improve the
biological performance of cell-free extract, culture medium or
metabolite on the target. Examples are surfactants, mineral or
vegetable oils, and other auxilaries. Further examples are listed
by Knowles, Adjuvants and additives, Agrow Reports DS256, T&F
Informa UK, 2006, chapter 5.
[0331] Suitable thickeners are polysaccharides (e.g. xanthan gum,
carboxymethyl cellulose), inorganic clays (organically modified or
unmodified), polycarboxylates, and silicates.
[0332] Suitable bactericides are bronopol and isothiazolinone
derivatives such as alkylisothiazolinones and benzisothiazolinones.
Suitable anti-freezing agents are ethylene glycol, propylene
glycol, urea and glycerin. Suitable anti-foaming agents are
silicones, long chain alcohols, and salts of fatty acids. Suitable
colorants (e.g. in red, blue, or green) are pigments of low water
solubility and water-soluble dyes. Examples are inorganic colorants
(e.g. iron oxide, titan oxide, iron hexacyanoferrate) and organic
colorants (e.g. alizarin-, azo- and phthalocyanine colorants).
Suitable tackifiers or binders are polyvinyl pyrrolidones,
polyvinyl acetates, polyvinyl alcohols, polyacrylates, biological
or synthetic waxes, and cellulose ethers.
[0333] When living microorganisms, such as Paenibacillus strains of
the invention in form of cells or spores, form part of the
compositions, such compositions can be prepared as compositions
comprising besides the active ingredients at least one auxiliary
(inert ingredient) by usual means (see e.g. H. D. Burges:
Formulation of Microbial Biopesticides, Springer, 1998). Suitable
customary types of such compositions are suspensions, dusts,
powders, pastes, granules, pressings, capsules, and mixtures
thereof. Examples for composition types are suspensions (e. g. SC,
OD, FS), capsules (e.g. CS, ZC), pastes, pastilles, wettable
powders or dusts (e.g. WP, SP, WS, DP, DS), pressings (e.g. BR, TB,
DT), granules (e.g. WG, SG, GR, FG, GG, MG), insecticidal articles
(e.g. LN), as well as gel formulations for the treatment of plant
propagation materials such as seeds (e.g. GF). Herein, it has to be
taken into account that each formulation type or choice of
auxiliary should not influence the viability of the microorganism
during storage of the composition and when finally applied to the
soil, plant or plant propagation material. Suitable formulations
are e.g. mentioned in WO 2008/002371, U.S. Pat. No. 6,955,912, U.S.
Pat. No. 5,422,107.
[0334] Examples for suitable auxiliaries are those mentioned
earlier herein, wherein it must be taken care that choice and
amounts of such auxiliaries should not influence the viability of
the microbial pesticides in the composition. Especially for
bactericides and solvents, compatibility with the respective
microorganism of the respective microbial pesticide has to be taken
into account. In addition, compositions with microbial pesticides
may further contain stabilizers or nutrients and UV protectants.
Suitable stabilizers or nutrients are e.g. alpha-tocopherol,
trehalose, glutamate, potassium sorbate, various sugars like
glucose, sucrose, lactose and maltodextrine (H. D. Burges:
Formulation of Microbial Biopesticides, Springer, 1998). Suitable
UV protectants are e.g. inorganic compounds like titanium dioxide,
zinc oxide and iron oxide pigments or organic compounds like
benzophenones, benzotriazoles and phenyitriazines. The compositions
may in addition to auxiliaries mentioned for compositions
comprising compounds I herein optionally comprise 0.1-80%
stabilizers or nutrients and 0.1-10% UV protectants.
[0335] The agrochemical compositions generally comprise between
0.01 and 95%, preferably between 0.1 and 90%, and in particular
between 0.5 and 75%, by weight of active substance. The active
substances are employed in a purity of from 90% to 100%, preferably
from 95% to 100% (according to NMR spectrum).
[0336] Examples for composition types and their preparation
are:
i) Water-Soluble Concentrates (SL, LS)
[0337] 10-60 wt % of a whole culture broth, cell-free extract,
culture medium or metabolite of the invention and 5-15 wt % wetting
agent (e.g. alcohol alkoxylates) are dissolved in water and/or in a
water-soluble solvent (e.g. alcohols) ad 100 wt %. The active
substance dissolves upon dilution with water.
ii) Dispersible Concentrates (DC)
[0338] 5-25 wt % of a whole culture broth, cell-free extract,
culture medium or metabolite of the invention and 1-10 wt %
dispersant (e.g. polyvinyl pyrrolidone) are dissolved in organic
solvent (e.g. cyclohexanone) ad 100 wt %. Dilution with water gives
a dispersion.
iii) Emulsifiable Concentrates (EC)
[0339] 15-70 wt % of a whole culture broth, cell-free extract,
culture medium or metabolite of the invention and 5-10 wt %
emulsifiers (e.g. calcium dodecylbenzenesulfonate and castor oil
ethoxylate) are dissolved in water-insoluble organic solvent (e.g.
aromatic hydrocarbon) ad 100 wt %. Dilution with water gives an
emulsion.
iv) Emulsions (EW, EO, ES)
[0340] 5-40 wt % of a whole culture broth, cell-free extract,
culture medium or metabolite of the invention and 1-10 wt %
emulsifiers (e.g. calcium dodecylbenzenesulfonate and castor oil
ethoxylate) are dissolved in 20-40 wt % water-insoluble organic
solvent (e.g. aromatic hydrocarbon). This mixture is introduced
into water ad 100 wt % by means of an emulsifying machine and made
into a homogeneous emulsion. Dilution with water gives an
emulsion.
v) Suspensions (SC, OD, FS)
[0341] In an agitated ball mill, 20-60 wt % of a whole culture
broth, cell-free extract, culture medium or metabolite of the
invention are comminuted with addition of 2-10 wt % dispersants and
wetting agents (e.g. sodium lignosulfonate and alcohol ethoxylate),
0.1-2 wt % thickener (e.g. xanthan gum) and water ad 100 wt % to
give a fine active substance suspension. Dilution with water gives
a stable suspension of the active substance. For FS type
composition up to 40 wt % binder (e.g. polyvinyl alcohol) is added.
[0342] vi) Water-dispersible granules and water-soluble granules
(WG, SG) 50-80 wt % of a whole culture broth, cell-free extract,
culture medium or metabolite of the invention are ground finely
with addition of dispersants and wetting agents (e.g. sodium
lignosulfonate and alcohol ethoxylate) ad 100 wt % and prepared as
water-dispersible or water-soluble granules by means of technical
appliances (e.g. extrusion, spray tower, fluidized bed).
[0343] Dilution with water gives a stable dispersion or solution of
the active substance. vii) Water-dispersible powders and
water-soluble powders (WP, SP, WS) 50-80 wt % of a whole culture
broth, cell-free extract, culture medium or metabolite of the
invention are ground in a rotor-stator mill with addition of 1-5 wt
% dispersants (e.g. sodium lignosulfonate), 1-3 wt % wetting agents
(e.g. alcohol ethoxylate) and solid carrier (e.g. silica gel) ad
100 wt %. Dilution with water gives a stable dispersion or solution
of the active substance.
viii) Gel (GW, GF)
[0344] In an agitated ball mill, 5-25 wt % of a whole culture
broth, cell-free extract, culture medium or metabolite of the
invention are comminuted with addition of 3-10 wt % dispersants
(e.g. sodium lignosulfonate), 1-5 wt % thickener (e.g.
carboxymethyl cellulose) and water ad 100 wt % to give a fine
suspension of the active substance. Dilution with water gives a
stable suspension of the active substance.
ix) Microemulsion (ME)
[0345] 5-20 wt % of a whole culture broth, cell-free extract,
culture medium or metabolite of the invention are added to 5-30 wt
% organic solvent blend (e.g. fatty acid dimethylamide and
cyclohexanone), 10-25 wt % surfactant blend (e.g. alcohol
ethoxylate and arylphenol ethoxylate), and water ad 100%. This
mixture is stirred for 1 h to produce spontaneously a
thermodynamically stable microemulsion.
x) Microcapsules (CS)
[0346] An oil phase comprising 5-50 wt % of a whole culture broth,
cell-free extract, culture medium or metabolite of the invention,
0-40 wt % water insoluble organic solvent (e.g. aromatic
hydrocarbon), 2-15 wt % acrylic monomers (e.g. methylmethacrylate,
methacrylic acid and a di- or triacrylate) are dispersed into an
aqueous solution of a protective colloid (e.g. polyvinyl alcohol).
Radical polymerization initiated by a radical initiator results in
the formation of poly(meth)acrylate microcapsules.
[0347] Alternatively, an oil phase comprising 5-50 wt % of a whole
culture broth, cell-free extract, culture medium or metabolite of
the invention, 0-40 wt % water insoluble organic solvent (e.g.
aromatic hydrocarbon), and an isocyanate monomer (e.g.
diphenylmethene-4,4'-diisocyanatae) are dispersed into an aqueous
solution of a protective colloid (e.g. polyvinyl alcohol). The
addition of a polyamine (e.g. hexamethylenediamine) results in the
formation of polyurea microcapsules. The monomers amount to 1-10 wt
%. The wt % relate to the total CS composition.
xi) Dustable powders (DP, DS)
[0348] 1-10 wt % of a whole culture broth, cell-free extract,
culture medium or metabolite of the invention are ground finely and
mixed intimately with solid carrier (e.g. finely divided kaolin) ad
100 wt %. [0349] xii) Granules (GR, FG)
[0350] 0.5-30 wt % of a whole culture broth, cell-free extract,
culture medium or metabolite of the invention are ground finely and
associated with solid carrier (e.g. silicate) ad 100 wt %.
Granulation is achieved by extrusion, spray-drying or fluidized
bed.
xiii) Ultra-Low Volume Liquids (UL)
[0351] 1-50 wt % of a whole culture broth, cell-free extract,
culture medium or metabolite of the invention are dissolved in
organic solvent (e.g. aromatic hydrocarbon) ad 100 wt %.
[0352] The compositions types i) to xiii) may optionally comprise
further auxiliaries, such as 0.1-1 wt % bactericides, 5-15 wt %
anti-freezing agents, 0.1-1 wt % anti-foaming agents, and 0.1-1 wt
% colorants.
[0353] Solutions for seed treatment (LS), suspoemulsions (SE),
flowable concentrates (FS), powders for dry treatment (DS),
water-dispersible powders for slurry treatment (WS), water-soluble
powders (SS), emulsions (ES), emulsifiable concentrates (EC) and
gels (GF) are usually employed for the purposes of treatment of
plant propagation materials, particularly seeds.
[0354] Preferred examples of seed treatment formulation types or
soil application for pre-mix compositions are of WS, LS, ES, FS, WG
or CS-type.
[0355] Typically, a pre-mix formulation for seed treatment
application comprises 0.5 to 99.9 percent, especially 1 to 95
percent, of the desired ingredients, and 99.5 to 0.1 percent,
especially 99 to 5 percent, of a solid or liquid adjuvant
(including, for example, a solvent such as water), where the
auxiliaries can be a surfactant in an amount of 0 to 50 percent,
especially 0.5 to 40 percent, based on the pre-mix formulation.
Whereas commercial products will preferably be formulated as
concentrates (e.g., pre-mix composition (formulation)), the end
user will normally employ dilute formulations (e.g., tank mix
composition).
[0356] Seed treatment methods for applying or treating the strains,
whole culture broths, cell-free extracts, culture media, compounds
of formula I and compositions of the invention, respectively, to
plant propagation material, especially seeds, are known in the art,
and include dressing, coating, filmcoating, pelleting and soaking
application methods of the propagation material. Such methods are
also applicable to the combinations according to the invention. In
a preferred embodiment, the strains, whole culture broths,
cell-free extracts, culture media, compounds of formula I, and
compositions of the invention, respectively, are applied or treated
onto the plant propagation material by a method such that the
germination is not negatively impacted. Accordingly, examples of
suitable methods for applying (or treating) a plant propagation
material, such as a seed, is seed dressing, seed coating or seed
pelleting and alike.
[0357] It is preferred that the plant propagation material is a
seed, seed piece (i.e. stalk) or seed bulb.
[0358] Although it is believed that the present method can be
applied to a seed in any physiological state, it is preferred that
the seed be in a sufficiently durable state that it incurs no
damage during the treatment process. Typically, the seed would be a
seed that had been harvested from the field; removed from the
plant; and separated from any cob, stalk, outer husk, and
surrounding pulp or other non-seed plant material. The seed would
preferably also be biologically stable to the extent that the
treatment would cause no biological damage to the seed. It is
believed that the treatment can be applied to the seed at any time
between harvest of the seed and sowing of the seed or during the
sowing process (seed directed applications). The seed may also be
primed either before or after the treatment.
[0359] Even distribution of the ingredients in the strains, whole
culture broths, cell-free extracts, culture media, compounds of
formula I, and compositions of the invention, respectively, and
adherence thereof to the seeds is desired during propagation
material treatment. Treatment could vary from a thin film
(dressing) of the formulation containing the combination, for
example, a mixture of active ingredient(s), on a plant propagation
material, such as a seed, where the original size and/or shape are
recognizable to an intermediary state (such as a coating) and then
to a thicker film (such as pelleting with many layers of different
materials (such as carriers, for example, clays; different
formulations, such as of other active ingredients; polymers; and
colourants) where the original shape and/or size of the seed is no
longer recognizable.
[0360] An aspect of the present invention includes application of
the strains, whole culture broths, cell-free extracts, culture
media, compounds of formula I, and compositions of the invention,
respectively, onto the plant propagation material in a targeted
fashion, including positioning the ingredients in the combination
onto the entire plant propagation material or on only parts
thereof, including on only a single side or a portion of a single
side. One of ordinary skill in the art would understand these
application methods from the description provided in EP954213B1 and
WO06/112700.
[0361] The strains, whole culture broths, cell-free extracts,
culture media, compounds of formula I and compositions of the
invention, respectively, can also be used in form of a "pill" or
"pellet" or a suitable substrate and placing, or sowing, the
treated pill, or substrate, next to a plant propagation material.
Such techniques are known in the art, particularly in EP1124414,
WO07/67042, and WO 07/67044. Application of the strains, whole
culture broths, cell-free extracts, culture media, compounds of
formula I and compositions, respectively, described herein onto
plant propagation material also includes protecting the plant
propagation material treated with the combination of the present
invention by placing one or more pesticide-containing particles
next to a pesticide-treated seed, wherein the amount of pesticide
is such that the pesticide-treated seed and the
pesticide-containing particles together contain an Effective Dose
of the pesticide and the pesticide dose contained in the
pesticide-treated seed is less than or equal to the Maximal
Non-Phytotoxic Dose of the pesticide. Such techniques are known in
the art, particularly in WO2005/120226.
[0362] Application of the strains, whole culture broths, cell-free
extracts, culture media, compounds of formula I and compositions of
the invention, respectively, onto the seed also includes controlled
release coatings on the seeds, wherein the ingredients of the
combinations are incorporated into materials that release the
ingredients over time. Examples of controlled release seed
treatment technologies are generally known in the art and include
polymer films, waxes, or other seed coatings, wherein the
ingredients may be incorporated into the controlled release
material or applied between layers of materials, or both.
[0363] Seed can be treated by applying thereto the strains, whole
culture broths, cell-free extracts, culture media, compounds of
formula I, and compositions of the invention, respectively, in any
desired sequence or simultaneously.
[0364] The seed treatment occurs to an unsown seed, and the term
"unsown seed" is meant to include seed at any period between the
harvest of the seed and the sowing of the seed in the ground for
the purpose of germination and growth of the plant.
Treatment to an unsown seed is not meant to include those practices
in which the active ingredient is applied to the soil but would
include any application practice that would target the seed during
the planting process.
[0365] Preferably, the treatment occurs before sowing of the seed
so that the sown seed has been pre-treated with the strains, whole
culture broths, cell-free extracts, culture media, compounds of
formula I and compositions of the invention, respectively. In
particular, seed coating or seed pelleting are preferred. As a
result of the treatment, the ingredients are adhered on to the seed
and therefore available for pest control.
[0366] The treated seeds can be stored, handled, sowed and tilled
in the same manner as any other active ingredient treated seed.
[0367] In particular, the present invention relates to a method for
protection of plant propagation material from pests and/or
improving the health of plants grown from said plant propagation
material, wherein the soil, wherein plant propagation material is
sown, is treated with an effective amount of a strain, cell-free
extract, culture medium, metabolite or composition of the
invention, respectively.
[0368] In particular, the present invention relates to a method for
protection of plant propagation material from pests, wherein the
soil, wherein plant propagation material is sown, is treated with
an effective amount of a strain, cell-free extract, culture medium,
metabolite or composition of the invention, respectively.
[0369] In particular, the present invention relates to a method for
protection of plant propagation material from harmful fungi,
wherein the soil, wherein plant propagation material is sown, is
treated with an effective amount of a strain, cell-free extract,
culture medium, metabolite or composition of the invention,
respectively.
[0370] In particular, the present invention relates to a method for
protection of plant propagation material from animal pests
(insects, acarids or nematodes), wherein the soil, wherein plant
propagation material is sown, is treated with an effective amount
of a strain, cell-free extract, culture medium, metabolite or
composition of the invention, respectively.
[0371] The user applies the compositions of the invention usually
from a predosage device, a knapsack sprayer, a spray tank, a spray
plane, or an irrigation system. Usually, the agrochemical
composition is made up with water, buffer, and/or further
auxiliaries to the desired application concentration and the
ready-to-use spray liquor or the agrochemical composition according
to the invention is thus obtained. Usually, 20 to 2000 liters,
preferably 50 to 400 liters, of the ready-to-use spray liquor are
applied per hectare of agricultural useful area.
[0372] When it comes to the treatment of plant propagation
material, especially seeds, the compositions disclosed herein give,
after two-to-tenfold dilution, active components concentrations of
from 0.01 to 60% by weight, preferably from 0.1 to 40%, in the
ready-to-use preparations. Application can be carried out before or
during sowing. Methods for applying a strain, cell-free extract,
culture medium, metabolite or composition of the invention,
respectively, onto plant propagation material, especially seeds,
include dressing, coating, pelleting, dusting, soaking and
in-furrow application methods of the propagation material.
Preferably, the strains, whole culture broths, cell-free extracts,
culture media, compounds of formula I or compositions of the
invention, respectively, are applied onto the plant propagation
material by a method such that germination is not induced, e.g. by
seed dressing, pelleting, coating and dusting.
[0373] When the strains of the invention are employed in crop
protection, wherein the strains are applied as foliar treatment or
to the soil, the application rates usually range from about
1.times.10.sup.6 to 5.times.10.sup.15 (or more) CFU/ha, preferably
from about 1.times.10.sup.7 to about 1.times.10.sup.13 CFU/ha, even
more preferably from 1.times.10.sup.9 to 5.times.10.sup.12
CFU/ha.
[0374] When the strains of the invention are employed in seed
treatment, the application rates with respect to plant propagation
material usually range from about 1.times.10.sup.1 to
1.times.10.sup.12 (or more) CFU/seed, preferably from about
1.times.10.sup.3 to about 1.times.10.sup.10 CFU/seed, and even more
preferably from about 1.times.10.sup.3 to about 1.times.10.sup.6
CFU/seed. Alternatively, the application rates with respect to
plant propagation material preferably range from about
1.times.10.sup.7 to 1.times.10.sup.16 (or more) CFU per 100 kg of
seed, preferably from 1.times.10.sup.9 to about 1.times.10.sup.15
CFU per 100 kg of seed, even more preferably from 1.times.10.sup.11
to about 1.times.10.sup.15 CFU per 100 kg of seed.
[0375] When cell-free extracts, culture media and/or compounds of
formula I are employed, the solid material (dry matter) are
considered as active components, e.g. to be obtained after drying
or evaporation of the extraction medium or the suspension medium in
case of liquid formulations. When employed in plant protection, the
amounts of active components applied are, depending on the kind of
effect desired, from 0.001 to 2 kg per ha, preferably from 0.005 to
2 kg per ha, more preferably from 0.05 to 0.9 kg per ha, and in
particular from 0.1 to 0.75 kg per ha. In treatment of plant
propagation materials such as seeds, e.g. by dusting, coating or
drenching seed, amounts of active components of from 0.1 to 1000 g,
preferably from 1 to 1000 g, more preferably from 1 to 100 g and
most preferably from 5 to 100 g, per 100 kilogram of plant
propagation material (preferably seeds) are generally required.
When used in the protection of materials or stored products, the
amount of active components applied depends on the kind of
application area and on the desired effect. Amounts customarily
applied in the protection of materials are 0.001 g to 2 kg,
preferably 0.005 g to 1 kg, of active components per [0376] cubic
meter of treated material.
[0377] According to one embodiment, individual components of the
composition of the invention such as parts of a kit or parts of a
binary or ternary mixture may be mixed by the user himself in a
spray tank or any other kind of vessel used for applications (e.g
seed treater drums, seed pelleting machinery, knapsack sprayer) and
further auxiliaries may be added, if appropriate.
[0378] If living microorganisms, such as the strains of the
invention, form part of such kit, it must be taken care that choice
and amounts of the components (e.g. chemical pesticidal agents) and
of the further auxiliaries should not influence the viability of
the microorganisms in the composition mixed by the user. Especially
for bactericides and solvents, compatibility with the respective
microorganisms has to be taken into account.
[0379] Various types of oils, wetters, adjuvants, fertilizer, or
micronutrients, and further pesticides (e.g. herbicides,
insecticides, fungicides, growth regulators, safeners,
biopesticides) may be added to the strains, cell-free extracts,
culture media, metabolites, compounds of formula I and composition
of the invention, respectively as premix or, if appropriate not
until immediately prior to use (tank mix). These agents can be
admixed with the compositions according to the invention in a
weight ratio of 1:100 to 100:1, preferably 1:10 to 10:1.
Preferably, a composition of the invention comprises a further
biopesticide. Even more preferably, a composition of the invention
comprises besides an auxiliary and at least one compound of formula
I, a microbial pesticide.
[0380] A pesticide is generally a chemical or biological agent
(such as a virus, bacterium, antimicrobial or disinfectant) that
through its effect deters, incapacitates, kills or otherwise
discourages pests. Target pests can include insects, plant
pathogens, weeds, mollusks, birds, mammals, fish, nematodes
(roundworms), and microbes that destroy property, cause nuisance,
spread disease or are vectors for disease. The term pesticides
includes also plant growth regulators that alter the expected
growth, flowering, or reproduction rate of plants; defoliants that
cause leaves or other foliage to drop from a plant, usually to
facilitate harvest; desiccants that promote drying of living
tissues, such as unwanted plant tops; plant activators that
activate plant physiology for defense of against certain pests;
safeners that reduce unwanted herbicidal action of pesticides on
crop plants; and plant growth promoters that affect plant
physiology to increase plant growth, biomass, yield or any other
quality parameter of the harvestable goods of a crop plant.
EXAMPLES
[0381] The present invention will be described in greater detail by
means of the following examples. The following examples are for
illustrative purposes and are not intended to limit the scope of
the invention.
Example 1: Isolation of Novel Bacterial Strains of the
Invention
[0382] Soil samples from a variety of European locations including
Germany were collected. By applying commonly known microbial
isolation procedures to these soils, the inventors obtained a
variety of bacteria that were further subjected to conventional
isolation techniques for providing pure isolates as described
herein.
[0383] Standard microbial enrichment technique (C. A. Reddy, T. J.
Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt, and L. R.
Snyder (eds.). Methods for General and Molecular Microbiology, Am.
Soc. Microbiol., Washington, District of Columbia) was followed to
isolate each type of bacteria.
[0384] The following strains have been isolated and deposited under
Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und
Zellkuturen (DSMZ) on Feb. 20, 2013: a) Lu16774 as deposited with
DSMZ having the deposit number DSM 26969 [0385] b) Lu17007 as
deposited with DSMZ having the deposit number DSM 26970 [0386] c)
Lu17015 as deposited with DSMZ having the deposit number DSM
26971.
Example 2--Characterization of Novel Bacterial Strains
Example 2.1: 16S-rDNA Sequencing
[0387] The 16S rRNA gene sequences of the Paenibacillus strains
were determined by direct sequencing of PCR-amplified 16S rDNA at
the DSMZ, Braunschweig, Germany.
[0388] Genomic DNA extraction was carried out using the
MasterPure.TM. Gram Positive DNA Purification Kit from Epicentre
Biotechnologies according to the manufacturer's instructions.
PCR-mediated amplification of the 16S rDNA and purification of the
PCR product was carried out as described previously (Int. J. Syst.
Bacteriol. 46, 1088-1092, 1996). Purified PCR products were
sequenced using the BigDye.RTM. Terminator v1.1 Cycle Sequencing
Kit (Applied Biosystems) as directed in the manufacturer's
protocol. Sequence reactions were electrophoresed using the 3500xL
Genetic Analyzer from Applied Biosystems. Sequence ambiguities may
be due to the existence of several cistrons encoding 16S rRNAs with
different sequences within a single genome (J. Bacteriol. 178(19),
5636-5643, 1996).
[0389] The resulting sequence data from the strains was put into
the alignment editor AE2
(http://iubio.bio.indiana.edulsoft/molbio/unixlae2.readme), aligned
manually according to the secondary structure of the resulting rRNA
molecule and compared with representative 16S rRNA gene sequences
of organisms belonging to the Firmicutes (Nucl. Acids Res. 27,
171-173, 1999). For comparison, 16S rRNA sequences were obtained
from the EMBL and RDP data bases.
[0390] The 16S rDNA sequences of the strains of the invention are
set forth in the Sequence Listing as indicated in Table 2.
TABLE-US-00003 TABLE 2 Sequence listing references of the 16S rDNA
of the Paenibacillus strains. Strain SEQ ID NO Lu16774 1 Lu17007 2
Lu17015 3
[0391] The 16S rDNA gene identity values in % were calculated by
pairwise comparison of the sequences within the alignment of the
sequences compared.
[0392] Comparison performed of only two sequences based on pairwise
sequence alignment are denoted herein as binary values. The other
values are based on a multiple sequence alignment of all sequences
within the comparison. Higher identity values from multi-sequence
comparisons result from the problem that the sequence data of the
compared sequences were of different length resulting in a shorter
alignment.
[0393] The % identity from pair-wise comparisons of the complete
rDNA sequences among the three novel strains Lu16774, Lu17007 and
Lu17015 was between 99.5 and 99.9% (Table 3, binary values).
TABLE-US-00004 TABLE 3 Identity in % of the complete 16S rRNA
sequences of the three novel Paenibacillus strains (binary values
in brackets). Identity of the complete 16S rRNA sequence of the
novel Paenibacillus strains (%) Strains Lu16774 Lu17015 Lu17007
Lu16774 -- Lu17015 99.7 (99.5) -- Lu17007 99.9 (99.8) 99.8 (99.5)
--
[0394] The comparison of the complete 16S rRNA sequence of the
three novel strains Lu16774, Lu17007 and Lu17015 with related taxa
(see FIG. 9) revealed a high percentage of identity to
Paenibacillus peoriae (type-strain DSM 8320) with 99.8%. The binary
values for pairwise-sequence alignments of P. peoriae with the
novel strains were as follows: Lu16774: 99.5%, Lu17007: 99.5%; and
Lu17015: 99.7% identity, respectively.
[0395] A final evaluation of species to which the novel
Paenibacillus strains Lu16774, Lu17015 and Lu17007 belong was based
on the 16S rRNA sequence data not possible.
[0396] The sequencing of the complete rDNA resulted for
Paenibacillus peoriae NRRL BD-62 in 100.0% identity to P. peoriae
(type strain DSM 8320) confirming the species designation P.
peoriae for this strain BD-62 (see FIG. 9).
[0397] The close relationship of all three novel Paenibacillus
strains Lu16774, Lu17007 and Lu17015 to P. peoriae was confirmed by
the comparison with the 16S rRNA sequence of P. peoriae strain
BD-62 which resulted in identity values of 99.8% (see FIG. 9).
[0398] For construction of the phylogenetic dendrogram operations
of the ARB package (Nucl. Acids Res. 35, 7188-7196, 2007) were
used: based on the evolutionary distance values the phylogenetic
tree was constructed by the neighbor-joining method (Jukes, T. H.
& Cantor C. R. (1969). Evolution of protein molecules. In
Mammalian protein metabolism, pp. 21-132. Edited by H. N. Munro.
New York: Academic press) using the correction of Jukes and Cantor
(Mol. Biol. Evol. 4, 406-425, 1987). The root of the tree was
determined by including the 16S rRNA gene sequence of Cohnella
thermotolerans into the analysis. The scale bar below the
dendrogram indicates 1 nucleotide substitutions per 100
nucleotides. The results are given in FIG. 10.
[0399] The phylogenetic dendrogram of these sequences (FIG. 10)
shows that the three novel strains Lu16774, Lu17007 and Lu17015 are
most-closely related to each other and that their closest relative
known to each of them was the Paenibacillus peoriae strain NRRL
BD-62.
Example 2.2: RiboPrint-Analysis
[0400] Standardized, automated ribotyping is performed using the
Qualicon RiboPrintersystem. The RiboPrinter system combines
molecular processing steps for ribotyping in a stand-alone,
automated instrument. The procedure includes cell lysis, digestion
of chromosomal DNA with restriction enzyme EcoRI, separation of
fragments by electrophoresis, transfer of DNA fragments to a nylon
membrane, hybridization to a probe generated from the rmB operon
from E. coli, chemiluminescent detection of the probe to the
fragments containing rm operon sequences, image detection and
computerized analysis of RiboPrint patterns (Food Technology 50(1),
77-81, 1996; Proc. Natl. Acad. Sci. USA 92, 5229-5233, 1995; Int.
Journ. Syst. Bact. 44(3), 454-460, 1994).
[0401] Ribotyping have been executed by the DSMZ, Germany with the
novel Paenibacillus strains Lu16774, Lu17007 and Lu17015 in
comparison to the P. peoriae strain BD-62 using the restriction
enzyme EcoRI. The resulting patterns have been compared using the
Software of the RiboPrinter system, the integrated DuPont
Identification Library as well as the BioNumerics Software (Applied
Maths, Belgium).
[0402] Similarity of all three novel strains to BD-62 was between
0.24 and 0.5 (FIG. 11). The three novel strains group in two
groups, first comprising Lu17015, whereas the second group
comprises the strains Lu16774 and Lu17007. None of the novel
strains has a similarity higher than 0.84 to any strain within the
DuPont Identification Library and was therefore not identified
automatically.
[0403] The strain BD-62 has been identified as Paenibacillus
peoriae based on the entry DUP-13142 of the DuPont identification
library (entry based on Paenibacillus peoriae DSM 8320).
Example 2.3: Morphological and Physiological Characterization
[0404] The strains were characterized at the DSMZ in analogy to
methods described in Gordon, R. E., Haynes, W. C. & Pang. C.
H.-N. (1973): The Genus Bacillus, Agriculture Handbook no. 427.
Washington D.C.: US Department of Agriculture. The results are
given in Table 4.
TABLE-US-00005 TABLE 4 Characterization Data of the Paenibacillus
strains of the invention and comparison to known Paenibacillus
peoriae strain NRRL BD-62. Paenibacillus strains Identification
Lu16774 Lu17007 Lu17015 BD-62 Characteristics cell form rod- rod-
rod- rod- shaped shaped shaped shaped width [.mu.m] 0.9-1.0 0.9-1.0
0.9-1.0 0.9-1.0 length [.mu.m] 3->5.0 3-5.0 3-5.0 2.5-5.0
ellipsoid spores + + + + swollen sporangium + + + + Catalase + + +
+ Oxidase - - - - anaerobic growth + + + + VP reaction + + + + pH
in VP-Medium 5.2 5.7 4.8 5.2 maximum temperature positive growth at
.degree. C. 40 40 40 40 negative growth at .degree. C. 50 50 50 50
Growth in: Medium pH 5.7 + + + + NaCl 2% + + + + NaCl 5% - - - -
NaCl 7% - - - - Acid formation from: D-Glucose + + + + L-Arabinose
+ + + + D-Xylose + + + + D-Mannitol + + + + D-Fructose + + + +
Raffinose + + + + Trehalose + + + - Glycerol + + + + Gas from
glucose + + + + Hydrolysis of starch + + + + gelatin + + + + casein
+ + + ? Tween 80 - - - - esculin + + + + Utilisation of citrate
n.g.* n.g. n.g. n.g. propionate n.g. n.g. n.g. n.g. N0.sub.3 to
N0.sub.2 + + + + Indole reaction - - - - Lecithinase + + + -
Phenylalanine - - - - desaminase Arginine dihydrolase - - - -
Lysozyme + + + + *n.g. = no growth.
[0405] Analysis of the cellular fatty acids performed at the DSMZ
resulted that all strains showed at typical profile for
Paenibacillus spp.
[0406] Using the available genetic, physiological and biochemical
data, it is shown that the strains Lu16774, Lu17007 and Lu17015
belong to the genus Paenibacillus. As the strains Lu16774, Lu17007
and Lu17015 as well as BD-62 do produce gas from glucose, none of
them belongs to Paenibacillus jamilae.
[0407] A phenotypic differentiation between Paenibacillus peoriae
and Paenibacillus polymyxa is primarily possible using
characteristics of acid production from certain substrates (Int. J.
Syst. Bacteriol. 43(2), 388-390, 1993; In. J. Syst. Bacteriol.
46(6), 988-1003, 1996). None of the novel strains did completely
match with its characteristics outlined in Table 4 completely to
any of these two species, but in sum of the available genetic,
physiological and biochemical data most likely point to the species
Paenibacillus peoriae and P. polymyxa or at least to another
species very closely related to Paenibacillus peoriae and P.
polymyxa.
[0408] Due to the multitude of Paenibacillus species described so
far, it is impossible to determine the correct taxonomic species of
the three isolates tested based on physiological and morphological
criteria from Table 4 (Rainer Borriss, Humboldt University Berlin,
unpublished results).
[0409] Nevertheless, it was not possible to completely determine
the species within this genus. The most closely related species and
strain was found to be Paenibacillus peoriae BD-62 based on
16S-rDNA analysis (see e.g. FIG. 11).
Example 2.4: Phylogenetic Analysis Based on Genes Coding for DnaN,
GyrB, RecF, RecN and RpoA
[0410] The nucleotide sequences of the genes coding for DnaN, GyrB,
RecF, RecN and RpoA have been extracted from complete genome
sequences or from public databases (Sequence listings as outlined
in Table 28).
[0411] The identity tables (FIGS. 12 to 16) have been generated
with an all against all approach where every sequence is aligned
with every other sequence. The sequence alignment was performed
with a program needle (EMBOSS package 6.6.0; Trends in Genetics 16
(6), 276-277). Standard parameters where used (gap creation 10.0;
gap extension 0.5). Identity Scores are calculated on the basis of
the alignments without taking any gaps into account.
[0412] For the phylogenetic trees (FIGS. 17 to 21), multiple
sequence alignments that have been performed with Clustal Omega
(version 1.2.0; Molecular Systems Biology 7: 539,
doi:10.1038/msb.2011.75). The phylogenetic trees are calculated by
maximum likelihood method with the software DnamI (implemented in
the Phylip 3.696 package; Felsenstein 1981,
http://evolution.genetics.washington.edu/phylip.html). The
dendrograms have been established using a F84 distance model while
applying a transition-transversion ratio of two (2). The trees are
plotted with the tool Dendroscope (http://dendroscope.org/).
TABLE-US-00006 TABLE 28 Sequence listing references of the dnaN,
gyrB, recF, recN and rpoA DNA sequences of the Paenibacillus
strains. Strain Gene SEQ ID NO Lu16774 dnaN 4 Lu17007 dnaN 5
Lu17015 dnaN 6 Lu16774 gyrB 7 Lu17007 gyrB 8 Lu17015 gyrB 9 Lu16774
recF 10 Lu17007 recF 11 Lu17015 recF 12 Lu16774 recN 13 Lu17007
recN 14 Lu17015 recN 15 Lu16774 rpoA 16 Lu17007 rpoA 17 Lu17015
rpoA 18
Example 2.5: Core Genome Comparisons and AAI Matrix
[0413] Genome comparisons have be performed using the software
package EDGAR of the university Gie.beta.en (BMC Bioinformatics 10,
154, 2009;
(https://edgar.computational.bio.unigiessen.de/cgi-bin/edgar.cgi).
The determination of the core genome, the phylogenetic dendrograms
on the basis of the complete genome sequences and the AAI matrix
values have been performed using the software package EDGAR.
Results are shown in FIG. 22.
Example 3: Growth (Fermentability) of Strains for In-Vivo Tests
[0414] For green-house and field trials, the Paenibacillus strains
were first grown on ISP2 plates (ready-to-use agar from BD [USA],
catalog number 277010). Afterwards, baffled shake flasks containing
liquid ISP2 medium were inoculated with a colony from the agar
plate and incubated for 5-7 days at 150 rpm and 25.degree. C.
Depending on the test, either whole culture broth, or the
centrifuged and H.sub.2O-washed cell pellet, or the supernatant was
applied to the plants. A scale-up to 10 L fermenters was
possible.
[0415] Paenibacillus strains were grown in ISP2 liquid media (10
g/L malt extract, 4 g/L Bacto yeast extract, 4 g/L glucose
monohydrate) for 6 days at 22.degree. C. at 150 rpm. OD.sub.600nm
indicating bacterial growth was measured at different time
points.
TABLE-US-00007 TABLE 5 Bacterial growth of Paenibacillus strains in
liquid ISP2 medium. OD at 600 nm Paenibacillus strain 0 d 3 d 6 d
Lu17007 0.011 3.110 3.050 BD-62 0.013 0.442 0.446
Example 4--In-Vitro Confrontation Assay for Antifungal Activity
[0416] Antagonistic activity of the Paenibacillus strains against
plant pathogens was shown in in-vitro confrontation assay. The
phytopathogenic fungi used are Sclerotina scerotiorum (SCLSCL),
Botrytis cinerea (BOTRCI), Alternaria sp. (ALTESP) and Phytophthora
infestans (PHYTIN).
[0417] As growth medium for BOTRCI, ALTESP, SCLSCL, ISP2 medium is
used comprising per litre: 10 g malt extract (Sigma Aldrich,
70167); 4 g Bacto yeast extract (Becton Dickinson, 212750); 4 g
glucose monohydrate (Sigma Aldrich, 16301); 20 g Agar (Becton
Dickinson, 214510), pH about 7, Aq. bidest. As growth medium for
PHYTIN, V8 medium is used comprising per litre: 200 ml of vegetable
juice, 3 g calcium carbonate (Merck Millipore, 1020660250); 30 g
Agar (Becton Dickinson, 214510), pH 6.8, Aq. bidest.
[0418] The Paenibacillus strains were point-inoculated on one side
of an agar plate. An agar block (approx. 0.3 cm.sup.2) containing
one actively growing plant pathogen was put in the center of the
plate. After incubating for 7-14 days at 25.degree. C., the growth
of the plant pathogen was examined, especially for inhibition
zones.
[0419] Thereafter, the agar plates are incubated at .degree. C. for
about 7-14 days before evaluation. Antibiosis is scored by
evaluation of the diameter of the fungi-free zone (zone of
inhibition). Competition is scored by comparing the diameter of the
growth of the fungal pathogen on plates with bacterial strains in
comparison to control plates. Mycoparasitism can be documented in
case the bacteria overgrows the fungal pathogen and also parasitize
the pathogens. This can be visualized by microscopy.
[0420] The novel Paenibacillus strains showed antifungal activity
against all tested plant pathogens.
TABLE-US-00008 TABLE 6 In-vitro confrontation assay results.
Diameter of zone of inhibition [mm] Paenibacillus strain PHYTIN
BOTRCI ALTESP SCLSCL Lu16774 8 2 2 2 Lu17007 8 8 5 2 Lu17015 8 5 5
2 BD-62 2 5 0 0
Example 5--Glasshouse Tests for Activity Against Plant Pathogenic
Fungi
Use Example 5.1: Activity Against Late Blight on Tomato Caused by
Phytophthora infestans with Protective Application
[0421] Commercially available young tomato seedlings ("Goldene
Konigin") were used for the described greenhouse trial. 2
replications (pots with 1 plant each) were used per treatment.
Plants were grown in commercially available substrate (Universal,
Floragard) at approx. 22.degree. C. in the greenhouse. The humidity
was controlled using a special device (.about.90% humidity). The
plants were sprayed to runoff with crude/whole culture broth of 6
days old cultures of the respective Paenibacillus strain (depending
on the setup) using a spray cabinet. Culture conditions for the
strains are described in Example 3. One day after application the
treated plants were inoculated with a suspension of sporangia of
Phytophthora infestans (PHYTIN). After inoculation, the trial
plants were immediately transferred to a humid chamber. The extent
of fungal attack on the leaves was visually assessed 5-7 days after
inoculation. Fungal attack in the untreated control was between
80-100% and set to 100% for comparison reason.
TABLE-US-00009 TABLE 7 Paenibacillus strain PHYTIN (% fungal
attack) Lu17007 4 Lu16774 20 BD-62 53
Use Example 5.2: Activity Against Grey Mold on Pepper Caused by
Botrytis cinerea with Protective Application
[0422] Commercially available young pepper seedlings ("Neusiedler
Ideal") were used for the described greenhouse trial. 2
replications (pots with 1 plant each) were used per treatment.
Plants were grown in commercially available substrate (Universal,
Floragard) at approx. 22.degree. C. in the greenhouse. The humidity
was controlled using a special device (.about.90% humidity). The
plants were sprayed to runoff with crude culture broth of 6 days
old cultures of the respective Paenibacillus strain (depending on
the setup) using a spray cabinet. Culture conditions for the
strains are described in Example 3. One day after application the
treated plants were inoculated with a suspension of spores of
Botrytis cinerea (BOTRCI). After inoculation, the trial plants were
immediately transferred to a humid chamber. The extent of fungal
attack on the leaves was visually assessed 5-7 days after
inoculation. Fungal attack in the untreated control was between
80-100% and set to 100% for comparison reason.
TABLE-US-00010 TABLE 8 Paenibacillus strain BOTRCI (% fungal
attack) Lu17007 2 Lu16774 16 Lu17015 20 BD-62 97
Use Example 5.3: Activity Against Early Blight on Tomato Caused by
Alternaria solani with Protective Application
[0423] Commercially available young tomato seedlings ("Goldene
Konigin") were used for the described greenhouse trial. 2
replications (pots with 1 plant each) were used per treatment.
Plants were grown in commercially available substrate (Universal,
Floragard) at approx. 22.degree. C. in the greenhouse. The humidity
was controlled using a special device (-90% humidity). The plants
were sprayed to runoff with crude/whole culture broth of 6 days old
cultures of the respective Paenibacillus strain (depending on the
setup) using a spray cabinet. Culture conditions for the strains
are described in Example 3. One day after application the treated
plants were inoculated with a suspension of spores of Alternaria
solani (ALTESO). After inoculation, the trial plants were
immediately transferred to a humid chamber. The extent of fungal
attack on the leaves was visually assessed 5-7 days after
inoculation. Fungal attack in the untreated control was between
80-100% and set to 100% for comparison reason.
TABLE-US-00011 TABLE 9 Paenibacillus strain ALTESO (% fungal
attack) Lu17007 3 Lu17015 16 BD-62 96
Use Example 5.4: Activity Against Soybean Rust on Soybean Caused by
Phakospora pachyrhizi with Protective Application
[0424] Commercially available young soybean seedlings (`Mentor`)
were used for the described greenhouse trial. 2 replications (pots
with 1 plant each) were used per treatment. Plants were grown in
commercially available substrate (Universal, Floragard) at approx.
22.degree. C. in the greenhouse. The humidity was controlled using
a special device (-90% humidity). The plants were sprayed to runoff
with crude culture broth of 2-6 days old cultures of Paenibacillus
spp. (depending on the setup) using a spray cabinet. One day after
application the treated plants were inoculated with a suspension of
spores of Phakopsora pachyrhizi (PHAKPA). After inoculation, the
trial plants were immediately transferred to a humid chamber. The
extent of fungal attack on the leaves was visually assessed 5-7
days after inoculation.
Use Example 5.5: Activity Against Fusarium Head Blight on Wheat
Caused by Fusarium graminearum with Protective Application
[0425] Commercially available young wheat seedlings were used for
the described greenhouse trial. 2 replications (pots with 1 plant
each) were used per treatment. Plants were grown in commercially
available substrate (Universal, Floragard) at approx. 22.degree. C.
in the greenhouse. The humidity was controlled using a special
device (.about.90% humidity). The plants were sprayed to runoff
with crude culture broth of 2-6 days old cultures of Paenibacillus
spp. (depending on the setup) using a spray cabinet. Culture
conditions are described in Example 3. One day after application
the treated plants were inoculated with a suspension of spores of
Fusarium graminearum (GIBBZE). After inoculation, the trial plants
were immediately transferred to a humid chamber. The extent of
fungal attack on the leaves was visually assessed 5-7 days after
inoculation.
Use Example 5.6: Activity Against Speckled Leaf Blotch on Wheat
Caused by Septoria tritici with Protective Application
[0426] Commercially available young wheat seedlings were used for
the described greenhouse trial. 2 replications (pots with 1 plant
each) were used per treatment. Plants were grown in commercially
available substrate (Universal, Floragard) at approx. 22.degree. C.
in the greenhouse. The humidity was controlled using a special
device (.about.90% humidity). The plants were sprayed to runoff
with crude culture broth of 2-6 days old cultures of Paenibacillus
spp. (depending on the setup) using a spray cabinet. Culture
conditions are described in Example 3. One day after application
the treated plants were inoculated with a suspension of spores of
Septoria tritici (SEPTTR). After inoculation, the trial plants were
immediately transferred to a humid chamber. The extent of fungal
attack on the leaves was visually assessed 21-28 days after
inoculation.
Use Example 5.7: Activity of the Paenibacillus Cells and of the
Supernatant Against Various Pathogens with Protective
Application
[0427] Whole culture broth from 6 days old cultures of
Paenibacillus strain Lu17007 was obtained according to Use Example
3 and used as in the experimental setup of Use Example 5.1 to 5.3.
Alternatively, such whole culture broth was filtered through a
filter with 0.2 .mu.m pore size to obtain the culture medium and
the crude cell fraction. The crude cell fraction could further be
washed three times with the original volumes of phosphate-buffered
saline to obtain washed cells.
[0428] The glasshouse trials were performed as described in the Use
Examples 5.1, 5.2 and 5.3 above for the respective pathogens
Phytophthora infestans, Botrytis cinerea and Alternaria solani. The
extent of fungal attack on the leaves was visually assessed 5-7
days after inoculation. Fungal attack in the untreated control was
between 80-100% and set to 100% for comparison reason.
TABLE-US-00012 TABLE 10 Paenibacillus culture % fungal attack by
component BOTRCI ALTESO PHYTIN Whole culture broth 0 2 7 Culture
medium 3 40 3 Crude cell fraction 0 5 4 Washed cells 1 10 1
Example 6--Enzymatic Tests
Use Example 6.1: Chitinase
Chitinase Test Solid Medium:
[0429] 2 g/l NaNO.sub.3, 1 g/l K.sub.2HPO.sub.4, 0.5 g/l
MgSO.sub.4, 0.5 g/l KCl, 0.2 g/l pepton, 15 g/l agar, 10 g/l chitin
from crab shells (Sigma-Aidrich C7170).
[0430] Test solid medium is autoclaved and filled into 9 cm Petri
dishes. Paenibacillus strains are inoculated in the center of the
plates and incubated for two days at 27.degree. C. Thereafter, the
plates are stained with a 1:3 diluted Lugol solution (Carl Roth
N052.2) for 5 to 10 min. Lugol solution is poured out and the
plates are photographed and evaluated. Growth of the different
strains was no more than 5-10 mm. Non-stained zones (correlating
with chitinase activity) varied from 0 mm (no activity; "-" in
Table 11) to several cm ("+" in Table 11).
Use Example 6.2: Cellulase
Cellulase Test Solid Medium:
[0431] 2 g/l NaNO.sub.3, 1 g/l K.sub.2HPO.sub.4, 0.5 g/l
MgSO.sub.4, 0.5 g/l KCl, 0.2 g/l pepton, 15 g/l agar, carboxymethyl
cellulose, sodium salt (Sigma-Aldrich 419273).
[0432] Medium is autoclaved poured into 9 cm Petri dishes.
Paenibacillus strains are inoculated in the center of the plates
and incubated for two days at 27.degree. C. After incubation plates
are stained with a 1:3 diluted Lugol solution (Carl Roth N052.2)
for 5 to 10 min. Lugol solution is poured out and plates
photographed.
Use Example 6.3: Amylase
Amylase Test Solid Medium:
[0433] 2 g/l NaNO.sub.3, 1 g/l K.sub.2HPO.sub.4, 0.5 g/l
MgSO.sub.4, 0.5 g/l KCl, 0.2 g/l pepton, 15 g/l agar, 10 g/l
soluble starch (Merck 1.01252).
[0434] Medium is autoclaved poured into 9 cm Petri dishes.
Paenibacillus strains are inoculated in the center of the plates
and incubated for two days at 27.degree. C. After incubation plates
are stained with a 1:3 diluted Lugol solution (Carl Roth N052.2)
for 5 to 10 min. Lugol solution is poured out and plates
photographed.
TABLE-US-00013 TABLE 11 Chitinase, cellulose and amylase activities
of Paenibacillus strains. Strain Chitinase Cellulase Amylase
Lu16774 + + - Lu17007 ++ + + Lu17015 + + + BD-62 - - - -, no
activity; (+), low activity; +, regular activity; ++, high
activity.
Example 7--Fusaricidin-Type Metabolites Obtained from Paenibacillus
Strains
Example 7.1: Large Scale Cultivation of Bacterial Isolates and
Extraction of Fusaricidin-Type Metabolites
a) Cultivation
[0435] The Paenibacillus strains were cultivated on agar plates
containing GYM medium (10 g/l glucose, 4 g/l yeast extract, 10 g/l
malt extract; pH 5.5, adjusted before autoclaving) and 20 g/l agar.
Cultivation was performed for 10 to 20 days at room temperature.
For maintenance agar slants with the same medium were used and
stored at 4.degree. C.
[0436] Small scale liquid cultures (250 ml GYM medium in 500 ml
flasks) were inoculated with 4-5 pieces of a well grown agar
culture and cultivated in an orbital shaker at 120 rpm at room
temperature (20-23.degree. C.).
[0437] Large scale fermentations were performed in 20 l fermenters
with 15 l GYM medium (total capacity of fermenters was not used
because of foam formation) inoculated with 250 ml well grown liquid
culture and fermentation was carried out at room temperature
(20-23.degree. C.) with agitation (120 rpm) and aeration (3 l/min)
for 5 to 8 days.
b) Extraction
[0438] One equal volume of isopropanol was added to the whole
culture broth (no separation of biomass from liquid culture was
performed). After agitation and incubation for 2 to 16 hours,
common table salt (sodium chloride--100 to 200 g/l) was added to
the mixture until phase separation of the organic and aqueous phase
was visible.
[0439] The isopropanol phase was concentrated in vacuo. The
resulting extract, still containing large amount of salt, was
dissolved in methanol, centrifuged for better precipitation of salt
residues, and the organic phase was concentrated again. This step
was repeated until no salt precipitate was present anymore.
c) Purification
[0440] i) Silica Gel Chromatography
[0441] 30 grams of extract were dissolved in methanol and bound to
50 g silica gel (Merck, K60, 70-230 mesh), dried at 40.degree. C.
and layered onto 1 kg of silica gel (column 10 cm diameter, 30 cm
high approx.).
[0442] Elution was carried out in four steps as following:
Step 1-4 l ethyl acetate Step 2-4 l ethyl acetate:methanol (3:1,
v/v) Step 3-7 l ethyl acetate:methanol (1:1, v/v) Step 4-4 l
methanol
[0443] The third fraction (intermediate 1), containing the active
compounds, was dried in vacuo and dissolved in 40% methanol (MeOH)
in 0.1% formic acid (FA) (concentration: 100 mg/ml). The other
fractions were discarded.
[0444] ii) Chromabond HR-X Fractionation
[0445] 20 ml of intermediate 1 was loaded onto a previously
equilibrated (with 40% MeOH in 0.1% FA) Chromabond HR-X cartridge
(Macherey-Nagel, 1000 mg, ref 730941). The cartridge was washed
with 100 ml 40% MeOH in 0.1% FA and eluted with 60 ml 70% MeOH in
0.1% FA. This intermediate 1-1 was then dried in vacuo.
[0446] iii) Preparative HPLC on a Sunfire C18 Column
[0447] Intermediate 1-1 was dissolved in DMSO (concentration: 200
mg/ml) and 300 .mu.l of intermediate 1-1 were chromatographed on a
Sunfire C18 column (19.times.250 mm, 5 .mu.m, Waters) as
follows:
16 min at 10 ml/min, isocratic 70% 0.2 FA; 30% acetonitrile (ACN),
1 min at 14 ml/min, gradient to 65% 0.2% FA; 35% ACN, 5 min at 14
ml/min, isocratic 65% 0.2% FA; 35% ACN.
[0448] Five fractions could be detected. All five resulting
fractions were dried in vacuo and dissolved in DMSO (concentration:
125 mg/ml). Further purification was performed using the same
column and isocratic conditions (flow: 10.5 ml/min) adjusted for
every fraction (12.5 mg per run):
[0449] Fraction 1: 69% 0.2 FA; 31% ACN; two peaks detected (1-1 and
1-2)
[0450] Fraction 2: 69% 0.2 FA; 31% ACN; two peaks detected (2-1 and
2-2)
[0451] Fraction 3: 69% 0.2 FA; 31% ACN; three peaks detected (3-1,
3-2 and 3-3)
[0452] Fraction 4/5: 67% 0.2 FA; 33% ACN; one peak detected
(4/5)
[0453] Fraction 6: 65% 0.2 FA; 35% ACN; two peaks detected (6-1 and
6-2)
[0454] The purity and quantity of the following samples was
sufficient for NMR analysis and structure elucidation: peaks 1-2,
2-1, 3-2, 4/5 and 6-1.
Example 7.2: Structural Elucidation of Novel Compounds 1A and
1B
[0455] From peak 2-1 of fraction 2, a mixture of compounds 1A and
1B (ratio about 3:7) was obtained as a brown oil
([.alpha.].sub.D.sup.25=+20.9 (c=0.6, DMSO-de)).
[0456] The molecular formula C.sub.47H.sub.78N.sub.10O.sub.12 of
the major component, compound 1B, was deduced from the HR-ESI-MS
spectrum which gave a peak at m/z 975.5863 [M+H].sup.+; ESI-MS:
975.6 (100%, [M+H].sup.+), 488.4 (51%, [M+2H].sup.2+).
[0457] Besides, the mixture also contained as minor component, the
lighter homologue 1A, and the mass difference between both
compounds was 14 amu. This observation was supported by a second
peak observed in the ESI-MS spectrum at m/z 961.6.
[0458] The NMR spectra (Table 12) included in addition to signals
of exchangeable protons between .delta. 6.83 and 8.58, resonances
of carbonyl in the range of .delta. 166.0-174.5 and methine signals
between .delta. 47.8 and .delta. 60.4 indicative for a peptide.
[0459] Extensive analysis of the 1D- and 2D-NMR data of compound 1B
revealed the presence of six amino acids including tyrosine (Tyr),
glutamine (Gln), alanine (Ala), two threonines (Thr1 and Thr2) and
isoleucine (Ile). Their sequence was found using two or three bonds
correlations across amide functions. Thus, COSY, NOESY (FIG. 2) and
HMBC (FIG. 3) spectra depicted correlations from the
nitrogen-proton of Thr2 at .delta. 8.58 to the signal of methine
proton of Thr2 at .delta. 3.84 and the carbonyl at .delta. 166.7 of
Tyr while the same relationship was noted between the
nitrogen-proton of Tyr at .delta. 8.52 and the signal of methylene
proton of Tyr at .delta. 2.60 and the carbonyl at .delta. 170.4 of
Ile. Furthermore, the methine hydrogen of Ile at .delta. 4.16 had a
strong correlation with the carbonyl signal of Ile at .delta. 170.4
and a weak contact with that of Thr1 at .delta. 168.6; the signal
of the methine proton at .delta. 5.30 of Thr1 correlated with the
carbonyl signal at .delta. 170.4 of Ala. Additionally to the
aforementioned correlations, others were displayed from the
N-proton at .delta. 7.27 of Ala to the methine proton at .delta.
4.20 of the same amino acid while this latter proton had the same
interaction with the carbonyl of its amino and the one of Gln.
Besides, a cross peak was revealed from the exchangeable proton at
.delta. 8.20 of Gln to the methine hydrogen at .delta. 3.87 of Gln
and the carbonyl of Thr2 at .delta. 170.6; these above-mentioned
data suggested the cyclodepsipeptidic structure for compound
1B.
[0460] This cyclodepsipeptide 1B contained a terminal guanidine
.beta.-hydroxy fatty acid attached to Thr1 since a key correlation
was observed between the signal of its .alpha.-methine proton at
.delta. 4.39 and the resonance of a carbonyl at .delta. 171.9; HMBC
contacts from that carbonyl at .delta. 171.9 to the
.alpha.-methylene protons at .delta. 2.35 and the .beta.-methine
proton at .delta. 3.77 were further observed as well as between the
methylene protons at .delta. 3.03 and the guanidine carbon at
.delta. 157.2. The side chain was deduced to contain twelve
methylene groups between the 1-hydroxy and the guanidine group on
the basis of the fragment ion observed in the APCI-MS-MS spectrum
of the parent [M+H].sup.+ ion at m/z 256.2. Likewise, this spectrum
provided information (FIG. 4b) which confirmed the connection
sequence of amino acids and led to elucidate the structure of
compound 1B as shown in FIG. 1.
[0461] Signals of a CH.sub.2 group at 2.80, 2.52/36.3 in the 1 D-
and 2D-spectra corresponded presumably to the CH.sub.2 group of
asparagine (Asn) in compound 1A. This conclusion was supported by
reported data (Heterocycles 53, 1533-1549, 2000) in conjunction to
fragments obtained from MS/MS of the parent peak at m/z 961.6 (FIG.
4a). Likewise, the latter analyses provided information (FIGS. 4a,
4b) which confirmed the connection sequence of amino acids in both
compounds and led to elucidate the structure of compounds 1A and 1B
as shown in FIG. 1.
Example 7.3: Structural Identification of Compounds 2A and 2B as
Fusaricidins C and D
[0462] From peak 1-2 of fraction 1, a mixture of compounds 2A and
2B (ratio about 1:1) was obtained as a brown oil. The molecular
formula of the heavier component, compound 2B, was determined to be
C.sub.46H.sub.76N.sub.10O.sub.12 on the basis of the low resolution
mass spectrometry. Analysis of the NMR data (Table 13) allowed to
identify compound 2B as fusaricidin D. The lighter component of the
mixture, compound 2A, was likewise identified as fusaricidin C, in
which the Gln residue of fusaricidin C is replaced by Asn.
[0463] The mass spectrometric fragmentation pattern of the parent
ions of m/z 961.6 and 947.6 for compounds 2B and 2A, respectively,
(FIGS. 5a, 5b) confirmed the length of the substituted fatty acid
side chain to be identical as in compound 1B. Fusaricidins C and D
have formerly been reported by Kajimura et al. (J. Antibiot. 50,
220-228, 1997).
Example 7.4: Structural Identification of Compound 3 as LI-F08b
[0464] From peak 6-1 of fraction 6, compound 3 was isolated as a
brown oil and its low resolution presented a peak at m/z 925.6
[M+H].sup.+ which, combined with NMR data (Table 14), led to the
molecular formula C.sub.44H.sub.80N.sub.10O.sub.11. Compound 3
showed similar features in the NMR spectra as compound 1B and
compound 2B (fusaricidin D) except for the presence of aromatic
signals (Table 14). Thus, characteristic resonances of a peptide
were observed namely ten signals of protons attached to nitrogen
between .delta. 6.89 and 8.49, eight resonances of carbonyl ranged
between .delta. 168.1 and 174.3, and six signals of N-methine
comprised between .delta. 48.0 and 59.5. A detailed analysis of the
HMQC, COSY and TOCSY spectra revealed the presence of six amino
acids including Gln, two units of Thr, two units of lie and Ala.
Furthermore, these spectra showed chemical shifts attributable to
the same .beta.-hydroxyl fatty acid with a terminal guanidine as in
compounds 1A, 1B and fusaricidins C (2A) and D (2B). The position
of this side chain was determined on the basis of a long range
correlation found on the HMBC spectrum between the proton signal of
N-methine at .delta. 4.44 of Thr1 and the carbonyl signal at
.delta. 172.1 of the fatty acid. The sequence of the amino acids
was deduced from NOESY interactions and the fragmentation pattern
(FIG. 6).
[0465] The combination of the NMR data (Table 14) and mass
spectrometry led to identify the metabolite compound 3 as LI-F08b,
herein also called fusaricidin LI-F08b, reported for the first time
by Kuroda et al. (Heterocycles 53, 1533-1549, 2000).
Example 7.5: Structural Identification of Compounds 4A and 4B as
LI-F06a and LI-F06b and of Compounds 5A and 5B as Fusaricidin A and
B, Respectively
[0466] From peak 4/5 of fraction 4/5, a mixture of two further
metabolites, compounds 4A and 4B (ratio about 1:3), was obtained
which gave two peaks at m/z 897.5 (4A) and 911.6 (4B) in the ESI-MS
spectrum, suggesting two further homologous cyclodepsipeptides.
Resonances indicative for peptides were observed in their NMR
spectra (Table 15) as well as those of a .beta.-hydroxyl fatty acid
terminating in a guanidine group. The fragmentation patterns of
both parent ions found for compounds 4A and 4B (FIGS. 7a, 7b)
allowed to determine the sequence of amino acids and to identify
the constituents of the mixture as LI-F06a (4A) and LI-F06b (4B),
respectively.
[0467] Obtained from peak 3-2 of fraction 3, the mixture of
compounds 5A and 5B (ratio about 1:3) was analyzed in the same
manner. The ESI mass spectrum of the mixture showed two peaks at
m/z 883.6 (5A) and 897.5 (5B) and the fragmentation patterns of
these parent ions (FIGS. 8a, 8b) in conjunction to NMR data (Table
16) allowed to identify the components as fusaricidin A (5A) and
fusaricidin B (5B). The data found for 4A, 4B, 5A and 5B matched
those previously reported. (J. Antibiot. 50, 220-228, 1997;
Heterocycles 53, 1533-1549, 2000).
TABLE-US-00014 TABLE 12 .sup.1H (DMSO-d.sub.6, 600 MHz) and
.sup.13C-NMR (DMSO-d.sub.6, 150 MHz) data of compounds 1A and 1B.
Compounds 1 Compound 1A Compound 1B *Pos. .delta..sub.H
.delta..sub.C Pos. .delta..sub.H .delta..sub.C Thr1 Thr1 NH 7.79
(br) -- NH 8.18 (br s) -- 1 -- 168.6 1 -- 168.6 2 4.46 (br d, 8.5)
56.4 2 4.39 (br d, 8.7) 56.9 3 5.30 (overlapped) 70.2 3 5.30 (m)
70.2 4 1.13 (overlapped) 16.6 4 1.13 (d, 6.4) 16.7 Ala Ala NH 7.22
(br) -- NH 7.27 (br s) -- 1 -- nf* 1 -- 170.4 2 4.13 (overlapped)
47.7 2 4.20 (m) 47.8 3 1.11 (overlapped) 17.8 3 1.17 (d, 7.1) 17.8
Asn Gln NH 8.33 (overlapped) -- NH 8.20 (br s) -- 1 -- 169.7 1 --
70.4 2 4.20 (1H, m) 50.6 2 3.87 (m) 53.2 3 2.52 (m), 36.3 3 1.96
(m), 2.08 (m) 26.2 2.80 (dd, 5.9, 15.1) 4 -- 172.5 4 2.08 (m), 2.18
(m) 32.0 5 -- -- -- -- 174.3 NH.sub.2 6.99 (br s), 7.42 (br s) --
NH.sub.2 6.83 (br s), 7.26 (br s) -- Thr2 Thr2 NH 8.50 (overlapped)
-- NH 8.58 (br s) -- 1 -- 170.6 1 -- 170.6 2 3.94 (m) 59.9 2 3.84
(m) 60.5 3 3.94 (m) 65.5 3 3.85 (m) 65.8 4 1.05 (br) 20.3 4 1.08
(overlapped) 20.0 Tyr Tyr NH 8.48 (overlapped) -- NH 8.52 (br s) --
1 -- nf 1 -- 166.7 2 4.60 (m) 54.2 2 4.51 (m) 54.5 3 2.60
(overlapped) 36.8 3 2.60 (m), 2.88 (m) 36.9 2.88 (overlapped) 4 --
127.7 4 -- 127.8 5 and 9 7.07 (d, 8.7) 130.2 5 and 9 7.06 (d, 8.5)
130.2 6 and 8 6.60 (overlapped) 114.7 6 and 8 6.60 (d, 8.5) 114.7 7
-- 155.9 7 -- 155.9 Ile Ile NH 7.28 (br s) -- NH 7.42 (br s) -- 1
-- nf 1 -- 170.4 2 4.16 (overlapped) 56.5 2 4.16 (br d, 8.5) 56.5 3
1.34 (overlapped) 37.2 3 1.34 (m) 37.2 4 1.34 (overlapped) 25.4 4
1.22 (m), 1.34 (m) 25.4 5 0.52 (overlapped) 14.4 5 0.53
(overlapped) 14.4 6 0.59 (overlapped) 11.4 6 0.61 (overlapped) 11.4
*FA FA 1 -- 171.9 1 -- 171.9 2 2.35 (overlapped) 43.1 2 2.35 (m)
43.3 3 3.77 (overlapped) 67.5 3 3.77 (m) 67.5 4 1.34 (overlapped)
36.8 4 1.34 (m) 36.9 5-12 1.19-1.30 (br s) 29.0-29.2 5-12 1.19-1.30
(br s) 29.0-29.2 13 1.25 (br s) 21.2 13 1.25 (br s) 21.2 14 1.43
(overlapped) 28.7 14 1.43 (m) 28.5 15 3.03 (overlapped) 40.6 15
3.03 (q, 6.6) 40.6 *Gu Gu NH nf -- NH 8.40 (br s) -- 16 -- 157.2 16
-- 157.2
TABLE-US-00015 TABLE 13 .sup.1H (DMSO-d.sub.6, 600 MHz) and
.sup.13C-NMR (DMSO-d.sub.6, 150 MHz) data of compounds 2A and 2B.
Compounds 2 = fusaricidins C and D Compound 2A = fusaricidin C
Compound 2B = fusaricidin D Pos. .delta..sub.H .delta..sub.C Pos.
.delta..sub.H .delta..sub.C Thr1 Thr1 NH 7.66 (d, 7.1) -- NH 8.17
(br s) -- 1 -- 168.5 1 -- 168.6 2 4.44 (br d, 8.9) 56.6 2 4.40 (br
d, 8.9) 57.0 3 5.31 (m) 70.2 3 5.30 (m) 70.3 4 1.13 (overlapped)
16.5 4 1.14 (overlapped) 16.7 Ala Ala NH 7.21 (br) -- NH 7.60 (br
s) 1 -- nf 1 -- 170.6 2 4.12 (m) 47.7 2 4.19 (m) 47.8 3 1.12
(overlapped) 17.8 3 1.17 (d, 7.2) 17.7 Asn Gln NH 8.26 (br) -- NH
8.08 (br s) -- 1 -- 169.7 1 -- 170.4 2 4.21 (m) 50.5 2 3.86 (m)
53.2 3 2.53 (overlapped), 36.3 3 1.98 (m), 2.09 (m) 26.1 2.80 (dd,
6.3, 15.0) 4 -- 172.6 4 2.10 (m), 2.18 (m) 31.9 5 -- -- 5 -- 174.3
NH.sub.2 nf -- NH.sub.2 6.84 (br s), -- 7.28 (br s) Thr2 Thr2 NH
8.52 (overlapped) -- NH 8.47 (overlapped) -- 1 -- 170.3 1 -- 170.6
2 3.85 (m) 60.5 2 3.94 (m) 59.9 3 3.86 (m) 65.8 3 3.92 (m) 65.7 4
1.09 (d, 5.7) 19.9 4 1.05 (d, 5.8) 20.2 OH-3 4.96 (br d, 4.2) --
OH-3 5.05 (d, 2.9) -- Tyr Tyr NH 8.46 (overlapped) -- NH 8.52
(overlapped) -- 1 -- nf 1 -- 172.3 2 4.60 (m) 54.2 2 4.52 (m) 54.6
3 2.63 (overlapped) 36.9 3 2.63 (m), 2.87 (m) 36.9 2.87
(overlapped) 4 -- 127.7 4 -- 127.7 5 and 9 7.08 (overlapped) 130.2
5 and 9 7.06 (d, 8.4) 130.2 6 and 8 6.60 (overlapped) 114.7 6 and 8
6.60 (d, 8.4) 114.7 7 -- 155.8 7 -- 155.8 OH nf -- OH 9.13 (br s)
-- Val Val NH 7.30 (overlapped) -- NH 7.42 (br s) -- 1 -- nf 1 --
170.3 2 4.12 (br s) 57.5 2 4.12 (br s) 57.5 3 1.59 (m) 30.9 3 1.59
(m) 31.0 4 0.56 (d, 6.4) 18.2 4 0.57 (d, 6.3) 18.3 5 0.35 (d, 6.5)
18.7 5 0.40 (d, 6.6) 18.7 FA FA 1 -- nf 1 -- 172.0 2 2.37
(overlapped) 43.1 2 2.37 (m) 43.3 3 3.79 (overlapped) 67.5 3 3.79
(m) 67.6 4 1.35 (overlapped) 36.9 4 1.35 (m) 36.9 5 1.22
(overlapped) 25.3 5 1.22 (br s) 25.3 6-12 1.20-1.27 (br s)
29.1-29.2 6-12 1.20-1.27 (br s) 29.1-29.2 13 1.26 (br s) 26.1 13
1.26 (br s) 26.1 14 1.44 (overlapped) 28.5 14 1.44 (m) 28.7 15 3.07
(overlapped) 40.7 15 3.07 (q, 6.7) 40.7 Gu Gu NH nf -- NH 7.60 (br
s) -- 16 -- 156.8 16 -- 156.8
TABLE-US-00016 TABLE 14 .sup.1H (DMSO-d.sub.6, 600 MHz) and
.sup.13C-NMR (DMSO-d.sub.6, 150 MHz) data of compound 3 being
Ll-F08b. Compound 3 = Ll-F08b Pos. .delta..sub.H .delta..sub.C Thr1
NH 7.55 (br s) -- 1 -- 168.1 2 4.44 (br d, 8.4) 56.6 3 5.33 (m)
70.2 4 1.15 (d, 6.5) 16.7 Ala NH 7.53 (br s) -- 1 -- 170.6 2 4.05
(m) 48.0 3 1.22 (br s) 17.2 Gln NH 7.93 (br s) -- 1 -- 170.5 2 3.94
(m) 52.7 3 1.98 (m), 2.09 (m) 26.5 4 2.12 (m), 2.20 (m) 31.9 5 --
174.3 NH.sub.2 6.89 (br s), 7.32 (br s) -- Thr2 NH 8.48 (br s) -- 1
-- 170.7 2 4.03 (m) 59.5 3 3.98 (m) 65.7 4 1.08 (d, 6.1) 19.8 Ile1
NH 8.49 (br s) -- 1 -- 172.5 2 4.15 (t, 7.6) 57.3 3 1.81 (m) 35.4 4
1.17 (m), 1.41 (m) 24.4 5 0.80 (t, 6.3) 10.6 6 0.81 (d, 7.2) 15.5
Ile2 NH 7.30 (br s) -- 1 -- 171.3 2 4.53 (m) 55.3 3 1.65 (m) 38.2 4
1.01 (m), 1.37 (m) 25.5 5 0.83 (t, 6.4) 11.4 6 0.70 (d, 7.4) 14.2
FA 1 -- 172.1 2 2.37 (d, 5.7) 43.4 3 3.77 (m) 67.6 4 1.37 (m) 36.9
5-12 1.20-1.28 (br s) 29.0-29.2 13 1.25 (br s) 26.2 14 1.43 (m)
28.7 15 3.03 (q, 6.7) 40.6 Gu NH 8.37 (br s) -- 16 -- 157.2
TABLE-US-00017 TABLE 15 .sup.1H (DMSO-d.sub.6, 600 MHz) and
.sup.13C-NMR (DMSO-d.sub.6, 150 MHz) data of compounds 4A and 4B.
Compounds 4 = Ll-F06a and Ll-F06b Compound 4A = Ll-F06a Compound 4B
= Ll-F06b Pos. .delta..sub.H .delta..sub.C Pos. .delta..sub.H
.delta..sub.C Thr1 Thr1 NH 8.31 (br) -- NH 7.59 (br s) -- 1 --
168.5 1 -- 168.4 2 4.40 (m) 56.9 2 4.44 (m) 56.7 3 5.30 (m) 70.5 3
5.32 (m) 70.3 4 1.14 (m) 16.6 4 1.15 (m) 16.6 Ala Ala NH nf -- NH
7.53 (br s) 1 -- 170.6 1 -- 170.7 2 3.97 (m) 47.9 2 4.07 (m) 48.0 3
1.15 (overlapped) 17.3 3 1.21 (d, 7.3) 17.4 Asn Gln NH 8.06 (br) --
NH 7.96 (br s) -- 1 -- 169.8 1 -- 170.7 2 4.28 (m) 50.5 2 3.93 (m)
52.9 3 2.55 (m), 36.9 3 1.97 (m), 2.10 (m) 26.5 2.75 (dd, 6.7,
15.1) 4 -- 172.6 4 2.12 (m), 2.21 (m) 32.0 5 -- -- 5 -- 174.4
NH.sub.2 nf -- NH.sub.2 6.88 (br s), -- 7.33 (br s) Thr2 Thr2 NH
8.54 (br) NH 8.48 (br) -- 1 -- 170.4 1 -- 170.6 2 3.91 (m) 60.5 2
4.02 (m) 59.7 3 3.92 (m) 65.6 3 3.99 (m) 65.7 4 1.09 (d, 6.4) 19.6
4 1.08 (d, 6.4) 19.8 Val Val NH 7.28 (m) -- NH 7.39 (m) -- 1 -- nf
1 -- 171.0 2 4.40 (overlapped) 57.3 2 4.39 (m) 57.0 3 1.83
(overlapped) 32.0 3 1.83 (m) 31.6 4 0.75 (d, 6.6) 18.1 4 0.74 (d,
6.6) 18.4 5 0.84 (overlapped) 19.3 5 0.80 (overlapped) 19.2 Ile Ile
NH 7.31 (overlapped) -- NH 7.23 (overlapped) -- 1 -- nf 1 -- 171.2
2 4.51 (overlapped) 55.5 2 4.51 (1H, m) 55.6 3 1.65 (overlapped)
38.1 3 1.65 (m) 38.1 4 1.02 (m), 1.36 (m) 25.4 4 1.02 (m), 1.36 (m)
25.5 5 0.82 (overlapped) 15.6 5 0.82 (overlapped) 15.6 6 0.72
(overlapped) 14.4 6 0.71 (overlapped) 14.3 FA FA 1 -- 172.1 1 --
172.2 2 2.44 (dd) 43.1 2 2.37 (m) 43.4 3 3.81 (m) 67.7 3 3.78 (m)
67.7 4 1.37 (overlapped) 36.9 4 1.37 (m) 36.9 5-12 1.22-1.24 (br s)
29.1-29.2 5 1.22-1.24 (br s) 29.1-29.2 13 1.25 (br s) 26.4 13 1.25
(br s) 26.2 14 1.43 (m) 28.5 14 1.43 (m) 28.5 15 3.03 (q, 6.7) 40.7
15 3.03 (q, 6.7) 40.7 Gu Gu NH nf -- NH 8.34 (br s) -- 16 -- 157.2
16 -- 157.2
TABLE-US-00018 TABLE 16 .sup.1H (DMSO-d.sub.6, 600 MHz) and
.sup.13C-NMR (DMSO-d.sub.6, 150 MHz) data of compounds 5A and 5B.
Compounds 5 = fusaricidins A and B, Ll-F04a and Ll-F04b Compound 5A
= fusaricidin A Compound 5B = fusaricidin B Pos. .delta..sub.H
.delta..sub.C Pos. .delta..sub.H .delta..sub.C Thr1 Thr1 NH 7.66
(br) -- NH 8.30 (d, 8.0) -- 1 -- 168.5 1 -- 168.4 2 4.46 (m) 56.6 2
4.40 (br d, 8.5) 57.0 3 5.32 (m) 70.4 3 5.31 (m) 70.3 4 1.16
(overlapped) 16.3 4 1.15 (d, 5.7) 16.6 Ala Ala NH 7.26 (br) -- NH
7.53 (br s) -- 1 -- 170.6 1 -- 170.6 2 4.00 (m) 47.8 2 4.10 (m)
47.9 3 1.15 (overlapped) 17.4 3 1.20 (d, 7.2) 17.5 Asn Gln NH 8.10
(br) -- NH 8.53 (d, 4.3) -- 1 -- 169.8 1 -- 170.6 2 4.28 (q, 6.6)
50.5 2 3.92 (m) 52.9 3 2.53 (m), 36.7 3 1.98 (m), 2.09 (m) 26.4
2.76 (dd, 6.6, 15.0) 4 -- 172.5 4 2.10 (m), 2.20 (m) 31.9 5 -- -- 5
-- 174.3 NH.sub.2 nf -- NH.sub.2 6.86 (br s), 7.30 (br -- s) Thr2
Thr2 NH 8.54 (br s) -- NH 8.46 (d, 6.9) -- 1 -- nf 1 -- 170.5 2
3.91 (m) 60.4 2 4.02 (m) 59.7 3 3.91 (m) 65.6 3 3.99 (m) 65.5 4
1.09 (d, 5.6) 19.6 4 1.07 (d, 6.0) 19.9 Val Val NH nf -- NH 7.29
(br s) -- 1 -- nf 1 -- 171.2 2 4.40 (m) 57.1 2 4.40 (m) 57.1 3 1.82
(m) 31.4 3 1.82 (m) 31.5 4 nf nf 4 0.76 (d, 6.6) 18.4 5 0.82 (d,
6.0) 19.1 5 0.81 (d, 6.2) 19.1 Val Val NH 8.41 (br s) -- NH 8.37
(d, 7.6) -- 1 -- 172.1 1 -- 173.1 2 4.13 (m) 58.3 2 4.23 (m) 57.8 3
2.02 (m) 29.7 3 1.99 (m) 30.2 4 0.86 (d, 6.7) 18.2 4 0.86 (d, 6.7)
18.2 5 0.84 (7.0) 19.3 5 0.84 (7.0) 19.3 FA FA 1 -- 172.1 1 --
172.0 2 2.37 (br d, 5.8) 43.4 2 2.34 (dd, 7.0, 13.5), 43.4 2.44
(dd, 4.9, 13.5) 3 3.80 (m) 67.6 3 3.80 (m) 67.6 4 1.37 (m) 36.9 4
1.37 (m) 36.8 5-12 1.22-1.25 (br s) 26.2-29.2 5-12 1.22-1.25 (br s)
26.2-29.2 13 1.25 (br s) 26.2 13 1.25 (br s) 26.2 14 1.43 (m) 28.7
14 1.43 (m) 28.5 15 3.03 (q, 6.7) 40.6 15 3.03 (q, 6.7) 40.6 Gu Gu
NH nf -- NH 8.47 (br s) -- 16 -- 157.2 16 -- 157.2
[0468] No hydrolysis experiments were carried out to determine the
configuration of the constituting amino acids. *Pos.=position;
FA=fatty acid; Gu=Guanidine; nf=not found. Legend applies also to
Tables 13 to 16
Example 8--Metabolites Produced by Paenibacillus Strains
Example 8.1: Production of Metabolites by Paenibacillus Strains
[0469] The presence of fusaricidins in general and in particular of
the known fusaricidins A, B, C, D, LI-F06a, LI-F06b and LI-F08b as
well as the novel fusaricidin-type compounds 1A and 1B was
determined for the Paenibacillus strains following the procedural
steps which are described in Example 7.1 above.
TABLE-US-00019 TABLE 17 Fusaricidin-type metabolite production of
the Paenibacillus strains. Compound/Fusaricidin 2A 2B 3 4A 4B 5A 5B
Strains 1A 1B C D LI-F08b LI-F06a LI-F06b A B Lu16774 + ++ ++ ++ ++
- - ++ ++ Lu17007 + ++ ++ ++ ++ + ++ ++ ++ Lu17015 ++ ++ ++ ++ ++
++ ++ ++ ++ BD-62 - - - - - - - - - Legend: -, compound not
detectable; +, compound detectable; ++, compound detectable at
higher amounts compared to scale +.
[0470] The whole culture broth of all of the novel Paenibacillus
strains Lu16774, Lu17007 and Lu17015 contained at least one the
fusaricidin-type metabolites identified in Example 7 (Table 17).
None of these fusaricidin-type metabolites were detected in the
whole culture broth of P. peoriae strain BD-62.
[0471] The whole culture broth of the novel Paenibacillus strains
Lu16774, Lu17007 and Lu17015 all contained the novel
fusaricidin-type compounds 1A and 1B. Further, the whole culture
broth of the novel Paenibacillus strains Lu16774, Lu17007 and
Lu17015 all contained the fusaricidins A, B, C and D as well as
LI-F08b. In addition, the whole culture broth of the novel
Paenibacillus strains Lu17007 and Lu17015 contained fusaricidins
LI-F06a and LI-F06b.
[0472] Compounds 1A and 1B were not detected in the whole culture
broth of the closely related P. peoriae strain BD-62. Fusaricidins
A, B, C and D, LI-F06a, LI-F06b and LI-F08b were also not in the
whole culture broth of P. peoriae strain BD-62.
Example 9: Activity of Metabolites by Paenibacillus Strains Against
Various Fungal Pathogens
[0473] The compounds 1A and 1B, fusaricidin A, B and D and were
obtained were used in the following experiments.
[0474] Fungal growth assays were performed in 96 well plates with
spore suspension of the pathogen Botrytis cinerea (BOTRCI, in YBA
[10 g Bacto peptone (Becton Dickinson 211677), 10 g yeast extract
(Becton Dickinson 212750), 20 g sodium acetate, ad 1000 mL aqua
bidest] or Alternaria solani (ALTESO, in YBG [10 g Bacto peptone
(Becton Dickinson 211677), 10 g yeast extract (Becton Dickinson
212750), 20 g glycerine 99%, ad 1000 mL aqua bidest]). Fusaricidins
and compounds 1A and 1B were dissolved and diluted in DMSO.
Different concentrations ranging from 60 .mu.M down to 0.3 .mu.M
were pipetted into the microtiter plate. An aqueous suspension of
10.sup.4 spores/ml was added. The plates were incubated at about
18.degree. C. Fungal growth was determined by measuring the optical
density at 600 nm in a microplate reader 3 and 7 days after the
inoculation of the spores and compared to the untreated control
(DMSO). IC.sub.50 (concentration [.mu.M] of the respective
metabolite required for 50% inhibition of fungal growth) has been
determined thereafter.
[0475] Notably, the compounds 1A and 1B showed the highest
antifungal efficacy with IC.sub.50 values of 0.4-0.6 .mu.M (Tab.
18).
TABLE-US-00020 TABLE 18 Antifungal growth inhibition of
Paenibacillus metabolites IC.sub.50 values Compound/Fusaricidin
Pathogen 2B 5A 5B (Evaluation 1A 1B Fus. D Fus. A Fus. B day)
Fungal growth inhibition (IC50 [.mu.M]) ALTESO (3 d) 0.6 0.6 1.1
1.3 1.1 ALTESO (7 d) 0.5 0.4 0.6 0.7 0.6 BOTRCI (7 d) 0.3 0.4 0.5
0.5 0.6 "--" means that growth inhibition in tested concentration
range not sufficient to determine IC.sub.50.
[0476] In addition, glasshouse trials were performed with Compounds
1A and 1B as described in the Use Examples 5.1 to 5.5 above for the
respective pathogens Botrytis cinerea (BOTRCI), Alternaria solani
(ALTESO), Phytophthora infestans (PHYTIN), Phakopsora pachyrhizi
(PHAKPA) and Fusarium graminearum (GIBBZE). The extent of fungal
attack on the leaves was visually assessed 5-7 days after
inoculation.
[0477] Notably, compounds 1A and 1B were effective in controlling
important fungal diseases on crop plants already at dose levels as
low as 7.2 ppm and showed higher antifungal efficacy than
Fusaricidin A, B and D (Tables 19 to 21).
TABLE-US-00021 TABLE 19 Antifungal efficacy of metabolites
determined in planta. % efficacy (% fungal attack) Metabolite
tested Conc. BOTRCI ALTESO PHYTIN PHAKPA GIBBZE Untreated -- 0
(100) 0 (100) 0 (100) 0 (100) 0 (100) Compound 1A 360 ppm 99 100 95
49 Compound 1A 36 ppm 97 74 Compound 1B 360 ppm 100 100 97 Compound
1B 36 ppm 97
TABLE-US-00022 TABLE 20 Efficacy of metabolites against late blight
on tomato caused by Phytophthora infestans with protective
application. % efficacy Metabolite tested Conc. (% fungal attack)
Untreated -- 0 (100) Fusaricidin A 7.2 ppm 15 Fusaricidin B 7.2 ppm
4 Fusaricidin D 7.2 ppm 0 Compound 1B 7.2 ppm 44
TABLE-US-00023 TABLE 21 Efficacy of metabolites against head blight
on wheat caused by Fusarium graminearum with protective
application. % efficacy Metabolite tested Conc. (% fungal attack)
Untreated -- 0 (100) Fusaricidin A 360 ppm 31 Fusaricidin B 360 ppm
0 Compound 1A 360 ppm 49
TABLE-US-00024 TABLE 22 Efficacy of metabolites against head blight
on wheat caused by Septoria tritici with protective application. %
efficacy Metabolite tested Conc. (% fungal attack) Untreated -- 0
(100) Fusaricidin D 360 ppm 50 Compound 1B 360 ppm 80
Example 10: Comparison of Activity of Paenibacillus polymyxa nov.
ssp. Plantarum Strains Lu16674 and Lu17007 of According to the
Invention with Paenibacillus polymyxa nov. ssp. Plantarum M-1
Against Various Pathogens in Glass House Trials
[0478] Whole culture broth from 6 days old cultures of
Paenibacillus strain Lu17007, Lu16674 and M1 was obtained according
to Use Example 3 and used as in the experimental setup of Use
Example 5.1 to 5.5. The glasshouse trials were performed as
described in the Use Examples 5.1 to 5.5 above for the respective
pathogens. The extent of fungal attack on the leaves was visually
assessed 5-7 days after inoculation.
[0479] Notably, the Paenibacillus strains Lu16774 and Lu17007 were
effective in controlling important fungal diseases on crop plants
even at high dilution factors and showed higher antifungal efficacy
than the closely related strain M-1 (Tables 22 to 27).
TABLE-US-00025 TABLE 22 Paenibacillus Dilution factor of BOTRCI %
efficacy strain whole culture broth (% fungal attack) Untreated 0
(100) Lu16674 1:10 95 M-1 1:10 86 Lu16674 1:50 76 Lu17007 1:50 98
M-1 1:50 51
TABLE-US-00026 TABLE 23 Paenibacillus Dilution factor of BOTRCI %
efficacy strain whole culture broth (% fungal attack) Untreated 0
(100) Lu17007 undiluted 92 M-1 undiluted 87 Lu17007 1:10 84 M-1
1:10 53 Lu17007 1:50 63 M-1 1:50 32
TABLE-US-00027 TABLE 24 Paenibacillus Dilution factor of ALTESO %
efficacy strain whole culture broth (% fungal attack) Untreated 0
(100) Lu16674 1:10 77 M-1 1:10 41
TABLE-US-00028 TABLE 25 Paenibacillus Dilution factor of PHYTIN %
efficacy strain whole culture broth (% fungal attack) Untreated 0
(100) Lu17007 1:10 83 M-1 1:10 42 Lu16674 1:50 13 Lu17007 1:50 30
M-1 1:50 0
TABLE-US-00029 TABLE 26 Paenibacillus Dilution factor of PHAKPA %
efficacy strain whole culture broth (% fungal attack) Untreated 0
(100) Lu17007 undiluted 94 M-1 Undiluted 87
TABLE-US-00030 TABLE 27 Paenibacillus Dilution factor of GIBBZE %
efficacy strain whole culture broth (% fungal attack) Untreated 0
(100) Lu17007 undiluted 70 M-1 undiluted 31 Lu16674 1:50 52 Lu17007
1:50 33 M-1 1:50 24
[0480] The documents as cited herein are incorporated by
reference.
BRIEF DESCRIPTION OF THE FIGURES
[0481] FIG. 1. Compounds 1A, 1B, 2A, 2B, 3, 4A, 4B, 5A and 5B.
[0482] FIG. 2. Key NOESY and COSY correlations of compound 1B.
[0483] FIG. 3. HMBC correlation of compound 1B.
[0484] FIG. 4. Fragmentation patterns a) of compound 1A and b) of
compound 1B.
[0485] FIG. 5. Fragmentation patterns a) of compound 2A
(fusaricidin C) and b) of compound 2B (fusaricidin D).
[0486] FIG. 6. Fragmentation pattern of compound 3 (LI-F08b).
[0487] FIG. 7. Fragmentation patterns a) of compound 4A (LI-F06a)
and b) of compound 4B (LI-F06b).
[0488] FIG. 8. Fragmentation patterns a) of compound 5A
(fusaricidin A) and b) of compound 5B (fusaricidin B).
[0489] FIG. 9 shows the percentage identity of the complete 16S
rDNA sequence of the Paenibacillus strains of the invention to
related taxa after multiple sequence alignment. Legend: *Strain
numbers: 1=Paenibacillus strain Lu16774; 2=Paenibacillus strain
Lu17015; 3=Paenibacillus strain Lu17007; 4=Paenibacillus peoriae
NRRL BD-62; 5=Paenibacillus anaericanus M H21; 6=Paenibacillus
brasiliensis PB172; 7=Paenibacillus campinasensis 324;
8=Paenibacillus chibensis JCM 9905; 9=Paenibacillus glucanolyticus
DSM 5162; 10=Paenibacillus hunanensis FeL05; 11=Paenibacillus
jamilae CECT 5266; 12=Paenibacillus knbbensis AM49;
13=Paenibacillus lactis MB 1871; 14=Paenibacillus lautus JCM 9073;
15=Paenibacillus macerans IAM 12467; 16=Paenibacillus massiliensis
2301065; 17=Paenibacillus pabuli HSCC 492; 18=Paenibacillus peoriae
DSM 8320 (BD-57); 19=Paenibacillus pini S22; 20=Paenibacillus
polymyxa IAM 13419; 21=Paenibacillus purispatii ES_MS17;
22=Paenibacillus sedininis GT-H3; 23=Paenibacillus terrae AM141;
24=Paenibacillus terrigena A35; 25=Paenibacillus timonensis
2301032; 26=Paenibacillus turicensis MOL722; 27=Paenibacillus
uliginis N3/975; 28=Cohnella thermotolerans CCUG 47242. Strains 6
to 28 are type strains for the respective species.
[0490] Similarities of the novel strains with Paenibacillus peoriae
(NRRL BD-62 and DSM 8320) have been marked in bold letters.
[0491] FIG. 10 shows a phylogenetic dendrogram calculated from the
% identity of 16S-rDNA sequences of the Paenibacillus strains of
the invention with other taxa (FIG. 9). The root of the tree was
determined by including the 16S rRNA gene sequence of Cohnella
thermotolerans into the analysis. The scale bar below the
dendrogram indicates 1 nucleotide substitutions per 100
nucleotides.
[0492] FIG. 11 shows the RiboPrint pattern obtained from samples of
the Paenibacillus strains of the invention in comparison to a
sample of the closely related P. peoriae strain BD-62 using
RiboPrinter Microbial Characterization System and a phylogenetic
dendrogram resulting therefrom.
[0493] FIG. 12 shows the percentage identity of the DNA sequence of
the dnaN gene of the Paenibacillus strains of the invention to
related Paenibacillus strains after multiple sequence
alignment.
[0494] Legend: *Strain numbers: 1=Paenibacillus strain Lu16774;
2=Paenibacillus strain Lu17007; 3=Paenibacillus strain Lu17015;
4=P. peoriae DSM 8320.sup.T=KCTC 3763.sup.T (GenBank acc. no.
AGFX00000000; J. Bacteriol. 194, 1237-1238, 2012); 5=P. polymyxa
1-43 (GenBank acc. no. ASRZ01000000; deposition no. GCMCC 4965; CN
102352332B); 6=P. polymyxa A18 (GenBank acc. no JWJJ00000000.1;
NCBI Project ID 225496); 7=P. polymyxa ATCC 842.sup.T=DSM
36.sup.T=KCTC 3858.sup.T (GenBank acc. no. AFOX00000000; J.
Bacteriol. 193(18), 5026-5027, 2011); 8=P. polymyxa CF05 (GenBank
acc. no. CP009909; Genome Announc 3(2):e00198-15.
Doi:10.1128/genomeA.00198-15); 9=P. polymyxa CICC 10580 (GenBank
acc. no. JNCB00000000; Genome Announc. 2(4):e00854-14.
doi:10.1128/genomeA.00854-14); 10=P. polymyxa DSM 365 (GenBank acc.
no. JMIQ00000000; J. Biotechnol. 195, 72-73, 2015); 11=P. polymyxa
E681 (GenBank acc. no. CP000154; GenomeNet Ref Seq NC_014483.2; J.
Bacteriol. 192(22), 6103-6104, 2010); 12=P. polymyxa M-1 (GenBank
acc. no. HE577054.1; GenomeNet Ref Seq NC_017542.1); 13=P. polymyxa
NRRL B-30509 (GenBank acc. no. JTHO00000000; Genome Announc. 2015
March-April; 3(2): e00372-15); 14=P. polymyxa SC2 (GenBank acc. no.
CP002213; J. Bacteriol. 193 (1), 311-312, 2011); 15=P. polymyxa
SQR-21 (GenBank acc. no. CP006872; GenomeNet Ref Seq NZ_CP006872.1;
Genome Announc. 2014 March-April; 2(2): e00281-14); 16=P. polymyxa
Sb3-1 (GenBank acc. no. CP010268; Genome Announc. 2015 March-April;
3(2): e00052-15); 17=P. polymyxa TD94 (GenBank acc. no.
ASSA00000000); 17=P. polymyxa WLY78 (GenBank acc. no.
ALJV00000000); P. terrae HPL-003 (GenBank acc. no. CP003107; NCBI
Ref Seq NC_016641.1); P. polymyxa CR1 (GenBank acc. no. CP006941;
Genome Announc. 2014 January-February; 2(1): e01218-13).
[0495] FIG. 13 shows the percentage identity of the DNA sequence of
the complete gyrB gene of the Paenibacillus strains of the
invention to related Paenibacillus strains after multiple sequence
alignment. Strain numbers are described in Legend to FIG. 12.
[0496] FIG. 14 shows the percentage identity of the DNA sequence of
the complete recF gene of the Paenibacillus strains of the
invention to related Paenibacillus strains after multiple sequence
alignment. Strain numbers are described in Legend to FIG. 12.
[0497] FIG. 15 shows the percentage identity of the DNA sequence of
the complete recN gene of the Paenibacillus strains of the
invention to related Paenibacillus strains after multiple sequence
alignment. Strain numbers are described in Legend to FIG. 12.
[0498] FIG. 16 shows the percentage identity of the DNA sequence of
the complete rpoA gene of the Paenibacillus strains of the
invention to related Paenibacillus strains after multiple sequence
alignment. Strain numbers are described in Legend to FIG. 12.
[0499] FIG. 17 shows the maximum likelihood dendrogram on basis of
the complete dnaN gene sequence of strains of the P. polymyxa
complex. The scale of 0.1 shown corresponds to 1% nucleotide
exchanges.
[0500] FIG. 18 shows the maximum likelihood dendrogram on basis of
the complete gyrB gene sequence of strains of the P. polymyxa
complex. The scale of 0.1 shown corresponds to 1% nucleotide
exchanges.
[0501] FIG. 19 shows the maximum likelihood dendrogram on basis of
the complete recF gene sequence of strains of the P. polymyxa
complex. The scale of 0.1 shown corresponds to 1% nucleotide
exchanges.
[0502] FIG. 20 shows the maximum likelihood dendrogram on basis of
the complete recN gene sequence of strains of the P. polymyxa
complex. The scale of 0.1 shown corresponds to 1% nucleotide
exchanges.
[0503] FIG. 21 shows the maximum likelihood dendrogram on basis of
the complete rpoA gene sequence of strains of the P. polymyxa
complex. The scale of 0.1 shown corresponds to 1% nucleotide
exchanges.
[0504] FIG. 22 shows the Amino Acid Index (AAI) matrix of
representative genomes of the P. polymyxa complex performed
according to Example 2.5. Strain numbers are described in Legend to
FIG. 12.
Sequence CWU 1
1
1811539DNAPaenibacillus polymyxa spp. plantarummisc_featureStrain
Lu16774 complete 16S rDNA 1tttgatcctg gctcaggacg aacgctggcg
gcgtgcntaa tacatgcaag tcgagcgngn 60ttatntagaa gcttgcttct anaattncna
gcggcggacg ggtgagtaac acgtaggcaa 120cctgcccaca agacagggat
aactaccgga aacggtagct aatacccgat acatcctttt 180cctgcatggg
agaaggagga aagncggagc aatctgtcac ttgtggatgg gcctgcggcg
240cattagctag ttggtggggt aanggcctac caaggcgacg atgcgtagcc
gacctgagag 300ggtgatcggc cacactggga ctgagacacg gcccagactc
ctacgggagg cagcagtagg 360gaatcttccg caatgggcga aagcctgacg
gagcaacgcc gcgtgagtga tgaaggtttt 420cggatcgtaa agctctgttg
ccagggaaga acgtcttgta gagtaactgc tacaagagtg 480acggtacctg
agaagaaagc cccggctaac tacgtgccag cagccgcggt aatacgtagg
540gggcaagcgt tgtccggaat tattgggcgt aaagcgcgcg caggcggctc
tttaagtctg 600gtgtttaatc ccgaggctca acttcgggtc gcactggaaa
ctggngagct tgagtgcaga 660agaggagagt ggaattccac gtgtagcggt
gaaatgcgta gagatgtgga ggaacaccag 720tggcgaaggc gactctctgg
gctgtaactg acgctgaggc gcgaaagcgt ggggagcaaa 780caggattaga
taccctggta gtccacgccg taaacgatga atgctaggtg ttaggggttt
840cgataccctt ggtgccgaag ttaacacatt aagcattccg cctggggagt
acggtcgcaa 900gactgaaact caaaggaatt gacggggacc cgcacaagca
gtggagtatg tggtttaatt 960cgaagcaacg cgaagaacct taccaggtct
tgacatccct ctgaccggtc tagagatagn 1020cctttccttc gggacagagg
agacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1080atgttgggtt
aagtcccgca acgagcgcaa cccttatgct tagttgccag caggtcaagc
1140tgggcactct aagcagactg ccggtgacaa accggaggaa ggtggggatg
acgtcaaatc 1200atcatgcccc ttatgacctg ggctacacac gtactacaat
ggccggtaca acgggaagcg 1260aagncgcgag gtggagccaa tcctagaaaa
gccggtctca gttcggattg taggctgcaa 1320ctcgcctaca tgaagtcgga
attgctagta atcgcggatc agcatgccgc ggtgaatacg 1380ttcccgggtc
ttgtacacac cgcccgtcac accacgagag tttacaacac ccgaagtcgg
1440tgaggtaacc gcaaggngcc agccgccgaa ggtggggtag atgattgggg
tgaagtcgta 1500acaaggtagc cgtatcggaa ggtgcggctg gatcacctc
153921545DNAPaenibacillus polymyxa spp. plantarummisc_featureStrain
Lu17007 complete 16S rDNA 2tttgatcctg gctcaggacg aacgctggcg
gcgtgcctaa tacatgcaag tcgagcgngn 60ttatntagaa gcttgcttct anataancta
gcggcggacg ggtgagtaac acgtaggcaa 120cctgcccaca agacagggat
aactaccgga aacggtagct aatacccgat acatcctttt 180cctgcatggg
agaaggagga aagacggagc aatctgtcac ttgtggatgg gcctgcggcg
240cattagctag ttggtggggt aawggcctac caaggcgacg atgcgtagcc
gacctgagag 300ggtgatcggc cacactggga ctgagacacg gcccagactc
ctacgggagg cagcagtagg 360gaatcttccg caatgggcga aagcctgacg
gagcaacgcc gcgtgagtga tgaaggtttt 420cggatcgtaa agctctgttg
ccagggaaga acgtcttgta gagtaactgc tacaagagtg 480acggtacctg
agaagaaagc cccggctaac tacgtgccag cagccgcggt aatacgtagg
540gggcaagcgt tgtccggaat tattgggcgt aaagcgcgcg caggcggctc
tttaagtctg 600gtgtttaatc ccgaggctca acttcgggtc gcactggaaa
ctggngagct tgagtgcaga 660agaggagagt ggaattccac gtgtagcggt
gaaatgcgta gagatgtgga ggaacaccag 720tggcgaaggc gactctctgg
gctgtaactg acgctgaggc gcgaaagcgt ggggagcaaa 780caggattaga
taccctggta gtccacgccg taaacgatga atgctaggtg ttaggggttt
840cgataccctt ggtgccgaag ttaacacatt aagcattccg cctggggagt
acggtcgcaa 900gactgaaact caaaggaatt gacggggacc cgcacaagca
gtggagtatg tggtttaatt 960cgaagcaacg cgaagaacct taccaggtct
tgacatccct ctgaccggtc tagagatagn 1020cctttccttc gggacagagg
agacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1080atgttgggtt
aagtcccgca acgagcgcaa cccttatgct tagttgccag caggtcaagc
1140tgggcactct aagcagactg ccggtgacaa accggaggaa ggtggggatg
acgtcaaatc 1200atcatgcccc ttatgacctg ggctacacac gtactacaat
ggccggtaca acgggaagcg 1260aagccgcgag gtggagccaa tcctagaaaa
gccggtctca gttcggattg taggctgcaa 1320ctcgcctaca tgaagtcgga
attgctagta atcgcggatc agcatgccgc ggtgaatacg 1380ttcccgggtc
ttgtacacac cgcccgtcac accacgagag tttacaacac ccgaagtcgg
1440tgaggtaacc gcaaggagcc agccgccgaa ggtggggtag atgattgggg
tgaagtcgta 1500acaaggtagc cgtatcggaa ggtgcggctg gatcacctcc tttct
154531547DNAPaenibacillus epiphyticusmisc_featureStrain Lu17015
complete 16S rDNA 3tgagtttgat cctggctcag gacgaacgct ggcggcgtgc
ctaatacatg caagtcgagc 60ggggttgntt agaagcttgc ttctaancaa cctagcggcg
gacgggtgag taacacgtag 120gcaacctgcc cacaagacag ggataactac
cggaaacggt agctaatacc cgatacatcc 180ttttcctgca tgggagaagg
aggaaagacg gagcaatctg tcacttgtgg atgggcctgc 240ggcgcattag
ctagttggtg gggtaaaggc ctaccaaggc gacgatgcgt agccgacctg
300agagggtgat cggccacact gggactgaga cacggcccag actcctacgg
gaggcagcag 360tagggaatct tccgcaatgg gcgaaagcct gacggagcaa
cgccgcgtga gtgatgaagg 420ttttcggatc gtaaagctct gttgccaggg
aagaacgtct tgtagagtaa ctgctacaag 480agtgacggta cctgagaaga
aagccccggc taactacgtg ccagcagccg cggtaatacg 540tagggggcaa
gcgttgtccg gaattattgg gcgtaaagcg cgcgcaggcg gctctttaag
600tctggtgttt aatcccgagg ctcaacttcg ggtcgcactg gaaactgggg
agcttgagtg 660cagaagagga gagtggaatt ccacgtgtag cggtgaaatg
cgtagatatg tggaggaaca 720ccagtggcga aggcgactct ctgggctgta
actgacgctg aggcgcgaaa gcgtggggag 780caaacaggat tagataccct
ggtagtccac gccgtaaacg atgaatgcta ggtgttaggg 840gtttcgatac
ccttggtgcc gaagttaaca cattaagcat tccgcctggg gagtacggtc
900gcaagactga aactcaaagg aattgacggg gacccgcaca agcagtggag
tatgtggttt 960aattcgaagc aacgcgaaga accttaccag gtcttgacat
ccctctgacc ggtctagaga 1020tagncctttc cttcgggaca gaggagacag
gtggtgcatg gttgtcgtca gctcgtgtcg 1080tgagatgttg ggttaagtcc
cgcaacgagc gcaaccctta tgcttagttg ccagcaggtc 1140aagctgggca
ctctaagcag actgccggtg acaaaccgga ggaaggtggg gatgacgtca
1200aatcatcatg ccccttatga cctgggctac acacgtacta caatggccgg
tacaacggga 1260agcgaaatcg cgaggtggag ccaatcctag aaaagccggt
ctcagttcgg attgtaggct 1320gcaactcgcc tacatgaagt cggaattgct
agtaatcgcg gatcagcatg ccgcggtgaa 1380tacgttcccg ggtcttgtac
acaccgcccg tcacaccacg agagtttaca acacccgaag 1440tcggtggggt
aacccgcaag ggagccagcc gccgaaggtg gggtagatga ttggggtgaa
1500gtcgtaacaa ggtagccgta tcggaaggtg cggctggatc acctcct
154741143DNAPaenibacillus polymyxa spp. plantarummisc_featureStrain
Lu16774 dnaN 4atgaagatta gcattctgaa aaacgttttg aacgaggcca
tacaacatgt atccaaagcg 60atatccagtc gaacgacaat tccaattttg agtggtatta
agctggatgt gaatcaccag 120ggagtcacac tgaccgccag cgatacagac
atctctattc aatcctttat tccgatggag 180gatggtgacc aaacggtcgt
tcagatcgaa caacccggca gtgtagtgct acccgctaaa 240ttctttgtcg
aaattatcaa aaagttgccg tctcaggaga tccgtatgga ggtaaaagac
300caattccaaa cctttatctc atccggtgct actgaaattc agatcgttgg
tttggaccct 360gaagaatttc cggtgcttcc caacattgaa gaaaatcaag
tgatctctgt gccaggtgat 420ttgcttaaaa atatgattaa acagacggta
ttctccatct ctacccatga aacgacacct 480attttgactg gtgtattgtg
gaatctggct gagggcgaat tgaaatttgt cgcaacggac 540cgccaccgcc
ttgccacccg cagcgctcat ttggagacgt ctgaaggctt gcgttttagc
600aatgttgtca ttgcaggcaa aacgctcaat gagctgagca gaattattcc
ggatcaaaat 660atgcttgtgg atatcgtcgt agcggacaat caggtattat
ttaaggtgga tcgcgtgtta 720ttttactctc gcatcttgga cggcacctat
cctgatactt ctagaattat tccgacttcc 780tacaaaacag aactgattgt
ggacacaaaa agtttgagcg agtctattga ccgtgcttat 840ttgctgtccc
gtgaggaaaa aacgaatatt gtaaaaatgc aatcgttgga aaacggtgat
900ctagagattt cctccagctc atctgaactt ggtaaagtgc gtgaggaagt
aaatgtatcc 960aaatttgagg gagagccact caaaatctcg ttcaactcca
aatatatgct cgacgtgctg 1020aaggtaattg acagcgagca gctgacgatt
gcttttaccg gcattatgag ccccattatt 1080ttaaaaccgg cagattccag
caatgcgctg tatatcatcc tgccatatcg cacaaccaac 1140tag
114351980DNAPaenibacillus polymyxa spp. plantarummisc_featureStrain
Lu16774 gyrB 5atggtcgaca aaatcgactt gtctgcggga gcttccggta
cacagaacgg agcttcagaa 60tatggcgcgg acgacattca agtgctcgaa gggcttgtgg
cagttcgcaa acggccgggc 120atgtacatcg ggagcaccag ttcttcggga
ctgcatcatt tggtatggga aattgtagac 180aacgcggtgg atgaacatct
cgccaagttt tgctctcgca ttgatatcac aatgcataag 240gacggttctg
ttacagtatc agacaacggg cgcggtattc ctacgggaat gcacaaaatg
300ggaattccta cgcctcaagt tgtattcacc attttgcacg ccggaggtaa
gtttggcggt 360tcgggatata aaaagtccgg gggtctgcat ggggtaggtg
cgtctgtaac gaacgctctt 420tcggaatggc ttgaagtgga aatctaccgg
gacggcaaga ttcaccgtca gcggtttgaa 480tattggcagg acaagaaggg
cgtggagcat gtcggtgaac cgaccacagg ccttgaagtg 540ctgggcaata
ctaacaagac gggctcgaaa attacattta aaccggatat tcgcgttttt
600cagtcaggaa ttcattttaa ctacgatacg ctggctgaac gccttcagga
gattgctttt 660ctgaattccg gccttcgtat tcagcttaaa gacgaacgca
gcggaaagtc agatgaatat 720ttttatgagg gtggagcaag tcagtttgtt
tcttttttga atgagggtaa ggatgtactg 780catgatgtta ttcactttaa
tgccgagaaa gaagacattg aagtggagat tgccatccaa 840tacaatgccg
gctacacaga gacgattgct tcgttcgtta actccattcc gacacgtggc
900gggggtacgc atgaaacagg cttcaaaacc gcttacactc gtatcatgaa
cgactatgca 960cgcaaaacag cgatgttgaa ggaaaaggat aaaaacctgg
aaggtaacga tctgcgtgag 1020ggtatgatgg ctgtaatcag tgtcaagatg
gccgaggttg aatttgtcgg tcagacaaaa 1080gatcagctgg gtagtgcttc
ggcgcggagt acagtggatg ccatcgtatc tgaacaaatg 1140cagcgctttt
tggaggaaaa tccacagata gcgcaaacct tgatcagaaa ggcagttcaa
1200gcatccaaag cgcgtgaagc tgcacgtaag gctcgggacg aaatgcgttc
tggcaagaaa 1260cgcagtgaaa gttctaattt gaatggcaaa ctgtcgcctg
cgcagtctaa ggattttaca 1320cgtaatgagt tatttatcgt ggaaggcgat
tcggctggag gatcggccaa acaaggacgg 1380gattccaaaa tccaggcaat
tttgccgtta aagggcaagc cgatgaatcc ggaaaaatca 1440aagttggcgg
atattatgaa aaatgatgag tatcgtgcga ttacggcagc gattggcgcg
1500ggggtaggaa ctgagttcac gctggaagac agcaattatt ccaaaatcat
cattatgacc 1560gatgcagata cagatggcgc gcacattcaa gtactgttgt
tgacgttctt ttatcggtac 1620atgaaagaac tcattgatgc aggacgcata
tttattgctc agccgccatt gtataaaata 1680acccgcaagt cgggtaagct
cgaaacggtt cgttatgcct ggactgacga gcagcttgat 1740aattacttaa
aagaatttgg acgaaatttt gagcttcaac gttataaagg actcggggag
1800atgaaccctg atcagttatg ggaaacgaca atgaatcccg agtcacgcac
cctgttgcgc 1860gttcagattg aggatgctgc caaagctgaa cgccgtgtgt
ccacattgat gggtgataag 1920gtggatccac gtaagcgctg gatcgtggaa
aacgtggatt tcacggaata cgtagagtag 198061116DNAPaenibacillus polymyxa
spp. plantarummisc_featureStrain Lu16774 recF 6gtgtttgtga
acaacattgt tttgcagcag taccggaact ataaacagct ggagctgaat 60gaattcgggc
ccgttaattt gctgatcgga caaaatgcgc aaggcaaaac gaatctggtt
120gaggcgattt ttgtattagc cttaactaaa agtcaccgaa cgtcccgtga
caaggaatta 180atttctttcg gggctacttc cacacatcta gctgctgatg
tggataagaa atacgggaaa 240atcagattgg atctctcgtt atccacacaa
ggcaaaaaag caaagatcaa cgggctagag 300cagcgaaagc tgagcgattt
tatcggttcg ttaaacgtgg tcatgtttgc gcccgaggat 360ctggaaattg
tcaaaggaac accgggggtt cgccgccggt ttcttgacat ggaaattgga
420caagttgcgc caggatattt gtatcatttg cagcaatatc agaaagtgct
ggttcagcgg 480aataacctgc tcaagcaagc ttgggggaaa gatatggcgt
ccgtgcagct gatgctggag 540gtatggaatg agcaacttgt tgagcatggt
gttaaaattg taaaaaagcg gaaacaattt 600ataacaaagc tacaaaagtg
ggcccaagcc attcatgaag ggattgcagg tgggacagaa 660gagttaaaat
tagcctatgt tccctctttc ggtgagccag aggaagaaga tgaagctgtc
720ttattggagc gatttatgat aaagttatcc caaatgaggg aacaggaaat
ccgccgtggc 780atgactttgg cgggacccca tcgtgatgat ttggcctttg
ccattaacgg cagagaagtg 840catacgtatg gctctcaggg gcagcagcgg
acgacggccc tgtctttgaa gctggccgaa 900atagaattaa ttcatgagga
aattggggag tatcctatcc tgctgctgga tgatgtattg 960tccgagctgg
acccctatcg tcagactcag ctgatcgaga ctttccaaag caaggtacag
1020acctttatca cggcaaccgg gattgagacg ttgaacgcag aacgacttaa
gggtgcccat 1080atttatcacg tccacgacgg gcatgtggaa cactaa
111671719DNAPaenibacillus polymyxa spp. plantarummisc_featureStrain
Lu16774 recN 7atgctggtca ctttgtctat acggaatttg gcagtcgtag
aagctgtcga tgttcatttt 60tataaaggat ttcatgtatt gagcggagaa actggtgctg
gtaaatccat tattatcgac 120gcacttgggc tgattgcggg cggcagggga
tctgctgatc tagtgcgtta cggatgtgat 180aaagccgaaa tggaagcctt
gtttgaattg ccggtcaaac atcccgtttg gaatacgttg 240gaggaacaag
ggattaaggc taatccagaa gagcatttgc tgattcgtcg agaacttaca
300gttcagggga aaagctcatc tcgaattaac ggtcagatgg ttaatttaac
gatgctgcgt 360gaggtaggtg agcaactcgt taatatccac gggcagcatg
agcatcaaag cttgctgcgt 420gcggatcgcc atcttgcgct gctggatacg
ttcggtgact cggtcattgg tccagtcaaa 480gcgctttacc gggagcgcta
caatgctttt gtcaaagcgg aaaaagaagt aagagaattg 540caaagctcca
gtcaaaaggc ttatcagcta ttggacatgt atcgcttcca attggaagag
600atcgctgcgg cggagttaaa attgggtgaa gatgaattat tggcagagga
acgggtcaag 660ctatcccata gtgagaaaat gatggatgga gtatcaggag
catacgagct gttaagtggc 720agaggtggtc tggatacggt caataacgtg
ttgtccagat taaatgatgt tcagagctac 780gacagtaaaa gccttcagcc
cattgcggag cagattcaat ctgctttcta tcagttggag 840gatgcagcgt
ttcaattacg ctcttatcgt gaggatattg aatttaatcc gggcaagctg
900catgaggtgg agcaacgttt gaatcaaatt accgggttac agcgaaaata
tggtgatagt 960atagagcaga ttttggaata ctatagccgt attgagcagg
aaaccgatct gttggaaaat 1020aaagatgagc ggctggagca gctcattgca
aagcgggatg agttgctttc gaatttgctg 1080gagattgctg aagagcttac
agaggcacgt gaaatttgtg ctgaagagct tgcagagcaa 1140gtagagcagg
aattaaaaga tcttcaaatg gaaagaacgt cactcaaggt gcgtattgat
1200ccaattgaag atccacgtgg atatgaatat aaaggtctaa aggtacgacc
taccaagcaa 1260gggatagata atgcggaatt tctgatttcg cccaatccag
gtgagccact tcgcccactc 1320ggtaaaatcg cttccggtgg tgagttatca
cgtatcatgt tggcgatgaa aagtattttt 1380gcgcgtcatg atcaaattcc
ggtgctcatt tttgacgagg tggataccgg ggtaagtggt 1440cgtgcagctc
agtccatagc cgagaagctt tatcgtttgt cttccgtttg tcaggtgttt
1500tccattactc atttgccgca ggtggcatgt atggcagatc atcagtacct
gattgagaaa 1560aatgttcatg acggacggac catgactcaa attgagggac
taacggagga aggtcgtgtt 1620aaggaattgg cacggatgct gggtggggta
gaaattaccg aaaaaacatt gcatcacgca 1680caggaaatgc tgaatttggc
ggaaggaaag aaagcctga 17198945DNAPaenibacillus polymyxa spp.
plantarummisc_featureStrain Lu16774 rpoA 8gtgatagaaa tcgaaaagcc
gaaaattgag acggttgacg tcaatgatga tggcacctat 60ggaaaattcg tagtagaacc
gctggaacgc ggatacggta cgacgcttgg gaactcgctt 120cgccgtattc
tgttatcctc gttaccgggg gcagcagtca catcggttca gatcgatggg
180gttctgcacg agtttgcaac ggttcccggt gtgaaggaag acgtaacgga
gatcattctg 240aacttgaaag ctttatcgct taaaatccac tcagatgaag
agaaagtact tgaaatcgat 300gcggaaggcg aaggagttgt aacggcaggt
gatatccgtg cggatagtga tgtggaaatt 360cttaatccgg atcttcacat
tgcaacgctc ggaccgggtt cgagacttca catgcgtatt 420tttgccaatc
gcggtcgcgg ttacgttaag caggatcgga ataaacgtga tgaccagccg
480atcggcgtca ttcccgtcga ctccatctac actccgattg cacgcgtgaa
ctacggcgta 540gaaaatacgc gtgtcggcca ggttacgaat tatgacaagc
tgacacttga ggtttggact 600gacggaacta ttcgtcctga agaagctgtg
agccttggag ccaaaatttt gaccgagcat 660gtgatgctat tcgtgggtct
cacggatgaa gcaaaagatg cagaaattat ggtcgaaaaa 720gaagaagaca
aaaaagaaaa agttcttgaa atgacgatcg aagagctgga tctctccgtc
780cgttcctata actgccttaa gcgcgctggt atcaatacgg tacaagaact
cacgactaaa 840tctgaagaag atatgatgaa ggtccgtaac ttgggtcgca
aatctttgga agaagtacaa 900gagaagctcg aggaacttgg tttaggactt
cgtacggaag aatag 94591143DNAPaenibacillus polymyxa spp.
plantarummisc_featureStrain Lu17007 dnaN 9atgaagatta gcattctgaa
aaacgttttg aacgaggcca tacaacatgt atccaaagcg 60atatccagtc gaacgacaat
tccaattttg agtggtatta agctggatgt gaatcaccag 120ggagtcacac
tgaccgccag cgatacagac atctctattc aatcctttat tccgatggag
180gatggtgacc aaacggtcgt tcagatcgaa caacccggca gtgtagtgct
acccgctaaa 240ttctttgtcg aaattatcaa aaagttgccg tctcaggaga
tccgtatgga ggtaaaagac 300caattccaaa cctttatctc atccggtgct
actgaaattc agatcgttgg tttggaccct 360gaagaatttc cggtgcttcc
caacattgaa gaaaatcaag tgatctctgt gccaggtgat 420ttgcttaaaa
atatgattaa acagacggta ttctccatct ctacccatga aacgacacct
480attttgactg gtgtattgtg gaatctggct gagggcgaat tgaaatttgt
cgcaacggac 540cgccaccgcc ttgccacccg cagcgctcat ttggagacgt
ctgaaggctt gcgttttagc 600aatgttgtca ttgcaggcaa aacgctcaat
gagctgagca gaattattcc ggatcaaaat 660atgcttgtgg atatcgtcgt
agcggacaat caggtattat ttaaggtgga tcgcgtgtta 720ttttactctc
gcatcttgga cggcacctat cctgatactt ctagaattat tccgacttcc
780tacaaaacag aactgattgt ggacacaaaa agtttgagcg agtctattga
ccgtgcttat 840ttgctgtccc gtgaggaaaa aacgaatatt gtaaaaatgc
aatcgttgga aaacggtgat 900ctagagattt cctccagctc atctgaactt
ggtaaagtgc gtgaggaagt aaatgtatcc 960aaatttgagg gagagccact
caaaatctcg ttcaactcca aatatatgct cgacgtgctg 1020aaggtaattg
acagcgagca gctgacgatt gcttttaccg gcattatgag ccccattatt
1080ttaaaaccgg cagattccag caatgcgctg tatatcatcc tgccatatcg
cacaaccaac 1140tag 1143101980DNAPaenibacillus polymyxa spp.
plantarummisc_featureStrain Lu17007 gyrB 10atggtcgaca aaatcgactt
gtctgcggga gcttccggta cacagaacgg agcttcagaa 60tatggcgcgg acgacattca
agtgctcgaa gggcttgtgg cagttcgcaa acggccgggc 120atgtacatcg
ggagcaccag ttcttcggga ctgcatcatt tggtatggga aattgtagac
180aacgcggtgg atgaacatct cgccaagttt tgctctcgca ttgatatcac
aatgcataag 240gacggttctg ttacagtatc agacaacggg cgcggtattc
ctacgggaat gcacaaaatg 300ggaattccta cgcctcaagt tgtattcacc
attttgcacg ccggaggtaa gtttggcggt 360tcgggatata aaaagtccgg
gggtctgcat ggggtaggtg cgtctgtaac gaacgctctt 420tcggaatggc
ttgaagtgga aatctaccgg gacggcaaga ttcaccgtca gcggtttgaa
480tattggcagg acaagaaggg cgtggagcat gtcggtgaac cgaccacagg
ccttgaagtg 540ctgggcaata ctaacaagac gggctcgaaa attacattta
aaccggatat tcgcgttttt 600cagtcaggaa ttcattttaa ctacgatacg
ctggctgaac gccttcagga gattgctttt 660ctgaattccg gccttcgtat
tcagcttaaa gacgaacgca gcggaaagtc agatgaatat 720ttttatgagg
gtggagcaag tcagtttgtt tcttttttga atgagggtaa ggatgtactg
780catgatgtta ttcactttaa tgccgagaaa gaagacattg aagtggagat
tgccatccaa 840tacaatgccg gctacacaga gacgattgct tcgttcgtta
actccattcc gacacgtggc 900gggggtacgc atgaaacagg cttcaaaacc
gcttacactc gtatcatgaa cgactatgca 960cgcaaaacag cgatgttgaa
ggaaaaggat aaaaacctgg aaggtaacga tctgcgtgag 1020ggtatgatgg
ctgtaatcag tgtcaagatg gccgaggttg aatttgtcgg tcagacaaaa
1080gatcagctgg gtagtgcttc ggcgcggagt acagtggatg ccatcgtatc
tgaacaaatg 1140cagcgctttt tggaggaaaa tccacagata gcgcaaacct
tgatcagaaa ggcagttcaa 1200gcatccaaag cgcgtgaagc tgcacgtaag
gctcgggacg aaatgcgttc tggcaagaaa 1260cgcagtgaaa gttctaattt
gaatggcaaa ctgtcgcctg cgcagtctaa ggattttaca 1320cgtaatgagt
tatttatcgt
ggaaggcgat tcggctggag gatcggccaa acaaggacgg 1380gattccaaaa
tccaggcaat tttgccgtta aagggcaagc cgatgaatcc ggaaaaatca
1440aagttggcgg atattatgaa aaatgatgag tatcgtgcga ttacggcagc
gattggcgcg 1500ggggtaggaa ctgagttcac gctggaagac agcaattatt
ccaaaatcat cattatgacc 1560gatgcagata cagatggcgc gcacattcaa
gtactgttgt tgacgttctt ttatcggtac 1620atgaaagaac tcattgatgc
aggacgcata tttattgctc agccgccatt gtataaaata 1680acccgcaagt
cgggtaagct cgaaacggtt cgttatgcct ggactgacga gcagcttgat
1740aattacttaa aagaatttgg acgaaatttt gagcttcaac gttataaagg
actcggggag 1800atgaaccctg atcagttatg ggaaacgaca atgaatcccg
agtcacgcac cctgttgcgc 1860gttcagattg aggatgctgc caaagctgaa
cgccgtgtgt ccacattgat gggtgataag 1920gtggatccac gtaagcgctg
gatcgtggaa aacgtggatt tcacggaata cgtagagtag
1980111116DNAPaenibacillus polymyxa spp.
plantarummisc_featureStrain Lu17007 recF 11gtgtttgtga acaacattgt
tttgcagcag taccggaact ataaacagct ggagctgaat 60gaattcgggc ccgttaattt
gctgatcgga caaaatgcgc aaggcaaaac gaatctggtt 120gaggcgattt
ttgtattagc cttaactaaa agtcaccgaa cgtcccgtga caaggaatta
180atttctttcg gggctacttc cacacatcta gctgctgatg tggataagaa
atacgggaaa 240atcagattgg atctctcgtt atccacacaa ggcaaaaaag
caaagatcaa cgggctagag 300cagcgaaagc tgagcgattt tatcggttcg
ttaaacgtgg tcatgtttgc gcccgaggat 360ctggaaattg tcaaaggaac
accgggggtt cgccgccggt ttcttgacat ggaaattgga 420caagttgcgc
caggatattt gtatcatttg cagcaatatc agaaagtgct ggttcagcgg
480aataacctgc tcaagcaagc ttgggggaaa gatatggcgt ccgtgcagct
gatgctggag 540gtatggaatg agcaacttgt tgagcatggt gttaaaattg
taaaaaagcg gaaacaattt 600ataacaaagc tacaaaagtg ggcccaagcc
attcatgaag ggattgcagg tgggacagaa 660gagttaaaat tagcctatgt
tccctctttc ggtgagccag aggaagaaga tgaagctgtc 720ttattggagc
gatttatgat aaagttatcc caaatgaggg aacaggaaat ccgccgtggc
780atgactttgg cgggacccca tcgtgatgat ttggcctttg ccattaacgg
cagagaagtg 840catacgtatg gctctcaggg gcagcagcgg acgacggccc
tgtctttgaa gctggccgaa 900atagaattaa ttcatgagga aattggggag
tatcctatcc tgctgctgga tgatgtattg 960tccgagctgg acccctatcg
tcagactcag ctgatcgaga ctttccaaag caaggtacag 1020acctttatca
cggcaaccgg gattgagacg ttgaacgcag aacgacttaa gggtgcccat
1080atttatcacg tccacgacgg gcatgtggaa cactaa
1116121719DNAPaenibacillus polymyxa spp.
plantarummisc_featureStrain Lu17007 recN 12atgctggtca ctttgtctat
acggaatttg gcagtcgtag aagctgtcga tgttcatttt 60tataaaggat ttcatgtatt
gagcggagaa actggtgctg gtaaatccat tattatcgac 120gcacttgggc
tgattgcggg cggcagggga tctgctgatc tagtgcgtta cggatgtgat
180aaagccgaaa tggaagcctt gtttgaattg ccggtcaaac atcccgtttg
gaatacgttg 240gaggaacaag ggattaaggc taatccagaa gagcatttgc
tgattcgtcg agaacttaca 300gttcagggga aaagctcatc tcgaattaac
ggtcagatgg ttaatttaac gatgctgcgt 360gaggtaggtg agcaactcgt
taatatccac gggcagcatg agcatcaaag cttgctgcgt 420gcggatcgcc
atcttgcgct gctggatacg ttcggtgact cggtcattgg tccagtcaaa
480gcgctttacc gggagcgcta caatgctttt gtcaaagcgg aaaaagaagt
aagagaattg 540caaagctcca gtcaaaaggc ttatcagcta ttggacatgt
atcgcttcca attggaagag 600atcgctgcgg cggagttaaa attgggtgaa
gatgaattat tggcagagga acgggtcaag 660ctatcccata gtgagaaaat
gatggatgga gtatcaggag catacgagct gttaagtggc 720agaggtggtc
tggatacggt caataacgtg ttgtccagat taaatgatgt tcagagctac
780gacagtaaaa gccttcagcc cattgcggag cagattcaat ctgctttcta
tcagttggag 840gatgcagcgt ttcaattacg ctcttatcgt gaggatattg
aatttaatcc gggcaagctg 900catgaggtgg agcaacgttt gaatcaaatt
accgggttac agcgaaaata tggtgatagt 960atagagcaga ttttggaata
ctatagccgt attgagcagg aaaccgatct gttggaaaat 1020aaagatgagc
ggctggagca gctcattgca aagcgggatg agttgctttc gaatttgctg
1080gagattgctg aagagcttac agaggcacgt gaaatttgtg ctgaagagct
tgcagagcaa 1140gtagagcagg aattaaaaga tcttcaaatg gaaagaacgt
cactcaaggt gcgtattgat 1200ccaattgaag atccacgtgg atatgaatat
aaaggtctaa aggtacgacc taccaagcaa 1260gggatagata atgcggaatt
tctgatttcg cccaatccag gtgagccact tcgcccactc 1320ggtaaaatcg
cttccggtgg tgagttatca cgtatcatgt tggcgatgaa aagtattttt
1380gcgcgtcatg atcaaattcc ggtgctcatt tttgacgagg tggataccgg
ggtaagtggt 1440cgtgcagctc agtccatagc cgagaagctt tatcgtttgt
cttccgtttg tcaggtgttt 1500tccattactc atttgccgca ggtggcatgt
atggcagatc atcagtacct gattgagaaa 1560aatgttcatg acggacggac
catgactcaa attgagggac taacggagga aggtcgtgtt 1620aaggaattgg
cacggatgct gggtggggta gaaattaccg aaaaaacatt gcatcacgca
1680caggaaatgc tgaatttggc ggaaggaaag aaagcctga
171913945DNAPaenibacillus polymyxa spp. plantarummisc_featureStrain
Lu17007 rpoA 13gtgatagaaa tcgaaaagcc gaaaattgag acggttgacg
tcaatgatga tggcacctat 60ggaaaattcg tagtagaacc gctggaacgc ggatacggta
cgacgcttgg gaactcgctt 120cgccgtattc tgttatcctc gttaccgggg
gcagcagtca catcggttca gatcgatggg 180gttctgcacg agtttgcaac
ggttcccggt gtgaaggaag acgtaacgga gatcattctg 240aacttgaaag
ctttatcgct taaaatccac tcagatgaag agaaagtact tgaaatcgat
300gcggaaggcg aaggagttgt aacggcaggt gatatccgtg cggatagtga
tgtggaaatt 360cttaatccgg atcttcacat tgcaacgctc ggaccgggtt
cgagacttca catgcgtatt 420tttgccaatc gcggtcgcgg ttacgttaag
caggatcgga ataaacgtga tgaccagccg 480atcggcgtca ttcccgtcga
ctccatctac actccgattg cacgcgtgaa ctacggcgta 540gaaaatacgc
gtgtcggcca ggttacgaat tatgacaagc tgacacttga ggtttggact
600gacggaacta ttcgtcctga agaagctgtg agccttggag ccaaaatttt
gaccgagcat 660gtgatgctat tcgtgggtct cacggatgaa gcaaaagatg
cagaaattat ggtcgaaaaa 720gaagaagaca aaaaagaaaa agttcttgaa
atgacgatcg aagagctgga tctctccgtc 780cgttcctata actgccttaa
gcgcgctggt atcaatacgg tacaagaact cacgactaaa 840tctgaagaag
atatgatgaa ggtccgtaac ttgggtcgca aatctttgga agaagtacaa
900gagaagctcg aggaacttgg tttaggactt cgtacggaag aatag
945141143DNAPaenibacillus epiphyticusmisc_featureStrain Lu17015
dnaN 14atgaagatca gcattctgaa aaacgttttg aacgaggcta tacaacatgt
atccaaagcg 60atatcaagtc ggactacgat tccaattctg agtggtatta agctggatgt
gaatcaccag 120ggagtaacgc tgaccgccag cgatacagac atctccattc
aatcctttat tccgatggag 180gatggtgacc aaactgttgt tcaggtcgaa
caacccggca gtgttgtgct gcctgccaaa 240ttctttgtcg aaattatcaa
aaagttgccg tcgcaggaga tccatatgga ggtaaaagac 300caatttcaaa
cctttatctc gtctggcgca actgaaattc agattgttgg cttggaccct
360gaagaattcc cggtgcttcc caacattgaa gaaaatcaag tcatctctgt
accaggagat 420ttacttaaaa atatgattaa acagacggta ttctccatct
ccacccacga aacgacaccg 480attttaactg gcgtgttgtg gaatctggct
gagggtgaat tgaagtttgt ggcaacggac 540cgccaccgcc ttgccacccg
tagcgctcat ttggagacgt ctgaaggctt gcgttttagc 600aatgttgtca
ttgcgggcaa aacgctgaat gagctgagca gaattattcc agatcaaaat
660atgcttgtgg atatcgtagt agcggacaat caggtattat tcaaagtaga
tcgggtgcta 720ttttattccc gcatcttgga cggcacctat cctgatactt
ctagaattat tccgacctcc 780tacaaaacag aactgattgt ggatacaaaa
agtttaagtg agtcaattga ccgtgcttat 840ttgctgtccc gtgaggaaaa
aacgaatatt gtaaaaatgc agtcgctgga aaatggcggt 900ttggagattt
cctctagttc ctctgagctt ggcaaagtgc gtgaggaagt aactgtgtcc
960aaatttgagg gagagccgct caaaatttcg ttcaactcta aatacatgct
cgacgtgctg 1020aaggtgattg acagcgagca gctgacgatt gcttttaccg
gcattatgag ccccattatt 1080ttaaaaccgg ctgattccag caatgcgctg
tatatcatcc tgccatatcg cacaaccaac 1140tga 1143151980DNAPaenibacillus
epiphyticusmisc_featureStrain Lu17015 gyrB 15atggtcgaca aaatcgactt
gtctgcggga gtgtccggca cacaaagcgg agcttcggaa 60tatggcgcgg acgacattca
agtgctcgaa gggcttgtgg cagttcgcaa acggccgggc 120atgtacatcg
ggagcaccag ttcttcgggg ctgcatcatt tggtatggga aattgtagac
180aacgcggtgg atgaacatct cgccaagttt tgctctcgca ttgatattac
aatgcataag 240gacggttccg ttacagtatc agacaacggg cgcggtattc
ctacgggaat gcacaaaacg 300ggaattccta cgcctcaggt tgtattcacc
attttgcacg ccggaggtaa gtttggcggt 360tcgggatata aaaaatccgg
gggtctgcac ggtgtaggtg cgtctgtaac gaacgctctt 420tcggaatggc
ttgaagtaga aatttaccgg gacggcaaga ttcaccgtca gcggtttgaa
480tattggcagg acaagaaggg cgtggagcat gtcggggaac cgaccacagg
ccttgaagtg 540ctgggcaata ctaacaagac gggctcgaaa attacattta
aaccggatat tcgtgttttt 600caggcaggca ttcattttaa ctacgatacg
ttggctgagc gccttcagga aattgctttt 660ctaaattcgg gccttcgtat
tcaacttaaa gacgaacgca gcggaaagtc agatgagtat 720ttttatgagg
gtggcgcaag tcagtttgtt gcttttctga atgagggcaa ggatgtgctg
780catgacgtta ttcactttaa tgccgagaaa gaagacattg aagtagagat
tgccatccag 840tacaatgctg gttatacaga gacgattgct tcgttcgtta
actccattcc gacacgtgga 900ggaggtacgc atgaaacggg attcaaaacc
gcttacactc gtgtcatgaa cgactatgcc 960cggaaaacgg tgatgttgaa
agaaaaggat aaaaacttgg agggcaacga tctacgtgag 1020ggcatgatgg
ctgtaatcag tgtcaagatg gctgaggttg aatttgtcgg ccagacaaag
1080gatcagctgg gaagcgcttc ggcacggagt acagtggatg ccatcgtatc
tgagcagatg 1140cagcgttttt tggaagaaaa tccgcagata gcacaaactt
tgatcaagaa ggcagttcaa 1200gcatccagag cacgtgaagc tgcacgtaaa
gctcgggatg aaatgcgttc cggtaaaaag 1260cgcagtgaaa gttccaattt
gaatggtaaa ctatcgcctg cgcagtccaa ggattttaca 1320cgtaatgagt
tgtttattgt ggaaggcgat tcggctggag gatcagccaa gcagggacgg
1380gattccaaaa ttcaggccat attgccgcta aagggcaagc cgatgaatcc
ggaaaaatcc 1440aaactggcgg atattatgaa gaatgatgag taccgtgcta
ttacagcagc tattggtgcg 1500ggagtaggaa cagagttttc gctggaagac
agcaattatt ccaaaatcat cattatgacc 1560gatgcagata cagatggtgc
gcacattcaa gtgctgttgt tgacgttctt ttatcggtac 1620atgaaagagc
ttattgatgc aggacgcata tttattgctc aaccgccatt gtataaaata
1680actcgaaagt cgggtaagct cgaaacggtg cgttatgctt ggactgacga
gcagcttgat 1740aattatttaa aagaatttgg acgaaatttt gagcttcagc
gctataaagg acttggggaa 1800atgaaccctg atcagttatg ggaaacaacg
atgaatcccg attcacgcac cttgctacgc 1860gttcagatag aggatgcagc
caaggctgaa cgcagggtgt ccactttgat gggtgataag 1920gtggatccgc
gcaagcgctg gatcgtggaa aacgtagatt ttacggaata cgtagagtag
1980161116DNAPaenibacillus epiphyticusmisc_featureStrain Lu17015
recF 16gtgtttgtga acaacattgt tttgcagcag taccggaact atgaacagct
ggagctgaat 60gaatttgggc ccgttaattt gctgatcgga caaaatgcgc agggcaaaac
gaatctggta 120gaggcaattt ttgtactggc tttaaccaaa agtcatcgaa
cgtcccgcga caaggagtta 180atctctttcg gggctacttc cactcaccta
gctgcggatg tggataaaaa atacgggaaa 240atcagactgg atctcgcgtt
atccacacaa ggcaaaaaag caaagatcaa cggactggag 300cagcgcaaac
tgagcgattt tatcggttcg ttaaatgtgg tcatgtttgc acctgaggat
360ctggaaattg tgaaaggaac accgggggtt cgccgccggt ttcttgacat
ggaaatcgga 420caggttgcgc caggatatct gtatcatttg cagcaatatc
agaaagtatt ggttcagcga 480aacaacctgc tcaagcaagc ttggggtaag
gatatggcgt cagtgcagct gatgctggag 540gtatggaatg agcaacttgt
tgagcatggt gttaaaattg ttaaaaagcg gaaacaattt 600ataacaaagc
tacaaaagtg ggctcaggcc attcatgaag ggatcgcagg tgggacagaa
660gagttaaaat taacctatgt tccctccttc agtgagccag aggaagaaga
tgaagctgtc 720ttattggagc gatttatgat aaagttatcc caaatgaggg
aacaggaaat ccgccgtggc 780atgactttgg cgggacccca tcgtgacgat
ttggcctttg ccattaacgg cagagaagtg 840catacgtatg gctctcaggg
gcagcagcgg acgacggccc tgtccttgaa gttggccgaa 900atagagttaa
ttcatgagga aattggtgaa tatcctgtct tgctgctgga tgatgttttg
960tccgagctgg acccctatcg tcagacccag ctgatcgaga ctttccaaag
caaggtacag 1020acctttatca cggcaaccgg ggttgagact ttgaacgcag
aacgactcaa ggatgccaat 1080atttatcacg tccacgacgg gcatgtggaa cactaa
1116171719DNAPaenibacillus epiphyticusmisc_featureStrain Lu17015
recN 17atgctggtca ctttgtctat acggaatttg gcggtcgtag aggccgtcga
tgttcatttt 60tataaagggt ttcatgtctt gagcggggaa acaggtgctg gtaaatccat
tattatcgat 120gcgcttggcc tgattgcagg cggtaggggc tctgctgatc
tagtgcgtta cggttgtgat 180aaagcagaga tggaagcctt gtttgagctg
ccggtaaaac atccagtttg gaaaacactt 240gaggaacaag ggattaaggc
caatgcggag gagcatttgc tgattcgtcg cgaacttacg 300gttcagggga
aaagctcttc tcgaattaac ggtcagatgg ttaatttaac gatgctgcgt
360gaggtaggtg agcagctcgt caatatccac gggcaacatg agcatcaaag
cctgctacgt 420gcagatcgcc atctggcgct gctggatacg ttcggtgatt
cggtgatcgg tccagtcaaa 480acgctttacc gtgagcgtta caatgctttt
gtcaaagcgg aaaaagaagt aagagaactg 540caaagctcca gtcaaaaggc
ttatcagctt ttggatatgt accgatttca attagaagag 600atcgctgcgg
cggagttgaa attgggagaa gatgaattat tggcagagga acgggtcaag
660ctatcccata gtgagaaaat gatggatggg atatcaggag catacgaact
gctaagcggc 720agaggtgggc tggatacgat caataacgta ttgtctagat
tgaacgatgt ccaaagctat 780gacagcaaaa gccttcagcc cattgcggag
cagattcagt ctgcttttta ccagttagag 840gacgcagcat tccaattacg
ttcttatcgt gaggatattg aatttaatcc aggcaagctg 900catgaagtgg
agcaacgttt gaatcaaatt accggattac agcgaaaata tggtgatagt
960atagagcaga ttttggaata ctatagccgt attgagcagg aaaccgatct
gctggaaaat 1020aaagatgagc ggctggagca gctcattgca aaacgggatg
agttgctttc cgatttgctg 1080gagatttcag aagagcttac agaagcacgt
gaaatttgtg ctgaagagct tgcagagcaa 1140gtggagcagg agttaaaaga
cctgcagatg gaaagaacgt cactcaaggt gcgcattgat 1200ccaattgaag
atccacgcgg atatgagtat aaaggtctga aggtaaggcc taccaagcaa
1260ggaattgata atgcggaatt tcttatttca cccaatccag gtgagccact
acgtccactt 1320ggtaagatcg cttcaggcgg tgagctatca cgtatcatgt
tggcgatgaa aagtattttt 1380gcgcgtcatg atcaaattcc agtactcatt
tttgacgagg tggataccgg ggtgagtggt 1440cgtgcagctc aatccattgc
cgagaagcta tatcgtttat cttccgtttg tcaggtgttt 1500tccattactc
atttgccaca ggtggcatgt atggcagatc atcagtacct tattgagaaa
1560aatgttcatg atggacggac catgactcaa attgagggac ttacggagga
cgggcgtgtc 1620aaggaattgg cacggatgct gggcggcgtg gaaattaccg
aaaaaacatt gcatcacgca 1680caggaaatgc tgaatttggc ggaaggaaag
aaagcctga 171918945DNAPaenibacillus epiphyticusmisc_featureStrain
Lu17015 rpoA 18gtgatagaaa tcgaaaagcc gaaaattgag acggttgacg
tcaatgatga tggcacctat 60ggaaaattcg tagtagaacc gctggaacgc ggatacggta
cgacgttggg aaactcgctt 120cgccgtattc tgttatcctc gttaccgggg
gcagcagtca catcggttca gatcgatggg 180gttctgcacg agtttgcaac
ggttcccggt gtgaaggaag acgtaacgga gatcattctg 240aacttgaaag
ctttatcgct taaaatccac tcggatgaag agaaagtact cgaaatcgat
300gcggaaggcg aaggagttgt aacggcagga gatatccgtg cggatagtga
tgtggaaatt 360cttaatccgg atcttcacat tgctacgctc ggaccgggtt
cgagacttca catgcgtatt 420tttgccaatc gcggtcgcgg ttacgttaag
caggatcgga acaaacgtga tgaccagccg 480atcggcgtca ttcccgtcga
ctccatctac actccgattg cacgcgtgaa ctacggcgta 540gaaaatacgc
gtgtcggcca ggttacgaat tacgacaagc tgacacttga ggtttggact
600gacggaagta ttcgtcccga ggaagcagtg agccttggag ccaaaatttt
gaccgagcat 660gtgatgttgt tcgtgggtct cacggacgag gcaaaagatg
ctgaaattat ggttgaaaaa 720gaagaagaca agaaagaaaa agttcttgaa
atgacgatcg aagagctgga tctctccgtc 780cgttcctata actgccttaa
gcgcgctggt atcaatacgg tacaagaact cacgactaaa 840tctgaagaag
atatgatgaa ggtccgtaac ttgggtcgca aatctttgga agaagtacaa
900gagaagctcg aggaacttgg tttaggactt cgtacggaag aatag 945
* * * * *
References