The purpose of this project is to explore the potential of rhizobacteria from natural ecosystems as biofertilizers to improve crop stress tolerance and reduce the use of inorganic fertilizers in sustainable agriculture.
Increasing water and nutrient use efficiency becomes increasingly important in sustainable agricultural systems due to environmental concerns with water conservation and inorganic fertilizers. Fresh water availability for irrigation is becoming limited while there is increasing demand on the use of recycled water for agricultural and horticultural production. However, irrigation with poor quality water has the potential of causing salinity stress. Increased attention is currently being directed toward the study of naturally-occurring microorganisms as biofertilizers to improve plant growth with low inputs and environmental impact. Bacteria in the microbial communities that co-exist within the plant can promote plant growth, induce plant defenses against herbivory and pathogens, assist in the acquisition of minerals and water, and enhance plant adaptation to poor soils with salinity and nutrient deficiency. Those bacteria are collectively referred to as Plant Growth Promoting Bacteria (PGPB). PGPB have great potential to increase plant productivity, while reducing fertilizer inputs and water use, resulting in greater economic sustainability for the grower and conservation of natural resources.
Some PGPB contain deaminase enzymes that utilize 1-Aminocyclopropane-1-carboxylic acid (ACC), a precursor of the phytohormone ethylene, as a nitrogen source. These PGPB suppress ethylene accumulation in the plant, which in turn promotes root growth, water and nutrient uptake (Saleem et al., 2007), as well as plant tolerance to abiotic stress, including salinity (Bal et al., 2013; Nadeem et al., 2007, 2010; Mayak et al. 2004; Gamalero et al., 2010; Siddikee et al., 2012). Several studies reported inoculation of plants with ACC-deaminase bacteria increased leaf relative water content (Ahmad et al., 2013; Nadeem et al., 2007, 2010), chlorophyll content (Bal et al., 2013; Mayak et al. 2004; Nadeem et al., 2007), and water use efficiency (Mayak et al. 2004) under salinity stress. We isolated ACC-deaminase bacteria, Burkholderia gladioli, from the roots of native grass species in the New Jersey Pine Barrens. Pine barrens are a unique type of ecosystem that have acidic, sandy and oligotrophic soils with low organic matter, a lack of nutrients, and poor water holding capacity. Over 50 native Poaceae grass species were documented in the New Jersey Pine Barrens, and the symbiosis of microorganisms with plants may support plant growth in such poor soils in this ecosystem. We had collected over 120 species/strains of bacteria from the New Jersey Pine Barrens and found that Burkholderia gladioli is among the most effective PGPB species for promoting plant stress tolerance. Our preliminary studies have demonstrated an increase in biomass production, nutrient uptake and plant tolerance to salinity stress in perennial ryegrass (Lolium perenne) when the roots were inoculated with B. gladioli (Cheng et al. 2016). This study will expand from our previous work with PGPB to investigate the roles of B. gladioli in improving root growth, nutrient use efficiency, and salinity tolerance in agronomic crops, such as maize (Zea mays), and horticultural crops, such as tomato (Solanum lycopersicum).
Maize was produced on over 85,000 acres in New Jersey in 2016, with the combined value of grain corn, sweet corn, and silage valued at over 53 million dollars. Tomatoes were grown on 3,000 acres in New Jersey in 2016, and valued at over 46 million dollars (USDA, 2016). Perennial ryegrass is valuable soil building pasture forage that is also commonly planted in orchards throughout the Northeast. Growers are continuously looking for ways to improve plant health, crop yields, and efficiency while reducing costs and environmental pollution. These challenges are exacerbated as climate changes produce unpredictable growing seasons and additional plant stressors. Incorporating naturally-occurring PGPB as biofertilizers in agricultural and horticultural systems is a sustainable approach to improve plant health by ameliorating stress and improving nutrient and water use efficiencies.
This study will provide further understanding of the roles of microorganisms native to the New Jersey Pine Barrens in sustainable agricultural systems and explore the potential of utilizing the naturally-occurring PGPB as biofertilizer to enhance plant growth and stress tolerance. Future research will screen all grass-associated PGPB collected from the New Jersey Pine Barrens and investigate the combined effects of various species of beneficial microorganisms on the growth of economically important plants.
1. To determine the effectiveness of B. gladioli for improving salinity tolerance in tomato and maize irrigated with saline water by evaluating growth and physiological factors, as well as nutritional status of plants affected by the ACC-deaminase bacteria inoculation under salinity stress. 1. To determine the effectiveness of B. gladioli for improving salinity tolerance in tomato and maize irrigated with saline water by evaluating growth and physiological factors, as well as nutritional status of plants affected by the ACC-deaminase bacteria inoculation under salinity stress.
2. To determine the effectiveness of B. gladioli for improving nutrient uptake efficiency in tomato, maize, and perennial ryegrass in reduced fertility conditions by evaluating growth and physiological factors, as well as nutritional status of plants affected by the ACC-deaminase bacteria inoculation.
- To determine the effectiveness of gladioli for improving salinity tolerance in tomato and maize irrigated with saline water by evaluating growth and physiological factors, as well as nutritional status of plants affected by the ACC-deaminase bacteria inoculation under salinity stress.
Plant materials and growth conditions. Seeds of tomato and maize will be planted into pots (6 cm in diameter and 50 cm deep) filled with sterile, calcined clay (Kottkamp et al. 2010). Plants will be established in a greenhouse and then transplanted to growth chambers (Environmental Growth Chamber, Chagrin Falls, OH) for bacterial inoculation and subsequent salinity and drought treatments. The controlled-environment growth chambers will be set to maintain 23/18 °C (day/night), 680 µmol.m-2.s-1 1 PAR, 60% relative humidity, and 12-h photoperiod. Plants will be irrigated daily with sterile water and fertilized with full-strength Hoagland nutrient solution (Hoagland and Arnon, 1950) every 3 d from emergence to first true leaves.
Bacterial preparation and inoculation. B. gladioli RU1 will be used to inoculate tomato and corn plants. Bacterial cultures will be revived from frozen stock vials stored at −80 °C by streaking on nutrient agar plates. Single colonies will be picked and inoculated in lysogeny broth and incubated at 23 °C on a water-bath shaker for 48 h. Bacterial suspensions will be centrifuged at 8000 gn for 10 min at 4 °C then re-suspended in deionized water. The centrifuge and re-suspension process will be repeated twice to remove the lysogeny broth. The prepared bacterial suspension was adjusted to optical density of 1.0. Plants will be inoculated by soil drenching with 30 mL prepared bacterial inoculum into each pot twice at an interval of 6 h. The control group for the bacterial inoculation treatment will be watered with 30 mL of deionized water. At 7 and 14 d of inoculation, bacteria will be isolated from roots of plants inoculated through soil drenching and ACC-deaminase activity of bacteria will be measured to ensure successful inoculation of both strains.
Salinity treatment and experimental design. Salinity treatment will be initiated 1 d following bacterial inoculation. Plants in each pot will receive 50 mL sterile NaCl solution daily for the duration of the experiment. NaCl treatment will be increased at 2 d intervals from 20, 40, 80, 160, to 250 mM to avoid initial salinity shock. Plants will be subjected to 250 mM salinity irrigation for 21 d. The experimental design will be a completely randomized design with two factors (salinity treatment and bacterial inoculation). Each treatment will consist of four replicates and three subsamples (containers) with a total of 12 containers (one plant in each container). Four replicates for each treatment will be placed in four different growth chambers, and containers of plants will be randomly placed inside each growth chamber. Additionally, all containers will be relocated among four growth chambers every 3 d to avoid possible confounding effects of chamber environmental variations.
Physiological analyses. Leaf electrolyte leakage (EL) will be measured as an indicator of cellular membrane stability according to the procedure by Blum and Ebercon (1981). Approximately 0.2 g fresh leaves will be collected, rinsed with deionized water to remove exogenous solutes, and placed in a test tube containing 30 mL deionized water. Tubes will be placed on a conical flask shaker for 12 h and the initial conductance (Ci) measured using a conductivity meter (model 132; YSI, Yellow Springs, OH). Leaf samples will be killed by autoclaving at 120 °C for 20 min and shaking for 12 h. The maximal conductance of killed tissue (Cmax) will then be measured. EL will be calculated using the formula (%) = (Ci/Cmax) ×100.
Relative water content (RWC) will be measured according to the procedure by Barrs and Weatherley (1962). Leaf RWC will be calculated based on leaf fresh weight (FW), turgid weight (TW), and dry weight (DW) using the formula (%) = 100 x [(FW – DW)/(TW – DW)]. FW of leaves will be determined with a mass balance immediately after detaching leaves from the plant. Samples will be wrapped in tissue paper and submerged in deionized water for 12 h at 4 °C. Leaf samples will then be removed from the water, blotted dry, and again weighed for TW. Following a drying period of 3 d at 80 °C, samples will be weighed a final time for DW. Leaf photochemical efficiency will be estimated by measuring chlorophyll fluorescence expressed as the ratio of variable to maximum fluorescence (Fv/Fm) with a fluorescence induction monitor (OS 1FL, Opti-Sciences, Hudson, NH). Leaves will be dark adapted for 30 min before Fv/Fm was measured.
ACC determination. ACC content will be determined according to the method of Concepcion et al. (1979). About 0.1 g of fresh leaf tissue will be ground into powder with liquid nitrogen and dissolved in 1.5 mL ethanol. The sample will then be centrifuged at 10,000 gn for 15 min at 4 °C and the supernatant will be evaporated in a vacuum at 50 °C. The sample will be added with 0.75 mL deionized H2O and 0.75 mL chloroform and then vortexed and centrifuged at 10,000 gn for 15 min at 4 °C. A 0.5 mL of the water phase extract will be transferred to a glass tube with rubber cap affixed, 10 μL x0.1 M HgCl2 will be added, and the volume brought up to 0.8 mL with water. A 0.2 mL ice cold mixture (v/v=2:1) of commercial bleach (8% NaOCl) and saturated NaOH will be injected by a syringe and the glass tube will be vortexed. Following 3 min incubation on ice, 1 mL air sample will be withdrawn using a syringe and injected into a gas chromatograph (GC-8A; Shimadzu Scientific Instruments, Columbia, MD) (Watkins and Frenkel, 1987).
Shoot and root growth analyses. Visual evaluation of plant quality (PQ) will be performed biweekly during the salinity treatment. PQ will be rated on a scale of 1 to 9, with 1 being brown and desiccated plants, 6 being the minimal acceptable level, and 9 being green and dense plants. Ratings will be based on parameters such as uniformity, visual attractiveness, leaf color, and canopy density (Beard, 1972). Shoot and root dry weights will be measured at 10 d and 20 d of salinity treatment. Roots will be washed free of fritted clay and severed from shoots by destructively sampling 10 different plants at 10 or 20 d of salinity treatment. All tissues will be dried at 80 °C for 3 d and weight measured using a mass balance. Root morphological parameters will be analyzed upon harvest at 20 d of salinity treatment. Roots will be washed free of calcined clay, stained with 1% crystal violet solution, and scanned with a digital scanner (Epson Expression 1680, U.S. Epson, Inc., Long Beach, CA) to generate high-definition digital images. Images will be analyzed using WinRHIZO Basic V.2002 software (Regent Instruments Inc., Quebec, QC, Canada) for root length, volume, surface area, and diameter.
Shoot and root nutrient analysis. Roots will be washed free of calcined clay and severed from shoots at 20 d after salinity initiation. They will be washed with deionized water and dried at 80 °C for 3 d. The dry plant samples will be ground with liquid nitrogen and passed through a 2 mm mesh sieve. Approximately 0.2 g samples will be analyzed for nutrient content in shoots and roots. Nitrogen content will be determined using the combustion method of Horneck and Miller (1998). The content of P, K, Ca, Mg, Mn, Fe, Cu, B, Al, Zn and Na will be measured by the dry ash method (Miller, 1997).
Statistical analysis. Main effects of salinity or bacterial inoculation and their interactions will be determined by analysis of variance according to the general linear model procedure of a statistical program (SAS version 9.0; SAS Institute, Cary, NC). Differences between treatment means will be separated by Fisher’s protected least significance difference (LSD) test at the 0.05 probability level
- To determine the effectiveness of B. gladioli for improving nutrient uptake efficiency in tomato, maize, and perennial ryegrass in reduced fertility conditions by evaluating growth and physiological factors, as well as nutritional status of plants affected by the ACC-deaminase bacteria inoculation.
Plant materials and growth conditions. Seeds of tomato, corn, and perennial ryegrass will be planted into pots (6 cm in diameter and 50 cm deep) filled with sterile calcined clay. Plants will be established in a greenhouse and then transplanted to growth chambers (Environmental Growth Chamber, Chagrin Falls, OH) for bacterial inoculation and subsequent salinity and drought treatments. The controlled-environment growth chambers will be set to maintain 23/18 °C (day/night), 680 µmol.m-2.s-1 1 PAR, 60% relative humidity, and 12-h photoperiod. Plants will be irrigated daily with sterile water and fertilized weekly with sterile full-strength Hoagland’s solution during plant establishment prior to the initiation of low fertility treatment.
Bacterial preparation and inoculation methods will be conducted as stated above.
Nutrient use efficiency experimental design. Nutrient use efficiency treatment will be initiated 1 d following bacterial inoculation. Plants in each pot will be subjected to two levels of fertility to determine if ACC-deaminase bacteria can improve plant growth with low fertility inputs. Plants of tomato, maize, and perennial ryegrass will be fertilized weekly with (1) Hoagland solution at 100% concentration as the control treatment (full strength) and (2) Hoagland solution at 25% concentration as the low fertility treatment. Full-strength Hoagland solution has been widely used for plant culture, including maize, perennial ryegrass (Smith et al., 1983) and tomato (Kaur et al., 2016). Kaur et al. (2016) showed that the low fertility level of 50% Hoagland solution resulted in significant reduction in plant height and fruit production.
The experimental design will be a completely randomized design with two factors (fertilizer levels and bacterial inoculation). Each treatment will consist of four replicates and three subsamples (containers) with a total of 12 containers (one plant in each container). Four replicates for each treatment will be placed in four different growth chambers, and containers of plants will be randomly placed inside each growth chamber. Additionally, all containers will be relocated among four growth chambers every 3 d to avoid possible confounding effects of chamber environmental variations.
Physiological analyses, ACC determination, shoot and root growth analyses, shoot and root nutrient analysis, and statistical analysis will be performed as stated above.
Education & Outreach Activities and Participation Summary
Outreach for this project will include publication in at least one peer reviewed journal, creation of a poster, and presentation at both Rutgers University and a professional conference, such as the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America (ASA, CSSA, SSSA) Annual Meeting, which hosts over 4,000 participants. This research will benefit growers in the Northeast by providing new information about PGPB biofertilizers that can help improve salinity tolerance and nutrient use efficiency in maize, tomato, and perennial rye grass, which are widely grown and economically important crops in the region. The findings of this study may be especially useful to producers who are growing on land where crop productivity is limited by insufficient soil fertility or high soil salinity, or where low quality or recycled water with elevated salinity levels is being used for irrigation.
Researchers studying the roles of PGBP, biofertilizers, phytobiomes, and plant stress physiology will also benefit from this investigation by gaining a better understanding of how ACC-deaminase bacteria influence plant growth. The isolation of B. gladioli from a native NJ Pine Barrens ecosystem and its application as a biofertilizer represents an innovative approach that utilizes native soil microorganisms for sustainable crop production. While outreach from this project will communicate the potential of B.gladioli as a biofertilizer for use in the Northeast, it will also provide insight for other researchers to investigate the roles of additional native microorganisms in natural ecosystems throughout the region, and how they might be applied in sustainable agriculture. With sufficient communication amongst researchers, the potential exists to create specific combinations of microorganisms that are performing various functions that support plant growth in natural ecosystems. When applied to agricultural systems, the combined effects of the various microbial populations could reduce fertilizer and water use, while improving salinity tolerance and disease resistance, ultimately producing higher yields with reduced inputs and less impact on the environment.
Rutgers Cooperative Extension has been facilitating the transfer of knowledge between researchers and producers since 1914. The proposed project specifically meets the goals of cooperative extension that are focused on enhancing and protecting environmental resources and ensuring economic growth and agricultural sustainability. Extension agents will gain additional strategies to recommend to growers who are trying to meet these objectives as further evidence on the potential of B. gladioli as a biofertilizer is evaluated.