Agrobacterium tumefaciens
Tumor caused by A. tumefaciens on rose (Agrios, 1988)By Alyssa CollinsTumor caused by A. tumefaciens on tomato (Schaad et al., 2001.)
A Class Project for
PP728 Soilborne Plant Pathogens
North Carolina State University
Department of Plant Pathology
 

                   Introduction

Agrobacterium tumefaciens, the cause of the economically important disease, crown gall, has also been studied for years because of its remarkable biology. The mechanism this bacterium uses to parasitize plant tissue involves the integration of some of its own DNA into the host genome resulting in unsightly tumors and changes in plant metabolism. A. tumefaciens prompted the first successful development of a biological control agent and is now used as a tool for engineering desired genes into plants.


Host Range and Distribution

Agrobacterium tumefaciens is cosmopolitan in distribution, affecting dicotyledonous plants in more than 60 different plant families. Crown gall can be found most often on stone fruit and pome trees as well as brambles and several species of ornamental plants.


IdentificationElectron micrograph of Agrobacterium (Agrios, 1988)

Agrobacterium tumefaciens is a member of the family Rhizobiaceae. These bacteria are Gram-negative and grow aerobically, without forming endospores. The cells are rod-shaped and motile, having one to six peritrichous flagella. Cells are 0.6-1.0 m m by 1.5- 3.0 m m and may exist singly or in pairs. In culture on carbohydrate-containing media, cells produce large amounts of extracellular polysaccharides, giving colonies a voluminous, slimy appearance.

Recently, a reclassification of the species of Agrobacterium has been undertaken by use of ribosomal RNA sequencing as a taxonomic tool. The resulting nomenclature places the former species, A. tumefacians biovar 1, A. radiobacter biovar 1, and A. rhizogenes biovar 1, within the new taxon: Agrobacterium tumefaciens.



Isolation
A. tumefaciens on Brisbane and Kerr medium 1A (Schaad et al., 2001.)
A. tumefaciens can be effectively isolated for identification from gall tissue, soil or water. Optimal gall tissue for isolation is white or cream-colored from a young, actively growing gall. The gall should be washed or surface sterilized using 20% household bleach, and rinsed several times in sterile water. Cut a few samples from different parts of the white tissue of the gall, and further divide samples into small pieces. Place these pieces into a culture tube containing sterile distilled water or buffer, vortex and allow to stand for at least 30 minutes. Using an inoculating loop, streak this suspension on Medium 1A (Schaad et al., 2001), and incubate at 25-27° C. Different strains will grow at different rates. One may also use this selective medium to detect A. tumefaciens in soil dilutions or irrigation water.

It should be noted, however, that the presence of A. tumefaciens cells in a sample does not necessarily dictate the existence of the crown gall-inciting strain in the sample. Only cells containing a specific plasmid (the Ti plasmid) can cause disease. A. tumefaciens strains lacking the plasmid live as rhizosphere-inhabiting bacteria without causing disease.


Tumor on rose stem (Horst, 1983)SymptomsCreamy white interior of tumor (Horst, 1983)

Crown gall manifests itself initially as small swellings on the root or stem near the soil line, and occasionally on aerial portions of the plant. Young tumors, which often resemble the callus tissue that results from wounding, are soft, somewhat spherical and white to cream colored. As tumors become older, their shape becomes quite irregular, and they turn brown or black. Tumors may be connected to the host surface by only a narrow bit of tissue, or may appear as a swelling of the stem, not distinctly separate. The tissue can be spongy and crumbling throughout the gall or can be woody and knot-like. Several tumors may occur on the same plant and may rot from the surface of the plant completely or partially, possibly developing repeatedly in the same area season after season. Additional symptoms include stunting, chlorotic leaves, and plants may be more susceptible to adverse environmental conditions and secondary infection.



Pathogen Life Cycle

Pathogenic strains of A. tumefaciens may live saprophytically in soil for up to two years. When a nearby host plant is wounded near the soil line by insect feeding, transplant injury or any other means the bacterium chemotactically moves into the wound site and between host cells. These bacteria then stimulate the surrounding host cells to rapidly and irregularly divide. The bacterium accomplishes this by inserting a piece of its own DNA into the host cells' chromosomes, causing overproduction of cytokinins and auxins which are plant growth regulators, and opines which serve as nutrients for the pathogen. The resulting tissue is undifferentiated with a white or cream color, and cells may have one or more nuclei. This tissue continues to enlarge and a tumor is formed on the root or stem of the plant, depending on original wound site. The bacteria occupy the intercellular spaces around the periphery of the gall and are not found in the center of the enlarging tumor. The tumor is not protected by an epidermis, leaving the tissue susceptible to secondary pathogens, insects and saprophytes. Degradation of the tumor by secondary invaders causes brown or black discoloration and releases A. tumefaciens cells back into the soil to be carried away by with soil or water, or remain in the soil until the next growing season. In perennial plants, part of the infected tissue may remain alive and inhabited by A. tumefaciens, which, even if the tumor has sloughed off, can persist to cause a new tumor the following season in the same place.



Disease Control

Introduction of pathogenic A. tumefaciens strains can be avoided by thorough inspection of nursery stock for crown gall symptoms. Susceptible varieties should not be planted in soils known to be infested with the pathogen. These soils should be planted in a monocotyledonous crop like corn or wheat for several years. Nursery stock should be certified crown gall-free and should be budded rather than grafted. If the threat of crown gall exists, all practices that wound tissue should be avoided and chewing insects should be controlled.

Preventative treatment of seeds or transplants with the non-pathogenic biocontrol organism Agrobacterium radiobacter is a relatively inexpensive and effective means of managing the development of crown gall in commercial operations. Application of this antagonist by soaking seeds or dipping transplants can prevent infection by most strains of A. tumefaciens due to the production of the antibiotic agrocin 84 by strain K84 of A. radiobacter. Some curative properties are exhibited by a commercially available mixture of 2,4-xylenol and metacresol in an oil-water emulsion when painted directly on established tumors. But this is rarely used due to labor and time constraints.


References

Agrios, G.N. 1988. Plant Pathology, 3rd Ed. Academic Press Inc., London. pp. 558-565.

Horst, R.K. 1983. Compendium of Rose Diseases. APS Press, St. Paul, MN. pp 23-25.

Schaad, N.W., J.B. Jones & W. Chun. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd Ed. APS Press, St. Paul, MN. pp. 17-35.



 

Links to other sites with information about Agrobacterium tumefaciens

Biology and Control of Crown Gall

Pictures of Crown Gall Symptoms



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Posted Spring 2001