In counting millions of bacteria from the soil, microbiologists use a technique called "dilution plating." A soil or root sample is mixedin a liquid and then diluted to a point that will allow the bacteria to grow as distinct colonies on selective or non-selective media.

BLACI( BOX RESEARCHSeeking answers to the effectiveness of bacterial inoculents.by MoNICA L. ELLIOTf, Ph.D.

TIE PLANT rootzone, oftenreferred to as,the rhizosphere, istruly a black box of the microbial

unknown (Bowen and Rovira, 1991).While extensive research has beenconducted on the roots of annualcrops such as com, wheat, and cotton(Pankhurst et a1., 1996), research onturfgrass roots and the soils or root-zone mixes they inhabit has only justbegun. When so few facts are known,myths are bound to develop. A com-mon myth is that golf course soils,especially putting greens, are sterileenvironments with few microbes. Wehave already learned that is not true(Mancino et a1., 1993; Liu et a1., 1995;Elliott and Des Jardin, 1999a). What wewant to learn now, if possible, is how

6 USGA GREEN SECTION RECORD

to manipulate microbial populations inthe rootzone.

TerminologyFirst, definitions of commonly used

terms are in order. Microbes refers tobacteria and fungi. In this article, onlybacteria will be discussed. Bacterianormally are single-cell organismswhose genetic material is not separatedfrom the rest of the cell by a membrane.In contrast, fungi and plant cells dohave their genetic material (nucleus)separated by a membrane. Colonyforming units (CFU) is the measure-ment used to describe bacterial popu-lations. In theory, each colony on aplate represents a single cell. Becausebacterial numbers are so large, they are

converted to log values. Instead ofstating there are 2,700,000 CFU, westate there are log106.4CFU.

Common bacterial groups referred toin this article are: 1)fluorscent pseudo-monads that glow in the dark with aUV light, 2) gram-negative bacteria,3) gram-positive bacteria, 4) heat-tolerant bacteria that survive a 10-minute soak in a 176°F water bath, and5) actinomycetes that look like fungi.Gram reactions (positive or negative)refer to a simple test that differentiatesbacteria, based primarily on their cellwall components. Most heat-tolerantbacteria produce a specialized cellcalled an endospore. Actinomycetesalso produce spores, but they are nottolerant of extreme heat. They are

often confused with fungi, but they arenot related. The five groups listed aboveare detected using selective media.

A sixth group of bacteria is referredto as total aerobic bacteria. They rep-resent all the bacteria that will grow ona non-selective medium. The counts forthe other groups will not add up tothe count for the "total" group. It iscritical to understand that this tech-nique of growing bacteria on mediaonly accounts for approximately 10%of the bacteria in the soil (Olsen andBakken, 1987). We have not learnedhow to grow most bacteria in thelaboratory. This is just one of manytechniques for examining bacterialpopulations. We use this techniquebecause we want to save many of thebacteria we isolate for future research.

Background ResearchIn a previous study we examined the

effects of four natural organic nitrogenfertilizers on bacterial populations ofan established (four years old) ber-mudagrass putting green (Elliott andDes Jardin, 1999a). For two yearsthese products, plus a synthetic organicfertilizer, were applied to the puttinggreen every two weeks at the normalSouth Florida annual rate of 18poundsN per 1,000 square feet. Significant dif-

A highly magnified look at actinomycetespores. They may look like fungi, butthey are bacteria. This group is thesource of most antibiotics.

ferences in bacterial populations due tofertilizer treatments were not observed.In other words, bacterial populationsremained the same despite usingnatural organic fertilizers, even the twofertilizers that had bacteria added tothem. There also were no consistentdifferences observed among the fer-tilizer treatments on turfgrass growthor quality (Elliott and Prevatte, 1997).

Every scientist wants to obtaindramatic, eureka!-type results, but wedid not. Why? Two reasons mayexplain our results. First, since the putt-ing green was maintained as a regulargreen, it was topdressed routinely(every two weeks). The topdressing

(80/20 mix) did contain bacteria; infact, more than the fertilizers. Second,since this was an established green,perhaps an optimum level of bacteriahad already been obtained.

New ResearchFor the next project, research started

at the beginning - the very beginningof putting green construction. Ques-tions to be considered were: Do brand-new greens have different bacterialpopulations? Would peat sources affectbacteria? Does fumigation kill all thebacteria in the rootzone mix? Whatbacteria rebound first after fumigation?What bacteria are associated with thebermudagrass sprigs?

At the research center, trencheswere dug for placement of 100-gallonLerio™ tree containers, 36 inchessquare and 18 inches deep, to buildmini-greens. A 6-inch layer of non-calcareous, washed river gravel wasplaced in the bottom of each container.No intermediate layer was added, asthe gravel and the two rootzone mixesevaluated complied with USGA recom-mendations. The rootzone mixes,matched for physical characteristics,contained either Canadian sphagnumpeat (85/15 mix) or Dakota reed sedgepeat (93/7 mix). The subplot or second

Mini-greens were constructed in 36-inch square, i8-inch deep containers at the University of Florida Research Center in FortLauderdale. Soil and turfgrass samples were taken throughout the experiment to determine population sizes of six bacterial groups.

NOVEMBER/DECEMBER 2000 7

*Values are based on gnam dry weight of ei1l1~reach component, rootzone m~, or plant material

JSand~one

Spha.~num peat alone

,R~ed sedge peat alone

Bennudagnasssprigs 4.5

o

4.1"'" '"

4.5

4.5

7.1

Total(;!

5.0

6.6'

8.4

5.8

6:3

8.0

factor in the experiment was fumi-gation type. The containers were eithernot fumigated (control) or were fumi-gated with methyl bromide (1 poundper cubic yard, injected) or metamsodium (2 gallons Metam 326 per 1,000square feet).

Soil and/or turfgrass samples wereobtained to determine population sizesof the six different bacterial groups forthe individual rootzone componentsprior to blending and each rootzonemix after blending, but before place-

ment in containers. Samples also wereobtained from each container just priorto fumigation, 10days after fumigation,25 days after fumigation, and eachmonth after planting of bermudagrassfor five months. Samples also wereobtained of the bermudagrass sprigsprior to planting. Protocol for theexperiment was based on previousbermudagrass research (Elliott and DesJardin, 1999b).

Bacterial populations associatedwith individual mix components, final

mixes, and bermudagrass sprigs arepresented in Table 1. The sand andsphagnum peat contained the lowestnumber of bacteria, with one group notdetected at all (fluorescent pseudo-monads). All bacterial groups weredetected in the reed sedge peat. Theywere also all detected on the grass.This is important to remember sincebermudagrass is vegetatively propa-gated and not seed propagated.

When the containers were sampledjust prior to fumigation, there were

Q.8

6.30

5.go1.0

TotalActinomycetes ""Heat-tolerantw.,/ u '~

4.45.35.2

4.9Gram-negative

" ""~' ','

loglOcolony fonning units of bacteria per gram dry weightof~oil*

Table 2Effect of fumigapon on bacterial populations in putting .green rootzone mixes. Since significant

differences were due to fuqtigations, results from the two mixes were combined 'for :~~Iysis.

,~~~~gati~n.10,daYs 8(ter fumigation

, ''''GQntiol(no fumigation)'MetamSodiumMethyl Bromide

",25 days after fumigationControl (no fumigation)

, Metam SodiumMetljyl Bromide

!'so.daYs after fwnigation, ~"Gotlir,61 (no fumigation)' 5.51 .

. M<rtanlSodium ' . ' J"~ 5.&,iMethylaromide .,4.4l 4~61. ,~~i;;':1\ ~:, ", 5.614.81

A, ",' ' ....•• , ".,;' ... " , %\ "'. '\'~'S\.~,\. ,;,' , ..... ~,(" ,Iii ~,i,:',,:, ",\,'!'":",,,::,, " ~'!ii 1)

'w.:*Valuesfollowed by a :0 are asignifi~t decrease (P = 0.05) from the Pre,"ffunigati9n'v.~lue~~ValuesJqlI,9we~i':;byah I'are:a' significant \\<

inctease over the pre-fumigationvCllues ..Ifvalues-have no '0 ,or. I; then' they, arc 'not sigriificantly'di~~~rttfromtft.e pre-ftunigation '~ues . .statistical comparison conducted using Dunnett's t-test, I'=0.Q5~' rtt" ;;J,>: ~ . '? '" ,

if iJd, " ,J-\'" ~ '~!_(;~\ ',' ~'(0:~~':' ,. , ,

8 USGA GREEN SECTION RECORD

greater numbers of Gram-negativebacteria and total bacteria in the sandisphagnum peat mix than in the sandireed sedge peat mix. The actinomycetesand heat-tolerant bacteria were greatestin the reed sedge peat mix. After fumi-gation, there was no overall significantdifference between rootzone mixes.Significant differences observed inbacterial populations were due to thefumigation method (Table 2). Ten daysafter fumigation, the least number ofeach bacterial population was oftenassociated with the metam sodiumfumigant. At 25 days after fumigation,the most interesting observation wasthat the fluorescent pseudomonadswere no longer detected in any of thecontainers fumigated with methylbromide. Although the bermudagrasshad been planted for one month at 50days after fumigation, the containerswere less than 50% grown-in. There-fore, we conducted a third post-fumi-gation sampling of just the soil. By thistime, all the bacterial populations hadrebounded and were either equal to orgreater than the pre-fumigation popu-lations.

When the results from the fourmonthly post-planting turfgrasssamples were evaluated, there were nosignificant differences due to type ofrootzone mix or fumigation method.Differences were due to the month thesamples were obtained. The highestcounts were for four and five monthspost-plant, whereas the lowest countswere for two and three months post-plant (Table 3).

ConclusionsAs we have demonstrated, bermuda-

grass putting greens are not devoid of

.~months post"plant>ill

.'.~mo~!hs post-plant l

'.'r" ;_'),~ ~~ .~'< \'~

:4 months post-plant

bacteria. Even newly constructedgreens rapidly build up bacterial popu-lations that include the most commongroups associated with soils.

While the goal of research projects isto answer questions, new questions willalso be raised. One concerns the appli-cation of general bacterial inoculantson the golf course. Some productsclaim to include 20 or more differentbacteria. Because these products arenot regulated and usually not evalu-ated, it is difficult to know what theconsumer has actually purchased. Ifa product's only claim is to increasebacterial populations in the soil, this isprobably a questionable benefit. Evenif a product has proven to be effectiveon bentgrass, it does not mean it willbe effective on bermudagrass. Also,some products contain more than justmicrobes. Often, nutrients or planthormones are part of the formulationalso.

The superintendent's best researchtool for determining if a product willbenefit a course is a piece of plywood.Simply place the plywood in the centerof the green before applying themicrobial inoculant product. Evaluatethis control plot in comparison to thearea treated with the product. Alter-natively, apply the product to only onehalf of each green. Unwilling to takethese chances? Then why are youtaking a chance with the product inthe first place?

ReferencesBowen, G. D., and A. 0. Rovira. 1991. Therhizosphere, the hidden half of the hiddenhalf. Pages 641-649 in Plant Roots - TheHidden Half. Y Waisel and U. Kafkafi, eds.Marcel Dekker, New York.

Elliott, M. L., and M. Prevatte. 1997.Effectof natural organic nitrogen fertilizers onhybrid bermudagrass growth. HortTech-nology 7:289-292.Elliott, M. L., and E. A. Des Jardin. 1999a.Effect of organic nitrogen fertilizers onmicrobial populations associated with ber-mudagrass putting greens. Biology andFertility of Soils 28:431-435.Elliott, M. L., and E. A. Des Jardin. 1999b.Comparison of media and diluents forenumeration of aerobic bacteria from ber-mudagrass golf course putting greens.Joumal of Microbiological Methods 34:193-202.Liu, L. X., T. Hsiang, K. Carey, and J. L.Eggens. 1995. Microbial populations andsuppression of dollar spot disease increeping bentgrass with inorganic andorganic amendments. Plant Disease 79:144-147.Mancino, C. E, M. Barakat, and A. Maricic.1993. Soil and thatch microbial populationsin an 80% sand:20% peat creeping bent-grass putting green. HortScience 28: 189-191.Olsen, R. A., and L. R. Bakken. 1987.Viability of soil bacteria: optimization ofplate-counting technique and comparisonbetween total counts and plate countswithin different size groups. MicrobialEcology 13:59-74.Pankurst, C. E., K. Ophel-Keller, B. M.Doube, and VVS.R. Gupta. 1996. Bio-diversity of soil microbial communitiesin agricultural systems. Biodiversity andConservation 5:197-209.

MONICA L. ELLIOTT, Ph.D., is anassociate professor of plant pathologywith the University of Florida at the FortLauderdale Research and EducationCenter. Her primary research interests aresoil-borne fungal pathogens and soil!rhizosphere microbiology.

NOVEMBER/DECEMBER 2000 9