Reactions of potato cultivars to Synchytrium endobioticum and implications for minimum tuber numbers in wart susceptibility tests

Results of wart susceptibility tests on cultivars submitted for UK National List Trials in 1984/1994 are analysed. In cultivars classified as Resistance Group 2 (RG2), the percentage of individual tubers giving RG2 reactions ranged from 1.5 to 76. For most such cultivars (87%), less than 50% of tubers gave RG2 reactions. The percentage of tubers giving susceptible reactions in cultivars classified as susceptible ranged from 10 to 100. For most susceptible cultivars (87%) , more than 60% of tubers reacted as susceptible. It is suggested that slightly susceptible cultivars in which 10-21% of tubers react as susceptible are likely to produce numerous winter sporangia but no wart tissue when grown in infested soil. Those in which > 67% of tubers react as susceptible are likely to produce varying quantities of wart tissue. There was no cultivar in which 22–62% of tubers gave susceptible reactions, indicating a possible natural break in the spectrum between slightly susceptible cultivars and those which produce wart tissue. It is suggested, on the basis of the evidence available. that the number of tubers per cultivar used for susceptibility testing could safely be reduced for most cultivars.     

Resistance of potato cultivars to Synchytrium endobioticum in field and laboratory tests, risk of secondary infection, and implications for phytosanitary regulations

Laboratory (Spieckermann) tests, pot tests and field tests provided concordant evidence for the partial nature of resistance of potatoes to pathotypes 1 (D1) and 6 (O1) of Synchytrium endobioticum . Susceptible potato cultivars produced large warts (> 16 mm in diameter) in Spieckermann tests and had low field resistance levels (1–6). Field-resistant cultivars (levels 7–9) produced small warts or no warts at all in Spieckermann and field tests. In pot tests, at low inoculum levels (1 sporangium per 25 g soil) susceptible cultivars still developed warts, whereas field-resistant ones did not develop any warts below 25 sporangia per g soil. Above 35 sporangia per g soil, 100% disease incidence was observed in susceptible cultivars but only minimal wart development in field-resistant ones. Tests with continuous cultivation of potato cultivars in infected soil during three consecutive years showed that field-resistant cultivars will not support build-up of inoculum in soil. It is concluded that field-resistant cultivars do not create a risk of secondary infection, the criterion given for resistance in EU Directive 69/464/EC.     

A novel technique using the Hendrickx centrifuge for extracting winter sporangia of <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis> from soil

A zonal centrifugation method, known as the Hendrickx centrifuge technique, was tested for routine detection of winter sporangia of Synchytrium endobioticum in soil. In four experiments the ability of the Hendrickx centrifuge to extract the sporangia from soil was compared with a method used by the Dutch Plant Protection Service, which is a modification of the recommended EPPO method. Naturally and artificially contaminated soil samples were used to study the recovery percentage of and variation in numbers of winter sporangia. The effects of soil type and inoculum density were studied. The Hendrickx centrifuge method, developed originally for extraction of free living nematodes from soil, performed better than the method used by the Dutch Plant Protection Service. This was due to a better extraction recovery (60% higher), a lower measurement error (50% lower) and a lower detection level (down to 0.02 sporangia g⁻¹ soil). The Hendrickx centrifuge method is much less labour-intensive than the method used by the Dutch Plant Protection Service. It can be used to extract many different organisms from soil, and DNA can be subsequently extracted from the supernatant for further PCR analysis. Inclusion of the Hendrickx centrifuge method in the official EPPO diagnostic protocol for regulated pests is recommended as an alternative method for detection of sporangia in soil.     

Assessment of the resistance of potato cultivars to Synchytrium endobioticum (Schilb.) Per. in Poland

In Poland the Plant Breeding and Acclimatization Institute is responsible for officially assessing the resistance to Synchytrium endobioticum of domestic potato breeding lines and cultivars from other countries. Cultivation of potato cultivars in Poland requires confirmation of resistance to potato wart disease. The official assessment uses the modified Glynne-Lemmerzahl method (laboratory tests) and pot tests. The full cycle of assessment of resistance to wart disease requires 52 seed potatoes per variety/breeding line. Forty two tubers are used in laboratory tests. To complete the laboratory tests the next 10 tubers are grown in pot tests (in soil with winter sporangia) during the vegetation season. The final results for domestic breeding lines of potato are available after 3 years of investigation. For cultivars from other countries the authorization of resistance to S. endobioticum takes approximately one year. The Polish breeders (breeding lines) or the breeder's representative (cultivars from other countries) receive the certificate only for lines/cultivars with laboratory and field resistance to S. endobioticum .     

Development of PCR-based Detection Methods for the Quarantine Phytopathogen <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis>, Causal Agent of Potato Wart Disease

PCR-based methods were developed for the detection and quantification of the potato pathogen Synchytrium endobioticum in soil extracts and in planta. PCR primers, based on the internal transcribed spacer region of the multi-copy gene rDNA were tested for specificity, sensitivity and reproducibility in conventional and real-time PCR assays. Soil extraction procedures compared included the Hendrickx centrifugation (HC) procedure, nested wet sieving (NWS) and a method used by the Plant Protection Service (PPS). The primers amplified a 472 bp product from S. endobioticum DNA, but did not amplify DNA from other potato pathogens, other plant pathogens, and related species. Standard cell disruption and DNA extraction and purification methods were optimized for amplification of S. endobioticum DNA from resting sporangia. DNA was successfully amplified from a single sporangium and equivalent DNA preparations from soil extracts. Low levels of target DNA in water did not amplify, possibly due to DNA loss during final purification steps. A real-time PCR assay, developed for soil-based extracts using primers and probe based on the rDNA gene sequences, involved co-amplification of target DNA along with an internal DNA fragment. Both conventional and real-time PCR methods performed well with HC- and NWS-extracts having a threshold sensitivity of 10 sporangia per PCR assay. Of the three soil extraction methods, only with the HC method could 100 g soil samples be efficiently processed in one single PCR assay. Such a high capacity assay could be useful for routine soil analysis in respect to disease risk assessments and to secure de-scheduling according to EPPO guidelines.     

Direct examination of soil for sporangia of Synchytrium endobioticum using chloroform, calcium chloride and zinc sulphate as extraction reagents

Fields infested with Synchytrium endobioticum can be descheduled when the soil is found free from sporangia of S. endobioticum . For direct examination, EPPO Standard PM 3/59 describes a soil extraction technique based on the use of a sieve shaker with six sieves. We compared recovery of sporangia between this (modified) method and an extraction method employed by the Dutch Plant Protection Service (PPS method). Recovery was determined using an inoculum dilution series: 125, 25, 5, 1, 0.2 or 0.04 sporangia per g soil. Extraction reagents used were chloroform and calcium chloride in the method described by EPPO, calcium chloride and zinc sulphate in the PPS method. At 125 sporangia per g soil, the mean density determined for the modified EPPO method was 228 sporangia per g soil when chloroform was used. Using calcium chloride, recovery percentage was higher for the modified EPPO method than for the PPS method (286, 136%, n.s. P  < 0.05). The advantage of the modified EPPO method was the larger soil volume to be processed; its disadvantages were use of complex equipment and noxious reagents (chloroform). Both extraction methods showed high variation in recovery between samples, making accurate estimation of sporangial densities in soil awkward.     

Effects of sanitation processes on survival of Synchytrium endobioticum and Globodera rostochiensis

The two quarantine pests Synchytrium endobioticum, the causal agent of potato wart disease and Globodera rostochiensis, the yellow potato cyst nematode are currently present in Germany. Winter sporangia of Synchytrium endobioticum and cysts of Globodera rostochiensis can be spread with waste from potato processing industries, if infected tubers are processed. The German Biowaste Ordinance prescribes sanitation of organic waste before it can be used on arable land as fertilizer or filling material. Sanitation parameters prescribed by the German Biowaste Ordinance include composting for 7 days at 65°C or 14 days at 55°C or pasteurisation for 60 min at 70°C. The effect of composting and pasteurisation processes on winter sporangia of Synchytrium endobioticum and cysts of Globodera rostochiensis was tested with varying time-temperature relations. Cysts of Globodera rostochiensis were killed by composting for 7 days at 50–55°C and by pasteurisation for 30 min at 70°C. In contrast, viable winter sporangia of Synchytrium endobioticum could be extracted from sample material after composting for 70 days at 30–45°C, composting for 21 days at 50–55°C and after composting for 12 days at 60–65°C. Likewise viable winter sporangia could be extracted after pasteurisation for 90 min at 70°C and heating in a water bath at 80°C and in a dry oven at 90°C for 8 h. The parameters prescribed in the German Biowaste Ordinance are sufficient to kill cysts of Globodera rostochiensis but not sufficient to kill winter sporangia of Synchytrium endobioticum in organic waste.     

Bobbelblad bij sla

Summary In The Netherlands symptoms of big vein were found in lettuce and endive (Cichorium endivia L.). In the roots of these plants sporangia ofOlpidium occurred. In the glasshouse healthy lettuce plants showed symptoms when planted in infected soil.Gedetacheerd bij het Proefstation voor de Groenteteelt in de Volle Grond in Nederland, Alkmaar.     

Oospores associated with tomato seed may lead to seedborne transmission of<Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Tomato fruits at the green mature stage were inoculated with a mixed sporangial suspension of A₁ and A₂ isolates ofPhytophthora infestans. Other fruits were inoculated with either A₁ or A₂ sporangia. Seeds were extracted from the blighted fruits and sown in soil or on agar media to test for the transmission of late blight to the emerging seedlings. Only 23 (0.09%) of approximately 25,000 seedlings developed symptoms. All blighted seedlings originated from fruits inoculated with mixed A₁ + A₂ sporangia. Isolates ofP. infestans recovered from the emerging blighted seedlings were seemingly of oosporic origin, as they differed phenotypically (mating type, virulence, sensitivity to metalaxyl) from the parent isolates used to inoculate the fruits. The results suggest that transmission ofP. infestans might occur by seeds extracted from fruits carrying oospores and less probably by seeds extracted from fruits having no oospores. http://www.phytoparasitica.org posting April 30, 2004.     

Development of detection systems for the sporangia of <Emphasis Type="Italic">Peronospora destructor</Emphasis>

A monoclonal antibody that recognises components of the wall of sporangia of Peronospora destructor was raised. Tests using spores of higher fungi and other species of mildew demonstrated the specificity of the monoclonal. The antibody was used to develop lateral flow devices for sporangia of P. destructor. A competitive lateral flow format was developed which could detect onion downy mildew sporangia. Five-microliter gold anti-mouse IgM solution pre-mixed with 10 μl of P. destructor monoclonal antibody (EMA 242) proved the optimal concentration for detection of sporangia of P. destructor when applied to sample pads of lateral flow devices. Limits of approximately 500 sporangia of P. destructor could be detected by the absence of a test line on the lateral flow device within test samples. Using a scanning densitometer improved the sensitivity of detection. Further development and validation of the test is required if it is to be used for risk assessments of onion downy mildew in the field.     

诱导疫霉菌产生游动孢子囊液体培养基的研制

研制了几种诱导疫霉菌产生游动孢子囊的蔬菜汁混合培养液,测定了各培养液诱导苎麻疫霉游动孢子囊的效果以及其对苎麻疫霉菌游动孢囊产生量、形态特征、游动孢子释放、卵孢子产生量、卵孢子的形态、菌丝生长和菌落形态等生物学性状的影响。试验结果显示,各配比培养液均能很好地诱导游动孢子囊的产生,且以较低浓度的培养液对孢子囊诱导效果较好,而高浓度则有利于卵孢子的产生,不同培养液中产生的游动孢子囊均能正常释放游动孢子。其中西红柿∶黄瓜=1∶2配比的培养液简单易行且诱导效果最好,是V₈C的理想替代培养液。     

Characterization of tuber blight‐suppressive soils from four provinces of the Ecuadorean Andes

Late blight, caused by the oomycete Phytophthora infestans, is a threat to potato‐cropping systems worldwide. In the Ecuadorian Andes, despite a high late blight incidence in foliage, tuber blight is rare. In this work, the hypothesis that Ecuadorian Andean soils are naturally suppressive to P. infestans tuber infection was evaluated. Soils from four potato‐growing regions were assessed for disease suppressiveness by determining the effects of soil heat treatment on P. infestans sporangia and their ability to infect potato slices after 1, 8, 15 and 30 days of exposure to soils. Tuber infection after inoculation with P. infestans‐infested soils was consistently lower during the evaluation period compared with heat‐treated soils. Fresh, untreated soils affected germination and viability of P. infestans sporangia in a site‐dependent manner. In addition, the effect of heat treatment on soil bacterial communities was assessed through terminal restriction fragment length polymorphism analysis of the 16S rDNA gene region. Heat treatment disrupted bacterial community composition, and a subset of terminal restriction fragments (TRF) was either positively or negatively correlated with tuber infection. Bacterial TRF negatively correlated with tuber infection corresponded in fragment size to taxa with known ability to inhibit pathogens and promote plant growth. Finally, bacterial isolates obtained from untreated soils, which inhibited P. infestans growth in vitro, represented 22–47% of isolates recovered, and matched classes predicted by the TRFs. This work represents a first step in understanding the mechanisms behind the low incidence of tuber blight in Andean potato‐cropping systems.     

Survival of Phytophthora infestans in Surface Water

ABSTRACT Coverless petri dishes with water suspensions of sporangia and zoospores of Phytophthora infestans were embedded in sandy soil in eastern Washington in July and October 2001 and July 2002 to quantify longevity of spores in water under natural conditions. Effects of solar radiation intensity, presence of soil in petri dishes (15 g per dish), and a 2-h chill period on survival of isolates of clonal lineages US-8 and US-11 were investigated. Spores in water suspensions survived 0 to 16 days under nonshaded conditions and 2 to 20 days under shaded conditions. Mean spore survival significantly increased from 1.7 to 5.8 days when soil was added to the water. Maximum survival time of spores in water without soil exposed to direct sunlight was 2 to 3 days in July and 6 to 8 days in October. Mean duration of survival did not differ significantly between chilled and nonchilled sporangia, but significantly fewer chilled spores survived for extended periods than that of nonchilled spores. Spores of US-11 and US-8 isolates did not differ in mean duration of survival, but significantly greater numbers of sporangia of US-8 survived than did sporangia of US-11 in one of three trials.     

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.     

Asexual Reproduction of Phytophthora capsici as Affected by Extracts from Agricultural and Nonagricultural Soils

ABSTRACT Formation of sporangia and zoospores in species of Phytophthora is known to be influenced by soil microbial and chemical composition. In Phytophthora capsici, the study of the relationship of soil chemical composition to production of sporangia and zoospores has been limited. P. capsici is a soilborne pathogen of a wide array of vegetable crops, including chile pepper (Capsicum annuum) in New Mexico. Production of sporangia and zoospores by P. capsici was evaluated in extracts of soils from three different environments in New Mexico: (i) agricultural environments with a long history of chile pepper cropping and occurrence of P. capsici (CP), (ii) agricultural environments with no history of chile pepper cropping and no occurrence of P. capsici (Non-CP), and (iii) nonagricultural environments consisting of forests and rangelands (Non-Ag). There was a significant difference in production of P. capsici asexual propagules, expressed as natural log (number of sporangia x number of zoospores), among the three environments (P = 0.0298). Production of propagules was 9 to 13% greater in Non-Ag than in CP or Non-CP environments. Stepwise multiple discriminant analysis and canonical discriminant analysis identified the edaphic variables Na, pH, P, organic matter content, and asexual propagule production as contributing the most to the separation of the three environments. Two significant (P < 0.0001) canonical discriminant functions were derived with the first function, accounting for approximately 75% of the explained variance. Based on the two discriminant functions, approximately 93, 86, and 89% of observations in CP, Non-CP, and Non-Ag environments, respectively, were classified correctly. Soils from agricultural and nonagricultural environments differentially influence production of sporangia and zoospores in P. capsici, and soil samples could be effectively classified into agricultural and nonagricultural environments based on soil chemical properties and the production of asexual propagules by P. capsici in soil extracts.     

A comparison of morphology,pathogenicity and restriction fragment patterns of mitochondrial DNA among isolates of Phytophthora porri Foister

Thirteen strains ofPhytophthora porri from five different hosts were compared with respect to their morphology, cardinal temperatures for growth, pathogenicity to leek and cabbage and restriction fragment patterns of mitochondrial DNA. Morphology of vegetative growth was rather similar in most isolates. Those characters which differed among isolates showed overlapping variability and could not be used to distinguish groups, with the exception of production of oogonia and sporangia and the antheridium type. Considerable differences were found in restriction patterns of mitochondrial DNA, isolates from the same host mostly showing identical patterns. Isolates from differentAllium species showed relatively similar restriction patterns if compared to the other isolates. Isolates fromBrassica oleracea proved to be a homogeneous group, quite different from the others with respect to restriction patterns, production of sporangia, production of oogonia, antheridium type and pathogenicity. One isolate, CBS 366.59, isolated from and pathogenic toA. porrum, deviated in many characters from the other isolates. It showed the restriction patterns ofPhytophthora nicotianae and also the high cardinal temperatures for growth typical for this species. The sporangia, however, were distinctly non-papillate and the majority of antheridia was of the paragynous type.     

Survival of Phytophthora meadii in Sri Lankan soils

In laboratory tests, mycelium and sporangia of Phytophthora meadii survived in soil for about 3 weeks, whereas chlamydospores survived for 12 weeks. When petioles of rubber (Hevea brasiliensis) colonized by P. meadii were buried in soil, chlamydospores formed in the tissues after about 2 weeks and P. meadii could be reisolated up to 22 weeks after burial. Colonized petioles buried in soil in a rubber plantation decayed more rapidly than those in laboratory tests, and attempts to reisolate P. meadii after 18 weeks were unsuccessful, although chlamydospores were still visible. P. meadii was isolated most readily from soil in a rubber plantation during epidemics of pod and leaf disease, suggesting that sporangia from these sources maintained soil inoculum at a high level. Inoculum detected in soil before pod and leaf infection may have arisen from subclinical infections or from chlamydospores surviving in petioles from the previous season.     

Bioassays using detached tobacco leaves to determine the sensitivity of Peronospora tabacina to fungicides

Two bioassay methods are described which use detached tobacco leaves to measure the sensitivity of Peronospora tabacina to systemic fungicides. Tobacco leaves (13–15 cm²), treated with fungicides before or after detachment from the plant, were inoculated with sporangia in water drops and, after incubation in beakers and Petri plates, the disease severity and/or production of sporangia was determined 4–7 days after treatment with the fungicides. Of 15 systemic fungicides applied to detached leaves, eight N-phenylamides at 0.066?1.0 μg ml^?1 controlled blue mould; metalaxyl was the most effective fungicide. Isolates of P. tabacina, collected in the field from tobacco plants grown in soil treated with metalaxyl, were not resistant to the fungicide applied to detached leaves prior to inoculation. The fungicide, applied to leaves before detachment, was used to measure the efficacy of five systemic N-phenylamide fungicides sprayed on the basal and unsprayed distal portions of the leaves. Blue mould was controlled on the basal portion of the leaf by all the fungicides at 0.66?1.0 μg ml^?1, but it required the application of 3–30 times more chemical on the basal portion to achieve comparable blue mould control on the distal part of the leaf.     

Species of Pythium in the Netherlands

A review is given on observations ofPythium spp. so far published. In personal investigations about 4000 isolates ofPythium species were obtained from soil, water, Daphnias and plants during 1966–1972. Special attention was paid to the progressive colonization of the newly reclaimed polder Zuidelijk Flevoland: the numbers of species per sample increased from 0 in 1966 to 13 in 1972. The percentage of samples which did not contain anyPythium decreased from 54 in 1968 to 0 in 1972. From Zuidelijk Flevoland altogether 21 species were isolated, from Oostelijk Flevoland 22, of which 19 occurred in both the polders. The soil depth down to 10 cm had little influence on the occurrence ofPythium species. From soils near Rhenen and Zutphen 12 and 19 species respectively were isolated, from flax and flaxfields 11, from forests 8, from roots of different plants 17 and from water 11. Besides species already previously recorded, 15 new records ofPythium species for the Netherlands are documented. Species with filamentous non-swollen sporangia preferentially occurred in water and wet soils while species with spherical sporangia or hyphal swellings dominated in cultivated soils, forests and in the roots of phanerogams. In 1968 species with filamentous non-swollen sporangia comprised 89% of the isolations from Zuidelijk Flevoland; this percentage decreased to 9 in 1972. The percentage of isolates with spherical sporangia or hyphal swellings from cultivated soils, forest soils or plant material was mostly over 90.P. rostratum Butler, though found in all kinds of soils, was often the only species which could be isolated from dry sandy soil samples.P. sylvaticum Campbell & Hendrix was the most common species in cultivated soils in the Netherlands (37% of the isolates in 1968–1972), followed byP. oligandrum Drechsler (22%) andP. paroecandrum Drechsler (11.5%).