Antimicrobials in Phytophthora isolation media and the growth of Phytophthora species

Many recently described Phytophthora species detected using high-throughput sequencing have never been isolated into culture. NARH is a commonly used isolation medium containing cornmeal agar with n ystatin 22.72 ppm, a mpicillin 100 ppm, r ifampicin 10 ppm, and h ymexazol 50 ppm. We investigated whether the antimicrobial compounds in this medium selectively inhibit growth of some Phytophthora species. Growth of 10 Phytophthora species from 10 Phytophthora clades was tested in NARH medium with antimicrobials in a range of concentrations, singly and in combination: nystatin 12.5–100 ppm, ampicillin 62.5–500 ppm, rifampicin 5–40 ppm, hymexazol 12.5–100 ppm and in addition, chloramphenicol 5–40 ppm. Two combinations of antimicrobials that supported good growth of the greatest number of species were selected: (a) nystatin 12.5 ppm, ampicillin 250 ppm, rifampicin 10 ppm, and hymexazol 12.5 ppm, and (b) these compounds with the addition of chloramphenicol 5 ppm. Subsequently the growth of 47 Phytophthora species from all clades was tested in media with these two combinations and standard NARH, both in aseptic culture and in plates with leaf baits infected from soils known to be infested with Phytophthora. Although growth of some Phytophthora species was better with the new combinations of antimicrobials in aseptic culture, suppression of competing microorganisms was best in standard NARH. Sensitivity to the NARH antimicrobials is not the reason for the difficulty or failure of isolating many species detected using high-throughput sequencing. NARH is a robust and appropriate medium for isolation of Phytophthora species from all clades.     

Comparative aggressiveness of standard and variant hybrid alder phytophthoras, Phytophthora cambivora and other Phytophthora species on bark of Alnus, Quercus and other woody hosts

Pathogenicity tests were carried out on the bark of Alnus glutinosa with 19 isolates of the standard (near-tetraploid) hybrid alder phytophthora, nine isolates representing its known heteroploid variants and 11 isolates of P. cambivora , a probable parent species of the hybrid. Over a 4-year period, 12 experiments were conducted on living alder logs incubated at 20°C. Most isolates of the standard hybrid and those of the 'Dutch variant' were highly aggressive to alder bark. Isolates of the 'Swedish', 'UK' and 'German variants', and of P. cambivora , were only weakly pathogenic. Also, isolates of P. fragariae , P. cinnamomi , P . sp. 'O-group', P. cryptogea , P. megasperma , P. gonapodyides and P. citricola were either weakly or nonpathogenic. Rates of lesion development were greatest on logs cut during July–October, slower on logs cut between November and March and zero on logs cut during April, indicating a strong seasonal effect. Other evidence indicated that lesion development was subject to critical thresholds of host resistance. The standard hybrid was nonpathogenic to the bark of four other hardwood and two conifer species, indicating that it is relatively host specific. In contrast, P. cambivora was an aggressive pathogen on live bark of Quercus and Castanea . The significance of these results is discussed.     

Molecular analysis of the major Phytophthora species on cocoa

The internally transcribed spacer (ITS) regions of the ribosomal RNA (rRNA) gene cluster of 161 isolates of Phytophthora species involved in pod rot, stem canker and leaf blight of cocoa were analysed to determine inter- and intraspecific variation in this disease complex. The species P. palmivora , P. megakarya , P. capsici , P. citrophthora and P. nicotianae could all be clearly distinguished by PCR amplification of the ITS region followed by restriction analysis with Hae III, Hinf I, Pvu II and Alu I. This method provided a relatively rapid identification procedure for these species, and was able to distinguish isolates that had previously been misidentified by morphological methods. Sequence analysis showed that the four main cocoa-associated species formed two distinct groups, one comprising P. capsici and P. citrophthora , and the other P. palmivora and P. megakarya . Detailed sequence analysis and comparison with published literature suggested that P. capsici isolates from cocoa may be closely related to P. tropicalis , a species recently described from Cyclamen and Dianthus .     

银杏疫病病原种的鉴定

 从引起银杏疫病的病原疫霉菌株中,选取2个代表菌株进行分类鉴定。根据病原菌形态特征、生理特性、寄主范围和菌体可溶性蛋白质电泳测定等,认为引起银杏疫病的病原疫霉为烟草疫霉(Phytophthora nicotianae Breda de Hann)。该病原菌寄主范围较广,生长温度大于35℃,异宗配合,自然条件下桂北银杏产区不能产生卵孢子,但在低温等不良环境下形成大量厚垣孢子。注意这些特性,对有效开展病害防治有十分重要的意义。     

Efficacy of indigenous Trichoderma harzianum in controlling Phytophthora leaf fall (Phytophthora palmivora) in Thai rubber trees

Journal of Plant Diseases and Protection - Phytophthora leaf fall disease in rubber trees is serious and is known to cause a reduction in both the quantity and quality of rubber latex in Thailand....     

Phytophthora species attacking strawberry and raspberry1

Phytophthora frugariae causes red core root rot of strawberries. Although the disease is probably most acute in northern Europe, serious outbreaks have been reported from a number of Mediterranean countries, especially France and Italy. Leather rot of fruit and crown rot, which are caused by P. cuctorum, can also be severe problems in warmer climates. Both fungi survive in soil for long periods, but the most common form of spread is in diseased planting material. Sensitive tests have been developed to detect red core in planting material, and been used to effect in certification schemes. Root rot of raspberry has gained prominence in the last 10 years in Europe as raspberry growing has expanded, but the disease has been known for many years in France. Nine species of Phyfophthora have been recovered from affected plants, but two of these, one with affinities with P. megasperma, and P. cumbiuora, are responsible for most major outbreaks. The other species are only troublesome where drainage is poor. Again, spread is mainly in infected material. Few raspberries are resistant to root rot, but some wild RubuP spp., blackberries and raspberry x blackberry hybrids are resistant and may be useful in breeding programmes.     

Fungicide resistance in soil-borne Phytophthora species1

Systemic fungicides such as the phenylamides and phosphonates have provided new opportunities for chemical control of soil-borne Phytophthora species. Potential problems in their use include biodegradation in soils, and the emergence of pathogen isolates resistant to these compounds. Resistance to phenylamides is well known in leaf-infecting Oomycetes, but less is known about the development and relative fitness of resistant isolates among populations of soil-borne Phytophthora species. Experiments with P. capsici and P. palmivora have shown that stable resistant and virulent isolates can be selected under laboratory conditions. Some mutants resistant to both metalaxyl and fosetyl-A1 have been recovered, with growth rates and sporulation equivalent to parental wild types. Selected mutants proved able to compete in vivo with fungicide-sensitive wild types in the absence of the compounds. More information is required to fully evaluate the implications of these results for chemical control of soil-borne Phytophthora species in the field. Résistance des Phytophthora du sol aux fongicides     

Electrophoretic study of three related Phytophthora species1

Morphological and cultural characters currently used for the classification of species of Phytophthora are inadequate to distinguish P. cryptogea, P. drechsleri and P. erythroseptica. In this study isolates of the above-mentioned species, including type and/or authentic cultures of P. cryptogea and P. drechsleri were compared. Morphological and cultural characters, together with electrophoretic patterns of both total mycelial proteins and isozymes (arylesterase and malate dehydrogenase), allowed the distinction of P. erythroseptica from the others. Authentic cultures of P. cryptogea and P. drechsleri showed distinctly different electrophoretic patterns of arylesterase and malate dehydrogenase isozymes and of total mycelial proteins. Some Italian isolates previously identified as P. cryptogea or P. drechsleri showed features of both species, therefore it was impossible to discriminate the two species solely on the basis of the electrophoretic patterns of mycelial proteins.     

Evaluation of Banksia species for response to Phytophthora infection

Phytophthora cinnamomi was the species isolated most frequently from soil associated with dead or dying proteaceous plants in the Adelaide region of South Australia. The association of P. citricola with diseased Banksia species in South Australia is reported for the first time. The response of a range of Banksia species to inoculation with P. cinnamomi and P. citricola was assessed. P. cinnamomi was generally more pathogenic than P. citricola . Inoculation of 10-month-old seedlings with colonized millet seed or zoospores showed that B. hookeriana and B. ashbyi were the most susceptible of the species tested, whereas B. coccinea , B. menziesii and B. prionotes were moderately susceptible. B. ericifolia , B. serrata , B. spinulosa var. collina and B. lemanniana showed tolerance. Similarly, 2–3-week-old seedlings of B. ericifolia , B. serrata and B. spinulosa var. collina inoculated in vitro showed little disease 6 and 12 days after inoculation, whereas B. baueri, B. baxteri , B. coccinea and B. solandri , as well as B. hookeriana and B. ashbyi , showed severe symptoms of disease after 6 days. Results suggested that the in vitro assay may have potential in the evaluation of breeding material. Development of infection was studied microscopically in 2–3-week-old seedlings of B. coccinea , B. menziesii , B. serrata and B. spinulosa var. collina inoculated in vitro with zoospores of P. cinnamomi . Roots of B. coccinea and B. menziesii were colonized rapidly and root tips became necrotic within 24 h and hypocotyls by day 5. Penetration was delayed in B. spinulosa var. collina , and callose deposition was delayed in B. coccinea . Necrosis of roots of B. serrata and B. spinulosa var. collina began 3 days after inoculation but rarely extended more than half way up the root by 9 days.     

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 obscura sp. nov., a new species of the novel Phytophthora subclade 8d

A new Phytophthora species was detected (i) in the USA, infecting foliage of Kalmia latifolia, (ii) in substrate underneath Pieris, and (iii) in Germany in soil samples underneath Aesculus hippocastanum showing disease symptoms. The new species Phytophthora obscura sp. nov. is formally named based on phylogenetic analysis, host range, Koch’s postulates and morphology. Phytophthora obscura is homothallic with paragynous antheridia and semipapillate sporangia. It is genetically closely related to P. syringae and P. austrocedrae and together these three species define a new Phytophthora subclade 8d, with significant support for all genetic loci analysed including seven nuclear genes and the mitochondrial gene coxII. The morphological and ecological characteristics are very similar to P. syringae, and it is likely that P. obscura was not described earlier because it was identified as P. syringae. Artificial inoculations indicated that horse chestnut, kalmia, pieris and rhododendron might be hosts, and Koch’s postulates were confirmed for kalmia from which it was isolated. This pathogen was named after its elusive nature since it has to date rarely been detected in the US and Germany.     

Detection of Phytophthora gallica in Bulgaria and co-existence with other Phytophthora species in a small river

Journal of Plant Diseases and Protection - Co-existence of four Phytophthora species in a small river in Bulgaria was established—P. plurivora, P. gonapodyides, P. chlamydospora and P....     

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.     

几种常用杀菌剂对马铃薯晚疫病防治效果比较研究

试验表明50%烯酰吗啉等九种杀菌剂对马铃薯晚疫病发病初期均有好的防效,药后7d防效均在81.5%以上。在马铃薯晚疫病初期施药,各药剂防效差异明显。第2次用药后7d,50%烯酰吗啉水分散粒剂、70%丙森锌可湿性粉剂、60%吡唑醚菌酯/代森联水分散粒剂、72%霜脲·锰锌可湿性粉剂对马铃薯晚疫病防治效果在83%以上,且最后增产效果均在40%以上。而70%甲基硫茵灵可湿性粉剂等其它5种药剂的防病效果只在70%左右,增产幅度也低。各药剂对马铃薯安全,无药害表现。建议向农民首推50%烯酰吗啉水分散粒剂防治马铃薯晚疫病,其次是72%霜脲·锰锌可湿性粉剂、70%丙森锌可湿性粉剂和60%吡唑醚菌酯/代森联水分散粒剂。     

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.     

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.     

Cross-reactivity of antiserum raised against Phytophthora fragariae with other Phytophthora species and its evaluation as a genus-detecting antiserum

Antiserum (anti-PfM) raised against mycelial suspensions of Phytophthora fragariae isolates reacted strongly with antigens from several Phytophthora species. Some cross-reactions with antigens from Pythium species were decreased by fractionating on an affinity column of Sepharose 4B bound to extracts of Fragaria vesca roots infected with P. fragariae. The affinity-purified anti-PfM retained its high cross-reactivity with the various Phytophthora species tested. It also detected infection of raspberry and strawberry roots by some Phytophthora species. This antiserum could, therefore, prove useful as a broad-spectrum Phytophthora-detecting antiserum.
Anti-PfM could not be made specific for P. fragariae because it was raised against components shown to be antigenically similar in all Phytophthora species tested. However, immunoblotting with the affinity-purified anti-PfM produced distinct patterns for P. fragariae, P. erythroseptica and P. cactorum: three serotypes were identified for the latter species. This antiserum might therefore prove useful in classifying Phytophthora species.     

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.