Method for Establishing a Bacterial Inoculum on Corn RootsFRANCISCO A. MENDEZ-CASTROt AND MARTIN ALEXANDER*
Laboratory of Soil Microbiology, Department ofAgronomy, Cornell University, Ithaca, New York 14853
Received 8 March 1982/Accepted 31 August 1982
Few bacteria from the corn rhizosphere grew in media with 50 jig of mancozebper ml. A mancozeb-resistant Pseudomonas strain from the rhizosphere wasserially subcultured in media containing mancozeb and spectinomycin until it wasresistant to 175 ,ug of mancozeb and 850 ,ug of spectinomycin per ml. Thepopulation of the pseudomonad added to soil fell to low numbers in 6 days inunamended or glucose-amended soil, but its numbers exceeded 105/g for at least12 days if the soil was supplemented with mancozeb. The numbers of thisorganism remained small on corn roots derived from untreated, inoculated seeds,but the population was two or more orders of magnitude greater on roots derivedfrom mancozeb-coated seeds. The abundance of the inoculum strain on the 3-cmportion of roots nearest the stem declined markedly after about 1 week, butapplying urea to the foliage reduced or prevented the decline. The numbers of thepseudomonad on segments of roots 3- to 6- and 6- to 9-cm from the stem werehigher on plants derived from the mancozeb-coated seeds. Applying spectinomy-cin to the foliage did not promote growth of the bacterium. This method isproposed as a means to establish an introduced bacterium on plant roots.
Microorganisms are beneficial to plant growthin many ways. By mineralizing organic forms ofnitrogen, phosphorus, and sulfur that roots can-not use as nutrients, the rhizosphere microfloraprovides inorganic ions that sustain plantgrowth. The rhizosphere inhabitants, in cultureat least but possibly also in nature, are known tosynthesize plant growth regulators (8) and prod-ucts toxic to soil-borne plant pathogens (24).Many studies have demonstrated that bacteriaassociated with the roots of nonlegumes mayalso bring about N2 fixation (3).Because of the benefits that the rhizosphere
inhabitants can provide, many attempts havebeen made to enhance the processes that thesemicroorganisms effect. One approach to bringabout such an enhancement is to inoculateplants with a species active in the particulartransformation. Particular attention has beengiven to nitrogen fixation; thus, Azotobacterchroococcum has been inoculated onto wheatseeds (18), Azospirillum sp. has been added tomillet (1), and Beijerinckia derxii has been ap-plied to wheat (19). Attempts have also beenmade to increase phosphorus uptake of plants byuse of inocula that contain bacteria which solu-bilize insoluble phosphates (13). Strains of Pseu-domonas (6), Bacillus, and Streptomyces (15)have also been applied to seeds of cereal or
t Present address: Escuela de Agronomia, UniversidadCentro-Occidental "Lisandro Alvarado," Apartado 400, Bar-quisimeto 3001, Venezuela.
vegetable crops to enhance plant growth. Inmost instances, no means was employed toselectively favor growth of the bacterium usedas inoculum, and it was assumed that the micro-organism would be able to establish itself on theroots in competition with the indigenous micro-flora solely because it was added to the seed inlarge numbers. It has recently been shown,however, that the growth or survival of Rhizobi-um phaseoli in the rhizosphere of beans may beenhanced by applying a fungicide and a fungi-cide-resistant strain of R. phaseoli to seeds (14,16). The fungicide, in addition to suppressingmany bacteria that would compete with theinoculum strain for plant exudates, markedlyreduces the population of protozoa that mightprey on the rhizosphere colonizers. As a result,nitrogen fixation and bean growth were in-creased.The present study was designed to determine
whether the application of a fungicide and afungicide-resistant bacterium would lead to en-hanced colonization of the roots of a cereal. Incontrast with Rhizobium spp., the bacterium inthis instance does not enter into the root andthus must survive prolonged interactions withother microorganisms growing on root surfaces.
MATERIALS AND METHODSObtaining resistant isolates. Dilutions of soil from the
rhizosphere of corn grown in Lima silt loam wereplated on nutrient agar containing mancozeb (Mn andZn salt of ethylene bis[dithiocarbamate]; 91.6% pure;
E. I. DuPont de Nemours & Co., Inc., Wilmington,Del.), thiram (tetramethylthiuram disulfide; 97% pure;Aldrich Chemical Co., Milwaukee, Wis.), spectinomy-cin (GIBCO Laboratories, Grand Island, N.Y.), orstreptomycin (Nutritional Biochemicals Corp., Cleve-land, Ohio) in a range of concentrations. The plateswere incubated for 48 h at 28°C, and then bacteriagrowing rapidly on plates which had the highest chemi-cal concentrations and few colonies were transferredto nutrient agar containing one of the antimicrobialagents. To obtain isolates of greater resistance, thebacteria were grown in nutrient broth for 48 h at 30°Con a rotary shaker, and the cells were collected bycentrifugation and washed three times with 0.1 Mphosphate buffer (pH 7.0). An inoculum containing 3.8x 109 to about 1 x 1010 of these washed cells per mlwas added to 100-ml Erlenmeyer flasks containing 50ml of nutrient broth supplemented with, in subsequenttransfers, mancozeb, streptomycin, thiram, and spec-tinomycin in increments increasing by 25, 50, 100, and100 p.g, respectively.The pseudomonad that was thus isolated was rou-
tinely grown in a medium (SMS) containing the follow-ing (per liter of distilled water): sucrose, 10 g; yeastextract, 0.5 g; Casamino Acids, 0.5 g; K2HPO4, 0.6 g;(NH4)2HP04, 0.4 g; MgSO4 * 7H20, 0.2 g; CaC12, 20mg; FeCl3, 2 mg; and agar, 15 g. The pH afterautoclaving was 7.0. When used, sugars, antibiotics,and fungicides were sterilized by filtration through0.22-,um GS filters (Millipore Corp., Bedford, Mass.)and then added to the agar media. Before sterilization,streptomycin and spectinomycin sulfates were dis-solved in demineralized water, mancozeb was dis-solved in 1% Na-EDTA solution, and thiram wasdissolved in chloroform.The bacteria were counted on nutrient agar or SMS
medium by the pour-plate method, the plates beingincubated for 72 h at 28°C. The medium in which thepseudomonad in soil or rhizosphere was counted con-tained the specific chemicals at the highest levels towhich the bacterium was resistant, which were 175 ,ugof mancozeb and 850 ,ug of spectinomycin per ml.
Effect of glucose and mancozeb amendment. Tripli-cate 25-g portions of Lima silt loam (pH 7.1) fromAurora, N.Y., were placed in 250-ml Erlenmeyerflasks. The soil was from the top 15 cm and was passedthrough a 0.2-mm sieve before use. The soil sampleswere inoculated with 3 x 104 pseudomonads per g andamended with (i) nothing, (ii) 1% glucose, (iii) 175 ,ugof mancozeb per g, or (iv) glucose and mancozeb. Thesoils were incubated at 28°C for 12 days at 35% of theirwater-holding capacity, and counts were made every 2days, using all the soil in each flask to prepare theinitial dilution. The counts were made on SMS agarand SMS agar containing 175 ,ug of mancozeb per ml.Root colonization. Pseudomonad cells from 5.0 ml of
a 48-h liquid culture were collected by centrifugation,washed three times with 0.1 M phosphate buffer (pH7.0), and suspended in 50 ml of a 40% aqueous gumarabic solution. This solution was mixed for 2 min with200 corn seeds (Northrup King variety PX20, pesticidefree), and the seeds were then allowed to dry for 1 h.Each seed was inoculated with 7 x 104 cells, and halfof the seeds were treated with mancozeb (about 2 mgper seed). Seven replicate groups, each with 20 seedsof similar size, were then placed in sterile plastic petridishes and covered with 100 g of Lima silt loam, and
the dishes were incubated for 12 days in a BiotronetteMark III environmental chamber (Lab Line Instru-ments, Melrose Park, Ill.) under 12 h of light providing3.0 ,uEinsteins/s per m2 at 28°C and 12 h of darkness at21°C. The soil moisture was kept at about 35% of thewater-holding capacity.Groups of 10 seedlings chosen at regular intervals
were gently removed from the soil with forceps, andthe seedlings were shaken to detach soil not stronglyadhering to the roots. The roots were then separatedfrom the seeds with scissors, and dilutions and countswere made separately for the seeds and for the entireroots. Dilutions prepared for every two seeds orseedlings were plated on SMS agar containing 175 ,ugof mancozeb per ml, and counts of the pseudomonadwere made on plates incubated at 28°C for 4 days.Movement of pseudomonad with roots. Four repli-
cate groups of seeds were inoculated with 700 cells perseed, treated with about 1.2 mg of mancozeb per seed,and sown in plastic pots (11.5 cm diameter by 7 cmhigh, one seed per pot) containing 600 g of soil. Theplants were grown for 26 days in the environmentalchambers, and counts of five plants were made atregular intervals. To perform the counts, the plantswere carefully removed from the pots with sterileforceps and gently shaken to dislodge soil not stronglyattached to the roots, and then the roots were cut into3-cm sections. The root pieces were placed in tubescontaining 10 ml of sterile distilled water, with no morethan 5 pieces of the same root section in a tube. Thesamples were mixed for 5 min at about 420 cycles permin with a Lab Line super mixer (Arthur Thomas,Philadelphia, Pa.), and dilutions were prepared andplated on SMS agar containing 175 ,ug of mancozeb perml. The dishes were incubated at 28°C for 4 daysbefore counting.On day 8 and at 4-day intervals thereafter, the
foliage of groups of the plants was sprayed twice witha solution of 0.1% Tween 80 in water or 0.1 M urea andTween 80. The pots were watered 3 h before eachspraying. The spraying was conducted with an atomiz-er, and both sides of the leaves were treated. Each leafreceived about 0.1 ml of liquid. To prevent drops fromthe sprayed leaves from falling onto the soil, we placedWhatman no. 3 filter paper disks around the stem ofeach plant above the soil. Root samples were preparedas described above, and the pseudomonads on the 3-cm portion of the roots nearest the stems and on the 3-to 6- and 6- to 9-cm segments were counted, usingSMS medium containing mancozeb.
Effect of seed and foliar treatment on introduced andindigenous bacteria. Uninoculated and inoculatedseeds treated or not treated with mancozeb were sownin soil in plastic pots, and the pots were placed atrandom in the environmental chambers and wateredevery 2 days. At 4-day intervals, the plants werewatered with 20 to 40 ml of a nutrient solution contain-ing 22.8 g of NH4NO3, 20.7 g of K2HPO4, and 19.7 g ofKH2PO4 per liter of distilled water (23). On day 5 aftergermination, each leaf was sprayed with about 0.1 mlof liquid. The seedlings derived from inoculated, fungi-cide-treated seeds received a 0.1% Tween 80 solutionor 0.1 M urea in the Tween 80 solution. The seedlingsgrowing from inoculated seeds not treated with man-cozeb were sprayed with 850 pLg of spectinomycin or1.0 mg of terrazole (5-ethoxy-3-trichloromethyl-2,2,4-thiodiazole) (Mallinckrodt Inc., St. Louis, Mo.) per ml
a Counts in millions of cells per gram. All other values are actual numbers.b ND, Not determined.
of the Tween 80 solution. The roots were removedfrom the soil, and counts of the introduced and nativebacterial populations on the 3-cm root portion nearestthe stem were made before and after the foliage wassprayed. Each treatment was replicated four times,and bacterial counts were made of each plant.
RESULTSIsolation of resistant bacteria. Dilutions of soil
from the corn rhizosphere were plated on tripli-cate plates of nutrient agar to which mancozeb,thiram, spectinomycin, or streptomycin hadbeen added. The colonies were counted after 3days of incubation at 30°C. At any one concen-tration of the four antimicrobial chemicals, man-cozeb permitted the growth of the fewest colo-nies (Table 1). Essentially the same results wereobtained for counts made from samples of thealfalfa and soybean rhizospheres, mancozeb be-ing the most effective of the chemicals againstthe bacteria from the rhizosphere of each plantspecies.
Six dissimilar colonies were selected from theplates, and these bacteria were subcultured re-peatedly in nutrient broth containing increasingconcentrations of the chemicals (first mancozeband then either spectinomycin or streptomycin)above the levels to which the bacteria werealready resistant. An isolate was chosen thatwas resistant to 175 ,ug of mancozeb and 850 ,ugof spectinomycin per ml. This bacterium was agram-negative, nonspore-forming, straight rod,0.5 to 0.7 ,um wide by 1.0 to 1.8 ,um long, thatwas motile and possessed 1 to 5 polar flagella. Itproduced a fluorescent pigment in King B medi-um and a yellow pigment in SMS medium. Itgave positive tests for catalase, urease, peroxi-dase, use of nitrate as a nitrogen source, nitriteand ammonium formation from nitrate, gelatinliquefaction, and the presence of poly-p-hydrox-ybutyrate granules. Gas was not produced fromseveral sugars, and N2 was not formed from
nitrate. These characteristics indicate that thebacterium is a strain of Pseudomonas (5, 17). Itwas designated Pseudomonas sp. strainMMR51.Pseudomonas sp. strain MMR51 retained its
viability when suspended in 0.1 M phosphatebuffer (pH 7.0) at 28°C on a rotary shakeroperating at 200 rpm. Thus, with a washedinoculum of 9.5 x 106 cells per ml derived from a48-h culture grown in SMS broth, the populationwas 2.1 x 106, 1.1 x 106, and 0.48 x 106/ml at 7,18, and 30 days, respectively. Hence, the bacte-rium is resistant to starvation.
Addition of glucose and mancozeb to soil. Todetermine whether this bacterium could becomeestablished if the indigenous soil community wassuppressed by an antimicrobial agent to whichthe added organism was resistant, a suspensionof Pseudomonas sp. strain MMR51 was intro-duced into soil that had been treated with glu-cose alone, mancozeb alone, a combination ofglucose and mancozeb, or no chemicals. Dilu-tions of the inoculated soils were plated, intriplicate, on SMS agar with and without manco-zeb. The total counts of bacteria able to grow onfungicide-free agar were initially low, the reasonfor which is unknown (Fig. 1). In soil receivingno mancozeb, counts of these bacteria roseslowly if no sugar was added but increased morerapidly and reached higher values if the soil wassupplemented with glucose. In soil treated withmancozeb, the total number of bacteria rosesomewhat but increased markedly if sugar wasalso added to the soil. The data also show thatPseudomonas sp. strain MMR51 fell to lownumbers by day 6 if the soil was not amendedwith mancozeb, although replication initially oc-curred when glucose was introduced into thesoil. In contrast, Pseudomonas sp. strainMMR51 populations were maintained at higherlevels in the mancozeb-treated soil, whether ornot glucose was also added.
FIG. 1. Populations of total bacteria and Pseudo-monas sp. strain MMR51 in unamended soil and in soilamended with glucose, mancozeb, or glucose plusmancozeb.
Colonization of roots. Colonization of cornseeds and roots was measured with seeds thatwere inoculated with the pseudomonad and ei-ther coated with mancozeb or left uncoated. Thedata show that the size of the Pseudomonas sp.strain MMR51 population on the seeds increasedduring the first few days and then decreased, butthe number of cells was far greater on themancozeb-treated than on the unamended seeds(Fig. 2). Similarly, the number of cells on rootsderived from mancozeb-coated seeds was fargreater than on roots from uncoated seeds;indeed, the population was often two orders ofmagnitude larger. Thus, coating the seeds withmancozeb favored the establishment and surviv-al of the resistant bacteria on both seeds androots.Movement of pseudomonad with roots. The
movement of bacteria from the inoculated seedwith the emerging root was determined withseeds treated or not treated with mancozeb. Thefoliage of some of the plants was sprayed withurea on day 8, and the spray was repeated
0
z.1o LH~~~~UTREATED
SEEDS SE
4
2 4 6 8 10 12
DAYS
FIG. 2. Colonization of corn roots and seeds byPseudomonas sp. strain MMR51 inoculated onto un-treated or mancozeb-treated seeds. The counts are pergram of seed or per gram of root system.
thereafter at 4-day intervals. The population ofPseudomonas sp. strain MMR51 on the 3-cmportion of mancozeb-treated roots nearest thestem (designated 0 to 3 cm) increased morequickly than that on roots derived from fungi-cide-free seeds (Fig. 3). However, the popula-tion sizes on both treated and untreated rootsdecreased after days 6 and 7. If urea was appliedto the foliage of plants derived from the manco-zeb-coated seeds, the extent of the decline innumbers was reduced, and a larger Pseudomo-nas sp. strain MMR51 population was main-tained. The pseudomonad was also found on the3- to 6- and 6- to 9-cm portions of the roots, andhere too its numbers were greater if the seedswere coated with the fungicide. The effect ofurea was evident also on the 3- to 6-cm seg-ments. The beneficial effect of urea may berelated to improved plant growth because ureain separate experiments was found to causestatistically significant increases (95% confi-dence interval) in plant weight at harvests madeon days 7, 11, 13, 17, and 21.
Effect of seed and foliar treatment on intro-duced and indigenous bacteria. Mancozeb-treat-ed and untreated seeds were inoculated with
ed seeds. The foliage of half of the corn plants emerg-ing from fungicide-coated seeds was sprayed withurea.
Pseudomonas sp. strain MMR51. After 5 days,the seedlings from the treated seeds weresprayed every 12 h with urea, and seedlingsgrowing from fungicide-free seeds were sprayedwith a solution containing urea and spectinomy-cn or a solution containing urea and terrazole.One group of seeds was not chemically treated.Each foliar spray was applied 4 times, andcounts were made on the 0- to 3-cm portion ofthe roots.The counts of total bacteria on the root seg-
ments were high if the seeds were not coatedwith the fungicide (Fig. 4). The total bacterialcount on the roots derived from the mancozeb-coated seeds was initially low, but then thenumbers increased but were still below those onseedlings arising from untreated seeds. The
00:,.0
U0z-j-Iw06,0-A
8 16 24DAYS
FIG. 4. Counts of total bacteria and Pseudomonassp. strain MMR51 on corn roots derived from manco-zeb-coated and uncoated seeds and of the pseudomo-nad on roots of plants having the foliage treated withurea, spectinomycin, or terrazole.
Pseudomonas sp. strain MMR51 populationcounted on SMS agar amended with mancozebwas again higher on plants derived from manco-zeb-treated seeds than it was on plants fromuncoated seeds, and in this instance, treatmentwith urea even more markedly enhanced thegrowth and survival of the pseudomonads. Al-though Pseudomonas sp. strain MMR51 wasspectinomycin-resistant, spraying the foliagewith this antibiotic resulted only in a slight andtransitory increase in numbers; in this instance,the agar medium used for counting containedboth mancozeb and spectinomycin. Similarly,application of terrazole, which presumably istranslocated downward, had only a transitoryeffect.
DISCUSSIONThese findings show that it is possible to
organisms that are resistant to a chemical thatinhibits the normal inhabitants of the root zone.It is not clear from the data presented whetherthe pseudomonad is favored because the inhibi-tor suppresses bacteria competing with the pseu-domonad for root and seed exudates or becausethe inhibitor suppresses protozoa or other pred-ators and parasites that feed on the pseudomo-nad, or both. However, the marked fall in num-bers of Pseudomonas sp. strain MMR51 afterthe initial period of proliferation is probably nota consequence of competition, because popula-tions of this bacterium do not decline readilywhen starved. The decline is similar to thatobserved in the bean rhizosphere for R. pha-seoli, a population decrease that has been as-cribed to predation by protozoa (14, 16). Theinability of Dobereiner and Divan Baldani (3) toobtain growth of streptomycin-resistant strainsof Azospirillum lipoferum and other bacteria oncorn roots may have resulted from their use of acell density far too large to show replication.Applying urea to the foliage also was benefi-
cial to the introduced pseudomonad. Vrany etal. (22) reported that the number of Pseudomo-nas putida in the rhizosphere of wheat wasincreased by applying urea to the foliage. Ureamay be beneficial because it alters the amountand composition of root exudates as a result ofits effects on photosynthesis (7, 10). Becauseurea and other foliar sprays alter the quantityand composition of the root exudates as well asthe microflora living on them (9, 20), such proce-dures may be a practical means of further favor-ing the growth or survival of an introducedbacterium.Members of the genus Pseudomonas may be
especially appropriate for inoculation becausesome are known to influence the development ofhigher plants (6) or to antagonize plant patho-gens (2). However, other microorganisms mightprove to be of greater practical value; for exam-ple, those that increase the availability of nitro-gen or phosphorus to plants, produce growthregulators, or act detrimentally on plant patho-gens. Bacteria that enhance plant growth are notdifficult to obtain (12).Mancozeb is known to decrease the total
number of bacteria in soil (4). The fungicide isnot known to be extensively metabolized bymicroorganisms in soil, but it is converted to theactive principle, ethylenethiuram monosulfide,which is itself persistent in soil (11, 21). Al-though mancozeb is useful for the purposesdescribed herein, other fungicides or antibioticsmight be appropriate candidates for suppressingthe rhizosphere flora. Thiram has already beenused for this purpose (14, 16).The approach described herein has major limi-
tations, however. Among the chief limitations
are the lack of appreciable movement of bacteriafrom the seed coat to distant portions of the rootsystem, the restricted movement of many organ-ic toxicants from the point of their introductioninto soil, and the high cost of fungicides andantibiotics. The problem of lack of movement ofthe inoculum organism from the seed could bealleviated by using an organism that persistsfrom year to year; in this way, the colonists ofthe distal roots would be cells derived from aninoculum added in a previous season. The re-stricted movement of many chemicals fromseeds to roots or downward after foliar applica-tion could be overcome by selecting chemicalsshowing basipetal translocation; such chemicalsare known. Finally, although fungicides andantibiotics are admittedly expensive, the com-pounds that are selected to enhance root coloni-zation might be those that are already applied toseeds or foliage for disease control.
This approach to enhance colonization is thuspotentially useful for increasing plant growth.Further work is needed, however, to definewhich processes should be enhanced, determinewhich organisms are most useful for the pur-poses described, find chemicals that will havethe needed properties, and devise means to alterthe physiology of the plant to enhance furtherthe beneficial action of the inoculated microor-ganism.
ACKNOWLEDGMENT
This work was supported in part by funds provided byUniversidad Centro-Occidental "Lisandro Alvarado," Bar-quisimeto, Venezuela.
LITERATURE CITED
1. Barber, L. E., S. A. Russell, and H. J. Evans. 1979. Inocu-lation of millet with Azospirillum. Plant Soil 52:49-57.
2. Cook, R. J., and A. D. Rovira. 1976. The role of bacteriain the biological control of Gaeumannomyces graminis bysuppressive soils. Soil Biol. Biochem. 8:269-273.
3. Ddbereiner, J., and V. L. Divan Baddani. 1979. Selectiveinfection of maize roots by streptomycin-resistant Azo-spirillum lipoferum and other bacteria. Can. J. Microbiol.25:1264-1269.
4. Donkche, B. 1974. Effets du "Mancozebe" sur la micro-flore des sols du vignoble bordelais. Premiers rdsultats.C. R. Acad. Sci. Ser. D 278:3011-3014.
5. Doudoroff, M., and N. J. Palleroni. 1974. Genus I. Pseu-domonas, p. 217-243. In R. E. Buchanan and N. E.Gibbons (ed.), Bergey's manual of determinative bacteri-ology. The Williams & Wilkins Co., Baltimore.
6. Eklund, E. 1970. Secondary effects of some pseudomo-nads in the rhizoplane of peat grown cucumber plants.Acta Agric. Scand. Suppl. 17:1-57.
7. Hinsvark, 0. N., S. W. Wittwer, and H. B. Tukey. 1953.The metabolism of foliar-applied urea. I. Relative rates ofC"402 production by certain vegetable plants treated withlabeled urea. Plant Physiol. 28:70-76.
8. Jackson, R. M., M. E. Brown, and S. K. Burlingham.1964. Similar effects on tomato plants of Azotobacterinoculation and application of gibberellins. Nature (Lon-don) 203:851-852.
9. Jadali, B. L. 1976. Biochemical nature of root exudates in
relation to root rot of wheat. III. Carbohydrate shifts inresponse to foliar treatments. Soil Biol. Biochem. 8:127-129.
10. Jalali, B. L., and K. H. Domsch. 1977. Effect of somefungitoxicants on the amino acid spectrum of wheat rootexudates. Phytopathol. Z. 90:22-26.
11. Kaars Slpesteln, A., and J. W. Vonk. 1970. Microbialconversions of dithiocarbamate fungicides, p. 799-804. InJ. Pochon and J. P. Voets (ed.), Action des pesticides etherbicides sur la microflore et la faunule du sol. FaculteitRijskuniversitet, Gent, Belgium.
12. Kloepper, J. W., and M. N. Schroth. 1981. Plant growth-promoting rhizobacteria and plant growth under gnotobi-otic conditions. Phytopathology 71:642-644.
13. Kundu, B. S., and A. C. Gaur. 1980. Establishment ofnitrogen-fixing and phosphate-solubilising bacteria in rhi-zosphere and their effect on yield and nutrient uptake ofwheat crop. Plant Soil 57:223-230.
14. Lennox, L. B., and M. Alexander. 1981. Fungicide en-hancement of nitrogen fixation and colonization of Pha-seolus vulgaris by Rhizobium phaseoli. AppI. Environ.Microbiol. 41:404-411.
15. Merriman, P. R., R. D. Price, J. F. Kollmorgen, T. Pig-gott, and E. H. Ridge. 1974. Effect of seed inoculationwith Bacillus subtilis and Streptomyces griseus on thegrowth of cereals and carrots. Aust. J. Agric. Res. 25:219-226.
16. Ramirez, C., and M. Alexander. 1980. Evidence suggest-ing protozoan predation on Rhizobium associated with
germinating seeds and in the rhizosphere of beans (Pha-seolus vulgaris L.). Appi. Environ. Microbiol. 40:492-499.
17. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966.The aerobic pseudomonads: a taxonomic study. J. Gen.Microbiol. 43:159-271.
18. Thompson, J. P. 1974. Seed inoculation of wheat andbarley with Azotobacter chroococcum in Queensland.Queensl. J. Agric. Anim. Sci. 31:129-138.
19. Thompson, J. P. 1974. Inoculation of wheat with Beijer-inckia derxii. Queensl. J. Agric. Anim. Sci. 31:139-144.
20. van Vuurde, J. W. L. 1978. The rhizosphere microflora ofwheat grown under controlled conditions. I. The effect ofsoil fertility and urea leaf treatment on the rhizoplanemicroflora. Plant Soil 50:447-460.
21. Vonk, J. W., and A. Kaars SlpesteQn. 1970. Studies onthe fate in plants of ethylenebisdithiocarbamate fungicidesand their decomposition products. Ann. Appi. Biol.65:489-496.
22. Vrany, J., V. Vancura, and M. Stanek. 1981. Control ofmicroorganisms by inoculation of seeds with Pseudomo-nas putida and by foliar application of urea. Folia Micro-biol. (Prague) 26:45-51.
23. Warncke, D. D., and S. A. Barber. 1974. Root develop-ment and nutrient uptake by corn grown in solutionculture. Agron. J. 66:514-516.
24. Wright, J. M. 1956. Biological control of a soil-bornePythium infection by seed inoculation. Plant Soil 8:132-140.
