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.     

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.     

Specific detection of Phytophthora cactorum in diseased strawberry plants using nested polymerase chain reaction

Validated protocols for DNA purification and PCR amplification are reported for detection of Phytophthora cactorum in diseased strawberry plants. To remove PCR inhibitors, necrotic strawberry tissues were soaked in 5% alconox solution for >12 h before DNA extraction, and the extracted genomic DNA was embedded in an agarose gel chamber and subjected to electrophoresis. The purified DNA was amplified reliably by PCR. Nested PCR was used to detect a portion of the rRNA gene of P. cactorum in samples. In the first round of PCR, primers ITS1 and ITS4 amplified fragments of varying sizes from total genomic DNA from diseased strawberry plants. In the second round of PCR, a 1:25 dilution of the first-round PCR products was used as template with two P. cactorum- specific primer pairs (BPhycacL87FRG and BPhycacR87RRG, which amplified a 340-bp fragment and a 480-bp fragment from the rRNA gene; and BPhycacL89FRG and BPhycacR176RRG, which amplified a 431-bp fragment). Validation tests using culture-based isolations as a standard for comparison indicated that the DNA purification and PCR primers and amplification protocols were reliable and specifically amplified a portion of the rRNA gene of P. cactorum from necrotic root, crown and petiole tissues of strawberry naturally infected by the pathogen.     

3株放线菌对致病疫霉抑制作用的比较研究

采用对峙培养法、菌悬液混合法和块茎切片法分别比较了3株拮抗放线菌Sy11、NB8和A5295对致病疫霉(Phytophthora infestans)的抑制作用及其对马铃薯晚疫病的预防效果。结果显示,放线菌活体菌株以NB8对致病疫霉菌丝生长的抑制作用最强(87.18%),Sy11次之(76.92%),A5295无明显抑制作用。3株拮抗株菌对致病疫霉游动孢子释放和孢子囊直接萌发的抑制作用均随菌液浓度下降而减弱。其中,Sy11的抑制作用最强,A5295最弱;其原液对游动孢子释放的抑制率分别为92.3%、79.0%和63.7%,对孢子囊直接萌发的抑制率分别为75.8%、72.4%和55.8%。在块茎切片上对马铃薯晚疫病的预防效果也以Sy11最高(75.90%),显著优于NB8和A5295。受Sy11和NB8抑制致病疫霉部分菌丝体形态发生畸变,而A5295对菌丝形态未表现出明显的致畸作用,表明Sy11防治马铃薯晚疫病潜力最大。     

Response of selected South Australian native plant species to Phytophthora cinnamomi

Thirty‐seven South Australian native plant species from 11 families, including 15 threatened species in the state (of which six are listed as threatened under the federal Environment Protection and Biodiversity Conservation Act 1999) were assessed for response to infection by Phytophthora cinnamomi. Seedlings, 3–6 months old and grown in a greenhouse, were inoculated by placing infested pine wood plugs in the potting mix, maintained in moist conditions and assessed for mortality and disease symptoms for between 3 and 10 months. Thirty species were found to be susceptible, of which nine were highly susceptible, 15 moderately susceptible and six slightly susceptible. Three species were found to be resistant and results for four species were inconclusive. Six of the 15 threatened, rare or locally endangered species tested (Eucalyptus viminalis var. viminalis, Correa aemula, C. calycina, Olearia pannosa ssp. pannosa, Pomaderris halmaturina ssp. halmaturina and Prostanthera eurybioides) were moderately susceptible, while two (Allocasuarina robusta and Pultenaea graveolens) were highly susceptible. Significant populations of at least five of the threatened species susceptible to the disease are located close to confirmed or suspected Phytophthora‐infested areas or growing in areas conducive for P. cinnamomi. An effective management strategy is therefore required to avoid extinction of such species due to infection by the phytophthora dieback pathogen.     

Two new species,Phytophthora nagaii sp. nov. and P. fragariaefolia sp. nov., causing serious diseases on rose and strawberry plants,respectively, in Japan

A new disease of rose was noticed in Chiba Prefecture of Japan in 1968, and the pathogen was initially identified as Phytophthora megasperma based on morphological characteristics. Similar Phytophthora isolates have since been collected from rose plants in Chiba, Kanagawa, and Shizuoka Prefectures. In 2005, several Phytophthora isolates were recovered from crowns of strawberry plants in Hokkaido Prefecture. These were considered to be members of a new species. In this study, we re-examined all these isolates using morphological and physiological studies and a multilocus phylogenetic analysis. The rose and strawberry isolates were mostly similar morphologically and physiologically, with some exceptions. The rose isolates differed significantly from P. megasperma sensu stricto and other related Phytophthora species. The rose and strawberry isolates had external proliferation of sporangia, characteristic funnel-shaped oogonia, predominantly paragynous antheridia, and fast growth rates of 10.5 mm/24 h at an optimum temperature of 28 °C. In the multilocus phylogenetic tree constructed using sequences from the rDNA ITS regions, rDNA LSU, and the translation elongation factor 1-α, β-tubulin and coxI genes, they formed a distinct monophyletic group in clade 7 with strong bootstrap support. The rose and strawberry isolates separated into two distinct groups. The results indicate that the rose and strawberry isolates constitute two separate species, designated here as Phytophthora nagaii and P. fragariaefolia.     

Root and crown rot of strawberry caused by Pythium helicoides and its distribution in strawberry production areas of Japan

Pythium species were isolated from seedlings of strawberry with root and crown rot. The isolates were identified as P. helicoides on the basis of morphological characteristics and sequences of the ribosomal DNA internal transcribed spacer regions. In pathogenicity tests, the isolates caused root and crown rot similar to the original disease symptoms. Multiplex PCR was used to survey pathogen occurrence in strawberry production areas of Japan. Pythium helicoides was detected in 11 of 82 fields. The pathogen is distributed over six prefectures.     

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.     

Comparison of serological, culture, and bait methods for detection of Pythium and Phytophthora zoospores in water

Two immunodiagnostic detection assay procedures were compared with two conventional assays for their sensitivity in detecting propagules of Pythium ultimum var. sporangiiferum , Pythium Group F, Phytophthora cactorum and P. cryptogea in dilution series in sterile distilled water. The most sensitive assay for all four species was the zoospore trapping immunoassay (ZTI). Conventional membrane filtration-dilution plating gave similar results to ZTI with the two Phytophthora spp., but was less sensitive in Pythium detection. Immunodiagnostic dipstick assays and conventional bait tests showed similar sensitivities in the dilution series, and were generally about two orders of magnitude less sensitive than ZTI. The four techniques were also compared for their detection efficacy with water samples collected from horticultural nurseries and in in situ tests of infected root zones of Chamaecyparis , tomato and Chrysanthemum . In these comparisons, ZTI was again the most sensitive test for water samples, although membrane filtration-dilution plating proved to be a more consistent test. Dipstick and baiting assays were the best techniques for in situ testing, and dipsticks provided epidemiologically valuable, quantitative data on pathogen propagule numbers.     

Effect of temperature and host genotype on the production of inoculum by Phytophthora fragariae var. fragariae from the roots of infected strawberry plants

A bioassay was used to monitor the release of inoculum in drainage water from strawberry plants inoculated with zoospores of Phytophthora fragariae var. fragariae. The fungus was detected in drainage water from plants that had been held at temperatures between 2 and 20 C. but not from plants held at 26°C. The lag phase before secondary inoculum was first released, the maximum and total amounts of inoculum released, and the length of time over which inoculum was released were all greater at the lower temperature regimes, especially those below 10 C. The results were consistent with observations on the effect of temperature on zoospore production from agar discs and on zoospore motility: more zoospores were produced at lower temperatures and they remained motile for longer. From this it is concluded that the inoculum detected consists mainly of motile zoospores. In most experiments with standardized suspensions c. 10-15 were sufficient to initiate infection of the plants in the bioassay. In general, more inoculum was produced by host genotype/fungal isolate combinations in which there were marked root rot symptoms than in combinations in which the host was resistant.     

Pathogenicity of four Pythium species to wheat, barley, peas and lentils

Pythium heterothallicum, P. irregulare, P. torulosum and P. ultimum var. sporangiiferum were compared for pathogenicity to seedlings of winter wheat, spring barley, lentils and peas in growth chambers at 5, 10, 15, 20 and 25 C. These four fungi are among the most commonly isolated Pythium species from wheat roots and wheat-field soils in eastern Washington and northern Idaho, USA, where wheat, spring barley, lentils and peas are grown in various rotations. Pathogenicity was determined in artificially infested soils (500 propagules per g) based on ability to cause pre-emergence death and post-emergence stunting of seedlings. P. ultimum var. sporangiiferum caused significant pre-emergence death of the wheat at 15–25 C, lentils at 10–25 C. and peas at 5 25 C. P. irregulare caused pre-emergence death only of peas and only at 5 C. With the possible exception of lentils at 25 C, P. heterothallcum and P. torulosum caused no pre-emergence death of any of the four plant species. None of the species caused pre-emergence death of spring barley. P. ultimum var. sporangiiferum caused the most post-emergence stunting of wheat, peas and lentils at 10 C and above. Pythium irregulare caused as much or more stunting than P. ultimum var. sporangiiferum on wheat, lentils and peas at 5 C, and was the most pathogenic species on barley at 10, 20 and 25 C. P. irregulare caused significantly more post-emergence stunting of wheat at 5 C with than without chaff (added as a food base for the pathogen); this was not offset by adding ammonium sulphate with the chaff.     

Pathogenicity and genetic variation of Phytophthora cactorum from silver birch and strawberry

Phytophthora cactorum strains isolated from necrotic stem lesions on Betula pendula seedlings or from Fragaria ananassa plants suffering from crown rot were pathogenic to their host plants. Only isolates from birch caused clear lesions on non-wounded bark of birch. P. cactorum isolates from birch were not detrimental to strawberry. Random Amplified Polymorphic DNA (RAPD) analysis revealed variation within P. cactorum, isolates from silver birch having different banding patterns than those from strawberry. UPGMA analysis clustered isolates from silver birch and strawberry plants into separate groups. The data show that the recent outbreak in Finland of P. cactorum in birch could not be caused by the import of strawberry plants affected by crown rot.     

Frequency of virulence phenotypes of Phytophthora fragariae in the field

The virulences of 102 single-zoospore cultures of Phytophthora fragariae from one field site were determined on a range of strawberry differentials, and used to assign the cultures to four clusters (I, II A, IIB, IIC) using cluster analysis. On cv. Favourite, which is susceptible to all known races of the pathogen, isolates in cluster I were recovered most frequently and had the narrowest spectrum of virulence. On cv. Saladin isolates in cluster IIB were more common and were pathogenic to this cultivar. However, c. 30% of the single zoospores from field isolates from Saladin were avirulent on this cultivar and belonged to cluster I. Hyphal-tip and single-zoospore cultures from selected field isolates in cluster IIB did not always have the same virulence phenotype as the parent isolates. One hyphal-tip culture from a field isolate in cluster IIB had a virulence phenotype (IID) which had not been recorded before in Europe, and it attacked some cultivars not previously affected by red core. When cultivars such as Saladin were inoculated with mixtures of zoospores from two isolates from different clusters, with and without the corresponding virulence factors, the isolate with the corresponding virulence factor was selected. However, on the universally susceptible cv. Favourite the results depended on the relative competitiveness of the isolates and not on virulence factors.     

Fusarium oxysporum Fo47 confers protection to pepper plants against Verticillium dahliae and Phytophthora capsici,and induces the expression of defence genes

The application of the nonpathogenic isolate Fusarium oxysporum 47 (Fo47) reduced the symptoms of verticillium wilt, phytophthora root rot and phytophthora blight in pepper plants. Botrytis cinerea was also tested on the leaves of plants treated with Fo47, but no protection was observed. Verticillium dahliae colonies cultured in the presence of Fo47 grew slower than control cultures, but Phytophthora capsici growth was unaffected by Fo47. At least part of the protection effect observed against V. dahliae could therefore be due to antagonism or competition. In order to search for induced resistance mechanisms, three defence genes previously related to pepper resistance were monitored over time. These genes encode a basic PR‐1 protein (CABPR1), a class II chitinase (CACHI2) and a sesquiterpene cyclase (CASC1) involved in the synthesis of capsidiol, a phytoalexin. These three genes were transiently up‐regulated in the roots by Fo47 in the absence of inoculation with the pathogen, but in the stem only CABPR1 was up‐regulated. In plants that were inoculated with V. dahliae after the Fo47 treatment, the three genes had a higher relative expression level than the control in both the roots and the stem.     

A new Phytophthora disease of the aerial parts of Eucalyptus species1

Aerial damage on transplanted seedlings of Eucalyptus spp. was recorded in Italy for the first time. In 1987 a widespread infection was detected in the eucalyptus nursery of the Ovile Farm, Rome. It occurred only on E. globulus and was subject to sprinkler irrigation. In 1988 the disease occurred exclusively on E. delegarensis and E. regnans. The agent, which attacks leaves, petioles and shoots, was identified from the morphological and physiological characteristics of the isolates obtained in each year as Phytophthora nicotianae var. nicotianae.     

Phylogenetic and morphological analyses of Pythium graminicola and related species

Isolates of Pythium graminicola and related species were differentiated using restriction fragment length polymorphism (RFLP) analyses of the internal transcribed spacer (ITS) regions of rDNA and the cytochrome c oxidase subunit II (COX II) gene. These sequences were used in subsequent phylogenetic analyses. Finally, the phylogenetic placement of species was compared to that determined from morphological characteristics. The 62 isolates tested were divided into seven groups, A–G, based on RFLP analysis of the rDNA-ITS region. In the RFLP analysis of the COX II gene, isolates were divided into groups similar to those based on ITS-RFLP. Groups A and B were each separated into two additional subgroups. Grouping of isolates based on RFLP analyses agreed with the morphological differentiation. Groups A, B, D, E, F, and G were identified as P. graminicola, P. arrhenomanes, P. aphanidermatum, P. myriotylum, P. torulosum, and P. vanterpoolii, respectively. Group C was closely related to group B based on phylogenetic analysis of the rDNA-ITS region and the COX II gene and is similar to P. arrhenomanes. Each of the other species occupied their own individual clades. Although P. arrhenomanes is morphologically similar to P. graminicola, our phylogenetic analyses revealed that it was evolutionarily distant from P. graminicola and more closely related to P. vanterpoolii. Our analysis also revealed that P. torulosum with smaller oogonia is more closely related to P. myriotylum with large oogonia than to P. vanterpoolii, which forms smaller oogonia and is morphologically similar to P. torulosum. P. aphanidermatum with large oogonia and aplerotic oospores was not related to the morphologically similar species P. myriotylum. Results suggest that P. graminicola and related species are phylogenetically distinct, and molecular analyses, in addition to morphological analyses, are necessary for the accurate taxonomic placement of species in this complex.     

Parasitism of macroconidia, chlamydospores and hyphae of Fusarium culmorum by mycoparasitic Pythium species

Parasitism of macroconidia and endoconidial chlamydospores of Fusarium culmorum by Pythium oligandrum was studied on water agar (WA), corn-meal agar (CMA) and glass slides. Loss of cytoplasmic content in F. culmorum spores was followed by complete degradation, and P. oligandrum produced an abundance of oogonia on the parasitized macroconidia. A simple method for assessing the relative aggressiveness of isolates is presented, based on the percentage of macroconidial cells devoid of cytoplasm. Parasitism of macroconidia by P. acanthophoron , P. oligandrum and P. periplocum , but not by the plant pathogenic species, P. tracheiphilum , was demonstrated by this method. Interactions between hyphae of P. oligandrum and F. culmorum on WA resulted in an increase in the number of oogonia of P. oligandrum and a decrease in the sporulation of F. culmorum . The ability of isolates of P. oligandrum , P. periplocum , P. acanthophoron and P. mycoparasiticum to suppress disease symptoms caused by F. culmorum on barley seedlings was demonstrated in a greenhouse test.     

Survival,distribution and genetic variability of inoculum of the strawberry red core pathogen,Phytophthora fragariae var. fragariae,in soil

An experimental field infested with Phytophthora fragariae var. fragariae (Pff) and used for strawberry red core fungicide and cultivar resistance trials until 1981 was surveyed for the presence of inoculum of the pathogen 11 and 12 years later. Alpine strawberries, highly susceptible to all races of Pff, were grown from true seed and planted as a bait crop on a 0·5 m‐spaced grid. Rapid and widespread red core infection was observed, which provided good evidence that oospores had survived in soil for this extended period. Site elevation and the distribution of red core infected plants showed a strong correlation, with a higher frequency of infected and dead plants in the lowest areas of the field. The race designation of 18 recovered isolates were determined and AFLP fingerprint patterns of some of these and their single‐spore derivatives were analysed. The isolates differed little in race type, and the majority were genetically identical at 433 AFLP loci. Races used to inoculate the site in the 1970s were recovered. The fingerprints of the single variant isolate matched that of an isolation made by Hickman in the 1950s, originally used to inoculate the site. Clearly Pff is a very stable and long‐lived pathogen able to retain its genetic integrity and lie dormant in soil for many years, ensuring its survival between epidemiologically favourable conditions which occur erratically.