Increase in Seedling Emergence and Dry Weight of Pigeon Pea in the Field with Chitin-supplemented Formulations of Bacillus subtilis AF 1

Summary Different formulations of a chitinolytic plant growth-promoting rhizobacterium, Bacillus subtilis AF 1, were evaluated for growth promotion of pigeon pea in the field. Alginate, peat, chitin/Aspergillus niger mycelium-supplemented peat, and spent compost formulations of B. subtilis AF 1 effectively increased the seedling emergence, plant height and dry weight in the field, with chitin-supplemented peat formulation being the most effective in two different cropping seasons viz., ‘kharif’ and ‘rabi’. As a seed treatment, chitin-supplemented peat formulation of B. subtilis AF 1 increased the emergence and dry weight of pigeon pea seedlings by 29 and 33%, in comparison to an increase of 21 and 30%, respectively by mid-exponential phase cells of B. subtilis AF 1. Colonization of the root system was assessed by the recovery of introduced ampicillin-resistant and chitin-degrading B. subtilis AF 1, on media containing ampicillin and colloidal chitin, from the rhizoplane during the cropping period.     

Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists

AIMS: Five bacterial strains belonging to Bacillus subtilis, Pseudomonas fluorescens and Ps. corrugata and two fungal strains belonging to Trichoderma viride and Gliocladium virens were evaluated for their efficacy in controlling sugar beet and cucumber damping-off caused by Pythium ultimum. METHODS AND RESULTS: The in vitro antagonistic activity of bacteria against various Pythium spp. was evaluated with dual cultures in various media. Pseudomonas strains inhibited the pathogen better than Bacillus strains. To identify potentially useful antagonist combinations, dual compatibility of antagonists was also evaluated, based on growth in two liquid media containing substrate previously used by other antagonists. Four pairs of bacteria were selected. Sugar beet damping-off biocontrol was attempted with bacterial seed treatments (individually and in pairs). Cucumber damping-off biocontrol was attempted with bacterial seed treatments and bacterial and fungal compost treatments. In sugar beet, satisfactory biocontrol was only achieved with Pseudomonas antagonists. Antagonist combinations did not show any superior biocontrol ability to individual antagonists and compatibility of bacteria in vitro did not correlate with compatibility in vivo. Bacterial seed treatments and fungal compost treatments failed to control cucumber damping-off. Better biocontrol in cucumber was achieved when bacterial antagonists were applied by drenching or by coating seed with bacteria in a peat carrier. CONCLUSIONS: Pseudomonas antagonists were superior to Bacillus antagonists in controlling damping-off in cucumber and sugar beet. Pseudomonas peat inocula maintained a good shelf-life 2 years after preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas peat formulations have the potential for development into commercial biopesticides.     

Production of fungal cell wall degrading enzymes by a biocontrol strain of Bacillus subtilis AF 1

Fungal cell wall degrading chitinases and glucanases attained significance in agriculture, medicine, and environment management. The present study was conducted to describe the optimum conditions required for the production of beta-1,4-N-acetyl glucosaminidase (NAGase) and beta-1,3-glucanase by a biocontrol strain of Bacillus subtilis AF 1. B. subtilis AF 1 was grown in minimal medium with colloidal chitin (3.0%) and yeast extract (0.3% YE ) and incubated at pH 7.0 and 30 degrees C on constant shaker at 180 rpm for 6 days produced highest amounts of NAGase. Presence of 0.5 mM of phenyl methyl sulfonyl fluoride (PMSF) and 0.04% of Tween 20 further improved the enzyme production. B. subtilis AF 1 grown in minimal medium with laminarin (1%) and yeast extract (0.3%) for 3 days produced maximum amount of beta-1,3-glucanase. These conditions can be further scaled-up for large-scale production of NAGase and beta-1,3-glucanase by B. subtilis AF 1.     

Production and detection of muramidase and acetylglucosaminidase from Agaricus bisporus

The production and regulation of extracellular bacteriolytic enzymes of Agaricus bisporus are being studied to understand better the nutrition of this fungus and to identify factors that regulate the selectivity of mushroom compost as a growth medium. Both muramidase (EC.3.2.1.17) and N -acetyl-β- D -glucosaminidase (β-GlcNAcase, EC.3.2.1.30) have been detected in liquid cultures of A. bisporus , and in cultures fruiting in sterile and non-sterile compost. A turbidometric assay, based on the decrease in optical density of suspended Bacillus subtilis bacterial cell walls, was used to measure muramidase production by A. bisporus . A colorimetric assay was used to measure β-GlcNAcase. Both bacteriolytic enzyme activities were produced on a range of sole carbon sources, including killed freeze-dried B. subtilis cells. Muramidase activity was highest in axenic compost cultures. Bacteriolytic enzyme activity peaked as the first group of fruit bodies was harvested in both sterile and non-sterile compost.     

Survival of Bacillus subtilis AF 1 in the bacterized peanut rhizosphere and its influence on native microflora and seedling growth

A mutant of Bacillus subtilis AF 1 (an antifungal strain with plant growth-promoting activity, resistant to 100 g streptomycin/ml) was isolated from pigeon pea rhizosphere and, when used in peanut bacterization, decreased fungal and bacterial numbers and increased the actinomycete population in the rhizosphere. Over 28 days, AF 1 declined by 0.9 log c.f.u. in sterilized soil and by 2.6 log c.f.u. in native soil. From the seedlings raised with peanut seeds bacterized with AF 1 at 6.5 log c.f.u./seed, 4.1 and 4.7 log c.f.u. of AF 1 were recovered from the rhizosphere and total underground parts, respectively. Bacterization with AF 1 had no significant growth-promoting or -inhibiting effect on the peanut.     

Biological control of collar rot disease with broad-spectrum antifungal bacteria associated with groundnut

Bacteria associated with 6 habitats of groundnut were evaluated for their broad-spectrum antifungal activity and suppression of collar rot (Aspergillus niger) of groundnut. Three hundred and ninety-three strains were tested against 8 fungal pathogens of groundnut including 5 necrotrophic fungi, Aspergillus flavus, A. niger, Rhizoctonia bataticola, Rhizoctonia solani, and Sclerotium rolfsii, and 3 biotrophic fungi, Cercospora arachidicola, Phaeoisariopsis personata, and Puccinia arachidis. Pseudomonas sp. GRS 175, Pseudomonas aeruginosa GPS 21, GSE 18, GSE 19, and GSE 30, and their cell-free culture filtrates were highly antagonistic to all the test fungi. The cell-free culture filtrates of these bacteria were fungicidal and induced mycelial deformations including hyphal bulging and vacuolization in necrotrophic fungi. The cell-free culture filtrates at 10% (v/v) concentration significantly inhibited the spore germination of biotrophic fungi. In the greenhouse, P. aeruginosa GSE 18 emerged as an effective biocontrol agent of collar rot closely followed by P. aeruginosa GSE 19. The bacterium applied as a seed treatment reduced the pre-emergence rotting and postemergence wilting by > 60%. Pseudomonas aeruginosa GSE 18 effectively colonized the groundnut rhizosphere, both in native and in A. niger infested potting mixtures. Ninety-day-old peat formulation of P. aeruginosa GSE 18 had biocontrol ability comparable with the midlog-phase cells. Pseudomonas aeruginosa GSE 18, tolerant to thiram, in combination with the fungicide had an improved collar rot control. The present study was a successful attempt in selection of broad-spectrum and fungicide tolerant biocontrol agents that can be a useful component of integrated management of collar rot.     

Development of formulations of biological agents for management of root rot of lettuce and cucumber

The effect of various carrier formulations of Bacillus subtilis and Pseudomonas putida were tested on germination, growth, and yield of lettuce and cucumber crops in the presence of Pythium aphanidermatum and Fusarium oxysporum f.sp. cucurbitacearum, respectively. Survival of B. subtilis and P. putida in various carriers under refrigeration (about 0 degree C) and at room temperature (about 22 degrees C) was also studied. In all carrier formulations, B. subtilis strain BACT-0 survived up to 45 days. After 45 days of storage at room temperature (about 22 degrees C), populations B. subtilis strain BACT-0 were significantly higher in vermiculite, kaolin, and bacterial broth carriers compared with other carriers. Populations of P. putida were significantly higher in vermiculite, peat moss, wheat bran, and bacterial broth than in other carriers when stored either under refrigeration (about 0 degree C) or at room temperature (about 22 degrees C) for 15 or 45 days. Germination of lettuce seed was not affected in vermiculite, talc, kaolin, and peat moss carriers, but germination was significantly reduced in alginate and bacterial broth carriers of B. subtilis compared to the non-treated control. Germination of cucumber seed was not affected by any of the carriers. Significantly higher fresh lettuce and root weights were observed in vermiculite and kaolin carriers of B. subtilis compared with P. aphanidermatum-inoculated control plants. Lettuce treated with vermiculite, and kaolin carriers of B. subtilis, or non-inoculated control lettuce plants had significantly lower root rot ratings than talc, peat moss, bacterial broth, and P. aphanidermatum-inoculated control plants. Growth and yield of cucumber plants were significantly higher in vermiculite-based carrier of P. putida than the other carriers and Fusarium oxysporum f.sp. cucurbitacearum-inoculated plants.     

Whole cells of Bacillus subtilis AF 1 proved more effective than cell-free and chitinase-based formulations in biological control of citrus fruit rot and groundnut rust

In foliar and postharvest biocontrol systems, the use of active metabolites produced by antagonistic microorganisms is advantageous compared with the use of living microorganisms. Chitinases, a major group of hydrolytic enzymes produced by biocontrol agents, are involved in the lysis of cell walls of pathogenic fungi. In the present study, an attempt was made to test the partially purified beta-1,4-N-acetylglucosaminidase (NAGase) of a biocontrol strain Bacillus subtilis AF 1 for control of rust in groundnut (caused by Puccinia arachidis) and soft rot in lemons (caused by Aspergillus niger). Four proteins of molecular mass 67, 40, 37, and 32 kDa were isolated from the culture filtrates of AF 1 by affinity chromatography, of which the 67-kDa protein has detectable chitinolytic ability. This protein (NAGase) effectively inhibited the in vitro growth of A. niger in microtitre plates. In the presence of NAGase, germination of urediniospores of P. arachidis was reduced by 96% compared with the control. In a detached leaf bioassay, NAGase reduced the rust lesion frequency by >60%. NAGase significantly reduced the incidence of soft rot in harvested lemon fruits. However, fresh cells and (or) alginate formulation of AF 1 were more effective than NAGase in control of both of the test plant - pathogen systems.     

Seed Bacterization with Bacillus subtilis AF 1 Increases Phenylalanine Ammonia-lyase and Reduces the Incidence of Fusarial Wilt in Pigeonpea

Cell free culture filtrate of a biocontrol PGPR strain of Bacillus subtilis showed a concentration-dependent growth and conidiation inhibition of Fusarium udum . the incitant of pigeonpea wilt. In vitro interaction of cell free filtrate of AF 1 induced bulb-like structures in the hyphae of F. udum . Seed treatment with B. subtilis significantly reduced the incidence of pigeonpea wilt, but not with the cell free culture filtrate of AF 1. Seeds bacterized with B. subtilis AF 1 showed an increase in phenylalanine ammonia-lyase (PAL) from day 1, while the peroxidase activity increased from day 6. The results are discussed in terms of induction of host plant resistance in biological control of plant pathogens.     

Biological control of fusarial wilt of pigeon pea by Bacillus brevis

A virulent strain of pigeon pea wilt pathogen was isolated from wilted pigeon pea plants and was identified as Fusarium oxysporum f. sp. udum. Many bacterial cultures showing antagonism to the pathogen were isolated from various ecological niches. When tested under pot and field conditions, development of fusarial wilt symptoms was prevented in pigeon pea seeds treated with one such antagonist, Bacillus brevis. A formulation of B. brevis with vermiculite as a carrier had a shelf life of at least 6 months. Bacillus brevis produced an extracellular antagonistic substance which induced swelling of the pathogen's hyphal tips, and cells were bulbous and swollen with shrunken and granulated cytoplasm. The antagonistic substance also inhibited germination of conidia, and was fungicidal to the vegetative mycelia of the pathogen. Comparison of the properties of our antagonistic substance with that of known antibiotics produced by B. brevis suggests that our antagonistic substance is a novel compound. The observations reported here indicate that this strain of B. brevis may have potential as a biocontrol agent against fusarial wilt in pigeon pea.     

Turnover of15N-labelled urea in various rotations of groundnut sorghum and pigeon pea crops

Summary A field experiment on N turnover in rotations of groundnut, sorghum and pigeonpea crops was conducted in an Indian Alfisol during 1978–80.¹⁵N-labelled urea N was applied @ 40 kg ha^–1 in 1978. In the first year, the groundnut utilized 19.5% of the applied labelled N, sorghum 25.5%, and pigeon pea 10.3%. More fertilizer N was removed through weeding than by crop uptake in sorghum and pigeon pea. The fertilizer N left behind in soil upto 40 cm depth was 44.4% in groundnut plots, 35.1% in sorghum plots and 40.1% in pigeon pea plots.The uptake in 1979 of the residual fertilizer N in the soil was 8.9% in sorghum, 8.3% in groundnut and 2.8% in pigeon pea. In 1980, it declined to less than 2% for pigeon pea and groundnut and to about 4% for sorghum.A balance sheet drawn at the end of each rotation showed that about 51.3% of the applied labelled N was accounted for in groundnut-sorghum-pigeon pea rotation, 70.9% in sorghumpigeon pea-groundnut, and 43.5% in pigeon pea-groundnut-sorghum.     

Formulation and Delivery of the Bacterial Antagonist Bacillus subtilis for Management of Lentil Vascular Wilt Caused by Fusarium oxysporum f. sp. lentis

Different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log₁₀ CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log₁₀ CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease caused by Fusariumoxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments the glucose and talc‐based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field application.     

Effect of spent mushroom compost tea on mycelial growth and yield of button mushroom (Agaricus bisporus)

Preliminary studies suggested that the use of compost tea made from spent mushroom substrate (SMS) may be regarded as a potential method for biologically controlling dry bubble disease in button mushroom. The aim of this study was to assess the effect of SMS compost tea on the host, the button mushroom, to ascertain whether the addition of these water extracts has a toxic effect on Agaricus bisporus mycelium growth and on mushroom yield. In vitro experiments showed that the addition of SMS compost tea to the culture medium inoculated with a mushroom spawn grain did not have an inhibitory effect on A. bisporus mycelial growth. The effect of compost teas on the quantitative production parameters of A. bisporus (yield, unitary weight, biological efficiency and earliness) was tested in a cropping trial, applying the compost teas to the casing in three different drench applications. Quantitative production parameters were not significantly affected by the compost tea treatments although there was a slight delay of 0.8-1.4 days in the harvest time of the first flush. These results suggest that compost teas have no fungitoxic effect on A. bisporus so that they can be considered a suitable biocontrol substance for the control of dry bubble disease.     

Enhanced rhizosphere competence of Trichoderma viride grown in solid state fermentation on corn cob residue

Abstract

A process has been disclosed in the present investigation for the production of a formulation of mycopesticide with enhanced shelf life of Trichoderma viride. A comparative evaluation of talc-based and corn cob formulations in the form of seed coat and soil treatment brought to light that the soil treatment significantly enhanced the plant growth performance of pea, green gram and pigeon pea. Among the two formulations, the corn cob formulation proved to be better as the rhizosphere competence of corn cob formulation was superior to that of talc formulation. Our results suggest that corn cob residue is a better substrate owing to high cellulase and spore production. Solid-state fermentation in batch cultures. The biocontrol ability of both formulations was tested against Fusarium in Cajanus species and it was found that the disease incidence was reduced by 86% when the corn cob formulation was given as soil application.     

Biological control of pre-harvest aflatoxin contamination in groundnut (Arachis hypogaea L.) with Bacillus subtilis G1

The antagonistic activity of Bacillus subtilis strain G1 was tested against various isolates of Aspergillus flavus in vitro. A talc-based powder formulation of B. subtilis strain G1 was prepared and evaluated to control A. flavus infection and aflatoxin B1 contamination in groundnut under greenhouse and field conditions. The results showed that B. subtilis strain G1 could inhibit the growth of all isolates of A. flavus tested in dual culture assay and the growth inhibition ranged from 93 to 100%. Results of greenhouse and field experiments indicated that B. subtilis strain G1 when applied to groundnut as seed treatment and soil application significantly suppressed A. flavus population in the soil, A. flavus infection and aflatoxin B1 content in kernels and increased the pod yield. These studies show that B. subtilis strain G1 has potential as a biocontrol agent for control of aflatoxin contamination in groundnut.     

Improving biocontrol of black vine weevil (<Emphasis Type="Italic">Otiorhynchus sulcatus</Emphasis>) with entomopathogenic fungi in growing media by incorporating spent mushroom compost

Amending a peat-based growing medium with 10% v/v spent mushroom compost, a source of fungal chitin and other nutrients, prolonged the persistence of entomopathogenic fungi (Metarhizium brunneum Petsch and Beauveria bassiana (Balsamo) Vuillemin; Hypocreales: Clavicipitaceae). This resulted in improved efficacy of M. brunneum against black vine weevil, Otiorhynchus sulcatus F. (Coleoptera: Curculionidae) larvae compared with using inoculum without spent mushroom compost. B. bassiana only controlled larvae when used in combination with spent mushroom compost (75?±?7% reduction in live larvae). Mixing entomopathogenic fungal inoculum with spent mushroom compost and growing medium was as effective in controlling black vine weevil larvae as using spent mushroom compost colonised with M. brunneum or B. bassiana in the growing medium (80?±?12% reduction in live larvae). The former method is preferable since it does not require production and storage of colonised spent mushroom compost, or registration of new substrate formulations of M. brunneum or B. bassiana.     

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.     

The effect of transition metal ions on the resistance of bacterial spores to hydrogen peroxide and to heat.

The presence of 10 microM-Cu2+ increased the lethal effect of hydrogen peroxide on spores of Clostridium bifermentans but not on those of Clostridium sporogenes PA 3679, Clostridium perfringens, Bacillus cereus or Bacillus subtilis var. niger. Cu2+ at 100 muM also increased the lethal effect of heat on spores of C. bifermentans but not on those of B. sutilis var. niger. The rate and extent of Cu2+ uptake by spores of C. bifermentans and B. subtilis var. niger were similar, but examination of unstained sections of spores by electron microscopy suggested that Cu2+ is bound by the protoplasts of spores of C. bifermentans but not of B. subtilis var. niger.     

Beneficial rhizospheric microorganisms mediated plant growth promotion and suppression of aflatoxigenic fungal and aflatoxin contamination in groundnut seeds

The potential of root‐colonising antagonistic microbial biocontrol agents was evaluated for their ability to improve plant growth and suppress aflatoxigenic fungal and aflatoxin contamination in groundnut. By considering root colonisation of groundnut seedlings, plant growth promotion and antagonism against aflatoxigenic Aspergillus flavus as preliminary criteria, eight rhizobacteria and nine Trichoderma spp. were selected and characterised for their beneficial traits. These strains gave varying results for IAA production, phosphate solubilisation, ACC deaminase, chitinase and siderophore production. Under laboratory and greenhouse conditions, these strains significantly (P < 0.05) suppressed seed‐borne and rhizospheric population of A. flavus and improved seed quality variables. However, cdELISA results revealed that none of the biocontrol strains were effective in reducing aflatoxin level in seed. Based on the overall performance, Pseudomonas fluorescens 2bpf, Bacillus sp. Bsp‐3/aM and Trichoderma atroviride UMDBT‐Dha.Tat8 were used for field trials in the form of talcum powder formulations. Under field conditions, biocontrol agents improved seedling emergence, plant biomass and pod yield. Seeds harvested from plots treated with biocontrol agents showed significant (P < 0.05) reduction in A. flavus infection and aflatoxin production after 6 months' storage. Use of microbial strains with multiple beneficial traits is advantageous in bioformulation development. Hence, in future, these formulations will play a major role as biofertilisers and biopesticides, which can reduce the usage of agrochemicals up to greater extents in groundnut production.     

Survival of a plasmid-bearing strain of Bacillus subtilis introduced into compost

Survival of Bacillus subtilis strain 168 containing plasmid pAB224, which carries a gene for tetracycline resistance, was studied in mushroom compost under mesophilic and thermophilic conditions. Stable populations of B. subtilis were maintained as spores in both sterile and fresh mushroom compost incubated at 37 degrees C. At 65 degrees C, the introduced B. subtilis populations declined during incubation but spores were still detectable after 28 d. Survival at the higher temperature was greater in fresh than in sterile compost. There was no apparent loss of plasmid pAB224 or plasmid-determined phenotype from the introduced B. subtilis population at either incubation temperature. The frequency of tetracycline resistance in the indigenous Bacillus population was very low (10(-5), but some tetracycline-resistant isolates contained plasmid DNA. Four plasmid DNA profiles were found associated with five Bacillus phenotypes, and some evidence for homology with pAB224 was found. However, pAB224 was found to be a suitable marker for release studies because it was easily recovered, readily distinguished from indigenous plasmids on agarose gels, and was maintained in compost-grown B. subtilis 168 in the absence of any selective pressure.