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.

A single nucleotide polymorphism in the translation elongation factor 1α gene correlates with the ability to produce fumonisin in Japanese Fusarium fujikuroi.

PCR-RFLP based on the translation elongation factor 1α (TEF) gene was developed to identify Fusarium fujikuroi in the Fusarium (Gibberella) fujikuroi species complex. Ninety-three strains, most of which were obtained from various sources in Japan, were identified as F. fujikuroi and their capability to produce fumonisin was investigated using an in vitro assay. Fumonisin production was detected in 50 strains isolated from maize, strawberry, wheat, and rice, whereas it was undetectable in 43 strains derived from rice seeds and rice seedlings carrying the bakanae disease, and from unknown sources. A single nucleotide polymorphism in the TEF gene (T618G) correlated with the ability to synthesize fumonisin.

Changes in the concentration of fumonisins in forage rice during the growing period, differences among cultivars and sites, and identification of the causal fungus.

To clarify the changes in rice fumonisin (FUM) concentrations, we conducted field cultivation of 10 forage rice cultivars and inoculation with fumonisin-producing fungal isolates. We cultivated 10 forage rice cultivars at the NARO Institute of Livestock and Grassland Science and one cultivar at two additional farmland sites in Japan in 2011 and 2012. Fusarium fujikuroi, which primarily infects plants shortly after heading, was inoculated on rice just after heading, and we sampled heads at the yellow-ripe and full-ripe stages to assess FUM concentrations. We found differences among cultivars in the FUM concentration and differences among the sites for the same cultivar, but no cultivar had high levels in leaves and stems. Fusarium fujikuroi was the main fumonisin producer. The FUM concentration in heads increases from <1 to 4760 µg/kg DM after the yellow-ripe stage. To control FUM levels, it is necessary to select low-FUM cultivars and manage the cultivation environment.

Changes in the concentrations of fumonisin, deoxynivalenol and zearalenone in corn silage during ensilage.

We assessed the production of the mycotoxins fumonisin, deoxynivalenol and zearalenone during the ensiling of corn. Corn was harvested at yellow-ripe or full-ripe stage and separated into the stem and leaf parts and the ear parts, including bracts. Each material was ensiled under five conditions: (1) no fungus added, anaerobic conditions; (2) no fungus added, aerobic conditions; (3) mycotoxin-producing fungus added, anaerobic conditions; (4) mycotoxin-producing fungus added, aerobic conditions; and (5) mycotoxin-producing fungus added to autoclaved material, aerobic conditions. After 40 days of ensilage, we analyzed the silage fermentative quality and mycotoxin concentration. The fermentative quality of all materials was good in treatments (1) and (3), because the pH < 4 increased the lactic acid content preventing mycotoxin levels from increasing. In treatments (2) and (4), fermentative quality of all materials was poor, and mycotoxin levels were slightly increased. In treatment (5), fermentative quality was poor, and mycotoxin levels were increased remarkably. These results indicate that mycotoxins are not produced under anaerobic conditions and are hardly produced under aerobic condition during the ensiling of corn. Our findings suggest that almost all mycotoxins in corn silage are produced pre-harvest.

Changes of fumonisin production in rice grain during ensiling.

We assessed fumonisin production during the ensiling of rice grain. Rice grain was harvested at the full-ripe stage and prepared as rough rice, crushed rough rice, brown rice or crushed brown rice. Each material was ensiled under six conditions: (1) no fungus, anaerobic; (2) no fungus, aerobic; (3) water added, anaerobic; (4) water and fumonisin-producing fungus added, anaerobic; (5) water and fumonisin-producing fungus added, aerobic; or (6) fumonisin-producing fungus added to autoclaved material, aerobic. After 40 days of ensilage, we analyzed the silage fermentative quality and fumonisin concentration. The fermentative quality of all materials was good in treatments (3) and (4) (pH < 4), reasonable in treatment (5) (pH = 5∼6) and unacceptable in treatments (1) and (2) (pH > 6.5). The fumonisin concentration was low in all materials in treatments (1) to (4), slightly increased in the three materials other than rough rice in treatment (5), and enormously increased in all materials in treatment (6). The results indicate that the fumonisin-producing fungus does not produce fumonisin in anaerobic conditions. It is important that an anaerobic condition be maintained during ensiling in order to reduce the fumonisin content in rice grain silage.

Culturable bacterial communities on leaf sheaths and panicles of rice plants in Japan.

Culturable bacterial communities on rice plants were investigated from 2001 to 2003. In total, 1,394 bacterial isolates were obtained from the uppermost leaf sheaths at 1 month before heading time and from leaf sheaths and panicles at heading time. The average culturable bacterial population on the leaf sheaths was larger at heading time than at 1 month previously. Furthermore, the population was significantly larger on panicles than on leaf sheaths, suggesting that the bacterial population is influenced by the organs of rice plants. Larger proportions of bacteria were obtained from the macerates of leaf sheaths after washing with phosphate buffer, and most culturable bacteria were verified to inhabit the inside or inner surface, rather than the outer surface, of the tissues. Verification of the bacterial composition based on 16S rRNA gene sequences revealed that genera of Sphingomonas, Microbacterium, Methylobacterium, and Acidovorax tended to be dominant colonizers on leaf sheaths, whereas Pseudomonas and Pantoea were isolated mainly from the panicles, indicating that leaf sheaths and panicles harbor distinct communities. Furthermore, the richness of bacterial genera was less on both leaf sheaths and panicles at heading time compared with that observed 1 month before heading time. Phylogenetic analyses using bacterial isolates belonging to the four dominant genera inhabiting leaf sheaths at heading time revealed that particular bacterial groups in each genus colonized the leaf sheaths.

Culturable leaf-associated bacteria on tomato plants and their potential as biological control agents.

Culturable leaf-associated bacteria inhabiting a plant have been considered as promising biological control agent (BCA) candidates because they can survive on the plant. We investigated the relationship between bacterial groups of culturable leaf-associated bacteria on greenhouse- and field-grown tomato leaves and their antifungal activities against tomato diseases in vitro and in vivo. In addition, the isolated bacteria were analyzed for N-acyl-homoserine lactone (AHL) and indole-3-acetic acid (IAA) production, which have been reported to associate with bacterial colonization, and resistance to a tomato alkaloid (alpha-tomatine). Leaf washings and subsequent leaf macerates were used to estimate the population size of epiphytic and more internal bacteria. Bacterial population sizes on leaves at the same position increased as the leaves aged under both greenhouse and field conditions. Field-grown tomatoes had significantly larger population sizes than greenhouse-grown tomatoes. Analysis of 16S rRNA gene (rDNA) sequencing using 887 culturable leaf-associated bacteria revealed a predominance of the Bacillus and Pseudomonas culturable leaf-associated bacterial groups on greenhouse- and field-grown tomatoes, respectively. Curtobacterium and Sphingomonas were frequently recovered from both locations. From the 2138 bacterial strains tested, we selected several strains having in vitro antifungal activity against three fungal pathogens of tomato: Botrytis cinerea, Fulvia fulva, and Alternaria solani. Among bacterial strains with strong in vitro antifungal activities, Bacillus and Pantoea tended to show strong antifungal activities, whereas Curtobacterium and Sphingomonas were not effective. The results indicated the differences in antifungal activity among predominant bacterial groups. Analysis of alpha-tomatine resistance revealed that most bacterial strains in the dominant groups exhibited moderate or high resistance to alpha-tomatine in growth medium. Furthermore, some Sphingomonas and Pantoea strains showed AHL and IAA production activities. Strain 125NP12 (Pantoea ananatis) showed particular alpha-tomatine resistance, and AHL and IAA production had the highest protective value (91.7) against gray mold. Thus, the differences of these physiological properties among dominant bacteria may be associated with the disease suppression ability of BCAs on tomato plants.

Cloning and characterization of a gene rpg1 encoding polygalacturonase of Rhizopus oryzae.

The polygalacturonase (PG)-encoding gene (rpg1) of Rhizopus oryzae, the causal pathogen of rhizopus rot of mulberry, was cloned and sequenced. PGs were partially purified from incubation mixture of 2% pectin medium and their N-terminal amino acid sequences were determined by a gas-phase protein sequencer. RT-PCR was performed using degenerate primers designed from the amino acid sequences, which resulted in part of a PG-encoding gene being obtained. By 3'-RACE and TAIL-PCR analyses, the entire region of the PG-encoding gene was cloned and sequenced. The structural gene comprised 1199 bp coding for 383 amino acids with a putative signal peptide of 26 amino acids, and the open reading frame was interrupted by single intron of 47 bp. Phylogenetic analysis using the deduced amino acid sequence revealed that R. oryzae RPG1 belonged to a clade consisting of exo-PGs of ascomycete fungi.