In the zone (Research)

The soil environment immediately around a root frequently has a larger number of microorganisms compared to soil just a few millimeters away from a root. This zone of influence is called the rhizosphere (Rovira, 1991), which is composed of many groups of organisms that are capable of affecting plant health beneficially and deleteriously (Schippers et al., 1987).

Putting greens are artificially constructed soils, built from a predetermined mixture usually composed of sand and organic matter (USGA Green Section staff, 1993). In the Southeastern U.S., newly built putting greens are often fumigated before planted. However, previous research shows microbial populations present before fumigation rebound quickly after fumigation (Elliott and Des Jardin, 2001; Elliott et al., 2004). Additionally, as the putting greens mature, thatch, root and shoot production will cause significant increases in organic matter (Gaussoin et al., 2006), which will promote microbial growth.

Natural materials, organic materials and microbial inoculants are used by the golf course industry because there’s an assumption few microbes are present in the turfgrass system or the “wrong” microbes are present. However, recent studies indicate turfgrass systems have extensive microbial populations (e.g., Bigelow et al., 2002; Elliott and Des Jardin, 1999; Elliott et al., 2004; Feng et al., 2002; Mercier, 2006) and diverse microbial communities (e.g., Mueller and Kussow, 2005; Sigler et al., 2001; Yao et al., 2006). Also, it’s unclear whether introduced bacteria can influence bacterial populations in the phyllosphere, thatch, rhizosphere soil or bulk soil (Hodges et al., 1993; Lynch, 2002; Mercier, 2006; Mueller and Kussow, 2005; Sigler et al., 2001).

The emphasis of the project described herein was on culturable bacteria because it’s culturable bacteria that are being exploited by the golf course industry. In other words, if you can’t grow bacteria in large quantities (by a company or directly on the golf course in fermentation tanks), they aren’t useful as products. While we know there’s a diverse microbial community present in turfgrass root systems, it’s not known which culturable fluorescent pseudomonad species or culturable bacilli species are present.

A joint project was undertaken by Auburn University, Clemson University and the University of Florida to examine bacterial populations and diversity in USGA putting greens during a three-year period after the greens were established. We’ve reported on the flux of the extensive bacterial populations present in putting greens (Elliott et al., 2004), the effect of nitrogen rate and root-zone mix on rhizosphere bacterial populations (Elliott et al., 2003), and the identification of a diverse group of denitrifying bacteria from putting greens (Wang and Skipper, 2004). This report summarizes which culturable bacterial genera and species were present and dominant in bentgrass and Bermudagrass putting greens in the Southeastern U.S. (Elliott et al., submitted).

Study sites

The bentgrass (Crenshaw) putting greens are located at the Charlotte (N.C.) Country Club Golf Course and Auburn (Ala.) University. The hybrid Bermudagrass (Tifdwarf) putting greens are located at the Cougar Point Golf Course in Kiawah Island, S.C., and the University of Florida in Fort Lauderdale. All four sites were fumigated with methyl bromide before planting the turfgrass. Putting greens at university sites (Alabama and Florida) are miniature versions of those on golf courses. All greens were managed in a manner typical for the region.

Rhizosphere sample

Four putting greens from each location were sampled four times a year (about every three months) for a minimum of three years in 1997 to 2000. Ten cores (0.40 inch by 4 inches) were collected per putting green to constitute a sample. Green tissue was removed from each core with a sterile razor blade. For each sample, turfgrass roots were separated from the root-zone mix, and all root material and rhizosphere soil was subjected to shaking in a sterile diluent. Aliquots of dilutions were spread plated onto duplicate plates of selective and nonselective media (Elliott et al., 2004). For enumeration of total aerobic bacteria and selection of bacteria for identification with GC-FAME, solidified 10 percent tryptic soy broth (10 percent TSBA), amended with 100 µg mL-1 cycloheximide to inhibit fungi, was used. For each sampling date, 40 bacterial isolates per green sampled were randomly selected from the 10 percent TSBA for future identification. An estimated 10,000 bacterial isolates were selected for identification during the course of this study.

ID bacteria isolates

Analysis of the bacterial isolates was conducted using the GC-FAME/Microbial Identification System (MIDI in Newark, Del.) at Auburn University or at the Multi-user Laboratory at Clemson University. Isolates were processed according to the protocol for aerobic bacteria of environmental origin (Sasser, 2006). Fatty acid peak profiles were analyzed using the Sherlock Standard Aerobe Libraries (MIS version 4.0, Microbial ID, www.midi-inc.com). According to literature provided by MIDI, strains with a similarity index of 0.50 or greater are considered a good match at the species level, whereas strains with a similarity index between 0.30 and 0.49 are considered a good match at the species level but indicates an atypical strain (Anonymous, 2005a). Because the bacterial species present in putting greens were largely unknown when this study was initiated, a similarity index of 0.30 or greater was used as the basis for identifying bacterial isolates.

Bacterial genera

A total of 9,216 bacterial isolates were analyzed using the GC-FAME/Microbial Identification System. Overall, there were 50, 57, 64 and 64 bacterial genera identified in Alabama bentgrass, North Carolina bentgrass, Florida Bermudagrass and Sout Carolina Bermudagrass, respectively. There were 76 genera identified at both Bermudagrass sites, with 13 unique to Florida, 13 unique to South Carolina and 50 common to both. There were 59 genera identified at both bentgrass sites, with three unique to Alabama, nine unique to North Carolina and 47 common to both. Forty genera were common to all four sites.

There were five genera that composed at least 1 percent of the isolates at all four sites (Bacillus, Clavibacter, Flavobacterium, Microbacterium and Pseudomonas, with Bacillus and Pseudomonas the dominant bacterial genera at each location. However, the percentage of isolates identified as Bacillus in the Bermudagrass sites was almost twice the number of isolates identified as Pseudomonas. At the bentgrass sites, Pseudomonas was either the dominant genus (North Carolina) or was equal to Bacillus (Alabama). This is consistent with the previously reported enumeration data that Bacillus is the dominant genus over Pseudomonas in the Bermudagrass rhizosphere, and that significantly greater numbers of fluorescent pseudomonads are found in the bentgrass rhizosphere than in the Bermudagrass rhizosphere (Elliott et al., 2004).

Arthrobacter composed a significant portion of the bacterial isolates at the bentgrass sites (9.1 percent at Alabama and 7.5 percent at North Carolina), with only Bacillus and Pseudomonas composing a greater percentage of the isolates identified. While Stenotrophomonas was identified from all four sites, it composed at least 1 percent of the isolates only at the Florida and South Carolina Bermudagrass sites.

Bacterial species

There were five species that composed at least 1 percent of the isolates at all four sites: Bacillus cereus, B. megaterium, Clavibacter michiganensis, Flavobacterium johnsoniae and Pseudomonas putida. Another three species – Agrobacterium radiobacter, B. pumilus and B. thuringiensis – composed at least 1 percent of the isolates at the Alabama, Florida and South Carolina sites, but not the North Carolina site. A fourth species, Comamonas acidovorans, composed at least 1 percent of the isolates at the NC, AL and Florida sites but not the South Carolina site. One species was common at the 1-percent level only to the Bermudagrass locations: Stenotrophomonas maltophilia. Four species were common at the 1-percent level only to the bentgrass locations: Arthrobacter ilicis, P. chlororaphis, P. fluorescens and P. syringae. Figures 1 to 4 illustrate examples of species composition for single dates at each study site.

Unidentifiable isolates

The number of unidentifiable isolates (similarity index of less than 0.30) was 50.1 percent for Florida Bermudagrass, 38 percent for South Carolina Bermudagrass, 34.3 percent for Alabama bentgrass and 32 percenty for North Carolina bentgrass (Table 1). These values fall within the range of unidentifiable isolates obtained in other studies using GC-FAME for identification purposes (Germida and Siciliano, 2001; Gooden et al., 2004; Kim et al., 2001/2002; Mahaffee and Kloepper, 1997; Poonguzhali et al., 2006; Siciliano and Germida, 1999). Thus, the number of unidentified isolates in this study, obtained from an artificially constructed soil, would appear to be similar to the number from field soils in the same states using the same identification system.

Why are some bacterial isolates not identified? The MIDI aerobe bacteria library includes fatty acid profiles for 695 environmental species, with usually 20 or more strains representing each species or subspecies (Anonymous, 2005b; Sasser, 2006). Our results and those of others illustrate that a significant number of bacteria isolated from bulk or rhizosphere soils aren’t part of the bacterial collection that’s the basis of the MIDI environmental species library. Any database is only as good as the data – in this case, fatty acid methyl ester profiles of bacterial isolates – accumulated within it. The unidentifiable isolates aren’t necessarily new species per se but simply might be species not represented in the MIDI database.

Taxonomic diversity

This is the first study to survey for a portion of the culturable, aerobic bacterial genera and species common to golf course putting greens in the southeastern U.S. It demonstrates there’s considerable taxonomic diversity present in the rhizosphere of putting greens, despite their intense management. Obviously, while we have identified some of the bacteria genera and species present in golf course putting greens, there still are many unidentified bacteria. Even less information is known regarding what these bacteria do in the turfgrass system. GCI

About the authors:

M.L. Elliott, Ph.D., is professor and associate center director in the department of plant pathology at the University of Florida in Fort Lauderdale; J.A. McInroy is a research associate in the department of entomology and plant pathology at Auburn University in Alabama; K. Xiong, J.H. Kim and H.D. Skipper, Ph.D., are professors in the department of entomology, soil and plant science at Clemson University in South Carolina; and E.A. Guertal, Ph.D., is a professor of turfgrass management in the department of agronomy and soils at Auburn University.

Acknowledgements: This research was supported, in part, by a grant from the USGA, and by the Agricultural Experiment Stations of Alabama, Florida and South Carolina. The authors gratefully acknowledge the cooperation of M. Stoddard and M. Pilo at the Charlotte Country Club and K. Bibler and K. Wiles at the Cougar Point Golf Club.

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