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.     

Damping-off of soybean in southern Brazil can be associated with different species of Globisporangium spp. and Pythium spp.

Damping-off of soybean is a common problem in some fields in southern Brazil. Frequently, Phytophthora sojae has been pointed out as the main causal agent. However, questions were raised as to whether other oomycetes could also be present. Hence, the objectives of this study were to isolate, identify, and determine the pathogenicity and aggressiveness of oomycetes isolated from soybean seedlings. Thirty-three isolates were obtained from 13 fields. Isolates were identified based on morphology and molecular methods. Nine species were recovered: Pythium conidiophorum (n = 14, 42.4%), Globisporangium rostratifingens (n = 7, 21.2%), G. irregulare (n = 2, 6.1%), G. spinosum (n = 1, 3.0%), G. ultimum var. sporangiiferum (n = 2, 6.1%), Pythium acanthicum (n = 2, 6.1%), P. deliense (n = 1, 3.0%), P. inflatum (n = 1, 3.0%), and P. torulosum (n = 3, 9.1%). Pathogenicity and aggressiveness were determined with two soybean cultivars (DM57i52 and NEO530) in a seed rot assay and a root rot assay. All isolates were pathogenic to soybean with variable levels of aggressiveness in both assays. The range of seed disease severity index varied from 7.5 to 100 for DM57i52 and from 23.1 to 100 for NEO530. The mean root disease severity index of the soybean cultivars ranged from 29.4 to 92.8. This is the first official report of G. irregulare, G. rostratifingens, G. spinosum, G. ultimum var. sporangiiferum, P. acanthicum, P. conidiophorum, P. deliense, P. inflatum, and P. torulosum causing damping-off of soybean in the state of Rio Grande do Sul in southern Brazil.     

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。     

A simple and effective inoculation method for Phytophthora and fungal species on woody plants

In inoculations carried out on buds, young shoots, and stems of woody plants in Norway, map pins contaminated with the pathogens to be tested proved to be convenient tools. The pins were inserted into the plant tissue and were left standing in the inoculation site throughout the experimental period. In the present investigation, the method was used successfully for one Phytophthora and four fungal species on different host plants. Inoculated plant parts developed clear symptoms, while no symptoms occurred where non‐contaminated pins were used. The advantages of this method are multiple: minimal physical damage to the tissue, inoculation points easily traced due to the relatively large top grip on the needles, and time saving as there is no need to cover with polyethylene or Parafilm after inoculation.     

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.     

Identification of Phytophthora spp. isolated from plants and soil samples on strawberry plantations in Poland

Journal of Plant Diseases and Protection - Crown and leather rot of strawberry caused by Phytophthora spp. are major soil diseases of cultivated strawberry...     

Pathogenicity and fungicide sensitivity of Pythium and Phytopythium spp. associated with soybean in the Huang-Huai region of China

Pythium and Phytopythium spp. cause seed decay, damping off, and root rot in soybean, wheat, and many other crops. However, their diversity and importance as pathogens, particularly in different crop rotation systems, are largely unknown. A survey was conducted in the Huang-Huai region, one of the main areas of soybean–wheat rotation farming in China. In 2016–2018, we collected 300 soybean seedlings and 150 field soil samples from several representative locations, and identified 26 Pythium and 6 Phytopythium spp. from 212 isolates, based on internal transcribed spacer 2 (ITS2) and cytochrome oxidase subunit 1 sequences. The pathogenicity of these isolates was evaluated by growing soybean and wheat seeds in dishes and pots containing oomycete cultures. We found that 12 Pythium spp. (but no Phytopythium spp.) showed high pathogenicity on soybean and/or wheat, and nine of them (75%) were highly pathogenic on both crops. Among the nine species, Pythium spinosum, Pythium ultimum, Pythium species 1 (tentatively designated as ‘Candidatus Pythium huanghuaiense’), Pythium aphanidermatum, and Pythium myriotylum were highly abundant among all isolates (15%, 10%, 9%, 8%, and 5%, respectively). Nine species were selected for testing of sensitivity to the fungicides metalaxyl and mefenoxam. The EC₅₀ values were all less than 10 μg/ml, indicating little resistance. Minimum inhibitory concentration values indicated isolates were about twice as sensitive to mefenoxam as to metalaxyl. These results provide a systematic understanding of Pythium and Phytopythium species associated with soybean in the Huang-Huai region, which is important for disease management and breeding programmes.     

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.     

Inhibition of polyamine biosynthesis in Phytophthora infestans and Pythium ultimum

Mycelial growth, polyamine concentrations and the activities of enzymes of polyamine biosynthesis and catabolism were examined in Phytophthora infestans and Pythium ultimum grown in the presence of difluoromethylornithine (DFMO), an inhibitor of omithine decarboxylase (ODC), and difluoro-methylarginine (DFMA), an inhibitor of arginine decarboxylase (ADC). Growth of P. infestans was reduced by DFMO and a mixture of DFMO + DFMA, but was increased by DFMA at concentratiotis of 5 MM and greater, Polyamine concentrations and ODC activity were significantly reduced in P. infestans grown on all inhibitor treatments. In contrast, growth, polyamine concentrations and enzyme activities were not affected in P, ultimum exposed to the inhibitors.
It seems unlikely that P. infestans and P. uttimum possess ADC activity, as neither of the products of ADC activity (agmatine and putrescine) could be detected in ADC assays. Although ODC from P. infestans was sensitive to DFMO, ODC from P. uttimum was insensitive to the inhibitor. Moreover, uptake of DFMO by P. infestans was three times greater than that observed by P. ultimum.     

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.     

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.     

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.     

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.     

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.     

A comparison of crop and non-crop plants as sensitive indicator species for regulatory testing

The effectiveness of regulatory non-target plant testing using crop species to predict the phytotoxicicity of herbicides to non-crop species was evaluated for eleven herbicides. These herbicides were representative of eight chemical classes and six modes of action. Data for non-crop plants from pre-emergence and post-emergence efficacy screening studies were compared with those for the most sensitive crop species defined by regulatory tests conducted to meet US EPA requirements. Testing under pre-emergence conditions for ten compounds indicated that for five of the compounds (K-815910, trifluralin, pyridyloxy A, pyridyloxy B and cyanazine), the most sensitive crop species was more sensitive than all the non-crop species evaluated. For metsulfuron-methyl, chlorimuron-ethyl, hexazinone and bromacil, only one of the non-crop species evaluated was more sensitive than the most sensitive crop species from regulatory tests. Data for the tenth compound, chloroacetamide, showed that four of 32 non-crop species tested in efficacy screens had at least one rate at which greater visual effects were observed than were observed for the most sensitive crop response in a regulatory test. The results of post-emergence exposure comparisons for five of the compounds (pyridyloxy A, cloransulam-methyl, chlorimuron-ethyl, cyanazine and hexazinone) indicated that the most sensitive crop species were more sensitive than all the non-crop species evaluated. Data for pyridyloxy B, metsulfuron-methyl and bromacil indicated that only one of the non-crop species evaluated was more sensitive than the most sensitive crop species. For trifluralin, three of the eight non-crop species were more sensitive than the most sensitive crop species. Data for K-815910 indicated that four of the fourteen non-crop species tested were marginally more sensitive than the most sensitive crop, but were within the same range of sensitivity. These results indicate that the current regulatory test batteries and methods using crop species effectively provide suitable sensitive indicator plants for the eleven diverse herbicides evaluated. This comparison indicates that crop species sensitivity to test substances is likely to be representative of non-crop herbaceous species response, regardless of chemical class, mode of action and magnitude or route of exposure.     

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.