Pathogenicity testing of shellfish hatchery bacterial isolates on Pacific oyster Crassostrea gigas larvae

Bacterial diseases are a major cause of larval mortality in shellfish hatcheries. Even with proper sanitation measures, bacterial pathogens cannot be eliminated in all cases. The pathogenicity of bacteria isolated from Pacific Northwest shellfish hatcheries to Pacific oyster Crassostrea gigas larvae was investigated. We found 3 highly pathogenic strains and 1 mildly pathogenic strain among 33 isolates tested. These strains appear to be members of the genus Vibrio. Although there have been many studies of bivalve bacterial pathogens, a standard method to assess bacterial pathogenicity in bivalve larvae is needed. Thus, we developed 2 methods using either 15 ml conical tubes or tissue culture plates that were employed for rapidly screening bacterial strains for pathogenicity to Pacific oyster larvae. The tissue culture plates worked well for screening both mildly pathogenic strains and LD50 (lethal dose) assays. This method allowed for non-intrusive and non-destructive observation of the oyster larvae with a dissecting microscope. The LD50 for the 3 highly pathogenic strains ranged between 1.6 and 3.6 x 10(4) colony forming units (CFU) ml(-1) after 24 h and between 3.2 x 102 and 1.9 x 10(3) CFU ml(-1) after 48 h.     

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.     

Toxicity of chitinase-producing Bacillus thuringiensis ssp. kurstaki HD-1 (G) toward Plutella xylostella

One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium. Of 14 strains that produced chitinase, B. thuringiensis ssp. kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study. This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30 degrees C for 2 days. The toxicities of B. thuringiensis ssp. kurstaki HD-1(G) and B. thuringiensis ssp. kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC(50)'s of 4.93 x 10(4) and 1.32 x 10(5) spores/ml, respectively. If the culture broth from B. thuringiensis ssp. kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC(50)'s were reduced to 6.23 x 10(3) and 7.60 x 10(4) spores/ml, respectively. The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B. thuringiensis ssp. kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy. The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae. A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B. thuringiensis ssp. kurstaki HD-1(G) supplemented with supernatant containing chitinase. Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.     

DNA hybridization assay for detection of gypsy moth nuclear polyhedrosis virus in infected gypsy moth (Lymantria dispar L.) larvae.

Radiolabeled Lymantria dispar nuclear polyhedrosis virus DNA probes were used in a DNA hybridization assay to detect the presence of viral DNA in extracts from infected larvae. Total DNA was extracted from larvae, bound to nitrocellulose filters, and assayed for the presence of viral DNA by two methods: slot-blot vacuum filtration and whole-larval squashes. To test the assays, neonate larvae were fed droplets containing a known concentration of L. dispar nuclear polyhedrosis virus and observed for up to 10 days to determine the percentage of infected larvae. The average percent mortalities were 88.0, 60.7, 26.0, and 5.3% for larvae fed droplets containing 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) polyhedral inclusion bodies (PIBs) per ml, respectively. Other larvae treated with the same virus concentrations were frozen at 2, 4, and 6 days postinoculation and examined by the hybridization techniques. The average percentage of slot blots containing viral DNA equaled 81.0, 58.0, 18.0, and 6.0% for larvae blotted 4 days after treatment with 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) PIBs per ml, respectively, and 89.9, 52.1, 26.6, and 6.0%, respectively at 6 days postinoculation. Thus, the hybridization results were closely correlated with mortality observed in reared larvae. Hybridization of squashes of larvae frozen 4 days after receiving the above virus treatments also produced accurate measures of the incidence of virus infection.     

DNA hybridization assay for detection of gypsy moth nuclear polyhedrosis virus in infected gypsy moth (Lymantria dispar L.) larvae

Radiolabeled Lymantria dispar nuclear polyhedrosis virus DNA probes were used in a DNA hybridization assay to detect the presence of viral DNA in extracts from infected larvae. Total DNA was extracted from larvae, bound to nitrocellulose filters, and assayed for the presence of viral DNA by two methods: slot-blot vacuum filtration and whole-larval squashes. To test the assays, neonate larvae were fed droplets containing a known concentration of L. dispar nuclear polyhedrosis virus and observed for up to 10 days to determine the percentage of infected larvae. The average percent mortalities were 88.0, 60.7, 26.0, and 5.3% for larvae fed droplets containing 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) polyhedral inclusion bodies (PIBs) per ml, respectively. Other larvae treated with the same virus concentrations were frozen at 2, 4, and 6 days postinoculation and examined by the hybridization techniques. The average percentage of slot blots containing viral DNA equaled 81.0, 58.0, 18.0, and 6.0% for larvae blotted 4 days after treatment with 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) PIBs per ml, respectively, and 89.9, 52.1, 26.6, and 6.0%, respectively at 6 days postinoculation. Thus, the hybridization results were closely correlated with mortality observed in reared larvae. Hybridization of squashes of larvae frozen 4 days after receiving the above virus treatments also produced accurate measures of the incidence of virus infection.     

The effect of some pesticides on the rhythmic activity of adductor muscle of fresh-water mussel larvae

The effect of some pesticides on the rhythmic and tonic activity of adductor muscle of fresh-water mussel (Anodonta cygnea) larvae was investigated in short-term experiments. In the examined concentration range, Dimecron-50 does not influence the rhythmic activity of adductor muscles, while high concentrations of Gramoxone (is greater than 1 ml/l), Thimet-10 G (is greater than 5 x 10(-1) g/l) and Hungaria L-7 (is greater than 10(-1) g/l) cause a slight increase in it. These substances also induce a prolonged tonic contraction of a small proportion (10%) of larvae. Hungaria L-7 makes an exception, causing considerable contracture (30%). The concentrations inhibiting the TA activation by 50% are: 6x 10(-1) ml/l Dimecron-50; 2.5 x 10(-1) ml/l Gramoxone; 10(-1) g/l Hungaria L-7 and 8x 10(-2) g/l Thimet-10 G, while the 50% inhibition of the nonspecific ACh activation is resulted by 2 x 10(-1) ml/l D-50. It is concluded that contamination of environmental waters with Dimecron-50, Thimet-10 G or Hungaria L-7 may be injurious also for Pelecypoda larvae, that may contribute to the unbalancing of the water ecosystem.     

Effect of probiotic bacteria on survival and growth of Cortez oyster larvae, Crassostrea corteziensis (Bivalvia: Ostreidae)

Disease control problems have major constraints in aquaculture production, and the use of probiotics in larviculture is a valid alternative to antibiotics. This study analyzed the effect of probiotic bacteria on survival and final size of Cortez oyster larvae Crassostrea corteziensis. Two different probiotic concentrations were evaluated, 1 x 10(4) and 1 x 10(5) CFU/ml of Lactic acid bacteria (strain NS61) isolated from Nodipecten subnodosus, and bacilli isolated from the white leg shrimp, Litopenaeus vannamei (Pseudomonas aeruginosa, strain YC58) and C. corteziensis (Burkholderia cepacia, strain Y021). Bacteria were added directly into culture tanks, starting the bioassays from veliger to pediveliger stages as follows: (1) Control, without probiotics; (2) lactic acid bacteria (Lb); (3) bacilli mix (Mb) in a proportion 1:1. Results showed a higher larval survival with Lb and Mb at a dose of 1 x 10(4) CFU/ml compared to the control group. Larvae exposed to Mb at 1 x 10(5) CFU/ml showed higher survival than Lb and control. Larval final size was not significantly increased with the tested probiotics, but larvae treated with Lb at 1 x 10(5) CFU/ml showed less survival rate than those treated at 1 x 10(4) CFU/ml. This study showed the beneficial effect of these probiotics, added individually or mixed in C. corteziensis larvae culture.     

Efficacy of Bacillus thuringiensis against Phyllocnistis citrella (Lepidoptera: Phyllocnistidae)

The purpose of this work was to analyze the efficacy of Bacillus thuringiensis Berliner on the control of Phyllocnistis citrella Stainton (Lepidoptera: Phyllocnistidae) in laboratory and field trials. In the laboratory, four B. thuringiensis were used: Dipel (commercial formulation) tested at the concentrations of 25 x 10(6) and 25 x 10(8) spores/ml and the isolates K, 6, and 15 (collections of the University of Azores) at the concentration of 25 x 10(6) spores/ ml. A surfactant solution of nonoxinol also was tested with or without the different suspensions of B. thuringiensis. Leaves of Citrus sinensis (L.) Osbeck with second or third instars of leafminer larvae were used in all tests. Bacterial suspensions were applied topically on the surface of intact leaf mines or by injection inside the mine, near the head of the leafminer. When injecting both concentrations of Dipel into the mines, mortality of the leafminers increased compared with the topical application, although no significant differences were observed. The addition of the nonoxinol to the Dipel suspension, applied topically, increased the effect of B. thuringiensis, but differences were not significant. The mortality of the leafminers treated only with the nonoxinol solution increased significantly 48 h after treatment, compared with the control group, suggesting an insecticidal effect of this surfactant when used at a concentration of 0.01%. All the tested B. thuringiensis were equally active against the leafminer, either when applied topically or by mine injection. Field trials showed a significant difference between larval mortality of the control group and the results observed at the trees treated with B. thuringiensis 48 h after treatments.     

Evaluation of Metarhizium anisopliae (Deuteromycota: Hyphomycetes) for control of broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae) in mulberry

A study on 12 entomopathogenic fungi for controlling broad mite (Polyphagotarsonemus latus (Banks)) in mulberry found that Metarhizium anisopliae CKM-048 was the most virulent strain in controlling both larvae and adult broad mites at the concentration of 2 x 10(8) conidia/ml. There was no ovicidal effect when tested with broad mite eggs. Median lethal concentrations (LC(50)) of M. anisopliae in killing larvae and adults were 8.7 x 10(6) and 1.3 x 10(7 )conidia/ml, respectively. Median lethal times (LT(50)) of larvae and adults were 2.4 and 3.8 days, respectively, at the concentration of 2 x 10(8) conidia/ml. The fungus was found to produce protease and chitinase. Scanning electron microscope (SEM) studies were done to monitor the infection steps of the fungus on broad mites. A greenhouse test on mulberry trees revealed that M. anisopliae could reduce the broad mite population within 4 days after treatment. However, after 7 days, its efficacy was decreased significantly.     

Laboratory evaluation of entomopathogenic fungi against larvae and adults of onion maggot (Diptera: Anthomyiidae)

Laboratory experiments were done to measure the susceptibility of larvae and adults of the onion maggot, Delia antiqua (Meigen) (Diptera: Muscidae: Anthomyiidae) to 27 isolates of entomopathogenic fungi from four genera [Beauveria Vuillemin, Lecanicillium (Petch) Zare & W. Gams, Metarhizium Sorokin, and Paecilomyces Bainier]. A novel bioassay was developed for D. antiqua larvae by using a diet based on mixed vegetable powder. When evaluated in a virulence screen, the fungal isolates caused less mortality of D. antiqua larvae than adults. Only three isolates caused > 50% mortality of larvae, whereas 12 isolates caused > 50% mortality of adults. Fungal species was a statistically significant factor affecting the mortality of larvae but not of adults. The fungal isolates causing the most mortality of larvae tended to belong to Metarhizium anisopliae (Metschnikoff) Sorokin. Two M. anisopliae isolates (389.93 and 392.93) were evaluated in dose-response bioassays. The median lethal concentrations of the isolates against larvae were 6.1 x 10(7) conidia ml(-1) for isolate 389.93 and 7.6 x 10(7) conidia ml(-1) for isolate 392.93. The emergence of adult flies from pupae was reduced at high concentrations of conidia (3.0 x 10(8) and 1.0 x 10(8) conidia ml(-1)). The median lethal concentrations of the isolates against adults were 1.7 x 10(7) and 4.0 x 10(7) conidia ml(-1), respectively. Some of the fungal isolates examined may have potential as biological control agents of larvae of D. antiqua and related species.     

Larval survival and development of susceptible and resistant Ostrinia nubilalis (Lepidoptera: Pyralidae) on diet containing Bacillus thuringiensis

Abstract 1 Larval survival and development of Dipel‐susceptible and ‐resistant strains of European corn borer, Ostrinia nubilalis (Hübner), were assayed using diets incorporating low doses of a commercial formulation of Bacillus thuringiensis var. kurstaki Berliner (Dipel ES). 2 Larval mortality, growth and development, pupation rate and pupal weight were not significantly different between Dipel‐susceptible and ‐resistant strains when larvae were reared on a nontoxic control diet. 3 Larval mortality of Dipel‐resistant larvae did not significantly change as Dipel concentration increased at the tested concentrations, whereas mortality of Dipel‐susceptible larvae increased dramatically as Dipel concentration increased. 4 Larval development was significantly delayed when larvae were fed diets containing low doses of Dipel. 5 Pupation rate and pupal body weight declined as Dipel concentration increased but it decreased faster for the susceptible strain than for the resistant strain.     

Screening and characterisation of potentially pathogenic bacteria associated with Atlantic cod Gadus morhua larvae: bath challenge trials using a multidish system

In intensive aquaculture systems, high concentrations of nutrients and high densities of fish larvae provide favorable conditions for opportunistic pathogenic bacteria to flourish. We screened potentially pathogenic bacterial strains isolated from moribund Atlantic cod Gadus morhua larvae, pollack Pollachius pollachius, coalfish Pollachius virens, Atlantic halibut Hippoglossus hippoglossus, rotifers, algae and water samples from different hatcheries. Three identical challenge experiments tested a total of 53 strains. A multidish system was used: cod eggs were placed in single wells, together with 2 ml of sterile seawater, and exposed to the bacterial cultures. Final bacterial concentrations in the wells were 10(6) and 10(4) CFU ml(-1). Eggs and larvae not exposed to bacteria were used as unchallenged controls. Challenged controls were exposed to Vibrio anguillarum strain 610. Eggs were challenged approximately 48 h prior to hatching and mortality was recorded daily throughout the yolk-sac period. In spite of the high challenge dose of 106 CFU ml(-1), only 5 bacterial strains tested caused higher mortality than the unchallenged controls. Four of these strains were identified by 16S rDNA and gyrase B gene (GyrB) sequencing as resembling V. anguillarum and 1 strain resembled Carnobacterium sp. Most of the larvae exposed to these strains died within 10 d of challenge. Serotyping of the strains resembling V. anguillarum gave inconclusive results. This indicates differences in serology compared to the serotypes O1, O2 and O3, associated with disease. Three bacterial strains seemed to have a slower infection rate, indicating a longer incubation period. The remaining 45 strains did not seem to have a negative effect on larval survival, suggesting that these are not primary pathogens.     

Evaluation of three formulations of Beauveria bassiana for control of lesser mealworm and hide beetle in Georgia poultry houses

Initial screening of 12 Beauveria bassiana (Balsamo) Vuillemin isolates against larvae of the lesser mealworm (Alphitobius diaperinus [Panzer]) resulted in the selection of two isolates, GHA and 707, for further testing under field conditions. Three formulations of each strain were prepared: an EC, a ground corn granular formulation, and waste product of fungal propagation containing spent media, mycelia, and unharvested conidia ("residue" formulation). Two field trials were conducted in commercial caged-layer houses in Georgia with 5-6 mo of manure accumulation and established populations of A. diaperinus and hide beetles (Dermestes maculatus DeGeer). In the first trial field, B. bassiana was applied a single time to the manure surface at either 10(9) (EC and granular cornmeal bait formulations) or 10(8) (residue formulation) fungal spores per square meter. In the second trial, two successive weekly treatments were applied, using a total of 6x the rate of application used in the first trial, Significant treatment effects were short-lived and only detected 2 wk after treatment in both trials. The granular formulations of both strains and the residue formulation of the GHA strain provided the greatest degree of suppression (60-90%) of beetle larvae. A laboratory bioassay confirmed that the granular bait was the most effective formulation. More frequent applications made earlier in the manure accumulation cycle may be necessary to achieve satisfactory control of these beetles.     

Immobilization in alginate as a technique for the preservation of Bacillus thuringiensis var. israelensis for long-term preservation

Technique for immobilization using sodium alginate as the matrix to preserve Bacillus thuringiensis var. israelensis isolates for long time storage was developed. Two strains of B. thuringiensis var. israelensis viz., VCRC B-17 and WHO standard strain IPS-82 were immobilized in alginate matrix and preserved at 4 degrees C and when tested both were found to have maintained excellent viability and mosquito larvicidal activity for 10 years. Mosquito larvicidal activity of B-17 and IPS-82 alginate beads, in term of LC(50) values before storage was 72.07 ng/ml and 47.07 ng/ml, respectively and after storage at 4 degrees C for a period of 1 to 10 years the values ranged from 69.88 to 73.86 ng/ml with a mean of 72.38 ng/ml and 45.32 to 48.60 ng/ml with a mean of 47.49 ng/ml, respectively. Similarly spore count of the beads of the respective strains was 4.37 x 10(8) and 3.33 x 10(10) CFU/mg before storage. After storage at 4 degrees C for a period of 1 to 10 years the counts of the beads of the respective strains ranged from 4.23 x 10(8) to 4.83 x 10(8) CFU/mg (mean of 4.49 x 10(8) CFU/mg) and 3.2 x 10(10) to 3.87 x 10(10) CFU/mg (mean of 3.54 x 10(10) CFU/mg). The alginate matrix immobilization technique has many advantages over free cells are that they enhance the stability of both spores and toxin against several physicochemical conditions and confer reduced susceptibility to contamination.     

Susceptibility of Ixodes scapularis (Acari: Ixodidae) to Metarhizium brunneum F52 (Hypocreales: Clavicipitaceae) using three exposure assays in the laboratory

An emulsifiable concentrate (EC) and granular (G) formulation of the entomopathogenic fungus, Metarhizium brunneum strain F52 (formerly Metarhizium anisopliae strain F52) were tested against unfed adults and nymphs of Ixodes scapularis Say in the laboratory. Three exposure methods; dip, surface contact, and direct spray application, and three exposure time intervals (3, 30, and 300 min) were used to evaluate the EC formulation. Application rates ranged from 2.6 x 10(2) to 2.6 x 10(8) conidia/cm2. The surface treatment was used for granular formulation with concentrations ranging from 2.3 x 10(5) to 2.3 x 10(7) conidia/cm2 for same three exposure times. Both the EC and G formulations of this fungus were highly pathogenic against I. scapularis adults and nymphs. Logistic regression analysis found formulation, spore concentration, time of exposure, and observation period were significant or highly significant factors influencing tick mortality. For adult I. scapularis, the spray application with the EC formulation of M. brunneum F52 resulted in a lower LC50 (5.9 x 10(4) conidia/cm2) at 30 min than surface exposure to the EC (LC50 = 1.3 x 10(6) conidia/cm2) or G formulation (LC50 = 8.1 x 10(5) conidia/cm2). At higher concentrations, fungal activity was evident in adult I. scapularis held at 5 degrees C suggesting the fungus may provide control in the cooler fall season. While the observed pathogenicity of a fungus against ticks can be dependent upon the bioassay assessment, we found nymphs and adults of I. scapularis to be highly susceptible to M. brunneum F52, regardless of the exposure method used.     

Toxicity of plant essential oils and their components against Lycoriella ingenua (Diptera: Sciaridae)

Plant essential oils from 20 plant species were tested for their insecticidal activity against larvae of Lycoriella ingenua (Dufour) (Diptera: Sciaridae) by using a fumigation bioassay. Good insecticidal activity (>90%) against larvae of L. ingenua was achieved with essential oils of caraway seed Carum carvi (L.)], lemongrass [Cymbopogon citratus (D.C.) Stapf.], mandarine (Citrus reticulate Blanco), nutmeg (Myristica fragrans Houtt), cade (Juniperus oxycedrus L.), spearmint (Mentha spicata L.), cumin (Cuminum cyminum L.), and thyme red [Thymus vulgaris (L.)] oils at 30 X 10-3 mg/1 air. Among them, caraway seed, spearmint, cumin, and thyme red essential oils were highly effective against L. ingenua at 20 x 10(-3) mg/ml air. Analysis by gas chromatography-mass spectrometry led to identification of 4, 9, 8, and 17 compounds from caraway seed, spearmint, cumin, and thyme red oils, respectively. These compounds were tested individually for their insecticidal activities against larvae of L. ingenua, and compared with the toxicity of dichlorvos. Carvacrol, thymol, linalool, cuminaldehyde, p-cymen, terpinen-4-ol, and carvone was effective at 10 x 10(-3) mg/l. The insecticidal activity of dichlorvos was 60% at 10 x 10(-3) mg/ml. Effects of four selected plant essential oils on growth of oyster mushroom, Pleurotus ostreatus, also were investigated.     

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.     

Pathogenicity of Beauveria bassiana,Metarhizium anisopliae (Deuteromycotina: Hyphomycetes), and other entomopathogenic fungi against Lygus lineolaris (Hemiptera: Miridae)

The pathogenicity of 32 fungal isolates from the genera of Beauveria, Verticillium, Paecilomyces, Metarhizium, Mariannaea, and Hirsutella to second-instar tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was tested under laboratory conditions. These isolates originated from various insect hosts and substrates from France, Denmark, Austria, Italy, Turkey, Syria, and the United States. A single exposure concentration (1 x 10(7) conidia/ ml) assay for each isolate was first conducted by immersing the insects in 10 ml of a fungal suspension for 5s. These were followed by concentration-mortality assays on five of the most pathogenic isolates using four test concentrations ranging from 2 x 10(4) to 2 x 10(7) conidia/ml. B. bassiana 726 (Bemisia-passaged GHA strain) was used as a standard for comparison in all of the assays. Among the test isolates, three produced mortality not significantly different from the water control. Mortality ranged from 35 to 98% among the other 29 isolates. The LC50 values of the five most pathogenic isolates ranged from 0.8 to 5.0 x 10(5) conidia/ml. The LT50 values for these isolates ranged from 6.0 to 6.9, 3.1 to 5.1, and 2.5 to 4.0 d for concentrations of 2 x 10(5), 2 x 10(6), and 2 x 10(7) conidia/ml, respectively. Two strains of B. bassiana (ARSEF 1394,5665) and one M anisopliae (ARSEF 3540) were more pathogenic to the nymphs than the standard, having significantly lower LC50 and LT50, values. Our results demonstrated that several genera of entomopathogenic fungi have promise as microbial control agents against L. lineolaris.     

Effection of Ocneridia volxemi Bolivar (Pamphaginae, Orthoptera) hoppers and adults by Beauveria bassiana (Deuteromycotina, hyphomycetes) conidia in an oil formulation

Ocneridia volxemi Bolivar (Pamphaginae, Orthoptera) is one of the important insects pest in the heigh plains of north east of Algeria. Larval and adult stages caused severe damage to the leaves of cereals and an other various crops. Laboratory studies were conducted to the determine the potential of the fungus Beauveria bassiana for the control of the pest of larval and adults. Positive results were obtaved when larvae and adults were sprayed with different concentration of fungus conidia (D1: 2 x 10(6) conidia/ml, D2: 5 x 10(5) conidia/ml, D3: 8 x 10(4) conidia/ml, D4: 10(2) conidia/ml). Concentration at D1 and D2 in an oil formulation showed respectively 100% and 90 % larval mortality at the first day of treatment and 100%-9% adults mortality at the five day of treatement. The results revealed that the rates of infection and mortality were significantly related to the concentration of conidia in the corresponding samples. Beauveria bassiana can be considerd as a promising biocontrol agents of Ocneridia volxemi may be an other locuts, but field experimentation is necessary to subtantiate these finding.     

Mortality of Delia floralis, Galleria mellonella and Mamestra brassicae treated with insect pathogenic hyphomycetous fungi

Abstract:  The susceptibility of Delia floralis eggs, neonates and larvae and the susceptibility of Galleria mellonella and Mamestra brassicae larvae to seven different Norwegian isolates of the insect pathogenic, hyphomycetous fungi Tolypocladium cylindrosporum , Metarhizium anisopliae and Beauveria bassiana , were investigated. Metarhizium anisopliae isolate ARSEF 5520 was highly virulent to G. mellonella larvae and caused 100% mortality when tested at a concentration of 3.6 × 10⁶ conidia/ml. The same M. anisopliae isolate was not virulent to D. floralis larvae. Isolates of T.cylindrosporum , were equally virulent to G. mellonella and D. floralis causing up to 36.0% mortality of larvae. It is suspected, however, that the use of grated rutabaga as a food source in the D. floralis bioassay reduced the fungal virulence of both M. anisopliae and T. cylindrosporum to D. floralis . Among three T. cylindrosporum isolates tested at a concentration of 1.0 × 10⁷ conidia/ml against eggs of D. floralis none of them reduced the hatching percentage. One isolate, ARSEF 5525 did, however, significantly reduce the longevity of neonates. Beauveria bassiana isolates ARSEF 5510 and ARSEF 5370 tested at a concentration of 1.0 × 10⁷ conidia/ml resulted in M. brassicae mortality levels of 70.0 and 55.0%, respectively. The B. bassiana isolate ARSEF 5557, however, was not virulent to M. brassicae . Among the three isolates tested against M. brassicae the two virulent isolates produced a red pigment, probably oosporein, when cultured in Sabouraud dextrose agar.