Identification of <Emphasis Type="Italic">Pratylenchus thornei</Emphasis>, the cereal and legume root-lesion nematode,based on SCAR-PCR and satellite DNA

Two different molecular tools for the diagnosis of the cereal and legume root-lesion nematode Pratylenchus thornei were developed. A randomly amplified DNA (RAPD) fragment specific to P. thornei was identified. After sequencing the fragment, longer primers were designed that complement the terminal sequences of the RAPD fragment, and this pair of specific primers was used to amplify the sequence-characterized amplified region (SCAR). Using the developed pair of SCAR primers, the SCAR fragment specific to P. thornei was easily amplified with DNA extracts obtained from different life stages of the nematode. The described SCAR-PCR-based assay has the potential to be optimized for routine practical diagnostic tests. In addition, the use of a species-specific satellite DNA sequence to distinguish P. thornei from other Pratylenchus spp. is discussed.     

Plants for planting; indirect evidence for the movement of a serious forest pathogen, <Emphasis Type="Italic">Teratosphaeria destructans</Emphasis>, in Asia

Fungal diseases caused by native pathogens and pathogens introduced with planting stock have a significant impact on exotic plantation forestry in the tropics. Teratosphaeria destructans (formerly Kirramyces destructans) is a serious pathogen causing leaf, bud and shoot blight diseases of Eucalyptus spp. in plantations in the sub-tropics and tropics of south-east Asia. This pathogen was first discovered in Indonesia in 1995 and has subsequently spread to Thailand, China, Vietnam and East Timor. The biology, ecology and genetics of this important pathogen have not been explored yet. The objective of this study was, thus, to determine the genetic diversity and movement of T. destructans throughout south-east Asia using multi-gene phylogenies and microsatellite markers. Out of nine gene regions only two microsatellite markers detected a very low nucleotide polymorphism between isolates; seven other gene regions, ITS, β-tubulin, EF1-α, CHS, ATP6 and two microsatellite loci, reflected genetic uniformity. The two polymorphic molecular markers resolved six haplotypes among isolates from Indonesia and only a single haplotype elsewhere in Asia. The low diversity observed among isolates in the region of the first outbreak is as expected for a small founder population. The spread of a single clone over large distances throughout the region supports the hypothesis of spread via the human-mediated movement of germplasm.     

Potential of Ypt1 and ITS gene regions for the detection of Phytophthora species in a lab‐on‐a‐chip DNA hybridization array

A novel DNA‐chip hybridization assay that uses the ras‐related GTP‐binding protein 1 gene (Ypt1) was developed for the identification of several devastating Phytophthora species. The hybridization was conducted in a portable microfluidic lab‐on‐a‐chip device for fast and accurate detection of 40 Phytophthora, two Pythium and one Phytopythium species. Moreover, the functionality of the Ypt1 region was examined in comparison to an array for the internal transcribed spacer (ITS) region by in silico modelling. The difference in species‐specific capture probe sequences was lower for the ITS than for the Ypt1 region. While ITS‐probes of Phytophthora ramorum, Phytophthora fragariae and Phytophthora lateralis cross‐reacted with up to 11 non‐target species, Ypt1‐probes were specific except for P. fragariae/Phytophthora rubi. First analyses of artificially inoculated Rhododendron leaves successfully demonstrated the usability of the respective capture probes for the Ypt1 and the ras‐related plant protein Rab1a gene region. The on‐chip hybridization enabled the detection of up to 1 pg μL^?1 target DNA depending on the species examined. Due to the complementarity of ITS and Ypt1 genetic features, the use of multiple loci is recommended to identify targets of different taxonomic rank.     

Meloidogyne luci,a new root‐knot nematode parasitizing potato in Portugal

In 2013, during a field survey conducted in Portugal on potato, Solanum tuberosum, an unusual esterase (EST) phenotype was detected in a root‐knot nematode (RKN) from potato roots collected in Coimbra. This Portuguese isolate was purified and maintained on tomato, S. lycopersicum, and morphological, biochemical and molecular characteristics were studied. Perineal pattern morphology was highly variable, similar to Meloidogyne ethiopica and not useful for identification. The EST phenotype, from young egg‐laying females, displayed three bands similar to the Brazilian M. luci (L3) and distinct from M. ethiopica (E3). Phylogenetic analyses of mitochondrial cytochrome oxidase subunit I and the mitochondrial DNA region between COII and 16S rRNA genes revealed that the Portuguese isolate grouped with M. luci isolates close to M. ethiopica isolates. However, considering the ITS1‐5.8S‐ITS2 region, the Portuguese isolate grouped with isolates of M. luci, M. ethiopica and M. hispanica, which limits the confidence of this region for M. luci diagnosis, and its differentiation from other species with morphological similarities. The M. luci pathogenicity to potato was also assessed in 16 commercial cultivars and compared with M. chitwoodi, considered to be a quarantine RKN species by EPPO. All potato cultivars were susceptible to both Meloidogyne species with gall indices of 5 and higher reproduction factor values ranging from 12.5 to 122.3, which suggests that M. luci may constitute a potential threat to potato production. In the present study, M. luci is reported for the first time attacking potato in Portugal.     

Quantification of Mirafiori lettuce big‐vein virus and its vector,Olpidium virulentus,from soil using real‐time PCR

Big vein disease of lettuce (Lactuca sativa) is an economically important disease transmitted through soil by Olpidium virulentus, and has occurred in most production areas worldwide. The disease is assumed to be caused by Mirafiori lettuce big‐vein virus (MiLBVV). To understand the dynamics of the virus and its vector, MiLBVV and O. virulentus were directly detected in soil. DNA and RNA were extracted from 5 g soil using a bead beating method, followed by purification using adsorption to a column. Detection and quantification were performed using real‐time PCR and a TaqMan probe that was prepared based on the CP region of MiLBVV and the rDNA‐ITS region of O. virulentus, respectively. Furthermore, using a visual assessment of the incidence rate of big vein disease on lettuce in agricultural fields, the C_t values of MiLBVV and O. virulentus from soil were also determined using real‐time PCR. The results showed that MiLBVV concentrations in the soil were high in the field, as also determined by a visual assessment of the incidence rate of big vein disease on lettuce. However, the amount of O. virulentus in soil was not directly correlated with the incidence of MiLBVV. From these results, it is suggested that the risk of lettuce crops developing big vein disease can be estimated using an index of the amount of MiLBVV in the soil.     

First report of <Emphasis Type="Italic">Ageratum enation virus</Emphasis> infecting <Emphasis Type="Italic">Crassocephalum crepidioides</Emphasis> (Benth.) S. Moore and <Emphasis Type="Italic">Ageratum conyzoides</Emphasis> L. in India

In October 2009, vein yellowing disease was observed on the weeds Crassocephalum crepidioides and Ageratum conyzoides in a subtemperate region in northern India. Ageratum enation virus (AEV), along with a nanovirus like satellite DNA 1, was found to be associated with both weeds. The isolates had 99% identity with each other and with an isolate of AEV reported from Zinnia elegans from this region. To the best of our knowledge, this report is the first of any begomovirus infection in C. crepidioides in India and the first on AEV infecting C. crepidioides worldwide and A. conyzoides in India.     

Characterisation of black locust isolates of <Emphasis Type="Italic">Peanut stunt virus</Emphasis> (PSV) from the Pannon ecoregion show the frequent occurrence of the fourth taxonomic PSV subgroup

Biological and molecular characterisation of ten Peanut stunt virus (PSV) isolates from Robinia pseudoacacia was carried out. The host range of these isolates was similar to that of the previously described PSV strains in most cases, but on Pisum sativum and Lens culinaris latent infection was induced. Variability in systemic symptoms was observed only on Nicotiana glutinosa. The partial RNA3 sequences were determined, including the carboxyl terminal region of the movement protein gene, the intergenic region, the entire coat protein gene and the 3′ untranslated region. Nucleotide sequence comparison of the coat protein genes showed 77.6–84.2% identity with most of the known PSV strains and 96.3–98.0% identity with PSV-Rp the typical member of subgroup IV. Phylogenetic analysis indicated the presence of the ancient homologous recombination in all of the examined black locust isolates and all the isolates were members of the fourth PSV subgroup. These results showed that the isolates of the fourth subgroup are widely distributed in black locust in this region.     

Characterization of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melongenae</Emphasis> isolates from eggplant in Turkey by pathogenicity,VCG and RAPD analysis

Fusarium wilt is an economically important fungal disease of common eggplant (Solanum melongena) cultivated in the eastern Mediterranean region of Turkey. Seventy-four isolates of Fusarium oxysporum isolated from diseased eggplant displaying typical Fusarium wilt symptoms were screened for pathogenicity on the highly susceptible cv. ‘Pala’. All the isolates tested were pathogenic to eggplant and designated as Fusarium oxysporum f. sp. melongenae (Fomg). Genetic diversity among a core set of 20 Fomg isolates that were selected based upon geographic locations, were characterized by using pathogenicity, vegetative compatibility grouping (VCG), and random amplified polymorphic DNA (RAPD) analysis. The area under the disease progress curve (AUDPC) was calculated for each Fomg isolate until 21 days after inoculation (DAI). The most virulent isolate was identified as Fomg10 based on AUDPC, disease severity and vascular discoloration measurements at 21 DAI. At this date, a good correlation was observed between disease severity and AUDPC values for all isolates (r = 0.73). UPGMA (unweighted pair group method with arithmetic average) cluster analysis of RAPD data using Dice’s coefficient of similarity differentiated all the Fomg isolates tested, and indicated considerable genetic variation among Fomg isolates, but isolates from the same geographic region were grouped together. There was no direct correlation between clustering in the RAPD dendrogram and pathogenicity testing of Fomg isolates. Twenty isolates of Fomg were assigned to VCG 0320.     

<Emphasis Type="Italic">Amaranthus leaf mottle virus</Emphasis>: 3′-end RNA sequence proves classification as distinct virus and reveals affinities within the genus <Emphasis Type="Italic">Potyvirus</Emphasis>

Amaranthus leaf mottle virus (AmLMV) was classified as a member of the genus Potyvirus on the basis of its particle morphology, serology, and biological properties (Casetta et al., 1986). Based on these properties, an Amaranthus viridis-infecting virus isolated in Spain, causing mottle and leaf blistering as well as reduced growth has been identified as AmLMV. The 3′ terminal genomic region of this and a reference isolate from Italy has been sequenced and reveals a 95% nucleotide identity between the two isolates. The sequenced part comprises the coat protein with 281 amino acids and 315 nucleotides of the 3′ untranslated region (UTR) preceding a polyadenylated tail. Pairwise comparisons and phylogenetic analysis of the nucleotide and deduced amino acid sequences of the CP and 3′ UTR of the cloned cDNAs with those of other potyviruses shows that AmLMV is a distinct potyvirus closely related to Potato virus Y.     

Identification of <Emphasis Type="Italic">Ditylenchus</Emphasis> species associated with Fabaceae seeds based on a specific polymerase chain reaction of ribosomal DNA-ITS regions

A technique based on the use of specific primers for polymerase chain reaction (PCR) was developed for the identification of the stem and bulb nematode belonging to the Ditylenchus dipsaci species complex. The internal transcribed spacer region ITS1 and ITS2, the gene 5.8 S and part of genes 18 S and 26 S of twenty populations of the D. dipsaci species complex belonging to both D. dipsaci sensu stricto and Ditylenchus sp. B (corresponding to populations of giant individuals associated to Vicia faba) and three congeneric species were amplified with two universal ribosomal primers. PCR-amplified DNA samples were digested with five restriction enzymes in order to reveal some polymorphism allowing the identification of D. dipsaci populations associated with Fabaceae seeds. The polymorphism among species was confirmed by the sequencing of the PCR products. A primer (DdpS2) was designed in a region conserved in all populations of both D. dipsaci sensu stricto and D. sp. B studied in the present work. The other Anguinidae species (except a few species from Central Asia associated to Astereaceae and D. sp. G associated to Plantago maritima) differ in two to four nucleotides at the 3′ extremity of this region. This sequence portion coincides with a TspEI restriction site. In combination with a primer located in the ribosomal region, this first primer is a good candidate for identification by PCR of populations of the D. dipsaci species complex found in Fabaceae seeds. A second primer (DdpS1) was designed in a similar way and was specific to D. dipsaci sensu stricto. The utility of these two sets of primers is discussed against the background of quarantine regulation.     

Consistent responses of yield and resistance of wheat cultivars to the root-lesion nematode,Pratylenchus thornei,in the Australian northern subtropical region,but not in the temperate southern region

To understand the yield response of cereal cultivars to Pratylenchus thornei, eight experiments were conducted within the subtropical northern, and temperate southern grain-producing regions of Australia. Wheat cultivars (Triticum aestivum) ranging from susceptible to moderately resistant to P. thornei were grown in Year 1 to establish a range of population densities. In Year 2 before sowing, P. thornei was quantified in each plot and six cereal cultivars were each grown on a similar range of population densities (average minimum to maximum of 3.4–60.6 P. thornei/g soil); P. thornei was quantified again at harvest. In the four experiments in the northern region there was a significant, negative logarithmic response of yield of the three most intolerant/susceptible cultivars as P. thornei population densities increased (yield decreased 172–479 kg/ha per unit increase in log_e-transformed P. thornei/g soil). The responsiveness of yield to increasing P. thornei population densities diminished as the tolerance and resistance of the cultivars improved. In the southern region, there was no relationship between yield and P. thornei in three experiments and minor, positive increases in one experiment (1.6 kg/ha per unit increase in P. thornei/g soil). Across both regions, the change in P. thornei population densities from sowing to harvest was logarithmic and positive, and generally greatest in the northern region. The contrast of responses of cereal cultivars between the regions, despite similar population densities of P. thornei, is indicative of the influence of the environment particularly on tolerance, therefore management with a regional focus is essential.     

Quick assessment of the invasiveness of non‐native plant species by using eco‐physiological parameters in Tram Chim National Park,Vietnam

Using eco‐physiological parameters, a quick assessment of the invasiveness of non‐native plant species was conducted in Tram Chim National Park, a Ramsar site that is located in the Mekong River Delta region of Vietnam. An investigation of non‐native species and a vegetation analysis were carried out along 25 line transects and in 50 quadrats by using the Braun–Blanquet method. The researchers identified 84 non‐native plant species but only 31 species were naturalized in the wetland ecosystems. Twenty of those 31 species with a high importance value index were screened by using a parameter that was obtained from chlorophyll a fluorescence measurement, the performance index. Five species were identified as invasive and five others were predicted to be potentially invasive. The first group of five species were: M imosa pigra, P anicum repens, E ichhornia crassipes, S alvinia cucullata and L eersia hexandra, which already had been confirmed as important weeds in the national park by previous studies. From the second group, two species ( L udwigia hyssopifolia and S accharum spontaneum) already are becoming prominent species in some locations. The three remaining species ( M onochoria hastata, I sachne globosa and M arsilea quadrifolia) are likely to become invasive in the future.     

Molecular variability among isolates of <Emphasis Type="Italic">Mycosphaerella graminicola</Emphasis>, the causal agent of septoria tritici blotch,in Argentina

The genetic structure and diversity of Mycosphaerella graminicola population were studied with ISSR molecular markers, using isolates from several locations of the Argentinean wheat region: subregion IV (SE of Buenos Aires Province) and II South (central part of Buenos Aires Province). Samples were taken from different bread wheat (Triticum aestivum) cultivars. A total of 126 isolates were subjected to molecular analysis to compare the genetic structure of the isolates from both wheat subregions. Ten ISSR primers were used: (GACA)₄; (AAC)₇; (ATC)₇; (AC)₉; (AAG)₇; (AG)₉; (AGC)₅; (CAG)₅, (GTG)₅ and (GACAC)₃. Eighty-four bands ranging from 200 bp to 8.000 were amplified. Eighty-one distinct haplotypes were identified and 43 isolates did not generate any amplification products. The highest number of polymorphic DNA fragments were produced using ISSR primers (ATC)₇ and (GTG)₅, which detected bands in 38 isolates. The molecular analysis revealed the existence of 81 different haplotypes among the 126 isolates studied. These results revealed a high degree of genetic diversity in the M. graminicola population in Argentina.     

Molecular characterization and PCR detection of the melon pathogen <Emphasis Type="Italic">Acremonium</Emphasis><Emphasis Type="Italic">cucurbitacearum</Emphasis>

Acremonium cucurbitacearum is a soil-borne pathogen that causes collapse of muskmelon and watermelon plants. Cluster analysis based on RAPD patterns, obtained from use of 25 primers, divided isolates of A. cucurbitacearum from Spain and USA into two major groups. Most isolates from the USA fell into group 1, however, genetic similarity was not highly correlated with geographical origins or with previously established VCG groups. Analysis of 5.8S-ITS sequences showed very little sequence variation among isolates of A. cucurbitacearum, most had identical 5.8S-ITS sequence. Nodulisporium melonis, previously reported to cause a similar disease in Japan, had a 5.8S-ITS sequence that was identical to that of isolate A-419 proposed as the type strain of A cremonium cucurbitacearum suggesting that the two fungal pathogens should be considered a single species. Phylogenetic analysis, based on the 5.8S-ITS region, indicated that A cremonium cucurbitacearum is a monophyletic taxon more closely related to Plectosphaerella cucumerina than to other species of the genus Acremonium. Based on the 5.8S-ITS nucleotide sequence, a polymerase chain reaction was designed and used for specific detection of A. cucurbitacearum in diseased plants.     

Molecular,physiological and pathogenic variability of <Emphasis Type="Italic">Pyrenochaeta lycopersici</Emphasis> associated with corky rot disease of tomato plants in Turkey

Isolates of Pyrenochaeta lycopersici, the causal agent of corky root rot on tomato plants, were assessed for physiological and genetic characteristics using conventional and molecular techniques. All isolates were able to produce necrosis on tomato roots and classified into temperature group according to the optimal growth temperatures. Specific-PCR assays and DNA sequence analysis of the ribosomal DNA (rDNA) internal transcribed spacer region confirmed the existence of both types (Type 1 and Type 2) of the pathogen among the isolates tested. All isolates were identified as Type 2 except for isolate Pl-4, which was classified as Type 1. Restriction fragment length polymorphism (RFLP) analysis with six enzymes resulted in three distinct banding patterns among the isolates depending on the length and restriction profiles of the rDNA intergenic spacer region. Inter-simple sequence-repeat analysis revealed a high level of genetic diversity among the isolates in agreement with the data of RFLP analysis. These results indicated that there were three different intraspecific groups among Turkish isolates of P. lycopersici. The presented study is the first attempting to characterize Turkish isolates of P. lycopersici. The results obtained will be useful in screening of tomato seedlings for resistance to P. lycopersici.     

PM 9/25 (1) Bactericera cockerelli and ‘Candidatus Liberibacter solanacearum’

Specific scope

This Standard describes a national regulatory control system for Bactericera cockerelli and the bacterial pathogen ‘Candidatus Liberibacter solanacearum’ the cause of zebra chip disease in potato. The scope is as follows:

• Exclusion from the EPPO region of B. cockerelli an efficient vector of ‘Ca. L. solanacearum’ within solanaceous crops (e.g. potato, tomato)
• Eradication of incursions of B. cockerelli
• Exclusion from the EPPO region of ‘Ca. L. solanacearum’ haplotypes A and B. Although reference will only be made to haplotypes A and B, the Standard would also apply to new non‐European haplotypes of ‘Ca. L. solanacearum’ which may have different host ranges, or which may be vectored more efficiently by psyllids which are widespread in the region.

The reduction of the risk of spreading ‘Ca. L. solanacearum’ haplotypes C, D and E to potato production systems and potatoes being moved within the EPPO region may be recommended in future when more information is available but is not covered in this Standard.

Specific approval

First approved in 2017‐09.     

White root rot disease of the lacquer tree <Emphasis Type="Italic">Toxicodendron vernicifluum</Emphasis> caused by <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

In early August 2010, lacquer trees (Toxicodendron vernicifluum) severely damaged by a root rot disease were found on plantations in Iwate, Japan. The causal agent was a fungus identified as Rosellinia necatrix, based on morphology and the sequence of the ribosomal DNA internal transcribed spacer region. The fungus was clearly pathogenic on T. vernicifluum root plantings. This report is the first of white root rot on T. vernicifluum.     

Identification of <Emphasis Type="Italic">Pythium tracheiphilum</Emphasis> as the causal agent of vascular necrosis of endive (<Emphasis Type="Italic">Cichorium endivia</Emphasis>) in Spain

Pythium isolates were recovered from endive plants (Cichorium endivia) showing vascular necrosis collected from commercial fields located in Castellón province (eastern Spain). They were identified as Pythium tracheiphilum on the basis of their phenotypical and molecular profile. Pathogenicity tests conducted with two P. tracheiphilum isolates, obtained from endive and lettuce (Lactuca sativa), respectively, in this region, confirmed that both isolates were pathogenic to endive, with no significant differences in virulence between them. This is the first report of vascular necrosis caused by P. tracheiphilum on endive in Spain.     

Occurrence of virulent root-knot nematode populations on tomatoes bearing the <Emphasis Type="Italic">Mi</Emphasis> gene in protected vegetable-growing areas of Turkey

Root-knot nematodes (Meloidogyne spp.; RKN) are one of the most important pathogens of vegetables in Turkey. Assessing the existing virulent RKN populations is of importance for pathogen mapping in the west Mediterranean region of Turkey. Therefore, 95 populations of RKN were collected from different protected vegetable-growing locations in the region. Pure cultures were obtained and identified by means of species-specific primers. Virulence of the populations against the Mi-1 gene conferring resistance to Meloidogyne incognita, M. javanica and M. arenaria was determined according to their egg masses and gall rating on resistant and susceptible tomato varieties. Results showed that seven populations of M. incognita and six populations of M. javanica were able to overcome the resistance controlled by the Mi-1 gene. The frequency of virulent populations of M. incognita and M. javanica collected from different protected-grown vegetables was 11.7% and 21.4%, respectively. To our knowledge, this is the first report of populations of RKN virulent to the Mi-1 gene in Turkey.