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Potential ofLecanicillium 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 4B1b Department of Agro-environmental Science, Obihrio University of Agriculture & Veterinary Medicine, Obihiro, Hokkaido 080-8555, JapancApplied Entomology Division, National Institute of Agricultural Science and Technology, Suwon 441-707, Republic of Koread Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japane Institut de recherche en biologie vgtale, Dpartement de sciences biologiques, Universit de Montral, Montral, QC, Canada H1X 2B2

a r t i c l e i n f o

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

a b s t r a c t

Fungi in thegenus Lecanicillium (formerly classified 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 path-

ogen. 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 develop-

ment of Lecanicillium hybrids through protoplast fusion may result in strains that inherit parental attri-

butes, thereby allowing development of hybrid strains with broader host range and other increased

benefits, such as increased viability. Such hybrids have demonstrated increased virulence against aphids,

whiteflies and the soybean cyst nematode. Three naturally occurring species of Lecanicillium, L. attenua-

tum, 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 ofSphaerotheca fuliginea, the causal agent of cucumber powdery mildew. These results

suggest that strains ofLecanicillium 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 antag-

onistic, parasitic and disease resistance inducing characteristics. However, to our knowledge, no Lecani-

cillium spp. have been developed for control of phytopathogens or phytoparasitic nematodes.

2008 Elsevier Inc. All rights reserved.

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 bio-

pesticides (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

et al., 2004; Ownley et al., 2004, 2008; Kavkov and Curn, 2005)

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 con-

trol agents of pest arthropods, plant parasitic nematodes and plant

pathogens.

2. Nomenclature ofLecanicillium

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 redefined 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 in-

clude L. attenuatum, L. lecanii, L. longisporum, L. muscarium and

L. nodulosum, which were all formerly classified as V. lecanii.

These recent reclassifications 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 sequenc-

ing, 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 specifically

known that the species in question was verified using the

new nomenclature.

0022-2011/$ - see front matter 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.jip.2008.01.009

* Corresponding author. Fax: +1 403 382 3156.

E-mail address: goettelm@agr.gc.ca (M.S. Goettel).

Journal of Invertebrate Pathology 98 (2008) 256261

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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). Lec-

anicillium spp. generally follow the typical pathway of pathogene-

sis of entomopathogenic mitosporic fungi: adherence of conidia to

the host cuticle; germination of conidia; penetration of the cuticle;

production of blastospores within the hemocoel; ramification ofthe mycelia and invasion of tissues causing death of the host;

and finally, 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 iso-

lated 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

whiteflies and thrips and Verticillin against whiteflies, aphids

and mites (Faria and Wraight, 2007). Strains ofL. longisporum have

been commercialized against aphids as Vertalec and whiteflies

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

identified as L. muscarium must have been a contaminant or misi-

dentification, as Vertalec has never been based on the smallerspored L. muscarium. Vertalec is based solely on strain CBS

102072 identified 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 ofLecanicillium are well known and important nemato-

phagous 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; Ngu-

yen et al., 2007). Lecanicillium spp. infect females, cysts and eggs

ofHeterodera glycines, the soybean cyst nematode (SCN), reducingnematode populations in laboratory and greenhouse studies

(Meyer et al., 1997). Mutant strains of an SCN active strain were in-

duced through UV radiation which resulted in increased efficacy

against this nematode (Meyer and Meyer, 1996).

Some reports indicated that immature eggs are more suscep-

tible to fungal attack than the mature eggs containing second-

stage 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 phytopatho-

genic 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 fungican 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 digita-

tum, 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 blas-

tospores to cucumber roots induced systemic resistance. L. musca-

rium pre-inoculated plants suffered significantly 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 melonFusar-

ium oxysporum f.sp. melonis (Fig. 1).

6. Genetic improvement using protoplast fusion

As previously mentioned, Mycotal (L. muscarium) and Verta-

lec (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 per-

formed using three strains ofLecanicillium spp. (as V. lecanii) to ob-

tain new strains possessing useful characteristics as biological

control agents (Aiuchi et al. 2004, 2008). From the combination

of VertalecMycotal, B-2Mycotal, 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 confirmed 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 genomicanalyses confirmed successful fusion and/or genetic recombina-

tion. These results demonstrated the usefulness of conducting

genomic analyses such as polymerase chain reaction-restriction

fragment length polymorphism and arbitrarily primed-PCR for dis-

covering nucleotides that exhibit high polymorphism in order to

ascertain success of protoplast fusion (Aiuchi et al., 2008).

Further studies were conducted to screen desirable Lecanicilli-

um hybrid strains that have a wide host range or increased efficacy

(Aiuchi et al., 2007). Initially, 43 hybrid strains were used in bioas-

says against the cotton aphid,Aphis gossypii. Of these, 30 strains in-

duced mortality equal to or higher than Vertalec (42%). Secondly,

50 hybrid strains were used in bioassays against the greenhouse

whitefly (Trialeurodes vaporariorum). Of these, 37 strains exhibited

an equal or higher infection rate as compared to that of Mycotal

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(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 re-

sults identified 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 efficient

nematode control agents. Three parental strains (Vertalec, Mycot-

al, and B-2) and their 162 hybrid strains were screened in green-

house pot tests against the soybean cyst nematode H. glycines.

Some of these hybrid strains reduced the density of SCN in the soiland suppressed damage to soybean plants. In particular, one hy-

brid strain, AaF42 (Vertalec: L. longisporum Mycotal: L. muscari-

um), reduced nematode egg density by 93% as compared with

the control providing excellent protection to soybean plants

(Fig. 3). Furthermore, this strain significantly reduced cyst and

egg densities compared to the parental strains.

Shinya et al. (2008b) also evaluated the effects of fungal culture

filtrates of the Lecanicillium hybrid strains on mature eggs, embry-

onated eggs (eggs fertilized but without development of juveniles),

and J2 of SCN and compared these effects to those of their parental

strains. The fungal culture filtrates of some hybrid strains inhibited

egg hatch of mature eggs. Furthermore, the fungal culture filtrates

of two hybrid strains, AaF23 and AaF42 (Vertalec: L. longispo-

rum Mycotal: L. muscarium), exhibited high toxicity against

embryonated eggs. However, most of the fungal culture filtrates

didnot inactivate J2. These results suggested that enzymes or other

active compounds in the fungal culture filtrates exhibit activity

against specific stages in the SCN life cycle. In addition, based on

a visual assessment of the morphological changes in eggs caused

by filtrates of each strain, there were differences between the hy-

brid strains and their respective parental strains with regard tothe 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 fol-

lowing: (i) the infection frequencies of eggs, (ii) the number of eggslaid, and (iii) the number of mature and healthy eggs. Subse-

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).

Fig. 2. Lecanicillium muscarium hybrid (B-2Mycotal

) 2aF43 producing conidiaon a cucumber leaf hair.

Fig. 3. Lecanicillium hybrid (Vertalec Mycotal) protected soybean plants from

soybean cyst nematode (Heterodera glycines) (left: control: right: treated plants).

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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 significantly reduced

the number of eggs laid and number of mature eggs in the pale yel-

low 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 efficacy 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 po-

tential 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 efficient against powdery mildew. Kim

et al. (2007) found no significant differences in activity against

cucumber powdery mildew with these same two isolates using

a leaf disk bioassay. The discrepancies between the results of Ask-

ary et al. (1998) and Kim et al. (2007) may be due to strain dif-

ferences of the S. fuliginea. Kim et al. (2007) found no difference

in activity against powdery mildew between conidia and blas-

tospores 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 significantly suppressed spore production

ofS. fuliginea compared to the water controls. For dual control as-

says, 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 significantly reduced

Fig. 4. Application of Vertalec

for dual managementof aphids andpowdery mildew on greenhouse cucumbers. (a)Aphidonly, (b)cucumber powderymildew (Sphaerothecafuliginea) 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 sup-

pressed 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 re-

duced 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 biopesti-

cides 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 efficacy and host range. There is

much potential for development of a single microbial control agent

based ona 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 characteris-

tics. However, to our knowledge, no Lecanicillium spp. have been

developed for control of phytopathogens or phytoparasitic

nematodes.

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