Antagonistic actions of Pythium oligandrum and Trichoderma harzianum against phytopathogenic fungi (Fusarium oxysporum and Pythium ultimum var. ultimum)

Abstract

The possible biological control of damping-off fungi, Fusarium oxysporum and Pythium ultimum by Pythium oligandrum or Trichoderma harzianum was in vitro investigated. Results of comparing the antagonistic activity of P. oligandrum and T. harzianum in dual plates against the tested phytopathogens indicated different degrees of antagonism. After 12 days of incubation colony of the phytopathogenic fungus was completely overgrown by the antagonist, except for the interaction between T. harzianum and F. oxysporum which showed no overgrowth or any hyphal penetration by the antagonist. However, growth and proliferation of F. oxysporum colony was repressed. T. harzianum and P. oligandrum produced chitinase and β-1,3-glucanase when they were grown on liquid culture medium supplemented with chitin or fungal dried mycelium as a sole carbon source, and enzyme production was higher by T. harzianum comparing with P. oligandrum under the same condition. Fungal dried mycelium of F. oxysporum was the most selective carbon source for enzyme production, on the other hand, chitinase production was significant locked when P. ultimum dried mycelium was used as a carbon source. Production of volatile compounds by P. oligandrum or T. harzianum against F. oxysporum and P. ultimum was examined using the inverted plates method. F. oxysporum was inhibited by the antagonist volatile compounds and it is inhibited 100% by increasing the amount of inoculum size. Production of potential biocontrol agents provided with economically features and working under field conditions are recommended.     

Integration of Pythium nunn and Trichoderma harzianum isolate T-95 for the biological control of Pythium damping-off of cucumber

Two biological control agents, Pythium nunn and Trichoderma harzianum isolate T-95, were combined to reduce Pythium damping-off of cucumber in greenhouse experiments lasting 3–4 weeks. T. harzianum T-95, a rhizosphere competent mutant, was applied to seeds and P. nunn was applied to pasteurized and raw soils naturally and artificially infested with Pythium ultimum. Some treatments were also amended with bean leaves to enhance the activity of P. nunn. The biological control of Pythium damping-off was evaluated in a Colorado soil (Nunn sandy loam) and an Oregon soil mix, which were replanted twice after 2 and 3 months. Interactions between P. nunn and T-95 were detected in the Colorado but not the Oregon soil. No consistent evidence of antagonism between P. nunn and T. harzianum was seen, and significant interactions were detected in the Colorado, but not the Oregon soil. In the first planting of some treatments, the combination of P. nunn and T. harzianum gave greater control of damping-off than either applied alone. P. nunn was most effective in soils that were pasteurized or amended with bean leaves. T. harzianum controlled Pythium damping-off in the Colorado, but not the Oregon soil. In both soils, disease declined over time in treatments amended with bean leaves but without P. nunn or T. harzianum added. This suppression was greater in the Colorado soil, which contained an indigenous population of P. nunn. This work demonstrates that two compatible biological control agents can be combined to give additional control of a soil-borne plant pathogen.     

Comparative Antagonistic Properties of Gliocladium virens and Trichoderma harzianum on Sclerotium rolfsii and Rhizoctonia solani—its Relevance to Understanding the Mechanisms of Biocontrol

An isolate of Trichoderma harzianum which is less effective than G. virens in suppressing S. rolfsii and R. solani was compared with G. virens for various mechanisms of antagonism in vitro, viz., antagonism in dual culture/hyphal parasitism, parasitism of sclerotia and antibiosis. G. virens and T. harzianum were equally effective in parasitizing the hyphae of R. solani. Only T. harzianum parasitized the hyphae of S. rolfsii, and the two antagonists were comparable with respect to antibiosis on the test pathogens. However, G. virens readily parasitized the sclerotia of the test pathogens and was found to be more effective than T. harzianum in destroying the sclerotia. Under SEM, G. virens was found to colonize, penetrate, and sporulate inside the sclerotia of the test pathogens.Parasitism of sclerotia is suggested as the principal mechanism of biological control of S. rolfsii and R. solani by G. virens.     

Do Pathogenic Fungi Have the Potential to Inhibit Biocontrol Fungi?

Metabolites produced by the soilborne plant pathogen Rhizoctonia solani (R-23) and Pythium ultimum (PuZ3) grown on cellophane disks placed on potato-dextrose agar (PDA), molasses brewer's yeast agar (MYA) and wheat bran extract agar (BEA) did not significantly affect the rate of growth of isolates of the antagonists Trichoderma viride (TS-I-R3. Tv-101), T. harzianum (Th-57, Th-87), T. hamatum (Tm-23, TRI-4), or Gliocladiun virens (GI-3, GI-21) when these antagonists were grown on the three agars containing pathogen metabolites. However. in some instances. density of antagonist mycelium growing on the agar media as well as the observable production of antagonist conidia on the agar media were reduced. Using four antagonists in liquid cultures of potato-dextrose broth (PDB) containing metabolites of the pathogens grown on bran extract broth, metabolites from R-23 significantly reduced mycelial dry weight of Th-87 and GI-21 but not that of TRI-4 and GI-3. On the contrary. metabolites of PuZ3 increased the mycelial dry weight of all four antagonists, Metabolites of R-23 reduced production of conidia of only TRI-4; metabolites of PuZ3 significantly reduced production of conidia of all four antagonists. Pathogen metabolites did not affect germination of conidia in the system used.     

Native Trichoderma strains isolated from Bangladesh with broad spectrum antifungal action against fungal phytopathogens

Nineteen Trichoderma isolates, collected from different locations in Bangladesh, were characterised through phenotypic, biochemical and molecular means. Besides, they were assessed for their antifungal action in vitro. The isolates were divided into three groups: T. asperellum, T. virens and T. harzianum. A dual culture assay and a culture filtrate assay against 6 phytopathogens revealed that 9 of the 19 isolates showed significant antifungal activities. The isolate T. harzianum TR05 showed the highest inhibition against Fusarium oxysporum, Rhizoctonia solani, Fusarium circinatum and Phomopsis vexans, followed by T. asperellum TR08 and T. virens TR06. TR08 had the highest inhibition against Sclerotium rolfsii and Pythium aphanidermatum, followed by TR05 and TR06. These findings were in agreement with their activities of extracellular hydrolytic enzymes, including chitinase, β-1,3-glucanase, and proteinase. Our results suggest that isolates TR05, TR06 and TR08 have the potential to be effective biocontrol agents against the phytopathogenic fungi.     

Comparative efficacy of thermophilic bacterium,Bacillus licheniformis (NR1005) and antagonistic fungi,Trichoderma harzianum to control Pythium aphanidermatum-induced damping off in chilli (Capsicum annuum L.)

Damping off is a very serious disease in many field crops including chilli and its biological control has gained significance in recent times due to ill effects of chemicals. The effect of thermophilic bacterium, Bacillus licheniformis was evaluated to control Pythium aphanidermatum induced damping off in chilli (Capsicum annuum L.) under laboratory as well as pot culture conditions. B. licheniformis suppressed the growth of P. aphanidermatum equally as Trichoderma harzianum under laboratory conditions by dual culture technique. B. licheniformis treatment was at par with T. harzianum when studied in pot cultures. There was 81.18% and 83.16% inhibition of disease causing P. aphanidermatum with respect to infected control pots with B. licheniformis and T. harzianum, respectively. B. licheniformis used in present study is thermophilic in nature and hence the study has relevance in the context of failure of T. harzianum at higher temperature.     

Flavonoids in roots of white clover: interaction of arbuscular mycorrhizal fungi and a pathogenic fungus

The effects of two arbuscular mycorrhizal fungi (AMF) (Glomus mosseae and G. claroideum) and a pathogenic fungus (Pythium ultimum) on the production of eight flavonoids in roots of two white clover (Trifolium repens L.) cultivars were evaluated. Quantification of AM and pathogenic fungi in the roots showed that the AM symbiosis significantly reduced P. ultimum biomass and in some cases prevented infection. The flavonoid productions in clover roots varied depending on the presence of beneficial and/or pathogenic fungi, fungal isolate or plant cultivar. Only plants colonized with G. claroideum showed detectable concentrations of either coumestrol or kaempferol (cultivar-dependant). In addition, inoculation with G. claroideum resulted in significantly higher concentrations of coumestrol in cv. Sonja and medicarpin in cv. Milo. A low production of coumestrol and kaempferol in mycorrhizal plants may be G. mosseae-specific. Only the concentrations of formononetin and daidzein increased in clover roots in response to infection with P. ultimum. These flavonoids are supposedly stress metabolites, synthesized or produced from glycosides in response to pathogen infection. However, the presence of one or both AMF significantly lowered the formononetin and daidzein concentrations, and overruled the inductive effect of P. ultimum. Therefore the antagonistic action of AM against the pathogen must take place through another mechanism.     

Effect of antimicrobial activity of Melaleuca alternifolia essential oil on antagonistic potential of Pleurotus species against Trichoderma harzianum in dual culture

Melaleuca alternifolia (tea tree) essential oil was investigated for its “in vitro” ability to control Trichoderma harzianum, a fungal contaminant that causes extensive losses in the cultivation of Pleurotus species. The antifungal activity of M. alternifolia essential oil and antagonist activities between Pleurotus species against three T. harzianum strains were studied in dual-culture experiments on an agar-based medium in which different concentrations of essential oil were incorporated. M. alternifolia essential oil at a concentration of 0.625 μL/mL, inhibited T. harzianum mycelial growth by 5.9–9.0%, depending on the strain. At the same concentrations P. ferulae and P. nebrodensis stimulated mycelial growth by 5.2–8.1%. All strains of T. harzianum were antagonistic to the Pleurotus species in the control. When essential oil was added to the substrate cultural, the antagonistic activity of T. harzianum against the Pleurotus species was weak (0.0625 μL of essential oil) or non-existent (0.125 μL of essential oil). M. alternifolia essential oil could be an alternative to the synthetic chemicals that are currently used to prevent and control T. harzianum in mushroom cultivation.     

Control of the bean rust fungus <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> by means of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: leaf disc assays on the antibiotic effect of spore suspensions and culture filtrates

Several species of the fungal genus Trichoderma act as antagonists of other fungi. A number of strains from the Trichoderma species T. harzianum Rifai are used as biological control agents for the control of soilborne as well as foliar plant pathogens. Six T. harzianum strains, five of them isolated from commercial preparations, were evaluated for their capability to control the bean rust fungus Uromyces appendiculatus (Pers. ex Pers.) Unger. Different kinds of leaf disc assays were performed with conidial spore suspensions and sterile culture filtrates of the T. harzianum strains. Great differences were observed concerning the efficacy of the Trichoderma strains to reduce the number of the uredial pustules developing after rust inoculation which followed the application of the particular Trichoderma strains. Efficacy values ranged from 1 to over 50%. Increasing spore or culture filtrate concentrations of the two most effective isolates T12 and T_U led to decreases in the number of developing uredial pustules. Culture filtrate applications had a protective but no curative effect. T12 spore suspensions maintained their disease reducing activity even when autoclaved. This and some other evidence for an antibiotic interaction between T. harzianum and U. appendiculatus are discussed. Handling Editor: Reijo Karjalainen.     

Evaluation of seed dressing and soil application formulations of Trichoderma species for integrated management of dry root rot of chickpea

The efficacy of the newly developed seed dressing and soil application formulations of Trichoderma viride, T. virens and T. harzianum were evaluated individually and in combinations under pot and field experiments for the management of dry root rot (Rhizoctonia bataticola) of chickpea (Cicer arientinum). In pot experiments, T. harzianum based seed dressing formulation, Pusa 5SD, and soil application formulations, Pusa Biogranule 5 (PBG 5) and Pusa Biopellet 10G (PBP 10G), were found to be effective in reducing dry root rot incidence in chickpea and increasing the seed germination, shoot and root lengths of the crop. Under field experiments, a combination of soil application of T. harzianum based PBP 10G and seed treatment with Pusa 5SD+carboxin was found to be the best by providing the highest seed germination, shoot and root lengths and grain yield and the lowest dry root rot incidence in chickpea.     

Combined effect of an arbuscular mycorrhizal fungus and a biocontrol bacterium against<Emphasis Type="Italic">Pythium ultimum</Emphasis> in soil

The combined effect of the arbuscular mycorrhizal (AM) fungusGlomus intraradices and the bacteriumBurkholderia cepacia onPythium ultimum was studied in a growth system with root-free soil compartments allowing examination of microbial interactions without direct interference from roots. Signature fatty acids were used to quantify individual microbial populations. Biomass and amount of energy reserves were estimated using specific phospholipid fatty acids (PLFA) and neutral lipid fatty acids (NLFA), respectively. Population density ofP. ultimum was estimated by colony forming units on selective media. Both biocontrol agents (BCAs) reduced population density ofP. ultimum. Biomass ofB. cepacia measured using signature fatty acids was inhibited by the presence of mycelium ofG. intraradices, which might explain why no additive effect onP. ultimum was found when combining the two BCAs. In conclusion, combiningG. intraradices andB. cepacia did not enhance the biocontrol efficacy againstP. ultimum.     

Synergistic effects of Trichoshield on enhancement of growth and resistance to downy mildew in pearl millet

Trichoshield, a talc formulation consisting of spores of Trichoderma harzianum, Trichoderma lignorum, Gliocladium virens and Bacillus subtilis was tested, following different application methods, for its ability to promote growth of pearl millet plants and to induce resistance to downy mildew of pearl millet. Under laboratory conditions, trichoshield seed treatment enhanced seed germination and seedling vigor of pearl millet significantly over the control; under greenhouse conditions vegetative growth parameters like height, fresh and dry weight, leaf area and number of tillers were significantly enhanced over the control: Trichoshield formulation offered greater protection against downy mildew in comparison with individual strains of T. harzianum, T. lignorum, G. virensand B. subtilis. Among the methods of application, foliar spray was found to be a more efficient delivery method than seed treatment or slurry treatment. Combinations of foliar spray with seed treatment and slurry treatment produced the same effect as foliar spray alone. Under field conditions, trichoshield treatment enhanced reproductive parameters like number of earheads, length and girth of earheads, 1000 seed weight and yield significantly over the control. Days required for 50% flowering was reduced by 4 days compared to the control. Yield enhancement of 28% over the control was highly significant. Trichoshield treatment offered protection ranging from 52 to 71% under field conditions, depending on the application method. However, the chemical fungicide metalaxyl Apron provided the highest protection against downy mildew, both under greenhouse and field conditions.     

Influence of bio-inoculants on the control of root rot and wilt disease of pyrethrum caused by Rhizoctonia solani

The effect of four bio-inoculants namely, Glomus aggregatum, Streptomyces sp., Bacillus subtilis and Trichoderma harzianum and a fungicide, Ridomil-mancozeb was evaluated on the biomass production and control of root rot and wilt disease under glasshouse conditions. Results showed that 21-day- prior inoculation with G. aggregatum was most effective where none of the treated plants produced disease symptoms and interestingly their growth was increased by 39.4%. The colonisation by G. aggregatum (>80%) also increased P concentration in shoot. While, similar treatment with Streptomyces sp., B. subtilis, T. harzianum and Ridomil-mancozeb individually failed to produce any significant effect over Rhizoctonia solani inoculated control, where all inoculated plant died, prematurely. The simultaneous and 3-day-post treatments of G. aggregatum were non-effective but simultaneous treatment with Streptomyces sp. produced 70% disease control, while B. subtilis and T. harzianum individually provided 50% control. Their effects were either better or at par when compared with the simultaneous treatment of Ridomil-mancozeb. In 3-day-post treatment, Streptomyces, B. subtilis and Ridomil-mancozeb individually provided 50% disease control, whereas T. harzianum was least effective as it could protect only 25% plants against infection. The results reveals that 15 days prior treatment of G. aggregatum can significantly controls the root rot and wilt disease of pyrethrum. Further, treatment of Streptomyces can also serve the next effective post infection control method.     

Ultrastructure of Mycoparasitism of Trichoderma, Gliocladium and Laetisaria Species on Botryodiplodia theobromae

Mycoparasitic activities of various isolates of Trichoderma viride, T. harzianum, T. hamatum, T. longibrachiatum, T. koningii, T. pseudokoningii, Gliocladium virens and Laetisaria arvalis were studied against a serious plant pathogen, Botryodiplodia theobromae by scanning electron microscopy (SEM). Macroscopic observations of fungal growth in dual-cultures revealed that most of the isolates made hyphal contact with the pathogen within 2 days after inoculation, leading to the inhibition in pathogen growth. However, T. viride Tv-4, T. hamatum and T. pseudokoningii inhibited pathogen growth before hyphal contact and exhibited an inhibition zone between the colonies of both fungi. SEM investigations demonstrated that in case of hyphal interaction, the firm binding of antagonists (T. viride Tv-1 & Tv-3, T. harzianum Th-1 & Th-2, T. longibrachiatum and L. arvalis) to B. theobromae hyphae established either by coiling around its hyphae, or by penetrating its hyphal cells by forming hooks, haustoria and appressoria-like structures which invariably led to cell disruption. Although T. koningii and G. virens Gv-2 & Gv-3 did not interact physically by way of coiling and penetration, they produced wall lytic enzymes or antifungal substances after coming in contact with B. theobromae which caused wrinkling, bursting and collapsing of pathogen mycelium. It is, therefore, suggested that the outcome of the interaction of antagonist and pathogen was most likely determined by initial hyphal contact that triggered a series of events in pathogen degradation.     

Production of Inter- and Intra-strain Hybrids of Trichoderma spp. by Protoplast Fusion and Evaluation of Their Biocontrol Activity Against Soil-borne and Foliar Pathogens

Protoplast fusion techniques were used for the production of new antagonistic strains of Trichoderma spp. Two British and two Italian strains of Trichoderma with different biocontrol potential against Botrytis cinerea, Sclerotinia sclerotiorum and Pythium ultimum, were treated with u.v. radiation and mutants resistant to hygromycin B or propiconzole were selected. Protoplasts were obtained from these mutants after digestion of young hyphae with Novozym 234 and used in inter- and intra-strain protoplast fusion experiments. Hybrids were obtained in 13 crosses isolated on fungicide-amended media by the inheritance of resistance markers from both parental strains. Selected, fast-growing, stable mutants were tested in biocontrol trials against P. ultimum on lettuce seedlings and B. cinera on grape bunches in comparison with their parental strains. Intra-strain hybrids derived from cross XIII were tested in vitro for their mycoparasitic ability on sclerotia of S. sclerotiorum. A high degree of variability in the biocontrol and the mycoparasitic ability of the fusants was observed but no significant increase in the activity was accomplished after fusion, the hybrids being generally less activewas accomplished after fusion, the hybrids being generally less active than their parental strains.     

Mycotic flora of the interdigital spaces of the human foot: A preliminary investigation

Culture filtrates of Trichoderma harzianum Rifai have been found to inhibit zoospore germination, germ tube elongation and mycelial growth of Pythium aphanidermatum causing the damping-off disease of tobacco in Nigeria. Further, the invasion of the hyphae of P. aphanidermatum by hyphae of T. harzianum has been demonstrated in mixed cultures of the two fungi. The results of investigations on the role of T. harzianum as an agent of biological control of the damping-off disease in sterilized and unsterilized soils are reported and discussed.     

Interaction of nutrients and four antifungal antibiotics in their effects on Pythium species in vitro and in soil

Summary Griseofulvin, streptomycin, aureomycin, and actidione, with and without nutrients, were tested againstPythium ultimum Trow (two strains),P. irregulare Buis., andP. rostratum Butler. Streptomycin was tolerated in the highest concentration, and actidione and aureomycin in the lowest.P. rostratum, a saprophytic species, was more resistant to the antibiotics than the pathogenic species, which were inhibited at low concentrations. The two strains ofP. ultimum behaved differently, the oogonial strain being more tolerant than the sporangial strain. Yeast extract was usually superior to sucrose or asparagine in counteracting the toxicity of antibiotics.Germination of sporangia ofPythium irregulare was studiedin vitro and in a natural soil in the presence of antibiotic and antibiotic-nutrient solutions. Very meager germination of sporangia occurred in soil amended with antibiotics. Actidione severely restricted germination in soil. Nutrients nullified the toxicity to varying degrees. Usually higher germination of sporangia occurredin vitro than in soil. Sub-lethal concentrations of antibiotics induced morphological abnormalities in germ tubes. Aureomycin caused a dendroid type of branching; actidione caused distortion, vacuolation, and vesiculation; griseofulvin caused lysis and septation; and streptomycin caused distortion.Germ tubes were lysed in a natural soil and in soils amended with antibiotics, but nutrients, in general, prevented early lysis at low concentrations of antibiotics. No resistant bodies were formed prior to the initiation of lysis of germ tubes.     

Biocontrol potential of Trichoderma species against mango malformation pathogens

Malformation disease of Mango (Mangifera indica L.) caused by Fusarium moniliforme var. subglutinans is one of the most destructive diseases, which is a major production constraint in the mango-growing regions of India. In this study, The bioagents Trichoderma viride (Tr1), Trichoderma virens (Tr2) and Trichoderma harzianum (Tr3) were evaluated in culture with the pathogens to monitor the antagonistic effect and their volatile compound and culture filtrates (non-volatile compound). It was found that all the three isolates of bioagents significantly checked the growth of F. moniliforme var. subglutinans. In dual culture, the best result was obtained with T. harzianum followed by T. virens and T. viride. A similar result was also observed in the case of culture filtrates ofTrichoderma spp. The results clearly showed that inhibition of the growth of the fusaria isolates by T. harzianum was significantly superior to T. viride andT.virens. In case of antifungal activity of volatile compounds released by Trichoderma isolates, it was also observed that T. virens was more superior to T.harzianum and T. viride.     

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.     

Use of Trichoderma harzianum cultured on ground mesocarp fibre of oil-palm as seed treatment to control Pythium aphanidermatum,a causal agent of damping-off of Chinese kale seedling

Trichoderma harzianum, isolate T 01-22, was cultured on either sorghum grains, ground mesocarp fibre of oil-palm or oil-palm shell, both amended with urea fertilizer (100:1, w/w). T. harzianum cultured on ground mesocarp fibre was then used to coat seeds of Chinese kale (Brassica alboglabra Bailey) to control damping-off of seedlings caused by Pythium aphanidermatum. Biomass of T. harzianum cultured on ground mesocarp fibre of oil-palm was more effective than Captan and Benomyl, but less effective than Metalaxyl, in controlling damping-off of Chinese kale seedlings. Viability of T. harzianum growing on sorghum grains was reduced significantly during 7 months storage, followed by that of T. harzianum cultured on ground mesocarp fibre and oil-palm shell, both amended with urea fertilizer (46-0-0) at 100:1 (w/w).