Neighbor-joining Method Research Articles

Macrophytes in Northern Himalayan Dal Lake of Kashmir valley identified through DNA barcoding shows high antioxidant potential

Aquatic macrophytes, the essential biomarkers of environmental conditions and long-term water quality indicators provide habitat to many invertebrates and also play a vital role in biodiversity conservation. Therefore, the current study was focused on identifying the new novel macrophytes of Dal Lake, their sequence characterization, and antioxidant profiling. Twenty different macrophyte leaf isolates were collected from Dal Lake and DNA was extracted from fresh and young leaf samples of different macrophytes followed by PCR amplification using Primer pairs K-Lab-Matk-F and K-Lab-Matk-R. The purified PCR products were outsourced for sequencing. Phylogenetic tree was constructed based on neighbour-joining (NJ) method. Moreover, antioxidant profiling of twenty different types of leaves of macrophytes was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The present investigation revealed the taxonomy of the species collected from Dal Lake based on MatK region. The current research findings identified six new macrophyte species viz., Trapa bicornis (Osbeck.), Lycopus uniflorus (Michx.), Carex vestita (Willd.), Potamogeton praelongus (Wulfen), Ceratophyllum platyacanthum (Cham), and Phragmites pusillus, showed antioxidant potential that can be used as superfoods, source of nutraceuticals, and pharmaceuticals. This research created a path for identifying bioactive compounds of newly identified macrophytes that can impart antioxidant and anticancer characteristics to these macrophytes.

Genetic variation of pest fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in different landscapes in Bogor

Spodoptera frugiperda is an invasive pest from the American continent that attacks corn (Zea mays) and rapidly invaded Africa and Asia. Two main factors that support migration and population distribution of this species are suitable habitats and human activities. To date, two genetic strains of S. frugiperda have been found in corn in Indonesia: the corn strain (CS) and the rice strain (RS). The most accurate gene markers to detect these strains are COI and Tpi, which are located in mitochondria and Z chromosome. This study aims to determine the existing strains of S. frugiperda and their distribution in various landscapes in Bogor Regency. The research was conducted from July 2020 to December 2021 in Bogor, West Java. Sampling of S. fungiperda was carried out from corn plants in Leuwisadeng, Pamijahan1, Pamijahan2, Kemang, Tenjolaya, Dramaga, Cigombong, Cijeruk, Tamansari, and Ciomas. Larval samples were collected and preserved using 96% ethanol, followed by DNA extraction, DNA amplification, electrophoresis, and DNA sequencing. Distribution data were analyzedusing QGIS and Google Earth Pro programs, and statistical analysis was performed using SPSS 22. Sequence data were edited using GeneStudio, aligned using ClustalW in BioEdit, and the phylogeny tree was reconstructed using the neighbor-joining method (bootstrap 1000x) using MEGA X. The obtained sequences were compared with sequences from the GenBank® database. The results showed the presence of two distinct strains of COI (COI-CSh4 and COI-RS) and one strain of Tpi (Tpi-C) in Bogor. The study found no relationship between thelandscape structure and genetic variation of S. frugiperda.

Characterization, Amplification, and Phylogenetic Analysis of Gossypium herbaceum Using rbcL Molecular Marker

People have used medicinal plants for centuries to produce traditional remedies that greatly interest modern health care. One of these plants, Gossypium herbaceum or commonly called Arabian cotton, has been used in various medicinal applications. Scientists are turning to DNA barcoding, a molecular technique that identifies species using standardized DNA regions. Objective: To evaluate samples of Gossypium herbaceum and their physical properties. Methods: DNA was extracted from the plant material, and its quality and quantity were checked. Using PCR and gel electrophoresis, amplification of the RBCL gene was done. Purification of the PCR products was done for DNA sequencing. After that, all the DNA sequences were compared with the available DNA sequences in public databases. The relationship between Gossypium herbaceum and other related species was evaluated using the neighbour-joining method for phylogenetic analysis. Results: The results showed a high percentage of pairwise nucleotide sequence identity with Gossypium richmond and Gossypium hirsutum. The study demonstrated the potential of DNA barcoding using the rbcL gene as a reliable method for identifying and confirming Gossypium herbaceum. Also, this study provides valuable insight into the phylogenetic relationships of this medicinal plant species. Conclusions: The findings support the conservation and appropriate use of medicinal plants and highlight the importance of ensuring the authenticity and quality of herbal products.

Genetic characterization of fish, Cynoglossus lingua Hamilton, 1822 found in Chandipur Coast of Odisha using COI and Cytochrome b genes

Aim: In this study we characterized the fish Cynoglossus lingua collected from Chandipur Coast of Odisha using the mtDNA markers cytochrome c oxidase I (COI) and cytochrome b (Cyt-b) genes. Methodology: Samples of flat fish were collected from Balaramgadi fish landing center, Chandipur sea beach, Odisha coast. The collected specimens were initially identified based on the morphology using standard taxonomic keys. Molecular characterization was accomplished through PCR amplification of COI and Cyt-b genes. The sequences were subjected to BLAST analysis and aligned using Clustal W for homology mapping. Phylogenetic status was inferred by the neighbor-joining method using MEGA v7.0. Results: The taxonomic keys and the COI and Cyt-b gene sequences identified the specimens as C. lingua. The COI and Cyt-b gene sequences showed 98% and 99% sequence similarity with C. lingua isolate FMU.CIFRI.FF-7 COI gene (GenBank accession: MN889532.1) and the C. lingua isolate FM.CIFRI.CYTB.FF-7 Cyt-b gene (GenBank accession MT362541.1), respectively. Interpretation: The study supports the reliability of combining classical taxonomic approaches with molecular analysis using COI and Cyt-b genes for the identification of C. lingua. Key words: Cynoglossus lingua, COI gene, Cytochrome b gene, DNA barcoding, Molecular taxonomy

Characterization of two novel HIV-1 second-generation recombinants (CRF01_AE/CRF07_BC) identified in Hebei Province, China.

The unique recombinant forms (URFs) of HIV-1 consist of a mixture of subtypes, and each URF has a unique breakpoint. In this study, we identified the near fulllength genome (NFLG) sequences of two novel HIV-1 URFs (Sample ID: BDD034A and BDL060) isolated during HIV-1 molecular surveillance in 2022 in Baoding city, Hebei Province, China. The two sequences were aligned with subtype reference sequences and CRFs from China using MAFFT v7.0, and the alignments were adjusted manually using BioEdit (v7.2.5.0). Phylogenetic and subregion trees were constructed using MEGA11 with the neighbor-joining (N-J) method. Recombination breakpoints were identified by SimPlot (v3.5.1) based on Bootscan analyses. Recombinant breakpoint analysis revealed that the NFLGs of BDD034A and BDL060 were composed of CRF01_AE and CRF07_BC, containing seven segments, respectively. For BDD034A, three CRF01_AE fragments were inserted into the CRF07_BC main framework, whereas for BDL060, three CRF07_BC fragments were inserted into the CRF01_AE main framework. The emergence of the CRF01_AE/CRF07_BC recombinant strains indicates that HIV-1 co-infection is common. The increasing genetic complexity of the HIV-1 epidemic in China warrants continued investigation.

Molecular Characterization of Influenza A/H3N2 Virus Isolated from Indonesian Hajj and Umrah Pilgrims 2013 to 2014.

The Hajj and Umrah are the annual mass gatherings of Muslims in Saudi Arabia and increase the transmission risk of acute respiratory infection. This study describes influenza infection among pilgrims upon arrival in Indonesia and the genetic characterization of imported influenza A/H3N2 virus. In total, 251 swab samples with influenza-like illness were tested using real-time RT-PCR for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and influenza viruses. Complete sequences of influenza A/H3N2 HA and NA genes were obtained using DNA sequencing and plotted to amino acid and antigenicity changes. Phylogenetic analysis was performed using a neighbour-joining method including the WHO vaccine strains and influenza A/H3N2 as references. The real-time RT-PCR test detected 100 (39.5%) samples positive with influenza with no positivity of MERS-CoV. Mutations in the HA gene were mainly located within the antigenic sites A, B, and D, while for the NA gene, no mutations related to oseltamivir resistance were observed. Phylogenetic analysis revealed that these viruses grouped together with clades 3C.2 and 3C.3; however, they were not closely grouped with the WHO-recommended vaccine (clades 3C.1). Sequences obtained from Hajj and Umrah pilgrims were also not grouped together with viruses from Middle East countries but clustered according to years of collection. This implies that the influenza A/H3N2 virus mutates continually across time.

Molecular characterization of Amblyomma geoemydae using CO1 mitochondrial gene to validate phenotypic taxonomical evaluation.

Animal ectoparasites are linked to the spread of serious medical and veterinary important pathogens. Our research intends to close the knowledge gap concerning the numerous ectoparasites that inhabit animals in Wayanad. Ectoparasites in animals brought to the veterinary dispensaries in Wayanad were retrieved and identified morphologically and molecularly. Using a high-quality stereomicroscope, the taxonomic features of the four following species were examined and identified: Haemaphysalis bispinosa, Rhipicephalus annulatus, R. microplus, and Amblyomma geoemydae. The important disease vector A. geoemydae was reported for the first time in Kerala. The important phenotypic characters of the highlighted species A. geoemydae are the edge of the basis capituli is circular without cornua, and the hypostomal dental formula is 2/2. The taxonomically identified four species were subjected to CO1 gene sequence analysis. The evolutionary relationship was inspected through the neighbour-joining method, and the phylogenetic tree was built through the Maximum Likelihood method. The present study has also estimated the diversity index of R. microplus, R. annulatus, H. bispinosa, and A. geoemydae. Among them, R. microplus 0.36638 have reported with the maximum diversity index score. The significance of the study is the presence of Lyme disease vector A. geoemydae, in the Wayanad District of Kerala, and it is the first report of the species from where an outbreak of Lyme disease occurred in 2013.

Genotyping of Environmental Isolates of Acanthamoeba in Hamadan, West of Iran.

Different genotypes of Acanthamoeba have been abundantly isolated in environmental samples such as water, soil, and dust, as well as in different hospital departments and eyewash stations. This protozoan is a potential hazard for immunocompromised patients and contact lens wearers. The aim of the present study was isolation and genotyping of environmental and corneal isolates of Acanthamoeba in Hamadan, west of Iran. During 2018-2020, a total of 104 environmental samples including, water, soil, and dust and 16 corneal scraping samples were collected and investigated for the presence of Acanthamoeba using morphological and molecular identification tools. Genotypes were determined using sequence analysis of the diagnostic fragment 3 (DF3) from Acanthamoeba-specific amplimer S1 (ASA.S1) gene. Phylogenetic tree was constructed with the MEGA7 software using Neighbor-Joining method. The presence of Acanthamoeba spp. was determined in 87.5% of water, 53.1% of soil, and 25% of dust samples. From 30 dust samples collected from eight wards of three hospitals, 7 (23.3%) were contaminated with Acanthamoeba. Sequencing analysis of environmental samples revealed that the T4 genotype was the most prevalent (92.6%) one. Genotypes T2 (1.9%), T2/T6 (1.9%), and mixed T4 and T2/T6 (3.7%) were also identified in environmental samples. Acanthamoeba was seen in none of the examined corneal scraping samples from patients with suspected keratitis. The widespread occurrence of this potentially pathogenic amoeba in most hospital wards and environmental resources and areas of the region highlights a strong need to increase awareness regarding this ubiquitous amoeba among susceptible individuals, such as immunocompromised patients and contact lens wearers.

First Report of Leaf Spot Caused by Phyllosticta capitalensis on Alpinia oxyphylla Miq. in China.

Alpinia oxyphylla Miq. is mainly distributed in Hainan, Guangdong and Guangxi provinces of China. Between July and August 2021, a leaf spot disease was observed in Ledong, Hainan Province, China (18°70'20.50″ N, 109°25'25.47″E) on A.oxyphylla. The incidence of infected leaves ranged from 8% to 10%, and the incidence rate of infected plants was about 50%. Symptoms appeared as primary yellow-brown withered spots on the diseased leaves, which further developed into irregular red-brown spots. The center of the lesions was gray-black, and the tissue was irregularly necrotic, ruptured or perforated, and there were yellow chlorotic halos around the edges of the lesions (Figure 1A). Tissues 5 mm in diameter were taken from the junction of diseased and healthy tissue for pathogen isolation, Successively, a total of 8 isolates were obtained from the affected leaves. Three single spore isolates (YZ-HN-001, YZ-HN-043 and YZ-HN-051) were obtained and confirmed to be identical based on morphological characteristics. Therefore, the representative isolate YZ-HN-001 was selected for morphological and molecular identification. On Potato Dextrose Agar(PDA), the colony was gray-white at first and gradually turned dark green to dark brown with lead gray on the back, growth was slow, and mycelium was short and dense (Figure 1B and Figure 1C). Pycnidia were epiphyllous, globose, brown (about 120-140 µm in diameter), and conidia were elliptical, colorless, single celled and smooth (8-12×4-7 µm) (Figure 1D). Molecular identification was performed by partially sequencing the internal transcribed spacer gene (ITS), 18S rRNA gene and the actin gene (ACT) by using the primers ITS1/ITS4 (White et al. 1990), EF4/Fungi5 (Khodaparase et al. 2005) and ACT-512F/ACT-783R (Carbone and Kohn. 1999). The sequences of the amplified fragments were deposited in GenBank, the ITS sequence (ON005130, 616 bp) showed 100% identity with Phyllosticta capitalensis strain CGMCC3.14345 (JN791605.1), the 18S rRNA sequence (ON005129, 541 bp) showed 99% identity with P. capitalensis isolate MUCC0029 (AB454185.1) and the ACT sequence (ON049348, 251 bp) showed 100% identity with P. capitalensis strain DZSN202005-2 (MW533248.1). A phylogenetic analysis was conducted in MEGA X using the neighbor-joining method and showed that isolate YZ-HN-001 clustered together with P. capitalensis (Figure 2). Based on the above morphological and molecular characteristics, the isolate was determined to be P. capitalensis. Pathogenicity tests were conducted in three replicates by inoculating surface-sterilized leaves of A. oxyphylla. The leaves were wounded and inoculated with colonized PDA plugs (5×5 mm) from 15-day-old cultures. Control leaves wounded in the same way and were inoculated with sterile PDA plugs (5×5 mm). Leaves were moisturized by spraying with sterile water every three days. After 20 days at room temperature (23 to 28℃), similar symptoms were observed in the inoculated leaves as in the field (Figure 1E), but no symptoms were observed on the control leaves (Figure 1F). The same P. capitalensis was reisolated in the inoculated leaves, confirming Koch's postulates. Phyllosticta capitalensis has been reported to cause leaf spots or black spots on various host plants around the world (Wikee et al. 2013), including on oil palm (Nasehi et al. 2020), tea plant (Cheng et al. 2019 ), and castor (Tang et al. 2020). Nevertheless, to our knowledge, this is the first report of leaf spot caused by P. capitalensis on A. oxyphylla worldwide.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Cosmos bipinnata in Northeastern China.

Cosmos bipinnata (Cav.) is a common garden flowering plant with high ornamental value in China, and which is also widely distributed as a landscaping plant in northeastern China. During the summer of 2020, powdery mildew on C. bipinnata was observed in the garden of Best West Fortune Hotel, Harbin, Heilongjiang Province, China. More than 70% of plants in the garden were affected but at low severity, with only a few older leaves showing yellowing and wilting. Powdery mildew colonies began as small white spots on the upper surface of both young and mature leaves, and then spread to entire leaves. Conidia collected from infected plant tissues were ellipsoid-ovoid to barrel-shaped, with distinct fibrosin bodies visible in their cytoplasm, and measured 20 to 28 × 14 to 19 μm with a length/width ratio of 1.5 to 2.0 (n = 30). Conidiophores were unbranched, straight, 92 to 230 × 9 to 15 μm in size, and produced four to six immature conidia in chains (n = 30). Foot cells of conidiophores were cylindrical and 40 to 70 μm long, with light constriction at the basal septum, and followed by one to three short cells. Fungal hyphae were septate, branched, flexuous to straight, and up to 5 μm wide with indistinct to slightly nipple-shaped appressoria. Chasmothecia were absent in the collected infected samples. The morphological characteristics were consistent with Podosphaera xanthii reported by Braun and Cook (2012). For molecular identification, total gDNA was extracted from fungal colonies on infected leaves of three collections separately. For each DNA sample, the part of LSU and ITS regions were amplified using primers LSU1/LSU2 and ITS1/ITS4 (Scholin et al. 1994; White et al. 1990), respectively. BLASTn analysis of the 617bp (OP218411) and 563bp (MW166865) amplicons revealed 99.67% to 100% sequence identity with respective rDNA sequences of P. xanthii isolates present in GenBank (LSU: KY860729 and MK439610; ITS: MT242593 and MK439611). The phylogenetic trees were constructed using the neighbor-joining method in MEGA 5.0. Based on the ITS rDNA phylogenetic tree, the sequences retrieved from the specimens clustered within a strongly supported clade with P. xanthii. On the basis of morphological characteristics and molecular analysis, the isolate was identified as P. xanthii. Koch's postulates were carried out to prove the pathogenicity of the isolate. A pathogenicity test was performed by dusting conidia from an infected leaf onto young leaves of five healthy C. bipinnata plants in the greenhouse with five non-inoculated plants as a control. Powdery mildew symptoms were observed on inoculated leaves after 7 days of inoculation at 20/25°C (night/day) and 75% relative humidity, whereas control plants remained asymptomatic. The fungus on inoculated leaves was morphologically identified as that observed on the original diseased leaves. P. xanthii has been reported on several hosts in China, such as Zinnia elegans, Verbena bonariensis and Melothria indica (Fan et al. 2022; Hong et al. 2021; Zhong et al. 2022). To our knowledge, this is the first report of P. xanthii infecting C. bipinnata in northeastern China, and the information presented in this note will assist the horticultural industry on developing management strategies for this disease in China.

First report of Diaporthe tulliensis causing leaf spot of betel vine

The betel vine (Piper betle) is grown in protected areas under the humid and shaded conditions required for the plant's growth. This damp, shady environment favours the spread of numerous diseases, particularly leaf spot disease (Chattopadhayay & Maity, 1990). During 2019–2022, diseased betel leaves were collected from the East Khasi Hills and Ri Bhoi districts of Meghalaya, India. The leaves had spots that were irregular in shape and size, light to dark brown and surrounded by a chlorotic yellow halo, which gradually spread to the leaf centre followed by yellowing, drying and drooping of the whole leaf. An area of approximately of 8 m2 was affected and 5–20% of the plants had symptoms, depending on the year. The number of leaf spots increased with temperature and humidity. To identify the causal agent, lesions (5-10 mm in diameter) were cut from ten infected leaves (Figure 1), surface disinfected with 70% ethanol, rinsed three times in sterile distilled water and then plated on potato dextrose agar (PDA) amended with 30mg/ml of streptomycin sulphate. The same colony type was obtained from all leaves which was white producing yellowish-green pigments and the reverse olive green to brown after seven days (Figure 2A-B) and after 30 days of incubation at 30°C, produced semi-immersed black pycnidial conidiomata which exuded pale yellow to black conidial droplets (Figure 2C). The α-conidia were one to two-celled, hyaline, oval to fusiform, guttulate and 5.3 × 2.5 μm (Figure 2D), conidiophores hyaline but no β- and γ-conidia were observed (Li et al., 2022), and conidiogenous cells were hyaline and septate (Figure 2E-F). The identification of the fungus was confirmed by sequence analysis using ITS1 and ITS4 primers (White et al., 1990) (GenBank Accession No. OQ402579). BLAST analysis showed 100% identity with sequences of Diaporthe tulliensis (e.g. MN911384). In phylogenetic analysis, the isolate in the current study clustered with D. tulliensis using the neighbour-joining method, showing 100% identity (Figure 3). To confirm pathogenicity, the isolate was grown in potato dextrose broth at 28°C for seven days, then inoculated onto five healthy betel leaves using pin-prick inoculation with 10 μl spore suspension (1×105 spores/ml). The untreated control did not show any symptoms, however, the inoculated plants had brown necrotic spots followed by yellowing after four days (Figure 4). On PDA, cultures isolated from infected plant leaf lesions exhibited the same morphology as D. tulliensis and similar conidia were discovered during microscopic analysis subsequently satisfying Koch's postulates. To the best of our knowledge, this is the first report of D. tulliensis as a fungal pathogen of betel leaves. Diaporthe tulliensis has been reported to cause leaf spots on coffee in China (Gorg et al., 2020) and on Parthenocissus tricuspidata in Taiwan (Huang et al., 2021), cacao pod rot in Puerto Rico (Serrato-Diaz et al., 2022), and stem canker of kiwifruit in China (Bai et al., 2017) and of jasmine in Taiwan (Hsu et al., 2023). The authors thank the Head of the Department of Botany, North-Eastern Hill University for providing laboratory facilities. The first author is also grateful to the Non-Net Fellowship, New Delhi, for financial support in the form of a research fellowship.

Musk mallow (Abelmoschus moschatus): a new host of Cucurbit aphid‐borne yellows virus

Musk mallow (Abelmoschus moschatus, family Malvaceae) is a medicinal plant cultivated across Asia, Africa and South America. It is found as a weed all over the Deccan region of India (Pawar & Vyawahare, 2017). The seeds of the plant are used for medicinal purposes and as a source of fragrant compounds (Gul et al., 2011). Musk mallow plants exhibiting yellowing of older leaves, midvein clearing, leaf reduction, mottling and slight downward curling (Fig. 1) were observed in October 2022 at the experimental research farm of the Indian Agricultural Research Institute in Pune, Maharashtra, India. The plants occurred as weeds and the disease incidence was 4% (c. 16 plants) in an eight-acre area. Ten diseased and four asymptomatic plants were sampled to ascertain the viruses associated with the symptoms. ELISA was done using antisera against Cucurbit aphid-borne yellows virus (CABYV) (DSMZ, Germany), Cucurbit yellow stunting disorder virus (CYSDV) (Arsh Biotech, India), Cucumber mosaic virus (CMV), Papaya ringspot virus (PRSV) and Zucchini yellow mosaic virus (ZYMV) (Agdia, USA). CABYV was detected in all diseased samples but CMV, CYSDV, PRSV and ZYMV were not detected. None of the five viruses were detected by ELISA in the asymptomatic samples. Total RNA was extracted from both diseased and asymptomatic samples using an RNeasy Plant Mini Kit (Qiagen, USA). Two-step RT-PCR assay was done using the extracted RNA and CABYV-specific primers which amplify c. 484 bp of the partial coat protein region (Boubourakas et al., 2006). RT-PCR was also performed using primers specific for CMV, CYSDV, PRSV, ZYMV, Beet pseudo-yellows virus and Cucumber vein yellowing virus. Amplicons of the expected size were obtained in all diseased plants with CABYV primers but not with primers for any of the other viruses. No amplification was obtained in the asymptomatic samples for any of the viruses. The amplicons of five samples were Sanger sequenced bidirectionally and found to be identical (GenBank Accession No. OP455156). A BLASTn search of the sequence revealed that it shared 100% identity with an Indian CABYV sequence from Praecitrullus fistulosus (OP455157). A phylogenetic tree was constructed using the neighbour-joining method (MEGA X program) with CABYV sequences from different hosts and geographies, and the sequence clustered with other Indian CABYV sequences (Fig. 2). CABYV primarily infects cucurbits but also infects other crops and weeds including three Malvaceae species, Abutilon theophrasti, Gossypium hirsutum and Malva parviflora (Lecoq et al., 2022). To our knowledge, this is the first report of CABYV infecting Abelmoschus moschatus in the world. The present finding is of epidemiological importance as it indicates the further broadening of the host range of CABYV and the possibility of infecting other economically important Malvaceae members, such as G. hirsutum which is grown for cotton in India. Furthermore, musk mallow plants could serve as an alternative / reservoir host for CABYV aiding its transmission to other commercial crops. The authors thank the Director, ICAR-IARI, New Delhi for providing lab facilities.

Classical morphology and DNA barcoding based identification of freshwater ectoparasite, Argulus foliaceus in rohu Labeo rohita

DNA barcoding coupled with classical morphological description have become promising approach for the species levelidentification. In the present study, 278 numbers of rohu Labeo rohita were screened, for Argulus infestation, amongwhich 167 fish (60.07%) were found infested with Argulus. Morphological attributes of the parasites were studied by lightmicroscope (LM) and Scanning Electron Microscope (SEM) which showed dorsoventrally flattened body comprising ofhead, thorax, abdomen, a pair of large compound eyes, suctorial organs with sclerotised support structures and two openingsof spermatheca at posterior end. Posterior incision of abdomen do not reach the mid-line and the cephalo-thoracic carapacehave not extended beyond the beginning of abdomen. Further validation was done using molecular tools for accurateparasite identification. The pair-wise genetic distance value, using Kimura-2 parameter showed a species level variationof 0.001 (1%) with that of A. foliaceus, while 0.083 and 0.052 (i.e. more than 2%) with that of A. indicus and A. japonicusrespectively. Phylogenetic tree generated using Neighbour-Joining (NJ) and Maximum-Likelihood (ML) methods, withKimura-2 parameter were also in agreement with pairwise distance values. The mitochondrial cytochrome c oxidase subunit1 (COI) sequences of A. foliaceus formed one cluster with the present studied samples, whereas sequences of A. japonicusformed a sister group. Integrating morphological and rapid DNA barcoding tools, the species was delineated as A. foliaceus. Keywords: Argulus foliaceus, COI gene, DNA barcoding, Labeo rohita, Morphology

Cross-species amplification and genetic variation among blackgram genotypes using SSR markers developed from mungbean DNA sequence scaffolds harbouring putative resistance genes

The cross species amplification of 97 mungbean derived resistance gene-SSR markers were investigated for diversity analysis in a set of 44 blackgram genotypes. A total of 68(70%) SSR markers showed amplification in blackgram. Our of 68 markers, thirty randomly selected markers were used to study the genetic variation among 44 blackgram genotypes varying for yellow mosaic disease (YMD) and powdery mildew disease (PMD) reaction. Thirty SSR primers collectively amplified 90 alleles in blackgram with an average of three alleles/locus. The polymorphic information content (PIC) of the SSR markers ranged from 0 to 0.86 with an average of 0.43. Cluster analysis based on UPGMA neighbour-joining method grouped the 44 genotypes into seven clusters. The genotypes NDU-1 and PU-19 were observed to be highly dissimilar with similarity coefficient of 0.27 in comparison to other genotypes. YMD and PMD resistant and susceptible genotypes could be differentiated by three (MRGSSR 12, MRGSSR 56, MRGSSR 77) and four SSR markers (MRGSSR12, MRGSSR 32, MRGSSR56 and MRGSSR65), respectively. Two of these markers viz.,  MRGSSR12 and MRGSSR56 were mutually effective in differentiating YMD and PMD resistant genotypes. These were located in mungbean scaffolds JJMO01002369 and  JJMO01001477 and exhibited homology with TMV resistance protein N and DNA damage-repair/toleration protein DRT100, respectively. Keywords: Blackgram, Cross species amplification, Resistance genes, Genomic-SSR, Polymerase chain reaction, Diversity analysis.

First report of Aspergillus niger causing fruit rot of bilimbi in Malaysia.

In July 2019 to November 2021, symptoms of fruit rot of Averrhoa bilimbi (commonly known as bilimbi) fruits were observed in Serdang (3°00'05.8"N 101°42'18.4"E) and Tanjong Karang (3°25'55.3"N 101°12'55.7"E), Selangor, Malaysia. External decay showed some yellowish to brownish-red discoloration and inside the fruit there was black powdery sporulation and a brownish decay that measured between 8 - 15 mm wide. Twenty random symptomatic fruits were collected from each location. Small pieces (5 mm) of infected tissues from the fruit rot were surface sterilized for 1 min in 0.5% NaOCl, washed twice with sterile distilled water and cultured onto potato dextrose agar (PDA) and peptone pentachloronitrobenzene agar (PPA). The plates were incubated at 28 ± 1oC under 12 hours light/dark for 7 days. The fungal colonies growing from the plates were purified using hyphal tip technique (Leyronas et al. 2012). A total of 42 fungal isolates were obtained, and the morphology characteristics of six isolates were matched that of Aspergillus niger. The A. niger isolates were further identified based genus and species-specific Internal Transcribed Spacer (ITS) sequencing. Primers ITS1/ITS4, were used to amplify and subsequently sequenced the ITS1-5.8S-ITS2 region (White et al. 1990). Primer ASAP1 (5'-CAGCGAGTACATCACCTTGG-3'), ASAP2 (5'-CCATTGTTGAAAGTTTTAACTGATT-3') was used for Aspergillus species confirmation, primer ASPU (5'-ACTACCGATTGAATGGCTCG-3') / Ni1r (5'-ACGCTTTCAGACAGTGTTCG-3') for A. niger species-specific, ASPU / Af3r (5'-CATACTTTCAGAACAGCGTTCA-3') for A. fumigatus specific-specific and ASPU / Fl2r (5'-TTCACTAGATCAGACAGAGT-3') for A. flavus specific-specific (Sugita et al. 2004). In general, Aspergillus niger isolates grew rapidly on PDA and were visibly white initially then appearing black and powdery on the second day of incubation (Figure 1A). Some isolates grew rapidly (0.71-0.85 cm/day) and have a cottony appearance. The conidia were appeared brown to black, globose and rough with diameter ranging between 4.1-5.2 µm (Figure 1B). The vesicles were hyaline, globose, and brown in color with measurement of 30-75 µm in diameter with uniseriate sterigmata (Figure 1C). The conidial head was brownish black in color and split into several irregular and regular columns of conidial chains (Figure 1D-E). The conidiophores were hyaline, and brown in color. Phylogenetic trees of ITS (Figure 2A) and ASAP sequences (Figure 2B) were constructed using a Neighbor-Joining method showing isolates Aspergillus niger #11, #15, #32, #33, #41 and #42 were grouped into the same clade as A. niger (accession no. MT446087). To examine virulence of A. niger, pathogenicity tests were performed three times by inoculating an asymptomatic fruit with six isolates of A. niger (isolate #11, #15, #32, #33, #41 and #42) and a single isolate for each species of Aspergillus aculeatus, Lasiodiplodia theobromae and Penicillium gerundense. Ten fruits were inoculated by placing a mycelial disc (6 mm) (Kouame et al. 2010) from a 5-day-old culture of each fungal colony while control fruits were non-inoculated with any fungal colony (10 fruits were inoculated with a sterile agar disc and 10 were non-inoculated, respectively). After 3 days, typical symptoms of Aspergillus fruit rot were observed on A. niger inoculated fruits, whereas the control fruits remained asymptomatic (Figure 1F-P). Aspergillus niger was reisolated and reidentified based on morphological and molecular characterization from the inoculated, symptomatic fruits, thus confirming Koch's postulates. A. niger causing widespread diseases in various plant and it is a common contaminant of food. This study shows A. niger to be highly virulent on bilimbi fruits and leads to reduction of fruit quality and its production. To our knowledge, this is the first report of A. niger causing fruit rot on bilimbi and future work on its pathogenesis may provide strategies for disease control against the pathogen.

Isolation and Characterization of Lignocellulolytic Bacteria from Municipal Solid Waste Landfill for Identification of Potential Hydrolytic Enzyme

The utilization of lignocellulose biomass as an alternative source of renewable energy production via green technology is becoming important, and is in line with sustainable development goal initiatives. Lignocellulolytic bacteria, such as Bacillus spp., can break down biomass by producing hydrolytic enzymes, which are crucial in the successful conversion of biomass or lignocellulosic material into renewable energy. This information gave rise to this study, where municipal solid waste sediments of a sanitary municipal solid waste landfill were sampled and screened, and lignocellulolytic bacteria were isolated and characterized. Samples were taken from four different locations at the Pulau Burung landfill site in Malaysia. Lignin and starch were used as sources of carbon to identify potential bacteria that exhibit multi-enzymatic activity. The growth rate and doubling time of bacterial isolates in lignin and starch were taken as the criteria for selection. Eleven bacterial isolates were screened for cellulase activity using iodine and Congo red dyes. The cellulase activity of these isolates ranged from 0.8 to 1.7 U/mL. We carried out 16S rRNA gene sequencing to identify the phyla of the selected bacterial isolates. Phylogenetic analysis was also conducted based on the 16S rRNA sequences of the bacterial isolates and related Bacillus species, and a tree was generated using the Neighbor-Joining method. In this study, Bacillus proteolyticus, Bacillus Sanguinis, Bacillus spizizenii, Bacillus paramycoides, Bacillus paranthracis and Neobacillus fumarioli were identified as promising bacteria capable of expressing lignocellulolytic enzymes and degrading the lignocellulosic biomass present in municipal solid waste.

Start codon targeted (SCoT) variability analysis and its association with hydroxy citric acid (HCA) in Garcinia gummi-gutta (L.) Roxb.

Hydroxycitric acid (HCA) is one of the proven natural antiobesity agents enriched in the fruits of Garcinia gummi-gutta (L.) Roxb. (Family: Clusiaceae). The present research work was carried out to evaluate the genetic variability among 35 candidate plus trees (CPTs) using HCA estimated through HPLC and start codon targeted (SCoT) molecular markers. The association analysis between phenotypic and genotypic traits was also conducted. The selected CPTs showed an average HCA content of 29.11 mg/g and Gar 17 had the highest (48.32 mg/g) followed by Gar 6 (45.48 mg/g). SCoT marker analysis revealed that 19 primers, out of 30 yielded a total of 151 bands with 66.89% polymorphic bands. Principal coordinate analysis (PCoA) organized the CPTs into the four quadrants of a scatterplot irrespective of HCA content. Dendrogram based on neighbour joining method proved its reproducibility by its bootstrapping values, and it has three clusters. STRUCTURE analysis opened the probability of two assumed subpopulations within the selected individuals. Association analysis based on a general linear model (GLM) agreed with the strong association of SCoT 5d allele with HCA content, which also supports the promising nature of Gar 6 as per previous findings. Analysis based on HCA and SCoT markers was effective in tracing out the genetic variabilities among the CPTs and the marker-trait association. The findings are the first in G. gummi-gutta, best of our knowledge.

Comparison of genetic diversity and phylogenetic structure of BRCA1 gene of some domestic and wild sheep breeds in different countries

BRCA1 gene plays an important role in DNA damage repair, cell cycle, and transcription process regulation; hence it’s called gate keeper. The current research aims to perform bioinformatics analyzes of the BRCA1 gene of different breeds of domestic and wild sheep from 49 breeds in 14 countries using the NCBI genome database. The desired sequences were aligned using MEGA11 software and a phylogenetic tree was drawn by Neighbor-Joining method. The number of mutations, nucleotide diversity, and haploid diversity were also analyzed using Dnaspv5 software. The analyses showed 296 polymorphisms, which led to the creation of 45 different haplotypes with a haplotype diversity of 0.035. Nucleotide diversity and average nucleotide differences among breeds were estimated as 0.259 and 0.052, respectively. The average genetic distance within the population of countries was calculated as 0.052. The amount of sequence conservation in this research was 0.313 on average, which indicates the high polymorphism of this gene and the emergence of new proteins. Tajima’s D value in Tajima’s neutrality test was −2.421, which was significant (p < 0.05). One of the reasons for the high genetic diversity in Iran’s wild sheep population is the existence of forests and open environments, which prevent genetic drift and reduce inbreeding.

Polymorphism of E6 and E7 Genes in Human Papillomavirus Types 31 and 33 in Northeast China.

Persistent infection with human papillomavirus (HPV) types 31 and 33 is an important causative factor for cervical cancer. The E6/E7 genes are key oncogenes involved in the immortalization and transformation of human epithelial cells. Genetic polymorphism may lead to differences in the virus' carcinogenic potential, the immune reaction of the host, and the potencies of vaccines. Few studies on HPV31/33 E6/E7 genetic polymorphism have been carried out. To study the genetic polymorphism of HPV31 and HPV33 E6/E7 genes in northeast China, these genes (HPV31 E6/E7, n = 151; HPV33 E6/E7, n = 136) were sequenced and compared to reference sequences (J04353.1, M12732.1) using BioEdit. Phylogenetic trees were constructed by the neighbor-joining method using MegaX. The diversity of the secondary structure was estimated using the PSIPred server. The positively selected sites were analyzed using PAML4.9. The major histocompatibility complex (MHC) class I and MHCII epitopes were predicted using the ProPred-I server and ProPredserver. B-cell epitopes were predicted using the ABCpred server. In the 151 HPV31E6 sequences, 25 (25/450) single-nucleotide mutations were found, 14 of which were synonymous mutations and 11 were nonsynonymous. In the 151 HPV31E7 sequences, 8 (8/297) nucleotide mutations were found, 3 of which were synonymous mutations and 5 were nonsynonymous. In the 136 HPV33E6 sequences, 17 (17/450) nucleotide mutations were observed, 7 of which were synonymous mutations and 10 were nonsynonymous. C14T/G (T5I/S) was a triallelic mutation. Finally, in the 136 HPV33E7 sequences, 9 (9/294) nucleotide mutations were observed, 3 of which were synonymous mutations and 6 were nonsynonymous. C134T/A (A45V/E) and C278G/A (T93S/N) were triallelic mutations. Lineage A was the most common lineage in both HPV31 and HPV33. In all of the sequences, we only identified one positively selected site, HPV33 E6 (K93N). Most nonsynonymous mutations were localized at sites belonging to MHC and/or B-cell predicted epitopes. Data obtained in this study should contribute to the development and application of detection probes, targeted drugs, and vaccines.

Genetic Diversity of Global Faba Bean Germplasm Resources Based on the 130K TNGS Genotyping Platform

Novel germplasm resources are the key to crop breeding, with their genetic diversity and population structure analysis being highly significant for future faba bean breeding. We genotyped 410 global faba bean accessions using the 130K targeted next-generation sequencing (TNGS) genotyping platform, resulting in a total of 38,111 high-quality SNP loci by high-standard filtering. We found the polymorphism information content (PIC) and Nei’s gene diversity were 0.0905–0.3750 and 0.0950–0.5000, with averages of 0.2471 and 0.3035, respectively. After evaluating the genetic diversity of 410 accessions using Nei’s gene diversity and PIC, on the basis of their geographical origin (continent) and structure-analysis-inferred subpopulations, we found that the faba bean accessions from Asia (except China) and Europe had rich genetic diversity, while those from the winter sowing area of China were low. The 410 faba bean accessions were divided into four subpopulations according to population structure analysis and clustering analysis based on Nei’s (1972) genetic distance using the neighbor-joining (NJ) method. However, the same subpopulation contained materials from different geographical origins, thereby indicating that the gene flow or introgression occurred among the accessions. Results from NJ clustering based on shared allele genetic distance indicated that the 410 accessions were divided into three groups according to their dissemination routes. The genetic diversity analysis results demonstrated that the genetic relationships among the faba bean groups with similar ecological environments and geographic origins in neighboring regions or countries were closer and frequently found within the same group, while genetic variation among individuals was the main source of their total genetic variation.