Effects of Powdery Mildew of Grape on Carbon Assimilation Mechanisms of Potted ‘Chardonnay’ Grapevines

Potted grapevines (Vitis vinifera L. ‘Chardonnay’) were inoculated with conidial suspensions of the grapevine pathogen causing powdery mildew of grape (GPM) (Uncinula necator (Schw.) Burr.). Leaves of inoculated and noninoculated vines were studied for the effects of varying light (PAR) and CO2 concentrations on factors affecting carbon assimilation. GPM reduced carboxylation effi ciency (k), net CO2 assimilation rate (A), stomatal conductance (gs), and internal CO2 concentration (Ci) under ambient CO2, Amax at >900 ppm CO2, stomatal limitations to A (lg), and photochemical effi ciency (φ) on diseased leaves, while having no effect on the CO2 compensation point (Γ) or the light compensation point (cp). GPM had no signifi cant effect on chlorophyll fl uorescence (Fv/Fm). Vasconcelos et al. (1994) found no increase in photosynthetic rate in the remaining leaves. Punching holes in the leaves of other crop species have been used to simulate the effects of damage by phytophagous arthropods (Boucher et al., 1987; Flore and Irwin, 1983; Poston et al., 1976). Stacey (1983) found that leaf removal on tomato plants largely approximated pest damage. Defoliation experiments have been inconsistent in approximating damage caused by foliar pathogens, as visual estimates of infection do not always adequately indicate the effects of a pathogen on photosynthetic and transpirational activities (Shtienberg, 1992). Measurements of chlorophyll fl uorescence have also been used to determine the health of photosynthetic mechanisms in plants (Buwalda and Noga, 1994; Krause and Weis, 1991) and have been correlated with end-product inhibition of leaf A due to damage to photosystem II (PSII) (Layne and Flore, 1993). Damaged leaves may exhibit less potential maximal photochemical effi ciency than uninfected leaves, depending on the nature of pathogen-induced foliar damage. These experiments were designed to determine the physiological effects of GPM infection on individual grape leaves regarding gas exchange and chlorophyll fl uorescence. Materials and Methods Plant material. Two-year-old dormant grapevines (V. vinifera L. ‘Chardonnay’, Dijon clone 96, grafted to Courdec 3309 rootstock) were planted in 19-L pots in a medium of 50% loam, 40% sand, and 10% peat. The plants were grown and maintained on a gravel pad outdoors at the Horticultural Teaching and Research Center, Michigan State University, East Lansing, during the 2002 growing season. Plants were thinned shortly after full bud burst to three shoots per vine. Vines were watered regularly to container capacity and fertilized monthly with a soluble fertilizer at a rate of 0.38 g N, 0.17 g P, and 0.32 g K (1.9 g Peter’s 20–20–20) per pot. Plants were largely fruitless; a few plants that did have fruit were retained to determine phenological stages during the growing season. Flower clusters were removed from all treatment plants prior to bloom. Laterals were removed as they appeared throughout the growing season. Two applications of Sevin [1-naphthyl N-methylcarbamate (carbaryl), Aventis, Bridgewater, N.J.] liquid were made as needed to control Japanese beetle (Popillia japonica Newman) infestations. All chemical applications were made at least 7 d before gas exchange measurements. Experimental design. Plants were blocked according to the fresh weight of the dormant, unpotted vines and arranged in a randomized complete block design with 32 blocks. Treatments were assigned randomly within blocks and wereas follows. 1) Plants inoculated with a conidial suspension of U. necator in distilled water (produced by soaking infected leaves of Marechal Foch (Kuhlmann 188-2) grapevines for ≈10 min and agitating to dislodge conidia) between the 5 mm berry (as determined from the nontreatPlant responses to foliar biotic and abiotic stresses may vary with the nature (causal element) and magnitude of the stress. Net CO 2 assimilation (A) by foliage is a critical factor infl uencing plant productivity, since ≥90% of plant dry matter is derived from C fi xed through photosynthesis (P n ) (Flore and Lakso, 1989). Therefore, factors that inhibit assimilation through P n may be detrimental to productivity. Photosynthesis in plants can be limited by biotic stresses in a variety of ways. Johnson (1987) divided the seven categories of foliar pest effects on plants as described by Boote et al. (1983) into two groups: a) those whose major effects are on solar radiation interception (tissue consumers, leaf senescence accelerators, stand reducers, and light stealers) and b) those whose major effects are on relative use effi ciency (photosynthetic rate reducers, assimilate sappers, and turgor reducers). Damage to the photosynthetic apparatus may occur by more than one of these effects; reductions in A caused by the effects of most foliar pathogens on photosynthetic activity result from a decrease in the photosynthesizing leaf area and/or its reduced effi ciency (Goodman et al., 1986; Shtienberg, 1992; Yarwood, 1967). Response patterns affecting reductions in P n and transpiration (E) have been related to the general type of trophic relationships involved (Shtienberg, 1992); powdery mildews tended to have more similar response patterns as compared to other foliar pathogens, for example. Powdery mildew of barley [Blumeria (syn. Erysiphe) graminis D.C. ex Merat f.sp. hordei Marchal] was associated with decreases in chlorophyll after 4 d of infection and loss of electron transport activity, with no loss of electron carrier concentration in remaining chlorophyll (Holloway et al., 1992). Powdery mildew of sugar beet (Erysiphe polygoni DC) infection was associated with inhibition of electron transport in noncyclic proteins, accompanied by alterations in chloroplast ultrastructure and reduction of enzyme activity (Magyrarosy et el., 1976). Carboxylation resistance increased in winter wheat infected by Blumeria (syn. Erysiphe) graminis D.C. ex Merat f.sp. tritici, with consequent negative effects on stomatal resistance, boundary layer resistance, and transport resistance (Rabbinge et al., 1985). Carbon assimilation was negatively affected by powdery mildews in all three studies. There does not appear to be a relationship between decreases in A and E among pathosystems; rather, E has been shown to increase, decrease, or stay the same in response to foliar pathogens, including those causing powdery mildew symptoms (Shtienberg, 1992). Grape leaves infected with Uncinula necator (Schw.) Burr. have demonstrated reduced photosynthetic rates compared to uninfected leaves (Lakso et al., 1982), due to destruction of palisade cells by the pathogen. Transpiration was not affected; consequently, water-use effi ciency was less in infected leaves. Field experiments have demonstrated negative effects of GPM on grapevine health during the season of infection, including decreased fruit quality (Gadoury et al., 2001; Ough and Berg, 1979; Pool et al., 1984) and fruit set (Chellemi and Marois, 1992). Multiseasonal effects include reduced vine size (as determined by cane pruning weights) and yield in susceptible varieties (Pool et al., 1984), or only with vine size in relatively resistant varieties (Gadoury et al., 2001). Defoliation experiments have been conducted on grapevines for a variety of reasons, including manipulation of fruit set, modifying the fruit microclimate, and to simulate pest damage. Grapevine responses to defoliation by removing whole leaves frequently include increased A by the remaining leaves (Candolfi Vasconcelos and Koblet, 1990, 1991; Hofäcker, 1978; Intrieri et al., 1997), although Candolfi HORTSCIENCE 39(7):1670–1673. 2004. Received for publication 30 June 2003. Accepted for publication 24 Nov. 2003. I thank James A. Flore and Adriana Nikoloudi for their technical assistance. 7761-Path.indd 1670 10/14/04 11:09:46 AM 1671 HORTSCIENCE 39(7) DECEMBER 2004 ment fruited vines) and 1200 growing degree days (GDD) (base 10 °C) stages using a hand sprayer and sprayed to runoff (James Olmstead, personal communication). Concentrations of conidia in suspension were not determined. This treatment was designated “Infected.” 2) Plants were sprayed with myclobutanil [αbutyl-α-(4-chlorophenyl)-1H-1,2,4, triazole1-propanenitrile (NOVA), Rohm and Haas, Philadelphia, Pa.] at bloom (16 June) and between the 5-mm berry stage and midseason (23 July, ≈1200 GDD) at a rate of 0.21 g myclobutanil/L H 2 O and sprayed to runoff. This treatment was designated “Noninfected”. Plants sprayed with myclobutanil were separated from inoculated plants by ≈10 m for 48 h to help eliminate the potential effects of drift and/or volatiles from affecting inoculated plants. Noninfected plants had levels of GPM ≤5% through the veraison period. Ten plants from each treatment were selected for gas exchange responses to varying CO 2 concentrations and photosynthetically active radiation (PAR) level measurements by the following criteria: The most recent fully expanded leaves on the longest shoot on each plant were examined just before veraison; leaf health was evaluated based on visual ratings of disease severity, expressed as a percentage of the leaf surface with visible GPM infection. The most recent fully expanded leaves from each of the 10 blocks which had both the healthiest noninfected leaves and an obviously infected, but otherwise undamaged (by insects, wind laceration, etc.) leaf, were selected for gas exchange measurements. Disease severity on infected leaves ranged from 60% to 90% infected leaf area. Most (>80%) leaves on infected vines had visible symptoms of GPM; overall disease severity was not assessed on a vine-by-vine basis, but was estimated to be 50% at the time of measurement. There were no visible disease symptoms on noninfected leaves. Plants selected for measurement were watered to pot runoff the previous day to ensure lack of water stress during measurements. Gas exchange measurements. Gas exchange measurements were conducted using a portable infrared gas analyzer (IRGA) (CIRAS-2, PP Systems, Amesbury, Mass.) fi tted with a leaf cuvette with light source (PLC6; PP Systems). Effects of CO 2 concentration were determined by gradually increasing CO 2 from 0 to 200 ppm at 50-ppm increments, and from 200 to 1000 ppm at 100-ppm increments at PAR = 1500, allowing the IRGA to equilibrate between each measurement using the onboard computer (PenCentra 130; Fujitsu PC Corp., Santa Clara, Calif.) and software (version 1.0, PP Systems, Amesbury, Mass.). Responses to changes in PAR were taken immediately afterward, using the same equipment and software, by reducing PAR from 2000 to 200 in 200 PAR increments, and from 200 to 0 in 50 PAR increments. Measurements were taken between 0900 and 1500 HR at 26 °C (±2 °C) air temperature. Plants were measured within each block according to their random placement to help alleviate the effects of natural diurnal variances in A (Downton et al., 1987). The data were analyzed by applying the Marquardt-Levenberg algorithm for nonlinear regression analysis for curve fi tting ( Layne and Flore, 1992, 1995; Marquardt, 1963). Parameters calculated from plant responses of A to variable PAR (light response curves) were: the light compensation point (cp), extrapolated from the data where A = 0, and quantum yield (φ), as determined by the slopes of the linear portion of the curves. Parameters calculated from plant responses of A to variable internal CO 2 concentration (C i ) were the CO 2 compensation point (Γ), extrapolated from the data where A = 0; carboxylation effi ciency (k), as determined by the slopes of the linear portion of the curves; stomatal limitation to A (l g ,), calculated according to the differential method of Jones (1985); and A max , the maximum A value at saturating CO 2 . A, g s , and C i at ambient CO 2 concentrations and saturating light conditions were also measured (A 360 , g s360 , and C i360 , respectively). Single leaf measurements were also performed on the most recent fully expanded leaf of the longest shoot on all plants in the plot over a period of 2 d to determine relationships, if any, between A and g s and C i , at PAR = 1000 and CO 2 = 375 ppm. Chlorophyll fl uorescence measurements. Three blocks were randomly selected for chlorophyll fl uorescence measurements. The longest shoot on each plant, also used for gas exchange measurements, was selected and each leaf evaluated for disease severity, expressed as the percentage of leaf area with visible GPM symptoms. A clip with a sliding window to admit or exclude light was attached to each leaf, and the leaf section was allowed to dark acclimate for ≥30 min. Chlorophyll fl uorescence was measured with a plant effi ciency analyzer (model PEA; Hansatech Instruments, Norfolk, U.K.). Fluorescence was expressed as the ratio of variable fl uorescence (F v ) to the maximum fl uorescence (F m ) (F v /F m ). Destructive harvest measurements. Plants from all 32 blocks were destructively harvested after leaf fall and cut into component plant parts (roots, trunk, shoots, and leaves), and fresh weights were measured. These plant parts were dried in a forced-air drying oven at 45 Fig. 1. CO 2 (A) and light (B) response curves of single leaves of potted Chardonnay grapevines infected or not infected with grape powdery mildew. PLANT PATHOLOGY 7761-Path.indd 1671 10/14/04 11:09:49 AM HORTSCIENCE 39(7) DECEMBER 2004 1672 °C for ≥2 weeks, and dry weights (biomass) were determined. Statistical analysis. Statistical analysis was performed using SAS statistical software (version 8.2; SAS Institute Inc., Cary, N.C.). Curve fi tting for nonlinear regression was performed using SigmaPlot software (version 8.01; SPSS Ltd., Chicago, Ill.).