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Isolation, Screening and Development of Biocontrol Agent of Endophytic Antagonistic Bacteria to Camellia Soft Rot Disease

Author: LiXueJiao
Tutor: ZhouGuoYing
School: Central South University of Forestry Science and Technology
Course: Microbiology
Keywords: Camellia oleifera soft rot disease Antagonistic Endophytic Bacteria Fermente Biological control
CLC: S476
Type: Master's thesis
Year: 2011
Downloads: 51
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Abstract

Camellia oleifera soft rot disease is one of dangerous diseases in Camellia region. In south China provinces it has caused the varying degree damage. The disease mainly damages camellia leaves and fruits. Camellia soft rot disease has become the supplement quarantine object in Anhui province. The suffering parts are decayed causing a lot of leaves and full-drop, even fallen leaves and fruits. Caused significant economic loss. At the nursery, camellia branches suffer pretty serious, even whole plant becomes to die.At present chemical prevention is the main control method on camellia soft rot disease. There is no biological control reports. This paper isolated a series of endophytic bacteria strains form the healthy Camellia leaves, signed L46. Carry on the physiological and biochemical experiments and molecular identification, it was judged that the strain had high relationship with the Bacillus amyloliquefaciens. The strain L46 was made wetter powder, and used it in the field control. Main results was reported as follows:(1) Separation and screening endogenous antagonist bacteria against Camellia Soft Rot disease.56 bacterial strains were isolated from the healthy leaves of Camellia oleifera collected from youxian, Hunan province. One strain inhibiting Camellia Soft Rot disease were screened out by flat confrontation test and liquid fermentation tests, signed L46. Through morphology observation, physiological and biochemical determination and molecular identification, the strain L46 was Bacillus amyloliquefaciens. In this paper L46 strains was the major object which was persevered in CCTCC, numbered CCTCC M 2010324. The strain L46 have teratogenic role to Camellia soft rot, causing hyphae fracturing and bresking.(2) Optimizing fermentation condition of endogenous antagonist bacteria L46 to Camellia Soft Rot disease. Single factor and orthogonal test were used in this paper. The optimal fermentation conditions of producing antibiotic products of L46 were given out. The best medium was NB, the best carbon source was lactose, the best nitrogen source were peptone and yeast cream. The optimal culture conditions of the bacteria for the antibacterial protein producing are pH7.0,30℃,7%,140r/min, NYBD medium with shaking for 72h. The design of orthogonal test L9(34) showed that the best media was lactose 2.0%, specially designed 0.8%, yeast extract 1.1%, NaCl 0.5%. At the optimal medium and cultivating conditions, determinate the bacteriostatic rate curve of L46 and OD600 curve of strain L46. It showed that, strain L46 has the biggest biomass after trained 36h, but had supreme antibacterial activities after 72h.(3) Development on wetter powder of endogenous antagonist bacteria L46 to Camellia Soft Rot disease. Single factor and orthogonal test used wettability and suspended rate as standards. It showed the best formula of wettab powders of strain L46:Kaolin 35.72%, ZnSO4 1.34%, STPP 3.57%, SDBS 8.93%, Ascorbic acid 0.44%, original powder of L46 50%. Various indicators of wetter powders of strain L46 have reached national standard. Through the study on the wetter powder storage time and temperature, it showed that the dry powder could be saved at least for 2-6 months, but the specific expiration date also needs for further verification. In three different experimental bases, studyed the wetter powder controling effect experiment, it showed that the effect of L46 wetter powder had the similar control effect with chemical pesticide.

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