Virulence of Hypocreales fungi to pecan aphids (Hemiptera: Aphididae) in the laboratory

There is need for efficacious biocontrol agents for aphids in commercial orchards. As a preliminary step to this end we determined the virulence of several Hypocreales fungi to pecan aphids. In the first experiment we tested the virulence of Isaria fumosorosea (ARSEF 3581) blastospores to three pecan aphids Monellia caryella, Melanocallis caryaefoliae, and Monelliopsis pecanis under laboratory conditions. Rates of 1 × 10⁷ or 1 × 10⁸ spores per ml were applied in 2 ml via a spray tower to 90 mm Petri dishes containing 10 aphids each. Mortality and mycosis were determined after 24, 48 and 72 h. Treatment effects were observed by 48 h post-application, and by 72 h the higher application rate caused >90% mortality and mycosis in M. caryella and M. caryaefoliae, whereas <70% was observed in M. pecanis.We conducted two subsequent experiments (Experiments 2 and 3), using the same methodology, to compare the virulence of several Hypocreales species and strains against the aphid of primary economic concern to most pecan growers, M. caryaefoliae. In Experiment 2, we compared blastospores and conidia of two I. fumosorosea strains (ARSEF 3581 and ATCC 20874 [= strain 97]). The blastospores of ARSEF 3581 and conidia of ATCC 20874 showed higher virulence than other treatments and thus were included in Experiment 3, which also compared the virulence of conidia of Beauveria bassiana (GHA strain) and Metarhizium anisopliae (F52 strain). Results in Experiment 3 indicated the highest virulence in I. fumosorosea 3581 blastospores and M. anisopliae (F52) followed by I. fumosorosea (20874) conidia. The detection of pathogenicity to pecan aphids establishes the potential for commercial usage and additional study. Results reported here will narrow treatments to test in future greenhouse and field trials.     

Relationship between virulence and repellency of entomopathogenic isolates of Metarhizium anisopliae and Beauveria bassiana to the termite Macrotermes michaelseni

Termites encounter a diverse array of potentially useful and harmful fungi in their subterranean habitats. These vary from symbiotic to harmful species with varying levels of virulence. How these hemiedaphic insects survive in habitats with infective fungi is not well understood. Possible mediation of olfactory signals in avoiding contact with entomopathogenic fungi has been explored by a number of workers. In the present study, we initially found that Macrotermes michaelseni detected a virulent isolate of Metarhizium anisopliae from some distance and avoided direct physical contact. We hypothesized that there may be a relationship between virulence and repellency of different isolates of M. anisopliae and Beauveria bassiana to the termite. We compared these for selected isolates of the two fungi. Positive correlations between the two parameters for both sets of isolates of the fungi were obtained. The results show an interesting co-evolutionary phenomenon in which the termite's response to either M. anisopliae or B. bassiana is directly related to potential harm these fungi can inflict on the insect and that the virulent strains are more likely to be recognized from some distance and avoided.     

Increased pathogenicity against coffee berry borer, Hypothenemus hampei (Coleoptera: Curculionidae) by Metarhizium anisopliae expressing the scorpion toxin (AaIT) gene

Coffee berry borer (CBB) is the Worlds most devastating coffee pest causing an estimated US$500 million worth of losses annually through damage and control costs. Beauveria bassiana and Metarhizium anisopliae have been employed to control this pest but their low virulence (slow kill and large inoculums) is an important factor constraining their use. M. anisopliae (AaIT-Ma549) has been modified to express the scorpion toxin (AaIT) in insect hemolymph and this greatly increased pathogenicity against Manduca sexta and Aedes aegypti. Here, we demonstrate that AaIT-Ma549 was also dramatically more virulent against CBB, and we provide a much more comprehensive analysis of infection processes and post-mortality development than in the previous research. We evaluated several spore concentrations (10¹ through 10⁷ spores/ml) of both the wild type and recombinant strain. At concentrations of 10¹, 10² and 10³ spores/ml, the recombinant strain significantly increased mortality of CBB by 32.2%, 56.6% and 24.6%, respectively. The medial lethal concentration (LC₅₀) was reduced 15.7-fold and the average survival time (AST) was reduced by 20.1% to 2.98 ± 0.1 days with 10⁷ spores/ml. This is the first occasion that an entomopathogenic fungus has been found to kill CBB in less than 3 days. However, AaIT-Ma549 produces significantly fewer spores on cadavers than the parental strain.     

Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi Beauveria bassiana and Metarhizium anisopliae

The effects of nutrient medium composition and temperature on the germination of conidia of the fungi Beauveria bassiana (strain AlG) and Metarhizium anisopliae (strain M-99) and their entomopathogenic activity have been studied. It was demonstrated that the presence of carbohydrates alone was sufficient for the spores of M. anisopliae M-99 to germinate, whereas the germination of B. bassiana AlG spores was inhibited by carbohydrates. Addition of KJ, ZnSO₄, or KBr into the Czapek medium increased the entomopathogenic activity of B. bassiana. The optimum temperature for spore germination was 20–35°C in both fungal species.     

Comparative virulence of Beauveria bassiana isolates against lepidopteran pests of vegetable crops

Forty-three isolates of the entomopathogenic fungus Beauveria bassiana were screened for virulence against second-instar larvae of diamondback moth (Plutella xylostella) (DBM), European corn borer (Ostrinia nubilalis) (ECB), corn earworm (Helicoverpa zea) (CEW), and fall armyworm (Spodoptera frugiperda) (FAW); 30 of these isolates were tested against beet armyworm (Spodoptera exigua) (BAW). Highly virulent isolates were also tested against black cutworm (Agrotis ipsilon) (BCW), and the most virulent isolate was also assayed against imported cabbage worm (Pieris rapae) (ICW) and cabbage looper (Trichoplusia ni) (CL). All lepidopteran species tested were susceptible to B. bassiana. Corn earworm and beet armyworm were most susceptible to fungal infection, and fall armyworm was least susceptible. Limited testing suggested low susceptibility of black cutworm and cabbage looper. B. bassiana isolate 1200 exhibited virulence against all pest species greater than or equal to commercial strain GHA of B. bassiana currently registered in the USA as BotaniGard®. In assays in which larvae were topically sprayed and maintained on the treated substrate for 24 h at 100% relative humidity, 6-day (25 °C) median lethal concentrations (LC₅₀s) of this isolate against CEW, BAW, DBM, FAW, ICW, ECB, CL, and BCW were 4, 5, 7, 11, 12, 98, 125, and 273 conidia/mm², respectively. The respective LC₅₀s of commercial strain GHA against these pest species were 9, 67, 97, 1213, 29, 1668, 541, and 3504 conidia/mm². Use of LC₅₀ versus median lethal concentration ratios (comparing LC₅₀s of each isolate to a “standard” strain) generated similar rankings of isolate virulence. Results from parametric ANOVAs of log LC₅₀ values followed by Tukey HSD multiple comparisons tests and those from Kruskal-Wallis nonparametric analyses followed by sequential Bonferroni tests for means comparisons were nearly identical.     

Inhibition of Metarhizium anisopliae in the alimentary tract of the eastern subterranean termite Reticulitermes flavipes

Reticulitermes flavipes workers were individually inoculated with 10,000 conidia of the entomopathogenic fungus Metarhizium anisopliae. After being kept in groups of 20 individuals for 1-6 d, histopathological approach showed that most of the inoculated conidia were groomed from the surface of the cuticle by nestmates within 24 h, and that a large number of conidia was subsequently found in different parts of the gut of the groomers. Our observations showed that, among thousands of conidia found in the termite’s gut, conidial germination never occurred in all inspected specimens, even when the conidia had the chance to bind to the surface of the cuticular lining of the gut. In addition, when termites were left for decomposition several days after death caused by an external infection of M. anisopliae, the hyphal growth was generalized in the body cavity of the cadaver, but was never observed in the lumen of the gut even 2 d post-mortem. Our observation suggests that the putative biochemicals involved in the termite’s gut defense against fungal pathogens are from multiple origins.     

Entomopathogenic fungi of Eurygaster integriceps Puton (Hemiptera: Scutelleridae): collection and characterization for development

Exploratory activities were done in Syria, Turkey, Iran, Uzbekistan, Kazakhstan, The Kyrghyz Republic, and Russia to locate entomopathogenic fungi of Eurygaster integriceps. Isolates from the entomopathogenic genera Beauveria, Paecilomyces, and Verticillium were collected. Beauveria bassiana was the most commonly recovered species. Thirty-one isolates of the 221 recovered were examined at 20, 25, 30, and 35 °C for 20 days for growth and sporulation. Growth and sporulation were generally highest at 25 °C. None of the isolates grew at 35 °C, and at 30 °C growth was retarded with no conidia being produced. Single- and multiple-concentration bioassays were conducted on greenhouse-grown wheat plants and in pine litter to evaluate virulence of fungi from several sources to E. integriceps. When tested at a single concentration, mortality after 15 days ranged from 66 to >95% in the litter assays and 50 to 91% in the plant assays. There was a distinct concentration response for most of the isolates tested in the multiple-concentration assay, particularly in the in-litter environment. In litter, mortality tended to develop earlier than in on-plant assays. Several isolates of B. bassiana and one Metarhizium anisopliae displayed consistently high virulence against E. integriceps and were more virulent than two commercial strains. Our results demonstrate the potential of entomopathogenic fungi for management of E. integriceps in overwintering sites and in wheat fields.     

西花蓟马高毒力虫生真菌筛选及其生防应用潜力的研究进展

西花蓟马是一种外来入侵的世界性害虫,对农林业危害巨大。查阅国内外相关文献,综述了当前防治西花蓟马的虫生真菌的种类、高毒力菌株的筛选及防治现状。现已知西花蓟马的寄生病原真菌有5种,包括蜡蚧轮枝菌(半知菌:丝孢目)、球孢白僵菌(半知菌:丝孢目)、金龟子绿僵菌(半知菌:丝孢目)、玫烟色棒束孢(半知菌:束梗孢目)和小孢新接霉。其中,球孢白僵菌、金龟子绿僵菌在西花蓟马的生物防治中应用最广,具有良好的开发应用潜力,部分防效好的虫生真菌已申请专利及实现工厂化生产。     

Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface

Conidial germination and the formation of appressoria are important events in the interactions between entomopathogenic fungi and their arthropod hosts. In this study, we demonstrate the effects of lipids extracted from tick epicuticle and the surface of a mammalian host (calf) on conidial germination and the development of appressoria in two subspecies of Metarhizium anisopliae, M. anisopliae var. anisopliae (M.an.an.-7) and M. anisopliae var. acridum (M.an.ac.-5), which have different levels of virulence toward ticks. Pentane extracts of epicuticles of ticks susceptible and resistant to fungal infection always stimulated the germination of M.an.an.-7 conidia and the development of their appressoria; whereas the effects of dichloromethane (DCM) extracts of tick epicuticle varied depending on the tick. The DCM extracts from most of the tick species and developmental stages stimulated conidial germination and/or the formation of appressoria in M.an.an.-7. However, a DCM extract of lipids from the most resistant tick, engorged Hyalomma excavatum female, inhibited the germination of M.an.an.-7 conidia. Conidia of the non-virulent M.an.ac.-5 did not germinate on agarose amended with any of the examined tick extracts. However, when the tick extracts were placed on bactoagar, conidial germination increased 7- to 8-fold. Extracts from the skin, hair and ear secretions of a calf stimulated conidial germination and the formation of appressoria in M.an.an.-7, but not M.an.ac.-5. This study demonstrates that lipids from tick epicuticles and mammalian skin selectively affect the germination of conidia of entomopathogenic fungi. The effects of these lipids may explain the variability in tick control these fungi provide for different hosts.     

Entomopathogenic fungi in cornfields and their potential to manage larval western corn rootworm Diabrotica virgifera virgifera

Entomopathogenic ascomycete fungi are ubiquitous in soil and on phylloplanes, and are important natural enemies of many soil-borne arthropods including larval western corn rootworm, Diabrotica virgifera virgifera, which is a major pest of corn. We measured the prevalence of Beauveria bassiana and Metarhizium anisopliae sensu lato in ten cornfields in Iowa, USA by baiting with larval insects. B. bassiana and M. anisopliae s.l. were present in 60% ± 6.3% and 55% ± 6.4% of soil samples, respectively. Subsequent laboratory bioassays found that some M. anisopliae s.l. strains collected from cornfields killed a greater proportion of D.v. virgifera larvae than a standard commercial strain.     

Soil properties affect the availability, movement, and virulence of entomopathogenic fungi conidia against puparia of Ceratitis capitata (Diptera: Tephritidae)

The Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae), is the major tephritid pest in the Mediterranean region. This insect may overwinter as pupae inside fruits or in soil. Therefore, infection with entomopathogenic fungi is a potentially useful control technique during the insect’s soil-dwelling stage. Entomopathogenic fungi have an important role in Integrated Pest Management programs as an alternative to conventional chemical control, but they have been usually selected on the basis of laboratory results with little regard to fungal ecology. In this work, we designed several experiments to study the availability and movement of the EF Beauveria bassiana (Balsamo) Vuill. and Metarhizium anisopliae (Metsch.) conidia in 16 soils differing widely in pH, texture, organic matter, and carbonate contents. Experiments of adsorption and drag of conidia by soil particles suspended in CaCl₂ solutions of different ionic strength showed B. bassiana conidia to be retained by clay particles, and this effect disappeared with increasing ionic strength. The availability of M. anisopliae conidia in the suspension tended to be lower for sandy than for clayey soils and was not influenced by ionic strength. Regardless of soil properties, over 90% of the added fungal propagules were recovered from the surface layer of columns of packed soils representing model combinations of texture (sandy or clayey) and pH values (acid or alkaline). However, retention of B. bassiana conidia in the surface layer was higher in clayey than in sandy soils, and the retention of M. anisopliae conidia in the surface layer was higher in sandy than in clayey soils. Finally, neither soil texture nor ionic strength affected the infectivity of conidia of both fungal strains to C. capitata puparia.     

Potential of Metarhizium anisopliae and Beauveria bassiana to control Dinoderus porcellus (Coleoptera: Bostrychidae) infesting yam chips

The beetle Dinoderus porcellus Lesne is a serious storage insect pest that causes important losses by destroying stocks of yam chips. In the aim to found an alternative control method to the use of synthetic insecticides for its management, the virulence of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin (isolate Bb115) and Metarhizium anisopliae (Metschnikoff) Sorokin (isolate Met 31) against adults of D. porcellus was evaluated under laboratory conditions (25 ± 2 °C and 70 ± 5% RH). Then, the effectiveness of the most virulent entomopathogenic fungus as biological agent against D. porcellus was assessed under farmer storage conditions. For each entomopathogenic fungus isolate, four conidial concentration (0, 10⁵, 10⁷, and 10⁹ conidia/mL) at the dose of 1 µL were inoculated topically on D. porcellus adults (3–5 days old). Observations focused on insect mortality, cadaver sporulation and weight loss of yam chips. Lethal dose and lethal time values were estimated using probit analysis. Both fungal isolates at all conidial dose caused more than 50% mortality on day 7, with the highest mortality (94.44%) achieved using B. bassiana at the 10⁹ conidia/mL. LT₅₀ values for B. bassiana and M. anisopliae isolates were 2.63 and 3.35 days, respectively, while their LT₉₀ values were 6.15 and 9.87 days, respectively. Yielding the lower LD₉₀ values and the highest rates of cadaver sporulation, B. bassiana isolate appeared as the most virulent against D. porcellus. After 3 months of storage, comparatively to the control, the B. bassiana isolate at the highest conidial dose (10⁹ conidia/mL) significantly reduced D. porcellus populations, and weight loss of yam chips. This study revealed the potential of B. bassiana and M. anisoplae isolates as biological control agent against D. porcellus for yam chips protection.     

Behavioral and electrophysiological responses of Coptotermes formosanus Shiraki towards entomopathogenic fungal volatiles

Termites adjust their response to entomopathogenic fungi according to the profile of fungal volatile organic compounds (VOCs). This study demonstrates the pathogenicity of Metarhizium anisopliae, Beauveria bassiana and Isaria fumosorosea (=Paecilomyces fumosoroseus) towards the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Using no-choice assays, M. anisopliae was found to be highly virulent (LT₅₀ 3.10 d) when compared to B. bassiana (LT₅₀ 6.62 d) and I. fumosorosea (LT₅₀ 12.39 d). Also using choice assays, the foraging behavior of C. formosanus was determined in the presence of pathogenic fungi. The highly pathogenic fungi (M. anisopliae) elicited a repellent response, causing most of the termites to forage in a safe zone farthest from the fungal source. This repellency resulted in relatively low mortality similar to the controls. The repellency of M. anisopliae conidia can be used to protect human belongings and timber from termites. While I. fumosorosea cultures were not repellent to C. formosanus workers, the termites were highly susceptible to infection. Electroantennographic responses of workers showed approximately 47% and 78% lower level of response to conidia of B. bassiana and I. fumosorosea, respectively, as compared to M. anisopliae. The VOC profile of repellent cultures of M. anisopliae mainly consisted of paraffins (60.97%), while the major proportion of the I. fumosorosea profile consisted of branched and cyclic alkanes (84.41%). From the above findings, we conclude that the incorporation of I. fumosorosea may increase the control potential of bait.     

Carbon catabolite repressor gene BbCre1 influences carbon source uptake but does not have a big impact on virulence in Beauveria bassiana

A gene (BbCre1, GenBank accession number EF108309) encoding a carbon catabolite repressor (CreA) with two Cys₂His₂ zinc finger regions and a nuclear localization signal was cloned from the entomopathogenic fungus Beauveria bassiana. Overexpression and antisense strategies were used to investigate the biological functions of this gene. Compared with the wild type, the conidial yield and colony growth of BbCre1-overexpression transformants were significantly decreased on the plates with xylose or ethanol as the sole carbon source. With glucose as the sole carbon source, a significant difference was observed in the activity of Pr1A-like protease among BbCre1-overexpression transformants, antisense-BbCre1 transformants and the wild type. However, bioassays showed that knockdown or overexpression of BbCre1 did not have a significant impact on the virulence of B. bassiana to aphids. These results imply that the fungus remains virulent, even when simpler, less expensive nutrients are available, i.e. glucose.     

Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana

Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae are being developed as alternatives to chemical insecticides. They infect insects by direct penetration of the cuticle using a combination of physical pressure and extracellular hydrolytic enzymes such as proteases and chitinases. Previously we found that overexpression of a subtilisin-like protease (Pr1A) or a chitinase (Bbchit1) resulted in increased virulence of M. anisopliae and B. bassiana, respectively. In this study, we found that a mixture of the B. bassiana Pr1A homolog (CDEP1) and Bbchit1 degraded insect cuticle in vitro more efficiently than either CDEP1 or Bbchit1 alone. Based on this we produced three plasmid constructs; (1) Bbchit1, (2) CDEP1, and (3) a fusion gene of Bbchit1 linked to CDEP1 each under the control of the constitutive gpd promoter from Aspergillus nidulans. B. bassiana transformants secreting the fusion protein (CDEP1:Bbchit1) penetrated the cuticle significantly faster than the wild type or transformants overexpressing either Bbchit1 or CDEP1. Compared to the wild type, the transformant overexpressing CDEP1 showed a 12.5% reduction in LT₅₀, without a reduction in LC₅₀. The LT₅₀ of the transformant expressing CDEP1:Bbchit1 was reduced by 24.9%. Strikingly, expression of CDEP1:Bbchit1 resulted in a 60.5% reduction in LC₅₀, more than twice the reduction obtained by overexpression of Bbchit1 (28.5%). This work represents a significant step towards the development of hypervirulent insect pathogens for effective pest control.     

Infection of Blissus antillus (Hemiptera: Lygaeidae) Eggs by the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana

This study determined the pathogenicity and virulence of Beauveria bassiana and Metarhizium anisopliae to eggs of the chinch bug Blissus antillus (Hemiptera: Lygaeidae). Eggs were inoculated under laboratory conditions by immersion in concentrations of 1 × 10⁴ and 5 × 10⁶ conidia/ml. Inoculated eggs were kept under controlled conditions. Evaluations were carried out daily for 20 days. M. anisopliae isolates were highly virulent to eggs, even at 1 × 10⁴ conidia/ml. All B. bassiana isolates tested were considered to be of low virulence or avirulent. The most virulent isolate tested was ESALQ 818 (M. anisopliae), which caused 96.7% infection, when eggs were immersed in suspensions of 1 × 10⁴ conidia/ml. Conidial production on infected eggs was observed to be highest for M. anisopliae isolate CG144, with a mean value of 11.6 × 10⁵ conidia/ml/egg. Infection of Blissus eggs oviposited on plant stems was greater when M. anisopliae isolate CG144 was formulated in mineral oil (63.5% mortality) than when formulated in Tween 80 (27.1% mortality).     

Losing the battle against fungal infection: suppression of termite immune defenses during mycosis

The dampwood termite, Zootermopsis angusticollis is known to generate humoral immune responses to the entomopathogenic fungus Metarhizium anisopliae. However, little is known about how the termite's cellular immune system reacts to fungal infection. To test the effect of conidia exposure on cellular immunity, we quantified the number and types of hemocytes in the hemolymph of naïve nymphs and compared their circulating counts with those of nestmates exposed to 0, 2 × 10³, 2 × 10⁶ or 2 × 10⁸ conidia/ml doses. These termites were then bled and their hemocytes counted on days 1, 2, 3, 4, 7 post-exposure. Our results show, first, that naïve Z. angusticollis nymphs have three different blood cell types tentatively identified as granular hemocytes, prohemocytes and plasmatocytes. In these individuals, plasmatocytes were on average 13.5 and 3.3 times more numerous than granular hemocytes and prohemocytes, respectively. Second, a full factorial general linear analysis indicated that hemocyte type, time elapsed since conidia exposure and conidia dosage as well as all their interactions explained 43% of the variability in hemocyte density. The numbers of prohemocytes and particularly plasmatocytes, but not granular hemocytes, appear to be affected by the progression of disease. The decline in hemocyte numbers coincided with the appearance of hyphal bodies and the onset of “sluggish” termite behavior that culminated in the insect's death. Hemocyte counts of infected males and females were affected to the same extent. Hence, M. anisopliae overtakes the cellular immune responses of Z. angusticollis mainly by destroying the host's most abundant hemocyte types.     

Insect-toxic secreted proteins and virulence of the entomopathogenic fungus Beauveria bassiana

Fungal virulence has been mostly associated with cuticle-degrading enzymes that can be regulated depending on nutrient conditions. However, few studies have related fungal virulence to insect-toxic secreted proteins. Here, we describe how the presence of secreted toxic proteins may be linked to conidial virulence, which can be affected by nutrient factors. In this study we evaluated: (1) the virulence of the conidia of four Beauveria bassiana strains (EABb 01/103-Su, EABb 01/12-Su, EABb 01/88-Su and EABb 01/110-Su) grown on three different media (malt extract agar (MA), Rice (Rice), Sabouraud dextrose agar (SDA) and harvested from the cadavers of fungal-infected Galleria mellonella larvae (CAD) and (2) the toxicity of the crude soluble protein extracts (CSPEs) obtained from Adamek’s liquid medium inoculated with these conidia. Conidial suspensions were obtained from the four media, assessed on G. mellonella larvae and used to produce CSPEs that were injected into healthy G. mellonella larvae. The larvae were also injected with conidia obtained from MA and CAD cultures to expose them to in vivo-secreted proteins. For all isolates, the CAD conidia were by far the most virulent, followed by conidia grown on SDA, Rice and MA. The injected CSPEs showed the same toxicity trends as the conidial suspensions. In addition, the outcomes of injection of the in vivo-secreted proteins showed that the toxic proteins secreted in vitro by the EABb 01/110-Su strain are not produced in vivo. However, the other strains produced toxic proteins both in vivo and in vitro, suggesting that these toxic proteins may be virulence factors involved in invertebrate pathogenesis.     

The development and multiple uses of a standardised bioassay method to select hypocrealean fungi for biological control of aphids

A technically standardised bioassay method was designed, evaluated and used to assess virulence and host range of hypocrealean fungi against aphids. A track mounted sprayer was used to apply conidia because hand held versions of the same sprayer can be used for field applications, thereby allowing the outcome from laboratory experiments to predict activity in the field accurately. Eighteen fungal isolates were assessed in single concentration bioassays against the black bean aphid Aphis fabae Scopoli. Isolates comprised commercially available mycoinsecticides (based on Beauveria bassiana and Lecanicillium longisporum) and isolates of B. bassiana, Lecanicillium spp., Paecilomyces fumosoroseus and Metarhizium anisopliae. Aphid mortality was in excess of 80% for 15 isolates, and HRI 1.72 (L. longipsorum), Z11 (P. fumosoroseus), Mycotech strain GHA (B. bassiana) and ARSEF 2879 (B. bassiana) were studied further. Multiple concentration bioassays identified HRI 1.72 as the most virulent isolate against A. fabae with significantly smaller LC₅₀ and LT₅₀ values compared to other isolates. A precise LC₅₀ value (2.95 × 10² conidia ml⁻¹) was calculated for HRI 1.72 using a second multiple concentration assay with smaller concentrations of conidia. The four isolates were applied at a single concentration (1 × 10⁸ conidia ml⁻¹) against Myzus persicae, A. fabae, Acyrthosiphon pisum, Metopolophium dirhodum, Sitobion avenae and Rhopalosiphum padi. A ranking of aphid susceptibility was obtained, such that S. avenae > M. persicae, A. pisum, A. fabae > R. padi. Results indicate the importance of standardising bioassay methods to reduce bioassay variability without compromising the ability to use the bioassay to investigate fungus–host interactions under varying abiotic and biotic conditions.