Arbuscular mycorrhizal fungi associated to Zea Mays L. plants in an agroecosystem of Atlántico, Colombia.

El maíz es considerado uno de los más importantes cultivos a nivel mundial. Como en muchos otros países, Colombia ha utilizado este cereal no solo como alimento para humanos y animales sino también para fabricar diferentes productos industriales. Las plantas de maíz están bien adaptadas a las diferentes condiciones climáticas y agroecológicas de Colombia, lo cual le permite estar ampliamente distribuido en todo el país. Algunas de sus adaptaciones naturales son atribuidas a la existencia de relaciones simbióticas con hongos micorrizógenos arbusculares (HMA), los cuales promueven la captación de nutrientes en especial de aquellos que tienen escasa movilidad tales como el fósforo (P) y el nitrógeno (N). Se identificaron HMA asociados a cultivos de maíz localizados en el municipio de Sabanalarga, Atlántico, Colombia. El número de esporas en 100 g de suelo se determinó mediante tamizado, siguiendo protocolos de centrifugación en sacarosa. El número de esporas por 100 g de suelo mostró diferencias estadísticas significativas durante los meses de muestreo (p<0,05). Se identificaron un total de 19 morfotipos correspondientes a doce especies del género Glomus, cinco del género Gigaspora y una especie para los géneros Acaulospora y Scutellospora. Se encontró correlación negativa entre temperatura y número de esporas; sin embargo no existió correlación entre el pH y las variables densidad de esporas, porcentaje de colonización y temperatura del suelo. Asimismo, se reportó bajo contenido de materia orgánica (0,99 %) y baja capacidad de intercambio catiónico (7,50 cmol.kgr-1suelo). Estos resultados, sumados al hecho de que este tipo de cultivos son grandemente dependientes de la actividad de hongos micorrizógenos, explican la densidad de esporas (400-1350 esp/100 g) y el elevado porcentaje de colonización (40-98 %) que fue encontrado durante el muestreo. Es claro que este cereal depende de la presencia de hongos micorrizógenos durante la toma de nutrientes.Corn is considered one of the most important cereal crops worldwide. As many other countries, Colombia has used this cereal not only to feed humans and animals but also to manufacture many different industrial products. Corn plants are well adapted in different climatic and ecological conditions in Colombia which allows it to be widely distributed throughout the country. Some of its natural adaptations are attributed to the existence of symbiotic relationships with Arbuscular Mycorrhizal fungi (AMF) which promote the nutrient uptake, especially those with known low mobility such as phosphorus (P) and nitrogen (N). AMFs associated to corn crops were identified in samples collected in the fields of the municipality of Sabanalarga (Atlántico, Colombia). The number of spores per 100 g of soil was determined by sieving following sucrose centrifugation standard protocols. The number of spores per 100 g of soil showed statistically significant differences during the months of sampling (p < 0.05). A total of 19 morphotypes, corresponding to twelve species of the genus Glomus, five of the genus Gigaspora, and one species of both genus Acaulospora and Scutellospora were identified. A negative correlation between temperature and number of spores was found but no correlation between pH and the spore density, percentage of colonization and soil temperature variables was found. Additionally, low organic matter content (0.99%) and low cation exchange capacity (7.50 cmol*soil-Kg-1) were reported. These results, in addition to the fact that this kind of crops are highly dependent of mycorrhizal fungi activity, explain the spore density (400-1350 spore/ 100 g) and the high percentage of colonization (40-98%) that were obtained during sampling. It is clear that this cereal crop depends on the presence of mycorrhizal fungi during nutrient uptake

Clathrin-dependent endocytosis is associated with RNAi response in the western corn rootworm, \u3ci\u3eDiabrotica virgifera virgifera\u3c/i\u3e LeConte

The cellular uptake of dsRNA after dietary exposure is critical for RNAi efficiency; however, the mechanism of its uptake in many insects remains to be understood. In this study, we evaluated the roles of the endocytic pathway genes Clathrin heavy chain (Chc), Clathrin adaptor protein AP50, ADP ribosylation factor-like 1 (Arf72A), Vacuolar H+ ATPase 16 kDa subunit (Vha16), and small GTPase Rab7 and putative sid-1-like genes (silA and silC) in RNAi response in western corn rootworm (WCR) using a two-stage dsRNA exposure bioassay. Silencing of Chc, Vha16, and AP50 led to a significant decrease in the effects of laccase2 dsRNA reporter, indicating that these genes are involved in RNAi response. However, the knockdown of either Arf72A or Rab7 did not suppress the response to laccase2 dsRNA. The silencing of the silC gene did not lead to a significant reduction in mortality or increase in the expression of V-ATPase A reporter. While the silencing of the silA gene significantly decreased insect mortality, significant changes in V-ATPase A expression were not detected. These results suggest that clathrin-dependent endocytosis is a biological mechanism that plays an important role during RNAi response in WCR adults. The fact that no definitive support for the roles of silA or silC in RNAi response was obtained support the idea that RNAi response varies greatly in different insect species, demanding additional studies focused on elucidating their involvement in this mechanism

Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, \u3ci\u3eDiabrotica undecimpunctata howardi\u3c/i\u3e (Barber)

Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), β-actin (Actin), β-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RTqPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species

Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, \u3ci\u3eDiabrotica undecimpunctata howardi\u3c/i\u3e (Barber)

Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), β-actin (Actin), β-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RTqPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species

Clathrin-dependent endocytosis is associated with RNAi response in the western corn rootworm, \u3ci\u3eDiabrotica virgifera virgifera\u3c/i\u3e LeConte

The cellular uptake of dsRNA after dietary exposure is critical for RNAi efficiency; however, the mechanism of its uptake in many insects remains to be understood. In this study, we evaluated the roles of the endocytic pathway genes Clathrin heavy chain (Chc), Clathrin adaptor protein AP50, ADP ribosylation factor-like 1 (Arf72A), Vacuolar H+ ATPase 16 kDa subunit (Vha16), and small GTPase Rab7 and putative sid-1-like genes (silA and silC) in RNAi response in western corn rootworm (WCR) using a two-stage dsRNA exposure bioassay. Silencing of Chc, Vha16, and AP50 led to a significant decrease in the effects of laccase2 dsRNA reporter, indicating that these genes are involved in RNAi response. However, the knockdown of either Arf72A or Rab7 did not suppress the response to laccase2 dsRNA. The silencing of the silC gene did not lead to a significant reduction in mortality or increase in the expression of V-ATPase A reporter. While the silencing of the silA gene significantly decreased insect mortality, significant changes in V-ATPase A expression were not detected. These results suggest that clathrin-dependent endocytosis is a biological mechanism that plays an important role during RNAi response in WCR adults. The fact that no definitive support for the roles of silA or silC in RNAi response was obtained support the idea that RNAi response varies greatly in different insect species, demanding additional studies focused on elucidating their involvement in this mechanism

Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, \u3ci\u3eDiabrotica undecimpunctata howardi\u3c/i\u3e (Barber)

Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), β-actin (Actin), β-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RTqPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species

Clathrin-dependent endocytosis is associated with RNAi response in the western corn rootworm, \u3ci\u3eDiabrotica virgifera virgifera\u3c/i\u3e LeConte

The cellular uptake of dsRNA after dietary exposure is critical for RNAi efficiency; however, the mechanism of its uptake in many insects remains to be understood. In this study, we evaluated the roles of the endocytic pathway genes Clathrin heavy chain (Chc), Clathrin adaptor protein AP50, ADP ribosylation factor-like 1 (Arf72A), Vacuolar H+ ATPase 16 kDa subunit (Vha16), and small GTPase Rab7 and putative sid-1-like genes (silA and silC) in RNAi response in western corn rootworm (WCR) using a two-stage dsRNA exposure bioassay. Silencing of Chc, Vha16, and AP50 led to a significant decrease in the effects of laccase2 dsRNA reporter, indicating that these genes are involved in RNAi response. However, the knockdown of either Arf72A or Rab7 did not suppress the response to laccase2 dsRNA. The silencing of the silC gene did not lead to a significant reduction in mortality or increase in the expression of V-ATPase A reporter. While the silencing of the silA gene significantly decreased insect mortality, significant changes in V-ATPase A expression were not detected. These results suggest that clathrin-dependent endocytosis is a biological mechanism that plays an important role during RNAi response in WCR adults. The fact that no definitive support for the roles of silA or silC in RNAi response was obtained support the idea that RNAi response varies greatly in different insect species, demanding additional studies focused on elucidating their involvement in this mechanism

Expression and Characterization of a Recombinant Endoglucanase From Western Corn Rootworm, in \u3ci\u3ePichia pastoris\u3c/i\u3e

ABSTRACT. The endoglucanase cDNA, Dvv-ENGase I, from western corn rootworm, Diabrotica virgifera virgifera LeConte was expressed using the GS115 methylotrophic strain of Pichia pastoris. The Dvv-ENGase I gene was cloned into the integrative plasmid pPICZαA under the control of AOX1, which is a methanol-inducible promoter. Positive clones were selected for their ability to produce the recombinant endoglucanase upon continuous methanol induction. The secreted recombinant insect endoglucanase Dvv-ENGase I has an apparent molecular mass of 29 kDa. The recombinant endo-1,4 -β-glucanase (ENGase) was able to digest the substrates: hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC), and Whatman No. 1 filter paper. A higher accumulation of reducing sugar was evident when the P. pastoris expression medium contained HEC (1%) instead of CMC (1%). An enzymatic activity band was detected after performing electrophoretic separation under nondenaturing conditions. The biological activity of the recombinant Dvv-ENGase I was influenced by the presence of protease inhibitors in the culture medium

Clathrin-dependent endocytosis is associated with RNAi response in the western corn rootworm, \u3ci\u3eDiabrotica virgifera virgifera\u3c/i\u3e LeConte

The cellular uptake of dsRNA after dietary exposure is critical for RNAi efficiency; however, the mechanism of its uptake in many insects remains to be understood. In this study, we evaluated the roles of the endocytic pathway genes Clathrin heavy chain (Chc), Clathrin adaptor protein AP50, ADP ribosylation factor-like 1 (Arf72A), Vacuolar H+ ATPase 16 kDa subunit (Vha16), and small GTPase Rab7 and putative sid-1-like genes (silA and silC) in RNAi response in western corn rootworm (WCR) using a two-stage dsRNA exposure bioassay. Silencing of Chc, Vha16, and AP50 led to a significant decrease in the effects of laccase2 dsRNA reporter, indicating that these genes are involved in RNAi response. However, the knockdown of either Arf72A or Rab7 did not suppress the response to laccase2 dsRNA. The silencing of the silC gene did not lead to a significant reduction in mortality or increase in the expression of V-ATPase A reporter. While the silencing of the silA gene significantly decreased insect mortality, significant changes in V-ATPase A expression were not detected. These results suggest that clathrin-dependent endocytosis is a biological mechanism that plays an important role during RNAi response in WCR adults. The fact that no definitive support for the roles of silA or silC in RNAi response was obtained support the idea that RNAi response varies greatly in different insect species, demanding additional studies focused on elucidating their involvement in this mechanism

Evaluation of reference genes for real-time quantitative PCR analysis in southern corn rootworm, \u3ci\u3eDiabrotica undecimpunctata howardi\u3c/i\u3e (Barber)

Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), β-actin (Actin), β-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RTqPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species