Identification And Phylogenetic Analysis Of Walnut-associated Pantoea Agglomerans

Walnut is an important economic tree in China, which has a long history and wide distribution. With the development of walnut industry, the cultivation of walnut is intensive and variety. Due to the cultivation technology limited and the genetic difference of walnut varieties. the walnut diseases become more and more serious. Walnut blight(WB) and Brown apical necrosis(BAN) caused by Pantoea agglomerans is important diseases of walnut.This paper mainly collected 54 strains as sampled from different locations and studied on the morphological observation, biochemical property and the taxonomic status using multi- locus sequence analysis(MLSA) and the genetic diversity using BOX-PCR and ERIC-PCR. The main results are as follows:(1) The colony of the strains were round with smooth surface, and it is oyster white at the beginning and becomes deep yellow in the later stage. The thalli grow in colonies, and the thallus was straight-rod- like, with a size of 0.9-3.0 ?m×0.6-1.2 ?m, and 4-6 peri- flagellum. The biochemical properties were as follows: gram negative, non- liquefied gel, hydrolyzation of starch, negative arginine dihydrolase, oxidative metabolism of glucose producing acid rather than gas, and nitrate reduction.(2) 16 S r DNA sequences of the tested strains were sequenced.Selected fusa, rpo B, gyr B,leuS that of four housekeeping genes for multilocus sequence analysis(MLSA)of the tested strains.MLSA divides the Pantoea agglomerans for test into 6 different evolution branches,the A group included all strains from SXS and GSC from GSL. The B group included all two strains from GZB. The C group consisted of NSA, NSB from SDJ, TSA from GSC, and all three strains from SDS. The D group included all three strains from GSQ. The E group consisted of GSA, GSB from GSL.The F group chonsisted of WSA from SDW and all four strains from SDT. MLSA is a reliable, stable and sensitive identification tool, which can be used to distinguish the species of Pantoea agglomerans. MLSA has the important significantion for the identification and phylogenetic, geographical and taxonomic studies of Pantoea agglomerans.(3) ERIC-PCR and BOX-PCR were performed on 54 strains of Pantoea agglomerans, and the size of the amplified products ranged from about 150 bp to 5000 bp. BOX-PCR and ERIC-PCR amplified a total of 399 bands, of which 328 bands were polymorphic, and the percentage of polymorphism was 82.21%, which showed a high level of po lymorphism. ERIC-PCR and BO X-PCR cluster analysis showed that the tested strains could be divided into 4 groups. The genetic diversity and genetic relationship of the fungus were demonstrated at the molecular level.ERIC-PCR and BOX-PCR are excellent tools for the identification of different groups of the Pantoea agglomerans populations.(4) The Ne value of the strains for test was 1.4275, the Nei's genetic diversity(H) was 0.2714, the Shannon information content(I) was 0.4282, indicating abundant polymorp hism of the 54 Pantoea agglomerans, and high genetic diversity level. The genetic identity among the 9 populations was 0.5956-0.9926, the genetic diversity index(Gst) among populations was 0.6932, and the genetic flow was 0.2213, which means that there was low genetic exchange among the Pantoea agglomerans populations for test, and low genetic flow would promote genetic differentiation. 62.82% genetic variation derives among the 9 populations of Pantoea agglomerans, and 37.18% variation derives within populations(Fst=0.6282). The genetic diversity of the Pantoea agglomerans may be related to the extensive geographical distribution of it and the regional cultivation of walnut.