Causal Factors of Macrophoma Rot Observed on Petit Manseng Grapes

Causal factors of Macrophoma rot observed on Petit Manseng grapes

Nicole Encardes

Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Master of Science in Life Science in Horticulture

Tony Wolf, Co-Chair Mizuho Nita, Co-Chair Anton Baudoin

April 28, 2020 Blacksburg, Virginia

Keywords: grapes, Macrophoma rot, Neofusicoccum ribis, sunlight exposure, infection, fungicide assay

Nicole Encardes ABSTRACT

Macrophoma rot is a general term for fruit rots of Vitis spp. caused by the fungus Neofusicoccum ribis (syn. Botryosphaeria ribis) or closely related or renamed taxa, including Botryosphaeria dothidea. While mainly observed as a fruit pathogen of muscadine grape, the disease has recently been observed on bunch grapes in Virginia. Isolates (N = 835) were collected from Petit Manseng fruit clusters from seven Virginia vineyards in 2018 and 2019. A subset of these isolates was sequenced using three primer sets (ITS, RPB2, and EF). The preliminary result showed a single taxonomic strain of N. ribis. A controlled inoculation study of Petit Manseng clusters verified that infection could occur anytime between bloom and 2 weeks post-veraison; however, both the mean cluster incidence and the severity of Macrophoma rot did not differ from each other at any growth stage during the season. A season-long cluster exposure experiment showed that any amount of sun exposure significantly increased Macrophoma rot severity compared to shaded clusters, and that full sun exposure was associated with greatest rot severity. This finding contravenes current management recommendations for Macrophoma rot, and it raises yet unanswered questions as to why exposed clusters are more susceptible to Macrophoma rot than are shaded clusters. An in vitro fungicide assay study using nine fungicides identified captan, thiophanate-methyl, and tetraconazole as potential candidates for management of Macrophoma rot which need to be investigated further. Causal factors of Macrophoma rot observed on Petit Manseng grapes

Nicole Encardes PUBLIC ABSTRACT

Macrophoma rot is a general term for fruit rots of grapes caused by the pathogenic fungi in the family Botryosphaeriaceae. The rot is mainly observed on Muscadine grapes, but recently more cases were found on a wine grape cultivar Petit Manseng in Virginia. Macrophoma rot symptoms begin as dark brown, circular lesions on the surface of the berry and look similar to sunburn and other fruit rots. As the disease progresses, the lesion envelopes the entire berry and black fruiting bodies develop. Severe cases may lead to crop loss. The same group of pathogens is also associated with rots on other crops including apple, pear, olive, and kiwis. Very little is known about the disease cycle and the control of Macrophoma rot, therefore, an investigation into this fungal pathogen was needed. Multiple studies with the wine grape variety Petit Manseng were conducted during the 2018- 2019 growing seasons, including a survey, leaf removal trial, and an inoculation study. Results showed that a species called Neofusicoccum ribis was found in vineyards across northern and central Virginia based on the genetic identification of fungal isolates collected at seven vineyards in those areas. Macrophoma symptoms were observed to be more prevalent and severe in more exposed clusters based on a leaf removal experiment. An artificial inoculation experiment revealed that grape clusters are susceptible to Neofusicoccum ribis at any time during the season. Based on the screening of nine fungicides, three chemicals (captan, thiophanate-methyl, and tetraconazole) showed promising results as possible management tools for Macrophoma rot. The knowledge collected will lead to an increase in understanding of this fungal pathogen and to further studies to manage Macrophoma rot.

Acknowledgement

I would like to thank my advisors, Dr. Tony Wolf and Dr. Mizuho Nita for their help and direction with these projects. I would like to thank lab members for their help with my experiments: Silvia Liggieri, Tremain Hatch, Diana McHenry, Dana Melby, Jonathan Ames, and Akiko Mangan as well as my emotional support, Peter Yupari. I would also like to thank my other committee member, Anton Baudoin for his support and guidance. Lastly, I would like to thank my source of funding: The Virginia Wine Board.

Table of Contents

List of Tables vi List of Figures viii Chapter 1: Introduction and Purpose of Study 1 References 12 Chapter 2: Prevalence of Macrophoma rot in Virginia Introduction 17 Methods and Materials 18 Results 21 Discussion 25 References 44 Chapter 3: Fruit Exposure and Expression of Rot and the Timing of Susceptibility to Infection Introduction 46 Methods and Materials Fruit Exposure and Expression of Rot 48 The Timing of Susceptibility to Infection 51 Results Fruit Exposure and Expression of Rot 54 The Timing of Susceptibility to Infection 56 Discussion Fruit Exposure and Expression of Rot 57 The Timing of Susceptibility of Rot 58 References 75 Chapter 4: In vitro Fungicide Assays to Determine effective Fungicides for Macrophoma rot Management Introduction 77 Methods and Materials 78 Results 80 Discussion 81 References 88 Chapter 5: Conclusions and Future Research 89

List of Tables

Table 2.1. Primer sequences for the EF-D, ITS, and RPB2 gene regions that were used to identify isolates as Neofusicoccum ribis. 27

Table 2.2. Recipe for PCR with AmpliTaq GoldTM 360 Master Mix used for sequencing the RPB2 gene region to identify isolates as Neofusicoccum ribis. 28

Table 2.3. Thermal Profile for PCR with AmpliTaq Gold 360 Master Mix used for sequencing the RPB2 gene region to identify isolates as Neofusicoccum ribis. 29

Table 2.4. Recipe for PCR with New England BioLabs Taq DNA Polymerase used for sequencing the ITS and EF1-α gene regions to identify isolates as Neofusicoccum ribis. 30

Table 2.5. Thermal Profile for PCR with New England Biolabs Taq DNA Polymerase used to sequence the ITS and EF-D gene regions to identify isolates as Neofusicoccum 31 ribis.

Table 2.6. The percentage of sequenced Neofusicoccum isolates out of collected isolates from each survey vineyard in 2018 and 2019. 32

Table 2.7. Neofusicoccum species from GenBank used in the phylogenetic analysis to produce a neighbor-joining tree. 33

Table 2.8. Primary chemistry for Petit Manseng survey vineyards at the time of harvest in 2018 and 2019a used to judge the maturity of the grapes including soluble solids (Brix), pH, and titratable acidity (TA). 35

Table 2.9. Macrophoma rot disease incidence (the presence or absence of Macrophoma- like symptoms) for Petit Manseng survey vineyards at the time of harvest in 2018 and 2019 measured as the percentage of clusters that had Macrophoma-like symptoms. 36

Table 2.10. Average mean daily monthly temperature, monthly rainfall, growing degree days (GDD), and ambient solar radiation at the Agricultural Research Extension Center (AREC) of Winchester (VA) from 1 April through 31 October in 2018 and 2019. 37 Table 3.1. Weekly primary chemistry among Petit Manseng leaf removal treatment in 2018 and 2019a used to judge the maturity of the grapes including soluble solids (Brix), pH, and titratable acidity (TA). 61

Table 3.3. Dates of inoculation (Inoc.), harvest (Harv.) and assessment (Assess.) of bagged Petit Manseng clusters as part of the inoculation trial in 2018 and 2019. 63

Table 3.4. Enhanced Point Quadrat Analysis (EPQA) averages for each treatment in the Petit Manseng leaf removal trial in 2018 and 2019 including occlusion layer number (OLN), cluster exposure layer (CEL), leaf exposure layer (LEL), cluster exposure flux availability (CEFA), and leaf exposure flux availability (LEFA) for comparison of equal treatment application between seasons. 64

Table 3.5. Components of yield among Petit Manseng leaf removal treatments in 2018 and 2019a for comparison of equal vine size between seasons. 65

Table 3.6. Primary chemistry among bagged Petit Manseng treatments in 2018 and 2019a used to judge the maturity of the grapes including soluble solids (Brix), pH, and titratable acidity (TA). 66

Table 4.1. List of fungicides and their active ingredients (A.I.) used for in vitro fungicide assays with the calculated concentration and dilutions needed for the serial dilutions performed during the protocol for making fungicide amended 24-well plates (Acumedia®, Neogen Company Lansing, MI) used in the in vitro fungicide assays. Mode of Action (MOA) include Quinone outside Inhibitors (QoI), Succinate-dehydrogenase inhibitors (SDHI), Host-Plant defense induction (HPDI), DeMethylation Inhibitors (DMI), Methyl Benzimidazole Carbamates (MBC), and multi-site inhibitors. 83

List of Figures

Figure 1.1. Conidial morphology of Neofusicoccum ribis produced in culture on water agar and pine needles. (Source: Slippers et al., 2004) 10

Figure 1.2. Black pycnidia on Macrophoma rot symptomatic Petit Manseng grapes (top left) and a developing lesion (top right) on a cluster found in a Virginia vineyard. 11

Figure 2.1. Geographic locations of Petit Manseng survey vineyards in northern and central Virginia. 38

Figure 2.2. All isolates that were collected from Petit Manseng survey vineyards in 2018 and 2019 that were sequenced using the ITS gene region for identification. 39

Figure 2.3. Neighbor-joining tree of 59 concatenated consensus sequences (EF, ITS, and RPB2). Numbers on the branches of the tree are bootstrap values from 100,000 resampling replicates (below 70 are not shown). Neofusicoccum buxi (CBS113714) was the outgroup. Neofusicoccum ribis is highlighted in red and the collected sample (NE0001) is highlighted in blue. Isolate NE0001 grouped with two N. ribis accessions (bootstrap = 95%), within a larger clade of N. batangarum (Scabby Canker of cactus pear) and N. umdonicola (bootstrap = 96%). Isolate NE0001 was 99.6% identical (1,326/1,331 bp) to N. ribis CBS121.26 and 100% identical (1,331/1,331 bp) to N. ribis CMW7773. 40

Figure 2.4. Brix versus Macrophoma rot disease incidence (presence or absence of Macrophoma-like symptoms in a cluster) in both 2018 and 2019 from Petit Manseng survey vineyards. 41

Figure 2.5. pH versus Macrophoma rot disease incidence (presence or absence of Macrophoma-like symptoms in a cluster) in both 2018 and 2019 from Petit Manseng survey vineyards. 42

Figure 2.6. Titratable Acidity (TA) versus Macrophoma rot disease incidence (presence or absence of Macrophoma-like symptoms in a cluster) in both 2018 and 2019 from Petit Manseng survey vineyards. 43

Figure 3.1. The mean cluster Macrophoma rot disease incidence (presence or absence of Macrophoma-like symptoms in a cluster) in 2018 and 2019 from the Petit Manseng leaf removal field trial. Measured as the percentage of clusters that showed Macrophoma-like symptoms. Treatments include full sun, half sun, and full shade as levels of sunlight exposure. Means sharing a letter are not significantly different (Tukey-adjusted comparisons). 67

viii

Figure 3.2. The mean cluster Macrophoma rot disease severity (percentage of cluster affected by Macrophoma-like symptoms) in 2018 and 2019 from the Petit Manseng leaf removal trial. Treatments include full sun, half sun, and full shade as levels of sunlight exposure. Means sharing a letter are not significantly different (Tukey-adjusted 68 comparisons).

Figure 3.3. The mean cluster Botrytis bunch rot disease severity (percentage of cluster affected by Botrytis like symptoms) in 2018 from the Petit Manseng leaf removal trial. Treatments include full sun, half sun, and full shade as levels of sunlight exposure. Means sharing a letter are not significantly different (Tukey-adjusted comparisons). 69

Figure 3.4. All isolates that were collected from clusters in the Petit Manseng leaf removal trial in 2018 and 2019 that were sequenced using the ITS gene region for identification. 70

Figure 3.5. The mean cluster Macrophoma rot disease incidence (the presence or absence of Macrophoma-like symptoms) in 2018 and 2019 from the Petit Manseng field inoculation trial. Measured as the percentage of clusters that showed Macrophoma-like symptoms. Treatments include bloom, pea-sized, veraison, and 2-weeks post veraison plus a control of no inoculation as times during the season that inoculations were performed. Means sharing a letter are not significantly different (Tukey-adjusted comparisons). 71

Figure 3.6. The mean cluster Macrophoma rot disease severity (the percentage of the cluster affected by Macrophoma-like symptoms) in 2018 and 2019 from the Petit Manseng field inoculation trial. Treatments include bloom, pea-sized, veraison, and 2-weeks post veraison plus a control of no inoculation as times during the season that inoculations were performed. Means sharing a letter are not significantly different (Tukey-adjusted comparisons). 72

Figure 3.7. All isolates that were collected from bagged Petit Manseng clusters in 2018 and 2019 that were sequenced using the ITS gene region for identification. 73

Figure 3.8. The mean cluster Botrytis bunch rot disease severity (percentage of cluster affected by Botrytis like symptoms) in 2018 from the Petit Manseng field inoculation trl. Treatments include bloom, pea-sized, veraison, and 2-weeks post veraison plus a control of no inoculation as times during the season that inoculations were performed. Means sharing a letter are not significantly different (Tukey-adjusted comparisons). 74

Figure 4.1. Sample layout of a 24-well plate (Acumedia®, Neogen Company Lansing, MI) used in the in vitro fungicide assays. Each fungicide was serial-diluted into 5 different concentrations and pipetted into the 24-well plate so that each concentration was pipetted into 4 wells per plate plus 4 wells containing media that wasn’t amended with a fungicide. Each plate had 1 fungicide and was divided into 2 sections for 2 different isolates. Each fungicide was repeated twice for each isolate meaning that 2 amended 24-well plates were made for each fungicide and for each pair of isolates. 86

Figure 4.2. The effect of fungicide a.i. concentration on the mean percent mycelial growth inhibition by isolates on a logarithmic scale. A total of 22 N. ribis isolates from Virginia were examined against nine different mode of action fungicides using fungicide-amended PDA in 24-well culture plate (Acumedia®, Neogen Company Lansing, MI) in 2019. There were 6 fungicide-concentration combinations per isolate which was repeated 4 times for each fungicide. Each dot represents the mean of the growth inhibition for the fungicide- concentration combination and the error bars are based on the standard error. All isolates are shown in each cell, although they overlap due to having similar means and cannot be discerned from one another. 87

Chapter 1: Introduction and Significance of the Study

Neofusicoccum ribis is an ascomycete fungus that belongs to the family

Botryosphaeriaceae. This genus was originally classified as one of Botryosphaeria species

(Botryosphaeria dothidea and Botryosphaeria ribis) with a Fusicoccum anamorph but has since been reclassified as Neofusicoccum based on the diversity of the large subunit and beta-tubulin genes (Crous, et al., 2006; Phillips, et al., 2008; Sakalidis, et al., 2013). Among species in the genus Neofusicoccum, N. ribis and N. parvum, are two common pathogenic species, and are often confused due to the similarity in the ITS gene region and colony morphology (Sakalidis, et al., 2011b). However, the classification based on the Genealogical Sorting Index (GSI) and the

RPB2 gene created the boundary between the two species (Sakalidis, et al., 2011b).

Neofusicoccum ribis mainly reproduces through budding from a complex asexual fruiting body called a pycnidium and the sexual stage is rarely found on hosts and hasn’t been replicated in the lab (Ma, et al., 2001). Within the pycnidium are layers of conidiophores which produce conidia. They are hyaline, aseptate and ellipsoidal in shape with rounded ends while the pycnidia are individual, small, and spherical and range from 16.5–24.0 μm in length and 3.5–6.5 μm in width (Ngobisa et al., 2013; Úrbez-Torres, et al., 2006; Wilcox, et al., 2015) (Figure 1.1). Rain or other wetting events stimulate the release of pycnidiospores, which are then spread by wind and splashing rain throughout the growing season to cause infection, but are mostly captured in the spring (Amponsah, et al., 2009; Wilcox, et al., 2015). The optimal temperature for germination of pycnidiospores is 25-30 qC in free water and they do not germinate well without free water

(Shafi, et al., 2018; Sutton, 1981; Tennakoon, et al., 2018a). When grown as a culture on petri dishes containing 4% potato dextrose agar (PDA), isolates developed a white aerial mycelium

which gradually turned green to dark green after a 4- to 5-days of incubation at room temperature

(Úrbez-Torres, et al., 2006).

Macrophoma rot caused by N. ribis, and closely related species, is a very important disease in muscadine grapes in the Southeastern United States. Symptoms on muscadines are the same as those on bunch grapes: circular, flat, sunken lesions form on the fruit and then expand to form a brown, soft rot across the entire fruit (Millholland, 1991). An example of the lesions found on bunch grapes can be seen in Figure 1.2. Infected fruit then shrivel and fall from the vine and become hollow shells covered in pycnidia (Millholland, 1991). These symptoms usually occur near berry maturity in muscadines as well as bunch grapes (Andersen, et al., 2003; Wilcox, et al., 2015). Because N. ribis can infect a wide variety of hosts, it’s believed that the pathogen can overwinter on other hosts as well as on mummified berries which makes it hard to control

(Andersen, et al., 2003).

Neofusicoccum ribis, and other closely related Neofusicoccum species, are commonly found in a canker in grapevines but can be found on a wide range of hosts, including the wild

Ribes species, such as currants and gooseberries, for which it is named (Phillips, et al., 2008).

Surface wounds, such as pruning cuts, are considered the point of entry for N. ribis, and thus, this pathogen is often isolated from failed graft unions (Billones-Baaijens, et al., 2013a; Niekerk, et al., 2006). Detection of N. ribis can be difficult at the time of grafting since the infected vines can be asymptomatic (Billones-Baaijens, et al., 2013a). Worldwide distribution of contaminated nursery stock may explain the extensive geographic distribution and host range (Sakalidis, et al.,

2013). Spores or mycelia can also be moved with pruning tools and soils that may contain infected tissues (Billones-Baaijens, et al., 2013a).

Pathogens in the Neofusicoccum genus have been found to be highly pathogenetic

(Billones-Baaijens, et al., 2013b). Infections on the grapevine cause a brown discoloration of the vascular tissues, often called streaking, which can be spread extensively within the infected trunk

(Niekerk, et al., 2006). In some cases, infected trunks may show a dark brown discoloration and water-soaked appearance (Niekerk, et al., 2006). In cross section, the discolored vascular system may appear as small black dots, and cross sections of the infected trunk or cordon may show wedge shaped necrotic wood sections that resemble symptoms of Eutypa dieback (Niekerk, et al., 2006). Sometimes the disease is called black dead arm which can be caused by N. ribis as well (Larignon, et al., 2001), but the name is often misused since other pathogens, Diaporthe ampelina (aka Phomopsis viticola), and Eutypa lata can also cause very similar symptoms.

Foliar symptoms include a mild chlorosis starting at the base of the shoot and moving up causing shoot dieback and premature leaf fall (Larignon, et al., 2001; Niekerk, et al., 2006). Over time, the plant declines and eventually dies from these symptoms (Larignon, et al., 2001). These symptoms have been exacerbated under drought conditions, which could mean that N. ribis symptoms could be increased under similar conditions (Galarneau, et al., 2019).

The discolored vascular system and necrotic wood symptoms from trunk diseases, including Botryosphaeria dieback, have been theorized to be caused by secondary metabolites produced by the fungi. Neofusicoccum parvum has been shown to produce fatty acids and they may be the reason for the symptoms seen within the wood of the grapevine (Salvatore, et al.,

2018). Phytotoxic metabolites have also been found to be produced by N. parvum and may produce the canopy symptoms observed (Abou-Mansour, et al., 2015; Burruano, et al., 2016).

Although these phytotoxic metabolites have been collected from fungal isolates and tested for phytotoxicity, it is still uncertain whether or not these metabolites when produced in the wood of

the vine, are actually the compounds responsible for the symptoms we can see, or if something else is responsible (Abou-Mansour, et al., 2015). There have also been genes in the leaves of the vine that have been found to be activated by infection even when no canopy symptoms have been seen, suggesting a connection between trunk and canopy disease response via the vascular system soon after the initial infection (Czemmel, et al., 2015).

Neofusicoccum ribis is often associated as a trunk disease pathogen. Examples are recently found stem and branch canker in Cannabis sativa or shoot blights in pistachio.

However, in apples, olives, kiwis, pears, almonds, and grapes, they produce a fruit rot (Alberti, et al., 2018; Michailides, 1991; Ma, et al., 2001). In apples, the fruit disease is called white rot which causes a soft, lighter colored rot compared to surrounding tissue (Brown, 1986).

Symptoms usually appear in midsummer, 4-6 weeks before harvest, and are very similar to those of black rot caused by B. obtusa (Brown, 1986). Wounding is necessary for infection, but rot doesn’t occur until sugar content reaches 10.5% (Brown, 1986). The pathogen can also cause postharvest decay in apples (Jurick, et al., 2013).

In olives it causes drupe rot, which was only discovered recently (Chattaoui, et al., 2011).

Symptoms include brown spots on the surface of the fruit that then start to depress and lead to mummification of the fruit and premature senescence (Chattaoui, et al., 2011). These symptoms are similar to those found in grapes. Neofusicoccum ribis causes ripe rot in kiwis, which doesn’t always produce clear external symptoms (Koh, et al., 2003). When present, there is a sunken portion of the fruit surface with water-soaked flesh seen when the skin is removed (Koh, et al.,

2003). Internal symptoms include milky flesh with a dark green margin that spread concentrically as the fruit ripens, with symptoms usually occurring on over-ripe fruit (Koh, et al.,

2003). In pears, fruit rot symptoms include soft, dark brown, slightly sunken, circular spots

surrounded by irregular margins on the surface of the fruit (Garibaldi, et al., 2012). Internally the fruit decays, rots, and brown and fruit falls prematurely, similar to that of olives (Garibaldi, et al.,

2012).

Neofusicoccum ribis has also been found to cause post-harvest rots as well. In apples it causes a decay in fruit storage and fruit has a brownish lesion that was soft, dry and leathery but without formation of pycnidia (Jurick, et al., 2013). In both mangoes and avocados, it causes a stem end rot (Hartill, 1991; Sakalidis, et al., 2011a). The pathogen causes a soft, watery rot that starts on the stem end of the fruit and spreads throughout in both mangoes and avocados (Hartill,

1991; Sakalidis, et al., 2011a). The pathogen establishes asymptomatically in the field but is triggered by stress like cold storage (Sakalidis, et al., 2011a). Neofusicoccum ribis has also been linked to mango decline (Sakalidis, et al., 2011a).

The disease cycle of Neofusicoccum ribis is not fully understood, but the disease cycle for other diseases caused by various species in the Botryosphaeriaceae family, such as

Botryosphaeria dieback on grapevine, have been well studied. Botryosphaeria Spp. infect mainly through wounds (Wilcox, et al., 2015). In the example of Botryosphaeria dieback, they can infect through pruning wounds soon after the vine is pruned in the winter or in-season pruning and trimming events (Wilcox, et al., 2015). Pycnidiospores are disseminated from pycnidia on nearby grapevine trunks, dead wood of debris left in the vineyard from the previous season, or other hosts surrounding the vineyard (Amponsah, et al., 2009; Wilcox, et al., 2015).

Infection can also occur through infected rootstocks or scion woods when they are grafted (Wilcox, et al., 2015). The pathogen can remain dormant or endophytic, and vines can remain asymptomatic (Wilcox, et al., 2015). Infection timing studied in blueberry stem canker showed that the most prevalent time of season for infection was in the spring (May and June),

and there was an incubation period of 4-6 weeks before symptom development (Creswell, et al.,

1988). Conditions favorable for disease development are temperatures ranging from 59-98⁰F and disease incidence tends to increase after rain events (Wilcox, et al., 2015).

The most common way to manage Botryosphaeria dieback on grapevine is through cultural methods. It is recommended to avoid injury to trunks and to practice proper pruning to avoid unnecessary wounds during the dormant season (Brown, 1986). The removal of diseased wood is also recommended, and then the grower can look to fungicide protection of the wounds that had to be made (Brown, 1986). There are very few fungicides that are approved and recommended to control the spread of Botryosphaeria pathogens throughout the world, and some have been shown to reduce lesion growth (Amponsah, et al., 2011). In Amponsah, et al. (2011), vines were inoculated with Neofusicoccum luteum and then treated with a variety of fungicides belonging to a wide range of modes of action (i.e., FRAC groups). Some fungicides, including flusilazole, carbendazim, tebuconazole, prochloraz, procymidone, iprodione, fenarimol, thiophanate methyl, chlorothalonil and mancozeb, affected lesions by restricting their growth, but others did not slow down the infection (Amponsah, et al., 2011). Carbendazim and tebuconazole have been tested to control wood cankers caused by Neofusicoccum spp. as wound protectants, but they can pose a problem of phytotoxicity when applied in doses large enough to offer proper protection (Bester, et al., 2007; Niekerk, et al., 2006; Tennakoon, et al., 2018b).

Biological control methods have recently been looked at as a way of control for other pathogens in the Neofusicoccum genus, namely Neofusicoccum parvum which is very closely related to N. ribis. Two bacteria, Bacillus subtilis and Pantoea agglomerans, have shown promise of reducing wood necrosis in French and Tunisian grapes treated with them together

(Rezgui, et al., 2017).

Copper nanoparticles grown in aqueous media as a alternative to fungicides have been studied in an in vitro experiment with an unspecified Neofusicoccum spp., and two Fusarium spp. The results showed great promise in reducing mycelial growth by causing the mycelia to produce intercellular reactive oxygen species which cause damage to the mycelial cells (Pariona, et al., 2019).

In previous studies, most of the observations were on muscadine grapes or in a controlled laboratory setting. In general, there is very little research done on Macrophoma rot, especially with Vitis vinifera grapes grown in the field. Neofusicoccum ribis is known to be dispersed through rainwater and is thought to infect trunks through pruning wounds, but it’s not known when fruit infection occurs (Amponsah, et al., 2009). Symptoms on berries develop later in the season, usually after veraison, but infection could occur weeks ahead of the symptom development.

Leaf removal is a common practice in all grape growing regions to open up the canopy to expose the fruit to more sunlight. It has been shown that it can reduce disease and possibly enhance the quality of the grapes for winemaking, although disease control is the most helpful result of this canopy management practice (Lemut et al., 2015; Tardaguila, et al., 2010). This practice, along with others including shoot thinning, hedging, and cluster thinning, open up the canopy so that leaves and clusters dry faster, pesticide sprays cover better, and sunlight can enter into the canopy (Percival, et al., 1994; Smart, et al., 1991). Different levels and different timings of leaf removal have been debated to get the outcomes desired by of growers, but in practice it still comes down to how the grower has been doing it in the past or how they’ve been taught personally (Lemut et al., 2015; Tardaguila, et al., 2010). It also contributes to the philosophy of vine balance, or the ratio of vegetative growth to reproductive growth being within optimal

ranges during the growing season, to produce grapes in the fashion desired for making a quality wine, which varies from variety to variety and amongst growers in different regions of the world.

In general, there seems to be only positive connotations when it comes to leaf removal as a canopy management practice.

Leaf removal is a great way to control fungal diseases because the canopy can dry faster after morning dew or rain. One good example of a positive influence of canopy management is against a disease called Botrytis gray mold or bunch rot. The pathogen Botrytis cinerea thrives in continued wet conditions and especially in shaded canopies (Wilcox, et al., 2015); thus, the risk of disease development increases if grape canopy is not well managed. Conversely, it has been anecdotally observed that an increase in direct sunlight exposure of the clusters may increase the development of Macrophoma rot symptoms as the season progresses. Better fungicide coverage on exposed clusters typically helps management of other pathogens, thus, the increase in

Macrophoma may indicate that the timing of fungicide application or the material may not be appropriate.

Pesticides are used for disease control in all facets of agriculture, specifically fungicides being the most widely used as fungal diseases are the most prevalent. Fungicides control the spread of fungal pathogens by restricting mycelial growth or by keeping spores from germinating

(Agrios, 2004). The method by which they work is known as the mode of action that is how the fungicide produces a toxic reaction of the chemical that produce responses inside the pathogen to achieve the desired result of control (Ware and Whitacre, 2004). The Fungicide Resistance

Action Committee (FRAC), which was formed in the 1980’s (FRAC, 2019), classifies modes of action into groups. Each group is provided with a FRAC number. For example, if two chemicals belong to the same FRAC group, these two are the same in terms of mode of action, regardless of

their chemical compositions. Growers can use these groupings to plan their pest management program by rotating through modes of action to minimize risk of development resistance to one group. This method is heavily advised to them as a way to plan fungicides sprays, as some modes of action are already seeing a reduction in efficacy. Winegrape growers in Virginia use a variety of FRAC number groups seasonally to control multiple pathogens prevalent in the mid-

Atlantic. It is important also to follow the label instruction on fungicides when mixing to the desired concentration because if the guidelines aren’t followed, then resistance can develop more quickly, lowering the efficacy these fungicides have against disease control.

The goal of this thesis is to lay the groundwork of the research and knowledge of

Macrophoma rot on grape vines in Virginia. The objectives for each chapter include determining the prevalence of N. ribis in different locations across Northern and Central Virginia, studying the relationship between disease incidence and severity and sunlight exposure, finding the time of the season during which N. ribis infects, and screening fungicides that can potentially be used for control of Macrophoma rot.

Figure 1.1. Conidial morphology of Neofusicoccum ribis produced in culture on water agar and pine needles. (Source: Slippers et al., 2004)

Figure 1.2. Black pycnidia on Macrophoma rot symptomatic Petit Manseng grapes (top left) and a developing lesion (top right) on a cluster found in a Virginia vineyard.

References

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Chapter 2: Prevalence of Macrophoma Rot in Virginia

Introduction

Macrophoma rot caused by Neofusicoccum ribis, and closely related species, is a very important disease in muscadine grapes in the Southeastern United States. Symptoms on muscadines are the same as those on bunch grapes: circular, flat, sunken lesions form on the fruit and then expand to form a brown, soft rot across the entire fruit (Millholland, 1991). Because N. ribis can infect a wide variety of hosts, it’s believed that the pathogen can overwinter on other hosts as well as on mummified berries which makes it hard to control (Andersen, et al., 2003).

The damage done by these symptoms make the grapes unusable and are usually culled before the winemaking process. This can cause a significant loss of crop yield depending on the severity of the infection.

Macrophoma rot is considered to be caused by fungal species belong to the family

Botryosphaeriaceae such as Botryosphaeria ribis, Botryosphaeria dothidea, and Neofusicoccum ribis. Other species within the family Botryosphaeriaceae have also been found to cause trunk diseases. Identification of Botryosphaeriaceae based on multilocus sequence typing (MLST) is necessary because N. ribis and closely related species are similar in visual symptoms, growth characteristics in culture, and single genetic markers cannot distinguish species (Sakalidis, et al.

2013). After being reclassified, and then differentiated, the clearest way to confirm the identity of Macrophoma rot is through the growth in culture for genetic identification using multiple genetic markers.

There are no documented cases of Macrophoma rot infections in Virginia, but growers have seen symptoms on Petit Manseng grapes that resemble those of Macrophoma rot. These symptoms are very similar to those of other grape pathogens so further investigation of these symptoms was needed, which is the purpose of this study.

The objective of this study was to determine the geographic range of N. ribis in Northern and Central Virginia through a combination of symptom observation and genetic sequencing on disease samples. Information from this study will be used to inform growers on the prevalence of

Macrophoma rot and to aid in their seasonal decision making on disease control.

Materials and Methods

Seven vineyards in northern and central Virginia were selected based on the acreage of cultivar Petit Manseng. This cultivar was chosen for the survey because an outbreak of

Macrophoma rot was confirmed in Petit Manseng vineyard at the Alson H. Smith Agricultural

Research and Extension Center (AHS AREC) (Figure 2.1).

Vineyards were visited every 10 days from veraison until harvest for the 2018 season and twice during the 2019 season, also between veraison and harvest, or August and October. At each site, scouting of the disease, collection of symptomatic berries, and collection of berry sample for juice analysis were conducted. Within each Petit Manseng block, the same two rows were used for the sampling over time. A total of 100 clusters were randomly chosen during each visit to rate disease incidence. Clusters were distributed throughout the entire block to be representative of the entire canopy, so clusters in all positions within the canopy were chosen.

During the season, clusters were visually rated incidence of Macrophoma-like. At harvest,

clusters were rated for disease incidence and also for disease severity by assessing the percentage of the cluster with Macrophoma-like disease symptoms.

Average berry weight, soluble solids (Brix), pH, and titratable acidity were also measured at each visit based on 100 apparently healthy berries randomly collected from the predesignated two rows. Berries were weighed on a scale and that number divided by the sample size to get the average berry weight. Once weighed, the berries were crushed in the bag and then pressed through a potato ricer to express the juice. The juice was transferred to a 50 mL conical tube and centrifuged to pelletize insoluble solids. Soluble solids were measured using an Atago Pocket refractometer, pH was measured using a Thermo Scientific Orion 3 Star pH meter, and titratable acidity was measured with a Metrohm 848 Titrino Plus and was titrated to an endpoint pH of

8.00.

Berries exhibiting Macrophoma-like symptoms were collected for causal pathogen isolation. The lesions found on the berries were dissected into approximated 4 mm2 pieces, and surface sterilized in a following manner: soaked in 70% ethanol for 30 seconds; soaked in 10% bleach for 1 minute 30 seconds; then rinsed with distilled for 1 minute 30 seconds. The surface sterilized tissue was dried for 2 minutes in the laminar flow hood, and placed in one well of a 24- well plate (Acumedia®, Neogen Company Lansing, MI) filled with potato dextrose agar (PDA)

(Acumedia®, Neogen Company Lansing, MI). Each berry was dissected into four pieces and three berries were selected per visit resulting in twelve isolates created. Isolates in the 24-well plates were incubated at 25°C for 3 days then phenotypic characteristics, including color and growth patterns of the mycelia were recorded. Isolates exhibiting light grey to dark grey mycelia and a dense mycelial growth pattern that filled the entire plate were ideal. Each isolate was transferred to antibiotic-amended (streptomycin and chloramphenicol, 100 mg/ml each) quarter

strength PDA plates to maintain a pure culture. Transferred isolates were then grown in an incubator at 25°C. Once the dish was fully covered in mycelium, the mycelium was harvested by wetting the plate with distilled water and scraping the mycelium off of the PDA using a bent probe. The mycelia were dried off and placed into a MP FastPrep®-24 2-mL tube with appropriate beads and stored at -80°C overnight in preparation for DNA extraction.

After at least 12 hours in -80°C, the mycelia were homogenized using a MP Biomedicals

FastPrep®-24 Tissue and Cell Homogenizer for 60 sec at 4.5 m/s and then centrifuged at 11,000 x g for 1 min. DNA was extracted using a modified Plant Tissue (Mini) Protocol from the

Bioline ISOLATE II Plant DNA Kit (#BIO-52070). The DNA was eluted in ultrapure water and stored at -20qC.

To delineate species among and within the genera Neofusicoccum, Botryosphaeria, and others, a multilocus sequence typing (MLST) approach was used. Three PCRs for three gene regions – RNA polymerase II subunit RPB2 (RPB2 or rpb2), internal transcribed spacer of the rDNA (ITS), and translational elongation factor 1-α(EF1-α) – were done for a subset of isolates selected previously based on phenotypic characteristics that resembled N. ribis grown in culture

(Table 2.1). PCRs for RPB2 were done using AmpliTaq GoldTM 360 Master Mix and its suggested thermal profile (Table 2.2 and 2.3) and PCRs for ITS and EF1-α were done using New

England Biolabs Taq DNA Polymerase and its suggested thermal profile (Table 2.4 and 2.5).

PCR product was visualized using gel electrophoresis on a 1.5% agarose gel with

5μL GelRed and Tris/Borate/EDTA buffer (TBE). The gel was photographed by a FOTODYNE unit with the FOTO/Analyst Investigator to observe the amplicons. Amplicon length was confirmed with Bioline’s HyperLadder™ 50 bp. PCR products were then purified using a

modified Spin Protocol from the QIAGEN MinEluteo PCR Purification Kit (#28004, Handbook

March 2008). DNA sequencing of purified amplicons was performed at the Virginia

Biocomplexity Institute (Virginia Tech, Blacksburg, VA). Consensus sequences were edited, concatenated, and then aligned in Geneious 11.1.5 (https://www.geneious.com). A neighbor- joining tree was built in Geneious Tree Builder using the Tamura-Nei genetic distance model.

Bootstrap sampling was performed with 100,000 replicates. Neofusicoccum buxi (CBS113714) was the outgroup, which is a taxon outside the group of interest and is used as a reference when determining the evolutionary relationships within the ingroup (“Reading Trees: a quick review.”,

2004). The alignment of 59 concatenated sequences was 1,331 base pairs (bp); prior to concatenation, EF-α was 270 bp, ITS 520 bp, and RPB2 541 bp.

Results

Macrophoma-like disease symptoms were observed in each surveyed vineyard in both

2018 and 2019 after veraison. Dark brown, circular lesions were found in the berries which ranged in size from covering about a quarter of the berry surface to enveloping the entire berry.

An example of the lesions found can be seen in Figure 1.2 within the previous chapter. Pycnidia forming on the lesions were seen in a few examples closer to harvest.

2018: DNA sequences for all three gene regions were obtained for 18 isolates; all 18 were 100% identical at every site. DNA sequences for RPB2 gene region were obtained for all

18 isolates while sequences for EF-α gene region were obtained for 9 isolates; isolates were

100% identical. Each vineyard produced at least one isolate that was sequenced and found to be

N. ribis. When separated into each individual vineyard, SCSO and VVW had the largest

percentage of sequenced isolates identified as N. ribis in 2018 while SHV and WGV had the lowest percentage (Table 2.6). Isolates were also sequenced to determine other pathogens that were found on clusters exhibiting these symptoms (Figure 2.2).

2019: DNA sequences for 90 isolates for RPB2 were obtained, but no isolates collected from survey vineyards were identified as N. ribis (Table 2.6). Isolates were also sequenced to determine other pathogens that were found on clusters exhibiting these symptoms (Figure 2.2).

Because all sequences came back 100% identical, only one of these sequences (NE0001) was included in the alignment, along with 58 from GenBank (see Table 2.7). Isolate NE0001 grouped with two N. ribis accessions (Figure 2.3, bootstrap = 95%), within a larger clade of N. batangarum (Scabby Canker of cactus pear) and N. umdonicola (bootstrap = 96%). Isolate

NE0001 was 99.6% identical (1,326/1,331 bp) to N. ribis CBS121.26 and 100% identical

(1,331/1,331 bp) to N. ribis CMW7773.

The primary chemistry, Brix, pH, and TA, were recorded from the 100-berry sample after each visit to each vineyard to examine their potential effect on Macrophoma-like symptom development. When the effect of location, sampling date, and their interactions were examined in

2018, sampling date was found to be significant on all three measurements (P ≤ 0.05). Brix and pH tended to increase over the course of the season, and TA tended to decrease as the berry matured. At some locations in 2018, Brix decreased at the end of the season, probably due to frequent rain events (Table 2.8). Brix and pH did not differ among seven sampled locations; however, there was a significant effect on TA (P = 0.001), where the vineyard LV resulted in significantly higher TA than the vineyard SCSO. The interaction between location and date was

not significant (P > 0.05), indicating the change over time did not differ among locations with these three measurements.

The primary chemistry was sampled in 2019 at all seven locations at either one or two time points. When the effect of the year was compared using the harvest time measurements, both Brix and pH were significantly affected (P ≤ 0.05), but TA was not. Similar to 2018, Brix and pH increased during the season while TA decreased. There was no significant effect of the location on pH (P > 0.05), but Brix and TA were affected (P ≤ 0.05). Brix at vineyard SHV was significantly lower than the other locations and the TA at vineyard LV was significantly higher than other locations at harvest like in the previous year.

When tracked across the 2018 season, the primary chemistry of each site was not abnormal compared to expected values for the Petit Manseng variety in the Northern Virginia area with the weather experienced in 2018 (Table 2.8). When compared to disease development across the season from veraison to harvest, it does not seem to show an increase in Macrophoma- like disease symptoms as the °Brix increases, and the same is true for pH and titratable acidity

(Figures 2.4, 2.5, and 2.6).

Disease incidence was tracked across the season in 2018 and several times in 2019. The mean disease incidence across the seven surveyed vineyards was greatest at harvest (Table 2.9).

Disease incidence ranged from 2% to 87% in 2018 and 2% to 82% in 2019. The effect of location was significant in 2018 (χ2 = 128.6, P < 0.001) where vineyard CV had a significantly higher disease incidence than all the other surveyed vineyard, with the exception of vineyard

SHV. Also, vineyard VVW had a significantly lower overall disease incidence than other vineyards, except vineyard SHV.

The disease incidence at each location increased from the first assessment to the second assessment, then generally decreased toward the end of the season. This observation is attributed to the fact that vineyards that showed the greatest decrease removed diseased berries before harvest. Vineyards WGV and SHV showed the most dramatic decrease with WGV going from

40% to 18% incidence and SHV going from 63% to 18% incidence during the last two assessments made during the 2018 season. The effect of the date was also significant (χ2 = 11.1,

P < 0.001). In some locations, a general decline of disease incidence was observed, which was reflected in the significant interaction of location and date (χ2 = 41.2, P < 0.001), indicating that trends over sampling time point were different among the seven surveyed locations.

When we compared the disease incidence at harvest for the season, the effects of year (χ2

= 7.7, P = 0.001) and location were significant (χ2 = 210.8, P < 0.001). Disease incidence among the seven sampled vineyards was higher in 2018 than 2019. The difference in weather between these two seasons was significant in rainfall and could be a contributing factor to the changes seen in disease incidence (Table 2.10). Disease incidence at the last sampling time point of vineyard CV was significantly (P ≤ 0.05) higher than other locations.

When a non-parametric correlation, Kendall’s tau, was estimated among Brix, pH, TA, and disease incidence, disease incidence at the time of harvest was significantly negatively correlated to pH (P = 0.01), indicating that there was a general trend where disease incidence increased as pH increased. There were general negative trends among Brix, pH, and TA, but none were significant (P > 0.05).

Discussion

To our knowledge, this is the first study to confirm Macrophoma rot caused by

Neofusicoccum ribis in Virginia vineyards. N. ribis was isolated from all seven surveyed vineyards where Macrophoma-like symptoms were observed. We were able to record symptoms at veraison, which indicates the infection took place before our sampling time, and symptom development in addition to the production of pycnidia on the surface suggests that protection against Macrophoma-like disease is necessary. (Brown, 1986; Kummang, et al., 1996a;

Kummang, et al., 1996b).

Macrophoma-like symptoms were found in each vineyard surveyed in both 2018 and

2019, but in 2019 none of the collected isolates that were sequenced were identified as N. ribis.

2018 had more rain events than 2019, which could have provided more instances for the disease to spread through water splash and infect (Table 2.10) (Amponsah, et al., 2009). Without knowing at exactly which time of the season the initial infection of Macrophoma rot occurs, it can’t be said for certain, but wet weather is needed for spore germination and the growing season of 2019 was drier than average (Shafi, et al., 2018; Sutton, 1981; Tennakoon, et al., 2018a).

The decrease in Macrophoma-like symptoms seen in 2019 can’t be wholly attributed to the weather as some vineyards surveyed were under new management during the 2019 season and the disease management programs have changed based on the difference in seasons and disease pressure to include different modes of action in their pesticide rotation and reducing pesticide applications if they weren’t needed. Disease management programs also changed due to the difference in weather between seasons and could have caused the decrease as well. All vineyards also harvested earlier in 2019 versus 2018 due to weather conditions (Table 2.9), so

disease development might have continued and reached the same levels as 2018 if the grapes were allowed to hang on the vines longer.

The symptoms seen and presented in Figure 1.2 in the previous chapter, relate to the genetic sequences isolated. Macrophoma-like symptoms are very similar to other diseases, including ripe rot, black rot, white rot, bitter rot, Phomopsis, and also the environmental injury sunburn (Hoffman, et al., 2004; Rustioni, et al., 2014; Smith, et al., 2014). This makes it hard to distinguish diseases in the vineyard no matter how experienced the scouter is. The only certain way to know what disease is present is to bring samples back to the lab and have them genetically sequenced to relate the visuals back to the disease identification. The disease incidence seen was only of Macrophoma-like symptoms, so the observations from 2019 cannot be related to Macrophoma rot as none of the sequences of isolates collected in 2019 were identified as N. ribis. As seen in the symptoms observed in the survey vineyards in both 2018 and 2019 presented in Figure 1.2, the visual symptoms can to Macrophoma rot in some instances, but they also include other diseases including Phomopsis cane and leaf spot, black rot, and sunburn at different developmental stages (Smith, et al., 2014). The beginning stages of

Macrophoma rot disease development include lesions that closely resemble Phomopsis lesions and sunburn on the berry and can be easily mistaken causing mismanagement of the disease

(Erincik, et al., 2001; Rustioni, et al., 2014; Smith, et al., 2014). In later stages, where the berry shrivels and produces pycnidia, the symptoms closely resembles the final stages of black rot which can cause confusion (Hoffman, et al., 2004; Smith, et al., 2014).

Table 2.1. Primer sequences for the EF-D, ITS, and RPB2 gene regions that were used to identify isolates as Neofusicoccum ribis.

Gene Primer Sequence (5’ – 3’) Product size (bp) Reference EF1-D Forward CAT CGA GAA GTT CGA GAA GG 270 Carbone et al. 1999 Reverse TAC TTG AAG GAA CCC TTA CC ITS Forward CTT GGT CAT TTA GAG GAA GTA A 520 Gardes & Bruns 1993 Reverse TCC TCC GCT TAT TGA TAT White et al. 1990 RPB2 Forward GGT AGC GAC GTC ACT CCC 541 Sakalidis et al. 2011b Reverse GGA TGG ATC TCG CAA TGC G

Table 2.2. Recipe for PCR with AmpliTaq GoldTM 360 Master Mix used for sequencing the RPB2 gene region to identify isolates as Neofusicoccum ribis. Reagent Volume (μl) per 1 reaction Final Concentration Ultrapure water 9

AmpliTaq GoldTM 360 Master Mix (2X) 12.5 1X

Forward primer (10 μM) 0.75 300 nM

Reverse primer (10 μM) 0.75 300 nM

Template (DNA) 2

Total volume 25

Table 2.3. Thermal Profile for PCR with AmpliTaq Gold 360 Master Mix used for sequencing the RPB2 gene region to identify isolates as Neofusicoccum ribis. Step Temperature ℃ Duration Initial Denaturation 95 10 min

Denaturation 95 30 s

Annealing 55 45s

Extension 72 45s

Total Cycles (35)

Final Extension 72 7 min

Table 2.4. Recipe for PCR with New England BioLabs Taq DNA Polymerase used for sequencing the ITS and EF1-α gene regions to identify isolates as Neofusicoccum ribis. Reagent Volume (μl) per 1 reaction Final Concentration Ultrapure water 16.375

NEB Standard Taq Reaction Buffer (10X) 2.5 1X

dNTPs (10 mM) 0.5 0.2 mM

MgCl2 (25 mM) 2 2 mM

Forward primer (10 μM) 0.75 300 nM

Reverse primer (10 μM) 0.75 300 nM

Taq DNA Polymerase (5 U/μl) 0.125 0.025 U/μl

Template (DNA) 2

Total volume 25

Table 2.5. Thermal Profile for PCR with New England Biolabs Taq DNA Polymerase used to sequence the ITS and EF-D gene regions to identify isolates as Neofusicoccum ribis. Step Temperature ℃ Duration Initial Denaturation 95 5 min

Denaturation 95 30 s

Annealing 55 45s

Extension 68 45s

Total Cycles (35)

Final Extension 68 5 min

Table 2.6. The percentage of sequenced Neofusicoccum isolates out of collected isolates from each Petit Manseng survey vineyard in 2018 and 2019. 2018 2019 Vineyard # of Isolates % N. ribisa # of Isolates % N.ribisa CV 40 5.00 24 0.00

GMV 36 8.33 24 0.00

LV 35 8.57 24 0.00

SCSO 36 11.11 12 0.00

SHV 36 2.78 12 0.00

VVW 36 11.11 24 0.00

WGV 32 3.12 24 0.00 a Isolates identified through sequencing of the EF-D, ITS, and RPB2 gene regions.

Table 2.7. Neofusicoccum species from GenBank used in the phylogenetic analysis to produce a neighbor-joining tree. GenBank Accession Culture Species Collection Number EF ITS RPB2 Neofusicoccum algeriense CBS719.85 KX464646 KX464151 KX464000 Neofusicoccum andinum CBS117452 DQ306264 DQ306263 KX464001 Neofusicoccum arbuti CBS116131 KF531792 AY819720 KX464003 Neofusicoccum australe CBS110865 KX464661 AY343408 KX464005 Neofusicoccum australe CBS114792 KX464670 AY343396 KX464006 Neofusicoccum batangarum CMW28315 FJ900652 FJ900606 FJ900614 Neofusicoccum batangarum CMW28320 FJ900654 FJ900608 FJ900616 Neofusicoccum batangarum CMW28363 FJ900653 FJ900607 FJ900615 Neofusicoccum batangarum CMW28637 FJ900655 FJ900609 FJ900617 Neofusicoccum buxi CBS113714 KX464677 KX464164 KX464009 Neofusicoccum cordaticola CMW13992 EU821868 EU821898 EU821928 Neofusicoccum corticosae CBS118099 KX464681 KX464168 KX464012 Neofusicoccum corticosae CBS120081 KX464682 DQ923533 KX464013 Neofusicoccum cryptoaustrale CBS122813 FJ752713 FJ752742 KX464014 Neofusicoccum cryptoaustrale CBS122814 FJ752710 FJ752740 KX464015 Neofusicoccum hongkongense CERC2967 KX278155 KX278050 KX278281 Neofusicoccum kwambonambiense CBS123639 EU821870 EU821900 EU821930 Neofusicoccum kwambonambiense CBS123641 EU821889 EU821919 EU821949 Neofusicoccum kwambonambiense CBS123643 EU821894 EU821924 EU821954 Neofusicoccum kwambonambiense MUCC157 EU339516 EU339522 EU339565 Neofusicoccum kwambonambiense MUCC210 EU339515 EU301016 EU339564 Neofusicoccum lumnitzerae CMW41469 KP860724 KP860881 KU587925 Neofusicoccum luteum CBS562.92 KX464690 KX464170 KX464020 Neofusicoccum luteum CBS655.77 KX464680 KX464167 KX464011 Neofusicoccum macroclavatum CBS114497 KX464695 AF452534 KX464021 Neofusicoccum macroclavatum CBS118223 DQ093217 DQ093196 KX464022 Neofusicoccum mangiferae CBS118532 DQ093220 AY615186 KX464023 Neofusicoccum mangroviorum CMW42486 KP860741 KP860898 KU587938 Neofusicoccum microconidium CERC3497 KX278158 KX278053 MF410203 Neofusicoccum nonquaestitum CBS126655 GU251295 GU251163 KX464025 Neofusicoccum occulatum CBS256.80 KX464696 KX464175 KX464026 Neofusicoccum occulatum CBS128008 EU339509 EU301030 EU339558 Neofusicoccum occulatum CMW9070 AY615156 AY615164 EU339556 Neofusicoccum occulatum MUCC232 EU339510 EU301031 EU339559 Neofusicoccum occulatum MUCC270 EU339508 EU339529 EU339557 Neofusicoccum parvum CBS123650 KX464708 KX464182 EU821944

Neofusicoccum parvum CBS123652 KX464710 KX464184 EU821951 Neofusicoccum parvum CCF109 KC507809 KC507812 KC507803 Neofusicoccum parvum CMW6235 AY615128 AY615136 EU339569 Neofusicoccum parvum CMW9071 AY236880 AY236938 EU339571 Neofusicoccum parvum MUCC124 EU339518 EU339544 EU339567 Neofusicoccum parvum MUCC673 EU339520 EU339553 EU339570 Neofusicoccum pistaciae CBS595.76 KX464676 KX464163 KX464008 Neofusicoccum pistaciarum CBS113083 KX464712 KX464186 KX464027 Neofusicoccum protearum CBS114176 KX464720 AF452539 KX464029 Neofusicoccum ribis CBS121.26 AY236879 AF241177 KX464030 Neofusicoccum ribis CMW7773 AY236878 AY236936 EU339555 Neofusicoccum sp. karanda MUCC125 EU339514 EU339525 EU339563 Neofusicoccum sp. karanda MUCC247 EU339513 EU301028 EU339562 Neofusicoccum stellenboschiana CBS121114 EF445385 EF445354 KX464043 Neofusicoccum stellenboschiana CBS121116 EF445387 EF445356 KX464044 Neofusicoccum terminaliae CBS125263 GQ471780 GQ471802 KX464045 Neofusicoccum terminaliae CBS125264 GQ471782 GQ471804 KX464046 Neofusicoccum umdonicola CBS123646 EU821875 EU821905 EU821935 Neofusicoccum umdonicola CBS123647 EU821885 EU821915 EU821945 Neofusicoccum ursorum CBS122811 FJ752709 FJ752746 KX464047 Neofusicoccum viticlavatum CBS112878 AY343342 AY343381 KX464048 Neofusicoccum vitifusiforme CBS110887 AY343343 AY343383 KX464049 NE0001 NE0001 none yet none yet none yet

GMV 23.1 3.3 10.41 26.1 2.85 9.81

LV 19.7 2.95 9.66 24.8 2.85 11.19

SCSO 18.4 3.01 6.17 21.7 2.93 10.07

SHV 19.4 3.47 8.55 18.7 2.85 9.15

VVW 21.9 3.04 6.5 25.9 2.96 7.46

WGV 20.6 3.44 10.01 27.3 2.88 9.00 a Only healthy berries were collected for primary chemistry analysis. Analysis conducted on juice from 60 berries per replicate.

Table 2.9. Macrophoma rot disease incidence (the presence or absence of Macrophoma-like symptoms) for Petit Manseng survey vineyards at the time of harvest in 2018 and 2019 measured as the percentage of clusters that had Macrophoma-like symptoms. Macrophoma Disease Harvest Date Incidence % Vineyard 2018 2019 2018 2019 CV 9/29 9/12 87 82

GMV 9/20 9/11 14 19

LV 10/1 9/11 25 4

SCSO 9/26 9/17 28 4

SHV 9/22 9/12 18 2

VVW 9/20 9/11 2 8

WGV 9/26 9/12 25 13

April May June July August September October Sum Temperature (°C) 10.0 20.3 21.4 23.4 22.8 20.6 13.9 Rainfall (mm) 83 242 262 148 127 211 39 1112

2018 GDD (base 10 °C) 65 332 353 433 435 338 161 2117 Solar radiation (MJ/m2) 502 539 515 588 532 275 340 Temperature (°C) 13.7 19.1 21.5 24.7 23.1 21.5 14.3 Rainfall (mm) 84 127 59 120 59 16 103 568

2019 GDD (base 10 °C) 136 279 352 470 420 366 149 2172 Solar radiation (MJ/m2) 365 425 506 521 468 368 243 Data were recorded at the Winchester (Virginia Tech, Alson H. Smith Jr. AREC) weather station and summarized by the Network for environment and weather application (newa.cornell.edu).

Figure 2.1. Geographic locations of Petit Manseng survey vineyards in northern and central Virginia.

Figure 2.2. All isolates that were collected from Petit Manseng survey vineyards in 2018 and 2019 that were sequenced using the ITS gene region for identification.

Figure 2.4. Brix versus Macrophoma rot disease incidence (presence or absence of Macrophoma-like symptoms in a cluster) in both 2018 and 2019 from Petit Manseng survey vineyards.

Figure 2.5. pH versus Macrophoma rot disease incidence (presence or absence of Macrophoma- like symptoms in a cluster) in both 2018 and 2019 from Petit Manseng survey vineyards.

Figure 2.6. Titratable Acidity (TA) versus Macrophoma rot disease incidence (presence or absence of Macrophoma-like symptoms in a cluster) in both 2018 and 2019 from Petit Manseng survey vineyards.

References

Amponsah, N. T., Jones, E., Ridgway, H. J., and Jaspers, M. V. 2009. Rainwater Dispersal of Botryosphaeria Conidia from Infected Grapevines. New Zealand Plant Protection. 62:228-233. Andersen, P. C., Crocker, T. E., and Breman, J. 2003. The Muscadine Grape. EDIS New Publications RSS, School of Forest Resources and Conservation. HS763. Brown II, E. A. 1986. Botryosphaeria Diseases of Apple and Peach in the Southeastern Unites Stated. Plant Disease. 70:480. Carbone, I., and Kohn, L. M. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia. 91:553-556. Erincik, O., Madden, L. V., Ferree, D. C., and Ellis, M. A. 2001. Effect of growth stage on susceptibility of grape berry and rachis tissues to infection by Phomopsis viticola. Plant Disease. 85:517-520. Gardes, M., Bruns, T. D. 1993. ITS primers with enhanced specificity for basidiomycetes-- application to the identification of mycorrhizae and rusts. Molecular Ecology. Apr; 2:113-118. Hoffman, L. E., Wilcox, W. F., Gadoury, D. M., Seem, R. C., and Riegel, D. G. 2004. Integrated control of grape black rot: Influence of host phenology, inoculum availability, sanitation, and spray timing. Phytopathology. 94:641-650. Kummuang, N., Diehl, S. V., Smith, B. J., and Graves, Jr., C. H. 1996a. Muscadine grape berry rot disease in Mississippi: Disease epidemiology and crop reduction. Plant Disease. 80:244–247. Kummuang, N., Smith, B. J., Diehl, S. V., and Graves, Jr., C. H. 1996b. Muscadine grape berry rot disease in Mississippi: Disease identification and incidence. Plant Disease. 80:238– 243. Millholland, R. D. 1991. Muscadine Grapes: Some Important Diseases and Their Control. Plant Disease. 75:113. "Reading Trees: a quick review." Understanding Evolution. University of California Museum of Paleontology. 2004 . Rustioni, L., Rocchi, L., Guffanti, E., Cola, G., and Failla, O. 2014 Characterization of Grape (Vitis vinifera L.) Berry Sunburn Symptoms by Reflectance. Journal of Agricultural and Food Chemistry. 62:3043−3046.

Chapter 3: Fruit Exposure and Expression of Rot and the Timing of Susceptibility to Infection

Introduction

Fruit Exposure and Expression of Rot. Leaf removal is a common practice in many grape growing regions to open up the canopy to expose the fruit to more sunlight. It has been shown that it can reduce disease and possibly enhance the quality of the grapes for winemaking, although disease control is the most helpful result of this canopy management practice (Lemut et al., 2015; Tardaguila, et al., 2010). This practice, along with others including shoot thinning, hedging, and cluster thinning, open up the canopy causing leaves and clusters to dry faster, pesticide sprays to cover better, and sunlight to enter into the canopy easier (Percival, et al.,

1994; Smart et al., 1991). Different leaf removal levels do cause different levels of sunlight exposure, so even though the industry can vary which level of leaf removal is ideal, especially when it comes to varietal, site location, and climate differences, it’s not known which exact level of exposure is the most beneficial.

Leaf thinning is an effective means to manage fungal growth because the canopy can dry faster after morning dew or rain basically eliminating some of the environmental conditions needed for a pathogen to germinate on any part of the canopy depending on the pathogen and propagule. Most, but not all, fungi need specific periods of wetness to germinate and start growing to produce symptoms in the vine either on leaves, fruit, or shoots (Wilcox, et al., 2015).

In comparison, Macrophoma rot does also need wet conditions to germinate, but once those conditions are gone, it can thrive without continued wetness. It has been anecdotally

observed that an increase in direct sunlight exposure of the clusters causes an increase in the development of Macrophoma rot symptoms as the season progresses. This is uncharacteristic of many other fungi that thrive in the more shaded canopies that aren’t managed as precisely.

The objective of this study was to observe the expression of rot symptoms in relation to different levels of leaf removal used in the viticulture industry in Virginia. Panels consisting of 5 vines each within an experimental vineyard were chosen for a randomized complete block design study. The information will be used to advise growers on non-fungicide-related ways to control the prevalence and expression of Macrophoma rot.

The Timing of Susceptibility to Infection. Many fungicide sprays are applied with the disease cycle, time of season, and weather conditions in mind to properly control a variety of pathogens (Wilcox, et al., 2015). Most sprays are timed around when the initial inoculation by a pathogen takes place to manage disease before the infection occurs, as well as control sprays throughout the rest of the season if the initial spray timing was off or weather conditions caused a high disease pressure. The disease cycle of Macrophoma rot hasn’t been fully studied to determine at which point in the season initial infection takes place.

Each disease has a cycle that consists of different stages including reproduction, dispersal, growth, and dormancy (De Wolf and Isard, 2007). To fully understand the disease, the disease cycle must be studied. The cycle for each individual pathogen is mainly determined by the temperature, environment, and the host with dormancy and dispersal less important (De Wolf and Isard, 2007). This is due to the fact that temperature, environment, and the host can be monitored and used as predictors for disease severity and prevalence more easily than dispersal

and dormancy. Knowledge of each stage of the disease cycle is integral to maintaining a proper disease management plan.

The objective of this study was to determine the time of the season in which N. ribis infects the berry. Individual vines within the same row were chosen as experimental blocks and clusters were chosen from each vine to be used for this randomized complete block design study.

Information from this study will be used to inform growers of the optimal time of the season to use fungicide sprays as a means of control for Macrophoma Rot.

Materials and Methods:

Fruit Exposure and Expression of Rot. To assess sunlight exposure on disease development, the vineyard at the Alson H. Smith Agricultural Research and Extension Center

(AREC) in Winchester, VA was used. Experimental units were 5-vine panels of mature Petit

Manseng grafted to 420-A rootstock. The experiment was arranged in a randomized complete block design with three treatments and six replicates per treatment. Each panel contained five vines, totaling 60 vines per treatment. The three treatments included a control where no leaves were removed from the canopy (Full Shade), a moderate exposure where 2-3 leaves per shoot were removed from the fruit zone (Half Sun), and full exposure where 5-6 leaves were removed from the fruit zone per shoot (Full Sun). Treatments were applied by hand at E-L 29 (fruit set).

Panels were interspersed between non-treatment panels that were maintained by vineyard crew..

Other canopy management procedures (ex: shoot thinning, fruit thinning, hedging, etc.) were also handled in the same manner as the rest of the vineyard. Suckers, basal and lateral shoots were removed throughout the season to maintain fruit exposure levels during the season.

Point Quadrat Analysis (PQA) was performed each season at E-L 32-33 (bunch closure) to document treatment impacts on cluster exposure. A metal probe was inserted 25 times, or every 15-20 cm, into the canopy at the fruit zone for each panel (five insertions per vine) and each contact was recorded. If the probe came into contact with a stem, it was not counted.

Insertions with no contacts were recorded as gaps. Leaf layer number (LLN) was calculated from this by summarizing all leaves contacted by the number of insertions per panel.

Berry samples were taken weekly in the 2019 season, but not during the 2018 season because of a change in protocol, from veraison (E-L 35) until harvest to monitor primary chemistry (Table 3.1). This was done to track berry maturity but also to observe possible difference in primary chemistry between treatments. One hundred berries were taken randomly per treatment from multiple clusters throughout each experimental panel to get a representative sample among experimental blocks. These samples were then weighed and prepared as previously described in Chapter 2 to measure Brix, pH, and titratable acidity (TA).

Fruit was harvested when berry samples attained 20 qBrix in 2018 and 28 qBrix in 2019 and Macrophoma-like symptoms were assessed at harvest. Fruit was harvested at a lower Brix levels due to the suboptimal weather in 2018 to achieve the maturity level usually obtained for ideal wine production (Table 2.10). The cluster weights per vine and then fruit was combined by panel. The fruit was stored at 4qC for up to 3 days until clusters could be rated for Macrophoma- like or other disease symptoms.

One hundred clusters were chosen per panel and rated for disease incidence and severity in the same way described in the previous chapter. Clusters harvested from the AREC vineyard were also rated for Botrytis bunch rot and for non-specific rots which could have included ripe

rot, sour rot, and others. From these clusters, Macrophoma rot diseased berries were chosen from each panel based on the presence of Macrophoma-like symptoms to be used in the fungal isolation procedure in the same way as described in Chapter 2. During the 2018 season, three berries displaying Macrophoma-like symptoms were randomly sampled per panel resulting in 72 fungal isolates per treatment A single symptomatic berry per panel was randomly chosen in 2019 resulting in six isolates per treatment. PCR products of the ITS gene region were sequenced for the total of 24 samples. In addition, the MSLT-based identification of 2 N. ribis isolates was conducted as described in Chapter 2. Berry samples were also taken from clusters harvested from each panel. Sixty berries were collected per plot at harvest, weighed and prepared, and Brix, pH, and titratable acidity were measured.

The effect of leaf removal treatments on incidence and severity of Macrophoma rot and

Botrytis bunch rot was examined using the generalized regression in JMP (Pro, ver. 14, SAS

Institute, Cary, NC). The binomial and normal distribution was assumed for cluster disease incidence and severity, respectively. If the treatment was significant, Tukey’s honest significant difference (HSD) method was used for multiple comparison of the three means.

The Timing of Susceptibility to Infection. To assess the susceptibility to infection, 16 Petit

Manseng vines of the AHS AREC vineyard were chosen based on their uniform age and size.

The location of the vines differed between 2018 and 2019, but vines were selected within the same planting area. On each vine, five clusters were chosen, one for each treatment, and bagged with a paper bag (Demeter, Laizhou Demeter Imports and Exports Co., Ltd., Shandong, China) at the start of bloom with the vines serving as blocks and the clusters serving as individual experimental units. The experiment was a randomized complete block design, with the clusters being assigned treatments at random during the time of inoculation in 2018 and in 2019; clusters

were randomly assigned treatments at the time of bagging. There were 5 treatments both years, each representing a different inoculation timing throughout the season including a control

(bagged, but no inoculation), bloom (E-L 23), pea-sized (E-L 31), veraison (E-L 35), and 2- weeks post-veraison (E-L 36) (Table 3.3). With each vine having one cluster from each treatment, there were 16 clusters per treatment in total.

For each inoculation in 2018, a spore suspension was prepared using an isolate collected from the AHS AREC vineyard in 2017 and its identity as Neofusicoccum ribis confirmed by the plant pathology clinic on Virginia Tech’s main campus through genetic sequencing. Inoculations were done in 2019 with isolate NE0403 collected from the AHS AREC vineyard in 2018 and its identity was confirmed as Neofusicoccum ribis by the Virginia Biocomplexity Institute (Virginia

Tech, Blacksburg, VA) through genetic sequencing. The isolate was transferred to multiple antibiotic-amended ¼ PDA plates (Chapter 2) and grown under a UV light to induce pycnidia development. In 2019, the plates were also amended with pieces of grapevine canes harvested the season before and stored at -20qC. The shoots were cut into pieces small enough to fit inside the plates and surface-sterilized in 0.6% sodium hypochlorite for 90 seconds and then rinsed in distilled water for another 90 seconds before being wrapped in aluminum foil and autoclaved.

The pieces were then added to the plates. The isolate was transferred to plates containing the autoclaved shoots and grown under UV light in the same manner.

The spore suspension was made from plates incubated for approximately 14 days by flooding the surface of the plate with 1 mL of distilled water and scraping the pycnidia from the plate with a bent probe, being careful not to break the surface of the media. The suspension was then transferred to a 1.5 mL Eppendorf tube and the pycnidia were pulverized by a glass rod to break them open and release the spores into the solution. Once the spores were released, the

suspension was filtered through 4 layers of cheesecloth into a 15 mL conical tube and rinsed with

10 mL of distilled water to remove mycelia and help the spores filter through the cheesecloth. A hemocytometer was used to estimate the number of spores per volume of water by counting the spores that appear on the grid matrix of the hemocytometer for 20 μL of suspension and the suspension was diluted with distilled water to a concentration of 1 x 104 A suspension with a volume of 100 mL was prepared for each inoculation and was the volume needed to inoculate all clusters at a particular time point.

Inoculations were performed in the afternoon to avoid the hottest part of the day. The suspension was transferred into a hand atomizer for inoculation. For each inoculation, clusters were randomly chosen, one per vine, on the day of inoculation. The paper bags were removed, and each cluster received 6 sprays of inoculum, or about 6 mL. This was enough to wet the cluster but not cause drips. Once the clusters were inoculated, a plastic bag containing a damp paper towel was placed around the cluster and the original paper bags were placed over the plastic bags to prevent overheating of the cluster. The clusters were then flagged with the appropriate color of flagging tape to indicate that they had been inoculated. The plastic bags were kept on the clusters overnight and removed the following morning. This resulted in 18-20 hours of wetness for each cluster. When the plastic bags were removed, a new paper bag was placed on the cluster, leaving the flagging tape on the shoot. Clusters then remained bagged and were harvested when other Petit Manseng vines in the vineyard attained commercial harvest parameters (Table 3.2).

The harvested clusters were brought to the lab to be weighed and were then stored at 4qC until they were rated for disease incidence and severity within 3 days of harvest in the same manner as earlier in this chapter. Each cluster was rated for disease incidence (presence of

disease symptoms) and severity (how much of the cluster was apparently affected by each disease). Macrophoma rot, Botrytis bunch rot, and non-specific rots were all rated separately by symptoms. Diseased berries were collected from each cluster and lesions found on the berries were dissected and sterilized in a sterilized laminar flow hood and placed in one well of a 24- well plate filled with potato dextrose agar (PDA) (Acumedia®, Neogen Company Lansing, MI), selected and grown out under UV light to promote mycelial growth, mycelia harvested, DNA extracted, and PCR products of ITS gene region were sequenced for the total of 155 samples. In addition, the MSLT-based identification of 114 N. ribis isolates were conducted as described in

Chapter 2. A 60-berry sample experimental unit was also taken from the clusters to be weighed and prepared to measure Brix, pH, and titratable acidity in as described in Chapter 2.

The effect of timing of inoculation on disease incidence and severity of Macrophoma rot was examined using the generalized regression in JMP (Pro, ver. 15, SAS Institute, Cary, NC) adjusted based on the percent reduction of mycelial growth from the control. The binomial and normal distribution was assumed for cluster disease incidence and severity, respectively. If treatment was found to be significant, Tukey’s honest significant difference (HSD) method was used for multiple comparisons of the means.

Results

The weather during the 2018 and 2019 seasons was very different from one another causing differences in data collected. The 2018 growing season was much wetter and a little cooler than that of 2019 (Table 2.10).

Fruit Exposure and Expression of Rot. The EPQA showed that occlusion layer number was similar among treatments, but slightly greater in 2019 than in 2018 (Table 3.4). Both

seasons, leaf layer number was greater for the full shade treatments and lesser for the full sun treatments, but in 2019, the leaf layer number of all three treatments were greater than the previous year. The interior leaf percentage and exterior cluster percentage were similar in both years showing that cluster exposure was similar in both years. The percentage of gaps measured was lower in 2019 which can be attributed to the higher leaf layer number recorded that year.

The components of yield among treatments for a given year also showed that each treatment was similar to the others in vine size and production (Table 3.5). Within each year, there was no significant difference between the average number of clusters per vine, yield per vine, or berry weight among treatments. There was a difference of cluster number and yield weight between years due to heavier cluster thinning performed in 2019. The average cluster weight was similar among treatments but was significantly different between years.

Except for Brix in 2019, fruit exposure treatment did not impact fruit chemistry. Full exposure was associated with lower Brix than the other two treatments in 2019 (Table 3.2).

When comparing primary chemistry between years, pH wasn’t significantly affected, while Brix and TA were (P ≤ 0.05). Brix and pH tended to increase over the course of the season, and TA tended to decrease as the berry matured, and both Brix and TA was significantly higher in 2019 than in 2018.

In both years, Macrophoma rot incidence was greater with more as sunlight exposure.

Mean disease incidence ranged from 12% to 48% in 2018 and 55% to 67% in 2019 (Figure 3.1).

In both years, the effect of treatment was significant (F = 82.7; P ≤ 0.0001) and all three treatments were significantly different in 2018 and the full sun treatment was significantly different from the other two in 2019. Full sun exposure treatment was associated with the

greatest disease incidence at harvest, while the full shade treatment had the lowest incidence in both years. When comparing the disease incidence at harvest for the season, the effect of year was significant (F = 298.9; P ≤ 0.0001). Disease incidence among treatments was higher in 2019 than in 2018 and the treatment effect wasn’t the same between the two years.

Disease severity showed a similar response to exposure. Mean disease severity ranged from 0.9% to 6.6% in 2018 and 6.7% to 11.7% in 2019 (Figure 3.2). When comparing the disease severity at harvest for the season, the effect of year was significant (χ2 = 87.1; P ≤

0.0001). Disease severity among treatments was higher in 2019 than in 2018 and was greatest for the full sun treatment than the other two treatments.

In both years, the effect of treatment was significant (χ2 = 18.76; P ≤ 0.0001) with all 3 treatments being significantly different from one another in both 2018 and 2019.

Botrytis disease incidence and severity were also estimated in both years. Mean disease incidence ranged from 89.83% to 99.99% in 2018 and mean disease severity ranged from 25.3% to 52.76% (Figure 3.3). No disease symptoms of Botrytis were found in 2019. The effect of the treatment was significant in 2018 for both incidence (χ2 = 34.6; P ≤ 0.0001) and severity (F =

61.1; P ≤ 0.0001) with all three treatments being significantly different from each other for both incidence and severity.

Fungal isolates collected from diseased berries that had Macrophoma-like symptoms were sequenced to confirm the presence of Neofusicoccum ribis in the vineyard and a multitude of other pathogens (Figure 3.4). The largest percentage of isolates identified as not N. ribis, belonged to the Botrytis family (6).

The Timing of Susceptibility to Infection. Primary chemistry samples that were taken at harvest were analyzed and showed that all treatments were similar in Brix, pH, and titratable acidity (Table 3.6). All isolates (114) collected from inoculated clusters at harvest and were used for genetic sequencing were 100% identical to N. ribis, confirming that the symptoms that were observed can be attributed to the isolates that were used in the inoculation.

Macrophoma-like disease symptoms were found in all inoculated clusters and in addition, about 38% and 93% in all non-inoculated control clusters in 2018 and 2019 respectively (Figure

3.5). Disease incidence ranged from 38% to 100% across all treatments in 2018 and 93% to

100% across all treatments in 2019 (Figure 3.5). The effect of treatment was significant for 2018

(F = 42.3137; P ≤ 0.0001) and 2019 (F = 10.353; P ≤ 0.0001). The pea-sized treatment was greater than the other treatments in 2018 but in 2019 only the control and veraison treatment resulted in significantly lower disease from the other treatments.

Disease severity ranged from 2.7% to 56% across all treatments in 2018 and from 39% to

83% across all treatments in 2019 (Figure 3.6). The effect of treatment was significant in both

2018 (χ2 = 2333.584; P ≤ 0.0001) and 2019 (χ2 = 687.934; P ≤ 0.0001) adjusted based on the control. Mean disease severity of the pea-sized treatment was significantly different from the other treatments in 2018, and both fruit set but in 2019 the pea-sized treatments were only significantly different from the control and none of the treatments were significantly different from each other, just the same as disease incidence.

Fungal isolates collected from diseased berries that had Macrophoma-like symptoms were sequenced to confirm that 75% of sequenced isolates were Neofusicoccum ribis or

Botryosphaeria spp. in the inoculated clusters. 38 isolates were also subjected to the MLST that

confirmed the presence on N. ribis. Of the other 25% there were four other plant pathogenic fungal species (Figure 3.7). The largest percentage of isolates identified as not N. ribis, belonged to the Alternaria genus.

Discussion

Fruit Exposure and Expression of Rot. This study shows that there is a relationship between sunlight exposure and Macrophoma-like symptom development. This relationship isn’t fully understood, but in studies involving growing N. ribis on plates in the lab, the isolates used tended to favor producing in UV light to produce spores (Slippers, et al., 2004). This is something seen in the inoculation study discussed in this chapter as well, so the increased exposure to UV light in the vineyard could contribute to the increased incidence and severity of

Macrophoma-like symptoms as well. There was a significant difference between the levels of leaf removal and subsequent sunlight exposure so that suggests that any amount of sunlight exposure can increase the incidence and severity of Macrophoma-like symptoms. There is most likely a threshold of exposure where it makes a difference and that would need to be studied further. Because of this, canopy management strategies like leaf removal and possibly even hedging can be used as a cultural method of disease control by limiting these activities or adjusting them to suit the needs of the individual vineyard (Percival, et al., 1994; Smart, et al.,

1991).

There was an increase in the incidence and severity of Macrophoma-like symptoms from

2018 to 2019 and this could be attributed to the difference in weather between seasons (Table

2.10). There wasn’t a difference in sunlight except for September 2019, but disease symptoms

recorded could have actually been sunburn mistaken as Macrophoma-like symptoms due to how similar they can look (Rustioni, et al., 2014). The difference between seasons could also be due to the large amount of Botrytis bunch rot seen in 2018 compared to none found in 2019 (Figure

3.3). The Botrytis infection could have covered a percentage of Macrophoma-like symptoms and kept them from being properly observed and recorded (McClellan, et al., 1973).

There are of course also human differences that could have been made between seasons like differences in the timing of the application of treatments. The 2019 season started earlier than the 2018 season as well as the project itself starting later in 2018 due to scheduling. All of this could be due to slight differences between seasons. Based on all of this, the experiment should be repeated with these points in mind to see if a similar season to either 2018 or 2019 produces the same results.

The Timing of Susceptibility to Infection. Based on both year’s data, there seems to be no real correlation between treatments and incidence and severity of Macrophoma-like symptoms.

There was a very positive correlation with the pea-sized fruit inoculation timing in 2018, but the significant difference between treatments didn’t carry over into 2019 even though the same inoculation time did result in a similar level of incidence and severity. This means that growers can’t be advised on what time of the season this pathogen is most active even though previous research done with muscadines have led to a more concrete understanding of the disease cycle

(Brown, 1986; Kummang, et al., 1996a; Kummang, et al., 1996b). Single berry inoculations could be used to test susceptibility in a different way to come to a clearer conclusion on the susceptibility to infection (Gabler, et al., 2001; Gabler, et al., 2006).

There could be a few explanations for the differences seen between seasons. Two to three of the bags used in 2018 fell off and had to be replaced or were lost by the end of the season.

This could have been human error in the initial placement of the bags as they were done by a team versus only one person in 2019, or due to the weather in 2018 having more rain and windy conditions associated with it.

Because of the weather differences causing 2018 to be a much wetter season, the amount of botrytis bunch rot seen in the vineyard and within the bags used made it difficult to identify

Macrophoma-like symptoms (Table 2.10 and Figure 3.8) (McClellan, et al., 1973). The botrytis was able to get into the bags, along with insects that could have brought in different diseases, so incidence and severity observed in 2018 could have been closer to what was seen in 2019 without the subsequent botrytis infection.

In 2019, the tissue used in the growing media to produce spores was switched from pine needles to pieces of healthy grapevine shoots in an effort to produce more spores. The effort was successful in producing more spores but could have increased the pathogenicity of these spores due to the tissue being from the target species used in the inoculation trial.

The timing of the inoculations was done at the same phenological stage each year, but the time that the plastic bags containing a wet paper towel were left on the clusters was done within a certain range and therefore not exactly the same duration each time. This could have contributed to a difference between treatments, but not one that can be defined as it isn’t known if exceeding the wetness hours needed for germination can compromise the process of germination (Shafi, et al., 2018; Sutton, 1981; Tennakoon, et al., 2018a).

Based on this study, the experiment should be repeated with these points taken into consideration to see if a similar season to either 2018 or 2019 produces the same results.

Table 3.1. Weekly primary chemistry among Petit Manseng leaf removal treatment in 2019a used to judge the maturity of the grapes including soluble solids (Brix), pH, and titratable acidity (TA).

Date Treatment 100 Berry Weight (g) pH Brix TA 3-Sep Full 123.64 2.95 24.10 13.02 50 117.70 2.96 24.70 12.08 Shade 120.22 2.97 24.90 12.27 12-Sep Full 123.12 2.91 27.80 8.95 50 115.92 2.93 28.30 8.45 Shade 112.40 2.92 28.20 8.84 19-Sep Full 115.88 2.99 28.10 8.84 50 108.74 3.00 28.60 9.04 Shade 113.81 2.99 28.90 9.15 25-Sep Full 114.76 3.00 29.00 8.63 50 111.57 3.04 29.00 8.61 Shade 109.45 3.04 29.20 8.60 a Only healthy berries were collected for primary chemistry analysis. Analysis conducted on juice from 60 berries per replicate.

Table 3.2. Primary chemistry among Petit Manseng leaf removal treatments in 2018 and 2019a used to judge the maturity of the grapes including soluble solids (Brix), pH, and titratable acidity (TA). Treatment 2018 2019 Brix pH TA (g/L) Brix pH TA (g/L) Full Sun 20.70 3.05 6.46 27.53a 3.20 6.91

Half Sun 20.23 3.05 6.55 28.23b 2.75 6.81

Full Shade 20.88 3.05 6.52 28.37b 3.22 7.12

Significanceb ns ns ns p=0.0126 ns ns

Year p<0.0001 ns p=0.0046 p<0.0001 ns p=0.0046

Y*Tc ns ns ns ns ns ns a Only healthy berries were collected for primary chemistry analysis. Analysis conducted on juice from 60 berries per replicate. bSignificance p>F; ns=not significant at 0.05 level. Comparison of means among treatments made using Tukey- Kramer HSD test. Means not sharing the same letter within a column are significantly different (≤0.05). cY*T = Year*Treatment.

Table 3.3. Dates of inoculation (Inoc.), harvest (Harv.) and assessment (Assess.) of bagged clusters as part of the Petit Manseng inoculation trial in 2018 and 2019. 2018 2019 Treatmenta Inoc. Harv. Assess. Inoc. Harv. Assess. Control n/a 10/9 10/11 n/a 9/30 10/3

Bloom 6/5 10/9 10/11 5/30 9/30 10/3

Pea-sized 6/21 10/9 10/11 6/12 9/30 10/3

Veraison 8/15 10/9 10/11 7/31 9/30 10/3

2-weeks 8/30 10/9 10/11 8/14 9/30 10/3 post aTreatments signify the time of season the inoculation took place, control being there was no inoculation.

Treatment OLN CEL LEL CEFA LEFA OLN CEL LEL CEFAc LEFAc Full Sun 1.5066a 0.1345a 0.1494a 0.5695a 0.5357a 1.8133a 0.2095a 0.1078a

Half Sun 2.5800b 0.4055b 0.2195ab 0.3835b 0.4380b 2.7133b 0.5111b 0.2341b

Full Shade 3.3133c 0.7957c 0.3451b 0.1844c 0.3407c 3.1000c 0.8675c 0.2972b

Significance p < 0.0001 p < 0.0001 p = 0.0565 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001

Year ns ns ns ns ns ns ns ns

Y*Tb ns ns ns ns ns ns ns ns aSignificance p>F; ns=not significant at 0.05 level. Comparison of means among treatments made using Tukey-Kramer HSD test. Means not sharing the same letter within a column are significantly different (≤0.05). bY*T = Year*Treatment cThere was no light data collected in 2019 so CEFA and LEFA could not be calculated.

Table 3.5. Components of yield among Petit Manseng leaf removal treatments in 2018 and 2019a for comparison of equal vine size between seasons. Treatment 2018 2019 Clusters/ Yield/ Single Clusters/ Yield/ Single vine vine (kgs) berry vine vine (kgs) berry weight (g) weight (g) Full Sun 50.27 8.41 1.21 29.57 4.72 1.16

Half Sun 50.53 8.29 1.18 31.73 5.51 1.10

Full Shade 50.63 7.07 1.15 32.73 5.21 1.15

Significanceb ns ns ns ns ns ns

Year p<0.0001 p<0.0001 ns p<0.0001 p<0.0001 ns

Y*Tc ns ns ns ns ns ns aOnly healthy berries were collected for primary chemistry analysis. Analysis conducted on juice from 60 berries per replicate. bSignificance p>F; ns=not significant at 0.05 level. Comparison of means among treatments made using Tukey- Kramer HSD test. Means not sharing the same letter within a column are significantly different (≤0.05). cY*T = Year*Treatment.

Table 3.6. Primary chemistry among bagged Petit Manseng treatments in 2018 and 2019a used to judge the maturity of the grapes including soluble solids (Brix), pH, and titratable acidity (TA). Treatment 2018 2019 Brix pH TA (g/L) Brix pH TA (g/L) Control 16.9 3.07 6.05 28.2 3.29 6.13

Bloom 17.0 3.07 6.82 29.1 3.35 5.91

Pea-sized 18.2 3.10 6.65 29.1 3.32 6.05

Veraison 17.4 3.14 5.74 28.9 3.40 5.68

2-weeks 17.8 3.13 5.32 29.0 3.46 5.40 post Significanceb ns ns ns ns ns ns

Year p<0.0001 p=0.0002 ns p<0.0001 p=0.0002 ns

0.67a

0.59b 0.55b 0.47a

0.32b

0.12c

10.88a

9.58a

6.60a 6.75b

3.52b

0.94c

52.76a

37.81b

25.30c

Figure 3.4. All isolates that were collected from clusters in the Petit Manseng leaf removal trial in 2018 and 2019 that were sequenced using the ITS gene region for identification.

1.00a 1.00a 1.00a 1.00a 0.93a 0.93a 0.93a 0.87a

0.68ab

0.38b

81.87a 77.18a

68.43a 69.33a

55.62a

39.68b

17.81b 12.81bc 10.93bc

2.69c

Figure 3.7. All isolates that were collected from bagged Petit Manseng clusters in 2018 and 2019 that were sequenced using the ITS gene region for identification.

6.56a 6.25a

5.38a

3.75a

0.00a

Figure 3.8. The mean cluster Botrytis bunch rot disease severity (percentage of cluster affected by Botrytis like symptoms) in 2018 from the Petit Manseng field inoculation trial. Treatments include bloom, pea-sized, veraison, and 2-weeks post veraison plus a control of no inoculation as times during the season that inoculations were performed. Means sharing a letter are not significantly different (Tukey-adjusted comparisons).

References

Brown II, E. A. 1986. Botryosphaeria Diseases of Apple and Peach in the Southeastern Unites Stated. Plant Disease. 70:480. De Wolf, E. D., and Isard, S. A. 2007. Disease Cycle Approach to Plant Disease Prediction. Annual Review of Phytopathology. 45:1, 203-220. Gabler, F. M., and Smilanick, J. L. 2001. Postharvest Control of Table Grape Gray Mold on Detached Berries with Carbonate and Bicarbonate Salts and Disinfectants. American Journal of Enology and Viticulture. 52:13-20. Gabler, F. M., Fassel, R., Mercier, J., and Smilanick, J. L. 2006. Influence of temperature, inoculation interval, and dosage on biofumigation with Muscodor albus to control postharvest gray mold on grapes. Plant Disease. 90:1019-1025. Kummuang, N., Diehl, S. V., Smith, B. J., and Graves, Jr., C. H. 1996a. Muscadine grape berry rot disease in Mississippi: Disease epidemiology and crop reduction. Plant Disease. 80:244–247. Kummuang, N., Smith, B. J., Diehl, S. V., and Graves, Jr., C. H. 1996b. Muscadine grape berry rot disease in Mississippi: Disease identification and incidence. Plant Disease. 80:238– 243. Lemut, M.S., P. Sivilotti, L. Butinar, J. Laganis, and U. Vrhovsek. 2015. Pre-flowering leaf removal alters grape microbial population and offers good potential for a more sustainable and cost-effective management of a Pinot Noir vineyard. Australian Journal of Grape and Wine Research. 21:439–450. McClellan, W. D., and Hewitt, W. B. 1973. Early Botrytis Rot of Grapes: Time of Infection and Latency of Botrytis cinerea Pers. In Vitis vinifera L. Phytopathology. 63:1151-1157. Percival, D. C., Fisher, K. H., and Sullivan, J. A.1994. Use of fruit zone leaf removal with Vitis vinifera L. cv. Riesling grapevines. II. Effect on fruit composition, yield, and occurrence of bunch rot (Botrytis cinerea Pers.:Fr.). American Journal of Enology and Viticulture. 45:133-140. Rustioni, L., Rocchi, L., Guffanti, E., Cola, G., and Failla, O. 2014 Characterization of Grape (Vitis vinifera L.) Berry Sunburn Symptoms by Reflectance. Journal of Agricultural and Food Chemistry. 62:3043−3046.

Shafi, A., Ridgway, H. J., Jaspers, M. V., and Jones, E. E. 2018. Factors influencing virulence and conidial production of Neofusicoccum species on grapevine shoots. European Journal of Plant Pathology. 153:1067–1081. Slippers, B., Crous, P. W., Denman, S., Coutinho, T. A., Wingfield, B. D., and Wingfield, M. J. 2004. Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea. Mycologia, 96: 83–101. Smart, R., and Robinson, M. 1991. Sunlight into Wine. A Handbook for Winegrape Canopy Management. Winetitles, Adelaide. Sutton, T. B. 1981. Production and Dispersal of Ascospores and Conidia by Physalospora obtusa and Botryosphaeria dothidea in Apple Orchards. Phytopathology 71:584-589. Tardaguila, J., F. Martinez de Toda, S. Poni, and M.-P. Diago. 2010. Impact of early leaf removal on yield and fruit and wine composition of Vitis vinifera L. Graciano and Carignan. American Journal of Enology and Viticulture. 61:372–381. Tennakoon, K. M. S., Ridgway, H. J., Jaspers, M. V., & Jones, E. E. 2018a. Factors affecting Neofusicoccum ribis infection and disease progression in blueberry. European Journal of Plant Pathology. 151:87–99. Wilcox, W. F., et al., editors. 2015. Compendium of Grape Diseases, Disorders, and Pests. 2nd ed., The American Phytopathological Society.

Chapter 4: In vitro Fungicide Assays to Determine effective Fungicides for Macrophoma rot Management

Introduction

The fungicide mode of action is the method by which fungicides inhibit the growth or spread of the target fungal pathogen. The fungicide has an active chemical ingredient that produces a toxic reaction within the target pathogen that produces physical response, such as reduced growth, inability to germinate, or death of cells, to achieve the desired result of control

(Ware and Whitacre, 2004). The Fungicide Resistance Action Committee (FRAC), which was formed in the 1980’s (FRAC, 2019), classifies modes of action into groups. Each group is provided with a FRAC number. For example, when two chemicals belong to the same FRAC group, these two are the same in terms of mode of action, regardless of their chemical compositions. Winegrape growers in Virginia use a variety of FRAC number groups to control the variety of pathogens prevalent in the Mid-Atlantic.

There are no fungicides currently registered for use to control Macrophoma rot; therefore, growers have limited options in the event of Macrophoma rot outbreak. In addition, there is very little information available on fungicide efficacy against Macrophoma rot. Growers are advised to use cultural practices such as leaf removal to create environmental conditions that do not favor development of Macrophoma rot.

The objective of this study was to evaluate the efficacy of the modes of action and concentration to the growth of N. ribis in vitro.

Materials and Methods

Nine fungicides were chosen to be screened for their efficacy against N. ribis in 2019.

The selection was based on the mode of action and pathogens listed on the label (Table 4.1).

Isolates were grown on a 24-well plate (Acumedia®, Neogen Company Lansing, MI) with wells

16 mm in diameter and 3.4 mL in total volume. Each well was filled with 1 mL Potato Dextrose

Agar (PDA) amended with the fungicide at different concentrations. The concentration of each fungicide was determined based on its active ingredient (a.i.) (Table 4.1). A stock solution of each fungicide was made by mixing a pre-determined amount of the fungicide with distilled water in a 1.5 mL tube. Powdered and pelleted fungicides were weighed on sterile weigh boats and liquid fungicides were pipetted into the tubes before adjusting the volume with distilled water to 750 μL and mixing with the pipette tip. A serial dilution was performed within 50 mL conical tubes filled with 15 mL PDA at 65°C to make amended PDA with concentrations of 100,

10, 1, 0.1, and 0.01 ppm. The fungicide stock solution was pipetted into the first PDA filled tube and mixed in with a clean pipette tip. PDA was then transferred from one tube to the next, mixing after each transfer to achieve the desired concentrations before being pipetted into a 24- well plate. Each well was filled with 1mL of amended PDA of the desired concentration with a row of wells that contained PDA that wasn’t amended with fungicide to be used as a negative control. Figure 4.1 is an example of the plate layout used for each assay. Plates were then left to solidify in a sterile laminar flow hood for at least 12 hours before being used. There were two internal replications of each fungicide and concentration combination per experimental run, and there were two independent experimental runs. Thus, there were a total of four data points per fungicide and concentration combination per isolate.

A total of 22 N. ribis isolates from multiple sites across Virginia were selected for the assay. Species identification was made based on EF-α, ITS, and RPB2 genes (Chapter 2). These isolates were grown on ¼ PDA plates amended with two antibiotics [streptomycin and chloramphenicol (100 mg/ml each)] for 8 days or until mycelia covered the surface of the plate, which could take up to 14 days. In a sterile laminar flow hood, 4 mm plugs of the plate were made using a sterile cork borer. The mycelium plug was then transferred onto the center of each well of the pre-made fungicide amended 24-well plates. Plates were sealed with parafilm and placed in an incubator (Precision™ Low Temperature BOD Refrigerated Incubator, Fisher

Scientific) set to 25°C. After 72 hours, the mycelial diameter was measured with a digital caliper.

Since the 4-mm mycelium plug was placed at the center of the well, and the diameter of the well was 16 mm, the maximum length of growth was 6 mm. The mean percent mycelial growth inhibition was calculated as the mean of the difference between the mycelial growth of the negative control (0 ppm) and the treatment, divided by the mycelial growth of the negative control.

The effective fungicide concentration to reduce the mean mycelial growth rate by 50%

(EC50) for each isolate was calculated by fitting the mean percent mycelial growth inhibition against the log transformed fungicide concentrations using the nonlinear model in JMP Pro (ver.

15, SAS Institute, Cary NC). The calculated EC50 values were filtered through two thresholds:

(i) mean mycelial growth inhibition at the highest fungicide concentration was more than 50% of the zero-control; (ii) P-value of the EC50 parameter estimation (i.e., inflection point) was less than 0.05. A total of five non-linear models (logistic with three or four parameters, probit with four parameters, and Gompertz with three and four parameters) were fitted to the data. Based on

the AIC (Akaike Index of Criterion (Akaike, 1974)), R-square value, and the number of successfully fitted model, logistic and Gompertz with three parameters were examined.

Results

The mean percent mycelial growth inhibition ranged from 0% to 100% and differed greatly among tested fungicides and 22 tested isolates (Figure 4.2). With captan and thiophanate- methyl, seven and twelve isolates, respectively, resulted in higher than 50% mean mycelial growth inhibition at 100 ppm. With mancozeb, polyoxin-D, and tetraconazole, the mean mycelial growth inhibition of seven isolates reached 50%. With azoxystrobin and polyoxin-D, one and three isolates showed the mean percent mycelial growth of 50% at 100 ppm. None of isolates exhibited 50% or higher mean percent mycelial growth inhibition with copper octanoate and potassium phosphite even at 100 ppm.

Two non-linear models, Gompertz and logistic with three parameters, were fitted to the data to estimate EC50, and the best fit models for each fungicide-isolate combination were shown in Table 4.2. Not all of the fungicide-isolate combinations that showed higher than 50% mean mycelial growth inhibition resulted in the successful model fit probably because some isolates were only inhibited at 100 ppm. The average EC50 of seemingly sensitive isolates for each fungicide are azoxystrobin = 0.25, boscalid = 6.87, captan =18.92, mancozeb = 12.95, polyoxin-D = 19.90, tetraconazole = 190.45, thiophanate-methyl = 12.78.

Discussion

Three fungicides showed the most promise in controlling the growth of mycelia: Captan, thiophanate-methyl, and tetraconazole, with thiophanate-methyl and tetraconazole having the greatest reduction in growth compared to the control. These fungicide, and possibly other fungicides within their mode of action group could be field tested as a control method in vineyards to reduce the potential impact of N. ribis-induced disease. All three are already registered for use against other grape diseases so they are available for use by growers, although not yet officially for Macrophoma rot.

Tetraconazole is a demethylase inhibitor (DMI) and has been shown to be more effective after the infection occurs and that could have lent itself to the inhibition seen here (Wong, et al.,

2007). Captan was originally used as a preplant fungicide for seeds as well as used for foliar applications as well (Pandey, et al., 2006). Thiophanate-methyl is very effective against root rot and has been used on fruit rots as well (Taneja, et al., 1982).

None of the isolates were inhibited more that 50% by copper octanoate or potassium phosphite. Copper octanoate is used frequently as an organic fungicide in home gardens as well as to manage mildew diseases. It has been shown to be very effective against fruit rots and copper nanoparticles have been shown to be even more effective so it could be possible that

Neofusicoccum isolates may have some resistance to copper compounds because of how often that they are used in viticulture (Civardi, et al., 2015). Potassium phosphite is part of the host resistance inducer of action FRAC group but has been shown to be effective against other fruit rots (Rebollar-Alviter, et al., 2010).

There were six isolates that didn’t exhibit 50% or higher mean percent growth inhibition with any of the fungicides tested. NE0169, NE0177, NE0211, NE0265, NE0542, and NE0547 are all from different vineyards across Virginia and have nothing in common other that the fact that they are Neofusicoccum isolates and were isolated during the same season so it is unknown as to why they each have a resistance to all of the fungicides tested.

There was some trouble in regard to measuring the fungicides because they’re not designed to be used at such a small quantity. Many were of a thicker consistency that wasn’t suited for pipetting, while some of the granular fungicides couldn’t dissolve properly in such a small quantity of water and in such a small tube. Pipetting into the plates was also troubling sometimes as pipettes aren’t meant for transferring media and when done in larger quantities, can damage the pipette. Normally, filters are used on pipette tips to protect them, but the PDA media couldn’t be pipetted when filtered tips were used. In the beginning, plates that were left to cool and solidify were also getting contaminated which lead to a change in sanitizing technique. This experiment should be repeated with isolates collected from other areas where Macrophoma-like symptoms are found.

MOA Fungicide A.I. Liquid Suspension Stock Dilution A.I Concentration μg/mL or PPM in Volume μL of Suspension Adjusted ppm Agar (mL) used QoI Abound azoxystrobin 0.093 mL / 0.75 mL 124619 100 12.5 10 SDHI Endura boscalid 0.251 g / 0.75 mL 335513 100 12.5 3.7 Multi-site Captan Gold 80 WDG captan 0.281 g / 0.75 mL 374817 100 12.5 3.3 Multi-site Cueva copper octanoate 0.007 mL / 0.75 mL 9586 100 12.5 130.4 Multi-site Dithane 75DF Rainshield mancozeb 0.269 g / 0.75 mL 359479 100 12.5 3.5 HPDI Prophyt potassium phosphite 0.188 g / 0.75 mL 251635 100 12.5 4.9 DMI Mettle tetraconazole 0.045 mL / 0.75 mL 59913 100 12.5 20.8 MBC Topsin M thiophanate-methyl 0.251 g / 0.75 mL 335513 100 12.5 3.7 Chitin Synthase PhD polyoxin D 0.251 / 0.75 mL 335513 100 12.5 3.7

Table 4.2. EC50 value estimate based on non-linear models for each fungicide – isolate combination with the mean mycelial growth inhibition at the highest fungicide concentration more than 50% of the zero-control and P-value of the EC50 parameter estimation (i.e., inflection point) less than 0.05. Inflection Point Wald Chi- Fungicide Isolate Model Std Error p-value EC50 AIC R2 Estimate squared Azoxystrobin 168 Gompertz 3P -1.91 0.14 196.67 <.0001 0.25 83.69 0.95 Boscalid 248 Gompertz 3P 1.91 0.01 35051.27 <.0001 6.87 -146.53 1.00 Captan 135 Gompertz 3P 2.53 0.01 156327.46 <.0001 12.65 -101.30 1.00 140 Logistic 3P 2.84 0.66 18.70 <.0001 17.13 -119.95 1.00 180 Gompertz 3P 2.49 0.00 475559.72 <.0001 12.22 -105.90 1.00 190 Logistic 3P 2.23 0.00 1592240.05 <.0001 9.37 -194.59 1.00 218 Logistic 3P 2.43 0.00 981345.39 <.0001 11.41 -161.57 1.00 246 Gompertz 3P 3.92 0.81 23.27 <.0001 50.72 64.59 1.00 Mancozeb 136 Logistic 3P 2.66 0.17 243.10 <.0001 14.37 -120.81 1.00 140 Logistic 3P 2.29 0.04 3405.27 <.0001 10.00 -126.91 1.00 248 Logistic 3P 2.53 0.01 157820.09 <.0001 12.64 -173.95 1.00 395 Logistic 3P 2.69 0.51 28.17 <.0001 14.80 74.61 0.99 Polyoxin D 161 Logistic 3P 2.68 0.18 231.71 <.0001 14.68 -144.75 1.00 179 Gompertz 3P -1.85 0.21 75.49 <.0001 0.26 92.86 0.88 180 Logistic 3P 2.42 0.04 4368.01 <.0001 11.37 -170.99 1.00 395 Logistic 3P 3.97 0.93 18.18 <.0001 53.28 -167.16 1.00

Tetraconazole 395 Logistic 3P 5.25 0.00 <.0001 190.45 91.65 0.93 Thiophanate- 121 Gompertz 3P 2.06 0.00 1256670.80 <.0001 7.97 -95.15 1.00 methyl 136 Gompertz 3P 0.08 0.01 36.45 <.0001 1.18 -168.39 1.00 140 Gompertz 3P 3.72 1.33 7.81 0.01 41.46 78.13 0.99 141 Logistic 3P 1.91 0.41 21.30 <.0001 6.84 -174.45 1.00 190 Logistic 3P 2.38 0.27 75.08 <.0001 10.92 75.09 1.00 220 Gompertz 3P 2.09 0.02 18063.77 <.0001 8.21 -116.08 1.00 391 Logistic 3P 2.30 0.67 11.96 0.00 10.10 83.48 0.98

Figure 4.1. Example layout of a 24-well plate (Acumedia®, Neogen Company Lansing, MI) used in the in vitro fungicide assays. Each fungicide was serial-diluted into 5 different concentrations and pipetted into the 24-well plate so that each concentration was pipetted into 4 wells per plate plus 4 wells containing media that weren’t amended with a fungicide used as the control. Each plate had 1 fungicide and was divided into 2 sections for 2 different isolates. Each fungicide was repeated twice for each isolate meaning that 2 amended 24-well plates were made for each fungicide and for each pair of isolates. This example shows isolate #140 on a plate amended with thiophanate-methyl inoculated on September 10, 2019.

Figure 4.2. The effect of fungicide a.i. concentration on the mean percent mycelial growth inhibition by isolates on a logarithmic scale. A total of 22 N. ribis isolates from Virginia were examined against nine different mode of action fungicides using fungicide-amended PDA in 24- well culture plate (Acumedia®, Neogen Company Lansing, MI) in 2019. There were 6 fungicide-concentration combinations per isolate which was repeated 4 times for each fungicide. Each dot represents the mean of the growth inhibition for the fungicide-concentration combination and the error bars are based on the standard error. All isolates are shown in each cell, although they overlap due to having similar means and cannot be discerned from one another.

References

Akaike, H. 1974. A new look at the statistical model identification. IEEE Transactions on Automatic Control. 19:716–723. Civardi, C., Schubert, M., Fey, A., Wick, P., Schwarze1. F. W. M. R. 2015. Micronized Copper Wood Preservatives: Efficacy of Ion, Nano, and Bulk Copper against the Brown Rot Fungus Rhodonia placenta, PLoS One. 10:eCollection. FRAC, 2019. About FRAC. http://www.frac.info/. Pandey, K. K., Pandey, P. K., and Mishra, K. K. 2006. Bio-efficacy of fungicides against different fungal bioagents for tolerance level and fungistatic behavior. Indian Phytopathology. 59:68-71. Rebollar-Alvitera, A., Wilson, L. L., Madden, L. V., and Ellis, M. A. 2010. A comparative evaluation of post-infection efficacy of mefenoxam and potassium phosphite with protectant efficacy of azoxystrobin and potassium phosphite for controlling leather rot of strawberry caused by Phytophthora cactorum. Crop Protection. 29:349-353. Taneja, M., and Grover, R. K. 1982. Efficacy of benzimidazole and related fungicides against Rhizoctonia solani and R. bataticola. Annals of Applied Biology. 100:425-432. Ware, G., and Whitacre, D., 2004. Modes of action of insecticides. In. The Pesticide Book. Willoughby, OH: MeisterPro Information Resources, 188-202. Wong, F., and Midland, S. 2007. Sensitivity distributions of California populations of Colletotrichum cereale to the DMI fungicides propiconazole, myclobutanil, tebuconazole, and triadimefon. Plant Disease. 91:1547-55.

Chapter 5: Conclusions and Future Research

This study focuses on Neofusicoccum ribis, causal agent of the disease Macrophoma rot, and its presence and possible management tools in the Virginia wine region. Different areas within the region were surveyed for its presence in one project; two others looked at its prevalence and susceptibility under the normal climate conditions of the region; while a fourth explored the possibility of management through fungicide applications. Neofusicoccum ribis was found in all areas surveyed which spanned the majority of the Virginia wine region. This brings up the possibility that it could be found in grapevine growing regions with similar climates as well.

When leaf thinning was introduced into the vineyard where N. ribis was previously found, the increase in sunlight produced an increase in the prevalence of Macrophoma-like symptoms.

This relationship could be due to increased UV exposure, but it isn’t understood. Inoculation through spore suspension was also introduced into the vineyard and confirmed that N. ribis spores can be spread through rain events, such as the ones that are common throughout the growing season in this region. Although timing of inoculation was done originally to confirm the previous theory and there was no correlation between seasons, this could lead to more studies that look into at what point in the season infection is likely to occur.

Out of the nine fungicides trialed in this study, three showed promise as a method of control for the mycelial growth of N. ribis. Although there was reduced mycelial growth, this was only seen in the higher concentrations used and not at all in the lower concentrations.

These studies should be reproduced as the differences in the weather between seasons in this region were great, and results may be skewed to extremes because of it. In the future, more varieties of bunch grapes should be surveyed. The symptoms may appear different in red grapes

versus the white varieties similar to the one observed in this study. Due to the results seen with leaf thinning, further studies should focus on learning more about the relationship between increased sunlight and increased disease incidence. This could be done by measuring light intensity, row orientation, or UV light intensity in correlation with rating disease symptoms throughout the season and at harvest. New locations should be used for both leaf thinning and susceptibility experiments to observe a wider range of microclimates and weather conditions between seasons. Further fungicide studies should focus more on in field applications rather than in vitro assays to assess how the fungicides manage N. ribis in a practical setting. All experiments should be repeated during a variety of seasons and weather conditions to increase the knowledge of N. ribis in the Virginia wine region.

In conclusion, Neofusicoccum ribis was found in Virginia vineyards across Northern and

Central Virginia, Macrophoma rot symptoms increased as sunlight exposure increased, no inoculation timing had the greatest disease incidence or severity across both seasons, and multiple fungicides were identified as potential controls for Macrophoma rot. This information can be used as the groundwork for Macrophoma rot knowledge in Virginia, but the potential for future research topics is expansive.