Screening,Identification And Greenhouse Biological Control Of Streptomyces Sp.Activity Against Xanthomonas Oryzae Pv.oryzae

Rice bacterial blight is one of the most serious rice diseases in the world,caused by bacterial plant pathogen Xanthomonas oryzae pv.oryzae?Xoo?which lead to the quantity and quality of rice.It is the top ten with highest yield loss.In areas where the diseases is not serious,rice production will be reduced by about 20-30%,but in some areas which highly prevalent,rice production will be reduced by 80-100%.Currently,only a few rice varieties are resistant to this disease but not enough for resistance to many diseases.Using biocontrol is a good choice for disease control.Streptomyces have a long history of biological control of plant diseases and have been extensively studied.They can decompose complex mixtures of plant that are important to the ecosystem and the most of them are found in spores.Many of secondary metabolites antibiotics are produced in the stationary phase.Streptomyces as biocontrol agents have efficiency and safety for humans and other non-target organisms.In this study is screening and identification antagonists Streptomyces against the bacterial pathogen of Xanthomonas oryzae pv.oryzae?Xoo?1.Soil samples were taken from different places from Zhejiang Normal University,for example,grasslands,roots of tree,garbage area and the canteen area which obtained 65 strains.Streptomyces strains were co-cultured with Xoo,and then 8Streptomyces strains were obtained by preliminary screening.Re-screening again by Oxford cup method,the strongest effect of the inhibition zone was Streptomyces strain Sr-63 which isolated from roots of tree which diameter was 46.48±2.32 mm and it was obtained by fermentation with 50 ml of Gauze's synthetic medium No.1.The phylogenetic analysis using MEGA software indicated that strain could be confirmed that the defense was belonging to the genus Streptomyces and it was closely related to Streptomyces roseoverticillatus.For the nitrogen source,the strain utilizes peptone,tryptone,L-glutamic acid,L-asparagine,potassium nitrate?KNO₃?,L-isoleucine.For carbon sources,the strain utilizes glucose,sucrose,raffinose,rhamnose,?-lactose,mannose,galactose,xylose,inositol and arabinose are used.The most suitable Na Cl concentration is 1%and it could survive to above 2%Na Cl.The most suitable acid-base was p H 6-7 and it could survive in p H 3-11.The optimum temperature for growth was between 28-37? and the viable culture temperature was 20-43?.2.Antimicrobial effect of Streptomyces sp.Sr-63 strain on rice bacterial blight in different media which incubated with 100 m L in different broth media for 3-10days at 28? on rotary shaked at 180 rpm.Gauze's synthetic medium No.1:the biggest of inhibition zone was 5 d culture which diameter was 46.50±1.05 mm.PDA's improvement medium:the biggest of inhibition zone was 6 d culture which diameter was 45.50±0.58 mm.PD medium:the biggest of inhibition zone was 9 d culture which diameter was 41.75±0.96 mm.Hiskey medium:the biggest of inhibition zone was 5 d culture which diameter was 46.00±0.82 mm.GYM medium:the biggest of inhibition zone was 7 d culture which diameter was 27.75±0.50 mm.ISP2 medium:the biggest of inhibition zone was 9 d culture which diameter was 34.00±0.82 mm.SCA medium:the biggest of inhibition zone was 9 d culture which diameter was32.75±0.96 mm.From the above results,it could be concluded that the best antimicrobial effect on the Sr-63 strain was Gauze's synthetic medium No.1.3.Antibacterial and antifungal test with fermentation filtrate of Streptomyces sp.Sr-63 strain,The best effect of antagonizing with bacterial pathogens in vitro was Xanthomonas oryzae pv.oryzicola which diameter was 25.33±0.76 mm.And the best effect of antagonizing with fungal pathogens in vitro was Fusarium solani which the bacteriostatic rate averaged was 12.62%.Fermentation filtrate stability test:the fermentation broth was influence on the UV light?40W?,after the irradiation time reached 18 h,the inhibition was decreased by 57.35%.But it has no significant effect on fluorescent lamps.Temperature stability test:it also has an influence on the heat temperature,when the temperature reached 90-100°C,the inhibition was reduced by59.39%.4.The optimization of fermentation filtrate of Streptomyces sp.Sr-63 strain,the ingredients of Gauze's synthetic medium No.1 was optimized.The optimization of carbon source:the inhibition zone of soluble starch was the largest with 36.00±1.26mm of diameter.At a concentration of 5%soluble starch,the diameter was the largest at 54.33±1.75mm.Optimization of nitrogen source:the inhibition of KNO₃ was largest and the diameter was 37.17±0.98 mm.At a concentration of 0.3%KNO₃,the diameter was largest at 46.33±2.07 mm.The Na Cl concentration was 0.5%,and the diameter of the zone of inhibition was 44.33±0.82 mm.The acid-base optimization was p H 7 which diameter was 43.17±1.17 mm.Optimization of culture time was 5 d culture which diameter was 46.50±1.05 mm.Culture temperature optimization was 28? which diameter was 46.33±1.21 mm.Optimization of RPM speed was 160 rpm which diameter was 43.50±1.64 mm.From the above optimization of fermentation,the diameter of the inhibition zone reached 48.83 mm±1.17 mm.5.Greenhouse biological control experiment:using the fermentation broth of Streptomyces sp.Sr-63 strain for controlling six rice species susceptible to rice bacterial blight disease,five of them were rice species from Thailand?Thai black glutinous rice,Chai Nat 2,Khaoluang,KDML 105,RD6?and one chinese rice specie?Zhonghua 11?.The best result of biological control was method 1?spray the fermentation filtrate first,then inoculate the pathogen?,especially in Zhonghua 11,KDML105,Khaoluang and Chai Nat 2,the lesion inhibition ratio has reached more than 90%which the lesion inhibition ratios were 95.35%,94.39%,94.14%,and92.86%respectively.Second,using the method 5?Before planting rice,water the fermentation broth in to the soil and then inoculate the bacteria?,the inhibitory effect was great,especially in KDML105 and Khaoluang which the lesion inhibition ratios were 80.14%and 75.83%respectively.Comparing the fermentation broth with bismerthiazol which was a type of bactericides,except for Thai black glutinous rice,fermentation broth of strain Sr-63 has a better control effect than bismerthiazol.From the results was showed that strain Sr-63 was a good preventing with rice bacterial blight disease.