No abstract

Though information exists regarding the pathogenesis of the shot-hole disease (SH) in flowering cherry (FC), there has been a lack of research focusing on SH management. Therefore, here, we investigated the inhibitory activities of antagonistic bacteria against SH pathogens both in vitro and in vivo as well as their biochemical characteristics and bioactive compounds. Two biosurfactant-producing bacterial antagonists, identified as Bacillus velezensis strains JCK-1618 and JCK-1696, exhibited the best effects against the growth of both bacterial and fungal SH pathogens in vitro through their cell-free culture filtrates (CFCFs). These two strains also strongly inhibited the growth of the pathogens via the action of their antimicrobial diffusible compounds and antimicrobial volatile organic compounds (VOCs). Crude enzymes, solvent extracts, and biosurfactants of the two strains exhibited antimicrobial activities. Liquid chromatography/electrospray ionization time-of-flight mass spectrometric analysis of the partially purified active fractions revealed that the two antagonists produced three cyclic lipopeptides, including iturin A, fengycin A, and surfactin, and a polyketide, oxydifficidin. In a detached leaf assay, pre-treatment and co-treatment of FC leaves with the CFCFs led to a large reduction in the severity of the leaf spots caused by Epicoccum tobaicum and Bukholderia contaminans, respectively. In addition, the two antagonists produced indole-3-acetic acid, siderophore, and a series of hydrolytic enzymes, along with the formation of a substantial biofilm. To our knowledge, this is the first report of the antimicrobial activities of the diffusible compounds and VOCs of B. velezensis against the SH pathogens and their efficiency in the biocontrol of SH.

The shot-hole disease (SH) is one of the most common and important diseases affecting the flowering cherry (FC; Prunus × yedoensis Matsumura; ‘Somei-yoshino’) trees in South Korea every year, resulting in premature defoliation and reduced flowering in the following year. However, pathogens associated with the disease remain unknown, which has rendered disease management challenging. Here, the pathogens associated with SH, their biochemical characteristics, and their host range were elucidated. Detached leaf and in planta assays revealed that two biofilm-forming bacteria, namely Burkholderia contaminans (Bc) and Pseudomonas syringae pv. syringae (Pss), caused SH of FC trees. These pathogens were recorded for the first time as the causes of SH of FC trees in South Korea. Additionally, the two pathogens induced similar disease symptoms in several stone fruits belonging to the genus Prunus, including peach (P. persica), plum (P. salicina), and apricot (P. mume), with peach being the most susceptible. These results indicate that Bc and Pss caused SH on FC trees and presented a broad spectrum of hosts. Furthermore, Xanthomonas arboricola pv. pruni, the causative agent of leaf spot on stone fruits, incited brown spots and shot holes on FC leaves. Therefore, FC trees are susceptible to infections by various pathogenic bacteria, including Bc, Pss, and Xap. These findings will be of great importance as a reference for effective management of SH in the face of possible cross-infection between Prunus species in the future.

Flowering cherry (FC, Prunus x yedoensis Matsumura; Somei-yoshino cherry) is an ornamental tree, planted across South Korea and producing stunning flowers in spring. The seasonal blooms are annually celebrated during cherry blossom festivals in many locations across the country. The leaf spot disease is among the most common and important diseases affecting FC trees every year, resulting in premature defoliation and reduced flowering of cherry blossoms in the following year. In May 2018, brown spots (2 to 5 mm), circular to irregular and with dark borders were observed on FC leaves in Hadong, Gyeongsangnamdo, South Korea (35°07'48.9"N, 127°46'53.8"E), with a disease incidence of 55%. Single lesions often coalesced and were sometimes perforated, leaving shot holes. Sampled leaves were surface sterilized with 1% NaOCl for 1 min and 70% ethanol for 30 s, and then rinsed twice with sterile distilled water. About 2-mm-long infected leaf pieces from the margins of lesions were put onto water agar (WA, 1.5% agar) plates and incubated at 25oC for 72 h. Mycelia grown from symptomatic tissue were transferred to PDA plates, and five similar fungal isolates were obtained from hyphal tips. They produced a strong reddish-orange diffusible pigment on PDA after 5 d and exudates after 8 d. Conidia were globular to pear-shaped, dark, verrucose, multicellular, and 14.8 to 23.5 μm in diameter (av. = 18.7 μm, n = 30) for isolate JCK-CSHF10. These morphological characteristics were consistent with the Epicoccum genus. Three loci, ITS, tub2, and rpb2, from three isolates JCK-CSHF8, JCK-CSHF9, and JCK-CSHF10 were amplified using the primer pairs ITS1F/LR5 (Gardes and Bruns 1993; Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), and RPB2-5F2/RPB2-7cR (Liu et al. 1999; Sung et al. 2007), respectively. The ITS, tub2, and rpb2 sequences of the three isolates were deposited in Genbank (MW368668-MW368670, MW392083-MW392085, and MW392086-MW392088, respectively), showing 99.6 to 100% identity to E. layuense (E33), a later synonym for E. tobaicum (Hou et al. 2020). The phylogenetic tree using concatenated sequences of the three loci placed the three isolates in a cluster of E. tobaicum (CBS 232.59, CGMCC 3.18362, and CBS 384.36; Hou et al. 2020). Taken together, the three isolates were identified as E. tobaicum. The pathogenicity of JCK-CSHF10 was tested on 15 healthy leaves on three FC trees (cv. Somei-yoshino, 1.2 m in height) kept in a greenhouse. Five-mm-diameter plugs from 7-d-old fungal cultures grown on PDA or mycelia-free PDA plugs as controls were placed on the abaxial side of a leaf at three points, previously wounded by a sterile needle (Zlatković et al. 2016). Inoculation sites were covered with moist cotton plugs. Trees were then covered with a clear plastic bag and maintained in high humidity at 25oC in darkness for 24 h, followed by a 12-h photoperiod. Brown spots appeared on inoculated leaves after 7 d, identical to those observed in the field, while control leaves remained symptomless. This experiment was repeated three times. A fungus with the same morphology as JCK-CSHF10 was recovered from lesions, thus confirming Koch’s postulates. E. layuense (syn. E. tobaicum) has been reported as a leaf spot-causing agent on Perilla sp. (Chen et al. 2017) and Camellia sinensis (Chen et al. 2020). To date, there is no report on the occurrence of E. tobaicum from leaf spots on FC. To our knowledge, this is the first report of E. tobaicum causing leaf spot on FC in South Korea.

Gynostemma pentaphyllum, also known as Jiao-Gu-Lan, is a perennial liana belonging to the family Cucurbitaceae. This plant has been used as a traditional folk medicine for the treatment of various human diseases. In May 2020, G. pentaphyllum cultivated at the Research Station of Medicinal Plants at Tam Dao, Vinh Phuc, Vietnam was affected severely by root rot disease, resulting in leaf yellowing, wilting, and death. Based on morphology and sequence analysis of the ITS region, the pathogen was identified as Globisporangium attrantheridium. Koch's postulates were fulfilled. This is the first report of Gl. attrantheridium as a pathogen causing root rot on G. pentaphyllum. This finding will be of importance for effective disease management.