In 2015 and 2016, three major strawberry cultivation regions in Gyeongnam province were investigated for Fusarium wilt disease symptoms. The main strawberry production areas (Sukok, Hadong, and Sancheong) used well established soil cultivation systems for cultivars such as Jangee, Sulhyang, Maehyang, Santa, and Amaka. During the cultivation season, Fusarium wilt disease symptoms were documented, including irregular leaf development, yellowish leaf and decay in the crown part of the plant. The Fusarium wilt infected plants were collected from the sites and systematically identified.

The crown parts with disease lesions were cut (0.5 × 0.5 cm) and their surface sterilized by 1% sodium hypochlorite for 30 s and 70% ethanol for 30 s. The segments were continuously washed 5 times with sterilized water then dried for 10 min under clean bench, placed on water agar medium (WA; agar 20 g per 1 l) for 3 days at 28°C. The grown hypha was transferred to a potato dextrose agar (PDA; potato dextrose 24 g, agar 20 g per 1 l) for 5 days at 28°C. From the three cultivation regions of Sukok, Hadong, and Sancheong, a total of 110 pathogens were isolated as the symptoms.

The isolates were transferred from WA to PDA and then incubated for 5 days at 28°C. The contamination was observed for 5 days and the mycelium color of the uncontaminated isolates was observed. The isolates having the same color were collected and classified as different phenotype groups. Prior to preparing spore suspensions of the pathogen hypha classified by color, the spore suspensions were poured into 10 ml of sterilized water into a mold-cultured plate. Optical microscope (Carl Zeiss A.G., Oberkochen, Germany) observations (×400) were performed to determine the spores’ type and morphology and a 50 μm meter scale bar was used.

Total genomic DNA was extracted at the mycelium using cetyl trimethyl ammonium bromide (CTAB) protocol (Porebski et al., 1997). The extracted DNA from each isolate was amplified with F. oxysporum f. sp. fragariae specific primers; FofraF and Fofra R (Suga et al., 2013). The PCR conditions were as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and at 72°C for 15 s. The final extension temperature was set at 72°C for 5 min. The complete ITS region of ribosomal RNA was amplified with the primers ITS1 and ITS4 (Kistler et al., 1987; White et al., 1990) then sequencing was done using Solgent Co. (Daejeon, Korea). Sequencing similarities were compared to previously deposited sequences in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

ITS, intergenic spacer (IGS), translation elongation factor1-α (TEF-1α), and β-tubulin were employed for PCR-RFLP analysis. PCR was used with PCR PreMix (Bioneer, Daejeon, Korea) including 10 pmol of the primer sets and 50 ng of fungal genomic DNA. ITS1 and ITS4 (Kistler et al., 1987; White et al., 1990) for ITS and PCR conditions were as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 1 min, 50°C for 1 min, and at 72°C for 1 min and the final extension temperature was set at 72°C for 10 min for ITS. To amplify IGS, FIGS and FIGS2 primers were used and the PCR conditions were as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 40 s, 52°C for 40 s, and at 72°C for 40 s and the final extension temperature was set at 72°C for 10 min. Primers, TEF fusarium F and fusarium R, and PCR condition was following settings; initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 1 min, 58°C for 1 min, and at 72°C for 2 min and the final extension temperate was at 72°C for 10 min for TEF-1α. For β-tubulin gene, primers FBETA F and FBETA R was used (Table 1) and PCR condition was following settings; initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 40 s, 62°C for 40 s, and at 72°C for 30 s and the final extension temperate was at 72°C for 10 min. The amplified PCR products were subjected to enzymatic cut with the restriction enzymes AluI, HhaI, HinP1I, and HpyCH4V (NEB, Hitchin, UK) according to protocol in 20 μl total volume. The restriction reaction mixtures were incubated at 37°C for 4 h. To remove the enzyme activity continuously the reaction tubes were incubated at 70°C for 15 min. The DNA fragments were then loaded on 3% agarose gel at 120 V or 200 mA for 40 min in 1× TAE buffer. The gels were stained with ethidium bromide (EtBr) for visualization. The product fragments were formulated as ‘0’ or ‘1’. The 0 signified absence while 1 signified the presence of a particular band. Based on PCR-RFLP analysis, unweighted pair group method with arithmetic average (UPGMA) method dendrograms were determined via genetic variance between the isolates and then discriminant analysis and construction according to linear combinations of the variables was used to determine the maximum separation between the isolates using XLSTAT program (www.xlstat.com) (Gang et al., 2015).

Fusarium wilt diseased strawberries were collected in the Gyeongnam region during the strawberry cultivation period in 2015. The samples were collected from three locales—Sukok, Hadong, and Sancheong and the cultivated types were either high cultivation or soil cultivation. Strawberry cultivars were varieties including Sulhyang, Maehyang, Janghee, Santa, and Amaka. The infected plant showed typical Fusarium wilt disease symptoms (Supplementary Fig. 1). As a result, 38 isolates were isolated from Sukok, 29 from Hadong, and 43 from Sancheong (Supplementary Table 1). When they were classified into cultivated form, 36 isolates were cultivated in high cultivation and 74 isolates were cultivated in soil cultivation. When they were classified based on cultivar, 13 isolates from Maehyang, 67 isolates from Sulhyang, 24 isolates from Janghee, and 3 isolates were isolated from Santa and Amaka (Table 2).

The isolates were classified into five phenotypes. The criterion for classification was the mycelium color of isolated pathogens. The 110 isolated pathogens were classified into 5 different hypha phenotypes and the colors of hyphae were as follows: dark red, light violet, light yellow, white, and dark pink (Fig. 1). The spores of the isolates were classified based on morphological characteristics. Microconidia and macroconidia of the same shape and size as those of F. oxysporum f. sp. fragariae were confirmed. Generally, microconidia have 5-12 × 2.3-3.5 μm and macroconidia have 23-54 × 3-4.5 μm (Supplementary Fig. 2). Furthermore, the results of ITS sequencing for all isolates showed 99-100% similarity with standard F. oxysporum f. sp. fragariae strain (Supplementary Fig. 3).

The species-specific primer sets were used to identify the isolated pathogens, Fofra F and Fofra R for F. oxysporum f. sp. fragariae (Suga et al., 2013). PCR results showed a single amplicon (233 bp) (Supplementary Fig. 4) from each region’s agriculture land and confirmed 38 isolates from Sukok, 30 isolates from Hadong and 42 isolates from Sancheong. In total 110 isolated pathogens were identified as F. oxysporum f. sp. fragariae.

A total of 110 isolates were analyzed by PCR-RFLP for evidence of genetic variation. When the restriction enzyme was applied to the gene amplified from the ITS region, various PCR bands were obtained (Fig. 2). AluI-ITS generated 4 types of diversity pattern of the pathogen. Furthermore HhaI-ITS showed 7 types of diversity pattern, HinP1-ITS indicated 6 types of diversity pattern and HpyCH4V-ITS revealed 4 types of diversity pattern, respectively (Supplementary Fig. 5). The PCR-RFLP results for the IGS and TEF1-α regions revealed two or three patterns. AluI-IGS, HhaI-IGS, HpyCH4V-IGS generated 3 types of diversity pattern; however, HinP1-IGS indicated only 1 pattern and separated the three groups (Supplementary Fig. 6). AluITEF1-α, HhaI-TEF1-α showed 3 types diversity pattern and HinP1-TEF1-α, HpyCH4V-TEF1-α revealed 2 types diversity pattern (Supplementary Fig. 7). In the β-tubulin region, 2 types of pattern were observed when the AluI restriction enzyme was treated, and all of the remaining patterns showed a uniform pattern (Supplementary Fig. 8). PCR-RFLP patterns were analyzed with the XLSTAT program. When examined, it was confirmed that three of the four genes were differentiated into three groups except for the β-tubulin domain, but the β-tubulin domain could be divided only into two groups (Supplementary Fig. 9). When analyzed, it was divided into five groups. To find a correlation genetic diversity of the pathogen and among the criteria such as strawberry cultivated, cultivated cultivar, cultivated region and phenotype. Only cultivation regions showed a reliable correlation grouping among the criteria (Fig. 3, Table 3). Specifically, 95.2% of samples in Sancheong were found only in group 5 and 76.7% of samples in Hadong were detected in group 4 (Table 3, Supplementary Fig. 10). Principal component analysis (PCA) was conducted to clarify the cause analysis of the genetic classification of the pathogen isolates. Three groups were divided in PCA and the grouping was divided by the cultivation regions (Fig. 4).