Towards a best practice methodology for the detection of Phytophthora species in soils

The genus Phytophthora contains species that are major pathogens worldwide, affecting a multitude of plant species across agriculture, horticulture, forestry, and natural ecosystems. Here, we concentrate on those species that are dispersed through soil and water, attacking the roots of the plants, causing them to rot and die. The intention of this study was to compare the soil baiting protocol developed by the Centre for Phytophthora Science and Management (CPSM) with two other baiting methods used in Australia. The aim was to demonstrate the effectiveness of each protocol for soil baiting Phytophthora species in different substrates. Three experiments were conducted: the first to test the sensitivity of each method to detect Phytophthora cinnamomi, the second to test the effect of substrate type (sand or loam), and the third to test the detection of species (P. cinnamomi, P. multivora, or P. pseudocryptogea). The specificity of different plant species baits was compared within and between the methods. Substrate type influenced isolation in all methods; however, the CPSM method was superior regardless of substrate, albeit slower than one of the other methods for one substrate. Comparing bait species between the three methods, Quercus ilex was the most attractive bait for P. cinnamomi, particularly in the CPSM method. The choice of protocol affected the isolation associated with each bait type. Overall, the multiple bait system used by CPSM was shown to provide the most sensitive and reliable detection of Phytophthora species from soil samples.     

Morphological and molecular characterization of Phytophthora species associated with root and crown rot of pomegranate in Iran

The purpose of this research was to identify the pathogens causing root and crown rot in major pomegranate-growing areas of Iran. Infected tissue samples were collected from trees with symptoms from 49 pomegranate orchards in four provinces of Iran: Fars, Markazi, Isfahan, and Kohgiluyeh va Boyer-Ahmad. In total, 23 Phytophthora spp. isolates were obtained, which were identified as P. cryptogea species complex (12 isolates) and P. cinnamomi (11 isolates) based on morphological characters. Molecular confirmation of identification was performed by inference of phylogeny of ITS-rDNA regions, β-tubulin gene, and the mitochondrial gene cytochrome c oxidase subunit 1. The results of our phylogenetic analysis confirmed the morphological identification of P. cinnamomi isolates and placed them in Clade 7c of Phytophthora. In addition, the P. cryptogea species complex isolates, despite morphological similarities, were in fact four distinct species including P. cryptogea sensu stricto (two isolates), P. pseudocryptogea (one isolate), P. sp. kelmania (one isolate), and P. erythroseptica (six isolates). This is the first report of pomegranate root and crown rot caused by P. cinnamomi and P. cryptogea species complex.     

Phosphorous acid treatments control Phytophthora diseases in Australia1

Treatments with a partially neutralized formulation of phosphorous acid containing potassium phosphite were assessed for control of Phytophthora diseases in subtropical and temperate crops in Australia. In Queensland, trunk injections of phosphite (10% solution) controlled severe root rot (Phytophthora cinnamomi) of avocado trees and resulted in the recovery of trees. Single pre-harvest sprays (2.5 kg ha^-1) of phosphite controlled root and heart rot (P. cinnamomi) of pineapples. Foliar sprays of phosphite (64 g per tree) controlled root rot (P. nicotianae var. parasitica) and trunk canker (P. citrophthora) of mandarin trees. In Victoria, a foliar spray of phosphite (300 g ha^-1) reduced root rot (P. clandestina) of subterranean clover and increased dry matter by 1.96 to 5.11 t ha^-1. Trunk injections of phosphite (10% solution) controlled trunk rot (P. cactorum) of peach trees and foliar sprays (10 kg ha^-1) reduced severity of root rot (P. nicotianae var. nicotianae) of tomatoes.     

Molecular analysis of Phytophthora diversity in nursery‐grown ornamental and fruit plants

The genetic diversity of Phytophthora spp. was investigated in potted ornamental and fruit tree species. A metabarcoding approach was used, based on a semi‐nested PCR with Phytophthora genus‐specific primers targeting the ITS1 region of the rDNA. More than 50 ITS1 sequence types representing at least 15 distinct Phytophthora taxa were detected. Nine had ITS sequences that grouped them in defined taxonomic groups (P. nicotianae, P. citrophthora, P. meadii, P. taxon Pgchlamydo, P. cinnamomi, P. parvispora, P. cambivora, P. niederhauserii and P. lateralis) whereas three phylotypes were associated to two or more taxa (P. citricola taxon E or III; P. pseudosyringae, P. ilicis or P. nemorosa; and P. cryptogea, P. erythroseptica, P. himalayensis or P. sp. ‘kelmania’) that can be challenging to resolve with ITS1 sequences alone. Three additional phylotypes were considered as representatives of novel Phytophthora taxa and defined as P. meadii‐like, P. cinnamomi‐like and P. niederhauserii‐like. Furthermore, the analyses highlighted a very complex assemblage of Phytophthora taxa in ornamental nurseries within a limited geographic area and provided some indications of structure amongst populations of P. nicotianae (the most prevalent taxon) and other taxa. Data revealed new host–pathogen combinations, evidence of new species previously unreported in Italy (P. lateralis) or Europe (P. meadii) and phylotypes representative of species that remain to be taxonomically defined. Furthermore, the results reinforced the primary role of plant nurseries in favouring the introduction, dissemination and evolution of Phytophthora species.     

Phytophthora species distribution in South African citrus production regions

Several Phytophthora spp. are known to cause a range of symptoms on citrus, resulting in significant crop losses worldwide. In South Africa, Phytophthora remains a destructive citrus disease, but the species and their distribution have not been well documented. A total of 162 Phytophthora isolates was collected from 60 citrus orchards in seven provinces of South Africa (Eastern Cape, Kwazulu-Natal, Limpopo, Mpumalanga, Northern Cape, North West and Western Cape). Isolates were identified to the species level through PCR-RFLP (restriction fragment length polymorphism) analyses of the internal transcribed spacer region. The identity of a subset of the isolates was confirmed using morphological and sequence analyses. Phytophthora nicotianae was the predominant species (76 % of isolates) and occurred in 80 % of the orchards in all of the provinces, followed by P. citrophthora (22 % of isolates in 28 % of orchards). The P. citrophthora isolates were further subdivided into two previously identified subgroups, G1 and G2, with most (69 %) of the isolates belonging to the G1 subgroup. Other Phytophthora species included P. multivora in the Western Cape Province, and an unknown species in the Eastern Cape Province with high sequence similarity (98 %) to a putative new species submitted to GenBank as Phytophthora taxon Sisuluriver. Phytophthora palmivora, a known citrus pathogen, was not identified. Most of the P. nicotianae isolates (79 %) were of the A1 mating type. The P. citrophthora isolates were mostly sterile (64 %), including most of the G1 isolates (81 %). The remaining G1 isolates (19 %) belonged to the A1 mating type, whereas almost all G2 isolates belonged to the A2 mating type except for one isolate that was sterile.     

Characterization of accessions and species of Macadamia to stem infection by Phytophthora cinnamomi

Phytophthora cinnamomi is a major pathogen in most macadamia plantations worldwide. Due to stem lesions, stem cankers and leaf defoliation, it results in loss of productivity and tree death. This study examined accessions of the four Macadamia species and their hybrids, produced via rooted stem cuttings or germinated seeds, for susceptibility to stem canker and necrotic lesions caused by P. cinnamomi. Plants were wound‐inoculated with agar containing P. cinnamomi. The symptoms produced in inoculated plants were used to characterize host susceptibility variation within and among the population. Lesion length and severity of stem canker were recorded. The four species and hybrids differed significantly in stem canker severity (P < 0.001) and lesion length (P = 0.04). Macadamia integrifolia and M. tetraphylla hybrids were the most susceptible. Macadamia integrifolia had the greatest stem canker severity and the most extensive lesions above and below the site of inoculation. Restricted lesion sizes were observed in M. ternifolia and M.  jansenii. The effects of basal stem diameter and the method of propagation either from cuttings or from seed were not significant. The genetic variation in the reaction of macadamia accessions to stem infection by P. cinnamomi is discussed.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

Collar rot caused byPhytophthora citrophthora on pear trees in Greece

A severe crown rot of pear trees of cultivar ‘Kondoula’ grafted on quince rootstock was observed in Greece. Isolations from the affected tissues repeatadly yielded aPhytophthora sp. that was determined by morphological and physiological characteristics to beP. citrophthora. The pathogenicity of two of theP. citrophthora isolates was tested by inoculating trunks of 2-year-old pear trees by mycelial agar disks. Thirty-two days after inoculation all inoculated trees were infected. Although the pear isolates could not be differentiated from isolates ofP. palmivora orP. nicotianae based on isozyme profiles of α-esterase or lactate dehydrogenase, RAPD profiles with one selected primer differentiated the pear isolates from the other species and revealed an electrophoretic banding pattern similar to that of aP. citrophthora standard. This is the first report ofP. citrophthora on pear trees in Greece.     

<Emphasis Type="Italic">Pythium</Emphasis> and <Emphasis Type="Italic">Phytophthora</Emphasis> species associated with root and stem rots of kalanchoe

Pythium and Phytophthora species were isolated from kalanchoe plants with root and stem rots. Phytophthora isolates were identified as Phytophthora nicotianae on the basis of morphological characteristics and restriction fragment length polymorphism (RFLP) analysis of the rDNA-internal transcribed spacer regions. Similarly, the Pythium isolates were identified as Pythium myriotylum and Pythium helicoides. In pathogenicity tests, isolates of the three species caused root and stem rots. Disease severity caused by the Pythium spp. and Ph. nicotianae was the greatest at 35°–40°C and 30°–40°C, respectively. Ph. nicotianae induced stem rot at two different relative humidities (60% and >95%) at 30°C. P. myriotylum and P. helicoides caused root and stem rots at high humidity (>95%), but only root rot at low humidity (60%).     

Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgl^–1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgl^–1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.     

Pathogenicity of 21 newly described Phytophthora species against seven Western Australian native plant species

The pathogenicity of some Phytophthora species recently described from Western Australia, together with P. cinnamomi as a control, was tested against seven Western Australian native plant species in the glasshouse. Host species were Banksia grandis, B. littoralis, B. occidentalis, Casuarina obesa, Corymbia calophylla, Eucalyptus marginata and Lambertia inermis. Twenty‐two Phytophthora species were grown on a vermiculite, millet seed and V8 substrate and used as soil inoculum when the plant hosts were approximately 3 months old. Pathogenicity was assessed after 6 weeks and plants were scored for death, root damage, and percentage reduction of shoot growth compared with control plants. The pathogenicity of P. cinnamomi was confirmed. Phytophthora niederhauserii was shown to be similar to P. cinnamomi in pathogenicity and of concern ecologically. Other species that killed one or more hosts were P. boodjera, P. constricta, P. elongata, P. moyootj and P. rosacearum, while P. condilina, P. gibbosa, P. gregata, P. litoralis and P. ‘personii’ caused significant reduction to shoot and/or root growth, but did not kill plants. Host species susceptible to the highest number of Phytophthora species were B. grandis, B. littoralis, B. occidentalis and E. marginata. No Phytophthora species tested killed C. calophylla.     

Oomycete species associated with Theobroma cacao crops in Colombia

The study of oomycetes associated with crops is highly important due to the economic losses they might cause. In cacao, the genus Phytophthora has been extensively studied, but little is known about other genera and species of oomycetes associated with this plant. This study aimed to determine the oomycetes’ diversity present in Colombian cacao crops. A total of 146 isolates were obtained from diseased plants and soil in 11 departments. Analysis of internal transcribed spacer (ITS) and cytochrome oxidase subunit I (coxI) sequences was performed along with the assessment of morphological characteristics. Nine species were identified, distributed in four genera: Phytophthora (P. palmivora, 54%; P. nicotianae, 1%), Phytopythium (Phy. chamaehyphon, 15%; Phy. cucurbitacearum, 9%; Phy. vexans, 7%; Phy. helicoides, 1%), Globisporangium (G. splendens, 3%), and Pythium (Py. delicense, 1%; Py. inflatum,1%). Additionally, an unidentified and possibly new species of Phytophthora (5%) and three unidentified species of Phytopythium (3%) were found. This is the first report of Globisporangium, Phytopythium, and Pythium in cacao crops of Colombia and the first report of the species Phy. chamaehyphon in the country. Interestingly, some isolates of Phytopythium spp. were isolated from necrotic leaves and vascular section of stems, which may suggest a role in cacao diseases traditionally associated with Phytophthora. Also, it is proposed that the new species of Phytophthora may be contributing significantly to black pod disease in Colombian cacao crops, and we highlight that the study of P. palmivora is urgent because of its distribution all over the country.     

Isolation and pathogenicity of Phytophthora species from declining Rubus anglocandicans

Rubus anglocandicans is the most widespread and abundant blackberry species within the European blackberry (Rubus fruticosus) aggregate in Western Australia (WA). European blackberry is also one of the 32 Weeds of National Significance in Australia. A disease recorded as ‘blackberry decline’ was first observed in some blackberry sites in WA in 2006. A disease survey was conducted in the Manjimup‐Pemberton region along the Warren and Donnelly River catchments in WA between 2010 and 2012. Phytophthora amnicola, P. bilorbang, P. cryptogea, P. inundata, P. litoralis, P. multivora, P. taxon personii, P. thermophila and a P. thermophila × amnicola hybrid were recovered from declining and adjacent decline‐free sites, as well as from streams and rivers. Phytophthora cinnamomi was isolated from dying Banksia and Eucalyptus species from two non‐decline sites. Of these species, P. bilorbang and P. cryptogea were more pathogenic than the others in under‐bark inoculations using excised stems (primocanes), in planta primocane inoculations in blackberry growing wild in native forest stands, and in glasshouse pot trials. It was concluded that blackberry decline is a complex syndrome and Phytophthora species, in particular P. bilorbang and P. cryptogea, together with temporary inundation, are major biotic and abiotic factors contributing to blackberry decline.     

Phytophthora diversity and the population structure of Phytophthora ramorum in Swiss ornamental nurseries

Invasive oomycete pathogens have been causing significant damage to native ecosystems worldwide for over a century. A recent well‐known example is Phytophthora ramorum, the causal agent of sudden oak death, which emerged in the 1990s in Europe and North America. In Europe, this pathogen is mainly restricted to woody ornamentals in nurseries and public greens, while severe outbreaks in the wild have only been reported in the UK. This study presents the results of the P. ramorum survey conducted in Swiss nurseries between 2003 and 2011. In all 120 nurseries subjected to the plant passport system, the main P. ramorum hosts were visually checked for above ground infections. Phytophthora species were isolated from tissue showing symptoms and identified on the basis of the morphological features of the cultures and sequencing of the ribosomal ITS region. Phytophthora was detected on 125 plants (66 Viburnum, 58 Rhododendron and one Pieris). Phytophthora ramorum was the most frequent species (59·2% of the plants), followed by P. plurivora, P. cactorum, P. citrophthora, P. cinnamomi, P. cactorum/P. hedraiandra, P. multivora and P. taxon PgChlamydo. The highest incidence of P. ramorum was observed on Viburnum × bodnantense. Microsatellite genotyping showed that the Swiss P. ramorum population is highly clonal and consists of seven genotypes (five previously reported in Europe, two new), all belonging to the European EU1 clonal lineage. It can therefore be assumed that P. ramorum entered Switzerland through nursery trade. Despite sanitation measures, repeated P. ramorum infections have been recorded in seven nurseries, suggesting either reintroduction or unsuccessful eradication efforts.     

Identification and differentiation of Phytophthora by electrophoresis of mycelial proteins and isoenzymes1

Total proteins (both native and denatured) and three isoenzyme systems (esterases, malate dehydrogenases, superoxide dismutases) were analysed for eight Phytophthora species and for eight strains of P. nicotianae. The results obtained showed low intraspecific variability (62-100% of similarity index) for P. nicotianae and allowed the characterization of P. capsici, cinnamomi, cryptogea, megakarya, megasperma, nicotianae, palmivora and citrophthora by the polyacrylamide gel electrophoretic profiles of native proteins and isoenzymes. The interspecific variability between these eight species is high (27-49% of similarity index). The results obtained support the use of these macromolecular criteria as an aid to the identification of Phytophthora spp.     

Molecular and Pathogenicity Characteristics of Phytophthora nicotianae Responsible for Root Necrosis and Wilting of Pepper (Capsicum annuum L.) in Tunisia

Isolates of Phytophthora from pepper, produced in Tunisia, were characterised according to molecular and pathogenicity criteria. Polymerase chain reaction amplification of the ITS1 region in the ribosomal DNA resulted in different sized fragments. The pepper isolates and P. nicotianae yielded a fragment of 310bp that distinguished it from P. capsici with a fragment of 270bp. The ribosomal RNA gene amplicons of both internal transcribed spacers and the 5.8 S of the pepper Phytophthora and P. nicotianae were digested with 8 endonucleases. The patterns generated, with the 2 enzymes that cut, were identical for both taxa. This molecular analysis corroborated the morphological and biological characteristics and suggests strongly that the isolates of Phytophthora from pepper belong to the species P. nicotianae. Inoculation of pepper, tomato, eggplant and tobacco plants with the isolates of P. nicotianae from pepper showed they were highly pathogenic on pepper but not on tobacco, while their pathogenicity was weak on tomato and eggplant and was associated with atypical symptoms not observed in the field. These pathogenicity tests suggest that pepper isolates of P. nicotianae are particularly adapted to their host and may thus constitute a forma specialis of P. nicotianae.     

Diversity of Phytophthora species in natural ecosystems of Taiwan and association with disease symptoms

In 2013 a survey of Phytophthora diversity was performed in 25 natural and seminatural forest stands and 25 rivers in temperate montane and subtropical lowland regions of Taiwan. Using baiting assays, 10 described species and 17 previously unknown taxa of Phytophthora were isolated from 71.5% of the 144 rhizosphere soil samples from 33 of 40 tree species sampled in 24 forest stands, and from 19 rivers: P. capensis, P. citrophthora, P. plurivora, P. tropicalis, P. citricola VII, P. sp. × botryosa‐like, P. sp. × meadii‐like and P. sp. occultans‐like from Clade 2; P. palmivora from Clade 4; P. castaneae and P. heveae from Clade 5; P. chlamydospora and P. sp. forestsoil‐like from Clade 6; P. cinnamomi (Pc), P. parvispora, P. attenuata nom. prov., P. flexuosa nom. prov., P. formosa nom. prov., P. intricata nom. prov., P. × incrassata nom. prov. and P. × heterohybrida nom. prov. from Clade 7; P. sp. palustris and five new hybrid species from Clade 9. The A1 mating type of Pc was widespread in both montane and lowland forests and rarely associated with disease, whereas the A2 mating type was limited to lowland forests and in some cases causing severe dieback. Most other Phytophthora species were not associated with obvious disease symptoms. It is concluded that (i) Taiwan is within the centre of origin of most Phytophthora taxa found, (ii) Pc A2 is an introduced invasive pathogen, and (iii) interspecific hybridizations play a major role in speciation and species radiations in diverse natural ecosystems.     

Phomopsis husk rot of macadamia in Australia and South Africa caused by novel Diaporthe species

Phomopsis husk rot (PHR) in macadamia is a disease of economic importance in major commercial production areas in Australia and South Africa. Effective control of PHR is hindered by limited knowledge about its aetiology and epidemiology. The diversity and pathogenicity of more than 50 isolates of Diaporthe associated with PHR in macadamia orchards in Australia and South Africa was assessed. Multilocus phylogenetic analyses of DNA sequences of the ITS, tef1α, and tub2 gene loci revealed four novel clades that are described as Diaporthe australiana sp. nov., D. drenthii sp. nov., D. macadamiae sp. nov., and D. searlei sp. nov. Pathogenicity tests with representative isolates found that all four species caused PHR of varying severity between and within species, as well as between the two macadamia cultivars HAES 344 and HAES 816. The Australian species, D. australiana, was the most aggressive species compared with the three South African species. This study improves our understanding of the aetiology of PHR in macadamia and paves the way for more effective disease management.     

Phytophthora lateralis discovered in an old growth Chamaecyparis forest in Taiwan

The geographic origins of the invasive Phytophthora species, P. lateralis and P. ramorum are unknown. In 2008 soil samples were collected in an old growth yellow cedar (Chamaecyparis obtusa var. formosana) stand in the Ma‐kau Ecological Park in north eastern Taiwan and subjected to Phytophthora baiting procedures at 18°C. Cedar needle baits yielded isolates of a slow growing Phytophthora culture from one soil sample, together with P. cinnamomi. Phytophthora bisheria sp. nov. was obtained from another sample. The slow growing isolates conformed closely to P. lateralis in the morphology of their sporangia and chlamydospores, growth–temperature relationships, absence of gametangia and their ITS and cox II sequences. The isolates’ sporangia were partially caducous, with short pre‐formed pedicels of ca. 3–5 μm, a highly unusual feature in a non‐papillate Phytophthora. The isolates also produced multicellular stromata on cedar decoction agar. Small morphological and molecular differences were observed between the Taiwan‐isolates and Oregon‐control isolates. Taiwan may lie within the geographic centre of origin of P. lateralis. By analogy Japan may also lie within the natural range of P. lateralis; and Japan, along with Taiwan and Yunnan, could be an origin for the closely related P. ramorum.     

Genetic variation and host specificity of <Emphasis Type="Italic">Phytophthora citrophthora</Emphasis> isolates causing branch cankers in Clementine trees in Spain

Considerable tree losses have been observed during the past few years in Spain due to Phytophthora branch canker of clementines caused by Phytophthora citrophthora. The emergence of this disease led to the speculation that either the pathogen has evolved increasing its aggressiveness or specificity to clementines. A total of 134 isolates of P. citrophthora collected from 2003 to 2005 in 135 citrus orchards in Spain and 22 reference isolates were analyzed genotypically and phenotypically to determine the structure of the population. Genotypic diversity was evaluated by means of Inter-Simple Sequence Repeat (ISSR) markers. Among the phenotypic characteristics examined, sporangial characters, sexual behavior, growth rates and colony morphology of the isolates at different temperatures were studied. The aggressiveness and host-specificity of selected isolates were evaluated by pathogenicity tests on sweet oranges and clementines under field conditions. Phytophthora branch canker of clementines was associated mainly with one genotype (P-1), which included 88% of the isolates obtained from branches. Strains isolated years before the first disease outbreak clustered also with this major genotype, thus it may be considered as a predominant population. Thirteen other minor genotypes were determined, but most contained only one isolate. Although there was wide variation in the morphological and physiological characters, all Phytophthora isolates obtained from branch cankers were sexually sterile and showed a characteristic petalloid colony pattern. As in previous greenhouse studies, pathogenicity tests under field conditions demonstrated that clementines and their hybrids were more susceptible to P. citrophthora than sweet oranges. However, no evidence was found to support the hypothesis that the emergence of the disease was associated with more aggressive or host-specific forms of P. citrophthora.