Soil bacteria selected for suppression of Rhizoctonia solani, and growth promotion, in bedding plants

SoilBiot.Biochem.Vol. 26, No. 9, pp. 1249-1255,1994

Copyright0 1994 ElsevierscienceLtd Printedin Great Britain.All rightsreserved 0038-07 I7/94 $7.00 + 0.00

003%0717(94)E0034-Y

SOIL BACTERIA SELECTED FOR SUPPRESSION OF RHIZOCTONIA SOLANI, AND GROWTH PROMOTION, IN BEDDING PLANTS A. R. HARRIS,‘*D. A.

SCHISLER,‘~ R. L. CQRRELL’

and M. H.

RYDER’

‘CSIRO Division of Soils and *CSIRO Biometrics Unit, Private Bag No. 2, Glen Osmond, SA 5064, Australia (Accepted II February 1994) Summary-Over

5000 bacterial isolates were obtained by dilution plating of potting media that were suppressive to damping-off disease caused by RhizoctoniasotaniAG 8 in seedlings of Celosia argentea. These isolates were screened for inhibition of AG 8 in vitro,then 91 selected isolates were screened further for their ability to suppress the effects of R. solani AG 8 in seedlings of Capsicumannuum and Celosia argentea grown in pasteurized potting medium in a glasshouse. Fourteen isolates selected from the above screenings then were screened similarly for suppression of damping-off caused by R. solani AG 4 in seedlings of Capsicum, Celosia, Petuniaand Viola. Three isolates reduced disease more consistently than the others. B. subtilisA13, an isolate previously reported to control R. solaniin bedding plants, did not reduce damping-off in any host plant species. These three isolates were compared in two experiments over a range of doses for growth promotion, and suppression of AG 4, in Capsicum.Although there were few dose responses, when data for all dose rates were combined, all isolates increased dry weights of Capsicum shoots in the absence of R. solani. In one of the experiments, all but one isolate also increased seedling survival in the absence of R. solani. Two bacterial isolates and the fungicide quintozene reduced damping-off caused by AG 4. The selected bacterial isolates exhibit potential for biological suppression of damping-off in bedding plants in nursery potting media.

1971; Kommedahl and Windels, 1978; Howell and Stipanovic, 1979; Geels and Schippers, 1983; Homma and Suzui, 1989; Tschen et al., 1989; Zhang et al., 1990). Several isolates of Pseudomonas spp and Bacillus spp did not control damping-off in zinnia grown in soil-less mix (Lumsden and Locke, 1989). Pseudomonas spp can promote plant growth in the absence of pathogens (Elad et al., 1987; Lifshitz et al., 1987), and Bacillus spp promote growth of some plant genera in steamed potting medium, especially under low nutrient regimes (Broadbent et al., 1977). We describe glasshouse experiments to test the efficacy of bacteria, isolated from nursery potting media, for control of two anastomosis groups of R. solani on seedlings grown in a pasteurized potting medium. We also describe the direct effect of the bacteria on seedlings in pasteurized potting medium with no added pathogens.

INTRODUCIION

A wide range of bedding plants is susceptible to damping-off diseases throughout the world (Stephens et al., 1983, Cline et al., 1988). Nurseries commonly suffer losses due to death of seedlings or increased

production costs. Many nurseries reduce the incidence of damping-off in container-grown seedlings by pasteurization or fumigation of potting media and pots, combined with good nursery hygiene. However, fungicides often still need to be drenched into potting media after seeding to prevent or contain disease outbreaks. The major causes of damping-off in bedding plants are Rhtzoctontu solani Kuhn [teleomorph = Thanatephorus cucumeris (Frank) Donk] and Pythium spp (Cline et al., 1988). Damping-off attributed to Rhizoctoniu in the Ohio bedding plant industry apparently is caused primarily by R. soluni anastomosis group 4 (AG 4) (Stephens et al., 1982), but isolates from South Australian nurseries sity (Schisler et al., 1993).

show greater diver-

Several

MATERIALS AND METHODS

researchers have reported suppression of R. solani by soil bacteria (Broadbent et al.,

*Author for correspondence. TPresent address: National Center for Agricultural Utilization Research, USDA-ARS, 1815 N. University Street, Peoria, IL 61604, U.S.A.

Source and culture of Rhtzoctonia bolates R. solani AG 8 isolate R-21 was obtained from roots of diseased wheat seedlings from Avon, South Australia. This isolate is also pathogenic on capsicum (syn. bell pepper, Capsicum annuum L. ‘Green Giant’) (Harris et al., 1993a), celosia dwarf (Celosia argentea L. var. crtstata ‘Fiery Feather’) (Schisler and Ryder,

1249

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A. R. HARRISet al.

1991, Brussels sprouts (Brassica oleraceu L. ‘Lunet’), silver beet (Beta oulgaris L.) and ornamental lupin (Lupinus angustifolius L. ‘Russell mixed’) (unpubl. data). R. soluni AC 4 isolate DlBl was collected by baiting a potting medium from a plant nursery near Adelaide, South Australia (Schisler et al., 1993). Isolates of pathogenic R. soluni were cultured on sterilized wheat bran or millet seed (Harris et al., 1993a). The wheat bran was sieved to obtain the 250-500 pm fraction, washed with deionised water, and sterilized before adding inoculum of R. soluni. Potting medium and punnets

Seedlings were grown in a commercial potting medium that consisted of sphagnum peat moss, sand and rice hulls (70: 15 : 15 vol), plus basic fertilizers containing macro- and micronutrients. Plastic punnets (seedling trays) each consisted of six tapered cells ca 3.5 x 3.9 cm at the top, 5.0 cm deep, and cu 55 cm3 capacity. Source and preliminary screening of bacterial isolates

A bioassay was used to identify soils that were suppressive to Rhizoctonia and therefore were more likely to contain high numbers of organisms suppressive to Rhizoctonia. The bioassay was made on 53 samples of potting media that were obtained from 32 nurseries or potting mix suppliers in South Australia. Samples included freshly prepared soil-less potting media, fresh potting media amended with previously cropped media and pasteurized, and currently or previously cropped media. Air-dried to slightly moist samples were added individually to an air-dried, y-irradiation-sterilized (5 Mrad) potting medium (described in previous section) at a rate of 10: 90 (v/v). In this way, soil microbial properties, but not chemical and physical properties, were transferred to a uniform soil background. Potting media were moistened with sterile deionized water to give soil water potentials of ca -0.1 MPa (- 1 bar). The potting media were then kept at 21°C in loosely tied plastic bags for 1 week. For each treatment, three replicate plastic cups (125 cm3 capacity, 70 mm dia, 50mm tall) were filled with potting medium, and eight millet seeds infested with R. soluni isolate R-21 were banded 2.7 cm below the soil surface. After perforated lids were put on the cups, they were placed in a constant temperature root air bath (21°C) in a glasshouse, and kept for an additional 7 days. Ten seeds of Celosia argentea then were added to each cup and the seeds covered with sterile coarse river sand. Seedlings were grown for 34 weeks, and scored for total plant stand and total shoot dry weights. Roots of seedlings from individual cups that showed no sign of root disease (plant survival and shoot weights similar to control cups without added pathogen) were selected for dilution plating. The cups that were selected represented microbial components from 13 different nurseries. Seedling roots were shaken gently, and appropriate dilutions of rhizosphere soils and

root macerates were plated for aerobic bacteria, actinomycetes, heat-resistant microbes and pseudomonads as described by Schisler and Ryder (1991). Over 5000 isolates from dilution plates then were screened for suppression of R. solani R-21 by an in oitro bioassay (Schisler et al., 1991). Isolates that inhibited R-21 in vitro, or were recovered in high numbers (based on similar colony type) from treatment pots that exhibited biological suppression of Rhizoctonia damping-off, were stored in 15% glycerol at -70°C. When needed, bacteria were plated on l/2 or l/5-strength tryptic soy agar in Petri dishes and grown at 25°C. Bacillus subtilis Al3 was obtained from P. Barkley (nee Broadbent), N.S.W. Agriculture, Rydalmere, Australia to compare with our bacterial isolates for disease suppression and plant growth promotion. Isolate Al3 has been reported to control some isolates of R. soluni (Broadbent et al., 1971), and originally was obtained from lysed hyphae of Sclerot ium rolfsii (Turner and Backman, 1991). Al3 is used commercially in a formulated seed inoculant (Turner and Backman, 1991) for application at ca lo* viable spores g-’ of seed, and also is registered for biological control of soil fungi in field crops. In our experiments, unformulated bacteria were used. Screening of bacterial isolates for suppression of R. solani AG 8 on Capsicum and Celosia seedlings R. sofani isolate R-21 was cultured on millet seed (Harris et al., 1993a). Two infested millet seeds were placed on a 2-cm3 layer of moist, sterilized sand in the bottom of each punnet cell. In the control with no R. soluni treatment, autoclaved infested millet seeds were added. Punnets were then filled with pasteurized uninfested potting medium. Punnets were seeded with either Capsicum annuum ‘Green Giant’ (Capsicum) or Celosia argentea var. cristata ‘Fiery Feather’ (Celosia) using a commercial vacuum drum seeding machine. Punnets to be seeded with Capsicum were run through the seeding machine twice to give 14 seeds per punnet cell. The other punnets were passed through the seeding machine once to give l-5 Celosiu seeds per punnet cell. Punnets were top dressed with fine vermiculite and watered to saturation. Each of 91 bacterial isolates was suspended in sterile deionized water to make a concentration of ca 3 x 10’ bacterial cells ml-‘, based on spectrophotometer measurements. For each isolate, 2.25 ml of the suspension was pipetted onto the surface of the potting medium in each cell of four replicate punnets (i.e. 7 x 10” bacteria per punnet cell or 5 x 10’ bacteria g-’ dry wt of potting medium). The fungicide propamocarb (Previcur, Schering AC, Alexandria, Australia), though not usually considered to be effective against Rhizoctonia, was included as a treatment of Capsicum, but not Celosia, seedlings because it is used in plant nurseries against unspecified dampingoff pathogens. The propamocarb was diluted to 1.78 ml 1-l deionized water, and tolclofos-methyl

Bacteria suppress Rhizoctoniaand stimulate seedlings

1251

(Rizolex, Sumitomo Chemical Co., Osaka, Japan) and benomyl (Benlate, Du Pont, N. Sydney, Australia) were each diluted to 594 mg of 50% wettable powder 1-i deionized water plus 296 ~1 of non-ionic organic surfactant (Agral 60, ICI Australia Ltd). Each fungicide suspension was pipetted onto the surface of the potting medium at a dose of 4.3 ml per punnet cell, i.e. 4.6 mg a.i. (active ingredient) of propamocarb or 1.3 mg a.i. of tolclofos-methyl or benomyl per punnet cell. After allowance for drainage, the amounts of active ingredients retained would have been ca 1.5 times the recommended commercial rates. Each fungicide treatment had four replicate punnets, and there were 20 for controls with R. solani and 16 for controls without R. solani. Punnets were arranged in a completely randomized design on benches in a glasshouse. Seedlings were grown in ambient light during August-September (winter-spring) 1988, and glasshouse temperatures ranged from 25 to 28°C. The potting medium in the punnets was watered until draining twice weekly with a nutrient solution consisting of 6.7 g KN03, 3.6 g Ca(NO,),4H,O, 6.0 g NH,N03, 120 mg H,BO, and 15 mg Na,MoO, in 50 1. deionized water, and on other days with deionized water. Root and hypocotyl samples from randomly selected damped-off seedlings were surface-disinfested, placed on neutral Dox yeast (NDY) medium (Warcup, 1950) in Petri dishes, and kept for 2 days in darkness at 25°C to confirm that the pathogen was associated with disease symptoms. After 26 days, the standing seedlings were counted and excised at soil level, and the tops were dried at 60°C and weighed. Initial analysis of data indicated that shoot dry weights were unduly influenced by variation in seedling numbers, partially due to seeding variation induced by the drum seeder. Thus, data were normalized by adjusting the log weights of shoots using a covariate of log number of plants. This covariate halved the error for Celosia and reduced it by 20% for the Cupsicum data set. Treatment means were compared with the control + pathogen treatment and assessed by Fisher’s protected least significant difference (PLSD) (Table 1).

Table 1. Effect of R. sold AC 8 isolate R-21, alone or in combination with selected bacterial isolates or fungicides, on dry weights of shoots of Capsicm and Celosia seedlings

Suppression of R. solani AG 4 on four host plant species

Control + AG 4 Control - AG 4 Propamocarb B. hbrilis Al 3 NA4 NAIO NA27 NA31 NA40 NA41 NA48 NA56 NA58 NB12 NB13 NB16 NB64

Bacterial isolates that promoted Capsicum or Celosia survival or growth in the presence of R. solani AG 8 or Pythium ultimum var. sporangiiferum (Harris and Schisler, unpubl. data) were selected to test their ability at a lower dose to also suppress R. solani on four host plant species chosen to represent a range of dicotyledonous families (Solanaceae, Amaranthaceae and Violaceae). Although the R. solani AG 8 isolate is pathogenic on bedding plants, AG 4 is more likely to cause damping-off in nurseries (Stephens et al., 1982; Schisler et al., 1993) so was used in this and subsequent experiments. R. soiuni isolate D 1Bl on wheat bran substrate was mixed with potting medium at 0.21% v/v, and 7 cm3

Dry weight of shoots (mg per punnet) (corrected for number of seedlings per punnet) Treatment 191 483’. 216 557” 518** 304** 326** 212 180 345.. 284** 255. 270..

Control + R. solani AG 8 Control - R. sold AG 8 Propamocarb Tolclofos-methyl Benomyl NA27 NA31 NA48 NA77 NB8 NB12 NB16 NB 64

53 152** NT’ 159** 13911 68 53 40 135** 40 73 99’ 50

Each value is a mean from four punnets (except 20 punnets for ‘Control + R. sold AG 8’. 16 for ‘Control - R. sohi AG 8’). *and ** represent significant differences from the ‘Control + R. solani AG 8’ treatment at P = 0.05 and P = 0.01, respectively. TNT = Not tested.

of this mix was added to the bottom of each punnet cell. In the control treatment with no R. solani, sterilized uninfested bran was added at the same dose as before to four replicate punnets per host plant species. All punnets were then filled with pasteurized uninfested potting medium. Six seeds of Capsicum, Celosia, Petunia (P. hybrida Vilm. ‘Colour Parade’) or Viola (I/. tricolor L. var. hortensis ‘Bambini’) were sown on top of the potting mix in each punnet cell, and covered with ca 7 cm3 of sterilized, washed coarse sand. Each of 14 bacterial isolates including B. subtilis Al3 (Table 2), was suspended in sterile deionized water to make a concentration of ca 10’ bacteria ml-‘, based on direct counts with a microscope. Table 2. Survival of seedlings of four host plant species grown in potting medium inoculated with R. sold AG 4 isolate DlBl alone or in combination with bacterial isolates or a fungicide No. of plants survived per punnet Treatment

PLSD

(P < 0.05)

Capsicum

Celosia

Petunia

Viola

14.5 33.5’ 22.0 18.0 19.8 19.0 18.5 25.5’ 25.5’ 20.0 11.3 10.5 10.0 12.0 19.5 a.3 11.5

10.3 27.3’ 14.0 2.3 6.0 12.0 5.8 9.5 10.8 10.0 2.0 1.3 1.5 0.8 4.3 2.3 3.0

5.5 26.3* 17.58 11.3 IS.08 17.3. 10.3 21.8’ 19.3* 25.59 15.5. 11.8 18.0’ 14.8’ 15.81 15.3. a.3

3.0 25.3’ 3.0 2.5 2.3 4.0 2.0 15.5’ 13.0’ 16.5’ 3.5 2.3 3.0 6.0 2.8 1.8 5.8

1.9

7.9

1.9

1.9

Each value is a mean from four punnets, each sown with 36 seeds. *Significantly greater than ‘Control + AG 4’ for same host plant species (P < 0.05)

A. R. HARRB et al.

1252

Table 3. Effect of bacterial antagonists on dry weight of shoots of Capsicnm seedlings in pasteurized potting medium without added R. sold Antagonist None B. subtiiisAl 3

NA31 NA40 NA41 PLSD (P < 0.01)

mg per punnet

(% increase)

mg per plant

(% increase)

(1Y7) (23.7) (22.1) (14.0)

11.3 12.9 13.9 13.7 12.9

(13.9) (23.1) (20.7) (14.1)

378 426 468 462 431 51

1.5

Each value is a mean from 15 punnets (except nine punnets for controls), each sown with 36 seeds.

Propamocarb was diluted to 1.78 ml 1-l deionized water. A bacterial or fungicide suspension was pipetted onto the surface of the potting medium at a dose of 2.25 ml per punnet cell in four replicate punnets for each host plant species. The dose of each bacterial isolate was equivalent to cu 2 x IO6bacteria g-i dry wt of potting medium. The applied dose of propamocarb was 2.4mg a.i. per punnet cell, which is the recommended commercial rate. Punnets were arranged in a randomized completeblock design in a glasshouse. The host plants were randomized within blocks and the microbial treatments randomized within host plant sub-blocks. Seedlings were grown in a glasshouse at a mean of 25°C (range 15-34°C) for cu 3 weeks during February-March (summer-autumn) 1990, by which time most seedlings had four true leaves and damping-off had ceased. The potting medium in the punnets was watered and fertilized as described earlier. The standing and collapsed seedlings were counted separately, and standing seedlings were excised, dried and weighed, as described above. Data for each variable were subjected to 2-way analysis of variance (ANOVA). One variable (number of plants survived) required a weighted analysis to give uniform residuals, so each observation was weighted by the reciprocal of its estimated variance. The difference between treatment means and the control + pathogen treatment were compared by Fisher’s protected least si~ifi~t difference. Suppression of R. solani AC 4, and growth promotion, in Capsicum

Two similar experiments were made to test the ability of selected bacterial isolates to stimulate shoot growth of Capsicum seedlings in the absence of added R. soiani, and to test the efficacy of different doses of the bacterial isolates to suppress damping-off caused by R. solani AG 4. The bacterial isolates were selected for superior biological control of AG 4 in the previous experiment, and for plant growth promotion and biological control of Pythium ultimum var. sporungiiferum (Harris and Schisler, unpubl. data). In the first experiment (Table 3), R. solani isolate DlBl on wheat bran substrate was mixed with potting medium at 0.21% v/v, and 7 cm3 of this mix was added to the bottom of each punnet cell in half of the punnets. For the control without R. solani

treatment, autoclaved uninfested wheat bran was added similarly to the bottom of nine punnets. All punnets then were filled with pasteurized uninfested potting medium. Six seeds of Cupsic~m were sown 0.5-l cm deep in each punnet cell and covered with 0.5 cm of sand. Bacterial isolates B. subtilis A13, NA3 1, NA40 and NA41 were suspended in sterile deionized water to make concentrations of 104, 105.s, 107, 108.5and 10” bacterial cells ml-‘, and 2.25 ml of a suspension was pipetted onto the surface of the potting mix in each cell of three replicate punnets for each dose. Each microbial isolate was also added at the same five doses to the remainder of the punnets without R. solani, to determine the effects of the microorganisms directly on plant growth. The fungicide quintozene (PCNB) (Terraclor, Uniroyal Australia Pty Ltd, Melbourne, Australia) was pipetted onto the surface of each punnet cell at a dose of 11.3 mg cell-’ (2.25 ml of a solution of 5 g I-‘). The applied dose was 8.4 mg a.i. per punnet cell, which approximates the recommended commercial rate. Fungicide and control treatments each had nine replicate punnets. Punnets were arranged in a randomized complete block design with three blocks. The potting medium in the punnets was watered and fertilized as described above. Plants were grown in a glasshouse at a mean of 25°C (range 21-30°C) for 20 days during March-April (autumn) 1990. In the second experiment (Table 4), R. solani isolate D 1Bl on wheat bran substrate was mixed with potting medium at 0.28% v/v, and 7 cm3 of this mix was added to the bottom of each punnet cell. The same four bacterial isolates were suspended in sterile

Table 4. Mean number of surviving seedlings and dry weights of shoots of Capsimmin pasteurized potting medium after addition of bacteria1 isolates but no added R. sokmi Dry weight of shoots Antagonist None B. subtilirA13 NA31 NA40 NA41 PLSD

No. of plants survived per punnet

mg per punnet

mg per plant

31.6 33.9 33.2 32.9 33.5

499 613 624 597 616

15.7 18.1 18.9 18.3 18.4

1.4 (P < 0.05)

(P
(P
Each value is a mean from 15 punnets (except nine punnets for controls), each sown with 36 seeds.

Bacteria suppress Rhizoctonia and stimulate seedlings deionized water to make concentrations of lo’,‘, 104, lo’.‘, 10’ and lo’.’ bacterial cells ml-‘, and 2.25 ml of a suspension was pipetted onto each punnet cell. In addition to quintozene, propamocarb was included at a dose of 2.4 mg a.i. per punnet cell, as described in the previous section. Other methods were the same as in the first experiment. Seedlings were grown in a glasshouse at a mean of 25°C (range 19-30°C) for 4 weeks during June-July (winter) 1990. In both experiments, the standing and collapsed seedlings were counted separately, then the standing seedlings were excised, dried and weighed as described above. For each variable, the means for the five doses were subjected to regression analysis, but, because no dose responses were observed, the means for each treatment (across all doses) were calculated and subjected to ANOVA. Treatment means (from 15 punnets each) were compared by Fisher’s protected least significant difference.

1253

was a negative response in survival and shoot weights per punnet to increasing doses of B. subtilis Al3 (data not presented). The same two variables showed a positive response to increasing doses of NA31, but decreased at the highest dose. There were no dose-responses in the absence of R. solani. When data for all dose rates were combined and analysed for differences between antagonist treatments, all bacterial isolates increased dry weights of Cupsicum shoots in the absence of added pathogen in both experiments (Tables 3 and 4). In the second experiment (Table 4) all isolates except NA40 also promoted seedling survival in the absence of added pathogen (P < 0.05). When R. solani was co-inoculated to the potting medium, no bacterial treatment in the first experiment had a significant (P < 0.05) effect on seedling survival or shoot dry weight (data not presented). In the second experiment, only quintozene increased seedling survival or shoot dry weight in the presence of R. solani (P < 0.05).

RESULTS DISCUSSION

In the initial large screening experiment (Table 1), 35 of 91 bacterial isolates co-inoculated with R. soluni AG 8 increased the total dry weight of Capsicum shoots per punnet compared with R. solani AG 8 alone (P < 0.05). Twenty-four of the isolates showed an increase in total dry weight of Capsicum shoots per punnet that exceeded the P = 0.01 level. For Celosia seedlings, 12 bacterial isolates increased the total shoot dry weight per punnet at the P = 0.05 level, and four of these were significant at the P = 0.01 level. Results for fungicides and eight of the bacterial isolates are reported in Table 1. Bacterial isolates NA3 1, NA40 and NA41 reduced damping-off caused by R. solani AG 4 more consistently in Capsicum, Petunia and Viola than other isolates including B. subtifis Al 3 (P < 0.05) (Table 2). All three isolates suppressed damping-off in the four host plant species, and all effects except NA41 in Capsicum were significant (P < 0.05). There was a significant (P c 0.001) effect of plant species on damping-off at the used dose of R. solani AG 4, with Viola the most susceptible and Capsicum the least susceptible. There also was a significant (P < 0.001) treatment x plant species interaction, and Petunia was most amenable to biological control of damping-off, as 10 of 14 bacterial isolates suppressed disease. Across all four plant species, isolates NA31, NA40 and NA41 suppressed damping-off (P < O.OOl),but B. subtilis Al3 and propamocarb did not. The only treatments that increased shoot dry weights per punnet were NA4, NAlO, NA41, NA58 and propamocarb, all on Petunia (data not presented). In the first of the two dose-response experiments, there was no relationship between dose of bacterial antagonist and seedling survival or shoot dry weight, either with or without added R. solani. In the second experiment, in the presence of R. solani only, there

From the initial screening of 91 bacterial isolates for suppression of R. solani AG 8 (Table l), and subsequent screening for suppression of Pythium ultimum var. sporangiiferum on Capsicum (Harris and Schisler, unpubl. data), 14 isolates were screened against R. solani AG 4 on four host plant species (Table 2). Of these, isolates NA31, NA40 and NA41 suppressed damping-off caused by AG 4 in seedlings of Capsicum, Petunia and Viola at doses equivalent to cu 2 x lo6 bacteria gg’ dry weight of potting medium. The four host plant species tested, which represented three dicotyledonous families, differed in susceptibility to R. soluni AG 4 isolate DlBl (Table 2). Although many treatments showed increased seedling survival, the dearth of increases in shoot dry weights was probably due to crowding and competition between surviving seedlings. The number of viable seeds in each experiment is indicated by the number of plants that survived in the control treatment with no pathogen or bacterial antagonist. In the dose-response experiments, there were some increases in shoot growth and seedling survival due to the bacterial antagonists (across all doses). The bacterial isolates B. subtilis Al 3, NA31, NA4O and NA41 apparently stimulated shoot growth of Capsicum seedlings in pasteurized potting medium with no added pathogen. Plant growth promotion has been reported for Bacillus spp (Broadbent et al., 1977) and Pseudomonas spp (Elad et al., 1987; Lifshitz et al., 1987). The general lack of responses to dose of each bacterial isolate suggests that small doses were generally as effective at plant growth promotion as large doses. There was some evidence that large doses of B. subtilis A 13 or NA3 1 may even be detrimental, but the negative dose-responses were observed in only one experiment and only in the presence of R. solani. The growth promotion at low doses, and the absence

1254

A. R. HAIWS et al.

of an improved response to increased doses of the three selected bacterial isolates, suggest that even small amounts of inoculum (IO* bacteria g-i dry wt of potting medium) are sufficient to colonize and stimulate whole seedlings. We do not know why only NA31 reduced damping-off in these two doseresponse experiments, after NA40 and NA41 also gave good biological control in earlier experiments. The efficacy of the bacterial isolates may be improved if they colonize the seedlings before the pathogen, either by prior inoculation or by placement of antagonist inoculum close to the seeds or germinating seedlings. Further research is needed to determine whether these isolates of bacteria can protect plants in non-pasteurized soils or against other damping-off fungi. Attempts to identify the three bacterial isolates by different methods have given inconsistent results, so their identity is still unknown. NA31 is a Gram-variable motile rod. NA40 and NA41 are Gram-negative motile rods, and both isolates are oxidase negative. All three isolates were obtained from bait plants grown in a pasteurized potting medium from one nursery. In vitro tests (unpubl. data) showed that NA40 and NA41 inhibited hyphal growth of R. solani AG 4 and AGX on various agar media. NA31 retarded hyphal growth of both fungi, but inhibition zones were overgrown after about 2 days. These inhibitions could be due to either diffusible or volatile antibiotics. A strain of Pseudomonas fluorescetzs produces the which is active against antibiotic pyrrolnitrin, R. solani (Howell and Stipanovic, 1979). We observed only weak or temporary inhibition of R. solani hyphal growth on King’s B agar without Fe3+, and the inhibition was no greater than with Fe3+, indicating that siderophores are not involved in inhibition by these three bacterial antagonists in this medium. Another possible mechanism of biological control is induced resistance in the host plants (Wei et al., 1991), and further research is needed to determine which of these mechanisms is important with these three bacterial isolates in this system in vivo. The three selected isolates of antagonistic bacteria suppressed damping-off caused by R. solani AG 4 better than B. subtilis A13, and stimulated growth of Cap&cum as much as A13. Isolate Al3 was reported to control R. solarti (Broadbent et al,, 1971), but the anastomosis group was not stated. Lumsden and Locke (1989) found that several unspecified isolates of Bacillus spp did not control damping-off caused by R. solani AG 4 in zinnia seedlings grown in a soil-less potting mix. Although our three selected bacterial isolates did not suppress R. solam or promote shoot growth as much as two binucleate R~izocto~ia isolates (Harris et al., 1993b, 1994; Harris, 1994), the bacteria may be useful biological control agents under certain environmental conditions, They also may be used in mixtures with other antagonists to improve the versatility of biological control products.

The addition of a biological control agent to potting media may provide economical, prolonged protection of seedlings against damping-off caused by R. sofani. thank Incitec Ltd, Brisbane, Queensland and the Horticultural Research and Development Corp., Australia for financial support, and Falg Nurseries Pty Ltd, Uraidla, South Australia for supplying potting medium and punnets. We are also grateful to R. 0. Rowden of Incitec Ltd and P. G. Adkins for technical assistance. Acknowledgements-We

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