Takuya Shiba

Resistance to the rice leaf bug, Trigonotylus caelestialium, is conferred by Neotyphodium endophyte infection of perennial ryegrass, Lolium perenne

Neotyphodium fungal endophytes form mutualistic symbiotic associations with many grasses of the subfamily Pooideae, including important forage and turfgrass species. This relationship provides a competitive advantage to the host plant by increasing abiotic/biotic stress tolerance, such as its resistance to drought, diseases, and insect pests. The insect deterrent effects of endophytes are now receiving attention in Japan, as insect pests growing in meadows are causing problems in adjacent rice paddies. One of the most serious problems is the kernel spotting of rice grains caused by the rice leaf bug, Trigonotylus caelestialium Kirkaldy (Heteroptera: Miridae), which reproduces on Lolium species grown as forage. To determine the potential of Neotyphodium endophytes to reduce the invasion of rice crops by T. caelestialium from adjacent Lolium crops, we carried out choice and no‐choice feeding tests using endophyte‐infected and endophyte‐free clonal perennial ryegrass (Lolium perenne L.) (Poaceae). Our experiments revealed that the presence of the Neotyphodium endophyte strongly deterred the feeding of both first‐instar larvae and adults of T. caelestialium. These results show the potential of Neotyphodium endophytes to reduce the number of T. caelestialium in forage fields and grasslands, and thus to reduce the damage to rice grains caused by this insect pest.

Fungal loline alkaloids in grass–endophyte associations confer resistance to the rice leaf bug, Trigonotylus caelestialium

Plant symbiotic fungi (endophytes) of the genus Neotyphodium [anamorphs, asexual derivatives of Epichloë (Ascomycota: Clavicipitaceae)] often associate with grass species of the subfamily Pooideae, which includes important forage and turf species. These endophytes are known to produce a range of alkaloids that enhance their hosts resistance to insects or are toxic to grazing animals. Among the alkaloids, loline alkaloids (saturated 1‐aminopyrrolizidines) are generally observed in the highest concentrations in many Neotyphodium–grass symbiotic associations, and are known to be toxic to insects but not to mammals. Some Neotyphodium‐infected grasses have enhanced resistance to rice leaf bug, Trigonotylus caelestialium (Kirkaldy) (Heteroptera: Miridae), one of the major pests for rice production in Japan. Our laboratory experiments quantified the effects of purified loline (N‐formylloline) and in planta synthesis of loline alkaloids by meadow fescue [Lolium pratense (Huds.) S.J. Darbyshire (syn. Festuca pratensis Huds.)]–Neotyphodium uncinatum (Gams, Petrini & Schmidt) Glenn, Bacon & Hanlin and Italian ryegrass (Lolium multiflorum Lam.)–Neotyphodium occultans Moon, Scott & Christensen associations on the development and survival of T. caelestialium. No‐choice feeding assays with laboratory populations of the insect revealed that their growth was significantly decreased by the infected grasses, and the effect was greater for N. uncinatum than for N. occultans, in keeping with differences in N‐formylloline concentrations in the plants. Artificial feeding of N‐formylloline through feeding sachets indicated that the chemical has an adverse effect on survival of larvae, even at the lowest concentration tested (50 µg/g), which was considerably lower than the typical concentrations in many Neotyphodium–grass associations. The results confirmed the ability of Neotyphodium‐infected forage grasses to control T. caelestialium propagation in meadows, which may cause damages to nearby rice paddies.

Detection and diagnosis of rice-infecting viruses

Rice-infecting viruses have caused serious damage to rice production in Asian, American, and African countries, where about 30 rice viruses and diseases have been reported. To control these diseases, developing accurate, quick methods to detect and diagnose the viruses in the host plants and any insect vectors of the viruses is very important. Based on an antigen–antibody reaction, serological methods such as latex agglutination reaction and enzyme-linked immunosorbent assay have advanced to detect viral particles or major proteins derived from viruses. They aid in forecasting disease and surveying disease spread and are widely used for virus detection at plant protection stations and research laboratories. From the early 2000s, based on sequence information for the target virus, several other methods such as reverse transcription-polymerase chain reaction (RT-PCR) and reverse transcription-loop-mediated isothermal amplification have been developed that are sensitive, rapid, and able to differentiate closely related viruses. Recent techniques such as real-time RT-PCR can be used to quantify the pathogen in target samples and monitor population dynamics of a virus, and metagenomic analyses using next-generation sequencing and microarrays show potential for use in the diagnosis of rice diseases.

Evaluating the fungal endophyte Neotyphodium occultans for resistance to the rice leaf bug, Trigonotylus caelestialium, in Italian ryegrass, Lolium multiflorum

Fungal endophytes of the genus Neotyphodium (Ascomycota: Clavicipitaceae) form symbiotic associations with many grass species of the subfamily Pooideae, including some important forage and turf grasses from the genus Lolium. Some Neotyphodium‐infected grasses have enhanced resistance to the rice leaf bug, Trigonotylus caelestialium (Kirkaldy) (Heteroptera: Miridae), one of the major pests in rice production in Japan. The insects can grow on Lolium grasses and thus cause a conflict of interest between production of forage and rice. Endophyte‐mediated resistance to T. caelestialium is likely to be conferred by N‐formylloline, one of the major loline alkaloids (saturated 1‐aminopyrrolizidines) observed in some grass–Neotyphodium associations. We checked the feasibility of using Neotyphodium occultans, the native endophyte of Italian ryegrass (Lolium multiflorum Lam.), to reduce T. caelestialium, as this endophyte produces N‐formylloline, but no known mammalian toxins. To this end, we conducted choice and no‐choice feeding assays using T. caelestialium first instars and 35 N. occultans‐infected Italian ryegrass plants with different in planta N‐formylloline concentration. Some N. occultans‐infected ryegrass showed significant resistance to T. caelestialium. The preference and the survival of this insect were considerably lower on infected plants, and the higher the in planta N‐formylloline concentration, the greater the adverse effects on the insect. The incremental risk of death of the insect was assumed to be due to intake of the chemical, and in addition, to the decreased amount of feeding caused by its insect‐deterrent effect. These results indicated that the use of the N. occultans–grass associations has potential to reduce T. caelestialium propagation in ryegrass crops. The breeding of N. occultans‐infected Italian ryegrass cultivars with high N‐formylloline concentrations should be an effective way to develop pasture grasses with a greater protective effect against T. caelestialium.

Enhanced Resistance to Four Species of Clypeorrhynchan Pests in Neotyphodium uncinatum Infected Italian Ryegrass

ABSTRACT Particular alkaloids produced by Neotyphodium endophytes show toxicity to invertebrates. Italian ryegrass (Lolium multiflorum Lamarck) cultivars and strains that are symbiotic with Neotyphodium endophytes have been recently established in Japan. N. uncinatum-infected Italian ryegrass lines accumulate N-formylloline, a type of loline alkaloid (1-aminopyrrolizidine) showing neurotoxicity to herbivorous insects. This study investigated the toxicity of N-formylloline and resistance of N. uncinatum-infected Italian ryegrass to vascular-sap feeding Clypeorrhynchan pests. When four vascular-sap feeding insects: Laodelphax striatellus (Fallén) (Homoptera: Delphacidae), Sogatella furcifera (Horváth) (Homoptera: Delphacidae), Cicadulina bipunctata (Melichar) (Homoptera: Cicadellidae), and Nephotettix cincticeps (Uhler) (Homoptera: Cicadellidae) fed on N. uncinatum-infected Italian ryegrass, significant decreases in survival rate were observed for three phloem-sap feeders but not for a xylem-sap feeder, N. cincticeps. This result suggests an uneven distribution of N-formylloline among plant tissues. A potency assay for N-formylloline using a Parafilm feeding sachet and a quantitative analysis of N-formylloline in plant showed a concentration-dependent lethal effect of N-formylloline on all four tested vascular-sap feeders. Our results strongly suggest that N. uncinatum-infected plants can control some Clypeorrhynchan pests in crop fields.

Quantitative analysis of Rice stripe virus in a transovarial transmission cycle during the development and reproduction of its vector, Laodelphax striatellus

The amount of Rice stripe virus (RSV) maintained through transovarial transmission was analyzed during the development and reproduction of its vector, Laodelphax striatellus. Reverse transcription quantitative PCR analysis was used to quantify RNA expressed from the RSV coat protein (CP) gene as an estimate of RSV content in nymphs and adults of L. striatellus at various developmental stages. The 18S ribosome RNA gene of L. striatellus was chosen as the reference for calculating RSV CP expression using the comparative Ct method. Based on the CP transcript levels, the amount of RSV did not differ significantly throughout the nymphal stage or between adult females of different ages; however, RSV content tended to increase slightly as males became older. The average RSV content in males was 1.30–2.49 times that in females. The amount of RSV in L. striatellus adults was compared between generations. The RSV content of female adults did not differ significantly between the parent and progeny populations three of three different females. L. striatellus grown to adults on a susceptible cultivar and five RSV-resistant cultivars were compared to analyze whether the amount of RSV varied among cultivars. Although the amount of RSV in L. striatellus adults differed significantly among the six rice cultivars evaluated, the difference seemed independent of whether resistance genes were present. In addition, the percentage of viruliferous insects was similar among cultivars.

Seed Transmission of Endophytic Fungus, Neotyphodium occultans, in Cross Breeding of Italian Ryegrass (Lolium multiflorum) Using Detached Panicle Culture, and Comparison with Situations in Interspecific/Intergeneric crossings including Festuca species

Neotyphodium species are seed-transmitted endophytic fungi that form mutualistic (symbiotic) associations with grasses of the subfamily Pooideae, and their presence can increase stress tolerance of host grasses. Italian (annual) ryegrass (Lolium multiflorum) clones infected with N. occultans were pollinated by pollen from annual ryegrass and perennial ryegrass (L. perenne) using detached panicles, and from tall fescue (Festuca arundinacea) by conventional bagging, as part of studies on the use of this endophytic fungus to enhance productivity of this important forage grass and also of interspecific/intergeneric hybrids. In mating involving L. multiflorum, 64–100% seed transmission ratios of the fungus were observed among 13 cross-combinations, along with one exceptional case of no infection. In mating with L. perenne 53–100% transmission was observed, whereas less than 30% transmission was observed in crosses involving F. arundinacea. The results indicated that the symbiont can be seed transmitted through mating using detached panicles, and the possibility of poor compatibility between N. occultans and some L. multiflorum genotypes as well as with L .perenne and F. arundinacea.