Mating-type diversity
All 317 isolates obtained from 99 fields in 7 prefectures were paired with the standard type strains (A1; EPC201522sh1 and A2; EPC2017K01sh), and also grown as single cultures. By observing the sexual structures in the reacting zone between colonies for pairing and in single cultures, four kinds of mating types were categorized: heterothallic A1 and A2 types, and self-fertile (SF) strains of A2 and A1/A2 mating types (Figs. 1 and 2). Overall, 8 and 200 out of the 317 isolates were heterothallic A1 and A2 mating type strains, respectively, without self-fertilization in single culture. The remaining 109 isolates produced oogonia and oospores in the single cultures. However, 103 and 6 isolates strongly reacted with EPC2017K01sh or both EPC2017K01sh and EPC201522sh1, producing much more oospores in the contact zones than in single cultures. Therefore, these isolates were defined as SF A2 and SF A1/A2 types, respectively (Table 1).
The A1 type strains were detected in Kagoshima and Miyazaki Prefectures, especially in Ehime, whereas the A2 type was prevalent in other countries. The A2 type strains were detected in almost all fields of the surveyed prefectures. Although the detection frequency of the SF strains compared to the A2 type strains, they were detected in many fields of all surveyed prefectures except for Saitama Prefecture. In Miyazaki and Ehime Prefectures, the fields only with the SF strains were found. The highest detection frequency of the SF strains was in Fukui Prefecture. The mating type ratios did not vary between different years.
Composition of mating types in different taro fields and lesions
In 64 taro fields, where two or more isolates were collected, mating-type complexity was found. Multiple mating types were present in half of the fields (Table 3). Exclusively heterothallic A2 type strains were found in 30 fields, whereas only SF A2 type strains occurred in three fields. There was no field with exclusive occurrence of A1 type strains. On the other hand, multiple infestation was found in two fields with heterothallic A1 and A2 types, 22 fields with heterothallic A2 and SF A2, two fields with heterothallic A2, SF A2 and A1/A2, three fields with heterothallic A1, A2 and SF A2, and two fields with heterothallic A1, A2, SF A2 and A1A2 (Table 3).
Two to three isolates per leaf were recovered from 13 different leaves in five fields of Kagoshima Prefecture (Table 4). Eight leaves were infected with multiplemating types; one leaf was infected with heterothallic A1 and SF A2, and 7 leaves were infected with heterothallic A2 and SF A2.
Segregation of mating type by purification of the isolates
To assess the reliability and stability of mating-type, we conducted single hyphal tip, zoosporangium and zoospore isolation from five SF A2 and two SF A1/A2 isolates, and two isolates each of heterothallic A1 and A2 types (Table 5). SF isolates segregated different mating types by the isolations, were re-obtained from all of SF isolates except for the KS17Ai3-2 isolate. On the other hand, the heterothallic isolates of A1 and A2 types did not segregate different mating types after purification of the isolates.
In the single hyphal tip isolation, heterothallic A2 and SF A2 isolates were obtained from four out of five SF A2 isolates. All single hyphal tip isolates from the KS17Ai3-2 isolate were heterothallic A2 type. In SFA1/A2 isolates, heterothallic A1, and SF A1 and A2 types were obtained from the KS16TaYo2 isolate. whereas the he KS16TaOki5 isolate produced SF A1 and A2 types but not heterothallic type.
In the single zoosporangium isolation, SF A2 isolates strongly trended to produce heterothallic A1 and A2 types. All single zoosporangium isolates form MS28041, KS17Ai3-2 and EPC201534 isolates were heterothallic A2 type. In the EPC201527 isolate, 75% of the obtained isolates were heterothallic A1 type. The variation of the obtained isolates was also found in SF A1/A2 type. The KS16TaYo2 isolates produced heterothallic A1 and A2 types, and SF A1 and A2 types.
In the single zoospore isolation, both of SF A2 and A1/A2 types produced heterothallic A1 and/or A2 types. In the MS28101 and EPC201527 isolate, SF A2 type was only obtained.
Comparison of pathogenicity between mating types
To compare the pathogenicity between mating types, we selected one isolate of each mating type from Kagoshima, Miyazaki and Ehime prefecture, except for SFA1 type, to conduct a pathogenicity test (Table 6). The size of the lesions after inoculation on taro leaves was measured to determine the pathogenicity. Three isolates, one heterothallic A2 isolates from Miyazaki, and two SF A2 isolates from Miyazaki and Kagoshima, respectively, expressed strong pathogenicity and produced about 10 mm diam. lesions. Five isolates, including that three heterothallic A1 isolates, one SF A2 isolate from Ehime and one SF A1/A2 isolate from Kagoshima, demonstrated a weak or even no pathogenicity producing lesions with less than 3 mm diameter. The other isolates showed moderate virulence (Table 6).
The growth rates on V8 agar showed variability between the isolates, but no relationship was found between the growth rate and the virulence to taro. Moreover, to evaluate the stability of the mating-type in these isolates during the pathogenicity tests, the recovered isolates from the lesion were tested for mating type. Heterothallic A1 and A2 isolates did not change the mating types (Table 6). Although several SF isolates changed SF A1 and A2 to SF A1/A2 type, and SF A1/A2 to SF A2 type, the characteristic of the self-fertilization did not lose.