Isolation of native cadmium-tolerant bacteria and fungi from cacao (Theobroma cacao L.) - Cultivated soils in central Colombia.

Cadmium (Cd) is one of the most toxic heavy metals for human health. It can be present in multiple food products, including cocoa cultivated in regions with high soil Cd concentrations. A strategy to minimize Cd uptake by cacao trees is using cadmium-tolerant bacteria (CdtB) and filamentous fungi (CdtF) to reduce Cd availability in soils. We isolated culturable CdtB and CdtF from different locations in a cocoa-producing region in central Colombia. A total of 42 CdtB and 30 CdtF morphotypes were isolated from locations with varying natural Cd concentrations. In vitro characterizations showed that in addition to their resistance to Cd, bacteria and fungi are involved in the nutrient cycling of N, P, and C in the soil. Bacterial morphotypes from genera Pseudomonas and Burkholderia grew in concentrations up to 140 mg kg-1 Cd. Among the isolated fungi, P. igniaria, Metarhizium sp., and Annulohypoxylon sp. were the most resistant, with the highest average Cd bioaccumulation, Cd remotion, and tolerance. We present new information about the native culturable bacterial morphotypes associated with cacao plants and, to the best of our knowledge, this is the first report of Cd-TF associated with cacao crops. Our results expand the knowledge about culturable CdtB and CdtF in cacao-cultivated soils and their interaction with key soil elements, with the potential to develop integrated soil management strategies.

ALTA PRESENCIA DE CADMIO RESULTA EN BAJA DIVERSIDAD DE HONGOS FORMADORES DE MICORRIZAS ARBUSCULARES ASOCIADOS A CACAO (Theobroma cacao L.) / High cadmium concentration resulted in low arbuscular mycorrhizal fungi community diversity associated to cocoa (Theobroma cacao L.)

RESUMEN Los hongos formadores de micorrizas arbusculares (HFMA) son simbiontes obligados presentes en la rizósfera de plantas de cacao y la diversidad de sus comunidades se modifica, dependiendo de diversos factores como la presencia de cadmio (Cd) en el suelo. La persistencia de HFMA en suelos enriquecidos naturalmente con Cd podría ser un indicador de su capacidad para tolerar esta condición. Esta investigación caracterizó la estructura de la comunidad de HFMA locales presentes en la rizósfera de plantas de cacao en dos suelos con baja (B-Cd: 0,1 mg kg-1) y alta (A-Cd: 20,9 mg kg-1) concentración de Cd. Esporas de HFMA se identificaron mediante claves taxonómicas y su abundancia, riqueza y diversidad se determinó en muestras de suelo originales y después de su multiplicación mediante cultivos trampa. Las comunidades de HFMA se compararon usando análisis de componentes principales (ACP) e índices de diversidad alfa y beta. Los resultados indican que A-Cd presentó valores significativamente menores de abundancia (21 %), riqueza (20 %) y diversidad (11 %) de morfoespecies de HFMA con respecto a B-Cd. Las dos comunidades de HFMA presentaron cinco de siete géneros en común, pero solo cuatro de las 23 morfoespecies descritas se encontraron en ambas comunidades. El análisis de diversidad beta y el ACP determinaron baja similaridad y tasa de recambio entre las comunidades de HFMA. La dominancia de Diversispora spurca, Rhizoglomus sp. y Claroideoglomus etunicatum en A-Cd sugiere que estas morfoespecies son estrés-tolerantes y candidatos potenciales para el desarrollo de estrategias de mitigación en suelos con Cd.

ABSTRACT Arbuscular mycorrhizae fungi (AMF) are obligate symbionts present in rhizosphere of cocoa plants and their community diversity is modified depending on several factors, such as cadmium (Cd) presence in soil. AMF persistence on Cd natural enriched soils might be an indicator of their tolerance and their potential in biotechnological applications. In this research we characterized local AMF community structure present in cocoa rhizosphere soils with low (B-Cd: 0.1 mg kg-1) and high (A-Cd: 20.9 mg kg-1) natural Cd concentrations. AMF spore identification was carried out using taxonomic keys and their abundance, richness and diversity were determined in original samples and after multiplication process using onion trap cultures. AMF communities were compared using alpha and beta diversity indexes and principal component analysis (PCA). The results indicated that A-Cd presented significative lower values of abundance (21 %), richness (20 %) and diversity (11 %) of AMF morphospecies in comparison with B-Cd. Both AMF communities presented five of seven genera in common, but only four of 23 morphospecies described were found in two communities. Low similarity and turnover were found among AMF communities throughout beta diversity analysis and PCA. Dominance of Diversispora spurca, Rhizoglomus sp. and Claroideoglomus etunicatum in A-Cd suggests that these morphospecies are stress-tolerant and they are potential candidates for the development of mitigation strategies in cocoa plants under Cd stress.

A new method for the inoculation of Phytophthora palmivora (Butler) into cacao seedlings under greenhouse conditions.

BACKGROUND: The black pod disease affects cacao plantations worldwide; it is caused by the oomycete species of the genus Phytophthora. The resistance of cacao plants to the black pod is commonly evaluated by artificial inoculation of the pathogen and the monitoring of the disease symptoms. However, it is difficult to identify resistant plants because the commonly used methods for the inoculation of the pathogens produce inconsistent results. Therefore, this study aimed to develop an efficient and reliable method to evaluate the resistance of Theobroma cacao seedlings to the infection by Phytophthora palmivora. RESULTS: Seedlings of different cacao genotypes were inoculated with P. palmivora under greenhouse conditions using the previously reported inoculation methods and a newly proposed method, the agar-water solution method. While none of the previously reported methods was effective, the agar-water solution method ensured a 100% seedling infection under greenhouse conditions. The proposed agar-water methodology is fast, simple and reproducible. Furthermore, the evaluation of this method in susceptible (CCN-51) and tolerant (SCA-6) T. cacao genotypes produced the expected contrasting results. CONCLUSIONS: The agar-water solution method presented in this study is an efficient alternative inoculation protocol for the identification of cacao genotypes that are resistant to black pod under greenhouse conditions.

Impact of potato cultivation and cattle farming on physicochemical parameters and enzymatic activities of Neotropical high Andean Páramo ecosystem soils.

The Andean Páramos are high mountain ecosystems whose soils are essential for the management of South American water resources, but research on anthropic impacts to these soils is currently minimal and insufficient. The objective of this study was to evaluate the impacts of potato (Solanum tuberosum) cultivation and livestock on the physicochemical parameters and enzymatic activities that determine the soil quality of the Neotropical high Andean Páramo ecosystem in the Nevados National Natural Park (Nevados NNP) in Colombia. It was hypothesised that sites with potato crops and livestock farming would exhibit significant changes in soil physicochemical parameters and enzymatic activities compared with Páramo sites that have been conserved without agriculture. Samples were collected from soils under potato cultivation, livestock and Páramo (subject to the lowest degree of human intervention possible), on three farms in the El Bosque District at three different altitudes (Buenos Aires, El Edén and La Secreta) during two seasons (dry and rainy). The results showed that none of the physical parameters under study presented statistically significant differences due to the type of use (livestock, potato crop or Páramo), season of sampling (dry or rainy season) or altitude (different farms). The chemical parameters that statistically significantly differed due to land use were organic carbon, cation exchange capacity, calcium, potassium, and ammonium and those that showed statistically significant differences associated with the sampling timing were organic carbon, nitrogen, cation exchange capacity, total carbon, C/N and nitrate. Additionally, there were differences in organic carbon due to the altitude of the farms. With respect to enzymatic activities, those of ß-glucosidase, phosphodiesterase and urease significantly decreased in soils under potato cultivation and livestock relative to those of Páramo, but those of acid phosphatase and protease increased significantly under potato cropping and livestock. The activities of ß-glucosidase, acid phosphatase, alkaline phosphatase, phosphodiesterase and protease were higher during the dry season than the rainy season, and the activities of ß-glucosidase, acid phosphatase and urease decreased statistically in the lower-altitude farm (La Secreta). These decreases in enzymatic activities are attributable to changes in the organic carbon of the soil. This study provides a novel insight on the relationships between land use and the physicochemical parameters and enzymatic activities of Páramo soils (which have been minimally studied to date) at different altitudes and during different seasons. The results suggest that changes in agricultural practices should be implemented to maintain the organic carbon of soil and, therefore, its enzymatic activities.

Evaluación de diferentes métodos de extracción de ARN a partir del hongo nativo Xylaria sp / Evaluation of different RNA extraction methods from the native fungus Xylaria sp

RESUMEN La extracción de ARN de calidad constituye el primer paso para el análisis de la expresión génica. Sin embargo, su obtención no es sencilla debido a la susceptibilidad de esta molécula a la presencia de contaminantes como ARNasas, proteínas y polisacáridos. Adicionalmente, debido a la diversa composición de la pared celular de los hongos se requiere optimizar los procesos de extracción de ARN para organismos específicos. Este estudio evalúo el uso de diferentes metodologías de homogeneización de tejido (nitrógeno líquido y liofilización) y extracción de ARN (Trizol, CTAB y RNeasy mini kit) a partir del hongo nativo ascomiceto Xylaria sp. Se determinó la pureza, concentración e integridad del ARN obtenido por medio de espectrofotometría y electroforesis. Adicionalmente, se diseñaron cebadores de referencia para el gen β-Tubulina a partir del alineamiento de secuencias de este gen obtenidas de diferentes ascomicetes. Estos cebadores fueron utilizados para evaluar si el ARN extraído es amplificable mediante RT-PCR. Se determinó que la homogeneización de tejido por medio de liofilización generó mayores rendimientos de extracción independientemente del protocolo de extracción utilizado; sin embargo, éstos alteraron la integridad del ARN. Se obtuvo un ARN con mayor pureza con el protocolo CTAB y un mayor rendimiento con el RNeasy mini kit. Los resultados indican que el ARN extraído, independientemente de la metodología de homogeneización y extracción utilizada, es amplificable mediante RT-PCR. No obstante, se recomienda homogeneizar el tejido con nitrógeno líquido y extraer con RNeasy mini kit por la brevedad del protocolo de extracción y calidad obtenida.

ABSTRACT Obtaining high quality RNA is the first step for gene expression analysis. However, the low stability of this molecule and high presence of contaminants such as RNases, proteins and polysaccharides may trouble extractions. Fungi cell wall composition is highly diverse; therefore optimizing RNA extraction procedures is necessary when studying specific organisms. In this study, different methods of tissue homogenization (liquid nitrogen and lyophilization) and RNA extraction (Trizol, CTAB and RNeasy mini kit) were assessed with a native ascomycete, Xylaria sp. RNA purity, concentration and integrity were determined by spectrophotometry and electrophoresis. In addition, a set of housekeeping gene primers was designed targeting the β-tubulin gene. The primers were used to determine if the RNA extracted allowed RT-PCR amplification. It was demonstrated that homogenization of tissue by lyophilization allowed higher yields of RNA regardless of the extraction protocol used, however, the RNA integrity was affected. The higher RNA purity was obtained using CTAB and the higher yields using the RNeasy mini kit. The extracted RNA is amplifiable by RT-PCR regardless of the homogenization and extraction methodology used. However, it is recommended to homogenize the tissue with liquid nitrogen and to extract RNA with the RNeasy mini kit due the shortness and efficiency of these protocols.