• The Korean Journal of Mycology
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• v.21 no.4
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• pp.323-331
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• 1993

The protoplast formation and intergeneric protoplast fusion between Gliocladium virens and Trichoderma harzianum were attempted to obtain fusants. Protoplast formation was the most effective when the strains were treated with concentration of 5 mg/ml of Novozyme 234 and Cellulase at $25^{\circ}C$ for 3 hours in phosphate buffer, pH 6.5, supplemented with 0.6 M sorbitol as osmotic stabilizer. Auxotrophic mutants of G. virens G88 did not grow in minimal medium and benomyl resistant T. harzianum T95 from wild types, however, was selected by treatment with UV light as genetic marker to isolate fusants. When the intergeneric protoplast fusion between G. virens G88 and T. harzianum T95 was carried out using 30% PEG 4000 containing 10 mM $CaCl_{2}$, and 50 mM glycine (pH 8.5) as fusogenic agent at $25^{\circ}C$ for 10-15 min, the fusion frequency was $0.8{\times}10^{-4}$. Fusants obtained from intergeneric protoplast fusion were spontaneously segregated into va rious strains by continous culture on complete medium. Several intergeneric hybrids were classified into three types: parent-like hybrids, segregants, and recombinants.

• Korean Journal Plant Pathology
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• v.11 no.4
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• pp.287-291
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• 1995

Biocontrol agents, Gliocladium virens G872B and Pseudomonas putida Pf3, were compatible each other in colonizing cucumber rhizosphere, which contributed to a long-term inhibition of cucumber Fusarium wilt. G872B colonized successfully on the cucumber root system, irrespective of the introduction of Pf3. Pf3 also colonized well in the cucumber rhizosphere regardless of the presence of G872B. The individual strains effectively suppressed cucumber wilt up to 56 days after transplanting. The combined treatment of G872fB and Pf3 provided a long-term protection of about 80 days with the efficacy greater than that obtained by any individual strains under greenhouse conditions. These results suggest that the colonization of the biological control agents in the rhizosphere could be correlated directly to Fusarium wilt-suppressive potentials.

• Journal of Microbiology and Biotechnology
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• v.7 no.3
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• pp.161-166
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• 1997

Nuclei were isolated from the protoplasts of Trichoderma harzianum T95 and treated with colchicine, a polyploid inducer. The nuclei were transferred into the protoplast of multi-auxotrophic Gliocladium virens G88 which cannot grow in minimal medium. The protoplast of G. virens G88 carrying the transferred nuclei were regenerated in a regeneration minimal medium containing $17{\mu}g/ml$ of chloroneb as a haploid inducer. Six intergeneric hybrids between G. virens and T. harzianum were isolated from the regeneration minimal medium. The hybrids could be classified into three types according to morphology, those with an isozyme pattern, those with an protein band and those with an randomly amplified polymorphic DNA(RAPD) pattern produced by random primers and repetitive sequences. The first group was identified to be a haploid recombinant, the second group a heterokaryon, and the third appeared to be petite.

• The Plant Pathology Journal
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• v.17 no.1
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• pp.52-56
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• 2001

This study was undertaken to separate and identify antifungla substances produced by Gilocladium virens G1, a biocontrol agent used for the control of plant diseases caused by Rhizoctonea solani. The culture of G. virens G1 effectively inhibited the growth of R. solani, Colletotrichum gloeosporioides, and Phytophthora capsici, but less that of Fusarium oxysporum. The n-hexane extract of the G. virens culture, which was used for the purification of responsible substances, strongly inhibited R. solani and C. gloeosporioides, but not P. capsici, although the n-butanol extract was effective on all of the pathogens tested. An antifungal substance was purified using the n-hexane extract by Silica gel column chromatography and HPLC. The substance was examined for purity by HPLC and for nature by UV spectrometry, which differed from known antibiotic compounds such as gliotoxin, viridin and gliovirin. The antifungal substance was very liphophilic based on its solvent-solubility and Rf values on TLC, and more inhibitory to C. gloeosporioides than other fungal pathogens tested.

• Mycobiology
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• v.28 no.4
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• pp.180-184
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• 2000

In order to find an environment-friendly method to suppress astragal stem rot caused by the isolates of Rhizoctonia solani AG 1 and AG 4, we tested an antagonistic fungus Gliocladium virens G1 was evaluated as a biocontrol agent and estimated inorganic compounds and organic materials were tested for their effect of the disease suppression. G. virens G1 effectively inhibited mycelial growth in a dual culture and caused mycelial lysis in the culture filtrate. No adverse effect was observed when examined for seed germination and seedling growth. Promoted seedling growth was observed with the seed treatment. Seeds of astragal plant were germinated higher in the sterile soil than the natural soil. Of 14 inorganics tested, alum, aluminum sulfate and calcium oxide significantly suppressed the mycelial growth and sclerotial germination. Milled pine bark and oak sawdust also suppressed the mycelial growth. Soil amended with 1% of G. virens G1 composted with pine bark (w/v) significantly controlled astragal stem rot in the glasshouse experiments.

• The Korean Journal of Mycology
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• v.22 no.3
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• pp.276-280
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• 1994

To obtain hybrids producing antagonisms and plant growth promoting effects by intergeneric nuclei transfer, the nuclei were isolated from the protoplasts of Trichoderma harzianum T95 and treated with colchicine. The nuclei were tranferred into protoplast of multi-auxotrophic Gliocladium virens G88 which cannot grow in minimal medium. The nuclei tranferred into protoplasts of G. virens G88 were selected on the regeneration minimal medium containing chloroneb as a haploid inducer. Low transfer frequency of 0.08% was observed with three chemical treatment, however no segregants were found in the intergeneric nuclei transfer. The various types of hybrids with different morphology were detected when different concentration of chloroneb were treated. These morphologies were classified as parental, recombinant and petite type.

• The Korean Journal of Mycology
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• v.27 no.3 s.90
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• pp.191-197
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• 1999

The phylogenetic position of Gliocladium and its related taxa were investigated, using the neighbor-joining method of the sequences from internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal DNA (rDNA). It was focused especially on the generic concept by comparing with the related genera such as Trichoderma, Hypocrea, Verticillium, Penicillium and Talaromyces. Gliocladium species and its related genus were divided into three groups by the phylogenetic analysis using the neighbor-joining method. The first group includes Penicillium-like strains such as Penicillium, Tararomyces, Verticillium and one species of Gliocladium (G. cibotii JCM 9203 and JCM 9206). Especially, Gliocladium cibotii JCM 9203 is thought to be the similar species with Verticillium bulbillosum JCM 9214. Between these two species, Gliocladium cibotii and Verticillium bulbillosum, the intraspecies concept needs to examined with culture condition. and morphological properties. The second group includes two species Verticillium, Verticillium tricorpus and Verticillium albo-atrum which extracted from the GenBank database in NCBI (National Center for Biotechnology Information). Trichoderma-like strains, such as Trichoderma, Hypocrea and several species of Gliocladium are included in the third group. Also, Gliocladium penicillioides IFO 5869 and Gliocladium catenulatum ATCC 10523 formed the subgroup of Trichoderma-like strains. The species of Gliocladium were dispersed in Trichoderma-like and Penicillinum-like group, and only one species of Gliocladium cihotii used in our study was located in Penicillium-like genus group. The species of Verticillium appeared in all three groups and the species of Trichoderma formed the monophylogeny with Hypocrea (telemorph). Also, Gliocladium virens was grouped with Trichoderma harzianum with a high bootstrap value, supporting that Gliocladium virens is to be placed in Trichoderma. The results suggest that Gliocladium is polyphyletic, and is more Trichoderma-like than Penicillium-like.

• Korean Journal Plant Pathology
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• v.12 no.2
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• pp.255-265
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• 1996

토양에 존재하는 진균인 Gliocladium virens는 식물병을 감소 또는 방제할 수 있는 생물학적인 특성에 의하여 G. virens는 지난 수십년간 실용가능성이 큰 생물학적 방제균(또는 길항균)으로 집중적으로 연구되었다. 이 균이 식물병의 발생을 감소시키는 생물적 방제효과는 항생작용, 중복기생, 근권에서의 생존과 집단번식, 뿌리표면에서의 정착 등에 의한 것으로 분류되고 있다. 특히, 항생물질인 gliotoxin, gliovirin, viridin 등은 Rhizoctonia solani 및/또는 Pythium spp. 등에 항생효과가 뚜렷하고, 식물병의 발생과 직접적인 상관관계를 나타내고 있어 G. virens의 식물병의 방제에 관련된 중요한 작용기작으로 제시되어 있다. 또한, 근권에서 이균의 생존과 집단증식 및 뿌리표면에서의 정착은 식물병의 방제와 상관관계를 나타낼 수 있는 중요한 작용기작으로 제시되고 있다. 그러나 이균이 R. solani 등에 기생하는 현상은 식물병의 생물적 방제의 직접적인 연관관계를 나타내고 있지 않다. G. virens을 이용의 생물적 방제효과를 증진시키기 위한 방법으로 다음과 같은 두 가지 방법을 들 수 있다. 첫째, 길항효과가 높은 G. virens 균주를 선발하기 위하여 여러 종류의 토양에서 길항력이 높은 G. virens의 선발이 지난 수십년간 진행되고 있다. 또한, 특정 길항효과를 발현하는 유전자를 G. virens의 염색체에 도입하고 이를 발현시킴으로써 생물적 방제효과를 증진시키는 것으로 이러한 방법은 1980년 후반부터 진행되고 있다. 둘째, G. virens의 길항효과가 최대의 효율로 발현될 수 있도록 최적의 미세환경을 갖추고 있으며 농민이 편리하게 사용할 수 있는 G. virens의 운송매체의 개발이 중요하다. 운송매체의 개발에 의한 'Glioguard'는 G. virens의 포자를 alginate 입자에 제형화한 것으로서 미국에서 시판되고 있다. Aldicarb, metalaxyl, atrazine 등의 농약을 분해할 수 있는 능력은 G. virens의 다른 생물적 특성중의 하나이다. 특히, parathion을 분해할 수 있는 Flavobacterium sp.의 유전자(opd)가 G. virens의 염색체에 도입되여 발현될 수 있는 방법이 제시되었으며, 이는 G. virens을 이용한 토양에서의 특정한 농약의 분해효율을 증진시킬수 있는 가능성을 제시한 것이다. 그러나, G. virens를 이용한 농약의 생물적 분해에 관한 연구는 기초단계로 평가되고 있으며, 포장에서 이를 실용화하기 위해서는 향후 지속적인 연구가 필요하다.

• Korean Journal Plant Pathology
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• v.11 no.4
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• pp.292-297
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• 1995

We developed an in vitro assay method for evaluating plant growth promotion and providing an evidence that the growth promotion is rendered by growth enhancing factors. The amendment of culture filtrates of Trichoderma harzianum T95 and Gliocladium virens G872 and G872B in Murashige and Skoog (MS) agar medium enhanced the cotyledon growth of cucumber in terms of fresh weight and primary leaf area of cucumber cotyledon cuttings, of which the treatment of G. virens G872B was the most effective. The mycelial culture filtrate of G872B was more effective in the growth promotion than its conidial germling filtrate. The addition of 1% sucrose in MS mineral medium with 0.1% culture filtrates of the antagonists (T95 and G872B) was optimum for enhancing the effect of the filtrates on the growth of cotyledon cuttings in vitro. When cucumber seeds treated with G872B, Pseudomonas putida Pf3 or the G872B-Pf3 mixture were planted in a greenhouse, the rate of seed germination, biomass of shoot and root, and yield of cucumber fruits were increased, especially by G872B or the G872B-Pf3 mixture. Correspondingly, cucumber fruit yields in early to middle-cycles of harvest were significantly greater in the plots of G872B than the control and Pf3-treated plots, and the final yield was highest in the plots of the G872B-Pf3 mixture applications.

• The Plant Pathology Journal
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• v.18 no.5
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• pp.279-283
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• 2002

Biological control of Fusarium udum causing wilt disease of pigeonpea was studied in vitro, as well as, in vivo. Aspergilluspavus, Anergillus niger, Bacilius licheniformis (strain-2042), Gliocladium virens, Peniciliium citrimum, and Trichoderma harzianum, which were found to be the most potent ones in inhibiting the radial colony growth of the test pathogen, were used as biological control by amending their inocula at diffeyent concentrations in pots and in pathogen-infested soil in the fields. Maximum reduction of the wilt disease was observed with G. vireos both in pots and in the fields. The population of E. udum was found to be markedly reduced when the antagonists were applied in the soil. The study establishes that G. virens can be exploited for the biological control of wilt disease at field level.