Application method affects the distribution and efficacy of rhizobacteria suppressive of downy brome (Bromus tectorum)

Application method affects the distribution and efficacy of rhizobacteria suppressive of downy brome (Bromus tectorum)

Soil Biol. Biochem. Pergamon 003s0717(95)ooo53-4 Vol. 27, No. 10, pp. 1271-1278, 1995 Copyright 0 1995 ElsevierScience Ltd Printed in Great Britain...

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Soil Biol. Biochem.

Pergamon

003s0717(95)ooo53-4

Vol. 27, No. 10, pp. 1271-1278, 1995 Copyright 0 1995 ElsevierScience Ltd Printed in Great Britain. All rights reserved 0038-0717/95 $9.50 + 0.00

APPLICATION METHOD AFFECTS THE DISTRIBUTION AND EFFICACY OF RHIZOBACTERIA SUPPRESSIVE OF DOWNY BROME (BROMUS TECT0RUA4) MARK MAZZOLA,‘*

PHILLIP W. STAHLMAN’ and JAN E. LEACH’

‘Department of Plant Pathology, Throckmorton Hall, Kansas State University, Manhattan, KS 66506-5502, U.S.A. and *Kansas State University Agricultural Research Center-Hays, 1232 240th Avenue, Hays, KS 67601-9228, U.S.A. (Accepted 24 March 1995)

Summary-Rhizobacteria that are capable of suppressing plant growth in a species-specific manner have potential as bioherbicides. Three bacterial strains, Pseudomonas putida strain FH160, Stenotrophomonus maltophilia strain FH131, and Enterobacter taylorae strain FH650, have been reported to suppress the growth of downy brome (Bromus tectorum L.). These strains were evaluated in the greenhouse using various application methods for the ability to colonize the rhizosphere and to inhibit downy brome emergence or biomass production. When the biocontrol strains were incorporated into the soil profile more consistent colonization of the downy brome rhizosphere by all three strains resulted relative to that achieved by either wheat seed or soil surface applications. Downy brome emergence was reduced when any of the bacterial strains were applied as a soil surface application or a wheat seed treatment. Wheat biomass production was not enhanced when plants were grown individually in soils treated with any of the three biocontrol strains. When wheat was interplanted with downy brome, increases in wheat biomass were obtained with certain bacterial treatments relative to untreated controls suggesting that these rhizobacteria diminish the competitive ability of downy brome. Application method affects the population size and distribution of the biocontrol strain in the soil profile and downy brome rhizosphere, and thus, has a marked influence on efficacy of rhizobacteria for weed control

INTRODUCTION

Downy brome (Bromus tectorum L.) commonly exists in winter wheat stands at densities of lo&400 plants m - ’and can cause wheat yield reductions that range between 15-40% (Rydrych, 1974). Chemical herbicides provide inconsistent control of downy brome (Cook and Veseth, 1991). Cultural measures, such as crop rotation, do not limit downy brome seed dispersal, and are ineffective when other small grains are included in the rotation (Cook and Veseth, 1991). Therefore, it is necessary to develop alternative management strategies, including biological control measures, that will selectively control downy brome in winter wheat production systems. Inhibition of plant growth by deleterious rhizobacteria (DRB) can be species- or cultivar-specific (Gardner et al., 1984; Fredickson and Elliott, 1985; Schippers et al., 1987). Thus, DRB may have the potential to inhibit the growth or competitiveness of weed species without having negative effects on the growth and yield of a crop plant. Several rhizosphereinhabiting bacteria were identified that selectively inhibit root elongation or seed germination of downy *Author for correspondence, presently at: USDA Agricultural Research Service, Root Disease and Biological Control Research Unit, 365 Johnson Hall, Washington State University, Pullman, WA 99164-6430,U.S.A.

brome, but which have no apparent deleterious effect on the growth and development of winter wheat (Kennedy et al., 1991). In field experiments, Kennedy et al. (1991) obtained reductions in downy brome biomass of between 18-54% when specific bacterial strains were applied to the soil surface. In similar studies, strains capable of inhibiting downy brome root elongation reduced weed biomass and increased wheat grain yields by l&33% (Harris and Stahlman, 1990). As with many biological systems for pest management, a major impediment to the implementation of a biological weed control strategy that employs plant-associated microorganisms is the inconsistent performance of microbial agents under field conditions. Suppression of downy brome by individual strains of rhizobacteria was site-specific (Kennedy ef al., 1991), and inconsistent weed control was observed in field studies (Tranel ef al., 1993). The erratic performance of biocontrol rhizobacteria in the field is common, and can be attributed to several factors including the loss of ecological competence, inability of the biocontrol agent to produce the active compound at the appropriate time and place, and variable root colonization by the introduced strain (Weller, 1988; O’Sullivan and O’Gara, 1992; Weller and Thomashow, 1993). The relationship between rhizosphere colonization and the plant-suppressive

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effects of DRB in agricultural soils has received little attention. However, the delivery of the natural pesticides produced by a DRB including hydrogen cyanide (Bakker and Schippers, 1987; Alstrom and Burns, 1989) toxins (Tranel et al., 1993) and organic acids (Loper and Schroth, 1986) probably will require that the bacterium effectively colonize the rhizosphere of the target plant after it is released into the soil profile. The method utilized to introduce a biocontrol organism into an agro-ecosystem can significantly affect any subsequent rhizosphere colonization by the introduced strain. Rhizosphere colonization by fluorescent pseudomonads applied as a seed or seedpiece inoculant can be quite variable, and typically, many roots may not be colonized by the introduced strain (Bull et al., 1991; Loper et al., 1984). In contrast, rhizobacteria suppressive toward the take-all pathogen Gaeumannomyces graminis var. tritici colonized wheat roots more uniformly after spray-inoculation of soils (Mazzola et al., 1992). Bacteria are sensitive to rapid fluctuations in temperature and moisture, conditions that are common on the soil surface. Thus, bacterial strains applied directly to the soil surface may not survive in sufficient numbers to provide control of the target pest. Fundamental to the adoption of pest control measures is the ability to integrate them into current crop management practices. A broadcast soil surface application of a biocontrol agent would enable producers to utilize microbial herbicides as a preplant or postemergence treatment. Use of the biocontrol agent would be limited to a preplant application if the organism has to be incorporated into the top l&l2 cm of the soil profile. Seed application is an attractive means of introducing bioherbicides into the agroecosystem. However, this method of application requires that the organism initially colonizes roots emerging from the seed of the crop plant, and then colonizes the rhizosphere of adjoining target weeds to populations that are sufficient to inhibit plant growth. Our objective was to assess, under greenhouse conditions, the effect of application method on the distribution and survival of three downy brome-suppressive rhizobacteria in soil and the rhizosphere, and on the efficacy of these strains in reducing emergence and growth of downy brome. MATERIALS AND METHODS

weeds downy brome, Japanese brome (Bromus japonicus Thunb. ex. Murr.) or jointed goatgrass (Aegilops cylindrica Host). Each strain was resistant to tetracycline and carbenicillin at concentrations of 10 and 100 pg ml-‘, respectively. Spontaneous mutants of each strain possessing resistance to rifampicin were obtained by selecting colonies that exhibited vigorous growth after repeated transfer onto Pseudomonas agar F (Difco Laboratories, Detroit, MI, USA.) containing rifampicin (100 pg ml - ‘). Growth characteristics in terrific broth (TB) (Tartoff and Hobbs, 1987) and Pseudomonas minimal salts medium (Bolton and Elliott, 1989) and colony morphology of the mutants were similar to that of the parental strains. The rifampicin-resistant derivatives of strains FH13 1, FH160 and FH650 were designated FHl3lR, FH160R and FH650R, respectively, and were used in these studies. Application of bacteria

Individual bacterial strains were introduced into the soil system either as a surface application, incorporation into soil, or as a wheat seed treatment. Strains were grown overnight in TB with aeration at 28°C. Cells were collected by centrifugation, washed twice, and resuspended in sterile water. The individual strains were incorporated into separate soil samples to obtain populations of from lo5 to 10’ cfu g - ’ soil (Mazzola et al., 1992). Soils with a population of lo5 cfu g - ’soil of one of the biocontrol strains were used in experiments to assess biocontrol efficacy, but were not included in studies examining the distribution and survival of the introduced strains in soil and the rhizosphere. Bacteria were applied to the soil surface by pipetting 1 ml of a cell suspension (approximately lo9 cfu ml - ‘) of the respective strains directly onto the soil surface (6.28 cm’) of each plant-growth container (described below). Seeds were inoculated by resuspending the individual strains in a 1.5% methyl cellulose solution and applying the individual strains to wheat seeds (cv. Karl). Treated seeds were air-dried in a laminar flow hood prior to planting and populations of each strain were determined by placing treated seed in 20 ml of sterile distilled water, vortexing for 5 min, and plating serial dilutions of the seed wash onto Pseudomonas agar F amended with 40 ,ug rifampicin ml -I and 75 pg cycloheximide ml--‘. Populations of introduced strains at the time of planting were between 1 x 10’and 5 x lOacfu seed - ‘. Experimental design and sampling procedures

Bacterial strains and media Enterobacter taylorae strain FH650, Pseudomonas putida strain FH160 and Stenotrophomonas maltophilia [ = Xanthomonus maltophilia (Palleroni and

Bradbury, 1993)] strain FH131 were used in these studies. All three strains were isolated from the rhizosphere of downy brome or adjacent soil. All had been shown in field studies (Harris and Stahlman, 1990) to reduce growth of the winter annual grass

All experiments were conducted in natural Kennebec silt loam. Untreated soil, or soil that had been treated with one of the bacterial strains as described above, was dispensed into conical plastic tubes [20.7 x 4cm (top dia.); Stuewe & Sons Inc., Corvallis, OR, U.S.A.] with each tube (soil cell) containing 100 g soil (dry wt). Untreated soils were planted with (a) wheat seed that had received one of the bacterial treatments, (b) untreated wheat seed or

Distribution of downy brome-suppressive bacteria wheat seed followed by application of 1 ml per soil cell of the appropriate bacterial suspension distributed over the soil surface. Soils with incorporated bacteria were planted with untreated wheat seed. Two wheat seeds were planted in thecenter of each soil cell at a depth of 2 cm. At the same soil depth, eight downy brome seeds were arranged in a circle at a distance of approximately 1.5 cm from the wheat seeds. Each soil cell planted to wheat and downy brome represented one experimental unit. Treatments were arranged in a completely randomized design with 20 soil cells per treatment. Plants were grown in the greenhouse for 30 days at 17 f 3°C without supplemental lighting. During the initial experiment, plants were watered from below for the duration of the experiment by submerging the bottom 5 cm of the plastic tubes into water. The biocontrol strains failed to effectively colonize the soil profile when bacteria were applied to the soil surface and watered from below. Therefore, in all subsequent studies, plants were watered from above with 10 ml of tapwater at 0, 12, 18 and 24 days after planting to facilitate dispersal of the introduced strains into the soil profile. Downy brome and wheat emergence were assessed at 10 days after planting, and the appropriate number of wheat and downy brome seedlings were randomly harvested and discarded so that for the duration of the growth period each soil cell contained one wheat and four downy brome seedlings. Soil and rhizosphere populations of the introduced strains were determined every 10 days. At each sampling, five soil cells were randomly selected for each treatment. Soil samples were collected from the top 3 cm of the soil profile for all bacterial treatments. For seed and surface treatments of S. maltophiliu FH13lR and P. putidu FHl60R, an additional soil sample was obtained from the bottom 3 cm (12-l 5 cm depth) of the soil profile. Soil (100 mg dry wt) was suspended in 10 ml sterile water and populations of the introduced strain were determined by plating serial dilutions of the soil suspension onto Pseudomonas agar F (two plates for each dilution) containing 40 pg rifampicin ml - ’and 75 pg cycloheximide ml _ ‘. These antibiotic concentrations were determined to be sufficient to suppress growth of the resident soil microflora. Colonies were enumerated after 48 h at 28°C. Rhizosphere populations of the introduced strains were estimated for each wheat plant and for one randomly-selected downy brome plant in each soil cell. The wheat root segments 3-5 and 7-9 cm below the seed were excised from the primary root (R) and either the-2A or -2B seminal root, the roots that emerge from the seed immediately following the primary root (Klepper et al., 1984). For downy brome, the root segments 2-6 and 8-12 cm below the seed were excised from the primary root and served as the sampling units. Root segments were placed in 10 ml sterile water, vortexed for 60 s, and serial dilutions of the root (c) untreated

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wash were plated onto selective media and incubated as described above. Potential direct effects of the three bacterial strains on growth of wheat were assessed in separate experiments. Treatments were as described above with 10 soil cells per treatment and two wheat seeds planted in each cell. Wheat was grown individually in conical tubes rather than interplanted with downy brome. Plants were harvested at 30 days after planting and wheat biomass was determined as described below. Determination of plant biomass

After 30 days of plant growth, five soil cells were harvested randomly for each treatment. All plants were removed from the tube, soil was discarded and roots were washed to remove all adhering soil. Plants were dried in an oven at 65°C for 3 days, and plant dry weights for wheat and downy brome were determined. Data analysis

Data were analyzed with SAS, using the general linear models procedure, including ANOVA and the LSD test (P = 0.05). Log transformation of bacterial population data was performed prior to analysis. All experiments were conducted twice. RESULTS

Soil populations

Regardless of application method, each soil cell contained an initial total population of between lo* and lo9 cfu of one of the biocontrol strains. The population size of the introduced strain in the top 3 cm of the soil profile declined gradually over time, and the magnitude of this decrease in population size was dependent upon strain and inoculation technique (Table 1). The population of the introduced strains in the upper 3 cm of soil fluctuated the least when strains had been incorporated into the soil profile. When applied to the soil surface, P. putidu FH 160R exhibited no significant change in population over 30 days, but S. maltophilia FH131R and E. taylorae FH650R demonstrated a decline in population size, and at 30 days after planting were significantly smaller than their respective populations at 10 days after planting. In comparison to other methods, applying these rhizobacteria as a wheat seed inoculant generally resulted in smaller populations of the introduced strains in the upper soil profile (Table 1). When introduced as a soil surface treatment, S. maltophiliu FH13lR and P. putidu FH160R were detected at a depth of 12-15 cm within 10 days after planting. In contrast, when applied to wheat seeds S. maltophiliu FH131R was not detected at the 12-15 cm soil depth until 20 days after planting, and then at a population of less than 10’cfu g- ’soil. Although P. putidu FH160R was detected in the lower portion of the soil profile by 10 days postplanting when applied as a wheat seed treatment, its population was

Mark Mazzola et al.

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Table 1. Populations at various days after planting of E. ~a$wae FH650R (.I?. /.), P. pufida FHl6OR (P. p.) and S. mahophilia FHl31R (S. m.) at a depth of O-3 cm or 12-15 cm in Kennebec silt loam

Application method’ Seed

Strain S. m. P. p. E. 1.

Incorporation

s. tn. P. p. E. 1.

Surface

s. In. P. p. E. I.

Loe cfu cell ’ ai planting

(Iog cfu g - ’soil) - 12-l 5 cm soil depth

Po*tion t&3 cm soil depth 10 days

20 days

10 days

20 days

8.57 8.66 8.54

4.64aBt S.llabA 5.58bcdC

5.05bB 5.16bcA 4.39aB

3.92bA 4.54cA 3.07aA

30 days

OaA 2.92bA ND$

0.50aA 3.33bA ND

8.96 9.02 8.93

5.50bcAB 5.95cdA 6.17deA

6.13dB 5.82cdA 5.96dA

5.15deA 5.65efA 5.9OfA

ND ND ND

ND ND ND

8.74 8.88 8.82

S.85cdB 5.74cdA 6.66eB

5.98dB 5.24bcA 5.74cdA

5.20deA 5.04cdA 5.5OdefA

4.6lcA 5.03cB ND

3.93bA 4.92cB ND

30 days 3.96aB 4.68aB ND ND ND ND 4.86aA 4.07aA ND

*Seed = bacteria were applied as a wheat seed treatment; incorporation = bacteria were applied as an atomized mist and distributed uniformly throughout the soil sample by mixing; and Surface = bacteria were applied by pipetting the cell suspension onto the soil surface. tFor a given soil depth or root segment, means within a column that are followed by the same lowercase letter or wthin a row followed by the same uppercase letter do not differ significantly. $ND = Not determined.

significantly smaller than that observed at the same sampling time when this strain was applied to the soil surface (Table 1). At 30 days, there were no significant differences in the population sizes of the introduced bacteria among any of the strains or application methods. Wheat rhizosphere populations of introduced strains

The population of S. maltophilia FH 13 1R on the root segment 3-5 cm below the seed was significantly smaller than that of P. putida FH160R by 10 days regardless of application method, and was smaller than the population of E. taylorae FH650R except where bacteria had been applied as a wheat seed treatment (Table 2). Of the three biocontrol strains, only S. maltophiliu FH131R exhibited an increase in wheat rhizosphere population between 10-20 days. By 20 days the population of S. maltophilia FH131R was larger than that of both P. putida FH160R and E. taylorae FH650R for all application methods (Table 2). Thereafter, the population of each strain on the wheat root segment 3-5 cm below the seed declined at least 90%. At 30 days, the population of E. taylorae FH650R was smaller than that of S. maltophiliu FH131R and P. putidu FH160R where bacteria had been applied as a wheat seed treatment (Table 2).

Initial colonization of the wheat root segment 7-9 cm below the seed by S. maltophilia FH131R was restricted relative to either P. putida FH160R or E. tavlorae FH650R when applied as a soil surface treatment (Table 2). However, the rhizosphere population of S. maltophilia FH131R continued to increase, and by 30 days it was present in the rhizosphere at a significantly larger population than P. putida FH160R. When applied as a wheat seed inoculant, colonization of the 7-9 cm root segment during the initial 20 days of plant growth was comparable for all three strains, and by 30 days, the rhizosphere population of E. taylorue FH650R was smaller than that of S. maltophiliu FH131R and P. putida FHl60R. Downy brome rhizospherepopulations of the introduced strains

The population dynamics of the introduced strains in the rhizosphere of downy brome exhibited patterns which were similar to those observed for the respective strains in the wheat rhizosphere. S. maltophilia FH 13 1R and E. taylorae FH650R generally colonized the downy brome rhizosphere more slowly than P. putidu FH160R; however, by 20 days the populations of S. maltophilia FH 13 1R and E. taylorae FH650R on

Table 2. Rhizosphere populations at various days after planting ofE. taylorae FH650R (E. t.), P. putida FHl60R (P. p.) and S. malrophilia FHl3lR (S. m.) on the wheat root segments 3-5 and 7-9 cm below the seed Population on the wheat mot segment(log cfu cm - I root) 3-5 cm soil depth 7-9cm soil depth

Application method*

Strain

10 days

20 days

-_-_ 30 days

IO days

20 days

30 days

Seed

S. m.

4.77bB 5.03cc 4.39aB

5.34bC 4.58aB 4.72aB

3.76bcA 3.70bA 1.OOaA

3.33bcA 2.64abA 2.93abA

4.39cA 3.67bcA 4.17~8

3.80bcA 3.19bA 2.OlaA

5.30dB 5.73eB 5.83eB

6.27dC 5.58bcB 5.73cB

4.64eA 4.OOcA 4.44deA

ND ND ND

ND ND ND

ND ND ND

5.38dB 5.95eB 5.97eC

6.38dC 5.85cB 5.37bB

4.48deA 4.3ldA 4.46deA

2.33aA 4.81dB 4.06cdB

2.04aA 3.24bA 2.27aA

4.69dB 3.18bA 4.34cdB

P. p. E. t.

Incorporation

S. m. P. p. E. t.

Surface

S. m. P.p. E. r.

*See footnotes to Table 1

Distribution

of downy

brome-suppressive

bacteria

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Table 3. Rhizosphere populations at various days after planting of E. ra~loroe FH650R (E. I.), P. parida FH16OR (P, p,) and S. mafrophilia FHl3lR (S. tn.) on the downy brome root segments 2-6 and 8-12cm below the seed PopnIrtIanondownybmmeraotaagmant(lagefnenl-’mot) 2-6 cm soil depth 8-12 cm soil depth Application method*

Strain

10 days

20 days

30 days

10 days

20 days

30 days

Seed

s. m.

2.88aA 3.72bB 2.57aB 4.llbA 4.94cB 4.0lb 4.52bcA 5.09cA 5.18cB

4.62bB 2.99aAB 3.10aB 5.7OcB 4.66bB 4.43bB 5.52cB 6.OOcB 5.24bcB

3.93bB 2.34aA 1.62aA 4.9lcAB 3.85bA 24OaA 4.56bcA 4.82cA 1.91aA

OaA 1.89bA 2.26bA 4.03cA 499dB 4.42cdB OaA 3.95cA 3.86cB

0.74aA 4.14deB 3.77cdE 5.59fB 4.7Oei-B 4.49deB 2.97cB 3.03cA 2.00bA

3.65bcB 2.87abAB 2.56aA 4.97dB 3.98cA 2.95abA 4.14cdc 3.93cA 24OaA

P. p. E. I.

s. m.

Incorporation

P. p. E. r.

s. tn.

Surface

P.p. E. t.

*See footnotes to Table 1.

the root segment 2-6 cm below the seed were each comparable to or larger than that of P.putida FH160R (Table 3). With the exception of the wheat seed treatment, the population size of strain E. taylorae FH650R on the root segment 2-6 cm below the seed was smaller than that of both S. maltophilia FH 13 1R and P. putidu FH160R by 30 days. When applied as a wheat seed inoculant or to the soil surface S. maltophilia FH 13 1R was not detected on the 8-l 2 cm root segment until 20 days postplanting (Table 3). In contrast, at 10 days P.putida FH160R and E. taylorae FH650R were recovered from downy brome root segments at a depth of 8-12 cm at a population of lo* to lo4 cfu cm - ’root. When incorporated into soil, the population of the introduced strains on the 8-12 cm root segment was similar to that detected on the root segment 2-6 cm below the seed (Table 3). Of the three application methods, soil incorporation treatments resulted in the largest population recovered from the 8-12 cm root segment for each of the introduced strains (Table 3). Plant emergence

When incorporated into Kennebec silt loam at an initial population of approximately lo5 or 10’cfu g- ’ soil, all strains failed to significantly reduce downy brome emergence (Table 4). Downy brome emergence

was reduced by as much as 33% when these bacteria were applied either to the soil surface or as a wheat seed treatment. Wheat emergence was unaffected by any of the bacterial treatments (data not shown). Plant biomass

Downy brome biomass was not reduced significantly by any of the bacterial treatments (Table 4). When wheat was interplanted with downy brome, significant increases in wheat biomass were observed only when bacteria had been incorporated into soil (Table 4). Incorporation of S. maftophilia FH131R into soil at either 10’ or 10’ cfu g-l soil resulted in increased wheat dry weight, while strain P. putidu FH 160R provided an increase in wheat biomass when incorporated into soil at an initial population of 1O’cfu g - I soil. Regardless of application method, E. tuylorue FH650R failed to provide a significant increase in wheat biomass. Enhanced growth of wheat in soils treated with any of these rhizobacteria was not observed when wheat was grown individually rather than interplanted with downy brome (Table 5). DISCUSSION

Many previous investigations have the population dynamics of introduced

Table 4. Effect of E.raylorae FH650R (E. f.), P. putida FHlCOR (P. p.) and S. mnlrophilia FH13lR (S. m.) on wheat and downy brome biomass production, and downy brome emergence Application method* Control Seed Seed Seed Incorp. Incorp. Incorp. Incorp. Incorp. Incorp. Surface Surface Surface

Strain

s. m. lo5 lo5 10J 10’ 10’ 10’

P. p. P. p. s. In. P. p. P. p. s. tn. P. p. P. p. s. tn. P. p. P. p.

Average wheat dry wt (mg) 182ab$ 194abc 173a 176a 213~ 190abc 205bc 214c 215~ 208bc 180ab 203lx 173a

Average downy brome dry wt (ma) 13.5a 13.4a 13.8a 14.0a 12.7a 10.9a 13.4a ll.Oa Il.Xa 12.5a 13.3a 14.Oa 16.Oa

Downy brome emeraencet 6.5a 4.4c 5.4b 5.4b 6.0ab 6.lab 6.6a 6.0ab 5.9ab 6.7a 5.7b 5.3b 5.3b

*See footnotes to Table 1. Incorp. IO5and Incorp. IO’indicate that strains were established at an initial population of 10”and 10’cfu g - I, respectively. tValue is the mean number of downy brome plants per cell at 10 days postplanting, with 20 cells per treatment and each cell initially planted with 8 downy brome seeds. fMeans within a column that are followed by the same letter do not differ significantly.

monitored biocontrol

Mark Mazzola et al.

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Table 5. Effect of E. ta~lorae FH65OR (E. 1.). P. pucida FHI60R (P. p.) and S. ntaltophilia FH131R (S. m.) on wheat biomass production Application method* Control Seed Seed Seed Incorp. Incorp. Incorp. Incorp. Incorp. Incorp. Surface Surface Surface

Strain S. m. P. p. E. I.

101 IO’ 10s 10’ 10’ IO’

s. in. P. p. E. f.

s. in. P. p. E. I.

s. m. P. p. E. I.

Average plant weight (mg)t 216 203 217 212 221 206 220 215 228 223 229 194 223

*See footnotes to Table 1. Incorp. 10’and Incorp. 10’indicate that strains were established at an initial population of IO’and 1O’cfu g I, respectively. tThere was no significant (P = 0.05) difference detected between any of the means.

rhizobacteria and characterized the environmental conditions and microbial traits important to successful colonization after introduction into soil systems (Parke, 1991; Kloepper and Beauchamp, 1992; Weller and Thomashow, 1994). However, although many investigators have examined a particular method of bacterial application, few have evaluated multiple application methodologies in a single growing system and related this to biocontrol activity. Our studies demonstrate that the method of application can be an essential element in achieving successful biological weed control with plant-suppressive rhizobacteria. The method by which biocontrol strains were introduced into soils had a significant effect on their distribution in the soil profile and their efficacy in suppression of downy brome emergence. Interestingly, when applied as a wheat seed treatment (approximately lo* cfu seed- I), all three biocontrol strains successfully colonized the downy brome rhizosphere to a population size of between lo)-lo4 cfu cm ’root, and the introduced strains were present at populations of lo’-10” cfu g-’ soil even at a depth of 15 cm. The biocontrol strains were not detected on the downy brome root segments 8-12 cm below the seed prior to recovery of the strain from bulk soil at a depth of 12-l 5 cm. In addition, the population dynamics of the introduced strains in the rhizosphere mirrored the population dynamics of the introduced strains in bulk soil. Efficient dissemination of the introduced strains through the soil profile was achieved only in the presence of percolating water. Therefore, it is likely that downy brome roots were colonized by bacterial cells that were washed from treated wheat seeds into the soil profile, rather than resulting entirely from contact between downy brome roots and wheat roots that had been colonized by the biocontrol strain. Among the three application techniques, wheat seed treatment was the least efficient method to ensure colonization of the downy brome rhizosphere by the introduced strains. Wheat seed treatment resulted in the greatest variability in rhizosphere populations, the greatest number of uncolonized roots, and the smallest

maximum soil and rhizosphere populations for each of the introduced strains. Application as a seed inoculant was also found by Wei et al. (1993) to be a less efficient means, relative to soil drench application, for establishing the plant growth-promoting rhizobacterium P. putida strain 34-13 in the rhizosphere of cucumber. The ability to rapidly colonize the rhizosphere of the host plant and to persist in competition with indigenous rhizosphere colonists is an essential microbial trait for successful control of soilborne plant diseases with microorganisms (Weller, 1988). Likewise, rhizosphere competence is an important characteristic of any potential bioherbicide that functions by releasing into the rhizosphere phytotoxins that inhibit root development. Although all three introduced strains persisted in soil in similar numbers, downy brome rhizosphere populations of E. taylorae FH650R consistently demonstrated a more rapid decline in numbers. In general, the population of E. taylorae FH650R in the downy brome rhizosphere was 1-2 orders of magnitude smaller than the population size of S. maltophilia FH 13 1R and P. putida FH 160R by 30 days after planting. These findings suggest that E. taylorae FH650R is less capable of competing with the indigenous rhizosphere microflora and, therefore, will not be a suitable organism for providing consistent weed control. Our experiments demonstrate that application method influences the ability of these rhizobacteria to provide effective biological weed control. Wheat seed and soil surface treatments of any of the biocontrol strains provided a significant reduction in downy brome emergence. These strains failed to reduce plant emergence when incorporated into the soil profile at a population of either 10’or 10’cfu g- ’soil. When the relative distribution of the biocontrol strains and their populations are considered, these results are not unexpected. All treatments, with the exception of soil incorporation at 10’ cfu g-’ soil, were applied to obtain an initial population of between lo8 and lo9 cfu soil cell ’for the introduced strain. Therefore, when applied to wheat seed or to the soil surface, populations of the biocontrol strains were localized in proximity to downy brome seeds, while the soil incorporation treatment dispersed the introduced strain uniformly throughout the soil profile. This suggests that the ability of a given biocontrol strain to suppress downy brome emergence, not surprisingly. may be concentration dependent. Although the differences observed in downy brome biomass production between treatments were not statistically significant, certain treatments did enhance wheat biomass production. In the absence of downy brome, the presence of these strains did not result in increased wheat biomass. In addition, although bacterial treatments did not significantly reduce downy brome biomass, those treatments that had the lowest average downy brome dry weights generally had enhanced wheat biomass. These increases in wheat

Distribution of downy brome-suppressive bacteria

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