Identification And Molecular Detection Of The Causal Agent Of Bacterial Leaf Spot Of Corn

Bacterial leaf spot of corn is a class of leaf spot diseases caused by various bacterial species. The pathogens and disease symptoms of the bacterial leaf spots exhibit a wide range of diversity. In recent years, the occurrence of bacterial leaf spot disease is increasing in the fields of normal corn varieties and sweet corn in most corn production regions of China. Bacterial leaf spot of corn has shown the characteristics of fast spread, rapid occurrence, and difficult to be controlled. This disease has impacted on maize production in some regions. However, this disease was rarely tudied in the world.In this study, the pathogenicity and cultural characteristics of the pathogens were studied to determin the cause agents of the severe chlorosis and spot symptoms on corn leaves.This study was conducted to investigate the causal agent of the severe chlorosis and spot symptoms on corn leaves Based on the results of conventional morphological observation, physiological and biochemical characterization, analyses of whole-cell fatty acids and 16S rDNA sequence, this strain of bacterium was identified to be Bacillus megaterium. At the same time, a rapid detection of Pantoea ananatis was established using the known specific primers.Using the universal primers L1/L2 for the bacterial 16S-23S rDNA ITS, the ITS fragment of the B. megaterium was amplified with the size of 356 bp. Based on the homologous sequence analysis using NCBI Blast homology search, specific primers BaF1/BaR1 were desighed using premier5.0 package. This primer pair was able to amplify a 125 bp fragment from the target bacterium, but not from the bacterial strains other than B. megaterium, which indicated that the primers are highly specific. The sensitivities of the primers for amplifying B. megaterium-specific products from the bacterial suspension and isolated genomic DNA were 3×104 CFU/mL and 16 pg/μL, respectively. A rapid detection of B. megaterium with molecular technique was thus established using the specific primer pair BaF1/BaR1.The bacterial strains whose colony morphology and cultural characteristics were the same as B. megaterium and Pantoea ananatis were selected from the isolations products of diseased leaf tissues of bacterial leaf spot disease. A 125 bp fragment was amplified from 10 strains which were isolated from Zhejiang,Hainan and Yunnan province by the specific primers BaF1/ BaR1, respectively, and these strains were identificated to be B. megaterium. However, A 363bp fragment was amplified from the other 7 strains which isolated from Zhejiang, Hainan and Beijing by the specific primers PanITS2-1F/PanITS2-1R, respectively, and these strains were identificated to be P. ananatis.