Effect of VvLaeA on the growth and development of Beauveria bassiana / 生物工程学报

It is unclear how VvLaeA functions in regulating the growth and development of Volvariella volvacea (Bull. ex. Fr.) Sing.. Firstly, bioinformatics analysis of VvLaeA was carried out in this study. Subsequently, the Vvgpd promoter and the open reading frame (ORF) fragment of VvlaeA were amplified and fused by polymerase chain reaction (PCR). The fusion fragment was cloned into the pK2 (bar) plasmid. The recombinant construct pK2(bar)-OEVvlaeA was transfected into Beauveria bassiana by Agrobacterium tumefaciens-mediated transformation. Finally, the growth and development of the transformants were examined. The results showed that VvLaeA shared a low homology with similar proteins in other fungi. Compared with the wild type, the colony diameter of the transformant was significantly increased. However, the pigment deposition, conidial yields and germination rates were significantly decreased. The overexpression strains were more sensitive to stresses than that of the wild type. Further studies showed the conidial cell wall properties of the transformants were altered, and the expressions of genes related to the conidial development were significantly down-regulated. Collectively, VvLaeA increased the growth rate of B. bassiana strains and negatively regulated the pigmentation and conidial development, which shed a light for the functional identification of straw mushroom genes.

Applying different infective structures of Beauveria bassiana to Coffea arabica infested with Hypothenemus hampei (Coleoptera: Curculionidae) under field

Coffee is the most appreciated beverage in worldwide; Brazil is the largest producer and exporter of this commodity. Organochlorine endosulfan was banned from the country in 2013 due to its teratogenic agent-related features. Since then, coffee plantations have experienced increased Hypothenemus hampei infestation rates. The aim of the current study is to assess variations in the rates of Coffea arabica fruits brocaded by H. hampei after the application of entomopathogenic fungal species Beauveria bassiana IBCB66. Experiments were carried out with 'Catuaí' and 'Mundo Novo' cultivars between 2018 and 2020, during the borer transit period. Three experiments were carried out based on the application of the aforementioned fungal species on the investigated coffee plant species, both by spraying and sprinkling, at 30-day intervals; 10 fruits were collected per face of each useful plant in each repetition. The experiment has followed a randomized blocks design with five treatments, including the control, and five repetitions, each. Beauveria bassiana Ecobass (IBCB66) wettable powder spray, at the concentration of 2 × 1013·ha­1, was used in experiments I and II. On the other hand, the mix used in experiment III was prepared with blastospores at concentration of 5 × 1012·ha­1 blastospores + 0.1% Silwet. The sprinkling process in all three experiments has used dry aerial conidia at concentration of 2 × 1013·ha­1. Collected data were subjected to analysis of variance (ANOVA), which was followed by Fisher's test at 5% probability level, in the SISVAR software. More than 35,000 fruits were assessed. In addition to variations between experiments, results have evidenced that the rate of brocaded fruits remained high.

Late effects of Beauveria bassiana on larval stages of Aedes aegypti Linneo , 1762 (Diptera: Culicidae) / Efeitos tardios de Beauveria bassiana nos estágios larvais de Aedes aegypti Linneo, 1762 (Diptera: Culicidae)

Aedes aegypti is a culicide that has gained relevance over the years due to its ability to transmit various viruses that cause diseases in humans that all the years cause high mortality rates in the world population. The main problem is that Ae. aegypti has managed to establish and maintain a close relationship with humans and their habitat, which is why the search for alternatives to control vector populations becomes imperative. The objective of the present work was to study the effects of two Beauveria bassiana strains on Aedes aegypti. Third instar larvae of Ae. aegypti in 250 mL plastic containers were inoculated with the GHA and NB3 strains at different concentrations (1.5 × 104 , 1.5× 105 , 1.5 × 106 and 1.5 × 107 conidia/mL). The NB3 strain presented highest mortality values with 63% in the highest concentration i.e., 1.5 × 107 , while for the GHA strain the highest mortality value was 30.7% at the same concentration. The results showed significant difference in mortality with respect to the strain and days post treatment (P = 0.0001), but not with respect to the conidial concentration (P = 0.634). The average mortality of larvae per day for the NB3 for different concentrations ranged from 20 to 25 larvae per day, while for the GHA daily mortality ranged from 5 to 12 larvae. In post-treatment mortality, the highest mortality was recorded in the third stage larvae for the NB3, while for GHA the highest percentage mortality was observed in individuals who managed to reach the adult state. The findings of the current research depicted the noteworthy role of B. bassiana for the management of an important vector of human disease.

O Aedes aegypti é um culicida que vem ganhando relevância ao longo dos anos devido à sua capacidade de transmitir diversos vírus causadores de doenças em humanos que ao longo dos anos ocasionam altas taxas de mortalidade na população mundial. O principal problema é que Ae. aegypti tem conseguido estabelecer e manter uma relação próxima com o homem e seu habitat, por isso a busca por alternativas para o controle das populações de vetores torna-se imperativa. O objetivo do presente trabalho foi estudar os efeitos de duas cepas de Beauveria bassiana sobre Ae. aegypti. Larvas de terceiro instar de Ae. aegypti em recipientes plásticos de 250 mL foram inoculados com as cepas GHA e NB3 em diferentes concentrações (1.5 × 104, 1.5 × 105, 1.5 × 106 e 1.5 × 107 conídios/mL). A cepa NB3 apresentou os maiores valores de mortalidade com 63% na concentração mais alta, ou seja, 1.5 × 107, enquanto para a cepa GHA o maior valor de mortalidade foi 30.7% na mesma concentração. Os resultados mostraram diferença significativa na mortalidade com relação à cepa e dias pós-tratamento (P = 0.0001), mas não com relação à concentração de conídios (P = 0.634). A mortalidade média de larvas por dia para o NB3 para diferentes concentrações variou de 20 a 25 larvas por dia, enquanto para o GHA a mortalidade diária variou de 5 a 12 larvas. Na mortalidade pós-tratamento, a maior mortalidade foi registrada nas larvas de terceiro estágio para o NB3, enquanto para o GHA o maior percentual de mortalidade foi observado em indivíduos que conseguiram atingir o estado adulto. Os resultados da pesquisa atual retratam o papel notável de B. bassiana no manejo de um importante vetor de doenças humanas.

Late effects of Beauveria bassiana on larval stages of Aedes aegypti Linneo, 1762 (Diptera: Culicidae) / Efeitos tardios de Beauveria bassiana nos estágios larvais de Aedes aegypti Linneo, 1762 (Diptera: Culicidae)

Aedes aegypti is a culicide that has gained relevance over the years due to its ability to transmit various viruses that cause diseases in humans that all the years cause high mortality rates in the world population. The main problem is that Ae. aegypti has managed to establish and maintain a close relationship with humans and their habitat, which is why the search for alternatives to control vector populations becomes imperative. The objective of the present work was to study the effects of two Beauveria bassiana strains on Aedes aegypti. Third instar larvae of Ae. aegypti in 250 mL plastic containers were inoculated with the GHA and NB3 strains at different concentrations (1.5 × 104, 1.5× 105, 1.5 × 106 and 1.5 × 107 conidia/mL). The NB3 strain presented highest mortality values with 63% in the highest concentration i.e., 1.5 × 107, while for the GHA strain the highest mortality value was 30.7% at the same concentration. The results showed significant difference in mortality with respect to the strain and days post treatment (P = 0.0001), but not with respect to the conidial concentration (P = 0.634). The average mortality of larvae per day for the NB3 for different concentrations ranged from 20 to 25 larvae per day, while for the GHA daily mortality ranged from 5 to 12 larvae. In post-treatment mortality, the highest mortality was recorded in the third stage larvae for the NB3, while for GHA the highest percentage mortality was observed in individuals who managed to reach the adult state. The findings of the current research depicted the noteworthy role of B. bassiana for the management of an important vector of human disease.

O Aedes aegypti é um culicida que vem ganhando relevância ao longo dos anos devido à sua capacidade de transmitir diversos vírus causadores de doenças em humanos que ao longo dos anos ocasionam altas taxas de mortalidade na população mundial. O principal problema é que Ae. aegypti tem conseguido estabelecer e manter uma relação próxima com o homem e seu habitat, por isso a busca por alternativas para o controle das populações de vetores torna-se imperativa. O objetivo do presente trabalho foi estudar os efeitos de duas cepas de Beauveria bassiana sobre Ae. aegypti. Larvas de terceiro instar de Ae. aegypti em recipientes plásticos de 250 mL foram inoculados com as cepas GHA e NB3 em diferentes concentrações (1.5 × 104, 1.5 × 105, 1.5 × 106 e 1.5 × 107 conídios/mL). A cepa NB3 apresentou os maiores valores de mortalidade com 63% na concentração mais alta, ou seja, 1.5 × 107, enquanto para a cepa GHA o maior valor de mortalidade foi 30.7% na mesma concentração. Os resultados mostraram diferença significativa na mortalidade com relação à cepa e dias pós-tratamento (P = 0.0001), mas não com relação à concentração de conídios (P = 0.634). A mortalidade média de larvas por dia para o NB3 para diferentes concentrações variou de 20 a 25 larvas por dia, enquanto para o GHA a mortalidade diária variou de 5 a 12 larvas. Na mortalidade pós-tratamento, a maior mortalidade foi registrada nas larvas de terceiro estágio para o NB3, enquanto para o GHA o maior percentual de mortalidade foi observado em indivíduos que conseguiram atingir o estado adulto. Os resultados da pesquisa atual retratam o papel notável de B. bassiana no manejo de um importante vetor de doenças humanas.

Late effects of Beauveria bassiana on larval stages of Aedes aegypti Linneo, 1762 (Diptera: Culicidae)

Abstract Aedes aegypti is a culicide that has gained relevance over the years due to its ability to transmit various viruses that cause diseases in humans that all the years cause high mortality rates in the world population. The main problem is that Ae. aegypti has managed to establish and maintain a close relationship with humans and their habitat, which is why the search for alternatives to control vector populations becomes imperative. The objective of the present work was to study the effects of two Beauveria bassiana strains on Aedes aegypti. Third instar larvae of Ae. aegypti in 250 mL plastic containers were inoculated with the GHA and NB3 strains at different concentrations (1.5 × 104, 1.5× 105, 1.5 × 106 and 1.5 × 107 conidia/mL). The NB3 strain presented highest mortality values with 63% in the highest concentration i.e., 1.5 × 107, while for the GHA strain the highest mortality value was 30.7% at the same concentration. The results showed significant difference in mortality with respect to the strain and days post treatment (P = 0.0001), but not with respect to the conidial concentration (P = 0.634). The average mortality of larvae per day for the NB3 for different concentrations ranged from 20 to 25 larvae per day, while for the GHA daily mortality ranged from 5 to 12 larvae. In post-treatment mortality, the highest mortality was recorded in the third stage larvae for the NB3, while for GHA the highest percentage mortality was observed in individuals who managed to reach the adult state. The findings of the current research depicted the noteworthy role of B. bassiana for the management of an important vector of human disease.

Resumo O Aedes aegypti é um culicida que vem ganhando relevância ao longo dos anos devido à sua capacidade de transmitir diversos vírus causadores de doenças em humanos que ao longo dos anos ocasionam altas taxas de mortalidade na população mundial. O principal problema é que Ae. aegypti tem conseguido estabelecer e manter uma relação próxima com o homem e seu habitat, por isso a busca por alternativas para o controle das populações de vetores torna-se imperativa. O objetivo do presente trabalho foi estudar os efeitos de duas cepas de Beauveria bassiana sobre Ae. aegypti. Larvas de terceiro instar de Ae. aegypti em recipientes plásticos de 250 mL foram inoculados com as cepas GHA e NB3 em diferentes concentrações (1.5 × 104, 1.5 × 105, 1.5 × 106 e 1.5 × 107 conídios/mL). A cepa NB3 apresentou os maiores valores de mortalidade com 63% na concentração mais alta, ou seja, 1.5 × 107, enquanto para a cepa GHA o maior valor de mortalidade foi 30.7% na mesma concentração. Os resultados mostraram diferença significativa na mortalidade com relação à cepa e dias pós-tratamento (P = 0.0001), mas não com relação à concentração de conídios (P = 0.634). A mortalidade média de larvas por dia para o NB3 para diferentes concentrações variou de 20 a 25 larvas por dia, enquanto para o GHA a mortalidade diária variou de 5 a 12 larvas. Na mortalidade pós-tratamento, a maior mortalidade foi registrada nas larvas de terceiro estágio para o NB3, enquanto para o GHA o maior percentual de mortalidade foi observado em indivíduos que conseguiram atingir o estado adulto. Os resultados da pesquisa atual retratam o papel notável de B. bassiana no manejo de um importante vetor de doenças humanas.

Producción de unidades infectivas de Isaria fumosorosea (Hypocreales: Cordycipitaceae) a partir de aislados nativos del noreste de México mediante 3 estrategias de propagación / Production of infectious units of Isaria fumosorosea ( Hypocreales: Cordycipitaceae) from different indigenous isolates of northeastern Mexico using 3 propagation strategies

El objetivo del presente estudio fue evaluar la producción de blastosporas y conidios de diferentes aislados nativos de México del hongo entomopatógeno Isaria fumosorosea y de una cepa de colección mediante diferentes técnicas de propagación. En la producción de blastosporas se utilizaron 2 medios de cultivo líquidos (sumergidos), uno a base de casaminoácidos y el otro a base de peptona de colágeno como fuentes de nitrógeno, con glucosa como fuente de carbono en ambos. Para la producción de conidios, los hongos se cultivaron en agar papa dextrosa, a partir de esos cultivos se prepararon suspensiones de 1 x 10(6) conidios/ml para inocular matraces con caldo dextrosa Sabouraud, para iniciar así la fase líquida del cultivo bifásico, denominado también precultivo. Posteriormente con el precultivo y las suspensiones de conidios se inocularon bolsas con granos de arroz, que se incubaron durante 14 días para el cultivo bifásico y para la fermentación sólida, respectivamente. El aislado HIB-23 fue el que logró la más elevada concentración de blastosporas obtenida en el cultivo sumergido: 4,90 x 10(8) blastosporas/ml en el medio casaminoácidos; y en el medio con peptona de colágeno se obtuvieron 2,15 x 10(8) blastosporas/ml. La máxima producción de conidios en fermentación sólida la logró la cepa Pfr-612 (1,58 x 10(9) conidios/g), mientras que la máxima en cultivo bifásico correspondió al aislado HIB-30 (9,00 x 10(6) conidios/g). La fermentación sólida resultó ser el método más efectivo, con un promedio de 1,09 x 10(9) conidios/g, mientras que el cultivo bifásico fue el menos efectivo, con un promedio de 2,76 x 10(6) conidios/g. Para la producción de blastosporas en los medios sumergidos no se obtuvo diferencia significativa alguna.

The aim of this study was to evaluate the production of blastospores and conidia of different native isolates and a strain of Isaria fumosorosea using different propagation techniques. Two liquid culture media of casamino acids and peptone as nitrogen sources and glucose as carbon source for both media cultures were respectively used in the production of blastospores, while for the production of conidia, the fungi were grown in potato dextrose agar; from these cultures, solutions of conidia to a concentration of 1 x 10(6) per milliliter were prepared to inoculate flasks with Sabouraud dextrose broth for the liquid phase of the biphasic culture, also known as preculture. Subsequently, rice grain bags were inoculated with the preculture and the conidia solutions, which were incubated for 14 days for solid fermentation and biphasic culture, respectively. The HIB-23 isolate recorded a concentration of 4.90 x 10(8) blastospores/ml in the casamino acid medium, while a concentration of 2.15 x 10(8) blastospores/ml was obtained in the peptone collagen medium. For the Pfr-612 strain, the conidia production in solid-state fermentation was 1.58 x 10(9) conidia/g, and for HIB-30 in the biphasic culture of 9.00 x 10(6) conidia/g. Solid-state fermentation proved to be the most effective method with an average of 1.09 x 10(9) conidia/g, whereas the biphasic culture was the least effective method with 2.76 x 10(6) conidia/g; no significant difference was reported for the submerged production media.

THE POWER OF THE SMALL: THE EXAMPLE OF Paracoccidioides brasiliensis CONIDIA / O poder do pequeno: o exemplo dos conídios do Paracoccidioides brasiliensis

SUMMARYResearch on Paracoccidioides brasiliensis has centered in the yeast cell probably because of the lack of distinctive features in the mycelium. In 1942 and for the first time, lateral conidia were noticed in the fungus' hyphae. Later on, Brazilian, Venezuelan and Argentinean researchers described "aleurias" when the fungus was grown in natural substrates. In 1970 authors became interested in the conidia and were able to obtain them in large numbers and treat them as individual units. Their shape and size were defined and the presence of all the elements of a competent eukaryotic cell were demonstrated. Conidia exhibited thermal dimorphism and, additionally, when given intranasally to BALB/c male mice, they converted into yeasts in the lungs and produce progressive pulmonary lesions with further dissemination to other organs. Studies on the phagocyte-conidia interaction were revealing and showed that these versatile structures allow a better understanding of the host- P. brasiliensisinteractions.

RESUMOA investigação sobre Paracoccidioides brasiliensis tem-se centrado na célula de levedura, provavelmente devido à falta de características distintas no micélio. Em 1942 e, pela primeira vez, conídios laterais foram notados nos hifas dos fungos. Mais tarde, pesquisadores brasileiros, venezuelanos e argentinos descreveram "aleurias" quando o fungo foi cultivado em substratos naturais. Em 1970, os autores se interessaram pelos conídios e foram capazes de obtê-los em grande número e tratá-los como unidades individuais. A sua forma e tamanho foram definidos, e a presença de todos os elementos de uma célula eucariótica competente foram demonstrados. Conídios apresentam dimorfismo térmico e, além disso, quando administrados por via intranasal a camundongos BALB/c machos, são convertidos em leveduras nos pulmões e produzem lesões pulmonares progressivas com posterior disseminação para outros órgãos. Estudos sobre a interação de fagócitos-conídios foram reveladores e mostraram que estas estruturas versáteis permitem melhor compreensão das interacções entre hospedeiro e P. brasiliensis.

Effect of surfactants and temperature on germination and vegetative growth of Beauveria bassiana

Three non-ionic surfactants: Tween20, Tween80 and Breakthru® were screened for their effects on spore germination and mycelial growth rates and for their influence on three isolates of Beauveria bassiana spore germination at various temperatures. Tween20 and Tween80 were compatible with all the B. bassiana isolates in the germination studies, but inhibited germination at higher surfactant concentrations, irrespective of the conidial concentrations. Breakthru® had an inhibitory effect on germination even at the lowest concentration of 0.1% on all the B. bassiana isolates. The effects of the surfactants on spore germination did not correspond with their effects on colony growth. Conidial viability within the same formulation declined significantly with increases in temperature, irrespective of the surfactant. The optimal temperature for conidial germination of B. bassiana isolates was approximately 25 °C with an upper limit at 30 °C. Isolate 7320 was identified as the least affected by the different surfactants. This isolate was able to germinate rapidly in a broad temperature range of 25–30 °C after 24 h, this characteristic being an essential factor in controlling house fly populations in poultry houses.

Paracoccidioides lutzii: estudo de alguns mecanismos de patogenicidade

A paracoccidioidomicose (PCM) é uma doença granulomatosa sistêmica, causada por Paracoccidioides spp., (P. brasiliensis e P. lutzii), geograficamente, limita-se a América Latina com as áreas endêmicas estendendo-se desde o México até a Argentina, constituindo uma das micoses sistêmicas de maior incidência na região, afetando principalmente trabalhadores rurais. O maior número de pacientes com PCM tem sido reportado principalmente no Brasil, Colômbia e Venezuela. A incidência real desta micose encontra-se subestimada no Brasil e pouco se conhece em relação a nova espécie descrita - P. lutzii. A maioria dos estudos em P. lutzii foram focados em genética, especiação e na geração de novos antígenos para melhorar a especificidade e sensibilidade dos testes sorológicos. Atualmente, as preparações antigênicas tradicionais, preparadas a partir de isolados de P. brasiliensis, são ineficientes. Raros são os trabalhos focados na biologia de P. lutzii e nos fatores de virulência que podem ser comparados com P. brasiliensis nos modelos experimentais. A nossa proposta de estudo foi avaliar alguns aspectos in vitro e in vivo relacionados com a patogenicidade e destacamos: a fagocitose e a morte intracelular de P. lutzii por macrófagos, peritoneais, de camundongos Knockouts (KO) e selvagens para PRRs (TLR2, TLR4 e Dectina) e ativadores intracelulares (MyD88 e NALP3). Paralelamente a este estudo, animais foram infectados com leveduras de P. lutzii e comparados com os modelos de infecção já estabelecidos com leveduras (Pb18) e conídios (ATCCPb60855) de P. brasiliensis. Nossos dados indicam que similar ao que ocorre com P. brasiliensis a fagocitose de P. lutzii depende de TLR2, TLR4 e Dectina- 1, resultados semelhantes também foram observadas na expressão de moléculas envolvidas na co-estimulação e a apresentação de antígenos (MHC II, CD80 e CD86)...

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides spp. (P. brasiliensis and P. lutzii), geographically, is limited to Latin America with endemic areas from Mexico to Argentina, as one of the systemic mycoses with the highest incidence in the region, mainly affecting rural workers. The largest number of patients with PCM has been mainly reported in Brazil, Colombia and Venezuela. The true incidence of this mycosis is underestimated in Brazil and little is known about the new species described - P. lutzii. Most studies in P. lutzii were focused on genetics, speciation and the generation of new antigens to improve the specificity and sensitivity of serological tests. Currently, traditional antigenic preparations, prepared with isolates of P. brasiliensis, are inefficient. There are few studies focused on P. lutzii biology and virulence factors that can be compared with P. brasiliensis in experimental models. Our study aimed to evaluate some in vitro and in vivo aspects related to pathogenicity: phagocytosis and intracellular killing of P. lutzii by peritoneal macrophages from knockouts (KO) for PRRs (TLR2, TLR4 and Dectin) and intracellular activators (MyD88 and NALP3). In addition, animals were infected with P. lutzii yeast and compared with the well-established models of infection with yeast cells (Pb18) and conidia (ATCC Pb60855) from P. brasiliensis. Our data indicate that similarly to what happens with the phagocytosis of P. brasiliensis, P. lutzii phagocytosis is dependent on TLR2, TLR4 and Dectin-1. Other molecules, involved in co-stimulation and presentation of antigens such as MHC II, CD80 and CD86 were also shown to participate in the P. lutzii-host interaction...

Production of Beauveria bassiana Air Conidia by Means of Optimization of Biphasic System Technology

Air conidia production of Beauveria bassiana, strain CA-603 was studied based on modified diphasic system. The biomass yield obtained in the first phase based on submerged cultivation of fungus was processed using methodology providing different contact with air space. Our study indicated that productivity of the second stage of diphasic system is had inversely proportional dependence on depth of liquid fungal biomass. Increase of biomass depth is significantly decreased production of air conidia. Two methodology of biomass processing extending contact biomass with air space including distribution of fungal material on surface of hygroscopic paper and starch packaging peanuts were investigated. The novel substrates provided optimal contact between the submerged fungal biomass and the air, and overall, conidial production was directly proportional to the total area of air-to-fungal surface. Technologies based on the starch peanuts and hygroscopic paper were the most productive in comparison to the common technology where the submerged culture was transferred to flat containers. The advantages and disadvantages of these different production methods are discussed.

Interaction between Paracoccidioides brasiliensis conidia and the coagulation system: involvement of fibrinogen

The infectious process starts with an initial contact between pathogen and host. We have previously demonstrated that Paracoccidioides brasiliensis conidia interact with plasma proteins including fibrinogen, which is considered the major component of the coagulation system. In this study, we evaluated the in vitro capacity of P. brasiliensis conidia to aggregate with plasma proteins and compounds involved in the coagulation system. We assessed the aggregation of P. brasiliensis conidia after incubation with human serum or plasma in the presence or absence of anticoagulants, extracellular matrix (ECM) proteins, metabolic and protein inhibitors, monosaccharides and other compounds. Additionally, prothrombin and partial thromboplastin times were determined after the interaction of P. brasiliensis conidia with human plasma. ECM proteins, monosaccharides and human plasma significantly induced P. brasiliensis conidial aggregation; however, anticoagulants and metabolic and protein inhibitors diminished the aggregation process. The extrinsic coagulation pathway was not affected by the interaction between P. brasiliensis conidia and plasma proteins, while the intrinsic pathway was markedly altered. These results indicate that P. brasiliensis conidia interact with proteins involved in the coagulation system. This interaction may play an important role in the initial inflammatory response, as well as fungal disease progression caused by P. brasiliensis dissemination.

Propuesta de un método para evaluar resistencia genética en Schizolobium parahybum (Vell) Blake (Pachaco) frente a Ceratocystis spp: evidencias preliminares de resistencia en Ecuador / Proposal for a method to evaluate genetic resistance in Schizolobium parahybum (Vell) Blake (Pachaco) against ceratocystis spp: preliminary evidence of resistance in Ecuador

Se condujo una investigación en el Trópico Húmedo Ecuatoriano (THE), dirigida a encontrar un método eficiente para evaluar la resistencia genética en árboles de Schizolobium parahybum (pachaco) frente al complejo Ceratocystis: C. paradoxa, C. moniliformis, y C. fimbriata. Se estudiaron dos métodos basados en el empleo de dos tipos de tejidos vegetales: a) tejidos de ramas laterales, y 2) tejidos de corteza fustal. Se emplearon cinco rodales de pachaco, tres de los cuales son considerados de introducción original de la especie forestal al THE desde la amazonía, y dos que son descendientes de los primeros. Los resultados permitieron definir que el método basado en tejidos de corteza fustal, fue el más eficiente y logísticamente viable. La metodología final aplicada, consistió en extraer corteza desde árboles adultos, reducirla a secciones pequeñas de 1,5 cm x 4 cm (6 cm2) y mantenerlas en una cámara húmeda durante 96 horas. Una vez distribuidas las secciones de corteza, se inocularon con 0,45mL-1 de una suspensión calibrada a razón de 30.000 unidades de infección (ascosporas, conidias y micelio). Para la evaluación, se empleó una escala arbitraria de 0 a 4 que permitió estimar el crecimiento de micelio y número de peritecios para cada uno de los hongos. Esta metodología permitió discriminar entre árboles: resistentes (0,0 a 1,0), moderadamente resistentes (1,1 a 2,0), susceptibles (2,1 a 3,0), y muy susceptibles (3,1 a 4,0), lo cual la hace viable para futuros trabajos de selección de individuos y mejoramiento genético de la especie.

A research was conducted in the Humid Tropics of Ecuador (THE), aimed at finding an efficient method to evaluate genetic resistance in Schizolobium parahybum (Pachaco) trees against Ceratocystis complex: C. paradoxa, C. moniliformis and C. fimbriata. We studied two methods based on the use of two types of plant tissues: a) tissue of lateral branches, and 2) stem bark tissues. Five forest of pachaco were used, three of which are considered original introduction of forestry specie to THE from the Amazon, and two who are descendants of the former. The results allowed to define the method based on stem bark tissue was the most efficient and logistically feasible. The final methodology applied, consisted in to remove bark from mature trees, reducing it to small sections of 1.5 cm x 4 cm (6cm2) and maintained in a moist chamber for 96 hours. Once distributed the sections of bark, were inoculated with 0.45mL-1 of a suspension calibrated at a rate of 30.000 units of infection (ascospores, conidia and mycelium). For evaluation, we used an arbitrary scale from 0 to 4, which allowed to estimate the growth of mycelium and perithecia number for each of the fungi. This methodology allows us to discriminate between trees: resistant (0.0 to 1.0), moderately resistant (1.1 to 2.0), susceptible (2.1 to 3.0), and very susceptible (3.1 to 4 , 0), which makes it viable for future selection of individuals and breeding of the forest species.

Cellulase activity in solid state fermentation of palm kernel cake with Trichoderma sp

Aims: The effect of different types of fungal inocula to the cellulase activity measured on palm kernel cake (PKC) was studied. Methodology and Results: Isolate Pro-A1 which was identified as Trichoderma sp. was selected as a potential producer of cellulase via solid state fermentation technique (SSF). Two types of PKCs were used; raw PKC (containing residual oil) and defatted PKC. The PKCs were inoculated with different concentrations of conidia and varying amounts (g) of solid mycelia plugs (SMP) for SSF. The effect of ultrafiltered crude fungal filtrate (CFF) as inocula was also being tested. The highest cellulase activity of 2.454 FPU/mL was detected with 60% (wt/wt) SMP applied to the raw PKC. Conversely, 2.059 FPU/mL of cellulase activity was measured when 80% (wt/wt) of SMP was applied to the defatted PKC which is 62.3% higher than the untreated defatted PKC; and more than 100% increase in enzymatic activity compared to raw PKC. The cellulase activity in the SSF inoculated with 8 x 106 conidia /mL and 12 x 106 conidia /mL were 1.704 FPU/mL for raw PKC and 1.856 FPU/mL for defatted PKC, an enhancement of about 46% from uninoculated batch. Inoculation with CFF bears corresponding maximum improvement of the cellulase activity on both PKCs of 13.58% (raw) and 2.86% (defatted). Conclusion, significance and impact of study: The current study proves that Trichoderma sp. in the form of SMP can enhance the cellulase activity on PKCs effectively with more than 100% increment. Fungal conidia are also a better choice in enhancing cellulase activity of Trichoderma sp. permitted that the PKC used is devoid of oil. From this study, Trichoderma sp. holds the potential of converting lignocellulosic materials into products of commercial and industrial values such as glucose and other biofuels.

Silencing gene of TLR4 down-regulates the effect of TLR2 signal transduction in RAW264.7 cells to anti-Aspergillus fumigatus conidia stimulation / 中华微生物学和免疫学杂志

Objective To study the role of TLR2 and TLR4 signal transduction in RAW264.7 monocyte-macrophages stimulated by Aspergillus fumigatus conidia,and to investigate the expression of TLR2 signal transduction after silencing gene of TLR4.Methods Macrophages were randomly divided into normal group ( N group),normal+stimulated with Aspergillus fumigatus conidia ( N +Af group ),normal + transfected with TLR4-siRNA [ TLR4 (RNAi) group ],normal+transfected with TLR4-siRNA +stimulated with Aspergillus fumigatus conidia[ TLR4(RNAi) +Af group].RT-PCR and Western blot were used to assay expression levels of TLR2,TLR4,MyD88 mRNA and pro-inflammatory cytokines TNF-α protein when macrophages were stimulated 12 h by Aspergillus fumigatus conidia after tranfected 24 h with TLR4-siRNA by technology of RNAi.Results ( 1 ) Compared with N group,the expression of TLR2,TLR4,MyD88 mRNA and TNF-αprotein in N+Af group significantly increased before silencing gene of TLR4.(2) Silencing efficiency of macrophates was up to 83％ after transfected with TLR4-siRNA.(3)The expression of TLR2,MyD88 mRNA in TLR4 (RNAi) group significantly decreased contrast with normal group.Meanwhile the expression of TLR2,MyD88 mRNA and TNF-α protein also obviously reduced in TLR4(RNAi) +Af group when compared with N +Af group.Compared with TLR4 (RNAi) group,the expression of MyD88 mRNA in TLR4 (RNAi) +Af group significantly increased.However,the expression of TLR2 mRNA and TNF-α protein have no significant change after silencing gene of TLR4.Conclusion Signaling pathway of TLR2 and TLR4 in macrophages was activated by given stimulus of Aspergillusfumigatus conidia and exerted the effect of anti-Aspergillus fumigatus spores stimulation through the release of pro-inflammatory cytokines TNF-α.Meanwhile,silencing gene of TLR4 down-regulate the effect of TLR2 signal transduction in RAW264.7 cells to anti-Aspergillus fumigatus conidia stimulation,and it found that TLR4 played an more important role by contrast with TLR2.

Optimization of Solid-State Fermentation for Improved Conidia Production of Beauveria bassiana as a Mycoinsecticide

The production of conidia of entomopathogenic Beauveria bassiana by solid-state fermentation was studied for the development of a biocontrol agent against aphid Myzus persicae. The optimal conditions for conidia production on polished white rice were 40% moisture content, 25degrees C culture temperature, 2-day-old seeding culture grown in 3% corn meal, 2% rice bran, 2% corn steep powder medium, initial conidia concentration of 107 conidia/g in the wet rice, 10% inoculum size, and use of a polyethylene bag as a container. The polyethylene bag containing inoculated rice was hand-shaken every 12 hr during fermentation. Using optimal conditions, the maximum conidia production obtained was 4.05 g conidia/100 g dry rice after 14 days of cultivation, a rate 2.83 times higher than conidia yield of pre-optimization.

Esporulação de Pyricularia grisea em diferentes meios de cultura e regimes de luz / Sporulation of Pyricularia grisea on different culture media and light regimes

Diferentes fatores influenciam o crescimento e a esporulação dos fungos, tais como: composição do meio de cultivo, temperatura e luz. O objetivo do trabalho foi determinar o melhor meio de cultivo e regime de luz para a esporulação de Pyricularia grisea. Um isolado deste, obtido da cultivar de trigo 'BRS 194', foi repicado por disco micelial em oito meios de cultivo (farinha integral de arroz, aveia, centeio, cevada, trigo, triticale; BSA e extrato de tomate) e submetido a três regimes de luz (escuro, fotoperíodo de 12h e luz contínua). As placas permaneceram por 10 dias, em câmara de crescimento sob temperatura de 23-25°C. Em seguida, o número de conídios foi determinado em câmara de Neubauer. O meio de cultivo à base de farinha integral de trigo propiciou a maior esporulação do fungo em todos os regimes de luz, e a maior esporulação ocorreu em regime de luz contínua para todos os meios de cultivos testados. Os meios de arroz e BSA propiciaram uma menor produção de conídios pelo fungo.

A set of conditions is necessary for the growth and sporulation of fungi, particularly the composition of culture media, temperature and light. The objective of this research was to determine the ideal culture medium and light regime for the sporulation of P. grisea. Micelial discs of an isolate, obtained from cultivar 'BRS 194' of wheat, were transferred to eight culture media (rice wholemeal, oat, rye, barley, wheat, triticale, PSA and tomato sauce) and kept in the dark, under continuous light and 12h of light. In a growing chamber, for 10 days at 23-25°C. Then, the number of conidia was determined in a Neubauer chamber. The higher sporulation, regardless of light regime, was observed on wheat wholemeal culture medium. The continuous light provided higher sporulation this fungus in all culture medium. The lowest production of conidia was observed on rice wholemeal and PSA culture media.

Adiaspiromicose pulmonar humana / Human pulmonary adiaspiromycosis

A adiaspiromicose é uma doença fúngica sistêmica que acomete usualmente roedores e raramente atinge o homem. É causada pelo fungo Emmonsia crescens e ocorre após a inalação da forma contagiante (conídios). Embora estas formas não se multipliquem nem se disseminem no organismo humano, induzem uma reação inflamatória crônica granulomatosa de padrão miliar que pode levar a falência respiratória e morte. Apresentamos aqui um caso de adiaspiromicose pulmonar humana em paciente imunocompetente que exibia infiltrado intersticial pulmonar difuso ao exame de imagem e fora diagnosticado mediante biópsia pulmonar.

Adiaspiromycosis is a systemic fungal disease that usually affects rodents and rarely infects humans. It is caused by the fungus Emmonsia crescens and occurs after inhalation of its contagious form (conidia). Although these forms neither multiply nor spread in the human body, they cause a chronic granulomatous inflammatory reaction of miliary pattern, which may lead to respiratory failure and death. In this study we present a case of human pulmonary adiaspiromycosis in an immunocompetent patient that showed a diffuse pulmonary interstitial infiltrate diagnosed by pulmonary biopsy.

Production of Aerial Conidia of Lecanicillium lecanii 41185 by Solid-State Fermentation for Use as a Mycoinsecticide

The production of aerial conidia of Lecanicillium lecanii 41185, a highly virulent fungus, by solid-state fermentation was studied for use as a biocontrol agent against aphids. Among several agro-industrial solid media, steamed polished rice was found to produce the highest amount of aerial conidia. The optimal conditions for aerial conidia production were determined to be a 28.5% moisture content in the rice, 25degrees C culture temperature, rice pH of 6.0, 75% ambient relative humidity, 4-dold seeding culture, 0.6% KNO3, and 12 d of culture time. The conidia yield increased from 5.7 x 10(9) conidia/g polished rice to 18.2 x 10(9) conidia/g polished rice following application of these optimized conditions.

Biological Characteristics of Paecilomyces cicadae LB Strain / 微生物学通报

The effect of carbon source,nitrogen source,temperature,moisture,pH and light on the fungal growth,conidia production and conidia germination of Paecilomyces cicadae LB were studied.For the fun-gal growth and conidia production,the optimum carbon sources were soluble starch and glucose,while the optimum nitrogen source was peptone.For the fungal growth and spore germination of P.cicadae,the opti-mum temperature was 25?C~27?C,for the conidia production,the optimum temperature was 25?C.Conidia geminated at RH 90%~100%,but did not below 90%.When RH reaches to 100% or the conidia were in wa-ter,the germination rate was the highest.The range of pH for the fungal growth,conidia production and co-nidial germination was 4~10,while the optimum pH for the fungal growth was 6 and 6~7 for the conidia production and conidial germination.The light treatment significantly influenced fungal conidia production.The lethal temperature for the fungal conidia was 55?C remaining 10 minutes.The present results suggest the isolate LB can adapt to nutrition and environment more widely,and has greater potential as biological control factor against of pest.

Determinação da variabilidade em isolados de Colletotrichum lindemuthianum por meio de marcadores morfológicos e culturais / Determination of variability in isolates of Colletotrichum lindemuthianum based on morphological and cultural markers

Colletotrichum lindemuthianum (teleomorfo Glomerella cingulata f. sp. phaseoli) apresenta ampla variabilidade genética, demonstrada por suas características morfológicas. Com este trabalho, objetivou-se caracterizar, por meio de marcadores morfológicos, diferentes isolados de C. lindemuthianum e identificar marcadores morfológicos com uso potencial em análises genéticas. Foram avaliados os seguintes caracteres morfológicos e culturais: cor e textura das colônias, compatibilidade vegetativa e sexual, índice de velocidade de crescimento micelial (IVCM), diâmetro colonial (DC), capacidade de esporulação (CE), dimensões e formas conidiais, dimensões dos ascósporos, formação de estruturas reprodutivas e formação de anastomoses entre hifas e conídios. Os resultados obtidos demonstraram que os isolados de C. lindemuthianum possuem ampla variabilidade genética para todas as características avaliadas e que a forma do conídio pode ser usada como marcador morfológico em análises genéticas.

Colletotrichum lindemuthianum (teleomorfo Glomerella cingulata f. sp. phaseoli) presents wide genetic variability, demonstrated by its morphological traits. The objective of this study was to characterize morphological markers in different isolates of C. lindemuthianum and, to identify useful morphological markers in genetic analyses. The following morphological and cultural traits were evaluated: color and texture of the colonies, vegetative and sexual compatibility, micelial growth index (MGI), colonial diameter (CD), esporulation capacity (EC), conidia dimensions and form, ascospores dimensions and formation of reproductive structures. The data showed wide genetic variability for all traits and that conidial form can be used as morphological marker in genetic analysis.