Hydrogen Cyanide Sensitivity in Bacterial Pathogens of Cyanogenic and Non-Cyanogenic Plants

RUST, L. A., W. E. FRY, and S. V. BEER. 1980. Hydrogen cyanide sensitivity in bacterial pathogens of cyanogenic and non-cyanogenic plants. Phytopathology 70:1005-1008. The cyanide sensitivity of bacterial pathogens of cyanogenic and nongenerally were more sensitive to HCN than were the xanthomonads tested. cyanogenic plants was determined by observing the effects of HCN on their Among xanthomonads, strains of Xanthomonas manihotis, a pathogen of growth rates in buffered nutrient broth. The HCN concentration that the highly cyanogenic plant, cassava (Manihot esculenta), appeared to be reduced the growth rate of each strain to 50% of its growth rate in the most tolerant. None of the bacteria appeared to either metabolize HCN or absence of cyanide (EC5o) was calculated and used to compare strains, to adapt markedly to it. In contrast to fungal pathogens of cyanogenic Bacterial pathogens of cyanogenic plants appeared to be only slightly more plants, bacterial pathogens of cyanogenic plants are not distinctly more tolerant of HCN than were bacterial pathogens of non-cyanogenic plants; tolerant of HCN than are those of non-cyanogenic plants. these differences were not statistically significant (P= 0.05). Pseudomonads Cyanogenic glycosides are produced by over 1,000 plant species, location, and donor of each strain were tabulated (Table 1). but their role is a subject of speculation (25). One function may be Pseudomonads were maintained on Difco nutrient agar (Difco to protect plants from pests (14,23). However, simple correlations Laboratories, Detroit, MI 43210) while xanthomonads were between glycoside content and resistance to a given pathogen have maintained on potato-dextrose agar plus 1% glucose (PDA +). not been consistently identified (16,28,29). This medium was made from 500 ml of water in which 200 g of Studies of the copperspot disease of the cyanogenic plant, potato tuber tissue had been autoclaved, 20 g Difco Bacto agar, 30 g birdsfoot trefoil (Lotus corniculatus L.) reveal that the fungal glucose, and water added to bring the total volume to I L. pathogen Stemphylium loti Graham, is tolerant of cyanide (HCN) HCN sensitivity. The sensitivity of bacteria to HCN was (17). Tolerance is probably due to an HCN-inducible enzyme, determined by observing its effects on the growth rate of each formamide hydro-lyase (FHL), that detoxifies cyanide to strain. Growth was determined turbidimetrically. Preliminary formamide (9-11). FHL also was found in Gloeocercospora inoculum consisted of two loopfuls of cells from a sorghi Bainand Edgerton, a pathogen of the cyanogenic plant sorghum culture which had grown on an agar slant for 24 hr at 27 C. This (Sorghum bicolor (L.) Moench), indicating that this enzyme is inoculum was placed into 25 ml of buffered nutrient broth plus 1% likely to be responsible for HCN tolerance in that fungus. Fry and (w/ v) glucose (BNB+)in a 125-mi Erlenmeyer flask. BNB+ consists Munch (12) hypothesized that pathogens of cyanogenic plants cope of 8 g Difco nutrient broth plus 10 g glucose per L in 0.1 M with HCN if it is released. An association between fungal potassium phosphate buffer, pH 6.8. This culture, which was pathogens of cyanogenic plants and the ability to produce FHL has designated the primary inoculum, was incubated at 27-29 C on a since been demonstrated. Of 31 species of fungi tested, every rotary shaker operated at 250 cpm. After 15 hr, 10 ml of the culture aggressive pathogen of a cyanogenic plant has a metabolic means of was removed and added to 10 ml of fresh BNB+ in a sterile sidearm coping with HCN (8). Furthermore, in vivo detoxification of HCN (nephelo) culture flask made from a 250-ml Erlenmeyer flask with via FHL is indicated in the pathogenesis of sorghum by G. sorghi an 18 mm X 150-mm test tube as a sidearm. This culture was (19-21) in which the pathogen apparently is exposed to HCN designated the secondary inoculum and was maintained under during disease development, but can tolerate the concentrations conditions described for the primary inoculum. Secondary encountered. inoculum was used to initiate the sensitivity studies when its The purpose of our work was to determine if a similar turbidity, measured at 620 nm with a Bausch & Lomb Model 20 relationship exists for bacterial pathogens, that is, to determine if Spectronic colorimeter (Bausch & Lomb Inc., Rochester, NY bacterial pathogens of cyanogenic plants are more tolerant of H CN 14650), attained an optical density (OD) of 0.9 or greater. than bacterial pathogens of non-cyanogenic plants. A preliminary The effect of HCN on growth of the different bacteria was report of this work has been made (24). determined by using the sidearm flasks as described above. Each flask contained 8 ml fresh BNB+, 1 ml of the secondary inoculum MATERIALS AND METHODS and 1.0 ml of HCN solution. Flasks were plugged with sterile rubber stoppers immediately after addition of the HCN. At the Bacteria. Six species of bacteria in two genera were studied. At beginning of an assay, each sidearm flask contained a total volume least two strains of each species were tested. Pseudomonas syringae of 10 ml of a BNB+ culture of exponentially growing cells with an and Xanthomonas manihotis are pathogens of the cyanogenic OD620nm = 0.10±+ .03 at a desired final HCN concentration ranging plants sorghum and cassava ( Manihot esculenta Crantz), from zero to 0.30 mM. Three concentrations of HCN and a zerorespectively (3,18). P. lachrymans, P. tabaci, X. malvacearum, and HCN control were tested in each experiment. Each treatment was X. vesicatoria were isolated from plants not known to be replicated once or twice within an experiment. Cultures were cyanogenic. Strains of P. syringae isolated from wheat (Triticum incubated under conditions described for the inoculum and aestivum L.) and corn (Zea mays L.), which are not highly turbidity was determined periodically. Experiments routinely were cyanogenic, also were tested. The strains used, host of isolation, terminated when the OD of the control (no HCN) flask had increased from the initial OD by 0.60 or more units. 0031 -949X/80/1 0100504/$03.00/0 When the average increase in 0D 620 nm (A~ 0D 620 nm) of the two @1980 The American Phytopathological Society flasks containing no HCN was 0.60 ± .01, the AX 0D 620 nm for each Vol. 70, No. 10, 198