Use of LAMP for Assessing Botrytis cinerea Colonization of Bunch Trash and Latent Infection of Berries in Grapevines.

A real-time loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Botrytis cinerea in grapevine bunch trash, immature berries, and ripening berries. A simple method for the preparation of crude extracts of grape tissue was also developed for on-site LAMP analysis. When tested with 14 other fungal species frequently found in grapevines, the LAMP assay was specific and sensitive to a B. cinerea DNA quantity of 0.1 ng/µL. The sensitivity was further tested using bunch trash samples with B. cinerea colonization levels between 6 and 100% and with bulk-berry samples composed of 4 pathogen-free berries or 4 berries among which 25 to 100% had been inoculated with B. cinerea. The LAMP assay detected the lowest B. cinerea colonization level tested in bunch trash and in immature and mature berries in less than 20 min. In single-berry experiments, LAMP amplified B. cinerea DNA from all artificially inoculated individual immature and mature berries. No amplification occurred in B. cinerea-free material. The real-time LAMP assay has the potential to be used as a rapid on-site diagnostic tool for assessing B. cinerea colonization in bunch trash and B. cinerea latent infections in berries, which represent critical stages for decision-making about disease management.

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.

Reduction of Botrytis cinerea Colonization of and Sporulation on Bunch Trash.

Botrytis bunch rot (BBR) of grapevine, caused by Botrytis cinerea, is commonly managed by fungicide (FUN) sprays at flowering (A), at prebunch closure (B), at veraison (C), and before harvest. Applications at A, B, and C are recommended to reduce B. cinerea colonization of bunch trash and the production of conidia during berry ripening. The effects of these applications were previously evaluated as reductions in BBR severity at harvest rather than as reductions in bunch trash colonization and sporulation by B. cinerea. This study investigated the effects of FUNs (a commercial mixture of fludioxonil and cyprodonil), biological control agents (BCAs; Aureobasium pullulans and Trichoderma atroviride), and botanicals (BOTs; a commercial mixture of eugenol, geraniol, and thymol) applied at different timings (A, B, C, or ABC) compared with a nontreated control (NT) on B. cinerea bunch trash colonization and sporulation in vineyards. The ability of B. cinerea to colonize the bunch trash (as indicated by B. cinerea DNA content) and sporulate (as indicated by the number of conidia produced under optimal laboratory conditions) was highly variable, and this variability was higher between years (2015 to 2018) than among the three vineyards and three sampling times (i.e., 1 week after applications at A, B, and C). B. cinerea sporulation on bunch trash was significantly lower in plots treated with FUN than in NT in only 3 of 18 cases (3 vineyards × 2 years × 3 sampling times). FUN applications, however, significantly reduced B. cinerea colonization of bunch trash compared with NT; for colonization, BCA efficacy was similar to that of FUN, but BOT efficacy was variable. For all products, colonization reduction was the same with application at A versus ABC, meaning that the effect of an early season application lasted from flowering to 1 week after veraison. These results indicate that the early season control of B. cinerea is important to reduce the saprophytic colonization of bunch trash, especially when the risk of BBR is high.

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.

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.