Aspects of the use of honeybees and bumblebees as vector of antagonistic micro-organisms in plant disease control

Aspects of the use of honeybees and bumblebees as vector of antagonistic micro-organisms in plant disease control J.J.M. van der Steen 1, C.J. Langerak 2, C.A.M. van Tongeren 2 & A.J. Dik 3 1 Applied Plant Research, Research Unit Bees, Wageningen University and Research Centre, Ambrosiusweg 1, 5081 NV Hilvarenbeek, The Netherlands; 2 Plant Research International, Wageningen University and Research Centre, Bornsesteeg 65, 6708 PD Wageningen, The Netherlands; 3 Applied Plant Research, Research Unit Glasshouse Horticulture, PO Box 8, 2670 AA Naaldwijk, The Netherlands Honeybees (Apis mellifera L.) and bumblebees (Bombus terrestris L.) are used for pollination in agriculture and horticulture. The morphological and behavioural characteristics of bees make them good pollinators. Thanks to this, bees may also be used as vector of antagonistic micro-organisms for plant disease control, both preventive and curative. To determine the practical consequences of this way of plant disease control, research has started on the two main aspects: the impact of the antagonist on the vector itself and the impact on the transmission of Botrytis aclada into seeds of onion. Preliminary tests for method development have been carried out to determine the impact on honeybees and bumblebees and on the dissemination of the antagonist over the flowering plants. Results: Trichoderma harzianum and PGBY1 have no negative impact in the brood of honeybees and bumblebees. Honeybees are rather effective vectors to transfer the antagonist Ulocladium atrum to onion flowers and this resulted in transfer into the seeds. Keywords: antagonists, seed transmission, organic seed, Apis mellifera, Bombus terrestris, Trichoderma harzianum, PBGY1, Allium cepa, Botrytis aclada, Ulocladium atrum, ARSA, PLYS-agar Several plant pathogenic micro-organisms enter the plant through open flowers and can infect the seeds in that way. Spores of antagonistic micro-organisms present on flowers can successfully compete with the possible pathogens. Disseminating antagonistic micro-organisms by honeybees and bumblebees can reduce plant infections. Kovach et al. (2000). showed in a 4-years study that the dissemination of Trichoderma harzianum 1295-22 to control Botrytis cinerea on strawberry (Fragaria ananassa), both honeybees and bumblebees could effectively transport T. harzianum 1295-22 from the hive into the flowers. This resulted in a better control of Botrytis than the spray application of T. harzianum 1295-22. Similar results were obtained by Yu & Sutton (1997) in the control of B. cinerea in raspberry using Gliocladium roseum. By transporting antagonists it is unavoidable that this antagonistic material also enters the brood nest of the bees. Since a healthy brood nest is an essential stimulant for the foraging activities of the bees and since fungi and yeast may be able to cause infections of the brood, the effect of two antagonistic micro-organisms is tested on the honeybee and the bumblebee brood. This study is divided into two parts: Part 1: the impact of the antagonistic micro-organisms T. harzianum T39 and PBGY1 on the brood of the honeybee (A. mellifera L.) and the bumblebee (B. terrestris L.). Part 2: the dissemination by honeybees and the detection of the antagonistic micro-organism Ulocladium atrum in onion seeds (Allium cepa) for the control of Botrytis aclada infection in onion seeds. MATERIAL AND METHODS Part 1 The impact of the antagonistic micro-organisms T. harzianum T39 and PBGY1 on the brood of the honeybee (A. mellifera) and the bumblebee (B. terrestris) Honeybee (A. mellifera) brood test Honeybee worker brood development takes 21 days egg: 3 days, larva: 6 days and pupa: 12 days. The brood develops in a fixed cell in a fixed location on the comb. Therefore infection and checking of the subsequent impact on the development can be carried out with marked brood cells. In two colonies, per colony 25 cells containing eggs, 25 cells containing larvae aged 0 to 4 days and 25 cells containing larvae aged 4 to 6 days were selected. The position of these cells on the PROC. NETH. ENTOMOL. SOC. VOLUME 15 2004 41

42 INSECTS AS VECTORS frame was marked on a transparent (overhead) sheet. To each selected cell, 5 µl sucrose solution 12.5 ± 2.5% containing T. harzianum T39 / µl was administered (2.67 g Trichodex / litre). The same was done in two colonies in which sucrose solution 12.5 ± 2.5% containing 10 7 spores of PBGY1 / ml was applied. The two control colonies were treated similarly with 5 µl sucrose solution 12.5 ± 2.5% per cell. The sucrose concentration was based on the food offered to larvae in an artificial honeybee rearing (Wittmann 1982). The concentrations of T. harzianum T39 and PBGY1 tested, were based on the concentrations that had showed to reduce B. cinerea infections in cucumber and tomato (Dik et al. 1999). According to test scheme 1, treated brood was removed from the cell, rinsed 3 times in 10 ml sterile demineralised water and subsequently homogenised. This material was cultivated on PDA medium or KIMMIG medium for respectively PBGY1 and Trichoderma cultivation. The PDA and KIMMIG mediums were incubated for at least 7 x 24 h at 35 ± 2 C. Bumblebees (B. terrestris) brood test Bumblebee worker brood development takes 30 days egg: 6 days, larva: 14 days, pupa: 10 days (Rothe 1995, Van der Steen, unpublished). The brood development starts with a cluster of about 10 eggs. This egg clump is situated on top of a pupa cell. During the development separate cells are constructed for individual larvae. This results in a chaotic brood nest in which the location of cells changes all along. Therefore administration of T. harzianum and PBGY1 and checking of the subsequent impact on the development is carried out by infecting the entire brood nest and checking old larvae and adults according to test scheme 2. Prior to the administration of the test suspensions the queen was removed in order to stop the oviposition after treatment of the brood nest. The brood nest of two colonies was sprinkled twice with 2 ml demineralised water containing T. harzianum T39 (2.67 g Trichodex / litre). The same was done to two colonies with demineralised water solution containing 10 7 spores of PBGY1 / ml. The two control colonies were Test scheme 1. Treatment and sampling of honeybee brood Day Treatment and sampling of the brood Brood phase at sampling, 5 larvae/sample 0 Dripping the micro-organisms in the cell 4 Sampling of brood treated in the larval phase 0 to 3 days (L1-L3) Larvae aged 4 to 6 days 6 Sampling of brood treated in the egg phase Larvae aged 3 to 6 days 8 Sampling of brood treated in the larval phase 4 to 6 days (L4-L5) Pupae aged 6 to 8 days 12 Sampling of brood treated in the larval phase 0 to 3 days (L1-L3) Pupae aged 6 to 9 days 17 Sampling of brood treated in the egg phase Pupae aged 8 to 11 days 23 Check on the emergence of the treated brood Test scheme 2. Treatment and sampling of bumblebee brood Situation in the brood nest during administrations of test suspensions Eggs in sealed cells Larvae aged 1 to 9 Larvae aged 10 to 14 Pupae in sealed Adults Day Action days in sealed cells days in open cells cells 0 Queen removal 0 First No exposure Indirect exposure Direct exposure No exposure vector administration 7 Brood check 5 larvae 7 Second Larvae aged 1 to 9 Larvae aged 10 to 14 Pupae in sealed cells Pupae in sealed Adults administration days in sealed cells days in open cells cells Indirect exposure Direct exposure No exposure No exposure vector 14 Brood check 5 larvae 21 Adult check 5 adults 21* Removal of all workers 28 Adult check 5 adults 28** Removal of all workers 35 Adult check 5 adults * Start emergence of adults that were exposed to the test suspension in the larval phase; ** Start emergence of adults that were exposed to the test suspension in the egg phase

J.J.M. VAN DER STEEN, C.J. LANGERAK, C.A.M. VAN TONGEREN & A.J. DIK 43 treated similarly with sterile demineralised water. The concentration of T. harzianum T39 and PBGY1 tested, were based on the concentrations that had showed to reduce B. cinerea infections in cucumber and tomato (Dik et al. 1999) According to test scheme 2, treated brood was removed from the cell, rinsed 3 times in 10 ml sterile demineralised water and subsequently homogenised. The adults that had been exposed to T. harzianum T39 and PBGY1 during their development were rinsed in 10 ml sterile demineralised water and subsequently 20 µl of this water was smeared on PDA medium or KIMMIG medium for respectively PBGY1 and Trichoderma cultivation. Of the rinsed workers, the fatbody was dissected and inoculated on PDA medium or KIMMIG medium. The PDA and KIMMIG mediums were incubated for at least 7 x 24 h at 35 ± 2 C. Part 2 The dissemination by honeybees and detection of the antagonistic micro-organism U. atrum in onion seeds (A. cepa) for the control of B. aclada infection of seeds This study was part of a study to optimize organic seed production. In two gauze tents of (8 x 8 x 2.5 m) with 48 flowering onion plants honeybees were introduced. The honeybees were housed in MiniBeuten, were queen right and had brood in all phases. The introducing honeybee colonies and the provision of these colonies with micro-organisms is described in test schemes 3 and 4. In this study both the preventive and curative action of the dissemination of the pathogenic B. aclada and the antagonistic U. atrum 385 by honeybees is tested. J. Köhl of Plant Research International kindly provided an isolate of this antagonist. In order to provide the honeybees with Botrytis and Ulocladium, an adapted pollen dispenser type BeeBooster was placed in front of the MiniBeute. The micro-organisms were mixed with cellulose powder (Cellulos microcryst PH101) in a ratio of 1 ml suspension of the micro-organism (10 6 conidia / ml) + 1 g cellulose powder. This resulted in a dry powder that could easily be picked up by the honeybees as they pass over it. Portions of 1 g powder were placed in the BeeBooster (test schemes 3 and 4) daily. Test scheme 3 (Tent 2). Scheme of the subsequent placing of honeybee colonies in tent 2 and the provision with Ulocladium and Botrytis. Date Activity Action 24 June 2003 Introduction of colony E + Botrytis aclada (2 x 1 g) Infection action 25, 26, 27, 28 June 2003 Adding Botrytis aclada into BeeBooster (1 g) 1 July 2003 Removal of colony E from the tent 1 July 2003 Introduction colony F 8 July 2003 Removal of colony F 8 July 2003 Introduction of colony G + Ulocladium atrum (1 g) Curative action 9, 10 July 2003 Adding Ulocladium atrum into BeeBooster (1 g) 15 July 2003 Removal of colony G 15 July 2003 Introduction colony H 2 July 2003 Removal of colony H * (1 ml suspension 5.10 6 conidia/ml in 1 g cellulose) Test scheme 4 (Tent 3). Scheme of the subsequent placing of honeybee colonies in tent 2 and the provision with Ulocladium and Botrytis. Date Activity Action 24 June 2003 Introduction of colony A + Ulocladium atrum (2 x 1 g) Preventive action 25, 26, 27, 28 June 2003 Adding Ulocladium atrum into BeeBooster (1 g) 1 July 2003 Removal of colony A from the tent 1 july 2003 Introduction colony B 8 July 2003 Removal of colony B 8 July 2003 Introduction of colony C + Botrytis aclada (1 g) Infection action 9, 10, 12 July 2003 Adding Botrytis aclada into BeeBooster (1 g) 15 July 2003 Removal of colony C 15 July 2003 Introduction colony D 2 July 2003 Removal of colony D * (1 ml suspension 5.10 6 conidia/ml in 1 g cellulose)

44 INSECTS AS VECTORS The day, the bee colonies A and E were introduced in the two tents, the onion umbels varied in flowering. Five different stages of flowering of the umbels were distinguished (each umbel consisted of numerous individual flowers): I, no open flowers; II, 20-50% of flowers just opening; III, 50-100% of flowers completely open; IV, 50% open and 50% deteriorating and V, 100% deteriorating and filling of the ovary. To relate the efficiency of transmission of the fungi to the degree of flowering, 10 umbels of each flowering stage were marked and at the time of seed harvest used as a reference to classify the maturity of the bulk of the umbels. Two days after introduction of the clean bee colonies B and F, 10 closed, open or deteriorating flowers representative for each maturity class were collected from 5 marked umbels and analysed for presence of either B. aclada or U. atrum. Seed-set in the umbels with lowest maturity (class I) was completed eight weeks after introduction of the bee colonies A and E. All umbels of all maturity classes were harvested at the same time. Three mixed umbel samples per maturity class were created for the two tents (material set 1). Prior to combining umbels of similar maturity, umbels of 6 plants (tent 2) and of 14 plants (tent 3) were kept separately as material set 2. Seeds of both material sets were cleaned in a standard way. Collected flower material and cleaned seeds were analysed on PLYS (Presly) for B. aclada and on ARSA (Pryor et al.) for U. atrum. RESULTS & DISCUSSION Part 1 The impact of the antagonistic micro-organisms T. harzianum T39 and PBGY1 on the brood of the honeybee (A. mellifera) and the bumblebee (B. terrestris) Honeybee brood Dripping 5 µl sucrose solution 12.5% into brood cells of honeybees did not influence the development of the brood. Suspensions of T. harzianum T39 and PBGY1 in sucrose solution 12.5% did also not affect the normal development of the brood. In none of the sampled brood phases, T. harzianum and PBGY1 was found. The study showed that these antagonistic microorganisms did not infect the brood in the egg phase, larval phase aged 0 to 4 days and in the larval phase 4 to 6 days. What exactly limits or inhibits these micro-organisms is not studied but in a honeybee colony among others, the acidity of the food material (royal jelly) produced in the pharyngeal glands, the peroxide and high sugar concentration in the honey, and propolis have strong antibiotic actions. Bumblebee brood Dripping 2-ml sterile demineralised water on the brood nest did not influence the development of the brood. Suspensions of Trichoderma harzianum T39 and PBGY1 in demineralised water did also not affect the normal development of the brood. In none of the sampled brood phases, Trichoderma harzianum and PBGY1 was found. Prior to inoculations of the larval and adult material, the pupae and adults were stored at 21 C for at least 2 days. The study showed that these antagonistic micro-organisms did not infect the brood in the egg phase, larval phase aged 0 to 4 days and in the larval phase 4 to 6 days. What exactly limits or inhibits these micro-organisms is not studied. In bumblebee colonies no natural resistance against micro-organisms is described. Nevertheless in the laboratory rearing unit, the conditions (29 C, 40-60% RH) are such that microorganisms and fungi seldom develop in the brood. An exception is the chalkbrood fungus Ascosphaera apis that can develop rapidly in the bumblebee brood in case the colony is offered pollen containing this fungus. Part 2 The dissemination by honeybees and detection of the antagonistic micro-organism U. atrum in onion seeds (A. cepa) for the control of B. aclada infection of seeds Preventive and curative control effects in onion seed of U. atrum against B. aclada Analysis of flower material after the first introduction of B. aclada in tent 2 and of U. atrum in tent 3 showed only presence of B. aclada in open flowers of umbels from class III whereas U. atrum was only found in deteriorating flowers originating from class IV umbels. This indicated that the bees had transferred the pathogenic and antagonistic fungi from the BeeBooster into the flowering plants. Apparently there seems to be a preference of B. aclada for young open flowers whereas U.

J.J.M. VAN DER STEEN, C.J. LANGERAK, C.A.M. VAN TONGEREN & A.J. DIK 45 atrum prefers older and deteriorating flowers, which fits with the saprophytic nature of this antagonist (Kessel et al. 2001). Infection levels of B. aclada in seeds produced under a curative control regime (Table 1) are generally lower than those produced under a preventive control regime (Table 2). As found before in checks on flowers, there is a tendency that umbels with a lower maturity and having more open flowers at the time that contaminated bees were foraging, are more susceptible to the pathogen than more mature umbels with a relative low number of open flowers. The opposite is found for the colonisation of seeds with U. atrum. Both in the curative and in the preventive test situation (Tables 1 and 2) higher levels are found in seeds from umbels with a starting deterioration of the flowers at the time that the bees were actively searching for nectar or honey. It would be speculative to state that lower levels of B. aclada found in combination with higher levels of U. atrum is due to a curative or preventive effect of the antagonist. Reason for this reserve is that no sufficient quantitative data are available from the flower checks half-way the experiment and no control series were included in the experiment of seed producing plants, which got only visits of bees with a pathogen contamination. Nevertheless, we proved that honeybees are rather effective vectors to transfer an antagonist into onion seed in a simple way. Table 1. Infection levels of Ulocladium atrum and Botrytis aclada in seeds produced under a curative control regime (test scheme 3). Seed samples tested differed in maturity at harvest. Maturity class % seed infection with Ulocladium % seed infection with Botrytis set 1: n=110 set 2: n=3x200 set 1: n=150 set 2: n=3x200 class I - * 8.8 - * 5 class II 16.3 18.0 0 0.2 class III -* 23.0 - * 0 class IV 35 30.0 0 0.6 class V 50 44.5 0 0 *no seeds available Table 2. Infection levels of Ulocladium atrum and Botrytis aclada in seeds produced under a preventive control regime (test scheme 4). Seed samples tested differed in maturity at harvest. Maturity class % seed infection with Ulocladium % seed infection with Botrytis set 1: n=6x40 set 2: n=2x200 set 1: n=6x60 set 2: n=2x200 class I 15.0** 32.5 35.8*** 13.8 class II -* 22.3 -* 9.3 class III 41.5 35.3 5.6 8.3 class IV 54.5 49.7 1.7 8.0 class V -* 36.5 - * 4.5 *no seeds available; **n=2x40 seeds; ***n=2x60 seeds REFERENCES Dik, A.J., Koning, G. & Köhl J. 1999. Evaluation of microbial antagonists for biological control of Botrytis cinerea stem infection in cucumber and tomato. Eur. J. Pl. Path. 105: 115-122. Dustmann, J.H. 1987. Biologische Abwehrmechanismen eines Bienenvolkes gegen Krankheiten und Schädlingen. ADIZ 21(1): 2-8. Kessel, G.J.T., Haas, B.H. de, Lombaers van der Plas, C.H., Ende J.E. van den, Pennock-Vos, M.G., Werf, W. van der & Kohl, J. 2001. Comparative analysis of the role of substrate specificity in biological control of Botrytis elliptica in lily and Botrytis cinerea in cyclamen with Ulocladium atrum. Eur. J. Pl. Path. 107: 3 273-284. Kovach, J., Petzoldt, R. & Harman, G.E. 2000. Use of honey bees and bumble bees to disseminate Trichoderma harzianum 1295-22 to strawberries for Botrytis control. Biol. Contr. 18: 235-242. Presly, A.H. 1985. Methods for inducing sporulation of some Botrytis species occurring on onions and leeks. Trans. Brit. Mycol. Soc. 85: 621-624.

46 INSECTS AS VECTORS Pryor, B.M., Davis, R.M. & Gilbertson, R.L. 1994. Detection and eradication of Alternaria radicina on carrot seed. Plant Disease 78: 5, 452-456. Rothe, U. 1995. Die Entwicklung von Völker der Dunklen Erdhummel (Bombus terrestris L.) unter Zuchtbedingungen. Praktikum 1995 am Ambrosiushoeve. Forschungszentrum für Insektenbestäubung und Bienenzucht die Niederlande. Southwick, E.E. 1992. Physiology and social; physiology of the honey bees. In: The hive and the honeybee, pp. 171-196. Dadant and Sons ISBN 0-915698-09-9. Steen, J.J.M. van der. 2001. Het effect van Trichoderma harzianum T39 op het broed van honingbijen (Apis mellifera L). Rapport Praktijkonderzoek Plant en Omgeving sector Bijen. Steen, J.J.M. van der. 2001. Het effect van PBGY1 op het broed van honingbijen (Apis mellifera L.). Rapport Praktijkonderzoek Plant en Omgeving sector Bijen.. Wittmann, D. 1982. Entwicklung von Testverfahren und Experimeten zur Beurteilung von Insekticidwirkungen auf Bienenlarven. Dissertation Eberhard Karl Universität Tübingen. Yu, H. & Sutton, J.C. 1997. Effectiveness of bumblebees and honeybees for delivering inoculum of Gliocladium roseum to raspberry flowers to control Botrytis cinerea. Biol. Contr. 10: 113-122.