Journal of Invertebrate Pathology 98 (2008) 256261

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Journal of Invertebrate Pathology

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Potential of Lecanicillium spp. for management of insects, nematodes and plant diseases
Mark S. Goettel a,*, Masanori Koike b, Jeong Jun Kim c, Daigo Aiuchi b, Ryoji Shinya d, Jacques Brodeur e
a

Lethbridge Research Centre, Agriculture and Agri-Food Canada, P.O. Box 3000, 5403 1st Avenue South, Lethbridge, Alta., Canada T1J 4B1 Department of Agro-environmental Science, Obihrio University of Agriculture & Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan c Applied Entomology Division, National Institute of Agricultural Science and Technology, Suwon 441-707, Republic of Korea d Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan e Institut de recherche en biologie vgtale, Dpartement de sciences biologiques, Universit de Montral, Montral, QC, Canada H1X 2B2
b

a r t i c l e

i n f o

a b s t r a c t
Fungi in the genus Lecanicillium (formerly classied as the single species Verticillium lecanii) are important pathogens of insects and some have been developed as commercial biopesticides. Some isolates are also active against phytoparasitic nematodes or fungi. Lecanicillium spp. use both mechanical forces and hydrolytic enzymes to directly penetrate the insect integument and the cell wall of the fungal plant pathogen. In addition to mycoparasitism of the plant pathogen, the mode of action is linked to colonization of host plant tissues, triggering an induced systemic resistance. Recently it was demonstrated that development of Lecanicillium hybrids through protoplast fusion may result in strains that inherit parental attributes, thereby allowing development of hybrid strains with broader host range and other increased benets, such as increased viability. Such hybrids have demonstrated increased virulence against aphids, whiteies and the soybean cyst nematode. Three naturally occurring species of Lecanicillium, L. attenuatum, L. longisporum, and an isolate that could not be linked to any presently described species based on rDNA sequences have been shown to have potential to control aphids as well as suppress the growth and spore production of Sphaerotheca fuliginea, the causal agent of cucumber powdery mildew. These results suggest that strains of Lecanicillium spp. may have potential for development as a single microbial control agent effective against several plant diseases, pest insects and plant parasitic nematodes due to its antagonistic, parasitic and disease resistance inducing characteristics. However, to our knowledge, no Lecanicillium spp. have been developed for control of phytopathogens or phytoparasitic nematodes. 2008 Elsevier Inc. All rights reserved.

Article history: Received 23 January 2008 Accepted 30 January 2008 Available online 13 March 2008 Keywords: Lecanicillium longisporum Lecanicillium muscarium Sphaerotheca fuliginea Verticillium lecanii Heterodera glycines Aphids Cucumber powdery mildew Entomopathogenic fungi Multiple control

1. Introduction Mitosporic fungi such as Beauveria bassiana, Lecanicillium spp. (formerly Verticiliium lecanii), Metarhizium anisopliae and Isaria fumosorosea (formerly Paecilomyces fumosoroseus) are well known entomopathogens that have been commercially developed as biopesticides (Goettel et al., 2005; Faria and Wraight, 2007). However, recently it has been discovered that these entomopathogenic fungi may also demonstrate activity against fungal plant pathogens (Kang et al., 1996; Verhaar et al., 1997, 1998; Askary et al., 1998; Dik et al., 1998; Benhamou and Brodeur, 2000, 2001; Miller urn, 2005) et al., 2004; Ownley et al., 2004, 2008; Kavkov and C which suggests that such entomopathogens have potential to be developed as biopesticides with multiple roles. Here we review the potential of Lecanicillium spp. as multipurpose microbial control agents of pest arthropods, plant parasitic nematodes and plant pathogens.
* Corresponding author. Fax: +1 403 382 3156. E-mail address: goettelm@agr.gc.ca (M.S. Goettel). 0022-2011/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.01.009

2. Nomenclature of Lecanicillium Until recently, the form genus Verticillium contained a wide variety of species with diverse host ranges including arthropods, nematodes, plants and fungi (Zare and Gams, 2001). The genus has been recently redened using rDNA sequencing, placing all insect pathogens into the new genus Lecanicillium (Zare et al., 2000; Gams and Zare, 2001; Zare and Gams, 2001). These include L. attenuatum, L. lecanii, L. longisporum, L. muscarium and L. nodulosum, which were all formerly classied as V. lecanii. These recent reclassications bring forth the possibility that several different species were actually involved in previous studies. There is also evidence that in recent literature, some authors have simply replaced the genus name Verticillium with Lecanicillium without conducting the necessary rDNA sequencing, adding to the confusion (Sugimoto et al., 2003; Koike et al., 2007a). In this review we will refer to the former name, Verticillium lecanii, as Lecanicillium spp. unless it is specically known that the species in question was veried using the new nomenclature.

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3. Lecanicillium spp. as pathogens of insects and mites Species of Lecanicillium have a wide host range and have been isolated from a variety of insect orders (Zare and Gams, 2001). Lecanicillium spp. generally follow the typical pathway of pathogenesis of entomopathogenic mitosporic fungi: adherence of conidia to the host cuticle; germination of conidia; penetration of the cuticle; production of blastospores within the hemocoel; ramication of the mycelia and invasion of tissues causing death of the host; and nally, production of conidia on the surface of the cadaver (Askary et al., 1999). Some Lecanicillium spp. strains are known to produce toxic metabolites in vitro, which may be implicated in the ability of the fungus to overcome its host (Claydon and Grove, 1982; Gindin et al., 1994). At least 15 products based on Lecanicillium spp. have been, or are in the process of being commercialized with various trade names for use against a variety of pests in numerous countries worldwide (Faria and Wraight, 2007). L. attenuatum has been isolated from a caterpillar in Poland, leaf litter in the US (Zare and Gams, 2001) and an aphid in S. Korea (Kim et al, 2007) while L. lecanii is primarily a pathogen of soft scale insects (Zare and Gams 2001). L. nodulosum substrates include numerous insects, mites and decaying wood (Zare and Gams 2001). Lecanicillium muscarium has a broader host range and has been isolated from a range of substrates, mainly insects and fungi and has been commercialized as the biopesticides Mycotal against whiteies and thrips and Verticillin against whiteies, aphids and mites (Faria and Wraight, 2007). Strains of L. longisporum have been commercialized against aphids as Vertalec and whiteies and thrips as Vertirril. Isolates received by Zare and Gams (2001) as Vertalec proved to be based either on L. longisporum or L. muscarium leading them to speculate that initially Vertalec was based on L. muscarium and later changed to L. longisporum or that different strains are sold in different countries under the same product name. However, the isolate labeled Vertalec which was identied as L. muscarium must have been a contaminant or misidentication, as Vertalec has never been based on the smaller spored L. muscarium. Vertalec is based solely on strain CBS 102072 identied by Zare and Gams (2001) as L. longisporum while Mycotal is based solely on L. muscarium (W. Ravensberg, pers commun.). 4. Lecanicillium spp. as pathogens of plant parasitic nematodes Species of Lecanicillium are well known and important nematophagous fungi with potential for development as biopesticides against phytoparasitic nematodes. For instance, L. psalliotae, L. antillanum, and other Lecanicillium spp. infect the eggs of the root-knot nematode Meloidogyne incognita (Gan et al., 2007; Nguyen et al., 2007). Lecanicillium spp. infect females, cysts and eggs of Heterodera glycines, the soybean cyst nematode (SCN), reducing nematode populations in laboratory and greenhouse studies (Meyer et al., 1997). Mutant strains of an SCN active strain were induced through UV radiation which resulted in increased efcacy against this nematode (Meyer and Meyer, 1996). Some reports indicated that immature eggs are more susceptible to fungal attack than the mature eggs containing secondstage juveniles (J2) (Chen and Chen, 2003; Irving and Kerry, 1986; Kim and Riggs, 1991). Furthermore, Meyer et al. (1990) demonstrated that one strain of Lecanicillium sp. (as V. lecanii) decreased the number of viable SCN eggs from yellow females, whereas the viability of eggs from cysts was not affected. This strain also reduced the viability of SCN eggs without colonization of the egg; however, no such effect was observed in other strains.

5. Lecanicillium spp. as plant pathogen antagonists/parasites Lecanicillium spp. have activity against numerous phytopathogenic fungi including powdery mildews (Verhaar et al., 1997, 1998; Askary et al., 1998; Dik et al., 1998; Miller et al., 2004), rusts (Spencer and Atkey, 1981; Leinhos and Buchenauer, 1992) green molds (Benhamou and Brodeur, 2000) and Pythium (Benhamou and Brodeur, 2001). Fungi that may control plant pathogenic fungi can act through antibiosis and mycoparasitism (Kiss, 2003). Some Lecanicillium isolates act as mycoparasites, attaching to powdery mildew mycelia and conidia, producing enzymes such as chitinase, that allow penetration of the mildew spores and hyphae, killing the pathogen (Askary et al., 1997). Leinhos and Buchenauer (1992) demonstrated that several Lecanicillium spp. were able to penetrate and colonize uredial sori of Puccinia coronata. In Penicillium digitatum, the mode of action was attributed to changes in host cells prior to contact by the Lecanicillium spp. (Benhamou and Brodeur, 2000) while in P. ultimatum, in addition to mycoparasitism of the plant pathogen, the mode of action was linked to colonization of host plant tissues, triggering a plant defense reaction (Benhamou and Brodeur, 2001). Hirano et al. (in press) found that applying L. muscarium blastospores to cucumber roots induced systemic resistance. L. muscarium pre-inoculated plants suffered signicantly fewer lesions and reduced disease severity compared with non-inoculated plants. Kusunoki et al. (2006) and Koike et al. (2007b) found that root treatment with L. muscarium reduced disease incidence and wilting score in other soil-borne disease combinations such as tomato Verticillium dahliae, Japanese radishV. dahliae, and melonFusarium oxysporum f.sp. melonis (Fig. 1). 6. Genetic improvement using protoplast fusion As previously mentioned, Mycotal (L. muscarium) and Vertalec (L. longisporum) are strains commercialized for insect control. Strain B-2 of L. muscarium, which was isolated from the peach aphid (Myzus persicae) in Japan, has high epiphytic ability on cucumber leaves (Koike et al., 2004). Protoplast fusion was performed using three strains of Lecanicillium spp. (as V. lecanii) to obtain new strains possessing useful characteristics as biological control agents (Aiuchi et al. 2004, 2008). From the combination of Vertalec Mycotal, B-2 Mycotal, and B-2 Vertalec, many hybrid strains were detected. Nit (nitrate non-utilizing) mutants (Correll et al., 1987) were used for visually selecting protoplasts. The morphological characteristics of the hybrid strains differed from those of their parental nit mutants. Furthermore, genomic analyses were done to ascertain the success of protoplast fusion. These conrmed protoplast fusion in genomic DNA but not in mitochondrial DNA (mtDNA). In both analyses, they observed a uniform biased tendency of the banding pattern, depending on the combination of the parental strains. Some of these genomic analyses conrmed successful fusion and/or genetic recombination. These results demonstrated the usefulness of conducting genomic analyses such as polymerase chain reaction-restriction fragment length polymorphism and arbitrarily primed-PCR for discovering nucleotides that exhibit high polymorphism in order to ascertain success of protoplast fusion (Aiuchi et al., 2008). Further studies were conducted to screen desirable Lecanicillium hybrid strains that have a wide host range or increased efcacy (Aiuchi et al., 2007). Initially, 43 hybrid strains were used in bioassays against the cotton aphid, Aphis gossypii. Of these, 30 strains induced mortality equal to or higher than Vertalec (42%). Secondly, 50 hybrid strains were used in bioassays against the greenhouse whitey (Trialeurodes vaporariorum). Of these, 37 strains exhibited an equal or higher infection rate as compared to that of Mycotal

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Fig. 1. Effects of Lecanicillium muscarium on prevention of damage caused by Fusarium wilt of Melon (Fusarium oxysporum f.sp melonis) (left: control, center: Pathogen inoculation, right: Lecanicillium muscarium treatment before pathogen inoculation).

(36.2%). Finally, 50 hybrid strains were applied to cucumber leaves in order to test strain viability under low humidity conditions (ca. 13% RH). Two weeks after application, 17 hybrid strains exhibited viabilities equal to or higher than B-2 (1.5 103 cfu/cm2). These results identied hybrid strains whose parental characteristics had not only recombined but also whose pathogenicity or viability had improved, with a hybrid isolate even producing conidia on a leaf hair (Fig. 2). Finally, 13 candidate hybrid strains were selected that exhibited improved qualities, and these hybrid strains can be expected to be highly effective as biological control agents. Shinya et al. (2008a) investigated whether the protoplast fusion technique was an effective tool for development of more efcient nematode control agents. Three parental strains (Vertalec, Mycotal, and B-2) and their 162 hybrid strains were screened in greenhouse pot tests against the soybean cyst nematode H. glycines. Some of these hybrid strains reduced the density of SCN in the soil and suppressed damage to soybean plants. In particular, one hybrid strain, AaF42 (Vertalec: L. longisporum Mycotal: L. muscarium), reduced nematode egg density by 93% as compared with the control providing excellent protection to soybean plants (Fig. 3). Furthermore, this strain signicantly reduced cyst and egg densities compared to the parental strains. Shinya et al. (2008b) also evaluated the effects of fungal culture ltrates of the Lecanicillium hybrid strains on mature eggs, embryonated eggs (eggs fertilized but without development of juveniles),

Fig. 3. Lecanicillium hybrid (Vertalec Mycotal) protected soybean plants from soybean cyst nematode (Heterodera glycines) (left: control: right: treated plants).

Fig. 2. Lecanicillium muscarium hybrid (B-2 Mycotal) 2aF43 producing conidia on a cucumber leaf hair.

and J2 of SCN and compared these effects to those of their parental strains. The fungal culture ltrates of some hybrid strains inhibited egg hatch of mature eggs. Furthermore, the fungal culture ltrates of two hybrid strains, AaF23 and AaF42 (Vertalec: L. longisporum Mycotal: L. muscarium), exhibited high toxicity against embryonated eggs. However, most of the fungal culture ltrates did not inactivate J2. These results suggested that enzymes or other active compounds in the fungal culture ltrates exhibit activity against specic stages in the SCN life cycle. In addition, based on a visual assessment of the morphological changes in eggs caused by ltrates of each strain, there were differences between the hybrid strains and their respective parental strains with regard to the active substances produced by Lecanicillium spp. against the embryonated eggs. As a result of promoting recombination of whole genomes via protoplast fusion, several hybrid strains may have enhanced production of active substances that are different from those produced by their parental strains. It was concluded that natural substances produced by Lecanicillium hybrids are important factors involved in the suppression of SCN damage. Finally, Shinya et al. (2008c) investigated the pathogenicity and mode of action of Lecanicillium hybrid strains AaF23 and AaF42 to the sedentary stages of SCN. Three different sedentary stages (pale yellow female, yellow brown cyst, and dark brown cyst) of SCN were treated with the Lecanicillium hybrids and incubated on water agar. After 3 weeks incubation, eggs were investigated for the following: (i) the infection frequencies of eggs, (ii) the number of eggs laid, and (iii) the number of mature and healthy eggs. Subse-

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quently, the fecundity of SCN treated with the Lecanicillium hybrids was investigated in greater detail. It was found that some strains had a greater ability to infect eggs and this signicantly reduced the number of eggs laid and number of mature eggs in the pale yellow females. This study indicates that Lecanicillium hybrids are more effective against the females than the cysts of SCN and that the hybrid strains may act on the nematode in multiple ways. In conclusion, some of the hybrid strains exhibited enhanced biocontrol efcacy by protoplast fusion. Therefore, the protoplast fusion technique may be a potentially valuable tool for developing Lecanicillium strains for commercialization as microbial control agents. 7. Lecanicillium spp. for multiple pest management: powdery mildew and aphids Three species of Lecanicillium, L. attenuatum, L. longisporum, and an isolate (DAOM 198499) that could not be linked to any presently described species (Zare and Gams, 2001) based on rDNA sequences (Bissett, pers. commun.) have been shown to have potential to control aphids as well as suppress the growth and spore production of Sphaerotheca fuliginea (Askary et al., 1998; Kim et al., 2007, 2008). Askary et al. (1998) compared the activity of three strains of Lecanicillium spp. (DAOM 198499, 216596 and Vertalec (all described as V. lecanii)) against Macrosiphum euphor-

biae and S. fuliginea and concluded that strain DAOM 198499 was superior in its dual activity against powdery mildew and potato aphids. They reported that Vertalec had good activity against aphids, but it was not as efcient against powdery mildew. Kim et al. (2007) found no signicant differences in activity against cucumber powdery mildew with these same two isolates using a leaf disk bioassay. The discrepancies between the results of Askary et al. (1998) and Kim et al. (2007) may be due to strain differences of the S. fuliginea. Kim et al. (2007) found no difference in activity against powdery mildew between conidia and blastospores in the three Lecanicillium spp. tested in leaf disk bioassays. In follow up studies using potted whole plant bioassays (Kim et al., 2008), Vertalec was applied onto cucumber plants that had been infested with either cotton aphids, S. fuliginea spores, or both. Irradiation-inactivated Vertalec was also applied to an identical series of cucumber plants as a control. The Vertalec was highly pathogenic to adult aphids with an LT50 of 7 days. The irradiated Vertalec did not affect aphid survival. Application of either active or irradiated Vertalec signicantly suppressed spore production of S. fuliginea compared to the water controls. For dual control assays, Vertalec applications were made one day after infestation of both aphid and S. fuliginea on cucumber plants. Fifteen days after the Vertalec treatments, the numbers of surviving aphids and the production of powdery mildew spores were signicantly reduced

Fig. 4. Application of Vertalec for dual management of aphids and powdery mildew on greenhouse cucumbers. (a) Aphid only, (b) cucumber powdery mildew (Sphaerotheca fuliginea) only, (c) aphid and powdery mildew, (d) aphid, powdery mildew plus Vertalec application.

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compared with the water control. The presence of aphids also suppressed S. fuliginea spore production. Further studies under greenhouse conditions supported the concept of using Vertalec for dual control of aphids and cucumber powdery mildew. (Kim et al., unpublished). Vertalec treatment reduced powdery mildew and cotton aphid populations on cucumber leaves which were previously inoculated with aphids, powdery mildew or both (Fig. 4). 8. Conclusions It is evident that the potential of Lecanicillium spp. as biopesticides extends well beyond their development as bioinsecticides. Naturally occurring isolates of L. longisporum have activity against aphids and powdery mildew. Through protoplast fusion, hybrids can be developed with improved efcacy and host range. There is much potential for development of a single microbial control agent based on a Lecanicillium sp. which could be effective against several plant diseases, pest insects and plant parasitic nematodes due to its antagonistic, parasitic and disease resistance inducing characteristics. However, to our knowledge, no Lecanicillium spp. have been developed for control of phytopathogens or phytoparasitic nematodes. References
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