Selection And Mechanism Of Action Of Antagonist Yeast Against Bacterial Fruit Blotchbiosynthetic Genes

Bacterial fruit blotch(BFB) caused by Acidovorax avenae subsp.citrulli(Schaad et al.) Willems et al.(Aac) is a serious disease threatening the watermelon industry.So far, there are no available commercial cultivars of cucurbit crops resistant to the disease. Although some bacteroicides could be used to control BFB,consumers concern over the bactericides contamination to environment and residues of bacteroicides in watermelon. Therefore,it is necessary to develop alternative control methods to reduce environmental risks and raise consumer confidence.Biological control is considered an ecologically sound and environmentally friendly approach for management of BFB on hami melon.A study was undertaken to screen antagonistic yeast strains,to evaluate the biocontrol potential of selected yeast strains,to identify selected yeast strain 0732-1,to characterize cultural conditions for production of antibacterial substances(ABS) by the yeast strain 0732-1 and mechanisms involved in biocontrol of BFB by the yeast strain 0732-1.1.Four hundred and sixty-three yeast strains were isolated from leaves or flowers of plants collected from Wuhan of Hubei,Xinyang of Henan,Shihezi of Xinjiang in China for against BFB.Through the sequential screening procedure(in vitro test and in vivo test),a yeast strain designated as 0732-1 was selected as a biocontrol agent for BFB of hami melon.2.Treatment of hami melon seeds with cell-free cultural filtrates of the yeast strain 0732-1 resulted in a significant reduction in severity of seedling blight caused by seedborne Aac,and the efficacy was not significantly different(P＞0.05) from that of chemical seed treatments including streptomycin sulfate(0.1%,w/v) and hydrochloric acid(2%,v/v).Spraying concentrations of the yeast strain 0732-1 at 1×10⁹cfu/ml and at 1×10⁷ cfu/ml were significantly capable of reducing disease index of BFB and the efficacy was not significant after incubation for 15 d in field test.Efficacy of the yeast strain 0732-1 against BFB on hami melon was maintained when applied before pathogens, but when applied simultaneously with or after Aac,biological control efficacy was significantly reduced.In addition,the yeast strain 0732-1 had no effect on promotion of hami melon growth when it cultures of this yeast strain was drench-applied to roots of hami melon. 3.Based on morphological,cultural and physiological characteristics and analysis of the DNA sequence of the internal transcribed spacer of ribosomal DNA(The GenBank accession numbe EU380207),the yeast strain 0732-1 was identified as Pichia anomala Kurtzman.4.The yeast strain 0732-1 could use six kinds of nitrogen sources and eight kinds of carbon sources for growth,and could utilize each of fructose,glucose,sucrose,maltose, ethanol and glycerin for ABS production.The optimum carbon source was fructose, followed by glucose or sucrose.Peptone was the optimum nitrogen source for ABS production.Potassium phosphate(KH₂PO₄) could enhance the yeast strain to produce ABS.5.The yeast strain 0732-1 could effectively inhibit growth of 21 plant pathogens including 7 pathogens isolated from hami melon.This result suggests that the yeast strain 0732-1 might be an agent with potential to control other diseases of hami melon.6.Culture factors affecting ABS production by the yeast strain 0732-1 were studied. Results showed that the optimum medium was PSB and the optimized with the initial pH value ranging from 5.0 to 11.0,the amount of PSB being 80 ml in 250 ml flasks,the inoculum being 10%(v/v);the temperature being 20℃and the culture time being 96 h.7.Results showed that ABS produced by the yeast strain 0732-1 was heat-stable (121℃,25 min),resistant to UV irradiation,active under pH4.0.This ABS could not purified by precipitation with(NH₄)₂SO₄(20-90%,w/v) or by extraction with benzene, acetone,chloroformm,tetrachloride,ethanol and ethyl acetate.It could be partially distilled out.8.Production of ABS by the yeast strain 0732-1 was found to be the most important mechanism responsible for suppression of BFB caused by Aac.Meanwhile,results showed that the yeast cells of 0732-1 could survive on hami melon leaves and on hami melon fruits.Inoculation 1×10⁹cfu/cm²,the population quantity of the yeast strain 0732-1 decreased to 1.2×10⁵ cfu/cm² after 15 days on hami melon leaves,and to 1×10⁷ cfu/cm² after 10 days on hami melon fruits.Additionally,treatment of hami melon leaves with the yeast strain 0732-1 could enhance activity of a few defense-related enzymes including peroxidase,phenylalanine anmmonia-lyase and catalase,induction of defense response might be another mechanism involved in suppression of BFB by the yeast strain 0732-1.These results laid a solid foundation to use the yeast strain 0732-1 to control BFB of hami melon.