In vitro and in vivo antagonism of biocontrol agents against Colletotrichum lindemuthianum causing bean anthracnose

Bean anthracnose caused by Colletotrichum lindemuthianum is a serious seed borne disease. For devising an effective management strategy, the efficacy of different bioagents, viz. Trichoderma viride, Trichoderma harzianum, Trichoderma hamatum and Gliocladium virens conducted under in vitro and in vivo conditions revealed maximum inhibition of mycelial growth in dual culture (59.48%) and inverted plate (55.98%) with T. viride. All the bioagents overgrew the pathogen and the principal mechanism of mycoparisitism observed was coiling, brusting and disintegration of pathogen hyphae. Culture filtrate from T. viride was found best as it completely inhibited radial growth at 25 and 50% concentration and reduced the spore germination of test fungus significantly. However, lower concentrations of culture filtrate from all bioagents showed little effect on spore germination. Seed application of bioagents was found better as compared to soil application. A maximum increase in seed germination and inhibition of seed borne infection was observed with T. viride followed by T. harzianum under pot culture conditions. T. viride has the maximum potentiality to suppress the spore germination, mycelial growth, seed borne infection of C. lindemuthianum and increased seed germination when compared with the other biocontrol agents.     

Screening of some wild fungal isolates for cellulolytic activities

Six wild fungal strains, Trichoderma viride, T. harzianum, Gliocladium virens, Aspergillus terreus, A. niger and Tiarosporella phaseolina , isolated from decomposed jute stacks and diseased jute stem, were tested for their cellulolytic and hemicellulolytic activities and compared with T. reesei MCG 77. Filter paper cellulase production by all these wild strains were lower than those produced by T. reesei while some strains ( T. viride, T. harzianum and G. virens ) possessed carboxymethyl cellulase, β-glucosidase, xylanase and β-xylosidase activities comparable to T. reesei. A. terreus and A. niger produced 3·2 and 1·2 times respectively, greater β-glucosidase activity compared to T. reesei when grown on microcrystalline cellulose.     

Diversity of facultatively anaerobic microscopic mycelial fungi in soils

The numbers of microscopic fungi isolated from soil samples after anaerobic incubation varied from tens to several hundreds of CFU per one gram of soil; a total of 30 species was found. This group is composed primarily of mitotic fungi of the ascomycete affinity belonging to the orders Hypocreales (Fusarium solani, F. oxysporum, Fusarium sp., Clonostachys grammicospora, C. rosea. Acremonium sp., Gliocladium penicilloides, Trichoderma aureoviride, T. harzianum, T. polysporum, T. viride. T. koningii, Lecanicillum lecanii, and Tolypocladium inflatum) and Eurotiales (Aspergillus terreus, A. niger, and Paecilomyces lilacimus), as well as to the phylum Zygomycota, to the order Mucorales (Actinomucor elegans, Absidia glauca, Mucor circinelloides, M. hiemalis, M. racemosus, Mucor sp., Rhizopus oryzae, Zygorrhynchus moelleri, Z. heterogamus, and Umbelopsis isabellina) and the order Mortierellales (Mortierella sp.). As much as 10-30% of the total amount of fungal mycelium remains viable for a long time (one month) under anaerobic conditions.     

Evaluation of the effects of chemical versus biological control on Botrytis cinerea agent of gray mould disease of strawberry

This study investigates on effects of four fungicide and six isolate from Trichoderma and Gliocladium on Botrytis cinerea agent grey mold of strawberry under library and greenhouse condition. The effect of four fungicides i.e. benomyl, dichlofluanid, captan and triadimenol on B. cinerea was studied in the laboratory condition by method mixed poison to culture medium. It was shown that the fungicide including benomyl, triadimenol, dichlofluanid and captan were able to inhibit mycelial growth of B. cinerea on PDA plate with EC50 of 0.16, 1.42, 3.40 and 7.73 ppm respectively. These fungicides delayed myceliogenic germination of sclerotia at 1000 ppm, while exhibiting no fungicidal effect. Moreover, the antagonistic effects of six fungi including Trichoderma koningii (T21), T. viride (T4), T. harzionum (T5), T. viride (T2), G. virens (G2), G. virens (G8) on B. cinerea were assessed. This assessment was done under library condition and its results as follows: The antagonistic mechanism occurred through branching at the end of B. cinerea hyphae, hyphal contact, coiling, vacuolization and lyses. Volatile metabolites of T. koningii (T21) and non-volatile metabolites of G. virens (G2 and G8) and T. koningii (T21) caused maximum inhibition of the fungal growth. Trichoderma spp and G. virens were able to colonize and sporulate on sclerotia and caused their lysis within 7-21 days. In greenhouse, a completely randomized design with 11 treatments (4 chemical and 6 biological and one untreated control) each replicated five times were used for the comparison. Greenhouse studies revealed that application of fungicides i.e. captan, dichlofluanid, triadimenol and benomyl reduces disease severity by 42, 45, 48 and 52% respectively. The fungal antagonists reduce the grey mold disease severity between 5-42%. All treatments caused a decline in post harvest disease, as the most effective treatment of chemical control was benomyl with 68.33% and for the biological treatment this was T. koningii (T21) with 56%.     

Antimycotic Compounds from the Plant Pathogen Rhizoctonia solani and its Antagonist Trichoderma harzianum

The soilborne rhizosphere-competent fungal biocontrol agent Trichoderma harzianum isolate Th008 secreted trichodermin (MW = 292) and a small peptide (MW = 876) in culture. These compounds were antagonistic in culture to the mycelial growth of the soilborne fungal pathogen Rhizoctonia solani isolate 2B-12, which is highly virulent to soybean ( Glycine max )seedlings. When 100mg of dried autoclaved mycelial mat of R. solani was added to 200 ml liquid cultures of T. harzianum , the quantity of antimycotic compounds secreted by the latter was 3.5 times greater than that of the antagonist alone. R. solani secreted a coumarin derivative (MW = 313) in liquid culture, which inhibited the mycelial growth of T. harzianum ; however, inhibition of the growth of the antagonist required a greater concentration than that for the antimycotic compounds produced by the antagonist against the pathogen. The inclusion of 100 mg of dried autoclaved mycelial mat of T. harzianum in a 200 ml liquid culture of R. solani did not affect the quantity of the antimycotic compound produced by the pathogen.     

Effects of some carbon and nitrogen sources on spore germination,production of biomass and antifungal metabolites by species of Trichoderma and Gliocladium virens antagonistic to Sclerotium cepivorum

Conidia of Trichoderma pseudokoningii (IMI 322662) and T. viride (IMI 322659) were incubated in 1% bacteriological peptone at 25° C for 20 h and more than 95% of the spores germinated. In the same medium, only 35% of the conidia of Gliocladium virens (G20) and T. viride (IMI 322663) germinated but when 1% glucose was added, germination was increased to 70%. In the presence of glucose as a carbon source, maximal biomass production of G. virens (G20) after seven days at 25°C was obtained with either potassium nitrate or L‐alanine as the nitrogen source, whereas the Trichoderma isolates needed an organic nitrogen source. With L‐alanine as a nitrogen source, glucose, galactose and sucrose were readily utilized for biomass production by all fungal isolates. Maltose utilization by G. virens (G20) and T. pseudokoningii was incomplete after 21 days incubation, whereas glucose utilization was complete by this time. G. virens, T. pseudokoningii and T. viride (IMI 322663) produced antifungal metabolites which were effective at reducing radial growth of Rhizoctonia solani, Botrytis cinerea as well as S. cepivorum. The metabolites produced by G. virens were very active against all three pathogens but the metabolites produced by T. pseudokoningii and T. viride (IMI 322663) were less active. T. viride (IMI 322659) was a very poor antifungal metabolite producer.     

Expression of the red fluorescent protein DsRed-Express in filamentous ascomycete fungi

The recently reported red fluorescent protein DsRed from the reef coral Discosoma sp. represents a new marker that has been codon-optimized for high expression in mammalian cells. To facilitate expression of DsRed in ascomycete fungi, we used the clone pDsRed-Express (Clontech) for constructing a plasmid vector, pPgpd-DsRed, containing the constitutive Aspergillus nidulans glyceraldehyde 3-phosphate (gpd) promoter. This vector was used for co-transformation of Penicillium paxilli, Trichoderma harzianum and Trichoderma virens (syn. Gliocladium virens) together with either pAN7-1 or gGFP, both containing a gene for hygromycin resistance for transformant selection. In addition, gGFP contains a green fluorescent protein (GFP) gene for expression in Ascomycetes. Expression of DsRed-Express was obtained in all three fungi, indicating that DsRed can be used as a highly effective vital marker in Ascomycetes. Dual marked transformants expressed both DsRed-Express and GFP in the same mycelium and were used for non-quantitative comparison of the intensity of the fluorescence using confocal laser scanning microscopy.     

Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum

Protoplasts were isolated from Trichoderma harzianum strain PTh18 using lysing enzymes and self-fusion of T. harzianum protoplasts was carried out using polyethylene glycol in STC buffer. The fused protoplasts of T. harzianum were regenerated and 15 self-fusants were selected to study the chitinase production and biocontrol activity. High chitinase activity was measured in the culture filtrates of most of the self-fusants (87%) than the parent. Among the fusants, the strain SFTh8 produced maximum chitinase with a two-fold increase as compared to the parent strain. All the self-fusants exhibited increased antagonistic activity against Rhizoctonia solani than the parent. The crude chitinase preparation of SFTh8 lysed the mycelia of T. harzianum, Trichoderma viride and Trichoderma reesei and released the protoplasts in higher number than the crude chitinase preparation of parent strain PTh18.     

Tricalcium phosphate solubilizing abilities of Trichoderma spp. in relation to P uptake and growth and yield parameters of chickpea (Cicer arietinum L.)

Nine isolates of Trichoderma spp. were investigated for their ability to solubilize insoluble phosphate in Pikovskaya's broth and were compared with an efficient phosphate-solubilizing bacterium Bacillus megaterium subsp. phospaticum PB that was used as the reference strain. All 9 Trichoderma isolates were found to solubilize insoluble tricalcium phosphate to various extents. Trichoderma viride (TV 97) (9.03 microg x mL(-1)), Trichoderma virens (PDBCTVs 12) (9.0 microg x mL(-1)), and Trichoderma virens (PDBCTVs 13) (8.83 microg x mL(-1)) solubilized 70% of that solubilized by the reference strain Bacillus megaterium (12.43 microg x mL(-1)). Pot culture and field evaluations with Trichoderma harzianum (PDBCTH 10), Trichoderma viride (TV 97), and Trichoderma virens (PDBCTVs 12) using chickpea (Cicer arietinum L.) 'Annegeri-1' as the test plant and rock phosphate as the phosphorus source showed significantly increased P uptake in plants treated with Trichoderma harzianum (PDBCTH 10) followed by Trichoderma virens (PDBCTVs 12) and Trichoderma viride (TV 97). Inoculation of Trichoderma spp. also showed increased growth and yield parameters of chickpea compared with the uninoculated controls under both glasshouse and field conditions.     

Diversity of facultatively anaerobic microscopic mycelial fungi in soils

The numbers of microscopic fungi isolated from soil samples after anaerobic incubation varied from tens to several hundreds of CFU per one gram of soil; a total of 30 species was found. This group is composed primarily of mitotic fungi of the ascomycete affinity belonging to the orders Hypocreales (Fusarium solani, F. oxysporum, Fusarium sp., Clonostachys grammicospora, C. rosea, Acremonium sp., Gliocladium penicilloides, Trichoderma aureoviride, T. harzianum, T. polysporum, T. viride, T. koningii, Lecanicillum lecanii, and Tolypocladium inflatum) and Eurotiales (Aspergillus terreus, A. niger, and Paecilomyces lilacimus), as well as to the phylum Zygomycota, to the order Mucorales (Actinomucor elegans, Absidia glauca, Mucor circinelloides, M. hiemalis, M. racemosus, Mucor sp., Rhizopus oryzae, Zygorrhynchus moelleri, Z. heterogamus, and Umbelopsis isabellina) and the order Mortierellales (Mortierella sp.). As much as 10–30% of the total amount of fungal mycelium remains viable for a long time (one month) under anaerobic conditions.     

Molecular characterization and identification of biocontrol isolates of Trichoderma spp

The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as "Trichoderma harzianum" Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.     

Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host.

For monitoring chitinase expression during mycoparasitism of Trichoderma harzianum in situ, we constructed strains containing fusions of green fluorescent protein (GFP) to the 5'-regulatory sequences of the T. harzianum nag1 (N-acetyl-beta-d-glucosaminidase-encoding) and ech42 (42-kDa endochitinase-encoding) genes. Confronting these strains with Rhizoctonia solani led to induction of gene expression before (ech42) or after (nag1) physical contact. A 12-kDa cut-off membrane separating the two fungi abolished ech42 expression, indicating that macromolecules are involved in its precontact activation. No ech42 expression was triggered by culture filtrates of R. solani or by placing T. harzianum onto plates previously colonized by R. solani. Instead, high expression occurred upon incubation of T. harzianum with the supernatant of R. solani cell walls digested with culture filtrates or purified endochitinase 42 (CHIT42, encoded by ech42) from T. harzianum. The chitinase inhibitor allosamidin blocked ech42 expression and reduced inhibition of R. solani growth during confrontation. The results indicate that ech42 is expressed before contact of T. harzianum with R. solani and its induction is triggered by soluble chitooligosaccharides produced by constitutive activity of CHIT42 and/or other chitinolytic enzymes.     

Effects of elevated salt concentrations on growth, sporulation and pigmentation of Trichoderma spp

On agar media supplemented with NaCl (5% = 856 mM), seven Trichoderma species aggregates and an isolate intermediate between T. harzianum and T. viride (T. 410) grew more slowly, sporulated poorly, and there was no characteristic pigmentation of the colonies. NaCl or KCl concentrations inducing 'albinization' differed with different isolates. CaCl2 (90-270 mM) reversed the anti-sporulating effect of NaCl (856 mM) on T. 410, and stimulated conidiation in media with no NaCl added. Gliocladium virens but not G. roseum, reacted like Trichoderma to NaCl. Fourteen other fungal genera offered various reactions. Since 856 mM NaCl decreases osmotic potential (eta) of the media used from-1.2 to -41 bars, the influence of eta on T. 410 was examined. After 3 to 4 days, maximal growth was observed between-1.2 and -10 bars. Growth was reduced to 50% at about -30 bars. No growth occurred at about -90 bars. No pigmentation was observed at -32 bars (NaCl) and -41 bars (KCl). Abundant sporulation and pigmentation occurred at -31 bars with CaCl2 as sole electrolyte added. Na+ was toxic at high concentration. Results are discussed in view of possible use of Trichoderma in biological control.     

Gallotannin hydrolysis by immobilized fungal mycelia in a packed bed bioreactor.

Hydrolysis of gallotannin to gallic acid by immobilized mycelia of Aspergillus niger MTCC 282, Aspergillus fischerii MTCC 150, Fusarium solani MTCC 350 and Trichoderma viride MTCC 167 in a packed bed bioreactor was studied. Fungal mycelia preinduced with 5 g L-1 gallotannin were immobilized in calcium alginate gel (1.5%) and the resultant beads were packed in a column to a bed volume of 175 mm3. Gallotannin dissolved in distilled water was passed through the column and the eluate was recycled after adjusting pH to 6 with ammonium hydroxide (10%). Maximum hydrolysis of gallotannin was recorded by immobilized mycelia of F. solani and T. viride at 35 degrees and 45 degrees C after 175 and 60 min of residency period respectively. Optimum substrate concentration required for maximum hydrolysis was 10 g L-1 at pH 5 for both the fungi. Immobilized mycelia of A. niger and A. fischerii revealed maximum operational stability. Loss of activity after eighth run was in the order of-A. niger (no loss), A. fischerii (7.5%), F. solani (18%) and T. viride (18%). Stability in terms of retention of enzyme activity after 150 days of storage at 4 degrees C was A. niger (58%), A. fischerii (26.8%), F. solani (83%) and T. viride (85.1%).     

Management of postharvest disease of mango anthracnose incited by Colletotrichum gleosporioides

Abstract

Among the seven fungal (Gliocladium virens, Trichoderma hamatum, T. harzianum, T. koningii, T. longibrachiatum, T. pseudokoningii and T. viride) and two bacterial (Bacillus subtilis and Pseudomonas fluorescens) antagonists screened against C. gloeosporioides under in vitro conditions, T. harzianum exhibited maximum inhibition followed by Pseudomonas fluorescens at 5 days after incubation. These fungal and bacterial antagonists were selected for application to fruits infected with pathogens. Fruits inoculated with C. gloeosporioides were dipped in spore/cell suspensions of fungal/bacterial antagonists and kept for different durations. The fungal antagonists T. harzianum and P. fluorescens were effective in checking the spread of pathogens on fruits compared with the pathogen-inoculated control.     

Utility of biocontrol agents in the suppression of seed borne pathogenic mycoflora and their effect on seed quality in sorghum (Sorghum bicolor (L.) Moench)

Abstract

The scope of biocontrol agents in the control of sorghum grain molds was investigated both in vitro as well in vivo. Their utility in enhancing the germination and seedling vigor of mold-infected seeds were assessed. The biocontrol agents, Trichoderma viride, T. harzianum, T. hamatum, T. koeningii and Pseudomonas fluorescens (isolates collected from the state of Tamil Nadu, India) were efficient in checking the growth of mold pathogens when grown in Potato Dextrose Agar (PDA) as dual cultures. The bacterial bioagent P. fluorescens was most efficient in checking the growth of all the test pathogens viz., Fusarium moniliforme, Curvularia lunata and Alternaria alternata (66.8, 68.5 and 64.9% inhibition of colony growth respectively) followed by other bioagents for their antagonistic effect. The in vivo effectiveness of the bioagents were enhanced when combined with other components like host-resistance, cultural and chemical control strategies, paving the way for a possible Integrated Disease Management. The percent incidence and severity of molds were lowest (19.44 and 13.5% respectively) with the treatment T₆ (seeds harvested at physiological maturity + propiconazole @ 0.02% + P. fluorescens @ 1 × 10⁸ cfu ml^?1) that recorded maximum 100 seed weight (2.905 g), followed by 2.871 g in T₂ (seeds harvested at physiological maturity + P. fluorescens). The effect of bioagents in enhancing the germination and seedling vigor was most promising. Of all the treatments, P. fluorescens resulted in maximum germination (88%) and seedling vigor (2635.60 vigor index), followed by treatment with T. viride (86.6% and 2478.47 respectively).     

Biocontrol of Damping-off Diseases Caused by Rhizoctonia solani and Pythium ultimum with Alginate Prills of Gliocladium virens, Trichoderma hamatum and Various Food Bases

Alginate prills were formulated with the biomass of isolates of Gliocladium virens and Trichoderma spp. and various food bases (wheat bran, corn cobs, peanut hulls, soy fiber, castor pomace, cocoa hulls and chitin). Alginate prills with G. virens (Gl-21) biomass and all food bases except cocoa hull meal significantly reduced the damping-off of zinnia in a soil-less mix caused by Rhizoctonia solani and Pythium ultimum. The prills with bran, soy fiber, castor pomace or chitin resulted in stands similar to those in the non-infested control. In soil, prills with all the food bases and Thrichoderma hamatum (TRI-4) biomass controlled the damping-off of cotton caused by R. solani and gave stands comparable to, or better than, those in the non-infested control soil. Prills with all the food bases resulted in a proliferation of Gl-21 in a soil-less mix and of Gl-21 and TRI-4 in soil. Prills with food bases and TRI-4 biomass reduced the survival of R. solani in infested beet seed to less than 30%, with bran and chitin being the most effective food bases; prills with Gl-21 biomass and all food bases also reduced the survival of R. solani in beet seed, but not as much as did prills with TRI-4 biomass. In prills containing wheat bran, soy fiber or chitin, the biocontrol isolate Th-58 (T. harzianum) was almost as effective as TRI-4, but isolate Gl-3 (G. virens) was less effective. There was no significant interaction between the biocontrol fungus and the food base. The results suggest that the intrinsic properties of a selected fungus isolate are more important than some formulation variables in biocontrol.     

Biological Control of Macrophomina phaseolina Charcoal Rot of Sunflower and Mung Bean

Infection by Macrophonina phaseolina was substantially reduced following treatment of sunflower and mungbean seeds with Trichoderma harzianum, Gliocladium virens, Paecilomyces lilacinus or Streptomyces sp. which gave promising control of charcoal rot disease. Treatment of mungbean seeds with Rhizobium meliloti also gave good disease control.     

被内生真菌侵染的禾草提取液对真菌的抑制作用

以带有与不带有Neotyphodium属内生真菌的醉马草Achnatherum inebrians、披碱草Elymus dahuricus和野大麦Hordeum brevisubulatum的草粉浸提液对细交链孢Alternaria alternata、根腐离蠕孢Bipolaris sorokiniana、燕麦镰孢Fusarium avenaceum和绿色木霉Trichoderma viride进行了抑菌活性研究。结果表明:披碱草、醉马草和野大麦草粉浸提液对细交链孢、根腐离蠕孢、燕麦镰孢和绿色木霉的菌落生长、孢子萌发率和芽管长度均有一定的抑制作用。而披碱草中的Neotyphodium可显著增强披碱草草粉浸提液对细交链孢、燕麦镰孢、绿色木霉菌落生长及对细交链孢和根腐离蠕孢孢子萌发及燕麦镰孢芽管长度的抑制作用;醉马草中的Neotyphodium显著增强了醉马草草粉浸提液对燕麦镰孢、绿色木霉菌落生长和芽管长度,以及细交链孢、根腐离蠕孢和燕麦镰孢孢子萌发的抑制作用;野大麦中的Neotyphodium显著增强了野大麦草粉浸提液对绿色木霉菌落生长、孢子萌发和芽管长度的抑制作用。     

Antimicrobial activities of various essential oils against foodborne pathogenic or spoilage moulds

The use of essential oils in the food industry, as natural sanitizing agents, requires the definition of optimal conditions. The aim of the present work was to evaluate some antimicrobial activity parameters as mycelial growth inhibition, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of six essential oils against Aspergillus niger, Aspergillus terreus,Chaetomium globosum, Penicillium chrysogenum, Penicillium pinophilum, Trichoderma harzianum and Trichoderma viride. The antimicrobial activity of essential oils was monitored by the macrodiluition technique. The mycelial growth inhibition, fungistatic and fungicidal concentrations were recorded for each strain that showed sensitivity to the essential oils. The essential oils of catnip, cinnamon, tea tree and thyme essential oils exhibited a large spectrum antimicrobial activities; those of clary sage and laurel inhibited the mycelial growth in a few fungal strains. The essential oils of cinnamon and thyme had the lowest MIC and MFC values against all the fungi assayed, followed by catnip, tea tree, clary sage and laurel. The use of these natural products rather than, the currently used antifungal chemicals, may be of interest given that: i) essential oils are of natural origin which means they are safer for human health and the environment and ii) there is less chance that the pathogenic microorganisms will develop resistance.