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Made available in DSpace on 2022-04-29T08:33:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-04-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) In the Neotropics, leaf-cutting ants are pests of great importance to various crops. The natural defenses and complexity of these ants reduce the impact of potentially useful pathogens for biological control. Furthermore, several types of research with entomopathogenic fungi usually focus on only a few isolates of well-known species, like Beauveria bassiana and Metarhizium anisopliae, in addition to not always use isolates obtained from leaf-cutting ants. Here, we evaluated the pathogenicity of several filamentous fungi to workers of the leaf-cutting ant Atta sexdens rubropilosa. For that, we used fungi previously isolated in an extensive isolation study in Atta spp. reproductive females. Conidia suspensions of Aspergillus flavus, B. bassiana, Isaria fumosorosea and Purpureocillium lilacinum were prepared to a final concentration of 108 conidia mL− 1. In addition, we included sterilized distilled water as control and commercial B. bassiana as standard. Ant workers were immersed in these treatments for eight seconds. The number of dead workers was counted daily. Several fungal isolates killed more, or faster than the standard B. bassiana, showing that they may have a better potential for use in leaf-cutting ants´ biological control. Additionally, the present study reports for the first time the pathogenicity of P. lilacinum and I. fumosorosea towards Atta workers. Thus, we showed that an extensive and specific prospection is of importance for the development of biopesticides for leaf-cutting ants control. Departamento de Proteção Vegetal Faculdade de Ciências Agronômicas Universidade Estadual Paulista (UNESP), SP Universidade Federal do Piauí (UFPI), Piauí Departamento de Biologia Geral e Aplicada Instituto de Biociências de Rio Claro Universidade Estadual Paulista (UNESP), SP Departamento de Proteção Vegetal Faculdade de Ciências Agronômicas Universidade Estadual Paulista (UNESP), SP Departamento de Biologia Geral e Aplicada Instituto de Biociências de Rio Claro Universidade Estadual Paulista (UNESP), SP FAPESP: 2008/07032-7

Exotic invasive whiteflies (Hemiptera: Aleyrodidae) in India cause direct and indirect yield losses in agriculture, horticulture and forestry crop plants. Around 25 years ago, the spiralling whitefly, Aleurodicus dispersus Russell invaded and established on many host plants including economically important crops in India. Recently, within a span of five years, seven whiteflies invaded India viz., solanum whitefly, Aleurothrixus trachoides (Back) reported to breed on 37 plant species; rugose spiraling whitefly (RSW), Aleurodicus rugioperculatus Martin found breeding on 40 host plants; nesting whiteflies, Paraleyrodes bondari Peracchi on 34 host plants and P. minei Ιaccarino infest about 25 host plants; legume feeding whitefly, Tetraleurodes acaciae (Quaintance) infesting 5 host plants; palm infesting whitefly, Aleurotrachelus atratus Hempel on 4 host plants and woolly whitefly, Aleurothrixus floccosus (Maskell) infesting guava. These invasive species are native to the Neotropical region, mostly from Central America and the Caribbean. Extensive spread along the coastal regions and gardens near the backwater of India is predicted owing to the favorable weather factors and availability of host plants. Species of exotic whiteflies with similar habits co-exist in more or less the same niche and have a similar pattern of growth and development. The intensity of infestation of RSW on coconut, banana and oil palm, the woolly whitefly on guava and the palm infesting whitefly and nesting whiteflies on coconut was severe. The exotic aphelinid parasitoid, Encarsia guadeloupae Viggiani (Hymenoptera: Aphelinidae), a predator Pseudomallada astur (Banks) (Neuroptera: Chrysopidae) and the entomopathogenic fungus, Isaria fumosorosea Wize (Hypocreales: Clavicipitaceae) play a major role in reducing the population of these invasives. The most insidious spread of these species in India is likely mediated by humans through the movement of infested seedlings and plant materials. Extensive surveys revealed that these species spread rapidly in the large geographical region of India mostly through transportation of infested seedlings. This study reports a major expansion of the geographic and host range and the patterns of co-occurrence, damage and economic impact of these exotic species in India and their natural enemies.

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) is considered as a highly invasive and destructive agricultural pest due to its strong dispersal and adaptive capacity. We evaluated the susceptibility of B. dorsalis pupae to six entomopathogenic fungi (Isaria fumosorosea IFCF01, Beauveria bassiana Bb02, B. bassiana Bb04, Metarhizium anisopliae Ma01, M. anisopliae Ma04, and M. anisopliae Ma09) in the laboratory through impregnation method (immersing fly pupae into conidial suspension). Results indicated that fungus treatment during the pupal stage of B. dorsalis, not only caused significant mortality of target pupae (4.4–14.4% mortality), but also influence the survival of the emerged adults (38.9–72.8% mortality on the 10 days post-emergence). Among the six entomopathogenic fungi, M. anisopliae Ma04 presented the highest virulence against B. dorsalis. Results of virulence bioassay indicated that the LC50 values of M. anisopliae Ma04 against B. dorsalis declined from 5.2 × 1028 to 5.2 × 107 conidia/ml over a 1–10-day period post adult emergence, and the LT50 values decreased from 5.25 to 2.78 days with the concentrations of conidial suspension increasing from 1.0 × 108 to 1.0 × 1010 conidia/ml. Therefore, M. anisopliae Ma04 have a greater potential for B. dorsalis control.

12 páginas, 4 figuras y 2 tablas., The pine engraver beetle, Ips acuminatus Gyll, is a bark beetle that causes important damages in Scots pine (Pinus sylvestris) forests and plantations. As almost all higher organisms, Ips acuminatus harbours a microbiome, although the role of most members of its microbiome is not well understood. As part of a work in which we analysed the bacterial diversity associated to Ips acuminatus, we isolated the strain Arthrobacter sp. IA7. In order to study its potential role within the bark beetle holobiont, we sequenced and explored its genome and performed a pan-genome analysis of the genus Arthrobacter, showing specific genes of strain IA7 that might be related with its particular role in its niche. Based on these investigations, we suggest several potential roles of the bacterium within the beetle. Analysis of genes related to secondary metabolism indicated potential antifungal capability, confirmed by the inhibition of several entomopathogenic fungal strains (Metarhizium anisopliae CCF0966, Lecanicillium muscarium CCF6041, L. muscarium CCF3297, Isaria fumosorosea CCF4401, I. farinosa CCF4808, Beauveria bassiana CCF4422 and B. brongniartii CCF1547). Phylogenetic analyses of the 16S rRNA gene, six concatenated housekeeping genes (tuf-secY-rpoB-recA-fusA-atpD) and genome sequences indicated that strain IA7 is closely related to A. globiformis NBRC 12137T but forms a new species within the genus Arthrobacter; this was confirmed by digital DNA-DNA hybridization (37.10%) and average nucleotide identity (ANIb) (88.9%). Based on phenotypic and genotypic features, we propose strain IA7T as the novel species Arthrobacter ipsi sp. nov. (type strain IA7T = CECT 30100T = LMG 31782T) and suggest its protective role for its host., The project was funded by Czech Science Foundation project GACR 19-09072S. ZSS received a grant from the Junta de Castilla y León, Spanish Regional Government, and LIGD received a grant supported by Junta de Castilla y León (Escalera de Excelencia CLU-2018-04), co-funded by the P.O. FEDER of Castilla y León 2014-2020.

Understanding the interaction between pathogen virulence and host responses is very important for selecting virulent isolates of entomopathogenic fungi that control an economically important pest, such as the date palm dust mite, Oligonychus afrasiaticus (McGregor). In this study, the virulence of Metarhizium anisopliae (ARSEF 8453 and ARSEF 9374), Beauveria bassiana (ARSEF 6884 and ARSEF 8037), and Isaria fumosorosea (ARSEF 9602 and ARSEF 7284) was evaluated by virulence-regulating traits and host detoxification-related enzymatic analysis. The results revealed significant differences in virulence among all tested isolates of entomopathogenic fungi. The most virulent isolate of B. bassiana ARSEF 6884 (LT50 = 6.3 days; LC50 = 2.7 × 106 conidia ml−1) observed in the current study revealed the highest activity of cuticle-degrading protease Pr1 (2.12 ± 0.10 μmol NA ml−1 min−1) compared with the least virulent isolate of I. fumosorosea ARSEF 7284 (LT50 = 13.1 days; LC50 = 7.9 × 107 conidia ml−1). Virulent isolates successfully infected the target host and revealed lower activities of antioxidant (catalase and superoxide dismutase) and detoxification (glutathione S-transferases) enzymes at late stage infection. In conclusion, isolates ARSEF 6884 and ARSEF 9374 were found to be promising for the development of an environmentally friendly management strategy to control date palm dust mites.

The European large raspberry aphid, Amphorophora idaei (Borner) (Hemiptera: Aphididae), is one of the most important pests of the raspberry, causing significant economic damage due to its high mobility and its role as a vector of various viral plant pathogens. In the absence of effective control solutions, 16 strains of entomopathogenic fungi comprising six genera, i.e. Isaria fumosorosea, Lecanicillium longisporum, Lecanicillium muscarium, Beauveria bassiana, Metarhizium anisopliae and Metarhizium brunneum (formally known as Metarhizium anisopliae var. anisopliae) were screened for their comparative virulence against A. idaei. Three different concentrations of fungal conidial suspensions (1 × 105, 1 × 106 and 1 × 108 ml−1) of M. brunneum, I. fumosorosea and L. longisporum were sprayed on detached raspberry leaves infested with A. idaei under laboratory conditions, and further trials were conducted in the glasshouse. The results showed that these entomopathogenic fungal strains were able to reduce the number of A. idaei. However, virulence varied significantly between different fungal strains/species. The most virulent species were L. longisporum and I. fumosorosea, followed by M. brunneum. All species of B. bassiana showed least virulence. The virulence of three selected strains M. brunneum BNL102, I. fumosorosea FE 9901 and L. longisporum Vertalec were dependent on dose and insect life stage. LT50 values of A. idaei exposed to pre-treated leaves with entomopathogenic fungi were relatively lower than those of aphids that received direct inocula by spraying. Similarly, significant reduction in A. idaei populations were observed in the glasshouse. Overall, this study demonstrates the effectiveness of fungal strains that could be commercially exploited against A. idaei.

Entomopathogenic fungi are essential participants in ecosystems that help regulate arthropod communities. Many of them have a symbiotic-endophytic relationship with plants, endowing them with insecticidal or insect-repellent traits, which could be exploited for insect control in agriculture. Among insects, aphids are one of the most harmful taxa; they damage plants directly by sucking plant sap or indirectly by transmitting economically important viruses. Plant infestation by aphids depends on prefeeding activities related to host identification, particularly leaf walking and short probing. Although several aspects of aphid prefeeding activities have been studied in different hosts, this behavior has not been examined on plants colonized by entomopathogenic fungi. Here, we studied the effects of three strawberry endophytic entomopathogenic fungi, namely, Beauveria bassiana, Isaria fumosorosea and Metarhizium anisopliae var. robertsii, on the prefeeding activity of the aphid Myzus persicae by monitoring aphids feeding on leaves of fungus colonized and uncolonized plants. Our analysis revealed that the endophytes significantly affected aphid prefeeding behavior, which was typified by a reduction of the expected mean number of short probes, an avoidance to make a second probe, and a high propensity for abandoning the leaf. We discuss possible mechanisms of fungal interference with aphid activity.

A field experiment was carried out to evaluate the effectiveness of synthetic and natural preparations in protecting potato against Leptinotarsa decemlineata (Colorado Potato Beetle). Seven substances and microorganisms with proved or potential toxic effect on the pest were used: Neem extract (Azadirachta indica), suspensions of Beauveria bassiana Balsamo (Vuillemin) (Hypocreales: Cordycipitaceae) and Isaria fumosorosea Wize (Hypocreales: Cordycipitaceae) entomopathogenic fungal spores, natural pyrethrin and three chemical insecticides (lambda-cyhalothrin, thiamethoxam and imidacloprid). Plots where no protection measures were applied served as control. Chemical insecticides were more effective in controlling L. decemlineata larvae than natural preparations. It was found that chemical insecticides protected potato yield more effectively than the preparations containing natural pyrethrin or spores of entomopathogenic fungi. Tuber yield from the plots protected with entomopathogenic fungi and Neem extract was significantly higher than that on unprotected plots but still lower than the yield obtained from the plots protected with a chemical preparation.

The lady beetle Thalassa montezumae and the entomopathogenic fungus Isaria fumosorosea (Ifr) were assessed alone and in combination to suppress green croton scale, Phalacrococcus howertoni, populations on croton plants using laboratory bioassays and greenhouse cage studies. The acquisition of Ifr blastospores by beetle larvae (3rd instar) and adults during contamination in well plates was used to simulate exposure to direct spraying and subsequent possible fungal infection was assessed. Spore dispersal by the insects was determined after the blastospore-contaminated T. montezumae life stages roamed on agar plates for 24 h by counting the number of colony-forming units (CFUs) produced in the plates. There were no significant differences in survival times at 14 days post-treatment between beetle larvae and adults exposed to Ifr and those exposed to water only. Mean survival time of larvae exposed to Ifr was 14 days and water 12 days, whereas for adults it was 13 days compared to 13 days, respectively. Plates with Ifr blastospore-contaminated T. montezumae adults roaming on the agar surface displayed significantly more fungal trails as CFUs compared to plates with larvae. In greenhouse cage studies, the mean mortality rates of the scale exposed to beetle larvae, either alone (80.8%) or in combination with Ifr (89.1%), were not significantly different. Scale mortality rates in the fungus-only (60.5%) and beetle larvae-only treatments were statistically similar. The treatment with both biocontrol agents had a significantly higher scale mortality rate compared to the treatment with Ifr only. Therefore, spraying Ifr prior to releasing T. montezumae is an effective and compatible biological control strategy for management of the green croton scale on croton plants.

The entomopathogenic fungus Isaria fumosorosea Wize, formerly known as Paecilomyces fumosoroseus is reported as a promising biocontrol agent for controlling subterranean termites, particularly those belonging to the family Rhinotermitidae. In Malaysia, the family Rhinotermitidae includes two species of subterranean termites with extremely high economic importance; namely Coptotermes curvignathus Holmgren, and the Asian Subterranean Termite (Coptotermes gestroi Wasmann). To comprehend the potential control of this soil-dwelling fungus against these subterranean termites in Malaysia, an investigation was carried out by testing the pathogenecity of 11 isolates against these termite species. All isolates showed pathogenic potential against the termite (Mortality rate of C. curvignathus: 84.4%; C. gestroi: 67.3%). Isolate PF49 was the most effective against both species of termites and was further tested for its virulence and mycosis. The LC50 values of PF49 against C. curvignathus and C. gestroi were 7.55 × 103 and 1.09 × 102 conidia/ml, respectively. The average number of days required to complete the mycosis process in C. curvignathus and C. gestroi were 4.7 and 8 days, respectively. These fungi are believed useful for protecting living trees, plants, wood, wood structures, and other cellulosic materials susceptible to termite infestation and damage.

Diaphorina citri (Kuwayama) is a global pest of citrus that transmits the bacteria associated with the disease, Huanglongbing. Entomopathogenic fungi and the parasitoid Tamarixia radiata (Waterston) are important biological control agents of this pest and likely to interact in D. citri populations. As a basis for interaction studies, we determined the susceptibility of nymphs and adults of D. citri and adults of the parasitoid T. radiata to six fungal isolates from the species Beauveria bassiana s.l. (Bals.-Criv.) Vuill. (isolates B1 and B3), Metarhizium anisopliae s.s. (Metsch.) (Ma129 and Ma65) and Isaria fumosorosea Wize (I2 and Pae). We conducted experiments evaluating infection levels in all three insect groups following inoculation with a series of conidial concentrations (1 × 104–1 × 108 conidia mL−1). Results showed that D. citri nymphs and T. radiata were more susceptible to fungal isolates than D. citri adults. Overall, B. bassiana and M. anisopliae isolates caused the greatest infection compared with I. fumosorosea isolates in all three groups of insects. Isolates B1 (B. bassiana) and Ma129 (M. anisopliae) infected a greater proportion of adults and nymphs of D. citri, respectively. Both isolates of B. bassiana caused greater infection in T. radiata compared with isolates of the other fungal species. We propose that isolates B1 and Ma129 are the strongest candidates for control of D. citri. Our results represent the first report of entomopathogenic fungi infecting T. radiata, and the basis for future studies to design a biological control programme that uses both agents more efficiently against D. citri populations.

Efficacy of the five native entomopathogenic fungi (EPFs), Beauveria bassiana, Isaria fumosorosea, Lecanicillium muscarium, Metarhizium anisopliae, and Simplicillium lamellicola, against adults of the stored-grain insect pests, Sitophilus granarius and Sitophilus oryzae (Coleoptera: Curculionidae), was evaluated under laboratory conditions at two different temperatures. Conidial suspensions (1 × 108 conidia ml−1) of the fungi were directly applied to both the pests in Petri dishes (2 ml per dish), using a Potter spray tower. All the dishes were incubated both at 20 and 25 °C in 16 h light/8 h dark and in 75 ± 5% relative humidity (RH). Dead individuals were counted daily, following treatments, for 7 days. Lethal time values (LT50 and LT90) for EPFs were calculated. The results demonstrated that the mortality rates varied according to both the temperature and the tested EPFs. The highest effect among EPFs at (1 × 108 conidia ml−1) concentration on S. granarius at 20 °C at the end of day 7 was showed by I. fumosorosea (92.69%) and M. anisopliae (90.35%), followed by the other EPFs. Similarly, M. anisopliae and I. fumosorosea were the most effective ones with 90.48 and 84.21% mortality rates, respectively, at 25 °C. However, while M. anisopliae (85.68%) showed the highest effect among all the EPFs applied on S. oryzae at 20 °C, B. bassiana with a mortality rate of 93.66% was the most effective one at 25 °C. LT50 values for I. fumosorosea and M. anisopliae were 2.75 and 2.88/days, respectively, and LT90 values were 4.17 and 4.47/days, respectively, at 20 °C for S. oryzae. However, LT50 values for M. anisopliae on S. granarius in both temperatures were the lowest. This study indicated that M. anisopliae and I. fumosorosea had a significant potential as a biological control agent against S. granarius and S. oryzae. Further studies are necessary to evaluate the efficacy of the isolate on the pests under storage conditions.

The Beauveria bassiana (Bals.) Vuill. and Isaria fumosorosea (Wize) entomopathogenic fungi were either cultured on the medium composed of glucose and potato dissolved in water treated with permanent magnets or their cultures were exposed to the magnetic field. In both cases, the magnetic field provided increase in the linear growth of the fungi and their pathogenicity against the Tenebrio molitor (Col., Tenebrionidae) larvae. The effects depended on the fungus species, the applied magnets and the time of water treatment with magnets. The I. fumosorosea fungus was more sensitive to both ways of treatment than the B. bassiana one.

Spotted (Diabrotica undecimpunctata howardi) and striped (Acalymma vittata) cucumber beetles are significant pests of cucurbits, and organic management options are limited. Beetle feeding results in aesthetic damage and reduction in marketable yields as well as transmission of bacterial wilt resulting in plant mortality. Biopesticides are formulated from naturally occurring organisms such as fungi and bacteria that are pathogenic or toxic to insect pests and may be used in organic production systems. These products are thought to have lower risk to non-target organisms including mammals and beneficial insects. This research examined the potential of novel microbial products and fungal organisms to reduce cucumber beetle damage on melons. Laboratory bioassays were performed to evaluate the efficacy of a bacterial extract of Chromobacterium subtsugae and two common strains of entomopathogenic fungi, Beauveria bassiana and Isaria fumosorosea, on cucumber beetles in a controlled environment. Trials were performed against adult beetles in organic field experiments on muskmelon (Cucumis melo L. cv. reticulatus Ser.). Prior to testing in the field, anti-feedant effects were observed when beetles were exposed to leaves with Chromobacterium subtsugae and Beauveria bassiana in the laboratory. However, in field applications, we did not observe a reduction in beetle populations below the economic threshold levels, and subsequently, there was no yield increase compared to the control. Additional research is needed to improve biopesticide efficacy through formulation and timing of delivery, determine efficacy on multiple life stages of the pest, and understand interactions of microbial biopesticides in the environment and potential for non-target impacts in organic systems.

The longevity of Asian citrus psyllid, Diaphorina citri, infected by an entomopathogenic fungus, Isaria fumosorosea, was studied by microcalorimetry. Experimental results of microcalorimetry suggest that in the process of metabolism of D. citri heat or heat flow declined exponentially. The metabolism of D. citri inhibited by increasing temperature, and the longevity of D. citri decreased gradually with respect to temperature. The longevity of D. citri almost reaches 500 h at 25 °C and D. citri died within 130 h at 41 °C under high-temperature exposure. The same phenomenon, decrease in longevity and metabolism inhibition, was observed under the low-temperature exposure. The supercooling point of insects can be derived from a cooling curve plotting the temperature versus time by using microcalorimetry. The heat released due to phase change can be determined by using microcalorimeter. Our results indicated that microcalorimetry can be used to measure the metabolism of herbivore insects.

The filamentous fungus, Isaria fumosorosea, is a promising insect biological control agent. Chitinases have been implicated in targeting insect cuticle structures, with biotechnological potential in insect and fungal control. The I. fumosorosea chitinase gene, Ifchit1, was isolated and determined to encode a polypeptide of 423 amino acids (46 kDa, pI = 6.53), present as a single copy in the I. fumosorosea genome. A split marker transformation system was developed and used to construct an Ifchit1 gene knockout. The ΔIfchit1 strain displayed minor alterations in mycelial growth on diverse media at 26 °C compared to the wild type and complemented (ΔIfchit1::Ifchit1) strains; however, colony morphology was affected, and the mutant strain had a temperature sensitive phenotype (32 °C). Although sporulation was delayed for the mutant, overall conidial production was almost twice than that of wild type. Biochemical assays indicated decreased chitinase activity during growth in Czapek-Dox liquid media for the ΔIfchit1 strain. Insect bioassays using diamondback moth, Plutella xylostella, larvae revealed decreased infectivity, i.e., increased LC 50 (threefold to fourfold) and a significantly delayed time to death, LT 50 from 3 to 6 days, for the ΔIfchit1 strain compared to the wild type and complemented strains. These data indicate an important role for the Ifchit1 chitinase as a virulence factor in I. fumosorosea.

Aphids are one of the major insect pests of greenhouses and field crops worldwide. Several species of entomopathogenic fungi (EPF), including Lecanicillium spp. Beauveria bassiana, Isaria fumosorosea and Metarhizium anisopliae, have been developed commercially to control aphid species. However, there are no commercial products based on EPF that have been registered in Iraq. This study, therefore, was conducted to evaluate the virulence of four isolates of EPF, including, B. bassiana, Lecanicillium lecanii, M. anisopliae and Chaetomium globosum to select the highly virulent ones against the greenhouse aphids. Although the isolate of L. lecanii had the highest virulence against all tested aphid species in the laboratory bioassay, Myzus persicae Sulz and Aphis gossypii Gloy were more susceptible than Aphis fabae and Macrosiphum euphorbiae to infection with L. lecanii. Mortality caused by L. lecanii 7 days post-treatment with 108 conidia ml−1 was 100%. Further experiments in the greenhouses showed that L. lecanii significantly reduced the density of both M. persicae and A. gossypii 5 days after fungal applications. This isolate shows a promise for development of an alternative control agent against M. persicae and A. gossypii in Iraq.

Conidia of the entomopathogenic fungus Isaria fumosorosea are used to control insect pests in crops. Commercially available mycoinsecticides manufactured with this fungus are produced on a large scale via solid-state cultures (SSC). In order to favour gaseous exchange in SCC, texturizers can be added to increase porosity fraction (epsilon). This work presents results of water hyacinth (Eichhornia crassipes) as a novel texturizer. A mixture of parboiled rice (PR), with a epsilon = 0.23, was used as a substrate, which was then mixed with water hyacinth (WH amendment) as a texturizer at different proportions affecting epsilon. Strains CNRCB1 and ARSEF3302 of I. fumosorosea yielded 1.6 (1.49-1.71) x 10(9) and 7.3 (7.02-7.58) x 10(9) conidia per gram of initial dry rice after 8 days, at epsilon values of 0.34 and 0.36, respectively. Improvement of conidial yields corresponded to 1.33 and 1.55 times, respectively, compared to rice alone using WH amendment in the mixtures PR:WH (%) at 90-10 and 80-20. In addition, infectivity against Galleria mellonella larvae was maintained. This is the first report of the use of water hyacinth as a texturizer in SSC, affecting epsilon, which is proposed a key parameter in conidia production by I. fumosorosea, without affecting conidial infectivity.

The emergence of insecticide resistant insect pests is of significant concern worldwide. The whitefly, Bemisia tabaci, is an important agricultural pest and has shown incredible resilience developing resistance to a number of chemical pesticides. Entomopathogenic fungi such as Isaria fumosorosea offer an attractive alternative to chemical pesticides for insect control, and this fungus has been shown to be an effective pathogen of B. tabaci. Little is known concerning the potential for the development of resistance to I. fumosorosea by B. tabaci. Five generations of successive survivors of B. tabaci infected by I. fumosorosea were assayed with I. fumosorosea. No significant differences in susceptibility to I. fumosorosea, number of ovarioles, or ovipostioning were seen between any of the generations tested. Effects of I. fumosorosea and cell-free ethyl acetate fractions derived from the fungus on the B. tabaci fat body, ovary, and vitellogenin were also investigated. These data revealed significant deformation and degradation of ovary tissues and associated vitellogenin by the fungal mycelium as well as by cell-free ethyl acetate fungal extracts. These data indicate the lack of the emergence of resistance to I. fumosorosea under the conditions tested and demonstrate invasion of the insect reproductive tissues during fungal infection.

The application of entomopathogenic fungi against weevils of the genus Otiorhynchus spp. might represent an environmentally friendly management option for biological control of these insects, which have recently increased in their importance as pests in a variety of horticultural plants. As only little information is available on the susceptibility of different weevils of this genus towards various fungal species, laboratory experiments were conducted to evaluate the control potential of three different commercially available entomopathogenic fungi (Beauveria bassiana, Isaria fumosorosea, Metarhizium anisopliae) against adults of O. sulcatus, O. raucus, O. dieckmanni, O. rugosostriatus and O. crataegi. Mortality rates were dependent on fungal and weevil species, respectively. Application of Naturalis® containing B. bassiana as the active ingredient caused a significantly higher mortality rate than evident in control groups in four of the five Otiorhynchus species assessed. Adults of O. rugosostriatus were apparently not susceptible to any of the fungi tested. Efficacy of a single application of Naturalis® against adult O. sulcatus was also assessed in three independent semi-field experiments. Weevils got infected during the first 14 days after application, but mortality rates did not substantially increase until 2 months after application. Even 6 months after application, mortality of weevils was caused by the respective applied B. bassiana strain, which was confirmed via an amplification of isolate-specific microsatellite markers. Thus, a single application of a B. bassiana-based product in the field could have long-term effects on population densities of respective weevils.

SEVIM, ALI/0000-0003-2472-599X WOS: 000346176700013 Eurygaster integriceps (Hemiptera: Scutelleridae) is one of the most serious pest of wheat and other cereal crops throughout the Near and Middle East including Turkey. To contribute biological control of this pest, we investigated the culturable bacterial flora of E. integriceps and their virulence against it. We also tested different entomopathogenic fungi (Isaria fumosorosea ARSEF8356, Beauveria bassiana ARSEF8356, Metarhizium brunneum ARSEF8671 and Nomurae rileyi ARSEF1670) against this pest under laboratory conditions. Bacterial isolates were characterized based on morphological, biochemical, physiological and molecular characteristics (16S rRNA sequencing). the isolates were identified as Pantoea sp. (S1, S5, S7, S8, S10 and S11), P. agglomerans (S2, S3 and S4), Pseudomonas sp. S6 and Micrococcus luteus S9. the highest mortality within bacterial isolates was observed from Pantoea sp. S1, P. agglomerans S4 and Pantoea sp. S7 with 100%. Mortality of other bacterial species ranged from 33% to 88%. the highest mortality among the tested entomopathogenic fungi was obtained from Isaria fumosorosea ARSEF 8333 with 100%. Mortality of other fungi ranged from 33% to 50%. Consequently, Pantoea sp. S1, P. agglomerans S4, Pantoea sp. S7, Beauveria bassiana ARSEF 8356 and Isaria fumosorosea ARSEF 8356 seem to be promising candidates in the control of E. integriceps.

The efficacy of silver generated larvicide with the help of entomopathogenic fungi, Isaria fumosorosea (Ifr) against major vector mosquitoes Culex quinquefasciatus and Aedes aegypti. The Ifr-silver nanoparticles (AgNPs) were characterized structurally and functionally using UV-visible spectrophotometer followed by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy and Fourier transform infrared (FTIR) spectra. The optimum pH (alkaline), temperature (30 °C) and agitation (150 rpm) for AgNP synthesis and its stability were confirmed through colour change. Ae. aegypti larvae (I–IV instars) were found highly susceptible to synthesized AgNPs than the larvae of Cx. quinquefasciatus. However, the mortality rate was indirectly proportional to the larval instar and the concentration. The lethal concentration that kills 50 % of the exposed larvae (LC50) and lethal concentration that kills 90 % of the exposed larvae (LC90) values of the tested concentration are 0.240, 0 0.075.337, 0.430, 0.652 and 1.219, 2.210, 2.453, 2.916; 0.065, 0.075, 0.098, 0.137 and 0.558, 0.709, 0.949, 1.278 ppm with respect to 0.03 to 1.00 ppm of Ifr-AgNPs against first, second, third and fourth instars of Cx. quinquefasciatus and Ae. aegypti, respectively. This is the first report for synthesis of AgNPs using Ifr against human vector mosquitoes. Hence, Ifr-AgNPs would be significantly used as a potent mosquito larvicide.

Conidia production and quality from mycoinsecticides in solid-state cultures (SSC) are frequently inferred from superficial culture (SC) results. Both parameters were evaluated for two Isaria fumosorosea strains (ARSEF 3302 and CNRCB1), in SC and SSC, using culture media with the same chemical composition. For both strains, conidia production was higher in SC than SSC in terms of conidia per gram of dry substrate. Germination in both strains did not show significant differences between SC and SSC (>90 %). Similarly, conidia viability in ARSEF 3302 strain did not show differences at early stages between SC and SSC, but was higher in SC compared to SSC in the late stage of culture; in contrast, conidia from CNRCB1 strain did not differ between both culture systems. Some infectivity parameters improved in conidia from SSC, compared to SC at the early stages, but these differences disappeared at the final stage, independently of the strain. Both strains showed decreased conidia production when 26 % O2 pulses were applied; nevertheless, conidiation in SSC was two orders of magnitude more sensitive to oxidant pulses. In SC with 26 % O2 pulses, conidia viability for both strains at early stages, was higher than in normal atmospheric conditions. Infectivity towards Galleria mellonella larvae was similar between conidia from normal atmosphere and oxidant conditions; notably, for the strain ARSEF 3302 infectivity decreased at the final stage. This study shows the intrinsic differences between SC and SSC, which should be considered when using SC as a model to design production processes in SSC.

The pathogenicity of local isolates of the entomopathogenic fungi (EPF) Beauveria bassiana (Balsamo) Vuillemin, Isaria fumosorosea (Wize) (formerly Paecilomyces fumosoroseus (Wise) Brown & Smith) and Metarhizium anisopliae (Metschnikoff) Sorokin (Ascomycota: Hypocreales) were evaluated under laboratory conditions against pupae and adults of Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) via different routes of exposure. Average mortality of pupae after immersing them into spore suspensions was in general low and ranged from 18.7 to 23.9 % depending on fungal species and dose applied. However, adults obtained from treated pupae appeared to have higher mortality rates compared to control with all fungi and doses tested. Adults’ average mortalities ranged from 41.9 to 88.0 % after exposing them to EPF either by feeding on a mixture of conidial suspension and artificial food or by bodily contact with conidia, depending on fungal species, dose and method of exposure. The proportion of cadavers, pupae or adults, that produced visible signs of mycosis ranged from 40.4 to 73.3 % with the exception of those that were exposed to M. anisopliae. In that case, none of the insects that died after exposure to conidia of M. anisopliae developed visible mycelium on their surface. Moreover, confining treated with untreated adults indicated horizontal conidial transmission in some cases. Our results indicate that EPF might be a promising biological control agent for the Mediterranean fruit fly and could be utilised with different modes of application: soil application against pupae and cover or bait sprays against adults.

Isaria fumosorosea frequently causes mycosis of agricultural pests in the hot semiarid and dry tropical regions of Mexico. Because temperature tolerance restricts the use of fungal biopesticides, we investigated two isolates from these areas for possible development into mycoinsecticides for use in hot weather agricultural zones. We studied the effects of culture system (solid or submerged cultures) and temperature on the fungal growth, extracellular enzyme production, pathogenicity, and thermotolerance of the produced propagules. Between 20 and 28 °C, the specific growth rates of the isolate PCC were higher on solid media, but in the submerged culture, the isolate P43A grew faster even at temperatures of up to 34 °C. On solid media, P43A produced 1.5-fold more proteases than PCC, but in the submerged culture, both strains had similar activities. Under the same culture conditions, PCC produced a blastospore:conidia ratio of 1:2, and P43A produced a ratio of 1:5. PCC aerial conidia had the shortest Lethal Time 50 (LT(50), the time to reach 50 % mortality) against Galleria mellonella larvae, but LT(50) was equal for the aerial conidia and the submerged propagules of P43A and PCC. The submerged and aerial propagules of P43A were more thermotolerant than those of PCC. Each isolate performed differently in each culture system, and we concluded that the intended production method should be included as a criterion for screening of entomopathogenic fungus. We found that thermotolerance is a specific characteristic of an isolate from a given species. Because of its specific characteristics, P43A shows more promise for the development of a submerged conidia-based mycoinsecticide for foliar application in aqueous form in hot climate regions.

Rhipicephalus microplus (Canestrini) is an ectoparasite accountable for great economic losses. The use of entomopathogenic fungi to control arthropods has shown promising responses. The present study evaluated the virulence of Isaria farinosa (Holmsk.) Fr., Isaria fumosorosea (Wize) Brown and Smith, and Purpureocillium lilacinum (=Paecilomyces lilacinus) (Thom.) Samson to engorged females, eggs, and larvae of R. microplus. There were four treatment groups (10(5), 10(6), 10(7), and 10(8) conidia ml(-1)) and the control group (water and Tween 80, 0.1% v/v). The treatment was based on immersion of the specimen in 1 ml of the suspension or control solution. The study observed changes in egg viability and larval mortality after treatment. The results showed that I. farinosa, P. lilacinum, and I. fumosorosea caused alterations in the biological parameters of R. microplus ticks. I. fumosorosea presented the greatest potential to control R. microplus engorged females in vitro, causing a 49% decrease in nutritional index. All fungal isolates presented significant reduction in the egg production index. I. farinosa reduced the hatching percentage if the eggs were treated with the two highest conidial concentrations. All conidial concentrations of I. fumosorosea were able to reduce the hatching percentage significantly. All tested isolates showed pathogenicity toward unfed R. microplus larvae. As far as we know, this is the first study reporting the effect in vitro of I. farinosa, I. fumosorosea, and P. lilacinum to different developmental stages of R. microplus ticks.

Wireworms of the genus Agriotes (Coleoptera: Elateridae) are severe and widespread agricultural pests affecting numerous crops throughout the world and causing damage of economic importance. Between 2008 and 2011, a survey of the occurrence of natural microbial antagonists of wireworms was conducted. Altogether, 3,420 wireworms and two dead beetles from agricultural habitats and grassland of various parts of Germany and 700 lab-reared wireworms from Italy were screened for the presence of disease. Of these, 283 selected dead and moribund Agriotes specimens were diagnosed by light and electron microscopy for insect pathogens; 66 wireworms contained unspecified bacterial infections; and 86 specimens displayed fungal growth with 25 infected by the entomopathogenic taxa Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea, Isaria spp. and Lecanicillium sp. Nematodes were observed in 29 dead individuals, and 141 individuals showed no distinguishable pathogens. In one dead Agriotes larva, an intracellular bacterium was identified as a new Rickettsiella pathotype named ‘Rickettsiella agriotidis’. The survey resulted in a natural infection rate of 0.66 %. The possible use of these pathogens as biocontrol agents is discussed.

The Asian citrus psyllid Diaphorina citri Kuwayama is a destructive insect pest in citriculture, because it is an efficient vector of the proteobacteria 'Candidatus Liberibacter asiaticus' (Las), 'Ca. L. africanus' (Laf) and 'Ca. L. americanus' (Lam). These bacteria cause the 'huanglongbing' disease or 'greening' or 'yellow dragon' disease. The disease kills the plant and reduces fruit production. This insect pest is susceptible to entomopathogenic fungi, and we report the use of different strains of Metarhizium anisopliae, Cordyceps bassiana and Isaria fumosorosea against the nymphs and adults of D. citri under field conditions. The fungi were applied four times using a concentration of 2 £ 10 13 conidia/ha with a time interval of 15 days between applications. The percentage of control of Cb 108, Ma 65, Ma 14 and Ifr 4 was 60, 50, 40 and 35% in nymphs, and 50, 50, 42 and 22% in adults, respectively. Metarhizium anisopliae, C. bassiana and I. fumosorosea applied on Persian lime groves are more effective in reducing higher density of nymphs than adults of D. citri.

The pathogenicity of 19 hypocrealean entomopathogenic fungi from seven different genera in adult Aedes aegypti was tested. All fungi proved to be pathogenic, and Isaria fumosorosea, Lecanicillium muscarium, Lecanicillium psalliotae, Metarhizium anisopliae, Metarhizium lepidiotae, Metarhizium majus, Metarhizium frigidum, Paecilomyces carneus, and Paecilomyces lilacinus caused total mortality within 15 days of exposure of mosquitoes to the fungal culture. All fungi developed on dead individuals. The high susceptibility of adults to most tested strains underlines the interest of entomopathogenic fungi-especially those of the genera Metarhizium, Isaria, Paecilomyces and Lecanicillium--for biological control of A. aegypti.

Long-term persistence of entomopathogenic fungi as biopesticides is a major requirement for successful industrialization. Corn oil carrier was superior in maintaining germination rates of Isaria fumosorosea SFP-198 conidia during exposure to 50°C for 2 h, when compared with other oils, such as soybean oil, cottonseed oil, paraffin oil, and methyl oleate. The corn oil-based conidial suspension (91.6% germination) was also better in this regard than conidial powder (28.4% germination) after 50°C for 8 h. Long-term storage stabilities of corn oil-based conidial suspension and conidial powder at 4 and 25°C for 24 months were investigated, based on the correlation of germination rate with insecticidal activity against greenhouse whiteflies, Trialeurodes vaporariorum. Viability of conidia in corn oil was more than 98.4% for up to 9 months of storage at 25°C, and followed by 23% at 21 months. However, conidial powder had only 34% viability after 3 months of storage at 25°C, after which its viability rapidly decreased. The two conidial preparations stored at 4°C had better viabilities than those at 25°C, showing the same pattern as above. These results indicate that corn oil-based conidial suspension can be used to improve conidial persistence in long-term storage and be further applied to the formulation of other thermo-susceptible biological control agents.

Production and biocontrol efficacy of culture filtrates containing cuticle degrading enzymes from three isolates of Isaria fumosorosea against diamondback moth (Plutella xylostella) was observed during this study. This fungus when cultured in liquid medium having different carbon sources showed maximum biomass production when 1% chitin was added as carbon source. These isolates when grown in liquid culture conditions having 1% chitin as source produced cuticular degrading enzymes (proteases (Pr1 and Pr2), chitinases, chitosanase, and lipase) in a sequential manner and the production of these enzymes differed from control. Biocontrol assays with P. xylostella showed that the culture filtrates of I. fumosorosea were potent antifeedants because reduction in the feeding rate and body weight of the larvae was observed. Similarly, reduction in rates of successful pupation, adult emergence was observed when the culture filtrates were applied topically. At the end of the test period, the lowest ST50 value (1.57 ± 0.20 days) was recorded for insect groups treated with culture filtrates from isolate IF28.2 when compared to the control. In view of the need for safer and environmentally friendly pest management tools, the present study can help in the development of enzyme-based biopesticides against P. xylostella.

The whiteflies Bemisia tabaci (Gennadius) and Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) are major crop pests throughout the world. Although extensive research about biological control of whitefly by parasitoids and predators has been conducted, also entomopathogenic fungi can be considered as potential biological control agents. Surveys for entomopathogenic fungi were carried out in organic and conventional horticultural crops in greenhouses and open fields in Buenos Aires and Corrientes provinces, Argentina. These surveys resulted in the recovery and isolation of the following fungi from whiteflies: Lecanicillium lecanii (Zimmerm.) Zare & W. Gams, L. muscarium (Petch) Zare & W. Gams, L. longisporum (Petch) Zare & W. Gams, Isaria fumosorosea Wize and I. javanica (Frieder. & Bally) Samson & Hywel-Jones. Pathogenicity tests were conducted against T. vaporariorum nymphs using a conidial suspension (1 × 107 conidia/ml) of the fungi. A mortality rate between 26.6% and 76.6% was obtained at 7 days post-infection. These are the first records of natural infections in the southernmost region of the South American continent of L. lecanii, L. muscarium, L. longisporum and Isaria javanica (Ascomycota: Hypocreales) on T. vaporariorum and also the first report of I. fumosorosea on B. tabaci.

Background RNA interference (RNAi) technology shows a great potential in controlling agricultural pests, despite the difficulty of introducing exogenous dsRNA/siRNA into target pests. Isaria fumosorosea is a common fungal pathogen of the B-biotype Bemisia tabaci (whitefly), which is a widespread pest. Entomopathogenic fungi directly penetrate the cuticle and invade insect hemocoel. Application of I. fumosorosea expressing dsRNA of whitefly immunity-related gene may aid in developing RNAi technology to effectively control whiteflies. Methods A dsRNA expression plasmid, psTLR7, was constructed by introducing the Toll-like receptor 7 (TLR7) gene of B-biotype whitefly to the silent vector, pSilent-1. The plasmid psTLR7 was transferred into the protoplast of the I. fumosorosea strain IfB01. Then, the recombinant strain was screened out based on the biological stability and bioactivity against whitefly. Results A genetically stable recombinant strain IfB01-TRL7 was screened out. The impact of IfB01-TRL7 against whitefly TRL7 gene was validated by qPCR. Lower expression levels of the TLR7 gene was observed in the whiteflies infected by the recombinant strain. The bioassay results indicated that compared to IfB01 strain, IfB01-TRL7 increased the mortality of whitefly nymphs, and decreased and shortened the values of LC50 and LT50, thus indicating higher virulence of IfB01-TRL7. Conclusion The expression of the dsRNA of whitefly TLR7 gene in recombinant I. fumosorosea strain successfully knocked down the host target gene by infecting the nymphs and enhanced the whiteflies mortality. The present study will give insight to new application of RNAi technology for more effective biocontrol of this pests. Electronic supplementary material The online version of this article (doi:10.1186/s12896-015-0170-8) contains supplementary material, which is available to authorized users.

The bioactivity of three kinds of multi-walled carbon nanotubes (MWCNT) towards the conidia of entomopathogenic fungus Isaria fumosorosea was examined in an in vitro study. Commercial—raw and functionalized—carboxylated MWCNT were applied. A fungal conidia suspension was placed in contact with dispersed MWCNT over different time-periods. After contact with the nanomaterial, the conidia were cultured on dishes and both the linear vegetative mycelium growth and the sporulation and germination of the spores derived from the culture were investigated. Also, the pathogenicity of the conidia after contact with MWCNT was examined in relation to test larvae. No fungistatic activity of MWCNT relative to I. fumosorosea conidia was demonstrated. Conidia contact with MWCNT resulted in the following changes in vital processes in the subsequent culture compared to the control standard culture: (1) raw MWCNT limited mycelium inoculation, but the growth rate observed later in the log-phase was more intense; (2) after 24-h conidia contact with all MWCNT types, the mycelium sporulated the most intensively; longer contact resulted in sporulation process limitation. Germination of conidia after contact with the MWCNT was not significantly modified. Raw MWCNT potentiated conidia pathogenicity towards test insects. It was observed that carboxylation of MWCNT reduces the bioactivity of this nanomaterial towards the investigated conidia.