Rhizobacterial colonization of bermudagrass by Bacillus spp. in a Marvyn loamy sand soil
Graphical abstract
Beneficial characteristics of rhizobacteria may explain why inoculation of grass results in growth promotion. Colonization and persistence in grass was determined using antibiotic-resistant mutants.
Introduction
Plant-microbe interactions have been extensively studied, and the understanding of these relationships are increasingly important for continued crop production and protection. Microbes, including plant growth-promoting rhizobacteria (PGPR) in the rhizosphere have been among the most heavily studied soil organisms because of their intimate association with plant, root, and soil health (Singh et al., 2011; Calvo et al., 2014). Previous PGPR studies noting beneficial genera for growth promotion or antibiosis have been more prevalent than studies on ecological or population dynamics. Rhizobacteria and endophytes are associated with nearly every plant, yet relatively few plant species have been studied in detail in relation to plant colonization (Ryan et al., 2008). PGPR increase plant growth and root architecture through improved nutrient cycling, production of plant hormones, reducing or preventing pathogens, as well as changes to plant-water regulations (Compant et al., 2005; Ryan et al., 2008; Calvo et al., 2014; Coy et al., 2014; Gagné-Bourque et al., 2015). While most PGPR work has focused on plant-microbe interactions in the rhizosphere and rhizoplane, PGPR colonization is not restricted to these areas. PGPR or plant growth-promoting endophytic bacteria (PGPEB) typically colonize the endorhizosphere within the root cortex or vascular tissue and move into plant foliage (Baldani and Döberreiner, 1980; Lalande et al., 1989; van Peer and Schippers, 1989; van Peer et al., 1990; Gagné-Bourque et al., 2013; Gagné-Bourque et al., 2015; Santoyo et al., 2016). In addition, bacterial endophytes may also colonize the phyllosphere, anthosphere, or spermosphere (Sturz et al., 2000).
Within the study of plant-microbe interactions of grasses, endophytes are commonly encountered and almost exclusively refer to fungi in cool-season species (Carroll, 1988; Funk et al., 1993; Held and Potter, 2012). However, a more appropriate definition of endophytes refers to both fungi and bacteria that complete all or part of their life cycle within the tissues of plants that result in unapparent or asymptomatic infection of plant tissue with no disease symptoms (Sturz et al., 2000; Santoyo et al., 2016). This broader definition of endophyte is important for turfgrass because bacterial endophytes have been previously isolated from three warm-season grasses, kallar grass (Leptochloa fusca L.), saltmarsh grass (Spartina alterniflora Loisel), and switchgrass (Panicum virgatum L.), as well as the cool-season C3 model grass Brachypodium distachyon (McClung et al., 1983; Reinhold-Hurek and Hurek, 1998; Gagné-Bourque et al., 2013; Gagné-Bourque et al., 2015). The presence of bacterial endophytes in these grasses is likely an indicator that they are more prevalent and are likely to occur in other species, but have been overlooked.
Endophytic Bacillus spp. are commonly reported in corn, cotton, cucumber, grape, peas, soybean, and spruce (Lalande et al., 1989; McInroy et al., 1992; Bell et al., 1995; Hallman et al., 1997; Shishido et al., 1999; Reva et al., 2002; Berg et al., 2005; Durham, 2013). Species within Bacillus that have previously been shown to be endophytic, including B. amyloliquefaciens, B. endophyticus, B. firmus, B. insolitus, B. licheniformis, B. megaterium, B. pumilus, and B. subtilus. The capacity of bacteria to establish internal plant populations within the vascular system may be advantageous, as it allows the bacteria to be in constant contact with plant cells, offers protection from competition with other soil microbes and environmental extremes, which may increase persistence (Shishido et al., 1999; Reinhold-Hurek and Hurek, 1998; Santoyo et al., 2016). Additionally, the nutrient rich, low oxygen environments within the plant and rhizosphere offer optimal condition for nitrogenase activity to fix nitrogen for plant use (Reinhold-Hurek and Hurek, 1998; Sevilla and Kennedy, 2000). Further, PGPR isolated from internal plant organs are sometimes biochemically distinct and are more effective plant colonizers (van Peer et al., 1990). The biochemical changes to PGPR strains from endophytic colonization may increase the efficacy of PGPR. This is likely a result from close associations with plant activities and defenses, demonstrating the adaptability of bacteria to find ecological purposes that form intimate, positive relationships with plants that aid in plant growth or protection from other microbes or abiotic stress (Shishido et al., 1999; Compant et al., 2005).
Some endophytic bacteria induced systemic resistance in plants, which alters, increases, or prevents stress from disease, insects, and nematodes by altering plant signaling compounds that include jasmonic acid, salicylic acid, or ethylene pathway (van Loon et al., 1998; Kerry, 2000; Sturz et al., 2000; Crow, 2014; Coy et al., 2017). While the endophytic plant colonization of bacilli-bacteria is well documented (Reva et al., 2002; Gagné-Bourque et al., 2013, Gagné-Bourque et al., 2015; Durham, 2013), considerable knowledge gaps remain on the levels of colonization and application frequencies, as well as persistence of PGPR for plant growth and protection, especially in perennial cropping systems. Production of turf crops are limited by nutrients, water, temperature, and pests. Nitrogen, in the forms of ammonium NH4+ and nitrate NO3− are the most important nutrients for sustaining plant growth in turfgrass, and are abundantly applied to amenity grass (Frank and Guertal, 2013a). Losses of nitrogen in fertilized turfgrass can be as high as 50% (Barber, 1995; Horgan et al., 2002; Frank and Guertal, 2013a). Avenues of loss are leaching, volatilization, and denitrification which releases nitrous oxide, a greenhouse gas leading to environmental concerns (NRC, 1993). Use of PGPR and other microbial inoculants could allow for a reduction of nitrogen fertilizer rates if they can improve nutrient uptake and efficiency while reducing greenhouse gas emissions (Calvo et al., 2013). Phosphorus, in the form of orthophosphate (PO43−), is the third most important nutrient for turfgrass growth and is often applied during turfgrass establishment for increasing seedling vigor and growth (Beard, 1973; Frank and Guertal, 2013b). In turfgrass and other crops, there are growing environmental concerns over runoff (Steward et al., 2006; Bierman et al., 2010) and leaching (Erickson et al., 2005; King et al., 2006). Forms of phosphorus vary by regions and soil conditions. Aluminum and iron phosphate forms are commonly encountered in the northern and southeastern United States, and calcium phosphate common in the western United States (Frank and Guertal, 2013b). The production of siderophores from bacterial metabolites has been linked to growth promotion by increasing chlorophyll content, disease suppression, and bioremediation (Sharma and Johri, 2003; Sayyed et al., 2013; Calvo et al., 2014).
Using three strains of two Bacillus spp., we assessed rhizobacterial colonization in the economically important bermudagrass system. Coy et al. (2014) noted growth promotion in bermudagrass with select rhizobacterial blends, yet the colonization and persistence of rhizobacteria as well as the mechanisms for growth promotion are not clearly defined. For future development of rhizobacterial products for turfgrass, understanding fluctuations of bacterial populations over time will benefit efforts to develop application frequencies or intervals in a perennial crop. Additional research was conducted to identify the beneficial characteristics of select rhizobacterial strains to provide a better understanding of the mechanisms which may be used for growth promotion. Rhizobacterial strains were evaluated for nitrogenase activity, phosphate solubilization, and siderophore production.
Section snippets
Bacterial strains and rifampicin marking of bacteria
Rifampicin-resistant mutants of PGPR strains were created and tested for persistence and colonization in soil and in planta. While there are many marking systems for bacteria, the rifampicin marking system is advantageous as it is an inexpensive marking tool that is uncommonly found in soil bacteria (McInroy et al., 1992, McInroy et al., 1996). Further, it allows for the isolation and culturing of marked bacterial strains from a non-sterile environment in the presence of native soil microbes.
Colonization of rifampicin resistant bacteria in bermudagrass
All rhizobacterial strains were recoverable within 24 h of inoculation of bermudagrass and for the duration of the experiment (Fig. 1). Populations of all three strains were epiphytic and endophytic in foliage and roots. Temporary loss of antibiotic resistance was observed with all endophytic bacterial isolates for each strain at some point during the experiment. No bacterial colonies grew from the non-treated control samples on RTSA plates; however, fungal isolates were often observed.
Discussion
This study is the first documenting colonization and persistence of bacterial endophytes associated with growth promotion in an amenity grass. Previously bacterial endophytes were only reported from saltmarsh and bioenergy grasses (McClung et al., 1983; Reinhold-Hurek and Hurek, 1998; Gagné-Bourque et al., 2013). In field conditions, populations of all three PGPR strains rapidly colonized rhizosphere soil, as well as external and internal plant tissues. Generally, bacterial populations
Acknowledgements
John McInroy provided technical assistance and training on the development of the antibiotic resistance mutation bacterial lines used in this research. This paper is based upon work supported in part by the Alabama Agricultural Experiment Station through Hatch funding from the National Institute of Food and Agriculture, United States Department of Agriculture.
References (63)
- et al.
Importance of Acermonium endophytes in turfgrass breeding and management
Agric. Ecosyst. Environ.
(1993) - et al.
A modified single solution method for the determination of phosphate in natural waters
Anal. Chim. Acta
(1962) - et al.
Temporary loss of antibiotic resistance by marked bacteria in rhizosphere of spruce seedlings
FEMS Microbiol. Ecol.
(2002) - et al.
Life in grasses: diazotrophic endophytes
TrendsMicrobiol.
(1998) - et al.
Plant growth-promoting bacterial endophytes
Microbiol. Res.
(2016) - et al.
Universal chemical assay for the detection and determination of siderophores
Anal. Biochem.
(1987) - et al.
Growth promoting influence of siderophore-producing Pseudomonas strains GRP3A and PRS9 in maize (Zea mays L.) under iron limiting conditions
Microbiol. Res.
(2003) - et al.
Endophytic colonization of spruce by plant growth-promoting rhizobacteria
FEMS Microbiol. Ecol.
(1999) - et al.
Efficient soil microorganisms: a new dimension for sustainable agriculture and environmental development
Agric. Ecosyst. Environ.
(2011) - et al.
Endophytic colonization of potato (Solanum tuberosum L.) by a novel competent bacterial endophyte, Pseudomonas putida strain P9, and its effect on associated bacterial communities
Appl. Environ. Microbiol.
(2009)
Host plant specificity in the infection of cereals with Azospirillum spp
Soil Biol. Biochem.
Soil Nutrient Bioavailability: A Mechanistic Approach
Turfgrass: Science and Culture
Endophytic bacteria in grapevine
Can. J. Microbiol.
Impact of plant species and site on rhizosphere-associated fungi antagonistic to Verticillium dahlia Kleb
Appl. Environ. Microbiol.
Phosphorus runoff from turfgrass as affected by phosphorus fertilization and clipping management
J. Environ. Qual.
Microbial-based inoculants impact nitrous oxide emissions from an incubated soil medium containing urea fertilizers
J. Environ. Qual.
Agricultural uses of plant biostimulants
Plant Soil
Fungal endophytes in stems and leaves: from latent pathogen to mutualistic symbiont
Ecology
Endophytic colonization of Vitis cinifera L. by a plant growth promoting bacterium, Burkholderia sp. strain PsJN
Appl. Environ. Microbiol.
Rhizobacterial inoculants increase root and shoot growth in ‘Tifway’ hybrid bermudagrass
J. Environ. Hortic.
Bacterial inoculant treatment of bermudagrass alters ovipositional behavior, larval and pupal weights of the fall armyworm (Lepidoptera: Noctuidae)
Environ. Entomol.
Effects of a commercial formulation of Bacillus firmus I-1582 on golf course bermudagrass infested with Belonolaimus longicaudatus
J. Nematol.
Isolation and identification of aerobic nitrogen-fixing bacteria from soil and plants
Characterization of Root Colonization by the Biocontrol Bacterium Bacillus firmus Strain GB126
Phosphate Solubilization Potential of Rhizosphere Fungi Isolated from Plants in Jimma Zone, Southwest Ethiopia
International Journal of Microbiology
Phosphorus and potassium leaching under contrasting residential landscape models established on sandy soil
Crop Sci.
Nitrogen research in turfgrass
Potassium and phosphorus research in turfgrass
Isolation and characterization of indigenous endophytic bacteria associated with leaves of switchgrass (Panicum virgatum L.) cultivars
J. Appl. Microbiol.
Accelerated growth rate and increased drought stress resilience of the model grass Brachypodium distachyon colonized by Bacillus subtilis B26
PLoS ONE
Cited by (21)
Evaluation of the anti-oomycete bioactivity of rhizosphere soil-borne isolates and the biocontrol of soybean root rot caused by Phytophthora sojae
2022, Biological ControlCitation Excerpt :Bacillus SN337 could reduce the harmful effects caused by P. sojae and improve the soil environment as well as the health status of soybean plants. The bacterial colonization is crucial for the biocontrol effects of biocontrol microorganisms such as Bacillus species (Coy et al., 2019). Therefore, in this study, the strain SN337 was rationally supposed to effectively colonize surface and endorhizosphere of soybean root or influence on soil rhizosphere for playing sustainable biocontrol roles.
Endophytic halotolerant Bacillus velezensis FMH2 alleviates salt stress on tomato plants by improving plant growth and altering physiological and antioxidant responses
2021, Plant Physiology and BiochemistryCitation Excerpt :Overall, external colonization of the rhizoplane was significantly greater than internal colonization of the plant components (Fig. 2). These findings were in concordance with results reported by Coy et al. (2019) in which rhizosphere and rhizoplane colonization by B. sphaericus AP282 were significantly greater than endorhizal populations. Subsequently, B. velezensis FMH2 could be considered as epiphytic and endophytic strain in foliage and roots, thereby it could be admitted as a PGPB.
Improving the Efficiency of the Indoor Air Purification from Formaldehyde by Plants Colonized by Endophytic Bacteria Ochrobactrum sp.
2023, Water, Air, and Soil PollutionThe impact of commercially available microbial inoculants on bermudagrass establishment, aesthetics, and function
2022, Crop, Forage and Turfgrass Management