First report of seedling damping off in forage ryegrass caused by Pythium aphanidermatum and P. periilum in Japan.

Italian ryegrass (Lolium multiflorum Lam.) and perennial ryegrass (L. perenne L.) are important for hay fields and grazing lands across Japan, with nearly 70,000 ha production, the largest share in forage grass cultivation. In August 2018, damping off of seedlings of both species was observed about 2wk after seeding in Tochigi Prefecture, central region of Japan. Roots were brown and decayed drastically with browning of basal stem. Nearly 90% of the row seedling stands were eradicated in some fields, especially ones seeded from August to early September, when the soil and air temperatures were around 25-30 ˚C. Six Pythium-like isolates were obtained by isolation from surface-sterilized diseased hypocotyls (1-2cm) placed on water agar. Six isolates were purified as single hyphal tips and deposited at the NARO genebank (https://www.gene.affrc.go.jp/index_en.php), with accession no. MAFF101946-101951. Two of them, MAFF101946 and 101948 were used for detailed study. The isolates were grown in the dark on clarified V8 juice agar for 10 days to produce oogonia. The oogonia of MAFF101946 were globose, colorless, smooth, 21 to 30 µm in size, and had 1(- 2) antheridia. Oospores were mostly aplerotic, and oogonia walls were 1.1 to 2.3 µm thick. The oogonia of MAFF101948 were globose, colorless, smooth, 19 to 27 µm in size, and had 2-5 antheridia. Oospores were mostly plerotic, and oogonia walls were 1.7 to 4.2 µm thick. The morphology of the MAFF101946 matched that of P. aphanidermatum (Edson) Fitzp.(abbr. PA) and the MAFF101948 matched P. periilum Drechsler (abbr. PP) (Van der Plaats-Niterink, 1981). DNA sequences of cox1 (Robideau et al, 2011) and rDNA-ITS regions (White et al,1990) of each isolate were analyzed. The cox1 sequences of the MAFF101946 and 101948 (GenBank Accession No. LC548774 and LC548775) matched 100% (680/680 bp) with that of PA (HQ708486) and PP (HQ708781), respectively. The rDNA-ITS sequence of the MAFF101946, LC592700, matched PA (772/777 bp; HQ643439), and the MAFF101948 (LC592701) matched PP (HQ643740), with 99% similarity (764/773 bp). The optimal growth temperature was 35 ˚C for both. Their pathogenicity was confirmed by seeding two Italian (cv. Inazuma and Minamiaoba) and perennial (cv. Yatsuyutaka and Natsugoshi-pere) ryegrasses cultivars in cell trays containing commercial potting mixture inoculated with the isolates. Barley grains autoclaved with a half volume of water and incubated with each isolate at 25 ˚C for about 2 wk were added to inoculate the potting mixture (5%, v/v). Separate trays were used for each isolate to avoid cross-contamination, sown with 60 seeds were sown per cv. and the trays were placed in a light thermostat chamber under the daily cycle of 16 hr light at 30 ˚C and 8 hr dark at 25 ˚C. About 3 wk later, seedlings on the inoculated soil exhibited the symptoms, but not in control (no inoculum) plots. Both inoculated organisms were re-isolated from the diseased plants to confirm their pathogenicity. PA was more aggressive with grater percent damping off compared to PP. Both species are known as pathogens of diverse plants including grasses and legumes (Abad et al, 1994; Ao et al, 2018), but to our knowledge, this is the first report of seedling damping off caused by these Pythium species in forage ryegrass in Japan. With the increased duration of hot, humid condition across temperate regions due to global warming, the damping off may become a problem in hay fields and pasture and resistance breeding for these pathogens may be needed.

Genetic Diversity and Structure of Lolium Species Surveyed on Nuclear Simple Sequence Repeat and Cytoplasmic Markers.

To assess the genetic diversity and population structure of Lolium species, we used 32 nuclear simple sequence repeat (SSR) markers and 7 cytoplasmic gene markers to analyze a total of 357 individuals from 162 accessions of 9 Lolium species. This survey revealed a high level of polymorphism, with an average number of alleles per locus of 23.59 and 5.29 and an average PIC-value of 0.83 and 0.54 for nuclear SSR markers and cytoplasmic gene markers, respectively. Analysis of molecular variance (AMOVA) revealed that 16.27 and 16.53% of the total variation was due to differences among species, with the remaining 56.35 and 83.47% due to differences within species and 27.39 and 0% due to differences within individuals in 32 nuclear SSR markers set and 6 chloroplast gene markers set, respectively. The 32 nuclear SSR markers detected three subpopulations among 357 individuals, whereas the 6 chloroplast gene markers revealed three subpopulations among 160 accessions in the STRUCTURE analysis. In the clustering analysis, the three inbred species clustered into a single group, whereas the outbreeding species were clearly divided, especially according to nuclear SSR markers. In addition, almost all Lolium multiflorum populations were clustered into group C4, which could be further divided into three subgroups, whereas Lolium perenne populations primarily clustered into two groups (C2 and C3), with a few lines that instead grouped with L. multiflorum (C4) or Lolium rigidum (C6). Together, these results will useful for the use of Lolium germplasm for improvement and increase the effectiveness of ryegrass breeding.