Ultraviolet-B Radiation Damage on Kentucky Bluegrass II: Hormone Supplement Effects

High ultraviolet-B (UV-B; 290-320 nm wavelength) radiation may signifi cantly contribute to the quality decline and death of kentucky bluegrass (Poa pratensis L.) sod during summer transplanting. Antioxidants and protective pigments may be involved in plant defense against oxidative stress caused by UV-B. Selected exogenous hormones may alleviate UV-B damage by upregulating plant defense systems. The objectives of this study were to determine if exogenous hormone or hormone-like substances could alleviate UV-B damage to ‘Georgetown’ kentucky bluegrass (Poa pratensis L.) under greenhouse conditions. The hormone salicylic acid at 150 mg·m–2 and the hormone-containing substances, humic acid (HA) at 150 mg·m–2 and seaweed extract (SWE) at 50 mg·m–2, were applied to plugs of kentucky bluegrass and then subjected to UV-B radiation (70 μmol·m–2·s–1). The UV-B irradiation stress reduced turf quality by 51% to 66% and photochemical effi ciency by 63% to 68% when measured 10 or 12 days after initiation of UV-B. Endogenous alpha-tocopherol (AT) and antioxidant enzymes (superoxide dismutase (SOD) and catalase) were reduced by UV-B stress. Anthocyanin content was increased from day 1 to 5 and then decreased from day 5 to 10 of continuous UV-B irradiation. Application of SA and HA + SWE enhanced photochemical effi ciency by 86% and 82%, respectively, when measured 10 or 12 days after UV-B initiation. In addition, application of the hormonal supplements increased AT concentration, SOD, catalase activity, and anthocyanin content when compared to the control at 10 days after UV-B initiation. Bluegrass with greater AT concentration and SOD and catalase activity exhibited better visual quality under UV-B stress. The results of this study suggest that foliar application of SA and HA + SWE may alleviate decline of photochemical effi ciency and turf quality associated with increased UV-B light levels during summer. The vagaries of the turfgrass and landscaping industry often dictate that kentucky bluegrass sod be harvested, transported, and transplanted during the summer. Frequently, increased respiratory heating during storage, transport, and subsequent exposure to high UV-B during transplanting can cause shock (Giese et al., 1997). The shock often results in bleached, inactive turfgrass leaves. Plants better adapted to resist UV-B induced photo-bleaching usually contain more robust screening (pigment) and scavenging (antioxidant) protection systems (Mackerness, 2000). Resistance to UV-B damage most likely involves both avoidance and tolerance mechanisms. Day et al. (1992) noted conifers are effi cient avoiders of UV-B damage due to higher needle levels of fl avonoid and related phenolic pigments (anthocyanins) that strongly absorb UV and transmit nondamaging longer wavelengths. Most herbaceous species do not peroxidase activity and phenolic content in Pisum sativum. Exogenous application of SA reduced visual injury and loss of photochemical effi ciency of kentucky bluegrass and creeping bentgrass under UV-B stress (Schmidt and Zhang, 2001). It has been documented that application of alkaline extracts of the seaweed Ascophyllum nodosum Jol. (SWE), in combination with acid extracts of the humic acid (HA) component of leonardite enhanced antioxidant activity (Zhang and Schmidt, 1999), photochemical effi ciency (Zhang et al., 2003), and improved resistance to UV-B induced damage in kentucky bluegrass (Schmidt and Zhang, 2001). This SWE source contains ≈70 mg·kg of the cytokinin, zeatin riboside (Zhang and Ervin, 2004). Higher cytokinin levels in tissues undergoing abiotic stress have been associated with greater antioxidant enzyme activities resulting in less photosynthetic damage (Liu and Huang, 2002). Numerous HA sources have been shown to contain indole acetic acid (IAA) and exhibit auxin-like activity (Canellas et al., 2002; Muscolo et al., 1998; O’Donnell, 1973). Increased endogenous amounts of cytokinins and auxins have been associated with membrane stabilization leading to protection of photosynthetic activity during osmotic and temperature stress (Nooden and Leopold, 1988; Aldesuquy, 2000). Given this evidence, our hypothesis was that application of SA or HA + SWE, before UVB exposure, would up-regulate plant defense mechanisms resulting in greater tolerance of kentucky bluegrass to UV-B. Our objectives were to determine if these substances would improve endogenous antioxidant concentrations and antioxidant activities in kentucky bluegrass and be associated with greater UV-B tolerance as determined by photochemical effi ciency, visual quality, and pigment content responses. Materials and Methods The detailed experimental procedures, including fi eld growth conditions of harvested plant material, subsequent greenhouse growing conditions, UV-B treatments, data collection, measurement, and statistical protocols were described in a companion paper (Ervin et al., 2004). Experiment 1. Treatments for this study were 1) SA at 150 mg·m, 2) HA at 150 mg·m + SWE at 50 mg·m, and 3) control. Three replications were arranged in randomized complete blocks on the greenhouse bench. Salicylic acid (Research Organics; Cleveland, Ohio), seaweed extract (Acadian Seaplants Limited, Dartmouth, Nova Scotia, Canada) and HA (Plant Wise Biostimulants, Inc. Louisville, Ky.) are each available as dry powders. Solutions were made by mixing the products with water. Salicylic acid solution required addition of a surfactant (Aqua-Gro, Aquatrols Corporation, Cherry Hill, N.J.) at 0.05% to encourage uniform coverage. Light absorbance of the two treatment solutions was measured across the UV spectral range (250 to 400 nm) and results are presented as percent absorbance at each wavelength (Fig. 1). The SA-treatment solution had 20% appear to have such robust screening systems. However, it has been established that exposure of herbaceous plants to UV radiation will up-regulate the formation of fl avonoids (Salisbury and Ross, 1991). Additionally, application of a green-pigmented colorant that strongly absorbs in the UV range (250 to 400 nm) has been shown to improve avoidance of UV-B damage in kentucky bluegrass (Ervin et al., 2004) and creeping bentgrass (Agrostis stolonifera L.; Schmidt and Zhang, 2001). Further, by exogenously applying ascorbic acid and alpha-tocopherol, endogenous alphatocopherol and antioxidant enzyme activities increased and directly correlated with greater tolerance of UV-B by kentucky bluegrass (Ervin et al., 2004). Greater tolerance to oxidative stress caused by high UV-B may involve hormonal signals to up-regulate antioxidant enzyme activity. One of the implicated hormones is the phenolic compound, salicylic acid (SA). Numerous studies have shown that SA is a key signaling compound involved in the activation of certain plant defense mechanisms including induction of pathogenesis-related genes and antioxidant enzymes (Mackerness, 2000; Jordan, 1996). Recently, Clarke et al. (2002) noted that SA reduced heat-induced oxidative damage and increased catalase, glutathione reductase and peroxidase activity in Phaseolus vulgaris. Sanjay et al. (2001) indicated SA increased HORTSCIENCE 39(6):1471–1474. 2004. Received for publicatio 14 Apr. 2003. Accepted for publication 31 Dec. 2003. This Research was conducted as part of USDA-CSREES project no. VA-135660. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by Virginia Tech or the U.S. Dept. of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable. Assistant professor. Research scientist. 7834-Turf.indd 1471 9/20/04 12:31:43 PM HORTSCIENCE VOL. 39(6) OCTOBER 2004 1472 to 30% absorbance across the UV-B spectra, while maximum absorbance of 20% occurred at 290 to 300 nm for the HA + SWE solution. The treatments were sprayed onto kentucky bluegrass foliage using 60-mL syringes with needles on 15 Mar. 2001. The treated turfgrass was not irrigated for 24 h. Twenty-four hours after treatment the plugs were placed under artifi cial UV-B radiation (70 μmol·m·s) provided by three 40-W UV-B fl uorescent lamps (UVB-313, Cleveland, Ohio). The plugs were spaced evenly (2.5 cm), kept 0.5 m below the UV-B source, and grown under continuous UV-B in a greenhouse maintained at 22 ± 2 C. Daylength averaged 11 h. Plugs were subjected to UV-B from 16 to 26 Mar. and irrigated three times a week to prevent moisture stress. On 27 Mar., the plugs were removed from UV-B treatment and placed under a mist system for recovery. Photochemical effi ciency and turf quality (based on a visual scale of 1 to 9, with 9 indicating the best quality) scores (as an indication of the degree of visual injury or lack thereof) were determined as in previous trials. Experiment 2. Treatments remained the same as in Expt. 1. The plugs were treated on 31 May and then placed under continuous UVB irradiation from 1 through 12 June 2001. On 13 June, UV-B treatment ceased and the plugs were set under a mist system for recovery. Photochemical effi ciency and turf quality scores were determined as in previous trials. Experiment 3. Treatments remained the same as those in Expts. 1 and 2. However, there were four replications instead of three. The plugs were treated on 24 Jan. 2002 and placed under continuous UV-B from 25 Jan. through 4 Feb. 2002. On 5 Feb., UV-B irradiation stress was discontinued and the plugs were placed under a mist system for recovery. Photochemical effi ciency and turf quality scores were determined as in previous trials. Antioxidant concentration and activity (AT, SOD, catalase, and ascorbate peroxidase) were measured by leaf samples which were taken from each experimental unit at 1, 5, and 10 d after UV-B initiation, immediately frozen with liquid N 2 , and stored at –80 C until laboratory analyses could be performed as detailed in the companion paper (Ervin et al., 2004). Anthocyanin content was determined following the procedure of Rabino and Mancinelli (1986). Briefl y, anthocyanin was extracted from leaf samples (100 mg FW) by periodically shaking samples over a 48 h period at 4 C in acidic (1% HCL) methanol. The absorbance of the extract, clarifi ed by fi ltration, was measured at 530 and 657 nm using a spectrophotometer (BioMate 3, Thermo Spectronic, Rochester, N.Y.). The formula A – (0.25 × A) was used to calculate anthocyanin content. Statistical analyses. Measured variables were subjected to a two-factor ANOVA using the general linear models procedure in the Statistical Analysis System (SAS Institute, 1990). Factor one included three levels of supplement treatments and factor two included three levels of experimental time periods (i.e., Expts. 1, 2, and 3). Supplement × experiment interactions were nonsignifi cant for three of four sampling dates, therefore, quality and photochemical effi ciency data are presented as means analyzed over all three experimental periods. Mean differences were ascertained using Fisher’s protected l sd (P = 0.05).