The Genus Agrobacterium

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CHAPTER 3.1.4 ehT suneG mui retcaborgA

ANN G. MATTHYSSE

Introduction Sinorhizobium meliloti and A. tumefaciens (Hooykaas et al., 1982). When plasmids from The genus Agrobacterium is a group of Gram- Rhizobium phaseoli were introduced into A. negative soil bacteria found associated with tumefaciens strain C58 (a biotype 1 strain); see plants. Many members of this group cause dis- also Taxonomy; the resulting bacteria were able ease on plants. Infections of wound sites by to form nitrogen-ﬁxing nodules on bean roots. Agrobacterium tumefaciens cause crown gall This outcome suggests that all of the chromo- tumors on a wide range of plants including most somal genes required for the interaction of R. dicots, some monocots, and some gymnosperms. phaseoli with plants were present on the A. tume- Infections by A. rhizogenes cause hairy root dis- faciens chromosomes (Martinez et al., 1987; see ease. A. vitis causes tumors and necrotic lesions also Genetics, Chromosomal). Along similar on grape vines and is commonly found in the lines, the nodC gene on the sym plasmid of S. xylem sap of infected plants. Despite the general meliloti can be induced when this plasmid is perception that most of the agrobacteria cause present in A. tumefaciens but not when the disease, the member of this group most often plasmid is transferred to E. coli, Xanthomonas isolated from soil, A. radiobacter, is avirulent. campestris or Pseudomonas savastanoi (Yelton et al., 1987). All of these results suggest that the chromosomal genes of agrobacteria and rhizobia Phylogeny are so closely related, they can substitute for each other. Sequencing of the genomes of Earlier studies using physiological characteristics S. meliloti and A. tumefaciens is currently in such as ability to grow on various carbon sources progress and should help to elucidate the rela- placed the agrobacteria with the rhizobia in the tionship between these bacteria. family Rhizobiaceae. More recent studies of both 16S rDNA and other chromosomal gene DNA sequence homologies suggest that these two groups of bacteria are indeed closely related Taxonomy (Willems et al., 1993). Both physiological characteristics and 16S rDNA sequence data place The genus is divided into species largely based these bacteria in the α subgroup of the Proteo- on pathogenic properties, although other physi- bacteria. They appear to be closely related to ological characteristics correlate with pathogenic members of the genus Brucella. properties. The major species are A. radiobacter On the basis of genomic organization the agro- (nonpathogenic), A. tumefaciens (the causative bacteria appear to form a unique group within agent of crown gall tumors), A. rhizogenes (the the α2 subgroup of the Proteobacteria (Jumas- causative agent of hairy root disease), and A. Bilak et al., 1998); see also Genetics; Biovar 1 vitis (the causative agent of tumors and necrotic strains and A. rubi have both a circular and a disease on grapevines). There are also less well linear chromosome. Biovar 2 and 3 strains also studied proposed species such as A. rubi isolated have 2 chromosomes, but both appear to be lin- from cane galls on Rubrus species. ear. Large plasmids (200–400 kb) are present in Agrobacteria also have been divided into most strains. Rhizobia, although closely related biotypes (biovars) based on physiological pro- to agrobacteria, appear to lack the linear chro- perties. Biovar 1, which includes most strains of mosome present in biovar 1 agrobacteria. A. tumefaciens, has no growth factor require- Genetic experiments suggest that at least some ments and will grow in the presence of 2% NaCl. members of the rhizobia are closely related to Most strains produce 3-ketolactose. All biovars agrobacteria. The gene order on the circular produce acid from mannitol and adonitol. Biovar chromosome appears to be conserved between 1 bacteria also produce acid from dulcitol, jim_3-1-4.fm Page 92 Thursday, October 6, 2005 5:19 PM

92 A.G. Matthysse CHAPTER 3.1.4

melizitose, ethanol, and arabitol. Some biovar 1 vines, from the xylem sap of infected plants. The strains are able to grow at 37°C. However, they bacteria are not numerous in older galls and may may lose the Ti plasmid, which is required for be easier to isolate from the surrounding soil virulence, when grown at this temperature. Bio- than from the tumor tissue. var 2 includes most strains of A. rhizogenes. Agrobacteria grow readily in culture on com- These bacteria require biotin for growth. They plex or defined media (Table 1). Nutrient agar fail to grow in the presence of 0.5% NaCl or at (with or without yeast extract [0.5%]) or yeast 37°C. Some biovar 2 strains can grow on tartrate mannitol agar will support the growth of most producing alkali. Biovar 3 strains include most strains. Some strains require B vitamins for A. vitis strains. Some authors also include some growth, usually 0.2 mg/liter each of biotin, pan- A. tumefaciens strains in this group. Like biovar tothenic acid and/or nicotinic acid. Many strains, 1 strains, these bacteria will grow in the presence including most A. rhizogenes isolates, are sensi- of 2% NaCl but generally do not grow at 37°C. tive to salt and will not grow on media such as Both biovar 2 and 3 strains fail to produce 3- Luria-Bertani agar because this medium con- ketolactose. Biovar 3 strains can produce alkali tains too much NaCl. The colonies are generally from tartrate. Some biovar 3 strains require white or slightly cream or pale pink in color. No biotin for growth (Table 2). Selective growth distinctive pigment is produced. Large amounts media for various biovars have been reported of extracellular polysaccharide may be produced and are described in the section on isolation of on some media giving the colonies a watery agrobacteria (Table 1). Biovars 1 and 3 contain appearance. The bacteria grow at a moderate both strains with wide and others with narrow rate. A. tumefaciens will usually require 2 to 4 host ranges (Kerr et al., 1977b). days to form colonies on complex media. Some strains of A. rhizogenes are slow growing and may require as much as 1 week to form colonies Habitat on complex media. Optimal growth temperature for most strains Agrobacteria usually are found in soil in associ- is between 25 and 28°C, although the optimal ation with roots, tubers, or underground stems. temperature for plant infection may be lower The bacteria also cause tumors from which they (22°C). can be isolated. Tumors may be prevalent on Selective media may be used to isolate grafted plants at the graft junction; examples Agrobacteria (Table 1). include grapes, roses, poplars, and fruit trees. In some cases, the bacteria can be isolated from the xylem of infected plants. Thus it is often possible to isolate A. vitis from the xylem of infected Identification grapevines. Although agrobacteria are generally isolated Agrobacteria have been traditionally identified from cultivated soils and plants, biovars 1 and 2 as Gram-negative bacteria that don’t produce can be found in association with roots from fluorescent pigment on King’s B medium and do uncultivated plants of the natural savanna and produce tumors (or hairy roots) when inoculated tall grass prairie which has never been cultivated onto test plants. The test plants most often used (Bouzar et al., 1987). As is the case in most other are tomato, sunflower, Datura spp., Kalanchoë field studies of agrobacteria, the majority of daigremontiana (also called Bryophyllum), these isolates were nonpathogenic. Schroth et al., tobacco, and Nicotiana glauca (Figs. 1–4). These 1971 were able to isolate agrobacteria from plants respond relatively readily and rapidly to almost every soil they tested in California by inoculation of Agrobacterium strains by produc- using selective media and enrichment culture ing tumors in as few as 10 days. Sugar fermenta- methods. Thus the bacteria appear to be widely tions and production of ketolactose also have distributed regardless of the plants previously been used in identification of agrobacteria (Table grown in the location. However, the number of 2). In recent years, lipid and fatty acid profiles bacteria pathogenic for a crop grown in a partic- have been used to identify both virulent and ular location was greater if the same crop had avirulent agrobacteria (Jarvis et al., 1996; Bouzar formerly been grown in that location. et al., 1993a). Polymerase chain reaction (PCR) has also been used in identification and to distin- guish pathogenic from nonpathogenic strains. Isolation The PCR primers chosen from vir genes such as virD2 (See Genetics) can be used to identify Agrobacteria can be isolated from soil obtained potentially pathogenic strains (Haas et al., 1995). from the vicinity of infected plants, from galls Pathogenic strains have been identified by their formed by the bacteria, or, in the case of grape- ability to grow on different opines, and the jim_3-1-4.fm Page 93 Thursday, October 6, 2005 5:19 PM

CHAPTER 3.1.4 The Genus Agrobacterium 93

Table 1. Media for growth of agrobacteria. General Media General Media Luria Agar (for biovar 1 and some biovar 3 strains) Dissolve salts in the order given; adjust pH to 7.2; add after Tryptone 10g autoclaving 10ml of sterile 20% glucose or sucrose. Yeast extract 5g NaCl 5g Selective media (Kerr, 1986)* Water 1 liter Selective Medium of Biovar 1* 3M NaOH 1ml L(-) Arabitol 3.04g Agar 14g K2HPO4 1.04g

Yeast Mannitol Agar (for all biovars) KH2PO4 0.54g

Mannitol 10g NH4NO3 0.16g

Yeast extract 1g MgSO4 ≅ 7H2O 0.25g K2HPO4 0.5g Sodium taurocholate 0.29g CaCl2 0.2g Water 1 liter NaCl 0.2g 1% Crystal violet 2ml ≅ MgSO4 7H2O 0.2g Agar 15g FeCl3 0.01g Water 1 liter Add after autoclaving 10ml of 2% cyclohexamide and 10ml

Agar 15g of 1% Na2SeO3 ≅ 5H2O. On this medium colonies of agrobacteria are white, circular, raised, and glistening. Adjust to pH 7.0. For biovar 2 add biotin, calcium They may become mucoid. pantothenate, and nicotinic acid, all at 200μg/liter. Selective Medium for Biovar 2* Mannitol glutamate agar (for all biovars) (Roberts et al., Erythritol 3.05g

1974) K2HPO4 1.04g

Mannitol 10g KH2PO4 0.54g

L-Glutamic acid 2g NH4NO3 0.16g

KH2PO4 0.5g MgSO4 ·7H2O 0.25g NaCl 0.2g Sodium taurocholate 0.29g MgSO4 ·7H2O 0.2g Yeast extract 0.01g Biotin 0.002g Malachite green 0.005g Water 1 liter Water 1 liter Agar 15g Agar 15g

Adjust pH to 7.0 before autoclaving. Add after autoclaving 10ml of 2% cyclohexamide and 10ml ≅ H4 Minimal Medium (for biovars 1 and 3, biovar 2 will grow of 1% Na2SeO3 5H2O. On this medium colonies of very slowly on this medium) (Matthysse et al. 1976) agrobacteria are white, circular, raised, and glistening. They may turn brown a they age. NH4Cl 5g

NH4NO3 1g Selective Medium for Biovar 3*

Na2SO4 2g Adonitol 4.0g

K2HPO4 3g K2HPO4 0.9g

KH2PO4 1g KH2PO4 0.7g

MgSO4 ·7H2O 0.1g NaCl 0.2g Water 1 liter MgSO4 0.2g Yeast extract 0.14g Dissolve salts in the order given; adjust pH to 7.2; add 10ml Boric acid 1.0g of sterile 20% glucose after autoclaving. Water 1 liter AB Minimal Medium Agar 15g K HPO 3g 2 4 Adjust pH to 7.2 before autoclaving. After autoclaving add NaH PO 1g 2 4 10ml of 2.5% cyclohexamide, 1ml of 8% NH Cl 1g 4 triphenyltetrazolium chloride, 1ml of 2% D-cycloserine, MgSO ·7H O 0.3g 4 2 and 1ml of 2% trimethoprin. On this medium colonies of KCl 0.15g agrobacteria have dark red centers with white edges. CaCl2 0.005g

FeSO4 ·7H2O 0.0025g Water 1 liter *Note that these media are only semi-selective. Other organisms may grow. Additional tests are necessary to positively identify an isolate as Agrobacterium. jim_3-1-4.fm Page 94 Thursday, October 6, 2005 5:19 PM

94 A.G. Matthysse CHAPTER 3.1.4

Table 2. Traits used for identiﬁcation of biovars of Agrobacterium. Characteristic Biovar 1 Biovar 2 Biovar 3 A. rubi Growth on selective medium 1Aa Yes 2Eb Yes RSc Yes Growth factor requirements None Biotin Biotin, some strains Biotin, pantothenic acid, nicotinic acid 3-Ketolactose production Most strains No No No Growth on 2% NaCl Yes No Yes Yes Growth at 37°C Yes No No Yes Acid production from mannitol Yes Yes Yes Yes Adonitol Yes Yes Yes Yes Erythritol No Yes No No Dulcitol Yes Yes No No Melizitose Yes No No No Ethanol Yes No No No Arabitol Yes No No No Alkali production from tartrate No Yes Yes No Data from Kerr (1986).

formation of particular opines by tumors caused by various strains has been used to group these strains. In general, grouping by sugar fermenta- tions, fatty acid proﬁles, PCR, opine production and utilization, and genome organization all reach similar conclusions so that no one method of identiﬁcation of agrobacterial species or biovars is preferable.

Preservation

The bacteria can be stored as stabs into vials of nutrient agar (all biovars) or Luria agar (biovars 1 and 3) at room temperature essentially indeﬁ- nitely (more than 10 years). They can also be stored frozen in 25% glycerol at –70°C. Liquid cultures of biovars 1 and 3 can be spun down, resuspended in phosphate buffered saline con- taining 1 mM MgSO4, and stored in the refriger- ator for approximately 10 weeks.

Physiology General Agrobacteria are Gram-negative, nonspore– forming, short rods. They can use glucose as a carbon source, growing aerobically. Table 1 lists agrobacterial growth media formulations and Fig. 1. The stem of a tobacco plant wounded at two places Table 2 lists characteristics of different biovars. and inoculated with A. tumefaciens. The tumors are shown at 6 weeks after inoculation. Opines: Production and Utilization Crown gall tumors produce speciﬁc substances crown gall tumor cells on the T DNA. These (often substituted L-amino acids) called opines. genes are usually expressed constitutively in the The production of opines is catalyzed by tumor tissue. Typical opines result from conden- enzymes encoded by genes introduced into sation reactions between compounds already jim_3-1-4.fm Page 95 Thursday, October 6, 2005 5:19 PM

Fig.CHAPTER 2. Carrot 3.1.4 root discs (A) uninocu- The Genus Agrobacterium 95 lated, (B) inoculated with A. tumefaciens, and (C) and (D) inoculated with A. rhizogenes. The discs are shown after 5 weeks incubation.

Fig. 3. A leaf of Bryophyllum daigremontiana (also called Fig. 4. A leaf of Bryophyllum daigremontiana (also called Kalanchoë daigremontiana) inoculated with A. tumefaciens. Kalanchoë daigremontiana) inoculated with A. rhizogenes. The site on the back right was inoculated with a strain lacking Note that the roots formed at the wound sites are branching the Ti plasmid. Tumors are shown after 4 weeks growth. and ageotropic. The leaf is shown 5 weeks after inoculation.