Trichoderma Harzianum Isolate Research Articles

Biological control of Fusarium oxysporum f. sp. phaseoli by Trichoderma harzianum and its use for common bean seed treatment

Biological control of seed-borne pathogens has shown to enhance germination and physiological quality of seeds. The objectives of this study were to evaluate the in vitro antagonistic effect of five Trichoderma harzianum isolates (CEN287, CEN288, CEN289, CEN290, and CEN316) against Fusarium oxysporum f. sp. phaseoli (Foxy) and test its potential use in seed treatment. Initially, pathogen and antagonists were grown in paired cultures at 25oC, from which samples were assessed using scanning electron microscopy (SEM). Then, clean or Foxy-infected seeds were treated with conidial suspension of the antagonists. Percent of Foxy-infected seeds and normal seedlings were evaluated at seven and nine days of incubation, respectively. All but one Trichoderma isolate (CEN290) inhibited Foxy mycelial growth. SEM analysis revealed that only one Trichoderma isolate (CEN287) showed parasitic interaction with Foxy. Two isolates (CEN287 and CEN316) significantly reduced the Foxy incidence and enhanced seed germination, though less effective than the fungicide mixture (carboxin + thiram). A principal component analysis indicated the importance of volatile metabolites in reducing Foxy incidence on common bean seeds. CEN287 Trichoderma harzianum isolate formed a single group due to its increase in germination rate of Foxyinfected seeds.

Biological control of sorghum anthracnose with rhizosphere and rhizoplane fungal isolates from perennial grasses of the Western Ghats of India

Sorghum is an important drought tolerant crop cultivated for food and fodder purposes. Anthracnose disease caused by Colletotrichum graminicola is a major constraint in sorghum productivity in India. Certain antagonistic fungi, that were isolated in the previous study from the rhizosphere and rhizoplane of perennial grasses in India, were studied for their antagonism in vitro to C. graminicola, root colonization ability and rhizosphere competence. Out of 138 isolates tested, 89 were antagonistic. Fifteen fungal isolates with greater than 70 % in vitro inhibition zone to the pathogen tested positive for root and rhizosphere colonization abilities. Three isolates – Chaetomium globosum isolate 57, Trichoderma harzianum isolate 184 and Fusarium oxysporum (NSF isolate 9) with prominent biocontrol potentials were tested for the control of sorghum anthracnose in greenhouse and field. Chaetomium globosum, Trichoderma harzianum and Fusarium oxysporum isolates decreased seedling mortality, and incidence and severity of disease at different growing stages. They promoted plant growth (dry biomass- 45.3, 40.0 and 46.7 %) and increased yield (grain biomass- 33.3, 23.8 and 49.2 %) respectively, over control in field. The population of the above fungi in soil was moderately high at harvest stage. The present investigation revealed that fungal isolates from rhizosphere and rhizoplane of perennial grasses could be employed to manage anthracnose and enhance plant growth and yield potentialities in sorghum, at the same time.

CONTROL OF FUSARIUM WILT OF BANANA BY USING TRICHODERMA HARZIANUM AND RESISTANT BANANA CULTIVARS

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one the most damaging diseases of banana. The disease has spread throughout all banana growing areas in Indonesia. No effective control method is available to overcome the disease. This research was conducted to study the ability of two Trichoderma harzianum isolates, TH UH and TH13, and resistant banana cultivars to control fusarium wilt disease both in the glasshouse and in a field trial. Four banana cultivars were used in this study, i.e., 'PisangPanjang', 'KepokKuning', 'Puju' and 'Barangan'. An in vitro trial showed that T. harzianum isolates TH UH and TH 13 could inhibit the growth of tropical race 4 of the Foc isolate Bnt2. However, Foc inoculated banana seedlings treated with T. harzianum TH UH and TH 13 did not show any reduced disease intensity in the glasshouse trial. After 14 months of observations in a field trial, the disease only occurred in the banana cultivar 'Barangan' treated with T. harzianum and in the untreated control. The disease did not appear in the cultivars 'PisangPanjang', 'KepokKuning' and 'Puju' treated with T. harzianum or in the untreated controls. The results suggest that T. harzianum is not effective in controlling fusarium wilt of banana and only resistant banana cultivars such as 'PisangPanjang', 'KepokKuning' and 'Puju' can be cultivated to tolerate the disease. This research was funded by ACIAR CP2004/034.

Effect ofTrichoderma harzianumon Mineral Component and Antioxidant Activity of Tomato Fruits

Tomato is one of the important vegetable crops with good nutritional quality and antioxidant activity. Soilborne pathogen Rhizoctonia solani that infect tomato plant reduced the uptake of mineral nutrient from the soil and ultimately reduced their quality. Trichoderam spp. is the most potent biocontrol agent of this pathogen used for the management of this pathogen. In this experiment we studied the effect of Trichoderma-plant-R. Solani interaction on mineral content, nutritional quality and antioxidant capacity of extract of tomato fruits. We used two Trichoderma harzianum isolates i.e. BHU51 and BHU105 and their consortium for the study. Results clearly showed that the Trichoderma treated plant fruits had greater mineral content and antioxidant activity than pathogen inoculated control. The consortium of Trichoderma treated plants fruits had the maximum mineral content and their quality, it might be due to the synergistic effect of Trichoderma isolates that reduced the incidence of R. solani in better ways than single Trichoderma isolate.

English

  In an attempt to construct superior Trichoderma harzianum isolates for improving β-glucosidase productivity, protoplast fusion technique was applied. After application of different mutagenic treatments, twenty mutants were chosen to be tested for their resistance or sensitivity against four antifungal agents. Out of them, four isolates were selected on the basis of their response to antifungal agents and their productivities of carboxymethylcellulase (CMCase) and β-glucosidase to be introduced into intraspecific protoplast fusion experiments using two different methods (PEG and electrofusion). Three crosses were carried out among the selected four isolates. Results showed that, the number of fusants obtained after electrofusion were more than those obtained after polyethylene glycol (PEG) method. In addition, high productivities of CMCase and β- glucosidase were obtained after electrofusion in the three crosses. The applied protoplast electrofusion method proved to be a good and effective method for obtaining T.  harzianum fusants with higher productivity of β- glucosidase enzyme.   Key words: Trichoderma, protoplast fusion, electroporation, β-glucosidase.

Determination of physiological features of mutant Trichoderma harzianum isolates and antagonistic activity against plant pathogen Fusarium sp

Determination of physiological features of mutant Trichoderma harzianum isolates and antagonistic activity against plant pathogen Fusarium sp

Effect of Trichoderma isolates on tomato seedling growth response and nutrient uptake

Trichoderma species are commonly used as biological control agents against phytopathogenic fungi and some isolates are able to improve plant growth. In this study, the effects of three Trichoderma isolates including Trichoderma harzianum isolate T969, T. harzianum isolate T447 and Trichoderma sp. isolate T in tomato seedling vigor and their nutrient uptake via two inoculants introduction methods (inoculating seed with Trichoderma spore suspension and inoculating nursery soil with Trichoderma fortified wheat) were examined. Seed germination rate was not affected by Trichoderma application, but shoot height, shoot diameter, shoot fresh and dry weight and root fresh and dry weight in tomato seedlings were interestingly (p ≤ 0.05) increased when sown in Trichoderma sp. T and T. harzianum T969 fortified soil and when compared to the control. The soil amended by Trichoderma sp. T and T. harzianum T969 had marked increase in leaf number and leaf area (p ≤ 0.05). Chlorophyll content increased in seedling grown in Trichoderma sp. T amended soil as well as in Trichoderma sp. T and T. harzianum T969 coated seed. A dramatic increase (p ≤ 0.05) in the concentrations of Ca 2+ , Mg 2+ , P and K + were recorded in the seedling shoot and root among T. harzianum T447 soil amended treatment

Effect of Trichoderma spp. inoculation on the chemical composition and in vitro digestibility of wheat straw

To evaluate the cultural ability of some Trichoderma isolates on wheat straw and the influence of their exogenous enzyme activities on chemical compositions as well as in vitro digestibility and upgrading of the nutritive value of lignocellulolytic materials, sterilized and non sterilized wheat straw were inoculated withTrichoderma isolates (including Trichoderma harzianum isolate T447, T. hamatumisolate T 614, T. hamatum isolate T625 and T. harzianum isolate T 969). The experimental design used in this study was complete randomized design (CRD) through factorial experiment with 2 factors (factor A = effects of sterilization, factor B = effects of Trichoderma spp.) in three replicates for each treatment. Effects of the Trichoderma isolates on the substrate neutral detergent fiber (NDF), acid detergent fiber (ADF) and pH as well as in vitro dry matter (DM) digestibility (IVDMD), in vitro organic matter (OM) digestibility (IVOMD) and in vitro digestible OM in DM (IVDOMD) were analyzed. The obtained results showed that sterilization of wheat straw could decrease pH, NDF and ADF (P<0.05) but not IVDMD, IVOMD and IVDOMD. The isolates of T447 and T969 showed higher ability in improving the nutritive value of wheat straw. Comparatively, higher reduction in pH was recorded by T614 inoculating sterilized wheat straw. The least NDF content were observed in treated non-sterilized wheat straw with T447, also treated sterilized wheat straw with T447 showed maximum reduction in NDF content. Moreover, higher change in IVDMD, IVOMD and IVDOMD were obtained by T447 grown in sterilized wheat straw substrate. Key words: Trichoderma, wheat straw, dry matter digestibility, organic matter digestibility, digestible organic matter in dry matter.

Successive Construction of ?-Glucosidase Hyperproducers of Trichoderma Harzianum Using Microbial Biotechnology Techniques

In an attempt to construct superior Trichoderma harzianum isolates for improvement β-glucosidase productivity, induction of mutants was applied. After application of UV irradiation and Ethyl methane sulfonate (EMS), 461 isolates were obtained, out of them 99 after UV application and 362 isolates after EMS treatments. Five isolates (two after UV application and three after EMS treatments) were selected on the basis of their highly productivity of both enzymes to be treated with doses of colchicine (0.1% and 0.2%) as a second step of induction mutation. After colchicine treatments, 191 isolates were obtained, out of them 40 isolates after treating the wild type strain, 70 isolates after treating the two UV induced-mutants with colchicine and 81 isolates after treating the three EMS induced-mutants with colchicine .These isolates were tested for their CMCase and β-glucosidase productivities. One isolate (D1/4) proved to be the highest producer for the two enzymes, since it produced 160% and 186% CMCase and β-glucosidase, respectively, more than the original strain. In addition, highest DNA content and also highest amounts of CMCase and β-glucosidase were obtained after EMS-treatments followed by colchicine application.

Trichoderma harzianum in combination with sheep manure amendment enhances soil suppressiveness of Fusarium wilt of tomato

The effect that the biocontrol agent Trichoderma harzianum (isolate Jn14) in combination with an amendment of sheep manure has on the soil suppressiveness of Fusarium wilt of tomato was investigated over a 28-month period. A combination of T. harzianum and organic amendment at concentrations (w:w) of 6 and 10% reduced tomato wilt by 21–36 % and 29–36% respectively, after 0–28 months of soil incubation. When the amendment was added at concentration of 2%, the wilt was suppressed only after 18–28 months. A combination of T. harzianum and the amendment at 6% also increased tomato plant fresh weights by 52% after 28 months, and the 10% amendment increased fresh weights by 56, 40, and 63%, after 18, 24, and 28 months respectively, compared to the experimental controls. Organic amendment at the higher concentrations further stimulated T. harzianum populations, enhanced microbial activity against Fusarium oxysporum in the soil and reduced pathogen populations. Without T. harzianum , the organic amendment at a concentration of 10% reduced disease by only 22, 24, and 23% and only after 18, 24 and 28 months of soil incubation respectively, compared with the controls. However, tomato wilt was not reduced at a 2% manure concentration in less than 12 months of incubation. Organic amendment alone at 6 and 10% reduced the pathogen population by 25% and 37% respectively after 28 months of soil incubation compared with the control. T. harzianum produced fungitoxic metabolites that reduced mycelial growth of Fusarium by 37% and conidium germination by 55% when the pathogen was grown on potato dextrose agar amended with a T. harzianum culture filtrate.

Biological control of Sclerotinia sclerotiorum (Lib.) de Bary with Trichoderma harzianum and Bacillus amyloliquefaciens

The survival of Sclerotinia sclerotiorum, to a large extent depends on the production and viabilty of sclerotia. Reduction in the production of sclerotia is essential in the control of the pathogen. A local Trichoderma harzianum isolate and two Bacillus amyloliquefaciens isolates were evaluated in vitro and in vivo as potential biological control agents (BCAs) against S. sclerotiorum. The study showed that T. harzianum and B. amyloliquefaciens inhibited the growth and production of mycelia and sclerotia. The local isolates, T. harzianum and B. amyloliquefaciens appeared to exhibit mycoparasitism and antibiosis, respectively, in the in vitro study. As antagonists, these isolates protected over 80% of tomato, squash and eggplant seedlings inoculated with S. sclerotiorum. The efficacy of T. harzianum and B. amyloliquefaciens compared with two commercial products, PlantShield and SoilGard in the control of S. sclerotiorum was similar or slightly lower depending on the crop plant.

Trichoderma harzianum and its Metabolite 6‐Pentyl‐alpha‐pyrone Suppress Fusaric Acid Produced by Fusarium moniliforme

AbstractThe interaction of the pathogen Fusarium moniliforme and two antagonistic Trichoderma harzianum isolates was studied especially with respect to their secondary metabolites fusaric acid (FA) and 6‐pentyl‐alpha‐pyrone (6PAP). Among 10 isolates of F. moniliforme screened for FA production on maize kernels, the isolate 8 accumulated the highest amount of FA (678 μg/g). Mycelial growth and production of FA by isolate 8, determined in different liquid media revealed that the highest biomass and FA were produced in Czapek Dox Broth (CDB) followed by Richard’s solution. The amount of FA per gram mycelial dry weight reached its maximum in CDB and Richard’s solution after 14 days of incubation. Mycelial growth and conidia production of both Trichoderma isolates (T16 and T23) were retarded by increasing concentrations of FA in agar medium. At FA concentration of 300 mg/ml the radial mycelial growth of the isolates T16 and T23 were retarded by 32.5% and 45%, respectively. Conidia production was diminished in a similar extent as mycelial growth. Both T. harzianum isolates were capable to degrade FA in potato dextrose broth medium, particularly when lower doses of FA were present. In the presence of 50 mg/ml FA in the culture medium, the isolates T23 and T16 reduced FA by 51.4% and 88.4%, respectively, 9 days post‐inoculation. The antifungal metabolite 6PAP, isolated from T. harzianum T23 cultures, was introduced at different concentrations into 2‐day‐old cultures of F. moniliforme. After further 5 days of incubation of F. moniliforme in the presence of 6PAP, the FA contents per gram mycelial dry weight were significantly decreased compared to control cultures where 6PAP was absent. Dosages of 300 and 400 mg/l of 6PAP in the cultures retarded FA accumulations by 62.5% and 77.2%, respectively. The current results, however, provided the first evidence for activity of 6PAP, as a Trichoderma secondary metabolite, on degrading/synthesis suppression of the Fusarium toxin FA.

Management ofMeloidogyne incognita inArtemisia pallens with bio-organics

The sedentary endoparasitic root-knot nematode [RKN],Meloidogyne incognita (Kofoid and White) Chitwood, is worldwide an economically important agri-pest, reducing the yield and quality of crops. The traditional method of RKN control is based mainly on chemical nematicides, which will not be frequently available from 2005 onwards; their use is highly objectionable due to their major contribution to ground water contamination, and dangers to the environment and to human and animal health. Consequently, new and environmentally safe tools for RKN management are urgently needed. The objective of the present investigation was to evaluate different bio-organic wastes on RKN infestation and the growth / oil yield ofArtemisia pallens Wall. The tested wastes were: 1. Distillation waste ofMentha arvensis (menthol mint); 2.Murraya koengii (meethi neem / curry leaf); 3.Cymbopogon flexuosus (lemongrass); 4.C. martinii (palmarosa); 5.C. winterianus (citronella); 6.Pelargonium graveolens (geranium); 7.Pogostemon patchouli (patchouli); 8.Tagetes minuta (marigold); 9. Vermicompost ofM. arvensis, Chrysanthemum cinerariaefolium (pyrethrum),Tagetes minuta andC. winterianus; 10.Trichoderma harzianum isolate U; 11. VA fungiGlomus aggregatum; G. fasciculatum. The results obtained with these different bio-organics were compared with three controls: untreated — uninoculated, untreated — inoculated and the chemical pesticide carbofuran. Results revealed a considerable enhancement of herbage biomass, flower bud and oil yields when plants were treated with the distillation waste ofC. martinii, C. winterianus, M. koengii, M. arvensis, vermicompost ofC. cinerariaefolium, T. minuta, M. arvensis and bio-agentT. harzianum. Least nematode infections were recorded with the distillation waste ofM. koengii, C. martinii, C. flexuosus, and vermicompost ofT. minuta, C. cinerariaefolium andM. arvensis. The results revealed that these environmentally sound bio-organics could be used for replacement of chemical nematicides.

Production of hydrolytic enzymes by Trichoderma isolates with antagonistic activity against Crinipellis perniciosa, the causal agent of witches' broom of cocoa

Two isolates of Trichoderma, which reduce the incidence of witches'broom disease caused in cocoa by Crinipellis perniciosa, were evaluated for their potential to produce hydrolases in liquid medium. Very low or no hydrolytic activity was produced in the absence of any substrate. The activities of chitinase, N-acetylglucosaminidase, b-1,3-glucanase, total cellulase, endoglucanase, aryl- b-glucosidase, b-glucosidase, protease and amylase increased dramatically within 72-120 h of growth in the presence of specific substrates. Except for N-acetylglucosaminidase and b-glucosidase Trichoderma harzianum isolate 1051 produced the largest amounts of hydrolases. The possible involvement of these enzymes in the antagonistic interaction between Trichoderma and C. perniciosa is discussed.

Inhibitory Potential of Selected Biocontrol Agents and Plant Pathogenic Fungi against Arthrobotrys musiformis : an in vitro Evaluation

An isolate of Arthrohotrys musiformis isolated previously was screened for its effectiveness against some potent plant pathogenic fungi and biocontroJ agents. An in vitro assessment of the degree of inhibition suffered by A. musiformis in presence of fellow fungi indicated that this effective nematode predator could grow well in presence of all the fungi tested except Trichoderma harzianum isolates which inhibited the growth of A. musiformis .

Complete DNA Sequence and Analysis of a Mitochondrial Plasmid in the Mycoparasitic Trichoderma harzianum Strain T95

A circular plasmid called pThr1, with a monomer size of 2.6 kb, was identified in the mitochondria of a Trichoderma harzianum isolate. Hybridization studies using cloned plasmids revealed no DNA sequence similarity between the plasmid and the mitochondrial genome of the isolate. The complete sequence of the plasmid was determined, and the sequence analysis revealed that it contained a single long open reading frame of 1818 bp. Sequence comparisons indicated that the derived amino acid sequence of the ORF exhibited similarity to the reverse transcriptases of the circular Mauriceville and Varkud retroplasmids of Neurospora spp. and the linear pFOXC2 and pFOXC3 retroplasmids of Fusarium oxysporum strains. In the regions of homology all of the seven conserved amino acid blocks characteristic of RTs could be found.

Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches' broom disease

BackgroundSeveral Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes has been considered as the main mechanism involved in the antagonistic process. However, although Trichoderma strains were found to impair development of Crinipellis perniciosa, the causal agent of cocoa plant witches' broom disease, no fungal strain is available for effective control of this disease. We have then undertaken a program of construction of hydrolytic enzyme-overproducing Trichoderma strains aiming improvement of the fungal antagonistic capacity. The protease of an indian Trichoderma isolate showing antagonistic activity against C. perniciosa was purified to homogeneity and characterized for its kinetic properties and action on the phytopathogen cell wall.ResultsA protease produced by the Trichoderma harzianum isolate 1051 was purified to homogeneity by precipitation with ammonium sulfate followed by hydrophobic chromatography. The molecular mass of this protease as determined by SDS-polyacrylamide gel electrophoresis was about 18.8 kDa. Its N-terminal amino acid sequence shares no homology with any other protease. The purified enzyme substantially affected the cell wall of the phytopathogen C. perniciosa. Western-blotting analysis showed that the enzyme was present in the culture supernatant 24 h after the Trichoderma started to grow in casein-containing liquid medium.ConclusionsThe capacity of the Trichoderma harzianum protease to hydrolyze the cell wall of C. perniciosa indicates that this enzyme may be actually involved in the antagonistic process between the two fungi. This fact strongly suggest that hydrolytic enzyme over-producing transgenic fungi may show superior biocontrol capacity.

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.

Beneficial Effects of Solid Matrix Chemo-priming in Okra.

Two field trials at Stillwater and Bixby, OK, evaluated the efficacy of solid matrix priming techniques, alone or in combination with fungicide seed treatment on seedling emergence and reduction of damping-off of okra in field soil naturally infested with Pythium ultimum. The following treatments were evaluated: thiram + carboxin (chemo-primed) (commercially applied), biological seed treatment (bio-primed) (Trichoderma harzianum isolate OK-110, 1 g suspended in 1% carboxymethylcellulose [CMC]), untreated seed (control), and a 1% CMC control. Chemo-primed seeds had a more uniform and faster emergence compared with untreated seeds at both field sites. Within 3 days, 92 and 78% of chemo-primed seeds had emerged at Stillwater and Bixby, respectively, compared with 84 and 71% emergence in the untreated control. Mean emergence of chemo-primed seeds was lower (P ≤ 0.05) than the untreated control. Chemo-primed seeds had greater vigor (P≤ 0.05) at both locations compared with either fungicide-treated or priming alone, at both locations. There were no differences (P ≤ 0.05) in yield among treatments at both locations. P. ultimum was consistently isolated from damped-off seedlings and surrounding soil at both locations. Isolates of P. ultimum were more pathogenic on okra in laboratory tests than isolates of Rhizoctonia spp., Fusarium spp., and other Pythium spp. also isolated from seed or soil.

Characterization of an amylase produced by a Trichoderma harzianum isolate with antagonistic activity against Crinipellis perniciosa, the causal agent of witches’ broom of cocoa

An isolate of Trichoderma harzianum showing antagonistic activity against Crinipellis perniciosa, the causal agent of the witches’ broom disease of cocoa, produces substantial amounts of hydrolytic enzymes. An amylase purified from isolate 1051 had a molecular mass of about 68.7 kDa. Maximal activity against soluble starch was determined at pH 4.0 and 60°C. The K m and V max values were 3.5 mg ml −1 and 1.67 mg min −1 of reducing sugar. The end products were mostly malto-oligosaccharides. The enzyme also hydrolyzed glycogen, amylopectin, maltotriose, and maltotetraose, but not pullulan or cellobiose. Maltose was only barely hydrolyzed. The purified amylase exerted a discrete hydrolytic effect on the C. perniciosa cell wall in vitro as observed by scanning electron microscopic analysis. While Fe 3+, Al 3+, Zn 2+, and Cu 2+ were effective in inhibiting the purified amylase, Mn 2+ considerably enhanced the activity. Ca 2+, Mg 2+, and Co 2+ showed no substantial effect on enzyme activity.