Fusarial wilt control and growth promotion of pigeon pea through bioactive metabolites produced by two plant growth promoting rhizobacteria

The bioactive metabolites produced by two plant growth promoting rhizobacteria strains, a Pseudomonas aeruginosa strain RRLJ 04 and a Bacillus cereus strain BS 03, which showed growth promotion and disease control in pigeon pea against Fusarium udum, were isolated and screened for their efficacy to control fusarial wilt of pigeon pea under gnotobiotic and nursery condition. Bioactive metabolites viz., BM 1 and BM 2 from RRLJ 04 and BM 3 from BS 03 also showed in vitro antibiosis against F. udum. Seeds treated with 50 μl seed^?1 of BM 1, 30 μl seed^?1 of BM 2 and 70 μl seed^?1 of BM 3 and grown in pathogen infested soil showed suppression of wilt disease besides growth enhancement. Per cent disease control was 90 % with BM 2 application as compared to 87 and 83 %, respectively in BM 1 and BM 3 after 90 days of growth. BM 2 treated plants were more resistant to the pathogen as compared to the other fractions tested. Mycelial dry weight was found to be reduced on treatment with the bioactive metabolites. Formation of chlamydospore-like structures was observed in the pathogen mycelium treated with BM 3. The analytical studies confirmed that two of these metabolites are phenazine derivatives.     

南极沉积物来源抗菌细菌的筛选及抑菌物质的鉴定

【背景】科学研究表明,由于南极环境条件特殊,微生物资源丰富,有望筛选出功效显著的抗菌微生物。【目的】以黄瓜枯萎病致病菌木贼镰刀菌(Fusarium equiseti)为指示菌,从南极沉积物中分离筛选具有拮抗作用的细菌菌株并对其抑菌物质进行初步鉴定。【方法】应用平板对峙法和琼脂扩散法分别对样品和发酵液进行初筛和复筛,筛选出对F. equiseti抑菌效果最强的菌株,基于形态学、生理生化、分子生物学分析,对该菌株进行鉴定。之后,对目标菌株发酵上清中的抑菌物质进行抑菌谱研究,并对其抑菌成分进行温度和pH的稳定性检测,通过硫酸铵沉淀的方法初步鉴定发酵液中的抑菌物质。【结果】从南极沉积物样品中共分离纯化出62株细菌,有5株具有较好的抑菌效果,其中抑菌效果最强的菌株鉴定为枯草芽孢杆菌斯氏亚种(Bacillus subtilis subsp.spizizenii),命名为JYM35。抑菌谱检测结果显示,菌株JYM35对丝瓜枯萎病致病菌层生镰刀菌(Fusarium proliferatum)和辣椒枯萎病致病菌F. equiseti具有较强的抑菌效果,对长豆褐腐病致病菌笄霉属(Choanephora)有较明显的拮抗作用,对水产致病菌副溶血弧菌(Vibrio parahaemolyticus)和溶藻弧菌(V.alginolyticus)也有一定的拮抗作用。菌株JYM35发酵上清中所含的抑菌物质热稳定性强且耐碱不耐酸,硫酸铵沉淀后可初步确定其抑菌物质隶属蛋白类。【结论】菌株JYM35是一株产蛋白类活性物质的广谱型抑菌菌株,对枯萎病致病菌的拮抗作用最强。因此具有较好的开发利用价值。     

Two rhizobacterial strains,individually and in interactions with Rhizobium sp., enhance fusarial wilt control,growth, and yield in pigeon pea

A Pseudomonas aeruginosa strain, RRLJ 04, and a Bacillus cereus strain, BS 03, were tested both individually and in combination with a Rhizobium strain, RH 2, for their ability to enhance plant growth and nodulation in pigeon pea (Cajanus cajan L.) under gnotobiotic, greenhouse and field conditions. Both of the rhizobacterial strains exhibited a positive effect on growth in terms of shoot height, root length, fresh and dry weight, nodulation and yield over the non-treated control. Co-inoculation of seeds with these strains and Rhizobium RH 2 also reduced the number of wilted plants, when grown in soil infested with Fusarium udum. Gnotobiotic studies confirmed that the suppression of wilt disease was due to the presence of the respective PGPR strains. Seed bacterization with drug-marked mutants of RRLJ 04 and BS 03 confirmed their ability to colonize and multiply along the roots. The results suggest that co-inoculation of these strains with Rhizobium strain RH 2 can be further exploited for enhanced growth, nodulation and yield in addition to control of fusarial wilt in pigeon pea.     

Chitin-supplemented formulations improve biocontrol and plant growth promoting efficiency of Bacillus subtilis AF 1

Formulations of a chitinolytic biocontrol and a plant growth promoting Bacillus subtilis AF 1 were prepared in peat, in peat supplemented with either 0.5% chitin or Aspergillus niger mycelium, or in spent compost obtained from Agaricus bisporus cultivation and were evaluated for biocontrol of two fungal pathogens and plant growth promoting activities on pigeon pea and groundnut. A steady increase in cell numbers of introduced B. subtilis AF 1 was observed in all the formulations at 30 degrees C. The increase in cell numbers was about 5.0 log units. Peat or spent compost inoculated with physiologically active and dormant states of B. subtilis AF 1 showed different time period requirements to attain maximum cell numbers. The presence of chitin or A. niger (in peat) or A. bisporus (in spent compost) as supplement in the carrier material improved the multiplication of B. subtilis AF 1. When used as seed treatments, formulations of AF 1 in peat supplemented with chitin or chitin-containing materials showed better control of A. niger (causing crown rot of groundnut) and Fusarium udum (causing wilt of pigeon pea) than AF 1 culture alone, in both groundnut and pigeon pea. Bacillus subtilis AF 1 formulations promoted seed germination and biomass of both groundnut and pigeon pea even under pathogen pressure. Survival of AF 1 on fresh culture-treated and formulation product-treated plants was similar in pathogen-infested soil.     

Biological activity of phenylpropionic acid isolated from a terrestrial Streptomycetes.

The strain ANU 6277 was isolated from laterite soil and identified as Streptomyces sp. closely related to Streptomyces albidoflavus cluster by 16S rRNA analysis. The cultural, morphological and physiological characters of the strain were recorded. The strain exhibited resistance to chloramphenicol, penicillin and streptomycin. It had the ability to produce enzymes such as amylase and chitinase. A bioactive compound was isolated from the strain at stationary phase of culture and identified as 3-phenylpropionic acid (3-PPA) by FT-IR, EI-MS, 1H NMR and 13C NMR spectral studies. It exhibited antimicrobial activity against different bacteria like Bacillus cereus, B. subtilis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, P. flourescens, Staphylococcus aureus and some fungi including Aspergillus flavus, A. niger, Candida albicans, Fusarium oxysporum, F. udum and Penicillium citrinum. The antifungal activity of 3-PPA of the strain was evaluated in in vivo and in vitro conditions against Fusarium udum causing wilt disease in pigeon pea. The compound 3-PPA is an effective antifungal agent when compared to tricyclozole (fungicide) to control wilt caused by F. udum, but it exhibited less antifungal activity than carbendazim.     

苏云金芽胞杆菌抗菌脂肽初步鉴定及抑菌活性测定

植物枯萎病是影响作物生长的重要因素，利用植物内生菌拮抗病原菌生长，从而降低其危害程度是目前研究的热点。本研究从健康的番茄植株中筛选分离得到一株对番茄枯萎病病原菌有较强拮抗作用的内生细菌B-R1,通过形态学、生理生化以及分子生物学检测分析，鉴定该菌株为苏云金芽胞杆菌(Bacillus thuringiensis)。为进一步探索该菌株的生防作用，首先将菌株发酵后离心，得到发酵上清液，采用盐酸沉淀法对其活性物质进行粗提，并检测其对大肠埃希菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)以及尖孢镰刀菌(Fusarium oxysporum)的抑菌活性。通过薄层色谱法(TLC)、傅里叶红外光谱(FI-TR)、高分辨液相色谱-质谱联用仪(LC-MS)分析并鉴定活性物质的结构。结果表明，苏云金芽胞杆菌对尖胞镰刀菌有良好的抑制作用，结构分析初步鉴定抗菌物质中含有丰原素(fengycin),属于脂肽类抗生素。     

A stable plasmid vector and control of its copy number in Bacillus brevis 47, a protein-producing bacterium.

A low-copy-number plasmid vector, pHY481, was constructed by combining a macrolide resistance gene of a Staphylococcus aureus plasmid with a cryptic plasmid found in a Bacillus brevis strain isolated from soil. The plasmid introduced into B. brevis 47, an extensively investigated protein-producing bacterium, was maintained very stably in the absence of selective antibiotics. A Bacillus megaterium alpha-amylase gene subcloned into pHY481 was retained much more stably in B. brevis 47 than one subcloned into a plasmid of S. aureus origin. B. brevis 47 mutants were also isolated in which the copy number of pHY481 was amplified about 10-fold. The copy number of pHY481 with the inserted amylase gene also increased in the mutants. As a result, a severalfold-higher amount of the enzyme was produced in the mutants compared with that produced in wild-type B. brevis 47. Thus, the plasmid vector constructed here and the copy-number mutants of B. brevis 47 are useful for cloning foreign genes and performing genetic engineering in the protein-producing bacterium.     

A stable plasmid vector and control of its copy number in Bacillus brevis 47, a protein-producing bacterium

A low-copy-number plasmid vector, pHY481, was constructed by combining a macrolide resistance gene of a Staphylococcus aureus plasmid with a cryptic plasmid found in a Bacillus brevis strain isolated from soil. The plasmid introduced into B. brevis 47, an extensively investigated protein-producing bacterium, was maintained very stably in the absence of selective antibiotics. A Bacillus megaterium alpha-amylase gene subcloned into pHY481 was retained much more stably in B. brevis 47 than one subcloned into a plasmid of S. aureus origin. B. brevis 47 mutants were also isolated in which the copy number of pHY481 was amplified about 10-fold. The copy number of pHY481 with the inserted amylase gene also increased in the mutants. As a result, a severalfold-higher amount of the enzyme was produced in the mutants compared with that produced in wild-type B. brevis 47. Thus, the plasmid vector constructed here and the copy-number mutants of B. brevis 47 are useful for cloning foreign genes and performing genetic engineering in the protein-producing bacterium.     

向日葵枯萎病拮抗芽胞杆菌的筛选与生防评价

目的筛选向日葵枯萎病拮抗芽胞杆菌菌株并研究其抗菌谱,探讨环境条件对菌株抑菌活性的影响并通过植物栽培完成生防评价。方法从向日葵根际土壤中选择性分离芽胞杆菌,通过5点对峙法确定1株高效拮抗菌株,进行鉴定后,测定其抑菌谱;单因素实验探讨环境条件对抑菌活性的影响;向日葵发芽实验完成生防评价。结果确定1株高效的枯萎病拮抗菌株WBFL-1,经鉴定为枯草芽胞杆菌,该菌对镰刀菌属具有广谱抗性,其抑菌活性的最佳条件是温度40℃,pH值7.0,接种量150μL,发酵时间48 h。生防评价表明该菌对枯萎病的拮抗效果显著。结论该菌可为农作物枯萎病的生物防治提供有效菌种储备。     

Production, purification, and characterization of antifungal metabolite from Pseudomonas aeruginosa SD12, a new strain obtained from tannery waste polluted soil

A new strain, SD12, was isolated from tannery waste polluted soil and identified as Pseudomonas aeruginosa on the basis of phenotypic traits and by comparison of 16S rRNA sequences. This bacterium exhibited broad-spectrum antagonistic activity against phytopathogenic fungi. The strain produced phosphatases, cellulases, proteases, pectinases, and HCN and also retained its ability to produce hydroxamate-type siderophore. A bioactive metabolite was isolated from P. aeruginosa SD12 and was characterized as 1-hydroxyphenazine ((1-OH-PHZ) by nuclear magnetic resonance (NMR) spectral analysis. The strain was used as a biocontrol agent against root rot and wilt disease of pyrethrum caused by Rhizoctonia solani. The stain is also reported to increase the growth and biomass of Plantago ovata. The purified compound, 1-hydroxyphenazine, also showed broad-spectrum antagonistic activity towards a range of phytopathogenic fungi, which is the first report of its kind.     

烟草青枯病劳尔氏菌与拮抗菌对根系分泌物的竞争作用

[目的]研究青枯病病原菌与拮抗菌的营养特性及其对烟草根系分泌物的响应差异,对提高拮抗菌定殖能力、有效生物防控烟草青枯病具有非常重要的意义。[方法]本研究通过筛选与鉴定贵州烟区青枯病病原菌株及拮抗菌株,通过Biolog表型芯片技术分别检测病原菌与拮抗菌的特征性碳、氮源,利用气质联用(GC-MS)检测烟草主栽品种K326根系分泌物的主要物质,在此基础上进行病原菌与拮抗菌对其利用能力、利用强度以及共培养的研究。[结果]经鉴定,分离、筛选到的病原菌株和拮抗菌株分别为青枯劳尔氏菌(Ralstonia solawacearum)和枯草芽孢杆菌(Bacillus subtilis);在含量为0.01μg/mL以上的根系分泌物中,12种物质的含量从高到低排序为:果胶>葡萄糖>木糖>阿拉伯糖>半乳糖>核糖>蔗糖>苯甲酸>果糖=D-甘露醇>棕榈酸>富马酸,果胶含量最高且明显高于其他物质;拮抗菌(LX4)对碳源利用能力高于病原菌(Rs)的碳源有阿拉伯糖、木糖和核糖,分别是病原菌利用能力的1.22、1.95和2.17倍;前12 h拮抗菌利用果糖强度高于病原菌,不同碳源共培养24 h后LX4对gfp-Rs(绿色荧光蛋白标记后的青枯病病原菌)抑制率为18.34%(阿拉伯糖)、53.23%(木糖)、63.53%(核糖)和52.09%(果糖)。[结论]拮抗菌对烟草根系分泌物的利用不及病原菌,但在特定碳源条件下拮抗菌能够利用根系分泌物中的某些碳源产生某种拮抗物质抑制病原菌,拮抗菌与病原菌之间同时存在利用性竞争和干扰性竞争关系,研究结果为进一步研究烟草青枯病的生物防控提供了新的理论依据。     

Biochemical and molecular characterization of a rhizobitoxine-producing <Emphasis Type="Italic">Bradyrhizobium</Emphasis> from pigeon pea plants

Out of a total of 8 bacterial strains isolated from the root nodules of pigeon pea plants grown in arid region, five were identified as rhizobia based on biochemical test and confirmed by 16S rDNA sequencing. PCR based screening for the rtxA gene (involved in biosynthesis of rhizobitoxine) revealed that the gene was present in one strain identified biochemically and genetically as belonging to species Bradyrhizobium (BS KT-24). The strain was resistant to phosphomycin, nalidixic acid, kanamycin, gentamicin and neomycin but sensitive towards streptomycin and spectinomycin. Bioinformatic-tool-guided phylogenetic analysis of rtxA gene revealed its distinctiveness from other known rtxA genes (present in B. japonicum, B. elkanii and Xanthomonas oryzae). The rhizobitoxine producing strain BS KT-24 is considered to exhibit better survival and nodulation protection besides competitiveness for pigeon pea and other legumes grown under abiotic stress and, thus, be a candidate in practical aspect of rhizobitoxine production by rhizobium and its application as rhizobial inoculants.     

铜绿假单胞菌XQ17提取物对革兰阳性菌的拮抗作用

从临床分离的1株铜绿假单胞菌能够产生拮抗物质,对其粗提物的初步研究表明,能有效杀灭受试的多数革兰阳性菌。通过比较其基本特征,与已知的铜绿假单胞菌产生的其他抗菌物质如Pyocyanin、Phenazine-1-carboxamide、Pseudomonic acid、pyocin等有明显区别,提示可能是一种具有潜在应用价值的新抗菌物质。     

Characterization of several bovine rumen bacteria isolated with a xylan medium

Dehority, B. A. (Ohio Agricultural Research and Development Center, Wooster). Characterization of several bovine rumen bacteria isolated with a xylan medium. J. Bacteriol. 91:1724-1729. 1966.-Studies were conducted to characterize eight strains of bacteria isolated from bovine rumen contents, by use of a medium containing xylan as the only added carbohydrate source. Based on morphology, biochemical reactions, nutritional requirements, and fermentation products, five of the eight strains were identified as Butyrivibrio fibrisolvens. Many properties of the remaining three strains resembled Bacteroides ruminicola; however, propionic acid was consistently found as a fermentation product. When the type strains for B. ruminicola subsp. ruminicola and B. ruminicola subsp. brevis were compared with the present isolates, it was found that propionic acid was a normal fermentation product for the type strain B. ruminicola subsp. ruminicola when grown in a 40% rumen fluid-0.5% glucose broth. Production of propionic acid was markedly reduced for all strains when grown in a 20% rumen fluid-1% glucose broth. The three remaining strains were thus placed in the species B. ruminicola, and further classified into the subspecies ruminicola (one strain) and brevis (two strains) on the basis of their requirement for hemin. Although the type strain of B. ruminicola subsp. brevis did not produce propionic acid, both of the present isolates classified as this subspecies produced substantial amounts. One strain of B. ruminicola subsp. brevis had an absolute requirement for volatile fatty acids. Either isobutyric or dl-2-methylbutyric acid would satisfy this requirement, whereas isovaleric acid was ineffective. It is of interest that xylan-fermenting bacteria isolated from 10(-7) and 10(-8) dilutions of rumen contents by use of a xylan medium are similar to the xylan fermenters isolated at the same dilutions with a nonselective medium.     

Bacterial antagonists of Sunflower (Helianthus annuus L.) fungal pathogens

Bacteria isolated on nutrient agar and King's medium B from sunflower leaves, crown and roots inhibited in vitro growth of the leaf spot and wilt pathogens Alternaria helianthi, and Sclerotium rolfsii, respectively, and also the root rot pathogensRhizoctonia solani and Macrophomina phaseolina. Antagonistic bacteria from leaves were mainly actinomycetes and pigmented Gram-positive bacteria, while those from roots and crowns were identified asPseudomonas fluorescens-putida, P. maltophilia, P. cepacia, Flavobacterium odoratum andBacillus sp. In soil bioassays, when used as seed inoculum in the presence ofS. rolfsii, P. cepacia strain N24 increased significantly the percentage of seedling emergence. Bacterial strains which exhibited broad spectrum in vitro antagonistic activity were tested for colonisation of sunflower roots, when used as a seed inoculum. Good colonisers (10⁴ to 10⁶ bacteria/g root) were consistent in their ability to reduce disease and fungal wilt. A seedling having a primary root length < 5 cm with fewer lateral roots, necrosed cotyledons or crown and a wilted shoot indicated its diseased status. On an average, only 30% of seedlings were diseased when treated with the antagonistic strains, in the presence of the pathogen, while 60% of the seedlings were diseased in the presence of the pathogen alone. In microplots treated with strain N24, only 1 to 3% of the seedlings were wilted, while 14% of the seedlings were wilted in the presence of the pathogen alone. The results obtained show that bacterial antagonists of sclerotial fungi can be used as seed inocula to improve plant growth through disease suppression     

Response of endophytic bacterial communities in banana tissue culture plantlets to Fusarium wilt pathogen infection

Endophytic bacteria reside within plant hosts without having pathogenic effects, and various endophytes have been found to functionally benefit plant disease suppressive ability. In this study, the influence of banana plant stress on the endophytic bacterial communities, which was achieved by infection with the wilt pathogen Fusarium oxysporum f. sp. cubense, was examined by cultivation-independent denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA directly amplified from plant tissue DNA. Community analysis clearly demonstrated increased bacterial diversity in pathogen-infected plantlets compared to that in control plantlets. By sequencing, bands most similar to species of Bacillus and Pseudomonas showed high density in the pathogen-treated pattern. In vitro screening of the isolates for antagonistic activity against Fusarium wilt pathogen acquired three strains of endophytic bacteria which were found to match those species that obviously increased in the pathogen infection process; moreover, the most inhibitive strain could also interiorly colonize plantlets and perform antagonism. The evidence obtained from this work showed that antagonistic endophytic bacteria could be induced by the appearance of a host fungal pathogen and further be an ideal biological control agent to use in banana Fusarium wilt disease protection.     

Effect of cultival practices and soil treatments on incidence of wilt disease of pigeon pea

Summary The incidence of wilt disease of pigeon pea (Cajanus cajan (L.) Millsp.) caused byFusarium udum under soil treatments with various substances was studied under field conditions. The disease incidence was highly suppressed under mixed cropping withCrotalaria medicaginea. Phygon XL was found to be the most effective fungicide to reduce the incidence of the disease. The incidence of wilt disease also decreased in case of soil amended with the leaves ofC. medicaginea. Wilt incidence increased in the soil amended with the roots of pigeon pea.     

Extracellular Chitinases of Fluorescent Pseudomonads Antifungal to Fusarium oxysporum f. sp. dianthi Causing Carnation Wilt

Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.     

百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究

【目的】筛选对百合枯萎病具有抑菌活性的拮抗细菌,对其抑菌活性物质进行初步分离纯化分析。【方法】以强致病力的百合尖孢镰刀菌(Fusarium oxysporum)为靶菌,采用系列稀释法和平板对峙法初筛拮抗细菌,并通过产铁载体能力、水解酶活性、土壤定殖力等多种生防特性指标进行复筛,结合形态学特征、生理生化指标和16S rRNA基因序列比对鉴定其分类地位;利用百合尖孢镰刀菌作为靶菌进行活性追踪,结合酸沉淀、快速柱色谱、HPLC等分离纯化手段,对菌发酵液中的抑菌活性物质进行纯化分析。【结果】在64株百合根际细菌和386株海洋细菌中进行初筛,得到9株对百合镰刀菌具有较强拮抗活性的菌株,最后筛选了1株拮抗活性较强且产铁载体能力和水解酶活性、土壤定殖能力较高的菌株11B91,鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其抑菌活性物质初步推测可能为iturin和fengycin脂肽类化合物。【结论】菌株11B91在百合枯萎病的生物防治中具有潜在的应用价值,证实海洋来源的微生物也具有防治陆地植物病原菌的潜力,为植物病害的防治拓宽了思路。     

Characterization of five fungal endophytes producing Cajaninstilbene acid isolated from pigeon pea [Cajanus cajan (L.) Millsp

Five fungal endophytes (K4, K5, K6, K9, K14) producing Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid) were isolated from the roots of pigeon pea [Cajanus cajan (L.) Millsp.]. CSA is responsible for the prominent pharmacological activities in pigeon pea. The amount of CSA in culture solution varied among the five fungal endophytes. K4 produced the highest levels of CSA (1037.13 μg/L) among the endophytes tested after incubation for five days. Both morphological characteristics and molecular methods were used for species identification of fungal endophytes. The five endophytic isolates were characterized by analyzing the internal transcribed spacer (ITS) rRNA and β-tubulin genes. The K4, K5, K9 and K14 strains isolated from pigeon pea roots were found to be closely related to the species Fusarium oxysporum. K6 was identified as Neonectria macrodidym. The present study is the first report on the isolation and identification of fungal endophytes producing CSA in pigeon pea. The study also provides a scientific base for large scale production of CSA.