Biological characteristics of Gliocladium virens WJGV2、TLGV22 and the mass production of chlamydospore formulation for disease control

• Main Authors: Sheng-Chi Chu, 朱盛祺
• Other Authors: Dean Der-Syh Tzeng
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/60176376705632462927

碩士 === 國立中興大學 === 植物病理學系 === 92 === Biological characteristics of Gliocladium virens WJGV2 and TLGV22 and the mass production of chlamydospore formulation for disease control Sheng-Chi Chu The investigation was aimed to establish technique platform for exploring the Gliocladium/Trichoderma microbial resources for plant disease control application. A primed goal attempted was to produce the robustious chlamydospore biomass, rather than the conidial formulation, by a traditional stirrer tank fermentor. A total of seven antagonistic Gliocladium/Trichoderma strains with superior growth and conidiation characteristics were screened for the competence of chlamydospore production and plant growth promotion. Gliocladium strains WJGV2 and TLGV22 were among them the best in promoting the growth of cabbage seedlings and in chlamydospore productivity. The two strains are both rhizosphere isolates from rice. They both showed superior antagonisity on Rhizoctonia solani AG1, AG4, and Pythium aphanidermatum. They also expressed great hydrolytic activities on macro biomolecules including starch, protein, chitin and cellulose, whereas not on pectin or phospholipid. The morphological characteristics of mycelial growth and conidiation indicated the 2 strains accorded well the species characteristics of Gliocladium virens described by Domsh, Harman and Kubicek. The internal transcribed spacer (ITS) region rDNA of the two strains that include ITS1, 5.8S rDNA, and ITS2, was cloned and sequenced. The result obtained from sequence analysis indicated the cloned sequences from these 2 test strains were 652 bp in length which shared 99-100 % identity as compared to that of the 9 existing Gliocladium virens (syn. Trichoderma virens ) registed in GenBank of NCBI. The high identity of full length rDNA ITS region comparing to that known in GenBank further strengthen the view that to two test strains are members of G. virens. For mass production of chlamydospore formulation, improvement of cultured medium constituents was attempted. In a flask shaking culture system, the amendment of carbohydrate-enriched growth factor appeared to increase the chlamydospore production greatly; whereas the amendment of N-enriched growth factors Y & F, on the contrary, greatly reduced it. The yield of chlamydospore in flask culture system reached 1.5X108 /ml 7 days after inoculation, and size of the produced spores ranged from 9.2 to 10.8 μm in diameter. The viable chlamydospores in the culture dropped to 107 /ml level gradually after 6 months storage at 4℃, indicating the need of protective measurement in the formulation process. In 50 L stirrer tank fermentor system, the improved broth medium developed in flask culture system was found repeatable; the yield of chlamydospore reached 108 level as expected. However, the production protocol takes 8-9 days operation. A chlamydospore formation stimulation factor (CSF) was found useful in initiating the sporulation process. The timing and dosage of CSF application had been optimized. With the introduction of 0.25 units CSF at the starting point of the culture process, the chlamydospore yield reached the expected 108 /ml level in 5 days. The improved protocol established significantly shortened the production time and greatly improved the spore uniformity. The usefulness of the established technique platform have been testified in 50 L tank system with the use of 2 strains of Trichoderma spp. and 2 strains of Gliocladium spp., and as well with test strain WJGV2 in a 750L tank fermentor. The liquid formulation produced can be applied by foliar spray, soil drenching and seed soaking/coating. With the application by soil drenching and/or seed soaking of WJGV2 and TLGV22 each at appropriate concentration, substantial growth promotion on cabbage and rice has been demonstrated. In the case of cabbage, the seed soaking with WJGV2 formulation at 50X dilution led to 115.2 % increase in fresh weight 10 weeks after treatment. Whereas for rice, the same treatment led to 58.9 % increase in plant height 2 weeks after application. In a greenhouse system, the application of both strains was also shown effective in controlling Rhizoctonia solani AG4 and Pythium aphanidermatum seedling damping off infections on cabbage. For cabbage seeded on substrates artificially inoculated with R. solani AG4, seed soaking with WJGV2 at 50X dilution led to a 38.9 % increase in survival; likewise, soil drenching with TLGV22 at 2000X in dilution led to a 33.4 % survival increase. As for seedling damping off caused by P. aphanidermatum, soil drenching by WJGV2 and TLGV22 led to 30 % and 21.2 %, increase respectively, in percent survival. The chlamydospore formulation developed from the 2 strains thus appeared to be effective as biofungicide in the control of these 2 important soil-borne fungal pathogens. Moreover, the possible application of these 2 attempted biofungicides in the control of sheath blight on rice (R. solani AG1) was also investigated. The test formulation was applied by dripping as liquid formulation and by spraying as floatable granule formulation. The applied biofungicide treatment significantly reduced the sheath blight infection and resulted in substantial yield increases. Their potential for the control of rice sheath blight appeared to be promising and worth great attention.