Classification and Identification of Marine Penicillium Species Based on ITS Sequences of rDNA

[ Objective ] The aim of this study is to identify 6 marine fungi species by analyzing ITS nucleotide sequence, which had been primarily identified as penicillium based on morphological characteristics. [ Method ] The ITS regions of these species were cloned by molecule biology method and phylogenetic analyzed using ClustalX1.83 software. [ Result] The ITS regions of these species were sequenced, and phylogenetic analysis between the yield sequences and the ITS sequences assessed in GenBank showed that the 6 strains all belonged to penicillium. [ Condusion] The present study suggests ITS sequence analysis could not be used as an only proof, but it is a very useful supplementary tool for the classification and identification of marine peniciUium combined with morphological characteristics.

Phylogenetic Relationships of Caragana(Fabaceae) by the Use of nrITS Sequences

[Objective] The aim was to explore the phylogenetic relationships of Cara- gana （Fabaceae） by the use of nrlTS sequences. [Method] Internal transcribed spac- er （ITS） sequences of nuclear ribosomal DNA （nrDNA） from 29 taxa of Caragana species and seven close relatives （all belong to Astralinae （Adens） Benth） were used for phylogenetic analysis. [Result] Length of the entire ITS region ranges from 611 to 614 bp in Caragana species. The aligned sequences nrlTS of Caragana are 655 bp, and 170 sites are variable, with 107 phylogenetically informative sites. The phylogenetically informative sites are 16.3% of the total aligned sequences. The ITS sequences data are useful to resolve some relationships at lower taxonomic levels within Caragana. The Caragana Fabr. is not a monophyletic group with very close connection with Calophaca tianschanica. The ITS data revealed that the species of Sect. tragacanthoides were dispersed in MP tree or ME tree. Although the morphol- ogy of C. ordosica is similar to C. tibetica, the ITS results revealed an unexpectedly close relativeship to C. roborovskyi. The ITS data also indicate C. davazamcii, C. korshinskii, C. intermedia, and C. microphylla are different species. [Conclusion] ITS sequences have an important reference value in exploring the relationships of Cara- gana.

Identification and rDNA-ITS Sequence Analysis of a New Anthracnose Pathogen of Dracaena fragrans in Yunnan Province

In this study, identification and rDNA-ITS sequence analysis of an anthracnose pathogen on Dracaena fragrans were carried out. [Method] D. fra-grans leaves with lesions were used as experimental materials to isolate anthrac-nose pathogen. Morphological observation, rDNA-ITS amplification and sequence analysis were performed to identify the pathogen strain. [Result] Caonidia of the iso-lated anthracnose pathogen were straight or curved, el iptic to crescent, with 2-5 oil droplets, 7.5-20 × 4.5-5 μm. According to molecular phylogenetic analysis, the iso-lated pathogen strain was identified as a new species, which was named Col-letotrichum dracaena-fragrantis sp. nov. [Conclusion] This study provided theoretical basis for the prevention and control of anthracnose.

ISSR Marker and ITS Sequence Study of Melampsora Larici-populina

To compare the differences in intertranslation space of ribosomal DNA （ITS） of Melampsora larici-populina, between the isolates from China and isolates from other countries, this study investigated ITS sequences and ITS polygenetic tree based on 11 isolates that were collected from 5 races in different parts of China. The results indicated that there was no difference among the ITS sequences of 11 isolates from China. The ITS sequence of isolates from China was more homogeneous with that of isolates from Britain compared with France, Germany, and Canada. Intersimple sequence repeat （ISSR） markers were also used to study the genetic division of Melampsora larici-populina, and the results showed that the 11 tested isolates could be divided into Western population and Northern population. Genetic diversity index of race C2 was significantly different from that of races C4, C3, and C1, and no significant differences were observed among the other races. Pathogenicity division of races must not harmonize with their genetic division, except race C2. The ITS region is conservative, and ITS sequence is not fit for studying the differences that existed among the races. ISSR marker can be used for intraspecies population study, and Melampsora larici-populina in China can be divided into two populations.

Pharmacognosy research and identification of Euphorbia prostrata Ait.

Background:Euphorbia prostrata Ait.is an annual herb widely distributed in the southern region of China with great medical values on Anti-inflammation,insect repellent,treatment of diarrhea.Despite its extensive uses as a traditional Chinese medicine,no systematic research on the identification of E.prostrata has been reported.Methods:The study aimed to establish an accurate identification system for E.prostrata through traditional pharmacognostical methods,including botanical origin,morphological characters,medicinal material characters,microscopic characters,physicochemical parameters determination,phytochemical screening,and DNA barcoding analysis.Results:Physicochemical results show that this plant likely contains flavonoids,anthraquinones,and other substances.The ITS loci of the nuclear genome and psbA-trnH loci of the chloroplast genome were selected and evaluated,which were the most variable loci.Conclusion:The findings of this study are expected to contribute to the development of species identification,as well as provide references for authenticity identification,genetic relationship analysis,and further utilization of E.prostrata.

Screening of Decolorizing Fungi for Rose Bengal and Water-soluble Colour Paste and Its Decolorization Conditions

The strain No. 2 which was isolated from the soil through enrichment culture was used as the experimental material. It was cultured in liquid medium to research decolorizing effect to Rose Bengal and soluble color paste under the different conditions of different media,carbon sources,nitrogen sources,initial pH values and culture temperatures. The results revealed that the optimum decolorizing conditions were using bean juice medium and PDA medium as the minimal medium,sucrose as the carbon source,and ammonium nitrate as the nitrogen source,initial pH 6.0-8.0. In addition,the strain was primarily identified as Aspergillus flavus according to its morphous and ITS sequence analysis.

Preliminary Studies on Identification of Lycium Linn.Germplasm Resources by nrDNA ITS Sequencing

[Objective] The study aimed to identify woltberry （Lycium Linn.） germplasm resources at molecular level by analyzing the nrDNA ITS sequence. [Method] Genomic DNA from woltberry leaves extracted by modified CTAB method were regarded as templates for PCR amplification by specific primer, clone and sequencing. [Result] The nrDNA ITS sequences were obtained and then differentiated among three tested materials. [Conclusion] PCR amplification and sequencing on nrDNA ITS is a feasible approach to identify different woltberry germplasm resources.

A New Method of Identification on Edible Lycium Linn. Germplasm Resources——nrDNA ITS Sequencing

[ Objective ] The study aimed to identify Lycium Linn. at molecular level. [ Method ] The nrDNA ITS sequence of 5 edible Lycium Linn. germplasm resources were investigated. [ Result ] The nrDNA ITS regions of five edible Lycium Liun. germplasm resources were sequenced. The whole sequences varied from 628 bp to 632 bp, with the average length of 630 bp. Total 79 variation sites were observed in the sequences, which accounts for 12. 5%. [ Conclusion] Sequence analysis based on nrDNA sequencing provides a new approach to identify edible Lycium Linn. germplasm resources.

Pharmacognostical Studies of Richardia scabera L.

Objective:To provide a scientific basis for identifying Richardia scabra with the help of pharmacognosy parameters,which has never been done before.Methods:Roots,stems,and leaves of R.scabra were collected for pharmacognostic studies involving source identification,character observation,microscopic evaluation,phytochemical screening,ultraviolet spectrum analysis,as well as DNA barcoding analysis.Results:The results showed that it had strong microscopic characters,so it could be used to identify R scabra.Phytochemical study showed the presence of carbohydrates,tannins,and proteins from water extract,and the data related to moisture,ash content,and molecular pharmacognosy were obtained.Conclusion:The established method in this study is easy and accurate,which provides a reference basis for its further development and utilization.

Identification of two Skeletonema costatum-like diatoms by fluorescence in situ hybridization

A harmful algae bloom (HAB) is a dense aggregation of algae in a marine or aquatic environment that can result in significant environmental problems. To forecast the occurrence of HAB, development of a rapid and precise detection method is urgently required. In this study, two Skeletonema costatum-like diatoms (SK-1 and SK-2), were identified morphologically under a light microscope, and detected using fluorescent in situ hybridization (FISH). Strain SK-1 was isolated from a frequently HAB affected area of the East China Sea, and strain SK-2 from an aquatic farm in Qingdao, China. Fluorescent DNA probes were designed that were complementary to the ITS sequence (including 5.8S rDNA) of strain SK-1. After hybridization, strong green fluorescence was observed in cells of strain SK-1 under an epifluorescence microscope; however, no such fluorescence was observed with strain SK-2, which indicates that probes hybridized only the DNA of the target strain, SK-1, in species-specific manner, and that the two strains do not belong to a same species. This finding was confirmed by ITS sequence analysis. The FISH technique used in this study was sensitive, simple, and rapid, and is a promising tool for detecting target HAB species in natural environments.

Sequence analysis and typing of Saprolegnia strains isolated from freshwater fish from Southern Chinese regions

Saprolegniasis,caused by Saprolegnia infection,is one of the most common diseases in freshwater fish.Our study aimed to determine the epidemiological characteristics of saprolegniasis in Chinese regions of high incidence.Saprolegnia were isolated and identified by morphological and molecular methods targeting the internal transcribed spacer(ITS)ribosomal DNA(rDNA)and building neighbor-joining(NJ)and maximum parsimony(MP)phylogenetic trees.The ITS sequences of eight isolated strains were compared with GenBank sequences and all strains fell into three clades:CLADE1(02,LP,04 and 14),CLADE2(S1),and CLADE3(CP,S2,L5 and the reference ATCC200013).Isolates 02 and LP shared 80%sequence similarity with S.diclina,S.longicaulis,S.ferax,S.mixta,and S.anomalies.Further,isolates 04 and 14 shared 80%similarity with S.bulbosa and S.oliviae.Finally,extremely high ITS sequence similarities were identified between isolates S1 and S.australis(100%);CP and S.hypogyna(96%);and S2,L5,ATCC200013 and S.salmonis(98%).This research provides insights into the identification,prevention and control of saprolegniasis pathogens and the potential development of effective drugs.

The diversity of soil culturable fungi in the three alpine shrub grasslands of Eastern Qilian Mountains

To understand the diversity of culturable fungi in soil at alpine sites, Rhododendron fruticosa shrubland, Salix cupularis fruticosa shrubland, and Dasiphoru fruticosa shrubland of the Eastern Qilian Mountains were selected to investigate. Three methods, including tradi- tional culturing, rDNA internal transcribed spacer （ITS） sequence analysis, and economical efficiency analysis, were carried out to estimate the diversity of soil culturable fungi of these three alpine shmblands. A total of 35 strains of culturable fungi were cultured by dilution plate technique and were analyzed by rDNA ITS sequence. The diversity indices such as species abundance （S）, Shannon-Wiener index （H）, Simpson dominance index （D）, and Pielou evenness index （3） of Rhododendron fruticosa shrubland, Salix cupularis fruticosa shrubland, and Dasiphoru fruticosa shrubland were ranged between 16 and 17, 2.66-2.71, 0.92, 0.95~）.97 respectively. The results showed that the diversity of soil fungi were abundant in these three types of alpine shrub grasslands, while further study should be done to explore their potential value.

Cryptic genetic diversity and host specificity of Bothriocephalus acheilognathi Yamaguti,1934(Eucestoda:Bothriocephalidea)

In this report, genetic variation and phylogeny of B. acheilognathi were analyzed based on 96 samples collected from 20 fish host species in 29 different localities by using the rDNA internal transcribed spacer （ITS） sequences. The ITS1 and ITS2 sequences of B. acheilognathi varied among 561-639 bp and 559-648 bp, and ten simple sequence repeat loci （microsatellites） were detected in the ITS regions, which contributed to significant sequence length variation. Phylogenetic analyses revealed 4 genetic clades （A, B, C, D） in samples with significantly different fish host spectra and geographic distribution. Clade A possessed a wide host range and geographic distribution, including all the samples ofB. acheilognathi determined in previous report. Clades B, C, and D only infected the small cyprinid fishes Opsariichthys bidens and Zacco platypus, and were limited to different locality. Interesting, Clades A and D were detected coexisting in same water body, and even in same fish host O. bidens in Danjiangkou Reservoir. The relatively higher genetic divergence and wide geographic distribution orB. acheilognathi parasitic in O. bidens indicated that O. bidens is likely to be the primary host. Sympatric occurrence of the two genetically distinct clades suggests the possibility of allopatric speciation and second encounter events.