Association of Slugs with the Fungal Pathogen Epichlo ¨E Typhina
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Annals of Applied Biology ISSN 0003-4746 RESEARCH ARTICLE Association of slugs with the fungal pathogen Epichloe¨ typhina (Ascomycotina: Clavicipitaceae): potential role in stroma fertilisation and disease spread G.D. Hoffman & S. Rao Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331, USA Keywords Abstract Arion subfuscus; choke disease; Dactylis glomerata; Deroceras reticulatum; fungal Epichloe¨ spp. are endophytes of grasses, and form epiphytic external stromata ecology; mycophagy; Prophysaon andersoni. on flowering tillers. E. typhina was first noticed infecting Dactylis glomerata (= orchardgrass, cocksfoot) stands in the Willamette Valley in 1996, and Correspondence soon became the primary factor limiting the longevity of seed production G.D. Hoffman, Department of Crop and Soil fields. Several species of slugs are present in these fields, and we investigated Science, Oregon State University, Corvallis, OR 97331, USA. Email: their role in E. typhina biology. Pre-dawn surveys of D. glomerata fields in [email protected] 2009 and 2010 found Prophysaon andersoni and Arion subfuscus slugs feeding on the fungal stromata. When unfertilised and fertilised immature stromata Received: 9 August 2012; revised version predominated, approximately 80% of the individuals of these two species accepted: 17 February 2013. that were seen on plants were found on the stromata. As the majority of stromata reached maturity the presence of these species on stromata declined doi:10.1111/aab.12024 to between 20–40%. The common agricultural slug pest, Deroceras reticulatum, was on stromata only 20% of the time early in the season, and declined to <5% at stromata maturity. Observations of frass from slugs determined that the most common constituent was the food sources upon which the slug species was usually found during these surveys. Typically 100% of the frass from P. andersoni and A. subfuscus contained stroma material, compared to 25% for D. reticulatum. Spermatia, and ascospores later in the season, were commonly seen in the frass of slugs that consumed stromata. Some slugs that had no stroma material in their frass appeared to have consumed spermatia and ascospores from the leaf surface. A multiple-choice laboratory test confirmed the different proportional preferences of P. andersoni and D. reticulatum for stroma (0.72 vs 0.20) and leaf (0.07 vs 0.38), respectively. Two laboratory multiple-choice tests, and a field survey, found that P. andersoni preferred unfertilised and immature stroma over mature stroma. D. reticulatum is the most common and abundant slug in Willamette Valley grass seed fields, yet it is the least likely to move spermatia between unfertilised stromata, or ascospores to uninfected plants. P. andersoni and A. subfuscus are mycophagous, frequently transport viable spermatia and ascospores in their frass; yet they are generally confined to field edges. Data and observations suggest the role of slugs in the epidemiology of E. typhina is small compared to other factors. Introduction in the Willamette Valley of western Oregon (Pfender & Alderman, 2006). Epichloe¨ (Ascomycotina: Clavicipi- Epichloe¨ typhina (Pers.:Fr.) Tul. is the primary factor taceae) endophytes are of considerable interest in agri- limiting the longevity of productive Dactylis glomerata cultural research, as pathogens in cultivated grass seed L. (= orchardgrass, cocksfoot) seed production fields production fields (Pfender & Alderman, 1999), as causal 324 Ann Appl Biol 162 (2013) 324–334 © 2013 Association of Applied Biologists G.D. Hoffman & S. Rao Slug consumption of Epichloe¨ stromata agents of livestock toxicosis (Belesky & Bacon, 2009), and of stroma fertilisation have recently been described. as beneficial organisms which reduce insect herbivory as Ascospores released from early maturing stromata can well as increase drought tolerance of host plants (Siegel fertilise late emerging stromata (Alderman & Rao, 2008). et al., 1990; Schardl, 1996; Schardl et al., 2009). During Spermatia dislodged from a stroma by air pressure driven the vegetative growth phase of the host plant, Epichloe¨ water mist, mimicking wind-blown rain, can fertilise is characterised by intercellular hyphal growth with lit- adjacent stromata (Kaser, 2009). tle to no penetration of the host cell wall (Christensen Slugs are serious pests in the grass seed cropping et al., 2002). When the host grass enters the reproductive systems in the Willamette Valley, OR, USA, particularly phase, branched hyphal masses (stromata) form epiphyti- on emerging grass seedlings and in no-till systems (Gavin cally on grass culms, and occasionally on vegetative tillers et al., 2008; Anderson et al., 2010; Mellbye et al., 2011). (Schardl, 1996; Christensen et al., 2008). After stroma fer- The introduced Deroceras reticulatum (Muller),¨ known as tilisation there is a proliferation of white fungal hyphae either the grey garden slug, or grey field slug, is the most leading to a thickening of the stroma and eventual forma- common slug in Willamette Valley agronomic systems tion of perithecia and ascospores. Growth of the stroma (Dreves & Fisher, 2012; personal observation). Other slug mechanically inhibits grass inflorescence development species, particularly those in the genus Arion, are present and production of viable grass seed; this syndrome is in smaller numbers in many grass seed fields. Slugs seen known as choke disease (Kirby, 1961; Bucheli & Leucht- on stromata during choke surveys raised the question of mann, 1996). E. typhina does not produce compounds whether they could be involved in the cycle of stroma toxic to livestock (Leuchtmann et al., 2000), and infected fertilisation, ascospore dispersal and the infection of new plants are not a problem in forage production or grazing. plants. E. typhina, was first recorded in Oregon in 1996, and We investigated the association between the orchard- was likely introduced from Europe where the species is grass pathogen E. typhina and slugs in seed production native (Alderman et al., 1997). By 2000, approximately fields in the Willamette Valley. We documented within 90% of orchardgrass seed production fields in Oregon the plant feeding sites of three slug species: D. reticulatum; were infected by the fungus (Pfender & Alderman, the native slug Prophysaon andersoni Cooper (reticulated 2006). It appears that seed yield loss is proportional to taildropper); and the introduced slug Arion subfuscus Dra- the percentage of flowering tillers choked (Large, 1954; parnaud (dusky Arion). The frass from these slugs was Pfender & Alderman, 2006). While E. typhina spread examined to identify diet components, and confirm that quickly through the Willamette Valley, infecting orchard slug location on the plant during night feeding peri- grass for experimental studies has proven to be difficult ods corresponded to the food consumed. To determine (S.C. Alderman, personal communication). if feeding site locations were due to a true food pref- The sexually reproducing Epichloe¨ are bipolar het- erence, we ran multiple-choice feeding preferences tests erothallic, and are obligate out crossers (White & Bult- in a controlled environment. Corroborative tests deter- man, 1987). Stroma of sexual Epichloe¨ species produce mined the field preference of P. andersoni for stromata of haploid spermatia and receptive hyphae of one of two different developmental stages. This information allowed possible mating types (MAT1-1-1 or MAT1-2-1) (Chung us to make predictions on the role of these slugs in the & Schardl, 1997). It appears that only one mating type infection biology of E. typhina. is found within a host plant (Schardl, 1996). The mei- otically derived haploid ascospores produced within the perithecia are ejected at maturity (Chung & Schardl, 1997; Methods and materials Leyronas & Raynal, 2008). E. typhina is a Type 1 reproduc- Field sampling tive system fungus (White, 1988), requiring ascospores to spread the fungus outside of the host and into susceptible In 2009, we focused our slug surveys on three individuals; it is not transmitted vertically through seed. orchardgrass fields in the mid-Willamette Valley, two For sexual reproduction in Epichloe¨ species, spermatia containing P. andersoni. We began the slug survey shortly produced on the stroma must be transported to a stroma of after the onset of stromata formation (early May). the opposite mating type for gamete transfer and meiosis Sampling was from 5:00 am to 6:30 am on cloudy or to occur. Female flies in the genus Botanophila (Diptera: foggy mornings, at approximately 2-week intervals. The Anthomyiidae) transfer viable spermatia from one stroma exceptions were two 10:00 pm to 12:00 am sampling to another in the process of female feeding, defecation, events (14 May and 3 June), done to compare night and egg laying (Kohlmeyer & Kohlmeyer, 1974; Bultman with the subsequent early morning feeding periods. We et al., 1995, 1998). Spermatia are not carried by wind attempted to record the location of 30–40 individuals (Bultman et al., 1995); however other mechanisms of each species seen on orchardgrass plants during Ann Appl Biol 162 (2013) 324–334 325 © 2013 Association of Applied Biologists Slug consumption of Epichloe¨ stromata G.D. Hoffman & S. Rao each sampling period. Because D. reticulatum was most The frass generally consisted of a digested, unidentifiable common, we stopped recording information on this granular appearing substance and a portion of undigested species after 40 observations, as we continued