Introduction
Cucurbitaceae plants are among the most important vegetable crops cultivated worldwide and are the second-largest economically important horticultural family after Solanaceae. Agricultural products from cucurbit plants are known for their high nutritional value and are a crucial source of vitamins (Paris, 1989; Salehi et al., 2019). In Korea, the production of cucumber and watermelon fruits is approximately 254,276 and 672,914 tons per year, respectively (Seo and Kim, 2017). Cucurbit plants are susceptible to various diseases caused by viruses, bacteria, nematodes, and fungi (Song et al., 2018; Kwak et al., 2021). Soil-borne pathogens such as Fusarium species and root-knot nematodes are extremely difficult to control in cucurbit plants (Seo and Kim, 2017). Fusarium species causing Fusarium wilt is one of the most harmful pathogens to the cucurbit family. Fusarium oxysporum, F. solani, F. graminearum, F. moniliforme, F. proliferatum, F. sambucinum, and F. semitectum have been reported to cause diseases in cucurbit plants (Kim and Kim, 2004). In Korea, cucurbit plants such as cucumber and watermelon are mostly cultivated in greenhouses. Unfortunately, greenhouse facilities also protect pathogens from harsh winter conditions. Therefore, cultivation fields and greenhouses will continue to be infected due to the capability of Fusarium species to persist for long periods (Seo and Kim, 2017). Hence, accurately identifying pathogenic Fusarium species against cucurbit plants to effectively implement control measures is crucial. To this end, we aimed to identify and classify pathogenic Fusarium species in cultivated Korean cucurbit plants in this study.
Materials and Methods
Fungal isolates
Typical symptoms of Fusarium wilt disease in cucurbit plants include yellowing, withering, and wilting leaves. Upon excision of the infected plant stem, browning of vascular tissues was observed due to colonization by the fungal pathogen. Symptomatic tissue samples in this study were collected from cultivated cucurbit plants that exhibited the aforementioned symptoms. Thirty-six fungal isolates belonging to the Fusarium family were obtained from the National Institute of Horticulture and Herbal Science (NIHHS) germplasm fungal collection. Each isolate was re-cultured on potato dextrose agar (Difco Laboratories, Detroit, MI, USA) and carnation leaf agar (Difco Laboratories, Detroit, MI, USA) at 25℃ for 10 days in the dark. The potato dextrose agar (PDA) culture was used as source material for molecular and pathogenicity analyses, whereas carnation leaf agar (CLA) was used for morphological examination.
Morphological and molecular identification
The morphology of the fungal isolates was examined from the CLA culture using a light microscope (Carl-Zeiss, Oberkochen, Germany). The morphological characteristics examined included the shape and size of the conidia, septa formation, conidiophores, and colony appearance on the medium. For molecular identification, the isolate was identified using an internal transcribed spacer of ribosomal DNA (ITS-rDNA), elongation factor-1α (EF-1α), and beta-tubulin (β-tub) gene marker sequences. Amplification of gene markers, ITS-rDNA, EF-1α, and β-tub was performed using the primer pairs ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Glass and Donaldson, 1995), and Bt2a/Bt2b (Carbone and Kohn, 1999) respectively. Polymerase chain reaction (PCR) amplification was performed using the GoTaq DNA polymerase kit (Promega, Madison, WI, USA) following the suggested standard protocol. A phylogenetic tree was constructed using MEGA-X bioinformatics software (Kumar et al., 2018).
Pathogenicity test
Pathogenicity tests were performed on two weeks old of watermelon (var. Speedggul), cucumbers (var. baekdadaki), and bottle gourds (var. dongjanggoon). Microconidia were harvested by flooding the isolated culture PDA with sterile water and scraped with a sterile triangle scraper before filtering with double layers of sterile cheesecloth. The root systems of the plant host seedlings were inoculated with a conidial suspension (1 × 106 conidia·µL-1) for 30 min before returning to the soil pot. Inoculated seedlings were then transferred to a greenhouse (temperature: 30℃, daylight: 14 h) for observation. Control plants were inoculated with sterile water. Fungal pathogens from the symptomatic seedling root system were re-isolated and their morphological characteristics and molecular identities were re-identified to fill Koch’s postulate.
Results and Discussion
The characteristics of each of the 36 selected Fusarium species were examined and grouped based on morphologies described by Hyun et al. (2019) and Booth (1971). Following the morphological assessment, molecular identification was performed to confirm the identity of the isolates and their groups. A BLASTN and phylogenetic analysis results showed that the selected fungal isolates were clustered into three groups: F. oxysporum, F. solani, and F. equiseti (Fig. 1). Among the 36 isolates examined, 23 were identified as F. oxysporum, nine as F. solani, and four as F. equiseti. However, as a species complex, Fusarium species were indistinguishable as pathogenic and non-pathogenic species based on morphological and molecular identification using housekeeping gene markers alone. Therefore, pathogenicity tests were conducted to assess the pathogenicity of these isolates (Fig. 2). Among the 36 fungal isolates examined, six of them are non-pathogenic to any of the cucurbit plants host used, whereas the remaining 30 isolates exhibited a pathogenicity trait range from low to highly virulence (Table 1). Approximately 72% of the isolates selected are pathogenic to watermelon, 38% pathogenic to bottle gourd and only 27% pathogenic to cucumber. This result indicated that generally, the watermelon plant is more susceptible to Fusarium pathogen infection compared to bottle gourd and cucumber plants. Therefore the implementation of disease management should be given more attention to the watermelon cultivation area.
Fig. 1. Phylogenetic tree constructed based on concatenated sequences of ITS-rDNA, EF-1α, and β-tub of 36 fungal isolates from National institute of horticulture and herbal science (NIHHS). Reference sequences of Fusarium oxysporum, F. solani, F. graminearum, F. sambucinum, and F. equiseti were obtained from Guan et al. (2020), Li et al. (2015), Lu et al. (2014), Stefańczyk et al. (2016), and Zhang et al. (2017), respectively. The tree rooted to Colletotrichum kahawae has been reported by Sharma and Shenoy (2014).
Five of the F. solani isolates (11-117, 14-130, 17-554, 17-555 and 17-556) that wew isoluded from symptomatic tissues of cucumber) were found to be highly pathogenic to both cucumber and watermelon plants. This result may indicate that besides F. oxysporum, F. solani species may also pose a greater threat to the cucurbit agricultural production in Korea. Another interesting finding in this study was the identification of F. oxysporum and F. equiseti as causal agents to wilt disease in bottle gourd plants. Result show that four F. oxysporum (14-065, 14-066, 14-077, 19-050) and one F. equiseti (14-079) isolates are highly pathogenic to bottle guard plant. Interestingly, this is the first time F. equiseti identified pathogenic to bottle gourd in Korea. Bottle gourds are widely used as rootstocks for grafted watermelons in Korea because of their resistance to Fusarium infection (Lee et al., 2010). However, this result suggests that the bottle gourd resistance to Fusarium infection is slowly waning. Therefore, the identification of these fungal species as pathogenic to bottle-gourd plants is crucial for both farmers and researchers. The geographical distribution of pathogenic F. oxysporum and F. equiseti species to grafted watermelons will allow implementing effective management strategies to manage Fusarium wilt disease in a specific region. This also provides crucial information to develop a quick and efficient identification protocol to identify Fusarium species that cause wilt disease in grafted watermelon. Bottle-gourd resistance against Fusarium invasion is waning. This information is also crucial for assessing new cucurbit species or cultivars that are highly resistant to Fusarium species and serve as rootstocks for grafted watermelon in the future.
Conclusion
In this study, F. oxysporum, F. solani, and F. equiseti were problematic to cultivated cucurbit plants in Korea. Among this group, F. solani was shown to be highly pathogenic to both watermelon and cucumber plants, whereas F. equseti was identified as a novel pathogen to bottle gourd. A high number of F. oxysporum isolates together with F. equiseti, which have been identified as pathogenic to bottle gourds, may indicate that bottle gourd resistance to Fusarium species is waning and a new rootstock for grafted watermelon is needed.
Acknowledgments
This work was performed with the support of the "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01435702), Rural Development Administration, Republic of Korea.
Authors Information
Walftor Bin Dumin, http://orcid.org/0000-0001-9952-8208
You-Kyung Han, National Institute of Horticultural and Herbal Science, Researcher
Jong-Han Park, National Institute of Horticultural and Herbal Science, Senior researcher
Yeoung-Seok Bae, National Institute of Horticultural and Herbal Science, Senior researcher
Chang-Gi Back, http://orcid.org/0000-0001-5665-4730
