Final report for LNE20-407R
Project Information
Turfgrass sod production is an important component of our diverse agricultural landscape in the Northeast. In New Jersey, there are 28 sod producers growing approximately 8,960 acres of turf, with annual revenues of $46.4 million in sales and a total economic impact of $66.2 million. Sod growers are continuously looking for ways to improve both plant and soil health while minimizing inputs of water and nutrients to reduce costs and environmental pollution. Some bacteria co-exist within the roots of plants and in soil rhizospheres and promote plant growth and efficient acquisition of minerals and water while increasing carbon storage in the soil. These bacteria are collectively referred to as plant growth promoting rhizobacteria (PGPR). Our previous work had identified several novel strains of PGPR, BurkholderiaNJPB that are native to New Jersey and are effective in improving turfgrass growth under reduced fertility and water deficit conditions in greenhouse and growth chamber trials. However, optimizing this approach for field conditions required additional investigation.
The objectives of this project were:
- To evaluate which PGPR strains are most effective for improving drought tolerance in field applications and to determine effective methods of PGPR inoculation for cool season grasses under field conditions.
- To quantify the presence and density of bacteria in creeping bentgrass roots that have been inoculated with PGPR and to identify which amendments may help to improve PGPR inoculation.
- To test the optimized inoculants on working sod farms to improve drought stress tolerance and reduce water use in cool season grass production.
Creeping bentgrass plots were established in field soil at the Rutgers Horticultural Research Farm II during the 2020 and 2021 growing seasons. The plants were inoculated with two individual strains of PGPR separately or with a combination of the two strains using a foliar spray or soil drench method. The plants were then subjected to a period of drought stress (60% ET) and recovery upon re-watering. The soil drench method using a combination of the two strains was found to be most effective at improving drought tolerance and post-drought recovery, resulting in improved turf quality, NDVI, and canopy density.
To evaluate whether different amendments could be used to improve inoculation efficiency, an experiment was conducted in which biochar, zeolites, or humic acid were added to plants receiving PGPR treatments. The presence of bacteria in the roots of inoculated plants was confirmed with PCR analysis every two weeks and it was found that the addition of zeolites to the planting medium was most effective at enhancing bacteria colonization of the roots, and that the addition of humic acid to the liquid inoculant also improved efficacy.
Field trials were also conducted at two sod farms, where the combined PGPR strains were used to inoculated non-irrigated plots of Kentucky bluegrass/tall fescue sod in a 0.01% humic acid solution using the soil drench method. The inoculated plots demonstrated improved turf quality, canopy density and chlorophyll content during drought stress or post-drought recovery.
Three field tours were conducted at the research farm as part of Rutgers Turfgrass Field Days, with attendance by the NJ Cultivated Sod Association, researchers, and turfgrass industry professionals. The results were also shared through presentations at the annual meeting for the Crop Science Society of America.
1) To identify effective and efficient colonization methods of turfgrass with Burkholderia strains from New Jersey Pine Barrens natural ecosystems;
2) To determine effectiveness of Burkholderia strains for promoting turf performance, sod quality, and soil health for different turfgrass species under management regimes with reduced irrigation and fertility in the Northeast.
The information from this project will be applicable for turfgrass sod farms with a goal of reducing inputs for water and fertilizers.
Research
Experiment 1: Drought Stress Trials (2020, 2021)
Plant materials and growth conditions:
Creeping bentgrass (Agrostis stolonifera cv. Penncross) plots (1mx1.3m) were established in field soil at the Rutgers University Horticultral Research Farm and maintained at a height of 1.2 cm during the 2020 and 2021 growing seasons. Plants were inoculated twice before the start of drought stress and once again upon re-watering using a soil drench method. A rainout shelter was used to cover the plots to simulate drought stress.
Application timing and frequency:
Bacterial inoculant suspensions at the density of 1 x 105-107 cfu/mL (effective dose found in our growth chamber tests) mixed in humic acid (0.01%) was watered into field plots as soil drenching in sufficient volume to moisten the upper 2-inch root zone or as foliar spray. Inoculants were applied at three time points: 1) two weeks before deficit irrigation treatments were imposed 2) The day prior to starting deficit irrigation, and 3) at the time of re-watering following deficit irrigation. The control plants were treated with humic acid (0.01%) without bacteria inoculum in the same volume as bacteria inoculants and were compared to a commercially available inoculant (Quantum Growth). Individual strains and combinations of strains were evaluated.
Inoculation Treatments
Foliar Spray
- Humic Acid
- WSF23 + Humic Acid
- WSF14 + Humic Acid
- WSF23 + WSF14 + Humic Acid
- Quantum Growth (commercially available inoculant)
Soil Drench
- Humic Acid
- WSF23 + Humic Acid
- WSF14 + Humic Acid
- WSF23 + WSF14 + Humic Acid
- Quantum Growth (commercially available inoculant)
Deficit Irrigation Treatments:
- Well-Watered Control: Plants were irrigated(100% ET)
- Drought Stress: Deficit Irrigation (60% ET) for 49 days
- Recovery: Drought-stressed plants were re-watered for 28 days
Measurements:
- Turf Quality
- Soil water content using TDR
- MSR (NDVI, SI)
- Light Box (Canopy Density, DGCI)
- Thermal Images
- Rooting characteristics
- Bacteria colonization or viability
Visual turf quality (TQ) was measured at seven day intervals throughout the experimental period. Turf was rated on a scale of 1 to 9, with 1 being brown and desiccated turf, 6 being the minimal acceptable level, and 9 being green and dense turf. Turf quality ratings were based on uniformity, visual attractiveness, leaf color, and canopy density.
A TDR moisture probe was used to take weekly soil mositure measurements from each plot to monitor soil water content throughout the deficit irrigation period.
Weekly radiometry based objective assessment was conducted using a handheld multispectral radiometer (MSR model CT1000; Crop Scan Inc., Fargo, ND). The MSR measures canopy reflectance characteristics (510-1700 nm) which were used to calculate normalized difference vegetation index (NDVI) and stress index (SI) of the plants.
Light box digital images were collected weekly the experimental period. Image analysis with Sigma Scan software was used to evaluate canopy density (% green cover) and dark green color index (DGCI) throughout the growing season.
Canopy temperature is a good indicator of plant physiological health for stress tolerance, as it estimates transpirational cooling. Canopy temperature was measured using an infrared thermal camera (FLIR). Thermal images werer captured weekly during the trial, and imagery data will be processed using computer software.
Root samples were collected twice for analysis of bacterial inoculation efficiency. The presence of bacterial inoculants in plant tissues are currently determined by both bacterial isolation and quantitative real-time polymerase chain reaction (qPCR) analysis that was designed based on signature sequences of the 16S rDNA in BurkholderiaNJBP.
Root samples were also collected at the conclusion of the field season to be evaluated for morphological characteristics such as root length, volume, surface area, diameter, and total biomass.
Experiment 2: Seedling Establishment Trials (2021)
Kentucky bluegrass and creeping bentgrass were seeded in field plots at Rutgers Horticultural Research Farm II (New Brunswick, NJ) in September 2021. The plots were managed following typical golf fairway (creeping bentgrass) and athletic field/home lawn (Kentucky bluegrass) management regimens, including mowing fertilization, irrigation, and pest management. Plots were treated with a soil drench of WSF23+WSF14 in a 0.01% humic acid solution or with just the 0.01% humic acid solution as a control. Each treatment was replicated in four plots (3 x 4‘ each) arranged in a randomized complete block design. Plots were well irrigated prior to and during the treatments to allow effective bacterial inoculation.
Physiological Analysis
- Weekly radiometry based objective assessments were conducted using a handheld multispectral radiometer (MSR model CT1000; Crop Scan Inc., Fargo, ND). The MSR measures canopy reflectance characteristics (510-1700 nm) which were used to calculate normalized difference vegetation index (NDVI), stress index (SI), and leaf area index (LAI).
- Light box digital images were collected during the experimental period and analyzed using SigmaScan Pro 5 software to calculate canopy density (percent green cover) and dark green color index (DGCI).
Statistical Analysis
The effects of the inoculation treatments were determined by analysis of variance (ANOVA) according to the general linear model procedure of SAS (version 9.4; SAS institute, Cary, NC, USA). Differences between the means were distinguished by Fisher’s protected least significant difference (LSD) test at the 0.05 probability level.
Experiment 3: Reduced Fertility Trials (2021)
Trials were initiated on established Kentucky Bluegrass plots to evaluate the effects of inoculation with WSF23+WSF14 on turfgrass performance under reduced fertility conditions. . Plots were treated with a soil drench of WSF23+WSF14 in a 0.01% humic acid solution or with just the 0.01% humic acid solution as a control. Plots were well irrigated prior to and during the treatments to allow effective bacterial inoculation.
Fertility treatments were applied at three different rates using a slow release organic fertilizer (8-2-4) consistent with fall fertility programs for turfgrass in September, 2022.
- 0 Fertility: No supplemental fertilizer applied
- ½ Rate Fertility: 8-2-4 Fertilizer applied at a rate of 0.5 lbs. N per 1000 sq. ft.
- Full Rate Fertility: 8-2-4 Fertilizer applied at a rate of 1.0 lbs. N per 1000 sq. ft.
Each treatment was replicated in four plots (3 x 4‘ each) arranged in a randomized complete block design.
Physiological Analysis
- Weekly radiometry based objective assessments were conducted using a handheld multispectral radiometer (MSR model CT1000; Crop Scan Inc., Fargo, ND). The MSR measures canopy reflectance characteristics (510-1700 nm) which were used to calculate normalized difference vegetation index (NDVI), stress index (SI), and leaf area index (LAI).
- Light box digital images were collected during the experimental period and analyzed using SigmaScan Pro 5 software to calculate canopy density (percent green cover) and dark green color index (DGCI).
- Nutrient analysis was conducted on shoot and leaf tissue five weeks after the initial fertility treatments and inoculation, with samples collected from each of the treated and control plots.
Statistical Analysis
The effects of the inoculation treatments were determined by analysis of variance (ANOVA) according to the general linear model procedure of SAS (version 9.4; SAS institute, Cary, NC, USA). Differences between the means were distinguished by Fisher’s protected least significant difference (LSD) test at the 0.05 probability level.
Experiment 4: Optimizing Application Rate for Improving Drought Stress in Field Conditions (2022)
Plant materials and growth conditions:
Creeping bentgrass (Agrostis stolonifera cv. Penncross) plots (1mx1.3m) were established in field soil at the Rutgers University Horticultural Research Farm and maintained at a height of 1.2 cm during the 2022 growing season. Plants were inoculated two weeks before the onset of drought stress and every two weeks thereafter throughout the growing season using the soil drench method. A rainout shelter was used to cover the plots to simulate drought stress (60% ET).
Inoculation treatments:
- P. aspalathi strains ‘WSF23’ and ‘WSF14’ were applied at varying rates using the soil drench method to determine the most effective concentration for improving drought stress tolerance under field conditions. A concentration of 1.0 x 107 CFUs was found to be effective in previous growth chamber trials, so this was used as a starting point for field concentrations. Additional 1.5x and 2.0x concentration treatments were evaluated in this experiment. All treatments (including the non-inoculated control) were mixed with humic acid (0.01%) and sprayed onto field plots, then watered in with 0.25” of water. The inoculation treatments were:
-
- Non-inoculated control
- 1.0 x 107 CFUs (‘WSF23’ + ‘WSF14’)
- 1.5 x 107 CFUs (‘WSF23’ + ‘WSF14’)
- 2.0 x 107 CFUs (‘WSF23’ + ‘WSF14’)
Irrigation treatments:
-
- Drought Stress: Deficit Irrigation (60% ET) for 35 days
- Post-stress Recovery: Drought-stressed plants were re-watered for 14 days (100% ET)
Measurements:
The following weekly measurements were collected according to the procedures described above (Experiment 1)
- Turf Quality
- Soil water content using TDR
- MSR (NDVI, SI)
- Light Box (Canopy Density, DGCI)
- Thermal Images
- Rooting characteristics
- Bacteria colonization or viability
Experiment 5: Drought Stress and Post-Drought Recovery Trials on Sod Farms (2022)
Field trials were conducted on two collaborating sod farms in Central New Jersey (Tri-County Turf, Cream Ridge, NJ and Reid Sod Farm, Freehold, NJ). Research plots (1m x 1.3m) were marked out on established Kentucky bluegrass/tall fescue sod being grown on non-irrigated field soil. The plots were inoculated with ‘WSF23’+‘WSF14’ every two weeks starting in early July in four replications at each of the two field sites. Four replicates of non-inoculated plots were also evaluated as controls. Bi-weekly measurements of turf quality, canopy density, and chlorophyll content (using a Field Scout chlorophyll meter) were collected from July through October 2022. Moderate drought conditions were experienced on both farms during the growing season and no supplemental irrigation was applied to the plots, except when the inoculants were applied. Non-inoculated plots received an equal amount of water without the bacterial inoculant to control for the variable of applied water.
Experiment 6: Optimizing Inoculation Efficiency and Efficacy Methods Using Soil Amendments (2022)
Creeping bentgrass (cv. Penncross) sod plugs were collected from the Rutgers Horticultural Research Farm II and transplanted into PVC tubes filled with sand. The roots were trimmed completely before transplanting and the plants were then grown in controlled environment growth chambers. PGPR inoculants of ‘WSF23’ +’WSF14’ were applied in combination with different soil amendments to determine if inoculation could be enhanced with their use. Two weeks after the initial inoculation, the roots from each treatment group were sampled weekly for a period of 4 weeks. The roots were analyzed for the presence of the inoculated PGPR using PCR analysis.
Bacteria inoculation treatments:
- Control: Plants were grown in the sand without any other treatments
- Bacteria inoculation: bacteria suspension (OD600 =1.0, approximately 1.0 x 107 cfu), was watered into the pots filled with the sand (ACCd)
Amendment treatments:
- Biochar + Bacteria inoculation: Biochar was mixed in the sand at 0.8 g per kg sand in each pot, and plants were inoculated with bacteria suspension (Biochar + ACCd)
- Zeolite + Bacteria inoculation: Zeolites were mixed in the sand at 10% in volume in each pot, and plants were inoculated with bacteria suspension (Zeolite + ACCd) (Bigelow, 2004)
- Humid acid + Bacteria inoculation: humid acid (0.01%) was mixed into the bacteria suspension and watered into the pots filled with sand (HA + ACCd)
- ACC+ Bacteria inoculation: 3 mM ACC (this is the concentration of ACC used in bioassay of bacteria ACC-deaminase enzyme activity) was added to the bacteria suspension and watered into the pots filled with sand (ACC + ACCd)
Each treatment was repeated in 16 pots (4 replicates x 4 weeks of measurements).
Experiment 1: Drought Stress Trials (2020, 2021)
Plots inoculated with the combination of bacterial strains (WSF14+WSF23) using the soil drench method exhibited increased drought tolerance and post-drought recovery in 2020. Preliminary data analysis indicates greater turf quality, NDVI, canopy density, and DGCI in these plots. Figures 1-3 illustrate these differences throughout the growing season.
In 2021, field trials were repeated to verify the results of the previous season.
In 2021, plots inoculated with the single strain WSF23 applied as a soil drench did not significantly differ from non-inoculated plots during the 4-week drought stress period. However, treatment with WSF23 did enhance the recuperative ability of drought stressed turf, as WSF23 promoted higher Turf Quality, increased NDVI, lower Stress Index, and greater Leaf Area Index relative to non-inoculated plots after 2 weeks of re-watering. Figures 4-7.
Comparatively, turf that was inoculated with the single strain WSF14 applied as a soil drench had higher NDVI and a lower stress index than non-inoculated plots after 4-weeks of drought stress, but did not exhibit significant differences during the recovery period.
Combining the two strains (WSF23+WSF14) and applying them as a soil drench resulted in synergistic effects, such that drought stress tolerance and post-drought recovery were both improved relative to non-inoculated plots. After four weeks of drought stress, plots inoculated with the combination treatment (WSF23+WSF14) demonstrated higher Turf Quality, increased NDVI, lower Stress Index, and greater Leaf Area Index than non-inoculated plots. Inoculation with the combination of strains also resulted in greater capacity for recovery upon rewatering. After a 2-week recovery period, the plots treated with WSF23+WSF14 demonstrated improvements in all measured parameters relative to non-inoculated plots.
To evaluate the colonization efficiency of bacteria, roots were sampled from the plots to examine the presence and quantity or density of the bacterial species that comprises WSF23 and WSF14 (P. aspalathi in plant tissues). The presence of bacterial inoculants in plant tissues was determined by both bacterial isolation and quantitative real-time PCR (qPCR) that was successfully designed based on signature sequences of the 16S rDNA in P. aspalathi. Both sequencing analysis and bacterial streaming from plant tissue were able to confirm successful inoculation using both the foliar spray and soil drench method, with the soil drench method demonstrating higher levels of bacterial streaming. Figures 8-9
These findings are consistent with measurements from the previous season and suggest that the combination treatment (WSF23+WSF14) applied as a soil drench is the most effective method for improving drought stress tolerance and recovery for sod producers.
Experiment 2: Seedling Establishment Trials (2021)
Creeping Bentgrass Establishment
Creeping bentgrass plots treated with WSF23+WSF14 did not exhibit significant differences from control plots during fall establishment with regards to the parameters measured, including NDVI, Stress Index, Leaf Area Index, Percent Green Cover, and Dark Green Color Index.
Kentucky Bluegrass Establishment
Kentucky Bluegrass plots treated with WSF23+WSF14 did not exhibit significant differences from control plots during fall establishment with regards to the parameters measured, including NDVI, Stress Index, Leaf Area Index, Percent Green Cover, and Dark Green Color Index.
Experiment 3: Reduced Fertility Trials (2021)
Kentucky Bluegrass plots treated with WSF23+WSF14 did not exhibit significant differences from control plots during fall fertility treatments with regards to the parameters measured, including NDVI, Stress Index, Leaf Area Index, Percent Green Cover, and Dark Green Color Index. Results from the nutrient analysis did not indicate significant differences in nutrient content amongst the inoculation treatments.
Experiment 4: Optimizing Application Rate for Improving Drought Stress in Field Conditions (2022)
All three concentrations of bacterial inoculant (1.0, 1.5, and 2.0 x) resulted in improvements in drought stress tolerance and post-drought recovery of creeping bentgrass. The 1.5 x concentration demonstrated the greatest degree of enhancement, with improvements in Turf Quality, NDVI, Stress Index, and Leaf Area Index parameters over the greatest period of time. Figures 10-13
Experiment 5: Drought Stress and Post-Drought Recovery Trials on Sod Farms (2022)
Inoculated sod retained higher turf quality and greater canopy density during drought stress at location 1 (Reid’s Sod Farm), with significantly higher values 14 days after the initial inoculation. Figures 14-15
Inoculated sod also demonstrated higher chlorophyll content during drought stress and post-drought recovery. Chlorophyll content at location 1 (Reid’s Sod Farm) was significantly higher in inoculated plots at 14, 28, and 42 days after the initial inoculation. Figure 16 This period of time correlates to the onset of drought stress on the farm. At location 2 (Tri-County Turf), enhanced chlorophyll content was observed during the post-drought recovery period (105 days after the initial inoculation. Figure 17
Experiment 6: Optimizing Inoculation Efficiency and Efficacy Using Soil Amendments
Of the treatments tested, inoculation efficacy was most enhanced with the addition of zeolites. Roots inoculated with ‘WSF23+WSF14’ combined with zeolites were the only ones to retain bacterial colonization for the full 4-week evaluation period. Additionally, the addition of zeolites resulted in the highest percentage of total confirmed inoculations at 94%. Figures 18-19 The ACC treatments are not included because the presence of ACC reduced root growth to the extent that samples were not able to be obtained.
The novel PGPR strains, P. aspalathi 'WSF23' and 'WSF14' were able to improve drought stress tolerance and post-drought recovery in cool season turfgrass, suggesting that inoculation with these PGPR strains can help to reduce water use while maintaining turf quality.
The greatest improvements in growth and drought stress tolerance were observed when both PGPR strains were combined (WSF23+WSF14) and applied as a soil drench treatment.
The concentration was optimized at 1.5 x 107 CFUs of bacteria for field conditions.
The addition of zeolites to the soil resulted in greater inoculation efficacy and efficiency.
Education & Outreach Activities and Participation Summary
Educational activities:
Participation Summary:
The results from the first field season were presented as a poster at the American Society of Agronomy(ASA)/Crop Science Society of America (CSSA)/Soil Science Society of America (SSSA) Annual Meeting, which was held virtually in November, 2020. The poster won first place in the Graduate Student Competition in the C5 Turfgrass Management Division and was viewed by 250 industry professionals and researchers. The abstract is published in the proceedings for this event. 2020 ASA Poster - Bill Errickson
This poster was also presented at the Rutgers Turfgrass Symposium in February 2021, with the abstract published in the proceedings for the event, which was attended by 100 industry professionals and researchers.
The following field events were held in 2021 and 2022, to demonstrate this project and communicate the results to professional sod growers and turf managers.
Rutgers Turfgrass Research Field Days
- 7/28/21: Improving Drought Stress Tolerance with Rhizobacteria Field Demonstration for 50 members of the NJ Cultivated Sod Association
- 7/27/21: Improving Drought Stress Tolerance with Rhizobacteria Field Demonstration for 250 Turfgrass Managers and Industry Professionals
- 7/26/22 Improving Drought Stress Tolerance with Beneficial Microbes Field Demonstration for 300 Turfgrass Managers and Industry Professionals
Two oral presentations were also delivered at the 2021 ASA/CSSA/SSSA Annual Meeting, which was held in Salt Lake City, UT in November, 2021. The presentations both won first place in their respective Graduate Student Competitions of the C5 Turfgrass Genetics Division and the C2 Crop Physiology Division with 150 industry professionals and researchers in attendance. The abstracts are published in the proceedings for this event. 2021 Crop Science Presentation C2 - Errickson 2021 Crop Science Presentation C5 -Errickson
One oral presentation and one poster presentation were delivered at the 2022 ASA/CSSA/SSSA Annual Meeting, which was held in Baltimore, MD in November 2022. The oral presentation and poster both won first place in their respective Graduate Student Competitions of the C5 Turfgrass Genetics Division with 250 industry professionals and researchers in attendance. The abstracts are published in the proceedings for this event. 2022 Crop Science Oral Presentation 2022 Crop Science Poster
Learning Outcomes
Reduce water use or improve irrigation efficiency.
Incorporate plant health products and biostimulants into their operation.
Project Outcomes
Information Products
- Colonization Stability and Efficacy of Plant Growth Promoting Rhizobacteria in Creeping Bentgrass (Conference/Presentation Material)
- Rhizobacteria Inoculation and Colonization for Promoting Plant Growth in Cool Season Turfgrass (Conference/Presentation Material)