A New Approach: Determining cyt b G143A Allele Frequency in Zymoseptoria tritici by Digital Droplet PCR.

Z. tritici first appeared in Italy later than in northern-central European countries. QoIs fungicides currently play a role in STB control, used in combination with Demethylation Inhibitors (DMIs) or Succinate dehydrogenase Inhibitors (SDHIs). In this study, we set up a fast, sensitive, and accurate ddPCR protocol in order to investigate the presence and frequency of G143A substitution, causing a reduction in strobilurins' efficacy in Z. tritici. The best PCR conditions for the clear separation of positive and negative droplets were identified. The lowest wild-type and resistant alleles frequencies were accurately determined on samples consisting of mixed DNAs from monoconidial cultures of Z. tritici and were expressed as fractional abundance. The protocol was tested by determining the copy number and frequency of alleles on gDNA purified in three Italian Z. tritici field populations representative of different fungicide management strategies. For the first time, the determination of allele concentration and the frequency of a mutation involved in Z. tritici fungicide resistance was carried out by employing digital PCR. This new approach provides a diagnostic tool that is rapid and able to detect very low G143A substitution percentages, which is very useful for fungicide resistance detection at early stages, thus, informing field management strategies for contrasting STB disease.

Double-Stranded RNA Targeting Dicer-Like Genes Compromises the Pathogenicity of Plasmopara viticola on Grapevine.

Downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevine, attacking all green parts of the plant. The damage is severe when the infection at flowering stage is left uncontrolled. P. viticola management consumes a significant amount of classical pesticides applied in vineyards, requiring efficient and environmentally safe disease management options. Spray-induced gene silencing (SIGS), through the application of exogenous double-stranded RNA (dsRNA), has shown promising results for the management of diseases in crops. Here, we developed and tested the potential of dsRNA targeting P. viticola Dicer-like (DCL) genes for SIGS-based crop protection strategy. The exogenous application of PvDCL1/2 dsRNA, a chimera of PvDCL1 and PvDCL2, highly affected the virulence of P. viticola. The reduced expression level of PvDCL1 and PvDCL2 transcripts in infected leaves, treated with PvDCL1/2 dsRNA, was an indication of an active RNA interference mechanism inside the pathogen to compromise its virulence. Besides the protective property, the PvDCL1/2 dsRNA also exhibited a curative role by reducing the disease progress rate of already established infection. Our data provide a promising future for PvDCL1/2 dsRNA as a new generation of RNA-based resistant plants or RNA-based agrochemical for the management of downy mildew disease in grapevine.

Genome Sequence Resource for Stemphylium vesicarium, Causing Brown Spot Disease of Pear.

Stemphylium vesicarium is the causal agent of several plant diseases as well brown spot of pear (BSP), which is one of the most economically important fungal diseases in European pear-production areas. In addition to the relevance of the economic impact, conidia spread widely from plant material infected by the pathogen can trigger respiratory allergy. Here, we report the first genome of a S. vesicarium strain, 173-1a13FI1M3, isolated from pear and sensitive to the mostly used fungicide classes currently authorized in Europe against BSP. The availability of this draft genome could represent a first important step in understanding the physiology and the infection mechanism of the pathogen. Furthermore, this contribution could be fundamental in order to design more effective and sustainable strategies to control the disease.

General phytoplasma detection by a q-PCR method using mycoplasma primers.

Phytoplasmas and mycoplasmas are bacteria belonging to the class Mollicutes. In this study, a fine tuning of quantitative polymerase chain reaction (qPCR) with a universal mycoplasma primer pair (GPO3F/MGSO) targeting the 16S rRNA gene was carried out on phytoplasmas. The dissociation curves of DNAs from Catharanthus roseus phytoplasma-infected micropropagated shoots and from phytoplasma field-infected plant samples showed a single peak at 82.5 °C (±0.5) specifically detecting phytoplasmas belonging to several ribosomal groups. Assay specificity was determined with DNA of selected bacteria: 'Candidatus Liberibacter solanacearum', Xylella fastidiosa, Ralstonia solanacearum and Clavibacter michiganensis. No amplification curves were observed with any of these tested bacteria except 'Ca. L. solanacearum' that was amplified with a melting temperature at 85 °C. Absolute quantification of phytoplasma titer was calculated using standard curves prepared from serial dilutions of plasmids containing the cloned fragment GPO3F/MGSO from European stone fruit yellows phytoplasma. Phytoplasma copy number ranged from 106 to 103 according with the sample. The sensitivity evaluated comparing plasmid serial dilutions resulted 10-6 for conventional PCR and 10-7 for qPCR. The latter method resulted therefore able to detect very low concentrations of phytoplasma in plant material.

Differences in the efficacy of carboxylic acid amide fungicides against less sensitive strains of Plasmopara viticola.

BACKGROUND: Plasmopara viticola is controlled by fungicides with different modes of action, including carboxylic acid amides (CAAs). The aim of this study was to evaluate differences in the response of CAA-resistant P. viticola strains towards CAAs. RESULTS: The G1105S mutation affects all four CAAs, but with different impacts. While this confirms that they have the same mode of action, it shows that differences between CAAs can occur. CONCLUSION: Further molecular modelling and docking studies are needed to gain a better understanding of the different behaviours reported here. © 2015 Society of Chemical Industry.

Genetic diversity analysis of the cucurbit powdery mildew fungus Podosphaera xanthii suggests a clonal population structure.

The sexual stage of Podosphaera xanthii is rarely found worldwide. However, chasmothecia are frequently recorded in northern Italy, suggesting the presence of an actively mating population. With the aim of investigating the genetic structure of the Italian population with respect to populations from other countries, genetic diversity analysis was performed both on 92 isolates from European and American countries by multilocus sequence typing (MLST) and on 59 isolates by amplified fragment length polymorphism (AFLP) methods. Mating type frequencies were tested for random mating and two-locus linkage disequilibrium (LD) analysis was performed. Results showed very low levels of genetic diversity: MLST showed no variations in eight housekeeping gene fragments and, accordingly, UPGMA dendrogram from AFLP data showed a high similarity (0.91-1.00 simple matching similarity coefficient) between isolates. Moreover, the random mating test showed no deviations from mating-type 1:1 ratio in the Italian population but deviations were observed in populations from Europe and American countries while two-locus LD analysis showed the presence of significant LD. The results suggest that the populations of P. xanthii are likely to be predominantly clonal, and asexual reproduction, producing a huge amount of conidia, appears to be the predominant type of reproduction of the species.

New rapid DNA extraction method with Chelex from Venturia inaequalis spores.

The objective of this study was to develop a rapid method to isolate DNA from Venturia inaequalis spores for use in diagnostic DNA mutation analysis. Chelex-100 resin was evaluated and compared with a well established DNA exctraction method, utilizing CTAB in order to have a robust comparison. In this research we demonstrated that Chelex-100 efficiently makes extraction of the DNA from V. inaequalis spores available for direct use in molecular analyses. Also, the quantity and quality of extracted DNA were shown to be adequate for PCR analysis. Comparatively, the quality of DNA samples isolated using Chelex method was better than those extracted using CTAB. In conclusion, the Chelex method is recommended for PCR experiments considering its simplicity and cost-effectiveness.

Sensitivity to strobilurin fungicides of Italian Venturia inaequalis populations with different origin and scab control.

BACKGROUND: Venturia inaequalis (Cooke) Winter with reduced sensitivity to strobilurins has been reported in several countries, including Italy. This study aimed to characterise the sensitivity to strobilurins of three different types of V. inaequalis population: (a) wild types; (b) from commercial orchards satisfactorily managed with strobilurins; (c) from an experimental orchard with control failures by trifloxystrobin and kresoxim-methyl. In vitro sensitivity tests included antigerminative activity on population conidia and mycelial growth inhibition on monoconidial isolates. Cleaved amplified polymorphic sequence (CAPS) analysis was used for the detection of G143A substitution. RESULTS: Wild-type populations showed EC(50) values lower than 0.031 mg L(-1), while those of orchards with good performance by strobilurins presented EC(50) values never higher than 0.063 mg L(-1). Samples with scab control failures showed a strongly reduced population sensitivity. Similar differences were confirmed in monoconidial isolates. The G143A substitution was always detected in low-sensitivity populations, only sometimes in well-controlled populations and generally not in wild types. CONCLUSIONS: In vitro sensitivity assays were able to discriminate the three population types with different scab management, while the qualitative PCR analysis (CAPS) was only partially reliable. High sensitivity differences among V. inaequalis populations with good and poor field control by strobilurins were observed.