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Oxalate production by virulent but not by hypovirulent strains of Endothia parasitica
Physiological
Plant Pathology
(1983)
23, 369-376
Oxalate production by virulent strains of Endothia parasitica
but not by hypovirulent
EVELYN A. HAVIR Department
of Biochemistry
and Genetics
and SANDRA L. ANAGNOSTAKIS Department (Acceptedfor
of Plant
Pathology
publication
and Botany,
The Connecticut
Agricultural
Experimnt
Station,
New Haven,
CT 06504
June 1983)
Three virulent strains of Endofhia parasitica when grown on potato dextrose agar containing methionine and biotin (PDAmb) or minimal media containing glucose supplemented with either glycolate or glyoxylate produced 10-12, 5660 and 20-40 mg oxalate g-r dry weight of fungus respectively. Oxalate accumulation was detected the fourth day after inoculation when the strains were grown on PDAmb or the glycolate medium but not until day 7 on the glyoxylate medium. In the early stages of growth (up to 0.1 g dry weight per colony) the total amount ofoxalate excreted into the medium was proportional to the dry weight ofthe fungus but there was little or no correlation at later stages of growth. Oxalate was detected at the edge of the fungal colony, which is important if oxalate is to play a role in pathogenicity. Under identical conditions three hypovirulent strains, derived from the virulent ones by the introduction of cytoplasmic determinants for hypovirulence, produced no detectable oxalate.
INTRODUCTION Endothiuparusitica
is the fungus that causes chestnut tree blight. Its control, a goal of many research groups [I, 4,9] will be facilitated by knowing the mechanism by which the pathogen attacks its host. For a number of other fungi, such as Sclerotium rolfsii [S, 2.21, Sclerotiniu sclerotiorum [N, 161 and Aspergillus niger [8], production of oxalic acid is important in pathogenicity. Hodgkinson has summarized the pathways of oxalate biosynthesis in the fungi [IO]. Oxalic acid is believed to function in two ways: first, it combines with calcium from host tissue, thus physically weakening it; second, it lowers the pH of the host tissue providing a favourable condition for the action of degradative enzymes such as polygalacturonase [6]. Englander & Corden in 1971 [7] reported that crystals of calcium oxalate were formed by E. parasitica in agar medium. More recently, McCarroll & Thor [25] showed that oxalic acid was present at the advancing edge of cankers caused by this fungus on chestnut trees. However, no further investigation has been made of the formation ofoxalate and its role in this host:pathogen relationship. This study was undertaken to determine how factors such as time, colony size, and media affect oxalate production by E. parasitica in vitro. The oxalate formation of three virulent strains of varying origin was contrasted with that of three hypovirulent strains derived from them [3]. 0048-4059/83/060369
+ 08 $03.00/O
0
1983 Academic
Press Inc.
(London)
Limited
370
E. A. Havir and S. L. Anagnostakis
MATERIALS AND METHODS Growth offungi Virulent E. parasitica strains were: 67, ATCC
no. 38753, an Italian strain; 42, ATCC no. 38751, a Connecticut strain isolated in 1975; 155, ATCC no. 38755, a Connecticut strain isolated in 1977. Curative converts of these strains with Italian cytoplasmic determinants for hypovirulence were: 748, (67[1]) ; 752, (42[1]); 747, (155[1]) [3], respectively. All strains were grown and stored on potato dextrose agar containing methionine and biotin (PDAmb), Difco PDA with methionine (100 mg 1-l) and biotin (1 mg 1-l). The tests of special substrates were made on minimal medium containing lactate and tannic acid (MMLT) agar [2]. The pH was adjusted to 5.6 (the pH of Difco PDAmb) and the carbon source was either 0.10 M glucose, 0.05 M glucose + 0.05 M potassium glycolate or 0.05 M glucose + O-05 M sodium glyoxylate. Agar media were dispensed to approximately 75 ml per 150 x 15 mm sterile disposable Petri dish. Two discs of cellophane, DuPont 193-PUDO, cut a little less than 150 mm diameter, were autoclaved wet and placed on the surface of the agar. Plugs (3 mm diameter) were cut with a cork borer from the edge of rapidly expanding mycelium in PDAmb, and placed on the cellophane in the centre of each dish, mycelium-side up. Dishes were wrapped in ParafilmB and incubated at 27.C in a seed germinator with 12 h of white fluorescent light per day. Extraction
and precipitation
of oxalate
Fungal colonies were measured daily and growth rates were expressed as diameter increase (mm) per day. At the time of sampling, the mycelium was scraped from the surface of the cellophane and dried at 35 “C for dry weight analysis. The cellophane was stripped away, and agar from under the colony was transferred to a small beaker and covered with 0.5 N HCl. The contents of the beaker were incubated at 30°C with gentle shaking. After 2 h, the HCl was removed and fresh HCl added for another 2 h of extraction. The two extracts were combined and an aliquot was mixed with calcium acetate buffer (O-23 M, pH 4.5) to precipitate calcium oxalate. The suspension was stored at 10°C overnight, and the precipitate, collected by centrifugation, was dissolved by the addition of l-4 ml of O-4 N H&SO, (depending on the volume of the precipitate). In order to dissolve very heavy precipitates, the solutions were heated to 40 “C for 20-30 min. Two controls were performed to ensure quantitative recovery of oxalate. To check completeness of extraction of some samples, a third addition of HCl was made and assayed as above. Oxalic acid (10 pmol) was also added to some samples when a precipitate of calcium oxalate was not observed. The recovery of the added oxalate was determined by the procedure described. Determination
of oxalate by high pe$ormance
liquid chromatography
(HPLC)
The oxalate in a 5-20 pl sample of the dissolved precipitate was determined by HPLC on a Perkin-Elmer 3B Liquid Chromatograph equipped with an Aminex HPX-87 column (Bio-Rad Corp) (300 x 7.8 mm) with O-015 N H,SO, as eluting agent. The flow rate was 0.8 ml min-1 at a temperature of 25°C; detection was by UV absorbancy at 2 10 nm. The peak area of oxalate, as calculated by a Hewlett-Packard
Oxalate
production
by Endothia
parasitica
3390A Integrator, was proportional range of 0.5-30 nmol.
to concentration
371 of authentic
oxalic
acid
in the
Location of oxalate formation within the colony Endothia parasitica strain 155 was grown for 4 days on cellophane over PDAmb agar or MMLT plus glycolate media. The cellophane was then removed and the media sectioned as follows: a core (10 mm diameter) was removed from the centre and concentric rings were cut from the plates: 5-15 mm from the centre, 15-25 and 25-32 mm (PDAmb) or 25-28.5 mm (MMLT + glycolate). The oxalate was extracted from the agar and precipitated as described above. IdentiJication of oxalate by X-ray di$raction A culture of strain 155 was grown for 7 days on cellophane over PDAmb. The cellophane and mycelium were removed and a 5 cm diameter plug of agar was taken from the plate. The plug was melted in 50 ml of demineralized water and centrifuged. The supernatant was decanted, the pellet suspended in 15 ml boiling water and the suspension recentrifuged. After two more such washings, a fine white crystalline material was recovered. It was transferred to a microscope slide and scanned in a Philips recording X-ray diffractometer. RESULTS Iden@ation of oxalic acid and conditions for quantitative determination Crystals were observed embedded in the agar on which certain strains of E. parasitica We identified the crystalline material as calcium oxalate by X-ray were grown. diffraction and HPLC. The oxalate was usually completely extracted from the agar media by two extractions with HCl. Only when oxalate levels exceeded 3 mg per culture was there appreciable oxalate in the third extraction. No interference with precipitation was observed on the addition of authentic oxalate to extracts. Recovery of the added oxalate was >90%. Formation of oxalate by cultures grown on PDAmb Three virulent and three hypovirulent strains of E. parasitica were grown on replicate plates of PDAmb. Oxalate production by all three virulent strains on PDAmb were similar over a 2-week period, i.e. oxalate production was first noted by day 4, increased sharply to day 8 and then remained constant. In contrast, none of the hypovirulent strains produced detectable oxalate, except for single samples of strains 748 and 752. These isolated samples did not signal the beginning of a rise in oxalate production similar to that shown by the virulent strains (Fig. 1). In general, hypovirulent strains have a somewhat slower growth rate than do the virulent strains both in the laboratory and in the host (Anagnostakis and D. Aylor, unpublished data). In order todetermine whether the lack ofoxalate production by the hypovirulent strains was due to slower growth, or whether oxalate production begins only after cultures obtain a certain size or weight, the data were replotted to show milligrams of oxalate produced on a dry weight basis (Fig. 2). Again, all the data points for the three virulent strains lie on the same curve in the early stages of growth.
372
E. A. Havir and S. L. Anagnostakis
0para 2
4
Days
[748
6
8
after
FIG. 1. Production of oxalate by virulent (O), 752 (A), 747 (IJ)] strains of E.
2.5
IO
12
inocirlafion
[67 (@), 42 (A), 155 (a)] grown on PDAmb.
and
hypovirulent
2.0 G r 2 -0 ox 0 5 ‘0 I-
1.5
I.0
0.5
0 0
o-05
0.10
o-15
Dry weight
FIG. 2. Total weight. Virulent PDAmb (symbols
0.20
(g )
amount of oxalate produced by cultures (67, 42, 155) and hypovirulent (748, as in Fig. 1).
of E. parasitica as a function of dry 752, 747) strains were grown on
The scatter about this line became pronounced as the cultures aged (beyond 8 days for the virulent strains). The hypovirulent cultures eventually exhibited dry weight gains equivalent to those of the virulent but did not produce oxalate, except in the few isolated samples mentioned above. A similar result was obtained when the total amount of oxalate produced was plotted against colony diameter. E$ect on oxalate production
of glucose, glyoxylate,
and glycolate added to minimal
medium
When cultures were grown on minimal medium agar with glucose as the sole carbon source, no oxalate was produced by either virulent or hypovirulent strains. To determine possible pathways of oxalate production in E. parasitica, the glucose concentration was decreased and either glycolate or glyoxylate was added. The growth rates of the different strains on these media were identical with those on PDAmb.
Oxalate
production
by Endothia
parasitica
373
70) 60 5 -$ L
50
6
40
;
30
2 2 -0 2
20
i
0
IO 0 2
FIG. 3. Production strains of E. parasitica
4
6
8
Days
after
inoculation
IO
12
of o&ate by virulent (67, 42, 155) and hypovirulent grown on MMLT plus glycolate. Symbols as in Fig. 1.
(748,
752, 747)
(748,
752,
6 30 Tm r 20 g z
IO
O
0 2
4
6
Days after FIG. 4. Production strains of E. parasifica
8
IO
12
inoculation
of oxalate by virulent (67, 42, grown on MMLT plus glyoxylate.
155) and hypovirulent Symbols as in Fig. 1.
747)
Glycolate and glyoxylate were selected for testing because in fungi glycolate can be converted to glyoxylate and then oxalate [IO]. The formation of oxalate by virulent strains 67 and 155 on glycolate medium was similar while strain 42 showed a lag period of 2 days (Fig. 3). This contrasted with the identical behaviour of all three strains on PDAmb (Fig. 1). Again, no consistent accumulation of oxalate by any of the hypovirulent strains was found. The levels of oxalate reached on the tenth day by all virulent cultures on glycolate-containing medium exceeded the levels reached on PDAmb for the same time period by at least fivefold. Oxalate accumulation occurred 3-4 days later on glyoxylate-containing media than on PDAmb or glycolate-containing media (Fig. 4). Also, the total amounts of oxalate produced were lower than those on glycolate after 8 days growth. However, as mentioned above, fungal growth with glyoxylate was as good as with PDAmb, minimal medium with glucose, or minimal medium with glucose and glycolate. Again, as in all the other experiments, no appreciable amounts of oxalate could be detected
374
E. A. Havir and S. L. Anagnostakis
in the extract from the glyoxylate medium on which hypovirulent grown. Location of oxalate formation
strains had been
in the colony
Oxalic acid production must be associated with the advancing edge of the mycelium of a pathogen if it contributes significantly to pathogenicity. To determine the pattern of oxalate formation by strains of E. parasitica agar media on which strain 155 had been grown (either PDAmb or MMLT with glycolate) were cut into concentric rings. Oxalate was present in all of the sections on both media. Thus, oxalate was produced and was detected at the edge of the colony as well as under the older mycelium. Oxalate formation is therefore not only associated with mature or older and senescent sections of a colony, but also with rapidly growing hyphae. The conclusion is corroborated by the observation that oxalate accumulation begins very early in the growth of the mycelium (Fig. 2). DISCUSSION
Our results corroborate, in part, and extend the earlier studies of Englander & Corden [7] and McCarroll & Thor [1.5], but there are important differences. First, we showed that the most rapid production of oxalate by virulent strains of E. parasitica occurred during the most rapid growth whereas Englander & Corden [7] suggested that oxalate accumulation was correlated with poor growth. Second, the hypovirulent strains derived from the virulent ones accumulated no oxalate under identical conditions whereas two hypovirulent strains of the fungus grown in vitro by McCarroll & Thor [15] produced about one-half the amount produced by two virulent strains. This amount of oxalate was about one-tenth the oxalate measured in our experiments. For E.parasitica and other fungi in which oxalate synthesis has been studied, the carbon source of the medium influences the accumulation of oxalate to a great degree (see Figs 1, 3, 4 and [22]). For example, Armentrout et al. [.5] showed that Schlerotium rolfsii when grown on glucose produced no oxalate and had relatively few microbodies, the site of oxalate formation. Endothia parasitica also did not produce oxalate on glucose but the presence or absence of microbodies was not determined. Although glyoxylate is not the only possible precursor of oxalate [II], it was surprising that E. parasitica produced more oxalate on glycolate media than on glyoxylate media since a common sequence of reactions is glycolate--glyoxylateoxalate [ZU, 131. It is possible that glycolate is more readily taken up than is glyoxylate, although growth was equal on both media. The results with E. parasitica are in agreement with those of Maxwell & Bateman [12] who found much higher oxalate produced by Schlerotium rolfsii on glycolate than on glyoxylate-containing media. The growth of Schlerotium rolfsii in the glyoxylate medium was much greater than in the glycolate medium, however. The time course of oxalate production by E. parasitica was similar to that of Schlerotium rolfsii [12], i.e. the highest accumulation occurred early in the growth of the culture, 2-4 days for Schlerotium rolfsii and 4-7 days for E. parasitica (Figs 1 and 3). However, Schlerotium rolfsii and Schlerotinia sclerotorium [12, 141 produced almost ten times as much oxalate in vitro as E. parasitica. Such differences may not be significant since the first two are basidiomycetes and the third an ascomycete and since the
Oxalate
production
by Endothia
parasitica
375
hosts of the organisms are so dissimilar. Schlerotium rolfsii and Schlerotinia sclerotorium are primarily pathogens of the soft tissues of annual plants such as sunflower and bean. Virulent strains of E. parasitica can attack bark tissue and overwinter on its host, the chestnut tree, and resume growth whenever conditions are favourable. However, hypovirulent strains grow very little, or for a restricted time, when inoculated alone into chestnut trees. Why the hypovirulent strains do not produce oxalate has not been investigated. It is an important observation since it is the only biochemical change known that is brought about by the introduction of the cytoplasmic determinants for hypovirulence. Three general possibilities exist. One is that the hypovirulent strains are not able to transport some vital metabolite into the site of oxalate biosynthesis or, less likely, they are not able to transport oxalate out of their cells. Second, some enzyme(s) of biosynthesis may be lacking, or there may be an enzyme system present which breaks down oxalate as soon as it is formed. Third, all the relevant enzymes for oxalate biosynthesis may be present but their activity is suppressed. We do not as yet have data to allow us to choose among these possibilities. Our laboratory study has demonstrated large differences in oxalate production between virulent and hypovirulent strains grown in vitro. This information may now be useful in designing experiments to study the host-pathogen interaction in vivo. We wish to thank Dr Charles Frink for the X-ray diffraction determination and Dr Israel Zelitch for helpful discussions. The skilful technical assistance of Marie Maier on all phases of this work and of Carol Barbesino and Ellen Hennessey for the oxalate determinations is greatly appreciated. REFERENCES 1. ANAGNOSTAWS, S. L. (1982). Biological control of chestnut blight. Science 215,46647 1. 2. ANAGNOSTAKIS, S. L. (1982). An improved defined medium for growth of Endothia parasitica. Mycologia 74,826-830. 3. ANAGNOSTAKIS, S. L. & DAY, P. R. (1979). Hypovirulence conversion in En&&in parasitica. Phytopathology 69, 12261229. 4. ANDERSON, P. J. (1914). The Morphology and Life History of the Chestnut Blight Fungus. The Commission for the Investigation and Control of the Chestnut Tree Blight Disease in Pennsylvania, Bulletin F, State Printer, Harrisburgh, Pennsylvania, 44 pp. and XIX. 5. ARMENTROUT, V. N., Craves, L. B., JR. & MAXWELL, D. P. (1978). Localization of enzymes of oxalate biosynthesis in microbodies of Sclerotium rolf$ii. Phytopathology 68, 1597-1599. 6. BATEMAN, D. F. & BEER, S. V. (1965). Simultaneous production and synergistic action of oxalic acid and polygalacturonase during pathogenesis by Sclerotium rolfsii. Phytopathology 55, 204-2 11. 7. ENGLANDER, C. M. & CORDEN, M. E. (1971). Stimulation of mycelial growth of .&dothia porcsitica by heavy metals. Applied Microbiology 22, 1012-1016. 8. GIBSON, I. A. S. (1953). Crown rot, a seedling disease of groundnuts caused by Aspergillus niger. Transactions British Mycological Society 36, 198-209. 9. GRENTE, J. (1981). Les variants hypovirulents de 1’Endothia parasitica et la lutte biological contra le chancre du chltaignier. Ph.D. Thesis Universite de Bretagne Occidentale, Brest, France, 195 pp. 10. HODGKINSON, A. (1977). Oxalic acid metabolism in lower plants. In Oxalic Acid in Biology and Medicine. Academic Press, New York. 11. MAXWELL, D. P. (1973). Oxalate formation in Whetzelinia sclerotiorum by oxaloacetate acetylhydrolase. Physiological Plant Pathology 3, 279-288. 12. MAXWELL, D. P. & BATEMAN, D. F. (1968). Influence of carbon source and pH on oxalate accumulation in culture filtrates of Sclerotium rolfsii. Phytopathology 58, 1351--1355. 13. MAXWE].r, D. P. & BATEMAN, D. F. (1968). Oxalic acid biosynthesis by Sclerotium rolfsii. Phytopathology 58, 1635-1642.
E. A. Havir and S. L. Anagnostakis
376 D. P. & Lumsden, bean and in culture. 15. MCCARROLL, D. R. & THOR, 14. MAXWELL,
infected
R.
D.
(1970).
Phytopathology
acid
production
acid Ed. by J. Luckok & C. Smith, pp. 60-63. West Virginia University Press, 16. NOYES, R. D. & HANCOCK, J. G. (1981). Role of oxalic acid in Physiological Plant Pathology IS, 123-132. parasitica.
In Proceedings
of
E. (1978).
Oxalic
the American
by
Sclnotinia sclerotiorum in
60, 1395-1398.
The
Chestnut
role
of oxalic
Symposium,
in the pathogen&s W. L. MacDonald, Morgantown. the Sclerotinia wilt
of Endothia F. D. Ceck, of sunflower.
Plant Pathology
(1983)
23, 369-376
Oxalate production by virulent strains of Endothia parasitica
but not by hypovirulent
EVELYN A. HAVIR Department
of Biochemistry
and Genetics
and SANDRA L. ANAGNOSTAKIS Department (Acceptedfor
of Plant
Pathology
publication
and Botany,
The Connecticut
Agricultural
Experimnt
Station,
New Haven,
CT 06504
June 1983)
Three virulent strains of Endofhia parasitica when grown on potato dextrose agar containing methionine and biotin (PDAmb) or minimal media containing glucose supplemented with either glycolate or glyoxylate produced 10-12, 5660 and 20-40 mg oxalate g-r dry weight of fungus respectively. Oxalate accumulation was detected the fourth day after inoculation when the strains were grown on PDAmb or the glycolate medium but not until day 7 on the glyoxylate medium. In the early stages of growth (up to 0.1 g dry weight per colony) the total amount ofoxalate excreted into the medium was proportional to the dry weight ofthe fungus but there was little or no correlation at later stages of growth. Oxalate was detected at the edge of the fungal colony, which is important if oxalate is to play a role in pathogenicity. Under identical conditions three hypovirulent strains, derived from the virulent ones by the introduction of cytoplasmic determinants for hypovirulence, produced no detectable oxalate.
INTRODUCTION Endothiuparusitica
is the fungus that causes chestnut tree blight. Its control, a goal of many research groups [I, 4,9] will be facilitated by knowing the mechanism by which the pathogen attacks its host. For a number of other fungi, such as Sclerotium rolfsii [S, 2.21, Sclerotiniu sclerotiorum [N, 161 and Aspergillus niger [8], production of oxalic acid is important in pathogenicity. Hodgkinson has summarized the pathways of oxalate biosynthesis in the fungi [IO]. Oxalic acid is believed to function in two ways: first, it combines with calcium from host tissue, thus physically weakening it; second, it lowers the pH of the host tissue providing a favourable condition for the action of degradative enzymes such as polygalacturonase [6]. Englander & Corden in 1971 [7] reported that crystals of calcium oxalate were formed by E. parasitica in agar medium. More recently, McCarroll & Thor [25] showed that oxalic acid was present at the advancing edge of cankers caused by this fungus on chestnut trees. However, no further investigation has been made of the formation ofoxalate and its role in this host:pathogen relationship. This study was undertaken to determine how factors such as time, colony size, and media affect oxalate production by E. parasitica in vitro. The oxalate formation of three virulent strains of varying origin was contrasted with that of three hypovirulent strains derived from them [3]. 0048-4059/83/060369
+ 08 $03.00/O
0
1983 Academic
Press Inc.
(London)
Limited
370
E. A. Havir and S. L. Anagnostakis
MATERIALS AND METHODS Growth offungi Virulent E. parasitica strains were: 67, ATCC
no. 38753, an Italian strain; 42, ATCC no. 38751, a Connecticut strain isolated in 1975; 155, ATCC no. 38755, a Connecticut strain isolated in 1977. Curative converts of these strains with Italian cytoplasmic determinants for hypovirulence were: 748, (67[1]) ; 752, (42[1]); 747, (155[1]) [3], respectively. All strains were grown and stored on potato dextrose agar containing methionine and biotin (PDAmb), Difco PDA with methionine (100 mg 1-l) and biotin (1 mg 1-l). The tests of special substrates were made on minimal medium containing lactate and tannic acid (MMLT) agar [2]. The pH was adjusted to 5.6 (the pH of Difco PDAmb) and the carbon source was either 0.10 M glucose, 0.05 M glucose + 0.05 M potassium glycolate or 0.05 M glucose + O-05 M sodium glyoxylate. Agar media were dispensed to approximately 75 ml per 150 x 15 mm sterile disposable Petri dish. Two discs of cellophane, DuPont 193-PUDO, cut a little less than 150 mm diameter, were autoclaved wet and placed on the surface of the agar. Plugs (3 mm diameter) were cut with a cork borer from the edge of rapidly expanding mycelium in PDAmb, and placed on the cellophane in the centre of each dish, mycelium-side up. Dishes were wrapped in ParafilmB and incubated at 27.C in a seed germinator with 12 h of white fluorescent light per day. Extraction
and precipitation
of oxalate
Fungal colonies were measured daily and growth rates were expressed as diameter increase (mm) per day. At the time of sampling, the mycelium was scraped from the surface of the cellophane and dried at 35 “C for dry weight analysis. The cellophane was stripped away, and agar from under the colony was transferred to a small beaker and covered with 0.5 N HCl. The contents of the beaker were incubated at 30°C with gentle shaking. After 2 h, the HCl was removed and fresh HCl added for another 2 h of extraction. The two extracts were combined and an aliquot was mixed with calcium acetate buffer (O-23 M, pH 4.5) to precipitate calcium oxalate. The suspension was stored at 10°C overnight, and the precipitate, collected by centrifugation, was dissolved by the addition of l-4 ml of O-4 N H&SO, (depending on the volume of the precipitate). In order to dissolve very heavy precipitates, the solutions were heated to 40 “C for 20-30 min. Two controls were performed to ensure quantitative recovery of oxalate. To check completeness of extraction of some samples, a third addition of HCl was made and assayed as above. Oxalic acid (10 pmol) was also added to some samples when a precipitate of calcium oxalate was not observed. The recovery of the added oxalate was determined by the procedure described. Determination
of oxalate by high pe$ormance
liquid chromatography
(HPLC)
The oxalate in a 5-20 pl sample of the dissolved precipitate was determined by HPLC on a Perkin-Elmer 3B Liquid Chromatograph equipped with an Aminex HPX-87 column (Bio-Rad Corp) (300 x 7.8 mm) with O-015 N H,SO, as eluting agent. The flow rate was 0.8 ml min-1 at a temperature of 25°C; detection was by UV absorbancy at 2 10 nm. The peak area of oxalate, as calculated by a Hewlett-Packard
Oxalate
production
by Endothia
parasitica
3390A Integrator, was proportional range of 0.5-30 nmol.
to concentration
371 of authentic
oxalic
acid
in the
Location of oxalate formation within the colony Endothia parasitica strain 155 was grown for 4 days on cellophane over PDAmb agar or MMLT plus glycolate media. The cellophane was then removed and the media sectioned as follows: a core (10 mm diameter) was removed from the centre and concentric rings were cut from the plates: 5-15 mm from the centre, 15-25 and 25-32 mm (PDAmb) or 25-28.5 mm (MMLT + glycolate). The oxalate was extracted from the agar and precipitated as described above. IdentiJication of oxalate by X-ray di$raction A culture of strain 155 was grown for 7 days on cellophane over PDAmb. The cellophane and mycelium were removed and a 5 cm diameter plug of agar was taken from the plate. The plug was melted in 50 ml of demineralized water and centrifuged. The supernatant was decanted, the pellet suspended in 15 ml boiling water and the suspension recentrifuged. After two more such washings, a fine white crystalline material was recovered. It was transferred to a microscope slide and scanned in a Philips recording X-ray diffractometer. RESULTS Iden@ation of oxalic acid and conditions for quantitative determination Crystals were observed embedded in the agar on which certain strains of E. parasitica We identified the crystalline material as calcium oxalate by X-ray were grown. diffraction and HPLC. The oxalate was usually completely extracted from the agar media by two extractions with HCl. Only when oxalate levels exceeded 3 mg per culture was there appreciable oxalate in the third extraction. No interference with precipitation was observed on the addition of authentic oxalate to extracts. Recovery of the added oxalate was >90%. Formation of oxalate by cultures grown on PDAmb Three virulent and three hypovirulent strains of E. parasitica were grown on replicate plates of PDAmb. Oxalate production by all three virulent strains on PDAmb were similar over a 2-week period, i.e. oxalate production was first noted by day 4, increased sharply to day 8 and then remained constant. In contrast, none of the hypovirulent strains produced detectable oxalate, except for single samples of strains 748 and 752. These isolated samples did not signal the beginning of a rise in oxalate production similar to that shown by the virulent strains (Fig. 1). In general, hypovirulent strains have a somewhat slower growth rate than do the virulent strains both in the laboratory and in the host (Anagnostakis and D. Aylor, unpublished data). In order todetermine whether the lack ofoxalate production by the hypovirulent strains was due to slower growth, or whether oxalate production begins only after cultures obtain a certain size or weight, the data were replotted to show milligrams of oxalate produced on a dry weight basis (Fig. 2). Again, all the data points for the three virulent strains lie on the same curve in the early stages of growth.
372
E. A. Havir and S. L. Anagnostakis
0para 2
4
Days
[748
6
8
after
FIG. 1. Production of oxalate by virulent (O), 752 (A), 747 (IJ)] strains of E.
2.5
IO
12
inocirlafion
[67 (@), 42 (A), 155 (a)] grown on PDAmb.
and
hypovirulent
2.0 G r 2 -0 ox 0 5 ‘0 I-
1.5
I.0
0.5
0 0
o-05
0.10
o-15
Dry weight
FIG. 2. Total weight. Virulent PDAmb (symbols
0.20
(g )
amount of oxalate produced by cultures (67, 42, 155) and hypovirulent (748, as in Fig. 1).
of E. parasitica as a function of dry 752, 747) strains were grown on
The scatter about this line became pronounced as the cultures aged (beyond 8 days for the virulent strains). The hypovirulent cultures eventually exhibited dry weight gains equivalent to those of the virulent but did not produce oxalate, except in the few isolated samples mentioned above. A similar result was obtained when the total amount of oxalate produced was plotted against colony diameter. E$ect on oxalate production
of glucose, glyoxylate,
and glycolate added to minimal
medium
When cultures were grown on minimal medium agar with glucose as the sole carbon source, no oxalate was produced by either virulent or hypovirulent strains. To determine possible pathways of oxalate production in E. parasitica, the glucose concentration was decreased and either glycolate or glyoxylate was added. The growth rates of the different strains on these media were identical with those on PDAmb.
Oxalate
production
by Endothia
parasitica
373
70) 60 5 -$ L
50
6
40
;
30
2 2 -0 2
20
i
0
IO 0 2
FIG. 3. Production strains of E. parasitica
4
6
8
Days
after
inoculation
IO
12
of o&ate by virulent (67, 42, 155) and hypovirulent grown on MMLT plus glycolate. Symbols as in Fig. 1.
(748,
752, 747)
(748,
752,
6 30 Tm r 20 g z
IO
O
0 2
4
6
Days after FIG. 4. Production strains of E. parasifica
8
IO
12
inoculation
of oxalate by virulent (67, 42, grown on MMLT plus glyoxylate.
155) and hypovirulent Symbols as in Fig. 1.
747)
Glycolate and glyoxylate were selected for testing because in fungi glycolate can be converted to glyoxylate and then oxalate [IO]. The formation of oxalate by virulent strains 67 and 155 on glycolate medium was similar while strain 42 showed a lag period of 2 days (Fig. 3). This contrasted with the identical behaviour of all three strains on PDAmb (Fig. 1). Again, no consistent accumulation of oxalate by any of the hypovirulent strains was found. The levels of oxalate reached on the tenth day by all virulent cultures on glycolate-containing medium exceeded the levels reached on PDAmb for the same time period by at least fivefold. Oxalate accumulation occurred 3-4 days later on glyoxylate-containing media than on PDAmb or glycolate-containing media (Fig. 4). Also, the total amounts of oxalate produced were lower than those on glycolate after 8 days growth. However, as mentioned above, fungal growth with glyoxylate was as good as with PDAmb, minimal medium with glucose, or minimal medium with glucose and glycolate. Again, as in all the other experiments, no appreciable amounts of oxalate could be detected
374
E. A. Havir and S. L. Anagnostakis
in the extract from the glyoxylate medium on which hypovirulent grown. Location of oxalate formation
strains had been
in the colony
Oxalic acid production must be associated with the advancing edge of the mycelium of a pathogen if it contributes significantly to pathogenicity. To determine the pattern of oxalate formation by strains of E. parasitica agar media on which strain 155 had been grown (either PDAmb or MMLT with glycolate) were cut into concentric rings. Oxalate was present in all of the sections on both media. Thus, oxalate was produced and was detected at the edge of the colony as well as under the older mycelium. Oxalate formation is therefore not only associated with mature or older and senescent sections of a colony, but also with rapidly growing hyphae. The conclusion is corroborated by the observation that oxalate accumulation begins very early in the growth of the mycelium (Fig. 2). DISCUSSION
Our results corroborate, in part, and extend the earlier studies of Englander & Corden [7] and McCarroll & Thor [1.5], but there are important differences. First, we showed that the most rapid production of oxalate by virulent strains of E. parasitica occurred during the most rapid growth whereas Englander & Corden [7] suggested that oxalate accumulation was correlated with poor growth. Second, the hypovirulent strains derived from the virulent ones accumulated no oxalate under identical conditions whereas two hypovirulent strains of the fungus grown in vitro by McCarroll & Thor [15] produced about one-half the amount produced by two virulent strains. This amount of oxalate was about one-tenth the oxalate measured in our experiments. For E.parasitica and other fungi in which oxalate synthesis has been studied, the carbon source of the medium influences the accumulation of oxalate to a great degree (see Figs 1, 3, 4 and [22]). For example, Armentrout et al. [.5] showed that Schlerotium rolfsii when grown on glucose produced no oxalate and had relatively few microbodies, the site of oxalate formation. Endothia parasitica also did not produce oxalate on glucose but the presence or absence of microbodies was not determined. Although glyoxylate is not the only possible precursor of oxalate [II], it was surprising that E. parasitica produced more oxalate on glycolate media than on glyoxylate media since a common sequence of reactions is glycolate--glyoxylateoxalate [ZU, 131. It is possible that glycolate is more readily taken up than is glyoxylate, although growth was equal on both media. The results with E. parasitica are in agreement with those of Maxwell & Bateman [12] who found much higher oxalate produced by Schlerotium rolfsii on glycolate than on glyoxylate-containing media. The growth of Schlerotium rolfsii in the glyoxylate medium was much greater than in the glycolate medium, however. The time course of oxalate production by E. parasitica was similar to that of Schlerotium rolfsii [12], i.e. the highest accumulation occurred early in the growth of the culture, 2-4 days for Schlerotium rolfsii and 4-7 days for E. parasitica (Figs 1 and 3). However, Schlerotium rolfsii and Schlerotinia sclerotorium [12, 141 produced almost ten times as much oxalate in vitro as E. parasitica. Such differences may not be significant since the first two are basidiomycetes and the third an ascomycete and since the
Oxalate
production
by Endothia
parasitica
375
hosts of the organisms are so dissimilar. Schlerotium rolfsii and Schlerotinia sclerotorium are primarily pathogens of the soft tissues of annual plants such as sunflower and bean. Virulent strains of E. parasitica can attack bark tissue and overwinter on its host, the chestnut tree, and resume growth whenever conditions are favourable. However, hypovirulent strains grow very little, or for a restricted time, when inoculated alone into chestnut trees. Why the hypovirulent strains do not produce oxalate has not been investigated. It is an important observation since it is the only biochemical change known that is brought about by the introduction of the cytoplasmic determinants for hypovirulence. Three general possibilities exist. One is that the hypovirulent strains are not able to transport some vital metabolite into the site of oxalate biosynthesis or, less likely, they are not able to transport oxalate out of their cells. Second, some enzyme(s) of biosynthesis may be lacking, or there may be an enzyme system present which breaks down oxalate as soon as it is formed. Third, all the relevant enzymes for oxalate biosynthesis may be present but their activity is suppressed. We do not as yet have data to allow us to choose among these possibilities. Our laboratory study has demonstrated large differences in oxalate production between virulent and hypovirulent strains grown in vitro. This information may now be useful in designing experiments to study the host-pathogen interaction in vivo. We wish to thank Dr Charles Frink for the X-ray diffraction determination and Dr Israel Zelitch for helpful discussions. The skilful technical assistance of Marie Maier on all phases of this work and of Carol Barbesino and Ellen Hennessey for the oxalate determinations is greatly appreciated. REFERENCES 1. ANAGNOSTAWS, S. L. (1982). Biological control of chestnut blight. Science 215,46647 1. 2. ANAGNOSTAKIS, S. L. (1982). An improved defined medium for growth of Endothia parasitica. Mycologia 74,826-830. 3. ANAGNOSTAKIS, S. L. & DAY, P. R. (1979). Hypovirulence conversion in En&&in parasitica. Phytopathology 69, 12261229. 4. ANDERSON, P. J. (1914). The Morphology and Life History of the Chestnut Blight Fungus. The Commission for the Investigation and Control of the Chestnut Tree Blight Disease in Pennsylvania, Bulletin F, State Printer, Harrisburgh, Pennsylvania, 44 pp. and XIX. 5. ARMENTROUT, V. N., Craves, L. B., JR. & MAXWELL, D. P. (1978). Localization of enzymes of oxalate biosynthesis in microbodies of Sclerotium rolf$ii. Phytopathology 68, 1597-1599. 6. BATEMAN, D. F. & BEER, S. V. (1965). Simultaneous production and synergistic action of oxalic acid and polygalacturonase during pathogenesis by Sclerotium rolfsii. Phytopathology 55, 204-2 11. 7. ENGLANDER, C. M. & CORDEN, M. E. (1971). Stimulation of mycelial growth of .&dothia porcsitica by heavy metals. Applied Microbiology 22, 1012-1016. 8. GIBSON, I. A. S. (1953). Crown rot, a seedling disease of groundnuts caused by Aspergillus niger. Transactions British Mycological Society 36, 198-209. 9. GRENTE, J. (1981). Les variants hypovirulents de 1’Endothia parasitica et la lutte biological contra le chancre du chltaignier. Ph.D. Thesis Universite de Bretagne Occidentale, Brest, France, 195 pp. 10. HODGKINSON, A. (1977). Oxalic acid metabolism in lower plants. In Oxalic Acid in Biology and Medicine. Academic Press, New York. 11. MAXWELL, D. P. (1973). Oxalate formation in Whetzelinia sclerotiorum by oxaloacetate acetylhydrolase. Physiological Plant Pathology 3, 279-288. 12. MAXWELL, D. P. & BATEMAN, D. F. (1968). Influence of carbon source and pH on oxalate accumulation in culture filtrates of Sclerotium rolfsii. Phytopathology 58, 1351--1355. 13. MAXWE].r, D. P. & BATEMAN, D. F. (1968). Oxalic acid biosynthesis by Sclerotium rolfsii. Phytopathology 58, 1635-1642.
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376 D. P. & Lumsden, bean and in culture. 15. MCCARROLL, D. R. & THOR, 14. MAXWELL,
infected
R.
D.
(1970).
Phytopathology
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production
acid Ed. by J. Luckok & C. Smith, pp. 60-63. West Virginia University Press, 16. NOYES, R. D. & HANCOCK, J. G. (1981). Role of oxalic acid in Physiological Plant Pathology IS, 123-132. parasitica.
In Proceedings
of
E. (1978).
Oxalic
the American
by
Sclnotinia sclerotiorum in
60, 1395-1398.
The
Chestnut
role
of oxalic
Symposium,
in the pathogen&s W. L. MacDonald, Morgantown. the Sclerotinia wilt
of Endothia F. D. Ceck, of sunflower.