Abstract
Cytospora canker is a serious fungal disease of many shade, fruit, and ornamental tree species in the urban forest, orchards, and nurseries. Because Cytospora species are difficult to identify and their host ranges are poorly understood, it is not known if disease occurrence on one host poses a threat to other host species. Cytospora isolates were collected from aspen (Populus tremuloides) (Cytospora chrysosperma), green ash (Fraxinus Pennsylvanica) (Cytospora pruinosa), Siberian elm (Ulmus pumila) (Cytospora sacculus), alder (Alnus spp.) (Cytospora umbrina), cottonwood (Populus spp.) (Cytospora chrysosperma), and multi- and single-stemmed willow (Salix spp.) (Cytospora fugax). These isolates were inoculated into drought-stressed aspen, green ash, Siberian elm, thinleaf alder (A. tenuifolia), eastern cottonwood (P. deltoides), and single-stemmed willow. Ash, alder, and elm isolates were host specific. Aspen and cottonwood isolates were pathogenic only on aspen and cottonwood trees. Isolates from multi-stemmed willows caused cankers on aspen only and not single-stemmed willows. However, Cytospora spp. isolates collected from a single-stemmed willow were pathogenic on trees cloned from that willow. Water potential, as a covariate, did not explain variation in canker size among trees. Thus, Cytospora fungi that are host specific may not be a threat to other nearby tree species.
Cytospora canker, caused by fungi in the genus Cytospora Ehrenb.:Fr., is a worldwide problem and affects more than 70 species of woody shrubs and trees (Agrios 1997). Cytospora refers to the anamorphic (asexual) stage of the causal fungi commonly found forming cankers. Numerous species of Cytospora are listed as causing Cytospora canker. However, species identification is difficult, even for the professional plant pathologist because Cytospora fruiting and vegetative structures, as well as spore size, vary greatly. A poor understanding of the host range of each species also contributes to the difficulty of identification (Waterman 1955; Spielman 1985). Thus, it is not known if Cytospora isolates found on diverse woody hosts in a localized region are the same, are different strains of a single species, or are different species. Consequently, in the urban forest or nursery, it is not known if disease occurrence on one woody plant species poses a threat to other tree species.
The objective of this study was to determine if Cytospora isolates collected from six common urban and riparian tree species in northern Colorado, United States, would show single- or multiple-host specificity A fungal isolate was considered to be either pathogenic or nonpathogenic on a particular host based on whether or not it caused tissue disruption as evidenced by a canker larger than the noninoculated control.
MATERIALS AND METHODS
Four experiments were conducted during 1997 and 1998 to test the pathogenicity of Cytospora isolates collected from six hardwood tree species. In three experiments, inoculations were conducted on drought-stressed trees. One final experiment 1) addressed the pathogenicity of fungal isolates from multi- versus single-stemmed willows, and 2) attempted to determine if the infection of cottonwoods by elm isolates that occurred in one out of three experiments could be repeated.
Plant Material
All experimental trees were grown in 19-L (5-gal) black plastic pots containing a potting mix composed of 25% sand, 20% peat, 5% composted manure and sawdust, and 50% clay loam. Green ash (Fraxinus pennsylvanica cv ‘Patmore’ Marsh.), eastern cottonwood (P. deltoides cv ‘Siouxland’, Bartr.), and multi-stemmed willow (Salix spp.) were clonal plant material, while thinleaf alder (Alnus tenuifolia Nutt.), aspen (Populus tremuloides Michx), and Siberian elm (Ulmus pumila L.) were seedlings (Table 1). Willows were obtained as cuttings taken from a mature single-stemmed willow in Fort Collins, Colorado. Cuttings were rooted in Fafard No. 4-P Growing Mix (Conrad Fafard, Inc., Agawam, MA) in the greenhouse. The five other tree species were purchased from various nurseries. All trees were maintained outside under partial shade until fungal pathogenicity studies began. At study initiation, all trees were two to four years old and had an average height of 1.7 m (5.6 ft) and an average diameter of 1.6 cm (0.6 in.) measured 15 cm (6 in.) above the soil line.
Trees and fungi used in Cytospora pathogenicity studies.
Fungal Isolates
A total of 18 Cytospora isolates were used in the four experiments (Table 1). For the first three experiments, 12 isolates of Cytospora were tested. Two isolates from each of six host tree species were obtained during 1995 and 1996 from infected bark tissue. Isolates from aspen, green ash, and Siberian elm were identified as Cytospora chrysosperma (Pers.:Fr.) Fr., Cytospora pruinosa Fr.:Fr., and Cytospora sacculus (Schw.) Gvrit., respectively. These isolates from urban trees species were collected from landscapes in Fort Collins, Colorado. The isolates from alder, cotton-wood, and multi-stemmed willow were identified as Cytospora umbrina (Bonord.) Sacc, Cytospora chrysosperma (Pers.:Fr.) Fr., and Cytospora fugax (Bull.:Fr.) Fr., respectively. These isolates from riparian tree species were collected in the Poudre Canyon northwest of Fort Collins. In the final experiment, six willow isolates of C. fugax were obtained from a single-stemmed willow in Fort Collins. This tree was the parent plant from which all the willows were cloned. Cytospora isolates from multi-stemmed willow did not produce significantly larger cankers than controls on the single-stemmed willow trees in the first three experiments. For this reason, new Cytospora isolates were collected to determine if these isolates were pathogenic on the cloned single-stemmed willow material.
Potato dextrose agar (PDA) and a defined glucose medium (Kastirr 1985) served as media for the initial isolations. All isolates were transferred to petri dishes containing PDA and grown in the laboratory under light at 23°C ± 2°C (73.4°F) for one week prior to inoculation. Isolates were maintained on PDA slants at 5°C (41 °F). Asexual and sexual fruiting bodies on the original plant material from which the isolates were obtained were used to identify Cytospora species using Spielman’s (1984) monograph on Valsa and Cytospora species. The identification of the willow species was from asexual and sexual fruiting bodies on multi-stemmed willow, but only asexual fruiting bodies were found on the single-stemmed willow.
Induction of Drought Stress
All trees in the first three experiments and half of the trees in the final experiment were drought stressed for two weeks prior to and for four weeks following inoculation. Drought stress was achieved by withholding moisture from trees until leaves reached predawn water potentials of -1.5 to -2.5 MPa. Predawn water potential (the measure of water stress in the tree) was measured daily on one leaf from each tree by means of a pressure chamber (Spomer 1985). Supplemental water (approximately 250 to 500 mL [8.5 to 17 oz]) was added as needed to maintain trees at approximately the same level of drought stress. Half the trees in the final experiment were well-watered prior to and for four weeks following inoculation. Predawn water potential was measured weekly on one leaf from each tree by means of a pressure chamber.
Inoculations
Stems were surface disinfested with 95% ethanol and allowed to dry before wounding. Trees were wounded by removing a bark disk to the xylem with an 8-mm-diameter (0.3-in.) flame-sterilized leather punch. Each tree received four wounds. Each wound was located 10 to 15 cm (4 to 6 in.) above and 90° clockwise around the stem from the one below. A 6-mm-diameter (0.2 in.) plug of mycelium from an actively growing colony of a Cytospora isolate was placed into each of the three wounds with the mycelium facing the wound. The fourth wound received a 6-mm-diameter plug of sterile PDA as a control. All wounds were wrapped with wax film for two weeks following inoculation, after which the film was removed.
Canker size measurements were taken four weeks after inoculation. Canker size was recorded as the sum of horizontal and vertical dimensions of discolored bark measured through the center of each wound. Isolations were attempted from 20% of the inoculation sites to confirm presence of the fungus. Surface-disinfested chips of bark and xylem tissue were placed in petri dishes containing PDA amended with streptomycin sulfate at 100 mg/L.
Experimental Design
All experiments were conducted in a glass house with shade cloth in which temperatures were maintained at 22°C to 30°C (71.6°F to 86°F). The first three experiments used a randomized block split-plot design and consisted of two replications per experiment. Each block (replication) consisted of six plots, and each plot contained four trees, for a total of 24 trees per replication. Each plot within a replication contained one tree species. Each of the four trees within a plot received four wounds. Three wounds were inoculated with a different Cytospora isolate, while the fourth received sterile agar and served as a control. The isolates and controls were randomly chosen and placed into the wounds on the four trees in each plot. Using this protocol, all six tree hosts were inoculated with 12 fungal isolates. To adjust for unequal variances and skewed data, canker measurements were log10 transformed. To determine whether there were differences among isolates or host tree species, data were analyzed by analysis of covariance using SAS software (PROC MIXED, SAS Institute 1996). Water potential varied among trees within a species and was included as a covariate. When host tree species by isolate interactions were significant, paired comparisons using Student’s t-tests were performed to detect differences among isolates for each host tree species. Because canker size did not significantly differ between isolates obtained from the same host tree species, the two isolates were pooled in the statistical analysis. One isolate each from green ash, alder, and cottonwood was not used after the first experiment due to inconsistent canker development. These isolates were replaced with other isolates for the remaining experiments.
In the final experiment, twelve single-stemmed willows and six cottonwood trees were used in a split-plot design with three replications. A plot for willow consisted of two willow trees inoculated with six single-stemmed willow isolates as either drought-stressed or well-watered treatments (Table 1). For cottonwood, a plot consisted of a single cottonwood tree inoculated with the two elm fungal isolates, as either drought-stressed or well-watered treatments. This resulted in six plots each (two treatments by three replications) for both willow and cottonwood trees. Each of the two willow trees within a plot received four wounds. Three of the wounds were inoculated with different Cytospora isolates, while the fourth received sterile agar and served as a control. Each cottonwood tree in a plot received five wounds. Four wounds were inoculated, two wounds each with one of the two different Cytospora isolates, while the fifth received sterile agar and served as a control. The isolates and controls were randomly chosen and placed into the wounds on tree(s) in each plot. Data analysis was handled as previously described.
RESULTS
Canker size in the first three experiments was dependent upon the host tree species-fungal isolate combination. Aspen and cottonwood isolates each produced significantly (P ≤ 0.05) larger cankers than controls on aspen and cottonwood trees in all three experiments (Figures 1 and 2). Only on their respective hosts did the ash and alder isolates produce significantly (P ≤ 0.05) larger cankers than controls (Figures 1 and 2). This occurred in all three experiments with ash isolates. However, significantly larger cankers than controls occurred only in the first experiment for alder isolates. Nevertheless, mean canker size for alder isolates was always greater than control canker size. Elm isolates produced significantly (P ≤ 0.05) larger cankers than controls on elm trees in the first and third experiments as well as on cottonwood trees (Figure 1). Although isolates from multi-stemmed willows failed to produce significantly larger cankers than the controls on single-stemmed willow trees in any of the three experiments, they did, produce significantly (P ≤ 0.05) larger cankers on aspen trees (Figure 2). Water potential, as a covariate, did not explain variation in canker size among trees in any experiment.
Canker size on three urban hardwood species four weeks post-inoculated with two Cytospora isolates from six tree species. Measurements were log10 transformed, n = 4; isolate data were pooled. Asterisks indicate means, which are significantly different than that of controls (P ≤ 0.05).
Canker size on three riparian hardwood species four weeks post-inoculated with two Cytospora isolates from six tree species. Measurements were log10 transformed, n = 4; isolate data were pooled. Asterisks indicate means, which are significantly different than that of controls (P ≤ 0.05).
On the single-stemmed willow trees in the final experiment, five of the six Cytospora isolates from single-stemmed willows produced significantly (P ≤ 0.05) larger cankers than controls on drought-stressed trees (Figure 3). Three of these five Cytospora isolates produced significantly (P ≤ 0.05) larger cankers than controls on watered trees (Figure 3).
Canker size on single-stemmed Salix spp. four weeks post-inoculated with six willow isolates from single-stemmed willow and subjected to drought stress. Measurements were log10 transformed, n = 3. Asterisks indicate means, which are significantly different than that of controls (P ≤ 0.05).
On the cottonwood trees in the final experiment, neither Cytospora isolate from elm produced cankers on cottonwood trees. There also was no significant difference in canker size between drought-stressed and well-watered treatments with elm isolates on cottonwood trees.
In all experiments, reisolations to confirm the presence of the fungus were 50% positive for drought-stressed trees and 73% positive for well-watered trees. All reisolations from nonexpanding wounds were negative. This finding indicates the fungi placed in the wounds were the cause of the canker expansion.
DISCUSSION
Cytospora canker size was dependent upon the host tree species-fungal isolate combination. This result is consistent with previous reports indicating that many species of Cytospora parasitize diverse woody plant hosts (Helton and Konicek 1961), while other Cytospora species are host specific (Helton and Moisey 1955; Farr et al. 1989; Proffer and Hart 1994). There appears to be host specificity among the species of Cytospora examined in the current study, and future research is needed to determine the effect of these Cytospora isolates on tree species other than those tested.
The involvement of predisposing environmental stresses in the ability of Cytospora fungi to infect hosts is not well understood. Cytospora fungi are mostly opportunistic pathogens and attack hosts that are stressed and of poor vigor (Schreiner 1931; Hinds and Stewart 1965; Schoenweiss 1967; Hinds 1985; Sinclair et al. 1987; Biggs 1989; Guyon et al. 1996; McIntyre et al. 1996). Helton (1961b) and Dhanvantari (1978) reported that low-temperature injury could predispose stone fruit trees to infection by Cytospora.
Among drought-stressed trees, ash isolates were host specific, and although pathogenicity occurred in only one of three experiments, it appears that alder isolates were host specific as well. The lack of canker development in two of the three experiments with alder suggests that possibly a stress other than drought is involved in pathogenesis. Although elm isolates were pathogenic on cottonwood trees in the first experiment, results from the final experiment indicated they were not. One might assume that the canker development on elm trees was a result of variation in environmental stresses that we did not monitor. Based on preliminary tests that were positive, and the fact that elm trees were the only other host on which cankers developed, it seems reasonable to conclude that elm isolates are host specific.
The range of tree hosts of Cytospora chrysosperma is not completely defined from our study. This species is often reported as the pathogen associated with Cytospora canker on aspen, cottonwood, and willow trees (Christensen 1940; Hinds 1985; Walla and Conway 1986; Sinclair et al. 1987). Therefore, one might expect isolates of Cytospora from aspen, cottonwood, and willow to behave similarly on these hosts. Although aspen and cottonwood isolates each produced significantly larger cankers than controls on drought-stressed aspen and cottonwood trees, multi-stemmed willow isolates caused cankers on aspen trees only. Had willow isolates not infected aspen trees, the conclusion could be made that they were perhaps saprophytic, and therefore not capable of being pathogenic on trees other than multi-stemmed willow. The results, however, are similar to those Helton (1961a) observed in a host range study in which a willow isolate (Salix spp.) did not cause cankers on golden willow (Salix alba). Additionally, Helton found five out of ten Cytospora isolates failed to induce cankers on trees of the same or closely related species from which they were isolated, and only two of the ten isolates produced their largest cankers on the host species or variety from which they were isolated. Clearly there are important factors other than host specificity that affect Cytospora canker development, and they appear to be related to the type and timing of stress.
The lack of canker development by multi-stemmed willow isolates on single-stemmed willow trees was explained by the final experiment. Data from the test indicated that single-stemmed willow isolates could cause cankers on single-stemmed willow trees. Thus, multi-stemmed willow isolates are host specific and will not infect single-stemmed willows.
Numerous species of Cytospora are listed in the literature as causing Cytospora canker, yet only a few have been tested in pathogenicity studies. These facts, combined with the broad host range of Cytospora spp., make questionable any conclusions regarding which species of Cytospora occur in a local region. Because species identification of Cytospora is difficult due to variation in fruiting and vegetative structures and spore size, examining differences among isolates from various hosts by means of host range and pathogenicity studies is important. The host specificity exhibited by ash isolates, as well as host preference of aspen and cottonwood isolates, indicates these fungi have specific host ranges. The possible host specificity of alder and elm isolates, and the host specificity displayed by willow isolates, lends further support to host specificity.
Cytospora species described on the six hosts we studied are numerous, but recent studies (Spielman 1985; Farr et al. 1989) have reduced the number of species by 80% to 90%. These reduced number of species are listed below; those in bold we found on our plant material. Cytospora species on alder are C. sacculus, C. umbrina, and C. chrysosperma; ash species are C. fugax, C. leucosperma, C. minuta, C. pruinosa, C. sacculus, and C. annually; poplar species are C. chrysosperma, C. leucosperma, C. lecostoma, C. nivea, and C. sacculus; willow species are C. chrysosperma, C. fugax, C. germanica, C. leucosperma, C. nivea, and C. sacculus; and elm species are C. carbonacea, C. chrysosperma, C. leucosperma, and C. sacculus.
Techniques for characterizing races, varieties, and species of Cytospora are needed to further examine host-fungus species relationships. This study supports the contention that molecular techniques to examine differences among host-specific Cytospora spp. should be used in conjunction with host range and pathogenicity studies.
There appears to be both host-specific and host-nonspecific Cytospora species associated with Cytospora canker of hardwood trees. Knowledge of host specificity may aid nursery managers, landscape planners, and arborists in tree species selection. Planting trees that are attacked only by host-specific Cytospora near hosts susceptible to other Cytospora would be a good preventive practice in nurseries and landscapes. Knowing the specificity of a Cytospora fungus causing cankers on a tree would allow arborists to decide if the fungus is a threat to nearby trees and what actions are needed.
Acknowledgments
Partial financial support for this study was provided by the Colorado Agricultural Experiment Station, Colorado State University, journal article number 157541. We appreciate the statistical advice of J.R. zumBrunnen, mycological advice of E Brent Reeves, Jr. and for reviewing the manuscript, John E. Hendrix, J.R Hill, and Howard E Schwartz.
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