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A.M. Romero, Fitopatología, Facultad de Agronomía Universidad de Buenos Aires. Av. San Martín 4453, Buenos Aires 1417, Argentina (e-mail: firstname.lastname@example.org).
Aims: Plant growth-promoting (PGP) activity of two Azospirillum strains and their effects on foliar and vascular bacterial diseases were evaluated on fresh market and cherry tomato.
Methods and Results: Tomato seeds were inoculated with A. brasilense Sp7 or Azospirillum sp. BNM-65. Four-week-old plants were challenge-inoculated with Clavibacter michiganensis subsp. michiganensis (bacterial canker) or with Xanthomonas campestris pv. vesicatoria (bacterial spot). Azospirillum-induced PGP was greater on cherry than on fresh-market tomato. Cherry tomato was more resistant to bacterial canker but more susceptible to bacterial spot than the fresh-market tomato. Canker severity was not affected by Azospirillum seed treatments. However, leaf- and plant-death were delayed on Azospirillum-treated plants compared with nontreated controls. Azospirillum increased the bacterial spot severity on cherry but not on fresh-market tomato.
Conclusions: PGP was observed on both tomato genotypes, although growth effects were larger on cherry tomato. Also, Azospirillum treatments may alter tomato susceptibility to bacterial diseases.
Significance and Impact of the Study: The interaction between PGP rhizobacteria like Azospirillum spp., not known to induce systemic resistance, with plant pathogens distantly located is frequently overlooked. This work demonstrates the importance of this kind of evaluation.
Bacterial canker and bacterial spot are economically important diseases of tomato (Shirakawa et al. 1991; Stall 1993); both can be seed-transmitted or are endemic ( Jones et al. 1986; Fatmi and Schaad 2002). Bacterial spot can be caused by at least three species of the reclassified ‘Xanthomonas campestris pv. vesicatoria’ complex: X. vesicatoria, X. axonopodis pv. vesicatoria and X. gardnerii (Vauterin et al. 1995; Jones et al. 1998). They infect all above-ground-plant parts resulting in leaf, stem and fruit lesions, defoliation and yield loss. Resistant cultivars to some races of the pathogen are available, although they are not used in Argentina, so control relies heavily on preventive copper sprays. Unfortunately, these sprays are not very effective and in addition pose severe environmental problems. Bacterial canker is caused by Clavibacter michiganensis subsp. michiganensis, which invades the xylem resulting in a systemic vascular disease. Infected plants wilt and subsequently die. There are no commercial cultivars resistant to bacterial canker, and because of the location of the bacteria, preventive chemical treatments are ineffective. In Argentina, fresh-market tomato is seriously affected by bacterial spot in the field and by bacterial canker in greenhouse production systems. As cherry tomato production is recent, there is no information about its sanitary status.
The increased concern of consumers about the impact of agrochemicals on the environment and food safety is leading to the development of alternative approaches for crop production, including biological and ecological strategies. Certain strains of rhizosphere bacteria, called plant growth promoting rhizobacteria (PGPR), stimulate plant growth mainly by directly affecting plant metabolism and/or the availability of nutrients (Bashan and Levanony 1990). Other PGPR strains promote plant growth indirectly by suppressing soil-borne pathogens, or by stimulating plant natural defences, by a mechanism called induced systemic resistance (ISR) (Kloepper et al. 1993). In some countries like China PGPRs are extensively applied to more than 20 million hectares of crops (Zehnder et al. 2001).
Azospirillum sp. is possibly the most studied PGPR bacteria. The first Azospirillum inoculants are already available in Europe for maize and in South Africa for maize and wheat (Dobbelaere et al. 2001). Their use may reduce the costs and quantities of pesticide and fertilizer applications, especially in developing countries like Argentina. However, field results are often inconsistent (Bashan and Holguin 1997). There are many biotic and abiotic factors that can alter PGPR results, i.e. environmental conditions, plant genotype and interactions with other microorganisms (Bashan and Holguin 1997). There apparently are complex interactions among plant genotypes and Azospirillum bacterial strains (Bashan and Holguin 1997; Saubidet and Barneix 1998). Moreover, some strains can become pathogenic under stress conditions (Bashan 1998). Azospirillum spp. are not known as ISR activating bacteria (Bashan and de-Bashan 2002b), although there are some reports of its biocontrol activity. Azospirillum can reduce the incidence and severity of damping off caused by Rhizoctonia solani, possibly by bacterial colonization of the sclerotia (Gupta et al. 1995). Bashan and de-Bashan (2002a) reported a protective effect of Azospirillum brasilense on tomato when applied as a foliar treatment prior to inoculation with the pathogen Pseudomonas syringae pv. tomato, causal agent of bacterial speck. Tomatoes were also protected when both microorganisms were co-inoculated on seed. The mechanism may involve pathogen displacement, in a process called pre-emptive competitive exclusion (Wilson et al. 2002).
An important issue frequently overlooked is the possible interaction between the effects of Azospirillum and phytopathogens distantly located on a plant. Azospirillum root colonization alters the physiology and metabolism of the plant (Bashan and Holguin 1997). Also, the quantity and quality of host root exudates can be modified on diseased plants, which may have an effect on PGPRs colonization and metabolite production. Thus, plant susceptibility to pathogens and ability of Azospirillum to compete with other microorganisms in the rhizosphere can be either increased or decreased. Crops often are exposed to plant pathogens and, for this reason, careful studies must be done to evaluate potential interactions with the most common pathogens before releasing a PGPR strain into the environment.
To increase our knowledge about the complex interactions between Azospirillum spp. and plant diseases, experiments reported in this paper were conducted. The objectives were to determine the effect of Azospirillum-plant growth promotion on the development of bacterial diseases on tomato, determine if this effect was tomato genotype-Azospirillum strain dependent, and if it was affected by the pathogen colonization characteristics. As Azospirillum PGPR activity was already determined on fresh-market tomato (Bashan et al. 1989), another objective was to evaluate the PGPR activity of Azospirillum on cherry tomato, as yet not studied in this respect.
Materials and methods
Two Azospirillum spp. strains were used: Azospirillum brasilense Sp7 (ATCC 29145) and Azospirillum sp. BNM-65, which was isolated from soil in Río Cuarto; Córdoba Province, Argentina (Banco Nacional de Microorganismos, Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, Argentina). The strains were chosen for their positive PGP activity on cherry tomato [Lycopersicon esculentum Mill. var. cerasiforme (Dunal) A. Gray] on previous studies (data not shown). Bacteria were cultured on nitrogen-free NFb semi-solid medium (Döbereiner et al. 1995) supplemented with 1 g l−1 ammonium chloride for 44 h at 30 ± 2°C (1–2 × 108 CFU ml−1) at 150 rpm. Cells were harvested by centrifugation (10 000 g for 10 min) and washed twice in sterile tap water (STW). Washed cells were suspended in STW (1–2 × 107 CFU ml−1) and this suspension was used as inoculum. Bacterial concentration was verified by dilution plating on Congo Red plates (Rodríguez and Cáceres 1982).
Plant growth conditions and Azospirillum inoculation
Experiments were conducted on tomato (L. esculentum Mill.) fresh-market cultivar ACE 55 (Asgrow) and cherry hybrid Supersweet 100 (Rogers). Seeds were surface disinfested with 0·2% commercial detergent for 10 min, exhaustively washed with tap water to eliminate detergent, suspended in 2% sodium hypochlorite for 10 min and rinsed 6–7 times in STW. Finally, seeds were suspended in STW for 30 min. All disinfestation steps were done under agitation. Seeds were aseptically transferred to a sterile filter paper to remove excess water, and then inoculated by immersion in one of the two Azospirillum sp. suspensions for 30 min with agitation. Seeds suspended in STW were used as controls. The mean number of bacteria adhering to the seed (1 × 105 CFU per seed) was determined by serial dilution plating on Congo Red medium and nutrient agar. Bacterized seeds were pregerminated in the dark at 28°C in a humid chamber. After 48–72 h, one germinated seed was planted in each 300 ml plastic pot containing an oven-sterilized mixture of commercial potting substrate (GemTM, Agroquímicos Larroca SRL, Buenos Aires, Argentina), perlite and soil (2 : 1 : 1). Pots were placed in the greenhouse under natural temperature and light conditions. Plants were watered daily with tap water. The experiment was repeated twice with 10 replicates per treatment.
Tomato rhizosphere colonization by Azospirillum spp. Three plants from each genotype treated with BNM-65 or nontreated were randomly harvested 56 days after planting (DAP). The root system was gently shaken to remove nonrhizosphere soil. Root samples, separated from their shoots, were placed in plastic bags with sterilized tap water and sonicated in an ultrasonic bath (XB2 Grant Instruments, Shepreth, UK) for 10 min to extract rhizosphere and rhizoplane micro-organisms. Sonicated solutions were serially diluted and 100 μl of each dilution was inoculated by triplicate into tubes containing NFb semisolid medium. Tubes were incubated at 30°C and Azospirillum presence was determined by the formation of a rising pellicle on the medium surface and light microscopy to verify the presence of the typical motile cells. The most probable number (MPN) expressed as CFU g−1 of root dry weight (dw) was calculated as described by Döbereiner et al. (1995). As this technique cannot discriminate between the inoculated Azospirilla and those indigenous in the soil, the results are referred to as Azospirillum spp.
Tomato plants were challenged with either a strain of C. michiganensis subsp. michiganensis, Cm9, or a mixture of three strains of the X. campestris pv. vesicatoria complex: Xv1, Xv9 and Xv12. X. campestris pv. vesicatoria strains were isolated from field tomatoes showing bacterial spot symptoms in Buenos Aires province, Argentina. All of the strains can degrade starch, and based on genotypic fingerprints (rep-PCR using BOX primers; Louws et al. 1995) Xcv 1 corresponds to X. vesicatoria (Vauterin et al. 1995), while the other two strains remain to be placed in the new classification scheme. Cm9 was isolated from a wilting tomato plant in a production greenhouse in Buenos Aires. The pathogenicity of the isolates was tested on tomato and Cm9 was also infiltrated on Mirabilis jalapa leaves, which showed a characteristic hypersensitive response 24 h later. All strains were cultured on modified yeast dextrase carbonate (YDC) medium (Ritchie and Dittapongpitch 1991) at 26°C for 48 h.
Four-week-old plants were inoculated either with Cm9 or the Xv mix. Bacteria were suspended in sterile distilled water (SDW) and their concentrations were adjusted using a spectrophotometer. Cm9 was inoculated by removing the first true leaf of each plant with a flamed-sterilized scalpel immersed in the bacterial suspension (108 CFU ml−1); for pathogen-free control plants the scalpel was immersed in SDW. To inoculate plants with Xv1, Xv9 and Xv12, each strain was diluted and added in equal proportion to a flask with SDW to a final concentration of 5 × 106 CFU ml−1. Finally, 0·01% Silwet L-77 (Loveland Industries Inc., Greeley, CO, USA) was added (Romero et al. 2001). The aerial part of the plants was immersed in the inoculum suspension for 20 s. Pathogen-free control plants were immersed in SDW with Silwet. After inoculation, all plants were covered with plastic bags for 72 h and kept at 25°C in a growth chamber with a 12 h photoperiod, before being returned to the greenhouse.
Evaluation of plant growth promotion and disease development
The number of fully developed true leaves was counted 30 DAP. Plants were harvested from five randomly selected pots 56 DAP, and plant height was measured. The root system of each plant was gently separated from bulk soil and washed under tap water. Shoots and roots were clipped and placed in paper bags at 65°C for 72 h to determine dry weight.
Disease severity was evaluated every 3–4 days for a total of 6–8 observations. Plants inoculated with C. michiganensis subsp. michiganensis were evaluated using a 0–7 rating scale, where 0, no disease; 1, less than half of a leaf wilted; 2, between one half of a leaf but <25% of the leaves wilted; 3, between 25 and 49% of the leaves wilted; 4, between 50 and 74% of the leaves wilted; 5, same as 4 but some apical leaves wilted; 6, >75% of the leaves wilted; 7, plant dying or dead. The proportion of the fifth (ACE 55) or sixth (Cherry Supersweet 100) leaf covered with spots was evaluated on plants inoculated with the X. campestris pv. vesicatoria complex. For both diseases, the area under disease progress curve (AUDPC) was calculated (Campbell and Madden 1990).
The number of fully developed true leaves was counted 12 and 15 days after challenge inoculations (DAI). Plant height was measured 12 DAI.
Experimental design and statistical analysis
Plants were arranged in a completely randomized design. Analysis of variance was performed using the general linear models (GLM) procedure of SAS (SAS Institute, Cary, NC, USA), and means were compared using Fisher's protected least significant difference.
Effect of Azospirillum on plant growth
Cherry tomato plants had more leaves and were taller than fresh-market tomato plants, 30 and 56 days after sowing, respectively (P < 0·0001). For both cultivars, plants bacterized with Azospirillum had more leaves, were taller, and had higher shoot and root dw, than nonbacterized plants (P < 0·0001; Table 1). The effect was most evident on cherry tomato. On this hybrid, plant height, and shoot and root dw were almost doubled on bacterized plants compared with nonbacterized controls, while on fresh-market tomato only shoot dw was almost doubled. For fresh-market tomato, there was no Azospirillum spp.-strain effect, however, cherry tomato shoot growth (height and dry weight) was promoted more by Sp7 than by BNM-65.
Table 1. Effect of Azospirillum-seed treatments on the morphology and biomass of plants of fresh-market tomato ACE 55 and cherry tomato Supersweet 100. Tomato seed was inoculated with Azospirillum sp. BNM-65, Azospirillum brasilense Sp7, or left as nontreated controls
Leaves 30 DAP*
Shoot height 56 DAP
Dry weight 56 DAP
% of control
% of control
% of control
% of control
*DAP, days after planting.
† For each tomato genotype, means with the same letter within a column are not significantly different, based on comparisons with Fisher's Protected Least Significant Differences at P = 5%.
Rhizosphere colonization by Azospirillum spp. was higher on cherry Supersweet 100 than on fresh market ACE 55 plants (3·3 × 105 ± 0·7 × 105 CFU g−1 and 4·5 × 104 ± 0·4 × 104 CFU g−1, respectively). No Azospirillum spp. were detected on control plants on either tomato genotype.
Bacterial spot development
Disease developed sooner and reached higher severity levels on cherry Supersweet 100 than on fresh market ACE 55 tomato (P = 0·0016). There was no difference in bacterial spot severity among treatments for the fresh-market tomato (P = 0·2160; Fig. 1). On cherry tomato, disease severity varied with treatments (P < 0·0001). Cherry tomato plants bacterized with Azospirillum sp. had severity values higher than nonbacterized controls, reaching a final value of AUDPC at least twice as high as nonbacterized controls (Fig. 1). There was no difference associated with Azospirillum strains.
Azospirillum plant-growth promotion was still evident on challenged plants 15 DAI on cherry tomato. At that time, evaluated leaves had 10% (controls) to 25% (Azospirillum-treatments) of their area covered with spots. Azospirillum-bacterized plants were taller and had more leaves than nonbacterized controls (P = 0·0001 and P = 0·0074, respectively) (Table 2). Sp7-treated plants were taller than BNM-65-treated plants. Defoliation was severe in 18 DAI but BNM-65 treated plants still had more leaves than controls. A similar situation was observed on fresh-market tomato, although plant growth promotion was less evident than on cherry tomato. Azospirillum-bacterized plants were still slightly taller and had more leaves than nonbacterized controls (P = 0·0545 and P = 0·0495, respectively) 15 DAI (Table 2).
Table 2. Effect of challenge inoculation with bacterial spot or bacterial canker agents on height and number of leaves of tomato plants seed-inoculated with Azospirillum sp. BNM-65, Azospirillum brasilense Sp7, or left as nontreated controls. Plants of fresh-market tomato Ace 55 and cherry tomato Supersweet 100 were challenge-inoculated with pathogens 30 days after sowing
Height (15 DAI*)
Leaves (15 DAI)
Leaves (18 DAI)
Height (15 DAI)
Leaves (15 DAI)
% of control
% of control
% of control
% of control
% of control
*DAI, days after inoculation.
†, For each tomato genotype, means with the same letter within a column are not significantly different, based on comparisons with Fisher's Protected Least Significant Differences at P = 5%.
Bacterial canker development
Fresh-market tomato was much more susceptible to C. michiganensis pv. michiganensis than cherry tomato (P = 0·0001). For both tomato genotypes, there was no difference in AUDPC among seed treatments (Fig. 2).
Plant growth effect of Azospirillum was still noticeable 15 DAI, when plants had <25% of the leaves wilted. At that time, Azospirillum-bacterized plants were taller and had more leaves than nonbacterized plants, for both tomato genotypes tested (Table 2). Plant death was delayed on Azospirillum-treated plants. Twenty-five DAI 60% of the fresh-market tomato control plants were dead vs 20–30% of the plants bacterized with Sp7 or BNM-65, respectively (Fig. 3). All fresh-market tomato control plants were dead 30 DAI after inoculation, while 40–30% of the plants pre-treated with Sp7 or BNM-65 were still alive. Because cherry tomato was more resistant to bacterial canker, most of the plants were still alive at the end of the experiment (6 weeks after inoculation). At that time, 20–30% of the Azospirillum-treated plants were dead vs 60% of the controls (Fig. 3).
Cherry tomato is a specialty crop with increasing production during recent years. Although PGP activity of A. brasilense on fresh-market tomato was reported previously (Bashan et al. 1989), experimental data remains scarce for the efficacy of different PGPR species or strains, or susceptibility to diseases on cherry tomato. We have verified plant growth promotion activity of A. brasilense Sp7 and Azospirillum sp. BNM-65 on fresh market and, for the first time, on cherry tomato. There were differences in plant growth between the two tomato genotypes. Both Azospirillum strains evaluated in this study had been selected for their PGP activity on cherry tomato. Thus, the greater stimulation on the cherry genotype could have been caused by a plant genotype-Azospirillum strain specificity, as was observed in other crops (Bashan and Holguin 1997; Saubidet and Barneix 1998). It could also be caused by the greater root colonization by Azospirillum spp. on that genotype, which could likewise be caused by a cherry tomato-Azospirillum specificity.
Cherry tomato was more susceptible to the X. campestris pv. vesicatoria complex, causal agent of bacterial spot, having disease severity values twice as great as the fresh-market tomato. Mirroring what happened with plant growth promotion, Azospirillum-treated cherry tomato had greater levels of bacterial spot severity than the nontreated controls. For fresh-market tomato, there was no detectable change in bacterial spot severity on Azospirillum-treated plants compared with controls. Bashan and de-Bashan (2002b) in experiments with fresh-market tomato and a bacterial foliar disease, also reported that seed treatments with A. brasilense did not alter the development of bacterial speck caused by P. syringae pv. tomato. Based on these two experiences, it is possible that seed treatments with Azospirillum do not affect development of foliar diseases on fresh-market tomato. Another possibility is that the severity of foliar diseases increases when growth is stimulated (as in cherry tomato treated with Azospirillum), simply because more tissue is available. In addition, this extra tissue may be more susceptible. Azospirillum can alter plant physiology, presumably by the production of several phytohormones [especially Indole acetic acid (IAA), but also gibberelins, cytokinines and ethylene; Dobbelaere et al. 1999]. Also, the release of undefined signal molecules might interfere with plant metabolism (Bashan and Holguin 1997), or even with plant defence pathways.
Fresh-market ACE 55 was more susceptible to bacterial canker, caused by C. michiganensis subsp. michiganensis, than was cherry tomato Supersweet 100. There was no effect of Azospirillum seed-treatment on disease development for either tomato genotype, however, we observed a delay in leaf death and plant mortality on plants that had been bacterized with Azospirillum. Root inoculation with Azospirillum sp. has been claimed to improve plant growth under water stress conditions (Bashan and Holguin 1997; Creus et al. 1998); for example inoculated Sorghum bicolor had a reduced leaf senescence under osmotic stress, indicating an improved water uptake by roots (Dobbelaere et al. 2001). These effects could be caused by an increase in the density and length of root hairs, and thus the root surface area, and enhancement of root hydraulic conductivity (Dobbelaere et al. 2001). Our results could be explained by similar mechanisms, as tomato plants affected by bacterial canker were under water stress, having their water movement impaired because of colonization of the vascular tissue by the bacterial pathogen.
In this study, we have demonstrated the PGP activity of two Azospirillum strains on cherry tomato, and that this stimulation can modify the susceptibility of plants to bacterial diseases. The outcome of this interaction depends on the tomato genotype and the colonization characteristics of the pathogen. For fresh-market tomato, susceptibility to bacterial foliar diseases, i.e. bacterial spot (this study) and bacterial speck (Bashan and de-Bashan 2002b), was not altered by Azospirillum-seed treatments, while cherry tomato susceptibility to bacterial spot increased. In contrast, susceptibility to a vascular pathogen was not altered by Azospirillum treatment on the tomato genotypes tested in this study. However, seed inoculation with these PGPR bacteria delayed leaf and plant death, which could be beneficial in a field situation. In our experiments, plants were under severe biotic stress caused by optimal inoculation conditions with the pathogen. In a field situation, it is likely that bacterial canker disease pressure would not be as great, thus a delay in plant death could be beneficial towards attaining higher yields.
The authors would like to thank Dr David F. Ritchie and Dr Marcelo Soria for their valuable comments while reviewing the manuscript, and Marcela Nores and Ignacio Negri Aranguren for their assistance in the laboratory and greenhouse. Support for this project came from UBACYT 01/G 009.