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 Copyright, Nicole Encardes Causal factors of Macrophoma rot observed on Petit Manseng grapes 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 v 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 3.2. 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). 62 vi 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 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 84 estimation (i.e., inflection point) less than 0.05. 2 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).