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Rhizobium rhizogenes=Agrobacterium rhizogenes

Pathogen profile created by Miranda Ganci

Requirement for PP 728 Soilborne Plant Pathogens, Fall 2012

Department of Plant Pathology, North Carolina State University

Introduction

Infectious hairy root disease is caused by Rhizobium rhizogenes and it occurs on many dicotyledonous plants. It was first identified as a pathogen of economic importance on apples in the early 20th century (8). The bacterium was formally named Agrobacterium rhizogenes in 1942 (1). Recently, it was discovered through the use of molecular tools that A. rhizogenes was closely related to Rhizobium spp.. Interestingly, species of Rhizobium and Agrobacterium are capable of horizontal gene transfer through the exchange of Root-inducing (Ri) plasmids (extrachromosomal structures that carry genes) in-vitro. Additionally, wild-type strains of symbiotic bacteria were isolated from root nodules of leguminous Yellow Pea-bush (Sesbania cannabina) and identified as close relatives of A. tumefaciens(=Rhizobacterium radiobacter)(7). The original classification of the bacterium as Agrobacterium was based on the pathogenicity of disease causing strains. Phylogenetic relationships based on the comparison of 16S rDNA analysis have elucidated the similarity between Agrobacterium and Rhizobium which led to the reclassification of Agrobacterium spp. as Rhizobium spp (9).

In nature, there are both pathogenic and non-pathogenic strains of R. rhizogenes. Pathogenic strains are capable of causing hairy root and crown gall. Crown gall is usually associated with Rhizobium radiobacter (formerly known as Agrobacterium tumefaciens). Bacteria are capable of gene transfer and thus some strains of R. rhizogenes have acquired the ability to cause crown galls but R. rhizogenes are most commonly known for causing hairy root (1). Crown gall, usually caused by R. radiobacter, can be an economically important disease of many plants. Non-pathogenic strains of R. rhizogenes can be used as a biological control agent against pathogenic strains of R. radiobacter. Allen Kerr identified the K84 strain of R. rhizogenes (formerly known as A. radiobacter). He discovered that if the K84 strain of non-pathogenic R. rhizogenes was present in high population densities in the soil with pathogenic R. radiobacter then the incidence of crown gall could be significantly reduced. Kerr identified the antibiotic agrocin 84, produced by R. rhizogenes, as the method of control (3). The commercially available biological control agent, Galltrol A, contains R. rhizogenes strain K84.

Currently, the study of R. rhizogenes in plant pathology is not centered around the role of the bacterium as a pathogen. R. rhizogenes is economically important in genetic engineering, as its plasmids are commonly used as vectors for gene transfer and it has many plant hosts. Researchers are studying the use of secondary metabolites of R. rhizogenes and secondary metabolites produced by plants induced by R. rhizogenes for commercial application.

Common Bean Genetically Transformed with Rhizobium rhizogenes

Photo Courtesy of Federico Sanchez, Universidad Nacional Autónoma de México

Soybean Plant Without Rhizobium rhizogenes Infection

Photo Courtesy of Peter M. Gresshoff, The University of Queensland

   

Soybean Plant With Rhizobium rhizogenes Infection

Photo Courtesy of Peter M. Gresshoff, The University of Queensland


Host Range and Distribution

The plant families Solanaceae, Rosaceae, Fabaceae, Crassulaceae, Caesalpinaceae, Brassicaceae, Polygonaceae, and Asteraceae are susceptible to hairy root caused by R. rhizogenes. Laboratory experiments have shown that many plants can serve as hosts including monocots and primitive dicots (6). Hosts which are phenol accumulators tend to be more sensitive to infection by R. rhizogenes (1). R. rhizogenes has a world-wide distribution but it cannot grow above 30°C in culture.

Tobacco Roots Showing Symptoms of Hairy Root Disease

Potato Plant Showing Symptoms of Hairy Root Disease

Photo Credit: Potato Gene Engineering Network

Tobacco Plant Showing Symptoms of Hairy Root Disease

Photo Credit: Adriana M. Alippi, Facultad de Ciencias Agrarias y Forestales, Argentina

 


Isolation

Selective media can be used to isolate R. rhizogenes from roots, soil, and tumors. R. rhizogenes is resistant to toxic amounts of tellurite (K2TeO3). One example of a selective media is 2E-Te, containing erythritol (see Table 1) with 320mg/L K2TeO3. R. rhizogenes and R. radiobacter have similar colony morphology on tellurite-selective media. Colonies have circular morphology and are black in color with a metallic shine (4). However, the components in the 2E-Te media are more selective for R. rhizogenes than R. radiobacter.

Table 1: 2E-Te, Rhizobium rhizogenes Selective Media (4)

Component

Rate per liter

Erythritol

3.05 g

NH4NO3

0.16 g

KH2PO4

0.54 g

K2HPO4

1.04 g

MgSO4, 7H2O

0.25 g

Sodium taurocholate

0.29 g

1% Yeast extract

1 ml

0.1% Malachite green

5 ml

Agar

15 g

 

After autoclaving:

 

K2TeO3

0.32 g

Rhizobium rhizogenes Colony Morphology on Tellurite Amended Medium

Excerpted from, "Rapid and Efficient Methods to Isolate, Type Strains and Determine Species of Agrobacterium spp. in Pure Culture and Complex Environments"

2% cycloheximide

1 ml

   

To isolate R. rhizogenes from soil or plant material a series of isolations must be performed. First, the soil or plant material must be combined with sterile distilled water and macerated. Aliquots should be plated on the selective 2E-Te media with tellurite. Plates should be incubated for 3-4 days at 28°C and then colonies with typical morphology should be selected and submerged in sterile distilled water. Bacteria should then be plated on selective media, 2E, without tellurite. Colonies should be selected once again based on morphology and a series of isolations should be performed to ensure the selection of a pure culture (4).

Pathogenic strains of Rhizobium can be isolated from soil, tumors, or roots by utilizing genome extraction kits and PCR. However, species and biovar specific primers are currently in development for soil and plant isolations (4).


Identification

R. rhizogenes is a capsule forming gram-negative bacterium with a polar flagellum. R. rhizogenes was first identified based on the ability to induce hairy root growth in plants. In the 1970’s, it was discovered that gene transmission into the plant via the Ri-plasmid is responsible for hairy root growth and that the Ri-plasmids are highly variable amongst strains. After that discovery, hairy growth on roots was deemed unreliable as an identification method for all strains of the bacteria. It was determined that strains could be identified by their enzymatic abilities and classified into separate biovars. Biovars within a genus are separated by chromosomal genes. R. rhizogenes is characterized as biovar 2 (4).


Symptoms

Hairy root disease is characterized by an overabundant growth of adventitious roots at the site of infection (usually on the stems of plants near and immediately below the soil surface). Symptoms of hairy root disease may also include wrinkled leaves. Hairy root disease symptoms have been divided into 2 categories, ‘simple or fibrous hairy-root’ and ‘woolly-knot form of hairy root.’  Simple hairy root disease is identified as stated above where as the woolly-knot form is characterized by the initial formation of a smooth tumor on the plant stem followed by an outgrowth of adventitious roots from the tumor surface. The high variability of the plasmids in R. rhizogenes strains is the reason that many strains are capable of inducing excessive root growth as well as tumor growth (1).

Foliar Wilt Caused by Rhizobium rhizogenes infection on Mulberry

Photo Credit: William M. Brown Jr.

Gold Mound Duranta Plant Showing Symptoms of Hairy Root Disease

Photo Credit: University of Florida Distance Diagnostic and Identification System

 

Roots of a Young Mulberry Tree Showing Symptoms of Hairy Root Disease

Photo Credit: William M. Brown Jr.

 

Ecology and Life Cycle

R. rhizogenes are soil dwelling bacteria which live in the rhizosphere of many plant roots.  Optimum growth occurs at pH above 4 and 20-28°C. Many live their entire lives as saprophytes (non-pathogenic) and do not cause damage to plants. They can exist as biofilms on plant root surfaces. The strains which contain Ri-plasmids with virulence genes are considered plant pathogens. However, due to the ease of horizontal transfer of plasmids via conjugation, it is difficult to distinguish pathogenic strains from non-pathogenic strains.

Pathogenic strains of R. rhizogenes are capable of inducing hairy root growth on their hosts. When a plant is wounded it releases compounds which are sensed by the bacterium in the soil. R. rhizogenes is attracted towards the plant wound and it can transfer its DNA into the host cell via transfer of a portion of the root-inducing (Ri) plasmid. The transferred DNA (T-DNA) is integrated into the plant cell genome. After integration the plant produces an abundance of growth hormones and opines which are beneficial for growth of R. rhizogenes. It is thought that the virulence genes of R. rhizogenes are activated by the lignin forming compounds in some plant cell walls (2).

R. rhizogenes is characterized by very interesting and important interactions with plants. The species contains strains which are economically important as plant pathogens, as tools for genetic engineering, as biological control agents, and for use in commercial production of plant and bacteria secondary metabolites. The changing nomenclature can be confusing for researchers however this organism will be heavily studied in the future.


Links to Other Sites

The Microbial World: Biology and Control of Crown Gall

Bacteria confused with rhizobia, including Agrobacterium taxonomy


Selected References

1.      De Cleene, M., and De Ley, J. 1981. The host range of infectious hairy root. The Botanical Review. 47:147-194.

2.      Gafni, Y., and Levy, Y. 2005. Coniferyl alcohol, a lignin precursor, stimulates Rhizobium rhizogenes A4 virulence. Current Microbiology. 50:262-265.

3.      Kerr, A. 1980. Biological control of crown gall through production of agrocin 84. Plant Disease. 64:25-30.

4.     Shams, M., Campillo, T., Lavire, C., Muller, D., Nesme, X., and Vial, L. 2012. Rapid and efficient methods to isolate, type strains and determine species of Agrobacterium spp. in pure culture and complex environments, Biochemical Testing, Dr. Jose C. Jimenez-Lopez (Ed.), ISBN: 978-953-51-0249-6, InTech, Available from: http://www.intechopen.com/books/biochemical-testing/rapid-and-efficient-methods-to-isolate-type-strains-and-determine-species-of-agrobacterium-spp-in-pu

5.     McClure, N.C., Ahmadi, A., and Clare, B.G. 1998. Construction of a range of derivatives of the biological control strain Agrobacterium rhizogenes K84: a study of factors involved in biological control of crown gall disease. Applied and Environmental Microbiology. 64:3977-3982.

6.      Porter, J.R. and Flores, H. 1991. Host range and implications of plant infection by Agrobacterium rhizogenes. Critical Reviews in Plant Sciences. 10:387-421.

7.      Pulawska, J. 2010. Crown gall of stone fruits and nuts, economic significance and diversity of it’s causal agents: tumorigenic Agrobacterium spp.. Journal of Plant Pathology. 92:S1.87-S1.98.

8.      Riker, A. J., Banfield, W.M., Wright, W.H., Keitt, G.W., and Sagan, H.E. 1930. Studies on infectious hairy root of nursery Apple trees. Journal of Agricultural Research. 41:507-540.

9.      Young, J.M., Kuykendall, L.D., Martínez-Romero, E., Kerr, A., and Sawada, H. 2001. 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 undicolade Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicolaand R. vitis. International Journal of Systematic and Evolutionary Microbiology. 51:89-103.