Bioprotection of Olive Trees Against Verticillium Wilt by Pomegranate and Carob Extracts.

Bioprotection using plant extracts is an environmentally friendly strategy in crop protection. Effective control of Verticillium wilt of olive (Olea europaea; VWO), caused by Verticillium dahliae, has proven challenging due to the ineffectiveness of chemicals, which makes it necessary to search for new control tools. Thus, the aim of this study was to evaluate the effect of pomegranate (Punica granatum) and carob (Ceratonia siliqua) extracts against VWO. Extracts derived from pomegranate peels and carob pods and leaves were obtained using ethanol, methanol, or ethyl acetate as solvents. A targeted analysis of their metabolite composition was performed using QTRAP Ultra High-Performance Liquid Chromatography with Mass Spectrometry (QTRAP UHPLC‒MS). Remarkably, gallic acid was detected in all extracts at a high concentration. The effect of the extracts on the mycelial growth and on the germination of conidia and microsclerotia of V. dahliae was evaluated by in vitro sensitivity tests at various doses: 0 (control), 3, 30, 300 and 3,000 mg of extract/liter. Extracts obtained with ethanol or methanol significantly reduced the viability of V. dahliae structures when applied at the highest dose, while those obtained with ethyl acetate were ineffective across all doses. The most effective extracts, as determined in vitro, were then evaluated against the disease in olive plants. Potted plants of cv. Picual were treated by spraying (foliar application) or irrigation (root application) of extracts at 3,000 mg of extract/liter, followed by inoculation with V. dahliae. The results indicated that foliar applications were ineffective, while root treatments with pomegranate peel or carob leaf extracts were more effective in reducing disease severity, regardless of solvent, compared to that of the untreated control.

First Report of Verticillium Wilt caused by Verticillium dahliae on Chilli in South Korea.

Chili pepper (Capsicum spp.) is an important crop in South Korea and is widely used in Korean cuisine, cultivated across a land area of roughly 29.8 thousand hectares, with a total production of 69 thousand tons (Lee et al., 2005; Statista, 2022). In September 2020, two farmer fields in Samcheok (37.444039°N, 129.135875°E; 37.633738°N, 128.911731°E), Gangwon province, South Korea, it is observed that chili pepper leaves showing yellowing and wilting symptoms, with an estimated disease incidence of approximately 10-15%. To identify the causal agents six affected plants were brought to lab. All the plants exhibited vascular discoloration in stem and root. After surface sterilizing small pieces of discolored tissue in 1% NaOCl for 30 s and rinsing twice in sterile distilled water, the tissue pieces were placed on water agar and incubated at 25°C for 10 days. Six pure isolates with consistent morphological characteristics were obtained by single spore isolation. Two representative isolates, NC17601 and NC20845 were selected for identification based morphological and molecular characters. Colonies on potato dextrose agar (PDA) during 10 days of incubation at 25°C in the dark were cottony white initially but progressively became dark as the formation of melanized microsclerotia. Conidiophores were hyaline, vertically branched or not, and had 2 - 4 phialides per node. Phialides were subulate and tapering from base to tip. The colonies produced abundant conidia, which were hyaline, single celled, smooth walled, cylindrical to oval, clustered on phialides, and 3.8 - 7.2 ×2.1 - 3.9 ㎛(mean ± SD: 5.2 ± 0.7 × 2.8 ± 0.5). Microsclerotia were aggregated form, various size and shape, and brown. These are the typical morphology of Verticillium dahliae (Inderbitiz et al. 2011; Yu et al. 2016). The molecular identification was later confirmed through PCR amplification, and sequencing targeting the translation elongation factor 1 alpha (TEF), actin (ACT), tryptophan synthase (TS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes using the primer sets described by Inderbitiz et al. (2011). The resulting sequences were deposited in GenBank with accession numbers LC761935 to LC761942. The maximum likelihood tree based on combined sequences of ACT, GPD, TEF and TS was inferred using RAxML- HPC2 on XSEDE as implemented on the CIPRES web server. The phylogenetic tree showed that the isolates were sit together with V. dahliae isolates (Ex-type PD322, PD227 and PD502). Pathogenicity tests using two isolates (NC17601 and NC20845) were conducted in the greenhouse, where 10 two weeks old seedlings per isolates (cv. Bigstar) were root-tip cut and then soaked in a fungal spore suspension of 107 conidia ml-1 for 1 h, while 10 seedlings were treated with sterile distilled water as a control. All the treated plants were maintained at 25°C (night)/ 25°C (Day) under natural light. After three weeks, all inoculated plants exhibit growth stunting with vascular discoloration in the stem and roots as compared to asymptomatic control plants. The isolates of V. dahliae were consistently re-isolated from discolored root tissues and identified based on morphological characteristics, thus fulfilling Koch's postulates. In South Korea, V. dahliae has been reported to cause wilt disease in various crops, including Kimchi cabbage and radish (Dumin et al. 2020; Choi et al. 2023). To the best of our knowledge, this is the first report that V. dahliae causing Verticillium wilt of chili pepper in South Korea. Overall, Verticillium dahliae is considered to be a significant threat to agriculture in South Korea, and efforts are being made to develop effective control strategies to mitigate its impact on crops.

First Report of Verticillium dahliae causing Verticillium Wilt on Radish in South Korea.

Radish (Raphanus sativus L.) is an important crop grown in Korea primarily for its use in kimchi, and is cultivated on an area of about 23,000ha. In September 2016, radish leaves were observed with yellowing and wilting symptoms in Gangneung (37.55406°N, 128.84871°E) and Jeongseon (37.42895°N, 128.85882°E), Gangwon province, South Korea. Disease incidence was estimated at approximately 10% in two fields, respectively. About 30% of radish plants (cv. Gwangdongyeoreum) with foliar symptoms exhibited vascular discoloration in the roots. Small pieces of discolored root tissue were surface sterilized in 1% NaOCl for 30s, and then rinsed in sterile water. The tissue pieces were placed on water agar and incubated at 25°C for 10 days. Eight isolates were obtained through single spore isolation, and a representative isolate NC16557 (from Gangneung) was selected for identification. After 14 days, colonies on potato dextrose agar (PDA) were 3.7 cm in diameter and becoming dark due to the formation of microsclerotia. Aerial hyphae were smooth-walled, 3 to 4 µm wide. Conidiophores were erect or slanted, verticillately branched or unbranched, and hyaline. Conidia were hyaline, smooth-walled, non-septate, cylindrical to oval, 4.8 to 6.6 × 2.6 to 3.4 µm (mean 5.7 × 2.9 µm, l/w =2.0, n =80). Microsclerotia were immersed in the agar, composed of rounded, brown-pigmented cells and elongate or irregular in shape, 42 to 60 µm diam. Based on morphology, NC16557 isolate was tentatively identified as Verticillium dahliae (Hawksworth and Talboys, 1970; Isaac, 1967). To confirm taxonomic placement, DNA extracted from mycelia of the same isolate was PCR amplified and sequenced targeting internal transcribed spacer (ITS) regions of rDNA, translation elongation factor 1 alpha (TEF), actin (ACT), tryptophan synthase (TS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes (Inderbitzin et al., 2011). The sequences were deposited in GenBank with accession numbers MZ723402, and MZ735720-MZ735723, respectively. The sequencing results showed 100% (ITS=479/479, TEF=500/500, ACT=185/185, and TS=295/295) and 99.61% (GPD=257/258) similarity with the sequences of V. dahlia type strain PD322 (LR026889, HQ414624, HQ206921, HQ414909, and HQ414719) by BLAST. Based on the morphology and multigene sequence analysis, the isolate was identified as V. dahliae. Pathogenicity of two isolates (NC16557 and NC16547) was carried out in the greenhouse using ten 10-day-old seedlings (cv. Gwangdongyeoreum) by root-tip cutting and then soaking the roots in a fungal spore suspension of 106 conidia mL-1 for 1 hour. Ten seedlings were treated with sterile distilled water as a control. All plants were placed in the greenhouse at 15°C (night)/25°C (day) with natural light. After 6 weeks, all inoculated plants showed vascular discoloration in the roots while control plants remained asymptomatic. However, the above-ground symptoms of inoculated plants, such as yellowing and wilting, were indistinguishable from control plants. V. dahliae was consistently re-isolated from the symptomatic root tissues and the pathogen identity was confirmed by observing morphological characteristics. Verticillium wilt of radish has been reported from China(Yan et al., 2019). In Korea, this is the first report that V. dahliae causes Verticillium wilt of radish, although Dumin et al. (2020) already reported on Verticillium wilt of Chinese cabbage in Gangwon province, the main production area of Chinese cabbage and radish in summer. With these findings in Chinese cabbage and now radish, it will be critical to identify or develop Verticillium-resistant varieties and other management strategies for the stable production of these crops in Korea.

First Report of Verticillium Wilt Caused by Verticillium dahliae Infection on Chinese Cabbage in Korea.

Chinese cabbage (Brassica rapa L.) is one of the most important vegetables in Korea due to its role as the main ingredient for the making of Kimchi. In June 2014, disease symptoms of leaves wilt, dry, and drop off on Chinese cabbage were observed in a Chinese cabbage farm located at Taebeak (37°26'50.7"N 128°95'50.0"E), Gangwon province, Korea. This disease was observed on approximately 35% of the plants in the field, causing an almost 10% decrease in total production. At the early stage of infection, the color at the edge of the plant foliage changed from green to yellow. As the disease progressed, infected leaves wilted, dried off, and detached from the plant. Soft rot that occurred at the base of the leaf stem and root tissues caused the infected leaves to dry and fell off the plant. To identify the causal agent, a small piece of infected leaf tissues was sterilized with 1% sodium hypochlorite solution for 1 min and rinsed with sterile water before it was transferred onto potato dextrose agar (PDA) media. The plates were then incubated at 25°C for 10 days in the dark. Fungal colonies grown on PDA media were of white-creamy in color with an abundance of mycelia and later develop into black color due to the formation of microsclerotia embedded in the media. Microscopic examination showed conidiophores and phialides were both appeared in a verticillate arrangement, whereas conidia were hyaline, smooth-walled, and ellipsoidal to oval with average size 5.4×2.5 µm (n=100). Microsclerotia appeared in elongate to an irregularly spherical shape and greatly variable in size. The morphological attributes of the fungal isolate described above were comparable to the characteristics of Verticillium dahliae Kleb. (V. dahliae) described by Hawksworth and Talboys (1970), and V. dahliae isolated from Chinese cabbage in Japan reported in Kishi (1998). Pathogenicity test was performed by soaking twelve individual Chinese cabbage seedlings for 15 min into fungal pathogen conidial suspension (1x106 conidium/ml) before transferred into soil tray. The same number of non-inoculated seedlings on the soil tray was used as a control. Inoculated and control plants were then covered with a plastic bag for 24 hours to maintain high humidity before transferred into the greenhouse (25°C). Seven days post-inoculation (dpi), treated plant leaves turned yellow, and soft rot was observed. At 10-dpi, plant leaf tissues dried off and severe soft rot occurred. Pathogenicity test was repeated three times and consistent results were obtained. The re-isolated fungal pathogen from the inoculated plants showed identical morphological characteristics to the original isolates, thus fulfilling Koch's postulates. For further identification, PCR amplification targeting Internal Transcribed Spacer (ITS) and RNA polymerase II gene (RPB2) regions were performed (Liu et al., 1999; White et al., 1990). Each PCR product was sequenced and deposited in the GenBank under the accession LC549667 and LC061275, respectively. Sequence analysis using BLAST showed that the nucleotide sequences of ITS and RPB2 DNA fragments are 99-100% identical to the reference strain of V. dahliae available in the NCBI database (MG585719, HE972023, XM_009652520 and DQ522468, respectively). Therefore, based on the results of morphological and molecular analyses, the fungal pathogen isolated from Chinese cabbage in this study was identified as V. dahliae and deposited in the National Institute of Horticultural and Herbal Science germplasm collection (NIHHS 13-252). Recently, due to high demand and a more competitive price, more Chrysanthemum farmers in Korea switch their crops to Chinese cabbage. Interestingly, the occurrence of V. dahliae infection was also reported to occur in Chrysanthemum plants in Korea (Han et al. 2007), which indicates a serious problem ahead to these farmers. Therefore, in this current study, the identification of V. dahliae pathogenic to Chinese cabbage will provide vital knowledge for the development of disease management strategies to minimize the loss of crop production. To our knowledge, this is the first report that V. dahliae causes Verticillium wilt disease on Chinese cabbage in Korea.