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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In an attempt to isolate the ascomycete Cryphonectria 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, mating-type, saprotroph
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, 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 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 virulence (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.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). 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
).
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). 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 x COP26-5 and ph1113 x 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 x COP26-5 and ph1113 x COP26-5 was chosen randomly for the analysis of ascospores. Ascospores from the C. parasitica crosses M1115 x M1297, M1115 x MB117, and M1297 x 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 1000x 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 presence 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Ascospore dimensions – We analyzed the ascospores obtained from the two putative C. radicalis and the three C. parasitica crosses. Both "species" had two-celled 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.
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) 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 idiomorph-specific 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 non-specific 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.
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 of Swiss C. parasitica strains, as they had no bands in common (Fig. 3 ).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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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 x 3–4 µm, whereas C. parasitica ascospores range from 7–11 x 3.5–5 µm (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 x COP26-5 and ph1113 x 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 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 mating-type 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 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present adress: Institut für Forstbotanik, Georg-Austust-Universität Göttingen, Büsgenweg 2, D-37077 Götingen, Germany. 
Accepted for publication June 14, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Anagnostakis SL., 1988 Cryphonectria parasitica, cause of chestnut blight Adv Plant Pathol 6:123-136
Anagnostakis SL., 1995 The pathogens and pests of chestnuts Adv Bot Res 21:125-145
Anderson PJ, Anderson HW., 1912 The chestnut blight fungus and a related saprophyte Phytopathology 2:204-210
Arie T, Christiansen SK, Yoder OC, Turgeon BG., 1997 Efficient cloning of ascomycete mating-type genes by PCR amplification of the conserved MAT HMG box Fungal Genet Biol 21:118-130[Medline]
Barr ME., 1978 The Diaporthales of North America with emphasis on Gnomonia and its segregates. Mycol Mem 7 Lehre, Germany: J. Cramer. 232 p
Bissegger M, Heiniger U., 1991 Chestnut blight (Cryphonectria parasitica) north of the Swiss alps Eur J For Path 21:250-252
Bissegger M, Rigling D, Heiniger U., 1997 Population structure and disease development of Cryphonectria parasitica in European chestnut forests in the presence of natural hypovirulence Phytopathology 87:50-59[Medline]
Bissegger M, Sieber TN., 1994 Assemblages of endophytic fungi in coppice shoots of Castanea sativa Mycologia 86:648-655
Boerboom JHA, Maas PWT., 1970 Canker of Eucalyptus grandis and E. saligna in Surinam caused by Endothia havanensis Turrialba 20:94-99
Brasier CM., 1983 The future of Dutch elm disease in Europe. In: Burdekin DA, ed. Research on Dutch elm disease in Europe. London, UK: HMSO Forestry Commission Bulletin 60:96-104
Brasier CM., 1991 Ophiostoma novo-ulmi sp. nov., causative agent of current Dutch elm disease pandemics Mycopathologia 115:151-161
Chen B, Chen C-H, Bowman BH, Nuss DL., 1996 Phenotypic changes associated with wild-type and mutant hypovirus RNA transfection of plant pathogenic fungi phylogenetically related to Cryphonectria parasitica Phytopathology 86:301-310
Choi GH, Nuss DL., 1992 Hypovirulence of chestnut blight fungus conferred by an infectious viral cDNA Science 257:800-803
Coppin E, Debuchy R, Arnaise S, Picard M., 1997 Mating-types and sexual development in filamentous ascomycetes Microbiol Mol Biol R 61:411-428
Day PR, Dodds JA, Elliston JE, Jaynes RA, Anagnostakis SL., 1977 Double-stranded RNA in Endothia parasitica Phytopathology 84:528-534
Elliston JE., 1982 Hypovirulence Adv Plant Pathol 1:1-33
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Hodges CS., 1980 The taxonomy of Diaporthe cubensis Mycologia 72:542-548
Hodges CS., Alfenas AC, Ferreira FA., 1986 The conspecificity of Cryphonectria cubensis and Endothia eugeniae Mycologia 78:334-350
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Myburg H, Wingfield BD, Wingfield MJ., 1999 Phylogeny of Cryphonectria cubensis and allied species inferred from DNA analysis Mycologia 91:243-250
Petri L., 1917 [Studies on ink disease of chestnut.] Extracted from Annali del R. Istituto superiore forestale nazionale, Volume Secondo Florence, Italy: Laboratorio di Patologia e Fisiologia del R. Istituto superiore forestale di Firenze. 181 p. [In Italian.]
Prospero S, Conedera M, Heiniger U, Rigling D., 1998 [Survival and sporulation of the chestnut blight fungus Cryphonectria parasitica on cut and stacked Castanea sativa stems.] Monti e Boschi 3/4:44–50. [In Italian.]
Rigling D., 1995 Isolation and characterisation of Cryphonectria parasitica mutants that mimic a specific effect of hypovirulence-associated dsRNA on laccase activity Can J Bot 73:1655-1661
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Torsello ML, Davis DD, Nash BL., 1994 Incidence of Cryphonectria parasitica cankers on scarlet oak (Quercus coccinea) in Pennsylvania Plant Dis 78:313-315
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indicates death of one tree