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.     

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.     

Diversity and populations of Phytophthora,Phytopythium and Pythium species recovered from sediments in an agricultural run‐off sedimentation reservoir

Agricultural run‐off sedimentation reservoirs are an emerging aquatic system of critical importance to plant biosecurity, water and environmental sustainability. Oomycete pathogens such as Phytophthora and Pythium species in irrigation water have been demonstrated to pose significant risks to ornamental crops, but little is known about their diversity and populations in sediments of agricultural irrigation systems. This study investigated the oomycete communities including Phytophthora (Ph.), Phytopythium (Pp.) and Pythium (Py.) species in sediments at various depths of an agricultural run‐off sedimentation reservoir in Virginia during the winters of 2011 and 2015. The recovery of these oomycetes declined sharply with sediment depth from surface to 0·8 m and none was recovered from sediments deeper than 1·4 m. A total of 47 oomycete species were recovered, with all four species of Phytophthora and five of Phytopythium exclusively from the surface. Recovered species included many important plant pathogens such as Ph. nicotianae, Ph. pini, Ph. tropicalis, Pp.  vexans, Py. irregulare and Py. monospermum. These results underline the importance of decontaminating sediments excavated from top layers (0–1·4 m) of the sedimentation reservoir before reuse in plant production.     

Characterisation and detection of Pythium and Phytophthora species associated with grapevines in South Africa

Replant and decline diseases of grapevines not only cause quantitative and qualitative yield losses, but also result in extra costs when vineyards have to be replanted. This study investigated the role of Pythium and Phytophthora in the decline syndrome in South Africa by determining (1) the species associated with nursery and established vines, and (2) pathogenicity of Ph. sp. niederhauserii and P. vexans relative to known grapevine pathogens. Quantitative real-time PCR (qPCR) assays were also developed for detection of the most prevalent oomycete groups. In total, 26 Pythium and three Phytophthora species were identified from grapevine nurseries and established vineyards. The most common infections in sampled nursery vines were caused by P. vexans (16.7%), followed by P. ultimum var. ultimum (15.0%) and P. irregulare (11.7%). In established vineyards, P. irregulare (18.0%) and P. vexans (6.2%) were also among the three most prevalent species, along with P. heterothallicum (7.3%). Three Phytophthora species were also identified from the sampled established vines, of which Ph. cinnamomi (5.1%) was predominant, followed by Ph. sp. niederhauserii (1.1%). In established vineyards a higher incidence and more diverse species composition was observed in spring and winter, than in summer. Pathogenicity studies showed that some Ph. sp. niederhauserii and P. vexans isolates were as aggressive as the known grapevine pathogens Ph. cinnamomi and P. irregulare. Sensitive qPCR assays were developed for the detection of P. ultimum var. ultimum, P. irregulare, P. vexans and the genus Phytophthora. These assays will be invaluable in limiting pathogen dispersal through screening of nursery material. This is especially important since pathogenic species were also isolated from healthy looking vines in nurseries.     

Phytophthora boehmeriae boll rot: a new threat to cotton cultivation in the mediterranean region

A boll rot of cotton (Gossypium hirsutum L.) was observed for the first time in Greece in August 1993 in Larissa and Volos counties, and in August and September 1995 in Trikala and Phthiotis counties. Fungi of the genusPhytophthora were isolated from diseased plants. Morphological characteristics of the pathogen were recorded on mounts made directly from the infected tissues or after growth of the isolated fungus on corn meal agar or sterile distilled water. Colony morphology, growth rates, features of asexual and sexual structures and maximum growth temperatures were examined. APhytophthora species new to Europe,Phytophthora boehmeriae Sawada, attacking cotton bolls, was identified. The pathogenicity of the isolates was confirmed by artificial inoculations of detached cotton bolls. Analysis of α-esterase isozymes revealed unique banding patterns for isolates ofP. boehmeriae compared with those ofP. cactorum andP. parasitica, which arePhytophthora species with similar morphology.     

Phytophthora hydropathica,a new pathogen identified from irrigation water,Rhododendron catawbiense and Kalmia latifolia

A new species of Phytophthora, previously referred to as taxon Dre II, is named Phytophthora hydropathica. It is heterothallic, but all isolates recovered to date are of the A1 compatibility type. Plerotic oospores are produced. Its sporangia are usually obpyriform and are nonpapillate and noncaducous. Isolates of P. hydropathica had nearly identical single‐strand conformation polymorphism (SSCP)‐based DNA fingerprints that are distinct from those of all existing species. Their closest relatives are P. parsiana and P. irrigata. This new species is able to grow at relatively high temperatures, with an optimum of 30°C and a maximum of 40°C. It was frequently isolated from irrigation water during warm summers. This species caused leaf necrosis and shoot blight of Rhododendron catawbiense and collar rot of Kalmia latifolia at two nurseries where irrigation reservoirs yielded P. hydropathica. Its potential impact on other horticultural crops is discussed.     

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].     

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.     

Targets for pathology research in protected crops

Due to the need to diminish the amount of pesticides used, alternative ways of controlling fungal diseases have to be developed. Foliar diseases have been managed mainly by chemicals, but research has been started to develop integrated control programmes. Data from studies on epidemiology, in combination with nutritional and climatic management and use of partial resistant cultivars will lead to a reduced use of chemicals. Models used are: Botrytis cinerea in gerbera and roses, and powdery mildew in roses and cucumbers. Research has been intensified on biological control of these pathogens. In closed systems, with recirculation of the nutrient solution, soil-borne fungi can cause serious problems. Not only do diseases known from traditional cultural systems, like fusarium wilt of carnation, fusarium crown and root rot of tomato and Phytophthora and Pythium spp. in several crops occur, but also new problems like a new Phytophthora sp. and Gnomonia sp. in roses and a Cylindrocladium sp. in Spathiphyllum spp. To prevent introduction of pathogens in recirculation systems, emphasis is put on developing an integrated disease management programme by using disease-free planting material, disease-free irrigation water, strict hygienic measures, resistant cultivars, methods of disinfecting the nutrient solution and biological control. Research over the last 5–6 years has revealed good prospects for biological control, especially in closed systems with a limited amount of substrate. Wilt in carnation, caused by Fusarium oxysporum f.sp. dianthi can be prevented effectively by adding a non-pathogenic isolate of F. oxysporum and/or Pseudomonas spp. There are indications that non-pathogenic isolates of F. oxysporum are also effective against wilt disease in other crops. An isolate of Trichoderma harzianum appears to be very effective against fusarium crown and root rot of tomato. More applications of this, and other, biocontrol agents seem possible. However, translation of the results of research to practical application and registration is still very difficult.     

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.     

Usefulness of single copy genes containing introns in Phytophthora for the development of detection tools for the regulated species P. ramorum and P. fragariae

Introns are generally highly polymorphic regions within genes and were proven to be of great interest for discriminating among phylogenetically-close Phytophthora species. Phytophthora ramorum and P. fragariae are considered as quarantine pathogens by the European Union and accurate detection tools are therefore necessary for their monitoring. From introns located in different single copy genes (GPA1, RAS-like, and TRP1), we developed a series of PCR primers specific to P. ramorum and P. fragariae. The specificity of these primers was successfully checked with a wide collection of Phytophthora isolates and a protocol was developed to detect both pathogens directly in infected plant tissues. These genes should be of particular interest for the development of additional species-specific detection tools within the Phytophthora genus.     

Identification of Phytophthora species on fruit crops in Turkey by polyacrylamide gel electrophoresis1

To identify 11 Phytophthora isolates obtained from lemon, peach and apple trees, and from strawberry plants, in the Mediterranean region of Turkey, protein, acetylesterase, peroxidase and catalose band patterns of these isolates and the same bands of 10 identified isolates of Phytophthora citrophthora, P. cinnamami and P. cactarum were compared. These 11 regional isolates were identified on the basis of the similarity of their protein and enzyme band patterns to those of identified Phytophthora isolates. It was concluded that protein band patterns could be used for identification of Phytophthora species. With enzyme band patterns, however, it may be possible to identify at species level but it is more practical to use this method only for identification of subspecies.     

Extracts of the endemic resurrection plant Haberlea rhodopensis stimulate in vitro growth of various Phytophthora spp. pathogens

The Phytophthora pathogens are among the most important pests in the modern agriculture and forestry. This makes them a subject of intensive studies, including laboratory experiments with in vitro culture on various plant-derived media. Here we show that leaf extracts from the resurrection endemic plant Haberlea rhodopensis stimulates significantly the in vitro growth of various isolates from several Phytophthora species. The extracts stimulate the formation of oospores in some heterothallic species. In this respect, the idea to propose Haberlea extracts as a potential ingredient of culture media for Phytophthora spp. maintenance under controlled conditions or as a component of detection systems for Phytophthora presence in nature seems quite attractive and promising.     

Managing Phytophthora ramorum in the Netherlands1

Phytophthora ramorum came to the Netherlands in 1993. Despite initially not seeming to pose a high risk, findings in California showed its potential destructive impact on ecosystems. A programme began in the Netherlands to eliminate P. ramorum from nurseries and surveys in the natural environment were held to obtain information to determine a strategy for dealing with the disease. About 1100 nurseries are inspected annually by NAKtuinbouw under the auspices of the Plant Protection Service and measures are taken according to EC directives. The percentage of infected nurseries decreased steadily during recent years, from 4% in 2002/2003 to 0.5% in 2004/2005. Surveys in the natural environment show that P. ramorum occurs on 2% of the sites with Rhododendron and therefore it was concluded that an elimination scenario is not realistic. A programme based on containment measures supported by an extension programme was put into place with its effects being monitored by the Plant Protection Service. 12 years of observing P. ramorum show that the risk for indigenous trees and shrubs in the Netherlands is very limited. Spread from infected rhododendrons to other potential hosts, even at heavily infected Rhododendron sites, hardly takes place although some infected Quercus rubra trees have been found. Recently several new Phytophthora species were found in natural environments in Europe and California, mainly as a result of intensive P. ramorum surveys. As well as P. ramorum, the Phytophthora spp. P. kernoviae, P. numerosa and P. pseudosyringae pose risks, indicating the need for a more general approach against Phytophthora diseases. As a result, a new protocol for detection and identification of Phytophthora spp. both as a group and individually is being developed and workers are asking whether these Phytophthora species could be managed together.     

<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%).     

Ilyonectria palmarum sp. nov. causing dry basal stem rot of Arecaceae

During surveys conducted in 2010–2013, a complete breakage or bending of the trunk and a dry basal stem rot were observed on containerised Brahea armata, B. edulis, Howea forsteriana and Trachycarpus princeps plants in different nurseries located in eastern Sicily (southern Italy). A cylindrocarpon-like species was consistently obtained from diseased palm tissues, while known pathogens of these hosts such as Ganoderma, Phytophthora and Thielaviopsis were not found associated with symptomatic tissues or isolated on standard or selective media. A total of 40 cylindrocarpon-like isolates were collected and characterised based on morphology and DNA phylogeny. Multigene analyses based on the β-tubulin, histone H3, translation elongation factor 1-α, and the internal transcribed spacers (ITS1, 5.8S, ITS2) genes facilitated the identification of a new species, described here as Ilyonectria palmarum. The pathogenicity of one representative isolate collected from each palm species was tested on plants cultivated under nursery conditions and in a growth chamber. All isolates were pathogenic to B. armata, B. edulis, H. forsteriana, and T. princeps and symptoms identical to that observed in nurseries were reproduced. Dry basal stem rot and stem bending caused by Ilyonectria palmarum represents a potentially serious problem for nurseries cultivating containerised palms.     

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.     

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.     

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.     

Specific and Sensitive Detection of Phytophthora nicotianae By Simple and Nested-PCR

Phytophthora nicotianae Breda de Haan is one of the most important soil-borne plant pathogens. The identification of this pathogen based on morphological or physiological characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Based on this concept, conventional and single closed tube nested-PCRs were developed for the specific and sensitive detection of P. nicotianae. Two new specific primers, designed from the spacer regions ITS1 and ITS2, internal to the nucleotide sequence flanked by universal primers ITS4 and ITS6, were used. To evaluate the specificity of the method, 36 morphologically characterized isolates were tested. A positive reaction, characterized by an amplification product of 737 bp, was shown by all P. nicotianae isolates and two P. nicotianae/cactorum hybrids. No amplification product was observed when other Phytophthora species and genera were assayed. The sensitivity of this method was analysed by serial dilutions of a defined amount of fungal DNA in a healthy root extract. Nested-PCR was at least 1000 times more sensitive than conventional PCR. In addition, samples from different infection sites, origins and crops, samples from nutrient solution, water and the rockwool used in hydroponic cultures, were analysed to validate this method.