Selection of a highly virulent fungal isolate, Metarhizium anisopliae CLO 53, for controlling Hoplia philanthus

The June beetle, Hoplia philanthus Füessly (Coleoptera: Scarabaeidae), has become a widespread and destructive insect pest of lawns, sport turf, pastures, and horticultural crops in Belgium. The virulence of 34 entomopathogenic fungal isolates from the genera Metarhizium, Beauveria, and Paecilomyces to third-instar H. philanthus was tested in bioassays by dipping larvae in 10(7)conidia/ml suspensions. Two isolates of Metarhizium anisopliae (CLO 53 and CLO 54) caused maximally 90% mortality 10 weeks post-inoculation while other isolates only caused mortalities between 10 and 62%. The virulence of M. anisopliae CLO 53 was further tested by exposing H. philanthus larvae to conidial serial concentrations of 10(4)-10(9)conidia/g sandy soil for up to 11 weeks at 15, 20 or 25 degrees C. Mortality was dependant on the fungal concentration, exposure time, and temperature. Eleven weeks after inoculation, the LC50 values for this isolate ranged from 1.3 to 4.0 x 10(6), 1.0 to 3.2 x 10(5), and 2.5 x 10(4) to 10(5)conidia/g soil at 15, 20, and 25 degrees C, respectively. The LT50 values for this isolate ranged from 3.5 to 21.7, 2.4 to 18.7, and 2.9 to 16.1 weeks at concentrations of 10(9) and 10(4)conidia/g soil at 15, 20, and 25 degrees C, respectively. In glasshouse pot experiment with perennial ryegrass (Lolium perenne L.), the isolate CLO 53 caused mortalities of 50 and 88% of H. philanthus larvae 10 weeks after application of 10(4) and 10(6)conidia/cm(2) soil surface, respectively. The present results suggest that the Belgian isolate CLO 53 has excellent potential for biological control of H. philanthus.     

Food consumption by Chilo partellus (Lepidoptera: Pyralidae) larvae infected with Beauveria bassiana and Metarhizium anisopliae and effects of feeding natural versus artificial diets on mortality and mycosis

Second and third instar Chilo partellus larvae were infected with Beauveria bassiana and Metarhizium anisopliae (both at 1x10(8)conidia/ml) and daily consumption of maize leaves was measured. Infection by the fungi was associated with reduced mean daily food consumption. Reduction in food consumption became evident 3-4 days after treatment with the fungi for second instar larvae and 4-5 days for third instar larvae. Four conidial concentrations, 1x10(5), 1x10(6), 1x10(7), and 1x10(8)conidia/ml, were tested against second instar larvae. Food consumption dropped by 70-85% when the second instar larvae were treated with the fungi at 1x10(8)conidia/ml. Reduction in food consumption by C. partellus larvae infected with B. bassiana and M. anisopliae may offset the slow speed of kill of the fungi. The effect of artificial versus natural diets on mortality and mycoses of second instar larvae treated with the fungi at 1x10(8)conidia/ml was determined. Larvae provided with artificial diet suffered little mortality and mycoses than larvae provided with maize leaves. The LT(50) was longer for larvae provided with artificial diet.     

Selection of a Highly Virulent Fungal Isolate,Metarhizium anisopliae Ma TB 101, for Control of Taro Beetle,Papuana uninodis (Coleoptera: Scarabaeidae)

Fungal isolates (31 Metarhizium anisopliae var. anisopliae , five M. anisopliae var majus , three Beauveria bassiana and four B. brongniartii ) originating from a wide range of geographical locations, insect species and soil types were tested against Papuana uninodis (Coleoptera: Scarabaeidae). In a first test series, spores were applied topically to third-instar larvae and adults. The most effective strain against P. uninodis larvae and adults was Ma TB 101, a M. anisopliae isolate from adult P. woodlarkiana . For adults, strain Ma FI 384, a M. anisopliae from Popillia japonica , was almost equally effective. The 11 most effective isolates (nine M. anisopliae var. anisopliae , one B. brongniartii and one B. bassiana ) with LT values of less than 6 weeks in 50 adults and/or less than 4 weeks in larvae were tested for their efficacy against adults and larvae of P. uninodis by application of spores to soil (107 spores/g). Ma TB 101 was significantly more effective against both adults and larvae (LT ca. 10 days) than all other isolates (LT > 50 50 3 weeks). Two other M. anisopliae strains, Ma F 248 from soil and Ma FI 384 from P.japonica , were more effective than most isolates in adults. The latter three M. anisopliae isolates were tested in a concentration series against third-instar larvae and adults. Mortality was concentration related. Isolates Ma F 248 and Ma FI 384 did not achieve 50% mortality within the test period at concentrations lower than 107 spores/g of soil or feed. For concentrations of Ma TB 101 from 1 107 to 2 105 spores/g the LT ranged from 13 to 30 days in adults and 12 to 24 50 days in third-instar larvae. Accordingly, concentrations causing 50% mortality (LC ) for Ma 50 TB 101 were significantly lower than for the two other M. anisopliae isolates tested.     

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.     

Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (Anamorphic fungi) on the German cockroach (Blattodea: Blattellidae)

Virulence of Metarhizium anisopliae (Metschnikoff) Sorokin strain EAMa 01/121-Su against the German Cockroach, Blatella germanica (L.), was determined using four concentrations ranging from 4.2 x 10(6) to 4.2 x 10(9) spores per milliliter. The LD50 value was 1.4 x 10(7) spores per milliliter (56,000 spores per cockroach) and LT50 values were 14.8 days and 5.3 days for 4.2 x 10(8) and 4.2 x 10(9) spores per milliliter, respectively. An experiment was conducted to evaluate whether a fungal transmission could exist among infected and healthy cockroaches. Percentage mortality at a ratio of 1:10 of infected to unexposed cockroaches was 87.5% and LT50 was 12.2 days, which indicated the potential of this strain to be horizontally transmitted and to rapidly spread the infection in the insect population. The effect of a sublethal dose (ca. LD60) of M. anisopliae EAMa 01/121-Su strain, applied topically on German cockroaches, was studied by reciprocal crossing. Othecal production, oothecal hatchability, and nymphal production declined upon exposure to M. anisopliae EAMa 01/121-Su strain. The mean number of oothecae laid by female was progressively and significantly reduced by fungal treatment from second oviposition period onwards. Oothecal hatch of fungally challenged females was reduced by 46-49%, oothecal viability by 48-85%, and nymphal production by 22-35%. Only treated females showed an effect on oothecal production, oothecal hatch, and nymphal production, although oothecal hatch was also governed by treated males at a higher significance level. Our results on virulence and horizontal transmission of fungal conidia of M. anisopliae EAMa 01/121-Su strain and its sublethal reproductive effects on German cockroach females are discussed in terms of its potential to decrease the pest status of B. germanica in the short and long terms.     

Susceptibility of Agrilus planipennis (Coleoptera: Buprestidae) to Beauveria bassiana and Metarhizium anisopliae

The susceptibility of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) to selected strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin was evaluated through bioassays with direct immersion or foliar exposure under laboratory conditions. Results showed that A. planipennis adults were susceptible to B. bassiana and M. anisoplae. Significant time-mortality response was found for each isolates. Isolate B. bassiana GHA killed A. planipennis adults at a faster rate compared with other isolates tested, with the lowest average time-to-death values. The LC50 values estimated under direct immersion method ranged from 1.7 x 10(5) to 1.9 x 10(7), 3.5 x 10(4) to 5.3 x 10(5), and 4.1 x 10(3) to 2.9 x 10(5) conidia/ml for B. basissiana and from 3.2 x 10(6) to 1.1 x 10(7), 4.5 x 10(3) to 4.5 x 10(5), and 1.4 x 10(2) to 1.2 x 10(5) conidia/ml for M. anisopliae at 4, 5, and 6 d after treatment, respectively. By days 5 and 6, B. bassiana GHA outperformed all other isolates tested except ARSEF 7234, followed by ARSEF 7152, 6393, and 7180. Significant concentration-mortality response was also observed for two B. bassiana GHA formulations, BotaniGard ES and Mycotrol O, and M. anisopliae F52 when insects were treated through foliar exposure. The LC50 values ranged from 114.5 to 309.6, 18.4 to 797.3, and 345.3 to 362.0 conidia/cm2 for BotaniGard, Mycotrol, and M. anisopliae F52, respectively. Based on the results of these bioassays, the efficacy of both B. bassiana GHA formulations and M. anisopliae F52 were similar against adult A. planipennis. The potential use of entomopathogenic fungi for management of A. planipennis in North America is discussed.     

Evaluation of entomopathogenic fungus Metarhizium anisopliae against soil-dwelling stages of cabbage maggot (Diptera: Anthomyiidae) in glasshouse and field experiments and effect of fungicides on fungal activity

The effect of two isolates of the entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin (389.93 and 392.93) on root-feeding stages of cabbage root fly, Delia radicum (L.), was studied under glasshouse and field conditions. In glasshouse studies, the effect of drenching a suspension of conidia (concentration 1 x 10(8) m(-1), 40 ml per plant, applied on four occasions) onto the base of cabbage plants infested with D. radicum eggs was compared with mixing conidial suspension into compost modules (concentration 1 x 10(8) ml(-1), 25 ml per plant) used to raise seedlings. Drench application reduced the mean number of larvae and pupae recovered per plant by up to 90%, but the compost module treatment had no statistically significant effect. Both application methods reduced the emergence of adult flies from pupae by up to 92%. Most conidia applied as a drench application remained in the top 10-cm layer of compost. Applications of the fungicides iprodione and tebuconazole, which are used routinely on brassica crops, were compatible with using M. anisopliae 389.93 against D. radicum under glasshouse conditions, even though these fungicides were inhibitory to fungal growth on SDA medium. In a field experiment, drench applications of M. anisopliae 389.93 to the base of cauliflower plants at concentrations of 1 x 10(6) to 1 x 10(8) conidia ml(-1) did not control D. radicum populations, although up to 30% of larval cadavers recovered supported sporulating mycelium. Drench applications often exhibited considerable lateral movement on the soil surface before penetrating the ground, which may have reduced the amount of inoculum in contact with D. radicum larvae.     

Evaluation of Metarhizium anisopliae (Metsch) Sorok. to target larvae and adults of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) in soil and fiber band applications

The aim of this work has been to evaluate in the laboratory the potential of entomopathogenic fungi against adults and larvae of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) through fiber band application and a potted plant bioassay with soil application, respectively. Our previous findings revealed that Metarhizium anisopliae EAMa 01/58-Su isolate was the most virulent against neonate larvae of the buprestid. In the present work, M. anisopliae EAMa 01/58-Su isolate has been also shown to be highly virulent against adult beetles by immersion in a conidial suspension; thus it was selected to accomplish our objectives. When adult beetles were stimulated to climb 100 x 200 mm non-woven commercial fiber bands impregnated with conidia of M. anisopliae EAMa 01/58-Su isolate, total mortality rates varied from 85.7% to 100.0%; whereas no significant correlation was detected between the time needed to cross the band (mean value 648.7+/-22.4s) and the time of death, with mean average survival time ranging between 10.3 and 16.0 days, compared to 28 days of the controls. Potted seedlings (5-6 months old) of cherry plum (Prunus myrobalana Lois.), a commonly used apricot rootstock, were used to study the efficacy of soil treatment with M. anisopliae EAMa 01/58-Su isolate against neonate C. tenebrionis larvae. The soil inoculation with M. anisopliae EAMa 01/58-Su isolate had a significant effect on the mean number of dead larvae recovered from the roots, with mean mortality ranging from 83.3% to 91.6%; whereas no significant differences were detected between the three fungal doses. In all cases, dead larvae found within roots exhibited external signs of fungal growth. Hence, it may be possible to use M. anisopliae EAMa 01/58-Su isolate in a biocontrol strategy targeting both adults and larvae of C. tenebrionis.     

Attempts to control cabbage root flies Delia radicum L. and Delia floralis (Fall.) (Dipt., Anthomyiidae) with entomopathogenic fungi: laboratory and greenhouse tests

One laboratory and three greenhouse experiments were conducted to study the pathogenicity and efficacy of Finnish isolates of entomopathogenic hyphemycetous fungi against cabbage root flies. In Petri dishes, exposure to 1.5 × 10¹⁰ spores of Metarhizium anisopliae per dish caused 40–50% mortality of undifferentiated second- and third-stage larvae of Delia floralis , and 1 × 10⁷ spores per dish caused 40–50% mortality of Delia radicum larvae. In one greenhouse test, 1 × 10⁸ and 1 × 10⁹ spores of M. anisopliae and Paecilomyces fumosoroseus reduced the root damage of head cabbage by 20–70% compared with untreated controls, although this was not accompanied by significant reductions in the number of pupae. Only M. anisopliae consistently grew out of larvae and pupae of D. floralis during incubation that followed their recovery from the soil at the end of an experiment testing different formulations of M. anisopliae and Beauveria bassiana , but the frequency of the latent infections of the pest by M. anisopliae was not associated with reduced severity of damage to seedlings of head cabbage.     

Sporulation of Metarhizium anisopliae and Beauveria bassiana on Coptotermes formosanus and in vitro

The sporulation of 22 total isolates of Metarhizium anisopliae and Beauveria bassiana was quantified on cadavers of the Formosan subterranean termite, Coptotermes formosanus. Conidial production increased significantly over 11 days post-death. Effects of isolates of M. anisopliae and B. bassiana on in vivo sporulation were significant. Although the overall effects of fungal species on in vivo sporulation were not significant, the interactions between fungal species and certain times post-death were significant, indicating different sporulation patterns between the two fungal species. B. bassiana isolates could be categorized into a group with high total sporulation (day 11) and low quick sporulation (on days 2 and 3), while M. anisopliae isolates fell into another group with high quick sporulation and low total sporulation. This could give M. anisopliae an advantage over B. bassiana in termite microbial control due to termite defensive social behaviors. Conidial production was significantly higher in vitro than in vivo. In vitro and in vivo sporulation differed by as much as 89x and 232x among the selected isolates of B. bassiana and M. anisopliae, respectively. Correlation between in vivo and in vitro conidial production was positive and significant. This may allow preliminary in vitro screening of a large number of isolates for high in vivo sporulation.     

Virulence of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) on Anastrepha ludens (Diptera: Tephritidae): laboratory and field trials

Twenty isolates of the fungus Metarhizium anisopliae (Metsch). Sorkin (Ma) were evaluated to determine their virulence against last instar and adult emergence of Mexican fruit fly, Anastrepha ludens (Loew). Larvae were exposed by immersion in a conidial suspension at a concentration of 10(8) UFC/ml under laboratory conditions. Larvae and pupae cumulative mortality rates ranged from 37.9 to 98.75%. Thirteen isolates caused mortality rates > 83.7%, and their LT50 values ranged from 1.8 to 6.2 d. The Ma2, Ma8, and Ma16 isolates were evaluated at seven different concentrations ranging from 10(1) to 10(7) UFC/ml, showing LC50 values from 3.7 to 4.8 x 10(5) UFC/ml. In a field-cage experiment, 200 ml of a conidial suspension of Ma2, at a concentration of 2.5 x 10(6) UFC/ml, was applied on 2,500 cm2 soil surface (2 x 10(5) UFC/cm2). The fungus reduced adult emergence, 22% fewer adults emerging in a sandy loam soil, and 43% fewer in loam soil, compared with the controls. M. anisopliae may offer a preferable alternative to chemicals as a biological control agent against A. ludens.     

绿僵菌及其与dsRNA混合使用对褐飞虱的防治效果

褐飞虱Nilaparvata lugens Stl是我国重要的迁飞性水稻害虫,本文研究了金龟子绿僵菌Metarhizium anisopliae及其与dsRNA混合使用对褐飞虱的防治效果。绿僵菌悬浮液1.6×108孢子/m L至8×106孢子/m L对褐飞虱2龄、4龄和成虫进行喷药,发现1.6×107孢子/m L对各个虫态虫龄均有良好致死效果,并且成虫和4龄若虫均好于2龄若虫。在交配行为上来看,绿僵菌处理过的褐飞虱成虫活跃度非常低,从配对开始一直到交配结束的各个阶段都受到明显影响,处理组3 h的交配率只有3.70%,而对照组的交配率为24.44%。还把褐飞虱几丁质合成酶基因A的dsRNA与绿僵菌混合使用防治褐飞虱2龄和4龄若虫,结果表明0.5μg/μL ds CHSA与绿僵菌混合使用的防治效果最好,2龄若虫的死亡率为89.63%,4龄若虫的死亡率达到93.94%。而0.2μg/μL ds CHSA与绿僵菌的混合,对2龄和4龄若虫的致死率为65.56%-76.52%。研究结果为褐飞虱的生物防治提供了新的思路。     

Hirsutella thompsonii and Metarhizium anisopliae as potential microbial control agents of Varroa destructor,a honey bee parasite

The potential of Hirsutella thompsonii Fisher and Metarhizium anisopliae (Metschinkoff) as biological control agents of the parasitic mite, Varroa destructor Anderson and Trueman was evaluated in the laboratory and in observation hives. In the laboratory, time required for 90% cumulative mortality of mites (LT(90)) was 4.16 (3.98-4.42) days for H. thompsonii and 5.85 (5.48-7.43) days for M. anisopliae at 1.1 x 10(3) conidia mm(-2). At a temperature (34+/-1 degrees C) similar to that of the broodnest in a honey bee colony, Apis mellifera L., H. thompsonii [LC(90)=9.90 x 10(1) (5.86-19.35) conidia mm(-2) at Day 7] and M. anisopliae [LC(90)=7.13 x 10(3) (2.80-23.45) conidia mm(-2) at Day 7] both showed significant virulence against V. destructor. The applications of H. thompsonii to observation hives resulted in significant mortality of mites, and reduction of the number of mites per bee 21 and 42 days post-treatments. The treatments did not significantly affect the mite population in sealed brood. However, the fungus must have persisted because infected mites were still observed [82.97+/-(0.6)%] 42 days post-treatment. In addition, the fungus was found to sporulate on the host. A small percentage [2.86+/-(0.2)%] of dead mites found in the control hives also showed fungal infection, suggesting that adult bees drifted between hives and disseminated the fungus. H. thompsonii was harmless to the honey bees at the concentrations applied and did not have any deleterious effects on the fecundity of the queens. Microbial control with fungal pathogens provides promising new avenues for control of V. destructor and could be a useful component of an integrated pest management program for the honey bee industry.     

Susceptibility of the boll weevil to Steinernema riobrave and other entomopathogenic nematodes

The susceptibility of the boll weevil (BW), Anthonomus grandis Boheman, to Steinernema riobrave and other nematode species in petri dishes, soil (Hidalgo sandy clay loam), and cotton bolls and squares was investigated. Third instar weevils were susceptible to entomopathogenic nematode (EN) species and strains in petri dish bioassays at 30 degrees C. Lower LC(50)'s occurred with S. riobrave TX- 355 (2 nematodes per weevil), S. glaseri NC (3), Heterorhabditis indicus HOM-1 (5), and H. bacteriophora HbL (7) than H. bacteriophora IN (13), S. riobrave TX (14), and H. bacteriophora HP88 (21). When infective juveniles (IJs) of S. riobrave were applied to weevils on filter paper at 25 degrees C, the LC(50) of S. riobrave TX for first, second, and third instars, pupae, and 1-day-old and 10-days-old adult weevils were 4, 5, 4, 12, 13, and 11IJs per weevil, respectively. The mean time to death, from lowest to highest concentration, for the first instar (2.07 and 1.27days) and second instar (2.55 and 1.39days) weevils were faster than older weevil stages. But, at concentrations of 50 and 100IJs/weevil, the mean time to death for the third instar, pupa and adult weevils were similar (1.84 and 2.67days). One hundred percent weevil mortality (all weevil stages) occurred 3days after exposure to 100IJs per weevil. Invasion efficiency rankings for nematode concentration were inconsistent and changed with weevil stage from 15 to 100% when weevils were exposed to 100 and 1IJs/weevil, respectively. However, there was a consistent relationship between male:female nematode sex ratio (1:1.6) and nematode concentration in all infected weevil stages. Nematode production per weevil cadaver increased with increased nematode concentrations. The overall mean yield of nematodes per weevil was 7680IJs. In potted soil experiments (30 degrees C), nematode concentration and soil moisture greatly influenced the nematode efficacy. At the most effective concentrations of 200,000 and 400,000IJs/m(2) in buried bolls or squares, higher insect mortalities resulted in pots with 20% soil moisture either in bolls (94 and 97% parasitism) or squares (92 and 100% parasitism) than those of 10% soil moisture in bolls (44 and 58% parasitism) or squares (0 and 13% parasitism). Similar results were obtained when nematodes were sprayed on the bolls and squares on the soil surface. This paper presents the first data on the efficacy of S. riobrave against the boll weevil, establishes the potential of EN to control the BW inside abscised squares and bolls that lay on the ground or buried in the soil.     

Parasitism of Helicotylenchus lobus by Pasteuria penetrans in Naturally Infested Soil

The population density of Helicotylenchus lobus and the percentage of the population with spores of Pasteuria penetrans were determined for 10 monthly intervals in naturally infested turf grass soil at Riverside, California. The percentage of nematodes with attached spores ranged from 40% to 67%. No relationship was found between nematode density and the percentage of nematodes with spores. The mean and maximum numbers of spores adhering per nematode with at least one spore ranged from 2 to 8 and 7 to 66, respectively. The mean number of spores per nematode (based on total number of H. lobus) was correlated with the percentage of nematodes with spores. Spores adhered to both adult and juvenile H. lobus. Between 9% and 32% of the nematodes with spores had been penetrated and infected by the bacterium. Many infected nematodes were dead, but mature spores were also observed within living adult and juvenile H. lobus that exhibited no apparent reduction in viability and motility. Spore and central endospore diameters of this P. penetrans isolate were larger than those reported for the type isolate from Meloidogyne incognita, but transmission and scanning electron microscopy did not reveal significant morphological differences between the two isolates. Spores of the isolate associated with H. lobus did not adhere to juveniles of M. incognita.     

Host Range and Ecology of Isolates of Pasteuria spp. from the Southeastern United States

Isolates of Pasteuria penetrans were evaluated for ecological characteristics that are important in determining their potential as biological control agents. Isolate P-20 survived without loss of its ability to attach to its host nematode in dry, moist, and wet soil and in soil wetted and dried repeatedly for 6 weeks. Some spores moved 6.4 cm (the maximum distance tested) downward in soil within 3 days with percolating water. The isolates varied greatly in their attachment to different nematode species and genera. Of five isolates tested in spore-infested soil, three (P-104, P-122, B-3) attached to two or more nematode species, whereas B-8 attached only to Meloidogyne hapla and B-I did not attach to any of the nematodes tested. In water suspensions, spores of isolate P-20 attached readily to M. arenaria but only a few spores attached to other Meloidogyne spp. Isolate P-104 attached to all Meloidogyne spp. tested but not to Pratylenchus scribneri. Isolate B-4 attached to all species of Meloidogyne and Pratylenchus tested, but the rate of attachment was relatively low. Isolate P-Z00 attached in high numbers to M. arenaria when spores were extracted from females of this nematode; when extracted from M. javanica females, fewer spores attached to M. arenaria than to M. javanica or M. incognita.     

Epizootiological studies of Amblyospora camposi (Microsporidia: Amblyosporidae) in Culex renatoi (Diptera: Culicidae) and Paracyclops fimbriatus fimbriatus (Copepoda: Cyclopidae) in a bromeliad habitat

The epizootiology of Amblyospora camposi was studied in a natural population of Culex renatoi, a bromeliad-inhabiting mosquito, and its intermediate host, Paracyclops fimbriatus fimbriatus, over a 2-year period. Twenty Eryngium cabrerae plants were sampled monthly from January 2003 to January 2005 and the prevalence of A. camposi in P.f. fimbriatus and Cx. renatoi populations was determined. The monthly prevalence rates of meiospore infections in Cx. renatoi larvae never exceeded 5.5% and was detected in 50% of the monthly samples. Meiospores were available in plants over the course of the study at a mean concentration of 2 x 10(4) meiospores/ml. Within each plant the parasite was maintained by horizontal transmission. P.f. fimbriatus with vegetative stages and mature spores were found regularly in bromeliads suggesting efficient meiospore infectivity to field copepod populations. The mean concentration of spores from copepods found in plants was 8 x 10(2) spores/ml. Infections in copepods were detected in 54% of the monthly samples with a prevalence rate ranging from 0.55 to 17.4% and an overall average of 5.1%. Vegetative stages in fourth instar mosquito larvae (probably derived from the horizontal pathway via spores formed in copepods) were detected in 12.5% of the monthly samples with an overall prevalence rate of 1.1%. Infections in female and male adults were detected in 20.8% of the monthly samples with an overall average of 4.1% and 6.8%, respectively.     

Laboratory evaluation of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against puparia and adults of Ceratitis capitata (Diptera: Tephritidae)

Laboratory experiments were done to measure the pathogenicity of 10 autochthonous isolates of Beauveria bassiana (Balsamo) Vuill. and of five Metarhizium anisopliae (Metsch.) Sorok. toward puparia and adults of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Although all isolates applied via inoculation of the fungal suspensions on the ventral surface of the abdomen were pathogenic to adults, with mortality rates ranging from 30 to 100% and average survival times (ASTs) from 6.5 to 8.6 d, when C. capitata puparia were immersed in the conidial suspensions, only B. bassiana Bb-1333 and EABb 01/103-Su and M. anisopliae EAMa 01/58-Su isolates caused >50% mortality of puparia. In a second series of bioassays conducted on five selected isolates, adults were sprayed with four 10-fold concentrations ranging from 1.0 x 10(5) to 1.0 x 10(8) colony-forming units (cfu)/ml. The median lethal concentrations (LC50) of the four most virulent isolates ranged from 4.9 x 10(5) to 2.0 x 10(6) cfu/ml with estimated time to kill 50% of the insects ranging from 4.6 to 5.3 d. The effect of a sublethal dose (ca. LD50) of either B. bassiana EABb 01/103-Su or M. anisopliae EAMa 01/58-Su isolate was studied by reciprocal crossing. Treatment with B. bassiana reduced fecundity and fertility at 6, 8, and 10 d after treatment, with fecundity and fertility reductions ranging from 20.0 to 71.2% and from 33.6 to 60.0%, respectively. These reductions occurred in pairing combinations of treated females with either treated or nontreated males. M. anisopliae was more effective in reducing fecundity and fertility at 6 d after treatment, with the reduction varying from 58.4 to 72.1% and from 28.6 to 45.9%, respectively. In addition, the first oviposition was significantly delayed for 1 d in females treated by either fungal species. The above-mentioned five selected isolates were assayed against C. capitata puparia treated as late third instars in sterilized soil at 25'C under three moisture conditions (-0.1, -0.01, and -0.0055 MPa). At -0.01 MPa, all isolates were low pathogenic to C. capitata puparia, whereas significant differences in the puparia mortality occurred between isolates at -0.1 and -0.0055 MPa. The highest pupal mortalities ranged from 52.5 to 70.0%, as a function of soil moisture and were caused by EAMa 01/58-Su and Bb-1333 isolates.     

Factors affecting the toxicity of Bacillus thuringiensis var. israelensis and Bacillus sphaericus to fourth instar larvae of Chironomus tepperi (Diptera: Chironomidae)

Laboratory bioassays (48h duration, 25+/-1 degrees C) were used to determine the toxicity of Bacillus thuringiensis var. israelensis (B.t.i.) and Bacillus sphaericus to fourth instar larvae of Chironomus tepperi, a major pest of rice in southern Australia. Bioassays were conducted using different combinations of larval ages and densities to determine if these factors affected toxicity. The effects of temperature and substrate type on B.t.i. toxicity were also investigated. Tests were conducted using a commercial B.t.i. formulation (VectoBac WDG, 3000ITU/mg), a spore/crystal mixture derived from the VectoBac WDG strain, and VectoLex WDG, a commercial B. sphaericus formulation (650ITU/mg). VectoBac WDG was highly toxic to fourth instar C. tepperi in bioassays using a sand substrate (LC(50) 0.46mg/L, older larvae); younger fourth instar larvae were more susceptible (LC(50) 0.20mg/L). Increasing larval densities (from 10 to 30 per bioassay cup) increased LC(50) values for both age groups, significantly so in the case of older larvae (higher density LC(50) 0.80mg/L). Use of a soil substrate increased the LC(50) value (older larvae, 10 per cup) to 0.99mg/L. Similar differences in toxicity relative to larval age and substrate type were found in bioassays using the B.t.i. spore/crystal mixture. VectoBac WDG and the spore/crystal mixture both showed similar (approximately 6-fold) declines in activity between 30 and 17.5 degrees C. At lower temperatures (between 17.5 and 15 degrees C), activity of the spore/crystal mixture declined much more rapidly than that of VectoBac WDG. VectoLex WDG showed very low toxicity to C. tepperi larvae, and the overall impact of larval age and density was relatively minor (LC(50) values 1062-1340mg/L). Autoclaving VectoLex WDG did not substantially reduce its toxicity (LC(50) 1426mg/L), suggesting that formulation additives (i.e., surfactants and other adjuvants) are responsible for much of the toxicity occurring at the high product concentrations required to cause C. tepperi mortality. Whilst VectoLex WDG was ineffective against C. tepperi, VectoBac WDG has the potential to provide selective control of this rice pest at economically viable application rates.     

Metarhizium anisopliae host-pathogen interaction: differential immunoproteomics reveals proteins involved in the infection process of arthropods

Metarhizium anisopliae is an entomopathogenic fungus well characterized for the biocontrol of a wide range of plagues. Its pathogenicity depends on the secretion of hydrolytic enzymes that degrade the host cuticle. To identify proteins involved in the infection process and in host specify, immunoproteomic analysis was performed using antiserum produced against crude extract of M. anisopliae cultured in the presence of Rhipicephalus (Boophilus) microplus and Dysdercus peruvianus cuticles. Spots detected using antisera produced against M. anisopliae cultured in cuticles and spore surface proteins, but not with antiserum against M. anisopliae cultured in glucose, were identified so as to give insights about the infection process. An MS/MS allowed the identification of proteases, like elastase, trypsin, chymotrypsin, carboxypeptidase and subtilisin (Pr1A, Pr1I and PR1J), chitinases, DNase I and proline-rich protein. Chymotrypsin and Pr1I were inferred as host specific, being recognized in D. peruvianus infection only. This research represents an important contribution to the understanding the adaptation mechanisms of M. anisopliae to different hosts.