We conducted various assays to elucidate the diversity and pathogenic characteristics of Fusarium spp., which cause Fusarium wilt in minor legumes in Korea. The results confirmed that not only the well-known Fusarium spp. but also species that had not been reported in Korea so far currently caused Fusarium wilt in legumes in Korea.
By sampling wilted minor legumes and isolating the fungi, several fungal genera were obtained, including Fusarium spp., Colletotrichum spp., Macrophomina spp., Rhizoctonia spp., Pythium sp., Phytophthora sp., and Lasiodiplodia sp. Among these fungal genera, Fusarium spp. accounted for 71% of the recovered isolates, indicating that this genus was the dominant and major causal pathogen of wilt symptoms. A previous study involving Fusarium wilt of soybean in Korea from 2014 to 2016 reported that Fusarium spp., Colletotrichum sp., Rhizoctonia sp., Macrophomina sp., Phytophthora spp., and Calonectria ilicicola have been isolated from the soybean; in addition, the Fusarium genus isolation rate was 79% [12]. These results are consistent with this study, probably attributable to the fact that the cultivation area and environment were almost the same in both studies. On the other hand, in a Chinese study of soybean-related Fusarium spp., the isolation of Fusarium spp., Alternaria sp., Aspergillus sp., Botryosphaeria sp., Colletotrichum sp., Corynespora sp., and Diaporthe sp. have been reported [19]. And only Fusarium spp. and Colletotrichum sp. were similar to the species isolated in a Korean study [12]. This is thought to be because of differences in the distribution and dominant species of fungal pathogens depending on region, environment, and cultivation method.
Among the various fungal genera mentioned above, Fusarium is one of the most important plant pathogens owing to its wide host range and it being the main causal pathogen of wilt disease [19]. However, as some Fusarium spp. are similar, identifying these isolates at the species level remains confusing and complicated [20]. Therefore, herein, the Fusarium isolates were identified at the species complex level by observing their morphological features, and genetic analysis was additionally performed to identify them at the species level. The 29 Fusarium isolates were classified into 4 species complexes: FOSC, FSSC, FFSC, and FIESC. In a similar previous study conducted in Korea [12], the Fusarium strains isolated from soybeans differed from the current study only in being identified as F. graminearum species complex and not as FIESC. Species identification through the molecular sequencing of the fungal DNA revealed 14 Fusarium spp. in this study. A much greater variety of Fusarium spp. were isolated than the five species from soybeans in a previous study [12]. Among the 14 species, F. oxysporum was the most common with 7 isolates. These results indicate that F. oxysporum remains the major causal pathogen of Fusarium wilt in legumes.
The NC20-745 isolate was not clearly distinguished from F. phaseoli and F. crassistipitatum owing to the lack of difference in the nucleotide sequence for molecular identification. However, according to Aoki, et al. [21], F. crassistipitatum is distinguished from F. phaseoli by its yellowish colonies on potato dextrose agar (PDA). Therefore, the NC20-745 isolate has been identified as F. phaseoli based on these morphological features. The major pathogen of Fusarium wilt is reportedly, F. oxysporum; however, various other Fusarium spp. have also been frequently reported as wilt and root rot pathogens [12, 16, 22, 23]. In the UK, F. solani, F. coeruleum, F. oxysporum, F. redolens, F. avenaceum, F. graminearum, F. sambucinum, and F. equiseti have been isolated from legumes exhibiting root rot symptoms [24]. Furthermore, in China, F. fujikuroi, F. proliferatum, F. luffae, and F. sulawense have been isolated from the rotten pods of soybean [25]. As such, the Fusarium spp. involved in legume wilt and root rot are very diverse. As this distribution is primarily affected by the cultivation environment, it is necessary to investigate the diversity of Fusarium spp. and understand the characteristics of such fungal pathogens to prevent future disease outbreaks.
However, because not all of these isolated Fusarium spp. are pathogenic, a pathogenicity test was performed to distinguish between the pathogenic and nonpathogenic isolates. The results revealed that four isolates of F. fujikuroi (100%), five of F. oxysorum (71%), one of F. solani, F. proliferatum, and F. ipomoeae (50%), two of F. vanettenii (100%), and one of F. phaseoli, F. concentricum, F. oblongum, and F. azukicola (100%) had a disease severity of > 2.5. Whereas F. falciforme (three isolates), F. metavorans (two isolates), F. proliferatum (one isolate), and F. ferrugineum (one isolate) were nonpathogenic. Thus, not all the 14 isolated Fusarium spp. were pathogenic, and even the same species showed different pathogenicity depending on the isolate. Arias et al.[16] reported that only one isolate of the 14 F. oxysporum isolates from infected soybeans caused the root rot disease; and that other Fusarium spp. also showed considerable differences in pathogenicity by isolate. These results are consistent with the results of this study. Moreover, according to the Fusarium spp., the degree of disease severity also differed in different studies. In the US, F. graminearum is the most pathogenic followed by F. virguliforme, F. proliferatum, F. sporotrichioides, and F. solani [16]. However, in China, F. proliferatum is reportedly the most pathogenic followed by F. fujikuroi, F. sulawense, and F. luffae. [25] These results indicate complex differences among different countries, including those in the dominant Fusarium spp., types of cultivated legumes, cultivation environment, and cropping system. Therefore, it is advisable that an in-depth investigation of the diversity and pathogenic traits of Fusarium spp. according to their isolation regions, crops, soil, and cropping system should be conducted in Korea.
Based on the results of the pathogenicity assay, 19 Fusarium isolates were selected whose host range was investigated on 7 leguminous and 2 gramineous plants. Most Fusarium isolates were evaluated as having a broad host range. Most Fusarium isolates did not cause wilt disease in corn, but two isolates of F. fujikuroi and one isolate of F. proliferatum caused wilt disease in all the nine crops, including corn, and were thus evaluated as having the widest host range. Similarly, a previous study by Amatulli et al.[26] reported that F. fujikuroi and F. proliferatum had a wide host range, including corn, asparagus, fig, onion, palm, pine, and rice. Therefore, according to the results of this and the current study, it can be concluded that there are at least 14 host species of F. fujikuroi and F. proliferatum. Furthermore, all Fusarium spp. belonging to FFSC caused wilt disease in rice. However, F. fujikuroi reportedly causes bakanae disease in rice. Therefore, we additionally performed immersion inoculation [27] to confirm whether F. fujikuroi isolated from legumes can cause bakanae disease, which showed that all the four F. fujikuroi isolates increased the height of rice and caused bakanae disease (data not shown). This is consistent with the results of the previous study conducted by Choi, et al.[12], which confirmed that F. fujikuroi isolated from soybeans had caused bakanae disease in rice. Three isolates of F. oxysporum, known as the representative pathogen of Fusarium wilt, and one isolate of F. azukicola, reported for the first time in Korea, also caused wilt disease in all seven legumes. With respect to F. azukicola, Aoki, et al.[23] reported that eight strains of F. azukicola isolated from Japan also caused root rot in adzuki beans, kidney beans, mung beans, and soybeans. Thus, it is likely to become a problematic fungal pathogen in the near future. As such new pathogens may exist in the future, continuous pathogen identification and host range monitoring are highly recommended.
Currently, research regarding the Fusarium wilt of legumes in Korea is insufficient; hence, information regarding the existing Fusarium pathogens is lacking. Therefore, this study revealed some previously unreported Fusarium wilt pathogens and their pathogenic characteristics and host range. The results of this study can be used for future research in effective Fusarium wilt management strategies, including breeding of wilt-resistant varieties and cultivation control methods such as crop rotation.