Suspension Cultures Inoculated with Erwinia amylovora

HSU, S., and R. N. GOODMAN. 1978. Production of host-specific, wilt-inducing toxin in apple cell suspension cultures inoculated with Erwinia amylovora. Phytopathology 68: 351-354. A wilt-inducing toxin was detected in filtrates prepared of the virulent bacteria paralleled production of the toxin in from apple cell suspension cultures (ACSC) which had been the suspension cultures. The virulent strain of the bacterium inoculated with Erwinia amylovora. The toxin induced rapid produced high amounts of toxin in living ACSC, but little or wilting of susceptible host (apple) but not of nonhost plants none was produced in heat-killed ACSC or extracts of (tomato and tobacco). The toxin was produced by a virulent ACSC. strain but not by an avirulent strain of the pathogen. Growth Erwinia amylovora (Burrill) Winslow et al., the causal initiated from pieces of callus tissue placed in SH liquid organism of fire blight of apple and pear, produces a toxic medium and incubated at 26 C on a horizontal, rotary polysaccharide on infected host tissue (2, 3). The toxin, shaker at a speed of 110 rpm. The ACSC were initially which is called amylovorin or fire blight toxin, induces grown in 500-ml Erlenmeyer flasks containing 100 ml of wilting in many hosts but not in nonhost plants (3). SH liquid medium and these were used as stock cultures Because of its host specificity, the toxin may be used to for all experimental suspension cultures. Most of the evaluate varietal resistance of apple and pear to the fire experimental suspension cultures were grown in 250-ml blight bacterium. The toxin has not yet been produced in flasks and were started by transferring 10 ml of 14-dayan artificial medium. Preparation of the toxin in our old stock suspension cultures into flasks which contained * laboratory usually requires immature apple or pear fruits 50 ml of fresh SH liquid medium. Stock suspension which are available only at certain times of the year. Even cultures were transferred at 14-day intervals. these green fruits become less effective in yielding toxin as Bacterial cultures and toxin production.--Virulent they ripen. If the toxin is produced as a result of (E9) and avirulent (E8) strains of E. amylovora were used interaction between the host tissues and the pathogen, it (4) in these studies. The isolate E8 is a mutant of E9; should be possible to employ tissue culture techniques for however, in its inability to produce toxin and cause toxin production. The purpose of this study was to symptom development it is similar to several other determine whether the fire blight toxin could be produced avirulent isolates in our collection and a number of in apple cell suspension cultures (ACSC) inoculated with avirulent isolates obtained from Dr. Mortimer Starr, E. amylovora. University of California, Davis, 95616 (1,5). Suspensions of the bacterial cells were prepared from 24-hr cultures MATERIALS AND METHODS grown at 28 C on Difco nutrient agar supplemented with 0.5% yeast extract and 1% dextrose, and were adjusted to Apple callus tissue and cell suspension 108 cells/ml. The ACSC were grown for 14 days in SH cultures.-Attempts were made to grow stem and petiole liquid medium in 250-ml flasks and then were inoculated tissues of several cultivars (Jonathan, Red Delicious, with 1 ml of bacterial suspension. Culture filtrates were Malling-26) of apple (Pyrus malus) on various tissue collected at various times after inoculation by filtration culture media. Calli were induced but these grew poorly through Whatman No. 1 filter papers, centrifuging at on all the media tested. Callus tissues of the fire-blight5,000 g for 30 mmn, and finally by passage through a susceptible cultivar Antonovka (obtained from the School of Forestry, University of Missouri, Columbia) Millipore filter (0.45 /Am). The filtrates were tested for however, were found to grow well on Schenk and toxin activity (wilt induction) by the excised apple shoot Hildebrandt's medium (SH medium) (6) and were used (5 cm in length) bioassay procedure (3). The length of the tderntsmissue were grown o apple shoot used was crucial to the sensitivity of the throughout the study. Callus tissues were grown on SH bioassay. Stoffl (7) found that the apical 5 cm of Jonathan agar medium in 160-ml prescription bottles ' at 26 ± 2 apple shoots 5, 10, and 20 cm in length wilted in 1.5, 4.5, and were subcultured at 1-mo intervals. The ACSC were and 10.0 hr, respectively, when exposed to 100 g!g/ml amylovorin. 00032-949X/78/000 058$03.OO/0 Copyright © 1978 The American Phytopathological Society, 3340 It was found that excised apple leaves with petioles Pilot Knob Road, St. Paul, MN 55121. All rights reserved, attached also could be used to determine toxin activity.