Effective, consistent, and rapid non-contact application methods for seedling basal stem infection by Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary, an economically devastating soil-borne fungal pathogen known to cause disease across a wide range of plants, produces long-term inoculum called sclerotia that can either germinate carpogenically by ascospores infecting above-ground plant parts or myceliogenically to infect stem base and roots. Typically for research purposes S. sclerotiorum diseases are initiated by direct contact methods, using S. sclerotiorum mycelium agar plugs wrapped around the stem or sclerotia placed directly beneath root mass. However, reproducible non-contact methods leading to basal stem infection are not currently available. Therefore, the objective of this study was to develop effective non-contact protocols that consistently generate basal plant stem infection from S. sclerotiorum in the soil. Using three host plant species (canola, lupin, and lettuce) we determined two methods that reliably produced basal stem infection. The first method, where mycelial agar plugs were positioned just below the soil surface at a distance of 5 mm from each seedling, led to 100% infection in all plants. The second method used pathogen-infested soil by mixing the soil with dry inoculum in the form of a powder prepared from mycelium-colonized organic substrates. Four substrates consistently produced 100% seedling infection at four days after inoculation (DAI); wheat bran, wheat grain, red rice, and hulled millet. In contrast, chia, canary, sesame, and ryegrass seed substrates resulted in less than 50% seedling infection at 10 DAI and infection levels did not progress further. The two soil inoculation methods outlined in this study will enhance future research on the progression of S. sclerotiorum diseases, with the potential to screen disease-resistant host genotypes to basal S. sclerotiorum infection, and in particular to test the effectiveness of soil applications of fungicides or biocontrol agents against S. sclerotiorum basal infection.

Pathogenicity and fungicide sensitivity of Phytophthora parvispora, a new pathogen causing gummosis and root rot disease on citrus trees.

In 2021, pomelo (Citrus grandi) trees grown in Tuyen Quang and Phu Tho in northern Vietnam suffered from leaf yellowing, gummosis on stems, brown rot on fruit, and black rot on roots. Based on morphological and sequence analysis of the ITS and cox1 gene regions, the pathogen causing gummosis and root rot of citrus trees was identified as Phytophthora parvispora. Pathogenicity assays using mycelial plugs and zoospore suspension showed that P. parvispora induces disease symptoms on both the upper and lower parts of various citrus trees, including pomelo, orange (C. sinensis), and lime (C. aurantiifolia). This is the first report of P. parvispora as the causative agent of gummosis and root rot on various citrus trees in South-East Asia as well as in Vietnam. Further, P. parvispora was sensitive to all tested fungicides, including mancozeb, chlorothalonil, fosetyl aluminium, potassium phosphonate, and dimethomorph. These findings will have important implications for the effective management of gummosis and root rot disease of citrus trees.

Drought stress-responsive abscisic acid and salicylic acid crosstalk with the phenylpropanoid pathway in soybean seeds.

Crosstalk between hormones and secondary metabolites regulates the interactions between plants and stress. However, little is known about the effects of hormone crosstalk on the concentration of flavonoids in seeds. In this study, we identified abscisic acid (ABA) as a negative regulator of flavonoid accumulation in soybean seeds under drought-stress conditions. Alterations in flavonoid accumulation at several intensities of water stress, followed by a recovery period, were measured during the soybean seed-filling stage. Low soil moisture (SM 10%) significantly decreased the total flavonoid content in seeds. The decline in flavonoid content was proportional to the severity of drought stress and was dependent on the activities of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), two key phenylpropanoid pathway enzymes. The expression of phenylalanine ammonia-lyase 1 (GmPAL1), chalcone isomerase 1A (GmCHI1A), and chalcone synthase 8 (GmCHS8) was associated with phenolic and flavonoid accumulation in soybean seeds of plants subjected to drought stress. Interestingly, the expression levels of GmCHS8 were highly correlated with flavonoid levels under drought stress and water recovery conditions. Cinnamic acid, which is a biosynthesis precursor shared by both phenylpropanoid metabolism and salicylic acid (SA) biosynthesis, decreased under drought stress conditions. Notably, exogenous ABA suppressed the expression of GmPAL1, which encodes the first rate-limiting enzyme in the phenylpropanoid biosynthesis pathway and affects downstream products such as SA and flavonoids. In conclusion, drought stress altered the phenylpropanoid-derived compounds, at least with regard to flavonoid and SA accumulation in seeds, which was regulated by antagonistic interactions with ABA.

First Report of Epicoccum tobaicum Associated with Leaf Spot on Flowering Cherry in South Korea.

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 (Zlatkovic 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.

First Report of Shot-Hole on Flowering Cherry Caused by Burkholderia contaminans and Pseudomonas syringae pv. syringae.

Shot-hole disease (SH) is one of the most common and important diseases affecting 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 and Pseudomonas syringae pv. syringae-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 fruit belonging to the genus Prunus, including peach (Prunus persica), plum (P. salicina), and apricot (P. mume), with peach being the most susceptible. These results indicate that B. contaminans and P. syringae pv. syringae 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 fruit, incited brown spots and shot holes on FC leaves. Therefore, FC trees are susceptible to infections by various pathogenic bacteria, including B. contaminans, P. syringae pv. syringae, and X. arboricola pv. pruni. These findings will be of great importance as a reference for effective management of SH in the face of possible cross-infection between Prunus spp. in the future.

Identification, Characterization, and Efficacy Evaluation of Bacillus velezensis for Shot-Hole Disease Biocontrol in Flowering Cherry.

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.