Final report for GNE17-149
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 spp., 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 spp. are 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 expands on our previous work with PGPB to investigate the roles of Burkholderia spp. 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).
This project evaluated the effects of a native strain of endophytic ACC deaminase producing Burkholderia bacteria on promoting the growth of maize (Zea mays cv. ‘American Dream’) and tomato (cv. ‘Big Beef’) seedlings in saline and reduced fertility environments. The Burkholderia strain was used to inoculate the roots of maize and tomato seedlings, which were then subjected to salinity stress (150 mM NaCl) or reduced fertility treatments in growth chamber trials. Physiological and growth parameters of inoculated plants were compared to non-inoculated controls by measuring electrolyte leakage EL, shoot and root biomass, and root length, surface area, volume, and diameter. Inoculated maize seedlings subjected to salinity stress demonstrated significantly higher membrane stability, shoot biomass, root biomass, root volume, and root diameter compared to non-inoculated plants. Increases in shoot and root biomass as well as root length, surface area, and volume , as well as reduced ethylene concentrations were also observed for inoculated tomato seedlings grown in saline conditions. Inoculated maize, tomato, and perennial ryegrass plants under reduced fertility conditions exhibited changes in root morphology relative to non-inoculated plants including increased root length, surface area, and volume. Perennial ryegrass plants also demonstrated increased shoot growth with inoculation under low fertility conditions. Future studies focused on optimizing the bacteria/host/stress interaction and understanding the mechanisms involved in the bacteria-mediated stress tolerance response will facilitate the adaptation of PGPR inoculants for field application.
1. To determine the effectiveness of Burkholderia sp. 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 Burkholderia sp. 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.
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.
Salinity is a major stress limiting plant growth by imposing cellular and physiological damages including osmotic stress, ion toxicity, and nutrient disturbances (Alshammary et al., 2004). Salinity stress may cause changes in hormone metabolism in plants, including the promotion of ethylene production (Morgan and Drew, 1997). Increases in the production of an ethylene precursor, ACC, are associated with salinity stress in leaves and roots of various plant species (Arbona et al., 2003; Ghanem et al., 2008; Gómez‐Cadenas et al., 1998). Excessive ethylene in plants exposed to stressors limits shoot and root growth (Abeles et al., 2012). Approaches that can suppress accumulation of stress-induced ethylene may be effective to mitigate stress damages and subsequent yield reductions. Another abiotic stress for plants growing in poor soils or low-fertilizer input systems is inadequate soil nutrients; under those scenarios increasing nutrient use efficiency is important to maintain plant productivity. Using PGPB as a biofertilizer has been found to be a promising approach for improving stress tolerance and maintaining adequate plant nutrition.
Some PGPB such as Burkholderia spp. contain deaminase enzymes that utilize ACC as a nitrogen source, breaking down ACC and reducing ACC availability for ethylene synthesis (Saleem et al., 2007). The inoculation of plants with ACC-deaminase producing bacteria was reported to promote shoot and root growth under salinity stress in rice (Oryza sativa) (Bal et al., 2013), maize (Zea mays), wheat (Triticum aestivum) (Nadeem et al., 2010), tomato (Solanum lycopersicum) (Mayak et al. 2004), cucumber (Cucumis sativus) (Gamalero et al., 2010), and red pepper (Capsicum annuum) (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), and water use efficiency (Mayak et al. 2004) under salinity stress. Despite reports on ACC-deaminase bacteria enhancing plant tolerance to salinity stress, there is a wide range of variability in the effectiveness of different species or sources of PGPB. Limited work has been done exploring the benefits of bacteria forming symbiosis with plants adapted to poor quality soils, such as in the New Jersey Pine Barrens. In addition, some PGPB, such as Bacillus spp. are reported to improve nutrient uptake and assimilation of N, P and K (Wu et al. 2005). However, the nutritional effects of ACC-deaminase bacteria for promoting plant growth and reducing fertilizer inputs are not well documented and deserve further investigation.
This study provides further understanding of the roles of microorganisms native to the New Jersey Pine Barrens in sustainable agricultural systems and explores the potential of utilizing the naturally-occurring PGPB as biofertilizers 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.
Materials and methods:
- To determine the effectiveness of Burkholderia sp. 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 were planted into pots (tomato: 10 cm square pots; maize: 10 cm diameter round pots, 50 cm deep) filled with sterile, calcined clay (Kottkamp et al. 2010). Plants were established in a greenhouse and then moved to growth chambers (Environmental Growth Chamber, Chagrin Falls, OH) for bacterial inoculation and subsequent salinity and low fertility treatments. The controlled-environment growth chambers were set to maintain 23/18 °C (day/night), 680 µmol.m-2.s-1 1 PAR, 60% relative humidity, and 12-h photoperiod. Plants were irrigated with DI water and fertilized with Hoagland nutrient solution (Hoagland and Arnon, 1950).
Bacterial preparation and inoculation. A native strain of Burkholderia sp. with high ACCd activity was used to inoculate tomato and maize plants. Bacterial cultures were revived from frozen stock vials stored at −80 °C by streaking on nutrient agar plates. Single colonies were picked and inoculated in luria broth and incubated at 23 °C on a water-bath shaker for 48 h. Bacterial suspensions were centrifuged at 8000 gn for 10 min at 4 °C then re-suspended in deionized water. The centrifuge and re-suspension process was repeated twice to remove the luria broth. The prepared bacterial suspension was adjusted to optical density of 1.0. Plants were inoculated by soil drenching prepared bacterial inoculum into each pot twice at an interval of 24 h (50 mL for tomatoes and 200 mL for corn). The control groups for the bacterial inoculation treatments were watered with an equivalent amount of deionized water.
Salinity treatment and experimental design. Salinity treatment was initiated 3 d following bacterial inoculation. Tomato plants received 50 mL 150 mM NaCl solution every other day for the duration of the experiment, while maize plants received 200mL. NaCl treatment were increased at 2 d intervals from 20, 40, 80, to 150 mM to avoid initial salinity shock. Plants were be subjected to 150 mM salinity irrigation for 21 d (tomato) and 28 d (corn). The experimental design was a completely randomized design with two factors (salinity treatment and bacterial inoculation). Each treatment consisted of four replicates with and three subsamples for tomatoes and two subsamples for corn. The four replicates for each treatment were placed in four different growth chambers, and containers of plants were randomly placed inside each growth chamber. Additionally, all containers were rerandomized and relocated within the growth chambers every 3 d to avoid possible confounding effects of chamber environmental variations.
Physiological analyses. Leaf electrolyte leakage (EL) was measured as an indicator of cellular membrane stability according to the procedure by Blum and Ebercon (1981). Approximately 0.2 g fresh leaves were collected, rinsed with deionized water to remove exogenous solutes, and placed in a test tube containing 35 mL deionized water. Tubes were placed on a conical flask shaker for 16 h and the initial conductance (Ci) was measured using a conductivity meter (model 132; YSI, Yellow Springs, OH). Leaf samples were killed by autoclaving at 120 °C for 20 min and shaking for 16 h. The maximal conductance of killed tissue (Cmax) was then measured. EL was calculated using the formula (%) = (Ci/Cmax) ×100.
Relative water content (RWC) was measured according to the procedure by Barrs and Weatherley (1962). Leaf RWC was 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 was determined with a mass balance immediately after detaching leaves from the plant. Samples were wrapped in tissue paper and submerged in deionized water for 24 h. Leaf samples were then removed from the water, blotted dry, and again weighed for TW. Following a drying period of 3 d at 80 °C, samples were weighed a final time for DW. Leaf photochemical efficiency was 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 were dark adapted for 30 min before Fv/Fm was measured.
ACC determination. ACC content was determined according to the method of Concepcion et al. (1979). Approximately 0.1 g of fresh leaf tissue was ground into powder with liquid nitrogen and dissolved in 1.5 mL ethanol. The sample was then centrifuged at 10,000 gn for 15 min at 4 °C and the supernatant was evaporated in a vacuum at 50 °C. The sample was combined 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 aliquot of the water phase extract was transferred to a glass tube with rubber cap affixed, 10 μL x0.1 M HgCl2 was 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 was injected by a syringe and the glass tube was vortexed. Following 3 min incubation on ice, a 1 mL air sample was 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 (VQ) was performed weekly during the salinity treatment. VQ was 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 were based on parameters such as uniformity, visual attractiveness, leaf color, and canopy density (Beard, 1972). Shoot and root dry weights were measured at the conclusion of the stress treatments. Roots were washed free of fritted clay and severed from shoots by destructively sampling. All tissues were dried at 80 °C for 3 d and their weights were measured using a mass balance. Root morphological parameters were analyzed upon harvest by scanning with a digital scanner (Epson Expression 1680, U.S. Epson, Inc., Long Beach, CA) to generate high-definition digital images. Images were 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 were washed free of calcined clay and severed from shoots at the conclusion of the stress treatments and dried at 80 °C for 3 d. The dry plant samples were ground with a mortar and pestle and passed through a 2 mm mesh sieve. Samples were analyzed for whole plant nutrient content. Nitrogen content was 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 was measured by the dry ash method (Miller, 1997).
Statistical analysis. Main effects of salinity or bacterial inoculation and their interactions were 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 were separated by Fisher’s protected least significance difference (LSD) test at the 0.05 probability level
- To determine the effectiveness of Burkholderia sp. 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, maize, and perennial ryegrass were planted into pots (6 cm in diameter and 50 cm deep) filled with sterile calcined clay.
Seeds of tomato, maize, and perennial ryegrass were planted into pots (tomato and perennial ryegrass: 10 cm square pots; maize: 10 cm diameter round pots, 50 cm deep) filled with sterile, calcined clay (Kottkamp et al. 2010). Plants were established in a greenhouse and then moved to growth chambers (Environmental Growth Chamber, Chagrin Falls, OH) for bacterial inoculation and subsequent low fertility treatments. The controlled-environment growth chambers were set to maintain 23/18 °C (day/night), 680 µmol.m-2.s-1 1 PAR, 60% relative humidity, and 12-h photoperiod. Plants were irrigated with DI water and fertilized with Hoagland nutrient solution during plant establishment prior to the initiation of low fertility treatment.
Bacterial preparation and inoculation methods were conducted as stated above.
Nutrient use efficiency experimental design. Nutrient use efficiency treatment was initiated 3 d following bacterial inoculation. Plants in each pot were 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 were 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 was a completely randomized design with two factors (fertilizer levels and bacterial inoculation). Each treatment consisted of five replicates with three subsamples for tomato, four replicates with two subsamples for corn, and ten replicates for perennial ryegrass. All replicates for each treatment were placed in a growth chamber, and containers of plants were randomly arranged within each growth chamber. Additionally, all containers were rotated within the growth chambers every 7 d to avoid possible confounding effects of chamber environmental variations.
Physiological analyses, ACC determination, shoot and root growth analyses, nutrient analysis, and statistical analysis were performed as stated above.
Maize Salinity Stress
Maize plants inoculated with ACCd bacteria demonstrated improved salinity tolerance through the promotion of increased membrane stability and shoot and root growth. Inoculation of plants subjected to salinity stress resulted in reduced electrolyte leakage (fig. 3), as well as an increase in shoot biomass (fig. 13), root biomass (fig. 14), root volume (fig. 16), and root diameter (fig. 18) compared to non-inoculated controls. No significant differences were found in ethylene content in inoculated maize plants under salinity stress, (fig. 19), nor were significant differences found in nutrient composition in the inoculated plants (table 1). A full representation of the data is included in figures 3, 6, 9, 12, 13-19, and table 1.
Maize Low Fertility
Maize plants inoculated with ACCd bacteria demonstrated improved root growth in low fertility conditions compared to non-inoculated controls. Root volume (fig. 16) and root diameter (fig. 18) were significantly increased in inoculated plants under low fertility stress. While trends were observed for reduced ethylene content in inoculated maize, no significant differences were found (fig. 19), nor were significant differences found in nutrient composition in the inoculated plants (table 1). A full representation of the data is included in figures 2, 5, 8, 11, 13-19, and table 1.
Tomato Salinity Stress
Tomato plants inoculated with ACCd bacteria demonstrated improved salinity tolerance through the promotion of increased shoot and root growth. Inoculation of plants subjected to salinity stress resulted in an increase in shoot biomass (fig. 32), root biomass (fig. 33), root length (fig. 34), root volume (fig. 35), and root surface area (fig. 36) compared to non-inoculated controls. Inoculated plants also had lower ethylene concentrations compared to non-inoculated plants under salinity stress (fig. 38). No significant differences found in nutrient composition in the inoculated plants (table 2). A full representation of the data is included in figures 22, 25, 28, 31-38, and table 2.
Tomato Low Fertility
Tomato plants inoculated with ACCd bacteria demonstrated improved low fertility tolerance through the promotion of leaf hydration status and root growth. Inoculation of plants subjected to low fertility stress resulted in higher leaf relative water content (fig. 24) and root length (fig. 34) compared to non-inoculated controls. Trends for increased shoot biomass, root biomass, root surface area and volume were also observed, though these results were not significant. Similarly, inoculated plants show trends for increases in several nutrients, including N, P, K, Ca, Mg, and S, but no significant differences (table 2). A full representation of the data is included in figures 21, 24, 27, 30, 32-36, and table 2.
Perennial Ryegrass Low Fertility
Perennial ryegrass plants inoculated with ACCd bacteria demonstrated improved low fertility tolerance through the promotion of membrane stability status, shoot growth, and root morphology. Inoculation of plants subjected to low fertility stress resulted in lower electrolyte leakage (fig. 40) and increased shoot biomass (fig. 47), root volume (fig. 50), and root surface area (fig. 51) compared to non-inoculated controls. Trends for increased visual quality and root biomass as well as reduced ethylene content were also observed, though these results were not significant. A full representation of the data is included in figures 39-53 and table 3.
Burkholderia sp. with ACC-deaminase enzymes improved growth in low-fertility conditions by promoting changes to root morphology. ACC-deaminase bacteria protected plants from membrane damages associated with salinity stress, while promoting shoot and root growth in saline conditions. Improved root growth associated with ACCd bacteria can increase the water and nutrient uptake capacity of maize, tomato, or perennial ryegrass in low nutrient or salinity stress conditions by allowing the plant to explore a larger volume of soil. Plant growth promoting rhizobacteria with ACCd activity may provide growers with additional biological methods to mitigate low-fertility and salinity stress in maize, tomato, and perennial ryegrass.
Education & Outreach Activities and Participation Summary
Outreach achievements include an accepted abstract and oral presentation in the C-2 Crop Physiology Division at the 2018 ASA, CSSA International Meeting in Baltimore, MD, with over 4,000 participants as well as an abstract and poster presentation for the 2019 Rutgers University Plant Science Symposium. Additionally, aspects of this project were included in a webinar that was presented for the USDA Northeast Climate HUB Graduate Climate Adaptation Partners Fellowship. Furthermore, we hope to include the results in a future journal publication.
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 ryegrass, 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 can 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. Outreach from this project will communicated the potential of Burkholderia as a biofertilizer for use in the Northeast and provided 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.
In order to make these bacteria available and accessible to growers, we are beginning to work on developing the best methods of inoculation for different crops, comparing seed inoculation vs soil drench systems to apply the bacteria to the plants. Optimizing this component will be an essential element towards getting these microorganisms into growers’ fields and improving food production. Several industry partners who can help to make this technology available on a larger scale have expressed interest in our work, as we proceed to bring our efforts to fruition. We are also planning to work with growers to trial these applications in their fields. This can help to increase yields, while reducing water and fertilizer use, improving economic sustainability and efficiency for the grower, while conserving natural resources.
One of the research challenges that still remains involves further developing our understanding of the mechanisms behind the plant-bacteria interaction and the associated conference of enhanced growth and stress tolerance. In order to investigate these mechanisms, we are planning to conduct research into the changes in plant hormone levels, metabolites, RNA expression, and proteins that occur in relation to bacterial inoculation and abiotic stress. A second challenge involves the applied aspect of this work as it relates to sustainable agriculture in regards to developing field studies that build on our growth chamber trials. In the growth chamber, we are able to control many variables that exist in the field, especially the presence of native soil microbes that may compete or interact with our beneficial bacteria strains. Additional research will also focus on the effects of inoculation on crop yields. While our initial screening focused on seedlings and produced encouraging results, it will now be important to determine if these beneficial effects will translate to increased crop yields. Optimizing field inoculation protocols and testing our bacteria strains in the field will be a new and exciting direction for this research to bring it closer to adoption in growers' fields.