First Report of Pre-Harvest Soft Rot of Peach Fruit (Prunus persica) Caused by Enterobacter mori in China.

Abstract

Peach (Prunus persica L. Batsch) is one of the major fruit crops of China and a rich source of vital nutrients and fibers. In July 2019, a peach orchard was visited at Pinggu District, Beijing, China. The average temperature and relative humidity at the sampling site were 28±2 °C and 75±2 % respectively. In the orchard, unripe and near to ripe peach fruits cv. 'China Pearl' were observed with soft brown patches (3-4×2-3cm) on the surface. The samples (n=25) were collected based on typical symptoms. When the rotten part of the fruit was pressed, a liquid oozed out of the fruit emitting an unpleasant odor. The disease incidence of fruit soft rot was 22 %. The percent incidence was calculated based on total number of infected fruits divided by total number of fruits examined. Infected fruit tissues were excised into small pieces (5mm) and surface disinfested with 1% NaClO for 25s and rinsed twice with sterilized distilled water (SDW). These pieces (4-5 pieces per sample) were macerated with 1.5 ml SDW in a 2 ml tube. Later, 5 µl supernatant was streaked on Luria Bertani (LB) agar plates and incubated at 28 ºC for 3 days. Oozing liquid (50 µl) was also inoculated on 100ml LB broth and incubated for 24 h and 5 µl was streaked on LB agar medium and incubated at 28 ºC. Purified colonies were obtained by re-streaking 3 times. The isolate formed creamy to light yellowish, irregular, round, rough or smooth-looking colonies on LB medium and pink colonies on Eosin Methylene blue. The bacterium was rod shaped, measuring 0.8 to 1.4 μm in length and 0.2 to 0.5 μm in width (Fig 1) observed using a scanning electron microscope (Hitachi-SU8010). Morphological characteristics were similar to previously described characteristics of Enterobacter spp. (Zhu et al. 2011; Nishijima et al. 1987). Genomic DNA was extracted from all twenty-five isolates by using TIANamp Bacteria DNA Kit (Tiangen-Biotech, Beijing, China) according to the manufacturer's instructions. The 16S ribosomal RNA gene of 25 isolates was amplified by using universal primers (27F:5'-AGAGTTTGATCCTGGCTCAG-3',/1492R:5'-CTACGGCTACCTTGTTACGA-3'), and rpoB gene (F2: 5'-AACCAGTTCCGCGTTGGCCTGG-3', R2: 5'-CCTGAACAACACGCTCGGA-3') (Mollet et al., 1997). Sequences for EPT-1 were submitted to GenBank (accessions MN548761 (16S rRNA), MN594495 (rpoB)). Accessions MN548761 and MN594495 had 99.35 and 99.77% sequence identity with E. mori (GenBank accessions KF747680, GQ406571). Maximum Likelihood phylogenetic tree constructed (1000 replicates) using Mega X (Kumar et al. 2018) indicated that isolate EPT-1 clustered with E. mori sequences. To confirm the pathogenicity, medium-sized (n=60) surface disinfested ripe peach fruits cv. 'China Pearl' were wound inoculated with 5 µl suspension of EPT-1 (107cfu/ml) and the control fruit were treated with 5 µl sterilized water. The fruit were kept in sterilized plastic box and incubated at 28 ºC for 7 days at 70% relative humidity, and the pathogenicity test was repeated 3 times. Symptoms similar to the original fruit samples were observed on all inoculated fruit (Fig 2). The pathogen was re-isolated, the colonies obtained from the oozing liquid were similar to those of infected fruit tissues and identified as E. mori based on morphological and sequencing analysis. Previously, E. mori has been reported to cause bacterial wilt on Morus alba L. in China (Zhu et al. 2011). To our knowledge, this is the first report of E. mori causing soft rot of peaches in China.