J. Bakonyi

Characterisation of Isolates of Phytophthora infestans From Hungary

A total of 36 single-lesion isolates were collected from 9 crops of potato and 13 of tomato in different regions of Hungary in the past decade, particularly in 1998. These were analysed for mating type, sensitivity to metalaxyl, allozyme genotype at glucose-6-phosphate isomerase and peptidase loci and genotype at 24 loci detected using the multilocus RFLP probe RG57. The ratios of the mating types A1 to A2 were 8 : 9 and 4 : 15 among isolates recovered from potato and tomato, respectively. Resistance to metalaxyl was found more frequently among isolates from potato and in the A1 mating type. The populations were not clearly differentiated on the basis of host origin. All isolates were homozygous (100/100) at the locus for glucose-6-phosphate isomerase. Unlike in other European countries, the most common peptidase allele was 96. Genotypes at the peptidase locus were 96/96 (50%), 96/100 (27.7%) and 100/100 (16.6%). In addition, one isolate from 1993 and another from 1998, were defined as 83/96, a genotype that had not been described elsewhere. The 18 RG57 fingerprints that were observed among 36 isolates, with one exception, seem to be unique to Europe. On the basis of combined genotypic traits, 20 multilocus genotypes were designated. These data, which reveal a remarkable variability with unique genotypes of the late blight pathogen, suggest that migration and sexual and/or asexual recombination have a role in the recent evolution of the pathogen in Hungary.

Phenotypic and molecular characterization of species hybrids derived from induced fusion of zoospores of Phytophthora capsici and Phytophthora nicotianae

Phenotypes of species hybrids created from in vitro fusion of zoospores from Phytophthora nicotianae and P. capsici were characterized and compared. The species hybrids were created as part of a study of sources of genetic variation in populations of the parent species that are pathogenic over a similar range of plants. Four isolates of species hybrids proved to be similar to both P. capsici and P. nicotianae in relation to vegetative and reproductive morphologies. As in a previous study, DNA of P. capsici was detected more readily than that of P. nicotianae in all hybrid isolates. In the present study, DNA of P. nicotianae was detected in three of four hybrids by hybridization of RAPD-PCR products with species-specific DNA from P. nicotianae. By thermal denaturation analyses, DNA melting temperatures and GC contents of parent species and species hybrids were similar. The mean GC content of 47.2% was similar to GC contents reported for other Phytophthora spp. Additionally, the distributions of GC-rich regions of hybrids were more similar to the distribution in P. capsici than in P. nicotianae. By these molecular analyses, the hybrids were shown to be more similar to P. capsici than to P. nicotianae. Even though interspecific somatic fusion is likely to occur rarely under natural conditions, it could contribute to the genetic diversity of heterothallic species of Phytophthora.

Phytophthora niederhauserii sp. nov., a polyphagous species associated with ornamentals, fruit trees and native plants in 13 countries

A non-papillate, heterothallic Phytophthora species first isolated in 2001 and subsequently from symptomatic roots, crowns and stems of 33 plant species in 25 unrelated botanical families from 13 countries is formally described here as a new species. Symptoms on various hosts included crown and stem rot, chlorosis, wilting, leaf blight, cankers and gumming. This species was isolated from Australia, Hungary, Israel, Italy, Japan, the Netherlands, Norway, South Africa, Spain, Taiwan, Turkey, the United Kingdom and United States in association with shrubs and herbaceous ornamentals grown mainly in greenhouses. The most prevalent hosts are English ivy (Hedera helix) and Cistus (Cistus salvifolius). The association of the species with acorn banksia (Banksia prionotes) plants in natural ecosystems in Australia, in affected vineyards (Vitis vinifera) in South Africa and almond (Prunus dulcis) trees in Spain and Turkey in addition to infection of shrubs and herbaceous ornamentals in a broad range of unrelated families are a sign of a wide ecological adaptation of the species and its potential threat to agricultural and natural ecosystems. The morphology of the persistent non-papillate ellipsoid sporangia, unique toruloid lobate hyphal swellings and amphigynous antheridia does not match any of the described species. Phylogenetic analysis based on sequences of the ITS rDNA, EF-1α, and β-tub supported that this organism is a hitherto unknown species. It is closely related to species in ITS clade 7b with the most closely related species being P. sojae. The name Phytophthora niederhauserii has been used in previous studies without the formal description of the holotype. This name is validated in this manuscript with the formal description of Phytophthora niederhauserii Z.G. Abad et J.A. Abad, sp. nov. The name is coined to honor Dr John S. Niederhauser, a notable plant pathologist and the 1990 World Food Prize laureate.

Phytophthora × pelgrandis Causes Root and Collar Rot of Lavandula stoechas in Italy

In 2007, Phytophthora isolates with atypical morphological and biological characteristics were found associated with root and collar rot of potted plants of Stoechas lavender (Lavandula stoechas) in an ornamental nursery in Italy. A polyphasic approach, including morphological and cultural observations, sequencing the ITS-rDNA region, the Pheca and the mitochondrial coxI genes, multiplex PCRs with primers specific for P. nicotianae or P. cactorum, as well as random amplified polymorphic DNA-polymerase chain reaction, was used to characterize these isolates. On the basis of morpho-cultural and molecular analyses, the isolates from Stoechas lavender were identified as Phytophthora × pelgrandis, a natural hybrid of P. nicotianae × P. cactorum previously reported in other European countries, the Americas, and Taiwan, as a pathogen of ornamentals and loquat plants. In pathogenicity tests using potted plants of Stoechas lavender, the P. × pelgrandis isolates, similarly to the parental species P. nicotianae, induced the symptoms observed on plants with natural infections and were reisolated only from artificially inoculated plants. Dispersal of P. × pelgrandis on this host could exacerbate the damage caused by Phytophthora root and collar rot, of which the main causal agent presently is P. nicotianae on lavender in Europe. Application of hygienic measures is important to reduce the proliferation and spread of the Phytophthora hybrids.

Six new Phytophthora species from ITS Clade 7a including two sexually functional heterothallic hybrid species detected in natural ecosystems in Taiwan.

During a survey of Phytophthora diversity in natural ecosystems in Taiwan six new species were detected. Multigene phylogeny based on the nuclear ITS, ß-tubulin and HSP90 and the mitochondrial cox1 and NADH1 gene sequences demonstrated that they belong to ITS Clade 7a with P. europaea, P. uniformis, P. rubi and P. cambivora being their closest relatives. All six new species differed from each other and from related species by a unique combination of morphological characters, the breeding system, cardinal temperatures and growth rates. Four homothallic species, P. attenuata, P. flexuosa, P. formosa and P. intricata, were isolated from rhizosphere soil of healthy forests of Fagus hayatae, Quercus glandulifera, Q. tarokoensis, Castanopsis carlesii, Chamaecyparis formosensis and Araucaria cunninghamii. Two heterothallic species, P. xheterohybrida and P. xincrassata, were exclusively detected in three forest streams. All P. xincrassata isolates belonged to the A2 mating type while isolates of P. xheterohybrida represented both mating types with oospore abortion rates according to Mendelian ratios (4–33 %). Multiple heterozygous positions in their ITS, ß-tubulin and HSP90 gene sequences indicate that P. xheterohybrida, P. xincrassata and P. cambivora are interspecific hybrids. Consequently, P. cambivora is re-described as P. xcambivora without nomenclatural act. Pathogenicity trials on seedlings of Castanea sativa, Fagus sylvatica and Q. suber indicate that all six new species might pose a potential threat to European forests.

Multiple new cryptic pathogenic Phytophthora species from Fagaceae forests in Austria, Italy and Portugal

During surveys of Phytophthora diversity in natural and semi-natural Fagaceae forests in Austria, Italy and Portugal, four new cryptic species were isolated from rhizosphere soil samples. Multigene phylogeny based on nuclear ITS, ß-tubulin and HSP90 and mitochondrial cox1 and NADH1 gene sequences demonstrated that two species, P. tyrrhenica and P. vulcanica spp. nov., belong to phylogenetic Clade 7a, while the other two species, P. castanetorum and P. tubulina spp. nov., clustered together with P. quercina forming a new clade, named here as Clade 12. All four new species are homothallic and have low optimum and maximum temperatures for growth and very slow growth rates at their respective optimum temperature. They differed from each other and from related species by a unique combination of morphological characters, cardinal temperatures, and growth rates. Pathogenicity of all Phytophthora species to the root system of their respective host species was demonstrated in soil infestation trials.

Nothophytophthora gen. nov., a new sister genus of phytophthora from natural and semi-natural ecosystems

During various surveys of Phytophthora diversity in Europe, Chile and Vietnam slow growing oomycete isolates were obtained from rhizosphere soil samples and small streams in natural and planted forest stands. Phylogenetic analyses of sequences from the nuclear ITS, LSU, β-tubulin and HSP90 loci and the mitochondrial cox1 and NADH1 genes revealed they belong to six new species of a new genus, officially described here as Nothophytophthora gen. nov., which clustered as sister group to Phytophthora. Nothophytophthora species share numerous morphological characters with Phytophthora: persistent (all Nothophytophthora spp.) and caducous (N. caduca, N. chlamydospora, N. valdiviana, N. vietnamensis) sporangia with variable shapes, internal differentiation of zoospores and internal, nested and extended (N. caduca, N. chlamydospora) and external (all Nothophytophthora spp.) sporangial proliferation; smooth-walled oogonia with amphigynous (N. amphigynosa) and paragynous (N. amphigynosa, N. intricata, N. vietnamensis) attachment of the antheridia; chlamydospores (N. chlamydospora) and hyphal swellings. Main differing features of the new genus are the presence of a conspicuous, opaque plug inside the sporangiophore close to the base of most mature sporangia in all known Nothophytophthora species and intraspecific co-occurrence of caducity and non-papillate sporangia with internal nested and extended proliferation in several Nothophytophthora species. Comparisons of morphological structures of both genera allow hypotheses about the morphology and ecology of their common ancestor which are discussed. Production of caducous sporangia by N. caduca, N. chlamydospora and N. valdiviana from Valdivian rainforests and N. vietnamensis from a mountain forest in Vietnam suggests a partially aerial lifestyle as adaptation to these humid habitats. Presence of tree dieback in all forests from which Nothophytophthora spp. were recovered and partial sporangial caducity of several Nothophytophthora species indicate a pathogenic rather than a saprophytic lifestyle. Isolation tests from symptomatic plant tissues in these forests and pathogenicity tests are urgently required to clarify the lifestyle of the six Nothophytophthora species.

Genetic diversity and origins of the homoploid type hybrid Phytophthora×alni

ABSTRACT Assessing the process that gives rise to hybrid pathogens is central to understanding the evolution of emerging plant diseases. Phytophthora ×alni, a pathogen of alder, results from the homoploid hybridization of two related species, Phytophthora uniformis and Phytophthora ×multiformis. Describing the genetic characteristics of P. ×alni should help us understand how reproductive mechanisms and historical processes shaped the population structure of this emerging hybrid pathogen. The population genetic structure of P. ×alni and the relationship with its parental species were investigated using 12 microsatellites and one mitochondrial DNA (mtDNA) marker on a European collection of 379 isolates. Populations of P. ×alni were dominated by one multilocus genotype (MLG). The frequency of this dominant MLG increased after the disease emergence together with a decline in diversity, suggesting that it was favored by a genetic mechanism such as drift or selection. Combined microsatellite and mtDNA results confirmed that P. ×alni originated from multiple hybridization events that involved different genotypes of the progenitors. Our detailed analyses point to a geographic structure that mirrors that observed for P. uniformis in Europe. The study provides more insights on the contribution of P. uniformis, an invasive species in Europe, to the emergence of Phytophthora-induced alder decline. IMPORTANCE Our study describes an original approach to assess the population genetics of polyploid organisms using microsatellite markers. By studying the parental subgenomes present in the interspecific hybrid P. ×alni, we were able to assess the geographical and temporal structure of European populations of the hybrid, shedding new light on the evolution of an emerging plant pathogen. In turn, the study of the parental subgenomes permitted us to assess some genetic characteristics of the parental species of P. ×alni, P. uniformis, and P. ×multiformis, which are seldom sampled in nature. The subgenomes found in P. ×alni represent a picture of the “fossilized” diversity of the parental species.

First Report of Phytophthora citricola on False Cypress in Hungary

In May 2005, an estimated 10 to 15% mortality of various cultivars of false cypress (also named Lawson cypress or Port-Orford-cedar [Chamaecyparis lawsoniana]) with severe wilt was observed in field stands of an ornamental nursery in western Hungary. Wilted plants had rot-associated reduction of their root system. Root discoloration and occasional chlorosis of lower leaves commenced on potted 3-year-old plants that were held in the open air for 10 to 12 months before planting. Four species of Phytophthora (P. lateralis, P. eriugena, P. hibernalis, and P. cinnamomi) have been reported on this host (2). Direct plating of discolored roots from the most susceptible cultivar (Silver Globus) onto a selective potato dextrose agar or carrot agar medium yielded pure cultures that developed white, stellate colonies with sparse aerial mycelia. The hyphal growth was optimal at 25°C, but the growth above 32°C and below 4°C was completely inhibited. Single, terminal sporangia on simple (occasionally sympodial) sporangiophores formed abundantly in nonsterile soil filtrate but not in agar. Sporangia, 31 to 67 μm (59.1 ± 9.3 μm) long and 25 to 39 μm (31.5 ± 4.0 μm) wide, were noncaducous and semipapillate, variable in shape, mostly obpyriform, rarely obovoid, ovoid-ellipsoid and spherical or bifurcated and distorted, and the exit pore was narrow (7.2 ± 0.8 μm). No external or internal proliferation and no hyphal swellings or chlamydospores were observed. The isolates were homothallic with smooth-walled oogonia (27.3 ± 3.4 μm in diameter) and paragynous antheridia. The oospores (24.7 ± 2.1 μm in diameter) were plerotic. The morpho-physiological features suggested that our isolates belonged to Waterhouses Group III, and in particular, represented P. citricola. This was confirmed by cellulose acetate electrophoresis of malate dehydrogenase; the isozyme pattern of false cypress isolate was identical to that of the ITS-sequenced (NCBI Accession No. AY366193) P. citricola isolate from a Hungarian alder forest (1). Pathogenicity tests on four 3-year-old potted false cypress (cv. Silver Globus) plants in the greenhouse resulted in rapidly developing (within 2 weeks) sunken, necrotic lesions at the stem base around the site of wound inoculation with a 5-mm-diameter mycelial agar plug. After 12 weeks, each inoculated plant wilted and died. The causal agent was consistently reisolated from necrotic tissues. In Hungary, P. citricola was first isolated and identified from alder forest soil (1). Nonetheless that false cypress has been listed as the host of P. citricola in Norway and Poland (3,4), to our knowledge, this report is the first definitive description of this Phytophthora sp. on this host. References: (1) J. Bakonyi et al. Plant Pathol. 52:807, 2003. (2) D. C. Erwin and O. K. Ribeiro. Pages 282-287 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) V. Talgø V. and A. Stensvand. Grønn kunnskap e 7(101G):1, 2003. (4) K. Wiejacha et al. Page 45 in: Improvement and Unification of Plant Disease Diagnostics. Abstracts of International Workshop, Skierniewice, Poland, 2004.