Assessment of the biocontrol potential of a Trichoderma viride isolate: Part II: Protection against soft rot and basidiomycete decay

A previous paper reported on the establishment of a field and fungal cellar trial set up to determine the biocontrol potential of a specific Trichoderma isolate against wood decay fungi. This paper reports on the analyses used to examine the protective effect of the selected isolate, and presents results indicating an initial protective effect against both basidiomycetes and soft rot fungi. The parameters assessed in the field and fungal cellar trials were soft rot decay, basidiomycete decay, Trichoderma colonisation, moisture content and nitrogen content. The results of these analyses show that the introduction of a biological control agent has had a significant effect on moisture content, decay and nitrogen content. A protective effect has been observed against soft rot and basidiomycete decay fungi in field samples.     

Biological treatments for the control of decay in tree wounds.: Field tests

In an attempt to control fungi causing decay a range of micro-organisms, selected after laboratory tests, was inoculated into wounds made in the trunks of beech trees. The most successful micro-organism was an isolate of Trichoderma viride, which, over a period of 4 years, maintained significantly lower colonisation by decay fungi at both sites - c. 15% of the level found in uninoculated wounds. T. viride could be reisolated from the majority of wounds into which it had been inoculated up to 4 years later. No other antagonist persisted very effectively. Inoculation of T. viride in glycerol improved its establishment but malt extract and sucrose had no such effect. Both the addition of glycerol to wounds and the wrapping of them in black PVC sheet increased natural colonisation by T. viride. The use of PVC but not glycerol reduced numbers of decay fungi but not through the agency of T. viride present by natural infection, as this proved generally ineffective in the control of decay fungi. Another isolate of T. viride persisted considerably better when covered with a wound sealant.     

Enhancing biological control of basal stem rot disease (<Emphasis Type="Italic">Ganoderma boninense</Emphasis>) in oil palm plantations

Basal Stem Rot (BSR) disease caused by Ganoderma boninense is the most destructive disease in oil palm, especially in Indonesia and Malaysia. The available control measures for BSR disease such as cultural practices and mechanical and chemical treatment have not proved satisfactory due to the fact that Ganoderma has various resting stages such as melanised mycelium, basidiospores and pseudosclerotia. Alternative control measures to overcome the Ganoderma problem are focused on the use of biological control agents and planting resistant material. Present studies conducted at Indonesian Oil Palm Research Institute (IOPRI) are focused on enhancing the use of biological control agents for Ganoderma. These activities include screening biological agents from the oil palm rhizosphere in order to evaluate their effectiveness as biological agents in glasshouse and field trials, testing their antagonistic activities in large scale experiments and eradicating potential disease inoculum with biological agents. Several promising biological agents have been isolated, mainly Trichoderma harzianum, T. viride, Gliocladium viride, Pseudomonas fluorescens, and Bacillus sp. A glasshouse and field trial for Ganoderma control indicated that treatment with T. harzianum and G. viride was superior to Bacillus sp. A large scale trial showed that the disease incidence was lower in a field treated with biological agents than in untreated fields. In a short term programme, research activities at IOPRI are currently focusing on selecting fungi that can completely degrade plant material in order to eradicate inoculum. Digging holes around the palm bole and adding empty fruit bunches have been investigated as ways to stimulate biological agents.     

Biological control of root-rot of Coleus forskohlii Briq. using microbial inoculants

The root-rot or wilt of Coleus forskohlii is a very serious soil-borne disease caused by Fusarium chlamydosporum. A field study was undertaken to study the possibility of controlling the disease using three biocontrol agents viz., Glomus mosseae, Pseudomonas fluorescens, Trichoderma viride, singly and in combination. Planting of coleus cutting was done in wilt sick soil. Inoculation with Trichoderma viride + Glomus mosseae gave the best result in controlling the disease. The same treatment also resulted in maximum growth, yield and root forskolin concentration of coleus. Plants treated with T. viride + G. mosseae showed a disease severity index of 33.28% compared to uninoculated control plants, which had a maximum disease severity index of 85.5%. The fungicide Emisan (0.2%) was not as effective as the biocontrol agents in controlling the pathogen.     

Biological control of wood decay in an open tropical environment with Penicillium sp. and Trichoderma viride

The ability of Penicillium sp. and Trichoderma viride to retard the decay of obeche (Triplochiton scleroxylon) wood in the field for 11 months (January–November) covering dry and wet seasons in a tropical environment was investigated using the ‘graveyard’ method. Inoculation of stakes with Gloeophyllum sp. or G. sepiarium (decay fungi) 24 h after treatment with T. viride or Penicillium sp. in January (dry season) did not increase decay after 11 months. Total inhibition of the decay fungi was indicated since the low weight losses of stakes was not markedly different from losses in control stakes biologically treated but not exposed to decay fungi. Inhibition of the activities of other unidentified field fungi was also indicated because weight losses were greater in uninoculated and untreated stakes. However, decay occurred in stakes biologically treated in January but later exposed to decay fungi in May, after the dry season. A repeat application of T. viride treatment in May, to stakes earlier treated in January, markedly reduced decay following exposure to decay fungi in September (wet season). A similar Penicillium sp. application was not as effective as T. viride application because unlike the T. viride protected stakes, decay was greater in stakes twice protected with Penicillium sp. but not exposed to decay fungi. The results indicate that repeated application of biocontrol agents may be important for controlling wood decay following the adverse effect of the dry season.     

绿色木霉对生物降解和生物防治的影响机理与应用研究进展

绿色木霉(Trichoderma viride)是一种广泛分布于自然界的有益微生物,具有高效生物降解和生物防治功能。研究表明,绿色木霉在代谢过程中会产生纤维素酶、几丁质酶、木质素过氧化物酶等一系列水解酶类,能够高效降解环境中的有机物质,同时,作为农业上常见的生防菌,具有抑制病原菌、促进植物生长、提高土壤肥力等有益功能。基于此,围绕绿色木霉生物功能最新研究进展,从绿色木霉生物降解和生物防治角度,探究绿色木霉实际应用和作用机理,以期为充分利用绿色木霉资源提供依据,并对本领域未来的发展方向和应用前景进行展望。     

Ecological management of tropical sugar beet (TSB) root rot (Sclerotium rolfsii (Sacc.) by rhizosphere Trichoderma species

Trichoderma species are collected from different location of sugarbeet growing areas of Tamil Nadu and it is effective against Sclerotium rolfsii pathogen caused by sugarbeet ecosystems. Out of thirty-one isolates of Trichoderma viride and four isolates of Trichoderma harzianum collected and tested for their antagonistic activity against S. rolfsii by dual culture technique, one isolate was found to be effective T. viride (TVB1) that recorded the maximum (73.03%) inhibition on the mycelial growth recording only 2.40 cm growth as against 8.90 cm in the control. The isolates of T. harzianum THB-1 recorded 71.19% mycelial growth reduction over control. The colonisation behaviour of T. viride (TVB1) revealed that it completely over grew on pathogen within 48 h after interaction with the pathogen, and speed of growth on pathogen was also high and it possesses a higher level of competitive saprophytic ability. The best four isolates of TVB1, TVB-2, TVB-3 and TVB31 and two isolates of T. harzianum THB-1 and THB-2 were compared with other species of Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma koningii and Chaetomium globosum and tested under in vitro condition. BA of neem cake at 150 kg ha^?1 + T. viride isolate (TVB1) at 2.5 kg/ha recorded least root rot disease incidence of 17.05% which accounted for 75.37% disease reduction over control and highest recorded maximum root yield 65.73 t ha^?1 and increasing sugar content.     

Biocontrol of onion white rot by application of Trichoderma species formulated on wheat bran powder

In vitro, Trichoderma album, Trichoderma harzianum, Trichoderma koningii, Trichoderma viride and Trichoderma virens showed antagonistic effect against the most pathogenic isolate (Sc2) of Sclerotium cepivorum, the cause of onion white rot disease. Five Trichoderma preparations of each Trichoderma sp. were prepared on wheat bran powder to be used for controlling white rot disease of onion. Greenhouse and field experiments followed the same trend where T. harzianum and T. koningii were the most effective in reducing the incidence and severity of white rot disease compared with the control. Trichoderma species preparations caused promotion to vegetative parameters of onion plants in pots and increase bulb productivity in filed. In this regard, T. harzianum and T. koningii were the most effective. A positive correlation was found between the biocontrol activity of Trichoderma species preparations and enhancement of peroxidase, polyphenoloxidase and chitinase enzymes in onion plants to resist infection with S. cepivorum.     

Studies on Biological Control of Postharvest Rot in Yams (Dioscorea spp.) using Trichoderma viride

From in vitro and in vivo screening tests for antagonism by isolates of Trichoderma against postharvest pathogens of yams (Dioscorea spp.), an isolate of Trichoderma viride Pers. ex S.F. Gray was selected as the most promising candidate for the biocontrol of postharvest rot of yams. Inoculation of white yam (Dioscorea rotundata Poir.) with conidiaspores of T. viride and subsequent storage of the tubers under the ambient environment conditions of a traditional yam barn, resulted in a drastic reduction in the frequency of occurrence of the normal tuber surface mycoflora over a 4‐month (December‐April) storage period. Trichoderma viride on the other hand, maintained a high frequency of occurrence during the same period. Furthermore, whereas up to 52.0%) rot was found among groups of tubers that were artificially inoculated with the postharvest pathogens of yams, Aspergillus niger Van Tiegh., Botryodiplodia theobromae Pat. or Penicillium oxalicum Currie and Thom, and also the group that was not inoculated with any organism (control), among groups of tubers that were inoculated with T. viride the rot was either totally suppressed or only a low percentage of rot occurred. The significance of the findings is discussed in relation to yam storage especially by farmers with limited resources.     

Tropical field assessment of combined Trichoderma viride Proteus sp. and urea protective treatment of wood against biodeterioration

Two previous reports showed that decay of wood blocks was inhibited by treatment with combined phosphate buffer (pH7) and Trichoderma viride or urea and ureolytic Proteus sp. A field assessment of the two treatment strategies was therefore carried out in the tropical rainforest for 24 months using the “grave yard method”. The groundline region of stakes treated with buffer/T. viride succumbed to biodegradation after 8 months as indicated by loss of resistance to compression, occurrence of basidiomycete and soft-rot cavities. This was attributed to leaching due to the heavy rainfall, soil microflora and the failure of T. viride to spread due to the dry season effect at the onset of the trials. No signs of degradation were evident until 18 months in stakes treated with urea and Proteus sp. The addition of T. viride to the urea/Proteus treatment system caused a marked improvement as no signs of biodegradation were observed throughout the duration of the field trial. As the concentration of ammonia declined, the hitherto suppressed T. viride began to spread and “fill in the gap”. It can be concluded that the triple combination of urea, Proteus sp. and T. viride, can significantly extend the service life of timber in the field.     

Saccharification of used paper with different cellulases

Various used paper materials have been exposed to the action of cellulases from Penicillium funiculosum, Trichoderma reesei, Trichoderma viride and Aspergillus niger. A 2 h incubation period showed cellulase from T. viride the most active except for office paper that was maximally degraded by A. niger cellulase. Cellulase mixtures increased saccharification while sequential treatment with cellulases from T. reesei and P. funiculosum increased biodegradation at values between 15% and 190%. The maximum increase of saccharification (190%) was obtained when T. reesei cellulase initiated the sequential treatment of newspaper relative to the sole action of P. funiculosum cellulase on this non-pretreated and pretreated material.     

Wood biodeterioration control potential of Acalypha hispida leaf phenolic extract in combination with Trichoderma viride culture filtrate

The phenolic extract of Acalypha leaves inhibited growth of Gloeophyllum sepiarium and Pleurotus sp. (test wood-rot fungi) in potato dextrose agar, starch agar, starch glucose agar, carboxyl methyl cellulose agar and carboxyl methyl cellulose glucose agar. Fungicidal or fungistatic concentration of the extract (10–14 mg/ml) depended on the medium. However a lower concentration of the extract (8–10 mg/ml) in combination with Trichoderma viride culture filtrate caused a similar inhibitory pattern. Degradation of obeche (Triplochiton scleroxylon), mahogany (Khaya ivorensis) and walnut (Lovoa trichilioides) by the test fungi was limited or prevented by extract treatment of 8–10 mg/g wood. A similar inhibitory effect again occurred when a combination of T. viride filtrate and lower extract concentration (6–8 mg extract per gram of wood) was used. On-going wood decay was limited or halted by a combined treatment involving 8–12 mg extract per gram of wood depending on the fungal residence period. Treated stakes exposed to 6 months of tropical wet season retained resistance to fungal attack including soft rot. The phenolic extract of A. hispida may prove useful in an integrated chemical and biological approach to wood treatment.     

Trichoderma species for biocontrol of soil-borne plant pathogens of pasture species

Soil-borne plant pathogens such as Rhizoctonia solani (Kuhn), Pythium ultimum (Trow) and Sclerotinia trifoliorum (Eriks) can reduce grass and forage legume establishment. The potential for biocontrol of these pathogens by Trichoderma fungi was evaluated. Following dual culture assays, nine Trichoderma isolates (five of Trichoderma atroviride and one each of Trichoderma hamatum, Trichoderma koningiopsis, Trichoderma viride and Trichoderma virens) were chosen for assessment in pot experiments. In the presence of R. solani, perennial ryegrass (Lolium perenne L.) emergence was increased by 60–150% by two isolates of T. atroviride and by 35–212% by the isolate of T. virens, with the increase depending on growing medium and amount of pathogen inoculum. Red clover (Trifolium pratense L.) emergence in the presence of S. trifoliorum was significantly increased by two T. atroviride isolates and the T. hamatum isolate. In the presence of P. ultimum, white clover (Trifolium repens L.) emergence was increased by 25–42% by one isolate of T. atroviride and the T. hamatum isolate. However, for all three pasture species, some Trichoderma isolates reduced seedling emergence. Seedling growth (shoot and root fresh weight/plant) of the three pasture species was significantly increased by one or more T. atroviride isolates. On the basis of these results for both disease reduction and growth promotion, four T. atroviride isolates were selected for field assessment as biocontrol agents of soil-borne pathogens of pasture species.     

Use of plant extracts and biocontrol agents for the management of brown spot disease in rice

Fifty plant extracts, four oil cakes and eight antagonistic organisms were tested against Bipolaris oryzae (Cochliobolus miyabeanus), the causal agent of brown spot disease of rice. In vitro studies indicated that two leaf extracts, Nerium oleander and Pithecolobium dulce exerted the higher percent inhibition to mycelial growth (77.4, 75.1%) and spore germination (80.3, 80.0%) of B. oryzae. Among the four oil cake extracts tested in vitro against B. oryzae, neem cake extract showed the maximum inhibition percent to mycelial growth (80.18%) and spore germination (81.13%) of the pathogen followed by mahua cake extract, castor and gingelly cake extract. Trichoderma viride (Tv2) was significantly effective in inhibiting the mycelial growth (62.92%) and spore germination (77.03%) of the pathogen followed by Trichoderma harzianum (Th5) and Trichoderma reesei (Tr3). The promising leaf extracts, oil cake extracts and antagonistic microorganisms were further evaluated for their efficacies in disease management under glasshouse and field conditions. In glasshouse studies, post-infectional spraying of rice plants with neem cake extract, N. oleander leaf extract and T. viride (Tv2) was significantly effective in reducing the incidence of brown spot of rice by 66, 52 and 45 percent respectively. Two rounds of spraying of rice plants with neem cake extract, N. oleander leaf extract and T. viride (Tv2) in the field at initial appearance of disease and 15 days later reduced the incidence of brown spot (70, 53 and 48% disease reduction respectively) and increased the yield by 23, 18 and 15 percent respectively.     

Addition of aluminum oxide microparticles to Trichoderma viride My preculture enhances cellulase production and influences fungal morphology

Morphological engineering techniques have recently become popular, since they are used to increase the production of a variety of metabolites and enzymes when fungi are grown in submerged cultures. This study aimed to facilitate cellulase production by adding aluminum oxide to Trichoderma viride My precultures.

The results showed that the highest cellulase activity was achieved when aluminum oxide at 10 g/L was used, and the activities of cellulase for filter paper and endoglucanase activity assays increased from 519.11 to 607.35 U/mL by 17.1%, and from 810.08 U/mL to 917.59 U/mL by 13.3%, compared with the control, respectively. Addition of aluminum oxide decreased the size of T. viride My pellets and increased the final pH. The changes in pellet diameter after the addition of different concentrations of aluminum oxide were fitted using a modified exponential decay model, which could precisely predict the pellet size by controlling aluminum oxide concentration.

The optimum concentration of microparticles, and therefore pellet size, could significantly improve cellulase production, which is an encouraging step towards commercial cellulase production.

     

Identification of Critical Stage for Disease Development and Biocontrol of Alternaria Blight of Indian Mustard (Brassica juncea)

Fungicides mancozeb and carbendazim caused 100% reduction in mycelial growth of Alternaria brassicae over control in vitro while 1% (w/v) aqueous bulb extract of Allium sativum and leaf extract of Acacia nilotica caused significant reductions. In dual culture, GR isolate of Trichoderma viride performed the best among the test isolates of Trichoderma, causing 81%, 82% reduction in mycelial growth of A. brassicae over control. Performance of isolates SI‐2, P and SI‐1 of T. viride were at par (P < 0.01) with that of GR isolate. Spraying of A. brassicae at different ages of the mustard host plant identified 75 days after sowing (d.a.s.) as the most critical age of the mustard plant for development of Alternaria blight severity on the crop with 45 d.a.s. being the next most important one. Mancozeb was the best among all the treatments, resulting in the lowest disease severity on leaves of mustard at both Sewar and Ludhavai as also the lowest A‐value (area under disease progress curve). Performance of bulb extract of A. sativum in checking the disease severity on leaves and pods was at par (P < 0.05) with mancozeb. The GR isolate of T. viride was at par with mancozeb in checking blight severity on mustard leaves at Sewar while performance of the bioagent was significantly (P < 0.05) inferior to the chemical fungicide at Ludhavai. Performance of the bioagent isolate GR of T. viride in checking the disease severity on pods was at par (P < 0.05) with mancozeb at both Sewar and Ludhavai, the treatment recording the lowest A‐value on pods. While application of bulb extract of A. sativum resulted in highest seed yield at Sewar in 2001–2002, the bioagent isolate GR of T. viride did so at Ludhavai, both the treatments being at par (P < 0.05) with mancozeb and significantly higher than control. Application of bulb extract of A. sativum at 45 and 75 d.a.s. resulted in lowest blight severity on leaves and pods as also in highest seed yield among the different single and combination of treatments. Although disease severity in the treatment was at par (P < 0.05) with that in mancozeb, application of the plant extract at the two stages of crop growth resulted in significantly higher seed yield compared with the two applications of the chemical fungicide. However, application of the treatments singly only at 75 d.a.s., GR isolate of T. viride at 45 and 75 d.a.s., A. sativum 45 d.a.s. + T. viride 75 d.a.s., and T. viride 45 d.a.s. + A. sativum 75 d.a.s. resulted in seed yield at par (P < 0.05) with application of bulb extract of A. sativum at 45 and 75 d.a.s.     

Effect of <Emphasis Type="Italic">Trichoderma</Emphasis> spp. isolates for biological control of tan spot of wheat caused by <Emphasis Type="Italic">Pyrenophora tritici-repentis</Emphasis> under field conditions in Argentina

Trichoderma spp. have been used as biocontrol agents to protect plants against foliar diseases in several crops, but information from field assays is scarce. In the present work, experiments were carried out to determine the effect of six isolates of Trichoderma harzianum and one isolate of T. koningii on the incidence and severity of tan spot, caused by Pyrenophora tritici-repentis (anamorph: Drechslera tritici-repentis) under field conditions. Significant differences between years, wheat cultivars and treatments were found. In 2003, two of the isolates assayed (T5, T7) showed the best performance against the disease applied as seed treatments or sprayed onto wheat leaves at different stages. The application of six of the treatments on wheat plants significantly reduced disease severity by 16 to 35% in comparison with the control. Disease control provided by isolate T7 was similar to that provided by the fungicide treatment (56% reduction). This is the first report on the efficacy of Trichoderma spp. against tan spot under field conditions in Argentina.     

An Antifungal Protein Purified from Pearl Millet Seeds Shows Sequence Homology to Lipid Transfer Proteins

In the course of a search for antifungal proteins from plant seeds, we observed inhibition of mycelial growth of Trichoderma viride with extracts of pearl millet. We have identified several proteins with antifungal properties in the seeds of pearl millet. One of these proteins has been purified to homogeneity and characterized. The purified protein has a molecular mass of 25 kDa. The N-terminal sequence of the protein (25 residues) shows homology to non-specific lipid transfer proteins (LTPs) of cotton, wheat and barley. The purified LTP inhibited mycelial growth of T. viride and the rice sheath blight fungus, Rhizoctonia solani in vitro.     

Eco-friendly approaches for the management of fenugreek root rot

To evolve eco-friendly management of fenugreek root rot caused by Rhizoctonia solani, a field trial was conducted during Kharif 2002 and Rabi seasons of 2002–2003 and 2003–2004. Experiments were conducted with eight treatments and three replications in RBD using the variety CO-2. The pooled analysis of the three season data showed that seed treatment with Trichoderma viride at 4g/kg of seed + soil application of Trichoderma viride at 5 kg/ha + soil application of neem cake at 150 kg/ha (T₃) recorded a percent disease index (PDI) of 23.1 versus 65.5 PDI in the control which accounted for a disease reduction of 64.7%. It was on par with seed treatment with Trichoderma viride at 4g/kg of seed + soil application of T. viride at 5 kg/ha (T₂) which reduced the disease incidence by 62.3% (24.7 PDI). The chemical treatment used for comparison, i.e. seed treatment with carbendazim + soil drenching at 0.1% + soil application of neem cake at 150 kg/ha recorded the lowest PDI of 16.8 with 74.4% disease reduction. Among the various treatments T₃ gave a seed yield of 572.7 kg/ha followed by T₂ (555.7 kg/ha). Treatment T₇ recorded the highest yield of 578.7 kg/ha. In the control plot the recorded yield was only 359.3 kg/ha. Though T₃ was more effective at reducing the disease incidence than T₂, the C:B ratio was higher (1:9.1) in respect of T₂ than T₃, which gave a C:B ratio of only 1:3.9. Hence, seed treatment with T. viride at 4g/kg + soil application of T. viride at 5kg/ha is a cost effective, eco-friendly management strategy for fenugreek root rot.     

Biological activity and chemical isolation of root saponins of six cultivars of alfalfa (Medicago sativa L.)

Wheat, cheat and Trichoderma viride bioassays were used to establish the relationship between the content of biologically active saponins in the roots and the degree of winter dormancy and/or time of six cultivars of alfalfa over the period January to August, 1988. Wheat and cheat bioassay results indicated no significant difforences among cultivars, whereas T. viride was inhibited most by extracts of roots collected during months with high rainfall and rapid growth. Cheast seedling roots were inhibited 8–10% more than those of wheat seedling roots indicating that alfalfa root saponins were more effective as allelopathic compounds in preventing growth of cheat than that of wheat alone. An average of 14 different saponins per cultivar were separated by thin-layer chromatograms. Saponins, and the aglycones produced by acid hydrolysis of the May samples, were separated by thin-layer chromatography. The conclusion is that the amount, structure, and type of saponins present in alfalfa roots vary with time.