Laboratory bioassays to assess the pathogenicity of mitosporic fungi to Varroa destructor (Acari: Mesostigmata), an ectoparasitic mite of the honeybee, Apis mellifera

Biological Control 24 (2002) 266–276 www.academicpress.com

Laboratory bioassays to assess the pathogenicity of mitosporic fungi to Varroa destructor (Acari: Mesostigmata), an ectoparasitic mite of the honeybee, Apis mellifera Katie E. Shaw,a Gillian Davidson,b Suzanne J. Clark,a Brenda V. Ball,a,* Judith K. Pell,a David Chandler,b and Keith D. Sunderlandb b

a IACR-Rothamsted, Plant and Invertebrate Ecology Division, Harpenden, Herts AL5 2JQ, UK Department of Entomological Sciences, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK

Received 21 August 2001; accepted 4 February 2002

Abstract A laboratory bioassay was developed to measure the pathogenicities of isolates of mitosporic fungi to Varroa destructor, an ectoparasite of the European honeybee, Apis mellifera. Forty isolates of entomopathogenic fungi were assessed against V. destructor in a single-dose experiment (conidial concentration 1
108 ml1 ) at 25 °C and 100% RH. The fungal species were Verticillium lecanii (nine isolates), Hirsutella spp. (16 isolates), Paecilomyces spp. (three isolates), Beauveria bassiana (four isolates), Metarhizium spp. (six isolates), and Tolypocladium spp. (two isolates). All isolates could infect and kill V. destructor and 26 caused mean times to death of less than 100 h. Control (Tween-treated) mortality was 5% at 7 days post-treatment. Nineteen isolates were also examined for side effects against bees. Caged bees sprayed with conidial suspensions (1
108 ml1 ) of seven of these isolates died within 14 days. However, not all mortality could be attributed to fungal infection as confirmed by sporulation; the mortality of control bees was 27%. Nine isolates were selected for further examination against V. destructor at 30 °C and 40% RH to simulate the conditions in bee colonies. Of these, three isolates of M. anisopliae, one of V. lecanii, and one of B. bassiana killed 100% of V. destructor within 7 days at a conidial concentration of 1
108 ml1 . One isolate of M. anisopliae also killed 97% of V. destructor within 7 days at a conidial concentration of 1
106 ml1 . These results indicate that entomopathogenic fungi have potential as microbial control agents of V. destructor in honeybee colonies. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Varroa destructor; Apis mellifera; Metarhizium spp.; Verticillium lecanii; Beauveria bassiana; Hirsutella spp.; Paecilomyces spp.; Tolypocladium spp.; Entomopathogenic fungus; Microbial control; Pathogenicity

1. Introduction For decades, the varroa mite, Varroa destructor Anderson and Trueman (Acari: Mesostigmata), parasitizing the European honeybee, Apis mellifera L. (Hymenoptera: Apidae) was described as Varroa jacobsoni Oudemans. However, recent research has shown that the mites infesting Apis cerana (F.), the Eastern hive bee, throughout Asia are a complex of at least two species (Anderson and Trueman, 2000). Only one of these species, V. destructor, has extended its range to A. mellifera in Europe, the Americas, Africa, and Austral*

Corresponding author. Fax: +44-1582-760-981. E-mail address: [email protected] (B.V. Ball).

asia and is now causing severe damage to bee populations world-wide (De Jong et al., 1982; Oldroyd, 1999). Adult female V. destructor feed directly on the hemolymph of honeybee pupae and adults (De Jong et al., 1982) and can activate and transmit honeybee viruses (Ball, 1997). An increased prevalence of bee diseases and colony mortality are the usual results of infestation (Thomas, 1997), causing a decline in pollination efficiency and honey production (Matheson, 1994). At present, the most effective and widely used means of controlling V. destructor is with chemical acaricides (Ferrer-Dufol et al., 1991). However, the number of approved products available in individual countries is limited and populations of mites resistant to pyrethroid (Elzen et al., 2000) and organophosphorous (Spreafico

1049-9644/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 9 - 9 6 4 4 ( 0 2 ) 0 0 0 2 9 - 4

K.E. Shaw et al. / Biological Control 24 (2002) 266–276

et al., 2001) acaricides are already established and their distribution is increasing (Elzen et al., 1998; Milani, 1999). Repeated use of chemical treatments also risks the accumulation of residues in hive products (Wallner, 1999). Alternative means of controlling mite populations such as the removal of infested drone brood and the use of organic acids and volatile essential oils are labor-intensive, imprecise, and of limited efficacy (Fries, 1997; Thomas, 1997). These problems have highlighted the need for alternative control strategies including biological and microbial control. No natural enemies have been observed causing population declines of V. destructor in honeybee colonies. However, entomopathogenic fungi, which kill other mites and ticks in nature, have been identified as potential microbial biopesticides (Chandler et al., 2000, 2001). This paper describes laboratory experiments to measure the pathogenicity of mitosporic fungi to adult female V. destructor feeding on A. mellifera pupae. Forty isolates of fungi were tested against V. destructor in a single-dose bioassay at 25 °C/100% RH. Selected isolates that killed V. destructor were then examined further under conditions that more accurately reflected the environment of a honeybee colony (30 °C and 40% RH). Subsidiary experiments to measure the effect of selected isolates on bees are also described. In this paper, pathogenicity and virulence are used to describe the ability of an isolate to penetrate host defenses and kill V. destructor (Tanada and Fuxa, 1982) and virulence is measured by the mean time to death of the host.

267

(25
25
110 mm3 ) and supplied ad libitum with water, 65% sucrose solution (measured using a refractometer) in gravity feeders and pollen (Bailey, 1971). The bees were maintained at 30 °C for 1 week before treatment. 2.3. Sources of fungal isolates and preparation of inoculum Forty isolates of entomopathogenic fungi from six genera (Beauveria, Hirsutella, Metarhizium, Paecilomyces, Tolypocladium, and Verticillium) were tested in this study (Table 1). Stock cultures of the isolates were stored in liquid nitrogen (Chandler, 1994). Laboratory cultures were grown on Sabouraud’s dextrose agar (SDA) slants and maintained at 4 °C for up to 6 months. Subcultures for laboratory experiments were grown on SDA from slant cultures and incubated in darkness at either 26  1 °C for 21 days (Hirsutella isolates) or 23  1 °C for 14 days (all other isolates), prior to assay. Conidia were harvested in sterile 0.03% Tween 80 and suspensions were filtered through milk filters (Lantor (UK), Bolton, UK) to remove hyphal fragments. Conidia were counted in an improved Neubauer hemacytometer and aliquots were prepared at concentrations of 1
106 or 1
108 ml1 in sterile 0.03% Tween 80. Suspensions were held on ice for a maximum of 24 h before assay. Viability was assessed by measuring the germination of conidia on SDA after incubation for 24 h at 23 °C (Goettel and Inglis, 1997) and was never less than 93%. 2.4. Laboratory bioassay to measure the susceptibility of V. destructor to entomopathogenic fungi

2. Materials and methods 2.1. Source of V. destructor for laboratory bioassays Populations of V. destructor were established in four colonies of A. mellifera by introducing brood frames infested with mites. Numbers of V. destructor were monitored weekly and experiments were done between June and October when mites were abundant (estimated 1500 mites per colony). Adult female V. destructor for laboratory bioassays were collected by hand from sealed worker brood cells. White-eyed bee pupae were collected by hand from uninfested frames as a food source for the mites. Mites were held overnight on bee pupae in 1.5 ml Eppendorf tubes plugged with dental roll at 30 °C and 100% RH in darkness before treatment. Five V. destructor were placed on each bee pupa. 2.2. Source of adult A. mellifera for laboratory bioassays Combs containing mature worker bee pupae were removed from mite-free colonies and maintained overnight at 35 °C. Groups of 30 newly emerged adult bees (<18 h old) were transferred to wooden cages

Groups of five V. destructor were immersed for 10 s in 5 ml conidial suspension or 0.03% Tween 80 as a control. Excess suspension was removed by filtration under vacuum through filter paper (Whatman No. 1) and the mites plus the wetted filter paper were transferred to a 9 cm diameter petri dish. This was sealed with parafilm and kept on the laboratory bench for 1 h. Each group of five mites was then transferred to a 1.5 ml Eppendorf tube containing a fresh white-eyed honeybee pupa. The Eppendorf tubes were placed horizontally on racks inside clear polypropylene containers (293
202
130 mm3 ) with ventilated push-fit lids. Containers were maintained in the dark at 25 °C and 100% RH (humidity obtained by placing 1% water agar in two 9 cm petri dish bases in the bottom of the container) or at 30 °C and 40% RH (humidity obtained by dissolving 400 g potassium carbonate in 250 ml distilled water poured into the bottom of the container). The temperature and humidity within the containers were monitored using Tinytalk Data Loggers (RS Components, Corby, UK). Conditions within containers set at 25 °C and 100% RH ranged from 25.8 to 26.9 °C at 100% RH. Conditions within containers set at 30 °C and 40% RH ranged from 29.4 to

268

K.E. Shaw et al. / Biological Control 24 (2002) 266–276

Table 1 Isolates tested against V. destructor in the first bioassay, and where indicated, in the second bioassay against honeybees (2), and the third bioassay under colony conditions (3) Species Beauveria bassiana

IsolateA a

431.99 (T228) 432.99b 433.99c 434.99 (ARSEF2869)d (H1)e (H2)e (H3)e (H4)e (H11)e (H12)e

Host or source

Country

2

3

Acari Anthonomus grandis (Coleoptera: Curculionidae) – Bephratelloides cubensis (Hymenoptera: Eurytomidae)

Denmark USA – USA

U U U

U

Acari: Tarsonemidae Acari: Tarsonemidae Eriophyes piri (Acari: Eriophyidae) Abacarus hystrix (Acari: Eriophyidae) Stenotarsonemus fragariae (Acari: Tarsonemidae) Stenotarsonemus fragariae (Acari: Tarsonemidae)

Poland Poland Poland Poland Poland Poland

U

U

Hirsutella sp.

435.99 436.99 437.99 438.99 439.99 440.99

Hirsutella kirchneri

46.81 (IMI251256)f 47.81 (IMI257456)f

Abacarus hystrix (Acari: Eriophyidae) Abacarus hystrix (Acari: Eriophyidae)

UK UK

Hirsutella necatrix

49.81 (IMI252317)f

Abacarus hystrix (Acari: Eriophyidae)

UK

Hirsutella thompsonii

34.79 51.81g 71.82 73.82g 74.82g 75.82g 77.82g

Eriophyes guerreronis (Acari: Eriophyidae) Eriophyes guerreronis (Acari: Eriophyidae) Eriophyes guerreronis (Acari: Eriophyidae) Phyllocoptruta captrila (Acari: Eriophyidae) Eriophyes guerreronis (Acari: Eriophyidae) Colomerus novahebridensis (Acari: Eriophyidae) Eriophyes guerreronis (Acari: Eriophyidae)

Ivory Coast USA Jamaica USA USA New Guinea Jamaica

441.99 (ARSEF3297)d 442.99 (ARSEF4556)d 443.99 (T248)a 444.99 (ATCC38249)h 445.99i

Boophilus sp. (Acari: Ixodidae) Boophilus sp. (Acari: Ixodidae) Acari Hylobius pales (Coleoptera: Curculionidae) –

Mexico USA – – –

456.99 (DAT F001)j ;k

Adoryphorus couloni (Coleoptera: Scarabaeidae)

Australia

446.99 (CCFC002085)

Mycobates sp. (Acari: Mycobatidae)

Canada

Metarhizium anisopliae

Metarhizium flavoviridae

U

U U

U U

U U U U U

U U U

var. flavoviridae Paecilomyces farinosus

l

Paecilomyces fumosoroseus

409.96 447.99 (KVL319)a

Phenacoccus solani (Hemiptera: Pseudococcidae) Ixodes ricinus (Acari: Ixodidae)

USA –

Tolypocladium inflatum

448.99 (ARSEF3278)d

Mycobates sp. (Acari: Mycobatidae)

Canada

Tolypocladium niveum

449.99 (CCFC002081)m

Mycobates sp. (Acari: Mycobatidae)

Canada

Verticillium lecanii

1.72n 17.76 19.79o 30.79 31.79 450.99 451.99 452.99 453.99

Macrosiphoniella sanborni (Homoptera: Aphididae) Cecidophyopsis sp. (Acari: Eriophyidae) Trialeurodes vaporariorum (Homoptera: Aleyrodidae) Cecidophyopsis sp. (Acari: Eriophyidae) Cecidophyopsis sp. (Acari: Eriophyidae) Cecidophyopsis ribis (Acari: Eriophyidae) Acari: Orbatidae Tetranychus urticae (Acari: Tetranychidae) Acari

UK UK UK UK UK UK Poland – Canada

A

(IMI235048)f (ARSEF1367)d (CBS317.70A)p (CCFC006079)m

U

U U U U

U

U

U

U

Isolate number in the Horticulture Research International culture collection (isolate number from culture collection of origin). Kindly supplied by T. Steenberg, Danish Pest Infestation Laboratory, Skovbrynet 14, DK-2800, Lyngby, Denmark. b Isolate forms the active ingredient in the proprietary mycopesticide ‘BotaniGard’ (Mycotech Corporation, P.O. Box 4109, Butte, MT 59702, USA). c Isolate forms the active ingredient in the proprietary mycopesticide ‘Naturalis’ (Troy Biosciences Inc., 113 South 47th Ave., Phoenix, AZ 850433, USA). d Kindly supplied by the USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF), USDA-ARS Plant Protection Research Unit, U.S. Plant, Soil and Nutrition Laboratory, Tower Road, Ithaca, New York, 14853-2901, USA. e Kindly supplied by C. Tkaczuk, University of Podlasie, Department of Plant Protection, ul. Prusa 14, 08110 Siedlce, Poland. f Obtained from the CAB International Mycological Institute, Bakeham Lane, Egham, Surrey, UK. g Kindly supplied by C.W. McCoy, University of Florida, CREC, 700 Experiment Station Road, Lake Alfred, FL 33850, USA. h Obtained from the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia, 20110-2209, USA. i Isolate forms the active ingredient in the proprietary mycopesticide ‘Bio-Blast’ (Eco-Science Corporation, 17 Christopher Way, Eatontown, NJ 07724, USA). a

K.E. Shaw et al. / Biological Control 24 (2002) 266–276

269

31.4 °C at 43.7–45.4% RH. Bee pupae were replaced every 4 days (at 30 °C) or 6 days (at 25 °C). Dead mites (no movement or response to stimulus) were removed and incubated on 1% water agar at 23 °C. The presence of sporulating mycelia on mite cadavers was used as an indication of fungus-induced mortality.

and incubated on 1% water agar at 23 °C. The presence of sporulating mycelia on honeybee cadavers was used to indicate fungus-induced mortality.

2.5. Laboratory bioassay to measure the susceptibility of adult A. mellifera to entomopathogenic fungi

Three laboratory bioassay experiments were done to assess isolates of entomopathogenic fungi as potential microbial control agents of V. destructor as follows.

Young adult bees were treated with conidia suspensions of entomopathogenic fungi. Groups of 30, oneweek-old adult worker bees were removed from their wooden cages (see above), briefly anesthetized with carbon dioxide and then transferred to spray cages. Each spray cage was made from a 12.5 cm square of 6 mm thick Perspex with a hole 10 cm in diameter in the center (Ball et al., 1994). A 12.5 cm square of 0.711 mm galvanized wire mesh with an aperture of 2.46 mm (60% open area) was fixed to one side of the Perspex and another held to the other side with bulldog clips. The depth of the Perspex allowed the bees to move freely between the wire screens but prevented clustering. The bees were allowed to recover from anesthetization at room temperature (<1 h) and were then sprayed with suspensions of fungal conidia (1
108 ml1 ) using a hand-held plant mister. The concentration was estimated by spraying the same volume of water over a petri dish of the same diameter as the spray cage aperture and weighing the liquid deposited. It was calculated to be 3:49
109 conidia=cm2 . The bees were returned to their bioassay cages without anesthetization immediately after spraying. Bees killed during this transfer were discarded. For the first 24 h post-inoculation, the bioassay cages were placed inside clear polypropylene containers (293
202
130 mm3 ) with ventilated, push-fit lids and maintained in darkness at an average temperature of 32.6 °C. An average 72.3% RH was obtained using a solution of 55 g glycerol: 45 g distilled water in two 9 cm petri dish bases in the bottom of the container (Johnson, 1940). After 24 h, the glycerol solutions were removed and the cages were maintained at 30 °C and ambient humidity (average 19.8% RH) for the remainder of the experiment The numbers of living and dead honeybees in each cage were counted daily for 21 days. Dead bees (no movement or response to stimulus) were removed

2.6. Assessing isolates of entomopathogenic fungi as potential microbial control agents of V. destructor

2.6.1. Susceptibility of V. destructor to 40 isolates of entomopathogenic fungi Forty isolates of entomopathogenic fungi (Table 1) were assessed against adult female V. destructor at a single concentration of fungal conidia using the laboratory bioassay described above. This experiment was done as a ‘maximum challenge’ bioassay, i.e., with the highest concentration of conidia practical for the experiment. Bioassays with Hirsulella spp. were done at a concentration of conidia of 1
106 ml1 because the isolates of this fungus sporulated poorly in culture. All other isolates (referred to hereafter as ‘non-Hirsutella isolates’) sporulated well in culture and bioassays with these fungi were done at a concentration of 1
108 ml1 . Experiment 1 was done at 25 °C and 100% RH to provide optimal conditions for fungal infection. Mite mortality was assessed daily for 10 days. The experiment was done according to an alpha design (Patterson and Williams, 1976) with two replicates of four blocks of 10 isolates. A control was included in each block and blocks were run consecutively. Mites collected for each block of the experiment were divided among the 11 treatments per block to give at least 60 mites per fungal isolate and 200 mites for controls over the two replicates. 2.6.2. Susceptibility of A. mellifera to 19 isolates of entomopathogenic fungi Nineteen isolates of entomopathogenic fungi that killed V. destructor in the first bioassay were examined for side effects against adult honeybees (Table 1) using the bioassay procedure described above. Young adult worker bees were tested, as this would be the principal life stage exposed to a microbial biopesticide applied to a mite-infested bee colony. Fungal isolates were tested against bees at a single concentration of conidia of 1
108 ml1 . After

Table 1 (continued). j Kindly supplied by Biocare Technology Pty Ltd, RMB 1084 Pacific Highway, Somersby, NSW 2250, Australia. k Isolate forms the active ingredient in the proprietary mycopesticide ‘Biogreen’ (Bio-Care Technology Pty. Ltd., R.M.B.1084 Pacific Highway, Somersby, NSW 2250, Australia). Previously described as M. anisopliae but reclassified by Driver et al., 2000. l Isolate forms the active ingredient in the proprietary mycopesticide ‘PFR97’ (Thermo-Trilogy Corporation, 9145 Guildford Road, Suite 175, Columbia, MD 21046, USA). m Obtained from the Canadian Collection of Fungal Cultures, ECORC Room 1015, K.W. Neatby building, C.E.F. Ottawa, Ontario, Canada. n Isolate forms the active ingredient in the proprietary mycopesticide ‘Vertalec’ (Koppert Biological Systems, P.O. Box 155, 2650 AD Berkel en Rodenrijs, The Netherlands). o Isolate forms the active ingredient in the proprietary mycopesticide ‘Mycotal’ (Koppert Biological Systems, The Netherlands). p Obtained from the Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740AG, BAARN, The Netherlands.

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K.E. Shaw et al. / Biological Control 24 (2002) 266–276

an initial 24 h period at ca. 70% RH, treated bees were held at 30°C and ca. 20% RH. Mortality was assessed daily for 21 days. Eleven isolates were tested in the first bioassay and eight in the second. Thirty bees treated with 0.03% Tween 80 were included as controls in each bioassay. Thirty bees were treated per isolate. 2.6.3. Susceptibility of V. destructor to nine isolates of entomopathogenic fungi tested under colony conditions Nine isolates of entomopathogenic fungi that killed V. destructor in the first bioassay and grew in vitro at 30 °C (G. Davidson, unpublished observations) were examined further against adult female V. destructor in laboratory bioassays at 30 °C and 40% RH. These conditions more closely reflect those within the brood nest of a honeybee colony. The nine isolates (see Table 1) were assessed at conidial concentrations of 1
106 and 1
108 ml1 using the bioassay procedure described above. Mortality was assessed twice a day for 14 days. Beauveria bassiana 432.99, Hirsutella thompsonii 75.82, Metarhizium anisopliae 442.99, and Verticillium lecanii 453.99 were also bioassayed at 25 °C and 100% RH to allow comparison with the previous bioassays; these four isolates were designated reference isolates. A latin square design with three replicates of three blocks of three isolates was used. The fungal isolates were randomly allocated but with the constraint that at least one, and at most two, reference isolates would be run in each block. Each isolate was tested at both concentrations each time it was tested. Blocks were run consecutively, 1/ week. Controls were included in each block at each temperature. The total number of mites treated with each isolate was 60 and 180 for the control. 2.7. Data analysis 2.7.1. Susceptibility of V. destructor to 40 isolates of entomopathogenic fungi The mean times to death (MTD) of mites were calculated for both replicates of all 40 fungal isolates using the daily mortality data. Estimates of times to death for mites still alive at the end of the experiment (censored observations) were obtained using the method of Taylor (1973) implemented in the Genstat 5 library procedure CENSOR (Genstat 5 Committee, 1993). MTDs (transformed to natural logarithms) were analyzed according to a linear mixed model with three levels of random effects (replicate runs, blocks of isolates within runs, and individual isolates within blocks) using Restricted Maximum Likelihood (REML) (Patterson and Thompson, 1971). Each MTD was weighted by the inverse of its variance after adding an offset of unity to allow for MTDs with zero variance (i.e., when all the individuals died on the same day). The data for the 24 non-Hirsutella and the 16 Hirsutella isolates were analyzed separately because these two groups of fungi were

bioassayed at different concentrations of conidia. The isolates were ranked in order of average MTD (N ¼ 2). 2.7.2. Susceptibility of A. mellifera to 19 isolates of entomopathogenic fungi MTDs of honeybees were calculated for each fungal isolate on each occasion, as described above. 2.7.3. Susceptibility of V. destructor to nine isolates of entomopathogenic fungi tested under colony conditions MTDs of mites were calculated for each replicate of each fungal isolate at each concentration and transformed values (natural logarithms) analyzed using REML as described above. The MTD estimates from 25 to 30 °C were analyzed separately. Mean MTDs (N ¼ 3) for each isolate were calculated. 3. Results 3.1. Susceptibility of V. destructor to 40 isolates of entomopathogenic fungi The average mortality in control treatments for Experiment 1 was 5% at 7 days post-inoculation (dpi) (n ¼ 199) and there was no evidence of fungus-induced mortality. Control data were excluded from the analyses as MTD estimates based on 9/199 deaths at 7 dpi are not realistic. Natural mortality was considered unlikely to have an impact on fungus-induced mortality; therefore the pathogenicities of the fungal isolates were compared directly without reference to the control data. Varroa destructor was susceptible to all 40 fungal isolates examined in the initial bioassay (Tables 2 and 3). Mean MTDs varied significantly within the non-Hirsutella isolate group (Table 2) ðv223 ¼ 99:8; p < 0:001Þ and within the Hirsutella isolate group (Table 3) ðv215 ¼ 538:5; p < 0:001Þ. Of the 24 non-Hirsutella isolates examined, 23 killed 93–100% of individuals at 7 dpi (Table 2). The two most virulent isolates, M. anisopliae 441.99 and M. anisopliae 442.99, caused 100% mortality within 3 days. Mean MTDs for the non-Hirsutella isolates ranged from 44 to 82 h. Five out of the top 10 isolates were isolates of V. lecanii. Mean MTD values for the Hirsutella isolates were generally greater than for the non-Hirsutella isolates (Table 3), probably due to the different conidia concentrations used. Hirsutella thompsonii isolates 75.82, 77.82, and 71.82 and Hirsutella necatrix isolate 49.81 killed 88– 97% of V. destructor at 7 dpi (Table 3) and mycosis was confirmed in >78% of these cadavers. Mean MTDs of the Hirsutella isolates ranged from 84 to 289 h (Table 3). 3.2. Susceptibility of A. mellifera to 19 isolates of entomopathogenic fungi The young adult bees exhibited a large control mortality in the bioassay: control mortalities were 27% in

K.E. Shaw et al. / Biological Control 24 (2002) 266–276

271

Table 2 Pathogenicity of non-Hirsutella isolates applied to V. destructor at 1
108 ml1 at 25 °C and 100% RH Rank

Species

Isolate

n

Mean MTD (h)

95% C.I.

% Mortalitya at 7 dpib

% Mycosisc at 7 dpi

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

M. anisopliae M. anisopliae V. lecanii V. lecanii M. anisopliae V. lecanii V. lecanii M. anisopliae B. bassiana V. lecanii M. anisopliae P. fumosoroseus B. bassiana B. bassiana V. lecanii T. inflatum P. farinosus P. fumosoroseus M. flavoviridae V. lecanii B. bassiana V. lecanii T. niveum V. lecanii

441.99 442.99 19.79 450.99 445.99 1.72 17.76 444.99 432.99 30.79 443.99 409.96 431.99 433.99 31.79 448.99 446.99 447.99 456.99 453.99 434.99 451.99 449.99 452.99

65 60 60 65 60 60 60 70 60 65 60 70 60 60 60 60 65 65 60 60 70 65 60 60

44 44 51 55 56 56 56 57 58 58 58 60 60 60 62 67 71 73 73 74 77 78 78 82

(36, (39, (43, (47, (47, (47, (48, (51, (49, (49, (52, (50, (53, (53, (50, (51, (57, (56, (61, (45, (63, (56, (56, (52,

100 100 100 100 100 98 100 100 100 100 100 100 100 100 100 100 100 97 100 63 100 95 100 93

100 100 100 100 100 98 100 100 100 100 100 100 100 100 97 100 97 97 100 60 100 75 82 93

53) 50) 62) 65) 66) 67) 66) 65) 69) 69) 66) 71) 68) 69) 78) 88) 89) 95) 87) 123) 94) 108) 110) 127)

Isolates ranked by mean of mean times to death (MTD, N ¼ 2) (back transformed), with 95% confidence intervals. % Mortality ¼ 100 (total number dead/n), where n is the number of mites tested. b Days post-inoculation. c % Mycosis ¼ 100 (total number sporulated/n). a

Table 3 Pathogenicity of Hirsutella isolates applied to V. destructor at 1
106 ml1 at 25 °C and 100% RH ranked by mean of mean times to death (MTD, N ¼ 2) (back transformed), with 95% confidence intervals Rank

Species

Access.

n

Mean MTD (h)

95% C.I.

% Mortalitya at 7 dpib

% Mycosisc at 7 dpi

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

H. thompsonii H. thompsonii H. thompsonii H. necatrix H. kirchneri Hirsutella sp. H. thompsonii Hirsutella sp. H. thompsonii H. thompsonii Hirsutella sp. H. thompsonii Hirsutella sp. H. kirchneri Hirsutella sp. Hirsutella sp.

75.82 77.82 71.82 49.81 47.81 437.99 51.81 438.99 74.82 73.82 439.99 34.79 435.99 46.81 440.99 436.99

60 60 64 60 60 65 60 60 60 70 60 60 60 65 70 60

84 91 103 120 139 168 175 192 194 221 231 236 246 254 272 289

(70, 101) (82, 101) (81, 130) (108, 133) (114, 170) (131, 214) (135, 228) (155, 238) (136, 277) (165, 297) (177, 303) (180, 310) (189, 321) (203, 318) (219, 338) (258, 325)

93 97 88 97 68 62 47 30 47 30 23 27 27 15 14 12

93 78 84 88 62 46 45 25 40 27 17 23 20 9 13 3

a

% Mortality ¼ 100 (total number dead/n), where n is the number of mites tested. Days post-inoculation. c % Mycosis ¼ 100 (total number sporulated/n). b

blocks 1 and 2 at 14 dpi (Table 4). Therefore some caution is required when interpreting the results from this experiment, as there may be an interaction between fungus and non-fungus sources of mortality. Mortalities of bees treated with fungi ranged from 10 to 100% and

27 to 100% in blocks 1 and 2, respectively. Bee mortality reached 100% for seven of the isolates within 14 dpi. Low levels of mortality were observed with three isolates of fungi: V. lecanii 453.99, V. lecanii 17.76, and H. necatrix 49.81. Differences in the susceptibilities of the

272

K.E. Shaw et al. / Biological Control 24 (2002) 266–276

Table 4 Pathogenicity of 19 isolates tested against A. mellifera at 1
108 ml1 at 30 °C and ambient RH, ranked for two blocks in the order of the mean time to death (MTD, N ¼ 1) with associated SE Species

Isolate

n

MTDa (h)

SE (MTD)

Alive at end (>21 days)

% Mortality at 14 dpib

% Sporulation at 14 dpi

Block 1 M. anisopliae V. lecanii M. anisopliae B. bassiana P. fumosoroseus V. lecanii M. anisopliae V. lecanii Control V. lecanii V. lecanii H. necatrix

445.99 1.72 444.99 432.99 409.99 30.79 443.99 450.99 Tween 453.99 17.76 49.81

30 30 30 30 31 30 30 30 30 30 30 30

61 106 121 139 153 222 224 311 532 638 838 909

1 13 9 9 8 21 15 52 45 63 63 45

0 0 0 0 0 0 0 9 16 19 23 26

100 100 100 100 100 73 90 57 27 30 17 10

87 57 57 100 71 33 37 37 0 7 3 10

Block 2 H. thompsonii B. bassiana H. thompsonii B. bassiana M. anisopliae M. anisopliae V. lecanii H. thompsonii Control

71.82 431.99 77.82 433.99 442.99 441.99 19.79 75.82 Tween

29 30 28 30 29 30 30 30 30

57 91 148 156 246 259 394 479 610

3 3 32 13 27 38 46 62 61

0 0 3 0 3 6 11 15 18

100 100 89 97 79 73 47 43 27

90 87 14 97 62 60 37 20 0

a b

Mean time to death. Days post-inoculation.

bees to isolates of entomopathogenic fungi were observed between and within fungus species (Table 4). 3.3. Susceptibility of V. destructor to nine isolates of entomopathogenic fungi tested under bee colony conditions Bioassays were done against V. destructor with selected isolates (Table 1) at 30 °C and 40% RH to reflect the environment of a honeybee colony. Average control mortality at 7 dpi was 3% (n ¼ 180). There was no evidence of fungus-induced mortality in the controls. Therefore the pathogenicities of the fungal isolates were compared directly without reference to the control data. Varroa destructor was susceptible to all nine fungal isolates examined (Table 5). There were significant effects on mortality due to conidia concentration ðv21 ¼ 194:2; p < 0:001; Wald test) and fungal isolate ðv28 ¼ 586:4; p < 0:001Þ and a significant interaction between these two parameters ðv28 ¼ 18:3; p < 0:05Þ. Mortalities of fungus-treated mites at 7 dpi ranged from 15 to 100% at conidia concentrations of 1
108 ml1 and 2–97% at 1
106 ml1 . All isolates, except V. lecanii 453.99, killed more quickly at a concentration of 1
108 ml1 than 1
106 ml1 . At a concentration of 1
108 ml1 , M. anisopliae isolates 442.99, 443.99, and 444.99, V. lecanii isolate 17.76 and B. bassiana isolate 432.99 caused 100% mortality at 7 dpi (Table 5).

Hirsutella thompsonii isolates 75.82 and 49.81 and V. lecanii isolate 453.99 caused 30, 15, and 27% mortalities, respectively (Table 5). For bioassays done at 1
106 ml1 , M. anisopliae 442.99 caused 97% mortality at 7 dpi (Table 5). Mortalities induced by the other isolates at this concentration of conidia ranged from 80% (B. bassiana 432.99) to 2% (H. necatrix 49.81) (Table 5). MTD values at 1
108 ml1 ranged from 56 h (M. anisopliae 442.99) to 632 h (V. lecanii 453.99). MTD values at 1
106 ml1 ranged from 90 h (M. anisopliae 442.99) to 604 h (V. lecanii 453.99). MTD confidence intervals were wider at 1
106 ml1 than at 1
108 ml1 (Table 5). Bioassays against mites were also done with a subset of four reference isolates (M. anisopliae 442.99, B. bassiana 432.99, V. lecanii 453.99, and H. thompsonii 75.82) at 25 °C and 100% RH to compare with bioassays done the previous year in the first experiment. Average control mortality at 7 dpi for this bioassay was 6% (n ¼ 180) and there was no evidence of fungus-induced mortality in the controls. Bioassays done with reference isolates M. anisopliae 442.99 and B. bassiana 432.99 were comparable with those of the first experiment (Table 6). These isolates consistently killed large numbers of mites in both bioassays (Table 6). Mortality data for H. thompsonii 75.82 and V. lecanii 453.99 were more variable within and between the two bioassays, possibly because these isolates were less pathogenic than the

K.E. Shaw et al. / Biological Control 24 (2002) 266–276

273

Table 5 Pathogenicity of nine isolates tested against V. destructor at two concentrations at 30 °C and 40% RH, and ranked by mean of mean times to death at 106 ml1 , (MTD, N ¼ 3) (back transformed), with 95% confidence limits Species

Isolate

M. anisopliae

442.99

B. bassiana

432.99

M. anisopliae

444.99

M. anisopliae

443.99

V. lecanii

17.76

H. thompsonii

77.82

H. thompsonii

75.82

H. necatrix

49.81

V. lecanii a b

Concentration 8

10 106 108 106 108 106 108 106 108 106 108 106 108 106 108 106 108 106

453.99

n

Mean MTDa (h)

95% C.I.

% Mortality at 7 dpib

% Mycosis at 7 dpi

60 60 59 60 60 60 60 60 60 60 60 60 56 60 60 60 60 60

56 90 63 95 74 102 60 178 84 194 127 304 284 429 208 594 632 604

(53, 58) (81, 100) (61, 64) (79, 115) (69, 80) (85, 123) (57, 63) (94, 336) (81, 88) (118, 320) (85, 188) (141, 653) (123, 657) (103, 1792) (155, 279) (437, 807) (235, 1701) (340, 1073)

100 97 100 80 100 67 100 53 100 43 70 28 30 18 15 2 27 5

100 97 100 80 100 65 100 40 100 43 3 0 13 15 0 0 22 3

Mean time to death. Days post-inoculation.

Table 6 Pathogenicity of four reference isolates tested in both bioassays against V. destructor at 25 °C and 100% RH, with 95% confidence limits Species

Isolate

Concentration

Bioassay

n

Mean MTDa (h)

95% C.I.

% Mortality at 7 dpib

% Mycosis at 7 dpi

M. anisopliae

442.99

B. bassiana

432.99

V. lecanii

453.99

108 108 106 108 108 106 108 108 106 106 108 106

1 3 3 1 3 3 1 3 3 1 3 3

60 60 60 60 60 60 60 60 60 60 57 60

44 43 77 58 57 88 74 139 164 84 99 151

(39, 50) (40, 46) (70, 85) (49, 69) (53, 62) (79, 97) (45, 123) (86, 223) (103, 260) (70, 101) (78, 125) (100, 228)

100 100 100 100 100 100 63 67 42 93 60 65

100 100 100 100 100 100 60 67 38 98 35 55

H. thompsonii

a b

75.82

Mean time to death. Days post-inoculation.

others (Table 6). There was a significant effect on MTD of conidia concentration ðv21 ¼ 238:0; p < 0:001Þ and isolate ðv23 ¼ 146:1; p < 0:001Þ, but no interaction ðv23 ¼ 6:5; p > 0:05Þ. The reference isolates were also more virulent at 25 °C than at 30 °C. Overall, variability in MTD increased with increasing mean MTD and with increased temperature. Temperature had the least effect on the most virulent isolate, M. anisopliae 442.99.

4. Discussion Selecting the concentration and environmental conditions for the first bioassay was not straightforward, as there was no prior information on the susceptibility of V. destructor to entomopathogenic fungi. Although the

concentration of conidia in inocula was high and initial bioassay conditions favored fungal activity, the ability of all 40 fungal isolates to infect V. destructor was unexpected. The majority of the isolates examined in this study originated from acarine hosts, but V. destructor was also killed by isolates originating from insect hosts: Coleoptera (two isolates), Hymenoptera (one isolate), Homoptera (two isolates), and Hemiptera (one isolate). It is noteworthy too that V. destructor was susceptible to H. thompsonii, as this fungus is a specialist pathogen of eriophyid and tetranychid mites (Prostigmata) and has been reported only twice from other hosts; in both cases, mesostigmatid mites (Balazy and Wisniewski, 1982; McCoy, 1981). Varroa destructor was very susceptible to fungal pathogens in the laboratory. Although microorganisms

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have been visualized on the body surface of mites (Liu and Ritter, 1988), there have been no reports of their natural infection by fungi. The mite spends the vast majority of its life inside bee colonies and all its preadult life stages within sealed brood cells. Bees invest substantial resources in colony hygiene, and hence, the mite may be unlikely to encounter pathogens in its normal environment. This may partially account for its lack of resistance to pathogen challenge in the laboratory experiments reported here. Experiments with reference isolates indicated that fungi were less virulent at 30 °C and 40% RH than at 25 °C and 100% RH. Humidity and temperature have a major effect on fungal activity and were considered key variables in this study. The germination of conidia, for example, does not occur below 93% RH and is usually optimal at saturation (Chandler et al., 1994; Gillespie and Crawford, 1986). Humidity within honeybee colonies is considerably lower than this and so might reasonably be expected to be a significant constraint to fungal infectivity. The lowest ambient humidity in a honeybee colony occurs in the brood area, where it is normally 40–50% RH but increases to 70% RH during bouts of evaporative cooling (Simpson, 1961). The ability of fungi to infect V. destructor at 40% RH in our study is encouraging and can probably be attributed to the presence of favorable sites for attachment and penetration of the host cuticle. Varroa destructor is pubescent and has a prominent carapace; features likely to create areas on the body with high microclimatic humidity that allows infection at low ambient humidities. Moreover, the conditions within sealed brood cells, where the mite reproduces, and between the overlapping segments of the adult bee’s abdomen, where mites primarily attach to feed, may also create humid microclimates. Milner et al. (1997) reported that the in vitro germination of isolates of M. anisopliae was completely inhibited below 94% RH and yet the fungus was able to kill termites, Nasutitermes exitiosus (Hill) and Coptotermes acinaciformis (Froggatt), at ambient conditions as low as 86% RH (the lowest humidity at which the termites would survive) with no consistent effect of humidity on pathogenicity. It was concluded that the termites created a humid microclimate that was favorable for conidia germination under most ambient conditions. Our study suggests that the humidity conditions in bee colonies are also unlikely to prevent fungal infection of V. destructor. Temperature, however, is likely to have a significant impact on the ability of a mycopesticide to control V. destructor populations, influencing both the choice of fungal isolate for the active ingredient and the time of year it can be used. The temperature of a honeybee colony changes markedly between summer and winter. Colony temperatures in summer are largely independent of ambient temperatures (Liu et al., 1990). The brood

area is usually maintained at 32–37 °C (Free and Spencer-Booth, 1959; Jay, 1964; Powell, 1979) while broodless parts of the colony vary from 28 to 33 °C (Kaya et al., 1982). Many isolates of entomopathogenic fungi require moderate temperatures (15–27 °C) for optimal activity (Burges, 1981) and would be adversely affected by such conditions. There is a need therefore to identify fungal isolates that can infect V. destructor at high temperatures, particularly if they are to be effective in the brood area. The nine isolates of fungi tested against V. destructor in bioassays at 30 °C in our study were already known to grow in vitro at this temperature (G. Davidson, unpublished observations). Ongoing research continues to identify and characterize other isolates active at high temperatures. The fact that V. destructor seems very susceptible to fungal infection greatly increases the opportunities for obtaining these isolates. Highly aggressive pathogens may also be able to compensate partially for high temperature inhibition (Fargues and Rodriguez-Rueda, 1980). An alternative would be to apply a mycopesticide to honeybee colonies at times of year when the colony temperature is at its lowest. In winter, for example, honeybees remain active within the colony to generate heat and cluster together to reduce the rate of heat loss (Free and Spencer-Booth, 1959; Winston, 1987). In temperate regions, the temperature within a winter cluster is normally maintained at 20–30 °C (Free and Spencer-Booth, 1959; Simpson, 1961). Many isolates of entomopathogenic fungi are likely to be more effective at this temperature range. Further work will be required to identify the best time for fungus application. There is likely to be a balance between the requirements of the fungus and the pattern of population development of V. destructor. In this study, four isolates—M. anisopliae 442.99, 443.99, and 444.99 and B. bassiana 432.99—were very effective against V. destructor even at 1
106 conidia/ml and 30 °C and 40% RH, but also caused significant mortality of adult honeybees in maximum challenge bioassays. Verticillium lecanii 17.76 caused moderate levels of mortality in V. destructor at 1
106 conidia/ml and 30 °C and 40% RH and had very little impact on adult honeybees, and hence may be a better candidate to take forward for development as a microbial control agent. However, the results from the honeybee bioassays should be interpreted with caution due to high control mortality. This may partly be attributed to the conditions under which the bees were maintained after treatment, which favored the germination of conidia. Confining or crowding bees can induce the establishment of virus infections (Bailey et al., 1983) and wetting bees by spray application and maintaining them in a humid environment are unfavorable conditions for their survival. Butt and Goettel (2000) recommended that special care be taken when using bioassays of honeybees for risk assessment, as fungi that killed adult bees in

K.E. Shaw et al. / Biological Control 24 (2002) 266–276

laboratory bioassays had been shown, subsequently, to have minimal effects in whole-hive experiments. Similarly, an isolate of M. anisopliae that caused high levels of mortality in adult honeybees in a bioassay caused no adverse effects in a field experiment when bees were used to deliver the fungus to oilseed rape flowers for control of pollen beetles, Meligethes aeneus (F.) (Butt et al., 1994, 1998). For these reasons, further assessment of the impact on bees of isolates of fungi active against V. destructor is required using a formulation and mode of application more appropriate to field use. To further evaluate the potential of fungi as microbial control agents of V. destructor, dose–response assays of selected fungi against V. destructor and the impact of fungi on populations of V. destructor in honeybee colonies are also planned. Our study has shown that V. destructor is very susceptible to entomopathogenic fungi. Isolates of fungi were able to kill mites in laboratory bioassays done under the conditions of low humidity and high temperature that occur within honeybee colonies. There is therefore potential to develop entomopathogenic fungi as microbial control agents of V. destructor and the unique environment of a honeybee colony, being uniform throughout the world, would enable a biocontrol product to have a global application.

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