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  • Research note
  • Open Access

Biological control agent Rhizobium (=Agrobacterium) vitis strain ARK-1 suppresses expression of the essential and non-essential vir genes of tumorigenic R. vitis

BMC Research Notes201912:1

https://doi.org/10.1186/s13104-018-4038-6

  • Received: 8 October 2018
  • Accepted: 24 December 2018
  • Published:

Abstract

Objective

To gain insights into the virulence suppressive mechanism of a nonpathogenic strain of Rhizobium vitis ARK-1, we co-inoculated ARK-1 with a tumorigenic (Ti) strain of R. vitis to examine the expression of two essential virulence genes (virA and virG) and one non-essential gene (virD3) of the Ti strain at the wound site of grapevine.

Results

Co-inoculation of ARK-1 with a Ti strain VAT03-9 at a 1:1 cell ratio into grapevine shoots resulted in significantly lower expression of the virulence genes virA, virD3, and virG of VAT03-9 at 1 day after inoculation compared with those when shoots were inoculated only with VAT03-9. ARK-1 was not able to catabolize acetosyringone, which is the plant-derived metabolites inducing the entire vir regulon in Ti strains, suggesting the direct effect of ARK-1 on the induction of broad range of vir genes of R. vitis Ti strains.

Keywords

  • Rhizobium vitis
  • Grapevine crown gall
  • Biological control
  • Gene expression
  • Acetosyringone

Introduction

Grapevine (Vitis vinifera L.) crown gall is caused mainly by Rhizobium vitis (Ti) [syn. Agrobacterium vitis (Ti)], where “Ti” means “tumorigenic”. To avoid confusion, we follow the nomenclature for Rhizobium species adopted by Young et al. [1]. Crown gall is one of the most important diseases of grapevine around the world [2, 3]. Infected vines generally lose their productivity, and rapid decline can be associated with the infection of young vines.

The virulence (vir) genes and transfer DNA (T-DNA) are located mostly in large tumor-inducing plasmids (pTi). Rhizobium Ti strains transfer T-DNA and several virulence effector proteins into plant host cells, and this infection pathway is mediated by a bacterial type IV secretion system [4, 5]. The plant phenolics acetosyringone (AS) and α-hydroxyacetosyringone induce the entire vir regulon in Rhizobium as well as the formation of T-DNA intermediate molecules [4]. T-DNA transfer and processing require products of the several vir genes, which are named as virA to virE, and virG and located outside of the T-DNA coding region [47].

Previously, we have reported that a nonpathogenic R. vitis strain VAR03-1, which was isolated from grapevine in Japan and strongly inhibited tumor formation in tomato, grapevine, rose, sunflower, and apple [811]. Moreover, we isolated and identified nonpathogenic R. vitis strain ARK-1, which performed much better than VAR03-1 at inhibiting tumor formation in grapevine in greenhouse and field trials, as a new antagonistic strain [1216]. ARK-1 is also endophytic in grapevine [12]. When grapevine shoots were inoculated with a Ti strain that was not affected by ARK-1 in the antibiosis assay, ARK-1 was able to suppress tumor formation. [13]. In addition, dead cells of ARK-1 (autoclaved) and the culture filtrate (CF) of ARK-1 (without cells) were not able to inhibit tumor formation in grapevine [15]. When ARK-1 and a Ti strain was co-inoculated, the number of colony-forming unit (cfu) of the Ti strain was not affected from 1 to 5 days after inoculation (dai), but it was significantly reduced at 7 dai [13, 14].

Saito et al. [17] have reported that the suppressive activity of antagonistic and non-pathogenic R. vitis strain VAR03-1 on the virulence gene expression of Ti was found to be its CF in vitro. Consistent with our speculation, the cfu of Ti strain was temporarily reduced after incubation of CF prepared from the growth medium of VAR03-1. Interestingly, the suppressive activity was detected in the high molecular weight fraction (> 100 kDa) of CF, suggesting that the antagonistic effects of VAR03-1 on Ti are mediated by large particles released in the culture media [17]. On the other hand, the CF of ARK-1 did not show suppressive effect on both the tumor formation and the expression of vir genes in planta experiments [14].

Two different mechanisms (antibiotic compounds or quorum-sensing) of biological control of plant crown gall disease using antagonistic bacteria have been reported [810, 1723], but disease suppression mechanism of ARK-1 is different from these two mechanisms [13, 14, 16]. The biological control activity of ARK-1 is likely based on the suppression of some essential virulence genes [14, 16]. Two Rhizobium proteins, VirD2 and VirE2 expressed by virD and virE, respectively, are directly associated with the T-strand [46]. Co-inoculation of grapevine shoots with ARK-1 and Ti strain at a 1:1 cell ratio resulted in significantly lower expression of the virulence genes virD2 and virE2 of Ti strain at 1 dai than expression levels of these genes by a Ti strain inoculated by itself [14, 16]. When a non-pathogenic R. vitis strain VAR06-30, which is neither antagonistic against R. vitis (Ti) nor limit the development of crown gall of grapevine, was co-inoculated with a Ti strain, expression levels of virD2 and virE2 were not affected (Additional file 1: Table S1), [14].

At this moment, it remains unclear if ARK-1 suppresses the expression of the other vir genes including essential or non-essential genes. Two Rhizobium proteins, VirA and VirG are directly associated with the T-strand as essential vir genes. VirA molecule works as the sensor protein to recognize the plant signal molecule AS. VirG protein works as the response regulator, which activates all genes in the regulon [46]. On the other hands, there are some non-essential genes such as virD3 in vir regulon, which are not required for tumorigenicity on plants [24].

To gain insights into the virulence suppressive mechanism of ARK-1, we co-inoculated ARK-1 with a Ti strain to examine the expression of two essential virulence genes (virA and virG) and one non-essential gene (virD3) of the Ti strain at the wound site of grapevine.

Main text

Methods

Detection of vir genes’ mRNA using the RT-qPCR

Cell suspensions of the non-pathogenic strains ARK-1 (antagonistic and non-pathogenic), VAR06-30 (non-antagonistic and non-pathogenic), and VAT03-9 (tumorigenic) (Additional file 1: Table S1) were prepared from 48-h-old cultures on potato sucrose agar medium slants [12, 14, 15]. Supernatant of these cultures was discarded, then the surface of the slant was washed with distilled water to obtain a cell suspension. Cell concentration was adjusted to approximately 108 cells mL−1. Two mixed cell suspensions at a cell ratio of 1:1 (ARK-1 plus VAT03-9, VAR06-30 plus VAT03-9), and a VAT03-9 suspension were prepared. An inoculation method was followed the needle-prick method using grapevine seedlings grown from a seed (2 years old, cv. ‘Neo Muscat’, seeds obtained from grape clusters cultivated in NARO, Japan) [14]. Each seedling (one plant per pot) was inoculated once with one of the mixed cell suspensions or with only VAT03-9. Seedlings inoculated with sterile distilled water were used as the negative control. We grew the seedlings in a greenhouse at 20 to 35 °C with natural sunlight and collected the shoot samples that included the one wound site. One sample, which was 0.2 g fresh weight, was obtained per plant from five plants (i.e., n = 5) at 1 dai.

The basic information for the RT-qPCR procedures, which followed the methods of Bustin et al. [25] and Kawaguchi [14], are summarized in Table 1 and the Additional file 2: Table S2. A housekeeping gene pyrG of Ti strain VAT03-9 was amplified to be used as both for the standardization of amplified products and an internal control [14]. To amplify the pyrG gene of VAT03-9 alone and to not amplify the same gene of strains ARK-1 and/or VAR06-30, specific primers and probe were designed and confirmed that these primers/probes worked well [14].
Table 1

Oligonucleotide primers and probes used in the RT-qPCR analyses

Name

Primer or probe

5′-3′ nucleotide sequence

Target DNA

Reference

Accession no.

Design or supplier

virA-F

Primer

CTC GAC TGA AAG AAG GAC ATA CTC A

virA

This study

CP000637

Licensed Dual-Labeled Probes for qPCR (Sigma-Aldrich)

virA-R

Primer

CGC AAG AAG GTC TAT CAT AAG GAA G

virA

This study

CP000637

Licensed Dual-Labeled Probes for qPCR (Sigma-Aldrich)

virA-P

Probe

AAT GGT TCC GCT CGC AAG CAC CTT G

virA

This study

CP000637

Licensed Dual-Labeled Probes for qPCR (Sigma-Aldrich)

virD3-F1P

Primer

GTC AAC TCG GGG AAG TCG T

virD3

This study

CP000637

ProbeFinder (Roche Diagnostics)

virD3-R1P

Primer

CAG TTC AGT TCG ATC GCT TG

virD3

This study

CP000637

ProbeFinder (Roche Diagnostics)

virD3-P

Probe

TTC CTC TG

virD3

This study

CP000637

Universal ProbeLibrary probe #6 Cat. no. 04685032001 (Roche Diagnostics)

virG-F

Primer

CTG TCT CGG ATG CGA GTA CG

virG

This study

CP000637

Licensed Dual-Labeled Probes for qPCR (Sigma-Aldrich)

virG-R

Primer

CGG AGT ATC TAA CGA TCC ACG C

virG

This study

CP000637

Licensed Dual-Labeled Probes for qPCR (Sigma-Aldrich)

virG-P

Probe

TGA CCG CCG TAT CCG ATA GTC AGC AAT T

virG

This study

CP000637

Licensed Dual-Labeled Probes for qPCR (Sigma-Aldrich)

VAT03-9pyrG-F-2

Primer

GGT GAA GTC TTC GTC ACC GAC

pyrG

[14]

AB272142

Licensed Dual-Labeled Probes for qPCR (Sigma-Aldrich)

VAT03-9pyrG-R-2

Primer

CGC CCT GTG AAG CGT TCA TAG

pyrG

[14]

AB272142

Licensed Dual-Labeled Probes for qPCR (Sigma-Aldrich)

VAT03-9pyrG-P-2

Probe

CGG CGC AGA GAC CGA CCT TGA TCT TG

pyrG

[14]

AB272142

Licensed Dual-Labeled Probes for qPCR (Sigma-Aldrich)

Relative quantification of the virA, virD3, and virG genes’ mRNA concentrations was carried out using the ΔΔCt method [26] by the Ct values of pyrG gene’s mRNA as reference points [14] across three independent technical replications. Relative expression rate of each vir gene in grapevine inoculated with ARK-1 plus VAT03-9 and VAR06-30 plus VAT03-9 was reported as percentage of the expression of each gene in grapevine inoculated with only VAT03-9 strain. All measurements were taken at 1 dai. The means of five biological replicates were used as a measured relative expression level.

The effect of treatment, gene, and their interaction on the median adjusted gene expression rate was examined using the linear mixed model ANOVA [packages ‘car’ and ‘lme4’ in R, ver. 3.5 (http://www.r-project.org/)]. In the model, treatment, gene, and their interaction were considered as fixed effects, and experimental replication was considered as a random effect. For the effect of treatment, Tukey’s HSD (package ‘emmeans’ in R) was used as a post hoc multiple comparison method.

Catabolism test of AS

Cell suspensions (2 mL each) of ARK-1, VAR06-30, and VAT03-9 were prepared from 24-h-old cultures on King’s B medium [27] in a shaking incubator at 28 °C. The tubes with each culture were centrifuged at 4000 rpm for 15 min. A supernatant of these strains was discarded and the surface was washed with distilled water to obtain a cell suspension, which was suspended in AT minimal media solution [2830] with 0.2% mannitol as a carbon source (called ATM) with supplemental biotin (2 μg mL−1). The cell concentration was adjusted to approximately 2 × 108 cells mL−1. Two millilitre of cell suspensions were prepared in three tubes per strain. Three tubes containing 2 mL of ATM were also prepared as a negative control. The 2 μL of AS (Sigma-Aldrich, Germany) was dissolved in dimethyl sulfoxide (DMSO) to produce a 10 mM stock solution. The 2 μL of the stock AS solution was applied to each tube of ARK-1, VAR06-30, VAT03-9, or the negative control. Each tube was incubated in a shaking incubator at 28 °C for 24-h. Then, 1 mL of culture supernatant was obtained from each culture after centrifugation with 13,000 rpm for 5 min. Then, concentration of AS in each culture fluid was measured at UV 296 nm after 1.95 min using the high-performance liquid chromatography (HPLC, HITACHI LaChrom Elite, Japan) equipped with Kinetex 2.6 μm Biphenyl column (150 × 4.6 mm) (Phenomenex, USA). The experiment was repeated three times. The effect of treatment on the concentration of AS was examined with a linear mixed model ANOVA as described above.

Results

Suppressive effect on expression of the vir genes of the Ti strain by co-inoculation with ARK-1 in grapevine plants

The treatment factor significantly affected the median adjusted expression rate (F = 21.9, P < 0.01), but the effect of gene (F = 1.0, P = 0.38) and treatment and gene interaction (F = 0.09, P = 0.98) were not significant. The median adjusted expression rate of virA, virD3, and virG genes were significantly suppressed (P ≤ 0.05) by co-inoculation of ARK-1 with VAT03-9 (Fig. 1). On the other hand, no significant difference (P > 0.05) in the adjusted median expression rate of virA, virD3, and virG were observed when co-inoculation of VAR06-30 with VAT03-9 and inoculation of VAT03-9 alone were compared (Fig. 1).
Fig. 1
Fig. 1

Relative expression rate of the virA, virD3, and virG genes in bacterial cells of the Rhizobium vitis Ti strain in grapevine plants measured using the reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Values (%) represent the relative expression rate of the vir genes at 1 day after inoculation (dai) compared with the value when only Ti strain VAT03-9 was inoculated (“Ti strain alone”), which had a value of 100%. Each cell suspension or mixture (ARK-1 plus a Ti, VAR06-30 plus a Ti, and a Ti alone) at cell ratios of 1:1 was inoculated onto the stems of grapevine plants after wounding. Stem samples were harvested at 1 dai. The center bar of the boxplot is the median, the lower and upper horizontal bars are 25th and 75th percentile, and whiskers show 95% range. Boxes labeled with different letters indicate a significant difference from the other bars in same gene expression (P ≤ 0.05, Tukey’s HSD test)

Catabolization of AS by ARK-1

There were no significant differences in the concentrations of AS among each R. vitis strain (F = 2.2, P = 0.19). No significant reduction of AS content was observed compared with the negative control (Fig. 2).
Fig. 2
Fig. 2

The concentration of acetosyringone (AS) after incubation Rhizobium vitis Ti strain (VAT03-9) and non-pathogenic strains (ARK-1 and VAR06-30) with AT medium (ATM) or only ATM without a bacterial strain as negative control. The center bar of the boxplot is the median, the lower and upper horizontal bars are 25th and 75th percentile, and whiskers show 95% range

Discussions

In our study, treatment with ARK-1 suppressed the expression of virA, virD3, and virG by the Ti strain in grapevine. In comparison, VAR06-30, a non-antagonistic and non-pathogenic strain, did not suppress the expression of these genes. With results from this and a previous study [14], ARK-1 has shown to be able to suppress the expression of at least five different vir genes: virA; virD3; virG (this study); virD2; and virE2 [14]. ARK-1 suppresses both essential (virA, virD2, virE2, and virG) and non-essential (virD3) virulence genes of R. vitis Ti strains. Since ARK-1 suppresses the expression of virA and virG, which are the first two triggers of expression of all other vir genes in Rhizobium Ti strains [46], there is a possibility that all subsequent expression of vir genes can be suppressed.

We previously demonstrated that ARK-1 suppressed the expression of vir genes of Ti when the expression was triggered by AS in a liquid culture medium [14]. However, there was a possibility that the suppression of these vir genes was a consequence of quick catabolization of AS by ARK-1, thus, the sensor protein VirA was not able to detect AS to trigger subsequent vir gene expressions [46].

In catabolization test, however, all the tested R. vitis strains including ARK-1was not able to catabolize AS (Fig. 2). This result indicates that ARK-1 is not capable of metabolizing AS to interfere with a Ti strain’s vir genes. Therefore, ARK-1 has an ability to suppress the expression of vir genes via other mechanism(s).

Conclusions

ARK-1 suppressed expression of the virulence genes virA, virD3, and virG of a Ti strain at the wound site without catabolizing AS. The suppressive effect of ARK-1 was not specific to the essential virulence genes.

Limitations

We have carried out the inoculation with grapevines seedlings, not adult trees.

Abbreviations

CF: 

culture filtrate

AS: 

acetosyringone

RT-qPCR: 

reverse transcriptase quantitative polymerase chain reaction

DMSO: 

dimethyl sulfoxide

Declarations

Authors’ contributions

AK and YN conceived and designed the study. AK, TI, and MW conducted experiments. AK, MN, and YN contributed to data analysis and led the writing of the manuscript. AK wrote the first draft. AK, MN, and YN wrote the second draft, critically reviewed, revised and finalized the manuscript. AK is the principal investigator and guarantor. All authors read and approved the final manuscript.

Acknowledgements

We are very grateful to all laboratory personnel of WARC, AHS Jr. Agricultural Research and Extension Center, and Graduate School of Environmental and Life Science of Okayama University.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

All data generated or analyzed during this study are included in this published article. For other data, these may be requested through the corresponding author.

Consent to publish

Not applicable.

Ethics approval and consent to participate

Not applicable.

Funding

This research was supported by KAKENHI Grant 17H03778 from the Ministry of Education, Culture, Sports, Science and Technology of Japan to A. Kawaguchi and Y. Noutoshi. This funding worked in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

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Authors’ Affiliations

(1)
Western Region Agricultural Research Center, National Agriculture and Food Research Organization (NARO), 6-12-1 Nishifukatsu-cho, Fukuyama Hiroshima, 721-8514, Japan
(2)
AHS Jr. Agricultural Research and Extension Center, School of Plant and Environmental Sciences, Virginia Polytechnic Institute and State University, 595 Laurel Grove Rd, Winchester, VA 22602, USA
(3)
Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan

References

  1. Young JM, Kuykendall LD, Martínez-Romero E, Kerr A, Sawada H. A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. Int J Syst Evol Microbiol. 2001;51:89–103.View ArticleGoogle Scholar
  2. Burr TJ, Bazzi C, Süle S, Otten L. Crown gall of grape: biology of Agrobacterium vitis and the development of disease control strategies. Plant Dis. 1998;82:1288–97.View ArticleGoogle Scholar
  3. Burr TJ, Otten L. Crown gall of grape: biology and disease management. Annu Rev Phytopathol. 1999;37:53–80.View ArticleGoogle Scholar
  4. Gelvin SB. Traversing the cell: Agrobacterium T-DNA’s journey to the host genome. Front Plant Sci. 2012;3:52. https://doi.org/10.3389/fpls.2012.00052.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Nester EW. Agrobacterium: nature’s genetic engineer. Front Plant Sci. 2015;5:730. https://doi.org/10.3389/fpls.2014.00730.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Lacroix B, Citovsky V. The roles of bacterial and host plant factors in Agrobacterium-mediated genetic transformation. Int J Dev Biol. 2013;57:467–81.View ArticleGoogle Scholar
  7. McCullen CA, Binns AN. Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annu Rev Cell Dev Biol. 2006;22:101–27.View ArticleGoogle Scholar
  8. Kawaguchi A, Inoue K, Ichinose Y. Biological control of crown gall of grapevine, rose, and tomato by nonpathogenic Agrobacterium vitis strain VAR03-1. Phytopathology. 2008;98:1218–25.View ArticleGoogle Scholar
  9. Kawaguchi A, Inoue K, Nasu H. Inhibition of crown gall formation by Agrobacterium radiobacter biovar 3 strains isolated from grapevine. J Gen Plant Pathol. 2005;71:422–30.View ArticleGoogle Scholar
  10. Kawaguchi A, Inoue K, Nasu H. Biological control of grapevine crown gall by nonpathogenic Agrobacterium vitis strain VAR03-1. J Gen Plant Pathol. 2007;2007(73):133–8.View ArticleGoogle Scholar
  11. Kawaguchi A, Kondo K, Inoue K. Biological control of apple crown gall by nonpathogenic Rhizobium vitis strain VAR03-1. J Gen Plant Pathol. 2012;78:287–93.View ArticleGoogle Scholar
  12. Kawaguchi A. Biological control of crown gall on grapevine and root colonization by nonpathogenic Rhizobium vitis strain ARK-1. Microbes Environ. 2013;28:306–11.View ArticleGoogle Scholar
  13. Kawaguchi A. Reduction of pathogen populations at grapevine wound sites is associated with the mechanism of biological control of crown gall by Rhizobium vitis strain ARK-1. Microbes Environ. 2014;29:296–302.View ArticleGoogle Scholar
  14. Kawaguchi A. Biological control agent Agrobacterium vitis strain ARK-1 suppresses expression of the virD2 and virE2 genes in tumorigenic A. vitis. Euro J Plant Pathol. 2015;143:789–99.View ArticleGoogle Scholar
  15. Kawaguchi A, Inoue K. New antagonistic strains of non-pathogenic Agrobacterium vitis to control grapevine crown gall. J Phytopathol. 2012;160:509–18.View ArticleGoogle Scholar
  16. Kawaguchi A, Inoue K, Tanina K, Nita M. Biological control for grapevine crown gall using nonpathogenic Rhizobium vitis strain ARK-1. Proc Jpn Acad Ser B. 2017;93:547–60.View ArticleGoogle Scholar
  17. Saito K, Watanabe M, Matsui H, Yamamoto M, Ichinose Y, Toyoda K, Kawaguchi A, Noutoshi Y. Characterization of the suppressive effects of the biological control strain VAR03-1 of Rhizobium vitis on the virulence of tumorigenic R. vitis. J Gen Plant Pathol. 2018;84:58–64.View ArticleGoogle Scholar
  18. Burr TJ, Reid CL. Biological control of grape crown gall with nontumorigenic Agrobacterium vitis F2/5. Am J Enol Vitic. 1994;45:213–9.Google Scholar
  19. Chen F, Guo YB, Wang JH, Li JY, Wang HM. Biological control of grape crown gall by Rahnella aquatilis HX2. Plant Dis. 2007;91:957–63.View ArticleGoogle Scholar
  20. Staphorst JL, van Zyl FGH, Strijdom BW, Groenewold ZE. Agrocin-producing pathogenic and nonpathogenic biotype-3 strains of Agrobacterium tumefaciens active against biotype-3 pathogens. Curr Microbiol. 1985;12:45–52.View ArticleGoogle Scholar
  21. Wang HM, Wang HX, Ng TB, Li JY. Purification and characterization of an antibacterial compound produced by Agrobacterium vitis strain E26 with activity against A. tumefaciens. Plant Pathol. 2003;52:134–43.View ArticleGoogle Scholar
  22. Kaewnum SD, Zheng D, Reid CL, Johnson KL, Gee JC, Burr TJ. A host-specific biological control of grape crown gall by Agrobacterium vitis strain F2/5; its regulation and population dynamics. Phytopathology. 2013;103:427–35.View ArticleGoogle Scholar
  23. Kerr A. Biological control of crown gall through production of agrocin 84. Plant Dis. 1980;64:25–30.Google Scholar
  24. Vogel AM, Das A. The Agrobacterium tumefaciens virD3 gene is not essential for tumorigenicity on plants. J Bacteriol. 1992;174:5161–4.View ArticleGoogle Scholar
  25. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller MR, Nolan T, Pfaffl MW. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–22.View ArticleGoogle Scholar
  26. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002;30:e36.View ArticleGoogle Scholar
  27. King EO, Ward MK, Raney DE. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med. 1954;44:301–7.PubMedGoogle Scholar
  28. Abarca-Grau AM, Burbank LP, de Paz HD, Crespo-Rivas JC, Marco-Noales E, López MM, Vinardell JM, von Bodman SB, Penyalvera R. Role for Rhizobium rhizogenes K84 cell envelope polysaccharides in surface interactions. Appl Environ Microbiol. 2011;78:1644–51.View ArticleGoogle Scholar
  29. Morton ER, Fuqua C. UNIT 3D.1 laboratory maintenance of Agrobacterium. Curr Protoc Microbiol. 2012. https://doi.org/10.1002/9780471729259.mc03d01s24.View ArticlePubMedPubMed CentralGoogle Scholar
  30. Tempé J, Petit A, Holsters M, Van Montagu M, Schell J. Thermosensitive step associated with transfer of the Ti plasmid during conjugation: possible relation to transformation in crown gall. PNAS. 1997;74:2848–9.View ArticleGoogle Scholar

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