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Phytophthora drechsleri in remote areas of southeast Alaska
[ 379 ] Trans. Br. mycol. Soc. 91 (3), 379-384 (1988)
Printed in Great Britain
PHYTOPHTHORA DRECHSLERI IN REMOTE AREAS OF SOUTHEAST ALASKA By E. M. HANSEN AND P. B. HAMM Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, U.S.A. C. G. SHAW III USDA Forest Service, Rocky Mountain Forest and Range Experiment Station, Fort Collins, CO 80526, U.S.A. AND P. E. HENNON USDA Forest Service, Forest Pest Management, Juneau, AK 99802, U.S.A.
Phytophthora drechsleri was recovered from remote, undisturbed muskeg and forested sites and from one more accessible area of native vegetation in southeast Alaska. Identity of 10 isolates was confirmed by morphology and behaviour as well as electrophoretic comparison of proteins with those of known isolates. Oospores formed when Alaska isolates were mated with an Az isolate of P. drechsleri from California. Although recovered from soils and streams in forests dominated by declining Chamaecyparis nootkatensis, the fungus was not pathogenic to this tree species in inoculation tests. Its presence in this habitat and its limited pathogenicity suggest that P. drechsleri may be indigenous to the forests of northwestern North America. Phytophthora species are pathogens of many agricultural crops world-wide; however, little is known of their behaviour in native plant communities, and theories on their origins and evolution are largely speculative. Several species, including P. cinnamomi and Pi lateralis, are destructive in forests and other native vegetation, but the susceptibility of the hosts involved, the relatively recent origin of epidemics, and the consistent association of damage with human disturbance suggest that, in most cases at least, the fungi were introduced to the affected areas. Claims that pathogenic fungi are either indigenous or introduced are often controversial, but the question has practical significance for disease control programmes, especially opportunities for effective quarantine. It is also of more fundamental interest in elucidating the evolutionary history of pathogenic fungi and the search for disease resistance (Leppik, 1970). While investigating the causes of decline and mortality of Chamaecyparis nootkatensis (D. Don) Spach. (Alaska-cedar) in southeast Alaska (Shaw et al., 1985; Hennon, 1986; Hamm et al., 1988), we searched for potential pathogens including species of Phytophthora, The isolates were characterized, and their pathogenicity to Alaska-cedar was tested. MA TERIALS AND METHODS
Isolation of Phytophthora was attempted from soil and streams in six areas of southeast Alaska (Fig.
1): Peril Strait, Helm Bay, Slocum Arm, Wrangell Island, Prince of Wales Island, and in the vicinity of Juneau, the capital city. Three of these, Peril Strait, Helm Bay, and Slocum Arm were in remote undisturbed areas many kilometres from roads or permanent settlement and accessible only by boat or float plane.
Soil sampling In summer 1984, 162 one 1 soil samples were collected from the five areas; in summer 1985,77 samples were collected near Peril Strait and 25 near Slocum Arm. Each sample was taken within 0'5 m of the bole of a declining or healthy Alaskacedar. The soil organic fraction was separated by wetsieving (Ostrofsky, Pratt & Roth, 1977); 5 g (wet wt) portions of that fraction were placed in double styrofoam drinking cups and flooded with distilled water (Linderman & Zeitoun, 1977). Hymexazol (Z5 p.p.m.) was added to the baiting solution to inhibit Pythium (Hamm & Hansen, 1984) in 1984 but not in 1985 samples. Alaska-cedar foliage baits z-3 em long were floated in the cups for 6 d to trap Phytophthora from the soil, then transferred to amended cornmeal agar (CMP) containing 20 pg/ ml pimaricin and zoo pg/ml vancomycin in Petri dishes. Phytophthora colonies were subcultured for identification after 6 d. After the 1984 sampling different foliage baits were compared for their
Phytophthora drechsleri in Alaska
o
15 30 45 :miles
Prince of Wales Island
Fig.
1.
Sampling locations in Southeast Alaska: 1, Slocum Arm; 2, Peril Strait; 3, Wrangell Island; 4, Prince of Wales Island; 5, Helm Bay; 6, Juneau.
efficacy in recovering Phytophthora from zoospore suspensions. Two Phytophthora isolates recovered in the 1984 sampling were used. None of eleven species of native plants compared as baits was superior to Alaska-cedar. At the same time, apple, pear, lemon, and avocado fruits were compared as baits. Pears proved to be superior and were used in subsequent stream sampling.
Stream sampling In summer 1985, eight streams draining into Peril Strait and two entering Slocum Arm were sampled for the presence of Phytophthora. Seven of the 10 streams drained from bogs or muskegs with associated dead and dying Alaska-cedar. Streams were up to 4 m wide. Large green d'Anjou pears were placed individually in nylon mesh bags
E. M. Hansen and others anchored along the stream courses at 10 m intervals, starting just above tidewater. Each stream tributary was baited with two pears, one just above the mouth and another 10 m above that; between 33 and 41 pears were placed in each stream system. Pears were removed after 4-6 d and incubated outdoors at ambient temperature (15°C) for 3-7 d until distinct brown necrotic spots appeared. In November, 1987, a total of 77 pears was placed in 5 streams near Juneau. Pears were removed one week later, and incubated as before. Pears were dissected and small portions of tissue from the margins of brown areas were aseptically removed and transferred to CMP. Isolation plates were incubated at room temperature and inspected daily to separate fast-growing Pythium colonies from possible Phytophthora. All Phytophthora colonies were subcultured for identification. Species identification Isolates were maintained on CMP at 5°. To induce sporangia, colonies were grown 5 d in pea broth, washed thoroughly with distilled water, and then flooded with soil extract water for 24-48 h. Isolates also were grown on clarified V -8 agar with or without added Alaska-cedar foliage, grass leaves, or p-sitosterol and on cedar extract agar (Trione, 1974) made with Alaska-cedar foliage. Matings to induce oospore formation were attempted between Alaskan isolates and known A1 and A2 mating types of P. drechsleri Tucker, P. cambivora (Petri) Buisman, and P. cinnamomi Rands on clarified V8 agar. Morphological characteristics were compared with those described by Waterhouse (19 63). Electrophoretic protein patterns of Alaskan Phytophthora isolates were compared with those of other Phytophthora species found on conifers in the Pacific Northwest, including P. drechsleri, P. cinnamomi, P. megasperma, P. lateralis and P. pseudotsugae. Protein was extracted according to methods previously described (Hamrn & Hansen, 1983) and separated with sodium dodecyl sulphate (SDS) polyacrylamide gels. Pathogenicity testing Pathogenicity of Phytophthora isolates recovered from Alaska and P. drechsleri from Oregon was tested on 2- to 4-year-old Alaska-cedar and z-yearold Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings. Two isolates from each source were grown separately for 3 wk in cornmeal-sand (Hansen et al., 1979), then mixed to produce two composite inocula, one of Alaskan isolates and one of Oregon isolates. Cornmeal-sand inoculum was
combined 1: 16 with pasteurized potting mix (1: 1: 1, peat: vermiculite: sand); sterile cornmealsand was combined with potting mix as a control treatment. Six seedlings per treatment were then transplanted into 450-ml pots containing the inoculum mix. Pots were flooded with water for 65 h, then watered to saturation daily for 8 wk. The test was repeated. Root disease was evaluated on a scale of 0-4: 0 = no roots dead; 1 = 1-25 %, 2 = 26-50%, 3 = 51-75 %, and 4 = 76-100% of roots dead. Pathogenicity of three isolates of Phytophthora from Alaska and two of P. lateralis Tucker & Milbrath from Oregon to stems of Port-Orfordcedar (Chamaecyparis lawsoniana (A. Murr.) Parl.) and Alaska-cedar seedlings was also tested. Small portions of mycelium from 5-d-old pea broth cultures were placed in 1 em longitudinal cuts to the cambium on at least four seedlings per tree species, then coated with petrolatum to retard drying. The number of seedlings girdled after 8 wk was recorded by treatment. RESULTS
Phytophthora was isolated infrequently in 1984 and 1985, both years from the Peril Strait area but not at all from the other areas. In total, seven isolates were obtained from four different sites within 10 km of each other. In 1984 Phytophthora was recovered from four soil samples collected at two sites approximately 3 km apart on opposite sides of the strait. In 1985 Phytophthora was not recovered from any soil samples, but was isolated from two streams (three pears) entering Peril Strait. One stream flowed through an area of declining Alaskacedar, the other primarily through healthy forest. All pears in the two streams sampled at Slocum Arm were eaten by brown bears; no further isolations were attempted in that area. Phytophthora was isolated from 3 pears placed in one small stream (Wren Road Creek) near Juneau. All 10 isolates were identified as P. drechsleri. They produced non-papillate, ovoid to ellipsoid sporangia which were 41 ftm (40-65) long and 36 ftm (30-45) wide on long sporangiophores and which proliferated both internally and externally (Fig. 2). Oogonia were not formed in single-strain cultures on any medium but did form when the Alaskan isolates were paired with an A2 isolate of P. drechsleri from California (0. K. Ribeiro, isolate P545). Oogonia were 47 ftm (39-54 ftm) diam, and pigmented. Antheridia were amphigynous. Electrophoretic protein patterns were indistinguishable from those of P. drechsleri isolates obtained from diseased Douglas-fir seedlings in Oregon and Washington nurseries, but were readily separated
Phytophthora drechsleri in Alaska
A
SOlim
B
Fig.
2.
Sporangia of P. drechsleri from Alaska. Note internal proliferation and long sporangiophores,
from those of other Phytophthora species (Fig. 3)· Alaskan isolates of P. drechsleri caused no root rot on Douglas-fir and only slight damage to Alaska-cedar in pathogenicity tests. Although fine roots of cedar were regularly killed and the total root mass was reduced, damage was confined to < 10 % of the root system and did not differ significantly (t test; P = 0'05) from the uninoculated control. Oregon isolates of P. drechsleri significantly damaged Douglas-fir roots (average rating = 1'1) but not Alaska-cedar. Neither source of P. drechsleri girdled stems of Alaska-cedar or Port-Orford-cedar; Oregon isolates of P. lateralis girdled all inoculated seedlings of both species. DISCUSSION
This is the first report of Phytophthora drechsleri from Alaska, and only the second report of any Phytophthora species from that state: P. infestans was noted on potatoes on Wrangell Island (Cash, 1934). More significant than a range extension, however, is the habitat from which this fungus was isolated. Phytophthora species are seldom recovered (or looked for) outside agricultural or disturbed-
forest settings, and possibly never recovered from areas beyond historical human activity. The search for the Phytophthora gene centre has been controversial. Most attention has focused on P. cinnamomi and its world-wide distribution. Crandall & Filippo-Gravatt (1967) correlated disease outbreaks on all continents except Antarctica with the history of plant introductions and agricultural commerce over the past three centuries. They and others have hypothesized an origin in eastern Asia, a conclusion supported by more recent writers (Shepherd, 1975; Zentmeyer, 1980). Ko, Chang & Su (1978) subsequently isolated the fungus from native, apparently healthy vegetation in Taiwan. P. cinnamomi has also been reported on native vegetation in seemingly undisturbed areas in Australia (Pratt, Heather & Shepherd, 1973) but is more often associated with road building or other disturbance (Brown, 1976). Most recently P. cinnamomi was recovered from native vegetation in South Africa (Van Broembsen & Kruger, 1985). Phytophthora drechsleri is an important pathogen on many agricultural crops around the world (Anon., 1985) but, in contrast to P. cinnamomi, has attracted little speculation about its origin. The only previous report of P. drechsleri on native
E. M. Hansen and others
Fig. 3. Comparison of protein banding patterns of P. drechsleri isolates from Alaska (3 lanes on left) and from Oregon (3 lanes on right) as distinguished by electrophoresis on SDS polyacrylamide gels.
vegetation, from eastern Australia (Pratt & Heather, 1973), prompted the suggestion that it had accompanied P. cinnamomi in its migration from the Pacific Islands north of Queensland (Shepherd, 1975). In western North America, P. drechsleri causes root rot in conifer nurseries (Pratt et al., 1976; Harnm, Hansen & Sutherland, 1985) and other crops but was previously unknown as a forest inhabitant. A strong case can be made that P. drechsieri is indigenous to southeast Alaska. It was found many kilometres from roads, trails, or agricultural settlements, although local Indians foraged in the vicinity and used Alaska-cedar for wood and fibre. Mineral prospecting in the Peril Straits area has been sporadic with no sign of past activity near our study areas. Logging has been extremely limited, confined to small patches adjacent to the water. Some trees had been cut 50 yr earlier along the
lower reaches of one stream where we recovered Phytophthora, but the other sites were well separated and in virgin forest. The stream near Juneau, while near a road, was draining an area of undisturbed muskeg and forest. There has been regular sea traffic through Peril Strait between Sitka and Juneau, Alaska for one hundred years, and Russian traders sailing from St Petersburg stopped in Australia and Sitka on round-the-world voyages from 1813 on (Ivanshintsov, 1980). Poison Cove (site of one Phytophthora isolation) on Peril Strait, is a sheltered harbour and, despite its distance from the scheduled stops, is a conceivable point of introduction. Still, we are unaware of a Phytophthora report in any more remote, less disturbed location. An endemic pathogen is expected to cause minimal damage to the plant communities in which it has evolved (Leppik, 1970). Although P. drechsleri was associated with the widespread mortality of Alaska-cedar near Peril Strait, it was not a constant associate, was never isolated directly from dying cedar (Hennon, 1986), and caused only limited root necrosis on cedar in pathogenicity tests. Epidemiological evidence suggests instead that Alaska-cedar mortality is caused by complex environmental stresses (Hennon, 1986). It seems likely that P. drechsleri inhabits the bogs and adjacent forests common to this area, nibbling on rootlets of many plant species, perhaps including Alaska-cedar. Phytophthora drechsleri may, in fact, be as widespread in the forests of northwestern North America as it apparently is in eastern Australia (Pratt & Heather, 1973; Shepherd & Pratt, 1973). Moreover, geological evidence suggests a link between this disjunct distribution. The present west coast of North America has been built up from the collision over the last 200 million years of several small land masses (terranes) with the ancestral continent through the processes of microplate tectonics (Jones et al., 1982). Indeed, stratigraphic and palaeontological evidence indicates that the Alexander and Wrangellia terranes composing much of the Peril Strait area today originated in the South Pacific as parts of present-day eastern Australia and ancient island arcs (Jones et al., 1982; Gehrels & Saleeby, 1984). Microplate tectonics explains a number of biogeographical problems of the Pacific region (Nur & BenAvaham, 1979), including perhaps the recovery of .P. drechsleri in remote areas of eastern Australia and southeast Alaska. It is presumptuous in the zoth century to hypothesize fungal indigeny based on remoteness from human disturbance (Zentmeyer, 1980). Too much history has passed without a written record,
Phytophthora drechsleri in Alaska even in a place as secluded as southeast Alaska. Better evidence from genetic comparisons of these remote Phytophthora isolates with those from agricultural settings will further our understanding of speciation in this important genus (Hansen, 1986).
We thank Mark Nay for assistance with the isolations. Paper 2205, Forest Research Laboratory, Oregon State University, Corvallis, OR 97331, U.S.A.
REFERENCES
ANON. (1985). Phytophthora drechsleri. CMf Descriptions of Pathogenic Fungi and Bacteria No. 840. Kew: CAB International Mycological Institute. BROWN, B. M. (1976). Phytophthora cinnamomi in tropical rain forests of Queensland. Australian Plant Pathology Society Newsletter 5, 208. CASH,E. K. (1934). Alaskan fungi. Plant Disease Reporter 18,74-88. CRANDALL, B. S. & FILIPPO-GRAVATT, E. (1967). The distribution of Phytophthora cinnamomi. CEfBA 13, 57-7 8. GEHRELS, G. E. & SALEEBY, J. B. (1984). Paleozoic geologic history of the Alexander terrane in S.E. Alaska, and comparisons with other orogenic belts. Geological Society of America Abstract 16, 516. HAMM, P. B. & HANSEN, E. M. (1983). Phytophthora pseudotsugae, a new species causing root rot of Douglasfir. Canadian Journal of Botany 61, 2626-2631. HAMM, P. B. & HANSEN, E. M. (1984). Improved method for isolating Phytophthora lateralis from soil. Plant Disease 68, 517-519. HAMM, P. B., HANSEN, E. M. & SUTHERLAND, J. R. (1985). Phytophthora cryptogea and Phytophthora drechsleri associated with root-rotted Douglas-fir seedlings in British Columbia. Plant Disease 69, 361. HAMM, P. B., HANSEN, E. M., HENNON, P. E. & SHAW III, C. G. (1988). Pythium species from forest and muskeg areas of southeast Alaska. Transactions of the British Mycological Society 91, 385-388. HANSEN, E. M. (1986). Speciation in Phytophthora : Evidence from the P. megasperma complex. In Evolutionary Biology of the Fungi. (British Mycological Society Symposium Volume 12) (ed. A. D. M. Rayner, C. M. Brasier & D. Moore), pp. 325-337. Cambridge, U.K.: Cambridge University Press. HANSEN, E. M., HAMM, P. B., JULIS, A. J. & ROTH, L. F. (1979)· Isolation, incidence, and management of Phytophthora in forest tree nurseries in the Pacific Northwest. Plant Disease Reporter 63, 607-Ql1. HENNON, P. E. (1986). Pathological and ecological aspects of decline and mortality of Chamaecyparis nootkatensis in southeast Alaska. Ph.D. Thesis, Oregon State Univ., Corvallis, OR.
IVANSHINTOV, N. A. (1980). Russian Round-the-world Voyages, 1803-1849. Translated by G. R. Barratt. Ontario, Canada: Limestone Press. JONES, D. L., Cox, A., CONEY, P. & BECK, M. (1982). The growth of western North America. Scientific American 247, 70-84. Ko, W. H., CHANG, H. S. & Su, H. J. (1978). Isolates of Phytophthora cinnamomi from Taiwan as evidence for an Asian origin of the species. Transactions of the British Mycological Society 65, 496-499. LEPPIK, E. E. (1970). Gene centers of plants as sources of disease resistance. Annual Review of Phytopathology 8, 323-344· LINDERMAN, R. G. & ZEITOUN, F. (1977). Phytophthora cinnamomi causing root rot and wilt of nursery-grown native western azalea and salal. Plant Disease Reporter 61, 1°45-1°48. NUR, A. & BEN-AvAHAM, Z. (1979). Speculations on mountain building and the lost Pacifica continent. Journal of Physics of the Earth 26, 21-37. OSTROFSKY, W. D., PRATT, R. G. & ROTH, L. F. (1977). Detection of Phytophthora lateralis in soil organic matter and factors that affect its survival. Phytopathology 67, 79-84. PRATT, B. H. & HEATHER, W. A. (1973). Recovery of potentially pathogenic Phytophthora and Pythium spp. from native vegetation in Australia. Australian Journal of Biological Science 26, 575-582. PRATT, B. H., HEATHER, W. A. & SHEPHERD, C. J. (1973). Recovery of Phytophthora cinnamomi from native vegetation in a remote area of New South Wales. Transactions of the British Mycological Society 60, 197-204. PRATT, R. G., ROTH, L. F., HANSEN, E. M. & OSTROFSKY, W. D. (1976). Identity and pathogenicity of species of Phytophthora causing root rot of Douglas-fir in the Pacific Northwest. Phytopathology 66, 710-714. SHAW, C. G. III, EGLITIS,A., LAURENT, T. H. & HENNON, P. E. (1985). Decline and mortality of Chamaecyparis nootkatensis in Southeast Alaska, a problem of long duration but unknown cause. Plant Disease 69, 13-17. SHEPHERD, C. J. (1975). Phytophthora cinnamomi - an ancient immigrant to Australia. Search 6, 484-49°. SHEPHERD, C. J. & PRATT, B. H. (1973). Separation of two ecotypes of Phytophthora drechsleri Tucker occurring in Australian native forests. Australian Journal of Biological Sciences 26, 1095-11°7. TRIONE, E. J. (1974). Sporulation and germination of Phytophthora lateralis. Phytopathology 64, 1531-1533. VON BROEMBSEN, S. L. & KRUGER, F. J. (1985). Phytophthora cinnamomi associated with mortality of native vegetation in South Africa. Plant Disease 69,715-717. WATERHOUSE, G. (1963). Key to the species of Phytophthora De Bary. Mycological Papers 92. Kew: CAB International Mycological Institute. ZENTMEYER, G. A. (1980). Phytophthora cinnamomi and the diseases it causes. American Phytopathological Society Monograph 10, 1-96.
(Received for publication 1 April 1987 and in revised form 23 September 1987)
Printed in Great Britain
PHYTOPHTHORA DRECHSLERI IN REMOTE AREAS OF SOUTHEAST ALASKA By E. M. HANSEN AND P. B. HAMM Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, U.S.A. C. G. SHAW III USDA Forest Service, Rocky Mountain Forest and Range Experiment Station, Fort Collins, CO 80526, U.S.A. AND P. E. HENNON USDA Forest Service, Forest Pest Management, Juneau, AK 99802, U.S.A.
Phytophthora drechsleri was recovered from remote, undisturbed muskeg and forested sites and from one more accessible area of native vegetation in southeast Alaska. Identity of 10 isolates was confirmed by morphology and behaviour as well as electrophoretic comparison of proteins with those of known isolates. Oospores formed when Alaska isolates were mated with an Az isolate of P. drechsleri from California. Although recovered from soils and streams in forests dominated by declining Chamaecyparis nootkatensis, the fungus was not pathogenic to this tree species in inoculation tests. Its presence in this habitat and its limited pathogenicity suggest that P. drechsleri may be indigenous to the forests of northwestern North America. Phytophthora species are pathogens of many agricultural crops world-wide; however, little is known of their behaviour in native plant communities, and theories on their origins and evolution are largely speculative. Several species, including P. cinnamomi and Pi lateralis, are destructive in forests and other native vegetation, but the susceptibility of the hosts involved, the relatively recent origin of epidemics, and the consistent association of damage with human disturbance suggest that, in most cases at least, the fungi were introduced to the affected areas. Claims that pathogenic fungi are either indigenous or introduced are often controversial, but the question has practical significance for disease control programmes, especially opportunities for effective quarantine. It is also of more fundamental interest in elucidating the evolutionary history of pathogenic fungi and the search for disease resistance (Leppik, 1970). While investigating the causes of decline and mortality of Chamaecyparis nootkatensis (D. Don) Spach. (Alaska-cedar) in southeast Alaska (Shaw et al., 1985; Hennon, 1986; Hamm et al., 1988), we searched for potential pathogens including species of Phytophthora, The isolates were characterized, and their pathogenicity to Alaska-cedar was tested. MA TERIALS AND METHODS
Isolation of Phytophthora was attempted from soil and streams in six areas of southeast Alaska (Fig.
1): Peril Strait, Helm Bay, Slocum Arm, Wrangell Island, Prince of Wales Island, and in the vicinity of Juneau, the capital city. Three of these, Peril Strait, Helm Bay, and Slocum Arm were in remote undisturbed areas many kilometres from roads or permanent settlement and accessible only by boat or float plane.
Soil sampling In summer 1984, 162 one 1 soil samples were collected from the five areas; in summer 1985,77 samples were collected near Peril Strait and 25 near Slocum Arm. Each sample was taken within 0'5 m of the bole of a declining or healthy Alaskacedar. The soil organic fraction was separated by wetsieving (Ostrofsky, Pratt & Roth, 1977); 5 g (wet wt) portions of that fraction were placed in double styrofoam drinking cups and flooded with distilled water (Linderman & Zeitoun, 1977). Hymexazol (Z5 p.p.m.) was added to the baiting solution to inhibit Pythium (Hamm & Hansen, 1984) in 1984 but not in 1985 samples. Alaska-cedar foliage baits z-3 em long were floated in the cups for 6 d to trap Phytophthora from the soil, then transferred to amended cornmeal agar (CMP) containing 20 pg/ ml pimaricin and zoo pg/ml vancomycin in Petri dishes. Phytophthora colonies were subcultured for identification after 6 d. After the 1984 sampling different foliage baits were compared for their
Phytophthora drechsleri in Alaska
o
15 30 45 :miles
Prince of Wales Island
Fig.
1.
Sampling locations in Southeast Alaska: 1, Slocum Arm; 2, Peril Strait; 3, Wrangell Island; 4, Prince of Wales Island; 5, Helm Bay; 6, Juneau.
efficacy in recovering Phytophthora from zoospore suspensions. Two Phytophthora isolates recovered in the 1984 sampling were used. None of eleven species of native plants compared as baits was superior to Alaska-cedar. At the same time, apple, pear, lemon, and avocado fruits were compared as baits. Pears proved to be superior and were used in subsequent stream sampling.
Stream sampling In summer 1985, eight streams draining into Peril Strait and two entering Slocum Arm were sampled for the presence of Phytophthora. Seven of the 10 streams drained from bogs or muskegs with associated dead and dying Alaska-cedar. Streams were up to 4 m wide. Large green d'Anjou pears were placed individually in nylon mesh bags
E. M. Hansen and others anchored along the stream courses at 10 m intervals, starting just above tidewater. Each stream tributary was baited with two pears, one just above the mouth and another 10 m above that; between 33 and 41 pears were placed in each stream system. Pears were removed after 4-6 d and incubated outdoors at ambient temperature (15°C) for 3-7 d until distinct brown necrotic spots appeared. In November, 1987, a total of 77 pears was placed in 5 streams near Juneau. Pears were removed one week later, and incubated as before. Pears were dissected and small portions of tissue from the margins of brown areas were aseptically removed and transferred to CMP. Isolation plates were incubated at room temperature and inspected daily to separate fast-growing Pythium colonies from possible Phytophthora. All Phytophthora colonies were subcultured for identification. Species identification Isolates were maintained on CMP at 5°. To induce sporangia, colonies were grown 5 d in pea broth, washed thoroughly with distilled water, and then flooded with soil extract water for 24-48 h. Isolates also were grown on clarified V -8 agar with or without added Alaska-cedar foliage, grass leaves, or p-sitosterol and on cedar extract agar (Trione, 1974) made with Alaska-cedar foliage. Matings to induce oospore formation were attempted between Alaskan isolates and known A1 and A2 mating types of P. drechsleri Tucker, P. cambivora (Petri) Buisman, and P. cinnamomi Rands on clarified V8 agar. Morphological characteristics were compared with those described by Waterhouse (19 63). Electrophoretic protein patterns of Alaskan Phytophthora isolates were compared with those of other Phytophthora species found on conifers in the Pacific Northwest, including P. drechsleri, P. cinnamomi, P. megasperma, P. lateralis and P. pseudotsugae. Protein was extracted according to methods previously described (Hamrn & Hansen, 1983) and separated with sodium dodecyl sulphate (SDS) polyacrylamide gels. Pathogenicity testing Pathogenicity of Phytophthora isolates recovered from Alaska and P. drechsleri from Oregon was tested on 2- to 4-year-old Alaska-cedar and z-yearold Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings. Two isolates from each source were grown separately for 3 wk in cornmeal-sand (Hansen et al., 1979), then mixed to produce two composite inocula, one of Alaskan isolates and one of Oregon isolates. Cornmeal-sand inoculum was
combined 1: 16 with pasteurized potting mix (1: 1: 1, peat: vermiculite: sand); sterile cornmealsand was combined with potting mix as a control treatment. Six seedlings per treatment were then transplanted into 450-ml pots containing the inoculum mix. Pots were flooded with water for 65 h, then watered to saturation daily for 8 wk. The test was repeated. Root disease was evaluated on a scale of 0-4: 0 = no roots dead; 1 = 1-25 %, 2 = 26-50%, 3 = 51-75 %, and 4 = 76-100% of roots dead. Pathogenicity of three isolates of Phytophthora from Alaska and two of P. lateralis Tucker & Milbrath from Oregon to stems of Port-Orfordcedar (Chamaecyparis lawsoniana (A. Murr.) Parl.) and Alaska-cedar seedlings was also tested. Small portions of mycelium from 5-d-old pea broth cultures were placed in 1 em longitudinal cuts to the cambium on at least four seedlings per tree species, then coated with petrolatum to retard drying. The number of seedlings girdled after 8 wk was recorded by treatment. RESULTS
Phytophthora was isolated infrequently in 1984 and 1985, both years from the Peril Strait area but not at all from the other areas. In total, seven isolates were obtained from four different sites within 10 km of each other. In 1984 Phytophthora was recovered from four soil samples collected at two sites approximately 3 km apart on opposite sides of the strait. In 1985 Phytophthora was not recovered from any soil samples, but was isolated from two streams (three pears) entering Peril Strait. One stream flowed through an area of declining Alaskacedar, the other primarily through healthy forest. All pears in the two streams sampled at Slocum Arm were eaten by brown bears; no further isolations were attempted in that area. Phytophthora was isolated from 3 pears placed in one small stream (Wren Road Creek) near Juneau. All 10 isolates were identified as P. drechsleri. They produced non-papillate, ovoid to ellipsoid sporangia which were 41 ftm (40-65) long and 36 ftm (30-45) wide on long sporangiophores and which proliferated both internally and externally (Fig. 2). Oogonia were not formed in single-strain cultures on any medium but did form when the Alaskan isolates were paired with an A2 isolate of P. drechsleri from California (0. K. Ribeiro, isolate P545). Oogonia were 47 ftm (39-54 ftm) diam, and pigmented. Antheridia were amphigynous. Electrophoretic protein patterns were indistinguishable from those of P. drechsleri isolates obtained from diseased Douglas-fir seedlings in Oregon and Washington nurseries, but were readily separated
Phytophthora drechsleri in Alaska
A
SOlim
B
Fig.
2.
Sporangia of P. drechsleri from Alaska. Note internal proliferation and long sporangiophores,
from those of other Phytophthora species (Fig. 3)· Alaskan isolates of P. drechsleri caused no root rot on Douglas-fir and only slight damage to Alaska-cedar in pathogenicity tests. Although fine roots of cedar were regularly killed and the total root mass was reduced, damage was confined to < 10 % of the root system and did not differ significantly (t test; P = 0'05) from the uninoculated control. Oregon isolates of P. drechsleri significantly damaged Douglas-fir roots (average rating = 1'1) but not Alaska-cedar. Neither source of P. drechsleri girdled stems of Alaska-cedar or Port-Orford-cedar; Oregon isolates of P. lateralis girdled all inoculated seedlings of both species. DISCUSSION
This is the first report of Phytophthora drechsleri from Alaska, and only the second report of any Phytophthora species from that state: P. infestans was noted on potatoes on Wrangell Island (Cash, 1934). More significant than a range extension, however, is the habitat from which this fungus was isolated. Phytophthora species are seldom recovered (or looked for) outside agricultural or disturbed-
forest settings, and possibly never recovered from areas beyond historical human activity. The search for the Phytophthora gene centre has been controversial. Most attention has focused on P. cinnamomi and its world-wide distribution. Crandall & Filippo-Gravatt (1967) correlated disease outbreaks on all continents except Antarctica with the history of plant introductions and agricultural commerce over the past three centuries. They and others have hypothesized an origin in eastern Asia, a conclusion supported by more recent writers (Shepherd, 1975; Zentmeyer, 1980). Ko, Chang & Su (1978) subsequently isolated the fungus from native, apparently healthy vegetation in Taiwan. P. cinnamomi has also been reported on native vegetation in seemingly undisturbed areas in Australia (Pratt, Heather & Shepherd, 1973) but is more often associated with road building or other disturbance (Brown, 1976). Most recently P. cinnamomi was recovered from native vegetation in South Africa (Van Broembsen & Kruger, 1985). Phytophthora drechsleri is an important pathogen on many agricultural crops around the world (Anon., 1985) but, in contrast to P. cinnamomi, has attracted little speculation about its origin. The only previous report of P. drechsleri on native
E. M. Hansen and others
Fig. 3. Comparison of protein banding patterns of P. drechsleri isolates from Alaska (3 lanes on left) and from Oregon (3 lanes on right) as distinguished by electrophoresis on SDS polyacrylamide gels.
vegetation, from eastern Australia (Pratt & Heather, 1973), prompted the suggestion that it had accompanied P. cinnamomi in its migration from the Pacific Islands north of Queensland (Shepherd, 1975). In western North America, P. drechsleri causes root rot in conifer nurseries (Pratt et al., 1976; Harnm, Hansen & Sutherland, 1985) and other crops but was previously unknown as a forest inhabitant. A strong case can be made that P. drechsieri is indigenous to southeast Alaska. It was found many kilometres from roads, trails, or agricultural settlements, although local Indians foraged in the vicinity and used Alaska-cedar for wood and fibre. Mineral prospecting in the Peril Straits area has been sporadic with no sign of past activity near our study areas. Logging has been extremely limited, confined to small patches adjacent to the water. Some trees had been cut 50 yr earlier along the
lower reaches of one stream where we recovered Phytophthora, but the other sites were well separated and in virgin forest. The stream near Juneau, while near a road, was draining an area of undisturbed muskeg and forest. There has been regular sea traffic through Peril Strait between Sitka and Juneau, Alaska for one hundred years, and Russian traders sailing from St Petersburg stopped in Australia and Sitka on round-the-world voyages from 1813 on (Ivanshintsov, 1980). Poison Cove (site of one Phytophthora isolation) on Peril Strait, is a sheltered harbour and, despite its distance from the scheduled stops, is a conceivable point of introduction. Still, we are unaware of a Phytophthora report in any more remote, less disturbed location. An endemic pathogen is expected to cause minimal damage to the plant communities in which it has evolved (Leppik, 1970). Although P. drechsleri was associated with the widespread mortality of Alaska-cedar near Peril Strait, it was not a constant associate, was never isolated directly from dying cedar (Hennon, 1986), and caused only limited root necrosis on cedar in pathogenicity tests. Epidemiological evidence suggests instead that Alaska-cedar mortality is caused by complex environmental stresses (Hennon, 1986). It seems likely that P. drechsleri inhabits the bogs and adjacent forests common to this area, nibbling on rootlets of many plant species, perhaps including Alaska-cedar. Phytophthora drechsleri may, in fact, be as widespread in the forests of northwestern North America as it apparently is in eastern Australia (Pratt & Heather, 1973; Shepherd & Pratt, 1973). Moreover, geological evidence suggests a link between this disjunct distribution. The present west coast of North America has been built up from the collision over the last 200 million years of several small land masses (terranes) with the ancestral continent through the processes of microplate tectonics (Jones et al., 1982). Indeed, stratigraphic and palaeontological evidence indicates that the Alexander and Wrangellia terranes composing much of the Peril Strait area today originated in the South Pacific as parts of present-day eastern Australia and ancient island arcs (Jones et al., 1982; Gehrels & Saleeby, 1984). Microplate tectonics explains a number of biogeographical problems of the Pacific region (Nur & BenAvaham, 1979), including perhaps the recovery of .P. drechsleri in remote areas of eastern Australia and southeast Alaska. It is presumptuous in the zoth century to hypothesize fungal indigeny based on remoteness from human disturbance (Zentmeyer, 1980). Too much history has passed without a written record,
Phytophthora drechsleri in Alaska even in a place as secluded as southeast Alaska. Better evidence from genetic comparisons of these remote Phytophthora isolates with those from agricultural settings will further our understanding of speciation in this important genus (Hansen, 1986).
We thank Mark Nay for assistance with the isolations. Paper 2205, Forest Research Laboratory, Oregon State University, Corvallis, OR 97331, U.S.A.
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(Received for publication 1 April 1987 and in revised form 23 September 1987)