Mycologia, 101(1), 2009, pp. 129–135. DOI: 10.3852/07-203 # 2009 by The Mycological Society of America, Lawrence, KS 66044-8897
Phytophthora rosacearum and P. sansomeana, new species segregated from the Phytophthora megasperma ‘‘complex’’ Everett M. Hansen1
unrelated species. Hansen and colleagues (Hansen et al 1986) defined six distinct ‘‘subgroups’’ within P. megasperma. In this paper we describe two of these groups as new species. Phytophthora megasperma, as conceived by Drechsler (1931), was a homothallic species with unusually large oogonia and nonpapillate sporangia. It first was described as a pathogen on hollyhock (Althaea rosea), but the host range is broad (Erwin and Ribeiro 1996). Tompkins and colleagues (1936) extended the species concept to include isolates with smaller spores and from other hosts, and isolates from alfalfa, soybean and clover subsequently were included (Erwin 1965, Hildebrand 1959, Pratt 1981, Waterhouse 1963). The diversity within the expanded species was recognized through a series of formal and informal nomenclatural proposals calling for segregated species (Kaufmann and Gerdemann 1958), varieties (Hildebrand 1959, Waterhouse 1963) or forma speciales (Kuan and Erwin 1980, Pratt 1981) based on oospore size or host specificity. The resulting confusion triggered a reexamination of the species as broadly defined. Hansen and colleagues (1986) gathered a large group of isolates and compared them for growth behavior, pathogenicity, protein patterns and karyotype as well as morphology. Phytophthora megasperma was recognized as a complex of distinct ‘‘emerging biological species groups’’ distinguished largely on protein profiles, pathogenicity and cultural morphology. Hansen and Maxwell (1991) subsequently recognized the legume pathogens P. sojae Kauf. and Gerd., P. medicaginis Hansen & Maxwell and P. trifolii Hansen & Maxwell as species distinct from P. megasperma Drechsler. They noted at the time that other species remained to be segregated from the complex. Cooke and colleagues (2000) provided the first comprehensive molecular phylogeny for Phytophthora, arranging some 50 species among eight clades based on ITS DNA sequences. This work confirmed the polyphyly of P. megasperma sensu latu, with P. sojae, P. medicaginis (and P. trifolii) and P. megasperma s.s. placed in three clades. Brasier et al (2003) followed with a detailed phylogenetic examination of clade 6, the P. megasperma/P. gonapodyides clade. They recognized at least 11 phenotypically distinct taxa within the clade, most lacking formal nomenclatural designation.
Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331
Wayne F. Wilcox Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, New York 14456
Paul W. Reeser Wendy Sutton Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331
Abstract: Phytophthora megasperma sensu lato was a conglomeration of morphologically similar but phylogenetically unrelated species. In this paper we continue the segregation of species from the old P. megasperma complex, formally naming two previously recognized isolate groups. Isolates recovered from rosaceous fruit trees (especially apple and cherry) are in ITS clade 6, related to but distinct from P. megasperma sensu strictu. They are named here Phytophthora rosacearum. They have been referred to previously as the ‘‘AC’’ or ‘‘high temperature small oospore’’ group of P. megasperma. A second group of isolates, earlier called ‘‘soybean race non-classifiable’’, recovered from soybeans in Indiana and other Midwestern states, are morphologically similar to P. megasperma sensu strictu but unrelated to that species, falling in ITS clade 8. They are named here P. sansomeana. Isolates recovered from Douglas-fir seedlings in nurseries in the Pacific Northwest and various weedy hosts in New York State, referred to in earlier work as ‘‘P. megasperma DF1’’, appear to be conspecific with the soybean isolates, although they include certain ITS DNA polymorphisms. Both new species are supported by a combination of new and previously published morphological, growth and molecular data. Key words: apple-tree collar rot, clade 6, Douglas-fir root disease, ITS phylogeny, plant pathogens, soybean root disease INTRODUCTION
Phytophthora megasperma s.l. was a conglomeration of morphologically similar but phylogenetically Accepted for publication 16 September 2008. 1 Corresponding author. E-mail:
[email protected]
129
130
MYCOLOGIA leading edge of the colony and genomic DNA was extracted (Winton and Hansen 2001). DNA was amplified with primers DC6 and ITS4 (Cooke et al 2000). The products were sequenced with DC6, ITS4 and internal primers ITS2 and ITS3 (White et al 1990). Published sequences of other Phytophthora species were downloaded from GenBank, and distance-based phylogenetic analysis was performed in Clustal X v1.81 with neighbor joining tree building options. Support for internal branches was obtained from 1000 bootstrap replicates. The phylogenetic tree was generated with TreeView (Win32). TAXONOMY
Phytophthora rosacearum E.M. Hansen & Wilcox, sp. nov. Homothallica, oosporas in culturis procreans, antheridiis paragynis aut amphigynis, oogoniis medio diam 29–34 mm. Sporangiis in culturis aquosis procreatis, ovoideis ellipsoideis vel pyriformibus, sine papillis, 56 mm longis, media proportione longitudinis latitudinisque 1.5. Coloniis in agaro farina maydis crescentibus 6 mm in die ad 25 C. In Malus, Prunus.
FIGS. 1–8. Fruiting structures of Phytophthora rosacearum and P. sansomeana. 1–4. P. rosacearum. 1. Nested sporangia. 2. Nonpapillate sporangium. 3, 4. Oogonia and oospores, with paragynous antheridia. 5–8. P. sansomeana. 5. Nonpapillate sporangium with internal proliferation. 6. Nonpapillate sporangium. 7, 8. Oogonia and oospores, with paragynous (7) and amphigynous (8) antheridia. Bar 5 20 mm. MATERIALS AND METHODS
Morphological descriptions are based on comparisons of P. megasperma s.l. isolates (Hansen and Hamm 1983, Hansen et al 1986, Wilcox and Mircetich 1987), augmented by new observations as noted. Carrot agar (Brasier 1972) was the standard growth medium for oogonium morphology and growth rate; sporangia were produced on colonies grown in pea broth, washed, then incubated in soil water. Isolates were grown on CMA with beta-sitosterol for DNA extraction. After 2 wk a 5 mm plug was removed from the
Phytophthora rosacearum is homothallic, with predominately paragynous antheridia. Oogonia average 29–34 mm diam (range of isolate means). Sporangia are ovoid or obpyriform, nonpapillate, with a slight apical thickening, and about 56 mm long, with a length to width ratio of 1.5 (FIGS. 1–4). Sporangia form in water on loosely sympodial sporangiophores. Proliferation is typically internal, occasionally lateral from beneath the sporangium. Chlamydospores are not formed in agar. Radial growth on CMA at 25 C is about 6 mm/d, with a growth optimum at 30 C and a maximum temperature for growth at 36 C. A distinctive strongly petaloid colony pattern develops on carrot agar (illustrated in Hansen et al 1986). Holotype. OSU Isolate 52, from apple in California. Originally collected by John Mircetich as isolate 20-3-9. In Herbarium OSC (dried culture) and American Type Culture Collection MYA-4456. GenBank EU925376. Other isolates examined. OSU Isolate 55 from apricot in Maryland; Mircetich isolate 4-1-5. OSU Isolate 62 from cherry in California; Mircetich isolate 24-1-9. OSU Isolate 63 from cherry in California; Wilcox isolate 261-S1. OSU Isolate 65 from apple in California; Mircetich isolate 24-4-9. Etymology. The name is derived from Rosaceae, the family of fruit trees most frequently parasitized by this pathogen. Phytophthora rosacearum was referred to as the ‘‘high temperature/small oogonia’’ group of P. megasperma s.l. (Wilcox and Mircetich 1987) and the AC, or apple-cherry protein group (Hansen et al 1986) (TABLE I). Phytophthora rosacearum is an important pathogen of rosaceous fruit trees, including
HANSEN ET AL: TWO NEW SPECIES
131
morphologically similar species (TABLE II) by electrophoretic protein pattern (Hansen et al 1986), mitochondrial and chromosomal DNA restriction fragment length polymorphisms (Fo¨rster et al 1988, 1989, Fo¨rster and Coffey 1993) and ITS sequence (Fo¨rster et al 2000, Brasier et al 2003). Similar species.—P. rosacearum is morphologically similar to the species in Waterhouse (1963) group 5, hence its early misidentification as P. megasperma. It is most readily distinguished from P. megasperma s.s. and morphologically similar species by its distinctive strongly rosette-petaloid colony morphology when grown on CA (illustrated in Hansen et al 1986 and Brasier et al 2003), as well as its higher growth temperature. It differs from P. gonapodyides and other self-sterile species in ITS clade 6 by its homothallism and from the homothallic members of the clade by its distinctive colony morphology. Phytophthora sansomeana E.M. Hansen & Reeser, sp. nov. FIG. 9. Phylogenetic tree based on ITS sequences, showing positions of P. rosacearum and P. sansomeana among the clades of Phytophthora (Cooke et al 2000).
apple and cherry in California (Wilcox and Mircetich 1985a, b), apple in New York (Jeffers et al 1982, Wilcox unpubl), apricot in Maryland and peach in Ohio (Hansen et al 1986, Wilcox unpubl). Phylogeny.—P. rosacearum falls in ITS clade 6 (Cooke et al 2000) with P. megasperma s.s., P. gonapodyides and a number of other poorly identified taxa (Brasier et al 2003). ITS DNA sequences distinguish P. rosacearum from P. megasperma s.s. and other Phytophthora species (FIG. 9). It is readily distinguished from related species in clade 6 (Cooke et al 2000) and unrelated but
Homothallica, oosporas in culturis procreans, antheridiis pro parte maxima paragynis, oogoniis 37–45 mm diam. Sporangiis in culturis aquosis procreatis, ovoideis ellipsoideis vel pyriformibus, sine papillis, 54–59 mm longis, media proportione longitudinis latitudinisque 1.5–1.6. Coloniis in agaro farina maydis crescentibus 7 mm in die ad 25 C. In Glycine max, Pseudotsuga menziesii, et Daucus, Trifolium.
Phytophthora sansomeana is a homothallic species with predominately paragynous antheridia. Oogonia of soybean isolates average 39 mm diam (isolate means 37–41 mm). Oogonia of isolates from other hosts are somewhat larger, averaging 40–45 mm diam. Sporangia are ovoid or obpyriform, nonpapillate, and average 56 mm long, (isolate means 54–59 mm) with a usual length to width ratio of 1.5–1.6 (FIGS. 5–8). Sporangia form in water, on long, unbranched or loosely sympodial sporangiophores. Proliferation is
TABLE I. Concordance of previous reports of isolate groups formerly referred to Phytophthora megasperma and now included in P. rosacearum, P. sansomeana and P. megasperma sensu strictu Species P. rosacearum P. sansomeana
P. megasperma s.s.
Previous Designation
Reference
AC (apple-cherry) High/small DF DF1 Group1 D1 Soybean race non-classifiable BHR Low/large DF2 Group 2 AL2, D2
Hansen et al 1986 Wilcox and Mircetich 1987 Hansen et al 1986 Wilcox and Mircetich 1987 Hamm and Hansen 1987 Hansen and Hamm 1983 Reeser et al 1991 Hansen et al 1986 Wilcox and Mircetich 1987 Wilcox and Mircetich 1987 Hamm and Hansen 1987 Hansen and Hamm 1983
V 8 clover 37–46 mm aerial, tufted, no pattern 3.3 23 C V 8 alfalfa 33–43 mm dense aerial, no pattern 4.7 26 C V 7 soybean 35–40 mm dense aerial, no pattern 4.9 20 C V 6 various 39–56 mm moderately aerial, broadly patterned to no pattern 6.3 20–25 C 6.1 30 C Growth rate mm/day (25 C) Optimum temperature
V 8 soybean 39–45 mm moderately aerial, broadly petaloid 7–10.0 27 C V 6 apple, cherry 33–41 mm strongly petaloid Waterhouse Morphological Group Cooke et al (2000) ITS DNA clade Primary hosts Oogonium diam Colony pattern on carrot agar
P. rosacearum
P. sansomeana
P. megasperma s.s.
P. sojae
P. medicaginis
P. trifolii
MYCOLOGIA TABLE II. Distinguishing features of Phytophthora species segregated from P. megasperma sensu Waterhouse. Data compiled from this paper and Hansen et al 1986, Hansen and Maxwell 1991, Hamm and Hansen 1987, Hansen and Hamm 1983 and Wilcox and Mircetich 1987
132
typically internal, occasionally lateral from beneath the sporangium. Chlamydospores are not formed in agar. It is morphologically similar to other species of the P. megasperma complex sensu Waterhouse (1963) (TABLE II) but can be distinguished statistically by the combined features of oogonium and sporangium size (Hansen and Hamm 1983). Growth of soybean isolates on CMA averages 7 mm/d at 25 C, and isolates from other hosts grow up to 10 mm/d, with optimum growth at 25–27 C and a maximum growth temperature above 35 C. Colonies exhibit a moderately aerial, broadly petaloid pattern on carrot agar (illustrated in Hansen et al 1986). Holotype. OSU isolate 1819B, from soybean in Indiana, collected by Reeser, in Herbarium OSC (dried culture) and American Type Culture Collection MYA-4455. GenBank EU925375. Other isolates examined. OSU isolates 2516A, 2323, and 9284 from soybean in Indiana; collected by Reeser. OSU isolate 20, from a Douglas-fir seedling in Oregon; Hamm isolate B3A, ATCC 46374. OSU isolate 21, from a Douglas-fir seedling in Oregon, Hamm isolate B2-17, ATCC 46373. OSU Isolate 22, from a Douglas-fir seedling in Oregon, Hamm isolate 345, ATCC 46375. OSU Isolate 77, from a Douglas-fir seedling in Oregon, Hamm isolate 304. OSU isolate 44, from white clover in New York; Stack isolate WC1(75). OSU isolate 46, from wild carrot in New York; Stack isolate Car2(86). OSU isolate 72, from white cockle in New York; Stack isolate WC1(88). Etymology. The name commemorates Dr Eva Sansome, whose astute cytological observations established the diploid nature of Phytophthora and other oomycetes. P. sansomeana first was characterized based on isolates from Douglas-fir seedlings in nurseries in Oregon and weeds (white clover, wild carrot, white cockle) in alfalfa fields in New York. They were labeled variously P. megasperma DF, DF1, D1, and Group 1 (Hansen et al 1986, Hansen and Maxwell 1991, Hamm and Hansen 1987, Hansen and Hamm 1983) (TABLE I). More recently it was recognized that an unnamed Phytophthora species from soybeans in the Midwest was morphologically similar and shared growth and pathogenicity characteristics. Because P. sansomeana from soybean is better characterized ecologically, including pathogenicity, we have chosen an Indiana soybean isolate as type. Isolates of P. sansomeana from Oregon, New York and the Midwestern United States carry ds RNA (Kuhn, Collins, Hansen unpubl, Hacker et al 2005). Whisson et al (1993; their isolate P1330), and Fo¨rster and Coffey (1993; their group F1) distinguished P. sansomeana from similar species by RAPD and RFLP DNA analysis. Fo¨ rster et al (2000)
HANSEN ET AL: TWO NEW SPECIES distinguished an isolate of P. sansomeana (their P3163, OSU No. 72) from other species of Waterhouse groups V and VI with ITS sequence analysis. Phylogeny.—Phylogenetically P. sansomeana falls in ITS clade 8 (Cooke et al 2000). ITS DNA sequences distinguish P. sansomeana from P. megasperma s.s. and other Phytophthora species (FIG. 9). Isolates of P. sansomeana from Douglas-fir in Oregon, weeds in alfalfa fields in New York and from soybeans in the Midwest represent phylogenetically closely related populations (see DISCUSSION). Isolates are morphologically indistinguishable, have similar appearance in culture and exhibit similar pathogenicity on Douglasfir and soybean. Similar species.—P. sansomeana is morphologically similar to the species in Waterhouse’s group 5, hence its early misidentification as P. megasperma. It is most readily distinguished from P. megasperma and similar species by its faster growth, pathogenic behavior and host preferences (TABLE II). It differs from other species in ITS clade 8 by its homothallism, distinguishing it from the heterothallic P. drechsleri and P. cryptogea, and by faster growth with different colony morphology than P. medicaginis and P. trifolii. It does not exhibit the host specific pathogenicity of the latter two species. Pathogenicity.—In Jul 1990 root rot epidemics afflicted numerous soybean fields over a large part of Indiana, especially in the east-central counties. The disease affected certain cultivars with the Rps1-k gene for resistance to root rot caused by Phytophthora sojae Kauf. & Gerd. as well as some cultivars with ratereducing resistance (field tolerance) and some susceptible cultivars. Aboveground symptoms included whole plant wilting or yellowing and stunting. Lateral roots were discolored, rotted or missing, and taproots exhibited internal discoloration and frequently were rotted through below the soil line. Externally visible stem discoloration symptoms commonly associated with late season onset of Phytophthora sojae root rot usually were absent. Additional isolations from Indiana soybean fields were made in 1991 and 1992. A preliminary report was published (Reeser 1991) and new information is provided here. In soybean hypocotyl inoculation tests seedlings of all race-specific resistant differential cultivars suffered varying levels of mortality. Plants exhibited an overall wilt, in contrast to the girdling lesion typical of the hypersensitive response expressed in plants inoculated with isolates of P. sojae. Soybean seedlings grown in soil infested with the new Phytophthora species exhibited yellowing, stunting and root rot similar to that exhibited by diseased plants
133
from which the isolates were first recovered. Symptom development was greatest at 15 and 20 C. Uninoculated control plants remained healthy. The new Phytophthora species was re-isolated from diseased stems of hypocotyl-inoculated seedlings and from diseased roots of rootinoculated plants. Oospores were observed in squash mounts of infected roots. Douglas-fir isolates of P. sansomeana induced similar reactions in soybean hypocotyl and root inoculations as isolates from soybean and were aggressive pathogens of Douglas-fir seedlings in inoculation trials (Hamm and Hansen 1981, Hansen and Hamm 1983) and in a forest tree nursery (Hansen et al 1981, Hamm and Hansen 1987). Isolates from weeds in alfalfa fields in New York have pathogenicity characteristics on Douglas-fir and the legume hosts identical to the isolates from Douglas-fir (Hansen et al 1986). DISCUSSION
Phytophthora megasperma sensu Waterhouse (1963) illustrates the limitations of morphological taxonomy in this genus. Host specific pathogenicity exhibited by some isolate groups provided the first indication that this was not a homogeneous species; as molecular techniques and phylogenetic analysis have improved in recent years, the full complexity has become evident. After segregation of the unrelated species, including P. sansomeana described here, Phytophthora megasperma, as first described by Drechsler in 1931, remains a legitimate and useful species. It corresponds in large part to P. megasperma var. megasperma of Waterhouse (1963). P. megasperma s.s. causes a nonspecific root rot of many plant species, including Douglas-fir and alfalfa. It is also found in the absence of apparent disease in wet soils and water in forests in Europe (Brasier et al 1993, Hansen and Delatour 1999) and western North America (Hansen unpubl). It is closely allied phylogenetically in ITS clade 6 (FIG. 9) with P. rosacearum and the sterile P. gonapodyides (Brasier et al 2003, Cooke et al 2000). As pointed out by Brasier et al (2003) more taxa in this clade still are awaiting better understanding and nomenclatural designation. Similarly there remain isolate groups that have been referred to broadly as Phytophthora megasperma but which represent unrelated taxa. Isolates from asparagus were labeled P. megasperma (Boesewinkel 1974) but based on genetic evidence (Cooke et al 2000, Whisson et al 1993) represent a distinct species. Isolates identified as P. megasperma from rose (Nagai et al 1978) also have different protein patterns and behavior from P. megasperma s.s. and its known segregates (Hansen et al 1986).
134
MYCOLOGIA
While host specificity is an important feature of some Phytophthora species, host association must be used cautiously in identification. Phytophthoras from alfalfa, Douglas-fir and soybean exemplify the problem. Alfalfa fields may harbor P. medicaginis, P. megasperma s.s. and P. sansomeana. Isolates from Douglas-fir may be either P. megasperma s.s. or P. sansomeana, and now it is recognized that isolates from soybean may be either P. sojae or P. sansomeana. Phytophthora sansomeana as described here may represent two sibling species and a hybrid population. Isolates from weeds in alfalfa fields in New York and soybeans in the Midwest appear to represent related but distinct populations, based on ITS DNA polymorphisms. In ITS sequence electropherograms from Douglas-fir isolates however we observed two overlapping peaks (double peaks) at 7 of 8 polymorphic loci (of 826 loci sequenced; data not shown). At each double peak locus one nucleotide matched the corresponding single peak from soybean isolates and the other nucleotide matched the single peak from weed isolates. Similar double-peak patterns were observed in ITS sequences of an unidentified Phytophthora isolated from Abies Christmas trees in Michigan (Fulbright et al 2006). Double-peak patterns in ITS DNA sequences have been interpreted to indicate a hybrid origin of some Phytophthora species (e.g. Brasier et al 1999). Additional isolates must be collected and additional genes analyzed to resolve these points. At this time we consider all three groups of isolates to represent P. sansomeana based on their shared phylogeny, morphology, growth behavior and pathogenicity. LITERATURE CITED
Boesewinkel HJ. 1974. Phytophthora on asparagus in New Zealand. Plant Disease Reporter 58:525–529. Brasier CM. 1972. Observations on the sexual mechanism in Phytophthora palmivora and related species. Trans Br Mycol Soc 58:237–251. ———, Robredo F, Ferraz JFP. 1993. Evidence for Phytophthora cinnamomi involvement in Iberian oak decline. Plant Path 42:140–145. ———, Cooke DEL, Duncan JM. 1999. Origin of a new Phytophthora pathogen through interspecific hybridization. Proc Nat Acad Sci USA 96:5878–5883. ———, ———, ———, Hansen EM. 2003. Multiple new phenotypic taxa from trees and riparian ecosystems in Phytophthora gonapodyides-P. megasperma ITS Clade 6, which tend to be high-temperature tolerant and either inbreeding or sterile. Mycol Res 107:277–290. Cooke DEL, Drenth A, Duncan JM, Wagels G, Brasier CM. 2000. A molecular phylogeny of Phytophthora and related Oomycetes. Fungal Genet Biol 30:17–32.
Drechsler C. 1931. A crown rot of hollyhock caused by Phytophthora megasperma n. sp. J Wash Acad Sci 21:513– 526. Erwin DC. 1965. Reclassification of the causal agent of root rot of alfalfa from Phytophthora cryptogea to P. megasperma. Phytopathology 55:1139–1143. ———, Ribeiro OK. 1996. Phytophthora diseases worldwide. St Paul, Minnesota: APS Press. 562 p. Fo¨rster H, Coffey MD. 1993. Molecular taxonomy of Phytophthora megasperma based on mitochondrial and nuclear DNA polymorphisms. Mycol Res 97:1101–1112. ———, Cummings MP, Coffey MD. 2000. Phylogenetic relationships of Phytophthora species based on ribosomal ITS 1 DNA sequence analysis with emphasis on Waterhouse groups V and VI. Mycol Res 104:1055– 1061. ———, Kinscherf TG, Leong SA, Maxwell DP. 1988. Estimation of relatedness between Phytophthora species by analysis of mitochondrial DNA. Mycologia 80:466– 478. ———, ———, ———, ———. 1989. Restriction fragment length polymorphism of the mitochondrial DNA of Phytophthora megasperma isolated from soybean, alfalfa, and fruit trees. Can J Bot 67:529–537. Fulbright DW, Jacobs J, Stadt S, Catal M, Vettraino AM, Vannini A. 2006. Phytophthora root rot of Abies and Pinus in Michigan. In: Brasier C, Jung T, Osswald W, eds. Progress in research on Phytophthora diseases of forest Trees. Alice Holt Lodge, Farnham, Surrey, UK: Forest Research. p 155–156. Hacker CV, Brasier CM, Buck KW. 2005. A double-stranded RNA from a Phytophthora species is related to the plant endornaviruses and contains a putative UDP glycosyltransferase gene. J Gen Virol 86:1561–70. Hamm PB, Hansen EM. 1981. Host specificity of Phytophthora megasperma from Douglas-fir, soybean and alfalfa. Phytopathology 71:65–68. ———, ———. 1987. Identification of Phytophthora spp. known to attack conifers in the Pacific Northwest. NW Sci 61:103–109. Hansen EM, Delatour C. 1999. Phytophthora species in oak forests of north-east France. Ann Fo Sci 56:539–547. ———, Hamm PB. 1983. Morphological differentiation of host-specialized groups of Phytophthora megasperma. Phytopathology 73:129–134. ———, ———, Julis AJ, Roth LF. 1981. Isolation, incidence and management of Phytophthora in forest tree nurseries in the Pacific Northwest. Plant Disease Reporter 63:607–611. ———, Maxwell DP. 1991. Species of the Phytophthora megasperma complex. Mycologia 83:376–381. ———, Brasier CM, Shaw DS, Hamm PB. 1986. The taxonomic structure of Phytophthora megasperma: evidence for emerging biological species groups. Trans Br Mycol Soc 87:557–573. Hildebrand AA. 1959. A root and stalk rot of soybean caused by Phytophthora megasperma Drechsler var. sojae var. nov. Can J Bot 37:927–957. Jeffers SN, Aldwinkle HS, Burr TJ, Arneson PA. 1982. Phytophthora and Pythium species associated with crown
HANSEN ET AL: TWO NEW SPECIES rot in New York apple orchards. Phytopathology 72: 533–538. Kaufmann JJ, Gerdemann JW. 1958. Root rot of soybeans caused by Phytophthora sojae n. sp. Phytopathology 48: 201–208. Kuan TL, Erwin DC. 1980. Formae speciales differentiation of Phytophthora megasperma isolates from soybean and alfalfa. Phytopathology 70:333–338. Nagai Y, Takeuchi T, Watanabe T. 1978. A stem blight of rose caused by Phytophthora megasperma. Phytopathology 67:684–688. Pratt RG. 1981. Morphology, pathogenicity and host range of Phytophthora megasperma, P. erythroseptica and P. parasitica from arrow leaf clover. Phytopathology 71: 276–282. Reeser PW, Scott DH, Ruhl DE. 1991. Recovery of race nonclassifiable Phytophthora megasperma f. sp. glycinea from soybean roots in Indiana in 1990. Phytopathology 81: 1201. Tompkins CM, Tucker CM, Gardner MW. 1936. Phytophthora root rot of cauliflower. J Agric Res 53:685–692. Waterhouse GM. 1963. Key to the species of Phytophthora de Bary. Mycological Papers 92. Commonwealth Mycological Institute, Kew, UK.
135
Whisson SC, Howlett BJ, Liew ECY, Maclean DJ, Manners JM, Irwin JAG. 1993. An assessment of genetic relationships between members of the Phytophthora megasperma complex and Phytophthora vignae using molecular markers. Aust Syst Bot 6:295–308. White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: a guide to methods and applications. San Diego: Academic Press. p 315–321. Wilcox WF, Mircetich SM. 1985a. Effects of flooding duration on the development of Phytophthora root and crown rots of cherry. Phytopathology 75:1451– 1455. ———, ———. 1985b. Pathogenicity and relative virulence of seven Phytophthora spp. on mahaleb and Mazzard cherry. Phytopathology 75:221–226. ———, ———. 1987. Lack of host specificity among isolates of Phytophthora megasperma. Phytopathology 77:1132– 1137. Winton LM, Hansen EM. 2001. Molecular diagnosis of Phytophthora lateralis in trees, water and foliage baits using multiplex polymerase chain reaction. Forest Pathol 31:275–283.
