Aim: To examine the biocontrol activity of broad-range antagonists Serratia plymuthica IC1270, Pseudomonas fluorescens Q8r1-96 and P. fluorescens B-4117 against tumourigenic strains of Agrobacterium tumefaciens and A. vitis. Methods and Results: Under greenhouse conditions, the antagonists, applied via root soak prior to injecting Agrobacterium strains into the wounded stems, significantly suppressed tumour development on tomato seedlings. A derivative of P. fluorescens Q8r1-96 tagged with a gfp reporter, as well as P. fluorescens B-4117 and S. plymuthica IC1270 marked with rifampicin resistance, stably persisted in tomato tissues for at least 1 month. Mutants of P. fluorescens Q8r1-96 and S. plymuthica IC1270 deficient in 2,4-diacetylphloroglucinol or pyrrolnitrin production, respectively, also proficiently suppressed the tumour development, indicating that these antibiotics are not responsible for the observed biocontrol effect on crown gall disease. The volatile organic compounds (VOCs) produced by the tested P. fluorescens and S. plymuthica strains inhibited the growth of A. tumefaciens and A. vitis strains in vitro. Solid-phase microextraction-gas chromatography-mass spectrometry analysis revealed dimethyl disulfide (DMDS) as the major headspace volatile produced by S. plymuthica IC1270; it strongly suppressed Agrobacterium growth in vitro and was emitted by tomato plants treated with S. plymuthica IC1270. 1-Undecene was the main volatile emitted by the examined P. fluorescens strains, with other volatiles, including DMDS, being detected in only relatively low quantities. Conclusions: S. plymuthica IC1270, P. fluorescens B-4117 and P. fluorescens Q8r1-96 can be used as novel biocontrol agents of pathogenic Agrobacterium. VOCs, and specifically DMDS, might be involved in the suppression of oncogenicity in tomato plants. However, the role of specific volatiles in the biocontrol activity remains to be elucidated. Significance and Impact of the Study: The advantage of applying these antagonists lies in their multiple activities against a number of plant pathogens, including Agrobacterium.

We show that volatile organic compounds (VOCs) produced by rhizospheric strains Pseudomonas fluorescens B-4117 and Serratia plymuthica IC1270 may act as inhibitors of the cell-cell communication quorum-sensing (QS) network mediated by N-acyl homoserine lactone (AHL) signal molecules produced by various bacteria, including strains of Agrobacterium, Chromobacterium, Pectobacterium and Pseudomonas. This quorum-quenching effect was observed when AHL-producing bacteria were treated with VOCs emitted by strains B-4117 and IC1270 or with dimethyl disulfide (DMDS), the major volatile produced by strain IC1270. LC-MS/MS analysis revealed that treatment of strains Pseudomonas chlororaphis 449, Pseudomonas aeruginosa PAO1 or Ps. fluorescens 2-79 with VOCs emitted by strain IC1270 or DMDS drastically decreases the amount of AHLs produced by these bacteria. Volatile organic compounds produced by Ps. chlororaphis 449 were able to suppress its own QS-induction activity, suggesting a negative interaction between VOCs and AHL molecules in the same strain. Quantitative RT-PCR analysis showed that treatment of Ps. chlororaphis 449 with VOCs emitted by cells of IC1270, B-4117 or 449 itself, or with DMDS, leads to significant suppression of transcription of AHL synthase genes phzI and csaI. Thus, along with AHLs, bacterial volatiles might be considered another type of signal molecule involved in microbial communication in the rhizosphere.

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.

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