Guy Mercadier

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l−1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 108 spores ml−1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 108 spores ml−1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 108 spores ml−1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.

Effect of liquid culture media on morphology, growth, propagule production, and pathogenic activity of the Hyphomycete, Metarhizium flavoviride

Two isolates of Metarhizium spp. were studied for propagule production, because of their pathogenic activity towards locusts and grasshoppers (Mf189 = M. flavoviride (or M. anisopliae var. acridum) strain IMI 330189, and Mf324 = M. flavoviride strain ARSEF324). Both isolates were grown in seven different liquid media, which have been developed for mass production of various Hyphomycetes, considered as candidates for microbial control of noxious insects. Shake-flask experiments were carried out at 28 °C in the dark. Production was quantified for 72 h and the effects of the tested media were evaluated on propagule concentration, morphology and pathogenicity. Based on preliminary experiments, all tested media were supplemented with 0.4% Tween 80 to avoid the formation of pellets and to produce unicellular propagules. Submerged propagule yields were higher withMf189 than with Mf324 in all seven media. While high concentrations of propagules (1.4 to 2.4 × 108 propagules ml-1 for MF189 and1.4 to 8.3 × 107 propagules ml-1 for Mf324) were produced in four media (Adamek, Catroux, Jackson, and Jenkins–Prior media), production of propagules was lower in the three other media (Goral, Kondryatiev, and Paris media). Both isolates produced oblong blastospore-like propagules, except in Kondryatiev medium in which they provided ovoid propagules. In this case, Mf189 submerged propagules looked like aerial conidia, but scanning observations did not demonstrate a typical conidiogenesis via phialides. In Kondryatiev medium, Mf324 submerged propagules were significantly smaller than aerial conidia. Infection potential of submerged propagules was assayed on Schistocerca gregaria. Second-instar larvae fed for 48 h on fresh wheat previously contaminated by a spraying suspension of each inoculum titrated at 107 propagules ml-1. All seven media produced submerged propagules that were highly infectious for S. gregaria larvae. Shake flask culture assays permitted us to select three low-costmedia, Adamek, Jenkins–Prior, and Catroux for improving scale-up of liquid fermentation focused on mass-production of Metarhizium propagules for mycoinsecticides devoted to locust control.

Evaluation of an entomopathogenic fungus, Paecilomyces fumosoroseus (Wize) Brown and Smith (Deuteromycota: Hyphomycetes) obtained from Formosan subterranean termites (Isop., Rhinotermitidae)

Abstract:  An isolate of Paecilomyces fumosoroseus was obtained from Coptotermes formosanus collected in Hong Kong, and a commercially available isolate of Metarhizium anisopliae, were both tested against C. formosanus shipped live from China. Survivorship of termites treated with a suspension of 5 × 105M. anisopliae conidia/ml and kept alone declined more rapidly than for those treated at the same concentration of P. fumosoroseus conidia. At a 5 × 106 conidia/ml concentration, no significant differences in terms of termite survivorship were observed between the two fungal species. However, among termites kept in groups of 10 after treatment, those sprayed with P. fumosoroseus conidia at either 5 × 105 or 5 × 106 conidia/ml had significantly lower survivorship than those sprayed with M. anisopliae conidia. All the cadavers of termites treated with P. fumosoroseus and kept alone sporulated and among grouped termites 29% of the cadavers sporulated. By comparison, 53% of the cadavers of termites treated with M. anisopliae and kept alone sporulated, and only 4% of the cadavers of treated termites kept in groups sporulated.

A distributed delay routine-based simulation model of Beauveria bassiana conidial stability in response to environmental stressors

Using published data and equations on therelationship between spore longevity of theentomopathogenic hyphomycetes, Metarhiziumanisopliae var. acridum and Beauveria bassiana (Balsamo) Vuillemin(Deuteromycota: Hyphomycetes) and temperatureand moisture content, a model of sporeviability was constructed based on adistributed-delay routine. The model ismodified via average spore survival time or byincluding an additional attrition (mortality)rate. The model was parameterized usingpublished values from studies on M. a.var. acridum spores, and output comparedfavorably with germination data and with apreviously-developed model. After initializingthe model using parameter estimates of B.bassiana spores from the laboratory andpublished data on changes in (1) spore viabilitywith respect to temperature and moisturecontent, and (2) spore moisture content withrespect to temperature and relative humidity,the model was run using daily min/maxtemperature and relative humidity data andcompared with data from four field experimentsof Mycotech B. bassiana isolate GHAsprayed on canteloupe plants. For two of theexperiments, observed viability trends werecompared to model outputs using weather datafrom both a weather station and fromwithin-canopy temperature and humidity probes. Output using weather station data fitobservations much better than output usingwithin-canopy probe data. For the tworemaining sets of field data, both earlier inthe season, only weather station data wereavailable and the resulting output fitobservations poorly. An attrition rate of 98%was needed to fit output to field data early inthe growing season, and a rate of 74% wasneeded for data collected four weeks later. These attrition rates can be consideredestimates for the proportion of spores dyingfor reasons other than temperature and relativehumidity, and they were attributed largely toUVB radiation due to the more open canopyearlier in the season.

Within-day variation in continuous hive weight data as a measure of honey bee colony activity*

Hourly weight data, from 4 honey bee hives placed on balances linked to dataloggers, were divided into two independent parts: (1) daily running average and (2) detrended weights, obtained by subtracting the running average from raw data. Weekly changes in running average weights, WCRAW, were correlated with food store changes but not adult or brood weights. Detrended weights showed daily fluctuation due to water and foraging bee movement and were modeled using sine curves, which fit all weekly subsets. Adult and brood populations, measured independently, were expressed as colony consumption rates via published per capita rates, and those consumption rates were correlated with sine amplitudes. Amplitudes were more sensitive to hive activity than WCRAW and unlike WCRAW detected high activity when foraging success was masked by high consumption Estimating food store changes with WCRAW and colony consumption with amplitudes reveals hive growth and activity without disturbing bees.ZusammenfassungWir analysierten hier wie stündlich aufgenommene Gewichtsdaten Hinweise auf dynamische Veränderungen in Honigbienenvölkern liefern können. Das Wiegen von Völkern ist zwar eine schnelle und eingriffsfreie Massnahme, andererseits aber muss in regelmässigen Abständen und mit ausreichender Präzision gewogen werden, wenn man zuverlässige Informationen über Stockaktivität und Volksdynamik erhalten will. In dieser Studie wurden Bienenvölker in 2005 und im Frühjar 2006 in zweiwöchigen Abständen inspiziert. Die einzelnen Waben wurden gewogen und das Gewicht der verdeckelten Brut, der erwachsenen Bienen und der eingelagerten Futtervorräte wurden bestimmt. Die Völker waren auf elektronischen Waagen aufgestellt und mit Dataloggern verbunden. Stündlich registrierten Gewichtsdaten wurden aufgeteilt in einen Mittelwert über 25 Stunden hinweg, als Langzeitmittel, und einen trendfreien Gewichtswert, als Kurzzeitinformation. Letzterer gibt die Abweichung der Rohdaten für die jeweilige Stunde von dem laufenden Mittelwert für die entsprechende Stunde an. Laufende Mittelwerte wurden auch benutzt, um die Wöchentliche Änderung im Laufenden Mittel (WCRAW, übersetzt WÄLM) zu berechnen, indem Wochenmittel von dem der jeweils folgenden Woche subtrahiert wurden. Die WCRAW-Daten wurden mit denen der Inspektionsdaten von 2005 für Gewichte der verdeckelten Brut und der adulten Bienen, sowie der Futtervorräte verglichen, und diese Vergleichsergebnisse wurden gegen die der in 2006 vorgenommenen Inspektionen verglichen. An die trendfreien Gewichtsdaten wurde eine Sinuskurve angepasst und die Parameter dieser Kurvenanpassungen wurden für WCRAW-ähnliche Analysen benutzt, ausser dass hier die Daten über die Brut- und Adultpopulationen in Verbrauchsraten umgerechnet worden waren.Die WCRAW-Daten zeigten eine gute Korrelation mit den täglichen Veränderungen im Gewicht der Futtervorräte (Tab. I), aber nicht mit den Gewichtswerten für die Brut und für adulte Bienen. Die trendfreien Daten zeigten klare aber variable Tagesverlaufsmuster. Sinuskurven konnten an alle trendfreien Daten angepasst werden und diese wiesen stets eine Periodik zwischen 22 und 26 Stunden auf. Die Futtergewichtsdaten und die geschätzten Werte der Verbrauchsraten wurden für 2005 und 2006 mit den jeweiligen täglichen Amplituden der Sinuskurven und den WCRAW-Schätzwerten verglichen (Abb. 6 und 7). Die Amplituden und die Gewichte der meisten Komponenten zeigten eine signifikante Beziehung (Tab. I). Insbesonders die Beziehung zwischen den Verbrauchsraten für die Brut und adulte Bienen war signifikant mit der jeweiligen Amplitude korreliert (Abb. 8) und kann damit wichtige Informationen über den Volkszustand geben. Die Kombination der Wägungen mit der kontinuierlichen Datalogger-Information wurde hier benutzt, um Rückschlüsse über die Sammelaktivität, Wasserbewegungen, Schwarmvorgänge, Ruhezustände, sowie über Veränderungen in Futtervorräten und Futterverbrauch zu treffen. Diese Methode ist damit in der Lage, sowohl die Notwendigkeit invasiver Inspektionen zu reduzieren, als auch die Information aus solchen Inspektionen umfassender auszuwerten.

Impact of a treatment of Beauveria bassiana (Deuteromycota: Hyphomycetes) on honeybee (Apis mellifera) colony health and on Varroa destructor mites (Acari: Varroidae)*

In 2 experiments bee colonies in southern France were treated with conidia of a Beauveria bassiana isolate collected from Varroa mites in the region. Objectives were to evaluate treatment effect on colony weight, adult bee mass, and capped brood and honey, and on Varroa fall onto sticky boards. Treatments included conidia formulated with either wax powder or wheat flour, flour alone, or control. Treatment did not affect colony health. Colonies treated with conidia and wax powder had higher mite fall compared to controls while those treated with conidia and wheat flour did not. The proportion fallen, infected mites in both conidia treatments was higher than controls for up to a week. Higher mite fall and infection rates were observed in treated hives in the 2nd, smaller experiment. The relationships between dosage and proportion fallen, infected mites, and between ambient temperature and infection duration, were examined. Future experiments will explore Varroa control using conidia.ZusammenfassungIn zwei Experimenten wurden Honigbienenvölker in Südfrankreich mit Konidiensporen eines aus Varroamilben der Region gesammelten Isolates behandelt. Ziel war die Erfassung der Auswirkung der Behandlung auf das Gewicht der Völker, das Gesamtgewicht an adulten Bienen, auf die verdeckelte Brut und auf die Honigmenge, sowie auf den Totenfall von Varroamilben auf klebrige Bodeneinlagen. Die Behandlungen umfassten Formulierungen von Konidiensporen entweder mit Wachspulver oder mit Weizenmehl, sowie Weizenmehl alleine und unbehandelte Kontrollvölker. Die Behandlungen hatten keinen Einfluss auf die Gesundheit der Bienenvölker. Die mit Konidiensporen und Wachspulver behandelten Völker hatten einen höheren Milbentotenfall als die Kontrollvölker, bei den mit Konidiensporen und Weizenmehl behandelten Völkern war dies nicht der Fall. Der Anteil an infizierten Milben im Totenfall war bei beiden Behandlungen mit Konidiensporen über den Zeitraum von etwa einer Woche höher als bei den Kontrollen. Ein erhöhter Milbentotenfall und erhöhte Infektionsraten wurden auch in dem zweiten, kleineren Experiment gefunden. Die Beziehung zwischen der Dosierung und dem Anteil infizierter Milben im Totenfall und zwischen der Umgebungstemperatur und der Infektionsdauer wurden untersucht. Zukünftige Experimente sollen die Möglichkeit einer Behandlung der Varroose mittels Konidiensporen untersuchen.

Using balances linked to dataloggers to monitor honey bee colonies

Beekeepers sometimes weigh hives to monitor honey flow, and researchers weigh hives to obtain data on the weight of honey reserves, brood and adult bees (e.g., McLellan, 1977; Szabo and Lefkovitch, 1991; Harbo, 1993). Using an outdoor balance of sufficient precision and linked to a datalogger, hive weight can be measured frequently and thus used to observe changes due to external factors such as experimental treatments or weather, to internal factors such as swarming, and to control for differences between colonies. Using two such balances, we evaluated (1) growth of two experimental hives, (2) the magnitude and duration of rainfall effects on hive weight, (3) weight changes associated with swarming and queen loss, and (4) amplitude and periodicity of daily weight change cycles. On 26 March 2004, two packages of bees were established in an apiary with three pre-existing colonies at the European Biological Control Laboratory, France. One package, containing approximately 1 kg of adult ‘Frère Adam’ (Buckfast) bees and 1 queen, was placed in each of two painted, 10-frame, wooden Dadant brood boxes (56 l capacity) (Ickowicz, Bollène, France). The hives were covered with telescoping lids and a weight was placed on top. The mass of each hive without bees was about 21 kg. The two hives were placed on top of two planks resting on outdoor stainless steel electronic balances (TEKFA model B2418, Galten, Denmark). The balances had a 100 kg maximum capacity, a precision of 10 g and an operating temperature range of -30o to 70o C. The balances were linked to 12-bit dataloggers (Hobo U-12 External Channel datalogger, Bourne, MA, USA) and powered by a solar panel (BP Solar model 1230, Mimeure, France). The weighing system had an overall precision of about 30 g. Hourly weight was recorded starting at midnight on 15 June 2004 and continuing until 4 PM on 8 June 2005. Hourly temperature and rainfall data were obtained from Meteo France (Aix-enProvence, France). To obtain information on the weights of the different parts of each colony, hive weight was divided into (1) the ‘non-colony’ component consisting of the previously-weighed physical structure, e.g., brood box, lids, super, hive base, frames with foundation comb, and (2) the ‘colony’ component, consisting of the adult bees, brood, honey, pollen and wax (other than foundation). Every two weeks the adult bee mass was estimated by weighing each brood box frame and the super separately, after shaking them free of bees, with a portable electronic balance (Kern & Sohn model 12K 1N, Balingen, Germany), and subtracting the combined weight of the frames and other non-colony components from the current running average weight. Colony mass other than adult bees (i.e. honey, wax, brood, and pollen) was calculated by subtracting the weight of the adult bees, wooden frames, and foundation combs from the hive weights. The data from the balances were examined in terms of the raw data and their two components: (1) the longer-term changes, estimated using the 25-hour running average weight, and (2) short-term changes, estimated using the hourly de-trended weight. The hourly running average was calculated by averaging for each hour the raw colony weight over the preceding 12 hours, the following 12 hours, and that hour (for a total of 25 hours). The running average can be used to examine general trends in colony growth and long-term effects of different treatments, but it is not considered further here. Hourly detrended weight was calculated by subtracting the running average weight for each hour from the raw data for that hour. Raw data were used to examine hive weight changes associated with particular events such as rainfall and swarming. Using the balances, we were able to show that the magnitude of weight changes due to rainfall was correlated with rainfall intensity, and the duration of the weight change (the time it took for the hive to return to pre-rainfall levels) was 10–34 hours. Swarm departures were observed with precision: in 2005 colony 1 produced swarms of 2.36, 1.40 and 0.95 kg on 27 April, 5 May and 7 May, respectively. Assuming all bees had full foreguts, about 20% of the swarm weight could be attributed to honey (Harbo 1993), yielding a total loss of about 0.94 kg honey and 3.77 kg bees. Colony 2 did not produce swarms. Changes in hourly detrended data were largely a function of the movement of foraging bees and of the gain and loss of water in the form of nectar, drinking water, bee respiration and evaporative cooling (Gary, 1992). Honey gains would cause the running average to increase since honey tends to stay in the nest longer than 25 hours, and would be accounted for in the running average data rather than the detrended hourly data. Using forager traps at the hive entrance, Danka et al. (1986) found that the O R I G I N A L A RT I C L E

Effects of multiple applications of a Beauveria based biopesticide on Varroa destructor (Acari: Varroidae) densities in honey bee (Hymenoptera: Apidae) colonies.

W. G. Meikle, G. Mercadier, F. Annas and N. Holst European Biological Control Laboratory, USDA ARS, Campus International de Baillarguet, CS 90013 Montferrier sur Lez, 34988 St. Gely du Fesc, France. Present address: Kika de la Garza Subtropical Agricultural Research Center, USDA ARS, 2413 E. Highway 83, Weslaco, TX 78596, USA. ADAPRO LR-CRALR, Maison des Agriculteurs, Mas de Saporta CS 30012, 34875 Lattes, France. Danish Institute of Agricultural Sciences, Flakkebjerg, 4200 Slagelse, Denmark.