Sitosterol-mediated Antioxidant Regulation to Enhance Heat Tolerance in Creeping Bentgrass

Heat stress symptoms in cool-season plants are characterized by loss of chlorophyll (Chl) and membrane stability, as well as oxidative damage. The objectives of this study were to determine whether foliar application of β-sitosterol, a naturally occurring plant metabolite, may promote heat tolerance by suppressing heat-induced leaf senescence as indicated by the maintenance of healthy turf quality (TQ), and Chl and membrane stability; and to determine its roles in regulating antioxidant metabolism in creeping bentgrass (Agrostis stolonifera). ‘Penncross’ plants were exposed to heat stress (35/30 °C day/night) optimal temperature conditions (nonstressed control, 22/17 °C day/night) for a duration of 28 days in environment-controlled growth chambers. Plants were foliar-treated with β-sitosterol (400 µM) or water only (untreated control) before heat stress, and at 7-day intervals through 28 days of heat stress. Plants treated with β-sitosterol had significantly greater TQ and Chl content, and significantly less electrolyte leakage (EL) than untreated controls at 21 and 28 days of heat stress. Application of β-sitosterol reduced malondialdehyde (MDA) content significantly at 21 and 28 days of heat stress, and promoted the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) from 14 through 28 days of heat stress. β-Sitosterol effectively improved heat tolerance through suppression of leaf senescence in creeping bentgrass exposed to heat stress in association with the alleviation of membrane lipid peroxidation and activation of the enzymatic antioxidant system.

Field Quantification of Ammonia Emission following Fertilization of Golf Course Turfgrass in Sub/Urban Areas

Low cost and favorable handling characteristics make urea (46-0-0) a leading nitrogen source for frequent, foliar N fertilization of golf course putting greens in season. Yet few field investigations of resulting NH3 volatilization from putting greens have been directed. Meanwhile, NH3 emissions degrade air and surface water quality. Our objective was to quantify NH3 volatilization following practical, low-N rate, and foliar application of commercial urea-N fertilizers. Over the 2019 and 2020 growing seasons in University Park, PA, USA, an industrial vacuum pump, H3BO3 scrubbing flasks, and sixteen dynamic flux chambers were employed in four unique experiments to measure NH3 volatilization from creeping bentgrass putting greens (Agrostis stolonifera L. ‘Penn G2’) in the 24 h period ensuing foliar application of urea based-N at a 7.32 or 9.76 kg/ha rate. Simultaneous and replicated flux chamber trapping efficiency trials showing 35% mean NH3 recovery were used to adjust NH3 volatilization rates from treated plots. Under the duration and conditions described, 3.1 to 8.0% of conventional urea N volatilized from the putting greens as NH3. Conversely, 0.7 to 1.1% of methylol urea liquid fertilizer (60% short-chain methylene urea) or 0.7 to 2.2% of urea complimented with dicyandiamide (DCD) and N-(n-butyl) thiophosphoric triamide (NBPT) volatilized as NH3.

Adaptability to High Temperature and Stay-Green Genotypes Associated With Variations in Antioxidant, Chlorophyll Metabolism, and γ-Aminobutyric Acid Accumulation in Creeping Bentgrass Species

High temperature limits the cultivation and utilization of cool-season plants in many regions worldwide. Recently, extreme hot waves swept across the globe in summer, leading to enormous economic loss. The evaluation and identification of genotypic variation in thermotolerance within species are critical to breeding for environmental adaptation and also provide potential materials to explore thermo-resistant mechanism in plants. Forty-two accessions of creeping bentgrass (Agrostis stolonifera), which is a cool-season perennial grass for turf and ecological remediation, were collected from 15 different countries. Physiological traits, namely, chlorophyll (Chl) content, electrolyte leakage, photochemical efficiency, performance index on absorption basis, leaf relative water content, and osmotic potential were used to evaluate the heat tolerance of these materials in controlled growth chambers and field during summer. Stay-green and early-aging genotypes were selected to further reveal the potential mechanism of tolerance to senescence and heat damage associated with alterations in Chl metabolism, antioxidant and photosynthetic capacity, and endogenous γ-aminobutyric acid (GABA). Findings showed that there were significant genetic variations in physiological traits among 41 materials in response to high temperature stress. The 13M, PROVIDENCE, and LOFTS L-93 were the top three accessions with superior tolerance to heat and summer stress than other materials in terms of laboratory and field tests. In response to heat stress, the stay-green genotype PROVIDENCE exhibited significantly higher photochemical efficiency, net photosynthetic rate, transpiration rate, and water use efficiency than the heat-susceptible W6 6570. Delayed leaf senescence in relation to less Chl loss was detected in the PROVIDENCE associated with maintenance of significantly higher expression levels of Chl-anabolic genes (AsCHLH, AsPBGD, and AsPOR) and lower Chl-catabolic gene AsPPH under heat stress. Genetic attributes, such as better capacity to scavenge reactive oxygen species and higher endogenous GABA content could play positive roles in alleviating heat-induced senescence, oxidative damage, and metabolic disturbance in the PROVIDENCE.

First Report of Summer Patch of Creeping Bentgrass Caused by Magnaporthiopsis poae in Southeastern China

Creeping bentgrass (Agrostis stolonifera L.) is an important cool-season perennial turfgrass that has been widely used on golf courses across China. In July 2017, an unknown disease outbreak caused damages on seven of the 18 putting greens of creeping bentgrass at Jiuqiao golf club in Hangzhou city of Zhejiang province, day-time high temperatures were consistently above 35°C during the disease development. Symptoms appeared in tan irregular patches of 5 to 20-cm diameter, exhibiting chlorosis and foliar dieback in most part. Necrotic roots were frequently observed in diseased areas and colonized with ectotrophic hyphae under a microscope. Similar symptoms and signs were reported on creeping bentgrass caused by Magnaporthiopsis poae (Landschoot & Jackson) J. Luo & N. Zhang on golf courses in Beijing (Hu et al. 2017). Fifteen disease samples were collected from seven putting greens. Dark root tips were cut, surface sterilized in 0.6% sodium hypochlorite (NaClO) for 5 min, washed twice with sterilized water, air dried for 1 min and placed on potato dextrose agar (PDA) containing each of 50 mg L-1 ampicillin, streptomycin sulfate, and tetracycline. Plates were incubated in the dark at room temperature for 4 days, and 10 fungal isolates with similar morphology as described by Clarke and Gould (1993) were consistently recovered from the diseased root tips. DNA of two representative isolates was extracted and amplified with primers ITS 5/ITS 4 (White et al. 1990). PCR products were sequenced (deposited in GenBank as MZ895215 and MZ895216), and BLAST analysis showed 99.17% similarity to M. poae (accession number: DQ528765). Six plastic pots (15 cm height × 15 cm top diameter × 10 cm bottom diameter, three replicates for each isolate) were seeded with creeping bentgrass and placed in the greenhouse for two months of plant growth before inoculation. The pathogenic inoculum was prepared by inoculating autoclaved oat seeds with M. poae isolates, followed by two weeks of incubation at 25°C. About 25 mg M. poae-infested oat seeds were placed 10 cm below the soil surface in the root zone of creeping bentgrass. Non-infested oat seeds were inoculated on healthy creeping bentgrass as controls. Pots were placed in a growth chamber with a 12-h day/night cycle at 35/28°C and watered daily to keep high soil moisture. Disease symptoms (foliar dieback and necrotic roots) were noted 3 weeks after inoculation. M. poae was consistently recovered from the roots of inoculated turf and identified molecularly as described above, fulfilling Koch’s postulates. To our knowledge, this is the first report of summer patch on creeping bentgrass caused by M. poae in southeastern China. This research demonstrates a wider distribution of M. poae and will be an important step towards the development of management strategies for summer patch control in China.

Inhibition of oomycetes by the mixture of maleic acid and copper sulfate

After the discovery of the protective activity of Bordeaux mixture against plant disease caused by oomycetes, copper compounds have been used for over a century as a significant plant protection strategy. However, application of excessive copper can cause adverse effects through long-term heavy metal accumulation in soils. Therefore, it is necessary to develop new strategies to reduce or replace copper in pesticides based on organic and low-input farming systems. Organic acids are eco-friendly in nature. In this study, we tested the antifungal and anti-oomycete activity of maleic acid (MA) and copper sulfate (CS) against thirteen plant pathogens. Treatment of MA and CS mixture showed strong anti-oomycetes activity against Phytophthora cambivora, P. capsici and P. cinamomi. Moreover, the concentration of CS in the activated mixture of MA and CS was lower than that in the activated CS only, and the mixture showed synergy or partial synergy effects on the anti-oomycete activity. Application of a wettable powder formulation of MA and CS mixture (MCS 30WP; 26.67 % MA and 3.33 % CS) exhibited excellent protective activities in pot experiments with control values of 73 % Phytophthora blight on red pepper, 91 % damping-off on cucumber, and 84 % Pythium blight on creeping bentgrass, which are similar to those of the CS wettable powder formulation (6.67 % CS) containing two times the CS content in MCS 30WP. These observations suggest that the synergistic effect of the MA and CS combination is a sustainable alternative for effective management the destructive oomycete diseases.

Occurrence of the Lance Nematode Hoplolaimus stephanus Infecting Bentgrass Agrostis stolonifera in Georgia, USA

A high population of lance nematodes Hoplolaimus spp. were found associated with creeping bentgrass (Agrostis stolonifera L.) in May 2019 in Georgia, USA. The nematode was pathogenic to bentgrass as its population increased by over 3-fold 180 days after inoculation under greenhouse conditions. Morphological measurements of body and stylet lengths of both mature females and males were similar to a grass population of H. stephanus from South Carolina. DNA sequence analyses of the D1-D3 expansion segments of the 28s gene identified the nematode as H. stephanus. The DNA sequence of the nematode was 99.7% identical to a H. stephanus isolate from South Carolina. Also, the PCR method using a species-specific primer set confirmed the identity of H. stephanus. To the best of our knowledge, this is the first report of H. stephanus Sher, 1963, infecting creeping bentgrass in Georgia.

Investigating Chemical and Biological Control Applications for Pythium Root Rot Prevention and Impacts on Creeping Bentgrass Putting Green Rhizosphere Bacterial Communities

Pythium root rot (PRR) is a disease that can rapidly devastate large swaths of golf course putting greens, with little recourse once symptoms appear. Golf courses routinely apply preventative fungicides for root diseases, which may be altering the rhizosphere microbiome leading to unintended impacts to plant health. A multi-year field trial was initiated on a ‘T-1’ creeping bentgrass (Agrostis stolonifera L. cv. ‘T-1’) putting green in College Park, Maryland to evaluate preventative PRR management for disease suppression and impacts to rhizosphere bacterial communities. Fungicides commonly used to prevent PRR and a biological fungicide were repeatedly applied to experimental plots throughout the growing season. Rhizosphere samples were collected twice annually from each plot to evaluate rhizosphere bacterial communities through amplicon sequencing and monitor biological control organism populations via qPCR. Cyazofamid was the only treatment to suppress PRR in both years compared to the control. Fosetyl-Al on a 14 d interval and Bacillus subtilis QST713 also reduced PRR severity in 2019 compared to the non-treated control. Treatments did not significantly alter bacterial communities, however seasonal environmental changes did. Repeated rhizosphere targeted applications of B. subtilis QST713 appear to have established the bacterium into the rhizosphere, as populations increased between samples, even after applications stopped. These findings suggest that QST713 may reduce pathogen pressure when repeatedly applied and can reduce fungicide usage during periods of low PRR pressure.

Transfer of resistance alleles from herbicide-resistant to susceptible grass weeds via pollen-mediated gene flow

The objective of this paper was to review the reproductive biology, herbicide-resistant (HR) biotypes, pollen-mediated gene flow (PMGF), and potential for transfer of alleles from HR to susceptible grass weeds including barnyardgrass, creeping bentgrass, Italian ryegrass, johnsongrass, rigid (annual) ryegrass, and wild oats. The widespread occurrence of HR grass weeds is at least partly due to PMGF, particularly in obligate outcrossing species such as rigid ryegrass. Creeping bentgrass, a wind-pollinated turfgrass species, can efficiently disseminate herbicide resistance alleles via PMGF and movement of seeds and stolons. The genus Agrostis contains about 200 species, many of which are sexually compatible and produce naturally occurring hybrids as well as producing hybrids with species in the genus Polypogon. The self-incompatibility, extremely high outcrossing rate, and wind pollination in Italian ryegrass clearly point to PMGF as a major mechanism by which herbicide resistance alleles can spread across agricultural landscapes, resulting in abundant genetic variation within populations and low genetic differentiation among populations. Italian ryegrass can readily hybridize with perennial ryegrass and rigid ryegrass due to their similarity in chromosome numbers (2n=14), resulting in interspecific gene exchange. Johnsongrass, barnyardgrass, and wild oats are self-pollinated species, so the potential for PMGF is relatively low and limited to short distances; however, seeds can easily shatter upon maturity before crop harvest, leading to wider dispersal. The occurrence of PMGF in reviewed grass weed species, even at a low rate is greater than that of spontaneous mutations conferring herbicide resistance in weeds and thus can contribute to the spread of herbicide resistance alleles. This review indicates that the transfer of herbicide resistance alleles occurs under field conditions at varying levels depending on the grass weed species.

Improving Soil Moisture Assessment of Turfgrass Systems Utilizing Field Radiometry

The need for water conservation continues to increase as global freshwater resources dwindle. Turfgrass mangers are adapting to these concerns by implementing new tools to reduce water consumption. Time-domain reflectometer (TDR) soil moisture sensors can decrease water usage when scheduling irrigation, but nonuniformity across unsampled locations creates irrigation inefficiencies. Remote sensing data have been used to estimate soil moisture stress in turfgrass systems through the normalized difference vegetation index (NDVI). However, numerous stressors other than moisture constraints impact NDVI values. The water band index (WBI) is an alternative index that uses narrowband, near-infrared light reflectance to estimate moisture limitations within the plant canopy. The green-to-red ratio index (GRI) is a vegetation index that has been proposed as a cheaper alternative to WBI as it can be measured using digital values of visible light instead of relying on more costly hyperspectral reflectance measurements. A replicated 2 × 3 factorial experimental design was used to repeatedly measure turf canopy reflectance and soil moisture over time as soils dried. Pots of ‘007’ creeping bentgrass (CBG) and ‘Latitude 36’ hybrid bermudagrass (HBG) were grown on three soil textures: United States Golf Association (USGA) 90:10 sand, loam, and clay. Reflectance data were collected hourly between 07:00 and 19:00 using a hyperspectral radiometer and volumetric water content (VWC) data were collected continuously using an embedded soil moisture sensor from soil saturation until complete turf necrosis by drought stress. The WBI had the strongest relationship to VWC (r = 0.62) compared to GRI (r = 0.56) and NDVI (r = 0.47). The WBI and GRI identified significant moisture stress approximately 28 h earlier than NDVI (p = 0.0010). Those metrics also predicted moisture stress prior to fifty percent visual estimation of wilt (p = 0.0317), with lead times of 12 h (WBI) and 9 h (GRI). By contrast, NDVI provided 2 h of prediction time. Nonlinear regression analysis showed that WBI and GRI can be useful for predicting moisture stress of CBG and HBG grown on three different soil textures in a controlled environment.

Detection of Pythium spp. in golf course irrigation systems

Many Pythium spp. are causal agents of diseases of turfgrasses. Pythium spp. disseminate through irrigation systems in agricultural settings, and this study provides evidence that Pythium spp. also disseminate through golf course irrigation. Water samples were collected from irrigation heads and water sources at ten golf courses in Missouri and Kansas. Samples were collected from 2018 to 2019 in April, July, and October. Phosphorus, nitrogen, and chloride concentrations were measured from irrigation head samples to determine if these parameters influence frequency of Pythium spp. detected. Pythium spp. were detected in samples through baiting and membrane filtration. Cultures were isolated on PARP media and DNA was extracted from putative Pythium isolates. The ITS region was PCR amplified and sequenced. Phylogenetic trees were constructed using representative sample sequences, sequences from seven morphologically identified reference isolates of Pythium, and similar Genbank accessions. Detected Oomycete species include Lagenidium giganteum, Pythium biforme, P. insidiosum, P. marsipium, P. plurisporium, and Saprolegnia hypogyna. Twenty-one clades lacked species-level resolution, and fourteen of these clades were associated with Pythium species. Clades A, C, D, E, I, and M contain Pythium species that cause root and crown rot on creeping bentgrass. Detected Pythium communities were dependent on the detection method used and sampling source. Pythium frequency and diversity were highest in April 2019. Sample temperature, sampling site, chloride, and nutrient concentrations did not influence Pythium frequency in samples. Irrigation systems using surface water sources contained at least three Pythium spp. over the course of two years.