Isaria Farinosa Research Articles

Rearing tomato whitefly and field evaluation of modified and unmodified conidia of Beauveria bassiana, Isaria farinosa, Metarhizium anisopliae and low rates of chlorpyrifos under tropical conditions.

The whitefly ( Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a serious pest of a wide range of economically important agricultural and ornamental crops in all agro-ecological regions in the world. This study evaluated the efficacy of conidia of entomopathogenic Beauveria bassiana (BB 315), Isaria farinosa (ARSEF 5081) and Metarhizium anisopliae (Ma 275.86DC), against whitefly ( Bemisia tabaci ) under tropical climate. Two forms of conidia: (a) modified conidia (conidia with improved ecological competence), and (b) unmodified conidia of the three isolates were subjected to heat treatment (45°C) for 5 days. Germination rates of these conidia before and after their exposure to heat were compared at 25 °C on Sabouraud Dextrose Agar (SDA, aw= 0.995) to assess resilience to heat stress. Further, assessment of germination potentials of conidia was conducted at three aw levels (0.995, 0.98 and 0.96: corresponding to different levels of water availability). Thereafter, they were tested against B. tabaci infestation on tomato plants and insect densities after the treatments were compared with the effect of low rate chlorpyrifos. The conidia germinated differently on SDA at the three a w levels and the rate of germination of modified conidia of each isolate was significantly higher at 0.98 a w (F 2, 16 =0.31, P=0.001). Modified conidia of B. bassiana appeared more tolerant to temperature stress. All the isolates significantly reduced pest incidencerelative to control after 14 days and there was no significant difference (P>0.05) between the effect of modified and unmodified conidia against B. tabaci in the field. The level of control achieved using the conidia of the three fungal isolates was comparable with the chlorpyrifos treatment. Key Words : Bemisia tabaci, entomopathogen, water activity

Występowanie oraz potencjał infekcyjny grzybów entomopatogenicznych w glebach z pól uprawnych, łąk i siedlisk leśnych

Summary The aim of the study was to compare the species composition and the intensity of occurrence of entomopathogenic fungi in the soil of arable fields and adjacent grassland and forest habitats. The soil and litter samples were collected in October 2012 from different habitats: mixed forest, farmland and meadow. Fungi were isolated from soil by means of two methods: trap insects (larvae of Galleria mellonella) and selective medium. A total five entomopathogenic fungal species: Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea, Isaria farinosa and Conidiobolus sp., were isolated by means of the first method, while only three first species of fungi by means of selective medium. It was found, that usually one fungal species dominated in each of the surveyed habitats. On average in the litter and forest soil the most infectious units formed fungus B. bassiana, in the soil of farmlands M. anisopliae, while in the meadow soil in a similar intensity I. fumosorosea and B. bassiana occurred. The richest species composition of entomopathogenic fungi was found in soils from meadows but the highest density of their colony forming units was found in soil samples collected from the small-scale arable fields.

Prevalence of Beauveria pseudobassiana among entomopathogenic fungi isolated from the hard tick, Ixodes ricinus

Human and animal disease-transmitting hard ticks (Acari: Ixodidae) are of great concern for public health and animal farming. Alternatives to tick control by chemical acaricides are urgently needed, and one intensively evaluated biocontrol strategy is based on the use of tick-pathogenic filamentous fungi. An indispensable prerequisite for the development of tick-derived fungal isolates into registered myco-acaricides is their sound taxonomic characterisation. A set of fungal strains isolated from ixodid ticks in the Republic of Moldova was genetically characterised at the genus and species level together with further tick-derived fungal isolates from different geographic locations in Europe and North America. In a previous study, the same isolates had been assigned to the species Beauveria bassiana. Using a recent molecular taxonomic approach based on phylogenetic reconstruction from both internal transcribed spacer (ITS) and protein-encoding gene sequences, all fungi investigated were conclusively assigned to one of the two “hyphomycete” genera, Beauveria or Isaria (Ascomycota; Hypocreales; Cordycipitaceae). Within the genus Isaria, two species, Isaria farinosa and Isaria fumosorosea, were equally represented. Within the genus Beauveria, the comparatively rare species Beauveria pseudobassiana was found to strongly prevail among the isolates from Moldova, and one of the two tick-derived Beauveria strains from North America could be assigned to this species as well. In particular, the previous classification as B. bassiana could not be confirmed for any of the characterised tick pathogens from Europe and North America. The data presented here lend support to the hypothesis that within the genus Beauveria specific adaptation to ticks might have occurred within the species B. pseudobassiana. To test this hypothesis, a more extensive molecular taxonomic survey carefully reconsidering previous taxonomic assignments of tick-derived fungal isolates is needed.

Growth and sporulation of entomopathogenic Beauveria bassiana, Metarhizium anisopliae, Isaria farinosa and Isaria fumosorosea strains in relation to water activity and temperature interactions

This study examined six strains of Beauveria bassiana s.l. and Isaria farinosa, one strain of Isaria fumosorosea and five strains of Metarhizium anisopliae s.l. to identify the ability for (1) growth and (2) sporulation under interacting environmental factors of water activity (aw) and temperature stress. Growth on Sabouraud Dextrose Agar (SDA; water activity, aw = 0.995) or SDA modified with glycerol to 0.98, 0.96 and 0.94 aw was measured at four different temperatures (25, 30, 35 and 37°C). All M. anisopliae strains grew at 25–35°C and 0.995 aw while only two strains tolerated extreme water stress at 0.94 aw.Three strains of B. bassiana were able to grow at 25–37°C and 0.995 aw. Only one strain of I. farinosa was able to grow at 25–37°C and 0.995 aw. Aw and temperature interactions resulted in different strain-dependent responses, in terms of growth and sporulation. Only one strain of I. farinosa and three of M. anisopliae grew at 0.94 aw and none of the B. bassiana strains tolerated such water stress. At 0.96 and 0.94 aw and 35–37°C, sporulation by all the strains of the three species were significantly affected. Under elevated temperatures and drought stress, very few of these strains of entomopathogenic fungi are able to grow and sporulate. Indeed, the B. bassiana strains were unable to tolerate the extreme conditions examined. Resilience to such abiotic interactions is critical for selecting strains for formulations. Tolerance to water and temperature stress could be good criteria for selection of strains with secondary spread potential for use as part of an integrated pest management system where secondary cycling may be important, especially in sub-tropical and tropical environments.

담배가루이(Bemisia tabaci )에서 분리한 곤충병원성진균의 동정 및 병원성 검정

Entomopathogenic fungi were isolated from Bemisia tabaci in an Oriental melon field, and their growth characteristics, factors related to a natural outbreak, and infectivity against Bemisia tabaci, Tetranychus urticae, and Frankliniella intonsa were investigated. The isolates had erect conidiophores bearing whorls of 4-6 phialides with a swollen base where cylindrical conidia of were attached. The isolates were identified as Isaria fumosorosea on the basis of morphological characteristics and an ITS sequence with 99% similarity. I. fumosorosea IFs-08 grew well on Sabouraud dextrose agar+yeast extract medium(3.2 mm/day/); it grew better at than at . The isolates of I. fumosorosea-IFs were highly infective and killed 93.9-96.7% B. tabaci, 84.9-92.0% T. urticae, and 81.5-84.4% F. intonsa in bioassay, whereas three isolates (Isaria tenuipes, Isaria farinosa, and Isaria fumosorosea) from KACC showed a low infectivity of 10-20%. To the best of our knowledge, this is the first report of I. fumosorosea isolated from B. tabaci in Korea.

Phyllosphere mycobiota on garden ponds plants

Investigations were conducted on calamus, common cattail, soft rush, yellow iris and white water lily plants in twenty ponds in Malopolska and Podkarpacie Regions. Mycobiota existing in the phyllosphere caused discolouring and necroses of leaves and shoots. 88 species of mycobiota were identified and isolated from the diseased tissues. Dominant were <em>Alternaria alternata, Epicoccum nigrum</em> and <em>Isaria farinosa</em>. Fungi of genera: <em>Aspergillus, Botrytis, Chaetomium, Cladosporium, Fusarium, Ilyonectria, Mortierella, Mucor, Penicillium, Phialophora, Phoma, Pleustomophora, Sordaria, Trichoderma</em> and <em>Umbelopsis</em> were also numerous. The monophagous and the polyphagous were identified.

Characterization and phylogeny of Isaria spp. strains (Ascomycota: Hypocreales) using ITS1‐5.8S‐ITS2 and elongation factor 1‐alpha sequences

The elongation factor 1-alpha (EF1-α) and the internal transcribed spacer (ITS) regions ITS1 and ITS2 (ITS1-5.8S-ITS2) sequences were used to characterize and to identify Isaria isolates from Argentina, Mexico, and Brazil, as well as to study the phylogenetic relationships among these isolates and other related fungi from the order Hypocreales. The molecular characterization, which was performed by PCR-RFLP of EF1-α and ITS1-5.8-ITS2 genes, was useful for resolving representative isolates of Isaria fumosorosea, Isaria farinosa, and Isaria tenuipes and to confirm the taxonomic identity of fungi from Argentina, Mexico, and Brazil. The phylogenetic analyses showed three clades corresponding to three families of Hypocreales. The genus Isaria was confirmed as polyphyletic and in family Cordycipitaceae, Isaria species were related to anamorphic species of Beauveria, Lecanicillium, and Simplicillium and to teleomorphic Cordyceps and Torrubiella. Therefore, EF1-α and ITS1-5.8S-ITS2 genes were found to be powerful tools for improving the characterization, identification, and phylogenetic relationship of the Isaria species and other entomopathogenic fungi.

Molecular cloning of a novel subtilisin-like protease (Pr1A) gene from the biocontrol fungus Isaria farinosa

Fungal proteases, such as subtilisin-like serine protease (Pr1) and trypsin-like serine protease (Pr2), play an important role in penetration through the host cuticle in entomopathogenic fungi, including Isaria farinosa. In the present study, one gene of I. farinosa subtilisin-like protease (Ifa-pr1A), showing high homology to a Beauveria bassiana cuticle-degrading protease, was amplified by rapid amplification of cDNA ends PCR. The resulting full-length cDNA displayed an open reading frame (ORF) of 960 bp, encoding a protein of 319 amino acids. The Ifa-Pr1A protein was expressed in Escherichia coli to verify its protease activity. The recombinant Ifa-Pr1A protein exhibited high enzymatic activity according to the enzyme assay using a synthetic substrate. The expression profiles of Ifa-pr1A and Ifa-pr1H (another subtilisin-like protease gene from I. Farinosa) were analyzed at different induction times by adding cuticle material to the media. Quantitative reverse transcriptase PCR (RT-PCR) analysis revealed that Ifa-pr1A transcripts increased 58,800-fold at 12 h post-induction, whereas Ifa-pr1H transcripts peaked at 12 h with only an 11-fold increase, indicating that Ifa-pr1A may be a major gene of cuticle-degrading proteases in I. farinosa. Furthermore, Ifa-pr1A gene expression under in vivo conditions showed a clear upward trend during the early stages of the infection process. These results suggest that Pr1A cloned from I. farinosa is a potential virulence factor for the development of engineered biopesticides.

Efficacy of entomopathogenic hypocrealean fungi against Periplaneta americana

The American cockroach Periplaneta americana, one of the worlds' most important urban insect pests was tested with entomopathogenic fungi. Most promising Metarhizium anisopliae, Metarhizium robertsii and Beauveria bassiana killed nymphs (≥81.7% mortality, 25days after treatment), and these fungi developed on all dead insects. Other fungi tested were less virulent (Metarhizium frigidum and Purpureocillium lilacinum) or avirulent (Isaria cateniobliqua, Isaria farinosa, Simplicillium lanosoniveum, Sporothrix insectorum and Tolypocladium cylindrosporum). Intrageneric and intraspecific variability of fungal activity was detected. Adults were highly susceptible, and oothecae proved to be more resistant than nymphs and adults to infection with M. anisopliae IP 46. Findings of the study underscore the potential of fungi as biocontrol agents against this pest.

High concentrations of biological aerosol particles and ice nuclei during and after rain

Abstract. Bioaerosols are relevant for public health and may play an important role in the climate system, but their atmospheric abundance, properties, and sources are not well understood. Here we show that the concentration of airborne biological particles in a North American forest ecosystem increases significantly during rain and that bioparticles are closely correlated with atmospheric ice nuclei (IN). The greatest increase of bioparticles and IN occurred in the size range of 2–6 μm, which is characteristic for bacterial aggregates and fungal spores. By DNA analysis we found high diversities of airborne bacteria and fungi, including groups containing human and plant pathogens (mildew, smut and rust fungi, molds, Enterobacteriaceae, Pseudomonadaceae). In addition to detecting known bacterial and fungal IN (Pseudomonas sp., Fusarium sporotrichioides), we discovered two species of IN-active fungi that were not previously known as biological ice nucleators (Isaria farinosa and Acremonium implicatum). Our findings suggest that atmospheric bioaerosols, IN, and rainfall are more tightly coupled than previously assumed.

Plant and fungal diversity in gut microbiota as revealed by molecular and culture investigations.

BackgroundFew studies describing eukaryotic communities in the human gut microbiota have been published. The objective of this study was to investigate comprehensively the repertoire of plant and fungal species in the gut microbiota of an obese patient.Methodology/Principal FindingsA stool specimen was collected from a 27-year-old Caucasian woman with a body mass index of 48.9 who was living in Marseille, France. Plant and fungal species were identified using a PCR-based method incorporating 25 primer pairs specific for each eukaryotic phylum and universal eukaryotic primers targeting 18S rRNA, internal transcribed spacer (ITS) and a chloroplast gene. The PCR products amplified using these primers were cloned and sequenced. Three different culture media were used to isolate fungi, and these cultured fungi were further identified by ITS sequencing. A total of 37 eukaryotic species were identified, including a Diatoms (Blastocystis sp.) species, 18 plant species from the Streptophyta phylum and 18 fungal species from the Ascomycota, Basidiomycota and Chytridiocomycota phyla. Cultures yielded 16 fungal species, while PCR-sequencing identified 7 fungal species. Of these 7 species of fungi, 5 were also identified by culture. Twenty-one eukaryotic species were discovered for the first time in human gut microbiota, including 8 fungi (Aspergillus flavipes, Beauveria bassiana, Isaria farinosa, Penicillium brevicompactum, Penicillium dipodomyicola, Penicillium camemberti, Climacocystis sp. and Malassezia restricta). Many fungal species apparently originated from food, as did 11 plant species. However, four plant species (Atractylodes japonica, Fibraurea tinctoria, Angelica anomala, Mitella nuda) are used as medicinal plants.Conclusions/SignificanceInvestigating the eukaryotic components of gut microbiota may help us to understand their role in human health.

Pathogens of bark beetles (Curculionidae: Scolytinae) and other beetles in Bulgaria

The presence and diversity of beetle pathogens associated with different tree species in Bulgaria was investigated. In total, 818 specimens belonging to 22 beetle species were examined. Pathogens occurred in 9 host species. The infections were found in the gut (virus, nematodes, protozoans, microsporidia) and haemolymph (nematodes) of the infected insects. The following pathogen species: ItEPV, Beauveria bassiana, B. brongniartii, Isaria farinosa, Gregarina typographi, Gregarina spp., Chytridiopsis typographi, Chytridiopsis sp., and nematodes were revealed. Insects with mycoses were mummified and filled up with fungal structures, formed pseudosclerotium. Most pathogens were established in bark beetles (Scolytinae). For the first time, the fungus B. bassiana was reported in Tomicus piniperda, Orthotomicus longicollis, O. erosus, X. spinole, Taphrorychus villifrons and Phylobius sp. in Bulgaria. We also present the first records of gregarines in O. longicollis, Acanthocinus aedilis, Rhagium inquisitor, Pyrochroa coccinea, and of the microsporidium Chytridiopsis sp. in O. longicollis.

Total synthesis of farylhydrazones A and B

Herein we report a concise total synthesis of farylhydrazones A and B, naturally occurring phenylhydrazones recently isolated from cultures of the Cordyceps-colonizing fungus Isaria farinosa, completed in six and five steps respectively starting from 2-nitrobenzoic acid. The synthesis is completely scalable, and highly convergent—making it adaptable for the preparation of analogues and investigation into the biological activity of these unique natural products.

Isolation and characterisation of Isaria farinosa and Purpureocillium lilacinum associated with emerald ash borer, Agrilus planipennis in Canada

Entomopathogenic fungi of the genera Isaria and Purpureocillium were recovered from infestation sites of emerald ash borer (EAB) in Southern Ontario, Canada. Isolates were identified using morphological characters and by sequencing the ITS1-5.8S-ITS2 ribosomal DNA gene and partial β-tubulin gene. Phylogenetic analysis and constructed trees based on the ITS and β-tubulin gene explicitly confirm isolates L66B, SY17-a and LHY46-a as Isaria farinosa and B3A, B59A and SY45B-a as Purpureocillium lilacinum. Pathogenicity was tested in the laboratory against adult EAB using a single concentration (2.0×107 conidia/ml) applied topically to adults. Controls included three commercial isolates: Isaria fumosorosea LRC176, Metarhizium brunneum LRC187 and Beauveria bassiana strain GHA. The native isolates I. farinosa L66B and P. lilacinum SY45B-a killed 75 and 50% of the beetles 14 days post-inoculation. Although these indigenous entomogenous fungi were less virulent compared with the commercial isolates, yearly isolation from EAB populations suggests they are one of the natural mortality factors of EAB in Canada.

Occurrence and diversity of fungal entomopathogens in soils of low and high Arctic Greenland

Knowledge of the occurrence, distribution and diversity of pathogens of insects and arachnids (entomopathogens) in the Arctic is very limited. Climate change is expected to affect Arctic terrestrial arthropod communities and therefore also host–pathogen interactions, given that entomopathogens are present. We conducted a survey of fungal entomopathogens in soil samples collected at four localities in Greenland; two at low Arctic sites (Ritenbenk and Disko Island) and two at sites in the high Arctic (Zackenberg and Danmarkshavn). Fungi were isolated from soil samples using larvae of the insect species Galleria mellonella (Lepidoptera) and Tenebrio molitor (Coleoptera) as baits providing evidence that the fungal isolates were indeed entomopathogenic. Five fungal species (Ascomycota; Hypocreales) were found: Isaria fumosorosea Wize, Isaria farinosa (Holmsk.) Fr., Beauveria bassiana (Bals.) Vuill., Beauveria pseudobassiana Rehner and Humber and Tolypocladium inflatum W. Gams (syn. = T. niveum). I. farinosa was found at all four localities, while I. fumosorosea was detected in single samples at each of three localities including both high Arctic sites. Only the locality on Disko Island revealed B. bassiana, whereas B. pseudobassiana was isolated at the three other sites. T. inflatum was only found on Disko Island and only isolated with T. molitor as a bait insect. The results document that fungal entomopathogens are widely distributed in the soil environment in Greenland. Entomopathogens should therefore be included in future studies of arthropod ecology in the Arctic.

Factors affecting the occurrence of entomopathogenic fungi in soils of Slovakia as revealed using two methods

Soil samples from 901 sampling sites in Slovakia were collected during 2008 and the presence of entomopathogenic fungal species from the order Hypocreales was evaluated. Entomopathogenic fungi were detected in 430 samples by the Galleria mellonella bait method (GBM) and in 627 soil samples using selective medium (SM) containing cycloheximide and dodine. There was a significant difference between the ability of these methods to detect the fungi. Four species were detected by the GBM: Beauveria bassiana (31% of samples), Metarhizium anisopliae (8%), Isaria farinosa (6%), and Isaria fumosorosea (6%). Only three species were detected with SM: B. bassiana (36% of samples), M. anisopliae (37%), and I. fumosorosea (9%). In addition, some samples that were positive using SM were negative with the GBM and vice versa, implying that these methods are not equivalent for the determination of the occurrence and distribution of entomopathogenic fungi. Our results indicated that B. bassiana preferred natural habitats, particularly forests, though it was also frequently detected in other habitats. I. farinosa was strictly limited to forest habitats and I. fumosorosea was most frequently detected in soil samples from hedgerows. M anisopliae was observed primarily in field and meadow samples, and its occurrence was not negatively affected by intensive agriculture. It was most frequently found in lowland soils with a neutral or alkaline pH. Detection of B. bassiana and I. farinosa significantly increased with increasing altitude and decreasing soil pH. Both fungi also preferred soils with high organic content.

Below- and aboveground abundance and distribution of fungal entomopathogens in experimental conventional and organic cropping systems

The below- and aboveground communities of fungal entomopathogens (Ascomycota: Hypocreales) were investigated in an experimental conventional and organic vegetable cropping system over two seasons in Denmark. The experimental design allowed for evaluating differences between farming practices in the occurrence of soil-borne fungal entomopathogens and as natural infections aboveground in arthropod hosts. Belowground, Metarhizium anisopliae dominated the agricultural field with Beauveria bassiana, Metarhizium flavoviride, and Isaria fumosorosea being present at lower frequencies. Abundances of M. anisopliae were not different between conventional and organic soils. Aboveground, B. bassiana was the most common fungal entomopathogen in arthropod host cadavers. Infections of M. flavoviride, Isaria farinosa, and Gibellula spp. were also recorded. Most mycosed cadavers were recovered in August and September from conventionally farmed plots. Cadaver sizes ranged from 1.9 to 23.6mm with means of 3.7–4.1mm. Among host functional groups, predators dominated (47–63%) over herbivores, parasitoids, and nectar-feeders. No occurrence of M. anisopliae was recorded among >200 mycosed cadavers aboveground. The results therefore suggest that M. anisopliae is not involved in direct below- and aboveground interactions in the investigated agroecosystem. Conservation biological control strategies targeting M. anisopliae should thus be solely for controlling soil-dwelling pests in temperate regions as this fungus is unlikely naturally to infect aboveground hosts in the agroecosytem.

Effects of some fungicides on Isaria farinosa, and in vitro growth and infection rate on Planococcus citri

The effects of some fungicides used against citrus diseases, on mycelial growth and conidial germination of Isaria farinosa (Holmsk.) Fries [Sordariomycetes: Hypocreales] and also on the pathogenicity of the fungus on citrus mealybug, Planococcus citri (Risso), were determined. Systemic fungicides such as tebuconazole, penconazole and nuarimol were the most effective as regards both conidial germination and mycelial growth. Protective fungicides such as captan, chlorothalonil, mancozeb and propineb inhibited conidial germination at between 1 and 5 μg ml−1 concentration, but captan, chlorothalonil and propineb did not inhibit the mycelial growth at 5,000 μg ml−1. Mancozeb inhibited mycelial growth between 2,500 and 5,000 μg ml−1. Sulphur and copper oxychloride did not inhibit the fungus even at very high concentrations. Sulphur, copper oxychloride, fosetyl-al, chlorothalonil and carbendazim did not decrease the mortality percentage caused by I. farinosa. Tebuconazole, penconazole and mancozeb were the most effective and respectively reduced the mortality from 83% to 33%, 28% and 30% in the ovisacs, from 81% to 29%, 27% and 29% in the 1st instar larvae, and from 84% to 34% in the adult females.

Characterization of a hydrophobin of the ascomycete Paecilomyces farinosus

The entomopathogenic ascomycete Paecilomyces farinosus (alternative name Isaria farinosa) synthesized a hydrophobin, irrespective of being grown in submerged or surface culture. The protein was extracted using trifluoroacetic acid and purified using preparative HPLC and SDS-PAGE. Partial sequences were obtained using ESI-MS/MS. The peptides were used as a start to apply a 'template switching oligo' protocol to elucidate the complete open reading frame of P. farinosus hydrophobin 1 (pfah1). The deduced protein sequence comprised 107 amino acids (10.7 kDa) including a 16 amino acid long hydrophobic signal peptide, showed a calculated pI of 4.56, and was interrupted by one intron. Phylogenetic analyses revealed relationships to hydrophobins of the ascomycetes Magnaporthe grisea and Metarhizium anisopliae. Based on solubility, hydropathy pattern and phylogeny PfaH1 was assigned to the class Ia hydrophobins.

Laboratory evaluation of the effectiveness of the entomopathogen; Isaria farinosa, on citrus mealybug, Planococcus citri

Citrus mealybug Planococcus citri (Risso) (Hemiptera: Pseudococcidae) is one of the main pests on citrus trees. Biological control of the pest is based on the release of hymenopterous parasitoids and coccinellid predators at present. The effectiveness of entomopathogen fungus Isaria farinosa (Holmsk.) Fries ([Sordariomycetes: Hypocreales] (Syn: Paecilomyces farinosus), as an alternative biological control agent on citrus mealybug, was investigated using four different inoculum densities and different relative humidities (RH). The entomopathogen caused 89.39% mortality in ovisacs, 84.07% mortality in second larval stage, 84.53% mortality in adult females, and 78.71% mortality in first larval stage at 95% RH and at 1 × 108 conidia ml−1 inoculum concentration. Percent mortalities were between 50 and 60 in ovisac, first and second larval stage at 95% RH and at 1 × 107 conidia ml−1. Percent mortality was decreased parallel to the decrease in humidity level and inoculum densities; however, the fungus caused significant infection in 70% and 80% RH. These results demonstrated that the effectiveness of the entomopathogen is promising for biocontrol of citrus mealybug.