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Department of Plant Pathology, Iowa State University, Ames, Iowa 50010
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODS AND MATERIALSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Ceratocystis fimbriata is a widely distributed, plant pathogenic fungus that causes wilts and cankers on many woody hosts. Earlier phylogenetic analyses of DNA sequences revealed three geographic clades within the C. fimbriata complex that are centered respectively in North America, Latin America and Asia. This study looked for cryptic species within the North American clade. The internal transcribed spacer regions (ITS) of the rDNA were sequenced, and phylogenetic analysis indicated that most isolates from the North American clade group into four host-associated lineages, referred to as the aspen, hickory, oak and cherry lineages, which were isolated primarily from wounds or diseased trees of Populus, Carya, Quercus and Prunus, respectively. A single isolate collected from P. serotina in Wisconsin had a unique ITS sequence. Allozyme electromorphs also were highly polymorphic within the North American clade, and the inferred phylogenies from these data were congruent with the ITS-rDNA analyses. In pairing experiments isolates from the aspen, hickory, oak and cherry lineages were interfertile only with other isolates from their respective lineages. Inoculation experiments with isolates of the four host-associated groupings showed strong host specialization by isolates from the aspen and hickory lineages on Populus tremuloides and Carya illinoensis, respectively, but isolates from the oak and cherry lineages did not consistently reveal host specialization. Morphological features distinguish isolates in the North American clade from those of the Latin American clade (including C. fimbriata sensu stricto). Based on the phylogenetic evidence, interfertility, host specialization and morphology, the oak and cherry lineages are recognized as the earlier described C. variospora, the poplar lineage as C. populicola sp. nov., and the hickory lineage as C. caryae sp. nov. A new species associated with the bark beetle Scolytus quadrispinosus on Carya is closely related to C. caryae and is described as C. smalleyi.
Key words: Microascales, Scolytus quadrispinosus, speciation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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). Hosts of C. fimbriata include Eucalyptus spp., Mangifera indica (mango), Theobroma cacao (cacao), Coffea arabica (coffee), Hevea brasiliensis (rubber tree), Platanus spp. (sycamore or plane tree), Prunus spp. (almond and other stone fruits) and Populus spp. (aspen and other poplars). Nonwoody hosts include Colocasia esculenta (taro) and Ipomoea batatas (sweet potato), from which the species originally was described (Halsted 1890). The geographic range and genetic diversity of C. fimbriata is similarly impressive, although most of the diversity in the species is found in the Americas (Baker et al 2003, Barnes et al 2001, CABI 2001, Harrington 2000, Steimel et al 2004). Ceratocystis albifundus, a closely related species, is native to Africa (Roux et al 2000, Wingfield et al 1996).
Webster and Butler (1967) concluded that in-terfertility and lack of morphological differences precluded recognition of additional species within C. fimbriata, but sequences of the internal transcribed spacer region (ITS) of the nuclear ribosomal DNA and other genetic analyses show that there are several subgroups or clades within C. fimbriata (CABI 2001, Harrington 2000). One of these major clades seems to be centered in Latin America, where C. fimbriata infects numerous native and nonnative hosts. Two of the members of the Latin American clade, the sweet potato pathogen C. fimbriata sensu stricto and the sycamore pathogen C. platani (Walter) Engelbrecht & Harrington, are found in eastern North America and elsewhere (Baker et al 2003, Engelbrecht et al 2004, Engelbrecht and Harrington 2005). Other members of the Latin American clade include the cacao pathogen C. cacaofunesta Engelbrecht & Harrington, a Xanthosoma pathogen in the Caribbean, and various Central and South American populations (Baker et al 2003, Engelbrecht and Harrington 2005, Harrington 2000, Marin et al 2003, Thorpe et al 2005). A second clade occurs on fig (Ficus carica) and taro in Japan and the Pacific (Harrington 2000, Thorpe et al 2005), and the recently described C. pirilliformis I. Barnes & M. J. Wingf. from Australia (Barnes et al 2003) and Africa (Roux et al 2004) and C. polychroma M. van Wyk, M. J. Wingf. & E.C.Y. Liew from Indonesia (van Wyk et al 2004) are also in the Asian clade based on rDNA sequence analysis (Harrington unpublished, Thorpe et al 2005). Members of a third clade infect Populus spp., Carya spp., Quercus spp., and Prunus spp. in North America (Harrington 2000). A morphologically similar species, C. variospora (R.W. Davidson) C. Moreau, was described from Quercus in the eastern USA (Davidson 1944, Hunt 1956), but some have considered this species and Rostrella coffaea to be synonyms of C. fimbriata (Upadhyay 1981, Webster and Butler 1967, Zimmermann 1900).
This study applies analyses of allozymes, DNA sequences, interfertility tests, host specialization and morphology to identify cryptic species among isolates of the C. fimbriata complex from North America using the phylogenetic species concept (Harrington and Rizzo 1999). This concept recognizes species as populations or lineages with unique phenotypic characters, such as morphology and host specialization.
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METHODS AND MATERIALS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Further attempts were made during summer 2001 to collect isolates of Ceratocystis spp. from Iowa. We examined and obtained isolates from recently wounded Quercus spp. at the Yellow River State Forest in northeastern Iowa. An additional isolate was obtained from a bleeding canker on the European species Q. robur in an experimental planting at the Iowa State University (ISU) research farm at Rhodes. At another site south of Boone, samples were taken from a C. ovata (shagbark hickory) tree with crown dieback and a C. cordiformis tree with recent attacks by a wood-boring beetle (Agrilus sp.). Ceratocystis species were not isolated from either hickory tree initially, but when the same trees were resampled 2 wk later, the wounded bark was found to be extensively colonized and isolates were recovered. Isolates also were obtained from C. cordiformis trees in a forest stand in Coggan, in northeastern Iowa, where an outbreak of the hickory bark beetle (Scolytus quadrispinosus Say) was ongoing; isolates were obtained from beetle galleries and from discolored wood associated with beetle attacks. Additional isolations were obtained from logging wounds on C. cordiformis and P. serotina (black cherry) trees in the same stand. Another outbreak of S. quadrispinosus was located near Cambria, in southern Iowa, and an isolate was obtained June 2002 from a C. ovata tree infested with the beetle.
In 2002 trees were wounded artificially at four locations in Iowa. Wounds were created at breast height (1.4 m) on the main stem by removing a 6 x 6 cm patch of bark with a flame-sterilized hatchet, then bruising the bark with the back of the hatchet along two sides of the wound to loosen the bark. Each site was revisited approximately 10 d after wounding, and samples were taken from the wound face and from bark surrounding the wound. Isolations were made with the carrot disk method of Moller and DeVay (1968). At the first site, north of Ogden, one tree from each of six species (P. serotina, Q. macrocarpa [bur oak], C. ovata, Celtis occidentalis [hackberry], Populus grandidentata [big-tooth aspen] and Tilia americana [basswood]) was wounded in mid-June. Isolates of a Ceratocystis sp. were recovered from all but the Carya ovata and Celtis occidentalis trees. At a second site near Lucas one tree each of five species was wounded at the end of June, and isolates of Ceratocystis were recovered from Q. macrocarpa, Carya ovata and Celtis occidentalis, but no Ceratocystis was recovered from Prunus serotina and Ulmus rubra (red elm). The third site, near Ames, contained both upland and bottomland species, and Prunus serotina, Q. macrocarpa, Carya ovata, Ostrya virginiana (ironwood), Juglans nigra (black walnut), Gleditsia triacanthos (honeylocust), Fraxinus pennsylvanica (green ash), and Populus deltoides (cottonwood) were wounded in mid-July, but Ceratocystis was isolated only from O. virginiana. A fourth site north of Boone was visited in August, wounds were made on various hardwood species, and isolations were attempted 10 d later, but Ceratocystis was not recovered.
Representative isolates from the above collections were selected for DNA sequence and allozyme analyses, inoculation studies and mating experiments (TABLE I). Additional isolates representing the Latin American and Asian clades of C. fimbriata were used for allozyme analysis (TABLE II). Isolates C904, C1062 (CMW 4081), C1083 (CMW 4110) and C1360 (JC 6885) of the African species C. albifundus were supplied by J. Roux and used as an outgroup taxon in DNA sequence and allozyme analyses.
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. The PCR primers, reagents and cycling conditions were as previously described (Harrington et al 2001). Sequencing was performed at the ISU DNA Sequencing and Synthesis Facility using the PCR primers. Sequences were aligned manually by adding gaps, and parsimony analysis was performed with PAUP 4.0b10 (Swofford 2002). Ceratocystis albifundus was used as outgroup taxon, and the ingroup was considered to be monophyletic. Of 714 total aligned characters, including gaps, 217 were ambiguously aligned and excluded from the analysis, 110 remaining sites were variable, and of these, 32 were parsimony informative. Except for gaps between ingroup and outgroup, there were only single-base gaps, which were treated as a fifth character. A maximum parsimony heuristic search was performed with all characters having equal weight. Starting trees were obtained through stepwise addition, and tree-bisection-reconnection was used. Bootstrap analysis with 2000 replications of heuristic searches was used to determine support for internal branches.
Allozyme analysis.— –
One hundred ten isolates of C. fimbriata, including representatives from all three geographic clades, and three isolates of the outgroup taxon, C. albifundus, were tested for allozyme variation (TABLES I
and II). Cultures were grown 14 d in 125 mL Erlenmeyer flasks containing 30 mL of MYB at room temperature. Enzymes were extracted from mycelial mats onto paper wicks and stored at –80 C until electrophoresis (Zambino and Harrington 1992), which was performed on 12% starch gels (Harrington et al 1996). Buffers and electrophoresis conditions are shown (TABLE III). With few exceptions (TABLE I), enzymes were extracted and tested for allozyme activity at least twice. Isolates C1418 and C1410 were included in each gel as reference isolates. For each allozyme, electromorphs were designated by numbers in order of decreasing anodal migration, and the electromorphs were considered to be alleles. These data were used to develop an uncorrected "p" distance matrix, and phenograms were generated with neighbor joining and UPGMA (unweighted pair group method with arithmetic mean) with PAUP 4.0b10. The neighbor joining tree was rooted to C. albifundus.
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, Witthuhn et al 2000b). Thus MAT-2 (self-fertile) and MAT-1 (self-sterile) progeny are recovered from selfings of MAT-2 strains. Most field isolates are MAT-2, and pairing experiments are hampered in that MAT-2 strains are usually self-fertile. Some self-fertile MAT-2 strains produce mutant sectors that lack the ability to produce protoperithecia and perithecia, and these MAT-2 sectors are self-sterile and function poorly as females in crosses (Engelbrecht and Harrington 2005, Harrington and McNew 1997, Harrington et al 2002).
MAT-2 tester strains that were used in pairings were obtained by subculturing sterile sectors that arose spontaneously from fertile, selfing isolates. The presence of the MAT-2 gene in these tester strains was confirmed with PCR (Witthuhn et al 2000b). All MAT-2 testers were self-sterile, except isolates C1959 and C1483, which produced deformed perithecia that could be distinguished readily from normal perithecia produced in successful pairings with other strains. The MAT-1 testers were obtained by recovering single ascospore progeny from self-fertile isolates.
The MAT-1 testers were used as recipients (females), and MAT-2 testers served as donors (males). MAT-2 testers also were paired with each other, but no combination resulted in an interfertile cross. Both male and female cultures were grown on MYEA plates at room temperature. After 5 d male testers were flooded with 15 mL sterile, deionized water and scraped with a sterile spatula to suspend spores and mycelial fragments. Female testers were 5 d old colonies, which received 1 mL of a conidial suspension from the male tester at the edge of the expanding female colony. Spermatized cultures were allowed to grow 7 d at room temperature (24 C) before initial evaluation with a dissecting microscope. Ambiguous reactions were re-inspected after an additional 4 d. When perithecia were found they were examined at 400x or 1000x with a compound microscope to see whether normal ascospores had formed.
Four host cross-inoculations.— – We first tested whether isolates from the four main, host-associated lineages exhibited host specialization to four hosts: Populus tremuloides, Q. rubra (red oak), Prunus serotina and Carya illinoensis (pecan). Nine inoculation treatments, consisting of two isolates from each of the four major lineages and a control, were applied to each host.
Lateral roots of P. tremuloides were dug from a clone near Johnston, Iowa, and were maintained under mist until root sprouts appeared (Benson and Schwalbach 1970). Sprouts were harvested when they were 3–6 cm tall, dipped in 1000 ppm IBA (indolebutyric acid) and placed in peat pellets to root (Snow 1938). The plants were inoculated at 5–6 mo after rooting. Carya illinoensis seed (Sheffield’s Seed Company, Locke, New York) were cold-stratified for 4 mo then germinated in Kimpak paper (Kimberly-Clark Corporation, Irving, Texas) in a growth chamber set to a 16 h day (30 C)/8 hour night (20 C) cycle. After 14 d the germinated seeds were transferred to 4-inch pots in the greenhouse and inoculated after 3–4 mo. Seed of Q. rubra (Sheffield’s Seed Co.) were cold-stratified for 6 wk, planted directly to 4-inch pots in the greenhouse, and the seedlings were inoculated after 2.5–3 mo. Half-sib seedlings of Prunus serotina were dug from under a tree near Ames, Iowa, planted in 4-inch pots, and grown 3–4 mo before inoculation.
Before inoculation, plants were grown on greenhouse benches in a mixture of 50% perlite, 50% Peat-lite mix (Fafard, Aawam, Massachusetts). All plants received a slow release fertilizer (Osmocote 19-6-12) at the time of sowing and biweekly feedings with liquid fertilizer (Miracle-Gro EXCEL 21-5-20). Artificial light was used to maintain a 16 h day. Plants were transferred to growth chambers 7 days before inoculation, where they were maintained on a 16/8 h light/dark cycle at 25 C. Each experiment (a single host) was performed with a randomized complete block design with six blocks, and six replications per treatment. The C. illinoensis seedlings were inoculated on greenhouse benches using the same experimental design. All experiments were repeated.
Inoculum was prepared from 7 d old cultures on MYEA plates (Baker et al 2003). Sterile water was added to the plates, the colonies were scraped and the suspension was filtered through four layers of sterile cheesecloth. Inoculum primarily comprised endoconidia, which were adjusted to 1.0 x 106 spores per mL with a hemacytometer. Control inoculum was prepared by flooding sterile MYEA plates with water, scraping and filtering the resulting solution through sterile cheesecloth.
Plants were prepared for inoculation by making a downward-slanting horizontal cut through the bark and into the xylem of each stem with a sterile razor blade. Immediately after wounding 0.2 mL of inoculum was introduced into the wound with a syringe (21-gauge needle), and each wound was wrapped with parafilm. Plants were watered daily, and any mortality occurring before the end of the experiment was recorded and the plants harvested. Populus tremuloides and C. illinoensis plants were harvested at 40 d, while Q. rubra and Prunus serotina plants were harvested after 30 d. At harvest a shallow cut was made along the stem above and below the inoculation point, without cutting into the xylem, and the length of cankers (phloem necrosis) was recorded. A slightly deeper cut then was made, exposing the xylem tissue so that the total length of xylem discoloration could be measured. The fungus was re-isolated from inoculated plants by placing discolored tissue between carrot slices (Moller and DeVay 1968).
Length of xylem discoloration was analyzed by host plant, source of inoculum, experiment (within host), host x source interaction and source x experiment (within host) interaction using a multi-factorial analysis of variance (ANOVA) with controls excluded. For each inoculated host ANOVA indicated significant variation (P = 0.05) due to the two experiments (within host), so each experiment then was analyzed separately with one-way ANOVA. When the ANOVA indicated significant variation among isolates (without controls), then Duncan’s multiple range test was used to separate means, including the controls. Statistics were performed with SAS statistical software (SAS Institute, Cary, North Carolina).
Prunus virginiana and Quercus macrocarpa cross-inoculations.— – An additional experiment was performed in which two hosts, P. virginiana (common chokecherry) and Q. macrocarpa, were inoculated with isolates from the oak and cherry lineages: two isolates from the four-host inoculation experiment (C1009 from Quercus, and C821 from Prunus), two additional isolates from the oak lineage, six Iowa isolates from the cherry lineage, and isolate C1965 from P. serotina in Wisconsin.
One-year-old bareroot seedlings obtained from the Iowa State Forest Nursery were grown in the greenhouse 6 wk after bud break in 6-inch pots in greenhouse soil amended with Osmocote slow-release fertilizer. Inoculations were performed in the greenhouse as described above using a completely random design with nine replications per treatment. Plants were harvested 37 d after inoculation. Length of xylem discoloration for each inoculated host was analyzed separately as described above.
Host range of hickory isolates.— – Four species of Carya and two species from the related genus Juglans were inoculated with isolates from the hickory lineage. Bareroot seedlings (2 y old) of C. cordiformis, C. ovata, C. illinoensis, J. nigra (black walnut), and J. cineria (butternut) were grown in 2-gallon pots as described above and inoculated in a growth chamber with four isolates of C. fimbriata collected from C. cordiformis. Inoculations were performed 67 d after planting (50 d after first flush for Juglans spp. and 40 d after first flush for Carya spp.). The experiment used a completely randomized design, with five replicates per treatment. Plants were harvested 6 wk after inoculation and evaluated for linear extent of xylem discoloration. The experiment was repeated with the same hosts and procedures, except that Carya plants were planted 7 d before the Juglans species in an effort to ensure that plants would be in a similar growth stage at the time of inoculation. Two-way ANOVA and Duncan’s multiple range tests were used as described above.
Host range of aspen isolates.— – Two inoculation experiments were performed in a growth chamber to test the susceptibility of five Populus species to four isolates from the aspen lineage. In the first experiment, P. tremuloides, P. nigra (European black poplar), P. balsamifera (balsam poplar), and P. trichocarpa (black cottonwood) were inoculated. The P. tremuloides plants were generated from root sprouts and inoculated 3 mo later. All other plants were 3 mo old rooted cuttings from dormant twigs. The P. tremuloides sprouts were grown in 4-inch pots, while the other hosts were grown in 6-inch pots. Fertilization, care and inoculation procedures were as described above. A completely randomized design was used, with five replicates per treatment.
The experiment was repeated with an additional host, P. deltoides (eastern cottonwood). The P. tremuloides plants for the second experiment were 9 mo old; the other hosts were 4–5 mo old and were generated by rooting greenwood cuttings. The first experiment was harvested at 7 wk, and the second experiment was harvested after 5 wk. For each experiment, a two-way ANOVA was used to analyze the effects of isolate, host and isolate x host interaction. For each experiment there was no significant interaction between host and isolate, so the results from the four isolates were combined and one-way ANOVA was performed to compare the response of each host. Duncan’s multiple range test was used to separate means in each experiment.
Morphology.— – Isolates were grown on MYEA at room temperature (approximately 23 C) and lighting 5–12 d before measurements. Measurements of endoconidia and endoconidiophores were made after 4–7 d growth, while perithecia and ascospores were measured after 7–10 d. Aleurioconidia were measured from cultures that had grown 7–20 d. Material to be measured was mounted in lactophenol cotton blue and observed with Nomarsky interference microscopy (Olympus BH-2 microscope), photographed with a Kodak DC 120 digital camera and analyzed with Openlab digital imaging software (Improvision Inc., Lexington, Massachusetts). Perithecia were measured with an eyepiece reticule at 200x or 400x magnifications. For most structures 10 observations were recorded per isolate; when measuring endoconidia, however, 20 conidia were measured per isolate. Some structures were rare or hard to locate in a few isolates, and fewer observations were made.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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only in the minor branches, those without bootstrap support. Four host-associated lineages were evident in all of the MP trees and in a neighbor joining analysis of the same dataset (not shown). All isolates from diseased Populus spp. grouped in a strongly supported branch. Similarly most isolates from Carya spp. grouped into a single lineage with 99% bootstrap support (FIG. 1). Four Quercus isolates formed a separate lineage (89% bootstrap support) with a Betula isolate from Japan and the holotype specimen of C. variospora (BPI 595631) (FIG. 1). Nearly all Prunus isolates grouped into a single lineage (96% bootstrap support) with a few isolates from Quercus, Populus, Carya, Celtis occidentalis and T. americana, all wound associated; as well as a Betula platyphylla isolate from Japan. An isolate (C1965) collected from a wounded Prunus serotina tree in Wisconsin did not group into any of the four host-associated lineages. These groups henceforth are referred to as the aspen, hickory, oak, cherry and cherry-Wisconsin lineages.
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and 3). There was substantial variation in electromorphs among isolates from the North American clade. The Latin American clade was supported only weakly in both analyses, and there was little variation among the isolates tested. There was no bootstrap support for the Asian clade, but two distinct lineages were apparent, one comprising isolates from Ficus carica in Japan and the other from Colocasia esculenta in the Pacific.
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and 3). Six isolates of the hickory ITS lineage formed a sublineage within the hickory lineage, and these two sublineages are designated here as hickory A and hickory B.
Pairings.— –
Self-sterile MAT-2 tester strains from the cherry, hickory B, aspen and oak lineages were used to spermatize MAT-1 testers of other representative isolates of the North American clade (TABLE IV). The MAT-2 testers formed successful, interfertile pairings (perithecia producing abundant, normal ascospores) only with MAT-1 testers from their respective lineages. When a MAT-2 tester from the hickory B sublineage was used, perithecia and abundant ascospores were formed with MAT-1 testers from both the hickory A and hickory B sublineages. MAT-2 testers from C578 and C856, which are from the cherry lineage, paired with most other cherry testers but did not pair with MAT-1 testers from T. americana. Conversely the MAT-2 tester from T. americana mated with MAT-1 testers from the same host (C1954 and C1959) but not with other testers of the cherry lineage. The MAT-1 testers from the Wisconsin Prunus isolate C1965 and the two Japanese isolates failed to mate with any MAT-2 tester.
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). When observed microscopically these perithecia were filled mainly with cellular debris, apparently from aborted asci and ascospores. Ascospores, when present, were uncommon and often misshapen. These pairings were interpreted as partial interfertility due to interspecific crossing, consistent with the interpretation of interspecific pairings between other species of Ceratocystis (Engelbrecht and Harrington 2005; Harrington and McNew 1997, 1998). Some pairings between testers also resulted in sterile perithecia that lacked ascospores. Four host cross-inoculations.— – The analysis of variance for the four-host inoculation experiment revealed a significant effect on linear extent of discoloration for each of the main factors, with the host plant showing the greatest effect (F = 35.92; P < 0.0001). Experiment (within host) was the second largest source of error (F = 27.98; P < 0.0001). There was also a significant host x source (of isolate) interaction (F = 21.67; P < 0.0001). Consequently xylem discoloration then was analyzed separately for each host and each experiment.
Isolates of the aspen and hickory lineage caused dramatically more discoloration (FIG. 4) on P. tremuloides and C. illinoensis, respectively, than did isolates from the other lineages. Hickory isolate C682 caused no more discoloration than the controls in both Populus experiments and was suspected to have deteriorated and lost pathogenicity. Thus hickory isolate C684 was substituted for C682 in the inoculation of other hosts.
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). When the experiment was repeated, however, isolates of the oak lineage caused significantly more discoloration in Quercus than did the controls and the isolates from the other hosts, and the cherry isolates caused more discoloration than did isolates from aspen or hickory. Similarly, when P. serotina seedlings were inoculated, isolates of the oak lineage caused discoloration similar to that caused by isolates from the cherry lineage in one experiment, but only the cherry isolates caused substantial discoloration in P. serotina in a second experiment. In both experiments on P. serotina isolates from the aspen and hickory lineages induced significantly less discoloration when compared to isolates from the cherry lineage.
Prunus virginiana and Quercus macrocarpa cross-inoculations.— –
Because of the ambiguous results seen when inoculating Q. rubra and P. serotina, another experiment was performed in which Q. macrocarpa and P. virginiana were inoculated with isolates from the oak and cherry lineages. For each host inoculations with all isolates resulted in significantly greater discoloration than was seen in plants that received control inoculations (FIG. 5). Significant differences among the isolates also were seen when the controls were excluded from the analysis (Q. macrocarpa: F = 2.51, P = 0.0105; P. virginiana: F = 3.27, P = 0.0012). However there was no evidence of host specialization, as isolates from each lineage produced similar amounts of discoloration in each host (FIG. 5).
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) the two Juglans species had significantly more discoloration than the three Carya species. In the second experiment more discoloration was seen in J. cineria than in any of the other hosts, but the amount of discoloration was not significantly different than that seen in J. nigra, C. cordiformis, or C. illinoensis (FIG. 6).
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) Populus trichocarpa was the most susceptible host, followed by P. balsamifera; P. tremuloides and P. nigra were less susceptible. The trend was similar in the second experiment, with significantly more discoloration in P. trichocarpa and P. balsamifera than in other hosts.
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), but this structure was absent in isolates of C. fimbriata ss and C. platani.
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and Hunt (1956) reported that the ostiolar hyphae of Ceratocystis variospora are shorter than those of C. fimbriata isolates, and we found that the type specimen of C. variospora and isolates of the oak lineage had ostiolar hyphae considerably shorter than those of isolates from the Latin American clade (TABLE V). Isolates of the cherry lineage and the cherry-WI isolate also had relatively short ostiolar hyphae, although only the cherry-WI isolate had ostiolar hyphae that were consistently as short as those seen in isolates of the oak genotype.
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), slightly smaller than the range found in C. fimbriata ss and C. platani (5.5–7.0 x 3.5–5.5 µm). Hunt (1956) reported ascospores 4–6 x 2.5–3.5 µm in C. variospora and 4.5–8 x 2.5–5.5 µm for C. fimbriata.
As is typical for Ceratocystis spp. the studied isolates produced two or three anamorphs, which are accommodated in the genus Thielaviopsis (Paulin-Mahady et al 2002). Flask-shape phialides (and the endoconidia produced from them) of isolates in the North American clade were similar in dimension to those reported by Hunt (1956) and Webster and Butler (1967) for C. fimbriata and C. variospora. Isolates from the hickory A sublineage conspicuously lacked flask-shape phialides (TABLE V).
All isolates in the North American clade produced a second endoconidial stage with doliiform conidia from wide-mouth phialides, but within the Latin American clade only isolates from Platanus produced these structures (TABLE V). This second type of endoconidiophore often was found clustered around the bases of perithecia produced in culture and in samples of naturally colonized plant tissue. Wide-mouth phialides were generally shorter (12–65 µm) than flask-shape phialides, similar to earlier reports (Webster and Butler 1967). Doliiform conidia were 4.5–19.5 µm long x 3.5 to 9.5 µm wide and often were found in long chains. Webster and Butler (1967) reported that doliiform conidia "are at first hyaline, becoming subhyaline to light brown with age"; however we observed a change in the color of doliiform conidia only among isolates from the aspen lineage. Doliiform conidia from the aspen lineage frequently expanded in size after emerging from their phialides and developed into thick-walled, melanized chlamydospores.
Aleurioconidia were 8.5–26 µm long x 6.5–17.5 µm wide, were produced blastically and accumulated in chains. No obvious differences were seen in the size of aleurioconidia among isolates of the various lineages, but isolates from the hickory A sublineage did not produce aleurioconidia.
These differences in morphology are incorporated into the emended descriptions of C. variospora and the newly recognized taxa in the North American clade.
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TAXONOMY |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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Endoconidiophora variospora Davids., Mycologia 36:303. 1944.
Ophiostoma variosporum (Davids.) Arx, Antonie van Leeuwenhoek 18:212. 1952.
EMENDED DESCRIPTION: Cultures on malt yeast-extract agar hyaline at first with a fluffy appearance, becoming brown, gray or olive-green after 2–4 days, undersurface of agar turning dark; radial growth 18 mm at 5 d; odor sweet, often with banana scent. Hyphae hyaline to pale brown, often terminating as endoconidiophores. Perithecia (FIG. 8) with bases superficial to partially immersed, bases black or rarely brown, globose, 130–350(425) µm diam, unornamented or with undifferentiated hyphae attached; possessing a collar at the base of the neck 51–80 µm wide; necks black, slender, up to 830 µm long, 25–50 µm diam at base and 12- µm at the hyaline tip; ostiolar hyphae (FIGS. 9, 10) hyaline, 10–20 in number, 1–2 µm wide (Hunt), 22–50 µm long, tapering to a blunt tip; asci not seen; ascospores (FIG. 11) with outer cell wall forming a brim, hat-shaped, 3.5–6.0 x 3.0–5.0 µm. Endoconidiophores of two types; one flask-shape, hyaline to light brown, septate with conidiophores 52–198 µm long, conidiogenous cell 37–66 µm long, width 4.5–7.0 µm at base and 2.5–4.5 µm at the mouth; producing hyaline endoconidia (FIGS. 12, 13) 6.0–30.0 x 2.5–5.0 µm; the other endoconidiophores shorter, 32–90 µm long, not tapering, often flared at mouth, conidiogenous cell 16–38 µm long, width (3.0) 4.0–5.5 µm at base and 4.5–7.5 µm at mouth; producing doliiform endoconidia (FIGS. 14, 15), hyaline 5.5–10.0 x 5.0–8.0 µm. Aleurioconidia (FIG. 16) produced blastically, singly or in chains, orange-brown to brown, ovoid or obpyriform, smooth, 9.0–6.5 x 7.5–14.0 µm.
SPECIMEN EXAMINED: HOLOTYPE: USA. WEST VIRGINIA: Moorefield, from cambium side of Quercus prinus bark, May 1943, M.E. Fowler, BPI 595631.
CULTURES EXAMINED: USA. MINNESOTA: Ramsey County, from sapwood of Q. ellipsoidalis stumps cut 2–3 wk previously, 1955 or 1956, R. Campbell, isolate C1009 (= CBS 773.73, ATCC 12861). Ramsey County, from sapwood of Q. ellipsoidalis stumps cut 2–3 wk previously, 1955 or 1956, R. Campbell, from isolate C1483 (= ATCC 12866). IOWA: Harper’s Ferry, from wound on Q. alba stem, Jul 2001, J.A. Johnson, isolate C1843 (= CBS 114715, BPI 843737). IOWA: Rhodes, from bleeding canker on Q. robur, Sep 2001, J.A. Johnson, isolate C1846 (= CBS 114714, BPI 843738).
Comments: This species is similar to Ceratocystis fimbriata sensu stricto (the sweet potato pathogen) but differs in the production of doliiform conidia from wide-mouthed phialides, and it differs from C. fimbriata, C. cacaofunesta and C. platani in its shorter ostiolar hyphae and slightly smaller ascospores. The presence of a distinct collar at base of perithecial necks distinguishes C. variospora from C. fimbriata, C. cacaofunesta, C. platani, C. albifundus, and C. polychroma. Cultures of the recently described C. pirilliformis from Australia were not available at the time of study, but the description by Barnes et al (2003) includes the presence of a collar at the base of the perithecial necks, as in C. variospora. Dimensions of ostiolar hyphae were not given for C. pirilliformis, but the ostiolar hyphae illustrated were up to 60 µm long (Barnes et al 2003), longer than those observed in isolates of C. variospora. Although C. variospora and C. pirilliformis are morphologically similar, the ITS sequences of isolates of C. pirilliformis are distinct from those of C. variospora (Thorpe et al 2005). Ceratocystis variospora differs from C. albifundus and C. moniliformis in the production of aleurioconidia and from C. moniliformis in the absence of ornamentation on the perithecial bases.
Ceratocystis variospora was described by Davidson based on fruiting structures found on the inner bark of chestnut oak (Quercus prinus) in West Virginia 1 wk after the bark was removed from a living tree (Davidson 1944). It also has been collected from Q. ellipsodalis stumps in Minnesota (Campbell 1957), from a wound on Q. alba in Iowa and from a bleeding canker on Q. robur, also in Iowa. Isolates from Minnesota and Iowa had similar ITS sequences and were sexually interfertile, and the ITS sequence amplified from DNA extracted from the holotype specimen of C. variospora was also similar. A morphologically similar isolate (C1709) from a sporulating mat on a log of Betula platyphylla in Japan has a similar ITS sequence, but the Japanese isolate is not interfertile with the oak isolates from North America. Other isolates from wounds of various hardwoods in Iowa and a Prunus sp. in Wisconsin were morphologically indistinguishable from the Quercus isolates of C. variospora, but they differed in ITS sequence and allozyme electromorphs and the Quercus isolates were in a separate intersterility group. Most of these isolates from hosts other than Quercus formed ostiolar hyphae longer than 50 µm, longer than those found in the Quercus lineage of C. variospora, but no ostiolar hyphae longer than 50 µm were seen in the Prunus isolates C1822 and C 1841. Also host-specialization of isolates to Quercus spp. or Prunus spp. could not be demonstrated clearly. For the present, all these isolates are considered C. variospora, but the emended description of C. variospora is based solely on the Quercus isolates.
Ceratocystis populicola J. A. Johnson and Harrington, sp. nov. FIGS. 17–25
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Cultures on malt yeast agar hyaline to white initially, becoming darker, and turning brown or olive-green after 2–4 d, radial growth 17–21 mm at 5 d; cultures smell sweet or of banana oil. Perithecia on MYEA fully formed after 4–6 d, scattered on surface of agar or with bases partially submerged. Perithecia (FIG. 17) with black bases, globose, 110–275 µm diam; unornamented or with undifferentiated hyphae attached, possessing a collar at the base of neck, necks black, emerging from collars, hyaline at tip, slender, up to 665 µm long, 24–45 µm diam at base and 13–30 µm at hyaline tip; ostiolar hyphae hyaline, slender, tapered to a blunt tip, 42–75 µm long (FIG. 18). Asci not seen; ascospores (FIG. 19) with outer cell wall forming a brim, hat-shape, 4.5–6.5 x 3.0–5.0 µm. Endoconidiophores of two types; one flask-shaped, hyaline to light brown, septate with conidiophores 45–200 µm long, conidiogenous cell 35–85 µm long, width 3.5–7.0 µm at base and 3.5–4.5 µm at mouth; producing hyaline endoconidia 10– 33 x 2.0–5.0 (5.5) µm (FIGS. 20, 21); the other endoconidiophores shorter, not tapering, often flared at mouth; often produced in masses around perithecial bases (FIG. 17); conidiophores 17–95(125) µm long, conidiogenous cell 12–40 µm long; width 3.5–6.0 µm at base and 3.5–8.5 µm at tip of conidiogenous cell; producing doliiform endoconidia, hyaline at first, 6.5–12.0 x 3.5–.5 µm (FIGS. 22), often becoming swollen and melanized with thick walls (FIGS. 23, 24), 8.0–13.5 x 6.0–10.5 µm. Aleurioconidia (FIG. 25) produced blastically, singly or in chains, orange-brown to brown, ovoid or pyriform, smooth, 9.0–18.5 x 8.0–17.5 µm.
HOLOTYPE: CANADA. QUEBEC: from Populus tremuloides, E. Smalley, BPI 843723, from isolate C685 (= CBS 115161).
CULTURES EXAMINED: CANADA. QUEBEC: from Populus tremuloides, E. Smalley, isolate C685 (= CBS 115161). USA. SOUTH DAKOTA: Black Hills, from P. tremuloides, 1980, T.E. Hinds, isolate C89 (= CO 301, = CBS 114725). COLORADO: from P. tremuloides, T.E. Hinds, isolate C1485 (= ATCC 24096). POLAND. KÓRNIK: from canker on Populus hybrid, Aug 1976, J. Gremmen, isolate C947 (= ATCC 36291). from canker on Populus hybrid, Aug 1976, J. Gremmen, isolate C995 (= CBS 119.78).
Etymology. populicola, Latin = on Populus.
Comments: This species is similar to C. fimbriata ss but differs in the production of doliiform conidia from wide-mouth phialides. The distinct collar at the base of perithecial neck distinguishes C. populicola from C. fimbriata, C. cacaofunesta, C. platani, C. polychroma and C. albifundus. C. populicola differs from C. variospora and C. pirilliformis in the production of chains of swollen, melanized chlamydospores from wide-mouth phialides. Ceratocystis populicola differs from C. albifundus and C. moniliformis in the production of aleurioconidia and from C. moniliformis in the absence of ornamentation on the perithecial bases.
In our inoculations only isolates of C. populicola were capable of causing disease in Populus spp. Distinctive, target-shape cankers caused by C. fimbriata have been noted on P. tremuloides in Minnesota (Manion and French 1967, Wood and French 1963), Pennsylvania, much of the western USA, including Alaska (Hinds 1972, Hinds and Laurent 1978) and Quebec, Manitoba and Saskatchewan in Canada (Zalasky 1965). All these reports are believed to be of C. populicola. It is likely that the pathogen is present wherever Populus tremuloides naturally occurs. Hybrid poplars were found infected at plantations in Poland (Gremmen and de Kam 1977, Przybyl 1984b), and isolates from these plantations are C. populicola. An additional report from Quebec (Vujanovic 1999) describes C. fimbriata infecting rooted cuttings of P. balsamifera, a host found susceptible to C. populicola in our inoculations.
Ceratocystis caryae J.A. Johnson and Harrington, sp. nov. FIGS. 31–33
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Cultures on malt yeast agar hyaline to white initially, becoming darker, and turning brown, gray or olive-green after 2–4 d, culture texture varying from fluffy to felty, undersurface of agar turning dark. Cultures with a sweet scent, often smelling like banana oil. Perithecia on MYEA fully formed after 4–6 d; perithecia scattered or clumped on surface of agar or with bases partially submerged. Perithecia with bases black, globose or broadly obpyriform, 135–340 µm diam; unornamented or with undifferentiated hyphae attached; occasionally with collar at apex 48–103 µm wide; necks black, tapering to a hyaline tip, up to 950 µm long, 25–52 µm diam at base and 15–30 µm at tip; ostiolar hyphae hyaline, slender, tapered to a blunt tip, 32–80 µm long. Asci not seen; ascospores with outer cell wall forming a brim, hat-shape, 4.0–6.0 x 3.5–4.5 µm. Endoconidiophores of two types; one flask-shaped, hyaline to light brown, septate with conidiophores 42–510 µm long, conidiogenous cell 33–80 µm long, width 3.8–7.5 µm at base and 3.2–4.8 µm at the mouth; producing hyaline endoconidia 8.5–27.0(43.0) x 2.5–6.0 µm (FIGS. 31, 32); the other endoconidiophores shorter, not tapering, often flared at mouth; often produced in masses around perithecial bases, conidiophores 40–100 µm long, conidiogenous cell 15–55 µm long; width 5.0–6.5(7.0) µm at base and 5.5–8.0 µm at tip of conidiogenous cell; producing hyaline doliiform endoconidia, 6.0–13.5(16.0) x 5.5–9.5 µm. Aleurioconidia produced blastically, singly or in chains, orange-brown to brown, ovoid or pyriform, smooth, 9.0–21.5 x 8.5–16.5 µm (FIG. 33).
HOLOTYPE. USA. IOWA: Coggan, from Carya cordiformis (Wangenh.) K. Koch, Aug 2001, J.A. Johnson, BPI 843735, from isolate C1829 (= CBS 114716).
CULTURES EXAMINED: USA. IOWA: Coggan, from Carya cordiformis, Aug 2001, J.A. Johnson, isolate C1829 (= CBS 114716). Clayton County, from C. cordifomis, Sep 1998, T.C. Harrington, isolate C1412 (= BPI 843728). Clayton County, from C. cordifomis, Sep 1998, T.C. Harrington, isolate C1413. Boone County, from C. ovata, Jun 2001, J.A. Johnson, isolate C1827 (= CBS 115168). Boone County, from C. cordiformis, Jul 2001, J. A. Johnson, isolate C1845. Ames, from Ostrya virginiana, Aug 2002, J.A. Johnson, isolate C1971.
Etymology. caryae, Latin = on Carya.
Comments: This species is morphologically similar to C. variospora but differs in the length of the ostiolar hyphae. C. caryae differs from C. fimbriata ss in the production of doliiform conidia from wide-mouthed phialides, and from C. fimbriata, C. cacaofunesta, C. platani, C. polychroma and C. albifundus in the presence of a collar subtending the perithecial neck. The doliiform conidia and aleurioconidia of C. caryae are larger than those reported for C. pirilliformis (Barnes et al 2003). C. caryae differs from C. moniliformis in the absence of ornamentation on the perithecial bases. C. caryae lacks the melanized doliiform conidia seen in C. populicola. All isolates of C. caryae sensu stricto have been recovered from Carya spp., Ulmus spp. or Ostrya virginiana.
Ceratocystis smalleyi J.A. Johnson and Harrington, sp. nov. FIGS. 26–30
Culturae odore dulci bananae carentes. Perithecia basibus atris, globosa, 100–300 µm diam, aliquando collari basim colli circumdante; collum atrum, gracile, usque ad 570 µm longum, diametro ad basim 22–80 µm et ad apicem 15–40 µm; hyphae ostioli hyalinae, graciles, 55–100 µm longae. Ascosporae 4.0–6.0 x 3.5–5.0 µm. Endocondidiophora hyalina ad fusca, uniformia, brevia, cellulaconidiogena saepe dilatati versus apicem, endoconidiis doliiformibus, hyalinis, 7.5–13.5(16.0) x 5.5–9.5 µm. Aleurioconidia non visa.
Cultures on malt yeast agar hyaline to white initially, becoming darker and turning brown, gray or olive-green after 2–4 d, often with lighter colored gray to white patches, undersurface of agar turning dark, many isolates sectoring readily. Radial growth 21 mm at 5 d; cultures may have a sweet scent, but the banana odor typical of C. caryae is absent. Perithecia on MYEA fully formed after 4–6 d, often fruiting in concentric rings; perithecia on surface or with bases partially submerged. Perithecia (FIG. 26) with bases black, globose or broadly obpyriform, 100–300(350) µm diam; unornamented or with undifferentiated hyphae attached; occasionally with collar at apex 42–73(85) µm wide; necks black, tapering to a hyaline tip, up to 570 µm long, 22–80 µm diam at base and 15–37 µm at tip; ostiolar hyphae (FIG. 27) hyaline, slender, tapered to blunt tip, 55–100 µm long. Asci not seen; ascospores (FIG. 28) with outer cell wall forming a brim, hat-shape, 4.0–6.0 x 3.5–5.0 µm. Endoconidiophores (FIG. 29) of one type, not tapering, often flared at mouth; commonly produced in masses around perithecial bases, conidiophores multicellular, 35–105 µm long, conidiogenous cell 22–65 µm long; width 4.0–6.0 µm at base and 4.0–7.5 µm at tip of conidiogenous cell; producing doliiform to cylindrical hyaline endoconidia (FIG. 30), 7.5–31.5 x 4.0–7.5 µm.
HOLOTYPE. USA. WISCONSIN: Hickory Ridge, from Carya cordiformis, 1993, E. Smalley, BPI 843722, from isolate C684 (= CBS 114724).
CULTURES EXAMINED: USA. WISCONSIN: Hickory Ridge, from Carya cordiformis, 1993, E. Smalley, isolate C684 (= CBS 114724). La Crosse, from C. cordifomis, 1986, E. Smalley, isolate C682. Evansville, from C. ovata, 1993, E. Smalley, isolate C683. IOWA: Clayton County, from C. cordiformis, Sep 1998, T.C. Harrington, isolate C1410. Clayton County, from C. cordiformis, Sep 1998, T.C. Harrington, isolate C1411. Coggan, from C. cordiformis, Aug 2001, J.A. Johnson, isolate C1828. Coggan, from C. cordiformis, Aug 2001, J.A. Johnson, isolate C1839. Coggan, from C. cordiformis, Aug 2001, J.A. Johnson, isolate C1840. Coggan, from C. cordiformis, Aug 2001, J.A. Johnson, isolate C1842. Coggan, from C. cordiformis, Aug 2001, J.A. Johnson, from isolate C1844. Cambria, from C. cordiformis, Aug 2002, J. A. Johnson, isolate C1952.
Etymology. smalleyi, named after the late Eugene Smalley, who associated this fungus with Scolytus quadrispinosus and brought the new taxon to our attention.
Comments: This species differs from C. caryae in the absence of cylindrical conidia from flask-shape phialides and in the absence of aleurioconidia. All isolates of C. caryae from wounded Carya spp. or Ostrya virginiana are closely related to C. smalleyi based on ITS sequence analysis and allozyme banding patterns; they behave similarly in inoculation tests, and they appear to be sexually interfertile. The isolates from wounds produce pink ascospore masses, while ascospore masses of C. smalleyi are white to cream. Perithecia of C. smalleyi do not consistently produce a distinct collar at base of perithecial necks, but such swellings can be seen in at least some perithecia of all isolates, as they can in perithecia of C. caryae, C. variospora and C. populicola. Eugene Smalley first isolated the fungus from a tree that had been attacked by the hickory bark beetle (Scolytus quadrispinosus), and he later made collections in association with the beetle from other locations in Wisconsin (pers comm). We later collected isolates from northeastern and south-central Iowa. Isolates have been made from hickory bark beetle egg galleries, from stained wood surrounding galleries and from discolored sapwood associated with beetle attacks from previous years.
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSTAXONOMYDISCUSSIONLITERATURE CITED |
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). Pairings between mutant MAT-2 tester strains that had lost the ability to self and MAT-1 testers provided evidence of many biological species, but not all of these biological species are formally recognized in this study.
To delimit species under the phylogenetic species concept supported by Harrington and Rizzo (1999), a lineage should have a unique combination of phenotypic characters. The taxa in the North American clade can be distinguished from the Latin American clade of C. fimbriata by their slightly smaller ascospores and the collar present at the base of the perithecial necks. The taxa within the North American clade are distinguished from each other by a number of minor morphological characters, presence or absence of conidial states and by host range. Inoculation experiments distinguished some of the host-associated lineages, with strong evidence for host specialization shown by isolates from the aspen and hickory lineages. Isolates from the oak and cherry lineages showed little to no evidence of host specialization, so these are retained as a single species.
The name C. variospora is available for the oak lineage. Ceratocystis variospora originally was reported in West Virginia on the inner bark of Q. palustris collected for tanning and later was collected in Minnesota from a fresh stump of Q. ellipsoidalis (Davidson 1944, Campbell 1957). The ITS sequence generated from the holotype specimen was similar to that of the Minnesota isolates and Iowa isolates from Q. alba, a native tree, and Q. robur, a European species. The isolate collected from Q. alba was recovered from a wound, while the isolate from Q. robur was isolated from a bleeding canker in a small experimental planting where many of the Q. robur trees showed severe cankering.
We also are applying the name C. variospora to the lineage containing isolates from wounds on Prunus and other hardwood species. The cherry and oak lineages could be separated based on differences in ITS sequences, allozymes and interfertility, but they could not be consistently distinguished through morphology or host specialization. Isolates from a Tilia tree were typical of the cherry lineage in ITS sequence, allozymes and morphology but testers from these isolates were able to mate only with themselves. Isolates from Betula platyphylla logs in Japan also were morphologically similar to USA isolates from the oak and cherry lineages of C. variospora, but they were intersterile with the USA isolates and with each other. The ITS sequence and allozyme electromorphs of the Wisconsin isolate from Prunus were unique, but this isolate is morphologically indistinguishable from C. variospora and behaved similarly in inoculations of Quercus and Prunus.
Previous observations of C. fimbriata in North America have focused on mortality of infected trees, but C. variospora appears to be more common and occur on more hosts as a relatively innocuous wound colonizer. The cherry lineage of
