SEARCH

SEARCH BY CITATION

Keywords:

  • Azospirillum brasilense;
  • Burkholderia phytofirmans;
  • ethylene;
  • PGPB;
  • Pseudomonas putida;
  • transgenic plants

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study showed that various rhizosphere bacteria producing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD), which can degrade ACC, the immediate precursor of ethylene in plants, and thereby lower plant ethylene levels, can act as promising biocontrol agents of pathogenic strains of Agrobacterium tumefaciens and A. vitis. Soaking the roots of tomato (Solanum lycopersicum) seedlings in a suspension of the ACCD-producing Pseudomonas putida UW4, Burkholderia phytofirmans PsJN or Azospirillum brasilense Cd1843 transformed by plasmid pRKTACC carrying the ACCD-encoding gene acdS from UW4, significantly reduced the development of tumours on tomato plants injected 4–5 days later with pathogenic Agrobacterium strains via wounds on the plant stem. The fresh mass of tumours formed by plants pretreated with ACCD-producing strains was typically four- to fivefold less than that of tumours formed on control plants inoculated only with a pathogenic Agrobacterium strain. Simultaneously, the level of ethylene evolution per amount of tumour mass on plants pretreated with ACCD-producing bacteria decreased four to eight times compared with that from tumours formed on control plants or plants pretreated with bacteria deficient in ACCD production. Moreover, transgenic tomato plants expressing a bacterial ACCD were found to be highly resistant to crown gall formation relative to the parental, non-transformed tomato plants. The results support the hypothesis that ethylene is a crucial factor in Agrobacterium tumour formation, and that ACCD-produced rhizosphere bacteria may protect plants infected by pathogenic Agrobacteria from crown gall disease.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The genus Agrobacterium belongs to the Rhizobiaceae family of α-Proteobacteria and includes plant pathogens that cause crown gall and hairy root diseases. The soilborne bacterium Agrobacterium tumefaciens induces tumours (crown galls) in more than 1000 different dicotyledonous and some monocotyledonous plant species, including many economically important plants. Strains of Agrobacterium vitis are the predominant cause of crown gall tumours of grape in various grape-growing regions worldwide (Tzfira & Citovsky, 2008).

The pathogenicity of Agrobacterium strains results from the presence of Ti (tumour-inducing) plasmids. During crown gall induction, a specific segment (T-DNA) of the Ti-plasmid is transferred and integrated into the plant host cell genome. The T-DNA possesses oncogenic (onc) genes which are expressed in transformed plants and are involved in phytohormone synthesis. Two genes (iaaM and iaaH) code for indole-3-acetic acid (IAA, auxin) synthesis, ipt is a cytokinin biosynthesis gene, and gene 6b increases the auxin sensitivity of the transformed plant cells. The delivery, insertion and subsequent expression of these onc genes in(to) the plant genomic DNA leads to a phytohormonal imbalance in the infected plants, altering the normal rate of cell division and resulting in tumorous growth (Tzfira & Citovsky, 2008).

In addition to the well-known roles of auxin and cytokinin in crown gall formation and morphogenesis, the plant hormone ethylene, which functions as a regulator in many aspects of plant life, also plays an important role in this process. Galls induced by A. tumefaciens produce very high ethylene concentrations, suggesting that tumour-induced ethylene is a controlling factor in gall development (Aloni & Ullrich, 2008). Ethylene levels produced in crown galls are up to 140 times higher than in wounded but uninfected control stems of tomato, reaching a maximum at 5 weeks after infection (Aloni et al., 1998; Wächter et al., 1999). It was also suggested that the high auxin levels induced by the T-DNA-encoded oncogenes stimulate ethylene production (Aloni & Ullrich, 2008), as IAA is well known to activate the transcription of the enzyme ACC synthase. The vigorous ethylene synthesis in galls is enhanced by high levels of auxin and cytokinin (Wächter et al., 1999). These studies demonstrate that there is a critical role for ethylene in determining crown gall development and morphogenesis. Thus, the T-DNA-encoded oncogenes, namely iaaH, iaaM and ipt, trigger a cascade of the following phytohormones: auxin, cytokinin, ethylene, abcisic acid and jasmonic acid, which, together with gene 6b expression-dependent flavonoid accumulation, promote crown gall growth (Veselov et al., 2003; Gális et al., 2004).

Many plant-growth-promoting bacteria (PGPB) produce the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD) [EC 4·1·99·4], which can cleave ACC, the immediate precursor of ethylene in plants, to α-ketobutyrate and ammonia (Honma & Shimomura, 1978). Glick et al. (1998) proposed a model in which a PGPB containing ACCD attached to the plant tissue provides a sink for ACC from the plant tissue and thereby reduces ethylene synthesis, promotes root elongation and reduces the plant’s stress symptoms. Recently, it was demonstrated that A. tumefaciens strain C58 partially lost its ability to induce crown gall tumours on tomato plants upon either being transformed with an ACCD gene, acdS, from the PGPB strain Pseudomonas putida UW4, or upon being co-inoculated with this natural ACCD-producing strain (i.e. P. putida UW4). In both types of experiments, it was observed that the presence of ACCD was inhibitory to tumour development on both tomato and castor bean plants (Hao et al., 2007).

The objectives of the present work were to: (i) confirm that the production of ACCD is indeed an important trait for the ability of rhizosphere bacteria to protect plants against crown gall formation, (ii) ascertain whether this effect is specific for A. tumefaciens strains or if it can be implemented for the control of A. vitis strains as well, (iii) determine if ACCD-producing strains can serve as biocontrol agents for the prevention of crown gall disease when the PGPB strain is applied to plants prior to infection by pathogenic Agrobacterium, (iv) assess whether the pretreatment of plants with ACCD-producing strains influences the level of ethylene in crown galls and stems of plants infected by Agrobacterium, and (v) determine if transgenic plants that produce bacterial ACCD are more resistant to crown gall disease than non-transformed plants.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains and media

Bacterial strains used in this work are listed in Table 1. Luria-Bertani broth (LB) or Luria-Bertani agar (1·5% w/v, LA) (Ausubel et al., 1994) and potato dextrose agar (PDA) were used for the experiments.

Table 1.   Bacterial strains used in this work
StrainRelevant characteristicsOrigin, sourceReferences
Agrobacterium tumefaciens C58Nopaline pTiCherry crown gall, USASciaky et al., 1978
A. tumefaciens Sh-1Octopine/agropine pTiVitis vinifera cv. Tsolikouri stem, Republic of GeorgiaDandurishvili et al., 2009
Agrobacterium vitis S4Vitopine pTiV. vinifera cv. Sárfehér crown gall, HungarySzegedi et al., 1988
A. vitis Tm4Octopine/cucumopine pTiV. vinifera cv. Téli Muskotály crown gall, HungarySzegedi et al., 1988
Azospirillum brasilense Cd1843Non-ACCD-producing strainRhizosphere of maize, FrancePenot et al., 1992
A. brasilense Cd1843/pRKTACCA. brasilense Cd1843, transformed by plasmid pRKTACC carrying the acdS gene from P. putida UW4Glick laboratory constructHolguin & Glick, 2003
Burkholderia phytofirmans PsJNACCD-producing strainOnion roots, CanadaSessitsch et al., 2005
B. phytofirmans PsJN acdS-ACCD-deficient deletion mutant of PsJNGlick laboratory constructSun et al., 2009
Pseudomonas fluorescens B-4117Potential biocontrol agent of crown gall disease of grapeSoil from a botanical garden, GeorgiaKhmel et al., 1998
Pseudomonas putida UW4ACCD-producing strainRhizosphere of reeds, CanadaGlick et al., 1995
P. putida UW4 acdS-ACCD-deficient mutant of UW4Glick laboratory constructLi et al., 2000
Serratia plymuthica IC1270Broad-range biocontrol agentRhizosphere of grapevine, UzbekistanOvadis et al., 2004

Plants

Tomato (Solanum lycopersicum) cvs M82D and UC82B, plus line 8338 [a transgenic derivative of the UC82B expressing a bacterial ACCD encoded by the acdS gene from Pseudomonas strain 6G5 under control of the 35S promoter (Klee et al., 1991)] were used in greenhouse experiments, since this plant species is universally susceptible to tumour formation induced by pathogenic agrobacteria.

Tumorigenicity and biocontrol assays in the greenhouse

Tomato (cv. M82D) seeds were planted in small plastic trays (8 × 12 × 6 cm) and grown for 3–4 weeks at 25°C in a growth chamber with a controlled environment: 26°C, 80% relative humidity, light at 300 μE m−2 s−1 and a daily photoperiod of 14 h. Bacterial inoculants were prepared by growing the bacteria in LB medium overnight at 28°C with shaking, then washing twice with tap water. Roots of 4-week-old tomato seedlings with two to three true leaves grown in trays in the nursery were soaked overnight in an aqueous suspension of the tested bacterium (∼108 cells mL−1) or tap water. The plants were then transferred to soil in 2·5-L pots, placed in the greenhouse and infected 4–5 days later with pathogenic strains of either A. tumefaciens or A. vitis (∼108 cells mL−1 in sterile distilled water). For inoculation, plants were scratched and the pathogenic agrobacteria (10 μL) were injected into stems wounded in the middle of second and third internodes, about 5–7 and 9–10 cm above the root neck, respectively. A similar procedure was used to test transgenic line 8338, which produced bacterial ACCD, and non-transgenic (cv. UC82B) tomato plants for resistance to crown gall disease. However, the seedlings grown first in the nursery and then transferred to the greenhouse as described above were directly infected with A. tumefaciens or A. vitis, so the step of seedling pretreatment by root soaking with the ACCD-producing strains was omitted. Crown gall formation was recorded 4–5 weeks after pathogen inoculation. Disease index was rated in terms of tumour fresh mass.

Ethylene measurement

The procedure described by Aloni et al. (1998) was used. The level of ethylene in crown galls and internodes excised from tomato plants (cv. M82D) treated as described above was detected with an FID gas chromatograph (Varian 3300, USA), with a Poropak column, oven temperature of 80°C and carrier gas N2 at 100 mL min−1. For ethylene measurements, the excised tumours or internodes were held in 30-mL tubes closed with rubber stoppers. To reduce the effect of wounding caused by the cuts made in the stem during the excision of the crown galls and internode pieces, tissues were cut from plants and first incubated for 2 h in open tubes before ethylene measurement was started, then the tubes were closed and incubated for an additional 3 h before starting the measurement of the ethylene level in 1-mL air samples. Evolution of ethylene from non-transformed tomato cv. UC82B and its transgenic derivative 8338 was generally measured as described above; following the protocol used by Klee et al. (1991), whole leaves instead of internodes were enclosed in sealed containers and 1·0-mL gas samples were withdrawn after 1 h. The calculation for ethylene production was: units = amount of ethylene (p.p.m.) × volume of incubation tubes (30 cm3)/incubation time (3 h) × fresh weight (g) of plant tissue or tumours.

Statistical analysis

All statistical analyses were carried out using jmp8 software (SAS Institute Inc.). Data were analysed by one-way analysis of variance (anova). Mean comparisons were made by the Tukey-Kramer honestly significant difference (HSD) multiple range test at < 0·05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Suppression of crown gall development by ACCD-producing bacteria

The ACCD-producing strains Pseudomonas putida UW4, Burkholderia phytofirmans PsJN and Azospirillum brasilense Cd1843/pRKTACC, carrying the acdS gene from UW4 under the tetracycline resistance (tet) promoter (Table 1), were tested for the ability to protect tomato plants (cv. M82D) against pathogenic Agrobacterium strains. The formation of tumours was strongly inhibited when the roots of 4-week-old tomato seedlings grown in the nursery were soaked in a suspension of an ACCD-containing bacterium and then transferred into the greenhouse and infected 4–5 days later by injection into a wound on the stem with a pathogenic Agrobacterium strain. Average fresh mass of tumours formed by A. tumefaciens strain Sh-1 decreased from 0·4 g in control plants to about 0·1 g when tomato seedlings were pretreated with strains UW4, PsJN or Cd1843/pRKTACC prior to Agrobacterium injection. In the case of A. vitis S4, the average mass of tumours on control plants was three- to 10-fold less than in pretreated plants. The results indicate that the ability to produce ACCD is a necessary requirement for the tested strains to protect plants from gall development (Figs 1 and 2). It was noteworthy that a small but significant reduction in tumour mass was observed when roots were pretreated with non-ACCD-producing strains UW4acdS-, PsJNacdS and Cd1843. The reason for this residual activity of the non-ACCD-producing strains is not clear.

image

Figure 1.  Effect of ACC deaminase (ACCD)-producing bacteria on the formation of crown galls. Roots of 4-week-old tomato seedlings grown in the nursery were soaked in a suspension of the tested bacteria or tap water, then the seedlings transferred into the greenhouse and injected 4–5 days later with pathogenic Agrobacterium vitis S4 (A) or A. tumefaciens Sh-1 (B) via a wound on the stem. Variants are indicated as strain S4 or Sh-1, followed by the strain used for root soaking: Pseudomonas putida UW4, Burkholderia phytofirmans PsJN and Azospirillum brasilense Cd1843/pRKTACC, or their respective derivatives deficient in ACCD production: UW4acdS, PsJNacdS and Cd1843. Columns with the same letter are not significantly different according to Tukey’s HSD multiple range test at < 0·05.

Download figure to PowerPoint

image

Figure 2.  Crown gall formation on stems of tomato seedlings after roots were treated with Burkholderia phytofirmans PsJN (1), Azospirillum brasilense Cd1843/pRKTACC acdS+ (3), Pseudomonas putida UW4 (5) or their respective derivatives deficient in ACC deaminase production: PsJNacdS (2) Cd1843 (4) and UW4acdS (6), 4–5 days before Agrobacterium vitis S4 was injected into a wound on the stem. Control (7) roots were soaked in tap water and then plants infected with A. vitis S4 as above.

Download figure to PowerPoint

Ethylene level in crown galls formed on tomato plants pretreated with ACCD-producing bacteria

A dramatic increase in ethylene production is observed in tomato crown galls induced by A. tumefaciens in comparison to the level of ethylene in gall-less internodes of the same plant or in internodes of non-infected control plants (Aloni et al., 1998). The present study confirmed this observation, demonstrating that the level of ethylene per mass of internodes carrying galls, induced by strains A. tumefaciens Sh-1 or A. vitis S4 and excised from tomato plants 4 weeks after infection, was up to 10-fold greater than of internodes of control plants (Fig. 3) or of gall-less internodes of infected plants (data not shown). However, the level of ethylene per mass of internodes carrying galls induced by strains A. tumefaciens Sh-1 or A. vitis S4 on plants whose roots were pretreated with the ACCD-producing strain P. putida UW4 was significantly less than that of internodes carrying small galls formed on plants pretreated by bacteria deficient in ACCD production. The effect was found to be specific to the ACCD-producing strain, since no decrease in the level of ethylene per mass was observed in internodes carrying large galls normally induced by strains A. tumefaciens Sh-1 or A. vitis S4 formed on tomato plants pretreated with the ACCD-deficient mutant strain P. putida UW4acdS, or in internodes carrying galls formed on tomato plants pretreated with bacterial antagonists of Pseudomonas fluorescens B-4117 or Serratia plymuthica IC1270 (Fig. 3). As was shown recently, using of these antagonists for pretreatment of tomato seedlings by soaking of roots prior to injection of pathogenic Agrobacterium into the plant stem led to the formation of small tumours (Dandurishvili et al., 2009), similar in size to those that appeared on plants pretreated with ACCD-producing bacteria. Both of these antagonists produced only on a basal level of ACCD, comparable to that found in the UW4acdS mutant (data not shown).

image

Figure 3.  Ethylene levels in crown galls formed on tomato plants pretreated with ACC deaminase (ACCD)-producing bacteria. Plants were treated as described in Fig. 1 and variants are indicated as strain S4 or Sh-1, followed by the strain used for root soaking. Ethylene was measured by a gas chromatograph. Units = amount of ethylene (p.p.m.) × volume of incubation tubes (30 cm3)/incubation time (3 h) × weight of tumours or internodes of untreated tomato (control). Columns with the same letter are not significantly different according to Tukey’s HSD multiple range test at < 0·05.

Download figure to PowerPoint

Resistance of ACCD-producing transgenic tomato to crown gall disease

To investigate whether the protection that treatment of tomato plants with ACCD-producing rhizobacteria afforded against crown gall formation could be provided to plants by transforming them with a bacterial ACCD, tomato cv. UC82B and its transgenic line 8338 were used. Evaluation of ethylene from young leaves of wild-type plants and line 8338 was performed before inoculation with the Agrobacterium strains. The level of ethylene production from leaves of line 8338 was 0·56 ± 0·1 units vs. 5·37 ± 1·09 units from leaves excised from non-transgenic plants. Seeds from the transgenic plants germinated normally and the plants were phenotypically indistinguishable from controls. This indicates that reduction of ethylene synthesis in these transgenic plants did not cause any apparent vegetative phenotypic abnormalities (Klee et al., 1991). On the other hand, a very strong suppression of crown gall formation was observed in transgenic plants infected by oncogenic strains of A. tumefaciens or A. vitis, relative to the parental tomato line. The average mass of tumours formed by strains A. tumefaciens C58 and Sh-1 or A. vitis S4 and Tm4 on wild-type tomato varied between 0·3 and 0·5 g, whilst the mass of tumours induced by the same strains on the transgenic tomato line expressing bacterial acdS gene was reduced five- to 10-fold (Fig. 4). These results directly show the critical role for ethylene in determining crown gall morphogenesis and support the idea that ethylene plays an important role in crown gall formation, possibly by interfering with auxin. On the other hand, ACCD, whether it is present as part of a PGPB or via a transgene in the plant, alters the balance of phytohormones essential for tumorigenesis in plants transformed by pathogenic Agrobacteria.

image

Figure 4.  Comparison of crown gall formation in ACC deaminase (ACCD)-producing transgenic tomato line 8338 and non-transformed tomato cv. UC82B. Four-week-old nursery-grown seedlings were transferred into the greenhouse and injected 4–5 days later with Agrobacterium tumefaciens C58 or Sh-1 or A. vitis S4 or Tm4 via a wound on the stem. Columns with the same letter are not significantly different according to Tukey’s HSD multiple range test at < 0·05.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

ACC is the immediate precursor of the phytohormone ethylene, an important mediator of stress responses and plant growth and development (Abeles et al., 1992). A model describing how ACCD-containing PGPB modulate plant ethylene levels has been described (Glick et al., 1998). Briefly, along with other small molecules, a portion of the ACC in plants is exuded from plant roots or seeds, and the bacteria attached to the surface of the seeds or roots can take up some of this ACC and cleave it through the activity of ACCD, thereby acting as a sink for ACC. Thus, ACCD can limit inhibitory ethylene levels, often associated with various stresses, such as flooding, drought, pathogens, high salinity, and the presence of organic and inorganic soil contaminants, while maintaining lower beneficial ethylene levels in plants (Stearns & Glick, 2003). This model is consistent with a wide range of experimental data (Glick et al., 2007).

In view of these observations, it is not surprising that inhibitors of ethylene synthesis, such as the enzyme ACCD, can suppress Agrobacterium-mediated tumour growth. The results of the work described here confirm and extend the previous observation of Hao et al. (2007) that bacterial ACCD being expressed in a pathogenic A. tumefaciens strain or being produced by a non-pathogenic rhizosphere bacterium (e.g. P. putida UW4) used for co-inoculation of the plant together with the pathogen can decrease crown gall formation. As shown here, not only co-inoculation with an ACCD-producing strain, but also root soaking with ACCD-producing bacteria helps plants to resist crown gall formation on stems infected by pathogenic Agrobacterium. The effect was observed with various ACCD producers, including naturally occurring strains of P. putida (UW4) and B. phytofirmans (PsJN), as well as a strain of A. brasilense (Cd1843) that expressed ACCD following transformation with a plasmid carrying an acdS gene. In all instances tested, the ability of an added bacterium to produce ACCD is the critical trait for the prevention of gall development; the ACCD-minus mutants UW4acdS and PsJNacdS, and the parental A. brasilense strain Cd1843 used for soaking of roots before infection were all unable to inhibit gall development induced by pathogenic Agrobacterium. The results support the idea that ACCD provided by PGPB prevents the overproduction of ethylene in the tumour tissues and in turn alters the balance of auxin and other hormones essential for tumorigenesis in plants infected by pathogenic Agrobacteria.

The level of ethylene in galls induced by strains of A. tumefaciens or A. vitis on plants whose roots were pretreated with the ACCD-producing bacteria was significantly less than that in galls formed on plants pretreated with bacteria deficient in ACCD production, including the ACCD-deficient mutant strain P. putida UW4acdS and bacterial antagonists P. fluorescens B-4117 or S. plymuthica IC1270. The latter two antagonists were found to protect tomato plants from tumour formation using a mechanism other than that of ACCD production (Dandurishvili et al., 2009).

The results obtained here support the hypothesis that ethylene plays an important role in crown gall formation and that ACCD provided by PGPB alters the balance between ethylene, auxin and other phytohormones essential for plant tumorigenesis. The transgenic tomato plants expressing a bacterial ACCD were used here to further sustantiate this hypothesis. These transgenic plants were highly resistant to crown gall formation in comparison to the parental, non-transformed tomato plants. These results also support the role of ethylene as a crucial factor in crown gall formation. Aloni et al. (1998), using the ethylene-insensitive tomato mutant Never ripe (Nr), observed that although tumours caused enhanced ethylene production in both Nr and control plants, since the Nr plants did not perceive the ethylene tumour formation was inhibited. Insensitivity of the Nr mutant to ethylene action in some sense mimics the effect of decreased ethylene levels in galls formed by plants treated with ACCD-producing bacteria or transgenic tomato expressing bacterial ACCD. Moreover, it is likely that such a plant would be damaged to a lesser extent by most pathogens, including Agrobacterium, than its wild-type counterpart.

The nontumorigenic strain A. radiobacter K84, producing the anti-agrobacterial compound agrocin 84, is used worldwide for biocontrol of crown gall disease caused by A. tumefaciens of the nopaline/agrocinopineA-type (Ryder & Jones, 1990). However, other A. tumefaciens strains, as well as strains of A. vitis, are insensitive to agrocin 84, rendering biological control of crown gall by K84 ineffective in those instances. This emphasizes the need for alternative antagonistic bacteria, and indeed, considerable effort has been invested in identifying new strains capable of controlling crown gall on grape. Most of these antagonists produce other types of agrocins or antibacterial compounds that are specifically inhibitory to pathogenic A. vitis (Burr et al., 1998; Otten et al., 2008). Some rhizosphere strains of P. fluorescens and S. plymuthica, which produce broad-range antibiotics that are efficient against various plant-pathogenic bacteria and fungi, significantly suppressed the development of crown galls on plants infected by pathogenic A. tumefaciens or A. vitis strains (Khmel et al., 1998; Dandurishvili et al., 2009). These results may help to explain the observed residual ability of non-ACCD-producing strains to reduce tumour mass (Fig. 1a and b) which could be related to production of as-yet unidentified compounds able to influence tumour. For instance, antimicrobial volatiles, shown to suppress growth of A. tumefaciens and A. vitis strains in vitro (Dandurishvili et al., 2009), may have been produced.

The data presented here support those of Hao et al. (2007) that either the cloning and expression of the ACCD-encoding gene from P. putida UW4 in A. tumefaciens strain C58 or the co-inoculation of these two strains into tomato internodes inhibits gall development. Additionally, these results indicate the prophylactic potential of ACCD-producing bacteria against crown gall formation, since the effect was observed when the treatment of plants with protecting and pathogenic bacterial strains was separated both spatially and temporally. In addition, the ACCD-producing strains demonstrated their ability to suppress crown galls induced by the related pathogen A. vitis which is still a problem to control, with only a limited number of bacteria showing biocontrol activity against this pathogen (Otten et al., 2008).

ACCD-producing B. phytofirmans strain PsJN was able to establish rhizosphere and endophytic populations associated with various plants, including tomato and grapevines, where it stimulated plant growth (Sessitsch et al., 2005), whilst P. putida UW4 and A. brasilense Cd1843 used in this work are not endophytes (B. Glick, unpublished data). Thus, both endophytic and rhizospheric bacteria are effective at controlling infection by agrobacteria.

To summarize, these results indicate that crown gall formation may be prevented by pretreatment of plant roots with ACCD-producing non-pathogenic bacteria before the plant stems become infected by pathogenic Agrobacterium strains, thereby suggesting a possible prophylactic role for ACCD-producing bacteria towards crown gall disease. Importantly, the ACCD strains exhibit a comparable level of protection against both A. tumefaciens and A. vitis, the species known as the main causative agent of crown gall disease on grape (Ryder & Jones, 1990; Burr & Otten, 1999). One of the main goals of this study was to investigate potential of ACCD-producing rhizosphere bacteria to protect sensitive plants specifically towards A. vitis-induced crown galls. Tomato was chosen as the test plant because it was found to be a universal host both for A. tumefaciens and A. vitis (Szegedi, 1985), able to give rapid and reproducible results. Also, ACCD-producing transgenic tomato plants were available, so the whole set of experiments could be carried out on the same test species. The potential of ACCD-producing strains as biocontrol agents of A. vitis was thus demonstrated, although on the vine the effect may be different. Clearly, a 3-year old grapevine is different from a 5-week-old tomato seedling deliberately wounded only a few centimeters from the rhizosphere. Therefore long-term studies are needed to ascertain if (i) similar tumour inhibition occurs on grapevine and (ii) these bacteria can survive for a long period of time in the grape rhizospere and vessels.

The use of ACCD-containing bacteria may be viewed as a general antipathogenic agent. In this regard, plants treated with ACCD-containing bacteria (Wang et al., 2000; Barka et al., 2002) or transformed with exogenous ACCD genes (Lund et al., 1998; Robison et al., 2001) are also less susceptible to a range of other pathogenic agents.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research was supported by USAID-CDR grant no. TA-MOU-03-CA23-037. ES was supported by the Hungarian Scientific Foundation (OTKA) grant no. K-68053. We are thankful to Harry J. Klee (University of Florida, Gainesville, USA) for supplying transgenic tomato seeds, to Mrs Asya Weksler (ARO, Volcani Center, Israel) for help with gas chromatography and to Mr Udi Landau (HUJI, Israel) for help with statistical analyses.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Abeles FB, Morgan PW, Saltveit Jr ME, 1992. Ethylene in Plant Biology. New York, USA: Academic Press.
  • Aloni R, Ullrich CI, 2008. Biology of crown gall tumors. In: TzfiraT, CitovskyV, eds. Agrobacterium: From Biology to Biotechnology. New York, USA: Springer, 565591.
  • Aloni R, Wolf A, Feigenbaum P, Avni A, Klee HJ, 1998. The Never ripe mutant provides evidence that tumor-induced ethylene controls the morphogenesis of Agrobacterium tumefaciens-induced crown galls on tomato stems. Plant Physiology 117, 841847.
  • Ausubel FM, Brent R, Kingston RE et al. , 1994. Current Protocols in Molecular Biology. New York, USA: John Wiley & Sons Inc.
  • Barka EA, Gognies S, Nowak J, Audran J-C, Belarbi A, 2002. Inhibitory effect of endophyte bacteria on Botrytis cinerea and its influence to promote the grapevine growth. Biological Control 24, 135142.
  • Burr TJ, Otten L, 1999. Crown gall of grape: biology and disease management. Annual Review of Phytopathology 37, 5380.
  • Burr TJ, Bazzi C, Süle S, Otten L, 1998. Crown gall of grape: biology of Agrobacterium vitis and the development of disease control strategies. Plant Disease 82, 12881297.
  • Dandurishvili N, Szegedi E, Eliashvili P et al. , 2009. In-vitro and in-planta suppression of oncogenic strains of Agrobacterium vitis and Agrobacterium tumefaciens by bacterial biocontrol agents. In : EladY, MaurhoferM, KeelC, GesslerC, DuffyB, eds. Proceedings of the Meeting “Molecular Tools for Under-standing and Improving Biocontrol”, Interlaken, Switzerland . IOBC/WPRS Bulletin vol. 43, 225229.
  • Gális I, Kakiuchi Y, Simek P, Wabiko H, 2004. Agrobacterium tumefaciens AK-6b gene modulates phenolic compound metabolism in tobacco. Phytochemistry 65, 169179.
  • Glick BR, Karaturovíc D, Newell P, 1995. A novel procedure for rapid isolation of plant growth-promoting rhizobacteria. Canadian Journal of Microbiology 41, 533536.
  • Glick BR, Penrose DM, Li J, 1998. A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria. Journal of Theoretical Biology 190, 6368.
  • Glick BR, Todorovic B, Czarny J, Cheng Z, Duan J, McConkey B, 2007. Promotion of plant growth by bacterial ACC deaminase. Critical Reviews in Plant Sciences 26, 227242.
  • Hao Y, Charles TC, Glick BR, 2007. ACC deaminase from plant growth-promoting bacteria affects crown gall development. Canadian Journal of Microbiology 53, 12911299.
  • Holguin G, Glick BR, 2003. Transformation of Azospirillum brasilense Cd with an ACC deaminase gene from Enterobacter cloacae UW4 fused to the Tet(r) gene promoter improves its fitness and plant growth promoting ability. Microbial Ecology 46, 122133.
  • Honma M, Shimomura T, 1978. Metabolism of 1-aminocyclopropane-1-carboxylic acid. Agricultural and Biological Chemistry 42, 18251831.
  • Khmel IA, Sorokina TA, Lemanova NB et al. , 1998. Biological control of crown gall in grapevine and raspberry by two Pseudomonas spp. with a wide spectrum of antagonistic activity. Biocontrol Science and Technology 8, 4557.
  • Klee HJ, Hayford MB, Kretzmer KA, Barry GF, Kishore GM, 1991. Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants. The Plant Cell 3, 11871193.
  • Li J, Ovakim D, Charles TC, Glick BR, 2000. An ACC deaminase minus mutant of Enterobacter cloacae UW4 no longer promotes root elongation. Current Microbiology 41, 101105.
  • Lund ST, Stall RE, Klee HJ, 1998. Ethylene regulates the susceptible response to pathogen infection in tomato. The Plant Cell 10, 371382.
  • Otten L, Burr T, Szegedi E, 2008. Agrobacterium: a disease-causing bacterium. In: TzfiraT, CitovskyV, eds. Agrobacterium: From Biology to Biotechnology. New York, USA: Springer, 146.
  • Ovadis M, Liu X, Gavriel S, Ismailov Z, Chet I, Chernin L, 2004. The global regulator genes from biocontrol strain Serratia plymuthica IC1270: cloning, sequencing, and functional studies. Journal of Bacteriology 186, 49864993.
  • Penot I, Berges N, Guinguene C, Fages J, 1992. Characterization of Azospirillum associated with maize (Zea mays) in France, using biochemical tests and plasmid profiles. Canadian Journal of Microbiology 38, 798802.
  • Robison MM, Shah S, Tamot B, Pauls KP, Moffatt BA, Glick BR, 2001. Reduced symptoms of Verticillium wilt in transgenic tomato expressing a bacterial ACC deaminase. Molecular Plant Pathology 2, 135145.
  • Ryder MH, Jones DA, 1990. Biological control of crown gall. In: HornbyD, ed. Biological Control of Plant Pathogens. Wallingford, UK: CAB International Publishing, 4563.
  • Sciaky D, Montoya AL, Chilton MD, 1978. Fingerprints of Agrobacterium Ti plasmids. Plasmid 1, 238253.
  • Sessitsch A, Coenye T, Sturz AV et al. , 2005. Burkholderia phytofirmans sp. nov., a novel plant-associated bacterium with plant-beneficial properties. International Journal of Systematic and Evolutionary Microbiology 55, 11871192.
  • Stearns JC, Glick BR, 2003. Transgenic plants with altered ethylene biosynthesis or perception. Biotechnology Advances 21, 193210.
  • Sun Y, Cheng Z, Glick B, 2009. The presence of a 1-aminocyclopropane-1-carboxylate (ACC) deaminase deletion mutation alters the physiology of the endophytic plant growth-promoting bacterium Burkholderia phytofirmans PsJN. FEMS Microbiology Letters 296, 131136.
  • Szegedi E, 1985. Host range and specific l(+)tartrate utilization of biotype 3 of Agrobacterium tumefaciens. Acta Phytopathologica Academiae Scientiarum Hungaricae 20, 1722.
  • Szegedi E, Czakó M, Otten L, Koncz CS, 1988. Opines in crown gall tumors induced by biotype 3 isolates of Agrobacterium tumefaciens. Physiological and Molecular Plant Pathology 32, 237247.
  • Tzfira T, Citovsky V, 2008. Agrobacterium: From Biology to Biotechnology. New York, USA: Springer.
  • Veselov D, Langhans M, Hartung W et al. , 2003. Development of Agrobacterium tumefaciens C58-induced plant tumors and impact on host shoots are controlled by a cascade of jasmonic acid, auxin, cytokinin, ethylene and abscisic acid. Planta 216, 512522.
  • Wächter R, Fischer K, Gäbler R et al. , 1999. Ethylene production and ACC-accumulation in Agrobacterium tumefaciens-induced plant tumours and their impact on tumour and host stem structure and function. Plant Cell and Environment 22, 12631273.
  • Wang C, Knill E, Glick BR, Défago G, 2000. Effect of transferring 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA derivative CHA96 on their growth-promoting and disease-suppressive capacities. Canadian Journal of Microbiology 46, 898907.