Cryphonectria radicalis: rediscovery of a lost fungus

Mycologia, 94(1), 2002, pp. 105–115. q 2002 by The Mycological Society of America, Lawrence, KS 66044-8897

Cryphonectria radicalis: rediscovery of a lost fungus

Patrik J. Hoegger1 Daniel Rigling

INTRODUCTION

The fungal genera Cryphonectria Sacc. and Endothia Fr. contain 12 species (Barr 1978, Micales and Stipes 1987). Three species are known to be plant pathogens: C. parasitica (Murr.) Barr, C. cubensis (Bruner) Hodges and E. gyrosa (Schw.) Fr. (Boerboom and Maas 1970, Van Arsdel 1972, Roane et al 1974, 1986, Snow et al 1974, Hodges 1980, Hodges et al 1986, Myburg et al 1999). The others are considered to be saprotrophic (Roane et al 1986). Cryphonectria parasitica is the causal agent of chestnut blight. While recovery from this disease was observed in many Castanea sativa Mill. populations in Europe (Heiniger and Rigling 1994), C. parasitica virtually eliminated Castanea dentata (Marsh.) Borkh. in its natural range in North America (Anagnostakis 1987). At the beginning of this century, the emergence of chestnut blight in North America stimulated investigations on the saprotrophic species C. radicalis (Schw. ex Fries) Barr in Europe and North America, because of its close relationship to C. parasitica. These investigations revealed that C. radicalis was common in Europe (Shear 1912, Petri 1917, Shear et al 1917), North America (Anderson and Anderson 1912, Shear et al 1917) and Japan (Shear et al 1917). After the spread of chestnut blight, C. radicalis has not been found in North America, and it apparently has disappeared since (Elliston 1982, Torsello et al 1994, Anagnostakis 1995). Cryphonectria radicalis was not reported in Europe since the introduction of chestnut blight. We failed to find C. radicalis in several studies in southern Switzerland, including a study where cut and natural dead chestnut stems were sampled in five different chestnut stands (Prospero et al 1998, S. Prospero and D. Rigling pers comm). Also in Asia, C. radicalis appears to be rare or nonexistent, as it was never isolated in extended chestnut sampling campaigns in China and Japan (M. G. Milgroom pers comm). A hypothesis for the disappearance of C. radicalis suggested by Elliston (1982) is that C. radicalis has been ‘‘absorbed’’ by C. parasitica through hybridization. However, the indirect evidence presented by Elliston (1982) was circumstantial and no experimental matings between C. radicalis and C. parasitica were attempted to demonstrate the possibility of interspecific hybridizations. Another hypothesis, the displacement hypothesis, is

WSL Swiss Federal Research Institute, CH-8903 Birmensdorf, Switzerland

Ottmar Holdenrieder Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland

Ursula Heiniger WSL Swiss Federal Research Institute, CH-8903 Birmensdorf, Switzerland

Abstract: In an attempt to isolate the ascomycete Cr yphonectria parasitica (Diaporthales, Valsaceae) from dead chestnut stems, we obtained three C. radicalis strains. All three strains were isolated in areas of Switzerland with high chestnut blight incidence. To confirm our species designation, we compared the three C. radicalis strains to hypovirus (hv)-free and hv-infected C. parasitica strains. The comparison revealed several distinctive characteristics. On potato dextrose agar in the dark, the C. radicalis strains produced a fluffy mycelium and small droplets of a purple exudate giving the mycelium a light pinkish appearance. On corn meal medium in the dark, the C. radicalis strains caused a color change of the medium to purple, whereas the C. parasitica strains did not cause any color change. Ascospores from C. radicalis were significantly smaller than C. parasitica ascospores and their dimensions fit within other published size ranges. Southern hybridization analysis of the two species using nuclear and mitochondrial probes support their taxonomic separation. This separation is further supported by the lack of successful interspecific crosses. In virulence tests on chestnut trees, the C. radicalis strains exhibited very low virulence, comparable to highly hypovirulent hv-infected C. parasitica strains. Our results suggest that C. radicalis still coexists with C. parasitica although at a low frequency. Key Words: Castanea sativa, chestnut blight, Cryphonectria parasitica, Diaporthales, hypovirus, matingtype, saprotroph Accepted for publication June 14, 2001. 1 Corresponding author, Email: [email protected] Present adress: Institut fu¨r Forstbotanik, Georg-Austust-Universita¨t Go¨ttingen, Bu¨sgenweg 2, D-37077 Go¨tingen, Germany.

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106 TABLE I.

MYCOLOGIA Origin of the newly isolated C. radicalis strains WSLCC No.a

Year

ph1111

M2268

Winter 1995

ph1113

M2269

Winter 1995

COP26-5

M2270

Spring 1996

Strain

a

Origin Monthey, VS, Switzerland Monthey, VS, Switzerland S. Antonino, TI, Switzerland

TABLE II.

lence (hypovirulence) and a white culture morphology (Day et al 1977, Choi and Nuss 1992, Hillman et al 1995). The other C. parasitica strain was hypovirus (hv)-free. To our knowledge, this is the first reported isolation and detailed characterization of C. radicalis since the numerous reports at the beginning of the 20th century. MATERIAL AND METHODS

Fungal strains. TABLE I shows the origins of the three putative C. radicalis strains characterized in this study. S. Antonino is located in southern Switzerland, where chestnut blight has been present since at least 1948 (Ga¨umann 1951). In Monthey, chestnut blight was detected in the 1980‘s (Bissegger and Heiniger 1991). Incidence of chestnut blight was high at both locations. COP26–5 was isolated from a cut chestnut stem (Castanea sativa) that was piled in the forest for one year. The stem was living and not showing any signs of fungal growth on the bark at the time of the cut. Strains ph1111 and ph1113 were isolated from cut chestnut stems, but no information was available on the time of the cut. All three strains were obtained in an attempt to isolate C. parasitica. Isolations were performed on tannic acid malt extract agar (Rigling 1995). Only one old C. radicalis strain (M285) was available from the WSL culture collection. The C. parasitica strains M1275 (hv-free) and VSX (hv-infected) were used for comparison. M1115 (MAT-2) and M1297 (MAT-1) were used as C. parasitica mating-type tester strains (TABLE II).

Origin of the C. radicalis and C. parasitica strains used for comparison and the mating-type tester strains

Strain

Species

Year

M285a

C. radicalis

,1954

M1275a VSX

C. parasitica C. parasitica

1990 1996

Ticino, Switzerland Laboratory conversion

MB85 MB117 M1297a M1115a

C. C. C. C.

1991 1991 1976 1976

Weggis, Switzerland Murg, Switzerland Ticino, Switzerland Ticino, Switzerland

b

Latitude: 468 159 300 N, longitude: 68 569 300 E Latitude: 468 159 300 N, longitude: 68 569 300 E Latitude: 468 89 300 N, longitude: 88 599 00 E

WSLCC, culture collection of the WSL, Swiss Federal Institute, Phytopathology, CH-8903 Birmensdorf, Switzerland.

that the non-pathogenic C. radicalis has been displaced by its pathogenic relative, C. parasitica, when it was introduced into North America and Europe. The same explanation was suggested by Brasier (1983, 1991) for the successive replacement of Ophiostoma ulmi (Buism.) Nannf. by the more virulent O. novo-ulmi Brasier. The lack of recent isolation of C. radicalis prevented any evaluation of the two hypotheses. In an attempt to isolate C. parasitica from the bark of dead chestnut stems, we obtained three strains showing unusual culture morphology on potato dextrose agar. The unusual culture morphology proved to be a stable and reproducible trait. Based on an analysis of the literature, we hypothesized that the strains belonged to the species C. radicalis. The objective of this study was to identify the strains using reproducible methods and to give a proper description. We analyzed phenotypic traits such as culture morphology, ascospore dimensions, mating behavior, and virulence on chestnut plants. In addition, we performed nuclear and mitochondrial (mt) DNA hybridization experiments to analyze the genetic relationship of the three strains to C. parasitica isolates. An old C. radicalis strain from our culture collection and two C. parasitica strains were used for comparison. One of the C. parasitica strains was infected with Cryphonectria hypovirus 1, which confers reduced viru-

a

Coordinates

parasitica parasitica parasitica parasitica

Origin Italy?

Remarks Strain Biraghi 86, MCU 34, classified as Endothia fluens (Sow.) S. et S.b Hv-free Hv-infected (Strain M1275 converted with hypovirus from strain M784a) Hv-free, Hoegger et al (2000) Hv-free, Hoegger et al (2000) MAT-1 tester strain, hv-free MAT-2 tester strain, hv-free

From the culture collection of the WSL, Swiss Federal Institute, Phytopathology, CH-8903 Birmensdorf, Switzerland. Obtained from M. Orsenigo, Italy. Italian origin is most likely.

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Culture morphology. a) The strains were inoculated on potato dextrose agar (PDA, 39 g L21; Difco, Detroit, Michigan, USA) plates and incubated at 25 C in the dark. After one week the cultures were exposed for an additional week to diffuse daylight at room temperature on the laboratory bench. The culture morphology was assessed after dark and light incubation. For colony growth measurements strains were incubated on PDA for 4 d at 20 and 25 C in the dark in five replicates. Two perpendicular diameters of each colony were measured. b) Corn meal medium was prepared by adding 10 g of corn meal to 20 mL of distilled water in 100 mL-Erlenmeyer flasks and autoclaving for 20 min at 120 C, 1.2 bar (Shear and Stevens 1913). The cultures were incubated at 25 C in the dark for 7 wk. Two cultures per strain were grown. Mating experiments. Crosses were performed on autoclaved stem pieces of Castanea sativa in water agar as previously described for mating-type tests with C. parasitica (Anagnostakis 1988). The three putative C. radicalis strains were paired with each other, with C. radicalis strain M285 and with the C. parasitica mating-type tester strains M1115 and M1297. Incubation and spermatization were performed as described by Bissegger et al (1997). The C. parasitica tester strains were paired with each other as positive controls. Both C. parasitica tester strains and the three putative C. radicalis strains were also examined for their ability to produce perithecia alone in order to exclude self-fertilization. The culture morphology of the progeny from the two putative C. radicalis crosses that produced perithecia (ph1111 3 COP26-5 and ph1113 3 COP26-5) was examined on PDA. The ascospore suspension from one perithecium of each cross was plated on water agar and incubated for 20 h at 25 C in the dark. Twenty-five single, germinating ascospores from each cross were picked under the dissecting microscope, placed on PDA and incubated as described above to assess the culture morphology. Ascospore dimensions. One perithecium from each of the crosses ph1111 3 COP26-5 and ph1113 3 COP26-5 was chosen randomly for the analysis of ascospores. Ascospores from the C. parasitica crosses M1115 3 M1297, M1115 3 MB117, and M1297 3 MB85 (TABLE II) were used for comparison. The ascospores were stained with cotton blue in phenol (Erb and Matheis 1983) and the lengths and widths of 30 mature ascospores from each perithecium were measured under a light microscope using 10003 magnification. Virulence tests. Three year-old Castanea sativa plants grown from cuttings were used for the virulence tests. The plants were grown in the greenhouse under semi-controlled conditions (temperature range 10–28 C, relative atmospheric moisture range 60–98%). Inoculations were performed by making a hole in the bark with a cork borer (5 mm in diameter) and filling it with a plug of mycelium grown on PDA. The negative control consisted of a sterile PDA plug. The wounds were covered with masking tape to prevent desiccation. The axial length of the lesions was measured every two weeks until 20 wk after infection. One year after infection, the bark tissue of the trees was analyzed for the pres-

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ence of mycelial fans and reisolations of the fungi were performed. Hybridization with pMS5.1 and mtDNA from C. parasitica. Fungal DNA extraction, gel electrophoresis and non-radioactive hybridization procedures were as described in Hoegger et al (2000). To ensure equal loading on the gel, the DNA from each extraction was quantified using a DyNA Quant 200 fluorometer (Amersham Pharmacia Biotech, Sweden). All samples were run on the same gel. After hybridization with the C. parasitica DNA fingerprinting probe pMS5.1 (Milgroom et al 1992), the blot was stripped in 0.2 M NaOH, 0.1% SDS and reprobed with purified mtDNA of C. parasitica isolate EP67 (ATCC 38753). The mtDNA was prepared as described by Milgroom and Lipari (1993). Mating-type identification by PCR. To compare the mating system of the putative C. radicalis strains to C. parasitica we used two different PCR methods for mating-type determination. The presence of a conserved DNA binding motif, i.e., the high mobility group (HMG) box, in the MAT-2 idiomorph of several ascomycetes was demonstrated by Arie et al (1997). The presence or absence of the HMG box can be used to assign the mating-types MAT-2 or MAT-1, respectively, in C. parasitica (Hoegger et al 2000). In order to detect the presence of the HMG box in the three putative C. radicalis strains, PCR with C. parasitica primers CpHMG3 and CpHMG4 was performed as described in Hoegger et al (2000). Furthermore, we used two primer pairs (M1-GS1/M1-GS2-rev, M2-GS2/InvA5n) specific for the two MAT idiomorphs of C. parasitica which span a large portion of each idiomorph (Marra and Milgroom 1999). Positive and negative controls using DNA from the two C. parasitica mating-type testers were included in each PCR setup. RESULTS

Culture morphology on PDA. After one week in the dark, the culture morphologies of the C. radicalis strain M285 and the three putative C. radicalis strains ph1111, ph1113, and COP26-5 were similar to the white culture morphology of the hv-infected C. parasitica strain VSX. The white mycelium covered the whole Petri dish and no pigmentation or sporulation was observed. However, a light pinkish coloration of the mycelium in the outer half of the Petri dish was a striking characteristic that distinguished the putative C. radicalis strains from the hv-infected C. parasitica strain. This pinkish aspect was due to small droplets (0.10–0.30 mm in diameter) of a purple exudate on the hyphae, visible under the dissecting microscope. After exposure to light, the mycelium of M285 and of the three putative C. radicalis strains became yellow to orange within a few days. This orange pigmentation was similar to the hv-free C. parasitica strain M1275. But compared to the hv-free C. parasitica strain, less sporulation and a much more fluffy mycelium in the outer half of the Petri dish was

108 TABLE III.

MYCOLOGIA Colony growth of C. radicalis and C. parasitica strains on PDA 20 C a

Mean diameter (mm) C. radicalis strains M285 ph1111 ph1113 COP26-5 C. parasitica strains M1275 VSX a b

6 6 6 6

25 C Significance

b

a

Mean diameter (mm)

Significanceb

6 6 6 6

0.8 0.4 0.8 2.5

a b b b

54.7 65.7 65.7 64.4

0.7 0.3 0.7 1.2

a b b b

41.0 6 0.8 41.0 6 0.8

c c

44.9 6 1.9 48.0 6 1.3

c c

43.8 54.8 55.4 53.7

Mean colony diameter on PDA after four days 6 standard deviation, n 5 5. Strains with different lowercase letters have significantly different colony diameter at P , 0.01 (Mann-Whitney U test).

present. The area in the center, up to a half diameter of the Petri dish, was almost free of aerial mycelium. After four days of growth, colony diameters of the three putative C. radicalis strains ph1111, ph1113, and COP26-5 were significantly larger compared to all other strains. Although the difference was smaller, M285 diameters were still significantly larger compared to the C. parasitica strains. The differences in growth between the C. radicalis and C. parasitica strains were significant at 20 C and 25 C (TABLE III). Culture morphology on corn meal medium. On corn meal medium C. radicalis strain M285 and the three putative C. radicalis strains ph1111, ph1113, and COP26-5 showed an identical culture morphology. All four strains caused a color change of the medium from beige to purple (FIG. 1). They produced few but large pycnidia. Spores were extruded in long, thick masses. In contrast, the hv-free and hv-infected C. parasitica strains M1275 and VSX failed to produce any purple color in the corn meal medium (FIG. 1). M1275 produced numerous small pycnidia with small spore masses. The cultures of the hv-infected C. parasitica strain VSX produced similar looking pycnidia, but less than the hv-free strain M1275. Mating experiments. No perithecia were produced in the interspecific crosses between the putative C. radicalis and the C. parasitica strains. Perithecia of the putative C. radicalis strains were only observed in the pairings ph1111 3 COP26-5 and ph1113 3 COP26-5. As expected, mating of the C. parasitica tester strains resulted in abundant perithecia production when paired with each other, and no perithecia when paired with themselves (TABLE IV). The culture morphology of the 50 single ascospore progeny from the two successful crosses of the putative C. radicalis strains was identical to that of the parental strains.

Ascospore dimensions. We analyzed the ascospores obtained from the two putative C. radicalis and the three C. parasitica crosses. Both ‘‘species’’ had twocelled ascospores. The mean lengths and widths of the ascospores are shown in TABLE V. The ascospores from the putative C. radicalis crosses were significantly smaller than the ascospores from all three C. parasitica crosses. No significant differences were found between the ascospores of the two perithecia from the putative C. radicalis crosses, either in length or in width. Virulence tests. We compared the virulence of the three putative C. radicalis strains to C. radicalis strain M285, hv-free C. parasitica strain M1275 and hv-infected C. parasitica strain VSX on young chestnut trees. All strains except the hv-free C. parasitica strain M1275 exhibited a very low virulence (FIG. 2, TABLE VI). Two weeks after infection a small lesion was produced by all strains (FIG. 2). Subsequently, only the hv-free C. parasitica strain M1275 developed larger lesions, whereas the lesions of the other strains almost stopped growing. After three months, the mean lesion lengths produced by the latter strains were significantly larger than the negative controls but significantly smaller than the lesions produced by the hv-free C. parasitica strain M1275 (TABLE VI). Stromata were produced by all strains on all plants. However, only the hv-free C. parasitica strain M1275 produced spore tendrils. M1275 was also the only strain producing mycelial fans and causing tree mortality. None of the other strains caused tree mortality after one year (TABLE VI). From all lesions Cryphonectria sp. strains were reisolated, showing the same culture morphology on PDA as the initially inoculated strains. Mating-type identification by PCR. The presence of the HMG box in the MAT-2 idiomorph was deter-

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FIG. 1. Purple coloration of the corn meal medium by the C. radicalis strains. Cultures were incubated at 25 C in the dark for seven weeks. a. C. radicalis strain ph1111. b. C. radicalis strain ph1113. c. C. radicalis strain COP26-5. d. C. radicalis strain M285. e. Hv-free C. parasitica strain M1275. f. Hv-infected C. parasitica strain VSX.

110 TABLE IV.

MYCOLOGIA Perithecia production in mating experiments with C. radicalis and C. parasitica strains C. parasitica a

HMG box C. radicalis M285 ph1111 ph1113 COP 26-5 C. parasitica M1115 M1297

C. radicalis

M1297

M1115

n.d.b 1 1 2

n.d. 0/4 0/4 0/2

n.d. 0/4 0/4 0/2

1 2

4/4 0/4

0/4

COP 26-5 0/2c 1/4 1/4 0/2

ph1113

ph1111

0/2 0/4 0/4

0/2 0/4

a

Presence (1) or absence (2) of HMG box determined by PCR. Not determined. c Number of crosses with perithecia/total number of crosses. b

mined by PCR amplification. In the strains ph1111 and ph1113, a fragment with the same length as in C. parasitica strains (Hoegger et al 2000) was detected, indicating the presence of the HMG box. No fragment was amplified in strain COP26-5 (TABLE IV). The absence of the fragment suggests the absence of the MAT-encoded HMG box as in MAT-1 isolates of C. parasitica (Hoegger et al 2000). The PCR with the C. parasitica MAT idiomorphspecific primer pairs yielded inconclusive results. No PCR products corresponding to the sizes of the C. parasitica fragments were obtained from the three putative C. radicalis isolates. Under high stringent annealing conditions (66 C) the MAT-1 primers failed to amplify a fragment in the putative C. radicalis isolates. The MAT-2 primers weakly amplified a nonspecific 1 kb fragment in all putative C. radicalis isolates (not shown). Under less stringent conditions (56 C), the C. parasitica and the putative C. radicalis isolates yielded several fragments with both primer pairs which were not specific for mating-type but differed among the two ‘‘species’’ (not shown). Positive and negative controls in all PCR experiments were as expected. TABLE V.

Dimension of ascospores from C. radicalis and C. parasitica crosses Cross

C. radicalis strains ph1111 3 COP26-5 ph1113 3 COP26-5 C. parasitica strains M1115 3 M1297 M1115 3 MB117 M1297 3 MB85 a b

Hybridization with pMS5.1 and mtDNA from C. parasitica. The C. parasitica DNA fingerprinting probe pMS5.1 hybridized poorly to the digested DNA of the putative C. radicalis strains ph1111, ph1113 and COP26-5 (not shown). Only one band at 5.4 kb was observed in these strains. DNA fingerprints of Swiss C. parasitica strains typically have 7 to 12 bands (Hoegger et al 2000). Compared to C. parasitica DNA fingerprints, the signal of the single band from the putative C. radicalis strains was very faint. Hybridization of C. radicalis DNA with the purified C. parasitica mtDNA was also weak. In contrast with the single putative C. radicalis fingerprint band, the C. parasitica mtDNA probe hybridized to a number of mtDNA fragments from the putative C. radicalis isolates (FIG. 3). In strains ph1111 and ph1113 13 bands were detected and in strain COP26-5 12 bands (FIG. 3). The size of the fragments ranged from 2.5 kb to .20 kb. Strains ph1111 and ph1113 showed the same pattern. Between that pattern and that of COP26-5, 9 polymorphic and 8 identical bands were observed. The mtDNA patterns of the putative C. radicalis strains did not resemble the typical pattern

Mean length (mm)a

Significanceb (lengths)

Mean width (mm)a

Significanceb (widths)

6.3 6 0.6 6.7 6 0.6

a a

2.8 6 0.3 2.8 6 0.3

a a

9.2 6 0.5 8.2 6 0.6 9.0 6 0.6

b c b

4.4 6 0.3 3.5 6 0.3 3.8 6 0.4

b c d

Mean ascospore length or width 6 standard deviation, n 5 30 (from 1 perithecium per cross). Crosses with different lower case letters are significantly different for length or width at P , 0.01 (Mann-Whitney U test).

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FIG. 2. Canker development of C. radicalis and C. parasitica strains on 3-yr-old Castanea sativa plants. The mean lesion length (n 5 3) is shown. Bars indicate standard deviation. † indicates death of one tree.

of Swiss C. parasitica strains, as they had no bands in common (FIG. 3). DISCUSSION

The culture morphology on PDA of the isolates ph1111, ph1113 and COP26-5 provided the first piece of evidence that these isolates were not C. parasitica, but in fact C. radicalis isolates. All three isoTABLE VI.

lates showed a strong similarity to M285, a C. radicalis strain from the culture collection. The purely white culture morphology exhibited after incubation in the dark clearly distinguished them from the orange-pigmented cultures of hv-free C. parasitica strains. The most strikingly distinctive characteristic compared to the white, hv-infected C. parasitica strains was the production of purple droplets on the mycelium of the C. radicalis strains. These droplets seem to be

Virulence of C. radicalis in comparison to C. parasitica on three years old European chestnut trees Mean lesion length (mm)a

C. radicalis strains ph1111 ph1113 COP26-5 M285 C. parasitica strains M1275 (hv-free) VSX (hv-infected) Negative control

6 6 6 6

Significanceb

Tree mortality 3 months p.i.c

Tree mortality 12 months p.i.c

Mycelial fans 12 months p.i.d

11.5 11.5 7.6 13.2

a a a a

0/3 0/3 0/3 0/3

0/3 0/3 0/3 0/3

0/3 0/3 0/3 0/3

113.3 6 2.9 25.0 6 8.7 5.0 6 0.0

b a c

1e/3 0/3 0/3

3/3 0/3 0/3

3/3 0/3 0/3

31.7 41.7 33.3 30.0

Mean lesion length after three months 6 standard deviation, n 5 3. Strains with different lowercase letters have significantly different lesion lengths at P , 0.05 (Mann-Whitney U test). c Number of dead plants/total number of plants, p.i. 5 post infection. d Number of plants where mycelial fans were observed/total number of plants, p.i. 5 post infection. e Only distal parts of plant were dead. a

b

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FIG. 3. mtDNA restriction pattern of three C. radicalis strains and a representative C. parasitica strain. Southern blots of PstI-digested total DNA hybridized with DIG-labelled purified C. parasitica mtDNA and detected with the chemiluminescence method. Lane 1: C. parasitica strain M1265 from the WSL culture collection, lane 2, 3 and 4: C. radicalis strains ph1111, ph1113 and COP26-5. Lane on the left indicates size in Kb.

identical to the pigment first described by Shear and Stevens (1913) as ‘‘perilla purple’’ and later named endothine red by Sando (1919). We never observed such droplets in C. parasitica cultures. After exposure to light, C. radicalis strains were distinguishable from the white hv-free and the orange hv-infected C. parasitica strains by their fluffy yellow-orange cultures and ‘‘flat’’ center area. All C. radicalis strains showed larger colony diameters on PDA after 4 d compared to the C. parasitica strains. Chen et al (1996) also reported a higher growth rate for a C. radicalis strain compared to a C. parasitica strain. On corn meal medium, the two species could be distinguished from each other very easily. As described by Shear et al (1917) for C. radicalis, the strains ph1111, ph1113, COP26-5 and M285 showed a color change of the medium from light beige to purple. This characteristic was never observed with the C. parasitica strains. The lack of the purple pigment exudation by C. parasitica strains provides an important differential characteristic to C. radicalis, as pointed out earlier by Shear et al (1917) and Hawkins and Stevens (1917). Along with the culture morphology on PDA and corn meal medium, the ascospore dimensions further confirmed our classification of strains ph1111, ph1113 and COP26-5 to C. radicalis. Cryphonectria radicalis ascospores typically range from 6–10 3 3–4 mm, whereas C. parasitica ascospores range from 7– 11 3 3.5–5 mm (Roane et al 1986). In our measurements the C. radicalis ascospores were significantly smaller than the C. parasitica ascospores and their dimensions fit within the ranges from the literature. The results of the mating experiments and the HMG box PCR with the C. parasitica primers suggest that C. radicalis has a similar mating system as the closely related C. parasitica. Like several other filamentous ascomycetes, C. parasitica has a single mating-type locus containing one of two idiomorphs (Arie et al 1997, Coppin et al 1997, Marra and Milgroom 1999). Successful mating requires two strains of opposite mating-type, i.e., with different idiomorphs. In our experiments, mating between the C. radicalis strains was only successful in pairings where the HMG box was present in one strain but not in the other. Neither in the crosses where the HMG box was present in both strains nor in the self crosses were perithecia observed. The fact that only one of the four crosses of each of the two pairs ph1111 3 COP26-5 and ph1113 3 COP26-5 were successful suggests that the conditions used for the mating experiments were not ideal for C. radicalis. Strain M285, which never produced any perithecia, may also have lost its ability to mate during storage since 1954. More mating experiments with additional

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C. radicalis strains and more molecular markers are needed to better characterize the mating system. For such experiments, the strains characterized here could serve as a starting point to establish matingtype tester strains, with COP26-5 as a hypothetical MAT-1 and ph1111 as a hypothetical MAT-2 tester. The strong amplification of the HMG box in the C. radicalis strains ph1111 and ph1113 with the C. parasitica primers indicates a high similarity in this sequence among the two species as can be expected for a conserved motif (Arie et al 1997). Chen et al (1996) found almost no variation among the 18S ribosomal sequences of C. radicalis and C. parasitica. They concluded that within the genus Cryphonectria sp., these two species shared a common recent ancestor. However, the failure to amplify MAT specific fragments with the C. parasitica MAT idiomorph primers and the weak hybridization with the C. parasitica DNA fingerprinting probe pMS5.1 and with purified C. parasitica mtDNA showed that there are also considerable genetic differences between the two species. In C. parasitica isolates, probe pMS5.1 typically hybridizes to 6–17 restriction fragments (Milgroom et al 1992). Smaller numbers of fragments hybridizing to the fingerprinting probe were only observed in C. parasitica isolates from China (Milgroom et al 1996). In contrast, the probe pMS5.1 hybridized only to one fragment of the C. radicalis strains. The signal was very weak, although the same DNA quantities were loaded on the gel for the C. parasitica and C. radicalis strains. Considerable differences between C. radicalis and C. parasitica were also observed when the blots were hybridized with C. parasitica mtDNA. The patterns of the C. radicalis strains were very similar to each other. Again, hybridization was weak compared to C. parasitica. The weak hybridization is most probably due to low sequence similarity between the C. parasitica probes and the C. radicalis DNA and indicates genetic differences between C. radicalis and C. parasitica. In the virulence tests on chestnut trees, the C. radicalis strains exhibited very low virulence, comparable to highly hypovirulent hv-infected C. parasitica strains. The C. radicalis strains did not produce mycelial fans and did not cause any tree mortality. These results are in good agreement with the descriptions that C. radicalis is ‘‘. . . not an active parasite.’’ (Anderson and Anderson 1912) and is ‘‘. . . almost purely saprophytic.’’ (Shear et al 1917). Anderson and Anderson (1912) based their statement on virulence tests and the observation that this fungus never killed a tree of the thousands examined in regions in western Pennsylvania where it was extremely common on dead stumps and logs. They also never observed mycelial fans in C. radicalis lesions. Similarily, Shear et

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al (1917) never found any evidence for active parasitism in over one-thousand inoculations with C. radicalis from Europe and North America on Castanea sprouts. Our results suggest that C. radicalis and C. parasitica still exist sympatrically, with the latter species being much more frequent. One C. radicalis strain in this study was isolated in the post-epidemic area of C. parasitica in southern Switzerland with a high incidence of chestnut blight. The two others were found in an area where C. parasitica arrived only recently. Our findings, although not providing any conclusive evidence, support the displacement rather than the hybridization hypothesis. This is also supported by the fact that C. parasitica has the potential to occupy niches that are probably used by the saprotrophic C. radicalis: i.) Prospero et al (1998) found considerable saprotrophic activity of C. parasitica on dead chestnut stems and ii.) C. parasitica was found as an endophyte in chestnut trees together with saprotrophic and weakly pathogenic fungi (Bissegger and Sieber 1994). There may be other causes for the low frequency of C. radicalis isolations. As C. parasitica is only a weak pathogen on the resistant Asian chestnuts, it is not expected to displace C. radicalis in Asia. However, no C. radicalis strains were isolated in recent extended chestnut sampling campaigns in China and Japan (M. G. Milgroom pers comm). Perhaps C. radicalis has never been a very abundant species, or the unspectacular C. radicalis has not been displaced completely by the threatening C. parasitica in nature, but only in the attention of the scientific community. After the introduction of C. parasitica in North America and Europe, the interest of researchers focused on cankers on living or dying chestnut trees rather than on dead chestnut trees. A bias cannot be excluded as probably all isolations were performed in the search of C. parasitica exclusively. A better knowledge of the biology of C. radicalis and its niches should result in higher isolation efficiency. A systematic sampling of dead chestnut wood, i.e., stumps, logs or branches, could bring more light into the darkness of the whereabouts of C. radicalis. ACKNOWLEDGMENTS

We are very grateful to K. P. Lawrenz for his excellent assistance with the chestnut plants and the greenhouse facilities. We also thank D. M. Balmer for her help with the ascospore measurements. We greatly appreciate the helpful comments and criticisms made by the reviewers. LITERATURE CITED

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