Isolation And Identification Of A New Pathogen Pantoea Ananatis Causing Rice Leaf Blight

Rice as a major food crop in China is closely related to people’s lives,and the country’s rice cultivation area is widely distributed,Northeast China has fertile land,suitable temperature in summer,and sufficient sunshine.In recent years,it has been the main producing area for high-quality rice in China.However,Rice planting is often accompanied by a variety of pests and diseases,and rice yield is inevitably affected.For common rice diseases,cultivating and promoting the cultivation of rice disease-resistant varieties is the main means for comprehensive disease prevention,but this may lead to some new diseases.In recent years,rice diseases caused by Pantoea genus bacteria have been found in many regions in foreign countries,and pantotheca bacteria have also been reported to cause panicle diseases in rice in China.In the summer of 2016 and 2017,leaf wilt symptom was found in rice leaves in Dandong of Liaoning Province,Gongzhuling and Laxi of Jilin Province.The symptom of infected leaves was similar to that caused by Xanthomonas oryzae pv.oryzae infection.The symptom showed lesions on leaves extending the leaf margins,dehydration and dryness.Through pathogen isolation identification and pathogenicity testing of collected diseased leaves,it was determined that the disease was a rice leaf disease caused by P.ananatis.In addition,this study analyzed the biological characteristics of P.ananatis,and preliminary explored the interactions between P.ananatis and rice.The specific findings are as follows:1.In this paper,representative representative diseased leaves were collected from paddy fields,and 43 representative strains were isolated from the diseased leaves by dilution isolation.Through the physiological and biochemical tests of bacterial isolates,16S rDNA and gyrB gene sequence analysis,28 strains of bacteria were identified as Pantoea,of which 19 strains of P.nanatis,the bacteria can be isolated in each region of rice disease leaves.The isolated 43 bacterial isolates were inoculated on tobacco leaves.The results showed that all P.nanatis and P.gglomerans can cause tobacco allergic reactions.Six P.nanatis strains and four P.gglomerans strains originating from different regions were selected and inoculated with rice leaves using a leaf cutting method.Four P.nanatis strains could cause the symptoms of rice leaf blight,and they were derived from the inoculated rice leaves.P.nanatis can still be isolated,and Koch’s rule verification is completed.It is determined that P.nanatis is the pathogenic factor of the rice leaf diseases.This is the first report of rice leaf diseases caused by P.nanatis in China.2.P.nanatis can infect many host plants such as rice and maize,but there are no specific studies on the biological characteristics of P.ananatis.The effects of different conditions including temperature,pH and salt concentration on the growth of P.ananatis were tested in this paper.The results showed that the optimum temperature for P.ananatis was 28-32℃.If the temperature is too high or too low,the growth rate of the bacteria will be slowed down.The lethal temperature of the bacteria is 53℃;the pH value of the bacteria is suitable for the pH value of 5.7-7.4.Growth under weak and acidic conditions,weak alkaline conditions at pH above 8.2,slow growth,no growth under strong acid and alkaline conditions,and rapid growth of bacteria at salt concentrations of 1-3%.The higher the growth rate is,the lower the growth rate is,and the salt concentration cannot exceed 9%.It can not grow on Kan,Amp,Gm,Cep commonly used antibiotic plates.3.In order to explore the interaction between P.ananatis and rice,leaf was used to inoculate the leaves of rice plants.After inoculation,rice leaves were collected at 0,12,24,48,and 72 h.RNA was extracted using Trizol method.The relative expression levels of defense genes(RR1b and RR5)were detected by qRT-PCR at different time points.The results showed that the relative expression levels of RR1b and RR5 genes were gradually increased after 24 h of inoculation.At 72 h after inoculation,the expression of RR1b gene was up-regulated by 18-fold,and the expression of RR5 gene was up-regulated by 23-fold.The change trend of PAL gene was not clear.This indicates that the bacteria can induce regulation of defense gene expression after invading rice leaves.