Differential cultivars for aggressive pathotypes of Synchytrium endobioticum in Ukraine1

The use of a internationally agreed standard differential set of potato cultivars to identify aggressive pathotypes of Synchytrium endobioticum is reviewed. In recent years, many new cultivars and hybrids have been tested in Ukraine for their reaction to the Carpathian pathotypes (11, 13, 18 and 22). Because of the specificity and stability of their reactions, and their availability, some of these potato cultivars are now recommended as part of a new differential set for use in Ukraine, especially with respect to the Carpathian pathotypes.     

Quarantine pests of potato in Ukraine and requirements for their control1

Three principal quarantine pests (Phthorimaea operculella, Globodera rostochiensis and Synchytrium endobioticum) attack potato in restricted areas within Ukraine. Their biology is briefly presented. A complex of phytosanitary and plant protection measures is in place to prevent movement, multiplication and further spread of these quarantine pests.     

National control measures for Synchytrium endobioticum1

Although Synchytrium endobioticum has been found in almost all potato-growing countries of the EPPO region, it is absent from the great majority of fields. A few plots scattered across the potato-growing areas have been ‘scheduled’ as infested but potatoes are not grown on them. The disease is accordingly hardly ever seen. This is the result of continent-wide multiyear campaign of potato wart control within countries, according to which it is prohibited to grow potatoes on infested plots, and only resistant cultivars may be grown in a safety zone around them. S. endobioticum can persist for several decades in soil, as resting spores, but it is now dying out in plots that have been subject to the control campaign for a sufficiently long period. Procedures are now being agreed for officially ‘descheduling’ such plots.     

Interlaboratory tests for resistance to Synchytrium endobioticum in potato by the Glynne‐Lemmerzahl method

Synchytrium endobioticum is a major quarantine pathogen of potato causing potato wart disease. In Europe, the pathotypes 1(D1), 2(G1), 6(O1) and 18(T1) are the most widespread and occur locally in almost all countries. Resistance to this disease in potato cultivars is tested for in the majority of the EU countries by the Glynne‐Lemmerzahl method. This paper describes the results of testing two different protocols of this method in five laboratories of three different countries (Germany, Poland, and the Netherlands). The four pathotypes 1(D1), 2(G1), 6(O1) and 18(T1) were tested mainly on the cultivars described in the EPPO Standard PM 7/28 Synchytrium endobioticum. For pathotype 1(D1) and in most cases also for pathotype 18(T1), the results of the cultivars tested were identical to the rating in the EPPO Standard for both protocols. For pathotypes 2(G1) and 6(O1), the cultivars Désirée, Delcora and Miriam showed different results between laboratories as well as between the two protocols. In conclusion, further research is needed to develop one harmonised methodology for resistance testing of potato cultivars to the pathotypes 2(G1), 6(O1) and 18(T1) and to develop a new differential set of potato cultivars for the identification of pathotypes of S. endobioticum.     

Variability of Synchytrium endobioticum in the Carpathian region of Ukraine1

The appearance of aggressive pathotypes of Synchytrium endobioticum in the Carpathian area of Ukraine is reviewed. An analysis is made of the factors that favoured their appearance in this area. Possibilities for distinguishing the pathotypes by characters other than their behaviour on differential cultivars are considered.     

Synchytrium endobioticum – risk from biogas plants?

Synchytrium endobioticum is one of the most important pathogens of potato and is known for its persistent propagation structures. Under favourable conditions, infection of highly susceptible potato cultivars leads to clearly visible cauliflower‐like tissue warts, the typical symptom of potato wart disease. However, unfavourable infection conditions or low infection pressure may result in symptoms being overlooked. Thus, the introduction of pathogen structures into stages of the processing industry cannot be ruled out. As the amounts of processed potato products continue to rise, phytosanitary risks from processing discarded potatoes and potato waste in biogas plants have to be considered. Hence, the resilience of resting spores against mesophilic anaerobic digestion was analysed in stirred tank reactors. Laboratory‐scale results show that S. endobioticum not only withstands mesophilic anaerobic digestion but also subsequent storage of the digestate for at least 4 weeks. Large numbers of viable resting spores were detectable by microscopic assessment in all samples. Viability was proved and verified additionally by bioassay. Consequently, potatoes, potato waste and processing water from potato processing industries used in biogas plants pose a phytosanitary risk if the accruing digestates are returned as fertilizer to arable land.     

Biotechnology of production of virus-free seed potatoes in Bukovina (Ukraine)1

In the past, the Ukrainian Plant Quarantine Research Institute used to produce true-to-type virus-free potato cultivars by in vitro propagation for its research on pathotypes of Synchytrium endobioticum and Globodera spp. Use of virus-free seed potatoes has considerably declined in the 1990s as potato production has passed almost entirely into private hands. The Institute now has a role to fill in supplying virus-free seed potatoes to local growers and has greatly increased its production in consequence. The techniques used for seed-potato production in Bukovina are described.     

The Longevity of Resting Sporangia of Synchytrium endobioticum (Schilb.) Perc. in Soil1)

Resting sporangia of the potato wart disease pathogen Synchytrium endobioticum (Schilb.) Perc. which persist in soil can be extracted from soil by a wet-sieving and chloroform centrifugation method and counted. Soils from the sites of old wart outbreaks have been checked for resting sporangia. Sampling procedures, patterns and errors are being assessed.     

History of potato wart disease in Europe – a proposal for harmonisation in defining pathotypes

Potato wart disease, caused by the chytridiomycete Synchytrium endobioticum, was first introduced into Europe in the late 19th century. It spread quickly, and today is reported in 15 European countries. Initially, only one pathotype was found, and the disease was efficiently controlled using resistant cultivars. In 1941, however, formerly resistant cultivars showed wart formation in the field simultaneously in Germany and South Bohemia (Czech Republic), indicating the occurrence of new pathotypes. New pathotypes have since been reported from Germany, The Netherlands, Czech Republic, Ukraine and Canada. Today the pathogen is present in The Netherlands (only in fields for ware and starch potatoes) but restricted to two demarcated areas and subject to official control. Outside these areas, the pathogen is absent. For pathotyping, different countries have used different sets of differential cultivars, and the usual system of numerical coding of pathotypes has not been consistently followed. In this review we propose a new standardised code to be used for the 43 pathotypes currently known and described in Europe. The code is a combination of a numerical and letter code, combining the two terminologies used by former West and East Germany, respectively. We also plead for harmonisation in the choice of differential cultivars used for pathotype identification. The set of differentials described in the international standard for diagnosis of S. endobioticum issued by the European and Mediterranean Plant Protection Organisation (EPPO), should serve as a basis. Through close collaboration of European countries dealing with new pathotypes of potato wart disease, a final agreed upon set of differentials, combined with a set of reference isolates, should ultimately be established, allowing a clear distinction between the most important pathotypes occurring in Europe.     

Improved real-time PCR assay for detection of the quarantine potato pathogen, <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis>, in zonal centrifuge extracts from soil and in plants

Real-time PCR was used for quantitative detection of the potato pathogen, Synchytrium endobioticum, in different substrates: zonal centrifuge extracts, warts and different plant parts of potato. Specific primers and a TaqMan probe, designed from the internal transcribed spacer region of the multi-copy rDNA gene were tested in extracts from artificially and naturally infested soil. Co-amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable by guarding against false negative results. A calibrations curve was created by spiking zonal centrifuge fractions of clean soil samples with a dilution series of winter spores. The Taqman assay was also performed on infected potato plant material (stolons) along with the detection of the cytochrome oxidase gene as a potato endogenous control. Sensitivity of the TaqMan assay was improved at least 100-fold and proved to be reliable for accurate diagnosis of the disease.     

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.     

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.     

The Relation Between Field and Laboratory Susceptibility of Potato Cultivars to Wart Disease1)

Potato wart disease has been effectively controlled legislatively by the growing of resistant cultivars. The testing of new potato seedlings for plant breeders is undertaken so that susceptibles can be removed from the breeding program at an early stage. In the laboratory, gradation of resistance and susceptibility is noted. The range of reactions is divided into 6 arbitrary groups, 3 with necrosis and 3 without (Hills , 1965), and Hille's infection patterns have been interpreted as a set of assessment keys. Susceptible cultivars show a variation in degree of susceptibility, but infections persist even though there may be a necrotic hypersensitive reaction of the host. With some less resistant cultivars, the fungus can become established in the host and complete its life cycle, but infected tissue mostly necroses and is sloughed off. These less resistant cultivars (intermediates) grow away from infection when grown on in pots. The cultivar Red Craigs' Royal showed no infection when inoculated in soil tanks (3 soil types) with heavy watering or when grown on infested land. All evidence collected gives indication that intermediate cultivars are field resistant and not affected to any measurable extent by Synchytrium endobioticum (Schilb.) Perc.     

Efficacy of chemical plant protection products on potato in Ukraine1

The principal potato pests controlled with plant protection products in Ukraine are Leptinotarsa decemlineata and Phytophthora infestons. Results are presented of trials of insecticides and fungicides against these pests. As a result, Sumi-alfa 5% EC (a.s. esfenvalerate), Karate water-soluble granules (a.s. lambda-cyhalothrin) and Betabytroid 2.5% EC (a.s. beta-cyfluthrin) were recommended for full-scale testing against Colorado beetle in Ukraine. Sul'fokarbation and EKhR 19517 were recommended for full-scale testing against potato blight.     

Resting-spore germination inSynchytrium endobioticum

The resting spores ofS. endobioticum function as a prosorus in germination. The contents of the resting spore are extruded as a vesicle. Within this vesicle one single sporangium develops. Germinating resting spores resemble resting spores which have been parasitized by certain fungi, but it is obvious from several facts presented that no parasites were present. On the basis of the mode of germination described,S. endobioticum should be transferred from the subgenusMesochytrium to the subgenusMicrosynchytrium.     

Effects of fungicide treatments of potato sprouts on assessment of resistance to Synchytrium endobioticum (Schilb.) Perc. using the Glynne–Lemmerzahl method

Five different active fungicides (pencycuron, mancozeb, copper oxychloride, azoxystrobin, and a mixture of carboxine and thiuram) were compared to protect potato sprouts from rotting when they are being tested using the Glynne–Lemmerzahl method. Sprouts were treated before or after inoculation with fresh warts of Synchytrium endobioticum. No symptoms of potato wart disease were observed on sprouts treated before inoculation. For sprouts treated after inoculation, symptoms of potato wart disease were recorded with all tested fungicides except azoxystrobin. In the case of pencycuron, symptoms were observed only when the concentration of fungicide was decreased to 20 mL L^?1. Among the active substances tested, the best results were obtained for copper oxychloride after inoculation using doses recommended by manufacturers.     

Composting to sanitize plant‐based waste infected with organisms of plant health importance

The potential for using the composting process to sanitize plant waste infected with one of three plant pathogens was investigated using bench‐scale composting equipment. Two of these pathogens, the potato wart disease fungus Synchytrium endobioticum and Potato spindle tuber viroid (PSTVd) are currently subject to European quarantine regulations. The third, Polymyxa betae, a parasite of sugar beet, is regulated in some European countries when in association with Beet necrotic yellow vein virus (BNYVV), the causal organism of rhizomania disease of sugar beet. Survival of test organisms following various combinations of compost temperature, exposure time and moisture was determined using RNA‐based detection methodology and/or plant‐based bioassays. Mathematically definable relationships between compost treatment (temperature/time) and organism viability were identified for P. betae and S. endobioticum; these give some indication of the practicality of using composting for dealing with infected wastes. However, for PSTVd, the considerable variability in measured susceptibility of the viroid to the composting process meant that no such definable relationship could be determined and further work would be needed to extrapolate to practical situations.     

Euphresco Sendo: An international laboratory comparison study of molecular tests for <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis> detection and identification

An international test performance study (TPS) was organised to generate validation data for three molecular Synchytrium endobioticum tests: van den Boogert et al. (European Journal of Plant Pathology 113, 47–57, 2005), and van Gent-Pelzer et al. (European Journal of Plant Pathology, 126, 129-133, 2010) for the detection of S. endobioticum, and the pathotype 1(D1) identification test described by Bonants et al. (European Journal of Plant Pathology, 143, 495-506, 2015). Two TPS rounds were organised focussing on different test matrices, i.e. round 1: warted potato tissue, and round 2: resting spore suspensions. When using the tests for detection and identification of S. endobioticum in warted potato tissue, no significant differences were observed for diagnostic sensitivity, diagnostic specificity, overall accuracy, analytical sensitivity and robustness. When using the tests for detection and identification of S. endobioticum in resting spore suspensions, the van den Boogert and van Gent-Pelzer tests significantly outperform the Bonants test for diagnostic sensitivity and diagnostic specificity. For overall accuracy and analytical sensitivity, the van Gent-Pelzer significantly outperforms the van den Boogert and Bonants tests and is regarded as the test of choice when identifying S. endobioticum from resting spores. Tests regarded fit for purpose for routine testing of wart material and resting spore suspensions are proposed for the update of EPPO standard PM7/28(1) Synchytrium endobioticum.     

Occurrence of potato wart disease (Synchytrium endobioticum) in Bulgaria: identification of pathotype(s) present

Potato wart disease caused by Synchytrium endobioticum was detected for the first time in Bulgaria in 2004. Laboratory tests, pot tests and field tests were carried out to determine which pathotype is present, specifically in Prodanovtsi near the town of Samokov. In addition, potato cultivars were tested for levels of resistance, in order to be used in buffer zones surrounding infested fields. Based on the results obtained in this study, it was found that most probably pathotype 8(F1) is present in Bulgaria. Most of the commercial potato cultivars tested were found to be susceptible to this pathotype. The local cultivars Koral and Bor were resistant, but these are not very important cultivars commercially. The potato cultivars Panda, Désirée and Amorosa were recommended for cultivation in the fields adjacent to infested plots.     

Future management of internal quarantine pests of potato in Ukraine1

A strictly regulatory approach has been taken in the past with respect to quarantine pests present with limited distribution in Ukraine. Leptinotarsa decemlineata had at one time this status, but the regulatory strategy against it was not successful, the pest became widespread and was deleted from the quarantine list. It is suggested that an approach based on pest risk analysis, relating the management strategy more closely to the risk specifically arising from each pest, could be more successful in future against the principal internal quarantine pests of potato in Ukraine: Phthorimaea operculella, Globodera rostochiensis and Synchytrium endobioticum.