Development of quantitative detection method for Meloidogyne incognita by qPCR / Desenvolvimento do método de detecção quantitativo para Meloidogyne incognita por qPCR

The root-knot nematode (Meloidogyne spp.) is the most important plant-parasitic nematode genus, they are the most common and destructive pathogens in this group. They produce some of the most drastic symptoms in plants and can significantly reduce the yield of crops. In order to achieve deploy an efficient method of plant-parasitic nematode management, is necessary an identification and quantification accurate and reliable of plant-parasitic nematodes. The aim of this study was to analyze samples in qPCR to detect and quantify M. incognita, in the field samples, comparing different methods of extraction of DNA and its efficacy in establishing the number of individuals. For this purpose the effectiveness of different DNA methods of extraction was compared through the values of CT intervals. For standard curve and method comparisons, we used nematodes multiplied in a greenhouse and carefully separated in the specific quantities of the experiments. For the number of individuals experiment field samples previously counted under an optical microscope were used. The DNA extraction was made from 100 nematodes by the methods: CTAB, Phenol: Chloroform and commercial kit (PureLink® Genomic DNA Kit, Invitrogen). In the comparative analysis using the three methods of DNA extracting from 100 nematodes, it was observed that commercial kit and CTAB methods obtained CT values similar. The CTAB method of extraction, showed less variation in the repeats and greater linearity of standard curve in comparison with other methods tested. So, it was possible to quantify the samples through the CT value intervals, established from different numbers of individuals (1, 10, 25, 100, 250, 500 and 750), in field samples. This study demonstrated that qPCR technique is an alternative sensitive and reliable for the quantification of M. incognita to support laboratories of diagnose and field survey.

Os nematoides-das-galhas (Meloidogyne spp.) é o gênero de fitonematoide mais importante, são os patógenos mais comuns e destrutivos deste grupo. Eles produzem alguns dos sintomas mais drásticos nas plantas e podem reduzir significativamente o rendimento das culturas. Para conseguir implantar um método eficiente de manejo de nematoides parasitas de plantas, é necessária a identificação e quantificação precisa e confiável dos fitonematoides. O objetivo deste estudo foi analisar amostras em qPCR para detectar e quantificar M. incognita, em amostras de campo, comparando diferentes métodos de extração do DNA e sua eficácia no estabelecimento do número de indivíduos. Para este propósito, a eficácia de diferentes métodos de extração de DNA foi comparada através dos valores dos intervalos de Ct. Para comparações padrão de curvas e métodos, usamos nematoides multiplicados em casa de vegetação e cuidadosamente separados nas quantidades específicas dos experimentos. Para o número de indivíduos, foram utilizadas amostras de campo previamente contadas sob um microscópio óptico. A extração de DNA foi realizada a partir de 100 nematoides, pelos métodos: CTAB, Phenol: Clorofórmio e kit comercial (PureLink® Genomic DNA Kit, Invitrogen). Na análise comparativa utilizando os três métodos de extração de DNA a partir de 100 nematoides, observou-se que o kit comercial e os métodos de CTAB obtiveram valores de CT semelhantes. O método de extração CTAB apresentou menor variação nas repetições e maior linearidade da curva padrão em comparação com os demais métodos testados. O coeficiente de correlação (R2) da curva padrão foi de 0,98 indicando uma relação linear entre o valor de Ct e a quantidade de padrões de DNA variando de 90 a 0,00009 ng.µL-1. Assim, foi possível quantificar as amostras através dos intervalos de valores de CT, estabelecidos a partir de diferentes números de indivíduos (1, 10, 25, 100, 250, 500 e 750), em amostras de campo. Este estudo demonstrou que a técnica de qPCR é uma alternativa sensível e confiável para a quantificação de M. incognita, para apoiar laboratórios de diagnóstico e levantamentos de campo.

Study of differential expression of miRNAs in lung tissue of mice submitted to experimental infection by Paracoccidioides brasiliensis.

MicroRNAs (miRNAs) are small single stranded RNA sequences involved in post-transcriptional regulation of different biological and physiological processes. Paracoccidioidomycosis (PCM) is an infection caused by Paracoccidioides brasiliensis, and it is a major cause of mortality due to systemic mycoses in Brazil. To date, there have been few reports on the role of miRNAs in the immune response against fungi, especially PCM. The objective of this study was to evaluate the differential expression of miRNAs related to the inflammatory response associated with pulmonary infection by P. brasiliensis. For this purpose, lungs from BALB/c mice, intravenously infected with P. brasiliensis (2.7×107 yeast cells/ml, n = 12) and noninfected BALB/c mice (n = 8), were collected at the 28 and 56 day after infection. The lung parenchyma presented a great number of yeast cells, granulomas, and edema at 28 days and a framework of resolution of the inflammatory process after 56 days. The mRNAs gata-3, ror-γt, foxp3, and IL-6 were positively regulated at the moment at the 56 day, while the TGF-ß1 mRNA was positively regulated at both moments. The miRNAs 126a-5p, 340-5p, 30b-5p, 19b-3p, 221-3p, 20a-5p, 130a-3p, and 301a-3p, 466k presented the greatest increase in expression levels 28 days after infection, and the miRNAs let-7f-5p, let-7a-5p, 5p-26b, let-7e-5p and 369-3p, 466k presented a greater increase in levels of expression 56 days after infection. This study shows a set of differentially expressed miRNAs possibly involved in the immune response in mice during pulmonary infection by P. brasiliensis.

Morphological, biochemical, and molecular characterization of Meloidogyne spp. populations from Brazilian soybean production regions / Caracterização morfológica, bioquímica e molecular de populações de Meloidogyne spp. de regiões brasileiras produtoras de soja

ABSTRACT: Soybean is a commodity of great economic importance worldwide, particularly in Brazil, world’s second largest producer. Nematodes, especially those of the Meloidogyne genus, severely limit productivity. Identification of nematode species is important for effective soybean management. Here, 26 populations of root-knot nematode (Meloidogyne spp.) from 15 municipalities in the states of Bahia, Mato Grosso, Goias, and Minas Gerais were characterized based on the morphology of the female perineal region, esterase profile, and identification based on amplification of specific regions of the population genome. Among the Meloidogyne spp. populations obtained, M. incognita and M. javanica, were identified. No mixed populations were present in the samples. Diagnosis based on molecular analysis was shown to be reliable and the fastest for characterization of nematode populations compared to other methods analyzed.

RESUMO: A soja é uma commodity de grande importância econômica em todo mundo, especialmente no Brasil, segundo maior produtor mundial. Os nematoides, em especial os do gênero Meloidogyne, causam grandes limitações na produtividade. A identificação das espécies de nematoides é uma informação importante para o manejo adequado. Neste trabalho, 26 populações do nematoide das galhas (Meloidogyne spp.), provenientes de municípios do estado da Bahia, Mato Grosso, Goiás e de Minas Gerais, foram caracterizados com base na morfologia da região perineal das fêmeas de Meloidogyne spp., seu perfil de esterase e identificação baseada em amplificação de regiões específicas do genoma dessas populações. Entre as populações de Meloidogyne spp. obtidas, identificou-se M. incognita e M. javanica. Não houve presença de populações mistas nas amostras analisadas. O diagnóstico baseado em análise molecular se mostrou mais rápido e confiável comparado ás outras análises.

Analysis of genes that are differentially expressed during the Sclerotinia sclerotiorum-Phaseolus vulgaris interaction.

The fungus Sclerotinia sclerotiorum (Lib.) de Bary, one of the most important plant pathogens, causes white mold on a wide range of crops. Crop yield can be dramatically decreased due to this disease, depending on the plant cultivar and environmental conditions. In this study, a suppression subtractive hybridization cDNA library approach was used for the identification of pathogen and plant genes that were differentially expressed during infection of the susceptible cultivar BRS Pérola of Phaseolus vulgaris L. A total of 979 unigenes (430 contigs and 549 singletons) were obtained and classified according to their functional categories. The transcriptional profile of 11 fungal genes related to pathogenicity and virulence were evaluated by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Additionally, the temporal expression profile obtained by RT-qPCR was evaluated for the following categories of plant defense-related genes: pathogenesis-related genes (PvPR1, PvPR2, and PvPR3), phenylpropanoid pathway genes (PvIsof, PvFPS1, and 4CL), and genes involved in defense and stress-related categories (PvLox, PvHiprp, PvGST, PvPod, and PvDox). Data obtained in this study provide a starting point for achieving a better understanding of the pathosystem S. sclerotiorum-P. vulgaris.

Exogenous application of methyl jasmonate induces a defense response and resistance against Sclerotinia sclerotiorum in dry bean plants.

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen that causes a disease known as white mold, which is a major problem for dry bean (Phaseolus vulgaris L.) and other crops in many growing areas in Brazil. To investigate the role of methyl jasmonate (MeJA) in defending dry bean plants against S. sclerotiorum, we used suppression subtractive hybridization (SSH) of cDNA and identified genes that are differentially expressed during plant-pathogen interactions after treatment. Exogenous MeJA application enhanced resistance to the pathogen, and SSH analyses led to the identification of 94 unigenes, presumably involved in a variety of functions, which were classified into several functional categories, including metabolism, signal transduction, protein biogenesis and degradation, and cell defense and rescue. Using RT-qPCR, some unigenes were found to be differentially expressed in a time-dependent manner in dry bean plants during the interaction with S. sclerotiorum after MeJA treatment, including the pathogenesis-related protein PR3 (chitinase), PvCallose (callose synthase), PvNBS-LRR (NBS-LRR resistance-like protein), PvF-box (F-box family protein-like), and a polygalacturonase inhibitor protein (PGIP). Based on these expression data, the putative roles of differentially expressed genes were discussed in relation to the disease and MeJA resistance induction. Changes in the activity of the pathogenesis-related proteins ß-1,3-glucanase, chitinase, phenylalanine ammonia-lyase, and peroxidase in plants after MeJA treatment and following inoculation of the pathogen were also investigated as molecular markers of induced resistance. Foliar application of MeJA induced partial resistance against S. sclerotiorum in plants as well as a consistent increase in pathogenesis-related protein activities. Our findings provide new insights into the physiological and molecular mechanisms of resistance induced by MeJA in the P. vulgaris-S. sclerotiorum pathosystem.

Phospholipase gene expression during Paracoccidioides brasiliensis morphological transition and infection.

Phospholipase is an important virulence factor for pathogenic fungi. In this study, we demonstrate the following: (i) the Paracoccidioides brasiliensis pld gene is preferentially expressed in mycelium cells, (ii) the plb1 gene is mostly up-regulated by infection after 6 h of co-infection of MH-S cells or during BALB/c mice lung infection, (iii) during lung infection, plb1, plc and pld gene expression are significantly increased 6-48 h post-infection compared to 56 days after infection, strongly suggesting that phospholipases play a role in the early events of infection, but not during the chronic stages of pulmonary infection by P. brasiliensis.

BEAUVERIA BASSIANA: quercetinase production and genetic diversity

Beauveria bassiana genetic diversity and ability to synthesize quercetin 2,3-dioxygenase (quercetinase) were analyzed. B. bassiana isolates, obtained from Brazilian soil samples, produced quercetinase after induction using 0.5 g/L quercetin. B. bassiana ATCC 7159 (29.6 nmol/mL/min) and isolate IP 11 (27.5 nmol/ml/min) showed the best performances and IP 3a (9.5 nmol/mL/min) presented the lowest level of quercetinase activity in the culture supernatant. A high level of polymorphism was detected by random amplified polymorphic DNA (RAPD) analysis. The use of internal-transcribed-spacer ribosomal region restriction fragment length polymorphism (ITS-RFLP) did not reveal characteristic markers to differentiate isolates. However, the ITS1-5.8S-ITS2 region sequence analysis provided more information on polymorphism among the isolates, allowing them to be clustered by relative similarity into three large groups. Correlation was tested according to the Person's correlation. Data of our studies showed, that lower associations among groups, level of quercetinase production, or geographical origin could be observed. This study presents the production of a novel biocatalyst by B. bassiana and suggests the possible industrial application of this fungal species in large-scale biotechnological manufacture of quercetinase.

Extracellular Paracoccidioides brasiliensis phospholipase B involvement in alveolar macrophage interaction.

BACKGROUND: Phospholipase B (PLB) has been reported to be one of the virulence factors for human pathogenic fungi and has also been described as necessary for the early events in infection. Based on these data, we investigated the role of PLB in virulence and modulation of the alveolar pulmonary immune response during infection using an in-vitro model of host-pathogen interaction, i.e. Paracoccidioides brasiliensis yeast cells infecting alveolar macrophage (MH-S) cells. RESULTS: The effect of PLB was analyzed using the specific inhibitor alexidine dihydrochloride (0.25 µM), and pulmonary surfactant (100 µg mL-1), during 6 hours of co-cultivation of P. brasiliensis and MH-S cells. Alexidine dihydrochloride inhibited PLB activity by 66% and significantly decreased the adhesion and internalization of yeast cells by MH-S cells. Genes involved in phagocytosis (trl2, cd14) and the inflammatory response (nfkb, tnf-α, il-1ß) were down-regulated in the presence of this PLB inhibitor. In contrast, PLB activity and internalization of yeast cells significantly increased in the presence of pulmonary surfactant; under this condition, genes such as clec2 and the pro-inflammatory inhibitor (nkrf) were up-regulated. Also, the pulmonary surfactant did not alter cytokine production, while alexidine dihydrochloride decreased the levels of interleukin-10 (IL-10) and increased the levels of IL-12 and tumor necrosis factor-α (TNF-α). In addition, gene expression analysis of plb1, sod3 and icl1 suggests that P. brasiliensis gene re-programming is effective in facilitating adaptation to this inhospitable environment, which mimics the lung-environment interaction. CONCLUSION: P. brasiliensis PLB activity is involved in the process of adhesion and internalization of yeast cells at the MH-S cell surface and may enhance virulence and subsequent down-regulation of macrophage activation.