Physiological differences among isolates of Phytophthora cinnamomil
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Depcrrtmenr qfBotat7.y crnd Microbiology, A~rb~rrn Ut~iversityAgriclrlt~lralExperiment Station, Alrbirrn, Alabnmn 36830 Accepted May 29, 1975 KELLEY.W. D. 1975. Physiological differences among isolates of Phy~ophthoracinnamonli. Can. J . Microbiol. 21: 1548-1552. Significant differences in amylase, P-glucosidase, and phosphatase activities were observed among four Phytopl~thorcrcinnamomi isolates grown in nutrient-amended sterilized soil for 20 days. Amylase pH optima for the four isolates were within a relatively narrow range; at pH 5.5 each isolate was within 90% of its peak activity. Isolates SB-216-1, 1-281. and C-39eachexhibited maximal P-glucosidase activity at pH 5.0 and maximal phosphatase activity a t pH 5.0-5.5. Maximal activity for these two enzymes of isolate A-7725 occurred a t pH 3.5. In timed experiments, isolates 1-281 and A-7725 exhibited greater amylase activities than did the other two isolates. For 0-glucosidase, greatest activity was observed for SB-216-1; activity of 1-281 was intermediate and least activity was observed for isolates A-7725 and C-39. Isolates SB-216-1 and 1-281 exhibited greatest phosphatase activities; isolate C-39 was intermediate in activity, and A-7725 was least active. Results indicate that significant differences exist among the isolates tested and that these differences can be quantitatively measured by the methods described. KELLEY,W. D. 1975. Physiological differencesamongisolatesofPhyroph~/~ora cinnnmomi. Can. J. Microbiol. 21: 1548-1552. Des differences significatives dans les activitks de I'amylase, de la P-glycosidase e t de la phosphatase furent observees chez quatre isolats de Phytophthora cintlan~ornimis en culture dulant 20jours dans un sol amende en ilements nutritifs et sterilise. Les pH optima de I'amylase pourles quatre isolats se situent i I'interieurd'une Ctendue relntivement etroite; B p H 5.5, chaque isolat se situe dans les 90% de son activite maximale. Les isolats SB-216-1, 1-281 et C-39 prksentent chacun une activite maximale pour lap-glycosidase i p H 5.0, et pour la phosphatasei pH 5.0-5.5. L'activitC maximale d e c e s deux enzymes chez I'isolat A-7725 se situe a pH 3.5. Dans des experiences de temps mesures, les isolats 1-281 et A-7725 ont presente des activitks plus grandes pour I'amylaseque lesdeux autres isolats. Pour lap-glucosidase, laplusgrande activitk a ete observee chez SB-216-1; I'activitC de 1-281 Ctait intermediaire e t la moins grande activiti fut observee chez les isolats A-7725 et (2-39. Les isolats SB-216-1 e t 1-281 ont present6 les plus grandes activites pour la phosphatase; I'isolat C-39 etait intermediaire et le A-7725 fut le moins actif. Ces resultats indiquent qu'il existe des differences significatives chez les isolats testes et qile ces differences peuvent i t r e mesurees q~~antitativement par les mkthodes decrites. [Tmduit par le journal]
Introduction Although Phytophthora cinnamomi Rands is pathogenic on a wide variety of hosts (10, 15), few studies have been done on physiological differences among isolates of this fungus. Haasis et al. (4) compared morphology of sexual and asexual structures and growth rates, sporangial production, and colony development on solid media among and between P. cinnamomi isolates representing the two mating types, and concluded that differences observed were within ranges expected for a single biological species.
Similar results were obtained by Chee and Newhook (1) when they compared P. cinnamomi isolates for vegetative growth and sporulation under a range of temperatures and pH values. In another study (2), they found considerable variability among P. cinnamomi isolates in respect of mating ability and responses to physical and chemical factors, and concluded that strain variability in sexual reproduction exists within the genus. Among the wide range of plants attacked by P. cinnamomi, no host specificity has been demonstrated for various isolates in laboratory tests (1, 16). This does not necessarily preclude ~-
'Received February 23, 1975.
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~
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KELLEY: DIFFERENCES AMONG P. CINNAMOMI ISOLATES
the existence of physiologically different isolates but, rather, may be indicative of the versatility of a given isolate. Although the studies referred to above show that differences among isolates of P. cinnarnorni may occur, no information is available on enzymatic activities of different isolates of the pathogen. Such information could be useful in choosing various isolates for use in studying the ecology of this fungus. Procedures for determining quantitatively enzymatic activities of fungi in soil are available (12-14), and they provide an excellent tool for use in studying activities of P. cinnarnorni enzyme systems. This study was conducted to determine if differences in enzymatic activities exist among four P. cinnarnorni isolates grown in sterilized soil cultures.
Materials and Methods The four P. cinnatnotni isolates used and their sources were SB-216-1, an isolate from Persea americana Mill., provided by G . A. Zentmyer, University of California, Riverside; C-39 (IMI 124492), an isolate from Eucalyptus marginata Donn ex Sm., provided by J. F. Titze, Forest Research Institute, Kelmscott, Western Australia; and 1-281, an isolate from Pinus echinata Mill., in Georgia, and A-7725, an isolate from soil from the Gatlinburg, Tennessee area, both provided by D. H. Marx and W. A. Campbell, U.S.D.A. Forestry Sciences Laboratory, Athens, Georgia. Soil used was a loamy sand from a littleleaf-diseased shortleaf pine stand in the Auburn University Forestry Plots, Lee County, Alabama. Preparation of Soil Cctltures Cultures were prepared in 250-mI Erlenmeyer flasks containing 100 g oven-dry weight (ODW) of soil. Soil moisture was adjusted to 20% ODW and the flasks were autoclaved for 1 h at 121 "C, followed by an additional 30-min autoclaving 24 h later. When cool, each flask received 10 ml of a nutrlent solutlon that provided 300 mg of dextrose, 1.78 mg of inorganic phosphorus (K2HP04), and 2.79 mg of NO3-N(KN03)/100g ovendry soil. Flasks were then stored for 48 h before inoculation. Inocula of P. cintcatnon~iwere prepared from 14- to 18-day-old cultures growing on oatmeal - V-8 juice agar. For each isolate, one half the mycelial mat and agar in a petri dish was blended for 30 s in 80 ml of sterile water in a semimicroblendor. The suspension was decanted into 200 ml of sterile water in a 500-ml flask. Two millilitres of the resulting suspension was used to inoculate each flask. Before inoculation, flasks were divided into five groups of 30 flasks each. One group served as uninoculated controls and the other four groups were each inoculated with one of the P. cinnan~omiisolates. Inoculum was pipetted in a line across the diameter of the soil in the flask. Soil in two flasks from each group was removed and air-dried at room temperature (about 25 "C) immediately
1549
after inoculation and a t 2-day intervals thereafter throughout a 20-day incubation period. Soil thus prepared was used to determine changes in enzymatic activities with time after inoculation. In addition, on the 14th day of the incubation period soil in seven additional flasks from each group was removed, composited within groups, air-dried, and stored for use in determining enzyme p H optima. Air-dried soil was stored in darkness over a dessicant at 5 "C until used in analyses. Enzyme Activity Analyses Enzymatic activities referred to in this paper were determined in air-dried soil. Thus, the terms amylase, P-glucosidase, and phosphatase refer to the sum total of enzyme systems present that were capable under the conditions defined of hydrolyzing a-1,Cglucan linkages (and to a lesser extent r-1,6-glucan linkages) in soluble potato starch, P-u-glucosidic linkages in arbutin, and orthophosphoric monoesters in phenyl disodium phosphate, respectively. Analytical procedures used were modifications of the methods described by Hofmann (5). Soil from 14-day-old pure cultures of the four P. cinnancomi isolates was used to determine effects of hydrogen ion concentrations on enzymatic activities. A range of pH values from 2.5 t o 6.5 was obtained using formate (pH 2.5-3.5), acetate (pH 4.0-5.0), and citrate or phosphate (pH 5.5--6.5) molar buffers. Enzymatic analyses were performed a t each pH for each of the three enzyme systems measured, and included controls without substrates and controls with twice-autoclaved soil. Phosphate buffers were not used in determining phosphatase activities. Subsequent enzymatic activities were determined at the optimum pH for the particular enzyme system being measured. All analyses were run in duplicate and were repeated at least twice. T o determine amylase activity, 5 g of air-dried soil was placed in a 50-mI Erlenmeyer flask a n d 1.5 ml of toluene was added; the mixture was shaken and allowed to stand for 15 min. To this were added 5 ml of water, 5 ml of buffer, and 5 ml of a 2% (wlv) soluble potato starch solution. The flask was stoppered and placed in an incubator at 37 "C for 5 h. After incubation 20 ml of distilled water was added, the flask was shaken, and a 10-mI aliquot of the suspension was centrifuged at 3500 rpnl for 20 min. One millilitre of the clear supernatant was then analyzed for reducing groups by a modified Somogyi method as developed by Nelson (8). Amylase activity is expressed as reducing groups released (in micrograms of glucose equivalents) per gram of soil when a reaction mixture of the composition given above was incubated for 5 h at 37 "C. Beta-glucosidase activity was determined for 5 g of air-dried soil. The substrate used was 5 ml of a 5% (wlv) solution of arbutin (hydroquinone-P-D-glucopyranoside) and incubation time was 4 h ; otherwise the procedure was as described for amylase activity. Beta-glucosidase activity is expressed as micrograms of glucose released per gram of soil when the reaction mixture was incubated for 4 h at 37 "C. The procedure for determining phosphatase activity was the same as those above through the centrifugation step, except that 5 ml of 0.031 M phenyl disodium phosphate served as the substrate and incubation time was
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CAN. J. MICROBIOL. VOL. 21, 1975
FIG. 1. Effect of hydrogen ion concentrations on enzymatic activities of four P. cinnarnorni isolates grown in sterilized soil cultures for 14 days. (A). Amylase activity. (B). Beta-glucosidase activity. (C). Phosphatase activity. The symbols are 0, P. cinnarnotniSB-216-1; 0 , P. cinnarnomi A-7725; 0, P. cinnarnorni 1-281 ; B, P. cinnarnorni C-39. 3 h. After centrifugation, 1 ml of the clear supernatant was added to a 50-ml volumetric flask containing 20 ml of distilled water and 5 ml of pH 9.6 borate buffer (5). One millilitre of a dye solution (200 mg of 2,6-dibromoquinone chlorimide in 95% ethyl alcohol up to 100 ml) was added to the flask. After a 20-min period for the color to develop, the solution was brought to volume with water and the optical density was determined at 630 nm. Phosphatase activity is expressed as micrograms of phenol released per gram of soil when the reaction mixture was incubated for 3 h at 37 "C. All data were analyzed for analysis of variance, and means were compared for significance by Duncan's new multiple-range test. Except where otherwise specified, all differences referred to in this paper were significant at the 1% level of probability.
Results Optimal pH Valuesfor Enzymatic Activities Amylase activity (Fig. 1A) increased with pH values from 4.0 to 5.0, 5.5, and 6.0 for P. cinnamomi isolate C-39, SB-216-1, and A-7725 and 1-281, respectively, then declined at higher p H values. Except for isolates A-7725 and 1-28 1, amylase activity decreased less than 15% at 0.5 pH unit either side of the recorded optimum. Activity of isolate A-7725 decreased by 35% at p H 6.5, and a decrease of 45% was observed for isolate 1-281 at pH 4.5. Data indicate that all isolates would be within 80% of maximum amylase activity at any value between pH 5.0 and 6.0. Beta-glucosidase activity (Fig. 1B) for isolates SB-216-1, 1-281, and C-39 was maximum at p H
5.0; for isolate A-7725 maximal activity was observed a t p H 3.5. Beta-glucosidase activity for all isolates declined rapidly toward either extreme of the pH range used. Average decline for the three isolates with maximal activities a t p H 5.0 was 79% at pH 6.5 and 58% at pH 3.5; activity for isolate A-7725 at pH 2.0 and 4.5 was 35% below that observed a t pH 3.5. Optimal p H values for phosphatase activities (Fig. 1C) of the four isolates were similar t o those observed for P-glucosidase, except that declines were not as rapid a t pH values o n either side of the observed optima. Optimal activity for isolates 1-281 and C-39 occurred a t p H 5.5; optimal activity for SB-216-1 occurred at pH 5.0. As with P-glucosidase activity, phosphatase activity for isolate A-7725 was optimum at pH 3.5. Activity for this isolate decreased steadily with increasing p H values above the optimum. Enzymatic Activity vs. Time Amylase activity (Fig. 2A) was not detected for any isolate until after the 4th day of incubation; no amylase activity was detected for isolate SB-216-1 until after the 6th day. Generally, greatest increases in amylase activity for all isolates occurred between days 4 and 10, after which time more gradual increases were observed through the 20th day of incubation. An exception t o this was isolate C-39, where a
KELLEY: DIFFERENCES AMONG P. ClNNAMOMI ISOLATES 1.00 1100 1200
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t > 100
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> ; ,000 U
I0
a 400
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> k ,100 900
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-
-
W
; ,,, s ul
a
200
-
I00
-
OD-
2
4
6
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10
12
14
16
18
20
DAYS AFTER INOCULATION
22
DAYS AFTER INOCULATION
FIG.2. Enzymatic activities in relation to incubation time for four P. cinnarnomi isolates grown in sterilrzed soil cultures. (A). Amylase activity. (B). Beta-glucosidase activity. (C). Phosphatase activity. The symbols are as in Fig. 1.
gradual decrease in activity was observed from the 10th through the 20th day. Beyond the 12th day of incubation, significant differences in amylase activities of the four isolates were observed. Activities of isolates 1-281 and A-7725 were both significantly higher than those observed for isolates SB-216-1 and C-39. Average mean activity of isolates SB-2 16- 1 and C-39 from the 14th through the 20th days was 37% of that observed for the other two isolates. Beta-glucosidase activity (Fig. 2B) of isolates A-7725 and C-39 increased sharply between the 4th and 10th days, after which time activity remained relatively constant for the remainder of the incubation period. Beta-glucosidase activity of isolates 1-28 1 and SB-216- 1 increased steadily throughout the incubation period, and in both cases the observed increases in activity with time approached linearity. Significant differences were observed between isolates as early as the 2nd day, and a definite pattern was evident as early as the 6th day. Activity of isolate SB-2 16- 1 was significantly greater than the activities of the other three isolates from the 6th day onward; difference between isolates SB-216- 1 and 1-281 on the 10th day was significant at the 5% level. From the 10th day onward, activity of isolate 1-281 was significantly higher than the activities of isolates C-39 and A-7725. Except for the 8th day, no significant differences were observed between isolates C-39 and A-7725. On the 20th day, activities of isolates 1-281, C-39, and A-7725 were, respectively, 77,
42, and 38% of the activity observed for isolate SB-216-1. Changes in phosphatase activity with time for the four isolates (Fig. 2C) were similar to those observed for P-glucosidase. Greatest increase for all isolates occurred between the 4th and 10th days of the incubation period. As with P-glucosidase, phosphatase activity for isolates C-39 and A-7725 changed little from the 10th through the 20th days. Activities of isolates SB-216-1 and 1-281 continued to increase beyond the 10th day of incubation, but at a lesser rate. Phosphatase activity for isolate 1-281 was significantly higher than the activities for the other isolates on days 6 through 12; on days 14 through 20 no significant differences were observed between isolates SB-216-1 and 1-281. Also on days 12 through 20, activity for isolate C-39 was significantly less than that observed for either SB-216-1 or 1-281. Activity for isolate A-7725 was significantly less than the activities of the other three isolates on days 10 through 20. On the 20th day greatest activity was observed for isolate 1-281, and activities of SB-216-1, C-39, and A-7725 were 97, 68, and 43% of that amount, respectively.
Discussion Results from pH optima determinations indicate that differences were small between isolates SB-216- 1 , 1-281, and C-39 for the three enzyme systems studied; pH optima for these isolates approximated the pH of the soil used in the
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CAN. J. MICROBIOL. VOL. 21, 1975
study (pH 5.4). However, p H optima of isolate A-7725 for P-glucosidase and phosphatase were well below those of the other three isolates. Thus, these two enzyme systems would be expected t o function well below peak efficiency in soil under conditions other than acid. For example, phosphatase activity of isolate A-7725 at p H 5.5 (Fig. 2C) was only 30% of that observed at p H 3.5. Such reductions in enzyme efficiencies may be significant in survival and pathogenicity of this isolate in soils with values of p H 5.0 and higher. Enzymatic activities observed in the timed experiment also indicated that significant physiological differences exist among the four P. cinnnmo~viisolates, and demonstrated that these differences can be quantitatively measured by the methods described. Similar analyses of other enzyme systems could be included to obtain a more comprehensive view of physiological differences. The enzymatic activities referred to in this paper represent those amounts produced by the isolates when grown under carefully controlled, identical conditions, thus legitimate comparisons of activities between isolates could be made. Under other conditions one would expect different responses in enzymatic activities, particularly with respect to substrates used t o -grow the fungi., and the ~ o s s i b l eactivation of other enzyme systems. I n this study' enzymatic analyses were 'Onducted with air-dried soil instead of with fresh mycelial preparations for several reasons. Enzimes prodiced and released by organisms in the soil often are adsorbed by soil particles (3, 6, 7, 9, and the they are more resistant to inactivation than those in the free state (3, 11). Thus, measuring the activity of a n enzyme produced by a fungus grown in soil more closely approximates the total produced over a period of time than would be possible, for example, in a liquid culture system where free enzymes are much more susceptible to inactivation. I n addition, measurements of enzyme activities directly in soil eliminates the critical temperature requirements of purified preparations from mycelium. Perhaps more importantly, analyses of enzymatic activities in air-dried soil can be repeated over extended periods of time with little change in activities.
Results of this investigation revealed significant differences in enzymatic activities of f o u r P. cinnamomi isolates. It is suggested that the methods used could be valuable in selecting isolates of a given fungus for ecological studies. I. C H E E ,K . H., and F. J. NEWHOOK.1965. Variability in Phytopl~tlrornci~rtlrrmomiRands. N. 2. J . Agric. Res. 8: 96-103. 2. CHEE,K. H., and F. J. NEWHOOK.1965. Variability in sexual reproduction of Phytopllthorn cintlatnomi Rands. N. Z. J . Agric. Res. 8: 947-950. 3. E N S M I N G EL. R , E., and J. E . GIESEKING. 1942. Resistance of clay-adsorbed protein to proteolytic hydrolysis. Soil Sci. 53: 205-209. 4. HAASIS,F. A., R. R. NELSON,and D. H. MARX.1964. Morphological and physiological characteristics of mating types of Plzy/ophtl7orcr cit~tzntnomi. Phytopathology, 54: 1145-1 151. 5. HOFMANN,E. 1963. Die analyse von Enzymen in Boden. 111 Moderne Methoden der Pflanzenanalyse. Vol. 6. Edited by M. V. Tracey et nl. Springer-Verlag, Berlin. pp. 416423. 6. MCLAREN,A. D. 1954. The adsorption and reactions of enzymes and proteinson kaolinite. 11. The action of chymotrypsin on lysozyme. Soil Sci. Soc. Am. Proc. 18: 170-174. 7. MCLAREN,A. D., and E. F. ESTERMANN. 1956. T h e adsorption and reactions of enzymes and proteins o n kaolinite. 111. The isolation of enzyme-substrate complexes. Arch. Biochem. Biophys. 61: 158-173. 8. NELSON,N. 1964. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153: 375-380. 9. P I N C KL. . A,, and F. E. A L L I S O N1961. . Adsorption and release of urease by and from clay minerals. Soil Sci. 91: 183-388. 10. PODGER,F. D. 1972. Pl~ytopl~thorocitltlntnotni, a c,L,se of lethal disease i n indigenous plant munities in Western Australia. Phyto~athology. . . -. 62: 972-98 I . I I. PORTER,L. K. 1965. Enzymes. Part 2.111 Methods of soil analysis. Editerl by C. A. Black et a / . American society of Agronomy, Madison, Wis. pp. 1536-1549. 12. R O D R I G U E Z - K A R A R. N A .1969. Enzymatic interactions of Sclerotirrtn rolfsii and Tritlloclertna viride in mixed soil cultul.e. Phytopathology, 59: 910-921. 13. RODRIGUEZ-KARANA, R., and E. A. CURL.1968. Saccharase activity of Sclero/ilrm rofiii in soil and the mechanism of antagonistic action by Triclloder~ntlviride. Phytopathology, 58: 985-992. 14. S K U J I N SJ,. J . 1967. Enzymes in soil. 117 Soil biochemistry. Editeclby A. D. McLaren and G. H. Peterson. Marcel Dekker Inc.. New York. pp. 371414. 15. T H O R NW , . A,, and G. A. ZENTMYER. 1954. Hosts of Pl~ytopl~tl~orcr cint~crtnomi.Plant Dis. Rep. 38: 47-52. G. A. 1952. Research on avocado root 16. ZENTMYER, rot. Calif. Avocado Soc. Yearbook. 37: 103-106.
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Depcrrtmenr qfBotat7.y crnd Microbiology, A~rb~rrn Ut~iversityAgriclrlt~lralExperiment Station, Alrbirrn, Alabnmn 36830 Accepted May 29, 1975 KELLEY.W. D. 1975. Physiological differences among isolates of Phy~ophthoracinnamonli. Can. J . Microbiol. 21: 1548-1552. Significant differences in amylase, P-glucosidase, and phosphatase activities were observed among four Phytopl~thorcrcinnamomi isolates grown in nutrient-amended sterilized soil for 20 days. Amylase pH optima for the four isolates were within a relatively narrow range; at pH 5.5 each isolate was within 90% of its peak activity. Isolates SB-216-1, 1-281. and C-39eachexhibited maximal P-glucosidase activity at pH 5.0 and maximal phosphatase activity a t pH 5.0-5.5. Maximal activity for these two enzymes of isolate A-7725 occurred a t pH 3.5. In timed experiments, isolates 1-281 and A-7725 exhibited greater amylase activities than did the other two isolates. For 0-glucosidase, greatest activity was observed for SB-216-1; activity of 1-281 was intermediate and least activity was observed for isolates A-7725 and C-39. Isolates SB-216-1 and 1-281 exhibited greatest phosphatase activities; isolate C-39 was intermediate in activity, and A-7725 was least active. Results indicate that significant differences exist among the isolates tested and that these differences can be quantitatively measured by the methods described. KELLEY,W. D. 1975. Physiological differencesamongisolatesofPhyroph~/~ora cinnnmomi. Can. J. Microbiol. 21: 1548-1552. Des differences significatives dans les activitks de I'amylase, de la P-glycosidase e t de la phosphatase furent observees chez quatre isolats de Phytophthora cintlan~ornimis en culture dulant 20jours dans un sol amende en ilements nutritifs et sterilise. Les pH optima de I'amylase pourles quatre isolats se situent i I'interieurd'une Ctendue relntivement etroite; B p H 5.5, chaque isolat se situe dans les 90% de son activite maximale. Les isolats SB-216-1, 1-281 et C-39 prksentent chacun une activite maximale pour lap-glycosidase i p H 5.0, et pour la phosphatasei pH 5.0-5.5. L'activitC maximale d e c e s deux enzymes chez I'isolat A-7725 se situe a pH 3.5. Dans des experiences de temps mesures, les isolats 1-281 et A-7725 ont presente des activitks plus grandes pour I'amylaseque lesdeux autres isolats. Pour lap-glucosidase, laplusgrande activitk a ete observee chez SB-216-1; I'activitC de 1-281 Ctait intermediaire e t la moins grande activiti fut observee chez les isolats A-7725 et (2-39. Les isolats SB-216-1 e t 1-281 ont present6 les plus grandes activites pour la phosphatase; I'isolat C-39 etait intermediaire et le A-7725 fut le moins actif. Ces resultats indiquent qu'il existe des differences significatives chez les isolats testes et qile ces differences peuvent i t r e mesurees q~~antitativement par les mkthodes decrites. [Tmduit par le journal]
Introduction Although Phytophthora cinnamomi Rands is pathogenic on a wide variety of hosts (10, 15), few studies have been done on physiological differences among isolates of this fungus. Haasis et al. (4) compared morphology of sexual and asexual structures and growth rates, sporangial production, and colony development on solid media among and between P. cinnamomi isolates representing the two mating types, and concluded that differences observed were within ranges expected for a single biological species.
Similar results were obtained by Chee and Newhook (1) when they compared P. cinnamomi isolates for vegetative growth and sporulation under a range of temperatures and pH values. In another study (2), they found considerable variability among P. cinnamomi isolates in respect of mating ability and responses to physical and chemical factors, and concluded that strain variability in sexual reproduction exists within the genus. Among the wide range of plants attacked by P. cinnamomi, no host specificity has been demonstrated for various isolates in laboratory tests (1, 16). This does not necessarily preclude ~-
'Received February 23, 1975.
----
~
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Otago on 08/31/14 For personal use only.
KELLEY: DIFFERENCES AMONG P. CINNAMOMI ISOLATES
the existence of physiologically different isolates but, rather, may be indicative of the versatility of a given isolate. Although the studies referred to above show that differences among isolates of P. cinnarnorni may occur, no information is available on enzymatic activities of different isolates of the pathogen. Such information could be useful in choosing various isolates for use in studying the ecology of this fungus. Procedures for determining quantitatively enzymatic activities of fungi in soil are available (12-14), and they provide an excellent tool for use in studying activities of P. cinnarnorni enzyme systems. This study was conducted to determine if differences in enzymatic activities exist among four P. cinnarnorni isolates grown in sterilized soil cultures.
Materials and Methods The four P. cinnatnotni isolates used and their sources were SB-216-1, an isolate from Persea americana Mill., provided by G . A. Zentmyer, University of California, Riverside; C-39 (IMI 124492), an isolate from Eucalyptus marginata Donn ex Sm., provided by J. F. Titze, Forest Research Institute, Kelmscott, Western Australia; and 1-281, an isolate from Pinus echinata Mill., in Georgia, and A-7725, an isolate from soil from the Gatlinburg, Tennessee area, both provided by D. H. Marx and W. A. Campbell, U.S.D.A. Forestry Sciences Laboratory, Athens, Georgia. Soil used was a loamy sand from a littleleaf-diseased shortleaf pine stand in the Auburn University Forestry Plots, Lee County, Alabama. Preparation of Soil Cctltures Cultures were prepared in 250-mI Erlenmeyer flasks containing 100 g oven-dry weight (ODW) of soil. Soil moisture was adjusted to 20% ODW and the flasks were autoclaved for 1 h at 121 "C, followed by an additional 30-min autoclaving 24 h later. When cool, each flask received 10 ml of a nutrlent solutlon that provided 300 mg of dextrose, 1.78 mg of inorganic phosphorus (K2HP04), and 2.79 mg of NO3-N(KN03)/100g ovendry soil. Flasks were then stored for 48 h before inoculation. Inocula of P. cintcatnon~iwere prepared from 14- to 18-day-old cultures growing on oatmeal - V-8 juice agar. For each isolate, one half the mycelial mat and agar in a petri dish was blended for 30 s in 80 ml of sterile water in a semimicroblendor. The suspension was decanted into 200 ml of sterile water in a 500-ml flask. Two millilitres of the resulting suspension was used to inoculate each flask. Before inoculation, flasks were divided into five groups of 30 flasks each. One group served as uninoculated controls and the other four groups were each inoculated with one of the P. cinnan~omiisolates. Inoculum was pipetted in a line across the diameter of the soil in the flask. Soil in two flasks from each group was removed and air-dried at room temperature (about 25 "C) immediately
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after inoculation and a t 2-day intervals thereafter throughout a 20-day incubation period. Soil thus prepared was used to determine changes in enzymatic activities with time after inoculation. In addition, on the 14th day of the incubation period soil in seven additional flasks from each group was removed, composited within groups, air-dried, and stored for use in determining enzyme p H optima. Air-dried soil was stored in darkness over a dessicant at 5 "C until used in analyses. Enzyme Activity Analyses Enzymatic activities referred to in this paper were determined in air-dried soil. Thus, the terms amylase, P-glucosidase, and phosphatase refer to the sum total of enzyme systems present that were capable under the conditions defined of hydrolyzing a-1,Cglucan linkages (and to a lesser extent r-1,6-glucan linkages) in soluble potato starch, P-u-glucosidic linkages in arbutin, and orthophosphoric monoesters in phenyl disodium phosphate, respectively. Analytical procedures used were modifications of the methods described by Hofmann (5). Soil from 14-day-old pure cultures of the four P. cinnancomi isolates was used to determine effects of hydrogen ion concentrations on enzymatic activities. A range of pH values from 2.5 t o 6.5 was obtained using formate (pH 2.5-3.5), acetate (pH 4.0-5.0), and citrate or phosphate (pH 5.5--6.5) molar buffers. Enzymatic analyses were performed a t each pH for each of the three enzyme systems measured, and included controls without substrates and controls with twice-autoclaved soil. Phosphate buffers were not used in determining phosphatase activities. Subsequent enzymatic activities were determined at the optimum pH for the particular enzyme system being measured. All analyses were run in duplicate and were repeated at least twice. T o determine amylase activity, 5 g of air-dried soil was placed in a 50-mI Erlenmeyer flask a n d 1.5 ml of toluene was added; the mixture was shaken and allowed to stand for 15 min. To this were added 5 ml of water, 5 ml of buffer, and 5 ml of a 2% (wlv) soluble potato starch solution. The flask was stoppered and placed in an incubator at 37 "C for 5 h. After incubation 20 ml of distilled water was added, the flask was shaken, and a 10-mI aliquot of the suspension was centrifuged at 3500 rpnl for 20 min. One millilitre of the clear supernatant was then analyzed for reducing groups by a modified Somogyi method as developed by Nelson (8). Amylase activity is expressed as reducing groups released (in micrograms of glucose equivalents) per gram of soil when a reaction mixture of the composition given above was incubated for 5 h at 37 "C. Beta-glucosidase activity was determined for 5 g of air-dried soil. The substrate used was 5 ml of a 5% (wlv) solution of arbutin (hydroquinone-P-D-glucopyranoside) and incubation time was 4 h ; otherwise the procedure was as described for amylase activity. Beta-glucosidase activity is expressed as micrograms of glucose released per gram of soil when the reaction mixture was incubated for 4 h at 37 "C. The procedure for determining phosphatase activity was the same as those above through the centrifugation step, except that 5 ml of 0.031 M phenyl disodium phosphate served as the substrate and incubation time was
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FIG. 1. Effect of hydrogen ion concentrations on enzymatic activities of four P. cinnarnorni isolates grown in sterilized soil cultures for 14 days. (A). Amylase activity. (B). Beta-glucosidase activity. (C). Phosphatase activity. The symbols are 0, P. cinnarnotniSB-216-1; 0 , P. cinnarnomi A-7725; 0, P. cinnarnorni 1-281 ; B, P. cinnarnorni C-39. 3 h. After centrifugation, 1 ml of the clear supernatant was added to a 50-ml volumetric flask containing 20 ml of distilled water and 5 ml of pH 9.6 borate buffer (5). One millilitre of a dye solution (200 mg of 2,6-dibromoquinone chlorimide in 95% ethyl alcohol up to 100 ml) was added to the flask. After a 20-min period for the color to develop, the solution was brought to volume with water and the optical density was determined at 630 nm. Phosphatase activity is expressed as micrograms of phenol released per gram of soil when the reaction mixture was incubated for 3 h at 37 "C. All data were analyzed for analysis of variance, and means were compared for significance by Duncan's new multiple-range test. Except where otherwise specified, all differences referred to in this paper were significant at the 1% level of probability.
Results Optimal pH Valuesfor Enzymatic Activities Amylase activity (Fig. 1A) increased with pH values from 4.0 to 5.0, 5.5, and 6.0 for P. cinnamomi isolate C-39, SB-216-1, and A-7725 and 1-281, respectively, then declined at higher p H values. Except for isolates A-7725 and 1-28 1, amylase activity decreased less than 15% at 0.5 pH unit either side of the recorded optimum. Activity of isolate A-7725 decreased by 35% at p H 6.5, and a decrease of 45% was observed for isolate 1-281 at pH 4.5. Data indicate that all isolates would be within 80% of maximum amylase activity at any value between pH 5.0 and 6.0. Beta-glucosidase activity (Fig. 1B) for isolates SB-216-1, 1-281, and C-39 was maximum at p H
5.0; for isolate A-7725 maximal activity was observed a t p H 3.5. Beta-glucosidase activity for all isolates declined rapidly toward either extreme of the pH range used. Average decline for the three isolates with maximal activities a t p H 5.0 was 79% at pH 6.5 and 58% at pH 3.5; activity for isolate A-7725 at pH 2.0 and 4.5 was 35% below that observed a t pH 3.5. Optimal p H values for phosphatase activities (Fig. 1C) of the four isolates were similar t o those observed for P-glucosidase, except that declines were not as rapid a t pH values o n either side of the observed optima. Optimal activity for isolates 1-281 and C-39 occurred a t p H 5.5; optimal activity for SB-216-1 occurred at pH 5.0. As with P-glucosidase activity, phosphatase activity for isolate A-7725 was optimum at pH 3.5. Activity for this isolate decreased steadily with increasing p H values above the optimum. Enzymatic Activity vs. Time Amylase activity (Fig. 2A) was not detected for any isolate until after the 4th day of incubation; no amylase activity was detected for isolate SB-216-1 until after the 6th day. Generally, greatest increases in amylase activity for all isolates occurred between days 4 and 10, after which time more gradual increases were observed through the 20th day of incubation. An exception t o this was isolate C-39, where a
KELLEY: DIFFERENCES AMONG P. ClNNAMOMI ISOLATES 1.00 1100 1200
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t > 100
-
-
> ; ,000 U
I0
a 400
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> k ,100 900
-
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-
-
W
; ,,, s ul
a
200
-
I00
-
OD-
2
4
6
8
10
12
14
16
18
20
DAYS AFTER INOCULATION
22
DAYS AFTER INOCULATION
FIG.2. Enzymatic activities in relation to incubation time for four P. cinnarnomi isolates grown in sterilrzed soil cultures. (A). Amylase activity. (B). Beta-glucosidase activity. (C). Phosphatase activity. The symbols are as in Fig. 1.
gradual decrease in activity was observed from the 10th through the 20th day. Beyond the 12th day of incubation, significant differences in amylase activities of the four isolates were observed. Activities of isolates 1-281 and A-7725 were both significantly higher than those observed for isolates SB-216-1 and C-39. Average mean activity of isolates SB-2 16- 1 and C-39 from the 14th through the 20th days was 37% of that observed for the other two isolates. Beta-glucosidase activity (Fig. 2B) of isolates A-7725 and C-39 increased sharply between the 4th and 10th days, after which time activity remained relatively constant for the remainder of the incubation period. Beta-glucosidase activity of isolates 1-28 1 and SB-216- 1 increased steadily throughout the incubation period, and in both cases the observed increases in activity with time approached linearity. Significant differences were observed between isolates as early as the 2nd day, and a definite pattern was evident as early as the 6th day. Activity of isolate SB-2 16- 1 was significantly greater than the activities of the other three isolates from the 6th day onward; difference between isolates SB-216- 1 and 1-281 on the 10th day was significant at the 5% level. From the 10th day onward, activity of isolate 1-281 was significantly higher than the activities of isolates C-39 and A-7725. Except for the 8th day, no significant differences were observed between isolates C-39 and A-7725. On the 20th day, activities of isolates 1-281, C-39, and A-7725 were, respectively, 77,
42, and 38% of the activity observed for isolate SB-216-1. Changes in phosphatase activity with time for the four isolates (Fig. 2C) were similar to those observed for P-glucosidase. Greatest increase for all isolates occurred between the 4th and 10th days of the incubation period. As with P-glucosidase, phosphatase activity for isolates C-39 and A-7725 changed little from the 10th through the 20th days. Activities of isolates SB-216-1 and 1-281 continued to increase beyond the 10th day of incubation, but at a lesser rate. Phosphatase activity for isolate 1-281 was significantly higher than the activities for the other isolates on days 6 through 12; on days 14 through 20 no significant differences were observed between isolates SB-216-1 and 1-281. Also on days 12 through 20, activity for isolate C-39 was significantly less than that observed for either SB-216-1 or 1-281. Activity for isolate A-7725 was significantly less than the activities of the other three isolates on days 10 through 20. On the 20th day greatest activity was observed for isolate 1-281, and activities of SB-216-1, C-39, and A-7725 were 97, 68, and 43% of that amount, respectively.
Discussion Results from pH optima determinations indicate that differences were small between isolates SB-216- 1 , 1-281, and C-39 for the three enzyme systems studied; pH optima for these isolates approximated the pH of the soil used in the
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CAN. J. MICROBIOL. VOL. 21, 1975
study (pH 5.4). However, p H optima of isolate A-7725 for P-glucosidase and phosphatase were well below those of the other three isolates. Thus, these two enzyme systems would be expected t o function well below peak efficiency in soil under conditions other than acid. For example, phosphatase activity of isolate A-7725 at p H 5.5 (Fig. 2C) was only 30% of that observed at p H 3.5. Such reductions in enzyme efficiencies may be significant in survival and pathogenicity of this isolate in soils with values of p H 5.0 and higher. Enzymatic activities observed in the timed experiment also indicated that significant physiological differences exist among the four P. cinnnmo~viisolates, and demonstrated that these differences can be quantitatively measured by the methods described. Similar analyses of other enzyme systems could be included to obtain a more comprehensive view of physiological differences. The enzymatic activities referred to in this paper represent those amounts produced by the isolates when grown under carefully controlled, identical conditions, thus legitimate comparisons of activities between isolates could be made. Under other conditions one would expect different responses in enzymatic activities, particularly with respect to substrates used t o -grow the fungi., and the ~ o s s i b l eactivation of other enzyme systems. I n this study' enzymatic analyses were 'Onducted with air-dried soil instead of with fresh mycelial preparations for several reasons. Enzimes prodiced and released by organisms in the soil often are adsorbed by soil particles (3, 6, 7, 9, and the they are more resistant to inactivation than those in the free state (3, 11). Thus, measuring the activity of a n enzyme produced by a fungus grown in soil more closely approximates the total produced over a period of time than would be possible, for example, in a liquid culture system where free enzymes are much more susceptible to inactivation. I n addition, measurements of enzyme activities directly in soil eliminates the critical temperature requirements of purified preparations from mycelium. Perhaps more importantly, analyses of enzymatic activities in air-dried soil can be repeated over extended periods of time with little change in activities.
Results of this investigation revealed significant differences in enzymatic activities of f o u r P. cinnamomi isolates. It is suggested that the methods used could be valuable in selecting isolates of a given fungus for ecological studies. I. C H E E ,K . H., and F. J. NEWHOOK.1965. Variability in Phytopl~tlrornci~rtlrrmomiRands. N. 2. J . Agric. Res. 8: 96-103. 2. CHEE,K. H., and F. J. NEWHOOK.1965. Variability in sexual reproduction of Phytopllthorn cintlatnomi Rands. N. Z. J . Agric. Res. 8: 947-950. 3. E N S M I N G EL. R , E., and J. E . GIESEKING. 1942. Resistance of clay-adsorbed protein to proteolytic hydrolysis. Soil Sci. 53: 205-209. 4. HAASIS,F. A., R. R. NELSON,and D. H. MARX.1964. Morphological and physiological characteristics of mating types of Plzy/ophtl7orcr cit~tzntnomi. Phytopathology, 54: 1145-1 151. 5. HOFMANN,E. 1963. Die analyse von Enzymen in Boden. 111 Moderne Methoden der Pflanzenanalyse. Vol. 6. Edited by M. V. Tracey et nl. Springer-Verlag, Berlin. pp. 416423. 6. MCLAREN,A. D. 1954. The adsorption and reactions of enzymes and proteinson kaolinite. 11. The action of chymotrypsin on lysozyme. Soil Sci. Soc. Am. Proc. 18: 170-174. 7. MCLAREN,A. D., and E. F. ESTERMANN. 1956. T h e adsorption and reactions of enzymes and proteins o n kaolinite. 111. The isolation of enzyme-substrate complexes. Arch. Biochem. Biophys. 61: 158-173. 8. NELSON,N. 1964. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153: 375-380. 9. P I N C KL. . A,, and F. E. A L L I S O N1961. . Adsorption and release of urease by and from clay minerals. Soil Sci. 91: 183-388. 10. PODGER,F. D. 1972. Pl~ytopl~thorocitltlntnotni, a c,L,se of lethal disease i n indigenous plant munities in Western Australia. Phyto~athology. . . -. 62: 972-98 I . I I. PORTER,L. K. 1965. Enzymes. Part 2.111 Methods of soil analysis. Editerl by C. A. Black et a / . American society of Agronomy, Madison, Wis. pp. 1536-1549. 12. R O D R I G U E Z - K A R A R. N A .1969. Enzymatic interactions of Sclerotirrtn rolfsii and Tritlloclertna viride in mixed soil cultul.e. Phytopathology, 59: 910-921. 13. RODRIGUEZ-KARANA, R., and E. A. CURL.1968. Saccharase activity of Sclero/ilrm rofiii in soil and the mechanism of antagonistic action by Triclloder~ntlviride. Phytopathology, 58: 985-992. 14. S K U J I N SJ,. J . 1967. Enzymes in soil. 117 Soil biochemistry. Editeclby A. D. McLaren and G. H. Peterson. Marcel Dekker Inc.. New York. pp. 371414. 15. T H O R NW , . A,, and G. A. ZENTMYER. 1954. Hosts of Pl~ytopl~tl~orcr cint~crtnomi.Plant Dis. Rep. 38: 47-52. G. A. 1952. Research on avocado root 16. ZENTMYER, rot. Calif. Avocado Soc. Yearbook. 37: 103-106.
