screening for plant growth–promoting rhizobacteria to promote early soybean growth

DIVISION S-3—SOIL BIOLOGY & BIOCHEMISTRY

Screening for Plant Growth-Promoting Rhizobacteriato Promote Early Soybean Growth

A. J. Cattelan, P. G. Hartel,* and J. J. Fuhrmann

ABSTRACTAlthough many studies have been conducted to identify the specific

traits by which plant growth-promoting rhizobacteria (PGPR) pro-mote plant growth, usually they were limited to studying just one ortwo of these traits. We selected 116 isolates from bulk soil and therhizosphere of soybean [Glycine max (L.) Merr.] and examined themfor a wide array of traits that might increase early soybean growthin nonsterile soil (PGPR traits). A subsample of 23 isolates, all butone of which tested positive for one or more of these PGPR traits, wasfurther screened for traits associated with biocontrol, (brady)rhizobialinhibition, and rhizosphere competence. Six of eight isolates positivefor 1-aminocyclopropane-l-carboxylate (ACC, a precursor of ethyl-ene) deaminase production, four of seven isolates positive for sidero-phore production, three of four isolates positive for p-l,3-glucanaseproduction, and two of five isolates positive for P solubilization in-creased at least one aspect of early soybean growth. One isolate,which did not share any of the PGPR traits tested in vitro exceptantagonism to Sclerotium rolfsii and Sclerotinia sclerotiorum, alsopromoted soybean growth. One of the 23 isolates changed bradyrhizo-bial nodule occupancy. Although the presence of a PGPR trait invitro does not guarantee that a particular isolate is a PGPR, theresults suggest that rhizobacteria able to produce ACC deaminaseand, to a lesser extent, |J-l,3-glucanase or siderophores or those ableto solubilize P in vitro may increase early soybean growth in nonster-ile soil.

THE MECHANISMS by which PGPR promote plantgrowth are not fully understood, but are thought to

include: (i) the ability to produce or change the concen-tration of the plant hormones indoleacetic acid (IAA;Mordukhova et al., 1991), gibberellic acid (Mahmoudet al., 1984), cytokinins (Tien et al., 1979), and ethylene(Arshad and Frankenberger, 1991; Glick et al., 1995);(ii) asymbiotic N2 fixation (Boddey and Dobereiner,1995; Kennedy et al., 1997); (iii) antagonism againstphytopathogenic microorganisms (e.g., Fusarium spp.;Scher and Baker, 1982) by production of siderophores(Scher and Baker, 1982), (3-1,3-glucanase (Fridlender etal., 1993), chitinases (Renwick et al., 1991), antibiotics(Shanahan et al., 1992), and cyanide (Flaishman et al.,1996); and (iv) solubilization of mineral phosphates andother nutrients (Sperber 1958a, 1958b; De Freitas et al.,1997). Many of the studies with PGPR show plantgrowth promotion, but only under gnotobiotic condi-

A.J. Cattelan and P.O. Hartel, Dep. of Crop & Soil Sciences, 3111Plant Sciences Bldg., Univ. of Georgia, Athens, GA 30602-7272; J.J.Fuhrmann, Dep. of Plant and Soil Sciences, Univ. of Delaware, New-ark, DE 19717-1303. Received 19 June 1998. *Corresponding author([email protected]).

Published in Soil Sci. Soc. Am. J. 63:1670-1680 (1999).

tions (Tien et al., 1979; Shenbagarathai, 1993; Glick etal., 1995) or in potting media (Polonenko et al., 1987;Fuhrmann and Wollum, 1989b) where these bacteria donot compete with the normal array of soil microor-ganisms.

There are some cases where PGPR may promoteplant growth in nonsterile soil by controlling fungal dis-eases. The addition of a siderophore-producing Pseu-domonas putida converted a Fusarium-conducive soilinto a Fusarium-suppressive soil for the growth of threedifferent plants (Scher and Baker, 1982). An isolateof Pseudomonas cepacia, positive for (3-1,3-glucanaseproduction, decreased the incidence of diseases causedby Rhizoctonia solani, Sclerotium rolfsii, and Pythiumultimum (Fridlender et al., 1993). Similarly, five fluores-cent Pseudomonas isolates, each positive for antibioticproduction, promoted potato (Solarium tuberosum L.)growth in nonsterile soil (Kloepper and Schroth, 1981).

In addition to the previously described PGPR traits,some rhizobacteria can promote plant growth indirectlyby affecting symbiotic N2 fixation, nodulation, or noduleoccupancy. In a competition study where soybean wascoinoculated with Bradyrhizobium japonicum USDA110 and USDA 118 and one of 17 different isolates ofrhizobacteria, nine rhizobacterial isolates increased theweights of nodules formed by B. japonicum USDA 110and three rhizobacterial isolates increased the numberof nodules (Polonenko et al., 1987). Similarly, Fuhr-mann and Wollum (1989b) found three fluorescentpseudomonads that consistently increased nodule occu-pancy of the more efficient B. japonicum USDA 110over the less efficient B. japonicum USDA 123 andUSDA 31 (now B. elkanii) in soybean grown in a pottingmedium with low Fe availability. Presumably thesepseudomonads increased nodule occupancy because oftheir siderophore production. Growth factors such asvitamins might also indirectly affect the growth of(brady)rhizobia in the rhizosphere (Derylo and Skorup-ska, 1993; Streit and Phillips, 1996). Because somestrains of the genus Bradyrhizobium do not producebiotin (Holt et al., 1994), these bradyrhizobial strainsmay benefit when grown with biotin-producing bacteria.

In addition to these direct and indirect traits, PGPRmust also be rhizosphere competent and able to survivein soil. Traits associated with rhizosphere competence

Abbreviations: ACC, 1-aminocyclopropane-l-carboxylate; ANOVA,analysis of variance; DAP, days after planting; IAA, indoleaceticacid; FAME, fatty acid methyl ester; PGPR, plant growth-promotingrhizobacteria; TSA, trypticase soy broth solidified with agar; TSB,trypticase soy broth.

1670

CATTELAN ET AL.: SCREENING FOR RHIZOBACTERIA TO PROMOTE EARLY SOYBEAN GROWTH 1671

and survival in soil include an ability to tolerate a reason-able range of abiotic factors including temperature, pH,and moisture (Sylvia et al., 1998). Furthermore, PGPRshould not be associated with traits attributed to delete-rious rhizobacteria. These include production of cellu-lase and pectinase (Ulrich, 1976; Chaterjee and Starr,1980). Cyanide production is an ambiguous trait and issometimes associated with deleterious as well as benefi-cial rhizobacteria (Bakker and Schippers, 1987; Alstromand Burns, 1989).

We screened 116 bacteria isolated from bulk soil andsoybean rhizosphere for putative PGPR traits that mightpromote some aspect of early soybean growth such asshoot height, root length, shoot and root dry weight,and nodule number and dry weight. The traits we testedwere production of siderophores, IA A, chitinase, (3-1,3-glucanase, ACC deaminase, and cyanide, P solubiliza-tion, biotin prototrophy, asymbiotic N2 fixation, and in-hibition of growth of three pathogenic soil fungi. Asubsample of 23 isolates, all but one of which testedpositive for one or more of the above traits, was furtherscreened for production of volatile antifungal com-pounds, inhibition of growth of eight strains of (brady)-rhizobia, production of cellulase and pectinase, andgrowth at three different temperatures and at pH 5.5and 7.0. The 23 isolates were tested in nonsterile soilunder greenhouse or lightroom conditions for promo-tion of early soybean growth. A further subsample ofPGPR, which promoted some aspect of early soybeangrowth, was coinoculated with B. japonicum USD A 110to determine their ability to alter nodule occupancy.

MATERIALS AND METHODSOrigin of Bacteria

A subsample of 116 bacterial isolates was selected from asample of 1131 isolates from bulk soil and the rhizosphereof soybeans (Cattelan et al., 1998). Subsample isolates wereinitially selected on the basis of fatty acid methyl ester(FAME) identification. From a collection of 572 isolates witha similarity index >0.300 for FAME profile match, 60 isolatesrepresenting 15 different species from eight different generawere selected. The most common genera were Pseudomonasor Burkholderia (39 isolates), Bacillus (eight isolates), andAlcaligenes (five isolates). Whenever possible, different spe-cies within each genus were selected. In order to maximizediversity among the remaining 559 unknown isolates, the iso-lates were assessed for Gram reaction and differences in cellu-lar and colony morphology. Because the rhizosphere tends toselect for Gram-negative bacteria over Gram-positive bacteria(Kloepper et al., 1992; Gilbert et al., 1993), an additionalGram-positive and 55 Gram-negative isolates were selected.This yielded a total of 105 Gram-negative and 11 Gram-posi-tive isolates. Pseudomonas putida PH6 (Fuhrmann and Wol-lum, 1989a, 1989b) was selected as a PGPR control. The 116isolates were then screened for traits that might be associatedwith ability to function as PGPR. Each in vitro test was repli-cated three times.

In Vitro TestsSiderophore production was determined in solution as de-

scribed by Schwyn and Neilands (1987) except the mediumwas O.lx trypticase soy broth (TSB; Difco Laboratories, De-

troit, MI). Indoleacetic acid production was determined asdescribed by Brick et al. (1991) except that the medium wasO.lX TSB solidified with agar (15 g L~'; TSA).

Chitinase production was determined as described by Ren-wick et al. (1991) in a defined medium composed of (g L~'):colloidal chitin (Reid and Ogrydziak, 1981), 8.0; NH4NO3,0.78; K2HPO4,0.80; KH2PO4,0.20; MgSO4 • 7H20,0.20; CaCl2,0.06; NaCl, 0.10; Na2MoO4 • 2H2O, 0.002, ZnSO4 • 7H2O,0.00024; CuSO4 • 5H20,0.00004; CoSO4 • 7H20,0.010; MnSO4 •4H2O, 0.003; Na2FeEDTA, 0.028; H3BO3, 0.005; and agar, 15.Magnesium sulfate and CaCl2 were autoclaved separately andadded to the medium after autoclaving. Biotin (5 jjig L""1) andp-aminobenzoic acid (10 (jig L"1) were filter-sterilized (0.2-u.m) and were added to the medium after autoclaving.

|3-l,3-glucanase production was determined as described byRenwick et al. (1991) in the previously defined medium, exceptthe C source was (3-1,3-glucan (5 g L~'; laminarin, SigmaChemical Co., St. Louis, MO). Production of ACC deaminasewas determined as described by Glick et al. (1995) by thepreviously described defined medium, except the C sourceswere sucrose (5.0 g L~!), mannitol (5.0 g L"1), and sodiumlactate (0.5 mL of a 5.4 M solution), and the N source wasACC (5.0 g L""1)- Cyanide production was determined as de-scribed by Bakker and Schippers (1987) in 0.1 X TSA amendedwith glycine (4.4 g L^1) and FeCl3 • 6H2O (0.3 mM; Gastric,1975). Solubilization of P was determined as described byKatznelson and Bose (1959), except the medium was O.lxTSA. Biotin prototrophy was determined by growing the iso-lates in 0.25 X Bacto Biotin Assay Medium (Difco Lab., De-troit, MI).

Putative, free-living, N2-fixing bacteria were screened in theACC deaminase defined medium, except the N source waseliminated and the agar was reduced to 1.75 g L"1 (Day andDobereiner, 1976). The isolates that grew after being sequen-tially transferred 10 times to the same medium were consid-ered presumptive positive for N2 fixation. To confirm nitroge-nase activity, positive isolates were tested for their ability toreduce acetylene to ethylene (Dart et al., 1972) after 24 h inthe same defined medium amended with 100 mg of yeastextract L"1.

To assess the ability of the 116 isolates to inhibit fungi, eachisolate was tested under conditions of low and high Fe (tosuppress siderophore production; Scher and Baker, 1982;Fuhrmann and Wollum, 1989a) against three different fungiwith the circle method (Da Luz, 1990). The fungi — Sclerotiumrolfsii, Fusarium oxysporum, and Sclerotinia sderotiorum —were obtained from the culture collection of R.W. Roncadori(University of Georgia, Athens). Briefly, bacterial isolateswere seeded in a 5.0-cm-diameter circle on a O.lX TSA platethat was either unamended or amended with 0.1 mM FeCl3.After 24 h at room temperature, a 7-mm plug of each funguswas placed on the center of the circle. Plates with S. rolfsiiand F. oxysporum were incubated at 28°C for 6 and 8 d,respectively, and plates with S. sderotiorum were incubatedat 22°C for 5 d. Fungal growth inhibition was assessed bymeasuring the mycelial radial growth.

A subsample of 23 isolates, all but one of which displayedstrong tendencies for one or more of the aforementioned traits,was selected from the 116 isolates and, with the PGPR controlP. putida PH6, were further tested for production of volatileantifungal compounds, ability to inhibit growth of eight strainsof (brady)rhizobia, production of cellulase and pectinase, andability to grow at 18, 28, and 37°C and pH 5.5 and 7.0.

The ability to produce volatile compounds inhibiting S.rolfsii, F. oxysporum, and S. sderotiorum was determined intwo-compartment Petri plates containing O.lX TSA inocu-lated with a 7-mm, O.lx TSA disc with each fungus on one

1672 SOIL SCI. SOC. AM. J., VOL. 63, NOVEMBER-DECEMBER 1999

side and a lawn of bacterial cells from each isolate spread onthe other side (Gagne et al., 1991). Fungal growth was assessedby measuring the radial growth of the mycelium.

The isolates were tested for their ability to inhibit growthof eight strains of (brady)rhizobia: Bradyrhizobium elkaniiUSDA 31, 76, and 94; B. elkanii SEMIA 587 and 5079; B.japonicum USDA 110 and 123; and Rhizobium fredii USDA205. Bradyrhizobium japonicum USDA 110 was obtainedfrom A.G. Wollum (North Carolina State Univ., Raleigh), B.elkanii SEMIA 587 and 5079 were obtained from FundagaoEstadual De Pesquisa Agropecuaria (FEPAGRO, Porto Al-egre, Brazil), R. fredii USDA 205 was obtained from theUSDA culture collection (Beltsville, MD), and B. elkaniiUSDA 31, 76, and 94 were from our own culture collection.Tests were conducted in 0.1 X TSA plates inoculated with alawn of bacterial cells from each of the 23 isolates in one halfand perpendicular streaks of each (brady)rhizobial strain inthe other half. One to four (brady)rhizobial strains werestreaked from a lightly turbid cell suspension in 0.15 M NaCl.The (brady)rhizobial strains were separated from the cell lawnby a distance of 2 mm. The linear inhibition of the (brady)rhi-zobial growth was measured after 7 d at 28°C. The controltreatment was (brady)rhizobial inoculation without bacte-rial inoculation.

Cellulase production was determined in M9 medium(Miller, 1974) amended with yeast extract (1.2 g Lr1) andcellulose (10 g L~'; Sigmacell Type 101, Sigma Chemical; Sa-manta et al., 1989). After 8 d of incubation at 28°C, isolatessurrounded by clear halos were considered positive for cellu-lase production. Pectinase production was determined in thesame M9 medium except the cellulose was replaced with pectin(4.8 g L~'). After 2 d of incubation at 28°C, the plates wereflooded with 2 M HC1 (T. Denny, 1997, personal communica-tion), and isolates surrounded by clear halos were consideredpositive for pectinase production (Andro et al., 1984).

The ability of each bacterial isolate to grow at 18, 28, and37°C was determined in 0.1 x TSB. A 100-jjiL sample of a cellsuspension, adjusted to an absorbance of 0.030 at 600 nm,was transferred into 10 mL of 0.1 X TSB contained in 50-mLErlenmeyer flasks and the contents periodically measuredspectrophotometrically at 600 nm for 24 h. The ability of thebacterial isolates to grow at two different pHs was determinedin a similar manner. The isolates were grown in 0.1 X TSB atpH 7.0 (natural pH of the medium) and pH 5.5 (adjusted with1 M HC1). The temperature was kept at 28°C.

In addition to the PGPR control P. putida PH6, one isolate,LN1116, was consistently negative for all the PGPR traits invitro except antagonism to S. sclerotiorum and S. rolfsii, andit was selected as the negative bacterial control for the short-term plant tests.

Short-Term Plant TestsThe Ap horizon of Appling sandy loam (clayey, kaolinitic,

thermic, Typic Kanhapludults) was obtained from the Univer-sity of Georgia Plant Sciences Farm near Watkinsville, GA.The Ap horizon of Dothan loamy sand (fine-loamy, kaolinitic,thermic Plinthic Kandiudults) was obtained from the South-east Georgia Branch Experiment Station near Midville, GA.

Both soils were passed through a 2-mm sieve and stored atroom temperature until used. The soils were analyzed for pH,total organic C, and selected nutrients according to standardprocedures (Plank, 1989) at the University of Georgia SoilTesting and Plant Analysis Laboratory (Table 1). The gravi-metric water contents (kg water kg"1 soil) of the soils at -0.01and -0.03 MPa were determined by the pressure plate method(Klute, 1986). Soil water content was determined gravimetri-cally by oven drying the soil overnight at 105°C.

In all experiments, seeds of soybean cv. Lee were wettedin 20.0 M ethanol (95%) for 5 s and surface-sterilized in 0.21 MNaOCl for 5 min. The seeds were washed once in steriledistilled water, soaked for 10 min in 0.01 M HC1 (to removetraces of NaOCl; Abdul-Baki, 1974), and washed five timesin sterile, distilled water to remove traces of HC1. On the lastrinse, seeds were left to imbibe water for 1 h. Seeds weregerminated between pieces of sterile, moist filter paper at28°C for 2 d.

All 23 bacterial isolates and P. putida PH6 were grown on0.1 X TSA overnight at 28°C. The cells were suspended in0.1 M MgSO4 (pH 7.0) to give an absorbance of 0.55 at 600nm («109 cells mL^1). Except for the nodule occupancy experi-ment, germinated seeds were immersed in the cell suspensionof each isolate or into sterile 0.1 M MgSO4 (nonbacterialcontrol) for 5 min. In the case of the nodule occupancy experi-ment, germinated seeds were immersed in equal volumes ofcell suspensions of the bacterial isolates and B. japonicumUSDA 110. Three seeds were planted per pot, except for thenodule occupancy experiment where four seeds were planted.The soil surface was covered with 1 cm of sterile sand toreduce aerial contaminants. In the lightroom experiments,plants were grown in =500 g (dry weight basis) of Applingsandy loam or Dothan loamy sand (P-solubilization experi-ment only) contained in 600-mL D-pots (Stuewe and Sons,Corvallis, OR) with a light intensity of 550 jj,M photon m~2

s~', a 16/8-h light/dark cycle, and day/night temperatures of26 ± 1 and 24 ± 1°C, respectively. In the single greenhouseexperiment, plants were grown in 1740 g (dry weight basis)of Appling sandy loam contained in 2-L polyethylene pots.Water was added daily to each soil to attain a water contentequivalent to —0.03 MPa for all experiments except for theFusarium antagonism experiment, where the soil water con-tent was equivalent to —0.01 MPa. After emergence, plantswere thinned to one plant per pot. Each treatment was repli-cated four times in a completely randomized design. At har-vest, the shoots were cut off and the roots were washed gentlyto minimize nodule loss. Nodules were separated from theroots and counted. Roots, shoots, and nodules were oven driedat 50°C and weighed after 2 d.

For bacterial isolates positive for IAA or ACC deaminaseproduction, plants were sampled at 14 d after planting (DAP)and plant shoot height was measured. Roots were stored informalin-acetic acid-alcohol (Postek et al., 1980, p. 296) androot length was estimated by the modified line intersectmethod (Tennant, 1975). Roots were oven dried at 50°C andweighed. For bacterial isolates positive for nitrogenase activ-ity, plants were harvested at 20 DAP and total N of plant shootswas determined by the micro-Kjeldahl method (Plank, 1992).

Bacterial isolates positive for P solubilization were tested

Table 1. Some chemical and physical characteristics of Appling and Dothan soils.

Soil

ApplingDothan

PH

5.55.6

Org. C

910

Total N

g kg ' ————0.560.52

NO3-N

2.31.8

NHj-N

1.41.8

P

— mg kg-' -196

K

6753

Mg

2752

Ca

230431

Water contentat -0.03 MPa

0.120.13


in Dothan loamy sand amended with 54 mg of K2SO4 and 92mg of CaHPO4 kg~l soil. Plants were sampled at 18 DAP.Total shoot P and N contents were determined by colorimetryand micro-Kjeldahl methods, respectively (Plank, 1992). Se-rology of the nodules from the control treatment was deter-mined by an ELISA assay (Fuhrmann and Wollum, 1985) forthe B. elkanii USDA 31, 76, and 94, and B. japonicum USDA110 and 123 serogroups.

Bacterial isolates positive for 3-1,3-glucanase, cyanide, sid-erophore, or chitinase production or positive for antagonismto F. oxysporum were tested in Appling sandy loam that waskept cooler (day and night temperatures of 23 ± 1 and 21 ±1°C, respectively) and at higher matric potential (-0.01 MPa)than the other experiments. These conditions are better suitedfor the development of root rot caused by Fusarium spp. thanare warmer and drier environments (Sinclair and Backman,1989). Because antagonism against F. oxysporum was the mostvariable of the three in vitro fungal antagonism tests, thisfungus was chosen for the plant tests. To add F. oxysporumto the soil, the fungus was grown for 4 d on 0.1X TSA, scrapedfrom the surface with an inoculating loop, and sonicated for1 h in 200 mL of distilled water. After adding the suspensionto air-dried soil, the fungal counts were determined by seriallyplating a portion of the solution on 0.1X TSA. The final countof F. oxysporum in soil was =1.7 X 103 colony-forming unitsg"1. The light and dark cycle was 14 and 10 h, respectively.Isolates positive for chitinase production were also tested inthe presence and absence of chitin. After inoculation, eachseed was coated with = 11.4 |j,g of ground (250 (xm), autoclavedchitin. Plants were sampled at 14 DAP.

Eight bacterial isolates that produced significant effect(s)in the previous plant-growth and nodulation experiments orexhibited in vitro antagonism against strains of Bradyrhizo-bium spp. were tested, along with P. putida PH6, for theirability to alter nodule occupancy. Plants were sampled at 28DAP. Serology of the nodules from the tap root was deter-mined by an ELISA assay for the B. elkanii USDA 31, 76,and 94 and B. japonicum USDA 110 and 123 serogroups forall treatments. Each treatment was replicated five times in acompletely randomized design.

Clustering of bacterial FAME profiles was determined withCANOCO, a program for canonical community ordination(Microcomputer Power, Ithaca, NY). Because of the possibil-ity that some unknown isolates that increased early soybeangrowth could be clones of each other, these isolates were alsotentatively identified with Biolog (Biolog, Hayward, CA) andribosomal typing (RiboPrinter Microbial Characterization sys-tem, Qualicon, Wilmington, DE). In the case of ribotyping,the restriction enzyme was EcoRl.

Statistical AnalysisAll fungal and bradyrhizobial antagonism data were ana-

lyzed by analysis of variance (ANOVA) and treatment meanswere separated by the Tukey's test with the SAS statisticalpackage (SAS Institute, 1988). All plant test data were alsoanalyzed by ANOVA but treatment means were separatedby the Fisher's least significant difference test.

RESULTSIn Vitro Tests

Of the 116 isolates, 22 (19%) showed strong tenden-cies for one or more of the PGPR traits tested, and one,LN1116, was negative for all traits (Table 2). The PGPRcontrol P. putida PH6 was negative for all the traits

except biotin prototrophy. A majority of the isolates(19 of 23, 83%) were Gram-negative rods. Four isolatesproduced IAA, eight produced ACC deaminase, fivesolubilized P, five exhibited low levels of nitrogenaseactivity, seven produced siderophores, two producedchitinase, four produced [3-1,3-glucanase, and five pro-duced cyanide. Only isolate LN1116 was a biotin auxo-troph. All isolates were negative for cellulase and pec-tinase production (data not shown). With regards totemperature, all isolates grew better at 28 than at 18°C.Most of the isolates also grew better at 28 than at 37°C,but at least four isolates grew at virtually the same rateat both temperatures. With regards to pH, only LN1116grew better at pH 5.5 than at pH 7.0, and at least nineother isolates grew equally well at both pHs. Althoughfive isolates apparently fixed N2, they exhibited low ni-trogenase activities (0.05-0.11 nM of ethylene tube'1h^1) compared with the positive control, Azospirillumbrasilense ATCC 29145 (173 nM of ethylene tube"1 h~').

All isolates, including P. putida PH6, inhibited thegrowth of S. rolfsii and S. sclerotiorum at both low andhigh Fe when the isolates and fungi were not physicallyseparated (Table 3). Ten isolates significantly inhibitedthe growth of F. oxysporum at low Fe and eight at highFe. Five of the ten isolates were positive for siderophoreproduction, four for (3-1,3-glucanase, and three for cya-nide production. Four of the ten isolates were positivefor two traits and two were negative for all traits (includ-ing chitinase). When the fungi and isolates were physi-cally separated in two-compartment plates, 18 of 23isolates inhibited S. rolfsii, 17 inhibited S. sclerotiorum,and two inhibited F. oxysporum.

With regards to (brady)rhizobial inhibition, 22 iso-lates inhibited at least one strain of (brady)rhizobia(Table 4). Only one isolate, LC3116, did not inhibitany of the eight strains tested. Bradyrhizobium elkaniiUSDA 31, 76, and 94 were not inhibited by any ofthe isolates.

Short-Term Plant TestsOf the 11 isolates positive for hormone production,

four isolates significantly increased soybean growth at14 DAP: LN3212 and LW2301 increased shoot dryweight, root length, and root dry weight; GN1201 in-creased root length and dry weight; and GW2306 in-creased shoot and root dry weight (Table 5). Interest-ingly, the negative bacterial control, LN1116, alsoincreased root dry weight. Except for LN1116, all theisolates that affected plant growth were positive forACC deaminase production; none of the isolates thatwere positive for IAA production affected plant growth.Three of the isolates that were positive for ACC deami-nase were also positive for (3-1,3-glucanase or sidero-phore production or both.

None of the five isolates positive for nitrogenase activ-ity significantly promoted soybean growth at 20 DAPand, in fact, one isolate (LN1310) significantly decreasednodule dry weight (data not shown). Of the five isolatespositive for P solubilization, two significantly affectedsome aspect of soybean growth in a P-deficient soil at

Tabl

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Sel

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raits

of

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1102

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1107

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2118

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1101

GW

2103

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1206

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1212

GN

2214

GW

3202

GW

3205

GN23

10G

N23

23G

W23

06LC

1118

LC31

16LN

1101

LN11

16LN

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LN32

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LW23

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Table 3. In vitro antagonism of three soil fungi by 24 bacterial isolates. Antagonism was measured by the inhibition of fungal radialgrowth on 0.1 X trypticase soy agar under conditions of no physical separation (single compartment plates) with high and low Fe inthe medium and physical separation (two-compartment plates).

Sclerotium rolfsii

No separation

Isolate

GN1102GN1107GN2118GW1101GW2103GS1206GN1201GN1210GN1212GN2214GW3202GW320SGN2310GN2323GW2306LC1118LC3116LN1101LN1116LN1118LN3212LN1310LW2301PH6Control

Low Fe

0*0*0*0*0*0*0*0*0*0*0*0*0*0*1*2*1*2*4*1*0*0*0*0*

34

High Fe

0*0*0*0*0*0*0*0*0*0*0*0*0*1*2*2*2*2*4*2*1*0*0*0*

35

Separation

161711*10*3*

1614*7*

13*1713*10*14*15*14*4*

1814*13*9*

12*9*

14*1720

Fusarium oxysporum

No separation

Low Fe

—————— Fungal7*

3029

9*33325*

3119*3422*14*2522*7*

303232363516*307*

3027

High Fe

radial growth,7*

3028

8*333213*33243320*13*252312*343233353216*3212*3227

Separation

mm —————1513*141415141514151515151414141514151514141413*1516

Sclerotinia sclerotiorum

No separation

Low Fe

0*0*0*0*0*1*0*0*0*0*1*1*1*1*1*1*0*4*4*2*0*1*0*0*

38

High Fe

0*0*0*0*0*1*0*0*0*0*1*0*0*3*0*1*0*2*6*2*0*2*0*0*

38

Separation

25*2723*23*17*2725*21*23*2724*24*23*25*24*19*282624*25*24*24*262729

*, Significantly different from the control according to Tukey's test (a = 0.05).

18 DAP (Table 6). The PGPR control P. putida PH6and isolate GN1212 showed significantly higher shootP content, but this was offset in the case of GN1212by a significant decrease in shoot dry weight. IsolateGN2214 significantly increased root dry weight, but thiswas offset by significantly lower nodule weight and num-

ber. However, it did not decrease shoot N content. Theserological reaction of 35 nodules from the nonbacterialcontrol treatment was tested to check for the distribu-tion of bradyrhizobial strains in the Dothan loamy sand.Of the 35 nodules tested, seven (20%) aligned with B.elkanii USD A 31, six (17%) with B. elkanii USD A 76,

Table 4. In vitro antagonism of Bradyrhizobium elkanii, B. japonicum, and Rhizobium fredii by 24 bacterial isolates. Antagonism wasmeasured by the inhibition of growth on 0.1 X trypticase soy agar.

Bradyrhizobium elkanii

Isolates

GN1102GN1107GN2118GW1101GW2103GS1206GN1201GN1210GN1212GN2214GW3202GW3205GN2310GN2323GW2306LC1118LC3116LN1101LN1116LN1118LN3212LN1310LW2301PH6Control

USDA 31

0000000000000000000000000

USDA 76

0000000000000000000000000

USDA 94

0000000000000000000000000

SEMIA 587

———— Zone000

£25*00

15*NDt

00

21*15*

9*8*

15*6*0000

19*0

23*00

SEMIA 5079

of inhibition, mm ———00

20*15*

£25*0

11*ND

00

£25*20*2i*23*20*

>25*00

19*19*

£25*£25*£25*

10

B. japonicum

USDA 110

00

20*£25*£25

022*

£25*00

£25*£25*

23*£25*>25*£25*

00

22*>25*£25*>25*£25*

40

USDA 123

£25*17*

a25*£25*£25*

17*£25*£25*£25*a25*£25*£25*£25*£25*£25*£25*

0£25*£25*£25*£25*225*£25*a25*

0

R. fredii

USDA 205

00

10*12*

£25*0

13*£25*

00

£25*£25*

21*£25*£25*£25*

00

£25*£25*£25*£25*£25*

20

*, Significantly different from the control according to Tukey's test (a = 0.05).t ND = not determined.


Table 5. Effect of 11 bacterial isolates, each positive for the production of the plant-growth regulator indoleacetic acid (IAA) (GW2103,GW3202, GN2323, and LC1118) or l-aminocyclopropane-l-carboxylate (ACC, a precursor of ethylene) deaminase (GW1101, GS1206,GN1201, GN1212, GW2306, LN3212, and LW2301) on growth and nodulation of soybean cv. Lee. Pseudomonas putida PH6 wasthe plant growth-promoting rhizobacteria control and LN1116 was the bacterial control negative for both traits tested.

Treatment

GW1101GW2103GS1206GN1201GN1212GW3202GN2323GW2306LC1118LN3212LW2301LN1116PH6ControlFisher's LSD

Shootheight

mm11011198

10198

10712511410098

12199

10210620

Shoot dryweight

mg plant '244227208230191169240281*220298*302*23621217284

Root length

cm plant"1

926743680

1062*542656800914729

1098*1012*934835619337

Root dryweight

mg plant'1

958483

112*668991

123*83

137*123*114*957038

Nodules

no. plant"1

1920122212171819141215171619

NSt

Nodule dryweight

mg plant ]

8154

113695665

128

129

*, Significantly different from the control (a = 0.05).t NS = means not significantly different from each other (a = 0.05).

10 (29%) with B. elkanii USD A 94, and none with eitherB. japonicum USDA 110 or 123. Fourteen nodules(40%) did not react with any of the sera tested, andtwo (6%) reacted with more than one.

When soybeans were inoculated with one of 12 iso-lates, each positive for (3-1,3-glucanase, cyanide, sidero-phore, or chitinase production or the ability to inhibitthe growth of Fusarium in vitro, and grown in soil inocu-lated with F. oxysporum for 14 d, only isolate LN3212increased all aspects of soybean growth. IsolatesGW3205 and GN1212 also increased nodule dry weight(Table 7). Again, the bacterial control isolate LN1116,negative for all traits, increased shoot dry weight. Theremaining isolates had no effect on soybean growth.

Of the eight bacterial isolates tested in the noduleoccupancy experiment plus the PGPR control P. putidaPH6, only one isolate, LC3116, changed nodule occu-pancy at 28 DAP (Table 8). In this case, LC3116 in-creased nodule occupancy with bradyrhizobial strainsbelonging to the serogroup B. elkanii USDA 31. How-ever, this increased nodule occupancy was at the ex-pense of other native bradyrhizobial serotypes, not sero-type B. japonicum USDA 110. None of the isolatessignificantly altered nodule occupancy by B. japonicumUSDA 110 and none promoted soybean growth (datanot shown).

When the initial 116 soil and rhizobacterial isolateswere compared by FAME profiles, distinct clusteringwas observed among the isolates (Fig. 1). The sevenisolates that increased some aspect of early soybeangrowth were closely associated with Pseudomonas spp.FAME analysis had previously identified three of theisolates as P. cepacia (GN1201), P. pickettii (GN2214),and P. chlororaphis (GN1212); the remaining four iso-lates were unknown. Biolog and ribotyping analyseswere unable to identify any of these unknown isolates.However, ribotyping did show that the unknown isolateswere different (data not shown).

DISCUSSIONSeven of the 23 isolates selected for the plant-growth

experiments significantly increased at least one aspectof early soybean growth, and five significantly increasedat least two aspects. All seven isolates were originallyfrom the soybean rhizosphere. Many of these sevenisolates shared common characteristics: six were positivefor ACC deaminase, four were positive for siderophoreproduction, three were positive for (3-1,3-glucanase pro-duction, and two for P solubilization. Previous researchsuggests that bacteria possessing these traits can in-crease plant growth. Glick et al. (1995) observed that

Table 6. Effect of five bacterial isolates, each positive for P solubilization, on growth, nodulation, and shoot P and N content of soybeancv. Lee. Pseudomonas putida PH6 was the plant growth-promoting rhizobacteria control and LN1116 was the bacterial controlnegative for the trait tested.

Treatment

GN1102GN1107GN1212GN2214LC3116LN1116PH6ControlFisher's LSD

Shoot dry Root dryweight weight

527580418*536507574449547118

524516376728*639622352500183

Nodulesno. plant '

101295*

18139

136

Nodule dryweight

mg plant"1

2521148*

3023292612

Shoot Pcontent

, _,6 8

1.241.301.55*1.201.341.241.52*1.250.18

Shoot Ncontent

14.918.519.620.515.218.020.117.63.3

*. Significantly different from the control (a = 0.05).


Table 7. Effect of 12 bacterial isolates, each positive for the production of f)-l,3-glucanase, cyanide, siderophores, or chitinase, on growthand nodulation of soybean cv. Lee. Whether or not the isolate inhibited the growth ofFusarium oxysporum in vitro is also noted. Isolatespositive for chitinase production were also tested with or without chitin. Pseudomonas putida PH6 was the plant growth-promotingrhizobacteria control and LN1116 was the bacterial control negative for all traits tested.

F. oxysporumTreatment inhibition

p-glucanase productionGW3205 +LN3212 +

Cyanide productionGN1212 +

Siderophore productionGN1201 +

p-glucanase + siderophore productionGW2306 +LW2301 +

Siderophore + cyanide productionGW3202 +GN2323 +

Chitinase productionGN1210 without chitinGN1210 with chitinLN1310 without chitinLN1310 with chitin

None of the above traitsGN1102 +GW1101 +LN1116PH6Control without chitinControl with chitinFisher's LSD

Shoot dryweight

————— mg plant ~'

157173*

157

135

145136

147145

142158153131

148149162*14810814153

Root dryweight

8290*

74

60

6173

7866

64637465

65667062517534

Nodules

no. plant '

817*

12

7

129

78

3997

8799538

Nodule dryweight

mg plant''

5*6*

6*

3

44

43

1432

3233113

*. Significantly different from the control without chitin (a = 0.05).

seven bacterial strains positive for ACC deaminase pro-duction promoted canola (Brassica napus L.) seedlingroot elongation under gnotobiotic conditions. 1-Amino-cyclopropane-1-carboxylate deaminase might act tostimulate plant growth and root elongation in particularby sequestering and hydrolyzing ACC from germinatingseeds. This would lower the level of ACC and conse-quently the level of ethylene in seeds. For many plantspecies, ethylene stimulates germination and breaks thedormancy of the seeds (Esashi, 1991). However, if theethylene concentration remains high after germination,root elongation (as well as symbiotic N2 fixation in legu-minous plants) is inhibited (Jackson, 1991). In the caseof 3-1,3 glucanase production, Fridlender et al. (1993)

observed that an isolate of P. cepacia with strong 3-1,3glucanase production decreased the incidence of threefungal diseases in three different plants grown in non-sterile soil. More than 50% of strains that controlledtake-all \Gaeumannomyces graminis (Sacc.) Arx & D.Olivier] of wheat (Triticum aestivum L.) produced3-glucanases (Renwick et al., 1991). In the case of sider-ophore production, a siderophore-producing P. putidaconverted a Fusarium-conducive soil to a Fusarium-suppressive soil (Scher and Baker, 1982). Similarly, inoc-ulation of chickpea (Cicer arietinum L.) and soybeanseed with a siderophore-producing fluorescent pseu-domonad resulted in increased seed germination,growth, and yield of the plants (Kumar and Dube, 1992).

Table 8. Effect of nine bacterial isolates on nodule occupancy of soybean cv. Lee inoculated with B. japonicum USDA 110. Isolateswere chosen based on a significant effect in previous plant growth and nodulation tests or in vitro antagonism to Bradyrhizobiumstrains. Pseudomonas putida PH6 was the plant growth-promoting rhizobacteria control.

SerogroupTreatment

GN2118GW2103GN1201GN2214GN2310LC3116LN1101LN3212PH6Control!Fisher's LSD

USDA 31

293031334460*3847413522

USDA 76

13141121136

20151312

NS§

USDA 94

14131524131915171410NS

USDA 110

7496

1455535

NS

No reaction

434342t283717*3325384422

Multiplereaction

558

12197

111175

Nodulestested

738685757571738468

108

*, Significantly different from the control (a = 0.05).t Includes the only nodule that reacted with the USDA 123 serogroup.I Inoculated with B. japonicum USDA 110.§ NS = not significantly different from each other (a = 0.05).


+ 2.5

L LW2301-0.5

Fig. 1. Two-dimensional plot of principal components analysis of fattyacid methyl ester (FAME) profiles of the bacterial isolates testedplus the plant growth-promoting rhizobacteria (PGPR) control,Pseudomonas putida PH6. The x- and v- axes are in Euclideandistance. Isolates are identified as (i) increasing some aspect ofsoybean growth (solid triangle), (ii) not increasing some aspectof soybean growth (open square), and (iii) not tested in plant(open circle).

The only trait tested here that changed some aspectof soybean growth independently of ACC deaminaseactivity was P solubilization. Some P-solubilizing organ-isms have been reported as plant growth promoters,but rigorous proof is lacking (De Freitas et al., 1997;Whitelaw et al., 1997). Interestingly, isolate LC3116,which increased nodule occupancy with serogroup B.elkanii USDA 31, was also positive for P solubilization.The results suggest that rhizobacteria able to reduce theconcentration of ethylene and to a lesser extent, produce3-1,3 glucanase or siderophores or able to solubilize P,may be important in promoting early soybean growth.

Under our conditions, isolates positive only for theproduction of IAA, chitinase, or cyanide did not en-hance soybean growth. The evidence that IAA, chi-tinase, or cyanide promotes plant growth in nonsterilesoil is scanty. Abbass and Okon (1993) hypothesizedthat IAA and other plant hormones were responsiblefor increased growth of canola, tomato (Lycopersiconesculentum Mill.), and wheat (Triticum turgidum L.) innonsterile soil inoculated with Azotobacter paspali, butthey did not establish that A. paspali actually producedthese hormones. Tien et al. (1979) did establish thatAzospirillum brasilense produced IAA, gibberellins,and cytokinins and that this bacterium could increasethe number of lateral roots and root hairs in pearl millet[Pennisetum glaucum (L.) R. Br.], but this was onlyunder gnotobiotic conditions. There are some reportsthat rhizobacteria that overproduce IAA inhibit rootelongation; this is attributed to the stimulation of ethyl-ene synthesis by IAA (Xie et al., 1996; Glick et al.,1998). Although chitinase has been proposed as a mech-anism of fungal antagonism (Potgieter and Alexander,1966; Renwick et al., 1991; Kurek and Jaroszuk, 1997),this has only been tested in vitro. Chitinase may havebeen involved in increased nodulation and shoot dry

weight of soybean when the seeds were coated withchitin and coinoculated with Streptomyces griseus andantibiotic-resistant B. japonicum, but the authors attrib-uted the PGPR mechanism to antibiotic production (Liand Alexander, 1988). Overproduction of cyanide maycontrol fungal diseases in wheat seedlings, but the exper-iments were only done in vitro (Flaishman et al., 1996).

It was unclear if asymbiotic N2 fixation was a PGPRtrait in our experiments. The nitrogenase activity of ourfive N2-fixing isolates was very low when compared withthe nitrogenase activity of the positive control, A. brasi-lense ATCC 29145. It may be that high nitrogenaseactivity is necessary for good plant growth promotion.However, low nitrogenase activity is not unusual forsome N2-fixing bacteria (Lifshitz et al., 1986; Al-Mallahet al., 1989).

Surprisingly, the bacterial control LN1116, an isolatethat did not share any of the in vitro PGPR traits testedexcept antagonism against S. rolfsii and S. sclerotiorum,also promoted at least two aspects of soybean growth.Why this isolate is also a PGPR is unclear. There maybe some other PGPR trait(s) for which we did not test(e.g., gibberellic acid production). The PGPR control,P. putida PH6, was also negative for most of the in vitrotraits tested, including siderophore production. This iso-late has been selected as a PGPR for soybean in partbecause it is believed to produce siderophores (Fuhr-mann and Wollum, 1989a, 1989b). Our inability to showsiderophore production for P. putida PH6 may be be-cause we used 0.1X TSB and the original authors (Fuhr-mann and Wollum, 1989a, 1989b) used King's mediumB (King et al., 1954). King's medium B is designedspecifically for enhancing pigment production by pseu-domonads. Pseudomonas putida PH6 and GN1212 in-creased the shoot P content in the P-solubilization ex-periment, but this seemed to be a concentration effectbecause the two isolates yielded the lowest shoot weightand P. putida PH6 did not solubilize P in vitro.

None of the nine isolates tested increased noduleoccupancy by B. japonicum USDA 110. While othershave observed beneficial changes in nodule occupancywith B. japonicum USDA 110 over other, less efficientbradyrhizobial strains in potting mixes (Polonenko etal., 1987; Fuhrmann and Wollum, 1989b), such changeshave not been observed in nonsterile soil. One isolate,Acinetobacter baumannii LC3116, did increase the per-centage of nodules containing strains of the serogroupB. elkanii USDA 31, but these bradyrhizobial strainsare considered inefficient (Israel, 1981). It is importantto note that B. elkanii USDA 31 is a poor competitorwhen coinoculated with B. japonicum USDA 110 (Fuhr-mann and Wollum, 1986b; Weiser et al., 1990), andunderstanding how a poor competitor can become astrong one may be important for understanding rhizo-sphere competition. None of the isolates, includingLC3116, significantly affected nodule occupancy withB. japonicum USDA 110.

All 23 isolates inhibited S. rolfsii and S. sclerotiorumin vitro when the bacteria and fungi were not physicallyseparated. This was somewhat surprising for S. rolfsiibecause in a similar study (Cattelan, 1994), only six of

CATTELAN ET AL.: SCREENING FOR RH1ZOBACTERTA TO PROMOTE EARLY SOYBEAN GROWTH 1679

32 rhizosphere isolates (19%) inhibited this fungus. Theinhibition we observed for F. oxysporum is similar tothat observed by Kurek and Jaroszuk (1997). Becausemost of the fungal antagonism in vitro was similar be-tween low- and high-Fe media for eight of 10 isolates,it is unlikely that siderophore production is involvedin this antagonism. When the isolates were physicallyseparated and they inhibited one or more of the fungitested, the most likely mechanism is the production ofone or more volatile compounds (Gagne et al., 1991;Flaishman et al., 1996). Based on our results, it is unclearwhether fungal inhibition in vitro was associated withpromoting soybean growth. In our plant test, only oneof 10 isolates positive for Fusarium inhibition promotedsoybean growth in nonsterile soil inoculated with F. oxy-sporum.

The presence of any of the PGPR in vitro traits testedhere does not guarantee that a particular isolate is aPGPR nor does their absence guarantee that it is not.However, given that six of eight isolates positive forACC deaminase, four of seven isolates positive for sid-erophore production, three of four isolates positive for(3-1,3-glucanase production, and two of five isolates pos-itive for P solubilization in vitro increased some aspectof early soybean growth in nonsterile soil, our resultssuggest that these traits are more worthy of screeningfor plant growth promotion than isolates positive forbiotin prototrophy or production of IAA, chitinase, andcyanide. Finally, there are other traits for which we didnot test (e.g., gibberellic acid production) that may beassociated with PGPR. Further screening is necessaryto establish traits definitively associated with PGPR,especially in nonsterile soil.

ACKNOWLEDGMENTSWe thank J.M. Bain, H.R. Boerma, S. Brooks, M.L. Ca-

brera, O. Finlay-Moore, C. Golt, J.W. Gray, T. Olexa, L.H.Pratt, J.A. Rema, N. Stern, and D.A. Zuberer for their techni-cal assistance. Alexandre Cattelan was supported by a scholar-ship from the Brazilian Agricultural Research Corporation(EMBRAPA) and the Inter-American Development Bank(BID). The research was supported by EMBRAPA, BID, andRegional Research funds to Project S-262.

screening for plant growth–promoting rhizobacteria to promote early soybean growth

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