Assessment of QoI and CAA fungicide resistance of Plasmopara viticola populations in vineyards of the Great Lakes region in the United States of America

The biotrophic oomycete Plasmopara viticola (Berk & Curt.) is the causal organism of downy mildew, one of the most destructive grapevine diseases throughout the world. This pathogen can severely infect all green tissues including leaves, stems, and clusters. In a severe epidemic, complete defoliation and 100% yield loss can occur under the ineffective management programme (Kassemeyer et al. 2015). The management strategies of this disease include planting resistance cultivars, proper drainage, good aeration to minimize leaf wetness, pruning the infected shoots. However, the effective management mainly relies on numerous prophylactic fungicide applications. Several multisite fungicides such as mancozeb, folpet, chlorothalonil, copper compounds and single-site fungicides such as quinone outside inhibitors (QoI) and carboxylic acid amide (CAAs) have been widely used for GDM management throughout the worldwide (Gisi and Sierotzki, 2015). However, frequent use of these single-site fungicides can lead to resistance development in P. viticola populations and ultimately leads to infective chemical management (Hermann and Stenzel, 2019) (Table 1).


Introduction
The biotrophic oomycete Plasmopara viticola (Berk & Curt.) is the causal organism of downy mildew, one of the most destructive grapevine diseases throughout the world. This pathogen can severely infect all green tissues including leaves, stems, and clusters. In a severe epidemic, complete defoliation and 100% yield loss can occur under the ineffective management programme (Kassemeyer et al. 2015). The management strategies of this disease include planting resistance cultivars, proper drainage, good aeration to minimize leaf wetness, pruning the infected shoots. However, the effective management mainly relies on numerous prophylactic fungicide applications. Several multisite fungicides such as mancozeb, folpet, chlorothalonil, copper compounds and single-site fungicides such as quinone outside inhibitors (QoI) and carboxylic acid amide (CAAs) have been widely used for GDM management throughout the worldwide (Gisi and Sierotzki, 2015). However, frequent use of these single-site fungicides can lead to resistance development in P. viticola populations and ultimately leads to infective chemical management (Hermann and Stenzel, 2019) ( Table 1).

Materials and Methods
The objectives of this study were to i) conduct survey to collect GDM samples from vineyards across MI, IN and NY in 2019 and 2020 and test for QoI and CAA resistance using sequencing ii) develop TaqMan assays to detect G1105S mutations responsible for CAA resistance in P. viticola.
A survey was conducted in 21 vineyards across three states including Michigan, Indiana, and New York. A total of 130 leaf samples were collected in 2019 and 2020. Leaf discs with P. viticola were cut and stored in petri plates at -20ºC. The DNA extraction was conducted using two methods: i) MagMax kit with Kingfisher method ii) crude DNA extraction with chelex extraction. These samples were tested for the presence of QoI and CAA resistance through sanger sequencing.

Results
The results showed that higher level of QoI resistance was found from P. viticola samples collected from juice and wine grapes collected in MI, NY, and IN. However, CAA resistance was only detected from P. viticola samples collected from wine grapevines from NY (Fig. 3).  To evaluate the limit of detection of the assay, purified DNA of CAA-resistant and CAA-sensitive P. viticola isolates were serially diluted ranging from 1 ng to 100 ag and loaded separately into a reaction. Three biological replicate reactions were used to construct the standard curve plots of the log of concentration and cycle of threshold. The assay showed sensitivity upto 0.01ng of DNA with R 2 value = 0.946, and 0.993 for wild-type allele and mutatnt allele, respectively (Figs. 4 and 5).  To test the specificity of this assay, a total of sixteen P. viticola isolates were tested using Biorad qPCR machine. The accuracy of the assay was confirmed by sanger sequencing. The results showed 100% accuracy in distinguishing both type of alleles (

Conclusions
Recent survey of many states in the United States showed that widespread resistance of QoI/ Frac 11 in P. viticola populations was present, however, high level of CAA resistance was detected in NY. Both juice and wine grapes had high resistance to QoIs but CAA resistance was only found on wine grapes.
The TaqMan-probe based assay developed in this study showed accurate and consistent detection of G1105S mutation in P. viticola. This assay will be tested for specificity using a downy mildew species on various hosts. The validation of the assay will be performed on samples collected by using different techniques such as spore traps, cotton swabs, and Toughspots. This assay can be a promising tool for rapid detection of CAA resistance by diagnostic laboratories that could help in timely and efficient disease management of grape downy mildew