Vol. 16, No. 3 Printed in U.S.A.
INFwTON AND ImMUNITY, June 1977, p. 1027-1028 Copyright C 1977 American Society for Microbiology
Replica-Plating Virulence Assay for Agrobacterium tumefaciens A. G. MATTHYSSE,* S. RATLIFF, AND J. SALBERG Department ofBotany, University of North Carolina, Chapel Hill, North Carolina 27514
Received for publication 3 December 1976
Replica plating onto wounded tobacco leaves provides a convenient method for screening for avirulent mutants of Agrobacterium tumefaciens. Extracellular infections of wounded dicotyledenous plants with the gram-negative bacterium Agrobacterium tumefaciens result in the development of crown gall tumors. Both tumorigenic and non-tumorigenic strains ofthe bacterium are known. The virulence of Agrobacterium strains has usually been assayed by inoculating stems, leaves, or storage roots with the bacterium and scoring for the presence (or absence) and size ofthe resulting tumors (4). Each of these assay systems requires making a separate inoculation for each bacterial strain to be studied. One assay system developed by Lippincott and Heberlein (3), using primary pinto bean leaves, allows the relation between the number of bacteria inoculated and the number of tumors formed to be studied. The number of tumors induced is proportional to the number of bacteria inoculated. We have been interested in modifying this leaf assay to allow more rapid screening for mutants of Agrobacterium that are avirulent. For this purpose, we have developed a replica-plating assay using tobacco leaves. Plants. Nicotiana tabacum L. variety Coker 319 was grown in the greenhouse, with one plant per pot. Bacteria. A. tumefaciens strains 15955 (virulent) and 6467 (avirulent) were obtained from the American Type Culture Collection. They were grown on 0.8% nutrient broth (Difco) and plated on 1.0% nutrient agar (Difco), with 9 to 40 colonies per plate. Plates were incubated for 3 to 5 days at 25°C before use. Occasionally they were stored at 4°C for 1 to 2 weeks. No effect of age of colonies or cold storage was observed. Similar results have been obtained with A. tumefaciens virulent strains 23308 (obtained from the American Type Culture Collection) and A6 (obtained from A. Braun). Inoculation. The upper surface of tobacco leaves was washed with tap water and wounded with carborundum (no. 600 silicon carbide) as described by Lippincott and Heberlein (2). The carborundum was then washed off with tap
water, and the leaves were allowed to dry for 15 to 30 min. A nutrient agar plate containing colonies of A. tumefaciens was replica plated on to sterile velvet and from the velvet onto a fresh plate of nutrient agar and onto the upper surface of the tobacco leaf (1). One to eight leaves on each plant were inoculated. Scoring. After inoculation, the tobacco plants were allowed to continue to grow for 10 to 30 days. They were watered from the bottom. The inoculated leaves were then picked. Tumors were scored by their raised appearance on the surface of the leaf as described by Lippincott and Heberlein (2) for pinto bean and tobacco leaves. The number of tumors was the same at 10 and 30 days, but the tumors were smaller at 10 days, so low-power magnification was necessary to score them; at 30 days tumors were visible to the unaided eye. The inoculation sites on the upper surface of the leaves were identified by dipping the leaf in a dilute (0.1%) solution of toluidine blue in tap water. This stained the inoculation sites dark blue. The tumors were photographed by turning the leaf over and covering it with 0.1% toluidine blue so that the normal surface of the leaf (except for some veins) was covered and the tumors stood out above the surface of the dye solution. Effect of age of plants and of leaves. Young tobacco plants (less than 3 months old) gave variable results. The percentage of replicaplated virulent colonies that gave rise to tumors averaged 60%, with a range of 30 to 100% in different experiments. Older tobacco plants (more than 5 months old) gave less variable results. The average percentage of virulent colonies causing tumors was 90%, with a range of 70 to 100% in different experiments. This variability could be reduced still further by using only the older leaves on the plant (about 20 cm long, 8th to 15th node from the bottom of the plant). Using only these leaves, 98 + 2% (range, 95 to 100%) of the virulent colonies replica plated onto the leaf gave rise to tumors (more than 500 colonies were tested). No aviru1027
1028
NOTES
INFECT. IMMUN.
FIG. 1. Tobacco leaf inoculated by replica plating 30 days previously with tumorigenic (T, strain 15955) and non-tumorigenic (N, strain 6467) Agrobacterium tumefaciens. The leaf was stained with 0.1 % toluidine blue. (A) The top side of the leafshowing the stained inoculation sites where the colonies occurred on the petri plate. The line at the bottom is 1 cm long. The leaf grew and expanded after inoculation, spreading the sites apart and increasing the area covered by each colony. (B) The bottom side of the leaf covered with 0.1% toluidine blue so that only the raised tumors and some veins show above the dye as white. The sites of the inoculating colonies shown in (A) are indicated by circles. The letters indicate the strain used to make the colony.
lent colonies replica plated onto the leaf gave rise to tumors (200 colonies were tested). Since one can use 40 colonies per plate, 1 plate per leaf, and 8 leaves per tobacco plant, more than 1,000 colonies could be screened using 4 tobacco plants. Figure 1 shows the tumors on a tobacco leaf inoculated with a petri plate on which virulent and avirulent Agrobacteria were grown on specific sites. At the inoculation site of each of the virulent colonies (labeled T), a tumor was present. No tumors were present at the inoculation sites of the avirulent colonies (labeled N). Thus, it appears that replica plating onto wounded tobacco leaves is a reliable method of screening Agrobacterium for avirulent mu-
tants. This research was supported by Public Health Service grant CA 18604 from the National Cancer Institute to A.G.M. LITERATURE CITED 1. Lederberg, J., and E. M. Lederberg. 1952. Replicaplating and indirect selection of bacterial mutants. J. Bacteriol. 63:399-406. 2. Lippincott, J. A., and G. T. Heberlein. 1965. The induction of leaf tumors by Agrobacterium tumefaciens. Am. J. Bot. 52:396-403. 3. Lippincott, J. A., and G. T. Heberlein. 1965. The quantitative determination of the infectivity of Agrobacterium tumefaciens. Am. J. Bot. 52:856-863. 4. Lippincott, J. A., and B. B. Lippincott. 1975. The genus Agrobacterium and plant tumorigenesis. Annu. Rev. Microbiol. 29:377-405.
INFwTON AND ImMUNITY, June 1977, p. 1027-1028 Copyright C 1977 American Society for Microbiology
Replica-Plating Virulence Assay for Agrobacterium tumefaciens A. G. MATTHYSSE,* S. RATLIFF, AND J. SALBERG Department ofBotany, University of North Carolina, Chapel Hill, North Carolina 27514
Received for publication 3 December 1976
Replica plating onto wounded tobacco leaves provides a convenient method for screening for avirulent mutants of Agrobacterium tumefaciens. Extracellular infections of wounded dicotyledenous plants with the gram-negative bacterium Agrobacterium tumefaciens result in the development of crown gall tumors. Both tumorigenic and non-tumorigenic strains ofthe bacterium are known. The virulence of Agrobacterium strains has usually been assayed by inoculating stems, leaves, or storage roots with the bacterium and scoring for the presence (or absence) and size ofthe resulting tumors (4). Each of these assay systems requires making a separate inoculation for each bacterial strain to be studied. One assay system developed by Lippincott and Heberlein (3), using primary pinto bean leaves, allows the relation between the number of bacteria inoculated and the number of tumors formed to be studied. The number of tumors induced is proportional to the number of bacteria inoculated. We have been interested in modifying this leaf assay to allow more rapid screening for mutants of Agrobacterium that are avirulent. For this purpose, we have developed a replica-plating assay using tobacco leaves. Plants. Nicotiana tabacum L. variety Coker 319 was grown in the greenhouse, with one plant per pot. Bacteria. A. tumefaciens strains 15955 (virulent) and 6467 (avirulent) were obtained from the American Type Culture Collection. They were grown on 0.8% nutrient broth (Difco) and plated on 1.0% nutrient agar (Difco), with 9 to 40 colonies per plate. Plates were incubated for 3 to 5 days at 25°C before use. Occasionally they were stored at 4°C for 1 to 2 weeks. No effect of age of colonies or cold storage was observed. Similar results have been obtained with A. tumefaciens virulent strains 23308 (obtained from the American Type Culture Collection) and A6 (obtained from A. Braun). Inoculation. The upper surface of tobacco leaves was washed with tap water and wounded with carborundum (no. 600 silicon carbide) as described by Lippincott and Heberlein (2). The carborundum was then washed off with tap
water, and the leaves were allowed to dry for 15 to 30 min. A nutrient agar plate containing colonies of A. tumefaciens was replica plated on to sterile velvet and from the velvet onto a fresh plate of nutrient agar and onto the upper surface of the tobacco leaf (1). One to eight leaves on each plant were inoculated. Scoring. After inoculation, the tobacco plants were allowed to continue to grow for 10 to 30 days. They were watered from the bottom. The inoculated leaves were then picked. Tumors were scored by their raised appearance on the surface of the leaf as described by Lippincott and Heberlein (2) for pinto bean and tobacco leaves. The number of tumors was the same at 10 and 30 days, but the tumors were smaller at 10 days, so low-power magnification was necessary to score them; at 30 days tumors were visible to the unaided eye. The inoculation sites on the upper surface of the leaves were identified by dipping the leaf in a dilute (0.1%) solution of toluidine blue in tap water. This stained the inoculation sites dark blue. The tumors were photographed by turning the leaf over and covering it with 0.1% toluidine blue so that the normal surface of the leaf (except for some veins) was covered and the tumors stood out above the surface of the dye solution. Effect of age of plants and of leaves. Young tobacco plants (less than 3 months old) gave variable results. The percentage of replicaplated virulent colonies that gave rise to tumors averaged 60%, with a range of 30 to 100% in different experiments. Older tobacco plants (more than 5 months old) gave less variable results. The average percentage of virulent colonies causing tumors was 90%, with a range of 70 to 100% in different experiments. This variability could be reduced still further by using only the older leaves on the plant (about 20 cm long, 8th to 15th node from the bottom of the plant). Using only these leaves, 98 + 2% (range, 95 to 100%) of the virulent colonies replica plated onto the leaf gave rise to tumors (more than 500 colonies were tested). No aviru1027
1028
NOTES
INFECT. IMMUN.
FIG. 1. Tobacco leaf inoculated by replica plating 30 days previously with tumorigenic (T, strain 15955) and non-tumorigenic (N, strain 6467) Agrobacterium tumefaciens. The leaf was stained with 0.1 % toluidine blue. (A) The top side of the leafshowing the stained inoculation sites where the colonies occurred on the petri plate. The line at the bottom is 1 cm long. The leaf grew and expanded after inoculation, spreading the sites apart and increasing the area covered by each colony. (B) The bottom side of the leaf covered with 0.1% toluidine blue so that only the raised tumors and some veins show above the dye as white. The sites of the inoculating colonies shown in (A) are indicated by circles. The letters indicate the strain used to make the colony.
lent colonies replica plated onto the leaf gave rise to tumors (200 colonies were tested). Since one can use 40 colonies per plate, 1 plate per leaf, and 8 leaves per tobacco plant, more than 1,000 colonies could be screened using 4 tobacco plants. Figure 1 shows the tumors on a tobacco leaf inoculated with a petri plate on which virulent and avirulent Agrobacteria were grown on specific sites. At the inoculation site of each of the virulent colonies (labeled T), a tumor was present. No tumors were present at the inoculation sites of the avirulent colonies (labeled N). Thus, it appears that replica plating onto wounded tobacco leaves is a reliable method of screening Agrobacterium for avirulent mu-
tants. This research was supported by Public Health Service grant CA 18604 from the National Cancer Institute to A.G.M. LITERATURE CITED 1. Lederberg, J., and E. M. Lederberg. 1952. Replicaplating and indirect selection of bacterial mutants. J. Bacteriol. 63:399-406. 2. Lippincott, J. A., and G. T. Heberlein. 1965. The induction of leaf tumors by Agrobacterium tumefaciens. Am. J. Bot. 52:396-403. 3. Lippincott, J. A., and G. T. Heberlein. 1965. The quantitative determination of the infectivity of Agrobacterium tumefaciens. Am. J. Bot. 52:856-863. 4. Lippincott, J. A., and B. B. Lippincott. 1975. The genus Agrobacterium and plant tumorigenesis. Annu. Rev. Microbiol. 29:377-405.
