Click on any
image for a more
detailed view

Pathogen Biology

Agrobacterium tumefaciens is a rhizoplane bacterium whose characteristics are Gram-negative, strictly aerobic, bacilliform rods measuring 1 x 3 µm, and whose nutritional requirements are non-fastidious. The rods bear flagella that are arranged subpolarly around the cylindrical circumference of the cell, referred as circumthecal flagellation (Figure 5). When A. tumefaciens cells perceive plant phenolic compounds, the virulence genes that are located in the resident Ti (tumor-inducing) plasmid are expressed, resulting in the formation of a long flexuous filament called the T pilus (see next section). The activation of VirA also shuts off motility of the circumthecal flagella, presumably when A. tumefaciens cells attach to plant cells. Attachment to the plant cells is a prerequisite for initiating the transfer of the T-DNA into the plant cell. Both the circumthecal flagella and the T pilus play an essential role in virulence, presumably by bringing the bacterial cell to its target followed by attachment to the plant host, respectively.

Figure 5.

Agrobacterium tumefaciens biotypes and biovars

Based on some distinct phenotypic differences, A. tumefaciens isolates were originally classified into three biotypes or biovars (biotype I, II and III; biovar 1, 2, and 3). Biotype I or biovar 1 strains produce 3-ketosugars and usually have wide host ranges. Biotype II or biovar 2 strains mainly classify as the hairy root-forming organism, A. rhizogenes. Biotype III or biovar 3 isolates are mainly confined to grapevines, prefer L-tartaric acid over glucose and produce polygalacturonase. Because grapevine isolates formed a distinct group verified by DNA homology studies and were frequently limited in host-range to grapevines, biovar 3 strains have been reclassified into one species, A. vitis. Agrobacterium rubi strains infect canes of the genus Rubus, representing blackberry and raspberry.

Ti plasmid and virulence genes

Experimental inoculation of an assay host plant such as Jimson weed (Datura stramonium) results in tumor formation within two weeks (Figure 6). Virulence and the host-range of A. tumefaciens are conferred by a large extrachromosomal DNA element designated as the Ti plasmid (for tumor-inducing) that resides in all virulent strains of this pathogen. The Ti plasmid is approximately 200 kilobases in length and is comprised of a covalently closed, double-stranded DNA circular molecule.

Figure 6.

Upon recognition of plant signals in the form of dimethoxyl phenolic compounds released from plant wounds, virulence gene, virA, encodes histidine kinase (VirA) that phosphorylates the response regulator (VirG), which in turn transcriptionally activates the remaining vir genes of the vir regulon. The products of the vir genes carry out the processing of the T-DNA and functions for T-DNA transfer to the plant cell. Also, perception of the plant phenolic compounds by VirA switches off A. tumefaciens cell motility. In addition, certain chromosomal genes (e.g., chv, att genes) of A. tumefaciens are also involved in virulence. Most of them have a role in the attachment of A. tumefaciens to plant cells, in promoting signal molecule recognition, and in the regulation of certain vir and T-DNA genes.

T-DNA transfer from A. tumefaciens to plants is aided by a long flexuous appendage known as the T pilus. The T-DNA is delivered as a single-stranded DNA molecule coupled to a VirD2 protein into the plant cell. The T-DNA is integrated into the plant chromosome as evidenced by in situ hybridization using the T-DNA as the probe (Figure 6). The T-DNA genes are expressed by plant transcriptional machinery.

The products of the T-DNA genes catalyze the formation of auxin, cytokinin and opines. Profuse production of auxin and cytokinin in the transformed cells results in abnormal cell division, cell enlargement, and uncontrolled growth of the infected plant tissues. Opines are utilized as specific nitrogen and carbon sources by A. tumefaciens. The opines produced by the transformed plant cells provide unique nutritional sources for the pathogen, and certain opines promote transfer of the Ti plasmid between Agrobacterium strains. Certain opines such as octopine and agrocinopine serve as inducers of the Ti plasmid conjugative transfer system.

Injury to young tissues leads to infection

Wounds made by cultivating practices include accidental, localized injuries caused by mowing and disking machines for weed removal in orchards and vineyards. The wounds are an open invitation for the initiation of the crown gall disease of the roots and the base of trees and vines. Also, tissues injured by frost are prone to infection by A. tumefaciens. In plants where A. tumefaciens resides systemically throughout the plant, such as in Marguerite daisy, grapevines and chrysanthemum, frost injury can cause a linear or confluent array of small tumors along the vascular system of the infected plant (Figure 2). Galls on subterranean parts originated from initial planting of diseased or infected stock (Figure 7). Wounds made by grafting and pruning before transplanting are also susceptible to crown gall infection.


Figure 2.	Figure 7.

Crown gall tumors are often fleshy and easily detached

Fresh crown galls are relatively sturdy and hard. As these galls age beyond one year, they appear convoluted with cavities and become friable. Insects such as earwigs commonly reside within these cavities. It is difficult to isolate A. tumefaciens from aged galls when most of the diseased plant tissue is dead. Aged galls are easily detached. Fresh galls are the best sources of this pathogenic bacterium.

Crown gall causes stunting of growth

Both loss of yield and stunting of growth may occur when seedlings or young cuttings are infected in the early stages of plant growth. The lack of vigor, reduction in foliage, and water stress are associated with chronically diseased root systems. When more mature tree crops become infected, secondary growths will appear from the root systems near the trunk. These "suckers" are a good sign that the root system is infected.