The electrophoretic patterns of proteins extracted from ...

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CANADIAN JOURNAL O F BOTANY. VOL. 48, 1970

Apple shoots were exposed to ammonia vapors until the first traces of tissue discoloration became apparent. At that time the ammonia treatment was terminated and the level of ammonia absorbed by 5 cm long apical stem sections was measured. The average of four such experiments revealed that 220 pg of ammonia was absorbed. hi^ level of ammonia is of the same order of magnitude as was evolved from tissue infected with E. amylovora (Table I). F~~~ these results it was concluded that the ammonia formed in the stem tissue infected with

E. amylovora is a necrotoxin in the twig blight of apple. 1. COOK,A. A., and R. E. STALL.1969. Necrosis in leaves induced by volatile materials produced in vitro by bacteria. Phytopathology, 59: 259-260. 2. LOVREKOVICH, L., H. LOVREKOVICH, and R. N. GOODMAN. 1969. The role of ammonia in the wildfire disease of tobacco caused by Pseudomorzas tabaci. Phytopathology, 59: 1713-171 6. 3. LOVREKOVICH, L., H. LOVREKOVI~H, and R. N. GOODMAN. 1970. Ammonia as a necrotoxin in the hypersensitive reaction caused by bacteria in tobacco leaves. Can. J. ~ o t48: . 167-171.

The electrophoretic patterns of proteins extracted from spores and mycelium of two Drechslera species1 W. A. SHIPTON~ AND W. C. MCDONALD Carlada Departmer~tof Agricult~rre,Research Statior~,Winrzipeg, Manitoba Received October 1, 1969 SHIPTON,W. A., and W. C. MCDONALD. 1970. The electrophoretic patterns of proteins extracted from spores and mycelium of two Drechslera species. Can. J. Bot. 48: 1000-1002. Electrophoresis of protein extracts from spores and mycelium of two Drechslera species ( D . erythrospilum and D. teres) was carried out on polyacrylamide gels. Spores were harvested from V-8 juice agar and mycelium from V-8 juice broth cultures. One or two band differences were found between electrophoretic patterns of spore extract and mycelial extract depending on the species. Interspecies differences in patterns were no greater than intraspecies differences. The results are discussed, and it is suggested that the method of polyacrylamide gel electrophoresis of proteins is unsuitable as an aid to fungal taxonomy in its present state of development.

Introduction One of the latest techniques to be proposed as an aid to fungal taxonomy is the electrophoresis of fungal proteins. In a previous paper (4), it was found that the conclusions made from comparisons among electrophoretic patterns of uredospore proteins of rust species, formae speciales, and races depended on the method of assessment. Most authors have apparently performed their studies on the assumption that proteins derived from the reproductive and vegetative structures are the same. To our knowledge, only two investigators have examined the correctness of this assumption (1, 2), and their data apply to some Fusarium species only. With plants it has been found that different organs may yield proteins (enzymes) which are apparently different (5). Although one cannot safely extrapolate from lcontribution No. 394. "resent address: James Cook University of North Queensland, Townsville, Queensland, Australia.

higher plants to fungi, their data together with those of Hall (2) indicated that the relationship between proteins from spores and mycelium should be examined more closely. Materials and Methods Cultures of Dreck.~leraerytlirospilum (Drechs.) Shoem. and D. teres (Sacc.) Shoem. were simultaneously grown on 10yo V-8 juice broth and 10% V-8 juice agar at 16OC in the light for 8 days. Spores were harvested from cultures on agar by flooding them with a Tween-20 solution and gently brushing the conidiophores. Mycelium was collected from broth cultures and washed with a Tween20 solution. Microscopic examination showed that there was little mycelium in the spore samples and that the harvested myceliu~n was virtually spore-free. In both cases, free water was removed from the samples by centrifugation. Spores were disrupted as previously described (4), while the mycelium was ground to a powder in dry ice with a Inortar and pestle. Proteins were extracted in 0.02 M tris-HC1 buffer (pH 6.7), and were dialyzed overnight against buffer at 1 OC, after cell debris had been removed by centrifugation. The proteins were then lvo~hilizedand sus~endedin 10% sucrose. of gel columns &d the details of electroThe

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phoresis and staining of the gels were as described previously (4). About 330 pg of protein was layered on each gel column, and the protein patterns were assessed using the technique of protein mixtures (4). Conclusions are based on data derived from four replicates.

Observations Agreement among the replicates was complete when the technique of protein mixtures was used. The data obtained for D. erythrospilurn and D. teres are given in Fig. 1. Although most bands in patterns from spore extracts appear to have corresponding bands in patterns from mycelial extracts, there is a two-band difference in the patterns of spores and mycelium of D. erythrospilurn and a one-band difference between the 1.0

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SPORE EXTRACT

MYCELIAL EXTRACT

extracts of D. teres. There are also differences in the width and color intensity of a number of bands, and occasionally two bands in one pattern appear to correspond with one in the other. A comparison of protein patterns from the extracts of spores of the two species revealed a two-band difference (one light and one medium color intensity band). Discussion There are obvious difficulties in growing and harvesting sufficient quantities of spores and mycelium of some fungi under identical conditions. The reasons for more profuse conidial production on agar than on broth cultures has 1.0

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SPORE EXTRACT

MYCELIAL EXTRACT

D. erythrospilum

FIG.1. Electrophoretic patterns of proteins extracted from spores and mycelium of D. erythrospilurn and D. teres. Arrows identify bands which appeared to have no homologue. Black bands were darkly stained, open boxes represent lightly stained bands, and striped boxes represent bands with an intermediate color intensity.


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CANADIAN JOURNAL O F BOTANY.

never been satisfactorily explained, although there are several theories (3). In view of our present knowledge, it is unsafe to state unreservedly that the data constitute conclusive proof of a difference between the electrophoretic patterns of extracts from spores and mycelium, but it is equally unsafe to assume that extracts from spores and mycelium give a similar pattern. The data of Hall (2) support our view, in that conidia of Fusarium solani (App. & Wr.) Wr. emend. Snyd. & Hans. were found to influence the number of catalase bands. Both spores and mycelium were harvested from the same plates and two extra bands of catalase activity were identified in conidial extracts under certain conditions. There appears to be no objection to comparing protein patterns of extracts from asporous cultures or from spores, but variable amounts of spores and mycelium could mask the real differences. It was noted by Clare et al. (I), however, that with F. oxysporum Schlecht. emend. Snyd. & Hans. changes in the numbers of spores did not influence the protein patterns. Pattern differences between extracts from spores and mycelium of D. erythrospilum were as great as the differences in the patterns between extracts from spores of the two species. For the technique of gel electrophoresis of proteins to be

VOL. 48.

1970

of taxonomic value, interspecies differences must be greater than intraspecies differences. This has generally been found to be true, but at present there appears to be too much scope for differences in interpretation of the same data. Shipton and Fleischmann (4) recorded the type of disparity that could arise when three currently used methods of assessment were used, and they noted several reasons why the same protein may show different Ep values (Ef = migration of protein bandslmigration of marker dye). We question the value of the method as a taxonomic aid in its present state of development. 1. CLARE,B. G., N. T. FLENTJE, and M. R. A T K ~ S O N . 1968. Elcctrophorctic pattcrns of oxidoreductases and other proteins as criteria in fungal taxonomy. Aust. J. Biol. Sci. 21 : 275-295. 2. HALL,R. 1967. Proteins and catalase isoenzvmes from ~~isaririnz solnni and their taxonomic sibificance. Aust. J. Biol. Sci. 20: 419-428. 3. HAWKER,L. E. 1966. Environmental influences on reproduction. In The fungi: An advanced treatise. Vol. 2. Edited by G. C. Ainsworth and A. S. Sussman. Academic Press Inc., New York. pp. 435-469. 4. SHIPTON,W. A,, and G. FLEISCHMANN. 1969. Taxonomic significance of protein patterns of rust species and formae speciales obtained by disc electrophoresis. Can. J. Bot. 47: 1351-1358. 5. WENNSTROM, J., and E. D. GARBER.1965. The genus Collinsin. XXVII. Separation of esterases and acid phosphatases in extracts from twelve species and one interspecific hybrid by starch-gel zone electrophoresis. Bot. Gaz. 126: 223-225.

Phaeoseptouia contoutae sp. nov. on Pinus contouta from Alberta1 J. A. PARMELEE Plant Research Institute, Depart~ne~zt of Agriczihure, Ottawa, Canada AND

Y . HIRATSUKA Forest Research Laboratory, Department of Fisheries and Forestry, Calgary, Alberta Received January 23, 1970 PARMELEE, J. A., and Y. HIRATSUKA. 1970. Phaeoseptoria corztortae sp. nov. on Pinus corztorta from Alberta. Can. J. Bot. 48: 1002-1004. A fungus, Phaeosepforia corzrortae sp. nov., is described and illustrated on the needles of Pinus corltorta Dougl. from Alberta. The type specimen shows partial association with the needle-cast fungus Davisomycella ampla (Davis) Darker but the nature of the association is not known.

A sphaeropsidaceous fungus showing partial association with the needle-cast fungus Davison~ycellaampla (J. J. Davis) Darker (= HypolContribution No. 752, Plant Research Institute, Department of Agriculture, Ottawa, Canada.

derinella ampla (J. J. Davis) Dearn.) was recently received for identification from southwestern Alberta. It showed general morphological similarities to a group of Fungi Imperfecti, including Hendersonia pinicola Wehm', which was described as associated with Lophoderniella con-