A simple and rapid DNA extraction method from leaves of grapevine suitable for polymerase chain reaction analysis

Piccolo, ra Lo; Alfonzo, Antonio; Conigliaro, Gaetano; Moschetti, Giancarlo; Burruano, Santa; Barone, Amalia

doi:10.5897/ajb11.3023

Cited by 25 publications

(20 citation statements)

References 28 publications

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“…Each leaf disk was stored in a microcentrifuge tube with 0.4 g CaCl 2 covered with a layer of sterile cotton. Tubes with leaf disks were maintained at 4 °C until DNA extraction using a modified cetyl trimethyl‐ammonium bromide (CTAB) extraction procedure (Lo Piccolo et al ., ). DNA samples were stored at −20 °C.…”

Section: Methodsmentioning

confidence: 97%

Cryptic species and population genetic structure of Plasmopara viticola in São Paulo State, Brazil

et al. 2019

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Downy mildew (Plasmopara viticola) is one of the most important diseases in grape-growing areas worldwide, including Brazil. To examine pathogen population biology and structure, P. viticola was sampled during the 2015/16 growing season from 516 lesions on nine grape cultivars in 11 locations in subtropical areas of São Paulo State, Brazil. For identification of cryptic species, a subsample of 130 isolates was subjected to cleaved amplified polymorphic sequence (CAPS) analysis, and for 91 of these isolates the ITS1 region was sequenced. These analyses suggest that the population of P. viticola in São Paulo State consists of a single cryptic species, P. viticola clade aestivalis. Seven microsatellite markers were used to determine the genetic structure of all 516 P. viticola isolates, identifying 23 alleles and 55 multilocus genotypes (MLGs). Among these MLGs, 34.5% were clonal and represented 93% of the isolates sampled. Four dominant genotypes were present in at least five different locations, corresponding to 65.7% of the isolates sampled. Genotypic diversity (Ĝ = 0.21-0.89) and clonal fraction (0.58-0.96) varied among locations (populations). Most populations showed significant deviation from Hardy-Weinberg expectations; in addition, excess of heterozygosity was verified for many loci. However, principal coordinate analysis revealed no clusters among locations and no significant isolation by distance was found, suggesting high levels of migration. The results indicate that downy mildew epidemics result from multiple clonal infections caused by a few genotypes of P. viticola, and reproduction of P. viticola in São Paulo State is predominantly asexual.

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Section: Methodsmentioning

confidence: 97%

Cryptic species and population genetic structure of Plasmopara viticola in São Paulo State, Brazil

et al. 2019

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“…For each isolate, three downy mildew lesions excised with a 10-mm cork borer were ground in liquid nitrogen, and DNA was extracted using a modified cetyl trimethyl ammonium bromide (CTAB) method ( Lo Piccolo et al, 2012 ). The genomic DNA was resuspended in 40 μL of Tris-EDTA (TE) buffer (5 mM) and stored at −20°C.…”

Section: Methodsmentioning

confidence: 99%

The Climate-Driven Genetic Diversity Has a Higher Impact on the Population Structure of Plasmopara viticola Than the Production System or QoI Fungicide Sensitivity in Subtropical Brazil

et al. 2020

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Downy mildew, caused by Plasmopara viticola, is the main disease affecting vineyards in subtropical Brazil. Here, we collected 94 P. viticola isolates from four organic and conventional vineyards in the two main grape-growing states of Brazil to evaluate the sensitivity to the quinone outside inhibitor (QoI) azoxystrobin by pheno-and genotyping assays. The impact of location, production system and sensitivity to QoI fungicides on the population genetics and structure of P. viticola was determined using 10 microsatellite markers. Cytochrome b sequencing revealed that 28 and 100% of the isolates from vineyards under organic and conventional management carried the G143A mutation, respectively. The G143A mutation was associated with high levels of azoxystrobin resistance. Three out of the 94 isolates analyzed carried the M125I alteration, not previously described in P. viticola, which was associated with a five-fold reduction in azoxystrobin sensitivity compared to wild-type isolates. Haplotype network analysis based on cytochrome b gene sequences suggested that the Brazilian populations are more closely related to the European than the North American population. A total of six haplotypes were identified, with two of them carrying the G143A mutation. Microsatellite analysis revealed high allelic and genotypic variation among the four populations. Population differentiation analyses indicated that state of origin directly influences the population biology of P. viticola, while production system and QoI sensitivity have little effect. Great genetic diversity, sexual reproduction and high levels of admixture were observed in Rio Grande do Sul State. In contrast, populations in São Paulo State were dominated by a few clonal genotypes, and no admixed genotype was detected between the two genetic pools identified in the state. This study raises the hypothesis that winter weather conditions influence the overwinter survival strategy with profound effects in the population biology of P. viticola.

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“…Sporulating lesions of downy mildew from inoculated leaves were excised using a 10‐mm cork borer, stored in a 2 ml tube containing 0.4 g CaCl 2 covered with a layer of sterile cotton and maintained at 4°C (Camargo et al ., 2019). For each isolate, three leaf discs were ground in liquid nitrogen, followed by DNA extraction using a modified cetyl trimethyl ammonium bromide (CTAB) method (Lo Piccolo et al ., 2012). The cytochrome b ( cytb ) and cellulose synthase 3 ( CesA3 ) encoding genes were partially amplified using the primer pairs CB 279F (Chen et al ., 2007) with StrobiR (Burbidge et al ., 2002) and Cesa3F with Cesa3R (Zhang et al ., 2017), respectively (Table 2).…”

Section: Methodsmentioning

confidence: 99%

Multiple resistance of Plasmopara viticola to QoI and CAA fungicides in Brazil

Santos

Fraaije

Garrido³

et al. 2020

Plant Pathology

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Grapevine downy mildew, caused by Plasmopara viticola, is commonly controlled by quinone outside inhibitor (QoI) and carboxylic acid amide (CAA) fungicides. QoI resistance is conferred by a mutation resulting in a G143A alteration in cytochrome b, while CAA resistance is associated with a G1105S mutation in cellulose synthase 3. The aims of this study were to (a) verify the presence of QoI and CAA resistance in P. viticola in Brazil, (b) develop a single nucleotide polymorphism (SNP) detection pyrosequencing assay for quantitative detection of G143A, and (c) determine the geographic prevalence and distribution of G143A and G1105S in Brazilian field populations. P. viticola isolates were sampled from two vineyards reporting failures of QoI and CAA control. Their growth was unaffected by azoxystrobin and mandipropamid, and resistance was associated with the G143A and G1105S mutations, respectively. The SNP detection pyrosequencing assay developed for G143A detection was precise and accurate. Pyrosequencing assays revealed that G143A and G1105S frequencies varied among the 66 field populations and were positively correlated with the number of QoI and CAA applications, respectively. The G143A mutation was observed in 60 populations, 39 of them with frequencies over 90%. G1105S was detected in 20 populations with frequencies ranging from 18.3% to 100%, and which also carried the G143A mutation. Multiple resistance to QoI and CAA in P. viticola was confirmed for the first time in South America. Future resistance monitoring surveys and management strategies must be adopted to prevent further increase in QoI and CAA resistance in Brazil.

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A simple and rapid DNA extraction method from leaves of grapevine suitable for polymerase chain reaction analysis

Cited by 25 publications

References 28 publications

Cryptic species and population genetic structure of Plasmopara viticola in São Paulo State, Brazil

Cryptic species and population genetic structure of Plasmopara viticola in São Paulo State, Brazil

The Climate-Driven Genetic Diversity Has a Higher Impact on the Population Structure of Plasmopara viticola Than the Production System or QoI Fungicide Sensitivity in Subtropical Brazil

Multiple resistance of Plasmopara viticola to QoI and CAA fungicides in Brazil

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