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Comparison of soluble proteins of Gaeumannomyces graminis var. tritici and Phialophora spp. by polyacrylamide gel electrophoresis
78
Myrol. Res. 94 (I):78-82 (1990) Printed in Great Britain
Comparison of soluble proteins of Gaeumannomyces grarninis var. fritici and Phialophora spp. by polyacrylamide gel electrophoresis
ERNA M. C. MAAS* Small Grain Centre, Private Bag X 29, Bethlehem 9700, South Africa
E L R I T H A VAN Z Y L , P. L. S T E Y N AND J. M. K O T Z E Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa
Comparison of soluble proteins of Gaeumannomyces graminis var. tritici and Phialophora spp. by polyacrylamide gel electrophoresis. Mycological Research 94 (1):78-82 (1990). Soluble proteins of Gaeumannomyces graminis var. tritici (Ggt), Phialophora graminicola, P. zeicola, Phialophora sp. and Magnaporthe rhizophila were compared by polyacrylamide gel electrophoresis (PAGE). Taxonomic relationships were quantified by computerassisted analysis of spectrophotometric data. All three Ggt isolates and the two Phialophora sp. isolates formed separate clusters, closely related to each other. M . rhizophila was related to a third cluster formed by two P. graminicola isolates. The remaining P. graminicola isolates clustered at different levels with the Ggt-Phialophora sp. group. P. zeicola related poorly to these groups. The tendency of Ggt and Phialophora sp. isolates to form separate groups suggests that PAGE can be used to differentiate between morphologically similar fungi. Key words: Polyacrylamide gel electrophoresis, Take-all, Gaeumannomyces, Phialophora, Numerical analysis.
The taxonomy of Gaeumannomyces graminis (Sacc.) v. Arx & Olivier var. h-itici Walker (Ggt), and fungi associated with take-all disease is based on a range of characteristics of which ~roductionof the teleomorph is probably the most important. Failure of some isolates to produce perithecia, as well as the fact that several of these fungi are closely related and can be distinguished only on detailed characters such as hyphopodium shape and ascospore size, complicate identification. Much confusion also surrounds the Pkialopkora state of the varieties of G. graminis (Walker, 1981). The principle of resolving taxonomic problems by molecular techniques has become increasingly important in recent years with the development of polyacrylamide gel electrophoresis of soluble proteins. It has frequently been reported to aid in bacterial (Kersters & de Ley, 1980), as well as in fungal classification (Snider & Kramer, 1974). Thus, by using electrophoresis, Abbott & Holland (1975) showed that Ggt was more closely related to G. grarninis var. avenae (Tumer) Dennis than either were to G. graminis var. graminis. The purpose of this study was to evaluate polyacrylamide gel electrophoresis as a means of distinguishing between morphologically similar fungi, and to simultaneously determine the relationship between Ggt and related taxa. Present address: Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002.
Particular emphasis was placed on the computer-assisted analysis of data obtained through spectrophotometric evaluation of protein patterns of the isolates.
MATERIALS A N D M E T H O D S Ovigin of isolates Twelve morphologically similar isolates were used in the present study. Three Ggt isolates, as well as two unidentified isolates (referred to as Pkialopkora sp. - J. W. Deacon, pers. comm.) were obtained by plating wheat root segments on 2% water agar supplemented with 250 p.p.m. chloramphenicol. Ggt was positively identified by the production of perithecia on artificially infected wheat seedlings. Hyphopodia and phialospores resembling those described for Pkialophora sp. (Walker, 1981) were produced by the two unknown isolates. Isolates of Magnaporthe rkizopkila Scott & Deacon (PREM 45952), Pkialopkora zeicola Deacon & Scott (PREM 45754) and P. graminicola (Deacon) Walker (PREM 48865) were supplied by the Plant Protection Research Institute in Pretoria. The isolate of P. graminicola was deposited by P. Landschoot, U.S.A. in the Institute's National Collection of Fungi - in Pretoria. Four other P. graminicola isolates were obtained from R. W. Smiley, U.S.A. (respectively numbered 59, 60, 197, and 5 7-84).
79
Ema M. C. Maas and others Culture methods and sample preparation
Isolates were cultured on potato dextrose agar (PDA) and each isolate inoculated in triplicate into 100 ml nutrient broth (0.1% meat extract, 0.2% yeast extract, 0.5 % peptone, 0.8% NaCl and 1% glucose) in 250ml Ehrlenmeyer flasks with cotton wool stoppers. To allow for sufficient mycelium development, flasks were incubated for 10 d at 25 OC. After this period the mycelial mats were collected, placed in 4 % KOH for 10 min (Marchant, 1966), decanted into 0.5 mm mesh sieves and rinsed under running tap water for I min. Mats were then placed on several layers of towelling paper and blotted dry. Of each fungal isolate, 0.3 g mycelium was immediately used for protein extractions. As repeated subculturing was found to affect protein patterns, the remaining mycelium was placed in sterile Petri dishes and frozen for further extractions. Protein extractions
The pre-weighed mycelium was ground in a mortar and pestle for 1min in 4 ml extraction buffer (pH 6.8) consisting of 25 ml 0.25 M Tris, 2.05 g sodium dodecyl sulphate (SDS), 10 ml glycerol, 5 ml 2-mercaptoethanol, 0.1 ml 1 % bromophenol blue solution and distilled water to 100 ml. The resultant slurry was decanted into vaccine bottles, stoppered lightly and heated in a boiling water bath for 2 rnin (Laemmli, 1970). Concomitantly, 0.01 g lysozyme and 0.01 g thyroglobulin, used as reference proteins (Kersters & de Ley, 1975) were dissolved in 10 ml extraction buffer and treated as described before. The extracts were transferred to Eppendorf microcentrifuge tubes (2 ml) and centrifuged for 15 min at 12000 rev. min-l. New extractions were made from frozen mycelium for each electrophoretic run. Two electrophoretic runs consisting of two gels per run were compared for the purpose of this study. Electrophoresis
The discontinuous dodecyl sulphate (SDS) buffer system described by Laemmli (1970) was used as a basis for one dimensional slab gel electrophoresis in a Protean 11 vertical electrophoresis unit. The gel slabs (160 x 200 x 1.5 mm) consisted of 10% acrylamide resolving gel at pH 8.8, and 4 % stacking gel at pH 6.8, into which a 15-well comb was placed before polymerization. After polymerization, the comb was removed and the wells were flushed with electrode buffer at pH 8.3. The buffer consisted of 9 g Tris-Base, 43 g glycine, 3 g SDS and distilled water to 3 1. Each well then received 5 vl glycerol :1% bromophenol blue solution (v/v 50 :50) as tracking dye, 5 ul lysozyme solution, and 20 ~1 protein extract of each fungus. One lane on each gel received only ) served as the thyroglobulin (30 ul) and lysozyme (5 ~ 1 and reference lane for spectrophotornetric tracings. Electrophoresis, carried out under constant voltage was started at 75 V until the blue tracking dye penetrated the resolving gel, after which the voltage was increased to 150 V. Each run lasted approximately 6 h. After electrophoresis, gels were removed, fixed and stained for 2 h in a solution consisting of 100 ml
10% acetic acid and 100 ml filtered stain concentrate (2.5 g Brilliant Blue R in I 1 95 % ethanol). Destaining was done in four steps for periods of 2-3 h in the following solutions : 200 ml 95 % ethanol and 300 ml 5 % acetic acid; 150 ml 95 % ethanol and 350 m l 5 % acetic acid; repeat of second step; 100 ml 95% ethanol and 400 ml 5 % acetic acid. After sufficient destaining, the gels were stored in a solution consisting of 100 ml 95% ethanol, 200 ml 5 % acetic acid and 200 ml water. Spectrophotometric evaluation
The principle of spectrophotornetric evaluation and numerical analysis as described by Kersters & de Ley (1975) was used with minor variations. Destained gels were placed between two clean glass plates and each lane (representing one fungal isolate), as well as the reference lane, were scanned using a Beckrnann DU-8 spectrophotometer. The spectrophotometer was set so that tracings represented three times the length of each lane. Each tracing was thus approximately 40 crn in length. The spectrophotornetric tracings (in which the x- and y-axes represented distance and absorbance respectively) of the reference lane of each gel was first divided into 180 spaces by placing it over a computer-drawn sliding scale on a light table. Starting from the bottom, position 12 and 180 were represented by the lysozyme and thyroglobulin peak respectively. The spectrophotornetric tracings of the fungal isolates were then each placed over the divided reference spectrophotornetric tracing on a light table and divided into 180 spaces, with the lysozyme peak being the point of concurrence. After this, the extinction, expressed as the height in mm of each of the 180 positions, was measured and used as data for numerical analysis. Compensation of spectrophotornetric scans
Compensation as described by Kersters & de Ley (1975) was unnecessary when two gels from the same run were analysed and compared since clustering levels for isolates of the same species ranged between r = 0.95 and 0.99. When gels from different runs were compared, compensation was achieved by calculating the average position number of the 12-16 most prominent bands from each of the tracings, and moving each band to its calculated position. This was effected by the omission or addition, respectively, of the lowest extinction value in the valley floor between bands. After compensation, clustering levels between isolates of a species ranged between r = 0.90 and 0.95. According to Kersters & de Ley (1975) correlation coefficients ranging between r = 0.82 and 0.92 are sufficiently high to allow comparison. Numerical analysis
The Pearson product-moment correlation coefficient r (Sokal & Sneath, 1963) between any pair of spectrophotornetric tracings was calculated, using the data already mentioned. The resultant r-matrix was transformed to a distance matrix and clustered by the unweighted average pair group method (UPGMA) (Zupan, 1982). The distance values of all clustering levels were again transformed to r-values and a dendrogram
Electrophoresis of Gaeumannomyces and Phialophora Fig. I. Spectrophotornetric tracings (reduced approx. 8 x ) of protein patterns of Gaettmannomyces, Phialophora and related genera. Absorbance and distance are represented on the y- and x-axes respectively. L, lysozyrne; T, thyroglobulin.
1
t
1
P . graminicola 60
V
P. graminicola 57-84
V
P. graminicola 197
1 I!
P. zeicola
Phialophora sp. 168b
1 1 I
P. graminicola 59
1
1 r~ G . graminis var. tritici 712
Reference proteins
M . rhizophila
81
Erna M. C. Maas and others compiled for the various isolates. All calculations were performed using the IBM-Mainframe computer of the Institute for Computer Sciences, University of Pretoria.
RESULTS
Fig. 3. Dendrogram illustrating the relationship within and between isolates of Gaeumannomyces, Phialophora and related genera, obtained through numerical analysis of electrophoretic data. P. graminicola PREM 48865 P. graminicola (60)
The spectrophotometric tracing (reduced approximately 8 x ) G . graminis var. rrilici (714) of each isolate, as well as the electrophoretically obtained protein patterns, are illustrated in Figs 1 and 2 respectively. G . graminis var. rritici (712) Under strict standardized conditions of growth, extraction and electrophoresis, protein patterns were distinctive and C.graminis var. tririci (716) reproducible over the two electrophoretic runs (two gels per P. graminicola (197) run). Visual examination of the gels revealed a high degree of similarity in protein band occurrence and position between Phialophora sp. (186b) the various isolates. Differences were related to the presence or absence of a few protein bands, protein concentration and Phialophora sp. (186a) banding pattern. A dendrogram, showing the correlation within and between M.rhizophila PREM (45952) species as established by numerical analysis of data from one P. graminicola (59) gel, is presented in Fig. 3. The three isolates of Ggt formed a tight separate cluster to which two of the P. graminicola P. graminicola ATCC 60239 (5744) isolates (60 and 197) were each separately related at r = 0.97 and 0.95 respectively. The two unidentified Phialophora P. zeicola (261)PREM 45754 isolates (168a and 168b) formed another tight cluster (r = 0-70 0.80 0.90 0.95), which in turn was related to the Ggt/P. graminicola group at r = 0.94. One P. graminicola isolate (PREM 48865) was separately related to this group at r = 0.89. A third tight cluster was produced by the last two P. graminicola isolates (59 DISCUSSION and 57-84), to which M. rhiwphila was related at a correlatio? The protein patterns of the various isolates were very similar, level of r = 0.90. This group was related to the Ggt/P. yet consistent and distinct differences were found to occur. graminicola group at r = 0.85. P. zeicola joined the whole This is in agreement with the report by Snider & Krarner differences (1974), Reynolds, Weinhold & Morris (1983) and Hansen et al. cluster at the low level of r = 0.73. Although - slight in correlation values occurred between dendrograms from (1986). different gels, the basic clustering pattern remained intrinsically At the onset of this study it was presumed that the P. the same. graminicola isolates used were homologous. However, the low level of association found between these isolates highlights the heterogeneity that has developed in the concept of the Fig. 2. Protein patterns of Gaeumannomyces, Phialophora and related latter species and the difficulty in separating isolates genera obtained through polyacrylamide gel electrophoresis. A, P. morphologically. This low level of association was verified in zeicola; B, C, Phialophora sp. (168b and 168a);D, E, F, Ggt (716, 714 a recent report by Smiley (1987), according to which the and 712); G, P. graminicola (197); H , P. graminicola (57-84);I, P. isolates used in the present study were incorrectly identified graminicola (60);J , P. graminicola (59);M. rhiwphila; L, P. graminicola because they do not resemble the anamorph of G. cylindrosporus (PREM 48865); M, reference lane (L*,lysozyrne, T, thyroglobulin). Hornby, Slope, Gutteridge & Sivanesan, viz. P. graminicola, isolated from plants by Jackson & Landschoot (1986). In a later publication, Landschoot & Jackson (1987) reclassified P. graminicola isolates 57-84 and 59 as the Phialophora state of a yet undesaibed Magnaporthe sp. That report corroborates the findings of the present study, in which these isolates showed an affinity to M . rhizophila, and always formed a tight separate cluster, whereas two other isolates (60 and 197) were more closely related to Ggt. Electrophoresis is useful in delimiting species only when the intraspecies difference is smaller than the interspecies dfference (Shipton & MacDonald, 1970). The very high levels of similarity observed between the Ggt and Phialophora sp. isolates is, thus, indicative of greater intraspecies relationship and suggest that PAGE can successfully be used to distinguish between morphologically and ecologically related fungi. No report concerning electrophoresis of Ggt has appeared
Electrophoresis of Gaeurnannomyces and Phialophora since the study b y Abbott & Holland (1975). The present investigation, as far as could be ascertained, is also the first report o n electrophoresis-aided fungal taxonomy in which computer-assisted analysis of data, obtained through spectrophotometric evaluation of polyacrylamide gels, has been used. The advantages attached t o the latter procedure include: (i) the objective gathering of data, (ii) the insensitivity of the correlation coefficients t o concentration, (iii) the assessment of similarities and dissimilarities through the evaluation of the protein pattern as a whole and not merely of individual protein bands, and (iv) the ability of the ~ ~ e c t r o p h o t o m e t et or monitor subtle differences undetectable to the human eye. A further advantage of electrophoresis is that it can be performed relatively rapidly. According to Smiley (1987), the taxonomic delimination of Phialophora spp. is quite uncertain and, in view of the new species involved in turfgrass diseases, rapid diagnostic procedures will b e required for positive identification of the agents of patch disease. Finally, it is suggested that electrophoresis can assist in verifying established taxonomy of fungal groups or isolates b y providing additional biochemical criteria. This study was financed b y the Department of Agriculture and Water Supply and the South African Wheat Board. W e thank D r F. C. Wehner for suggestions during the preparation of the manuscript and Professor J. J. Joubert for supplying the computer programme for numerical analysis. The assistance of M r J. Maritz, Professor K. Kersters and D r J. J. Bezuidenhout in the implementation of the computer programmes for cluster analysis, is gratefully acknowledged.
REFERENCES ABBOTT, L. K. & HOLLAND, A. A. (1975). Electrophoretic patterns of Gaeumannomyces graminis. Australian Journal of Botany 23, 1-12. HANSEN, E. M., BUSIER, C. M., SHAW D. S. & HAMM, P. B. (1986). The taxonomic structure of Phytophthora megasperma: (Received for publication 28 July 1988)
82
evidence for emerging biological species groups. Transactions of the British Mycological Society 87, 557-573. JACKSON, N. & LANDSCHOOT, P. J. (1986). Gaeumannomyces cylindrosporus associated with diseased turfgrass in Rhode Island. Phytopathology 76, 654. KERSTERS, K. & DE LEY, J. (1975). Identification and grouping of bacteria by numerical analysis of their electrophoretic protein patterns. lournal of General Microbiology 87,333-342. KERSTERS, K. & DE LEY, J. (1980). Classification and identification of bacteria by electrophoresis of their proteins. In Microbiological Classification and Identification (ed. M. Goodfellow & R. G. Board), pp. 273-297. Academic Press: London, U.K. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680-685. LANDSCHOOT, P. J. &JACKSON, N. (1987).The Phialophora state of a Magnaporthe sp. causes summer patch disease of Poa pratensis and P. annua L. Phytopathology 77, 119 (Abstr.). MARCHANT, R. (1966). Wall structure and spore germination in Fusarium culmorum. Annals of Botany 30, 821-830. REYNOLDS, M., WEINHOLD, A. R. & MORRIS, T. J. (1983). Comparison of anastomosis groups of Rhizoctonia solani by polyacrylamide electrophoresis of their soluble proteins. Phytopathology 73, 903-906. SHIPTON, W.A. & McDONALD, W. C. (1970). The electrophoretic patterns of proteins extracted from spores and mycelium of two Drechslera species. Canadian Journal of Botany 48, 1000-1002. SMILEY, R. W. (1987). The etiologic dilemma concerning patch disease of bluegrass turfs. Plant Disease 71, 774-781. SNIDER, R. D. & KRAMER, C. L. (1974). Polya~r~lamidegel electrophoresis and numerical taxonomy of Taphrina caemlescens and T. deformans. Mycologia 66, 743-753. SOKAL, R. & SNEATH, P. H. A. (1963). Principles of Numerical Taxonomy. W. H . Freeman and Co: San Francisco, U.S.A. WALKER, J. (1981). Taxonomy of take-all fungi and related genera and species. In Biology and Control of Take-all (ed. M. J . C. Asher & P. J. Shipton). Academic Press: London, U.K. ZUPAN, J. (1982). Cltrstering of Large Data Sets (ed. D. Bawden). Research Studies Press: Chichester, U.K.
Myrol. Res. 94 (I):78-82 (1990) Printed in Great Britain
Comparison of soluble proteins of Gaeumannomyces grarninis var. fritici and Phialophora spp. by polyacrylamide gel electrophoresis
ERNA M. C. MAAS* Small Grain Centre, Private Bag X 29, Bethlehem 9700, South Africa
E L R I T H A VAN Z Y L , P. L. S T E Y N AND J. M. K O T Z E Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa
Comparison of soluble proteins of Gaeumannomyces graminis var. tritici and Phialophora spp. by polyacrylamide gel electrophoresis. Mycological Research 94 (1):78-82 (1990). Soluble proteins of Gaeumannomyces graminis var. tritici (Ggt), Phialophora graminicola, P. zeicola, Phialophora sp. and Magnaporthe rhizophila were compared by polyacrylamide gel electrophoresis (PAGE). Taxonomic relationships were quantified by computerassisted analysis of spectrophotometric data. All three Ggt isolates and the two Phialophora sp. isolates formed separate clusters, closely related to each other. M . rhizophila was related to a third cluster formed by two P. graminicola isolates. The remaining P. graminicola isolates clustered at different levels with the Ggt-Phialophora sp. group. P. zeicola related poorly to these groups. The tendency of Ggt and Phialophora sp. isolates to form separate groups suggests that PAGE can be used to differentiate between morphologically similar fungi. Key words: Polyacrylamide gel electrophoresis, Take-all, Gaeumannomyces, Phialophora, Numerical analysis.
The taxonomy of Gaeumannomyces graminis (Sacc.) v. Arx & Olivier var. h-itici Walker (Ggt), and fungi associated with take-all disease is based on a range of characteristics of which ~roductionof the teleomorph is probably the most important. Failure of some isolates to produce perithecia, as well as the fact that several of these fungi are closely related and can be distinguished only on detailed characters such as hyphopodium shape and ascospore size, complicate identification. Much confusion also surrounds the Pkialopkora state of the varieties of G. graminis (Walker, 1981). The principle of resolving taxonomic problems by molecular techniques has become increasingly important in recent years with the development of polyacrylamide gel electrophoresis of soluble proteins. It has frequently been reported to aid in bacterial (Kersters & de Ley, 1980), as well as in fungal classification (Snider & Kramer, 1974). Thus, by using electrophoresis, Abbott & Holland (1975) showed that Ggt was more closely related to G. grarninis var. avenae (Tumer) Dennis than either were to G. graminis var. graminis. The purpose of this study was to evaluate polyacrylamide gel electrophoresis as a means of distinguishing between morphologically similar fungi, and to simultaneously determine the relationship between Ggt and related taxa. Present address: Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002.
Particular emphasis was placed on the computer-assisted analysis of data obtained through spectrophotometric evaluation of protein patterns of the isolates.
MATERIALS A N D M E T H O D S Ovigin of isolates Twelve morphologically similar isolates were used in the present study. Three Ggt isolates, as well as two unidentified isolates (referred to as Pkialopkora sp. - J. W. Deacon, pers. comm.) were obtained by plating wheat root segments on 2% water agar supplemented with 250 p.p.m. chloramphenicol. Ggt was positively identified by the production of perithecia on artificially infected wheat seedlings. Hyphopodia and phialospores resembling those described for Pkialophora sp. (Walker, 1981) were produced by the two unknown isolates. Isolates of Magnaporthe rkizopkila Scott & Deacon (PREM 45952), Pkialopkora zeicola Deacon & Scott (PREM 45754) and P. graminicola (Deacon) Walker (PREM 48865) were supplied by the Plant Protection Research Institute in Pretoria. The isolate of P. graminicola was deposited by P. Landschoot, U.S.A. in the Institute's National Collection of Fungi - in Pretoria. Four other P. graminicola isolates were obtained from R. W. Smiley, U.S.A. (respectively numbered 59, 60, 197, and 5 7-84).
79
Ema M. C. Maas and others Culture methods and sample preparation
Isolates were cultured on potato dextrose agar (PDA) and each isolate inoculated in triplicate into 100 ml nutrient broth (0.1% meat extract, 0.2% yeast extract, 0.5 % peptone, 0.8% NaCl and 1% glucose) in 250ml Ehrlenmeyer flasks with cotton wool stoppers. To allow for sufficient mycelium development, flasks were incubated for 10 d at 25 OC. After this period the mycelial mats were collected, placed in 4 % KOH for 10 min (Marchant, 1966), decanted into 0.5 mm mesh sieves and rinsed under running tap water for I min. Mats were then placed on several layers of towelling paper and blotted dry. Of each fungal isolate, 0.3 g mycelium was immediately used for protein extractions. As repeated subculturing was found to affect protein patterns, the remaining mycelium was placed in sterile Petri dishes and frozen for further extractions. Protein extractions
The pre-weighed mycelium was ground in a mortar and pestle for 1min in 4 ml extraction buffer (pH 6.8) consisting of 25 ml 0.25 M Tris, 2.05 g sodium dodecyl sulphate (SDS), 10 ml glycerol, 5 ml 2-mercaptoethanol, 0.1 ml 1 % bromophenol blue solution and distilled water to 100 ml. The resultant slurry was decanted into vaccine bottles, stoppered lightly and heated in a boiling water bath for 2 rnin (Laemmli, 1970). Concomitantly, 0.01 g lysozyme and 0.01 g thyroglobulin, used as reference proteins (Kersters & de Ley, 1975) were dissolved in 10 ml extraction buffer and treated as described before. The extracts were transferred to Eppendorf microcentrifuge tubes (2 ml) and centrifuged for 15 min at 12000 rev. min-l. New extractions were made from frozen mycelium for each electrophoretic run. Two electrophoretic runs consisting of two gels per run were compared for the purpose of this study. Electrophoresis
The discontinuous dodecyl sulphate (SDS) buffer system described by Laemmli (1970) was used as a basis for one dimensional slab gel electrophoresis in a Protean 11 vertical electrophoresis unit. The gel slabs (160 x 200 x 1.5 mm) consisted of 10% acrylamide resolving gel at pH 8.8, and 4 % stacking gel at pH 6.8, into which a 15-well comb was placed before polymerization. After polymerization, the comb was removed and the wells were flushed with electrode buffer at pH 8.3. The buffer consisted of 9 g Tris-Base, 43 g glycine, 3 g SDS and distilled water to 3 1. Each well then received 5 vl glycerol :1% bromophenol blue solution (v/v 50 :50) as tracking dye, 5 ul lysozyme solution, and 20 ~1 protein extract of each fungus. One lane on each gel received only ) served as the thyroglobulin (30 ul) and lysozyme (5 ~ 1 and reference lane for spectrophotornetric tracings. Electrophoresis, carried out under constant voltage was started at 75 V until the blue tracking dye penetrated the resolving gel, after which the voltage was increased to 150 V. Each run lasted approximately 6 h. After electrophoresis, gels were removed, fixed and stained for 2 h in a solution consisting of 100 ml
10% acetic acid and 100 ml filtered stain concentrate (2.5 g Brilliant Blue R in I 1 95 % ethanol). Destaining was done in four steps for periods of 2-3 h in the following solutions : 200 ml 95 % ethanol and 300 ml 5 % acetic acid; 150 ml 95 % ethanol and 350 m l 5 % acetic acid; repeat of second step; 100 ml 95% ethanol and 400 ml 5 % acetic acid. After sufficient destaining, the gels were stored in a solution consisting of 100 ml 95% ethanol, 200 ml 5 % acetic acid and 200 ml water. Spectrophotometric evaluation
The principle of spectrophotornetric evaluation and numerical analysis as described by Kersters & de Ley (1975) was used with minor variations. Destained gels were placed between two clean glass plates and each lane (representing one fungal isolate), as well as the reference lane, were scanned using a Beckrnann DU-8 spectrophotometer. The spectrophotometer was set so that tracings represented three times the length of each lane. Each tracing was thus approximately 40 crn in length. The spectrophotornetric tracings (in which the x- and y-axes represented distance and absorbance respectively) of the reference lane of each gel was first divided into 180 spaces by placing it over a computer-drawn sliding scale on a light table. Starting from the bottom, position 12 and 180 were represented by the lysozyme and thyroglobulin peak respectively. The spectrophotornetric tracings of the fungal isolates were then each placed over the divided reference spectrophotornetric tracing on a light table and divided into 180 spaces, with the lysozyme peak being the point of concurrence. After this, the extinction, expressed as the height in mm of each of the 180 positions, was measured and used as data for numerical analysis. Compensation of spectrophotornetric scans
Compensation as described by Kersters & de Ley (1975) was unnecessary when two gels from the same run were analysed and compared since clustering levels for isolates of the same species ranged between r = 0.95 and 0.99. When gels from different runs were compared, compensation was achieved by calculating the average position number of the 12-16 most prominent bands from each of the tracings, and moving each band to its calculated position. This was effected by the omission or addition, respectively, of the lowest extinction value in the valley floor between bands. After compensation, clustering levels between isolates of a species ranged between r = 0.90 and 0.95. According to Kersters & de Ley (1975) correlation coefficients ranging between r = 0.82 and 0.92 are sufficiently high to allow comparison. Numerical analysis
The Pearson product-moment correlation coefficient r (Sokal & Sneath, 1963) between any pair of spectrophotornetric tracings was calculated, using the data already mentioned. The resultant r-matrix was transformed to a distance matrix and clustered by the unweighted average pair group method (UPGMA) (Zupan, 1982). The distance values of all clustering levels were again transformed to r-values and a dendrogram
Electrophoresis of Gaeumannomyces and Phialophora Fig. I. Spectrophotornetric tracings (reduced approx. 8 x ) of protein patterns of Gaettmannomyces, Phialophora and related genera. Absorbance and distance are represented on the y- and x-axes respectively. L, lysozyrne; T, thyroglobulin.
1
t
1
P . graminicola 60
V
P. graminicola 57-84
V
P. graminicola 197
1 I!
P. zeicola
Phialophora sp. 168b
1 1 I
P. graminicola 59
1
1 r~ G . graminis var. tritici 712
Reference proteins
M . rhizophila
81
Erna M. C. Maas and others compiled for the various isolates. All calculations were performed using the IBM-Mainframe computer of the Institute for Computer Sciences, University of Pretoria.
RESULTS
Fig. 3. Dendrogram illustrating the relationship within and between isolates of Gaeumannomyces, Phialophora and related genera, obtained through numerical analysis of electrophoretic data. P. graminicola PREM 48865 P. graminicola (60)
The spectrophotometric tracing (reduced approximately 8 x ) G . graminis var. rrilici (714) of each isolate, as well as the electrophoretically obtained protein patterns, are illustrated in Figs 1 and 2 respectively. G . graminis var. rritici (712) Under strict standardized conditions of growth, extraction and electrophoresis, protein patterns were distinctive and C.graminis var. tririci (716) reproducible over the two electrophoretic runs (two gels per P. graminicola (197) run). Visual examination of the gels revealed a high degree of similarity in protein band occurrence and position between Phialophora sp. (186b) the various isolates. Differences were related to the presence or absence of a few protein bands, protein concentration and Phialophora sp. (186a) banding pattern. A dendrogram, showing the correlation within and between M.rhizophila PREM (45952) species as established by numerical analysis of data from one P. graminicola (59) gel, is presented in Fig. 3. The three isolates of Ggt formed a tight separate cluster to which two of the P. graminicola P. graminicola ATCC 60239 (5744) isolates (60 and 197) were each separately related at r = 0.97 and 0.95 respectively. The two unidentified Phialophora P. zeicola (261)PREM 45754 isolates (168a and 168b) formed another tight cluster (r = 0-70 0.80 0.90 0.95), which in turn was related to the Ggt/P. graminicola group at r = 0.94. One P. graminicola isolate (PREM 48865) was separately related to this group at r = 0.89. A third tight cluster was produced by the last two P. graminicola isolates (59 DISCUSSION and 57-84), to which M. rhiwphila was related at a correlatio? The protein patterns of the various isolates were very similar, level of r = 0.90. This group was related to the Ggt/P. yet consistent and distinct differences were found to occur. graminicola group at r = 0.85. P. zeicola joined the whole This is in agreement with the report by Snider & Krarner differences (1974), Reynolds, Weinhold & Morris (1983) and Hansen et al. cluster at the low level of r = 0.73. Although - slight in correlation values occurred between dendrograms from (1986). different gels, the basic clustering pattern remained intrinsically At the onset of this study it was presumed that the P. the same. graminicola isolates used were homologous. However, the low level of association found between these isolates highlights the heterogeneity that has developed in the concept of the Fig. 2. Protein patterns of Gaeumannomyces, Phialophora and related latter species and the difficulty in separating isolates genera obtained through polyacrylamide gel electrophoresis. A, P. morphologically. This low level of association was verified in zeicola; B, C, Phialophora sp. (168b and 168a);D, E, F, Ggt (716, 714 a recent report by Smiley (1987), according to which the and 712); G, P. graminicola (197); H , P. graminicola (57-84);I, P. isolates used in the present study were incorrectly identified graminicola (60);J , P. graminicola (59);M. rhiwphila; L, P. graminicola because they do not resemble the anamorph of G. cylindrosporus (PREM 48865); M, reference lane (L*,lysozyrne, T, thyroglobulin). Hornby, Slope, Gutteridge & Sivanesan, viz. P. graminicola, isolated from plants by Jackson & Landschoot (1986). In a later publication, Landschoot & Jackson (1987) reclassified P. graminicola isolates 57-84 and 59 as the Phialophora state of a yet undesaibed Magnaporthe sp. That report corroborates the findings of the present study, in which these isolates showed an affinity to M . rhizophila, and always formed a tight separate cluster, whereas two other isolates (60 and 197) were more closely related to Ggt. Electrophoresis is useful in delimiting species only when the intraspecies difference is smaller than the interspecies dfference (Shipton & MacDonald, 1970). The very high levels of similarity observed between the Ggt and Phialophora sp. isolates is, thus, indicative of greater intraspecies relationship and suggest that PAGE can successfully be used to distinguish between morphologically and ecologically related fungi. No report concerning electrophoresis of Ggt has appeared
Electrophoresis of Gaeurnannomyces and Phialophora since the study b y Abbott & Holland (1975). The present investigation, as far as could be ascertained, is also the first report o n electrophoresis-aided fungal taxonomy in which computer-assisted analysis of data, obtained through spectrophotometric evaluation of polyacrylamide gels, has been used. The advantages attached t o the latter procedure include: (i) the objective gathering of data, (ii) the insensitivity of the correlation coefficients t o concentration, (iii) the assessment of similarities and dissimilarities through the evaluation of the protein pattern as a whole and not merely of individual protein bands, and (iv) the ability of the ~ ~ e c t r o p h o t o m e t et or monitor subtle differences undetectable to the human eye. A further advantage of electrophoresis is that it can be performed relatively rapidly. According to Smiley (1987), the taxonomic delimination of Phialophora spp. is quite uncertain and, in view of the new species involved in turfgrass diseases, rapid diagnostic procedures will b e required for positive identification of the agents of patch disease. Finally, it is suggested that electrophoresis can assist in verifying established taxonomy of fungal groups or isolates b y providing additional biochemical criteria. This study was financed b y the Department of Agriculture and Water Supply and the South African Wheat Board. W e thank D r F. C. Wehner for suggestions during the preparation of the manuscript and Professor J. J. Joubert for supplying the computer programme for numerical analysis. The assistance of M r J. Maritz, Professor K. Kersters and D r J. J. Bezuidenhout in the implementation of the computer programmes for cluster analysis, is gratefully acknowledged.
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