Isolation of Pythium oligandrum from Egyptian soil and its mycoparasitic effect on Pythium ultimum var. ultimum the damping-off organism of wheat

Pythium oligandrum Drechsler bearing spherical sporangia with complex subglobose elements was isolated for the first time in Egypt from agricultural field soil cultivated with alfalfa (Trifolium alexandrinum) in El-Minia, Egypt. This fungus was found to be an active bio-control agent against P. ultimum var. ultimum, the damping-off organism of wheat. In agar plates, P. oligandrum parasitized P. ultimum var. ultimum hyphae with the aid of thin haustorial branches or infection pegs, eventually leading to host destruction. Incorporation of P. oligandrum into carboxymethylcellulose seed coating successfully eliminated pre-emergence damping-off of wheat caused by P. ultimum var. ultimum, whereas Post-emergence damping-off was prevented by adding inocula of P. oligandrum to the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Antagonistic effect of two isolates of Trichoderma harzianum against postharvest pathogens of tomato (Lycopersicon esculentum)

Pathogenicity test of all fungi (14 different isolates) isolated from both infected tomato fruits and the surface wash of other healthy fruits had different pathogenicity rates. The genus Rhizopus sp. was the most pathogenic one followed by Fusarium sp. Trichoderma harzianum isolates (T3 and T4) had a different pattern of antagonism against the tested pathogens. In dual plate test of the antagonistic action of T3 and T24 against the postharvest pathogens, clear zone size ranged between 1 and 4 and 3 and 6 mm by T3 and T24, respectively. The antagonists (T3 and T24) didn't show inhibition zone against Rhizopus sp., but they could overgrow it by 100% after 9 days of incubation. Mostly, all the other postharvest isolates showed high degree of overgrowth by T3 than T24. The two antagonists failed to overgrow Aspergillus species except for A. niger (3) which was overgrown by T3. Volatile and non-volatile metabolite tests indicated that mycelial growth of Penicillium stekii was significantly inhibited by T3 and T24 more than the other tested pathogens. The inhibition of A. niger (1) was 12% by non-volatile metabolites of T24 produced after 1 day incubation, and reached to 97% inhibition by the metabolites of 3 days. Interestingly, inhibition of Aspergillus sp. by volatile compounds of T3 and T24 was 2% and 20%, respectively, whereas the inhibition of the same pathogen by non-volatile compounds reached 75% and 87%, respectively. The results of slice assay clearly indicate that T. harzianum (T3 and T24) could provide a complete protection to tomato slices from the infection of the tested pathogens. After 3 days of incubation, Trichoderma suppressed the linear growth of these pathogens on tomato slices and the percentage of suppression was significant and ranged between 80 and 100%, except with Rhizopus sp. the suppression reached 33% only.     

Proteins Related to the Biocontrol of Pythium Damping-off in Maize with Trichoderma harzianum Rifai

Induced resistance has been evidenced as one of mechanisms of Trichoderma to control plant diseases, however, no study showed the change of host proteomics in Trichoderma-induced resistance of maize against damping-off caused by Pythium ultimum Trow. The mechanism of Trichoderma harzianum Rifai for controlling maize seedling disease caused by Pythium ultimum Trow was investigated firstly by proteome technique and the result suggested that T. harzianum strain T22 was not only able to promote seedling growth but also protein accumulation. One-dimensional electrophoresis assay showed that more bands appeared on the gel with T22 or T22 combined with P. ultimum （T22 ＋ P. ultimum） treatment than with other treatments. Enzyme assay showed that two chitinases of the root sample were more activated in the treatments with T22 than in the other treatments without T22. Proteins in the seedling roots from the various treatments were separated through protein extraction and 2-D electrophoresis technique. In the seedlings produced from the T22-treated seeds, there were 104 up-regulated proteins and 164 down-regulated proteins relative to the control, and 97 and 150, respectively, aftel treatment with T22 ＋ P. ultimum; however, with P. ultimum alone the values were much lower than with the other two treatments. The correlation coefficient values were 0.72, 0.51 and 0.49 for the comparison of protein spot distribution on gel among control with T22, P. ultimum and T22 ＋ P. ultimum, respectively. So it seemed that P. ultimum infection was more effective than T22 in interfering with the host proteome profile. Furthermore, analysis with MALDITOF-MAS showed that some important proteins associated with defensive reactions were identified in T22 or T22 ＋ P. ultimum treatments, including endochitinase, pathogenesis-related protein PRMS （pathogenesis-related maize seed）, GTP-binding protein, isoflavone reductase and other proteins related to respiration. All those proteins are probably part of the network of resistance or development-related proteins. Interestingly, P. ultimum treatment resulted in elimination of pathogenesis-related protein PRMS on gel, and therefore damping-off could be in part attributed to inhibition of the expression of this protein by P. ultimum infection. Some unknown proteins are also related to the defensive reaction of the host.     

Antagonistic activity and hyphal interactions of Trichoderma spp. against Fusarium proliferatum and F. oxysporum in vitro

Trichoderma is a well-known antagonist against soilborne plant pathogens. However, the species and even various isolates have different biocontrol potential. To evaluate the antagonistic activities of Trichoderma harzianum, T. harzianum strain T100 (T100), T. viride and T. haematum against Fusarium oxysporum and F. proliferatum, we used dual culture and productions of volatile and non-volatile metabolites in three different phases in vitro. An analysis of the data in dual culture tests represented T. viride, T. haematum and T100 as effective antagonists of Fusarium while T100 was the only fungus being able to lyse the confronting mycelia. Similar results were obtained in the volatile metabolites tests also. In contrast with the two previous tests, the non-volatile metabolites produced by T. harzianum inhibited Fusarium mycelial growth the most, and T100 acted moderately. It was also clearly showed that the antagonistic effect of Trichoderma spp. was more on F. proliferatum than on F. oxysporum. Finally, because Trichoderma spp. was most effective in the second phase, we recommend to use T100 against F. proliferatum at the initial stages of infection as its mycoparasitism on F. oxysporum was observed microscopically through forming apressoria structures without any coiling around the pathogen.     

Biological control of Fusarium oxysporum f. sp. radicis-cucumerinum by some Trichoderma harzianum isolates

The purpose of this study was to investigate the effects of isolates T₂₂, T₉ and T₆ of Trichoderma harzianum on isolate F₄₂ of Fusarium oxysporum f. sp. radicis-cucumerinum. The effect of T. harzianum isolates on controlling disease was examined under greenhouse conditions. Results showed that these three isolates, respectively, had the high effect on inhibition of pathogen growth. In examining the severity of disease in the greenhouse, it was found that isolate T₂₂ had the greatest effect on controlling the pathogen. The results of volatile compounds showed that these isolates, respectively, were effective in reducing mycelial growth of isolate F₄₂. The highest peroxidase activity was observed on the fourth day in isolate T₆ and the highest phenylalanine ammonia lyase enzyme activity was observed on the fifth day in isolate T₂₂. Based on the results, isolate of T₂₂ showed the greatest effect on the induction of resistance against F₄₂ and may be a successful agent for biological control of root and stem rot of cucumber.     

Seed biopriming with novel strain of Trichoderma harzianum for the control of toxigenic Fusarium verticillioides and fumonisins in maize

Fusarium verticillioides is one of the most important fungal pathogens in maize causing both pre- and post-harvest losses and also capable of producing Fumonisins. In the present study attempts have been made for screening potential T. harzianum from native rhizosphere and to study its effect on Fusarium ear rot disease, fumonisin accumulation in different maize cultivars grown in India. Eight isolates of T. harzianum were isolated and T. harzianum isolate Th-8 exhibited better antifungal activity than carbendizim. Th-8 was formulated in different solid substrates like wheat bran, paddy husk, talcum powder and cornstarch. Maize seeds of kanchan (moderately resistant), pioneer (resistant) and sweet corn (susceptible) were selected for laboratory and field studies and these seeds were treated with a conidial suspension of T. harzianum at the rate of 1 × 10⁸ spore/ml and formulation at the rate of 10 g/kg. Treated seeds were subjected to evaluate F. verticillioides incidence, seed germination, seedling vigour and field emergence, yield, thousand seed weight and fumonisin production. It was found that the pure culture of T. harzianum was more effective in reducing the F. verticillioides and fumonisin incidence followed by Talc formulation than the carbendizim treated and untreated control. Formulations of T. harzianum were effective at reducing the F. verticillioides and Fumonisin infection and also increasing the seed germination, vigour index, field emergence, yield, and thousand seed weight in comparison with the control.     

Biocontrol efficacy of Trichoderma spp. against sesame wilt caused by Fusarium oxysporum f. sp. sesami

Sesame (Sesamum indicum L.) is one of the most important oilseed crops in Egypt and worldwide. It is being infected with many pathogens, among these pathogens Fusarium oxysporum f.sp. sesami (Zap.) Cast is causing severe economic losses on sesame. In this study, antagonistic capability of 24 isolates of Trichoderma spp. was assessed in vitro against F. oxysporum f.sp. sesami. Two strains; T. harzianum (T9) and T. viride (T21) were revealed to have high antagonistic effect against F. oxysporum f.sp. sesami in vitro with inhibition percentage about 70 and 67%, respectively. These two isolates proved to have high ability to control Fusarium wilt disease under greenhouse conditions. The highest reduction in disease severity was achieved with T. viride followed by T. harzianum with reduction in disease severity about 77 and 74%, respectively. This study revealed that the time of application of bioagents is a decisive factor in determining the efficacy of Trichoderma isolates to control Fusarium wilt of sesame. It was revealed that the highest reduction in the disease severity was achieved when either Trichoderma viride or T. harzianum were applied 7 days before challenging with the F. oxysporum f.sp. sesami.     

The effect of Trichoderma harzianum and T. koningii on the control of tan spot (Pyrenophora tritici-repentis) and leaf blotch (Mycosphaerella graminicola) of wheat under field conditions in Argentina

The effect of six isolates of Trichoderma harzianum and one isolate of T. koningii on the incidence and severity of tan spot (Pyrenophora tritici-repentis) and leaf blotch of wheat (Mycosphaerella graminicola) was evaluated under field conditions. Significant differences between wheat cultivars, inoculum types and growth stages were found. Three of the isolates tested (T2 for M. graminicola, T7 for P. tritici-repentis and T5 for both of them) showed the best performance in controlling leaf blotch and tan spot when coated onto seed or sprayed onto wheat leaves at different growth stages, with significant severity reduction up to 56%. At tillering, six of the isolates reduced the severity of P. tritici-repentis and M. graminicola compared to the control by up to 39% and 12-53%, respectively. In some experiments, the biocontrol preparation (T2 and T5) gave a level of disease control similar to that obtained with Tebuconazole (70 and 48%, respectively). The effect of Trichoderma against P. tritici-repentis was also observed at the heading stage, when six of the treatments reduced disease severity by 16-35%. This is the first report on the efficacy of Trichoderma spp. against wheat necrotrophic pathogens under field conditions in Argentina.     

Cloning and heterologous expression of SS10, a subtilisin-like protease displaying antifungal activity from Trichoderma harzianum

Trichoderma harzianum parasitizes a large variety of phytopathogenic fungi. Trichoderma harzianum mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. A gene ( SS10 ) encoding a subtilisin-like protease was cloned from T. harzianum T88, a biocontrol agent effective against soil-borne fungal pathogens. The full-length cDNA was isolated by 5' and 3' rapid amplification of the cDNA ends. The coding region of the gene is 1302 bp long, encoding 433 amino acids of a predicted protein with a molecular mass of 45 kDa and a pI of 6.1. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) (EC 3.4.21.) and had a predicted active site made up of the catalytic residues Asp 187, His 218 and Ser 376. Northern experiments demonstrated that SS10 was induced in response to different fungal cell walls. Subtilisin-like protease gene SS10 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. The enzyme activity culminates (17.8 U mL⁻¹) 60 h after induction with galactose. The optimal enzyme reaction temperature was 50 °C and the optimal pH was 8. The subtilisin-like protease exerted broad-spectrum antifungal activity against Alternaria alternata, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum and Cytospora chrysosperma .     

Trichoderma harzianum antagonistic activity and competition for seed colonization against seedborne pathogenic fungi of sunflower

This study investigated the antagonistic effects of Trichoderma harzianum isolate (TRIC8) on mycelial growth, hyphal alteration, conidial germination, germ tube length and seed colonization by the seedborne fungal pathogens Alternaria alternata, Bipolaris cynodontis, Fusarium culmorum and F. oxysporum, the causes of seedling rot in over 30% of sunflowers. The antagonistic effect of TRIC8 on mycelial growth of pathogens was evaluated on dual culture that included two inoculation assays: inoculation of antagonist at 48 h before pathogen (deferred inoculation) and inoculation at the same time with pathogen (simultaneous inoculation). TRIC8 inhibited mycelial growth of the fungal pathogens between 70·67 and 76·87% with the strongest inhibition seen with deferred inoculation. Alterations in hyphae were observed in all pathogens. Conidial germination of F. culmorum was inhibited by most of the fungal pathogens (38·28%) by TRIC8. Inhibition of germ tube length by the antagonist varied from 31·83 to 37·67%. In seed colonization experiments, TRIC8 was applied in combination with each pathogen to seeds of a sunflower genotype that is highly tolerant to downy mildew. Seed death was inhibited by TRIC8 and the antagonist did not allow growth of A. alternata, B. cynodontis and F. culmorum on seeds and inhibited the growth of F. oxysporum at the rate of 58·32%.     

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.     

Structural and mechanistic analysis of engineered trichodiene synthase enzymes from Trichoderma harzianum: towards higher catalytic activities empowering sustainable agriculture

Trichoderma spp. are well-known bioagents for the plant growth promotion and pathogen suppression. The beneficial activities of the fungus Trichoderma spp. are attributed to their ability to produce and secrete certain secondary metabolites such as trichodermin that belongs to trichothecene family of molecules. The initial steps of trichodermin biosynthetic pathway in Trichoderma are similar to the trichothecenes from Fusarium sporotrichioides. Trichodiene synthase (TS) encoded by tri5 gene in Trichoderma catalyses the conversion of farnesyl pyrophosphate to trichodiene as reported earlier. In this study, we have carried out a comprehensive comparative sequence and structural analysis of the TS, which revealed the conserved residues involved in catalytic activity of the protein. In silico, modelled tertiary structure of TS protein showed stable structural behaviour during simulations. Two single-substitution mutants, i.e. D109E, D248Y and one double-substitution mutant (D109E and D248Y) of TS with potentially higher activities are screened out. The mutant proteins showed more stability than the wild type, an increased number of electrostatic interactions and better binding energies with the ligand, which further elucidates the amino acid residues involved in the reaction mechanism. These results will lead to devise strategies for higher TS activity to ultimately enhance the trichodermin production by Trichoderma spp. for its better exploitation in the sustainable agricultural practices.     

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.     

Cloning and heterologous expression of aspartic protease SA76 related to biocontrol in Trichoderma harzianum

Trichoderma harzianum is a soil-borne filamentous fungus that exhibits biological control properties because it parasitizes a large variety of phytopathogenic fungi. The production of hydrolytic enzymes appears to be a key element in the parasitic process. Among the enzymes released by Trichoderma, the aspartic proteases play a major role. A gene (SA76) encoding an aspartic protease was cloned by 3' rapid amplification of cDNA ends from T. harzianum T88. The coding region of the gene is 1,593 bp long, encoding a polypeptide of 530 amino acids with a predicted molecular mass 55 kDa and a pI of 4.5. The catalytic aspartic residues characteristic of aspartic proteases are conserved with an active-site motif (DSG); however, the DSG in the N-terminal lobe is unusual in that Ser replaced Thr. Northern blot analysis indicated that SA76 was induced in response to different fungal cell walls. Aspartic protease SA76 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. The enzyme activity culminates (10.5 U mL(-1)) 72 h after induction with galactose. The temperature optimum of the enzyme was 45 degrees C and its pH optimum was 3.5. The culture supernatant of the S. cerevisiae strain that expressed the aspartic protease SA76 was able to inhibit the growth of five phytopathogenic fungi. The inhibition of mycelial growth varied between 7% and 38%.     

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.     

Biological Control of Phytophthora Root Rot of Pepper Using Trichoderma harzianum and Streptomyces rochei in Combination

A combination of two compatible micro‐organisms, Trichoderma harzianum and Streptomyces rochei, both antagonistic to the pathogen Phytophthora capsici, was used to control root rot in pepper. The population of the pathogen in soil was reduced by 75% as a result. Vegetative growth of the mycelium of P. capsici was inhibited in vitro on the second day after P. capsici and T. harzianum were placed on the opposite sides of the same Petri plate. Trichoderma harzianum was capable of not only arresting the spread of the pathogen from a distance, but also after invading the whole surface of the pathogen colony, sporulating over it. Scanning electron microscopy showed the hyphae of P. capsici surrounded by those of T. harzianum, their subsequent disintegration, and the eventual suppression of the pathogen's growth. Streptomyces rochei produced a zone of inhibition, from which was obtained a compound with antioomycete property secreted by the bacteria. When purified by high‐pressure liquid chromatography, this compound was identified as 1‐propanone, 1‐(4‐chlorophenyl), which seems to be one of the principal compounds involved in the antagonism. A formulation was prepared that maintained the compound's capacity to inhibit growth of the pathogen for up to 2 years when stored at room temperature in the laboratory on a mixture of plantation soil and vermiculite. The two antagonists, added as a compound formulation, were effective at pH from 3.5 to 5.6 at 23–30°C. The optimal dose of the antagonists in the compound formulation was 3.5 × 10⁸ spores/ml of T. harzianum and 1.0 × 10⁹ FCU/ml of S. rochei. This is the first report of a compound biocontrol formulation of these two antagonists with a potential to control root rot caused by P. capsici.     

Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM

A novel 36-kDa endochitinase named chit36 has been isolated and characterized from Trichoderma harzianum Rifai TM. Partial amino acid sequences from the purified protein were used to clone the fungal cDNA, based on polymerase chain reaction with degenerate primers. The complete open reading frame encodes a 344-amino acid protein which shows 84% similarity to a putative chitinase from Streptomyces coelicolor. Chit36 was overexpressed under the pki1 constitutive promoter from Trichoderma reesei via biolistic transformation of T. harzianum TM. Stable transformants showed expression and endochitinase activity of chit36 in glucose-rich medium. Culture filtrates containing secreted CHIT36 as the sole chitinolytic enzyme completely inhibited the germination of Botrytis cinerea conidia. Growth of Fusarium oxysporum f. sp. melonis and Sclerotium rolfsii were significantly inhibited on agar plates on which the Trichoderma transformants had previously been grown.     

Antagonistic potential of Gluconacetobacter diazotrophicus against Fusarium oxysporum in sweet potato (Ipomea batatus)

Gluconacetobacter diazotrophicus, an endophytic diazotroph also encountered as rhizosphere bacterium, is reported to possess different plant growth promoting characteristics. In this study, we assessed the biocontrol potential of G. diazotrophicus under in vitro conditions with soil-borne plant pathogenic Fusarium oxysporum. The possible compounds involved in the biocontrol involves 2,4-diacetylphloroglucinol, Pyrrolnitrin and Pyoluteorin. Thin layer chromatography analysis revealed that G. diazotrophicus produced the antibiotic, Pyoluteorin which helped in the suppression of soil-borne pathogenic. The volatile compounds of G. diazotrophicus also inhibited the growth of F. oxysporum. Tests for the production of hydrogen cyanide indicated that G. diazotrophicus does not produced HCN.     

Co-entrapment of Trichoderma harzianum and Glomus sp. within alginate beads: impact on the arbuscular mycorrhizal fungi life cycle

Aims: This study was performed to explore the compatibility and applicability of plant beneficial micro‐organisms (i.e. Trichoderma harzianum MUCL 29707 and Glomus sp. MUCL 41833) co‐entrapped in alginate beads. Methods and Results: Spores of Glomus sp. and conidia of T. harzianum were immobilized in alginate beads and the impacts of the saprotrophic fungi on the presymbiotic and symbiotic phase of the arbuscular mycorrhizal (AM) fungi evaluated under strict in vitro culture conditions. Our results demonstrated the capacity of both micro‐organisms in combination to regrowth outside the calcium alginate coating. The presence of T. harzianum did not hinder the AM fungal development but rather stimulated its spore production and fitness. Conclusions: The combination of T. harzianum MUCL 29707 with Glomus sp. MUCL 41833 in alginate beads may represent a reliable alternative inoculum formulation for application in sustainable agriculture. Significance and Impact of the Study: The entrapment in the alginate beads of two fungi (i.e. a saprotroph and a symbiont) having beneficial effects on plants represents a promising formulation for the development of inoculants adapted to field application.