Hydroalcoholic Extracts from Pleurotus ostreatus Spent Substrate with Nematocidal Activity against Nacobbus aberrans Phytonematode and the Non-Target Species Panagrellus redivivus.

Pleurotus ostreatus, an edible mushroom widely consumed worldwide, generates a by-product known as spent mushroom substrate (SMS). This material has demonstrated biological activity against agricultural crop pathogens. In this study, we evaluated the nematocidal effectiveness of hydroalcoholic extracts (T5, T2, AT5, and AT2) derived from SMS of P. ostreatus against (J2) of the phytonematode Nacobbus aberrans and assessed their potential toxicity towards the non-target nematode Panagrellus redivivus. Among these extracts, AT5 exhibited the highest efficacy against N. aberrans and was the least toxic against P. redivivus. Liquid-liquid partitioning yielded the AQU fraction, which showed significant nematocidal activity against J2 (75.69% ± 8.99 mortality), comparable to chitosan. The GC-MS analysis revealed the presence of several compounds, including palmitic acid, linoleic acid, and 2,4-Di-tert-butylphenol. These findings are consistent with studies confirming the antagonistic effectiveness of these compounds against phytonematodes. Additionally, all extracts exhibited toxicity against P. redivivus, with T2 being the most toxic. Our findings demonstrate that while the AT5 extract displays antagonistic effectiveness against both N. aberrans and P. redivivus, it was the least toxic among the extracts tested. Thus, SMS of P. ostreatus holds potential as a source of nematocidal compounds, which could offer significant benefits for agricultural pest control.

In Vivo Production, Development and Storage of Oscheius myriophila (Nematoda: Rhabditida) in Galleria mellonella (Lepidoptera: Pyralidae).

Entomopathogenic nematodes have been used in biological control for some time and are an alternative for the control of insect pests, but during their implementation, situations have arisen that can be improved. These vary with each species and include their production and storage. Oscheius myriophila, an entomopathogenic nematode (EPN), was monitored for its performance when produced in vivo, as well as its development using Galleria mellonella larvae, using the MC5-2014 strain isolated from soil samples in the municipality of Tepalcingo, Morelos, México. For a study with native strains of EPNs, a wide range of tests must be conducted because the required conditions can be very specific. In vivo production was quantified at initial infective juvenile (IJ) inocula of 50, 100 and 500, and we obtained the same production for the three inocula. The life cycle of the EPNs lasted 12 days, and two generations were observed in which adults were found at days 5 and 9. Both evaluations were performed at a temperature of 27 °C in G. mellonella larvae. In addition, the temperatures of 8, 12, 20 and 24 °C were evaluated for their storage, and we observed that the EPNs can be kept for at least 6 months, maintaining a survival rate of 58.67% and a good infective capacity at a temperature of 12 °C, remaining above 60%.

Potential control of the infective stage of Taenia pisiformis using Bacillus thuringiensis GP526 strain.

The GP526 strain of Bacillus thuringiensis has been referred as an in vitro helminthicide on various stages of Dipylidium caninum and Centrocestus formosanus. Our study addresses the in vitro ovicidal activity of GP526 strain spore-crystal complex on Taenia pisiformis eggs, evaluating induced damage microscopically. The eggs exposed to the total extract containing spores and crystals show damage after 24 hours, with loss of integrity on the eggshell, and an ovicidal activity of 33% at 1mg/ml. The destruction of the embryophore was observed after 120 h with a 72% of ovicidal activity at 1 mg/ml. The LC50 was 609.6 µg/ml, dose that causes a 50% of lethality on the hexacanth embryo, altering the oncosphere membrane. The spore-crystal proteins were extracted, and the protein profile was obtained by electrophoresis, finding a major band of 100 kDa suggestive of an S-layer protein, since an S-layer was immunodetected in both, spores and extracted proteins. The protein fraction containing the S-layer protein presents adhesion to the T. pisiformis eggs, and 0.4 mg/ml of the protein induces a lethality of 21.08% at 24 h. The characterization of molecular mechanisms of ovicidal activity will be an important contribution, so the characterization of the proteins that make up the extract of the GP526 strain, would be useful to support the biological potential for control of this cestodiasis and other parasitosis. B. thuringiensis is shown as a potent helminthicide on eggs, with useful potential for biological control of this cestodiasis.

Histometric and morphological damage caused by Serratia marcescens to the tick Rhipicephalus microplus (Acari: Ixodidae).

Rhipicephalus microplus tick is the ectoparasite causing the greatest economic losses in the livestock industry. Multi-resistance in ticks is increasing, generating the inefficiency of traditional ixodicides, for which biological control has been proposed as an alternative. In this work, we analyze the histomorphological damage caused by the bacterial strain EC-35 on Rhipicephalus microplus. The ixodicidal effect of EC-35 total protein was evaluated on larval or adult ticks comparing with the commercial ixodicide coumaphos 0.02% as a control. Female ticks were processed using the paraffin-embedding technique and stained with hematoxylin-eosin. Also, the pathogenicity of EC-35 was evaluated by capillary feeding and coelom inoculation tests. The identification of the bacterium was performed using the molecular markers 16S RNA and rpoB, by PCR and sequencing technique, and the evolutionary distance was analyzed by Bayesian phylogenetic inference. No differences were observed in the perimeter and area of larvae treated with EC-35 or Coumaphos. The thickness of the integument decreased a 65% with the EC-35 treatment (6.01 ± 0.6 µm) and of 30% in coumaphos (12.04 ± 1.2 µm) in larvae compared with the control group (18.41 ± 2 µm), while no difference was found in adult ticks. The capillary feeding test and coelom inoculation with EC-35 showed an inhibition of reproductive potential of 99.8 ± 7 and an oviposition Inhibition 97 ± 3.02%. The EC-35 strain was genetically related to Serratia marcescens, concluding that these bacteria caused high mortality, oviposition Inhibition, and integument thinning and drastic loss of histoarchitecture in R. microplus tick larvae.

Characterization of insecticidal Cry1Cb2 protein from Bacillus thuringiensis toxic to Myzus persicae (Sulzer).

The toxins produced by Bacillus thuringiensis (Bt) are well known for their insecticidal activity against Lepidoptera, Diptera and Coleoptera; however, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. We describe the aphicidal effect of Cry toxin from Bt strain GP919 against one of the most pernicious hemipterans in the agricultural environment, Myzus persicae. The mortality bioassay shows that the strain cause mortality rates above 80% at concentration of 10 ng/µl with a LC50 of 9.01 ng/µl; whereas it showed no lethal toxicity against the lepidopteran Spodoptera frugiperda. The mayor protein (∼130 kDa) expressed by this strain was subjected to purification, solubilization and trypsin digestion, the band of âˆ¼ 65 kDa which was obtained from trypsin digestion was purified by ion-exchange chromatography and was used to feed the aphid. The bioassay shows mortality rates above 85% at concentration of 10 ng/µl and the LC50 was 6.58 ng/µl. The resulting fragment from the digestion was identified by mass spectrometry and the candidate protein showed an overall 100% amino acid sequence identity to the reported Cry1Cb2 (WP 033698561.1) protein from Bt. Koch's postulated also was carried out with the GP919 strain and also, we document the signs of infection caused by this strain. This is the first report of a Cry1Cb2 protein that is toxic to a sucking insect and this protein may become a promising environmentally friendly tool for the control of M. persicae and possible also for other sap sucking insect pests.

Characterization of a strain of Serratia sp. with ixodicide activity against the cattle tick Rhipicephalus microplus.

Cattle ticks are considered the most important ectoparasite in the livestock industry. Rhipicephalus microplus causes economic losses both through direct feeding on livestock and through disease transmission. Reports of the failure of chemical ixodicides to control this tick have led to a search for control alternatives, such as bacteria with ixodicide activity. The objective of this work was to select a bacterial strain with ixodicide activity against R. microplus. In total, 83 bacterial strains were isolated from soil and dead R. microplus specimens, and all strains were evaluated against larvae in a screening test. Bacteria with ixodicide activity were evaluated in larvae and engorged adult female ticks. The larvae were challenged using the larval immersion test (LIT) with 20 µg/mL total protein. The median lethal concentration (LC50) for larvae was obtained by using nine total protein concentrations. Engorged adult female ticks were challenged using the adult immersion test (AIT) with six protein concentrations. We evaluated adult mortality on day 10, oviposition rate on day 14 and hatching rate on day 40 after challenge. Only one bacterial strain (EC-35) showed ixodicide activity against larvae and adult R. microplus. The highest larval mortality, 52.3%, occurred with a total protein concentration of 40 µg/mL, and the LC50 was 13.9 µg/mL of protein. In adults, a total protein concentration of 10 µg/mL had the highest mortality (55%), oviposition inhibition (50.9%) and reproductive potential inhibition (52.5%). However, there was no significant effect on hatching. The 16S rRNA gene sequence showed 99% identity of EC-35 with Serratia sp.

Nematicidal and ovicidal activity of Bacillus thuringiensis against the zoonotic nematode Ancylostoma caninum.

Ancylostoma caninum is a gastrointestinal parasite that affect dogs and humans, considered a worldwide public health problem. The control of these parasitosis is increasingly difficult due to the development of multi-drug resistance. Bacillus thuringiensis is an insecticidal, spore forming bacterium, often species specific. The strain GP526 of B. thuringiensis has toxic effect on the cestode Dipylidium caninum and the trematode Centrocestus formosanus, both of them zoonotic parasites. The high degrees of specificity and environment safe make B. thuringiensis suitable for use against pathogen parasites, especially those resistant to synthetic chemical insecticides. The objective of the current work was to evaluate the in vitro effect of B. thuringiensis on Ancylostoma caninum. Spore-crystal mixture of the strains was co-incubated with 120 adult nematodes (males, non-pregnant females and pregnant females) or with 4800 eggs. GP526 showed a nematicidal effect with an LT50 of 35.8 h and an LC50 of 60 µg/ml. It also showed an ovicidal effect with an LC50 of 94.9 µg/ml. Histological analyses showed detachment of the cuticle and of the uterus in adult females, and vacuolization with destruction of the eggs. The effects of GP526 strain were comparable to those of albendazole, allowing us to propose GP526 for A. caninum control, in both, the adult stage at a gastrointestinal level, and in egg stage in the environment. In addition, GP526 can be proposed as a potential broad spectrum antiparasitic drug.

Bacillus thuringiensis Cry1Ab Domain III ß-22 Mutants with Enhanced Toxicity to Spodoptera frugiperda (J. E. Smith).

The fall armyworm, Spodoptera frugiperda, is an invasive maize pest that has spread from the Americas into Africa and Asia and causes severe crop damage worldwide. Most populations of S. frugiperda show low susceptibility to Bacillus thuringiensis (Bt) Cry1Ab or Cry1Ac toxins, which have been proved to be effective against several other lepidopteran pests. In addition, S. frugiperda has evolved resistance to transgenic maize expressing Cry1Fa toxin. The specificity and toxicity of Cry toxins are determined by their binding to different larval midgut proteins, such as aminopeptidase N (APN), alkaline phosphatase (ALP), and cadherin (CAD), among other proteins, by means of exposed domain II loop regions and also by the domain III ß-sheets ß-16 and ß-22. Here, we analyzed different Cry1Ab mutants with mutations in the domain III ß-22 region. Alanine-scanning mutagenesis of this region revealed that all mutants showed increased toxicity against a nonsusceptible Cry1Ab S. frugiperda population. Further analysis of the mutant toxin Cry1AbS587A (bearing a mutation of S to A at position 587) revealed that, compared to Cry1Ab, it showed significantly increased toxicity to three other S. frugiperda populations from Mexico but retained similar toxicity to Manduca sexta larvae. Cry1AbS587A bound to brush border membrane vesicles (BBMV), and its higher toxicity correlated with higher binding affinities to APN, ALP, and CAD recombinant proteins. Furthermore, silencing the expression of APN1 and CAD receptors in S. frugiperda larvae by RNA interference (RNAi) showed that Cry1AbS587A toxicity relied on CAD expression, in contrast to Cry1Ab. These data support the idea that the increased toxicity of Cry1AbS587A to S. frugiperda is in part due to an improved binding interaction with the CAD receptor.IMPORTANCESpodoptera frugiperda is an important worldwide pest of maize and rice crops that has evolved resistance to Cry1Fa-expressing maize in different countries. Therefore, identification of additional toxins with different modes of action is needed to provide alternative tools to control this insect pest. Bacillus thuringiensis (Bt) Cry1Ab and Cry1Ac toxins are highly active against several important lepidopteran pests but show varying and low levels of toxicity against different S. frugiperda populations. Thus, the identification of Cry1A mutants that gain toxicity to S. frugiperda and retain toxicity to other pests could be of great value to produce transgenic crops that resist a broader spectrum of lepidopteran pests. Here, we characterized Cry1Ab domain III ß-22 mutants, and we found that a Cry1AbS587A mutant displayed increased toxicity against different S. frugiperda populations. Thus, Cry1AbS587A could be a good toxin candidate to produce transgenic maize with broader efficacy against this important insect pest in the field.

Oscheius myriophila (Nematoda: Rhabditida) isolated in sugar cane soils in Mexico with potential to be used as entomopathogenic nematode.

A survey of entomopathogenic nematodes was conducted in sugar cane crops in a total of 14 soils, and positive results were obtained for strain MC5-2014 in the municipality of Tepalcingo, Morelos, in soil with a sandy loam texture and a pH of 6.4. Species determination was performed via morphological and morphometric techniques by searching for a tubular stoma with a swollen cylindrical pharyngeal body and a metacorpus in the basal part. The range of body length (L) was 750 to 1200 µm in females and 720 to 910 µm in males, while the corresponding maximum widths (W) of the body were 30 to 60 µm and 20 to 30 µm, respectively. Males exhibited bursa with a 1 + 1 + 3 + 3 distribution of papillae, and females exhibited a vulva located at the mid-body. For molecular identification, the ITS region of ribosomal DNA was used. Virulence tests (LC50) were conducted with Galleria mellonella, and a value of 4.732 was obtained for infective juveniles (IJs). Taking taxonomic and molecular characteristics into account, the isolate was determined to be Oscheius myriophila. The isolation of this strain represents the first geographic report of O. myriophila in Mexico, and it should be noted that the cultivation of sugar cane occurs with constant application of insecticides, herbicides, fungicides, and fertilizers as well as harvesting activities such as burning of the crop for harvest. The O. myriophila isolate has the potential to be used in the future as a method of biological control in our country.A survey of entomopathogenic nematodes was conducted in sugar cane crops in a total of 14 soils, and positive results were obtained for strain MC5-2014 in the municipality of Tepalcingo, Morelos, in soil with a sandy loam texture and a pH of 6.4. Species determination was performed via morphological and morphometric techniques by searching for a tubular stoma with a swollen cylindrical pharyngeal body and a metacorpus in the basal part. The range of body length (L) was 750 to 1200 µm in females and 720 to 910 µm in males, while the corresponding maximum widths (W) of the body were 30 to 60 µm and 20 to 30 µm, respectively. Males exhibited bursa with a 1 + 1 + 3 + 3 distribution of papillae, and females exhibited a vulva located at the mid-body. For molecular identification, the ITS region of ribosomal DNA was used. Virulence tests (LC50) were conducted with Galleria mellonella, and a value of 4.732 was obtained for infective juveniles (IJs). Taking taxonomic and molecular characteristics into account, the isolate was determined to be Oscheius myriophila. The isolation of this strain represents the first geographic report of O. myriophila in Mexico, and it should be noted that the cultivation of sugar cane occurs with constant application of insecticides, herbicides, fungicides, and fertilizers as well as harvesting activities such as burning of the crop for harvest. The O. myriophila isolate has the potential to be used in the future as a method of biological control in our country.

Mass production of a S-layer protein of Bacillus thuringiensis and its toxicity to the cattle tick Rhipicephalus microplus.

The most commonly used biopesticides to control agricultural, forest and insect vectors of human diseases are derived from the bacterium Bacillus thuringiensis, which begins to produce Cry and Cyt insecticidal proteins during the onset of the sporulation phase. Some B. thuringiensis strains also produce S-layer proteins that are toxic to certain pests. S-layer proteins are the most abundant proteins in bacteria and archaea. This proteins' key trait to design high performace processes for mass production is their continuous expression during the vegetative phase, unlike Cry and Cyt, which are restricted to the sporulation phase. In this work, a S-layer protein expressed by the GP543 strain of B. thuringiensis that is toxic to the cattle tick Rhipicephalus microplus was mass produced using the batch culture fermentation technique. In addition, the spore-protein complex showed a mortality rate of 75% with a dose of 300 µg·mL-1 on adult females of R. microplus after fourteen days. The lethal concentration 50 was 69.7 µg·mL-1. The treatment also caused a decrease of 13% in the weight of the mass of oviposited eggs with 200 µg·mL-1 of the spore-protein complex and inhibition of the hatching of eggs from 80 to 92%. Therefore, this could be a good option for controlling this parasite. The advantages of S-layer protein synthesis are focused on the production of a new generation of proteins in pest control. This is the first report on the mass production of an S-layer protein that is responsible for toxicity.

Enhanced Tolerance against a Fungal Pathogen and Insect Resistance in Transgenic Tobacco Plants Overexpressing an Endochitinase Gene from Serratia marcescens.

In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the corpse of a Diatraea magnifactella lepidopteran, which is an important sugarcane pest. The chitinase gene SmchiC amplified from the S. marcescens genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco (Nicotiana tabacum L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus Botrytis cinerea and to the pest Spodoptera frugiperda was evaluated: both the activity of chitinase as well as the resistance against B. cinerea and S. frugiperda was significantly higher in transgenic plants compared to the wild-type.

Effects of Bacillus thuringiensis strains virulent to Varroa destructor on larvae and adults of Apis mellifera.

The sublethal effects of two strains of Bacillus thuringiensis, which were virulent in vitro to Varroa destructor, were measured on Apis mellifera. The effects of five concentrations of total protein (1, 5, 25, 50 and 100µg/mL) from the EA3 and EA26.1 strains on larval and adult honey bees were evaluated for two and seven days under laboratory conditions. Based on the concentrations evaluated, total protein from the two strains did not affect the development of larvae, the syrup consumption, locomotor activity or proboscis extension response of adults. These same parameters were also tested for the effects of three concentrations (1, 10 and 15µg/kg) of cypermethrin as a positive control. Although no significant differences were observed after two days of treatment with cypermethrin, a dose-response relationship in syrup consumption and locomotor activity was observed. A significant reduction in the proboscis extension response of the bees treated with cypermethrin was also observed. Therefore, in contrast to cypermethrin, our results indicate that the EA3 and EA26.1 strains of B. thuringiensis can be used in beehives to control V. destructor and reduce the negative effects of this mite on colonies without adverse effects on the larvae and adults of A. mellifera. Additionally, the overuse of synthetic miticides, which produce both lethal and sublethal effects on bees, can be reduced.

Therapeutic use of Bacillus thuringiensis in the treatment of psoroptic mange in naturally infested New Zealand rabbits.

Bacillus thuringiensis is a bacteria known for its bioinsecticidal toxins and it has been proposed as an alternative in the treatment of several parasites that infect domestic animals (helminths, ticks, mites). In this work, we evaluated the clinical efficiency of the Bacillus thuringiensis GP532 strain in the treatment of six rabbits naturally infested with the P. cuniculi mite. GP532 extract (10mg/ml) was applied by aspersion in both pinna, with a second application after seven days, and the therapeutic effect was measured in both qualitative and quantitative manner. GP532 application resulted in a decreased infestation rate, which was observed as early as 3days post-treatment. At day 14, a decrease from 4.66±0.61 to 0.50±0.10 in the left pinna and from 1.66±0.21 to 0.66±0.16 (P<0.05) in the right pinna was observed. This response was comparable to the commercial drug Ivermectin, which induced a decreased infestation rate from 4.00±0.51 to 0.16±0.10 in the left pinna and from 4.66±0.80 to 0.25±0.11 in the right pinna (P<0.05). At day 30 post-treatment, GP532 decreased the total infested area by 76.80±16.06%, whereas Ivermectin resulted in a 97.41±0.99% decrease. Neither treatment produced irritation or macroscopic lesions. Our results show that the B. thuringiensis GP532 strain has a therapeutic potential in the treatment of psoroptic mange in rabbits.

Anthelmintic Effect of Bacillus thuringiensis Strains against the Gill Fish Trematode Centrocestus formosanus.

Parasitic agents, such as helminths, are the most important biotic factors affecting aquaculture, and the fluke Centrocestus formosanus is considered to be highly pathogenic in various fish species. There have been efforts to control this parasite with chemical helminthicides, but these efforts have had unsuccessful results. We evaluated the anthelmintic effect of 37 strains of Bacillus thuringiensis against C. formosanus metacercariae in vitro using two concentrations of total protein, and only six strains produced high mortality. The virulence (CL50) on matacercariae of three strains was obtained: the GP308, GP526, and ME1 strains exhibited a LC50 of 146.2 µg/mL, 289.2 µg/mL, and 1721.9 µg/mL, respectively. Additionally, these six B. thuringiensis strains were evaluated against the cercariae of C. formosanus; the LC50 obtained from the GP526 strain with solubilized protein was 83.8 µg/mL, and it could be considered as an alternative control of the metacercariae and cercariae of this parasite in the productivity systems of ornamental fishes.

Acaricidal effect and histological damage induced by Bacillus thuringiensis protein extracts on the mite Psoroptes cuniculi.

BACKGROUND: The mite Psoroptes cuniculi is a common worldwide ectoparasite and the most frequently found in rabbit farms. It causes significant economic losses on commercial rabbit breeding associated with poor leather quality, reduced conception rates, weight loss, poor growth and death. Several strategies have been proposed for the treatment of mange caused by this mite, ranging from the use of acaricides, entomopathogenic fungi, essential oils and vaccines. However, therapy and control of both human scabies and animal mange are still based mainly on the use of drugs and chemicals such as ivermectin, which involves disadvantages including genotoxic and cytotoxic effects, resistance and environmental damage. Bacillus thuringiensis is a bacterium, innocuous for human being, domestic animals and plants that produces highly biodegradable proteins, and has been used worldwide for biological control. The aim of this work was to find an alternative treatment based on biological control for scabies caused by Psoroptes cuniculi, using protein extracts from strains of Bacillus thuringiensis. METHODS: P. cuniculi mites were obtained from naturally infected New Zealand rabbits, and different doses of protein from B. thuringiensis were added to the mites. We measured mortality and obtained the median lethal concentration and median lethal times. For histological analysis, the mites were fixed in 10% formalin, processed according to the paraffin embedded tissue technique. Sections were stained with hematoxylin-eosin to observe the general histological structure. RESULTS: We report here for the first time evidence about the in vitro acaricidal effect caused by the strain GP532 of B. thuringiensis on the mite Psoroptes cuniculi, with an LC50 of 1.3 mg/ml and a LT50 of 68 h. Histological alterations caused by B. thuringiensis on this mite, included the presence of dilated intercellular spaces in the basal membrane, membrane detachment of the peritrophic matrix and morphological alterations in columnar cells of the intestine. CONCLUSIONS: Since this mite is an obligate ectoparasite that affects rabbits, goats, horses, cows and sheep, B. thuringiensis protein extracts are proposed as a potential treatment for biological control of mange in farm animals.

Identification of a new Alcaligenes faecalis strain MOR02 and assessment of its toxicity and pathogenicity to insects.

We report the isolation of a bacterium from Galleria mellonella larva and its identification using genome sequencing and phylogenomic analysis. This bacterium was named Alcaligenes faecalis strain MOR02. Microscopic analyses revealed that the bacteria are located in the esophagus and intestine of the nematodes Steinernema feltiae, S. carpocapsae, and H. bacteriophora. Using G. mellonella larvae as a model, when the larvae were injected with 24,000 CFU in their hemocoel, more than 96% mortality was achieved after 24 h. Additionally, toxicity assays determined that 1 µg of supernatant extract from A. faecalis MOR02 killed more than 70% G. mellonella larvae 96 h after injection. A correlation of experimental data with sequence genome analyses was also performed. We discovered genes that encode proteins and enzymes that are related to pathogenicity, toxicity, and host/environment interactions that may be responsible for the observed phenotypic characteristics. Our data demonstrates that the bacteria are able to use different strategies to colonize nematodes and kill insects to their own benefit. However, there remains an extensive group of unidentified microorganisms that could be participating in the infection process. Additionally, a nematode-bacterium association could be established probably as a strategy of dispersion and colonization.

Effects of passages through a suitable host of the fungus, Metarhizium anisopliae, on the virulence of acaricide-susceptible and resistant strains of the tick, Rhipicephalus microplus.

The aim of this work was to assess the virulence of strain M379 of the fungus, Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) after different passages through a suitable host and at different concentrations for the control of both acaricide-susceptible and resistant strains of the tick, Rhipicephalus (formerly Boophilus) microplus Canestrini (Ixodida:Ixodidae) in vitro. The highest value of LC50 for the susceptible strain corresponded to zero passage with 7.68 × 107 conidia/ml followed by the fourth passage with 2.68 × 107, which reduced 2.87-fold the lethal concentration. When comparing LC50 values of the fourth vs. the seventh passage (2.59 × 105 conidia/ml), the lethal concentration was reduced 103.47-fold by the seventh passage. In addition, in the resistant strain the LC50 highest value corresponded to zero passage with 4.95 × 107 conidia/ml followed by the fourth passage with 7.86 × 106, which reduced 6.30-fold the lethal concentration. When comparing LC50 values of the fourth vs. the seventh passage (1.04 × 105 conidia/ml) in the resistant strain, the lethal concentration was reduced 75.58-fold by the seventh passage. These results suggest that the number of passages on M. anisopliae through a suitable host increased its virulence on both R. microplus strains. When comparing LC50 of the zero passage through a suitable host of both acaricide-susceptible and resistant strains, the highest LC50 values corresponded to the susceptible strain with 7.68 × 107 conidia/ml followed by the resistant one with 4.95 × 107, showing that on the resistant strain the lethal concentration is reduced by 1.55-fold. When comparing the fourth passage, the highest values of LC50 corresponded to the susceptible strain with 2.68 x 107 conidia/ml followed by the resistant one with 7.86 × 106 conidia/ml, showing for the resistant strain a 3.41-fold reduced lethal concentration. Moreover, when comparing the seventh passages, the highest values of LC50 corresponded to the susceptible strain with 2.59 × 105 followed by the resistant with 1.04 × 105 conidia/ml, revealing for the resistant strain a 2.49-fold reduced lethal concentration. These results suggest that the resistant strain needs a lower concentration of conidia than the susceptible strain. In this case, the acaricide-resistant strain is more susceptible to M. anisopliae of zero- and seven-passage strains.

Evaluation of Bacillus thuringiensis pathogenicity for a strain of the tick, Rhipicephalus microplus, resistant to chemical pesticides.

The pathogenicity of four native strains of Bacillus thuringiensis against Rhipicephalus (Boophilus) microplus (Canestrine) (Acari: Ixodidae) was evaluated. A R. microplus strain that is resistant to organophosphates, pyrethroids, and amidines, was used in this study. Adult R. microplus females were bioassayed using the immersion test of Drummond against 60 B. thuringiensis strains. Four strains, GP123, GP138, GP130, and GP140, were found to be toxic. For the immersion test, the total protein concentration for each bacterial strain was 1.25 mg/ml. Mortality, oviposition, and egg hatch were recorded. All of the bacterial strains had significant effects compared to the controls, but no significant differences were seen between the 4 strains. It is evident that these B. thuringiensis strains have a considerable detrimental effect on the R. microplus strain that is resistant to pesticides.