Ann E. Hajek

Ecology of Terrestrial Fungal Entomopathogens

Fungal pathogens are capable of causing sensational levels of mortality in insect populations. As early as about 1000 AD, sericulturists in Asia reported Beauveria bassiana infections in silkworms (Steinhaus, 1956). The “germ theory of disease,” the concept that microbes can cause disease, was first experimentally proven by Agostino Bassi in 1834 working with B. bassiana and silkworms. From the late 1800s through 1925, research on the potential use of fungi for insect control was conducted. In recent years, largely due to our present knowledge of the hazards and inefficiencies of dependence on synthetic chemical pesticides for insect control, interest in developing fungal pathogens for control purposes has increased dramatically (Roberts and Hajek, 1992; Vandenberg, 1993). Although abundant research on use of entomopathogenic fungi for control has been conducted, there are major gaps in our understanding of the basic ecology of these fungal species, in part due to the complexity of host/pathogen/environment interactions and the diversity of host/pathogen systems to be studied. Lack of successful control in some systems and difficulties in adapting some species for typical control practices has promoted a shift in research emphasis. At present, some research efforts are directed toward understanding the factors leading to fungal infection in insects in order to investigate the potential for manipulating these systems to enhance levels of infection and promote development of epizootics (disease outbreaks).

Ecology and management of exotic and endemic Asian longhorned beetle Anoplophora glabripennis

1 The Asian longhorned beetle is native to China and Korea, and was found for the first time outside its native habitat in the U.S.A. in 1996, with subsequent detections being made in Canada and several European countries. 2 We review the taxonomy, distribution, basic biology, behaviour, ecology and management of endemic and exotic Anoplophora glabripennis, including information that is available in the extensive Chinese literature. 3 This species has caused massive mortality of Populus species in China and models have demonstrated that it could become established in many locations worldwide. 4 Anoplophora glabripennis is polyphagous but prefers Acer, Salix and Populus, section Aigeiros. 5 Although A. glabripennis adults do not disperse far when surrounded by host trees, they have the potential to fly more than 2000 m in a season. 6 Volatile organic compounds from preferred host trees are attractive to A. glabripennis and this attraction is heightened by drought stress. Males and females orientate to a volatile released by female A. glabripennis and males attempt to copulate after contacting a sex pheromone on the female cuticle. 7 At present, A. glabripennis is being (or has been) eradicated from areas where it has been introduced. After detection, extensive surveys are conducted and, if breeding populations are detected, at the very least, infested trees are removed and destroyed. Close attention is paid to imported solid wood packaging material to prevent new introductions. 8 Standard practice to control A. glabripennis in China is to spray insecticides in tree canopies. In North America, largely as a preventative measure, systemic insecticides are injected into trees. Entomopathogenic fungi have been developed for the control of A. glabripennis, and entomopathogenic nematodes, coleopteran and hymenopteran parasitoids and predatory woodpeckers have been investigated as biocontrol agents. 9 Ecological control of A. glabripennis in China involves planting mixtures of preferred and nonpreferred tree species, and this practice can successfully prevent outbreaks.

Entomopathogenic Fungi as Bioinsecticides

As early as 900 A.D., it was known in the Orient that fungi could grow in insects (Steinhaus, 1975). The pioneering work of Bassi with Beauveria bassiana in silkworms in 1834 proved that fungi could actually cause infectious diseases in insects. From the 1880s through the early 1900s, the spectacular epizootics caused by entomopathogenic fungi—fungi-infecting insects—led to studies of their potential use for pest control. Interest in fungi as pest control agents waned, however, as chemical insecticides were used more frequently. More recently, owing to the myriad difficulties that have been gradually encountered in the development and use of chemical insecticides, the field of biological control has been undergoing a renaissance. In particular, our knowledge of entomopathogenic fungi is at present increasing rapidly.

Control of Invasive Soybean Aphid, Aphis glycines (Hemiptera: Aphididae), Populations by Existing Natural Enemies in New York State, with Emphasis on Entomopathogenic Fungi

Abstract This study evaluated the diversity and abundance of existing natural enemies of soybean aphid, Aphis glycines L., under field conditions in New York State, with emphasis on entomopathogenic fungi. In 2003, five soybean fields were occasionally sampled to estimate abundance and species composition of entomopathogenic fungi. During 2004, five soybean fields and adjacent buckthorn were sampled weekly. Seven species of aphid pathogenic fungi were found, including Pandora neoaphidis (Remaud. et Henn.) Humber, Conidiobolus thromboides Drechsler, Entomophthora chromaphidis Burger et Swain, Pandora sp., Zoophthora occidentalis (Thaxter) Batko, Neozygites fresenii (Now.) Remaud. et Keller, and Lecanicillium lecanii, (Zimm.) Gams et Zare. P. neoaphidis was the most abundant species, causing 84% infection in an outbreak aphid population in 2003, after which the aphid population crashed. In 2004, we found the first aphids with fungal infections in late June to midJuly. Mycosis was strongly associated with aphid density, especially during increasing aphid populations. In agreement, epizootic levels of infection were associated with subsequent declines in aphid populations. There was variability among fields; in three of five fields sampled in 2004, the chances for infection were more than five times higher than in the other two fields. High infection levels were found on aphids on buckthorn over 3 wk in autumn. Three species of aphidiid parasitoids from A. glycines were documented: Aphidius sp. and two Praon species. Both genera were the first records from A. glycines in the United States. Coccinellids were the most abundant predator, followed by syrphids and cecidomyiids, whereas anthocorids and chrysopids were less common.

Isolation and characterization of Entomophaga maimaiga sp. nov., a fungal pathogen of gypsy moth, Lymantria dispar, from Japan.

Abstract A Japanese zygomycete pathogen from the gypsy moth, Lymantria dispar (Lepidoptera: Lymantriidae), is described as Entomophaga maimaiga sp. nov. (Entomophthorales: Entomophthoraceae). This fungus belongs to the Entomophaga aulicae species complex. Isolates from the E. aulicae species complex were tested for infectivity and pathogenicity toward larvae of the gypsy moth, and only those isolates from gypsy moths were pathogenic to gypsy moth larvae. Electrophoretic studies of isoenzymes indicated that the Japanese gypsy moth isolates differed from other Japanese and North American strains of the E. aulicae species complex. Among the isolates of E. maimaiga from Japan, ARSEF 1400 appeared most promising for biological control use; it caused 90–95% mortality in third instar larvae after an average of 5.2 days and can be cultured in both protoplast and mycelial vegetative stages. In host range trials, ARSEF 1400 infected only a few of the lepidopteran species tested. Among these alternate hosts, mortality was the greatest (92.5%) for the Douglas-fir tussock moth, Orgyia pseudotsugata, another economically important lymantriid.

Methods for Rearing the Asian Longhorned Beetle (Coleoptera: Cerambycidae) on Artificial Diet

Abstract The Asian longhorned beetle, Anoplophora glabripennis (Motschulsky), was recently introduced to the United States and has the potential to destroy many urban and forest trees. A successful artificial diet and rearing protocol are urgently needed, because research with this wood-boring beetle can be conducted only in the confined areas of quarantines. We compared larval growth and adult parameters using three artificial diets, one developed in China for A. glabripennis and two developed for other members of the Lamiini. The only difference in performance of larvae and adults reared on the three diet types was that nondiapausing larvae reared on Monochamus carolinensis (Olivier) diet needed less time to pupate than nondiapausing larvae on A. glabripennis diet. We further evaluated substituting the phloem–cambium of sugar maple, Acer saccharum Marshall, with sawdust or cellulose. Males grew fastest on diets with sawdust or phloem–cambium and remained as pupae for the shortest period of time on A. glabripennis diet. Females grew faster on diets with cellulose than sawdust and lived longest on A. glabripennis diet. The published A. glabripennis artificial diet, modified by increasing the water content from 50.0 to 64.6% (wt:wt) and substituting the phloem–cambium component with cellulose, was the optimal diet tested. A rearing protocol used to maintain our colony is included.

Field studies of control of Anoplophora glabripennis (Coleoptera: Cerambycidae) using fiber bands containing the entomopathogenic fungi Metarhizium anisopliae and Beauveria brongniartii

Abstract The Asian longhorned beetle, Anoplophora glabripennis, was first found attacking urban street trees in the United States in 1996 and in Canada in 2003. This tree-killing invasive insect has long been a major pest in China and is difficult to control because immature stages live within wood and long-lived adults are often located high in tree canopies. A microbial control product (Biolisa Kamikiri) consisting of non-woven fiber bands impregnated with cultures of an entomopathogenic fungus, Beauveria brongniartii, is marketed in Japan for control of a congeneric orchard pest. Replicated field trials were conducted in Anhui, China to compare Biolisa Kamikiri with similarly prepared bands containing Metarhizium anisopliae for control of A. glabripennis. One fungal band was placed at 2–2.5 m height, around the stem or major scaffold branch on each of 40 willow trees (Salix spp.) per plot, with five plots for each fungal treatment and five control plots. Adult beetles collected from fungal-treated plots 7–22 days after bands were attached to trees died faster than adults from control plots. Beetles exposed to B. brongniartii bands consistently died faster than controls throughout this period, while results from plots with M. anisopliae bands were not as consistent in differing from controls. Numbers of adult beetles from plots of each fungal species dying in <10 days were greater than controls (16% of beetles) but did not differ between fungal treatments (34–35%). Oviposition in fungal-treated plots was approximately half that in control plots. Locations of adult beetles and oviposition scars within tree canopies were quantified to determine optimal locations for band placement. Most adult beetles were found >3.5-m high in trees, with adults in B. brongniartii-treated plots higher within trees than adults in other plots.

Decline in virulence of Entomophaga maimaiga (Zygomycetes: Entomophthorales) with repeated in vitro subculture

Abstract Repeated subculture of Entomophaga maimaiga protoplasts in liquid media resulted in aberrant morphology after 50 passages. Bioassays demonstrated an increase in disease incubation time after 15 in vitro passages. Associated with increased incubation time, percentage mortality caused by E. maimaiga declined with repeated subculture (r2 = 0.62). After prolonged subculture, E. maimaiga produced only resting spores in many cadavers, while fungal lines that had not been repeatedly subcultured produced conidia or both conidia and resting spores after host death. After 2 months of in vitro culture, the rate of subculturing had an impact on virulence, while the absolute length of time in axenic culture did not.

Ants defend aphids against lethal disease

Social insects defend their own colonies and some species also protect their mutualist partners. In mutualisms with aphids, ants typically feed on honeydew produced by aphids and, in turn guard and shelter aphid colonies from insect natural enemies. Here we report that Formica podzolica ants tending milkweed aphids, Aphis asclepiadis, protect aphid colonies from lethal fungal infections caused by an obligate aphid pathogen, Pandora neoaphidis. In field experiments, bodies of fungal-killed aphids were quickly removed from ant-tended aphid colonies. Ant workers were also able to detect infective conidia on the cuticle of living aphids and responded by either removing or grooming these aphids. Our results extend the long-standing view of ants as mutualists and protectors of aphids by demonstrating focused sanitizing and quarantining behaviour that may lead to reduced disease transmission in aphid colonies.

Soil as an environment for winter survival of aphid-pathogenic Entomophthorales

The survival of Pandora neoaphidis was studied for both discharged primary conidia and hyphal bodies inside aphid cadavers after storage on moist soil at different temperatures. The activity of the inoculum was quantified by the ability to produce replicate conidia as well as the ability to infect aphids. No effect of inoculum type was found. Conidia were produced after storage for at least 32 days at 20C, 64 days at 10C, and 96 days at 5C. Inoculum retained the ability to initiate infections in aphids after storage for at least 14 days at 20C, 32 days at 10C, and 64 days at 5C. Morphological studies of the inoculum suggest that P. neoaphidis may survive unfavorable conditions as thick-walled conidia also known as loricoconidia. Furthermore, P. neoaphidis and Conidiobolus obscurus were documented for the first time in field-collected soil in early spring by baiting the soil with aphids. We hypothesize that germination of overwintering inoculum is stimulated by host-induced factors since inoculum apparently responded to the presence of aphids. 2003 Elsevier Science (USA). All rights reserved.