Electron microscopy

Copper grids (3.09 mm) (Agar Scientific Ltd.) were prepared for electron microscopy by formvar coating. A clean, dry cover slip was dipped into a 1.5% formvar in chloroform solution, allowed to dry in the vapour and, after scoring with a sharp blade, the form var coat was floated onto a water surface. Grids were placed onto the formvar and the coated grids removed from the water with heavy aluminium foil. A phage suspension ( approximately 10® p.f.u./m l) was centrifuged (5,000 rpm, 15 min) in an MSE Hi-Spin 21 and the supernatant recentrifuged (13,000 rpm, 90 min). The pellet was resuspended in 2% (w/v) phosphotungstic acid (pH 7.0) and 10 /d placed onto a formvar coated grid, blotted to rem ove excess liquid and allowed to dry in air. Grids were examined using a Jeol Electron Microscope at 60 kV.

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Bacterial samples for examination were grown, without shaking, at 27°C for 18 h and 10 /xl placed onto a formvar coated grid for 1 min. The liquid was removed with blotting paper and 1 0 jxl stain added and removed immediately before allowing the grid to air dry.

Bacterial cells (10 ml) containing pHCP2 were grown at 30°C for 18 hours in DDA containing maltose (0.2% ), MgSC>4 (10 mM) and ampicillin. The cells were centrifuged in an MSE Chilspin (5,000 rpm, 10 min, room temperature) and resuspended in 1 ml DDA containing lOmM MgS0 4- Lambda::Tn5 was added at a concentration o f 10^ p.f.u./m l and incubated at 30°C for 30 min. The cells were recentrifuged (5,000 rpm, 10 min, room temperature) and washed in 0.1 M sodium citrate before recentrifugation and resuspension in 10 ml LB. The suspension was incubated at 30°C for 30 to 45 min, recentrifuged and resuspended in 1 ml DDA. Samples (100 /¿I) were spread onto each o f three DDA plates containing kanamycin sulphate to select for the presence o f the transposon, and the plates incubated for 24 to 48 h at 30°C. Kamamycin resistant colonies were patched onto a number of plates containing minimal media and a range o f amino acids. The ability to grow on supplemented plates allowed identification o f a number o f auxotrophies.

Transduction o f plasmids or cosmids into E.coli via Lambda was essentially as described above, with the following differences; growth o f E.coli did not require the addition o f Ap, and after incubation to allow expression of antibiotic resistance (if appropriate) cells were plated onto media containing appropriate antibiotics.

All phages isolated were tested for the ability to transduce certain markers between strains o f Erwinia. The assay was carried out essentially as for the production o f auxotrophic mutants, except that the auxotrophic mutants were grown in kanamycin initially. The phage to be tested was added at a known multiplicity o f infection (m .o .i.) which was varied to offer the greatest chance o f success. Adsorption was carried out at 25 °C for varying amounts of time and after a final wash in minimal medium the cells were plated onto minimal agar containing glucose as the carbon source. As well as the ability to transduce chromosomal genes between strains the ability to transduce plasmids was also tested, the transduced plasmid (pHCP2) being selected on LB containing ampicillin. The frequencies o f generalised transduction were calculated after subtracting the number o f spontaneous prototrophic colonies from control plates (as above without the addition o f phage).

2.14 Phage adsorption assay

Bacterial cells were grown at 27°C overnight. To 10 ml bacterial cells (approx. 1.0 x 1 0® c.f.u./m l) phage lysate was added to a final titre o f approx. 1 0 9 p .f.u ./m l, and the mixture incubated at 27°C. At time intervals o f 0 , 10, 20, 30, 40 and 60 min, 1 0 0n\ o f the m ixture was removed and diluted 1 0 0 fold in ice cold phage buffer, followed by centrifugation in an MSE Chilspin (4,000 rpm, 10 m in, 4°C). These supernatants were titred on a permissive host.

2.15 The production o f phage resistant mutants

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o f culture was gently mixed with 1 0 0n\ of a high titre lysate of phage (approx.

1 0 * 0 p.f.u./m l), a soft agar overlay was made and incubated at 25°C for 48 to 76

hours. After this time colonies th at grew were tested for phage resistance by spotting 10 n\ o f phage onto a law n o f the cells. To confirm that this phage resistance was not due to lysogeny, each colony was grown overnight at 30°C and its supernatant spotted onto a law n o f wild type bacteria. Lack o f phage action indicated a phage resistant mutant an d not a lysogen.

2.16 Cross testing of the mutants w ith other phages

A number o f phage lysates (10 /¿l) were spotted onto each o f 40 lawns, each seeded with one phage resistant m utant. Phage sensitivity, resistance and decreased efficiency o f plating (e.o.p.) was noted in each case.

2.17 Inoculation o f miniplants

Growth o f potato (Solarium tuberosum) plants (cultivar Maris Bard, used because of its particular susceptibility to blackleg infection) was as described by Hinton et at. (1989a). When plants were approximately S to 10 cm tall and the stems approximately 2 mm thick, virulence tests were performed. Phage resistant mutants were patched onto LB agar and incubated at 27°C for 48 hours. Cells from each plate were inoculated into the top third o f a plant stem using a sterile cocktail stick and the wound immediately covered with Vaseline. Inoculated plants were placed inside polythene bags and exposed to a 16 h light regime and 80 to 1 0 0% humidity at 20°C for 5 to 7 days. After th is time blackleg symptoms were scored using a numerical index; 0, no reaction; 1, slight browning around inoculation site; 2, slight blackening around inoculation site; 3, small black rot spreading from the inoculation

site; 4, medium black rot spreading from inoculation site; 5, large black rot and stem collapse (Fig. B l). Phage resistant mutants which gave symptoms o f 1 and 2 were retested on several further occasions on both micropropagated plants and on tuber grown plants (approx. 40 cm tall). Mutants which continued to give such symptoms were termed reduced virulence (Rvi-) mutants.

2.18 Tuber maceration

Bacterial cells were grown in LB (27°C, overnight), washed in diluting buffer and diluted in this buffer to a final density o f approx. 1.0 x 107 c.f.u./m l. Potato tubers (Maris Bard) were surface sterilized by immersion in 5% chloros (5 min), rinsed with sterile distilled water and air dried. Using a 200 n\ micropipette tip, 10 n\ of bacterial cell suspension (1 . 0 x 1 0 7 c.f.u./m l) was injected into an intact tuber, to a depth o f IS mm. Replicates o f the same suspension were inoculated up to S times into the same tuber. This procedure was repeated for a bacterial suspension o f 1.0 x 10^ c .f.u ./m l. The tubers were placed in a Forma Scientific anaerobic incubator and covered with black polythene bags to reduce exposure to light. The tubers were incubated (20°C, 6 days) and sliced along the axis o f injection, and the diam eter of the rot measured (mm). Each strain was inoculated on 12 occasions and an average diameter taken.