Phytophthora betacei, a new species within Phytophthora clade 1c causing late blight on Solanum betaceum in Colombia.

Over the past few years, symptoms akin to late blight disease have been reported on a variety of crop plants in South America. Despite the economic importance of these crops, the causal agents of the diseases belonging to the genus Phytophthora have not been completely characterized. In this study, a new Phytophthora species was described in Colombia from tree tomato (Solanum betaceum), a semi-domesticated fruit grown in northern South America. Comprehensive phylogenetic, morphological, population genetic analyses, and infection assays to characterize this new species, were conducted. All data support the description of the new species, Phytophthora betacei sp. nov. Phylogenetic analyses suggest that this new species belongs to clade 1c of the genus Phytophthora and is a close relative of the potato late blight pathogen, P. infestans. Furthermore, it appeared as the sister group of the P. andina strains collected from wild Solanaceae (clonal lineage EC-2). Analyses of morphological and physiological characters as well as host specificity showed high support for the differentiation of these species. Based on these results, a complete description of the new species is provided and the species boundaries within Phytophthora clade 1c in northern South America are discussed.

The expansion of Phytophthora clade 8b: three new species associated with winter grown vegetable crops.

Despite its association with important agricultural crops, Phytophthora clade 8b is a poorly studied group of species. The clade currently consists of three officially described species (Phytophthora porri, P. brassicae and P. primulae) that are host-specific pathogens of leek, cabbages and Primula spp., respectively. However, over the past few decades, several other clade 8b-like Phytophthoras have been found on a variety of different host plants that were all grown at low temperatures in winter seasons. In this study, a collection of 30 of these isolates was subjected to a phylogenetic study using two loci (the rDNA ITS region and the mitochondrial cox1 gene). This analysis revealed a clear clustering of isolates according to their host plants. To verify whether these isolates belong to separate species, a detailed morphological study was conducted. On the basis of genetic and morphological differences and host specificity, we now present the official description of three new species in clade 8b: Phytophthora cichorii sp. nov., P. dauci sp. nov. and P. lactucae sp. nov. Two other groups of isolates (Phytophthora taxon castitis and Phytophthora taxon parsley) might also represent new species but the data available at this time are insufficient for an official description. This brings Phytophthora clade 8b to a group of six species that are all host-specific, slow-growing and specifically infect herbaceous crops at low temperatures.

Phylogenetic relationship of Phytophthora cryptogea Pethybr. & Laff and P. drechsleri Tucker.

The phylogeny and taxonomy of Phytophthora cryptogea and Phytophthora drechsleri has long been a matter of controversy. To re-evaluate this, a worldwide collection of 117 isolates assigned to either P. cryptogea, P. drechsleri or their sister taxon, Phytophthora erythroseptica were assessed for morphological, physiological (pathological, cultural, temperature relations, mating) and molecular traits. Multiple gene phylogenetic analysis was performed on DNA sequences of nuclear (internal transcribed spacers (ITS), ß-tubulin, translation elongation factor 1α, elicitin) and mitochondrial (cytochrome c oxidase subunit I) genes. Congruence was observed between the different phylogenetic data sets and established that P. drechsleri and P. cryptogea are distinct species. Isolates of P. drechsleri form a monophyletic grouping with low levels of intraspecific diversity whereas P. cryptogea is more variable. Three distinct phylogenetic groups were noted within P. cryptogea with an intermediate group providing strong evidence for introgression of previously isolated lineages. This evidence suggests that P. cryptogea is an operational taxonomic unit and should remain a single species. Of all the morphological and physiological traits only growth rate at higher temperatures reliably discriminated isolates of P. drechsleri and P. cryptogea. As a homothallic taxon, P. erythroseptica, considered the cause of potato pink rot, is clearly different in mating behaviour from the other two species. Pathogenicity, however, was not a reliable characteristic as all isolates of the three species formed pink rot in potato tubers. The phylogenetic evidence suggests P. erythroseptica has evolved from P. cryptogea more recently than the split from the most recent common ancestor of all three species. However, more data and more isolates of authentic P. erythroseptica are needed to fully evaluate the taxonomic position of this species.

Phytophthora parsiana sp. nov., a new high-temperature tolerant species.

As part of a study to examine the phylogenetic history of the taxonomically challenging species Phytophthora cryptogea and P. drechsleri, a distinct monophyletic group of isolates, previously described as P. drechsleri or P. cryptogea, were characterised. Analysis of their rDNA ITS sequences indicated that these isolates were distinct from P. drechsleri, P. cryptogea, and all members of Phytophthora ITS clades 1-8, clustering instead alongside basal groups previously described as clades 9 and 10. This group comprised six isolates all of which were isolated from woody plants, such as pistachio (Pistacia vera, Iran and USA), fig (Ficus carica, Iran), and almond (Prunus dulcis, Greece). Analysis of sequence data from nuclear (beta-tubulin and translation elongation factor 1alpha) and mitochondrial (cytochrome c oxidase subunit I) genes confirmed the ITS-based analysis as these isolates formed a distinct monophyletic group in all NJ trees. The isolates were fast growing with a relatively high optimum growth temperature of 30 degrees C and, in most cases, rapid colony growth even at 37 degrees C. The isolates produced complex colony patterns on almost all media, especially corn meal agar (CMA). Phylogenetic analysis and examination of all the other morphological and physiological data lead us to infer that this taxon has not been described previously. As this taxon was first isolated and described from Iran we propose that this taxon be formally designated as Phytophthora parsiana.

Wilt and Collapse of Cuphea ignea Caused by Phytophthora tropicalis in Italy.

The genus Cuphea (Lythraceae) includes approximately 250 species of annual, evergreen perennials and short shrubs native to Central and South America. During the springs of 2003 and 2004, 10% of the nursery stock of approximately 12,000 potted cigar-flowers (C. ignea A. DC) grown in a screenhouse at a commercial ornamental nursery near Piedimonte Etneo, Sicily, had symptoms of wilt, defoliation, and rapid collapse of the entire plant. These foliar symptoms were associated with a reduced root system, browning of the collar, and dark brown discolored roots. A Phytophthora species was consistently recovered by plating small pieces of rotted roots of symptomatic plants onto selective medium (3); pure cultures were obtained by single-hypha transfers. On potato dextrose-agar (PDA), cardinal temperatures for growth were 10 to 35°C and the optimum was 28 to 30°C. Sporangiophores were umbellate or in a close monoclasial sympodium and mean dimensions of sporangia were 52 × 26 mm, with a mean length/width ratio of 2:1. Sporangia produced on V8 juice agar (VJA) were ellipsoid, fusiform, or limoniform with a tapered base. They were papillate, occasionally bipapillate, caducous, with a long pedicel (as much as 150 µm). All isolates were mating type A1 determined by pairing with A2 reference isolates of P. palmivora (Butl.) Butl. and P. nicotianae Breda de Haan. Oogonia with amphigynous antheridia were formed on VJA after 10 to 15 days at 24°C in the dark. Occasionally, 10 of 15 isolates formed small chlamydospores on VJA. Electrophoretic patterns of total mycelial proteins and four isozymes (acid and alkaline phosphatase, esterase, and malate dehydrogenase) on polyacrylamide slab gels (3) of all Cuphea isolates were very similar to those of reference isolates of P. tropicalis M. Aragaki & J. Y. Uchida from Convolvulus cneorum L. (IMI 391714) and Rhamnus alaternus L., respectively. In addition, the Cuphea isolates were clearly distinct from reference isolates of other species including P. capsici Leon., P. citricola Sawada, P. citrophthora (R. E. Smith & E. H. Smith) Leon., P. nicotianae, and P. palmivora. On the basis of morphological cultural characters and the electrophoretic phenotype, the isolates were identified as P. tropicalis. Internal transcribed spacer (ITS) regions of rDNA sequences (2) confirmed the identification. Koch's postulates were fulfilled by testing three cigar-flower isolates, including isolate IMI 391709, on 10 6-month-old potted cuttings of Cuphea inoculated by applying a 10-ml zoospore suspension (2 × 104 zoospores/ml) to the crowns, incubated for 24 h at 100% relative humidity, and maintained in the greenhouse at 20 to 24°C. After 10 days, crowns and stems were brown and all plants wilted within 20 days. Ten control plants treated with water remained healthy. P. tropicalis was reisolated from infected tissues. The test was repeated with similar results. In Europe, P. tropicalis has been reported on Cyclamen persicum Mill. in Germany (4) and C. cneorum and R. alaternus in Italy (1), indicating a broad host range and spreading in ornamental nurseries. References: (1) S. O. Cacciola et al. Boll. Acc. Gioenia Sci. Nat. 31:57, 1999. (2) S. O. Cacciola et al. For. Snow Landsc. Res. 76:387, 2001. (3) D. C. Erwin and O. K. Ribeiro. Pages 39-41, 138-139 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul MN. 1996. (4) W. W. P. Gerlach and A. Schubert. Plant Dis. 85:334, 2001.

First Report of Brown Rot and Wilt of Fennel Caused by Phytophthora megasperma in Italy.

Fennel (Foeniculum vulgare Mill. var. azoricum (Mill.) Thell.) in the Apiaceae family is native to southern Europe and southwestern Asia. It is an economically important crop in Italy that produces approximately 85% of all fennel worldwide. The main producing regions are Apulia, Campania, Latium, and Calabria. During the late winter of 2004 in the Crotone Province of the Calabria Region, following heavy rains, patches of fennel plants with symptoms of brown, soft rot of the bulb-like structure formed by the thickened leaf bases, development of yellow leaves, stunting, and wilting of the entire plant were observed in fields. A homothallic Phytophthora sp. was isolated consistently from the brownish tissues of the stout stems and leaf bases of symptomatic plants using a selective medium (3). Pure cultures were obtained by single hyphal tip transfers. On potato dextrose agar (PDA), the diameter of oospores varied from 28 to 42 µm (mean = 36.3 ± 0.4). Antheridia were primarily paragynous. Sporangia were not produced on solid media but were formed in sterile soil extract solution. They were nonpapillate, noncaducous, ovoid and obpyriform (25 to 45 × 35 to 60 µm), and internally proliferating. Optimum and maximum temperatures for radial growth of the colonies on PDA were 25 and 30°C, respectively. At 25°C, radial growth rate was approximately 6 mm per day. On the basis of morphological and cultural characteristics, the isolates were identified as Phytophthora megasperma Drechsler. Electrophoretic patterns of mycelial proteins and four isozymes (acid and alkaline phosphatase, esterase, and malate dehydrogenase) on polyacrylamide gels of the fennel isolates were identical to those of reference isolates of P. megasperma of the BHR (broad host range) group included in P. gonapodyides-P. megasperma Clade 6 (1,3), but distinct from those of the isolates of other nonpapillate species included in Waterhouse's taxonomic group VI. Internal transcribed spacer (ITS) regions of rDNA sequences (2) confirmed that fennel isolates belonged to P. megasperma BHR group. Pathogenicity of a fennel isolate from Calabria (IMI 391711) was confirmed by pouring a zoospore suspension at 2 × 104 zoospores per ml on the soil of 10 3-month-old potted fennel plants. The soil of the inoculated and 10 control seedlings was flooded for 24 h. After 10 days, stems and leaf bases of the seedlings showed a brown rot. Chlorosis and wilting of all seedlings developed after 20 days. Controls inoculated with water did not develop any symptoms. The pathogen was reisolated from typical brown rot and tests were repeated with similar results. To our knowledge, this is the first report of P. megasperma causing disease on fennel. References: (1) S. O. Cacciola et al. For. Snow. Landsc. Res. 76:387, 2001. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) H. Masago et al. Phytopathology, 67:425, 1977.

Root and Foot Rot of Lantana Caused by Phytophthora cryptogea.

Lantana (Lantana camara L.) is an evergreen shrub in the Verbenaceae. In some countries, this plant has been declared a noxious weed. However, a number of sterile or near-sterile forms are cultivated as attractive flowered potted and garden plants. In early spring 2004, ≈4,000 potted, small trees of lantana grown in a screenhouse in a commercial nursery of ornamentals near Giarre, Sicily, showed symptoms of chlorosis, defoliation, and sudden collapse of the entire plant. These aboveground symptoms were associated with a reduced root system, rot of feeder roots, and brown discoloration of the base of the stem. A Phytophthora sp. was isolated consistently from roots and basal stems of symptomatic plants using the selective medium of Masago et al. (3). Cardinal temperatures for radial growth of pure cultures obtained by single hypha transfer were 2°C minimum, 25°C optimum, and 30 to 35°C maximum. Sporangia produced in the saline solution of Chen and Zentmyer (3) were obpyriform, persistent, and nonpapillate. All isolates were A1 mating type and differentiated oospores with amphigynous antheridia in dual cultures with A2 reference isolates of P. cryptogea Pethybr. & Laff. and P. drechsleri Tucker (3). Electrophoretic patterns of total mycelial proteins (3) of the isolates from lantana were very similar to those of reference isolates of P. cryptogea from different hosts, but clearly distinct from those of reference isolates of other species included in Waterhouse's taxonomic group VI (3). Indeed, isolates from lantana were identified as P. cryptogea on the basis of morphological and cultural characters as well as the electrophoretic phenotype. Sequences of internal transcribed spacer (ITS) regions of rDNA (1) confirmed the identification as P. cryptogea. Pathogenicity of a representative isolate from lantana (IMI 392045) was tested in a screenhouse by transplanting 20 6-month-old rooted cuttings of lantana in pots (12 cm in diameter) filled with infested soil; the soil was prepared by mixing steam-sterilized sandy loam soil at a concentration of 4% (vol/vol) with inoculum produced on a mixture of vermiculite and autoclaved oat seeds. Twenty control plants were transplanted in pots containing noninfested soil. The soil was saturated with water by plugging the pots' drainage holes for 48 h and watering. After 40 days, all plants except the controls showed symptoms of root and foot rot, and P. cryptogea was reisolated from infected tissues. To our knowledge, this is the first report of P. cryptogea on lantana. On this host and other species in the verbena family, only P. nicotianae van Breda de Haan (= P. parasitica Dastur) has been previously reported (2,3,4). A possible cause of the high incidence of this disease in the nursery was waterlogging due to heavy rain and excessive irrigation. References: (1) S. O. Cacciola et al. For. Snow Landsc. Res. 76:387, 2001. (2) M. L. Daughtrey et al. Compendium of Flowering Potted Plant Diseases. The American Phytopathological Society, St. Paul, MN, 1995. (3) D. C Erwin and O. K. Ribeiro. Pages 39-41, 84-95, 138-139 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (4) K. H. Lamour et al. Plant Dis. 87:854, 2003.

A New Phytophthora sp. Causing a Basal Canker on Beech in Italy.

In autumn 2001, bleeding cankers were observed on the basal portion of the trunk of a declining tree in a forest stand of European beech (Fagus sylvatica L.) in Latium (central Italy). A Phytophthora sp. was isolated consistently from infected trunk bark using whole apples as bait. Isolations were made from brown lesions that developed in the apple pulp around the inserted bark pieces. Pure cultures were obtained by using hyphal tip transfers. Colonies were stellate on V8 juice agar (V8A), uniform to slightly radiate on cornmeal agar, and cottony, without a distinct growth pattern on potato dextrose agar (PDA). On V8A, radial growth rates were 2.1, 4.8, and 4.5 mm/day at 10, 15, and 20°C, respectively. Colonies grew slowly at 5 and 25°C, but failed to grow at 30°C. On PDA, growth was 1.7 and 1.4 mm/day at 15 and 20°C, respectively. Catenulate hyphal swellings formed on solid and liquid media. Sporangia formed abundantly at 15°C, were ovoid to obpyriform, semipapillate, occasionally bipapillate, and had narrow exit pores (mean diameter = 5.4 µm). On V8A, pores were 40 to 50 µm in length and 25 to 40 µm in breadth. Isolates were homothallic with paragynous antheridia, oogonia were spherical with diameters from 32 to 35 µm, and oospores were plerotic with diameters from 20 to 30 µm. Electrophoretic banding patterns of mycelial proteins and isozymes (alkaline phospatase, esterase, glucose-6-phospate dehydrogenase, malate dehydrogenase, and superoxide dismutase) of beech isolates were distinct from those of reference isolates of semipapillate Phytophthora species, including P. citricola, P. hibernalis, P. ilicis (IMI 158964), P. psychrophila (CBS 803.95), and P. syringae from citrus fruits, whose identification had been confirmed on the basis of internal transcribed spacer (ITS)-restriction fragment length polymorphism (RFLP) patterns and sequences. Conversely, the electrophoretic phenotype and the ITS-RFLP pattern (and sequence) of the beech isolates were identical to those of a reference isolate (Ph24) from Quercus cerris, which was originally identified as P. syringae on the basis of morphological and cultural characters (1). However, the isolate Ph24 has been reexamined, and morphological and cultural characteristics as well as the ITS sequence would indicate that this isolate is a new species not yet formally described, for which the name P. pseudosyringae has been suggested (2). The pathogenicity of a beech isolate (IMI 390500) was compared to that of an Italian P. cambivora isolate from European chestnut by inoculating the stems of 16-month-old beech seedlings (10 replicates), which were placed at 18°C with a 12-h photoperiod. The beech isolate produced lesions averaging 2 cm long after 2 months, while those produced by the P. cambivora isolate averaged 3 cm. Control seedlings inoculated with sterile agar did not develop symptoms. The pathogen was reisolated from lesions to fulfil Koch's postulates. To our knowledge, this is the first report of this new Phytophthora sp. on beech in Italy. Conversely, the same species has been reported to be associated with decline of oak stands (1). References: (1) G. P. Barzanti et al. Phytopathol. Mediterr. 40:149, 2001. (2) T. Jung et al. Phytophthora pseudosyringae sp. nov., a new species causing root and collar rot of deciduous tree species in Europe. Mycol. Res. (In press).