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.     

Two Phytophthora species causing decline of wild olive (Olea europaea subsp. europaea var. sylvestris)

Since 2009, a severe decline leading to mortality has been observed affecting nearly 5 ha of a wild olive woodland of high ecological value in Seville, southern Spain. Phytophthora cryptogea and P. megasperma were consistently isolated from roots and rhizosphere of trees with symptoms sampled in 2009, 2011 and 2013. The isolates were identified on the basis of colony and reproductive structure morphology as well as temperature–growth relationships, and identification was further corroborated by their ITS and β‐tubulin sequences. Koch's postulates were demonstrated for both species on 1‐year‐old wild olives. Pathogenicity tests showed that both Phytophthora spp. are highly aggressive pathogens, although temperature–growth requirements for each species were distinct. As a consequence, the two species may be active in different seasons and their epidemiology may be differently influenced by global climate change, and they may show their active periods in different climatic scenarios. The climate change models for the Mediterranean Basin forecast a global temperature increase that favours the more thermophilic P. cryptogea. The high susceptibility to phytophthora root rot should not be disregarded in olive breeding programmes where wild olive is used as a source of resistance to verticillium wilt.     

Early survival of Quercus ilex subspecies from different populations after infections and co‐infections by multiple Phytophthora species

Forests in Europe are threatened by increased diversity of Phytophthora species, but effects on trees of simultaneous infections by Phytophthora and ecological consequences of their coexistence are unknown. This study explored variation in early survival of Quercus ilex to Phytophthora infections and assessed interactions between Phytophthora species when trees were co‐infected. Three Phytophthora species (P. cinnamomi, P. gonapodyides and P. quercina), seeds from 16 populations of Q. ilex (ballota and ilex subspecies) and two infection times were used as sources of variation in two experiments. The influence of Phytophthora species, Q. ilex subspecies and populations on plant germination and survival were analysed using generalized linear mixed models and survival analysis techniques. Germination rates were not influenced by Phytophthora spp. (P = 0.194) but by the subspecies and populations of Q. ilex (P < 0.001). In Phytophthora‐infested soils, Q. ilex subsp. ilex germinated at higher rates than Q. ilex subsp. ballota. Plant survival was strongly influenced by Phytophthora species (P < 0.001), not by the subspecies and populations of Q. ilex. Seedling mortality was reduced and delayed if a less virulent Phytophthora species infected plants prior to infection by a more virulent Phytophthora species. The results help to explain oak decline syndrome and the lack of natural and artificial regeneration of Q. ilex forests. Lack of interspecific variability of early survival to Phytophthora spp. discourages direct sowing for artificial reforestation programmes. Large, thick seeds, giving plants rapid growth, are advantageous traits when soils are infested with Phytophthora spp.     

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.     

<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%).     

Control of Phytophthora species in plant stock for habitat restoration through best management practices

Emergent plant pathogens represent one of the most significant threats to biodiversity, and exotic Phytophthora species have recently emerged as a serious problem in restored habitats in California and in nurseries producing the plant stock. It is hypothesized that ‘best management practices’ prescribed through a Phytophthora Prevention Programme (PPP) could be useful in minimizing phytophthora disease incidence. To understand the magnitude of the problem and the efficacy of the PPP, plants in restoration nurseries were evaluated for (i) the Phytophthora species assemblage present in the absence of the PPP, and (ii) the effectiveness of the PPP to reduce them. Sampling included 203 plants grown in the absence of the PPP, and 294 grown implementing the PPP. Only samples collected in the absence of the PPP were Phytophthora-positive, and cumulatively yielded 55 isolates from 13 different taxa, including 1 putative interspecific hybrid genotype. There were 21 novel Phytophthora–plant species combinations. The most common Phytophthora species was P. cactorum. Four plant species had the highest disease incidence, namely: Diplacus aurantiacus (50 ± 11.2%), Heteromeles arbutifolia (33 ± 9.6%), Ceanothus thyrsiflorus (30 ± 8.4%), and Frangula californica (30 ± 8.4%). Disease incidence in nurseries after the implementation of the PPP dropped to zero (P < 0.001), and was unaffected to any significant degree by nursery differences, or plant species tested. This study identifies a large number of novel ‘plant species × Phytophthora species’ combinations, and provides for the first time strong evidence that the PPP significantly reduced Phytophthora in plant stock for habitat restoration.     

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.     

Biocontrol of Phytophthora Root Rot of Angelica Trees by Enterobacter cloacae and Serratia ficaria Strains

Bacterial strains isolated from the rhizosphere of angelica trees were evaluated for their antagonistic activity against Phytophthora cactorum, a causal agent of Phytophthora root rot. Of these, three bacterial strains, designated as T-1-8, T-1-14 and T-1-23, strongly inhibited mycelial growth of P. cactorum ARE-862 in a dual-culture plate assay. Biocontrol activity of these strains was then examined by dipping root of young seedlings of angelica trees into a bacterial suspension. The incidence of Phytophthora root rot was markedly suppressed for at least 79 days in pot tests when treated seedlings were planted in naturally infested soil. The suppression was maintained through June of the next year. In addition, these strains significantly reduced the development of Phytophthora root rot up to 47 days in naturally infested field and up to 63 days (the last day of testing) in an artificially (moderately) infested field. Based on their main bacteriological properties, strain T-1-14 was identified as Enterobacter cloacae and T-1-8 and T-1-23 were identified as Serratia ficaria. Received 5 July 1999/ Accepted in revised form 25 October 1999     

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.     

Phytophthora root and crown rot of raspberry in Bulgaria

Root and crown rot of raspberry (Rubus idaeus L.) was observed in a plantation at the experimental station of small fruits in Kostinbrod, Bulgaria. Isolates ofPhytophthora spp. were obtained from diseased plants. Colony morphology, growth rates, features of asexual and sexual structures were studied and as a result twoPhytophthora species were identified:Phytophthora citricola Saw. andPhytophora citrophthora (R.E. Sm. & E.H. Sm.) Leonian. Their pathogenicity was confirmed in artificial inoculation experiments. The isozyme (-esterase) patterns ofP. citrophthora andP. citricola isolates from raspberry and from the collection of the CBS, Baarn the Netherlands were compared, using micro-gel electrophoresis. Both species are reported for the first time as pathogens of raspberry in Bulgaria. This is only the second report in phytopathological literature ofP. citrophthora on raspberry, the first being from Chile [Latorre and Munoz, 1993].     

Age‐related susceptibility of Eucalyptus species to Phytophthora boodjera

Phytophthora boodjera is a newly described pathogen causing damping off and mortality of Eucalyptus seedlings in Western Australian nurseries. This study evaluated the age‐related susceptibility of several taxa of mallee Eucalyptus to P. boodjera in sterilized washed river sand‐infestation pot trials. Phytophthora cinnamomi and P. arenaria were included for comparison. Seedlings of Eucalyptus taxa were inoculated at 0, 2, 4, 12 and 88 weeks with individual Phytophthora isolates. Pre‐emergent mortality in the presence of Phytophthora was almost 100%. Post‐emergent mortality was 50–100%, depending on isolate, compared to 0% for the control. Mortality was also high for inoculated 1 month‐old seedlings (46–68%) and root length of surviving seedlings was severely reduced. Death from root infection was not observed for seedlings inoculated at 12 and 88 weeks, but they developed root necrosis and reduced root dry weight compared to non‐inoculated controls. Phytophthora boodjera is a pre‐ and post‐emergent pathogen of mallee eucalypts. These eucalypts are susceptible to P. boodjera at all life stages tested, but the mortality rates declined with plant age. Similar results were obtained for P. cinnamomi and P. arenaria. The events leading to its recent appearance in the nurseries remain unknown and further investigations are underway to determine if this is an introduced or endemic pathogen. The approach used here to understand the impact of a Phytophthora species on multiple hosts at different seedling ages is novel and sets a benchmark for future work.     

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.     

大豆疫霉菌部分生物学特性及其药剂筛选研究

采用大豆疫霉菌在不同条件下菌丝生长速度法研究了大豆疫霉菌的部分生物学特性,并应用杀菌剂室内生测、盆栽药剂防效对药效作了评价。研究结果表明,大豆疫霉菌营养生长的最适温度为25～30℃;最适pH为6;光暗交替有利于该菌营养体的生长,在Rye或CA培养基上生长最快。室内药效测定结果表明,烯酰吗啉EC₅₀为0.165 4μg/mL,抑菌效果最好,甲霜灵、甲霜灵.锰锌和氟吗啉.锰锌的EC_{50分别为0.261 00、.451 0和0.984 2μg/mL,效果次之。盆栽试验结果表明,几种药剂在活体条件下对大豆疫病的防治效果较好,并有较长持效期。}     

Effect of nitrogen form and application method on incidence and severity ofPhytophthora crown and root rot of apple trees

The effect of ammonium nitrate broadcast as a soil or through irrigation, urea applied as a foliar spray, and monoammonium phosphate applied as a planting hole treatment on the incidence ofPhytophthora crown and root rot of apple trees was determined under orchard conditions in the Okanagan Valley of British Columbia, Canada. Results from the eight year study showed that ammonium nitrate applied as a single dose in spring at 240 g tree^–1 year^–1, as a split dose at 120 g tree^–1 each in spring and early autumn, and in irrigation water (fertigation) at 7.5 g tree^–1 wk^–1 for 10 wk year^–1 significantly increasedPhytophthora crown and root rot of Macspur on MM106 rootstock. There was no significant difference inP. cactorum infection between the unfertilized control and treatments with urea applied as a foliar spray at 1.0 kg 100 l^–1 of water in spring and early autumn, and monoammonium phosphate applied as a planting hole treatment at 1 g l^–1 of soil at planting time.     

Phytophthora pachypleura sp. nov., a new species causing root rot of Aucuba japonica and other ornamentals in the United Kingdom

Isolates of an unknown Phytophthora species from the ‘Phytophthora citricola complex’ have been found associated with mortality of Aucuba japonica in the UK. Based on morphological characteristics, growth–temperature relationships, sequences of five DNA regions and pathogenicity assays, the proposed novel species is described as Phytophthora pachypleura. Being homothallic with paragynous antheridia and semipapillate sporangia, P. pachypleura resembles other species in the ‘P. citricola complex’ but can be discriminated by its distinctively thick‐walled oospores with an oospore wall index of 0·71. In the phylogenetic analysis based on three nuclear (ITS, β‐tubulin, EF‐1α) and two mitochondrial (cox1, nadh1) DNA regions, P. pachypleura formed a distinct clade within the ‘P. citricola complex’ with P. citricola s. str., P. citricola E and P. acerina as its closest relatives. Phytophthora pachypleura is more aggressive to A. japonica than P. plurivora and P. multivora and has the potential to affect other ornamental species.     

Two novel and potentially endemic species of Phytophthora associated with episodic dieback of Kwongan vegetation in the south‐west of Western Australia

Two novel homothallic species of Phytophthora causing dieback of Kwongan vegetation in south‐west Western Australia are described here as Phytophthora arenaria sp. nov. and Phytophthora constricta sp. nov. DNA sequencing of the ITS rDNA and cox1 gene confirmed that P. arenaria and P. constricta are unique species residing in ITS clades 4 and 9, respectively. Phytophthora arenaria has been isolated from vegetation occurring on the northern sandplains which are warmer and drier than the southern sandplains from which P. constricta has been predominantly isolated, and both species appear morphologically and physiologically well adapted to the ecosystems in which they occur. Both species have been associated mainly with dead and dying Banksia species and the pathogenicity of both P. arenaria and P. constricta to Banksia attenuata was confirmed in this study. The combination of unique DNA sequences, including considerable variation in cox1 sequence data, thick oospore walls and physiological characteristics that appear to be adaptations favouring survival in the harsh Kwongan ecosystem suggest that these species may be endemic to Western Australia.     

Phytophthora rot of alpine delphinium caused by Phytophthora sp. kelmania

Severe stunting and root rot were observed on alpine delphinium plants (Delphinium elatum) from Aomori Prefecture, Japan, in 2010 and 2011. A Phytophthora isolate from the diseased crown was identified as Phytophthora sp. kelmania based on morphological characteristics and DNA sequence data. Inoculation of alpine delphinium plants with the isolate produced a similar root rot. Pathogenicity of the isolate on four species of plants that are known hosts for P. sp. kelmania was confirmed. We propose the name “Phytophthora rot” (eki-byo in Japanese) for the present new disease on alpine delphinium.

     

Paraphoma chlamydocopiosa sp. nov. and Paraphoma pye sp. nov., two new species associated with leaf and crown infection of pyrethrum

Two new pathogens of pyrethrum, described as Paraphoma chlamydocopiosa and Paraphoma pye, isolated from necrotic leaf lesions on pyrethrum plants in northern Tasmania, Australia, were identified using morphological characters, phylogenetic analysis of the internal transcribed spacer (ITS), elongation factor 1‐α (EF1‐α) and β‐tubulin (TUB) genes, and pathogenicity bioassays. Bootstrap support in the combined and individual gene region phylogenetic trees supported the two species that were significantly different from the closely related P. chrysanthemicola and P. vinacea. Morphological characteristics also supported the two new species, with conidia of P. chlamydocopiosa being considerably longer and wider than either P. chrysanthemicola or P. vinacea, and P. pye being distinct in forming bilocular pycnidia. Glasshouse pathogenicity tests based on root dip inoculation resulted in P. chlamydocopiosa and P. pye infecting the crown and upper root tissues of pyrethrum plants, and significant reduction in biomass 2 months after inoculation. Both of these Paraphoma species caused leaf lesions during in vitro and in vivo bioassays 2 weeks after foliar spray inoculation. Although P. chlamydocopiosa and P. pye were shown to be crown rot pathogens, they were also commonly isolated from leaves of diseased plants in pyrethrum fields of northern Tasmania.     

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.     

Phytophthora oleae sp. nov. causing fruit rot of olive in southern Italy

A homothallic Phytophthora species was found to be consistently associated with a rot of mature fruits of two local cultivars of olive (Olea europaea) in Calabria, southern Italy. The phylogenetic analysis of sequences of the ITS1‐5.8S‐ITS2 region and cox1 gene enabled its identification as a new species of clade 2, with a basal position compared to previously described subclades. The new species is described formally with the epithet Phytophthora oleae, referring to the natural matrix from which it was isolated. A unique combination of molecular and morphological characters clearly separates P. oleae from other already described Phytophthora species. This new species produced semipapillate, occasionally bipapillate, persistent sporangia on simple sympodially branching sporangiophores as well as globose and smooth‐walled oogonia, paragynous antheridia and spherical, plerotic oospores. The pathogenicity of P. oleae was confirmed in inoculation trials on fruits of three olive cultivars, including the two local cultivars from which the pathogen had been isolated.