Editorial: RNAi Based Pesticides

3openInternationalInternational coauthor/editoropenAndrás Székács; Azeddine Si Ammour; Michael L. MendelsohnSzékács, A.; SI AMMOUR, A.; Mendelsohn, M.L

A Real-Time PCR Assay for the Quantification of Plasmopara viticola Oospores in Grapevine Leaves

Grapevine downy mildew caused by Plasmopara viticola is one of the most important diseases in vineyards. Oospores that overwinter in the leaf litter above the soil are the sole source of inoculum for primary infections of P. viticola; in addition to triggering the first infections in the season, the oospores in leaf litter also contribute to disease development during the season. In the current study, a quantitative polymerase chain reaction (qPCR) method that was previously developed to detect P. viticola DNA in fresh grapevine leaves was assessed for its ability to quantify P. viticola oospores in diseased, senescent grapevine leaves. The qPCR assay was specific to P. viticola and sensitive to decreasing amounts of both genomic DNA and numbers of P. viticola oospores used to generate qPCR standard curves. When the qPCR assay was compared to microscope counts of oospores in leaves with different levels of P. viticola infestation, a strong linear relationship (R2 = 0.70) was obtained between the numbers of P. viticola oospores per gram of leaves as determined by qPCR vs. microscopic observation. Unlike microscopic observation, the qPCR assay was able to detect significant differences between leaf samples with a low level of oospore infestation (25% infested leaves and 75% non-infested leaves) vs. samples without infestation, and this ability was not influenced by the weight of the leaf sample. The results indicate that the qPCR method is sensitive and provides reliable estimates of the number of P. viticola oospores in grapevine leaves. Additional research is needed to determine whether the qPCR method is useful for quantifying P. viticola oospores in grapevine leaf litter

Quantification of Botrytis cinerea in Grapevine Bunch Trash by Real-Time PCR

Quantification of colonization of grape bunch trash by Botrytis cinerea is crucial for Botrytis bunch rot (BBR) control. A previously developed quantitative polymerase chain reaction (qPCR) method was adapted to quantify B. cinerea DNA in grape bunch trash, and a colonization coefficient (CC) was calculated as the ratio between the DNA concentrations of B. cinerea and of Vitis vinifera. CC values increased linearly with the number of conidia of B. cinerea or the quantity of mycelium of B. cinerea added to the bunch trash increased. CC values also increased linearly in bunch trash samples containing increasing percentages of B. cinerea-colonized bunch trash; in the latter samples, CC values were correlated with subsequent assessments of B. cinerea colonization of trash (as determined by plating on agar) and sporulation on the trash (as determined by spore counts after incubation in humid chambers). The qPCR assay was also validated using trash collected from bunches treated or not treated with fungicides in three vineyards in two seasons. CC values reflected the reduction in sporulation and in latent infections of mature berries caused by fungicide application. The qPCR assay enables rapid, specific, sensitive, and reliable quantification of the degree of colonization of bunch trash by B. cinerea, which makes it a useful tool for studies of the epidemiology and management of BBR

Signs of Silence: Small RNAs and Antifungal Responses in Arabidopsis thaliana and Zea mays

Plant small RNAs (sRNAs) are pivotal regulators of gene expression, which are crucial in maintaining genome integrity and flexibility during development, abiotic and biotic stress responses. Current evidence suggests that sRNAs might be inherent to the sophisticated plant innate immune system battling bacteria. However, the role of sRNAs during antifungal plant defences is less clear. Therefore, this chapter investigates the sRNA‐mediated plant antifungal responses against the hemibiotrophic fungi Colletotrichum higginsianum and Colletotrichum graminicola in their respective compatible hosts Arabidopsis thaliana and Zea mays. A phenotypic and metabolomic analysis of A. thaliana sRNA mutants in response to C. higginsianum infection was performed, showing a hormonal and metabolic imbalance during fungal infection in these plants. To find whether fungal-induced sRNA could directly regulate defence genes in an agricultural important plant model, the expression of maize miRNAs in response to C. graminicola leaf and root infections was investigated. The results revealed the tissue‐specific local and systemic adaptation of the miRNA transcriptome, where only a few miRNAs were targeting defence pathways. The general picture presented here points towards a role of sRNAs as fine‐tuners of genetic and metabolomic defence response layers. This chapter also further discusses the potential of utilizing sRNA‐based fungal control strategies

Development of Real-Time Isothermal Amplification Assays for On-Site Detection of Phytophthora infestans in Potato Leaves

Real-time loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays were developed targeting the internal transcribed spacer 2 region of the ribosomal DNA of Phytophthora infestans, the potato late blight causal agent. A rapid crude plant extract (CPE) preparation method from infected potato leaves was developed for on-site testing. The assay's specificity was tested using several species of Phytophthora and other potato fungal and oomycete pathogens. Both LAMP and RPA assays showed specificity to P. infestans but also to the closely related species P. andina, P. mirabilis, P. phaseoli, and P. ipomoeae, although the latter are not reported as potato pathogen species. No cross-reaction occurred with P. capsici or with the potato pathogens tested, including P. nicotianae and P. erythroseptica. The sensitivity was determined using P. infestans pure genomic DNA added into healthy CPE samples. Both LAMP and RPA assays detected DNA at 50 fg/μl and were insensitive to CPE inhibition. The isothermal assays were tested with artificially inoculated and naturally infected potato plants using a Smart-DART platform. The LAMP assay effectively detected P. infestans in symptomless potato leaves as soon as 24 h postinoculation. A rapid and accurate on-site detection of P. infestans in plant material using the LAMP assay will contribute to improved late blight diagnosis and early detection of infections and facilitate prompt management decisions

Exploit biodiversity in viticultural systems to reduce pest damage and pesticide use, and increase ecosystems services provision: the BIOVINE Project

Organic vineyards still rely on large external inputs to control harmful organisms (i.e., pests). The BIOVINE project aims to develop natural solutions based on plant diversity to control pests and reduce pesticide dependence. The capability of plants of increasing the ecosystem resistance to pests and invasive species is a well-known ecosystem service. However, monocultures (including vineyards) do not exploit the potential of plant diversity. BIOVINE aims to develop new viticultural systems based on increased plant diversity within (e.g., cover crops) and/or around (e.g., hedges, vegetation spots, edgings) vineyards by planting selected plant species for the control of arthropods, soil-borne pests (oomycetes, fungi, nematodes), and foliar pathogens. Candidate plants will be identified by a literature review, and the selected ones will be tested in controlled environment or small-scale experiments. The ability of the selected plants to: i) attract or repel target arthropod pests; ii) conserve/promote beneficials; iii) control soil-borne pests by means of biofumigation; iv) carry mycorrhizal fungi to the vine root system to increase plant health (growth and resistance); and v) control foliar pathogens by reducing the inoculum spread from soil, will be investigated. New viticultural systems able to exploit plant diversity will then be designed based on results of BIOVINE activities, following a design-assessment-adjustment cycle, which will then be tested by in-vineyard experiments in France, Italy, Romania, Slovenia, Spain and Switzerland for a 2-year period. Innovative viticultural systems should represent an improved way for pest control in organic viticulture, meanwhile they should positively affect functional biodiversity and ecosystem services. New control strategies may provide financial opportunities to vine growers and lower their reliance on pesticides

Molecular characterization of geminivirus-derived small RNAs in different plant species

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interaction

Molecular characterization of geminivirus-derived small RNAs in different plant species

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions