First Report of Pantoea agglomerans Causing Brown Apical Necrosis of Walnut in China.

Abstract

Brown apical necrosis (BAN) of walnut (Juglans regia L.) causes premature fruit drop and yield losses and has been reported to be an important walnut production problem in Spain, Italy, France, and Turkey (1,2). A number of organisms have been associated with BAN on walnut: Xanthomonas arboricola pv. juglandis, Fusarium spp., and Alternaria spp. (3). Since the spring of 2007, BAN was observed in 50 to 60% of the trees in walnut orchards in Taian City and Laiwu City, Shandong Province, China. Surface-disinfested tissue from premature walnut fruits was placed onto potato dextrose agar. Alternaria spp., X. arboricola pv. juglandis, and Pantoea agglomerans (formerly Enterobacter agglomerans) were isolated 76, 35, and 45% of the time, respectively. The P. agglomerans cultures formed a yellow lawn and were rod shaped with the body length of 1.5 to 3.0 μm, width of 0.5 to 1.0 μm, and four to six flagella. In biochemical tests, these bacteria were gram negative, lactose positive, and indole negative. Genomic DNA was extracted from one HXJ isolate and the 16S rRNA gene sequence (GenBank Accession No. HM016799) was obtained using universal primers 27F and 1492R. HM016799 had 99% sequence identity with P. agglomerans accessions in GenBank (GU477762, GQ494018, FJ756355, and AB004757). To confirm pathogenicity, HXJ isolate (108 CFU·ml-1) was inoculated at the bottom of the stigma within 5 days after florescence (DAF) and in premature fruit wounded with a needle within 30 DAF in 2008 to 2010. Stigmas injected with only sterile water served as controls. The bacteria were inoculated into three replicate 9-year-old plants of the walnut cv. Xiangling. Forty nuts on each plant were inoculated. The plants were grown in Shandong Province, China (36°09'59″N, 117°13'30″E). Ten days after inoculation, typical internal BAN symptoms were observed on all treated nuts and the controls were still healthy. In the inoculated stigmas, necrosis of stigma and style spread to internal tissues and reached the kernel. In treated premature fruit, internal tissues became necrotic and blackish and eventually led to nut drop. The same bacterium was reisolated from the inoculated tissue. On the basis of morphological, physiological, and biochemical characteristics and sequencing of the 16S rRNA gene, the bacterium was identified as P. agglomerans. To our knowledge, this is the first report of P. agglomerans causing internal type BAN of walnut in China or worldwide.