Nominating, Molecular Detection About Bacterial Wilt In Mulberry And Horizontal Gene Transfer Analysis In Enterobacteriaceae

A new mulberry wilt disease was observed in summer of 2006, which caused severe economic lost in mulberry plantations in Hangzhou, Zhejiang, China and the disease severity increased year by year. For effective management of the disease, the exact bacterial pathogen needs to be determined and the scientific name has to be nomenclatured. An accurate, rapid and sensitive molecular detection method for the pathogen has to be established. Based on a series of phytopathological methods for the 6 representative strains, the pathogen of this new mulberry bacterial wilt disease was proved to be a new species in Enterobacter genus; a pair of specific primers based on the rpoB regions was designed to detect the pathogen; the bioinformatics tools was used to analyze and evaluate the diversity of Enterobacteriaceae based on the gene gain and loss results. The major results are listed as follows:(1). The results of traditional bacteriology tests, physiological and biochemical analysis, electron microscope observation, pahogenicity and hypersensitive tests, biolog tests, ERIC-PCR analysis,16S rRNA and rpoB sequences analysis of the 6 representative strains show this pathogen is a new species in Enterobacter genus, which has not been reported before. Based on the results of DNA-DNA hybridization of a representative strain R18-2 (ZJUPD3) with the nine Enterobacter type strains, the pathogen was classified into Enterobacter cloacae complex and was nomenclatured as Enterobacter morus. Strain R18-2(ZJUPD3) has been deposited in culture collection center of China and BCCM/LMG Bacteria Collection of Belgium with the deposite ID CGMCC 1.10322T and LMG 25706T, respectivey.(2). A specific primer of the Enterobacter morus was designed within regions of the RNA polymeraseβ-subunit (rpoB) gene. An accurate, sensitive and rapid method was established and tested by performing on 9 type species of Enterobacter,4 E. morus,2 Ralstonia solanacearum strains,2 Pseudomonas syringae pv. mori strains and 50 unidentified epiphytic bacterial isolates from mulberry plants. The real-time PCR assays reliably detected one single cell in a 20μl reaction system, reflecting its strong sensitivity. Moreover, mulberry roots and stems were directly used for PCR detection and it was found that samples with E. morus (acquired by isolation) can be detected by this method whether it has obvious symptoms or not. The results indicated the sensitivity and specificity of the primer. This is the first report of establishing detection system for E. morus causing mulberry bacterial wilt and we applied a China national invention patent (Patent Application Number:200910157005.4).(3). Based on the horizontal gene transfer (HGT) analysis of Enterobacteriaceae, it can be concluded that gene gain and loss is the major force in Enterobacteriaceae evolution and the divergence is huge between core and pan genome. The number of core genome was only 164 while pan genome was more than 100,000. The size of the species core genome for a large number of sequenced strains was extrapolated by fitting the exponential decaying 2 functions to the amount of conserved genes while the number of strain-specific genes was extrapolated by fitting the Multiple Multiplicative Factor (MMF) model to the amount of strain-specific genes. We may speculate that the small genome endophyte in Enterobacteriaceae was first evolved with a gene loss event in this family. Then, species of pathogens were arised with continous HGT events in different evolutionary scale. The markov models suggested that more than 1/3 of genes in Enterobacteriaceae were horizontally transferred, which indicated that HGT played an important role in evolution. Positive selection analysis also revealed that the transferred genes were much easier to be influenced by positive selection pressure than those of the conserved genes.