GenBank Database Research Articles

Structure prediction, docking studies and molecular cloning of novel Pichia kudriavzevii YK46 metalloprotease (MetPr) for improvement of feather waste biodegradation

This study addresses the environmental risks associated with the accumulation of keratin waste from poultry, which is resistant to conventional protein degradation methods. To tackle this issue, microbial keratinases have emerged as promising tools for transforming resilient keratin materials into valuable products. We focus on the Metalloprotease (MetPr) gene isolated from novel Pichia kudriavzevii YK46, sequenced, and deposited in the NCBI GenBank database with the accession number OQ511281. The MetPr gene encodes a protein consisting of 557 amino acids and demonstrates a keratinase activity of 164.04 U/ml. The 3D structure of the protein was validated using Ramachandran's plot, revealing that 93% and 97.26% of the 557 residues were situated within the most favoured region for the MetPr proteins of template Pichia kudriavzevii strain 129 and Pichia kudriavzevii YK46, respectively. Computational analyses were employed to determine the binding affinities between the deduced protein and beta keratin. Molecular docking studies elucidated the optimal binding affinities between the metalloprotease (MetPr) and beta-keratin, yielding values of − 260.75 kcal/mol and − 257.02 kcal/mol for the template strains Pichia kudriavzevii strain 129 and Pichia kudriavzevii YK46, respectively. Subsequent molecular cloning and expression of the MetPr gene in E. coli DH5α led to a significantly higher keratinase activity of 281 ± 12.34 U/ml. These findings provide valuable insights into the potential of the MetPr gene and its encoded protein for keratin waste biotransformation, with implications for addressing environmental concerns related to keratinous waste accumulation.

First Report of Cucumber Green Mottle Mosaic Virus Infecting Zucchini (Cucurbita pepo) and Wax Gourd (Benincasa hispida) in China.

Cucumber green mottle mosaic virus (CGMMV) was first discovered in China in 2003 and caused an epidemic in 2005. In China, the virus has been reported in gourd crops including watermelons, cucumbers, melons, etc (Sui et al. 2019). In Shandong Province, China from September 2014 to 2017, approximately 30% of zucchini (Cucurbita pepo) and wax gourd (Benincasa hispida) plants in commercial cucurbit fields, the two most important cash crops, exhibited chlorosis, mosaic, and mottling symptoms suspected to be caused by a tobamovirus. To identify the causative pathogens, ten zucchini and 15 wax gourd samples were collected from the commercial cucurbit fields. Total RNA was extracted and all samples were tested using reverse transcription PCR (RT-PCR) with TobamodF/TobamodR and TobamodF2/TobamodR2 (Li et al. 2018a). Five common Cucurbitaceae viruses were also tested: cucumber mosaic virus, papaya ringspot virus, squash mosaic virus, watermelon mosaic virus, and zucchini yellow mosaic virus (Ali et al. 2012). All samples generated positive results using tobamovirus generic primers but were negative for the five common Cucurbitaceae viruses. Amplification products (880 bp) from all samples were inserted into pMD19-T and recombinant clones were selected for Sanger sequencing. The results showed that zucchini green mottle mosaic virus, CGMMV, and tobacco mosaic virus (TMV) were detected in zucchini samples. CGMMV and TMV were detected in the wax gourd samples. To confirm the presence of these viruses, RT-PCR was performed using specific primer pairs, including CGMMV-cpf/CGMMV-cpr (Chen et al. 2006), ZG-F/ZG-R (Li et al. 2018b), and TMV-CP-F/TMV-CP-R (Srivastava et al. 2015). CGMMV was detected in all samples, with four zucchini and nine wax gourds only containing CGMMV. Zucchini (n=4; CGZ1-CGZ4) and wax gourd (n=4; CGWX1-CGWX4) isolates were cloned into pMD19-T and sequenced bidirectionally. The BLASTn results confirmed the presence of CGMMV, and the sequencing results were processed using DNAMAN Version (Lynnon Biosoft, San Ramon, CA, USA) and submitted to the GenBank database (https://www.ncbi.nlm.nih.gov/). A phylogenetic tree based on the CGMMV coat protein (CP) was constructed using CGZ1-CGZ4 (OP779762-OP779765), CGWX1-CGWX4 (OP779766-OP779769), and representative CGMMV sequences from GenBank. Sequence analysis of the CP demonstrated that CGMMV-zucchini and -wax gourd isolates belonged to an independent branch of the Chinese muskmelon AH-FT197 isolate (KU175639) and had 100% identity with the AH-FT197 isolate. To confirm their infectivity, leaf sap extract of CGZ4 and CGWX4 in phosphate buffer (0.1 M, pH 7.0) was mechanically inoculated on leaves of virus-free zucchini seedlings (Cucurbita pepo cv. Zaoqingyidai, 4-leaf-stage, n = 10) or virus-free wax gourd seedlings (Benincasa hispida cv. Tiezhu 2, n = 10). Ten days after inoculation, all plants exhibited symptoms (systemic chlorosis, mosaic, and mottling) similar to those of diseased plants in the field. Control seedlings inoculated with phosphate buffer remained symptomless. RT-PCR analysis using the CGMMV-cpf/CGMMV-cpr primer confirmed that all ten zucchini or wax gourd seedlings were infected with CGMMV, and all the control plants were free from CGMMV. To the best of our knowledge, this is the first report on zucchini and wax gourd as natural hosts for CGMMV in China. CGMMV is a highly contagious seed-borne virus and further attention should be paid to its spread in cucurbit crops.

Improving the gold standard in NCBI GenBank and related databases: DNA sequences from type specimens and type strains.

Scientific names permit humans and search engines to access knowledge about the biodiversity that surrounds us, and names linked to DNA sequences are playing an ever-greater role in search-and-match identification procedures. Here, we analyze how users and curators of the National Center for Biotechnology Information (NCBI) are flagging and curating sequences derived from nomenclatural type material, which is the only way to improve the quality of DNA-based identification in the long run. For prokaryotes, 18,281 genome assemblies from type strains have been curated by NCBI staff and improve the quality of prokaryote naming. For Fungi, type-derived sequences representing over 21,000 species are now essential for fungus naming and identification. For the remaining eukaryotes, however, the numbers of sequences identifiable as type-derived are minuscule, representing only 739 species of arthropods, 1542 vertebrates, and 125 embryophytes. An increase in the production and curation of such sequences will come from (i) sequencing of types or topotypic specimens in museum collections, (ii) the March 2023 rule changes at the International Nucleotide Sequence Database Collaboration requiring more metadata for specimens, and (iii) efforts by data submitters to facilitate curation, including informing NCBI curators about a specimen's type status. We illustrate different type-data submission journeys and provide best-practice examples from a range of organisms. Expanding the number of type-derived sequences in DNA databases, especially of eukaryotes, is crucial for capturing, documenting, and protecting biodiversity.

Begomoviruses on two chili types in Southeast Sulawesi Indonesia: variation of symptom severity assessment and DNA-betasatellite identification

The association of viral satellite DNA with Begomoviruses influences symptom expression in infected plants. Normalized Difference Vegetation Index (NDVI) is an image processing method used to assess plant health based on the plant’s ability to absorb sunlight for photosynthesis. Therefore, this study aims to assess symptom severity based on symptom variation and NDVI, as well as to detect and identify the presence of beta-satellite DNA associated with chili plants. The study was conducted in North Kolaka Regency, Southeast Sulawesi Province, Indonesia. It involved observations and image recording of symptom variations in the sampled cayenne and big red chili plants, followed by the detection of beta-satellite DNA in the samples using the polymerase chain reaction (PCR). The results confirmed the presence of non-coding satellite DNA associated with the Pepper yellow leaf curl Indonesia virus (PepYLCIV) in all severe, mild, or asymptomatic plant samples. In the phylogenetic tree, the non-coding satellite DNA isolates from Southeast Sulawesi are included in the betasatellite group and exhibit different genetic characteristics from other isolates available in the GenBank database. The cayenne chili with severe symptoms had lower NDVI values than the mild and asymptomatic categories, indicating that this type of chili with severe symptoms had a lower ability to absorb light. Further studies are needed to characterize the alpha-satellite type in the Southeast Sulawesi region for potential use in cross-protection mechanisms against plant virus infections.

Artificial Intelligence: Using BLAST Algorithm for DNA Classification of Thicket Vegetation

Artificial Intelligence (AI) has revolutionized molecular ecology by enhancing DNA classification methods. This study explores the application of the Basic Local Alignment Search Tool (BLAST) algorithm, an AI-powered bioinformatics tool, for DNA classification within the unique ecosystem of Eastern Cape Thickets. Our focus was on leveraging BLAST's capabilities to identify and characterize plant species based on genetic sequences. We collected plant samples from diverse locations within the Eastern Cape Thickets and employed advanced DNA extraction and sequencing techniques to acquire a comprehensive dataset. BLAST analysis played a central role in comparing our sequences with the GenBank database to determine species identities and potential functions. The BLAST algorithm demonstrated remarkable efficiency in identifying known species, confirming their presence in the thicket ecosystem. Notably, BLAST's utility extended beyond species confirmation, as it unveiled sequences with limited matches, suggesting the existence of novel genetic elements specific to the region. By harnessing AI, BLAST facilitated accurate species classification and functional annotation. Our study showcases the potency of AI-driven DNA classification using the BLAST algorithm in elucidating the genetic makeup of the Eastern Cape Thickets. The algorithm's proficiency in decoding intricate genetic information, elucidating functional attributes, and predicting potential adaptations underscores its pivotal role in modern molecular ecology research. These findings contribute to the broader understanding of genetic diversity within this ecosystem and highlight the transformative impact of AI-driven bioinformatics tools on ecological studies and conservation strategies.

Isolation, characterization, and phylogenetic analysis of two new porcine parvovirus 1 isolates from Northern China

Porcine parvovirus (PPV) is a pathogen of infectious reproductive disease, which can cause stillbirth, mummification, embryo death, and infertility (SMEDI) syndrome in pigs. The objective of this study was to gain new insights into the evolution and phylogeny of the PPV1 genome. In this study, we isolated two new PPV1 (HLJ202108-Y and SDLC202109) from northern China and sequenced their whole genomes. The new isolates were found to have three amino acid substitutions (K195R, K562R, and S578P) in nonstructural protein 1. The VP2 amino acid site contained nine nonsynonymous substitutions, including six substitutions of the Kresse strain corresponding to the NADL-2 strain and three substitutions of A414S, S436T, and N555K. Genetic evolution analysis was conducted on 107 reference sequences available in the GenBank database, and 4–5 PPV1 taxa were defined. The new isolates were in the same phylogenetic cluster as strain 27a. The changes in the cluster, specifically marker amino acids, and their potential role in enhancing pathogenicity are discussed in this study. Furthermore, the evolutionary tree map results showed that the strains in China were evolving in two directions: one was becoming increasingly similar to early NADL-2 strains, while the other was evolving toward 27a-like strains. We also compared the proliferation ability of the isolated strains in susceptible cells by analyzing the multistep growth curves. The results showed that the virulence titer of the mutant strain was high. In summary, this study introduced the latest changes in PPV and discussed the virus characteristics that were considered to affect virulence.

Diversity of fungal endophytes isolated from the invasive plant Solanum rostratum

A culture-dependent method was used to isolate fungal endophytes from the leaves, stems, and roots of the invasive plant Solanum rostratum Dunal. growing in Xinjiang Province, China. All isolates were identified according to ITS (internal transcribed spacer) region of ribosomal DNA sequences and analyzed by Nucleotide BLAST according to NCBI GenBank and Mycobank database. Altogether 176 endophytic fungal isolates corresponding to 44 OTUs were identified, which were classified into 12 genera, with Penicillium (59.66%) and Aspergillus (23.29%) being the highly dominant genera. Ten endophytic isolates (OTU1, OTU15, OTU16, OTU21, OTU23, OTU25, OTU26, OTU30, OTU37 and OTU44) were identified as potential new species.

Diverse Bradyrhizobium spp. with Similar Symbiosis Genes Nodulate Peanut in Different Regions of China: Characterization of Symbiovar sv. Arachis.

A total of 219 rhizobial strains isolated from peanut grown in soils from six peanut croplands in Zhengyang county, Henan Province, were typed by PCR-RFLP of IGS sequences. Their phylogenetic relationships were refined on representative strains using sequence analyses of 16S rRNA genes, housekeeping genes (atpD, recA, glnII) and symbiosis genes (nodA, nodC and nifH). The 219 rhizobial isolates were classified into 13 IGS types, and twenty representatives were defined within eight Bradyrhizobium genospecies: B. guangdongense covering 5 IGS types (75.2% of total isolates), B. guangzhouense (2 IGS types, 2.7% total isolates), B. zhengyangense (1 IGS type, 11.3% total isolates) and five novel genospecies (5 IGS types, 0.9 to 3.2% total isolates). All representative strains had identical nodA, nodC and nifH sequences except for one nifH sequence. With this one exception, these sequences were identical to those of the type strains of Bradyrhizobium species and several Bradyrhizobium genospecies isolated from peanut in different regions of China. The nodC sequences of all strains showed < 67% similarity to the closest strains on the Genbank database indicating that they are representative of a novel Bradyrhiobium symbiovar. This study has shown that (1) diverse Bradyrhizobium spp. with similar symbiosis genes nodulate peanut in different regions of China. (2) Horizontal transfer of genes involved in nodulating peanut is common between Bradyrhizobium species in soils used to grow the crop in China. (3) The strains studied here are representative of a novel Bradyrhizobium symbiovar that nodulates peanut in China. We propose the name sv. arachis for this novel symbiovar indicating that the strains were isolated from Arachis hypogaea. Results here have practical implications in relation to the selection of rhizobial inoculants for peanut in China.

Molecular characterization of a novel benyvirus infecting wheat in China.

In this study, a novel positive single-stranded RNA (+ ssRNA) virus named wheat yellow stripe associated virus (WYSAV) was identified in wheat plants in China. Molecular characterization revealed that the complete genome of WYSAV is divided into two segments, RNA1 and RNA2, which are 6,460 and 4,935 nucleotides (nt) in length, excluding their respective poly(A) tails. RNA1 contains one large opening reading frame (ORF), encoding a replication-associated protein. RNA2 contains six ORFs, encoding a coat protein (CP), a coat protein readthrough domain protein (CP-RTD), triple gene block protein 1 (TGB1), triple gene block protein 2 (TGB2), triple gene block protein 3 (TGB3), and a cysteine-rich protein (CRP). Phylogenetic analysis showed that WYSAV is related to members of the genus Benyvirus in the family Benyviridae. Thus, WYSAV is proposed to be a new member of the genus Benyvirus. Wheat (Triticum aestivum L.) is one of the most important food crops and ranked third in the world in terms of production, only behind rice and maize [1]. During its growth cycle, wheat faces several biotic and abiotic stresses. Wheat soil-borne virus disease is an important disease that is difficult to control and causes severe yield loss in China each year [2]. The main pathogens causing wheat soil-borne virus disease are Chinese wheat mosaic virus (CWMV) and wheat yellow mosaic virus (WYMV), and their transmission vector is Polymyxa graminis [3-5]. Members of the viral family Benyviridae usually have two to five genomic RNA segments and are transmitted by root-infecting vectors belonging to the family "Plasmodiophoridae". Although few members of the family Benyviridae, of which beet necrotic yellow vein virus is the type member, have been identified [6], several recently identified viruses have been found to be phylogenetically related to benyviruses but are not classified as members of the family Benyviridae. These "unclassified benyviruses" include red clover RNA virus 1, Arceuthobium sichuanense virus 3, Dactylorhiza hatagirea beny-like virus, goji berry chlorosis virus [7], Guiyang benyvirus 1, Guiyang benyvirus 2, Mangifera indica latent virus [8], Rhizoctonia solani beny-like virus 1 [9], Sanya benyvirus 1 [10], and Sclerotium rolfsii beny-like virus 1 [11].In this study, we identified a novel + ssRNA virus in symptomatic leaf samples collected from cultivated wheat in the city of Zhumadian, Henan Province, China. We propose to name this virus "wheat yellow stripe associated virus" (WYSAV), and we have deposited its full-length sequence in the GenBank database under the accession numbers OQ547804 (RNA1) and OQ547805 (RNA2).

Whole genome structure and resistance genes in carbapenemase-producing multidrug resistant ST378 Klebsiella pneumoniae

Background Carbapenemase-producing Klebsiella pneumoniae (CPKP) is one of the most dangerous multidrug-resistant (MDR) pathogens in human health due to its widespread circulation in the nosocomial environment. CPKP carried by companion dogs, which are close to human beings, should be considered a common threat to public health. However, CPKP dissemination through companion animals is still under consideration of major diagnosis and surveillance systems.MethodsTwo CPKP isolates which were genotyped to harbor blaNDM-5-encoding IncX3 plasmids, were subjected to the whole-genome study. Whole bacterial DNA was isolated, sequenced, and assembled with Oxford Nanopore long reads and corrected with short reads from the Illumina NovaSeq 6000 platform. The whole-genome structure and positions of antimicrobial resistance (AMR) genes were identified and visualized using CGView. Worldwide datasets were downloaded from the NCBI GenBank database for whole-genome comparative analysis. The whole-genome phylogenetic analysis was constructed using the identified whole-chromosome SNP sites from K. pneumoniae HS11286.ResultsAs a result of the whole-genome identification, 4 heterogenous plasmids and a single chromosome were identified, each carrying various AMR genes. Multiple novel structures were identified from the AMR genes, coupled with mobile gene elements (MGE). The comparative whole-genome epidemiology revealed that ST378 K. pneumoniae is a novel type of CPKP, carrying a higher prevalence of AMR genes.ConclusionsThe characterized whole-genome analysis of this study shows the emergence of a novel type of CPKP strain carrying various AMR genes with variated genomic structures. The presented data in this study show the necessity to develop additional surveillance programs and control measures for a novel type of CPKP strain.

Phylogeny and Genetic Divergence among Sorghum Mosaic Virus Isolates Infecting Sugarcane.

Sorghum mosaic virus (SrMV, the genus Potyvirus of the family Potyviridae) is a causal agent of common mosaic in sugarcane and poses a threat to the global sugar industry. In this study, a total of 901 sugarcane leaf samples with mosaic symptom were collected from eight provinces in China and were detected via RT-PCR using a primer pair specific to the SrMV coat protein (CP). These leaf samples included 839 samples from modern cultivars (Saccharum spp. hybrids) and 62 samples from chewing cane (S. officinarum). Among these, 632 out of 901 (70.1%) samples were tested positive for SrMV. The incidences of SrMV infection were 72.3% and 40.3% in modern cultivars and chewing cane, respectively. Phylogenetic analysis showed that all tested SrMV isolates were clustered into three clades consisting of six phylogenetic groups based on 306 CP sequences (this study = 265 and GenBank database = 41). A total of 10 SrMV isolates from South America (the United States and Argentina) along with 106 isolates from China were clustered in group D, while the remaining 190 SrMV isolates from Asia (China and Vietnam) were dispersed in five groups. The SrMV isolates in group F were limited to Yunnan province in China, and those in group A were spread over eight provinces. A significant genetic heterogeneity was elucidated in the nucleotide sequence identities of all SrMV CPs, ranging from 69.0% to 100%. A potential recombination event was postulated among SrMV isolates based on CP sequences. All tested SrMV CPs underwent dominant negative selection. Geographical isolation (South America vs. Asia) and host types (modern cultivars vs. chewing cane) are important factors promoting the genetic differentiation of SrMV populations. Overall, this study contributes to the global understanding of the genetic evolution of SrMV and provides a valuable resource for the epidemiology and management of the mosaic in sugarcane.

Molecular characterization of a novel clade echovirus 3 isolated from patients with hand-foot-and-mouth disease in southwest China.

Echovirus 3 (E3) belongs to the species Enterovirus B. Currently, three nearly whole-genome sequences of E3 are available in GenBank in China. In this study, we determined the whole genomic sequences of six E3 strains isolated from the stools of patients with hand-foot-and-mouth disease in Southwest China in 2022. Their nucleotide and amino acid sequences shared 82.1%-86.4% and 96.6%-97.2% identity with the prototype Morrisey strain, respectively, and showed 87.1% and 97.2% mutual identity. The six E3 strains are not clustered with other Chinese strains and formed a novel subgenotype (C6) with the recent American and British strains. Recombination analyses revealed that intertype recombination had occurred in the 2 C and 3D regions of the six E3 strains with coxsackieviruses B5 and B4, respectively. This study augments the nearly whole-genome sequences of E3 in the GenBank database and extends the molecular characterization of this virus in China.

Isolation and Identification of Dextran-Producing Weissella cibaria from Pickled Sauerkraut

The presence of certain bacteria in food products are particularly desirable. Probiotic strains of lactic acid bacteria (LAB), which have a number of favorable characteristics, including antimicrobial properties that support the development of beneficial microflora, are among them. In addition, because of their biochemical activity, they have an impact on the dietary, nutritional, and organoleptic qualities of food products. Before being released into the market, each strain must undergo independent testing to ascertain its probiotic qualities and potency. The purpose of this study was to separate and identify the LAB strain (Weissella cibaria bacteria) at the molecular level from a sample of pickled Sauerkraut that might be employed in the dairy sector and maybe is regarded as a probiotic in the future. Directly from the can, the shredded picked sauerkraut was brined in 2.25% salt (NaCl) for 48 hours before being inoculated aseptically into deMan, Rogosa, and Sharpe agar (MRS) and incubated for two days at 37 °C. Gram’s reaction, catalase production, and sugar fermentation tests employing glucose, sucrose, maltose, lactose, and fructose were among the traditional assays performed on isolation. The sequence was acquired and checked against those in the GenBank database using the BLAST tool at the NCBI-GenBank database after the isolate was extracted using the AccuPrep Genomic DNA Extraction kit, amplified, and agarose gel electrophoresis. The isolated LAB strains were validated using SEM and a genetic PCR assay, and the matching rate of the two isolated strains was 99%. The obtained strains showed their ability to produce dextran by observing the viscous growth characteristic when they were grown in MRS medium supplemented with 15% sucrose. To identify the bacterium, many culture and biochemical tests were carried out. The overall study concluded that the strains of Weissella cibaria bacteria can be easily isolated from pickled Sauerkraut using the developed procedure with the possibility of using the dextran produced by the bacteria as a prebiotic in the food sector.

First report of Zymoseptoria tritici causing Septoria tritici blotch in wheat in Paraguay.

Wheat (Triticum aestivum) is the third most cultivated field crop in Paraguay; it is grown on over 450,000 hectares with an annual production of 927,776 tons (fao.org/faostat). In 1952, Septoria tritici blotch (STB) was associated with the fungus Septoria tritici solely based on microscopic observation of conidia (Viedma and Delgado 1987). However, no morphometric or molecular studies have been performed in Paraguay up to date. Over the following decades, STB epidemic outbreaks were recorded, with a reduction in wheat production of up to 70% (Viedma and Delgado 1987). During winter 2021, leaf blotch symptoms were observed with an incidence above 50% in wheat fields in Capitán Miranda, Itapúa, Paraguay. Scattered, spherical, buried, and light brown necrotic spots with dark edges were observed on the leaves. Pycnidia with prominent central ostiole were observed. Leaves with symptoms were washed with 1% sodium hypochlorite for 1 min, rinsed with sterile distilled water, and incubated in wet chambers to induce sporulation of the fungus. Pycnidia produced greyish to white cirri. Isolated conidia were thin, elongated, and hyaline, ranging from 26.9-72.7 × 1.5-2.9 μm with one to three septa. Monosporic colonies on potato dextrose agar (PDA, ; Difco laboratories, Detroit, MI) media varied in color from white to pink, dark gray to black, or black with stroma-like structures. Based on morphology, the fungus was characterized as Zymoseptoria tritici (Hoorne et al. 2002; Gilchrist-Saavedra et al. 2005). Fungal DNA was extracted from mycelia, and the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF1-α), 28S rRNA gene (LSU), actin gene (act), calmodulin (CaM) were amplified using ITS1/ITS4, EF1-728F/EF-2, LSU1Fd/ LR5, ACT-512F/ACT-783R, CAL-228F/CAL737R primers, respectively. PCR amplicons were sequenced at Macrogen (Seoul, Republic of Korea) and deposited in the NCBI GenBank database (ITS: OQ360718; TEF1-α: OQ999044, LSU: OQ996413, act: OQ999046, CaM: OQ999045). Sequences were aligned with several isolates of Septoria spp. previously reported (Verkley et al. 2013; Stukenbrock et al. 2012) using ClustalW. The alignments were concatenated with Bioedit (Hall 1999). The UPGMA method with 1,000 bootstrap replications, was used to construct the phylogenetic tree using MEGA11 with Readeriella mirabilis as the outgroup. The isolate from Paraguay grouped into the Zymoseptoria tritici clade with 96% bootstrap support. To confirm pathogenicity, ten wheat plants cv. Itapúa 80 were grown in pots for three weeks in growth chambers (22 ± 2°C; 16 h photoperiod). Subsequently, these plants were inoculated with 1×107 conidia ml-1 suspension, and ten non-inoculated plants served as control. Seven days after inoculation (DAI), symptoms were observed displaying oval necrotic lesions and approximately 14 DAI abundant pycnidia were observed on and around the lesions. Segments of symptomatic leaves were placed in moisture chambers overnight to enhance cirri development. Conidia were mounted on a slide and observed under the compound microscope. Individual cirrhus were transferred to plates containing PDA and produced colonies like those used in the inoculation (Hoorne et al. 2002). We confirmed that the causal agent of STB from wheat fields in Paraguay was Zymoseptoria tritici. This pathogen causes annual wheat disease epidemics in Paraguay; therefore, optimizing surveillance for early detection and understanding its distribution will improve integrated management.

&lt;i&gt;SIDERA TIBETICA&lt;/i&gt; (&lt;i&gt;HYMENOCHAETALES&lt;/i&gt;, &lt;i&gt;BASIDIOMYCOTA&lt;/i&gt;), A NEW SPECIES TO RUSSIA

The first findings of recently described polypore fungus Sidera tibetica are revealed in the Caspian lowland forests of the Samursky National Park in the Republic of Dagestan, Russia. Basidiomata collected on Pinus brutia var. eldarica were studied by both light microscopy and molecular methods. Detailed information on new localities and habitats of Sidera tibetica as well as the collection numbers of specimens deposited in the Mycological Herbarium of the Komarov Botanical Institute of RAS (LE) are provided. Two complete sequences of ITS1–5.8S–ITS2 nuclear ribosomal DNA for Caucasian specimens of S. tibetica have been obtained and submitted to the GenBank database. Newly generated sequences are formed a separate well-supported clade in the Maximum Likelihood phylogenetic analysis together with all available ITS nrDNA sequences of S. tibetica originated from Belarussian and Chinese collections including the reference sequence from a holotype.

New Data on Distribution of &lt;i&gt;Pleurotus abieticola&lt;/i&gt; (&lt;i&gt;Pleurotaceae&lt;/i&gt;, &lt;i&gt;Agaricales&lt;/i&gt;, &lt;i&gt;Basidiomycota&lt;/i&gt;) in Russia

Here, we present information about the findings of Pleurotus abieticola in the territory of the northeast of the European part of Russia and the western macroslope of the Northern Urals within the borders of the Komi Republic (Russia). P. abieticola is a rare species found in Europe (Czech Republic, Finland, Poland, Russia), Asia (China, Eastern Siberia, Far East, Korea) and North America (Alaska). In Russia, several locations are known in the Leningrad Region, Krasnoyarsk and Primorye Territories. Comparison of the ITS nucleotide sequences of several collected specimens of P. abieticola with the data deposited in the GenBank database showed 98.2–100% similarity with P. abieticola specimens from other parts of the range (Southern Siberia, Far East, China). The article presents a description of basidiomata, photographs of microstructures, clarified features of morpho-logy, and provides information on the distribution and ecology of this species.

Morphological Traits and Nuclear Genetic Diversity of Coptis sp. in Hoang Lien National Park, Lao Cai Province, Vietnam

Coptis is a medicinal plant genus in the Ranunculaceae family, and is also known as “Hoang Lien” in Vietnamese. It is a perennial herb that grows in some regions of the world. However, Coptis is endangered and faces global threats. This study aimed to characterize some main morphological characteristics and performed a phylogenetic analysis of 11 samples of Coptis sp. collected from Hoang Lien National Park using nuclear DNA sequence analyses. All sample species have unique morphological traits with distinct yellow rhizomes and basal leaves with five segments. The petiole measures 13-25 cm and is smooth, while the leaf blade is ovate, ranging from 7 to 15.5 cm in length and 5.5 to 14 cm in width. The leaves are subleathery, glabrous on the underside, and sparsely puberulous on the veins on the upper side. The molecular characterization of Coptis sp. genotypes was determined by ITS markers. The length of the ITS1-ITS2 sequences varied from 363 to 371 nucleotides. The average nucleotide composition was 17.11% A, 31.25% C, 32.247% G and 19.41% T, respectively. The comparison with the GenBank database showed that the samples had 95.71- 96.37% similarity with the species Coptis quinquesecta. The genetic distance among the 11 Coptis samples fluctuated from 0.00 to 0.017. A neighbor-joining tree was constructed to show the genetic relationships among Coptis samples. The results indicated that this endangered species had low levels of genetic diversity. The study has provided valuable information for genetic-based conservation of this rare endemic species and suggested some conservation strategies.

Evaluating the phylogenetic relationship of a dwarf honey bee through cytochrome oxidase subunit 1 (COX1) gene sequence databases

Dwarf honey bees, Apis florea, and Apis andreniformis are known as the potential wild pollinator in Asia. Apis florea was introduced to Indonesia, West Asia, and Africa. Based on the cytochrome oxidase subunit 1 (COX1) gene, introduced A. florea in Iran is suggested to come from India. Although the databases of the dwarf honey bee COX1 gene are available in GenBank, however, the phylogeny analysis was focused on the restricted area. Thus, this study aims to evaluate the phylogenetic relationship and genetic diversity of dwarf honey bees based on the COX1 gene sequence from GenBank databases. The COX1 gene sequence databases are collected from NCBI. The genetic diversity and phylogenetic relationship are analyzed using MEGAX. Alignment of 530 bp COX1 gene of dwarf honey bee showed that there are 60 nucleotide variations between A. florea and A. andreniformis. Our analysis indicated that the genetic diversity between A. andreniformis is low (0-0.19%). On the other hand, there are 28 nucleotide variations between A. florea and eight nucleotide sites that are suggested as a marker to differentiate A. florea1 India+Egypt+Iran and A. florea2 Thailand+China with 1.7-3.4% genetic distance. The high genetic distance of A. florea is supported by the separation sub-clade of A. florea1 and A. florea2 in the phylogenetic tree.

Species Diversity of Lycoperdaceae (Agaricales) in Israel, with Some Insights into the Phylogenetic Structure of the Family.

The diversity of Lycoperdaceae in Israel was studied. Molecular phylogenetic relationships within the family, and genus Lycoperdon in particular, were inferred using original ITS rDNA sequences of 58 samples belonging to 25 species from Israel and six other countries, together with 66 sequences stored in the GenBank database. The current molecular phylogenetic study recovered the family Lycoperdaceae as a monophyletic group, which was supported in both maximum likelihood and Bayesian analyses. The clades corresponding to the genera Apioperdon, Bovista, Calvatia, Disciseda, and Lycoperdon were revealed. The taxonomic structure of the named genera was partially resolved. Within the genus Lycoperdon, some species received significant statistical support; however, their relationships, as well as the problem of the genus monophyly, mostly remained questionable. As a result of a thorough literature survey, extensive sample collection, and studies of the material stored in the Herbarium of the Institute of Evolution, University of Haifa (HAI, Israel), fifteen species representing five genera were found in the territory of Israel. Six species, namely Apioperdon pyriforme, Bovista aestivalis, Calvatia candida, Lycoperdon decipiens, L. niveum, and L. perlatum, are new additions to the diversity of Lycoperdaceae in Israel. Detailed macro- and micromorphological descriptions, ecology, geography, and critical notes, together with light microscopy photos and SEM micrographs, are provided. In-depth discussion on some taxonomically challenging species is presented.

Sensitive detection of specific cell-free DNA in serum samples from sheep with cystic echinococcosis.

Developing more sensitive methods for the diagnosis of echinococcosis is essential. In this study PCR assay for sensitive detection of specific cell-free DNA (cfDNA) of Echinococcus granulosus sensu lato in the sera of the sheep naturally infected with echinococcosis was investigated. To extract cfDNA from 35 infected sheep, the modified phenol-chloroform method was used for two different volumes (0.5 and 2 ml) of serum samples. From each extracted sample, two DNA volumes (5 and 10 μl) were amplified using both standard PCR and semi-nested PCR targeting NADH dehydrogenase subunit I. Standard and semi-nested PCR on 0.5 ml of serum samples detected Echinococcus DNA in 8 and 12 out of 35 sheep, respectively; however, using 2 ml of serum samples, they detected 24 and 27 samples. By increasing the volume of template DNA, the PCRs could detect 29 and 33 out of 35 samples. The results were confirmed by sequencing of randomly selected PCR amplicons and comparing them with GenBank databases. Larger volumes of serum for DNA extraction, greater volumes of DNA template for PCR, and employing a semi-nested PCR protocol, increased the sensitivity of PCR to 95%. This approach can also be applied to the diagnosis of echinococcosis in humans.