"ELM - a" S v‘ I vs": i f: 1‘5 ._;‘ I:- m, ‘*-'-o.. 1007 This is to certify that the dissertation entitled Response of Pinot gris grapevines (Vitis vinifera L.) to infestation by potato leaflmoppers (Empoascafabae Ham's) presented by Marcel S. Lenz has been accepted towards fulfillment of the requirements for the PhD. degree in Horticulture ssor’s Signatfire 3,71ng 15,. 100} Date MSU is an afirmetive—action, equal-opportunity employer ..-,-.-.-.-._..— —.-i—V-’_.—.—.-.-o---—----.Ou----.—.—.—-—--u—.---v—~-v--vfit---0---u----u-----0-----—--v-- LIBRARY Michigan State Unive sity PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/07 p:/CIRC/DateDue.lndd-p.1 Response of Pinot gris grapevinos (Vm’s vimfera L.) to infestation by potato leafhoppers (Empoascafabae Harris) By Marcel S. Lenz A Dissertation Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 2007 ABSTRACT Response of Pinot gris grapevines (Vitis vimjfera L.) to infestation by potato Ieafhoppers (Empoascafabae Harris) By Marcel S. Lenz Potato leafltoppers (Empoascafabae Harris) can be pests in Michigan vineyards, but grapevine responses to infestations have not been well documented. In order to rectify this, experiments were conducted on potted grapevines at the whole-vine and single-leaf levels. Potted and fruitless Pinot gris (Vitis vimfera L.) grapevines grafted to rootstock 1103 Paulsen (V. berlwxlieri Planch. x V. rupesm's Scheele) were infested with various levels of potato leafhopper (PLH) nymphs for seven days. Infestation severity was directly related to the leaf symptoms of cupping and discoloration and inversely related to both shoot and leaf growth. Leafand root biomass tended to decline in response to infestation. Damage thresholds, defined as the number of insects necessary to cause damage to the plant (Pedigo et a1. 1986), were determined for leaf growth, shoot growth and leaf symptoms. A threshold of 0.5 PLH was found for leaf cupping, leaf size, shoot length and internode length; a threshold of 1.0 PLH was found for leaf discoloration and leaves per vine; a threshold of 3.0 PLH was found for internodes per vine and 4.5 PLH for root fresh mass, root dry mass and total vine dry mass. Although PLH infestations caused decreases in shoot growth, leaf growth and biomass accumulation, the vines were able to tolerate low infestation levels without measurable decline in growth. In addition, the vines were able to recover for some growth reductions during the post-infestation period. In order to investigate the impact of PLH infestation on grapevine leaf photosynthesis, PLH nymphs were caged on 2.5 cm2 portions of grapevine leaves and gas exchange was measured before and after the infestation period. PLH infestation level was inversely proportional to carbon assimilation (A), transpiration (E) and stomatal conductance (G.) and directly proportional to internal C02 concentration (C;). Decreases in A were correlated with decreased G. and increased C; and thus reductions in A were due to both stomatal and non-stomatal limitations. Damage thresholds existed for A, E, G. and Ci at most leaf positions. Threshold levels for the four leaf positions from apex to base respectively were: 1, 2, 2 and 8 for A; 1, l, 2 and none for E; l, 1, 2 and 8 for G.; and none, 4, 4 and 8 for C;. These studies show that: 1) PLH infestations reduce shoot growth, leaf growth and biomass accumulation of potted Pinot gn's grapevines; 2) PLH infestations reduce A, G. and E and increase Ci of grapevine leaves; 3) damage thresholds exist for growth and photosynthetic performance of potted Pinot gn's grapevines. Dedication This one goes out to all the seekers. iv Acknowledgments I would like to give thanks to my family: to my parents Uwe and Margit Lenz for everything they’ve done for my brother and me; to my brother Derek for always being my friend since even before the cord was cut; to my girlfriend Carrie for all the love and companionship she’s given to me. I would also like to thank the friends that I’ve made during graduate school for their help and for the good times we’ve had together: Conrad and Elzette Schutte, Mauricio and Marlene Canoles, Dan and Nicole Wampfler, Jon Treloar, Jay and Coryn Briggs, Jen Dwyer, Pat Dimanan, Nick and Kasey Wierzba, Dustin Stabile and Brian Hosmer. Finally, I would like to thank all of the members of my guidance committee for their patience and support: Drs. G. Stanley Howell, Rufirs Isaacs, James A Flore, David P. Miller and Annemiek Schilder. Table of Contents List of Tables ....................................................................................... viii List of Figures ...................................................................................... x List of Symbols, Units and Abbreviations ...................................................... xii Literature Review .................................................................................... 1 Literature Cited .................................................................................... 21 Chapter 1. Vegetative growth responses of Pinot gris (Via's vimfera L.) grapevine: to infestation by potato Ieafhoppers (Empoascafabae Harris). Abstract ............................................................................................. 29 Introduction ......................................................................................... 30 Materials and Methods .............................................................................. 33 General ................................................................................................... 33 Experiment I ............................................................................... 33 Experiment II .............................................................................. 38 Results ............................................................................................... 41 Experiment I ................................................................................. 41 Discussion .......................................................................................... 65 Literature Cited .................................................................................... 72 Chapter 2. Photosynthetic performance of Pinot gris (Vitis vimjfa-a L.) leaves in response to potato leafhopper (Empoascafabae Harris) infestation. Abstract ............................................................................................. 7 5 Introduction ......................................................................................... 77 Materials of Methods .............................................................................. 79 General ....................................................................................... 79 Experiment I .............................................................................. 82 Experiment II .............................................................................. 84 Results ............................................................................................... 87 Experiment I ................................................................................. 87 Experiment 11 .............................................................................. 95 Discussion .......................................................................................... 106 Literature Cited .................................................................................... 110 Conclusions and Future Research ............................................................... 112 Appendix 1: Pictures of cages used during single-leaf studies (Chapter 2). A side view, a quarter is shown for comparison; B. Top view, a quarter is shown for comparison; C. held in the open position, note: fabric on bottom half of cage only ........................ 115 Appendix II: Trend curves and R2 values for LR and AG curves .......................... 116 vii List of Tables Charger 1 Table 1. Treatment levels and their equivalents in terms of potato leafhoppers (PLH) per leaf and potato leafhopper hours (PLH-hrs.) per vine and per leaf (Experiment I) ....... 47 Table 2. Leaf cupping of Pinot gris grapevines in response to infestation by potato leafhopper (PLH) nymphs for seven days (Experiment I) .................................... 48 Table 3. Leaf discoloration of Pinot gris grapevines in response to infestation by potato leafhopper (PLH) nymphs for seven days (Experiment I) .................................... 48 Table 4. Leafproduction of Pinot gris grapevines in response to infestation by potato leafhopper (PLH) nymphs for seven days (Experiment I) .................................... 49 Table 5. Shoot production of Pinot gris grapevines in response to infestation by potato leaflropper (PLH) nymphs for seven days (Experiment I) .................................... 49 Table 6. Biomass production of Pinot gris grapevines in response to infestation by potato leafhopper (PLH) nymphs for seven days: 1. fresh biomass, 11. dry biomass (Experiment I) ...................................................................................... 50 Table 7. Damage thresholds for Pinot gris grapevines in response to infestation by potato leafhopper (PLH) nymphs for seven days in terms of PLH per leaf, PLH per vine and potato leafl'ropper hours (PLH-hrs.) per leaf and PLH-hrs per vine .................... 51 Table 8. Treatment levels and their equivalents in terms of potato leafhoppers (PLH) per vine and potato leafhopper hours (PLH-hrs.) per vine and per leaf (Experiment 11). . . ...58 Table 9. Leafproduction of Pinot gris grapevines prior to the potato leafhopper (PLH) infestation period (Time-l, Experiment 11) ..................................................... 59 Table 10. Shoot production of Pinot gris grapevines prior to the potato leafhopper (PLH) infestation period (Time-1, Experiment 11) ..................................................... 59 Table l l. Leafcupping of Pinot gris grapevines in response to infestation by potato leaflropper (PLH) nymphs for seven days (Time—2, Experiment I1) ........................ 60 Table 12. Leaf discoloration of Pinot gris grapevines in response to infestation by potato leafhopper (PLH) nymphs for seven days (Time-2, Experiment 11) ........................ 60 Table 13. Leafproduction of Pinot gris grapevines three days after the potato leafhopper (PLH) infestation period (Time-2, Experiment 11) ............................................ 61 viii Table 14. Shoot production of Pinot gris grapevines three days after the potato leafhopper (PLH) infestation period (T ime-2, Experiment 11) .............................. 61 Table 15. Leaf production of Pinot gris grapevines ten weeks after the potato leafhopper (PLH) infestation period (T ime-3, Experiment II) ............................................ 62 Table 16. Shoot production of Pinot gris grapevines ten weeks after the potato leafhopper (PLH) infestation period (T ime-3, Experiment 11) .............................. 62 Table 17. Biomass production of Pinot gris grapevines ten weeks after the potato leafhopper (PLH) infestation period: 1. fresh biomass, 11. dry biomass (Time-3, Experiment H) ...................................................................................... 63 Table 18. Damage thresholds for Pinot gris grapevines in response to infestation by potato leafhopper (PLH) nymphs for seven days in terms of PLH per leaf, PLH per vine and potato leafhopper ours (PLH-hrs.) per leaf and PLH-hrs. per vine (Time-3, Experiment 11) ...................................................................................... 64 Chapter 2 Table 19. Gas exchange of Pinot gris leaves in response to infestation by potato leaflroppers (PLH): I. apical leaf position ...................................................... 90 Table 20. Gas exchange of Pinot gris leaves in response to infestation by potato leaflroppers (PLH): II. apical-1 leaf position .................................................. 91 Table 21. Gas exchange of Pinot gris leaves in response to infestation by potato leaflroppers (PLH): III. MRFE leaf position .................................................... 92 Table 22. Gas exchange of Pinot gris leaves in response to infestation by potato Ieaflroppers (PLH): IV. cluster leaf position .................................................... 93 Table 23. Damage thresholds for gas exchange of Pinot gris in response to infestation by potato leathoppers (PLH) in terms of: I. PLH/leaf; II. PLH-hours/leaf .................... 94 Table 24. The efl‘ect of PLH infestations on dark respiration rate (Rd), uantum efficiency (0), light compensation point (cp), Am at 1400 urnol‘m'2*s' PAIL carboxylation efficiency (k), A"m at saturating ppm C02, and C02 compensation point (I‘) of Pinot gris leaves. Treatments are: 9 nymphs (PLH) or 0 (Control) ................ 105 ix List of Figures 9m}. Figure 1. Relationship between midrib length and leaf area of Pinot gris leaves. The midrib lengths of 69 leaves were measured then each leaf was scanned with a leaf area meter. A linear correlation analysis was performed and the relationship was used to estimate the leaf area of experimental vines ................................................... 44 Figure 2. The relationship between infestation severity and leaf cupping of Pinot gris grapevines. Leafcupping is expressed as the percentage of the total leaf number that exhibited downward cupping in response to PLH infestation. Infestation severity is expressed as the number of individuals in the infestation per vine ................................. 45 Figure 3. The relationship between infestation severity and leaf discoloration of Pinot gris leaves. Leafdiscoloration is expressed as the percentage of the total leaf number that exhibited discoloration in response to PLH infestation Infestation severity is expressed as the number of individuals in the infestation per vine ...................................... 46 Figure 4. Relationship between midrib length and leaf area of Pinot gris leaves. The midrib lengths of 469 leaves were measured then each leaf was scanned with a leaf area meter. A non-linear correlation was performed and the relationship was used to estimate the leaf area of experimental vines .............................................................. 55 Figure 5. The relationship between infestation severity and leaf cupping for Pinot gris grapevines. Leafcupping is expressed as the percentage of the total leaf number that exhibited downward cupping in response to PLH infestation. Infestation severity is expressed as the number of individuals in the infestation per leaf .......................... 56 Figure 6. The relationship between infestation severity and leaf discoloration of Pinot gris leaves. Leafdiscoloration is expressed as the percentage of the total leaf number that exhibited discoloration in response to PLH infestation. Infestation severity is expressed as the number of individuals in the infestation per leaf. ...................................... 57 Chester]. Figure 7. Light utilization of Pinot gris leaves prior to the PLH infestation period (day- l). Each value in the light response curves is the mean of four replicates. Standard errors of the means occur about each data point ...................................................... 99 Figure 8. Light utilization of Pinot gris leaves immediately after 42 hours of PLH infestation (day-3). Each value in the light response curves is the mean of four replicates. Standard error of the means occur about each data point .................................... 100 Figure 9. Light utilization of Pinot gris leaves seven days after a PLH infestation period of 42 hours (day-10). Each point in the light response curves is the mean of four replicates. Standard errors of the means occur about each data point ..................... 101 Figure 10. C02 utilization of Pinot gris leaves prior to the PLH infestation period (day- l). Each point in the AC; curves is the mean of four replicates. Standard errors of the means occur about each data point .............................................................. 102 Figure 11. C02 utilization of Pinot gris leaves immediately after the PLH infestation period (day-3). Each point in the AG curves is the mean of four replicates. Standard errors of the means occur about each data point ............................................... 103 Figure 12. C02 utilization of Pinot gris leaves seven days afier the PLH infestation period (day-10). Each point in the ACi curves is the mean of four replicates. Standard errors of the means occur about each data point ............................................... 104 List of Symbols, Units and Abbreviations Symbol PLH PLH-hrs %SS Parameter potato leafhopper potato leafhopper hours net C02 assimilation maximum rate of C02 assimilation sub-stomatal C02 concentration C02 compensation point quantum efficiency stomatal conductance transpiration carboxylation efficiency photosynthetically active radiation standard error most recently fully expanded leaf percent soluble solids xii Units umol C02 * m'2 "' s'l umol co. * m'2 r s" ul/l C02 uI/l C02 umol C02 fixed/umol photons mmol H20“ m’2 * s‘l mmol H20 * m“2 * s'l umol C02 fixed/ppm C02 umol photons "‘ m’2 '-" s’l °BRIX Literature Review Introduction Grapevines are one of the oldest known cultivated plants. Historical records indicate that cultivated Vitis vinifera L. originated in the region between the Black and Caspian seas (W inkler 1974, Mullins 1998) roughly 8,000 years ago (Mullins 1998). Grapevines are cultivated nearly everywhere in the world where human civilizations exist and where the climate is conducive to vine growth (Winkler 1974). Grapes are used for fresh fruit, raisins, juice, wine and as a seed oil extract. According to the Michigan Grape and Wine Industry Council’s website (2006), there are about 13,500 acres of vineyards in Michigan. Most of these are Concord and Niagara cultivars and are used for juice production. About 1,500 acres of this total are used for wine grapes making Michigan the eighth largest wine grape producer in the United States. About 58% of wine grapes in Michigan are vinifera; among these are Riesling, Chardonnay, Cabernet franc and Pinot gris. Since 1997 there has been a 24% increase in vineyard area, most of which is due to new vinifera plantings. Michigan vineyards are subjected to a variety of stresses. This region is considered a cool climate viticultural area, which means the growing season is short and vines in Michigan can suffer from freeze damage and both early and late season frosts (Howell 2001). Although rainfall is generally not lacking, high humidity can lead to much disease pressure, especially from fungi (Agrios 1997). In addition to about two dozen pathogens that can infest vines here, there are also about two dozen insect species that can infest Michigan vineyards (Isaacs et al. 2003). Among these insects, the potato leafhopper (PLH) is one of the most important. PLH infestations of winegrape vineyards can lead to leaf discoloration and deformation, and the cultivar Pinot gris is one of the more susceptible cultivars to such infestations (Isaacs, personal communication 2001, 2006). PLH infestations are commonly treated with pesticides at a cost of both time and money to growers (Wise et al. 2006). Although such resources are allocated to the control of PLH infestations, little is known about how the infestations actually affect grapevine productivity. Changes in leaf morphology and leaf color in response to infestations have been observed repeatedly (personal observation), but it is not clear how such symptoms afi‘ect vine growth. Knowing how PLH infestations affect vine growth would be valuable in making rational control decisions in the vineyard. Quantifying infestations in terms of their effects on vine growth and determination of damage thresholds could potentially reduce vineyard management costs. A better understanding of how PLH afi’ect grapevine productivity is necessary. To date, there are no scientific studies which document this relationship. However, there are many studies that show how PLH affect the growth of other host plant species such as alfalfa (Medicago sativa L.) (Womack 1984, Kabrick and Backus 1990, Ecale and Backus 1995b, Zhou and Backus 1999) and potato (Solarium tuberosum L.) (Walgenbach et al. 1985). These plant species generally exhibited stunted growth and reduced carbon assimilation when infestations were severe. If similar consequences occur in cases of PLH infestations of grapevines, grapevine crop balance might be altered, potentially leading to reduced vine productivity and vineyard profitability. Grapevine Balance The concept of grapevine balance dates back to the early 20‘ll century. The French viticulturist M. L. Ravaz is credited with discovering the relationship between vegetative and reproductive growth of grapevines within a given season. The relationship became known as the Ravaz Index and it is the ratio of fruit yield and vegetative yield as measured by flesh pruning weights (Ravaz 1911). The Ravaz Index is a method of quantifying events that already occurred during the previous growing season. In the 1920’s and 1930’s, the Michigan viticulturist Newton Partridge refined the Ravaz Index into a predictive model. He used the hindsight concept of the Ravaz Index and changed it to a model that could be used to estimate the potential yield of ripe fiuit on a vine for the coming season. Partridge showed that one could estimate the yield potential of the vine for the upcoming season based upon the flesh mass of pruned canes flom the previous growing season (Partridge 1925, 1926). Partridge observed that vines with heavier cane pruning weights had a greater capacity for larger yields the next growing season (Partridge 1925). Thus, pruning weights flom year-1 could be used to estimate the fluiting potential of year-2 (Partridge 1926). He also observed that fluit quality as defined by °BRIX was influenced by pruning and thinning methods (Partridge 1931). The observations made by Partridge are now known as the growth-yield relationship (Howell 2001). The growth-yield relationship is fundamentally about the accumulation, allocation and partitioning of vine resources between vegetative growth and reproductive yield. Today, the growth-yield relationship is often expressed as the amount of leaf area required to mature a given mass of fruit (Smart er al. 1990). The range of leaf area required to mature one gram of fruit can be wide (Smart 1985). According to a review by Howell, the range is 7-14 cm2 per gram fluit (Howell 2001). This wide range is partly due to factors that can influence leaf photosynthesis (Kriedemann 1977). Such factors include the environment, both abiotic (W arcing et al. 1968, Flore and Lakso 1989, Petrie er al. 2000c), and biotic (Mercader and Isaacs 2003, Lehman 2005, Nail and Howell 2005), and grapevine canopy management (Smart 1985). These factors can influence vine growth and productivity through their influences on leaf photosynthesis (Petrie et al. 2000b, Nail and Howell 2005). Photosynthesis The most fundamental physiological process that affects grapevine productivity is leaf photosynthesis (Kriedemann 1977). Over 90% of plant dry matter is derived flom atmospheric carbon fixed during photosynthesis (Flore and Lakso 1989). Grapevine photosynthesis occurs mainly in the chloroplasts of the leaf mesophyll cells (Kriedemann 1977), although other green tissues have been shown to be photosynthetically active (Kriedemann 1967, Pandey and Farmahan 1977). Sugar production via photosynthesis begins with the chloroplast. Triose phosphates are synthesized in the chloroplast then either stored as starch or exported flom the chloroplast to the cytosol where they can be reduced into sucrose. These sugars can then be loaded into the phloem, translocated through the plant and unloaded in actively growing tissues called “sinks”. Here, the sugars can be oxidized to produce energy compounds such as adenosine triphosphate (ATP), or reduced firrther to make starch, cellulose, tissues and organs (Taiz and Zeiger 1998) Photosynthetic rates can be measured directly as the amount of 02 evolved by the plant (Ladd Jr. 1964) or the amount of C02 assimilated by the plant (Wareing et al. 1968 Womack 1984, Baysdorfer and Bassham 1985, Smart et al. 1988, Flinn et al. 1990, Layne and Flore 1995, Edson et al. 1995a, Petrie et al. 2000c). Such gas exchange measurements can be conducted on whole plants (Layne and Flore 1995, Edson et al. 1995a) or individual leaves and leaf regions (Layne and Flore 1992, Edson et al. 1995a, Petrie er al. 2000c). Photosynthetic carbon assimilation can also be measured indirectly as biomass accumulation by the plant. Biomass accumulation can be measured as the size or mass of plants or portions of plants (Edson et al. 1995b, Miller et al. 1996b, Petrie et al. 2000b). Such techniques have lead to a greater understanding of the factors that can influence photosynthesis of grapevines and other plants. Several such factors that can influence photosynthesis include those of the abiotic environment. Abiotic Environment Several components of the abiotic environment are known to influence the rate of net photosynthesis (Pn) of plants. These include light (Flore and Lakso 1989), temperature (Flore and Lakso 1989), ambient C02 concentration (W arcing er a1. 1968, Stoev and Slavcheva 1979), water content of the soil and atmosphere (Flore and Lakso 1989), wind (Jackson 2000) and nutrient availability (Jackson 2000). Additionally, there are diurnal effects on photosynthesis and leaves will typically show a decline in Pn in the afternoon (Kriedemann and Smart 1971). The general photosynthetic reaction is C02 + H20 + light = C6H1205 + 02. From this equation it should be noted that the main substrates for plant photosynthesis are atmospheric C02 and water. In the presence of adequate light, and within a range of temperature optima, these substrates can be synthesized into carbohydrates with oxygen given off as a byproduct. Light provides the energy that drives photosynthesis. At low light levels, C02 losses due to respiration can exceed C02 gains due to photosynthesis (Kriedemann 1968, Stoev and Slavcheva 1979). As light levels increase, the light compensation point will be reached, defined as no net gain or loss in CO2 exchange by the leaf (Layne and Flore 1995). Beyond the light compensation point, the rate of grapevine leaf photosynthesis increased with increased light intensity striking the leaf (Kriedemann 1968, Wareing et a1. 1968, Kriedemann 1977, Smart er al. 1988). However, there is a point where photosynthetic rates no longer increase in response to increasing light (Kriedemann 1968), and this is referred to as the light saturation point. Grapevine leaves will typically reach the light saturation point around one third to one half full sunlight (Kriedemann 1977). In Michigan, this equates to 800-1000 |.tmol"‘m'2"‘s'l PAR (personal observation). Temperature is another abiotic influence on leaf photosynthesis. Temperature influences the rate of Pn over a range of light levels and C02 concentrations (Kriedemann 1968, Stoev and Slavcheva 1979), increasing up to a temperature optimum of 25°C for greenhouse grown vines and 30°C for field grown vines (Kriedemann 1968). Leaf Pn declined at temperatures exceeding about 35-40°C (Kriedemann 1968, Stoev and Slavcheva 1979). This decline in Pn due to excessive heat is likely due to enzyme destabilization, stomatal closure and tissue desiccation (Kriedemann 1977). C02 is one of two main substrates for sugar synthesis. Ambient CO2 levels influence the rate of carbon assimilation (Wareing et al. 1968, Stoev and Slavcheva 1979). Increasing ambient C02 concentration resulted in elevated rates of Pn over a range of light levels (Wareing er al. 1968). However, as with light, there is an optimum level of CO2 above which increasing C02 concentrations will not result in significant increases in Pn (Kriedemann 1977, Layne and Flore 1992, 1995). Water is the other main substrate for the reactions of photosynthesis. Water content of the soil and its influence on leaf water status can influence Pn (Flore and Lakso 1989). Soil water content can inhibit plant photosynthesis when there is either drought (Flore and Lakso 1989) or flooding (Davies and Flore 1986, Larson et al. 1991, Beckman et al. 1992, Blanke and Cooke 2004). In both cases, stomatal closure is involved in the inhibition of Pn (Bradford and Hsiao 1982, Davies and Flore 1986, Crane and Davies 1988, Flore and Lakso 1989, Larson er al. 1991, Blanke and Cooke 2004). However, C; is generally either not affected or it will increase in response to flooding (Larson et al. 1991, Beckman er al. 1992, Blanke and Cooke 2004) or drought (Flore and Lakso 1989). Photosynthesis is indirectly affected by airflow through the canopy. Wind can shift leaves in the canopy, allowing greater sunlight exposure of shaded leaves. As previously noted, low light levels can inhibit leaf photosynthesis (Kriedemann 1968). “(1nd can also reduce canopy moisture content by reducing humidity and drying wet leaves (English er al. 1990, Jackson 2000). High humidity in the canopy can facilitate fungal infections of the leaves (Agrios 1997) thus potentially reducing leaf photosynthesis (Lehman 2005, Nail and Howell 2005). These aforementioned components of the abiotic environment can influence plant photosynthesis. Thus, they can impact the carbohydrate status of the vine and vine productivity as well. Although humans can alter any of these environmental factors artificially, doing so is largely impractical for commercial purposes, perhaps with the exception of irrigation. However, modification of the vine via canopy management techniques can be used by the viticulturist in order to influence how the abiotic environment interacts with the vine. Canopy Architecture The canopy is defined as the shoot and leaf system (Shaulis and Smart 1974). It is all of the above ground portions of the grapevine. Canopy architecture refers to the relative amounts of each organ (shoots, leaves, fruit and wood) and also refers to how these tissues are organized in space. The architecture of the canopy can alter the microenvironment of the vine and thus influence how the abiotic environment interacts with the vine (Smart 1985). Thus, one can change the architecture of the canopy in order to optimize the influence of the abiotic environment on the vine and hence influence vine Pn. One of the most important aspects of canopy architecture is its impact on light interception. Light interception varies over different portions of the canopy indicating that vine architecture will influence light interception by the canopy (Smart 1973, Smart et al. 1990). Grapevine leaves absorb about 90% of the light that strikes them, with only about 5% reflected and 5% transmitted (Smart et al. 1988). Thus, inter-vine shading of the leaves can reduce carbon assimilation by the canopy. Leaves growing in the shade have been shown to have less photosynthetic potential than leaves growing in the sun (Smart er al. 1988, Intrieri et a1. 1995), and can be parasitic on vine carbohydrates below the light compensation point. In addition to the leaves, grapes have also been shown to be photosynthetically active (Kriedemann 1967, Pandey and F armahan 1977), thus fluit exposure to the light can also influence carbon assimilation of the vine. During the ripening period, shade reduced berry color change, mean berry weight, %SS and increased TA (Smart er a1. 1988), a consequence that could have been due to light, temperature or a combination of the two. Source/sink relations of grapevines In addition to the canopy architecture influence on light interception by leaves, it also affects photoassinrilate allocation and partitioning within the vine as it influences source/sink relations of the vine. The overall carbon budget of the vine can be described in terms of the ratio of carbon sources to carbon sinks. Sink strength has been defined as assimilate demand (Marcelis et al. 2004), and as the product of sink size and sink activity (Flore and Lakso 1989, Taiz and Zeiger 1998). Source strength is defined as assimilate supply (Marcelis et al. 2004). It should be noted that the relative strength of any given sink or source changes over the course of the season (Mullins er al. 1992). The main source of carbon used for growth and metabolism comes flom leaf photosynthesis (W ardlaw 1990). However, young leaves are net carbohydrate sinks and do not become net sources to the vine until they are one third to one halfof their final size (Hale and Weaver 1962). Many studies have been conducted showing how altering vine architecture can influence source/sink relations and thus carbohydrate accumulation, allocation and partitioning. The fluit is a major carbohydrate sink and by veraison will become the dominant sink. Altering crop level can affect carbohydrate translocation patterns of the vine (Hale and Weaver 1962) and also cause changes in leaf photosynthetic rates (Gucci et al. 1991, Edson et al. 1995a, Iacono et al. 1995, Petrie et al. 2000c). Reducing crop level caused decreased total fluit yields (Partridge 1931, Kaps and Cahoon 1989, Edson and Howell 1993, Edson et al. 1995b) but lead to increases in average berry size (Kaps and Cahoon 1989), cluster weight (Partridge 1931, Edson and Howell 1993, Edson et al. 1995b), berries/cluster (Edson and Howell 1993, Edson et al. 1995b), %SS of the fruit (Partridge 1931, Kaps and Cahoon 1989) and juice sugar concentration (Iacono et al. 1995). Increasing clusters per vine caused reductions in leaf area per vine, leaf area per gram fruit and leaf area per gram total vine dry mass (Edson and Howell 1993). In addition to crop level influences on reproductive growth, it also influences vegetative growth of the vine. Increasing crop level caused decreases in shoot length, internode length, leaf size and leaf area per vine (Edson et al. 1993, Edson and Howell 1993, Edson er al. 1995b), decreased leaf area per gram fluit (Edson and Howell 1993) and decreased leaf area per gram total vine dry mass (Edson and Howell 1993). These results were reflected in biomass partitioning which shows that increasing clusters per vine resulted in increased dry mass for fluit but reduced dry mass for leaves, shoots and roots, but with no apparent effect on total vine biomass (Edson and Howell 1993). Similar to reproductive sinks, the number and activity of vegetative sinks can also influence vine biomass partitioning. Shoot numbers are commonly varied by pruning and 10 by altering the number of nodes retained while pruning. As node number retained per vine increases, there is typically an increase in the number of shoots per vine (Smithyman et al. 1997, Smithyman eta1.1998, Miller and Howell 1998). This results in higher yields (Byme and Howell 1978, Miller et a1. 1993, Smithyman et al. 1997, Miller and Howell 1998), but reduced vegetative growth (Miller et al. 1993, Smithyman et a1. 1997, Smithyman et a1. 1998, Miller and Howell 1998). In some cases, higher yields are correlated with reduced fruit maturity as measured by fruit composition values (Byme and Howell 1978, Miller et al. 1993) A study varying shoot numbers within a population of fluitless potted Chambourcin (Joannes Seyve 26-205) grapevines showed that increasing the number of vegetative sinks also influenced biomass partitioning (Miller et al. 1996a, Miller et al. 1996c). As the number of shoots increased, individual shoots were shorter, had fewer and smaller leaves and shorter intemodes (Miller et al. 1996a, Miller et al. 1996c). This indicates within-vine competition; as the number of vegetative sinks increased, there were fewer vine resources allocated to each sink. However, the greater number of sinks did lead to higher vegetative growth on a per vine basis in terms of leaves per vine and total leaf area per vine (Miller et al. 1996a, Miller et al. 1996c). Finally, vines with higher shoot numbers had greater total dry mass by harvest (Miller et al. 1996b), indicating that vines with relatively few sinks were sink-limited. Defoliation is another means by which vine growth can be altered. Reducing source strength by defoliation can decrease the size of the leaf sink, at least temporarily, thus causing pronounced effects on vine growth. Mechanical defoliation of potted grapevines resulted in shorter internodes, smaller leaves and thinner shoots (Petrie et al. 11 2000b). Defoliation caused reductions in yield per vine (Koblet et al. 1994), cluster weight (Koblet et al. 1994, Petrie et al. 2000a), berry weight (Koblet et al. 1994, Petrie et al. 2000a), %SS (Mansfield and Howell 1981, Koblet et al. 1994, Petrie et al. 2000a), fruit sugar content (Iacono et al. 1995, Petrie et al. 2000a), increased titratable acidity (Koblet er al. 1994) and delayed berry maturation (Mansfield and Howell 1981). Defoliation also reduced total vine biomass accumulation (Petn'e et al. 2000b). These studies on defoliation show that removal of the principal source of vine sugars can result in fewer resources available for growth. However, the vine seems able to compensate to some degree for shifts in source/sink balance. Compensatory mechanisms The studies cited above show that varying the source/sink balance of grapevines leads to morphological changes in vine growth. When the balance is shifted, the relative allocation and partitioning of assimilate to vegetative and reproductive tissue is affected. However, in addition to changes in biomass, there are also physiological changes occurring in grapevines in response to shifis in source/sink balance. Leaan can be stimulated by reducing source strength or by increasing sink strength, a phenomenon known as photosynthetic compensation (Flore and Lakso 1989, Wardlaw 1990). Photosynthetic compensation has been demonstrated in several plant species. Reducing source strength by defoliation caused increased Pn for grapevines (Iacono et a1. 1995, Intrieri er al. 1997, Petrie et al. 2000c), alfalfa (Baysdorfer and Bassham 1985), cherry (Prunus cerasus L.) (Layne and Flore 1992, 1995) and both bean (Phaseolus vulgaris L.) and maize (Zea mays L.) (Wareing et a1. 1968). Reducing leaf area per vine 12 by varying the number of shoots retained resulted in no difi‘erences in vine dry mass throughout most of the season (Miller et al. 1996b), indicating that vines with low leaf area were operating at higher photosynthetic rates. Thus, it appears that leaves of grapevines and other plants can increase Pn in situations where source strength is insufficient to meet the energy demands of the sinks. Similarly, increasing the sink strength of grapevines can also stimulate leaf Pn. Situations where sink strength was increased by increasing crop level resulted in reductions in total leaf area but increases in leaf assimilation rates (Edson et al. 1995b, Petrie et al. 2000b) ultimately resulting in similar total vine biomass accumulation among vines that differed in crop level (Edson et al. 1995b, Petrie et al. 2000b). A study in which cluster numbers were varied on potted vines resulted in similar total leaf area per vine yet vines with higher crop level were able to ripen more fruit to similar maturity levels (Miller et al. 1996c). Since fruit maturity, as measured by %SS, pH and titratable acidity, was similar among vines that differed in leaf area per gram fruit, vines with less leaf area must have been operating at higher rates of leaf Pn (Miller et al. 1996c). Similar situations occurred where 12% of the main leaves were removed from grapevines but there 'were no differences in yields or fruit composition by the end of the study (Koblet et at 1994), and where half of the leaves were removed from shoots of Concord vines with no apparent affect on %SS, bud fruitfulness or subsequent vine size (Mansfield and Howell 1981). 13 The biotic environment and damage thresholds As suggested above, the abiotic environment directly and indirectly influences vine Pn. Additionally, Pn can also be influenced by the biotic environment. In Michigan, there are over two dozen pathogens that can cause disease to grapevines and there are also over two dozen insects that can infest grapevines (Isaacs et al. 2003 ). Every portion of the grapevine can be subjected to disease by pathogens or infestation by insects (Isaacs et al. 2003). Among the host of pathogens and insects known to affect grapevines in Michigan, some have been shown to infest grapevine leaves and reduce leaf Pn. These include powdery mildew (Uncinula necator Burr.) (Shtienberg 1992, Nail and Howell 2005), downy mildew (Plasmopara viticola Berk. and Curt. ex de Bary) (Lehman 2005), rose chafers (Macrodactylus subspinosus F.) (Mercader and Isaacs 2003) and Japanese beetles (Popillr’ajaponica Newman) (Mercader and Isaacs 2003). Another foliar-feeding organism of grapevines in Michigan is the leafhopper. The two most important species in Michigan are the Eastern grape leaflropper (Etytlrmneura comes Say) and the potato leafhopper (Empoascafabae Harris) (Isaacs et al. 2003). Both feed on the underside of grapevine leaves and can cause damage as they feed. Both are generalists and known to feed on several species of plants (DeLong 1931, Lamp et al. 1994, Martinson et al. 1994). In general, vinifera cultivars tend to be less tolerant than V. labrusca L. cultivars (Isaacs et a1. 2003). Although very little is known about how PLH infestations affect grapevines, the Eastern grape leafhopper (Erythroneura comes Say) reduced yields (Martinson et al. 1997), berry weights (Martinson et a1. 1997) and juice sugars (%SS) (Van Dine 1923) of Concord (Vitis labrusca L.) grapevines. l4 These various diseases and insects in Michigan vineyards are known to infest vine canOpies, and some are known to impact leaf photosynthesis. The concept of photosynthetic compensation discussed above is one example of the ability of grapevines to tolerate a degree of imbalance in source/sink relations, and this has been demonstrated by plant responses to abiotic defoliation discussed above (Layne and Flore 1992, Layne and Flore 1995, Petrie et al. 2000a, 2000b, 2000c). A second example is the concept of feedback inhibition, a situation where end-products of photosynthesis can inhibit further production of assimilate which is especially pronounced in response to low sink demand (Layne and Flore 1995). Although there are no examples in the literature of similar grapevine responses to biotic reductions in leaf area, the mechanism does seem to exist as evidenced by the affects of defoliation on Pn. The ability of plants to tolerate some level of stress is integral in the concept of thresholds. There have been several different thresholds described in the literature in regards to biotic stress (Pedigo et al. 1986, Pedigo and Higley 1992, Hunt et al. 2000). These thresholds focus on the ultimate goal of determining the economically acceptable levels of damage done to crops by pests, with damage being defined as “measurable loss in host utility” (Pedigo et a1. 1986). Before these economic thresholds can be defined, it is necessary to quantify plant growth in response to infestations by a specific pest; this has not been done with PLH infestations of grapevines to date. The damage threshold, also known as the damage boundary, is defined as the number of insects necessary to cause damage to the plant (Pedigo et al. 1986). Determination of damage thresholds is one of the objectives of the research conducted for this dissertation. 15 Enrpoascafabae Harris, the potato leafhopper The potato leafhopper (PLH) is a member of the family Cicadellidae which contains about 3,000 North American species (Bland and Jaques 1978). PLH are distributed throughout nearly all of the Eastern United States, and have been found on plants fi'om 220 species, 100 genera and 26 families (DeLong 1931, Lamp et al. 1989, Lamp er al. 1994). PLH have seven developmental stages beginning with the egg and ending with the adult. The eggs are about 1 mm long, elongate and whitish in color. In about 10 days the eggs hatch into nymphs. The nymphs progress through five stages of development called instars and during each instar the nymphs lack firnctional wings. The developing wings, called wingpads, gradually grow larger as the nymphs proceed through the instars eventually becoming fully functional wings by adulthood. The adults grow to be about 3 mm long, are an iridescent light-green color and have a life-span of about 28 days. PLH are not native to MI and do not overwinter here (DeLong 1931, Medler 1957, Shields and Testa 1999). They overwinter in the southern states along the coast of the Gulf of Mexico (Medler 1957, Shields and Testa 1999). As the weather warms during the early spring, they migrate north eventually arriving in Michigan in late May to early June (Carlson et al. 1992). As Fall temperatures cool, they complete their annual migration cycle by returning to the South (Shields and Testa 1999) or they die. Leafhopper infestations and plants PLH feed by using their needlelike mouthparts, called stylets, to probe plant tissues, lacerate cells and then ingest the fluids that are flushed from these cells. This feeding behavior can cause plants to deve10p ‘hopperburn’, a general yellowing and 16 wilting of plant tissues. Hopperbum is caused by mechanical damage fi'om probing and laceration, and chemical damage from the insect’s saliva (Medler 1941, Backus and Hunter 1989, Hunter and Backus 1989, Kabrick and Backus 1990, Ecale and Backus 1995a, Ecale and Backus 1995b). PLH feeding behavior has been termed ‘lacerate-and- flush’ in reference to their tendencies to rupture cells and tissues with their stylets and then ingest the fluids that are flushed out in the process (Backus and Hunter 1989, Hunter and Backus 1989, Kabrick and Backus 1990). Stylet probing of alfalfa (Medicago sativa L.) can cause abnormally enlarged and abnormally divided cells of the phloem (Medler 1941, Kabrick and Backus 1990, Zhou and Backus 1999) and vascular cambium (Kabrick and Backus 1990), disorganized phloem cells (Kabrick and Backus 1990, Zhou and Backus 1999), collapsed and constricted phloem (Kabrick and Backus 1990, Ecale and Backus 1995b, Zhou and Backus 1999), damaged mesophyll cells (Medler 1941, Hunter and Backus 1989) and damaged xylem (Ecale and Backus 1995b). This range of tissue damage is likely responsible for observations of the numerous altered physiological processes in alfalfa in response to PLH infestations. Assimilation and transpiration of alfalfa were both found to be inversely proportional to the number and duration of PLH exposed to alfalfa stems (Womack 1984). Stomatal conductance, assimilation and transpiration were all inversely proportional to the number of adult female PLH that infested alfalfa (Flinn and Hower 1984). Sugar translocation beyond fed-upon phloem tissues was reduced and sugar concentration upstream fi'om damaged tissue increased for alfalfa in response to PLH feeding (Flinn et al. 1990, Nielsen et al. 1990, Nielsen et al. 1999, Lamp et a1. 2001). 17 These studies indicate that PLH.damaged phloem tissue can act as a biological dam, inhibiting the flow of photoassimilates beyond the vicinity of stylet probing. Reductions in the rates of C02 assimilation and carbohydrate translocation mean that PLH feeding can reduce source strength of the host plants, at least temporarily and in the locality of feeding damage. When carbohydrate source strength is reduced, there will be fewer resources for plant growth and development and this can manifest as a general reduction in quantity and quality of yields. Infestations at the level of individual plants and plant populations of alfalfa showed height reduction (Poos and Johnson 193 6, Kindler et al. 1973, Faris et al. 1981, Flinn and Hower 1984, Hower and Flinn 1986), leaf and stem wilting (Harman et a1. 1995), reduced leaf number and reduction in both fresh and dry weights (Poos and Johnson 1936, Kindler et al. 1973, Flinn and Hower 1984, Hower and Flinn 1986, Flinn et al. 1990, Hutchins and Pedigo 1990). Changes in the nutritional value of alfalfa have been reported as reduced protein content (Poos and Johnson 1936, Kindler er al. 1973, Paris et al. 1981, Hower and Flinn 1986, Flinn et al. 1990, Hutchins and Pedigo 1990), fat content (Poos and Johnson 1936) and fiber content (Poos and Johnson 1936, Hutchins and Pedigo 1990). Infestations also caused delayed alfalfa growth within a growth cycle and regrowth failure in a cycle that followed severe infestations (Flinn et al. 1990, Hutchins and Pedigo 1990). Although damage by PLH can be severe, some evidence exists that plants can compensate for feeding damage. Walgenbach et al. (1985) observed partial recovery of assimilation rates for potato (Solanum mberosum L.) following PLH infestations. Infestation of alfalfa showed eventual phloem regrowth whereby new sieve tubes circumvented damaged areas of phloem (Zhou and Backus 1999). Another study on 18 alfalfa showed phloem damage due to PLH probing, but the phloem appeared normal again eight days after the infestation period (Ecale and Backus 1995b). These studies indicated a degree of tolerance and compensation of some host plants to PLH infestations. Compensation occurred as enhanced rates of photosynthesis and altered tissue growth. In conclusion, the responses of alfalfa to PLH infestations can be seen as set of symptoms occurring from the cellular level to the population level (Flinn and Hower 1984, Hower and Flinn 1986, Kabrick and Backus 1990, Ecale and Backus 1995b, Zhou and Backus 1999). Stylet probing directly damages alfalfa at the level of cells and tissues, but the indirect affects can extend throughout an individual plant and even to entire populations of plants (Flinn et al. 1990, Hutchins and Pedigo 1990). Carbon assimilation and sugar translocation can both be inhibited, leading to reduced vegetative growth and in some cases death (Womack 1984, Flinn et al. 1990, Hutchins and Pedigo 1990, Nielsen et al. 1990, Lamp et al. 2001). In general, probing damage can be thought of as reducing source strength of the host plant, which in turn can reduce both the quantity and quality of yields. Although studies tend to focus on alfalfa, possibly one might find similar responses to PLH by other host species, including grapevines. Quantifying potato leafhopper infestations of grapevines PLH infestations in Michigan vineyards are typically controlled by the use of one or more insecticide sprays (Wise er al. 2006). Pesticide application is a cost to growers and requires the use of fossil fuels and other non-renewable energy resources. Additionally, there is increasing public concern about the impact these chemicals will 19 have on human health and the environment. If pesticide use can be reduced while maintaining long-term productivity of the vineyard, growers could reduce their costs, resources would be conserved and any potential environmental impact of pesticide use could be limited. A better understanding of grapevine tolerance thresholds to PLH infestations is necessary in order to more efficiently use pesticides. Knowing the severity of PLH infestations required to reduce grapevine productivity and/or fiuit quality would be valuable in order to make rational control decisions in the vineyard. Dissertation Goals and Hypotheses The goals of the research for this dissertation were to: l) quantify PLH damage to grapevines in terms of vine vegetative growth, damage thresholds and recovery mechanisms; 2) characterize the impact of PLH infestations on leaf photosynthesis in terms of gas exchange rates, thresholds and recovery mechanisms. The specific hypotheses are: 1) PLH infestations will reduce grapevine leaf and shoot growth; 2) PLH infestations will reduce vine biomass accumulation; 3) there will be damage thresholds of PLH infestation below which no measurable reductions in growth or biomass accumulation will occur", 4) the vine will recover for growth reductions during the post- infestation period; 5) grapevine leaf photosynthesis will decrease in response to PLH infestation; 6) there will be damage thresholds for leaf gas exchange; 7) photosynthetic compensation to PLH infestations will occur for grapevine leaves. 20 Literature Cited Agrios, George N. 1997. Plant Pathology. Academic Press Limited. Backus, E. A and W. B. Hunter. 1989. Comparison of feeding behavior of the potato leafliopper Empoascafabae (Homoptera: Cicadellidae) on alfalfa and broad bean leaves. Environ. Entomol. 18(3): 473-480. Baysdorfer, C. and J. A Bassham. 1985. Photosynthate supply and utilization in alfalfa. A developmental shift from a source to a sink limitation of photosynthesis. Plant. Physiol. 77(2): 313-317. Beckman, T. G., R L. Perry and J. A. Flore. 1992. Short-term flooding affects gas exchange characteristics of containerized sour cherry trees. HortScience 27(12): 1297- 1301. Bland, R G. and H. E. Jaques. 1978. How to know the insects. Wm. C. Brown Company Publishers. Blanke, M. M. and D. T. Cooke. 2004. Effects of flooding and drought on stomatal activity, tranSpiration, photosynthesis, water potential and water channel activity in strawberry stolons and leaves. Plant Grth Regulation 42: 153-160. Bradford, K. J. and T. C. Hsiao. 1982. Stomatal behavior and water relations of waterlogged tomato plants. Plant Physiol. 70: 1508-1513. Byme, M. E. and G. S. Howell. 1978. Initial response of Baco noir grapevines to pruning severity, sucker removal and weed control. Am. J. Enol. Vitic. 29 (3): 192-198. Candolfi, M. P., M. Jennini, E. Carrera and M. Candolfi-Vasconcelos. 1993. Grapevine leaf gas exchange, plant growth, yield, fruit quality and carbohydrate reserves influenced by the grape leaflropper, Empoasca vitis. Entomol. Exp. App]. 69(3): 289-296. Carlson, J. D., M E. Whalon, D. A Landis and S. H. Gage. 1992. Springtime weather patterns coincident with long-distance migration of potato leafhopper into Michigan. Agric. For. Meteorol. 59: 183-206. Crane, J. H. and F. S. Davies. 1988. Periodic and seasonal flooding effects on survival, growth, and stomatal conductance of young rabbiteye blueberry plants. J. Am. Soc. Hortic. Sci. 113(4): 488-493. Davies, F. S. and J. A. Flore. 1986. Short-term flooding effects on gas exchange and quantum yield of rabbiteye blueberry (Vaccinium ashei Reade). Plant Physiol. 81: 289- 292. 21 DeLong, D. M. 1931. A revision of the American Species of Empoasca known to occur north of Mexico. USDA Technical Bulletin No. 231. Ecale, C. L. and E. A Backus. 1995a. Mechanical and salivary aspects of potato leaflropper probing in alfalfa stems. Entomol. Exp. Appl. 77(2): 121-132. Ecale, C. L. and E. A Backus. 1995b. Time course of anatomical changes to stem vascular tissues of alfalfa, Medicago sativa, from probing injury by the potato leafhopper, Empoascafabae. Can. J. Bot. 73(2): 288-298. Edson, C. E. and G. S. Howell. 1993. A comparison of vine architecture systems at different crop loads: leaf photosynthesis, vine yield and dry matter partitioning. Vitic. Enol. Sci. 48: 90-95. Edson, C. E., G. S. Howell and J. A Flore. 1993. Influence of crop load on photosynthesis and dry matter partitioning of Seyval grapevines. 1. Single leaf and whole vine response pre- and post-harvest. Am. J. Enol. Vitic. 44(2): 139-147. Edson, C. E., G. S. Howell and J. A Flore. 1995a. Influence of crop load on photosynthesis and dry matter partitioning of Seyval grapevines. II. Seasonal changes in single leaf and whole vine photosynthesis. Am. J. Enol. Vitic. 46(4): 469-477. Edson, C. E., G. S. Howell and J. A Flore. 1995b. Influence of cr0p load on photosynthesis and dry matter partitioning of Seyval grapevines. 11]. Seasonal changes in dry matter partitioning, vine morphology, yield, and fruit composition. Am. J. Errol. Vitic. 46(4): 478-485. English, J. T., A M. Bledsoe, J. J. Marois and W. M. Kliewer. 1990. Influence of grapevine canopy management on evaporative potential in the fruit zone. Am. J. Enol. Vitic. 41(2): 137-141. Faris, M. A, H. Baenziger and R P. Terlnme. 1981. Studies on potato leafhopper (Empoascafabae) damage in alfalfa. Can. J. Plant Sci. 61: 625-632. Flinn, P. W. and A A Hower. 1984. Effects of density, stage, and sex of the potato leaflropper, Empoascafabae (Homoptera: Cicadellidae), on seedling alfalfa growth. Can. Entomol. 116(11): 1543-1548. Flinn, P. W., A A Hower and D. P. Knievel. 1990. Physiological response of alfalfa to injury by Empoascafabae (Homoptera: Cicadellidae). Environ. Entomol. 19(1): 176- 181. Flore, J. A and A N. Lakso. 1989. Environmental and physiological regulation of photosynthesis in fruit crops. Hortic. Rev. 11: 111-157. 22 Gucci, R, C. Xiloyannis and J. A Flore. 1991. Gas exchange parameters, water relations and carbohydrate partitioning in leaves of field-grown Prunus domestica following fruit removal. Physiologia Planth 83: 497-505. Hale, C. R and R. J. Weaver. 1962. The effect of deve10pmental stage on direction of translocation of photosynthate in Vitis vinifera. Hilgardia: A Journal of Agricultural Science Published by the California Agricultural Experiment Station 33(3): 89-131. Harman, J. L., D. D. Calvin and R A Byers. 1995. Response of alfalfa, Medicago sativa, L., to sequential feeding by the potato leafhopper, Empoascafabae (Harris) (Homoptera: Cicadellidae). J. Kans. Entomol. Soc. 68(2): 159-168. Hibbs, E. T., D. L. Dahlman, and R. L. Rice, Richard 1964. Potato foliage sugar concentration in relation to infestation by the potato leafhopper, Empoascafabae (Homoptera: Cicadellideae). Ann. Entomol. Soc. Amer. 57(5): 517-521. Howell, G. S. 2001. Sustainable grape productivity and the growth: yield relationship: A review. Am. J. Enol. Vit. 52(3): 165-173. Hower, A. A. and P. W. Flinn. 1986. Effects of feeding by potato leaflmpper nymphs (Homoptera: Cicadellidae) on growth and quality of established stand alfalfa. J. Econ. Entomol. 79(3): 779-784. Hunt, T. E, L. G. Higley and L. P. Pedigo. 2000. A re-examination of economic injury levels for potato leafl10pper (Homoptera: Cicadellidae) on soybean. J. Entomol. Sci. 35(2): 97-104. Hunter, W. B. and E. A Backus. 1989. Mesophyll-feeding by the potato leafhopper, Empoascafabae (Homoptera: Cicadellidae): results from electronic monitoring and thin- layer chromatography. Environ. Entomol. 18(3): 465-472. Hutchins, S. H. and L. P. Pedigo. 1990. Phenological disruption and economic consequence of injury to alfalfa induced by potato leaflropper (Homoptera: Cicadellidae). J. Econ. Entomol. 83(4): 1587-1594. Iacono, F., M. Bertamini, A Scienza and B. G. Coombe. 1995. Differential effects of canopy manipulation and shading of Vitis vinifera L. cv. Cabernet Sauvignon. Leaf gas exchange, photosynthetic electron transport rate and sugar accumulation in berries. Vitis 34 (4): 201-206. Intrieri, C., G. Zerbi, L. MarchioL S. Poni and T. Caiado. 1995. Physiological response of grapevine leaves to light flecks. Scientia Horticulturae. 61: 47-59. Intrieri, C., S. Poni, B. Rebucci and E. Magnanini. 1997. Effects of canopy manipulations on whole-vine photosynthesis: results from pot and field experiments. Vitis 36(4): 167-173. 23 Isaacs, R, A Schilder, T. Zabadal and T. Weigle. 2003. A pocket guide for grape [PM scouting in the north central and eastern US. Michigan State University extension bulletin E-2889. Michigan State University Extension, Extension, E. Lansing, MI. Jackson, R S. 2000. Wine science: principles, practices and perception Academic Press, San Diego, CA Kabrick, L. R and E. A Backus. 1990. Salivary deposits and plant damage associated with specific probing behaviors of the potato leafhopper, Empoascafabae, on alfalfa stems. Entomol. Exp. Appl. 56: 287-304. Kaps, M. L. and G. A Cahoon. 1989. Berry thinning and cluster thinning influence vegetative growth, yield, fi'uit composition and net photosynthesis of ‘Seyval blanc’ grapes. J. Amer. Soc. Hort. Sci. 114(1): 20-24. Kindler, S. D., W. R Kehr, R L. Ogden and J. M. Schalk. 1973. Effect of potato leaflropper injury on yield and quality of resistant and susceptible alfalfa clones. J. Econ. Entomol. 66(6): 1298-1302. Koblet, W., M. C. Candolfi-Vasconcelos, W. Zweifel and G. S. Howell, G. S. 1994. Influence of leaf removal, rootstock, and training system on yield and hit composition of Pinot noir grapevines. Am. J. Enol. Vitic. 45(2): 181-187. Kriedemann, P. E. 1967. Observations on gas exchange in the developing Sultana berry. Aust. J. Biol. Sci. 21: 907-916. Kriedemann, P. E. 1968. Photosynthesis in vine leaves as a function of light intensity, temperature, and leaf age. Vitis 7: 213-220. Kriedemann, P. E. 1977. Vineleaf photosynthesis. Symposium international sur la qualite de la vendange le Cap Afrique du Sud. Kriedemann, P. E. and R E. Smart. 1971. Effects of irradiance, temperature, and leaf water potential on photosynthesis of vine leaves. Photosynthetica 5(1): 6-15. Ladd Jr., T. L. and W. A Rawlins. 1964. The effects of feeding of the potato leafhopper on photosynthesis and respiration in the potato plant. J. Econ. Entomol. 58: 628-633. Lamp, W. 0., M. J. Morris and E. J. Armbrust. 1989. Empoasca (Homoptera: Cicadellidae) abundance and species composition in habitats proximate to alfalfa. Environ. Entomol. 18(3): 423-428. Lamp, W. 0., G. R Nielsen and S. D. Danielson. 1994. Patterns among host plants of potato leafhoppers, Empoascafabae (Homoptera: Cicadellidae). J. Kans. Entomol. Soc. 67(4): 354-368. 24 Lamp, W. 0., G. R Nielsen, B. Quebedeaux and Z. Wang. 2001. Potato leaflropper (Homoptera: Cicadellidae) injury disrupts basal transport of “(z-labelled photoassimilates in alfalfa. J. Econ. Entomol. 94(1): 93-97. Larson, K. D., B. Schafl‘er and F. S. Davies. 1991. Flooding, leaf gas exchange and growth of mango in containers. J. Amer. Soc. Hort. Sci. 116(1): 156-160. Layne, D. R and J. A Flore. 1992. Photosynthetic compensation to partial leaf area reduction in sour cherry. J. Am. Soc. Hortic. Sci. 117(2): 279-286. Layne, D. R and J. A Flore. 1995. End-product inhibition of photosynthesis in Prrmus cerasus L. in response to whole-plant source-sink manipulation. J. Am. Soc. Hortic. Sci. 120(4): 583-599. Lehman, B. L. 2005. Downy mildew: host specialization and effects on photosynthesis and carbon partitioning in ‘Niagara’ grapevines. Masters thesis, Michigan State University, East Lansing, MI. Mansfield, T. K. and G. S. Howell. 1981. Response of soluble solids accumulation, fi'uitfirlness, cold resistance, and onset of bud growth to differential defoliation stress at veraison in Concord grapevines. Am. J. Enol. Vitic. 32(3): 200-205. Marcelis, L. F. M., E. Heuvelink, L. R. Hofinan-Eijer, J. Den Bakker and L. B. Xue. 2004. Flower and fruit abortion in sweet pepper in relation to source and sink strength. J. Exp. Bot. 55(406): 2261-2268. Martinson, T. E., T. J. Dennehy and C. J. Hoffman. 1994. Phenology, within-vineyard distribution, and seasonal movement of eastern grape leaflropper (Homoptera: Cicadellidae) in New York vineyards. Environ. Entomol. 23(2): 236-243. Martinson, T. E., R Dunst, A Lakso and G. English-Loeb. 1997. Impact of feeding injury by eastern grape leafhopper (Homoptera: Cicadellidae) on yield and juice quality of Concord grapes. Am. J. Enol. Vitic. 48(3): 291-302. Medler, J. T. 1941. The nature of injury to alfalfa caused by Empoascafabae (Harris). Ann. Entomol. Soc. Amer. 34: 439-450. Medler, J. T. 1957. Migration of the potato leafhopper. A report on a cooperative study. J. Econ. Entomol. 50: 493-497. Mercader, R J. and R Isaacs. 2003. Phenology-dependent efl‘ects of foliar injury and herbivory on the growth and photosynthetic capacity of nonbearing Vitis Iabrusca (Linnaeus) var. Niagara. Am. J. Enol. Vitic. 54(4): 252-260. 25 Miller, D.P., G. S. Howell and R. K. Striegler. 1993. Reproductive and vegetative response of mature grapevines subjected to differential cropping stresses. Am. J. Enol. Vitic. 44(4): 435-440. Miller, D. P., G. S. Howell and J. A Flore. 1996a. Effect of shoot number on potted grapevines. I. Canopy development and morphology. Am. J. Enol. Vitic. 47(3): 244-250. Miller, D. P., G. S. Howell and J. A Flore. 1996b. Effect of shoot number on potted grapevines. II. Dry matter accumulation and partitioning. Am. J. Enol. Vitic. 47(3): 251- 256. Miller, D. P., G. S. Howell and J. A Flore. 1996c. Influence of shoot number and crop load on potted Chambourcin grapevines. I. Morphology and dry matter partitioning. Am J. Enol. Vitic. 47(4): 380-388. Miller, D. P. and G. S. Howell. 1998. Influence of vine capacity and crop load on canopy development, morphology, and dry matter partitioning in concord grapevines. Am. J. Enol. Vitic. 49(2): 183-190. Mullins, M. G., A Bouquet and L. E. Williams. 1992. Biology of the grapevine. Cambridge University Press. Nail, W. R and G. S. Howell. 2005. Effects of timing of powdery mildew infection on carbon assimilation and subsequent seasonal growth of potted chardonnay grapevines. Am. J. Enol. Vitic. 56(3): 220-227. Nielsen, G. R, W. O. Lamp and G. W. Stutte. 1990. Potato leafllopper (Homoptera: Cicadellidae) feeding disruption of phloem translocation in alfalfa. J. Econ. Entomol. 83(3): 807-813. Nielsen, G. R, C. Fuentes, B. Quebedeaux, Z. Wang and W. O. Lamp. 1999. Alfalfa physiological response to potato leafhopper injury depends on leafhopper and alfalfa developmental stage. Entomol. Exp. Appl. 90(3): 247-255. Pandey, R M. and H. L. Farmahan. 1977. Changes in the rate of photosynthesis and respiration in leaves and berries of Vitis vinifera grapevines at various stages of berry development. Vitis. 16: 106-111. Partridge, N. L. 1925. Growth and yield of Concord grape vines. Proc. Am. Soc. Hort. Sci. 23:84-87). Partridge, N. L. 1926. The use of the growth-yield relationship in field trials with grapes. Proc. Am. Soc. Hort. Sci. 23: 131-134 Partridge, N. L. 1931. The influence of long pruning and thinning upon the quality of Concord grapes. Proc. Am. Soc. Hort. Sci. 28: 144-146 26 Pedigo, L. P., S. H. Hutchins and L. G. Higley. 1986. Economic injury levels in theory and practice. Annu. Rev. Entomol. 31: 341-368. Pedigo, L. P. and L. G. Higley. 1992. The economic injury level concept and environmental quality: a new perspective. Am. Entomol. 38(1): 12-21. Petrie, P. R, M. C. T. Trought and G. S. Howell. 2000a Fruit composition and ripening in Pinot noir (Vitis vinifera L.) in relation to leaf area. Australian J. of Grape and Wine Res. 6: 46-51. Petrie, P. R, M. C. T. Trought and G. S. Howell. 2000b. Growth and dry matter partitioning of Pinot noir (Vitis vinifera L.) in relation to leaf area and cr0p load. Australian J. of Grape and Wine Res. 6: 40-45. Petrie, P. R, M. C. T. Trought and G. S. Howell. 2000c. Influence of leaf ageing, leaf area and crop load on photosynthesis, stomatal conductance and senescence of grapevine (Vitis vimfera L. cv. Pinot noir) leaves. Vitis 39(1): 31-36. Poos, F. W. and W. H. Johnson. 1936. Injury to alfalfa and red clover by the potato leafhopper. J. Econ. Entomol. 29(2): 325-331. Ravaz, M. L. 1911. L’effeuellage de la vigne. Annales d L’Ecole Nationale d’agriculture de Montpellier. 11: 216-244. Shaulis, N. and R Smart. 1974. Grapevine canopies: management, microclimate and yield responses. Proc. XIX Int. Hort. Congress, September 1974, Warsaw. 254-265. Shields, E. J. and A M Testa. 1999. Fall migratory flight initiation of the potato leafhopper, Empoascafabae (Homoptera: Cicadellidae): observations in the lower atmosphere using remote piloted vehicles. Agric. For meteorol. 97(4): 317-330. Shtienberg, D. 1992. Effects of foliar diseases on gas exchange processes: a comparative study. Phytopathology 82: 760-765. Smart, R E. 1973. Sunlight interception by vineyards. Am. J. Enol. Vitic. 24(4): 141- 147. Smart, R E. 1985. Principles of grapevine canopy microclimate manipulation with implications for yield and quality: A review. Am J. Errol. Vitic. 36(3): 230-239. Smart, R E, S. M. Smith and R V. Winchester. 1988. Light quality and quantity effects on fruit ripening for Cabernet Sauvignon. Am. J. Enol. Vitic. 39 (3): 250-258. 27 Smart, R E., J. K. Dick, 1. M. Gravett and B. M. Fisher. 1990. Canopy management to improve grape yield and wine quality-principles and practices. S. Afr. Enol. Vitic. 11(1): 3-17. Smithyman, R P., G. S. Howell and D. P. Miller. 1997. Influence of canopy configuration on vegetative development, yield and fiuit composition of Seyval blanc grapevines. Am. J. Enol. Vitic. 48 (4): 482-490. Smithyman, R P., G. S. Howell and D. P. Miller. 1998. The use of competition for carbohydrates among vegetative and reproductive sinks to reduce fruit set and Botlytis bunch rot in Seyval blanc grapevines. Am. J. Enol. Vitic. 49(2): 163-170. Stoev, K. D. and T. Slavcheva. 1979. Influence of major ecological factors on photosynthesis of grape leaves. Sov. Plant Physiol. 26: 351-354. Taiz, L. and E. Zeiger. 1998. Plant physiology 2ml edition. Sinauer Associates, Inc. Van Dine, D. L. 1923. The effect of leaf-hepper injury on the sugar-content of grapes. J. Econ. Entomol. 16: 353-357. Walgenbach, J. F. and J. A Wyman. 1985. Potato leafhopper (Homoptera: Cicadellidae) feeding damage at various potato growth stages. J. Econ. Entomol. 78(3): 671-675. Wardlaw, J. F. 1990. The control of carbon partitioning in plants. New Phytol. 116(3): 341-3 8 1 . Wareing, P. F ., M. M. Khalifa and K. J. Treharne. 1968. Rate limiting processes in photosynthesis at saturating light intensities. Nature 220: 453-457. Wise, J. C, L. J. Gut, R Isaacs, A M. C. Schilder, G. W. Sundin, B. Zandstra, E. Hanson and B. Shane. 2006. 2006 Michigan Fruit Management Guide. Michigan State University extension bulletin E154. Womack, C. L. 1984. Reduction in photosynthetic and transpiration rates of alfalfa caused by potato leaflropper (Homoptera: Cicadellidae) infestations [Empoascafabae, Medicago sativa]. J. Econ. Entomol. 77(2): 508-513. Zhou, C. L Ecale and E. A Backus. 1999. Phloem injury and repair following potato leafhopper feeding on alfalfa stems. Can J. Bot. 77: 537-547. 28 Chapter 1 Vegetative growth responses of Pinot gris (Vitis vimfera L.) grapevines to infestation by potato leafhoppers (Emoascafabae Harris). Abstract Potato leaflroppers (Empoascafabae Harris) can be pests in Michigan vineyards, but grapevine responses to infestations by this insect have not been well documented. To rectify this, two experiments were conducted on potted and fruitless Pinot gris (Vitis vinifera L.) grapevines grafted to rootstock 1103 Paulsen (V. berlandieri Planch. x V. rupestris Scheele) and infested for seven days with a range of potato leafhopper (PLH) nymphs from 0.0-4.5 per leaf. Shoot growth, leaf growth and infestation symptoms of the leaves were quantified before and after the infestation period and biomass of vegetative vine structure was measured at the end of each experiment. Infestation severity was directly related to the leaf symptoms of cupping and discoloration and inversely related to both shoot and leaf growth. Both leaf and root mass declined in response to infestation level. Damage thresholds were observed for leaf symptoms, leaf growth and shoot growth for both experiments. Vrne recovery, as measured by the influence on growth reductions by infestation, was observed in a second experiment (Experiment 11). PLH infestations decreased shoot and leaf growth and vine biomass, but the vines were able to recover fiom low infestation levels in terms of leaf and shoot growth during the post-infestation period. 29 Introduction Potato leafhoppers, Empoascafabae Harris (PLH) can be grapevine pests in Michigan and the Great Lakes region, but grapevine responses to infestations have not been well documented. Knowing how PLH populations affect vineyard productivity would be a useful tool for vineyard management. Ifthere is a relationship between the severity of a PLH infestation and changes in the quantity or quality of grapevine growth, then damage thresholds could be defined. Damage thresholds have been defined as the number of insects necessary to cause damage to the plant (Pedigo et al. 1986). Such thresholds could be of value in making control decisions based upon estimated losses in vineyard productivity rather than the mere presence, absence or quantity of this insect. Utilization of such thresholds could decrease the use of insecticides, and thus reduce vineyard management costs and prevent any potential environmental pollution from insecticide use. An area of major importance in viticulture is carbohydrate source/sink relations. Shifts in source/sink relations can result in changes in vine growth Reducing carbohydrate source strength of potted Pinot noir (Vitis vinr'fera L.) by defoliation resulted in decreased rates of berry maturation (Petrie er al. 2000a) and reduced biomass accumulation (Petrie et al. 2000b). Mechanical defoliation of Niagara (Vitis labrusca L.) grapevines at bloom resulted in stunted shoots and lower dry mass of roots (Mercader and Isaacs 2003, Mercader and Isaacs 2004). Increasing vegetative sink number and competition by increasing shoot numbers of potted Chambourcin (J oannes Seyve 26-205) grapevines resulted in shoots that were shorter, had fewer and smaller leaves and shorter 3O internodes (Miller et al. 1996a). Similarly, increasing reproductive sink strength by retaining higher numbers of clusters on potted Seyval (Seyve-Villard 5-276) grapevines caused decreases in shoot length, leaf area and cluster weight (Edson et al. 1993). These studies show that altering carbohydrate source/sink relations can have profound affects on vine morphology. Decreasing the ratio of carbohydrate source strength to carbohydrate sink strength can lead to deficits in the vine resources available for plant growth and metabolism. Since the leaves are the main source of carbohydrate production for grapevines, disruption of leaf function by foliar-feeding insects could reduce source strength and limit vine growth, productivity and fiuit quality. PLH are foliar feeders of grapevines and thus infestations could lead to impaired leaf firnction. Although grapevine responses to PLH infestations have not been well established, the impact of infestations on other PLH host species has been studied extensively. The photosynthetic activity of alfalfa (Medicago sativa L.) and potato (Solanum tuberosum L.) leaves showed reduced rates of C02 assimilation, transpiration and stomatal conductance and disrupted translocation of sugars in response to PLH exposure (Hibbs et al. 1964, Ladd Jr. and Rawlins 1964, Womack 1984, Walgenbach and Wyman 1985, Flinn and Hower 1990, Nielsen et a1. 1990, Nielsen et al. 1999, Lamp er al. 2001). Infestations of individual alfalfa plants resulted in height reduction (Poos and Johnson 1936, Flinn and Hower 1984, Hower and Flinn 1986), leaf and stem wilting (Harman et al. 1995), reduced leaf number and decreases in both fresh and dry masses (Poos and Jolmson 1936, Flinn and Hower 1984, Hower and Flinn 1986, Flinn et al. 1990, Hutchins and Pedigo 1990). The studies described above show that PLH infestations can disrupt leaf function 31 of both alfalfa and potato, thereby reducing the strength of the main source of carbohydrate synthesis. Such source strength reductions can result in stunted growth of the infested plants. It seems likely that other host plants, including grapevines, will show similar responses to reductions in source strength due to PLH infestations. The goals of this research were: 1) to determine whether PLH infestations on grapevines will reduce the quantity of vegetative growth; 2) to determine whether there is a relationship between the symptoms of infestation and the quantity of growth; 3) to define damage thresholds for any changes in vegetative growth that might occur; and 4) determine whether vines can recover from PLH induced growth reductions. 32 Materials and Methods Two experiments were conducted to test the effects of PLH infestations on the growth and biomass accumulation of grapevines. This section will describe the materials and methods common to both experiments; this section will be followed by the unique details of each experiment. Plant material: The vines used for both studies were Pinot gris (Vitis vinifera L.) grafted to rootstock 1103 Paulsen (V. berlandr’eri Planch. x V. rupestris Scheele); the vines for Experiment I were three years old, those from Experiment 11 were two years old. For both experiments, the vines were grown in black plastic pots with ten liters of steam sterilized soil composed of 50% sandy loam, 30% Sphagnum peat and 20% washed sand. The vines were grown under natural light and watered as needed, usually three times per week. All flower clusters were removed; lateral shoots and non-count shoots were removed as they appeared. The vines were fertilized monthly with 600 ml of a solution made by mixing five tablespoons (about 60 g) of 15-30-15 fertilizer with 19 liters of water. This solution contained approximately 475 mg/l of both nitrogen and potassium, 950 mg/l of phosphorus and trace amounts of boron, copper, iron, manganese, molybdenum and zinc. Insect Source: Second and third instar PLH nymphs were used to infest the vines for this study. Adult PLH were collected fiom a field mixed with alfalfa (Medicago sativa L.) and clover (T nfolium pratense L.) using a canvas sweep net. These insects were placed into collapsible cages that measured 61 cm in length, width and height (3in Products, Inc., Gardena CA, model 1450D) and reared indoors on bean plants 33 (Viciafaba L.). The second and third instar nymphal offspring of these adults were used for the experiments. To collect nymphs for applying treatments, a piece of white valais sheers fabric was placed inside the body of an aspirator, then enough nymphs for one vine were aspirated and rendered temporarily immobile using C02 gas. While still immobile, the aspirator was dismantled and the fabric containing the nymphs was removed and placed on the soil at the base of the trunk. The vine was then enclosed in a bag made from this same fabric until insect removal occurred. This process was repeated until all the vines received the appropriate number of PLH nymphs. The fabric bags reduced the amount of photosynthetically active radiation by about 1/3 and reduced air flow only slightly (data not shown). Measurements: Every leaf was assessed visually for cupping. For Experiment 1, leaves were scored for cupping as a 1, 2 or 3. Looking down on the leaf, a score of 1 was given if the leaf was the normal flat to slightly concave shape. A score of 2 was given to a leaf that was slightly to moderately convex with margins curling inward towards the abaxial surface, but not overlapping the leaf. A score of 3 was given to a leaf that was severely convex with margins curling inward towards the abaxial surface and overlapping the central portions of the leaf. The same protocol was used for Experiment 11 except that leaves were scored as either a 1 (normal), 2 (slight cupping), 3 (moderate cupping) or 4 (severe cupping). For both experiments, the percentage of leaves cupped was quantified as the number of leaves showing any degree of cupping as a proportion of the total number of leaves. Discoloration was assessed visually and expressed as the percentage of tissue per leaf that was yellow to light green and given a score ranging fiom zero to 100% in 34 increments of ten percent. The percentage of leaves discolored was recorded as the number of leaves showing any amount of discoloration as a proportion of the total number of leaves. The variable ‘% non-zero leaf was the average percent discoloration per leaf of only those leaves that were discolored. Internode lengths and midrib lengths were measured with a ruler to the nearest millimeter. The length of an internode was the linear distance between two nodes and the length of a midrib was the linear distance between the basal and apical ends of the primary vein that bisects the leaf blade. Shoot length was the sum of the lengths of all intemodes for a given shoot. At the end of the experiment, biomass accumulation was measured as the fresh and dry mass of the leaves, shoots, wood and roots of each vine. The leaves included both the lamina and petiole, shoot mass included all of the current season’s shoot growth except the leaves, roots included all growth fiom the main trunk axis below the graft union excluding the main axis, and all remaining tissue was categorized as the wood. Total vine biomass was the sum of these four tissue types. Dry mass was attained fi'om plant material that was dried in an oven at 49°C until no further reduction in mass occurred. 35 Emma! Plant Material and Vine Training: On 23 June 2004, dormant vines were moved fi'om a coldroom at a temperature of 4°C into a greenhouse. Each vine was trained to three shoots and no pesticides were used. Three 1.5 m long bamboo stakes were inserted into the soil near the outer rim of the pots as trellises for the shoots. Prior to PLH infestation, the vines appeared healthy and normal showing no symptoms of nutritional imbalances or pest damage with the exception of very mild amounts of defoliation fi'om Japanese beetles (Popilliajaponica Newman) totaling well under 1% leaf area removed (data not shown). Measurements and Treatment Application: All data were collected in 2004. The first set was collected prior to treatment application on July 14til and 15til and included midrib lengths, internode lengths, leaf cupping and leaf discoloration. The treatment factor was the number of PLH nymphs per vine and there were six levels: 0, 25, 50, 75, 100 and 150 nymphs per vine. The PLH treatments were added to the vines on July 19‘” and removed from the vines and counted on July 26‘” for a total stress period of seven days. The second data set was collected on July 27"1 and 28''1 and included the same measurements as the first set. Destructive harvest for fresh masses occurred on August 26ml and dry masses were obtained on October 15‘“. Leaf Area Analysis: Leafarea was estimated by midrib length. During a previous study, midrib length and leaf area were measured on 69 leaves from the potted Pinot gris vines. For each leaf, midrib length was measured with a ruler and leaf area was measured with a leaf area meter (Li3000, LiCor Inc, Lincoln, NE). A linear 36 regression between these variables was significant (p<0.0001, R2=0.80), the relationship was y = 0.87x — 12.86, where y is leaf area and x is midrib length, and this equation was used to estimate leaf area for this study. Experimental Design and Statistical Analysis: The vines were arranged as a randomized complete block design with six treatments and seven blocks using initial leaf number per vine as the blocking factor. Midrib length, internode length, leaf cupping and leaf discoloration were analyzed by AN COVA using the pretreatment measurements for each respective variable as covariates. Biomass accumulation was measured at the end of the experiment and analyzed using ANOVA All statistical analyses were performed using SAS version 8 (SAS Institute, Cary NC). All figures were created using SigmaPlot 8.0 including best-fit equations and R2 values (Systat Software, Richmond CA). Herbicide Damage: Shortly after treatment removal, some vines were showing symptoms of herbicide damage including unusually short and thin intemodes and leaves that were very small and fan-shaped. All vines showing these symptoms at the time of the second set of measurements (July 27"I and 28‘”) were eliminated from the analysis of midribs, intemodes, leaf cupping and leaf discoloration. By August 12'“, all but four vines were showing these symptoms and the experiment was ended prematurely as a result of the prevalence of these symptoms. The symptoms of damage resembled those caused by 2,4—Dichlorophenoxyacetic acid, and we suspect that this herbicide caused the damage to the vines. Fresh and dry weight data were included in the results section, but these data should be viewed with caution due to this problem. 37 Exm‘ment 11 Plant Material and Vine Training: The vines were purchased fiom a nursery and each was weighed and labeled prior to potting. All data were collected in 2005. The vines were potted on June 29‘” (day-1) and prior to infestation, were normal in appearance with no symptoms of pest damage or nutritional imbalances. A 1.5 meter length of bamboo was then inserted into the soil at the edge of each pot for shoot training. Each vine was trained to one shoot. Measurements and Treatment Application: This experiment lasted for 114 days. At three times throughout the course of this experiment, the lengths of all intemodes and leaf midribs were measured and every leaf was scored for degree of cupping and amount of discoloration. On day 1 (June 29‘”), the vines were potted. The data for Time-1 were collected on days 27-29; included in this data set was the leaf area regression (see section for Leaf Area Analysis). On day 30, the leaf area of the experimental vines was measured and the vines were arranged into blocks based on leaf area per vine. On day 31, the treatments were randomly applied to each vine within a block, thus initiating the infestation period. There were six treatment levels: 0.0, 0.5, 1.0, 1.5, 3.0 and 4.5 PLH nymphs per leaf. The infestation period lasted for seven days, ending on day 38 when the PLH were aspirated off of each vine and counted. The data for Time-2 were collected on days 41-43, three days after the infestation was terminated. The data for Time-3 were collected on days 111-113, 73 days after the infestation was terminated. On day 114, the vines were destructively analyzed for biomass . accumulation. 38 LeafArea Analysis: Leaf area was estimated by midrib length. During Time-l, midrib length and leaf area were measured on 469 leaves from the potted Pinot gris vines. For each leaf, midrib length was measured with a ruler and leaf area was measured with a leaf area meter (Li3000, LiCor Inc, Lincoln, NE). A nonlinear regression between these variables was significant (p<0.0001, R2=0.95), the relationship was y = 0.0125x“””, where y is leaf area and x is midrib length. This equation was used to estimate leaf area for this study. Experimental Design and Statistical Analysis: This experiment was a randomized complete block design with repeated measures. The blocking factor was leaf area and the treatment factor was the number of PLH nymphs per leaf. Multiple comparison tests at the 5% level were used to examine pairwise differences among all means for all of the analyses using LSD. All midrib and internode length data were analyzed using a linear mixed model with treatment level and time as fixed effects and block as a random effect. The Shapiro- Wilk test was used to determine whether the residuals were normal; in cases where the residuals were not normal, the data were transformed. Log transformations were performed on shoot length and leaf area in order to normalize the residuals. Leafcupping and discoloration were analyzed using a linear mixed model with treatment level as a fixed effect and block as a random effect. Biomass data were analyzed by ANCOVA with initial pre-planting vine mass used as the covariate, treatment level was used as a fixed effect and block as a random effect. A log transformation was performed on the root flesh mass in order to normalize the residuals. With the exception of the figures, all statistical analysis was performed using SAS version 8 (SAS Institute, Cary NC). All 39 figures were created using SigmaPlot 8.0 including best-fit equations and R2 values (Systat Software, Richmond CA). Pest Problems and Spray Applications: A minor outbreak of powdery mildew (Uncinula necator Schw.) occurred and the vines were sprayed on days 56 and 62 with Compass and Terraguard respectively. Later in the season, the vines were showing mild symptoms of two-spotted spider mite (T etranyclms urticae Koch) damage and were sprayed with Floramite SC on day 90. 40 Results Expm'ment I Table 1 shows the treatment levels used in this study and their equivalents in terms of PLH per leaf, and the stress severity in terms of PLH-hours (PLH-hrs). PLH-hrs are the product of the number of PLH in the infestation and the number of hours of the infestation. PLH-hrs are expressed on a per vine and per leaf basis. In order to calculate leaf size and leaf area/vine, a regression was performed between midrib length and leaf area (Figure 1). There was a significant linear relationship between midrib length and leaf area at the 0.1% level with an adjusted R2 value of 0.80 (Figure 1). Leaf Symptoms: The typical PLH feeding symptoms of downward leaf cupping and yellow leaf discoloration both increased with infestation severity. Leaf cupping data are shown in Table 2. The average number of leaves that were cupped after the infestation period ranged from 5.1 for the Control to 27.4 for the highest infestation level, or 9.3% to 56.8% of the total leaves per vine that were cupped respectively. The average score of the cupped leaves ranged fi'om 2.01 (slight cupping) for the Control to 2.42 (moderate cupping) for the 150 PLH/vine level. Linear regressions between the number of PLH per vine and leaf cupping were all positive and significant at the 0.1% level. Table 3 shows leaf discoloration data. The number of leaves discolored after the infestation period ranged fiom 3.8 for the Control to 19.6 for the 150 PLH/vine level; or 6.9% to 40.5% of the total leaves per vine that were discolored respectively. The average percent discoloration of the discolored leaves ranged fiom 12.0% for the Control to 29.2% for the 150 PLH/vine level. Linear regressions between the number of PLH per 41 vine and leaf discoloration were all significant at the 0.1% level. A significant non-linear relationship existed between the number of PLH per vine and the percentage of leaves that were cupped at the 0.1% level with an adjusted R2 value of 0.68 (Figure 2). There was also a significant non-linear relationship between the number of PLH per vine and the percentage of leaves that were discolored at the 0.1% level with an adjusted R2 value of 0.54 (Figure 3). Shoot and Leaf Growth: Table 4 and Table 5 show how leaf and shoot growth were affected by PLH infestations. Infestation severity was inversely related to leaf number/vine and leaf area/vine (Table 4). Likewise, shoot length and intemodes/vine were both inversely proportional to infestation severity (Table 5). All four of these variables showed significant linear and quadratic regressions at the 0.1% level when plotted against treatment levels. Intemode length and leaf size were not significantly different among treatments and showed no clear trends in relation to infestation severity. Biomass Accumulation: Vine flesh and dry mass data showed few clear trends (Table 6). Based upon total flesh and total dry masses, vines that received 0, 25 or 50 PLH per vine tended to be larger than vines receiving 75, 100 or 150 PLH per vine, although statistically there were no significant differences between any level and the Control. A linear regression between leaf fi'esh mass and PLH per vine was significant at the 5% level with an R2 of 0.67, indicating that infestation severity resulted in a reduction in leaf fi'esh mass. Damage Thresholds: Table 7 shows damage thresholds for Experiment I. Leaves/vine, shoot length and intemodes/vine did not differ fi'om the Control until an infestation level of 25 PLH/vine. Leafcupping, leaf discoloration and leaf area per vine 42 did not differ from the Control until infestation severities reached 50 PLH/vine. Although biomass data showed no clear trends, leaf fresh mass was reduced at infestation severities of 75 and 150 PLH/vine, but not at 100 PLH/vine. Thus, the damage threshold for leaf fresh mass might have occurred between 75-150 PLH/vine. 43 70. 60 - 0 so - "a 3, 4o - C 5 3 3O .1 20 J —— y = 0.87x - 12.9 0 adj. R2=0.80 10- p