ITS-RFLP Analysis Research Articles

Characterization of Indigenous Microbial Communities in Vineyards Employing Different Agronomic Practices: The Importance of Trunk Bark as a Source of Microbial Biodiversity

Microbiomes are essential to viticulture and winemaking since various fungi and bacteria can exert positive and negative effects on grape health and wine quality. The current work evaluates the communities of culturable fungi and bacteria associated with Corvina vines derived from two vineyards from a similar terroir (within the Valpolicella DOC area, Italy) but on which different management practices were employed: organic and conventional farming. Samples of bark and grapes were collected in four spatial points for each vineyard. Populations of bark-associated microorganisms were monitored during ripening season (at veraison and at harvest time), and results were integrated with data from grape-associated microorganisms, sampled right before harvest. Culturable populations of fungi and bacteria were determined by plate counting on WL and PCA culture media. For fungi, biodiversity was also assayed on all samples through molecular methods, by ITS-RFLP analysis. Although this does not represent a comprehensive evaluation of the microbiome, since culturable and countable microorganisms only represent a portion of microbial biodiversity, our results emphasize the importance of vine trunk bark, not only as an interesting habitat to be characterized for monitoring microbial biodiversity in vineyards but also as a potential source of microbial viable species for further isolation.

Morphological and molecular intervention in identifying Phytophthora spp. causing leaf and nut fall in nutmeg (Myristica fragrans Houtt.)

Nutmeg, a major crop of India provides two important spices. During the monsoon season of 2011, a serious leaf and nut fall occurred in nutmeg plantations and Phytophthora ramorum was reported as the causal organism. P. ramorum being an alien species, the aetiology warranted a detailed investigation. Phytophthora isolated from different endemic locations were studied for morphological and molecular characterization using ITS, ITS-RFLP, MLST, SSCP and hybrid analysis. The isolates showed papillate, ovoid-obovoid sporangia with pedicel length > 20 μm which is distinct from the semi-papillate, caducous sporangia with short pedicel of <5 μm characteristic of P ramorum. The isolates failed to grow at 10 °C or at 37 °C and chlamydopsores were not produced in culture, a characteristic distinct to P. ramorun. MLST analysis placed the isolates in Clade 2 while P. ramorum belongs to Clade 8. P. ramorum has a unique set of morphological characteristics and a unique molecular sequence at the ITS region which delineate it from other Phytopthora species. Among the eight isolates studied, ITS region of six isolates (13–01–13-06) showed similarity to P. meadii and two isolates (13–55 and 98–68) to P. tropicalis. In SSCP, the isolates formed a separate group distinct from P.meadii of cocoa and cardamom. The double peaks in the ITS chromatograms indicated hybridity and further hybrid analysis placed them under P. citrophthora and P. meadii (Cit-Med) lineage. Thus due to the distinct differences from P. ramorum and evidences of shared lineage with P. citrophthora and P. meadii, we propose to call these isolates as P. citromeadii (13–01- 13-06) and P. citrocaptalis (13–55 and 98–68).

Molecular Characterization of Fusarium oxysporum Causing Chickpea Wilt by Internal Transcribed Spacer (ITS) Marker

Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris is one of the serious disease causes tremendous loss to crop throughout the world and has assumed serious proportions in the recent years. Wilt affected chickpea plant roots samples were collected from forty eight different regions of Maharashtra. The collected samples were characterized by designed ITS primers. The Fusarium oxysporum f. sp. ciceris (Foc) showed about 302 bp amplicon when subjected to PCR with these primers. Further the amplified product was digested with five restriction enzymes viz. AluI, EcoRI, HaeIII, MboI and MspI and restriction fragment length polymorphism (RFLP) pattern were analysed. A dendrogram derived from ITS-RFLP analysis of the rDNA region divided the Foc isolates into four clusters. The maximum genetic distance 0.75 exhibited by five Foc isolates viz. Foc-30, Foc-36, Foc-24, Foc-25 and Foc-48 belonging to Shahartakli, Shelur, Nashik, Satara, Kolhapur regions respectively and found to be more diverse among the other Foc isolates. Whereas isolate no. Foc-20, Foc-21 and Foc-8 from Ambajogai, Karjat and Rahata shown minimum genetic distance (0.13). This showed the genetic variability among the Foc isolates collected from different regions of Maharashtra.

Distribution and Phylogeny of Microsymbionts Associated with Cowpea (Vigna unguiculata) Nodulation in Three Agroecological Regions of Mozambique.

ABSTRACTCowpea derives most of its N nutrition from biological nitrogen fixation (BNF) via symbiotic bacteroids in root nodules. In Sub-Saharan Africa, the diversity and biogeographic distribution of bacterial microsymbionts nodulating cowpea and other indigenous legumes are not well understood, though needed for increased legume production. The aim of this study was to describe the distribution and phylogenies of rhizobia at different agroecological regions of Mozambique using PCR of the BOX element (BOX-PCR), restriction fragment length polymorphism of the internal transcribed spacer (ITS-RFLP), and sequence analysis of ribosomal, symbiotic, and housekeeping genes. A total of 122 microsymbionts isolated from two cowpea varieties (IT-1263 and IT-18) grouped into 17 clades within the BOX-PCR dendrogram. The PCR-ITS analysis yielded 17 ITS types for the bacterial isolates, while ITS-RFLP analysis placed all test isolates in six distinct clusters (I to VI). BLASTn sequence analysis of 16S rRNA and four housekeeping genes (glnII, gyrB, recA, and rpoB) showed their alignment with Rhizobium and Bradyrhizobium species. The results revealed a group of highly diverse and adapted cowpea-nodulating microsymbionts which included Bradyrhizobium pachyrhizi, Bradyrhizobium arachidis, Bradyrhizobium yuanmingense, and a novel Bradyrhizobium sp., as well as Rhizobium tropici, Rhizobium pusense, and Neorhizobium galegae in Mozambican soils. Discordances observed in single-gene phylogenies could be attributed to horizontal gene transfer and/or subsequent recombinations of the genes. Natural deletion of 60 bp of the gyrB region was observed in isolate TUTVU7; however, this deletion effect on DNA gyrase function still needs to be confirmed. The inconsistency of nifH with core gene phylogenies suggested differences in the evolutionary history of both chromosomal and symbiotic genes.IMPORTANCE A diverse group of both Bradyrhizobium and Rhizobium species responsible for cowpea nodulation in Mozambique was found in this study. Future studies could prove useful in evaluating these bacterial isolates for symbiotic efficiency and strain competitiveness in Mozambican soils.

Molecular Characterization and Phylogeny of Ditylenchus weischeri from Cirsium arvense in the Prairie Provinces of Canada.

Ditylenchus weischeri that parasitizes the weed Cirsium arvense (L.) Scop., 1772, (creeping thistle) was described in 2011 from Russia based on their morphology, ITS-RFLP analysis, and Hsp 90 gene sequence of a few individuals and one field collection of the plant. More recently, we found C. arvense parasitized by D. weischeri in the Prairie Provinces of Canada. Plant host preference for D. weischeri was also distinct from D. dipsaci (Kühn) Filipjev, 1936. In the current study, a comprehensive molecular analysis of many D. weischeri specimens from Canada is presented. Individuals from 41 C. arvense or yellow pea grain samples with seeds of C. arvense from the Prairie Provinces were sequenced for the internal transcribed spacer (ITS rDNA), large subunit (LSU) D2D3 28S rDNA, partial segment of small subunit (SSU) 18S rDNA, and the heat shock protein Hsp 90 gene. The analysis also included D. weischeri individuals from C. arvense from Russia and garlic with D. dipsaci from the Provinces of Ontario and Quebec in Canada. Available sequence data of Ditylenchus species retrieved from GenBank were used to phylogenetically position this species within the genus Ditylenchus . In all studied genes, several single-nucleotide polymorphisms between the Canadian D. weischeri and both Russian haplotype and individuals of D. weischeri from C. arvense from Russia were found. The sequences of ITS rDNA, LSU D2D3 28S rDNA, and Hsp 90 were used to construct separate dendrograms. For each of the three genes examined, D. weischeri was grouped separately from the other Ditylenchus . Ditylenchus samples from C. arvense was positioned to a single clade such as D. weischeri and distinct from D. dipsaci . With past reports of plant host preference and morphology, the results of this study provide further evidence for the fact that D. weischeri is distinct to be separated from D. dipsaci . Furthermore, minor differences in molecular divergence and morphology to the Russian haplotype and limited symptoms of disease on C. arvense in Prairie Canada suggest the Canadian and Russian populations of D. weischeri may be diverging.

Potential Role of Yeast Strains Isolated from Grapes in the Production of Taurasi DOCG.

Twelve samples of Aglianico grapes, collected in different locations of the Taurasi DOCG (Appellation of Controlled and Guaranteed Origin) production area were naturally fermented in sterile containers at room temperature. A total of 70 yeast cultures were isolated from countable WL agar plates: 52 in the middle of the fermentation and 18 at the end. On the basis of ITS-RFLP analysis and ITS sequencing, all cultures collected at the end of fermentations were identified as Saccharomyces (S.) cerevisiae; while, the 52 isolates, collected after 1 week, could be referred to the following species: Metschnikowia (M.) pulcherrima; Starmerella (Star.) bacillaris; Pichia (P.) kudriavzevii; Lachancea (L.) thermotolerans; Hanseniaspora (H.) uvarum; Pseudozyma (Pseud.) aphidis; S. cerevisiae. By means of Interdelta analysis, 18 different biotypes of S. cerevisiae were retrieved. All strains were characterized for ethanol production, SO2 resistance, H2S development, β-glucosidasic, esterasic and antagonistic activities. Fermentation abilities of selected strains were evaluated in micro-fermentations on Aglianico must. Within non-Saccharomyces species, some cultures showed features of technological interest. Antagonistic activity was expressed by some strains of M. pulcherrima, L. thermotolerans, P. kudriavzevii, and S. cerevisiae. Strains of M. pulcherrima showed the highest β-glucosidase activity and proved to be able to produce high concentrations of succinic acid. L. thermotolerans produced both succinic and lactic acids. The lowest amount of acetic acid was produced by M. pulcherrima and L. thermotolerans; while the highest content was recorded for H. uvarum. The strain of Star. bacillaris produced the highest amount of glycerol and was able to metabolize all fructose and malic acid. Strains of M. pulcherrima and H. uvarum showed a low fermentation power (about 4%), while, L. thermotolerans, Star. Bacillaris, and P. kudriavzevii of about 10%. Significant differences were even detected for S. cerevisiae biotypes with respect to H2S production, antagonistic activity and β-glucosidase activity as well as for the production of acetic acid, glycerol and ethanol in micro-vinification experiments.

First Report of Broccoli Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in Serbia.

In September 2012, soft rot symptoms on broccoli (Brassica oleracea L. var. italica Plenck) were observed in several commercial fields in the western part of Serbia. Following the first harvest, water-soaked areas developed on broccoli stem tissue and progressed into soft rot decay of entire plants. The incidence of disease was approximately 30%. In Serbia, broccoli is grown on smaller fields compared to other vegetables, but its production and consumption increased significantly in recent years. From the diseased tissue, shiny, grayish white, round colonies were isolated on nutrient agar. Six non-fluorescent, gram-negative, facultative anaerobic, oxidase-negative, and catalase-positive bacterial strains were chosen for further identification. All strains caused soft rot on potato and carrot slices and did not induce hypersensitive reaction on tobacco leaves. They grew at 37°C and in yeast salts broth medium containing 5% NaCl (2), did not produce acid from α-methyl glucoside, but utilized lactose and trehalose, and did not produce indole or lecitinase. Investigated strains formed light red, 1.5-mm-diameter colonies on Logan's medium (2), and did not produce blue pigmented indigoidine on glucose yeast calcium carbonate agar (2) nor "fried egg" colonies on potato dextrose agar. Based on biochemical and physiological characteristics (1) and ITS-PCR and ITS-RFLP analysis (4), the strains were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rRNA gene sequence from two strains (GenBank KC527051 and KC527052) showed 100% identity with sequences of P. carotovorum subsp. carotovorum previously deposited in GenBank (3). Pathogenicity of the strains was confirmed by inoculation of broccoli head tissue fragments. Three florets per strain were inoculated by pricking the petals with a syringe and hypodermic needle and depositing a droplet of bacterial suspension (approx. 1 × 108 CFU/ml) at the point of inoculation. Sterile distilled water was used as a negative control. Inoculated florets were placed in a sealed plastic container and incubated in high humidity conditions at 28°C. Tissue discoloration and soft rot developed around the inoculation point within 48 to 72 h. No symptoms developed on control florets. Identity of bacterial strains reisolated from inoculated plant tissues was confirmed by ITS-PCR using G1/L1 primers followed by digestion of PCR products with Rsa I restriction enzyme (4). In Serbia, P. carotovorum subsp. carotovorum has been isolated from potato, some vegetable crops, and ornamentals, but not from broccoli until now.

Sequence Variation in Two Protein-Coding Genes Correlates with Mycelial Compatibility Groupings in Sclerotium rolfsii

Populations of Sclerotium rolfsii, the causal organism of Sclerotium root-rot on a wide range of hosts, can be placed into mycelial compatibility groups (MCGs). In this study, we evaluated three different molecular approaches to unequivocally identify each of 12 previously identified MCGs. These included restriction fragment length polymorphism (RFLP) patterns of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) and sequence analysis of two protein-coding genes: translation elongation factor 1α (EF1α) and RNA polymerase II subunit two (RPB2). A collection of 238 single-sclerotial isolates representing 12 MCGs of S. rolfsii were obtained from diseased sugar beet plants from Chile, Italy, Portugal, and Spain. ITS-RFLP analysis using four restriction enzymes (AluI, HpaII, RsaI, and MboI) displayed a low degree of variability among MCGs. Only three different restriction profiles were identified among S. rolfsii isolates, with no correlation to MCG or to geographic origin. Based on nucleotide polymorphisms, the RPB2 gene was more variable among MCGs compared with the EF1α gene. Thus, 10 of 12 MCGs could be characterized utilizing the RPB2 region only, while the EF1α region resolved 7 MCGs. However, the analysis of combined partial sequences of EF1α and RPB2 genes allowed discrimination among each of the 12 MCGs. All isolates belonging to the same MCG showed identical nucleotide sequences that differed by at least in one nucleotide from a different MCG. The consistency of our results to identify the MCG of a given S. rolfsii isolate using the combined sequences of EF1α and RPB2 genes was confirmed using blind trials. Our study demonstrates that sequence variation in the protein-coding genes EF1α and RPB2 may be exploited as a diagnostic tool for MCG typing in S. rolfsii as well as to identify previously undescribed MCGs.

The pinewood nematode, Bursaphelenchus xylophilus, in Madeira Island

Abstract The environmental conditions in Madeira Island are favorable for the presence and dissemination of the pinewood nematode (PWN), Bursaphelenchus xylophilus. Five hundred Pinus pinaster wood samples were collected in several forest areas and PWN was detected in 22.8 % of the samples. Bursaphelenchus xylophilus isolates from Madeira Island displayed the species-specific diagnostic characters. A morphological variation in the female tail terminus was detected. In most females, the tail presented a broadly rounded terminus and, occasionally, a digitate terminus with a terminal nipple-like extension resembling a mucro. PCR ITS-RFLP analysis revealed that Madeira Island isolates exhibited patterns specific to the species B. xylophilus and similar to virulent isolates. Amplified ITS regions were further sequenced and no genetic diversity was found for this genomic region among 17 Portuguese isolates (Madeira Island and Continental Portugal). Phylogenetic analysis revealed that Portuguese isolates grouped with isolates from China, Korea and one isolate from Japan.

The occurrence and rapid discrimination of Fomes fomentarius genotypes by ITS-RFLP analysis

Sequence comparison of available Fomes fomentarius (L.) J. Kickx f. internal transcribed spacer (ITS) of ribosomal DNA sequences demonstrated genetic non-homogeneity of the species. Multiple sequence alignment indicated the presence of two genotypes with overall similarity of about 97% and a strong statistics support. Rapid and reliable method for discrimination of F. fomentarius genotypes based on restriction digestion of polymerase chain reaction (PCR)-amplified ITS sequences was developed. BseNI and SchI restriction endonucleases were found to clearly discriminate between two F. fomentarius genotypes. The method was used to study the variability in F. fomentarius isolates collected from natural forest reserves in Vihorlat Mountains (East Slovakia). In most localities both genotypes occur concurrently. The isolates belonging to the genotype A were found to be prevalent on beech (Fagus sylvatica), while genotype B tends to be found mainly on other hosts. The grouping of selected isolates was confirmed by sequence analysis. Our results indicate that F. fomentarius includes at least two sympatric cryptic species.

Occurrence and Identification of Yeast Species in Fermented Liquid Feed for Piglets

The major objective of the present study was to investigate the occurrence and identity of yeast species in fermented liquid feed (FLF) used for feeding piglets. In total, 40 different Danish farms were included in the analysis. The preparation and composition of FLF was found to be very heterogeneous with high variations in both yeast counts and yeast species composition. The yeast population varied between 6.0 × 10(3) and 4.2 × 10(7) cfug(-1) with an average yeast count of 8.7 × 10(6) ± 1.1 × 10(7) cfug(-1). A total of 766 yeasts were isolated and identified by conventional and/or molecular typing techniques. The predominant yeast species in the FLF samples were found to be Candida milleri (58.4%), Kazachstania exigua (17.5%), Candida pararugosa (6.40%) and Kazachstania bulderi (5.09%). No clear separation between isolates of C. milleri and Candida humilis could be obtained based on sequencing of the D1/D2 region of the 26S rRNA gene. The combined use of ITS-RFLP analysis and phenotypic criteria did meanwhile suggest a closer relationship with C. milleri than C. humilis.

Erratum

EPPO BulletinVolume 39, Issue 1 p. 84-84 Free Access Erratum This article corrects the following: Ditylenchus destructor and Ditylenchus dipsaci Volume 38Issue 3EPPO Bulletin pages: 363-373 First Published online: November 11, 2008 First published: 11 March 2009 https://doi.org/10.1111/j.1365-2338.2009.02241.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Since the publication of EPPO Standard PM 7/87 Ditylenchus destructor and Ditylenchus dipsaci, it has been brought to our attention that an error appeared in the volumes used in paragraph 2.2.1 of Appendix 1 for this standard. The corrected line of Appendix 1, paragraph 2.2.1 appears below. The Bulletin would like to apologise for this error. Appendix 1 ITS-RFLP analysis according to Wendt et al. (1993). 2.2.1 Master mix (provided within kit), 0.5 µM of each Vrain primer and 20 ng of DNA. Reference OEPP/EPPO (2008) EPPO Standard PM 7/87 Ditylenchus destructor and Ditylenchus dipsaci. Bulletin OEPP/EPPO Bulletin 38, 363– 373. Volume39, Issue1April 2009Pages 84-84 ReferencesRelatedInformation

ITS-RFLP analysis, an efficient tool for differentiation of Bursaphelenchus species

Abstract Restriction analysis of amplified ribosomal ITS sequences has provided species-specific fragment patterns for nematodes of several genera, including Bursaphelenchus. We used restriction enzymes RsaI, HaeIII, MspI, HinfI and AluI to produce ITS-RFLP reference profiles of 44 Bursaphelenchus species, including two intraspecific types in each of B. mucronatus and B. leoni. In addition, reference profiles of Aphelenchoides stammeri and Ruehmaphelenchus asiaticus were produced. Reference profiles of six species are shown here for the first time. Identical ITS-RFLP patterns were usually obtained from different isolates and from individual specimens of the same species. However, in the case of B. 'corneolus', B. lini, B. singaporensis and B. sexdentati, additional bands in the patterns of certain isolates or individual nematodes were observed which may be explained by ITS sequence microheterogeneity, i.e., the presence of ITS sequence variants within the number of rDNA tandem repeats. Since these 'extra' bands appeared only with one out of the five restriction enzymes employed, they did not seriously impair identification of species based on the overall reference patterns. ITS-RFLP analysis has proved valuable for differentiation of the pathogenic pine wood nematode, B. xylophilus, from related species. In many recent descriptions of new Bursaphelenchus species, ITS-RFLP profiles have been used as additional species identification criteria. Comparison of profiles from isolates of many different origins has provided new information on intraspecific types or genetically distinct provenances of several Bursaphelenchus species.

Ditylenchus destructor and Ditylenchus dipsaci

<i>Ditylenchus destructor</i> and <i>Ditylenchus dipsaci</i>

Prevalence and aetiology of Phytophthora foot rot of black pepper in Vietnam

Black pepper is a high value export crop in Vietnam. The production of black pepper, however, is reduced remarkably by Phytophthora foot rot disease. Although the disease was first reported in 1952, the identity of the causal organism has never been conclusively determined. Hence, the aim of this study was to identify the causal agent associated with Phytophthora foot rot epidemics in black pepper. A national survey of the disease and collection of samples were conducted in four major black pepper growing provinces. Phytophthora isolates obtained from the diseased roots, collars and leaves as well as the root-zone soil were identified by morphological characteristics and verified by ITS-RFLP analysis. Phytophthora capsici was determined as the main pathogen causing Phytophthora foot rot of black pepper in Vietnam based on disease symptoms, morphological characteristics, pathogenicity and ITS-RFLP analysis.

Morphometrical and molecular characterization of Bursaphelenchus species from Slovenia

Abstract The environmental conditions in Slovenia are relatively favourrable for the colonisation and spread of the pine wood nematode Bursaphelenchus xylophilus, a very dangerous pathogenic species which was recently identified in Europe in Portugal. To determine the presence of B. xylophilus in Slovenia and to introduce proper measures against its emergence and spread, a survey of Bursaphelenchus species was conducted in Slovenia from 2002 to 2005. Approximately 120 ha of conifer forests in Slovenia were surveyed for the presence of Bursaphelenchus species. In total, 206 wood samples were taken from the conifer forests. B. hofmanni, B. mucronatus were found for the first time in Slovenia, while B. xylophilus was not detected. Additionally, the species Aphelenchoides stammeri which is morphologically very similar to Bursaphelencus was found. All species have been described morphologically and characterized by ITS-RFLP analysis. Sequences of ribosomal DNA for B. hofmanni, B. mucronatus were analysed.

スペインの土壌から分離されたSporothrix schenckii のITS-RFLPによる検討

スペインの土壌から分離されたSporothrix schenckii のITS-RFLPによる検討

Spatial structure and richness of ectomycorrhizal fungi colonizing bioassay seedlings from resistant propagules in a Sierra Nevada forest: comparisons using two hosts that exhibit different seedling establishment patterns

In this study we analyzed the spatial structure of ectomycorrhizal fungi present in the soils as resistant propagules (e.g. spores or sclerotia) in a mixed-conifer forest in the Sierra Nevada, California. Soils were collected under old-growth Abies spp. stands across approximately 1 km and bioassayed with seedlings of hosts that establish best in stronger light (Pinus jeffreyi) or that are shade-tolerant (Abies concolor). Ectomycorrhizal fungi colonizing the roots were characterized with molecular techniques (ITSRFLP and DNA sequence analysis). Wilcoxina, five Rhizopogon species and Cenococcum were the most frequent of 17 detected species. No spatial structure was detected in the resistant propagule community as a whole, but P. jeffreyi seedlings had higher species richness and associated with seven Rhizopogon species that were not detected on A. concolor seedlings. We drew two conclusions from comparisons between this study and a prior study of the ectomycorrhizal community on mature trees in the same forest: (i) the resistant propagule community was considerably simpler and more homogeneous than the active resident community across the forest and (ii) Cenococcum and Wilcoxina species are abundant in both communities.

Spatial structure and richness of ectomycorrhizal fungi colonizing bioassay seedlings from resistant propagules in a Sierra Nevada forest: comparisons using two hosts that exhibit different seedling establishment patterns

In this study we analyzed the spatial structure of ectomycorrhizal fungi present in the soils as resistant propagules (e.g. spores or sclerotia) in a mixed-conifer forest in the Sierra Nevada, California. Soils were collected under old-growth Abies spp. stands across approximately 1 km and bioassayed with seedlings of hosts that establish best in stronger light (Pinus jeffreyi) or that are shade-tolerant (Abies concolor). Ectomycorrhizal fungi colonizing the roots were characterized with molecular techniques (ITS-RFLP and DNA sequence analysis). Wilcoxina, five Rhizopogon species and Cenococcum were the most frequent of 17 detected species. No spatial structure was detected in the resistant propagule community as a whole, but P. jeffreyi seedlings had higher species richness and associated with seven Rhizopogon species that were not detected on A. concolor seedlings. We drew two conclusions from comparisons between this study and a prior study of the ectomycorrhizal community on mature trees in the same forest: (i) the resistant propagule community was considerably simpler and more homogeneous than the active resident community across the forest and (ii) Cenococcum and Wilcoxina species are abundant in both communities.

Supplementary characters of Bursaphelenchus lini Braasch, 2004 (Nematoda: Parasitaphelenchidae) and remarks on this nematode

The description of Bursaphelenchus lini Braasch, 2004 is completed and corrected according to SEM micrographs taken from newly-found specimens obtained from Ningbo, China. Whereas the presence of three lateral lines outlined in the original description was confirmed, the number of caudal papillae pairs has to be corrected as being two only, one subventral preanal pair and one ventral postanal pair in front of the terminal bursa. The third ventrosublateral pair, originally described as closely posterior to the cloaca, was revealed to be an artefact. The rectangular and crystal like objects, which were observed in the posterior half of the body in several type specimens were not found in newly-found populations. Therefore, the presence of these rectangular objects has no diagnostic value. Affiliation of the newly-found specimens to the species B. lini was confirmed by ITS-RFLP analysis. As an additional biological character, B. lini has always been found accompanied by B. xylophilus.