Suppression of Growth Rate of Colony-Associated Fungi by High Fructose Corn Syrup Feeding Supplement, Formic Acid, and Oxalic Acid

Journal of Apiculturol Research and Bee World 47(2) 26-130 (2008) © IBRA 2008

ORIGINAL RESEARCH ARTICLE I B R A Suppression of growth rate of colony-associated fungi by high fructose corn syrup feeding supplement, formic acid, and oxalic acid.

Jay A. Yoder", Brady S. Christensen, Travis J . Croxall,Justin L. Tank and Diana Sammataro2. Department of Biology, Wittenberg University, Springfield, OH 45501 USA. Carl Hayden Honey Bee Research Center, United States Department of Agriculturc, Agricultural Research Service, Tucson, AZ 85719, USA.

Received 23 October 2007, accepted subject to revision 7 February 2008, accepted for publication 24 February 2008.

Corresponding author Email: jyoderwittenberg.edu -j- Mention of a proprietary product does not constitute an endorsement of use of the product by the U.S D.A

Summary

Select colony-associated fungi (bee isolates), Absidia sp., Ascosphaera apis, Aspergillus flavus, Fusarium sp., Penicillium glabrum, Mucor sp., showed a 40% reduction in radial growth rate with formic acid, a 28% reduction with oxalic acid, and a 15% reduction with fructose and high fructose corn syrup (HFCS) when grown on supplemented media at 30°C to mimic colony conditions. No effect, suppressing or promoting growth, was observed on other colony-associated fungi, Alternaria sp., Aspergillus niger, Cladosporium cladosporioides, Rhizopus sp. and Trichoderma sp., except 0.1 M formic and oxalic acid. Sensitivity to these compounds did not correlate with the fungus species being a slow- or fast-grower and sensitivity to one compound did not translate to sensitivity to another compound. Given the competitive nature and high-sporing (conidia) activity of these species, our results suggest that alteration or disruption of the colony \mycoflora can occur by use of these compounds. This may indicate a possible link between compound application and incidence of bee fungal pathogens. Supresión de la tasa de crecimiento de hongos asociados a las colonias mediante suplemento alimentario de sirope de maIz con alto contenido en fructosa, ácido fórmico y ácido oxálico.

Resumen Hongos asociados a las colonias (aislados de las abejas) como Absidia sp., Ascosphaera apis, Aspergillus flavus, Fusarium sp., Penicil!ium g!abrum, Mucor sp., mostraron una reducción del 40% en la tasa de crecimiento radial con ácido fórmico, del 28% con ácido oxálico y del 15% con fructosa y sirope de maIz con alto contenido en fructosa (SMACF) cuando crecieron el medio suplementado a 30°C para imitar las condiciones de )as colonias. No se observó ningün efecto de supresion o de promoción del crecimiento en otros hongos asociados a colmenas como A!ternaria sp., Aspergillus niger, Cladosporium c!adosporioides, Rhizo pus sp. y Trichoderma sp., excepto con ácidos fórmico y oxálico 0,1 M. La sensibilidad de estos compuestos no se correlaciona con la especie de hongo, además el crecimiento rápido o lento y la sensibilidad a un compuesto no se relaciona con la sensibilidad a otro. Dada la naturaleza competitiva y la alta actividad de esporulación (conidios) de estas especies, nuestros resultados sugieren que la alteración o interrupción de la micoflora de la colonia puede ocurrir mediante el uso de estos compuestos. Esto puede indicar una posible conexión ente la aplicación de estos compuestos y la incidencia de patógenos fingicos de abejas.

Keywords: colony-associated fungi, formic acid, oxalic acid, high fructose corn syrup

Fungus growth rates 127

Specifically, formic acid, oxalic acid and fructose (control for Introduction HFCS) were added to fungal media. Different formulations of HFCS from five different manufacturers were tested alone in a Numerous fungi are found in association with honey bee (Apis fungus growth study by measuring radial growth rates. mellifera) colonies and produce a diverse mycoflora favoured by the high relative humidity and stable conditions within the bee colony environment (Batra et 0/., 1973). Bee colonies are a suitable environment for fungi as the bees stabilize both the Materials and Methods relative humidity and temperature within the cluster where brood is reared. Some of these fungi are beneficial to the colony and Fungi, compounds and test conditions function to preserve stored pollen and bee bread while other Fungi were housed in permanent (-80°C in 10 % glycerol-distilled fungi are pathogenic and contribute to colony losses (Gilliam and water) freeze culture from Apis mellifera acquired in Arizona, Vandenberg, 1997). The majority of the fungi found in colonies USA (Benoit et al., 2004a). Pure cultures for testing were are assumed to be saprobes that originate from soil, litter and obtained by sub-culturing three rounds on potato dextrose agar organic debris that persist on nectar, pollen and colony debris. (PDA) in lOOxI 5mm petri dishes (Fisher: Pittsburgh, PA). Some fungi are harmful to bees, but immune mechanisms protect Samples were collected from the edges of 10 day old mycelium bees (Glibski and Buczek, 2003). Presumably, the activity of the that originated (first round of sub-culturing) from hyphal tips bees maintaining a relatively stable 30 35°C within the colony excised from the surface of an embedded, dead. autoclaved- cluster helps to keep fungal levels from becoming too high. Most sterilized bee that had been applied (20 pl aliquot) with thawed of these fungi are classified as mesophilic and high temperature storage culture to minimize potential negative effects of long- restricts their growth (Jennings and Lysek, 1999). term storage on the fungus by rearing from an actual host The bulk of bee colony fungi are accelerated spore (conidia)- Identification of fungi was confirmed by macroscopic (colony) and producing genera: once they are established, a fungus prevalent in microscopic (conidia) characteristics (Barnett and Hunter, 1998) one colony has the potential to spread to other colonies via and pure culture comparison to validate the integrity of strains, conidia carried by adult bees (Gliñski and Buczek, 2003). These i.e., production of assignable conidia and not unidentifiable fungi can also spread rapidly within the colony. In addition, the structures (Mycelia serilia) that can result from long term storage body surface of the bee is coated with numerous kinds of fungi on occasion. Incubation conditions were 30°C in total darkness. that are highly reminiscent and qualitatively match the kinds of The selection of 30°C was in order to simulate bee colony fungi that are found in the colony environment These conditions and to permit comparison with other bee colony components of the bees external mycoflora are also found on fungus work (Shaw et 01., 2002). Varroa mites that may be present within the colony (Benoit et al., Formic acid, oxalic acid and fructose were purchased 2004a). Fungi recovered from Arizona colonies include (majority commercially (> 99%: Sigma: St. Louis, MO) and diluted in double- of isolates): Absidio sp., Alternoria sp., Ascosphaero apis (causative distilled deionised (Dl) water to 0.1 M, 0.01 M and 0.001 M High organism of chalkbrood. Gilliam et o1., 1997), Aspergillus fIavus fructose corn syrup formulations were obtained from the (causative organism of stonebrood: Gliñski and Buczek, 2003), manufacturers and included: Tate and Lyle lsosweet 100 (TL 100). Aspergillus niger, Cladosporium c/odes porioides, Fusarium sp., Mucor Archer Daniels Midland Cornsweet 42 (ADM 42), Roquette Hi- sp., Penicillium glabrum, Rhizo pus sp. and Trichodermo sp. Sweet 55 (R 55), Mann Lake 55 HFCS (ML 55) and Mann Lake (mycoparasitic: Fisher and Cook, 1998) and are consistent with Sucrose Syrup (ML Sue). All were tested undiluted. PDA was the types of bee- and colony-associated fungi reported by Batra prepared according to directions from the manufacturer (Fisher) et o/(l973). This study seeks to gain insight into how certain by autoclave sterilization (121°C, 15 psi, 60 mm) and poured into organic acids applied in colonies for Vorroa control (e.g., formic sterile 100 x 5mm petri dishes in a laminar flow hood. FDA acid, Osterman and Currie, 2004: Amrine et al., 2007: oxalic acid, plates were used for an experiment once the agar solidified Rademacher and Harz, 2006) and a carbohydrate feeding (cooled to room temperature, 22-24°C), and plates were used supplement, high fructose corn syrup (HFCS) may alter the hive on the day that they were poured. environment and, thus, the composition of the colony mycoflora. Organic acids are administered either via sugar syrup, Growth rate determination, statistics sublimated vapours or directly into the colonies. HFCS is fed by and sample sizes commercial beekeepers during a dearth of nectar producing A 5mm id. well was punched out of a FDA plate aligning over the plants, and to stimulate brood rearing in the spring. When a top of the point of intersection of three lines (radiating from the fungus occupies a niche, any modification in its environment that centre with ± 40° separations) that had been scored on the affects its growth pattern has the potential of allowing other fungi bottom of the plate according to the trisecting line method to take over and exploit the substrate (Benoit et ol., 2004b). (Currah et ol., 1987; Baldrian and Gabriel, 2002). A I cm block There are various competitive mechanisms that fungi use to from an established pure culture mycelium was placed into the defend existing resources, which include the exploitation of well and then filled with 500 p1 of the test compound using a unoccupied substrate or the elimination of competitors, favouring sterile, calibrated glass micropipette (± 0.5% SE; Fisher): sterile Dl those that are more suited as a result of stresses from the water (diluent) served as a control. Measurements of the growth environment (Jennings and Lysek. 1999). These competitive rate of each fungus were taken daily for 5 days over the three interactions have the potential of fostering the growth of any one lines (thus. 3 measurements per plate per day) that had been particular, or group, of fungi, some of which could be pathogenic. scored on the bottom of the plate as the mycelium spread over

128 Yoder, Christensen. croxak, Tank, Sammataro

the agar surface. Rate of growth (K) was calculated according to Oxalic acid treatment equation I (Baldrian and Gabriel, 2002):- Those fungi that are shown in Table I experienced a significant (I) K (R.-R-,)/(t-to), reduction in growth rate (P < 0.05) at the lower concentrations where Ro and R are colony radii at initial (to) and elapsed (ti) of0.OIM and 0.00IM oxalic acid with the exception ofAbsidia sp. times (between beginning of linear, to, and stationary, ti, growth and Ascosphaera apis, but these are presented for consistency phases) as determined by a linear extrapolate from a plot of because of the suppression effect with the lower concentrations mycelium growth against time (time before to is the lag time). of formic acid. With oxalic acid treatments, similar responses were Growth rate was derived from the slope (expressed as mm/h). noted with Penicillium glabrum and Mucor sp., resulting in Each K represented the mean of 45 measurements, 15 per plate, consistent 1.3x reduction in growth rate (P > 0.05) without a replicated three times each using mycelia from seven separate dose response, but all three oxalic acid concentrations were pure cultures; that is, the same mycelium of a particular fungus effective at reducing the growth rate (Table I). The growth rate species was not used throughout the entire study. An analysis of of Fusarium sp. was suppressed the most (2. I x and 1.6x reduction variance (Duncan; ANOVA) was used to compare data respectively at 0. I M and 0.0 I Ni oxalic acid) compared to the (Microsoft Excel°° and MinitabTM). other fungi, though at 0.00 I M oxalic acid, no significant reduction was observed (P > 0.05). The most sensitive fungi to oxalic acid treatment were Fusarium sp., Penicillium glabrum and Mucor sp. using the extent of growth rate reduction and concentration Results effects.

Formic acid treatment High fructose corn syrup (HFCS) treatment Formic acid resulted in a growth rate reduction at 0. 1 H for at I The only four fungi that displayed negative effects on growth rate, fungi tested (ANOVA; P < 0.05). Results are given in Table I for resulting in less than 1.6x reduction, by fructose compared to the select group of I I fungi that showed a significant growth rates of the controls were Ascosphaera apis, Fusarium sp., reduction at lower concentrations of formic acid. This same group Penicillium glabrum and Mucor sp. (Table 2; P < 0.05). Other fungi, of fungi is presented in Table 2 for consistency with Table I At with no measurable effect, are shown in Table 2 for consistency lower concentrations of 0.01 M and 0.001 M formic acid, significant with results of formic and oxalic acid treatments presented in suppression in growth rate was observed compared to rate of Table I. Of the fungi affected, only Fusarium sp. and Mucor sp. controls for Absidia sp., Ascosphaera apis, Fusarium sp., Penicillium showed a lower growth rate at the lower concentrations of glabrum and Mucor sp. at 0.0 I M only (Table I; P < 0.05). Some 0.0 I M and 0.001 NI fructose. This was without a dose response fungi did not show a dose response; Ascosphaera apis (< I .5x (Table 2). However, fructose had the greatest suppression effect, reduction at all concentrations) and Penicillium glabrum approximately 2x, on Penicillium glabrum at 0. I M (P < 0.05). The (approximate 2x reduction in growth rate at all concentrations) most sensitive fungi affected by decreasing concentration and for example. Based upon the extent of lowering growth rate, the effect of the largest number of HFCS formulations were Fusarum most sensitive fungi to formic acid concentrations were Absidia sp. and Mucor sp. sp., Fusarium sp. and Penicil/ium g/abrum.

Table I. Effect of formic acid and oxalic acid on the average radial growth rate (Kr) on potato dextrose agar (PDA), 30°C, of Absidia sp. (ABS); Ascosphaera apis (ASC); Aspergillus flavus (ASP); Fusarium sp. (FUS); Penicillium glabrum (PEN); and Mucor sp. (MUC). Values followed by indicate significant differences from the control rate (ANOVA; P < 0.05).

Growth rate (mm/h;r) on PDA (±SE

0.lM 0.067 0,053 0.069 0.078 0.063 0.268

0.OIM 0.116 0,058 0.087 0.094 0.067 0.284

0.00I.M 0. 150 0.063 0.087 0.119 0.079 0.322

Oxalic

0.1 M 0.153 .-.. 0.058 0.086 0.076 0.099 0.248

9P J± 0201 0.074 0.081 0.101 0.106 0.267 1 0.00I.M 0.195 0.077 0.122 0.139 0.104 0.298

Fungus growth rates 129

Table 2. The effects of High Fructose Corn Syrup formulations and fructose on average radial growth rate (Kr) of fungi on potato dextrose agar (PDA) at 30°C. ABS, Absidia sp.; ASC, Ascosphaera apis; ASP Aspergi/Ius flavus; FUS, Fusarium sp.; PEN, Penicillium glabrum; NIUC, Mucar sp.; TL 100, Tate and Lyle lsosweet 100; ADM 42, Archer Daniels Midland Cornsweet 42; 155, Roquette Hi- Sweet 55; ML 55, Mann Lake 55 HFCS; and ML Suc, Mann Lake Sucrose Syrup. Values followed by are significantly different from the rate of the controls (ANOVA; P < 0.05).

Growth rate (mmlh; kr) on PDA (±SE<0.008), 30°C: Treatment ABS ASC ASP FUS PEN -- MUC Control 0.214 0.071 0.095 0.161 0.134 0.336 HFCS TL 100 0,223 0.067 0.113 0.144 - 0.148 - 0.279 ADM 42 0.207 0.059 0.088 p.147 0.122 0.314 R55 0.211 0.079 0.090 0.155 0.112 0.349 ML 55 0.198 0.064 0.106 - 0.139 0.139 0.26 1 MLSUC 0.210 0.084 0.097 0.172 0.130 0.294 Fructose 0.lM 0.207 - 0,059 0.096 05 0.116 0.277 0.OIM 0.220 0.069 0.102 0. 1 10 0.125 0.252 0.00IM 0.209 0.077 0.087 0.130 0.119 0.347

Control growth rates of fungi at 30°C varied, and when ranked (0. I M formic and oxalic acid) compared to the growth rates of statistically (P < 0.05) from fastest to lowest were: Rhizopus sp. controls. No enhanced growth was observed in response to the (0.381 mm/h) > Mucor sp. (0.336 mm/h) > Trichoderma sp. treatments either. Growth rates under control conditions were (0.267 mm/h) > Absidia sp. (0.214 mm/h) > Fusarium sp. (0. 161 fairly standard for these mesophilic fungi; characteristically mm/h) > Cladosporium cladosporioides (0. 147 mm/h) > Rhizopus sp., Mucar sp., Trichoderma sp. and Absidia sp. are typical Penicillium glabrum (0. 134 mm/h) > Alternaria sp. (0. 124 mm/h) > rapid growers. Similar growth rates for Alternaria sp., A. flavus and Aspergillus flavus (0.095 mm/h) > Aspergillus niger (0.082 mm/h) C. cladosporioides have been reported by Sautour et al. (2001), A. > Ascosphaera apis (0.07 I mm/h). niger and P glabrum by Benoit et al. (2004b) and Fusanum sp. by Brennan et al. (2003); the others represent new records for these fungal strains. We concluded that there is no connection between the differing growth rates of these fungi and their sensitivity Discussion toward the compounds tested. In addition, sensitivity to one of these compounds does not correlate with sensitivity to another Depending on the concentration used, formic acid, oxalic acid and compound. HFCS (and the fructose control) resulted only in suppression Fungi show considerable variation between different strains effects on fungal growth rates, or had no effect as a result of (e.g., origin of collection, substrate) of the same species (Barnett treatment. There was no evidence that formic acid, oxalic acid and Hunter, 1998; Fisher and Cook, 1998). Despite similar and HFCS promoted fungal growth, as none of the observed conditions within the colony environment, we emphasize that our growth rates was significantly higher than that of the controls. results for the Arizona strains of fungi that we examined can vary Of the II fungi examined, the subset of fungi that was the most among colonies. Such variations depend upon the composition of sensitive over the range of the concentrations tested to formic the regional mycoflora, geographic region, and extent or acid, oxalic acid and HFCS were: Absidia sp., Ascosphaera apis, frequency of application and prior exposure to the test Aspergillus flavus, Fusarium sp., Penicillium glabrum, and Mucor sp. compounds (formic acid, oxalic acid and HFCS). We also found Formic acid and oxalic acid have been demonstrated to have variations in growth response to the HFCS from each potent antifungal activity in culture conditions (Sahinler and Kurt, manufacturer. 2004; Yamaji et al., 2005), and growth on fructose is typically Implications from this study are that applications of formic and lower because it is less readily exploited and metabolized as a oxalic acid and HFCS, alone or in combination, have the potential carbon source than glucose (Solaiman and Saito, 1997; Jennings of altering the mycoflora balance in the bee colony. Conceivably, and Lysek, 1999). It was thus not surprising to find the negative enhancing or promoting distribution of Alternaria sp., Aspergillus effects on growth rate and lack of favourable growth when HFCS niger, Cladosporium c/adosporioides, Rhizo pus sp. and especially was added as a supplement under culture conditions. myco parasitic Trichoderma sp. could suppress growth of sensitive As a result of formic acid, oxalic acid and high fructose corn fungi and allow for expansion of non-sensitive fungi. Given the Syrup treatments, Alternaria sp., Aspergiilus niget Cladosporium high potential for spread of these colony fungi attributed to their cladaspariaides, Rhizo pus sp. and Trichaderma sp. showed no heavy spore-producing capacity and competitive interactions, Pronounced effect and did not display a statistically significant some of the fungi could expand quickly in an unoccupied niche drop in growth rate, except at the highest concentrations tested (Jennings and Lysek, 1999). Such differential growth rates would

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