rDNA ITS Region Sequence Research Articles

Morpho-molecular and Pathogenic Variability of Wilt of Lentil from Indo-Gangetic Plains of India

Background: Fusarium wilt in lentil (Lens culinaris L.) infected by Fusarium oxysporum is considered as one of the major biotic factors for low productivity in lentil. The present study was conducted to determine nine Fusarium isolates (W1-W8 and W10) which were isolated from different lentil growing fields of West Bengal, India and were characterized for their physio-morphological, biochemical and molecular traits in vitro. Methods: Nine Fusarium infected lentil plant samples were collected from 9 different places of West Bengal. Pathogen was isolated from lentil samples using PDA media followed by cultural, morphological, pathogenic and molecular variability studies for all the isolates of Fusarium. Type of pigmentation, sporulation, conidia size of each isolate was recorded by observing culture plate after complete growth of the mycelium. Pathogenicity test were done to check the aggressiveness of all the 9 isolates of Fusarium in three varieties of lentil (Maitri, HUL-57 and BM-6). The isolated fungal DNA was amplified with specific primer pair, Forward primer ITS 1 and Reverse primer ITS 4. Result: Morphological assessment of 9 isolates showed distinct identification characters and variability. The mycelia of all the isolates were septate, profusely branched with differential pigmentation, texture and margin.All the Fusarium isolates produced micro-and macro-conidia in pure culture within seven days after inoculation and showed different morphological characters in pathogenicity test, variety Maitri showed better germination (100% in W2, W4, W6, W7, W8 and W10) and less disease severity against most of the strains (75% in W1) as compared to other two varieties against and hence variety Maitri taken into consideration for greenhouse and field trials. Furthermore, identification of molecular variability was performed by sequence analysis of rDNA-ITS region of each isolate and sequencing of rDNA-ITS region supported the morphological study and confirmed the associated fungi at species level.

Damping-off of black ginger (<i>Kaempferia parviflora</i>) caused by <i>Pythium myriotylum</i> in Japan

Damping-off of black ginger (Kaempferia parviflora) seedlings was observed in Kyoto Prefecture in 2018. Fungal-like isolates were readily obtained from infected parts of tubers. These isolates caused similar symptoms after a soil drench of potted plants with individual isolates, which were identified as Pythium myriotylum based on morphological characteristics and sequences of rDNA ITS regions and coxI gene. This is the first report of a disease of black ginger caused by P. myriotylum in Japan, and we propose the Japanese name “tachigare-byou” (damping-off) of black ginger.

<p><strong>A new species of <em>Bunium</em> (Apiaceae: Apioideae) from Central Anatolia in Turkey: <em>Bunium sivasicum</em></strong></p>

A new species of Bunium from Central Anatolia (Turkey), Bunium sivasicum (Apiaceae), is described and illustrated. The taxonomic affinities of the new species are determined by detailed morphological investigation and molecular phylogenetic study. Nuclear rDNA-ITS region sequences are used to reveal the phylogenetic relationship of the new species. It belongs to the sect. Elegantia and is related to Bunium simplex, B elegans and B. verruculosum. The differences between the new species and its allies are discussed. Pollen characteristics and fruit surface features of the new species are investigated. The anatomical structure of the fruit is given in detail. In addition, the geographical distributions of the Bunium sivasicum and related taxa in Anatolia are also mapped.

Molecular authentication and differentiation of Dendrobium species by rDNA ITS region sequence analysis

Owing to their significant medicinal and edible values, the natural Dendrobium species have underdone over-collection and habitat destruction, and cultivated species emerged for candidates. However, these Dendrobium plants are similar in shape to be easily confused, leading to extreme difficulties for identification based on their morphological and chemical features. In this study, the rDNA ITS region sequence analysis was developed for rapid and accurate identification of thirteen wild and cultivated Dendrobium species belonging to two sections Formosae and Chrysotoxae. By cloning and sequencing the rDNA ITS region genes from 13 Dendrobium species, the phylogenetic relationships among them were analyzed. Results showed that the variation of the ITS region, together with the lengths and Guanine and Cytosine contents of ITS, 5.8s rDNA, ITS1 and ITS2 sequences occurred in the tested Dendrobium species, and which from section Chrysotoxae was higher than that from section Formsae. Phylogenetic analysis based on neighbor-joining and maximum p-arsimony trees indicated that the Dendrobium species of sections Formosae and Chrysotoxae could be well divided into two groups. A majority of Dendrobium species exhibited distinctive ITS2 secondary structures, while for those with close genetic relationships were similar. Therefore, the ITS2 region sequence analysis is simple, quick, and highly reliable that can be used as an effective tool for molecular identification and classification, as well as the reconstruction of the phylogeny of wild and cultivated Dendrobium species belonging to different sections.

Molecular phylogeny and bioprospecting of Endolichenic Fungi (ELF) inhabiting in the lichens collected from a mangrove ecosystem in Sri Lanka.

Endolichenic fungi (ELF) are unexplored group of organisms as a source for the production of bioactive secondary metabolites with radical scavenging activity, antilipase and amylase inhibitory activities. Endolichenic fungi in lichens collected from mangrove or mangrove associated plants are least known for their fungal diversity and potential to produce bioactive compounds. A total of 171 ELF strains were isolated from the lichens collected from mangrove and mangrove associated plants in Puttalam lagoon. Out of this collection, 70 isolates were identified using rDNA-ITS region sequence homology to the GenBank accessions and a phylogenetic analysis was performed. Commonly isolated genera of ELF from lichens were Aspergillus, Byssochlamys, Talaromyces, Diaporthe, Phomopsis, Endomelanconiopsis, Schizophyllum, Cerrena, Trichoderma, Xylaria, Hypoxylon, Daldinia, Preussia, Sordaria, Neurospora, and Lasiodiplodia. In the present study, the effectiveness of ethyl acetate extracts of the ELF isolates were investigated against antioxidant activity, antilipase activity and α-amylase inhibition activity in in-vitro conditions. The results revealed that the extracts of Daldinia eschscholtzii, Diaporthe musigena and Sordaria sp. had the highest radical scavenging activity with smaller IC50 values (25 μg/mL to 31 μg/mL) compared to the IC50 values of BHT (76.50±1.47 μg/mL). Antilipase assay revealed that 13 extracts from ELF showed promising antiobesity activity ranged between 25% to 40%. Amylase inhibitory assay indicated that the test extracts do not contain antidiabetic secondary metabolites.

Molecular Identification of Endophytic Fungi from Bark of Raru (Cotylelobium melanoxylon) that Produce the Antibacterial Compounds

<p class="IsiAbstrakIndo"><span lang="EN-GB">The Dipterocarpaceae plant, raru (</span><em><span lang="EN-GB">Cotylelobium melanoxylon</span></em><span lang="EN-GB">) is widespread in Southeast Asia. The bark of raru has been used by local communities in North Sumatera as antidiarrheal drugs due to its antibacterial compounds. The antibacterial activity of the </span><em><span lang="EN-GB">raru’s</span></em><span lang="EN-GB"> bark is induced by endophytic fungi that live in the region of the bark. This study aimed to identify the endophytic fungi-producer of antibacterial compounds in the bark of raru (</span><em><span lang="EN-GB">C. melanoxylon</span></em><span lang="EN-GB">) by means of molecular analysis. In general, endophytic fungi have the ability to inhibit the growth of pathogenic bacteria. Thirty-eight isolates of endophytic fungi were isolated from the bark of raru. Selection of isolates for antibacterial activity against </span><em><span lang="EN-GB">Escherichia coli</span></em><span lang="EN-GB"> ATCC 35218 and </span><em><span lang="EN-GB">Staphylococcus aureus</span></em><span lang="EN-GB"> ATCC 25923 used the dual culture assay. Selection using the dual culture assay yielded 6 endophytic fungal isolates that have the ability to inhibit the growth of test bacteria. EF10A sample was the most powerful isolate inhibiting the growth of both bacteria test. Those six bacteria molecularly identified used a sequence generated from ITS rDNA region. Based on rDNA ITS region sequences, isolate, the producers of the antibacterial compound were identified as </span><em><span lang="EN-GB">Talaromyces cellulolyticus, Penicillium purpurogenum, Aspergillus</span></em><span lang="EN-GB"> sp., </span><em><span lang="EN-GB">Trichoderma harzianum</span></em><span lang="EN-GB">, and </span><em><span lang="EN-GB">Aspergillus orizae</span></em><span lang="EN-GB">. The results of this study can be used by researchers to explore more potential endophytic fungi in raru plants (</span><em><span lang="EN-GB">C. melanoxylon</span></em><span lang="EN-GB">) as a source of medicine. The data obtained need to be supported by further research to isolate the bioactive compounds that can inhibit the growth of microbial pathogens.</span></p>

SHORT REVIEW ON DNA-FINGERPRINTING AND STANDARDIZATION OF HERBAL DRUGS

Medicinal plants play a key role in world health. In spite of new advances developed in modern medicines, herbal formulations are extensively used as therapeutic agents for treatment of several diseases. Herbal drug technology is used for converting botanicals materials into medicines. Standardization is a process of evaluating the quality & purity of crude drug by various parameters like morphological, microscopical, physical, chemical &biological observation. Fingerprint analysis approach is the most important in quality control of herbal medicines because of its accuracy and reliability. DNA fingerprinting is defined as the application of genetic/ molecular marker techniques which is used to identify cultivars. Fingerprinting is a process as it determines the concentrations of chemical substances in an herb by several methods like RFLP, AFLP, RAPD, SSR sequencing of rDNA-ITS region and DNA bar-coding. Development of fingerprints based on DNA markers is necessary for proper identification and standardization of plant species.

Characterization of a New Anastomosis Group (AG-W) of Binucleate Rhizoctonia, Causal Agent for Potato Stem Canker.

Two binucleate Rhizoctonia (BNR) isolates were recovered from potato cankered stems in Heilongjiang Province, northeastern China. Their cultural appearance on potato dextrose agar remained whitish as the cultures aged. White monilioid cells formed in the fluffy aerial hyphae, whereas no sclerotia appeared during the incubation. The two isolates could anastomose with each other, but they failed to anastomose with reference strains of BNR from AG-A to AG-Q, and AG-U. Analyses of restriction fragment length polymorphism (RFLP) of internal transcribed spacer of ribosomal DNA (rDNA-ITS) regions confirmed that these two isolates differed from the reference strains. The phylogenetic tree based on the sequences of rDNA-ITS regions showed that they were located in a distinct clade from other BNR AGs. These collective results suggested that the isolates recovered from potato in this study belonged to a new BNR AG designated as AG-W. Pathogenicity tests under glasshouse conditions revealed that both isolates were able to cause brown, dry, and slightly sunken lesions on potato subterranean stems. To our knowledge, this is the first report of the AG-W causing potato disease in China as well as worldwide.

First Report of Pythium Root and Stalk Rot of Forage Corn Caused by Pythium arrhenomanes in Japan.

Corn (Zea mays L.) is the most important forage crop in Japan. It was cultivated on 92,000 ha in 2011 and was mainly used as whole crop silage for cattle feed. In September 2009, a root and stalk rot disease was detected on corn plants cultivated in Tochigi, located in the central region of Japan. The symptoms of the disease included wilting of whole plants after the R5 (dent) stage (2) with drooping ears. Roots turned black and their number decreased. Further, the stalks became hollow and soft and harbored white hyphae. This tissue deterioration made machine harvest difficult. We obtained seven isolates of a Pythium-like organism by single hypha isolation from surface-sterilized pieces of diseased roots and stems on water agar and deposited one of the isolates at the NIAS genebank, Japan, under the accession no. MAFF511547. The isolate was grown in the dark on V8 juice agar medium for 10 days to produce oogonia. The oogonia were globose, light brown to yellow, smooth, 23.9 to 30.5 μm in size, and had 1 to 8 antheridia. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick. The morphology of the isolates was similar to that of Pythium arrhenomanes Drechsler and consistent with the species description (3). We analyzed the rDNA-ITS region sequences of the isolate as described by Kageyama et al. (1). The sequence (GenBank Accession No. AB903904) showed 99.1% (783/790 bp) similarity with that of P. arrhenomanes (AY598628). On the basis of morphological and rDNA sequence similarities, we identified the isolates obtained from corn as P. arrhenomanes. The pathogenicity of the isolate was confirmed by planting corn seedlings of the commercial Pioneer Brand hybrid 36B08 immediately after germination in five replicate pots containing soil mixed with 5% boiled barley grain by weight, incubated with or without the isolate for 7 days. After 10 days of incubation in a greenhouse at 20 to 25°C, only the inoculated plants exhibited symptoms of root and stalk rot. Since the inoculated organism was readily re-isolated from the diseased stems and roots, the pathogenicity of the isolate was confirmed. For field observation, the same hybrid of forage corn was sown in the fields in Nasushiobara, Tochigi, on 16 May 2011. The hybrid was sown in a row of 2 m, with 20 seeds planted at a distance of 10 cm with two replicates. For inoculum, the isolate was cultured on 5-cm-long wooden toothpicks, previously soaked in potato dextrose broth and placed on a V8 agar plate for 7 days at 25°C in the dark until covered by hyphae. The toothpicks were pierced into wounds made on the stems of corn plants, approximately 10 cm above the ground, using a thin iron needle. The wounds were about 2 mm in diameter and 2 cm deep. Field inoculation was conducted in late July at the R1 (silking) growth stage. Disease symptoms were observed in mid-September at R5, and only those plants that were inoculated with the toothpicks harboring the hyphae exhibited the typical stem rot symptoms. To our knowledge, this is the first report of root and stalk rot caused by P. arrhenomanes in forage corn in Japan.

First report of the protozoan parasite Perkinsus marinus in South America, infecting mangrove oysters Crassostrea rhizophorae from the Paraíba River (NE, Brazil)

The present work aimed to study the infection by Perkinsus sp. in the mangrove oysters Crassostrea rhizophorae from the estuary of the Paraíba River (Paraíba State, Brazil). Perkinsosis was detected by incubation of oyster gill pieces in Ray’s fluid thioglycollate medium. The monthly prevalence values were all above 70%, thus infection was not likely to be a transient event. Perkinsus sp. parasites isolated from eight oysters were propagated in vitro. PCR–RFLP analysis of in vitro cultured cells as well as the sequences of the rDNA ITS region allowed the identification of the in vitro propagated parasites as Perkinsus marinus. Phylogenetic analyses using rDNA ITS region sequences strongly supported the Perkinsus sp. from Paraíba in a monophyletic group with P. marinus. Thus, the results confirmed the species affiliation of Paraíba Perkinsus sp. as P. marinus. This is the first report of P. marinus in Brazil and South America and the first report of P. marinus naturally infecting C. rhizophorae.

Diversity and distribution of fungal communities in lakes of Antarctica

This study assessed the diversity and distribution of filamentous fungi obtained from water sampled from six lakes in the Antarctic Peninsula. One hundred and twenty-eight fungal isolates were purified and identified by analysis of nuclear rDNA ITS region sequences as belonging to 31 fungal different operational taxonomic units (OTUs). The most frequently isolated fungi were Geomyces pannorum and Mortierella sp.; these species occurred in six and three of the lakes sampled, respectively, and displayed the highest total colony-forming unit per L. Different species that have not been found to these lakes and/or had adapted to cold conditions were found. In general, the fungal community displayed low richness and high dominance indices. The species Cadophora cf. luteo-olivacea, Cadophora malorum, Davidiella tassiana, G.pannorum, Mortierella cf. alpina and Thelebolus cf. microsporus that were found in the lakes in question were also previously found in other cold ecosystems, such as Arctic, temperate and Alpine regions. The results of this study suggest the presence of an interesting aquatic fungal web, including symbionts, weak and strong saprophytes and parasite/pathogen fungal species. This aquatic web fungal may be a useful community model for further ecological and evolutionary studies of extreme habitats.

Identification of the medicinal plant Dipsacus asperoides from three other species in genus Dipsacus (Dipsaceae) by internal transcribed spacer of ribosomal deoxyribonucleic acid (rDNA ITS)

The internal transcribed spacer of ribosomal deoxyribonucleic acid (rDNA ITS including ITS1, 5.8S, and ITS2) of six populations of Dipsacus asperoides and two adulterants (Dipsacus japonicus and Dipsacus chinensis), were sequenced by polymerase chain reaction (PCR) products sequencing method and a phylogenetic tree was constructed by neighbor-joining method with Mega 3.1 software. The sequences of rDNA ITS region ranged from 596 to 602 bp. In comparison to the sequences among intraspecies of D. asperoides, variation of the rDNA ITS was significant among D. asperoides and its adulterants. Together with the reported rDNA ITS regions of Dipsacus mitis in GenBank (Accession No. AY236187), total of 122 variable sites and 29 informative sites were present among these interspecies. Notably, 14 of the total 29 informative sites were species-characteristic traits. Such a differentiation in the ITS sequence, could provide molecular markers to distinguish D. asperoides from the adulterants at the DNA level and to complement conventional morphological methods. And the phylogenetic analysis was conducted to obtain the information on variation and evolution of the four species. Based on sequence divergence and phylogenetic pattern, the results illustrated that D. asperoides was more closely related to D. mitis than to D. chinensis and to D. japonicus. Key words: Dipsacus, Dipsacus asperoides, phylogenetic analysis, internal transcribed spacer of ribosomal deoxyribonucleic acid (rDNA ITS).

Ceratocystis ficicola sp. nov., a causal fungus of fig canker in Japan

The causal fungus of Ceratocystis canker of fig in Japan is described as Ceratocystis ficicola sp. nov. This species is characterized by galeated ascospores and is similar to Ceratocystis fimbriata sensu lato. However, the perithecia of the new species are much larger than those of C. fimbriata. The fungus grows more rapidly than C. fimbriata sensu stricto at 25°C but more slowly at 30°C. Molecular analysis of the nucleotide sequences of rDNA ITS regions showed that C. ficicola is phylogenetically placed in the clade of C. fimbriata s. l. but is easily distinguishable from other species of C. fimbriata s. l.

Development of Species-Specific PCR Primers and Sensitive Detection of the Tylenchulus semipenetrans in China

Tylenchulus semipenetrans is the most economically important and widespread nematode pest of citrus in China. rDNA-ITS of 14 populations of T. semipenetrans which were collected from different citrus groves or Chinese fir ( Cunninghamia lanceolata) plantations in China were amplified and sequenced. The species-specific primers were designed for the first time to diagnosis T. semipenetrans based on the sequences of rDNA-ITS regions of geographic population above. The primers were sensitive to amplify the expected band size (297 bp) from DNA template of a single second-stage juvenile (J2) or different life stages of T. semipenetrans. No specific band was amplified from 15 non-target nematode species which were commonly found in citrus groves. Specificity and reliability of the primers were validated by further PCR amplification of 16 extra populations of T. semipenetrans collected from 4 provinces of China. The primers successfully detected a single J2 of T. semipenetrans within a whole nematode community comprising a large numbers of non-target nematode. The developed diagnostic technique can be used for accurate identification of T. semipenetrans and also as a decision tool for nematode management for citrus or Chinese fir in China.

Morphological and molecular analysis of genetic variability within isolates of Corynespora cassiicola from different hosts

Twenty-two isolates of Corynespora cassiicola obtained from cucumber, papaya, eggplant, tomato, bean, Vigna, sesame and Hevea rubber (Hevea brasiliensis) were analysed by morphological features, the differences of the ribosomal DNA internal transcribed spacer (rDNA-ITS) region sequence and the inter simple sequence repeat (ISSR) technique. Variability of morphological features was observed among the isolates. Pathogenicity tests showed that isolates from different hosts attacked Hevea rubber. Sequences of two outgroup taxa, C. proliferata and C. citricola, were downloaded from GenBank. The phylogenetic trees were constructed by using the rDNA-ITS region sequences from 24 Corynespora spp. isolates. In this analysis, the 24 sequences grouped into two clusters (A and B). Cluster A consists of sequences from all isolates of C. cassiicola; whereas cluster B consists of the two outgroup taxa, C. proliferata and C. citricola. However, the ITS region is conservative, and is not fit for studying differences among isolates. A total of 114 DNA fragments was amplified with 16 ISSR primers, among which 102 were polymorphic (89.5%). A dendrogram was created by the unweighted pair-group method with arithmetic averaging (UPGMA) analysis, and 22 isolates grouped into three clusters (C, D and E). Cluster C is composed of all of the Hevea rubber isolates, whereas cluster D is composed of nine isolates: four from papaya, five from cucumber, eggplant, bean, vigna and sesame. Cluster E is composed of two isolates from cucumber and tomato. These analyses showed that the genetic diversity was very rich among the tested isolates. There are no correlations between the morphological characteristics or rDNA-ITS region sequences of the 22 isolates and their host or geographical origin, but there is a link between ISSR clusters and their host origins. ISSR markers appear to be useful for intra-species population study in C. cassiicola.

<i>Ceratocystis ficicola</i> sp. nov., a causal fungus of fig canker in Japan

The causal fungus of Ceratocystis canker of fig in Japan is described as Ceratocystis ficicola sp. nov. This species is characterized by galeated ascospores and is similar to Ceratocystis fimbriata sensu lato. However, the perithecia of the new species are much larger than those of C. fimbriata. The fungus grows more rapidly than C. fimbriata sensu stricto at 25°C but more slowly at 30°C. Molecular analysis of the nucleotide sequences of rDNA ITS regions showed that C. ficicola is phylogenetically placed in the clade of C. fimbriata s. l. but is easily distinguishable from other species of C. fimbriata s. l.

ナス黒枯病菌の再同定

The pathogen of black blight of eggplant (Solanum melongena L.) was previously described as Corynespora melongenae Takimoto in Japan, but there was no Latin description of the fungus. We collected the fungus again in Shimane and Kochi prefectures, the western regions of Japan and reidentified them as C. cassiicola based on the morphological characteristics and sequences of rDNA-ITS regions after confirming their pathogenicity on eggplant. We propose to replace C. melongenae Takimoto nom. inval. with C. cassiicola as the pathogen of black blight in eggplant in Japan.

Molecular Identification of Fitzroya Cupressoides, Sequoia Sempervirens, and Thuja Plicata Wood Using Taxon-Specific RDNA-ITS Primers

The nuclear ribosomal DNA internal transcribed spacer (rDNA-ITS) region was PCR amplified and sequenced from the wood of three specimens of Fitzroya cupressoides, nine specimens of Sequoia sempervirens, and ten specimens of Thuja plicata. The full lengths of the ITS regions are 1110 bp for F. cupressoides, 1096 bp for S. sempervirens, and 1138 bp for T. plicata, and thus in the range of 975 bp to 1125 bp which is reported for members of the Cupressaceae. Length variation of ITS regions is due to differences in the length of the spacer region ITS1. Intraspecific variations of the sequences of rDNA-ITS regions were one bp in F. cupressoides, and 18 bp in S. sempervirens and T. plicata. Based on the interspecific sequence divergence of the ITS region, taxon-specific primers were designedfor the detection of F. cupressoides, S. sempervirens and T. plicata. The primer sequences were selected from the highly divergent ITS1 spacer. The specificity of the primers was checked by lengths and sequence of the amplicons, and the primers detected the target organism, solely, across 40 conifer species. Our data establish the molecular basis for DNA-based wood identification in these species.

Molecular Genetic Characterization of Olpidium virulentus Isolates Associated with Big-Vein Diseased Lettuce Plants.

Lettuce plants showing symptoms of lettuce big-vein disease were collected from fields in the Perth Metropolitan region of southwest Australia. When root extracts from each plant were tested by polymerase chain reaction (PCR) using primers specific to the rDNA internal transcribed spacer (ITS) region of Olpidium brassicae and O. virulentus, only O. virulentus was detected in each of them. The nucleotide sequences of the complete rDNA ITS regions of isolates from five of the root samples and 10 isolates of O. virulentus from Europe and Japan showed 97.9 to 100% identities. However, with the six O. brassicae isolates, their identities were only 76.9 to 79.4%. On phylogenetic analysis of the complete rDNA-ITS region sequences of the five Australian isolates and 10 others, the Australian isolates fitted within two clades of O. virulentus (I and II), and within clade I into two of its four subclades (Ia and Id). Japanese isolates had greatest sequence diversity fitting into both clades and into all of clade I subclades except Ib, while European isolates were restricted to subclades Ib and Id. When the partial rDNA-ITS region sequences of two additional southwest Australian isolates, four from Europe, and four from the Americas were included in the analyses, the Australian isolates were within O. virulentus subclades Ia and Id, the European isolates within subclade Ic, and the American isolates within subclades Ia and Ib. These findings suggest that there may have been at least three separate introductions of O. virulentus into the isolated Australian continent since plant cultivation was introduced following its colonization by Europeans. They also have implications regarding numbers of different introductions to other isolated regions. Lettuce big-vein associated virus and Mirafiori lettuce big-vein virus were both detected when symptomatic lettuce leaf tissue samples corresponding to the root samples from southwest Australia were tested using virus-specific primers in reverse transcription-PCR, so presence of both viruses was associated with O. virulentus occurrence.

Comparison of Ribosomal DNA ITS Regions Among Hippophae rhamnoides ssp. sinensis from Different Geographic Areas in China

Hippophae rhamnoides ssp. sinensis is of ecological and practical importance, PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the rDNA ITS region was conducted to estimate the genetic diversity of H. rhamnoides ssp. sinensis at 19 sites in Northern China. Restriction analysis divided the samples into seven RFLP patterns (I–VII), which implied that H. rhamnoides ssp. sinensis had a degree of genetic diversity. The genetic diversity of H. rhamnoides ssp. sinensis mainly lied within populations other than among populations, and the results also suggested that geographic distances did not have any clear effect on their genetic differentiation. The sequences of rDNA ITS region varied in length from 656 to 658 bp, which exhibited 95–100% sequence identity. Pairwise Kimura 2-parameter distances within these H. rhamnoides ssp. sinensis samples ranged from 0.27% to 5.95%. There were 48 variable sites within the entire ITS region, among them transitions were more frequent than transversions. The ITS1 and ITS2 transcripts of H. rhamnoides ssp. sinensis had conserved secondary structures, implying that the ITS region maybe under evolutionary pressure to maintain the RNA secondary structure involved in the post-transcriptional processing of rRNA.