First Report of Necrotic Disease Caused by Pantoea agglomerans on Plum (Prunus salicina) in China

Abstract

Prunus salicina L., commonly called Chinese plum or Japanese plum, is a small deciduous tree native to China and widely distributed in the world. Since spring of 2018, an unknown disease has affected commercial plums grown in an orchard located in Dazhou, Sichuan Province, China (31°40′41″N, 107°43′57″E). Disease symptoms in plum trees include water-soaked lesions on immature fruits, brown necrotic spots, and holes with a yellow halo in leaves, as well as dark brown oily cankers on twigs. To isolate the causal agent, surface-disinfested tissues from three separate symptomatic fruits, leaves, and twigs were placed onto nutrient agar (NA) plates and were incubated at 28°C. Pale yellow colonies with smooth margins and mucoid texture appeared after 12 h of incubation. Individual colonies were transferred two times to NA plates using the conventional streak plate techniques to obtain pure cultures. Purified bacteria cells were rod shaped, 1.5 to 3.0 μm long, and 0.5 to 1.0 μm wide. Three isolates (named CdjP1, CdjP2, and CdjP3) were used for further characterization. Biochemical tests using Gram stain, MacConkey agar, and Kovacs’s indole kit (Solarbio, Beijing, China) indicated that the three isolates were gram negative, lactose positive, and indole negative. The 16s rDNA fragment was amplified for the three strains using universal primers 27F and 1492R (Lane 1991) and sequenced (GenBank accession nos. MN542182, MN542183, and MN542184). BLASTn analysis revealed the nearly full-length (about 1,400 bp) 16S rDNA sequences of CdjP1, CdjP2, and CdjP3 shared over 99% identity with Pantoea agglomerans (MH101508) (identities 1,400/1,406, 1,407/1,412, 1,405/1,413), P. vagans (CP038853) (identities 1,397/1,406, 1,403/1,412, 1,402/1,414), and P. ananatis (KC178592) (identities 1,399/1,406, 1,405/1,412, 1,404/1,414), respectively. Multilocus sequence analysis has been shown to be a powerful molecular method for delineation of Pantoea species (Deletoile et al. 2009; Tambong et al. 2014). To identify the species of CdjP1, CdjP2, and CdjP3, six housekeeping genes (fusA, gyrB, leuS, pyrG, rlpB, and rpoB) of each strain were amplified and sequenced (GenBank accession nos. MN549363 to MN549380). Then a phylogenetic tree was constructed using Bayes inference methods based on fusA-gyrB-leuS-pyrG-rlpB-rpoB concatenated fragments (2,997 bp), which showed that CdjP1, CdjP2, and CdjP3 are most closely related to P. agglomerans. To fulfill Koch’s postulates, pathogenicity tests were conducted on 3-year-old plum trees (cv. Qinba) grown in Sichuan Province, China (31°40′53″N, 107°44′2″E). Immature plum fruits, leaves, and twigs were wounded with a sterile toothpick and were inoculated with 20 µl of bacterial suspension (10⁸ CFU/ml) of each isolate. Plants inoculated with sterile water were used as a control. All treatments and the control were repeated three times. The trees were then grown under natural conditions (monthly mean maximum temperature 32°C, monthly mean minimum temperature 23°C). After 2 weeks, all test-isolate-inoculated plum fruits, leaves, and twigs developed symptoms similar to those of the natural infections observed. No lesions were observed on the controls. Bacteria reisolated from the infected plum fruits, leaves, and twigs were similar in colony appearance to the original isolates, and the 16s rDNA fragments were identical to their original isolate sequences, thereby fulfilling Koch’s postulates. Based on colony morphology, biochemical tests, 16S rRNA gene sequencing data, and phylogenetic analysis, the pathogen was identified as P. agglomerans. In China, P. agglomerans has been reported to infect cotton, walnut, Vigna angularis, and Ziziphus jujuba (Lu et al. 2015; Ren et al. 2008; She et al. 2019; Yang et al. 2011). To our knowledge, this is the first report of P. agglomerans causing bacterial necrosis of plum in China or worldwide. This report expands the host range of P. agglomerans and will help to develop effective disease control strategies, as the etiology is known.