title / Screening pathogens for biocontrol of Varroa jacobsoni
/ DEFRA
project code / HH0813SHB
Department for Environment, Food and Rural Affairs CSG 15
Research and Development
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / Screening pathogens for biocontrol of Varroa jacobsoni
DEFRA project code / HH0813SHB
Contractor organisation and location / Horticulture Research International
Wellesbourne,
Warwick
CV35 9EF
Total DEFRA project costs / £ 410,229
Project start date / 01/06/98 / Project end date / 31/05/02
Executive summary (maximum 2 sides A4)
To tab in this section press the tab key and the Control key together
Press the DOWN arrow once to move to the next question.
CSG 15 (9/01) 3
Projecttitle / Screening pathogens for biocontrol of Varroa jacobsoni
/ DEFRA
project code / HH0813SHB
The varroa mite, Varroa destructor (previously termed Varroa jacobsoni) is an ectoparasite of the European honey bee, Apis mellifera. It originated in Asia but has spread world-wide in recent years and is causing severe damage to A. mellifera populations. At present, management of V. destructor is based on the use of chemical pesticides, but resistant mite populations are spreading and alternative methods of control are required. This project investigated entomopathogenic fungi and the bacterium Bacillus thuringiensis (Bt) as potential microbial control agents of V. destructor in a series of laboratory experiments. The project addresses DEFRA’s aim of sustainable development, and matches policy objectives to promote sustainable farming by reducing inputs of chemical pesticides and the exploitation of biological alternatives.
Varroa destructor has no known natural enemies, hence the project used candidate pathogens from other hosts (V. destructor cadavers were examined for evidence of disease in this project, but no pathogens were observed). Following a literature review of the fungal pathogens of Acari (which has now been published), 51 candidate isolates of entomopathogenic fungi were acquired from culture collections around the world and placed into cryostorage. A laboratory bioassay was developed to measure the susceptibility of adult female mites to entomopathogenic fungi and Bt. The assay was based on direct immersion and gave very low (<5 %) control mortalities. Eight strains of Bt were examined against V. destructor, but results were inconsistent and there was little evidence overall of pathogenicity. Forty isolates of anamorphic entomopathogenic fungi from six genera were also examined against V. destructor. These isolates were tested in ‘maximum challenge’ bioassays under conditions favourable to fungal infection (25oC and >95 % RH). Varroa destructor was found to be highly susceptible to entomopathogenic fungi in this system, with 19 isolates causing 100% mortality within 7 days post inoculation (dpi). Nine isolates of fungi were selected on the basis of their virulence to V. destructor and favourable growth at high temperatures (see below) and examined further against V. destructor in bioassays at 30oC / 40 % RH to simulate the conditions within the brood nest of a bee colony. Varroa destructor were still susceptible to infection under these testing conditions, and five isolates of fungi caused 100% mortality at 7 dpi. This result is particularly encouraging as humidity is normally the major limiting factor to fungal infection. Four of the fungal isolates were then investigated in a multiple dose bioassay at 30oC / 40% RH. The median lethal concentrations of the isolates ranged from 5.3 x 105 ml-1 (Metarhizium anisopliae 442.99) to 4.0 x 107 ml-1 (Verticillium lecanii 17.76). These findings are in press.
The high temperatures maintained within some areas of the honey bee colony could have a significant impact on the activity of fungal pathogens of V. destructor because most isolates of entomopathogenic fungi require moderate temperatures (20 - 30°C) for optimum growth and development. A series of experiments were therefore performed to investigate the thermal biology of the fungal isolates used in this study. The effect of temperature on fungal growth was studied by measuring the rate of colony extension on a solid medium. Cardinal temperatures for growth were determined using the Schoolfield et al. (1981) reformulation of the Sharpe and DeMichele (1977) model of poikilotherm development. Optimum temperatures ranged from 22.9oC (V. lecanii 19.79) to 31.1oC (Hirsutella thompsonii 34.79). Super-optimum temperatures (defined as the temperature above the optimum at which the colony extension rate was 10% of the maximum rate) ranged from 31.9oC (V. lecanii 19.79) to 43.2oC (V. lecanii 453.99). Optimum temperatures for in vitro germination were also calculated using the same model for four reference isolates selected in the bioassays. Optimum germination temperatures ranged from 26.4oC (Beauveria bassiana 432.99) to 29.8oC (H. thompsonii 75.82) and super-optimum temperatures ranged from 35.2oC (B. bassiana 432.99) to 46.5oC (V. lecanii 453.99). These data suggest that the thermal requirements of the fungal isolates examined against V. destructor are well matched to the temperatures in broodless areas of honey bee colonies in summer (25oC), and a subset of isolates will be able to function within drone brood areas where V. destructor preferentially reproduces (32.5oC). These findings have been submitted for publication. A method was also developed to measure the survival of fungal conidia at temperatures and humidities likely to be encountered in bee colonies, using the most virulent fungal isolate (M. anisopliae 442.99) as a model. The data suggest that the conidia of this isolate are unlikely to persist longer than 100h under brood nest conditions. The use of a nonpersistent, dose-dependent pathogen as investigated here will reduce the risk to non-target organisms.
A series of experiments was also performed to measure the effect of selected isolates of fungi against three non-target beneficial organisms (European honey bee Apis mellifera, seven spot ladybird Coccinella septempunctata, and the predatory mite Phytoseiulus persimilis) and representatives of a range of insect and mite orders, including Diptera (Delia antiqua), Homoptera (Aphis gossypii), Lepidoptera (Galleria mellonella), Coleoptera (Tenebrio molitor) and Acari (Tetranychus urticae). The bioassays were based on IOBC protocols to measure the effects of pesticides on non-target organisms or on bioassays of entomopathogenic fungi developed previously in DEFRA-funded research. The results of these bioassays were complex, with differences in virulence observed between and within fungal species. However isolates of fungi were identified that were virulent to V. destructor but had a low impact on non-targets including honey bees.
The movement of a fluorescent marker dye within bee populations in observation hives was monitored as an indication of the likely movement of fungal conidia in a bee colony. The dye was applied with a dispenser at the hive entrance. The bees reacted to the presence of the powder and local activity increased causing greater deposition. Almost all the bees entering the observation hive after a foraging trip passed through the brood nest area, and after six hours traces of dye were detected on more than half of young marked bees sampled, indicating effective transfer between foraging and nurse bees.
This study has shown that V. destructor is very susceptible to entomopathogenic fungi. Isolates of fungi killed varroa mites in laboratory bioassays under conditions of low humidity and high temperature that occur within bee colonies, and there would appear to be potential to develop entomopathogenic fungi as microbial control agents. Because the physical conditions inside honey bee colonies are similar everywhere, it is likely that an efficient control agent of V. destructor could be used successfully throughout the world.
The project has led to two refereed papers, and a third has been submitted. Nine articles have been published in the beekeeping press. Members of the project team have given 15 lectures to bee keeper associations in England and Wales including the British Bee Keepers Association, 12 poster presentations at national and international scientific meetings and bee keeping conventions, and oral presentations to the Parliamentary Under Secretary of State (Horticulture) and the President of the National Farmers Union.
CSG 15 (9/01) 3
Projecttitle / Screening pathogens for biocontrol of Varroa jacobsoni
/ DEFRA
project code / HH0813SHB
Scientific report (maximum 20 sides A4)
To tab in this section press the tab key and the Control key together
Press the DOWN arrow once to move to the next question.
CSG 15 (9/01) 3
Projecttitle / Screening pathogens for biocontrol of Varroa jacobsoni
/ MAFF
project code / HH0813SHB
1. INTRODUCTION
The varroa mite, Varroa destructor n. sp. (previously termed Varroa jacobsoni) is a highly damaging ectoparasite of the European honey bee Apis mellifera (Sammataro et al. 2000). It originates in Asia, but has extended its range worldwide. It was detected in the UK in 1992. Adult female mites feed on the haemolymph of adult bees and pupae, and in so doing they activate and transmit viral diseases which reduce the life expectancy of the bees and cause the colony to decline, particularly over winter (Ball, 1994a,b). Varroa destructor has had a major impact in all countries where it has become established. For example, the winter mortality of managed honey bee colonies in 1996 was estimated to be equivalent to a quarter of the global commercial population (Sanford, 1996). In the UK, V. destructor caused the loss of 30 – 50% of honey bee colonies in the south of England in the same period. The loss of honey bees on this scale is affecting the pollination of commercial crops and wild plants.
At present, the management of V. destructor is based on the use of chemical pesticides, but resistance has already been recorded (Hillesheim et al., 1996; Thomas, 1997; Elzen et al., 1998; Milani, 1999). In the UK for example, two pyrethroid-based acaricides are used, but pyrethroid resistance has been reported recently in the southwest of England (Anon, 2001). There is a need, therefore, for alternative, sustainable forms of V. destructor management.
This research project was developed following a DEFRA-funded desk study of the prospects for the biological control of V. destructor (DEFRA project HH0811SHB). Biological control technologies can offer an effective means of moving pest management strategies away from reliance on synthetic pesticides. However, little research has been done on the biological control of V. destructor, possibly because no natural enemies have been identified from V. destructor on A. mellifera or its original host, Apis cerana. Therefore, biological control will require natural enemies from other hosts. DEFRA project HH0811SHB identified entomopathogenic fungi as primary candidates for development as biocontrol agents of V. destructor, with the bacterium Bacillus thuringiensis (Bt) also worthy of consideration.
2. PROJECT OBJECTIVES
The overall aim of this project was to examine and characterise entomopathogens for their potential as biocontrol agents of V. destructor. The specific objectives of the project were as follows :
1) Obtain strains of fungi and Bt with potential for the biocontrol of V. destructor.
2) Quantify the virulence of acarine-active fungi and Bt to V. destructor in laboratory bioassays.
3) Measure the physiological characteristics of fungal biocontrol agents and relate these to hive conditions.
4) Quantify the virulence of candidate pathogens to selected non-target and beneficial organisms.
5) Determine the persistence and spread of potential biocontrol agents within the hive.
6) Initiate development of formulation and application strategies of potential biocontrol agents.
The project was based around a series of laboratory experiments done in Objectives 2, 3 and 4 to select ‘winning’ pathogens on the basis of virulence to V. destructor, potential to operate under bee colony conditions, and effects on non-target organisms. Pathogen selection was done as a step-wise process, with a high degree of feedback between Objectives 2, 3 and 4. However, for the purposes of this report, these Objectives are presented separately.
3. IDENTIFICATION AND COLLECTION OF ENTOMOPATHOGENS WITH POTENTIAL FOR THE BIOCONTROL OF VARROA DESTRUCTOR (Objective 1)
3.1 Candidate isolates of entomopathogenic fungi obtained from culture collections (years 1-4)
A literature review of the fungal biocontrol of Acari identified 58 species of fungi reported to infect over 70 species of Acari either naturally or in experiments (Chandler et al., 2000). Fifty one candidate isolates of fungi were acquired for experimentation from a variety of sources and were catalogued and placed in cryostorage in the HRI collection of entomopathogenic fungal cultures (Table 1).
Table 1: Fungal isolates obtained for this study. Details of the isolate sources are given in Shaw et al. (2002)
Species / Isolate / Host / Geographic originBeauveria bassiana / 322.89 / Hypothenemus hampei (Coleoptera : Hyponomeutidae) / Brazil
431.99 (T228) / Acari / Denmark
432.99 / Anthonomus grandis (Coleoptera : Curculionidae) / USA
433.99 / - / -
434.99 (ARSEF2869) / Bephratelloides cubensis (Hymenoptera : Eurytomidae) / USA
454.99 (I91636) / Acari / Oman
455.99 (DAT049) / - / -
460.99 / Acari / Israel
Hirsutella sp / 45.81 / - / -
435.99 (H1) / Acari : Tarsonemidae / Poland
436.99 (H2) / Acari : Tarsonemidae / Poland
437.99 (H3) / Eriophyes piri (Acari : Eriophyidae) / Poland
438.99 (H4) / Abacarus hystrix (Acari : Eriophyidae) / Poland
439.99 (H11) / Stenotarsonemus fragariae (Acari : Tarsonemidae) / Poland
440.99 (H12) / Stenotarsonemus fragariae (Acari : Tarsonemidae) / Poland
457.99 (339C) / Acari / Poland
Hirsutella kirchneri / 46.81 (IMI251256) / Abacarus hystrix (Acari : Eriophyidae) / UK
47.81 (IMI257456) / Abacarus hystrix (Acari : Eriophyidae) / UK
Hirsutella necatrix / 49.81 / Abacarus hystrix (Acari : Eriophyidae) / UK
Hirsutella thompsonii / 34.79 / Eriophyes guerreronis (Acari : Eriophyidae) / Africa
51.81 / Eriophyes guerreronis (Acari : Eriophyidae) / Africa
71.82 / Eriophyes guerreronis (Acari : Eriophyidae) / Jamaica
73.82 /
Phyllocoptruta captrila (Acari : Eriophyidae)
/ USA74.82 / Eriophyes guerreronis (Acari : Eriophyidae) / Africa
75.82 / Colomerus novahebridensis (Acari : Eriophyidae) / New Guinea
77.82 / Eriophyes guerreronis (Acari : Eriophyidae) / Jamaica
463.99 (DoCo Al) / Acari / Canada
Metarhizium anisopliae / 441.99 (ARSEF3297) / Boophilus sp. (Acari : Ixodidae) / Mexico
442.99 (ARSEF4556) / Boophilus sp. (Acari : Ixodidae) / USA
443.99 (T248) / Acari / Denmark
444.99 (ATCC38249) /
Hylobius pales (Coleoptera : Curculionidae)
/ -445.99 / - / -
456.99 (DATF001) / - / -
458.99 / - / -
459.99 / Acari / Israel
Metarhizium flavoviride / 203.84 / Nilaparvata lugens (Hemiptera : Delphacidae) / Brazil
Paecilomyces farinosus / 446.99 (CCFC002085) / Mycobates sp. (Acari : Mycobatidae) / Canada
Paecilomyces fumosoroseus / 409.96 / Phenacoccus solani (Hemiptera : Pseudococcisae) / -
447.99 (KVL319) / Ixodes ricinus (Acari : Ixodidae) / Denmark
Tolypocladium inflatum / 448.99 (ARSEF3278) / Mycobates sp. (Acari : Mycobatidae) / Canada
Tolypocladium niveum / 449.99 (CCFC002081) / Mycobates sp. (Acari : Mycobatidae) / Canada
Verticillium lecanii / 1.72 / Macrosiphoniella sanborni (Homoptera : Aphididae) / UK
12.74 (IMI79606) / Chloropulvinaria floccifera (Hemiptera : Coccidae) / Turkey
17.76 / Cecidophyopsis sp. (Acari : Eriophyidae) / UK
19.79 / Trialeurodes vaporariorum (Homoptera : Aleyrodidae) / UK
30.79 / Cecidophyopsis sp. (Acari : Eriophyidae) / UK
31.79 / Cecidophyopsis sp. (Acari : Eriophyidae) / UK
450.99 (IMI235048) / Cecidophyopsis ribis (Acari : Eriophyidae) / UK
451.99 (ARSEF1367) / Acari : Orbatidae / Poland
452.99 (CBS317.70A) / Tetranychus urticae (Acari : Tetranychidae) / -
453.99 (CCFC006079) / Acari / Canada
3.2 Examination of V. destructor cadavers for presence of entomopathogenic fungi (year 1)