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.

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.

Extratos aquosos de inhame (Dioscorea rotundata Poirr. ) e de mastruz (Chenopodium ambrosioides L. ) no desenvolvimento da lagarta-do-cartucho-do-milho Spodoptera frugiperda (J. E. Smith, 1797) / Aqueous extracts of yam (Dioscorea rotundata Poirr. ) and chenopodium (Chenopodium ambrosioides L. ) in the development of fall armyworm Spodoptera frugiperda (J. E. Smith, 1797)

RESUMO: Estudou-se o efeito de extratos aquosos de inhame (0; 5; 10; e 20% p/p) e de mastruz (0; 2; 4; 6; 8 e 10% p/p) na biologia da lagarta-do-cartucho. Secções de folhas de milho foram mergulhadas por 30 segundos em soluções de cada concentração; após a secagem, colocou-se em cada secção uma lagarta recém-eclodida. Foram avaliadas a viabilidade e a duração das fases larval e pupal, peso e comprimento das lagartas e pupas. Em relação ao extrato de inhame, a concentração de 20% causou maior influência na fase larval, sendo a viabilidade reduzida para 12%, com duração de 7 dias, diferindo da testemunha com 17 dias. O extrato da mesma planta a 10% causou 48% de mortalidade. Em todas as concentrações esse extrato também afetou a fase de pupa; na testemunha, 85% das pupas foram viáveis, enquanto nos demais tratamentos a viabilidade não excedeu a 25%. Para o peso e comprimento das lagartas, os resultados não foram significativos. Para o mastruz, o extrato a 20% causou influência na fase larval com baixa viabilidade e mortalidade logo nos primeiros seis dias de avaliação. Outras concentrações de mastruz não deferiram entre si nas fases larval e pupal. Verificou-se que a alimentação das lagartas com folhas tratadas com mastruz diminuiu o peso das pupas.

ABSTRACT: The effect of aqueous extracts of yam (0, 5, 10, and 20% h/h) and chenopodium (0, 2, 4, 6, 8 and 10% h/h) on the biology of fall armyworm was studied. Sections of maize leaves were dipped for 30 seconds in solutions of each concentration; after the section dried, a recently hatched caterpillar was placed onto each treated section. The viability and duration of the larval and pupal stages and the weight and length of the caterpillars and pupae were evaluated. For yam, the extract at 20% concentration caused the greatest influence on the larval stage of the insect, significantly reducing larval viability to 12%, with 7 day larval stage duration, differing from the control at 17 days. The extract of the same plant at 10% caused 48% larval mortality. At all concentrations, that extract also affected the pupal stage; in the control, pupal viability was 85%, whereas for the other concentrations the viability did not exceed 25%. No significant differences were observed for the weight and length of caterpillars. For chenopodium, the extract at 20% concentration caused influence on the larval stage, as it showed the lowest viability, causing mortality in six days. Other chenopodium concentrations did not show differences for the larval and pupal stages. Feeding caterpillars with leaves treated with the extract of chenopodium decreased pupal weight.

Peptaibol, secondary-metabolite, and hydrophobin pattern of commercial biocontrol agents formulated with species of the Trichoderma harzianum complex.

The production of bioactive polypeptides (peptaibiotics) in vivo is a sophisticated adaptation strategy of both mycoparasitic and saprotrophic Trichoderma species for colonizing and defending their natural habitats. This feature is of major practical importance, as the detection of peptaibiotics in plant-protective Trichoderma species, which are successfully used against economically relevant bacterial and fungal plant pathogens, certainly contributes to a better understanding of these complex antagonistic interactions. We analyzed five commercial biocontrol agents (BCAs), namely Canna(®) , Trichosan(®) , Vitalin(®) , Promot(®) WP, and TrichoMax(®) , formulated with recently described species of the Trichoderma harzianum complex, viz. T. afroharzianum, T. simmonsii, and T. guizhouense. By using the well-established, HPLC/MS-based peptaibiomics approach, it could unequivocally be demonstrated that all of these formulations contained new and recurrent peptaibols, i.e., peptaibiotics carrying an acetylated N-terminus, the C-terminus of which is reduced to a 1,2-amino alcohol. Their chain lengths, including the amino alcohol, were 11, 14, and 18 residues, respectively. Peptaibols were also to be the dominating secondary metabolites in plate cultures of the four strains obtained from four of the Trichoderma- based BCAs, contributing 95% of the UHPLC-UV/VIS peak areas and 99% of the total ion count MS peak area from solid media. Furthermore, species-specific hydrophobins, as well as non-peptaibiotic secondary metabolites, were detected, the latter being known for their antifungal, siderophore, or plant-growth-promoting activities. Notably, none of the isolates produced low-molecular weight mycotoxins.

Produção de conídios de Metarhizium anisopliae em meio sólido à base de resíduos agroindustriais / Production of conidia of Metarhizium anisopliae in solid media based on agroindustrial residues

As crescentes despesas com o custo do meio para o cultivo de Metarhizium anisopliae levantam a necessidade de averiguar a eficiência de alguns resíduos agroindustriais que, além de propriedades nutricionais importantes, possuem grande disponibilidade e baixo custo. Os experimentos foram conduzidos no Laboratório de Tecnologia das Fermentações, UNIOESTE, Campus de Cascavel, PR. Neste trabalho, foram avaliados como substratos arroz polido (meio padrão), arroz vermelho, arroz polido suplementado com melaço (2,5%, 5% e 10%), resíduo de cervejaria, resíduo de fecularia, arroz polido + resíduo de cervejaria (1:1) e arroz polido + resíduo de fecularia (1:1). Os experimentos foram realizados em triplicata, em frascos Erlernmeyer de 250 mL contendo 30 g do substrato (ou 15 g de cada, quando em mistura) e umidade de 70 ± 10%. Os frascos foram inoculados com 0,5 mL da suspensão de conídios (1 × 108 conídios/mL) e incubados em BOD (25 ± 1o C, fotofase 12h, 7 dias). Dentre os meios testados, verificou-se uma elevada produção de conídios no meio composto por arroz + resíduo de cervejaria (11,3 × 108 conídios/g de substrato) e no arroz (9,6 × 108 conídios/g). Os conídios produzidos no meio de arroz + resíduo de cervejaria apresentaram atividade inseticida frente a Spodoptera frugiperda semelhante a dos conídios produzidos no arroz. Este novo meio de cultivo poderá proporcionar uma redução de custo de aproximadamente 50%, representando uma alternativa viável para a produção do fungo.

Increasing costs of substrate for production of Metarizhiuma nisopliae raise the necessity of investigating the efficiency of some agro-industrial residues that, in addition to important nutritional properties, are widely available at low cost. The assays were conducted at the Fermentation Technology Laboratory, UNIOESTE, Cascavel, PR, Brazil. In this work, substrates such as polished rice (standard media), red rice, polished rice + sugar molasses (2.5%, 5% and 10%), brewery residue, cassava residue, polished rice + brewery residue (1:1) and polished rice + cassava residue (1:1) were evaluated. The experiments were carried out in triplicate, in Erlernmeyer flasks of 250 mL, containing 30 g of the substrate (or 15 g of each one, when as mixture) and moisture content of 70 ± 10%. The flasks were inoculated with 0.5 mL of conidia suspension (1 × 108 conidia/mL) and then incubated in controlled temperature and light (25 ± 1o C, 12 hours photophase, 7 days). High conidial production was verified in rice + brewery residue media (11.30 × 108 conidia/g of substrate and in the polished rice (9.59 × 108 conidia/g of substrate). The conidia produced in rice + brewery residue media showed an insecticidal activity against Spodoptera frugiperda similar to that attained with the conidia produced in rice. This new culture media may provide a reduction of approximately 50% in the production cost, thus presenting a viable alternative for the fungus production.