Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Abstract Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

Resumo As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.

Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.

Production of cold-adapted cellulase by Verticillium sp. isolated from Antarctic soils

Background: Cellulose can be converted to ethanol by simultaneous saccharification and fermentation (SSF). The difference between the optimal temperature of cellulase and microbial fermentation, however, has been identified as the critical problem with SSF. In this study, one fungal strain (AnsX1) with high cellulase activity at low temperature was isolated from Antarctic soils and identified as Verticillium sp. by morphological and molecular analyses. Results: The biochemical properties of crude AnsX1 cellulase samples were studied by filter paper cellulase assay. The maximum cellulase activity was achieved at low temperature in an acidic environment with addition of metal ions. Furthermore, AnsX1 cellulase demonstrated 54-63% enzymatic activity at ethanol concentrations of 5-10%. AnsX1 cellulase production was influenced by inoculum size, carbon and nitrogen sources, and elicitors. The optimal culture conditions for AnsX1 cellulase production were 5% inoculum, wheat bran as carbon source, (NH4)2SO4 as nitrogen source, and sorbitol added in the medium. Conclusions: Our present work has potential to enable the development of an economic and efficient cold-adapted cellulase system for bioconversion of lignocellulosic biomass into biofuels in future.

Evaluación de la virulencia de hongos entomopatógenos del pulgón del ciprés, de dos regiones ecológicas de Chile. II. / Evaluation of the virulence of entomopathogenic fungi of cypress aphid, from two ecological regions of Chile. II

El pulgón del ciprés Cinara cupressi, se considera uno de los insectos invasores más dañinos del mundo, ha provocado una gran mortalidad en especies Cupresáceas nativas y exóticas en varios países. En Chile, fue detectado el año 2003 y para el año 2008 esta plaga ya se encontraba distribuida en todo el país, afectando especies cupresáceas forestales exóticas como Cupressus macrocarpa y especies nativas, tales como, Austrocedrus chilensis (Ciprés de la Cordillera) y Fitzroya cupressoides (Alerce), que están categorizadas en el estado de conservación vulnerable y en peligro respectivamente. El área forestal de Chile ha potenciado y privilegiado el uso de controladores biológicos como parte del manejo integrado de plagas. Es por esto que se ha utilizado el parasitoide Pauesia juniperorum. Sin embargo, se han detectado bajos niveles de parasitismo. Se propone el uso de hongos entomopatógenos como alternativa y complemento al control biológico. Se prospectaron hongos entomopatógenos en la Región ecológica mediterránea per-húmeda y en la Región oceánica con influencia mediterránea. Como resultado se han identificado seis géneros de hongos descritos como patógenos de insectos. Sin embargo, bajo condiciones de laboratorio sólo cepas del género Verticillium fueron virulentas, causando sobre el 80 por ciento de mortalidad acumulada a los 7 días. No hubo diferencias significativas entre las cepas de Verticillium y un producto químico (pirimicarb) aplicado como control, aunque el hongo fue más lento. Las cepas más virulentas fueron identificadas como Verticillium lecanii, Ve 1 y Ve 2, con un TL50 de: 3.2 y 3.1 días y un DL50 : 1.24 y 1.37 conidias mL-1 respectivamente, sugiriendo el uso de estos hongos para controlar C. cupressi en Chile.

The cypress aphid, Cinara cupressi, is considered one of the most important invasive species causing high mortality in exotic and native species of Cupressaceae in several countries in the world. In Chile it was detected in 2003 and in 2008 was distributed throughout the country affecting the exotic forest species Cupressus macrocarpa and the native forest species Austrocedrus chilensis (Ciprés de la Cordillera) and Fitzroya cupressoides (Alerce), both classified as vulnerable and endangered species respectively. Efforts to their management have focused on biological control by using the parasitoid Pauesia juniperorum but until now it has not reached satisfactory control. We propose using entomopathogenic fungi, as alternative and complementary biocontrol. Entomopathogenic fungi were prospected in the ecological region Mediterranean per-humid and in the ecological region Oceanic with mediterranean influence in both colonies of C. cupressi. There were identified six genera of fungi described as insect pathogens. However, in laboratory assays only Verticillium strains were virulent, causing about 80 percent cumulative mortality at seven days. There were not significant differences among strains of Verticillium and chemical (pirimicarb) applied as control, although fungi were slower. The most virulent strains were two Verticillium lecanii, Ve 1 and Ve 2, with an LT50 of: 3.2 and 3.1 days and LD50 : 1.24 and 1.37 conidia mL-1 respectively, suggesting the use of these fungi to control C. cupressi in Chile.

Prospección de hongos entomopatógenos del pulgón del ciprés en dos regiones ecológicas de Chile: I / Prospection of entomopathogenic fungi of cypress aphid in two ecological regions of Chile: I

El pulgón del ciprés (Cinara cupressi), es una plaga exótica distribuida en todo el territorio continental chileno que ha provocado daños importantes tanto enespecies forestales introducidas como nativas. Se han estudiado algunos aspectos de la biología de estepulgón, mediante control biológico y productos químicos, los cuales no presentan actualmente resultadossatisfactorios. Durante el tiempo que esta plaga se ha establecido en Chile, no se ha realizado un estudio sistemático de hongos entomopatógenos asociados aeste pulgón, aunque se cuenta con antecedentes de un control promisorio en pulgones con estos agentes enotros países. Con el objetivo inicial de aislar en una primera fase del presente estudio hongos patogénicospotenciales para el control biológico de C. cupressi, iniciamos su búsqueda en dos regiones ecológicas del sur de Chile (Región ecológica Mediterránea Per-Húmeda y Región ecológica Oceánica con influencia Mediterránea), seleccionándose en cada una de ellas 6sitios de muestreo donde en cada uno se recolectaron 10 ramas con colonias de pulgones que fueron mantenidas en una cámara bioclimática (20 +/- 2 °C, 16:8 h) por 7 días, para estimular el desarrollo fúngico. Se aislaron integrantes de lo géneros Verticillium, Paecilomyces(ambos con mayor frecuencia de presencia), Fusarium y un entomophthoral en estudio.

The cypress aphid (Cinara cupressi) is an exotic plague distributed throughout the Chilean continental territory which has caused significant damage both inintroduced forest species as well as in native ones. Some characteristics as to the biology of this aphid have beenstudied by means of biological control and chemicals yet they have not revealed any satisfactory results up tonow. During the time of occurrence of this plague in Chile systematic study on entomopathogenic fungi associated to this aphid has failed to be accomplishedalthough information about a promissory control in aphids with these agents has been reported in other countries. In order to carry out an isolation of potential pathogenic fungi for the biological control of C.cupressiin the first phase of this present research, we began to look for them in two ecological regions from southernChile (Ecological Mediterranean Per-humid Region and Ecological Oceanic Region having Mediterraneaninfluence). Six sampling sites were selected in each of them while 10 branches infected with aphid colonies were kept in a bioclimatic chamber (20+2ºC, 16:8 h) for 7 days to stimulate fungal growth. Fungi of generaVerticillium, Paecilomyces (both showing the highest occurrence frequency), Fusarium and an entomophtoral under study were isolated.

Isolation and characterization of a chitinase gene from entomopathogenic fungus Verticillium lecanii / Isolamento e caracterização de um gene de quitinase do fungo entomopatogênico Verticillium lecanii

Entomopathogenic fungus Verticillium lecanii is a promising whitefly and aphid control agent. Chitinases secreted by this insect pathogen have considerable importance in the biological control of some insect pests. An endochitinase gene Vlchit1 from the fungus was cloned and overexpressed in Escherichia coli. The Vlchit1 gene not only contains an open reading frame (ORF) which encodes a protein of 423 amino acids (aa), but also is interrupted by three short introns. A homology modelling of Vlchit1 protein showed that the chitinase Vlchit1 has a (α/β)8 TIM barrel structure. Overexpression test and Enzymatic activity assay indicated that the Vlchit1 is a functional enzyme that can hydrolyze the chitin substrate, so the Vlchit1 gene can service as a useful gene source for genetic manipulation leading to strain improvement of entomopathogenic fungi or constructing new transgenic plants with resistance to various fungal and insects pests.

O fungo entomopatogênico Verticillium lecanii é um agente promissor no controle da mosca-branca e do pulgão. As quitinases secretadas por esse patógeno de insetos têm uma grande importância no controle biológico de doenças causadas por insetos. Um gene de endoquitinase Vlchit1 desse fungo foi clonado e expresso em Escherichia coli. O gene Vlchit contém não apenas um ORF que codifica uma proteína de 423 aminoácidos, mas também é interrompido por três pequenos introns. A modelagem de homologia da proteína Vlchit1indicou que a quitinase Vlchit1 tem uma estrutura (α/β) 8 TIM barrel. Testes de expressão e de atividade enzimática indicaram que Vlchit1 é uma enzima funcional que hidroliza quitina, portanto o gene Vlchit pode ser um gene útil para manipulação genética para melhoramento de cepas de fungos entomopatogênicos ou para a construção de novas plantas transgênicas com resistência a várias doenças causadas por fungos e insetos.