Selection of rhizosphere bacteria for biological control of Pythium aphanidermatum on hydroponically grown cucumber

BIOLOGICAL

CONTROL

2, 226-237 (1992)

Selection of Rhizosphere Bacteria for Biological Control of Pythium aphanidermatum on Hydroponically Grown Cucumber T. C. PAULITZ,’

T. ZHOU, AND L. RANKIN

Department of Plant Science, Macdonald Campus of McGill University, 21,111 Lakeshore Rd, Ste. Anne de Belleuue, Quebec, Canada H9X 3V9 Received April 8, 1992; accepted October 26, 1992

Six-hundred and four isolates of bacteria were cultured from the rhizosphere of cucumber (Cucumis sativus cv. Corona) grown in 34 agricultural and forest soils from Quebec. The isolates were tested for inhibition of mycelial growth and zoospore germination of Pythium aphanidermatum in vitro. Ninety-three isolates inhibited mycelial growth and 35 isolates, which reduced zoospore germination after 4.5 h to ~50% of the control treatment, were selected for further testing. Thirty-four of the 35 isolates were effective as washed bacterial cells in reducing zoospore germination, but the cell-free culture filtrates of only 3 isolates were effective. All 35 isolates significantly reduced zoospore attraction to capillary tubes filled with 3 mM aspartic acid + 0.05% agar in an attraction assay. These bacterial isolates were also tested in plant bioassays on cucumber seedlings of cv. Straight Eight grown in test tubes with nutrient solution and inoculated with zoospores of P. aphanidermatum and/or bacterial isolates. Inoculation with P. aphanidermatum alone reduced the root length to 6 7 % of the bacterium-free, pathogen-free control. The root lengths of plants inoculated with P. aphanidermatum plus 7 of the isolates ranged from 82 to 9 9 % of the bacterium-free, pathogen-free control and were not significantly different from that control. One isolate significantly increased root length, compared to the treatment with P. aphanidermatum alone. Three isolates, when applied to plants not inoculated with P. aphanidermatum, stimulated root growth to 134% of the bacterium-free, pathogen-free control. Five of the most effective isolates were tested on cucumber plants grown in aerated hydroponic culture for 3 weeks; all were effective root colonizers and were detected at population densities of lo”-lO’/cm” on the root surface after 3 weeks. The best isolate in the test tube bioassay also significantly increased the shoot dry weight and root volume of cucumber plants cv. Straight Eight or cv. Corona inoculated with P. aphanidermatum and grown

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in aerated hydroponic solution. This same isolate also decreased zoospore encystment and germination on the roots of cv. Straight Eight, 4 and 8 h after addition of zoospores to the hydroponic solution. The top 5 isolates in the plant bioassays, in terms of biocontrol ability, were also ranked among the top 5 isolates in the zoospore germination and/or the zoospore motility bioassays. Selection of isolates for inplanta testing based on their performance in zoospore bioassays may be a useful tool for prescreening large number of isolates, to increase the likelihood of identifying potential antagonists of zoosporic fungi. In general, in vitro assays were not useful predictors of the relative performance of isolates in planta. The combination of in vitro and in planta bioassays, however, was useful for selecting effective isolates and eliminating ineffective ones. o 1992 Academic

Inc.

INTRODUCTION

European-type gynoecious seedless cucumber (CUCUL.) is the second most important greenhouse vegetable in Canada after tomato. Approximately 3 million dozen were produced in Ontario in 1988, with lesser amounts in Quebec, British Columbia, and the Maritime provinces. Much of this production is in hydroponic or soilless systems, such as rock-wool, peat bags, sawdust, or nutrient film. These soilless media offer the potential of higher yields and quality, better control of nutrients, and reduction of soil-borne pathogens associated with soil media. However, Pythium root and stem rot of greenhouse cucumbers is still a serious problem in Quebec and other parts of Canada (Carrier, 1989; Favrin et al., 1988) and can be a potentially devastating problem on other greenhouse vegetables (Jenkins and Averre, 1983; Vanachter et al., 1983). Pythium can be mis sativus

226 Inc. reserved.

Press,

KEY WORDS: Biological control; Pythium aphanidermum; hydroponic; cucumber; Cucumis sativus cvs. Corona, Straight Eight; Pseudomonas spp.; Pseudomonas corrugata; Pseudomonas fluorescens; rhizobacteria.

BIOCONTROL

OF Pythium

ON GREENHOUSE

easily introduced into a soilless system through infested water or movement of contaminated soil from outside the greenhouse. Pythium ultimum and P. aphanidermaturn are the dominant pathogens in Quebec greenhouses (Paulitz, unpublished) and the latter produces zoospores which are well adapted to aquatic conditions. The lack of competing microorganisms in soilless culture exacerbates the problem, since Pythium species are pioneer colonizers and do not compete well with other microbes. Favorable moisture conditions, a susceptible succulent host, and a mechanism for the rapid dispersal of the pathogen throughout the culture system increase the severity of Pythium diseases in hydroponic culture (Zinnen, 1988). Although fungicides have shown some promise in controlling Pythium in hydroponics (Vanachter et al., 1983; Price and Fox, 1986; Gold and Stanghellini, 1985), phytotoxicity and residue problems must be overcome, none are registered for use in Canada on greenhouse cucumbers, and the small market precludes the development of new products. Surfactants that lyse zoospores may offer another means of disease control (Stanghellini and Tomlinson, 1987). Manipulation of light, temperature, and nutrient composition may also reduce the severity of disease (Funck-Jensen and Hockenhull, 1983; Gold and Stanghellini, 1985). Disinfestation of water with ultraviolet radiation is another promising method, but it is not effective in systems with high flow rates (Stanghellini et al., 1984). Filtration of water is also possible (Goldberg and Stanghellini, 1991a). Little research has been done to examine the potential of biological control in hydroponic and soilless systems. Closed, environmentally controlled greenhouses are more suitable for biological control than open fields, because of the uniform environmental conditions of the greenhouse, high economic value of the crop, and the ease of introducing the biocontrol agent in high populaAntagonistic rhizosphere-colonizing bacteria tions. could be introduced into the nutrient solution as a potential control for Pythium root rot in soilless culture. The purpose of this work was to select and evaluate rhizosphere-colonizing bacteria, isolated from cucumbers grown in soils collected from agricultural and forest sites in Quebec, for their ability to reduce Pythium root rot. In vitro tests and plant bioassays were developed to permit the evaluation of about 600 isolates of bacteria. The in vitro bioassays, based on the ability of the bacteria to inhibit mycelial growth, zoospore germination, and zoospore motility and chemotaxis were used to select 35 isolates for further testing in a plant bioassay conducted on cucumber seedlings growing in nutrient solution in test tubes. The five most effective isolates were further tested on plants growing in l-liter containers in an aerated hydroponic system.

MATERIALS

Isolation

227

CUCUMBERS

of Rhizosphere

AND

METHODS

Bacteria

Soil samples were collected from 34 different sites on 11 different soil types on the Emil Lods Research Centre and the Morgan Arboretum of Macdonald College, in April, 1990. The agricultural soils had previously been cropped with corn, wheat, alfalfa, timothy, birdsfoot trefoil, or grasses. The forest soils were collected from a mixed hardwood forest comprised mainly of sugar maple, beech, basswood, red oak, hickory, and white ash. The soils were classified as Farmington clay loam, Chateauguay clay loam, St. Bernard loam, Chicot fine sandy loam, St. Benoit light sandy loam, Bearbrook clay, St. Rosalie clay, Dalhousie clay, St. Damase fine sandy loam, St. Amable loamy sand, and upland sand. Soil from each sample was placed in 12.5-cm-diameter plastic pots, which were planted with three seeds of Cucumis sativus cv. Corona and thinned to one plant per pot after 1 week. The plants were grown for 3 weeks on a growth bench at 25°C under combined fluorescent-incandescent lighting, 16 h light per day. After 3 weeks, roots were removed from the pots, the excess soil was shaken off and the roots from each pot were placed in 99-ml sterile distilled water. The roots were sonicated in the flask for 30 s, 1 ml of the suspension was diluted, and 0.1 ml of the diluted suspension was spread onto each of three different agar media: nutrient agar (NA) (Difco), King’s medium B (KB) (King et al., 1954), or Luria Bertani (LB) medium (Maniatis et al., 1982). After 48 h, representative colonies were selected and streaked on nutrient agar to obtain single colonies. Six-hundred and four isolates were obtained and stored in nutrient broth + 10% glycerol (w/v) at -80°C. Spontaneous rifampicin-resistant strains of some isolates were selected by growing the isolates on nutrient agar amended with 100 pg/ml of rifampicin. These resistant strains were used for the hydroponic experiments. The 35 top-performing isolates were stained with Gram stain. The isolates were further identified using the Biolog GN Log microplates and the microcomputer data base (Biolog, Inc., Hayward, CA). Inhibition

of Mycelial

Growth

Isolates of bacteria were grown in nutrient broth for 24 h at 26°C. Three 50-~1 drops of bacterial suspension were placed equidistant from one another on the margins of NA, KB, or LB medium in lOO-mm-diameter plastic petri dishes. After 24 h, a 4-mm plug of P. aphanidermatum isolate 186 (from W. Jarvis, Agriculture Canada, Harrow, Ontario), obtained from a 24-h-old culture on V8 agar was placed in the center of the plates. After 24 h, the radii of the fungal colonies in the direc-

228

PAULITZ,

ZHOU,

tion of the bacterial colony and away from the colony were measured, and percentage inhibition was calculated by the following equation: 100 X (R, - &)I&, where R, is the maximum radius of the colony of P. aphanidermatum and R, is the radius of that part of the colony of P. aphanidermatum directly opposite the bacterial colony. Inhibition

of Zoospore Germination

Zoospores of P. aphanidermatum were produced by growing the fungus on V8 agar in petri plates at 26°C for 48 h. The agar culture was cut into l-cm-wide strips, and half of the strips were transferred to an empty petri plate. Both plates were flooded with 20-25 ml of sterile distilled water. After 0.5-l h, the water was removed and replaced with the same amount of water. The plates were incubated at 35°C under fluorescent lights for 18 h, then at 20°C for 4 h to stimulate zoospore release. The final concentration of zoospores ranged from lO,OOO80,000 zoospores/ml. Bacteria were cultured for 24 h in 5 ml of nutrient broth (NB) at 26°C on a rotary shaker at 200 rpm. An aliquot (0.1 ml) of the bacterial culture was placed in a 1.5-ml microcentrifuge tube, along with 0.9 ml of zoospore suspension. For the control treatment, 0.1 ml of NB was added instead of the bacterial suspension. The suspension was gently mixed by inverting the tube. A 30-~1 drop of the zoospore-bacteria mixture was placed in a ring on a ceramic ring slide (76 X 51 X 2.3-mm slide with 12 raised ceramic rings with inner diameters of 14 mm; Clay Adams, Division of Becton, Dickinson and Company, Parippany, NJ) and incubated for 2 h in a moist chamber. After 2 h, a drop of 95% ethanol was added to kill the zoospores. Zoospore cyst germination percentage was measured with a compound microscope by counting the number of germinated zoospores out of 100. Three drops were assayed for each of the 604 isolates. Seventy-four isolates, which limited zoospore germination to less than 40% of the control, were chosen for further zoospore inhibition assays. Thirty of these isolates also inhibited mycelial growth in the petri plate assay. Also included were 26 isolates that inhibited myCelia1 growth, but did not inhibit zoospore germination in the first assay. A second assay was performed as previously, except that germination was measured after 1.5 and 4.5 h. Thirty-five isolates, which inhibited zoospore germination to less than 30% of the control after 1.5 h and less than 50% of the control after 4.5 h, were selected for further testing. The zoospore germination assay was repeated once with the 35 isolates. The effects of washed bacterial cells and cell-free culture supernatants were also tested. One milliliter of each bacterial culture was centrifuged at 2000g for 15

AND

RANKIN

min, and the bacterium-free supernatant was removed. The pellet was resuspended to 1 ml with sterile distilled water. One-tenth milliliter of the supernatant or the washed bacterial suspension was added to 0.9 ml of the zoospore suspension and treated as described above. In tests with washed bacterial cells, 0.1 ml of water served as the control, and zoospore germination was measured after 8 h. For the supernatant test, 0.05 ml of NB was used in the control treatment, and germination was measured after 4 h. Both assays were repeated once. Inhibition

of Zoospore Motility

The effect of bacterial isolates on zoospore motility and chemotaxis was measured with the following bioassay. Capillary tubes (5-~1 micropipettes, Drummond Scientific Co., Broomall, PA) were filled with 3 mM asparatic acid mixed with 0.05% agar to increase the viscosity. Previous experiments had demonstrated that zoospores were attracted to this amino acid. An aliquot of zoospore suspension (1.8 ml, 50,000 zoospores/ml) was placed in a 5-cm-diameter glass petri dish, and 0.2 ml of bacterial culture (lo’-10’ cells/ml) was added to the suspension. For the control treatment, 0.2 ml of water or NB was added instead. Twenty minutes after the bacteria were mixed with the zoospores, the capillary tubes filled with aspartic acid were placed in the zoospore suspension. After 1 h, the tubes were removed and placed in a closed container containing formaldehyde. The number of zoospores in each 0.90-mm section of the tube was counted, starting from the end, using an ocular micrometer and a compound microscope. Six sections were counted for each tube. A motility index (MI) was calculated from the number of zoospores and the distance they had swum up the capillary tube using the following formula: MI = sum of [(Zi X i)], i = 1, . . . 6, where Zi = number of zoospores in each ith section, i is the section number, and section 1 is at the open end of the tube. The motility indices were subjected to log transformation for data analysis. This assay was performed twice using whole bacterial cultures and once with washed bacterial cells. Plant Bioassay in Test Tubes For the plant bioassays in test tubes, cucumber (CV. Straight Eight) was used. Seeds were germinated on moist filter paper for 2 days at 26°C in the dark and for 1 additional day in the light. Sterile test tubes (16 X 125 mm) were filled with plant nutrient solution, which consisted of 0.97 g Peters Hydro-sol fertilizer 5-11-26 (W. R. Grace, Fogelsville, PA) and 0.64 g Peters Ca(NO,), 15.5-0-O dissolved in 1 liter of sterile distilled water. Thirty-two of 35 bacterial isolates previously selected were grown as described above, and bacterial density was quantified by measuring optical density at

BIOCONTROL

OF Pythium

ON GREENHOUSE

640 nm and comparing to a standard curve. Isolates 12, 29, and 30 were not tested, because they produced a hard pellet in culture which was not easily broken up to give a uniform bacterial suspension. Bacteria were added to the tubes to give a final concentration of 1.0 X lo6 bacteria/ml of nutrient solution. Three-day-old cucumber seedlings were placed in the tubes, with the roots submerged in the nutrient solution. Two layers of Parafilm (American Can Co, Greenwich, CT) were wrapped around the top of the tube to secure the seedling. The tubes were incubated on a rotary shaker (80 rpm) on a growth bench at 26°C with 16 h of light (combined fluerescent-incandescent) per day. Twenty-four hours after the bacteria were added to the tubes, an aliquot of zoospore suspension was added to give a final concentration of 100 zoospores/ml. Each isolate was tested in two treatments: bacteria alone and bacteria + P. uphanidermatum. Two control treatments were used: no bacteria, no P. aphanidermatum (bacterium-free, pathogen-free control), and P. aphanidermatum only (Pythium-inoculated, bacterium-free control). Five replicate plants were used for each treatment. Sterile water was added daily to the tubes to replace the water that was transpired. After 8 days, the plants were removed, and the root length was measured using the line-intersect method of Newman (1966). This experiment was repeated once. Effect of Bacteria

on Attraction

of Zoospores to the Root

Seedlings of cucumber (cv. Straight Eight) were grown in test tubes in nutrient solution as described in the previous section. One day after being transferred to the test tubes, plants were treated with bacterial isolates to give a final concentration of lo6 cells/ml; they were inoculated with zoospores 1 day later (1000 zoospores/ ml). Four hours after the application of zoospores, roots were removed and fixed in FAA (13 ml formalin, 5 ml acetic acid, 200 ml 50% ethanol). The fixed roots were stained for 5 min with 0.5% malachite green, washed with distilled water, and then counterstained for 20 min with 0.001% acridine orange in a boric acid-borax buffer (pH = 8.6). Stained roots were observed under UV light with an epifluorescence microscope, and the number of zoospores on the surface of the root were counted. Each treatment had six replicate plants, and the experiment was repeated once. Root Colonization

by Bacteria

Cucumber seeds (cv. Corona) were germinated, placed in test tubes, and treated with bacterial isolates as described under Plant Bioassay in Test Tubes. Four days after treatment with bacteria, the plants were harvested and the roots were removed, placed in 9 ml sterile distilled water, and shaken with a votex mixer for 20 s.

229

CUCUMBERS

The roots were spread out on sterile paper towels and blotted to remove excess water. They were then placed on nutrient agar containing 100 fig/ml rifampicin and incubated for 48 h at 26°C. The total root length and the length of root colonized by bacteria, as determined by the presence of a visible colony on the agar, were measured by the line-intersect method (Newman, 1966). Pseudomonas putida NlR, a known root colonizer, was also tested. Effect of Selected Isolates on Cucumbers Hydroponic Culture

in Aerated

Seeds of cucumber cv. Straight Eight or cv. Corona were germinated on moist filter paper as previously described under Plant Bioassay in Test Tubes. The seedlings were then transferred to l-liter mason jars containing nutrient solution, with a lo-mm opening in the lid. The solution was replaced once a week with fresh nutrient solution. The volume of nutrient solution lost to transpiration was replaced daily with distilled water. Each jar was aerated with an aquarium pump, for 30 min every 6 h. Isolate 13, 15, 16, 27, or 35 was added to the jars 1 day after the plants were transferred, to give a final concentration of lo6 cells/ml. A zoospore suspension of P. aphanidermatum was added 24 h later to give a final concentration of 500 zoospores/ml of solution. Aeration was interrupted for 8 h after the addition of bacteria and 12 h after inoculation with P. aphanidermatum. After each weekly change of solution, two plugs (12 mm diameter) of P. aphanidermatum from a 48-hold culture on V8 agar were added to each jar. The treatments were no bacteria, no P. aphanidermatum (bacterium-free, pathogen-free control); P. aphanidermatum only (Pythium-inoculated, bacterium-free control); bacterial isolates alone; and bacterial isolates plus P. aphanidermatum. Each treatment was replicated five times, and the experiment was conducted three times, twice with cv. Straight Eight, and once with Corona. Bacterial population densities in the hydroponic solution were determined after 1 and 2 weeks by dilution plating on NA amended with rifampicin. At the end of the experiment, the population density of bacteria on the roots was determined by placing the drained root system in 200 ml of sterile distilled water, sonicating for 30 s, and dilution plating on NA amended with rifampitin. The total root volume was determined by measuring the amount of water displaced in a graduated cylinder. Bacterial population densities were expressed as cfu/cm3 of root. The population density of P. aphanidermatum in solution was determined after 1, 2, and 3 weeks in the first trial on cv. Straight Eight, by plating 0.5 ml of nutrient solution on Pythium-selective medium (Mircetich and Kraft, 1973). In the second trial on Straight Eight and in the trial on Corona, the popula-

230

PAULITZ,

tion densities were determined weeks. Statistical

ZHOU,

AND

RANKIN

after 4 h, 8 h, 24 h, and 3

Analysis

TABLE

Identification of Bacteria Isolated from the Rhizosphere of Cucumis sativus cv. Corona Grown in Quebec Soils Isolate

Because of the large number of isolates tested, zoospore bioassays and plant bioassays in test tubes could not be performed on all the isolates at once. Instead, the bioassays were run on groups of isolates. F tests showed no significant differences between the variances of the experiments, so the data were pooled. Data of the first and second (repeat) trial were also pooled for the same reason. Data were analyzed with SAS (SAS Institute, 1987), using the general linear models procedure, including the analysis of variance and Duncan’s multiple range test, P = 0.05. The data from the experiment of zoospore attraction to cucumber roots were not distributed normally and were analyzed with the KruskalWallis rank correlation test, followed by a multiple comparison (Daniel, 1990). The performance of the isolates in the different bioassays was compared using Spearman rank correlation procedure. This procedure was also used to determine whether the performance of the isolates in vitro could be used to predict the performance in planta. Plant mortality in the hydroponic experiment was analyzed at 95% confidence intervals derived from statistical tables based on the binomial distribution.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

RESULTS

Identification

of Bacterial

Isolates

With one exception, all of the 24 isolates identified were species of Pseudomonas (Table 1). Of those that could be identified to species, most were Pseudomonas corrugata or Pseudomonas fluorescens subgroup C or E. Inhibition

of Mycelial

Note. NI, no identification database; NT, not tested.

Identification NI Pseudomonas corrugata NT P. corruguta Pseudomonas sp. Pseudomonas sp. P. corrugata P. corrugata NI Escherichia vulneris P. cichorii Pseudomonas sp. P. corrugata P. corrugata P. fluorescens subgroup P. fluorescens subgroup NI NT Pseudomonas sp. Pseudomonas sp. P. fluorescens subgroup Pseudomonas sp. P. corrugata NI P. corrugata NI P. fluorescens subgroup P. corrugata NT NT P. corrugata P. corrugata P. corrugata NI P. corrugata

C C

C

E

because of lack of match with existing

Growth

Out, of 604 isolates, 93 (15.4%) inhibited the mycelial growth of P. aphanidermatum, as indicated by reduced fungal growth in the direction of the bacterial colony. In general, inhibition was evident on all three media. Of the 93 isolates inhibitory to mycelial growth, 30 also inhibited zoospore germination. Inhibition

1

of Zoospore Germination

Seventy-four of the 604 isolates (12.2%) inhibited zoospore germination to ~40% of the control treatment. Forty-four isolates inhibited only zoospore germination, while 30 isolates inhibited both mycelial growth and zoospore germination. When zoospore germination was tested at two different time points, 35 isolates inhibited germination to ~30% of the control after 1.5 h, and/ or ~50% after 4.5 h. The pooled germination from two

trials at 4.5 h is shown in Table 2. Fifty-one percent of these 35 isolates also inhibited mycelial growth on at least one of the agar media. When the culture supernatants were tested, only isolates 1,2, and 27 significantly reduced zoospore germination (data not shown). However, all except isolate 24 were effective as washed bacterial cells in reducing zoospore germination. Inhibition

of Zoospore Chemotactic

Motility

All 35 isolates tested significantly reduced zoospore motility, as measured by the number of zoospores and the distance which the zoospores swam into the capillary tubes (Table 2). When washed bacterial cells were tested, 23 of the 35 isolates inhibited zoospore motility.

BIOCONTROL

TABLE

OF Pythium

ON GREENHOUSE

231

CUCUMBERS

2

Effect of Selected Rhizobacteria on Mycelial Growth, Zoospore Germination, and Chemotactic Zoospore Motility of Pythium aphanidermatum Mycelial inhibition”

Isolate 1 2 3 4 5 6 7

8 9 10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25 26 27 28

29 30 31 32 33 34 35

Zoospore

NAor LB

KB

+ + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + -

germination (% control)*

Zoospore motility index (% control)’

22 66 75 62 66 41 55 54 47 50 32 35 47 53 77

*

0.5

*

28.2 0.4 18.2 1.5 6.4 5.2 17.0 0.4 2.3 0.3

59

* * *

50 61 77 63 52

59 68 32 62 77 17 68 54 53 34 64 37 62 60

* * * * * * * * * * *

* * * * * *

control 1

9

13

15

w

w w

8.9 28.8 w 13.5 w 0.6

22.9 w 1.4 3.5 w 0.4 12.6 12.6 1.4

1.1 w 2.1 0.6 0.4 w 1.7 2.6 w 6.3 1.3 w 5.0 0.7 5.6 0.5

16

24

26

27

32

35

NlR

Isolate

w

1.0 * * * * * * * * *

3

FIG. 1. Colonization of roots of Cucumis satiuus cv. Corona by selected rhizobacteria. Four days after application of bacteria, roots were harvested, suspended in sterile water, and vortexed. They were then rinsed in sterile water and plated on nutrient agar supplemented with rifampicin. The control treatment did not receive the bacteria. The percentage of root colonized by the bacteria was determined by a line-intersect method. Results presented are from the second trial, n = 4. The means were separated by the Duncan’s multiple range test (P = 0.05); means denoted by the same letters are not significantly different.

Distribution

of Bacteria

on Roots

The ability of bacterial isolates to colonize roots under hydroponic conditions varied with each isolate, ranging from above 80% of the total root length (isolates 1,27, and NlR) to less than 10% of the root length (isolate 26) (Fig. 1). Effect of Bacteria

on Attraction

of Zoospores to the Root

Isolates 13 and 15 significantly reduced the number of zoospores encysted on the roots of cv. Straight Eight after 4 h, to 56 and 33% of the nontreated bacteriumfree control plants (Fig. 2). Isolates 16, 27, and 35 also numerically reduced zoospore encystment on the root, but the reduction was not statistically significant. Plant Bioassay in Test Tubes

Note. All data are from the pooled results of two trials, n = 3. Duncan’s multiple range test was used to separate the means, P = 0.05. a Isolates marked + inhibited the radial growth of Pythiun uphunidermatum on agar by greater than 20%. Isolates marked - inhibited radial growth less than 20%. NA, nutrient agar; LB, Luria Bertani medium; KB, Kings medium B. b Isolates marked * significantly reduced the germination of zoospores compared to the nontreated control which had 95% germination. Whole-broth cultures were tested (culture filtrate + bacterial cells). ’ Motility index (MI) was calculated from the formula MI = sum of [(Z, X i)], i = 1, . . . 6, where 2, = number of zoospores in each ith section, i is the section number, and section 1 is at the open end of the 5.~1 capillary tube. The motility indices were log-transformed for data analysis. All isolates significantly reduced zoospore motility when applied as whole cultures. Isolates marked “w” did not inhibit motility when applied as washed bacterial cells.

The addition of 100 zoospores of P. aphanidermatum per milliliter reduced the root length of cucumber plants (CP) to 67% of the bacterium-free, pathogen-free control (C, Fig. 3). When bacterial isolates were added 1 day before zoospore inoculation, treatments with 7 of the isolates resulted in root lengths of 82-99% of the bacterium-free, pathogen-free control (C! in Fig. 3), and these bacterial treatments were not significantly different from that of control. Only inoculated plants treated with isolate 15 had significantly larger root systems, compared to the Pythium-inoculated, bacterium-free control (CP). When pathogen-free cucumber plants were treated with three isolates (13, 14, or 15), root lengths were about 134% of the bacterium-free, patho-

232

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AND

RANKIN

bacterial isolate and Pythium was not significant (P > 0.05).

inoculation

Effect of Selected Isolates on Cucumbers Hydroponic Culture

-

control

13

15

16

27

35

Isolate FIG. 2. Effect of selected rhizosphere bacteria on the attraction of zoospores of Pythium aphanidermatum to the roots of Cucumis satiuus cv. Straight Eight. Bacteria were applied to nutrient solution at lo6 bacteria/ml 24 h before zoospores of P. aphanidermatum were added at 1000 zoospores/ml. The control treatment did not receive bacteria. Observations were made 4 h after the addition of zoospores. The number of encysted or germinated zoospores on the P-mm-long root tips were counted. Mean separation was based on mean rank scores (Kruskal-Wallis test) of 48.5,31.7,23.5,42.7,33.4, and 33.0 for the control and isolates 13, 15, 16, 27, and 35, respectively.

gen-free control (Fig. 4). Two isolates (9 and 19) inhibited root growth, and 1 isolate (11) was inhibitory only on Pythium-inoculated plants. The interaction between

treatment

in Aerated

In the pooled experiments on cv. Straight Eight, 7 of 10 plants inoculated with P. aphanidermatum alone were dead at the end of the experiment. In treatments with isolates 13,X, 16,27, and 35,3/10,2/10,4/10,4/10, and l/10 plants were dead, respectively. At 95% confidence intervals based on binomial distribution, isolates 13, 15, and 35 significantly reduced plant mortality, compared to the Pythium-inoculated, bacterium-free control. However, in the trial on cv. Corona, no reduction in wilt and plant death was observed in the treatments with bacteria (data not shown). In general, inoculation with P. aphanidermatum significantly reduced shoot dry weight (Figs. 5a and 5b). This effect was greater on Corona, which was faster growing and more susceptible to P. aphanidermatum. The dry weight of Pythium-inoculated plants treated with isolate 15 was significantly greater than that of Pythium-inoculated, bacterium-free control plants, in both the first trial on Straight Eight and the trial on Corona. In the second trial on Straight Eight (data not shown), the average dry weight of Pythium-inoculated plants treated with isolate 15 was 1.64 g, compared to the dry weight of 1.31 gin the Pythium-inoculated bacte-

800

0 CPc

1 2 3 4 5 6 7 8 9101113141516171819202122~24252627293132393436

Isolate FIG. 3. Effect of 32 isolates of bacteria from the rhizosphere of cucumber on the root length of cucumbers grown in nutrient solution and infested with 100 zoospores/ml of Pythium aphanidermatum. Three-day-old seedlings were placed in nutrient solution containing lo6 bacteria/ ml. Twenty-four hours later, a zoospore suspension was added to the solution to give a final concentration of 100 zoospores/ml. The control bacterium-free treatment (C) did not receive bacteria or Pythium. Isolates marked * are significantly different from the Pythium-inoculated, control (CP). Duncan’s multiple range test was used to separate the means, P = 0.05. Results are pooled from two trials.

BIOCONTROL

0 CpCl

OF Pythium

ON GREENHOUSE

233

CUCUMBERS

2 3 4 5 6 7 6 91011131415161716192021222324252627283132333435

Isolate FIG. 4. Effect of 32 isolates of bacteria from the rhizosphere of cucumber on the root length of cucumber seedlings grown in nutrient solution. Three-day-old seedlings were placed in nutrient solution containing lo6 bacteria/ml and grown for 7 more days. Root length was calculated using a line-intersect method. Isolates marked * are significantly different from the control treatment that did not receive bacteria (C). Duncan’s multiple range test was used to separate the means, P = 0.05. Results are pooled from two trials.

rium-free control, but the difference was not statistically significant. In the first trial with Straight Eight, but not the second trial, treatment with isolates 13, 15, 27, and 35 resulted in plants with dry weights not significantly different from plants in the bacterium-free, pathogen-free control treatment. In the trial on cv. Corona, all Pythium-inoculated plants treated with bacteria had less dry weight than the bacterium-free, pathogen-free control. The effect of inoculation with P. aphanidermatum and treatment with bacterial isolates on root volume was similar to the effect on the shoot (Figs. 6a and 6b). In the first trial, Pythium-inoculated plants of Straight Eight treated with isolates 15 and 35 had root volumes intermediate between, but not significantly different from, the Pythium-inoculated, bacterium-free control and the bacterium-free, pathogen-free control. In the second trial on Straight Eight (data not shown), the average root volume of Pythium-inoculated plants treated with isolate 15 was 17.0 ml, compared to a root volume of 12.3 ml in the Pythium-inoculated bacteriumfree control, but the difference was not statistically significant. In the trial with Corona, only isolate 15 effectively reduced the loss of root volume due to inoculation with P. aphanidermatum. In all trials, the population density of introduced bacteria in the nutrient solution declined to 103-lo4 cfu/ml after 1 week, with no significant differences between the isolates. After 2 weeks, and one change of the nutrient solution, the population density declined to less than lo3 and could not be detected in some treatments. No

bacteria were detected in the nontreated controls. At the end of the experiments, the population density of bacteria on the roots ranged from 2.7 X lo4 to 3.08 X lo5 cfu/cm3 of root, with no significant differences between the cultivars or the isolates. The population density of P. aphanidermatum zoospores in the nutrient solution, originally added at 500 zoospores/ml, declined to approximately 35 cfu/ml after 4 h in the experiment with Straight Eight without bacteria (treatment P, Fig. 7). Treatment with bacterial isolates significantly reduced the population of zoospores in the solution. Isolates 15 and 16 also reduced the zoospore population density at 8 h, compared to the control treatment (P) without bacteria. By 24 h, the population density declined to less than 1 cfu/ml, and there were no significant differences between the treatments. After 3 weeks, at the end of the experiment, the population density of zoospores ranged between 5 and 35 cfu/ ml, with no significant differences between the treatments. Correlation

of Isolate Rankings

in Bioassays

No correlations existed between the ranks in the myCelia1 inhibition assay and the other in vitro bioassays. A significant negative correlation was present between the rankings in the zoospore germination assay using culture supernatants and the zoospore motility assay using whole bacterial cultures (R = -0.40, P = 0.025). The zoospore germination assay using culture supernatants was also positively correlated (R = 0.56, P = 0.0017) with the zoospore germination assay using

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ity, were used to select potential biocontrol bacteria active against P. aphanidermatum in a hydroponic system. Parke and King (1988) demonstrated that zoospore assays could be useful to select strains of bacteria to protect pea seeds against Aphanomyces euteiches. Lifshitz et al. (1986) also used a zoospore germination assay to select two strains of P. putida that inhibited zoospore germination of Phytophthora megasperma f. sp. glycinea. These two isolates did not inhibit mycelial growth on an agar medium and gave inconsistent levels of biocontrol when applied on soybean seeds. In our work, the correlation between isolate performance in zoospore bioassays and biocontrol performance on plants was not significant. However, zoospore bioassays may have a useful predictive value. The five best-performing isolates in planta were also ranked among the top isolates in inhibition of zoospore germination and/or motility. Therefore, zoospore bioassays may be useful as a

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Isoleto FIG. 5. Effect of selected bacterial isolates on the shoot dry weight of 3-week-old cucumbers grown in aerated hydroponic culture in l-liter glass jars. Plants were treated with bacteria to give a final concentration of lO”cells/ml of nutrient solution. In inoculated treatments, zoospores of P. uphanidermatum were added 24 h later, to give a final concentration of 500 zoospores/ml. The control treatments did not receive any bacteria. The nutrient solution was replaced with fresh solution at 1 and 2 weeks, and two l-cm plugs of P. uphanidermatum from a V8 culture were added to the replaced solution. Treatments followed by the same letters are not significantly different according to Duncan’s multiple range test, P = 0.05. (a) Cultivar Straight Eight, Trial 2. (b) Cultivar Corona.

washed bacterial cells. The zoospore germination and motility bioassays were not highly correlated with the biocontrol ability of isolates tested in planta in test tubes. However, it is interesting to note that of the top five performers in the in planta tests, two isolates (27 and 24) ranked first and third in the germination bioassay, and three isolates (27, 35, and 15) ranked second, third, and fourth, respectively, in the motility bioassay, tied with five other isolates. The mycelial inhibition test was negatively correlated (R = -0.38, P = 0.034) with the performance of isolates on Pythium-inoculated roots. The growth stimulation of root length by isolates on Pythium-free roots (Fig. 4) was positively correlated (R = 0.54, P = 0.0024) with their performance on Pythium-inoculated roots (Fig. 3). DISCUSSION Three in vitro bioassays, based on mycelial growth, zoospore germination, and chemotactic zoospore motil-

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Isolate FIG. 6. Effect of selected bacterial isolates on the root volume of 3-week-old cucumbers grown in aerated hydroponic culture in l-liter glass jars. Plants were treated with bacteria to give a final concentration of lo6 cells/ml of nutrient solution. In inoculated treatments, zoospores of P. aphanidermatum were added 24 h later, to give a final concentration of 500 zoospores/ml. The control treatments did not receive any bacteria. The nutrient solution was replaced with fresh solution at 1 and 2 weeks, and two l-cm plugs of P. aphanidermatum from a V8 culture were added to the replaced solution. Treatments followed by the same letters are not significantly different according to Duncan’s multiple range test, P = 0.05. (a) Cultivar Straight Eight, Trial 1. (b) Cultivar Corona.

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Isolate FIG. 7. Population density of zoospores of Pythium aphanidermatum 4 and 8 h after inoculation in a hydroponic system containing bacteria-treated and nontreated cucumber plants. P = Pythium only, no bacteria. Zoospores were originally added at 500 zoospores/ml, 24 h after plants of CV. Straight Eight were treated with bacterial cells. Population density of zoospores was determined by dilution plating on a Pythiun-selective medium and enumeration of the number of colony-forming units (cfu) per ml. Treatments from the same sampling time followed by the same letters are not significantly different according to Duncan’s multiple range test, P = 0.05.

screening tool to evaluate large numbers of isolates, without the danger of eliminating potentially useful bacteria. The top-performing isolates in the zoospores bioassay could then be tested in the more laborious seedling bioassay. Tests based on mycelial inhibition had little predictive value in determining which isolates were the best in planta. In fact, there was a slight negative correlation between these two assays, suggesting that isolates producing the most antibiotics on petri plates were the least effective biocontrol agents in the rhizosphere. The most effective biocontrol agents in our hydroponic system may control Pythium by mechanisms other than antibiosis, such as nutrient competition or induced resistance. Out. of 604 isolates tested in vitro, 32 were selected for further testing in a test tube plant bioassay. When 7 of these isolates were added to cucumber seedlings 1 day before inoculation with P. aphanidermatum zoospores, the root growth was not significantly different from the bacterium-free, pathogen-free control plants. Due to the high level of variability in the test tube plant bioassay, only one isolate (15) gave a significant increase in root growth over the bacterium-free control inoculated only with Pythium. This isolate was also the most effective in the longer-term hydroponic system and reduced plant death and increased shoot and root growth in most cases. Similar results were obtained in two fullscale greenhouse trials (unpublished data), suggesting that both the test tube and aerated hydroponic plant bioassay are useful for ranking and selecting isolates for

CUCUMBERS

235

further greenhouse testing. We believe that the test tube plant bioassay can be used to screen large numbers of isolates and is an important intermediate step between in vitro tests and greenhouse trials. Likewise, the aerated hydroponic system can be used to verify the effectiveness of isolates on more mature plants. The results of the trials in the aerated hydroponic system with both cucumber cultivars indicate that Corona is more susceptible to Pythium and faster growing than Straight Eight, but the ranking of the isolates was similar with both cultivars. Inexpensive, open-pollinated cucumber seeds (~$0.01 each) may be used to screen large numbers of isolates in the test tube plant bioassay, significantly reducing the cost compared to using hybrid seeds such as cv. Corona ($0.50 each). However, hybrid cultivars should be used in all greenhouse testing, since these are the standard cultivars used in greenhouse production. Three of the bacterial isolates caused growth promotion of cucumber roots when applied to pathogen-free plants. Numerous other studies have demonstrated that rhizosphere bacteria can stimulate plant growth (Burr and Caesar, 1984; de Freitas and Germida, 1990; Kloepper and Schroth, 1981; Lifshitz et al., 1987; Schippers et al., 1987). Van Peer and Schippers (1989) also reported a 20-40% increase in root growth in hydroponically grown cucumbers in the presence of plant growth promoting (PGP) Pseudomonas spp. This growth promotion may be due to production of hormone-like substances by the bacteria (Katznelson and Cole, 1965) or due to the effect of the fluorescent pseudomonads on other root microflora (Kloepper and Schroth, 1981), such as deleterious microflora (van Peer and Schippers, 1989). Two of the three growth-promoting isolates (13 and 15) were also among the top five performers on Pythium-inoculated plants. A significant correlation (R = 0.54, P = 0.0024) was found between the root growth of bacteria-treated, pathogen-free plants and bacteriatreated, Pythium-inoculated plants. This suggests that the reduction in root loss in inoculated plants treated with bacteria may simply be due to the growth-promoting effects of the bacteria overcoming or compensating for the root damage caused by Pythium. Two isolates, 9 and 19 were inhibitory to root growth when applied to noninoculated plants. These bacteria may be considered deleterious rhizobacteria or minor pathogens, possibly through the production of toxic metabolites. When applied to inoculated plants, they did not decrease root growth any more than the treatment with Pythium alone. However, isolate 11 was not inhibitory when added to noninoculated plants, but was inhibitory when added to inoculated plants, reducing the root growth more than treatment with Pythium alone. This suggests some synergistic interaction between this iso-

236

PAULITZ,

ZHOU,

late and Pythium, or that these bacteria predispose the plants to Pythium. All bacteria tested were capable of colonizing the roots under hydroponic conditions, but did not remain in high populations in the nutrient solution. They are adapted to grow on the root surface, and the level of plant exudates in the nutrient solution may be too dilute to support bacterial growth. These bacteria may reduce disease by interfering with the initial stages of zoospore attraction, attachment, encystement, and germination on the root. Isolate 15, the most effective isolate, reduced the number of zoospores encysted and germinated on the root surface after 4 and 8 h. Recent work by Jones et al. (1991) demonstrated that zoospores of P. aphanidermatum can accumulate around the root within 3-10 min, primarily in chemotactic response to amino acids. Bacterial isolates may interfere with this attraction by metabolizing the amino acids and reducing the concentration gradient. In the same study by Jones et al., zoospores encysted in the root hair zone of wheat within 14 min, possibly induced by chemoattractants, but also by surface interactions involving flagellar contact. These bacteria may also induce resistance to P. aphanidermatum and slow the internal spread of the fungus in the root and the production of secondary inoculum. Wei et al. (1991) recently reported that plant growth-promoting isolates of Pseudomonas applied to seeds induced resistance to Colletotrichum orbiculare on the leaves. However, more detailed studies are needed to confirm the mechanism of biocontrol in our system. Whatever the mechanism of growth increase, bacteria can be selected by in vitro and in planta bioassays to reduce the deleterious effects of P. aphanidermatum. To our knowledge, this is the first report of success in the use of bacteria as biocontrol agents against Pythium in hydroponic systems; other researchers have attempted to achieve biocontrol in hydroponic systems but without success (Goldberg and Stanghellini, 1991b). The efficacy of these isolates has also been confirmed in preliminary greenhouse experiments on rock wool (Paulitz, unpublished). However, a better understanding of the mechanisms of the biocontrol agent-host-pathogen interaction may lead to other bioassays that may be more efficient in selecting the most active isolates of bacteria from the myriad of potential antagonists in the rhizosphere. ACKNOWLEDGMENT This work was supported by a grant from the Canada-Quebec Subsidiary Agreement on Agri-Food Development and the Natural Sciences and Engineering Research Council of Canada.

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