Molecular-based race classification of Pyrenophora tritici-repentis causing tan spot of wheat in Japan

Tan spot, a foliar disease of Triticum spp. such as bread wheat (T. aestivum L.) and durum wheat (T. turgidum ssp. durum (Desf.) Husn.) caused by the filamentous fungus Pyrenophora tritici-repentis (Died.) Drechsler leads to serious losses of crop yield and quality in some areas in Japan. P. tritici-repentis is classified into eight races according to the combinations of three necrotrophic effectors, PtrToxA, PtrToxB, and PtrToxC encoded by ToxA, ToxB, and ToxC1, respectively. Race classification has been based on reaction of a differential variety to necrotrophic effectors, which is tested by inoculation. Recent identification of the Tox genes and development of specific DNA markers have enabled us to classify races of P. tritici-repentis collected in Japan by Tox gene genotyping. We found that 17 strains collected from Triticum spp. in Japan were mainly race 1 or 2, because they carried ToxA as a toxin gene by current race classification; wheat genotype tsn1 is resistant to ToxA. Establishment of wheat cultivars carrying tsn1 would be most effective for decreasing agronomic losses caused by the disease in Japan.


Introduction
A foliar disease of wheat (Triticum spp.) caused by the filamentous fungus Pyrenophora tritici-repentis (Died.)Drechsler, called tan spot or yellow spot.It occurs in almost all wheat-growing regions all over the world including Japan (Kamel et al. 2019, Lamari and Strelkov 2010, Nishi et al. 1993, Shi et al. 2022, Tsukiboshi 2005, Yoshimatsu and Kato 2003).The disease can lead to considerable yield loss up to 50% and/or red smudge on grains to deteriorate their quality on susceptible cultivars to the disease in favorable condition (Lamari and Bernier 1989, Rees et al. 1982, Schilder and Bergstrom 1994).Recently, the wheat cultivated area of Japan is about 220,000 ha, and yield is about 1 million t a year (https://www.maff.go.jp/j/ tokei/kouhyou/sakumotu/index.html,accessed April 7, 2023).Tan spot has become a serious problem in some areas of Japan.For reducing damages by the disease, crop rotation with rice, harvested residue removal and fungicides application are mainly done, but the most effective method for reducing disease is the development of genetically resistant varieties (Kariyawasam et al. 2018, Running et al. 2022).
Although information about Tox genes is available, knowledge of P. tritici-repentis races in Japan is insuffi-cient.In this study, we attempted to classify the strains present in Japan into races by genetic methods.

Fungus materials and genomic DNA extraction
We used 17 strains isolated from bread wheat (T.aestivum L.), durum wheat (T.turgidum ssp.durum (Desf.)Husn.), or Agropyron sp.registered as P. tritici-repentis at the Research Center of Genetic Resources, National Agriculture and Food Research Organization (NARO) (Table 1).Each strain was grown in potato dextrose broth (Becton, Dickinson and Co., Franklin Lake, NJ, USA), and genomic DNA was extracted in DNAs-ici-F extraction buffer (Rizo Inc., Tsukuba, Japan).

PCR amplification and sequencing
The species identity of the 17 strains was verified according to Marin-Felix et al. (2019) using DNA sequences of the internal transcribed spacer region (ITS) and the coding regions of the genes for glyceraldehyde-3-phosphate dehydrogenase (gapdh) and the second largest subunit of RNA polymerase II (rpb2).These regions were amplified in a PCR Thermal Cycler Dice (Takara Bio, Shiga, Japan) using AmpliTaq Gold 360 (Applied Biosystems, Waltham, MA, USA) at an initial 95°C for 10 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 60 s; and a final 72°C for 7 min.PCR primers are listed in Table 2.The PCR fragments were separated by electrophoresis in 2% agarose gel, visualized with Gel Red stain (Biotium, Fremont, CA, USA), and extracted with a Qiaquick Gel Extraction Kit (Qiagen, Hilden, Germany).They were sequenced by Fasmac Co. Ltd. (Kanagawa, Japan).The sequences were registered in the DNA Data Bank of Japan (DDBJ) with the accession numbers shown in Table 1.

Genotyping of Tox genes
Genomic DNA was amplified in a Thermal Cycler Dice using Quicktaq HS (Toyobo, Osaka, Japan) by quadruplex PCR (ToxA, ToxB, toxb, and chitin synthase 1 (CHS1) as an internal control) or duplex PCR (ToxB, toxb, or ToxC1 and CHS1 at an initial 95°C for 2 min, followed by 30 cycles of 94°C for 30 s, and 68°C for 60 s.The amplified PCR fragments were separated and visualized as above.

Species identification
All 17 strains listed in Table 1 formed a subclade together with P. tritici-repentis CBS 191.29 and were thereby confirmed as P. tritici-repentis (Fig. 1a).The pairwise distance was ranged 0.0005-0.0094(data not shown).The minimum was between MAFF 150089 and MAFF 150137, and the maximum was between MAFF 150080 and MAFF 306661.The positions of the 17 strains within the subclade did not reflect the wheat species or cultivar from which they were isolated, or the collection site, and year (Fig. 1b, Table 1).

Tox gene distribution
The 16 strains collected from bread wheat or durum wheat had ToxA-specific fragments, but not ToxB-and toxbspecific fragments in quadruplex or duplex PCR (Fig. 2a-2c, Table 1).The 6 strains of them, MAFF 150087, 150138, 305430, 306088, 306089 and 511122, also had ToxC1-specific fragments.ToxA is necessary and sufficient for production of PtrToxA, and ToxC1 is necessary but not sufficient for that of PtrToxC.So, PtrToxC is unproduced through expression of ToxC1 only.According to the current race classification by Faris et al. (2013), we inferred that the 10 strains of MAFF 150143, 150144, 150085, 150079, 150080, 150081, 150084, 150089, 150090 and 150137 with ToxA-specific fragments only belong to race 2 producing PtrToxA only, as well as that the 6 strains with ToxA-and ToxC1-specific fragments belong to race 2 or race 1 producing both PtrToxA and PtrToxC.MAFF 306661 collected from Agropyron sp. did not have ToxA-, ToxB-, toxb-, and ToxC1-specific fragments.We inferred that it belongs to race 4, which has no PtrToxs (Table 1).

Discussion
This is the first report of identification of tan spot pathogens in Japan as P. tritici-repentis by current genetic methods and the inference of their races from the presence of Tox genes (Figs. 1, 2, Table 1).17 strains were identified as P. tritici-repentis with ITS, gapdh and rpb2 sequence by current phylogenetic analysis according to Marin-Felix et al. (2019), a pairwise distances of them were very low and not characterized them by wheat species or cultivar from which they were isolated, or the collection site and year (Fig. 1, Table 1).No diversity was found ITS and β-tubulin gene in 10 strains of P. tritici-repentis from bread wheat in Mie Prefecture in Japan, although isolated year, site, and cultivar were differed (Hafez et al. 2022).We deduced from these reports that identification of P. tritici-repentis could be succeeded, but not clustered the collected information of 17 strains by phylogenetical analysis.
A race distribution of P. tritici-repentis in the world without East Asia including Japan was reported that race 1 was predominant in the Americas, Europe, North and South Asia and the Caucasus (Kamel et al. 2019).And race 2 was second predominant in the Americas and North and South Asia, but it was low frequency in Europe and the Caucasus (Kamel et al. 2019).In Australia and New Zealand, only races 1 or 2 were distributed (Antoni et al. 2010, Kamel et al. 2019).In Africa, a tendency of a race distribution of P. tritici-repentis were different from other areas, race 5 was predominant and race 6 was second predominant (Kamel et al. 2019).Hafez et al. (2022) used inoculation to analyze 10 isolates of P. tritici-repentis from bread wheat grown in Mie Prefecture in Japan and classified 8 isolates as race 1 and 2 isolates as race 2. Based on molecular analysis, we presume that the predominant race of P. tritici-repentis isolated from not only bread wheat but also durum wheat in Japan is race 1 or race 2, each of which has at least the ToxA gene and distributed in west area from Kanto region (Table 1, Fig. 2).Especially, 10 strains classified as race 2 were distributed around Hiroshima and Okayama Prefectures (Table 1, Fig. 2), because there is possibility that they were collected from a relatively confined location.In addition, this study inferred that MAFF 306661 from Agropyron sp. in Hokkaido was race 4. Race 4 has already been found from noncereal grasses in various areas in America with low frequency (Ali and Francl 2003).In the case of a distribution of wheat yellow mosaic virus in Japan, geographically distribution was found that pathotype I, II, and III were distributed in central, northern, and southern areas of Japan, respectively (Ohki et al. 2014).So, due to verify race identification with more strains of P. tritici-repentis from various collected sites and host wheats may lead complete understanding of race distribution in Japan.
The pathosystem between P. tritici-repentis and wheat has been studied in detail.In particular, hypersensitive reaction of the host leading to necrosis is mediated by PtrToxA and depends on the presence or absence of Tsn1 (an S/TPK-NBS-LRR gene) in the host (Faris et al. 2010).Wheat genotypes with Tsn1 are sensitive to PtrToxA, whereas null (tsn1) genotypes are insensitive (Faris et al. 2010).PtrToxC interacts with the wheat sensitivity gene Tsc1 to induce chlorosis, and the tsc1 genotype is insensitive (Effertz et al. 2002, Kariyawasam et al. 2018, Shi et al. 2022).Under the current distribution of P. tritici-repentis races, establishment of wheat cultivars with the tsn1 genotype is strongly required and of those with the tsc1 genotype is desirable as a direction of the crop breeding program to decrease agronomic losses caused by tan spot in Japan.In addition to introduce tsn1 and tsc1 in Japanese wheat cultivar(s) under the current situation, pyramiding of PtrToxB insensitive wheat genotype tsc2 with them might be better to be prepared for future invasions of new races of P. tritici-repentis.This breeding strategy will be possibility to confer a combined insensitivity for both necrosis and chlorosis by any PtrToxs.

Table 1 .
Materials used in this study a DNA sequences were registered in DDBJ.b + Tox gene-specific primers amplified a fragment; -they did not amplify a fragment.

Table 2 .
Sequences of primers used in this study