Trichoderma as biological control agent: scope and prospects to improve efficacy

World Journal of Microbiology and Biotechnology - Industrialisation, directly or indirectly, exposes humans to various xenobiotics. The increased magnitude of chemical pesticides and toxic heavy...     

Evaluation of Trichoderma harzianum strain T22 as biological control agent of Calonectria pauciramosa

The objective of this research was to evaluate Trichoderma harzianum strain T22 as a biocontrol agent of collar and root rot caused by different Calonectria pauciramosa isolates. Thus, the microsclerotia-forming ability and virulence of twenty C. pauciramosa isolates were assessed. Microsclerotia production varied partially among the isolates and dual culture with T22 on carnation leaf agar revealed isolates with both high and low microsclerotia-forming ability. Inoculation tests on red clover (Triflolium pratense) demonstrated its susceptibility to the pathogen. On red clover, the degree of virulence and T22 effects in controlling infections were highly variable among the isolates tested. A nursery trial performed on Feijoa sellowiana seedlings confirmed previous results, clearly indicating virulence variability among C. pauciramosa isolates. For three isolates tested in nursery trial, T22 effectiveness in controlling infection was inversely related to their degree of virulence. Overall, T. harzianum strain T22 showed good antagonist activity in reducing microsclerotia production on carnation leaf and the incidence and severity of collar and root rot on both selected hosts. This data could be crucial in developing integrated pest management strategies in ornamental plant nurseries.     

qRT-PCR quantification of the biological control agent Trichoderma harzianum in peat and compost-based growing media

To ensure proper use of Trichoderma harzianum in agriculture, accurate data must be obtained in population monitoring. The effectiveness of qRT-PCR to quantify T. harzianum in different growing media was compared to the commonly used techniques of colony counting and qPCR. Results showed that plate counting and qPCR offered similar T. harzianum quantification patterns of an initial rapid increase in fungal population that decreased over time. However, data from qRT-PCR showed a population curve of active T. harzianum with a delayed onset of initial growth which then increased throughout the experiment. Results demonstrated that T. harzianum can successfully grow in these media and that qRT-PCR can offer a more distinct representation of active T. harzianum populations. Additionally, compost amended with T. harzianum exhibited a lower Fusarium oxysporum infection rate (67%) and lower percentage of fresh weight loss (11%) in comparison to amended peat (90% infection rate, 23% fresh weight loss).     

The expression of extracellular fungal cell wall hydrolytic enzymes in different Trichoderma harzianum isolates correlates with their ability to control Pyrenochaeta lycopersici

Four isolates of Trichoderma harzianum (ThN3, Th11, Th12 and Th16) were selected for their ability to control the in vitro development of the tomato root pathogen Pyrenochaeta lycopersici. Analysis of the mechanisms involved in biocontrol showed that the formation of non-volatile metabolites appears to be one of those involved in biocontrol of P. lycopersici by all T. harzianum isolates tested. Nevertheless, the higher secretion of chitinases, both in number of isoenzymes and activity by the Th11 strain, correlated well with its higher ability to control this agent in laboratory and greenhouse experiments as compared to the other T. harzianum isolates tested. The secretion of beta-1,3-endoglucanases and/or proteases appeared to have less significance than endochitinases in the biological control of P. lycopersici.     

Physiological and biochemical characterization of Trichoderma harzianum, a biological control agent against soilborne fungal plant pathogens.

Monoconidial cultures of 15 isolates of Trichoderma harzianum were characterized on the basis of 82 morphological, physiological, and biochemical features and 99 isoenzyme bands from seven enzyme systems. The results were subjected to numerical analysis which revealed four distinct groups. Representative sequences of the internal transcribed spacer 1 (ITS 1)-ITS 2 region in the ribosomal DNA gene cluster were compared between groups confirming this distribution. The utility of the groupings generated from the morphological, physiological, and biochemical data was assessed by including an additional environmental isolate in the electrophoretic analysis. The in vitro antibiotic activity of the T. harzianum isolates was assayed against 10 isolates of five different soilborne fungal plant pathogens: Aphanomyces cochlioides, Rhizoctonia solani, Phoma betae, Acremonium cucurbitacearum, and Fusarium oxysporum f. sp. radicis lycopersici. Similarities between levels and specificities of biological activity and the numerical characterization groupings are both discussed in relation to antagonist-specific populations in known and potential biocontrol species.     

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.     

Biological spectrum of Trichoderma harzianum Rifai isolates to control fungal diseases of tomato (Solanum lycopersicon L.)

Three Trichoderma harzianum isolates viz., Th-Sks, Th-Ke and Th-Ar collected from respective states of India viz., Rajasthan, Kerala and Andhra Pradesh were evaluated for the management of six fungal diseases namely damping off, Fusarium wilt, Rhizoctonia wilt, early leaf spot, late blight and Septoria leaf spot in tomato. During in vitro analysis, T. harzianum isolates inhibited the pathogens’ growth. Isolate Th-Sks was the most virulent antagonist against all the test pathogens and exhibited maximum of 79.47% growth inhibition of Phytophthora infestans. Isolate Th-Sks proved most effective at suppression efficacy in the range of 95–100% and 91–100% against all diseases under glasshouse and in the field conditions, respectively. Tomato seeds treatment with isolate Th-Sks also promoted plant height (78.23 cm) and fruits yield (290 g/plant) during field trial and data were found to be not-significantly different from other isolates. Thus, it is concluded that isolate Th-Sks can be utilised as a biocontrol agent for management of fungal diseases in tomato.     

Marine isolates of Trichoderma spp. as potential halotolerant agents of biological control for arid-zone agriculture

The scarcity of fresh water in the Mediterranean region necessitates the search for halotolerant agents of biological control of plant diseases that can be applied in arid-zone agriculture irrigated with saline water. Among 29 Trichoderma strains previously isolated from Mediterranean Psammocinia sp. sponges, the greatest number of isolates belong to the Trichoderma longibrachiatum-Hypocrea orientalis species pair (9), H. atroviridis/T. atroviride (9), and T. harzianum species complex (7), all of which are known for high mycoparasitic potential. In addition, one isolate of T. asperelloides and two putative new species, Trichoderma sp. O.Y. 14707 and O.Y. 2407, from Longibrachiatum and Strictipilosa clades, respectively, have been identified. In vitro salinity assays showed that the ability to tolerate increasing osmotic pressure (halotolerance) is a strain- or clade-specific property rather than a feature of a species. Only a few isolates were found to be sensitive to increased salinity, while others either were halotolerant or even demonstrated improved growth in increasingly saline conditions. In vitro antibiosis assays revealed strong antagonistic activity toward phytopathogens due to the production of both soluble and volatile metabolites. Two marine-derived Trichoderma isolates, identified as T. atroviride and T. asperelloides, respectively, effectively reduced Rhizoctonia solani damping-off disease on beans and also induced defense responses in cucumber seedlings against Pseudomonas syringae pv. lachrimans. This is the first inclusive evaluation of marine fungi as potential biocontrol agents.     

Induction of trehalose in spores of the biocontrol agent Trichoderma harzianum

Spores of the biocontrol agent Trichoderma harzianum P1 produced in liquid media and harvested in the stationary sporulation stage SSS (after 60?h), had higher viability after slow (>4×) and fast drying (>12×) than their counterparts harvested in the exponential sporulation stage, ESS (after 30?h). The trehalose content of SSS spores was almost 20× higher than that of ESS spores (0.16 vs. 3.4?mg/100?mg, respectively). Heat shock (40?°C?×?90?min) effectively increased the trehalose content 2.5× with respect to untreated SSS spores. The trehalose content achieved in heat-treated SSS spores was almost 60% higher than the maximum reached by holding the spores under water-stress at 97% relative humidity prior to drying.     

The expression of genes involved in parasitism by Trichoderma harzianum is triggered by a diffusible factor

The mycoparasite Trichoderma harzianum has been extensively used in the biocontrol of a wide range of phytopathogenic fungi. Hydrolytic enzymes secreted by the parasite have been directly implicated in the lysis of the host. Dual cultures of Trichoderma and a host, with and without contact, were used as means to study the mycoparasitic response in Trichoderma. Northern analysis showed high-level expression of genes encoding a proteinase (prb1) and an endochitinase (ech42) in dual cultures even if contact with the host was prevented by using cellophane membranes. Neither gene was induced during the interaction of Trichoderma with lectin-coated nylon fibres, which are known to induce hyphal coiling and appressorium formation. Thus, the signal involved in triggering the production of these hydrolytic enzymes by T. harzianum during the parasitic response is independent of the recognition mediated by this lectin-carbohydrate interaction. The results showed that induction of prb1 and ech42 is contact-independent, and a diffusible molecule produced by the host is the signal that triggers expression of both genes in vivo. Furthermore, a molecule that is resistant to heat and protease treatment, obtained from Rhizoctonia solani cell walls induces expression of both genes. Thus, this molecule is involved in the regulation of the expression of hydrolytic enzymes during mycoparasitism by T. harzianum.     

Strain-specific SCAR markers for the detection of Trichoderma harzianum AS12-2, a biological control agent against Rhizoctonia solani, the causal agent of rice sheath blight

In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment.     

Soil microbial biomass influence on growth and biocontrol efficacy of Trichoderma harzianum

Hyphal growth and biocontrol efficacy of Trichoderma harzianum may depend on its interactions with biotic components of the soil environment. Effects of soil microbial biomass on growth and biocontrol efficacy of the green fluorescent protein transformant T. harzianum ThzID1-M3 were investigated using different levels of soil microbial biomass (153, 328, or 517 μg biomass carbon/g of dry soil). Hyphal growth of T. harzianum was significantly inhibited in soil containing 328 or 517 μg biomass carbon/g of dry soil compared with soil containing 153 μg biomass carbon/g. However, when ThzID1-M3 was added to soil as an alginate pellet formulation, recoverable populations of ThzID1-M3 varied, with the highest populations in soil containing 517 μg biomass carbon/g. When sclerotia of Sclerotinia sclerotiorum were added to soils (10 sclerotia per 150 g soil) with ThzID1-M3 (20 pellets per 150 g soil), colonization of sclerotia by ThzID1-M3 was significantly lower in the soil containing the highest level of biomass. Addition of alginate pellets of ThzID1-M3 to soils (10 pellets per 50 g) resulted in increased indigenous microbial populations (total fungi, bacteria, fluorescent Pseudomonas spp., and actinomycetes). Our results suggest that higher levels of microbial soil biomass result in increased interactions between introduced T. harzianum and soil microorganisms, and further that microbial competition in soil favors a shift from hyphal growth to sporulation in T. harzianum, potentially reducing its biocontrol efficacy.     

Morphological, Biochemical and Molecular Characterization of Trichoderma harzianum Isolates for their Efficacy as Biocontrol Agents

The random amplified polymorphic DNA (RAPD) procedure was used to examine the genetic variability among 8 isolates of Trichoderma harzianum , and their ability to antagonize Sclerotium rolfsii using a dual culture assay was correlated with RAPD profiles. Eight oligodeoxynucleotide primers were selected for the RAPD assays, which resulted in 86 bands for 8 isolates of T . harzianum . The data were entered into a binary matrix and a similarity matrix was constructed using the DICE similarity (SD) index. An unweighted pair grouping mathematical averaging (UPGMA) cluster based on SD values was generated using the NTSYS computer program. A mean coefficient of similarity obtained for pairwise comparisons was c. 30% and it showed that the variability among the isolates of T. harzianum was very high. Using the dual culture method in antagonism experiments, the T. harzianum isolates were classified in to antagonism classes. Further, T. harzianum isolates were screened for chitinase and β-1,3-glucanase activity. RAPD was efficient in demonstrating the high intraspecific genetic variation among isolates. The dendrogram did not show the grouping of isolates by their level of antagonism. Relationship among polymorphism existent, the aggressiveness and the origin of isolates were not found.     

Variability among strains of Trichoderma harzianum in their ability to reduce take-all and to produce pyrones

Antagonism tests on agar-plates and glasshouse screening indicated that three isolates of Trichoderma harzianum varied in their ability to antagonize the take-all fungus (Gaeumannomyces graminis var. tritici). Isolate 71 which was the most effective in suppressing take-all of wheat, produced two pyrones and other undetermined analogues. Isolates of T. koningii and T. hamatum shown to suppress take-all, produced a simple pyrone compound. Although T. harzianum isolates 70 and 73 did not produce any pyrones, they reduced the disease albeit to a much lesser extent than isolate 71; with isolate 73 showing distinct host growth promotion effects. It is proposed that the success of isolate 71 of T. harzianum was related to the pyrones it produces and that the ability of isolates 70 and 73 to reduce take-all may be related to mechanisms other than those involving antibiotics.     

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.     

Effect of culture conditions on spore shelf life of the biocontrol agent Trichoderma harzianum

The influence of pH, carbon:nitrogen (C:N) ratio, carbon content and harvesting time on spore attributes of the biocontrol agent Trichoderma harzianum was evaluated. The effect of these culture parameters on viability, shelf-life and ultrastucture was also assessed. pH was a key parameter to manipulate for both growth and sporulation, while carbon concentration and C:N ratio strongly affected spore production time. At fixed pH, the C:N ratio had a limited influence on production yield, but was critical for spore shelf-life. The highest spore longevity was found in a medium with a C:N ratio of 14 and a pH of 7.0, when most resulting spores were still alive after 45 d storage. These spores also remained viable during storage under a broad range of relative humidities, indicating that they would be more sustainable in the field.     

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).