Universally Primed Polymerase Chain Reaction Research Articles

Molecular Characterization of Ciborinia camelliae Kohn Shows Intraspecific Variability and Suggests Transcontinental Movement of the Pathogen.

Ciborinia camelliae Kohn is the causal agent of camellia flower blight. The fungus infects only the flowers of camellias. C. camelliae isolates obtained from symptomatic samples, collected in 13 different localities worldwide, were characterized by Multi-Locus Sequence Typing (MLST) using the following: (i) a nuclear ribosomal DNA internal transcribed spacer; (ii) subunit 2 of β-tubulin (β-TUB II), (iii) elongation factor 1-α (EF1α); and (iv) glycerol-3-phosphate dehydrogenase (GPDH). The variability of the strains was assessed using a universally primed-polymerase chain reaction (UP-PCR) with six universal primers. Gene sequence comparison showed high similarity among all the European strains and highlighted the diversity of the New Zealand and Chinese representative strains. The profiles obtained by UP-PCR confirmed the significant diversity of extra-European strains and identified subgroups within the European population. The presence of shared genetic profiles obtained from strains isolated in different countries (New Zealand and France) suggests the movement of strains from one location to another, which is probably due to the exchange of infected plant material. Moreover, our study shows the overall high intraspecific variability of C. camelliae, which is likely due to the sexual reproduction of the fungus, suggesting the risk of emergence of new pathotypes adapting to novel camellia varieties.

Genetic diversity evidence a mixed reproduction mode in Venturia oleaginea populations in Uruguay

Olive scab caused by Venturia oleaginea is one of the most important diseases of olive worldwide. The fungus infects young leaves and fruits by asexually produced conidia. The sexual stage and its importance in the epidemiology and genetics of the pathogen population is unknown. A collection of 52 fungal isolates was obtained from scabbed leaves collected in 16 orchards from three major regions in Uruguay. All isolates were identified as V. oleaginea by PCR with specific primers and sequence analysis of the TEF-1α gene region. Five colony morphotypes and different growing rates were observed on potato dextrose agar (PDA) medium. Universally primed polymerase chain reaction (UP-PCR) was successfully used for assessing the genetic diversity and population structure of the V. oleaginea isolates, whereas ISSR was unable to generate reproducible and polymorphic bands. Two populations genetically different were identified: [1] composed by 21 isolates mainly from Southern and South-Western regions; [2] composed by 31 isolates mainly from South-Eastern and Southern regions. Both populations showed a moderate gene diversity expressed as hNei = 0.163 and 0.212 for population [1] and [2], respectively. There was evidence of linkage disequilibrium in both populations, rejecting the null hypothesis of random mating as the predominant reproduction mode, a typical result of clonal populations. However, all isolates were genotypically different, an indirect evidence of recombination. We conclude that V. oleaginea populations in Uruguay present a mixed mode of reproduction, a characteristic of many pathogens that are difficult to control.

A Universally Primed-Polymerase Chain Reaction (UP-PCR) Marker to Discriminate Clonostachys rosea ACM941 from Related Strains.

Clonostachys rosea strain ACM941 is an effective biocontrol agent against several crop diseases including Fusarium head blight. In anticipation of its increased relevance going forward, the development of a reliable DNA-based molecular marker to track it is essential. Universally primed-PCR (UP-PCR) has been used successfully to differentiate other C. rosea strains. Herein, the development of a UP-PCR marker for ACM941 is described. A combination of two primers (AS15 and L45) produced a ~450 bp fragment that was unique to ACM941 compared to other commercial biocontrol agents. Primers subsequently designed based on the obtained fragment also produced a similarly unique band from ACM941 alone. BLAST analysis of the amplified sequence did not yield any homologous sequence in available online databases or within the closely related C. rosea IK726 and CBS125111 strains’ genomes. The specificity of this marker for ACM941 was validated against ten additional C. rosea strains isolated from Canada, with ACM941 producing the brightest band. Taken together, these results imply that the UP-PCR primers AS15 and L45 and the amplified fragment can be used to detect and monitor the ACM941 strain after its release into the environment.

Genetic diversity in Dactylonectria pauciseptata associated with black foot disease in New Zealand

The fungal genera Ilyonectria and Dactylonectria are causal agents of black foot disease affecting grapevines. A New Zealand survey of grapevines symptomatic for this disease was conducted in 2005, and 11 D. pauciseptata isolates were recovered. The incidence of this disease has increased over the last decade so a multi-gene approach was used to accurately identify these isolates and to determine whether they could infect grapevine roots in vitro. Eight of the original 11 D. pauciseptata isolates remained viable and were recovered from storage. DNA sequencing, universally primed polymerase chain reaction (UP-PCR) and a detached root assay were used to characterise these eight isolates. Phylogenetic analyses of ITS, β-tubulin, EF1-α and histone gene regions showed a monophyletic clade using the rRNA gene, EF1-α and histone gene, but paraphyletic based on β-tubulin. UP-PCR showed genetic diversity, with five major groups (P<0.01), with major groups concordant with the β-tubulin phylogeny. Pathogenicity tests with two common rootstocks showed that isolates produced lesions. Two distinct groups of D. pauciseptata were identified. Both groups are capable of infecting grapevine roots.

Identification and Genetic Diversity of Formae Speciales of Setosphaeria turcica in China.

Setosphaeria turcica is an important pathogen that causes northern leaf blight of corn and sorghum. In total, 188 isolates were isolated from diseased leaves of maize and sorghum in China. However, differences in morphology were not apparent among S. turcica isolates derived from different hosts. The formae speciales were identified as S. turcica. f. sp. zeae and S. turcica. f. sp. sorghi. Universally primed polymerase chain reaction (UP-PCR) analysis led to the conclusion that genetic differences were present among these formae speciales and divided these isolates into five clades. The isolates of each clade belonged to the same forma specialis, and different formae speciales were assigned to different clades. The results of the present study demonstrated that pathogenic specialization of the S. turcica isolate correlated more closely with genetic diversity than geographical distribution. Therefore, combined with assays for pathogenicity, UP-PCR analysis yields an enhanced understanding of the formae speciales of S. turcica.

Spatial distribution of Neofusicoccum species within a rootstock mother vine indicates potential infection pathways

Past studies have shown that the major source of infection of young grapevine plants by Botryosphaeriaceae fungi were through the use of infected rootstock and scion cuttings. To investigate the potential infection pathways of Botryosphaeriaceae species within a rootstock mother vine, three genotyping studies using universally-primed polymerase chain reaction (UP-PCR) were conducted with two Neofusicoccum species, namely N. luteum and N. parvum. The investigations identified genotypes of the fungal isolates in trunk and shoot infections of the same mother vine. Results showed that the trunk and shoot isolates from the same vine were of the same or different genotypes, suggesting multiple infections from different inoculum sources. This study further showed that the Neofusicoccum isolates recovered from the surfaces of the cuttings were of the same or different genotypes from those isolated from adjacent internal tissues, again suggesting multiple sources of external inoculum. Investigations into the spatial distribution of Botryosphaeriaceae fungi within an entire dormant cane also showed that multiple species and genotypes were distributed along the cane but most isolates were sited within the bark, being less frequently in the wood, which suggested that they were latent on surface tissues. Since some adjacent wood and bark infections were caused by the same genotypes, this indicated that wood infection may have originated from the bark. These fungi appear to cause latent infections in the bark of dormant cuttings which are used in plant propagation, thus providing an additional infection pathway for a disease that is known to show obvious symptoms only in older vineyards.

Genetic Diversity and Population Structure of Fusicladium effusum on Pecan in the United States.

Fusicladium effusum causes pecan scab, which is the most destructive disease of pecan orchards in the United States. Conidia of the pathogen are spread by rain splash and wind. The fungus is pathogenically diverse; yet there is no information on its genetic diversity or population genetics. Universally primed polymerase chain reaction (UP-PCR) was used to investigate the genetic diversity and population structure on a hierarchical sample of 194 isolates collected from 11 orchard locations from Florida to Texas, consisting of three to four isolates from each of five to six trees at each location. Genetic variation was high throughout the region, with all but nine of the multilocus haplotypes being unique. Nei's average gene diversity ranged from 0.083 for a population from Mississippi to 0.160 for a population from Kansas. An analysis of molecular variance of the hierarchically sampled populations found that the majority of the genetic variability (82.6%) occurred at the scale of the individual tree and only relatively small amounts among populations in trees from an orchard (5.0%) or within groups (i.e., orchard location populations) (12.5%). The results suggest little population differentiation in F. effusum in the southeastern United States, although φpt values of genetic distance for pairwise comparisons indicated some populations could be differentiated from others. There was evidence of linkage disequilibrium in certain populations, and the common occurrence of asexual reproduction in F. effusum could lead to measurable linkage disequilibrium under certain circumstances. However, the degree of genetic diversity and the scale over which diversity is distributed is evidence that F. effusum undergoes regular recombination despite no known sexual stage.

A comparison of UP‐PCR and RAPD markers to study genetic diversity of Fusicladium effusum (G. Winter), cause of pecan scab

SummaryFusicladium effusum infects pecan causing yield loss, but no information is available on the genetic diversity of F. effusum. Randomly amplified polymorphic DNAs (RAPDs) and universally primed polymerase chain reaction (UP‐PCR) were compared to detect polymorphisms on a group of 20 isolates of F. effusum from 11 geographical locations in the southeastern USA. Two tests (run 1 and 2) of both the RAPD and UP‐PCRs were conducted to assess the repeatability of the methods, and the markers scored on agarose gels. In addition, the UP‐PCR markers from run 1 were scored using an automated capillary system. Both RAPDs and UP‐PCR markers detected a high level of polymorphism among the scored markers (92 and 91% of RAPD markers, and 86 and 87% of manually scored UP‐PCR markers in run 1 and 2 were polymorphic, respectively; 93% of UP‐PCR markers were polymorphic when scored using the automated system). Unweighted paired group method of arithmetic averages (UPGMA) analysis showed both RAPDs and UP‐PCR markers individually identified each isolate, producing three groupings, but only the groupings based on run 1 and 2 of the UP‐PCR contained the same isolates. Bootstrap analysis based on the Dice coefficient produced phenograms from the UP‐PCR data with weak to moderate node support (≥54) for the primary branch, but no support for the RAPDs data (≤34). A Mantel test of runs 1 and 2 using RAPDs or UP‐PCR showed good agreement (r = 0.8761 and 0.8289, p < 0.0001), but poor agreement between RAPDs and UP‐PCR. UP‐PCR results based on the interisolate Dice coefficients showed a weak to strong association with distance. Based on these results, both RAPDs and UP‐PCR markers were capable of demonstrating polymorphisms and identifying relationships among isolates of F. effusum; however, UP‐PCR markers appear to be more reliable.

Atypical Microorganisms in Expressed Prostatic Secretion from Patients with Chronic Prostatitis/Chronic Pelvic Pain Syndrome: Microbiological Results from a Case-Control Study

Objectives: To investigate the microorganisms in expressed prostatic secretions (EPS) of both chronic prostatitis patients and normal young male adults and to determine which microorganisms are associated with the degree of intraprostatic inflammation. Methods: Specific polymerase chain reaction (PCR) technology was performed to confirm Neisseria gonorrhoeae, Mycobacterium tuberculosis, Mycoplasma and Chlamydia trachomatis as well as human papilloma virus (HPV), herpes simplex virus type 2 (HSV-2) and cytomegalovirus (CMV). Universal primer PCR technology was carried out to detect 16S bacteria rDNA, followed by cloning and sequencing of the entire 16S rDNA product. Results: The total number of bacteria and/or viruses detected by PCR assays was significantly associated with disease severity (p < 0.001). The white blood cell count and lecithin level was significantly correlated with the number of detected bacteria and/or viruses (p = 0.001 and p = 0.046, respectively). 17 bacterial isolates were identified from 14 EPS samples by 16S rDNA sequencing. Conclusions: Various microorganisms including Ureaplasma urealyticum, C. trachomatis, CMV, HPV and HSV-2 were identified in the EPS from patients with type III prostatitis. HPV infection may be associated with the degree of intraprostatic inflammation.

16S rRNA Gene-targeted TTGE in Determining Diversity of Gut Microbiota during Acute Diarrhoea and Convalescence

The human gut microbiota play a vital role in health and nutrition but are greatly modified during severe diarrhoea due to purging and pathogenic colonization. To understand the extent of loss during and after diarrhoea, faecal samples collected from children (n=21) suffering from acute diarrhoea and from their healthy siblings (n=9) were analyzed by 16S rRNA gene-targeted universal primer polymerase chain reaction (PCR), followed by temporal temperature gradient gel electrophoresis (TTGE). The gut microbiota decreased significantly as indicated by the number of TTGE bands at day 0 of acute diarrhoea [patients vs healthy siblings: 11±0.9 vs 21.8±1.1 (mean ± standard error), p<0.01]. The number of bands showed a steady increase from day 1 to day 7; however, it remained significantly less than that in healthy siblings (15±0.9, p<0.01). These results suggest that appropriate therapeutic and post-diarrhoeal nutritional intervention might be beneficial for the early microbial restoration and recovery.

Genetic and pathogenic diversity of Neofusicoccum parvum in New Zealand vineyards

Genetic diversity of 50 isolates of Neofusicoccum parvum, the predominant species of the Botryosphaeriaceae recovered from grapevines displaying symptoms of dieback and decline in New Zealand, was compared to that of isolates from Australia, South Africa, and California. The eight universally primed polymerase chain reaction (UP-PCR) primers distinguished 56 genotypes, with only four clonal pairs found. Seven main groups were identified in a neighbour-joining (NJ) tree with isolates from different regions and vineyards of New Zealand, Australia, and California distributed in different groups, indicating a high level of intra and intervineyard genetic variation. All of the South African isolates were positioned in a separate UP-PCR group, indicating that these isolates were less related to the other N. parvum isolates. When compared to fungi that reproduce sexually the genetic diversity and Shannon diversity indices were low (0.076–0.249; 0.109–0.367, respectively), genetic identity levels were high (0.76–0.95), and genetic distance levels were low (0.04–0.27). The large number of genotypes and the low number of clones in the New Zealand N. parvum populations may be explained by parasexual recombination as anastomosis was observed between nonself pairings. Pathogenicity tests using isolates from different UP-PCR groups inoculated onto either green shoots or 1-y-old grapevines detected virulence diversity, indicating intra and intervineyard variation between isolates, however, no correlation was detected between UP-PCR group and virulence.

Genetic variation inSpilocaea oleaginapopulations from New Zealand olive groves

Olive leaf spot caused by the fungus, Spilocaea oleagina, is the most important leaf disease of olives in many olive-growing regions worldwide with yield losses of up to 20%. The genetic structure of S. oleagina populations was investigated with universally primed-polymerase chain reaction (UP-PCR) techniques. Ninety-eight S. oleagina isolates were collected from 12 known and 4 unknown cultivars from olive groves in five New Zealand regions. UP-PCR profiles based on 159 markers were used to compute genetic distances between pairs of individuals. Low levels of gene and genotypic diversity were detected in all populations, with 76% of the loci being polymorphic and with Nei’s diversity indices ranging from 0.0234 to 0.1393. Analysis of molecular variance showed small but significant (P = 0.001) variations among regions, although most of the molecular variability (87%) was found within populations. Clustered analysis showed no evidence of grouping according to geographic origin of the isolates. The low level of genetic diversity found within and among populations indicates that reproduction for this fungus is predominantly by asexual means and that any effective control strategies are likely to be useful in all or most New Zealand olive groves.

Assessment of genetic diversity in isolates ofCiborinia camelliaeKohn from New Zealand and the United States of America

DNA fingerprinting with the universally primed polymerase chain reaction (UP-PCR) technique was used in an initial study to determine the degree of genetic diversity of Ciborinia camelliae populations within New Zealand and the United States of America. The UP-PCR band patterns from 27 isolates of C. camelliae collected from Wellington, Palmerston North, Wanganui and Christchurch in New Zealand and three isolates each from Georgia, Oregon and Virginia in the United States of America were analysed by two statistical methods. Cluster analysis indicated two distinct groupings, with isolates from New Zealand being less genetically diverse (9% polymorphic bands) than those from the United States of America (17% polymorphic bands). Canonical variates analysis showed that the majority (87%) of genetic diversity between locations was associated with the difference in UP-PCR band patterns between the two countries, with only 9% being associated with the genetic diversity within the countries. Therefore, for the isolates of C. camelliae examined, those from New Zealand were different from the small group of isolates obtained from the United States of America, but the genetic diversity in populations of C. camelliae within each country was low.

Evolutionary relationships within 'pygmaeus' group microphallids using genetic analysis and scanning electron microscopy.

There are four species of 'pygmaeus' microphallids, namely Microphallus pygmaeus, M. piriformes, M. pseudopygmaeus and M. triangulatus (Trematoda: Microphallidae) which are parasites of marine birds and their sporocysts give rise to transmissible metacercariae inside littoral gastropods (mostly littorines). Universally primed polymerase chain reaction (UP-PCR) showed no apparent pattern between genetic diversity of the metacercariae as estimated by genomic banding profiles and their geographic region or molluscan host species. At the same time UP-PCR product cross-hybridization showed that M. pseudopygmaeus and M. triangulatus are genetically very similar, indicating that these taxa represent one species complex. In contrast, M. pygmaeus and M. piriformes are genetically well separated from each other and also from the pseudopygmaeus-triangulatus complex. Scanning electron microscopy of ventral spines, and analyses of spine angles and the number of teeth per spine, showed that all species differed significantly from one another. It was concluded that M. piriformes represents the original western member of the 'pygmaeus' group. Microphallus pygmaeus probably diverged from M. piriformes as it progressively specialized for sea duck final hosts. Microphallus pseudopygmaeus and M. triangulatus diverged from each other and the piriformes-pygmaeus ancestral line relatively recently. Microphallus pseudopygmaeus specialized for adoption of a wide range of gastropod host species and M. triangulatus developed morpho-functional specialization associated with final host exploitation.

Low genetic variation detected in New Zealand populations of Phaeomoniella chlamydospora

Phaeomoniella chlamydospora is considered to be the causal agent of Petri disease. This disease causes decline of grapevines in most grape growing regions of the world. Genetic variation within 39 New Zealand isolates of Phaeomoniella chlamydospora was compared to six isolates from Italy using randomly amplified polymorphic DNA (RAPD), randomly amplified microsatellites (RAM), amplified fragment length polymorphism (AFLP), and universally primed polymerase chain reaction (UP-PCR). Using each method, genetic variation within New Zealand and Italian isolates of P. chlamydospora was shown to be low, with a maximum of seven genetic groups identified by each primer. The greatest amount of genetic variation was shown using AFLP analysis, with 21 different groups identified. RAPD, AFLP and UP-PCR primers detected inter-vineyard, intra-vineyard and intra-vine variation of New Zealand isolates. A subset of five New Zealand and one Italian isolate was further investigated using mycelial compatibility groups (MCGs). One MCG was identified, supporting low genetic variation within P. chlamydospora isolates. Low genetic variation within the New Zealand and the Italian populations suggests that asexual reproduction predominates, and the presence of intra-vineyard and intra-vine variation in New Zealand indicates that multiple introductions have occurred.

Biochemical and molecular characterization of Bacillus amyloliquefaciens, B. subtilis and B. pumilus isolates with distinct antagonistic potential against Xanthomonas campestris pv. campestris

Fifty‐one Bacillus isolates were characterized by fatty acid methyl ester (FAME) analysis; universal primer polymerase chain reaction (UP‐PCR) fingerprinting; production of secondary metabolites and antagonistic activity against Xanthomonas campestris pv. campestris (causal agent of black rot in cabbage) in vitro and in vivo. Based on FAME analysis and/or PCR fingerprinting, the isolates were clustered into three different groups, named as Bacillus amyloliquefaciens, B. subtilis and B. pumilus. Seed treatment with Bacillus spp. generally reduced germination of seeds and incidence of black rot, but no relationship was found between the results of in vitro and in vivo experiments. The B. amyloliquefaciens group contained isolates that were generally the most effective at reducing attack of black rot in vivo. The metabolic profiles of these isolates suggested that they produced surfactin, iturin, bacillomycine and/or azalomycin F. Isolates belonging to the B. subtilis group were mostly able to synthesize surfactin and arthrobactin. Surfactin, amphomycin, arthrobactin and valinomycin were generally found in culture extracts of isolates belonging to the B. pumilus group. No effect on growth of the pathogen was detected when the activity of filtered culture extracts and selected metabolites produced by the three different Bacillus species was tested in vitro against X. c. pv. campestris. However, inhibition was seen when bacterial liquid cultures were used. When the ability to colonize cabbage endophytically was examined for seven selected isolates with different antagonistic potential against black rot, it was found that the ability was related to the species and not to the antagonistic activity of the isolates.

Genetic relatedness of Trichoderma sect. Pachybasium species based on molecular approaches.

Molecular approaches including internal transcribed spacer (ITS) sequences of ribosomal DNA, universal primer polymerase chain reaction (UP-PCR) fingerprinting, and DNA-DNA hybridization were used to study the genetic relatedness of species within Trichoderma sect. Pachybasium. In the analysis of ITS and 5.8S sequences of ribosomal DNA, parsimony analysis demonstrated that forty-one strains were distributed into five main groups supported by high bootstrap values. The species of Trichoderma sect. Pachybasium were clustered into groups I, II, and IV, with the strains of Trichoderma fasculatum and Trichoderma strictipile forming a separate branch, an independent group V. Some species within each group showed nearly identical sequence differences (fewer than 1-3 bp). UP-PCR and DNA-DNA hybridization were further used to clarify the genetic relatedness of these species with highly similar ITS sequences. Highly similar or identical UP-PCR profiles and high values of DNA complementarity (>70%) were observed among some species, Trichoderma hamatum and Trichoderma pubescens; Trichoderma croceum, Trichoderma polysporum and Trichoderma album, Trichoderma crassum and Trichoderma flavofuscum; and Trichoderma strictipile and Trichoderma fasciculatum. Although every species can be differentiated morphologically, the species showed highly similar molecular characteristics in the above cases, indicating that they could be conspecific. However, in some cases (Trithoderma longipile, T. crassum and T. flavofuscum; Trichoderma fertile and Trichoderma minutisporum; Trichoderma tomentosum, Trichoderma inhamatum and Trichoderma harzianum) there were discriminative patterns of UP-PCR and (or) low levels (<50%) of DNA-DNA hybridization; even their ITS sequences were similar, suggesting a closely phylogenetic relationship.

Intraspecific Variation in Gamma-Radiation Resistance and Genomic Structure in the Filamentous Fungus Alternaria alternata: A Case Study of Strains Inhabiting Chernobyl Reactor No. 4

This is probably the first report on intraspecific variation in radiation resistance for filamentous fungi. It was revealed that natural (“field”) strains of the filamentous fungus Alternaria alternata are extremely variable in response to gamma-irradiation ranging from supersensitive to highly resistant to radiation. At the same time nearly all strains originating from the highly radiation-polluted reactor of the Chernobyl (Ukraine) Nuclear Power Plant possessed high radiation resistance. The genome structure of strains studied by universally primed polymerase chain reaction (UP-PCR) was found to be well conserved in “reactor” but not in “control” strains. The “reactor” strains appear to be genetically adapted to this high radiation habitat by means of selection, thus providing a natural source of genetically homogeneous fungal lineages.