Evolução da população do fungo nematófago Pochonia chlamydosporia -isolado PcMR, após inoculação, no solo e na raiz do tomateiro cultivado em estufa

O fungo Pochonia chlamydospoia é um potencial agente de controlo biológico dos nemátodes-das-galhas-radiculares. Com este trabalho, pretendeu-se avaliar a eficácia de inoculações de clamidósporos no solo, no estabelecimento de uma população do fungo no solo e na raiz de tomateiro em estufa com níveis de densidade iguais ou superiores aos considerados como necessários para um eficaz controlo dos nematodes-das-galhas-radiculares. Ao longo de dois anos de ensaio, fórum efetuadas inoculações do isolado PcMR e avaliada a densidade de fungo no solo e na raiz, As inoculações efetuadas permitiram estabelecer uma população de P. chlamydosporia no solo e atingir os valores de densidade pretendidos. No entanto, os valores pretendidos para colonização da raiz pelo fungo foram atingidos apenas no primeiro ano. Foi igualmente demonstrada a capacidade do fungo em se manter no solo durante longos períodos de tempo mesmo na ausência de cultura e em condições adversas de humidade e temperatura. /ABSTRACT: Pochonia chlamydosporia is a potential root-knot nematode biological control agent. The aim of this work was to evaluate the effectiveness of chlamydospore inoculations at the soil, for the establishment at both soil and greenhouse tomato root, of a fungus population in density levels equal or superior to those considered as needed for an effective control of root-knot nematode. Along two years, several inoculations using the Portuguese isolate PcMR were made and the density of fungus at the soil and roots studied. These inoculations allowed the establishment of a population of P. chlamydosporia at the soil and achieve the desired density values. However, only in the first year of assay, the desired values of root colonization by fungus were achieved. lt was also demonstrated that P. chlamydosporia can survive for itself at the soil for a long period of time even in the absence of plant culture and in adverse moist and temperature conditions.

The biocontrol fungus Pochonia chlamydosporia shows nematode host preference at the infraspecific level

A RAPD-PCR assay was developed and used to test For competitive variability in growth of the nematode biological control fungus Pochonia chlamydosporia. Saprophytic competence in soil with or without tomato plants was examined in three isolates of the fungus: RES 280 (J), originally isolated from potato cyst nematode (PCN) cysts; RES 200 (1) and RES 279 (S), both originally isolated from root knot nematode (RKN) eggs. Viable counts taken at 70 d indicated that I was the best saprophyte followed by S, with J the poorest. RAPD-PCR analysis of colonies from mixed treatments revealed that there was a cumulative effect of adding isolates to the system. This Suggested that the isolates did not interact and that they may occupy separate niches in soil and the rhizosphere. To investigate parasitic ability, soils were seeded with two isolates of the fungus: J and S, singly or in combination. Tomato or potato plants were grown in these soils; free of nematodes, or inoculated with PCN or RKN, and incubated for 77 d. The abundance of the PCN isolate J in PCN cysts was significantly greater than that of the RKN isolate S but in RKN egg masses, S was significantly more abundant than J. RAPD-PCR analysis of colonies from mixed treatments confirmed that J was more abundant than S ill PCN cysts whereas the converse was observed on RKN egg masses. This substantiates the phenomenon of nematode host preference at the infraspecific level of P. chlamydosporia and highlights its relevance for biological control of plant parasitic nematodes.

The biocontrol fungus pochonia chlamydosporia shows nematode host preference at the infraspecific level

A RAPD-PCR assay was developed and used to test For competitive variability in growth of the nematode biological control fungus Pochonia chlamydosporia. Saprophytic competence in soil with or without tomato plants was examined in three isolates of the fungus: RES 280 (J), originally isolated from potato cyst nematode (PCN) cysts; RES 200 (1) and RES 279 (S), both originally isolated from root knot nematode (RKN) eggs. Viable counts taken at 70 d indicated that I was the best saprophyte followed by S, with J the poorest. RAPD-PCR analysis of colonies from mixed treatments revealed that there was a cumulative effect of adding isolates to the system. This Suggested that the isolates did not interact and that they may occupy separate niches in soil and the rhizosphere. To investigate parasitic ability, soils were seeded with two isolates of the fungus: J and S, singly or in combination. Tomato or potato plants were grown in these soils; free of nematodes, or inoculated with PCN or RKN, and incubated for 77 d. The abundance of the PCN isolate J in PCN cysts was significantly greater than that of the RKN isolate S but in RKN egg masses, S was significantly more abundant than J. RAPD-PCR analysis of colonies from mixed treatments confirmed that J was more abundant than S ill PCN cysts whereas the converse was observed on RKN egg masses. This substantiates the phenomenon of nematode host preference at the infraspecific level of P. chlamydosporia and highlights its relevance for biological control of plant parasitic nematodes.

Estudo químico e avaliação da atividade antimicrobiana de fungos endofíticos associados à Paepalanthus chiquitensis (eriocaulaceae) e efeitos de moduladores químicos epigenéticos no cultivo de Pochonia chlamydosporia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene cloning, molecular modeling, and phylogenetics of serine protease P32 and serine carboxypeptidase SCP1 from nematophagous fungi Pochonia rubescens and Pochonia chlamydosporia

The fungi Pochonia chlamydosporia and Pochonia rubescens are parasites of nematode eggs and thus are biocontrol agents of nematodes. Proteolytic enzymes such as the S8 proteases VCP1 and P32, secreted during the pathogenesis of nematode eggs, are major virulence factors in these fungi. Recently, expression of these enzymes and of SCP1, a new putative S10 carboxypeptidase, was detected during endophytic colonization of barley roots by these fungi. In our study, we cloned the genomic and mRNA sequences encoding P32 from P. rubescens and SCP1 from P. chlamydosporia. P32 showed a high homology with the serine proteases Pr1A from the entomopathogenic fungus Metarhizium anisopliae and VCP1 from P. chlamydosporia (86% and 76% identity, respectively). However, the catalytic pocket of P32 showed differences in the amino acids of the substrate-recognition sites compared with the catalytic pockets of Pr1A and VCP1 proteases. Phylogenetic analysis of P32 suggests a common ancestor with protease Pr1A. SCP1 displays the characteristic features of a member of the S10 family of serine proteases. Phylogenetic comparisons show that SCP1 and other carboxypeptidases from filamentous fungi have an origin different from that of yeast vacuolar serine carboxypeptidases. Understanding protease genes from nematophagous fungi is crucial for enhancing the biocontrol potential of these organisms.

Sequencing and functional analysis of the genome of a nematode egg-parasitic fungus, Pochonia chlamydosporia

Pochonia chlamydosporia is a worldwide-distributed soil fungus with a great capacity to infect and destroy the eggs and kill females of plant-parasitic nematodes. Additionally, it has the ability to colonize endophytically roots of economically-important crop plants, thereby promoting their growth and eliciting plant defenses. This multitrophic behavior makes P. chlamydosporia a potentially useful tool for sustainable agriculture approaches. We sequenced and assembled ∼41 Mb of P. chlamydosporia genomic DNA and predicted 12,122 gene models, of which many were homologous to genes of fungal pathogens of invertebrates and fungal plant pathogens. Predicted genes (65%) were functionally annotated according to Gene Ontology, and 16% of them found to share homology with genes in the Pathogen Host Interactions (PHI) database. The genome of this fungus is highly enriched in genes encoding hydrolytic enzymes, such as proteases, glycoside hydrolases and carbohydrate esterases. We used RNA-Seq technology in order to identify the genes expressed during endophytic behavior of P. chlamydosporia when colonizing barley roots. Functional annotation of these genes showed that hydrolytic enzymes and transporters are expressed during endophytism. This structural and functional analysis of the P. chlamydosporia genome provides a starting point for understanding the molecular mechanisms involved in the multitrophic lifestyle of this fungus. The genomic information provided here should also prove useful for enhancing the capabilities of this fungus as a biocontrol agent of plant-parasitic nematodes and as a plant growth-promoting organism.

A metabolomic approach to study the rhizodeposition in the tritrophic interaction: tomato, Pochonia chlamydosporia and Meloidogyne javanica

A combined chemometrics-metabolomics approach [excitation–emission matrix (EEM) fluorescence spectroscopy, nuclear magnetic resonance (NMR) and high performance liquid chromatography–mass spectrometry (HPLC–MS)] was used to analyse the rhizodeposition of the tritrophic system: tomato, the plant-parasitic nematode Meloidogyne javanica and the nematode-egg parasitic fungus Pochonia chlamydosporia. Exudates from M. javanica roots were sampled at root penetration (early) and gall development (late). EMM indicated that late root exudates from M. javanica treatments contained more aromatic amino acid compounds than the rest (control, P. chlamydosporia or P. chlamydosporia and M. javanica). 1H NMR showed that organic acids (acetate, lactate, malate, succinate and formic acid) and one unassigned aromatic compound (peak no. 22) were the most relevant metabolites in root exudates. Robust principal component analysis (PCA) grouped early exudates for nematode (PC1) or fungus presence (PC3). PCA found (PC1, 73.31 %) increased acetate and reduced lactate and an unassigned peak no. 22 characteristic of M. javanica root exudates resulting from nematode invasion and feeding. An increase of peak no. 22 (PC3, 4.82 %) characteristic of P. chlamydosporia exudates could be a plant “primer” defence. In late ones in PC3 (8.73 %) the presence of the nematode grouped the samples. HPLC–MS determined rhizosphere fingerprints of 16 (early) and 25 (late exudates) m/z signals, respectively. Late signals were exclusive from M. javanica exudates confirming EEM and 1H NMR results. A 235 m/z signal reduced in M. javanica root exudates (early and late) could be a repressed plant defense. This metabolomic approach and other rhizosphere -omics studies could help to improve plant growth and reduce nematode damage sustainably.

Estudio de la interacción tritrófica: tomate, Meloidogyne javanica y Pochonia chlamydosporia

Los nematodos fitopatógenos y en particular los agalladores causan graves pérdidas al tomate, un cultivo de gran importancia económica en España, la Unión Europea y en todo el mundo. El manejo de enfermedades por nematicidas químicos y fumigantes está muy limitado por la prohibición en la Unión Europea y a escala mundial del uso de muchos nematicidas químicos y de fumigantes como el bromuro de metilo. Los suelos supresivos a nematodos son ejemplos de control biológico natural que incluyen antagonistas de nematodos con potencial para su uso en el manejo de dichos patógenos vegetales. Nuestro grupo posee una dilata experiencia en estudio de la biología y en particular en el análisis del modo de acción del hongo parásito de huevos de nematodos, Pochonia chlamydosporia. En este artículo incluimos un resumen de nuestros estudios sobre presencia de P. chlamydosporia en suelos agrícolas. A continuación abordamos el estudio de aspectos celulares y moleculares de la infección de huevos de nematodos por P. chlamydosporia. Nuestro grupo fue pionero al demostrar que los hongos nematófagos se comportan como endófitos colonizando las raíces de mono y dicotiledóneas. Recientemente hemos secuenciado el genoma de P. chlamydosporia. El estudio de sus relaciones filogenómicas y el análisis de sus familias génicas apoyan el comportamiento multitrófico del hongo. Finalmente aportamos nuestros resultados del análisis metabolómico de la interacción tomate-nematodo agallador-P. chlamydosporia. Nuestra actual hipótesis de trabajo es que el estudio de dicha interacción por técnicas de análisis molecular masivo abren nuevas vías al manejo sostenible de nematodos bloqueando su comunicación con la planta y activando o modulando las defensas de los cultivos por hongos antagonistas como P. chlamydosporia.

Endophytic colonization of barley (Hordeum vulgare) roots by the nematophagous fungus Pochonia chlamydosporia reveals plant growth promotion and a general defense and stress transcriptomic response

Plant crop yields are negatively conditioned by a large set of biotic and abiotic factors. An alternative to mitigate these adverse effects is the use of fungal biological control agents and endophytes. The egg-parasitic fungus Pochonia chlamydosporia has been traditionally studied because of its potential as a biological control agent of plant-parasitic nematodes. This fungus can also act as an endophyte in monocot and dicot plants, and has been shown to promote plant growth in different agronomic crops. An Affymetrix 22K Barley GeneChip was used in this work to analyze the barley root transcriptomic response to P. chlamydosporia root colonization. Functional gene ontology (GO) and gene set enrichment analyses showed that genes involved in stress response were enriched in the barley transcriptome under endophytism. An 87.5 % of the probesets identified within the abiotic stress response group encoded heat shock proteins. Additionally, we found in our transcriptomic analysis an up-regulation of genes implicated in the biosynthesis of plant hormones, such as auxin, ethylene and jasmonic acid. Along with these, we detected induction of brassinosteroid insensitive 1-associated receptor kinase 1 (BR1) and other genes related to effector-triggered immunity (ETI) and pattern-triggered immunity (PTI). Our study supports at the molecular level the growth-promoting effect observed in plants endophytically colonized by P. chlamydosporia, which opens the door to further studies addressing the capacity of this fungus to mitigate the negative effects of biotic and abiotic factors on plant crops.

Some isolates of the nematophagous fungus Pochonia chlamydosporia promote root growth and reduce flowering time of tomato

The fungal parasite of nematode eggs Pochonia chlamydosporia is also a root endophyte known to promote growth of some plants. In this study, we analysed the effect of nine P. chlamydosporia isolates from worldwide origin on tomato growth. Experiments were performed at different scales (Petri dish, growth chamber and greenhouse conditions) and developmental stages (seedlings, plantlets and plants). Seven P. chlamydosporia isolates significantly (P < 0.05) increased the number of secondary roots and six of those increased total weight of tomato seedlings. Six P. chlamydosporia isolates also increased root weight of tomato plantlets. Root colonisation varied between different isolates of this fungus. Again P. chlamydosporia significantly increased root growth of tomato plants under greenhouse conditions and reduced flowering and fruiting times (up to 5 and 12 days, respectively) versus uninoculated tomato plants. P. chlamydosporia increased mature fruit weight in tomato plants. The basis of the mechanisms for growth, flowering and yield promotion in tomato by the fungus are unknown. However, we found that P. chlamydosporia can produce Indole-3-acetic acid and solubilise mineral phosphate. These results suggest that plant hormones or nutrient ability could play an important role. Our results put forward the agronomic importance of P. chlamydosporia as biocontrol agent of plant parasitic nematodes with tomato growth promoting capabilities.

Chitosan enhances parasitism of Meloidogyne javanica eggs by the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol.

Uso de agentes microbianos e químico para o controle de Meloidogyne incognita em soja

Nematoides de galhas constituem importante grupo de patógenos da cultura da soja e o manejo integrado é uma das principais medidas de controle que visam à redução de perdas econômicas. Neste trabalho foi avaliada a eficácia dos fungos Paecilomyces lilacinus (Thom.) Samsom e Pochonia chlamydosporia (Goddard) Zare & Gams (sinonímia Verticillium chlamydosporium), de um produto comercial à base de Bacillus sp. (Nemix) e do nematicida químico Aldicarb no controle de Meloidogyne incognita em soja, variedade M-SOY 6101. O experimento foi realizado em casa-de-vegetação no delineamento experimental de blocos casualizados com nove tratamentos (três produtos biológicos usados no tratamento de sementes com ou sem a aplicação em pós-emergência, Aldicarb aplicado apenas em pós-emergência e duas testemunhas) e quatro repetições. Aldicarb reduziu o número de ovos e de juvenis do nematoide. P. lilacinus foi o mais atuante dos agentes biológicos, favorecendo a manutenção da quantidade de matéria seca da raiz de soja e reduzindo o número de ovos. O produto Nemix e P. chlamydosporia somente tiveram ação efetiva na redução do número de ovos do nematoide. Com base nos resultados, foi possível concluir que o agente químico e os agentes biológicos avaliados neste trabalho tiveram moderada atividade no controle de M. incognita em soja.

Agentes microbianos no controle de nematóides e fungos fitopatogênicos de soja e sua compatibilidade com agroquímicos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Agressividade comparada de Pratylenchus brachyurus com P. zeae e eficácia de métodos de controle de nematoides em cana-de-açúcar

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biocontrole de nematoides com fungos

Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV