Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt

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.

Filtrates from cultures of endophytic fungi isolated from leaves of Copaifera oblongifolia (Fabaceae) affect germination and seedling development differently

Abstract Interactions between endophytic fungi (EFs) and their host plants range from positive to neutral to negative. The results of such interactions can vary depending on the organ of the infected host plant. EFs isolated from the leaves of some species of plants have potential for use as agents to inhibit seed germination and control invasive plants. The objectives of this study were to identify EFs present in the leaves of Copaifera oblongifolia and to evaluate the role of these fungi in seed germination and seedling development. A total of 11 species of EFs were isolated, which were identified using the internal transcribed spacers (ITS) sequence of the nuclear ribosomal DNA. The isolated species of EFs are generalists and probably are transmitted horizontally. Laboratory tests revealed that filtrates of these fungal isolates differently affect seed germination and seedling development of C. oblongifolia. The species Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum and Phomopsis sp. negatively affected seed germination, with N. parvum standing out for its negative effects, inhibiting seedling germination and survival in 89 and 222%, respectively. In addition, Cochliobolus intermedius negatively affected seedling development. Thus, the combined use of N. parvum and C. intermedius, or products from the metabolism of these microorganisms, in the control of invasive plants deserves attention from future studies.

Resumo As interações entre fungos endofíticos (FEs) e suas plantas hospedeiras variam de positivas, neutras a negativas. Os resultados destas interações podem variar dependendo do órgão da planta hospedeira infectada. FEs isolados de folhas de algumas espécies de plantas têm potencial para serem usados como agentes inibidores da germinação de sementes e no controle de plantas invasoras. Os objetivos deste estudo foram identificar os FEs presentes nas folhas de Copaifera oblongifolia e avaliar o papel destes fungos na germinação das sementes e no desenvolvimento das plântulas. Um total de 11 espécies de FEs foi isolado das folhas de C. oblongifolia e identificado através da sequência dos espaçadores internos transcritos do DNA ribossomal nuclear. As espécies de FEs isoladas são generalistas e provavelmente devem ser transmitidas horizontalmente. Os resultados dos testes de germinação mostraram que filtrados destes isolados fúngicos podem afetar diferentemente a germinação das sementes e o desenvolvimento das plântulas de C. oblongifolia. As espécies Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum e Phomopsis sp. afetaram negativamente a germinação das sementes de C. oblongifolia. Dentre estas espécies devemos destacar que N. parvum reduziu a germinação e a sobrevivência das plântulas em 89 e 222%, respectivamente. Além disso, Cochiliobolus intermedius afetou negativamente o desenvolvimento das plântulas. Assim, o uso combinado de N. parvum e C. intermedius, ou de produtos do metabolismo destas espécies de fungos, têm potencial para serem usados no manejo de plantas invasoras.

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.(AU)

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.(AU)

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.

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.

Filtrates from cultures of endophytic fungi isolated from leaves of Copaifera oblongifolia (Fabaceae) affect germination and seedling development differently / Fungos endofíticos isolados de folhas de Copaifera oblongifolia (Fabaceae) afetam diferentemente a germinação e o desenvolvimento das plântulas

Abstract Interactions between endophytic fungi (EFs) and their host plants range from positive to neutral to negative. The results of such interactions can vary depending on the organ of the infected host plant. EFs isolated from the leaves of some species of plants have potential for use as agents to inhibit seed germination and control invasive plants. The objectives of this study were to identify EFs present in the leaves of Copaifera oblongifolia and to evaluate the role of these fungi in seed germination and seedling development. A total of 11 species of EFs were isolated, which were identified using the internal transcribed spacers (ITS) sequence of the nuclear ribosomal DNA. The isolated species of EFs are generalists and probably are transmitted horizontally. Laboratory tests revealed that filtrates of these fungal isolates differently affect seed germination and seedling development of C. oblongifolia. The species Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum and Phomopsis sp. negatively affected seed germination, with N. parvum standing out for its negative effects, inhibiting seedling germination and survival in 89 and 222%, respectively. In addition, Cochliobolus intermedius negatively affected seedling development. Thus, the combined use of N. parvum and C. intermedius, or products from the metabolism of these microorganisms, in the control of invasive plants deserves attention from future studies.

Resumo As interações entre fungos endofíticos (FEs) e suas plantas hospedeiras variam de positivas, neutras a negativas. Os resultados destas interações podem variar dependendo do órgão da planta hospedeira infectada. FEs isolados de folhas de algumas espécies de plantas têm potencial para serem usados como agentes inibidores da germinação de sementes e no controle de plantas invasoras. Os objetivos deste estudo foram identificar os FEs presentes nas folhas de Copaifera oblongifolia e avaliar o papel destes fungos na germinação das sementes e no desenvolvimento das plântulas. Um total de 11 espécies de FEs foi isolado das folhas de C. oblongifolia e identificado através da sequência dos espaçadores internos transcritos do DNA ribossomal nuclear. As espécies de FEs isoladas são generalistas e provavelmente devem ser transmitidas horizontalmente. Os resultados dos testes de germinação mostraram que filtrados destes isolados fúngicos podem afetar diferentemente a germinação das sementes e o desenvolvimento das plântulas de C. oblongifolia. As espécies Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum e Phomopsis sp. afetaram negativamente a germinação das sementes de C. oblongifolia. Dentre estas espécies devemos destacar que N. parvum reduziu a germinação e a sobrevivência das plântulas em 89 e 222%, respectivamente. Além disso, Cochiliobolus intermedius afetou negativamente o desenvolvimento das plântulas. Assim, o uso combinado de N. parvum e C. intermedius, ou de produtos do metabolismo destas espécies de fungos, têm potencial para serem usados no manejo de plantas invasoras.

Filtrates from cultures of endophytic fungi isolated from leaves of Copaifera oblongifolia (Fabaceae) affect germination and seedling development differently / Fungos endofíticos isolados de folhas de Copaifera oblongifolia (Fabaceae) afetam diferentemente a germinação e o desenvolvimento das plântulas

Interactions between endophytic fungi (EFs) and their host plants range from positive to neutral to negative. The results of such interactions can vary depending on the organ of the infected host plant. EFs isolated from the leaves of some species of plants have potential for use as agents to inhibit seed germination and control invasive plants. The objectives of this study were to identify EFs present in the leaves of Copaifera oblongifolia and to evaluate the role of these fungi in seed germination and seedling development. A total of 11 species of EFs were isolated, which were identified using the internal transcribed spacers (ITS) sequence of the nuclear ribosomal DNA. The isolated species of EFs are generalists and probably are transmitted horizontally. Laboratory tests revealed that filtrates of these fungal isolates differently affect seed germination and seedling development of C. oblongifolia. The species Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum and Phomopsis sp. negatively affected seed germination, with N. parvum standing out for its negative effects, inhibiting seedling germination and survival in 89 and 222%, respectively. In addition, Cochliobolus intermedius negatively affected seedling development. Thus, the combined use of N. parvum and C. intermedius, or products from the metabolism of these microorganisms, in the control of invasive plants deserves attention from future studies.(AU)

As interações entre fungos endofíticos (FEs) e suas plantas hospedeiras variam de positivas, neutras a negativas. Os resultados destas interações podem variar dependendo do órgão da planta hospedeira infectada. FEs isolados de folhas de algumas espécies de plantas têm potencial para serem usados como agentes inibidores da germinação de sementes e no controle de plantas invasoras. Os objetivos deste estudo foram identificar os FEs presentes nas folhas de Copaifera oblongifolia e avaliar o papel destes fungos na germinação das sementes e no desenvolvimento das plântulas. Um total de 11 espécies de FEs foi isolado das folhas de C. oblongifolia e identificado através da sequência dos espaçadores internos transcritos do DNA ribossomal nuclear. As espécies de FEs isoladas são generalistas e provavelmente devem ser transmitidas horizontalmente. Os resultados dos testes de germinação mostraram que filtrados destes isolados fúngicos podem afetar diferentemente a germinação das sementes e o desenvolvimento das plântulas de C. oblongifolia. As espécies Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum e Phomopsis sp. afetaram negativamente a germinação das sementes de C. oblongifolia. Dentre estas espécies devemos destacar que N. parvum reduziu a germinação e a sobrevivência das plântulas em 89 e 222%, respectivamente. Além disso, Cochiliobolus intermedius afetou negativamente o desenvolvimento das plântulas. Assim, o uso combinado de N. parvum e C. intermedius, ou de produtos do metabolismo destas espécies de fungos, têm potencial para serem usados no manejo de plantas invasoras.(AU)

Filtrates from cultures of endophytic fungi isolated from leaves of Copaifera oblongifolia (Fabaceae) affect germination and seedling development differently / Fungos endofíticos isolados de folhas de Copaifera oblongifolia (Fabaceae) afetam diferentemente a germinação e o desenvolvimento das plântulas

Interactions between endophytic fungi (EFs) and their host plants range from positive to neutral to negative. The results of such interactions can vary depending on the organ of the infected host plant. EFs isolated from the leaves of some species of plants have potential for use as agents to inhibit seed germination and control invasive plants. The objectives of this study were to identify EFs present in the leaves of Copaifera oblongifolia and to evaluate the role of these fungi in seed germination and seedling development. A total of 11 species of EFs were isolated, which were identified using the internal transcribed spacers (ITS) sequence of the nuclear ribosomal DNA. The isolated species of EFs are generalists and probably are transmitted horizontally. Laboratory tests revealed that filtrates of these fungal isolates differently affect seed germination and seedling development of C. oblongifolia. The species Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum and Phomopsis sp. negatively affected seed germination, with N. parvum standing out for its negative effects, inhibiting seedling germination and survival in 89 and 222%, respectively. In addition, Cochliobolus intermedius negatively affected seedling development. Thus, the combined use of N. parvum and C. intermedius, or products from the metabolism of these microorganisms, in the control of invasive plants deserves attention from future studies.

As interações entre fungos endofíticos (FEs) e suas plantas hospedeiras variam de positivas, neutras a negativas. Os resultados destas interações podem variar dependendo do órgão da planta hospedeira infectada. FEs isolados de folhas de algumas espécies de plantas têm potencial para serem usados como agentes inibidores da germinação de sementes e no controle de plantas invasoras. Os objetivos deste estudo foram identificar os FEs presentes nas folhas de Copaifera oblongifolia e avaliar o papel destes fungos na germinação das sementes e no desenvolvimento das plântulas. Um total de 11 espécies de FEs foi isolado das folhas de C. oblongifolia e identificado através da sequência dos espaçadores internos transcritos do DNA ribossomal nuclear. As espécies de FEs isoladas são generalistas e provavelmente devem ser transmitidas horizontalmente. Os resultados dos testes de germinação mostraram que filtrados destes isolados fúngicos podem afetar diferentemente a germinação das sementes e o desenvolvimento das plântulas de C. oblongifolia. As espécies Curvularia intermedia, Neofusicoccum parvum, Pseudofusicoccum stromaticum e Phomopsis sp. afetaram negativamente a germinação das sementes de C. oblongifolia. Dentre estas espécies devemos destacar que N. parvum reduziu a germinação e a sobrevivência das plântulas em 89 e 222%, respectivamente. Além disso, Cochiliobolus intermedius afetou negativamente o desenvolvimento das plântulas. Assim, o uso combinado de N. parvum e C. intermedius, ou de produtos do metabolismo destas espécies de fungos, têm potencial para serem usados no manejo de plantas invasoras.

Analysis of microbial diversity in the root of Astragalus mongholicus / Análise da diversidade microbiana em raízes de astrágalo

The dry root of Astragalus mongholicus has therapeutic effects such as tonifing the middle - jiao, replenishing qi, solidifing the surface, promoting diuresis, dispelling sepsis outwards and nourishing muscle. There are some slices having black spots after slicing the root of astragalus. The diversity of endophytic fungi between slices with black spots and normal slices was analysed in this paper. The endophytic fungal sequences obtained by high-throughput sequencing were 298,044 and 297,396, and the 116 OTU subsets obtained after clustering belonged to 3 phyla, 9 classes, 22 orders, 38 families and 46 genera. The dominant classes were Eurotiomycetes and Leotiomycetes. The dominant order is Eurotiales and Helotiales. The dominant families are Helotiales_fam_Incertae_sedis and Aspergillaceae. The dominant genera are Cadophora and Aspergillus. There are some peculiar fungal flora in both normal slices and spotted slices. The study on endophytic fungi diversity of astragalus slices will provide some help for drug development of this plant.

A raiz seca de astrágalo tem a função de tonificar o zhongjiao, qi benéfico, superfície sólida, diurético, remoção externa de veneno e fortalecimento muscular. Depois de cortar o astrágalo em fatias finas, algumas fatias têm pontos pretos. Neste trabalho foi analisada a diversidade de fungos endofíticos em fatias de ponto negro versus fatias normais. O sequenciamento de alto rendimento resultou em 314.998 e 315.051 sequências de fungos endofíticos, respectivamente, que após agrupamento resultaram em 116 subgrupos de OTU, pertencentes a três filos, nove classes, 22 ordens, 38 famílias e 46 gêneros. As classes dominantes são Eurotiomycetes e Leotiomycetes. A ordem principal é euclides e hilllows. As famílias predominantes foram aspergilaceae e aspergilaceae. Os gêneros predominantes foram leucostilla e aspergillus. Tanto as seções normais como as secções manchadas apresentam uma flora fúngica distinta. O estudo da diversidade de fungos endofíticos em fatias de astrágalo pode contribuir para o desenvolvimento de fármacos para esta planta.

Endophytic bacteria Bacillus safensis and Pseudomonas hibiscicola and their ability to increase rice seedling growth

Endophytic bacteria Bacillus safensis RS95 and Pseudomonas hibiscicola RS121 were evaluated for their ability to promote the growth of rice seedlings and produce indole-acetic acid (IAA) and siderophores and to solubilize phosphates. 'Guri' rice seeds were immersed in bacterial endophyte cell suspensions (separated and two-strain mixed), as well as in Escherichia coli DH5α, phosphate-buffered saline (PBS) and water treatments (negative controls). Seeds were sown on agar-water in Petri plates placed vertically at an angle of 65°. The ability of plant growth-promoting endophytic bacteria (PGPEB) to produce IAA and siderophores was determined by Salkowski colorimetric and chrome azurol S (CAS) assays, respectively. Mineral phosphate solubilization activity was calculated by inoculating the endophytes onto medium containing insoluble phosphate. PGPEB showed a positive effect on the growth of rice seedlings, causing a mean growth of shoots and primary-roots of 60 and 67%, respectively. Bacterial strains also showed positive traits for IAA and siderophore production, as well as phosphate-solubilization activity

Metagenomic study of the communities of bacterial endophytes in the desert plant Senna Italica and their role in abiotic stress resistance in the plant / Estudo metagenômico das comunidades de endófitos bacterianos na planta do deserto Senna Italica e seu papel na resistência ao estresse abiótico na planta

Plant leaves and roots are home to diverse communities of bacteria, which play a significant role in plant health and growth. Although one of the most unfriendly environments for plant growth is deserts, desert plants can influence their surrounding microbial population and choose favorable bacteria that encourage their growth under these severe circumstances. Senna italica is known for its excellent medicinal values as a traditional medical plant, but little is known about its associated endophytic bacterial community under extreme conditions. In the present study, metagenomic sequencing of 16S rRNA was used to report the diversity of endophytic bacterial communities associated with the leaves and roots of the desert medicinal plant Senna italica that was collected from the Asfan region in northeast Jeddah, Saudi Arabia. Analyses of the 16S rRNA sequences at the taxonomic phylum level revealed that bacterial communities in the roots and leaves samples belonged to five phyla, including Cyanobacteria, Proteobacteria, Actinobacteria, Firmicutes, and unclassified phyla. Results indicated that the most common phyla were Cyanobacteria/Chloroplast and Actinobacteria. Analysis of the 16S rRNA sequences at the taxonomic phylum level revealed that bacterial communities in the roots and leaves samples belonged to twelve genera at the taxonomic genus level. The most abundant ones were highlighted for further analysis, including Okibacterium and Streptomyces found in Actinobacteria, which were the dominant genus in roots samples. However, Streptophyta found in Cyanobacteria/Chloroplast was the dominant genus in leaf samples. Metagenomic analysis of medicinal plants leads to identifying novel organisms or genes that may have a role in abiotic stress resistance in the plant. The study of endophytic microbiome taxonomic, phylogenetic, and functional diversity will better know innovative candidates that may be selected as biological agents to enhance agricultural and industrial processes, especially for crop desert agricultural improvement.

As folhas e raízes das plantas abrigam diversas comunidades de bactérias, que desempenham um papel significativo na saúde e no crescimento das plantas. Embora um dos ambientes mais hostis para o crescimento de plantas sejam os desertos, as plantas do deserto podem influenciar a população microbiana circundante e escolher bactérias favoráveis ​​que encorajem seu crescimento sob essas circunstâncias severas. Senna italica é conhecida por seus excelentes valores medicinais como planta medicinal tradicional, mas pouco se sabe sobre sua comunidade bacteriana endofítica associada em condições extremas. No presente estudo, o sequenciamento metagenômico de 16S rRNA foi usado para relatar a diversidade de comunidades bacterianas endofíticas associadas às folhas e raízes da planta medicinal do deserto Senna italica que foi coletada na região de Asfan no nordeste de Jeddah, Arábia Saudita. Análises das sequências de rRNA 16S no nível taxonômico do filo revelaram que as comunidades bacterianas nas amostras de raízes e folhas pertenciam a cinco filos, incluindo Cyanobacteria, Proteobacteria, Actinobacteria, Firmicutes e filos não classificados. Os resultados indicaram que os filos mais comuns foram Cyanobacteria/Cloroplast e Actinobacteria. A análise das sequências de rRNA 16S no nível taxonômico do filo revelou que as comunidades bacterianas nas amostras de raízes e folhas pertenciam a doze gêneros no nível taxonômico de gênero. Os mais abundantes foram destacados para análise posterior, incluindo Okibacterium e Streptomyces encontrados em Actinobacteria, que foram os gêneros dominantes nas amostras de raízes. No entanto, Streptophyta encontrado em Cyanobacteria/Chloroplast foi o gênero dominante nas amostras de folhas. A análise metagenômica de plantas medicinais leva à identificação de novos organismos ou genes que podem ter um papel na resistência ao estresse abiótico na planta. O estudo da diversidade taxonômica, filogenética e funcional do microbioma endofítico conhecerá melhor os candidatos inovadores que podem ser selecionados como agentes biológicos para melhorar os processos agrícolas e industriais, especialmente para o melhoramento agrícola do deserto.

Alternative use of endocavitary probe to guide minimally invasive partial nephrectomy: is it reasonable?

Purpose: To describe the use of endocavitary ultrasound probe as an auxiliary tool when performing partial nephrectomy in cases of endophytic renal tumors, to standardize the method, and to report the preliminary results achieved with this technique. Methods: Fifteen patients diagnosed with completely endophytic underwent partial nephrectomy with the use of an endocavitary ultrasound probe. This article describes the technique involved in partial nephrectomy and details the preparation of the endocavitary ultrasound probe to ensure its safe use. Results: All the patients had a RENAL score between 8 and 11. The median time of warm ischemia was 26 and 18 minutes for laparoscopic or robot-assisted surgery, respectively. The median duration of surgery was 150 minutes, and the median console time was 145 minutes for the laparoscopic and robot-assisted surgery groups, respectively. The median estimate of blood loss was 200 mL. Only three patients in the laparoscopic group had focal positive surgical margins. There were no cases of infection at the site of probe entry. Conclusions: Intraoperative use of an endocavitary ultrasound probe for partial nephrectomy is possible and a safe alternative to the excision of endophytic tumors when neither robotic probes nor laparoscopic probes are available.

Microorganisms from corn stigma with biocontrol potential of Fusarium verticillioides / Microrganismos do estigma do milho com potencial de biocontrole de Fusarium verticillioides

The mycotoxigenic fungus Fusarium verticillioides is the primary maize pathogen and causes the maize stalk and ear rot diseases with significant economic losses. Furthermore, the excessive use of fungicides to control F. verticillioides constitutes threats to the environment and human health. Thus, sustainable alternatives such as biological control are needed to minimize the hazards associated with the current method. Although much is known about the vulnerability of the maize silks as a gateway for several fungal pathogens invading the developing grains, studies on the chemical properties of silk extracts and their resident microbiota are scarce. This study isolated and characterized bacteria and fungi that colonize the maize stigma to assess new potential biocontrol agents. The samples were collected from maize fields in the Brazilian localities of Sete Lagoas-MG, Sidrolândia-MS, Sertaneja-PR, and Goiânia-GO. One hundred sixty-seven microorganisms were isolated, 46% endophytic and 54% epiphytic. First, the antagonist activity was evaluated by the agar disc diffusion method performed in triplicate, and 83% of the isolates showed antagonist activity against F. verticillioides. Then, the 42 most efficient isolates were identified based on the partial sequencing of the bacterial 16S rRNA gene and fungi ITS region. The bacteria belong to the genera Bacillus (57.1%), Burkholderia (23.8%), Achromobacter (7.1%), Pseudomonas (2.4%), and Serratia (2.4%), while the fungi are Penicillium (2.4%), Candida (2.4), and Aspergillus (2.4%). The results showed that microorganisms from maize stigma might represent new promising agents for F. verticillioides control.

O fungo micotoxigênico Fusarium verticillioides é o principal patógeno do milho e causa doenças do colmo e da podridão da espiga com perdas econômicas significativas. Além disso, o uso excessivo de fungicidas no controle de F. verticillioides constitui uma ameaça ao meio ambiente e à saúde humana. Assim, alternativas sustentáveis, como o controle biológico, são necessárias para minimizar os riscos associados ao método atual. Este estudo isolou e caracterizou bactérias e fungos que colonizam o estigma do milho para avaliar novos agentes de biocontrole em potencial. As amostras foram coletadas em campos de milho nas localidades brasileiras de Sete Lagoas-MG, Sidrolândia-MS, Sertaneja-PR e Goiânia-GO. Cento e sessenta e sete microrganismos foram isolados, 46% endofíticos e 54% epifíticos. O teste de antagonismo empregando a técnica de disco de difusão em meio sólido, mostrou que 83% dos isolados apresentaram atividade antagonista contra F. verticillioides. Em seguida, 42 isolados mais eficientes foram identificados a partir do sequenciamento parcial do gene 16S rRNA bacteriano e da região ITS de fungos. Os isolados bacterianos pertencem ao gênero Bacillus (57,1%), Burkholderia (23,8%), Achromobacter (7,1%), Pseudomonas (2,4%) e Serratia (2,4%), enquanto os fungos são Penicillium (2,4%), Candida (2.4), e Aspergillus (2,4%). Os resultados mostraram que microrganismos do estigma do milho podem representar novos agentes promissores para o controle de F. verticillioides.

Evaluation of the incidence of endophytic fungi in leaves, stems and roots of bean plants in the State of Paraná / Avaliação da incidência de fungos endofíticos em folhas, caules e raízes de plantas de feijão no Estado do Paraná

The present study aimed to isolate endophilic fungi from plant tissues (leaves, stems and roots) of beans, in addition to identifying them at the genus level. The experiment was conducted at IDR-Londrina. Bean samples were collected at the Londrina Research Station and at UENP-Bandeirantes. The leaves, stems and roots were separated; fragments of 5 mm diameter were cut and disinfested. They were later transferred to BDA culture medium. The plates were incubated at room temperature for seven days for growth of the microorganisms present. The presence and absence, colonization rate (TC) for each tissue and gender identification of fungi were evaluated. The experiment was conducted in a completely randomized design, with five replications. Forty-eight endophilic isolates were found, 13 in root, 17 in stem and 18 in leaf. CT ranged from 17-100% (leaf), 17-83% (stem) and 16-100% (root). The genus Alternaria sp. and Cladosporium sp. have greater specificity with the leaf and stem, Penicillium sp. root and stem. The genera Fusarium sp. and Aspergillus sp. are found in all organs, Curvularia sp., Nigrospora sp. and Thichoderma sp. demonstrated leaf specificity, Macrophomina sp. the root, and Colletotrichum sp. to the stem. Thus, the identified fungi present potential for future studies in their application in the biocontrol of various plant diseases.

O presente estudo teve como objetivo realizar o isolamento de fungos endofíticos dos tecidos vegetais (folhas, caules e raízes) de feijão, além de identifica-los a nível de gênero. O experimento foi conduzido no IDR-Londrina. Amostras de feijão foram coletadas na Estação de Pesquisa de Londrina e na UENP-Bandeirantes. Foram separadas as folhas, caules e raízes, cortados fragmentos de 5 mm diâmetro e desinfestados. Posteriormente foram transferidos para meio de cultura BDA. As placas foram incubadas à temperatura ambiente por sete dias, para crescimento dos microrganismos presentes. Foram avaliados a presença e ausência, taxa de colonização (TC) para cada tecido e identificação a nível de gênero dos fungos. O experimento foi conduzido em delineamento inteiramente casualizado, com cinco repetições. Encontrou-se 48 isolados endofíticos, sendo 13 em raiz, 17 em caule e 18 em folha. A TC variou de 17-100% (folha), de 17-83% (caule) e 16-100% (raiz). O gênero Alternaria sp. e Cladosporium sp. apresentam especificidade maior com a folha e caule, o Penicillium sp. com raiz e caule. Os gêneros Fusarium sp. e Aspergillus sp. são encontrados em todos os órgãos, Curvularia sp., Nigrospora sp. e Thichoderma sp. demonstraram especificidade em folha, Macrophomina sp. a raiz, e Colletotrichum sp. ao caule. Dessa forma, os fungos identificados apresentam-se com potencial para futuros estudos em sua aplicação no biocontrole de diversas doenças de plantas.

Estudo de métodos naturais para redução de doenças fúngicas em sementes nativas da região Amazônica / Study of natural methods for reducing fungal diseases in native seeds in the Amazon region

Existe uma preocupação na produção de alimentos com a segurança sanitária desde o processo de cultivo até a sua expedição aos centros comerciais. Uma das maiores contaminações em sementes por suas características físicas e químicas, é a contaminação fúngica devida à acidez e umidade dos frutos. As doenças fúngicas entre elas, antracnose, vassoura de bruxa e podridão parda são consideradas importante doenças em pós colheita, ocorrendo, principalmente, sob condições de alta umidade relativa do ar e temperaturas elevadas (26ºC a 28ºC). Dentre as sementes da região Amazônica, destacam-se sementes de (cacau- Theobroma cacao L., pupunha- Bactris gasipaes, tucumã da Amazonas-Astrocaryum aculeatum), que apresentam problemas de contaminação fúngica durante o sistema de cultivo. Métodos de biocontroles com ação antimicrobiana: bactérias endofíticas, rizobactérias e fungo Trichoderma ssp, entre outros estão sendo estudados para a redução destas contaminações fúngicas em estudos in vitro e no campo. O objetivo deste trabalho é apresentar uma revisão bibliográfica sobre a utilização de métodos de biocontrole como uma alternativa promissora no manejo de doenças de plantas na fase de pós-colheita, com excelentes resultados em culturas de grande importância econômica. Assim novas alternativas ecologicamente sustentáveis demonstram a possibilidade de os produtos estudados serem utilizados no manejo da antracnose na pós-colheita.(AU)

There is a concern in food production with health security from cultivation process to its dispatch to redistribution centers. One of the biggest contaminations in seeds due to their physical and chemical characteristics, is the fungal contamination due to the acidity and humidity of the fruits. Fungal diseases including anthracnose, bruca's broom and brownrot are considered important diseases in post-harvest, occurring mainly under conditions of high relative humidity and high temperatures (26ºC to 28ºC). Among the seeds of the Amazon region, seeds of de (cacau- Theobroma cacao L., pupunha- Bactris gasipaes, tucumã da Amazonas- Astrocaryum aculeatum ), that present problems fungals contamination during in their growing cultures sistems. Biocontrol methods with antimicrobial action: endophytic bacteria, rhizobacteria and the fungus Trichoderma ssp, among others, are being studied to reduce these fungal contaminations in in vitro and field studies. The objective of this work is to present a bibliographic review on the use of biocontrol methods as an alternative that is promising in the management of plant diseases in the post-harvest phase, with excellent results in crops of great economic importance. Thus, new ecologically sustainable alternatives demonstrate the possibility of the studied products to be used in the management of anthracnose in the post-harvest period.(AU)

Biosurfactants synthesized by endophytic Bacillus strains as control of Moniliophthora perniciosa and Moniliophthora roreri

ABSTRACT: Ecuador is responsible for up to 70 % of the worlds production of fine flavor cocoa. Although fungal diseases have reduced cocoa production, ecofriendly alternatives have been implemented to control the spread of phytopathogens. This study aimed to explore the potential of endophytic strains isolated from fine flavor Nacional cocoa trees to synthetize compounds with antimicrobial and surface active properties. It was determined that under in vitro conditions, seven strains (DS03, DS07, DS18, DS23, DS31, DS34 and DS50) exhibited antifungal activity that inhibited the mycelial growth of Moniliophthora roreri and Moniliophthora perniciosa. The strains were identified based on their morphological (pattern forming Bacilli) and genetic characteristics using the 16S SSU rRNA gene sequences. Furthermore, we determined that the endophytic strains contained genes that encoded the lipopeptides Fengycin (fenA, fenB, fenC, fenE), Iturin (ituB, ituC, ituD), and Bacyllomicin D (bamC, bamD). The production of biosurfactant compounds was determined by a reduction in surface tension in the mineral medium. Additionally, the biosurfactant crude extract (BCE) was tested for its inhibitory activity against M. perniciosa and M. roreri. The minimal inhibitory concentration (MIC) was determined to be 0.07 and 0.035 mg mL1, respectively, in both cases in a fungistatic action mode. The swelling granulation and fragmentation of both phytopathogens hyphae were observed using the BCE treatment. These results suggested the biocontrol capacity of the endophytic strains, which under in vitro conditions, have the ability to synthesize antifungal and surface active compounds that might be used as an ecological alternative in cocoa plants to prevent infection by phytopathogens.

Megalopodidae (Insecta, Coleoptera): new occurrence and host plant records for Brazil

Abstract The Brazilian Megalopodidae fauna consists of 147 species from 11 genera, of which Mastostethus Lacordaire is the most diverse, representing about half of the species. Nevertheless, knowledge about species distributions and their relationships with host plants is still scarce. Since this group has endophytic immatures and bore into branches, they are economically important as they mainly damage Solanaceae species. Thus, this study sought to determine the Megalopodidae species that occur in forest fragments of southwestern and western Paraná and associate them with their host plants. For this purpose, we used material collected with Malaise traps and active manual sampling to determine associations with host plants. A total of 170 specimens of 11 species were sampled, of which Pseudhomalopterus carinatus Pic, 1920 was the most abundant and collected in all fragments. Four species were sampled exclusively through manual collections and associated with their host plants, all of which were new association records for Brazilian megalopodides. Mastostethus pantherinus Lacordaire, 1845 was associated with an Asteraceae for the first time. Additionally, Mastostethus minutusMonrós, 1947 is a new record for Brazil.

Megalopodidae (Insecta, Coleoptera): new occurrence and host plant records for Brazil

Abstract The Brazilian Megalopodidae fauna consists of 147 species from 11 genera, of which Mastostethus Lacordaire is the most diverse, representing about half of the species. Nevertheless, knowledge about species distributions and their relationships with host plants is still scarce. Since this group has endophytic immatures and bore into branches, they are economically important as they mainly damage Solanaceae species. Thus, this study sought to determine the Megalopodidae species that occur in forest fragments of southwestern and western Paraná and associate them with their host plants. For this purpose, we used material collected with Malaise traps and active manual sampling to determine associations with host plants. A total of 170 specimens of 11 species were sampled, of which Pseudhomalopterus carinatus Pic, 1920 was the most abundant and collected in all fragments. Four species were sampled exclusively through manual collections and associated with their host plants, all of which were new association records for Brazilian megalopodides. Mastostethus pantherinus Lacordaire, 1845 was associated with an Asteraceae for the first time. Additionally, Mastostethus minutusMonrós, 1947 is a new record for Brazil.

Biosurfactants synthesized by endophytic Bacillus strains as control of Moniliophthora perniciosa and Moniliophthora roreri

Ecuador is responsible for up to 70 % of the world’s production of fine flavor cocoa. Although fungal diseases have reduced cocoa production, eco–friendly alternatives have been implemented to control the spread of phytopathogens. This study aimed to explore the potential of endophytic strains isolated from fine flavor Nacional cocoa trees to synthetize compounds with antimicrobial and surface active properties. It was determined that under in vitro conditions, seven strains (DS03, DS07, DS18, DS23, DS31, DS34 and DS50) exhibited antifungal activity that inhibited the mycelial growth of Moniliophthora roreri and Moniliophthora perniciosa. The strains were identified based on their morphological (pattern forming Bacilli) and genetic characteristics using the 16S SSU rRNA gene sequences. Furthermore, we determined that the endophytic strains contained genes that encoded the lipopeptides Fengycin (fenA, fenB, fenC, fenE), Iturin (ituB, ituC, ituD), and Bacyllomicin D (bamC, bamD). The production of biosurfactant compounds was determined by a reduction in surface tension in the mineral medium. Additionally, the biosurfactant crude extract (BCE) was tested for its inhibitory activity against M. perniciosa and M. roreri. The minimal inhibitory concentration (MIC) was determined to be 0.07 and 0.035 mg mL−1, respectively, in both cases in a fungistatic action mode. The swelling granulation and fragmentation of both phytopathogens’ hyphae were observed using the BCE treatment. These results suggested the biocontrol capacity of the endophytic strains, which under in vitro conditions, have the ability to synthesize antifungal and surface active compounds that might be used as an ecological alternative in cocoa plants to prevent infection by phytopathogens.

Biosurfactants synthesized by endophytic Bacillus strains as control of Moniliophthora perniciosa and Moniliophthora roreri

Ecuador is responsible for up to 70 % of the worlds production of fine flavor cocoa. Although fungal diseases have reduced cocoa production, eco–friendly alternatives have been implemented to control the spread of phytopathogens. This study aimed to explore the potential of endophytic strains isolated from fine flavor Nacional cocoa trees to synthetize compounds with antimicrobial and surface active properties. It was determined that under in vitro conditions, seven strains (DS03, DS07, DS18, DS23, DS31, DS34 and DS50) exhibited antifungal activity that inhibited the mycelial growth of Moniliophthora roreri and Moniliophthora perniciosa. The strains were identified based on their morphological (pattern forming Bacilli) and genetic characteristics using the 16S SSU rRNA gene sequences. Furthermore, we determined that the endophytic strains contained genes that encoded the lipopeptides Fengycin (fenA, fenB, fenC, fenE), Iturin (ituB, ituC, ituD), and Bacyllomicin D (bamC, bamD). The production of biosurfactant compounds was determined by a reduction in surface tension in the mineral medium. Additionally, the biosurfactant crude extract (BCE) was tested for its inhibitory activity against M. perniciosa and M. roreri. The minimal inhibitory concentration (MIC) was determined to be 0.07 and 0.035 mg mL−1, respectively, in both cases in a fungistatic action mode. The swelling granulation and fragmentation of both phytopathogens hyphae were observed using the BCE treatment. These results suggested the biocontrol capacity of the endophytic strains, which under in vitro conditions, have the ability to synthesize antifungal and surface active compounds that might be used as an ecological alternative in cocoa plants to prevent infection by phytopathogens.(AU)