U.S. patent application number 14/426806 was filed with the patent office on 2015-10-08 for compositions and methods for controlling plant-parasite nematode.
This patent application is currently assigned to BAYER CROPSCIENCE LP. The applicant listed for this patent is BAYER CROPSCIENCE LP. Invention is credited to Eder Leonardo Sastoque Cala, Joseph W. Kloepper, Katheryn Kay Scott Lawrence, Jennifer Lynn Riggs, Juan David Castillo Russi.
Application Number | 20150282488 14/426806 |
Document ID | / |
Family ID | 49226562 |
Filed Date | 2015-10-08 |
United States Patent
Application |
20150282488 |
Kind Code |
A1 |
Riggs; Jennifer Lynn ; et
al. |
October 8, 2015 |
COMPOSITIONS AND METHODS FOR CONTROLLING PLANT-PARASITE
NEMATODE
Abstract
An entity having anti-nematodal activity derived from Bacillus
firmus bacteria selected from the group consisting of isolated
biosurfactant, isolated protease enzyme, isolated amylase, isolated
lipase, and isolated cellulase, is disclosed. The disclosure also
provides for compositions comprising the same, methods of making
the same, methods of controlling plant-parasite nematode, and
methods of protecting plants.
Inventors: |
Riggs; Jennifer Lynn;
(Raleigh, NC) ; Cala; Eder Leonardo Sastoque;
(Opelika, AL) ; Kloepper; Joseph W.; (Auburn,
AL) ; Lawrence; Katheryn Kay Scott; (Auburn, AL)
; Russi; Juan David Castillo; (Auburn, AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYER CROPSCIENCE LP |
Research Triangle Park |
NC |
US |
|
|
Assignee: |
BAYER CROPSCIENCE LP
Research Triangle Park
NC
|
Family ID: |
49226562 |
Appl. No.: |
14/426806 |
Filed: |
September 10, 2013 |
PCT Filed: |
September 10, 2013 |
PCT NO: |
PCT/US13/58866 |
371 Date: |
March 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61700054 |
Sep 12, 2012 |
|
|
|
Current U.S.
Class: |
424/94.61 ;
424/780; 424/94.6; 424/94.63; 435/198; 435/202; 435/209;
435/220 |
Current CPC
Class: |
C12N 9/20 20130101; C12N
9/2417 20130101; C12Y 302/01001 20130101; C12N 9/2414 20130101;
C12Y 304/00 20130101; A01N 63/10 20200101; C12N 9/48 20130101; C12Y
301/01003 20130101; C12N 9/2437 20130101; C12N 9/52 20130101; C12Y
302/01004 20130101 |
International
Class: |
A01N 63/02 20060101
A01N063/02; C12N 9/28 20060101 C12N009/28; C12N 9/52 20060101
C12N009/52; C12N 9/20 20060101 C12N009/20; C12N 9/42 20060101
C12N009/42 |
Claims
1. An entity having anti-nematodal activity derived from a
bacterium having anti-nematodal activity, wherein the entity is at
least one selected from the group consisting of an isolated
biosurfactant, an isolated protease, an isolated amylase, an
isolated lipase, and an isolated cellulase.
2. The entity according to claim 1, wherein the bacterium is of the
species Bacillus firmus.
3. The entity according to claim 2, wherein the strain of Bacillus
firmus is strain GB-126.
4. The entity according to claim 1, wherein the entity is at least
one biosurfactant obtainable by a method comprising: A. obtaining a
medium in which the bacterium has been grown, B. precipitating
biosurfactant from the medium.
5. The entity according to claim 1, wherein the entity has
anti-nematodal activity against Rotlyenchulus reniformis.
6. A composition having anti-nematodal activity comprising at least
one entity according to claim 1.
7. The composition according to claim 6, wherein the bacterium is
of the species Bacillus firmus.
8. The composition according to claim 7, wherein the strain
bacterium of species Bacillus firmus is strain GB-126.
9. The composition according to claim 6, wherein the composition
has anti-nematodal activity against Rotlyenchulus reniformis.
10. The composition according to claim 6 comprising at least one
isolated biosurfactant.
11. The composition according to claim 10, wherein the
biosurfactant concentration is greater than 0.5 ppm.
12. The composition according to claim 10, wherein the
biosurfactant concentration is at least 1 ppm.
13. The composition according to claim 10 wherein the biosurfactant
concentration is from 1 to 2 ppm.
14. The composition according to claim 10, wherein the
biosurfactant concentration is at least 2 ppm.
15. The composition according to claim 6 comprising at least one
acid precipitate from a culture medium in which the bacterium was
grown.
16. The composition according to claim 15, wherein the at least one
acid precipitate is obtainable by a method comprising: (a) adding
an acid to a medium in which a bacterium has been grown to generate
an acid precipitate, and (b) isolating the acid precipitate from
the medium.
17. The composition according to claim 16, wherein the medium is
obtained by growing a bacterium at 37.degree. C. for 48 h in a
flask comprising containing a minimal salt medium supplemented with
yeast extract and glucose.
18. The composition according to claim 16, wherein the at least one
acid precipitate is generated by adding a mineral acid to the
medium to obtain a final pH of 2.0.
19. The composition according to claim 18, wherein the mineral acid
is HCl.
20. The composition according to claim 16, wherein the at least one
acid precipitate is isolated from the medium by centrifugation and
extracting the acid precipitate with dicloromethane or
methanol.
21. A method of controlling plant-parasitic nematode comprising
applying an entity according to claim 1 to a plant, seed material,
and/or an area on which a plant grows.
22. The method according to claim 21, wherein the plant-parasitic
nematode is Rotylenchulus reniformis.
23. The method according to claim 21, wherein the plant is
cotton.
24. A method of protecting a plant comprising applying a entity
according to claim 1 to a plant, seed material, and/or an area on
which a plant grows.
25. An extract of a Bacillus firmus culture having the following
characteristics: anti-nematodal activity and/or presence of a
biosurfactant.
26. An extract of a Bacillus firmus culture having the following
characteristics: anti-nematodal activity and/or activity selected
from the group consisting of protease activity, amylase activity,
lipase activity, and cellulase activity.
27. An entity according to claim 1, capable of being used to
protect a plant.
28. An entity according to claim 1, capable of being used to
control at least one plant-parasitic nematode.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/700,054, filed Sep. 12, 2012, the content
of which is herein incorporated by reference in its entirety.
FIELD
[0002] An entity having anti-nematodal activity derived from
Bacillus firmus bacteria selected from the group consisting of
isolated biosurfactant, isolated protease enzyme, isolated amylase,
isolated lipase, and isolated cellulase, is disclosed. The
disclosure also provides for compositions comprising the same,
methods of making the same, methods of controlling plant-parasite
nematode, and methods of protecting plants.
BACKGROUND
[0003] Bacteria are an important group of natural antagonists of
plant-parasitic nematodes. Bacteria are distributed broadly, have
diverse modes of action, and have broad host ranges (Tian, B. et
al.). They exhibit diverse modes of action against nematodes that
include parasitism, production of toxins, antibiotics, or lytic
enzymes; induce systemic resistance, and promote plant health
(Aatlen, P. M. et al.; Kerry, B. R. 2000; Kerry, B. R. 1987;
Siddiqui, Z. A. et al.; Stirling, G. R. 1991; Tian, B. et al.; Van
Loon, L. C. et al.). Furthermore, bacteria can be in direct contact
with the entrance sites of the nematodes and influence root
exudates that can affect the nematode development (Sikora, R. A.
1992). The genera Pasteuria, Pseudomonas, and Bacillus have shown
promising potential for nematode biocontrol (Meyer S. L. F;
Siddiqui, Z. A. et al.; Stirling, G. R.; Tian, B. et al.).
[0004] Bacillus firmus strain GB-126 is a nematode biocontrol agent
registered initially in a bionematicide in Israel under the trade
name of BIONEM.RTM. WP (Blachinsky, D. et al.; Keren-Zur et al.).
This formulation was shown to reduce galling index caused by
Meloidogyne spp. on cucumber and tomato plots (Keren-Zur et al.).
Also, under field conditions, suppression of Meloidogyne spp. was
observed within 2 months of transplanting cucumbers and continued
through the end of the experiments (Giannakou, O. I. et al., 2004).
Control provided by B. firmus GB-126 was less effective than the
soil fumigant dazomet. However, its combination with soil
solarization improves nematode control giving results similar to
dazomet use (Giannakou, O. I. et al. 2007). Furthermore, when B.
firmus GB-126 was evaluated in tomato seedlings in the greenhouse,
it reduced gall formation by 91%, final nematode population by 76%,
and the number of M. incognita eggs by 45% (Terefe, M. et al.).
[0005] In other study, a formulation of B. firmus that contains
seaweed extract (BIONEM.RTM. L) was able to reduce Helicotylenchus
spp. and Tylenchorhynchus spp. in golf greens (Wick, R. L. 2006).
Furthermore, synergism of B. firmus with other nematode biocontrol
agents has been reported to improve nematode reduction (Mendoza, A.
R. et al. 2009). In banana, B. firmus was evaluated against R.
similis and applied in combination with F. oxysporum and P.
lilacinus, which reduced the infection of this migratory
endoparasitic nematode (Mendoza, A. R. et al. 2009). Under in vitro
conditions B. firmus was evaluated against the plant parasitic
nematodes Radopholus similis, Meloidogyne incognita, and
Ditylenchus dipsaci. Bacillus firmus produced bioactive secondary
metabolites that were toxic to these nematode juveniles and reduced
egg hatching (Mendoza, A. R. et al. 2008).
[0006] Thus, previous studies have demonstrated the antagonistic
effect of bioactive secondary metabolites of Bacillus firmus GB-126
against plant parasitic nematodes. Nevertheless, the types of
secondary metabolites and enzymatic properties involved and the
role of possible induction of plant resistance have not been
evaluated. Fewer and fewer traditional nematicide are available
today and many that are available are very expensive or not
environmentally and user friendly. Identification of a biological
means to protection plants from pathogenic nematodes fits well into
today's integrated pest management platform for agriculture.
Understanding the single or multiple modes of actions of a
biocontrol microbe can assist in the optimization of the growth of
the microbe prior to formulating into a commercial product. Studies
that evaluate the purpose of the various secondary metabolites can
define which of the many metabolites should be optimized during the
growth of the microbe. There are means available to optimize the
growth media of the microbe to enhance production of one metabolite
or another.
[0007] An object of the present invention is to provide a novel
entity having anti-nematodal activity derived from Bacillus firmus
bacteria selected from the group consisting of isolated
biosurfactant, isolated protease, isolated amylase, isolated
lipase, and isolated cellulase, is disclosed. Another object is to
provide for compositions comprising the same. Another object is to
provide for methods of making the same.
[0008] A further object is to provide for methods of controlling
plant-parasite nematode, and methods of protecting plants.
SUMMARY
[0009] In an aspect, the disclosure provides for an a novel entity
having anti-nematodal activity derived from Bacillus firmus
bacteria selected from the group consisting of isolated
biosurfactant, isolated protease, isolated amylase, isolated
lipase, and isolated cellulose.
[0010] In an aspect, the disclosure provides for a novel
composition having anti-nematodal activity comprising at least one
entity derived from Bacillus firmus bacteria selected from the
group consisting of an isolated biosurfactant, an isolated
protease, an isolated amylase, an isolated lipase, and an isolated
cellulase.
[0011] In an aspect, the disclosure provides for an extract of a
Bacillus firmus culture having the following characteristics:
anti-nematodal activity and presence of a biosurfactant.
[0012] In an aspect, the disclosure provides for an extract of a
Bacillus firmus culture having the following characteristics:
anti-nematodal activity and activity selected from the group
consisting of protease activity, amylase activity, lipase activity,
and cellulase activity.
[0013] In an aspect, the disclosure also provides for a method of
making the same.
[0014] The disclosure also provides for a method of controlling
plant-parasitic nematode by applying an entity or composition as
described herein to to a plant, the seed material, or the area on
which the plant grows. In an aspect, the disclosure provides for a
method of protecting a plant by applying an entity or composition
as described herein to a plant, the seed material, or the area on
which the plant grows.
[0015] In an aspect, the plant-parasitic nematode is Rotlyenchulus
reniformis.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 sets forth the effects of B. firmus metabolites at
100% and 50% concentration on R. reniformis eggs as compared to
water and media control.
[0017] FIG. 2 sets forth the effects of B. firmus metabolites at
100% and 50% concentration on R. reniformis second stage juveniles
as compared to water and media control.
[0018] FIG. 3 sets forth tests of biosurfactant production of B.
firmus GB-126. (A) represents the positive emulsification of
kerosene. (B) represents halo formation due to cell lysis in blood
agar. (C) represents positive oil drop collapse test.
[0019] FIG. 4 sets forth the effect of biosurfactant produced by B.
firmus on R. reniformis second stage juveniles after 30 minutes of
inoculation (P<0.05) as compared to BP media and water controls
and B. firmis bacteria.
[0020] FIG. 5 sets forth the effect of purified biosurfactant
produced by B. firmus at different concentrations (ppm) on R.
reniformis second stage juveniles after 30 minutes of inoculation
under in vitro conditions (P.ltoreq.0.05).
[0021] FIG. 6 sets forth the photos of second stage juveniles of R.
reniformis form (control and after application of 2 ppm B. firmis
biosurfactant) observed under SEM in in vitro trial.
[0022] FIG. 7 sets forth photos of growth of cotton plants after
systemic resistance trial of B. firmus GM-126 and S. maracescens
against R. reniformis in cotton plants.
[0023] FIG. 8 sets forth the enzyme reaction test of B. firmus
GB-126: (A) represents production of proteases, (B) represents
production of amylases, (C) represents production of cellulases,
and (D) represents production of chitinases.
[0024] FIG. 9 sets forth R. reniformis life stages with cotton
seeds treated with B. firmus GB-126 at 7.times.10.sup.6 cfu/seed
under greenhouse conditions in autoclaved soil (P.ltoreq.0.05).
DETAILED DESCRIPTION
[0025] In an aspect, the disclosure provides for an a novel entity
having anti-nematodal activity derived from Bacillus firmus
bacteria selected from the group consisting of isolated
biosurfactant, isolated protease, isolated amylase, isolated
lipase, and isolated cellulase.
[0026] Anti-nematodal activity is understood as meaning any
activity useful in controlling or killing nematodes, such as
reducing nematode egg hatching or paralyzing juveniles of
nematodes. Testing for anti-nematodal activity is done both
in-vitro and in-vivo through laboratory and greenhouse procedures.
In-vitro testing can involve testing eggs and/or juvenile nematodes
in selective media in small 96-well plates and measurements done by
microscopic evaluations. Proper control treatments would be
combined to monitor and compare activity. Under greenhouse studoes
soil was autoclaved using two 90 minute cycles at 130.degree. C. at
1.0 kg/cm.sup.3 pressure with a 24 hour cool down between cycles to
remove any natural competition for the microflora. The vermiform
stages were extracted from the soil by the modified gravity
screening and sucrose centrifugation-flotation. Eggs stages were
extracted from cotton roots by shaking the root system in a 1%
NaOCl solution for four minutes at 120 rpm. The nematode suspension
was collected and rinsed with water through a 25 .mu.m sieve.
Females in roots were stained with acid fuschin to facilitate
enumeration of the females invading the root. Variables measured
included plant height, shoot and root weights, females and eggs per
gram of root, and the number of vermiform life stages in soil.
[0027] In an aspect, the strain of Bacillus firmus is GB-126. In an
aspect, the entity described herein has anti-nematodal activity
against Reniform nematode, Rotlyenchulus spp.; Dagger nematode,
Xiphinema spp.; Lance nematode, Hoplolaimus spp.; Pin nematode,
Paratylenchus spp.; Ring nematode, Criconemoides spp.; Rootknot
nematode, Meloidogyne spp.; Sheath nematode, Hemicycliophora spp.;
Spiral nematode, Helicotylenchus spp.; Stubbyroot nematode,
Trichodorus spp.; Cyst nematode, Heterodera spp.; Sting nematode,
Belonolaimus, spp.; and/or Stunt nematode, Tylenchorhynchus
spp.
[0028] In an aspect, the entity as described herein is a
biosurfactant obtainable by a method comprising: obtaining a medium
in which Bacillus firmus bacteria has been grown and precipitating
the biosurfactant from the medium.
[0029] Biosurfactant is understood to mean microbially produced
surface-active compounds. They are amphiphilic molecules with both
hydrophilic and hydrophobic regions causing them to aggregate at
the interfaces between fluids with different polarities such as
water and hydrocarbons (Jennings E. R. et al., 2000).
[0030] Multiple initial tests were conducted to determine the
production of biosurfactant from B. firmus. The bacterium was grown
under blood agar, where a positive production of biosurfactant is
indicated by a transparent halo around the bacterial colony.
Additional testing, B. firmus was cultured on nutrient broth at
30.degree. C. for 24 hours. Subsequently, the living cells were
recovered by centrifugation at 5,181 xg for 15 minutes, and cells
were washed twice with NaCl 0.85% (w/v) and later suspended in 5 ml
of NaCl0.85% (w/v). They were used to inoculate 45 ml of saline
Davis minimal broth with an inoculum ratio of 1% (v/v). The
composition was K.sub.2HPO.sub.4 5.23 g/l, KH.sub.2PO.sub.4 1.91
g/l, MgSO.sub.4 0.09 g/l, (NH4)2SO.sub.4 1 g/l, as well as 1 ml/l
of trace elements solution (CoCl.sub.3 20 mg/l, H.sub.3BO.sub.3 30
mg/l, ZnSO.sub.4 10 mg/l, Cu.sub.2SO.sub.4 1 mg/l,
Na.sub.2MoO.sub.4 3 mg/l, FeSO.sub.4 10 mg/l and MgSO.sub.4 2.6
mg/l). Cultures were incubated at 30.degree. C. .+-.2 at 150 rpm
for 3 days. Again B. firmus living cells were separated from the
supernatant by centrifugation (20 minutes at 4000 xg). The
supernatant was filtered through a Millipore filter 0.45-0.22 .mu.m
to obtain the final bacterial biosurfactant product. This final
product was autoclaved twice for 30 minutes at 120.degree. C. at 1
kg/cm.sub.3 pressure to kill all the bacterium's living cells and
inactivate its enzymes. emulsifying activity of the cell-free
supernatant was evaluated by mixing 0.5 ml with 0.5 ml of kerosene
and 4 ml of distilled water to a disposable culture tube
(borosilicate glass 16.times.150 mm) The negative control consisted
of distilled water and kerosene, and the positive control consisted
of distilled water, kerosene, and Triton X-100 (100 mg/ml). Each
tube was agitated in a vortex for 1 min and was left to stand for
24 hours. The height of the emulsification ring was then measured
in millimeters and compared to that of the chemical emulsifier. If
there was positive production of surfactant, the kerosene
emulsified it and produced foam. The third test consisted of an oil
drop collapse in which one drop of the supernatant was placed on
parafilm paper and a drop of oil was placed on top of it. If the
drop of oil increased its diameter compared to the media control,
the bacteria was considered to have produced a biosurfactant.
Finally, the biosurfactant product was again tested under in vitro
conditions in 96 well-plate against second stage juveniles of R.
renifonnis. Volumes of 100 .mu.L of the treatments we transferred
to each well, which contained approximately 16 juveniles of R.
renifonnis. Treatments for this trial were i) water control, ii)
BPM control, iii) biosurfactant 100%, and iv) B. firmus
15.times.107 cfu/ml. Each treatment was replicated 8 times, and the
entire trial was repeated twice. The number of juveniles paralyzed
or dead was recorded 30 minutes after inoculation. Data were
analyzed on SAS 9.1 (SAS Institute Inc.).
[0031] In an aspect, the medium in which Bacillus firmus bacteria
has been grown is obtained by growing B. firmus bacteria
aerobically in medium. There are numerous mediums in which the
bacteria can grow on, for example, a minimal salt medium
supplemented with yeast extract and glucose. In an aspect, for
biosurfactant isolation, bacterial cells are removed from the
surfactant-containing medium by centrifugation, and the
biosurfactant is precipitated from the supernatant by adding an
acid. The acid precipitates are recovered by centrifugation and are
extracted with a solvent. After precipitation with an acid, the
crude fraction dissolved in the solvent is evaporated, and the
final purified biosurfactant product is diluted in distilled water
at specific concentrations.
[0032] In an aspect, the entity as described herein is an isolated
protease. Protease is understood to mean any polypeptides or
complex of polypeptides or fragments of polypeptides having
protease activity. Production of protease enzymes were evaluated by
use of milk agar assay. A clear halo following 24 hr period of
incubation is recorded as a positive indication for the production
of proteases.
[0033] In an aspect, the entity as described herein is an isolated
amylase. Amylase is understood to mean any polypeptides or complex
of polypeptides or fragments of polypeptides having amylase
activity. Production of amylase enzymes were evaluated by use of
starch agar assay. A clear halo following 24 hr period of
incubation is recorded as a positive indication for the production
of amylases.
[0034] In an aspect, the entity as described herein is an isolated
lipase. Lipase is understood to mean any polypeptides or complex of
polypeptides or fragments of polypeptides having lipase activity.
Production of lipase enzymes were evaluated by use of trybutirin
agar assay. A clear halo following 24 hr period of incubation is
recorded as a positive indication for the production of
lipases.
[0035] In an aspect, the entity as described herein is an isolated
cellulase. Cellulase is understood to mean any polypeptides or
complex of polypeptides or fragments of polypeptides having
cellulase activity. Production of cellulase enzymes were evaluated
by use of carboximetil cellulose (CMC) agar agar assay. A clear
halo following 24 hr period of incubation is recorded as a positive
indication for the production of cellulases.
[0036] In an aspect, the disclosure provides for a novel
composition having anti-nematodal activity comprising at least one
entity derived from Bacillus firmus bacteria selected from the
group consisting of an isolated biosurfactant, an isolated
protease, an isolated amylase, an isolated lipase, and an isolated
cellulase.
[0037] In an aspect, the strain of Bacillus firmus is GB-126. In an
aspect, the composition described herein has anti-nematodal
activity against Rotlyenchulus reniformis.
[0038] In an aspect, the composition described herein comprises an
acid precipitate from a culture medium in which the Bacillus firmus
bacteria was grown. In an aspect, the acid precipitate described
herein is obtainable by a method comprising: adding an acid to a
medium in which a Bacillus firmus bacteria has been grown to
generate an acid precipitate, and (b) isolating the acid
precipitate from the medium. In another aspect, the acid
precipitate has anti-nemadodal activity.
[0039] In an aspect, the medium in which Bacillus firmus bacteria
has been grown is obtained by growing B. firmus bacteria
aerobically in medium. There are numerous mediums in which the
bacteria can grow on, for example, a minimal salt medium
supplemented with yeast extract and glucose. In an aspect, for
biosurfactant isolation, bacterial cells are removed from the
surfactant-containing medium by centrifugation, and the
biosurfactant is precipitated from the supernatant by adding an
acid. The acid precipitates are recovered by centrifugation and are
extracted with a solvent. After precipitation with an acid, the
crude fraction dissolved in the solvent is evaporated, and the
final purified biosurfactant product is diluted in distilled water
at specific concentrations.
[0040] In an aspect, the composition as described herein comprises
of an isolated biosurfactant. In an aspect, the isolated
biosurfactant concentration in the composition as described herein
is greater than 0.5 ppm. In an aspect, the isolated biosurfactant
concentration in the composition described herein is at least 1
ppm. In an aspect, the isolated biosurfactant concentration in the
composition described herein is from 1 to 2 ppm. In an aspect the
isolated biosurfactant concentration in the composition described
herein is at least 2 ppm. In an aspect, the isolated biosurfactant
concentration in the composition described herein is from 1 to 5
ppm. In an aspect, the isolated biosurfactant concentration in the
composition described herein is from 2 to 5 ppm.
[0041] In an aspect, the composition as described herein is an
isolated protease.
[0042] In an aspect, the composition as described herein is an
isolated amylase.
[0043] In an aspect, the composition as described herein is an
isolated lipase.
[0044] In an aspect, the composition as described herein is an
isolated cellulase.
[0045] In an aspect, the the disclosure provides for an extract of
a Bacillus firmus bacteria having the following characteristics:
anti-nematodal activity and presence of a biosurfactant.
[0046] In an aspect, the the disclosure provides for an extract of
a Bacillus firmus bacteria having the following characteristics:
anti-nematodal activity and activity selected from the group
consisting of protease activity, amylase activity, lipase activity,
and cellulase activity.
[0047] Extract shall refer to any fraction extracted from reference
material.
[0048] The disclosure also provides for a method of controlling
plant-parasitic namatode by applying an entity or composition as
described herein to a plant, the seed material (e.g. grains, seeds
or vegetative propagation organs such as tubers or shoot parts with
buds) or the area on which the plants grow (e.g. the area under
cultivation).
[0049] In an aspect, the disclosure provides for a method of
protecting a plant by applying an entity or composition as
described herein to a plant, the seed material, or the area on
which the plant grows.
[0050] All plants and plant parts can be treated in accordance with
the invention. Plants are to be understood as meaning in the
present context all plants and plant populations such as desired
and undesired wild plants or crop plants (inclusive of naturally
occurring crop plants). Crop plants can be plants which can be
obtained by conventional plant breeding and optimisation methods or
by biotechnological and recombinant methods or by combinations of
these methods, inclusive of the transgenic plants and inclusive of
the plant varieties protectable or not protectable by plant
breeders' rights. Plant parts are to be understood as meaning all
aerial and subterranean plant parts and organs of the plants such
as shoot, leaf, flower and root, examples which may be mentioned
being leaves, needles, stalks, trunks, flowers, fruiting bodies,
fruits, seeds, roots, tubers and rhizomes. The plant parts also
include vegetative and generative propagation material, for example
cuttings, tubers, rhizomes, seedlings and seeds.
[0051] In an aspect, the plant selected from the group consisting
of agronomic crops, corn (field, seed & popcorn), sorghum,
wheat, barley, rye, oats, rice, forage grasses, soybeans, canola,
peanuts, cotton, alfalfa, fruting vegetables (peppers &
tomatoes), melons, squash, pumpkins, cucumbers and potatoes.
[0052] In an aspect, the plant-parasitic nematode is Reniform
nematode, Rotlyenchulus spp.; Dagger nematode, Xiphinema spp.;
Lance nematode, Hoplolaimus spp.; Pin nematode, Paratylenchus spp.;
Ring nematode, Criconemoides spp.; Rootknot nematode, Meloidogyne
spp.; Sheath nematode, Hemicycliophora spp.; Spiral nematode,
Helicotylenchus spp.; Stubbyroot nematode, Trichodorus spp.; Cyst
nematode, Heterodera spp.; Sting nematode, Belonolaimus, spp.;
and/or Stunt nematode, Tylenchorhynchus spp.
[0053] In an aspect, the plant is cotton. In an aspect the plant is
agronomic crops. In an aspect, the plant is corn (field, seed &
popcorn). In an aspect, the plant is sorghum. In an aspect, the
plant is wheat. In an aspect, the plant is barley. In an aspect,
the plant is rye. In an aspect, the plant is oat. In an aspect, the
plant is rice. In an aspect, the plant is forage grasses. In an
aspect, the plant is soybeans. In an aspect, the plant is canola.
In an aspect, the plant is peanuts. In an aspect, the plant is
cotton. In an aspect, the plant is alfalfa. In an aspect, the plant
is fruting vegetables (peppers & tomatoes). In an aspect, the
plant is melon. In an aspect, the plant is squash. In an aspect,
the plant is pumpkin. In an aspect, the plant is cucumber. In an
aspect, the plant is potato.
[0054] The following examples serve to illustrate certain aspects
of the disclosure and are not intended to limit the disclosure.
EXAMPLES
[0055] In vitro tests against R. reniformis.
[0056] An initial test was conducted to evaluate the effect of the
metabolites produced by B. firmus against second stage juveniles
and egg hatching of R. reniformis. Eggs of R. reniformis were
extracted from cotton roots by shaking the root system in a 1%
NaOCl solution for four minutes at 120 rpm. The suspension of eggs
was collected and rinsed with water through a 25 .mu.m sieve (8).
Eggs were rinsed with streptomycin sulfate (300 mg/L) and
chlortetracycline (12.5 mg/L) for bacterial disinfection, then with
metalaxyl (25 mL/L) and iprodine (20 mL/L) for fungal disinfection,
then finally with distilled water. For the second stage juvenile
trial, the eggs were placed in a modified Baerman dish on a slide
warming tray at 27.degree. C. Second stage juveniles were hatched
after three days. To obtain the bacterial metabolite, Bacillus
firmus GB-126 was grown in 50 mL of Tryptic Soy Broth (TSB)
(BACTO.TM.) for four days and then placed in 50 mL plastic tubes
and centrifuged for 20 minutes at 4000x g. The supernatant was
collected and filtered through a Millipore filter 0.45-0.22 .mu.m
to obtain the final bacterial metabolite product (CFE). In vitro
trials were conducted on 96-well plates, where volumes of 100 .mu.L
of the treatments were transferred to each well, which contained
approximately 16 juveniles or 20 eggs of R. reniformis. Treatments
were i) water control, ii) TSB media control, iii) metabolite 100%,
and iv) metabolite 50%. Each treatment had 6 replications, and the
entire trial was repeated twice. The number of eggs hatched
releasing juveniles was recorded at 0, 24, 48, and 72 hours after
inoculation. For the second stage juveniles, number of moving and
paralyzed nematodes was recorded at 0, 1, 2, 4, 6, and 12 hours
after inoculation. Data were analyzed on SAS 9.1 (SAS Institute
Inc.) using the GLIMMIX procedure where the distributional
assumption was evaluated with the student panel graphs. Dunnett's
option was used to assess the differences with the water and TSB
controls.
[0057] Table 1 demonstrates the statistical significance of FIG.
1.
TABLE-US-00001 TABLE 1 Statsical Comparison; P < 0.05 (Hr after
Combining) Treatments 24 48 72 Water Control vs CFE100% 0.11 0.03
0.03 Water Control vs CFE 50% 0.11 0.04 0.03 Media Ctl vs CFE 100%
0.24 .ltoreq.0.001 .ltoreq.0.001 Media Ctl vs CFE 50% 0.28
.ltoreq.0.001 .ltoreq.0.001
[0058] Table 2 demonstrates the statistical significant of FIG.
2.
TABLE-US-00002 TABLE 2 Statsical Comparison; P < 0.05 (Hr after
Combining) Treatments 1 2 4 6 12 Control vs CFE 100% .ltoreq.0.001
.ltoreq.0.001 .ltoreq.0.001 .ltoreq.0.001 .ltoreq.0.001 Control vs
CFE 50% .ltoreq.0.005 .ltoreq.0.009 .ltoreq.0.001 .ltoreq.0.001
.ltoreq.0.001 Media Ctl vs CFE 100% 0.04 0.003 .ltoreq.0.001
.ltoreq.0.001 0.02 Media Ctl vs CFE 50% 0.12 0.024 .ltoreq.0.001
.ltoreq.0.001 0.03
[0059] Enzyme characterization of B. firmus.
[0060] Different enzymatic properties of B. firmus GB-126 were
evaluated to test their capacity to degrade different media.
Production of enzymes was evaluated as positive when a transparent
halo was formed around the bacterium culture. Bacillus firmus
GB-126 was grown for 24 hours on milk agar to test the production
of proteases, starch agar for the production of amylases,
carboximetil cellulose (CMC) agar for the production of cellulases,
chitinase agar for the production of chitinases, and trybutirin
agar for the production of lipases. The CMC agar and chitinase agar
required the application of 5 ml of congo red to stain the media
and a transparent halo after 24 hours of culturing the bacteria. In
the case of the starch media, culture was stained with lugol.
[0061] Determination of production of biosurfactant by B.
firmus.
[0062] Three initial tests were conducted to determine the
production of biosurfactant from B. firmus. In the first test, the
bacterium was grown under blood agar, where a positive production
of biosurfactant is indicated by a transparent halo around the
bacterial colony. For the second and third tests, B. firmus was
cultured on nutrient broth at 30.degree. C. for 24 hours.
Subsequently, the living cells were recovered by centrifugation at
5,181 xg for 15 minutes, and cells were washed twice with NaCl
0.85% (w/v) and later suspended in 5 ml of NaCl0.85% (w/v). They
were used to inoculate 45 ml of saline Davis minimal broth with an
inoculum ratio of 1% (v/v). The composition was K.sub.2HPO.sub.4
5.23 g/l, KH.sub.2PO.sub.4 1.91 g/l, MgSO.sub.4 0.09 g/l,
(NH.sub.4).sub.2SO.sub.4 1 g/l, as well as 1 ml/l of trace elements
solution (CoCl.sub.3 20 mg/l, H.sub.3BO.sub.3 30 mg/l, ZnSO.sub.4
10 mg/l, Cu.sub.2SO.sub.4 1 mg/l, Na.sub.2MoO.sub.4 3 mg/l,
FeSO.sub.4 10 mg/l and MgSO.sub.4 2.6 mg/l). Cultures were
incubated at 30.degree. C. .+-.2 at 150 rpm for 3 days. Again B.
firmus living cells were separated from the supernatant by
centrifugation (20 minutes at 4000x g). The supernatant was
filtered through a Millipore filter 0.45-0.22 pm to obtain the
final bacterial biosurfactant product. This final product was
autoclaved twice for 30 minutes at 120.degree. C. at 1 kg/cm.sup.3
pressure to kill all the bacterium's living cells and inactivate
its enzymes.
[0063] In the second test, emulsifying activity of the cell-free
supernatant was evaluated by mixing 0.5 ml with 0.5 ml of kerosene
and 4 ml of distilled water to a disposable culture tube
(borosilicate glass 16.times.150 mm). The negative control
consisted of distilled water and kerosene, and the positive control
consisted of distilled water, kerosene, and Triton X-100 (100
mg/ml). Each tube was agitated in a vortex for 1 min and was left
to stand for 24 hours. The height of the emulsification ring was
then measured in millimeters and compared to that of the chemical
emulsifier. If there was positive production of surfactant, the
kerosene emulsified it and produced foam. The third test consisted
of an oil drop collapse in which one drop of the supernatant was
placed on parafilm paper and a drop of oil was placed on top of it.
If the drop of oil increased its diameter compared to the media
control, the bacteria was considered to have produced a
biosurfactant.
[0064] Finally, the biosurfactant product was again tested under in
vitro conditions in 96 well-plate against second stage juveniles of
R. reniformis. Volumes of 100 .mu.L of the treatments were
transferred to each well, which contained approximately 16
juveniles of R. reniformis. Treatments for this trial were i) water
control, ii) BPM control, iii) biosurfactant 100%, and iv) B.
firmus 15.times.10.sup.7 cfu/ml. Each treatment was replicated 8
times, and the entire trial was repeated twice. The number of
juveniles paralyzed or dead was recorded 30 minutes after
inoculation. Data were analyzed on SAS 9.1 (SAS Institute Inc.) as
described previously.
[0065] Purification and production of biosurfactant and in vitro
evaluations at different concentrations against R. reniformis.
[0066] For biosurfactant production, B. firmus GB-126 was grown
aerobically on minimal salt medium containing (per liter)
KH.sub.2PO.sub.4 (2.0 g), K.sub.2HPO.sub.4 (5.0 g),
(NH.sub.4).sub.2SO.sub.4 (3.0 g), NaNO.sub.3 (2.0 g), NaCl (0.1 g),
MgSO.sub.4 H.sub.2O (0.2 g), 0 FeSO.sub.4 7H.sub.2O (0.01 g),
CaCl.sub.2 (0.01 g), and 1 ml of a trace element solution. The
stock solution of trace elements contained (per liter) ZnSO.sub.4
7H.sub.2O (2.32 g), MnSO.sub.4 4H.sub.2O (1.78 g), H.sub.3BO.sub.3
(0.56 g), CuSO.sub.4 5H.sub.2O (1 g), Na.sub.2MoO.sub.4 7H2O (0.39
g), CoCl.sub.2 6H.sub.2O (0.42 g), EDTA (1 g), NiCl.sub.2 6H.sub.2O
(0.004g), and KI (0.66 g). The medium was supplemented with 0.05%
yeast extract (Vater, J. et al.) Glucose was added as a carbon
source at a concentration of 2% (wt/vol). The medium pH was 7.1 to
7.2. The organism was grown at 37.degree. C. for 48 h in 2-liter
Erlenmeyer flasks containing 800 ml of medium and shaken at 200 rpm
in a shaker incubator.
[0067] For biosurfactant isolation, bacterial cells were removed
from the surfactant-containing medium by centrifugation (13000x g
for 15 min at 4.degree. C.). The biosurfactant was precipitated
from the supernatant by adding 6 N HCl to obtain a final pH of 2.0.
The acid precipitates were recovered by centrifugation (13000x g
for 15 min at 4.degree. C.) and were extracted with dichloromethane
or methanol (lipopeptide fraction). When methanol was used as the
solvent, the extract was neutralized immediately to avoid formation
of methyl esters. After precipitation with HCl, the crude fraction
dissolved in methanol or dichloromethane was evaporated in a rotary
evaporator (Model Buchi R) under a vacuum pump (Model Gem 8890)
(Vater, J. et al.). The final purified biosurfactant product was
diluted in distilled water at concentrations of 2 ppm, 1 ppm, 0.5
ppm, 0.2 ppm, 0.1 ppm, and 0.02 ppm. These concentrations were
evaluated, compared to distilled water control under in vitro
conditions on second stage juveniles as described above, with 8
replications per treatment, and repeated twice.
[0068] Greenhouse trials.
[0069] To evaluate if B. firmus GB-126 induces systemic resistance
the following treatments were in tested in a split root system.
Treatments consisted of i) water control without nematodes, ii)
water control with nematodes, iii) B. firmus 1.times.10.sup.7
cfu/mL, iv) B. firmus 1.times.10.sup.6 cfu/mL, or v) Serratia
marcescens 1.times.10.sup.7 cfu/mL. Stoneville 5458 B2RF cotton
seeds were germinated in potting mixing soil under greenhouse
conditions. Emerging root radicals approximately 2.5 cm in length
were split with a razer blade. At 5 days after planting (DAP), soil
was removed from the roots and divided into two equal halves.
Plants were planted in 960 cm.sup.3 pots with each root half in a
different cup. At 7 days after splitting the roots, a suspension of
50 mL of treatments was applied on the left side of the root. Five
days later, the right side of the root was inoculated with 500
second stage juveniles of R. reniformis. The trial was harvested 45
DAP, and plant height, root fresh weight, and number of females and
eggs per gram of root were measured. Each treatment had 6
replications and the entire trial was repeated twice.
[0070] To evaluate the response of R. reniformis to cotton seeds
treated with B. firmus GB-126, a trial in autoclaved soil was
conducted in the Plant Science Research Center (PSRC) of Auburn
University. Cotton seeds from cultivar Stoneville 5458 B2RF were
treated with B. firmus GB 126 by the manufacturer in a liquid seed
dresser Hegel 1 (Hege Maschinen GmbH, Germany). Presence of the
bacterium in the seed was confirmed by culturing the treated seed
on Triptic Soy Agar (TSA) adjusted to pH 8.0 and recording the
growth after 16 hours. The soil was a Decatur silty clay loam
(sand-silt-clay: 17.5-51.3-31.2%; nitrogen: 0.16%; organic matter:
2.2; pH 7.24) from the Tennessee Valley Research and Extension
Center (TVREC) near Belle Mina, Ala. The soil was autoclaved using
two 90-minute cycles at 130.degree. C. at 15 psi with a 24 hour
cool down between cycles. Seed treatments were as follows: i)
untreated seed with nematodes; ii) imidacloprid (500 g ai/100 kg) a
standard insecticide; iii) B. firmus (7.times.10.sup.4 cfu/seed)
plus imidacloprid (500 g ai/100 kg); iv) B. firmus
(7.times.10.sup.5 cfu/seed) plus imidacloprid (500 g ai/100 kg);
and v) B. firmus (7.times.10.sup.6 cfu/seed) plus imidacloprid (500
g ai/100 kg). The standard insecticide seed treatment imidacloprid
was included because this insecticide is commonly applied as a seed
treatment and was tested to determine if it has any effect on B.
firmus or R. reniformis life stages.
[0071] Rotlyenchulus reniformis vermiform life stages were
extracted from the soil by modified gravity screening and sucrose
centrifugation-flotation. Eggs were extracted from cotton roots by
shaking the root system in a 1% NaOCl solution for four minutes at
120 rpm. The nematode suspension was and rinsed with water and
collected on a 25 .mu.m sieve. Females in roots were stained with
acid fuschin to facilitate enumeration of the females invading the
root. Vermiform life stages and eggs were counted under an inverted
TS 100 Nikon microscope at 40.times. magnification. Females
embedded in the root systems were quantified at 5.times.
magnification utilizing the Nikon SMZ800 compound microscope.
Variables measured were plant height, shoot and root weight,
females and eggs per gram of root, and the number of vermiform life
stages in 500 cm.sup.3 of soil. Greenhouse average temperature
where plants were grown was 29.degree. C. Soil moisture was
maintained between 40-60% of the maximum water holding capacity.
Data were analyzed in SAS 9.1 (SAS Institute Inc.). The
distributional assumption was evaluated with the student panel
graphs of the GLIMMIX procedure. Dunnett's option was used to
assess the differences with the untreated control.
[0072] In Vitro and Greenhouse Results
[0073] In the first in vitro trial, R. reniformis egg hatch was
reduced at 48 and 72 hours, when eggs were exposed to B. firmus
metabolites at 100% and 50%, when compared to the water and media
control (P.ltoreq.0.01) (FIG. 1). Furthermore, paralysis of second
stage juveniles of R. reniformis observed within one hour of
inoculation in 100% and 50% metabolite through 12 hours when all
the second stage juveniles were paralyzed (P.ltoreq.0.01) (FIG. 2).
No differences were observed between the water and the media
controls in these trials (P.ltoreq.0.99).
[0074] Biosurfactant production was confirmed by the emulsification
of kerosene, oil drop collapse, and halo formation in blood agar
(FIG. 3). Biosurfactant and living cells of B. firmus at a
concentration of 15.times.10.sup.7 cfu/mL paralyzed all the second
stage juveniles within 30 minutes after inoculation when compared
to the BP media and water controls (P.ltoreq.0.01) (FIG. 3). There
were no differences between the two controls (P.ltoreq.0.99).
Finally, in the last in vitro trial where the pure B. firmus
biosurfactant was evaluated at different concentrations, the
biosurfactant at 2 ppm and 1 ppm paralyzed 100% and 45.9%, of the
second stage juveniles of R. reniformis, respectively, within 30
minutes (FIG. 4).
[0075] These two concentrations produced an increase in paralysis
of the second stage juveniles compared to the water control
(P.ltoreq.0.001). Biosurfactant concentrations of 0.5 ppm, 0.2 ppm,
0.1 ppm, and 0.02 ppm did not paralyze second stage juveniles and
were not different from the water control (P.ltoreq.0.932). When
second stage juveniles from the water control and 2 ppm treatments
were observed under SEM, no mechanical damage to the cuticle was
observed (FIG. 5A, B). The enzymatic profile of Bacillus firmus
GB-126 indicated a high enzymatic activity for proteases, amylases,
and cellulases forming a transparent halo in milk agar, starch
agar, and CMC agar, respectively, within 24 hours. In contrast, no
production of chitinases was observed under chitinase agar (FIG.
7).
[0076] Bacillus firmus GB-126 inhibits the hatch of R. reniformis
eggs and paralyzes second stage juveniles under in vitro conditions
using secondary metabolites from this bacterium and also living
cells at a concentration of 15.times.10.sup.7cfu/ml. In greenhouse
trials, B. firmus GB-126 applied as a seed treatment at a rate of
7.times.10.sup.6 cfu/seed reduced number of R. reniformis females
in the root and juveniles in soil within the first 30 days of
planting. The effect of the insecticide imidacloprid, which is used
as a seed treatment and formulated with this bacterium, did not
show any nematicidal activity.
[0077] The results demonstrate that the mode of action of B. firmus
GB-126 against nematodes is a secondary metabolite toxic to
nematodes. This secondary metabolite is a biosurfactant which is
responsible for the paralysis of R. reniformis juveniles and
inhibition of egg hatch under in vitro conditions. Bacillus firmus
GB-126 biosurfactant needs a minimum concentration of 1 ppm to
paralyze second stage juveniles within 30 minutes.
[0078] The presence of enzymes (amylases, cellulases, and
proteases) suggests the possibility that B. firmus GB-126 can have
other modes of action against R. reniformis and other nematode
species during different stages of the life cycle. The production
proteases by this bacterium can affect on egg hatching and cause
nematode paralysis.
[0079] The induction of systemic resistance trial indicated cotton
plants treated with B. firmus GB-126 (1.times.10.sup.6 cfu/ml) was
taller than control with nematodes (P.ltoreq.0.05) and S. marcecens
(1.times.10.sup.9 cfu/ml) treatment (P.ltoreq.0.01). There were no
differences in left or right root fresh weights or the number of R.
reniformis females and eggs among the treatments (P.ltoreq.0.99)
(FIG. 6). In contrast, B. firmus GB-126 at a rate of
7.times.10.sup.6 cfu/seed reduced the number of females per gram of
root (P.ltoreq.0.001) and juveniles per 500 cm.sup.3 of soil 30
days after planting (P.ltoreq.0.01) (FIG. 8). The insecticide
imidacloprid did not have any effect on cotton plant growth or R.
reniformis life stages. Induction of systemic resistance of B.
firmus GB-126 in cotton plants was not observed at the
concentrations evaluated.
[0080] In summary, the biocontrol activity of B. firmus GB-126
observed in previous trials under greenhouse and field conditions
where eggs and juvenile stages were reduced can be explained
because the bacterium is producing a biosurfactant that is toxic to
the plant-parasitic nematode. No ISR was observed at rates tested.
However, B. firmus GB-126 possibly has other mechanisms of action
against R. reniformis due to the presence of proteases that can be
deleterious to the nematode.
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* * * * *
References