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.     

Evaluation of fungal culture filtrate containing chitinase as a biocontrol agent against Helicoverpa armigera

AIMS: To evaluate the biocontrol efficacy of culture filtrate containing chitinase from Trichoderma harzianum against Heliothis. METHODS AND RESULTS: T. harzianum was cultured by submerged fermentation using colloidal chitin as sole carbon source. The ability of the culture filtrate to hydrolyse colloidal chitin indicated the presence of chitinase as one of its components. Biocontrol assay on Heliothis showed that the culture filtrate is a potent antifeedant as it reduced the feeding rate and body weight of the larvae. It reduced the successful pupation and increased larval and pupal mortality in a dosage-dependent manner when applied topically. The highest mortalities (70%) were recorded for groups treated with 2000 U ml(-1) chitinase activity. The percentage of adult emergence was zero for the highest chitinase concentration (2000 U ml(-1)) tried. CONCLUSIONS: The studies showed that the culture filtrate containing chitinase from T. harzianum is capable of negatively affecting the growth and metamorphosis of Heliothis larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: In view of the need for safer and environmentally friendly pest management tools, the present study could help in the development of enzyme-based biopesticides against Heliothis.     

Encapsulation of Trichoderma harzianum conidia as a method of conidia preservation at room temperature and propagation in submerged culture

The objective of the present work was to develop a method for the preservation of T. harzianum conidia at room temperature and the immobilised conidia propagation in submerged culture. This was accomplished by immobilising the strain in sodium alginate capsules (white capsules) and subsequently propagating them in a column bubble reactor (green capsules). Three capsule diameters were tested (micro, medium and large capsules), which were produced by emulsion internal gelation and dripping methods. Tested variables were the immobilised conidia propagation in submerged culture for free conidia production, the immobilised conidia viability throughout the time (two years), the resistance of the encapsulated conidia to the UV irradiation of short and long wavelength, and the antagonistic effect of the encapsulated T. harzianum against four phytopathogenic fungi. It was found that the medium capsules (1.5?±?0.3?mm) favoured the massive production of released conidia in submerged culture and that the higher the density of conidia per capsule, the greater the protection against the ultraviolet irradiation. Regarding the conidia preservation in calcium alginate, a viability loss of around 30% was observed two years after storage at environmental temperature in both white and green capsules; along the two years that the viability of conidia was analyzed, the purity of the formulation was corroborated. The results presented here show the efficacy of the green and white capsules for T. harzianum preservation at room temperature for a long period of time.     

Colonisation of sclerotia of Sclerotinia sclerotiorum by a fungivorous nematode

Aphelenchoides saprophilus nematodes fed on sclerotia, mycelium, and alginate-formulated pellets of Sclerotinia sclerotiorum, mycelium of Trichoderma harzianum, and mixed fungal cultures. As many as 500 nematodes were found inside individual sclerotia. Results suggest potential impacts of fungivory on S. sclerotiorum and its ecological interactions with plant hosts and biocontrol fungi.     

Control of Root Rot and Wilt caused by Pythium myriotylum in Kangaroo Paw (Anigozanthos)

Control of root rot and wilt on Kangaroo Paw (Anigozanthos) caused by Pythium myriotylum was tested, using artificial inoculations, in greenhouse experiments. Disease incidence in the inoculated but non‐treated plots was 74% in Expt A (1999) and 46% in Expt B (2000). Disease incidence was significantly reduced by treatments with fenamidon (26 and 70%) and Terraclor SuperX (65 and 41%) in Expts A and B, respectively, in comparison with the non‐treated plots. Disease incidence was reduced with Ridomil Gold by 7 and 48% in Expts A and B, respectively; with Dynone by 22 and 41% in Expts A and B, respectively; with Tachigaren by 20% in both experiments; and with Trichoderma harzianum treatment, disease incidence was reduced by 9% only in Expt B. Yields (recorded only in Expt B) obtained in plots inoculated with P. myriotylum were significantly lower than in the non‐inoculated control plots. The most efficient treatment was fenamidon, with 324% more flowers than in the non‐treated control. The number of flowers in terraclor SuperX, Ridomil Gold, Dynone and T. harzianum treatments was higher by 285, 249, 234 and 235% than in the non‐treated control. In vitro tests demonstrated that the most effective doses, causing 50% inhibition of mycelial growth (ED₅₀), were 0.09, 0.4, 0.4 and 4 μg/ml for the fungicides fenamidon, Ridomil Gold (mefenoxam), Terraclor SuperX (etridiazole + quintozene) and Dynone (prothiocarb), respectively.     

Fate of transformed Trichoderma harzianum in the phylloplane of tomato plants

A propiconazole-resistant Trichoderma harzianum strain with high phylloplane survival capability was transformed with the E. coli hygromycin B phosphotransferase gene (hph), coding for hygromycin B resistance. Four transformants were analysed for survival ability on the phylloplane of tomato plants grown under glasshouse conditions in comparison with their prototype and a yellow, hygromycin B-sensitive mutant. Over 2 weeks, the four transformants showed higher survival rates in comparison with the wild-type strain. The yellow mutant TF3/973 did not significantly differ in survival from the transformants. Both hygromycin B resistance and mitotic stability of transformants were evaluated during growth in vitro and after reisolation from tomato phylloplane. Hybridization patterns with the complete plasmid indicated that all four transformants were mitotically stable after several rounds of vegetative growth without selective pressure and during 2 weeks on tomato plants. None of the transformants had lost the ability to grow in the presence of both propiconazole and hygromycin B after growth under the same conditions. The results are discussed in relation to risk assessment of the release of transgenic fungi.     

木霉防治灰霉病的研究进展

植物病害的生物防治是降低化学农药用量、减少环境污染的一种有效方式,木霉是现在普遍应用且生防潜力巨大的灰霉病防治真菌。目前,已经对防治灰霉的木霉菌株的筛选、应用及生防机制进行了大量而深入的研究。木霉的生防机制分为直接生防机制和间接生防机制,前者主要指木霉与灰霉病菌直接作用过程中所涉及的重寄生、抗生和营养竞争,后者是木霉通过诱导植物产生系统抗性来防治灰霉。本文对木霉直接防治灰霉病以及诱导植物产生系统抗性防治灰霉病所涉及的互作模式、信号传导途径以及所引起的防御反应进行综述,旨在通过机制的深入研究能够找到进一步提高木霉生防效果的技术方案。     

Optimization of culture conditions for production of the anti-tubercular alkaloid hirsutellone A by Trichoderma gelatinosum BCC 7579

AIMS: This work aimed to optimize the culture conditions for production of a novel and potent anti-tubercular alkaloid, hirsutellone A, by the saprophytic soil fungus Trichoderma gelatinosum BCC 7579. METHODS AND RESULTS: The fungus was initially cultured in shake flasks at 25 degrees C in the potato dextrose broth (PDB) supplemented with various carbon and nitrogen sources and mineral salts to select suitable medium for mycelial growth and hirsutellone A production. Cultivation conditions were further optimized by adjusting initial pH and changing temperature levels to maximize the production of hirsutellone A. The optimal condition that increased the production of hirsutellone A from 19.04 mg l(-1), obtained from basal condition, to 610.55 mg l(-1) and reduced the cultivation time from 40 to 6 days was to cultivate in a shaker at 200 rev min(-1) at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with initial pH of 7. Production of hirsutellone A in larger-scale using a 5-l batch fermenter was also completed yielding 958 mg l(-1) of hirsutellone A within 6 days. CONCLUSIONS: The suitable culture conditions for hirsutellone A production by T. gelatinosum BCC 7579 was the cultivation in 5-l fermenter at 25 degrees C in PDB plus 20 g l(-1) soluble starch, 10 g l(-1) peptone and 2.5% (v/v) salt solution with an initial pH of 7. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of hirsutellone A in a fermenter to obtain a high yield and reduce an incubation period will become very useful in anti-tubercular drug development process in the future.     

培养条件对一株木霉产纤维素酶过程影响的研究

采用固态发酵和连续监测正交实验结果的方法,研究了培养温度、初始pH值、液料比和接种量对一株木霉(Trichodermasp.)发酵过程中微晶纤维素酶活、CMC酶活和滤纸酶活的影响及影响程度。指出液料比在整个发酵过程中是对产酶影响最大的因素,温度在发酵初期影响较大,初始pH和接种量的影响均不显著。总体看来,培养温度、初始pH值、液料比和接种量分别为30℃、4、7和5%是比较合适的。     

螺旋纤维床固定化生物反应器同时产酶降解壳聚糖的研究

采用多孔聚酯泡沫固定里氏木霉,在鼓泡柱固定化反应器中同时产酶降解壳聚糖。结果表明通过控制降解时间可以得到不同平均聚合度的降解产物。在28℃,pH4.8,通气量3vvm条件下,利用固定化反应器,在30d内连续进行10批同时产酶降解试验,结果发现壳聚糖酶活力和壳聚糖降解率能保持稳定。每批产生的壳聚糖酶活力平均达到0.15u/mL以上,壳聚糖平均降解率为73%。     

Biological control of Dutch elm disease: Larvicidal activity of Trichoderma harzianum, T. polysporum and Scytalidium lignicola in Scolytus scolytus and S. multistriatus reared in artificial culture

A method is described for the culture of larvae of Scolytus multistriatus and Scolytus scolytus on an artificial medium following exposure to cultures of microorganisms. In control cultures, a natural mortality rate of 21.2% was found for S. multistriatus and 17.6% for S. scolytus. The effects of Trichoderma harzianum, T. polysporum and Scytalidium lignicola on fifth instar larvae of S. scolytus and S. multistriatus reared on the artificial medium were studied. The fungi were larvicidal and larval mortality was increased to more than 80% by inoculation of the larvae with the fungi. Another fungus, Phomopsis oblonga, had little effect on larvae of S. scolytus. The results are discussed in relation to mechanism of pathogenicity of the fungi and their potential use in the control of Dutch elm disease. It is proposed that with modifications, the method is applicable to other bark beetle pests.     

Potential of an antagonistic bacterium isolate obtained fromLentinus lepideus basidiospores as a biocontrol agent

TheBurkholderia sp. isolate 87-11 obtained from basidiospores ofLentinus lepideus was antagonistic against severalPythium andRhizoctonia isolates. The bacterium was tested against soilborne diseases of five plants caused byP. aphanidermatum andR. solani by soil and seed application, and its potential as a biocontrol agent is discussed.     

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.     

Medium components and culture conditions affect the thermotolerance of aerial conidia of fungal biocontrol agent Beauveria bassiana

AIMS: To produce more thermotolerable conidia of Beauveria bassiana, a well-known fungal biocontrol agent, by optimizing the medium components and culture conditions. METHODS AND RESULTS: The conidia produced on media including 0.5-6% glucose, sucrose or starch as carbon source and 50-300-microg ml(-1) Cu2+, Zn2+, Mn2+ or Fe3+ as additive to Sabouraud dextrose medium at 15-30 degrees C, pH 4-8 or KCl-adjusted water availabilities were exposed to 30-min wet heat stress at 48 degrees C. The medium components for conidial production with greatly enhanced thermotolerance included 4% glucose as optimum or 1% starch as alternative for the carbon source and < or =50-microg ml(-1) Mn2+ for the metal additive. The culture conditions were optimized as 25 degrees C and pH 5-6. Conidial thermotolerance decreased remarkably when sucrose and Fe3+ or Cu2+ were used in the cultures, but altered slightly when 50-200-microg ml(-1) Zn2+ were included. CONCLUSIONS: The tolerance of B. bassiana conidia to the thermal stress was significantly affected by the medium composition and culture conditions under which the conidia were produced. SIGNIFICANCE AND IMPACT OF THE STUDY: Proper treatment of small grains as mass production substrates for more glucose release and supplement of glucose or 50-microg ml(-1) Mn2+ are possible means to enhancing conidial thermotolerance and field persistence for improved insect control.     

Production of laccases by Pleurotus ostreatus in submerged fermentation in co‐culture with Trichoderma viride

Aims: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co‐cultures with Trichoderma viride as a function of infection time and agitation rate. Methods and Results: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co‐cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co‐cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. Conclusions: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. Significance and Impact of the Study: The evaluation of culture parameters that could influence Trichoderma–basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co‐cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.     

Colonization of Sclerotinia sclerotiorum sclerotia by a biocontrol isolate of Trichoderma harzianum,and effects on myceliogenic germination

Sclerotia of Sclerotinia sclerotiorum were incubated on cultures of Trichoderma harzianum. Myceliogenic germination decreased by 50% within 1 day and continued to decrease over time. Quantitative PCR showed a decrease in Sclerotinia DNA for older sclerotia, but not fresh sclerotia. Trichoderma DNA increased and persisted inside older sclerotia but not fresh sclerotia.     

Production of cellulases in batch culture using a mutant strain of Trichoderma reesei growing on soluble carbon source

Trichoderma reesei Rut-C30 is a highly derepressed mutant which synthesised active cellulases in culture media containing glucose and lactose as the only carbon sources. The maximum biomass, filter paper and specific filter paper activities for cell growth on 20 g glucose l^–1 were 20 g dry cell wt l^–1, 1.9 FPU ml^–1 and 4.8 FPU mg^–1 protein respectively, while on 40 g glucose l^–1 were 25 g dry cell wt l^–1, 4.5 FPU ml^–1 and 6.2 FPU mg^–1 protein, respectively. This strain had a higher specific filter paper activity (6.2 FPU mg^–1 protein) than was produced by other T. reesei mutants (3.6 FPU mg^–1 protein).     

Optimizing nutritional conditions for the liquid culture production of effective fungal biological control agents

Spores of fungal pathogens of weeds and insects are unique in their ability to actively infect and kill their pest host. While these capabilities are advantageous in terms of their use as a contact biological control agent, or biopesticide, they also require special consideration during spore production. Directed approaches to medium optimization must consider not only spore yield but also spore qualities such as desiccation tolerance, stability as a dry preparation, and biocontrol efficacy. Nutritional conditions during culture growth and sporulation should direct the accumulation of appropriate endogenous reserves so that newly formed spores possess these advantageous qualities. Studies with the bioherbicidal fungus Colletotrichum truncatum and with the bioinsecticidal fungus Paecilomyces fumosoroseus have demonstrated the impact of nutrition on spore ‘fitness’ for use as a biological control agent. The optimization strategy used in these nutritional studies as well as a comparison of the results are presented. Received 06 February 1997/ Accepted in revised form 29 May 1997     

Studies on the population dynamics and integrated management of Fusarium oxysporum f. sp. psidi (Fop), a cause of guava (Psidium guajava) wilt

To assess the impact of non-host crops intercropping, bioagents and oil cakes, on population dynamics of Fusarium oxysporum f. sp. psidii (Fop) and wilt of guava. Lowest population of Fop was recorded in garlic followed by intercropping with marigold with reduction of 84.9 and 83.9%, respectively. Bioagents viz; Aspergillus niger, Trichoderma viride and Trichoderma harzianum reduced the Fop population significantly, with the lowest in T. harzianum followed by A. niger. Garlic bulb extract exhibited maximum inhibition of Fop growth (2.7 cm) followed by marigold (2.4 cm), respectively, over control. Neem cake significantly reduced population of Fop, closely followed by mahua cake, over control. Integration of neem cake + T. harzianum + garlic reduced the Fop population significantly, over control, followed by neem cake + T. harzianum + marigold but neem cake + T. harzianum + marigold reduced wilt disease significantly in comparison to neem cake + T. harzianum + garlic inter-cropping.