Growth Promotion of Switchgrass by Bacterial Endophyte Pantoea agglomerans Strain PaKM Isolated from Seeds

Research Article

J Pathol & Microbiol. 2016; 1(2): 1007.

Growth Promotion of Switchgrass by Bacterial Endophyte Pantoea agglomerans Strain PaKM Isolated from Seeds

Kim-Dura S1, Lowman S1,2, Zhang S¹ and Mei C1,2,3*

1Center for Sustainable and Renewable Resources, Institute for Advanced Learning and Research, USA

2Departments of Horticulture, Virginia Polytechnic Institute and State University, USA

3Forest Resources and Environmental Conservation, Virginia Polytechnic Institute and State University, USA

*Corresponding author: Chuansheng Mei, Center for Sustainable and Renewable Resources, Institute for Advanced Learning and Research, Danville, USA

Received: May 27, 2016; Accepted: June 27, 2016; Published: June 28, 2016


Switchgrass is one of the most promising bioenergy crops for the US. It produces relatively high biomass yield and can grow on marginal lands. However, some traits need to be improved, including stand establishment and stress tolerance. We previously reported that Burkholderia phytofirmans PsJN promoted growth of switchgrass cv. Alamo. However, PsJN was found to be genotype specific in promoting switchgrass growth. In an effort to identify endophytes which have a broad spectrum of growth promotion with more efficiency, we isolated a bacterium from surface-sterilized switchgrass seeds. The bacterium was identified as a Pantoea agglomerans species and named strain PaKM. Our experiments demonstrated that PaKM significantly boosts switchgrass growth/vigor and root system development, and increases biomass yield under in vitro, growth chamber, greenhouse, as well as field conditions. Not only does PaKM promote growth of switchgrass Alamo, but also 8 other switchgrass cultivars tested. PaKM also enhanced salt and drought stress tolerance in vitro. Assays demonstrated that PaKM has the ability to solubilize phosphate and produces high levels of auxin. In summary, PaKM is an efficient growth promoter of switchgrass over a broad spectrum of genotypes and has potential application with low input and sustainable production systems on marginal lands.

Keywords: Bacterial endophyte; Broad spectrum of growth promotion; Auxin level; Mechanisms; Pantoea agglomerans strain PaKM; Switchgrass


While the economies of developing and developed countries continue to grow and improve, petroleum oil demand will only increase. As consumption of fossil fuels causes environmental pollution and global warming, it is imperative to find clean alternative energy sources, especially renewable energy for the future. Bioenergy is one such source as it is synthesized by higher plants and alga by utilizing sunlight and carbon dioxide from the atmosphere. The use of bioenergy not only has potential to greatly reduce greenhouse gas emissions in an environmentally friendly way, but it is expected to also boost rural economies. Switchgrass is considered one of the most promising bioenergy crop candidates in the US because it is a perennial warm-season grass native to North America, can grow in poor soil, and requires little fertilizer and pesticide inputs. However, consistent improvements in biomass yield and sustainability are needed, and poor stand establishment in the first year is an issue. Much effort is directed towards overcoming these challenges to develop a low input and sustainable perennial feedstock production system capable of producing reliably high biomass yields on marginal lands.

There are many approaches to improve switchgrass biomass yields including conventional breeding and biotechnology. However, these approaches may take a decade or longer to develop new cultivars. The use of beneficial endophytes, which reside in plant tissues and cause no apparent harm, is a practical and feasible way to increase switchgrass biomass yield and enhance stress tolerance [1,2]. Also, beneficial endophytes are naturally occurring, and their widespread use will likely face few restrictions. Endophytes generally promote host plant growth, enhance nutrient uptake, increase stress tolerance, and inhibit plant pathogen growth [1,3,4]. These benefits are wide ranging across a broad spectrum of plants. For example, one of the most studied plant growth promoting endophytes, Burkholderia phytofirmans strain PsJN, stimulates growth of many plant species, including potato, tomato, grapevine, and switchgrass [5-10]. Many other examples have been reported for endophyte-mediated plant growth promotion. Endophytic Klebsiella oxytoca strain GR-3 promoted the growth of Typha australis, a semi-aquatic grass [11]. Two isolates, PS4 and PS27, closely related to Pseudomonas rhodesiae and Pantoea ananatis respectively, successfully colonized pepper roots and significantly promoted plant growth and enhanced root fresh weight by 73.9% and 41.5% respectively [12].

Research on bacterial endophytes in switchgrass has increased in recent years. For example, Gagne-Bourgue et al. [13] isolated and characterized 31 bacterial endophytes from leaves of three switchgrass cultivars. Xia et al. [14] characterized a total of 307 bacterial isolates from surface sterilized switchgrass shoots, roots and seeds, and tested their abilities to influence plant growth. Ker et al. [4] isolated 8 bacterial strains from Cave-in-Rock rhizomes and inoculated switchgrass seeds with a mixture of 8 strains in a low N field and observed 40% yield increase in the first establishment year. We have tested Burkholderia phytofirmans strain PsJN in switchgrass and found that PsJN significantly promoted switchgrass cv. Alamo growth under in vitro, growth chamber, and greenhouse conditions [9], as well as in the field [10]. However, the growth promotion was genotype specific, as the upland switchgrass cv. Cave-in-Rock exhibits no growth promotion by PsJN [9]. There have been several reports regarding genotypic specificity of endophytes [15,16]. Therefore, exploring beneficial endophytes having a broad spectrum of growth promotion is imperative and has potential in practical applications in sustainable bioenergy feedstock production. In this study, we report the isolation and identification of a bacterial endophyte, PaKM, from surface-sterilized switchgrass seeds, and demonstrated its growth promotion under in vitro, greenhouse and field conditions. We also characterized its possible mechanisms of growth promotion.

Materials and Methods

PaKM isolation and identification

A yellow bacterium was found in surface-sterilized switchgrass seeds growing on MS medium (M519, PhytoTechnology Laboratories, Overland Park, KS, USA). After its ability to enhance switchgrass growth was confirmed, a single colony was isolated and sent to Midi Labs in Newark, DE for species identification with 16S rDNA gene sequence similarity and FAME matches.

Switchgrass seed sterilization

Switchgrass (Panicum virgatum L.) cvs. Alamo and Cave-in-Rock seeds were purchased from Warner Brothers Seed Co. (Lawton, OK, USA). Other cultivars were kindly provided by Dr. Bingyu Zhao (Department of Horticulture, Virginia Tech). Switchgrass seed surface-sterilization followed our previous report [9].

Bacterial endophyte culture conditions

The cultures were streaked on Luria Broth (LB) solid medium. Inoculum was produced by transferring one loop of PaKM from 1-day-old cultures to 5 ml LB broth in a 15-ml culture tube, followed by incubation at 28°C on a shaker (200 rpm) overnight. Five ml of the overnight PaKM cultures were added to 45 ml LB medium in a 250- ml Erlenmeyer flask and grown to 0.7 at OD600. Bacterial cells were then collected by centrifugation at 3,500 rpm for 7 min at 4°C, and re-suspended in PBS buffer (10 mM NaH2PO4, 0.8% NaC1, pH 6.5) after which the OD600 was adjusted with PBS buffer to 0.5.

PaKM tagged with GFP for visualization of colonization

In order to visualize colonization of PaKM in plants, PaKM wild type was transformed with plasmid p519ngfp (ATCC® Cat. No. 87453, Manassas, Virginia, USA) using electroporation. The transformants were selected on solid LB medium supplemented with 50 mg/l kanamycin and cultured at 37°C for 2 days. The colonies with GFP were chosen under a fluorescent microscope.

PaKM colonization

The plants inoculated with PaKM-GFP were examined under a fluorescent stereomicroscope equipped with a GFP filter (BP460- 490) (Model SZXILLD2-100; Olympus, Tokyo, Japan) to observe colonization. For bioassays, the control and PaKM-GFP inoculated plants were surface-sterilized with 0.032 M sodium hypochlorite for 1 min, then washed 4X with sterile deionized, distilled water (ddH2O). Fifty μl of the final wash were plated on solid LB medium to confirm effectiveness of surface sterilization. Root, leaf and sheath parts were then separated, weighed, and ground with mortar and pestle in 1 ml sterile ddH2O. The homogenates were then centrifuged at 2000 rpm for 3 min, and the supernatant diluted to 1:10, 1:100, and 1:1000 with sterile ddH2O. Fifty μl samples of the serially diluted solutions were spread on solid LB medium. The plates were incubated for 2 days at 28oC in the dark, and the number of GFP-positive colonies determined under fluorescence stereomicroscopy.

PaKM inoculation and plant growth conditions

Surface-sterilized seeds were germinated on wet filter paper in petri-dishes for 4-6 days at 25°C under white fluorescent light (67 μmol m-2 s-1), 16 h photoperiod. The root tips of the young seedlings were cut prior to PaKM inoculation to facilitate bacterial penetration, and seedlings were inoculated by soaking in PaKM solution (0.5 of OD600) for 1 min. Control seedlings were treated with PBS buffer alone. The treated seedlings were blot-dried with sterile paper towel, placed in GA7 Magenta vessels (Sigma-Aldrich) containing 50 ml of MS medium, 30 g/l maltose and 3 g/l phytogel, pH 5.8. In each vessel, 5 seedlings were placed and grown for 25-30 days in the incubator as above. Root and shoot length, and seedling fresh weight were then determined, and the plants transferred to a soil mix composed of 2/3 Miracle-Gro Potting Mix (Scotts Miracle-Gro Company, Marysville, Ohio, USA) and 1/3 Arabidopsis growing media (Lehle Seeds, Round Rock, Texas, USA). Plants were grown in 72-cavity trays in a growth chamber at a 28/22°C day/night temperature, 16 h photoperiod (white fluorescent bulbs at 88 μmol m-2 s-1) for 30 days, or 4-gallon pots in the greenhouse.

Salt and drought tolerance

Salt and drought tolerances were tested in vitro using PaKM inoculated and non-inoculated Alamo seedlings. Seed sterilization, germination and PaKM inoculation were described above. Inoculated seedlings and controls were then placed in salt stress medium (MS + 100 mM NaCl) or drought stress medium (MS + 7.52 g/L of D-mannitol, which leads to -0.4 MPa), and grown in a growth chamber under the above conditions for 40 days.

Field trial experimental design

The low fertility soil site was chosen at Walden Farm, a historic tobacco farm near Danville, Virginia, USA. Levels of P, K, Ca, and Mg were rated Low, Medium -, Low +, and High, respectively [10]. Additionally, no fertilizers were applied before or during the trial, and only one initial watering was performed at the time of transplanting. A paired experimental design was carried out to reduce soil and environmental variation. The distance between plants and the space between rows were both at 0.76 meter. Seedlings were inoculated following above procedure on July 3, 2012 and transplanted in the field on August 20, 2012. During the second year, root and shoot growth was determined on June 17, 2013 (n=10) after digging up the entire plants and washing roots with tap water. Roots were then blot dried with paper towels, and fresh weights of roots and shoots determined. The plants were then dried at ambient indoor environment (21°C and 35% humidity) for two weeks, and dry weights recorded. Fourteen pairs of aboveground parts were harvested on December 04, 2013 after the plants were dormant.

Root morphology

To determine the effect of PaKM inoculation on root growth and morphology, bacterized and non-bacterized Alamo plants were transplanted in 4-gallon pots containing Miracle Gro® potting mix on March 28, 2013 in a temperature controlled greenhouse at the Institute for Advanced Learning and Research in Danville, Virginia and the entire plant harvested on May 14, 2013. Roots were washed, and fresh and dry weights of roots and shoots determined. The numbers of lateral roots per cm on seminal roots were estimated by counting lateral roots in a 3 cm portion of a randomly selected seminal root and dividing by 3 [10]. The number of seminal roots was also counted on PaKM and control plants as well.

Growth promotion of different switchgrass cultivars by PaKM

Seven different switchgrass cultivars (Forestburg, Nebraska, Shawnee, Blackwell, Shelton, Sunburst and Canthage) were tested for plant growth promotion by PaKM in vitro. Seed surface sterilization and growth conditions were described previously [9].

Auxin quantification

The auxin quantification method was modified from Patten and Glick’s protocol [17]. PaKM bacteria were cultured in 4 ml of LB medium at 200 rpm, 30°C overnight. Twenty μl of overnight bacterial cultures were aliquoted into 4 ml of LB medium containing 100 μg/ ml of L- tryptophan. The cultures were incubated at 30oC for 2 days and centrifuged at 14,000 rpm for 10 min. The supernatant (0.5 ml) was mixed well with 2 ml of Salkowski reagent (15 ml of concentrated H2SO4, 25 ml of ddH2O and 0.75 ml of 0.5 M FeCl3.6H2O) and kept at room temperature for 20 min before measuring absorbance at 540 nm using Beckman Coulter Multimode DTX 880 (Beckman Coulter, Inc., Fullerton, CA, USA). Indole Acetic Acid (IAA) standard curve was prepared using 0.5 ml of IAA at different concentrations (0, 20, 40, 60, 80, 100 μg/ml) following the same procedure described above. The auxin concentration was expressed as μg IAA/mg fresh weigh of bacterial cells.

ACC (1-Aminocyclopropane-1-Carboxylate) deaminase activity

The ACC deaminase activity was measured following Penrose and Glick’s protocol [18]. The absorbance was measured at 540 nm with a Beckman Coulter Multimode DTX 880.

Phosphate solubilization and soluble P quantification

One μl of overnight growing PaKM was placed in the center of Pikovskaya’s agar medium plate, incubated at 30°C and a clear zone (halo) was measured at 6 days. Soluble P was also quantified with Murphy and Riley [19] method. Briefly, one hundred μl of overnight PaKM culture was added to 3.9 ml of NBRIP liquid medium [20] and grown at 30°C, 200 rpm for 3 days. Then liquid culture was centrifuged at 14,000 rpm for 10 min, and soluble P in the supernatant was quantified at 840 nm. The soluble P standard curve was established with KH2PO4.

Statistical analyses

Statistical analyses were performed using student’s t-test except for field experiment. For the field trial, a student’s paired t-test was carried out to reduce soil and environmental variation. Values were assigned to each group and reported at 95%, 99%, or 99.9% confidence levels.


PaKM isolation and identification

The bacterium isolated from surface sterilized seeds was characterized as a yellow gram negative rod shaped bacterium. Based on data from 16S rDNA gene sequence similarity and FAME matches done by Midi Labs in Newark, DE, it was identified to belong to species Pantoea agglomerans (Table 1) and labeled as strain PaKM.