Neighbor-joining Algorithm Research Articles

First report of Colletotrichum fructicola causing anthracnose on Pouteria campechiana in China.

Pouteria campechiana (Kunth) Baehni (=Lucuma nervosa A. DC.) is a fruit crop planted in southern China (Gao et al. 2019). It is originally from Central America, and also grown there commercially as well as in some American states (Fadzilah et al. 2018). In March 2019, a leaf spot disease was found on P. campechiana in Baoshan, Yunnan, China. Field surveys were done in a 0.06 ha orchard in Yunnan Province. Leaf spots were found on 90% of six-year-old plants in this field and were observed in other planting areas. The symptoms initially appeared as small, round, brown spots. As the disease developed, the center of the lesions was sunken with a dark brown border (Fig. 1). Under severe conditions, some spots were joined into larger irregular spots, and even whole leaves died. The disease severity of different plants varied, and some leaves showed only a few brown spots while others showed many spots. Small fragments of diseased tissues (3×3 mm) were disinfected in 75% ethanol for 10 s, 1% NaClO for 1 min, and rinsed three times in sterilized water. Then, tissues were placed onto potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 days. Fungal isolates with similar morphology were consistently recovered from diseased tissues. The 25 colonies were initially cottony, pale white to pale gray on the upper side and greyish-green with black zonation on the underside of plates. Conidia were single-celled and hyaline, aseptate, straight, and cylindrical, with rounded ends (Fig. 1B). The length and width of 200 conidia were measured for two representative isolates, DHG-1 and DHG-2, and these averaged 14.48 × 5.59 μm and 14.92 × 5.57 μm. Appressoria were ovoid, sometimes clavate, brown, averaged 7.47 × 5.86 μm and 7.25 × 5.85 μm (n=30). Brown and globose ascocarp were observed on the leaves of Pouteria campechiana. Asci were unitunicate, thin-walled, 6-8 spored, clavate, averaged 51.53×13.01 μm and 50.21 × 13.32 μm (n=30). Ascospores were hyaline, one-celled, slightly curved to curved with obtuse to slightly rounded ends, averaged 14.64×5.97 μm and 15.19 × 6.23 μm (n=30). These two isolates were selected for molecular identification. DNA was extracted from mycelia with the DNA secure Plant Kit (TIANGEN, Biotech, China). For further molecular identification, the internal transcribed spacer (ITS), partial actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), and the Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) gene (ApMat) genes of the strains (DHG-1, DHG-2) were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF1/GDR1, T1/Bt-2b, and AM-F/AM-R (Weir et al. 2012; Silva et al. 2012), respectively.The sequences were obtained and compared with GenBank and they all showed over 99% identity to the type strain of Colletotrichum fructicola ICMP 18581 (Accession nos. JX010165, JX010033, JQ807838, FJ907426, JX010405, JX009866, and FJ917508) (Weir et al. 2012). A phylogenetic tree based on the combined ITS, ACT, CAL, CHS-1, TUB2, GAPDH and ApMat sequences using the Neighbor-joining algorithm revealed that the isolates were C. fructicola (Fig. 2). The sequences were deposited into GenBank with accession MN955541, MN955542, and MN966581 to MN966592. Pathogenicity tests were conducted on eighteen healthy and tender leaves of six 1-year-old P. campechiana plants in a greenhouse. The experiment was repeated twice. The length and width of the inoculated leaves were between 8-13 cm × 2.5-3.6 cm. The epidermis of each tested leaf was lightly scratched in six separate areas with a sterilized needle. Each isolate was inoculated onto at least three wounded leaves by placing 20 μL of a conidial suspension (106 conidia/mL) on the wound sites. Control leaves were also wounded and inoculated with distilled water. All the plants were then sprayed with distilled water and covered with plastic bags. After 10 days, initial symptoms appeared as circular and deep yellow spots. After a few more days, the spots became brown, enlarged to up to 4.0 mm which was similar to symptoms observed in the field, whereas controls remained symptomless. Koch's postulates were fulfilled by re-isolation of C. fructicola from diseased leaves, and identification confirmed by sequencing. Colletotrichum fructicola has been associated with anthracnose on mango, apple, pear and cassava (Oliveira et al. 2018). To our knowledge, this is the first report of C. fructicola associated with anthracnose of P. campechiana worldwide. These results will provide crucial information for future epidemiological studies and for management of this disease.

Genetic diversity and population structure of elite drought tolerant bread wheat (Triticum aestivum L.) genotypes

The objective of this study was to assess genetic diversity and population structure of 47 bread wheat genotypes obtained from the International Maize and Wheat Improvement Centre (CIMMYT) using 10 polymorphic simple sequence repeat (SSR) markers. Data was subjected to analysis for generating a dissimilarity matrix by the Jaccard index for clustering by the Neighbour-joining algorithm on DARwin 6.5 software. GenAlex Software was used to analyse the number of detected alleles (Na), number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), genetic distance (GD), genetic identity (GI), gene flow (Nm), fixation index (F), Shannon’s Information Index (I), Analysis of molecular variance (AMOVA) and polymorphic information content (PIC). Results revealed that, at the locus level, marker Xgwm 132 had the highest Na (21), Ne (14.5), Ho (1.0) and He (0.94), while at the population level, Population III had the highest Na (21), Ne (5.59), He (0.83), and I (1.78). The mean PIC recorded was 0.80, and ranged from 0.63 (Wmc 78) to 0.93 (Xgwm 132). AMOVA revealed significant differences in genetic variation allocated within individuals (60%), across different individuals (37%) and across populations (3%) (P < 0.001). Four populations were distinguished based on pedigrees with GD ranging from 0.01 (Populations III and IV) to 0.31 (Populations II and III), while GI ranged from 0.74 (Populations II and III) to 0.99 (Populations III and IV). The selected markers successfully distinguished test genotypes with the most informative marker being Xgwm 132. Populations II and III were most distinct, thus suitable for parental selection and further drought tolerance breeding.

Streptomyces coryli sp. nov., isolated from hazelnut orchard soil

A novel actinobacteria, isolate A7024T, was isolated from commercial hazelnut orchard soil sample which was collected at Duzce, West Black Sea region, Turkey. A polyphasic taxonomic study was carried out to determine the status of this isolate. The phylogenetic tree reconstructed using the neighbour-joining algorithm based on 16S rRNA gene sequences indicated that isolate A7024T was positioned within the members of the genus Streptomyces with the highest sequence similarity (97.7 %) to Streptomyces cadmiisoli ZFG47T. The organism formed an extensively branched substrate and aerial hyphae which generated irregular rod-shaped spores with smooth-surfaces. The cell wall of strain A7024T contained ll-diaminopimelic. Glucose, mannose and ribose were detected as whole-cell sugars. Its polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, three unidentified phospholipids and three unidentified glycolipids. Major menaquinones were MK-9(H6) and MK-9(H4). The major cellular fatty acids were iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. Strain A7024T had a genome size of 9.0 Mb with a genome G+C content of 71.5 mol%. The low level of 16S rRNA gene similarity, 19.3 ± 2.3% digital DNA-DNA hybridization and 76.94 % average nucleotide identity values, as well as some different phenotypic characteristics allowed the strain to be distinguished from the closely related type strains. Therefore, it is concluded that strain A7024T represents a novel species of the genus of Streptomyces, for which the name Streptomyces coryli sp. nov. is proposed. The type strain is A7024T (=DSM 42066T=KCTC 29102T=NRRL B-24888T).

Mitochondrial ribosomal genes as novel genetic markers for discrimination of closely related species in the Angiostrongylus cantonensis lineage

The Angiostrongylus cantonensis lineage (Nematoda: Metastrongyloidea) consists of the closely related species A. cantonensis, Angiostrongylus malaysiensis, and Angiostrongylus mackerrasae. Various genetic markers have been used for species discrimination in molecular phylogenetic studies of this lineage. However, despite showing potential in other organisms, mitochondrial 12S and 16S ribosomal RNA (rRNA) genes have not been used for Angiostrongylus species discrimination. Therefore, this study assessed these genes’ suitability for inter- and intraspecies discrimination in the A. cantonensis lineage. The ultimate aim was to provide a novel genetic marker to support existing phylogenies. Sixty adult Angiostrongylus spp. worms from four geographic locations in Thailand were identified morphologically before molecular identification with 12S and 16S rRNA genes. Neighbor-joining and maximum likelihood algorithms were used for phylogenetic analyzes, and sequence variation was calculated to determine whether the genes could be used to discriminate among species. Furthermore, sequence variation was compared among previously used genetic markers to evaluate the robustness of the 12S and 16S rRNA genes as markers. Using both markers, the A. cantonensis lineage formed a monophyletic clade with a clear separation between A. cantonensis, A. malaysiensis, and A. mackerrasae. From our representative A. cantonensis and A. malaysiensis specimens, the genetic distance between the two clades was 6.8% –7.9% and 7.9% –10.0% for 12S and 16S rRNA genes, respectively, which is sufficient interspecific genetic variation for species discrimination. Higher levels of genetic variation were observed for the 16S rRNA gene, with 12 haplotypes and an intraspecific variation ≤2.2%. Thus, as a genetic marker, the 16S rRNA gene is comparable to mitochondrial protein-coding genes, which are commonly used in intra-level Angiostrongylus spp. studies. In conclusion, mitochondrial 12S and 16S rRNA genes can discriminate among closely related species in the A. cantonensis lineage, and they represent novel genetic markers for supporting existing phylogenies and verifying the phylogenetic position of A. mackerrasae.

Molecular Barcoding Reveal the Existence of Mole Crabs Emerita emeritus in North Coast of Central Java

The existence of mole crabs had been reported from the southern coast of Central Java. However, no mole crab data was available from the northern coast Central Java. This study aimed to figure out the existence of mole crabs in the northern coast of Central Java as revealed from the cytochrome c oxidase 1 (COI) barcoding. Mole crabs samples collected in Sendang Sikucing Beach Kendal. Taxonomic status of the samples was inferred from sequences similarity test using basic local alignment search tool to conspecific sequence deposited in GenBank. Pairwise genetic distances were calculated based on the Kimura 2-parameter model. A phylogenetic tree had reconstructed in molecular evolution genetic analysis (MEGA) software based on neighbor-joining algorithm. Branching reliability was obtained from 1000 bootstraps replication while branching polarity was obtained from the out-group comparison. The mole crab samples from the northern coast of Central Java had unambiguously identified as Emerita emeritus based on high sequences similarities (98.27%-99.70%), low genetic distances (0.002-0.005), and their monophyly with Emerita emeritus (KR047035) in GenBank. Pairwise analysis among each possibility pair of samples had genetic distances ranged between 0.000 and 0.005, indicated that all sample belong to single species. The data provide the first record of the existence of Emerita emeritus in the northern coast of Central Java. Our data on the existence of E. emeritus in the northern coast of Central Java is among essentials information as a scientific basis in creating policy for the management of sustainable use of the mole crabs in the areas.

Occurrence and molecular characterization of Pepper veinal mottle virus from solanaceous crops

ABSTRACT Potyviruses affect yield quantity and quality of solanaceous crops and a better understanding of their molecular properties is important for adequate disease management. Occurrence and molecular characterization of potyviruses associated with three solanaceous crops were studied. Thirty-four naturally infected leaves of pepper (Capsicum annuum L.), tomato (Solanum lycopersicum L.) and African eggplant (S. aethiopicum L.) were obtained and presence of potyviruses determined using degenerate primers targeted at the virus coat protein (CP) gene. Products were sequenced, and identities determined using the nucleotide option of the Basic Local Alignment Search Tool. Relationships between isolates were derived from phylogenetic trees using neighbor-joining algorithms. Positive amplifications were obtained only from pepper and tomato samples while sequence verification of amplicons identified only Pepper veinal mottle virus (PVMV) from eight isolates. Nucleotide and amino acid identities of PVMV CP gene ranged from 91% to 98% and 98% to 100%, respectively, confirming low genetic diversity within virus populations. Phylogenetic analyses clustered isolates into two sub-clades, indicating the presence of distinct PVMV strains. New CP-associated polymorphisms were identified in PVMV and the virus was established as the most prevalent naturally occurring potyvirus with low genetic diversity in the infected solanaceous crops. This information can be used toward the development of adequate management strategies for potyvirus infections in solanaceous crops.

Genetic diversity and similarity of pear (Pyrus communis L.) cultivars in Central Europe revealed by SSR markers

The Hungarian pear gene bank, located and founded in Keszthely in 1981, contains 210 pear cultivars including regional cultivars, cultivars of foreign origin and standard commercial cultivars. There are some cultivars with synonym names in the pear gene bank and in other pear growing areas within the Hungary. The aim of our work was to systematically analyse the genotypes of Hungarian cultivars in the pear gene bank and to set up a robust protocol for molecular identification and the interpretation of data. Eighty-eight cultivars were analysed employing eight SSR primers resulting in a total of 216 alleles. Seventy-seven cultivars were thoroughly analysed. Among the samples 29 were considered to be diploids and 59 triploids. A genetic diversity analysis was computed based on a Neighbour-Joining algorithm and combined with a PCA indicating close genetic relationship and an overall high amount of genetic diversity among the samples tested. Similarities and very close relations were verified in our studies between different pear cultivar variants: 'Korai szagos' A and B, which were planted with the same name in the gene bank. Six different 'Császár körte' and three 'Köcsög körte' cultivars were compared. It was important to establish how close their relationship was. Some cultivars originating from the same regions were compared. The 'Mezőkövesdi 2′ and '3′ are in the same main branch, however their distance is larger, the number of common alleles is less than those of the two 'Erdélyi körte' cultivars.

Characterization of the Auxin Efflux Transporter PIN Proteins in Pear

PIN-FORMED (PIN) encodes a key auxin polar transport family that plays a crucial role in the outward transport of auxin and several growth and development processes, including dwarfing trees. We identified a dwarfing pear rootstock ‘OHF51’ (Pyrus communis), which limits the growth vigor of the ‘Xueqing’ (Pyrus bretschneideri × Pyrus pyrifolia) scion, and isolated 14 putative PbPINs from the pear Pyrus bretschneideri. The phylogenic relationships, structure, promoter regions, and expression patterns were analyzed. PbPINs were classified into two main groups based on the protein domain structure and categorized into three major groups using the neighbor-joining algorithm. Promoter analysis demonstrated that PbPINs might be closely related to plant growth and development. Through quantitative real-time PCR (qRT-PCR) analysis, we found that the expression patterns of 14 PbPINs varied upon exposure to different organs in dwarfing and vigorous stocks, ‘OHF51’ and ‘QN101’ (Pyrus betulifolia), indicating that they might play varying roles in different tissues and participated in the regulation of growth vigor. These results provide fundamental insights into the characteristics and evolution of the PINs family, as well as the possible relationship between dwarfing ability and auxin polar transport.

Microbial Diversity Assessment in Milkfish Culture Ponds

Aims: To determine bacterial diversity in milkfish culture ponds that contain different life-cycle stages of the milkfish (pond A: fry, pond B: juveniles and pond C: adults) by DNA sequence analysis of organisms and compare that microbial diversity to organisms found in soil adjacent to the ponds.
 Study Design: Comparative metagenomic study of aquatic and terrestrial biodiversity based on DNA sequence analysis of water and soil DNA.
 Place and Duration of Study: SEADEC milkfish ponds in Tingnauan, Iloilo. Philippines. All water and soil samples were collected over a three-day period. 
 Methodology: DNA sequence analysis of nucleic acids extracted from water samples collected from the three types of milkfish ponds along with soil adjacent to the ponds. DNA was extracted and PCR was performed using the 11F-1492R primer pair to amplify 16S rRNA gene. Purified 16S rDNA amplicons were cloned in using the TOPO-TA cloning kit for DNA sequencing. 16s rRNA gene sequences were analyzed with the use of software tools at the National Center for Biotechnology Information website and imported into the ARB phylogenetic analysis software. Distance matrices were exported using the neighbor-joining algorithm in ARB, in the form of PHYLIP-formatted lower triangular matrices. The distance matrices were then used to calculate Shannon-Weaver and Simpson diversity indices to evaluate the richness and evenness of the sampled populations. Rarefaction curves were determined to evaluate sampling efficiency.
 Results: Rarefaction curves indicated that the sampling effort was sufficient to reveal the majority of phyla present in the sample. Shannon-Weaver and Simpson indices suggested that the diversities of all the groups were statistically different from each other. It was observed that pond A was least diverse, followed by pond C and pond B. The soil was most diverse. DNA sequence analysis identified the various species of bacteria in soil and water.
 Conclusion: All three pond communities were significantly different in diversity. This study did not identify any significant human pathogens such as Vibrios, Salmonella or Shigella. Bacterial diversity of sites decreased in the following order: soil > fry pond > fingerling pond > adult pond.

Determination and phylogenetic analysis of the complete mitochondrial genome of Bubalus bubalis Linnaeus, 1758 breed Nili-Ravi (Artiodactyla: Bovidae)

Nili-Ravi buffalo (Bubalus bubalis Linnaeus, 1758 breed Nili-Ravi, NRB) is a famous water buffalo breed in the world. It is the first time that the complete mitochondrial genome sequence of the NRB was reported. The total length of the mtDNA is 16,356 bp, It contains the typical structure, including 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes, and 1 non-coding control region (D-loop region). The overall composition of the mtDNA was estimated to be 33.11% for A, 26.45% for T, 26.55% for C, and 13.89% for G. Phylogenetic analyses using neighbor-joining (N-J) computational algorithms showed that the analyzed 18 Ruminantia species are divided into four major clades: Bovidae, Cervidae, Giraffidae, and Atilocapridae. In addition, our work confirmed that NRB has a close genetic relationship with B. bubalis isolate India 4.

SNP Mining by Genome Resequencing of 30 Apple Varieties in Shandong Province

In this article, we carried out genome resequencing and SNP mining for cultivated apples in Shandong Province for the sake of the rapid identification of apple varieties, germplasm evaluation, and utilization. Genomic DNA was extracted immediately from leaves of each sample, and Paired-end Illumina genomic libraries were prepared and sequenced on an Illumina Hiseq 4 000 platform following the manufacturer's instructions. Resequencing of the 31 apple genomes generated a total of 363 Gb high-quality cleaned sequences, with an average of 12.5 Gb per accession that represented approximately 15.9x coverage of the apple genome. The data volume fully meets the needs of downstream analysis and SNP mining. When we used the nucleotide mismatch parameter from 1~12, the mapping rate gradually increased to saturation. There was a highly significant correlation ( p <0.0001) between the total mapping rate, mapping rate of pair-end data, and mismatch parameter. Univariate fourth-order equation (regression coefficient  r >0.99) were predicted. As the mismatch rate increases, the accuracy of mapping decreases; the genome coverage gradually increases, and heterozygous sites' accuracy gradually increases. In this study, two algorithms were used in SNP mining. The intersection was further taken based on the 'chromosome+site information' as the eigenvalues to obtain a highly reliable single nucleotide variant dataset. A total of 374 404 SNP locus were detected. On average, one variation can be identified from 1 896 bp. The accuracy of the Sanger verification test is as high as 98.1%. Annotation analysis shows that among the 373 763 SNPs, 25 047 (6.7%) are located in the gene coding region, 143 269 (38.27%) are located in the intergenic region, and 179 426 (47.92%) are located in the 2 kb region upstream or downstream of the corresponding genes. Among the coding region SNPs, 13 422 are non-synonymous, while 11 625 are synonymous variations. The ratio of non-synonymous to synonymous SNP is 1.15: 1. Using the filtered 4DTV sites, population clustering analysis results constructed using neighbor-joining algorithms are in line with the trend of the classification of cultivated apples in Shandong province.

Characterization of Beta Chain Fibrinogen (FGB) Gene From Maleo (Macrocephalon Maleo S. Muller 1846) Tuva Village Gumbasa Sub-District Sigi Regency Central Sulawesi

The FGB gene is a gene that plays a role in synthesizing b-fibrinogen proteins and is often used as a molecular marker because it is useful for studying bird phylogenetics. The purpose of this study was to describe the character sequences of the FGB gene in Maleo birds (Macrocephalon maleo S. Muller 1864). This study uses laboratory exploratory methods. Alignment is done using the MEGA6 program (Clustal W). Phylogeny trees are constructed based on the Neighbor-Joining algorithm and the Juke-Cantor evolution model of the MEGA6 program. The sample in this study was 0.3 ml of blood from Maleo birds and members of the Megapodiidae group species used as a comparison. The results of this study indicate the length of the FGB 576 bp sequence and was identified with a base composition of 32.2% A, 30.4% T, 17.0% C and 20.3% G. Nucleotide composition of the sequence of Adenine-rich FGB genes. Analysis of the FGB gene sequence phylogeny tree produced a tree topology that was quite good and had the power to split at the interspecies level and place the maleo bird in its own line compared to other Megapoda genera.
 

Phylogenetic analysis of human papillomavirus proteins

Objectives Human papillomaviruses (HPVs) have evolved over millions of years, with over 200 serotypes already identified. HPVs cause oral, cervical, and any other keratinocyte cancers and are of 2 groups based on the risk of carcinogenesis; high-risk and low-risk HPVs. Differences in the evolution of high-risk and low-risk HPVs and their association with carcinogenesis in accordance with each protein expressed by HPVs are unclear. To reduce oral carcinogenesis by HPVs, information regarding evolutionary changes in HPV proteins and the mechanism underlying the acquisition of the ability for oral carcinogenesis of HPVs is needed. This study aimed to evaluate the carcinogenic potential of HPVs via phylogenetic analysis of HPVs proteins. Study Design Eight proteins of alpha HPVs were selected, namely, E1, E2, E4, E5, E6, E7, L1, and L2. The complete amino acid sequences of these 8 HPV proteins were downloaded from the UniProtKB database. HPVs were classified into 2 groups on the basis of high or low clinical risk for cancer in accordance with the International Committee on Taxonomy of Viruses (ICTV). Phylogenetic analysis of HPV genomes was performed by using Molecular Evolutionary Genetics Analysis version 7 (MEGA7) and the neighbor-joining algorithm. Bootstrap analysis of 1000 replicates was performed on each tree to determine the confidence levels. Fisher's exact test was performed for statistical analysis, with P Results Each protein from different HPV strains consistently showed evolutionary divergence into distinct lineages. Multiple sequence alignment revealed amino acid profiles showing conserved independent lineage specific substitutions. Phylogenetic analysis revealed that HPV proteins evolved in a similar manner. Conclusions The present results provide an overview of the characteristics of HPV proteins and their evolutionary changes, potentially explaining the pathogenesis of oral cancer caused by HPVs.

VCF2PopTree: a client-side software to construct population phylogeny from genome-wide SNPs.

In the past decades a number of software programs have been developed to infer phylogenetic relationships between populations. However, most of these programs typically use alignments of sequences from genes to build phylogeny. Recently, many standalone or web applications have been developed to handle large-scale whole genome data, but they are either computationally intensive, dependent on third party software or required significant time and resource of a web server. In the post-genomic era, researchers are able to obtain bioinformatically processed high-quality publication-ready whole genome data for many individuals in a population from next generation sequencing companies due to the reduction in the cost of sequencing and analysis. Such genotype data is typically presented in the Variant Call Format (VCF) and there is no simple software available that directly uses this data format to construct the phylogeny of populations in a short time. To address this limitation, we have developed a user-friendly software, VCF2PopTree that uses genome-wide SNPs to construct and display phylogenetic trees in seconds to minutes. For example, it reads a VCF file containing 4 million SNPs and draws a tree in less than 30 seconds. VCF2PopTree accepts genotype data from a local machine, constructs a tree using UPGMA and Neighbour-Joining algorithms and displays it on a web-browser. It also produces pairwise-diversity matrix in MEGA and PHYLIP file formats as well as trees in the Newick format which could be directly used by other popular phylogenetic software programs. The software including the source code, a test VCF file and a documentation are available at: https://github.com/sansubs/vcf2pop.

Diversity of Streptomyces spp. from mangrove forest of Sarawak (Malaysia) and screening of their antioxidant and cytotoxic activities

Streptomycetes have been the center of attraction within scientific community owing to their capability to produce various bioactive compounds, for instance, with different antimicrobial, anticancer, and antioxidant properties. The search for novel Streptomyces spp. from underexplored area such as mangrove environment has been gaining attention since these microorganisms could produce pharmaceutically important metabolites. The aim of this study is to discover the diversity of Streptomyces spp. from mangrove in Sarawak and their bioactive potentials — in relation to antioxidant and cytotoxic activities. A total of 88 Streptomyces isolates were successfully recovered from the mangrove soil in Kuching, state of Sarawak, Malaysia. Phylogenetic analysis of all the isolates and their closely related type strains using 16S rRNA gene sequences resulted in 7 major clades in the phylogenetic tree reconstructed based on neighbour-joining algorithm. Of the 88 isolates, 18 isolates could be considered as potentially novel species according to the 16S rRNA gene sequence and phylogenetic analyses. Preliminary bioactivity screening conducted on the potential novel Streptomyces isolates revealed significant antioxidant activity and notable cytotoxic effect against tested colon cancer cell lines (HCT-116, HT-29, Caco-2, and SW480), with greater cytotoxicity towards SW480 and HT-29 cells. This study highlighted that the Sarawak mangrove environment is a rich reservoir containing streptomycetes that could produce novel secondary metabolites with antioxidant and cytotoxic activities.

First Report of Leaf and Stem Spot on Common Bean Caused by Stemphylium lycopersici in China

Common bean (Phaseolus vulgaris L.) is one of the most economically important vegetable crops in China. In October 2018, leaf and stem spot symptoms were observed on P. vulgaris in Pozhuang County, Anqiu City, Shandong Province, China. The disease incidence ranged from 15 to 20%, causing large economic losses to growers. Lesions were initially small (1 to 2 mm in diameter), scattered, reddish-brown spots on leaves and stems. As the disease progressed, lesions expanded and merged, resulting in withering of the whole leaves and gradually drying of the whole plant. To isolate the pathogen, diseased tissue (5 × 5 mm) was cut from the margins of lesions and surface disinfected with 70% ethanol for 15 s followed by 0.1% mercuric dichloride for 2 min and then rinsed four to five times with sterile distilled water. The specimens were then transferred to potato dextrose agar plates and incubated at 28°C for 5 or 7 days. Nineteen fungal isolates with similar morphological characteristics were obtained. The colony of G9RS, a representative isolate, was white to brownish in obverse and deep red in reverse with a vivid red mycelial pigmentation that diffused into the culture medium. The conidia produced on potato carrot agar (PCA) as the standard medium were solitary, oblong, constricted at the main transverse septum, and measured 36.4 ± 7.3 × 16.5 ± 2.6 μm (n = 50). For molecular characterization, genomic DNA was extracted by CTAB (Niu et al. 2010), and sequences of the internal transcribed spacer (ITS) regions (White et al. 1990) and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes were amplified from isolates using the primer pairs ITS1/ITS4 and gpdf/gpdr (Câmara et al. 2002), respectively. The ITS and GPD sequences of isolate G9RS were submitted to GenBank (MN386223 and MN393479, respectively). BLASTn analysis of the sequences showed 99% identity with Stemphylium lycopersici (KX858848 and KR911809). Phylogenetic analysis (MEGA 7) using the neighbor-joining algorithm showed that the current isolate was clustered with S. lycopersici isolates (NO 425 and EGS46-001). Based on morphological and molecular characteristics, the isolate G9RS was identified as S. lycopersici. A pathogenicity test was conducted on intact plants according to Koch’s postulates. Conidia were collected from 21-day-old PCA cultures grown at 28°C under black light blue irradiation (365 nm) with a 12-h light/dark cycle, suspended in sterilized distilled water at 1.0 × 10⁵ conidia/ml, and the conidial suspension applied to ten 50-day-old bean plants (3 ml/plant). Ten plants inoculated with sterile distilled water served as controls. All samples were incubated in a growth chamber at 28°C. After 8 to 10 days, inoculated plants developed symptoms similar to original diseased plants in the field, whereas control plants remained symptomless. The same fungus was successfully reisolated from inoculated leaves and reidentified based on morphology and molecular evidence. The assay was conducted twice. Although S. lycopersici has been reported to cause leaf spot symptoms on other hosts, such as lettuce (Liu et al. 2019), potato (Yang et al. 2018), eggplant (Yang et al. 2017), Sansevieria (Kee et al. 2018), and pepino (Nasehi et al. 2016), to our knowledge, this is the first report of leaf and stem spot on common bean in China. Because the common bean is one of the most intensively planted crops in Shandong, this pathogen could have serious economic impact and may warrant development of integrated management strategies.

Exploring the Genetic Diversity and Population Structure of Turkish Laurel Germplasm by the iPBS-Retrotransposon Marker System

Laurel is a medicinally important plant and is known to the world for its essential oil. Turkey is the main market in the laurel leaf trade by sharing about 90% of the world trade. Here we made an effort to elucidate genetic diversity and population structure of 94 Turkish laurel genotypes collected from 26 provinces and four geographical regions using inter-primer binding site (iPBS) retrotransposon markers. A total of 13 most polymorphic primers were selected which yielded 195 total bands, of which 84.10% were found polymorphic. Mean polymorphism information content (PIC) was (0.361) and diversity indices including mean effective number of alleles (1.36), mean Shannon’s information index (0.35) and overall gene diversity (0.22) revealed the existence of sufficient amount of genetic diversity in the studied plant material. Most diversity was found in genotypes collected from the Mediterranean region. Analysis of molecular variance (AMOVA) revealed that most of the variation (85%) in Turkish laurel germplasm is due to differences within populations. Model-based structure, principal coordinate analysis (PCoA) and neighbor-joining algorithms were found in agreement and clustered the studied germplasm according to their collection provinces and regions. This is a very first study exploring the genetic diversity and population structure of laurel germplasm using iPBS-retrotransposon marker system. We believe that information provided in this work will be helpful for the scientific community to take more interest in this forgotten but the medicinally important plant.

Betaine accumulation suppresses the de-novo synthesis of ectoine at a low osmotic concentration in Halomonas sp SBS 10, a bacterium with broad salinity tolerance.

The study aims to find out osmoadaptive mechanism used to overcome the salinity stress by Halomonas sp SBS 10 isolated from the saltern crystallizer ponds of the Sambhar Salt Lake and its taxonomic position using neighbor-joining algorithm. The strain SBS 10 was tested for accumulation of two major compatable solutes betaine and ectoine and was observed that osmoprotection in the strain SBS 10 is achieved by the accumulation of betaine or by the de-novo synthesis of betaine or ectoine. Amount of endogenous content of the betaine and ectoine per milligram of cell biomass was estimated to be 581µg, 587µg, 588µg, 617µg, and 761µg for betaine and 1.52µg, 2.74µg, 3.14µg, 3.50µg, and 52.67µg for ectoine, when exposed to 5, 10, 15, 20 and 25% of NaCl concentration. Results obtained from HPLC analysis showed that the betaine accumulation suppresses the de-novo synthesis of ectoine partially at low NaCl concentration in the growth medium. However, at a high NaCl concentration, the ectoine concentration increases abruptly as compared to the betaine. This indicates that the ectoine accumulation is transcriptionally up-regulated by the salinity stress. Phylogenetic analysis based on the neighbor-joining algorithm included the strain SBS 10 in the genus Halomonas of the family Halomonadaceae belonging to the class Gammaproteobacteria. Most closely related type strain was found to be Halomonas gudaonensis SL014B-69T (98.2% similarity). Ultrastructure characteristics showed the strain to be non-spore forming rod, 0.3-0.4 × 0.75-1.65 μm in size and motile with the help of peritrichous flagella.

Histoplasma capsulatum and Pneumocystis jirovecii coinfection in hospitalized HIV and non-HIV patients from a tertiary care hospital in Mexico

BackgroundHistoplasma capsulatum and Pneumocystis jirovecii are respiratory fungal pathogens that principally cause pulmonary disease. Coinfection with both pathogens is scarcely reported. This study detected this coinfection using specific molecular methods for each fungus in the bronchoalveolar lavage (BAL) of patients from a tertiary care hospital. Materials and methodsBAL samples from 289 hospitalized patients were screened by PCR with specific markers for H. capsulatum (Hcp100) and P. jirovecii (mtLSUrRNA and mtSSUrRNA). The presence of these pathogens was confirmed by the generated sequences for each marker. The clinical and laboratory data for the patients were analyzed using statistical software. ResultsThe PCR findings separated three groups of patients, where the first was represented by 60 (20.8%) histoplasmosis patients, the second by 45 (15.6%) patients with pneumocystosis, and the last group by 12 (4.2%) patients with coinfection. High similarity among the generated sequences of each species was demonstrated by BLASTn and neighbor-joining algorithms. The estimated prevalence of H. capsulatum and P. jirovecii coinfection was higher in HIV patients.

Short Communication: Update phylogenetic information of the local varieties of cloves (Syzygium aromaticum) from North Maluku, Indonesia based on ITS sequences data

Abstract. Sundari, Nurhasanah, Mas'ud A, Amin M, Arumingtyas EL, Azrianingsih R. 2019. Update phylogenetic information of the local varieties of cloves (Syzygium aromaticum) from North Maluku, Indonesia based on ITS sequences data. Biodiversitas 20: 1510-1515. Clove (Syzygium aromaticum L. Merr & Perry) is a native spice plant species in Indonesia, especially Maluku and North Maluku. The phylogenetic relationship among local varieties of cloves in North Maluku, Indonesia is currently unclear; therefore molecular verification research is required for further conservation and breeding programs. This study aims to identify the taxonomic position and phylogenetic relationship among two valuable local varieties of cloves rom North Maluku, i.e., ’Afo’ and ’Sibela’, based on ITS sequences data. Afo clove is considered as the oldest clove native to Ternate island, and Sibela clove is the oldest clove native to Bacan Island. The phylogenetic analysis using both Maximum Parsimony and Neighbour Joining algorithms confirmed the monophyletic group of cloves (S. aromaticum) and separated to outgroup of Eugenia species. Both phylogenetic trees produced three clades with the same pattern and supported by strong bootstrap values of 99-100%.