Spray-Induced Silencing of Grape Powdery Mildew Genes to Reduce Powdery Mildew Growth

Powdery mildews are widespread pathogens of grapevine that are difficult to control. Resistance has emerged against all current fungicides and health consequences associated with extensive use of sulfur are beginning to surface. Therefore, the grape industry is in great need of new methods of limiting powdery mildew disease. The potential of spray induced gene silencing (SIGS) in agricultural pest control has been recently realized. The method is also useful in characterizing gene function. The efficiency of SIGS has been demonstrated to control the growth of Fusarium in barley and Botrytis in several plant host species. With the first year of AVF funding support, we showed that SIGS can also be effective in silencing powdery mildew genes, resulting in reduced powdery mildew growth and reproduction. We optimized dsRNA design, application method, dosage, timing of application and powdery mildew growth assessment for testing of SIGS against powdery mildew target genes in both powdery mildew G. orontii -Arabidopsis and powdery mildew E. necator-grapevine systems. With the continued support of AVF in Year 2, we screened powdery mildew genes prioritized to impact metabolic and regulatory pathways critical to powdery mildew colonization, growth, and reproduction. All selected G. orontii target genes had homologs in E. necator and are conserved among powdery mildews. dsRNA against individual target genes were designed, applied exogenously and the growth of powdery mildew was quantified. We had a 60% success rate in identifying efficacious novel targets, with reductions in powdery mildew proliferation ranging from 2- to 5-fold compared to the controls. The initial screening of target genes was done using the Arabidopsis-powdery mildew system as it was faster than grapevine. We then selected 6 genes that showed significant reduction in G. orontii growth on Arabidopsis via SIGS and tested them using the grapevine system. SIGS against each of these six E. necator genes showed significant reduction in powdery mildew growth and reproduction on grapevine. This growing season, we are testing SIGS against two genes that showed maximum reduction in powdery mildew growth on grapevine in research field trials, performed with the assistance of cooperator UCCE viticulture advisor George Zhuang. dsRNAs are biodegradable, flexible, and specific, with reduced negative environmental and health impacts compared with existing fungicides giving them excellent potential as future powdery mildew disease mitigation agents. Introduction of this research to vineyard managers, growers, owners and other stakeholders at the UC Davis Continuing and Professional Education course ‘Current Wine and Winegrape Research’, held in February 2019, and the ‘8th Annual Vineyards & Wineries Continuing Education Class Series’ organized by Napa County Farm Bureau Foundation and the Ag Commissioner’s Office in November 2019 was well received, and productive discussions ensued. We have successfully developed the methodology to identify novel powdery mildew targets that when silenced reduce powdery mildew disease of grapevine and are translating and testing this approach in the vineyard in a highly timely manner.

Persistence of Fungicide Resistance in Grape Powdery Mildew

The fitness of Erysiphe necator resistant to QoI/FRAC 11 fungicides was examined in terms of growth under various environmental stresses and overwintering survival and persistence. Eight Oregon E. necator isolates from the 2017 growing season were used. Four isolates were G143A mutants/QoI resistant and four were wild type/QoI sensitive, with two isolates each of MAT1-1 and MAT1-2 mating types. To analyze fitness under stress, a series of experiments were run where isolates were exposed to 10-32oC and germination, generation time, and mating were measured. The G143A mutant isolates had significantly greater germination at 14oC while all other temperatures had similar germination and growth. These results suggest that there may be a higher probability of G143A mutants infecting grapes in the early season and that QoIs should not be used during the early spring. The interaction of temperature and UVA and UVB radiative stress was examined, with results currently inconclusive as to whether there is a growth difference between wild type and G143A isolates. The same isolates were used in mating studies. Resulting chasmothecia have not fully matured and are still being observed. Previously designed real-time polymerase chain reaction (RT-PCR) primers for mating types were modified and optimized and determined to be poor at identifying mating type ratios in mixed samples. A digital droplet PCR was developed to quantify mating type ratios in mature chasmothecia, and will be used for future mating type analysis. Leaf swab samples were collected over 2018 and 2019 from 11 commercial vineyards untreated with QoIs. Samples showed increased G143A frequency from May to September. Chasmothecia sampling showed decreased G143A frequency in spring, suggesting fitness costs associated with overwintering.

The Role of Low-Molecular Weight Fungal Metabolites in Eutypa Dieback Grapevine Trunk Disease

This project focuses on understanding the fundamental chemistries that grapevine trunk disease fungi causing Eutypa canker and dieback employ in infecting and causing damage to grapevines. We will also develop treatments for GTDs by blocking the chemistries used by the pathogenic fungi. As part of this two-year project we will also disseminate our findings on both GTD causal systems and the treatments we develop to vineyard owners/managers and other interested parties. Our three Objectives for the two-year project are:
A) To better understand the role of iron-binding compounds produced by the mixed-consortia fungi involved in Eutypa dieback, with particular focus on how these compounds generate hydroxyl radicals – the most damaging form of ‘free radical’ found in biological systems – which we propose leads to stem/wood necrosis.
B) To use our enhanced understanding of Eutypa dieback in Year 2 of this research to develop treatments and management strategies for the control of Eutypa GTD.
C) To disseminate and interpret our research results to vineyards personnel to allow a better practical understanding of how Eutypa dieback chemistries function in causing the disease, and also to disseminate results of the treatment systems we will be designing to counteract the chemistries of the iron-binding compounds produced by Eutypa-consortia fungi.

We currently have been able to conduct research on the project for only 6 months, because of a funding start in June of 2019, and the time needed to ramp-up equipment and recruit a graduate student with appropriate background for the project. To date we have been able to grow three Eutypa consortia fungi under conditions of low nutrients and low iron, and demonstrate that these fungi will produce low molecular weight compounds iron-binding compounds. To produce these compounds, which have been shown to be capable of generating highly damaging hydroxyl radicals, it was necessary to simulate the low-iron and low nutrient condition inside the grapevine trunk. This has not been demonstrated previously because prior research on ironbinding compounds from these fungi was conducted using artificial media containing conventional laboratory media containing high sugar and high iron. Continuing in Year 1, we will chemically characterize the compounds and conduct assays for redox-cycling and hydroxyl radical generation. Our preliminary evidence so far indicates that several of the isolated compounds have these important properties, and thus they are ideal candidates for GTD pathogenic factors. We are also working currently with grapevine wood block assays to investigate soft rot activity as part of the pathogenicity process. These investigations are important because they provide us with targets for developing the chelating/antioxidant treatments that are the focus of our Year 2 research on controlling GTD fungi. Once these treatments have been developed and successfully tested in the lab, we are already preparing to move them into test in experimental vineyard tests we are currently developing this spring as an extension of this project. Dissemination of our results and outreach activities will continue throughout the project period.

Controlling Grapevine Trunk Diseases in California

A total of 20 vineyards belonging to 10 counties in California were sampled during summer 2019. Cordon, trunk and root tissue were taken from vines using non-destructive methods in order to isolate, analyze and study endophytic bacterial communities between healthy and diseased vines exhibiting typical trunk disease symptoms. A collection of over 1,750 isolates was obtained, from which 27.43% has been screened for their potential antifungal effect against the main GTD-causing pathogens in vitro. A set of 80 bacterial isolates was selected after a first screening against Neofusicoccum parvum, exhibiting >40% of inhibition of the pathogen mycelium. Phylogenetic analyses showed that 57 isolates belong to the genus Bacillus, 8 isolates to Variovorax, 6 isolates to Pseudomonas, 3 isolates to Stenotrophomonas, 2 isolates to Pantoea, 2 isolates to Lysobacter, 1 isolate to Lysinibacillus and 1 to Klebsiella. These isolates are currently being tested using the same methodology against other important GTD pathogens (Diplodia seriata, Eutypa lata, Diaporthe ampelina, Phaeomoniella chlamydospora, Phaeoacremonium minimum and Ilyonectria liriodendri) and a second selection will be performed with bacterial isolates exerting the higher percentages of inhibition against the majority of pathogens in order to be tested in greenhouse and field experiments. Furthermore, a fungicide trial was set over the summer of 2019 in three different nurseries (Winters, Wasco and Bakersfield), including chemical and biological products, using a vacuum chamber to infiltrate the fungicides through the vessels of dormant cuttings prior the grafting process. Fourteen treatments were included, and vines were grafted. Callus formation was evaluated 18 days after the treatments and results indicate frequencies of callusing ratings were similar among treatments, being in average 18.1% of rootstocks showing 100% of callusing, 48.0% showing 80-99%, 22,4% showing 60-79%, 7.7% showing 40-59% and 4.9% showing below 40%. Plants were further planted in pots according to each nursery protocols and kept in the facilities for approximately 3 months. In October 2019, actively growing vines were transported to the UC Davis Department of Plant Pathology field station located in Davis, CA, to be planted in the field under a completely randomized block design. In one of the nurseries, treated vines were planted in the ground after the callusing. The rooting of these plants will be evaluated in March during their transplantation to the field. Trunk disease incidence and severity will be evaluated yearly during the summer season.

Deep Sequencing for Trunk Disease Diagnostics

The aim of this multi-year project was to develop rapid and cost-effective diagnostic methods for detection, identification, and quantification of trunk pathogens in asymptomatic and symptomatic grape wood. Healthy vines are essential for the successful establishment and sustainability of all grape production systems. Since wood pathogens may remain asymptomatic in young, non-stressed vines, propagation material may contain latent fungal infections and may become symptomatic after planting and serve as a source of inoculum for further infections of potentially clean plants. Methods of virus detection and eradication have been crucial in ensuring that the material in germplasm repositories and clean plant programs is free of known viruses. There remains much to be developed in terms of fungal pathogen detection. Our laboratories have developed comprehensive genomic information on several ascomycetes associated with the most common and aggressive trunk diseases, which provides the unprecedented opportunity for the implementation of new sequencing based diagnostic tools that take advantage of Next Generation Sequencing (NGS) technologies. By allowing the testing of mother plants in foundation blocks and propagation material in nurseries, we expect that the applications of deep sequencing diagnostics will help establish a certification program for trunk pathogen-free germplasm and reduce the amount of trunk pathogens introduced into vineyards at planting as well as the incidence of young vine decline. Deep-sequencing diagnostics will also help identify disease-causing organisms associated with diseased vines in older vineyards.

In the 1st year of the project (2015 – 2016) we collected diseased wood material from commercial vineyards and characterized the associated fungal pathogen species using traditional methods, such as morphological and sequence-based identification of purified fungal colonies. We used these samples to determine how effective ITS-sequencing, meta-genome sequencing and metatranscriptome sequencing approaches are in identifying and quantifying pathogenic species directly in planta. Data simulations allowed us to determine what mapping algorithm was the most specific and sensitive in detecting trunk pathogens both qualitatively and quantitatively. All NGS methods we tested were in agreement with traditional diagnostic methods, but also allowed us to detect simultaneously multiple pathogen species with no need of hands-on sample culturing and colony purification. Additionally, unlike traditional diagnostics, which are strictly qualitative, NGS approaches allowed us to determine the relative abundances of the different infecting species. This work was published in Molecular Plant Pathology (Morales-cruz et al., 2017). Among all methods
tested, ITS-seq is still the most cost-effective until library preparation costs for RNA and DNA-seq do not decline significantly. For this reason, ITS-seq was chosen for further protocol optimization to improve sensitivity and specificity for diagnostics purposes. In the second year of the project (2016-2017), we (a) confirmed that NGS allows the detection with high specificity of actively infecting pathogens when vines are experimentally infected with individual pathogen strains; (b) established that NGS detection is quantitative and allows to differentiate between diseased and healthy vines; (c) developed a protocol for testing dormant cuttings and started testing cuttings provided by a commercial nursery. In the 2016-2017 funding cycle, we also developed a new DNA extraction protocol that reduced the time required for processing and the amounts of sample, reagents and waste. In the second phase on the project, our effort focused on the development and optimization of a new set of optimized primers for ITS-seq designed specifically to target the ITS of grapevine trunk pathogens. The primers as well as the method are publicly available and described in a peer-reviewed article published in December 2018 (Morales-cruz et al., 2018).

In summary, in these five years we have:
1. Applied NGS to trunk pathogen diagnostics and demonstrated that NGS provides qualitatively and quantitatively accurate simultaneous identification of multiple trunk pathogens directly from grapevine wood samples (Morales-Cruz et al., 2017 Mol Plant Pathol).
2. Developed a new protocol with optimized diagnostic markers for NGS ITS-seq diagnostics of trunk diseases, which is publicly available and described in detail in Morales-Cruz et al. (2018; BMC Microbiology).

We publish all protocols, which can be used freely by diagnostic and research labs. We are now seeking funds to survey propagation material and determine the association between pathogen contamination in propagation material and trunk disease incidence in young vineyards.

Investigation of the impact of grapevine red blotch-associated virus (GRBaV) on grapevine health and subsequent grape and wine composition and style

Since its identification in 2011, grapevine red blotch disease has been found to be wide spread in the United States. This disease is caused by grapevine red blotch virus (GRBV, in 2017 the name changed from grapevine red blotch-associated virus (GRBaV)) infection of grapevines. Over the past four years, we have investigated the impact of GRBV on grape composition and resulting wine quality across varieties, rootstocks, seasons and sites. This investigation completed a two-year study on Cabernet Sauvignon (CS) and Merlot (ME) grape varieties, examining the impact of GRBV on grapes at harvest and on the resulting wine composition. Additionally, the effect of rootstock on disease expression was also investigated as the CS grapevines were grafted onto two different rootstocks: 110R and 420A.
Grapes through development were analyzed for sugar and anthocyanin content, as well as pH and TA levels. The phenolic profiles of harvested grapes and final wines were analyzed by RP-HPLC and a modified protein precipitation (PP) assay. Volatile profiles of harvested grapes and final wines were analyzed using HS-SPME-GC-MS. Results from these analyses were compared to the sensory evaluation of the four wine treatments through a trained panel.
Consistently through the four years, symptomatic grapevines (RB (+)) resulted in lower sugar content, less anthocyanin accumulation, and higher titratable acidity (TA) when compared to asymptomatic grapevines (RB (-)). Due to these observations, chaptalization was used to adjust the soluble sugars (SS) (and therefore ethanol content of final wines) of the diseased grapes (RB (+) S) at crush to be the same as the healthy grapes. Chaptalization could potentially determine if ethanol concentration differences between wines made from RB (-) and RB (+) grapes were contributing to phenolic differences in the wines. In another treatment, diseased grapes were harvested a second time (RB (+) 2H) when the SS reached similar values as RB (-) grapes.
The results of this investigation confirm previous findings, that disease expression is dependent on genotypic and environmental factors. Anthocyanin and sugar accumulation were significantly lower in RB (+) grapes than RB (-), yet, the level of disease impact varied from rootstock, variety and location. Vine physiological measurements and phenolic profile analysis on harvested grapes indicated a potential larger impact on RB (+) vines on 420A rootstocks. In general a second harvest was successful at increasing anthocyanin, some phenolic, and volatile concentrations in infected grapes. The phenolic extraction during fermentation was followed and revealed that longer hang time (RB (+) 2H) increased extraction more than chaptalization (RB (+) S). Chemical analysis of the final wines indicated that both mitigation strategies (chaptalization and sequential harvesting) alleviated some of the differences due to disease status. This indicate that both ethanol content and cell wall integrity may play a role in phenolic extractability during winemaking.

Assessing Fungicide Resistance of grape powdery mildew in Wine, Table and Raisin Grapes

The 2018 assessment campaign processed >3000 samples from the Western US grape production
regions. Analysis of these ToughSpot samples for resistance to quinone outside inhibitor (QoI)
fungicides (FRAC Group 11) using the G143A qPCR assay indicated there is still widespread
resistance throughout Western grape production regions (71% samples with the G143A) but that
resistance is decreasing. Examination of samples from the Willamette Valley of Oregon indicates
that when growers rotate away from using QoI and DMI, the G143A allele occurrence significantly
decreases in the population (31% with G143A). These results were confirmed when isolate and
field sample DNA underwent genotyping by sequencing analysis of the cytb gene. These results
are similar to the QoI resistance observed throughout Oregon in 2015 and 2016. Analysis of these
samples using various molecular techniques and fungicide resistance bioassays to determine
resistance to demethylation inhibitor (DMI) fungicides (FRAC group 3) and succinate
dehydrogenase inhibitor (SDHI) fungicides (FRAC group 7) is ongoing. We have identified 7 E.
necator isolates that are resistant to at least one SDHI fungicide with only one isolate resistant to
more than one SDHI. Sequencing results indicate only one isolate has a known genetic mutation,
thus further sequencing is required. The Y136F qPCR assay for DMI resistance readily detects
the Y136F mutation associated with DMI resistance and can be used to determine DMI resistance
in single spore isolates. Its use on field samples is difficult to interpret because of the multiple
genotypes associated with DMI resistance. A leaf sample collection method was developed that
decreases sample collection and processing time by 40% and is cheaper to process. In addition, a
bark sampling method was developed that agrees with early spring G143A frequency, thus
allowing growers to assess their risk to QoI resistance during the dormant season. Both assays
will be made available to the industry in 2019. Using this project and Oregon Wine board funding
as a foundation for demonstrating grower support, we obtained funding from USDA-SCRI
program to significantly expand research efforts to develop regional networks for managing and
mitigating fungicide resistance.

Interaction of red blotch virus (GRBV) and deficit irrigation on grapevine water relations, disease development, and vine productivity

The second year of a field experiment with two irrigation treatments – wet (W) and dry (D) – and two vine disease statuses – healthy (GRBV-) and infected (GRBV+) – was continued in a commercial vineyard to understand the interaction between GRBV infection and deficit irrigation on disease development, vine productivity, and fruit quality. W vines were irrigated at 100% of crop evapotranspiration (ETc), while D vines received water at 50% ETc. Within each irrigation treatment, GRBV- and GRBV+ vines (split-plot) that were previously identified in early 2017 based on symptomology data from 2016. The identified vines were confirmed as GRBV+ and GRBV- by PCR-based assays. GRBV- vines from 2017 were re-tested in early 2018 to confirm disease status.
In both years, measurements of vine water status (midday stem water potential; stem) were made at regular intervals throughout the growing season beginning just after berry set until just before harvest. Similarly, disease severity was recorded every week after the first symptom appearance was observed on GRBV+ vines. At harvest, berry samples were collected for berry size and compositional analyses; and vine yield and yield components were determined.
With respect to vine water relations and gas exchange, there were no significant interactions among experimental treatments. Irrigation treatment and disease status both impacted these aspects of vine physiology, but they acted independently, with water deficits consistently reducing vine water status, and GRBV infection consistently increasing it. In other words, GRBV infection had the same effect on vine water status (stem water potential) under both well-watered and deficit conditions. However, the significant impact of GRBV infection on vine water status only arose post-veraison – at the same time that foliar symptoms became visible. The increase in post-veraison water status under GRBV+ conditions was likely a consequence of reduced stomatal conductance, which in turn reduced net photosynthesis.
Berry development was similarly impacted by the treatments independently, with consistently larger berries in W and GRBV+ vines. This was observed at nearly every sample date in each year, but differences between vines of different disease status only became significant post-veraison. TSS were also only significantly different between GRBV- and GRBV+ vines post-veraison, and there was a limited impact of irrigation treatment. In contrast, pH and TA were more variable among treatments and years, suggesting that GRBV has a limited effect on organic acid metabolism.
Irrigation treatment and disease status impacted skin and seed phenolic concentrations to varying degrees over two seasons. Whereas irrigation treatment and disease status impacted skin phenolic concentration together, disease status alone impacted seed phenolic concentration. In both years, skin anthocyanin concentration was increased with deficit irrigation – in both GRBV- and GRBV+ vines – but only increases in GRBV- vines were statistically significant. Conversely, skin tannin and iron-reactive phenolic concentrations were variably affected by treatments, and results were not consistent between years. In seeds, there were no effects of the irrigation treatments, but disease status significantly reduced both tannin and iron-reactive phenolic concentrations. This effect was consistent between years. All together, these results
2
suggest that the genetic control of phenolic metabolism by GRBV infection is stronger than the environmental control due to vine water deficits. Furthermore, experimental results suggest that keeping vines well-watered may mitigate some of the negative effects of GRBV infection, but ultimate changes in secondary metabolism due to GRBV infection may necessitate using infected fruit for different wine programs (e.g. rosé and/or sparkling) or blending with lots from healthy vineyards.
In contrast to 2017 data on disease severity, significant differences on rate of disease progression as well as disease severity were observed in 2018 between the wet and dry treatments. The vines in wet irrigation treatment showed significantly low disease severity at harvest and two weeks prior to harvest. The differences were observed as significant increase in vine canopy in irrigation treatment compared to vines in dry treatment; as a result percent of symptomatic leaves in wet treatment vines were less compared to dry treatment vines. Even though, the virus expression remained same (symptomatic) within wet and dry treatments, it would be informative to assess the status of virus (quantity) within each treatment. Furthermore, the carry over effect of less severe vines on vine health as well as fruit qualities would be an additional information on long-term management of GRBV infected vines. On this regard, we wanted to continue the project for another year, where the focus will be on virus quantification, vine health, and fruit quality as a result of carry over effects within less severe vines while keeping all treatments as is.

Understanding and managing the trunk disease Esca

Summary: Vine surgery (also known as trunk renewal) has been shown to be effective against
Eutypa dieback, but trunk pathogens cause mixed infections. Vineyards are thus likely to be
affected by more than one trunk disease. Furthermore, Eutypa dieback is not even the most
common trunk disease in California. It is important to also test the efficacy of vine surgery
against Esca because infections by two common Esca pathogens, Phaeomoniella chlamydospora
and Phaeoacremonium minimum, can occur at the base of the trunk, either during the
propagation process in the nursery or potentially by infections through roots after being planted
in the vineyard. To accomplish this objective, we established a replicated field trial in a
Sauvignon blanc vineyard in Lake County, CA. Vine surgery was done on vines with leaf
symptoms on shoots growing from only one cane, the idea being that the infection might be
restricted to that portion of the vasculature. In February 2018, we chain-sawed such vines above
the graft union and noted the presence of wood symptoms in 98 total data vines, which were
located among three experimental blocks in the vineyard. The vast majority of the 98 data vines
had wood symptoms, although they were restricted to less than 10% of the cross-section of the
trunk. This observation is important and is not so discouraging because a previous study of vine
surgery, combined with annual fungicide treatments of the cut trunk, showed that a higher
proportion of vines with wood symptoms on less than 20% of the trunk cross-section had normal
vigor and yields 8 years later, compared to vines with more advanced wood symptoms. In the 3rd
year after vine surgery (2020), the data vines will be fully trained and (assuming they are
healthy) are likely to be producing normal yields. After all, this is the goal of vine surgery, to
return vines to productivity and faster than would be possible with a replant. In January 2020,
we will submit a proposal to evaluate the progress of vine surgery, based on presence/absence of
leaf symptoms, vine growth, yields, and juice quality. The second objective of our study was to
identify the Basidiomycete wood-rotting fungi that cause Esca. For many years, there was an
untested assumption that these fungi infected vines after they were already infected by
Ascomycetes Phaeomoniella chlamydospora or Phaeoacremonium species. Our pathogenicity
tests show the wood-rotting fungi are pathogens when inoculated to vines alone. Because the
Basidiomycetes are assumed to be secondary pathogens, management practices have not been
evaluated against them. The most widespread Basidiomycete was Fomitiporia polymorpha.
This species was previously reported from one vineyard in California, so our finding expands its
range within the state. We described a new Basidiomycete species Inonotus vitis, which
represents the first report of Inonotus on grape in the Americas. Pathogenicity studies will be
established in January 2019 to evaluate whether I. vitis is pathogenic. If it is, then it might make
sense to include this species (along with F. polymorpha) in evaluations of pruning-wound
protectants.

Deep sequencing for trunk disease diagnostics

The aim of this multi-year project is to develop rapid and cost-effective diagnostic methods
for detection, identification, and quantification of trunk pathogens in asymptomatic and
symptomatic grape wood. Healthy vines are essential for the successful establishment and
sustainability of all grape production systems. Since wood pathogens may remain
asymptomatic in young, non-stressed vines, propagation material may contain latent fungal
infections and may become symptomatic after planting and serve as a source of inoculum
for further infections of potentially clean plants. Methods of virus detection and eradication
have been crucial in ensuring that the material in germplasm repositories and clean plant
programs is free of known viruses. There remains much to be developed in terms of fungal
pathogen detection. Our laboratories have developed comprehensive genomic information
on several ascomycetes associated with the most common and aggressive trunk diseases,
which provides the unprecedented opportunity for the implementation of new sequencingbased
diagnostic tools that take advantage of Next Generation Sequencing (NGS)
technologies. By allowing the testing of mother plants in foundation blocks and propagation
material in nurseries, we expect that the applications of deep sequencing diagnostics will
help establish a certification program for trunk pathogen-free germplasm and reduce the
amount of trunk pathogens introduced into vineyards at planting as well as the incidence of
young vine decline. Deep-sequencing diagnostics will also help identify disease-causing
organisms associated with diseased vines in older vineyards.
In the 1st year of the project (2015 – 2016) we collected diseased wood material from
commercial vineyards and characterized the associated fungal pathogen species using
traditional methods, such as morphological and sequence-based identification of purified
fungal colonies. We used these samples to determine how effective ITS-sequencing, metagenome
sequencing and meta-transcriptome sequencing approaches are in identifying and
quantifying pathogenic species directly in planta. Data simulations allowed us to determine
what mapping algorithm was the most specific and sensitive in detecting trunk pathogens
both qualitatively and quantitatively. All NGS methods we tested were in agreement with
traditional diagnostic methods, but also allowed us to detect simultaneously multiple
pathogen species with no need of hands-on sample culturing and colony purification.
Additionally, unlike traditional diagnostics, which are strictly qualitative, NGS approaches
allowed us to determine the relative abundances of the different infecting species. This
work was published in Molecular Plant Pathology (Morales-cruz et al., 2017). Among all
methods tested, ITS-seq is still the most cost-effective until library preparation costs for
RNA and DNA-seq do not decline significantly. For this reason, ITS-seq was chosen for
further protocol optimization to improve sensitivity and specificity for diagnostics purposes.
In the second year of the project (2016-2017), we (a) confirmed that NGS allows the
detection with high specificity of actively infecting pathogens when vines are experimentally
infected with individual pathogen strains; (b) established that NGS detection is quantitative
and allows to differentiate between diseased and healthy vines; (c) developed a protocol for
testing dormant cuttings and started testing cuttings provided by a commercial nursery. In
the 2016-2017 funding cycle, we also developed a new DNA extraction protocol that
reduced the time required for processing and the amounts of sample, reagents and waste.
In the 3rd and 4th year of the project, our effort focused on the development and
optimization of a new set of optimized primers for ITS-seq designed specifically to target
the ITS of grapevine trunk pathogens. The primers as well as the method are publicly
available and described in a peer-reviewed article published in December 2018 (Moralescruz
et al., 2018).
In summary, in these four years we have:
1. Applied NGS to trunk pathogen diagnostics and demonstrated that NGS provides
qualitatively and quantitatively accurate simultaneous identification of multiple trunk
pathogens directly from grapevine wood samples (Morales-Cruz et al., 2017 Mol
Plant Pathol).
2. Developed a new protocol with optimized diagnostic markers for NGS ITS-seq diagnostics of trunk diseases, which is publicly available and described in detail in
Morales-Cruz et al. (2018; BMC Microbiology).