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.

Two distinct Epichloë species symbiotic with Achnatherum inebrians, drunken horse grass.

Achnatherum inebrians, colloquially known as drunken horse grass, is associated with livestock toxicity in northern China. Epichloë gansuensis (Eg) was described from endophyte isolates from A. inebrians in Sunan County, Gansu Province, whereas a morphologically distinct variety, E. gansuensis var. inebrians (Ei), was described based on two isolates from A. inebrians seeds collected in Urumqi County, Xinjiang Province. Genome sequencing and alkaloid analyses also distinguish these taxa; the Ei isolates produce neurotropic lysergic acid amides (ergot alkaloids), and an Eg isolate produces paxilline (an indole-diterpene alkaloid). To better elucidate the taxonomic diversity of Epichloë spp. symbiotic with A. inebrians, we surveyed eight populations in Xinjiang, Gansu and Inner Mongolia provinces of China and analyzed their genotypes by multiplex PCR for alkaloid biosynthesis genes and mating-type genes. Genotypes consistent with Ei were present in all eight populations, of which they dominated seven. The Ei isolates were all mating type A and tested positive for the ergot alkaloid gene, dmaW. In contrast Eg isolates were all mating type B and had the indole-diterpene gene, idtG. The genome was sequenced from an Ei isolate from seeds collected in Xiahe County, Gansu, and compared to that of the varietal ex type isolate from Urumqi. Alkaloid genes and four different housekeeping genes were nearly identical between the two sequenced Ei isolates and were distinct from a sequenced Eg isolate. Phylogenetic analysis placed Ei, Eg and Epichloë sibirica into respective subclades of a clade that emanated from the base of the Epichloë phylogeny. Given its chemotypic, genotypic, morphological and phylogenetic distinctiveness, its widespread occurrence in rangelands of northern China, and its importance in livestock toxicity, we propose raising Ei to species rank as Epichloë inebrians.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci.

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.