First Report of strain YL19 causing crown infection pockets in strawberry in Liaoning Province, China.

Strawberry (Fragaria × ananassa Duch.) is an important fruit crop in China. Typical crown infection pockets symptoms were observed on the infected strawberry in Liaoning province, China (121°60'E, 38°90'N) in the autumn of 2017. The disease incidence was estimated to be around 5 to 10 %, but could reach 30 to 40 % in some heavily affected plastic tunnels. The infected plants early displayed water-soaked symptoms on the abaxial leaf surface and subsequently developed reddish-brown shaped stripes and coalesced lesions on the adaxial leaf surface around the main veins (Fig. 1-A, 1-B). Several variable-size (0.3-0.8 mm in diameter) pockets were observed inside the crown tissues after dissection (Fig. 1-C). The diseased plants rarely reached fruiting and were easily broken between the crown tissue and the stem, and would eventually die. To identify the causal agent of this disease, the several surface-disinfested infected main veins and crown tissues were individually ground in sterile water and plated on sucrose peptone agar(SPA) medium (Hayward 1960) with 10-fold serial dilutions and incubated at 25℃. A number of yellow colonies grew on the medium at the 104 dilution 7 days after plating (Fig. 1-D) in all specimens. The colonies were aerobic, yellow, viscous, smooth, and gram-negative, which is a typical characteristic of . To confirm identity of the causal bacteria, 18 colonies selected randomly were subjected to polymerase chain reactions (PCR) for the amplification of the (Sahin et al. 2010), , , and (C Manceau et al. 2011), respectively. The results showed that the 18 colonies are identical. The , , , and sequences of this isolate were deposited in GenBank with accession numbers MT513132.1, MW233896, MW233897, and MW233895, respectively. BLAST searches with sequences of this isolate , , , and revealed 97.7%, 96.4%, 97.8%, and 97.3% similarity with the corresponding sequences of strain NBC2815 (LT853880.1), respectively. The resulting concatenated data set of was used to build a Multilocus Sequence Analysis (MLSA) by maximum likelihood criteria (Fig. 2). The sequences of the isolate from Liaoning clustered in the clade containing the type strain of NBC2815, indicating that it belongs to . Thus, the bacterial strain from Liaoning was designated as strain YL19. To fulfill Koch's postulates, the base of leaf petioles of disease-free strawberry plants were syringe-infiltrated inoculated with bacterial suspension (2×10 CFU) prepared from colonies of YL19 washed from SPA plates. The inoculated and control (treated with sterile water) were placed in a chamber (25/20℃day/night,≥90% relative humidity(RH), 12/12 h photoperiod) for three months. After one month, water-soaked symptoms were observed in the crown tissues of all YL19-inoculated plants. Two months after inoculation, a significant crown pocket similarly to initial symptoms observed in the field was developed on all inoculated plants. No symptoms were observed in the control plants. The bacteria were re-isolated from the symptomatic leaves, petioles and crowns, and confirmed as YL19 by the above mentioned morphological and molecular analyses. Pathogenicity tests were conducted three times and the same results were obtained. It was reported that usually causes angular leaf spot, a serious bacterial disease in many strawberry production regions worldwide. The typical symptoms of angular leaf spot caused by include reddish-brown, irregular spots on the upper leaf surface, water-soaked lesions developed along leaf veins. Although angular leaf spot caused by has been reported in Tianjin and Taiwan province, China (Wang et al. 2017; Wu et al. 2020), there is no report about the symptoms that infection pockets on crowns caused by strain YL19 as described above. This result indicated that YL19 is different from the other two strains reported in China or the disease caused by YL19 could be a severe case of angular leaf spot and vascular decline or collapse in strawberry (Bradbury, 1977). . To the best of our knowledge, these results showed a previously unreported new strain YL19 of is the causal agent of crown infecton pocket in strawberry in China, it may lead to serious losses to the local strawberry industry. This report will assist in developing management measures for this disease promptly.