U.S. patent application number 10/847623 was filed with the patent office on 2007-04-26 for novel strain of bacillus as a bioinoculant.
This patent application is currently assigned to COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH. Invention is credited to Mansoor Alam, Mahendra Pandurang Darokar, Alok Kalra, Abdul Khaliq, Togarati Padmapriya, Om Parkash Dhawan, Suman Preet Singh Khanuja, Abdul Samad, Abdul Sattar, Ajit Kumar Shasany, Ashutosh Kumar Shukla, Poovappallivadakethil Viswanathan Nair Ajaya Kumar, Mohammad Yaseen, Mohammad Zaim.
Application Number | 20070092491 10/847623 |
Document ID | / |
Family ID | 34957925 |
Filed Date | 2007-04-26 |
United States Patent
Application |
20070092491 |
Kind Code |
A1 |
Sattar; Abdul ; et
al. |
April 26, 2007 |
NOVEL STRAIN OF BACILLUS AS A BIOINOCULANT
Abstract
The present invention relates to the selection and development
of superior strain of Bacillus spp for improving plant growth and
health by inhibiting pathogenic fungi
Inventors: |
Sattar; Abdul; (Lucknow,
IN) ; Alam; Mansoor; (Lucknow, IN) ; Khaliq;
Abdul; (Lucknow, IN) ; Preet Singh Khanuja;
Suman; (Lucknow, IN) ; Kalra; Alok; (Lucknow,
IN) ; Samad; Abdul; (Lucknow, IN) ; Shasany;
Ajit Kumar; (Lucknow, IN) ; Darokar; Mahendra
Pandurang; (Lucknow, IN) ; Shukla; Ashutosh
Kumar; (Lucknow, IN) ; Padmapriya; Togarati;
(Lucknow, IN) ; Yaseen; Mohammad; (Lucknow,
IN) ; Parkash Dhawan; Om; (Lucknow, IN) ;
Zaim; Mohammad; (Lucknow, IN) ; Viswanathan Nair
Ajaya Kumar; Poovappallivadakethil; (Lucknow, IN) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
COUNCIL OF SCIENTIFIC &
INDUSTRIAL RESEARCH
|
Family ID: |
34957925 |
Appl. No.: |
10/847623 |
Filed: |
May 18, 2004 |
Current U.S.
Class: |
424/93.4 ;
435/252.31 |
Current CPC
Class: |
C12N 1/20 20130101; Y10S
435/832 20130101 |
Class at
Publication: |
424/093.4 ;
435/252.31 |
International
Class: |
A01N 63/00 20060101
A01N063/00; C12N 1/20 20060101 C12N001/20 |
Claims
1-20. (canceled)
21. An isolated Bacillus spp bacterial strain having accession
number MTCC-5130 on deposit with the Microbial Type Culture
Collection & Gene Bank, Institute of Microbial Technology,
Chandigarh, India, wherein said strain enhances plant growth and
inhibits growth of plant fungal pathogens.
22-40. (canceled)
Description
FIELD OF INVENTION
[0001] The present invention relates to the selection and
development of superior strain of Bacillus spp for improving plant
growth and health by inhibiting pathogenic fungi
BACKGROUND OF INVENTION
[0002] Crops under cultivation suffer from many diseases caused by
plant pathogenic fungi. One particularly damaging plant
phytopathogenic fungus is Rhizoctonia solani which is widely
distributed and causing common diseases of greenhouse-grown crops,
field crops, vegetables, ornamentals, and fruits throughout the
world. It also causes root rot and wilt disease of pyrethrum and
geranium. Other detrimental fungal plant pathogens include
Sclerotinia sclerotiorum, Thielavia basicola, Fusarium oxysporum,
causing wilt, Pythium aphanidermatum causing lethal yellowing and
damping off in numerous plants.
[0003] The incidence of various kinds of fungal diseases cause
considerable damage to the medicinal and aromatic plants (MAPS) in
different part of India. The occurrence in severe form may either
kill emerging seedlings or reduce plant growth and adversely affect
the crop yield. Among fungal pathogens, species of Rhizoctonia,
Sclerotinia, Fusarium, Thielavia, Pythium, Helminthosporium,
Curvularia, Alternaria and Colletotrichum are most important and
common. They produce different kinds of diseases such as stem rot
and twig blight (Sclerotinia sclerotiorum) on periwinkle, Egyptian
henbane and Ammi majus; root rot & wilt (Rhizoctonia solani)
diseases on pyrethrum, leaf blight (Curvularia andropogonis);
lethal yellowing (Pythium aphanidermatum) and collar rot (Fusarium
moniliforme) on Java citronella; damping-off (Pythium dissotocum),
collar rot (Rhizoctonia solani) and leaf blight (Alternaria
alternata) on opium poppy, stolon and root rot (Thielavia
basicola), wilt (Fusarium oxysporum), leaf blight (Corynespora
cassiicola) on mints and anthracnose (Colletotrichum acutatum) and
wilt (Rhizoctonia solani) on geranium;. (Alam et al 1983, Indian
Phytopath. 367: 480-483; ibid 1992, Plant Disease 43:10578-1061;
ibid 1994, Plant Pathology 43:1057-1061; ibid 1996, Indian
Phytopath. 49:94-97; Sattar et al. 1993, Indian J. Plant Pathol 10:
10-11; ibid 1999, Indian J. Plant Pathol. 17:74-76; ibid 2002, J.
Mycol. & Pl. Pathol. 32: 31-34).
[0004] The use of huge amount of fertilizers and chemical
pesticides for maintaining the high productivity of crop has become
fatal to human and animal health. They also poses many other
serious problems including i) development of resistant strains of
pathogen (Schwinn et al., (1991) "Control with Chemicals" Advances
in Plant Pathology: vol. 7: Phytophtohora infestans, the Cause of
Late Blight of Potato, Ingram et al., eds., Academic Press, 8:
255-266) ii) build up of harmful residues in the edible plant and
iii) non-target side effect of beneficial micro flora. It is
desirable to replace them with biopesticides derived from the
microorganisms. They are as effective as broad-spectrum chemical
pesticides, easily degradable and have low cost production. They
are a distinct possibility for the future and can be successfully
exploited in modem agriculture without affecting our precious
ecosystem.
[0005] Plant growth promoting rhizobacteria (PGPR) exert a
beneficial effect on the plant by causing plant growth promotion
and/or suppressing plant pathogen population to avoid infection.
Efforts to select and apply PGPR for control of specific soilbome
fungal pathogens have been reviewed (Kloepper, 1993; Glick and
Bashan, 1997 Biotechnology Advances 15, 353-378). In most of the
cases, activity is due to production of metabolites such as
antibiotics, hydrogen cyanide, iron-chelating siderophores, and
cell wall-degrading enzymes, which directly inhibit the pathogen.
Plant growth promotion by PGPR may also be an indirect mechanism
leading to a reduction in the probability of a plant contracting a
disease when the growth promotion results in shortening the time
that a plant is in a susceptible state. An alternative mechanism
for biological control by PGPR is by induced systemic resistance.
Bacillus subtilis and few other Bacillus spp. are used as
biocontrol agents of fungal diseases caused by different plant
pathogens (Swinbume et al. (1975) Trans. Brit. Mycol. Soc.
65:211-217, Baker et al. (1983) Phytopathology 73:1148-1152, Singh
and Deverall, (1984) Trans. Br. Mycol. Soc. 83:487-490, Cook (1987)
Proceedings Beltwide Cotton Production--Mechanization Research
Conference, Cotton Council, Memphis, pp. 43-45, Gueldner, et al.,
(1988) J. Agric. Food Chem. 36:366-370, Pusey et al. (1988) Plant
Dis. 72:622-626, Ferreira et al. (1991) Phytopathology 81:283-287,
Sholberg et al. (1995) Can. J Microbiol. 41:247-252, Asaka, and
Shoda, (1996), Appl. Environ. Microbiol. 62:4081-4085). McKeen et
al. (1986) Phytopathology 76:136-139 and Pusey and Robins (1991)
U.S. Pat. No. 5,047,239 disclose control of post harvest fruit rot
using B. subtilis. Among different Bacillus spp (B. subtilis, B.
megaterium B. cereus, B. polymyxa and B. pumilus), B. subtilis is
most exploited as biocontrol agent because it is considered to be a
safe and potential biological control agent due to high thermal
tolerance, rapid growth in liquid culture, ready formation of
resistant spores. Handelsman (1991) U.S. Pat. No. 5,049,379
disclose that Zwittermicin-A producing B. cereus control damping
off in alfalfa and soybeans by inhibiting root rot fungus. A
Bacillus subtilis GBO3 strain is commercially used as seed dresser
under the names KODIAC..TM.. HB. or GUS 2000..TM.. by Gustafson,
Inc. Plano, Tex. 75093 (EPA Reg. No. 7501-146, 1992). This product
is available as a 2.75% powder formulation containing not less than
5.5.times10. sup. 10 viable spores per gram and is to be applied at
a rate ranging from 2-4 ounces per 100 pound of seed. The bacteria
is said to colonize the developing root systems and compete with
pathogens that would attack the roots. Huang et. al (1993), Can. J.
Microbiol. 39: 227-233 investigated antagonistic behavior of two
strains of Bacillus cereus; alf-87A & B-43 against Sclerotinia
sclerotiorum, the causal agent of basal pod rot & end rot
disease on dry pea. The vegetative growth & ascosporic
germination of S. sclerotiorum are inhibited by diffusible
metabolite produced by alf-87A but are unaffected by strain B-43.
The spraying on pea plants at the pod development stage with
alf-87A reduce the incidence of basal rot. The treatment of
soybeans with B. cereus has been shown to improve soybean yield at
field site (Osbum et al. 1995 Am. Phytopathol. Soc. 79: 551-556).
Chen et al (2002) Chinese J. Biol. Control 18:45-46 report that
antagonistic activity of B-916 strain of B. subtilis against R.
solani is due to proteins because addition of ammonium sulphate in
culture solution destroys its antagonistic ability. The application
of B. subtilis reduced the stem canker disease caused by R. solani
and common scab disease caused by Streptomyces scabies up to 63%
and 70%, respectively. Liu et al. (1995) U.S. Pat. No. 5,403,583
disclosed a Bacillus sp., ATCC 55000 and a method to control the
fungal plant pathogen, Rhizoctonia solani. Leifert et al. (1995),
J. Appl Bacteriol. 78:97-108, reported the production of
anti-Botrytis and anti-Alternaria antibiotics by two Bacillus
strains, B. subtilis CL27 and B. pumilus CL 45. The whole broth and
cell-free filtrates were active against Botrytis and Alternaria in
in vitro tests and were active against Botrytis in in vivo small
plant tests. Leifert et al. (1997) U.S. Pat. No. 5,597,565
disclosed that B. subtilis, B. pumilus and B. polymyxa are
effective against post harvest disease caused by Alternaria
brassicicola and Botrytis cinerea. They also disclose the presence
of antibiotics produced in the cell-free culture filtrate and their
activity at different pH values, but they do not identify these
compounds. The compounds from B. subtilis lose activity at low pH,
while the activity from the B. pumilus extracts occurs only at pH
values below 5.6. Leifert, et al. (1998) U.S. Pat. No. 5,780,080
disclose that the growth of Botrytis cinerea and Alternaria
brassicicola causing post-harvest disease is inhibited by applying
isolates of Bacillus pumilus NCIMB 40489 and Bacillus subtilis
NCIMB 40491 to cabbage at temperatures of about 20.degree C.
[0006] Bacilli are known to produce antifungal and antibacterial
secondary metabolites (Korzybski, et al., 1978 "Section C:
Antibiotic isolated from the genus Bacillus (Bacilliaceae)" In:
Antibiotics--Origin, nature and properties, American Society for
Microbiology, Washington D.C. Vol. III, pp. 1519-1661). The
chemical nature of antibiotics produced by Bacillus spp. are
peptide by the action of which they inhibit the growth of fungal
plant pathogens in the microenvironment (Katz and Demain 1977
Bacteriological Reviews, 41, 449-474; Singh & Deveral 1984;
McKeen et al. 1986; Utkhede et al (1986) Can. J. Microbiol. 32:
963-967; Wilson et al. (1989) Annual Review of Phytopathology. 27,
425-441, Hiraoka et al., (1992) J. Gen. Appl. Microbiol.
38:635-640.). Islam and Nandi (1985) J. Plant Dis. Protect.
92:241-246, disclose a Bacillus sp. with antagonism to Drechslera
oryzae, the causal agent of rice brown spot. The same authors,
Islam and Nandi (1985) J. Plant Dis. Protect. 92(3):233-240, also
disclose in-vitro antagonism of Bacillus sp. against Drechslera
oryzae, Alternaria alternata and Fusarium roseum. They discussd
three components in the culture filtrate. The most active
antibiotic was highly soluble in water and methanol with a UV peak
at 255 nm and a shoulder at 260 mn, that proved to be a
polyoxin-like lipopeptide. Loeffler et al. (1986) J. Phytopathology
115:204-213, disclose B. subtilis, B. pumilus, B. licheniformis,
and B. coagulans strains that produce various antibiotics with
antifungal and antibacterial activity. B. pumilus produces
bacilysin and iturin A. Bacilysin is a very small compound with a
molecular weight of 270, that inhibits only yeast. The iturins,
which are soluble in polar solvents, have broad antifungal and
antibacterial activity. McKeen et al. (1986), have shown that
antibiotics similar to the low molecular weight iturin cyclic
polypeptides contribute to the fungicidal activity of B. subtilis.
Rossall's (1991) U.S. Pat. No. 5,061,495 provides a novel
antibiotic from B. subtilis that is 63,500 Dalton, precipitates at
a pH below 5 and has activity against gram positive bacteria and
fungi (Botrytis and Erysiphe). Rossall's (1994) U.S. Pat. No.
5,344,647 discloses Bacillus subtilis strains with broad
anti-fungal activity. Stabb et al. (1994), Applied Environ.
Microbiol. 60: 4404-4412 have identified different strains of B.
subtilis, B. cereus, B. mycoides, B. thuringiensis that exhibit
antifungal activity. These strains have been shown to produce
zwittermicin-A and/or kanosamine (Milner et al. 1996, Applied
Environ. Microbiol. 62: 3061-3066), that are effective against
damping off disease caused by Phytophthora medicagenis, P.
nicotianae, Pythium aphanidermatum or Sclerotinia minor.
Zwittermicin-A is a water soluble, acid stable linear aminopolyol
molecule (He et al. 1994, Tetrahedron Lett. 35: 2499-2502) with
broad spectrum activity against many fungal and bacterial plant
pathogens. Kanosaminealso inhibits a broad range of fungal plant
pathogens and a few bacterial species (Milner et al. 1996).
[0007] Germida, et al. U.S. Pat. No. 6,015,553 disclosed Bacillus
subtilis strain AQ743 that produces a metabolite exhibiting
pesticidal activity against corn rootworm. Hassanein and El-Goorani
(1992) J. Plant Pathol. 133: 239-246 reported that application of
B. subtilis on wounded caster bean plants 30 min. before or
simultaneously with inoculation of Agrobacterium tumefaciens,
resulted in good control of crown gall without any phytotoxic
injury or growth retarding side effect.
[0008] So in the present invention systematic experiments were
planned to isolate and select superior strain of Bacillus strain
for promoting the growth of medicinal and aromatic plants as well
as inhibiting the growth of plant pathogenic fungi.
OBJECTS OF THE INVENTION
[0009] The main object of the present invention relates a novel
strain of Bacillus species having Acccession No. MTCC 5130.
[0010] Yet another object of the present invention relates to the
novel strain as Bioinoculant for plant growth promotion
[0011] Still another object of the present invention relates to use
of bacterial strain for antifungal activities and capability of
reducing fungal infection in medicinal and aromatic plants.
[0012] Another object of the present invention relates to use of
the bacterial strain for the control of fungal diseases selected
from root rot and wilt disease.
SUMMARY OF THE INVENTION
[0013] The present invention provides a novel and potential strain
of Bacillus spp, designated herein as Bacillus spp strain MTCC
5130, which is highly effective in promoting the growth of a plant
and inhibiting the growth of a wide range of plant pathogenic
fungi.
[0014] The invention provides a composition of a biologically
active Bacillus spp strain MTCC 5130 in promoting the growth of
treated plant and inhibiting the growth of a wide range of plant
pathogenic fungi. The composition is effective to promote the
growth of pyrethrum and geranium plant and inhibit infection of
Rhizoctonia solani causing root rot & wilt disease on pyrethrum
and geranium plant. The invention also encompasses a method for
protecting pyrethrum and geranium plants from root rot and wilt
disease caused by Rhizoctonia solani by applying to the plant or
its environment (rhizosphere).
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention relates to the selection and
development of a superior strain of Bacillus spp isolated from a
soil at Central Institute of Medicinal and Aromatic Plants (CIMAP),
Lucknow, India, where field experiments on geranium (Pelargonium
graveolens) were conducted. The selected strain improves plant
growth and health, particularly geranium and pyrethrum
(Chrysanthemum cinerarifoloum). Further, the invention is related
to inhibition of growth of pathogenic fungi by the newly selected
Bacillus spp MTCC 5130 strain and has been found to be highly
effective in protecting pyrethrum from root rot and wilt disease
caused by Rhizoctonia solani (PyRhl) and rose-scented geranium from
wilt disease caused by Rhizoctonia solani (GRhl). The invention
also includes methods of treatment for the control of root rot and
wilt disease in pyrethrum plants by using bacterial strain as such
or in delivery medium.
[0016] Accordingly, the main embodiment of the present invention
relates to a novel Bacillus spp bacterial strain having accession
No MTCC-5130, deposited at Institute of Microbial Technology,
Chandigarh, India, said bacteria having following characteristics:
[0017] Morphological characteristics: [0018] Cell shape: Spherical
colonies [0019] Cell size: 2-3 mm [0020] Cell arrangement: rod
arrangement [0021] Gram stain: Positive [0022] Motility: Yes [0023]
Pigment: Absent [0024] Capsule: Absent [0025] Spores: Endospore
formation [0026] Physiological properties [0027] Behaviour to
oxygen:Aerobic or facultative anaerobic [0028] Conditions for
growth: [0029] pH-5.6-6.5 [0030] Temperature -25.+-.2.degree. C.
[0031] Biochemical properties: [0032] Solvent tolerance test:
Butanol Positive [0033] Indole test: Negative [0034] Cytochrome
oxidase Test: Positive [0035] Growth under culture conditions:
[0036] Potato Dextrose Agar (PDA): The bacterial isolate was found
to have spherical colonies about 2-3 mm. in diameter, flattened and
mucoid in texture. These were gram positive rods. When the staining
for endospore formation was carried out the isolate was found to be
forming endospores in the centre of the cells. This isolate was
found to be similar to Bacillus on the basis of endospore position.
[0037] Potato Dextrose Broth (PDB) (liquid Medium): The strain grew
profusely and sporulated very extensively. [0038] Genotype
characteristics:
[0039] DNA was isolated and its nucleotide composition was
determined from its melting temperature (91.2.degree. C.). The G+C
content was calculated by the equation: % G+C=T-69.107/0.41. The
G+C content in the DNA of strain NP1010 was 510.24, a value close
to that of Geobacillus thermoleovorans (55%).
Amplification and 16S rRNA Gene Sequence Analysis
[0040] The partial 16S rRNA gene was amplified, then subjected to
cycle sequencing with protocol of MicroSeq 500 Kit procured from
Perkin Elmer Applied Bio systems (USA). The amplified product was
sequenced using the forward sequencing reaction mix. The DNA
sequence was searched for homology using BLAST search engine at
NCBI site (ncbi.nlm.nih.gov) and FASTA (ebi.ac.uk). Maximum
similarity (E value 2e-11, Score 78) was with Gamma proteobacterium
AKB16 16S ribosomal RNA gene, partial sequence (Accession no
AY083466.). In the FASTA homology search most of the hits were for
Bacillus spp in addition to Gamma proteobacteria. The partial rDNA
sequencing provided a characteristic 16S rDNA not having complete
homology with any bacteria of the database but showed similarity
towards Gamma proteobacteria and Bacillus spp. TABLE-US-00001 (SEQ
ID NO: 41) 1 TAATGTCGGT GGTGCGTTCA ACATACGTAA GCTAAGTGGA AAAGACGGGA
ATGCCGTCTT TCGACGCCAA GTGGTGGATG GGCGAGCAAT ATGCGGGCAA TTCGTTCGCA
AGATCGGGAC AATCTTGGGA AATTGGGGTC AACATTGGAC GGCCGCCCGA ATTGTACGGC
CTAAGATACA AAAGGCGGTC CTGGTCATTA TCCATAGACG GATTTGTGGT GTACCAGTCA
GCCGCCGAGG CAATGGTCTA TTAAGGTAAA GACGTGCAGT TGATTCGAGA GGGCGACTGG
TTATATCGGG ATCGAGATAA TGTTTAAATC TTCATGGGAG GTAGTAGCAG GGAACTCCTT
TTAACCGATT AAAGCTCCAT TGAGTAATTT TTTTTCAAGC GACCAAGGCC CCTCGCTTTC
AAAGTCTTTC CCCCCCAGGG AAAAATAAAC GGTGCCCCAA AACAAGGGGG GGATTTCCGT A
471.
[0041] Another embodiment of the present invention relates to novel
Bacillus spp bacterial strain having accession No MTCC-5130 capable
of enhancing plant growth and inhibiting fungal pathogens infecting
the plants.
[0042] Another embodiment of the present invention relates to the
strain MTCC 5130 wherein said strain has plant growth promoting
activity by inhibiting fungal pathogens for medicinal and aromatic
plants.
[0043] Still another embodiment of the present invention relates to
medicinal and aromatic plants wherein the medicinal and aromatic
plants are selected from group consisting of Pelargonium graveolens
and Chrysanthemum cinerarifolium and other related aromatic and
medicinal plant species.
[0044] Yet another embodiment of the present invention relates to
fungal pathogens wherein fungal pathogens are selected from group
consisting of Rhizoctonia spp., Fusarium spp., Pythium spp.,
Helminthosporium spp., Curvularia spp., Alternaria spp.,
Colletotrichum spp., Corynespora spp, and Thielavia spp.
[0045] Yet another embodiment of the present invention relates to
fungal pathogens wherein fungus pathogens are selected from group
consisting of Rhizoctonia solani, Fusarium oxysporum, Fusarium
semitectum, Pythium aphanidermatum, Helminthosporium carbonum,
Curvularia andropogonis, Alternaria alternata, Colletotrichum
acutatum, Colletotrichum capsici, Colletotrichum gloeosporiodes,
Corynespora cassiicola, and Thielavia basicola.
[0046] Another embodiment of the present invention relates to the
inhibition of fungal pathogens by strain wherein strain inhibits
Rhizoctonia solani inhibited in the range of about by 40-75%,
inhibits Fusarium oxysporum in the range of about 70 to 80,
inhibits Fusarium semitectum in the range of about 65 to 75%,
inhibits Pythium aphanidermatum in the range of about 10-30%,
inhibits Helminthosporium carbonum in the range of about 50 to 65%,
inhibits Curvularia andropogonis in the range of about 65 to 80%,
inhibits Alternaria alternata in the range of about 75 to 90%,
inhibits Colletotrichum acutatum in the range of about 70-80%,
inhibits Colletotrichum capsici in the range of about 60-75%,
inhibits Colletotrichum. gloeosporiodes in the range of about
50-65%, inhibits Corynespora cassiicola in the range of about
40-55%, and inhibits Thielavia basicola in the range of about
50-65%.
[0047] Still another embodiment of the present invention relates to
the inhibition of the fungal pathogens wherein inhibition of
Rhizoctonia solani is about 55%, inhibition of Fusarium oxysporum
is about 73%, inhibition of Fusarium semitectum is 68%, inhibition
of Pythium aphanidermatum is about 20%, inhibition of
Helminthosporium carbonum is about 55%, inhibition of Curvularia
andropogonis is about 70%, inhibition of Alternaria alternata is
about 83%, inhibition of Colletotrichum acutatum is about 76%,
inhibition of Colletotrichum capsici is about 65%, inhibition of
Colletotrichum. gloeosporiodes is about 58%, inhibition of
Corynespora cassiicola is about 48%, and inhibition of Thielavia
basicola is about 58%.
[0048] Yet another embodiment of the present invention relates to
the strain wherein said strain is effective in reducing the spore
germination of fungal pathogens is in the range of about
90-100%.
[0049] One more embodiment of the present invention relates to the
spore germination reduction wherein the reduction in spore
germination of fungal pathogens is about 95%. Another embodiment of
the present invention relates to the strain wherein strain MTCC
5130 is effective in increasing the plant yield in the range of
about 90-100%.
[0050] Still another embodiment of the present invention relates to
the strain MTCC-5130 wherein said strain is effective in increasing
the plant yield by about 95%.
[0051] Yet another embodiment of the present invention relates to
the strain MTCC 5130 wherein said strain is effective in enhancing
the yield of plant in the range of about 290 to 370 g/pot herb
yield when used alone or in combination with other
bioinoculants.
[0052] One more embodiment of the present invention relates to the
strain MTCC 5130 wherein said strain is effective in enhancing the
yield of plant to about 298.5 g/pot herb yield when used alone.
[0053] One more embodiment of the present invention relates to the
strain MTCC 5130 wherein said strain is effective in enhancing the
yield of plant to about 346 g/pot herb yield when used in
combination with other bioinoculants.
[0054] Yet another embodiment of the present invention relates to
the strain MTCC 5130 wherein said strain initiates plant rooting
within 20-30 days.
[0055] Still another embodiment of the present invention relates to
strain MTCC 5130 wherein said strain initiates plant rooting within
22-35 days.
[0056] One more embodiment of the present invention relates to
strain MTCC 5130 wherein said strain provides 100% survival of
plants.
[0057] Yet another embodiment of the present invention relates to
strain MTCC 5130 wherein said strain lowers the percentage
infection on plants is in the range of about 60-90%.
[0058] One more embodiment of the present relates to a strain MTCC
5130 wherein said reduction of percentage infection on plants in
the range of about 50-70%.
[0059] The following examples are given by way of illustration of
the present invention and should not be construed to limit the
scope of the present invention.
EXAMPLES
Example 1
Isolation of a Novel Bacterial Strain, Bacillus spp MTCC-5130
[0060] Bacteria were isolated from rhizosphere soil and from root
tissue of geranium (cv. Bar bourn) growing in the experimental
fields of CIMAP at Lucknow (India).
[0061] Rhizospheric soil and root materials were placed in a test
tube. Ten volumes of phosphate saline buffer (PSB, pH 7.3; Wollum,
1982) were added and the tube was vortexed for 1 minute. A dilution
series (10.sup.-1 to 10.sup.-8) was made using PSB. One hundred
.quadrature.1 of each dilution was plated onto petri dishes
containing PDA. Plates were incubated at 25.sup..quadrature.C. in a
growth chamber.
I. Isolation of MTCC-5130 Strain of the Present Invention from
Soil.
[0062] The bacterial strain of the present invention was isolated
from rhizospheric soil of a geranium plant. The samples were
collected randomly from the geranium experimental fields at CIMAP,
Lucknow to a depth of 0-2-cm, mixed thoroughly and stored at
5.degree. C. in polythene bags. The representative samples were
suspended in phosphate buffered saline (PBS) solution, serially
diluted and streaked onto agar medium, with a wire loop. A number
of different common agar media were used to culture the bacteria,
such as nutrient glucose agar (NDA; Difco, Detroit, Mich.),
nutrient-broth yeast extract agar (NBY), and potato dextrose agar
(PDA), the recipes for which are provided in Schaad (Schaad, 1988,
page 3). Colonies appeared on the medium in about 1-5 days at
25.+-.1.degree. C. in dark. The colonies appearing to be Bacillus
were sub-cultured on fresh PDA plants. The cultures were later
purified by a single spore isolation technique and maintained onto
PDA slants at 25.degree. C.
Bacterial Growth Media
[0063] All bacterial growth media were prepared using distilled
water and sterilized by autoclaving prior to use. All bacterial
samples were handled using standard aseptic laboratory techniques
to maintain purity.
[0064] PDA (Potato-Dextrose-Agar): Potato infusion (200 g/l),
dextrose (20 g/l and agar 18 g/l. This medium is available
commercially from Hi-Media Laboratory, Difco Co. Potato Dextrose
Broth (PDB) was made in the same manner except that agar was
omitted.
[0065] NA (Nutrient Agar) medium: 3 g/l beef extract, 5 g/l peptic
digest of animal tissue and 1.5 g/l agar (HI-Media Laboratory
Bombay. India) in distilled water. (pH 6.8).
[0066] Delivery medium: The delivery medium comprising
vermicompost/sawdust was moistened with water and was sterilized by
steam sterilization prior to use. Sterilization was typically
performed by autoclaving twice, each time for 60 minutes.
Harvesting of Bacterial Growth
[0067] Aliquot of 1.5 L Potato-Dextrose Broth (PDB) dispensed 200
ml each in 500 ml Erlenmeyer flasks was inoculated with 100 ml of
stock culture and incubated on shaker with a speed 200 rpm at 25
degree C. for three days. Spores were harvested by centrifugation
at 3000 rpm for 10 minutes. Supernatant then decanted off and the
concentrated spores suspension was washed and used directly to
inoculate delivery medium in the ratio of 1:100. Spores of strain
MTCC-5130 were also produced by growing culture for 7-10 days on
solid medium (for example on PDA) The spores were harvested from
culture in petridishes by scrapping the surface of the agar into
distilled water. The suspension of spores in water was mixed
directly into the delivery medium.
Fungal Pathogens
[0068] The cultures of fungal pathogens were obtained from the
infected tissues of various medicinal and aromatic plants. They
were maintained onto Potato-Dextrose-Agar or Corn meal-Agar slants
under mineral oil at 20.degree. C. in the culture collection of
Department of Microbiology and Plant Pathology, CIMAP, Lucknow,
India. Pathogenicity of each of the cultures was established on
host the under glasshouse conditions.
Example 2
Isolate Characterization and Identification
[0069] The strain was characterized morphologically by Gram
staining; biochemically by indol test, solvent tolerance test and
oxidase test; genetically by randomly amplified polymorphic DNA
analysis and 16S rDNA sequencing. All the test together proved that
the isolate is a new strain of endospore forming Bacillus with
close proximity to Gamma proteobacteria.
[0070] The isolate identified above as having antifungal activity
against wide range of plant pathogenic fungi was further
characterized using conventional methods.
[0071] The Bacillus MTCC-5130 strain of present invention is a Gram
positive, spore-forming, aerobic, flagellate bacterium, which
exhibits potent antifungal properties against a wide range of plant
pathogenic fungi. This strain shows the characteristics of Bacillus
spp nearer to the species Bacillus subtilis.
Morphological Analysis
[0072] Different morphological parameters such as size, shape and
colony characteristics were studied. The bacterial isolate was
found to have spherical colonies about 2-3 mm. in diameter,
flattened and mucoid in texture. These were gram positive rods.
When the staining for endospore formation was carried out the
isolate was found to be forming endospores in the centre of the
cells. This isolate was found to be similar to Bacillus on the
basis of endospore position. [0073] Biochemical analysis: [0074]
Solvent tolerance test:
[0075] The isolate MTCC-5130 showed sensitivity towards chloroform
and acetone and insensitivity towards butanol, a characteristic
observed in gram positive Bacillus subtilis taken as control (Table
1). TABLE-US-00002 TABLE 1 Solvent tolerance test for MTCC-5130 E.
coli (CA Bacillus subtilis New isolate Solvents 8000) (MTCC-121)
MTCC-5130 Acetone + - - Butanol + + + Chloroform - - -
Indole Test:
[0076] The new isolate was tested for indole production along with
E. coli as a positive control and was negative for indole
production.
Oxidase Test Assay:
[0077] The new isolate was analyzed and showed the presence of the
enzyme cytochrome oxidase like Bacillus subtilis as positive
control.
Example 3
16S rDNA Sequence and RAPD Analysis
Isolation of Bacterial Genomic DNA (Mini Prep Method):
[0078] Bacterial cells were grown in NB (5 ml) at 28.degree. C.,
overnight. Culture (1.5 ml.) was centrifuged in microfuge tube at
10,000 rpm for 3 minutes. Pellet was resuspended in 567
.quadrature.1 TE buffer by repeated pipetting. Thirty .quadrature.1
10% SDS and 3 .quadrature.1 of 20 mg/ml Proteinase K, were added,
mixed and incubated for 45-60 min. at 37.degree. C. Hundred
.quadrature.1 of 5 M NaCl was added and mixed thoroughly. Then 80
.quadrature.1 of CTAB/NaCl solution (CTAB 10% NaCl 0.7 M) was
added, mixed and incubated for 10 min. at 65.degree. C. Equal
volume of chloroform: Isoamyl alcohol (24:1) was added, mixed and
centrifuged to 10,000 rpm for 5 min. The supernatant was
transferred to a fresh tube. Equal volume of Phenol: Chloroform:
Isoamyl alcohol (25:24:1 saturated with TE pH 8.0) was added, mixed
and centrifuged for 5 min. The supernatant was transferred to a
fresh tube. Isopropanol (0.6 volume) was added and mixed gently
until DNA precipitated. The precipitate was then washed with 1 ml
of 70% ethanol. After centrifugation for 5 min. at 10,000 rpm
supernatant was discarded and the pellet was dried briefly in a
lyophilizer. The pellet was resuspended in 20 .mu.l of autoclaved
double distilled water and 2 .mu.l was checked on 0.8% agarose gel
for yield and purity.
Partial Sequencing of 16S rDNA
[0079] About 25 ng of genomic DNA was amplified following the
protocol of MicroSeq 500 Kit procured from Perkin Elmer Applied Bio
systems (USA). The amplified product was sequenced using the
forward sequencing reaction mix. The DNA sequence was searched for
homology using BLAST search engine at NCBI site (ncbi.nlm.nih.gov)
and FASTA (ebi.ac.uk). Maximum similarity (E value 2e-11, Score 78)
was with Gamma proteobacterium AKB 16 16S ribosomal RNA gene,
partial sequence (Accession no AY083466.). In the FASTA homology
search most of the hits were for Bacillus spp in addition to Gamma
proteobacteria. The partial rDNA sequencing provided a
characteristic 16S rDNA not having complete homology with any
bacteria of the database but showed similarity towards Gamma
proteobacteria and Bacillus spp. TABLE-US-00003 (SEQ ID NO: 41) 1
TAATGTCGGT GGTGCGTTCA ACATACGTAA GCTAAGTGGA AAAGACGGGA ATGCCGTCTT
TCGACGCCAA GTGGTGGATG GGCGAGCAAT ATGCGGGCAA TTCGTTCGCA AGATCGGGAC
AATCTTGGGA AATTGGGGTC AACATTGGAC GGCCGCCCGA ATTGTACGGC CTAAGATACA
AAAGGCGGTC CTGGTCATTA TCCATAGACG GATTTGTGGT GTACCAGTCA GCCGCCGAGG
CAATGGTCTA TTAAGGTAAA GACGTGCAGT TGATTCGAGA GGGCGACTGG TTATATCGGG
ATCGAGATAA TGTTTAAATC TTCATGGGAG GTAGTAGCAG GGAACTCCTT TTAACCGATT
AAAGCTCCAT TGAGTAATTT TTTTTCAAGC GACCAAGGCC CCTCGCTTTC AAAGTCTTTC
CCCCCCAGGG AAAAATAAAC GGTGCCCCAA AACAAGGGGG GGATTTCCGT A 471.
Randomly Amplified Polymorphic (RAPD) DNA Analysis
[0080] The strain of Bacillus spp MTCC-5130 showing morphological
characteristics nearer to Bacillus subtilis was compared through
RAPD with a strain of Bacillus subtilis (MTCC 121). Polymerase
chain reactions (PCRs) were carried out in 25 .quadrature.1 volume.
A reaction tube contained 25 ng of DNA, 0.2 unit of Taq DNA
polymerase, 100 .quadrature.1 each of dNTPs, 1.5 mM MgCl.sub.2 and
5 p mol of decanucleotide primers. The amplifications were carried
out using a thermal cycler (MJ Research, USA). The amplified
products were loaded in 1.2% agarose gel containing
0.5.quadrature.g ml.sup.-1 of ethidium bromide and photographed by
Polaroid system. The primers used have been listed in Table 2 and
Table 3.
[0081] Following Primers Were used in the Study: TABLE-US-00004
TABLE 2 MAP primers (Synthesized in the laboratory) Primer SEQ ID
S. No. (5 pmole/reaction) Sequence NO: 1. MAP 01 5' GTCCAATGAG 3' 1
2. MAP 02 5' AGGATACGTG 3' 2 3. MAP 03 5' AAATCGGAGC 3' 3 4. MAP 04
5' AAGATAGCGG 3' 4 5. MAP 05 5' GGATCTGAAC 3' 5 6. MAP 06 5'
TTGTCTCAGG 3' 6 7. MAP 07 5' GTCCTACTCG 3' 7 8. MAP 08 5'
GTCCTTAGCG 3' 8 9. MAP 09 5' TGCGCGATCG 3' 9 10. MAP 10 5'
AACGTACGCG 3' 10 11. MAP 11 5' GCACGCCGGA 3' 11 12. MAP 12 5'
CACCCTGCGC 3' 12 13. MAP 13 5' CATCCCGAAC 3' 13 14. MAP 14 5'
GGACTCCACG 3' 14 15. MAP 15 5' AGCCTGACGC 3' 15 16. MAP 16 5'
CTATCGCCGC 3' 16 17. MAP 17 5' CGGGATCCGG 3' 17 18. MAP 18 5'
GCCAATTCCG 3' 18 19. MAP 19 5' CCCTGCAGGC 3' 19 20 MAP 20 5'
CCAAGCTTGC 3' 20
[0082] TABLE-US-00005 TABLE 3 Primer set - OPO (Procured from
Operon Technologies, USA) Primer S. No. (5 pmole/reaction) Sequence
SEQ ID NO: 1 OPO 1 5' GGCACGTAAG 3' 21 2. OPO 2 5' ACGTAGCGTC 3' 22
3. OPO 3 5' CTGTTGCTAC 3' 23 4. OPO 4 5' AAGTCCGCTC 3' 24 5. OPO 5
5' CCCAGTCACT 3' 25 6. OPO 6 5' CCACGGGAAG 3' 26 7. OPO 7 5'
GACCACTGAC 3' 27 8. OPO 8 5' CCTCCAGTGT 3' 28 9. OPO 9 5'
TCCCACGCAA 3' 29 10. OPO 10 5' TCAGAGCGCC 3' 30 11. OPO 11 5'
GAGAGGAGGT 3' 31 12. OPO 12 5' CAGTGCTGTG 3' 32 13. OPO 13 5'
GTCAGAGTCC 3' 33 14. OPO 14 5' AGCAGAGCTC 3' 34 15. OPO 15 5'
TGGCGTCCTT 3' 35 16. OPO 16 5' TCGGCGGTTC 3' 36 17. OPO 17 5'
GGGTTATGCC 3' 37 18. OPO 18 5' CTCGCTATCC 3' 38 19. OPO 19 5'
GGTGCACGTT 3' 39 20. OPO 20 5' ACACACGCTG 3' 40
[0083] Analysis with these primers could show 11.1% similarity with
the tested Bacillus subtilis indicating the possibility of the
strain as a different species of Bacillus and a new species not
having any representation in the public database.
[0084] So the strain was resolved to a species of Bacillus and
named as Bacillus spp MTCC-5130 and referred all over the
specification in this name.
Example 4
Treatment with Bacillus spp MTCC-5130 to Initiate Early Rooting in
Geranium Cuttings:
[0085] Geranium cuttings treated with Bacillus strain MTCC-5130,
initiated rooting after 22-25 days of treatment, which was 5-7 days
earlier than untreated control cuttings. The results were further
compared with IBA treatment and it was confirmed that Bacillus spp
MTCC-5130 treatment gave better results than IBA. Hundred percent
survival was observed when the plants developed from MTCC-5130
treated cuttings were transplanted, while plants of untreated
cuttings showed 5-20% mortality depending on the time or period of
cutting preparation (Table 4). TABLE-US-00006 TABLE 4 Effect of
Bacillus spp MTCC-5130 treatment on root initiation and survivality
of geranium cuttings Treatments Root initiation % survivality &
date (days after) Normal Two node Apical 20-11-01 B1 25 100 70.0
100.0 IBA 27 100 69.2 95.8 Control 32 87.5 60.0 83.3 05-12-01 B1 26
100 100 100.0 IBA 28 100 100 100.0 Control 35 90 90.0 95.8 B1 =
Bacillus spp MTCC-5130, IBA = Indole butyric acid
Example 5
[0086] Effect of Bacillus spp MTCC-5130 on the growth and
productivity of geranium under glasshouse conditions.
[0087] Effect of Bacillus spp MTCC-5130 was tested alone and in
different combinations to test their effectiveness on the plant
growth and productivity of geranium. It produced 298.5 g/pot herb
yield when treated alone. The increase was recorded to be 95% over
untreated control (153.1 g/plant). The double combinations, the
treatment of present strain of invention with G. aggregatum also
performed best (310 g/pot) and increased herb yield by 102.9%. In
combinations of three bio-inoculants, G. aggregatum+Bacillus
spp.+Streptomyces sp. produced 346 g/pot herb yield which was
recorded to be 126.2% more than untreated control. Thus, treatment
of Bacillus spp MTCC-5130 performed effectively with other
bio-inoculants in the improvement of the productivity of geranium
over untreated control.
Example 6
Antifungal Activity Bioassay:
Screening of Various Strains of Bacillus/Pseudomonas to Determine
Potential of Their Antagonistic Activity Against Plant
Pathogens:
[0088] The colonies of bacteria were assayed for antifungal
activity. One such assay, referred to herein as a streak test, was
conducted by first streaking single colonies of bacterial isolates
on PDA. The sample was incubated for about 2-5 days, followed by
addition of a plug of fungal pathogen to the previously incubated
culture, at a specified distance from the bacterial streaks. The
resulting culture was examined for areas in which growth of
pathogen was inhibited. Rhizoctonia solani was used as
representative fungal pathogen for screening antifungal activity of
different isolates of B. spp. Few more fungal pathogens, such as
Fusarium, Curvularia, Alternaria, and Colletotrichum against which
antifungal activity of different bacterial isolates were assessed
further. During screening as described above resulted in the
initial identification of four isolates of Bacillus which are
capable of promoting plant growth and inhibiting mycelial growth of
Rhizoctonia solani. The tests were performed on two different
media, potato dextrose agar (PDA) and nutrient agar (NA). One
isolate designated herein as MTCC-5130 inhibited mycelial growth of
R. solani by at least 50% in comparison to growth of R. solani
under control conditions. In additional screening experiments the
antagonistic effect of bacterial isolates inhibiting the growth of
other plant pathogenic fungi belonging to group of Fusarium,
Helminthosporium, Curvularia, Alternaria, and Colletotrichum
evaluated.
[0089] Bacillus/Pseudomonas strains of different origin were
screened for potential antagonistic activity in vitro by following
common dual culture technique on PDA, where inocula of test
organisms have shown inhibition zone in between colonies of
antagonist and pathogen 6-day after inoculation and percent growth
inhibition was determined. Based on our results Bacillus spp CIMAP
B1 was selected as the most potential strain among the tested
antagonists. It has also been observed that the bacterial strains
produced lytic effect on the mycelia of test plant pathogens (Table
5). TABLE-US-00007 TABLE 5 Screening of different strains of
Bacillus sp. and Pseudomonas sp. showing inhibition zone against
plant pathogenic fungi. Inhibition zone (in mm) against the growth
of Rhizoctonia Antagonist Colletotrichum Fusarium Curvularia
Alternaria solani Strain acutatum oxysporum andropogonis alternata
(Pyre.) Bacillus spp 25 20 32 15 25 CIMAP.B1 Bacillus spp 32 20 --
20 07 CIMAP.B2 Bacillus spp 20 20 30 15 -- CIMAP.B3 Bacillus spp 12
15 05 15 10 CIMAP.B4 Bacillus spp 05 07 -- 11 10 CIMAP.B5
Pseudomonas 30 15 12 15 10 sp.Psf.sub.1 Bacillus spp. CIMAP B1 =
MTCC 5130 [Bacillus spp. CIMAP B1 was deposited at the
International depository, Institute of Microbial Technology
(IMTECH), Chandigarh, India and was given Accession No.
MTCC-5130]
Example 8
Characterization of MTCC-5130 Strain
In vitro Testing of Antagonistic Activity of Strain MTCC-5130
[0090] The antagonistic activity of strain Bacillus spp MTCC-5130
was tested in vitro by following common dual cultures technique on
PDA (Morton D T and N. H. Stroube 1955, Phytopathology 45: 419-420)
where inhibition zones in between the colonies of antagonist and
pathogen was measured 6 days after inoculation and percent growth
inhibition was determined. The newly isolated strain of Bacillus
spp MTCC-5130 was able to inhibit growth of Rhizoctonia solani by
50-55%, Fusarium oxysporum by 73%, Fusarium semitectum by 68%,
Pythium aphanidermatum by 20%, Helminthosporium carbonum by 55%,
Curvularia andropogonis by 70%, Alternaria alternata by 83%,
Colletotrichum acutatum by 76%, Colletotrichum capsici by 65%,
Colletotrichum. gloeosporiodes by 58.00%, Corynespora cassiicola by
48%, and Thielavia basicola by 58% in vitro (Table 6).
TABLE-US-00008 TABLE 6 In vitro growth inhibition of fungal
phytopathogens of some important medicinal & aromatic plants by
the new strain Bacillus spp CIMAP-B1 Inhi- S. bition No (%)
Pathogens Disease Host 1. 55.00 Rhizoctonia solani(PyRh1) Root rot
& Pyrethrum wilt 2. 52.00 Rhizoctonia solani(GRh1) Wilt
Geranium 3. 76.00 Colletotrichum acutatum Anthracnose " 4. 65.00
Colletotrichum capsici Leaf blight Indian basil 5. 58.00
Colletotrichum Leaf spot Aloe gloeosporiodes 6. 50.00 Rhizoctonia
solani(OPRh1) Collar rot Opium poppy 7. 20.00 Pythium
aphanidermatum Yellowing Java citronella 8. 70.00 Curvularia
andropogonis Leaf blight " 9. 83.00 Alternaria alternata Leaf spot
Menthol mint 10. 48.00 Corynespora cassiicola Leaf blight " 73.00
Fusarium oxysporum Wilt " 11. 68.00 Fusarium semitectum " " 12.
55.00 Helminthosporium carbonum Leaf blight " 13. 58.00 Thielavia
basicola Stolon & " root rot
Example 9
[0091] Inhibition of Spore Germination of Fungal Plant Pathogens by
Culture Filtrate of Bacillus spp MTCC-5130
[0092] The 4-day-old-culture filtrate of Bacillus spp MTCC-5130 was
evaluated against spore germination inhibition of fungal plant
pathogens such as Alternaria. alternata, Colletotrichum acutatum
and Colletotrichum capsici. The results indicated that more than
95% spore germination inhibition was recorded at 80% dilution of
culture filtrate. Thus, culture filtrate can be used for the
management of diseases caused by these fungi in future (Table 7).
TABLE-US-00009 TABLE 7 Effect of Bacillus spp CIMAP B1 culture
filtrate on the spore germination of plant pathogenic fungi Spore
germination (%) Inhibition over control after 6 h. (%) CF Conc.
Alternaria Colletotrichum Alternaria Colletotrichum (%) alternata
capsici alternata capsici 0 (control) 100 100 -- -- 10 69 68 31 32
20 59 56 41 44 80 06 08 94 97 80 09 05 91 95
Example 10
In vivo Testing of the Bacillus spp CIMAP B1 on Rhizoctonia solani
Causing Root Rot and Wilt Disease of Pyrethrum.
[0093] The pyrethrum seedlings were treated with the strain of
Bacillus spp MTCC-5130 of the present invention and exposed to the
fungal pathogen, Rhizoctonia solani causing root rot and wilt
disease. Initially they were observed to show growth characteristic
similar to the untreated unexposed control plants. Later, seedlings
treated with Bacillus spp CIMAP-B1 and exposed to the inoculum of
fungal pathogen, Rhizoctonia solani failed to produce typical
symptoms of the disease and also produced plants showing growth
characteristics superior to the untreated, unexposed plants. The
untreated plant inoculated with R. solani produced typical symptoms
of the disease (Table 8). TABLE-US-00010 TABLE 8 Influence of
Bacillus spp MTCC-5130 strain on the root rot & wilt disease of
pyrethrum caused by Rhizoctonia solani: Plants S. Infected Rating
of NO. Treatments (%) DSI* Effectiveness 1 Untreated control 0.0 0
-- 2 Treated control (R. solani) 100 4 -- 3 B1 + R. solani
.sup.21-day-prior 100 4.0 Non-effective .sup.Simultaneous 50 1.6
Effective .sup.5-day-post 50 2.0 Less Effective 4
Ridomil-mancozeb.sup.+ R. solani .sup.21-day-prior 100 4.0
Non-effective .sup.Simultaneous 50 1.5 Effective .sup.5-day-post 70
2.8 Less Effective B1 = Bacillus spp MTCC-5130 strain *Disease
severity index was calculated on 0-4 scales disease rating for
scoring root rot and wilt symptoms under epiphytotic conditions in
the glasshouse wherein 0 = no visible reaction; 1 = infection
restricted up to 1 cm length on the collar region; 2 = infection
more than 1 cm length on the collar region and starts spreading in
the root and shoot; 3 = infection spreads to roots and stem
followed by pronounced rotting symptoms at the collar region # as
well as on the stem leading to premature drying of lower leaves of
infected plants; 4 = pronounced rotting symptoms visible on roots,
collar region and stem leading to premature drying and death of
infected pants. Disease severity index was calculated on 0-4 scales
as described by Trivedi et al. 2001.
[0094] The spray of cell free culture filtrate of Bacillus spp
MTCC-5130 on the foliage of geranium in the commercial fields
significantly reduced by fungal infection caused by Colletotrichum
acutatum. The strain Bacillus spp MTCC-5130 of the present
invention produce vegetative cells or spores for incorporation into
a delivery medium. The composition comprising the vegetative cells
and spores of Bacillus spp MTCC-5130 and the delivery medium has a
long shelf life and is suitable for delivering the antagonist to
plants for effective control of fungal phytopathogens.
ADVANTAGES OF THE INVENTION
[0095] Bacillus spp MTCC-5130 strain is capable of promoting the
growth of geranium and pyrethrum and inhibiting the growth of wide
range of plant pathogenic fungi including Rhizoctonia, Fusarium,
Pythium, Helminthosporium, Curvularia, Alternaria, Colletotrichum,
Corynespora and Thielavia which have been causing different kinds
of diseases on agricultural, horticultural and medicinal and
aromatic crops. A thorough perusal of review of literature reveals
that no such strain of Bacillus spp has been obtained. The Bacillus
spp MTCC-5130 strain has moreover novelty in showing growth
promotion activity on plant as exemplified in geranium and
pyrethrum and growth inhibition of dark spore pathogenic fungi,
which are cosmopolitan in distribution. Therefore, it can be
utilized as plant growth promoter as well as biocontrol agent
against several plant pathogenic fungi of many important crops.
This new strain multiplies on simple delivery medium and is cost
effective and can be exploited commercially. It is non hazardous
and ecofriendly in nature.
[0096] So the present invention pertains to the isolation of a
number of rhizobacteria from the soil and identification of a new
Bacillus spp strain referred to as Bacillus spp MTCC-5130. This
strain is shown to exhibit strong antagonism towards a wide range
of fungal pathogens that cause various kinds of plant diseases such
as damping-off, root rot, stem rot, collar rot, twig blight, leaf
blight and anthracnose as shown in. As such, this B. spp strain is
suitable as biocontrol agent that can be used to protect plants
against infection by these fungal pathogens. Thus, B. spp CIMAP B
sub.1 strain is useful in methods for reducing the susceptibility
of plant to fungi infection. The biologically pure culture of B.
spp MTCC-5130 strain is capable of promoting the growth of plant
and inhibiting the growth of a wide range of plant pathogenic
fungi. This new stain of B. spp., called MTCC-5130 was isolated
from the soil of geranium (Pelargonium graveolens) planted in the
experimental fields of CIMAP, Lucknow, UP, India and is capable of
promoting the growth of plant and inhibiting the growth of a wide
range of plant pathogenic fungi. The invention also provides
information on the characterization of the strain B. spp MTCC-5130
exhibiting unique 16S rDNA sequence. The novel strain of B. spp, B.
spp MTCC-5130 has been found to promote the growth of geranium and
pyrethrum over untreated control. The novel strain of B. spp, B.
spp MTCC-5130, has been found to inhibit growth of Rhizoctonia
solani by at least 50-55%, Fusarium oxysporum by at least 93%,
Fusarium semitectum by at least 88%, Pythium aphanidermatum by at
least 20%, Helminthosporium carbonum by at least 55%, Curvularia
andropogonis by at least 70%, Alternaria alternata by at least 83%,
Colletotrichum acutatum by at least 76%, Colletotrichum capsici by
at least 65%, Colletotrichum gloeosporiodes by at least 58.00%,
Corynespora cassiicola by at least 48%, and Thielavia basicola by
at least 58% in vitro. The present invention also provides a method
for evaluating antifungal activity of B. spp MTCC-5130, in vitro
against wide range of fungal pathogens of medicinal and aromatic
plants.
REFERENCE CITED
[0097] TABLE-US-00011 U.S. Patent Documents 4250170 February, 1981
Kawaguchi et al. 5047239 September, 1991 Pusey and Robins 5049379
September, 1991 Handelsman et al. 5061495 October, 1991 Rossall
5215747 June, 1993 Hairston., et al. 424/93. 5344647 September,
1994 Rossall. 5403583 April, 1995 Liu., et al. 424/93. 5597565
January, 1997 Leifert et al. 5650372 July, 1997 Branly, et al.
5753222 May, 1998 Marrone, et al. 424/93. 5780080 July, 1998
Leifert et al. 504/117. 6004774 December, 1999 Marrone, et al.
435/41. 6245551 March, 1999 Lehman et al. 435/252.5 6291426
September, 2001. Heins, et al. 6524998 February, 2003 Kloepper, et
al
Other References
[0098] 1. Alam et al. (1983), "Leaf blight and leaf spot disease of
Java citronella caused by Curvularia andropogonis" Indian
Phytopath. 36: 480-483 [0099] 2. Alam et al.(1992), "Lethal
yellowing of Java citronella (Cymbopogon winterianus) caused by
Pythium aphanidermatum" Plant Dis 43:10578-1061 [0100] 3. Alam, et
al. (1994), "Collar rot and wilt: a new disease of Java citronella
(Cymbopogon winterianus) caused by Fusarium moniliforme Scheldon"
Plant Pathology 43:1057-1061 [0101] 4. Alam et al. (1996),
"Damping-off, a new disease of opium poppy caused by Pythium
dissotocum" Indian Phytopath. 49:94-97 [0102] 5. Aksaka, O. and
Shoda, M., (1996), "Biocontrol of Rhizoctonia solani damping-off of
tomato with Bacillus subtilis RB14" Appl. Environ. Microbiol.
62:4081-4085 [0103] 6. Baker et al. (1983), "Inhibitory effect of
Bacillus subtilis on Uromyces phaseoli and on development of rust
pustules on beans leaves" Phytopathol. 73:1148-1152 [0104] 7. Bland
et al., (1995), "Iturin-A, an antifungal peptide produced by
Bacillus subtilis" Proc. Plant Growth Regulation Soc. Am.
22.sup.nd:102-107 [0105] 8. Brenner, D. J. (1984),"Bergey's Manual
of Systematic Bacteriology (9th) Edition" (Ed. by Krieg, N. R.
& Holt, J. G.) pp. 408-420. Williams and Wilkins, Baltimore
[0106] 9. Chen et al (2002), "Antagonism to the plant pathogenic
fungi of Bacillus subtilis B-916 and its exudate Chinese J. Biol.
Control 18:45-46 [0107] 10. Cook C. G. et al., (1987), "Effect of
treatment with Bacillus species on cotton rot traits, yield and
Phymatotrichum root rot" Belt wide Cotton Production Research
Conferences, Proceedings Jan. 4-8, 1987, Dallas, Tex., pp. 43-45
[0108] 11. Ferreira et al. (1991), "Biological control of Eutypha
lata on grapevine by an antagonistic strain of Bacillus subtilis"
Phytopathology 81: 283-287 [0109] 12. Glick, B. R., and Bashan, Y.
(1997). Genetic manipulation of plant growth-promoting bacteria to
enhance biocontrol of phytopathogens. Biotechnology Advances 15,
353-378. [0110] 13. Gueldner, R. C. et al., (1988), "Isolation and
Identification of Iturins as Antifungal Peptides in Biological
Control of Peach Brown Rot with Bacillus subtilis," J. Agric. Food
Chem. 36:366-370 [0111] 14. Hassanein and El-Goorani (1992), "The
effect of Bacillus subtilis on in vitro growth and pathogenicity of
Agrobacterium tumefaciens" J. Plant Pathol. 133: 239-246 [0112] 15.
He et al. (1994), "Zwittermycin A. an antifungal and plant
protection agent from Bacillus cereus" Tetrahedron Lett. 35:
2499-2502 [0113] 16. Hiraoka et al., (1992), "Characterization of
Bacillus subtilis RB14, coproducer of peptide antibiotics iturin A
and surfactin" J. Gen. Appl. Microbiol. 38:635-640 [0114] 17. Huang
et.al (1993), "Bacterial suppression of basal pod rot and end rot
of dry peas caused by Sclerotinia sclerotiorum" Can. J.
Microbiol.39: 227-233 [0115] 18. Islam K. Z. and Nandi, B, (1985),
"Control of brown spot of rice by Bacillus megaterium" J. Plant
Dis. Protect. 92: 241-246 [0116] 19. Islam K. Z. and Nandi,
B.(1985), "Inhibition of some fungal pathogens of hose phylloplane
by Bacillus megaterium" J. Plant Dis. Protect. 92: 233-240 [0117]
20. Katz, E. and Demain (1977), "The Peptide Antibiotics of
Bacillus" Bacteriological Reviews, 41: 449-474 [0118] 21. Kloepper,
J. W. 1993. Plant growth-promoting rhizobacteria as biological
control agents. In `Soil Microbial Ecology--Applications in
Agricultural and Environmental Management,` ed. F. B. Metting, Jr.,
pp. 255-274 (Marcel Dekker, Inc., New York). [0119] 22. Korzybski,
T. et al. (1978), "Section C: Antibiotic isolated from the genus
Bacillus (Bacilliaceae)" In: Antibiotics--Origin, nature and
properties, American Society for Microbiology, Washington D.C. vol.
III, pp. 1519-1661 [0120] 23. Leifert et al., (1995), "Antibiotic
production and biocontrol activity by Bacillus subtilis CL 27and
Bacillus pumilus CL45" J. Appl. Bacteriol. 78: 97-108 [0121] 24.
Loeffler, W. et al. (1986), "Antifungal Effects of Bacilysin and
Fengymycin from Bacillus subtilis F-29-3. A Comparison with
Activities of Other Bacillus Antibiotics" J. Phytopathol.
115:204-213 [0122] 25. McKeen et al. (1986), "Production and
partial characterization of antifungal substances antagonistic to
Monilinia fructicola from Bacillus subtilis" Phytopathology
76:136-139 [0123] 26. Milner et al. (1996), "Production of
Kanosamine by Bacillus cereus UW85" Appl. Environ. Microb.
62:3061-3065 [0124] 27. Osburn et al. (1995), "Effect of Bacillus
cereus UW85 on the yield of soybean at two field sites in
Wisconsin" Am. Phytopathol. Soc. 79(6):551-556 [0125] 28. Pusey et
al.(1988), "Pilot tests and commercial production and application
of Bacillus subtilis (B-3) of post harvest control of peach brown
rot" Plant Dis. 72: 622-626 [0126] 29. Raupach et al. (1998),
"Mixtures of Plant Growth-Promoting Rhizobacteria Enhance
Biological Control of Multiple Cucumber Pathogens". Phytopathology.
88:1158-1164 [0127] 30. Schaad, N. W., Ed. (1988), "Laboratory
guide for identification of plant pathogenic bacteria", 2nd Ed.,
APS Press, Minneapolis, Minn., pp. 23, 60-80. [0128] 31. Schroth,
M. N., et al. (1983), "In selections from the prokaryotes, a
handbook on habitats, isolation and identification of bacteria",
(Starr, M. P., Ed.) Springer-Verlag, New York, N.Y. [0129] 32.
Sattar A. and Alam M. (1993), "Sclerotinia collar rot of
Trachyspermum ammi" Indian J. Plant Pathol. 10: 10-11 [0130] 33.
Sattar et al. (1999), "Collar rot: a new disease of opium poppy
caused by Rhizoctonia solani" Indian J. Plant Pathol. 17:74-76
[0131] 34. Sattar et al. (2002), "Anthracnose diseases of geranium
caused by Colletotrichum acutatum in northern Indian plains" J.
Mycol. & Pl. Pathol. 32: 31-34 [0132] 35. Sholberg et al.
(1995), "Biocontrol of post harvest disease of apple using Bacillus
sp. isolated from stored apples" Can. J. Microbiol.41: 247-252.
[0133] 36. Singh, V. and Deverall, B. J.(1984), "Bacillus subtilis
as a control agent against fungal pathogens of citrus fruit" Trans.
Br. Mycol. Soc. 83: 487-490. [0134] 37. Stabb et al. (1994),
"Zwittermycin A-producing strains of Bacillus cereus from diverse
soils" Appl. Environ. Microbiol. 60:4404-4412. [0135] 38. Tsuge et
al., (1995),"Characterization of Bacillus subtilis YB8, co producer
of lipopeptide surfactin and plipastatin B1" J. Gen. Appl.
Microbiol. 41:541-545. [0136] 39. Schwinn et al., (1991) "Control
with Chemicals" Advances in Plant Pathology: vol. 7: Phytophthora
infestans, the Cause of Late Blight of Potato, Ingram et al., eds.,
Academic Press, San Diego. 8:255-266 [0137] 40. Swinburne et al.
(1975), "Production of antibiotics by Bacillus subtilis and their
effect on fungal colonists of apple leaf scars" Trans. Brit. Mycol.
Soc. 65:211-217. [0138] 41. United States Environmental Protection
Agency (EPA), (1992) "Pesticide Fact Sheet--Bacillus subtilis
GB03". [0139] 42. Utkhede et al (1986), "In vitro Inhibition of
plant pathogens . . . ". Can. J. Microbiol. 32: 963-967 [0140] 43.
Wilson, et al. (1989), "Biological Control of Post harvest
Diseases" Annual Review of Phytopathology 27: 425-441 [0141] 44.
Wollum, A. G., (1982) "Cultural Methods for Soil Microorganisms,"
in Methods of Soil Analysis part 2, Second Edition, American
Society of Agronomy/Soil Science Society of America, Madison, Wis.,
pp. 785 [0142] 45. Yamada et al., (1990), "Biological activity of
antifungal substances produced by Bacillus subtilis" J. Pesticide
Sci. 15:95-96
Sequence CWU 1
1
41 1 10 DNA Artificial Sequence Primer MAP 01 1 gtccaatgag 10 2 10
DNA Artificial Sequence Primer MAP 02 2 aggatacgtg 10 3 10 DNA
Artificial Sequence Primer MAP 03 3 aaatcggagc 10 4 10 DNA
Artificial Sequence Primer MAP 04 4 aagatagcgg 10 5 10 DNA
Artificial Sequence Primer MAP 05 5 ggatctgaac 10 6 10 DNA
Artificial Sequence Primer MAP 06 6 ttgtctcagg 10 7 10 DNA
Artificial Sequence Primer MAP 07 7 gtcctactcg 10 8 10 DNA
Artificial Sequence Primer MAP 08 8 gtccttagcg 10 9 10 DNA
Artificial Sequence Primer MAP 09 9 tgcgcgatcg 10 10 10 DNA
Artificial Sequence Primer MAP 10 10 aacgtacgcg 10 11 10 DNA
Artificial Sequence Primer MAP 11 11 gcacgccgga 10 12 10 DNA
Artificial Sequence Primer MAP 12 12 caccctgcgc 10 13 10 DNA
Artificial Sequence Primer MAP 13 13 catcccgaac 10 14 10 DNA
Artificial Sequence Primer MAP 14 14 ggactccacg 10 15 10 DNA
Artificial Sequence Primer MAP 15 15 agcctgacgc 10 16 10 DNA
Artificial Sequence Primer MAP 16 16 ctatcgccgc 10 17 10 DNA
Artificial Sequence Primer MAP 17 17 cgggatccgg 10 18 10 DNA
Artificial Sequence Primer MAP 18 18 gccaattccg 10 19 10 DNA
Artificial Sequence Primer MAP 19 19 ccctgcaggc 10 20 10 DNA
Artificial Sequence Primer MAP 20 20 ccaagcttgc 10 21 10 DNA
Artificial Sequence OPO 1 21 ggcacgtaag 10 22 10 DNA Artificial
Sequence OPO 2 22 acgtagcgtc 10 23 10 DNA Artificial Sequence OPO 3
23 ctgttgctac 10 24 10 DNA Artificial Sequence OPO 4 24 aagtccgctc
10 25 10 DNA Artificial Sequence OPO 5 25 cccagtcact 10 26 10 DNA
Artificial Sequence OPO 6 26 ccacgggaag 10 27 10 DNA Artificial
Sequence OPO 7 27 gaccactgac 10 28 10 DNA Artificial Sequence OPO 8
28 cctccagtgt 10 29 10 DNA Artificial Sequence OPO 9 29 tcccacgcaa
10 30 10 DNA Artificial Sequence OPO 10 30 tcagagcgcc 10 31 10 DNA
Artificial Sequence OPO 11 31 gagaggaggt 10 32 10 DNA Artificial
Sequence OPO 12 32 cagtgctgtg 10 33 10 DNA Artificial Sequence OPO
13 33 gtcagagtcc 10 34 10 DNA Artificial Sequence OPO 14 34
agcagagctc 10 35 10 DNA Artificial Sequence OPO 15 35 tggcgtcctt 10
36 10 DNA Artificial Sequence OPO 16 36 tcggcggttc 10 37 10 DNA
Artificial Sequence OPO 17 37 gggttatgcc 10 38 10 DNA Artificial
Sequence OPO 18 38 ctcgctatcc 10 39 10 DNA Artificial Sequence OPO
19 39 ggtgcacgtt 10 40 10 DNA Artificial Sequence OPO 20 40
acacacgctg 10 41 471 DNA Bacillus sp. 41 taatgtcggt ggtgcgttca
acatacgtaa gctaagtgga aaagacggga atgccgtctt 60 tcgacgccaa
gtggtggatg ggcgagcaat atgcgggcaa ttcgttcgca agatcgggac 120
aatcttggga aattggggtc aacattggac ggccgcccga attgtacggc ctaagataca
180 aaaggcggtc ctggtcatta tccatagacg gatttgtggt gtaccagtca
gccgccgagg 240 caatggtcta ttaaggtaaa gacgtgcagt tgattcgaga
gggcgactgg ttatatcggg 300 atcgagataa tgtttaaatc ttcatgggag
gtagtagcag ggaactcctt ttaaccgatt 360 aaagctccat tgagtaattt
tttttcaagc gaccaaggcc cctcgctttc aaagtctttc 420 ccccccaggg
aaaaataaac ggtgccccaa aacaaggggg ggatttccgt a 471
* * * * *