Antagonistic potential of Gliocladium virens and Trichoderma longibrachiatum to phytopathogenic fungi

Three isolates of Gliocladium virens (G1, G2 and G3) and two of Trichoderma longibrachiatum (T1 and T2) were screened against isolates of three soilborne plant pathogens namely Rhizoctonia solani, Sclerotium rolfsii and Pythium aphanidermatum. G. virens exhibited stronger hyperparasitism and wider biological spectrum than T. longibrachiatum. Further, similarities as well as variation was observed in the ability of the various isolates to invade the test pathogens in dual culture. For the hyperparasites, acidic pH range (5.0 to 5.5) favoured both growth and spore germination. The hyperparasites made direct contact with the pathogens followed by varied modes of attack invariably leading to cell disruption. Antagonists, G1 and G3 revealed strong antibiosis while T2 showed moderate effect. All the isolates produced enhanced levels of lytic enzymes adaptively and there were marked differences among them. However, no correlation was observed between these attributes and the hyperparasitic potential of the various isolates in dual culture. The relevance and the role of enzymes and toxic metabolite(s) in the antagonism of G. virens and T. longibrachiatum to these pathogens are discussed.     

Isolation,characterisation and antifungal activity of marine actinobacteria from Goa and Kerala,the west coast of India

In total, 53 marine actinobacteria were isolated from the soils of six different locations in Goa and Kerala, on the west coast of India. All the isolates were screened for their antifungal properties against some phytopathogenic fungi by dual culture experiments. Among the 53 actinobacterial isolates, five isolates inhibited the growth of phytopathogens, namely Colletotrichum falcatum, Thielaviopsis paradoxa and Fusarium semitectum. But none of them were effective against Aspergillus niger, Aspergillus candidus and Aspergillus flavus. The antifungal activity of the actinobacteria was tested by food poisoning techniques, using four different concentrations (0.5%, 1.0%, 1.5% and 2.0%) of cell-free culture filtrates, which showed promising activity (almost 100% inhibition) against three pathogenic and one non-pathogenic fungi at 2% extract concentration. A comparison of the antifungal activity of the actinobacteria was also made with three commercial fungicides, namely hexaconazole, thiophanate methyl and propiconazole. The identity of the antagonistic actinobacteria was confirmed based on the morphological, cultural, biochemical, chemo-taxonomical and physiological characteristics. Among 5 antagonistic isolates, three antagonistic isolates were assigned to the genus Streptomyces, Nocardiopsis (1) and Saccharopolyspora (1).     

Genetic and functional characterization of a Bacillus sp. strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds

AIMS: A bacterial strain producing antifungal compounds active against the plant pathogenic fungi Fusarium, Rhizoctonia and Sclerotinia has been characterized and shown to control Rhizoctonia root rot of soya bean. METHODS AND RESULTS: The metabolites excreted by Bacillus BNM 122 remained active after autoclaving, were resistant over a wide pH range and to hydrolytic enzymes. By (1)H-NMR and thin-layer chromatography analyses surfactin and iturin-like compounds were partially identified. Moreover, soya bean seeds bacterization with BNM 122 in a compost-based formulation was as effective controlling Rhizoctonia solani as pentachloronitrobenzene. According to its 16S rDNA sequence BNM 122 was closely related to Bacillus amyloliquefaciens and Bacillus subtilis. PCR analysis of the 16S-23S rRNA intergenic spacer region and repetitive sequence-based PCR (rep-PCR) genomic fingerprinting revealed a close genetic relationship to B. amyloliquefaciens. However, by physiological characterization using API tests, this strain resembled more B. subtilis. CONCLUSIONS: This is the first report describing the co-production of surfactin and iturin-like compounds by a putative strain of B. amyloliquefaciens. The synergistic effect of both lipopetides is a remarkable trait for a candidate biocontrol agent. SIGNIFICANCE AND IMPACT OF THE STUDY: This kind of research has relevance in order to minimize the use of synthetic fungicides and surfactants, contributing to the preservation of the environment.     

Genetic Diversity in Pseudomonads Associated with Cereal Cultures Infected with Basal Bacteriosis

The genetic properties of 45 pseudomonad strains isolated from cereal cultures exhibiting symptoms of basal bacteriosis have been investigated. Considerable genetic diversity has been demonstrated using DNA fingerprints obtained by amplification with REP, ERIC, and BOX primers. Restriction analysis of the 16S–23S internal transcribed spacer (ITS1) allowed the strains to be subdivided into two major groups. In a phylogenetic tree, the ITS1s of these groups fell into two clusters, which also included the ITS1 of Pseudomonas syringae (“Syringae” cluster) and the ITS1 of P. fluorescens, P. tolaasii, P. reactans, P. gingeri, and P. agarici (“Fluorescens” cluster) from the GenBank database. Comparison of the ITS1 divergence levels within the “Fluorescens” cluster suggests expediency of treating P. tolaasii, P. reactans, various P. fluorescens groups, and, possibly, P. gingeri and P. agarici as subspecies of one genospecies. The intragenomic heterogeneity of ITS1s was observed in some of the pseudomonad strains studied. The results of amplification with specific primers and subsequent sequencing of the amplificate suggest the possibility of the presence of a functionally active syrB gene involved in syringomycin biosynthesis in the strains studied.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 537–544.Original Russian Text Copyright © 2005 by Bobrova, Milyutina, Troitskii.     

Bromelain, a cysteine protease from pineapple (Ananas comosus) stem, is an inhibitor of fungal plant pathogens

Aim: This study aimed to evaluate the effect of bromelain, a cysteine protease isolated from pineapple (Ananas comosus), on growth of several agronomically important fungal pathogens. Methods and Results: Purification of bromelain from pineapple stems was carried out by chromatography techniques, and its antimicrobial activity was tested against the fungal pathogens Fusarium verticillioides, Fusarium oxysporum and Fusarium proliferatum by broth microdilution assay. A concentration of 0·3 μmol l^?1 of bromelain was sufficient for 90% growth inhibition of F. verticillioides. The capability of bromelain to inhibit fungal growth is related to its proteolytic activity. Conclusions: The study demonstrates that stem bromelain exhibits a potent antifungal activity against phytopathogens and suggests its potential use as an effective agent for crop protection. Significance and Impact of the Study: The results support the use of a natural protease that accumulates at high levels in pineapple stems as alternative to the use of chemical fungicides for crop protection.     

Novel and rapid agar plate methods for in vitro assessment of bacterial biocontrol isolates’ antagonism against multiple fungal phytopathogens

Biological control of plant diseases with antagonistic bacteria is a promising alternative to conventional chemical control strategies. In vitro screening for inhibition of mycelial growth of phytopathogenic fungi by bacterial isolates is the first step in selecting putative bacterial biocontrol agents. Dual culture plate assay is the most common method involved in this first-line selection process. However, it needs independent agar plates to test antagonism by a specific bacterial isolate against each of the fungal phytopathogen. Two modified in vitro antagonism tests are proposed here. Antagonistic activity of a putative biocontrol bacterial strain against four different fungal phytopathogens could be assessed in a single agar plate simultaneously. A comparison of the new methods with conventional dual culture plate assay was also done. The proposed methods are easy to perform and results of antagonism are obtained rapidly. Results of fungal inhibition were qualitatively comparable with that generated through dual culture plate assay. Quantity of resources such as agar medium and plates required for the modified antagonistic assays is several folds less than that required for dual culture plate assay.     

Trichoderma asperelloides antagonism to nine Sclerotinia sclerotiorum strains and biological control of white mold disease in soybean plants

Sclerotinia sclerotiorum is an important plant pathogen with worldwide distribution that causes severe economic losses of various agricultural crops such as soybean. This fungus is normally controlled with synthetic chemical fungicides that pose risks to the environment, and can be harmful to human health, and they can also induce resistance in pests. The aim of this study was to investigate the potential of Trichoderma asperelloides as a biocontrol agent towards white mold disease on soybeans crops. The antagonism of two strains of T. asperelloides (T25 and T42) isolated from soil samples was determined in-vitro by dual-culture confrontation testing on nine S. sclerotiorum strains obtained from sclerotia collected on diseased soybean plants. The mycelial growth and inhibition of carpogenic and ascospore germination by T. asperelloides extracts, as well as the efficacy of these on white mold control in soybeans were evaluated. Both strains of T. asperelloides exhibited high potential of antagonism. Methanolic and ethyl acetate extracts of the two T. asperelloides strains showed excellent growth inhibition (60–100%) on all of the pathogens tested. The ethyl acetate extracts of both T. asperelloides strains exhibited the highest efficacy against carpogenic germination, decreasing by 20–30% the number of ascospores per apothecium. Strains of T. asperelloides tested were more efficient in controlling white mold than two commercial products made from Trichoderma harzianum. The new strains of T. asperelloides have potential for successful biological control of white mold disease of soybean crops in the field.     

Proteomic analysis of the phytopathogenic fungus Botrytis cinerea during cellulose degradation

The ascomycete Botrytis cinerea is a phytopathogenic fungus infecting and causing significant yield losses in a number of crops. Moreover, in the last few years, B. cinerea has been adopted as an important model system in molecular phytopathology. In spite of these contributions, the molecular basis of the infection cycle remains unclear. Proteomic approaches have revealed significant information about the infective cycle of several pathogens, including B. cinerea. The main aim of this study is to make available a proteomic database containing a significant number of identified proteins from B. cinerea. In brief, three independent B. cinerea cultures supplemented with carboxymethylcellulose were used, and the extracted proteins were independently separated by 2‐D PAGE to obtain the proteome map from B. cinerea. Two hundred and sixty‐seven spots were selected for MALDI TOF/TOF MS analysis, resulting in 303 positive identifications, mostly representing unannotated proteins. Identified proteins were then classified into categories using the PANTHER classification system ( www.pantherdb.org ), showing the relevance of protein metabolism and modification process and oxidoreductase activity. Since cellulose is one of the major components of the plant cell wall, many of the identified proteins may have a crucial role in the pathogenicity process. In brief, this proteomic map of B. cinerea will be a useful basis for exploring the proteins involved in the infection cycle, which will in turn provide new targets for crop diagnosis and focused fungicide design.     

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5–7, but the aspartic proteases were inactive across a pH range of 3–10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi.     

In vitro auxin production by Balansia epichloë

Balansia epichloë, a systemic plant pathogen isolated from Sporobolus poiretii, was shown to produce the plant growth regulators 3-indole acetic acid, 3-indole ethanol, 3-indole acetamide and methyl-3-indole carboxylate when grown on a medium containing tryptophan. When grown on a tryptophan deficient medium 3-substituted indole derivatives were not detected. However, extracts of the medium in lower doses increased and in higher doses inhibited the growth of wheat coleoptiles.