Effect of core cultivation, fertility, and plant growth regulators on recovery of voided creeping bentgrass greens canopies following annual bluegrass control via methiozolin

Abstract Methiozolin is commonly used for the safe and selective removal of annual bluegrass from creeping bentgrass golf greens. Studies were conducted in 2013 and 2014 with the objective of assessing fertility programs consisting of synthetic fertilizers and biostimulants, with and without the plant growth regulator trinexapac-ethyl, to aid putting green canopy recovery following annual bluegrass removal via methiozolin. Additional studies were conducted to compare recovery of creeping bentgrass following an aggressive core aerification event with fertility programs with and without methiozolin. In all cases, the addition of 7 kg ha−1 of N-P-K from fertilizer or biostimulant biweekly to greens increased turfgrass recovery time by 1 to 3 wk compared to a standard green’s fertility program alone. Creeping bentgrass treated with biostimulants recovered equivalent to or quicker than creeping bentgrass treated with synthetic fertilizer (SF) in all cases. In the presence of methiozolin treatments, trinexapac-ethyl reduced time to 90% recovery (T90) by 0.25 to 0.5 wk at two locations, and increased T90 recovery time by 0.1 wk at one location. Otherwise, plots treated with SF plus trinexapac-ethyl were equivalent to plots treated with SF only. Methiozolin slowed turfgrass recovery time at one location where severe drought stress occurred but not at the other location that did not experience drought stress. These results suggest that turf managers should increase fertilizer treatments but will not need to discontinue trinexapac-ethyl use to maximize creeping bentgrass recovery following annual bluegrass control with methiozolin. These data also suggest that methiozolin has the potential to negatively affect creeping bentgrass recovery when drought stress is experienced.


Introduction
Annual bluegrass regularly infests creeping bentgrass putting greens and ultimately can outcompete the desirable creeping bentgrass in climates that are conducive to annual bluegrass growth.This is problematic due to the propensity of annual bluegrass to disrupt the uniformity of the playing surfaces of creeping bentgrass putting greens, thus reducing the trueness of ball roll on these surfaces (Rana and Askew 2018).The plant growth regulators paclobutrazol and flurprimidol are commonly used on creeping bentgrass putting greens due to the ability of the herbicides to selectively reduce annual bluegrass populations over time (Johnson andMurphy 1995, 1996).However, methiozolin is the only herbicide labeled for postemergence control of annual bluegrass in creeping bentgrass putting greens.Although methiozolin typically controls annual bluegrass over several weeks, it may interact with environmental factors and cause annual bluegrass to decline rapidly, leaving voids in the putting surface (Petrovsky 2019;Venner et al. 2012).
When voids occur on putting greens, golf course superintendents strive to quickly repair the area through various fertility programs, seeding, and sodding (Jones and Christians 2012;Rossi and Grant 2009;Walker et al. 2003).Most commonly, superintendents use soluble nitrogen fertilizers that are ammonia-or nitrate-based.Another common practice of superintendents is to add micronutrient supplements to fertility programs to improve color and overall quality.Biostimulants are a group of products that are derived from natural sources such as seaweed, humic acids, and other organic sources (Kahn et al. 2009;Schmidt et al. 2003).These products contain not just nitrogen, phosphorus, and potassium, but also a vast array of compounds such as auxins, cytokinins, and gibberellins-hormones that are important for mediating plant growth (Xu and Huang 2010;Zhang et al. 2003Zhang et al. , 2013;;Zhang and Ervin 2004).
Biostimulants have been shown to increase the percentage of creeping bentgrass cover that is nearly equivalent to commercial fertility programs following aeration (Bigelow et al. 2010).When applied to creeping bentgrass plants under summer stress, Xu and Huang (2010) found that plots treated with biostimulant products had between 22% and 100% greater turfgrass quality than nontreated checks, and plots sprayed with the plant growth regulator trinexapac-ethyl (TE).Biostimulants and TE maintained up to 76% and 56% more chlorophyll content, respectively, in leaves compared to leaves in nontreated turf (Xu and Huang 2010).Additionally, many biostimulants can improve overall turfgrass vigor, rooting, and shoot growth (Acuña et al. 2022;Xu and Huang 2010;Zhang and Ervin 2008).
It is well established that root-inhibiting herbicides can reduce the speed at which turfgrasses can spread or recover from injury or disease.Beck et al. (2013) found that recovery time of bermudagrass (Cynodon dactylon L. Pers.) from spring dead spot (Ophiosphaerella spp.) was lengthened when treated with rootinhibiting herbicides compared to a nontreated check.The use of root-inhibiting preemergence herbicides at sprigging increased the time required for bermudagrass establishment in sod production (Brosnan et al. 2014).To date, no peer-reviewed literature exists investigating the options for putting green recovery following rapid annual bluegrass removal with methiozolin.The objectives of this research were to 1) determine the influence of increased fertility programs from SFs alone or with TE or from biostimulants on speed of putting green recovery following rapid annual bluegrass removal with methiozolin or on creeping bentgrass removed via core cultivation, and 2) to determine whether methiozolin interacts with various fertility programs to influence the speed of creeping bentgrass recovery on golf greens following core cultivation.

Materials and Methods
Two separate studies were conducted at various locations in Virginia to assess fertility programs in conjunction with methiozolin use on golf greens.In one study, voids were created in the putting surface via rapid removal of annual bluegrass using methiozolin.This study will be referred to as the annual bluegrass removal (ABR) study.In the second study, voids in the putting green canopy were created using a core-cultivator (Greens Aerator; The Toro Company, Bloomington, MN) to remove approximately 30% of a pure creeping bentgrass canopy.This study will be referred to as the core-cultivation (CC) study.

Annual Bluegrass Removal Study
The ABR study was conducted at the Virginia Tech Golf Course in Blacksburg, VA (37.23°N, 80.44°W), at three sites between 2013 and 2014 on two separate greens, both constructed in the 1950s and originally seeded with 'Pennlu' and 'C-19 Congressional' creeping bentgrass.Studies were initiated at two sites (ABR1 and ABR2) on March 22, 2013, and one site (ABR3) on March 27, 2014.All greens were maintained at 4 mm mowing height.The purpose of the ABR study was to create voids in the putting green canopy via rapid control of annual bluegrass to allow assessment of creeping bentgrass recovery rate as influenced by fertility, biostimulant, and plant growth regulator (PGR) programs.Initial annual bluegrass cover averaged 48%, 47%, and 65% at ABR1, ABR2, and ABR3, respectively, with standard deviations of 5% to 7% depending on location.Rapid removal of annual bluegrass was achieved by treating the entire study area with methiozolin applied at 3,000, 500, and 500 g ai ha −1 on a biweekly interval at initiation.This methiozolin program completely controlled annual bluegrass at all sites within 45 d, with no apparent injury to creeping bentgrass.
In order to assess creeping bentgrass recovery as influenced by fertility programs, a randomized complete block design study was initiated at each of the three sites.Four treatments were replicated three times at each site.A more detailed description of fertility programs is provided in Table 1, but generally, treatments can be described as 1) increased fertility using SF, 2) increased fertility via SF plus TE applied every 200 growing degree days with a base of 0 C (GDD 0 ), 3) increased fertility via biostimulants, and 4) no increased fertility treatment.
"Increased fertility" treatments are additional treatments added to a superintendent's standard fertility program on each green (Table 1).Six Virginia golf course superintendents, representing a range of golf course designs and budgets and one company sales representative, were consulted to determine an appropriate amount of macronutrient increase assuming a 40% loss of putting green turf cover.Based on these discussions, the macronutrients increase in each treatment, regardless of source, was biweekly treatments of 7 kg N ha −1 , 7 kg P ha −1 , and 7 kg K ha −1 .These treatments were repeated until complete turf recovery and included five treatments at ABR1 and ABR2 and four treatments at ABR3.Increased fertility treatments were initiated at the first sign of canopy voiding, which occurred on April 14, 2013, at ABR1 and ABR2; and May 2, 2014, at ABR3.Digital images were collected with an 8.0-megapixel Canon Digital Rebel XT camera (Canon U.S.A., Inc., Melville, NY) at the following settings: F16, ISO100, white balance set to fluorescent, and 2-s shutter speed.The camera was mounted to a wooden 68-cm × 51-cm × 68-cm box that functioned to completely exclude natural light.The interior of the box was equipped with four 13-watt compact fluorescent light bulbs to ensure image uniformity.Images were analyzed for percent green pixels using SigmaScan Pro 5.2 software (Systat Software, Inc., San Jose, CA), which optimizes green color detection (Karcher and Richardson 2003).Normalized difference vegetation index (NDVI) was assessed using a multispectral analyzer (GeoScout GLS-400; Holland Scientific, Inc., Lincoln, NE) on each rating date for the duration of the study.
To standardize the influence of varying annual bluegrass population levels, percentage cover of green pixels was converted to a percentage recovery of green turf using Equation 1: where Y is the percentage recovery of voided turf on a given date, P i is the observed percentage green pixels in plot i on a given date, and C i is the observed percentage of green pixels in plot i on the initial observation date.To control for variance structure in measurements taken over time, percentage recovery data from each plot were subjected to the hyperbolic function using the NLIN procedures with SAS software (version 9.2; SAS, Cary, NC) with Equation 2: where Y is the percentage recovery of voided turf, X is time in weeks, i is an estimated parameter that approximates the rate of recovery as time approaches zero, and a is an estimated parameter that approximates the upper asymptote of percentage turf recovery.Using the estimated parameters, the hyperbolic function was used to determine the time required in weeks to reach 75% (T 75 ) and 90% (T 90 ) recovery.These T 75 and T 90 values and NDVI data were determined to be normal using the NORMAL option in the UNIVARIATE procedure (with SAS software) and Shapiro-Wilk statistic, and homogeneity was assessed by visually inspecting plotted residuals.Data were subjected to ANOVA using the GLM procedure with sums of squares partitioned to reflect the effect of cultural treatments and trial sites, which were considered random.Mean squares associated with trial interactions were used to test for significance of treatment effects (McIntosh 1983) In order to assess creeping bentgrass recovery as influenced by methiozolin and cultural treatment programs, a split-plot study was initiated at each site, designated CC1 and CC2.Treatments were replicated three times at each site.Main plots consisted of fertility treatments, and subplots consisted of two rates of methiozolin.Subplots contained either no methiozolin or methiozolin applied at 500 g ai ha −1 six times at 2-wk intervals beginning on March 23, 2013, andMarch 27, 2014.Fertility programs were the same as outlined in the ABR Study, and can be found in Table 1.Superintendent-administered fertility programs applied to the entire green can be found in Table 2.
Fertility treatments were repeated at 2-wk intervals beginning immediately prior to core-cultivation on April 14, 2013, and May 2, 2014, and lasted until turfgrass was completely recovered and included six treatments at CC1 and seven treatments at CC2.Digital images, NDVI, and cover ratings were taken as outlined in the ABR Study.However, the spectrum analyzer used at the CC1 and CC2 locations had a calibration error that compromised data integrity.Thus, NDVI from CC1 and CC2 will not be presented.Statistical analyses were also performed as outlined previously, with the exception that sums of squares were further partitioned to evaluate effects of main plots, subplots, and interactions of these with each other and with trial.

Annual Bluegrass Removal Study
The interaction of location by treatment was significant for T 75 (P < 0.0001) and T 90 (P < 0.0001); therefore, data are presented separately by location (Table 3).Differences between locations were likely due to inconsistencies in the performance of both biostimulant and SF plus TE.For example, the biostimulant treatment had the fastest T 75 in ABR2 and ABR3 but was equivalent to that of SF in ABR1 (Table 3).The T 90 of SF plus TE was equivalent to that of SF alone at ABR1 and ABR2 but required more time at ABR3 (Table 3).Additionally, plots that received biostimulants were damaged due to mower scalping at ABR3 and had lower NDVI than plots receiving no additional fertility.This damage likely created the location interaction at one of the rating dates (Table 4).Despite these differences, some consistencies in responses between locations were noted.For example, biostimulant and SF treatments reduced 75% and 90% recovery times compared to the no-additional-fertility check at all locations (Table 3).
The T 75 times ranged from 2.5 to 7.3 wk depending on treatment and location (Table 3).In general, T 75 required more time in 2013 than in 2014.Biostimulant treatments required significantly less time than all other treatments at two of three locations.Synthetic fertilizer alone was equivalent to that of SF plus TE at all locations except ABR3 where the two differed in T 75 recovery time by about 3 d (Table 3).In 2013, all additional fertility treatments reduced T 75 recovery time by at least 2.4 wk at ABR1 and 1.8 wk at ABR2 (Table 3).The biostimulant program reduced T 75 recovery time by 2.5 to 2.8 wk at these two locations compared to the noadditional-fertility program ( When plots reached T 90 , actual putting green canopy coverage was approximately 96% and was commercially acceptable (>95% coverage; data not shown).Some differences were noted between trends in T 90 response times and those of T 75 .For example, biostimulant and SF programs were equivalent for T 90 times and generally averaged about 6 to 6.5 wk in 2013 and 4.5 wk in 2014 (Table 4).These two treatments reduced the time required for 90% recovery by more than 2 wk compared to the no-additional-fertility check at two sites in 2013 and about 1 wk at one site in 2014 (Table 4).The addition of TE to SF did not increase recovery time at two of three locations.Trinexapac-ethyl reduces cellular elongation and may increase the time required to sufficiently mask voids in turfgrass canopies due to reduced vertical plant growth (Ervin and Zhang 2007;Rademacher 2000).In low heights of cut like those on a putting green, vertical growth rate is of minimal benefit in masking canopy voids, and TE has been shown to have minimal impact on lateral recovery in some studies due to increased levels of cytokinin production leading to increased tiller production after several sequential applications (Ervin et al. 2002;Ervin and Zhang 2007).Variability in recovery time between sites could be partially attributed to turfgrass cultivar as has been demonstrated in previous studies (Jones and Christians 2009;Karcher et al. 2005).
At ABR1 and ABR2, plots that received no additional fertility had lower NDVI than all plots that received additional fertility at 6 wk after initial treatment (WAIT) and 8 WAIT, respectively (Table 4).

Core-Cultivation Study
The interaction of location by methiozolin by treatment was significant for T 75 (P < 0.0001) and T 90 (P < 0.0001).Therefore, data are presented separately by methiozolin treatment and location.The interaction between CC1 and CC2 could be attributed to a mid-season irrigation malfunction on the CC2 putting green, which lasted for approximately 4 wk.Two out of four irrigation   a Abbreviations: ABR1, annual bluegrass removal study location 1; ABR2, annual bluegrass removal study location 2; ABR3, annual bluegrass removal study location 3; SF, synthetic fertilizer; T 75 , time required in weeks to recover 75% of putting green turf voided by annual bluegrass removal by methiozolin; T 90 , time in weeks to recover 90% of voided turf.b Fertility programs were added to standard greens management programs and consisted of additional nitrogen, phosphorus, and potassium applied at 7, 7, and 7 kg ha −1 every 2 wk starting when canopy voids first appeared on the putting green.Trinexapac-ethyl was applied at 0.048 kg ai ha −1 and repeated at 200 growing degree days at base 0 C.More information about products applied appears in Table 1.c Means within a column followed by the same letter are not significantly different (P ≤ 0.05).
188 Venner et al.: Golf green recovery heads surrounding the putting green failed, and were not immediately noticed, as the green was aggressively core cultivated and topdressed to meet study objectives.It was not until heat stress became evident that the issue was discovered and corrected.As a result of this severe stress, turf quality in most plots fell to below 3 on a 1-to-9 scale where 6 is considered minimally acceptable (data not shown).Methiozolin-treated plots recovered more slowly than those not treated with methiozolin at CC2, a phenomenon that did not occur at CC1 (Table 5).Despite issues with irrigation, trends in 75% and 90% recovery as influenced by fertility treatments were generally consistent across locations (Table 5).
Recovery times for T 75 ranged from 2.2 to 4.7 wk depending on treatment, location, and methiozolin rate (Table 5).All fertility and PGR treatments recovered more quickly than treatments that received no additional fertility at all sites.When methiozolin was applied, 75% creeping bentgrass recovery required an additional 1.5 wk on average at CC2 but no additional time at CC1 (Table 5).Due to the general immobility of methiozolin in soil, substantial root loss is likely required to elicit creeping bentgrass injury from methiozolin in most cases (Brosnan et al. 2013;Flessner et al. 2015).Murphy et al. (1993) observed a 16% decrease in root weight density following cultivation, most likely due to root severing and trauma to the soil.Furthermore, when soil has reduced moisture, roots are more susceptible to injury than when soil is wet (Murphy et al. 1993).At CC1 where drought stress did not occur, methiozolin had no impact on creeping bentgrass recovery rate (Tables 5 and 6).The combined effects of core cultivation and drought at CC2 may have caused temporary root loss, and methiozolin may have prevented new root development.
Creeping bentgrass treated with SF reached T 75 at the same time as creeping bentgrass treated with biostimulants at both locations regardless of methiozolin use (Table 5).Trinexapac-ethyl influence on creeping bentgrass T 75 recovery time was independent of methiozolin use but dependent on location.At CC1, TE plus SF improved creeping bentgrass T 75 recovery time equivalent to the best performing treatments (Table 5).At CC2, where the drought injury occurred, TE programs were inferior to SF in the absence of methiozolin and both SF and biostimulant in the presence of methiozolin (Table 5).The location dependency of TE programs could be attributed to the increased levels of plant hormones contained in biostimulant products and growth regulation imparted by the TE application.Although commonly reported in warm-season turfgrass species like bermudagrass, increases in root growth as a direct result of TE application are inconsistent (Beasely et al. 2005;McCann and Huang 2007;Wherley and Sinclair 2009).Wherley and Sinclair (2009) and Beasley et al. (2005) did not observe changes in creeping bentgrass or Kentucky bluegrass root growth following applications of TE but postulated that rooting increases are more likely directly related to nitrogen application.
Although recovery trends were evident at T 90 and canopy coverage had reached approximately 95%, differences between sites became more evident, particularly at CC2 with methiozolin (Table 6).All fertility treatments recovered between 1.3 and 3.3 wk more rapidly than the no-additional-fertilizer treatment.When biweekly treatments of methiozolin were applied, biostimulant and SF treatments improved T 90 recovery time by approximately 0.5 wk compared to SF plus TE (Table 6).At CC2 where the drought stress occurred, biostimulant treated plots achieved 90% recovery 0.7 wk faster than SF and 0.4 wk faster than SF plus TE (Table 6).When methiozolin was not applied, plots treated with    Abbreviations: CC1, core-cultivation site 1; CC2, core-cultivation site 2; SF, synthetic fertilizer; T 90 , time required in weeks to recover 90% of putting green turf that was subject to 30% canopy removal via core-cultivation.
b Fertility programs were added to standard greens management programs and consisted of additional nitrogen, phosphorus, and potassium applied at 7, 7, and 7 kg ha −1 every 2 wk starting when canopy voids first appeared on the putting green.Trinexapac-ethyl was applied at 0.048 kg ai ha −1 and repeated at 200 growing degree days at base 0 C.More information about products applied appears in Table 1.
c Means within a column, followed by the same letter are not significantly different (P ≤ 0.05).
d An asterisk (*) following any mean indicates that methiozolin significantly impacted recovery time (P ≤ 0.05) within a given fertility program and location.

Weed Technology 189
biostimulant and SF reached 90% recovery at equivalent times but 1 to 3 wk faster than plots that did not receive additional fertility (Table 6).In all cases, increasing fertility reduced turfgrass recovery time relative to the standard program.These data suggest that turfgrass managers can improve recovery time of creeping bentgrass greens by 1 to 3 wk by adding 7 kg ha −1 of N-P-K to existing greens fertility programs.Methiozolin reduced turfgrass recovery time at one location where a severe drought occurred.These data suggest that turfgrass stress should be avoided when methiozolin programs are used for annual bluegrass control.Creeping bentgrass treated with biostimulants recovered equivalent to or faster than SF in all cases.In the presence of methiozolin treatments, TE reduced T 90 recovery time by 0.25 to 0.5 wk at two locations and increased T 90 recovery time by 0.1 wk at one location.Otherwise, plots treated with SF plus TE were equivalent to SF-only plots.Further research needs to be conducted to better explain how methiozolin may affect root recovery following stress-induced root loss.

Practical Implications
Since its recent registration, methiozolin has been used extensively to control annual bluegrass (Poa annua) in creeping bentgrass (Agrostis stolonifera) putting greens by golf course superintendents.Although treatment programs are designed to control annual bluegrass slowly to avoid compromising putting quality, environmental conditions, or overapplication of methiozolin can hasten the speed of control.Voided putting surfaces must be rapidly repaired or rejuvenated.Our work indicates that methiozolin does not slow the lateral spread of creeping bentgrass as measured by canopy cover, with the exception of one location where dry conditions prevailed.Additionally, our work demonstrates that biostimulant and synthetic fertilizer applications speed expansion of creeping bentgrass to cover canopy voids on greens.Furthermore, turf managers can continue or initiate plant growth regulator treatments using TE during the void recovery phase with little impact to time required for complete canopy recovery.This research contributed to language on the methiozolin label prohibiting intensive mechanical disturbance, such as core aerification, and allowing use of TE in conjunction with methiozolin treatments.

Table 1 .
Products used in annual bluegrass removal and core-cultivation studies.a,b

Table 3 )
. Although macronutrient concentrations were equivalent between biostimulant and SF programs, the biostimulants likely provided plants with various hormones and micronutrients that elicited immediate growth responses as opposed to the SF program, which provided necessary nutrients but required time to produce such hormones.The (Bigelow et al. 2010;Tucker et al. 2006ishedonline by Cambridge University Press reducing overall plant senescence.Increased fertility imparted by biostimulant products could speed turfgrass recovery by increasing turfgrass rooting, thereby allowing more shoots to be produced and increasing cover more rapidly(Bigelow et al. 2010;Tucker et al. 2006).

Table 2 .
Superintendent-administered fertility programs for both annual bluegrass removal and core-cultivation studies during the duration of each study.a a Abbreviations: ABR1, annual bluegrass removal study location 1; ABR2, annual bluegrass removal study location 2; ABR3, annual bluegrass removal study location 3; CC1, core-cultivation study location 1; CC2, core-cultivation study location 2.

Table 3 .
Time required in weeks to reach 75% and 90% recovery of voided putting green turf following rapid removal of annual bluegrass with methiozolin and as influenced by various fertility and plant growth regulator programs at locations ABR1, ABR2, and ABR3.a,c

Table 4 .
Influence of fertility and plant growth regulator program following rapid annual bluegrass removal on putting green normalized difference vegetative index averaged over three locations at 1 and 6 WAIT and separated by location at 8 WAIT.a,cFertility programs were added to standard greens management programs and consisted of additional nitrogen, phosphorus, and potassium applied at 7, 7, and 7 kg ha −1 every 2 wk starting when canopy voids first appeared on the putting green.Trinexapac-ethyl was applied at 0.048 kg ai ha −1 and repeated at 200 growing degree days at base 0 C.More information about products applied appears in Table1.
b c Means within a column followed by the same letter are not significantly different (P ≤ 0.05).

Table 5 .
Time required to reach 75% recovery of voided putting green turf following core-cultivation as influenced by methiozolin and various fertility and plant growth regulator programs at CC1 and CC2 sites.a,c,dFertilityprogramswereadded to standard greens management programs and consisted of additional nitrogen, phosphorus, and potassium applied at 7, 7, and 7 kg ha −1 every 2 wk starting when canopy voids first appeared on the putting green.Trinexapac-ethyl was applied at 0.048 kg ai ha −1 and repeated at 200 growing degree days at base 0 C.More information about products applied appears in Table1.Means within a column followed by the same letter are not significantly different (P ≤ 0.05).
b c

Table 6 .
Time required to reach 90% recovery of voided putting green turf following core-cultivation as influenced by methiozolin and various fertility and plant growth regulator programs at CC1 and CC2 sites.