Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA

The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.     

Mitochondrial DNA Sequence Divergence among Meloidogyne incognita,Romanomermis culicivorax,Ascaris suum,and Caenorhabditis elegans

Mitochondrial DNA sequences were obtained from the NADH dehydrogenase subunit 3 (ND3), large rRNA, and cytochrome b genes from Meloidogyne incognita and Romanomermis culicivorax. Both species show considerable genetic distance within these same genes when compared with Caenorhabditis elegans or Ascaris suum, two species previously analyzed. Caenorhabditis, Ascaris, and Meloidogyne were selected as representatives of three subclasses in the nematode class Secernentea: Rhabditia, Spiruria, and Diplogasteria, respectively. Romanomermis served as a representative out-group of the class Adenophorea. The divergence between the phytoparasitic lineage (represented by Meloidogyne) and the three other species is so great that virtually every variable position in these genes appears to have accumulated multiple mutations, obscuring the phylogenetic information obtainable from these comparisons. The 39 and 42% amino acid similarity between the M. incognita and C. elegans ND3 and cytochrome b coding sequences, respectively, are approximately the same as those of C. elegans-mouse comparisons for the same genes (26 and 44%). This discovery calls into question the feasibility of employing cloned C. elegans probes as reagents to isolate phytoparasitic nematode genes. The genetic distance between the phytoparasitic nematode lineage and C. elegans markedly contrasts with the 79% amino acid similarity between C. elegans and A. suum for the same sequences. The molecular data suggest that Caenorhabditis and Ascaris belong to the same subclass.     

Ascaris suum cytochrome b5, an adult-specific secretory protein reducing oxygen-avid ferric hemoglobin

The anaerobic parasitic nematode Ascaris suum has an oxygen-avid hemoglobin in the perienteric fluid, the biological function of which remains elusive. Here, we report that Ascaris cytochrome b₅ is expressed specifically in the intestinal parasitic stage and is secreted into the perienteric fluid, thus co-localizing with Ascaris hemoglobin. We also found that cytochrome b₅ reduces Ascaris non-functioning ferric methemoglobin more efficiently than mammalian methemoglobin. Furthermore, a computer graphics model of the electron transfer complex between Ascaris cytochrome b₅ and Ascaris hemoglobin strongly suggested that these two proteins are physiological redox partners. Nitric oxide has been reported to react easily with oxygen captured in hemoglobin to form nitrate, but not toxic free radicals, which may result in production of methemoglobin for the cytochrome b₅ to regenerate functional ferrous hemoglobin. Therefore, our findings suggest that Ascaris cytochrome b₅ is a key redox partner of Ascaris hemoglobin, which acts as an antioxidant.     

Comparative sequence analysis of citrate synthase and 18S ribosomal DNA from a wild and mutant strains of Aspergillus niger with various fungi

A mutation was induced in Aspergillus niger wild strain using ethidium bromide resulting in enhanced expression of citric acid by three folds and 112.42 mg/mL citric acid was produced under optimum conditions with 121.84 mg/mL of sugar utilization. Dendograms of 18S rDNA and citrate synthase from different fungi including sample strains were made to assess homology among different fungi and to study the correlation of citrate synthase gene with evolution of fungi. Subsequent comparative sequence analysis revealed strangeness between the citrate synthase and 18S rDNA phylogenetic trees. Furthermore, the citrate synthase movement suggests that the use of traditional marker molecule of 18S rDNA gives misleading information about the evolution of citrate synthase in different fungi as it has shown that citrate synthase gene transferred independently among different fungi having no evolutionary relationships. Random amplified polymorphic DNA (RAPD-PCR) analysis was also employed to study genetic variation between wild and mutant strains of A. niger and only 71.43% similarity was found between both the genomes. Keeping in view the importance of citric acid as a necessary constituent of various food preparations, synthetic biodegradable detergents and pharmaceuticals the enhanced production of citric acid by mutant derivative might provide significant boost in commercial scale viability of this useful product.

Abbreviations

CS - Citrate synthase, CA - Citric acid, RAPD - Random amplified polymorphic DNA, TAF - Total amplified fragments, PAF - Polymorphic amplified fragments, CAF - Common amplified fragments.     

Molecular Characterization of Meloidogyne christiei Golden and Kaplan, 1986 (Nematoda,Meloidogynidae) Topotype Population Infecting Turkey Oak (Quercus laevies) in Florida

Meloidogyne christiei isolated from turkey oak, Quercus laevies, from the type locality in Florida was characterized using isozyme profiles and ribosomal and mitochondrial gene sequences. The phenotype N1a detected from a single egg-laying female of M. christiei showed one very strong band of malate dehydrogenase (MDH) activity; however, no esterase (EST) activity was identified from macerate of one or even 20 females per well. Phylogenetic relationships within the genus Meloidogyne as inferred from Bayesian analysis of partial 18S ribosomal RNA (rRNA), D2-D3 of 28S rRNA, internal transcribed spacer (ITS) rRNA, and cytochrome oxidase subunit II (COII)-16S rRNA of mitochondrial DNA (mtDNA) gene fragments showed that M. christiei formed a separate lineage within the crown group of Meloidogyne and its relationships with any of three Meloidogyne clades were not resolved.     

Comparison of Sequences from the Ribosomal DNA Intergenic Region of Meloidogyne mayaguensis and Other Major Tropical Root-Knot Nematodes

The unusual arrangement of the 5S ribosomal gene within the intergenic sequence (IGS) of the ribosomal cistron, previously reported for Meloidogyne arenaria, was also found in the ribosomal DNA of two other economically important species of tropical root-knot nematodes, M, incognita and M. javanica. This arrangement also was found in M. hapla, which is important in temperate regions, and M. mayaguensis, a virulent species of concern in West Africa. Amplification of the region between the 5S and 18S genes by PCR yielded products of three different sizes such that M. mayaguensis could be readily differentiated from the other species in this study. This product can be amplified from single juveniles, females, or egg masses. The sequences obtained in this region for one line of each of M. incognita, M. arenaria, and M. javanica were very similar, reflecting the close relationships of these lineages. The M. mayaguensis sequence for this region had a number of small deletions and insertions of various sizes, including possible sequence duplications.     

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).     

A PCR Assay to Identify and Distinguish Single Juveniles of Meloidogyne hapla and M. chitwoodi

Random amplified polymorphic DNA (RAPD) bands that distinguish Meloidogyne hapla and M. chitwoodi from each other, and from other root-knot nematode species, were identified using a series of random octamer primers. The species-specific amplified DNA fragments were cloned and sequenced, and then the sequences were used to design 20-mer primer pairs that specifically amplified a DNA fragment from each species. Using the primer pairs, successful amplifications from single juveniles were readily attained. A mixture of four primers in a single PCR reaction mixture was shown to identify single juveniles of M. hapla and M. chitwoodi. To confirm specificity, the primers were used to amplify DNA from several isolates of M. hapla that originated from different crops and locations in North America and also from isolates of M. chitwoodi that differed in host range. In characterizing the M. hapla isolates, it was noted that there was a mitochondrial DNA polymorphism among isolates for cleavage by the restriction endonuclease DraI.     

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer''s protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit''s protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.     

Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum,Giardia lamblia,and Cyclospora cayetanensis,the Major Causes of Traveler’s Diarrhea

This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler’s diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×10³ oocysts for C. parvum, >1×10⁴ cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.     

Development of Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Azumiobodo hoyamushi (Kinetoplastea)

Ascidian soft tunic syndrome (AsSTS) caused by Azumiobodo hoyamushi (A. hoyamushi) is a serious aquaculture problem that results in mass mortality of ascidians. Accordingly, the early and accurate detection of A. hoyamushi would contribute substantially to disease management and prevention of transmission. Recently, the loop-mediated isothermal amplification (LAMP) method was adopted for clinical diagnosis of a range of infectious diseases. Here, the authors describe a rapid and efficient LAMP-based method targeting the 18S rDNA gene for detection of A. hoyamushi using ascidian DNA for the diagnosis of AsSTS. A. hoyamushi LAMP assay amplified the DNA of 0.01 parasites per reaction and detected A. hoyamushi in 10 ng of ascidian DNA. To validate A. hoyamushi 18S rDNA LAMP assays, AsSTS-suspected and non-diseased ascidians were examined by microscopy, PCR, and by using the LAMP assay. When PCR was used as a gold standard, the LAMP assay showed good agreement in terms of sensitivity, positive predictive value (PPV), and negative predictive value (NPV). In the present study, a LAMP assay based on directly heat-treated samples was found to be as efficient as DNA extraction using a commercial kit for detecting A. hoyamushi. Taken together, this study shows the devised A. hoyamushi LAMP assay could be used to diagnose AsSTS in a straightforward, sensitive, and specific manner, that it could be used for forecasting, surveillance, and quarantine of AsSTS.     

Development of species-specific primers for identification of Biomphalaria arabica,the intermediate host of Schistosoma mansoni in Saudi Arabia

Schistosoma mansoni is mediated through the intermediate host Biomphalaria arabica which lives in Saudi Arabia. Molecular characterization and identification of this intermediate host are important for epidemiological studies of schistosomiasis. The present work aimed to determine the molecular variations among the populations of B. arabica found in Southern part of Saudi Arabia, and to develop species-specific primers for identification of these snails as a first step in the development of multiplex PCR for simultaneously identifying the snails and diagnosing its infections in a single step. Five populations of Saudi B. arabica snails were collected from freshwater bodies. Three populations were collected from Asser and two populations were collected from AL-Baha. Genomic DNA was extracted from snails and was amplified using five different RAPD–PCR primers. The banding patterns of amplified materials by primers P1 and P5 were identical in all populations. However, the rest primers displayed intra-specific differences among populations with variable degrees. Largest sizes of RAPD–PCR products were cloned into TA cloning vector as a preparatory step for DNA sequence analysis. After sequencing, similarity searches of obtained DNA sequences revealed that there are no similar sequences submitted to genebank data bases and its associated banks. The results obtained will be helpful in the development of simultaneous identification of B. arabica snails and diagnosis of S. mansoni infection within it in a single step by an implementation of multiplex PCR.     

Secondary structure models of 18S and 28S rRNAs of the true bugs based on complete rDNA sequences of Eurydema maracandica Oshanin, 1871 (Heteroptera,?Pentatomidae)

The sequences of 18S and 28S rDNAs have been used as molecular markers to resolve phylogenetic relationships of Heteroptera for two decades. The complete sequences of 18S rDNAs have been used in many studies, while in most studies only partial sequences of 28S rDNAs have been used due to technical difficulties of amplifying the complete lengths. In this study, we amplified the complete 18S and 28S rDNA sequences of Eurydema maracandica Oshanin, 1871, and reconstructed the secondary structure models of the corresponding rRNAs. In addition, and more importantly, all of the length variable regions of 18S rRNA were compared among 37 families of Heteroptera based on 140 sequences, and the D3 region of 28S rRNA was compared among 51 families based on 84 sequences. It was found that 8 length variable regions could potentially serve as molecular synapomorphies for some monophyletic groups. Therefore discoveries of more molecular synapomorphies for specific clades can be anticipated from amplification of complete 18S and 28S rDNAs of more representatives of Heteroptera.     

Genetic Diversity of Schistosoma haematobium Eggs Isolated from Human Urine in Sudan

The genetic diversity of Schistosoma haematobium remains largely unstudied in comparison to that of Schistosoma mansoni. To characterize the extent of genetic diversity in S. haematobium among its definitive host (humans), we collected S. haematobium eggs from the urine of 73 infected schoolchildren at 5 primary schools in White Nile State, Sudan, and then performed a randomly amplified polymorphic DNA marker ITS2 by PCR-RFLP analysis. Among 73 S. haematobium egg-positive cases, 13 were selected based on the presence of the S. haematobium satellite markers A4 and B2 in their genomic DNA, and used for RFLP analysis. The 13 samples were subjected to an RFLP analysis of the S. haematobium ITS2 region; however, there was no variation in size among the fragments. Compared to the ITS2 sequences obtained for S. haematobium from Kenya, the nucleotide sequences of the ITS2 regions of S. haematobium from 4 areas in Sudan were consistent with those from Kenya (> 99%). In this study, we demonstrate for the first time that most of the S. haematobium population in Sudan consists of a pan-African S. haematobium genotype; however, we also report the discovery of Kenyan strain inflow into White Nile, Sudan.     

Phylogenetic analysis of the genus Sorghum based on combined sequence data from cpDNA regions and ITS generate well-supported trees with two major lineages

Background and Aims

Wild Sorghum species provide novel traits for both biotic and abiotic stress resistance and yield for the improvement of cultivated sorghum. A better understanding of the phylogeny in the genus Sorghum will enhance use of the valuable agronomic traits found in wild sorghum.

Methods

Four regions of chloroplast DNA (cpDNA; psbZ-trnG, trnY-trnD, trnY-psbM and trnT-trnL) and the internal transcribed spacer (ITS) of nuclear ribosomal DNA were used to analyse the phylogeny of sorghum based on maximum-parsimony analyses.

Key Results

Parsimony analyses of the ITS and cpDNA regions as separate or combined sequence datasets formed trees with strong bootstrap support with two lineages: the Eu-sorghum species S. laxiflorum and S. macrospermum in one and Stiposorghum and Para-sorghum in the other. Within Eu-sorghum, S. bicolor-3, -11 and -14 originating from southern Africa form a distinct clade. S. bicolor-2, originally from Yemen, is distantly related to other S. bicolor accessions.

Conclusions

Eu-sorghum species are more closely related to S. macrospermum and S. laxiflorum than to any other Australian wild Sorghum species. S. macrospermum and S. laxiflorum are so closely related that it is inappropriate to classify them in separate sections. S. almum is closely associated with S. bicolor, suggesting that the latter is the maternal parent of the former given that cpDNA is maternally inherited in angiosperms. S. bicolor-3, -11 and -14, from southern Africa, are closely related to each other but distantly related to S. bicolor-2.     

Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii,Ae. triuncialis and Ae. cylindrica,and their molecular organization

Background and Aims Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U^tU^tC^tC^t) and Ae. cylindrica (D^cD^cC^cC^c) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting.Methods The flow karyotypes obtained after the analysis of 4'',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA.Key Results FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C^t, T6U^tS.6U^tL-5C^tL, 1C^c and 5D^c could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2–5. This identified a partial wheat–C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C–2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected.Conclusions The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.     

Detection and Identification of Bursaphelenchus Species with DNA Fingerprinting and Polymerase Chain Reaction

We have evaluated the potential of DNA-based methods to identify and differentiate Bursaphelenchus spp. and isolates. The isolation of a DNA probe, designated X14, and development of a DNA fingerprinting method for the identification and differentiation of Bursaphelenchus species and strains is described. Polymerase chain reaction (PCR) amplification of DNA isolated from Bursaphelenchus species using two primers derived from the sequence of the cloned repetitive DNA fragment X14 resulted in multiple band profiles. A 4-kb fragment thus amplified from B. xylophilus DNA was not amplified from B. mucronatus or B. fraudulentus DNA. In addition to this fragment, several other fragments are amplified from the three species. The banding patterns obtained allowed species identification and may have value in determining taxonomic affinities.     

Phylogenetic Relationships Among Selected Heteroderoidea Based on 18S and ITS Ribosomal DNA

In a study of relationships among selected cyst-forming and noncyst-forming species of Heteroderoidea, combined sequences comprised of DNA from part of the conserved 18S ribosomal RNA gene (rDNA) plus the complete ITS rDNA segment were more similar to analyses based on the ITS data alone than to analyses based on the 18S data alone. One of the two noncyst-forming species, Ekphymatodera thomasoni, grouped with cyst-forming species of Heteroderoidea. Bilobodera flexa, also a noncyst-forming species, was separated from all the other taxa by a long branch. Afenestrata koreana, with a weakly sclerotized cyst, grouped closely with H. bifenestra. These observations suggest that phylogenetic analyses using molecular data may aid in our understanding of the evolution of cyst formation in nematodes, including the possibility of secondary loss. The usefulness of molecular phylogenetic analyses in nematodes may depend more on the particular selection of taxa than on mere addition of data from additional genes.     

Identification of parasite DNA in common bile duct stones by PCR and DNA sequencing

We attempted to identify parasite DNA in the biliary stones of humans via PCR and DNA sequencing. Genomic DNA was isolated from each of 15 common bile duct (CBD) stones and 5 gallbladder (GB) stones. The patients who had the CBD stones suffered from cholangitis, and the patients with GB stones showed acute cholecystitis, respectively. The 28S and 18S rDNA genes were amplified successfully from 3 and/or 1 common bile duct stone samples, and then cloned and sequenced. The 28S and 18S rDNA sequences were highly conserved among isolates. Identity of the obtained 28S D1 rDNA with that of Clonorchis sinensis was higher than 97.6%, and identity of the 18S rDNA with that of other Ascarididae was 97.9%. Almost no intra-specific variations were detected in the 28S and 18S rDNA with the exception of a few nucleotide variations, i.e., substitution and deletion. These findings suggest that C. sinensis and Ascaris lumbricoides may be related with the biliary stone formation and development.