Rapid and Specific Detection of Virulent Pseudomonas avellanae Strains by PCR Amplification

Specific oligonucleotides, based on hrpW (hypersensitive response and pathogenicity) gene sequences encoding harpin protein in phytopathogenic bacteria, were designed to detect and identify virulent strains of Pseudomonas avellanae by polymerase chain reaction (PCR). A population of virulent P. avellanae strains, isolated in central Italy (Viterbo region), was assessed with hrpW-derived primers, producing a specific band of about 350 base pairs in length. This target was successfully amplified from purified genomic DNA, from bacterial culture and from hazelnut bark tissue. No amplification was obtained when the PCR assay was performed on other plant-pathogenic species from the following genera Agrobacterium, Erwinia, Brenneria, Pseudomonas, Ralstonia, Xanthomonas or from hazelnut-associated bacteria, indicating the specificity of these primers. Moreover DNA from strain ISPaVe-MCB-596, isolated from north Italy (Piedmont region) and belonging to the less aggressive population of P. avellanae, did not amplify in PCR. The PCR assay with the primers described here provides a rapid, specific and sensitive diagnostic method for virulent P. avellanae strains and a useful tool to evaluate the progress of sanitation of the area.     

Genetic Relatedness among Pseudomonas avellanae, P. Syringae pv. Theae and P.s. pv. Actinidiae, and their Identification

A total of 37 strains of Pseudomonas avellanae, P. syringae pv. theae and P.s. pv. actinidiae, including pathotype and reference strains, obtained from all the countries where these pathogens have been reported, were compared by means of ARDRA, repetitive PCR using ERIC, BOX and REP primer sets, whole-cell protein analysis, biochemical and nutritional tests, and pathogenicity tests. P. syringae pathovar type strains representing six genomospecies sensu Gardan et al. (1999), were also included for comparison in UPGMA cluster analysis of repetitive PCR data and SDS-PAGE of protein extracts. Among the 12 endonucleases used in ARDRA, only Tru 9I differentiated P. avellanae from P.s. pv. theae and P.s. pv. actinidiae. UPGMA cluster analysis of repetitive PCR genomic fingerprints showed 65% similarity between P.s. pv. theae and P. avellanae and 50% between the latter species and P.s. pv. actinidiae. Strains of P.s. pv. actinidiae could be grouped according to their geographic origin. Similar results were obtained with SDS-PAGE cluster analysis. PCR amplification using primers PAV 1 and PAV 22 that were developed to detect P. avellanae in apparently healthy and visibly infected hazelnut specimens yielded a band of 762bp from all strains of P. avellanae, P.s. pv. theae and P.s. pv. actinidiae. All strains lacked the syrB gene. Based on these data, we suggest that P.s. pv. actinidiae should be included in the genomospecies 8 together with P. avellanae and P.s. pv. theae. Selected biochemical and nutritional tests could differentiate these groups of strains. Pathogenicity tests clearly indicated that each group is specifically pathogenic only on the host plant species from which it was originally isolated.The author is staff member of the Istituto Sperimentale per la Patologia Vegetale, Roma, Italy temporarily assigned to ISF.     

Evidence for separate origins of the two Pseudomonas avellanae lineages

Pseudomonas avellanae is the causal agent of hazelnut ( Corylus avellana ) decline, both in northern Greece and central Italy, and two lineages related to the geographical origins of the pathogen have previously been identified. Forty strains, obtained from all the areas where the disease has so far been observed, and representing six different subpopulations of the two lineages, were further assessed using insertion-sequence PCR genomic fingerprinting. The data previously obtained from repetitive-sequence PCR using ERIC and BOX primer sets and insertion-sequence PCR (IS50) were analysed using statistical methods, enabling genetic diversity and gene flow among the populations to be elucidated, as well as verifying the possible correlation between genetic diversity and geographical origin. The Mantel test performed with ERIC, BOX and IS50-PCR data revealed that the P. avellanae populations that are spatially distant from each other are also genetically dissimilar: gene flow estimates confirmed this. The present study supports the hypothesis that P. avellanae originated separately in Greece and Italy, and that the two lineages of the pathogen underwent separate local evolution.     

Pseudomonas syringae pv. coryli, the Causal Agent of Bacterial Twig Dieback of Corylus avellana

ABSTRACT Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.     

Phylogenetic relationships of Ralstonia solanacearum species complex strains from Asia and other continents based on 16S rDNA, endoglucanase, and hrpB gene sequences

The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AY464950 to AY465050     

Infections of Glomerella cingulata on olive in Italy1

Olive anthracnose, caused by strains or populations of Glomerella cingulata (anamorph Colletotrichum gloeosporioides) pathogenic for olive, was introduced into southern Italy before or during the 2nd World War presumably from Albania or Greece. In the following 20 years, severe outbreaks of fruit rot and dieback of twigs and branches were recorded in several areas of Puglia, Calabria, Sicilia and Sardegna. After the 1970s, the epidemics gradually regressed. At present, the disease is restricted to certain humid areas of southern Italy. Factors associated with this regression are discussed, including a supposed change in virulence of the fungus, possibly as a consequence of mixing of the introduced strains infecting olive trees with local, less pathogenic populations of G. cingulata. The first results of a comparison of olive isolates with isolates of the pathogen from citrus and Annona muricata in Calabria suggest that the population from olive is relatively homogeneous and can be distinguished from the population infecting other hosts by a number of morphological, pathogenic and biochemical characteristics.     

Molecular and phenotypic features of Pseudomonas syringae pv. actinidiae isolated during recent epidemics of bacterial canker on yellow kiwifruit (Actinidia chinensis) in central Italy

Pseudomonas syringae pv. actinidiae (Psa) was identified as the causal agent of severe epidemics of bacterial canker on Actinidia chinensis (yellow kiwifruit) in central Italy occurring during 2008–9. A total of 101 strains were obtained from infected leaves, twigs, branches and trunks of cvs Hort16A, Jin Tao and CK3. Outbreaks were also found on A. deliciosa cv. Hayward. A representative set of 21 strains were compared with other Psa strains isolated from previous outbreaks in Japan and Italy as well as with P. s. pv. syringae strains obtained from A. chinensis and with strains of genomospecies 8. Repetitive‐sequence PCR (rep‐PCR) typing using BOX and ERIC primer sets revealed that all Psa strains obtained during 2008–9 showed the same fingerprinting profile. This profile, however, was different from those of strains previously isolated in Japan and Italy. Multilocus sequence typing (MLST) of gapA, gltA, gyrB and rpoD revealed a higher genetic variability among the strains than rep‐PCR, with some of them showing the same sequence pattern although isolated from different areas, cultivars and years. None of the recently obtained strains possessed genes coding for phaseolotoxin or coronatine, and all had an effector protein, namely hopA1, differentiating them from the strains causing past outbreaks in Japan and Italy. All isolates were inhibited in vitro by copper‐based compounds, antibiotics, geraniol, citronellol and by a chitin‐based organic compound. The recent epidemics found in central Italy on yellow kiwifruit appear to have been caused by a different Psa population than those previously recorded in Japan, South Korea and Italy.     

Physiological, Biochemical and Molecular Analyses of an Italian Collection of Agrobacterium tumefaciens strains

Physiological, biochemical and molecular characteristics of Agrobacterium tumefaciens strains isolated in Italy from different host plants were analysed. Diseased plants were collected from several nurseries located in nine different regions. Out of 1293 strains isolated from 12 fruit tree and six ornamental plant species, a group of 120 strains was chosen as representative of the whole collection. The majority of the strains were biovar 2 (82.5%), agrocin 84 sensitive, and were isolated from stone fruit trees. Most of the strains identified as biovar 1 were isolated from ornamental plants and were insensitive to A. radiobacter antagonistic strain K84. Some strains that were isolated from Euonymus spp, Prunus GF 677 and Pyrus communis (pear) OHF tumours could not be allocated to any of the three Agrobacterium biovars. PCR-restriction fragment length polymorphism of the rrs gene plus the intergenic spacer was used for strain fingerprinting and characterisation. Results showed a wide genetic variability within the biovar 1 strains and homogeneity within the biovar 2 group. Biovar 2 strains from Sardinia were highly variable and differed from the biovar 2 strains isolated from the other regions of Italy.     

Pathogenic and genetic variability of Ralstonia solanacearum strains from the Philippines

In the Philippines, bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases affecting vegetables and banana. In this study, 89 strains of R. solanacearum isolated from various hosts were screened for their biovar, phylotype, pathogenicity, and genetic diversity. Foreign strains were included for comparison with these Philippine strains. Results of the biochemical and multiplex-PCR tests divided the Philippine strains into five biovars (1, 2, 3, 4, and N2) and three phylotypes (I, II, and IV). Three potato strains belonged to biovar N2/phylotype IV. Pathogenicity tests divided the strains into five pathogenicity types based on their virulence in tomato, potato, eggplant, sweet pepper, and tobacco. Strains classified as biovar N2 were weakly pathogenic to potato (pathogenicity type III) and almost all strains isolated from banana were not pathogenic to the test plants except potato (pathogenicity type V). The results of AFLP analysis divided the strains into four clusters. Cluster 1 was composed of strains isolated from solanaceous crops, ginger (Zingiber officinale), and Morus sp. from the Philippines and other Asian countries. Cluster 2 grouped the potato strains (biovar N2) from the Philippines and Japan and blood disease bacterium strains from Indonesia. Cluster 3 contained the local and foreign strains isolated from potato (biovar 2) and banana (biovar 1). Cluster 4 consisted only of the tomato strain from the USA.     

Genetic structure and fungicide sensitivity of Botrytis cinerea populations isolated from grapevine in northern Italy

Grey mould, caused by Botrytis cinerea, is a disease severely affecting grape production in northern Italy. However, little information is available on the variability of B. cinerea populations associated with grapevine. The mode of reproduction, sensitivity to fungicides, and for the first time in Italy, the genetic structure of B. cinerea populations isolated from grapevine in a northern Italian region are reported. Botrytis cinerea isolates (317) were completely genotyped for six microsatellite loci and characterized for the presence of the transposable elements Boty and Flipper, for the mating type and for resistance to cyprodinil, fludioxonil, boscalid and fenhexamid. All the isolates were found to belong to B. cinerea Group II, indicating the absence of B. pseudocinerea in the investigated areas. The populations possess a high genotypic diversity, different frequencies of transposable elements and a mixed mode of reproduction. At a regional level, B. cinerea populations belong to a large and interconnected pathogen population that includes the major grape‐growing districts. The populations were generally sensitive to fungicides, with a low proportion (8%) of isolates resistant to cyprodinil, fludioxonil and boscalid. A small genetic distance was found between B. cinerea populations. However, the populations geographically isolated from the others by a mountain range showed a small but statistically significant genetic differentiation and a different pattern of fungicide resistance. The results show that northern Italian B. cinerea populations possess a high evolutionary potential and adaptive capacity.     

Redefining the global populations of Pseudomonas syringae pv. actinidiae based on pathogenic,molecular and phenotypic characteristics

Knowing the population structure of a pathogen is fundamental for developing reliable phytosanitary legislation, detection techniques, and control strategies based on the actual aggressiveness and distribution of the pathogen. Currently, four populations of Pseudomonas syringae pv. actinidiae (Psa) have been described: Psa 1, Psa 2, Psa 3 and Psa 4. However, diagnostic assays specific for Psa populations do not detect Psa 4, the less virulent (LV) strains isolated in New Zealand. Similarly, multilocus sequence typing (MLST) of housekeeping genes, or broad Psa strain genome comparisons, revealed that Psa 4‐LV strains clustered separately from other Psa populations. In order to examine whether the placement of Psa 4 in the pathovar actinidiae was appropriate, various tests were carried out. It was shown that the Psa 4‐LV strains induced leaf and shoot wilting in Prunus cerasus, extensive necrotic lesions in Capsicum annuum fruits, and no significant symptoms in Actinidia deliciosa. Moreover, repetitive‐sequence PCR fingerprinting, type III secretion system effector protein genes detection and colony morphology clearly indicated the distinctiveness of Psa 4‐LV strains from the other three Psa populations. Rep‐PCR molecular typing revealed a high similarity of the Psa 4‐LV strains with members of Pseudomonas avellanae species. The Psa 4‐LV strains, most probably, belong to a new, still unnamed pathovar. It was concluded that the Psa 4‐LV strains isolated in New Zealand do not belong to the pathovar actinidiae, and, consequently, three Psa populations pathogenic to Actinidia spp. should currently include Psa 1, Psa 2 and Psa 3.     

Chromosomal and Plasmid Diversity of Agrobacterium Strains Isolated from Ficus benjamina Tumors

Agrobacteria were previously isolated from tumors developing on branches and aerial and hypogeous roots of weeping fig plants in Italy and in The Netherlands. A representative group of 48 strains was analyzed by PCR–RFLP of 16S and 16S + IGS ribosomal regions, PCR–RFLP of six Ti plasmid (pTi) regions and characterized for plasmid content. Two groups of agrobacteria were separated by cluster analysis of PCR–RFLP profiles of rrs gene: seventeen strains were similar to the new species Agrobacterium larrymoorei, while the remaining strains were included within the agrobacterium biovar 1 group. Sixteen different plasmid profiles from one to five plasmids were observed. In addition, 21 ribotypes and 20 pTi structures were arranged in many different combinations, showing that fig agrobacteria were characterized by a wide heterogeneity. A general lack of correlation between strain ribotypes and plasmid content was observed.     

Molecular characterization of Pseudomonas syringae isolates from fruit trees and raspberry in Serbia

Infection of fruit trees by Pseudomonas syringae is a potentially serious problem that may limit the establishment and sustained productivity of pome and stone fruit orchards in Serbia. To estimate possible diversity of Pseudomonas syringae fruit trees strains, we collected a set of strains in several areas of Serbia. The samples were taken from infected orchards with raspberry, plum, cherry, sour cherry, peach, pear and apple trees. Genetic diversity of P. syringae strains isolated from fruit trees was determined by using SpeI macrorestriction analysis of genomic DNAs by pulsed-field gel electrophoresis (PFGE) and REP-PCR. Molecular analysis showed that most of isolates had unique profiles, with the exception of isolates from plum and cherry that displayed profiles identical to each other and similar to P. syringae pv. morsprunorum. The study presented here clearly demonstrates the discriminative power of molecular techniques in enabling a detailed analysis of the genetic variations between strains of P. syringae from different pome and stone fruit hosts in Serbia.     

Biocontrol of Phytophthora Root Rot of Angelica Trees by Enterobacter cloacae and Serratia ficaria Strains

Bacterial strains isolated from the rhizosphere of angelica trees were evaluated for their antagonistic activity against Phytophthora cactorum, a causal agent of Phytophthora root rot. Of these, three bacterial strains, designated as T-1-8, T-1-14 and T-1-23, strongly inhibited mycelial growth of P. cactorum ARE-862 in a dual-culture plate assay. Biocontrol activity of these strains was then examined by dipping root of young seedlings of angelica trees into a bacterial suspension. The incidence of Phytophthora root rot was markedly suppressed for at least 79 days in pot tests when treated seedlings were planted in naturally infested soil. The suppression was maintained through June of the next year. In addition, these strains significantly reduced the development of Phytophthora root rot up to 47 days in naturally infested field and up to 63 days (the last day of testing) in an artificially (moderately) infested field. Based on their main bacteriological properties, strain T-1-14 was identified as Enterobacter cloacae and T-1-8 and T-1-23 were identified as Serratia ficaria. Received 5 July 1999/ Accepted in revised form 25 October 1999     

Genetic Variability and Population Structure of the Wheat Foot Rot Fungus, Fusarium culmorum, in Tunisia

The random amplified polymorphic DNA (RAPD) method was used to investigate the genetic variability and population structure of Fusarium culmorum isolated from wheat stem bases. A total of 108 isolates, representing seven geographically distinct populations, was collected from five climatic regions in Tunisia. Pseudo-allelic frequencies were estimated at each of the 25 putative RAPD loci analyzed by scoring for the presence or absence of amplified fragments; 92 haplotypes were found among the 108 strains. The analysis of the population structure did not reveal any trend with regard to geographic origin. Total gene diversity (H_T ^* = 0.318) was mostly attributable to diversity within populations (H_S ^* = 0.308). Analysis of molecular variance confirmed that most of the genetic variability was within populations. Genetic differentiation among populations was low to moderate (G_ST ^* ranged from 0 to 0.190 and averaged 0.041 over all loci). Cluster analysis with UPGMA using genetic distances did not reveal any spatial clustering of the isolates collected from the different geographic regions. Based on these results, we conclude that the F. culmorum isolates recovered from different regions in Tunisia might be part of a single population pool.     

Detection and Identification of <Emphasis Type="Italic">Phytophthora</Emphasis> Species in Southern Italy by RFLP and Sequence Analysis of PCR-amplified Nuclear Ribosomal DNA

In four neighbouring regions of southern Italy, Basilicata, Campania, Apulia and Calabria, pepper and zucchini plants showing Phytophthora blight symptoms, tomato plants with either late blight or buckeye rot symptoms, plants of strawberry showing crown rot symptoms and declining clementine trees with root and fruit rot were examined for Phytophthora infections by means of polymerase chain reaction (PCR) assays, using primers directed to nuclear ribosomal DNA (rDNA) repeat sequences. All diseased plants and trees examined tested positive. The detected fungal-like organisms were differentiated and characterized on the basis of primer specificity as well as through extensive restriction fragment length polymorphism (RFLP) and sequence analysis of PCR-amplified rDNA. Phytophthora capsici was identified in diseased pepper and zucchini plants, P. infestans was identified in tomato with late blight symptoms whereas buckeye rot-affected tomatoes and diseased strawberry plants proved to be infected by P. nicotianae and P. cactorum, respectively. Declining clementine trees were infected with P. citrophthora and P. nicotianae in about the same proportion. Also, thirty-one pure culture-maintained isolates of Phytophthora which had previously been identified in southern Italy by traditional methods but were never examined molecularly, were examined by RFLP and sequence analysis of PCR-amplified nuclear rDNA. Among these, an isolate from gerbera which had previously been identified by traditional methods only at genus level, was assigned to P. tentaculata. For the remaining pure culture-maintained isolates examined, the molecular identification data obtained corresponded with those delineated by traditional methods. Most of the diseases examined were already known to occur in southern Italy but the pathogens were molecularly detected and fully characterized at nuclear rDNA repeat level only from other geographic areas, very often outside Italy. A new disease to southern Italy was the Phytophthora blight of zucchini. This is also the first report on the presence and molecular identification of P. tentaculata from Italy.     

Presence of the invasive whitefly Aleurocanthus spiniferus (Hemiptera: Aleyrodidae) in Greece

Aleurocanthus spiniferus (Quaintance) (Hemiptera: Aleyrodidae), commonly known as the citrus (or orange) spiny whitefly, is an important pest of various economic crops such as citrus and tea and causes severe economic losses. It is reported for the first time in Greece, in the island of Corfu (North‐West Greece) on sweet orange trees (Citrus sinensis). Morphological and molecular data has been used for species identification. Sequences of the mitochondrial cytochrome oxidase I (COI) gene from Greek populations have been compared with sequences of the species from Italy and Montenegro, as well as with COI sequences retrieved from GenBank, to examine the genetic diversity of the species. Based on the preliminary results, it appears that the population from Corfu probably arose through several independent introductions.     

Phytophthora acerina sp. nov., a new species causing bleeding cankers and dieback of Acer pseudoplatanus trees in planted forests in northern Italy

A severe dieback of Acer pseudoplatanus trees was noticed in planted forest stands in northern Italy in 2010. Affected trees showed collar rot and aerial bleeding cankers along the stems, leading to crown dieback and eventually death. An unknown Phytophthora species was consistently isolated from necrotic bark and xylem tissue and from rhizosphere soil. Based on its unique combination of morphological and physiological characters and phylogenetic analysis, this new taxon is here described as Phytophthora acerina sp. nov. Phylogenetic analysis of ITS, cox1 and β‐tubulin gene regions demonstrated that P. acerina is unique and forms a separate cluster within the ‘P. citricola complex’, closely related to P. plurivora. Phytophthora acerina is homothallic with smooth‐walled oogonia, thick‐walled, mostly aplerotic oospores with a high abortion rate, paragynous antheridia, and persistent, morphologically variable semipapillate sporangia. Four to 5‐week‐old cultures produced globose to subglobose, appressoria‐like and coralloid hyphal swellings and characteristic stromata‐like hyphal aggregations. Optimum and maximum temperatures for growth are 25°C and 32°C, respectively. Genetic uniformity of all 15 studied isolates and the apparent absence of this species in the extensive surveys of nurseries, forests and seminatural ecosystems conducted in the previous two decades across Europe indicate a recent clonal introduction to northern Italy. Under‐bark inoculation tests demonstrated high aggressiveness of P. acerina to A. pseudoplatanus indicating that this pathogen might be a serious risk to maple plantations and forests in Europe.     

Efficacy of a bacterial preparation of <Emphasis Type="Italic">Aneurinibacillus migulanus</Emphasis> against downy mildew of cucumber (<Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>)

The reniform nematodes of the genus Rotylenchulus are semi-endoparasites of numerous herbaceous and woody plant roots and distributed in regions with Mediterranean, subtropical and tropical climates. In this study, we provide morphological and molecular characterisation of three out of 11 valid species of the genus Rotylenchulus: R. macrodoratus, R. macrosoma, and R. reniformis from Greece (Crete), Italy and Spain. The overall prevalence of reniform nematodes in wild and cultivated olives in Greece, Italy, and Spain was 11.5%, 19.0% and 0.6%, respectively. In Greece, R. macrodoratus and R. macrosoma were detected in cultivated olive with a prevalence of 8.2% and 6.2%, respectively, but none of them were found in wild olive. This is the first report of R. macrosoma in Greece. Only one reniform nematode species was detected in olive from Italy and Spain, viz. R. macrodoratus and R. macrosoma, respectively. The parasitism of R. macrosoma on hazelnut in northern Spain was also confirmed for the first time. This study demonstrates that R. macrodoratus and R. macrosoma have two distinct rRNA gene types in their genomes, specifically the two types of D2-D3 for R. macrosoma and R. macrodoratus, the two types of ITS for R. macrodoratus and the testing of the ITS variability in other R. macrosoma populations in different countries. Rotylenchulus macrosoma from Greece and Spain showed differences in nucleotide sequences in the ITS region and D2-D3 of 28S rRNA gene.