Laboratory evaluation of entomopathogenic fungi against the white grubs,Holotrichia oblita and Anomala corpulenta (Coleoptera: Scarabaeidae) from the field of peanut,Arachis hypogaea

We evaluated the pathogenicity of nine isolates of entomopathogenic fungi, including six isolates of Metarhizium anisopliae, one of Beauveria bassiana, and two of Beauveria brongniartii, against eggs and various larval instars of two scarab-beetle species, Holotrichia oblita and Anomala corpulenta, under laboratory conditions. The fungal isolates differed in pathogenicity. Generally, the isolates were more pathogenic to A. corpulenta than to H. oblita. Some of the isolates prevented egg hatching and caused larval death. Isolates M2-2 and Br5-8 caused 39 and 27% egg mortality, respectively, and produced 23 and 24% viewable fungal-infection rates in H. oblita. Three isolates had no significant effect on egg hatchability. Three isolates (Br5-8, Br232818, and M200614) caused about 40% mortality in H. oblita first instars. In A. corpulenta, all isolates except M200614 caused more than 60% egg mortality, and M2-2, Br232818 and Br5-8 caused egg-infection rates greater than 50%. M2-2 caused 47% infection and 100% mortality in first-instar larvae of A. corpulenta, while Br5-8 and Br232818 yielded over 80% mortality of the larvae. The three most virulent isolates, M2-2, Br232818 and Br5-8, were selected for further bioassays against second- and third-instar larvae. In addition, seven graduated concentrations of a Br5-8 conidial suspension were assayed against H. oblita second instars. Larval mortality was positively correlated with fungal dosage, and the LC₅₀ was 4.49×10⁶ conidia/mL. These three virulent isolates may be used to prevent H. oblita and A. corpulenta larval infestations in field crops.     

Infection of Blissus antillus (Hemiptera: Lygaeidae) Eggs by the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana

This study determined the pathogenicity and virulence of Beauveria bassiana and Metarhizium anisopliae to eggs of the chinch bug Blissus antillus (Hemiptera: Lygaeidae). Eggs were inoculated under laboratory conditions by immersion in concentrations of 1 × 10⁴ and 5 × 10⁶ conidia/ml. Inoculated eggs were kept under controlled conditions. Evaluations were carried out daily for 20 days. M. anisopliae isolates were highly virulent to eggs, even at 1 × 10⁴ conidia/ml. All B. bassiana isolates tested were considered to be of low virulence or avirulent. The most virulent isolate tested was ESALQ 818 (M. anisopliae), which caused 96.7% infection, when eggs were immersed in suspensions of 1 × 10⁴ conidia/ml. Conidial production on infected eggs was observed to be highest for M. anisopliae isolate CG144, with a mean value of 11.6 × 10⁵ conidia/ml/egg. Infection of Blissus eggs oviposited on plant stems was greater when M. anisopliae isolate CG144 was formulated in mineral oil (63.5% mortality) than when formulated in Tween 80 (27.1% mortality).     

Cosmopolites sordidus (Germar) susceptibility to indigenous Cameroonian Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates

Management of the banana root borer (BRB), Cosmopolites sordidus (Germar; Coleoptera: Curculionidae), remains a challenge in banana and plantain production worldwide. Synthetic pesticides remain the most widely used solution while mycoinsecticides are increasingly being recommended. In this study, we selected indigenous isolates of Beauveria bassiana and Metarhizium anisopliae collected from plantain fields in Cameroon, and tested them in the laboratory for their viability, pathogenicity and virulence against all C. sordidus life stages. Of 13 isolates initially screened for spore germination and pathogenicity to adult weevils in conidial suspension of 3.2 × 10⁸ conidia/ml, eight isolates with high to moderate germination and highest weevil mortality were selected for dose–response bioassays with four concentrations per isolate: 3.2 × 10², 3.2 × 10⁴, 3.2 × 10⁶ and 3.2 × 10⁸ conidia/ml. The virulent isolates from adult bioassays were tested with eggs, larva and pupae in conidial suspension of 3.2 × 10⁸ conidia/ml. Isolates performance depended on insect life stage with significantly high pathogenicity and virulence against larval, pupa and adult stages. The Beauveria isolate BIITAC6.2.2 caused the highest mortality rates followed by MIITAC1.1.5. Lethal times and lethal concentrations were relatively low for the three M. anisopliae isolates and three B. bassiana isolates which were the best isolates in almost all insect life stages. Apart from being effective in multiple life stages, these isolates were transmitted horizontally from one stage to another when eggs and pupae were treated. The implication of these findings for integrated management of the BRB, and potential biopesticides development and commercialization are discussed.     

The entomopathogenic nematode Steinernema kraussei and Metarhizium anisopliae work synergistically in controlling overwintering larvae of the black vine weevil,Otiorhynchus sulcatus,in strawberry growbags

Previously, the combination of reduced rate of entomopathogenic nematodes (EPN) and fungus caused additive or synergistic mortality to third-instar black vine weevil (BVW), Otiorhynchus sulcatus. In this study, we examined this interaction in unheated glasshouses during winter and compared a combination of commercial formulation of a cold-tolerant EPN, S. kraussei (Nemasys L?) and fungus Metarhizium anisopliae strain V275 against overwintering third-instar BVW. The combination of M. anisopliae with S. kraussei at a rate of 1×10¹⁰ conidia+250,000 nematodes/growbag resulted in additive or synergistic effects, providing 100% control of overwintering larvae.     

Synergism between Metarhizium anisopliae (Deuteromycota: Hyphomycetes) and Boric Acid against the German Cockroach (Dictyoptera: Blattellidae)

Mortality of German cockroaches, Blattella germanica (L.), caused by Metarhizium anisopliae (Metschnikoff) Sorokin strain AC-1 alone and in combination with different formulations of boric acid, was evaluated in laboratory bioassays. Topical application of M. anisopliae alone (8.96 × 10⁹ conidia/m²) required 28 days to cause >92% cockroach mortality (LT₅₀ = 10 days). In contrast, in combination with boric acid (topically applied as a dust or in drinking water), M. anisopliae killed cockroaches significantly faster than without boric acid. M. anisopliae conidial dust (8.96 × 10⁸ conidia/m²) with either 12.5% (w/w) boric acid dust or 0.1% (w/v) boric acid in drinking water killed 100% of the cockroaches in only 8 days (LT₅₀ = 5 days) and 10 days (LT₅₀ = 6 days), respectively, without compromising the fungus emergence from cadavers. Replacement of M. anisopliae with flour dust or heat-killed M. anisopliae conidia eliminated this effect, demonstrating that it was not the consequence of greater boric acid ingestion due to more extensive cockroach grooming upon exposure to M. anisopliae conidia. Moreover, injections of a low dose of M. anisopliae, which caused only 30% mortality, together with sublethal concentrations of boric acid into the cockroach hemocoel resulted in a doubling of mortality. Statistical analysis demonstrated a synergistic interaction between these two insecticides.     

Optimised fermentation conditions and improved collection efficiency using dual cyclone equipment to enhance fungal conidia production

Methods for enhancing conidial yield and for harvesting pure fungal conidia of entomopathogenic fungi were investigated. Fermentation conditions (liquid-to-solid ratio, MgSO₄·7H₂O, incubation temperature, inoculum sizes, KNO₃ and relative humidity) of Beauveria bassiana s.l. and Metarhizium anisopliae s.l. were optimised to increase the conidial yields that reached 11.2 mg/g and 24.5 mg/g, increases of 72% and 52% compared to the unoptimised yields of 6.5 mg/g and 16.1 mg/g, respectively. Three methods were compared for harvesting pure conidia of B. bassiana: dual cyclone equipment (DCE), sieving 200 and elution with 0.02% Tween-80 suspension. DCE performed the best, giving a conidial yield of 12.6 mg/g and 1.8 × 10¹⁰ conidia·g^–1. To further enhance the harvest efficiency, response surface methodology combined with a Box–Behnken design was employed, and the conidial yield of B. bassiana reached 20.9 mg/g, a total increase of 221% compared to the original conditions. Under these optimised harvest parameters, the conidial yield of M. anisopliae rose to 42.2 mg/g, an increase of 162%. The conidia of B. bassiana and M. anisopliae harvested in this way were pure, with no mycelial fragments or substrate visible in microscopic images.     

Effect of temperature and poultry litter in Beauveria bassiana (bals.) Vuill. and Metarhizium anisopliae (Metsch) virulence against the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

This study was carried out to evaluate the influence of temperature and poultry litter on germination vegetative growth virulence and conidial production of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates on larvae and adults of the lesser mealworm (Alphitobius diaperinus) (Panzer). The vegetative growth and conidial production were evaluated on culture media. Virulence was studied submerging larvae and adults in a conidial suspension (1 x 10(8) conidia/ml). All the experiments were carried out in growth chamber (26 degrees C and 32 degrees C and 14h photophase). Fungus-killed insects were daily collected and used for microscopic conidial counts. The poultry litter effect was evaluated by submerging the insects in a fungal suspension (10(8) conidia/ml) and then transferring them to cups containing poultry litter (new and used). B. bassiana isolates were more sensitive than M. anisopliae to high temperature because conidia viability, vegetative growth and virulence were negatively affected (P < 0.05). The conidial production was higher to B. bassiana in 26 degrees C (7 to 11 x 10(8) conidia/larval cadaver and 8 x 10(8) conidia/adult cadaver) (P < 0,05). Larval stage was about 10 times more sensitive to M. anisopliae at 26 degrees C than adults stage. Regarding B. bassiana, differences on sensitivity between larval stages and adults were not observed at this temperature. However, at 32 degrees C, larval stage was more sensitive for CB116 and UEL50 isolates. Mortality was higher when larvae and adults (15.7 and 66.7% respectively) were treated by B. bassiana and maintained on new poultry litter at 26 degrees C) (P < 0.05).     

Influence of fungicides and insecticides on the entomogenous fungus Metarhizium anisopliae a pathogen of the vine weevil,Otiorhynchus sulcatus

In the laboratory, the fungicides chlorothalonil and zineb prevented germination of Metarhizium anisopliae conidia when incorporated into Sabouraud dextrose agar (SDA) at the commercial concentration (based on the manufacturers’ recommended rates for horticultural crops). Twelve other fungicides and six insecticides had no effect on spore germination when applied at the same rate. Mycelial growth of M. anisopliae on SDA plates containing the recommended rate of all the pesticides (except propamocarb) was reduced compared with SDA alone. Two fungicides, benomyl and carbendazim, totally inhibited growth at 0.1 times the recommended rate. Growth was also completely prevented by the fungicides etridiazole, triforine and zineb, and the insecticides dichlorvos and hostathion, at 10 times the recommended rate. In a glasshouse experiment, a prophylactic drench of M. anisopliae conidia reduced vine weevil (Otiorhynchus sulcatus) populations on Impatiens plants by 88%. This level of control was not significantly reduced by subsequent application (7 days after egg infestation) of any of the pesticides at the recommended concentration. Larval control in pots treated with M. anisopliae plus any one of the 12 fungicides and four insecticides examined, ranged from 82% to 98%. The insecticide diazinon applied alone reduced larval numbers by 100%. Two other insecticides, dichlorvos and cypermethrin, and the fungicide pyrazaphos, also reduced weevil populations by over 50%. These experiments demonstrate the limitations of laboratory based in vitro screening programmes for assessing the chemical compatibility of M. anisopliae.     

Susceptibility of Ceratitis capitata Wiedemann (Diptera: Tephritidae) to Entomopathogenic Fungi and Their Extracts

The effectiveness of seven strains of entomopathogenic fungi against Ceratitis capitata adults was evaluated in the laboratory. Adults were susceptible to five of seven aqueous suspensions of conidia. Metarhizium anisopliae and strain CG-260 of Paecilomyces fumosoroseus were the most pathogenic fungi, with 10-day LD₅₀ values of 5.1 and 6.1 × 10³ conidia/fly, respectively, when applied topically. Sublethal effects on fecundity and fertility of the fungal-exposed females were also studied. The most effective fungus in reducing fecundity was P. fumosoroseus CECT 2705, with reductions on the order of 65% at 1 × 10⁶ conidia/fly. M. anisopliae and Aspergillus ochraceus also showed significant reductions of fecundity (40–50% for most of the assayed concentrations). Fertility was moderately affected by the fungi. M. anisopliae at 1 × 10⁶ conidia/fly was the most effective fungus, showing egg eclosion reduction of over 50% compared with the control. In addition, culture broth dichloromethane extracts from the entomopathogenic fungi were tested for insecticide activity against C. capitata, including effects on fecundity and fertility. The extract from M. anisopliae was the most toxic, resulting in about 90% mortality at a concentration of 25 mg/g of diet; under these conditions, fecundity and fertility of treated females were reduced by 94 and 53%, respectively, compared with untreated controls.     

Effects of successive subculturing on stability, virulence, conidial yield, germination and shelf-life of entomopathogenic fungi

Aims: To determine the stability and conidial yield of two strains of the entomopathogenic fungus Metarhizium anisopliae and one strain of M. brunneum, being developed for the control of insect pests. Methods and Results: The conidial yields and the shelf‐life of the conidia of two commercially viable strains of M. anisopliae V275 (=F52) and ARSEF 4556 and one strain of M. brunneum (ARSEF 3297) were determined after harvesting conidia from in vitro subcultures on Sabouraud dextrose agar (SDA) and broken basmati rice. The strains were stable and showed no decline in virulence against Tenebrio molitor, even when subcultured successively 12 times on SDA. Conidia‐bound Pr1 protease activity decreased in conidia harvested from SDA and mycosed cadavers after the 1st subculture, but increased in conidia produced on rice. The C:N ratio of conidia from mycosed cadavers was lower than that of conidia from rice or SDA. Irrespective of the number of subcultures, strain ARSEF 4556 produced significantly higher conidial yields than ARSEF 3297 and V275. The 12th subculture of V275 and ARSEF 3297 produced the lowest conidial yield. Shelf‐life studies showed that conidia of strain ARSEF 4556 had a higher conidial viability than V275 and ARSEF 3297 after 4 months, stored at 4°C. Conclusions: The current study shows that determining strain stability and conidial yield through successive subculturing is an essential component for selecting the best strain for commercial purposes. Significance and Impact of the Study: This is the first study to compare quality control parameters in the production of conidia on rice, and it shows that the level of Pr1 is comparatively high for inoculum produced on rice.     

Performance of a semiochemical‐baited autoinoculation device treated with Metarhizium anisopliae for control of Frankliniella occidentalis on French bean in field cages

The performance of an autoinoculation device was evaluated in field cage experiments for control of Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), in French bean, Phaseolus vulgaris L. var. Samantha (Fabaceae). Treatments consisted of a fungus‐treated device with and without semiochemical (Lurem‐TR), and a fungus‐free device as control; the fungus used was Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae). The overall mean number of conidia acquired by a single insect was higher in the fungus‐treated semiochemical‐baited device than in the device without semiochemical: 5.0 ± 0.6 × 10⁴ vs. 2.2 ± 0.4 × 10⁴ conidia per insect. The overall thrips mortality was also higher in the semiochemical‐baited device than in the device without the semiochemical: 59.3 ± 3.9 vs. 41.7 ± 3.5%. Conidial viability was not affected in the M. anisopliae‐treated device without semiochemicals, 7 days after treatment, whereas it was considerably decreased – from 81.0 ± 1.3 to 6.5 ± 1.1%, 2 and 7 days post‐inoculation – in the M. anisopliae‐treated semiochemical‐baited device. Thrips mortality was positively correlated with M. anisopliae conidial viability, and conidial viability was negatively correlated with conidial acquisition. This suggests that the semiochemical volatiles reduced the conidial viability, which in turn resulted in significant reduction in thrips mortality, despite the highest conidial acquisition in the semiochemical‐baited fungus‐treated device. Thrips density per plant was significantly reduced in both fungus‐treated treatments (with semiochemical: 8.7 ± 1.7 adults per plant; without semiochemical: 6.6 ± 1.4 adults per plant) compared with the fungus‐free control (19.8 ± 2.6 adults per plant). These results demonstrate the potential for an autoinoculation device strategy for the control of thrips, particularly in screenhouses.     

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.     

Cover crop and conidia delivery system impacts on soil persistence of Metarhizium anisopliae (Hypocreales: Clavicipitaceae) in sugarbeet

The sugarbeet root maggot, Tetanops myopaeformis (Röder), is a major North American pest of sugarbeet, Beta vulgaris L. Previous research suggests that moderate T. myopaeformis control is possible with the entomopathogen Metarhizium anisopliae (Metch.) Sorok. We conducted a three-year (2002–2004) experiment to assess impacts of oat, Avena sativa L. and rye, Secale cereale L., cover crops on persistence of corn grit-based granular or spray formulations of M. anisopliae isolate ATCC 62176 (i.e. MA 1200) applied at 8×10¹² viable conidia/ha in sugarbeet. More colony forming units (CFUs) were detected immediately after application [0 days after treatment (DAT)] in spray plots than granule-treated plots. However, 76–92% declines in CFUs per gram of soil occurred in spray plots within 30 DAT. Substantially (i.e. 83–560%) more rainfall occurred in June 2002 than during June of any other year. Subsequently, 71–670% increases in CFU concentrations occurred by 60 DAT in M. anisopliae granule-treated plots with oat or rye cover crops that year. CFU density increases were higher in cover crops in 2002, but no significant cover crop effects were detected. Conidia persisted for up to 30 DAT in M. anisopliae spray plots and 60 DAT in granule-treated plots in 2002; however, no increases occurred in the years with less June rainfall. Trends suggest that M. anisopliae aqueous sprays result in greater conidia concentrations than granules at sugarbeet plant bases in June during T. myopaeformis oviposition and larval establishment on host plants. Increases are possible when delivering conidia via granules, but high post-application rainfall could be necessary for conidia production.     

Combined use of entomopathogenic nematodes and Metarhizium anisopliae as a new approach for black vine weevil,Otiorhynchus sulcatus,control

Combined use of the entomopathogenic nematodes (EPNs), Heterorhabditis bacteriophora Poinar (Heterorhabditidae), Steinernema feltiae Bovien, and Steinernema kraussei Steiner (Steinernematidae) and the insect‐pathogenic fungus, Metarhizium anisopliae (Metsch.) Sorokin (Clavicipitaceae) was evaluated for control of third‐instar black vine weevil, Otiorhynchus sulcatus Fabricius (Coleoptera: Curculionidae). Black vine weevil larvae were exposed to various concentrations of M. anisopliae and EPNs and mortality was assessed weekly or at 3‐day intervals under laboratory and greenhouse conditions. The EPNs were added simultaneously, or 1 or 2 weeks after application of M. anisopliae. Throughout the experiments, the combined application of EPNs with M. anisopliae resulted in increased efficacy against black vine weevil. When the EPNs were applied 1 or 2 weeks after application of the fungus, 100% larval mortality was obtained, even when the biocontrol agents were used at reduced rates. The interactions observed suggest that EPN and M. anisopliae work together synergistically in potted Euonymus fortunei Blondy (Celastraceae) under greenhouse conditions and may provide a powerful and economically feasible approach for black vine weevil larval control.     

A new non-hydrophobic cell wall protein (CWP10) of Metarhizium anisopliae enhances conidial hydrophobicity when expressed in Beauveria bassiana

A cell wall protein, CWP10, resolved from the conidial formic acid extract of a Metarhizium anisopliae isolate, was characterized as a new 9.9-kDa protein with a 32-aa signal peptide with a central hydrophobic region (ca. 10 residues) at its N-terminus. This protein was proven neither to be hydrophobic nor glycosylated and encoded by a 363-bp, single-copy gene with three introns. CWP10 was existent in the conidial extracts of seven of 18 tested M. anisopliae isolates and much more abundant (immunogold-labeled) on conidial walls than in cytoplasm. Integrating the gene into a CWP10-absent strain of Beauveria bassiana led to excellent expression of CWP10 in aerial conidia, increasing net conidial hydrophobicity by 10.8% or adhesion to hydrophobic Teflon by 1.3-fold. However, the expressed protein had no effect on conidial tolerance to thermal and ultraviolet stresses. This is the first report on a non-hydrophobic cell-wall protein enhancing conidial hydrophobicity and adhesion of the fungal species.     

Resporulation of Metarhizium anisopliae granules on soil and mortality of Tenebrio molitor: Implications for wireworm management in sweetpotato

In Australia, sweetpotato (Ipomoea batatas L.) is vulnerable to root feeding insect pests such as wireworms (e.g., Agrypnus spp.). The number of registered insecticides to control these insect pests is limited and often pest pressure, for example by wireworms, is severe close to harvest, further limiting what insecticides can be applied. Incorporating biological control agents such as entomopathogenic fungi (e.g., Metarhizium anisopliae) into integrated pest management programmes may be feasible in sweetpotato. M. anisopliae has been shown to be effective in controlling more than 200 insects and it is able to reside and grow in the rhizosphere and rhizoplane, suggesting that M. anisopliae could be a promising candidate against soil insect pests. In the study presented here, M. anisopliae was formulated into calcium alginate granules fortified with nutrients. The resporulation of the fungal granules was tested on four different soil types in the laboratory. The biocontrol efficacy of the resulting fungal growth was also examined using larval mealworms, Tenebrio molitor as a model insect in the laboratory and the glasshouse. Our results indicated that sterilised soil favoured optimal fungal resporulation, although different soil types did not have a significant effect on fungal resporulation. The resulting fungal resporulation and growth on sterilised soil caused high mortality (up to 76%) of larval mealworms in the glasshouse, whereas the fungal granules applied to non-sterile soil demonstrated poor resporulation that led to low mortality (13%) of larval mealworms. The result of this study indicates that the manipulation of microbial populations in field soil is required to enhance the fungal growth and potential insect control against wireworms in the field.     

Virulence of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against red palm weevil,Rhynchophorus ferrugineus (Olivier)

Nineteen different isolates of the entomopathogenic fungi Beauveria bassiana sensu lato (s.l.) and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales), recovered from different soil samples (field crops, fruit orchards, vegetable fields and forests) and insect cadavers were tested against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) at two different spore concentrations (1 × 10⁷ and 1 × 10⁸ conidia mL^?1). Three isolates of B. bassiana and two of M. anisopliae gave >88 % larval and >75 % adult mortality of R. ferrugineus on their highest dose rate respectively. More sporulating cadavers (mycosis) resulted from a high dose rate compared to low dose on both life stages of R. ferrugineus. The current study confirmed the lethal action of B. bassiana and M. anisopliae isolates with mortality levels usually directly proportional to the conidial concentration. This study further confirmed that the isolates recovered from R. ferrugineus dead cadavers gave more mortality compared to the other sources. In the virulence assay two isolates of B. bassiana caused the highest percentage of both larval and adult mortality at all exposure intervals which suggest that they may be the most promising for use in sustainable management programs aimed at microbial control in date palm orchards.     

Comparative study between Larval Packet Test and Larval Immersion Test to assess the effect of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> on <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> tick larvae

Entomopathogenic fungi, such as Metarhizium anisopliae, for the control of arthropods, have been studied for more than 20 years. The aim of this study was to determine the best methodology to evaluate the in vitro effect of the fungus M. anisopliae on Rhipicephalus microplus tick larvae. We compared a modified Larval Packet Test (LPT) and a Larval Immersion Test (LIT). For the LPT filter papers were impregnated with 1 mL of M. anisopliae suspension in Triton X-100 at 0.02%, in concentrations of 10⁶, 10⁷ and 10⁸ conidia/mL and subsequently folded to include the larval ticks. LIT was performed by immersing the larvae in M. anisopliae suspensions for 5 min using the same three concentrations, then the larvae were placed on filter paper clips. For LPT, the LT₅₀ values obtained were 134.6, 27.2 and 24.8 days for concentrations of 10⁶, 10⁷ and 10⁸ conidia/mL; and the mortality after 21 days was 17.3, 17.6 and 38%, respectively. The LT₅₀ values of LIT were 24.5, 20 and 9.2 days with mortality after 21 days of 50.5, 64.7 and 98% for 10⁶, 10⁷ and 10⁸ conidia/mL, respectively. For the same conidia concentration, LIT showed a higher mortality in a shorter time interval when compared with LPT. These differences between the methods tested must be taking into account in further screening and effect studies with M. anisopliae. The set of results shown here could optimize the protocol used to identify M. anisopliae strains pathogenic against R. microplus.     

Virulence of the entomopathogenic fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> using soybean oil formulation for control of the cotton stainer bug, <Emphasis Type="Italic">Dysdercus peruvianus</Emphasis>

The cotton stainer bug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) is an insect pest that causes heavy losses in cotton plantations. The need to reduce the use of insecticides for control of this pest has increased steadily, and Metarhizium anisopliae (Ascomycota: Clavicipitaceae) could be an important biopesticide candidate to control this pest. The effect of M. anisopliae on D. peruvianus nymphs and adults using formulations with soybean oil and Agral^® was evaluated. Formulation using 10% soybean oil added to 10⁸ conidia mL^?1 (grown on used and reused rice) was the most effective for nymph and adult, causing 100% mortality 6 and 7 days after exposure, respectively. The SEM analysis of infected insects showed that M. anisopliae conidia were able to adhere anywhere on the exoskeleton, but were more abundant between the joints. Using the same rice for two batches of growth may be an option for improving commercial conidial production of M. anisopliae and may reduce overall costs. Its effect on D. peruvianus adults opens a new possibility for using this fungus as an alternative to chemical pesticides and the use of M. anisopliae in association with integrate pest management.     

Fecundity, longevity and establishment of Otiorhynchus sulcatus (Fabricius) and Otiorhynchus ovatus (Linnaeus) (Coleoptera: Curculionidae) from the Pacific North-west of the United States of America on selected host plants

1 The fecundity, longevity and establishment of Otiorhynchus sulcatus and Otiorhynchus ovatus from the Pacific North‐west U.S.A. was studied on five selected host plants: Picea abies‘Nidiformis’, Picea glauca‘Conica’, Taxus baccata, Rhododendron catawbiense‘Boursault’ and Fragaria×ananassa‘Totem’. 2 Teneral adults were used to study adult longevity and reproductive success. Leaves of these host plants were used for sustenance for 9 months. Larval establishment was studied by infesting potted host plants with eggs. 3 Fragaria×ananassa‘Totem’ produced the longest survival, shortest preoviposition time, the greatest number of eggs, and the highest fertility for adults of both species. Picea spp. were not good adult hosts for O. sulcatus. Taxus was a good adult host for O. sulcatus, but was a nonhost for adults and larvae of O. ovatus. 4 Adult hosts did not affect preoviposition time or egg viability with O. ovatus adults. With O. sulcatus, preoviposition time was greatly increased and egg viability was < 50% on Picea spp. 5 The best larval host was F.×ananassa‘Totem’ for O. sulcatus and P. glauca‘Conica’ for O. ovatus. Rhododendron was a poor larval host for both species. 6 When all of the studies on these two pests are considered, O. sulcatus appears to have varying host preferences from among its many geographical areas of occurrence whereas O. ovatus has a more universal host selection.