Cordyceps Javanica Research Articles

Effect of Host Size on Susceptibility of Melanotus Communis (Coleoptera: Elateridae) Wireworms to Entomopathogens.

Wireworms, the soil-borne larvae of click beetles (Coleoptera: Elateridae), are important crop pests throughout the world. In the eastern U.S., Melanotus communis larvae attack grain, root/ tuber, and vegetable crops. Our objectives were to characterize the pathogenicity and virulence of fungal and nematode entomopathogens on M. communis wireworms, and determine if wireworm size affected virulence. Pathogens tested included five entomopathogenic nematodes, Steinernema carpocapsae (All strain), S. feltiae (SN strain), S. riobrave (355 strain), Heterorhabditis bacteriophora (VS strain), and H. indica (HiHom1 strain); and two entomopathogenic fungi, Beauveria bassiana (GHA strain) and Cordyceps javanica (WF-GA17 strain). None of the pathogens tested caused >15% mortality at 7 or 14 days post-inoculation. Mortality was highest in S. carpocapsae (All strain); the other entomopathogens did not cause higher mortality than the untreated control. Overall, smaller wireworms were more susceptible than larger wireworms. Our results suggested that M. communis wireworms have defenses that limit the ability of the entomopathogens we tested to infect the wireworms. Conceivably, other entomopathogen strains or species may be more effective. Natural populations of entomopathogens may contribute to wireworm population reduction, but further studies are warranted before entomopathogens can be used for M. communis management.

Bt protein hasten entomopathogenic fungi-induced death of nontarget pest whitefly by suppressing protective symbionts

The risk assessment of Bacillus thuringiensis (Bt) crops on nontarget pests has received much attention. Despite the knowledge of various beneficial bacterial symbionts in pests, whether Bt proteins affect these symbionts and subsequently alter the pest's ecology remains largely unknown. The whitefly Bemisia tabaci is one of the most serious nontarget pests in Bt cotton. Here, we explored the Bt Cry1Ac protein-induced changes in whitefly symbiont abundance and the subsequent effects on whitefly response against a naturally prevalent entomopathogenic fungus Cordyceps javanica. The obligate symbiont ‘Candidatus Portiera aleyrodidarum’ (hereafter P. aleyrodidarum) as well as facultative symbionts ‘Candidatus Hamiltonella defensa’ (hereafter H. defensa), ‘Candidatus Cardinium hertigii’ (hereafter C. hertigii) and ‘Candidatus Rickettsia bellii’ (hereafter R. bellii) dominate the microbial community of whiteflies. The Bt exposure had no effects on H. defensa infected (H) and H. defensa-C. hertigii doubly infected (HC) whiteflies, but decreased the total copy number of symbionts as well as the R. bellii proportion in H. defensa-C. hertigii- R. bellii triply infected whiteflies (HCR). C. javanica caused whitefly adults 100 % mortality within 8 days. Without Bt protein exposure, HCR whiteflies survived significantly longer than H and HC whiteflies sprayed by C. javanica, suggesting that R. bellii confers protection. However, in Bt-exposed groups, C. javanica generated synchronous death of H, HC and HCR whiteflies. Specifically, in H and HC whiteflies, Bt protein-exposure showed no significant difference in progress of death caused by C. javanica. But in HCR whiteflies, Bt exposure hastened death induced by C. javanica, suppressing the R. bellii-conferred protection. This is the first report revealing that Bt protein altered symbiont community conferred adverse effects on nontarget pests, providing a new perspective for Bt risk assessment and biocontrol strategies of nontarget pests.

Development of Cordyceps javanica BE01 with enhanced virulence against Hyphantria cunea using polyethylene glycol-mediated protoplast transformation.

Cordyceps javanica has promising application prospects as an entomopathogenic fungus with a wide range of hosts. To enhance the virulence of C. javanica, a polyethylene glycol (PEG)-mediated protoplast genetic transformation system was constructed. Strains overexpressing the subtilisin-like protease genes CJPRB and CJPRB1 and the tripeptidyl peptidase gene CJCLN2-1 were constructed with this system, and the effects of these strains on Hyphantria cunea were tested. The aminoglycoside G418 was used at 800 μg ml−1 to screen the transformants. C. javanica hyphae were degraded with an enzyme mixture to obtain protoplasts at 1.31 × 107 protoplasts ml−1. The transformation of 2 μg of DNA into 1,000 protoplasts was achieved with 20% PEG2000, and after 6 h of recovery, the transformation efficiency was 12.33 ± 1.42 transformants μg−1 plasmid. The LT50 values of CJPRB, CJPRB1, and CJCLN2-1-overexpressing C. javanica strains were 1.32-fold, 2.21-fold, and 2.14-fold higher than that of the wild-type (WT) strain, respectively. The three overexpression strains showed no significant differences from the WT strain in terms of colony growth, conidial yield, and conidial germination rate. However, the infection rate of the CJPRB1 strain was faster than that of the WT strain, with infection occurring within 4–5 days. The CJCLN2-1 strain had a significantly higher mortality rate than the WT strain within 4–10 days after infection. A C. javanica genetic transformation system was successfully constructed for the first time, and an overexpression strain exhibited enhanced virulence to H. cunea compared with the WT strain.

Efficacy of soil-borne entomopathogenic fungi against subterranean termite, Coptotermes curvignathus Holmgren (Isoptera: Rhinotermitidae)

BackgroundCoptotermes curvignathus Holmgren (Isoptera: Rhinotermitidae) is a subterranean termite that poses serious damage to oil palm and rubber trees. Chemical pesticides could cause negative effect to human and the environment in long-term usage. The use of entomopathogenic fungi (EPF) to suppress the population of subterranean termites is in favour when compared to chemical pesticides because they do not harm to the environment and non-target organisms. The study aimed to isolate and identify the EPF from the soil using yellow mealworm larvae of Tenebrio molitor Linnaeus in the baiting method and assessed their efficacy against subterranean termite, C. curvignathus.ResultEleven EPF isolates were successfully isolated from the oil palm plantation in Universiti Putra Malaysia, namely: Aspergillus auricomus (UPM-A1C-1), A. caelatus (UPM-A1C-2), Metarhizium anisopliae var anisopliae (UPM-A2C-1, UPM-A3C-1, UPM-A3C-2, UPM-A5C-1 and UPM-A10C-1), Purpureocillium lilacinum (UPM-A2C-3 and UPM-A7C-1), Cordyceps javanica (UPM-A2C-5), and M. pinghaense (UPM-A13C-2). The identity of these EPF were confirmed by morphological and molecular characteristics. All EPF yielded 100% mortality in C. curvignathus in 10 days after inoculation (DAI), except UPM-A1C-1 and UPM-A1C-2 after exposure to 1 × 107 conidia ml−1. UPM-A2C-5 Cordyceps javanica yielded the highest mycelia formation (69%) after 6 DAI. The LT50 values varied from 3.90 to 7.75 days. UPM A2C-1 M. anisopliae var anisopliae showed the lowest LT50 (3.90 days), while UPM-A1C-1 Aspergillus auricomus showed the highest LT50 (7.75 days). The lowest LC50 value (1.49 × 105 conidia ml−1) was recorded in UPM A2C-1 M. anisopliae var anisopliae.ConclusionsThe present study confirmed the soilborne EPF with potential insecticidal activity against C. curvignathus. UPM-A2C-1 M. anisopliae var anisopliae was a potential biological control agent against Subterranean termite, C. curvignathus due to its virulence score and high percentage of mycelia formation after 6 DAI. The data reported in the present study, particularly using P. lilacinum, M. pinghaense, Aspergillus auricomus, A. caelatus and C. javanica with potential insecticidal activity against C. curvignathus, are new records.

Comparing Production and Efficacy of Cordyceps javanica With Cordyceps fumosorosea.

A newly discovered entomopathogenic fungus Cordyceps javanica (Friedrichs & Bally) Samson & Hywel-Jones (Hypocreales: Cordycipitaceae) strain Wf GA17 was compared with the commercial Cordyceps fumosorosea Wize (Hypocreales: Cordycipitaceae) Apopka 97 strain for liquid-culture production, formulation, insecticidal efficacy, and storage stability under laboratory conditions. We compared culture media with carbon:nitrogen (C:N) ratios of 10:1, 30:1, and 50:1 for these two isolates. A third strain, C. fumosorosea strain ARSEF 3581, had previously been optimized for liquid-culture production of blastospores at 10:1 C:N served as an added control. These seven cultures were processed by spray drying with skim milk powder, stored at 25oC to evaluate storage stability, and assayed for insecticidal activity against Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) neonates. Final blastospore concentrations were not significantly different among cultures, ranging from 4.47 to 9.88 × 108 spores/ml. Fungal biomass decreased and final glucose concentrations increased with increasing C:N ratios, indicating better fungal growth with higher nitrogen concentrations. Product yields from the spray dryer (grams per liter culture) increased with increased C:N ratios while spore concentrations decreased, ranging from 2.27 to 7.17 × 109 spores/g. There were no significant differences for insecticidal efficacy among the seven treatments. Spores produced in 10:1 C:N ratio media retained viability longer than spores produced in other media. Cost of ingredients decreased with increasing C:N ratios, such that the 30:1 media may yield the most economical product. The raw material cost needed for application was 1.4× greater for Wf GA17 compared with Apopka 97, a difference that could be erased by optimization of culture conditions.

DOPA-melanin, component and tolerance factor to heat and UV-B radiation in the conidia of two species of Cordyceps

ABSTRACT Melanins are heterogeneous pigments present in all biological kingdoms with many biological roles, mainly related to coping with environmental stress. In some species of fungi, melanin is involved in higher virulence against plants and animals; however, little is known about the presence and type of melanin found in entomopathogenic fungi (EF), used for biological pest control. In this work, we explore the presence of melanin in conidia from Cordyceps fumosorosea CHE-CNRCB 303 and Cordyceps javanica ARSEF 3302. Melanin from both species was purified and characterised by UV, Fourier transformed infrared resonance (FT-IR) and electron paramagnetic resonance (EPR). The DOPA-melanin pathway was established as the route used by the Cordyceps genus to synthesise the pigment, which presented the ability to reduce 2,2-diphenyl-1-picrylhydrazyl (DPPH), confirming its antioxidant capacity; furthermore, the melanised conidia of both Cordyceps species were more thermotolerant and resistant to UV-B radiation than non-melanised conidia. This is the first time that melanin has been characterised and its influence on conidia quality has been verified in two Cordyceps species, which are used extensively for pest control. This knowledge could improve both the persistence of conidia in the field and the effectivity in biological control.

Field Efficacy of Cordyceps javanica, White Oil and Spinetoram for the Management of the Asian Citrus Psyllid, Diaphorina citri.

Simple SummaryCitrus greening is devastating the citrus industry in Florida, and conventional synthetic pesticide applications are rapidly becoming unsustainable for the control of the vector, the Asian citrus psyllid (AsCP), Diaphorina citri. Laboratory experiments indicate that the entomopathogenic fungus Cordyceps javanica, alone and in combination with horticultural oils, may offer a more sustainable strategy to manage AsCP. The field study in this paper indicated that C. javanica alone, C. javanica mixed with white oil, and spinetoram (the chemical standard) mixed with white oil significantly suppressed AsCP populations by 60–90% and 61–83% 7 and 14 days after treatment, respectively, in 2018, compared to white oil only and the untreated control treatments. Only spinetoram + oil suppressed AsCP 100% up to 7 days after treatment in 2019. AsCP’s natural enemies, mostly lady beetles, were observed on fungal-treated citrus trees and the untreated control. Overall, C. javanica was as effective in suppressing AsCP adults as spinetoram for up to 14 days in field conditions and was compatible with the psyllids’ natural enemies. The use of C. javanica in citrus-integrated pest management is suggested.Citrus greening disease is devastating the citrus industry in Florida, and the conventional synthetic pesticide applications used to control the vector, the Asian citrus psyllid (AsCP), Diaphorina citri, are rapidly becoming unsustainable. Various laboratory experiments indicate that the entomopathogenic fungus Cordyceps javanica, alone and in combination with horticultural oils, may offer a more sustainable strategy for the management of AsCP. Field studies conducted in 2018 and 2019 in mature citrus indicated that C. javanica alone, C. javanica mixed with white oil, and the chemical standard spinetoram mixed with white oil significantly suppressed AsCP adult populations by 61–83% up to 14 days after treatment in 2018, although colony-forming units of C. javanica were still present on the leaves 21 days after treatment (DAT). Only spinetoram + oil significantly suppressed AsCP, by 100%, up to 7 DAT in 2019. Natural enemies of AsCP, including lady beetles, lacewing larvae and the parasitoid Tamarixia radiata, were observed in the fungal treatments and the untreated control. The AsCP suppression by C. javanica and its compatibility with beneficial organisms suggest the potential use of this entomopathogenic fungus in citrus-integrated pest management.

Entomopathogenicity of fungi in combination with Ricinus communis extract for the control of Aleurocanthus woglumi

AbstractCitrus is among the most important crops in the world, and has suffered losses of up to 80% due to the citrus blackfly, Aleurocanthus woglumi Ashby (Hemiptera: Aleyrodidae). The combined use of entomopathogenic fungi and plant extracts is a sustainable alternative to control this pest. The objectives of this study were to select entomopathogenic fungi to control A. woglumi and verify the insecticidal effect of the combined use of the selected fungi and aqueous extracts of castor bean leaves, Ricinus communis L. (Euphorbiaceae). Twenty fungal isolates obtained from field‐collected dead A. woglumi were identified and tested against the insect. Eight were identified as belonging to Fusarium incarnatum‐equiseti species complex, nine to Fusarium fujikuroi species complex, two to Cordyceps javanica (Bally) Kepler, B. Shrestha & Spatafora, and one to genus Akanthomyces. The five isolates that caused the maximum confirmed mortality were tested for compatibility with aqueous castor bean extract at 5, 10, and 20% (wt/vol) concentrations. Qualitative phytochemical analysis showed the presence of alkaloids, glycoside flavonoids, saponins, and tannins in the castor bean extract. Most of the isolates studied caused high confirmed mortality of citrus blackfly. In the compatibility tests, Fusarium volatile Al‐Hatmi, Sand.‐Den., SA Ahmed & de Hoog and Fusarium proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg combined with the 5% aqueous extract were the most suitable for testing against A. woglumi. Additive interactions were observed for the combined control agents. The highest value of confirmed mortality (98.2%) was observed for the combination of 5% aqueous castor bean extract and F. volatile (URM 8051), the efficacy of which should be confirmed by field tests.

Identification and Virulence of Cordyceps javanica Strain wf GA17 Isolated From a Natural Fungal Population in Sweetpotato Whiteflies (Hemiptera: Aleyrodidae).

A new strain of the entomopathogenic fungus, identified as Cordyceps javanica (Frieder. & Bally) Kepler, B. Shrestha & Spatafora (Hypocreales: Cordycipitaceae) wf GA17, was found naturally infecting the sweetpotato whitefly, Bemisia tabaci (Gennadius) MEAM1 in southern Georgia, US, in September 2017. The fungus was tested for pathogenicity and virulence in comparison with commercially available entomopathogenic fungal strains against several insect species in the laboratory. In specific, it was compared with Cordyceps fumosorosea (Wize) Kepler, B. Shrestha & Spatafora (Hypocreales: Cordycipitaceae) Apopka 97, Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) strain GHA, and Metarhizium brunneum Petch (Hypocreales: Clavicipitaceae) strain F52 for virulence against B. tabaci (4th instars) and cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae) (4th instars with or without wingbuds), on leaf-discs, and against last instars of pecan weevil Curculio caryae (Horn) (Coleoptera: Curculionidae) and citrus root weevil Diaprepes abbreviatus (L.) (Coleoptera: Curculionidae) in soil cups. Against B. tabaci, C. javanica exhibited higher mortality and mycosis development at 5 d post inoculation than other fungi. In assays against A. gossypii with and without wingbuds, C. javanica and C. fumosorosea had the highest mortality and mycosis levels and B. bassiana had the lowest; nymphs with wingbuds were more susceptible to some fungal infection than those without. Against C. caryae, B. bassiana was more effective than other fungi. For D. abbreviatus, B. bassiana also caused the highest mortality while M. brunneum had the lowest, with Cordyceps spp. being intermediate. Overall, the findings suggest high potential of the new strain, C. javanica wf GA17, for managing whiteflies and aphids, while it was not as effective as B. bassiana against the curculionids.

Isolation, fermentation, and formulation of entomopathogenic fungi virulent against adults of Diaphorina citri.

Mycopesticides are important for integrated management of the Asian citrus psyllid, Diaphorina citri. However, there are few reports on the fermentation and formulation for mycopesticides with high virulence against D. citri. From four different locations in South China, 12 fungal strains were isolated and classified into Beauveria bassiana (two isolates), Fusarium fujikuroi (five isolates), and Cordyceps javanica (five isolates) based on the phylogenetic analysis of ITS1-5.8S rDNA-ITS4 and morphology of colonies and conidia. Based on the highest initial virulence (86.52 ± 2.35%) and best growth characteristics, F-HY002-ACPHali was further optimized for biphasic fermentation (7.85 ± 2.62 × 109 g-1 dry substrate) with soybean meal + cottonseed flour (1:1) as the solid substrate and full light as the light/dark cycle. Furthermore, the oil-based conidial formulation was optimized with sesame oil as an effective carrier, which significantly enhanced conidial shelf life up to 16 weeks at 26 °C and 4°C, thermo-stress (50 °C) and UV-B stress resistance, and virulence against adults of D. citri with increased mortalities and decreased LT50 in comparison to that of unformulated conidia. Our results not only provide a valuable native strain with high virulence against adults of D. citri, but also imply the soybean meal + cottonseed flour as valuable solid substrate, full-light exposure as environmental stimuli for solid-state fermentation, and the sesame oil as an effective carrier for the formulation of the C. javanica. These findings will facilitate the development of a potential mycopesticide for the biological control of Asian citrus psyllid.

Evaluation of bioefficacy potential of entomopathogenic fungi against the whitefly (Bemisia tabaci Genn.) on cotton under polyhouse and field conditions

The whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), is becoming a serious problem on Bt cotton. It causes enormous crop loss through its direct feeding and as a vector of cotton leaf curl virus. Chemical-dependent management is harming the environment and increased insecticide resistance is often observed in the fields. Identification of most virulent strains of entomopathogenic fungi (EPF) is essential to serve as an important component of an IPM program for management of B. tabaci. Compared to B. tabaci adults, the nymphal stage is reported to be more susceptible to entomopathogens, and targeting nymphs also helps vector management. We evaluated the bioefficacy of EPF and chemical pesticides against nymphs of B. tabaci on Bt cotton under polyhouse and field conditions. The bioefficacy index (BI) was considered as a mechanism to select the most effective EPF strains for field evaluation. The highest nymphal mortality under polyhouse conditions was recorded for Metarhizium anisopliae NA-01299 (86.7%), Beauveria bassiana MT-4511 (85.1%), Cordyceps javanica IT-10498 (81.1%), IT-10499 (81%), and B. bassiana NA-0409 (78.2%) relative to other EPF strains, spiromesifen (69.6%), buprofezin (62.2%) and pyriproxyfen (52.7%) at 7-days-post-spray treatment (DAS). However, among all the EPF, the highest BI was recorded in C. javanica IT-10499 (77%), IT-10495 (75.4%), Fusarium verticillioides IT-10493 (74.6%), and B. bassiana MT-4511 (73.1%). The pooled data of two-year field trials (2017–18 & 2018–19) revealed that the highest nymphal mortality was recorded for MT-4511 (85%), IT-10499 (83.2%), and pyriproxyfen 10% EC (78.6%) at 7-DAS. The BI-based selection of EPF proved to be a useful predictor of field efficacy. A sequential spray of the selected EPF would be a vital approach for resilient and sustainable integrated management of the B. tabaci nymphal population under field conditions.

Principal component analysis of the biological characteristics of entomopathogenic fungi in nutrient-limited and cuticle-based media.

Media formulated with insect cuticle (0.5% and 1%; Sphequit Sph®), with a reduction in nutrients (¼ Sabouraud dextrose agar + yeast [SDAY]) and commercial media (potato dextrose agar, Sabouraud dextrose agar) were evaluated for the cultivation of Beauveria bassiana, Cordyceps javanica (Isaria javanica [Bally] Samson & Hywel-Jones), and Metarhizium robertsii. By using principal component analysis, it was determined that the ¼ SDAY and Sph formulations have greater advantages than commercial media for the development of fungi. The ¼ SDAY and Sph (0.5% and 1%) improved hydrophobicity, radial growth rate, germination, conidia yield, and virulence in B. bassiana; in M. robertsii, they favored conidia yield, germination, and virulence, and in C. javanica, the ¼ SDAY and Sph 0.5% media enhanced conidia yield, germination, radial growth rate, and virulence. We suggest that these formulations are an alternative to commercial culture media as they are cheaper and appropriate to improve the growth characteristics and virulence of the three strains evaluated. Some applications of culture media are suggested, and the importance of multivariate analysis as an exploratory tool to carry out the choice of culture media in a suitable way for the development of mycoinsecticides is also discussed.

Environmental Tolerance of Entomopathogenic Fungi: A New Strain of Cordyceps javanica Isolated from a Whitefly Epizootic Versus Commercial Fungal Strains.

Simple SummaryWhiteflies are significant pests of cotton and vegetables in southeastern USA. In previous studies, we isolated and identified a new strain of entomopathogenic fungus that caused epizootics among whiteflies in cotton fields of Southern Georgia, USA. The objective of this study was to test the level of tolerance of this new strain to environmental conditions as compared to commercial fungal strains. We exposed the new strain and three commercially available strains of biopesticides (BotaniGard, Met52, and PFR-97) to different temperatures and strong ultraviolet (UV) radiation before examining post-treatment viability and virulence against a common model organism for insect pathology, the greater wax moth larvae. We found that the new strain had similar levels of activity to commercial strains at moderate temperatures, but higher tolerance than PFR-97 to extremely low and high temperatures and strong UV intensity. These findings suggest that the new fungal strain has potential for commercial development as an alternative to PFR-97 for managing certain types of insect pests.A new strain of Cordyceps javanica (wf GA17) was observed causing widespread epizootics among whiteflies in Southern Georgia in 2017. The tolerance of conidia to environmental factors including variable temperature and ultraviolet (UV) light was compared between this strain and three commercial strains of entomopathogenic fungi (Metarhizium brunneum F52, Cordyceps fumosorosea Apopka97, and Beauveria bassiana GHA). Under 10–30 °C, C. javanica wf GA17 responded similarly to other fungi, with the highest virulence against Galleria mellonella at 25 °C, followed by 20, 30, and 15 °C; lowest virulence was observed at 10 °C. At 35 °C and 40 °C, C. javanica wf GA17 had lower tolerance than M. brunneum F52 and B. bassiana GHA, but was superior to C. fumosorosea Apopka97 in conidia viability and post-treatment virulence. After exposure to −20 °C for 56 d, C. javanica wf GA17 exhibited lower germination than M. brunneum F52 and lower virulence than M. brunneum F52 and B. bassiana GHA, but higher germination and virulence than C. fumosorosea Apopka97. Following exposure to strong UV light, viability and virulence of all fungi were reduced with increasing exposure periods. Increased environmental tolerance of C. javanica wf GA17 over C. fumosorosea Apopka97 suggests that the new strain could have applicability for commercial pest management.

Infection of Beauveria bassiana and Cordyceps javanica on different immature stages of Duponchelia fovealis Zeller (Lepidoptera: Crambidae)

Entomopathogenic fungi (EPF) can be viable options for biological control of agricultural pests. The selection of fungal strains is crucial for a successful pest control. The current study aimed to determine in the laboratory the susceptibility of different immature stages of Duponchelia fovealis Zeller (Lepidoptera: Crambidae) to EPF strains (Beauveria bassiana Bea111 and Cordyceps javanica Isa340). Immature stages of D. fovealis were sprayed with EPF concentrations from 2 × 103 to 2 × 109 conidia mL−1 and their viability and mortality were evaluated. Lethal time and concentration were estimated for pupae and each larval instar. Virulence depended on strain, dose, and insect life stage. The eggs were resistant to Bea111 and Isa340 strains. Mortality of larvae depended on the EPF strain and the larval instar, with the first instar being the most sensitive. Lethal concentrations for D. fovealis 1st instar larvae were 3.4 × 105 and 1.7 × 107 conidia mL−1 with Bea111 and Isa340, respectively. Isa340 performed better than Bea111 for controlling pupae. Scanning electron microscopy revealed the adhesion and penetration structures of both fungi in the 2nd instar infected larvae 6 h after inoculation, while fungal extrusion occurred between 54 and 94 h after spraying with Bea111 and Isa340, respectively.

Pigmentosins from Gibellula sp. as antibiofilm agents and a new glycosylated asperfuran from Cordyceps javanica.

In the course of our exploration of the Thai invertebrate-pathogenic fungi for biologically active metabolites, pigmentosin A (1) and a new bis(naphtho-α-pyrone) derivative, pigmentosin B (2), were isolated from the spider-associated fungus Gibellula sp. Furthermore, a new glycosylated asperfuran 3, together with one new (6) and two known (4 and 5) cyclodepsipeptides, was isolated from Cordyceps javanica. The pigmentosins 1 and 2 showed to be active against biofilm formation of Staphylococcus aureus DSM1104. The lack of toxicity toward the studied microorganism and cell lines of pigmentosin B (2), as well as the antimicrobial effect of pigmentosin A (1), made them good candidates for further development for use in combination therapy of infections involving biofilm-forming S. aureus. The structure elucidation and determination of the absolute configuration were accomplished using a combination of spectroscopy, including 1D and 2D NMR, HRMS, Mosher ester analysis, and comparison of calculated/experimental ECD spectra. A chemotaxonomic investigation of the secondary metabolite profiles using analytical HPLC coupled with diode array detection and mass spectrometry (HPLC–DAD–MS) revealed that the production of pigmentosin B (2) was apparently specific for Gibellula sp., while the glycoasperfuran 3 was specific for C. javanica.

Compatibility and Efficacy of the Parasitoid Eretmocerus hayati and the Entomopathogenic Fungus Cordyceps javanica for Biological Control of Whitefly Bemisia tabaci.

Biological control is an effective method for whitefly management compared to the potential problems caused by chemical control, including environmental pollution and the development of resistance. Combined use of insect parasitoids and entomopathogenic fungi has shown high efficiency in Bemisia tabaci control. Here, we assessed the impacts of an entomopathogenic fungus, Cordyceps javanica, on the parasitism rate of a dominant whitefly parasitoid, Eretmocerus hayati, and for the first time also compared their separate and combined potential in the suppression of B. tabaci under semi-field conditions. Six conidial concentrations of C. javanica (1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107 and 1 × 108 conidia/mL) were used to assess its pathogenicity to the pupae and adults of E. hayati. Results showed that the mortality of E. hayati increased with higher concentrations of C. javanica, but these higher concentrations of fungus had low pathogenicity to both the E. hayati pupae (2.00–28.00% mortality) and adults (2.67–34.00% mortality) relative to their pathogenicity to B. tabaci nymphs (33.33–92.68%). Bioassay results indicated that C. javanica was harmless (LC50 = 3.91 × 1010) and slightly harmful (LC50 = 5.56 × 109) to the pupae and adults of E. hayati respectively on the basis of IOBC criteria, and that E. hayati could parasitize all nymphal instars of B. tabaci that were pretreated with C. javanica, with its rate of parasitism being highest on second-instar nymphs (62.03%). Interestingly, the parasitoids from second and third-instar B. tabaci nymphs infected with C. javanica had progeny with increased longevity and developmental periods. Moreover, experimental data from 15 day semi-field studies indicate that combined application of C. javanica and E. hayati suppresses B. tabaci with higher efficiency than individual applications of both agents. Therefore, combined applications of C. javanica (1 × 108 conidia/mL) and E. hayati is a more effective and compatible biological control strategy for management of B. tabaci than using either of them individually.

Free conidia of entomopathogenic fungi modify quality traits by changing glutathione levels after an oxidant stimulus

Adverse environmental conditions (e.g. high temperature, high osmotic pressure, atmospheric changes, solar radiation, etc.) reduce the efficacy of conidia from entomopathogenic fungi used for biological control. Strategies to improve the quality traits of conidia include the introduction of sub-lethal stress treatments applied to fungal mycelium, which can improve the resistance of generated conidia to several stress factors. This work presents for the first time evidence that free conidia of Metarhizium robertsii Xoch8.1, Beauveria bassiana Tac1.1 and Cordyceps javanica CHE-CNRCB303, subjected directly to oxidant pulses (26% O2), had increased osmotic stress resistance compared to control conidia (maintained at 21% O2). In addition, germination and thermotolerance improved in conidia of M. robertsii Xoch8.1 and B. bassiana Tac1.1. Only one pulse was enough to observe these positive effects on the stress tolerance of the free conidia. However, the Cordyceps genus showed an extreme sensitivity to the treatment, since the quality diminished for Cordyceps fumosorosea ARSEF3302, and in the case of C. javanica CHE-CNRCB303 the treatment affected those tolerance features. In addition, changes in the level of glutathione in the conidia indicated that the treatment could generate oxidative stress, which affects quality. In conclusion, despite the low metabolic activity in free conidia in a latency state, these responded to 26% O2 pulses, modifying quality traits and in some cases, inducing cross protection in a species-dependent response. The information of this research could be tested in harvested conidia used in the preparation of formulations for biological control.

Novel combination of a biosurfactant with entomopathogenic fungi enhances efficacy against Bemisia whitefly.

Microbial surfactants are multifunctional surface-active molecules that have been overlooked in formulating microbial biopesticides. We report a novel approach using the biosurfactant rhamnolipid (RML) against the destructive cosmopolitan insect pest Bemisia tabaci, as well as the combined action of RML with aerial conidia of two entomopathogenic fungi, Cordyceps javanica and Beauveria bassiana. RML was also tested as a suspension agent to improve the recovery rate of conidia from solid substrate for fungal preparations. The recovery rate of conidia increased dramatically (two to five times) with RML compared with a standard surfactant (Tween 80). Spraying solutions of 0.075% and 0.1% (w/v) RML on B. tabaci third instar nymphs induced 100% mortality within 4days. Conidial suspensions at 5 × 106 conidia/mL amended with RML at 0.01% or 0.05% markedly increased nymphal mortalities and considerably reduced LC50 . Conidial suspensions of B. bassiana with 0.05% RML added were more effective against whitefly nymphs (87.3% mortality) than C. javanica + RML (51.4% mortality). Our results show that this bacterium-based RML improved the recovery rate of hydrophobic conidia, and that mixtures of RML with fungal spore suspensions increased their insecticidal activity. © 2019 Society of Chemical Industry.

Comparative effectiveness of bioassay methods in identifying the most virulent entomopathogenic fungal strains to control Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae)

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a serious pest of cotton that inflicts huge economic losses. Excessive use of chemical pesticides for its management causes environmental pollution and pesticide resistance. Six bioassay methods and ten entomopathogenic fungal strains (EPFs) were evaluated to find out the suitable bioassay method and the most virulent strain(s) for management of B. tabaci under laboratory and polyhouse conditions. The highest tenderness and survival period (> 30 days) of the leaves and increasing trend in nymphal mortality was recorded in a new modified polyhouse bioassay method (NMPBM). NMPBM was found to be effective, simpler, and less labor intensive for evaluating large numbers of EPF strains. Twelve newly isolated EPF strains were characterized based on their morphological and molecular characteristics. The highest whitefly nymphal mortality (at 107 conidia ml−1) was recorded by Beauveria bassiana (Bb)-4511 (95.1%), Bb-4565 (89.9%), and Metarhizium anisopliae-1299 (86.7%) at the seventh day post inoculation. However, the overall bioefficacy index was higher in Bb-4511 (78.1%), Cordyceps javanica (Cj)-102 (77.0%), and Cj-089 (75.4%) than other EPF strains. The lowest values of LC50 and LC90 were with Cj-089 and Bb-4511. The field deployment of effective formulation of these most virulent EPF strains might be helpful for managing B. tabaci populations and CLCuD incidence under insecticide resistance management programs.

Identification of a new Cordyceps javanica fungus isolate and its toxicity evaluation against Asian citrus psyllid.

The Asian citrus psyllid, Diaphorina citri Kuwayama, is the most serious pest of citrus worldwide. It acts as a vector for a group of phloem‐limited bacteria (Candidatus Liberibacter spp.) that causes Huanglongbing (HLB) disease. Thus, D. citri management is an important strategy against HLB, and biological control is currently considered as the most effective method because of the unsustainable and negative side effects of chemical control. Here, we isolated a new strain of entomopathogenic fungus, Cordyceps javanica (GZQ‐1), from one cadaver of D. citri adult based on its morphological and phylogenetic data. Five conidial concentrations of the C. javanica pathogen (1 × 103, 1 × 104, 1 × 105, 1 × 106, and 1 × 107 conidia/ml) were assessed against six life stages of D. citri (1st‐5th instar nymphs and adults). Results showed that C. javanica GZQ‐1 was highly pathogenic to D. citri nymphs (69.49%–90.87% mortality) and adults (69.98% mortality). The LC50 and LT50 values of C. javanica against 1st‐2nd instar (younger), 3rd‐4th instar (middle aged), 5th instar (older), and adults were 1.20 × 105, 1.10 × 106, 4.47 × 106, 8.12 × 106 conidia/ml and 4.25, 4.51, 5.17, 5.49 days, respectively. Moreover, glasshouse experiments indicated that this C. javanica GZQ‐1 caused higher infection rates of D. citri adults compared to two other fungal strains we previously isolated in the laboratory, Cordyceps fumosorosea (IF010) and Metarhizium anisopliae (CNGD7).