Original article

Effects of selected fungicides on in vitro spore germination and vegetative growth of aggregata

MS Goettel GM Duke GB Schaalje, KW Richards

Agriculture Canada Research Station, PO Box 3000, Main, Lethbridge, Alberta, Canada TIJ 4B1

(Received 8 August 1991; accepted 13 April 1992)

Summary — Five fungicides were studied for their in vitro effects on spore germination and vegeta- tive growth of Ascosphaera aggregata, the causative organism of chalkbrood in the alfalfa leafcutter bee, . The compounds were incorporated directly into liquid or agar growth medi- um. EC50s for inhibition of vegetative growth on an agar surface were estimated as follows: enilcona- zole, 0.034; Rovral (iprodione), 1.10; Benlate (benomyl), 1.16; and Ascocidin (N-glycosylpolifungin), 135.6 ppm (m/v). DFMO (α-DL-difluoromethyl omithine) stimulated growth resulting in an approximately 50% increased radius at 10 000 ppm compared to the control. Benlate inhibited spore germination; at the highest concentration only, Rovral inhibited germination while Ascocidin and enilconazole exhibited a slight effect. DFMO did not affect spore germination. Ascocidin and Benlate exhibited most effect on germling viability at 48 h followed by Rovral and enilconazole. DFMO had no effect on germling viabili- ty. Benlate, Rovral and enilconazole had the most pronounced effect on germ tube growth at 96 h. As- cocidin affected germ tube growth at the higest concentration; DFMO exhibited little to no effect. Com- parisons between in vitro results obtained in the present study with previously published in vivo results indicate that in vitro effects on spore germination most closely correlated with efficacy against chalk- brood in vivo, suggesting that compounds with activity against spore germination or germling viability may be the most promising candidates for use against chalkbrood.

Ascosphaera aggregata / Megachile rotundata / chalkbrood / chemical control / fungicide

INTRODUCTION ing up to 65% of bees (Kish et al, 1979; Stephen et al, 1981). The disease is use of contaminated nest- The alfalfa leafcutter bee, Megachile ro- spread through materials and adults that have tundata (F), is an important pollinator of ing by seed alfalfa in North America. Chalk- emerged from chalkbrood-infested materi- brood, a disease caused by the al (Vandenberg et al, 1980). Leafcutter Ascosphaera aggregata Skou is a serious bee larvae become infected after consum- threat to the leafcutter bee industry infect- ing A aggregata spores present in con-

* Contribution No 3879155 of the Lethbridge Research Station taminated pollen and nectar provisions lected fungicides recently studied for their in (Vandenberg and Stephen, 1982, 1983; vivo effects on chalkbrood in leafcutter bees McManus and Youssef, 1984). (Goettel et al, 1991). Results indicate that in Present control practices rely principally vitro activity does not necessarily translate to in vivo in reduction of chalkbrood on the decontamination of nesting materi- efficacy incidence in learfcutter bees. als and bee cells using sodium or calcium Compounds with were hypochlorite (Mayer et al, 1988) or heat activity against spore germination (Kish et al, 1979; Stephen, 1982). These most efficacious against chalkbrood. practices have provided some control; however, they have not stemmed the spread and increase in levels of the dis- MATERIALS AND METHODS ease, most probably due to the reintroduc- tion of spores from infested feral bee pop- ulations (Stephen, 1982). Fungicides, culture media and fungal inoculum Considerable effort has been spent searching for a compound which could be The compounds used were a 15% emulsifiable incorporated into cell provisions as a pro- concentrate of enilconazole (Janssen Pharma- Such a must be non- phylactic. compound ceutica), Benlate 50WP (benomyl, El duPont), toxic to bee larvae, reduce the incidence of Rovral 50WP (iprodione, Rhone Poulenc), As- chalkbrood, and must not be detrimental to cocidin (N-glycosylpolifungin, Warsaw Biotechni- bee behavior or development. Several lab- cal Research and Development Center), and oratory and field studies have evaluated DFMO (α-DL-difluoromethyl ornithine, Merrell- fungicides incorporated into the leafcutter Dow). Appropriate serial dilutions of fungicide were added to sterile Sabouraud dextrose broth bee larval food for chalkbrood provisions (SDBY) with 2% yeast extract supplemented control (Parker, 1984, 1985; Youssef and with streptomycin (50 μg•ml-1) and penicillin (25 McManus, 1985; Fichter and Stephen, IU•ml-1) to obtain final fungicide concentrations 1987; Parker, 1987, 1988; Youssef and of between 0.5 and 1 000 ppm (m/v) as re- Brindley, 1989; Mayer et al, 1990; Goettel quired. Fungicides were also incorporated into et al, 1991). However, these studies have Sabouraud dextrose agar with 2% yeast extract (SDAY) by suspending the chemical in 1 ml of provided inconsistent and even contradict- sterile distilled water, which was then added to results and no have been ory compounds sterile SDAY. Serial dilutions were performed on registered for control of chalkbrood in leaf- constantly stirred warm media, then pipetted cutter bees. Furthermore, in vivo assays into 60 x 15 mm Petri plates. are time-consuming and rely on the pres- Leafcutter bee cells from a 1989 population ence of leafcutter bee larvae or eggs which of bees near Brooks, Alberta with a 15% inci- are only available in the summer and there- dence of chalkbrood were supplied by the Cana- fore severely restrict the number of com- dian Alfalfa Leafcutter Bee Cocoon Testing Cen- tre, Brooks, Alberta. Cadavers were removed pounds that can be screened in a year. from the cells and spores of A aggregata were A possible way of facilitating the search harvested as follows. Up to 50 cadavers and a for a suitable compound would be to initial- magnetic stirrer bar were placed onto the sur- ly screen numerous compounds for their face of a concave fine mesh plastic screen (12 which was built the effects on A aggregata in vitro. Promising squares•cm-1) by glueing screen onto a 1.5 cm x 8.5 cm diameter agents could then be assayed in vivo. In plastic ring. The ring was then placed in a 9 cm diame- the we results of present paper present ter glass Petri dish, covered, sealed with Para- the in vitro effects on A aggregata spore film and placed on a magnetic stirrer. Cadavers germination and vegetative growth of se- were stirred until all ascomata were scraped off the surface. The spores were collected from be- viability was measured at 48, 72, and 96 h using neath the screen and were kept sealed at 4 °C a vital dye; germling lengths were measured at until needed. 96 h as follows. Spores from a stock suspension in SDBY were added to 100 of SDBY contain- Spore suspensions were prepared by com- μl either 10 or 100 of bining spores with a drop of SDBY and crushing ing 0, 0.1, 1, ppm (m/v) fungi- cide in 1.5 ml tubes to obtain final between 2 sterile glass slides to eliminate spore microcentrifuge concentrations of 1 x 106 Tubes balls. Spores were then suspended in SDBY in spores•ml-1. were with with Parafilm a sterile microcentrifuge tube and spore concen- purged CO2, wrapped and incubated at 30 °C for 24 h. trations were determined using an improved Spore germina- Neubauer hemocytometer. The inoculum for the tion rates were determined by counting numbers of versus vegetative growth assays was prepared by purg- germinated non-germinated spores. Tubes were then returned to the incubator. At ing a 200-μl spore suspension in a microcentri- 24-h intervals was fuge tube with CO2 from a pressurized gas cylin- thereafter, germling viability der and incubating at 30 °C for 12 h. The measured using fluorescein diacetate (FDA) (So- a stock solution of FDA contain- germinated spores were spread on SDAY plates derstrom, 1977); supplemented with cycloheximide (30 μg•ml-1), ing 1 mg•ml-1 acetone was used to produce a streptomycin (50 μg•ml-1) and penicillin (25 working solution of 5 μl of stock FDA•ml-1 deion- IU•ml-1) in order to inhibit growth of bacteria and ized water. Viability assessments were performed black yeast-like fungi (Trichosporonoides sp) by combining 5 μl FDA working solution with an amount of on a which are a frequent contaminant in spores (un- equal fungal suspension glass slides were then examined with a Nikon published observations). Single isolated germ- slide; fluorescence a di- lings were later transferred to SDAY plates not Optiphot microscope using supplemented with antimicrobial compounds chroic mirror of 510 nm, excitation filter of 450- 490 nm and a barrier filter of 520 nm. Percent via- and incubated at room temperature. bility was determined by counting the number of fluorescing germlings versus the total number of spores and germlings observed at 400 x in the Vegetative growth assays same field of view using phase contrast micro- scopy. Germ tube lengths of the 10 longest germ SDAY plates containing various concentrations tubes in 10 fields of view were measured using of test fungicides were inoculated with a 5-mm an ocular micrometer. Germ tube lengths were inverted plug of A aggregata obtained from the measured from 1 replicate only. periphery of 4-5-day-old cultures. The plates Initially the experiment used one tube per treat- were sealed with Parafilm and randomly placed ment testing all fungicides at the 4 concentrations in a container which was incubated at 28 °C and on the same day and was repeated 4 times. a daily photoperiod of 16 h light 8 h dark for 2 Large variability in results, in part due to contami- weeks. Measurements of growth were made by nation with Trichosporonoides sp, made meaning- averaging the minimum and maximum radiuses ful analysis of the germination and viability data of mycelial growth on each plate, minus the not possible. As replicating treatments testing all width of the inoculum plug. An initial trial used 5 fungicides in one experiment was precluded due concentrations of each fungicide and a control, to the time required for the viability determination, non-fungicide treated culture medium with 10 the experiment was repeated using 4 replications replicate plates for each treatment. An additional of 4 concentrations of a specific fungicide; each trial used adjusted fungicide concentrations as fungicide was tested on a separate day and in- necessary. cluded a control. Germination rates were then cor- rected for controls as described below.

Spore germination, germling viability and growth assays Statistical analyses

Spores were germinated in liquid media contain- The vegetative growth assay data for all fungi- ing various concentrations of fungicide; spore cides except DFMO were analyzed by fitting the germination was measured at 24 h and germling nonlinear model : teraction. Because the DFMO data were all close to 100% viability with little variability, they were not included in the analysis. The control treatments for each of the fungicides were not where r is the observed radius, e is the base of significantly different; therefore no correction natural logarithms, c is the concentration of the was applied to the viability rates. Pairwise com- fungicide, and a, βi, and μi are parameters to be parisons of viability rates among fungicides for estimated. The parameter a is the maximum each concentration were carried out using the achievable radius for all fungicides, μi is the nat- least significant difference procedure at the 0.01 ural logarithm of the EC50 (effective concentra- level of significance. tion for 50% reduction in for i radius) fungicide Mean germ tube lengths were computed, but and βi is a coefficient determining the rate of de- could not be compared statistically because only cline of the curve for i. The data for all fungicide one replicate (tube) was available per fungicide- as well as the control data were fitted fungicides concentration combination. For a rough guide- simultaneously. The control data were used line, pairwise differences between fungicides to estimate a. nonlinear only Weighted regres- were denoted as being ’significant’ in table II if NLIN sion via the procedure of SAS was used the difference between their mean germ tube to fit the function, estimate the parameters and lengths was over 3 times greater than the stan- their standard errors, and test for differences dard error of their difference (computed using among the log(EC50) values. their respective within-tube variances). Untransformed spore germination data were analyzed using an ANOVA model that included effects for fungicide, concentration, and their in- RESULTS teraction. Significant and often large differences among the control treatments for each fungicide indicated that the data needed to be corrected for daily variation in germination rates. Correct- Vegetative growth assays ed average germination rates for concentration j of fungicide i were computed as : The effects of fungicide concentrations on vegetative growth are summarized in fig- ure 1. Using the logistic function, EC50s in where g’ij is the corrected germination rate, gij is ppm (confidence limits) were estimated as the observed germination rate averaged over follows: enilconazole, 0.034 (0.0262, replicates, and gio is the observed average ger- 0.0435); Rovral, 1.10 (0.767, 1.588); Ben- mination rate for the control replicates run on late, 1.16 and Ascocidin, the same day as fungicide i. Pairwise compari- (0.214, 6.278); 135.6 DFMO stimulated sons of corrected germination rates among (76.96, 238.79). fungicides (i and k) for each concentration (j) growth resulting in an approximately 50% were carried out by dividing the value of : increased radius at 10 000 ppm compared to the control.

by its approximate standard error, and evaluat- ing the ratio at the 0.01 level using the normal Spore germination, germling distribution. The standard error was obtained viability and growth assays using the delta method (Rao, 1973) and the re- sidual mean squared error from the ANOVA. The effects of fungicide concentration on hour data were Forty-eight germling viability at 24 h are summarized transformed using the arcsin-square root trans- spore germination in 2. The dose tested was formation to achieve variance homogeneity, and figure maximum analysed using an ANOVA model that included 100 ppm because higher concentrations of effects for fungicide, concentration, and their in- the wettable powders obscured viewing of the spores and therefore made germina- Comparison between in vivo tion and viability assessments not possi- and in vitro effects ble. Benlate inhibited spore germination; at the concentration Rovral in- highest only, of the to in hibited germination, while Ascocidin and Ranking fungicides according vitro and in vivo efficacy reveals that ef- enilconazole exhibited a slight effect. fects on spore germination most closely DFMO did not affect germination. correlated with efficacy against chalkbrood The effects of concentration fungicide (table II). The effects of fungicides on veg- on after 48 h are sum- germling viability etative growth on an agar surface gave the marized in 3. Ascocidin and Benlate figure most contradictory results as far as com- exhibited most effect followed antifungal parisons with in vivo effects are con- Rovral and enilconazole. DFMO had no by cerned; enilconazole was superior on agar effect on Viabilities at 72 and 96 h viability. whereas it was ineffective against chalk- showed large variability, in part due to con- brood caused by A aggregata in vivo tamination with Trichosporonoides sp; (Goettel et al, 1991). meaningful analysis of the data was not possible and these data are not presented. The effects of concentration fungicide DISCUSSION on germ tube growth are summarized in table I. Benlate, Rovral, and enilconazole had the most pronounced effect on germ Comparisons between in vitro results ob- tube growth. Ascocidin affected germ tube tained in the present study with in vivo re- growth only at the highest concentration; sults obtained previously (Goettel et al, DFMO showed little effect. 1991) indicate that in vitro antifungal activi- ty does not necessarily translate to in vivo growth on an agar surface (fig 1) and inhi- efficacy in reduction of chalkbrood inci- bition to germling growth and viability dence in leafcutter bees (table II). Among equivalent to Rovral (fig 3; table I), it was the 5 compounds tested, in vitro effects on not effective against chalkbrood in vivo spore germination most closely correlated (Goettel et al, 1991). This suggests that with efficacy against chalkbrood in vivo the compound was either inactivated in the suggesting that compounds with activity gut or was not absorbed into the hemo- against spore germination or germling via- coel; since this compound does not inhibit bility may be the most promising candi- spore germination and initial germ tube dates for use against chalkbrood. growth, the fungus could penetrate the gut Although the addition of antimicrobials and escape the antifungal activity. in sensitivity tests should be avoided as It was previously demonstrated that they can themselves influence the test mi- DFMO actually increased levels of chalk- croorganism, this could not be avoided brood (Goettel et al, 1991). As DFMO has with A aggregata as this organism does a wide range of antimicrobial activity (Bi- not sporulate in vitro; spores obtained from tonti and McCann, 1987), this effect was chalkbrood cadavers were contaminated attributed to the inhibition of possible A ag- with bacteria and a Trichosporonoides sp. gregata antagonists (Goettel et al, 1991). Bacteria were adequately controlled with The results in the present study reveal that antibacterials; however, as cycloheximide DFMO has no effect on spore germination, may affect A aggregata, it was not used, germling viability and initial germling and thus Trichosporonoides sp remained a growth, but it stimulates vegetative growth contaminant and this precluded observa- at higher concentrations. Specific inhibition tions after 48 h. Nevertheless, as the re- of ornithine decarboxylase with DFMO in- sults presented are corrected for controls, hibits germination, mycelial growth and it would be expected that any nonsynergis- sporulation of fungi (Slocum and Galston, tic influence due to the addition of the anti- 1987). The in vitro results with A aggregata microbials would also be corrected for. suggest that it may possess an alternate or Rovral, Benlate and Ascocidin exhibited unique pathway for polyamine synthesis or efficacy in all in vitro parameters tested a DFMO insensitive ornithine decarboxy- (figs 1-3, table I). Both Rovral and Benlate lase as has been shown to occur in other had previously been shown to reduce organisms (Bitonti and McCann, 1987) and chalkbrood incidence in laboratory bioas- warrants further investigation. says (Fichter and Stephen, 1987; Goettel The comparisons between in vitro and et al, 1991) and in field trials, albeit only in vivo results suggest that activity against marginally (Parker, 1987; Mayer et al, spore germination is of paramount impor- 1990). Ascocidin has not been field-tested tance in efficacy against chalkbrood. Un- against A aggregata; however, it is used in fortunately, very few compounds have for of in Poland control been identified which are sporicidal to A honey bees (A Hartwig, Warsaw Agricultu- aggregata and could be incorporated into ral University, personal communication). leafcutter bee provisions at concentrations Enilconazole exhibited only slight spori- nontoxic to bees (Stephan et al, 1982; Kish cidal activity (fig 2), as is characteristic of and Panlasigui, 1985). Results from the sterol inhibitors in general (Ragsdale, present study suggest that in vitro screen- 1977). Even though it exhibited the highest ing for candidate compounds for control of antifungal activity against vegetative chalkbrood should pay special attention to effects on spore germination and germling sont résumés dans la figure 1. Les EC50 viability. (concentration efficace pour une réduction radiale de 50%) ont été estimées à 0,034 pour l’énilconazole, à 1,10 pour le Rovral, ACKNOWLEDGMENTS à 1,16 pour le Benlate et à 135,6 ppm pour l’Ascocidin. Le DFMO a stimulé la crois- sance, un accroissement radial We thank Janssen Pharmaceutica, Dupont, provoquant de 50% à 10 000 au té- Rhone Poulenc, A Hartwig, Warsaw Agricultural ppm par rapport University, and R St Leger, Boyce Thompson In- moin. Pour les tests de germination des stitute, for providing the fungicides; S Simms of spores, de viabilité et de croissance des the Canadian Alfalfa Leafcutter Bee Cocoon propagules, les spores ont été mises a Testing Centre for providing chalkbrood cadav- germer dans des milieux liquides conte- ers; D Gaudet and L Kawchuk for critically re- nant diverses concentrations de fongici- the manuscript. Partial for this viewing funding des; la germination des spores a été me- study was provided by Janssen Pharmaceutica. surée à 24 h et la viabilité des propagules jusqu’à 96 h avec le diacétate de flurores- céine comme colorant vital; la longueur Résumé — Action de fongicides sur la des propagules a été mesurée à 96 h. Le germination in vitro des spores et la Benlate a inhibé la germination des spo- croissance végétative d’Ascosphaera res; à la concentration la plus élevée, seul aggregata. On a étudié les effets de 5 pro- le Rovral a inhibé la germination, tandis duits sur la in vitro des spores germination que l’Ascocidin et l’énilconazole ont montré et la croissance végétative d’Ascosphaera un léger effet (fig 2); le DFMO n’a pas af- du couvain de la aggregata, agent plâtré fecté la germination des spores. L’Ascoci- rotundata. Les 5 mégachile, Megachile din et le Benlate ont été les plus efficaces étudiés étaient : un concentré produits en ce qui concerne la viabilité des propa- émulsifiable d’énilconazole à 15% (Jans- gules à 48 h, suivis par le Rovral et l’énil- sen le Benlate 50WP Pharmaceutica), (bé- conazole (fig 3). Le DFMO n’a eu aucune El le Rovral 50WP nomyl, duPont), (ipro- action sur la viabilité des propagules. Le dione Rhône-Poulenc), l’Ascocidin (N- Benlate, le Rovral et l’énilconazole sont les glycosylpolifungin, Centre de Développe- produits qui ont le plus inhibé la croissance ment et de Recherche Biotechnologique du tube germinatif à 96 h (tableau I). L’As- de Varsovie) et le DFMO (α-DL- cocidin a inhibé la croissance du tube ger- difluoromethyl ornithine, Merrell-Dow). Des minatif à la concentration la plus forte; le dilutions appropriées en série ont été ajou- DFMO a eu comparativement peu d’effet. tées à un bouillon dextrosé (SDBY) ou à La comparaison entre les résultats in vitro une gélose dextrosée de Sabouraud présentés ici et les résultats in vivo publiés (SDAY) stériles avec un extrait de levure à précédemment (Goettel et al, 1991, Apido- 2% afin d’obtenir les concentrations finales logie 22, 509-522) montre que les effets in en fongicides voulues, comprises entre 0,5 vitro sur la germination des spores sont les et 1000 ppm (m/v). Pour les études de plus étroitement corrélés avec l’efficacité in croissance végétative, de l’inoculum d’A vivo contre le couvain plâtré (tableau II). aggregata a été placé sur des plaques de Ceci laisse penser que les produits actifs gélose renfermant diverses concentrations contre la germination des spores ou la via- de fongicides et la croissance a été mesu- bilité des propagules peuvent être les can- rée 2 semaines plus tard. Les effets des didats les plus prometteurs pour lutter fongicides sur la croissance végétative contre le couvain plâtré. Ascosphaera aggregata / Megachile ro- Sporenkeimung, Lebensfähigkeit der tundata / couvain plâtré / lutte chimi- Keimlinge und Wachstumsstudien wurden que / fongicide Sporen in flüssigen Medien mit verschiede- nen Konzentrationen der Fungizide zur Keimung gebracht; die Sporenkeimung Zusammenfassug — Wirkungen ausge- wurde nach 24 Stunden gemessen und die wählter Fungizide auf die Sporenkei- Lebensfähigkeit der Keimlinge nach einer mung in vitro und auf das vegetative Zeit von bis zu 96 Stunden bestimmt wobei Fluoreszin-Diazetat als be- Wachstum von Ascosphaera aggrega- Vitalfärbung ta. Es wurden fünf Präparate hinsichtlich nutzt wurde; die Länge der Keimlinge ihrer in vitro-Wirkung auf die Sporenkei- wurde nach 96 Stunden gemessen. Benla- te hemmte die Rovral mung und das vegetative Wachstum von Sporenkeimung; Ascosphaera aggregata, dem Erreger der hemmte die Keimung nur in der höchsten Kalkbrut bei Megachile rotundata studiert. Konzentration, während Ascocidin und Enilconazole nur einen Effekt Es handelte sich um folgende Präparate: geringen DFMO beeinflußte die Enilconazole in einem zeigten (Abb 2); 15%igen emulgier- nicht. Ascocidin und Ben- baren Konzentrat (Janssen Pharmaceuti- Sporenkeimung von Rovral und Benlate 50WP El late, gefolgt Enilconazole, ca), (Benomyl, DuPont), die auf die Lebens- Rovral 50WP (Iprodione, Rhöne Poulenc), zeigten größte Wirkung fähigkeit der Keimlinge nach 48 Stunden Ascocidin (N-Glycosylpolifungin, Warsaw (Abb 3). DFMO zeigte keine Wirkung auf Biotechnical Research and Development die Lebensfähigkeit der Keimlinge. Benla- Centre), und DFMO (α-DL-difluoromethyl te, Rovral und Enilconazole waren die Ver- seria- Ornithine,