First Report of Enterobacter roggenkampii and E. cloacae complex sp. Caused Tomato Pith Necrosis in Guangdong Province, China.

Abstract

Tomato (Solanum lycopersicum L.) is an important greenhouse and field-grown vegetable. During 2019 to 2021, a new bacterial pith necrosis broke out in tomato producing areas in China. The disease incidence rate in the field was approximately 10% to 30% in a few tomato planting areas of Guangdong province, and even 100% in Dianbai distinct, Maoming city. Diseased plants showed yellowing of the lower leaves, brown vascular tissues, and wilting along with brown necrotic spots and a large number of adventitious roots on the stem. Diseased plants were collected, and short fragments of the diseased stems were sterilized with 75% alcohol for 2 minutes, washed with sterile water twice, and stripped the cortex (Fang 1998). Dilutions of xylem specimen soaking solution were plated onto the TTC medium (peptone 10.0 g, acid hydrolyzed casein 1.0 g, glucose 5.0 g, agar 15.0 g, distilled water 1000 mL, 0.5% 2, 3, 5-triphenyltetrazolium chloride, pH7.0), and cultured at 28℃ for 24 h. Three pink single colonies (A2 from Guangzhou (113°21' E, 23°9' N), Guangdong, and K6, and K7 from Maoming (110°55' E, 21°25' N), Guangdong) were selected and purified. Strains A2, K6, and K7 were Gram-negative, motile, and showed white fluidal colonies with pink center on TTC medium, and white, round, and smooth-surface colonies on NA medium (peptone 10 g, beef extract 3 g, sodium chloride 5 g, agar 15 g, distilled water 1000 mL, pH7.0) at 28℃ for 24 h. Three strains could utilize citrate, sorbitol, lactose and arginine, and were negative for methylred reaction test, determination of phenylalanine amino acid deaminase, lysine decarboxylase, urease, soluble starch decomposition and gelatin liquefaction, whereas were positive for Voges-Proskauer test, which conformed to the characteristics of genus Enterobacter (Davin-Regli et al. 2019). To determine the species of the Enterobacter isolates, partial sequences 16S rDNA, gyrB, and rpoB of strain A2, K6, and K7 were amplified. The PCR products were purified, sequenced, and deposited to GenBank. The BLASTN analysis of 16S rDNA, rpoB and gyrB sequences showed strain A2 (MW785888, OL364948, OL364943) was 99.20%, 99.17% and 98.57% identity with E. roggenkampii DSM16690, respectively, strain K6 (MW785890, OL364950, OL364945) was 99.73%, 99.63%, 99.63% identity with E. cloacae complex sp. N13-01531, and strain K7 (MW785893, OL364951, OL364946) was 99.8%, 98.81%, 98.99% identity with the E. roggenkampii Ed-982 and Ek140. Nucleotide sequences of 3 strains were aligned using ClustalW program, and neighbor-joining method (NJ) was used in the construction of a phylogenetic tree using MEGA7 program. Phylogenetic trees based on gyrB sequence, rpoB sequence, and the concatenated sequence of 16S rDNA-rpoB-gyrB and rpoB-gyrB showed strain A2 and K7 were clustered to E. roggenkampii, strain K6 was clustered to E. cloacae complex sp. The roots of tomato material 'Moneymaker' at stage of 4-5 true leaves were cut and irrigated 10 mL bacterial suspension (OD600=0.6) of strains A2, K6, and K7, respectively. As a control, the tomato roots were treated with 10 mL sterile water. All plants were incubated at 30°C. The experiments were conducted with 20 tomato seedlings for each tested strain and control, and repeated twice. All plants inoculated showed yellowing in the lower leaves 6-7 days after inoculation (DAI), subsequently the stems of some plants were rotten, along with bacterial pus in the internodes. The plants wilted, and stems were hollow 20 DAI, which is similar to the field symptoms. No symptoms were observed in control plants. Strains were successfully reisolated from wilting plants, and identified as A2, K6, and K7, respectively, based on gyrB sequence analysis, fulfilling Koch's postulates. Zhou et al. (2021) reported that E. roggenkampii caused bacterial wilt of mulberry in Guangxi, China. Chen et al. (2021) reported E. asburiae caused tomatoes pith necrosis in Fujian and Zhejiang, China. To our knowledge, this is the first report of E. roggenkampii and E. cloacae complex sp. causing bacterial pith necrosis of tomato. Further research would focus on exploring the pathogenic mechanism of the pathogen, and providing reference of controlling the disease.