Biologia, Bratislava, 61/Suppl. 21: S543—S616, 2006 Section Zoology DOI: 10.2478/s11756-007-0100-x

Aphid-pathogenic Entomophthorales (their taxonomy, biology and ecology)

Marek Barta & Ľudovít Cagáň

Department of Plant Protection, Slovak University of Agriculture, Tr. A. Hlinku 2,SK–94901, Nitra, Slovakia; e-mail: [email protected]

Abstract: Fungi of the order Entomophthorales (Zygomycota, Zygomycetes) constitute a group of mostly insect pathogenic species, which attracts attention of insect pathologists because of their high capacity for use in biological control of pest insects. This work primarily focuses on entomophthoralean species attacking aphids. The Entomophthorales are considered major pathogens of aphids in nature. In fact, the fungi are the only pathogens that regularly and effectively can affect aphid populations in natural ecosystems as well as in agroecosystems. Up to now, 33 entomophthoralean species organized into 9 genera have been recorded in aphid hosts. All fungal species are listed and organised by genus in the work. Descriptions are presented for all the fungal genera and species, including a nomenclature genesis, a geographical distribution, a host specificity/host range, a capability of cultivation in vitro, and possible prospects for their use in biological control strategies. A key to identification of the pathogens is provided as well. A general life cycle outline of the pathogens is followed by fundamental facts on biology and ecology of the fungi and analysis of primary factors that are involved in the study of epizootiology of infectious diseases in insect populations. The abiotic and biotic elements of the environment, which interfere in the pathogen–host interactions, are also underlined. In the last chapter, several examples are reviewed when the Entomophthorales were used against aphids within the four strategies of biological control, namely classical biological control, inoculation biological control, inundation biological control, and conservation biological control. The aim of this review is to cover the present status of knowledge about the natural enemies of aphids and to stimulate an interest of insect pathologists in this group of entomopathogenic fungi. Key words: Aphids, Entomophthorales, biological control.

1. Introduction and certain advances have recently been made in oth- ers. The fungi have been studied intensively in labora- Fungi of the order Entomophthorales constitute a group tories and tested in a small-scale in the field. However, of mostly zoopathogenic species and because of their there are some shortcomings of the Entomophthorales high capacity for use in biological control of pest in- as biocontrol agents coming from a fragility of infective sects in agriculture they have attracted attention of in- conidia, dormancy and an asynchronous germination of sect pathologists recently. The biological strategies of resting spores, as well as a complexity of infection cycle integrated pest management (IPM) reckon on the util- requiring successively: a contact between an infective isation of various groups of natural enemies. The En- spore and a host cuticle, germination, a penetration of tomophthorales are apt to be employed as prospective germ tube through a cuticle and, finally, a sporulation. biocontrol agents of several groups of agricultural pests. Moreover, all these phases are highly dependent upon It is especially because of their short infection cycle, a external conditions. high reproductive rate, as well as obvious and dramatic Interest in using the insect pathogens as control epizootics that may occur almost overnight. One of agents within IPM programs has generated a research particularities of the Entomophthorales, which enable in the development of microbial insecticides. Much them to spread fast, is an active projecting of conidia of this research has been focused on selection, isola- from conidiophores. Also, the Entomophthorales have tion, development, and production of highly pathogenic other characteristics making them valuable mortality species/strains with genetic characteristics favouring agents of insects. Most of the species are closely host their storage and usage. This orientation has enabled specific, so they pose no significant threat to non-target the pathogens to be applied to target hosts as re- organisms. Some of them are capable of being cultured placements for insecticides. Although some successes or produced in the laboratory or mass-scale. A tech- were noted, the majority of attempts to use the fun- nology of mass production is managed for some species gal pathogens as a substitute for insecticides resulted

c 2006 Institute of Zoology, Slovak Academy of Sciences S544 M. Barta & Ľ. Cagáň in inconsistent responses. The pathogens were treated be useful to students of aphids’ natural enemies, re- mostly as direct substitutes for chemical insecticides searchers in insect pathology and microbial control of and were applied without an adequate understanding aphids, as well as to persons interested in the produc- of their population biology or their interactions within tion of microbial insecticides. a local environment. Consequently, this lack of under- standing caused the field of epizootiology to be a major 2. Fungal diseases of arthropods research priority in the insect pathology. Nowadays, the fungal biological control is an exciting and rapidly de- Fungal diseases in insect populations are common veloping research area with implications for plant pro- and widespread. They can often destroy insect pop- ductivity, food production, as well as animal and human ulations in spectacular epizootics and thus attract health. This area has an interdisciplinary character and man’s attention. Many of these fungi are consid- includes ecology, genetics, pathology, physiology, mass ered an important factor regulating pest insect pop- production, formulation, and application strategies. In ulations. Insect pathologists have been interested in the light of new knowledge of host/pathogen biology the fungal pathogens of their respective host groups and the environmental factors affecting biology, vari- for over 100 years. Until now at least 90 genera ous strategies of fungal exploitation have been devel- and more than 700 species of entomopathogenic fungi oped and discussed recently, including classical, conser- have been identified as closely associated with inver- vation, inoculation, and inundation biological controls. tebrates, predominantly insects, but only 10 of them Aphids belong to important pests of crops. They have been or are currently being developed for in- cause crop losses by extracting phloem sap mostly from sect control (e.g., Roberts & Humber, 1981; Car- leaves and stems of host plants, and excluding honey- ruthers & Soper, 1987; Hajek & St. Leger, dew. In addition, aphids are one of the most active vec- 1994; Wraight et al., 2001; Bateman & Chap- tors of various plant viruses. Their feeding can result ple, 2001). Within the wide kingdom of fungi, virtu- in deformation, wilting, even in necrosis of host tissues ally every major fungal taxonomic group but higher depending on an infestation level. Thanks to their high Basidiomycetes and dematiaceous Hyphomycetes has variability and specificities of their life cycle, aphids members pathogenic to insects (Roberts & Hum- may create forms resistant against frequently applied ber, 1981). Entomopathogenic species are distributed pesticide groups. The development of pesticide resis- mostly among the classes Chytridiomycetes, Oomyce- tance in the aphid populations highlights the impor- tes, Trichomycetes, Zygomycetes, and Hyphomycetes. tance of biological control as the pest management tac- As a matter of fact, most species attacking terrestrial tic. Many farmers and food producers have been inter- insects belong to the class Hyphomycetes or the or- ested in biological strategies of pest management with der Entomophthorales in the class Zygomycetes, while the intention of producing residue-free provisions and those attacking aquatic insects are generally from the eliminating the pesticide detrimental effects on human Chytridiomycetes and Oomycetes (Butt & Goettel, health. 2000). Although fungal pathogens share much in com- Aphids seem to be excellent hosts for studying mon with viruses, bacteria, and protozoa, there are entomopathogenic fungi, including the Entomophtho- many excellent differences and the most fundamental rales. As plant feeders, they are a significant and fre- is the route of infection. Fungal entomopathogens al- quent component of entomofauna in agroecosystems. most always attack their hosts by direct penetrating Nearly 4,000 species of aphids distributed all over the the exoskeleton and do not require ingestion with sub- world and occupying various climatic regions, mostly sequent infection through the gut wall. In contrast, vi- the temperate regions, guarantee a high species diver- ral, bacterial, and protozoan pathogens of insects or sity of the group of fungi. As individuals, aphids are other arthropods infect their host primarily via the di- small and inconspicuous; however, they live in colonies gestive tract. They must be ingested and infect the host and frequently become incredibly numerous. Existence through the gut wall. Most bacterial entomopathogens of aphids in dense colonies gives optimal conditions for infect their hosts through the gut, although bacteria fast disease dispersal and a background for epizooti- could also gain access to the haemocoele through cutic- ology studies. Moreover, aphid colonies can be rela- ular wounds. The difference in the mechanisms of hosts’ tively simply manipulated and reared in laboratories infection implies that insects with sucking mouth parts for bioassay purposes. (the orders Homoptera and Heteroptera) are not at- The aim of this monograph is to cover the present tacked by bacteria, viruses or protozoa and fungi are status of knowledge about fungal natural enemies of only significant microbial pathogens of the hosts (Car- aphids. The chapters of the monograph thoroughly ruthers & Soper, 1987; Humber, 1991). summarize all relevant information necessary for study- Aphids can be attacked by entomopathogens of ing aphid pathology, including the taxonomy of fungal two different classes. They are the Zygomycetes and pathogens, a list of fungal species, a key to pathogens’ Hyphomycetes, but the entomophthoralean fungi of identification, biology and ecology of the fungi, and a the class Zygomycetes are the major fungal pathogens potential of the fungi for biological control. It should of aphids (Humber, 1991). Entomopathogenic Hy- Aphid-pathogenic Entomophthorales S545 phomycetes includes hundreds of species, but just a (Evans, 1989). Entomophthoralean conidia are of a rel- few of them are specific to aphids. Verticillium lecanii atively larger size with mucus layer on their surface and (Zimmermann) Viégas is one of the most important hy- they sporulate and germinate quickly. A relatively small phomycetous parasites of aphids. It can, however, at- number of conidia are produced per cadaver, but fewer tack a broad spectrum of insects both in tropical and conidia are required for infection initiation (Pell et al., temperate regions, but it is distributed mainly in trop- 2001). Conversely, hyphomycete fungi, in general, tend ical regions (Hall, 1984; Humber, 1991). The fungus to have wide host ranges and epizootics usually occur can rarely affect aphids under field conditions (e.g., only in insect populations in the soil (Keller & Zim- Feng et al., 1990; Humber, 1991; Hatting et al., mermann, 1989). Conidia are smaller and produced in 1999a). Besides V. lecanii, Beauveria bassiana (Bal- higher quantities per cadaver, not actively discharged, samo) Vuillemin can also seldom infect aphids in natu- usually without mucus on surface, and many conidia ral sites (e.g., Feng et al., 1990; Humber, 1991; Hat- are required for infection (Pell et al., 2001). ting et al., 1999a). Both the fungi, V. lecanii and B. Entomophthorales are considered a major patho- bassiana, have been successfully commercialised and genic group of sap sucking aphids. Bacterial and proto- registered for use in greenhouses against whiteflies, zoan infections have not been demonstrated in aphids aphids, and thrips (Wraight et al., 2001). (Latgé & Papierok, 1988; Humber, 1991), but some viruses have been found, which decrease the longevity of infected aphid individuals (D’Arcy et al., 1981). How- 3. Entomophthoralean infections ever, no viral epizootics in aphid populations have ever of arthropods been reported (Latgé & Papierok, 1988).

Fungi of the order Entomophthorales occur worldwide, mostly in terrestrial habitats except for members of the 4. Current status in the taxonomy genus Ancylistes occurring in waters (Balazy, 1993). of Entomophthorales At present the order Entomophthorales consists of five families with a total of 20 genera and 200–300 species Based on the presence of zygospores and a coenocytic (Keller, 1987a, 1991, 1997; Balazy, 1993; Eilen- mycelium, which may become divided by septae into berg, 2002). Fungi of this order have been identi- segments and often be fragmented into hyphal bodies, fied from many insect orders, including Diptera, Ho- the order Entomophthorales is placed in the phylum moptera, Lepidoptera, Coleoptera, Heteroptera, Hy- Zygomycota and the class Zygomycetes. Within Zy- menoptera, Orthoptera, and Dermaptera (Keller, gomycetes, the order Entomophthorales is characterised 1987a, 1991; Balazy, 1993). However, the fungi are by the repetitive discharge of conidia and the ability to also identified from mites (Acarina) (Keller, 1997) actively discharge conidia (Waterhouse, 1973). and recently some species have also been identified from The classification of the families and genera within Collembola (Apterygota) (Steenberg et al., 1996; the Entomophthorales has been disputed recently. The Keller & Steenberg, 1997). order Entomophthorales is, in all probability, mono- Fungi of the order Entomophthorales possess sev- phyletic (Jensen et al., 1998) and at present the eral noteworthy characteristics favouring their use in bi- following five families are recognised in the order ological control. In comparison with the Hyphomycetes, (Fig. 1): Completoriaceae (Humber), Meristacraceae the Entomophthorales are able to establish epizootics (Humber), Ancylistaceae (v. Thieghem), Entomoph- quickly due to their great capacity for multiplication thoraceae (Nowakowski), and Neozygitaceae (Ben-Ze’- and expansion in the host populations (Latgé et al., ev et Kenneth) (Eilenberg, 2002). Only the lat- 1983; Eilenberg, 2002). Their conidia are forcibly dis- ter three families contain arthropod-pathogenic species charged from conidiophores, which develop on the host (Humber, 1989; Keller, 1999). The family Neozygi- surface from hyphal bodies by penetrating and elongat- taceae has not been distinguished by Balazy (1993) ing through the cuticle. The entomophthoralean fungi and all the Neozygites species have been organized by may multiply as protoplasts and/or hyphal bodies af- him within the family Entomophthoraceae. The fam- ter having invaded a host and the hyphal bodies can ilies in the order Entomophthorales are distinguished develop into thick-walled spores (resting spores) in- primarily on the basis of nuclear characters (Hum- side a cadaver. These resting spores allow the fungus ber, 1981, 1984, 1987, 1989; Balazy, 1993). The fam- to survive adverse conditions and persist in the envi- ilies Entomophthoraceae and Neozygitaceae consist ex- ronment (Papierok & Hajek, 1997). The Entomoph- clusively of insect and mite pathogens, while only a thorales are characterized by a high specificity towards few insect pathogenic species are found in Ancylis- particular host species and generally by a low negative taceae. Recently, Keller & Petrini (2005) split the impact towards non-target organisms (Latgé & Pa- family Entomophthoraceae into three subfamilies, En- pierok, 1988). Close associations with foliar insect or tomophthoroideae, Erynioideae, and Massosporoideae. mite hosts is another characteristic of the group of fungi Further, the family Basidiobolaceae, traditionally ac- S546 M. Barta & Ľ. Cagáň

KINGDOM

PHYLUM

CLASS

ORDER

FAMILY

Fig. 1. Systematic position of the Entomophthorales within the Kingdom of Fungi [accepted by the Dictionary of Fungi (KIRK et al., 2001)]. cepted as a member of the order Entomophthorales, and several species have been described several times, was elevated to a new order Basidiobolales, with one resulting in many synonyms. family Basidiobolaceae, one genus Basidiobolus and five Since the first entomophthoralean species was de- species. However, a taxonomic affiliation of this group scribed there have arisen much confusion and equiv- of fungi is still uncertain. Some phylogenetic studies us- ocation as regards the nomenclatural and taxonomic ing nuclear SSU rDNA sequences show that the genus issues, and many of them still have not been solved Basidiobolus does not belong to the Entomophthorales completely. Over the years, however, the classification and confirm that it is more related to chytrids from the in the Entomophthorales has drawn great attention of Chytridiales and the Neocallimasticales (Nagahama et many taxonomists and during the last few years the tax- al., 1995; Jensen et al., 1998; James et al., 2000; Tan- onomy has undergone a revision and changed dramati- abe et al., 2000; Tehler et al., 2003). Other molec- cally. At the beginning of the investigation on the En- ular phylogenies based on the data from six gene re- tomophthorales these fungi were first classified into the gions place Basidiobolus close to the Entomophthorales class Basidiomycetes, but ten years later Nowakowski (James et al., 2006). The discrepancies primarily come correctly classified them into the class Zygomycetes from analyses of sequences of different fungal genes. The (Balazy, 1993). Four basic monographs of Entomoph- final position of Basidiobolus may have to await further thoraceae were published in the second half of the phylogenetic analysis, using sequences from additional 19th century and at the beginning of the 20th cen- genes. tury (Fresenius, 1858; Nowakowski, 1883; Thax- Main characters for generic classification include ter, 1888; Lakon, 1919). The Thaxter’s monograph a mode of discharge of primary conidia, a structure of became the most influential and had many followers. conidial wall, a number of nuclei per conidium, a shape Thaxter (1888) employed one genus Empusa for all of primary and secondary conidia, a mode of formation insect attacking species and the genus comprised three of secondary conidia, a presence of cystidia and rhi- subgenera: Empusa, Entomophthora,andTriplospo- zoids, and a pathobiology (Humber, 1981, 1987, 1989; rium. Thaxter further accepted the genera Basidiobo- Keller, 1987a, 1991, 1999). However, it has not always lus, Conidiobolus, Completoria,andMassospora as be- been of general acceptance which groups of characters longing to Entomophthoraceae. Lakon’s classification define genera in the Entomophthorales. Remaudiere´ (Lakon, 1919) was limited to the insect-pathogenic & Keller (1980) preferred the shape of primary coni- species of the family and was basically an emended ver- dia to be the main criterion for generic classification, sion of Nowakowski’s system (Ben-Ze’ev & Kenneth, while Humber (1981) recommended that primary coni- 1982a). Essentially, until 1963 only the genera Ento- dium shape should be secondary to nucleation, branch- mophthora and Massospora were recognised for insect- ing conidiophores and mode of discharge of primary pathogenic species (MacLeod, 1963). Few years later, conidia. Recent results of molecular analyses (Hajek another genus, Strongwellsea,wasdescribed(Batko et al., 2003) support the importance of primary coni- & Weiser, 1965). While the genera Massospora and dial shape in generic grouping. A total of 14 ento- Strongwellsea comprised only a few well-defined species, mopathogenic genera are present in the three families. Entomophthora consisted of nearly 100 species mor- Eilenberg (2002) listed 236 species altogether in the phologically rather diverse. Batko (1964b, c, d, e, 14 genera. Though, the number of species can be dis- 1966b), aware of the heterogeneity within the genus, puted. There is plenty of insufficiently described species was the first who laid the basis for a new classification Aphid-pathogenic Entomophthorales S547

Table 1. Different classifications of arthropod-pathogenic fungi in the order Entomophthorales.

BEN-ZE’EV &KENNETH BALAZY (1993) HUMBER (1989, 1997) KELLER (1987a, 1991, (1982a, b) 1999), KELLER &PETRINI (2005)

Ancylistaceae Ancylistaceae Ancylistaceae Ancylistaceae Conidiobolus Conidiobolus Conidiobolus1 Conidiobolus1 subg. Conidiobolus subg. Conidiobolus subg. Delacroixia subg. Delacroixia subg. Capillidium subg. Capillidium

Entomophthoraceae Entomophthoraceae Entomophthoraceae Entomophthoraceae Entomophaga Entomophaga Entomophaga Entomophthoroideae10 Entomophthora Batkoa Batkoa2 Entomophaga Massospora Entomophthora Entomophthora Batkoa Strongwellsea Eryniopsis3 Eryniopsis3 Entomophthora Erynia5 Massospora Massospora Eryniopsis3 subg. Zoophthora Strongwellsea Strongwellsea Erynioideae10 subg. Neopandora Zoophthora5 Zoophthora5 Orthomyces4 subg. Erynia subg. Zoophthora Pandora Strongwellsea subg. Furia subg. Neopandora Erynia Zoophthora5 Tarichium7 subg. Erynia Furia Pandora (a form genus) subg. Furia Tarichium7 Erynia Triplosporium6 Tarichium7 (a form genus) Furia Entomophthora8 (a form genus) Massosporoideae10 (nomina provisoria) Neozygites6 Massospora Tarichium7 (a form genus)

Neozygitaceae Neozygitaceae Neozygites Neozygites Apterivorax11

Basidiobolaceae9 Basidiobolaceae9 Basidiobolaceae9 Basidiobolus Basidiobolus Basidiobolus

1 KELLER (1987, 1999) and HUMBER (1989, 1997) do not split Conidiobolus into three subgenera; 2 New genus described in 1989 (HUMBER, 1989); 3 New genus described in 1984 (HUMBER, 1984); 4 New genus described in 1998 (STEINKRAUS et al., 1998b); 5 KELLER and HUMBER do not use Zoophthora as a genus name for all uninucleate genera; HUMBER elevated all the subgenera to the generic rank; 6 BEN-ZE’EV &KENNETH (1982a) prefer the generic name Triplosporium to the name Neozygites;BALAZY (1993) left the Neozygites genus in the family Entomophthoraceae; 7 A form genus for fungi with unknown conidial stage; 8 A provisional group of insufficiently described species; 9 Recent phylogenetic studies showed that the genus did not belong to the Entomophthorales; 10 KELLER &PETRINI (2005) split the family Entomophthoraceae into three subfamilies; 11 New genus described in 2005 (KELLER &PETRINI, 2005). by splitting this large genus into several genera. Since mophaga into Entomophaga and Batkoa was accepted his system had certain abnormalities and shortcom- (Balazy, 1993; Keller, 1999), the split of Erynia ings, it was not accepted. Consequently, Remaudiere´ into Erynia, Furia and Pandora was formerly consid- & Hennebert (1980), Remaudiere´ & Keller (1980) ered as premature and not accepted (Keller, 1991, and Humber (1981, 1984) revised the Batko’s system 1999). Recently, the genera separated from the com- and defined seven genera. These corrections found a mon genus Erynia have been accepted (Keller & wide acceptance. At the same time the main morpho- Petrini, 2005). Balazy (1993) also accepted the split logical and taxonomic criteria used in the Entomoph- but the genera were treated as subgenera of Zooph- thorales were revised and evaluated (Humber, 1981; thora. Ben-Ze’ev & Kenneth, 1982a). Later, more genera At present four systems of the Entomophthorales were proposed by splitting the genera Entomophaga, classification are normally used, referred to as Ben- Erynia (Humber, 1989) and Neozygites (Ben-Ze’ev Ze’ev and Kenneth’s classification, Balazy’s classifica- et al., 1987). The split of Neozygites into Neozygites tion, Keller’s classification, and Humber’s classification and Thaxterosporium was not justified and was re- (Table 1). The system proposed by Humber (1989, fused (Keller, 1997). Whereas the split of Ento- 1997) is followed in this work. S548 M. Barta & Ľ. Cagáň

5. Aphid-specific Entomophthorales 1. Pandora neoaphidis (Remaudi`ere et Hen- nebert) Humber This chapter contains a complete list of entomophtho- Mycotaxon 34: 452 (1989) ralean species, which have been recorded in aphid hosts. Pathogens not attacking aphid hosts in nature but only S: infecting them after artificial inoculation in laboratory Entomophthora aphidis Hoffman in Fresenius sensu Thaxter, Mem. Boston Soc. Nat. Hist. 4: 152–189 (1888) are also included (e.g., Pandora delphacis (Hori) Hum- Basionyms (B): Erynia subgenus Neopandora neoaphidis ber and Conidiobolus destruens (Weiser et Batko) Ben- Remaudi`ere et Hennebert ex Ben-Ze’ev et Kenneth, Myco- Ze’ev). Thirty-three pathogenic fungi altogether orga- taxon 14: 461–462 (1982) nized into three families are listed herein. The fungi Erynia neoaphidis Remaudi`ere et Hennebert, Mycotaxon are arranged into families and a description of each 11: 307–312 (1980) species is focused on a nomenclature genesis and his- Zoophthora subgenus Neopandora neoaphidis (Remaudi`ere tory with its synonyms [predominantly adopted from et Hennebert) Balazy, Flora of Poland, Fungi (Mycota) 24: Balazy (1993)] , a distribution throughout the world, 175–176 (1993) a host specificity, a capability of cultivation in vitro, and possible prospects for use in biological control. The species was originally discovered by Thaxter (1888) in the USA and the fungus was incorrectly inter- changed with a taxon Entomophthora aphidis Hoffman 5.1. Description of fungal species in Fresenius [today known as a basionym of the species Zoophthora aphidis (Hoffman in Fresenius) Batko] for Remaudiere` Hennebert Family: Entomophthoraceae Nowakowski nearly 100 years ( & , 1980). Thaxter Bot. Ztg. 34: 216–222 (1877) (1888), in his influential monograph, applied Genus: Pandora Humber the name Empusa (Entomophthora) aphidis to the fun- Mycotaxon 34: 451–453 (1989) gal pathogen of aphids, which produced conidia and whose resting spore stage might be assumed to exist but were not found. In addition to an incorrect ap- Synonyms (S): Zoophthora subgenus Pandora Batko (pro plication of the generic name Empusa to the fungus parte), Acta Mycol. 2: 18–19 (1966) (excluding the desig- [this generic name first applied to the fungal pathogens nated type, Entomophthora aphidis Hoffmann in Fresenius, by Cohn (1855) was properly rejected by Fresenius which is a species of Zoophthora sensu stricto) (1856) since it had been already used for orchids by Erynia subgenus Neopandora Ben-Ze’ev et Kenneth, Myco- Lindley and therefore Fresenius suggested a generic taxon 14: 460–462 (1982) name Entomophthora], the mycologist also incorrectly Zoophthora subgenus Neopandora Ben-Ze’ev et Kenneth (sensu Balazy, 1993), Flora of Poland, Fungi (Mycota) 24: treated his fungus as synonymous with Entomophthora 171–172 (1993) aphidis Hoffman in Fresenius, although the primary de- scription of this organism by Hoffmann was originally based on brown roughened resting spores and a conidial The genus was designated by Humber (1989) after stage of this species was not found or described (Re- a critical revision of four Batkoan subgenera of the maudiere` & Hennebert, 1980). Batko (1966b) was Zoophthora genus. Involving primary and secondary unaware that this name had been misapplied for yet generic characters as well as general pathobiology of the undescribed fungus and typified his subgenus Pandora fungi, the Batkoan subgenus Pandora was redescribed (organized in the genus Zoophthora Batko) by Ento- and raised to a generic rank. Originally, 16 species were mophthora aphidis Hoffman in Fresenius as Zoophthora included in this genus typified by Pandora neoaphidis (Pandora) aphidis (HoffmaninFresenius)Batko. (Remaudi`ere et Hennebert) Humber. However, Balazy After nearly 100 years, Remaudiere` & Hen- (1993) suggested to maintain the subgenus Neopan- nebert (1980) successfully rediscovered Hoffman’s fun- dora Ben-Ze’ev et Kenneth as one of four subgenera of gus and demonstrated that Thaxter misapplied the the genus Zoophthora Batko. The subgenus Neopandora Hoffman’s specific name. The taxonomists used the was formerly proposed by Ben-Ze’ev & Kenneth presence of capilliconidia in the rediscovered Hoff- (1982b) as a subgenus of the genus Erynia Nowakowski, mann’s E. aphidis and not in the Thaxter’s fungus to emend. Humber et Ben-Ze’ev. Balazy (1993) syn- place these two fungi into separate genera. The two onymized one species with a new one, excluded two different species were redescribed, the Thaxter’s mis- species and added another ten new species to the sub- applied species under a new name as Erynia neoaphidis genus. Keller & Petrini (2005) states 31 species of Remaudi`ere et Hennebert, and the Hoffmann’s E. the genus Pandora. Out of the group of species five aphidis as Zoophthora aphidis (HoffmaninFresenius) are pathogenic to aphids. Four species are obligatory Batko. Humber & Ben-Ze’ev (1981) emended a sense pathogens of aphids, while one is a natural pathogen of of the genus Erynia Nowakowski, rejected Zoophthora plant- and leaf-hoppers but can artificially kill aphids as a separate genus for species forming capilliconidia, as well. and placed both those species in the same genus as Aphid-pathogenic Entomophthorales S549

Erynia neoaphidis Remaudi`ere et Hennebert and Ery- as a principal aphid enemy within the colonies of ce- nia aphidis (Hoffman in Fresenius) Humber et Ben- real aphids (e.g., Dean & Wilding, 1971; Dedryver, Ze’ev. Consequently, the description of the genus Ery- 1978, 1981, 1983; Coremans-Pelseneer et al., 1983; nia Nowakowski emend. Humber et Ben-Ze’ev was Papierok & Havukkala, 1986; Ozino et al., 1988; emended again and all recognized species of the genus Feng et al., 1990, 1992a; Feng & Nowierski, 1991; were reclassified into four subgenera. E. neoaphidis was Steenberg & Eilenberg, 1995; Basky & Hopper, transferred into a new subgenus Neopandora (Ben- 2000; Hatting et al., 1999a, 2000; Štalmachová & Ze’ev & Kenneth, 1982b). Humber (1989) revised Cagáň, 2000; Hemmati et al., 2001b; Abdel-Mallek the familial and generic criteria of the Entomophtho- et al., 2003; Abdel-Rahman & Ali, 2003), pea aphid rales and elevated subgenera of the genus Erynia to a (e.g., Wilding, 1975; Milner, 1982, 1985; Picker- generic rank. E. (Neopandora) neoaphidis was allocated ing et al., 1989a; Pickering & Gutierrez, 1991; toanewgenusPandora. Finally, Balazy (1993) reclas- Behrens, 1993; Cagáň & Barta, 2001; Majchrow- sified again the species into the subgenus Neopandora icz, 2001), black bean aphid (e.g., Gustafsson, 1969; Ben-Ze’ev et Kenneth of the genus Zoophthora Batko Majchrowicz, 1979; Wilding & Perry, 1980), cab- as Zoophthora (Neopandora) neoaphidis (Remaudi`ere et bage aphid (Lowe, 1963, 1968; Sivčev, 1991, 1992), Hennebert) Balazy. black cherry aphid (Klingen & Jaastad, 2003), or As far as the morphological and physiological prop- green peach aphid (e.g., Shands et al., 1972; Elkass- erties are concerned, the species exhibits a certain de- abany et al., 1992; Kish et al., 1994; McLeod et al., gree of variability (Keller, 1991; Barta, 2004). This 1998; McLeod & Steinkraus, 1999; Dara & Semt- taxon is usually considered to be a species complex ner, 2001). (Humber, 1983; Balazy, 1993), though there is no The fungus can be easily isolated and cultivated evidence for this (Humber, 1991). Recent molecular on solid media based on Sabouraud’s dextrose agar en- analysis failed to yield intraspecific polymorphisms in riched with cow’s milk and egg yolk (Keller, 1991) P. neoaphidis isolates (Tymon et al., 2004), which is as well as in liquid media (Latgé et al., 1983; Li et in contrast to the results of Rohel et al. (1997) or al., 1993). The species probably does not produce rest- Sierotzki et al. (2000). ing spores as they have never been observed. There is P. neoaphidis is a common aphid pathogen with a only a single unsupported report on resting spores in distribution throughout the world, although it is less P. neoaphidis produced in vitro (Uziel & Kenneth, frequent in tropical regions. It has been long inves- 1986). tigated as a potential biological control agent (Pell P. neoaphidis is greatly regarded as a potential et al., 2001). In Poland, the fungus was recorded in agent for biological control of aphid populations and 23 aphid species in many localities all over the coun- several attempts have been made to introduce the fun- try (Balazy, 1993). In Slovakia, the fungus infected gus into the aphid populations in laboratory or field 48 aphid species, which made 73% of all the aphid (e.g., Wilding, 1981; Latteur & Godefroid, 1983; species found during a survey to be susceptible to the Wilding et al., 1986a, b, 1990; Shah et al., 2000a). Entomophthorales (Barta & Cagáň, 2006). In Fin- However, a mass production, an inoculum storage, and land, the fungus was recorded in nine aphid hosts an inoculum formulation of the fungus are still obsta- (Papierok, 1989). A period of pathogen’s appear- cles of its commercial use (Dedryver, 1981; Latgé et ance in the nature is usually from the second half al., 1983; Shah et al., 1998, 1999, 2000b). Recently, a of May till the first frost in the autumn (Balazy, new prospective substrate to produce the granular cul- 1993; Barta & Cagáň, 2006). In Switzerland, the tures of P. neoaphidis has been designed (Li & Feng, pathogen was identified from 23 aphid species (Keller, 2003). 1991) and it was considered to be the most important aphid pathogenic fungus (Keller & Suter, 1980). 2. Pandora nouryi (Remaudi`ere et Hennebert) The taxon Entomophthora aphidis was recorded in Humber 92 aphid species in France (Thoizon, 1970). How- Mycotaxon 34: 453 (1989) ever, a list of records was prepared before the re- vision of the taxon by Remaudiere` & Hennebert (1980) and the erection of new species E. neoaphidis. S: Erynia exitialis Hall et Dunn sensu Gustafsson 1965, P. neoaphidis infects a great diversity of aphid species, Lantburkshögskolans Annaler 32: 102–212 (1965) including some important pestiferous species, and is B: Erynia nouryi Remaudi`ere et Hennebert, Mycotaxon 11: generally considered to be the most important agent 313–316 (1980) responsible for fungal epizootics in the host popula- Zoophthora nouryi (Remaudi`ere et Hennebert) Ben-Ze’ev et Leatherdale Thoizon Mil- Kenneth, Phytoparasitica 9: 40–41 (1981) tions (e.g., , 1970; , 1970; Erynia subgenus Neopandora nouryi Remaudi`ere et Hen- ner Mi˛etkiewski van der Geest , 1981a; & , 1985; nebert ex Ben-Ze’ev et Kenneth, Mycotaxon 14: 462 (1982) Humber, 1991; Keller, 1991; Balazy, 1993; Steen- Zoophthora subgenus Neopandora nouryi (Remaudi`ere et berg & Eilenberg, 1995; Eilenberg, 2002; Barta Hennebert) Ben-Ze’ev et Kenneth sensu Balazy, Flora of & Cagáň, 2006). This species is regularly recorded Poland. Fungi (Mycota) 24: 195 (1993) S550 M. Barta & Ľ. Cagáň

Until 1980 this species was not separated from the The pathogen was described from the aphid Acyrtho- collections identified as “Entomophthora aphidis”in siphon kondoi (a type host for the species) in Aus- the sense of Pandora neoaphidis or as “Entomophthora tralia in 1977 and named Erynia kondoiensis Milner exitialis”. Remaudiere` & Hennebert (1980) revised by Milner et al. (1983). The taxon was successfully these groups of pathogens and erected a new species separated from an aphidicolous group closely related to Erynia neoaphidis to include collections previously re- P. neoaphidis. Apart from differences in the morphol- ferred to as Entomophthora aphidis Hoffman in Fre- ogy and the fatty acid content, it may be distinguished senius sensu Thaxter. Entomophthora exitialis Hall et from P. neoaphidis, P. nouryi,andP. delphacis by spe- Dunn (Hall & Dunn, 1957) was regarded as a nomen cific enzyme mobility. P. kondoiensis is very similar confusum and consequently rejected (Remaudiere` & to P. neoaphidis but has smaller primary spores and Hennebert, 1980). Those collections of E. exitialis the mycelium contains a much higher proportion of C having ovoid primary spores less than 18 µminlength 12:0 fatty acids. In Australia, P. kondoiensis causes epi- and forming resting spores (i.e., Entomophthora exi- zootics in the A. kondoi populations, but it was rarely tialis Hall et Dunn sensu Gustafsson) were renamed observed in the sympatric Acyrthosiphon pisum Harris, Erynia nouryi Remaudi`ere et Hennebert. Subsequently, 1776. Conversely, A. pisum was frequently infected with this revision was accepted by other taxonomists (Re- P. neoaphidis. It was suggested that the fungus could be maudiere` & Keller, 1980; Humber & Ben- Ze’ev, imported into New Zealand and the USA for the biolog- 1981; Milner, 1981a; Milner et al., 1983). During ical control of A. kondoi. Other aphid species, A. pisum, next years, the taxon was reclassified and removed to Therioaphis trifolii Monell, 1882, Hyperomyzus lactu- the genus Pandora (Humber, 1989) or Zoophthora in cae (L., 1758) and Myzus persicae Sulzer, 1776, were the subgenus Neopandora (Balazy, 1993). also infected artificially (Milner et al., 1983; Balazy, A new species, Entomophthora terrestris Gres 1993). et Koval, was described from Pemphigus fuscicornis Outside of Australia, the pathogen was described (Koch, 1857) in Ukraine (Gres & Kovaľ, 1982). This in China from M. persicae where it occurred epizoot- incompletely described species resembles P. nouryi. ically in the aphid population on tobacco (Fan et al., Keller & Petrini (2005) accepted this taxon and pro- 1991). posed a new combination, Pandora terrestris (Gres et Cultures of the fungus can be established on media Koval) S. Keller. containing Sabouraud’s agar, egg yolk and milk (Mil- P. nouryi is known from Europe, the USA, and ner et al., 1983). Australia. It was mainly identified on the root dwelling Pemphigus bursarius (L., 1758), but also from the leaf 4. Pandora uroleuconii Barta et Cagáň aphids Aphis fabae Scopoli, 1763 and Aphis umbrella Mycotaxon 88: 79–80 (2003) (Börner, 1950). The first aphid species is considered as the typical host (Gustafsson, 1965; Milner, 1981a; This fungal species was described from the cadavers of Balazy, 1993). In Poland, the fungus was identified Uroleucon aeneum (Hille Ris Lambers, 1939) in Slo- from P. bursarius and Pemphigus phenax Börner et vakia. The fungus was regular pathogen of the aphid in Blunck, 1916 (Balazy, 1993; Majchrowicz, 1999). In late spring and early summer, although the epizootic Slovakia, the species infected natural colonies of Aphis level of mycosis was not observed (Barta & Cagáň, acetosae L., 1761 (Barta & Cagáň, 2006). Generally, 2003a). Despite some characteristic features in exter- P. nouryi is a rare entomopathogen in the aphid pop- nal symptoms of disease, P. uroleuconii is microscop- ulations (Remaudiere` & Hennebert, 1980). Hum- ically very similar to P. neoaphidis and other species ber (2001) states isolates of P. nouryi in “USDA-ARS closely related to P. neoaphidis,namelyP. delphacis. Collection of entomopathogenic fungal cultures” with The pathogen is distinguished from the other species origins in France [on Therioaphis maculata (Buckton, of the genus Pandora by absence of rhizoids, different 1899)] and the USA (on aphids from potatoes), or Aus- size of primary conidia, growth on standard media and tralia (on Acyrthosiphon kondoi Shinji, 1938). probably high host specialization. The fungus develops in cultures on media contain- ing Sabouraud’s agar, egg yolk and milk (Gustafsson, 5. Pandora delphacis (Hori) Humber 1965; Remaudiere` & Hennebert, 1980; Milner et Mycotaxon 34: 452 (1989) al., 1983; Balazy, 1993). B: Entomophthora delphacis Hori, Entomol. Mag. (Tokyo) 3. Pandora kondoiensis (Milner) Humber 3: 81 (1906) Mycotaxon 34: 453 (1989) Erynia delphacis (Hori) Humber, Mycotaxon 13: 212 (1981) Zoophthora subgenus Neopandora delphacis (Hori) Balazy, Flora of Poland, Fungi (Mycota) 24: 183–185 (1993) B: Erynia kondoiensis (Milner), in Milner, Mahon et Brown, Austral. J. Bot. 31: 183 (1983) The species Pandora delphacis was first described as Zoophthora subgenus Neopandora kondoiensis (Milner) Balazy, Entomophthora delphacis Hori and isolated from its Flora of Poland, Fungi (Mycota) 24: 195–196 (1993) hosts Nilaparvata lugens (St˚al) and Sogatella furcifera Aphid-pathogenic Entomophthorales S551

(Horváth, 1899) (Homoptera: Delphacidae) in Japan at Genus: Erynia (Nowakowski ex Batko) Re- the beginning of the last century (Hori, 1906). Aphids maudi`ere et Hennebert emend. Humber do not belong to the ecological host range of the species, Mycotaxon 34: 448–449 (1989) but they can be attacked after artificial inoculation with pathogen’s conidia (Shimazu, 1977). S: Zoophthora subgenus Erynia Nowakowski ex Batko, Acta P. delphacis bears a strong morphological resem- Mycol. 2: 18 (1966) blance to P. neoaphidis, but differs in a complete Erynia (Nowakowski ex Batko) Remaudi`ere et Hennebert, lack of rhizoids on any of its hosts. Further, the iso- Mycotaxon 11: 301 (1980) lates of P. delphacis grow considerably more rapidly Erynia subgenus Erynia Batko ex Ben-Ze’ev et Kenneth, on a wider variety of nutritionally simple or complex Mycotaxon 14: 459 (1982) media and sporulate more intensively over a longer This generic name was first published in a report on period of time than any cultures of P. neoaphidis. the meeting of Polish physicians and biologists by These species also differ in a range of their hosts Nowakowski in 1881. Nowakowski later rejected this Humber and in mobility of specific enzymes ( , 1981, generic name in favour of Brefeld’s Entomophthora Ben-Ze’ev Kenneth Milner 1991; & , 1982b; et (Humber, 1981, 1989). Believing that Nowakowski’s Remaudiere` Hennebert al., 1983). & (1980) con- later disuse of Erynia removed this name in terms sidered the differences in growth of cultures between of nomenclatural priority, Batko (1964b, 1966b) pro- those two species to be insignificant and regarded posed the genus Zoophthora consisting of four sub- Erynia delphacis (= P. delphacis)asanomen du- genera, with Erynia as one of them. Remaudiere` Remaudiere` bium. This was fully accepted by & & Hennebert (1980) redefined Erynia Nowakowski, Keller (1980) as well. P. delphacis was not men- altering both Nowakowski’s generic description and Batko tioned by Batko in any of his papers ( 1964b, Batko’s subgeneric one. Consequently, the description c, 1966b) and this species was properly allocated to of Erynia Nowakowski was emended again (Humber Ben-Ze’ev the genus Erynia (subgenus Erynia)by & Ben-Ze’ev, 1981; Ben-Ze’ev & Kenneth, 1982b). Kenneth Humber & (1982b). (1989) in the sense of Whereas Humber (1989) concluded that the genus his emendations placed the fungus in the genus Pan- Erynia Nowakowski was a nomen nudum and had el- dora. evated all four Batkoan subgenera of Zoophthora to At present, the fungus is primarily known as a generic rank, Keller (1991) maintained the systematic pathogen of leaf- and planthoppers in rice paddies arrangement proposed by Remaudiere` & Hennebert in Southeast Asia and Indonesia where the pathogen (1980). On the other hand, Balazy (1993) supported causes regularly epizootics in the host populations preserving the Batko’s Zoophthora genus with the sub- Li Holdom Narayanasamy ( , 1988; et al., 1989; et genera based upon Ben-Ze’ev & Kenneth (1982b). Ambethgar Matsui al., 1992; , 1996; et al., 1998). At present, the genus in the sense of Humber includes 14 However, the pathogen has recently been recorded different species altogether (Humber, 1989; Balazy, in Spissistilus festinus (Say, 1830), a member of the 1993; Keller & Petrini, 2005). family Membracidae (Homoptera), in North Amer- Miller Harper ica ( & , 1987). Several strains of 6. Erynia erinacea (Ben-Ze’ev et Kenneth) Re- the fungus were isolated from Empoasca fabae (Har- maudi`ere et Hennebert ris) (Homoptera: Cicadellidae) in the USA and ci- Mycotaxon 11: 302 (1980) cadellids in Brazil (Humber, 2001). Balazy (1993) found a fungus of identical morphology on a spec- B: Zoophthora erinacea Ben-Ze’ev et Kenneth, Mycotaxon imen of Auchenorrhyncha (= Cicadoidea) in Ger- 10: 219–232 (1979) many. The most recent information says that P. del- Erynia subgenus Erynia erinacea (Ben-Ze’ev et Kenneth) phacis is capable to attack some species of the or- Remaudi`ere et Hennebert ex Ben-Ze’ev et Kenneth, Myco- taxon 14: 459 (1982) der Lepidoptera besides the homopterans. There is a Zoophthora subgenus Erynia erinacea Ben-Ze’ev et Kenneth record of a successful isolation of P. delphacis from sensu Balazy, Flora of Poland, Fungi (Mycota) 24: 155–156 the non-homopteran species Mamestra brassicae (L., (1993) 1758) (Lepidoptera: Noctuidae) in Japan (Humber, 2001). ThistaxonwasdescribedinAphis craccivora Koch, This fungus is able to infect some aphid species in 1854 on alfalfa in Israel in 1977 and 1978 and named laboratory as well (Shimazu, 1977; Milner et al., 1983; Zoophthora erinacea Ben-Ze’ev et Kenneth. It also Xu & Feng, 2000, 2002). Though it has never been caused different levels of mortality in the popula- observed infecting aphids in nature, the results of labo- tions of Aphis umbrella (Börner, 1950) and M. persi- ratory experiments showed even greater pathogenicity cae inhabiting Malva sp. and in the A. fabae popu- of P. delphacis for M. persicae than pathogenicity of lations in several localities. The aphid species Aphis P. neoaphidis isolates (Xu & Feng, 2002). The results spiraecola Patch, 1914 was successfully inoculated by indicate that P. delphacis has a potential as a valuable this pathogen in laboratory (Ben-Ze’ev & Kenneth, fungal agent for the control of aphids. 1979). According to the morphological features of the S552 M. Barta & Ľ. Cagáň taxon, Humber (1989) allocated it to the genus Ery- S: Zoophthora subgenus Zoophthora Batko, Acta Mycol. 2: nia and Balazy (1993) to the genus Zoophthora sub- 16–18 (1966) genus Erynia. The fungus was not recorded in any Erynia subgenus Zoophthora Ben-Ze’ev et Kenneth, Myco- other country until it was identified in A. umbrella taxon 14: 466–469 (1982) in Slovakia and Austria in 2003 (Barta & Cagáň, Zoophthora subgenus Zoophthora Batko (sensu Balazy 1993), Flora of Poland, Fungi (Mycota) 24: 147–149 (1993) 2006). Any attempts to isolate and cultivate the fungus The genus Zoophthora with its four subgenera (includ- on egg yolk medium and several rich agar media failed. ing the subgenus Zoophthora Batko synonymized here However, it started to grow very slowly on egg-yolk, with the genus Zoophthora (Batko) Remaudi`ere et Hen- Ben- which sustained the hope of future isolation ( nebert) was erected by Batko (1964b, 1966b) and Ze’ev Kenneth & , 1979). later revised by Remaudiere` & Hennebert (1980). Humber & Ben-Ze’ev (1981) emended a sense of the 7. Erynia conica (Nowakowski) Remaudi`ere et genus Erynia Nowakowski and rejected a separation of Hennebert Zoophthora Batko (sensu Remaudi`ere et Hennebert) Mycotaxon 11: 302 (1980) from the genus Erynia, which had been based on the presence of capilliconidia in the former one or on any B: Entomophthora conica Nowakowski,Pam.Akad.Umiej. other criteria. The genus Zoophthora was rejected as a Kraków, Wydz. Mat.-Przyr. 8: 155 (1883) later synonym for the genus Erynia and the species of Empusa subgenus Entomophthora conica Nowakowski ex Zoophthora were placed in Erynia. On the other hand, Thaxter, Mem. Boston Soc. Nat. Hist. 4: 186–187 (1888) an opinion to preserve the Batkoan intrageneric dif- Zoophthora conica (Nowakowski) Batko, Bull. Acad. Pol. ferentiation was practically adopted with some correc- Sci., Ser. Sci. Biol. 12: 404–405 (1964) Ben-Ze’ev Kenneth Zoophthora subgenus Erynia conica (Nowakowski) Batko ex tions by & (1982b). The my- Batko, Acta Mycol. 2: 18 (1966) cologists, however, accepted a priority of the genus Erynia subgenus Erynia conica (Nowakowski) Remaudi`ere Erynia Nowakowski over Zoophthora Batko and trans- et Hennebert ex Ben-Ze’ev et Kenneth, Mycotaxon 14: 459 ferred the genus Zoophthora Batko as a subgenus to (1982) Erynia Nowakowski emend. Humber et Ben-Ze’ev. Sev- Zoophthora subgenus Erynia conica (Nowakowski) Batko in eral years later Humber (1989) came to the conclusion Balazy, Flora of Poland, Fungi (Mycota) 24: 154 (1993) that the name Erynia Nowakowski is a nomen nudum and cannot be applied as a valid generic name. At the E. conica was described by Nowakowski at the end of same time he elevated all four Batkoan subgenera to th Humber the 19 century ( , 1989). Nowadays this fun- the generic rank. In this sense, the subgenus Zooph- gus is known as a common pathogen in members of thora was upgraded to genus as Zoophthora Batko with the families Culicidae and Chironomidae around lakes, 13 species and Zoophthora radicans (Brefeld) Batko was ponds and on river or brook banks just over the water accepted as a type species of the genus. In his mono- surface in Europe, North America, Asia, and Australia. graph Balazy (1993) emphasised that it was reason- In Poland, it was observed from May during the vege- able to maintain the genus Zoophthora sensu Batko Balazy tation season in a number of localities ( , 1993). (1964b) with its four subgenera as emended by Ben- In Switzerland, E. conica infected unidentified speci- Ze’ev & Kenneth (1982b) and described a further 14 mens of Chironomidae from mid-August until the end new species within the subgenus Zoophthora (classified Keller of October ( , 1991). in the genus Zoophthora). In the literature, there has been only one record of At present, the genus encompasses a homogenous E. conica from aphids until now. The pathogen En- group of 33 species (Keller & Petrini, 2005), all tomophthora conica was observed in France on five of which are pathogenic for insects, with seven ones aphid species: Aulacorthum solani Kaltenbach, 1843, pathogenic for aphids. Out of the seven pathogens, two Brachycaudus klugkisti (Börner, 1942), Myzus ascaloni- are rather wide polyphagous organisms, while the re- cus Doncaster, 1946, Myzus cerasi (F., 1775), and maining ones are obligatorily aphidophagous (Hum- Thoizon Paramyzus heraclei Börner, 1933 ( , 1970). ber, 1989; Keller, 1991; Balazy, 1993). However, those identifications are at least dubious since no other authorities have yet confirmed the aphi- 8. Zoophthora aphidis (HoffmaninFresenius) Waterhouse dophagous properties of the fungus. & Remaudi`ere et Hennebert Brady (1982) in their “Key to the species of Ento- Mycotaxon 11: 285–294 (1980) mophthora s. lato” also questioned about the ability of E. conica to infect aphids. B: Entomophthora aphidis Hoffman in Fresenius, Abh. Senckenb. Natforsch. Ges. 2: 208 (1858) Tarichium aphidis (Hoffman in Fresenius) Cohn, Beitr. Biol. Genus: Zoophthora (Batko) Remaudi`ere et Hen- Pflanz. 1: 84 (1870) nebert nec Zoophthora aphidis (Hoffman in Fresenius) Batko, Bull. Mycotaxon 11: 301 (1980) Acad. Pol. Sci., Ser. Sci. Biol. 12: 323–324 (1964) Aphid-pathogenic Entomophthorales S553

Erynia aphidis (Hoffman in Fresenius) Humber et Ben- of September and that of October in the populations Ze’ev, Mycotaxon 13: 509 (1981) of sexuales, epizootics and high mortalities being some- Erynia subgenus Zoophthora aphidis (Hoffman in Fresenius) timesobservedaswell(Keller, 1991). It was also no- Humber et Ben-Ze’ev ex Ben-Ze’ev et Kenneth, Mycotaxon ticed that aphids with sporulating fungus are fixed by 14: 467 (1982) rhizoids to the underside of leaves, whereas aphids con- Zoophthora subgenus Zoophthora aphidis (HoffmaninFrese- taining resting spores are mainly found around the buds nius) Remaudi`ere et Hennebert (sensu Balazy 1993), Flora of Poland, Fungi (Mycota) 24: 211–216 (1993) in the leaf shoulders or sometimes along the main veins of the leaves (Keller, 1991). In Slovakia, the fungus in- This species was for the first time described under fects natural colonies of four aphid species (Aphis pomi the name Entomophthora aphidis Hoffman in Frese- De Geer, 1773, A. corni, R. padi,andM. cerasi) dur- nius only from its brown spherical resting spores ob- ing October and it was not observed during spring or served inside the cadavers of Anoecia corni (F., 1775) summer (Barta & Cagáň, 2004, 2006). In general, in France. A conidial stage of this species was not the species is considered a rather uncommon pathogen found or described by Hoffman (Remaudiere` & Hen- with distribution in Europe (Milner, 1981a; Barta nebert, 1980). Thaxter (1888) used a name of Em- & Cagáň, 2006) and until 1991 it was regarded as pusa (Entomophthora) aphidis for the most common monophagous for A. corni. fungal pathogen of aphids that forms conidia, but rest- Z. aphidis has probably a low value for biological ing spores have not been observed yet. Thus, until 1980 control and no efforts to exploit the fungus in this way the name Entomophthora aphidis Hoffman in Fresenius have been published. However, the fungus is easy to sensu Thaxter was incorrectly applied to another un- isolate and cultivate on Sabouraud’s agars with yolk described fungus which is currently known under the and milk (Keller, 1991; Balazy, 1993), but resting name Pandora neoaphidis (Remaudi`ere et Hennebert) spores are not produced in culture (Balazy, 1993). Humber. In 1980, Remaudiere` & Hennebert (1980) rediscovered the Hoffman’s fungus and confirmed that 9. Zoophthora phalloides Batko Thaxter had misapplied the Hoffman’s specific name for Acta Mycol. 2: 7–13 (1966) a different species. The problem of nomenclatural sta- tus of this species was solved by describing a new taxon B: Erynia phalloides (Batko) Humber et Ben-Ze’ev, Myco- (E. neoaphidis) as well as by redescribing the Hoffman’s taxon 13: 505 (1981) fungus as Zoophthora aphidis (HoffmaninFresenius) Erynia subgenus Zoophthora phalloides (Batko) Humber et Batko. During the next few years a taxonomic position Ben-Ze’ev ex Ben-Ze’ev et Kenneth, Mycotaxon 14: 467 (1982) of Z. aphidis was changed by some authors (Humber & Ben-Ze’ev, 1981; Ben-Ze’ev & Kenneth, 1982b), or Z. phalloides was first described as a pathogen of aphids was maintained (Remaudiere` & Keller, 1980; Hum- by Batko in Poland (1966a) and since then it has also ber, 1989; Keller, 1991). Balazy (1993) proposed to been recorded in North America, Australia and New refuse the name Z. aphidis (HoffmaninFresenius)sensu Zealand (Glare et al., 1985). Batko classified this new Batko for this taxon since Batko (1966b) typified his taxon in his subgenus Zoophthora belongingtoagenus subgenus Pandora by the name Z. aphidis.Balazy as- of the same name. Since the pathogen was described it cribed a correct sense of this taxon to Remaudiere` & has also been treated as a member of the genus Ery- Hennebert (1980). nia (Humber & Ben-Ze’ev, 1981) or the subgenus E. aphidis Hoffman in Fresenius was also inaccu- Zoophthora of the genus Erynia (Ben-Ze’ev & Ken- rately applied to other aphid pathogens, Zoophthora neth, 1982b). At present, the species is classified in canadensis (MacLeod, Tyrrell et Soper) Remaudi`ere et the genus Zoophthora (Humber, 1989; Keller, 1991), Hennebert, as a result of misidentification, and Pan- which was elevated to this rank from the Batkoan sub- dora nouryi (Remaudi`ere et Hennebert) Humber, which genus Zoophthora by Humber (1989). Morphologically, was not distinguished from Pandora neoaphidis (Re- the species is very similar to other aphid pathogenic maudi`ere et Hennebert) Humber. The problem was members of Zoophthora such as Z. radicans, Z. aphidis, solved by Remaudiere` & Hennebert (1980). and Z. occidentalis. However, some work was done to di- This pathogen is distributed in Western and Cen- vide isolates of broadly defined Z. radicans and Z. phal- tral Europe in aphids of the genus Anoecia infest- loides using spore dimensions, host specificity, growth ing leaves of Cornus sp. in autumn. The fungus was in vitro, or analyses of isoenzymes and fatty acid com- observed in several localities in Poland in 1933 and position as distinguishing criteria (Glare et al., 1987). 1980 (Balazy, 1993). However, attempts to find this The taxonometric analyses confirmed that these two pathogen in the populations of Anoecia sp. on Cornus species are distinct and also supported the traditional sanguinea L. in Poland, Romania, Eastern France or criterion for separating these species, the morphology Germany were unsuccessful for two decades (Balazy, of primary conidia, as satisfactory. The authors fur- 1993). In Switzerland, the pathogen was identified from ther divided the Z. phalloides isolates into two groups two aphid species, Anoecia corni and Rhopalosiphum on the basis of their geographical origins, either Euro- padi (L., 1758). Z. aphidis was found between the end pean or North American ones. According to Balazy S554 M. Barta & Ľ. Cagáň

(1993), Batko’s incomplete description of the taxon as onymous with E. radicans. Batko (1964b) and Re- well as inconsistencies between measurements originally maudiere` & Hennebert (1980) concluded that E. presented by Batko and that obtained by Balazy from sphaerosperma was an invalid name according to the the holotype material bring confusions into a reliable rules of the International Code of Botanical Nomen- identification of the taxon. Thus Balazy (1993) rec- clature. Thus, its first validly applied name was Em- ommended a careful re-examination of all the accessible pusa radicans Brefeld. Many efforts were made to split materials to solve this problem. the many-hosted E. sphaerosperma into more consistent Z. phalloides is an obligatorily aphidophagous fun- species or subspecies. Turian (1957) proposed two new gus and seems to be widespread but not common. As subspecies: E. sphaerosperma subspecies cicadelliphaga mentioned above, the species was described in Poland attacking cicadas and subspecies elateridiphaga attack- from the nettle aphid Microlophium carnosum (Buck- ing Elateridae (Coleoptera). Batko (1964b) split E. ton, 1876) (Batko, 1966a). Until now, it has been ob- sphaerosperma into two new combinations: Zoophthora served in Central and Western Europe, Israel, North radicans (Brefeld) Batko as a synonym for Brefeld’s America and Australia in different aphid species (Ben- E. radicans and Zoophthora phytonomi (Arthur) Batko Ze’ev et al., 1984; Glare et al., 1985). Remaudiere` (syn. Entomophthora phytonomi Arthur). Concurrently, et al. (1976b, 1981) reported the pathogen in aphids he used the former species as a type for his genus and in France. In Switzerland, the species was collected be- subgenus Zoophthora. In France, Remaudiere` et al. tween June and October from A. pisum, Macrosiphum (1976a) studied Entomophthora isolates belonging to rosae (L., 1758), Metopolophium festucae Theobald, the “sphaerosperma group” and concluded that the fun- 1917 and R. padi. It was never found in aphids in an- gus should be restricted to those isolates which attack nual crops but in meadows and natural sites (Keller, lepidopterous larvae, Diptera and other orders. And the 1991). In Denmark, the pathogen infected colonies of Entomophthora isolates from aphids were identified by R. padi on the winter host and not colonies on cere- the authors as morphologically similar to Entomoph- als (Nielsen et al., 2001a). Existence of differences in thora phalloides Batko. Using the same taxonomic prin- yearly life cycle of the pathogen compared to other ento- ciples as Remaudiere` et al. (1976a) did and follow- mophthoralean fungi identified on cereal aphids in Den- ing Batko’s classification (with some restrictions), Ben- mark was pointed out. A survey of entomopathogens Ze’ev & Uziel (1979) found that Z. radicans attack- in aphid populations infesting natural vegetation in ing the yellow pecan aphid, Monellia costalis (Fitch, France showed that Z. phalloides is better adapted to 1855), had the same spore dimensions and morphology cooler spring and autumn conditions and is rare during as E. sphaerosperma sensu stricto (Remaudiere` et al., hot summer period (Remaudiere` et al., 1981). Glare 1976a). Various morphological, physiological and bio- et al. (1986b) also stressed the pathogen’s adaptation to chemical criteria were studied by Glare et al. (1987) cool and moist climates. Records of the pathogen from in order to distinguish reliably the species Z. phal- Elatobium abietinum (Walker, 1849) in Iceland (Aus- loides and Z. radicans. Despite the taxon separations tar˚a et al., 1997) might support the opinion about its described above, the species is still considered to be a preference to cooler climate. species complex (Humber, 1983, 1991; Balazy, 1993). Fungus can be easily cultivated on artificial media Balazy (1993) made thorough studies on morphology, containing Sabouraud’s agar with milk, egg yolk, and biology and particularly host specificity of the genus yeast extract (Glare et al., 1987). Zoophthora, which enabled him to distinguish and de- scribe seven new species morphologically similar to Z. 10. Zoophthora radicans (Brefeld) Batko radicans. Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 324 (1964) Z. radicans is a very common and widespread species. As mentioned above, it has a wide host range B: Empusa radicans Brefeld, Bot. Ztg. 28: 186 (1870) including species of the orders Lepidoptera, Diptera, Entomophthora radicans Brefeld, Bot. Ztg. 35: 345 (1877) Homoptera, Coleoptera, Orthoptera, Thysanoptera, Erynia radicans (Brefeld) Humber, Ben-Ze’ev et Kenneth Milner Soper in Humber et Ben-Ze’ev, Mycotaxon 13: 509 (1981) Hemiptera, and Hymenoptera (e.g., & , Soper Ward Papierok Erynia subgenus Zoophthora radicans (Brefeld) Humber, 1981; & , 1981; et al., 1984; Ben-Ze’ev et Kenneth ex Ben-Ze’ev et Kenneth, Mycotaxon Glare & Milner, 1991; Keller, 1991). Although Z. 14: 467 (1982) radicans is recorded from numerous insect groups, the literature suggests that individual isolates are better Z. radicans was originally described as a pathogen of adapted to infecting taxonomically related insect hosts caterpillars, Pieris brassicae (L., 1758), by Brefeld (Papierok et al., 1984; Goettel et al., 1990). Some (1870) under the name Empusa radicans Brefeld. A strains are not able to infect species of the orders other similar species attacking leafhoppers, Entomophthora than that they were isolated from (e.g., Papierok et sphaerosperma, was described by Fresenius in 1858 and al., 1984; Magalhaes˜ et al., 1988). However, some redescribed by Thaxter (1888). The fungi reported studies identified isolates with seemingly broader host for many years under the name E. sphaerosperma Fre- ranges (Poprawski et al., 1992; Furlong & Pell, senius in different host species were treated as syn- 1996). According to Balazy’s opinion (Balazy, 1993), Aphid-pathogenic Entomophthorales S555 this species is a specialized pathogen infecting caterpil- Z. canadensis is known only from the populations lars of Pieris spp., and the application of this name to of the aphid S. piniradiatae in red pine plantations in pathogens of other insects is either incorrect or at least Ontario (Canada). doubtful. Culture of the pathogen was established on co- This fungus was recorded on many aphid species agulated hen’s egg yolk and Grace’s insect tissue cul- all over the world. It infects cereal aphids (e.g., Feng ture medium. However, growth on both media was ex- et al., 1990; Feng & Nowierski, 1991), aphids in tremely slow with no conidia or resting spores produced potato fields (Shands et al., 1972), or pea aphid (Pick- (MacLeod et al., 1979). ering et al., 1989a) in the USA and green peach aphid or cabbage aphid in Slovakia (Barta & Cagáň, 12. Zoophthora occidentalis (Thaxter) Batko 2006). Several aphid species were killed by the fungus Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 404 (1964) in Israel (Kenneth & Olmer, 1975; Ben-Ze’ev & Uziel Thoizon , 1979), in France ( , 1970), in Switzer- B: Keller Abdel-Mallek Empusa subgenus Entomophthora occidentalis Thaxter, land ( , 1991), and in Egypt ( Mem. Boston Soc. Nat. Hist. 4: 170–171 (1888) et al., 2003). It was also recorded in the black bean Erynia occidentalis (Thaxter) Humber et Ben-Ze’ev, Myco- aphid in Sweden (Gustafsson, 1969) and the cabbage taxon 13: 509 (1981) aphid in Yugoslavia (Sivčev, 1991, 1992). Erynia subgenus Zoophthora occidentalis (Thaxter) Hum- Z. radicans has a great potential for biological ber et Ben-Ze’ev ex Ben-Ze’ev et Kenneth, Mycotaxon 14: control. Isolates of this species were released against 467 (1982) the potato leafhopper, Empoasca fabae in Illinois (the USA) and epizootics occurred (McGuire et al., 1987a, Thaxter (1888) described a fungus infecting aphids b). The pathogen (an isolate from Israel) was also re- on gray birch, Betula populifolia Marsh, as Em- leased in Australia, where the fungus had never been pusa subgenus Entomophthora occidentalis Thaxter. recorded before, against the spotted alfalfa aphid The- He recorded this pathogen as frequently and com- rioaphis trifolii f. maculata. The fungus established well monly infecting the aphids in Maine (the USA). Batko in the area and was able to survive as resting spores en- (1964c) renamed this species as Zoophthora occidentalis abling it to persist from year to year and subsequently (Thaxter) Batko and placed it in the subgenus Zooph- spread on its own (Milner & Soper, 1981; Milner et thora (Batko, 1966b). Since further notes on the oc- al., 1982; Milner & Mahon, 1985). currence of this pathogen were scarce and, moreover, Culture of this species can be established on the biological and morphological characteristics of Z. Sabouraud’s agar supplemented with milk and egg yolk phalloides closely resembled those of Z. occidentalis, (Keller, 1991; Balazy, 1993). Mi˛etkiewski et al. (1981) published a comprehen- sive study to compare conidial morphology and mea- 11. Zoophthora canadensis (MacLeod, Tyrrell et surements of both the fungi. Balazy (1993) suggested Soper) Remaudi`ere et Hennebert to make a revision of all the materials concerning the Mycotaxon 11: 301 (1980) aphids identified as Entomophthora sphaerosperma, Z. radicans, Z. occidentalis and Z. phalloides,andtocom- pare these morphologically similar species. B: Entomophthora canadensis MacLeod, Tyrrell et Soper, Can. J. Bot. 57: 2663–2672 (1979) The species is distributed in North America, South Erynia canadensis (MacLeod, Tyrrell et Soper) Humber et America, Europe and Asia in several aphid species, Ben-Ze’ev, Mycotaxon 13: 509 (1981) mostly unidentified ones (Balazy, 1993). Z. occiden- Erynia subgenus Zoophthora canadensis (MacLeod, Tyrrell talis was the most common pathogen of the aphid M. et Soper) Humber et Ben-Ze’ev ex Ben-Ze’ev et Kenneth, persicae in Orono, Maine (the USA) in 1977 and it was Mycotaxon 14: 467 (1982) also collected from A. fabae on pigweed, Chenopodium album L. A morphologically similar strain was also This pathogen was first found in the populations of found in Poland in the aphids on Deschampsia ceaspi- the woolly pine needle aphid, Schizolachnus piniradia- tosa (L.) P. B. in the forest undergrowth (Mi˛etkiewski tae (Davidson, 1909) in Canada. During a study on life et al., 1981). Batko (1962) listed this pathogen among history and ecology of the aphid, a number of mycosed Polish entomopathogens from an aphid on nettle, Ur- aphids were observed in the summer of 1960. The fungal tica dioica L. However, it was only found in a few pathogen was incorrectly identified as Empusa subgenus aphids in one locality. Some individuals of aphids were Entomophthora aphidis Hoffman in Fresenius (Grob- killed by the pathogen in Israel (Ben-Ze’ev et al., ler et al., 1962). A continuing research into biology 1984) and Chile (Aruta & Carvillo, 1989). E. oc- of the aphid pathogen revealed the misidentification cidentalis was also reported from Anoecia sp. in France and a new fungal species, Entomophthora canadensis (Thoizon, 1970) and three individuals of Sitobion ave- MacLeod, Tyrrell et Soper was described (MacLeod et nae (F., 1775) were recorded to die from Z. occiden- al., 1979). One year later, Remaudiere` & Hennebert talis infection in south-west Idaho (the USA) (Feng (1980) classified this taxon in the genus Zoophthora. et al., 1990). Balazy (1993) states further three aphid S556 M. Barta & Ľ. Cagáň species, Capitophorus similis van der Goot, 1915, Im- In the past the generic name Entomophthora was patientium asiaticum (Nevsky) and Sitobion fragariae applied to practically all entomophthoralean species (Walker, 1848), as hosts for Z. occidentalis in Poland except Massospora Peck. Fresenius was the first to and Barta & Cagáň (2006) state one aphid species, use the name for the entomopathogenic fungi (Fre- Uroleucon tanaceti (L., 1758), infected by the pathogen. senius, 1856) instead of invalid Empusa (preoccu- Strains of this fungus develop and sporulate well on pied by the orchid Empusa Lindley) applied ear- artificial media containing egg yolk (Balazy, 1993). lier by Cohn (1855). The Nowakowski’s genus En- tomophthora (Nowakowski, 1883) was characterised 13. Zoophthora orientalis Ben-Ze’ev et Kenneth by branched conidiophores, and possession of both zy- Phytoparasitica 9 (1): 33–42 (1981) gospores/azygospores and cystidia. Thaxter (1888) suggested to use one genus, Empusa, for all insects at- B: Erynia orientalis (Ben-Ze’ev et Kenneth) Humber, Ben- tacking fungi. This genus comprised three subgenera, Ze’ev et Kenneth in Humber et Ben-Ze’ev, Mycotaxon 13: of which Entomophthora included the Nowakowski’s 509 (1981) species of Entomophthora. Batko (1964e) correctly pointed out that Entomophthora Fresenius is the later This pathogen is known only from the type locality in but valid synonym for the invalid generic name Em- Israel where it was observed and described from Aphis pusa Cohn, while Entomophthora Nowakowski or Em- Ben-Ze’ev Ken- citricola van der Goot, 1912 by & pusa (Entomophthora) Thaxter cannot be synonymized neth (1981). with the genus. Consequently, he restricted Entomoph- thora to three related species without rhizoids and with 14. Zoophthora anhuiensis (Li) Humber simple conidiophores producing bell-shaped oligonucle- Mycotaxon 34: 453 (1989) ate (< 10–12 nuclei) conidia. Finally, Remaudiere` & Keller (1980) emended Batko’s definition of Ento- B: Erynia anhuiensis Li, Acta Mycol. Sin. 5 (1): 1–6 (1986) mophthora s. str. (typified by Entomophthora muscae (Cohn) Fresenius) by removing the restriction regard- This fungus was briefly described as Erynia anhuien- ing rhizoids and transferred some species of the rejected sis from China where it caused epizootics in the pop- Culicicola genus to the Entomophthora s. str. (including ulations of the green peach aphid M. persicae in An- Entomophthora planchoniana). This modification was Li hui Province during late autumn and early winter ( , later fully accepted (Ben-Ze’ev & Kenneth, 1982a; Humber 1986). (1989) listed it among the species of the Humber, 1989; Balazy, 1993). Keller (1987a) listed Balazy genus Zoophthora in his sense. (1993) regards 11 species in this genus. Later, Balazy (1993) added the taxon as an intermediary form between Zooph- another two species. Since then four new species have thora and Furia as far as primary conidia and rhizoids been described and Keller (2002) has reviewed the are concerned, and between Zoophthora and Erynia in genus recently and five new species have been charac- terms of pseudocystidia. This species has never been terized. A total number of Entomophthora species is 21 Feng reported in other countries ( et al., 1998). (Keller & Petrini, 2005), with two aphid pathogenic Results of a bioassay of this fungus against M. per- species. sicae indicated that the pathogen could be a promising agent for microbial control and competitive with other 15. Entomophthora planchoniana Cornu Feng Xu Zoophthora species ( et al., 1998; et al., 2000). Bull. Soc. Bot. France 20: 189 (1873) Based on laboratory observations, the fungus is most adaptive to cool climate or cooler seasons with the best ◦ nec Empusa planchoniana (Cornu)? ex Thaxter, Mem. effectiveness at 15 C(Feng & Li, 2003). Boston Soc. Nat. Hist. 4: 165–166 (1888) The fungus can be isolated and maintained on Feng Sabouraud-milk-egg yolk medium ( et al., 1998). E. planchoniana was first observed and described as a natural enemy of aphids on elderberry (Cornu, 1873) Genus: Entomophthora Fresenius sensu Re- in southern France. In point of fact, E. planchoniana is maudi`ere et Keller likely to be the first entomophthoralean fungus ever de- Mycotaxon 11: 332 (1980) scribed from the aphid host. Although Cornu (1873) gave no additional data on the fungus, at present it is S: Empusa Cohn, Nova Acta Acad. Caes. Leop.-Carol. considered one of the most important fungal pathogen Germ. Nat. Curios. 25: 301–360 (1855) (nec Empusa Lind- of aphids in Europe. It affects many aphid species in ley, Bot. Regn. Sub. T: 825 (1824)) dry and moderately humid habitats (Balazy, 1993). Entomophthora Fresenius, Bot. Ztg. 14: 882 (1856) [nec En- It does not occur in dense, humid crops and seems to tomophthora Nowakowski, Pam. Akad. Umiej., Wydz. Mat.- Przyr. 8: 175–176 (1883)] prefer dry habitats like tall plants, bushes, and trees Lamia Nowakowski, Pam. Akad. Umiej., Wydz. Mat.-Przyr. often causing epizootics (Keller, 1987a). During sum- 8 (1883) mer and autumn the species was found on 15 different Culicicola Nieuwland, Am. Midl. Nat. 4: 378 (1916) aphid species in plenty of localities in Poland (Balazy, Aphid-pathogenic Entomophthorales S557

1993) or on another 15 aphid species in Finland (Pa- tive exploitation of the fungus in biological control of pierok, 1989). In Switzerland, the fungus infected 16 aphids. aphid species (Keller, 1987a) and during a survey of aphid pathogens in South Africa the fungus infected six 16. Entomophthora chromaphidis Burger et different aphid species (Hatting et al., 1999a). Swain Generally, the fungus is distributed worldwide J. Econ. Entomol. 11: 288 (1918) but primarily in Europe where it causes epizootics within the aphid populations (Humber & Feng, B: Culicicola chromaphidis (Burger et Swain) Batko, Bull. 1991). E. planchoniana has been reported as an im- Acad. Pol. Sci., Ser. Sci. Biol. 12: 404 (1964) portant natural enemy of cereal aphids in Europe (e.g., Dean & Wilding, 1971; Kenneth & Olmert, 1975; This Entomophthora species infecting aphids was re- Dedryver, 1981, 1983; Coremans-Pelseneer et al., ported as a significant mortality factor of the walnut 1983; Papierok & Havukkala, 1986; Ozino et al., aphid Chromaphis juglandicola (Kaltenbach, 1843) dur- 1988; Steenberg & Eilenberg, 1995; Štalmachová ing a period of exceptionally hot weather in southern & Cagáň, 2000; Eilenberg, 2002), in South and California (Burger & Swain, 1918). For many years, North Africa (Hatting et al., 1999a, 2000; Abdel- E. chromaphidis was considered to be a synonym for Mallek et al., 2003), in Australia (Hall et al., 1976), E. planchoniana (Gustafsson, 1965; MacLeod et al., as well as in the USA (Feng et al., 1991). The fungus is 1976; Waterhouse & Brady, 1982) until these two also parasiting pea aphid (Wilding, 1975; Pickering species were definitely separated and justified (Hum- & Gutierrez, 1991), A. kondoi in Australia (Milner ber & Feng, 1991). E. chromaphidis occurring in et al., 1980), Phorodon humuli (Schrank, 1801) (Krej- North America and Australia is characterized by hav- zová, 1979), several aphids in potato fields in the USA ing smaller primary conidia and larger nuclei, whereas (Shands et al., 1972), A. fabae (Gustafsson, 1969; E. planchoniana is more widely distributed (with pri- Wilding & Perry, 1980; Barta & Cagáň, 2002) marily European distribution) and has larger primary as well as Brevicoryne brassicae (L., 1758) (Sivčev, conidia and smaller nuclei. E. chromaphidis has been 1991, 1992). The fungus was also observed in “non- successfully isolated and cultivated (Holdom, 1983; agricultural aphids”, including Drepanosiphum aceri- Humber & Feng, 1991). In spite of aforementioned dif- num (Walker, 1848) in Switzerland (Keller, 1987b) ferences, Balazy (1993) prefers not to separate those or Elatobium abietinum in Iceland (Austar˚a et al., taxons and preserves them under the name E. plan- 1997; Nielsen et al., 2001b), as well as in different choniana. Recently, genetic analysis could not sepa- aphid species in Denmark (Eilenberg, 2002). In Slo- rate it from E. planchoniana (Jensen & Eilenberg, vakia, the fungus infected 27 aphid species and has the 2001). Keller (2002) considers E. chromaphidis a same seasonal distribution as P. neoaphidis but with valid species. no epizootics found in the host populations (Štalma- In addition to the type host C. juglandicola,the chová & Cagáň, 2000; Barta & Cagáň, 2006). In fungus was also identified from Metopolophium dirho- this country, E. planchoniana was recorded individu- dum (Walker, 1849), S. avenae, Schizaphis graminum ally or together with other entomophthoralean species (Rondani, 1852), and Diuraphis noxia (Kurdjumov, as a component of parasitic mycoflora in aphid colonies. 1913) (Feng at al. 1990, 1991; Feng & Nowierski, The disease never reached a dominant position in the 1992; Wraight et al., 1993) and M. persicae (Kish et parasitic mycoflora (Barta & Cagáň, 2006). al., 1994) in the USA and from A. kondoi in Australia For many years, E. planchoniana has been re- (Holdom, 1983). This taxon has never been reported ported not to grow in vitro and this was the main from Europe. obstacle for consideration of its use in biological control. Only Holdom (1983) succeeded in isolat- Genus: Entomophaga Batko emend. Humber ing strains morphologically similar to E. planchoni- Mycotaxon 34: 447–448 (1989) ana and he believed the species had been E. plancho- niana. The strains were isolated in a liquid medium S: Entomophaga Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. based on foetal calf serum, glucose and neopeptone. 12: 325–326 (1964) However, the identification of the isolates was cor- rected later to Entomophtora chromaphidis Burger et This genus, based on Entomophthora grylli Fresenius, Swain (Humber & Feng, 1991). Recently, Freimoser was proposed by Batko (1964b) to include species et al. (2001) have been successful in isolation and with multinucleate spores and simple conidiophores in vitro cultivation of the aphid pathogenic fungus but without rhizoids. Batko (1974) tried to distin- for the first time. The strains were isolated from guish the species Conidiobolus and Entomophaga on Ovatus crataegarius (Walker, 1850) and cultivated the basis of trophic relations, saprophytism or facul- on Grace’s insect tissue cell culture medium supple- tative zoopathogenicity in the first versus arthropod mented with additional nutriments. Although the cul- parasitism in the second group. However, no sufficient tures did not sporulate, it raised hopes for prospec- criteria were provided to distinguish reliably species S558 M. Barta & Ľ. Cagáň placed in the genera Entomophaga, Culicicola Nieuw- the subdivision of the genus Entomophaga into En- land or the morphologically similar genus Conidio- tomophaga and Batkoa as proposed by Humber since bolus Brefeld. Due to lack of these criteria, all the he considered this separation to be premature due species of Entomophaga and some Culicicola species to insufficiently researched criteria and tansferred all were transferred to the genus Conidiobolus by Re- the Humber’s Batkoa species to Entomophaga Batko. maudiere` & Keller (1980). New criterion based on Several years later, however, Keller (1999) accepted nuclear morphology of the fungi propounded by Hum- the genus Batkoa Humber with the Humber’s species. ber (1981) did provide a suitable criterion to differen- The genus consists of six obligatorily entomopathogenic tiate the Conidiobolus species with ancylistoid nuclei species (Humber, 1989; Balazy, 1993; Keller, 1999; from those of Entomophaga with easily staining ento- Keller & Petrini, 2005). Despite the fact that all mophthoroid nuclei. Humber (1989) listed 10 species of the six species differ morphologically from Ento- under this generic name. Balazy (1993) recognizes 14 mophaga, the current results of molecular analyses Entomophaga species in his monograph (with 2 new demonstrate that Batkoa is closely related to Ento- species) and recently two Eryniopsis species were trans- mophaga (Hajek et al., 2003). Two species have been ferred to the genus (Hajek et al., 2003). At present, the recorded from aphids. genus includes 17 species (Keller & Petrini, 2005). There is only one species that exhibits aphidophagous 18. Batkoa apiculata (Thaxter) Humber properties. Mycotaxon 34: 446 (1989)

17. Entomophaga pyriformis (Thoizon) Balazy Flora of Poland. Fungi (Mycota) 24: 119 (1993) B: Empusa apiculata Thaxter, Mem. Boston. Soc. Nat. Hist. 4: 163–164 (1888) Lamia apiculata (Thaxter) Lakon, Z. Angew. Entomol. 5: B: Entomophthora pyriformis Thoizon, Entomophaga 12: 173–174 (1919) 303–307 (1967) Culicicola apiculata (Thaxter) Batko, Bull. Acad. Pol. Sci., Ser. Sci. Biol. 12: 404 (1964) Entomophthora apiculata (Thaxter) Gustafsson, Lantbruk- Among the fungus-infected aphids collected in dif- shogsk. Ann. 31: 131–133 (1965) ferent parts of France, Thoizon (1967) found spec- Conidiobolus apiculatus (Thaxter) Remaudi`ere et Keller, imens of Rhopalosiphum insertum (Walker, 1849) at Mycotaxon 11: 330 (1980) (nec Conidiobolus apiculatus La Verne being killed by species with pyriform coni- (Thaxter) Remaudi`ere et Keller in Keller, Sydowia 40: 128– dia. The author described the fungus and named it 130 (1987)) S: Entomophthora pyriformis Thoizon. Under laboratory Entomophaga domestica Keller, Sydowia 40: 141–143 (1987) conditions the pathogen also infected R. padi, S. ave- nae,andA. fabae. Thoizon (1970) also recorded this fungus in Forda formicaria von Heyden, 1837, Tetra- This species was found in various insect species col- neura ulmi (L., 1758), Linosiphon galii (Mamontova, lected in Maine and North Carolina in the USA. Thax- 1961) and R. insertum. This aphidophagous species is ter (1888) suggested a provisional name of Empusa known only from the type locality and has not been apiculata for the fungus until more complete descrip- observed by other mycologists in aphid colonies. The tion of another three species, to which the taxon was taxon was not mentioned in other comprehensive tax- closely allied, was reported. Lakon (1919) transferred onomic works (Waterhouse & Brady, 1982; Hum- the species to the genus Lamia. Lamia Nowakowski, ber, 1989; Ben-Ze’ev & Kenneth, 1982a) with the however, was a later homonym of Lamia Endl. In exception of Balazy’s and Keller’s one (Balazy, 1993; Sweden, Gustafsson (1965) found an entomophagous Keller, 2006). Balazy classified this species as comb. species, identified as Entomophthora apiculata,ona nov. in the genus Entomophaga. number of Diptera and Psocoptera species. However, E. pyriformis grows quite readily and may be cul- conidia were smaller than those of the Thaxter’s tured on Sabouraud dextrose agar and on egg yolk with strains and, in addition, microconidia were observed. glucose (Thoizon, 1967). MacLeod & Müller-Kögler (1973) believed that the Swedish strains were hardly allied to E. apiculata Genus: Batkoa Humber and they had related them to Conidiobolus pseudococci Mycotaxon 34: 446–447 (1989) (Speare) Tyrrell et MacLeod and Conidiobolus corona- tus (Costantin) Tyrrell et MacLeod. Remaudiere` & Typified by Batkoa apiculata (Thaxter) Humber, the Keller (1980) revised the genus Conidiobolus and al- genus was established by Humber (1989) to distin- locted the species to this genus, classifying it as Co- guish certain species from the genus Entomophaga on nidiobolus apiculatus. Keller (1987a) misidentified a the basis of marked differences in the structure of pathogen of Diptera and larvae of Tenthredinidae (Hy- conidiophores and with the presence of rhizoids as menoptera) in Switzerland as C. apiculatus, a species supporting character. Keller (1987a, 1991) rejected with typical ancylistoid nuclei proving its affinity with Aphid-pathogenic Entomophthorales S559 the genus Conidiobolus (family: Ancylistaceae). How- as well. After investigating the Thaxter’s authentic col- ever, Humber (1989) re-examined Thaxter’s collec- lections of E. apiculata var. major, Humber (1989) tion and stated that all the materials identified by classed this species with the new genus Batkoa (family Thaxter as Empusa apiculata, which had been consid- Entomophthoraceae) on the basis of entomophthoroid ered a basionym for C. apiculatus, had had entomoph- nuclei of the taxon and other morphological features. thoroid nuclei. Consequently, he transferred the Thax- He also stated that E. apiculata var. major was a ba- ter’s species to the family Entomophthoraceae as a type sionym for Conidiobolus major (Thaxter) Remaudi`ere of the new genus Batkoa and suggested describing the et Keller. Keller (1987a) misidentified a pathogen of Keller’s fungus as a new Conidiobolus species. Further, spittlebugs (Homoptera: Cercopidae) in Switzerland as anewtaxon,Entomophaga domestica Keller, described C. major, a species with typical ancylistoid nuclei prov- by Keller (1987a) in Switzerland as a pathogen of sev- ing its affinity with the genus Conidiobolus (family: An- eral Diptera and Coleoptera species in glasshouse was cylistaceae). Humber (1989) suggested a need to de- treated as a synonym for Batkoa apiculata (Humber, scribe the Keller’s species with features of C. major 1989). as a new Conidiobolus species and Humber simultane- B. apiculata is a polyphagous species. It has been ously synonymized Keller’s new species Entomophaga reported from all continents as a pathogen of adult limoniae Keller, entomopathogen of Diptera (Limoni- insects of different groups (Balazy, 1993). Thoizon idae) in Switzerland, with B. major. Keller (1991) (1970) reported the species Entomophthora apiculata in described new Conidiobolus species in view of Humber’s several aphid species in various parts of France as well. taxonomic corrections but persisted in his opinion that Altogether, three aphid species were collected being in- E. limoniae is a separate species. fected with this pathogen: Macrosiphoniella mutellinae This species is reported from plenty of localities Börner, 1950, S. avenae and R. padi. It is also the only in North and South America, Europe and Asia as a record of the pathogen from aphids. pathogen in several insect species of different orders The fungus develops and sporulates well on ar- (Balazy, 1993). The fungus was recorded from aphid tificial media, especially those containing egg yolk only in Sweden (Gustafsson, 1965). and Sabouraud’s dextrose agar (Gustafsson, 1965; The fungus grows and develops well on media Keller, 1987a; Balazy, 1993). containing egg yolk and Sabouraud’s dextrose agar (Gustafsson, 1965; Balazy, 1993). 19. Batkoa major (Thaxter) Humber Mycotaxon 34: 446 (1989) Family: Neozygitaceae Ben-Ze’ev et Kenneth in Ben-Ze’ev et al. S: Empusa apiculata var. major Thaxter, Mem. Boston. Soc. Mycotaxon 28: 313–326 (1987) Nat. Hist. 4: 164–165 (1888) Lamia apiculata var. major (Thaxter) Lakon, Z. Angew. En- Genus: Neozygites Witlaczil tomol. 5: 174 (1919) Arch. Mikrosk. Anat. 24: 599–603 (1885) B: Entomophthora major (Thaxter) Gustafsson, Lantburtk- shogsk. Ann. 31: 133–134 (1965) S: Empusa subgenus Triplosporium Thaxter, Mem. Boston Conidiobolus major (Thaxter) Remaudi`ere et Keller, My- Soc. Nat. Hist. 4: 152 (1888) cotaxon 11: 331 (1980) (nec Conidiobolus major (Thaxter) Triplosporium (Thaxter) Batko, Bull. Acad. Pol. Sci., Ser. Remaudi`ere et Keller in Keller, Sydowia 40: 132–134 (1987)) Sci Biol. 12: 324–325 (1964) This fungus observed on naturally infected adults of Neozygites Remaudi`ere et Keller, Mycotaxon 11: 331–332 (1980) Ptilodactyla serricollis (Say, 1823) (Coleoptera: Ptilo- Thaxterosporium Ben-Ze’ev et Kenneth in Ben-Ze’ev et al., dactylidae) collected in North Carolina, the USA was Mycotaxon 28: 323 (1987) described by Thaxter (1888) and named as Empusa apiculata var. major, a variant of the species E. apicu- The genus comprises a relatively homogenous group of lata. Gustafsson (1965) found this pathogen infecting fungi. The nuclear structure and the nuclear behaviour aphid Myzodium modestum (Hottes, 1926), but he dis- during mitosis of these fungi differ from the other ento- tinguished it from the species E. apiculata due to clear mophthoralean species (Butt & Heath, 1988; Butt differences in size and shape of conidia and in size of & Humber, 1989). Therefore, the genus was classed resting spores. He suggested a new name Entomoph- with the distinct family Neozygitaceae proposed and thora major as a distinct species and not a variant of described by Ben-Ze’ev et al. (1987), which was also E. apiculata. Batko (1964c, d) classified this fungus maintained by Humber (1989) and Keller (1991, in the genus Culicicola because of the presence of rhi- 1997). This family has not been distinguished recently zoids and multinucleate spores borne on simple coni- by Balazy (1993), who left all Neozygites species or- diophores. King (1976a, b), according to his compre- ganized within the family Entomophthoraceae. hensive study of the genus Conidiobolus, believed that The genus was originally established by Wit- the species had belonged to this genus. This conclu- laczil (1885). Thaxter (1888), however, proposed sion was affirmed by Remaudiere` & Keller (1980) the subgenus Triplosporium (in the genus Empusa) S560 M. Barta & Ľ. Cagáň for species characterised by smoky conidia and black, stage of E. fresenii and that Triplosporium and Neozy- oval zygospores. Batko (1964b) elevated the Thaxter’s gites were synonymous. Rudraiah & Usman (1955) subgenus to the generic rank and listed three species reported Entomophthora sp. from the oleander aphid, in the genus according to Thaxter (1888). Based Aphis nerii Boyer de Fonscolombe, in India. Gustafs- on nomenclatural priority, Remaudiere` & Keller son, in a subsequent reference to this fungus (Gustafs- (1980) placed Triplosporium in synonymy under Neozy- son, 1969), referred to it as a new species Entomoph- gites. Humber et al. (1981) proposed to conserve the thora neri Gustafsson. MacLeod & Müller-Kögler name Triplosporium against Neozygites, but the pro- (1973) suggested that E. neri is not a new species but posal failed. Ben-Ze’ev & Kenneth (1982a) preferred the resting-spore stage of N. fresenii. Until 1980, when the Batko’s generic name Triplosporium after its emen- Remaudiere` & Keller (1980) replaced Triplospo- dation. At present, a validity of the generic name Neozy- rium (Thaxter) Batko with the genus Neozygites Wit- gites has been accepted by most taxonomic authorities laczil, this fungus was mentioned in the literature as (Humber, 1989; Keller, 1991, 1997; Balazy, 1993). Triplosporium fresenii (Nowakowski) Batko. Neozygites consists of a homogenous group of 17 N. fresenii is a widely occurring fungal pathogen different fungi attacking small pterygote insects (Ho- of aphids and has been recorded in various countries. moptera, Thysanoptera) and mites. Species of the genus It has been reported from Europe (e.g., Thoizon, Neozygites can be divided into two subgroups on the 1970; Dedryver, 1978; Keller, 1991; Sivčev, 1991, basis of their morphology and life cycle. All the aphi- 1992; Balazy, 1993; Steenberg & Eilenberg, 1995; dophagous species belong to the Neozygites fresenii- Barta & Cagáň, 2002), North America (e.g., Soper group, except for Neozygites lecanii, which also seems & MacLeod, 1963; Feng et al., 1990), Israel (Bit- to be closer related to this group, but deficiencies in ton et al., 1979), South Africa (Hatting et al., 1999a), its description make the obstacle for a definite deci- the South Pacific (Keller, 1997) and Australia (Mil- sion (Keller, 1997). One fungal species attacking an ner & Holdom, 1986). Aphids of the genus Aphis, apterygote insect, the springtail Sminthurus viridis (L., especially the species Aphis fabae (e.g., Gustafsson, 1758), and formerly attributed to the genus Neozygites 1965; Robert et al., 1973; Dedryver, 1978; Barta (Steenberg et al., 1996; Keller & Steenberg, 1997; & Cagáň, 2002, 2006) and Aphis gossypii Glover, 1877 Dromph et al., 2001) was recently placed in a new (e.g., Silvie & Papierok, 1991; Steinkraus et al., genus Apterivorax S. Keller proposed by Keller & 1991, 1995; Marti & Olson, 2006) are considered Petrini (2005). The genus Neozygites includes eight the major host organisms for the pathogen. N. fre- aphid-pathogenic species. senii causes widespread epizootics in the aphid pop- ulations throughout the world. Moreover, it is best 20. Neozygites fresenii (Nowakowski) Remau- adapted to hot, humid, even tropical conditions (Re- di`ere et Keller maudiere,` 1977; Steinkraus et al., 1991; Keller, Mycotaxon 11: 332 (1980) 1997), but the epizootics has been reported in Iceland as well (Austar˚a et al., 1997; Nielsen et al., 2001b). B: Empusa fresenii Nowakowski,Pam.Akad.Umiej.Kraków In Slovakia, the pathogen was identified in 24 aphid 8: 171–172 (1883) species and when compared with P. neoaphidis and E. Empusa subgenus Triplosporium fresenii Nowakowski ex planchoniana, this fungus appeared in host colonies one Thaxter, Mem. Boston. Soc. Nat. Hist. 4: 167–169 (1888) month later (usually during May) (Barta & Cagáň, Triplosporium fresenii (Nowakowski) Batko, Bull. Acad. 2006). Epizootiology of the fungus in annual cropping Pol. Sci., Ser. Sci. Biol. 12: 325 (1964) ecosystems has been well studied (Steinkraus et al., Entomophthora fresenii (Nowakowski) Gustafsson, 1991, 1993, 1996b, 1999; Barta & Cagáň, 2002), but Lantburkshogsk. Ann. 31: 141–142 (1965) its commercial exploitation is limited due to absence S: Neozygites aphidis Witlaczil, Arch. Mikrosk. Anat. 24: 599–603 (1885) of in vitro isolates. Infection by N. fresenii probably Entomophthora neri Gustafsson, Lantburkshogsk. Ann. 35: induces specific behavioural modifications of diseased 243 (1969) aphids (M. carnosum and A. fabae). Infected aphids just before their death moved from feeding sites, i.e., This species named and described as Empusa fresenii leaf blades to leaf petioles or plant stems when resting by Nowakowski (1883) was discovered in naturally spores are formed (Barta & Cagáň, 2002, 2006). infected aphids collected in Poland. This is the first de- Despite numerous attempts to grow N. fresenii on scribed member of the Neozygites genus. Later, Wit- artificial media it has not been isolated yet (Keller, laczil (1885) described an aphid infection observed 1997). in Aphis (Hyalopterus) arundinis F., 1775 as Neozy- gites aphidis, believing the pathogen to be a gregarine 21. Neozygites microlophii Keller protozoan. Thaxter (1888), unaware of the descrip- Sydowia 43: 82 (1991) tion by Witlaczil, placed E. fresenii in a new subgenus, Triplosporium. Giard (1888) was the first who noted Neozygites microlophii was common fungus and caused that Witlaczil had in fact described a resting-spore epizootics among dense populations of the nettle aphid Aphid-pathogenic Entomophthorales S561 species, Microlophium carnosum, in Switzerland where cadavers were hanging on their proboscises from the it was first discovered and described by Keller (1991). branches. After certain time, the cadavers either dried The fungus could be found within the aphid populations up and fell down to the soil or they liquefied and resting between mid-June and mid-July. Keller (1997) thinks spore mass spread out on a bark of the trees. Some de- that some collections identified as morphologically sim- tectable residues of the resting spore mass were found ilar N. fresenii in the past might belong to this species, on the trees even after one year (Barta & Cagáň, especially those from larger aphids. 2006). Resting spores of the pathogen were later identi- At present, the fungus is known from the above- fied in the nettle aphid in Poland as well (Balazy, mentioned aphid species in Israel and central Europe 1993). The species was regularly found in one locality (Balazy, 1993; Keller, 1997). in Slovakia during four consecutive years in May and June. Each year the fungus appeared together with N. 23. Neozygites lageniformis (Thaxter) Remau- fresenii and both the species influenced significantly di`ere et Keller the growth of the nettle aphid populations (Barta Mycotaxon 11: 322 (1980) & Cagáň, 2003b, 2006). The development of N. mi- crolophii infection within the colonies of nettle aphid B: Empusa subgenus Triplosporium lageniformis Thaxter, M. carnosum and the transmission of the infection to Mem. Boston. Soc. Nat. Hist. 4: 169 (1888) other aphids in agricultural landscape were studied in Triplosporium lageniformis (Thaxter) Batko, Bull. Acad. Slovakia (Barta & Cagáň, 2003b). Pol. Sci., Ser. Sci. Biol. 12: 403 (1964) Entomophthora lageniformis (Thaxter) MacLeod et Müller- The species has been known only from the type Kögler, Mycologia 65: 858 (1973) host. The species is incompletely described. Thaxter (1888) 22. Neozygites turbinata (Kenneth) Remaudi`ere described this fungus as Empusa (Triplosporium) la- et Keller geniformis Thaxter for the first time in the USA from Mycotaxon 11: 322 (1980) unidentified aphid species. It was suggested that N. la- geniformis frequently occurred in association with Z. B: Entomophthora turbinata Kenneth, Mycotaxon 6: 381– occidentalis. The species is known from the USA and 390 (1977) Chile on aphids Myzocallis coryli (Goeze, 1778) on Thaxterosporium turbinatum (Kenneth) Ben-Ze’ev et Ken- Corylus avellana L. and unidentified aphid species on neth in Ben-Ze’ev, Kenneth et Uziel, Mycotaxon 28: 313– Betula populifolia Marsh and Solidago sp. (Aruta & 326 (1987) Carvillo, 1989; Balazy, 1993). The pathogen was discovered in Israel on the aphid Pte- rochloroides persicae (Cholodkovsky, 1899) in 1977 and 24. Neozygites lecanii (Zimmermann) Balazy named Entomophthora turbinata Kenneth (Kenneth, Flora of Poland, Fungi (Mycota) 24: 139–140 (1993) 1977). The species was closely related to the genus Triplosporium (Thaxter) Batko, but no secondary coni- B: Empusa lecanii Zimmermann, Meded. Lands Plantent. dia were observed and both hyphal bodies and primary 44: 25–27 (1901) spores contained 7–11 nuclei. However, Triplosporium Triplosporium lecanii (Zimmermann) Ben-Ze’ev et Kenneth, Mycotaxon 14: 436 (1982) described by Batko (1964b) was characterised to have tetranucleate spores. Remaudiere` & Keller (1980) The species was first observed on Lecanium viridae (Gi- listed this fungus under the generic name Neozygites, rault, 1916) (Homoptera: Coccidae) in Java in 1901 and whereas Ben-Ze’ev et al. (1987) separated it as a rep- described as Empusa lecanii (Zimmermman, 1901). resentative of the new genus, Thaxterosporium Ben- The species is incompletely described and known only Ze’ev et Kenneth. Keller (1991), who revealed an- from South East Asia (Balazy, 1993; Keller, 1997). other species with a number of nuclei in spores and Humber (1991) listed this species among fungi para- hyphal bodies other than four, did not see a reason for siting aphids. a separate genus for N. turbinata and synonymized the genus Thaxterosporium with Neozygites. 25. Neozygites cinarae Keller In Switzerland, the species was observed in the Sydowia 49 (2): 137–138 (1997) colonies of the aphid Tuberolachnus salignus (Gmelin, 1790) on Salix sp. from the end of August till the be- The species was collected in a plantation of Picea abies ginning of November, where it caused strong epizootics Karst. within the colonies of the aphid Cinara pilicor- (Keller, 1991, 1997). A strong epizootic due to N. nis (Hartig, 1841) (the type host) in Switzerland be- turbinata was also observed in the colonies of the gi- tween the end of June and the beginning of July in ant willow aphid, T. salignus, feeding on branches of 1991–1995 and described as a new species by Keller willow trees in Slovakia during October. The infected (1997). N. cinarae is closely related to N. fresenii and aphids were filled with black ovoid resting spores and N. microlophii from the morphological and taxonomical no cadavers with sporulating fungus were found. The points of view. It can be distinguished mainly by larger S562 M. Barta & Ľ. Cagáň conidia, the number of nuclei in conidia, and a length Ben-Ze’ev & Kenneth (1982a) proposed to divide of capillary tube. Conidiobolus genus into three subgenera (Delacroixia, In Slovakia and Austria, some individuals killed Conidiobolus and Capillidium) on the basis of peculiar by Neozygites cinarae Keller were also observed in the forms of secondary conidial sporulation. Later, Balazy colonies of its type host aphid, C. pilicornis,onP. abies (1993) accepted this arrangement. twigs. The pathogen was recorded during July and Au- Most of the Conidiobolus species are saprophytes gust (Barta & Cagáň, 2006). in detritus, but some are also known from insect and vertebrate mycoses (Balazy, 1993). They are often iso- 26. Neozygites remaudierei S. Keller lated from soil samples (e.g., Coremans-Pelseneer Sydowia 58 (1): 53 (2006) et al., 1983; Ali-Shtayeh et al., 2002; Keller et al., 2003). This relatively new species was described from Myzo- King (1977) recognised 27 species in his list of callis coryli (the type host) in Switzerland. M. coryli is Conidiobolus species. Ben-Ze’ev & Kenneth (1982a) the only known host of the fungus. The species is sim- added a further four species to the genus including ilar to N. fresenii, but it can be distinguished by the three entomopathogenic species recorded earlier under number of nuclei in hyphal bodies and longer capillary the generic names Empusa or Entomophthora s.l. Af- tubes (Keller, 2006). ter detailed studies of karyological characteristics, two from among them (C. apiculatus and C. major)werein- 27. Neozygites slavi S. Keller cluded into the family Entomophthoraceae (under the Sydowia 58 (1): 54 (2006) generic name Batkoa)(Humber, 1989), whereas Ento- mophthora destruens BatkoetWeiserwastransferred This incompletely described species was discovered in to Conidiobolus (Ben-Ze’ev, 1982). Subsequently, four one specimen of Slavum esfandiarii Davatchi et Re- new species were described (Balazy et al., 1987; maudi`ere, 1957, being preserved in the aphid collection Keller, 1991; Balazy, 1993), so the actual number for 50 years. The specimen of aphid was collected in Iran of the mostly saprophytic species is 34. Out of nine (Keller, 2006). The fungus has not been observed yet parasitic Conidiobolus species five have been recorded in host colonies in nature. from aphids.

Family: Ancylistaceae J. Schröt. 28. Conidiobolus coronatus (Costantin) Batko Die Natürlichen Pflanzenfamilien 1: 92 (1893) Entomophaga, Mem. Hors., Ser. 2: 129 (1964)

Genus: Conidiobolus Brefeld emended Humber B: Boudierella coronata Costantin, Bull. Soc. Mycol. Fr. 13: Mycotaxon 34: 455–456 (1989) 40 (1897) Delacroixia coronata (Costantin) Saccardo et P. Sydow, The genus was established by Brefeld in 1884 (King Syll. Fung. 14: 457 (1899) & Humber, 1981) and it is typified by Conidiobolus Entomophthora coronata (Costantin) Kevorkian, J. Agaric. utriculosus Brefeld. The criterion used to define this Puerto Rico 21: 198 (1937) fungal genus from Entomophthora s.l. was predomi- Conidiobolus coronatus (Costantin) Srinivasan et Thiruma- nantly saprophytism of the Conidiobolus species in op- lachar, Mycopathol. Mycol. Appl. 24: 294–296 (1964) position to parasitism of the other species (Ben-Ze’ev Conidiobolus subgenus Delacroixia coronatus (Costantin) Kenneth Tyrrell et MacLeod, J. Invertebr. Pathol. 20: 12 (1972) & , 1982a). Some insect parasitic species Conidiobolus subgenus Delacroixia coronatus (Costantin) were, however, later transferred from Entomophthora s. Batko in Balazy, Flora of Poland. Fungi (Mycota) 24: 78–79 l. to Conidiobolus as well (Batko, 1964a; Srinivasan (1993) & Thirumalachar, 1964; Tyrrell & MacLeod, S: Conidiobolus villosus G. W. Martin, Bot. Gaz. 80: 311– 1972). King (1977) also noted certain morphological 318 (1925) similarity of several entomopathogenic Entomophthora s. l. species with the species Conidiobolus. Extending This fungus isolated from a culture of Agaricus campes- the King’s concept of Conidiobolus, Remaudiere` & tris Fr. more than 100 years ago was named as Keller (1980) proposed to classify all the entomoph- Boudierella coronata Costantin and described by Cos- thoralean forms with spherical, ovoid and pyriform, uni- tantin in 1897 (MacLeod & Müller-Kögler, 1973). tunicate and multinucleate conidia in the genus Coni- Since then it has been re-isolated from numerous diverse diobolus. Due to this taxonomic emendation a num- sources (Hall & Dunn, 1957; Gustafsson, 1965; ber of species now allocated in Entomophaga Batko MacLeod & Müller-Kögler, 1973; King, 1979; emended Humber and Batkoa Humber were listed un- Keller, 1987a; Feng et al., 1990). Also, it has been der the name of Conidiobolus until Humber (1989) treated under seven different names and belonged to emended the description of Conidiobolus Brefeld and four different genera. C. coronatus is one of two Co- provided a clear morphological and developmental sep- nidiobolus species listed by King (1977), which was aration of this genus from Entomophaga and Batkoa. not originally described as a species of Conidiobolus. Aphid-pathogenic Entomophthorales S563

Kevorkian (1937) transferred this species to Ento- Conidiobolus subgenus Conidiobolus obscurus (Hall et Dunn) mophthora genus mainly because of its ability to at- Remaudi`ere et Keller in Balazy, Flora of Poland. Fungi (My- tack insects. It was Batko (1964a) who correctly of- cota) 24: 73–74 (1993) fered to transfer Entomophthora coronata to the genus S: Empusa planchoniana Thaxter, Mem. Boston. Soc. Conidiobolus. Ben-Ze’ev & Kenneth (1982a) and Nat. Hist. 4: 165 (1888), [nec Entomophthora planchoniana Cornu, Bull. Soc. Bot. France 20: 189–190 (1873)] Balazy (1993) classed this pathogen with the subgenus Empusa thaxteriana Petch, Trans. Br. Mycol. Soc. 21: 34 Delacroixia, based largely on the ability to produce mi- (1937) (nom. inval.) croconidia from the primary or secondary conidium. At Entomophthora ignobilis Hall et Dunn, Hilgardia 27: 162 the present time the species is considered a species com- (1957) plex, and a rather great variability or intraspecific di- Entomophthora thaxteriana (Petch), Hall et Bell, J. Insect. versity requires a detailed study (Batko, 1974; Hum- Pathol. 5: 186 (1963) ber, 1983, 1991). Entomophaga thaxteriana (Petch) Batko, Bull. Acad. Pol. The fungus is distributed worldwide. It is recorded Sci., Ser. Sci. Biol. 12: 404 (1964) as a widespread soil saprophyte utilising a variety of substrates, including detritus, living plants, different This pathogen was first discovered on numerous aphids dead arthropods, and occasionally mammals (includ- of several genera by Thaxter in Maine (the USA) in ing humans) (MacLeod & Müller-Kögler, 1973; 1888. However, he incorrectly identified the fungus as King, 1979; Keller, 1987a; Balazy, 1993; Sajap et Empusa planchoniana (Cornu) Thaxter (MacLeod & al., 1997). Nevertheless, the species is known to cause Müller-Kögler, 1973). Petch (1937), who discov- diseases in insects as well. Besides other insect or- ered the mistake, renamed the fungus observed by ders, it was also identified from several aphid species Thaxter as Empusa thaxteriana Petch. After examina- in Poland (e.g., Batko, 1962, 1966a; Balazy et al., tion of the Thaxter’s and Petch’s materials, Hall & 1990; Balazy, 1993) and France (Thoizon, 1970; Pa- Bell (1963) concluded that E. thaxteriana and Ento- pierok, 1985), from the cabbage aphid in Yugoslavia mophthora ignobilis, a pathogen described on the aphid (Sivčev, 1991), Elatobium abietinum in Great Britain T. maculata by Hall & Dunn (1957), were the same (Austar˚a et al., 1997), from the cereal aphids in South and the correct name for the species was Entomoph- and North Africa (Hatting et al., 1999a; Abdel- thora thaxteriana (Petch) Hall et Bell. Batko (1964c) Mallek et al., 2003), or in the USA (Feng et al., subsequently transferred the species to the genus En- 1990), from the green peach aphid in Israel (Gindin tomophaga. Several years later, Humber (1978), dur- & Ben-Ze’ev, 1994), or from Macrosiphum euphor- ing his studies on the identities of some Entomophthora biae (Thomas, 1878) in the USA (Shands et al., 1972). species, revealed that the name of Entomophthora thax- Strains of various origin, saprophytes and pathogens, teriana was not validly published and was rejected by proved to be pathogenic for aphids. Their virulence, him as nomenclatural error because Petch provided nei- however, showed obvious differences (Papierok, 1985; ther the Latin description nor reference to a previously Papierok et al., 1993). In laboratory, saprophytic published Latin diagnosis. Humber suggested that En- species of Conidiobolus were as infective for aphids as tomophthora ignobilis Hall et Dunn is the correct name species were active under natural conditions, but were for the fungal pathogen. In a later study, Remaudiere` more rapid in the action, probably due to the produc- et al. (1979) demonstrated the synonymy of E. thax- tion of toxins (Papierok, 1986). Biochemical analy- teriana (E. ignobilis)andEntomophthora obscura,the sis of C. coronatus isolates has recently revealed the species described on T. maculata simultaneously with presence of toxic proteins in conidia and hyphae of the E. ignobilis by Hall & Dunn (1957). Keller & Re- species (Bogu`s & Scheller, 2002). maudiere` (1980) consequently transferred E. obscura The fungus can be easily isolated in a variety to the genus Conidiobolus, which was later confirmed of simple media (Gustafsson, 1969; Balazy, 1993; with nuclear cytology of the species (Humber, 1981). Gindin & Ben-Ze’ev, 1994). Utilization of the species C. obscurus is a very common and widespread for biological control is limited by the fact that it might pathogen specific only to aphids. It has been found in cause a human phycomycosis, rhinoentomophthoromy- a number of countries including the USA, Sweden, Fin- cosis (Pai et al., 1993; Ochoa et al., 1996). land, Norway, Iceland, Great Britain, France, Switzer- land, Poland, Slovakia, Russia, Australia, Egypt, or 29. Conidiobolus obscurus (Hall et Dunn) Re- South Africa (e.g., Gustafsson, 1969; Thoizon, 1970; maudi`ere et Keller Wilding & Perry, 1980; Milner & Soper, 1981; Mycotaxon 11: 330 (1980) Papierok & Havukkala, 1986; Papierok, 1989; Keller, 1987a; Feng et al., 1990; Balazy, 1993; Behrens, 1993; Voronina, 1997; Hatting et al., Štalmachová Cagáň Nielsen B: Entomophthora obscura Hall et Dunn, Hilgardia 27: 162 1999a, 2000; & , 2000; (1957) et al., 2001b; Abdel-Mallek et al., 2003; Klingen & Entomophaga obscura (Hall et Dunn) Batko, Bull. Acad. Jaastad, 2003). C. obscurus demonstrates tendency Pol. Sci., Ser. Sci. Biol. 12: 404 (1964) to cause epizootics, but not as markedly as the most S564 M. Barta & Ľ. Cagáň common aphid pathogen Pandora neoaphidis (Keller thromboides as a biological control agent for pestifer- & Suter, 1980; Barta & Cagáň, 2006). The fungus ous aphid species (e.g., Hall & Dunn, 1958; Milner is present within aphid colonies at low levels alongside & Soper, 1981; Wilding, 1981; Raj & Ramakrish- other fungi and the disease development usually does nan, 1982; Chudare, 1990; Hatting et al., 1999b). not reach epizootic character (Barta & Cagáň, 2006). Methods for a mass-production of resting spores of C. Besides other aphid species, the pathogen regularly in- thromboides were also developed (Matanmi & Libby, fects cereal aphids (e.g., Feng et al., 1990; Steen- 1976; Latgé et al., 1978a, b; Chudare, 1990). berg & Eilenberg, 1995; Hatting et al., 1999a, Entomopathogenic strains of this fungus misiden- 2000; Nielsen et al., 2001a), pea aphid (e.g., Wild- tified as Entomophthora thaxteriana were in the seven- ing, 1975; Behrens, 1993; Cagáň & Barta, 2001), ties of the last century widely studied in order to apply cabbage aphid (Sivčev, 1991, 1992), or black bean them in biological control of aphids (Humber et al., aphid (Gustafsson, 1969; Steenberg & Eilenberg, 1977). 1995). The species shows good growth on Sabouraud’s The fungus has been widely studied as a potential agar (Gustafsson, 1969). aphid control agent (Soper et al., 1975; Latgé, 1980; Latgé et al., 1983; Chudare, 1990; Voronina, 1997), 31. Conidiobolus osmodes Drechsler although a part of older data also concerns the species Am. J. Bot. 41: 571 (1954) Conidiobolus thromboides as a result of misidentifica- Humber tion ( et al., 1977). B: Conidiobolus subgenus Conidiobolus osmodes The culture of this pathogen can be established on Drechsler in Balazy, Flora of Poland. Fungi (Mycota) 24: Sabouraud’s dextrose agar or media based on egg yolk 71 (1993) and milk (Keller, 1987a). This species was originally described as a saprophyte 30. Conidiobolus thromboides Drechsler on plant detritus by Drechsler (1954). Later it was J. Wash. Acad. Sci. 43: 38 (1953) found to be an occasional pathogen of aphids in France (Remaudiere` et al., 1976b). Ben-Ze’ev & Kenneth S: Entomophthora virulenta Hall et Dunn, Hilgardia 27 (1980) found resting spores in the larvae of Hypera pos- (1957) tica (Gyllenhal, 1813) in Israel and succeeded in isolat- Culicicola virulenta (Hall et Dunn) Batko, Bull. Acad. Pol. ing C. osmodes from the infected larvae, which proved Sci., Ser. Sci. Biol. 12: 404 (1964) to be pathogenic to Hypera larvae and houseflies in B: Conidiobolus subgenus Conidiobolus thromboides Drech- laboratory bioassays. While the fungus was recorded sler in Balazy, Flora of Poland. Fungi (Mycota) 24: 72 (1993) as an occasional pathogen of aphids in France, it was recorded as a very common and destructive pathogen C. thromboides isbestknownasanaphidpathogen, of Hypera spp. in Israel, even considerably more than but it was first described as a saprophyte from plant other pathogens in the fields (Ben-Ze’ev & Kenneth, detritus in the USA (Drechsler, 1953). Few years 1980). In France, C. osmodes was found on few aphid later, another pathogen, Entomophthora virulenta,was species during cold weather and was able to occasion- described from the aphid Therioaphis maculata in Cal- ally manifest itself in populations at epizootic levels. ifornia (USA) (Hall & Dunn, 1957). Batko (1964c) Epizootics caused by the fungus was observed in a pop- transferred E. virulenta to its rather controversial genus ulation of Macrosiphoniella oblonga (Mordvilko, 1901) Culicicola. However, the Batko’s inclusion of E. vir- infesting chrysanthemums (Papierok & Coremans- ulenta to Culicicola was based on the erroneous de- Pelseneer, 1980). C. osmodes is considered to be the scription of this species as producing rhizoids (Humber only saprophytic species of the genus Conidiobolus that et al., 1977). Subsequently, morphological, biochemi- could play an important role in the regulation of in- cal and pathogenic characters were used to compare C. sect populations, whereas then isolation of other sapro- thromboides and E. virulenta. Results of these analyses phytic Entomophthorales from dead insects is acciden- gave evidence about a synonymy of both the species tal (Papierok, 1986). In literature there are several (Latgé et al., 1980). records of C. osmodes from aphid hosts. It was iso- The species has been recorded in Europe, North lated from R. padi and S. avenae (Latgé, 1975; Re- America, Asia, Australia, North and South Africa and maudiere` et al., 1981) and observed on S. avenae and isolated from diverse sources including soil detritus M. persicae in France (Remaudiere` et al., 1976b), and often dead insects, mostly aphids (e.g., Gustafs- the cabbage aphid in Yugoslavia (Sivčev, 1991), and son, 1969; Thoizon, 1970; Milner & Soper, 1981; undetermined aphids in Poland (Balazy, 1993). The Cheng & Long, 1987; Feng et al., 1990; Balazy, species has been recorded in North America, Australia, 1993; Steenberg & Eilenberg, 1995; Hatting et al., Israel and Europe, isolated from organic detritus and 1999a, 2000; Nielsen et al., 2001a; Abdel-Mallek et dead insects, mostly the Hypera spp. larvae and aphids al., 2003; Barta & Cagáň, 2006). A number of labo- (Balazy, 1993), or recently from the larvae of Tipula ratory or field tests have been conducted to assess C. paludosa Meigen, 1830 (Gökc¸e & Er, 2003). Aphid-pathogenic Entomophthorales S565

Theculturecanbeestablishedon“Entomophthora Nowakowski (1883). Thaxter (1888) did not regard complete medium (ECM)” (Ben-Ze’ev & Kenneth, Tarichium as a genus but merely as a group of Empusa 1980). species with unknown conidial stage, and Lakon (1919) proposed to use the name Tarichium as a “temporary- 32. Conidiobolus destruens (Weiser et Batko) auxiliary genus” for species known only from their Ben-Ze’ev resting spores. MacLeod & Müller-Kögler (1970) Mycotaxon 14: 289 (1982) placed Tarichium as a subgenus to the genus Ento- mophthora. However, this was not accepted later (Ben- B: Entomophthora destruens Weiser et Batko, Fol. Parasit. Ze’ev & Kenneth, 1982a). At present, Tarichium as a 13: 144–149 (1966) form-genus is generally admitted and considered to be a Conidiobolus subgenus Conidiobolus destruens (Weiser et provisional group without nomenclatural validity since Batko) Ben-Ze’ev in Balazy, Flora of Poland. Fungi (My- it is applied to the description of species, parasites of cota) 24: 65–66 (1993) insects and mites known only from their resting spores. The fungus was described as Entomophthora destruens Conidial stage of these species still has been unknown, Weiser et Batko parasiting on hibernating mosquitoes but is presumed to exist (Ben-Ze’ev & Kenneth, in natural shelters, caves, and wine cellars of Southern 1982a; Humber, 1989; Keller, 1991, 1999; Balazy, Moravia (Weiser & Batko, 1966). After a detailed 1993). The genus includes 37 entomophagous species study of the karyological characteristics of a species with one parasitic on aphids (Balazy, 1993). recorded earlier under the generic name Entomophthora s.l., E. destruens was transferred to the genus Conidio- 33. Tarichium atrospermum (Petch) Balazy bolus (Ben-Ze’ev, 1982). Flora of Poland, Fungi (Mycota) 24: 256–257 (1993) C. destruens seemed to be rather host-specific to Culex pipiens L., 1758 because in the caves it B: Entomophthora (Tarichium) atrosperma Petch, was infected, whereas Culiseta annulata (Schrank, Trans. Br. Mycol. Soc. 17: 172 (1932) 1776) and Anopheles maculipennis Meigen, 1818 were Entomophthora (Tarichium) atrosperma (Petch) MacLeod not (MacLeod & Müller-Kögler, 1973). In some et Müller-Kögler, Mycologia 62: 39 (1970) localities the fungus destroyed more than 85% of The species was observed in an unidentified aphid the mosquitoes. Identical cases were later demon- species in England and is known only from the type strated in other localities in Moravia, Bohemia, Slo- locality (Balazy, 1993). vakia, the Netherlands, Denmark, England and France (MacLeod & Müller-Kögler, 1973; Mi˛etkiewski & van der Geest, 1985; Eilenberg, 2002). The opin- 5.2. A key to aphid-pathogenic Entomoph- ion about strong specificity of C. destruens to C. pipiens thorales was, however, later displaced by the fact that strains of the fungus successfully infected in laboratory condi- The key to the entomophthoralean species parasiting tions two aphid species, Megoura viciae Buckton, 1876 aphids presented herein is a result of our endeavour and Aphis fabae (Krejzová, 1972a), two members of to assemble any information from literature relating to Isoptera (Krejzová, 1972b), and Lepidoptera (Krej- the taxonomy of the aphid-pathogenic Entomophtho- zová, 1971a). That was the only record of C. destruens rales. In the last decades considerable progress in the from aphids. Application of conidia to M. viciae and A. knowledge of the taxonomy of Entomophthorales has fabae resulted in 35% and 54% mortality, respectively been made and much literature concerning the aphid- (Krejzová, 1972a). The pathogenicity of C. destruens pathogenic Entomophthorales has been published. Un- to aphids and other insect groups, however, seems to fortunately, the publications deal with species of a spe- be doubtful and needs confirmation. cific genus as a whole or the order at generic levels. Pa- The pathogen is distributed in Central and West- pers including all the aphid-pathogenic species of this ern Europe on C. pipiens and can be isolated on egg particular group of fungi are still missing. This key is yolk (Weiser & Batko, 1966; Balazy, 1993). primarily a synthesis of the most influential taxonomic monographs on the Entomophthorales published re- Form-genus: Tarichium Cohn cently (Humber, 1991; Balazy, 1993; Keller, 1987a, Beitr. Biol. Pflanz. 1: 58–86 (1870) 1991, 1997, 1999, 2002, 2006). Syn.: Entomophthora (Tarichium) MacLeod et Müller- 1a Only spherical resting spores known ...... Kögler, Mycologia 62: 33–66 (1970) ...... Tarichium –aformgenus9 This genus was created to include species known only 1b Conidia present ...... 2 by their resting spores (Cohn, 1870) and is typified by 2a Primary conidia unitunicate with more than one Tarichium megaspermum Cohn. The genus Tarichium, nucleus, staining well or not in AO, but staining characterized by smooth-walled or warted azygospores well in FRS*; primary conidia bell-shaped, spheri- and inability to produce conidia, was later accepted by cal, pyriform or broadly ellipsoidal in shape; coni- S566 M. Barta & Ľ. Cagáň

diophores unbranched; rhizoids present or absent apical point; rhizoids monohyphal ...... 8 and pseudocystidia absent...... 3 8a Pseudocystidia distinctly two to several times 2b Primary conidia uninucleate and bitunicate; nuclei thicker than conidiophores, usually long and of- relatively large and staining readily with AO; pri- ten clavate, nodular or furcate at tips; secondary mary conidia elongate pyriform, fusiform or cylin- conidia like primary ones or spherical, and some- drical in shape; conidiophores branched; rhizoids times tetraradiate (in water habitat); rhizoids at and pseudocystidia usually present ...... 7 least twice as thick as conidiophores without dis- 3a Primary conidia typically bell-shaped with api- tinct terminal holdfast...... cal point (Lakon’s type: truncata-campaniformis); ...... Erynia (fam. Entomophthoraceae) 31 projected primary conidia surrounded by ruptured 8b Pseudocystidia little thicker than conidiophores conidial wall; conidia with 2 to 20 (30) nuclei stain- and tapering apically; tetraradiate secondary coni- ing well in AO; secondary conidia similar to pri- dia not present; rhizoids (may be absent) ribbon- mary ones but without the apical point ...... like 2–3 times thicker than conidiophores terminat- ...Entomophthora (fam. Entomophthoraceae) 10 ing in discoid or irregularly branched and spread- 3b Primary conidia spherical to pyriform with ≥ 4 ing holdfast ...... nuclei, without apical point and not surrounded ...... Pandora (fam. Entomophthoraceae) 32 by ruptured conidial wall...... 4 9 Dark brown-black spherical resting spores with a 4a Primary conidia relatively small, spherical with diameter of 38–45 µm are present inside of dead truncate papilla, smoky spore wall and with 4–8 aphids; episporium is covered with stubby conical nuclei not staining with AO; secondary conidia like spines ...... Tarichium atrospermum primary or amygdaliform capilliconidia on long dis- 10a Primary conidia more than 15 µminlength; tally bent capillary; resting spores spherical to el- primary conidia 14–23 × 12–19 µm on average lipsoidal with black/brown episporium ...... (MacLeod et al., 1976) [15.6–21.4 × 13.4–18...... Neozygites (fam. Neozygitaceae) 11 µm]* ...... Entomophthora planchoniana 4b Primary conidia relatively large, subspherical to 10b Primary conidia less than 15 µm in length; pri- pyriform with more than 8 nuclei, papilla obtuse mary conidia 14.4–12.3 µm on average (Humber or pointed ...... 5 &Feng,1991) ....Entomophthora chromaphidis 5a Nuclei small, not or just weakly staining with AO, 11a On Aphidinae ...... 12 more than 50 nuclei per conidium on average; sec- 11b On Lachninae, Pterocommatinae, Phyllaphidinae, ondary conidia like primary ones, or microconidia, or Pemphiginae ...... 15 or capilliconidia produced (not in aphidophagous 12a Length of primary conidia < 30 µm on average 13 species), weak rhizoids rarely present ...... 12b Length of primary conidia ≥ 30 µm on average 14 ...... Conidiobolus (fam. Ancylistaceae) 18 13a Primary conidia 18–22 × 14–18 µm [17–21 × 14– 5b Nuclei large, deeply staining with AO; secondary 17 µm, L/D* = 1.1–1.5], predominantly with 4 nu- conidia like primary ...... 6 clei; capilliconidia 20–27 × 11–14 µm, L/D = 1.5– 6a Hyphal bodies spherical to subspherical or elon- 2.4 [19.2–30.1 × 10–17 µm, L/D = 1.4–2.5]; capil- gated; nuclei deeply staining with AO and a di- lary tube 17–35 µm [20–66 µm] on average; hyphal ameter of 4–6 µm; conidiophores clavate without bodies spherical to subspherical, 14–17 µm; resting neck-like narrowing part or only with very short spores ellipsoid 30–41 × 18–23 µm [41–43 × 21–22] one (see 7b); rhizoids absent ...... (Keller, 1997) ...... Neozygites fresenii .....Entomophaga (fam. Entomophthoraceae) 23 13b Primary conidia 24–26 × 18–19 µm [35.6–36.2 × 6b Hyphal bodies irregular, rounded to amoeboid; nu- 29–30 µm, L/D = 1.2], predominantly with 5 nu- clei deeply staining with AO, a diameter of 3–5 clei; capilliconidia 30–34 × 12–15 µm, L/D = 2.3– µm; conidiophores simple, distended in apical part, 2.6 [26–30 × 11–14 µm, L/D = 2.1–2.3]; capillary gently narrowed and elongated into relatively long tube 150–170 µm [96–108 µm] on average; hyphal neck; rhizoids present or absent, if present then bodies spherical to subspherical, 17–22 µm; rest- monohyphal, ended with disc-like holdfast ...... ing spores ellipsoid 35–43 × 20–23 µm; on Mic- ...... Batkoa (fam. Entomophthoraceae) 24 rolophium spp. (Keller, 1997) ...... 7a Primary conidia fusiform or cylindrical; papilla ...... Neozygites microlophii conical, pointed or rounded, demarcated from coni- 13c Primary conidia 22.7–23.3 × 19.7–20.4 µm, L/D dial body by a collar; secondary conidia like pri- = 1.14–1.15; conidiophores with 8–11 nuclei, capil- mary ones or sickle- to crescent-shaped on long, liconidia 27.9–30.6 × 13.8–15.3 µm, L/D = 2.00– slender capillaries; rhizoids pseudorhizomorphs... 2.07; capillary tube 192–197 µm on average; resting ...... Zoophthora (fam. Entomophthoraceae) 25 spores unknown; on Myzocallis coryli (Keller, 7b Primary conidia pyriform, fusiform or subcylindri- 2006) ...... Neozygites remaudierei cal; papilla smoothly rounded, not clearly demar- 14 Primary conidia 28–32 × 17–22 µm; capilliconi- cated from conidial body; secondary conidia like dia 29–35 × 16–18 µm; on Myzocallis coryli and primary ones or spherical with or without small unidentified species (Keller, 1997) ...... Aphid-pathogenic Entomophthorales S567

...... Neozygites lageniformis ovoid or short ellipsoid, distinctly thicker of L/D 15a Resting spores with smooth episporium ...... 16 ratio of 2 or less than 2; capilliconidia unknown .. 15b Resting spores with ornamented episporium ...17 ...... 26 16a Primary conidia 19–23 × 12–17 µm with 8 nuclei 25b Primary conidia of one type, slender ellipsoid or on average; resting spores ellipsoid 32–35 × 21– cylindrical of the L/D always more than 2; capilli- 22 µm[38× 23 µm, L/D = 1.6–1.7]; [germ capil- conidia observed ...... 27 liconidia 33.3–33.6 µm, capillary tube 73 µmon 26 Primary conidia ovoid to short ellipsoid 12.6–30.8 average]; hyphal bodies spherical to subspherical, × 8.2–16.5 µm, L/D = 2 or long ellipsoid 17.1– 15–24 µm(Kenneth, 1977) ...... 33.3 × 5.9–12.9 µm, L/D = 3; resting spores 22–32 ...... Neozygites turbinata µm in diameter, exogenously produced; on Myzus 16b Primary conidia 24–31 × 18–21 µm [24–27 × 14– persicae Sulzer in China (Li, 1986) ...... 15 µm, L/D = 1.7], predominantly with 4 nuclei; ...... Zoophthora anhuiensis capilliconidia 32–34 × 14–17 µm, L/D = 2.0–2.5 27a Primary conidia 15–22 × 5.5–7.5 µm on average, [32–34 × 14–16 µm, L/D = 2.1–2.2]; hyphal bodies L/D = 2.4–3.0 [14.9–19.2 × 5.9–7.3 µm, L/D = spherical 20–22 µm; resting spores ellipsoid 34–36 2.5–2.6]; capilliconidia 13–22 × 5–7 µm, L/D = × 23–24 µm [32–24 × 22–23 µm, L/D = 1.5]; on 2.7–3.7 [18.1–18.9 × 5.1–5.6 µm, L/D = 3.4–3.6]; Cinara pilicornis (Hartig, 1841) (Keller, 1997) . resting spores 24–29 µm; (Remaudiere` & Hen- ...... Neozygites cinarae nebert, 1980) ...... Zoophthora radicans 17 Primary conidia unknown; resting spores 28–35 × 27b Primary conidia longer than 20 µm on average, 25–33 µm; pisporium brown with knobs of 2–3 µm their average length is usually above 25–35 µm, in diameter; on Slavum esfandiarii (Keller, 2006) conidia shorter than 20 µmdonotoccurorspo- ...... Neozygites slavi radically ...... 28 18a Multiplicative resporulation by microconidia pre- 28a Length of primary conidia seldom exceeds 30 µm, sent; hirsute resting spores ...... 19 their average length is closed in the range of 25–30 18b Microconidia absent; resting spores with more or µm, and average L/D ratio is less than 3.5 ....29 less smooth walls ...... 20 28b Primary conidia commonly longer than 30 µm, 19 Primary conidia 38–40 × 50–53 µm on average; their average length is closed in the range of 30–40 resting spores 30–32 µm on average (Keller µm, and average L/D ratio is over 3.5 ...... 30 1987a); microconidia 15 × 11 µm(Prasertphon, 29a Primary conidia 27–32 × 9–12 µm, L/D = 2.5– 1963)...... Conidiobolus coronatus 3.2 [29.0–30.1 × 12.9–15.1 µm, L/D = 2.0–2.3]; 20a Rhizoids present ...... 21 capilliconidia amygdaliform 20–23 × 10–12 µm, 20b Rhizoids absent...... 22 L/D = 1.9–2.2 [28.3–29.3 × 12.7–13.4 µm, L/D = 21 Primary conidia 22.5–31.0 × 18.0–24.0 µm(Wei- 2.2]; resting spores 37–40 µm [37.1–41.3 µm] (Re- ser & Batko, 1966) (on aphids artificially) maudiere` & Hennebert, 1980) ...... (Krejzová, 1972a) ...... Conidiobolus destruens ...... Zoophthora aphidis 22a Primary conidia 33.5–44 × 28–36 µm on average, 29b Primary conidia 23.7–34.8 × 6.3–10.3 µm, L/D = L/D = 1.2–1.3 [31.1–42.5 × 26.0–36.8 µm, L/D = 3.2–3.3; capilliconidia like plum fruit in shape 24.5– 1.1–1.2]; resting spores 34–38 µm [32.2–38.5 µm] 29.2 × 5.5–10.3 µm; capillary tubes 50–60 µm long; in diameter (Keller, 1987a) ...... resting spores unobserved; on Aphis citricola v. d...... Conidiobolus obscurus Goot in Israel (Ben-Ze’ev & Kenneth, 1981) .. 22b Primary conidia 25–37 × 22–30 µmonaver- ...... Zoophthora orientalis age; resting spores 13–37 µm in diameter; in cul- 29c Primary conidia 15–36.5 × 6.5–14.5 µm, L/D tures characteristic odour of benzene hexachloride = 2.5; capilliconidia strongly elongate somewhat (Balazy, 1993) ...... Conidiobolus osmodes crescent-shaped 33 × 9 µm; resting spores 29– 22c Primary conidia 24–32 × 17.5–26.5 µm [24.4–27.2 43 µmindiameter;onSchizolachnus piniradiatae × 18.6–21.0 µm, L/D = 1.3] on average; resting (Davidson, 1909) in Canada (MacLeod et al., spores 15–27 µmindiameter(Balazy, 1993) .... 1979) ...... Zoophthora canadensis ...... Conidiobolus thromboides 30a Primary conidia 32–48 × 11–14 µm; capilliconi- 23 Primary conidia pyriform 15–31 × 12–25 µm, L/D dia 18 × 7 µm; capillary tubes 80–120 µm long; = 1.17–1.50, with markedly long papillar neck; resting spores unknown; on different aphid species resting spores 12–25 µmindiameter(Thoizon, (Balazy, 1993); primary conidia cylindrical with 1967) ...... Entomophaga pyriformis parallel sides in outline, hemispherically convex 24a Primary conidia together with papillae 48–52 (57) papilla and broadly obtuse ends (Mi˛etkiewski et × 40–47 µm(Balazy, 1993)...... Batkoa major al., 1981) ...... Zoophthora phalloides 24b Primary conidia together with papillae 30–38 × 30b Primary conidia 30–35 × 7–8.5 µm, L/D = 4.2; 29–34 µm(Balazy, 1993) ...... Batkoa apiculata capilliconidia almond-shaped 17–25 × 6–7.7 µm; 25a Primary conidia of two types: cylindrical elongate capillary tubes 55–85 µm long; resting spores 24–30 with rounded ends of L/D ratio of about 3, and µm in diameter; on several aphid species (Balazy, S568 M. Barta & Ľ. Cagáň

1993); primary conidia ellipsoidally spindle-shaped 34a Primary conidia less than 18 µminlength;resting with a little convex sides, papilla widely conical spores present ...... 35 or slightly convex often with small sharp central 34b Primary conidia more than 18 µminlength;resting apiculus, top part of the spore tapering with some- spore unknown ...... 36 what blunt apex (Mi˛etkiewski et al., 1981)..... 35 Primary spores 15–16.6 × 8–10.5 µm[17× 11 µm, ...... Zoophthora occidentalis L/D = 1.6]; resting spores 24.9–30.4 µm [26–27 31a Primary conidia 12.6–20.5 × 7.1–13.4 µmonaver- µm] (Remaudiere` & Hennebert, 1980)...... age; L/D = 1.4–2.2; echinulate resting spores 27.7– ...... Pandora nouryi 37.1 µm [30.7–32.1 µm] in diameter (Ben-Ze’ev & 36a Primary conidia 21–32 × 11–14 µm, L/D = 1.7–2.3 Kenneth, 1979) ...... Erynia erinacea [21.5–28.1 × 10.8–14.5, L/D = 1.6–2.2 µm] (Re- 31b Primary conidia 27–80 × 12–14 µm; tetraradi- maudiere` & Hennebert, 1980) ...... ate secondary conidia can be produced; resting ...... Pandora neoaphidis spores 30–50 µmindiameter(Balazy, 1993); doc- 36b Primary conidia 16–21 × 10–12 µm; morpholog- umented only from aphids by Thoizon (1970) ... ically similar to P. neoaphidis, but hyphal bod- ...... Erynia conica ies more branched and up to 200 µm long, it 32a Rhizoids absent...... 33 also differs biochemically by fatty acid composi- 32b Monohyphal rhizoids present ...... 34 tion (mycelium contains 21–46% of C12:0 in con- 33a Primary conidia 26.6×15.5 µm on average; resting trast P. neoaphidis mycelium only contains 3–6% spores 24–28 µm in diameter [measurements taken of C12:0), or by isoenzyme patterns (Milner et from aphid host (Shimazu, 1977)]; (on aphids only al., 1983); pathogen of aphids documented only in artificially) ...... Pandora delphacis Australia and China ...... Pandora kondoiensis 33b Primary conidia 24.7–33.0 × 11.6–17.2 µmonaver- age, L/D = 1.8–2.3; resting spores unobserved; no * AO – aceto-orcein stain; FRS – Feulgen reaction stain; L/D – length/diameter ratio of conidia; [measurements ob- growth on Sabouraud’s agar; pathogen of Uroleu- tained by the authors (BARTA, 2004)] con aeneum (Hille Ris Lambers, 1939) in Slovakia ...... Pandora uroleuconii Aphid-pathogenic Entomophthorales S569

6. Biology and ecology of Entomophthorales

Although at present it is generally accepted that en- tomopathogenic fungi can invade and kill arthropod hosts, mechanisms involved in the pathogenesis were open to speculation in the past. For example, at the end of the 19th century an opinion was presented that fun- gal spores needed to be ingested during feeding the host and infection occurred only within the host (Samson et al., 1988). Now it is known that most entomopathogenic fungi invade their hosts via cuticle and that infection from ingested spores is a rare occurrence (e.g., Batko, Balazy Hajek St. Lager 1974; , 1993; & , 1994). Bio- Fig. 2. General schema of the Entomophthorales life cycle: a – logical and ecological aspects of the pathogenic process mature resting spores developed inside the host’s body and de- of Entomophthorales do not deviate from the universal posited in soil; b – resting spores forming germ conidia during concept of the pathogenesis of arthropod pathogenic spring; c – host infected by germ conidia, primary conidia or th replicate conidia; d – mature primary conidia produced on killed fungi. Since the second half of the 20 century much hosts; e – primary conidia producing replicate ones. work has been done to understand all aspects of biol- ogy and ecology of the Entomophthorales (for review see Pell et al., 2001) as it is of broad adoption that dia land on a host surface, they germinate the form- for successful exploitation of these fungi in biocontrol a ing germ tubes, which grow through a host’s integu- comprehensive understanding of their biology and ecol- ment (Batko, 1974). With some genera, e.g., Neozy- ogy is prerequisite. gites, Zoophthora (Glare et al., 1985; Latgé & Pa- pierok, 1988) or Entomophthora (Eilenberg et al., 6.1. Life cycle of aphid-pathogenic Entomo- 1995), the primary conidia are less or not infective and phthorales the secondary conidia, infective structures, are always produced. The infective conidium infects a new host Life cycle of the Entomophthorales is complex and at by direct penetration of integument. Once the fun- least two types of spores are usually involved (coni- gus has penetrated the cuticle and epidermis, it starts dia and resting spores) in the cycle. The biology and to multiply as protoplasts, cell-walled hyphal bod- life cycle of the Entomophthorales has been studied re- ies or coenocytic hyphae in hemolymph and attacks cently by several authors (e.g., Balazy et al., 1990; host tissues (Batko, 1974; Brobyn & Wilding, 1977; Keller, 1997, 2002; Eilenberg, 2002). A schema of Butt et al., 1990). The Entomophthorales life cycle can basic life cycle is shown in Figure 2. This schema in- follow one of two paths. An asexual path where a host cludes both asexual conidia and sexual zygospores – dies and conidiophores producing conidia emerge on the resting spores. However, it is necessary to point out host’s surface or a sexual path where zygospores are that there are many exceptions to the basic pattern. formed inside the host body. The fungi complete the Conidia, asexual spores, are fungal structures, which infection cycle within a few days, depending on tem- are responsible for infection initiation in hosts and in- perature, host, and pathogen species. Shortly after a fection spreading during the season when hosts are ac- host’s death conidiophores emerge on the host’s surface tive. Conidia are formed apically on conidiophores, and conidiogenesis starts resulting in the production which have emerged through epidermis and the cuti- of infective units, conidia (Batko, 1974). Resting cle of hosts and have formed a hymenial layer cover- spores play an important role in the survive periods ing mostly dorsal and lateral parts of aphid’s abdomen of Entomophthorales when hosts are not present or ac- (Batko, 1974; Brobyn & Wilding, 1977; Butt et tive. Resting spores are formed inside the aphid’s body al., 1990). The fully developed conidia are actively dis- (Balazy, 1993), although certain exceptions may oc- charged out of conidiophores by means of high hy- cur, e.g., in Z. anhuiensis (Li, 1986). Resting spores drostatic pressure generated in the conidiophores and may be sexual or asexual, depending on fungal species. are readily spread by air current in the environment Some species were observed not to produce resting (Batko, 1974). If conidia land on a non-host surface, spores at all. Moreover, there are species that are only they can produce replicate conidia (conidia of higher known in their resting spore stage (Balazy, 1993). The orders), i.e., a primary conidium originating from a soil environment is the main reservoir of resting spores dead host may produce a secondary conidium and this in nature (Coremans-Pelseneer, 1981; Nielsen et may produce a tertiary conidium. Theoretically, the al., 2003). After a period of inactivity the spores ger- process could continue until vitality of protoplasm is minate and produce infective conidia, which may ini- exhausted or a susceptible host is encountered. If coni- tiate infection in the host populations. In most cases, S570 M. Barta & Ľ. Cagáň the resting spores are a dormant structure and require discharge a tertiary conidium, and so on (e.g., Brobyn environmental stimulation for germination (Bitton et & Wilding, 1977; King & Humber, 1981; Eilen- al., 1979). berg, 2002). Infectivity of higher order conidia may Six main phases can be distinguished in the onto- be reduced. P. neoaphidis conidia loss rapidly infectiv- genesis of entomopathogenic species of the order En- ity at 100% relative humidity on cover glass due to tomophthorales (Batko, 1965 in Batko, 1974). (1) exhaustion of energy reserves from the production of Infection phase, when infection takes place through the successive generations of conidia in a short time the integument of a host. A fungal conidium sticks to (Brobyn et al., 1987). Some genera produce the sec- the insect cuticle, germinates and penetrates the integu- ondary conidia more or less similar to primary ones ment by mechanical and enzymatic action. (2) Lipoly- such as Pandora, Erynia, Entomophthora (e.g., Ben- tic phase and localized development phase,which Ze’ev & Kenneth, 1982a; Eilenberg et al., 1995; follow after pathogen’s penetration into a host organ- Humber, 1997; Keller, 1999). Other genera such as ism. At first the fungus grows slowly and does not Neozygites or Zoophthora produce capillary conidia – spread within the insect body. At this time the par- mostly almond-shaped spores borne always on long asite secretes very active lipases, which very actively tapering capillary conidiophores (e.g., Balazy, 1993; digest fat reserves. (3) Colonization of host body Keller, 1997). The capilliconidia are not actively dis- is characterised by a rapid fungal spread within the charged (Balazy, 1993; Keller, 1997). Some Coni- host body and by rapid proliferation of numerous hy- diobolus species produce microconidia, small globose phal bodies. As a result, the fungus penetrates into all conidia similar to primary ones in shape and produced tagmata of the host’s body irrespective of the infection on radially projecting microconidiophores outgrowing site. The fungus utilizes nutrients that were accumu- from the primary conidia. Microconidia are best known lated in part during the previous phase and from the in C. coronatus, in which 5 to 28 microconidia may be host’s hemolymph without active digesting the host’s formed per primary conidium (Prasertphon, 1963) tissues. (4) Proteolytic phase, which goes after the and not all C. coronatus strains can produce micro- colonization. The fungus secretes very active proteolytic conidia (King, 1976a). Sometimes primary conidia are enzymes digesting muscles and other tissues of insect regarded as dispersal fungal structures only infecting and destroying very rapidly the whole contents of the when conditions are favourable and secondary (repli- host’s body. Chorion of mature eggs in bodies of females cate) ones are the main infective structures (Glare,et resists the destructive action of proteolytic enzymes. al., 1985; Latgé & Papierok, 1988; Eilenberg et al., These eggs are often fully viable. (5) Phase of host’s 1995; Eilenberg, 2002). Conidia of the entomophtho- body mummification. After completion of proteoly- ralean pathogens are covered with adhesive gelatinous sis, hyphal bodies absorb rapidly the surrounding liquid mucilage (Brey et al., 1986; Boucias & Pendland, and grow quickly to form a spongy material. The con- 1991), which is mainly responsible for the attachment sistency of the body changes to rigid. (6) Sporulation. of spore to the integument (Latgé & Papierok, 1988; Fungal hyphae breach the host’s integument and grow Boucias & Pendland, 1991). While in Conidiobolus outside transformed into conidiophores forming coni- genus the mucilaginous material is present on the coni- dia, or the hyphal bodies conjugate in pairs and form dial surface (Latgé et al., 1986a), in the genus En- thin-walled resting spores. tomophthora the material is present between the outer- most and innermost conidial wall layers (Eilenberg et 6.1.1. Initiation of pathogenesis al., 1986). Infective secondary capilliconidia in the gen- Conidia are the only infection units of the Entomoph- era Zoophthora and Neozygites, also called non-adhesive thorales. Vegetative hyphae, even if in direct contact spores, are not covered with adhesive material, but at with the susceptible host, are not infective (Humber, their apices a drop of sticky liquid called haptor is pro- 1991). Conidium can be defined as an asexual, uni- duced (Keller, 1997). In this case capilliconidia ad- cellular, thin-walled infective unit containing one or here to the host’s cuticle by means of the sticky hap- more nuclei (MacLeod, 1963; Ben-Ze’ev & Ken- tors (Glare et al., 1985). Generally, all areas of aphid neth, 1982a). Infection begins by a germ tube out- cuticle are susceptible to a conidial adhesion (Brey et growing from an infective conidium and penetrating the al., 1986). host’s integument (e.g., Brobyn & Wilding, 1977; Conidia stuck to the host’s surface may germi- King & Humber, 1981; Butt et al., 1990; Hajek nate forming penetration hyphae (germ tube). Coni- &St.Lager, 1994). In appropriate conditions (e.g., dia usually start to germinate within 2–4 hours post- high humidity, available nutrients on cuticle surface, inoculation (at 20 ◦C and saturated humidity) (Brobyn factors necessary for recognition of susceptible host) a & Wilding, 1977) and only one viable germ tube may discharged conidium after landing on a suitable sub- develop per conidium. However, some conidia may give strate germinates to form a germ tube. In unsuitable rise to several abortive germ tubes or germ initials conditions the infection can be aborted and the coni- (Krejzová, 1977; Brey et al., 1986). In most genera, dium forms a replicate conidium. A primary conidium for example Zoophthora, Conidiobolus, Pandora, Ery- discharges a secondary conidium, which may in turn nia, Neozygites,orEntomophaga, a penetration of host Aphid-pathogenic Entomophthorales S571 cuticle is preceded by the formation of appressorium, Brey et al., 1986; Latgé & Papierok, 1988). A strat- from which an infection peg penetrates into the integu- egy for the cuticle penetration seems to be various, de- ment (Brobyn & Wilding, 1977; Hywel-Jones & pending on particular species. On the penetration site Webster, 1986; Butt, 1987; Butt et al., 1990; Ma- a circular entry may be formed, e.g., P. neoaphidis and galhaes˜ et al., 1990a, b, 1991; Wraight et al., 1990; Z. radicans (Butt et al., 1990; Wraight et al., 1990), Nadeau et al., 1996a; Dara & Semtner, 1998; Ha- or tetraradiate fissure, e.g., C. obscurus (Brobyn & jek et al., 2002b). However, a penetration with some Wilding, 1977), and finally triradiate penetration may species, e.g., P. neoaphidis and C. obscurus, can also develop, e.g., genus Entomophthora (Brobyn & Wild- occur without the production of appressoria (Brobyn ing, 1977; Eilenberg et al., 1995). Generally, a pen- & Wilding, 1977; Brey et al., 1986). The appressorial etration is most frequently observed on abdomen, to formation has not been reported for species of the gen- a lesser extent on thorax, and never on head or ap- era Entomophthora and Batkoa (Brobyn & Wilding, pendages (Brey et al., 1986). Germ tubes may grow 1977; Lambiase & Yendol, 1977). Appressoria repre- astray over the surface of cuticle and do not penetrate sent an adaptation to concentrating physical energy and it (Brobyn & Wilding, 1977; Brey et al., 1986). It is lytic enzymes over a very small area to make subsequent of interest that the number of penetrating germ tubes penetration more efficient (St. Leger, 1991; Hajek & is often low. In C. obscurus less than 10% of germinat- St. Leger, 1994; Bechinger et al., 1999). They are ing conidia penetrated the host’s cuticle (Brey et al., likely to be produced in response to the recognition 1986). that the substrate is suitable for infection (Butt et al., 1990). The formation of appressoria from P. neoaphidis 6.1.2. Invasion and colonisation processes during patho- conidia was therefore significantly higher on living sub- genesis strates than on inert cover glass (Dara & Semtner, As soon as the fungus has breached the cuticle and 1998). On the other hand, it was observed that higher reached the haemocoele, it begins to multiply and in- percentages of appressoria were formed from the conidia vade the host’s tissues (Brobyn & Wilding, 1977; of Entomophaga maimaiga Humber, Shimazu et Soper Kobayashi et al., 1984; Butt et al., 1990). The in- deposited on hard surfaces and no nutrients or chem- vading fungal hyphae separate to form large hyphal ical stimuli were required for appressorial formation. bodies, which subsequently start to multiply by bud- Nutrients could, however, stimulate the growth of germ ding (Latgé, 1980). Hyphal bodies are single, coeno- tubes (Hajek et al., 2002b). Not only the growth of cytic cells, which vary in shape from spherical (e.g., N. germ tubes, but also the germination of conidia is reg- fresenii) to hyphae-like (e.g., P. neoaphidis), prolifer- ulated by various nutritional and physicochemical fac- ate rapidly, and circulate readily in hemolymph to all tors (Kerwin, 1982; Uziel & Kenneth, 1999). For in- parts of the infected host (Brobyn & Wilding, 1977; stance, free aminoacids and monosacharides originating Kobayashi et al., 1984; Humber, 1991). However, from the honeydew and aqueous cuticular extracts of A. most entomophthoraceous and neozygitaceous species pisum favour the formation of germ tubes from the C. develop as wall-less protoplasts within the first stages of obscurus conidia (Sampedro et al., 1984; Boucias & pathogenesis and differentiate into walled hyphal bod- Latgé, 1988). Host cuticular lipids stimulate the ap- ies later (e.g., MacLeod et al., 1980; Kobayashi et al., pressorium and penetration tube formation of Erynia 1984; Butt et al., 1981, 1990; Keller, 1997). These conica on the susceptible host and do not inhibit a coni- pathogens probably switch to the protoplast form in a dial resporulation on the non-susceptible host (Nadeau response to contact with the nutrient rich hemolymph et al., 1996a). Moreover, certain self-inhibited germina- (Butt et al., 1990). The protoplasts may vary in size tion resulting from a high spore concentration on the and shape and they multiply easily by budding (Butt host’s cuticle was observed for C. obscurus (Brey et et al., 1981). Wall-less protoplasts are assumed to be im- al., 1986). It was hypothesized that the self-inhibition portant in the early stages of infection, because these of conidial germination due to crowding could be caused structures are undetectable by host’s defence mecha- by the CO2 produced when conidia respire (Cook & nisms (Dunphy & Nolan, 1982; Brey et al., 1986) Whipps, 1993). This phenomenon of self-inhibited ger- and disease may continue. The protoplastic behaviour mination was not observed for Entomophaga maimaiga by entomopathogens also offers other possible advan- conidia deposited on larval cuticle (Hajek et al., 2002a) tages. (1) The protoplastic form facilitates rapid fungal or Erynia conica conidia on microscope slides (Nadeau growth. Contrary to walled hyphal bodies, the proto- et al., 1996b). Conversely, a percentage of germination plasts feed on pinocytosis so that they feed more ef- was positively associated with conidial density on water ficiently. Moreover, (2) energy for rapid multiplying is agar (Hajek et al., 2002a). not expended in cell wall synthesis (Butt et al., 1981). Germ tube penetrates the aphid integument me- The first hyphal bodies/protoplasts are usually ob- chanically and enzymatically. Lipases, proteases, and served in a fat body (Brobyn & Wilding, 1977; Butt chitinases were reported being produced exocellularly et al., 1981; Kobayashi et al., 1984; Funk et al., 1993). by the fungi (e.g., Gabriel, 1968; Brobyn & Wild- The attachment of fungal structures to the fat body is ing, 1977; King & Humber, 1981; Latgé et al., 1982; probably due to the high concentration of energy found S572 M. Barta & Ľ. Cagáň there. The fat body is a source of nutrients for ini- ter 36 hours aphids become slow-moving and then soon tial fungal growth (Funk et al., 1993). Shortly after become immobile. At this infection stage hyphal bodies the fungus has established in host, most tissues are di- are still wall-less, but the cell wall formation has already gested, but usually tracheoles, gut, and embryos are started. Aphids usually die between 60 and 96 hours only marginally affected (Brobyn & Wilding, 1977; after inoculation. Up to 24 hours after death, cadavers Butt et al., 1990). It is assumed that the protoplas- change in colour and cell walls are completely regen- tic form lacks or ceases the production of enzymes re- erated. Up to next 24 hours hyphae penetrate through quired to digest the cuticle, so the cuticle-enveloped the cuticle to form conidiophores (Brobyn & Wild- tracheoles, gut, and embryos are only slowly digested ing, 1977; Kobayashi et al., 1984; Butt et al., 1990). (Butt et al., 1990). Most entomopathogens depend Host’s death is probably caused by destruction of on the continual feeding of the host to provide water tissues due to secretion of digestive enzymes, physio- and nutrients required by host and pathogen (Humber, logical starvation of the host when the fungus has con- 1991). When the host’s body is occluded and nutrients sumed all reserves, or one or more mycotoxins may be in hemolymph are exhausted, the protoplasts regener- produced that kill the host before the haemocoele may ate a cell wall (Butt et al., 1990). The cell wall regener- have been filled with fungal hyphae (Latgé et al., 1980; ation is probably triggered by specific stimulus/stimuli. Papierok & Coremans-Pelseneer, 1980; Roberts Entomophthora thripidum Samson, Ramakers et Os- & Humber, 1981; Hall & Papierok, 1982; Gillespie wald cultured in artificial media hides a specific factor & Claydon, 1989; Chudare, 1990). that autoinduces the differentiation of protoplasts into the hyphal growth 10–20 days after medium inocula- 6.1.3. Post-mortem processes of pathogenesis tion (Freimoser et al., 2003). In moribund aphids the Post-mortem stage of infection includes the develop- pathogens already fill the body cavity attacking any ment of sterile structures like pseudocystidia and rhi- organs and tissues that remained unaffected through zoids as well as reproductive units like conidia or resting the earlier developmental stages (Brobyn & Wilding, spores. 1977; Butt et al., 1990; Humber, 1991). Shortly after the death of insect, unbranched or After the body is completely filled with hyphal variously branched conidiophores grow out from hy- bodies, the structures start to differentiate into rhi- phal bodies and emerge through exoskeleton covering zoids, pseudocystidia, and conidiophores, however, the most part of the insect’s body except the mid-ventral factors that control the differentiation have been still region. Conidiophores breach the relatively thin cuticle unknown (Brobyn & Wilding, 1977; Butt et al., of abdomen and intersegmental membranes, but not 1990). This formation of specialised hyphal structures the thick cuticle of head and thorax. They break out is preceded by hyphal body concentration beneath the through the host’s cuticle, using a combination of enzy- cuticle and orientation perpendicularly to a surface matic and mechanical means. At a tip of mature coni- (Butt et al., 1990). Rhizoids emerge shortly before diophore a conidium starts to generate. Normally, as or after the host’s death and prior to conidiogenesis the protoplasm of conidiophore passes into the devel- (Brobyn & Wilding, 1977; King & Humber, 1981). oping conidium, the conidium enlarges until its mature They are formed from large, vacuolated hyphal bod- shape and size is attained. It is then separated from ies in the mid-ventral region of the insect (Butt et al., the conidiophore by a transverse septum (MacLeod, 1990). Rhizoids are broad, highly vacuolated, sterile hy- 1963; Brobyn & Wilding, 1977; Eilenberg et al., phae ending in more or less complex holdfast or series of 1986; Latgé & Papierok, 1988; Butt et al., 1990). An short adhesive appendages anchoring an infected aphid important property of most entomophthoralean genera to a substrate (King & Humber, 1981). Rhizoids se- is their ability to actively discharge conidia. The dis- crete a viscous substance (MacLeod, 1963) and ad- charge mechanism was described with different terms here firmly to a plant cuticle, but do not penetrate it (Ben-Ze’ev & Kenneth, 1982a; Balazy, 1993). Pas- (Brobyn & Wilding, 1977; Butt et al., 1990). Not sively detached are only some types of secondary coni- all species form rhizoids. Species of the genera Coni- dia: capilliconidia of the genera Neozygites, Zoophthora diobolus and Neozygites are known not to form rhizoids and Conidiobolus (Ben-Ze’ev & Kenneth, 1982a). (Keller, 1987a, 1991, 1997). However, C. obscurus was Ben-Ze’ev & Kenneth (1982a) distinguished three reported to form a few delicate rhizoids around mouth- different ways of conidial forcible discharge: a “round- parts and forelegs (Brobyn & Wilding, 1977). ing off” characteristic for most genera and “sporophore The lethal time of infected hosts is generally within cannon” in the genus Entomophthora. A discharge of a few days, depending on fungal species, strain, insect conidia in the genus Entomophthora was earlier de- host, and incubation temperature (Milner & Bourne, scribed as a “sporophore canon” when spores were dis- 1983; Schmitz et al., 1993). Adult aphids infected with charged with a stream of cytoplasm from the conidio- the Entomophthorales die 3–6 days after inoculation phore (Ingold, 1971; MacLeod et al., 1976; Humber, (at 20 ◦C) (Wilding, 1969, 1971; Dean & Wilding, 1981; Ben-Ze’ev & Kenneth, 1982a). In recent stud- 1971). Twenty-four hours (at 25 ◦C) after inoculation ies, however, this theory has been rejected and “round- with P. neoaphidis aphids are moving normally. But af- ing off” mechanism of discharge has been suggested Aphid-pathogenic Entomophthorales S573

(Eilenberg et al., 1986, 1995). The general pattern ing summer (Dustan, 1927). It is assumed that prob- of conidial discharge is as follows. Contents of fully de- ably a lowering of temperature or a short period of dry veloped conidium as well as conidiophore bearing the weather at a certain stage of infection development may conidium absorb water rapidly and osmotic pressure supply a stimulus necessary for the formation of these increases in both. However, at conidial maturity the spores (MacLeod, 1963). C. obscurus formed resting osmotic pressure is greater in the conidium and thus a spores in the aphid body at 2 and 6 ◦C and high humid- columella, by which conidium is joined to conidiophore, ity, but when the temperature was higher than 12 ◦C, is forced into the conidiophore. The pressure exerted by conidiophores and conidia were formed on the surface the columella upset is so strong that the outer of the two of the cadaver (Papierok, 1978). N. fresenii usually walls enclosing the conidium ruptures in a circle around forms resting spores in the host’s bodies during dry its base and the conidium is discharged into the air and and hot periods at the postepizootic phase of epizootic carried a considerable distance (MacLeod, 1963). The wave (Barta & Cagáň, 2002, 2003b). The physiologi- conidia can be discharged several millimetres away of cal state of host was also demonstrated that might stim- the cadavers (Six & Mullens, 1996; Hemmati et al., ulate the initiation of resting spore formation. In non- 2001a). The discharge intensity and discharge dynam- feeding aphids, probably unsuitable for optimal fungal ics are dependent on temperature. Within the range of development, the production of resting spores of Z. rad- 10–30 ◦C, a higher temperature results in a more rapid icans was induced (Feng & Johnson, 1991). In nature, discharge of primary conidia (Milner, 1981b; Glare cadavers filled with resting spores usually fall to the soil et al., 1986a; Morgan et al., 1995; Hemmati et al., where they remain during the winter. Early spring tem- 2001a). peratures induce asynchronous germination of resting Pseudocystidia are mostly unbranched or slightly spores and infective conidia are produced over a period branched sterile hyphae enlaced among conidiophores, of several weeks (Latgé & Papierok, 1988). but extended beyond the level of conidiophores (Bro- byn & Wilding, 1977; King & Humber, 1981). Their 6.1.4. Host’s defence responses to the pathogens function has been still a point of debate. They break Insect immunity is a remarkable research area, but lit- through the host’s cuticle before conidiophore arising tle is known of how insects recognize a fungus and their and facilitate the emergence of conidiophores. It is also immune responses are initiated. Commonly, there are assumed that pseudocystidia may help keep a layer of two fundamental defence mechanisms in insect organ- moist air over the body of infected insect, thereby pro- isms, the humoral and cellular responses (Ratcliffe longing a period during which conidia are discharged & Rowley, 1979; Butt et al., 1988; Hajek & St. (Brobyn & Wilding, 1977). Leger, 1994). Most species are able to form resting spores in- The humoral responses are usually characterised side the host body. Two types of resting spores are by melanin deposition on an invading organism. Lo- observed in the Entomophthorales, zygospores or azy- calised cuticular melanization adjacent to fungal ap- gospores. The Zygospores arise from conjugation of two pressoria and penetration pegs is often the first response hyphalbodies(e.g.,inN. fresenii), whereas conjugation detected in insects (Brobyn & Wilding, 1977; Brey is not evident in the production of azygospores (King et al., 1986; Butt et al., 1988, 1990; Humber, 1991). & Humber, 1981). The spores are characterised by a The melanization may alter the mechanical properties thick chitin-protein wall and contain a large oil droplet of cuticle, making it both more rigid (Anderson, 1985) rich in saturated fatty acids (Latgé et al., 1984a). The and more resistant to enzymatic degradation (Kuo & wall has two thick layers and spores lie free within a Alexander, 1967). Melanin is known to inhibit mi- thinner outer wall (episporium). The episporium may crobial enzymes (Kuo & Alexander, 1967) and is be hyaline (e.g., C. obscurus) or coloured (e.g., genus fungistatistic (Söderhäll & Ajaxon, 1982). However, Neozygites), and smooth (e.g., Z. aphidis) or variously the melanization often occurs too late or in insufficient ornamented (e.g., C. coronatus)(King & Humber, magnitude to prevent fungal infection (Butt et al., 1981). The thicker inner layer of the spore wall consists 1988; St. Leger, 1991). Most penetration pegs are able of three sub-layers of different density and structure to breach the melanin collars, deposited around them, (Chudare, 2001). Matured resting spores of C. throm- or grow faster than the melanin could be synthesized boides and Z. radicans are binucleate, but in activated and deposited. It was observed that Z. radicans pro- spores the number of nuclei is reduced to one by fusion duced a further peg from appressorium after the initial and before germination subsequent nuclear divisions infection peg had been completely arrested by a thick result in a multinucleate germ tube (McCabe et al., melanotic capsule (Butt et al., 1988). 1984). Zygospores of the genus Neozygites are also bin- Granulomatous encapsulation by haemocytes is ucleate (Keller, 1997). The circumstances that give the major cellular immune response of insects (Rat- rise to the development of resting spores have been cliffe & Rowley, 1979; Götz, 1986). During the en- imperfectly understood. It was observed that resting capsulation, granulocytes are attracted to the fungus spores start to form toward the end of the growing sea- in the hemolymph and may engulf it in the process son (Thaxter, 1888), but they were also formed dur- of phagocytosis. Then plasmatocytes are recruited and S574 M. Barta & Ľ. Cagáň form a pseudotissue, thus differentiating a granuloma, Perry, 1988; Perry & Fleming, 1989; Nielsen et al., in which the fungus may be lysed (Götz, 1986). As 2001a). In some species short (Ben-Ze’ev et al., 1990) a matter of fact, the encapsulation may only protect or no dormancy (Soper et al., 1975) was observed. The hosts against weakly virulent pathogens, whereas hy- breaking of dormancy is thought to be a very com- pervirulent ones overcome the encapsulation (Hung et plex process controlled by several factors (Ben-Ze’ev al., 1993). et al., 1990; Nielsen et al., 2001a) and for a population Defence responses against the entomophthoralean of resting spores as a whole, the spore germination is fungi are frequently restricted just to the cuticular asynchronous (Hajek & Humber, 1997). The timing of melanization at the sites of fungal penetrations and no the spore germination seems to be correlated with host, obvious cellular immune response mediated by haemo- pathogen, temperature, humidity, and light (Wallace cytesisobserved(Brobyn & Wilding, 1977; Brey et et al., 1976; Perry & Latgé, 1982; Perry & Flem- al., 1986; Butt et al., 1988, 1990). It is because most ing, 1989; Ben-Ze’ev et al., 1990; Thomsen et al., Entomophthorales employ a unique method of how to 2001; Nielsen et al., 2003). Exact requirements for ini- avoid cellular defence responses. They multiply during tiation of germination have not been completely clari- first stages of host colonisation like wall-less protoplasts fied. Not all resting spores germinate during one year (MacLeod et al., 1980; Kobayashi et al., 1984; Butt (Hajek & Humber, 1997), thus creating a reservoir et al., 1981; 1990; Keller, 1997) and therefore do not of resting spores in the soil. For instance, viable rest- attract haemocytes (Dunphy & Nolan, 1982). Sac- ing spores of Entomophaga maimaiga are still present charides and other components secreted by a fungus for in the soil about 11–12 years after epizootic outbreak assembly into cell walls are the key elicitors of insect im- (Hajek et al., 2000). Entomophthoralean resting spores mune responses (Dunphy & Nolan, 1982; Pendland have been tested whether they respond to the presence & Boucias, 1996; Beauvias et al., 1989). of host organisms by their activation from dormancy or stimulation to germination, but no such action has been 6.1.5. Winter survival of Entomophthorales in temper- found (Hajek & Eastburn, 2001). On the contrary, ate region the results of Nielsen et al. (2003) supported the hy- The thought that the Entomophthorales may survive pothesis that the germination of overwintering or qui- a period of adverse conditions for fungal development escent inoculum is stimulated by host-induced factors. in the form of thick-walled resting spores is gener- Weiser & Batko (1966) observed in C. destruens ally accepted (e.g., Balazy, 1993; Keller, 1987a, thick-walled conidia and described them as “loriconi- 1991, 1997). However, not all species have been docu- dia”. They were formed from primary conidia by thick- mented to produce spores. Among the aphid pathogenic ening their cell walls. The structures were supposed fungi resting spores have been described as occurring to substitute the function of typical resting spores. in vivo in all species with the exception of Z. phal- Nielsen et al. (2001a, 2003) observed conidia similar loides, P. neoaphidis, P. kondoiensis,andN. lageni- to those described as “loriconidia” produced on aphids, formis (Keller, 1991; Balazy, 1993; Nielsen et al., which were killed with P. neoaphidis and stored for one 2001a). It can be assumed that the survival strategy month on sterilised soil in darkness at 5 ◦C. From in depends on fungus species and a single species may vitro cultures the thick walled conidia were observed as have more than one possible means of survival avail- well. able. Besides resting spores, other ways of survival have Latteur & Randall (1986) found out that the been observed or supposed to exist, namely specialized primary conidia of P. neoaphidis were able to preserve hyphal bodies (Keller, 1987b; Feng et al., 1992b), viability for 6–8 months at 5 ◦C deposited on soil sur- conidia or “loriconidia” (Weiser & Batko, 1966; Lat- face under dark conditions. Conidia or hyphal bodies teur & Randal, 1986; Nielsen et al., 2003), or a sur- of P. neoaphidis depositedonsoilsurfacewereableto vival through continuous conidial infections in the an- produce replicate conidia or sporulate, depending on holocyclic aphid populations (Byford & Reeve, 1969; temperature, for 23–96 days and the inocula retained Wilding, 1973; McLeod et al., 1998). the ability to initiate infections in aphids after storage The main reservoir of resting spores is soil (Co- for 14–64 days (Nielsen et al., 2003). However, conidia remans-Pelseneer, 1981), but resting spores may on plant leaves remain their infectivity only up to one also be deposited on a tree bark (Hajek et al., 1998; month at 5 ◦C(Schofield et al., 1995) or two weeks Barta & Cagáň, 2006), or in dead aphids attached in field conditions (Brobyn et al., 1985). In general, at to a tree top (Soper & MacLeod, 1981; Barta & low relative humidity conidia lost their viability very Cagáň, 2006). Thick-walled resting spores are not di- fast. Conidia of the pathogen deposited on a glass sur- rectly infective, but they may germinate and form infec- face lost their viability after four hours at 20 ◦Cand tive conidia (Coremans-Pelseneer, 1981; Humber, 75% of relative humidity (Barta, 2004). 1991). Resting spores of most species to germinate have Hyphal bodies of P. neoaphidis in aphid cadavers to undergo a period of obligatory dormancy, which can were able to survive a period of several months under be broken by storage for 2–4 months in humid cold con- dry and cold conditions (Wilding, 1973; Courtois & ditions (Bitton et al., 1979; Perry & Latgé, 1982; Latteur, 1984; Latteur et al., 1985). Feng et al. Aphid-pathogenic Entomophthorales S575

(1992b) found peculiar spherical hyphal bodies distin- aphid populations was presented by Dedryver (1983). guishable from the regular ones. The spherical hyphal He classified three types of mycoses in the aphid popu- bodies appeared only in autumn and they were able to lations by putting into relationship the number of living overwinter inside aphid cadavers and produce infective aphids and aphids killed by the Entomophthorales in a conidia after winter. P. neoaphidis survived the winter population. In the first type, epizootic type, the num- in the form of hyphal bodies on plant substrates above ber of infected aphids increases in populations more ground in relatively dry environments, but not in the quickly, as compared with the number of living aphids. moist soil. Such a type of hyphal bodies has also been The number of living aphids diminishes and that of in- documented for another aphid pathogen. In autumn, fected aphids continues to increase to exceed a ratio of E. planchoniana produced thick-walled hyphal bodies infected/living aphids 1/1. In the second type of my- in cadavers of Drepanosiphum acerinum in Switzerland cosis, enzootic type, the number of infected aphids re- even though resting spores are known from this species main approximately proportional to the number of liv- (Keller 1987b). In spring, it was possible to infect ing aphids, both in the population growth period and healthy aphids with E. planchoniana from aphids con- in their diminution phase. In most cases, the ratio of taining the thick-walled hyphal bodies. infected/living aphids is 1/100 during the population Finally, P. neoaphidis was also suggested that it growth phase and 1/10 during their reduction. In the may survive winter month in anholocyclic aphid pop- third type of mycosis, there is a growth and then a re- ulations via continuous conidial infections (Byford duction in the number of living aphids, while that of & Reeve, 1969; Wilding, 1973; Remaudiere` et al., infected aphids remains constantly low. 1981; Dara & Semtner, 2001). Steinhaus (1954) divided the epizootic wave into three phases: the preepizootic phase, the period just 6.2. Epizootiology of Entomophthorales in preceding an increase in numbers of infected insects, aphid populations which results in the epizootic phase itself, the climac- tic portion of epizootic wave followed by postepizootic 6.2.1. Types of disease prevalence in aphid populations phase, which is characterised by at least a few surviving A disease, expressed as an unbalanced state of physio- insects giving chance for the population survival and a logical equilibrium of organism, can be caused by nu- new population development. merous biotic and abiotic factors. The insect epizootiol- ogy concentrates on infectious diseases brought about 6.2.2. Factors governing disease prevalence and deter- by infective (biotic) agents. However, there may be a mining disease character complex of factors, including environmental conditions, Primary factors that are involved in the initiation and contributing or predisposing of insect to a susceptibility development of epizootics of infectious diseases in insect to the biotic agents and thus they should be considered populations are a pathogen population, a host popu- in epizootiology (Fuxa & Tanada, 1987). lation, and an efficient means of pathogen’s transmis- The intensity and extent of the interaction between sion. Since the pathogen – host interaction exists within the host and pathogen populations determine the state an environment characterised by its abiotic and biotic of disease prevalence which is generally epizootic or en- properties, the elements of the environment also inter- zootic. The definition of epizootic disease could be ex- fere in a disease development. pressed by different ways. It can simply be said that there is an unusually large number of cases of disease in 6.2.2.1. Host populations host populations (Barr, 1979). However, to know what There are a number of aphid population properties is an unusually large number, it is necessary to have which can determine a disease character. The most knowledge about the past history of the disease for a significant in the epizootics of entomophthoralean dis- number of years in particular host populations (Fuxa eases seems to be susceptibility of host population to & Tanada, 1987). Enzootic disease is a case of usu- pathogens, density or behaviour of insect population, ally low prevalence of infections, which are constantly and relationships among different co-occurring host present in populations. A certain sort of balance and populations in the environment (Watanabe, 1987). coexistence between pathogen and host is characteris- Many reports point out differences between two or tic of enzootic disease (Van der Plank, 1975; Fuxa more isolated populations of insect species in respond- & Tanada, 1987). While epizootics are sporadic, time- ing to fungal pathogens. With respect to aphids, Pa- limited and characterized by a sudden change in preva- pierok & Wilding (1979) reported differences in re- lence and incidence of case of infection, enzootic disease sponse to infection by fungus C. obscurus between two levels are of long duration and the ratio of daughter clones of pea aphid. Likewise, Milner (1982, 1985) to parent infections approximately equals one. During determined two biotypes of pea aphid with distinct epizootics the ratio of daughter and parent infections susceptibility to P. neoaphidis in Australia. A cer- significantly exceeds one (Van der Plank, 1975). tain clonal variation of pea aphid in resistance to P. A clear-cut distinction between enzootic and epi- neoaphidis was also found by Ferrari et al. (2001). zootic state of entomophthoralean disease prevalence in Evidently, a variety of endogenous and exogenous fac- S576 M. Barta & Ľ. Cagáň tors is involved in aphid susceptibility, which may act ing & Perry, 1980; Chudare, 1990). Rather aggre- together, e.g., age, moulting, food, and environmental gate populations typical of aphids facilitate inoculum factors. Several reports show that insect larvae decrease dispersal within colonies and thus promote the rapid in susceptibility to fungal infection as they age; not all spread of disease (Soper & MacLeod, 1981). High stages of host development are of even susceptibility host densities in colonies increase the contact between to a mycosis. Nymphs of pea aphid are more suscepti- uninfected and infected hosts and enhance the prob- ble to a fungal infection than adults. Moreover, alate ability of horizontal transmission of infection (Stein- adults are more susceptible than apterous adults (Mil- haus, 1954; Chudare, 1990). However, the mean aphid ner, 1982, 1985; Lizen et al., 1985). Alate adults from density by itself is usually not appropriate indicator three to four days of age are the most resistant to in- for prediction of entomophthoralean infection develop- fection (Lizen et al., 1985). Moulting may remove pen- ment and the degree of host aggregation must be con- etrating fungus prior to the colonisation of insects if sidered (Soper & MacLeod, 1981). Aphids forming it occurs shortly after inoculation (Vey & Fargues, aggregate colonies on goldenrod stems with clumped 1977; Fargues & Rodriguez-Rueda, 1979). Suscep- spatial pattern within goldenrod fields were more vul- tibility demonstrated in laboratory may not relate to nerable to N. fresenii infection than aphids living in that in field, suggesting differences between physiolog- sparse colonies and forming more random spatial pat- ical and ecological susceptibility. For instance, aphids tern (Cappuccino, 1988). The effect of fungal dis- are not attacked by P. delphacis in nature, but they eases on spatial patterns of aphids was also studied. can be inoculated with the fungus in laboratory (Shi- No difference was detected in spatial distribution on mazu, 1977). As a matter of fact, susceptibility relates cereals between infected and living aphids (Feng & to both the physiology of insects and their behaviour. Nowierski, 1992). The presence of alternative hosts Behavioural habit of host may encourage or discour- in insect community may also contribute to an increase age the acquisition of conidia by insects (Roy et al., in inoculum density and dispersal, resulting in the in- 1998; Hajek, 2001). As far as aphid’s behaviour is con- creased infection in primary hosts. Sympatric aphid cerned, it was observed that M. dirhodum and D. noxia colonies of A. pisum and A. kondoi were observed to were predominantly infected by P. neoaphidis proba- be differentially infected by P. neoaphidis in California. bly because the aphid species inhabit more humid mi- While P. neoaphidis killed few individuals of A. kon- croenvironments in cereal crops such as undersurface of doi, the pathogen density increased in more susceptible lower leaves or tightly rolled recently developed leaves A. pisum. It was assumed that the low-level infections (Dean & Wilding, 1971; Dedryver, 1983; Feng et in A. kondoi might increase the inoculum available for al., 1991; Feng et al., 1992a). S. avenae primarily in- transmission to the highly susceptible A. pisum (Pick- festing the ears of cereals, the microhabitat with lower ering & Gutierrez, 1991). It is assumed that not humidity, was less infected by P. neoaphidis in com- only pestiferous aphids, but also non-pest aphids in in- parison to C. obscurus, which is more tolerant to lower sect communities may play a role of alternative hosts humidity (Wilding, 1969; Dean & Wilding, 1971; for the Entomophthorales (Steenberg & Eilenberg, Feng et al., 1992a). Bioassay with Z. radicans against 1995; Barta & Cagáň, 2003b). Keller & Suter Therioaphis trifolii f. maculata indicated that aphids (1980) showed that epizootics in pestiferous aphid pop- that starved for 24 hours during inoculation with pri- ulations on several crops began after a large proportion mary spores were less susceptible than those that were of various aphids on weeds within and around the crop removed from plants for just the period of exposure to had been infected. The influence of weeds on aphid en- the primary spore shower (Milner & Soper, 1981). emies including the Entomophthorales was studied in The results imply that during initial stages of infection England as well (Powell et al., 1986a). The competi- a highly nutritious environment is required for hyphal tion between two aphid species via entomophthoralean bodies to multiply rapidly in the hemolymph. A study pathogens was also discussed (Pope et al., 2002). Low- of fungi – parasitoids interference in aphid host revealed specific fungi like Z. radicans,whichcanbeisolated that parasitoid developing inside the aphid’s body may from various insect orders, could disperse theoretically predispose the host to the infection by P. neoaphidis among different insect populations in the environment. (Powell et al., 1986b). However, a cross-transmission of infection in labora- A dissemination of pathogens within a host pop- tory is usually scarce and has not been observed in na- ulation or among different host populations plays an ture (Milner & Mahon, 1985), since an adaptation important role in the development of epizootics. In ad- of fungal strains to the host taxonomically related to dition to the mechanical factors such as rain and wind the host from which they were isolated probably exists (see Section 6.2.2.4.), the biotic factors including pop- (Papierok et al., 1984). Nevertheless, some isolates of ulation density or the behaviour of infected individu- Z. radicans originating from non-Homopteran hosts es- als can also affect inoculum spreading. Aphids them- tablished their infectivity to aphids (Papierok et al., selves may serve as biotic agents for inoculum disper- 1984; Milner & Mahon, 1985). sal. Aphids may spread infection among colonies ei- Parasitic fungi act as the density-dependent mor- ther in the same crop or among different crops (Wild- tality factors if the pathogens operate under favourable Aphid-pathogenic Entomophthorales S577 weather conditions (Steinhaus, 1954; Watanabe, fungi on the behaviour of their hosts is a characteristic 1987) and a correlation between fungal infection and property of these fungi, this is still a part of unexplored host density was found (Wilding & Perry, 1980; area, especially in the aphid – pathogen systems. Feng et al., 1991). Since the threshold host density at Fungal pathogens may be distributed within the which the fungi start to be effective is generally high, host population or among the populations not only by epizootics in aphid populations usually occur at high infected hosts themselves, but also by parasites and host densities (densities over the economic threshold) predators active within the host colonies. Parasites and (Sivčev, 1991; Pell et al., 2001; Barta & Cagáň, predators of aphids come in contact with their prey and 2002). In Slovakia, N. fresenii attacked the black bean may acquire inoculum from the environment and thus aphid colonies only after they had become damag- serve as passive vectors of disease among populations ingtofieldbeanorsugarbeet. The action thresh- (Poprawski et al., 1992; Roy et al., 2001) (see Section old for field bean and sugar beet was when 5% of 6.2.2.3.). plants were infested with aphids, but the infection ap- peared as late as the infestation rate had reached 80% 6.2.2.2. Pathogen populations and 40% for field bean and sugar been, respectively Major biological properties of fungal pathogens in- (Barta & Cagáň, 2002). However, this is not always volved in causing diseases in insect populations are vir- true. P. neoaphidis epidemic in the pea aphid popula- ulence, pathogenicity, pathogens’ multiplication, patho- tions, for instance, was maintained at a density below gens’ population density, dispersal, spatial and tem- four hosts per stem (Pickering & Gutierrez, 1991). poral distribution, as well as persistence in nature For Schizolachnus piniradiatae (Davidson), a threshold (Tanada & Fuxa, 1987; Pell et al., 2001). level of 50–80 aphids per 30-cm branch of pine tree was Infectivity, the ability of fungus to attack the needed before an epizootic of Z. canadensis (Soper & host and cause infection, varies with different fun- MacLeod, 1981). gal pathogens (Papierok, 1982). A number of factors Specific behavioural modifications of infected in- are involved in infectivity, including physiology of host sects may have an effect on dissemination of the (e.g., defence mechanism), and physiology of fungus pathogen. For instance, it was observed in some host- (e.g., production of enzymes and toxins). Virulence, the pathogen systems that infected insects tend to move ability to invade and injure host’s tissues, is often mea- upwards just before death and die on the top of sured by a response of the host to a known inoculum plants (e.g., Batko, 1974; Müller, 1993). Similar phe- dose, mostly death from a median lethal concentration nomenon was observed for some Entomophthorales in LC50 (Papierok, 1982). Values of LC50 range from few aphid populations as well (Rockwood, 1950; Evans, to hundreds of conidia per mm2, depending upon fun- 1989; Jensen et al., 2001). In case of aphids, the po- gal species and isolates within particular species (e.g., sition of infected aphids on host plant depends in all Papierok & Wilding, 1979; Milner & Soper, 1981; probability on the aphid species (Roy et al., 2002). Oger & Latteur, 1985; Feng & Johnson, 1991; Xu In laboratory experiments, cadavers of S. avenae were & Feng, 2000; Shah et al., 2004; Barta, 2004). Strains found higher on wheat than uninfected individuals, but of certain fungal species differ in their virulence. Latgé this was not the case for pea aphid cadavers (Roy, 1997 et al. (1982, 1984b, 1986b) reported two groups of C. in Pell et al., 2001; Roy et al., 2002). Aphids killed obscurus strains with different aggressiveness to pea by C. obscurus were more often found on the lower aphid. Likewise, Papierok & Wilding (1981) demon- parts of plants, but it was ascribed to more favourable strated two types of strain of C. obscurus differing in microhabitat for infection at ground level (Wilding, their physiological and infective properties and Dumas 1975; Dedryver, 1981). Even infected aphids are sig- & Papierok (1989) isolated two strains of Z. radi- nificantly more likely found on the undersides of leaves cans on the same day in the same locality, which var- than healthy aphids, which can be benefiting for the ied greatly in their virulence against adult mosquitoes. fungus because of not only a better position for coni- Insect pathogenic fungi may produce toxins that en- dial dissemination but also a protection from insolation hance their pathogenicity (Papierok & Coremans- (Jensen et al., 2001). Killed aphids were also found Pelseneer, 1980; for review see Vey et al., 2001). A more likely off the plants than were healthy aphids rapidly replicating pathogen is generally more virulent (Jensen et al., 2001). Entomophthoralean fungi may than species that replicates slowly in the host, though influence hosts’ behaviour in several ways. Roy et al. other factors may also play an important role (Tanada (1999) noticed that infected aphids less likely dropped & Fuxa, 1987). from a plant in response to alarm pheromone, demon- Pathogen population density is one of the most im- strating a decrease in aphids’ mobility. Infected aphids portant factors that determine if a disease develops to were unable to respond to alarm pheromone, though epizootic. Most entomopathogens gain entry to haemo- they could still produce it. On the other hand, A. pisum coele by penetrating the host’s cuticle, using a com- infected by P. neoaphidis demonstrated a behavioural bination of hydrolytic enzymes and mechanical forces response to the presence of foraging coccinellids (Roy (Butt et al., 1990; Hajek & St. Leger, 1994). The et al., 2002). Despite the effect of entomophthoralean speed of kill is influenced by the number of infective S578 M. Barta & Ľ. Cagáň propagules in contact with the host cuticle (Butt & process. Air-dispersed conidia in the space above crops Goettel, 2000). Pathogen population density relates demonstrated infectivity to aphids (Steinkraus et al., to some characteristics inherent to pathogen such as re- 1999). In many studies the pathogen population den- productive rate and capacity to survive, and also some sity during epizootics was chosen by multiple regression environmental factors such as wind and rain (see Sec- analyses as one of the most important factors in fun- tion 6.2.2.4.) (Tanada & Fuxa, 1987). The time from gal disease prevalence (e.g., Wilding, 1975; Soper & the initial aphid contact with conidium to death of MacLeod, 1981; Coremans-Pelseneer et al., 1983; the host and subsequent fungal sporulation is relatively Cappuccino, 1988; Steinkraus et al., 1996b, 1999; short. This can be as short as 3–6 days, depending Hemmati et al., 2001b). on fungus species, host, and external conditions (e.g., In order to effectively utilize entomopathogens as Wilding, 1969; Dean & Wilding, 1971; Brobyn & agents for biocontrol of aphids, it is also necessary to Wilding, 1977; Steinkraus et al., 1993). Generally, understand the biotic and abiotic conditions, which relatively fewer numbers of conidia are produced per ca- makes the dispersal and spatial distribution of infection daver in the Entomophthorales, when compared with, easier. Pathogenic fungi rely mainly on the physical and for example, Hyphomycetes. On the other hand, fewer biotic factors for their dispersal and, in general, they conidia are required to initiate the entomophthoralean have a very limited capacity to disperse through their infection (Pell et al., 2001). Thousands of conidia can own actions (Tanada & Fuxa, 1987). However, the be released from a single host, with numbers depend- Entomophthorales possess conidiophores that forcibly ing on cadaver biomass and temperature at sporulating discharge conidia by hydrostatic pressure (Ben-Ze’ev (Wilding, 1971; Glare & Milner, 1991; Dromph et & Kenneth, 1982a), which effectively facilitates inocu- al., 1997; Hemmati et al., 2001a). About 3,000 of pri- lum spreading (Pell et al., 2001). Values for maxi- mary conidia of N. fresenii are discharged per infected mum discharge distance of primary conidia from aphid cotton aphid and approximately 3/4 of these conidia surface are generally between 6 and 9 mm (Hemmati enter the air, while 1/4 immediately hit the leaf, on et al., 2001a). The conidia of Entomophthora muscae which the cadaver is located (Steinkraus et al., 1993). (Cohn) Fresenius were even discharged up to 87 mm Conversely, on average 50,000 to 60,000 conidia can be from a killed fly and average discharge distance was discharged from larger aphid species (Wilding, 1971; positively related to a cadaver size (Six & Mullens, Glare et al., 1986a; Sivčev, 1993). In optimal condi- 1996). Conidia, when discharged into the air, are car- tions, the released conidia deposited on any surface ger- ried by wind currents, which are the most important minate within a few hours to penetrate the host integu- physical factor for dispersal (Pell et al., 2001). Pro- ment or to form secondary conidia (Wilding, 1971; duction of replicate conidia may also increase the dis- Brey et al., 1986; Steinkraus et al., 1993). At satu- persal capacity (Pell et al., 2001), but in some genera, rated humidity, the mean time for infection is 4.5 hours e.g., Zoophthora, Neozygites, certain types of higher- at 20 ◦C(Glare & Milner, 1991). The pathogen den- order conidia are not actively discharged (Ben-Ze’ev & sity or dosage can cause different types of disease that Kenneth, 1982a; Keller, 1997). Cadavers of aphids in turn could affect epizootiology. For example, heavy killed by some species are fixed to plant surface, which doses can cause mortality more quickly than light ones. also enhances chances for the dispersal of conidia (Pell Rapid mortality, on the other hand, reduces the replica- et al., 2001). Some species released conidia with slime tion of pathogen and the subsequent pathogen density coats that serve in attachment to insects as well as in when the insect host dies (Tanada & Fuxa, 1987). dispersal (Brey et al., 1986; Boucias & Pendland, Little is known about the critical inoculum threshold 1991). Modified behaviour of infected hosts may also of pathogen population at which an epizootic can de- contribute to more effective dispersal of pathogens (see velop in aphid populations. Aerobiology and density Section 6.2.2.1.). Spatial distribution of the infective in- of airborne conidia were studied in naturally occurring ocula of Entomophthorales was studied in agroecosys- epizootics. During N. fresenii epizootics in the cotton tems (Steinkraus et al., 1996b, 1999; Hemmati et al., aphid populations, the number of conidia present in the 2001b). Temporal distribution of infection units in the air is immense. Up to 90,000 of primary conidia per cu- host’s habitat is as important to the development of epi- bic metre of air can be collected above cotton fields and zootics as the spatial distribution. Susceptible host and active discharge of conidia can be observed mainly be- infective pathogen must make contact in both space and tween 01:00 and 03:00 a.m. (Steinkraus et al., 1996b, time for infection to occur (Tanada & Fuxa, 1987). 1999). In the cereal aphid populations, the highest num- The time from conidiogenesis start to the conidial dis- ber of P. neoaphidis primary conidia was recorded in charge may significantly influence the chance that in- the air over fields between 04:00 and 08:00 a.m. when fection occurs. The conidia of P. neoaphidis produced temperatures were between 10 and 16 ◦C and humidity between the first and the third hour of conidiogenesis was greater than 90% (Hemmati et al., 2001b). These are more infective than those produced later (Latteur studies show that conidiogenesis and sporulation take et al., 1985). Numbers of conidia of certain Entomoph- place when the external conditions such as temperature, thorales discharged in the air have characteristic daily humidity and light are the most appropriate for the (Wilding, 1970a; Harper et al., 1984; Milner et al., Aphid-pathogenic Entomophthorales S579

1984; Steinkraus et al., 1996b, 1999; Hemmati et al., further highlighted the importance of higher moisture 2001b) as well as seasonal patterns (Harper et al., on the underside of leaves for a longer persistence. In 1984). The diurnal periodicity in conidial production field conditions, the primary conidia of Z. radicans on depends on environmental conditions, mainly on light foliage or soil lost their viability within 24 hours (Fur- and relative humidity. The fungi produce conidia dur- long & Pell, 1997). Infectivity of C. obscurus conidia ing the early morning hours when light conditions, hu- deposited on the surface of non-sterile soil persisted, midity, and temperature are optimum. At this time an depending upon temperature, from two days to two inoculum transmission to susceptible hosts must occur, months. The lower temperature of storage (within a since in daylight the conidia succumb to ultra-violet ra- range of 5 to 20 ◦C), the higher persistence of infectiv- diation and higher temperature (Aoki, 1981; Harper ity was recorded (Latteur, 1980). The author specu- et al., 1984; Milner et al., 1984; Steinkraus et al., lates whether the conidia discharged in autumn could 1996b, 1999; Hemmati et al., 2001b; Wraight et al., survive winter and initiate a life cycle in spring. The 2003). During winter and early spring months the envi- conidia of C. coronatus survived on cotton leaves un- ronment is, theoretically, free of infective inocula. The der humid conditions for 70 days, but in glasshouse main source of the infective units are resting spores de- conditions they remained viable just for 10–14 days posited in the soil and/or on tree bark (Bitton et al., (Gindin & Ben-Ze’ev, 1994). Capilliconidia are more 1979; Coremans-Pelseneer, 1981; Perry & Latgé, resistant to ultraviolet radiation (Furlong & Pell, 1982; Hajek et al., 1998). The resting spores have to 1997; Uziel & Shtienberg, 1993) and lower humid- germinate to produce germ conidia and thus to provide ity (Steinkraus & Slaymaker, 1994) than conidia. If an adequate density of inoculum in the environment, abiotic conditions are not favourable for conidiogenesis, a fundamental prerequisite for disease initiation. Early the aphid cadavers desiccate and mycelium may tem- spring temperatures induce asynchronous germination porarily persist in this anhydrobiosis to rehydrate and of resting spores, which produce infective conidia over recover after a humid period comes (Glare & Milner, a period of several weeks. These spores infect the first 1991; Behrens, 1993). The resistant stages of the En- aphids occurring in crops (Perry & Latgé, 1982). The tomophthorales – the resting spores – end up in the soil resting spores germinate under certain environmental where they remain during the winter. Soil is a highly conditions after they have undergone a stage of dor- favourable environment for the entomopathogen sur- mancy (Bitton et al., 1979; Perry & Latgé, 1982; vival and is a natural reservoir of entomophthoralean Ben-Ze’ev et al., 1990) and the period of dormancy resting spores (Coremans-Pelseneer, 1981; Latgé is influenced by temperature and humidity (Perry & & Papierok, 1988; Nielsen et al., 2003). Aphids and Latgé, 1982). For instance, 4-monthly storage of C. other insects can be infected with soil samples collected obscurus resting spores at 4 ◦C and 95% of relative hu- in place where the mycoses were active during the pre- midity is necessary to break their dormancy (Latgé et vious growing season, which validates the theory of soil al., 1983). In Israel, it was observed that the resting as a pathogen reservoir (Latteur, 1977; Hajek et spores of N. fresenii were synchronized to germinate in al., 2000; Nielsen et al., 2001a, 2003; Mi˛etkiewski spring concurrently with a build-up of aphids on citrus & Tkaczuk, 2005). The persistence of these spores (Bitton et al., 1979; Ben-Ze’ev et al., 1990). in the soil has a long-term significance for epizootics. Pathogens may persist and survive in abiotic envi- Many resting spores do not germinate in the first year ronments (in the stage of resting spores or conidia) (e.g., after production and can remain viable in the soil for a Bitton et al., 1979; Latteur, 1980; Nielsen et al., very long time (Latgé & Papierok, 1988; Weseloh 2003) and biotic environments of host’s habitat (in the & Andreadis, 2002). stage of hyphal bodies) (Keller, 1987b; Feng et al., 1992b; Nielsen et al., 2003). The factors in the abiotic 6.2.2.3. Transmission of infection environment above the soil surface affecting most sig- Transmission may be defined as a process by which a nificantly the persistence of fungi are sunlight (mainly pathogen or parasite is passed from a source of infec- ultraviolet light), temperature and humidity. There is tion to a new host. This process can be either direct considerable information on the effect of these factors when the fungus is transferred from the infected host on the pathogens. In general, the entomophthoralean to the susceptible one without an intervention of any conidia are relatively fragile and short-lived, but they living agent or indirect when one or more species of can germinate quickly and produce replicate conidia, intermediate hosts or vectors are involved. The most thus prolonging their persistence (Aoki & Tanada, prevailing transmission form among fungal pathogens 1974; Pell et al., 2001). Brobyn et al. (1985) demon- is the direct one. The knowledge of transmission path- strated that the survival of P. neoaphidis primary coni- ways is fundamental to understanding disease dynam- dia on leaves was greater when conidia were deposited ics and insect epizootiology (Andreadis, 1987). Trans- on underside (over 7 days) than on the upper surfaces mission is normally divided into two categories accord- (up to 3 days) of leaves where the fungus was not pro- ing to a manner, in which the pathogen is transferred tected from sunlight. This is also in agreement with within the host population. Transmission is horizon- the results of Furlong & Pell (1997). The authors tal when the fungus is transferred from individual to S580 M. Barta & Ľ. Cagáň individual by physical contact and it is vertical when tively, trapped from air were infected by entomophtho- there is a direct transfer of the pathogen from parent ralean infection, predominantly by P. neoaphidis (Feng to its progeny (Kůdela & Polák, 1999). The Ento- & Chen, 2002; Chen & Feng, 2004a). The infected mophthorales rely on horizontal routes of transmission aphids were able to fly for five hours (9 km), to initiate and thus the fungi are dependent on host density for colonies on host plants after the flight and to trans- their survival and dispersal (Wilding & Perry, 1980; mit fungal infection to their progeny (Chen & Feng Pell et al., 2001). The reliance on horizontal trans- 2004b; Feng et al., 2004). However, a post-flight fecun- mission means that the pathogen must either maintain dity of diseased aphids was greatly reduced due to infec- itself within the living host or cadaver throughout the tion by P. neoaphidis (Chen & Feng, 2006). Infected year or produce a resistant stage which is capable of aphids themselves produce a lover number of progeny surviving in the environment when susceptible stages of than healthy ones (Baverstock et al., 2006). A pos- the host are absent. In such host-pathogen relationships itive correlation between numbers of infected aphids the disease prevalence is usually density-dependent and and live alate ones in colonies of pea aphid confirms increases steadily or dramatically during the host pop- this way of infection transmission (Cagáň & Barta, ulation culmination (Andreadis, 1987). Vertical mode 2001). There exists a hypothesis for dioecious aphids of transmission is not expected to be in the Entomoph- that fundatrices, occupying primary hosts, might be a thorales parasiting aphids, since embryos are not vul- source of fungal inocula for summer aphid generations nerable to the infection and a transovarial transmission on secondary hosts. However, this was not indicated in is therefore disabled (Butt et al., 1990). The factors af- a study on R. padi populations because of very poor fecting transmission of fungal pathogens of aphids were fungal activity in colonies of fundatrices during the discussed by Steinkraus (2006). springs. Fungi did not establish themselves in spring Dispersal or dissemination is defined as the capac- generations of fundatrices thus the spring generations ity of a fungus to spread and distribute itself within of aphids did not play a role in disease transmission to a host population and throughout the environment. new colonies on summer hosts – cereal crops (Barta & This process within pathogen-aphid system is partly Cagáň, 2004). This phenomenon was noticed for other discussed from a viewpoint of host (see Section 6.2.2.1.) dioecious species as well (Barta & Cagáň, 2006). Few and pathogen (see Section 6.2.2.2.). The successful long- studies considered whether entomophthoralean species term persistence of pathogens within a host popula- could move freely between different aphid species and tion is related to its ability to disperse. Pathogens spread fungal infection in the agroecosystem (Milner with low dispersal capabilities have a very low poten- et al., 1983; Steenberg & Eilenberg, 1995; Barta & tial for developing epizootics even though they may be Cagáň, 2003b). Arthropod enemies of aphids and ento- highly virulent or survive effectively in the environment mopathogenic fungi co-exist in large aphid populations (Tanada, 1963). There are several ways in which en- in the same habitats. There are examples in the litera- tomophthoralean fungi are disseminated within a host ture when hymenopteran parasitoids or predators acted population or among host populations in nature: by as passive vectors of entomophthoralean fungi to host their own actions, by the behaviour and movements of populations during foraging (Poprawski et al., 1992; infected hosts, by the behaviour of non-host carriers, Pell et al., 1997a; Roy et al., 2001), but there are also and by climatic and physical agents (e.g., Chudare, examples where this did not occur (Furlong & Pell, 1990; Jensen et al., 2001; Pell et al., 2001; Roy et 1996; Fuentes-Contreras et al., 1998). The extent al., 2001). The extent of entomophthoralean dissemi- to which parasites and predators contribute to the dis- nation depends presumably upon: (1) density of host semination of disease and development of epizootics population (an aggregation of population), since the varies with each individual host – parasite – pathogen higher density of host, the greater probability of hori- system. The production of resting spores during un- zontal transmition by contact of healthy and diseased favourable conditions ensures the survival of these fungi aphids; (2) concentration of fungal inocula in the envi- over periods when no susceptible hosts are present and ronment; (3) physiological state of host organism, and provides a mode of dispersal to new host populations (4) pathogen properties (Chudare, 1990). The forcible in subsequent years (Perry & Latgé, 1982). Actions discharge of conidia from infected insect is an impor- of climatic and physical agents may also result in an in- tant method of autodissemination among entomoph- creased prevalence of disease within a host population thoralean fungi (Pell et al., 2001). Discharged conidia by bringing the pathogen into contact with new hosts are anemochorous. They enter the air current above (Pell et al., 2001), or in a decreased prevalence of dis- crops and can be passively dispersed (Hemmati et ease by removal of inocula from the environment, which al., 2001b). Distinctive movements or migration of dis- the host population inhabits (Pell et al., 1997b). eased aphids also affect the pathogen dispersal within a host population (Rabasse & Robert, 1975; Wild- 6.2.2.4. Environmental factors ing & Perry, 1980; Chudare, 1990; Feng et al., Since Entomophthorales are transmitted horizontally in 2004; Chen & Feng, 2006). 30% and 35% of migrantes the environment, they are considerably dependent upon alatae of cereal aphids and green peach aphid, respec- environmental conditions. Environmental factors may Aphid-pathogenic Entomophthorales S581 influence directly or indirectly the trio of primary fac- sporulates at temperatures from 11 to 25 ◦C(Sivčev, tors contributing to the epizootics of insect diseases: 1993) and not above 30 ◦C(Sivčev, 1993; Sivčev & host population, pathogen population, and means of Manojlovic´, 1995), which corresponds with the re- transmission. Environmental factors do not act sepa- sults of Shah et al. (2002). The greatest numbers of P. rately but as a whole complex. Therefore, the analysis neoaphidis conidia per cadaver are released at a change- of single environmental factor on epizootics is not al- able temperature regime (Sivčev, 1993). The germina- ways successful. Environmental factors, especially cli- tion percentages of P. nouryi conidia on the surface of matic factors, act on the capacity of the pathogens to water-agar were significantly lower at 8 and 30 ◦Cthan survive in the biotope, pathogen germination, conidial at 15–25 ◦C(Li et al., 2006). Optimum temperatures for discharge, as well as on their hosts (e.g., Glare & Mil- the conidial germination of the Entomophthorales are ner, 1991; Hemmati et al., 2001a). The detrimental ef- between 18–20 ◦Cand25◦C(Wilding, 1971; Glare & fects of high temperature, desiccation, and sunlight on Milner, 1991; Li et al., 2006). Temperature may in- free pathogens are well known. Species with a resting fluence not only the number of conidia produced from spore stage in their life cycles are better able to survive aphid cadavers and the germination of conidia but also and persist in the environment (e.g., Wilding, 1969, the maximum discharge distance of conidia. Greater 1970b; Brobyn et al., 1985). numbers of conidia are produced and they are shoot off Temperature is one of the principal environmen- further at 18 ◦C, lower and higher temperatures reduc- tal factors influencing activity of any living organ- ing these properties (Glare & Milner, 1991; Dromph ism. Temperature is a factor that effectively influences et al., 1997; Hemmati et al., 2001a). Species of the conidiogenesis, germination of conidia, and incubation family Neozygitaceae are unusual in that they are com- time of entomophthoralean infection (e.g., Wilding, mon and more active in hot weather. The fact that 1971; Milner & Bourne, 1983; Milner & Lutton, Neozygitaceae are adapted to hot and humid condi- 1983; Glare & Milner, 1991). The study of these ef- tions has been reported by many researchers (Gustafs- fects is complicated because the developmental rates son, 1965; Steinkraus et al., 1991; Keller, 1997). of both pathogen and aphids are regulated by tem- High temperatures that could occur in nature have only perature. If the temperature favours a quick genera- rarely been used in laboratory tests. High temperatures tion time for aphids, but is above or below the op- may, for instance, occur on the soil surface on hot sum- timum for the pathogen, the insect population may mer days (Benz, 1987). Temperature conditions in the still build-up to crop-damaging levels. Conversely, if top few centimetres of soil may range over 40 ◦Cbe- temperatures favour a brief incubation period for the tween daylight and night hours, and temperatures over pathogen, but retard insect development, epizootics can 50 ◦C can occur (Carruthers & Soper, 1987). Un- result (Benz, 1987). Many data on the influence of fortunately, there are also only few data on the heat temperature on diseases were collected under labora- resistance of fungi. Krejzová (1971b) reported that tory conditions. The mean optimum temperature for the hyphal bodies of C. obscurus lost their viability at entomopathogenic fungi is normally between 20 ◦Cand 40 ◦C. Resting spores do not germinate when exposed 25 ◦C, with a maximum of about 35 ◦C and a minimum to a temperature of 60 ◦C for 240 minutes, or of 100 ◦C of about 5–10 ◦C(Zimmermann, 1986). The time to for 15 minutes. The resting spores of C. thromboides kill the pea aphid by P. neoaphidis and C. obscurus showed somewhat higher resistance to these temper- depends on temperature and the infection does not oc- atures. Transmission of inoculum in the environment cur at 0 ◦Cor30◦C. The time to kill varied from 5 may be influenced by temperature through sensitivity to 16 days at 20 and 10 ◦C, respectively (Voronina, of discharged conidia to higher temperature, desicca- 1968; Wilding, 1970b). The incubation period of dis- tion or insolation (e.g., Furlong & Pell, 1997). eases increases as temperature decreases within a cer- Humidity is another important environmental fac- tain range of temperature (Milner & Bourne, 1983) tor affecting the course of epizootics in insect popula- and the infectivity of fungi may also vary at different tions. A water-saturated or near-saturated environment temperature regimes (Feng et al., 1999; Shah et al., is essential for active conidial discharge, germination, 2002). C. coronatus grows at the temperatures of 16– and infection initiation. According to Millstein et al. 30 ◦C, with the optimum of 20 ◦C(Papierok, 1985), (1982, 1983), a conidial discharge occurred when rela- but strains isolated from human mycosis have the opti- tive humidity in the crop exceeded 91%. This result is mum at 37 ◦C, which is lethal for the other strains (Pa- in agreement with earlier observations that conidial dis- pierok et al., 1993). Strains of C. coronatus isolated charge in two entomophthoralean species, P. neoaphidis from the soil in Israel had the optimum temperature for and C. obscurus, increased with the increasing humid- spore production at 20 ◦Candforgrowthat30◦C(Ali- ity and that more conidia were produced from cadav- Shtayeh et al., 2002). Z. radicans in spotted alfalfa ers in contact with liquid water than from those in a aphid produced more conidia at 25 ◦C than at 15, 20 ◦C saturated atmosphere (Wilding, 1969, 1971; Voron- or 30 ◦C(Milner & Lutton, 1983) and similar results ina, 1968). Sporulation was almost completely inhib- of conidial production were observed for dry-formulated ited below 93% of relative humidity (Wilding, 1969; mycelium as well (Wraight et al., 2003). P. neoaphidis Sivčev & Manojlovic´, 1995), but when the humid- S582 M. Barta & Ľ. Cagáň ity rises again sporulation can recommence (Glare & negative effect of rain on inoculum dispersal may also Milner, 1991). Some fungi have adapted to daily peri- occur, since the rain may deplete the amount of fun- odical changes in humidity level. N. fresenii discharges gal inocula in the vicinity of aphids by washing them most of its primary conidia at night when the humid- off plant surfaces (Kish & Allen, 1978; Furlong & ity is higher (Steinkraus et al., 1999). Before day- Pell, 1997). Heavy rainfall lasting for over 30 minutes light, most sensitive N. fresenii primary conidia have removes significant numbers of P. neoaphidis conidia, germinated to form capilliconidia, which are more re- particularly from the upper surfaces of leaves. And 60 sistant to lower humidity (Steinkraus & Slaymaker, minutes of heavy rainfall is necessary to remove aphid 1994). Elliot et al. (2002) studying mycoses in mite cadavers from foliage. Conversely, light rain may com- populations pointed out that saturation deficits (low pensate for the negative effects of heavy one by enhanc- humidities) are mainly determining a horizontal trans- ing the sporulation and germination of spores (Pell et mission, while the process of infection is not so affected al., 1997b). by abiotic conditions. Viability of the primary spores Desiccation is one of the decisive factors for the of Z. radicans at different humidities was studied and persistence of pathogens in nature. Generally, fungal the best viability was recorded at the humidity above pathogens cannot survive desiccation and conidia are 95% (Griggs et al., 1999). An influence of leaf wetness the most vulnerable units to desiccation (Pell et al., on the infection of aphids was investigated by Milner 2001). Exposure of N. fresenii primary conidia to 75% & Bourne (1983). Establishment of epizootics in the of relative humidity for only one minute reduced sig- aphid populations is usually associated with periods of nificantly the germination and subsequent formation rains or just after periods of rains. Percentage of the of more resistant capilliconidia. However, capilliconidia conidial germination of P. nouryi was higher when coni- of the species remain infective even for two weeks at dia were deposited on leaf surface than on glass cover 75% of relative humidity (Steinkraus & Slaymaker, slips (Li et al., 2006). Wilding (1975) reported that 1994). Primary conidia of P. neoaphidis lost their vi- infections of the pea aphid were positively correlated ability after four hours at 20 ◦C and 75% of relative to the average rainfall recorded 12 days prior to dis- humidity (Barta, 2004). Uziel & Kenneth (1991) ease observations. A similar relationship was later ob- also found that the primary conidia could not tolerate served by Cagáň & Barta (2001). Others have also prolonged exposure to low relative humidity. Contrary tried to quantify this relationship. Missonnier et al. to conidia, hyphae are more resistant to desiccation. (1970) determined that for an enzootic of Entomoph- Aphid cadavers filled with hyphal bodies can be stored thora sp. to be sustained in an aphid population there desiccated at low temperature and preserve fungal vi- must be a minimum of 90% relative humidity for at ability for several weeks (Rockwood, 1950; Wild- least eight hours per day. To increase disease levels ing, 1973; Latteur et al., 1985). Desiccated mum- to epizootic proportions, the relative humidity must mies of killed aphids can also preserve fungal viability exceed 90% for 10 hours per day and there must be in nature. C. obscurus and P. neoaphidis infecting pea five hours of rain per day for three consecutive days. aphids were tolerant to periods of low humidity and This is in accordance with results of other studies on the fungi in aphid mummies could accumulate the ef- entomophthoralean epizootics (e.g., MacLeod, 1955; fects of short periods of high humidity for development Wilding, 1975; Elkinton et al., 1991; Feng et al., of conidia (Behrens, 1993). 1991, 1992a). Voronina (1971) used the hydrother- Positive stimulation of pathogens by light is rarely mal coefficient (sum of precipitation × 10.0/sum of av- reported. Ege (1965) confirmed a stimulating action erage temperature > 10 ◦C) to define ecological zones of the blue part of the light spectrum on certain En- in the former Soviet Union according to their ability tomophthorales of aphids. During conidiogenesis of P. to support epizootics of Entomophthora sp. in the pea neoaphidis, light increases the infectivity of conidia aphid populations. In those areas where the coefficient (Latteur et al., 1985). Under light conditions, a coni- exceeds 1.4, fungi regularly control the pea aphid pop- dial rate of discharge reached higher values. In dark, it ulations below economic levels. When the coefficient is was maintained longer, whereas in light it soon slowed between 1.0 and 1.4, fungi are still an important aphid after reaching the maximum (Wilding, 1971). The ger- mortality factor, but some crop damage may occur. In mination process of P. neoaphidis conidia was faster very dry areas, where the coefficient is bellow 1.0, the in light than in dark (Sivčev & Manojlovic´, 1995). pea aphid causes crop losses almost every year. The at- These reports contrast to a number of reports on the tempt to predict areas where epizootics will occur based negative effects of light, especially on persistence of on a measurement of moisture like that of Voronina conidia in nature (e.g., Oduor et al., 1996; Furlong was not followed. Those predictions may be of some & Pell, 1997). Ultraviolet radiation is by far the most worth, however, they cannot be treated as a rule. Ken- important factor in conidia mortality. Survival of the in- neth & Olmert (1975) reported collections of N. fre- fective units is rapidly reduced after several minutes of senii from very dry areas of Israel. E. planchoniana, exposure to ultraviolet radiation (Brobyn et al., 1985; P. neoaphidis,andZ. radicans were recorded from two Uziel & Shtienberg, 1993; Sivčev & Draganic´, desert localities in Mexico (Sanchez-Pena´ , 2000). The 1994; Furlong & Pell, 1997). Discharged spores were Aphid-pathogenic Entomophthorales S583 more persistent on lower than upper surfaces of leaves by P. neoaphidis is observed on pea cultivars with re- due to a lower UV exposure on underside (Brobyn et duced wax bloom (Duetting et al., 2003). al., 1985; Furlong & Pell, 1997). Since unprotected Pesticides are anthropogenic factors that are fre- protoplasm is easily destroyed by the impact of solar quent in the environment of insects and their pathogens, energy, the action of light may be destructive. Many so they should be regarded as epizootiologically rele- organisms have therefore evolved light-absorbing pro- vant factors. Much work has been done to investigate tective pigments to create a shield against the nega- the effect of different pesticides on some entomoph- tive light effects. For instance, resting spores of N. fre- thoralean fungi and mycosis prevalence (e.g., Smith & senii have black episporium, which effectively shields Hardee, 1996; Lagnaoui & Radcliffe, 1998; Hat- the spore from penetration of light (Ben-Ze’ev et al., ting et al., 1999b; McLeod & Steinkraus, 1999; 1990). Wells et al., 2000; Latteur & Jansen, 2002). Pes- Plants/crops themselves may also affect the dis- ticides affect mostly the conidial production and ger- ease prevalence and the development of infection pro- mination (Wilding, 1982; Perry & Latgé, 1983; cesses in insect populations inhabiting the crops. The Keller, 1986). However, the field effects of agrochemi- action consists in specific microclimatic conditions typ- cals are probably less severe than the effects in the labo- ical of particular crop architecture. The physiological ratory. In particular, this is true for pesticides that have properties of plants may influence insect diseases as a low persistence on leaf surface. Therefore, in practice, well, particularly through an effect on infection inocula the compatibility of entomopathogenic fungi with agro- deposited on plant surface. Wax bloom on pea plant chemicals is influenced mainly by the timing of appli- surface has an adverse effect on P. neoaphidis conidia cation and the coincidence of biological and chemical adhesion and germination. Greater infection of aphids agents (Zimmermann, 1986). S584 M. Barta & Ľ. Cagáň

in modern times is that of DeGeer who found parasitic 7. Potential of Entomophthorales infection on flies in 1779 (Roberts & Humber, 1981). for biological control After nearly 60 years, in 1834, the Italian Agostino Bassi, a pioneer laying the foundations for the study Although the Entomophthorales is an important and of infectious diseases, elucidated the fungal nature of worldwide-distributed group of insect pathogenic fungi, the white muscardine disease of silkworms (Hall & no species is currently produced commercially and aug- Papierok, 1982). In 1874, Louis Pasteur was the first mented for aphid control (Humber, 1991; Eilenberg, who expressed the idea of using a “mycelium” to con- 2002). The main problem is the production of infec- trol aphid populations (Latgé & Papierok, 1988). tive and stable biopreparations. These fungal ento- Another eminent scientist of that period who upheld mopathogens do have, however, further weak spots from insect pathology was Metchnikoff in Russia. He was the a viewpoint of practical use. For example, there are first who mass-produced a fungus, Metarhizium aniso- some disadvantages linked with a short lifespan of in- pliae (Metchnikoff) Sorokin, for control of the wheat fective inoculum in the environment, which makes tim- cockchafer and later the sugarbeet curculio (Samson ing of inundative applications difficult or impossible et al., 1988). A common parasite of houseflies described (Lacey et al., 2001). Nevertheless, some attempts to in 1855 by Cohn and named as Empusa muscae, subse- manipulate disease timing in the aphid populations in quently renamed as Entomophthora by Fresenius, was fields with overhead irrigation and fungicide application the first description of an entomophthoralean species have been made (MacLeod & Steinkraus, 1999). (Brady, 1981). At the end of the 19th century, much The fungal control agents are still believed to be ef- activity was directed towards the taxonomy of ento- fective and to serve as alternatives to insecticides when mopathogenic fungi. Probably the most significant tax- fungal strains are selected carefully. The most inter- onomic work on Entomophthorales was that of Thax- esting strains for use in augmentation biological con- ter (Thaxter, 1888). Then a period of intensive re- trol are those possessing a high infectivity, a high in- search on the use of entomopathogenic fungi in control- tensity of sporulation and a short infection cycle (Pa- ling agricultural pests followed. Many attempts were pierok, 1982). Strains selected for biocontrol should made to use these beneficial organisms in the control of not have too narrow specificity, being commercially ad- various pest insects, but due to varying and often disap- vantageous if a product has a relatively wide host range pointing results the initial enthusiasm decreased more within an insect group containing several pest genera. and more. During the last 30 years, there has been a sort However, the host range cannot be too wide and ob- of renaissance in the development of biological control viously must exclude beneficial insects as well as other methods and in the interest in fungal pathogens. This invertebrates and vertebrates (Samson et al., 1988). was promoted by the increased use of pesticides and Successful utilisation of the Entomophthorales requires, consequently by the finding of their negative environ- first of all, increased pathogen virulence and speed of mental impact. kill, then improved pathogen performance under envi- Several researchers experimented with the use of ronmental conditions, greater efficiency in their pro- entomophthoralean fungi as microbial control agents in duction, improvements in formulation enabling an ease thelateof19th century. The first documented case in of application, increased environmental persistence, ap- which a member of the Entomophthorales was exploited propriate incorporation into integrated pest manage- as a control agent dates back to 1895 in South Africa ment, and acceptance by growers and the general public (Samson et al., 1988). A fungus isolated from diseased (Lacey et al., 2001). Apart from the fungal properties locusts and believed to be a species of the Entomoph- mentioned above, these and other factors are necessary thorales was mass-produced for sale to farmers. Unfor- to consider with respect to classical or conservation bio- tunately, the cultures were later identified as Mucor sp. logical control (Shah & Pell, 2003; Shah et al., 2004). Petch (1925) deduced that the original field pathogen was Entomophthora grylli Fresenius, but the initial iso- 7.1. Historical lations were contaminated and the contaminant was distributed. And the reported field successes in fungal Invertebrate pathology is just a recently organized dis- distribution were probably due to natural epizootics of cipline, but literature on fungal parasites of insects and E. grylli. Attempts to introduce inoculum of the En- mites is extensive and long established. In modern his- tomophthorales to pest populations were conducted in tory, the entomopathogenic fungi have been known to North America in the first half of the 20th century. be significant under natural conditions for over 150 Inoculum of Erynia radicans was successfully released years. However, insect diseases were probably first ob- against the European apple sucker in apple orchards in served by sericulturists in the Orient and early Japanese Canada (Dustan, 1924 in Samson et al., 1988). Com- notes (about 900 AD) say about muscardine silkworms parable trials followed, among which those involving being used for the treatment of palsy or paralysis aphids can be mentioned: Myzus persicae on potatoes (Steinhaus, 1956, 1975). One of the earliest refer- (Harris, 1948) and the spotted alfalfa aphid (Hall ences to the parasitism of insects by entomopathogens & Dunn 1958). During the last three decades, many Aphid-pathogenic Entomophthorales S585

field or laboratory trials to control aphids using ento- an establishment against aphid pest. One of the ex- mophthoralean fungi with different results have been amples is the release of an Israeli isolate of Z. radicans published in literature. In most cases, the aphid popula- to control the spotted alfalfa aphid, Therioaphis tri- tions were not reduced sufficiently to prevent crop dam- folii, in Australia. The aphid caused serious damage age suggesting that the fungi were not acting quickly to Australian alfalfa after its discovery in 1977 (Mil- enough (e.g., Dedryver, 1979; Wilding, 1981; Latgé ner & Soper, 1981). Since surveys for native fungal et al., 1983; Latteur & Godefroid, 1983; Wilding pathogens of this aphid pest found no candidates, it was et al., 1986a, b, 1990; Chudare, 1990; Silvie et al., concluded that exotic Entomophthora species should be 1990; Shah et al., 2000a). evaluated for its control (Milner et al., 1980). Af- ter the successful selection of the most promising Z. radicans strain (from an extremely hot and dry Israeli 7.2. Strategies for biological control site), it was released into field populations of the spot- ted alfalfa aphid. The fungus dispersed rapidly into the In general, biological control (biocontrol) of pests in- fields where the releases were made and was later found cludes the use of living organisms toward a reduction several hundred kilometres away (Milner et al., 1982; of pest population. Biological control is kept within a Milner & Mahon, 1985). framework of integrated pest management (IPM) as de- Another example of pathogen establishment in a fined by Kogan (1998): “IPM is a decision support new environment was the introduction of N. fresenii system for the selection and use of pest control tactics, into the cotton aphid populations in the San Joaquin singly or harmoniously co-ordinated into a management Valley of California in 1994 and 1995 (Steinkraus & strategy, based on producers, society and the environ- Rosenheim, 1995; Steinkraus et al., 1998a, 2002). ment.” Biological control can be defined as “The use Cotton aphid in California lacked fungal pathogens. of living organisms to suppress the population density Summer releases of isolates from Arkansas, using dried or impact of a specific pest organism, making it less infected aphids, were moderately successful, resulting abundant or less damaging than it would otherwise be” in the spread of the pathogen from the release sites. (Eilenberg et al., 2001). This definition emphasizes Fungus activity continued until September or October, the fact that only “living organisms” are used. This but epizootics did not develop. Whether the pathogen should include insect viruses as well, whereas genes or persisted in California and caused long-term suppres- gene fragments and metabolites from organisms are ex- sion of the cotton aphid populations was not discussed. cluded. Eilenberg (2002) presents his opinion about in- There are four different strategies for biological troducing Danish strains of P. neoaphidis virulent to control: Classical biological control, Inoculation biolog- the green spruce aphid, Elatobium abietinum, colonis- ical control, Inundation biological control, and Conser- ing spruce plantations in Iceland. This pathogen has vation biological control (Eilenberg et al., 2001). In- never been observed on this aphid species in nature in oculation biological control and inundation biological Iceland (Austar˚a et al., 1997; Nielsen et al., 2001b). control are often mentioned under a common name of In Western Iceland the only fungus prevalent on this Augmentation biological control (Pell et al., 2001). aphid was E. planchoniana, while in Southeast Iceland only N. fresenii was prevalent. This geographical dis- 7.2.1. Classical biological control tribution correlates with the distribution of two dif- The principal objective of classical biological control is ferent populations of E. abietinum found in Iceland. permanent establishment of a biological control agent Both species established epizootics within their dis- for self-sustained long-term control. Classical biological tribution area (Nielsen et al., 2001b). The idea to control depends on finding and releasing an appropriate artificially release E. planchoniana and N. fresenii in biological control agent that is not native to the area Southeast or Western Iceland, respectively, in order to where the pest needs to be controlled. Public concern test whether they can establish, proliferate and regulate over the negative side-effects on non-target organisms the aphid populations was given as well (Eilenberg, after releasing and expected proliferation of biological 2002). agent may be an obstacle (Hajek & St. Leger, 1994; Besides efforts to introduce the entomophthoralean Eilenberg et al., 2001; Shah & Pell, 2003). The fungi into the aphid populations, other insect – fungus – methodcanbedefinedas“The intentional introduc- crop systems have been evaluated or tested for classical tion of an exotic, usually co-evolved, biological control biological control. For example, a Japanese strain of En- agent for permanent establishment and long-term pest tomophaga maimaiga was introduced to forests in the control”(Eilenberg et al., 2001). USA (Hajek et al., 1995; Pell et al., 2001) and Bul- Introduction of exotic diseases for classical biolog- garia (Pilarska et al., 2006) against Lymantria dis- ical control is only rarely possible, since most diseases, par (L., 1758), or a Brazilian strain of Neozygites flori- like their aphid hosts, are distributed world-wide. How- dana Fischer was introduced to cassava crops against ever, several examples can be described where exotic Mononychellus tanajore (Bondar, 1938) in Africa (El- entomophthoralean species have been introduced for liot et al., 2000). S586 M. Barta & Ľ. Cagáň

7.2.2. Inoculation biological control the populations of cereal aphids in fields in France. Al- The common feature of inoculation biological control though significant quantities of produced conidia and is that low levels of material are usually released. The favourable weather conditions were recorded, no pos- inoculative releases are carried out early in the season itive results were achieved. Latgé et al. (1982) and and it is expected that inocula will reproduce, spread in Silvie et al. (1990) used the fresh or dry mycelium of the environment and control host population for an ex- P. neoaphidis for aqueous spray of the aphid-infested tended period (Pell et al., 2001; Shah & Pell, 2003). plants in a glasshouse. Although the fungus sporu- Success of the method depends on the ability of the re- lated and was able to infect some aphids, population leased organisms to multiply and then reduce the tar- suppression through horizontal transmission was not get pest population. No permanent establishment of the achieved. Two Conidiobolus species, C. obscurus and biological control organisms is achieved (Eilenberg et C. thromboides, were studied extensively for use in the al., 2001). A definition of this method thus can be as azygospore-based mycoinsecticides against Myzus per- follows: “The intentional release of a living organism sicae on potatoes in the USA. However, these efforts as a biological control agent with the expectation that were abandoned as technically impractical (Humber, it will multiply and control the pest for an extended pe- 1991). Classical introductions with fungus-mummified riod, but not permanently”(Eilenberg et al., 2001). aphid cadavers as well as inundative applications of Due to the ability to establish quickly epizootics in tar- the marcescent mycelium of P. neoaphidis against ce- get pest populations, narrow host range, and potential real aphids were carried out in England (Wilding et to persist in target insect populations, the entomoph- al., 1990). Unfortunately, there were no significant ef- thoralean fungi fit very well in inoculation biological fects of treatment on the numbers of aphids. The ar- control. There are several examples where natural lev- tificial introduction of P. neoaphidis acted too slowly els of the entomophthoralean inocula are supplemented and unpredictably. In a recent study by Poprawski to effect aphid control in this way. & Wraight (1998), small pieces of sporulating P. The work of Hall & Dunn (1957, 1958) was the neoaphidis mycelium were inserted into rolled wheat first major attempt to manipulate a pathogen of the leaves with the colonies of Diuraphis noxia Kurdjumov, Entomophthora group for microbial control of aphids. 1913 in Idaho (the USA). The fungus sporulated gener- In California, three entomopathogenic species (E. coro- ously, 18% of aphids were infected, and the fungus even nata, E. virulenta,andE. exitialis) were successfully es- spread rapidly to uninoculated nearby tillers, but the tablished in the Therioaphis maculata populations on fungus was slow to spread to the rest of the field. alfalfa by placing fungal cultures directly in the fields. Dedryver (1979) dispersed local isolates of N. fresenii 7.2.3. Inundation biological control in living A. fabae. The infected, still living aphids were Contrary to classical or inoculation biological control in then released to colonies of theaphidonbeanplantsin inundation biological control the control agent do not a glasshouse maintained under a regime of controlled multiply or persist in the environment, but affects tar- relative humidity. The colony growth was arrested ef- get pests immediately after a mass-release. Generally, fectively with the pathogen. Infection between 80% and a large number of organisms are necessary to achieve a 90% was observed in the local strains of P. neoaphidis sufficient effect (Eilenberg et al., 2001; Shah & Pell, and N. fresenii released in aphid cadavers into A. fabae 2003). This can be expressed as “The use of living or- populations under field conditions. During warm and ganisms to control pests when control is achieved ex- dry seasons (over two years) the Entomophthorales in- clusively by the released organisms themselves”(Eilen- troduced into populations established briefly but they berg et al., 2001). The entomophthoralean fungi pos- failed to spread. However, in cool and moist seasons sess some properties allowing them to be used for in- (another two years of experiments) the fungi spread undation biological control (e.g., high virulence), while rapidly and the yield of beans in the treated plots was other properties (e.g., short lifespan of conidia) indi- higher in comparison with the untreated ones. In sim- cate that these fungi may fail as inundation biological ilar experiments, Wilding et al. (1986b) introduced control agents, especially in outdoor environment. In- P. neoaphidis into a field population of A. fabae us- door environment, like glasshouses, is the most promis- ing three different types of inocula – living laboratory- ing site for this strategy of biological control. In many infected aphids, triturated cadavers of fungus-killed examples, trials were carried out on a small scale and aphids, and a homogenate of the fungus grown on agar were not repeated making the differentiation between plates. The fungus applied as triturate cadavers estab- inoculation and inundation biological control difficult. lished infection in the host population as effectively as Generally, for the Entomophthorales it is more usual by distributing laboratory-infected aphids. More than that the intention is to inoculate the crop with the 70% infection was recorded but the fungus failed to fungus early in the season (it fully corresponds with multiply fast enough to protect the crop adequately. inoculation biological control) so that the fungus can The application of homogenate even failed to establish establish and multiply, keeping the aphid populations infection. Latteur & Godefroid (1983) introduced below the damage threshold (Pell et al., 2001). the mycelium of P. neoaphidis (produced in vitro)to Several aphid species were tested with different Aphid-pathogenic Entomophthorales S587 types of C. obscurus inoculum in laboratory experi- It should be remembered that these compounds could ments (Krejzová, 1972a). The application of resting have an adverse effect on the entomopathogenic fungi. spore suspension produced in submerged culture to A. The way of the pesticide activity may be different. In fabae on Sambucus nigra L. resulted in 20% mortal- addition to the direct toxicity for the fungi, the activ- ity. On the other hand, the application of conidia by ity or even survival in the environment can be threat- discharging them onto the aphids resulted in 40% mor- ened by decreasing or eradicating their natural hosts. tality in case of Megoura viciae on Vicia faba L., in Several studies have treated the sensitivities of ento- 55% mortality in A. fabae and in 100% mortality in mopathogenic fungi to a variety of pesticide groups and Aphis gossypii. Other fungal species, C. thromboides, the results achieved were different. Fungicides may re- C. coronatus,andC. destruens, varied in a percent- duce the prevalence of entomophthoralean pathogens age of mortality in laboratory experiments from 28% in the host populations (Öncüer & Latteur, 1979; to 88%. In the former Soviet Union, Chudare (1990) Pickering et al., 1989b; Smith & Hardee, 1996), applied suspension of the fresh or dry culture (conidia or even delay an initiation of epizootics (Wells et or resting spores) of C. obscurus into the populations of al., 2000). Pesticides may affect the germination of over ten different aphid species with the results of 71– conidia or inhibit the growth of hyphae (Yendol, 99% mortality. A biopreparation “Entomoftorin”was 1968; Lagnaoui & Radcliffe, 1998; Hatting et al., prepared on the basis of resting spores of a local strain 1999b). Entomopathogens showed their susceptibility of C. obscurus. The preparation could be stored at 4 ◦C to benomyl, tridemorph, maneb, chlorthalonil, bina- for more than eight years without a loss of viability pacryl, bitertanol, iprodione, mancozeb, captan, zineb, and virulence. After application, the 62–90% mortal- carboxin, etridiazole, triphenyltin hydroxide, dithiocar- ity of tested aphids was usually recorded, but the tem- bamate, and metalaxyl (e.g., Yendol, 1968; Fritz, perature of 20–24 ◦C and the relative humidity of 60– 1976; Öncüer & Latteur, 1979; Brandenburg & 80% were inevitable for the effectiveness (Chudare, Kennedy, 1983; Carruthers et al., 1985; Picker- 1982). In Russia, resting spores of the selected strain ing et al., 1989b; Smith & Hardee, 1996; Lagnaoui of C. obscurus were exploited to develop a bioprepara- & Radcliffe, 1998; Hatting et al., 1999b; McLeod tion called “Mycoaphidin” with high effectiveness for & Steinkraus, 1999; Wells et al., 2000; Latteur aphids (Voronina, 1997). In Denmark, Eilenberg & Jansen, 2002). The environment pollution by heavy (2002) recommends the preparations of P. neoaphidis metals could also be a restrictive factor of the devel- for inundation biological control of aphids in glasshouse opment and pathogenicity of entomophthoralean fungi crops. Attempts to control aphids in a glasshouse were (Tkaczuk, 2006). made by Shah et al. (2000a). Experimental prepara- tions based on the alginate granules and mycelial mats 7.2.3.1. Production and formulation of aphid-patho- of entomopathogenic P. neoaphidis caused up to a 14% genic Entomophthorales infection rate in the potato aphid Macrosiphum eu- It is obvious that large quantities of properly formu- phorbiae with foliar application or up to 36% infec- lated inoculum are inevitable for use in inundation bio- tions with soil applications (Shah et al., 2000a). How- logical control. As a matter of fact, not only the quan- ever, other spray applications of unformulated mycelial tity but also the quality of mass-produced inoculum is stage of P. neoaphidis did not provide adequate control prerequisite (Magan, 2001). Usually, the most effec- in glasshouse or field tests (Latteur & Godefroid, tive way of fungal inoculum propagation is carried out 1983; Silvie et al., 1990). in in vitro conditions. Therefore, in vitro cultivation is Use of entomophthoralean fungi for biocontrol a decisive step in the development of fungal bioprepa- by dispersing sporulating cadavers or moribund in- rations and their consecutive use in biological control. sects gives usually mixed results (e.g., Wilding, 1981; In vitro production of the Entomophthorales can dif- Wilding et al., 1990). In the south-east of the USA, fer significantly among genera or species, since nutri- a method for harvesting large numbers of N. fresenii tional requirements for growth are different (Latgé, killed cotton aphids from natural epizootics in commer- 1981). Conidiobolus grows quickly on standard media. cial cotton fields was developed (Steinkraus & Boys, Zoophthora, Pandora and Batkoa are little more de- 2005). An important aspect of N. fresenii is its inability manding for nutritional supplements. Entomophthora to in vitro culture on artificial media and high labour and Entomophaga need more complex media (Balazy, expenses as well as time consumption in case of in vivo 1993; Leite et al., 2003). At present, the prospects of laboratory production. Mature hyphal bodies or early producing Neozygites species in vitro for use as mycoin- stage conidiophores are able to survive up to 6 years in secticides are uncertain. Successful in vitro cultures of dried and frozen (−14 ◦C) aphid hosts (Vingaard et Neozygites have been reported only for species isolated al., 2003) and can be applied to the cotton field as a from mites and thrips (Butt & Humber, 1989; De- potential source of fungal inoculum. lalibera, 1996; Keller, 1997; Grundschober et al., Any fungus used for biological control of insects 1998; Leite et al., 2003). No Neozygites species infect- may meet a diverse range of pesticides applied to con- ing aphids have been isolated yet in vitro (Keller, trol the whole range of other pests on the same sites. 1997). Out of the group of 33 aphidophagous Ento- S588 M. Barta & Ľ. Cagáň mophthorales described here, 19 species are known to Principally because of the simplicity with which mem- grow and sporulate in vitro, two species can grow but bers of the genus produce resting spores in vitro. Soper not sporulate, six have not been isolated yet, another et al. (1975) produced C. thromboides resting spores six species are incompletely described or the isolation using media containing egg yolks, with yields approxi- has not been tried. mating 2–3 g of spores per egg yolk. The mass-produced Different types of fungal material have been tested dry resting spores could be stored at 4 ◦C for one year for mass production: conidia, hyphal bodies, mycelium, with no change in germination capacity. Liquid media or resting spores. Conidia of Entomophthorales, be- were also used for in vitro production of resting spores. cause of their short lifespan, are of little value for biolog- Semi-defined media for resting spores production of C. ical control (Coremans-Pelseneer, 1981). The struc- thromboides (Latgé et al., 1977, 1978a, b) and C. ob- tures are also sticky, making them difficult to harvest scurus (Latgé, 1980; Latgé & Perry, 1980; Latgé from cultures and suspend uniformly in water. In ad- et al., 1983; Remaudiére, 1983) were proposed. To dition, if during formulation or application, the mucus germinate the spores required overcoming dormancy, a surrounding conidia is lost, a vital adhesion mechanism period of about three months at 4 ◦C and 95% relative could be lacking (Pell et al., 2001). Numerous studies humidity. Unfortunately, attempts to use the in vitro- have already been focused on developing the effective produced resting spores of C. obscurus failed (Wilding methods for mass production of inoculum and they have et al., 1986a) mainly due to a low or asynchronous ger- especially targeted at hyphal stages or resting spores. mination of spores after their application. Hyphal bodies and resting spores, contrary to conidia, Any inoculum produced in vitro has to be formu- are a better type of inoculum, because they can be pro- lated for successful application, stabilisation, storage, duced industrially and may discharge infective conidia and improved efficacy in the fields (Hall & Papierok, for some time after their application to crops (Latgé et 1982; Samson et al., 1988; for review see Wraight et al., 1983). When hyphal stages (hyphal bodies or dried al., 2001). A new way of formulation of P. neoaphidis mycelium) are applied to the fields, the inoculum is ex- mycelium was designed (Shah et al., 1998). The hyphae pected to rehydrate and produce conidia in situ (Pell of the pathogen were immobilized in a matrix consist- et al., 2001). In nature, the mycelium of P. neoaphidis ing of sodium alginate and calcium chloride was used as produced in vitro can project conidia for at most three a gelling agent. Conidia from freshly produced alginate days after it has been sprayed onto plants (Latgé et beads caused 27 to 32% infection in pea aphid in labo- al., 1983). On the contrary, resting spores are typical of ratory bioassays. This type of formulation represents a asynchronous germination after application. The ger- promising technique for practical application of fungal mination usually lasts one month and the maximum mycelium. Viability of preparation was best maintained daily level of germination does not exceed 10% of the if stored at 10 ◦C(Shah et al., 2000b). In order to en- applied spores. At 20 ◦C the resting spores produced in hance infectivity of alginate granules to aphids, various vitro stay viable for one to two months after application additives were tested for their use during the formu- (Latgé et al., 1983). lation process (e.g., sucrose, potato starch, or chitin) Mass production and drying method for Z. radi- (Shah et al., 1999) and glasshouse trials using the al- cans mycelium, a “marcescence process”, were success- ginate granules gave promising results (Shah et al., fully developed (McCabe & Soper, 1985 in Pell et 2000a). Soil applications were more rapid in causing al., 2001; Li et al., 1993). This technique exploited the infections when compared with foliar applications. An- natural capacity of fungi to desiccate and rehydrate other novel method to produce granular cultures of P. with resumption of their normal development. The neoaphidis on broomcorn millets (Li & Feng, 2003; mycelium could be stored viable in the freezer for at Feng & Hua, 2005) gives a relatively cheap and effec- least one year. Research on the use of the most impor- tive alternative to the alginate granules. The produc- tant and most frequent aphid pathogen, P. neoaphidis, tion of inocula of good quality in sufficient quantities for as an inoculative or inundative biocontrol agent is of commercial applications and their formulation requires limited success because of problems with mass pro- further research in order to define the media require- duction, a lack of stable inocula for testing, and de- ments during production and to devise optimal formu- pendence on suitable environmental conditions for in- lating methods. However, the mass production and for- fection (Milner, 1997). Some work has been done to mulation themselves are just single steps among a wide develop media and conditions for optimised mass pro- range of research activities, which have to be done in duction of P. neoaphidis (Gray et al., 1990; Li et al., the development of commercial mycoinsecticides (Zim- 1993; Gray & Markham, 1997). The maximum sur- mermann, 1986). vival of P. neoaphidis mycelium produced in liquid cul- Several reviews have recently been presented on ture is one month at 4 ◦C and two weeks at 20 ◦Cand the progress in the field of fungal biotechnology, includ- non-swelling clay may improve this storage up to two ing answers to questions about a selection of fungal months at 4 ◦Cornearlyonemonthat20◦C(Latgé organisms and fungal strains for biological control, a et al., 1983). Aphid pathogens of the genus Conidiobo- choice of fungal propagule for mass production, factors lus were focused on the resting spore mass-production. governing in vitro growth and sporulation, and finally a Aphid-pathogenic Entomophthorales S589 mass production on either liquid or solid media as well species that occupy different host plants and do not in- as a production strategy, a formulation, and a storage teract directly can, theoretically, influence each other’s of industrial preparations (Samson et al., 1988; Bate- population growth rates, since they share fungal dis- man & Chapple, 2001; Wraight et al., 2001). Modes eases. As a result, if one species increases in abundance, of spray application of mycoinsecticides are discussed the infection rate may also increase and consequently in detail by Bateman & Chapple (2001). the second species may suffer from higher mortality. The interspecific interactions between nettle aphids and 7.2.4. Conservation biological control selected pestiferous aphid species occupying the same Conservation biological control is distinguished from ecosystem were studied (Barta & Cagáň, 2003b). In the other strategies in that any natural enemies are case of perennial cultures, it was also observed that not released. The principal object of the strategy is to economically unimportant aphid species developing in keep pests under economic threshold through protec- meadows in spring were important for the propagation tion and enhancement of biological control agents liv- of entomophthoralean fungi that subsequently affected ing naturally in the environment. Conservation prac- aphids in fields (Keller & Suter, 1980; Majchrow- tices include limited and selective use of pesticides but icz, 2001). If aphid populations in meadows were high also active processes such as providing refuges adjacent in the spring, P. neoaphidis and C. obscurus built up to to crops or within crops, facilitating transfer of natu- sufficient levels to regulate aphid populations in adja- ral enemies between crops or directly provisioning food cent fields (Keller & Suter, 1980). Conversely, when or shelter for natural enemies (Van Driesche & Bel- the spring populations of aphids were scarce, the ento- lows, 1996). The method can be defined as “Modifi- mophthoralean infections were low as well. Powel et cation of the environment or existing practices to pro- al. (1986a) found that entomophthoralean fungi were tect and enhance specific natural enemies or other or- more common at weedy edges of fields, and they sup- ganisms to reduce the effect of pest”(Eilenberg et posed that it was either because of higher humidity in al., 2001). The conservation biological control, some- weedy plots, or because of the spread of infection from times also called as habitat manipulation, searches for weed aphid species, or combination of both. effective indigenous natural enemies and applies man- Arthropod natural enemies including parasitoids agement practices that conserve and promote them in and predators conserved in the same sites where par- the field. In annual crops, conservation approaches have asitic fungi were conserved could improve the fungal potential, particularly by the provision of permanent chances to be transmitted locally or to be transmit- or semi-permanent refuges in the agroecosystem, i.e., ted directly to the pest populations in adjacent crops providing the environment similar to that in perennial (Roy & Pell, 2000). As mentioned above, the interac- crops (Pell et al., 2001; Shah & Pell, 2003). Sev- tions between entomophthoralean fungi and arthropod eral examples say about attempts to conserve the En- natural enemies in the environment may be not only tomophthorales in an agroecosystem. positive (Furlong & Pell, 1996, 2000; Pell et al., In annual cropping systems, particular attention is 1997a; Roy et al., 1998, 2001; Roy & Pell, 2000), but paid to habitat manipulation in order to provide per- also antagonistic (Powel et al., 1986b; Brobyn et al., manent or semi-permanent reservoirs of entomophtho- 1988; Pell et al., 1997a; Furlong & Pell, 2000). ralean fungi – alternative hosts for entomophthoralean Modifications of the existing practice are often pro- fungi in an agroecosystem. A fundamental idea of these posed as means to promote entomophthoralean infec- reservoirs is that insects inhabiting non-crop vegetation tions; for example, less spraying with chemical pesti- in the vicinity of crops may serve as alternative hosts for cides or irrigation are at least theoretically supportive fungal diseases, which can reduce populations of pests. to these fungi (Eilenberg, 2002). Irrigation has been From these habitats – foci of diseases – the fungal in- documented in several systems to artificially encourage ocula may spread out through biotic (e.g., co-occurring epizootic development through high relative humidity insects) and abiotic (e.g., wind and rain) transmission for sporulation and infection (Wilding et al., 1986b; agents and affect the insect populations in adjacent Pickering et al., 1989a; McLeod & Steinkraus, crops. These reservoirs could be managed by field mar- 1999). However, an excess of precipitation (irrigation) gin strips (Roy & Pell, 2000). Entomophthoralean may be undesirable by lowering the density of airborne fungi, to be encouraged effectively by habitat manipu- conidia and washing spores off the aphid cadavers, lation, must be able to replicate extensively, must per- which may consequently decrease the infection poten- sist for long periods of time in the reservoirs and must tial of Entomophthorales in the area (Kish & Allen, be able to disperse from the reservoirs into pest hosts 1978). on crops (Pell et al., 2001). Several aphid-pathogenic As described above, intentional modifications in species are known to overwinter in (alternative) hosts the conventional practice and environmental manipula- in hedges and forest borders (Keller, 1987b, 1998; tion may be supportive to the development and distri- Nielsen et al., 2001a). A number of non-pest aphids bution of entomophthoralean fungi within agroecosys- can share their fungal enemies with pestiferous aphid tem. A simple reliance on the natural occurrence of en- species (Barta & Cagáň, 2003b). Aphids of different tomopathogens is risky predominantly due to the un- S590 M. Barta & Ľ. Cagáň predictability of factors that govern epizootics. How- when the very first aphid pathogen E. planchoniana ever, there is an example of utilization of natural epi- was observed and described in aphid colonies in France. zootics in regulation of the cotton aphid, A. gossypii, This discovery started great interest of mycologists and a significant secondary pest of cotton in Arkansas (the entomologists in this amazing group of insect antago- USA). The entomophthoralean fungus N. fresenii of- nists. Plenty of papers have appeared dealing with their ten reduces the requirement for chemical control of the taxonomy, general biology, ecology and potential use in pest (Steinkraus et al., 1991, 1995) and the N. fre- plant protection. This literature offers enough informa- senii epizootics can be predicted at least one week in tion but it needs to be pooled. It is hoped that the advance by careful pest monitoring and diagnosis of present review provides the reader with adequate and aphid samples (Hollingsworth et al., 1995). If fun- useful information in one place that could stimulate his gal prevalence is 15% or higher, there is a strong like- or her interest in further study of these organisms. lihood that the aphid population will decline within The overview presented in this work enables us to a week, due to occurrence of epizootic. If it is 50%, identify areas where further research is necessary. Some there is a strong likelihood that the aphid population critical points can be seen in the classification. Modern will decline within few days. When the disease preva- molecular analyses should be applied to characterise lence exceeds 15%, growers are advised to refrain from more exactly those species that have a wide morpho- insecticide application, thus saving money, preserving logical variation. Traditional morphometric studies do beneficial insects and reducing environmental contami- not allow us to judge whether they are single taxons nation by pesticides (Steinkraus et al., 1996a, 1998a; or complexes of morphologically similar species. Molec- Steinkraus & Boys, 1997). ular phylogenetic studies using sequences of specific fungal genes would also contribute to the discussion 7.3. Biochemicals and Entomophthorales about correctness of Basidiobolus displacement from the order Entomophthorales. In addition, original de- Parasitic fungi are known to produce metabolites toxic scriptions of some species are not sufficient and require to insects and, therefore, are attracting attention as re-description. potential biological agents of insect pests (Clarkson Entomophthoralean fungi have the characteristic & Charnley, 1996). In Hyphomycetes several toxic that makes them valuable agents for biological con- compounds have been recognized and characterized as trol. Most of the species are closely host specific and promising but not species-specific (Khachatourians, they pose no threat to non-target organisms. However, 1996). The Entomophthorales are known as species- species/strains selected for biological control should not specific entomopathogens with a rather narrow host have too narrow specificity, because it is commercially range (Pell et al., 2001). Recently, a proteinaceous advantageous. Narrow specificity is often connected mycotoxin has been documented, isolated and charac- with difficulties in cultivation on artificial media and terised for C. coronatus (Bogu´s & Scheller, 2002). thus the mass production of inocula for is disabled. A This success has given a hope for a future use of en- closer view on host specificity with the application of tomophthoralean secondary metabolites as biorational molecular techniques is required. chemical agents with a rather high specificity. In Rus- The last but not least critical point is the prac- sia, even two mycotoxins isolated from C. obscurus have tical utilization of Entomophthorales in plant protec- been utilized to develop biopreparations called “My- tion against aphids. The main problem in augmenta- coaphidin T ”and“Entoks”(Voronina, 1997). The tion biological control seems to be the production of “Mycoaphidin” is characterized by high effectiveness; infective and stable biopreparation and more efforts 70–100% mortality was recorded after 1% concentra- should be put into research and development of com- tion of the biopreparation had been applied to various mercially accepted product. Some problems could also aphid species populations. Lower mortality (50–60%) appear in conservation strategy of biological control. was noted only for M. persicae. Much work has been done when studying biology and ecology of Entomophthorales. Factors controlling the development of pathogens, their sporulation and infec- 8. Conclusions tion process were thoroughly studied by many authors, but little is known about an important period of life We have provided an extensive review of the aphid cycle of several species – their winter survival and acti- pathogenic Entomophthorales covering their taxonomy, vation in spring. Since conservation strategy rely prin- biology, ecology and use in biological control. Much cipally on the present concentration of inoculum in na- work has been done since the first entomophthoralean ture, the closer research into this part of life cycle is species were described in insect hosts. It was in 1873 needed. Aphid-pathogenic Entomophthorales S591

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Appendix

Plate 1. Pandora neoaphidis (Remaudi`ereetHennebert)Humber:a–aclusterofhyphalbodies(aceto-orcein“AO”),adetailofhyphal body (AO); b – primary conidia with visible nuclei (AO); c – primary conidium with visible nucleus (on the left), secondary conidia type Ib (on the right) (AO), c, e – the same magnification; d – germinating conidia (AO), b, d – the same magnification; e – primary conidium forming secondary one, type Ib (on the left), secondary conidia type Ia (on the right) (AO); f – monohyphal rhizoids with disc-like endings; g – rhizoids growing in the ventral part of abdomen of killed aphid. Aphid-pathogenic Entomophthorales S605

Plate 2. Pandora nouryi (Remaudi`ere et Hennebert) Humber: a – primary conidia (aceto-orcein “AO”); b – hyphal bodies (AO); c, d, g – formation of resting spores (AO); e – premature resting spores (AO); f – mature resting spores (AO). S606 M. Barta & Ľ. Cagáň

Plate 3. Pandora uroleuconii Barta et Cagáň: a – killed aphids adhered to the plant by their proboscises; b – cadaver with released spores around it; c – hyphal bodies (aceto-orcein “AO”); d – pseudocystidia (psc) protruding over a conidial layer (AO); e – conidiophores with forming primary conidia; f – primary conidia (left side – AO); g – secondary conidia (sc) forming on primary conidia (AO). Aphid-pathogenic Entomophthorales S607

Plate 4. Entomophthora planchoniana Cornu: a, b – primary conidia with visible nuclei (aceto-orcein “AO”); c – secondary conidia with a rest of the primary ones (AO); d – developing conidiophores with visible nuclei (AO); e) conidiophores; f – branched hyphal body (AO); g – mycelium dissected from a killed cadaver. S608 M. Barta & Ľ. Cagáň

Plate 5. Zoophthora aphidis (Hoffman in Fresenius) Remaudi`ere et Hennebert: a, b – primary conidia with visible nuclei (aceto-orcein “AO”); c – primary conidia forming capilliconidia and a rest of primary conidium with a capilla (AO); d – detached capilliconidia (AO). Zoophthora radicans (Brefeld) Batko: e, f – primary conidia with visible nuclei (AO); g – primary conidium forming capilliconidium (AO); h – detached capilliconidia (AO); i – detached capilliconidia and rests of primary conidia with capillae (AO). Aphid-pathogenic Entomophthorales S609

Plate 6. Erynia erinacea (Ben-Ze’ev et Kenneth) Remaudi´ere et Hennebert: a, b – mature resting spores with echinulate walls (aceto- orcein “AO”). Zoophthora phalloides Batko: c – hyphal bodies; d – conidiophores; e – primary conidia. S610 M. Barta & Ľ. Cagáň

Plate 7. Neozygites fresenii (Nowakowski) Remaudi´ere et Keller: a – capilliconidia (aceto-orcein “AO”); b – detached capilliconidia (AO); c – capillary conidiophores and detached capilliconidia (AO); d – primary conidium forming three capillary conidiophores and a rest of primary conidium with a capilla (AO); e, f – premature resting spores (AO); g, h – mature resting spores with black episporium. Aphid-pathogenic Entomophthorales S611

Plate 8. Neozygites microlophii Keller: a – primary conidia; b – conidiophores with forming primary conidia; c – a rest of primary conidium with a capilla; d – detached capilliconidia; e – mature resting spores. Neozygites turbinata (Kenneth) Remaudi`ere et Keller: f – mature resting spores with a germ capilliconidium; g – resting spore with a germ capilla; h – resting spore with a germ capilliconidium; i – detached germ capilliconidia. S612 M. Barta & Ľ. Cagáň

Plate 9. Neozygites cinarae Keller: a – primary conidia; b – conidiophores; c – primary conidium germinating and forming a germ tube; d – primary conidia; e – primary conidium forming a capilliconidium and rests of primary conidia with capillary conidiophores; f – resting spores dissected from a host. Aphid-pathogenic Entomophthorales S613

Plate 10. Conidiobolus obscurus (Hall et Dunn) Remaudi`ere et Keller: a – primary conidia (aceto-orcein “AO”); b, c – conidiophores with forming primary conidia (AO); d – germinating conidia forming germ tubes or secondary conidia (AO); e – hyphal bodies (AO); f – mature resting spores with thick cell wall(AO); g – premature resting spores (AO). S614 M. Barta & Ľ. Cagáň

Plate 11. Conidiobolus thromboides Drechsler: a – primary conidia and germinating primary conidia(aceto-orcein “AO”); b – mono- hyphal rhizoid c – secondary conidia with a rest of the primary one (AO); d – primary conidium forming atop of simple conidiophore; f – germinating conidia forming successive conidia (AO). Aphid-pathogenic Entomophthorales S615

Plate 12. a – Pea aphid killed by P. neoaphidis, white circle of projected conidia around the cadaver; b – the common nettle aphid killed by P. neoaphidis;c–Uroleucon aeneum killed by P. neoaphidis; d, e – aphids killed by P. neoaphidis are fixed to plant by rhizoids forming on the ventral part of abdomen; f – a group of Uroleucon aeneum killed by P. uroleuconii; g – the rose aphid killed by E. planchoniana;h–Anoecia corni killed by Z. aphidis; i, j – the common nettle aphid killed by C. obscurus; k – the common nettle aphid killed by N. microlophii; l, m – cadavers of the black bean aphid filled with resting spores of N. fresenii on petioles of sugarbeet; n – a colony of the black bean aphid infected by N. fresenii; o – the common nettle aphid killed by N. fresenii. S616 M. Barta & Ľ. Cagáň

Plate 13. a, b – Cadavers of the giant willow aphid filled with resting spores of N. turbinata and hanging down a willow branch; c, d, e – rests of the aphid cadavers with black resting spores on willow branches after six months. Plates of medium with growing cultures of P. neoaphidis (f), C. thromboides (g), Z. radicans (h), and C. obscurus (i).