First Report of Necrotic Disease Caused by Pantoea agglomerans on Ziziphus jujuba in China

Abstract

Ziziphus jujuba Mill., called Chinese date, has been grown in China for over 4,000 years. Z. jujuba ‘Dongzao’ is an important economic cultivar in northern China. In the recent years, Z. jujuba ‘Dongzao’ has been introduced into southern China. In February 2018, a new disease with necrotic symptoms affecting the leaves of Z. jujuba ‘Dongzao’ was observed in Qingyuan city, Guangdong province, China. Initially, tips of young leaves blackened and showed necrotic spots, and then the necrotic spots expanded until the whole leaf withered. A symptomatic jujube tree was collected from the field. Leaf samples from the diseased plant were washed with tap water, and 0.5- × 0.1-cm sections of the symptomatic tissue sample were surface disinfested in 70% ethanol for 30 s and 1% sodium hypochlorite for 30 s and then rinsed with sterile water six times. The tissue was mashed and suspended in 100 μl of sterile distilled water and left to stand for 5 min; the suspension was streaked on Luria-Bertani (LB) agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.2). Plates were incubated for 24 h at 30°C. Bacterial colonies with small, irregular round, slimy, yellow colonies were isolated from the diseased leaves. Two isolates were randomly selected for pathogenicity assays. Two-year-old jujube trees were spray inoculated with a bacterial suspension (3 × 108 CFU/ml), with three jujube trees for one isolate. The negative control was spray inoculated with LB medium. The inoculated jujube plants were incubated at 22 to 26°C and exhibited the same symptoms as the diseased jujube plants in the field 3 days postinoculation. The bacterium was reisolated from the leaf tissues collected from the inoculated jujube plants. No symptoms were observed on control plants. Two isolates could utilize citrate, malonate, D-cellobiose, D-mannitol, glycerol, L-rhamnose, inositol, maltose, salicin, and sucrose but not D-tartrate, D-sorbitol, lactose, and raffinose. These two isolates also gave a positive reaction for gelatin liquefaction, tobacco hypersensitivity, nitrate reduction, and yellow pigment production but gave a negative reaction for H2S production and fluorescence on King’s medium B. The physiological and biochemical characteristics of these two isolates were same as those of Pantoea agglomerans strain ATCC27155. Using the universal bacterial 16S rDNA primer set 27F/1541R (Weisburg et al. 1991; Woese et al. 1983), 1,447-bp fragments were amplified from the two isolates. The sequences of the two fragments (GenBank accessions MK348973 to MK348974) shared 99% sequence identity with 16S rDNA of P. agglomerans strain TH81 (GenBank accession CP031649) and Pantoea vagans strain FDAARGOS_160 (GenBank accession CP014129). Furthermore, the phylogenetic analysis results based on sequences of five housekeeping genes (fusA, gyrB, pyrG, leuS, and rplB; GenBank accessions MK348963 to MK348972) established that the two isolates were P. agglomerans. To our knowledge, this is the first recorded report of necrotic disease caused by P. agglomerans on Z. jujuba in Guangdong province in China.