Biological Control of Cucumber Beetles
(Coleoptera: Chrysomelidae) and Cucurbit Wilt Disease
with Plant Growth-promoting Rhizobacteria

Geoffrey Zehnder

Department of Entomology, Extension Hall, Auburn University, AL 36849

Joseph Kloepper

Department of Plant Pathology, Life Science Bldg., Auburn University, AL 36849

Changbin Yao

Department of Entomology, Extension Hall, Auburn University, AL 36849

Sadik Tuzun

Department of Plant Pathology, Life Science Bldg., Auburn University, AL 36849

Oyette Chambliss

Department of Horticulture, Funchess Hall, Auburn University, AL 36849

Richard Shelby

Department of Plant Pathology, Life Science Bldg., Auburn University, AL 36849

Additional index words. Cucumis sativus, Cucumis melo, melon, muskmelon, induced systemic resistance, ISR

Abstract. Field and greenhouse studies were done to evaluate select strains of plant growth-promoting rhizobacteria (PGPR) for induction of resistance in cucumber against cucumber beetle feeding and the beetle-transmitted cucurbit wilt disease. In the field, cucumber plant growth and yields were significantly greater, and populations of cucumber beetles were significantly lower on PGPR-treated cucumber than on nontreated cucumber. In addition, PGPR treatment resulted in superior control of cucumber beetles and cucurbit wilt than weekly applications of esfenvalerate insecticide. In choice and no-choice greenhouse cage experiments, beetles carrying the wilt pathogen, Erwinia tracheiphila, were released and allowed to feed on PGPR-treated or nontreated cucumber plants. The incidence of beetle feeding damage and wilt disease was significantly lower on PGPR-treated cucumber plants than on nontreated plants. HPLC analysis demonstrated that cotyledons from PGPR-treated plants contained significantly lower concentrations of the cucumber beetle feeding stimulant cucurbitacin than nontreated plants. These results indicate that PGPR-induced resistance may be more effective than insecticides for control of cucumber beetles and cucurbit wilt, and that a mechanism for PGPR-induced resistance against cucumber beetle feeding may involve a change in the metabolic pathway for cucurbitacin synthesis.

Cucurbits grown in the midwestern and eastern United States are susceptible to feeding damage by the striped cucumber beetle [Acalymma vittatum (F.)] and the spotted cucumber beetle (Diabrotica undecimpunctata howardi Barber). The most serious direct injury is caused by adult feeding on cotyledons and stems of young plants (Brewer et al., 1987; Foster et al., 1995; Metcalf and Metcalf, 1993). Cucumber beetle feed

ing behavior is strongly influenced by a group of secondary plant compounds called cucurbitacins that occur mainly in the plant family Cucurbitaceae (Chambliss and Jones, 1966). Cucurbitacins are among the most bitter compounds known (Metcalf et al., 1980) and are toxic to most herbivores (David and Vallance, 1955; Nielson et al., 1977). An exception are the diabroticine cucumber beetles, which can consume cucurbitacins with

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out evidence of acute toxicity. Cucumber beetles are highly sensitive to cucurbitacins, and even concentrations as low as 0.001 mg can produce locomotory arrest and compulsive feeding (Metcalf, 1986). A strong positive correlation has been established between total cucurbitacin concentration in cucurbit plant tissue and the extent of feeding and aggregation by D. u. howardi and A. vittatum cucumber beetles (Ferguson et al., 1983; Metcalf et al., 1982).

In addition to feeding damage, striped and spotted cucumber beetles transmit cucurbit wilt, a vascular disease of cucurbits caused by the bacterium Erwinia tracheiphila (Smith) (Bradbury, 1970). Cucumber (Cucumis sativus L.) and muskmelon (C. melo L.) are the most susceptible host crops (Sherf and MacNab, 1986). The pathogen is transmitted by inoculation through feeding wounds made by cucumber beetles (Foster et al., 1995; Harrison et al., 1980). E. tracheiphila is thought to be entirely dependent on cucumber beetles for inoculation and dissemination in the field (Agrios, 1978). Cucumber beetles colonize spring-planted cucurbits shortly after plant emergence (Hofmaster, 1989; Yao et al., 1996). A positive, linear relationship has been established between cucumber beetle density on plants and disease severity (Yao et al., 1996). The primary control for cucurbit wilt involves insecticide application targeted against the cucumber beetle vectors. Foliar sprays are, however, not consistently effective for preventing the spread of cucurbit wilt disease because cucumber beetles are highly mobile and exhibit great interplant and interfield movement. In addition, insecticide sprays may be ineffective because of the low beetle population threshold for transmission of the disease (Yao et al., 1996).

Research over the past 2 decades indicates that many pathogens or chemical compounds may be used to elicit host defense mechanisms leading to reduced pathogen attack (Kuc, 1983, 1987). This phenomenon, called induced systemic resistance (ISR) (Tuzun and Kuc, 1985), operates through the activation of defense genes and leads to the accumulation of defense compounds at sites distant from the point of pathogen attack (Dean and Kuc, 1985). Cucumber has been used as a model for induced resistance (Hammerschmidt et

al., 1982; Kuc, 1983, 1987), and resistance of cucumber to cucurbit wilt has been demonstrated by previous inoculation of lower leaves with fungal, bacterial, and viral pathogens (Bergstrom, 1981). More recently, plant growth-promoting rhizobacteria (PGPR) have been investigated as nonpathogenic elicitors of ISR against various pathogens (Liu et al., 1995 a, 1995b; Van Peer et al., 1991; Wei et al., 1991; Zhou and Paulitz, 1994). PGPR are root-associated bacteria which represent a subset of total rhizosphere bacteria exerting beneficial effects on plants. PGPR may benefit the host by causing plant growth promotion and/or biological disease control (Kloepper et al., 1993). PGPR are ideal vehicles for delivering benefits to plants because they can be applied to seed or roots directly with subsequent colonization of plant roots.

In field experiments to evaluate several PGPR strains for control of cucumber angular leaf spot caused by Pseudomonas syringae pv. lachrymans, it was unexpectedly observed that PGPR provided protection against cucurbit wilt disease associated with the presence of large numbers of cucumber beetles (Wei et al., 1995). Here, we present a general summary of the results of field and greenhouse experiments designed to assess the effects of PGPR treatment on cucumber beetle population density and feeding damage, and on the incidence of cucurbit wilt disease (see Zehnder et al., 1997a, 1997b, for complete details).

Materials and methods

pgpr cultures and seed treatment. PGPR strains for evaluation were obtained from the Dept. of Plant Pathology, Auburn University. These included Pseudomonas putida strain 89B-61, Serratia marcescens strain 90-166, Flavomonas oryzihabitans strain INR-5, and Bacillus pumilus strain INR-7. Bacteria were identified using fatty acid analysis (Sasser, 1990) and were maintained at ­80 °C in tryptic soy broth (TSB) with 20% glycerol. For bioassay, cultures from storage were grown in tryptic soy agar and incubated for 24 h at 28 °C. A loopfull of bacteria was then transferred to 1-L flasks with TSB and shaken at 150 rpm (24 °C) for 24 h. PGPR suspensions were centrifuged at 6,000 gn for 5 min. Cucumber seeds were dipped

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into the pelleted bacterial cells or into distilled water (control) immediately before planting in 10-cm2 plastic pots containing sterilized Promix soilless mix. A dilute PGPR suspension [100 mL containing 108 colony forming units (cfu)/mL] was poured into each pot immediately after seeding. Seedlings were transplanted into the field at the second leaf stage.

Field experiments. 'Straight 8' cucumbers were transplanted into the field on 19 Apr. 1993 and 15 Apr. 1994 at the E.V. Smith Horticulture Substation, Shorter, Ala. Cucumbers were grown in fumigated, raised beds with black plastic mulch and drip irrigation. Fertilization and weed control were done according to local cucumber production practices. Fungicides were not applied. Treatment plots consisted of one row (10 m long) with 0.9 m plant spacing.

In 1993, a seven treatment x six replication randomized complete block design was used. Treatments included PGPR strains 89B-61, 90-166, INR-5, and INR-7, an insecticide control (weekly sprays of esfenvalerate at 56 g [a.i.]/ha by backpack sprayer), and a nontreated control.

In 1994, a four treatment x six replication randomized complete block was used. Treatments included the strains INR-7 and 90-166, the insecticide control, and the nontreated control.

Beginning with initial colonization of plants by beetles, numbers of striped and spotted cucumber beetles were recorded weekly on five randomly chosen plants per plot. Plant growth was assessed 50 d after emergence by recording the length of the main runner per plant on five randomly chosen plants per plot. Cucumbers were harvested at least twice weekly and weighed to determine fruit yield in each plot. Data were analyzed using a single-factor ANOVA and treatment means were compared using the Newman-Keuls test.

Greenhouse choice experiments with caged cucumber beetles. Cucumber seeds (treated with PGPR strain INR-7 or nontreated) were placed into plastic pots and plants were introduced in cages at the secnd to fourth true leaf stage (cotyledon leaves were still present). PGPR strains were cultured as previously described and applied to plants as a seed treatment and root drench (100 mL containing

108 cfu/mL) before transplanting.

Experiments were done in screen cages designed in a cross arrangement with four arms (Zehnder et al., 1997b). At the start of each experiment, 100 spotted cucumber beetles were confined on E. tracheiphila-infected cucumber plants in the center 'cage within a cage' for 48 h before doors were opened to allow beetles free access to all four cage arms.

PGPR-treated cucumber plants were placed in two arms/cage, and nontreated plants in the other two arms/cage; two cages were used for each experiment (four treatment replications per experiment, eight plants per replication). Arrangement of treatments among the four cage arms was randomized for each experiment. Experiments were repeated twice. PGPR and nontreated means were compared using Student's t test.

Greenhouse no-choice experiments with caged cucumber beetles. Cucumber seeds (treated with PGPR strain 90-166 or nontreated) were placed into plastic pots as described previously and plants were introduced in screen cages at the secnd to fourth leaf stage. This experiment was designed as a no-choice experiment in which screen cages contained either PGPR-treated or nontreated plants.

Cages were 1.0 x 0.5 x 0.5 m with a top access door. Spotted cucumber beetles were released (25 per cage) and allowed to feed on three E. tracheiphila-infected cucumber plants (not treated with PGPR) placed in the center of each cage for 48 h before five healthy PGPR-treated or nontreated plants were introduced into each cage.

An experiment was conducted separately for each of three cucumber cultivars. Each cage contained one replicate treatment (PGPR or nontreated control) and treatments were replicated four times. 'Poinsett' bitter (BI; lot 88-298A) and nonbitter (Bi; lot 85-882) seed were obtained from the Dept. of Plant Breeding, Cornell University, Ithaca, N.Y.

Wilt incidence was assessed by determining the percentage of wilted leaves per plant in each cage 17 to 23 d after introduction of infected beetles to test plants. Percentage data were analyzed using t test analysis after an arcsin transformation of the square root of each

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Table 1. Mean main runner length (cm/plant) in 1993 and fruit yield (kg/plot) in 1993 and 1994 of plant growth-promoting rhizobacteria-treated, insecticide control, and nontreated cucumbers, field tests.z

Mean main

runner length/ Mean fruit

plant (cm) wt/plot (kg)

Treatment 1993 1993 1994

89B-61 62.6 aby 37.3 a NTx

90-166 64.9 a 35.9 a 28.1 a

INR-5 65.5 a 32.7 ab NT

INR-7 65.6 a 37.1 a 26.5 b

Insecticide control 58.8 bc 29.4 ab 21.9 ab

Nontreated 55.9 c 27.3 b 20.8 bc

zSeeds treated with plant growth promoting rhizobacteria before planting, sprayed weekly with esfenvalerate insecticide at the rate of 56 g (a.i.)/ha (insecticide control), or not treated with rhizobacteria or insecticide.

yMeans within columns not sharing the same letter are significantly different (P < 0.05; Newman-Keuls test). Means derived from six replications; 10 plants per replication. Plant growth data taken 17 d after transplanting.

xNT = not tested.

as evidenced by the significantly longer runners on PGPR-treated plants compared with the nontreated controls (Table 1). Cucumber yield (fruit weight per plot) was significantly greater in most PGPR treatments compared with the nontreated control in 1993 (Table 1). In 1994, yield in the 90-166 treatment was significantly (35%) greater than in the nontreated control and 30% greater than the insecticide control (not statistically significant). Yields in the 1994 INR-7 treatment were 22% to 27% greater than in the insecticide and nontreated controls, respectively (Table 1), although differences were not significant. Treatment with PGPR resulted in significantly lower numbers of cucumber beetles, and lower incidence of cucurbit wilt compared with nontreated control plants and plants sprayed weekly with the insecticide esfenvalerate (Table 2).

Greenhouse choice experiments with caged cucumber beetles. Beetle feeding damage on cotyledons was 6- and 10-fold lower on PGPR-treated plants than on nontreated plants at the end of the first and secnd experiments, respectively (Table 3). A mean of 1.1 wilted leaves per plant was recorded on nontreated plants at the end of the 1st experiment, but wilt symptoms were not observed on PGPR-treated plants (Table 3). The mean number of wilted leaves per plant at the end of the second experiment was 2.6 on nontreated plants and only 0.3 on PGPR-treated plants.

percentage (proportion).

Cucurbitacin analysis. Fresh or frozen cotyledon leaves from PGPR-treated or nontreated plants were ground in 100% A.C.S. grade acetonitrile using a 1:2 (w:v) ratio for the 'Poinsett' BI plants, and a 1:1 (w:v) ratio for the 'Straight 8' plants. 'Poinsett' BI is a 'bitter' cucumber cultivar with high cucurbitacin content and 'Straight 8' is a commercial cultivar with moderate levels of cucurbitacin. Ground plant material was kept at room temperature for 30 min, then filtered through a 0.45 mesh nylon membrane filter. The supernatant was injected directly into the HPLC using a Waters C-18 Novapak 0.8 ¥ 10-cm column. The mobile phase was 60 methanol : 40 H2O with a 1-mL·min­1 flow rate. Cucurbitacin 'C', the putative sole cucurbitacin in cucumber (Rice et al., 1981), was detected at 230 nm with a Perkin Elmer LC-85B detector. Cucurbitacin 'C' was estimated by peak height measurement and comparison with cucurbitacin 'C' standards obtained from D. Lavie, Dept. of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel. Data were analyzed using a single factor ANOVA, and treatment means were compared using the least significant difference test.

Results

Field experiments. PGPR treatment stimulated cucumber plant growth in the 1993 experiments,

Table 2. Plant growth-promoting rhizobacteria-induced resistance against cucumber beetles and bacterial wilt disease in field-grown cucumbers.

Beetles/plant Wilted vines

(mean no.) (mean %)

Treatmentz 1993 1994 1994

89B-61 0.61 dey NTx NT

90-166 0.44 e 2.34 c 2.61 c

INR-5 0.56 de NT NT

INR-7 0.73 cd 2.96 bc 3.35 bc

Insecticide control 0.89 c 3.62 b 11.48 b

Nontreated 1.73 a 5.42 a 24.55 a

zSeeds treated with plant growth promoting rhizobacteria before planting, sprayed weekly with esfenvalerate insecticide at the rate of 56 g (a.i.)/ha (insecticide control), or not treated with rhizobacteria or insecticide.

yMeans within columns followed by the same letter are not significantly different (P > 0.05; Newman-Keuls test). Beetle counts (spotted and striped cucumber beetle species combined) made from visual examination of foliage and flowers on five plants per plot, 30 plants per treatment, and averaged over four sample dates in 1993 and six sample dates in 1994. Percentage of wilted vines was determined 64 days after planting by visual examination of 10 plants per plot, 40 plants per treatment.

xNT = not tested.

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Table 3. Choice experiments. Cucumber beetle feeding damage and incidence of bacterial wilt symptoms on plant growth-promoting rhizobacteria-treated and nontreated cucumber plants.z

Cotyledon Wilted

damage leaves/plant

(mean %)y (mean no.)

Treatment Expt. 1 Expt. 2 Expt. 1 Expt. 2

INR-7 6.3 ax 1.9 a 0.0 a 0.3 a

Nontreated 35.5 b 19.0 b 1.1 b 2.6 b

zSeeds treated with plant growth promoting rhizobacteria prior to planting.

yValues are the mean percentage of cotyledon leaf area per plant with feeding damage, estimated by visual assessment. Each of the two cotyledons per plant represented one-half of the total plant cotyledon leaf area.

xMeans within columns not sharing the same letter are significantly different (P < 0.05; Student's t test).

We were able to more closely observe PGPR effects on cucumber beetle feeding and spread of cucurbit wilt in the greenhouse experiments. The dramatic differences in beetle feeding damage and spread of cucurbit wilt between PGPR-treated and nontreated plants in both the choice and no-choice experiments led us to hypothesize that PGPR may induce a physiological change in plants that can be detected by cucumber beetles. We suspected that cucurbitacin was involved because of its strong influence on cucumber beetle feeding behavior. The HPLC analysis demonstrated that cucurbitacin levels were reduced as much as 44% on PGPR-treated plants compared with nontreated plants. Because of the strong correlation that has been established between cucurbitacin concentration in plant tissue and the extent of feeding and aggregation by cucumber beetles (Ferguson et al., 1983; Metcalf et al., 1982), it is conceivable that beetle feeding behavior in our experiments was influenced by the induced decrease in cucurbitacin.

A plausible mechanism for the effect on cucurbitacin may involve a shift in the plant metabolic pathway to produce other plant defense compounds. Such a change in metabolic pathway was observed in potato where fatty acid elicitors from Phytophthora infestans elicited the accumulation of sesquiterpenoid defense compounds (Tjamos and Kuc, 1982). This increase was associated with an induced shift in the terpenoid pathway which lead to reduced production of steroid glycoalkaloids that are used by the fungus. It has been established that the mechanism for cucurbitacin 'C' biosynthesis involves a metabolic

Greenhouse no-choice experiments with caged cucumber beetles. The mean percentage of wilted leaves from plants representing the 3 cucumber cultivars ranged from 52.8% to 85.3% on the nontreated plants, but only from 7.6% to 13.1% on the PGPR-treated plants (Table 4). Cultivar did not appear to greatly influence the difference in mean wilt values between PGPR and nontreated plants, although the difference was least in the experiment with 'Poinsett' BI.

Cucurbitacin analysis. The concentrations of cucurbitacin 'C' in cotyledons of 'Poinsett' BI and 'Straight 8' cucumber were significantly lower in PGPR-treated plants compared with nontreated plants (Table 5). 'Straight 8' exhibited the greatest reduction; an mean of 44% less cucurbitacin in INR-7-treated plants compared with nontreated plants.

Discussion

Results of field experiments demonstrated that PGPR treatment suppressed cucumber beetle populations and incidence of bacterial wilt, and resulted in higher cucumber yields, compared with nontreated plants. Because PGPR-treated plants exhibited faster growth than nontreated plants, it is not clear if the yield increase in the PGPR treatments is a result of growth promotion, biological disease and insect control, or a combination of both. The field results also indicate that, given our experimental conditions, the PGPR treatments were more effective than insecticides for control of cucumber beetle populations and incidence of cucurbit wilt.

Table 4. No choice experiments: mean percentage of wilted leaves/plant on plant growth-promoting rhizobacteria-treated or nontreated 'Poinsett' bitter (BI), nonbitter (bi) and 'Straight 8' cucumbers.z

Wilted leaves/plantz

(mean %)

Treatmenty Poinsett BI Poinsett bi Straight 8

90-166 13.1 ax 9.1 a 7.6 a

Nontreated 52.8 b 85.8 b 85.3 b

zSpotted cucumber beetles infected with E. tracheiphila were released into cages with either PGPR-treated or nontreated plants and allowed to feed for 17 to 23 d when the percentage of wilted leaves was determined.

ySeeds treated with plant growth promoting rhizobacteria before planting.

xMeans within columns not sharing the same letter are significantly different (P < 0.05; Student's t test).

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Table 5. Mean cucurbitacin C concentration in plant growth-promoting rhizobacteria-treated or nontreated 'Poinsett' and 'Straight 8' cucumbers.z

Mean cucurbitacin C

concn (mg·g­1)y

Treatment Poinsett BI Straight 8

INR-7 117.3 bx 27.1 c

INR-5 117.9 b 35.2 bc

Nontreated 158.6 a 48.4 a

zSeeds treated with plant growth-promoting rhizobacteria before planting.

yTwo cotyledons from one plant/replication for 'Poinsett', and four cotyledons from two plants/replication for 'Straight 8'.

xMeans within columns not sharing the same letter are significantly different (P < 0.05; lsd). Cucurbitacin 'C' values are mg cucurbitacin/g dry weight plant material.

shelf life would be required of a PGPR-based commercial product, it will most likely be necessary to use PGPR spore preparations in any commercial formulation. Commercial interest in PGPR is, therefore, focused on spore-forming bacteria. The technology for development of commercial PGPR-based seed treatment formulations exists, as evidenced by the commercial product Kodiak, a Bacillus subtilus strain produced by Gustafson, (Plano, Texas). We are currently working in cooperation with Gustafson to evaluate PGPR spore formulations as a PGPR-based cucurbit seed treatment. Preliminary results of recent field trials indicate that the commercially prepared PGPR formulations are effective for ISR against cucurbit wilt and other foliar diseases of cucurbits. We are hopeful that PGPR-based cucurbit seed treatments and PGPR inoculants designed for other vegetable crops will be commercially available in the near future.

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