Incidence, Identification, and Mycoparasitic Activity of Clonostachys epichloë, a Hyperparasite of the Fungal Endophyte Epichloë typhina

Fungi of the genus Epichloë (Clavicipitaceae, Ascomycota) cause choke disease in many grass species. The disease manifests itself as fungal stromata that form around developing inflorescences, thereby suppressing their maturation. Economic losses in agricultural production due to choke have long been known in the U.S.A. and France, but attempts to control choke disease have not been successful. The interaction between Epichloë typhina (Pers.) Tul. & C. Tul. and its naturally occurring fungal hyperparasite, Clonostachys epichloë (Speg.) Schroers (sexual morph Bionectria epichloë) was investigated in populations of the grass Puccinellia distans (L.) Parl. Fungal hyperparasites occur widely in nature, and many are successfully used commercially as biological control agents against plant pathogenic fungi. Microscopy of Epichloë stromata infected with C. epichloë revealed a lack of asci with ascospores in perithecia and damage to mycelia at sites colonized by C. epichloë. The ability of C. epichloë to colonize E. typhina was confirmed via two in vitro experiments. The percent inhibition of growth of E. typhina strains by C. epichloë varied from 18.40 to 46.50%, and the mycoparasite colonized up to 100% of Epichloë mycelia in a precolonization experiment. We discuss the possibility of using C. epichloë to control choke disease caused by E. typhina in grass populations.

Effect of some factors on the incidence of choke (Epichloë typhina) in grass seed stands in the Czech Republic

Choke of grasses caused by Epichloë typhina occurred in 2008 and 2009 in more than 25% of grass seed stands of timothy (Phleum pratense and P. nodosum); a lower incidence was reported in orchard grass (Dactylis glomerata). A decline in disease incidence in 2010 was caused partly by a reduction in the total area of grasses grown for seed production and partly by a decrease of older grass seed stands. The incidence of choke in timothy varieties Odenwälder and Pampas was very high; they differed statistically from the other 24 grown cultivars. No difference was found among 21 varieties of orchard grass. The highest incidence of choke was observed in varieties Comer and Dolina (timothy) and Amera (orchard grass). The incidence of choke in cultivated varieties of Phleum spp. and D. glomerata was affected by stand age (the incidence of the disease in timothy was recorded in 13% of stands in the first cropping year and in 50% of stands in the fifth cropping year). Choke disease occurred in all regions where the species were grown for seed.  

The fungus Epichloë typhina in populations of a halophyte Puccinellia distans: salinity as a possible inhibitor of infection

<em>Puccinellia distans</em> is a non-agricultural halophytic grass that has become another host plant for <em>Epichloe typhina</em>, hitherto not reported from Poland. In 1992 we noticed the first symptoms of choke disease in a single population of <em>P. distans</em> in central Poland. Since then we have observed choke disease in 5 populations of <em>P. distans</em> only in man-made habitats. These habitats are strongly anthropogenically salinated but they exhibit the pattern of species composition characteristic of natural salines. In this paper we test the hypothesis that the level of salinity affects the infection of <em>P. distans</em> by the fungus <em>E. typhina</em>. Seven plots were established in the field and each plot was divided into 25 subplots. Within each plot the level of infection in a spring generation of shoots was negatively correlated with salinity (common regression within the plots, beta = -0.674, df = 117, p &lt; 0.001). Negative correlation was also found in an autumn generation within the plots (beta = -0.682, df = 94, p &lt; 0.001) after excluding plot P in which the frequency of infected individuals was the lowest and equal only to 0.05. The proportion of individuals infected by the endophytic stage of the fungus in the populations was assessed using diagnostic polymerase chain reaction. The greatest percentage (98.3%) of infected individuals was found in the population growing in the habitat of the lowest salinity. The high salinity reduces the chance of <em>P. distans</em> to become infected, but may promote the stroma formation of <em>E. typhina</em> twice in the season. Disease expression in autumn clearly represents a misadaptation which could be explained by the fact that the species interactions described here appeared relatively recently as a result of human activity. This hypothesis requires further experimental verification.

Choke Disease Caused by Epichloë bromicola in the Grass Agropyron repens in Poland

Agropyron repens (synonym Elymus repens, couch grass) is a species native to Europe and Asia. In Poland, it is a common weed of crop fields. In May 2008, we noticed for the first time symptoms of choke disease (caused by Epichloë spp.) on A. repens at two localities in central Poland. The localities, Pakość (52°47.531′N, 18°06.118′E) and Dulsk (52°45.329′N, 18°20.518′E), are located 16 km apart from each other. The following year, we confirmed the occurrence of choke disease on couch grass at these localities. Stromata were formed on reproductive stems that did not produce inflorescences. They ranged from 16 to 31 mm long and were covered with perithecia 520 to 560 × 160 to 250 μm at a density of 35 to 45 per mm2. Asci measured 270 to 310 × 5.2 to 6.5 μm and ascospores were 225 to 275 × 1.5 to 1.7 μm (specimen deposited in ZT). Morphological characters match with the original description of Epichloë bromicola (4). One strain was isolated from stromatal tissue and the partial DNA sequence of tubB including introns 1 to 3 was obtained as previously described (2). In a phylogenetic analysis, the isolate (GenBank Accession No. GU325782) grouped with Epichloë isolates from other Agropyron spp. from Poland (A. intermedium) and Japan (A. ciliare and A. tsukushiense) and with an isolate from a Roegneria sp. (from China). Experimental mating tests involving isolates from A. intermedium and a Roegneria sp. indicated that these isolates were sexually compatible with Epichloë bromicola from Bromus erectus. Similarly, E. yangsii was compatible with E. bromicola. This suggests that Epichloë isolates from Agropyron, Roegneria, and Bromus hosts form a common mating population, and implies that under a biological species concept the phylogenetic definition of E. bromicola has to be broadened. Epichloë on A. repens has been previously found in Poland (1), Germany (3), Hungary, and Romania (specimen deposited in herbarium of ETH Zurich, ZT) based on incidental records or on herbarium specimens that have been listed under E. typhina. Our study, based on morphology, tubB sequence similarity, and mating compatibility, suggests that the fungus infecting A. repens in Poland is E. bromicola. References: (1) I. Adamska. Acta Mycol. 36:31, 2001. (2) D. Brem and A. Leuchtmann. Evolution 57:37, 2003. (3) J. Kohlmeyer and E. Kohlmeyer. Mycologia 66:77, 1974. (4) A. Leuchtmann and C. L. Schardl. Mycol. Res. 102:1169, 1998.

First Report of Dicyma pulvinata on Epichloë typhina and Its Potential for Control of E. typhina

The hyperparasite Dicyma pulvinata is reported for the first time on Epichloë typhina, which causes choke disease in orchardgrass seed production fields in the Willamette Valley, OR. In an orchardgrass field near Corvallis, OR, D. pulvinata was found on 7% of E. typhina stromata. Infected stromata had fewer perithecia and appeared shrunken, desiccated, and pale gray to grayish-white, in contrast to the orange colored noninfected stromata with mature perithecia. In greenhouse inoculations, D. pulvinata significantly (P < 0.05) reduced perithecial formation on E. typhina. However, under field conditions, a significant (P < 0.05) reduction in perithecial development occurred in one trial initiated in mid-May, but not in a second trial initiated in early June, although D. pulvinata sporulated on 92% of the inoculated stromata from each trial within 72 h after their incubation in moist chambers. Under field conditions, D. pulvinata may have potential as a biocontrol agent of E. typhina if applied when stromata start to emerge during mid late April to early May when rain and high humidity conditions are typical. However, development of D. pulvinata would likely be limited by low humidity conditions that often occur during mid- to late June in the Willamette Valley. There are currently no effective chemical or cultural controls for choke in orchardgrass. Accepted for publication 3 November 2009. Published 16 February 2010.

First Report of Choke Disease Caused by Epichloë typhina on Orchardgrass (Dactylis glomerata) in China

Orchardgrass or cocksfoot (Dactylis glomerata L.) has been widely cultivated as a forage crop in many provinces of China (1). It is also a native perennial forage grass, which grows at the edge of forests, shrubs, and mountainous grasslands in Xinjiang and Sichuan (2). In September of 2007, signs of choke disease on orchardgrass were observed in a native grassland under birch woodland near Altai City, Xinjiang, China. Stromata, which formed on culms of diseased grass, enclosing the inflorescence and leaf sheath, were 4.5 to 5.5 mm long, smooth or wrinkled, white and later becoming yellowish or yellow, tuberculate, dry, and covered with perithecia. Inflorescences surrounded by fungal stromata were choked and failed to mature, thus restricting seed production. Pure cultures were obtained by directly scraping stromata from the surface and incubating it on antibiotic potato dextrose agar (PDA). The colonies were cottony, white on the upper surface, and white to yellow on the reverse. The growth rate was 13 to 21 mm per week at 25°C on PDA. Conidia were hyaline, lunate to reniform, and measured 4.1 ± 0.5 × 2.2 ± 0.5 μm. They accumulated in small globose heads at the tips of conidiogenous cells and were produced singly on conidiophores of 13 to 33 μm long and 2.7 to 4.1 μm wide at the base. Internal transcribed spacer (ITS) sequence by BLAST search had 99% similarity with an Epichloë typhina isolate of orchardgrass in Spain (GenBank Accession No. AM262420.1). Cultural characteristics, microscopic examination, and phylogenetic analysis showed that this choke disease on D. glomerata was caused by the fungus E. typhina (Pers.) Tul. & C. Tul. as described by White (4). To our knowledge, this is the first report of E. typhina causing choke disease on orchardgrass in China. The pathogen has been identified in France, England, Germany, Sweden, Switzerland, and the United States (3,4) with the same symptoms as those reported here. In 1997, choke disease was found in 70% of the fields in the Willamette Valley of Oregon, with disease incidences ranging from 0.05 to 28%. It was predicted to increase and spread under the prevailing climatic conditions (3). This new disease report is to provide observational and diagnostic information to help with recognition and prevention of disease spread in orchardgrass cultivation regions of China. References: (1) X. R. Chao et al. Shandong Agric. Sci. 1:7, 2005. (2) S. X. Jia, ed. China Forage Plant Flora. China Agriculture Press, Beijing, 1987. (3) W. F. Pfender and S. C. Alderman. Plant Dis. 83:754, 1999. (4) J. W. White. Mycologia 85:444, 1993.