Beauveria Bassiana Isolates Research Articles

Generating thermotolerant colonies by pairing Beauveria bassiana isolates

Low thermotolerance in entomopathogenic fungi is the main impediment to their industrialization. This research, for the first time, describes the generation of a thermotolerant colony by pairing and subculturing (cycling) two Beauveria bassiana isolates without sexual reproduction. A mixture of B.bassiana ERL1578 and ERL1576 was inoculated on quarter-strength Sabouraud dextrose agar with yeast extract (¼SDAY). The paired culture (ERL1578+1576) was cycled three times to increase the frequency of possible hyphal fusion at the first cycle (c.5/5×10(5) conidia), followed by a heat treatment as a selection pressure. Two non-paired isolates served as controls. Two morphologically different colonies (BbHet1 and BbHet2) were isolated from the pairing. BbHet1 colony had the highest conidial yield. BbHet2 had the most rapid mycelial growth and produced sponge-like mycelial masses (the others were flat), and its conidia were darker than the non-paired colonies under a microscope (400×). BbHet2 conidia had 60.7% germination after exposure to 45°C for 60min (the others had <15%) without significant loss of virulence against Western flower thrips, Frankliniella occidentalis; however, there was a slight decrease in conidial yield. The new phenotypes formed suggested that a genetic variation happened as a result of heterokaryosis and/or recombination, more than environmental adaptation, when mixing different conidia. This methodology seems to be very useful for enhancing thermotolerance in fungi.

Susceptibility of Sitophilus zeamais (Mostch.) (Coleoptera: Curculionidae) to Beauveria bassiana and Metarhizium anisopliae

Evaluation of eleven Metarhizium anisopliae and six Beauveria bassiana isolates against the maize weevil Sitophilus zeamais was conducted under laboratory with the objectives of identifying the most virulent locally available fungal isolates, and determining the dose mortality response. The pathogenicity (virulence) of the entomopathogenic fungi was determined using LT50 and percent mortality at the conidial concentration of 1 x 108 ml-1. The most virulent isolates of M. anisopliae (PPRC-2, PPRC-14 and PPRC-51) and B. bassiana (PPRC-GG and PPRC-HH) caused 84.4% to 98.3% mortality to S. zeamais. These isolates had LT50 ranging from 3.8 to 4.3 days. The dose response study with the isolates PPRC-2, PPRC-14 and PPRC-51 at doses ranging from 1x 104 to 1x 108 conidia ml-1 revealed susceptibility of the S. zeamais with mortality ranging from 25 to 95.5%. There was an increase in the resulting LT50 from 4.0 to 11.9 days with a decrease in conidial concentration from 1x 108 to 1x 104 conidia ml-1. PPRC-2 showed the lowest LC50 (1.46x 10 conidia ml-1) followed by PPRC-14 and PPRC-51 (3.5x 105 and 3.9x 105 conidia ml-1, respectively). In conclusion, the isolates PPRC-2, PPRC-14, PPRC-51, PPRC-GG and PPRC-HH, can be considered as a potential candidates in the development of mycoinsectisdes against S. zeamais.

Improving photoprotection of Beauveria bassiana conidia for biological control of the cactus pest Dactylopius opuntiae in the semiarid region northeast of Brazil

A technical grade conidial preparation of the entomopathogenic fungus Beauveria bassiana isolate LCB63 was exposed to ultraviolet light (UV) from artificial and natural sources in the presence of various photoprotectants to develop a viable formulation to control Dactylopius opuntiae, a limiting pest of forage cacti in the Brazilian semiarid region. In the first experiment, Conidial viability was assessed in preparations containing water and liposoluble photoprotectants at a concentration of 1% and exposed to solar radiation for 0, 60, and 120 min. In another experiment, preparations containing different concentrations of the selected photoprotectants were exposed to natural and artificial UV radiation for 120 and 5 min, respectively. Insecticide activity of technical grade conidial preparations containing selected photoprotectants were evaluated against first instar nymphs of D. opuntiae. In general, no evaluated compounds showed deleterious effects on conidial germination, and they provided a strong degree of protection over a short period of time. One-percent concentrations of starch and skim milk resulted in low photoprotection over a longer irradiation period, while 1% Neo Heliopan AV, Neo Heliopan E1000 and Oxybenzone provided a moderate degree of protection. Increasing the concentration of Oxybenzone increased germination rate to 70%, while treatment with 1% Neo Heliopan E1000 maintained germination at 50%. A technical grade conidial preparation containing 1% Oxybenzone was effective in promoting D. opuntiae nymph mortality. These results suggested that addition of liposoluble photoprotectants could provide better photoprotection for B. bassiana LCB63 conidia in bioinsecticide formulations.

Characterization of a newly discovered Beauveria bassiana isolate to Franklimiella occidentalis Perganda, a non-native invasive species in China

In this study, a new virulent Beauveria bassiana isolate ( B. bassiana-CYT5) had been identified as a new member of the species B. bassiana. The B. bassiana-CYT5 isolate was compared with four other virulent B. bassiana isolates and found to be highly infectious and virulent against the Franklimiella occidentalis Perganda. The B. bassiana-CYT5 could approximately 93.08% mortality of F. occidentalis 6 days post inoculation in the concentration of 1 × 10 8 conidia/mL. The phylogenetic tree based on ITS and partial sequence of elongation factor 1-alpha (EF1-alpha) indicated that B. bassiana-CYT5 isolate was in a cluster of B. bassiana. Furthermore, B. bassiana-CYT5 isolate demonstrated high heat tolerance (60–100% relative germination) between 1-h and 2-h exposure at 37 °C, 38 °C, 39 °C and 40 °C, respectively. So our results suggested that B. bassiana-CYT5 isolate could be a new efficient biocontrol agent against F. occidentalis.

Effect of culture substrates on virulence of Beauveria bassiana (Ascomycota: Cordycipitaceae) conidia against the browntail moth, Euproctis chrysorrhoea (Lepidoptera: Lymantriidae)

We studied the effect of different solid substrates on virulence of two Beauveria bassiana isolates against the browntail moth, Euproctis chrysorrhoea (L.) (Lep.: Lymantriidae). Conidia produced on wheat grains, wheat flour, wheat bran, rice flour, rice bran, rice paddy, corn flour, millet, and Sabouraud's dextrose agar with 1% yeast extract (SDAY) as control were compared. There were significant differences among these substrates for their effects on the virulence of produced conidia. Applying 107 conidia/mL of B. bassiana EUT105, produced on rice bran caused the highest (84.9%) and on rice flour, the lowest (57.6%) mortalities. Bioassay on fifth-instar larvae using aerial conidia harvested from wheat grains, rice paddy, and SDAY indicated that conidia from wheat grains had the highest virulence while those from rice paddy, the lowest.

Genetic analyses place most Spanish isolates of Beauveria bassiana in a molecular group with word-wide distribution

BackgroundThe entomopathogenic anamorphic fungus Beauveria bassiana is currently used as a biocontrol agent (BCA) of insects. Fifty-seven Beauveria bassiana isolates -53 from Spain- were characterized, integrating group I intron insertion patterns at the 3'-end of the nuclear large subunit ribosomal gene (LSU rDNA) and elongation factor 1-alpha (EF1-α) phylogenetic information, in order to assess the genetic structure and diversity of this Spanish collection of B. bassiana.ResultsGroup I intron genotype analysis was based on the four highly conserved insertion sites of the LSU (Ec2653, Ec2449, Ec2066, Ec1921). Of the 16 possible combinations/genotypes, only four were detected, two of which were predominant, containing 44 and 9 members out of 57 isolates, respectively. Interestingly, the members of the latter two genotypes showed unique differences in their growth temperatures. In follow, EF1-α phylogeny served to classify most of the strains in the B. bassiana s.s. (sensu stricto) group and separate them into 5 molecular subgroups, all of which contained a group I intron belonging to the IC1 subtype at the Ec1921 position. A number of parameters such as thermal growth or origin (host, geographic location and climatic conditions) were also examined but in general no association could be found.ConclusionMost Spanish B. bassiana isolates (77.2%) are grouped into a major phylogenetic subgroup with word-wide distribution. However, high phylogenetic diversity was also detected among Spanish isolates from close geographic zones with low climatic variation. In general, no correlation was observed between the molecular distribution and geographic origin or climatic characteristics where the Spanish B. bassiana isolates were sampled.

Biological control of engorged female Haemaphysalis qinghaiensis (Acari: Ixodidae) ticks with different Chinese isolates of Beauveria bassiana

Seven isolates of the fungus Beauveria bassiana (B.b) were assessed for their lethality against Haemaphysalis qinghaiensis, a prevalent tick species in China. Fourteen days after exposure to the isolates B.bAT1, B.bAT5, and B.bAT7 (at 10(8) conidia mL(-1)), the mortality rate had reached 100%. The results indicated that these three B. bassiana isolates were highly virulent against the engorged female H. qinghaiensis ticks. The present study suggests that B. bassiana has potential for biocontrol applications to eradicate H. qinghaiensis.

Selection of native isolates of Beauveria bassiana (Ascomycota, Hypocreales) for the control of the coffee borer beetle Hypothenemus hampei (Scolytinae) in Brazil

Selection of native isolates of Beauveria bassiana (Ascomycota, Hypocreales) for the control of the coffee borer beetle Hypothenemus hampei (Scolytinae) in Brazil Hypothenemus hampei (coffee borer beetle or coffee berry borer) is the main pest of coffee Coffea spp. in the world. The aim of this study was to select native isolates of the ascomycete Beauveria bassiana for biological control of this pest. We collected 27 isolates on H. hampei from the state of Espírito Santo in Brazil. Isolates CCA-UFES/Bb-15, Bb-11, Bb-4 and Bb-18 were selected, with confirmed beetle mortality of &gt; 60% after spraying with a suspension of 105 conidia/mL. The median lethal concentration (LC50) of these isolates varied from 4.0 × 104 to 7.9 × 104 conidia/mL. The standard isolate (ESALQ-447) showed the highest conidiogenesis, with 8.5 × 106 conidia, followed by CCA-UFES/Bb-18, Bb-11, Bb-15 and Bb-4, all exceeding 4 × 106. Isolates CCA-UFES/Bb-4, Bb-11, Bb-15, and Bb-18 have a potential to control H. hampei.

Utilization of Carbohydrates by Beauveria Bassiana Isolates Obtained from Forest Pests

Carbohydrate utilization profiles of ten isolates of the entomopathogenic fungus Beauveria bassiana (Bals.-Criv.) Vuillemin were studied and compared to assist in determining their phenotypic characterization. Isolates were obtained in pure cultures from dead individuals of several forest pests collected from different regions in Bulgaria. Studies on utilization profiles were based on the acidification of twenty carbohydrates. The results indicate that sucrose, maltose and trehalose were assimilated at a high degree compared to esculin, arabinose and dul- citol. According to the results of a cluster analysis of the carbohydrate utilization profiles, the B. bassiana isolates were divided into two larger groupings. All isolates in the first larger cluster were obtained from the coleopteran insects - Stenomax aeneus, Ips typographus, I. sexdentatus and Dryocoetes autographus. Isolates from the other cluster were obtained from the lepidopteran larvae of Thaumetopoea pityocampa and Lymantria dispar, and from an adult of the coleopteran species Hylurgops palliatus. We determined that each B. bassiana isolate exhibited a different and specific carbohydrate utilization profile but differences at the p-level < 0.05 were significant among some of them. The most distinguishable was the isolate 560Bb obtained from T. pityocampa. Differences between the isolate 560Bb and the other nine B. bassiana isolates were highly significant at the p-level < 0.005. Isolate 433Bb obtained from a dead adult of I. typogra- phus was significantly different from five of the studied isolates at the p-level < 0.05.

In vivo passages of heterologous Beauveria bassiana isolates improve conidial surface properties and pathogenicity to Nilaparvata lugens (Homoptera: Delphacidae)

In entomopathogenic hyphomycetes, desired candidates against the brown planthopper, Nilaparvata lugens (a sap-sucking rice pest in Asia), are lacking. In this study, 21 Beauveria bassiana isolates from heterologous host insects showed low pathogenicity to third-instar nymphs sprayed at the high concentration of ∼1000 conidia/mm 2, causing only 2–23% mortalities. Of those, three isolates killed significantly more nymphs (up to 45–62%) after two in vivo passages but no more after further passage. Conidial hydrophobicity rates ( H r), zeta potentials ( P z), and subtilisin-like protease (Pr1) activities ( A p) of these isolates showed the same trends in the three host passages ( N: 0–3). In multivariate correlation, the variables N, H r and P z were found contributing 89% to the mortality variation ( r 2 = 0.89). Significant positive correlations were also found between H r and N ( r 2 = 0.64), P z and N ( r 2 = 0.52), A p and N ( r 2 = 0.51), H r and A p ( r 2 = 0.45), and P z and A p ( r 2 = 0.57), respectively. However, irregular changes of H r and P z occurred in four other isolates, whose pathogenicity to N. lugens was not enhanced by repeated host passages, resulting in no correlation between the variables. Our data indicate that the conidial surface properties H r and P z associated with cuticle adhesion reflect the heterologous host-induced adaptation and help to select fungal candidates against N. lugens from repeated in vivo passages.

Susceptibility of Tetranychus urticae koch. (Acari: Tetranychidae) to isolates of entomopathogenic fungus Beauveria bassiana

Bioassays with five isolates of the entomopathogenic fungus Beauveria bassiana (Bals.- Criv.) Vuillemin were conducted under laboratory conditions with a goal to estimate their virulence to the two-spotted spider mite Tetranychus urticae Koch. (Acari: Tetranychidae). Common bean plants with mites were treated by spraying conidial suspensions of isolates at concentrations of 106, 107 and 108 conidia/ml. Lethal effects of the fungal isolates were evaluated as percentages of cumulative daily mortality due to mycoses, corrected for mortality in the control variant. Virulence of the isolates was estimated based on values of the median lethal time (LT50) calculated by probit analysis for the variants treated with conidial suspensions at the concentration of 106 conidia/ml. The two-spotted spider mite was found susceptible to the examined isolates of the entomopathogenic fungus B. bassiana. Mycosis caused to T. urticae by the B. bassiana isolates 444 Bb and 445 Bb had fast lethal effect after treatment with conidial suspensions even at the concentration of 106 conidia/ ml. The mean mortality values of host individuals were 83.78 ? 3.62% and 68.49 ? 4.28% on the first day, respectively, and up to 100% in both variants on the fourth day. The isolates 426 Bb, 444 Bb and 445 Bb of B. bassiana were highly virulent to two-spotted spider mites with values of the median lethal time varied within overlapped narrow confidence intervals from 0.122 to 1.084 days (average value 0.162 days), from 0.117 to 1.398 days (average value 0.146 days) and from 0.106 to 1.162 days (average value 0.131 days), respectively. Significant differences regarding virulence of the three isolates at p-level &lt; 0.05 could not been proved. The other two examined isolates were distinctly less virulent to T. urticae than these three B. bassiana isolates.

A new microbial consortia containing entomopathogenic fungus, Beauveria bassiana and plant growth promoting rhizobacteria, Pseudomonas fluorescens for simultaneous management of leafminers and collar rot disease in groundnut

The virulence of 20 isolates of Beauveria bassiana (Balsamo) Vuillemin to larvae of the leafminer, Aproaerema modicella, was tested in the laboratory. Leafminer larvae were sprayed with a standard concentration of 1×108 condia/mL of each B. bassiana isolate. All the B. bassiana isolates tested were pathogenic to A. modicella and the mortality varied between 16.7 and 68.9%. Beauveria bassiana isolate B2 was found to be the most virulent followed by isolate B4 which resulted in 59% mortality. Beauveria isolate B2 was selected for dose–response mortality studies with four different doses (1×102, 1×104, 1×106 and 1×108 conidia/mL). Among the various doses tested, 1×108 conidia/mL produced the highest mortality (70.7%). In addition, morphogenesis of the insect pest in all stages, larval, pupal and adult was greatly affected due to fungal infection. Further, B. bassiana isolate B2 and two Pseudomonas fluorescens strains, TDK1 and Pf1 were tested alone and in combination for suppression of groundnut leafminer and collar rot disease and promotion of plant growth and yield both under glasshouse and field conditions. The mixture of B. bassiana and P. fluorescens strains significantly reduced the leafminer and collar rot disease incidences when applied as talc-based formulation through seed, soil and foliar application under glasshouse and field conditions.

Susceptibility of apple clearwing moth larvae, Synanthedon myopaeformis (Lepidoptera: Sesiidae) to Beauveria bassiana and Metarhizium brunneum

Apple clearwing moth larvae, Synanthedon myopaeformis (Lepidoptera: Sesiidae) were found to be susceptible to infection by two entomopathogenic fungi: an indigenous fungus isolated from S. myopaeformis cadavers and identified as Metarhizium brunneum (Petch); and Beauveria bassiana isolate GHA. In laboratory bioassays, larvae exhibited dose related mortality after exposure to both the M. brunneum and Beauveria bassiana with 7 day LC50's of 2.9×105 and 3.4×105 spores/mL, respectively. Larval mortalities caused by the two isolates at 1×106 spores/mL were not significantly different and 73% of the M. brunneum-treated, and 76% of the B. bassiana-treated larvae were dead 7 days post treatment, with LT50's of 5.5 and 5.1 days, respectively.

Comparative Tolerances of Various Beauveria bassiana Isolates to UV-B Irradiation with a Description of a Modeling Method to Assess Lethal Dose

The tolerances of 20 Beauveria bassiana isolates derived from host insects worldwide to UV-B irradiation were assessed quantitatively in multi-dose bioassays. Conidial suspensions of the isolates smeared on glass slides were exposed to the gradient UV-B doses of 0.1-1.6 J cm(-2) (D), which generated from 0.75 to 10.17 min irradiation of weighted 312-nm wavelength at 2.0-2.61 mW cm(-2). Irradiated conidia were then incubated for 24 h at 25 degrees C under saturated humidity. The ratio of germination at each dose over that in the blank control was defined as survival index (I (s)). For all isolates, the I (s) - D observations fit well with the survival model I (s) = 1/[1 + exp(a + bD)] (0.94 < or = r (2) < or = 0.99) generated widely spanned lethal doses of 0.154-0.928, 0.240-1.139, and 0.383-1.493 J cm(-2) for their losses of 50%, 75%, and 95% viabilities, respectively. These were far below the solar UV-B dose of 2.439 J cm(-2) measured in a sunny day during the summer. The large variation of UV-B tolerance among the isolates indicates a necessity to select UV-tolerant candidates for formulations applied to insect control during summer. The highly efficient bioassay method was developed to measure accurately the UV-B tolerances of fungal biocontrol agents as lethal doses.

Virulence of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to sweet potato weevil Cylas puncticollis and effects on fecundity and egg viability

AbstractThe virulence of eight isolates of Metarhizium anisopliae and four isolates of Beauveria bassiana (Ascomycota: Hypocreales) to adult Cylas puncticollis (Coleoptera: Curculionidae) was tested in the laboratory. Insects were sprayed with a standard concentration of 1.0 × 107 conidia mL−1 using Burgerjon’s spray tower. All the isolates tested were pathogenic to C. puncticollis. Mortality varied between 77.5% and 84.2% with isolates of B. bassiana and between 62.5% and 89.2% with isolates of M. anisopliae, 26 days post‐treatment. The lethal time to 50% mortality for the 12 isolates varied between 9.7 and 18.5 days. Four isolates, M. anisopliae International Centre of Insect Physiology and Ecology (ICIPE) 18 and ICIPE 62 and B. bassiana ICIPE 275 and ICIPE 114 were selected for dose–response mortality [lethal concentration to 50% mortality (LC50)] bioassays. Five concentrations (1.0 × 106, 3.0 × 106, 1.0 × 107, 3.0 × 107 and 1.0 × 108 conidia mL−1) of both fungal species were used. B. bassiana ICIPE 275 was the most active isolate with LC50 value of 0.7 × 106 conidia mL−1. The effect of fungal infection on feeding, fecundity and egg viability of C. puncticollis adult females was also investigated under laboratory conditions. M. anisopliae isolate ICIPE 18 and B. bassiana isolate ICIPE 114 were tested for feeding experiment using six concentrations (0, 1.0 × 106, 3.0 × 106, 1.0 × 107, 3.0 × 107 and 1.0 × 108 conidia mL−1). For reproduction potential (fecundity and egg viability) bioassays, five concentrations (0, 1.0 × 106, 3.0 × 106, 1.0 × 107 and 3.0 × 107 conidia mL−1) of M. anisopliae isolate ICIPE 18 were used. Adult sweet potato weevils (SPWs) treated with M. anisopliae at the concentrations of 3.0 × 107 and 1.0 × 108 conidia mL−1 consumed significantly less food than weevils in the control and B. bassiana treatments at all the concentrations, except at the higher concentration of 1.0 × 108 conidia mL−1, 14 days post‐treatment. Female weevils in the control treatments laid more eggs than fungus‐treated females. Percentage egg viability differences between controls and fungus treatments were significant at all the concentrations tested, 10 days post‐treatment. These results show that B. bassiana and M. anisopliae are pathogenic to SPWs and infection can reduce feeding, fecundity and egg viability.

Impact of a treatment ofBeauveria bassiana(Deuteromycota: Hyphomycetes) on honeybee (Apis mellifera) colony health and onVarroa destructormites (Acari: Varroidae)

In 2 experiments bee colonies in southern France were treated with conidia of a Beauveria bassiana isolate collected from Varroa mites in the region. Objectives were to evaluate treatment effect on colony weight, adult bee mass, and capped brood and honey, and on Varroa fall onto sticky boards. Treatments included conidia formulated with either wax powder or wheat flour, flour alone, or control. Treatment did not affect colony health. Colonies treated with conidia and wax powder had higher mite fall compared to controls while those treated with conidia and wheat flour did not. The proportion fallen, infected mites in both conidia treatments was higher than controls for up to a week. Higher mite fall and infection rates were observed in treated hives in the 2nd, smaller experiment. The relationships between dosage and proportion fallen, infected mites, and between ambient temperature and infection duration, were examined. Future experiments will explore Varroa control using conidia.

Desenvolvimento dos fungos Metarhizium anisopliae (Metschnikoff, 1879) Sorokin, 1883 E Beauveria bassiana (Balsamo) Vuillemin, 1912 sobre Ctenocephalides felis felis (Bouché, 1835)

The flea Ctenocephalides felis felis is a parasite that causes allergic dermatitis and also may transmit etiologic agents to domestic animals and humans. This study investigated by scanning electron microscopy the development of entomopathogenic fungi on flea cuticle. Fleas were exposed to conidia (10(8) ml(-1)) of Metarhizium anisopliae (isolate 959) or Beauveria bassiana (isolate 986). Following standard protocols for electron microscopy, the specimens were prepared 2, 15, 26 and 96 h after infection. The micrography revealed that 2 h after fungus exposure, conidia attachments encompassed the entire flea cuticle, especially on abdominal intersegmental membranes. The emergence of germ tubes and appressoria formation occurred at 15 h, thickening and branching of hyphae on the flea cuticle was noted at 26 h. Therefore, both of these fungal isolates were able to develop on cuticular surfaces of C. f. felis.

Variability in tolerance to UV-B radiation among Beauveria spp. isolates

Solar radiation, particularly the UV-B component, negatively affects survival of entomopathogenic fungi in the field. In an effort to identify Beauveria spp. isolates with promise for use in biological control settings with high insolation, we examined 53 Beauveria bassiana isolates, 7 isolates of 4 other Beauveria spp. and Engyodontium albus (= Beauveria alba). The origins of these fungi varied widely as to host/substrate and country, but approximately 30% of these isolates were B. bassiana from ticks in Brazil. A preliminary trial with three B. bassiana isolates (Bb 19, CG 310 and CG 481) at several UV-B dosages indicated that 2 h of weighted UV-B irradiance at 978 mW m −2 (providing a total dose of 7.04 kJ m −2) allowed separation of isolates into low, medium or high UV-B tolerance. This dose, therefore, was selected as a single dose to compare UV-B tolerances of all 60 Beauveria spp. isolates. There was high variability in tolerance to UV-B radiation among the B. bassiana isolates, ranging from virtually zero tolerance (e.g., Bb 03) to almost 80% tolerance (e.g., CG 228). In addition, surviving B. bassiana conidia demonstrated delayed germination; and this is likely to reduce virulence. Conidia of the other species were markedly more sensitive to UV-B, with E. albus (UFPE 3138) being the least UV-B tolerant. Among B. bassiana isolates originating from 0° to 22° latitudes, those from lower latitudes demonstrated statistically significant greater UV-B tolerances than those isolates from higher latitudes. Isolates from above 22°, however, were unaffected by latitude of origin. A similar analysis based on host type did not indicate a correlation between original host and UV-B tolerance. The identification in this study of several B. bassiana isolates with relatively high UV-B tolerance will guide the selection of isolates for future arthropod microbial control experiments.

Selection of entomopathogenic fungi for use in combination with sub-lethal doses of imidacloprid: perspectives for the control of the leaf-cutting ant Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae)

In the first part of this study, four isolates of the fungus Beauveria bassiana (Bals.) Vuillemin (LPP1, LPP2, CG05 and CG24) and one isolate of Metarhizium anisopliae (Metsch.) Sorokin (CG46) were tested against adult foragers of Atta sexdens rubropilosa. Ants were allowed to walk on filter paper discs, inside Petri dishes, previously impregnated with 1 ml of a conidia suspension (2 x 10(7) conidia ml(-1)), maintained at 80% RH and 26 degrees C for 24 h and subsequently, transferred to sterile Petri dishes, maintained at 23 degrees C, 80% RH, 24 h dark. The mean values of LT(50) for LPP2, LPP1, CG46, CG24 and CG05 were 3.5, 3.7, 3.8, 4.2 and 4.4 days, respectively. Control insects for all tests in this study showed less than 10% mortality. Experiments were carried out to test the toxicity of imidacloprid (IMI) to A. sexdens rubropilosa. Mortality was evaluated 10 days following a 24 h exposure to the insecticide. Percent mortality caused by 500, 200, 100 and 10 ppm IMI was 77.8, 56.7, 45.5 and 5.5 respectively. Insects treated with 10 ppm IMI were observed to have reduced locomotor activity 24 h after exposure to the insecticide. The LC(50) of IMI was 154.3 ppm. Subsequent tests were carried out to evaluate the combination of a sub-lethal dose of IMI (10 ppm) and infection by CG24 (1 x 10(7) conidia ml(-1)). Mortality due to fungal infection alone was 43.3%. Mortality of insects treated with IMI followed by exposure to the fungus was 64.3%. These results indicate that IMI significantly increases the susceptibility of ants to infection by B. bassiana CG24.

Effect of nutrition on growth and virulence of the entomopathogenic fungusBeauveria bassiana

Three isolates of the entomopathogen Beauveria bassiana along with one strain of Metarhizium anisopliae were cultured on seven media with different carbon/nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was evaluated via measurement of colony growth, spore yield, germination speed, conidial C/N ratio and Pr1 (a serine protease) activity. 'Osmotic stress' medium produced the lowest colony growth with low numbers of conidia in all isolates. However, these conidia showed a high germination rate and virulence. However, conidial Pr1 activity was low in some isolates. In most but not in all cases conidia from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1 activity compared with conidia from other media. However, in some instances we could not conclude that there was a relationship among germination rate, conidial Pr1 activity and virulence. C/N ratio of conidia was statistically different among various media and fungal isolates. Conidia with lower C/N ratio generally produced lower LT(50) (lowest median lethal time) values (more virulent). Insect-passaged conidia from different media had lower C/N ratio compared with similar conidia from artificial cultures. Therefore, they should be more virulent than in vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio are independent of host, it seems that host-related determinants such as insect cuticle and physiology and environmental conditions may influence host susceptibility and therefore fungal isolate virulence towards host insects.