Accepted Manuscript

Detection of presumptive mycoparasites associated with Entomophaga mai-

maiga resting spores in forest soils

Louela A. Castrillo, Ann E. Hajek

PII: S0022-2011(14)00177-3

DOI: http://dx.doi.org/10.1016/j.jip.2014.11.006

Reference: YJIPA 6614

To appear in: Journal of Invertebrate Pathology

Received Date: 8 September 2014

Accepted Date: 20 November 2014

Please cite this article as: Castrillo, L.A., Hajek, A.E., Detection of presumptive mycoparasites associated with

Entomophaga maimaiga resting spores in forest soils, Journal of Invertebrate Pathology (2014), doi: http://

dx.doi.org/10.1016/j.jip.2014.11.006

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers

we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and

review of the resulting proof before it is published in its final form. Please note that during the production process

errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Journal of Invertebrate Pathology

(Short communication)

Detection of presumptive mycoparasites associated with Entomophaga maimaiga resting

spores in forest soils

Louela A. Castrillo* and Ann E. Hajek

Department of Entomology, Cornell University, Ithaca, NY 14853-2601

*Corresponding author: Louela A. Castrillo (lac48@cornell.edu)

2

Abstract

The fungal pathogen Entomophaga maimaiga can provide high levels of control of the gypsy

moth, Lymantria dispar, an important forest defoliator. This fungus persists in the soil as resting

spores and occurs naturally throughout many areas where gypsy moth is established. Studies on

the spatial dynamics of gypsy moth population have shown high variability in infection levels,

and one possible biological factor could be the variable persistence of E. maimaiga resting

spores in the soil due to attacks by mycoparasites. We surveyed presumptive mycoparasites

associated with parasitized E. maimaiga resting spores using baiting and molecular techniques

and identified an ascomycete (Pochonia sp.) and oomycetes (Pythium spp.).

Key words: Entomophaga maimaiga; resting spores; mycoparasites; soil baiting; taxon-specific

primers; Lymantria dispar

3

1. Introduction

The fungal pathogen Entomophaga maimaiga can provide high levels of control of the gypsy

moth, Lymantria dispar, which is considered the most important forest defoliator in northeastern,

north central and mid-Atlantic United States. This fungus persists in the soil as double-walled

resting spores (azygospores) and occurs naturally throughout many areas where gypsy moth is

established. Recently, Hajek et al. (2013) reported the discovery of a chytrid mycoparasite in the

soil from oak forests that killed E. maimaiga resting spores, and this parasite could play a critical

role affecting the persistence of viable pathogen inocula and reducing the impact of E. maimaiga

on gypsy moth populations. Mycoparasites are common in the soil and it is possible that there

are other mycoparasitic fungi impacting prevalence of E. maimaiga resting spores in forest soils.

Mycoparasitic fungi and pseudofungi that derive most or all of their nutrients from other

fungi have been described from different fungal taxa, but are more commonly known from the

groups Ascomycota, Chytridiomycota, and Oomycota (Jeffries and Young, 1994). Some of these

fungi have been commercialized as biological control agents against plant pathogenic fungi, but

they could also negatively impact beneficial fungi such as mycorrhizae or entomopathogenic

fungi. In this study we conducted a survey of presumptive mycoparasites associated with E.

maimaiga resting spores in forest soils using a combination of soil-baiting and PCR techniques.

We used molecular techniques to be able to detect mycoparasitic fungi and pseudofungi that may

be in the latent or quiescent stage.

2. Materials and Methods

2.1 Baiting experiment

4

Entomophaga maimaiga resting spores were collected from infected gypsy moth cadavers

following the method described by Hajek et al. (2008). Approximately 7 x 106 resting spores

were placed in individual 20 μm mesh bags (6 x 6 cm) with sealed edges. The resting spores are

> 20 μm in diameter while many mycoparasites have stages < 20 μm in diameter. Soil samples

were collected in July 2013 from the upper 3 cm of the soil surface under numerous oak trees at

three locations with known histories of recent gypsy moth outbreaks: Allegany State Park

(42°2'33.53"N, 78°50'25.34"W) and Yellow Barn State Forest, New York (latitude 42.46644,

longitude 76.31798); and State Game Lands #64, Pennsylvania (latitude 41.773241, longitude -

77.656052). Soil samples taken within each site were mixed and 35 g subsamples were placed in

individual 200-ml plastic cups for immediate use. There were six subsamples for each site, plus

another three for Allegany State Park from soil collected under non-oak trees. Bagged resting

spores were placed between layers of soil in each cup and incubated at 15°C in the dark, with 2

ml of sterile distilled water added after 3 weeks to maintain moisture (Hajek et al., 2013).

After 60 days each bag was carefully rinsed under running distilled water, and contents of

the bag were washed and collected in 15 ml conical polypropylene tubes and centrifuged at 3400

x g for 10 min to collect the resting spores. Spores were washed twice with distilled water, once

with 70% ethanol, followed by two rinses in distilled water, and resuspended in 1 ml sterile

distilled water. Aliquots of resting spores were examined under a compound microscope at 200

to 400X to determine the presence and prevalence of mycoparasitism.

2.2 DNA extraction, PCR assay conditions and sequencing

Aliquots (500 μl) of resting spore suspensions were pelleted by centrifugation at 16000 x

g for 5 min and were resuspended in 400 μl lysis buffer with RNase A (Qiagen, Valencia, CA) in

5

a bead beating tube (BioSpec Products, Bartlesville, OK) with 1 g of 2.0 mm zirconia and 0.5 g

of 0.5 mm zirconia silica beads (BioSpec Products). Samples were homogenized for 1 min at

5000 rpm using a cell disrupter (Mini-Beadbeater-1; BioSpec Products) and DNA was extracted

using the DNeasy kit (Qiagen) following the manufacturer’s protocol. DNA was eluted in 50 μl

elution buffer, quantified using a spectrophotometer, and stored at -20°C until use.

To detect fungi and pseudofungi associated with the resting spores, PCR assays using

taxon-specific fungal/pseudofungal ITS primers were conducted using primers and conditions

reported by Nikolcheva and Barlocher (2004). The ITS primer for chytrids was also tested

against Gaertneriomyces semiglobifer (Chytridiomycetes: Spizellomyces), which was discovered

by Hajek et al (2013) attacking E. maimaiga resting spores in soils from Ohio. Primers and

annealing temperatures used are listed in Table 1. PCR products were visualized on a 1% agarose

gel stained with ethidium bromide. PCR assays were replicated at least twice.

To recover individual bands for sequencing, PCR products were separated on 1.5 to 3.0%

NuSieve agarose gel (Lonza, Rockland, ME) and representative bands were excised with a

scalpel and purified using a QiaQuick Gel Purification kit (Qiagen). Purified products were

quantified and submitted to Cornell Biotechnology Resource Center for sequencing. Sequencing

primers used for each taxon were the same as those for amplification. Both strands were

sequenced and the results aligned prior to submission to a BLAST search for highly similar

sequences.

3. Results and Discussion

More than 90% of the resting spores examined from the baiting experiment were

parasitized, as evidenced by the change in morphology in contrast to healthy resting spores (Fig.

6

1A). Parasitized spores were often misshapen and were filled with distinct fungal structures of

various forms (Fig. 1B, 1C, and 1D), indicating the possibility of more than one type of

mycoparasite in the samples. We also observed many resting spores devoid of cytoplasmic

contents. Prevalence of parasitism was high among the three sites, with up to 100% parasitism

among resting spores from subsamples from each sites. The high prevalence observed, however,

could be an artifact of the baiting set up. In nature prevalence will more likely vary due to a

combination of biotic and abiotic factors (e.g., diversity, spatial distribution and abundance of

mycoparasite inoculum, location in the soil, length of time in the soil, etc.). Nonetheless, the

baiting technique optimized the possibility of detecting various mycoparasites associated with E.

maimaiga resting spores.

The taxon-specific primers, except ITS4Zygo and ITS4Basidio, generated one or more

bands from all of the samples. BLAST queries of sequence data from representative bands from

each taxon group identified presumed mycoparasites as an ascomycete (Pochonia sp.; GenBank

accession no. KM491882) and three oomycetes (Pythium spp.; GenBank accession nos.

KM491883, KM491884, and KM491885). Pochonia sp. is closely similar to P. bulbillosa, which

attacks nematodes, and was detected in all three sites. Nematophagous fungi, like P. bulbillosa,

have been known to attack other fungi (Tzean and Estey, 1978), but these are reports of attacks

on fungal hyphae. Resting spores, however, are nutrient rich reservoirs formed by fungi for

survival during adverse conditions and are valuable substrates for other fungi capable of

attacking them and penetrating the thick walls (Jeffries and Young, 1994). Whether this

Pochonia sp. is an obligate mycoparasite or an opportunistic one cannot be determined without

biological assays to study interaction between the two fungi. Among the three Pythium species

or strains, Pythium spp. 1 and 3 were detected in both sites in New York, but Pythium sp. 2 was

7

only found in the Yellow Barn State Forest site. Several mycoparasitic species have been

described in this genus, a few with broad host ranges and capable of attacking resting spores

(Davanlou et al., 1999). We did not detect any mycoparasitic chytrids, including G. semiglobifer,

from the three sampling sites.

Other fungi detected were assumed to be non-mycoparasitic following analysis of

sequences obtained. These included different species of mushrooms and lichen-associated fungi,

most of which generated relatively faint bands compared to those generated by Pochonia and

Pythium spp. While our results showed the ability of primers to detect different fungi from DNA

co-extracted with mycoparasitized resting spores, the possibility exists that there may be other

mycoparasitic fungi that were not detected. The primer ITS4Zygo did not generate any band

from E. maimaiga while the primer for chytrids did, indicating lack of specificity of the latter

and limited use of the former. These primers amplify the ITS region, which may not always be

an adequate marker for higher taxa separation. The molecular assays, however, provided a

convenient method for conducting initial surveys from environmental samples. Further studies

are needed to isolate and to cultivate each putative mycoparasite to test interactions with E.

maimaiga and assess impact on this pathogen’s population dynamics in the field.

Acknowledgments

We thank Abby Finley and Jake Henry (Cornell University, Ithaca, NY) for technical assistance.

This work was supported by the Cornell University Agricultural Experiment Station (Hatch

funds) received from the National Institute of Food and Agriculture (NIFA), United States

Department of Agriculture (USDA). Any opinions, findings, conclusions, or recommendations

8

expressed in the publication are those of the authors and do not necessarily reflect the view of

NIFA or the USDA.

References

Davanlou, M., Madsen, A.M., Madsen, C.H., Hockenhull, J., 1999. Parasitism of macroconidia,

chlamydospores and hyphae of Fusarium culmorum by mycoparasitic Pythium species. Plant

Pathology 48, 352-359.

Hajek, A.E., Longcore, J.E., Simmons, D.R., Peters, K., Humber, R.A., 2013. Chytrid

mycoparasitism of entomophthoralean azygospores. Journal of Invertebrate Pathology 114, 333-

336.

Hajek, A.E., Burke, A.E., Nielsen, C., Hannam, J.J., Bauer, L.E., 2008. Nondormancy in

Entomophaga maimaiga azygospores; effects of isolate and cold exposure. Mycologia 100, 833-

842.

Jeffries, P., Young, T.W.K., 1994. Interfungal Parasitic Relationships. CAB International,

Wallingford, UK.

Nikolcheva, L.G., Barlocher, F., 2004. Taxon-specific fungal primers reveal unexpectedly high

diversity during leaf decomposition in a stream. Mycological Progress 3, 41-49.

9

Tzean, S.S., Estey, R.H., 1978. Nematode-trapping fungi as mycopathogens. Phytopathology 68,

1266-1270.

10

Figure 1

Healthy and mycoparasitized Entomophaga maimaiga resting spores. Parasitism was detected

by comparing morphology of healthy spores (A) versus caged resting spores used as baits in soil

samples from oak forests. Most of the resting spores used as bait were parasitized, misshapen

and filled with different fungal or pseudofungal structures (B, C, and D) indicating presence of

multiple mycoparasitic species.

11

Table 1. PCR and sequencing primers used in this study.

______________________________________________________________________________

_________________

Primer Sequence Annealing

temperature (°C)

______________________________________________________________________________

__________________

Forward:

ITS 5 5'-GGAAGTAAAAGTCGTAACAAGG

Reverse (taxon-specific):

ITS4Asco (Ascomycota) 5’-CGTTACTAGGGCAATCCCTGTTG

55

ITS4Basidio (Basidiomycota) 5”-GCGCGGAAGACGCTTCTC

58

ITS4Chytrid (Chytridiomycota) 5’-TTTTCCCGTTTCATTCGCCA

50

ITS4Oo (Oomycota) 5’-ATAGACTACAATTCGCC

49

ITS4Zygo (Zygomycota) 5’-AAAACGTATCTTCAAA

40

________________________________________________________________________________________________

10 µm!

B

A

C D

Healthy (inset) and parasitized Entomophaga maimaiga resting spores

Graphical Abstract

12

Highlights

•Entomophaga maimaiga causes epizootics in gypsy moth populations.

•Resting spores persist in the soil but numbers decline over time.

•Resting spores were used as bait in soil collected at bases of oaks after epizootics.

•Under optimal lab conditions, most resting spores used as bait were parasitized.

•Presumptive mycoparasites Pochonia sp. and Pythium spp. located with resting spores.