Abstract
Creeping bentgrass (Agrostis stolonifera L.) is a turfgrass species that is widely used on golf courses throughout the United States. In field settings, plants are often subjected to more than one stress at a time, and studying stresses independently is likely insufficient. Stresses, such as heat stress and salt stress, can affect plant hormone levels and, in turn, plant hormone levels can affect how well the plant tolerates stress. The objectives of the experiments were to determine if the levels of heat stress and salt stress used would be detrimental to creeping bentgrass health, and if applying plant growth regulators could improve plant health during stress. During the first experiment, creeping bentgrass was transplanted to hydroponics systems in two different growth chambers. One chamber was set to have day and night temperatures of 35 °C and 30 °C (heat stress), respectively, and the other had day and night temperatures of 25 °C and 20 °C, respectively. Within each chamber, one block received a 50 mM NaCl treatment (salt stress) and the other did not (control). The stress treatments were applied for 14 days. Results of the first experiment indicated that the treatments were sufficient to negatively affect creeping bentgrass growth and health as indicated by fresh shoot and root weights, tillering, electrolyte leakage, and total chlorophyll content (TCC). There were significant interactions of temperature × salt level detected for shoot and root weights and electrolyte leakage. Plants that were exposed to both heat stress and salt stress were more negatively affected than plants exposed to either heat stress or salt stress alone for all metrics except for tillering. The presence of salt reduced tillering regardless of the temperature regimen. During the second experiment, plants were treated the same, but the plant growth regulator (PGR) treatments were also applied. The PGR treatments consisted of two different gibberellic acid (GA) synthesis inhibitor products, 2,4-dichlorophenoxyacetic acid, two different rates of aminoethoxyvinylglycine (AVG), an ethylene synthesis suppressor, and plants that were not treated with the PGR. In addition to the measurements of plant health and growth, dry shoot and root weights were measured. For the TCC, there was a two-way interaction between temperature × PGR treatment. For electrolyte leakage, there was a three-way interaction between temperature × salt level × PGR treatment. Combined heat stress and salt stress negatively affected all plants regardless of PGR treatment, but there were differences between PGR treatments. Plants treated with AVG exhibited improved health and growth compared with the other PGR treatments. These plants had the highest shoot and root masses. Plants treated with GA synthesis inhibitors had the lowest shoot and root masses as well as the lowest TCC when subjected to stress.
Creeping bentgrass is a commonly used turfgrass species on golf courses, primarily the greens, tees, and fairways. Creeping bentgrass, when mowed at low heights, forms a dense cover and can provide excellent putting surfaces and good, dense, tight cover on tees and fairways. This species is adapted to a temperate climate, but it is widely used for putting greens in areas outside of its zone of adaptation, such as the southeastern United States.
Like most plants, creeping bentgrass turf is sessile; therefore, it is subject to environmental and biotic stresses. When plants sense stress, a relatively complex response is induced and may result in the plant altering its metabolism and physiology, including, but not limited to, altered phytohormone levels. At times, the plant response may not be considered sufficient to withstand the stress exposure because the turf may become discolored, weakened, and less capable of withstanding traffic or experience altered growth.
High temperatures, or heat stress, has been described as an important limiting factor of turfgrass growth (DaCosta and Huang 2013). The first changes in plants occur at the cellular level and include altered protein and biochemical syntheses, altered metabolism, and changes in plasma membrane fluidity (DaCosta and Huang 2013). The overall effect of these cellular changes is that carbon is not taken in and assimilated quickly enough to offset increased carbon use, resulting in decreased biomass (DaCosta and Huang 2013). Decreases in biomass attributable to heat stress have been observed in many plant species, including creeping bentgrass (Hasanuzzaman et al. 2013; Xu et al. 2004). Heat stress also reduces antioxidant enzyme levels of creeping bentgrass (Liu and Huang 2000). Liu and Huang (2000) found reduced levels of these enzymes resulted in greater lipid peroxidation, as evidenced by increased electrolyte leakage and lower total chlorophyll content (TCC).
Many putting greens are constructed on sand-based root zones to provide ample drainage. Because of the drainage characteristics of these root zones, irrigation events tend to be frequent applications with low volumes, which can result in salt accumulation. Alternatively, irrigation may be heavy and infrequent (to prevent disease occurrence and promote deeper rooting), which can also result in salt accumulation as the soil dries (Bell 2011). Irrigation sources may contain salts, and a relatively large amount of water (∼10 cm per 30 cm of sand root zone) is required to flush salt from the root zone. Reclaimed water use as an irrigation source on golf courses can also contain high amounts of salts (Harivandi et al. 1992). Similar to heat stress, salt stress can reduce the TCC content and overall growth of turfgrass species (Dai et al. 2009; Uddin et al. 2011).
In most field settings, it is likely that plants experience more than one stress at a time, and it is unknown how or if plants prioritize their response to multiple stresses. Changes in weather patterns caused by climate change are predicted to result in higher temperatures and increased periods of floods and drought (Dai 2013; Rosenzweig et al. 2001). If these predictions are correct, then salt stress may also become more prevalent because it is considered to co-occur with heat stress (Chaves et al. 2009).
Mittler (2006) reviewed several published studies that examined combined stresses and concluded that the plant response is unique to stress combinations, and that studying stresses independently of each other was insufficient. Mittler (2006) also noted that salinity stress and heat stress could interact and cause more plant damage and/or death than either alone. Some research has investigated the effects of combined stress on plant health, but there is still a knowledge gap.
Research has shown that gibberellic acid (GA) biosynthesis inhibition leads to greater stress tolerance. GA synthesis inhibitors are widely used in turfgrass systems to reduce shoot growth (to reduce mowing). The use of trinexapac-ethyl, a late-stage GA synthesis inhibitor, can result in darker green shoots and greater density and overall superior turfgrass quality (reviewed by DaCosta and Huang 2013). Ervin and Zhang (2007a) found that trinexapac-ethyl also increased the cytokinin level, which is another plant hormone. There is evidence that other GA synthesis inhibitors, such as triazoles and prohexadione calcium, a plant growth regulator (PGR) with the same mode of action as trinexapac-ethyl, also result in increased cytokinin levels (Ervin and Zhang 2007b).
As reviewed by Waskiewicz et al. (2016), exogenous GA applications tend to have a positive effect on plants subjected to salinity stress by improving growth and nutrient uptake in several species. Archard et al. (2006) found that Arabidopsis DELLA mutants had better salt tolerance than the wild-type mutants. They proposed that the enhanced survival was beneficial but was associated with growth and development costs.
Ethylene levels may also increase during heat stress, but this appears to be dependent on the heat tolerance of the species or cultivar. Less tolerant species tend to have higher rates of ethylene production during stress (Balota et al. 2004; Xu and Huang 2007). Based on available research, it seems ethylene is important for the plant heat stress response (Tao et al. 2015). Larkindale and Knight (2002) found that treating Arabidopsis with aminocyclopropane-1 carboxylic acid, an ethylene precursor, increased survival and reduced membrane damage caused by oxidants after heat treatment. However, Hays et al. (2007) and Xu and Huang (2007) found that damage caused by heat stress was lessened when aminoethoxyvinylglycine (AVG), an ethylene synthesis suppressor, was applied. Xu and Huang (2007) also found reduced levels of ethylene production in a heat-tolerant Agrostis species. These results indicate that ethylene signaling is probably important for the initial plant stress response, although excess ethylene production can result in increased leaf senescence during heat stress. Xu and Huang (2009) investigated the use of AVG on well-watered creeping bentgrass subjected to heat stress. They found that AVG only reduced ethlyene production and enhanced the heat tolerance of creeping bentgrass.
Ethylene can be either positive or negative during salt stress according to a recent review of the literature by Tao et al. (2015). In a study that examined two cultivars of rice (Oryza sativa L.) differing in salt tolerance, the salt-resistant cultivar produced higher amounts of ethylene than the susceptible cultivar (Quinet et al. 2010). Ethylene signaling appears to be necessary for the plant salt stress response and acclimation, but the mechanism is not currently known (Waskiewicz et al. 2016). However, the key ethylene synthesis enzymes (acetyl-CoA synthetase and 1-aminocyclopropane-1-carboxylate synthase) are upregulated in response to salt stress and may result in a negative plant response.
Auxins are also involved in salt stress. Under salt stress conditions, auxin levels tend to decrease, but this may vary by species and organs and over time (Javid et al. 2011). Auxins may reduce the toxic effects of salt stress, and auxin distribution within the root is affected by salt stress (Egamberdieva 2009; He et al. 2005; Wang et al. 2009). A recent study of creeping bentgrass cultivars showed that salt stress led to increased levels of the auxin indole-3-acetic (IAA) in leaves but decreased IAA in the roots of the salt-susceptible cultivar (Krishnan and Merewitz 2015). Many other studies investigating the effects of auxin on salt stress have focused on germination (Waskiewicz et al. 2016). However, a study of maize (Zea mays L.) found that a salt-tolerant cultivar maintained IAA levels in roots as compared with the control (Zorb et al. 2013).
As mentioned, trinexapac-ethyl is widely used on golf course turf, and its effect on turfgrass growth during periods of stress is relatively well-documented. Recently, two new GA synthesis-inhibiting products were introduced to the turfgrass market. Anuew™ (Cleary Chemicals, LLC, Alsip, IL, USA) is advertised as a longer-lasting GA synthesis inhibitor than trinexapac-ethyl, which is known to have shortened efficacy during warmer weather. The active ingredient (a.i.) is prohexadione calcium, which has a similar mode of action as trinexapac-ethyl (blocks dioxygenase enzyme function late during the GA synthesis pathway) (Sponsel and Hedden 2010). The other product that was recently introduced is Muskateer® (SePro Corp., Carmel, IN, USA), which is a combination product and has three a.i.s, flurprimidol, paclbutrazol, and trinexapac-ethyl. Flurprimidol and paclobutrazol are early-stage GA biosynthesis inhibitors. These GA synthesis inhibitor products were selected for use in this research because of their relatively recent introduction to the market. Additionally, Anuew™ was selected specifically because it is advertised as having longer efficacy when temperatures are high.
Because there have been variable results regarding whether ethylene has a positive or negative role in salt stress, AVG is a compound of interest that could be used to help determine the role of ethylene in salt stress and in combined heat stress and drought stress. AVG was included in the experiment because it is used commercially for other crops such as apple and pineapple production, and it could have potential as a turfgrass health product. 2,4-dichlorophenoxyacetic acid (2,4-D) is a common a.i. in many turfgrass herbicide products. It is a synthetic auxin that is generally safe to use on desirable turfgrass to control broadleaf weeds. It has a structure similar to IAA; in plants, it is biologically active as an auxin, but it is still unknown why it does not kill grasses (Song 2014). However, at low concentrations, 2,4-D is used to promote cell elongation and division (Song 2014). Additionally, 2,4-D has been used to promote root growth in maize cultures (Green and Phillips 1975). Because auxin signaling and transport appear to have important roles in the plant stress response, and because turfgrasses are not as susceptible to damage caused by higher concentrations of 2,4-D, it is possible that the application of 2,4-D could have a positive role in the creeping bentgrass response to heat and drought stress. However, it should be noted that the use of 2,4-D on creeping bentgrass is usually not advised when temperatures reach or exceed 30 °C.
The first objective of this research was to establish the combined effect of heat stress and salt stress on creeping bentgrass. It was hypothesized that the combined effects of heat stress and salt stress would be more detrimental to creeping bentgrass health than either stress alone. The second objective of this research was to evaluate the effects of various PGRs on creeping bentgrass growth to determine if they could alleviate or induce further damage caused by heat stress and salt stress and by heat stress and salt stress combined. It was hypothesized that GA-synthesis inhibitors would improve plant health during stress because, under normal circumstances, they promote high-quality healthy turf. It was also expected that AVG would increase plant stress tolerance by reducing excessive ethylene production caused by stress, and that 2,4-D could promote healthy rooting, thereby enhancing turfgrass health.
Materials and Methods
‘Penncross’ creeping bentgrass was germinated in a greenhouse mist room (located at 680 Vernon Tharp Street, Columbus, OH 43210) on potting mix (45% to 55% Canadian peatmoss, composted bark, perlite, and dolomite lime) at a seeding rate of 226.7 g per 92.9 m2. At 12 d postgermination (DPG), seedlings with similar growth and development were transferred to a hydroponics system. Seedlings were selected based on similar leaf counts and shoot heights, and deference was given to intact root systems to help ensure survival. The roots were dipped in water to remove excess potting soil before transfer.
The hydroponics system consisted of 24 plastic 5.7-L boxes spray-painted black (outside only) located in two different Conviron E15 growth chambers (12 boxes per chamber) (Conviron, Winnipeg, Manitoba, Canada). The interiors of the chambers were 0.77 m long, with a width of 1.85 m and a height of 1.1 m (1.59 m3). In each chamber, 16 GE cool white, high-output, 85-W, fluorescent lights (F72T12-CW-HO) were located 1.1 m above the bottom of the interior. There were 19 holes drilled into the top of each box lid, and one seedling was placed in each hole. Attached to the underside of the lid were two layers (each with a thickness of 6.35 mm) of washable, black, polyurethane foam to support the seedlings and exclude light from the root zone. The boxes were completely filled with one-quarter-strength Hoagland’s hydroponics growth solution (Arnon 1949). An 80-gallon air pump was placed in each growth chamber, and a 6.35-mm-diameter rubber tube was run to each box. In each box, the tube was connected to a 30.48-cm-long air stone to distribute air to each box.
The seedlings were allowed to acclimate to the hydroponics system for 3 d with 25/20 °C day/night temperatures and 12-h photoperiods. The light intensity was monitored with a Spectrum Technologies, Inc. Lightscout DLI light monitor every third day by holding it as close to plant height as possible throughout the chambers (Plainfield, IL, USA). The monitor indicated that the plants were receiving between 750 and 999 μmol⋅m−2⋅s−1 photosynthetic active radiation, consistent with the reported figures (Purdue Agriculture 2018).
On the third day of acclimation, all plants were trimmed to a height of 2.54 cm. On the fourth day (16 DPG), the one-quarter-strength Hoagland’s solution was replaced with fresh solution, and heat and salt stress treatments were initiated. The boxes were filled to 85% capacity (∼4.85 L) for the remainder of the trial run. The salt stress treatment consisted of one-quarter-strength Hoagland’s solution that had 50 mM sodium chloride (NaCl) added; the solution had electrical conductivity (EC) of 5.3 dS⋅m−1. The NaCl concentration used was based on previous turfgrass salt tolerance research (Alshammary et al. 2004; Qian et al. 2001). The no-salt treatment comprised one-quarter-strength Hoagland’s solution. The heat treatment was 35/30 °C day/night temperatures (25/20 °C day/night temperatures for the nonheat treatment). The temperature regimens were selected based on known ideal temperatures for growth and previous research of creeping bentgrass and heat stress (Larkindale and Huang 2004; Xu and Huang 2008; Xu et al. 2009). The stresses were applied for 14 d, and the one-quarter-strength Hoagland’s solution (with and without salt) was replaced every third day. The one-quarter-strength Hoagland’s solution temperature was monitored with an infrared thermometer. The temperature of the fresh solution ranged from 18.3 to 27.8 °C, but it was usually approximately 21 °C throughout the experiments.
At 30 DPG, the plants were harvested one block at a time. The plants were placed on paper towels for 30 min to help remove excess moisture from the roots. The number of tillers per plant was counted. Then, the shoots were separated from the roots, and their fresh weights were measured.
Electrolyte leakage is an indirect measurement of membrane stability, and it has been documented that electrolyte leakage increases during both heat stress and salt stress (Agari et al. 1998; Dionisio-Sese and Tobita 1998; Liu and Huang 2000; Verslues et al. 2006). Roots from each plant were rinsed with distilled, deionized water three times (5 mL of water each time) and then soaked in 10 mL of distilled, deionized water for 1 h. Then, the roots were placed on a shaker set at 100 rpm at room temperature for 20 h. After 24 h, an initial EC measurement was obtained using an Oakton COND6+ EC meter (Oakton Instruments, Vernon Hills, IL, USA) that had been calibrated according to the user manual (Liu and Huang 2000). Then, the samples were autoclaved in a Steris/Amsco Century SV-120 Scientific Prevacuum Sterilizer set (Steris Corporation, Mentor, OH, USA) for the L20 cycle (121 °C for 20 min). Thereafter, a second EC measurement was obtained. Then, the percentage of electrolyte leakage was calculated.
The chlorophyll content decreases during heat stress and salt stress were attributed to impaired 5-aminolevulinic acid, which is a chlorophyll biosynthesis precursor (Khan 2003; Santos 2004; Tewari and Tripathy 1998). To measure the chlorophyll content, 0.2 mg of fresh shoot was obtained from the top of the tallest leaf from each plant and placed in 5 mL of dimethylformamide. The shoots were soaked for 1 to 2 h in a dark at room temperature and then placed in a refrigerator for 24 to 48 h (until the leaves were white). Then, the samples were removed from the refrigerator and allowed to warm to room temperature. Samples were vortexed and transferred into quartz cuvettes. A Shimadzu ultraviolet-1800 spectrophotometer with ultraviolet Probe 2.43 software was used to measure light absorption (Shimadzu Corporation, Kyoto, Japan). Then, the TCC was calculated according to Wellburn (1994).
The experiment was performed four different times (replications). For runs 1 and 3, the northern chamber served as the heat treatment (35/30 °C day/night) and the southern chamber served as the nonheated treatment. The chambers were switched for runs 2 and 4. This was performed to account for differences between the two chambers. Inside, each chamber was divided into east and west blocks. Each block contained six boxes. For runs 1 and 4, the east blocks contained one-quarter-strength Hoagland’s solution with 50 mM NaCl, and the west blocks contained only the one-quarter-strength Hoagland’s solution. The opposite was true for runs 2 and 3. This was performed to account for blocking effects within the chambers and to prevent human error when changing the solutions.
The turfgrass mortality rate was 40% to 50% during every run and in each box. Because plant death occurred before treatment initiation, the cause of death was likely trauma from transplanting (prior trial runs were managed somewhat differently and had higher mortality rates; these were used to inform when and how to best transplant the seedlings). Therefore, the missing sub-sub-samples were considered missing completely at random. The remaining sub-subsample measurements were averaged (mean substitution) (Greenland and Finkle 1995; Heitjan and Basu 1996; Rubin 1976).
The experiment was set up as a 2 × 2 factorial with salt levels nested in blocks. The statistical analysis was conducted using SAS (SAS Institute, Inc., Cary, NC, USA). The general linear model was used to test the run (replication), chamber, temperature, run (replication) × chamber × temperature, salt level, and temperature × salt level. An analysis of variance was performed to determine if there were significant interactions (P ≤ 0.05). Fisher’s protected least significant difference tests were computed for the main effects (P ≤ 0.05).
For the second experiment, the seedlings were also transplanted to the hydroponics system at 12 DPG. However, these seedlings were only allowed 2 d of acclimation; at that point, they were trimmed to a height of 2.54 cm. At 15 DPG, PGR treatments were applied.
Treatment 1 was the GA synthesis inhibitor combination product Muskateer®. This product contains 5.6% flurprimidol, 5.6% paclobutrazol, and 1.4% trinexapac-ethyl. It was applied at the label-recommended rate of 1.773 L⋅ha−1 using a handheld, CO2-powered spray boom calibrated to spray 841.1 L⋅ha−1.
Treatment 2 was the GA synthesis inhibitor product Anuew™, which contained 27.5% prohexadione calcium (calcium 3-oxido-5-oxo-4-propionylcyclohex-3-enecarboxylate). It was applied at the label-recommended rate of 0.638 kg⋅ha−1 using the same spray boom. Treatment 3 was 2,4-D applied at a rate of 4.89 L⋅ha−1 using the same spray boom. This rate was derived from typical rates of 2,4-D used safely on creeping bentgrass when applied as a selective herbicide.
Treatments 4 and 5 were 10 μM and 25 μM solutions of AVG, respectively. These treatments were applied using a small handheld spray bottle. These solutions were applied at a rate of 6.25 mL solution/plant. The molarity of the solutions and rates of application were based on those used by Xu and Huang (2009). Treatment 6 did not receive a PGR treatment; instead, water was sprayed at a rate of 841.1 L⋅ha−1 using the spray boom.
At 16 DPG, heat and salt treatments, as previously described, were initiated. The one-quarter-strength Hoagland’s solutions with and without 50 mM of NaCl were changed every third day, and the boxes were filled to 85% capacity again.
This experiment was set up as a 2 × 2 × 6 factorial and repeated (replicated) six times. For runs 1, 3, and 5, the north growth chamber was kept at 25/20 °C (day/night) and the south chamber was kept at 35/30 °C (day/night). For runs 2, 4, and 6, the north chamber was set to the higher temperature treatment and the south was set for the lower temperature treatment. For runs 1, 2, and 5 the east blocks received the salt treatment and the west blocks did not. For runs 3, 4, and 6, the west blocks received the salt treatment and the east blocks did not. The PGR treatments (1–6) were randomized within each block and re-randomized within each block for each run.
At 30 DPG, the plants were harvested one block at a time. The tillers were counted on each plant, and fresh shoot and root weights were measured. To measure root electrolyte leakage and TCC, roots and shoots were processed as mentioned. Those measurements were conducted using the same methods and equipment as those previously described. However, for this objective, the remainder of the shoots were placed in paper bags and then placed in a drying oven set at 60 °C for 48 h. After 48 h, shoot dry weights were measured. After obtaining the second EC measurement, roots were placed in paper bags and then in the same drying oven for 48 h. Unfortunately, during run 1, it was discovered that the dried roots adhered to the paper bags. For subsequent runs, the filter papers were weighed and roots were placed onto the filter papers; then, the papers were folded before being placed in paper bags. After oven-drying, the filter papers with roots were weighed and the tare was subtracted.
Again, the turfgrass mortality rate was 40% to 50%, as previously described. Therefore, the missing subsamples were considered missing completely at random. The remaining subsample measurements were averaged (mean substitution) (Greenland and Finkle 1995; Heitjan and Basu 1996; Rubin 1976).
The experiment was setup as a 2 × 2 × 6 factorial with salt levels nested in blocks. The statistical analysis was conducted using SAS (SAS Institute, Inc.). The general linear model was used to test the run (replication), chamber, temperature, run (replication) × chamber × temperature, salt level, PGR treatment, temperature × PGR treatment, salt level × PGR treatment, and temperature × salt level × PGR treatment. An analysis of variance was used to test for significant interactions (P ≤ 0.05). Fisher’s protected least significant difference tests were performed for the main effects (P ≤ 0.05).
Results and Discussion
Heat and salt interacted to reduce shoot fresh weights. Plants that received the combined stress had the lowest weights, consistent with prior research that found two stresses typically resulted in poorer plant health than just one stress (Fig. 1) (Jiang and Huang 2001; Yiwei and Huang 2000). There was also a positive significant interaction of temperature × salt level for fresh root weights and creeping bentgrass growth plants exposed to both stresses and the lowest fresh root weights (Fig. 2). The combined stress significantly reduced fresh root weights. Younger and Lunt (1967) found that the presence of salt can result in increased root weight; however, it has also been documented that salt stress can result in reduced root growth and root weights (Asana and Kale 1965; Munns 2002).
Fresh shoot weight means. Error bars represent SEs. According to the analysis of variance, a significant interaction between temperature × salt level was detected at P = 0.05. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Fresh shoot weight means. Error bars represent SEs. According to the analysis of variance, a significant interaction between temperature × salt level was detected at P = 0.05. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Fresh shoot weight means. Error bars represent SEs. According to the analysis of variance, a significant interaction between temperature × salt level was detected at P = 0.05. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Fresh root weight means. Error bars represent SEs. According to the analysis of variance, a significant interaction between temperature × salt level was detected at P = 0.05. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Fresh root weight means. Error bars represent SEs. According to the analysis of variance, a significant interaction between temperature × salt level was detected at P = 0.05. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Fresh root weight means. Error bars represent SEs. According to the analysis of variance, a significant interaction between temperature × salt level was detected at P = 0.05. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Tiller counts were also performed to measure plant health. No significant interaction of temperature × salt level was detected (not shown). This may have been because of the relatively short duration of the stress period as compared with that of other research (Xu and Huang 2001). The temperature regimen was not significant either (not shown). However, salt significantly reduced tillering as compared with the absence of salt (Fig. 3). Salt has been known to reduce tillering in wheat (Triticum aestevum L.) and rice (Francois et al. 1994; Maas et al. 1994; Zeng et al. 2001).
Tiller count means as affected by salt level. Error bars represent the least significant difference at P = 0.05 according to Fisher’s protected least significant difference (LSD) test. LSD = 0.418, N = 48.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Tiller count means as affected by salt level. Error bars represent the least significant difference at P = 0.05 according to Fisher’s protected least significant difference (LSD) test. LSD = 0.418, N = 48.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Tiller count means as affected by salt level. Error bars represent the least significant difference at P = 0.05 according to Fisher’s protected least significant difference (LSD) test. LSD = 0.418, N = 48.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Electrolyte leakage was measured as a marker of cell membrane health and plant stress. Electrolyte leakage is an indicator of the health of cell membranes. Both heat stress and salt stress can disrupt membrane integrity (DaCosta and Huang 2013; Munns 2002). A positive significant interaction of temperature × salt level was detected (Fig. 4). The plants exposed to both stresses had the highest mean percentage of root electrolyte leakage (Fig. 4).
Percentage of electrolyte leakage means. Error bars represent SEs. According to the analysis of variance, a significant interaction between temperature × salt level was detected at P = 0.05. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Percentage of electrolyte leakage means. Error bars represent SEs. According to the analysis of variance, a significant interaction between temperature × salt level was detected at P = 0.05. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Percentage of electrolyte leakage means. Error bars represent SEs. According to the analysis of variance, a significant interaction between temperature × salt level was detected at P = 0.05. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
For TCC, there was no significant interaction detected for combined stress × salt level (not shown). However, the temperature level was significant, and plants subjected to the higher temperature regimen had lower vTCC (Fig. 5). Both stresses are known to cause oxidative stress, which can damage chlorophyll (Abdelgawad et al. 2016; DaCosta and Huang 2013). The reduction of the total chlorophyll caused by stress in this experiment was relatively small. It is possible that the stresses were not applied long enough to induce greater changes as seen during other research (Liu and Huang 2000).
Total chlorophyll content means as affected by temperature. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 0.061. N = 48.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Total chlorophyll content means as affected by temperature. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 0.061. N = 48.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Total chlorophyll content means as affected by temperature. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 0.061. N = 48.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
The run (rep) was significant for fresh shoot and root weights as well as tillers. Although seedlings were transplanted at 12 DPG for every run, there was some observed variance in seedling growth between runs.
The results indicated that both the heat and salt treatments negatively affected 30-d-old creeping bentgrass growth and health. Although significant interactions of the temperature regimen × salt level were only detected for fresh weights and percentage of electrolyte leakage, based on the results, heat stress and salt stress combined were more damaging than either stress alone (Fig. 6).
Creeping bentgrass grown without stress treatments (left) and with both heat stress and stress treatments (right) 30 d postgermination.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Creeping bentgrass grown without stress treatments (left) and with both heat stress and stress treatments (right) 30 d postgermination.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Creeping bentgrass grown without stress treatments (left) and with both heat stress and stress treatments (right) 30 d postgermination.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
In the second experiment with the inclusion of PGRs, there were no significant two-way or three-way interactions detected between temperature regimen × salt level × PGR treatment for fresh shoot weights. However, for all three main effects, there were significant differences (not shown). Consistent with the results found during the first experiment, salt stress, heat stress, and combined salt stress and heat stress reduced fresh shoot weights (not shown). Plants treated with both rates of AVG had the highest fresh shoot weights, whereas plants treated with GA synthesis inhibitors had the lowest (not shown). The fact that AVG-treated plants had the highest fresh shoot weights indicated that those plants were able to maintain some growth under stress conditions. Under the unstressed conditions, plants treated with AVG also had the highest fresh shoot weights, indicating that AVG might promote growth. This was also observed when measuring dry shoot weights (Fig. 7). It was expected that both Muskateer® and Anuew™ would have lower fresh shoot masses because, by design, they are meant to reduce shoot growth. Under unstressed conditions, these products did reduce fresh shoot mass; however, they produced a greater number of tillers than the untreated control. Under heat stress and salt stress conditions, however, these products had tiller counts similar to those of the untreated control. This indicates that these products perform well under ideal growing conditions; however, poor turfgrass growth can occur under stress.
Dry shoot weight means as affected by the plant growth regulator (PGR) treatment. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 7.91, N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Dry shoot weight means as affected by the plant growth regulator (PGR) treatment. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 7.91, N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Dry shoot weight means as affected by the plant growth regulator (PGR) treatment. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 7.91, N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
There were no two-way or three-way interactions detected for fresh or dry root masses. The main effects of the temperature regimen, salt, and PGR were significant. Again, salt stress and heat stress combined reduced root growth the most. Plants treated with both rates of AVG had the highest dry root masses, and plants treated with GA synthesis inhibitors had the lowest root masses (Fig. 8). Both trinexapac-ethyl and paclobutrazol have been shown to have no effects on mature turfgrass rooting, even under drought stress (Ervin and Koski 1998; Jiang and Fry 1998).
Dry root weight means as affected by the plant growth regulator (PGR) treatment. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 1.4. N = 20.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Dry root weight means as affected by the plant growth regulator (PGR) treatment. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 1.4. N = 20.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Dry root weight means as affected by the plant growth regulator (PGR) treatment. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 1.4. N = 20.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
The AVG-treated plants had consistently higher fresh root masses than the untreated controls across all stress levels (not shown). The differences became much less pronounced for the dry root masses under unstressed and combined stress conditions. AVG-treated plants had higher dry root masses than the control in the presence of heat stress and salt stress alone. Based on these results, it is likely that AVG does not promote root growth under unstressed conditions, but it helps maintain growth under heat stress and salt stress conditions. AVG has been cited as promoting root growth by reducing ethylene synthesis in maize in conjunction with exogenous applications of IAA (Mulkey et al. 1982). Pretreating creeping bentgrass with AVG before the application of auxin should be investigated further.
Similar to the results of the first experiment, heat had no significant effect on tillering; however, salt reduced tillering. There were very small differences in tiller counts among the PGR treatments when salt was not present. However, when salt was present, plants treated with both AVG rates had the highest tiller counts, and all other treatments were similar to the control (Fig. 9). AVG was able to maintain some growth under salt stress. These results are somewhat consistent with previous research regarding the effects of GA synthesis inhibitors on tillering. Beasley and Branham (2005, 2007) found that paclobutrazol (an a.i. in Muskateer®) and trinexapac-ethyl (also an a.i. in Muskateer® and similar to prohexadione calcium) applied to Kentucky bluegrass (Poa pratensis L.) increased tillering. Ervin and Koski (1998) found similar results for perennial ryegrass (Lolium perenne L.) using trinexapac-ethyl; however, others have found that trinexapac-ethyl had no effect on tillering (Lickfeldt et al. 2000).
Tiller count means as affected by the plant growth regulator (PGR) treatment. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 0.72. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Tiller count means as affected by the plant growth regulator (PGR) treatment. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 0.72. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Tiller count means as affected by the plant growth regulator (PGR) treatment. Error bars represent the least significant difference (LSD) at P = 0.05 according to Fisher’s protected LSD test. LSD = 0.72. N = 24.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
A significant three-way interaction was detected for percentage of root electrolyte leakage × temperature regimen × salt level × PGR treatment (Fig. 10). The presence of heat more dramatically increased electrolyte leakage as compared with salt, and the combination of the two resulted in the most electrolyte leakage. The stresses and their combination increased electrolyte leakage regardless of the PGR treatment. Differences between PGR treatments were significant, but they were smallest when no stress was applied. The AVG-treated plants had slightly lower electrolyte leakage percentages than the other PGR treatments. It is likely that despite enhancing root growth, the roots were stressed. The roots that were exposed to salt stress and plants were more sensitive to root temperatures than air temperatures (Xu and Huang 2001).
Percentage of the electrolyte leakage means. Error bars represent SEs. A significant interaction between temperature × salt level × PGR treatment was detected at P = 0.05. N = 6.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Percentage of the electrolyte leakage means. Error bars represent SEs. A significant interaction between temperature × salt level × PGR treatment was detected at P = 0.05. N = 6.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Percentage of the electrolyte leakage means. Error bars represent SEs. A significant interaction between temperature × salt level × PGR treatment was detected at P = 0.05. N = 6.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Regarding the TCC, a significant interaction was detected for temperature regimen × PGR treatment (Fig. 11). The high temperature regimen reduced the TCC regardless of the PGR treatment. Both rates of AVG and the plants that were not treated with PGR had the highest TCC. The GA synthesis inhibitors had the lowest TCC; this may be because phytotoxicity has been observed with the use of such products. Salt also significantly lowered TCC during the second experiment. Similar to the first experiment, significant differences were detected, but they were relatively small. Again, this could be attributable to the relatively short length of time (2 weeks) when the plants were subjected to stress as compared with other research (Liu and Huang 2000).
Total chlorophyll content means. Error bars represent SEs. A significant interaction between temperature × PGR treatment was detected at P = 0.05. N = 12.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Total chlorophyll content means. Error bars represent SEs. A significant interaction between temperature × PGR treatment was detected at P = 0.05. N = 12.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Total chlorophyll content means. Error bars represent SEs. A significant interaction between temperature × PGR treatment was detected at P = 0.05. N = 12.
Citation: HortScience 58, 4; 10.21273/HORTSCI16978-22
Many different plant stresses can cause many different plant species to increase ethylene production (Morgan and Drew 1997). Ethylene is considered a stress response hormone because its presence is generally required for the plant stress response (Tao et al. 2015). The ways in which different stresses affect ethylene production can vary in intensity, and the suddenness of the stress and excess ethylene production of the plant can reduce plant growth and increase senescence (Morgan and Drew 1997; Tao et al. 2015). The rates of AVG used during this experiment likely prevented some excess ethylene production in creeping bentgrass under stress conditions, but that ethylene biosynthesis was not completely impaired. It has also been reported that AVG had no effect on ethylene evolution when plants were not subjected to heat stress (Xu and Huang 2009). Similar to the results found by Liu and Huang (2000), AVG can alleviate salt stress by reducing ethylene production; these results are consistent with those of another study (Kahn et al. 2014).
Although 2,4-D is typically safe to use on creeping bentgrass at the rate used, a typical recommendation is to avoid using products that contain 2,4-D when the turfgrass is heat-stressed (Patton and Weisenberger 2016). It is also usually recommended that these products should not be applied when temperatures reach ∼30 °C (Patton and Weisenberger 2016). In this experiment it is likely that 2,4-D was slightly harmful. It is still unknown why monocots are tolerant of 2,4-D; however, several theories have been proposed (Song 2014). Although no injury was visible, negative effects were likely because there was slight growth and TCC reduction compared with the untreated plants. There was also increased electrolyte leakage compared with the other treatments.
GA synthesis inhibitors are widely used on creeping bentgrass. However, they are meant to be applied to healthy, actively growing turfgrass. When managed correctly, they can produce darker green turfgrass that is more tolerant of stress, likely because of increased levels of cytokinins (Ervin and Zhang 2007a). The timing of the application used for this experiment was likely inappropriate because transplanting was stressful (as evidenced by high mortality); typically, sequential applications are required to generate plant health benefits. In this experiment, applications were performed 24 h before stress initiation (Ervin and Zhang 2007a). The results indicated that suppressing growth via the use of GA synthesis inhibitors without generating any health benefits from the prolonged use of such PGRs has negative effects on creeping bentgrass health and growth. For plants treated with GA synthesis inhibitors that were not subjected to either heat stress or salt stress, the growth was reduced (which is the point of using such products), but electrolyte leakage and TCC were similar to those of all the other treatments, which indicated that the plants were healthy.
It has been noted that a reduction of GA typically improves plant salt tolerance (Sharan et al. 2017). However, although the link is not well-established, GA may be involved in positively regulating heat shock protein gene expression (Zhang and Wang 2011). However, the role of GA in heat stress and salt stress may be dependent on the timing of application. Because of the application timing (after transplanting and only 1 d before stress initiation), this experiment may help elucidate the roles of GAs, cytokinins, and/or the interaction GAs × cytokinins in either heat stress or salt stress or heat stress and salt stress combined. Applying sequential GA synthesis inhibitors before stress initiation would have helped us to examine the role these PGR products can have during plant heat stress and salt stress. It would also be reasonable to apply the various GA synthesis inhibitors individually and with a.i. combinations for late-stage and early-stage GA synthesis inhibition.
A positive result of this experiment is that AVG could be used as a turfgrass PGR to improve growth and health during periods of heat stress and salinity stress and heat stress and salinity stress combined. Although AVG is expensive, it is effective at low rates and has the potential to be used as an emergency application because it was efficacious only 1 d before the onset of stress. AVG is also a commercially available a.i. used on apples and pineapples at a cost of approximately $260/ha. The rates used in this research are not readily convertible to label rates, but this cost would not be unreasonable for many golf courses. Additionally, it might be effective on creeping bentgrass at rates lower than what were used in this research. However, more research is needed to measure physiological changes, if any, such as hormone levels, and to test the performance on mature creeping bentgrass under field conditions.
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