Mycologia. 79(5), 1987, pp. 707-720. 1987, by The New York Botanical Garden, Bronx, NY 10458 ON THE CONSPECIFICITY OF ENDOTHIA EUGENIAE AND CRYPHONECTRIA CUBENSIS J. A. MICALES,1 R. J. STIPES Department of Plant Pathologv, Physiology and Weed Science. Virginia Tech, Blacksburg, Virginia 24061 AND M. R. BONDE Foreign Plant Disease and Weed Science Research Unit, U.S. Department of Agriculture-Agricultural Research Service, Building 1301 Fort Detrick. Frederick, Maryland 21701 ABSTRACT Isolates of Endothia eugniae, a pathogen of clove (Syzygium aromaticum), and Cryphonectria cubensis, a pathogen of eucalyptus (Eucalyptus spp.), were examined with chemotaxonomic techniques, including thin-layer chromatography of fungal pigments and isozyme analysis. The bisanthraquinone pigments skyrin and oxyskyrin were isolated from mycelial extracts of each species. Isolates of E. eugeniae and C. cubensis could not be differentiated by polyacrylamide gel electrophoresis of buffer- soluble proteins. The organisms also shared alleles at 16 presumed genetic loci, as detected by isozyme analysis. This study confirms the conspecificity of E. eugeniae and C. cubensis. Key Words: Endothia eugeniae, Cryphonectria cubensis, clove canker, eucalyptus canker, chemotax- onomy, isozyme analysis, fungal pigments, bisanthraquinones. Cryphonectria cubensis (Bruner) Hodges, the ma and innate pycnidia on clove. Cross-inocu- causal agent ofeucalyptus canker. is an extremely lation studies have shown that both fungi will important pathogen of Eucalyptus spp. in the colonize clove and eucalyptus: the morphology tropical and subtropical regions of North and of the stroma is dependent on the host. The con- South America, Africa and the Pacific Islands (1, specificity of these organisms was suggested on 19). Eucalypts, which serve as a major source of the basis of these cross-inoculation experiments, timber and fuel in these regions, are rapidly in- cultural characteristics. and the electrophoretic fected and colonized by the fungus, even when patterns of buffer-soluble mycelial proteins and introduced to remote areas where they have not specific enzymes from four isolates (21). been previously planted. The source of inoculum Additional chemotaxonomic procedures can is uncertain. Hodges et al. (21) have concluded be utilized to study fungal relationships when that C. cubensis is conspecific with Endothia eu- morphological distinctions are unclear. One such geniae (Nutman & Roberts) Reid & Booth, an technique is isozyme analysis. This procedure, opportunistic pathogen of clove, Syzygium aro- as performed with starch gel electrophoresis, has maticum (L.) Men. & Perry (31, 32). The clove, been used to determine taxonomic and genetic which is distributed throughout the tropics, is relationships within a number of fungal genera, therefore serving as the source of inoculum for including Peronosclerospora (8), Agaricus (25, 26, eucalyptus canker. 35, 36) . Puccinia (10, 11) and Phytophthora (40). The morphologies of C. cubensis and E. eu- Enzymes which are coded by different alleles or geniae are quite dissimilar on their respective separate genetic loci frequently possess different hosts. Cryphonectria cubensis produces little or electrophoretic mobilities. Such differences are no stromal tissue and superficial pycnidia on eu- due to variations in the amino acid content of calyptus, while E. eugeniae forms a limited stro- the molecule, which in turn is dependent on the sequence of nucleotides in the DNA. Crossing experiments may be required to confirm genetic 1 Current address: U.S. Department of Agriculture, Forest Service, Forest Products Laboratory, One Gif- interpretation; of electrophoretic data, but cer- ford Pinchot Dr., Madison, Wisconsin 53705. rain banding patterns are readily recognizable 707 708 MYCOLOGIA from comparable studies in human and animal Pigment extraction and thin-layer chromatog- genetics (17). raphy. - Pigment extraction and thin-layer chro- Initial studies have used electrophoresis to ex- matography (TLC) were conducted as described amine the relationship of E. eugeniae and C. by Roane and Stipes (34) with some modifica- cubensis. Alfenas et al. (2) reported that banding tions. Isolates were grown on autoclaved white patterns of the enzymes esterase, peroxidase, cornmeal (34) for 14-32 da at 25 C. Pigmented hexokinase, and phosphoglucomutase varied areas of the medium were excised and extracted among five isolates of C. cubensis and were re- in absolute ethanol. The pigment extracts were lated to differences in geographic origin and vir- then decanted and stored at 4 C. Nonfluorescent ulence of the pathogen. Hodges et al. (21) deter- silica gel chromatography sheets (Eastman mined that two isolates of C. cubensis and two Chromagram 6061) were spotted with 30-70 µl of E. eugeniae displayed identical banding pat- of pigment extract. Purified solutions of skyrin terns for buffer-soluble mycelial proteins and the and oxyskyrin (1 × 10-2 M) were used as stan- enzymes peroxidase, glutamate oxaloacetate dards. Chromatography was conducted for ap- transaminase, and lactate dehydrogenase; some- proximately 20 min at ambient temperature (27 what more variation was detected for esterase. C). The positions and colors of spots were re- Another important chemotaxonomic tool is corded under fluorescent and ultraviolet light (254 the identification of fungal pigments. Closely re- nm). and Rf values were determined. lated organisms frequently produce identical pig- Polyacrylamide gel electrophoresis of buffer-sol- ment profiles. Isolates of C. cubensis and E. eu- uble proteins. -Polyacrylamide gel electropho- geniae typically form brilliant orange pigments resis (PAGE) was used to determine the banding in culture (21): the chemical composition of these patterns of buffer-soluble mycelial proteins of all pigments is unknown. Other species of Endothia isolates listed in TABLE I. Protein samples were and Cryphonectria are known to produce the bis- prepared from cultures grown in 0.5% glucose- anthraquinone pigments skyrin, oxyskyrin, and 0.1% yeast extract broth, pH 5.7 (38) for 8 da at rugulosin (34). These compounds have not been 25 C. Mycelia were harvested by vacuum filtra- reported in E. eugeniae or C. cubensis. tion and lyophilized overnight. The lyophilized The objective of this study was to clarify fur- mycelium was crushed into a fine powder in a ther the relationship of C. cubensis and E. eu- chilled mortar and pestle and stored at 0 C over geniae using isozyme analysis for fifteen addi- desiccant. Protein samples were prepared by sus- tional enzymes, general protein patterns, and pending 60 mg lyophilized mycelium/ml 0.1 M pigment identification. Eighteen isolates were Tris-Cl + 10% glycerol overnight at 4 C. The used in this study, four of which were used pre- suspension was centrifuged and further clarified viously by Hodges et al. (21). Four isolates of by filtration through a Swinney filter fitted with Cryphonectria havanessis (Bruner) Barr, a fun- a penicillin sensitivity disc (Schleicher and Schuell gus which also colonizes Eucalyptus spp. (13), Inc., No. 740-E). The protein concentration of were included in this study to determine whether each sample was determined by the Coomassie they belong to a separate species. A preliminary blue protein assay (9) using known concentra- account of this work has been reported (30). tions of BioRad Protein Standard II, a prepa- ration of bovine serum albumin, for the standard MATERIALS AND METHODS curve. The volume of sample equivalent to 30 Maintenance of fungal cultures and preparation µg protein was loaded onto the gel. Discontin- of inoculum -All cultures were stored at 4 C on uous, nondissociating vertical slab gel PAGE was 1% glucose-0.2% yeast extract agar slants (38). conducted as described by Hames (16) using a Five-mm mycelial plugs taken from the margins Tris-glycine buffer system. Two separate extrac- of 7- to 20-da-old cultures grown on 0.5% glu- tions of the lyophilized mycelium were analyzed. cose-0.1% yeast extract agar (38) at 25 C were The banding pattern of each sample was delin- used as inoculum. All isolates were monoconidi- eated with a densitometer; densitometric trac- al clones. The isolate numbers, sources, dates of ings, visual observations and manual measure- collection and hosts are listed in TABLE I. The ments were used to produce banding diagrams isolates previously used (21) are H184, H185. of each gel. Bands separated by 1 mm or more H187, and H188. were resolved as individual proteins. Simple MICALES ET AL.: ENDOTHIA 709 TABLE I ISOLATE NUMBERS, SOURCES, DATES OF COLLECTION AND HOSTS OF ENDOTHIA AND CRYPHONECTRIA SPECIES USED IN THIS STUDY Collec- Isolate Source of tion Species number collection date Host Cryphonectria coccolobii E157V Fort Lauderdale, FL 10/82 Coccoloba uvifera (L.) (Vizioli) Jacq. Micales & Stipes Cryphonectria cubensis H91 Lihue-Koloa Forest 3/80 Eucalyptus saligna Sm. (Bruner) Hodges Reserve, HI H137 Botanical Garden, 8/81 Eucalyptus sp. Hong Kong H151 Wynaud, Calicut, In- 11/82 Eucalyptus grandis Hill dia ex Maid H154 Uverito, Venezuela 1/83 Eucalyptus grandis H170 Tampa Bay, FL 3/82 Eucalypt us camaldu- lensis Dehn. H175 IMI279614 Mangombe, Camer- 4/83 Eucalyptus urophylla S. oon T. Blake H182 IMI268904 Jarrahdale, Australia 4/82 Eucalyptus marginata Donn ex Sm. H187 Aracruz, E. S., Brazil 12/83 Eucalyptus alba Reinw. ex Blume H188 Ceplac Itabuna, Bra- 12/83 Eucalyptus grandis zil Cryphonectria havanensis E40 CBSS05.63 Loandijli, Congo 1960 Eucalyptus