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.     

Histopathology of infection and colonization of Quercus ilex fine roots by Phytophthora cinnamomi

Quercus ilex is one of the European forest species most susceptible to root rot caused by the oomycete Phytophthora cinnamomi. This disease contributes to holm oak decline, a particularly serious problem in the ‘dehesas’ ecosystem of the southwestern Iberian Peninsula. This work describes the host–pathogen interaction of Q. ilex and P. cinnamomi, using new infection indices at the tissue level. Fine roots of 6‐month‐old saplings inoculated with P. cinnamomi were examined by light microscopy and a random pool of images was analysed in order to calculate different indices based on the measured area of pathogen structures. In the early stages of invasion, P. cinnamomi colonizes the apoplast and penetrates cortical cells with somatic structures. On reaching the parenchymatous tissues of the central cylinder, the pathogen develops different reproductive and survival structures inside the cells and then expands through the vascular system of the root. Some host responses were identified, such as cell wall thickening, accumulation of phenolic compounds in the middle lamella of sclerenchyma tissues, and mucilage secretion blocking vascular cells. New insights into the behaviour of P. cinnamomi inside fine roots are described. Host responses fail due to rapid expansion of the pathogen and a change in its behaviour from biotrophic to necrotrophic.     

Metabarcoding and development of new real‐time specific assays reveal Phytophthora species diversity in holm oak forests in eastern Spain

The evergreen holm oaks (Quercus ilex subsp. ilex and Q. ilex subsp. ballota) are the most representative tree species in the Iberian peninsula and the main tree species in oak‐rangeland ecosystems (dehesas). Oak decline in western, central and southern parts of Spain has been associated with root rot caused by Phytophthora cinnamomi for decades. However, Phytophthora species such as P.  quercina and P. psychrophila have recently been found associated with Quercus decline in eastern Spain where calcareous soils are predominant. Soil and root samples from two Quercus forests presenting decline symptoms in two different geographical areas in eastern Spain (Carrascar de la Font Roja and Vallivana) were analysed by amplicon pyrosequencing. Metabarcoding analysis showed Phytophthora species diversity, and revealed that an uncultured Phytophthora taxon, named provisionally Phytophthora taxon ballota, was the predominant species in both areas. In addition, a real‐time PCR assay, based on the pyrosequencing results, was developed for the detection of this uncultured Phytophthora taxon, and also for the detection of P. quercina. TaqMan assays were tested on soil and root samples, and on Phytophthora pure cultures. The new assays showed high specificity and were consistent with metabarcoding results. A new real‐time PCR protocol is proposed to evaluate the implication of different Phytophthora spp. in oak decline in eastern Spain.     

Calcium chelate is as effective as phosphite in controlling Phytophthora root rot in glasshouse trials

Species in the genus Phytophthora cause significant economic losses in crops and damage to forests and natural ecosystems worldwide. Currently, phosphite is the most effective chemical for disease management, but excessive phosphite concentrations can result in phytotoxicity in plants and the development of tolerance by the pathogen. Two newly developed metal chelates and phosphite (alone and in combination) were tested for their in vitro and in planta efficacy against Phytophthora cinnamomi. In glasshouse trials, 0.25% and 0.5% of each chemical treatment (phosphite, Ca chelate, Zn chelate) and Ca chelate + phosphite were used as a foliar application on 3-month-old seedlings of Banksia grandis (experiment not repeated) and Eucalyptus marginata, prior to inoculation with P. cinnamomi. All noninoculated control plants remained healthy, while significant root damage and reduction of dry root weights were observed for inoculated untreated plants. Individually, phosphite and Ca chelate significantly reduced root lesion development of P. cinnamomi compared to the control, with Ca chelate attaining superior results to phosphite at the same concentration. In combination, Ca chelate + phosphite had the largest reduction in root lesion development in both plant species; however, this result has not yet been replicated but did reflect previous in vitro results. The Zn chelate applications were not effective. Ca chelate has the potential to be developed as a fungicide to control Phytophthora species.     

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.     

Phenotyping Castanea hybrids for Phytophthora cinnamomi resistance

Castanea sativa is susceptible to Phytophthora spp., a serious root pathogen causing ink disease, while C. crenata and C. mollissima show resistance to infection. Interspecific controlled crosses were established for introgression of resistance genes from the resistant species into the susceptible C. sativa, and two mapping populations were created. Phytophthora cinnamomi resistance of each progeny was evaluated by root and excised shoot inoculation tests. The number of days of survival after root inoculation was the best discriminator of resistance to P. cinnamomi while the percentage of shoots with internal lesions was the symptom most associated with survival. The lesion progression rate in the excised shoot inoculation test was strongly and negatively correlated with survival in the root inoculation test. The excised shoot inoculation test appears to be a reliable approach for screening the resistance of chestnut genotypes to P. cinnamomi. Strong genetic correlations were obtained between survival and ink disease symptoms and among symptoms, indicating that common or linked genes might influence resistance to P. cinnamomi. The most resistant genotypes selected from this study will be tested for other commercial variables, such as ease of vegetative propagation and stock–scion compatibility.     

Prevalence of Phytophthora species in macadamia orchards in Australia and their ability to cause stem canker

In Australia, Phytophthora cinnamomi is the only species reported as the causal agent of stem canker and root rot in macadamia. In other countries, five Phytophthora species have been reported to cause diseases in macadamia, which led us to question if more than one Phytophthora species is responsible for poor tree health in macadamia orchards in Australia. To investigate this, samples were collected from the rhizosphere, stem, and root tissues of trees with and without symptoms, nurseries, and water sources from 70 commercial macadamia orchards in Australia. Phytophthora isolates were identified based on morphological characteristics and DNA sequencing. P. cinnamomi was the most predominant and widely distributed species, and was obtained from the different types of samples including symptomless root tissues. In addition to P. cinnamomi, only P. multivora was isolated from diseased tissue (stem canker) samples. Six other Phytophthora species were obtained from the rhizosphere samples: P. pseudocryptogea, P. citrophthora, P. nicotianae, P. gondwanense, P. sojae, and a new Phytophthora taxon. Only P. cinnamomi was obtained from macadamia nursery samples, while five Phytophthora species were obtained from water sources. Of the heterothallic Phytophthora species, mating type A2 isolates were dominant in P. cinnamomi isolates, whereas only mating type A1 isolates were obtained for P. nicotianae, P. pseudocryptogea, and P. citrophthora. Pathogenicity assays revealed that P. cinnamomi and P. multivora caused significantly larger stem and leaf lesions than P. citrophthora, P. nicotianae, and P. pseudocryptogea. Phytophthora sp. and P. sojae were nonpathogenic towards leaves and stems.     

Penetration of suberized periderm of a woody host by Phytophthora cinnamomi

The mechanisms by which Phytophthora cinnamomi zoospores infect inundated, above‐ground woody stem tissue are described. Using 4–6‐ and 18‐month‐old jarrah seedlings, the infection courts were identified and the invasion of the stems at sites of zoospore cyst binding were described. Stems were inoculated with a suspension of motile zoospores on the green stem/young periderm region. Light microscopy was used to examine penetration at sites of taxis, and fluorescent microscopy was used to examine penetration sites of seedlings with intact periderm. Two main infection courts were identified on stems: the emerging axillary shoot and the region of stem immediately surrounding an axillary shoot, where the periderm was thin or discontinuous. Invasion also occurred at sites where the developing shoot had not yet emerged but was at the stem surface. At these sites the pathogen also directly invaded through the thin‐walled phellem of the periderm surrounding the shoot. Zoospores of P. cinnamomi were not attracted to stomata on mature leaves or green stems. Penetration of the epidermal cell layer of the axillary bud leaf primordia was inter‐ and intra‐cellular; growth of hyphae in the periderm surrounding the shoot was intercellular; while in collenchyma it was inter‐ and intra‐cellular, being intercellular between polyphenolic‐rich cells. Exposed stem collenchyma was also directly invaded immediately adjacent to the young axillary shoot. Zoospores demonstrated taxis to sites of discontinuous periderm, similar to wounded areas where the outer protective layers of the plant are breached. This study presents the first evidence that P. cinnamomi is capable of intercellular penetration of suberized periderm.     

Potential of Ypt1 and ITS gene regions for the detection of Phytophthora species in a lab‐on‐a‐chip DNA hybridization array

A novel DNA‐chip hybridization assay that uses the ras‐related GTP‐binding protein 1 gene (Ypt1) was developed for the identification of several devastating Phytophthora species. The hybridization was conducted in a portable microfluidic lab‐on‐a‐chip device for fast and accurate detection of 40 Phytophthora, two Pythium and one Phytopythium species. Moreover, the functionality of the Ypt1 region was examined in comparison to an array for the internal transcribed spacer (ITS) region by in silico modelling. The difference in species‐specific capture probe sequences was lower for the ITS than for the Ypt1 region. While ITS‐probes of Phytophthora ramorum, Phytophthora fragariae and Phytophthora lateralis cross‐reacted with up to 11 non‐target species, Ypt1‐probes were specific except for P. fragariae/Phytophthora rubi. First analyses of artificially inoculated Rhododendron leaves successfully demonstrated the usability of the respective capture probes for the Ypt1 and the ras‐related plant protein Rab1a gene region. The on‐chip hybridization enabled the detection of up to 1 pg μL^?1 target DNA depending on the species examined. Due to the complementarity of ITS and Ypt1 genetic features, the use of multiple loci is recommended to identify targets of different taxonomic rank.     

Survival of Phytophthora ramorum in Rhododendron root balls and in rootless substrates

This study assesses the survival of Phytophthora ramorum in the root ball of Rhododendron container plants as well as in different rootless forest substrates and a horticultural potting medium. Following inoculation of the root balls, the aboveground plant parts stayed symptomless, whilst the pathogen could be recovered with a novel non‐destructive baiting assay from the root balls until at least 8 months post‐inoculation. Plating of surface‐sterilized roots and direct microscopic analysis confirmed the presence of P. ramorum in the roots. Phytophthora ramorum could also be baited from the root balls of symptomless Rhododendron plants from commercial nurseries, even 2 years after acquisition. Survival of P. ramorum in rootless media was assessed after burying disks of infected leaf material below the soil surface in columns filled with four different undisturbed forest substrates or a potting medium, and incubated at an outdoor quarantine facility. Phytophthora ramorum could be recovered at least 33 months after burial from all substrates, with a significant increase in recovery after the winter period. These data suggest the possibility for long‐term symptomless presence of P. ramorum in root balls of commercial Rhododendron plants as well as survival in potting medium and different forest substrates under western European climate conditions. Symptomless presence in root balls can contribute to latent spread of this pathogen between nurseries. The novel baiting test, being non‐destructive, simple and applicable to a relatively large number of plants, can offer a valuable tool to test plants for the presence of Phytophthora species in root balls.     

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.     

Interaction between root rot basidiomycetes and Phytophthora species on pedunculate oak

Forest declines are usually complex multifactorial phenomena that involve interactions between different factors. The possible interaction between different types of mycelial pathogens was investigated through artificial inoculation of oak seedlings, involving two root rot basidiomycetes, Collybia fusipes and Armillaria mellea, and two Phytophthora species, P. cinnamomi and P. cambivora. These pathogens were inoculated onto young Quercus robur saplings in greenhouse conditions, either alone or combining a root rot basidiomycete with a Phytophthora species. Three out of the four Phytophthora spp.*root rot basidiomycete combinations tested resulted in significantly greater damage to the oak host than the sum of the damages induced by the individual pathogens. This positive interaction could be significant in oak decline syndrome.     

Phytophthora acerina sp. nov., a new species causing bleeding cankers and dieback of Acer pseudoplatanus trees in planted forests in northern Italy

A severe dieback of Acer pseudoplatanus trees was noticed in planted forest stands in northern Italy in 2010. Affected trees showed collar rot and aerial bleeding cankers along the stems, leading to crown dieback and eventually death. An unknown Phytophthora species was consistently isolated from necrotic bark and xylem tissue and from rhizosphere soil. Based on its unique combination of morphological and physiological characters and phylogenetic analysis, this new taxon is here described as Phytophthora acerina sp. nov. Phylogenetic analysis of ITS, cox1 and β‐tubulin gene regions demonstrated that P. acerina is unique and forms a separate cluster within the ‘P. citricola complex’, closely related to P. plurivora. Phytophthora acerina is homothallic with smooth‐walled oogonia, thick‐walled, mostly aplerotic oospores with a high abortion rate, paragynous antheridia, and persistent, morphologically variable semipapillate sporangia. Four to 5‐week‐old cultures produced globose to subglobose, appressoria‐like and coralloid hyphal swellings and characteristic stromata‐like hyphal aggregations. Optimum and maximum temperatures for growth are 25°C and 32°C, respectively. Genetic uniformity of all 15 studied isolates and the apparent absence of this species in the extensive surveys of nurseries, forests and seminatural ecosystems conducted in the previous two decades across Europe indicate a recent clonal introduction to northern Italy. Under‐bark inoculation tests demonstrated high aggressiveness of P. acerina to A. pseudoplatanus indicating that this pathogen might be a serious risk to maple plantations and forests in Europe.     

Callose and β‐1,3‐glucanase inhibit Phytophthora cinnamomi in a resistant avocado rootstock

Phytophthora root rot (PRR) of avocado, caused by Phytophthora cinnamomi, is a significant threat to sustainable production wherever the crop is grown. Resistant rootstocks in combination with phosphite applications are the most effective options for managing this disease. Recently, the mechanisms underpinning PRR resistance have been investigated by the avocado community. Here, biochemical assays and confocal and scanning electron microscopy were used to investigate early defence responses in PRR resistant and ‐susceptible avocado rootstocks. Zoospore germination and subsequent hyphal growth for the pathogen were significantly inhibited on the surface of resistant avocado roots. When penetration occurred in the resistant R0.06 rootstock, callose was deposited in the epidermal cells, parenchyma and cortex of roots. In addition, β‐1,3‐glucanase was released early (6 h post‐inoculation, hpi) in response to the pathogen, followed by a significant increase in catalase by 24 hpi. In contrast, susceptible R0.12 roots responded only with the deposition of lignin and phenolic compounds incapable of impeding pathogen colonization. In this study, PRR resistance was attributed to a timely multilayered response to infection by P. cinnamomi.     

The morphology,behaviour and molecular phylogeny of Phytophthora taxon Salixsoil and its redesignation as Phytophthora lacustris sp. nov.

Since its first isolation from Salix roots in 1972, isolates of a sexually sterile Phytophthora species have been obtained frequently from wet or riparian habitats worldwide and have also been isolated from roots of Alnus and Prunus spp. Although originally assigned to Phytophthora gonapodyides on morphological grounds, it was recognized that these isolates, informally named P. taxon Salixsoil, might represent a separate lineage within ITS Clade 6. Based on phylogenetic analyses and comparisons of morphology, growth‐temperature relationships and pathogenicity, this taxon is formally described here as Phytophthora lacustris sp. nov. Isolates of P. lacustris form a clearly resolved cluster in both ITS and mitochondrial cox1 phylogenies, basal to most other Clade 6 taxa. Phytophthora lacustris shares several unusual behavioural properties with other aquatic Clade 6 species, such as sexual sterility and tolerance of high temperatures, that have been suggested as adaptations to riparian conditions. It appears to be widespread in Europe and has also been detected in Australia, New Zealand and the USA. It was shown to be weakly or moderately aggressive on inoculation to Alnus, Prunus and Salix. The extent of P. lacustris’ activity as a saprotroph in plant debris in water and as an opportunistic pathogen in riparian habitats needs further investigation. Its pathogenic potential to cultivated fruit trees also deserves attention because P. lacustris has apparently been introduced into the nursery trade.     

Elucidation of defence responses and signalling pathways induced in Arabidopsis thaliana following challenge with Phytophthora cinnamomi

Arabidopsis thaliana (Arabidopsis) Col-0 was inoculated with Phytophthora cinnamomi to assess the interaction and defence responses involved. Pathogen ingress and asexual reproduction occurred on root tissue but not leaf tissue. The colonisation of root tissue did not cause disease symptoms or plant death, indicating that Arabidopsis Col-0 was tolerant of the infection. The induction of several plant defence responses including the expression of defence-related genes were found, with differences displayed between inoculated root and leaf tissue. Arabidopsis defence-related gene mutant/over-expressing lines were also inoculated with P. cinnamomi but none of the lines tested exhibited a marked increase in susceptibility to the pathogen.     

Investigations into Encephalartos insect pests and diseases in South Africa and identification of Phytophthora cinnamomi as a pathogen of the Modjadji cycad

South Africa holds the greatest diversity of Encephalartos species globally. In recent years several reports have been received of Encephalartos species in the country dying of unknown causes. The aim of this study was to investigate the presence of, and identify the causal agents of, diseases of Encephalartos species in the Gauteng and Limpopo Provinces of South Africa. Plant material with symptoms and insects were collected from diseased plants in private gardens, commercial nurseries and conservation areas in these regions. Insects collected were identified based on morphology, and microbial isolates based on morphology and DNA sequence data. Insect species identified infesting cultivated cycads included the beetle Amorphocerus talpa, and the scale insects Aonidiella aurantii, Aspidiotus capensis, Chrysomphalus aonidum, Lindingaspis rossi, Pseudaulacaspis cockerelli, Pseudaulacaspis pentagona and Pseudococcus longispinus. Fungal species isolated from diseased plants included species of Diaporthe, Epicoccum, Fusarium, Lasiodiplodia, Neofusicoccum, Peyronellaea, Phoma, Pseudocercospora and Toxicocladosporium. The plant pathogen Phytophthora cinnamomi was identified from E. transvenosus plants in the Modjadji Nature Reserve. Artificial inoculation studies fulfilled Koch's postulates, strongly suggesting that P. cinnamomi is responsible for the deaths of these plants under field conditions.     

Effects of inoculum density,plant age and temperature on disease severity caused by pythiaceous fungi on several plants

Alfalfa, maize, sorghum and sugarbeet plants were inoculated with zoospores ofPhytophthora andPythium species in order to assess the effects of inoculum density, plant age and temperature on disease severity. Seedlings were grown axenically in test tubes and inoculated with zoospore suspensions. Disease severity was assessed by measuring the root growth and discoloration of treated and control seedlings. The incremental root length of all plants decreased and root discoloration increased as inoculum concentration of the pathogen increased. Changes were more intensive among low levels of zoospore concentrations and no significant differences in disease severity were found for inoculum densities higher than 10⁴ zoospores ml^-1. Disease severity was negatively related to plant age. Disease development on sugarbeet seedlings infected withPythium andPhytophthora species was affected by temperature, but the pattern of response was determined by the pathogen’s temperature preferences. The incremental root length decreased as temperature increased up to 25°C. The effect ofPythium dissimile andPhytophthora cactorum on root length was significantly lower at 35°C than at 25°C, whereasPythium aphanidermatum andPhytophthora nicotianae caused significant damage to roots even at 35°C. http://www.phytoparasitica.org posting Dec. 3, 2001.     

Contrasting canopy and fibrous root damage on Swingle citrumelo caused by ‘Candidatus Liberibacter asiaticus’ and Phytophthora nicotianae

Huanglongbing (HLB), associated with the phloem‐limited bacterium ‘Candidatus Liberibacter asiaticus’ (Las), is devastating trees in citrus orchards of Florida. Additionally, Phytophthora nicotianae, omnipresent in citrus soils, causes root rot that reduces water and nutrient uptake by fibrous roots. To investigate fibrous root damage and replacement and canopy size in relation to infection of fibrous roots by Las and P. nicotianae, rootstock seedlings of Swingle citrumelo (Citrus paradisi × Poncirus trifoliata) were inoculated with Las or P. nicotianae in two greenhouse pot trials. Phytophthora nicotianae caused root damage within 5 weeks post‐inoculation, which led to greater reduction of canopy size than for Las‐infected seedlings by the end of the experiment. Las increased accumulation of fibrous root biomass at 5 weeks post‐root trimming (wpt) in the 2014 trial and at 11 wpt in the 2015 trial. New root length was not consistently increased by Las. Reduced total leaf area of symptomless Las‐infected seedlings compared to noninoculated controls might be due to the combined effect of altered carbohydrate allocation between shoots and roots and altered leaf morphology.