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Organic Pest Management in Grapes

Over 25 years of working in vineyards with my colleagues at Bio Ag Services, I have learned a fair amount about managing pests organically. What follows are my opinions; not always verified by scientific experiments.


I try to minimize use of dusting sulfur because I am convinced that it increases problems with mites, leafhoppers and mealybugs. Probably this happens in part because the sulfur interferes with predators and parasites. But I suspect, along with many others, that sulfur is also hard on the vines and makes them either more attractive or more conducive to pest increase.

Mildew control without dusting sulfur is a challenge because dusting can give better coverage than water based sprays, especially when the canopy and bunches are closing, as in June. Dusting sulfur is also cheaper than other methods and more quickly applied, using less labor and fuel.

Wettable sulfur might have the same drawbacks as dusting sulfur, but to a lesser degree. The amount of sulfur used is much less, fuming is probably reduced, and the treatment interval is longer (10-14 days vs. 6-10 days). Most of my organic growers use 2-3 lbs. wettable sulfur until bloom.

I recommend applying about 5 lbs. of wettable sulfur close at budbreak or just before. Dr. Doug Gubler has shown that this suppresses the overwintering chasmothecia on the canes just as they would be releasing spores to infect the new green tissue. Lime sulfur applied in late dormancy works almost as well. These treatments do not depend on temperatures in the mildew index, which models conidial spore production on the green tissue.

After bloom I recommend using a preventive material such as Regalia or Sonata, tank mixed with an exterminant such as M-Pede or Oroboost. Nu Film P also seems to work well as the tank mix. M-Pede should not be used after fruit set on table grapes. Oroboost and Nu Film P should be used with care. I have not seen much problems with the waxy bloom, but some of my growers think they have.

Coverage is the key to success with these materials. The interval is similar to wettable sulfur, 10-14 days depending on the mildew index.

Dr. Gubler has demonstrated good mildew control with highly refined oils such as Stylet Oil and Purespray. Some workers also have evidence that oil suppresses mites and leafhoppers if timing is correct. Personally I am reluctant to use oil on vines because I fear it might be hard on the vines. But I have talked to several people who use it and have not heard of any problems with raisin or juice grapes. Oil should not be used on table grapes after fruit set.

If mildew starts to show up so an outbreak is feared, use a wash with wettable sulfur or Kaligreen. Use a high volume such as 200-300 gallons. Be careful with Kaligreen- I have seen very serious damage to some table grapes in applications a few weeks before veraison.

Kaligreen can also be used safely just before bloom. It cleans up any mildew that has started, even if it is not evident yet.

Mildew control can be ended once veraison is reached, or a couple of weeks earlier if weather is very hot. This particularly applies to raisin and juice grapes. More care should be taken if mildew is starting to show up before veraison. On most varieties, I do not see stem mildew as much of a threat. On susceptible varieties resumption of treatments when weather cools off in the fall, perhaps with dusting sulfur, may be more effective than treatments in mid-summer.


Mite problems arise in grapes when vines are stressed. Causes may be lack of water such as in sandy streaks, dust and heat along roads, or sulfur dust. Growers should do their best to solve the underlying problems with such tools as soil amendments, dust suppression or washing vines, and using alternatives to sulfur dust.

The second line of defense is biological control. The most important agents are predatory mites and six-spot thrips, but lacewings, minute pirate bugs, big-eyed bugs, black hunter thrips, and some spiders may also help. Flower thrips, which are also pests, can contribute significantly to mite control. Trophic relationships are complex, as most of these predators also eat each other. Endemic biocontrol is best, but supplemental releases of predatory mites or six-spot thrips may also help if timing is good and the habitat is not antagonistic (i.e. too hot).

Predatory mites are the main reason that mites are difficult to find in many vineyards. They are able to maintain mite populations at very low levels. They can also survive on alternate prey such as Willamette mites or Tydeid mites.

Releases of predatory mites should be aimed at maintaining populations. If suppression of a mite outbreak is the goal, large numbers will be needed and success is still not guaranteed.

Six spot thrips tend to appear when mite populations start to increase, although that increase may be only a few leaves with 10-50 mites. Six spot thrips also seem to be more able than predatory mites to tolerate hot weather. Six spot thrips can be released to suppress mite outbreaks, but success is dependent on releasing them early in the outbreak. As with predatory mites, success is not guaranteed.

Organic miticides are not nearly as dependable as conventional miticides. At best they suppress the mites, and the same materials may work fairly well in one situation, but fail in another. Coverage is one important issue- the applicator should drive slowly. Weather may also be a factor. We are trying to learn more about how to effectively use organic miticides. Here are some options:
Oils or soaps that smother or desiccate: JMS Stylet, Purespray Green; M-Pede. Pyrethrum: PyGanic. Best tank mixed with M-Pede, Oroboost, or Nu Film P. Specialty plant oils: Biomite, GC Mite, Ecotrol.


Organic leafhopper management includes monitoring immigration, evaluating biocontrol, and correctly timing treatment if needed.

Grape and variegated leafhoppers usually do not overwinter on vines because they like green foliage in the winter. They may overwinter on weeds, or on neighboring evergreen crops such as citrus, or in people’s yards on evergreen bushes. They tend to leave these overwintering sites before grape budbreak and temporarily feed on earlier leafing plants such as stonefruit or almonds, or on late winter weeds. From there they move to grapes after budbreak. They feed on grapes for several weeks before they start laying eggs.

Later generations may migrate from other vineyards. Leafhoppers will move long distances, through orchards, fallow or weedy fields, or other crops until they reach a vineyard.

By monitoring sides and corners of vineyards during migration times, a scout can find out where to watch most closely for leafhopper hatch.

Leafhopper biocontrol comes from a complex of beneficials. In my opinion the most important are Anagrus egg parasites, certain spiders including the yellow sac spider (Cheiracanthium), and lacewings. Ants also play a role; possibly important but this has not been studied. Anagrus is more effective on GLH but is still a key to VLH control. Yellow sac spider populations can be judged by noting their 3 types of nests: overwintering, day-resting, and egg-nests. When they are abundant enough, the number of leafhopper nymphs will decline or at least not increase very fast. Moderate populations of leafhoppers with good biocontrol may not need any further treatment.

Leafhopper control is easier when little or no dusting sulfur is applied. Studies have shown that Anagrus parasites live shorter lives with sulfur. I also think that vine stress caused by sulfur gives the leafhoppers some advantage.

Lacewings are the only biocontrol available from insectaries that are likely to help with leafhoppers, and releases are only moderately effective at best. Lacewings happily eat leafhoppers, but seem to be more attracted to honeydew producing prey such as aphids and mealybugs. Usually eggs or new larvae are released, which is laborious since they need to be placed on or near every vine. Finally, ants often eat most of the released eggs, which are not on the protective stalks that the mother lacewings would make. One advantage of lacewings is that large numbers of eggs can be purchased cheaply from insectaries.

Organic leafhopper insecticides have little or no residual action, so it is important to time the application to minimize the number of eggs, which are protected, and adults, which are harder to kill than nymphs. The best time for this is when the first nymphs to hatch are close to turning into adults. Leafhoppers mature from hatch to adult in 3-4 weeks. This takes about 435 degree days, with min-max 50.5-95oF.

I have had best success with PyGanic tank mixed with Oroboost, M-Pede, or Nu Film P.

Some workers have also reported that oil applied during the egg-laying period helps to control leafhoppers.
The new material Venerate from Marrone also shows promise on leafhoppers.

Grape Mealybugs

Grape mealybugs are rarely a serious problem in organic grapes in the San Joaquin Valley. No one has been able to adequately demonstrate the reason for this; presumably many conventional growers are using some chemicals that disrupt mealybug biocontrol. Actually, most conventional raisin and wine growers and many conventional table grape growers have few GMB problems.

Interestingly, even though organic and raisin growers have traditionally used a lot of dusting sulfur for mildew, GMB has usually not been a serious problem. This is in contrast with vine mealybug (see below).

The key to GMB control is biocontrol, which is supplied mainly by predaceous midge maggots and parasites in the family Encyrtidae, with help from lacewings and the tiny lady beetle, Nephus.

Vine Mealybugs

Vine mealybugs are a big challenge. Their introduction drove many organic grape growers to give up and go conventional. But new tools and knowledge have made it possible, in most cases, to adequately control VMB for organic grapes.

The tactics available for organic VMB control are pheromone disruption, stopping use of sulfur dust, biocontrol- both endemic and insectary-reared, ant control, and pyrethrum sprays. Venerate might also be good.

Pheromone disruption works well if VMB populations are very low. This is often the case in organic vineyards in spring, because biocontrol tends to be most effective in the fall, leaving low overwintering populations. However it will not happen if many VMB are overwintering on roots or in the holes of carpenter worms, which both act as refuges from biocontrol. In these cases, pheromones are probably not worth their high cost.

Pheromones offer good protection against new infestation in vineyards that have few or no mealybugs. If a few crawlers come in, for example carried by birds, when they mature as females they will not attract males, which normally can fly in from a quarter mile away. The unmated females will not lay eggs and the tiny infestation will die off.

Some research suggests that in addition to confusing VMB males, the pheromones increase parasitism.

Pheromones should be placed by mid-April, before males begin to fly.

Stopping dusting sulfur is a very important tactic in the control of VMB. In many cases we have seen disaster turn into a minor problem by taking this step alone. As with mites and leafhoppers, we suspect that sulfur both interferes with biocontrol and makes the vine more susceptible through stress.

The most important VMB biocontrol agents are Anagyrus and Coccidoxenoides parasites, Nephus beetles, predaceous midge maggots, and green and brown lacewings. All but the midges are available from insectaries, although Nephus is in limited supply. All are also endemic and will come in by themselves.

The main problem with VMB biocontrol is that it often comes too late. Anagyrus typically dominates in the fall, and is a very good searcher for stray mealybugs, but it does not even emerge from overwintering until late May or June, after two VMB generations. The four predators start earlier but need at least moderate VMB populations to survive. Working together, the parasites and predators may reduce the VMB to a very low level by late August, but by then much of the crop may be lost.

Insectary releases of all of these insects are undoubtedly helpful, but evaluating the effect of releases compared to the endemic populations is difficult. Releasing enough to make a decisive difference is expensive, hundreds of dollars per acre. Releasing smaller amounts might get them started earlier, make significant improvements in their numbers, or speed up control by a couple of important weeks.

Nephus beetles are excellent at controlling VMB populations under bark. They are not effective on VMB on canes, canopy, or bunches. They also work poorly on young vines without much covering bark. Older vines may show wet bark from VMB, but upon examination few VMB are surviving and the canes and bunches are clean; the beetles ate the VMB before they could move up from the trunks. Nephus beetles can be released as soon as VMB can be found and the insectary is ready, in May or early June.

Midges also work best under the bark or between bunches and wood. They are more consistently present on GMB, but sometimes quickly clean up VMB infestations. Possibly they work better on VMB when GMB is also present.

Anagyrus parasites start late but build up over a couple of months and eventually clean up most of the VMB not eaten by parasites. In August and September bunches may look heavily infested, but most of the VMB are parasitized. Anagyrus continues to work at low VMB levels and is a key to very low overwintering numbers. Insectary releases may start in May before the endemic Anagyrus start to emerge, and continue at intervals through June and July.

Coccidoxenoides parasites are smaller than Anagyrus and work on smaller VMB instars, especially under bark. They also start earlier. The mummies are not well attached to the substrate, so they tend to fall off when bark is peeled. This makes them difficult to monitor. They are not available from insectaries. Some vineyards with releases by researchers showed marked decreases in VMB populations over a couple of years.

Green lacewings are strongly attracted to VMB honeydew. For VMB control, they can be released as adults, because they will find the vines with infestations. They can be released when honeydew becomes visible on the trunks.

Brown lacewings have been shown by researchers to be good mealybug predators. They probably play a bigger role than realized; they are easily overlooked because they are nocturnal, the eggs are not on obvious stalks like green lacewings, and the larvae are easily confused with green lacewing larvae. They are also available from an insectary.

Ants play a major role in mealybug problems. Ants protect VMB from biocontrol, move them from vine to vine, and make spaces for mealybugs around roots. In one study in Coachella Valley, VMB virtually disappeared when Formica field ants were controlled.

Almost all local ant species tend VMB. Gray field ants are the most common and fire ants are the worst. Other common tenders include pavement ants, pyramid ants, thief ants, odorous house ants, crazy ants, honeypot ants, little black ants, and Argentine ants. This last is rare in San Joaquin Valley vineyards but much more common in northern and coastal California districts. It can be very effective at protecting mealybugs. Harvester ants are the only common SJV species not tending mealybugs.

Ants are difficult to control, especially in organic vineyards. We have good fire ant baits, but they are not organic and do not control field ants. Seduce ant bait is organic and sometimes moderately effective on field ants. The pieces are too large for fire ants. Bait stations using sugar water with boron can be used on Argentine ants. Dry boron baits can also be devised that are attractive to either fire ants or field ants, but unfortunately they are not legal because Solubor is registered as a fertilizer, not an insecticide.

Ants can be temporarily kept off vines with sticky barriers. Obviously this is very labor intensive.

VMB is difficult to control with sprays because large portions of the population are always protected in hidden places under bark, inside bunches, or on roots. The best to be hoped for is to slow down large migrations of small crawlers to the canopy. The same PyGanic tank mixes that control leafhoppers will also kill younger stages of VMB if they are exposed.


Caterpillars are usually not too difficult to control in organic vineyards. They typically have good biocontrol by spiders, lacewings, parasites, and other beneficial insects. Ants probably also play an important role, so if ants are controlled, watch carefully for increasing caterpillar problems.

Well-timed B.t. sprays are effective on OLR and leaffolders. They may need to be repeated two or more times to equal insecticides with longer residual. Skeletonizers are less susceptible to B.t. but repeated BT Dust applications will control the first 3 instars. Entrust (spinosad) is very effective on all of these worms, but should be avoided where possible because it is disruptive to biocontrol, especially to mealybug parasites. It is less disruptive if used early, before the middle of May. Venerate might also work well.

Impact of Seaweed Extract-Based Cytokinins and Zeatin Riboside on Creeping Bentgrass Heat Tolerance

Xunzhong Zhang and E.H. Ervin, Dep. of Crop and Soil Envi- ronmental Sciences, Virginia Polytechnic Institute and State Univ., Blacksburg, VA 24061. Received 10 May 2007. *Corresponding author (


Heat stress is the primary factor limiting summer performance of creeping bentgrass (Agrostis stolonifera L.) in many temperate to subtropical regions. Seaweed extract (SWE)-based cytokinins have been used to improve stress tolerance, but their specific effects on creeping bentgrass under supraoptimal temperatures are lacking. This study was designed to determine whether SWE-based cytokinins affect creeping bentgrass heat tolerance, and to compare effects of SWE-based cytokinins to those of a trans-zeatin riboside (t-ZR)-standard. Concen- trations of t-ZR in two SWE sources (referred to as Oce and Aca) were determined. Treatments were applied twice to creeping bentgrass at an equivalent t-ZR concentration of 10 μM. One week after the initial treatment, heat stress was imposed (35/25°C [day/night]) for 42 d. The Oce SWE, Aca SWE, and t-ZR treatments resulted in leaf t-ZR concentrations that were 39, 32, and 28% higher, respectively, relative to the control at 14 d of heat stress. The Oce SWE, Aca SWE, and t-ZR treatments also increased superoxide dismutase activity and  alleviated  the  decline  of turfgrass quality, photochemical efficiency, and root viability. Ashed SWE provided results similar to the water control. Beneficial effects of SWE on heat tolerance appear to be associated with their organic, especially cytokinin, components and not the mineral (ashed) fraction. Proper application of SWE-based cytokinins may be an effective approach to improve summer performance of creeping bentgrass.

Abbreviations: 6-BA, 6-benzyladenine; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; MDA, malondialdehyde; PBS, phosphate-buffered saline; PEc, canopy photochemical efficiency; ROS, reactive oxygen species; SOD, superoxide dismutase; SWE, sea- weed extract; TTC, 2,3,5-triphenyltetrazolium chloride; t-ZR, trans- zeatin riboside.

Creeping bentgrass (Agrostis stolonifera L.) is a primary cool season turfgrass species used on close-cut golf course greens, tees, and fairways. Creeping bentgrass was originally adapted to cool-humid climates, but is increasingly utilized in subtropical regions for golf course putting greens (Beard, 1973). Although heat tolerance has been improved in new cultivars, it is still often the primary factor limiting summer performance of creeping bentgrass (Fry and Huang, 2004; McCarty, 2005).

It has been documented that environmental stresses damage cells via oxidative injury. Heat stress may decrease leaf photo- synthetic rate and carbohydrate reserves (Xu and Huang, 2000). As Calvin cycle activity and photosynthetic electron transport efficiency are reduced by environmental stresses, excess energy may be diverted to oxygen molecules, resulting in over-produc- tion of toxic reactive oxygen species (ROS; Zhang and Schmidt, 1997). Reactive oxygen species damage many cell components such as lipids, proteins, and nucleic acids (Smirnoff, 1995). Plant antioxidant enzymes [such as superoxide dismutase (SOD)] and other metabolites protect plant cells by scavenging ROS. Heat stress tolerance of cool season grasses has been found to be associated with antioxidant activity (Huang et al., 2001; Fry and Huang, 2004).

High soil temperature is the primary factor leading to turfgrass decline during summer months (Xu and Huang, 2000). High soil temperature may damage roots, a primary site of cytokinin biosynthesis. Extensive research has identified cytokinins as a class of very effective antisenescence growth regulators (Woo et al., 2002). Cytokinins exhibit antisenescence properties that are related to their antioxidant activity (Musgrave, 1994; Zhang and Ervin, 2004). The repression of lipoxygenase by cytokinins also contributes to their overall antisenescence functions (Strivastava, 2002). Thimann (1987) found that cytokinins delay senescence processes probably by maintaining the integrity of the tonoplast membrane.

Goatley and Schmidt (1990) reported that foliar appli- cation of 6-benzyladenine (6-BA) delayed senescence of Kentucky bluegrass. Liu et al. (2002) noted that root- zone-injection of zeatin riboside increased endogenous cytokinins, antioxidant activity, and delayed senescence in heat-stressed creeping bentgrass. Exogenous 6-BA also increased root cytokinin level (Wang et al., 2003) and leaf total nonstructural carbohydrate concentration (Wang et al., 2006) in creeping bentgrass under heat stress.

Seaweed [Ascophyllum nodosum (L.) Le Jolis] extract (SWE) has been shown to contain biologically active con- centrations of natural cytokinins such as trans-zeatin ribo- side (t-ZR) and isopentenyl-adenine (Senn, 1987; Zhang and Schmidt, 1999; Zhang and Ervin, 2004). Foliar application of SWE has been shown to increase leaf cytokinin content and antioxidant activity, while reducing lipid peroxidation and delaying senescence of creeping bentgrass under drought stress (Zhang and Schmidt, 1999, 2000; Zhang and Ervin, 2004). Seaweed extract treatments have also been reported to increase turfgrass tolerance to salinity (Nabati et al., 1994) and ultraviolet B irradiation stress (Ervin et al., 2004). Zhang et al. (2003a) noted that applying SWE plus humic acid to Kentucky bluegrass 1 wk before sod harvest increased canopy photochemical efficiency (PEc) and recovery from postharvest heat injury.

Although it has been documented that this source of SWE contains cytokinins and has a positive impact on stress tolerance in turfgrasses, no research has reported on effects of differentially extracted sources of seaweed on leaf cytokinin levels and heat tolerance of turfgrasses. Furthermore, no research is available regarding relative contributions of organic (particularly cytokinins) and inorganic fractions (ash or mineral residues) in the SWE on turfgrass performance under heat stress. Therefore, this study was designed to quantify the amount of t-ZR in two differentially extracted seaweed sources, determine whether these SWEs affect creeping bentgrass physiological responses to supraoptimal temperature, and to compare effects of SWE-based t-ZR to those of a pure zeatin riboside and an ashed SWE.

Plant Culture and Cytokinin Treatment

This experiment was conducted in 2005 and repeated in 2006. ‘L-93’ creeping bentgrass was planted in conetainers (3.8-cm diameter, 21-cm depth; Stuewe and Sons, Inc., Corvallis, OR) filled with calcined clay in November 2004 and 2005 and grown under a greenhouse bench mist system with photosynthetically active radiation at 350 μmol m-2 s-1 (at 1400 h) and 24/20°C (day/night). The grass was fertilized with 0.5 g N m-2 (Nutriculture 20–20–20 with micronutrients; Plant Marvel, Chicago, IL) weekly and clipped at 9.5 mm.

Three months after planting, the grass received initial SWE and t-ZR treatments. Treatments included: (i) control (water); (ii) Acadian seaplants liquid extract (Aca SWE) containing 10 μM t-ZR; (iii) Ocean Organics liquid SWE (Oce WSE) containing 10 μM t-ZR; (iv) zeatin riboside (t-ZR) at 10 μM; and (iv) ashed Ocean Organics liquid SWE; mineral residues were mixed in water and applied (ashed control). The Aca SWE was supplied by Acadian Seaplants Inc. (Dartmouth, NS, Canada). It is a KOH-extracted liquid SWE concentrate with 14.4% solids. The Oce SWE, from Ocean Organics (Waldoboro, ME), is a KOH-extracted liquid SWE concentrate with 8% solids. The t-ZR standard was from Sigma (St. Louis, MO). The t-ZR concentration in the two sources of SWE was determined using indirect enzyme-linked immunosorbent assay (ELISA) method as described subsequently. The SWE concentrates were diluted with water to obtain treatment solutions containing 10 μM t- ZR. The same amount of Oce SWE was ashed at 500°C for 8 h, mineral residues were dissolved in water, and the solution was used for the treatment. Synthetic cytokinin (t-ZR) was dissolved in trace methanol, and then diluted with water, supplemented with 0.05% Tween 20. The SWE and t-ZR treatments were evenly applied to creeping bentgrass foliage with a sprayer delivering 784 L ha-1 at 290 kPa.

One week after initial treatment, grasses were transferred into a growth chamber with temperature of 35/25°C (day/ night), photosynthetically active radiation at 300 μmol s-1 m-2 with a 14-h photoperiod. The containers were placed in plastic trays filled with one-quarter strength Hoagland’s solution that was replaced weekly. Two weeks after heat stress initiation, treatments were re-applied. A total of 100 containers were arranged in a completely randomized design in the growth chamber with four replications in space and four container subsamples per treatment in time. This design allowed for the destructive sampling of four containers per treatment at 0, 14, 28, and 42 d of heat stress exposure.

At 0, 14, 28, and 42 d after initiation of heat stress, turfgrass quality and canopy PEc were measured. Conetainer subsamples were then removed from the growth chamber and destructively sampled; the collected leaf tissues were immediately frozen with liquid N and stored at −80°C for analysis of t-ZR, SOD activity, and lipid peroxidation. Roots were separated, washed, weighed, and then tested for root viability.

Turfgrass Quality and Canopy PEc
Turfgrass visual quality was rated based on a scale of 1 to 9, with 1 indicating complete death, 9 the best possible quality, and 6 minimum commercial acceptability. Canopy photochemical efficiency was measured with a chlorophyll fluorometer OS- 50II (Opti-Sciences, Tynsboro, MA) according to the methods of Zhang et al. (2003a).

Leaf t-ZR
Frozen leaf tissues (500 mg) collected from each container were used for t-ZR assay with indirect ELISA. Zeatin riboside was extracted and purified following the methods of Turnbull et al. (1997), with minor modifications (Zhang and Ervin, 2004). A recovery rate greater than 90% was obtained based on the internal standards.

Zeatin riboside was analyzed using indirect ELISA as described by Trione et al. (1985) with modifications (Zhang and Ervin, 2004). Briefly, wells of a 96-unit plate were coated with 100 μL per well of t-ZR conjugated to bovine serum albumin (BSA) (1:2000 dilution), incubated overnight at 5°C, emptied, and washed three times with phosphate-buffered saline (PBS)-Tween (PBS containing 0.05% Tween 20). The reaction was “blocked” with 200 μL of 1% BSA in PBS (37°C, 30 min) to prevent non-specific protein adsorption. After the plate was washed twice with PBS-Tween, 50 μL of the cytokinin extracts or standards and 50 μL of the antibody t-ZR3 (1:200 dilution) were added to the wells and the plates were incubated at 37°C for 60 min, emptied, and washed three times with PBS-Tween. The A and B rows in the plate were used to develop a standard curve. A series of t-ZR concentrations (0, 2.5, 5, 10, 25, 50 ng mL-1) were made from the stock solution. Appropriate dilutions were prepared for the SWE samples. Each standard or sample was repeated three times and the averages were used for data analysis.

To each well, 100 μL of a 1:1000 dilution of alkaline phos- phatase–labeled goat anti-mouse IgG (Sigma Chemical Co., St. Louis, MO) was added and the plates were incubated at 37°C for 60 min. After three washes with PBS-Tween, 100 μL of substrate solution (3 mg mL-1 of p-nitrophenyl phosphate in 10% diethanolamine buffer, pH 9.8, 0.5 mM MgCl2) was added to each well and the plates were incubated at 37°C for 50 min. The color reactions in each well were determined by measuring absorbance at 405 nm by an enzyme immunoassay micro- plate reader (Opsys MR, Thermo Labsystems, Chantilly, VA). Zeatin riboside concentration was calculated based on the standard curve after logic conversion of the data.

Leaf SOD Activity
The SOD in leaf tissue (100 mg fresh weight) was extracted according to Zhang et al. (2005) and determined according to the method of Banowetz et al. (2004). Briefly, to each well 125 μL reaction solution containing 50 mM Pipes buffers (pH 7.5), 0.4 mM o-dia- nisidine, 0.5 mM diethylenetriaminepentaacetic acid, and 26 μM riboflavin, and then 20 μL enzyme extract was added. Absorbance at 560 nm of solution mixture was read immediately after addition of enzyme extract on a microplate reader (Opsys MR). The reaction was initiated by switching on a circular fluorescent light (irradiance = 60 μmol m-2 s-1) and slowly rotating sample tubes under the light for 30 min at room temperature (25°C). Absorbance at 560 nm was measured again. Superoxide dismutase activity was calculated based on changes in absorbance and from the standard curve. Since SOD is presented on a protein basis, protein concentration for each sample was analyzed by the bicinchoninic method, with BSA serving as the standard (Smith, 1985).

Leaf Lipid Peroxidation
Lipid peroxidation in leaf samples was measured in terms of malondialdehyde (MDA) concentration which was determined according to Heath and Packer (1968) with some modifications. Briefly, fresh sample (100 mg) was homogenized in 5 mL 0.25% 2-thiobarbituric acid in 10% trichloroacetic acid using mortar and pestle. The homogenate was heated at 95°C for 30 min and the mixture was quickly cooled down in an ice bath, and then centrifuged at 10,000 g for 10 min. The absorbance of the supernatants was measured at 532 nm and 600 nm. The value for the nonspecific absorbance at 600 nm was subtracted from the readings at 532 nm. The blank was 0.25% 2-thiobarbituric acid in 10% trichloroacetic acid. The concentration of the MDA was calculated using MDA’s extinction coefficient at 155 mm-1 cm-1 and expressed as nanomoles per gram fresh weight.

Root Viability
Root viability was measured according to the procedure of Comas et al. (2000). Fresh roots (200 mg) were cleaned with distilled water, cut into 1-cm sections, and transferred to 30 mL glass tubes. To each tube, 5 mL 0.6% (w/v) 2,3,5-triphen- yltetrazolium chloride (TTC) in 0.05 M Na2HPO4–NaH2PO4 (pH 7.4) plus 0.05% Triton X-100 was added and the mixture was vacuum-infiltrated for 5 min to ensure infiltration of TTC. Samples were incubated at 30°C for 24 h, and rinsed twice with distilled water. Root samples were extracted in 5 mL 95% (v/v) for 5 min at 85°C. The solution extracted was brought up to a volume of 10 mL and 200 μL of the solution was transferred into a microplate and absorbance was measured at 490 nm on a microplate reader as described previously. Root viability was expressed as A490 g-1 fresh weight and converted to a percentage relative to the control at day 0.

Experimental Design and Statistical Analysis
A complete randomized design was used with four replications and five subsamples in time per treatment in both 2005 and 2006. Data were analyzed with ANOVA, using year and treat- ments as factors. Since the year × treatment interaction was not significant, the data from 2005 and 2006 were pooled. Separation of means was performed with a Fisher’s protected LSD test at a 5% probability level (SAS Institute, 2003).

SWE Concentrate t-ZR
As measured by ELISA, the Aca SWE and Oce SWE liquid concentrates contained 354 μM t-ZR (868 μg g-1 dry weight) and 343 μM t-ZR (1554 μg g-1 dry weight), respectively.

Leaf t-ZR Concentration
Exposure of creeping bentgrass to heat stress decreased leaf t-ZR concentration at day 14 (Fig. 1). Both Aca SWE and Oce SWE treatments consistently resulted in less decline of leaf t-ZR concentration under heat stress, when compared to the control at days 14, 28, and 42. Greater leaf t-ZR concentration due to t-ZR treatment was only measured at day 14. The Aca SWE, Oce SWE, and t-ZR treatments resulted in t-ZR concentrations that were 39, 32, and 28% higher, respectively, when compared to the control at day 14. The mineral fraction of the Oce SWE (SWE ash) did not impact leaf t-ZR concentration.

Turfgrass Quality and Canopy PEc
Heat stress caused a gradual decline of turfgrass visual quality (Fig. 2). The Aca SWE and Oce SWE treatments alleviated decline of turfgrass quality. A similar result was observed with t-ZR treatment. At day 42, Aca SWE, Oce SWE, and t-ZR treatments resulted in 34, 26, and 32% greater turfgrass quality, respectively, relative to the control. The SWE ash, however, failed to affect turfgrass quality. No PEc differences were measured for any of the treatments on days 0, 14, or 28. By day 42, however, the Aca SWE, Oce SWE, and t-ZR treatments had higher PEc relative to the SWE ash and control (Fig. 3).

Superoxide Dismutase Activity
Leaf SOD activity of the control increased rapidly in the first 14 d and then returned to day 0 levels on the last two sample dates (Fig. 4). The Aca SWE, Oce SWE and t- ZR treatments increased SOD activity when compared to the control, with Oce SWE having a greater effect when measured at day 28. The SWE ash did not impact SOD activity. At day 42, Aca SWE, Oce SWE, and t-ZR treat- ments increased SOD activity by 59, 56, and 41%, respec- tively, when compared to the control.

Lipid Peroxidation
Significant lipid peroxidation in terms of MDA concen- tration was found after 14 d of heat stress (Fig. 5). The Aca SWE, Oce SWE, and t-ZR treatments consistently reduced lipid peroxidation (as evidenced by lower MDA concentration) when compared to the control. The SWE ash did not affect MDA in all sampling dates except on day 28.

Root Mass and Viability
Root fresh weight increased in the first 14 d and then decreased in response to heat stress (Fig. 6). The treat- ments did not impact root fresh weight when measured at days 0 and 14, while only Aca SWE increased root fresh weight at days 28 and 42.

Heat stress caused a gradual decline of root viability (Fig. 7). On average, root viability was reduced by 60% during 42 d of heat stress. The Oce SWE treatment alleviated root viability decline when measured at days 28 and 42, while Oce SWE and t-ZR improved root viability at day 42 only. The SWE ash showed improved root viability relative to the control on day 42 only.

It has previously been documented that SWE contains cytokinins (Sanderson and Jameson, 1986; Tay et al., 1985; Zhang and Ervin, 2004). However, no studies have compared the concentration of cytokinins present in different commercial sources of SWE, nor has there been a comparison of SWE-based cytokinins with synthetic cytokinins using the same rates and turfgrass species under identical environments. In this study, large differences in t-ZR concentration were found in the two KOH-extracted SWEs on a dry weight basis. The Oce SWE contained t-ZR that was 79% higher than Aca SWE on a dry weight basis. When these two sources of SWE were applied at the same rate in terms of t-ZR concentration (10 μM), they had similar effects on leaf ZR concentration and performance of creeping bentgrass under heat stress. Influence of the SWEs on leaf t-ZR levels was similar to those of the t-ZR treatment at day 14 of heat stress, but not on the two subsequent sampling dates. Liu et al. (2002) also reported that exogenous treatment of heat-stressed creeping bentgrass with 10 μM t-ZR (by root-injection) resulted in greater endogenous cytokinins content. However, their results indicated a longer term response with endogenous cytokinins contents remaining greater than the control over the entire 56-d trial period whereas our data indicated an increase due to exogenous t-ZR only at day 14. Differences in longevity of response between the two studies may have been related to differing methods of t-ZR delivery as we foliar-applied and they root-injected. Root injection may have resulted in more efficient plant uptake and transpirational distribution of t-ZR to the leaf tissues. Foliar application of the two SWE-sources of t-ZR did result in greater maintenance of endogenous t-ZR contents for the duration of our heat- stress study period. These results are similar to what was found at the end of a drought-stress trial on SWE-treated creeping bentgrass (Zhang and Ervin, 2004). Is there a biologically relevant difference between root-injection or foliar delivery of synthetic t-ZR or between foliar delivery of synthetic t-ZR and SWE-based t-ZR on creeping bentgrass heat- or drought-stress tolerance? None of these studies were designed to specifically address these questions. Additionally, the two sources of SWE used in this study contain many other possible “plant-active” organic constituents, such as betaines (Blunden et al., 1986), which may have affected the extent and longevity of metabolic response. Specific studies designed to address these questions need to be conducted.

The results of this study also showed that SWE and t-ZR treatments delayed decline of turfgrass quality and PEc under heat stress. The data supports the hypothesis that improved maintenance of visual quality, PEc, and antioxidant levels of turfgrasses during abiotic stresses may be associated with increased plant cytokinin levels due to exogenous application of SWE (Zhang et al., 2003a, 2003b; Zhang and Ervin, 2004; Zhang and Schmidt, 1999, 2000).

The results of this study also indicate that the Aca SWE, when applied in nonashed form, increased leaf t- ZR concentration; the same SWE, when ashed before application, failed to affect leaf t-ZR concentration in creeping bentgrass subjected to heat stress. This is in agreement with the previous results by Zhang and Ervin (2004) with creeping bentgrass subjected to drought stress. It appears that beneficial effects of SWE on plant tolerance to abiotic stresses are due to its hormonal components, not the mineral fraction.

The data of this study indicate that the SWE and t-ZR treatments significantly increased leaf SOD activity. This is supported by previous studies which showed that SWE increased SOD activity in various turfgrass species under abiotic stresses (Ervin et al., 2004; Zhang and Schmidt, 1999, 2000; Zhang et al., 2003a, 2003b). Liu et al. (2002) noted that root injected t-ZR at 1 to 10 μM increased SOD activity in creeping bentgrass subjected to heat stress. The mechanisms of SOD response to exogenous cytokinins are not clear. It is possible that the increase in leaf cytokinin levels due to SWE and t-ZR applications may delay plant senescence and sustain the synthesis and/or function of antioxidant enzymes (such as SOD) and metabolites under stress. The SWE may also increase nitrate reductase activity (Durand et al., 2003) and synthesis of proteins and antioxidant enzymes. A high level of SOD activity would effectively suppress ROS toxicity and protect cells under abiotic stresses (Zhang and Schmidt, 1999). Plants with higher levels of antioxidant activity may have greater PEc and root viability, and thus produce more cytokinins.

Root viability, but not root fresh weight, was signifi- cantly increased by applying Oce SWE and t-ZR in this study. This suggests that SWE-based cytokinins and t-ZR may delay root senescence and improve root function under heat stress. Cytokinins are primarily synthesized in roots and transported into shoots via xylem. Endogenous cytokinins level may be reduced under heat stress due to impaired root function. The SWE-based cytokinins and t-ZR treatments may increase endogenous cytokinins level and thus delay root senescence.

In summary, SWE-based cytokinins and t-ZR, when applied at the same estimated ZR rate (10 μM), showed a number of similar effects on heat tolerance of creeping bentgrass. The beneficial effects of SWE on heat tolerance appear to be associated with its organic components, particularly cytokinins. Supplementation of cytokinins via SWE applications before significant heat stress periods appears to be an environmentally friendly approach to improving summer performance of creeping bentgrass.

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