Community composition of the entomopathogenic fungal genus Metarhizium in soils of tropical and temperate conventional and organic strawberry fields.

Studies on community composition and population structure of entomopathogenic fungi are imperative to link ecosystem functions to conservation biological control. We studied the diversity and abundance of Metarhizium spp. from soil of conventionally and organically farmed strawberry crops and from the adjacent field margins in two different climatic zones: Brazil (tropical) and Denmark (temperate), using the same isolating methods. In Brazilian strawberry soil, Metarhizium robertsii (n = 129 isolates) was the most abundant species, followed by M. humberi (n = 16); M. anisopliae (n = 6); one new taxonomically unassigned lineage Metarhizium sp. indet. 5 (n = 4); M. pingshaense (n = 1) and M. brunneum (n = 1). In Denmark, species composition was very different, with M. brunneum (n = 33) being isolated most commonly, followed by M. flavoviride (n = 6) and M. pemphigi (n = 5), described for the first time in Denmark. In total, 17 haplotypes were determined based on MzFG543igs sequences, four representing Danish isolates and 13 representing Brazilian isolates. No overall difference between the two climatic regimes was detected regarding the abundance of Metarhizium spp. in the soil in strawberry fields and the field margins. However, we found a higher Shannon's diversity index in organically managed soils, confirming a more diverse Metarhizium community than in soils of conventionally managed agroecosystems in both countries. These findings contribute to the knowledge of the indigenous diversity of Metarhizium in agricultural field margins with the potential to contribute to pest regulation in strawberry cropping systems.

Pathogens of the oak processionary moth Thaumetopoea processionea: Developing a user-friendly bioassay system and metagenome analyses for microorganisms.

The oak processionary moth (OPM) Thaumetopoea processionea is a pest of oak trees and poses health risks to humans due to the urticating setae of later instar larvae. For this reason, it is difficult to rear OPM under laboratory conditions, carry out bioassays or examine larvae for pathogens. Biological control targets the early larval instars and is based primarily on commercial preparations of Bacillus thuringiensis ssp. kurstaki (Btk). To test the entomopathogenic potential of other spore-forming bacteria, a user-friendly bioassay system was developed that (i) applies bacterial spore suspensions by oak bud dipping, (ii) targets first instar larvae through feeding exposure and (iii) takes into account their group-feeding behavior. A negligible mortality in the untreated control proved the functionality of the newly established bioassay system. Whereas the commercial Btk HD-1 strain was used as a bioassay standard and confirmed as being highly efficient, a Bacillus wiedmannii strain was ineffective in killing OPM larvae. Larvae, which died during the infection experiment, were further subjected to Nanopore sequencing for a metagenomic approach for entomopathogen detection. It further corroborated that B.wiedmannii was not able to infect and establish in OPM, but identified potential insect pathogenic species from the genera Serratia and Pseudomonas.

Suppression of a Spodoptera frugiperda (Sf9) cellular microRNA following Baculovirus infection and its role in the insect cell - virus interactions.

Baculoviruses have been extensively studied for their potential in microbial pest control, but the mechanisms behind their mode of action still need to be addressed. Here we report differential expression of a cellular miRNA, Sfr-miR-184, from Sf9 cells in response to Autographa californica multicapsid Nucleopolyhedrovirus (AcMNPV) infection. Our results showed that Sfr-miR-184 is down-regulated in AcMNPV-infected cells but not with UV-inactivated virus. Prohibitin gene was determined as a target of the miRNA, which was up-regulated following AcMNPV infection. Using synthetic miRNA mimic, we found that oversupply of the miRNA resulted in decreased transcript levels of the target gene. Results suggest that Sfr-miR-184 negatively regulate prohibitin transcripts in the host cells. Antibody-mediated inhibition and silencing of the prohibitin gene revealed significant reductions in virus DNA replication suggesting a possible role for prohibitin in the virus-host interaction. These findings highlight another molecular mechanism used by baculovirus to manipulate host cells for its replication.

Advancements in nonthermal physical field technologies for prefabricated aquatic food: A comprehensive review.

Aquatic foods are nutritious, enjoyable, and highly favored by consumers. In recent years, young consumers have shown a preference for prefabricated food due to its convenience, nutritional value, safety, and increasing market share. However, aquatic foods are prone to microbial spoilage due to their high moisture content, protein content, and unsaturated fatty acids. Furthermore, traditional processing methods of aquatic foods can lead to issues such as protein denaturation, lipid peroxidation, and other food safety and nutritional health problems. Therefore, there is a growing interest in exploring new technologies that can achieve a balance between antimicrobial efficiency and food quality. This review examines the mechanisms of cold plasma, high-pressure processing, photodynamic inactivation, pulsed electric field treatment, and ultraviolet irradiation. It also summarizes the research progress in nonthermal physical field technologies and their application combined with other technologies in prefabricated aquatic food. Additionally, the review discusses the current trends and developments in the field of prefabricated aquatic foods. The aim of this paper is to provide a theoretical basis for the development of new technologies and their implementation in the industrial production of prefabricated aquatic food.

Potential Impact of Chemical Fungicides on the Efficacy of Metarhizium rileyi and the Occurrence of Pandora gammae on Caterpillars in Soybean Crops.

Entomopathogenic fungi may play a crucial role in the regulation of caterpillar populations in soybean crops, either through natural occurrences or applied as mycopesticides. In the present work, we reported the naturally occurring entomopathogenic fungus Pandora gammae attacking the caterpillar Chrysodeixis includens, with infection rates in field trials ran in two consecutive years in the 10-35% range. As many chemicals are potentially harmful to entomopathogenic fungi, this work aimed to investigate the potential impact of two chemical fungicides (azoxystrobin + benzovindiflupyr and trifloxistrobina + prothioconazole) used to control soybean rust (Phakopsora pachyrhizi) on the natural occurrence of P. gammae and Metarhizium rileyi, as well as the efficacy of the latter fungus applied as different formulations against the soybean caterpillars Anticarsia gemmatalis and C. includens. Under laboratory conditions, fungicides used at field-recommended rates had a considerable negative impact on the germinability of M. riley on the medium surface, and all tested formulations did not protect conidia from damage by these chemicals. This harmful effect also impacted host infectivity, as the larval mortality owing to this fungus was reduced by 30-40% compared to that of the fungicide-free treatments. In field trials conducted in two subsequent years, unformulated and formulated M. rileyi conidia applied to soybean plants produced primary infection sites in caterpillar populations after a single spray. Spraying unformulated or formulated M. rileyi conidia following fungicide application on plants did not affect host infection rates over time. Moreover, the use of M. rileyi-based formulations or chemical fungicide did not interfere with the natural infection rates by P. gammae on its host, C. includens. Although a higher degree of exposure to non-selective fungicides can negatively affect fungal entomopathogens, a single foliar application of fungicides may be harmless to both M. rileyi and P. gammae in soybean fields. Additionally, this work showed that naturally occurring wasps and tachnids also play an important role in the regulation of A. gemmatalis and, notably, C. includens, with parasitism rates above 40-50% in some cases.

Insecticidal and growth inhibitory activity of gut microbes isolated from adults of Spodoptera litura (Fab.).

BACKGROUND: Spodoptera litura (Fab.) (Lepidoptera: Noctuidae) commonly known as tobacco caterpillar is a polyphagous pest that causes significant damage to many agricultural crops. The extensive use of chemical insecticides against S. litura has resulted in development of resistance. In order to find potential biocontrol agents, gut microbes were investigated for insecticidal potential. These microbes live in a diverse relationship with insects that may vary from beneficial to pathogenic. RESULTS: Enterococcus casseliflavus, Enterococcus mundtii, Serratia marcescens, Klebsiella pneumoniae, Pseudomonas paralactis and Pantoea brenneri were isolated from adults of S. litura. Screening of these microbial isolates for insecticidal potential against S. litura showed higher larval mortality due to K. pneumoniae and P. paralactis. These bacteria also negatively affected the development of insect along with significant decline in relative growth and consumption rate as well as efficiency of conversion of ingested and digested food of insect. The bacteria significantly decreased the reproductive potential of insect. Perturbations in the composition of gut microbiome and damage to gut epithelium were also observed that might be associated with decreased survival of this insect. CONCLUSIONS: Our study reveals the toxic effects of K. pneumoniae and P. paralactis on biology of S. litura. These bacteria may be used as potential candidates for developing ecofriendly strategies to manage this insect pest.

Susceptibility of embryos of Biomphalaria tenagophila (Mollusca: Gastropoda) to infection by Pochonia chlamydosporia (Ascomycota: Sordariomycetes).

Schistosoma mansoni is a heteroxenous parasite, meaning that during its life cycle needs the participation of obligatory intermediate and definitive hosts. The larval development occurs in aquatic molluscs belonging to the Biomphalaria genus, leading to the formation of cercariae, which emerge to infect the final vertebrate host. For this reason, studies for control of the diseases caused by digenetic trematodes often focus on combating the snail hosts. Thus, the objective of this study was to evaluate the susceptibility of Biomphalaria tenagophila embryos to the fungus Pochonia chlamydosporia (isolate Pc-10). The entire experiment was conducted in duplicate, with five replicates for each repetition (five egg masses/replicate), utilizing a total of 100 egg masses, with 20-30 eggs/egg mass. At the end of 15 days, the egg masses were evaluated under a stereomicroscope to analyze the hatching of B. tenagophila embryos in both experimental groups. After days of interaction, the exposure to the fungal hyphae bodies significantly impaired the viability of the B. tenagophila eggs, inhibiting the embryogenesis process by 83.7% in relation to the control group. Transmission and scanning electron microscopic images revealed relevant structural alterations in the egg masses exposed to the hyphae action of the fungus, interfering in the development and hatching of the young snails under analysis. These results indicate the susceptibility of B. tenagophila embryos to the fungus P. chlamydosporia (isolate Pc-10) and suggest the potential of Pc-10 to be used in the control of intermediate host, for its ovicidal capacity and for being an ecologically viable option, but in vivo experiments become necessary.

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.

Lack of avoidance of the fungal entomopathogen, Metarhizium brunneum, by male Agriotes obscurus beetles.

Fungal entomopathogens can greatly reduce the fitness of their hosts, and it is therefore expected that susceptible insects will be selected to avoid exposure to pathogens. Metarhizium brunneum is a fungal pathogen that can infect Agriotes obscurus, which in its larval form is a destructive agricultural pest and is repelled by the presence of M. brunneum conidia. Due to the subterranean nature of larval A. obscurus, recent research has focused on targeting adult A. obscurus with M. brunneum. No-choice and choice behavioural assays were conducted to determine if male adult A. obscurus avoid M. brunneum mycosed cadavers, or conidia applied to either food or soil. To further investigate the response of A. obscurus beetles to conspecific cadavers, the movement and behaviour of beetles placed at the centre of a semi-circular arrangement of mycosed or control cadavers was examined using motion tracking software. We found little evidence to suggest that A. obscurus male beetles avoid M. brunneum conidia or mycosed conspecific cadavers or alter their behaviour in their presence.

Field evaluation of Solenopsis invicta virus 3 against its host Solenopsis invicta.

Viruses have been used successfully as biocontrol agents against several insect pests but not ants. Laboratory tests have shown that Solenopsis invicta virus 3 (SINV-3) may be an effective natural control agent against its host, the red imported fire ant (Solenopsis invicta Buren). In this field trial, SINV-3 was released into 12 active S. invicta nests within a 0.088-hectare area in Florida and the impact on the ants monitored. SINV-3 was successfully transmitted, established, and multiplied within treated colonies reaching a maximum mean value of 8.71 × 108 ± 8.26 × 108 SINV-3 genome equivalents/worker ant 77 days after inoculation. SINV-3 was not detected in any of the nests in the control group. A 7-fold decrease in nests was observed in the SINV-3-treated group compared with the untreated control. A correspondingly significant decrease in S. invicta nest size also was observed over the course of the evaluation. Based on the nest rating scale, nest size among those treated with SINV-3 decreased from 3.92 ± 1.24 on day 0 to 1.67 ± 2.06 on day 77, which represents a 57.4% decrease in size. Conversely, the nest rating for the control group increased 9.3%, from 4.42 ± 1.24 on day 0 to 4.83 ± 2.12 on day 77. A follow-up survey of SINV-3-treated and -untreated plots conducted 9 months after initial treatment revealed that fire ant populations rebounded, but at a different rate. A total of 11 and 19 nests were detected in the SINV-3-treated and -untreated areas, respectively. SINV-3 was still detected in the treated area 1.8 years after the initial virus treatment and the virus had spread into the adjacent control plot. Results demonstrate that SINV-3 is an effective natural control agent against the invasive ant, S. invicta; the virus causes no known detrimental ecological impacts, is host specific, and sustained in the environment.

Different bees as vectors for entomovectoring with enhanced pollination and crop protection control: current practices, use cases and critical view on transport.

Honeybees, bumblebees and other insects have been used commercially for pollination for many years, and microbial biocontrol agents have also been widely used in pest control. Pollinators and formulations of microbial pest-control agents are routinely transported internationally on a large scale. A novel approach has been developed to use bees as vectors of microbial agents by inoculating the surface of the pollinators using dispensers in modified hives. This innovation extends the market for these products and results in better yields. A successful entomovector system requires selecting the vector pollinator most appropriate for the crop and location, based on various criteria, in combination with a registered microbial agent. Currently, pollinators and microbial agents are packed separately and combined at the point of use. Local sourcing of the pollinator in the system reduces the need for long-distance shipping of these live insects and may improve efficiency due to local adaptation; however, it will delay use and benefits of the system until research at each site/country catches up with the work already conducted in a few countries. In the meantime, clear guidance for innovative systems employing live insects could support the promising increase in food production.

Depuis plusieurs années, les abeilles mellifères, les bourdons et d'autres insectes sont commercialisés à des fins de pollinisation, parallèlement à l'utilisation désormais largement répandue de micro-organismes pour la lutte biologique contre les nuisibles. Les pollinisateurs et diverses formulations d'agents microbiens destinés à la lutte contre les nuisibles font régulièrement l'objet de transports internationaux à grande échelle. Une approche innovante mise au point récemment consiste à utiliser les abeilles en tant que vecteurs d'agents microbiens, en inoculant ces derniers sur la surface du pollinisateur par le biais de diffuseurs disposés dans les ruches modifiées à cette fin. Cette innovation amplifie le marché de ces produits et se traduit par un meilleur rendement. La réussite d'un système d'entomovection passe par la sélection judicieuse du vecteur pollinisateur en fonction des cultures à protéger et du site, sur la base de plusieurs critères, et par son utilisation en association avec un agent antimicrobien autorisé. À l'heure actuelle, les pollinisateurs et les agents microbiens sont conditionnés séparément et leur assemblage est effectué sur le site même d'utilisation. L'approvisionnement local en pollinisateurs au sein du système réduit la nécessité de transporter ces insectes vivants sur de longues distances et pourrait améliorer l'efficacité du système au moyen d'adaptations locales ; néanmoins, cela retardera la mise en oeuvre du système et l'obtention de résultats bénéfiques, le temps que la recherche conduite dans les sites ou les pays utilisateurs atteigne le niveau des avancées déjà enregistrées dans un petit nombre d'autres pays. En attendant, des directives claires en faveur des systèmes innovants basés sur l'utilisation d'insectes vivants pourraient oeuvrer à l'appui d'une croissance prometteuse de la production alimentaire.

El uso comercial de abejas melíferas, abejorros y otros insectos con fines de polinización tiene ya muchos años. También está muy extendido el empleo de agentes microbianos de control biológico como método de lucha contra las plagas. El transporte internacional y a gran escala de polinizadores y de ciertas formulaciones de plaguicidas microbianos es algo muy habitual. Ahora se ha concebido un novedoso planteamiento en el que se utilizan ápidos como vectores de agentes microbianos. Para ello, se impregna de estos agentes la superficie del insecto polinizador empleando dispensadores en colmenas modificadas. Tal innovación amplía el mercado de estos productos y depara mejores cosechas. Para que un sistema entomovectorial sea eficaz es preciso seleccionar el vector polinizador que mejor encaje con el cultivo y las condiciones locales atendiendo a diversos criterios y combinarlo con un agente microbiano registrado. Actualmente, los polinizadores y los agentes microbianos se embalan por separado y se combinan solo en el punto de aplicación. El aprovisionamiento del polinizador del sistema a partir de fuentes locales hace menos necesarios los envíos a larga distancia de estos insectos vivos y puede ofrecer más eficacia gracias al mayor grado de adaptación del insecto a las condiciones locales. Sin embargo, ello retrasará el uso del sistema y la obtención de los consiguientes beneficios, pues habrá que esperar a que en cada lugar o país se haya llevado el procedimiento tan lejos como se ha hecho hasta ahora en unos pocos países. Mientras tanto, la existencia de claras pautas sobre estos innovadores sistemas en que se emplean insectos vivos podría traducirse en un prometedor aumento de la producción alimentaria.

Long-Term Biocide Efficacy and Its Effect on a Souring Microbial Community.

Reservoir souring, which is the production of H2S mainly by sulfate-reducing microorganisms (SRM) in oil reservoirs, has been a long-standing issue for the oil industry. While biocides have been frequently applied to control biogenic souring, the effects of biocide treatment are usually temporary, and biocides eventually fail. The reasons for biocide failure and the long-term response of the microbial community remain poorly understood. In this study, one-time biocide treatments with glutaraldehyde (GA) and an aldehyde-releasing biocide (ARB) at low (100 ppm) and high (750 ppm) doses were individually applied to a complex SRM community, followed by 1 year of monitoring of the chemical responses and the microbial community succession. The chemical results showed that souring control failed after 7 days at a dose of 100 ppm regardless of the biocide type and lasting souring control for the entire 1-year period was achieved only with ARB at 750 ppm. Microbial community analyses suggested that the high-dose biocide treatments resulted in 1 order of magnitude lower average total microbial abundance and average SRM abundance, compared to the low-dose treatments. The recurrence of souring was associated with reduction of alpha diversity and with long-term microbial community structure changes; therefore, monitoring changes in microbial community metrics may provide early warnings of the failure of a biocide-based souring control program in the field. Furthermore, spore-forming sulfate reducers (Desulfotomaculum and Desulfurispora) were enriched and became dominant in both GA-treated groups, which could cause challenges for the design of long-lasting remedial souring control strategies. IMPORTANCE Reservoir souring is a problem for the oil and gas industry, because H2S corrodes the steel infrastructure, downgrades oil quality, and poses substantial risks to field personnel and the environment. Biocides have been widely applied to remedy souring, but the long-term performance of biocide treatments is hard to predict or to optimize due to limited understanding of the microbial ecology affected by biocide treatment. This study investigates the long-term biocide performance and associated changes in the abundance, diversity, and structure of the souring microbial community, thus advancing the knowledge toward a deeper understanding of the microbial ecology of biocide-treated systems and contributing to the improvement of current biocide-based souring control practices. The study showcases the potential application of incorporating microbial community analyses to forecast souring, and it highlights the long-term consequences of biocide treatment in the microbial communities, with relevance to both operators and regulators.

The microsporidium Nosema pyrausta as a potent microbial control agent of the beet webworm Loxostege sticticalis.

The microsporidium Nosema pyrausta is an important mortality factor of the European corn borer, Ostrinia nubilalis. The present study was aimed at N. pyrausta virulence testing to the beet webworm (BW), Loxostege sticticalis. This agricultural pest, L. sticticalis, was highly vulnerable to N. pyrausta. The parasite's spores were located in salivary glands, adipose tissue, and Malpighian tubules of the infected specimens. Infection was transmitted transovarially through at least 3 laboratory generations, in which BW fitness indices were lower than in the control, and moth emergence and fertility decreased prominently. Transovarial infection was most detrimental to female egg-laying ability, resulting in zero fertility in F3. When propagated in BW, the microsporidium tended to increase its virulence to L. sticticalis, as compared to the Ostrinia isolates. The parasite's ability to infect this host at low dosages and transmit vertically should guarantee its effective establishment and spread within BW populations. In conclusion, N. pyrausta is a promising agent against BW, which is a notorious polyphagous pest in Eurasia.

The effect of synthetic female sex pheromone on the transmission of the fungus Metarhizium brunneum by male Agriotes obscurus click beetles.

Autodissemination techniques can potentially be used to distribute insecticides, including microbial insecticides, to cryptic pests. This approach is reliant on the target insect either passing the pathogen passively to other insects or the pathogen cycling within the population after the initial host dies. Here we examine, in small scale experiments, whether male Agriotes obscurus click beetles passively transmit the spores of the fungus Metarhizium brunneum directly, or indirectly via the environment, and whether this is influenced by exposure to synthetic female pheromone. We found that the beetles did not avoid M. brunneum spores and that this behaviour was not affected by pheromone. Exposure to pheromone increased beetle movement and uptake of spores, but this did not result in an increase in infected beetles under our conditions. Beetles were able to transfer spores at high levels via environmental contamination. However, contamination of the environment declined rapidly after exposure to the spores. The results are discussed in the context of developing an autodissemination strategy for click beetles.

Evaluation of bioefficacy potential of entomopathogenic fungi against the whitefly (Bemisia tabaci Genn.) on cotton under polyhouse and field conditions.

The whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), is becoming a serious problem on Bt cotton. It causes enormous crop loss through its direct feeding and as a vector of cotton leaf curl virus. Chemical-dependent management is harming the environment and increased insecticide resistance is often observed in the fields. Identification of most virulent strains of entomopathogenic fungi (EPF) is essential to serve as an important component of an IPM program for management of B. tabaci. Compared to B. tabaci adults, the nymphal stage is reported to be more susceptible to entomopathogens, and targeting nymphs also helps vector management. We evaluated the bioefficacy of EPF and chemical pesticides against nymphs of B. tabaci on Bt cotton under polyhouse and field conditions. The bioefficacy index (BI) was considered as a mechanism to select the most effective EPF strains for field evaluation. The highest nymphal mortality under polyhouse conditions was recorded for Metarhizium anisopliae NA-01299 (86.7%), Beauveria bassiana MT-4511 (85.1%), Cordyceps javanica IT-10498 (81.1%), IT-10499 (81%), and B. bassiana NA-0409 (78.2%) relative to other EPF strains, spiromesifen (69.6%), buprofezin (62.2%) and pyriproxyfen (52.7%) at 7-days-post-spray treatment (DAS). However, among all the EPF, the highest BI was recorded in C. javanica IT-10499 (77%), IT-10495 (75.4%), Fusarium verticillioides IT-10493 (74.6%), and B. bassiana MT-4511 (73.1%). The pooled data of two-year field trials (2017-18 & 2018-19) revealed that the highest nymphal mortality was recorded for MT-4511 (85%), IT-10499 (83.2%), and pyriproxyfen 10% EC (78.6%) at 7-DAS. The BI-based selection of EPF proved to be a useful predictor of field efficacy. A sequential spray of the selected EPF would be a vital approach for resilient and sustainable integrated management of the B. tabaci nymphal population under field conditions.

Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis.

Modern agriculture demands for more sustainable agrochemicals to reduce the environmental and health impact. The whole process of the discovery and development of new active substances or control agents is sorely slow and expensive. Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis are specific toxins against caterpillars with a potential capacity to broaden the range of target pests. Site-directed mutagenesis is one of the most approaches used to test hypotheses on the role of different amino acids on the structure and function of proteins. To gain a better understanding of the role of key amino acid residues of Vip3A proteins, we have generated 12 mutants of the Vip3Af1 protein by site-directed mutagenesis, distributed along the five structural domains of the protein. Ten of these mutants were successfully expressed and tested for stability and toxicity against three insect pests (Spodoptera frugiperda, Spodoptera littoralis and Grapholita molesta). The results showed that, to render a wild type fragment pattern upon trypsin treatment, position 483 required an acidic residue, and position 552 an aromatic residue. Regarding toxicity, the change of Met34 to Lys34 significantly increased the toxicity of the protein for one of the three insect species tested (S. littoralis), whereas the other residue substitutions did not improve, or even decreased, insect toxicity, confirming their key role in the structure/function of the protein.

Sub-lethal effects of a Bt-based bioinsecticide on the biological conditioning of Anticarsia gemmatalis.

Bioinsecticides based on Bacillus thuringiensis (Bt) Berliner, 1915 are widely used to control lepidopteran in several crops. However, surviving insects exposed to the sub-lethal concentration of Bt-based bioinsecticides can suffer a multitude of effects on the biological conditioning known as hormesis. Here, we aimed to provide a clearer understanding of the biological conditioning of Anticarsia gemmatalis (Hübner, 1818), exposed to different concentrations of a Bt-based bioinsecticide, by assessing life table parameters over three generations. We defined five sub-lethal concentrations (LC5, LC10, LC15, LC20, and LC25) from the response curve estimate of A. gemmatalis. Deionized water was used as a control. We assessed the parameters of eggs-viability and the duration of the stages, incubation, larval, pre-pupal, pupal, adult, pre-oviposition and total biological cycle. Data were used to construct the fertility life table using the two-sex program. The survival curves showed greater variation in the proportion of individuals at each development stage using the LC25. The sub-lethal concentrations did not influence the incubation-eggs period, pre-pupal and pupal. However, the larval and adult stages using LC25 and LC10 were the most affected. Changes in sex ratio were observed using LC20 and LC5. The toxic effect of Bt-based bioinsecticide interfered mainly in the parameters of fertility, sex ratio, net reproduction rate (R0), and gross reproduction rate (GRR).

Effectiveness of Quaternary Ammonium in Reducing Microbial Load on Eggs.

Table eggs are an affordable yet nutritious protein source for humans. Unfortunately, eggs are a vector for bacteria that could cause foodborne illness. This study aimed to investigate the effectiveness of a quaternary ammonium compound (quat) sanitizer against aerobic mesophilic bacteria, yeast, and mold load on the eggshell surface of free-range and commercial farms and the post-treatment effect on microbial load during storage. Total aerobic mesophilic bacteria, yeast, and molds were enumerated using plate count techniques. The efficacy of the quaternary ammonium sanitizer (quat) was tested using two levels: full factorial with two replicates for corner points, factor A (maximum: 200 ppm, minimum: 100 ppm) and factor B (maximum: 15 min, minimum: 5 min). Quat sanitizer significantly (p < 0.05) reduced approximately 4 log10 CFU/cm2 of the aerobic mesophilic bacteria, 1.5 to 2.5 log10 CFU/cm2 of the mold population, and 1.5 to 2 log10 CFU/cm2 of the yeast population. However, there was no significant (p ≥ 0.05) response observed between individual factor levels (maximum and minimum), and two-way interaction terms were also not statistically significant (p ≥ 0.05). A low (<1 log10 CFU/cm2) aerobic mesophilic bacteria trend was observed when shell eggs were stored in a cold environment up to the production expiry date. No internal microbial load was observed; thus, it was postulated that washing with quat sanitizer discreetly (without physically damaging the eggshell) does not facilitate microbial penetration during storage at either room temperature or cold storage. Current study findings demonstrated that the quat sanitizer effectively reduced the microbial population on eggshells without promoting internal microbial growth.

An Empirical Test of the Role of Small-Scale Transmission in Large-Scale Disease Dynamics.

A key assumption of epidemiological models is that population-scale disease spread is driven by close contact between hosts and pathogens. At larger scales, however, mechanisms such as spatial structure in host and pathogen populations and environmental heterogeneity could alter disease spread. The assumption that small-scale transmission mechanisms are sufficient to explain large-scale infection rates, however, is rarely tested. Here, we provide a rigorous test using an insect-baculovirus system. We fit a mathematical model to data from forest-wide epizootics while constraining the model parameters with data from branch-scale experiments, a difference in spatial scale of four orders of magnitude. This experimentally constrained model fits the epizootic data well, supporting the role of small-scale transmission, but variability is high. We then compare this model's performance to an unconstrained model that ignores the experimental data, which serves as a proxy for models with additional mechanisms. The unconstrained model has a superior fit, revealing a higher transmission rate across forests compared with branch-scale estimates. Our study suggests that small-scale transmission is insufficient to explain baculovirus epizootics. Further research is needed to identify the mechanisms that contribute to disease spread across large spatial scales, and synthesizing models and multiscale data are key to understanding these dynamics.

Biocontrol of Alternaria alternata and Fusarium oxysporum by Trichoderma asperelloides and Bacillus paralicheniformis in tomato plants.

The objective of this work was to evaluate the effectiveness of the putative biocontrol agents (PBA) Bacillus paralicheniformis and Trichoderma asperelloides in vitro and in vivo to control two of the most important tomato plant diseases: vascular wilt (Fusarium oxysporum) and early blight (Alternaria alternata). The assessment of the in vitro interactions between the PBA and the phytopathogenic fungi was performed by dual confrontation assays. The biocontrol effectiveness of the individual and combined PBA treatments towards individual phytopathogen inoculations was evaluated in tomato plants. T. asperelloides was able to exert an outstanding mycoparasitic effect on both phytopathogenic fungi in the in vitro tests by hyphal strangulation and penetration. In addition, the individual PBA treatments were effective in the biocontrol of A. alternata and F. oxysporum in tomato plants reducing the plant disease severity in more than 53.8 and 66.7% for each of the pathogens, respectively. On the other hand, the combined use of the tested strains showed similar effectiveness in the biocontrol of A. alternata, but no synergism was observed. In addition, it was concluded that B. paralicheniformis protected the plants from the attack of A. alternata through the induction of the systemic resistance of the plant. This study demonstrated the effectiveness of the individual and combined use of the strains tested for the biocontrol of A. alternata and F. oxysporum in tomato plants.