Biocontrol of selected soilborne diseases of tomato and pepper plants

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Biocontrol of selected soilborne diseases of tomato and pepper plants W. Mao*, J. A. Lewis, R. D. Lumsden and K. P. Hebbar Biocontrol of Plant Diseases laboratov, US Department Research Service, Be/&vi//e, MD 20705, USA

of Agriculture,

Agricultural

Biocontrol of soilborne diseases of tomato caused by Rhizoctonia solani and Pythium ultimum alone or in combination with Sclerotium rolfsii and Fusarium oxysporum f. sp. lycopersiciwere studied in the greenhouse and field. Soilborne diseases of pepper caused by the first three pathogens were also studied alone or in combination with Phytophthora capsici. Tomato and pepper seeds were treated with biomass of Gliocladium virens (Gl-3) and Burkholderia cepacia (Bc-F), individually and in combination, and planted in pathogen-infested soilless mix. Seedling stands for tomato from treated seeds were comparable to that in non-infested soilless mix. Although seed treatments with individual biocontrol agents reduced damping-off in peppers, only the Gl-3 + Bc-F treatment resulted in stands

similar to the non-infested control. When healthy seedlings of both crops were transplanted into pathogen-infested soilisoilless mix in the greenhouse, and supplementary root drenches of suspensions of Gl-3, Bc-F, and Gl-3 +Bc-F were applied, the plant fresh weight was significantly greater and the disease severity (DSI) significantly less than for infested controls. When transplants were set out into infested field plots, the combined Gl-3 + Bc-F application resulted in greater fresh weight and lower DSI for pepper, and greater fruit yield for tomato than those obtained with either Gl-3 or Bc-F alone. 0 1998 Elsevier Science Ltd. All rights reserved Keywords:

biocontrol

agents; Burkholderia cepacia;

Capsicum anuum;

Gliocladium virens; Lycopersicon esculentum; fhytopththora solani; Sclerotium rolfsii; seed treatment; soil drench

Introduction The production of tomato and pepper fruit is of worldwide agricultural importance. In the United States alone, for example, the value of harvested tomatoes in 1990 was $1.6 billion with almost half the production from the state of Florida (Datnoff et al., 199.5; Chellemi et al., 1997). Also, in 1990, the value of chili peppers produced in New Mexico and of bell peppers produced in New Jersey was $230 and $45 million, respectively (Bosland and Lindsey, 1991). Fusarium oxysporum f. sp. lycopersici (FOL) is a highly destructive pathogen of both greenhouse and field grown tomatoes in warm vegetable production areas. The disease caused by this fungus is characterized by wilted plants, yellowed leaves, and minimal or absent crop yield. There may be a 30 to 40% yield loss in Florida alone (Jones et al., 1991; Chellemi et al., 1997). For peppers, the yield is reduced under warm and wet environmental conditions in the field by the blight fungus, Phytophthora capsici. This disease occurs worldwide and causes root and crown rot, and aerial blight of leaves, stems, and fruit of pepper, tomato and cucurbits. The average yield loss in New *Corresponding author. Tel.: +l 301 504 5356; fax: +l 301 504 5968; e-mail: [email protected]

capsici;

Fusarium Pythium ukimum;

oxysporum; Rhizoctonia

Jersey is 20 to 25% (Bowers et al., 1990; Biles et al., 1992). Both crops are also attacked by Rhizoctonia solani, Pythium spp. and Sclerotium rolfsii (Sherf and MacNab, 1986). Many strategies to control these diseases and others on tomato and pepper have been investigated in the field. However, the major component in integrated control (IPM) studies used with tomato and pepper culture involves soil injection with the fumigant methyl bromide (MB). The ban on MB use in 2001 could result in crop losses of $300 and $500 million in California and Florida, respectively. For example, in Florida alone, the elimination of MB could cause a loss of $127 million for the winter pepper crop (Sanchez, 1997) and a 46% loss in the tomato crop (Gilbreath et al., 1994). The implication of chemical fungicides in soil and water pollution and the evidence that MB is an ozone depletor has mandated the search for alternative approaches to disease control management (Ristaino and Thomas, 1997). A promising strategy for the replacement of chemicals has been the implementation of biocontrol technology, used individually or as an IPM component. The recent developments in the commercialization of biocontrol products has accelerated this approach (Lumsden et al., 1996; Fravel et al., 1998). Biocontrol preparations of both fungi and bacteria

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have been applied to seeds, seedlings, and planting media in several ways to reduce tomato and pepper diseases in the field with various degrees of success. Two of the major biocontrol agents which reduce soilborne diseases of various crops include isolates of the bacterium Burkholderia (Pseudomonas) cepacia and the fungus G. virens (Lumsden and Locke, 1989; Bowers and Parke, 1993; Howell and Stipanovic, 1995). Recently, biomass of isolates of these microorganisms was used as a seed treatment, alone and in combination, to control damping-off, root rot, and stalk rot of field and sweet corn caused by Pythium spp. and Z? graminearum (Mao et al., 1997; 1998). The objectives of this research were: (i) to demonstrate the pathogenic&y of selected isolates of R. solani, P ultimum, S. rolfsii, FOL and p capsici, individually and together, on seedlings of tomato and pepper; and (ii) to evaluate the combined effect of seed treatment and root drenching with biomass of isolates of B. cepacia and G. virens on disease incidence and severity of tomato and pepper caused by a mixture of the above pathogens in the greenhouse and field.

Materials and methods Microbial

cultures

The pathogenic fungi used in this study included isolates of Rhizoctonia solani Kuhn (AG-4) Pythium ultimum Trow., Sclerotium rolfsii Sacc., Phytophthora and Fusarium oxysporum f. sp. capsici Leonian, lycopersici (FOL) Snyder and Hansen. Biocontrol agents studied were the fungus Gliocladium virens ( = Trichodenna virens) Miller, Giddens and Foster, isolate Gl-3 and the bacterium Burkholderia cepacia ( = Pseudomonas cepacia) Palleroni and Holmes, isolate Bc-F. Fungi were maintained on potato dextrose agar (PDA) (Difco, Detroit, MI) and the bacterium on nutrient agar (NA) (Difco). The isolates of l? ultimum and I! capsici were provided by D. E. Mathre, Montana State University and J. B. Ristaino, North Carolina State University, respectively. The bacterium was isolated from corn rhizosphere by K. P. Hebbar of the Biocontrol of Plant Diseases Laboratory (BPDL). The isolate of FOL was provided by R. P. Larkin of the BPDL. The other isolates were from the collection of the BPDL. Preparation

of inocula

Pathogens l? ultimum, P capsici, and R. solani were grown separately on a medium of 600 ml Redi-Earth 3 CP (Scotts, Marysville, OH), 330 ml 60% V-8 juice (Campbell Soup Co., Camden, NJ), 10 g potato dextrose broth powder (Difco) and 0.6 g CaC03 in foil-covered polypropylene flats (12 x 23 x 45 cm “). The flats were autoclaved for 1 h on each of two successive days, PDA disks of the isolates were incorporated, and the flats were incubated for two weeks at 23 ) 2°C. This inoculum was used immediately. The pathogen FOL was similarly cultured in a wheat bran-water (l:l, w/w) medium. After incubation, the inoculum of FOL was air-dried for three days, milled in a blender, and sieved through a 3.36 mm screen

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(Mao et al., 1997). The pathogen S. rol’ii was grown on 9 cm diameter Petri plates containing 10 ml PDA. Plates were incubated in the light for four weeks at 23 +2”C and the sclerotia which formed were dislodged from the surface of the plates and used immediately (Lewis and Fravel, 1996). After incubation of the inocula, the viability of Z? ultimum and I? capsici was determined by a most probable number (MPN) method (Harris and Sommers, 1968). There were approximately 1 x lo5 colony forming units (cfu) per gram of inoculum of p ultimum and p capsici. The FOL inoculum contained approximately 1 x lo5 cfu g -r as determined by serial dilution on a peptone pentachloronitrobenzene (PCNB) medium. Sclerotial germination on PDA indicated more than 95% viability for inoculum of S. rolfsii. The viability of R. solani was not determined. The biocontrol fungus G. virens (Gl-3) was grown for 10 days by liquid fermentation in 15 1 carboys containing a molasses (3.0%)-brewer’s yeast (0.5%) medium (Papavizas et al., 1984). The biomass was filtered on muslin, air-dried overnight in a transfer hood, milled in a blender, sieved through a 425 urn screen, and stored at 4°C. The biomass of Gl-3 consisted mostly of chlamydospores and there were > 1 x lo6 cfu g -’ of biomass of the fungus as determined with a semi-selective medium (Papavizas and Lumsden, 1982). Biomass of B. cepacia (Bc-F) was prepared in 250 ml Erlenmeyer flasks containing 100 ml tryptic soy broth (TSB) (Difco). Flasks were placed on a rotary shaker (120 rev min -‘) for 48 h at 23 f 2°C. The TSB cultures contained approximately 1 x 10 lo cfu ml -’ as determined on a semi-selective medium (Burbage et al., 1982). Seed coating and root drenching

All biocontrol experiments involved seed treatment with Gl-3 and Bc-F, alone and in combination, followed by a corresponding root drenching with these microorganisms. Treatments were applied to seeds of tomato (Lycopersicon esculentum Mill. cv. Bonny Best) and pepper (Capsicum anuum L. cv. Cayenne Large Red Thick). Cultures of Bc-F were centrifuged at 61OOg for 10 min and the supernatant was decanted. 10 ml of an aqueous 0.8% solution of the xanthan gum sticker Keltrol (Kelco, Chicago, IL) was vortexed with 2.0 ml of the bacterial pellet and 2.0 g of the Gl-3 biomass, alone and in combination, for 1 min. This slurry was mixed with 10 g of tomato or pepper seeds and the treated seeds were shaken in a covered jar with sufficient Nitragen Sterile peat (Lipha Tech, Milwaukee, WI)-Pyrax (R. T. Vanderbilt, Norwalk, CT) mixture (1:4, w/w) to individually coat the seeds. Tomato seeds were coated with 1.4 x lo4 cfu and 3.8 x lo5 cfu per seed of Gl-3 and Bc-F, respectively; pepper seeds were coated with 8.6 x lo5 cfu and 5.0 x lo6 cfu per seed of Gl-3 and Bc-F, respectively. Coated seeds were stored at 4°C for no longer than five days before planting. For the non-pathogen control, untreated seeds contained all the constituents except the biomass. For root drenching, a suspension of 200 ml of an uncentrifuged bacterial culture and 30.0 g of fungus biomass in

Biocontrol of pepper and tomato diseases: W. Mao et al.

2.5 1 of a Ketrol solution (0.3% w/w) was prepared. The suspension contained 3.7 x 107 cfu and > 10” cfu ml - ’ of Gl-3 and Bc-F, respectively. Greenhouse

tests

The ability of various plant pathogens to incite damping-off of tomato and pepper seedlings was evaluated. Soilless mix (Redi-Earth 3 CP) was infested with inoculum of single pathogens and with a combination of inocula. Fresh inocula of p c&mum, I-1capsici, or R. solani were mixed with the soilless mix at 16.7 g, 16.7 g, or 13.3 g kg -’ of mix, respectively. Dry inoculum of FOL was added at 10.0 g kg ~ ’ of mix. Fresh sclerotia of S. rolfsii were added at 0.5 g kg ~’ of mix. For the combination of pathogen inocula, the same rates were used of inocula per kilogram of mix as described for each isolate. Non-infested soilless mix and mix infested with each pathogen or with a combination were placed in Polyfoam plug trays (TLC, Plymouth, MN) (25 x 50 cm*) containing 24 circular cells (6.4 x 7.1 cm*). Four trays of each mix were planted immediately with untreated tomato seeds and four trays were planted with untreated pepper seeds. Each cell was planted with one seed. Trays were incubated in the greenhouse at 23 _t 2°C with a 12 h photoperiod and were watered daily. Seedling stands for tomato and pepper plants were determined after 30 and 40 days growth, respectively, in non-infested mix and in mixes infested with the pathogens, alone and in combination. The biocontrol potential of Gl-3 and Bc-F, alone and in combination, applied as a seed treatment to reduce damping-off of tomato and pepper seedlings was evaluated in soilless mix infested with the pathogen combination. Trays of non-infested soilless mix were planted with untreated tomato and pepper seeds as a control. Four replicate trays of soilless mix were planted with tomato or pepper seeds for each of the four treatments (untreated or treated with Bc-F, GI-3 and the combination of Bc-Ft Gl-3). Additional controls included trays of infested soilless mix planted with pepper seeds commercially treated with thiram. Trays were incubated, and seedling stands were determined as previously indicated. The potential of the biocontrol agents used as a seed treatment and as a soil drench to reduce the incidence and severity of disease on plants, more mature than seedlings, was evaluated. Seedlings from untreated and biocontrol-treated tomato and pepper seeds planted in cells containing non-infested soilless mix and soilless mix infested with the combined pathogens were drenched with 20 ml of the corresponding biocontrol suspension at the time of seedling stand determination. Five replicate cells of tomato and pepper seedlings for each treatment were then transplanted into 15 cm diameter pots filled with a mixture (l:l, w/w) of the soilless mix and a natural loamy sand (pH 6.2-6.6) from a Beltsville, MD field plot. The soilless mix was either non-infested or infested with inoculum of combined pathogens. Controls included non-infested and pathogen-infested mixtures planted with seedlings from untreated seeds.

Pots were incubated in the greenhouse at 23 +2”C with a 12 h photoperiod and were watered daily. One and three weeks after transplanting, pots were treated with a 20-20-20 soluble fertilizer solution to provide 50 mg kg - ’ of nitrogen (N). Sixty days after transplanting, tomato and pepper plants were harvested and plant fresh weight (gram per plant) was determined. A combined visual disease severity index (DSI) to evaluate wilt, root rot, and necrosis for each plant was used. Each plant was graded on a scale of 1 to 10 based on the assessment of plant appearance (wilting, discoloration, and leaf, stem, and root rot) such that plants with a rating of 1 were healthy and those with a rating of 10 were dead.

Field tests

The efficacy of seed treatment application together with root drenches of the biocontrol agents Gl-3 and Bc-F, alone and in combination, on diseases of tomato and pepper transplants was evaluated in 1997 in two field tests in a loamy sand field plot at Beltsville. The field was seeded with rye (Secale cerealis L.) in the autumn of 1996, which was incorporated into the soil in the spring of 1997 to a depth of 15 cm, followed by the application of napropamide herbicide. Fifty 7.0 m long furrow rows, 10 cm deep and 1.0 m apart were established in the field. Inocula of the pathogenic fungi were prepared as previously described. Evenly distributed within 20 furrow rows. each was a mixture composed of 400 g soil, 1.0 g S. rolfsii sclerotia, 20 g R. sofani inoculum, 20 g P uftimum inoculum, and 60 g FOL inoculum. These rows and five control rows were planted with tomato transplants. Another 20 furrow rows were amended with a similar mixture containing 60 g p capsici inoculum instead of FOL inoculum. These rows and five control rows were planted with pepper transplants. Duplicate sets of plots were planted in early April and late May. Plants were grown from untreated and biocontroltreated seeds in non-infested and pathogen-infested soilless mix in plug trays as previously indicated for the greenhouse bioassay. After 30 and 40 days of growth for tomato and pepper seedlings, respectively, 20 ml of the appropriate drenches were applied to the cells. One day later, the root balls with soilless mix were carefully removed from the cells and transferred to the field in the furrow rows with 12 plants of each treatment per row and 0.5 m between plants. The field treatments consisted of non-treated and non-inoculated controls, Gl-3 and Bc-F treatments alone, and a combination treatment of Gl-3 +Bc-F. After setting the transplants, the field was irrigated and watered thereafter when necessary. No fungicide, insecticide, or additional herbicide was applied. A 20-20-20 liquid fertilizer application (58 kg ha ~ ’ ) was made 30 days after transplanting. After 90 days in the field, plant stand was recorded, plants were carefully uprooted and plant fresh weight and disease severity were determined as previously described for the greenhouse bioassay. In addition, tomato and pepper fruit yields were determined as the total

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al.

weight (kg) of all harvested fruit from each treatment row (12 plants). Statistical analyses All greenhouse experiments were performed twice with four or five replicates per treatment. There were five replicates per treatment in the field experiments. In the greenhouse and field, respectively, plug trays and furrow rows were arranged in a randomized complete block design. Statistical analyses were conducted using the general linear models procedure of SAS version 6.08 (SAS Inc., Cary, NC). Data per cent values were arcsine transformed before analyses. The field data for each set of the two plantings were analyzed separately but were not significantly different (P > 0.05).

Results Greenhouse tests The effect of the pathogens R. solani, l? ultimum, S. rolfsii, I! capsici, and FOL alone, and in combination, on the stands of tomato and pepper seedlings was determined after 30 and 40 days of growth, respectively (Figure 1). The stands (%) reflect the total influence of seed rot and pre- and post-emergence

damping-off. The tomato seedling stand in non-infested soilless mix was > 90%, whereas that in the soilless mix infested with all the pathogens was ~10%. The pathogens R. solani and P ultimum each significantly reduced (PI 0.05) tomato seedling stand compared to that in non-infested soilless mix, but S. rolfsii, l? capsici and FOL did not. The combination of all pathogens resulted in tomato stands significantly lower (PI 0.05) than those from each of the pathogens alone. The pathogen FOL on tomato did not incite more seedling damage than that observed because it causes root rot, blight and wilt on more mature plants than seedlings. I? capsici may not have incited greater seedling damage than observed because the soilless mix was not maintained at a low matric potential. All pathogen treatments (except FOL which does not cause disease on peppers) resulted in pepper seedling stands that were significantly lower (PsO.05) than those in the non-infested soilless mix (90%). Soilless mix infested with R. solani, I? ultimum, and a combination of all pathogens resulted in pepper stands of ~25%. The combination of all pathogens resulted in pepper seedling stands significantly lower (P
a

Tomato a

a

a

Pepper

Treatment Figure 1. Tomato and pepper seedling stands (%) after 30 and 40 days of growth, respectively, in non-infested (control) soilless mix and in soilless mix infested with inoculum of Rhizoctonia solani, Pyfhium ukimum, Sclerotium rolfsii, PhytophN?ora capsici, and Fusarium oxysporum f. sp. lycopersici (FOL), individually and in combination. For each crop, bar values with the same letter do not differ according to Duncan’s multiple range test (P~0.05).

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mix caused by the combined pathogens was determined after 30 and 40 days of growth for tomato and (Table I). For the pepper seedlings, respectively tomato bioassay, R cupsici inoculum was omitted and for the pepper bioassay, FOL inoculum was omitted. Tomato seed treatment with biomass of the fungus Gl-3 and the bacterium Bc-F, alone and in combination, resulted in greenhouse seedling stands (94 to 98%) comparable to that in non-infested soilless mix (90%) and significantly higher (P10.05) than that from untreated seeds (45%) in the soilless mix infested with pathogens. Somewhat similar results were obtained with the pepper seed treatments. However, although both Gl-3 and Bc-F significantly seedling stand (66 to 68%) improved pepper compared to that in pathogen-infested mix (49%) only seed treatment with a combination of the

Table 1. Effect of seed treatments with biomass of the biocontrol agents Gliocladium virens (GI-3) and Burkholderia cepacia (Bc-F), alone and in combination, on the damping-off of tomato and pepper seedlings caused by a pathogen mixture in a soilless mix in the greenhouse after 30 and 40 days of growth, respectively’ Plant stand (%) Treatment

Tomato YOa’ 45b

Control (no pathogens) Control (pathogens) Control (thiram)’ U-3 Bc-F Gl-3 + Bc-F ‘Tomato dtimum, were

pathogen5

the first three

‘Numbers

Sclerotium

fungi previously

multiple

range

seeds were

treated

85a 49c 57bc 66b 68b 79a

96a Y8a 94a

oxysporum

in each column

to Duncan’s ‘Pepper

were

and Fusarium

Pepper

r&ii,

Rhizoctoniu

f. sp. lycopersici mentioned

followed

(FCC);

solani, pepper

and Phytophthora

by the same letter

Pythium pathogens

capsici.

do not differ

according

test (PsO.05). with

thiram

commercially.

of pepper

and tomato

diseases:

W. Mao et al.

biocontrol agents resulted in a plant stand (79%) comparable to that from untreated seed in non-infested soilless mix (85%). Pepper seed treatment with the fungicide thiram did not increase seedling stand compared to untreated seed in pathogen-infested soilless mix. In the greenhouse, healthy seedlings from each treatment were transplanted from plug trays to plastic pots to evaluate disease on plants after 60 days of growth (Figure 2). The fresh weight of tomato and pepper plants from untreated seeds in non-infested soilless mix was almost 200 g and 80 g per plant compared to about 40 g per plant, respectively, from the pathogen-infested controls. Seed treatment and subsequent root drenching with Gl-3 and Bc-F, alone and in combination, resulted in plant fresh weights of > 130 g and > 80 g per tomato and pepper plant, respectively. Moreover, treatment with Gl-3 and the to that combination gave weights comparable obtained in the non-infested control. The DSI values of plants from the non-infested and pathogeninfested controls were ~3 and > 8, respectively. The DSI values of plants treated with Gl-3, Bc-F, and the combination as seed coatings and root drenches were ~5, < 7, and ~4, respectively. The use of Gl-3 + BcF reduced disease of tomato to such an extent that the DSI was comparable to that of healthy plants in the system employed. As with tomato, seed treatment and root drenching of pepper with either or both of the biocontrol agents significantly increased (P 5 0.05) plant weight over that of plants from the pathogeninfested control. Also, as observed with tomato, treatment with Gl-3 and with Gl-3 +Bc-F gave weights comparable to that of the non-infested control. The DSI values of pepper plants with the various treatments were similar to those of tomato plants, and the use of Gl-3 +Bc-F reduced disease of pepper to give

200 160 120 60 40 0

10

8 6 4 2 0 Control (no pathogens)

Control (pathogens)

Bc-F

GI-3

GI-3

+ Bc-F

Treatments Figure 2. Effect of seed treatment and root drenching with biomass of Gliocladium virens (GI-3) and Burkholderia cepacia (Bc-F), alone and in combination, on disease severity of tomato and pepper seedlings in the greenhouse. For each measured variable of each crop, bar values with the same letter do not differ according to Duncan’s multiple range test (P~0.05).

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Biocontrol

of pepper and tomato

a DSI comparable non-infested control.

to

diseases: W. Mao et

that

of plants

from

a/. the

Field tests Thirty day-old tomato and 40 day-old pepper seedlings developed in plug trays from untreated and treated seeds with the appropriate root drenches were transplanted into the designated furrow rows at two planting dates. Plant stand, plant fresh weight, DSI, and crop yield were determined on plants 120 days after seeding. The data for each parameter for pepper and for tomato at the two field plantings correlated well (r* > 0.80). Consequently, only data from the second planting are presented (Figure 3). With tomato, plant stand was significantly (P10.05)

E’

3.2

om .I$s

2.4

“m”o =

1.6

*aI 2:

0.8

5

reduced by cumulative disease when the pathogeninfested control (80%) was compared to the non-infested control plants (96%). The application of Gl-3 and Bc-F, alone and in combination, resulted in plant stands comparable to that of healthy plants from the non-infested control. Biocontrol agents also resulted in an increase of tomato plant fresh weight compared to that of plants in the pathogen-infested control, and the biocontrol agents significantly reduced (P10.05) the overall DSI. Treatment with Gl-3 and with Gl-3 +Bc-F resulted in DSI values similar to that of the non-infested control plants (Figure 3). In any biocontrol study in the field, the major criterion for efficacy is an increase in crop yield. In this study, the application of biomass of the fungus and the bacterium, alone and in combination,

0

Control (no pathogens)

C 0 n tro (pathogens)

I

0 l-3

EC-F

0 I-3

+ B c-f

Treatments Figure 3. Effect of seed treatment and root drench with biomass of Gliocladium virens (GI-3) and Burkholderia cepacia (Bc-F), alone and in combination, on the damping-off, fresh weight, disease severity, and yield of tomato and pepper plants in the field after 120 days of growth for the second planting test. For each measured variable of each crop, bar values with the same letter do not differ according to Duncan’s multiple range test (P 10.05).

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as a seed treatment and a root drench gave a tomato yield comparable to that of plants from the non-infested control and the yield of treated plants was significantly greater (P
also minimal. For example, the combined application of 7: hanianum +I? nunn was better than each alone for the reduction of Pythium damping-off of cucumber (Paulitz et al., 1990) and the combined and a application of Pseudomonas SPP. non-pathogenic Fusarium was better than each alone for the reduction of Fusarium wilt of flax (Lemanceau and Alabouvette, 1991). We showed that the combination of biocontrol agents (Gl-3 + Bc-F) was better than that of each individual with regard to an increase of tomato and pepper fruit yield and plant fresh weight and a decrease in DSI of pepper plants in the field. The choice of the biocontrol isolate (Gl-3) of G. virens and the isolate (Bc-F) of B. cepacia for the present trials in greenhouse and field was based on previous results not only with these species, but with these specific isolates. Various isolates of G. virens have been used effectively to control several diseases in field tests (Lumsden and Locke, 1989; Ristaino et al., 1994; Howell and Stipanovic, 1995) as have various isolates of Pseudomonas spp. and B. cepacia (Bowers and Parke, 1993; Reddy, 1996). The application of Gl-3 and Bc-F in this study was specifically investigated because of their ability to reduce corn diseases (Mao et al., 1997; 1998). Their efficacy to also reduce tomato and pepper diseases suggests a broad potential for the use of these two microorganisms. Also, it is most likely that biocontrol efficacy is determined by the manner in which the biocontrol agents are applied to the ecosystem as well as by the number of treatments. In this study, biocontrol agents were applied as biomass in seed treatments (Harman, 1991) and as biomass in a root drench (Mickler et al., 1995). Seed treatment is probably the most cost effective and easiest approach for many crops, and root drenches can be used with high value transplant crops (Paulitz et al., 1990). It is evident from these results that biocontrol agents are operative in the field and the combination of biocontrol agents applied by two methods provided the control of a disease complex. Various coating systems, concentrations of applied biocontrol agents, and shelf-life of the treatments are being investigated. Mention of a trademark proprietary product does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. Acknowledgements The authors thank C. Nguyen, D. Lopez, and S. Poch for their technical assistance. References Biles, C. L., Lindsey, D. L. and Liddell, C. M. (1992) Control of Phytophthora root rot of chili peppers by irrigation practices and fungicides. Crop Protection 11, 225-228 Bosland, P. W. and Lindsey, D. L. (1991) A seedling screen for Phytophthora root rot of pepper, Capsicum anuum. Plant Diseases 75, 1048- 1050 Bowers, J. H. and Parke, J. L. (1993) Epidemiology of Pythium damping-off and Aphanomyces root rot of peas after seed treat-

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ment with bacterial 83,1466-1473

diseases: W. Mao et al.

agents for biological control. Phytoputhology

Bowers, J. H., Sonoda, R. M. and Mitchell, D. J. (1990) Pahogen coefficient analysis of the effect of rainfall variables on the epidemiology of Phytophthora blight of pepper caused by Phytophthora cap&i. Phytopothologv 50, 1439-1446

Burbage, D. A., Sasser, M. and Lumsden, R. D. (1982) A medium selective for Pseudomonas cepacia. PhytopathologV 72, 706 Chellemi, D. O., Olson, S. M., Mitchell, D. J., Seeker, J. and McSorley, R. (1997) Adaptation of soil solarization to the integrated management of soilborne pests of tomato under humid conditions. Phytopathology 87, 250-258 Datnoff, L. E., Nemec, S. and Pernezny, K. (1995) Biological control of Fusarium crown and root rot of tomato in Florida using Trichoderma harzianum and Glomus intraradicis. Biological Control 5,427-431

Fravel, D. R., Connick Jr., W. J. and Lewis, J. A. (1998) Formulation of microorganisms to control plant diseases. In Formulation of Microbial Pesticides, Beneficial Microorganisms and Nematodes, ed. Burgess, H. D. Chapman and Hall, London (in press). Gilbreath, J. P., Jones, J. P. and Overman, A. J. (1994) Soilborne pest control in mulched tomato with alternatives to methyl bromide. Proceedings of Florida State Horticultural Society 107, 156-159

Harman, G. E. (1991) Seed treatments for biological control of plant disease. Crop Protection 10, 166-171 Harris, D. F. and Sommers, L. E. (1968) Plate dilution frequency technique for assay of microbial ecology. Applied Microbiology 16, 330-334

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