Mycoparasitism-related genes expression of Trichoderma harzianum isolates to evaluate their efficacy as biological control agent

The selection of new isolates of Trichoderma harzianum with high suppressive activity against Fusarium oxysporum is a suitable strategy to avoid the increase of chemical pesticides. In this study, 31 isolates of Trichoderma sp. were analyzed by RAPD-PCR and five isolates of T. harzianum (T-30, T-31, T-32, T-57 and T-78) were selected. The expression of genes encoding for NAGases (exc1 and exc2), chitinases (chit42 and chit33), proteases (prb1) and β-glucanases (bgn13.1) activities and their respective in vitro enzymatic activities were measured. Dual plate confrontation assays of the isolates against F. oxysporum were also tested. Different profiles of gene expression between the different T. harzianum isolates were related to enzymatic activities values and dual plate confrontation. In this work, the T. harzianum isolates T-30 and T-78 showed the greatest mycoparasitic potential against F. oxysporum, which could lead to improved biocontrol of this phytopathogen.     

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).     

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.     

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...     

A new xylanase from a Trichoderma harzianum strain

A new xylanase (XYL2) was purified from solid-state cultures of Trichoderma harzianum strain C by ultrafiltration and gel filtration. SDS-PAGE of the xylanase showed an apparent homogeneity and molecular weight of 18 kDa. It had the highest activity at pH 5.0 and 45°C and was stable at 50°C and pH 5.0 up to 4 h xylanase. XYL2 had a low K _m with insoluble oat spelt xylan as substrate. Compared to the amino acid composition of xylanases from Trichoderma spp, xylanase XYL2 presented a high content of glutamate/glutamine, phenylalanine and cysteine, and a low content of serine. Xylanase XYL2 improved the delignification and selectivity of unbleached hardwood kraft pulp. Received 02 February 1999/ Accepted in revised form 17 April 1999     

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.     

Control of Grapevine Grey Mould with Trichoderma harzianum T39

The efficacy of a formulation of Trichoderma harzianum T39 for control of grey mould (Botrytis cinerea) on grapevine was examined in 133 experiments conducted under diverse commercial conditions. The experiments were carried out between 1988 and 1994 in 19 countries and on 34 varieties. The average disease incidence in the untreated plots of all experiments was 42 2.3% (mean standard error). In general, the reduction of disease achieved by T. harzianum application was lower than that obtained by chemical fungicides: 36.3 2.7% disease reduction in biocontrol treatments and 52.3 2.6% in the exclusively chemical treatments. Control efficacy declined when the interval between application and assessment dates increased to 5 weeks. The experiments also included treatments in which T. harzianum was integrated with chemical fungicides, the two being applied alternately, and a reduced chemical treatment in which only chemicals were applied, and only at the times when chemicals were applied in the integrated treatment. The mean control efficacy in these treatments was 55.8 3.2% and 44.2 4.9% respectively. The roles of the integration of biological and chemical compounds in reducing pesticide residues in fruit at harvest and lowering the pressure towards development of fungicide-resistance populations of B. cinerea are discussed.     

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.     

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.     

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.     

Domestic wastewater as substrate for cellulase production by Trichoderma harzianum

Cellulase production using residues as substrate has been well described, as it is an interesting method of reducing the costs of processes, one of the main bottlenecks for the production of enzymes. This research describes for the first time the use of raw domestic wastewater, which is largely and continuously generated, as a culture base medium for cellulase production. The strain Trichoderma harzianum HBA03 was selected according to the highest activity produced for FPase (5.4 U/mL) and CMCase (8.2 U/mL). Peptone was selected as a nitrogen source and microcrystalline cellulose as the inducer for cellulase production, resulting in FPase activities of 5.6 and 5.0 U/mL and CMCase activities of 12.0 and 14.4 U/mL. The use of domestic wastewater as the culture medium led to an increase of 1.41 and 1.14 fold of FPase and CMCase production, respectively, compared to the synthetic medium. Production was also carried out in a bubble column reactor in which the maximum productivities achieved 10.2 U/L.h (FPase) and 64.6 U/L.h (CMCase). The presented results demonstrate the feasibility of the use of domestic wastewater for cellulases production, thereby contributing to the development of a sustainable process for reusing wastewater with a significant reduction in environmental impact.     

Selection of strain and optimization of mutanase production in submerged cultures of Trichoderma harzianum

Nineteen fungal strains belonging to different genera were tested for extracellular mutanase production in shaken flasks. The optimal enzymatic activity was achieved by Trichoderma harzianum F-470, a strain for which the mutanase productivity has not yet been published. Some of factors affecting the enzyme production in shaken flasks and aerated fermenter cultures have been standardized. Mandels mineral medium with initial pH 5.3, containing 0.25% mutan and inoculated with 10% of the 48-h mycelium, was the best for enzyme production. A slight mutanolytic activity was also found when sucrose, raffinose, lactose and melibiose were carbon sources. Application of optimized medium and cultural conditions, as well as use of a fermenter with automatic pH control set at pH 6.0 enabled to obtain a high mutanase yield (0.33 U/ml, 2.5 U/mg protein) in a short time (2-3 days). The enzyme in crude state was stable over a pH range of 4.5-6.0, and at temperatures up to 35 degrees C; its maximum activity was at 40 degrees C and at pH 5.5.     

Efficiency of Trichoderma harzianum as a biocontrol agent in combination with humate and chitosan against Meloidogyne incognita on tomato

Abstract

A pot trial was conducted to estimate the role of Trichoderma harzianum alone or in combination with two organic substances, potassium humate and chitosan in controlling Meloidogyne incognita on tomato. All treatments caused greater decreases in parameters of M. incognita in comparison to the control treatment (nematode only) and this led to noticeable enhancements in growth and yield of tomato. The lowest numbers of eggmasses, eggs/eggmass, galls/root, females/root, and second stage juveniles/250?g soil were recorded due to the combination of T. harzianum (10¹⁰ spore/ml) with chitosan and potassium humate after 120 days from the transplanting of tomato seedlings. Also, this treatment showed the best promotion for all tomato parameters (lengths and weights of shoots and roots, and productivity). So, mixing chitosan, potassium and T. harzianum is highly recommended to be used as an effective bio-nematicide against M. incognita on tomato plants.     

Transformation of a mycoparasitic Trichoderma harzianum strain with the argB gene of Aspergillus nidulans

A transformation system was developed for the mycoparasitic filamentous fungus Trichoderma harzianum, based upon complementation of auxotrophic mutants. Prototrophic transformants were obtained using plasmids pSal23 and pAN5-41B, carrying the Aspergillus nidulans argB gene, and both the Aspergillus nidulans argB and Escherichia coli lacZ genes, respectively.     

木霉对月季幼苗生长的影响

为了研制促进月季生长的木霉生防菌肥，采用本实验室分离鉴定的哈茨木霉T6施于月季盆栽土壤中，分析木霉菌对"十姐妹"月季硬枝扦插发芽、幼苗生长、防御酶活性和内源激素水平的影响。结果表明：施用哈茨木霉T6的月季硬枝出芽率和幼苗枝条长度分别是对照的1.4和1.82倍；并且枝条和叶的干重、枝条和叶的含水率均显著增加。此外显著提高了叶中3种防御酶SOD、POD、CAT的活性及6种内源激素IAA、GA₃、ZT、ABA、SA和JA的水平。说明木霉T6具有促进月季生长和提高其抗性的作用，研究结果为研制促进月季生长的生物菌肥提供理论依据。     

The molecular basis of shoot responses of maize seedlings to Trichoderma harzianum T22 inoculation of the root: a proteomic approach

Trichoderma spp. are effective biocontrol agents for several soil-borne plant pathogens, and some are also known for their abilities to enhance systemic resistance to plant diseases and overall plant growth. Root colonization with Trichoderma harzianum Rifai strain 22 (T22) induces large changes in the proteome of shoots of maize (Zea mays) seedlings, even though T22 is present only on roots. We chose a proteomic approach to analyze those changes and identify pathways and genes that are involved in these processes. We used two-dimensional gel electrophoresis to identify proteins that are differentially expressed in response to colonization of maize plants with T22. Up- or down-regulated spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry and nanospray ion-trap tandem mass spectrometry. We identified 91 out of 114 up-regulated and 30 out of 50 down-regulated proteins in the shoots. Classification of these revealed that a large portion of the up-regulated proteins are involved in carbohydrate metabolism and some were photosynthesis or stress related. Increased photosynthesis should have resulted in increased starch accumulation in seedlings and did indeed occur. In addition, numerous proteins induced in response to Trichoderma were those involved in stress and defense responses. Other processes that were up-regulated were amino acid metabolism, cell wall metabolism, and genetic information processing. Conversely, while the proteins involved in the pathways noted above were generally up-regulated, proteins involved in other processes such as secondary metabolism and protein biosynthesis were generally not affected. Up-regulation of carbohydrate metabolism and resistance responses may correspond to the enhanced growth response and induced resistance, respectively, conferred by the Trichoderma inoculation.     

Metarhizium (Nomuraea) rileyi as biological control agent

Metarhizium rileyi is an entomopathogenic fungus of Lepidoptera and infects many noctuids, which are key pests of agricultural crops. In this literature review, we summarise the state of art and use of this potential biological pest control agent. Pathogen–host interactions include conidial adhesion, invasion process, and enzymatic activity. Environmental factors that impact the productivity associated with this fungus such as environment temperature, humidity, and availability of spores are discussed. Recent research has focused on enzymatic aspects related to pathogenicity, in which the cause is not fully elucidated yet. More than 60 known host species, mainly lepidopterans, are listed. Non-susceptible species, including predatory insects, are also presented. The difficulty of mass production and the formulation are the principal obstacles in use of M. rileyi. We summarise the challenges necessary to better utilise this agent in biocontrol and suggest novel research approaches, including the development of commercial and technically viable formulations.