Nucleotide Sequence Analysis Research Articles

Diagnosis and molecular characterization of three allexiviruses infecting garlic crop in Saudi Arabia.

Forty-four samples of garlic plants showing virus-like symptoms were collected, during the growing season (2021-2022) from different locations in Qassim province, Saudi Arabia. These samples were analyzed by ELISA against the important Allium allexiviruses including garlic virus A (GarV-A), garlic virus B (GarV-B), garlic virus C (GarV-C), and Shallot virus X (ShVX). The obtained results showed that 22/44 (50%) samples were found to be infected with one of the tested viruses. Mixed infections were detected in 13/22 samples (59.1%) with more than one virus. However, 13.6%, 0% and 27.2% were detected as single infection with GarV-A, GarV-B and GarV-C respectively. RT-PCR amplification with general allexiviruses primer (750 bp) and specific primers for GarV-A (1330 bp), GarV-B (1216 bp) and GarV-C (1557 bp) were used to detect the respective viruses. The phylogenetic tree and nucleotide sequence analysis of one of each GarV-A (OQ397541), GarV-B (OQ397542) and GarV-C (OQ397543) with general primer for allexiviruses while one isolate of each GarV-A (ON155441), GarV-B (OR343811) and two isolates of GarV-C (ON155445, and ON155446) with specific primers showed their similarity with their respective viruses from GenBank. In host range study, Chenopodium amaranticolor, Nicotiana benthamiana, N. tabacum and Allium cepa expressed necrotic lesions, mosaic and yellowing symptoms respectively against GarV-A, GarV-B and GarV-C. To our knowledge, this is the first report of GarV-A, GarV-B, and GarV-C in Saudi Arabia.

First report of the whole‑genome sequence analysis of Fig badnavirus 2 from China.

A novel plant virus was identified in fig trees exhibiting ring spot symptoms through high-throughput sequencing (HTS). The complete genome sequence was successfully determined using PCR and RT-PCR techniques. The virus features a circular DNA genome of 7233 nucleotides (nt) in length, encompassing four open reading frames (ORFs). ORF1 and ORF2 encode hypothetical proteins, while ORF3 encodes a putative polyprotein with conserved domains, including a zinc finger, aspartic protease, reverse transcriptase (RT), and RNase H. ORF4 encodes a putative protein of unknown function. Comparative nucleotide sequence analysis of the RT + RNase H region reveals 84.46% and 78.82% identity with grapevine badnavirus 1 (GBV-1, MF781082.1) and fig badnavirus 1 (FBV-1, MK348055.1), respectively. Notably, this virus's genomic organization diverges from GBV-1 but is similar to FBV-1. Phylogenetic analysis demonstrates that the three isolates of this virus form a distinct clade within the badnaviruses. Based on genomic structure and phylogenetic relationships, this novel virus represents a new member of the genus Badnavirus and is proposed to be named "Fig badnavirus 2" (FBV-2).

Infection survey, molecular, pathogenicity, and morphological characteristics of Sarcocystis species naturally infected water buffaloes (Bubalus bubalis) in Egypt

Abstract Background Sarcocystosis is a parasitic disease found worldwide, resulting from various Sarcocystis species. The current research was carried out in three significant economic areas in Egypt: Greater Cairo, the Nile Delta, and Upper Egypt. It aimed to investigate the occurrence of Sarcocystis spp. in locally bred water buffaloes Bubalus bubalis. Methods To achieve this objective, 317 buffalos were slaughtered in different slaughterhouses in various regions of Egypt. Samples of heart, skeletal muscle, esophagus, and tongue were assessed using macroscopic and microscopic tests. Examination methods included direct optical observation of tissues as well as digestion and examination of the sediment obtained from the tissues. Additionally, ultrastructural features were analyzed using scanning and transmission electron microscopy. Molecular characterization was conducted through PCR, followed by nucleotide sequencing and phylogenetic analysis. Results A total of 317 slaughtered buffaloes were examined for Sarcocystis during the period from September 2021 to October 2023. The prevalence of infection was recorded with 229 out of 317 (72.2%) infected with Sarcocystis spp. The results also showed that the prevalence of Sarcocystis species in females was higher than males. Based on the age of carcasses, adults (> 2 years) had a higher infection rate compared to young ones (< 2 years). Regarding seasonal variation, the highest prevalence of infection was recorded during the summer followed by spring, and then autumn, while winter had the lowest prevalence of infection. Additionally, the skeletal muscle was the most susceptible organ to sarcocystosis (87.3%) followed by the esophageal muscle (8.3%), the tongue (4.4%), and no infection in the heart muscle. The use of scanning and transmission electron microscopy allowed the identification of S. fusiformis and S. cruzi in buffaloes in Egypt. Furthermore, the Sarcocystis 18 S rRNA genes from skeletal tissue samples were cloned and sequenced under accession numbers OQ507387, OQ507388, and OQ507389 for S. fusiforms, and one OQ507391 for S. cruzi. Conclusion The findings revealed a notably high prevalence of Sarcocystis infection (72.2%) in buffaloes from Egypt, with skeletal muscle identified as the organ most susceptible to the parasite. Two Sarcocystis species were detected: S. fusiformis and S. cruzi.

Genetic Diversity and Phylogenetic Analysis of the Endangered Transylvanian Pinzgau Cattle: A Key Resource for Biodiversity Conservation and the Sustainability of Livestock Production

Animal biodiversity is essential for maintaining the functionality of local food systems and ensuring sustainable livelihoods. Starting in 2000, the Food and Agriculture Organization of the United Nations (F.A.O.) has drawn attention to the decline in cattle populations, including the Transylvanian Pinzgau breed from Romania. Renowned for its hardiness, adaptability, and enhanced resistance to diseases and climate change, the Transylvanian Pinzgau is regarded as an important genetic asset for advancing livestock production. As a result, tracking genetic diversity has become a key focus in breeding programs, particularly for small, endangered local populations that play a vital role in regional agro-ecological systems. This research paper sought to assess the genetic diversity of a group of 24 head of cattle from the Transylvania region by analyzing two mtDNA markers, cytochrome b and D-loop sequences, both widely recognized for their relevance and importance in the analysis of genetic diversity of cattle and phylogenetic studies. The findings, derived through statistical analysis of nucleotide sequences using specialized software, indicated that the analyzed cattle are part of the ancestral T haplogroup, with a direct lineage tracing back to Bos taurus. This information can aid in developing crossbreeding programs focused on conserving essential genetic resources, improving other cattle breeds, and protecting biodiversity.

Gnathostoma doloresi in domestic pigs in the Republic of Palau, 2020–2022

Gnathostoma adult worms were found in the stomachs of domestic pigs in the Republic of Palau, with a prevalence of 0.7% (2/277) in the meat inspections conducted between 2020 and 2022. Based on morphological characteristics and nucleotide sequence analysis, the worms were identified as the adult stage of Gnathostoma doloresi Tubangui, 1925 (Rhabditida: Gnathostomatidae). These findings suggest the presence of this zoonotic nematode in Palau, which warrants further surveillance to determine its route of transmission in this island country.

Similarity of Human Mitochondrial DNA Nucleotide Substitution Spectra Reconstructed Over One and Many Generations

Using phylogenetic analysis of mitochondrial whole genome nucleotide sequences (mtDNA), allowing the study of genetic changes over many generations, a spectrum of nucleotide substitutions (along the L-strand of mtDNA) was reconstructed in European populations. The spectra of mtDNA nucleotide substitutions observed in a heteroplasmic state (at ≥1% and ≥5% levels) in first generation children were also analyzed. It was found that the spectra of nucleotide substitutions reconstructed over one and many generations do not differ practically in the main parameters: the distribution of pyrimidine and purine substitutions (with predominance of transitions TC), the ratio of the number of transitions and transversions. Analysis of the phylogenetic tree of mtDNA haplotypes in Europeans clearly revealed the influence of negative (purifying) selection on mitochondrial gene pools. It is suggested that the selective processes guiding the mtDNA evolution in one and many generations are of a similar nature, i.e., caused by negative selection. The problem of how mutations occur and spread in mitochondria of germ line cells is discussed.

Genetic variability of abundant littoral species of mesostigmatic mites (Acari, Mesostigmata) with different distributions from the seashores of Eurasia

The marine littoral acarofauna is of interest both in relation to the history of seas and as biological indicators of water quality, but it remains poorly studied. For the first time, we characterize the molecular genetic variability of four widespread, common and abundant species of littoral mesostigmatic mites: Phorytocarpais kempersi, Parasitidae; Halolaelaps celticus and Halolaelaps orientalis, Halolaelapidae; and Thinoseius spinosus, Eviphididae. Based on the nucleotide sequence analysis of nuclear ribosomal repeats (ITS), we studied mite collections from the coasts of the Caspian, Azov, Black, Baltic, White, Barents, Norwegian, Bering, Okhotsk, and Japan seas. The molecular identification of the species studied, based on DNA barcoding using ITS sequences, appears to be clear. We obtained genetic evidence for the separation of Halolaelaps celticus and H. orientalis as two distinct species, based both on clustering their ITS genotypes (separated by 6 parsimony-informative substitutions) and the geographic distributions of their genotypes. The high genetic similarity of the western and eastern Palaearctic populations in both arctic-adapted species, Phorytocarpais kempersi and Thinoseius spinosus, is explicable in terms of their rather recent contact on the northern coast of Eurasia during the last interglacial or Holocene climatic optimum. The high genetic similarity of Halolaelaps orientalis, a relatively thermophilous species, from the Mediterranean region (the Azov, Black, and Caspian seas) with a sample from the very distant Sea of Japan suggests a recent connection between the European and Pacific populations via the Indian Ocean.

Production of recombinant norovirus VP1 protein and its antigenic and immunogenic properties

Introduction. The importance of noroviruses in human infectious pathology and the danger of large epidemic outbreaks in organized groups determine the need to develop means of specific prevention of infection. The aim of the study was to obtain recombinant norovirus VP1 protein and analyze its immunogenic and antigenic properties . Materials and methods. Computer analysis of nucleotide and amino acid sequences, molecular cloning, polymerase chain reaction, electrophoresis of nucleic acids in agarose gel and proteins in polacrylamide gel, affinity chromatography, enzyme immunoassay. Results and discussion. A genetic construct encoding recombinant VP1 of the GII genotype norovirus with codons optimized for highly effective expression in Escherichia coli has been created. The strain of E. coli Rosetta 2 (DE3) has been transformed by genetic construct. VP1 expression was carried out in E. coli cells, conditions for its production, purification and renaturation were optimized. A purified soluble recombinant VP1 protein forms virus-like particles with a diameter of 30–50 nm. Immunization of BALB/c mice by protein lead to antibodies production with a titer greater than 1 : 1000. When evaluating antigenic properties, it was shown that human IgG, IgM, and IgA antibodies interact with recombinant VP1. The total antibody detection rate was 47,4%. The results indicate the possibility of using recombinant VP1 for development of domestic vaccine for the prevention of norovirus infection.

Molecular survey and phylogenetic analysis of bee pathogens; with a note first detection of Apis mellifera Filamentous Virus, Varroa destructor virus-1, Apis rhabdovirus-1, and Apis rhabdovirus-2 in Türkiye

Turkey is one of the most important bee centers in the world, but there is limited information about honey bee pathogens in the country. This study aimed to investigate bee pathogens using microscopic and molecular techniques (PCR, RT-PCR, nested-PCR, and sequencing) in Sivas province, the second-biggest beekeeping center in Turkey. For this purpose, bee samples were obtained from 149 bee colonies belonging to 74 local beekeepers in eight districts of the Sivas province. The bee samples were examined for pathogens, and at least one pathogen was detected in 148 bee colonies. In this study, a single infection was determined in 26.17% (39/149) of the bee samples, while the prevalence of co-infection was 73.15% (109/149). Nosema ceranae, Varroa destructor haplotype-K, Apis mellifera Filamentous Virus, Deformed wing virus, Varroa destructor virus-1, Lake Sinai virus-2, Apis rhabdovirus-1, and Apis rhabdovirus-2 were detected. Apis mellifera Filamentous Virus, Varroa destructor virus-1, Apis rhabdovirus-1, and Apis rhabdovirus-2 were found for the first time in Turkey with the current study. Furthermore, nucleotide sequence analysis and phylogeny relationships of bee pathogens obtained from samples were done, and sequences were deposited to GenBank. This study provides molecular data on the presence and prevalence of important bee pathogens in the region. As the prevalence of many pathogens has been determined, especially the existence of viral new species has been revealed, the effects of the species on beekeeping should be studied, and beekeepers should take effective control measures to reduce the effects of these pathogens.

First report of cucurbit yellow stunting disorder virus causing yellowing disease on major gourds in India

Cucurbitaceous crops are a key group of vegetables widely cultivated in India. Characterized yellowing symptoms along with interveinal chlorosis, mottling and downward curling were observed in ridge gourd, snake gourd, and bottle gourd along with the presence of whiteflies (Bemisia tabaci). To investigate the infecting virus, RT-PCR assays followed by sequencing and Transmission Electron Microscopy (TEM) analysis were performed. Sanger sequencing, nucleotide sequence analysis, and TEM imaging confirmed the association of cucurbit yellow stunting disorder virus (CYSDV), genus Crinivirus within the family Closteroviridae. This study reports the natural occurrence of CYSDV in ridge gourd, snake gourd, and bottle gourd for the first time in India.

Three-Year Monitoring of Microorganisms' Composition and Concentration in Atmospheric Aerosols of Novosibirsk City and Suburbs.

The atmospheric environment is formed under the influence of local and distant sources as a result of horizontal and vertical transport. In the present work, microbiological analysis of 604 samples of atmospheric aerosol collected in the period from September 2020 to September 2023 at four sites differing in anthropogenic load, located in Novosibirsk and the region, was carried out. Day and night aerosol samples were collected during 12 h every two weeks by filtration using Sartorius reinforced Teflon membranes, then sown on a set of nutrient media. The taxonomic affiliation of the isolated microbial isolates was determined based on phenotypic characteristics and analysis of 16S rRNA gene nucleotide sequences. Changes in the composition and concentration of culturable microorganisms depending on the season, time of day, and site of aerosol sampling were observed. In winter, lower fungi and bacteria of the genera Bacillus, Staphylococcus, Micrococcus dominated with an average concentration from zero to 12.5 CFU/m3 of aerosol. In the warm period, the concentration and diversity of cocci, spore-forming and non-spore-forming bacteria, actinomycetes, and fungi (up to 1970 CFU/m3), among which pathogenic microorganisms were found, increased sharply in aerosols. The use of 16S metabarcoding techniques has greatly expanded the range of aerosols' microbial diversity detectable.

Analysis of nucleotide sequences and expression levels of the ABI3 and FUS3 genes in Pisum sativum L. genotypes differs in seed storage protein content

Analysis of nucleotide sequences and expression levels of the ABI3 and FUS3 genes in Pisum sativum L. genotypes differs in seed storage protein content

Development of a bioinformatics tool for identifying members of the Apis mellifera gut microbiome

The methods of molecular genetics and bioinformatics analysis of DNA nucleotide sequences are reliable and modern tools for studying the microbiome of various organisms, including the gut microorganisms of Apis mellifera. The creation of accessible software for processing and analyzing sequencing data is highly relevant, as existing commercial solutions are often too expensive for widespread use in academic settings. Available free software typically has significant functional limitations, preventing comprehensive analysis. Researchers are often forced to use a combination of different software tools, which not only reduces data processing efficiency but also significantly increases the risk of errors during analysis. The rapid advancement of research on the A. mellifera gut microbiota, combined with the lack of accessible analytical tools for processing sequencing data, underscores the importance of developing software that automates the entire analysis process. The implementation of such a tool would not only simplify and accelerate sequencing data processing but also make it more accessible to a broader range of researchers, fostering the development of microbiome studies in honeybees and other organisms. The aim of the study was to develop bioinformatics software for automating the processing and analysis of nucleotide sequence data of the 16S rRNA gene and ITS region, to validate its performance, and to conduct a comparative evaluation based on the results of studying the gut microorganisms of A. mellifera. As part of the work, yeasts were isolated from the gut of A. mellifera, and DNA was extracted from these microorganisms. Amplification and sequencing of the 16S rRNA gene and ITS region were performed. Based on the obtained data, specialized bioinformatics software was developed, integrating all the necessary tools for comprehensive sequencing data analysis. The developed software was successfully validated on the obtained sequencing data. To assess its effectiveness, a comparative analysis was conducted with the widely recognized Geneious software, confirming the competitiveness and reliability of the developed tool.

Rabies in free-ranging capybaras (Hydrochoerus hydrochaeris) on Anchieta Island, Ubatuba, Brazil.

Between December 2019 and January 2020, three cases of rabies were reported in free-ranging capybaras on Anchieta Island, Ubatuba-SP, Brazil. This 8.28km² island is located 540m offshore from the mainland. Two of the capybaras exhibited signs of hindlimb paralysis, and one was found dead. Rabies was diagnosed using the direct fluorescent antibody test (dFAT), while RT-qPCR and phylogenetic analysis of nucleotide sequences confirmed the presence of the vampire bat rabies virus (RABV) strain. Although no visible bat bite marks were found on the capybaras, vampire bats are known to inhabit the island. Other wildlife tested negative for rabies during this period, and no further rabies outbreaks have been observed since. Environmental changes and human activities, such as the disturbance of bat roosting sites, may have contributed to the incident. The detection of rabies in capybaras suggests a potential spillover from a vampire bat reservoir. Further investigation is needed to determine whether capybaras act as dead-end hosts or play a role in maintaining the rabies transmission cycle.

Characterization of a mammalian orthoreovirus isolated from the large flying fox, Pteropus vampyrus, in Indonesia.

Fruit bats serve as an important reservoir for many zoonotic pathogens, including Nipah virus, Hendra virus, Marburg virus and Lyssavirus. To gain a deeper insight into the virological characteristics, pathogenicity and zoonotic potential of bat-borne viruses, recovery of infectious viruses from field samples is important. Here, we report the isolation and characterization of a mammalian orthoreovirus (MRV) from a large flying fox (Pteropus vampyrus) in Indonesia, which is the first detection of MRV in Southeast Asia. MRV was recovered from faecal samples of three different P. vampyrus in Central Java. Nucleotide sequence analysis revealed that the genome of the three MRV isolates shared more than 99% nucleotide sequence identity. We tentatively named one isolated strain as MRV12-52 for further analysis and characterization. Among 10 genome segments, MRV12-52 S1 and S4, which encode the cell-attachment protein and outer capsid protein, had 93.6 and 95.1% nucleotide sequence identities with known MRV strains, respectively. Meanwhile, the remaining genome segments of MRV12-52 were divergent with 72.9-80.7 % nucleotide sequence identities. Based on the nucleotide sequence of the S1 segment, MRV12-52 was grouped into serotype 2, and phylogenetic analysis demonstrated evidence of past reassortment events. In vitro characterization of MRV12-52 showed that the virus efficiently replicated in BHK-21, HEK293T and A549 cells. In addition, experimental infection of laboratory mice with MRV12-52 caused severe pneumonia with 75% mortality. This study highlights the presence of pathogenic MRV in Indonesia, which could serve as a potential animal and public health concern.

DETECTION OF CHLAMYDIA-LIKE MICROORGANISM WADDLIA CHONDROPHILA IN TICKS

Chlamydia-like microorganism Waddlia chondrophila (C. Chondrophila) is a new pathogen that causes miscarriages and abortions in humans and cattle. This bacterium is considered as a potential zoonotic agent. The main reservoirs and carriers of C. chondrophila are ixodic ticks, and determining their role remains the most difficult and interesting question to be solved in future studies.The purpose of this study was to identify chlamydia in ixodic ticks collected from cattle by molecular methods. Polymerase chain reaction (PCR) and sequencing were used to confirm the presence of the pathogen in tick samples collected from domestic animals.The analysis of nucleotide sequences showed that the DNA of the bacterium W. chondrophila was detected in the ticks Dermacentor marginatus, Hyalomma asiaticum, Hyalomma marginatum, Ixodes persulcatus, Hyalomma anatolicum. Among 156 tick samples collected from North Kazakhstan, West Kazakhstan, Zhambyl and Turkestan regions, 7% were positive for chlamydia-like microorganism W. chondrophila.The presence of DNA of the chlamydia-like W. chondrophila microorganism in ixodic ticks suggests that additional research is needed to study the potential role of ticks as carriers of these zoonotic bacteria.

Characteristics of the Klebsiella Pneumoniae Strain Isolated from a Positive Blood Culture of a Premature Newborn Baby According to the Results of Whole Genome Sequencing

Relevance. K. pneumoniae is a common nosocomial pathogen in pediatric hospitals, often characterized by the presence of a wide range of virulence factors and genetic determinants of antibiotic resistance. Аim. To analyze the results obtained during wholegenome sequencing of a Klebsiella pneumoniae strain isolated from a positive blood culture of a premature newborn. Materials and methods. An ESBL-producing strain of K. pneumoniae isolated from a positive blood culture of a newborn premature infant. Sequencing was performed on the MiSeq platform (Illumina). Analysis of DNA nucleotide sequences of the complete genome of K. pneumoniae was carried out using the website of the Center for Genomic Epidemiology. The search for genetic determinants of antibiotic resistance and virulence was carried out using online services. Results and its discussion. The resulting nucleotide sequence was 5,414,099 bp in length, and the proportion of GC nitrogenous bases was 57.3%. The isolated strain belonged to the sequence type ST3559, had 4 genes encoding the synthesis of enzymes that hydrolyze antibacterial drugs from the beta-lactam group, 2 genes providing resistance to quinolones/fluoroquinolones, 1 resistance gene each to trimethoprim, chloramphenicol, fosfomycin and aminoglycoside antibiotics. Most of the virulence factor genes identified in the studied strain ensure the recognition and absorption of iron ions necessary for the competitive functioning of the bacterial cell. K. pneumoniae possesses the acrA efflux pump gene and its regulators, as well as 4 prophage particles and 1 CRISPCas IE system. Conclusions. Whole-genome sequencing of the K. pneumoniae strain isolated from a positive blood culture of a premature newborn allows us to characterize in detail the causative agent of a generalized infection and detect a wide range of genetic determinants of virulence factors and antibiotic resistance. The ESBL-producing strain of K. pneumoniae, as the etiological agent of neonatal sepsis, was characterized by the presence of virulence genes, multidrug resistance, both due to genes encoding enzymes that hydrolyze antibiotics, and due to the presence of efflux pumps and their regulators. The use of the results of traditional cultural research methods together with high-throughput sequencing data is a promising area of scientific research and has a reserve of practical application in the field of clinical medicine, genetics of microorganisms, molecular epidemiology at the local and global levels

Genes of Streptomyces globisporus 1912-4Crt Encoding Chitin Catabolism Enzymes

Polysaccharide chitin is one of the most common biopolymers in nature. Chitin (when used as the sole source of energy, carbon, and nitrogen) has been shown to be a substrate sufficient to enable the growth and synthesis of secondary metabolites by the S. griseus NCIMB 8136 strain and some other streptomycetes. The species Streptomyces globisporus and S. griseus belong to the same lower hierarchical taxon (S. griseus clade). The aim was to find in the S. globisporus 1912-4Crt genome genes encoding proteins that are necessary for chitin fermentation and transmembrane transport of resulting products. Methods. The object of the study was a sequence of the S. globisporus 1912-4Crt genome (reference NZ_QWFA01000000.1, GenBank) on the server of NCBI (The National Center for Biotechnology Information). Streptomycete S. globisporus 1912 and its variants are producers of antibiotic landomycin E and carotenoids. Search for and analysis of nucleotide and amino acid sequences were performed using programs BLAST (Basic Local Alignment Search Tool) on the aforementioned server. Results. A set of genes that determine enzymes of chitin catabolism and Ngc-transporter proteins were identified in the S. globisporus 1912-4Crt genome. Genes encoding 10 endo-acting chitinases (from the GH18 and GH19 families) and 2 exo-acting hydrolases (GH20) were identified in the S. globisporus 1912-4Crt sequence. Several genes determining deacetylases that deacetylate both chitin and oligosaccharides were found in the genome of the strain. One gene that determines endo-acting chitosanase from the GH75 family was discovered there. The ability of the wild-type strain S. globisporus 1912 and a number of its variants (including S. globisporus 1912-4Crt) to ferment chitin was proven in vitro. Conclusions. A set of genes sufficient for chitin assimilation was established in the S. globisporus 1912-4Crt genome sequence. Streptomycetes from different clades (for example, strains of S. coelicolor (S. albidoflavus group) and S. griseus, S. globisporus (S. griseus group)) containing different complexes of chitinolytic enzymes could be suggested.

Isolation and identification of Cladosporium tenuissimum causing leaf blight on garden pea (Pisum sativum L.) in Telangana from southern India

Garden pea (Pisum sativum L.), is an important legume grown worldwide as a source of proteins and carbohydrates. Since pea crop is cultivated globally in cool temperate climatic conditions, increasing temperatures accompanied by high humidity favors fungal infections. A fungal infection causing leaf blight symptoms was frequently observed on the leaves of pea plants grown in greenhouse and field conditions. The blight symptoms showed a water-soaked and wilting appearance with curly leaf edges accompanied by greyish mycelial growth and chlorotic lesions. The disease incidence in field conditions ranged between 5 and 10 %, whereas in the controlled greenhouse conditions, it ranged from 40 to 45%. Two isolates of the fungus, Gp03 and Gp04 were isolated from Arkel pea leaves and purified from a single conidium each. According to Koch's postulates, the pathogenicity tests with these isolates using detached leaf assay and whole plant leaf assays confirmed the pathogen. Morphological and molecular analysis of nucleotide sequences of the D1-D2 region, translation elongation factor-1α (TEF) and actin (ACT) genes confirmed these isolates as members of the genus, Cladosporium. The phylogenetic relatedness of these isolates with other members of the Cladosporium genus revealed them as fungal strains of Cladosporium tenuissimum. Further, these isolates were also confirmed as C. tenuissimum by Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh in India, and provided the accession nos. MTCC 13581 and MTCC 13582 to the C. tenuissimum strains, Gp03 and Gp04 respectively. This is the first report of Cladosporium tenuissimum infecting garden pea in Telangana, India.

Calreticulin from Apostichopus japonicus relieves endoplasmic reticulum stress induced by Vibrio splendidus through autophagy

When organisms are exposed to external stimuli, misfolded proteins accumulate continuously, resulting in endoplasmic reticulum (ER) stress. Autophagy is of great significance for eliminating aggregated proteins and maintaining cellular homeostasis. However, the molecular mechanism of activating autophagy in response to ER stress in sea cucumber is remain unclear. In the current study, we demonstrated that the pathogen Vibrio splendidus can cause ER stress in Apostichopus japonicus coelomocytes and identified a Ca2+ binding partner calreticulin (designated as AjCRT), which increased with the occurrence of ER stress. The nucleotide sequence analysis showed that the open reading frame of AjCRT was 1242 bp and encoded a 413-amino-acid residue polyprotein with calreticulin domains. The spatial expression analysis revealed that AjCRT was ubiquitously expressed in all examined tissues with large magnitude in the coelomocytes and was minimally expressed in muscle. Furthermore, silencing AjCRT in vivo could significantly exacerbate ER stress induced by V. splendidus and resulted in the significant reduction of coelomocyte autophagy. These findings indicate a calreticulin-based mechanism that positively regulates autophagy in response to ER stress induced by pathogen infection. The results will provide a basis for understanding the way of host alleviating ER stress through autophagy, and pharmacological approaches may have potential for managing ER stress induced by pathogen and related cellular disorders.