Taxonomy and Enzymology of Molds*

Original article

Microbiology of pollen and bee bread : taxonomy and enzymology of molds*

M. Gilliam, D. B. Prest B. J. Lorenz

US Department of Agriculture, Agricultural Research Service, Carl Hayden Bee Research Center, 2000 E. Allen Road, Tucson, AZ 85719, USA (received 26-5-1988, accepted 18-8-1988)

Summary — One-hundred and forty-eight molds were isolated from the following samples of almond, Prunus dulcis, pollen : floral pollen collected by hand; corbicular pollen from pollen traps placed on colonies of honey bees, Apis mellifera, in the almond orchard; and bee bread stored in comb cells for one, three, and six weeks. The majority of molds identified were Penicillia (32%), Mucorales (21%), and Aspergilli (17%). In general, the number of isolates decreased in pollen as it was collected and stored by the bees. Each type of pollen sample appeared to differ in regard to mold flora and dominant species. Aureobasidium pullulans, Penicillium corylophilum, Penicillium crustosum, and Rhizopus nigricans were among the molds that may have been introduced by bees during collection and storage of pollen. Mucor sp., the dominant mold in floral pollen, was not found in corbicular pollen and bee bread. Tests for 19 enzymes revealed that most of the molds produced caprylate esterase-lipase, leucine aminopeptidase, acid phosphatase, phosphoamidase, B-glucosidase, and Macetyl-B-glucosaminidase. Thus, enzymes involved in lipid, protein and carbohydrate metabolism were produced by pollen molds. Molds could also contribute organic acids, antibiotics and other metabolites. pollen - bee bread - molds

Résumé — Microbiologie du pollen et du pain d’abeilles : taxonomie et enzymologie des moisissures. A l’aide de divers milieux microbiologiques possédant des pH différents, on a isolé 148 moisissures des échantillons suivants de pollen d’amandier, Prunus dulcis : pollen de fleurs récolté à la main; pollen en pelotes prélevé dans les trappes à pollen posées sur des colonies d’abeilles (Apis mellifica) dans un verger d’amandiers; et pain d’abeilles stocké dans les cellules des rayons durant une, 3 et 6 semaines. La majorité des moisissures identifiées sont des Penicillia (32%), des Mucorales (21 %) et des Aspergillia (17%). C’est le pollen de fleurs qui est le plus riche en isolats, mais le plus pauvre en espèces. En général le nombre d’isolats diminue dans le pollen quand il est récolté et stocké par les abeilles. Chaque type d’échantillon pollinique semble différer des autres par la flore de moisissures et les espèces dominantes. Puisque les moisissures sont identifiées d’après les besoins de croissance et la caractérisation microscopique et macroscopique des structures morphologiques, les données biochimiques ne proviennent pas des tests taxonomiques. On a

* Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products or vendors that may also be suitable. donc analysé 19 enzymes chez 78 isolats, représentant 28 espèces, par le système API ZYM. Aucune moisissure ne produit de trypsine, de 8-glucuronidase, ni de a-mannosidase. La plupart des moisissures produisent de la caprylate lipase-estérase, de la leucine aminopeptidase, de la phosphatase acide, de la phosphoamidase, de la 8-glucosidase et de la N-acétyle-l3-glucosaminidase. Les moisissures du pollen produisent donc des enzymes impliqués dans le métabolisme des protéines, des lipides et des glucides. Ces résultats suggèrent que la flore de moisissures du pollen en pelotes et du pain d’abeilles peut résulter d’inoculations microbiennes par les abeilles et de modifications chimiques du pollen dues aux substances ajoutées par les abeilles lors de la régurgitation du contenu du jabot et à la fermentation microbienne, qui permet à certaines espèces de survivre et à d’autres pas. Même si, dans nos échantillons, les moisissures étaient plus nombreuses que les espèces de Bacillus et les levures, le pollen est rarement envahi par elles. Parce qu’elles sont susceptibles de fournir des enzymes, des acides organiques, des antibiotiques et d’autres métabolites, les moisissures méri- tent des études plus approfondies.

pollen - pain d abeilles - moisissures

Zusammenfassung — Mikrobiologie von Pollen und Bienenbrot : Taxonomie und Enzymolo- gie des Schimmels. Unter Verwendung verschiedener mikrobiologischer Medien mit unterschied- lichem pH-Wert wurden 148 Schimmelpilze von den folgenden Proben von Mandelpollen (Prunus dulcis) untersucht : Blütenpollen (von Hand gesammelt), Pollenhöschen (aus Pollenfallen an Bie- nenvölkern [Apis mellifera] im Mandelbaumgarten) und Bienenbrot, das 1, 3 und 6 Wochen in der Wabe gespeichert war. Die am häufigsten auftretenden Schimmelpilze waren Penicillia (32%), Mucorales (21 °l) und Aspergilli (17%). Der Blütenpollen lieferte die meisten Isolate, aber die wenigsten Arten. Im all- gemeinen nahm die Anzahl der lsolate im Pollen mit dem Sammeln und Speichern durch die Biene ab. Jeder Pollentyp schien im Hinblick auf die Schimmelflora und die dominierenden Arten ver- schieden zu sein. Da die Schimmelpilze aufgrund ihrer Wachstumserfordernisse sowie mikrosltopischer wie makroskopischer Charakterisierung von morphologischen Strukturen identifiziert werden, erhält man von taxonomischen Untersuchungen keine biochemischen Daten. Daher wurden 78 Isolate, die 28 Arten repräsentieren, mit dem API ZYM-System auf 19 Enzyme analysiert. Kein Schimmel- pilze produzierte Trypsin, β-Glucuronidase oder a-Mannosidase. Die meisten Schimmelpilze produ- zierten Caprylat-Esterase-Lipase, Leucin-Aminopeptidase, saure Phosphatase, Phosphoamidase, β-Glucosidase und N-Acetyl-β-Glucosaminidase. Dies bedeutet, daß die untersuchten Pollen- schimmel Enzyme des Protein-, Fett- und Kohlehydratstoflwechsels produzieren. Diese Ergebnisse deuten darauf hin, daß die Schimmelflora im Höselpollen und im Bienenbrot ein Ergebnis folgender Einflüsse ist : mikrobielle Inokulation und chemische Veränderung des Pol- lens durch Zugabe von Honigmageninhalt durch die Biene; Drüsensekretion sowie mikrobielle Fer- menta6on, die manche Schimmelarten tolerieren, andere nicht. Obwohl Schimmelpilze in unseren Proben weit zahlreicher waren als Bacillus spp. oder Hefen, wurde der Pollen selten von Schimmel überwachsen. Die Schimmelpilze sollten als potentielle Spender von Enzymen, organischen Säu- ren, Antibiotika und anderer Metabolite intensiver untersucht werden.

Pollen - Dienenbrot - Schimmel

conducted on various floral and bee-col- Introduction lected (corbicular) pollens. The conver- sion of pollen to bee bread has often been Studies have shown for many years that postulated to be the result of microbial pollen and bee bread, that is pollen stored action, principally a lactic acid fermenta- in comb cells of the hive, differ biochemi- tion caused by bacteria and yeasts cally, and extensive analyses have been (Foote, 1957; Haydak, 1958). However, the chemical and biochemical changes foraging of honey bees are probably occurring in pollen as it is collected and unable to become established within the stored by honey bees, Apis mellifera, are bee or the hive. He found that Penicillia not clearly understood, and relatively little were the most common molds within the is known about the microbiology of pollen hive, Aspergilli occurred less frequently, and bee bread. and species of Mucor did not grow well on To better understand the nutrition of brood combs. honey bees, we studied the chemical, bio- Fungus-caused spoilage of provisions and chemical, microbiological composition and mortality of honey bees are rare of pollen from a single plant species be- (Batra et aL, 1973). Recently Gilliam and fore, during, and after storage in comb Vandenberg (1988) reviewed the literature cells. Previous on the papers subject by on fungi pathogenic or detrimental to researchers at the Carl Hayden Bee honey bees. Only Ascosphaera apis Research Center reviewed earlier work which causes chalkbrood disease in and reported results concerning yeasts honey bees is of economic importance. Bacillus (Gilliam, 1979a); ssp. (Gilliam, The pollen mold, Bettsia alvei, is not a 1979b); fatty acids, sterols, vitamins, titra- serious problem since it does not grow table acidity, minerals (Loper et al., 1980); well in cells that are filled with pollen and and amino selected protein content, acids, finished with a layer of honey on top enzymes, pH, and 10-hydroxy-!2-deca- (Skou, 1972). noic acid (Standifer et al., 1980). In all this Burri stated that is work, the same samples of almond, Pru- (1947) pollen germ- free in blossoms that have not as nus dulcis (Prunus communis), pollen opened as if were utilized. well in opened blossoms uncontami- nated by insect visitation or air currents. Even molds are known though widely Neither of the two studies for their abilities to and to microbiological degrade synthe- of pollen and bee bread (Chevtchik, 1950; size numerous compounds including the Pain and Maugenet, 1966) gave data on production of many materials important to molds, although Chevtchik (1950) men- the food and chemical industries, drug, tioned B. alvei as a consumer of have received scant attention in possible they api- lactic acid in bee bread. cultural research concerned with pollen and bee bread. Early mycological re- Arizan et al. (1967) isolated Absidia search recognized that certain molds are ramnosa, Aspergillus flavus, Aspergillus common saprophytes on and inside fumigatus, Aspergillus niger, Aspergillus honey bees and brood combs, but efforts terreus, Aspergillus versicolor, Mucor were concentrated on dead bees; combs, mucedo, Penicillium clavigerum, Penicil- particularly from dead colonies; and moldy lium purpurogenum, Rhizopus nigricans pollen (Betts, 1912; Burnside, 1927). and Trichothecium roseum from Indian Betts (1912) reported a species of Clado- corn pollen collected by machine. Sainger sporium as well as Mucor erectus in corbi- et aL (1978) reported that Altemaria cular pollen and Beftsia alvei, Eremascus alternata was the most common isolate in fertilis, Gymnoascus setosus, Oospora pollen from 3 herbaceous annual plants. favorum, and Penicillium crustaceum in Other molds isolated were Aspergillus pollen stored in combs. She noted that flavus, Aspergillus luchuensis, Aspergil- honey appeared to be immune to attacks lus nidulans, Aspergillus sulphureus, A. of molds. Burnside (1927) stated that versicolor, Cladosporium oxysporum, most of the fungi collected by widespread Epicoccum purpurascens, Fusarium oxysporum, Monilia fructigena, Monilia sito- cultures of isolates were lyophilized for preser- phila, Monilia sp., Rhizopus sp., R. nigri- vation until tests were conducted. Molds were tested and identified to cans and Trichoderma viride. Molds isola- according Neergaard (1945), DeVries (1952), Cooke (1959), Ames ted from bee bread by Egorova (1971) (1961), Booth (1961), Morton and Smith (1963), were A. flavus, A. versicolor, Mucor Ellis (1965), Raper and Fennell (1965), Raper alboalter, Penicillium granulatum, Penicil- and Thom (1968), Zycha et al. (1969), Kendrick lium solitum and olivecum. and Carmichael (1973), Samson (1974), von Sporotrichum Arx (1975) and McGinnis et al. (1986). The the results present paper reports Since molds, in contrast to bacteria and of the isolation and identification of molds yeasts, are identified on the basis of growth from almond floral pollen, corbicular pol- requirements and microscopic and macroscop- len, and bee bread stored in comb cells; ic characterization of morphological structures, biochemical data do not result from tests for of select- analyses enzymes produced by identification. Therefore, selected isolates were ed isolates; and comparison of species tested for 19 enzymes with the API ZYM sys- isolated with those previously reported tem (Analytab Products) using the methods of from honey bees in Arizona. Bridge and Hawksworth (1984). Suspensions of some isolates such as Mucorssp. and Alter- naria tenuis were sonicated for 1—10 min to separate fungal spores before inoculation into the API ZYM strips. Also, malt extract agar Materials and Methods and/or potato dextrose agar (Difco) were used to prepare inocula of some cultures when growth appeared to be less than optimal on and collect- Details concerning bee colonies Czapek solution agar. ions of pollen and bee bread are given by Gil- liam (1979a), Loper etal. (1980), and Standifer et al. (1980). The following samples of almond pollen were examined : fresh floral pollen collected by hand; corbicular pollen containing Results 99.8% almond pollen from pollen traps placed on bee colonies in the almond orchard; and bee bread stored in cells for 1, 3, and 6 weeks No attempt was made to determine whe- in newly drawn combs of colonies of bees ther spores or mycelial elements were maintained in a greenhouse. Each sample was isolated from and bee bread. How- divided into 4 sub-samples of approximately pollen molds were isolated on all 4 media 0.75 g each. Then each of the 4 sub-samples ever, was homogenized by hand in 2.5 ml of sterile used (Table I). Seventy-seven percent of 0.85% NaCi in a glass tissue grinder. The the isolates were from media incubated at in homogenates were plated (0.1 ml) duplicate 25°C, and 23% were from media at 37°C on acidified yeast extract-malt extract agar which is near the brood nest containing 1% glucose, pH 3.7—3.8 (Miller et temperature al., 1976); mycological agar with low pH (Difco, of 34°C (Dunham, 1929). However, the pH 4.8); nutrient agar (Difco) acidified with 0.1 optimum temperatures for most fungi are N HCI to pH 5.0; and eugon agar (Difco, pH in the range of 20-30°C (Alexopoulos, 7.0). One plate from each sub-sample was Isolations from floral incubated at 25°C and one at 37°C. All were 1962). pollen incubated under aerobic conditions except increased with decreasing pH of the eugon agar plates which were placed in 4% media. In contrast, the highest percent of C02. During a 2-week incubation period, plates isolations from corbicular pollen from the were examined for mold periodically growth. pollen trap was on acidified nutrient agar When molds appeared, they were trans- with a pH of 5.0. Few isolations were ferred to plates of solution agar or malt Czapek made from floral or corbicular pollen on extract agar (Difco) to allow time for sporulation with a of but this and to test for purity. These plates were incu- eugon agar pH 7.0, bated at 25°C under aerobic conditions. Pure situation changed with bee bread. The percent of isolations on various media gemmae (chlamydospores) and yellow was similar in bee bread samples stored globules, globose sporangia with finely for one week and for 3 weeks. These spinose walls, columella with a distinct results indicate that a variety of media collarette, and elliptical smooth sporangio- with different chemical compositions and spores. The other molds identified from pH values incubated at both 25°C and floral pollen were found in at least one 37°C aerobically and under C02 should additional pollen source. be used for of determining mycoflora pol- The second most isolated len and bee bread. frequently mold was Penicillium corylophilum. It was One-hundred forty-eight molds were associated with corbicular pollen and all isolated from pollen and bee bread, of bee bread sources but not with floral pol- which 139 were identified (Table II). Over- len. This was also the case for R. nigri- all, the majority of molds identified were cans. Other species which first appeared Penicillia (32%), Mucorales (21%), and in corbicular pollen and were then found Aspergilli (17%). Floral pollen yielded in bee bread were Aureobasidium pullu- the highest number of isolates but the lans, Cladosporium herbarum, Penicil- smallest number of species. In contrast, lium chrysogenum, and Penicillium crus- bee bread stored in comb cells for 3 tosum. Aspergillus niger was a frequent weeks had the fewest isolates but the isolate and was found in all types of pollen greatest number of different species. In samples except bee bread stored for one general, the number of isolates decreased week and was isolated most often from in pollen as it was collected and stored by bee bread stored for 6 weeks. Another bees. frequently encountered mold was Penicil- The most frequent isolate was Mucor lium cyclopium which was most abundant sp., associated exclusively with floral pol- in bee bread stored for one week and was len. All 19 isolates appeared similar, but found in all sample types except corbicu- species identification was not made. They lar pollen. Cladosporium cladosporioides were characterized by the production of was the only species isolated from all coenocytic mycelium, some sympodially sample types but was most prevalent in branched sporangiophores containing floral pollen. Molds isolated from more than one source of bee bread but not flo- bread stored for 6 weeks, A. niger was ral or corbicular pollen were Aspergillus most common. amstelodami, A. flavus, and Paecilo- Eight unidentified molds in Table 11 myces varioti. Alternaria tenuis was found were non-viable after lyophilization. We in floral pollen and bee bread stored for were also unable to assign the unidenti- one week, and Penicillium brevi-compac- fied Ascomycete to genus. It produced tum was in floral and corbicular but pollen ostiolate dark with unbranched not bee bread. Other than Chaetomium ascocarps terminal hairs. The asci were elatum which was found in bee bread cylindrical and contained 4 stored for 6 weeks, the remaining species ascospores. Ascospores were ellipsoidal to ovoid, smooth, non- that were identified appeared once in only and to brown. one type of pollen source other than floral apiculate, yellow-brown Since it was difficult to determine pollen. Thus, each type of pollen sample by light the were appeared to differ in regard to mold flora microscopy whether ascospores and dominant species since the predom- single or double-pored, scanning electron was used to reveal that the inant mold in almond floral pollen was microscopy Mucor sp.; in corbicular pollen, P. corylo- majority were single-pored, although were also philum and P. crustosum were most com- double-pored ascospores pres- mon; in bee bread stored in comb cells for ent. one week, P. cyclopium and P. corylophi- To determine enzymatic activity of the lum were the most numerous isolates; in molds from pollen and bee bread, bee bread stored for 3 weeks, there was attempts were made to test at least one no obvious dominant species; and in bee strain of each species and more strains of the species frequently isolated. However, also grown on various media, and the Cladosporium sphaerospermum did not enzymology was compared to inocula survive lyophilization after identification from Czapek solution agar. Results of and could not be tested. Thus, 78 isolates tests on inocula of the same strain pre- representing 28 species were each anal- pared on different media gave the same yzed for 19 enzymes. A total of 113 com- results except that the concentrations of plete tests were conducted owing to repli- one or two of the enzymes produced were cation of tests, use of additional media for in a few cases slightly higher when the preparing the inocula of strains which did growth medium was potato dextrose agar not grow well on Czapek solution agar, compared to malt extract agar. These dif- duplicate tests which were incubated as ferences could be related to improved long as 24 h, and tests comparing enzy- growth of the molds and/or to the media mology of spore and mycelial inocula of composition. Bridge and Hawksworth selected strains. Aspergillus amsteloda- (1984) also found some minor variations mi, Chaetomidium pilosum, Chaetomium with different media. We also noted a few elatum, Thielavia sepedonium, Xylohy- similar minor variations when the incuba- pha bantiana, and the unidentified Asco- tion time was extended for 24 h for strains mycete failed to grow well enough on of R. nigricans. Mycelial inocula produced Czapek solution agar to yield sufficient the same enzymes as spore suspensions, inocula for API ZYM tests and were there- although in smaller quantities. fore grown on potato dextrose agar and Results of enzymatic activities based malt extract and then tested for agar on identities of the isolates regardless of Other selected were enzymes. species the pollen source are shown in Tables III-VI. All 40 Penicillia tested produced and leucine aminopeptidase. One of the leucine aminopeptidase, acid phosphata- A. flavus strains tested was var. columna- se, phosphoamidase, and f3-glucosidase; ris and gave the same reactions as strains none produced cystine aminopeptidase, identified as A. flavus. Enzymes produced trypsin, a-galactosidase, f3-glucuronidase, in the highest concentrations (> 20 nmol) or a-mannosidase (Table 111). Most also by Aspergilli were N-acetyl-B-glucosamini- produced alkaline phosphatase, caprylate dase and fi-glucosidase by A. niger, alka- esterase-lipase, and N-acetyl-B-glucos- line phosphatase by A. flavus, and aminidase. Enzymes produced by Penicil- alkaline and acid phosphatases and B- lia in the highest concentrations (> 20 glucosidase by A. versicolor. nmol) were acid phosphatase by P. corylophilum, N acetyl-f3-glucosaminidase by All Murocales tested produced leucine P. cyclopium, and f3-glucosidase by P. aminopeptidase, and most produced crustosum. acid phosphatase and phosphoamidase All Aspergilli tested produced acid (Table V). Otherwise these molds pro- phosphatase, phosphoamidase, B-gluco- duced few enzymes. The only enzymes sidase, and N acetyl-f3-glucosaminidase; produced in high concentrations (> 20 none produced myristate lipase, trypsin, nanomoles) were phosphoamidase by all chymotrypsin, f3-glucuronidase, a-manno- strains of R. nigricans tested, acid phos- sidase, or a-fucosidase (Table IV). Most phatase by 3 of the 4 R. nigricans strains, also produced alkaline phosphatase, buty- and leucine aminopeptidase by Mucor rate esterase, caprylate esterase—lipase, racemosus. Of the other molds tested, most pro- dase by Alt. tenuis and T. sepedonium duced acid phosphatase and f3-glucosida- and by C. cladosporioides from 6-week se (Table VI). Alkaline phosphastase, bee bread; and a-fucosidase by C. cla- butyrate esterase, caprylate esterase— dosporioides from floral and corbicular lipase, leucine aminopeptidase, and pollen. phosphoamidase were produced by Enzymology of the 78 molds tested is 52—67% of them. Enzymes produced in presented in Table Vil on the basis of the the highest concentrations (> 20 nmol) by pollen sources of the isolates. No molds various molds are as follows : alkaline produced trypsin, f3-glucuronidase, or a- phosphatase by Aur. pullulans from one- mannosidase. Few produced myristate week bee bread; caprylate esterase—lip— lipase, and only one produced chymotryp- ase by C. herbarum from corbicular pollen sin; these isolates were from corbicular and by C. cladosporioides; leucine amino- pollen. Valine aminopeptidase and cystine peptidase by Aur. pullulans and Peyrone- aminopeptidase were not produced by lia sp.; acid phosphatase by Alt. tenuis, isolates from 3-week bee bread, nor was Arthrinium phaeospermum, Aur. pullu- the latter enzyme produced by molds from lans, C. cladosporioides, C. herbarum, corbicular or 6-week bee bread. Few Peyronelia sp., Scytalidium sp., and X. molds from other pollen sources produced bantiana; phosphoamidase by X. bantia- these two peptidases. Of the glycosi na; a-galactosidase by Aur. pullulans from dases, a-glucosidase and a-fucosidast. one-week bee bread and by C. cladospo- were not associated with molds from rioides from corbicular pollen; B-glucosi- either 3-week or 6-week bee bread and were produced by few isolates from other lum, P. crustosum, and R. nigricans. pollen sources. Both a- and B-galactosi- Conversely, Mucor sp., the dominant mold dases were produced by a higher percent in floral pollen, was eliminated in corbic- of molds tested from floral pollen than ular pollen and bee bread. Thus, as with from other pollen sources. No molds from yeasts (Gilliam, 1979a) and Bacillus ssp. corbicular pollen produced B-galactosi- (Gilliam, 1979b), the mold flora of corbic- dase. ular pollen and bee bread may be the However, most molds from all pollen result of microbial inoculations by bees and chemical in sources produced caprylate esterase- changes pollen resulting lipase, leucine aminopeptidase, acid from additions by bees from regurgitation phosphatase, phosphoamidase, B-gluco- of honey sac contents and secretions of sidase, and N acetyl-f3-glucosamidase. A glands as well as microbial fermentation high percent of the isolates (> 50%) from which allow some species but not others to survive. Even molds were more all sources gave positive reactions for though numerous than or Bacillus in alkaline phosphatase. This was also the yeasts ssp. our is case with butyrate esterase except for samples, pollen rarely overgrown by those molds from one-week bee bread. molds. Potential microbial spoilage of pol- Therefore, pollen molds produced len provisions may be controlled by anti- biotic substances the normal enzymes involved in protein, lipid, and produced by carbohydrate metabolism. microflora, bees, pollen, and/or honey. Klungness and Peng (1983) examined corbicular pollen and bee bread with scanning electron microscopy and found Discussion no visible evidence of digestion or dam- age to pollen grain walls. They concluded that microorganisms associated with bee Seventy percent of the molds identified bread do not cause destruction of pollen from almond pollen and bee bread were intine or the cytoplasm, that substances Aspergilli, Mucorales, and Penicillia. We bees add to pollen during collection and have previously isolated from apiarian storage function as a preservative, and sources in Arizona most of the species of that the regurgitation added to pollen Aspergilli and Penicillia found in pollen probably allows growth of some microor- and bee bread as well as Aur. pullulans, ganisms and inhibits the growth of others. C. claedosporioides, Mucorales, and They observed that the few fungal spores Peyronelia sp. (Gilliam and Prest, 1972, that germinated produced hyphae less 1977, 1987; Gilliam et al., 1974, 1977, than 10 pm in length. isolates in the 1988). Frequent present If microorganisms are responsible for study which are new records of molds fermentation and the accompanying from apiarian sources in Arizona are chemical changes of pollen stored in Alt. tenuis, Penicillium crustosum, and comb cells by honey bees, the molds may R. nigricans. be a component of the required microbial Molds which were not present in floral complement. They could contribute anti- pollen but were frequent isolates from cor- biotics, organic acids and enzymes, pro- bicular pollen and bee bread may have ducts for which they are utilized industrial- been introduced by the bees during col- ly. These compounds may limit the growth lection and storage. The most obvious of deleterious microorganisms and provi- examples are Aur. pullulans, P. corylophi- de enzymes for utilization of nutrients.

Indeed, we have found Aspergilli, Muco- ever, most other molds produced B-gluco- rales, Penicillia, and other molds in bee sidase which hydrolyzes carbohydrates bread and guts of worker bees which in- such as cellobiose, salicin, amygdalin, hibit the growth of the chalkbrood fungus and gentibiose. N Acetyl-f3-glucosamini- (Gilliam et aL, 1988). dase was also produced by most molds. This is involved in of Enzymology of our isolates revealed enzyme hydrolysis chitin. f3-Galactosidase which that the major phosphatase was acid hydrolyzes phosphatase, although alkaline phospha- lactose and a-glucosidase which hydro- and tase was produced by most isolates lyzes sucrose, maltose, trehalose, except the Mucorales. Molds apparently melizitose were not produced by most of the molds. are not able to participate in the initial breakdown of long-chain fatty acids as In summary, molds as normal micro- evidenced by the lack of myristate lipase. flora in pollen and bee bread have recei- However, they did produce butyrate es- ved little attention. However, our results rase and caprylate esterase—lipase indicate that because they represented which act on shorter chain fatty acids. 38% of the total number of microorga- Leucine aminopeptidase was the major nisms we isolated (Gilliam, unpublished aminopeptidase. Molds did not produce data), produced a variety of enzymes, and trypsin or chymotrypsin. Phosphoamida- are well known for production of seconda- se was produced by most isolates tested. ry metabolites such as antibiotics, pheno- Results for glycosidases revealed that lic compounds, terpenes, steroids, and Mucorales were quite unreactive. How- polysaccharides as well as enzymes, they merit more intensive study. Even if mold Mycological Papers, No. 83, Commonwealth spores that germinate in corbicular pollen Mycological Institute, Kew, England and bee bread produce short hyphae Bridge P.D. & Hawksworth D.L. (1984) The API and our results ZYM enzyme testing system as an aid to the (Klungness Peng, 1983), identification of Penicillium isolates. with selected isolates of various rapid genera Microbiol. Sci. 1, 232-234 confirmed those with Penicillia by Bridge Burnside C.E. asso- and Hawksworth that ino- (1927) Saprophytic fungi (1984) mycelia ciated with the honey bee. Mic;1. Acad. Sci. 8, cula produce th, same enzymes, 59-86 although in reduced amounts, as spore Burri R. (1947) Die Beziehungen der Bakterien suspensions of the same strain. With this zum Lebenszyklus der Honigbiene. Schweiz. publication and those on yeasts (Gilliam, Bienen-Ztg. 70, 273-276 1979a) and Bacillus ssp. (Gilliam, 1979b), Chevtchik V. (1950) Mikrobiologie pylov6ho we have now reported 77% of the total kvaseni. Publ. Fac. Sci. Univ. Masaryk 323, 103-130 isolates from our almond pollen and bee bread samples. The remaining microorga- Cooke W.B. (1959) An ecological life history of Aureobasidium Arnaud. nisms will be described in a future pullulans (deBary) publi- 12, 1-45 cation. Mycopathologia DeVries G.A. (1952) Contribution to the Know- lege of the Genus Cladosporium Link ex Fr. Centraalbureau voor Schimmelcultures, Baarn, The Netherlands Acknowledgments Dunham W.E. (1929) The relation of external temperature on the hive temperature during the summer. J. Econ. Entomol. 798-801 We thank Dr. L.N. Standifer of the Carl Hayden 22, Bee Research Center for the pollen samples, Egorova A.I. (1971) Preservative microflora in Dr. James T Sinski of the University of Arizona stored pollen. Veterinariya 8, 40-41 for providing scanning electron microscopy and Ellis M.B. (1965) Dematiaceous Hyphomy- consultation on the unidentified Ascomycete, cetes. Vi. Mycological Papers, No. 103, Com- and Drs. E.W. Herbert Jr., D.R. Jimenez, and monwealth Mycological Institute, Kew, England J.D. Vandenberg for reviewing the manuscript. Foote H.L. (1957) Possible use of microorganisms in synthetic bee bread production. Am. Bee J. 97, 476-478 Gilliam M. of and References (1979a) Microbiology pollen bee bread : the yeasts. Apidologie 10, 43-53 Gilliam M. (1979b) Microbiology of pollen and Alexopoulos C.J. (1962) Introductory Myco- bee bread : the genus Bacillus. Apidologie 10, logy. John Wiley and Sons, New York 269-274 Ames L.M. (1961) A monograph of the Chaeto- Gilliam M. & Prest D.B. (1972) Fungi isolated miaceae. US Army Res. Div. Ser. No. 2 from the intestinal contents of foraging worker honey bees, Apis mellifera. J. Invertebr. Arizan D., Popa A., Mitroiu P., Toma C., Serban Pathol. 20, 101-103 M., Crisan 1. & Dobre V. (1967) Conservation Gilliam M. & Prest D.B. 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