Demonstrating the Applicability of Proton Transfer Reaction Mass Spectrometry to Quantify Volatiles Emitted by the Mycoparasitic Fungus Trichoderma atroviride in Real Time: Monitoring of Trichoderma-Based Biopesticides.

The present study aims to explore the potential application of proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) for real-time monitoring of microbial volatile organic compounds (MVOCs). This investigation can be broadly divided into two parts. First, a selection of 14 MVOCs was made based on previous research that characterized the MVOC emissions of Trichoderma atroviride, which is a filamentous fungus widely used as a biocontrol agent. The analysis of gas-phase standards using PTR-ToF-MS allowed for the categorization of these 14 MVOCs into two groups: the first group primarily undergoes nondissociative proton transfer, resulting in the formation of protonated parent ions, while the second group mainly undergoes dissociative proton transfer, leading to the formation of fragment ions. In the second part of this investigation, the emission of MVOCs from samples of T. atroviride was continuously monitored over a period of five days using PTR-ToF-MS. This also included the first quantitative online analysis of 6-amyl-α-pyrone (6-PP), a key MVOC emitted by T. atroviride. The 6-PP emissions of T. atroviride cultures were characterized by a gradual increase over the first two days of cultivation, reaching a plateau-like maximum with volume mixing ratios exceeding 600 ppbv on days three and four. This was followed by a marked decrease, where the 6-PP volume mixing ratios plummeted to below 50 ppbv on day five. This observed sudden decrease in 6-PP emissions coincided with the start of sporulation of the T. atroviride cultures as well as increasing intensities of product ions associated with 1-octen-3-ol and 3-octanone, whereas both these MVOCs were previously associated with sporulation in T. atroviride. The study also presents the observations and discussion of further MVOC emissions from the T. atroviride samples and concludes with a critical assessment of the possible applications and limitations of PTR-ToF-MS for the online monitoring of MVOCs from biological samples in real time.

Characteristics of biological control and mechanisms of Pseudomonas chlororaphis zm-1 against peanut stem rot.

BACKGROUND: Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this plant disease. Biological control is one of the most promising plant disease control measures. In this study, Pseudomonas chlororaphis subsp. aurantiaca strain zm-1, a bacterial strain with potential biocontrol properties isolated by our team from the rhizosphere soil of Anemarrhena asphodeloides, was studied to control this plant disease. METHODS: We prepared extracts of Pseudomonas chloroaphis zm-1 extracellular antibacterial compounds (PECEs), determined their antifungal activities by confrontation assay, and identified their components by UPLC-MS/MS. The gene knockout strains were constructed by homologous recombination, and the biocontrol efficacy of P. chlororaphis zm-1 and its mutant strains were evaluated by pot experiments under greenhouse conditions and plot experiments, respectively. RESULTS: P. chlororaphis zm-1 could produce extracellular antifungal substances and inhibit the growth of Sclerotium rolfsii, the main pathogenic fungus causing peanut stem rot. The components of PECEs identified by UPLC-MS/MS showed that three kinds of phenazine compounds, i.e., 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and the core phenazine, were the principal components. In particular, 1-hydroxyphenazine produced by P. chlororaphis zm-1 showed antifungal activities against S. rolfsii, but 2-hydroxyphenazine did not. This is quite different with the previously reported. The extracellular compounds of two mutant strains, ΔphzH and ΔphzE, was analysed and showed that ΔphzE did not produce any phenazine compounds, and ΔphzH no longer produced 1-hydroxyphenazine but could still produce PCA and phenazine. Furthermore, the antagonistic ability of ΔphzH declined, and that of ΔphzE was almost completely abolished. According to the results of pot experiments under greenhouse conditions, the biocontrol efficacy of ΔphzH dramatically declined to 47.21% compared with that of wild-type P. chlororaphis zm-1 (75.63%). Moreover, ΔphzE almost completely lost its ability to inhibit S. rolfsii (its biocontrol efficacy was reduced to 6.19%). The results of the larger plot experiments were also consistent with these results. CONCLUSIONS: P. chlororaphis zm-1 has the potential to prevent and control peanut stem rot disease. Phenazines produced and secreted by P. chlororaphis zm-1 play a key role in the control of peanut stem rot caused by S. rolfsii. These findings provide a new idea for the effective prevention and treatment of peanut stem rot.

Bio-efficacy of Soil Actinomycetes and an Isolated Molecule 1,2-Benzenedicarboxylic Acid from Nonomuraea sp. Against Culex quinquefasciatus Say and Aedes aegypti L. Mosquitoes (Diptera: Culicidae).

Vector-borne diseases such as filariasis and dengue that contribute significantly to disease burden, death, poverty, and social frailty are still a major public healthcare problem worldwide. Currently, synthetic chemicals have been used in mosquito control programs. However, repeated use of chemical insecticides causes environmental pollution and harmful effects on non-target organisms. Therefore, alternative ecofriendly sources from biological source are urgently needed to manage mosquitoes. In this respect, the present study was aimed to evaluate mosquito larvicidal and pupicidal activities of 22 crude extracts of soil actinomycetes on Culex quinquefasciatus and Aedes aegypti and to identify the active molecule. Briefly, the crude ethyl acetate extract and fractions were tested at 62.5, 125, 250, and 500 ppm and 2.5, 5.0, 7.5, and 10.0 ppm concentrations on larval and pupal stages of Cx. quinquefasciatus and Ae. aegypti. The larval and pupal mortality was assessed after 24 h of treatment. Among the 22 isolates screened, Nonomuraea sp. VAS-16 exhibited significant larvicidal and pupicidal activities against the tested mosquito species. Among the 18 fractions screened, fraction-6 showed strong larvicidal and pupicidal activities with the LC50 and LC90 values of 9.1, 18.7, 9.82, and 22.85 ppm against the larvae and LC50 and LC90 values of 10.5, 23.1, 12.3, and 24.13 ppm against the pupae of Cx. quinquefasciatus and Ae. aegypti, respectively. Fascinatingly, the isolated compound 1,2-benzenedicarboxylic acid from fraction-6 at 0.5, 1.0, 1.5, and 2.0 ppm concentration recorded lower LC50 and LC90 values of 4.27, 14.90, 4.67, and 11.90 ppm against the larvae and LC50 and LC90 values of 4.58, 12.06, 5.36, and 13.07 ppm against the pupae of Cx. quinquefasciatus and Ae. aegypti, respectively. On the other hand, the compound recorded less ovicidal activity of 11.0% and 10.3% at 2 ppm against the eggs of Cx. quinquefasciatus and Ae. aegypti, respectively. The present study clearly shows that the crude extract and the compound from Nonomuraea sp. VAS-16 can be used as an effective biopesticide in integrated mosquito management program.

Bio-Preservative Potential of Microorganisms Isolated from Red Grape against Food Contaminant Fungi.

Fungal spoilage is one of the main reasons of economic losses in the food industry, especially in the wine sector. Consequently, the search for safer and new preservation techniques has gained importance in recent years. The objective of this study was to investigate the antifungal and anti-mycotoxigenic activity from 28 microorganisms (MO) isolated from red grape. The antifungal activity of a cell free supernatant of fermented medium by the isolated MO (CFS) was tested with the agar diffusion method and the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) assay. Additionally, different antifungal compounds from the CFS were identified and quantified (organic acids, phenolic compounds, and volatile organic compounds). Finally, the most active CFS were tested as red grape bio-preservative agents. Results evidenced that CFS fermented by the strain UTA 6 had the highest antifungal activity, above all isolates, and produced a wide pool of antifungal compounds. The use of UTA 6 CFS as bio-preservative agent showed a reduction of 0.4 and 0.6 log10 spores per gram of fruit in grapes contaminated by A. flavus and B. cinerea, respectively. Moreover, UTA 6 CFS treatment reduced the occurrence of aflatoxin B1 and fumonisin (B2, B3, and B4) production in grapes contaminated by 28-100%.

Aphytis lepidosaphes (Hymenoptera: Aphelinidae) as an Effective Parasitoid for Controlling the Lepidosaphes tapleyi (Williams).

<b>Background and Objective:</b> The guava long scale insect <i>Lepidosaphes tapleyi</i> (Williams) (Hemiptera: Diaspididae) is considered one of the main destructive pests of guava around the world. Biological control represents a sustainable alternative for saving control of <i>L. tapleyi</i>. The main objective of the present work was to study the seasonal activity and evaluate the impacts of climatic factors on populations of the parasitoid, <i>Aphytis</i> <i>lepidosaphes</i>, during two successive years (2017/2018 and 2018/2019) in Esna district, Luxor Governorate, Egypt. <b>Materials and Methods:</b> Estimation of the relationship between the population density of <i>L. tapleyi</i> and <i>A. lepidosaphes</i> activity, by using different models of correlation and regression analyses. The estimate of the effects of climatic factors (daily mean max. temp., min. temp., mean of % relative humidity and mean of dew point) on seasonal activity of the parasitoid, <i>A. lepidosaphes</i>, during two successive years (2017/2018 and 2018/2019). <b>Results:</b> The results showed that the relationship between the population density of <i>L. tapleyi</i> and <i>A. lepidosaphes </i>activity was positive during both years. Furthermore, simple regression analysis indicated that the abundance of <i>A. lepidosaphes </i>was more highly correlated with the <i>L. tapleyi</i> population density in each whole year during the two successive years. The percentages of explained variance EV (%) indicated that all tested variables, i.e. daily mean maximum temperature, minimum temperature, relative humidity and dew point were responsible for 76.26 and 65.40% of the changes in parasitoid, respectively. Furthermore, the dew point was the most effective variable for the change in the parasitoid populations by 33.61 and 18.62%. <b>Conclusion:</b> The results showed that <i>A. lepidosaphes </i>had three peaks of seasonal abundance over the entire year. As well, the activity of <i>A. lepidosaphes</i> was more highly correlated with the <i>L. tapleyi </i>population size over the two successive years.

Characterization of cultural traits and fungicidal activity of strains belonging to the fungal genus Chaetomium.

AIMS: Compare and characterize Chaetomium strains with special regard to their potentialities as biocontrol agents. METHODS AND RESULTS: Twelve strains of the fungal genus Chaetomium from diverse ecological niches were identified as belonging to six different species. Large differences were observed between the strains with regard to temperature requirements for mycelial growth and pigmentation of culture filtrates. Culture filtrates and ethyl acetate extracts were assayed for fungicidal effects against important phytopathogens both on agar media and in multiwell plates. The samples from Chaetomium globosum were particularly active against Botrytis cinerea, Pyrenophora graminea and Bipolaris sorokiniana, while those from C. cochliodes and C. aureum were inhibitory towards Phytophthora infestans, and P. infestans and Fusarium culmorum respectively. To narrow down the active principle, the most promising extracts were separated by preparative HPLC and the resulting fractions tested in bioassays. Chaetoglobosins were identified as active compounds produced by C. globosum. CONCLUSIONS: The bioassays revealed C. aureum and C. cochliodes as promising candidates for use in biocontrol. Both showed remarkably good activity against the prominent plant pathogen P. infestans. SIGNIFICANCE AND IMPACT OF THE STUDY: We provide the first systematic study comparing six different Chaetomium species with regard to their use as biocontrol agents.

Production and characterization of the 13 C/15 N single-labeled insecticidal protein Cry1Ab/Ac using recombinant Escherichia coli.

Synthetic Cry1Ab/Ac proteins expressed by genetically modified (GM) crops have a high potential to control insect pests without utilizing large amounts of chemical insecticides. Before these crops are used in agriculture, the environmental fate and interactions in the soil must be understood. Stable isotope-labeled Cry1Ab/Ac protein is a highly useful tool for collecting such data. We developed a protocol to produce 13 C/15 N single-labeled Cry proteins. The artificially synthesized gene Cry1Ab/Ac of Bt rice Huahui No. 1, which has been certified by the Chinese government to be safe for human consumption, was subcloned into pUC57, and the expression vector pET-28a-CryAb/Ac was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Next, 0.2 mM isopropyl thiogalactoside (IPTG) was added to these cells and cultured at 37°C for 4 h to induce the synthesis and formation of inclusion bodies in M9 growth media containing either [U-13 C] glucose (5% 13 C-enriched) or [15 N] ammonium chloride (5% 15 N-enriched). Then, Cry inclusion bodies were dissolved in urea and purified by affinity chromatography under denaturing conditions, renatured by dialysis, and further detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The purities of 13 C/15 N-labeled Cry proteins reached 99% with amounts of 12.6 mg/L and 8.8 mg/L, respectively. The δ 13 C and ä 15 N values of 13 C-labeled Cry protein and 15 N-labeled Cry protein were 3,269‰ and 2,854‰, respectively. A bioassay test revealed that the labeled Cry1Ab/Ac proteins had strong insecticidal activity. The stable isotope-labeled insecticidal Cry proteins produced for the first time in this study will provide an experimental basis for future metabolic studies on Cry proteins in soil and the characteristics of nitrogen (N) and carbon (C) transformations. Our findings may also be employed as a reference for elucidating the environmental behavior and ecological effects of BT plants and expressed products.

Indications of biopesticidal Bacillus thuringiensis strains in bell pepper and tomato.

Members of the Bacillus cereus group are common contaminants of vegetables. One potential source of contamination is the application of B. thuringiensis based biopesticides. Although evidence of the presence of biopesticidal strains on food products is scarce, this information is essential for assessing potential risks associated with the application of these biopesticides. In order to contribute to knowledge about the presence of biopesticidal B. thuringiensis strains in foodstuffs, we investigated the occurrence of B. thuringiensis on tomatoes and bell pepper. We analyzed 99 samples of fresh bell pepper for B. cereus group members, while 426 samples of tomatoes were tested by the competent food control laboratories of the federal states in Germany. The isolates recovered from these samples were further characterized in terms of their capability to produce parasporal crystals as well as enterotoxins. A possible correlation between the B. thuringiensis isolates and biopesticidal strains was investigated by multilocus sequence typing (MLST) and whole genome Single Nucleotide Polymorphism (wgSNP) analyses. The prevalence of B. cereus group members was 41% for bell pepper and 28% for tomato samples. Isolates recovered from these samples were dominated by B. thuringiensis (93% and 99%, respectively). All B. thuringiensis isolates carried the enterotoxin genes nheA, hblD and cytK-2. In a subset of 83 B. thuringiensis isolates analyzed by MLST, 99% of the isolates matched the sequence types (ST) 8 and 15, which are also shared by the biopesticidal strains B. thuringiensis kurstaki ABTS-351 and B. thuringiensis aizawai ABTS-1857. Of the 82 isolates assigned to ST 8 or ST 15, a selection of 42 isolates was further characterized by wgSNP analysis. Of these, seven isolates differed from strain ABTS-351 by ≤4 core SNPs and 18 isolates differed from strain ABTS-1857 by ≤2 core SNPs, indicating a relationship of these isolates with the respective biopesticidal strain. These isolates originated from samples with maximum colony counts of 5.3 × 103 cfu/g for bell pepper and 1.0 × 105 cfu/g for tomatoes.

Perfiles de utilización de sustratos carbonados de Cladorrhinum (Ascomycota) / Carbon-substrate utilization profiles by Cladorrhinum (Ascomycota)

Abstract Fungi from the genus Cladorrhinum (Ascomycota) are promising agents in the biocontrol of phytopathogens, in the promotion of plant growth, and in the production of enzymes with technological application. We analyzed comparatively the ability of 5 native strains of Cladorrhinum samala and Cladorrhinum bulbillosum with reference strains belonging to the same genus. We used 95 individual carbon sources available in microplates from the Biolog® FF system. Although most of the strains mainly used soluble carbohydrates, the metabolic profile was highly dependent upon each isolate and it revealed intraspecific physiological variability in Cladorrhinum species.

Resumen Los hongos del género Cladorrhinum (Ascomycota) son agentes prometedores en el biocontrol de fitopatógenos, la promoción del crecimiento de las plantas y la producción de enzimas con aplicación tecnológica. En este trabajo se analizaron comparativamente las habilidades de 5 cepas nativas pertenecientes a las especies Cladorrhinum samala y Cladorrhinum bulbillosum con cepas de referencia del mismo género. Se usaron 95 fuentes individuales de carbono, disponibles en microplacas de Biolog® FF system. Aunque la mayoría de las cepas utilizaron principalmente carbohidratos solubles, el perfil metabólico fue altamente dependiente de cada aislamiento y reveló variabilidad fisiológica intraespecífica en las especies de Cladorrhinum.

Deciphering the associated risk on soil microbes upon use of biopesticides in rice ecosystem.

Plant species, viz Cleistanthus collinus, Lantana camara, and Strychnos nux-vomica are being traditionally used for pest management in rice. However, limited investigation has been carried out to understand the toxic effect of these materials on soil microbes. Hot water extracts of these plants were evaluated for their effects on soil microbial population and enzyme activities along with neem oil and chlorpyrifos as check. Soil microbial population, viz bacteria, fungi, phosphate-solubilizing bacteria (PSB), and asymbiotic nitrogen fixers were unchanged after application of plant extracts. Maximum population of bacteria including PSB and asymbiotic nitrogen fixers were observed in control, whereas, S. nux-vomica, and C. collinus-treated soil had higher number of actinomycetes and fungal population, respectively. Soil microbial biomass did not vary differently among the plant extracts. Application of plant extracts did not alter dehydrogenase, ß-glycosidase, acid phosphatase, alkaline phosphatase, and urease content in soil. Secondary metabolites present in these plant extracts may be responsible for variable effects on soil microbes. Chlorpyrifos had a fleeting negative effect on soil microbes and enzymes in comparison to plant extracts. All the three plants did not have any negative effect on soil microbes and enzymes and can be safely recommended in rice pest management.

Pathogen suppression by microbial volatile organic compounds in soils.

There is increasing evidence that microbial volatile organic compounds (mVOCs) play an important role in interactions between microbes in soils. In this minireview, we zoom in on the possible role of mVOCs in the suppression of plant-pathogenic soil fungi. In particular, we have screened the literature to see what the actual evidence is that mVOCs in soil atmospheres can contribute to pathogen suppression. Furthermore, we discuss biotic and abiotic factors that influence the production of suppressive mVOCs in soils. Since microbes producing mVOCs in soils are part of microbial communities, community ecological aspects such as diversity and assembly play an important role in the composition of produced mVOC blends. These aspects have not received much attention so far. In addition, the fluctuating abiotic conditions in soils, such as changing moisture contents, influence mVOC production and activity. The biotic and abiotic complexity of the soil environment hampers the extrapolation of the production and suppressing activity of mVOCs by microbial isolates on artificial growth media. Yet, several pathogen suppressive mVOCs produced by pure cultures do also occur in soil atmospheres. Therefore, an integration of lab and field studies on the production of mVOCs is needed to understand and predict the composition and dynamics of mVOCs in soil atmospheres. This knowledge, together with the knowledge of the chemistry and physical behaviour of mVOCs in soils, forms the basis for the development of sustainable management strategies to enhance the natural control of soil-borne pathogens with mVOCs. Possibilities for the mVOC-based control of soil-borne pathogens are discussed.

Synthetic agrochemicals: a necessary clarification about their use exposure and impact in crop protection.

Synthetic pesticides are largely decried. A common attitude against the synthetic agrochemicals is to avoid, criticise or ban these substances. Along with chemical pesticides to defend crops from bioagressors are microorganisms, semiochemical and natural substances used as plant protection products including biocontrol agents (BCAs) and crop protection products in organic production. Nevertheless, a natural substance status does not confer or imply safety, security or absence of residues (in the context of plant protection). Although in this paper we do not consider the toxicological perspective of highly toxic chemicals with adverse effects on humans and non-target organisms sprayed on crops, we have applied ourselves to working on the safe use of synthetic agrochemicals. Thus, along with biopesticides (either BCA or others) allowed in organic farming, we show that some synthetic chemical pesticides may be used in safe manner. HIGHLIGHTS: • Synthetic agrochemicals are widely criticised. • Some pesticide usages are not sprayed on crops. • Some biocontrol agents are of synthetic origin.

Pesticidal natural products - status and future potential.

There is a long history of using natural products as the basis for creating new pesticides but there is still a relatively low percentage of naturally derived pesticides relative to the number of pharmaceuticals derived from natural sources. Biopesticides as defined and regulated by the US Environmental Protection Agency (EPA) have been around for 70 years, starting with Bacillus thuringiensis, but they are experiencing rapid growth as the products have got better and more science-based, and there are more restrictions on synthetic chemical pesticides. As such, biopesticides are still a small percentage (approximately US$3-4 billion) of the US$61.3 billion pesticide market. The growth of biopesticides is projected to outpace that of chemical pesticides, with compounded annual growth rates of between 10% and 20%. When integrated into crop production and pest management programs, biopesticides offer the potential for higher crop yields and quality than chemical-only programs. Added benefits include reduction or elimination of chemical residues, therefore easing export, enabling delay in the development of resistance by pests and pathogens to chemicals and shorter field re-entry, biodegradability and production using agricultural raw materials versus fossil fuels, and low risk to non-target organisms, including pollinators. Challenges to the adoption of biopesticides include lack of awareness and education in how to deploy their unique modes of action in integrated programs, testing products alone versus in integrated programs, and lingering perceptions of cost and efficacy. © 2019 Society of Chemical Industry.

Developing tools for evaluating inoculation methods of biocontrol Streptomyces sp. strains into grapevine plants.

The endophytic Streptomyces sp. VV/E1, and rhizosphere Streptomyces sp. VV/R4 strains, isolated from grapevine plants were shown in a previous work to reduce the infection rate of fungal pathogens involved in young grapevine decline. In this study we cloned fragments from randomly amplified polymorphic DNA (RAPD), and developed two stably diagnostic sequence-characterized amplified region (SCAR) markers of 182 and 160 bp for the VV/E1 and VV/R4 strains, respectively. The SCAR markers were not found in another 50 actinobacterial strains isolated from grapevine plants. Quantitative real-time PCR protocols based on the amplification of these SCAR markers were used for the detection and quantification of both strains in plant material. These strains were applied on young potted plants using two methods: perforation of the rootstock followed by injection of the microorganisms or soaking the root system in a bacterial suspension. Both methods were combined with a booster treatment by direct addition of a bacterial suspension to the soil near the root system. Analysis of uprooted plants showed that those inoculated by injection exhibited the highest rate of colonization. In contrast, direct addition of either strain to the soil did not lead to reliable colonization. This study has developed molecular tools for analyzing different methods for inoculating grapevine plants with selected Streptomyces sp. strains which protect them from fungal infections that enter through their root system. These tools are of great applied interest since they could easily be established in nurseries to produce grafted grapevine plants that are protected against fungal pathogens. Finally, this methodology might also be applied to other vascular plants for their colonization with beneficial biological control agents.

Evolution of the biocontrol active substances in the framework of the European Pesticide Regulation (EC) No. 1107/2009.

BACKGROUND: The use of biocontrol agents (BCA) is growing strongly with a market of €1.9 billion worldwide and €542 million in Europe, and an estimated annual growth of between 15% and 20%. Biocontrol substances use natural mechanisms as part of integrated pest management. With the exception of macro-organisms, substances or products comprising microorganisms, semiochemicals and natural substances depend on plant protection Regulation (EC) No. 1107/2009. RESULTS: When this regulation came into force, a number of BCA had already been approved. The total number of BCA rose during the period 2011-2018 to ∼ 60 substances in line with global growth in approved substances. This growth was not smooth or equal between biocontrol categories; the microorganisms category increased the most, with a doubling of the number of approved substances in use, followed by natural substances and semiochemicals. CONCLUSION: This positive development has been influenced by many factors such as scientific advances and environmental characteristics. However, some obstacles remain that limit the development of BCA (market size, variability in effectiveness, etc.). Regulatory measures could be a key to enhancing the development of biocontrol registrations. © 2018 Society of Chemical Industry.

Salmonella spp. em pontos críticos da cadeia de produção de hortaliças orgânicas no Estado de São Paulo: contribuição para avaliação de risco / Salmonella spp. at critical points in organic vegetables production chain in the state of São Paulo: contribution for risk assessment

Dados de vigilância epidemiológica apontam uma crescente associação entre o consumo de hortaliças e surtos de origem alimentar. São inúmeras as fontes de contaminação aos quais os vegetais estão sujeitos ao longo da cadeia produtiva. Estudos sugerem que práticas agrícolas, como o uso de adubo constituído por esterco animal e água de irrigação não tratada, podem aumentar o risco de contaminação por micro-organismos patogênicos. Com as restrições ao uso de pesticidas sintéticos no sistema orgânico de produção agrícola, agentes de controle biológico, como Bacillus thuringiensis (Bt) desempenham um importante papel para a garantia da produtividade. No entanto, recentemente, a segurança do uso de Bt passou a ser questionada em função da possibilidade de produzir enterotoxinas. Este estudo teve por objetivos levantar dados sobre práticas adotadas no cultivo de hortaliças orgânicas no Estado de São Paulo, Brasil e sobre as características microbiológicas de fertilizantes, água de irrigação, água de lavagem e alfaces nas etapas pré e pós-colheita, assim como avaliar a persistência e interações entre Bt e Salmonella em hortaliças, visando contribuir para avaliações de risco microbiológico mais adequadas. Na primeira parte do estudo, dez propriedades de cultivo orgânico certificadas foram visitadas para a obtenção de dados sobre práticas adotadas e para a coleta de amostras para análise microbiológica. As amostras foram submetidas à enumeração e identificação (gênero e espécie) de Enterobacteriaceae; pesquisa de Salmonella spp. por método convencional e qPCR; e enumeração de coliformes totais e Escherichia coli nas amostras de água. Na segunda fase da pesquisa, avaliou-se a persistência e as interações entre Bt e Salmonella Montevideo no pré e pós-colheita de espinafres. Por fim, bactérias epifíticas isoladas de hortaliças foram testadas quanto a capacidade de inibir bactérias do grupo Bacillus cereus e cepas de Salmonella enterica. As contagens de Enterobacteriaceae variaram de <1 a 7,2 ± 0,1 log UFC/g nos fertilizantes, de 4,1 ± 0,3 a 5,6 ± 0,3 log UFC/g nas alfaces coletadas nos canteiros, de 2,9 ± 0,6 a 5,3 ± 0,5 log UFC/g nas alfaces lavadas, de <1 a 3,5 ± 0,1 log UFC/mL nas amostras de água de irrigação e de <1 a 3,0 ± 0,3 log UFC/mL nas amostras de água de lavagem. Salmonella não foi isolada por cultivo em placa, mas foi detectada por qPCR em uma amostra de alface orgânica lavada. Utilizando MALDI-TOF MS, 45 espécies pertencentes a 24 gêneros bacterianos foram identificadas na cadeia produtiva de hortaliças orgânicas. Bt foi capaz de persistir nas folhas de espinafre nas etapas pré e pós-colheita e afetou a persistência de Salmonella durante o cultivo, mas não durante o armazenamento pós-colheita a 12 ºC. Não foi observada tendência de germinação dos esporos de Bt após a aplicação nos espinafres, reduzindo assim a possibilidade de multiplicação e produção de enterotoxinas. A bactéria epifítica Pseudomonas chlororaphis, isolada de hortaliça, foi capaz de inibir membros do grupo Bacillus cereus, incluindo cepas patogênicas e Bt em testes in vitro, sugerindo uma barreira biológica para o controle da multiplicação destes micro-organismos. Este estudo traz importantes informações sobre a segurança microbiológica de hortaliças orgânicas e de práticas agrícolas, evidenciando a importância de boas práticas para a promoção do alimento seguro. Os resultados constituem uma importante contribuição para o desenvolvimento de modelos de avaliação de risco microbiológico e prevenção de surtos de origem alimentar

Epidemiological surveillance data indicate a growing association between vegetable consumption and food-borne outbreaks. There are numerous sources of contamination to which plants are subjected throughout the production chain. Studies suggest that agricultural practices such as the use of manure fertilizer and untreated irrigation water may increase the risk of contamination by pathogenic microorganisms. With the restrictions on the use of synthetic pesticides in the organic farming system, biological control agents such as Bacillus thuringiensis (Bt), play an important role in ensuring productivity. However, the safety of Bt has recently been questioned due to the possibility of producing enterotoxins. This study aimed to gather information about the agricultural practices employed in the organic vegetables production fields and the microbiological characteristics of fertilizer, irrigation water, wash water, and lettuces in pre and post-harvest stages, and to evaluate the persistence and interactions between Bt and Salmonella on leafy greens, aiming to contribute for more adequate microbiological risk assessments. In the first part of the study, ten certified organic farms were visited to collect data on the farming practices and for collection of samples for microbiological evaluations. The samples were submitted to Enterobacteriaceae enumeration and identification (genus and species); Salmonella spp. by conventional method and qPCR; and enumeration of total coliforms and Escherichia coli in water samples. In the second part of the study, the persistence and interaction between Bacillus thuringiensis subsp Aizawai (Bt) and Salmonella Montevideo in the pre and post-harvest of spinach were evaluated. Finally, epiphytic bacteria isolated from vegetables were tested for their ability to inhibit growth of Bacillus cereus group members and Salmonella strains. Enterobacteriaceae counts ranged from <1 to 7.2 ± 0.1 log CFU/g in fertilizers, from 4.1 ± 0.3 to 5.6 ± 0.3 log CFU/g in lettuces collected from the fields, from 2.9 ± 0.6 to 5.3 ± 0.5 log CFU/g in washed lettuces, <1 to 3.5 ± 0.1 log CFU/mL in irrigation water and <1 to 3.0 ± 0.3 log CFU/mL in wash water. Salmonella was not isolated by plating but it was detected by qPCR in one sample of washed organic lettuce. Using MALDI-TOF MS, 45 species belonging to 24 bacterial genera were identified in the organic vegetable production chain. Bt was able to persist on pre and post-harvest of spinach and affected Salmonella persistence during cultivation, but not during the storage at 12 ºC. Bt spores showed no tendency to germinate during pre-harvest of spinach, thus reducing the probability of growth and production of enterotoxins. The epiphytic bacterium Pseudomonas chlororaphis isolated from one vegetable sample was able to inhibit members of the Bacillus cereus group, including pathogenic strains and Bt in in vitro tests, suggesting a biological barrier to control the multiplication of these microorganisms. These studies provide important information about the microbiological safety of organic vegetables and agricultural practices, highlighting the importance of good practices for the promotion of safe food. These data are fundamental for the development of microbiological risk assessment models and prevention of foodborne outbreaks

Toxic Peptides in Populations of Two Pergid Sawflies, Potential Biocontrol Agents of Brazilian Peppertree.

Determination of the safety of agents prior to release is one of the most important research goals in biological control. In addition to concerns for the safety of non-target plants, determination of the potential toxic properties of new agents needs to be assessed. Numerous phytophagous insects are defended by chemicals against the attack of natural enemies. Some of these defensive compounds could pose an environmental risk if an agent is released. Here, larval populations of two pergid sawflies, Heteroperreyia hubrichi and H. jorgenseni, were analyzed by LC-MS/MS to investigate whether they contain alleged toxic peptides. The first species is a potential candidate for biological control of the invasive weed Brazilian peppertree in Florida and Hawaii. The chemical analyses revealed the presence of the peptides pergidin (Perg), 4-valinepergidin (VPerg), dephosphorylated pergidin (dpPerg), lophyrotomin (LGln and LGlu). The effect of sawfly population for each species was significantly influencing peptide concentration. All peptides occurred at lower concentrations compared with purportedly toxic species of this sawfly family. However, the concentrations of the peptides are of concern for the welfare of wildlife and livestock that would be exposed to these species. These results demonstrate that release of this biological control agent in the invaded range may pose an environmental threat.

Conspecific and Heterogeneric Lacewings Respond to (Z)-4-Tridecene Identified from Chrysopa formosa (Neuroptera: Chrysopidae).

Green lacewings (Chrysopidae) are predators of soft-bodied pest insects and are among the most important biological control agents in crop protection. Chrysopa spp. are of special importance since, unlike most green lacewing species, adults are also predatory. The current study was undertaken in search of Chrysopa formosa compounds with semiochemical activity. Using coupled gas chromatography-electroantennography (GC-EAG), head and thorax extracts of C. formosa elicited EAG responses to a compound subsequently identified by coupled GC/mass spectrometry, microchemistry, chemical synthesis and GC peak enhancement as (Z)-4-tridecene. In field experiments, this compound decreased attraction of adult C. formosa to (1R,4aS,7S,7aR)-nepetalactol and that of Chrysoperla carnea species-complex to a ternary floral lure, with the inhibitory effect found to be dose-dependent. Our results suggest that (Z)-4-tridecene may serve as a general warning signal among multiple green lacewing species. Perspectives for potential practical applications are discussed.

Large-scale cultivation of the bumblebee gut microbiota reveals an underestimated bacterial species diversity capable of pathogen inhibition.

A total of 1940 isolates from gut samples of 60 bumblebees representing Bombus pascuorum, Bombus terrestris, Bombus lucorum and Bombus lapidarius was collected and identified through state-of the-art taxonomic methods. The bacterial species diversity in these Bombus species exceeded that suggested by phylotype analysis through 16S rRNA amplicon sequencing, and revealed that B. pascuorum and B. terrestris had a unique microbiota composition, each. Representatives of most phylotypes reported earlier and detected in the present study were effectively isolated, and included several novel bacterial taxa and species reported for the first time in the bumblebee gut. Isolates were screened in pectin degradation assays and growth inhibition assays against the honeybee pathogens Paenibacillus larvae, Melissococcus plutonius and Ascosphaera apis and the bumblebee parasite Crithidia bombi. While inhibitory activity against each of these pathogens was observed, only one single culture was able to degrade pectin and polygalacturonic acid in vitro. The availability of accurately identified microbial isolates will facilitate future evaluation of the functional potential of the bumblebee gut microbiota.

A novel strategy for producing compost with enhanced biopesticide properties through solid-state fermentation of biowaste and inoculation with Bacillus thuringiensis.

In the framework of a circular economy, organic solid wastes are considered to be resources useful for obtaining value-added products. Among other potential uses, biodegradable wastes from agricultural, industrial, and domestic sources are being studied to obtain biopesticides through solid-state fermentation (SSF), mainly at the laboratory scale. The suitability of biowaste (source-selected organic fraction of municipal solid waste) for use as a substrate for Bacillus thuringiensis (Bt) growth under non-sterile conditions in a 10 L SSF reactor was determined in this study. An operational strategy for setting up a semi-continuous process yielding a stabilised organic compost-like material enriched with Bt suitable for use as a soil amendment was developed. Concentrations of 1.7·107-2.2·107 and 1.3·107-2.1·107 CFU g-1 DM for Bt viable cells and spores, respectively, were obtained in the final material. As the results confirmed, Bt-enriched compost-like material with potential biopesticide properties can be produced from non-sterile biowaste.