Koya Sugawara

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.

Phylogenetic position of an arbuscular mycorrhizal fungus, Acaulospora gerdemannii, and its synanamorph Glomus leptotichum, based upon 18S rRNA gene sequence

We examined the phylogenetic position of an arbuscular mycorrhizal fungus which produces two types of spore,Acaulospora gerdemannii andGlomus leptotichum, based upon the DNA sequence of the 18S rRNA gene. DNA was extracted separately from bothGlomus-like orAcaulospora-like spores and partial 5′-terminus segments of 18S rRNA gene were amplified by the PCR method. Several clones derived from each spore type were sequenced and compared. The sequences from both spore types agreed well, confirming that these morphologically different spores were formed by the same fungus. Nucleotide substitutions were found among several clones, suggesting polymorphism of the rRNA gene in glomalean fungi. Further phylogenetic analysis based upon the whole sequence of the 18S rRNA gene showed thatA. gerdemannii may be within the order Glomales but is far from the fungi that have been analyzed and probably should be in a new family.

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.

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.

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.

Flowers for Neotyphodium endophytes detection : a new observation method using flowers of host grasses

Neotyphodium endophytes are vertically transmitted fungal symbionts of grasses. Being pest-repelling and growth-promoting agents for their hosts, and also potential mycotoxin producers, their detection in plants is important. Observation of chemically cleared flowers of infected grasses (Festuca arundinacea, F. pratensis, Lolium perenne, and L. multiflorum) using differential interference contrast microscopy revealed the existence of endophytes within immature ovaries of host plants. This observation method provides an accurate and easy way to detect and distinguish Neotyphodium endophytes in flowering host grasses and to investigate the seed transmission process, which is critical to their life cycle, and the practical use of infected plants.

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.

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.