Comparison of volatile blends and nucleotide sequences of two Beauveria bassiana isolates of different virulence and repellency towards the termite Macrotermes michealseni.

Isolates of the fungus Beauveria bassiana have different levels of virulence and repellency against the termite Macrotermes michaelseni. In the present study, we compared the volatile profiles and gene sequences of two isolates of the fungus with different levels of virulence and repellence to the termite. Gas chromatography-mass spectrometric analyses showed quantitative and qualitative differences in the composition of volatiles of the two isolates. The repellencies of synthetic blends of 10 prominent components that mimicked the volatiles of each of the two isolates were significantly different. Subtractive bioassays showed that the repellency of each isolate was due to synergistic effects of a few constituents. As previously reported for isolates of Metarhizium anisopliae, some differences also were found in the nucleotide sequences of the two isolates of B. bassiana, suggesting a genetic basis for the observed intra-specific differences in their repellency and virulence against the termite.

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 LT(50) values of 8.0-8.9 days. ICIPE 69, ICIPE 7 and ICIPE 20 had the lowest LC(50) values of 1.1 × 10(7), 2.0 × 10(7) and 3.0 × 10(7) 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.

Attraction response of adult Rhipicephalus appendiculatus and Rhipicephalus pulchellus (Acari: Ixodidae) ticks to extracts from Calpurnia aurea (Fabaceae).

Experiments were carried out to investigate the response of two tick species Rhipicephalus pulchellus Gerstaker, 1873 and Rhipicephalus appendiculatus Neumann, 1901 to three different extracts (acetone, aqueous and oil) of the dried leaves of Calpurnia aurea (Aiton) Benth in both an inverted glass tube and a dual choice T-olfactometer. The oil extract at 50 and 100mg/ml attracted 46.7% and 65.9% of R. appendiculatus, respectively, in the inverted glass tube assay, which was comparable to 47.8% of the attraction-aggregation-attachment pheromone (AAAP) used as positive control. At a dose of 100mg/ml the oil extract attracted 52.4% of R. pulchellus in the T-olfactometer bioassay. The relative attraction of both tick species to plant extract was also tested in semi-field plot experiments using a trap baited with different concentrations of emulsifiable extract of C. aurea. A dose of 100mg/ml attracted 52.2% of R. pulchellus and 44.4% of R. appendiculatus from a distance of 1m while 14.4% of R. pulchellus and 12.2% of R. appendiculatus were attracted from 5m distance at the same dose. Addition of CO(2) to the plant extract-baited-trap at the dose of 100mg/ml increased the range of attraction of adult R. pulchellus (44.4% from 5m distance) and up to 33.3% of adult R. appendiculatus tick from a distance of 4m. The results of this study suggest that extracts from C. aurea can potentially be used as baits in a trap for the control of ticks in the field.

Pathogenicity of entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana (Hypocreales: Clavicipitaceae) isolates to the adult pea leafminer (Diptera: Agromyzidae) and prospects of an autoinoculation device for infection in the field.

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and three isolates of Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) were evaluated for their pathogenicity to the adult pea leafminer, Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae), in the laboratory. Flies were contaminated with dry conidia through a velvet material wrapped around the inner side of a cylindrical plastic tube. All the isolates were pathogenic to the pea leafminer, causing mortality between 40 and 100% at 5 d after exposure. The lethal time for 50% mortality (LT(50)) ranged from 2.6 to 5.4 d, whereas the LT(90) values varied between 3.2 and 9.1 d depending on the isolate. An autoinoculation device was evaluated in cage field experiments using only one of the virulent isolates, M. anisopliae ICIPE 20. The device was loaded with 2-3 g of dry conidia. Mortality of up to 100% was observed in flies captured from fungus-treated cages held under laboratory conditions. The average number of spores picked up by a single fly visiting the device increased with days after inoculation. One day after the inoculation, adults picked up an average of 4.1 +/- 0.7 x 10(5) conidia and 39.6 +/- 4.0 x 10(5) conidia 5 d after inoculation. Depending on the sampling date, the LT(50) varied between 1.8 and 3.4 d. Results indicate that some isolates of B. bassiana and M. anisopliae are highly pathogenic to L. huidobrensis, suggesting a potential for their use in the control of this pest. They also suggest the possibility of L. huidobrensis suppression with fungi using an autoinoculation device.

Performance of a Metarhizium anisopliae-treated semiochemical-baited trap in reducing Amblyomma variegatum populations in the field.

Experiments were carried out to evaluate the efficacy of Metarhizium anisopliae (Metsch.) Sorok. (Ascomycota: Hypocreales)-treated semiochemical-baited traps for control of Amblyomma variegatum Fabriscius (Acari: Ixodidae) under field conditions. Unfed A. variegatum adults (118) were seeded in each 100-m plot and allowed to acclimatise for 3 days. On the fourth day (Day 4), an emulsifiable formulation of M. anisopliae (consisting of 49.5% sterile distilled water, fungal conidia, 49.5% corn oil and 1% Tween 80) titrated at 10(9)conidia ml(-1) was applied in semiochemical-baited traps (900 cm(2)) which were placed at five spots within the plot. The control and fungal treatments were repeated after 14 and 28 days soon after rotating the traps clockwise (45 degrees ) in order to cover different sections of the plot. In the control plots, emulsifiable formulation without fungus was applied in the semiochemical-baited traps. Six weeks after the initiation of the experiments, five semiochemical-baited traps (untreated) were deployed in each plot for 3 successive days to trap ticks in the treated and control plots. The percentage of ticks recovered in the fungus-treated plots were significantly lower (31.1+/-5.2%) than in the control plots (85.6+/-3%) (P<0.001), which represented a relative tick reduction of 63.7%. Mortality of 93.8+/-2.3% was observed among the ticks that were recovered from the field and maintained in the laboratory for 2 weeks; while only 3.3+/-0.9% died from the control plots. The results of this study open up the possibility of developing an environmentally friendly and low cost application strategy to control Amblyomma ticks.

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

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

The use of a semiochemical bait to enhance exposure of Amblyomma variegatum (Acari: Ixodidae) to Metarhizium anisopliae (Ascomycota: Hypocreales).

Experiments were conducted to explore the use of a semiochemical bait to enhance exposure of Amblyomma variegatum Fabricius (Acari: Ixodidae) to different formulations of the entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorok. (Ascomycota: Hypocreales). Initially, the relative efficacies of attraction-aggregation-attachment pheromone (AAAP), made up of o-nitrophenol, methyl salicylate and nonanoic acid in the ratio 2:1:8, 1-octen-3-ol and butyric acid, were evaluated in an olfactometer. Only AAAP and 1-octen-3-ol were found to elicit attractive responses to the tick. Simultaneous release of 1-octen-3-ol and AAAP together with CO(2) from a trap in semifield plots attracted up to 94.0+/-6% of adult ticks from a distance of 6m, and up to 24.0+/-5.1% from 8m. Formulations of M. anisopliae (dry powder, oil, and emulsifiable) applied within the trap baited with AAAP, 1-octen-3-ol and CO(2) resulted in high levels of contamination of the ticks attracted to the traps. However, 48h after autoinoculation, 89.1 and 33.3% of conidia were lost in dry powder and oil formulations, respectively. Emulsifiable formulation showed least loss of propagules (17.1%). Samples of ticks attracted to the baited traps were transferred to plastic basins containing grass and maintained for 5 weeks. The experiment was conducted in rainy and dry seasons. Emulsifiable formulation gave the highest relative tick reduction in both seasons: 54.7 and 46.5% in rainy and dry seasons, respectively, followed by oil formulation (32.0 and 23.8%) and powder formulation (38.0 and 24.4%).

Effect of soil temperature and moisture on survival and infectivity of Metarhizium anisopliae to four tephritid fruit fly puparia.

The infectivity of 4 isolates of Metarhizium anisopliae to puparia of Ceratitis capitata treated as late third-instar larvae in unsterilized soil was investigated in the laboratory under controlled temperature and moisture. At 20-30 degrees C, mortality in puparia was highest at water potential of -0.1 and -0.01 mega Pascal (MPa) and lowest at water potential of -0.0055 and -0.0035 MPa in all the isolates. In wetter soil however, isolates ICIPE 20 and 60 caused significantly higher mortality than ICIPE 18 and 69. The survival of conidia in drier soil (-0.1 MPa) was not adversely affected at all temperatures. However, in wet soil (-0.0035 MPa) there was drastic reduction in colony counts in ICIPE 18 and 69 at 25 and 30 degrees C but conidial density in ICIPE 20 and 60 remained at the initial level at 14 days after inoculation at all temperatures. When ICIPE 20 was evaluated against three other fruit fly species (Ceratitis cosyra, Ceratitis rosa, and Ceratitis fasciventris), significant reduction in adult emergence and higher pupal mortality occurred in C. cosyra and C. fasciventris than in C. rosa at a combination of 15 and 20 degrees C and -0.1 and -0.0035 MPa. However, at higher temperature and the same moisture level, the isolates were equally pathogenic across the 3 species. It is probable that in addition to pathogen cycling and multiplication from dead infected insects in the soil, a balance between microbial degradation and replenishment of inoculum of virulent isolates occur through fluctuations in, and intricate interactions between temperature and moisture levels. This study is indicative of the potential of using isolate ICIPE 20 for soil inoculation against pupariating third-instar larva of fruit flies, thus providing a novel alternative to chemical soil application.

Prospects for biological control of the western flower thrips, Frankliniella occidentalis, with the entomopathogenic fungus, Metarhizium anisopliae, on chrysanthemum.

The potential of Metarhizium anisopliae (Metsch.) Sorok. for the control of Frankliniella occidentalis (Pergande) on chrysanthemum cuttings was evaluated in greenhouse experiments. The fungus significantly reduced both the adult and larval populations of F. occidentalis, although the level of control of larval populations was much lower than for adults. Combined application of M. anisopliae and Methomyl (Lannate), however, resulted in a significant reduction of both the larval and adult stages. The use of both control agents might be helpful in reducing the selection pressure for resistance to chemical insecticides, thereby delaying or preventing the build-up of resistant populations in greenhouses.