Horizontal Transmission of Entomopathogenic Fungi by the Diamondback Moth

The relative potential of the pathogenic fungi Beauveria bassiana and Zoophthora radicans for use as autodisseminated biological control agents of the diamondback moth (Plutella xylostella) was compared. The LC₅₀ of B. bassiana conidia to third instar larvae was 499 conidia/mm² of leaf surface and individual cadavers of mycosed fourth instar larvae yielded a mean of 67.5 × 10⁶ (±7.5 × 10⁶) conidia. All concentrations of B. bassiana tested in inoculation chambers (0.24, 2.4, and 6.2 μg/mm²) induced 100% mortality in adult male moths within 7 days. The times to death and sporulation were concentration and exposure duration dependent. A standard procedure for inoculating male moths resulted in >85% mortality from Z. radicans and >93% mortality from B. bassiana. Pairing of inoculated males with clean moths of both sexes yielded higher rates of passive transmission of B. bassiana than Z. radicans, but there was no evidence for sexual transmission of either pathogen. Similarly, B. bassiana was more effectively transmitted from inoculated male moths to larvae foraging on whole plants. Single sporulating cadavers producing B. bassiana or Z. radicans conidia placed on plants infested with larvae resulted in a similar rate of transmission for both pathogens. However, an increase of the density of sporulating cadavers from one to three/plant increased Z. radicans transmission (greater than fourfold) but had no effect on B. bassiana transmission. Simultaneous inoculations of larvae with conidia of both fungi reduced the mortality induced by each pathogen, the reduction being most acute for B. bassiana-induced mortality. Inoculation of adults with both fungi showed that, at concentrations required for effective passive transmission to larvae, B. bassiana severely inhibited Z. radicans mycosis in adults.     

Infection byBeauveria bassiana ofLeptinotarsa decemlineata larvae as a consequence of fecal contamination of the integument followingper os inoculation

A study ofper os inoculated larvae was undertaken to determine if the entomopathogenic fungus,Beauveria bassiana, is able to infect the Colorado potato beetle,Leptinotarsa decemlineata, via the alimentary tract. Surface orper os inoculated larvae which were immediately surface sterilized post inoculation did not succumb to infection, whereas those larvae not sterilized became infected. Histological studies of fed or starved, agnotobiotic (with microbial flora) and axenic larvae revealed that conidia can germinate in the gut regardless of the presence of gut microflora. However, infection via the alimentary tract was never observed in fed larvae and only noted in a single starved individual. It is concluded that infections ofper os inoclated larvae occurred after surface contamination of the integument by viable conidia contained in the frass. The rate of food passage through the gut is probably important in preventingper os infections.     

Pathogenicity of Beauveria bassiana (Deuteromycotina: Hypomycetes) to Larvae of the Small Poplar Longhorn Beetle, Saperda populnea (Coleoptera: Cerambycidae)

The small poplar longhorn beetle, Saperda populnea is an important pest of Lombardy poplars (Populus nigra L.) in Turkey. A survey for natural entomopathogenic fungi of S. populnea larvae was made in Erzurum, Turkey, during the period 2004–2005. Larvae (13.5%) infected with a strain of the fungus Beauveria bassiana were found. The pathogenicity of B. bassiana strain 46 was conducted with different concentrations of conidia (10⁶, 10⁷ and 10⁸ conidia/ml) of this isolate on S. populnea larvae. The lowest concentration (10⁶ conidia/ml) caused about 56% mortality within 6 days. One hundred percent mortality was achieved after median lethal time (LT₅₀) of 4.6 and 4.4 days for 10⁷ and 10⁸ conidia/ml, respectively. There were no significant differences between median lethal times. This is the first record of natural infection of S. populnea larvae by B. bassiana.     

Susceptibility of different life stages of the European cherry fruit fly,Rhagoletis cerasi,to entomopathogenic fungi

The effects of six fungus isolates on the mortality of different life stages of the European cherry fruit fly, Rhagoletis cerasi (Diptera: Tephritidae), were assessed in a series of laboratory experiments to find an isolate suitable for biological control. In a first step, the effects of fungus treatments on mortality, mycosis and fecundity of adult flies at a concentration of 10⁷ conidia/ml were evaluated. All fungus isolates caused mycosis but virulence varied considerably among the isolates. Beauveria bassiana and Isaria fumosorosea caused 90–100% mortality and had the strongest influence on fecundity. Metarhizium anisopliae also induced high rates of mortality, while the pathogenicity of Isaria farinosa was low. The effects of lower conidia concentrations and the influence of the age of flies were assessed in a second step. Higher conidia concentrations generally resulted in a higher mortality. B. bassiana was most efficient at low concentrations. Young flies showed lower mortality rates than older flies but, sub‐lethal effects on eclosion rate of eggs were greater in younger flies. Finally, the effects on L₃ larvae were tested: none of the fungus isolates induced mortality in more than 25% of larvae. As L₃ larvae and pupae are not susceptible to fungus infection, field control of R. cerasi should be focused on adult flies.     

Formulation and application of the entomopathogenic fungus: Zoophthora radicans (Brefeld) Batko (Zygomycetes: Entomophthorales)

Aims: To isolate and formulate a native strain of Zoophthora radicans naturally infecting larvae of diamondback moth, Plutella xylostella, existing in South Australia and to provide evidence that formulation of the fungus is effective against P. xylostella larvae, and therefore, it could be used as a tool in pest management of this insect. Methods and Results: Dose–response bioassays using formulated and unformulated forms of the fungus strain were carried out against third instar larvae of P. xylostella. Results obtained have indicated a significant increase in the larval mortality when higher concentrations of a formulated form of the fungus strain were applied compared to the treatments with the unformulated form (85·0 vs 57·5% of larval mortality, respectively, at the top concentration of 10⁷ conidia/ml). The median lethal concentration (LC50) for a formulated form was 100 times less than that of the unformulated form when they were applied against the third instar larvae of P. xylostella. In addition, the formulation used in the present bioassays has preserved the viability of introduced fungus conidia for longer time in comparison with the unformulated conidia. Conclusions: The effective application of a formulated fungus strain against P. xylostella larvae constitutes the first step towards its use in pest management of this insect. Significance and Impact of the Study: The formulated fungus in inverted emulsion could be used as an alternative tool to insecticides in pest management of P. xylostella larvae because of the development of resistance to insecticides in the treated larvae.     

Passive vectoring of entomopathogenic fungus Beauveria bassiana among the wax moth Galleria mellonella larvae by the ectoparasitoid Habrobracon hebetor females

Females of the ectoparasitoid Habrobracon hebetor attack and envenomate numerous host individuals during oviposition. The vectoring of the entomopathogenic fungus Beauveria bassiana during the adhesion stage by ectoparasitoid females among the wax moth larvae Galleria mellonella was explored under laboratory conditions. Vectoring occurred both from infected parasitoids to wax moth larvae and from infected to healthy wax moth larvae by parasitoids. The efficacy of vectoring in both cases was dose dependent. Parasitoid females were unable to recognize infected larvae in a labyrinth test. In addition, the presence of H. hebetor females significantly (1.5–13 fold) increased the mycoses level in clusters of G. mellonella, with 40% of the larvae infected with fungal conidia. Envenomation by H. hebetor increased conidia germination on the cuticles of the wax moth larvae by 4.4 fold. An enhanced germination rate (2 fold) was registered in the n‐hexane epicuticular extract of envenomated larvae compared to that of healthy larvae. Both envenomation and mycoses enhanced the phenoloxidase (PO) activity in the integument of G. mellonella and, in contrast, decreased the encapsulation rate in hemolymphs. We hypothesize that changes in the integument property and inhibition of cellular immunity provide the highest infection efficacy of entomopathogenic fungi with H. hebetor.     

Use of quantitative PCR to understand within-host competition between two entomopathogenic fungi

Interactions between the entomopathogenic fungi Zoophthora radicans and Pandora blunckii infecting larvae of Plutella xylostella were investigated. This is the first report to quantify within-host growth of one fungus in the presence of another competing for the same host resource using quantitative PCR (qPCR) at regular time points during the infection process. In larvae inoculated only with Z. radicans, there was a cumulative increase in the quantity of Z. radicans DNA throughout the time course of infection. However, in dual-inoculated larvae, there was an initial accelerated rate of growth of Z. radicans compared to when it was inoculated alone, but by the time of host death it had been effectively excluded by P. blunckii. The implications of these results for co-existence of these fungal pathogens in the field are discussed.     

Competition and co-existence of Zoophthora radicans and Pandora blunckii, two co-occurring fungal pathogens of the diamondback moth, Plutella xylostella

The entomopathogenic fungi Zoophthora radicans and Pandora blunckii co-occur in field populations of Plutella xylostella and, therefore, are likely to interact during the infection process. We have investigated the possible outcomes of these interactions in the laboratory. Using four isolates, two of each fungal species, inter-specific interaction experiments were done in Petri dishes and on intact plants. In Petri dish experiments, larvae were inoculated directly using sporulating mats of mycelium, both species had the same opportunity to infect and only the relative concentration of conidia of each pathogen species applied was manipulated. In the intact plant experiments, larvae were placed onto fungus-contaminated plants, inoculation was passive and the probability of infection by either or both species of fungi depended on larval activity and proximity to inoculum. In the Petri dish experiment, the species with the largest concentration of conidia out-competed the other regardless of virulence, and results were similar in the intact plant experiment. The ecological implications for competition or co-existence of these two pathogens in the field are discussed.     

Evaluation of the fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes), a potential biological control agent of Lutzomyia longipalpis (Diptera, Psychodidae)

Visceral leishmaniasis is a zoonosis whose primary vector in Brazil is the sandfly Lutzomyia longipalpis Lutz & Neiva. Presently, efforts to control the vector have not been effective in reducing the prevalence of disease. A possible alternative to current strategies is the biological control of the vector using entomopathogenic fungi. This study evaluates the effects of the fungus, Beauveria bassiana (Bals.) Vuilleman, in different developmental stages of L. longipalpis. Five concentrations of the fungus were utilized ranging from 10⁴ to 10⁸ conidia/ml, with appropriate controls. The unhatched eggs, larvae and dead adults exposed to B. bassiana were sown to reisolate the fungus. The fungus was subsequently identified by polymerase chain reaction (PCR) and DNA sequencing. Exposure to B. bassiana reduced the number of eggs that hatched by 59% (P < 0.01). The longevity of infected adults was 5 days, significantly lower than that of the negative control which was 7 days (P < 0.001). The longevity of the adult sandfly exposed to the positive chemical (pyrethroid, cypermetherin) control was less than 1 day. The effects of fungal infection on the hatching of eggs laid by infected females were also significant and dose-dependent (P < 0.05). With respect to fungal post-infection growth parameters, only germination and sporulation were significantly higher than the fungi before infection (P < 0.001). The identity of the reisolated fungus was confirmed by automated DNA sequencing post-passage in all insect stages. These data show that B. bassiana has good pathogenic potential, primarily on L. longipalpis larvae and adults. Consequently, the use of this fungus in sandfly control programs has potential in reducing the use of chemical insecticides, resulting in benefits to humans and the environment.     

Effects of Selected Fungicides and the Timing of Fungicide Application on Beauveria bassiana-Induced Mortality of the Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Four fungicides used for controlling foliar diseases of potato (Solanum tuberosum) were evaluated under field and laboratory conditions for their effects on the infectivity and sporulation of Beauveria bassiana when used as a control for the Colorado potato beetle, Leptinotarsa decemlineata (CPB). We investigated the direct effects of fungicides on B. bassiana-induced CPB mortality and the effect of time between fungicide and B. bassiana application. Effects of fungicide on conidial survival in the soil and on foliage were examined in the field. Significantly more larval mortality was observed when larvae were sprayed with B. bassiana than with the water control. Fungicide had no significant effect on larval mortality in the field. In the laboratory, survival of larvae was significantly lower among larvae fed fungicide-treated foliage. B. bassiana-induced mortality in the laboratory was observed only when larvae were fed foliage treated with copper hydroxide or water. Larvae fed mancozeb- or chlorothalonil-treated foliage experienced high mortality regardless of B. bassiana treatment. While there was no significant effect of fungicide on B. bassiana sporulation on cadavers in the field, a pattern emerged that indicated higher proportions of cadavers producing conidia in plots sprayed with water or copper hydroxide than in plots sprayed with chlorothalonil or mancozeb. Survival of B. bassiana conidia in the soil and on foliage was significantly greater in plots treated with copper hydroxide or water than in plots treated with mancozeb or chlorothalonil. Fungicides such as copper hydroxide may be less deleterious to the fungus than mancozeb and chlorothalonil.     

Isolation of Beauveria species from Lebanon and evaluation of its efficacy against the cedar web-spinning sawfly, Cephalcia tannourinensis

Larvae of the cedar web-spinning sawfly, Cephalcia tannourinensis Chevin (Hymenoptera: Pamphiliidae), infected with a white fungus were collected from the Tannourine-Hadath El-Jebbeh cedar forest. Macro- and micro-morphological data based on the examination of colonies, conidiophores, and conidial shape of the fungus suggested a Beauveria species. Sequence analysis of the internal transcribed spacer regions of the isolated fungus showed that it is most closely related to isolates of B. bassiana Clade C. The present study showed that the isolated B. bassiana is a naturally occurring entomopathogenic fungus parasitizing the larvae of C. tannourinensis in Lebanon. Laboratory bioassays showed that B. bassiana caused high mortality of eggs and larvae. The infected eggs turned brownish in color, while larvae of the first instar ceased feeding and showed immobility and rigidity within 5 days and before sporulating conidial mat appeared on their cuticle. Second and third larval instars took longer time to show fungal sporulation: mortalities ranged between 85 and 100% within 7 days when treated with different conidial concentrations. The efficacy of control of C. tannourinensis using B. bassiana was higher or equal to the reference insect growth regulator, diflubenzuron, suggesting the possibility of its success as a biological control agent.     

The effect of Fusarium verticillioides on oviposition behaviour and bionomics of lepidopteran and coleopteran pests attacking the stem and cobs of maize in West Africa

Abstract The effect of Fusarium verticillioides Sacc. (Nirenberg) in maize stem and grain on the oviposition behaviour and bionomics of lepidopteran and coleopteran pests in West Africa was studied in olfactometer, greenhouse, and field trials in Benin. In a choice experiment, the pyralid Eldana saccharina (Walker) laid on average 31.9 eggs on inoculated maize stems vs. 9.2 and 7.8 on stems from plants, grown from hot‐water or fungicide treated seeds, respectively. For the pyralid Mussidia nigrivenella (Ragonot) the values were 42.1 in the inoculation and 7.8 eggs in the fungicide treatment. The survival of E. saccharina larvae was significantly higher from the inoculation than the hot‐water or fungicide treatments. Fecundity in the three treatments was 494, 307, and 268 eggs per female, respectively. In an olfactometer experiment, no significant differences were found in the time spent by the curculionid Sitophilus zeamais Motschulsky between the odor field with Fusarium‐infected grain and with uninfected grain, but both were significantly different from the controls. Significant differences were obtained for the nitidulid Carpophilus dimidiatus (Fabricius) with 50.5 and 35.8%, respectively, of the time spent in the Fusarium‐infected and non‐infected odor field. For both species, no differences were found between sexes. For S. zeamais, development time was significantly higher when reared on infected (37.2 days) than on non‐infected grain (34.4 days), whereas fecundity, expressed as number of progenies during the first 4 weeks, was lower on the inoculated grain (11 vs. 16 offspring per female). For C. dimidiatus the situation was the reverse: development time was shorter and fecundity was higher on infected grain (32.4 vs. 34.4 days, and 18 vs. 13 offspring per female, respectively). It was concluded that the higher pest densities found in the stem and ear of field grown maize was due to a higher attraction to and higher immature survival and adult fecundity on F. verticillioides infected plants.     

Efficacy of Beauveria bassiana (Bals.) Vuill. against the tarnished plant bug, Lygus lineolaris L., in strawberries

Beauveria bassiana has a high insecticidal potential to control the tarnished plant bug, Lygus lineolaris, a significant pest of strawberries. Screening experiments showed that L. lineolaris adults were susceptible to several B. bassiana isolates. Another screening test with Coleomegilla maculata, a natural enemy found in strawberries, was also performed in order to select the isolate having lower entomopathogenic impact on this insect. Based on data obtained from both insect species and on the ecozone origin of the B. bassiana isolates, INRS‐IP and INRS‐CFL isolates were selected for further experiments. The LC₅₀ values of these two isolates against L. lineolaris adults were 7.8 × 10⁵ and 5.3 × 10⁵ conidia/ml, and average survival time (AST) values were 4.46 and 4.37 days at a concentration of 1 × 10⁸ conidia/ml respectively. Results also indicated that L. lineolaris nymphs are susceptible to the selected isolates. During field experiments, using a randomized block design with four replicates, INRS‐IP and INRS‐CFL isolates were applied at two rates (1 × 10¹¹ and 1 × 10¹³ conidia/ha) weekly during a period of 4 weeks. These multiple applications triggered a significant reduction of L. lineolaris nymphal populations in strawberries. Twenty‐four days after the first application, a significant difference was observed between the mean population densities of surviving nymphs in all B. bassiana‐treated plots (less than one insect per five plants) compared with those in control plots (four insects per five plants). During the field experiment, persistence of insecticidal activity and viability of B. bassiana conidia were also monitored. The results showed the presence of viable and infective conidia up to 6 days after each application on strawberry foliage. Moreover, the multiple applications of B. bassiana at the rate of 1 × 10¹³ conidia/ha triggered a significant reduction in strawberry fruit injuries induced by L. lineolaris feeding behaviour compared with the control plots.     

Preliminary caged‐field trial,using the fungal pathogen Zoophthora radicans brefeld (zygomycetes: entomophthorales) against the diamondback moth Plutella xylostella L. (lepidoptera: Yponomeutidae) in the UK

Zoophthora radicans Brefeld was tested for control of Plutella xylostella L. in a caged‐field trial. No infection was seen in control cages, but between 36% and 68% of larvae in live samples, and 38% and 55% of larvae at the final harvest, were infected in inoculated cages, suggesting the biological control potential of this fungus.     

Management of entomopathogenic fungi in cultures of Tenebrio molitor (Coleoptera: Tenebrionidae)

Larvae of mealworms Tenebrio molitor L. (Coleoptera: Tenebrionidae) have been used as animal feed, but fungal pathogens rapidly downsize the populations, resulting in economic losses. In this work, we established an effective management strategy for fungal pathogens. An entomopathogenic fungus, Beauveria bassiana, was isolated from mealworm cadavers. The bioassay of some isolates of this species at >90% relative humidity revealed that the ERL1575 isolate had the highest virulence. At 20–30% RH, ERL1575 conidia when ingested produced 80% mortality but when sprayed topically produced only <10% mortality. Mealworms that had ingested conidia were exposed to 20, 25, 30 and 35°C and high humidity (>95%) for 5 days. This experiment produced about 90% mortality except at 35°C where mortality was <20%. When 40 fungicides were assayed against ERL1575, fluazinam (1000‐fold) and mancozeb (667‐fold) significantly inhibited conidial germination and/or hyphal growth. When fluazinam and mancozeb were added to the mealworm diet of conidia‐inoculated wheat bran, most were alive 3 days post application. However, 100% mortality resulted 3 days post application in the conidia‐inoculated wheat bran without any fungicides. In conclusion, B. bassiana isolates are pathogenic at <30°C when they are ingested by mealworms but fluazinam and mancozeb can be used for management to control the pathogen in their cultures.     

Selection of promising fungal biological control agent of the western flower thrips Frankliniella occidentalis (Pergande)

Aims: Larval stages of Frankliniella occidentalis are known to be refractory to fungal infection compared with the adult stage. The objective of this study was to identify promising fungal isolate(s) for the control of larval stages of F. occidentalis. Methods and Results: Ten isolates of Metarhizium anisopliae and eight of Beauveria bassiana were screened for virulence against second‐instar larvae of F. occidentalis. Conidial production and genetic polymorphism were also investigated. Metarhizium anisopliae isolates ICIPE 7, ICIPE 20, ICIPE 69 and ICIPE 665 had the shortest LT₅₀ values of 8·0–8·9 days. ICIPE 69, ICIPE 7 and ICIPE 20 had the lowest LC₅₀ values of 1·1 × 10⁷, 2·0 × 10⁷ and 3·0 × 10⁷ conidia ml^?1, respectively. Metarhizium anisopliae isolate ICIPE 69 produced significantly more conidia than M. anisopliae isolates ICIPE 7 and ICIPE 20. Internally transcribed spacers sequences alignment showed differences in nucleotides composition, which can partly explain differences in virulence. Conclusion: These results coupled with the previous ones on virulence and field efficacy against other species of thrips make M. anisopliae isolate ICIPE 69 a good candidate. Significance and Impact of the Study: Metarhizium anisopliae isolate ICIPE 69 can be suggested for development as fungus‐based biopesticide for thrips management.     

Effects of combining <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Nosema pyrausta</Emphasis> on the mortality of <Emphasis Type="Italic">Ostrinia nubilalis</Emphasis>

We tested the combined effect of the fungus Beauveria bassiana and the microsporidium Nosema pyrausta on the European corn borer larvae, Ostrinia nubilalis, in the laboratory. The first instar of O. nubilalis larvae was the most sensitive to the B. bassiana infection followed by the fifth, second, third, and fourth instar (LC₅₀s were 4.91, 6.67, 7.13, 9.15, and 6.51 × 10⁵ conidia/ml for the first to fifth instars, respectively). Mortality of each instar increases positively with concentration of conidia. When B. bassiana and N. pyrausta were used in combination, mortality increased significantly in all instars. Relative to the B. bassiana treatment alone, the B. bassiana + N. pyrausta treatment decreased the LC₅₀s by 42.16%, 37.63%, 21.60%, 27.11%, and 33.95% for the first to fifth instars, respectively. The combined effects of the two pathogens were mostly additive. However, at the two highest concentrations the pathogens interacted synergistically in the first and second instar. Individuals that survived the B. bassiana and B. bassiana + N. pyrausta treatments and developed into adults had significantly shorter lifespans and females oviposited fewer eggs than non-exposed insects. The effects on the longevity and the egg production were most pronounced at high concentration of B. bassiana conidia.     

Microbial control of Agrilus planipennis (Coleoptera: Buprestidae) with Beauveria bassiana strain GHA: field applications

The effects of Beauveria bassiana strain GHA, applied as BotaniGard ES, on newly colonised and well-established populations of emerald ash borer, Agrilus planipennis (Coleoptera: Buprestidae) were evaluated in the field using foliar and trunk sprays in Michigan in 2004–2005. Results from field trials at a newly colonised white ash site showed a 41% reduction in A. planipennis population in fungal-treated trees compared with that of untreated controls. In addition, fungal infection was also found in 20% of the larval population within 14 days of incubation under laboratory conditions. At a site with a well-established Agrilus planipennis population in green ash trees, larval density was reduced by 47% for trees treated with the fungus compared with that of the controls; 21% of larvae from the current generation were found infected after 14 days of laboratory incubation. Fungal-treated green ash trees also produced fewer adults emerging in the next generation, with a 63% reduction in adult density observed in treated trees compared to that of controls. As a result, fungal-treated trees sustained 42% less crown dieback than did controls. A. planipennis larval density was negatively correlated with trunk height above the ground, and positively correlated with log diameter. Results of laboratory leaf bioassays on A. planipennis adults showed that fungal conidia persisted well under field conditions, with mortality of 78–100% at 7 – days post – exposure for leaves collected between 2 and 264 h after application. Potential strategies for using B. bassiana strain GHA for managing A. planipennis are discussed.     

Isolation of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) from the Chagas disease vector, <Emphasis Type="Italic">Triatoma infestans</Emphasis> (Hemiptera: Reduviidae) in Argentina

A survey for natural entomopathogenic fungi of the Chagas disease vector Triatoma infestans was conducted in five provinces of Argentina since 2001. Nymphs (1.5%) and adults (3.3%) infected with a strain of the fungus Beauveria bassiana were found at Dean Funes, Córdoba province, Argentina. Field collected insects that died in the laboratory were maintained in moist chambers and incubated at 22 °C. Beauveria bassiana from infected insects was cultured on SDAY media. Pathogenicity tests were conducted with a conidial suspension (1 × 10⁷ conidia/ml) of this isolate on T. infestans adults. A mortality rate of 100% was obtained at 15 days post-infection. This is the first record of natural infection of T. infestans by B. bassiana.