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.     

Risk of dissemination of Clavibacter michiganensis ssp. sepedonicus with potato waste

To estimate the risk of dissemination of Clavibacter michiganensis ssp. sepedonicus through potato residues from processing industries, the various processes and the usage of residues from plants from different processing branches were analysed with regard to the effect they can have on the pathogen. The residues were classified into different risk categories, from category 0 (no risk of dissemination) to category 4 (high risk of dissemination). Residues not heated during processing and used in agriculture, e.g., as fertilizer, were pooled in the highest risk category 4. Residues that were sanitised before use in agriculture, e.g., by composting or pasteurisation, were still classified as probably high risk (risk category 3), as no information on these treatments concerning the inactivation of the pathogen was available so far. Therefore the effect of composting and pasteurisation under varying conditions was tested on samples (ready-made compost mould) contaminated with Clavibacter michiganensis ssp. sepedonicus. Viable bacteria could be extracted after all experiments via bioassay on eggplants, and cultivated on semi-selective media from plant sap forming characteristic colonies. The viable pathogen could be extracted after composting for 6 days at maximum temperatures at 70 °C, 13 days at 55 °C and 90 min pasteurisation at 70 °C. It can be concluded that these sanitation treatments are not sufficient to inactivate Clavibacter michiganensis ssp. sepedonicus and the previous classification of treated residues in category 3 (probably high risk) could thus be confirmed.     

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.     

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.     

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.     

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.     

Nematicidal potential of Artemisia annua and its main metabolites

Nematotoxic effect of an aqueous extract of Artemisia annua and its components caffeic acid, chlorogenic acid (5-caffeoylquinic acid, 5-CQA), artemisinin and the related semi-synthetic artesunate, was investigated on the root-knot nematode Meloidogyne incognita and the potato cyst nematode Globodera rostochiensis and on the virus-vector dagger nematode Xiphinema index. Juveniles of M. incognita and G. rostochiensis and females of X. index were exposed to 500, 250 and 125 μl ml^?1 solutions of the A. annua aqueous extract, caffeic acid, chlorogenic acid and artesunate and to 50 μl ml^?1 solution of artemisinin for 2, 4, 8 and 24 h. Egg masses of M. incognita and cysts of G. rostochiensis were exposed for 24, 48, 96 h and 1 or 2 weeks only to the extract solutions. Aqueous extract was highly effective on G. rostochiensis juveniles, whereas M. incognita juveniles were affected only at long exposure times. Adversely, egg hatch inhibition was strong on M. incognita and poor or minimal on G. rostochiensis. Females of X. index were sensitive only to long exposures to the highest extract concentration. Both caffeic and chlorogenic acid did not affect juveniles of M. incognita but were highly active on G. rostochiensis juveniles and X. index females even at the lowest concentration. Artesunate toxicity was almost zero on M. incognita and low on X. index females, but high on G. rostochiensis juveniles. Artemisinin solution was lethal to more than 50 % of G. rostochiensis juveniles within 24 h, but did not affect M. incognita juveniles and X. index females. Results suggest different roles of the tested compounds in the biocidal activity on each target nematode species. The extract of A. annua and its main phytochemicals seem to have a potential to be developed into new nematicidal formulates, though their activity should be validated in the soil.     

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.     

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.     

Definition and distribution of aggressive pathotypes of Synchytrium endobioticum in Ukraine1

Aggressive pathotypes of Synchytrium endobioticum have been detected in Ukraine since 1963, mainly in the mountainous western part of the country. Potato crops are regularly surveyed to detect possible outbreaks. It has been observed that the aggressive forms attack the above-ground parts of the potato plant, as well as the underground parts, and this can assist detection. Possible foci are investigated by planting a standard replicated test with a susceptible cultivar and a cultivar resistant to the common pathotype. The distribution of S. endobioticum in Ukraine is summarized. Outbreaks are particularly found in potato plots adjoining farm buildings.     

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.     

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.     

Methods of assessing the efficacy of non-volatile nematicides

In-vivo tests of non-volatile nematicides using a fungal-feeding nematode Aphelenchus avenae were shown to be more reliable than in-vitro tests. Phoxim and carbofuran were as effective as thionazin against the stem nematode Ditylenchus dipsaci in preventing eggs being laid, but they did not prevent nematodes from migrating from treated plants. Aldicarb and phoxim delayed the emergence of second-stage juveniles from cysts of the potato cyst nematode Globodera rostochiensis; phoxim at 8 mg kg^?1 of soil gave the same results as aldicarb at 2 mg kg^?1 of soil and both chemicals altered the sex ratio in the final population. A simple pot-test with G. rostochiensis in foul soil types was used to demonstrate the comparative effectiveness of non-volatile nematicides.     

Development of Globodera rostochiensis pathotype Ro1 in resistant and susceptible cultivars of potato1

Studies on the development of Globodera rostochiensis in potato cultivars with different levels of resistance have shown that both males and females of the nematode complete their whole cycle of development in susceptible cv. Svitanok Kyivskii in 61 days. In resistant cv. Vodograi, only males complete their development. Development of females is interrupted at the third larval instar, and mature females do not appear.     

Modifications in the potato rhizosphere during infestations of <Emphasis Type="Italic">Globodera rostochiensis</Emphasis> and subsequent effects on the growth of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Two controlled environment experiments were conducted to explore the hypothesis that invasion and damage caused to potato roots by the potato cyst nematode Globodera rostochiensis might result in quantitative or qualitative changes in the release of root exudates to subsequently affect the growth of Rhizoctonia solani (AG3) in the potato rhizosphere. The growth of five R. solani isolates was compared on media amended either with root exudates from G. rostochiensis-infested or uninfested potato (cv. Désirée) plants at different time intervals after the introduction of the nematodes. In Experiment 1, the growth of R. solani was higher on medium amended with potato root exudates from G. rostochiensis-infested compared to uninfested plants, collected 4, 6, 8 and 12 days after the G. rostochiensis treatments were administered. Similarly, in Experiment 2, R. solani isolates grew faster on medium amended with potato root exudates from G. rostochiensis-infested than uninfested plants. This trend was particularly pronounced at the 12-day collection. At this time, 49% of the G. rostochiensis juveniles in roots were found to belong to the juvenile moults J2 and J3, indicating that root exudates were modified during the earlier stages of juvenile invasion. Carbohydrate analysis of root exudates indicated significantly higher levels of sucrose in root exudates from G. rostochiensis-infested than uninfested plants, whereas no significant differences were found in total nitrogen content. The results are discussed to help elucidate the mechanism behind the disease complex found between G. rostochiensis and R. solani in previous field research.     

The use of isoelectric focusing as a tool in the identification and management of potato cyst nematode populations1

Isoelectric focusing on thin layers of polyacrylamide or agarose gels may be used to separate proteins from the potato cyst nematodes Globodera rostochiensis and G. pallida. General protein patterns may be used to identify the two species, even from single cysts. Densitometry of species-specific protein bands may be used to assess the proportions of G. rostochiensis and G. pallida in potato cyst nematode samples. Staining for the enzymes phosphoglucomutase and phosphoglucose isomerase also revealed species-specific patterns, and variation among G. pallida populations was observed. Thus calculations of coefficients of similarity based on six enzymes, phosphoglucose isomerase, phosphoglucomutase, glycerol-3-phosphate dehydrogenase, enolase, hexokinase and malate dehydrogenase and the construction of a dendrogram for several Northern Ireland populations indicated clear separations between G. pallida (Pa1), G. pallida (Pa3) and G. rostochiensis (Ro1). The use of the technique of isoelectric focusing as a routine research and advisory tool in nematology, and as a means of further understanding the genetic basis of pathotype schemes, is discussed.     

Plant parasitic nematodes as pests of potatoes in Poland1

Only two plant parasitic nematode species have practical importance in potato crops in Poland. They are Ditylenchus destructor and Globodera rostochiensis pathotype Ro1. Control of D. destructor is done permanently by the elimination of infested seed potatoes and sowing of cereals in the infested fields. Damage to potatoes is observed rather seldom. G. rostochiensis is widely distributed throughout Poland. It is controlled by non-host crops and by resistant potato cultivars. Chemical control of potato nematodes is not put into practice in Poland.     

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.     

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.     

Recovery of Ralstonia solanacearum from canal water in traditional potato-growing areas of Egypt but not from designated Pest-Free Areas (PFAs)

Surveys over three seasons of irrigation, drainage and artesian well water throughout the major potato-growing areas of Egypt indicated that Ralstonia solanacearum bv. 2 race 3 (phylotype II sequevar 1), cause of potato brown rot, was limited to the canals of the traditional potato-growing areas in the Nile Delta region, with positive findings more commonly associated with the network of smaller irrigation canals flowing through potato-growing areas. Pathogen populations in the canals of the Delta (~100–200 cfu l^?1) were generally variable throughout the year with presence linked to potato cultivation in the immediate area. The pathogen was not detected in irrigation or drainage water associated with potato cultivation in the newly reclaimed desert areas (designated as Pest-Free Areas, PFAs) or in the main branches of the Nile upstream from these areas. In vitro studies showed that temperature and microbial activity were the main factors affecting survival of the pathogen in canal water. In experiments at temperatures of 4, 15, 28 and 35°C, survival was longest at 15°C and shortest at 35°C. Survival at 4 and 28°C tended to be intermediate between these extremes as was survival when the bacterium was grown at fluctuating temperatures. Aeration, solarisation and pH variation between 4 and 9 appeared to have little effect on survival. Survival in autoclaved or filter-sterilised canal water was longer than in untreated water irrespective of other factors with survival times exceeding 300 days at 15°C in some experiments. Evidence is presented indicating that survival in water-saturated sediment may be longer than in the overlying water suggesting that sediment may provide a protective niche for the pathogen in some circumstances. The maximum survival time in non-sterile Egyptian canal water at high inoculum pressure was estimated to be up to 300 days at optimum temperature for survival (15–30°C) suggesting the potential for long-distance spread in Egyptian surface waters from sources of contamination.