Microbiota Characterization of Compost Using Omics Approaches Opens New Perspectives for Phytophthora Root Rot Control

Phytophthora root rot caused by Phytophthora nicotianae is an economically important disease in pepper crops. The use of suppressive composts is a low environmental impact method for its control. Although attempts have been made to reveal the relationship between microbiota and compost suppressiveness, little is known about the microorganisms associated with disease suppression. Here, an Ion Torrent platform was used to assess the microbial composition of composts made of different agro-industrial waste and with different levels of suppressiveness against P. nicotianae. Both bacterial and fungal populations responded differently depending on the chemical heterogeneity of materials used during the composting process. High proportions (67–75%) of vineyard pruning waste were used in the most suppressive composts, COM-A and COM-B. This material may have promoted the presence of higher relative abundance of Ascomycota as well as higher microbial activity, which have proved to be essential for controlling the disease. Although no unique fungi or bacteria have been detected in neither suppressive nor conducive composts, relatively high abundance of Fusarium and Zopfiella were found in compost COM-B and COM-A, respectively. To the best of our knowledge, this is the first work that studies compost metabolome. Surprisingly, composts and peat clustered together in principal component analysis of the metabolic data according to their levels of suppressiveness achieved. This study demonstrated the need for combining the information provided by different techniques, including metagenomics and metametabolomics, to better understand the ability of compost to control plant diseases.This work was supported by LIFE project AGROWASTE (LIFE10/ENV/ES/469) http://ec.europa.eu/environment/life/

Molecular diagnosis and biological control of Phytophthora root rot in pepper and Fusarium wilt in muskmelon

The southeast Spain is localized in an area with an important agricultural sector that is well-known for its high production of pepper and melon. These two crops are affected by Phytophthora root rot and Fusarium wilt, respectively. An early detection of the causal agents of these diseases is of paramount relevance to avoid their expansion in new fields and, to improve the management strategies for their control. For this reason, we developed a TaqMan system in order to detect and quantify Phytophthora nicotianae specifically by real time PCR (qPCR) in pure culture and environmental matrices such as soil, compost and plant tissues. With this system, we identified P. nicotianae as the current causal agent of Phytophthora root rot in “El Campo de Cartagena” and we characterized its population through the use of mitochondrial molecular markers and phenotypic traits. Moreover, the application of the TaqMan system along with a new approach, namely digital PCR (dPCR), proved to be an attractive alternative to quantify P. nicotianae accurately. The dPCR was not only able to detect the presence of low levels of P. nicotianae in environmental samples, but was also less susceptible than the qPCR to inhibitors present in the DNA plant extracts. We also evaluated the ability of a batch of composts made from agro-industrial wasteto control Fusarium wilt and Phytophthora root rot under greenhouse nursery conditions. The results showed positive correlations between enzymatic activities such as NAGase, chitinase and protease regarding Fusarium wilt suppression, and dehydrogenase activity related to Phytophthora root rot suppression. Besides, a deeper study was carried out in order to elucidate the mechanisms involved in Phytophthora root rot control. This was accomplished studying the physical and chemical characteristics of composts, including their organic matter composition, and analyzing their metagenome and metabolome. We observed that the involvement of certain antagonistic microbes such as Streptomyces, Bacillus, Zopfiella or Fusarium in the phenomenon of disease suppression was related to the degree of stabilization of the materials and thus, to the microbial activity achieved. These parameters provided an array of outcomes whose integration may be a promising approach to improve not only the characterization but also the identification of suppressive composts.Furthermore, vineyard pruning waste compost amended with Trichoderma hazianum showed better results controlling Fusarium wilt than the same compost without this BCA and compared with peat. The control exerted by T. harzianum itself as well as the changes induced by its presence in the bacterial community and in some chemical parameters may explain these results. The use of composts fortified with certain strains of BCA can be a good alternative controlling Fusarium wilt of melon seedlings at greenhouse nursery level. The use of in vivo tests to characterize the suppressive effect of composts was vital to obtain accurate conclusions due to the inability of in vitro tests to provide them.El sureste español se encuentra localizado en un área que destaca por su importante sector agrícola, reconocido por su alta producción de cultivos como el pimiento y el melón, los cuales se ven afectados por enfermedades causadas por patógenos del suelo, como la tristeza y la fusariosis vascular respectivamente. Una detección temprana de los agentes causantes de estas enfermedades es de gran importancia, para evitar su expansión a nuevos terrenos y mejorar su control mediante una gestión integrada. Por esta razón, desarrollamos un sistema TaqMan para detectar y cuantificar Phytophthora nicotianae específicamente mediante PCR a tiempo real (qPCR) en cultivo puro y en matrices ambientales como suelo, compost y tejido vegetal. Con este sistema se identificó P. nicotianae como el actual causante de la tristeza del pimiento en el “Campo de Cartagena” y su población fue caracterizada mediante el uso de marcadores moleculares y un estudio fenotípico. Además, la aplicación del sistema TaqMan junto con la nueva técnica de la PCR digital (dPCR), ha mostrado ser una alternativa interesante para cuantificar P. nicotianae con gran precisión. La PCR digital no solo fue capaz de detectar bajos niveles de P. nicotianae en muestras ambientales, sino que también mostró ser menos susceptible que la qPCR a los inhibidores presentes en las extractos de ADN de plantas. Por otra parte, evaluamos la capacidad de un grupo de compost de origen agroindustrial para controlar la fusariosis vascular del melón y la tristeza del pimiento bajo condiciones de semillero. Los resultados mostraron correlaciones positivas entre las actividades enzimáticas NAGasa, chitinase y proteasa con el control de la fusariosis vascular, y entre la actividad dehydrogenasa y la supresión de la tristeza del pimiento. Con el objetivo de clarificar los mecanismos de los compost involucrados en la supresión de la tristeza del pimiento, se llevó a cabo un estudio más profundo donde se analizaron las características físicas y químicas de los compost, incluyendo la composición de la materia orgánica, y también, su metagenoma y metaboloma. La involucración de ciertos antagonistas como Streptomyces, Bacillus, Zopfiella o Fusarium en el fenómeno de supresión estuvo influenciada por el grado de estabilización de los materiales y por tanto, los niveles de actividad microbiana observados. La integración de los resultados obtenidos mediante estas técnicas podría ser de gran interés para mejorar la caracterización e identificación de compost supresivos. Además, compost de poda de vid enmendados con Trichoderma harzianum mostraron mejores resultados para controlar la fusariosis vascular del melón en comparación con el mismo compost sin enmendar y la turba. El control ejercido por T. harzianum per se así como los cambios inducidos por su presencia en la comunidad bacteriana y en algunos parámetros químicos del compost, podría explicar los resultados obtenidos. El uso de compost fortificados con ciertas cepas de ACBs puede ser una buena alternativa para controlar la fusariosis vascular en semillero. El uso de test in vivo para caracterizar la capacidad supresividad de los compost fue vital para obtener conclusiones realistas debido a la incapacidad de los test in vitro para proporcionarlas

Microbiota Characterization of Compost Using Omics Approaches Opens New Perspectives for Phytophthora Root Rot Control.

Phytophthora root rot caused by Phytophthora nicotianae is an economically important disease in pepper crops. The use of suppressive composts is a low environmental impact method for its control. Although attempts have been made to reveal the relationship between microbiota and compost suppressiveness, little is known about the microorganisms associated with disease suppression. Here, an Ion Torrent platform was used to assess the microbial composition of composts made of different agro-industrial waste and with different levels of suppressiveness against P. nicotianae. Both bacterial and fungal populations responded differently depending on the chemical heterogeneity of materials used during the composting process. High proportions (67-75%) of vineyard pruning waste were used in the most suppressive composts, COM-A and COM-B. This material may have promoted the presence of higher relative abundance of Ascomycota as well as higher microbial activity, which have proved to be essential for controlling the disease. Although no unique fungi or bacteria have been detected in neither suppressive nor conducive composts, relatively high abundance of Fusarium and Zopfiella were found in compost COM-B and COM-A, respectively. To the best of our knowledge, this is the first work that studies compost metabolome. Surprisingly, composts and peat clustered together in principal component analysis of the metabolic data according to their levels of suppressiveness achieved. This study demonstrated the need for combining the information provided by different techniques, including metagenomics and metametabolomics, to better understand the ability of compost to control plant diseases

Evaluation of the removal of pathogens included in the Proposal for a European Directive on spreading of sludge on land during autothermal thermophilic aerobic digestion (ATAD)

21 páginas, 5 figuras, 5 tablasThe European Union is promoting a new legislation through the "Proposal for a Directive of the European Parliament and of the Council on spreading of sludge on land", with special interest on human pathogens. Autothermal thermophilic aerobic digestion (ATAD) is described in this Proposal as an advanced treatment capable of producing sanitized sludge. A one-stage ATAD digester with effective volume of 15-m3 was started up and studied over 19months for its ability to stabilize and sanitize municipal sludge. Salmonella spp., Escherichia coli, and Clostridium perfringens spores were cultivated and pathogenity genes invA and cpa PCR-amplified. Volatile solids removal was 38.0% and the pathogen content significantly decreased by completely eliminating Salmonella spp. and E. coli but not Clostridium perfringens spores (9.6×103sporesmL-1). To completely achieve the disinfection of the sludge, a mesophilic stage was introduced after the ATAD treatment. Denaturing gradient gel electrophoresis analysis showed differences in the structures of the bacterial and fungal communities between thermophilic, mesophilic and raw sludge. The results demonstrated that the ATAD technology had the capability to produce sludge suitable for agricultural application when the operational parameters were stable and a mesophilic stage was introduced. © 2012 Elsevier B.V.This work was supported by the JAE Programme from the Consejo Superior de Investigaciones Científicas (CSIC), Spain, and the Project 324/pc08/2-04.3 included in the Plan Nacional de I+D+i 2008–2011. We thank Dr. David Walter for language comments on the manuscript.Peer reviewe

Principal component analysis according to the metabolome obtained from the composts COM-A (green balls), COM-B (pink balls), COM-C (red balls), COM-D (yellow balls), and peat (blue balls), n = 6.

<p>Principal component analysis according to the metabolome obtained from the composts COM-A (green balls), COM-B (pink balls), COM-C (red balls), COM-D (yellow balls), and peat (blue balls), n = 6.</p

Chemical composition of the composts and peat revealed by ¹³C NMR.

<p>(A) Distribution of organic carbon functional groups: 0–45 ppm (aliphatic groups); 45–60 ppm (methoxy groups), 60–110 ppm (carbohydrate groups); 110–160 ppm (aromatic C structures), and 160–210 ppm (carboxyl and ester group). (B) Alkyl/<i>O</i>-alkyl ratio values.</p

A heat map illustrating the 54 metabolites that differ among the composts and peat.

<p>Colors indicate relative quantity of each metabolite.</p

Physical-chemical and biological properties of composts and peat.

<p>Physical-chemical and biological properties of composts and peat.</p

Most abundant fungal genera identified (>1% relative abundance) in the composts and peat.

<p>Most abundant fungal genera identified (>1% relative abundance) in the composts and peat.</p

Relative abundances of the fungal orders identified in the composts and peat.

<p>Relative abundances of the fungal orders identified in the composts and peat.</p