Neighbor-joining Method Research Articles

First report of bean common mosaic virus naturally infecting yam bean (Pachyrhizus erosus) in China.

Yam bean (Pachyrhizus erosus), a high-yielding leguminous root crop with good nutritional value, is widely cultivated in southern China. In 2020, P. erosus (cv. Mumashan) plants exhibiting irregular yellow leaves and malformed seed pods (Supplementary Fig S1) were observed at Ningbo city, Zhejiang Province, China. To determine the causal agent(s) of the disease, symptomatic leaves (n=4) were collected for electron microscopy negative staining. Virus particles with a length of about 700nm, similar to viruses in the genus Potyvirus, were observed via transmission electron microscope (TEM), suggesting the presence a potyvirus(es). To further confirm which potyvirus(es) infected yam bean, total RNA was extracted from leaf samples of a total of six plants, including four symptomatic plants and two asymptomatic plants using TRIzol reagent (Invitrogen Carlsbad, CA, USA) according to the manufacturer's instructions. RNA was reverse-transcribed into cDNA with M4-T as the 3'-anchoring primer by ReverTra Ace® kit (Toyobo, Japan). Sprimer/M4 Potyviridae specific primers (Chen et al., 2001) were used for PCR analysis. A ~1,700-bp-long product was amplified from four symptomatic plants using KOD FX enzyme (Toyobo, Japan). No such band was amplified from the two asymptomatic plants. The PCR product (~1.7kb) amplified from a single symptomatic plant was ligated into the pEASY®-Blunt Zero vector (TransGen Bio, Beijing, China) and sequenced (Sangon Bio, Shanghai, China). The amplicon showed 99% nucleotide sequence identities with bean common mosaic virus (BCMV) isolate NKY021 (KJ807819). Subsequently, the complete nucleotide sequences of this BCMV isolate (referred as BCMV-NB) was amplified by overlapping RT-PCR and rapid amplification of cDNA ends with primers (Supplementary Table S1) designed from the sequence of BCMV isolate NKY021. The BCMV-NB full genome (Accession No. OL871237) consists of 10,053 nucleotides excluding the poly(A) tail and contains a large open reading frame encoding a polyprotein of 3222 amino acids. BLASTn analysis showed that BCMV-NB shared a sequence identity of 96.4% with BCMV isolate HZZB011 (KJ807815). Phylogenetic tree generated by Neighbour-Joining method revealing the BCMV-NB isolate was grouped together with Chinese isolates from Glycine max (Supplementary Fig S1). To test the infectivity of BCMV-NB, virus-free yam bean (cv. Mumashan) and Nicotiana benthamiana seedlings were mechanically inoculated with sap extracted from the symptomatic leaves of a BCMV-NB-infected yam bean plant. The inoculated yam bean plants developed typical BCMV mosaic and chlorotic symptoms at 16 days post inoculation (dpi), while Nicotiana benthamiana had no obvious symptoms at 10 or 20 dpi (Supplementary Fig S1). BCMV infections were confirmed in yam bean plants (infection rate 6/6) and N. benthamiana plants (infection rate 8/8) by RT-PCR at 16 dpi and 10 dpi, respectively. Twelve further P. erosus plants (cv. Mumashan) were collected from a field in Ningbo city and tested by RT-PCR with BCMV-specific primer pair BCMV CP (+)/(-) (Supplementary Table 1). Eight out of the 12 samples tested positive for BCMV by PCR-gel electrophoresis (Supplementary Fig S1) and Sanger sequencing, suggesting a high incidence of BCMV infection in this field. BCMV infection in yam bean has been reported from Indonesia (Damayanti et al., 2008) and Peru (Fuentes et al., 2012). To the best of our knowledge, this is the first report of BCMV naturally infecting yam bean in China. Thus, special attention and appropriate management strategies are needed to minimize the damage caused by BCMV to yam bean crops in China.

Forensic implications of the seasonal changes in the rate of development of blowfly, Chrysomya megacephala (Fabricius) (Diptera, Calliphoridae)

Studies on the development rate of Chrysomya megacephala (Fabricius) suggested that the blowfly as a significant candidate for forensic investigations. Under natural ambient conditions development rate of C. megacephala in monsoon, winter and summer seasons indicated significant differences among seasons. The larvae began pupation at 92nd h in summer, 157th h in the monsoon season and 191st h in winter. Rapid larval growth in terms of length was observed in summer. During summer, the length of the larvae increased to a maximum of 13.9 mm at 54th h. Time taken for the emergence of the adult fly was 164, 249 and 311h in summer, monsoon and winter seasons respectively. Life table studies were conducted to assess the percentage survival and mortality by recording the survival rate of different development stages. Molecular diagnosis of species was done using COI gene. The analysis included molecular sequences of other samples of the same species from different regions of India. The neighborjoining method allowed us to identify the species at molecular level with precision and accuracy.

Identification of Potential Isolate Phosphate Solubilizing Bacteria Used 16S rRNA Gene

Plants always interact with microorganisms either negatively or positively during their life cycle. The Phosphate Solubilizing Bacteria (PSB) group is one example that has some beneficial effects on the plant. PSB can transform insoluble soil inorganic phosphates into soluble phosphorous so plants can absorb them. This research aimed to test the isolate's ability to solubilize phosphorous and use the 16S rRNA marker gene to identify it. Testing the ability of bacteria to solubilize phosphorous on Pikovskaya's agar medium used a qualitative method by observing the halo zone in the colony around it. Then the bacterial isolate was identified by analyzing the result of sequencing the 16S rRNA gene. The RZ02 bacterial isolate showed the highest performance in solubilizing phosphorous, with a 9.1 mm solubilization index. The gene of 16S rRNA was carried out by using primers 27F and 1492R with an amplicon size of 1500 base pairs. Sequencing analysis and construction of a phylogeny tree were conducted by the Mega X neighbor-joining method and showed 100% similarity with Pseudomonas aeruginosa OIS 481. The solubilization index indicated that the bacteria had a potential candidate to be one of the inoculant components for biofertilizer.

Isolation and screening of microorganisms for high yield of succinic acid production.

This study involves the isolation of succinic acid (SA)-producing microorganisms from different samples, including the rumen, sludge, soil, and wastewater. For primary screening, 29 isolates exhibited a zone of clearance around the colony, indicating acid production. For secondary screening using thin-layer chromatography, only two isolates symbolized SA production according to their Rf values. These two isolates were further identified as Bacillus velezensis and Enterococcus gallinarum by phylogenetic analysis using the neighbor-joining method. The high SA concentrations of 50.2 and 66.9g/L were produced by B. velezensis and E. gallinarum with an SA yield of 0.836 and 1.12g/g glucose, respectively. The high SA concentration from these newly isolated strains was achieved with a low formation of unwanted acids compared with those from Actinobacillus succinogenes ATCC 55618. Moreover, E. gallinarum was cultured in palm oil mill wastewater (POMW) and molasses, which were cheap substrates. The high SA production of 73.9g/L with low other acids (the ratio of SA to total acids=0.917) was achieved using POMW and molasses (80:20) as substrates.

Occurrence of Entomopathogenic Fungi from Natural Ecosystems in Phetchabun, Thailand and Their Virulence Against Brown Planthopper

Brown planthopper (BPH), Nilaparvata lugens Stål, is a serious insect pest of rice. The invasion of BPH causes heavy losses in rice production, both quality and quantity. The objective of this research was to search for Metarhizium spp., a genus of entomopathogenic fungi in the Clavicipitaceae family, from forest soil in three districts of Phetchabun Province, Thailand: Khao Kho, Lom Kao and Nam Nao to control BPH. Metarhizium spp. fungi were isolated by soil dilution plate technique and determined their efficacy using conidia suspension at the concentration of 1X108 conidia/mL on BPH at the 2-3th instar nymph fed on seedlings of susceptible rice cultivars, Taichung Native 1 (TN1). The most effective Metarhizium sp. isolate was identified using the ITS region of 18S rDNA sequencing, Basic Local Alignment Search Tool (BLAST) and evolutionary history. The result showed that 126 isolates of Metarhizium spp. were found and coded as PB-01 to PB-126. All isolates infected BPH nymph with 26.7 to 100% mortality within 6 days after contacting conidia suspension. The isolate of PB-75 showed the highest efficacy (100% BPH mortality) with the lethal times of 50% mortality (LT50) within 2.9 days. The species identification showed that the DNA sequence of Metarhizium sp. isolate, PB-75, was 98.6% similar to M. anisopliae Genbank ID JQ889704.1. Evolutionary history based on phylogenetic analysis using neighbor-joining and maximum parsimony methods confirmed that PB-75 formed the same phylogenetic clade with M. anisopliae Genbank ID JQ889704.1 and M. anisopliae var. anisopliae.

Evaluation of DNA barcodes in discriminating Quercus species from Lam Dong, Vietnam

DNA barcoding is a tool for species discrimination and identification which helps overcome the problem of identification that cannot be covered by morphological identification. Quercus is the second biggest genus of Fagaceae in Vietnam as well as Lam Dong province after Lithocarpus. However, the species discrimination study of the Quercus species in Vietnam and Lam Dong province has yet to be well uncovered due to ambiguous species boundaries and the lack of universal molecular markers. In this study, the DNA barcodes were tested to discriminate among the species of the Quercus genus in Lam Dong province. A total of sixteen and two samples of the Quercus and Lithocarpus genus (out-group) were tested using matK, rbcL, and internal transcribed spacer (ITS). Of which, the new sequences in this study were sequenced from six species and one unknown species of Quercus, the rest was retrieved from GenBank. The BLAST, neighbor-joining, and Bayesian methods were employed to examine species discrimination success. The results showed that ITS was an efficient single-locus barcode for Quercus species by yielding the highest rate of universality as well as the best discriminatory and authentication power among the barcodes examined. In addition, the combination of ITS+matK+rbcL achieved the highest species discrimination. Therefore, matK and rbcL should not be used as DNA barcodes for the species identification of Quercus, whereas the combination of three genes that were proposed in this study is the most suitable DNA barcode for identifying Quercus species in Lam Dong province.

Genetic characterization of Staphylococcus aureus isolated from Norway rats in Boston, Massachusetts.

Despite the importance of domesticated animals in the generation and transmission of antibiotic-resistant Staphylococcus aureus, the role of wild animals, specifically rodents, in the ecology of S. aureus remains unclear. We recovered and genotyped S. aureus isolates from wild Norway rats (Rattus norvegicus) in Boston, Massachusetts to examine genetic relationships between common human and animal S. aureus isolates in a large US metropolitan area. We collected and necropsied 63 rats from June 2016 to June 2017. Nasal, foot pad, fur, and fecal swabs were collected. Staphylococcus aureus was isolated using culture-based methods and polymerase chain reaction confirmation. S. aureus isolates were spa typed, tested for antibiotic susceptibility, and whole genome sequenced. Assembled sequences were uploaded to the Comprehensive Antibiotic Resistance Database to identify antibiotic resistance elements. A phylogenetic tree was constructed using the neighbor-joining method with the maximum composite likelihood distance in MEGA7. We recovered 164 Gram-positive bacterial isolates from Norway rats. Nineteen isolates from eight individual rats were confirmed as S. aureus (prevalence: 12.9% (8/63)). All S. aureus isolates were methicillin-susceptible S. aureus (MSSA), pvl-negative, and resistant to penicillin. Two isolates displayed resistance to erythromycin. Four different S. aureus spa types were detected (t933, t10751, t18202, and t189). Thirteen unique antibiotic resistance elements were identified, and all isolates shared genes mepR, mgrA, arlR, and S. aureus norA. Phylogenetic analysis if the 19 S. aureus isolates revealed they were genetically similar to four clades of S. aureus with similar resistance gene profiles isolated from both human- and animal-derived S. aureus, as well as formed a distinct phylogenetic cluster composed only of rat isolates. Wild rodents may serve as a reservoir or vector of antibiotic resistance genes in the urban environment with relevance for human and animal health.

Mitochondrial COI and Cytb gene as valid molecular identification marker of sandfly species (Diptera: Psychodidae) in China

The accurate identification of sandfly species is crucial because some species transmit medically significant diseases, including leishmaniasis, bartonellosis and sandfly fever. However, due to the high similarity of the external morphology in sandfly species, identification can only be performed using internal morphological characteristics after dissection, which is time consuming and requires highly experienced staff. Thus, the introduction of suitable molecular markers may solve these identification problems. This study screened suitable DNA barcodes to identify common sandfly species in China. The phlebotomine sandflies were collected from Sichuan, Henan and Hainan Provinces from 2014 to 2016. The species were identified by the morphological characteristics of the pharyngeal armature and spermatheca. The genomic DNA of sandfly was extracted individually, and mitochondrial DNA (mtDNA) cytochrome C oxidase subunit I (COI) and cytochrome B (Cytb) as well as the 18S subunit of ribosomal DNA (rDNA) were amplified using polymerase chain reaction (PCR). Additionally, intraspecific and interspecific differences (p-distance) were calculated to evaluate the feasibility of the three gene fragments as a DNA barcode. The phylogeny trees of all sandfly species in this study were constructed using neighbor joining (NJ) method. Six species were identified by the morphological features, belonging to Phlebotomus and Sergentomyia, as Ph. chinensis s. l., Ph. stantoni, Se. bailyi, Se. iyengari, Se. squamirostris, and Se. squamipleuris. Analysis based on three gene fragments revealed some degree of intraspecific polymorphism among these sandfly species in China. The largest intraspecific variation occurred in Ph. chinensis s. l. (mtDNA COI, p-distance = 0.042; mtDNA Cytb, p-distance = 0.071), but the 18S rDNA fragment showed a small variation (p-distance = 0.005). The ranges of interspecific p-distances for mtDNA COI and mtDNA Cytb were 0.138 - 0.231 and 0.128 - 0.274, respectively. However, the interspecific p-distances of 18S rDNA are relatively low ranging from 0.003 to 0.055. Both mitochondrial COI and Cytb gene fragments are valid molecular identification markers in theses sandfly species. The topological structure of phylogeny trees based on mtDNA COI, mtDNA Cytb and 18S rDNA genes were all consistent with morphological classification. And we also found there were significant intraspecies differences within Ph. chinensis s. l. (0.006-0.071) and Se. bailyi (0.002-0.032) based on mtDNA Cytb gene fragment. Sequence alignment data suggested that Ph. chinensis s. l. from Sichuan should be Ph. sichuanensis, and the sandfly specimen collected from Henan was Ph. chinensis s. s.. There could be cryptic species in Se. bailyi from China.

Homologues of octopine/vitopine synthase genes in natural GMOs

The process of horizontal gene transfer causes the appearance of natural genetically modified organisms. At the moment, it is known that over 7% of dicotyledonous plant species are naturally transgenic, i.e. nGMO [1]. These plants contain the genes of agrobacteria, which are integrated in the nuclear genome during infection. In some species of naturally transgenic plants, agrobacterial genes have been preserved for millions of years of evolution. Among these genes, genes encoding octopine/vitopine synthase (ocs/vis) can be distinguished [2].
 The study of homologues of octopin/vitopin synthase genes in naturally transgenic plants: their structures and diversity, products of encoded enzymes will allow us to establish the functions and evolutionary role of homologues in nGMO. Currently, bioinformatic and genetic engineering methods are used to solve these problems.
 ocs\vis-like were found in 7 species: Albizia julibrissin Durazz., Cenostigma pyramidale (Tul.) Gagnon G.P.Lewis, Paulownia fortunei (Seem.) Hemsl., Pterocarya stenoptera C.DC., Rehmannia glutinosa Steud., Santalum album L., Viscum album L. In total twenty one ocs/vis sequences are known in 17 nGMO species. Twenty sequences are intact. This may indicate the functional significance of these genes for nGMO.
 Phylogenetic analysis of currently known ocs/vis-like genes of Agrobacterium, Rhizobium and natural GMOs suggests that diversity of studied genes is wider, than it was estimated based on agrobacterial sequences. On the phylogenetic tree constructed by the neighbor-joining method, 6 clusters for ocs/vis can be distinguished. Three clusters contain nGMOs and agrobacteria, showing the relationship of the T-DNA sequences of nGMO with those of currently known strains of of Agrobacterium/Rhizobium. Three clusters contain only nGMOs. One of them consists of species that belong to the Cannabaceae family. Other clusters are heterogeneous. No significant ecological similarities were found among the studied species.
 The obtained results can be used to study the diversity of ancient and modern strains of agrobacteria, their host specificity and the possible role of their genes in plant evolution.
 The work was supported by the RSF, grant 21-14-00050 and Research Resource Center for molecular and cellular technologies of Saint Petersburg State University.

Scaling neighbor joining to one million taxa with dynamic and heuristic neighbor joining.

The neighbor-joining (NJ) algorithm is a widely used method to perform iterative clustering and forms the basis for phylogenetic reconstruction in several bioinformatic pipelines. Although NJ is considered to be a computationally efficient algorithm, it does not scale well for datasets exceeding several thousand taxa (>100 000). Optimizations to the canonical NJ algorithm have been proposed; these optimizations are, however, achieved through approximations or extensive memory usage, which is not feasible for large datasets. In this article, two new algorithms, dynamic neighbor joining (DNJ) and heuristic neighbor joining (HNJ), are presented, which optimize the canonical NJ method to scale to millions of taxa without increasing the memory requirements. Both DNJ and HNJ outperform the current gold standard methods to construct NJ trees, while DNJ is guaranteed to produce exact NJ trees. https://bitbucket.org/genomicepidemiology/ccphylo.git. Supplementary data are available at Bioinformatics online.

Identification of Euseius nicholsi and Euseius oolong (Acari: Phytoseiidae) by using integrative taxonomy

Several specimens of an Euseius sp. were collected from Bajiaozhai National Forest Park (Guilin, China). Preliminary morphological observations showed that the species is very similar to Euseius nicholsi (Ehara and Lee, 1971) which is frequently reported from China and other far eastern countries, as well as Euseius oolong Liao and Ho, 2018, a species recently described from Taiwan. However, some morphological differences such as the body size, reticulation on the dorsal plate, shape of calyx of spermatheca etc., existed between these two species. In order to ascertain whether these morphological differences are representing different or a single species belonging to the genus Euseius, molecular analyses were conducted by using ITS, 12S rDNA, and mitochondrial DNA COI markers. Results revealed very low levels of genetic divergence (ITS 0–1%, 12S rDNA 0–2.37%, COI 0–2%), based on the three molecular markers, between Euseius sp., Euseius nicholsi [collected from Institute of Zoology, Guangdong Academy of Sciences (IZGAS) and from National Center for Biotechnology Information (NCBI) data], and Euseius oolong (collected from IZGAS). In addition, the phylogenetic tree constructed by using the neighbor-joining method also showed that sequences of these three populations were clustered in a single clade and parallel to out-group Euseius ovalis. Therefore, it can be concluded that Euseius sp. probably belongs to Euseius nicholsi, and Euseius oolong is a junior synonym of this species. This study clearly demonstrates the importance of integrative taxonomy for the proper identification of phytoseiid mites, one of the most important groups of predators utilized for biological control all over the world.

Wolbachia infection in field-collected Aedes aegypti in Yunnan Province, southwestern China.

Wolbachia is gram-negative and common intracellular bacteria, which is maternally inherited endosymbionts and could expand their propagation in host populations by means of various manipulations. Recent reports reveal the natural infection of Wolbachia in Aedes Aegypti in Malaysia, India, Philippines, Thailand and the United States. At present, none of Wolbachia natural infection in Ae. aegypti has been reported in China. A total of 480 Ae. aegypti adult mosquitoes were collected from October and November 2018 based on the results of previous investigations and the distribution of Ae. aegypti in Yunnan. Each individual sample was processed and screened for the presence of Wolbachia by PCR with wsp primers. Phylogenetic trees for the wsp gene was constructed using the neighbour-joining method with 1,000 bootstrap replicates, and the p-distance distribution model of molecular evolution was applied. 24 individual adult mosquito samples and 10 sample sites were positive for Wolbachia infection. The Wolbachia infection rate (IR) of each population ranged from 0 - 41.7%. The infection rate of group A alone was 0%-10%, the infection rate of group B alone was 0%-7.7%, and the infection rate of co-infection with A and B was 0-33.3%. Wolbachia infection in wild Ae. aegypti in China is the first report based on PCR amplification of the Wolbachia wsp gene. The Wolbachia infection is 5%, and the wAlbA and wAlbB strains were found to be prevalent in the natural population of Ae. aegypti in Yunnan Province.

Skin infections caused by Streptococcus pyogenes after the Eid Al-Adha

Introduction: Streptococcus pyogenes is Gram-positive bacteria in chain form. Infections typically begin in the throat and skin, although they present with a many different clinical cases. This study aimed to present a different clinical manifestation related to S. pyogenes. Methods: In this study, swab samples were taken from patients who came to our center with skin lesions after Eid al-Adha. Identification was performed using conventional methods (bacitracin susceptibility test and PYR test) and VITEK 2 (bioMerieux, Marcy l’Etoile, France). Phylogenetic tree was generated using the Tamura Nei model of Neighbor-joining method in MEGA 6.0 program. Results: All of the isolated microorganisms were defined as S. pyogenes. Phylogenetic tree revealed that strains were highly related, but there were genetic differences between the strains. Conclusion: While Orf virus (Parapoxvirus) is the most common cause of skin lesions after Eid al-Adha, S. pyogenes was found to be the causative agent in our study. To our knowledge, cases of S. pyogenes skin infection due to religious practices have been reported for the first time.

Phylogenetic Analysis of Small Ruminant Lentiviruses Originating from Naturally Infected Sheep and Goats from Poland Based on the Long Terminal Repeat Sequences.

Previous gag and env sequence studies placed Polish small ruminant lentiviruses (SRLVs) isolated from sheep and goats in subtypes B1, B2, A1, A5, A12, A13, A16-A18, A23, A24 and A27. This study extended the genetic/phylogenetic analysis of previously identified Polish SRLV strains by contributing long terminal repeat (LTR) sequences. A total of 112 samples were analysed. Phylogenetic analyses were carried out on the LTR fragment using the neighbour-joining, maximum likelihood, and unweighted pair group method with arithmetic mean methods. Polish caprine and ovine LTR sequences clustered within group A and grouped in at least 10 clusters (subtypes A1, A5, A12, A13, A16-A18, A23, A24 and A27). Most of the Polish strains (78%) belonged to the same subtype by the indication of the gag, env and LTR genomic regions. Discrepancies in affiliation depending on the particular sequence were observed in 24 (21%) strains, most of which came from mixed-species flocks where more than one SRLV genotype circulated. Sequences of the LTR reflected subtype-specific patterns. Several subtype-specific markers were identified, e.g. a unique substitution of T to A in the fifth position of the TATA box in A17, A27, A20 and B3. This study provides valuable insights into the genetic diversity of SRLV field strains in Poland, their phylogenetic relationships and their position in the recently established SRLV classification. Our results confirmed the existence of the ten subtypes listed and the readier emergence of new SRLV variants in mixed-species flocks.

Bioinformatics Analysis of WRKY Family Genes in Erianthus fulvus Ness.

One of the most prominent transcription factors in higher plants, the WRKY gene family, is crucial for secondary metabolism, phytohormone signaling, plant defense responses, and plant responses to abiotic stresses. It can control the expression of a wide range of target genes by coordinating with other DNA-binding or non-DNA-binding interacting proteins. In this study, we performed a genome-wide analysis of the EfWRKY genes and initially identified 89 members of the EfWRKY transcription factor family. Using some members of the OsWRKY transcription factor family, an evolutionary tree was built using the neighbor-joining (NJ) method to classify the 89 members of the EfWRKY transcription factor family into three major taxa and one unclassified group. Molecular weights ranged from 22,614.82 to 303,622.06 Da; hydrophilicity ranged from (-0.983)-(0.159); instability coefficients ranged from 40.97-81.30; lipid coefficients ranged from 38.54-91.89; amino acid numbers ranged from 213-2738 bp; isoelectric points ranged from 4.85-10.06. A signal peptide was present in EfWRKY41 but not in the other proteins, and EfWRK85 was subcellularly localized to the cell membrane. Chromosome localization revealed that the WRKY gene was present on each chromosome, proving that the conserved pattern WRKYGQK is the family's central conserved motif. Conserved motif analysis showed that practically all members have this motif. Analysis of the cis-acting elements indicated that, in addition to the fundamental TATA-box, CAAT-box, and light-responsive features (GT1-box), there are response elements implicated in numerous hormones, growth regulation, secondary metabolism, and abiotic stressors. These results inform further studies on the function of EfWRKY genes and will lead to the improvement of sugarcane.

Mitochondrial DNAD-loop hyper-variable region 1 variability in Kurdish horse breed.

Kurdish horse is one of the most valuable horse genetic resources in the Middle East. To assess the genetic diversity of Kurdish horses, Mitochondrial DNA D-loop hyper-variable region1 (HVR1) was sequenced in 29 non-related Kurdish horses which were sampled from diverse geographic regions of Iran. Total DNA was extracted from the collected blood samples by modified salting out method. The HVR1 was amplified by PCR and then sequenced using ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit. Consequently, the sequences were trimmed to 294bp using BIOEDIT to become comparable with other reported HVR1 sequences in GeneBank. Sequence alignment was performed using CLUSTALW package. Haplotype and nucleotide diversity were estimated using DNASP5.10 and phylogenetic tree was constructed by neighbour joining method. Fourteen different haplotypes and 22 polymorphic sites were detected. Haplotype diversity, nucleotide diversity and Tajima D values were 0.901 ± 0.001, 0.01153 ± 0.0020 and -1.378, respectively. Kurdish horse showed a high haplotype and low nucleotide diversity. The compositional frequency of consensus sequences for base A was the highest (29.93%) compared to other three nucleotides (C = 28.91%, T = 26.53% and G = 14.63%). As expected, all of the detected Kurdish horse haplotypes belonged to haplogroup K (i.e., Kurdish horses). According to the phylogenetic analysis, Kurdish horses were genetically more closely related to Tibetan, Chinese, Bulgarian and Iranian native horse breeds, compared to other Asian horse breeds, but some traces of European horse breeds were detected in their maternal lines.

First report of Phytophthora blight caused by Phytophthora nicotianae on Daphne odora in China.

The variegated leaves and fragrant flowers of Daphne odora var. marginata Mak. make it a popular garden plant. In May 2020, we found diseased D. odora plants in a greenhouse at the Ganzhou Vegetable and Flower Research Institute, in southeast China; 72% of 1800 plants had Phytophthora blight-like symptoms-shrunken stems, black withered branches, wilted and dropped leaves (Fig 1a), and rotted and dark green roots. The root and stem tissue surfaces were disinfected with 75% ethanol for 30s followed by 0.1% HgCl2 for 1min, rinsed thrice with sterile water, and cultured on potato-dextrose agar (PDA) medium at 25°C. Mycelia from the diseased tissue were subcultured on fresh PDA medium, providing three colonies. White colonies (~4.1mm) were formed after 10 days at 25°C (Fig 1b). Sporangia and chlamydospores were induced by placing actively growing mycelia on PDA medium at 25°C for ~30 days and then at 45°C for ~3 days. Sporangia were ovoid to spherical and 19.33 × 20.99µm in size (Fig 1c), whereas chlamydospores were spherical and 15.68 × 16.10µm in size (Fig 1d). All three colonies resembled Phytophthora spp. Genomic DNA was extracted from isolates using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech [Shanghai] Co. Ltd.), and rDNA-ITS and β-tubulin were amplified and sequenced. BLAST analysis (GenBank) revealed that the ITS (Accession No. MZ676071) and β-tubulin (MZ748503) sequences of isolates shared the highest similarity (99-100%) with those of Phytophthora nicotianae (Duccio et al. 2015). A phylogenetic tree of the relationship between our isolate hjt3 and its close relatives within the P. nicotianae species was constructed using the MEGA X neighbor-joining method (Fig 2). The pathogen was identified as P. nicotianae based on morphological and molecular characteristics. Sequencing results of the three samples were consistent, all indicating P. nicotianae. A specimen (JXAU-H2020245) was deposited in the Herbarium of the College of Agronomy, Jiangxi Agricultural University. To confirm pathogenicity, 9-month-old healthy D. odora plants were used for stem and soil inoculation. Stems were cut ~5 cm from the soil with sterilized scalpels and inoculated with 0.8 cm diameter PDA plugs containing actively growing mycelia of isolate hjt3. The soil was sterilized and 0.8 cm PDA plugs containing actively growing mycelia were buried in the soil at ~5cm; the mycelia were in contact with the roots. Plants in both groups were treated equally; those inoculated with sterile PDA plugs served as controls. There were six plants in each group, with each experiment performed in triplicate. All plants were incubated in a greenhouse at 25-28°C. The stems shrank and began to rot rapidly after 7 days (Fig 3) and the branches turned black and withered within 2 weeks. After soil inoculation, the stems of the inoculated plants blackened and rotted in ~20 days (Fig 4) and the roots rotted and turned dark green (Fig 5). These symptoms rapidly spread to the branches. The control plants did not exhibit any symptoms. Reisolated colonies showed the same morphological traits as the isolates used for inoculation; no target colonies were isolated from the control plants. Phytophthora blight caused by P. nicotianae on D. odora has been reported in Italy (Garibaldi A, 2009) and Korea (Kwon et al. 2005). This is the first detection in China. Therefore, Phytophthora blight on D. odora caused by P. nicotianae should be monitored and controlled to promote the development of the D. odora industry.

First report of Taxus media branch blight caused by Neopestalotiopsis clavispora in China.

Taxus media Rehder is a high-value tree species and can be used for landscape afforestation and medicinal purposes considering its beautiful shape and secondary metabolite paclitaxel. In May 2019, branch blight of T. media was observed in plantations in Hangzhou, Zhejiang province, China. The lesions were brown, necrotic and sunken, and the branches were withering. Plants from five plantations were surveyed, and the disease incidence was 60%. Necrotic tissues from 5 plans from each plantation were cut into small pieces (5×5 mm), surface disinfected with 75% alcohol for 30 s and 2% sodium hypochlorite solution for 2-3 min, washed twice with sterile water, then dried on sterile filter paper. Branch pieces were aseptically transferred to potato dextrose agar (PDA) plates and incubated at 25℃ in the dark for 3 days. Five purified fungal cultures were obtained and the representative isolate (LA-01) that grew slightly more vigorously under the same condition was selected for morphological characterization and DNA sequence comparison. The colony was initially white and cottony, with circular growth, undulated edge, wavy surface and pale beige on the back. The conidia were long spindle-shaped, straight or slightly curved, 21-27 × 6-8 μm (n=50) in overall shape. They contained 5 cells with those at the apex and base hyaline. The second and third cells from the apex were darker (brown) than the fourth cell from the apex (pale brown). Two or three (most frequently observed) hyaline appendages were apparent at the apex, 16-31 μm in length. Morphological characteristics of the obtained isolate were consistent with those in the genus of Neopestalotiopsis (Solart et al. 2018). DNA of LA-01 was extracted from fresh mycelia using Minibest Universal Genomic DNA Extraction Kit (Takara, Toyoto, Japan) according to the manufacturer's instructions. The partial regions from the internal transcribed spacer (ITS), β-tubulin gene (TUB2), and translation elongation factor subunit 1-a gene (TEF1) were amplified and sequenced using universal primer pairs ITS5/ITS4, BT2A/BT2B, and EF1-526F/EF1-1567R (Maharachchikumbura et al. 2012), respectively. The resulting nucleotide sequences were individually subjected to BLAST searches in GenBank. The nucleotide sequences of ITS (MZ148633, 554 bp), TUB2 (MZ286974, 473 bp ), TEF1 (MZ286975, 1030 bp) of the isolate LA-01 showed 99.64% similarity to ITS sequence (MW512858.1), 99.35% similarity to TUB2 sequence (MH423961.1), 98.18% similarity to TEF1 sequence (KU096881.1). The multi-locus phylogenetic tree was constructed based on the combined ITS-TUB2-TEF1 sequences using MEGA 7 using the neighbor-joining method, indicating that LA-01 represents Neopestalotiopsis clavispora. Inoculation was done on branches of ten T. media plants in the field to verify the pathogenicity of LA-01. Two healthy branches of each plant were surface sterilized with 75% ethanol allowed to dry, and two small wounds on each branch were obtained by puncturing with a sterilized needle. One wound was inoculated with 5-mm-diameter PDA discs excised from 7-day-old cultures, and wrapped with parafilm; the other inoculated with sterile PDA plugs as control, and wrapped with parafilm. Disease symptoms developed on all N. clavispora-inoculated wounds 3 days after inoculation. The yellow brown lesions became apparent 20 days later, whereas the control wounds remained asymptomatic. The fungus N. clavispora was reisolated from the diseased lesions using the same methods above, fulfilling Koch's postulates. Previously, the species was reported to cause leaf spot disease on another Taxus tree species T. chinensis in China (Wang et al. 2019). This is the first report of T. media branch blight caused by N. clavispora worldwide. Special attention should be paid to the damage it may cause to T. media producers, and precautionary monitoring on T. media population will be a significant part of the conservation efforts in China.

Stemphylium lycopersici and Stemphylium solani improved antioxidant system of soybean under chromate stress

Ecologists around the world are giving great attention to the metal pollution of agronomic soil. Recently, several techniques have been employed to remediate heavy metals, but the use of microorganisms is cheap, less time-consuming, and easily available. In the current study, the endophytic strains, Cp1 and Cp2 were isolated from sterilized 1-5 cm long root and leaf segments of Chlorophytum comosum using Hagem media. To get pure colonies, the strains were repeatedly cultured on potato dextrose agar (PDA) media. The strains Cp1 and CP2 were identified as Stemphylium lycopersici and Stemphylium solani based on ITS sequencing and neighbor joining (NJ) method. Both strains showed a growth-promoting potential in soybean seedlings exposed to chromate (Cr) stress. Moreover, S. lycopersici and S. solani improved the Indole-3-acetic acid (IAA), flavonoids, phenolics, protein, and proline contents, whereas, lowered Salicylic acid (SA) production in the seedlings. The selected endophytic fungal strains also promoted the antioxidant system of soybean seedlings through enhanced production of ascorbic acid oxidase (AAO), catalases (CAT), peroxidase, and free radical scavenging enzymes. Both strains bio-transformed the toxic Cr-VI to less toxic Cr-III in the cultural filtrate as well as host plants. In fact, efficient uptake of Cr and its conversion by the isolated endophytic fungal strains could be used as a viable tool to remediate Cr contamination in agricultural soils.

Characterization of avain metapneumovirus subgroup C isolated from chickens in Beijing, China

Avian metapneumovirus (aMPV) is an important causative agent that causes acute respiratory disease and egg-dropping in chickens and turkeys. Here, we characterized an aMPV subgroup C (aMPV/C) from 320-day-old broiler breeder chickens with severe respiratory diseases in Beijing, China, as evidenced by RT-PCR typing and confirmation of the nucleoprotein (N) gene sequence. The N gene sequence of the aMPV/C strain (designated BJ17) exhibited no deletions or insertions and possessed 94.6% to 99.6% identity to those of published aMPV/C isolates. The phylogenetic tree of the nucleotide sequences constructed using the neighbor-joining clustering method showed that the BJ17 strain formed one cluster with other aMPV/C viruses and formed one subcluster with published Chinese aMPV/C isolates regardless of Muscovy duck or chicken origins. Comparative analysis of the N proteins showed that a unique amino acid residue D at position 110 might be associated with regional distribution due to its occurrence in all the Chinese aMPV/C isolates only. Strain BJ17 was successfully isolated by cultured Vero cell passage and further inoculated in 3-wk-old specific-pathogen-free chickens for the examination of pathogenicity. Animal experimental results showed that BJ17-inoculated chickens had severe respiratory diseases and inflammatory lesions, as demonstrated by pathological changes and aMPV antigen in the nasal turbinate, tracheae, and lung tissues. These results enrich the available information regarding the epidemiology and pathogenicity of aMPV/C in chickens, which may facilitate the development of effective measures against aMPV/C infection in China.