Interrelation of growth media and water activity in sclerotia characteristics of Aspergillus section Flavi

AIMS: To examine sclerotium characteristics of two Aspergillus flavus and two A. parasiticus strains at different growth media and water stress. METHODS AND RESULTS: The effects of growth media and water activity (0.999, 0.971, 0.955 and 0.937) on characteristics of sclerotia production (number, size and volume) of four isolates of Aspergillus section Flavi were examined. There was total inhibition under the driest conditions (0.955 and 0.937). When an osmotic potential of 0.971 was generated in Czapek agar (CD) and maize meal extract agar with sucrose and sodium nitrate (MMEA S/N), an increase in sclerotial size and volume was observed. The amount of sclerotia produced by cultures at 0.999 a(w) value was higher on CD. CONCLUSIONS: The data show that the sclerotia characteristics of A. flavus and A. parasiticus have been influenced by water availability and growth media composition. SIGNIFICANCE AND IMPACT OF THE STUDY: The information obtained shows that if we know the nutritional and water stress requirements for sclerotia production, it could be possible to develop effective prevention strategies to inhibit the survival of these fungi in grain.     

Factors Influencing Formation of Sclerotia in Grifola umbellate （Pers.） Pilaet Under Artificial Conditions

The effects of substrate composition and temperature on myceilal growth and sclerotium production in Grlfola umbellate （Pers.） Pilaet were Investigated In the present study. The Induction of sclerotla of G. umbellate was affected greatly by the type of medium, as well as the type of carbon source. Malt-extract agar was able to induce the production of sclerotia. The production of sclerotia was also observed when the carbon source in the GPC agar medium （glucose 20 g/L, peptone 6 g/L, corn steep liquor 10 g/L, and agar 15 g/L） was replaced with glycerol or mannitol. Altering the composition of the GPC medium with milk powder, thiamine hydrochlorlde, extract of Armlllarla mellea, active clay, dlatomite, kaolin, or arginlne did not induce the production of sclerotla. A temperature range of 18-25 ℃ was suitable for both mycellai growth and sclerotium formation. Glycerol significantly Induced slerotium formation on nutrient supplemented with sawdust substrates In bottle culture. 24S-Polyporusterone A and polyporusterone B were assayed In samples of natural and cultured sclerotla. Both natural and cultured sclerotla contained 24S- polyporusterone A and polyporusterone B.     

Reserve substances inPaxillus involutus sclerotia

Summary The ectomycorrhizal fungus,Paxillus involutus, produces sclerotia in culture. These can be induced to form on agar medium by exposing mycelium grown at 25°C to various temperatures between6°C and 15°C. Sclerotia formed at 10°C and above were large and covered with drops of exudate, while those formed at 6°C or 8°C were very small and did not produce an exudate. Mature sclerotia were bounded by a compact rind and contained abundant storage reserves. Histochemistry of the larger sclerotia showed large quantities of protein stored as protein bodies in the cytoplasm, lipid present as small droplets, glycogen granules stored in the cytoplasm and polyphosphate present as small granules in the cytoplasm and in the protein bodies. Energy dispersive X-ray microanalysis confirmed the presence of phosphate in the granules and was used to map its distribution throughout the sclerotium. The smaller sclerotia induced at 8°C and below on the same medium had the same basic structure and composition, but lacked the complex phenolic cell network found in large sclerotia, and had abundant extracellular polysaccharides. The rind was not well developed and these small sclerotia are interpreted to have been arrested at an early stage of development.     

Production of phytase under solid-state fermentation using Rhizopus oryzae: novel strain improvement approach and studies on purification and characterization

Present study introduces linseed oil cake as a novel substrate for phytase production by Rhizopus oryzae. Statistical approach was employed to optimize various medium components under solid state fermentation (SSF). An overall 8.41-fold increase in phytase production was achieved at the optimum concentrations (w/w, mannitol, 2.05%; ammonium sulfate, 2.84% and phosphate, 0.38%). Further enhancement by 59% was observed due to a novel strain improvement approach. Purified phytase (～34 kDa) showed optimal temperature of 45 °C, dual pH optima at 1.5 and 5.5 and possesses high catalytic efficiency (2.38×10(6) M(-1) s(-1)). Characterization study demonstrates the phytase as highly thermostable and resistant to proteolysis, heavy metal ions, etc. Furthermore, an improved HPLC method was introduced to confirm the ability of phytase to degrade phytic acid completely and was found to be an efficient method.     

Enhancement of sporulation in species of Bipolaris, Curvularia, Drechslera, and Exserohilum by growth on cellulose-containing substrates

Nine species of Bipolaris, Curvularia, Drechslera, and Exserohilum were compared for sporulation on agar media and for enhancement of sporulation by growth on four cellulose-containing substrates (index card, filter paper, cheesecloth, cotton fabric). On two natural and one synthetic agar media, sporulation varied from profuse to nonexistent among three isolates of each species. Growth of all species on cellulose substrates resulted in large and significant increases in sporulation. Growth on index card pieces often provided the greatest increases, but no single substrate was superior for all species, and significant substrate × isolate interactions were observed within species. Overlay of filter paper onto whole colonies in agar plates resulted in 2 to 18-fold increases in sporulation for eight of nine species and production of spores in sufficient quantity for most experimental purposes. Overlay of soil dilution plates with filter paper to promote sporulation of colonies enabled detection of B. spicifera, B. hawaiiensis, C. lunata, and E. rostratum at relatively low population levels (≤1.3 × 10³ colony-forming units per gram of soil) in samples of a naturally infested soil. Results indicate that enhancement of sporulation by growth of species of Bipolaris, Curvularia, Drechslera, and Exserohilum on cellulose substrates may facilitate (i) their identification in culture, (ii) production of spores at relatively high concentrations, and (iii) detection and enumeration of these fungi in soil.     

Factors affecting germination, mycoparasitism, and survival of Sporidesmium sclerotivorum.

Macroconidia of Sporidesmium sclerotivorum, a mycoparasite of Sclerotinia spp., germinated after 3 days in soil adjacent to sclerotia of S. minor and on membrane filters placed on soil containing sclerotia. Germination increased with time up to 18 days and with concentration of sclerotia. Conidia as distant as 9 mm from single sclerotia germinated. Germination of conidia was maximum on a sclerotial agar medium in the range of pH 5 to pH 7. Cultivation of S. sclerotivorum parasitically on living sclerotia proceeded optimally in moist, fine quartz sand amended with 1 to 2% (w/w) sclerotia and 0.07% (w/w) CaCO3, at 25 degrees C. Infection of sclerotia in sand reached 100% by 5 weeks. Conidia production paralled infection resulting in logarithmic increase in numbers; a maximum of 3 x 10(5) to 4 x 10(5) conidia/g was reached in 6 to 12 weeks. Viability of air-dried sand-sclerotial cultures of S. sclerotivorum was reduced after 1 and 6 days, but viability was undiminished in air-dried soil. Sporidesmium sclerotivorum survived in moist and air-dried soils stored at room temperature for 15 months.     

THE EFFECT OF SOME PHYSIOLOGICAL AND ENVIRONMENTAL FACTORS ON SCLEROTIAL ASPERGILLI

Rudolph , Emanuel D. (Ohio State U., Columbus.) The effect of some physiological and environmental factors on sclerotial Aspergilli. Amer. Jour. Bot. 49(1): 71–78. Illus. 1962.—The effect of varying conditions of carbon-nitrogen balance, temperature, pH, and light upon the formation of sclerotia by 6 species of Aspergillus (A. alliaceus, A. avenaceus, A. flavus, A. quercinus, A. sclerotiorum and A. wentii) was studied. On Czapek's agar, optimal growth as well as maximum production of sclerotia and conidia took place at high sucrose and nitrate concentrations. In general, fewer sclerotia were formed with glucose than with sucrose, and very poor growth took place with lactose. Sclerotia were formed best at temperatures that were optimal or below optimal for mycelial growth. The ranges of pH through which sclerotia were formed were narrower than those through which conidia and mycelia were formed. Light had no effect upon sclerotium formation. The formation of sclerotia in A. alliaceus was found to represent the strand-type development. A number of UV-induced strains and a spontaneous mutant strain of A. alliaceus showing varying amounts of sclerotium and conidium production are characterized. It is suggested that the sclerotia in Aspergillus are sterile stromata.     

The role of a fadA ortholog in the growth and development of Colletotrichum graminicola in vitro and in planta

A transposon-based split-marker protocol was used to produce insertional mutations in the fadA ortholog of the maize anthracnose pathogen Colletotrichum graminicola. The mutants grew more slowly in culture, produced fewer oval spores, produced fusiform rather than falcate phialospores, lost their normal clockwise spiral growth pattern in culture, and were significantly reduced in their pathogenicity to maize stalks and leaves. The differential effect of the fadA mutation on oval spore versus phialospore production suggests that there are differences in the signaling pathways that regulate these two types of sporulation. It has been suggested that oval spores function in anthracnose lesion extension. In maize stalks, production of oval spores appeared to be relatively unaffected in the mutant strains, but production of vegetative hyphae and elongation of primary lesions were both reduced. This suggests that vegetative hyphae play a more important role than oval spores in primary lesion development. However, production of discontinuous secondary lesions in maize stalks infected by mutant strains did not appear to be seriously affected, and thus oval spores may play a more important role in that process.     

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.     

暗褐网柄牛肝菌菌核形成相关基因分析

【背景】暗褐网柄牛肝菌(Phlebopus portentosus)是第一个能够人工栽培的食用牛肝菌,人工栽培过程中,不同菌株会形成数量不等的菌核。【目的】探明不同菌株产核差异机制。【方法】采集多菌核(JH1)、寡菌核(JH2)菌株的成熟菌核及无菌核(JH3)菌株培养相同时间的菌丝体进行转录组测序,分析差异表达基因对菌核形成的作用和功能。【结果】KEGG富集分析显示,JH2 vs. JH1互比,苯丙氨酸、酪氨酸和色氨酸的生物合成,精氨酸和脯氨酸代谢,半胱氨酸及蛋氨酸代谢显著富集;JH3 vs. JH1互比,乙醛酸和二羧酸代谢显著富集;JH3 vs. JH2互比,谷胱甘肽、乙醛酸和二羧酸代谢显著富集。菌核形成相关基因分析显示,从JH2 vs. JH1、JH3 vs. JH1和JH3 vs. JH2的差异表达基因中分别筛选到69、118和82条与信号转导、感知刺激、防御、碳水化合物活性酶等有关的基因,其中碳水化合物活性酶基因数量最多。三个比较组共有的碳水化合物活性酶基因在JH1中的表达量高于JH2、JH3,表明JH1更能充分利用底物营养以形成更多菌核。【结论】本研究从转录组水平初步分析了暗...     

Intrafungal distribution of aflatoxins among conidia and sclerotia of Aspergillus flavus and Aspergillus parasiticus

This research examines the distribution of aflatoxins among conidia and sclerotia of toxigenic strains of Aspergillus flavus Link and Aspergillus parasiticus Speare cultured on Czapek agar (21 days, 28 degrees C). Total aflatoxin levels in conidia and sclerotia varied considerably both within (intrafungal) and among strains. Aspergillus flavus NRRL 6554 accumulated the highest levels of aflatoxin (conidia: B1, 84000 ppb; G1, 566000 ppb; sclerotia: B1, 135000 ppb; G1, 968000 ppb). Substantial aflatoxin levels in conidia could place at risk those agricultural workers exposed to dust containing large numbers of A. flavus conidia. Cellular ratios of aflatoxin B1 to aflatoxin G1 were nearly identical in conidia and sclerotia even though levels of total aflatoxins in these propagule types may have differed greatly. Aflatoxin G1 was detected in sclerotia of all A. flavus strains but in the conidia of only one strain. Each of the A. parasiticus strains examined accumulated aflatoxin G1 in both sclerotia and conidia. These results are examined in the context of current evolutionary theory predicting an increase in the chemical defense systems of fungal sclerotia, propagules critical to the survival of these organisms.     

Survival of Escherichia coli O157:H7 in broth and processed salami as influenced by pH, water activity, and temperature and suitability of media for its recovery.

The survival of unheated and heat-stressed (52 degrees C, 30 min) cells of Escherichia coli O157:H7 inoculated into tryptic soy broth (TSB) adjusted to various pHs (6.0, 5.4, and 4.8) with lactic acid and various water activities (a(w)s) (0.99, 0.95, and 0.90) with NaCl and incubated at 5, 20, 30, and 37 degrees C was studied. The performance of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), and modified eosin methylene blue agar in supporting colony development of incubated cells was determined. Unheated cells of E. coli O157:H7 grew to population densities of 10(8) to 10(9) CFU ml-1 in TSB (pHs 6.0 and 5.4) at an a(w) of 0.99. Regardless of the pH and a(w) of TSB, survival of E. coli O157:H7 was better at 5 degrees C than at 20 or 30 degrees C. At 30 degrees C, inactivation or inhibition of growth was enhanced by reduction of the a(w) and pH. A decrease in the a(w) (0.99 to 0.90) of TSB in which the cells were heated at 52 degrees C for 30 min resulted in a 1.5-log10 reduction in the number of E. coli O157:H7 cells recovered on TSA; pH did not significantly affect the viability of cells. Recovery was significantly reduced on MSMA when cells were heated in TSB with reduced pH or a(w) for an increased length of time. With the exception of TSB (a(w), 0.90) incubated at 37 degrees C, heat-stressed cells survived for 24 h in recovery broth. TSB (a(w), 0.99) at pH 6.0 or 5.4 supported growth of E. coli O157:H7 cells at 20 or 37 degrees C, but higher numbers of heated cells survived at 5 or 20 degrees C than at 37 degrees C. The ability of unheated and heat-stressed E. coli O157:H7 cells to survive or grow as affected by the a(w) of processed salami was investigated. Decreases of about 1 to 2 log10 CFU g-1 occurred soon after inoculation of salami (pHs 4.86 and 4.63 at a(w)s of 0.95 and 0.90, respectively). Regardless of the physiological condition of the cells before inoculation into processed salami at an a(w) of either 0.95 or 0.90, decreases in populations occurred during storage at 5 or 20 degrees C for 32 days. If present at < or = 100 CFU g-1, E. coli O157:H7 would unlikely survive storage at 5 degrees C for 32 days. However, contamination of salami with E. coli O157:H7 at 10(4) to 10(5) CFU g-1 after processing would pose a health risk to consumers for more than 32 days if storage were at 5 degrees C. Regardless of the treatment conditions, performance of the media tested for the recovery of E. coli O157:H7 cells followed the order TSA > modified eosin methylene blue agar > MSMA.     

Laboratory bioassays of entomopathogenic fungi for control of Delia radicum (L.) larvae

Laboratory soil bioassays were performed at economic field rates for in-furrow (3.85 x 10(6)spores/g dry soil) and broadcast (3.85 x 10(5)spores/g dry soil) applications with three isolates of Metarhizium anisopliae (F52, ATCC62176, and ARSEF5520) and one isolate of Beauveria bassiana (GHA). All isolates tested were infective to second instar Delia radicum (L.). The conditionally registered M. anisopliae isolate (F52) performed best killing an average of 85 and 72% of D. radicum larvae at the high and low concentration, respectively. The mean LC50 and LC95 of F52 against second instar D. radicum was 2.7 x 10(6) and 1.8 x 10(8)spores/g dry soil, respectively. The use of F52 in an integrated management program is discussed.     

Glycoconjugates for the recognition of Bacillus spores

Carbohydrates act as ligands in many biological processes, including the folding and secretion of proteins, cell-cell recognition, adhesion, and sporulation in the Bacillus genus. Fluorescent-labeled disaccharide glycoconjugates have been applied to evaluate binding to bacterial spores assuming that the spore surface is covered with carbohydrates. This study has shown that specific recognition of bacterial spores is based on interactions between disaccharide glycoconjugates acting as ligands and monosaccharide units expressed on the exterior of bacterial spores. Using fluorophore-assisted carbohydrate electrophoresis (FACE), carbohydrates that are expressed on the exterior of the spores were enumerated. The findings have an impact on how to improve ligand selection, essential for sensor development. In addition, the findings provide new information for inhibition of bacterial spores, and in general, demonstrate how carbohydrates function as recognition signals in nature.     

Rapid sporulation of Bacillus anthracis in a high iron, glucose-free medium

Spores are the infectious form of Bacillus anthracis (BA), causing cutaneous, inhalation and gastrointestinal anthrax. Because of the possible use of BA spores in a bioterrorism attack, there is considerable interest in studying spore biology. In the laboratory, however, it takes a number of days to prepare spores. Standard sporulation protocols, such as the use of ‘PA broth’, allow sporulation of BA to occur in 3 to 5 days. Another method employs growth of BA on plates in the dark for several days until they have efficiently sporulated. In efforts to determine the effect of iron on gene expression in BA, we grew BA Sterne strain 7702 in a minimal defined medium (CDM; Koppisch et al., 2005) with various concentrations of iron and glucose. As part of our initial observations, we monitored BA sporulation in CDM via light microscopy. In glucose-free CDM containing 1.5 mM Fe(NO₃)₃ (CDM-Fe), > 95% of the BA sporulated by 30 h; a far shorter time period than expected. We pursued this observation and we further characterized spores derived from PA and CDM-Fe media. Purified spores derived from PA or CDM-Fe had similar morphologies when viewed by light or electron microscopy, and were equally resistant to harsh conditions including heat (65 °C), ice and fresh 30% H₂O₂. Spore viability in long term cold storage in water was similar for the two spore preparations. Extracted spore coat proteins were evaluated by SDS-PAGE and silver staining, which revealed distinct protein profiles for PA and CDM-Fe spore coat extracts. ELISA assays were done to compare the interaction of the two spore preparations with rabbit antiserum raised against UV-killed Sterne strain 7702 spores prepared in PA medium. Spores from both media reacted identically with this antiserum. Finally, the interaction and fate of spores incubated with macrophages in vitro was very similar. In summary, BA spores induced in CDM-Fe or in PA medium are similar by several criteria, but show distinct extractable coat proteins. CDM-Fe liquid medium can be used for rapid production of BA spores, and could save considerable time in spore research studies.     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

Application of a simple yeast extract from spent brewer's yeast for growth and sporulation of Bacillus thuringiensis subsp. kurstaki: a Physiological Study

The suitability of using a simple brewer's yeast extract (BYE), prepared by autolysis of complete beer slurry, for growth and sporulation of Bacillus thuringiensis kurstaki was studied in baffled shake flasks. In a standard buffered medium with 2.5% (w/v) glucose and 1% (w/v) brewer's yeast extract, growth of B. t. kurstaki resulted in a low biomass production with considerable byproduct formation, including organic acids and a concomitant low medium pH, incomplete glucose utilization and marginal sporulation, whereas growth in the same medium with a commercial laboratory-grade yeast extract (Difco) resulted in a high biomass concentration, complete glucose utilization, relatively low levels of byproducts and complete sporulation (2.6 × 10⁹ spores/ml). When glucose was left out of the medium, however, growth parameters and sporulation were comparable for BYE and commercial yeast extract, but absolute biomass levels and spore counts were low. Iron was subsequently identified as a limiting factor in BYE. After addition of 3 mg iron sulphate/l, biomass formation in BYE-medium more than doubled, low byproduct formation was observed, and complete sporulation occurred (2.8 × 10⁹spores/ml). These data were slightly lower than those obtained in media with commercial yeast extract (3.6 × 10⁹spores/ml), which also benefited, but to a smaller extent, from addition of iron.     

Incubation period, spore egestion and horizontal transmission of Nosema fumiferanae (Microsporidia: Nosematidae) in spruce budworm (Choristoneura sp., Lepidoptera: Tortricidae): the role of temperature and dose

Various instars of Choristoneura occidentalis were fed with a range of doses of Nosema fumiferanae and reared at 20, 24 and 28 degrees C to determine the influence of temperature and dose on the time to spore egestion and the number of spores egested in the frass. When larvae were fed in the third stadium, as few as 10(2) spores per larva initiated infection, and both onset of spore egestion and the number of spores egested were affected by a complex relationship between temperature and inoculation dose. Onset of spore egestion varied from 11 to 15 days postinoculation. At 20 degrees C, the onset was delayed and spore production decreased with increasing inoculation dose whereas at higher temperatures spores were first egested at the lowest dose and spore production increased with dose. When larvae were fed spores in the fifth and sixth stadium, no spores were egested because pupation occurred before completion of the incubation period. To assess the effect of temperature on horizontal transmission, Choristoneura fumiferana larvae fed with 10(4) N. fumiferanae spores per larva were reared with uninfected larvae at 15, 20 and 25 degrees C. At 15 degrees C, we observed the highest degree of horizontal transmission, defined by the largest change in N. fumiferanae prevalence, even though the density of spores available for horizontal transmission was the lowest. Infected adults eclosed later than uninfected adults and the time to eclosion was also dependent on sex and temperature. We relate our experimental findings to consequences for horizontal and vertical transmission of N. fumiferanae in spruce budworm populations.     

Specific and sensitive detection of Nosema bombi (Microsporidia: Nosematidae) in bumble bees (Bombus spp.; Hymenoptera: Apidae) by PCR of partial rRNA gene sequences

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees. Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N. bombi (323bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 10(7) down to 10 spores diluted in 100 microl of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 10(6) spores down to 1 spore per PCR). Though the N. bombi-specific primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi-non-specific primer pair ITS-f2/r2, which amplifies a short fragment of approximately 120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to be more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy.