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Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato
Biological Control 18, 208 –215 (2000) doi:10.1006/bcon.2000.0837, available online at http://www.idealibrary.com on
Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato Pajand Nejad 1 and Paul A. Johnson Plant Pathology and Biocontrol Unit, SLU, P.O. Box 7035, SE-750 07 Uppsala, Sweden Received April 25, 1999; accepted March 17, 2000
To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively. ©
2000 Academic Press
Key Words: Brassica napus; Lycopersicon lycopercicum; Pseudomonas spp.; Serratia spp.; Verticillium dahliae; Fusarium oxysporum; biological control; tomato; oilseed rape.
INTRODUCTION
Reports on nonsymbiotic nitrogen fixation by Acetobacter spp. in sugarcane, Herbaspirillum spp. in rice, and Azospirillum spp. in cereals (Do¨bereiner, 1988, 1992; Do¨bereiner et al., 1993; Cavalcante and Do¨bereiner, 1988; App et al., 1980; Gillis et al., 1989; Boddey et al., 1991; Urquiaga et al., 1992; Baldani and Do¨bereiner, 1980; Baldani et al., 1986) have stimulated appreciation for the importance of endophytic bacteria in plant production and crop protection (Hollis, 1951; Poon et al., 1977; Mahaffee and Kloepper, 1994; Misaghi and Donndelinger, 1990; Sturz and Christie, 1 Current address: Liggargatan 3C 75420, Uppsala, Sweden. Email: [email protected].
1049-9644/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
1995; Pleban et al., 1995). Other examples include the work of Kempe and Sequira (1983), which showed that two endophytic bacteria were able to suppress development of bacterial wilt in potato, and the work of Mahaffee and Kloepper (1994), which provided further evidence that bacterial residents of plant tissues can be involved in the general defense response in plants such as cotton, cucumber, and potatoes. Against plant pathogenic fungi on different agar media and in potato, in pilot experiments, we observed high growth-suppressing activities of selected endophytically isolated bacteria. The results suggested to us that certain of our plant-inhabiting isolates may have a considerable capacity for suppressing plant pathogens, not only those pathogens interacting with their respective host plants but also pathogens interacting with other plants. Should this be the case, certain endophytically isolated bacteria may play significant roles in plant–pathogen interactions or types of such interactions. This idea is consistent with the finding that endophytically resident bacteria are known to be inhabitants of several plant species (Trevet and Hollis, 1948; Hollis, 1951; Mundt and Hinkle, 1976; Cameron, 1970). However, the extent of their involvement in plant growth promotion, control of vascular pathogens, and control of development of systemic infection caused by such pathogens remains unclear. Nevertheless, our increasing understanding of the contributions of endophytically resident bacteria to plant–pathogen interactions makes biological control by means of endophytes an attractive possibility, especially as an alternative to control of vascular wilt diseases by fungicidal compounds, whose efficacy has proven to be unsatisfactory, particularly for Verticillium dahliae Kleb. and Fusarium oxysporum f. sp. lycopersici. However, few studies so far have actually shown that endophytic bacteria are able to significantly control vascular wilt diseases. Mahaffee and Kloepper (1994) have shown that biological control by endophytic bacteria is possible and can involve induced resistance to soilborne pathogens.
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Also, inoculation with some bacterial endophytes has been demonstrated to reduce disease incidence and symptoms of F. oxysporum in cotton (Chen et al., 1995) and V. dahliae in oilseed rape (Nejad, 1995). In addition, work is in progress for utilizing transformed forms of a corn xylem-colonizing endophyte, Clavibacter xyli subsp. cynodontis, to control the European corn borer (Kostka et al., 1988; Dimock et al., 1989). The overall objective of this study was to begin exploring the possibility that some species of endophytic bacteria can, for more than one plant species, contribute fundamentally not only to the suppression of soilborne diseases but also to the enhancement of plant growth, where enhancement of plant growth includes not only suppression of disease but also growth promotion that is infection independent and therefore occurs even if a plant is entirely uninfected by pathogens. Among the crucifers, oilseed rape in particular suffers throughout the world from disease caused by wilt pathogens, such as V. dahliae. This fact, and the importance of oilseed rape in the world’s agricultural economy, its multiple uses, its global crop production, and its genetic potential (Nejad, 1994), suggests that this plant is appropriate for case studies of biological control of wilt diseases by bacterial endophytes. The tomato plant also suffers seriously from disease caused by wilt pathogens, particularly the pathogen F. oxysporum. With this in mind, our specific objective was to find, within oilseed rape plants, endophytic bacterial isolates that can provide significant biological control of soilborne diseases not only for oilseed rape but also for tomato and that can enhance plant growth for both of these plant species. MATERIALS AND METHODS
Bacterial Isolation Healthy looking young oilseed rape plants, wild and cultivated, growing in fields near Uppsala, were collected for bacterial isolation. The bacteria were isolated from the internal tissues of roots and stems, which were individually disinfected by soaking each in 5.25% sodium hypochlorite and then adding 1 ml of Tween 20 (polyoxyethylene sorbitan monolaurate) for 10 –15 min, depending on the tissue, followed by immersion in 70% ethanol for 1–2 min. They were then rinsed three times in sterile distilled water (SDW) to clear them of alcohol before obtaining bacterial isolates by two different techniques from the internal tissues of their surface-disinfected roots and stems. Trituration. This technique allows endophytic bacteria to be selectively isolated from vascular tissue in consistently high numbers (Hallmann et al., 1997). Since one key to success in isolating and studying
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endophytic bacteria is surface sterility (Hallmann et al., 1997), sterility checks were carried out for each sample to monitor the efficiency of the disinfestation procedure. For these checks, either 0.1 ml of the last wash was transferred to Tryptic soy agar (TSA) or, alternatively, 0.1 ml of the final wash was transferred to 9.9 ml Tryptic soy broth (TSB), incubated at room temperature on a shaker, and spread onto a TSA agar plate. After 48 h, if no bacterial growth occurred in the sterility check, the recovered bacteria were considered to be endophytes. The tissue (5 g fresh weight) was then macerated in 100 ml phosphate-buffered saline solution (PBS, per liter: 8 NaCl, 0.2 KH 2PO 4, 1.1 Na 2HPO 4, and 0.2 KCl, pH 7.4) with mortar and pestle under aseptic conditions. After filtering the slurry through sterile cotton cloth, the filtrate was diluted appropriately, plated onto a sterile enrichment medium of Rovira (1956) with some modifications (The medium used contained, g liter ⫺1 distilled water: 2 K 2HPO 4, 2.5 (NH 4) 2HPO 4, 0.25 MgSO 4 䡠 7 H 2O, 0.5 NaCl, 0.5 CaCl 2, 0.05 FeCl 3, 0.5 proteose peptone, 0.5 yeast extract, 10 glucose, 15 Difco bactoagar), and left for incubation at room temperature (20 ⫾ 2°C) until the growth of colonies became visible. Imprinting of plant tissue. The disinfecting procedure for this method of isolation was similar to that of the trituration method, with the following differences. Stems, 2–3 cm long, were excised from plants, surface disinfested, and dried with moistened paper (SDW). With a flamed scalpel, stems were cut longitudinally into two pieces. The pieces were aseptically transferred and pressed onto three different concentrations of TSA within the agar (TSA 10, TSA 30, and TSA 50), left for 1 h, and then removed. (Here TSA 10, for example, contains 10 g Tryptic Soy Broth (Difco Ltd.) and 12 g Technical Agar (Oxoid Ltd.) in 1000 ml distilled water; TSA 30 contains 30 g.) The agar plates were incubated at either of two temperature ranges, 5–10°C or 10 – 15°C, to obtain a spectrum of bacteria with different temperature preferences. Bacterial colonies that appeared frequently and looked morphologically different were randomly selected for further study. After each isolate had been recultured and checked for purity, one loopful was suspended in sterile tap water containing 20% glycerol (pH 7) and then frozen at ⫺80°C for long-term storage. For short-term storage, the isolate cultures were freeze-dried at 4°C. Before use in tests and experiments, stored cultures were given a day to acclimate to experimental conditions. For those isolates that both promoted plant growth and reduced incidence of wilt diseases in gnotobiotic and greenhouse experiments, some preliminary identification was carried out using fatty acid methyl ester analysis. We found that isolates PN and PP closely resemble frequently occurring plant-associated genera,
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such as Pseudomonas and Serratia (Mahaffee and Kloepper, 1994). Fungal Pathogens The pathogens V. dahliae and F. oxysporum in our studies were isolated at the Plant Pathology Department of SLU from naturally infected oilseed rape and tomato plants from southern Sweden. Before proceeding with bioassays, the pathogenicity was confirmed for V. dahliae and F. oxysporum in the test cultivars Casino and Dansk export, respectively. This was done by culturing the fungus on Potato Dextrose Agar (PDA) at room temperature for about 2 weeks and blending 41 plate per plant with sterile tap water in an electric mixer. The resulting fungal suspension was applied as a soil treatment and as a seed treatment to 2-week-old oilseed rape plants and 3-week-old tomato plants. Typical symptoms could be observed 2–3 weeks after soil treatment and 5 weeks after seed treatment. For further confirmation, infected leaves were removed 40 – 45 days after infection and placed onto filter paper moistened with SDW in large petri dishes with glass lids. After incubation, microsclerotia and conidia of the pathogens that appeared were identified under the light microscope. In Vitro Screenings Effects on seed germination. One hundred sixty isolates were tested for seed emergence on Brassica napus seeds by placing them onto sterile filter paper moistened with SDW in petri plates (two plates with 20 seeds/plate). Seeds were treated with bacterial suspensions (20 l/seed) and left to incubate at room temperature. Control plates were arranged in a similar way, except that they were treated with SDW only. For each isolate, effects on seed germination were measured by counting the number of fully germinated seeds per plate and comparing that with that of the control plates. After 4 days, we measured seedling length for each plate to obtain an average, which was compared with that of the control plates. Effects on fungal mycelial growth. Bacterial isolates that exhibited clearly measurable effects on seed germination and seedling growth (stem and root) were then tested in vitro on agar media against fungi. Deepfrozen cultures of bacteria were revived on King’s medium B agar (KBA; King et al., 1954) and recultured on KBA after checking purity. Disks of fresh culture of the fungus (5 mm diameter) were cut out and placed in the center of a 9-cm petri plate with PDA. Also, to determine whether antimicrobial activity is substrate dependent, we included three other agar media (cornmeal agar, CMA, malt extract agar, MEA, green bean agar, GBA). Bacterial cultures were inoculated in either of two ways: a bacterial suspension was streaked in two parallel strips each 2 cm long on either side of the
fungal agar disc or the suspension was seeded as spots, one spot at each of the four corners of the plate. Fungal growth for individual and dual bacterial cultures was compared with fungal growth in control plates with the fungus only. This experiment was repeated to check the consistency of the results. Production of volatile metabolite. Using a modification of the procedure of Millar and Higgins (1970), we assayed the amount of HCN produced by bacteria grown on KBA with or without glycine (4.4 g 䡠 L ⫺1). In the lid of each petri dish, we placed filter paper strips soaked in picric acid solution (2.5 g of picric acid, 12.5 g of Na 2CO 3, and 1 L of water). The dishes were then sealed with Parafilm. After incubation at 28°C for 48 h, the presence of a colored zone around the bacteria indicated the production of HCN. For each of the endophytic strains, reactions were scored as weak (yellow to light brown), moderate (brown), or strong (reddish brown). Also, to determine whether the strains produce a volatile metabolite other than HCN, we grew the bacteria on KBA and the fungi on PDA. When both the bacteria and the fungi had visible growth, we placed the bacterial agar plate on top (inverted) of the fungal agar plate and sealed the plates with Parafilm to avoid contamination and to keep gases from escaping. If the region of fungal infection is found to be limited compared to that of the control in such an experiment, then, presumably, the only cause can be the production of volatile metabolites by the endophytic bacteria. Greenhouse Screenings of Bacterial Effects First screening. For evaluating plant growth promotion, 302 bacterial isolates were tested on oilseed rape. Of these isolates, we selected 144 based upon their superior plant growth promotion and disease suppression, which were then tested on tomato. For these screenings, seeds were sown in 10-cm pots filled with commercial soil (“Hasselfors” containing g 䡠 m ⫺3: 180 N, 210 K, 250 Mg, 9.0 Fe, 2.0 Cu, 0.7 Mo, 75 P, 2500 Ca, 120 S, 5.0 Mn, 0.8 B, and 2.0 Zn; sand mixture in volume ratio ⫽ 4:1). After 1 week, the pots were thinned to one plant per pot and watered regularly. The pots were placed in a greenhouse with controlled temperatures. The experiments described here and in the following two sections were completely randomized designs with 10 replications (one pot each) per treatment. Day/night temperatures were, respectively, 22 ⫾ 2 and 20 ⫾ 2°C for oilseed rape plants and 26 ⫾ 2 and 24 ⫾ 2°C for tomato. A photoperiod of 16 h and a relative humidity within the range of 70 – 80% was maintained. When needed, Phillips HPI-T mercury halogen lamps (400 W) provided additional light. Seven to 10 days after bacterial soil inoculations, a fertilizer (Superba S liquid fertilizer, 6.5% N, 1.0% P,
BIOCONTROL OF WILT DISEASES USING ENDOPHYTIC BACTERIA
4.7% K plus microelements; Supra Ceres, Landskrona, Sweden) was provided regularly twice a week. To provide uniform exposure to light and temperature, twice a week experimental pots were rotated on the bench. For further avoidance of the effects of environmental variation between plants, extra plants (untreated) were set out on the margins of the experimental plants. The above screenings resulted in a selection of 21 bacterial isolates for further evaluation of plant growth promotion and disease suppression using soil and seed applications. They were chosen for their generally good performance in suppression of disease and promotion of plant growth. Soil application. The tested bacterial isolates were individually cultured from deep-frozen vials on King’s medium B agar at 20°C for 24 – 48 h. A single colony of each bacterial isolate was then cultured in a 250-ml Erlenmeyer flask containing 100 ml TSB and incubated on a shaker at room temperature for 74 h at 120 rpm. Each broth culture was diluted with TSB to 200 ml before being applied as a soil drench for each 2- to 3-week-old oilseed rape plant (25 ml per plant). To measure plant growth effects, we compared leaf surface parameters (i.e., length and width of fourth and fifth leaves) or shoot dry weight of isolate-treated plants against control plants. Shoot dry weight was recorded after air-drying in an oven at 80°C overnight. Of the 21 isolates selected for further evaluation, 15 of the better performers were chosen for the experiments reported in this paper. These bacterial strains were inoculated either into the soil or as a seed treatment. For soil treatment, the 2- to 3-week-old bacterial-treated seedlings were removed from pots and transplanted into individual pots (12.5 ⫻ 14.5 cm) containing three layers. Each plant was transferred with all soil attached and adhering to its roots. The bottom layer (one-third) of each pot consisted of greenhouse soil. The second layer (one-third) consisted either of V. dahliae-infested soil or F. oxysporum-infested soil (41 of the fully grown fungi on an agar plate). The surface layer (one-third) consisted again of the greenhouse soil. Healthy plants (treated with either SDW or TSB) and diseased plants (treated with either V. dahliae or F. oxysporum only) served as controls. The plants were not watered on the day of treatment. Seed application. Two mixed cultures were included in all seed treatment experiments. The four isolates in these cultures, PF, PK, PH, and PN, were arbitrarily chosen. The isolates were cultured from deep-frozen isolates on KBA at room temperature for 48 h. For each cultured isolate, one loopful of bacterial lawn from the agar surface was scraped and then transferred to a test tube containing 20 ml of sterile water. For all isolates, suspensions were prepared identically (final concentration approx 10 9 colony-forming units/ml). For the mixed cultures, 10 ml of one
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culture was combined with 10 ml of another. Then, the bacterial suspension of each individual culture and each mixed culture was poured into a container with seeds of either tomato or oilseed rape. After 20 min of soaking, the content of each container was decanted and the seeds were dried under a fan for about 14 h. Seeds treated with SDW or TSB were used as controls. The bacteria-treated oilseed rape and tomato seeds, after drying, were sown in pathogen-contaminated soils as described above. Each experiment was continued for 10 –15 weeks. The plants were observed regularly for visible disease symptoms. For oilseed rape, disease symptom appearance for each leaf was scored by estimating its area of symptom appearance based upon a disease rating scale that ranged from 0 (no suppression) to 1.0 (complete suppression). Healthy control plants generally had 15 leaves. Maximum symptoms were assumed to exist for a plant when 12 of its leaves had succumbed to the disease, inasmuch as such plants were effectively dead. Therefore, the symptom appearance for a plant was the number of diseased leaves divided by 12. For all control diseased plants (untreated with bacteria), symptom appearance was 1.0. Hence, 1.0 minus symptom appearance is an appropriate index of disease suppression. For tomato, we measured both symptom incidence and plant fresh weight. Finding that the two parameters were strongly correlated, we chose fresh weight as the index of symptom appearance. We computed mean disease suppression for tomato using the formula S ⫽ (T ⫺ D)/(H ⫺ D), where S is an index of disease suppression, T is the mean fresh weight (g) of treated plants, H is the mean fresh weight of healthy plants, and D is the mean fresh weight of control diseased plants. Here, disease suppression is the ability of an endophytic bacteria to suppress the effect of a disease on the fresh weight, which is a realistic index of disease suppression when, as in the case of this experiment, H is significantly greater than D and symptom incidence and plant fresh weight are strongly correlated. The above formula for disease suppression also takes into account the possibility that an endophytic bacteria can enhance plant growth (mean fresh weight) by means beyond that of disease suppression. Only when such is the case, as was true for treatment with some of our bacteria, can S take values greater than 1. In cases in which S is greater than 1, disease suppression is complete, and the value S ⫺ 1 is an index of the extent to which treatment with the bacteria can promote plant growth independent of the occurrence of disease. For each experiment that evaluated the performance of bacterial isolates, a one-tail, two-sample t test was used to obtain a confidence interval for testing the hypothesis that at the time of evaluation the experimental value of a treatment (i.e., plants treated with a given isolate) was greater than that for the control.
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TABLE 1 Plant Growth Promotion of Oilseed Rape by Endophytes— Length and Width of Fourth and Fifth Leaves after Bacterial Inoculation of Nonsterile Greenhouse Soil in the Absence of Pathogens Fourth leaf
Fifth leaf
Treatment
Aver. length (cm)
Aver. width (cm)
Aver. length (cm)
Aver. width (cm)
Isolate PP PN PO PV TSB control Water control
19.75* 19.3* 17.45 17.4 14.85 14.3
15.3* 14.57* 13.4 13.1 11.5 11.1
21.75* 20.6* 20.0* 19.8* 16.15 15.45
14.85* 13.8* 13.9* 13.3 11.25 10.55
* Significantly different from both controls at level 0.01.
RESULTS
In Vitro Screenings Effects on seed germination. From experiments using gnotobiotic systems, we found for oilseed rape that some of our bacterial isolates had no apparent effects, whereas others, when applied individually, caused not only suppression of seed germination (in vitro) and seedling growth but also root necrosis. Yet, other isolates had a strong potential for promoting seedling growth (stem and root). Hence, growth-regulating substances might have been involved. Effects on fungal mycelial growth. Isolates used in gnotobiotic assays were tested in vitro for inhibition of mycelium growth of fungi. Some of the isolates also suppressed mycelium growth of F. oxysporum (e.g., PL and PP). For V. dahliae, approximately 41% of the isolates exhibited strong inhibition, 33% moderate inhibition, 15% weak inhibition, and 11% no inhibition. For both pathogens, these results varied when tested on different agar media. Hence, antimicrobial activity appeared to be substrate dependent. However, for V. dahliae, some isolates, such as PK, PM, and PP, showed strong activity on more than one medium, and isolate PP was growth suppressive on all nutrient media. Production of volatile metabolite. Performance of selected endophytic bacteria in production of volatile metabolite was characterized as follows. Following 48 h incubation, most isolates were HCN negative. On KBA, with or without glycine, only the isolate PC showed detectable cyanide production by changing color from yellow to brown around its colonies. Consistent with this result, our experiments concerning volatile metabolites indicate that most isolates that were effective against wilt pathogens in oilseed rape and
tomato plants produced fungal-inhibitory volatile metabolites other than hydrogen cyanide. Greenhouse Screenings of Bacterial Effects Soil application. From experiments with soil treatment, isolates PP and PN expressed their promotion of plant growth for oilseed rape through significant enhancement of leaf size within a week after inoculation (Table 1). The experimental increases in leaf size for test plants treated with PP and PN over those treated with the TSB control were 33.3 and 27.0%, respectively. Compared to the water control, the respective increases were 39.4 and 32.8%. Significant increases in leaf size by these two isolates and isolate PO compared to controls continued after 2 weeks. Also, after inoculation of the test cultivar, significant increases in shoot dry weight occurred for all isolates included in Table 1. In another soil treatment experiment (Fig. 1), using the same isolates, the shoot dry weight and plant length were significantly increased by bacterial treatment in greenhouse soil, both in the presence and in the absence of V. dahliae. Seed application. Over several weeks under nonsterile conditions, all individual and dual-isolate cultures shown in Fig. 2 significantly suppressed and delayed symptoms of disease in oilseed rape caused by V. dahliae. In Fig. 2, the mean disease suppression for all control plants treated with broth or SDW on the 100th day of final observation showed severe disease symptoms in at least 12 true leaves. In contrast, symptoms for the isolates PX, PF, and PA hardly appeared on the 5th true leaf. As in oilseed rape, in tomato all tested isolates significantly suppressed and delayed symptoms caused by F. oxysporum (Fig. 3). All bacterial isolates included in Fig. 4 increased shoot dry weight for tomato and oilseed rape compared to control plants. On both tomato and oilseed rape, all isolates not only increased shoot dry weight but also
FIG. 1. Plant growth promotion of Brassica napus by endophytes—shoot dry weight after bacterial inoculation of plant roots in nonsterile greenhouse soil in the presence and absence of Verticillium dahliae. Asterisks denote significance at P ⫽ 0.05.
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FIG. 2. Effects of endophytic bacteria on incidence of wilt disease caused by Verticillium dahliae in oilseed rape. Bars indicate 99% confidence intervals. P ⱕ 0.001.
increased plant height. Expansion of leaf size was also observed. The two dual-isolate cultures performed no better on average than the other isolates for both tested plants. All bacterial isolates included in Fig. 5 allowed the plants to significantly increase shoot fresh weight compared to control diseased plants at the end of the experimental period of 100 days. In fact, isolates PF, PA, and PN yielded fresh weight that was greater than that of untreated healthy plants. Treatment by coinoculation of endophytic isolates (mixed inoculum) resulted not only in suppressed disease symptoms (Fig. 3) but also in increased shoot fresh weight (Fig. 5). However, for both attributes, those isolates were less effective than isolates that were applied individually.
FIG. 4. Plant growth promotion in terms of shoot dry weight of (A) tomato and (B) oilseed rape under greenhouse conditions after seed bacteria treatment with bacterial isolates in the absence of pathogens. Bars indicate 99% confidence intervals. P ⱕ 0.001.
The specific objective of this study was to find naturally occurring endophytic bacterial isolates within the tissues of a variety of plants and to rank them with respect to their capacities to (a) enhance the growth of oilseed rape and tomato plants when uninfected with wilt pathogens and (b) provide these plants with bio-
logical control against disease when infected. To our knowledge, this paper is the first to report finding such endophytic bacteria, which significantly improve growth and development of oilseed rape and tomato and control their soilborne diseases: V. dahliae in oilseed rape and F. oxysporum f. sp. lycopersici. in tomato. Two of our endophytes, PA and PF, achieved such control, and several others reduced disease incidence and severity of the two pathogens by at least 75%. Some endophytes also resulted in significant increase in shoot dry/fresh weight, plant height, and leaf size in
FIG. 3. Effects of endophytic bacteria on incidence of Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici in tomato. Bars indicate 99% confidence intervals. P ⱕ 0.001.
FIG. 5. Effects of endophytic bacterial isolates on shoot fresh weight of tomato in the presence of Fusarium oxysporum f. sp. lycopersici. Bars indicate 99% confidence intervals. P ⱕ 0.001.
DISCUSSION
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both the presence and the absence of the pathogen in the soil. However, although this was true for oilseed rape and tomato in our greenhouse experiments, the performance of our isolates for different cultivars under various field conditions has yet to be investigated. Also, the observed effects of the endophytes may have largely been circumstantial. The effects may have been beneficial only as a consequence of (i) the particular microflora to which the experimental plants had been exposed before bacterial inoculation, (ii) the stage at which the plants were inoculated, and (iii) the dosage (inoculum concentration). However, the dosage was not so high as to eliminate or mask isolate-dependent effects. Our experiments were not intended to clarify specific mechanisms by which the endophytes promoted growth and protected the host from the diseases caused by V. dahliae and F. oxysporum. We did, however, find that most of our effective isolates produced microbialinhibitory volatiles other than hydrogen cyanide, albeit that HCN may well be a possible and perhaps frequent mechanism by which bacteria suppress plant pathogens (Ross and Ryder, 1994; Maurhofer et al., 1994). From a more general perspective, a basic reason for finding our bacterial isolates effective in suppressing soilborne diseases caused by Verticillium and Fusarium in oilseed rape and tomato, respectively, might be that they were isolated from within vascular tissue (conductive tissue). In accord with the ideas of Hallmann et al. (1997), they may well follow the same path as soilborne pathogens to colonize the vascular tissue of a plant. In other words, endophytically resident bacteria may be strategically at the right place and at the right time for suppression of vascular wilt diseases, not only with regard to antagonistic effects, such as antibiosis and competition for nutrients and infection sites, but also with regard to optimal timing of, for example, induced resistance. Further, because endophytically resident bacteria probably have the unique ability to survive inside plants with little or no microbial competition, they may provide the plant with reliable suppression of vascular diseases (Misaghi and Donndelinger, 1990). By mutually enhancing one another’s fitness values, the endophytic bacterium and its host plant can coevolve in a manner that leads to intimate and effective means of communication and cooperation in combating and suppressing vascular (wilt) diseases. Finally, microfloral populations already resident within the host plant may well influence and be influenced by rhizosphere bacteria. Hence, Sturz and Christie (1995) have proposed that endophytically competent microflora, such as endophytically resident bacteria, may mediate the establishment of biocontrol agents within the rhizosphere.
ACKNOWLEDGMENTS The authors thank Professor B. Gerhardson for supervision and critical comments on the manuscript. The study is part of the Microbial Antagonism against Fungi program, financed by the Foundation for Strategic Environmental Research (MISTRA).
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BIOCONTROL OF WILT DISEASES USING ENDOPHYTIC BACTERIA cynodontis containing a delta-endotoxic gene from Bacillus thuringensis subsp. kurstaki (BT). Phytopathology 78, 1540. [Abstract] Mahaffee, W. F., and Kloepper, J. W. 1994. Applications of plant growth-promoting Rhizobacteria in sustainable agriculture. In “Soil Biota—Management in Sustainable Farming Systems” (C. E. Pankhurst, B. M. Double, V. V. S. R. Gupta, and P. R. Grace, Eds.), pp. 23–31. CSIRO Adelaide, Australia. Maurhofer, M., Sacherer, P., Keel, C., Haas, D., and De`fago, G. 1994. Role of some metabolites produced by Pseudomonas fluorescens strain CHA0 in the suppression of different plant diseases. In “Improving Plant Productivity with Rhizosphere Bacteria” (M. H. Ryder, P. M. Stephens, and G. D. Bowen, Eds.), pp. 117–119. Proc. of IIIrd Int. Workshop on Plant Growth-Promoting Rhizobacteria, March 7–11, 1994, CSIRO Adelaide, Australia. Millar, R. L., and Higgins, V. J. 1970. Association of cyanide with infection of birdsfoot trefoil by Stemphylium loti. Phytopathology 60, 104 –110. Misaghi, I. J., and Donndelinger, C. R. 1990. Endophytic bacteria in symptom-free cotton plants. Phytopathology 80, 808 – 811. Mundt, J. O., and Hinkle, N. F. 1976. Bacteria within ovules and seeds. Appl. Environ. Microbiol. 32, 694 – 698. Nejad, P. 1994. “Global Agricultural Crop Production under Different Environments—Genetic Potential and Level of Sustainability.” Masters thesis, Swedish University of Agricultural Sciences, Department of Crop Production Science. Uppsala, Sweden.
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Nejad, P. 1995. “Potential of Rape Bacteria to Influence Brassica napus L. and Control of Its Wilt Pathogen, Verticillium dahliae.” Masters thesis, Swedish University of Agricultural Sciences. Department of Plant Pathology. Uppsala, Sweden. Pleban, S., Ingel, F., and Chet, I. 1995. Control of Rhizoctonia solani and Sclerotium rolfsii in the greenhouse using endophytic Bacillus spp. Eur. J. Plant Pathol. 101, 665– 672. Poon, E. S., Huang, T. C., and Kuo, T. T. 1977. Possible mechanism of symptom inhibition of bacterial blight of rice by an endophytic bacterium isolated from rice. Bot. Bull. Acad. Sinica 18, 61–70. Ross, I. L., and Ryder, M. H. 1994. Hydrogen cyanide production by a biocontrol strain of Pseudomonas corrugata: Evidence that cyanide antagonises the take-all fungus in vitro. In “Improving Plant Productivity with Rhizosphere Bacteria” (M. H. Ryder, P. M. Stephens, and G. D. Bowen, Eds.), pp. 131–133. Proc. of IIIrd Int. Workshop on Plant Growth-Promoting Rhizobacteria, March 7–11, 1994. CSIRO Adelaide, Australia. Sturz, A. V., and Christie, B. R. 1995. Endophytic bacterial systems governing red clover growth and development. Ann. Appl. Biol. 126, 285–290. Trevet, I. W., and Hollis, J. P. 1948. Bacteria in the storage organs of healthy plants. Phytopathology 38, 960 –967. Urquiaga, S., Cruz, K. H. S., and Boddey, R. M. 1992. Contribution of nitrogen fixation to sugar cane: Nitrogen-15 and nitrogen balance estimates. Soil Sci. Soc. Am. Proc. 56, 105–114.
Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato Pajand Nejad 1 and Paul A. Johnson Plant Pathology and Biocontrol Unit, SLU, P.O. Box 7035, SE-750 07 Uppsala, Sweden Received April 25, 1999; accepted March 17, 2000
To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively. ©
2000 Academic Press
Key Words: Brassica napus; Lycopersicon lycopercicum; Pseudomonas spp.; Serratia spp.; Verticillium dahliae; Fusarium oxysporum; biological control; tomato; oilseed rape.
INTRODUCTION
Reports on nonsymbiotic nitrogen fixation by Acetobacter spp. in sugarcane, Herbaspirillum spp. in rice, and Azospirillum spp. in cereals (Do¨bereiner, 1988, 1992; Do¨bereiner et al., 1993; Cavalcante and Do¨bereiner, 1988; App et al., 1980; Gillis et al., 1989; Boddey et al., 1991; Urquiaga et al., 1992; Baldani and Do¨bereiner, 1980; Baldani et al., 1986) have stimulated appreciation for the importance of endophytic bacteria in plant production and crop protection (Hollis, 1951; Poon et al., 1977; Mahaffee and Kloepper, 1994; Misaghi and Donndelinger, 1990; Sturz and Christie, 1 Current address: Liggargatan 3C 75420, Uppsala, Sweden. Email: [email protected].
1049-9644/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
1995; Pleban et al., 1995). Other examples include the work of Kempe and Sequira (1983), which showed that two endophytic bacteria were able to suppress development of bacterial wilt in potato, and the work of Mahaffee and Kloepper (1994), which provided further evidence that bacterial residents of plant tissues can be involved in the general defense response in plants such as cotton, cucumber, and potatoes. Against plant pathogenic fungi on different agar media and in potato, in pilot experiments, we observed high growth-suppressing activities of selected endophytically isolated bacteria. The results suggested to us that certain of our plant-inhabiting isolates may have a considerable capacity for suppressing plant pathogens, not only those pathogens interacting with their respective host plants but also pathogens interacting with other plants. Should this be the case, certain endophytically isolated bacteria may play significant roles in plant–pathogen interactions or types of such interactions. This idea is consistent with the finding that endophytically resident bacteria are known to be inhabitants of several plant species (Trevet and Hollis, 1948; Hollis, 1951; Mundt and Hinkle, 1976; Cameron, 1970). However, the extent of their involvement in plant growth promotion, control of vascular pathogens, and control of development of systemic infection caused by such pathogens remains unclear. Nevertheless, our increasing understanding of the contributions of endophytically resident bacteria to plant–pathogen interactions makes biological control by means of endophytes an attractive possibility, especially as an alternative to control of vascular wilt diseases by fungicidal compounds, whose efficacy has proven to be unsatisfactory, particularly for Verticillium dahliae Kleb. and Fusarium oxysporum f. sp. lycopersici. However, few studies so far have actually shown that endophytic bacteria are able to significantly control vascular wilt diseases. Mahaffee and Kloepper (1994) have shown that biological control by endophytic bacteria is possible and can involve induced resistance to soilborne pathogens.
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Also, inoculation with some bacterial endophytes has been demonstrated to reduce disease incidence and symptoms of F. oxysporum in cotton (Chen et al., 1995) and V. dahliae in oilseed rape (Nejad, 1995). In addition, work is in progress for utilizing transformed forms of a corn xylem-colonizing endophyte, Clavibacter xyli subsp. cynodontis, to control the European corn borer (Kostka et al., 1988; Dimock et al., 1989). The overall objective of this study was to begin exploring the possibility that some species of endophytic bacteria can, for more than one plant species, contribute fundamentally not only to the suppression of soilborne diseases but also to the enhancement of plant growth, where enhancement of plant growth includes not only suppression of disease but also growth promotion that is infection independent and therefore occurs even if a plant is entirely uninfected by pathogens. Among the crucifers, oilseed rape in particular suffers throughout the world from disease caused by wilt pathogens, such as V. dahliae. This fact, and the importance of oilseed rape in the world’s agricultural economy, its multiple uses, its global crop production, and its genetic potential (Nejad, 1994), suggests that this plant is appropriate for case studies of biological control of wilt diseases by bacterial endophytes. The tomato plant also suffers seriously from disease caused by wilt pathogens, particularly the pathogen F. oxysporum. With this in mind, our specific objective was to find, within oilseed rape plants, endophytic bacterial isolates that can provide significant biological control of soilborne diseases not only for oilseed rape but also for tomato and that can enhance plant growth for both of these plant species. MATERIALS AND METHODS
Bacterial Isolation Healthy looking young oilseed rape plants, wild and cultivated, growing in fields near Uppsala, were collected for bacterial isolation. The bacteria were isolated from the internal tissues of roots and stems, which were individually disinfected by soaking each in 5.25% sodium hypochlorite and then adding 1 ml of Tween 20 (polyoxyethylene sorbitan monolaurate) for 10 –15 min, depending on the tissue, followed by immersion in 70% ethanol for 1–2 min. They were then rinsed three times in sterile distilled water (SDW) to clear them of alcohol before obtaining bacterial isolates by two different techniques from the internal tissues of their surface-disinfected roots and stems. Trituration. This technique allows endophytic bacteria to be selectively isolated from vascular tissue in consistently high numbers (Hallmann et al., 1997). Since one key to success in isolating and studying
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endophytic bacteria is surface sterility (Hallmann et al., 1997), sterility checks were carried out for each sample to monitor the efficiency of the disinfestation procedure. For these checks, either 0.1 ml of the last wash was transferred to Tryptic soy agar (TSA) or, alternatively, 0.1 ml of the final wash was transferred to 9.9 ml Tryptic soy broth (TSB), incubated at room temperature on a shaker, and spread onto a TSA agar plate. After 48 h, if no bacterial growth occurred in the sterility check, the recovered bacteria were considered to be endophytes. The tissue (5 g fresh weight) was then macerated in 100 ml phosphate-buffered saline solution (PBS, per liter: 8 NaCl, 0.2 KH 2PO 4, 1.1 Na 2HPO 4, and 0.2 KCl, pH 7.4) with mortar and pestle under aseptic conditions. After filtering the slurry through sterile cotton cloth, the filtrate was diluted appropriately, plated onto a sterile enrichment medium of Rovira (1956) with some modifications (The medium used contained, g liter ⫺1 distilled water: 2 K 2HPO 4, 2.5 (NH 4) 2HPO 4, 0.25 MgSO 4 䡠 7 H 2O, 0.5 NaCl, 0.5 CaCl 2, 0.05 FeCl 3, 0.5 proteose peptone, 0.5 yeast extract, 10 glucose, 15 Difco bactoagar), and left for incubation at room temperature (20 ⫾ 2°C) until the growth of colonies became visible. Imprinting of plant tissue. The disinfecting procedure for this method of isolation was similar to that of the trituration method, with the following differences. Stems, 2–3 cm long, were excised from plants, surface disinfested, and dried with moistened paper (SDW). With a flamed scalpel, stems were cut longitudinally into two pieces. The pieces were aseptically transferred and pressed onto three different concentrations of TSA within the agar (TSA 10, TSA 30, and TSA 50), left for 1 h, and then removed. (Here TSA 10, for example, contains 10 g Tryptic Soy Broth (Difco Ltd.) and 12 g Technical Agar (Oxoid Ltd.) in 1000 ml distilled water; TSA 30 contains 30 g.) The agar plates were incubated at either of two temperature ranges, 5–10°C or 10 – 15°C, to obtain a spectrum of bacteria with different temperature preferences. Bacterial colonies that appeared frequently and looked morphologically different were randomly selected for further study. After each isolate had been recultured and checked for purity, one loopful was suspended in sterile tap water containing 20% glycerol (pH 7) and then frozen at ⫺80°C for long-term storage. For short-term storage, the isolate cultures were freeze-dried at 4°C. Before use in tests and experiments, stored cultures were given a day to acclimate to experimental conditions. For those isolates that both promoted plant growth and reduced incidence of wilt diseases in gnotobiotic and greenhouse experiments, some preliminary identification was carried out using fatty acid methyl ester analysis. We found that isolates PN and PP closely resemble frequently occurring plant-associated genera,
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such as Pseudomonas and Serratia (Mahaffee and Kloepper, 1994). Fungal Pathogens The pathogens V. dahliae and F. oxysporum in our studies were isolated at the Plant Pathology Department of SLU from naturally infected oilseed rape and tomato plants from southern Sweden. Before proceeding with bioassays, the pathogenicity was confirmed for V. dahliae and F. oxysporum in the test cultivars Casino and Dansk export, respectively. This was done by culturing the fungus on Potato Dextrose Agar (PDA) at room temperature for about 2 weeks and blending 41 plate per plant with sterile tap water in an electric mixer. The resulting fungal suspension was applied as a soil treatment and as a seed treatment to 2-week-old oilseed rape plants and 3-week-old tomato plants. Typical symptoms could be observed 2–3 weeks after soil treatment and 5 weeks after seed treatment. For further confirmation, infected leaves were removed 40 – 45 days after infection and placed onto filter paper moistened with SDW in large petri dishes with glass lids. After incubation, microsclerotia and conidia of the pathogens that appeared were identified under the light microscope. In Vitro Screenings Effects on seed germination. One hundred sixty isolates were tested for seed emergence on Brassica napus seeds by placing them onto sterile filter paper moistened with SDW in petri plates (two plates with 20 seeds/plate). Seeds were treated with bacterial suspensions (20 l/seed) and left to incubate at room temperature. Control plates were arranged in a similar way, except that they were treated with SDW only. For each isolate, effects on seed germination were measured by counting the number of fully germinated seeds per plate and comparing that with that of the control plates. After 4 days, we measured seedling length for each plate to obtain an average, which was compared with that of the control plates. Effects on fungal mycelial growth. Bacterial isolates that exhibited clearly measurable effects on seed germination and seedling growth (stem and root) were then tested in vitro on agar media against fungi. Deepfrozen cultures of bacteria were revived on King’s medium B agar (KBA; King et al., 1954) and recultured on KBA after checking purity. Disks of fresh culture of the fungus (5 mm diameter) were cut out and placed in the center of a 9-cm petri plate with PDA. Also, to determine whether antimicrobial activity is substrate dependent, we included three other agar media (cornmeal agar, CMA, malt extract agar, MEA, green bean agar, GBA). Bacterial cultures were inoculated in either of two ways: a bacterial suspension was streaked in two parallel strips each 2 cm long on either side of the
fungal agar disc or the suspension was seeded as spots, one spot at each of the four corners of the plate. Fungal growth for individual and dual bacterial cultures was compared with fungal growth in control plates with the fungus only. This experiment was repeated to check the consistency of the results. Production of volatile metabolite. Using a modification of the procedure of Millar and Higgins (1970), we assayed the amount of HCN produced by bacteria grown on KBA with or without glycine (4.4 g 䡠 L ⫺1). In the lid of each petri dish, we placed filter paper strips soaked in picric acid solution (2.5 g of picric acid, 12.5 g of Na 2CO 3, and 1 L of water). The dishes were then sealed with Parafilm. After incubation at 28°C for 48 h, the presence of a colored zone around the bacteria indicated the production of HCN. For each of the endophytic strains, reactions were scored as weak (yellow to light brown), moderate (brown), or strong (reddish brown). Also, to determine whether the strains produce a volatile metabolite other than HCN, we grew the bacteria on KBA and the fungi on PDA. When both the bacteria and the fungi had visible growth, we placed the bacterial agar plate on top (inverted) of the fungal agar plate and sealed the plates with Parafilm to avoid contamination and to keep gases from escaping. If the region of fungal infection is found to be limited compared to that of the control in such an experiment, then, presumably, the only cause can be the production of volatile metabolites by the endophytic bacteria. Greenhouse Screenings of Bacterial Effects First screening. For evaluating plant growth promotion, 302 bacterial isolates were tested on oilseed rape. Of these isolates, we selected 144 based upon their superior plant growth promotion and disease suppression, which were then tested on tomato. For these screenings, seeds were sown in 10-cm pots filled with commercial soil (“Hasselfors” containing g 䡠 m ⫺3: 180 N, 210 K, 250 Mg, 9.0 Fe, 2.0 Cu, 0.7 Mo, 75 P, 2500 Ca, 120 S, 5.0 Mn, 0.8 B, and 2.0 Zn; sand mixture in volume ratio ⫽ 4:1). After 1 week, the pots were thinned to one plant per pot and watered regularly. The pots were placed in a greenhouse with controlled temperatures. The experiments described here and in the following two sections were completely randomized designs with 10 replications (one pot each) per treatment. Day/night temperatures were, respectively, 22 ⫾ 2 and 20 ⫾ 2°C for oilseed rape plants and 26 ⫾ 2 and 24 ⫾ 2°C for tomato. A photoperiod of 16 h and a relative humidity within the range of 70 – 80% was maintained. When needed, Phillips HPI-T mercury halogen lamps (400 W) provided additional light. Seven to 10 days after bacterial soil inoculations, a fertilizer (Superba S liquid fertilizer, 6.5% N, 1.0% P,
BIOCONTROL OF WILT DISEASES USING ENDOPHYTIC BACTERIA
4.7% K plus microelements; Supra Ceres, Landskrona, Sweden) was provided regularly twice a week. To provide uniform exposure to light and temperature, twice a week experimental pots were rotated on the bench. For further avoidance of the effects of environmental variation between plants, extra plants (untreated) were set out on the margins of the experimental plants. The above screenings resulted in a selection of 21 bacterial isolates for further evaluation of plant growth promotion and disease suppression using soil and seed applications. They were chosen for their generally good performance in suppression of disease and promotion of plant growth. Soil application. The tested bacterial isolates were individually cultured from deep-frozen vials on King’s medium B agar at 20°C for 24 – 48 h. A single colony of each bacterial isolate was then cultured in a 250-ml Erlenmeyer flask containing 100 ml TSB and incubated on a shaker at room temperature for 74 h at 120 rpm. Each broth culture was diluted with TSB to 200 ml before being applied as a soil drench for each 2- to 3-week-old oilseed rape plant (25 ml per plant). To measure plant growth effects, we compared leaf surface parameters (i.e., length and width of fourth and fifth leaves) or shoot dry weight of isolate-treated plants against control plants. Shoot dry weight was recorded after air-drying in an oven at 80°C overnight. Of the 21 isolates selected for further evaluation, 15 of the better performers were chosen for the experiments reported in this paper. These bacterial strains were inoculated either into the soil or as a seed treatment. For soil treatment, the 2- to 3-week-old bacterial-treated seedlings were removed from pots and transplanted into individual pots (12.5 ⫻ 14.5 cm) containing three layers. Each plant was transferred with all soil attached and adhering to its roots. The bottom layer (one-third) of each pot consisted of greenhouse soil. The second layer (one-third) consisted either of V. dahliae-infested soil or F. oxysporum-infested soil (41 of the fully grown fungi on an agar plate). The surface layer (one-third) consisted again of the greenhouse soil. Healthy plants (treated with either SDW or TSB) and diseased plants (treated with either V. dahliae or F. oxysporum only) served as controls. The plants were not watered on the day of treatment. Seed application. Two mixed cultures were included in all seed treatment experiments. The four isolates in these cultures, PF, PK, PH, and PN, were arbitrarily chosen. The isolates were cultured from deep-frozen isolates on KBA at room temperature for 48 h. For each cultured isolate, one loopful of bacterial lawn from the agar surface was scraped and then transferred to a test tube containing 20 ml of sterile water. For all isolates, suspensions were prepared identically (final concentration approx 10 9 colony-forming units/ml). For the mixed cultures, 10 ml of one
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culture was combined with 10 ml of another. Then, the bacterial suspension of each individual culture and each mixed culture was poured into a container with seeds of either tomato or oilseed rape. After 20 min of soaking, the content of each container was decanted and the seeds were dried under a fan for about 14 h. Seeds treated with SDW or TSB were used as controls. The bacteria-treated oilseed rape and tomato seeds, after drying, were sown in pathogen-contaminated soils as described above. Each experiment was continued for 10 –15 weeks. The plants were observed regularly for visible disease symptoms. For oilseed rape, disease symptom appearance for each leaf was scored by estimating its area of symptom appearance based upon a disease rating scale that ranged from 0 (no suppression) to 1.0 (complete suppression). Healthy control plants generally had 15 leaves. Maximum symptoms were assumed to exist for a plant when 12 of its leaves had succumbed to the disease, inasmuch as such plants were effectively dead. Therefore, the symptom appearance for a plant was the number of diseased leaves divided by 12. For all control diseased plants (untreated with bacteria), symptom appearance was 1.0. Hence, 1.0 minus symptom appearance is an appropriate index of disease suppression. For tomato, we measured both symptom incidence and plant fresh weight. Finding that the two parameters were strongly correlated, we chose fresh weight as the index of symptom appearance. We computed mean disease suppression for tomato using the formula S ⫽ (T ⫺ D)/(H ⫺ D), where S is an index of disease suppression, T is the mean fresh weight (g) of treated plants, H is the mean fresh weight of healthy plants, and D is the mean fresh weight of control diseased plants. Here, disease suppression is the ability of an endophytic bacteria to suppress the effect of a disease on the fresh weight, which is a realistic index of disease suppression when, as in the case of this experiment, H is significantly greater than D and symptom incidence and plant fresh weight are strongly correlated. The above formula for disease suppression also takes into account the possibility that an endophytic bacteria can enhance plant growth (mean fresh weight) by means beyond that of disease suppression. Only when such is the case, as was true for treatment with some of our bacteria, can S take values greater than 1. In cases in which S is greater than 1, disease suppression is complete, and the value S ⫺ 1 is an index of the extent to which treatment with the bacteria can promote plant growth independent of the occurrence of disease. For each experiment that evaluated the performance of bacterial isolates, a one-tail, two-sample t test was used to obtain a confidence interval for testing the hypothesis that at the time of evaluation the experimental value of a treatment (i.e., plants treated with a given isolate) was greater than that for the control.
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TABLE 1 Plant Growth Promotion of Oilseed Rape by Endophytes— Length and Width of Fourth and Fifth Leaves after Bacterial Inoculation of Nonsterile Greenhouse Soil in the Absence of Pathogens Fourth leaf
Fifth leaf
Treatment
Aver. length (cm)
Aver. width (cm)
Aver. length (cm)
Aver. width (cm)
Isolate PP PN PO PV TSB control Water control
19.75* 19.3* 17.45 17.4 14.85 14.3
15.3* 14.57* 13.4 13.1 11.5 11.1
21.75* 20.6* 20.0* 19.8* 16.15 15.45
14.85* 13.8* 13.9* 13.3 11.25 10.55
* Significantly different from both controls at level 0.01.
RESULTS
In Vitro Screenings Effects on seed germination. From experiments using gnotobiotic systems, we found for oilseed rape that some of our bacterial isolates had no apparent effects, whereas others, when applied individually, caused not only suppression of seed germination (in vitro) and seedling growth but also root necrosis. Yet, other isolates had a strong potential for promoting seedling growth (stem and root). Hence, growth-regulating substances might have been involved. Effects on fungal mycelial growth. Isolates used in gnotobiotic assays were tested in vitro for inhibition of mycelium growth of fungi. Some of the isolates also suppressed mycelium growth of F. oxysporum (e.g., PL and PP). For V. dahliae, approximately 41% of the isolates exhibited strong inhibition, 33% moderate inhibition, 15% weak inhibition, and 11% no inhibition. For both pathogens, these results varied when tested on different agar media. Hence, antimicrobial activity appeared to be substrate dependent. However, for V. dahliae, some isolates, such as PK, PM, and PP, showed strong activity on more than one medium, and isolate PP was growth suppressive on all nutrient media. Production of volatile metabolite. Performance of selected endophytic bacteria in production of volatile metabolite was characterized as follows. Following 48 h incubation, most isolates were HCN negative. On KBA, with or without glycine, only the isolate PC showed detectable cyanide production by changing color from yellow to brown around its colonies. Consistent with this result, our experiments concerning volatile metabolites indicate that most isolates that were effective against wilt pathogens in oilseed rape and
tomato plants produced fungal-inhibitory volatile metabolites other than hydrogen cyanide. Greenhouse Screenings of Bacterial Effects Soil application. From experiments with soil treatment, isolates PP and PN expressed their promotion of plant growth for oilseed rape through significant enhancement of leaf size within a week after inoculation (Table 1). The experimental increases in leaf size for test plants treated with PP and PN over those treated with the TSB control were 33.3 and 27.0%, respectively. Compared to the water control, the respective increases were 39.4 and 32.8%. Significant increases in leaf size by these two isolates and isolate PO compared to controls continued after 2 weeks. Also, after inoculation of the test cultivar, significant increases in shoot dry weight occurred for all isolates included in Table 1. In another soil treatment experiment (Fig. 1), using the same isolates, the shoot dry weight and plant length were significantly increased by bacterial treatment in greenhouse soil, both in the presence and in the absence of V. dahliae. Seed application. Over several weeks under nonsterile conditions, all individual and dual-isolate cultures shown in Fig. 2 significantly suppressed and delayed symptoms of disease in oilseed rape caused by V. dahliae. In Fig. 2, the mean disease suppression for all control plants treated with broth or SDW on the 100th day of final observation showed severe disease symptoms in at least 12 true leaves. In contrast, symptoms for the isolates PX, PF, and PA hardly appeared on the 5th true leaf. As in oilseed rape, in tomato all tested isolates significantly suppressed and delayed symptoms caused by F. oxysporum (Fig. 3). All bacterial isolates included in Fig. 4 increased shoot dry weight for tomato and oilseed rape compared to control plants. On both tomato and oilseed rape, all isolates not only increased shoot dry weight but also
FIG. 1. Plant growth promotion of Brassica napus by endophytes—shoot dry weight after bacterial inoculation of plant roots in nonsterile greenhouse soil in the presence and absence of Verticillium dahliae. Asterisks denote significance at P ⫽ 0.05.
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FIG. 2. Effects of endophytic bacteria on incidence of wilt disease caused by Verticillium dahliae in oilseed rape. Bars indicate 99% confidence intervals. P ⱕ 0.001.
increased plant height. Expansion of leaf size was also observed. The two dual-isolate cultures performed no better on average than the other isolates for both tested plants. All bacterial isolates included in Fig. 5 allowed the plants to significantly increase shoot fresh weight compared to control diseased plants at the end of the experimental period of 100 days. In fact, isolates PF, PA, and PN yielded fresh weight that was greater than that of untreated healthy plants. Treatment by coinoculation of endophytic isolates (mixed inoculum) resulted not only in suppressed disease symptoms (Fig. 3) but also in increased shoot fresh weight (Fig. 5). However, for both attributes, those isolates were less effective than isolates that were applied individually.
FIG. 4. Plant growth promotion in terms of shoot dry weight of (A) tomato and (B) oilseed rape under greenhouse conditions after seed bacteria treatment with bacterial isolates in the absence of pathogens. Bars indicate 99% confidence intervals. P ⱕ 0.001.
The specific objective of this study was to find naturally occurring endophytic bacterial isolates within the tissues of a variety of plants and to rank them with respect to their capacities to (a) enhance the growth of oilseed rape and tomato plants when uninfected with wilt pathogens and (b) provide these plants with bio-
logical control against disease when infected. To our knowledge, this paper is the first to report finding such endophytic bacteria, which significantly improve growth and development of oilseed rape and tomato and control their soilborne diseases: V. dahliae in oilseed rape and F. oxysporum f. sp. lycopersici. in tomato. Two of our endophytes, PA and PF, achieved such control, and several others reduced disease incidence and severity of the two pathogens by at least 75%. Some endophytes also resulted in significant increase in shoot dry/fresh weight, plant height, and leaf size in
FIG. 3. Effects of endophytic bacteria on incidence of Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici in tomato. Bars indicate 99% confidence intervals. P ⱕ 0.001.
FIG. 5. Effects of endophytic bacterial isolates on shoot fresh weight of tomato in the presence of Fusarium oxysporum f. sp. lycopersici. Bars indicate 99% confidence intervals. P ⱕ 0.001.
DISCUSSION
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both the presence and the absence of the pathogen in the soil. However, although this was true for oilseed rape and tomato in our greenhouse experiments, the performance of our isolates for different cultivars under various field conditions has yet to be investigated. Also, the observed effects of the endophytes may have largely been circumstantial. The effects may have been beneficial only as a consequence of (i) the particular microflora to which the experimental plants had been exposed before bacterial inoculation, (ii) the stage at which the plants were inoculated, and (iii) the dosage (inoculum concentration). However, the dosage was not so high as to eliminate or mask isolate-dependent effects. Our experiments were not intended to clarify specific mechanisms by which the endophytes promoted growth and protected the host from the diseases caused by V. dahliae and F. oxysporum. We did, however, find that most of our effective isolates produced microbialinhibitory volatiles other than hydrogen cyanide, albeit that HCN may well be a possible and perhaps frequent mechanism by which bacteria suppress plant pathogens (Ross and Ryder, 1994; Maurhofer et al., 1994). From a more general perspective, a basic reason for finding our bacterial isolates effective in suppressing soilborne diseases caused by Verticillium and Fusarium in oilseed rape and tomato, respectively, might be that they were isolated from within vascular tissue (conductive tissue). In accord with the ideas of Hallmann et al. (1997), they may well follow the same path as soilborne pathogens to colonize the vascular tissue of a plant. In other words, endophytically resident bacteria may be strategically at the right place and at the right time for suppression of vascular wilt diseases, not only with regard to antagonistic effects, such as antibiosis and competition for nutrients and infection sites, but also with regard to optimal timing of, for example, induced resistance. Further, because endophytically resident bacteria probably have the unique ability to survive inside plants with little or no microbial competition, they may provide the plant with reliable suppression of vascular diseases (Misaghi and Donndelinger, 1990). By mutually enhancing one another’s fitness values, the endophytic bacterium and its host plant can coevolve in a manner that leads to intimate and effective means of communication and cooperation in combating and suppressing vascular (wilt) diseases. Finally, microfloral populations already resident within the host plant may well influence and be influenced by rhizosphere bacteria. Hence, Sturz and Christie (1995) have proposed that endophytically competent microflora, such as endophytically resident bacteria, may mediate the establishment of biocontrol agents within the rhizosphere.
ACKNOWLEDGMENTS The authors thank Professor B. Gerhardson for supervision and critical comments on the manuscript. The study is part of the Microbial Antagonism against Fungi program, financed by the Foundation for Strategic Environmental Research (MISTRA).
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