Journal of Invertebrate Pathology 114 (2013) 333–336
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Chytrid mycoparasitism of entomophthoralean azygospores Ann E. Hajek a,⇑, Joyce E. Longcore b, D. Rabern Simmons b, Kenlyn Peters a, Richard A. Humber c a
Department of Entomology, Cornell University, Ithaca, NY 14853-2601, USA School of Biology and Ecology, University of Maine, Orono, ME 04469-5722, USA c USDA-ARS Biological Integrated Pest Management Research Unit, Ithaca, NY 14853-2901, USA b
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Article history: Received 23 May 2013 Accepted 2 October 2013 Available online 15 October 2013 Keywords: Azygospores Chytridiomycota Gypsy moth Entomophaga maimaiga Gaerntneriomyces semiglobifer Biological control
a b s t r a c t Mycoparasitism – when one fungus parasitizes another – has been reported to affect Beauveria bassiana and mycorrhizal fungi in the field. However, mycoparasitism of any fungi in the Order Entomophthorales has never been reported before now. The majority of entomophthoralean species persist as resting spores (either zygospores or azygospores) in the environment and dormant entomophthoralean resting spores (whether formed as zygospores or azygospores) are thought to be especially well adapted for survival over long periods due to their thick double walls. Entomophthoralean resting spores can accumulate in the soil as large reservoirs of inoculum which can facilitate the onset and development of epizootics. We report parasitism of azygospores of the gypsy moth pathogen Entomophaga maimaiga caged in soil from southern Ohio by the chytrid fungus Gaertneriomyces semiglobifer. G. semiglobifer had previously been isolated from soil samples from North America, Europe and Australia or horse manure from Virginia. After isolation and identification of G. semiglobifer, azygospores of E. maimaiga exposed to zoospores of G. semiglobifer exhibited high levels of mycoparasitism and G. semiglobifer was subsequently reisolated from mycoparasitized resting spores. We discuss the importance of this finding to the epizootiology of insect diseases caused by entomophthoralean fungi. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Mycoparasitism has been defined as the direct attack of a fungal cell and utilization of nutrients from that fungus by another fungus, the mycoparasite (Viterbo and Horwitz, 2010). While mycoparasitism is considered beneficial for human endeavors when it leads to control of fungal pathogens causing plant disease, the opposite can be true when mycoparasites attack mycorrhizal fungi or insect pathogenic fungi. An example of mycoparasitism of an entomopathogenic fungus is the ascomycete Syspastospora parasitica (Tulasne) Cannon & Hawksworth attacking Beauveria bassiana (Bals.-Criv.) Vuill. growing on a Colorado potato beetle (Leptinotarsa decemlineata (Say)) cadaver (Posada et al., 2004). Also, the chytrid Spizellomyces punctatum (Koch) Barr seems to be a weak facultative mycoparasite of azygospores of Gigaspora margarita Becker & Hall, a mycorrhizal member of the Glomeromycota (Schenck and Nicolson, 1977). However, mycoparasites have not been documented attacking azygospores of entomophthoralean fungi. Entomophthoralean fungi are known for their ability to cause epizootics in arthropod populations (Pell et al., 2001). These obligate pathogens usually persist in the environment as soil-borne resting spores (zygospores or azygospores), which have
⇑ Corresponding author. Fax: +1 607 255 0939. E-mail address:
[email protected] (A.E. Hajek). 0022-2011/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2013.10.002
double-layered walls several microns thick. The persistent spores can undergo prolonged dormancy and do not require exogenous nutrients for germination. The ecology of entomophthoralean resting spores and the conditions favoring their germination have been studied little in nature, although we know that for Entomophaga maimaiga, an important fungal pathogen of the gypsy moth (Lymantria dispar (L.)), the majority of azygospores are located in the top 0–3 cm of soil, often at the bases of trees that had been occupied by its hosts (Hajek et al., 1998). During bioassays investigating germination of E. maimaiga azygospores, we observed that some azygospores were dead and infected with a mycoparasitic chytrid, which was isolated and which is identified here as Gaertneriomyces semiglobifer (Chytridiomycetes: Spizellomycetales). We also report results from bioassays examining mycoparasitism of E. maimaiga azygospores by G. semiglobifer.
2. Methods 2.1. Initial detection and isolation of mycoparasite We collected samples from the top 3 cm of soil within 10 cm of the bases of white oaks (Quercus alba L.) on Telegraph Hill, Wayne National Forest, Lawrence Co., Ohio (38.78643, 82.63980, elevation 261 m; Shelocta-Latham soil) in April 2008 and maintained samples in landscape-cloth lined plastic boxes in the woods. In July 2009, we
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enclosed 35 g of soil in each of three lidded clear plastic deli cups (4.3 cm ht. 10.5 cm diam.) with water added to field capacity. We collected cadavers of late instar L. dispar larvae whose bodies were filled with E. maimaiga azygospores from trunks of red oaks (Quercus rubra L.) in Rothrock State Forest, Huntingdon Co., Pennsylvania, in July 2009. Cadavers were soaked overnight in distilled water, macerated with a glass rod and the resulting suspension was filtered through cheesecloth into a 50 mL centrifuge tube. The suspension of azygospores was centrifuged for 5 min at 250g, decanted, 30 mL distilled water were added and the tube was vortexed; this procedure was repeated 2 times for a total of 3 washes. On 31 July we sealed approximately 1 105 azygospores into 6 6 cm, 20 ll mesh nylon bags (Sefar Filtration, Depew, New York); the packets of azgospores were placed in the middle of the soil sample cups, 4 ml distilled water were added to each cup, and these set-ups were incubated at 15 °C and 14:10 (L:D). Azygospores were evaluated after 49 d, and a mycoparasite was isolated from parasitized azygospores as described below. During viewing infected azygospores mounted in distilled water on a microscope slide we noted the discharge of what appeared to be chytrid zoospores. We washed these zoospores onto a 90 mm Petri dish of PmTG agar medium (Barr, 1986; peptonized milk, 1 g/L; tryptone, 1 g/L; glucose, 5 g/L; agar 5 g/L with penicillin, 200 mg/L; streptomycin sulfate, 200–500 mg/L added after autoclaving). We spread the zoospores on the plate and later transferred thalli that grew from the zoospsores onto clean plates of PmTG and allowed them to develop into colonies. We established stock cultures on PmTG slants in screw-topped culture tubes, transferred them at 100 d intervals, and stored them at 5 °C between transfers. The culture obtained, JEL623, is stored in the Longcore Collection (University of Maine) and has been deposited in the USDA-ARS Collection of Entomopathogenic Fungal Cultures (Ithaca, NY) as ARSEF 12091. Following the PCR and sequencing protocols described by Simmons (2011), we extracted DNA from
isolate JEL623 with Whatman FTAÓ cards (Whatman Inc., Piscataway, New Jersey; Borman et al., 2006) and amplified nuclear large subunit (nucLSU) ribosomal DNA (rDNA) with the primer pair LR0R/LR5 (Rehner and Samuels, 1994; Vigalys and Hester, 1990). We assembled chromatograms in Geneious 4.8.4 (Drummond et al., 2010). 2.2. Bioassay with azygospores Desiccated late instar L. dispar cadavers containing E. maimaiga azygospores were collected on 26 June 2012 from trunks of Q. rubra on Clark Farm Rd., Galeton, Pennsylvania. We microscopically examined azygospores at 200X; approximately 50% were mature (C4–C5 = thick, double-layered wall, containing several large lipid droplets; Hajek et al., 2008). Ten cadavers were soaked in distilled water overnight, macerated, and filtered through a stack of 500, 250, 125, 63 and 20 lm 75 mm diameter sieves (Gilson Company, Inc., Lewis Center, Ohio). Each layer of sieves was rinsed with distilled water, and azygospores were collected on the 20 lm sieve. Azygospores in 14.5 ml distilled water containing 50 mg gentamicin were incubated at room temperature for 12 days to allow further maturation until approximately 70% of the azygospores were rated with a maturity of C4–C5. We placed a 5 5 mm square of agar covered with thalli of isolate JEL623 in the center of each of three 8 cm diameter Petri dishes containing 1% water agar and added 1 ml of the azygospore suspension to each dish. As controls, we added azygospores to 1% agar Petri dishes without colonies of isolate JEL623. Petri dishes were sealed and incubated at 15 °C, 60% relative humidity (RH) and 14:10 h (L:D). On days 1 and 3, we added 1 ml of sterile water to each Petri dish. Viewing through an inverted microscope at 200X, we counted 20 azygospores at each of five randomly chosen locations on each Petri dish every day for eight days and ranked them as immature (
