Neighbor-joining Method Research Articles

Sequence Analysis of some species in the genus Amaranthus L. using Ribulose-1.5 - bisphosphate carboxylase/oxygenase (rbcL) genes

Bioinformatics is a novel method to investigate evolutionary relationships and genetic diversity among species. This study assessed the evolutionary patterns and genetic diversity of Amaranthus species using ribulose-1.5-bisphosphate carboxylase/oxygenase (rbcL gene). A total of 20 full FASTA-format rbcL gene sequences for accessions of Amaranthus tricolor, A. dubius, A. cruentus, A. viridis, and A. spinosus from different regions were retrieved from the National Center for Biotechnology Information. Multiple sequence alignment was used to determine genetic variations. A phylogenetic tree was constructed using neighbor-joining clustering method while a distant matrix was created using Tamura-Nei genetic distance model. From position 1 to 22 in nucleotide alignment, only accession KX082771.1 from USA, aligns. The exclusive alignment in this region suggests an exceptionally conserved sequence among species, implying a common genetic origin. However, from position 23 to 50, nine different accessions namely MN192576.1, MN204800.1, EF590496.1, GQ248546.1, OQ737000.1, MG246937.1, MN104855.1, MN104858.1, and MN04859.1 align. The phylogenetic tree representing the evolutionary relationships offers insights into the genetic diversity and relatedness of these species within specific regions. The genetic distance matrix shows that sequences within the same region share relatively high similarity scores, indicating a genetic connection likely influenced by their geographic proximity. However, sequences from different regions, exhibit moderate genetic differences, emphasizing the genetic diversity across geographically distinct regions. The observed genetic diversity and evolutionary relationships among accessions of Amaranthus revealed by rbcL genes highlight the unique genetic features of these species in different regions, and therefore should be explored for their genetic improvement and conservation purposes.

First Report of Root Rot Caused by Fusarium incarnatum on Mongolian Snake gourd in China.

First Report of Root Rot Caused by Fusarium incarnatum on Mongolian Snake gourd in China.

Monitoring and Genetic Characterization of Historical Grapevine Varieties (V. vinifera ssp.) from Styria in Slovenia

The aim of this research work was to find historical varieties that existed in this area before phylloxera and to identify them on the basis of historical written sources and genetic analyses. At the beginning of the 19th century, around 300 grape varieties were cultivated in Styria. Between 2020 and 2022, old vineyards were monitored at 115 locations in Styria (between the Mura and Sava rivers) in Slovenia. The directly collected samples (340 grapevine accessions) were determined by molecular analysis with 24 SSR markers. A total of 66 different genotypes were detected. After comparison with the available databases, 29 historical varieties and 37 unknown historical genotypes were identified. Several parameters were calculated to evaluate the usefulness of the selected loci in this work, and a dendrogram representing the genetic similarities between the origins was created using the neighbor-joining method to investigate possible ancestry relationships in the sample set. The most common historical varieties were ‘Belina’ (‘Heunisch weiss’), ‘Vrbovec’ (‘Tantovina Eihenblaetrige’), ‘Ranfol’ (‘Ranfol beli’), and ‘Pelesovna’ (‘Vulpea’). Varieties from the current variety list were also frequently found, such as ‘Frankinja’ (‘Blaufraenkisch’) and ‘Žametovka’ (‘Kavčina črna’). In a few locations, one of the most important red varieties from the beginning of the 20th century was also found in this area (alongside ‘Frankinja’ and ‘Žametovka’), i.e., ‘Vranek’ (‘Zimmettraube balu’). At that time, this variety was planted in multi-variety vineyards and was preserved, but its importance in single-variety vineyards quickly declined due to female flower. In addition, genetic analyses have shown that 37 unknown historical genotypes have been found in this area. These genotypes need to be described ampelographically and technologically evaluated in the future. Most of the vegetative offsprings of these genotypes have already been transferred to the Meranova gene bank, where they can be accurately described ampelographically under the same pedoclimatic conditions.

Exploring Aeolothrips spp. Diversity: A Morpho-Molecular Examination of Aeolothrips collaris and Aeolothrips intermedius (Thysanoptera: Aeolothripidae)

This study investigated the evolutionary relationship between Aeolothrips collaris Priesner, 1919 and Aeolothrips intermedius Bagnall, 1934 within the Thysanoptera: Aeolothripidae family. Notably, A. collaris DNA barcode was made for the first time and compared with other Aeolothrips species in GenBank. While revealing a close genetic association (82-83%) between these species, the analysis using the Neighbor-Joining method clustered A. collaris with A. albicinctus Haliday, 1836 and A. ericae Bagnall, 1920, while A. albicinctus grouped alongside A. fasciatus (Linnaeus 1758) and A. duvali Moulton, 1927 GenBank BlastN analysis supported the expected placement of A. intermedius. These insights highlight significant genetic connections but suggest the necessity for a broader examination involving more species and gene regions. Expanding this research could yield a comprehensive understanding of the intricate taxonomic relationships within this thrips genus, setting the groundwork for future investigations into thrips species evolution and taxonomy.

DNA barcoding, phylogeography and evolutionary dynamics of Chrysichthys auratus

This study embarked on an exploration into the genetic structure and evolutionary history of the Chrysichthys auratus species, leveraging PCR amplification, phylogenetic trees, and haplotype networks. Specific DNA segments were successfully amplified and visualized through electrophoresis. Newly obtained sequences were Bank into GenBank and given accession numbers (OR730807-OR730808-OR730809). The Neighbor-Joining method provided insights into the evolutionary relationships among taxa, further augmented by bootstrap values and the Tamura 3-parameter method. A comprehensive geographical haplotype network showcased pronounced genetic differentiation, especially between remote populations. Nonetheless, shared haplotypes between proximate regions indicated either ancestral genetic connections or ongoing gene flow. Employing the COI-DNA barcodes, an in-depth understanding of intra- and inter-populational genetic diversity was achieved. The study's findings unravel the intricate genetic landscape and evolutionary dynamics of C. auratus, offering novel perspectives into its demographic history across its vast native habitat.

Concatenated ScaA and TSA56 Surface Antigen Sequences Reflect Genome-Scale Phylogeny of Orientia tsutsugamushi: An Analysis Including Two Genomes from Taiwan.

Orientia tsutsugamushi is an obligate intracellular bacterium associated with trombiculid mites and is the causative agent of scrub typhus, a life-threatening febrile disease. Strain typing of O. tsutsugamushi is based on its immunodominant surface antigen, 56-kDa type-specific antigen (TSA56). However, TSA56 gene sequence-based phylogenetic analysis is only partially congruent with core genome-based phylogenetic analysis. Thus, this study investigated whether concatenated surface antigen sequences, including surface cell antigen (Sca) proteins, can reflect the genome-scale phylogeny of O. tsutsugamushi. Complete genomes were obtained for two common O. tsutsugamushi strains in Taiwan, TW-1 and TW-22, and the core genome/proteome was identified for 11 O. tsutsugamushi strains. Phylogenetic analysis was performed using maximum likelihood (ML) and neighbor-joining (NJ) methods, and the congruence between trees was assessed using a quartet similarity measure. Phylogenetic analysis based on 691 concatenated core protein sequences produced identical tree topologies with ML and NJ methods. Among TSA56 and core Sca proteins (ScaA, ScaC, ScaD, and ScaE), TSA56 trees were most similar to the core protein tree, and ScaA trees were the least similar. However, concatenated ScaA and TSA56 sequences produced trees that were highly similar to the core protein tree, the NJ tree being more similar. Strain-level characterization of O. tsutsugamushi may be improved by coanalyzing ScaA and TSA56 sequences, which are also important targets for their combined immunogenicity.

First Report of Chocolate Spot Caused by Botrytis eucalypti on Faba bean in China.

Faba bean (Vicia faba) is one of the characteristic economic crops in Qinghai Province of China, which has multiple uses as grain, vegetable, fodder, fertilizer and medicine. Chocolate spot is a critical disease of faba bean in the world, and it is widely spread in all production areas of Qinghai. In August 2021, a severe occurrence of chocolate spot was found in a faba bean field in Xunhua County, Qinghai Province (35°52'N, 102°22'E, alt. 1890m). All plants in the field were affected by this disease. A voucher specimen was deposited in the Herbarium of Plant Pathology, College of Agricultural and Forestry Sciences at Qinghai University under accession No. PY015. The pathogen infected the leaves and stems, causing small irregular red spots to appear, which later coalesce into larger spots and faded green lesions appear around the spots. Diseased leaf pieces 5 mm2 were surface sterilized with 75% ethyl alcohol for 30s, 1.2% NaOCl for 30s, and rinsed three times with sterile water. They were then plated on potato dextrose agar (PDA) at 22℃ for 10 days in the dark. Fungal colonies are initially white, then gray, and have produced spores by 5 days. Conidia are clusters, ellipsoidal or ovoid, 9-14 × 6-9 μm. The conidiophore is straight, terminally enlarged, septate, 300-1500 μm long, 8-13 μm wide. No sclerotia were observed during culture. DNA of the strain PY015 was extracted by CTAB method. Molecular identification was first performed using the universal region of ITS (ITS1/ITS4). The PCR product was sequenced, the sequence was deposited in GenBank under the accession number OR739575. The results showed 100% similarity to Botrytis spp. (KX301016, MT250940, LC519322) in BLAST search. Molecular characterization was continued using five specific primer pairs: RPB2 (DNA-dependent RNA polymerase subunit II, RPB2-5F/RPB2-7cR), NEP1 and NEP2 (necrosis and ethylene-inducing proteins, NEP1for/ NEP1revB and NEP2forD/NEP2revD), HSP60 (heat-shock protein 60, HSP60for/HSP60rev), G3PDH (glyceraldehyde-3-phosphate dehydrogenase, G3PDHfor/G3PDHrev). The sequences of PY015 were deposited in GenBank (accession numbers: OR731179, OR731180, OR731181, OR731182, OR731183), and all five sequences showed 100% similarity to Botrytis eucalypti YZU171088 (accession numbers: MH614610 MH614611, MH614612, MH614613 MH614614). A phylogenetic tree based on these five genes was constructed using Mega7.0 (1000 bootstrap replicates, neighbor-joining method), and PY015 was placed in the same clade as YZU171088 with 100% bootstrap values. Morphological and molecular biological results confirmed that isolate PY015 was B. eucalypti. To fulfill Koch's postulates, the spore suspension (2 × 105 conidia/ml) was sprayed on healthy faba bean (Yun-122) plants at the 10-leaf stage, while an equal amount of sterile distilled water was applied to controls. After 7 days, the inoculated plants showed symptoms consistent with field infection and B. eucalypti was re-isolated using the same protocol, while the control remained asymptomatic. The pathogenicity test was repeated twice. The same isolates were recovered from symptomatic leaves and identified by NEP1 sequence. B. eucalypti was morphologically and molecularly identical to the original isolates, completing Koch's postulates. Currently, Botrytis fabae, Botrytis fabiopsis, and Botrytis cinerea are the main pathogens of chocolate spot on faba bean that have been identified and reported nationally and internationally. B. eucalypti is a new species discovered from eucalyptus in southern China in 2016, and its current hosts are only eucalyptus and citrus. To our knowledge, the present study is the first report of chocolate spot caused by B. eucalypti on faba bean in China.

Whole genome analysis of Human Mastadenovirus D causing Keratoconjunctivitis in India - A multicentre study.

Human mastadenovirus (HAdV) types 8, 37, 64 have been considered the major contributors in Epidemic keratoconjunctivitis (EKC) epidemics, but recent surveillance data have shown the involvement of emerging recombinants, including HAdV-53, HAdV-54, and HAdV-56. In our initial work, positive samples for adenovirus revealed that our strains were closer to HAdV-54 than HAdV-8. Hence, the current study aimed to use whole genome technology to identify the HAdV strain correctly. Oxford Nanopore technique was used, wherein a Targeted sequencing approach using long-range PCR amplification was performed. Primers were designed using HAdV-54 (AB448770.2) and HAdV-8 (AB897885.1) as reference sequences. Amplicons were sequenced on the GridION sequencer. Sequences were annotated using Gatu software, and similarities with standard reference sequence was calculated using Bioedit software. The phylogenetic tree was built after alignment in MEGA v7.0 using Neighbour joining method for each of the genes: Penton, Hexon, and Fiber. The effect of novel amino acid changes was evaluated using the PROVEAN tool. The Recombination Detection Program (RDP) package Beta 4.1 was used to identify recombinant sequences. Of the five samples sequenced, OL450401, OL540403, and OL540406 showed nucleotide similarity to HAdV-54 in the penton region. Additionally, OL450401 showed a statistically significant recombination event with HAdV-54 as minor and HAdV-8 as major parents. This was further supported by phylogenetic analysis as well. In the present study, we have found evidence of a shift from HAdV-8 towards HAdV-54, thus stressing the need for surveillance of HAdVs and to stay updated on the rise of new recombinants.

Effect of Bacillus aryabhattai extract addition on growth and productivity of Pleurotus ostreatus ((Jacq ex Fr.) P. Kumm)

Pleurotus ostreatus (White oyster mushroom) is one of the most widely cultivated mushrooms in the world. Nutrients provided in the media are one of the most important factors affecting the growth of mushrooms. The present study mainly focuses on the isolation of mushroom growth-promoting (MGP) bacteria and studies its effects on the growth of mushroom fruiting bodies. Bacillus aryabhattai was isolated from the growth media of Pleurotus ostreatus, identified based on 16S rDNA sequencing and phylogenetic tree construction by the neighbor-joining method using MEGA version 6.06 and its effect on fruiting body growth of the P. ostreatus was investigated. The results of this research strongly suggest that the addition of specific bacteria supernatant into the mushroom growth media has beneficial applications for mushroom production.

Insights into measles virus: Serological surveillance and molecular characterization

BackgroundMeasles has been a significant public health concern in Pakistan, especially in the Khyber Pakhtunkhwa (KPK) province, where sporadic and silent epidemics continue to challenge existing control measures. This study aimed to estimate the prevalence and investigate the molecular epidemiology of the measles virus (MeV) in KPK and explore the vaccination status among the suspected individuals. MethodsA cross-sectional study was conducted between February and October 2021. A total of 336 suspected measles cases from the study population were analyzed for IgM antibodies using Enzyme-Linked Immunosorbent Assay (ELISA). Throat swabs were randomly collected from a subset of positive cases for molecular analysis. Phylogenetic analysis of MeV isolates was performed using the neighbor-joining method. The vaccination status of individuals was also recorded. ResultsAmong the suspected participants, 61.0% (205/336) were ELISA positive for IgM antibodies, with a higher prevalence in males (64.17%) compared to females (57.04%). The majority of cases (36.0%) were observed in infants and toddlers, consistent with previous reports. The majority of IgM-positive cases (71.7%) had not received any dose of measles vaccine, highlighting gaps in vaccine coverage and the need for improved immunization programs. Genetic analysis revealed that all MeV isolates belonged to the B3 genotype, with minor genetic variations from previously reported variants in the region. ConclusionThis study provides valuable insights into the genetic epidemiology of the MeV in KPK, Pakistan. The high incidence of measles infection among unvaccinated individuals highlights the urgency of raising awareness about vaccine importance and strengthening routine immunization programs.

Molecular Genetic Diversity of Manioc (Manihot esculenta Crantz) Using Simple Sequence Repeated Markers (SSR) in the Kara Region of Togo

Cassava (Manihot esculenta Crantz), a plant of the future, is one of the main crops with high genetic potential. Therefore, the knowledge of its genetic variability would be of great importance for its effective use in genetic improvement programs. The objective of this work was to characterize the genetic diversity of cassava cultivars present in the Kara region of Togo. Thus, a total of 91 cassava leaf samples collected in the 7 prefectures of the Kara region were analyzed using 7 pairs of SSR microsatellite primers. Fifty-three alleles were detected with an average of 7.57 alleles per locus. The polymorphic information content ranged from 0.63 to 0.83 with an average of 0.74, indicating a high level of marker polymorphism. Molecular analysis of variance revealed that the majority of variability occurred within individuals and accounted for 99 % of the total variation between individuals. Weak genetic differentiation (Fst=0.011) was also observed between populations. The genetic structuring model based on the Neighbour-Joining algorithm method divided the individuals into 6 groups independently of local names. These results indicate the presence of high genetic resource variability in the studied populations. Therefore, it would be important to implement management strategies to better conserve cassava genetic resources and to facilitate the identification of successful cultivars.

Coat protein of alfalfa mosaic alfamovirus (AMV) from Türkiye: genetic inference and in silico docking analysis for potential antiphytoviral purposes

In 2021, a study was conducted in the Denizli region of Türkiye to investigate the phylogenetic relationship and presence of alfalfa mosaic alfamovirus (AMV) infecting pepper plants exhibiting viral disease symptoms. A total of 57 samples were collected, of which twenty-four tested positive for AMV with reverse transcriptase polymerase chain reaction (RT-PCR) assays. Samples from pepper plants displaying virus symptoms gave positive bands on the agarose gel, while healthy plants yielded negative results. One of the positive samples was randomly selected, cloned and sequenced. This sequence of the Denizli AMV isolate (753 bp) was recorded in the GenBank database with accession number OQ845956. The nucleotide sequence showed a high nucleotide consensus of 97%-99% compared with the nucleotide sequences of the same variants from different origins in GenBank. According to the phylogenetic tree generated through the Neighbour Joining (NJ) method, this AMV isolate belongs to the same group as Iranian isolates from various of hosts. Furthermore, in silico docking analysis of the coat protein (CP) of the AMV isolate and promising 12 essential oil compounds was performed to enable potential antiviral drug development. Docking study showed that eucalyptol, eugenol and carvacrol can make important contributions to the advancement of drug-based strategies for the managing of plant viruses by interacting with the virus coat protein of high binding energies, -5.3, -5.2 and -5.0 kcal mol-1, respectively. Although the presence of AMV in Denizli province has been reported previously, this study reports the phylogenetic relationships and docking analysis of the new AMV isolate in pepper crops.

Molecular Epidemiology of HIV-1 Subtypes and Primary Antiretroviral Resistance Profiles in Northern Cyprus: First Data Series.

The distribution of human immunodeficiency virus-1 (HIV-1) subtypes indicates difference from region to region and in risk groups acquiring the disease worldwide. Although subtype C is more in terms of total cases, subtype B is dominant in certain regions, especially in western and central Europe. Molecular epidemiological studies are essential for the control, effective treatment, and understanding in transmission routes of HIV-1 infection. This study aims to determine the molecular epidemiology and antiretroviral drug resistance profiles of HIV-1 in northern Cyprus. The study involved 71 naive HIV-positive patients diagnosed in northern Cyprus between 2016 and 2022. HIV-1 subtypes and circulating recombinant forms (CRFs) were identified by phylogenetic analysis (neighbor-joining method) of pol gene sequences. Drug resistance mutations were analyzed using the World Health Organization (WHO) lists of mutations for surveillance. The Stanford University HIVdb program was used to interpret drug resistance mutations. In our study, 40 of 71 samples were successfully sequenced. Subtype B of HIV-1 was dominant with a rate of 52.5%, followed by CRF02_AG (20%) and G (7.5%) subtypes. The rate of subtype B (71.4%) in northern Cyprus was significantly higher than in the other country of origin (p = .028). Antiretroviral drug resistance was found in 15% of the sequenced serum samples. Nucleoside/nucleotide reverse transcriptase inhibitor (NRTI), non-nucleoside nucleotide reverse transcriptase inhibitor (NNRTI), and protease inhibitor (PI) resistance rates were 10% (4/40), 7.5% (3/40), and 2.5% (1/40), respectively. According to the results, it is noteworthy that the dominant subtype circulating in northern Cyprus is the B subtype, and CRFs were detected at a higher rate than expected.

Choice of Metric Divergence in Genome Sequence Comparison.

The paper introduces a novel probability descriptor for genome sequence comparison, employing a generalized form of Jensen-Shannon divergence. This divergence metric stems from a one-parameter family, comprising fractions up to a maximum value of half. Utilizing this metric as a distance measure, a distance matrix is computed for the new probability descriptor, shaping Phylogenetic trees via the neighbor-joining method. Initial exploration involves setting the parameter at half for various species. Assessing the impact of parameter variation, trees drawn at different parameter values (half, one-fourth, one-eighth). However, measurement scales decrease with parameter value increments, with higher similarity accuracy corresponding to lower scale values. Ultimately, the highest accuracy aligns with the maximum parameter value of half. Comparative analyses against previous methods, evaluating via Symmetric Distance (SD) values and rationalized perception, consistently favor the present approach's results. Notably, outcomes at the maximum parameter value exhibit the most accuracy, validating the method's efficacy against earlier approaches.

Contribution to the Molecular Phylogeny of Anthribidae (Coleoptera: Curculioniodea) Inferred from COI Sequences

Fungus weevils, Anthribidae, is a relatively small family, including about 3861 species worldwide, of the diverse superfamily Curculioniodea. Currently, three subfamilies are recognized within Anthribidae (Anthribinae, Choraginae and Urodontinae); however, this subfamily division is somewhat controversial and not accepted by all authors of the field. In this study, we constructed family level evolutionary histories within Anthribidae by using COI sequences and discussed the phylogenetic relations between these three subfamilies and also between tribes. Estimates of evolutionary divergence between sequences used in this study are calculated and the overall average divergence was 0,274 where minimum was 0,133 and maximum was 0,365. Phylogenetic and evolutionary histories were inferred by using 34 COI sequences representing most of the tribes and subfamilies currently recognized within Anthribidae. The phylogenetic trees were inferred by using Neighbor-Joining (NJ) and Maximum Likelihood (ML) methods. Both method provided quite similar evolutionary histories. According to the phylogenetic trees, Anthribinae + Coraginae members together constitute a monophyletic clade, which makes currently recognized Anthribinae paraphyletic and Coraginae polyphyletic. Urodontinae seems to form a monophyletic clade with respect to used COI sequences. Tribal classification which fully seem to depends on morphological characters are useful for practical taxonomic purposes but most tribes (those represented with more than one species/sequence) does not constitute monophyletic clades.

Occurrence of 'Candidatus Phytoplasma asteris'-Related Strains infecting Chinaberry (Melia azedarach) Showing Chlorotic Leaf Symptoms on Hainan Island of China.

Chinaberry (Melia azedarach), belonging to the family of Meliaceae, is an ornamental tree distributes across southern of China. In the autumn of 2021, In an area of 400 acres located in Wanning city of Hainan Province, a tropical island in China, with coordinates of 110°28'42.72″E, 19°2'9.96″N, about 20 % (100) of the chinaberry trees showed disease symptoms included chlorotic leaves. The disease symptoms were consistent with infections by a phloem-limited prokaryotic pathogen phytoplasma. The samples of six symptomatic and three asymptomatic were collected for pathogen detection. To identify the pathogen, total nucleic acids were extracted from 0.10 g fresh leaf tissues from the diseased and healthy plant using CTAB DNA extraction method based on Doyle and Doyle. Three primer pairs of R16mF2/R16mR1, secAfor1/secArev3 and fTuf1/rTuf1 were used for specific identification of phytoplasma conserved gene fragments of 16S rDNA, secA and tuf, PCR amplification. Target PCR bands were amplified from the DNA of six diseased chinaberry samples, but not from the DNA of the healthy samples. The products of amplified were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The phytoplasma gene sequences of 16S rRNA, secA and tuf were obtained and all the sequences were identical with the length of 1336 bp, 710 bp and 955 bp, respectively. Representative sequence data for strain MaCL-hn were deposited in Genbank under accession Nos. OR438638 (16S rDNA), OR513089 (secA) and OR860415 (tuf). The phytoplasma strain identified in the study was described as chinaberry chlorotic leaf (MaCL) phytoplasma, MaCL-hn strain. BLAST search based on 16S rRNA genes showed that 43 strains in 16SrI group 'Candidatus Phytoplasma asteris' showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on secA genes showed that 9 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on tuf genes showed that 21 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. RFLP analysis based on iPhyClassifier indicated that the MaCL-hn strain was a member of 16SrI-B subgroup with a similarity coefficient 1.00 to the reference phytoplasma strain (AP006628). Phylogenetic tree was constructed based on 16S rRNA by MEGA 11.0 using neighbor-joining (NJ) method with 1000 bootstrap value. The results showed that the MaCL-hn strains were clustered into one clade with 16SrI group 'Ca. Phytoplasma asteris' related strains with 99 % bootstrap value. Multilocus sequence analysis (MLSA) based on the concatenated sequences with the length of 3001 bp including the sequences of 16S rRNA, secA and tuf showed that the MaCL-hn strains were clustered into one clade with the phytoplasma strains in the group with 100 % bootstrap value. To our knowledge, this is the first report that chinaberry can be infected by 'Ca. Phytoplasma asteris'-related strains belonging to 16SrI-B subgroup on Hainan Island of China. This finding in the study will contribute to the epidemic monitoring and the preventive management of the phytoplasmas and their related diseases.

First Report of Bulb Rot Disease of Banana Caused by Klebsiella variicola in China.

Banana (Musa spp.) is an economically important fruit and food crop globally as well as in China. In March 2023, a bulb rot disease was observed on more than 20% of cultivated dwarf bananas in a plantation in Wuming County of Guangxi Province, a major hub of banana production in China. Infected plants showed crackles at the basal part of stem and were relatively dwarf, while yellowing of the leaves was not observed. When the rhizomes were cut open, water-soaked lesions with a yellow or black margin can be seen in the bulb. In severe infections, the internal tissue became dry or wet rot, and there was typical dark-brown cavity formation in the bulb. The rot was limited to the bulb. To isolate the causal agent, dissected diseased tissues (5×5 mm) were surface sterilized with 75% ethanol (30 s) and 2% NaClO (3 min), followed by three rinses with sterile water. The sterilized sections were soaked in 2 mL of sterile water and shaken for 5 min in a vortex oscillator. The suspension was streaked on Luria-Bertani (LB) agar medium, and incubated at 28℃ for 24 h. Single colonies were re-streaked three times to obtain purified isolation. Twelve pure bacterial cultures with similar morphology were isolated from three plants taken from the field. The bacterial colonies were yellowish white, mucoid, round, and raised with translucent surfaces on the LB agar plate. Three strains Gxkv1, Gxkv2 and Gxkv3 were selected for further analyses. The 16S rDNA gene (GenBank Accession OR461756, PP094726 and PP109349) were amplified using primer pair 27F/1492R (Frank et al. 2008). Comparing 16S sequences against GenBank showed 99.86%-100% sequence identity to Klebsiella variicola strain (MZ475068) for the three isolates Gxkv1 (1,398/1,398 bp), Gxkv2 (1,398/1,396 bp) and Gxkv3 (1,398/1,398 bp). A multilocus phylogenetic analysis was conducted by neighbor-joining method (1,000 bootstrap values) based on three housekeeping gene sequences of gyrA (GenBank Accession No. OR515493, PP105747, PP105748), rpoB (OR515494, PP105751, PP105752 ) and infB (OR515495, PP105749, PP105750) genes which were amplified by gyrA-A/gyrA-C, CM31b/CM7 and infB867F/infB1819R primer sets, respectively (Rosenblueth et al. 2004). The results of phylogenetic analysis showed the three strains belong to the K. variicola clade. A pathogenicity test was conducted on six healthy 3-month-old dwarf banana plants by spraying 10 mL of bacterial suspensions of Gxkv1 (108 CFU/mL) into the rhizome which wounded with a sterilized needle; another six healthy control plants were sprayed with 10 mL of sterile water. Following inoculation, the plants were placed in a greenhouse at 28-32°C. After 30 days, all inoculated plants showed symptoms similar to those observed in the field, while the control plants remained healthy. Bacteria were successfully reisolated from the symptomatic tissues and identified to be K. variicola by PCR mentioned above. K. variicola has been reported to cause rhizome rot of banana in India (Loganathan et al. 2021), and to cause plantain soft rot in Haiti (Fulton et al. 2021). Besides, previous reports from China only showed K. variicola causing banana sheath rot (Fan et al. 2015, Sun et al. 2023). To our knowledge, this is the first report of bulb rot disease of banana caused by K. variicola in Guangxi Province, China. This finding will provide important information for studying the epidemiology and management of this pathogen.

Genetic Diversity of White-spotted Rabbitfish (Siganus canaliculatus) on Different Seagrass Habitats in Inner Ambon Bay, Indonesia Based on Mitochondrial CO1 Sequences.

This study aims to analyse the genetic diversity of Siganus canaliculatus in the Inner Ambon Bay (IAB) waters. DNA of S. canaliculatus specimens collected from IAB was extracted from tissues using a Tissue Genomic DNA Mini Kit, and partial CO1 genes were amplified using pair of universal primers. Genetic distances were determined by Kimura 2-parameter, and phylogenetic trees were constructed using the neighbour-joining method in MEGA 10.2.2 software. Arlequin software was used to analyse Fixation Index (Fst) and Analysis of Molecular Variance (AMOVA). There are three SNPs of S. canaliculatus from IAB that distinguish GenBank sequence data from S. canaliculatus. In Tanjung Tiram population group, contained three specific 677 (A), 679 (G), 703 (T) sites and two 693 (G), 714 (A) sites for the Nania population. Haplotype and nucleotide diversity of each population range from 0.000 to 1,000 and 0.000 to 0.004. Intra- and inter-population genetic differentiation were 21.19% dan 78.81%, respectively. Intra- and inter-population genetic distances were in range of 0.40-1.13 and 0.00-0.37, respectively. The pattern and direction of tidal currents as a link or barrier to spatial distribution and connectivity of S. canaliculatus larvae between seagrass habitats, as well as the presence of different anthropogenic pressures in each seagrass habitat, are thought to influence the genetic characteristics (genetic diversity, genetic variation, genetic differentiation and genetic distance) of S. canaliculatus populations in IAB waters. The results of this study provide information about the urgency of habitat-based fisheries management to support sustainable utiliation.s.

Ipomoea obscura Represents a New Host of Phytoplasma Belonging to 16SrII Group Associated with Witches'-Broom Symptoms in China.

Phytoplasmas are phloem-limited plant pathogenic prokaryotes which can not be cultured in vitro. The pathogens could cause various plant symptoms such as witches'-broom, virescence, and leaf yellows. Ipomoea obscura is a valuable plant species belonging to the family Convolvulaceae, mainly used as a traditional Chinese medicine used to treat diseases such as dehydration and diuresis. In western countries it is commonly referred to as 'obscure morning glory'. During 2020 to 2021, plants showing abnormal symptoms including witches'-broom, internode shortening, and small leaves were found in Hainan Province, a tropical island of China. Approximately 30 % of I. obscura plants in the sampling regions which spanned 400 acres, showed symptoms. In order to identify the associated pathogen, six symptomatic samples and three asymptomatic samples were collected and total DNA were extracted from 0.10 g fresh plant leaf tissues using CTAB DNA extraction method. 16S rRNA and secA gene fragments, specific to phytoplasmas, were PCR amplified using primers R16mF2/R16mR1 and secAfor1/secArev3. The target PCR bands were obtained from the DNA of six symptomatic samples, whereas not from the DNA of the asymptomatic samples. The PCR products of phytoplasma 16S rRNA and secA gene obtained from the diseased samples were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The 16S rRNA and secA gene sequences identified in the study were all identical with the length of 1330 bp (GenBank accession: OR625212) and 720 bp (OR635662) respectively. According to methods and protocols of phytoplasma identification and classification (Wei and Zhao, 2022), the phytoplasma strain identified in the study was described as Ipomoea obscura witches'-broom (IoWB) phytoplasma, IoWB-hnld strain. The partial 16S rRNA gene sequence of IoWB showed 100 % sequence identity over the full 1330 bp sequence to phytoplasmas belonging to 16SrII group like cassava witches'-broom phytoplasma (KM280679). The BLAST search of the 720 bp partial secA gene fragment of IoWB showed 100% sequence identity for the full sequence to phytoplasmas belonging to 16SrII group like 'Sesamum indicum' phyllody phytoplasma (OQ420657). RFLP analysis based on the 16S rRNA gene using iPhyClassifier demonstrated that the IoWB strain was a member of 16SrII-A subgroup with the similarity coefficient 1.00 to the reference phytoplasma strain (L33765). Phylogenetic analysis based on 16S rRNA and secA genes by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value indicated that IoWB-hnld was clustered into one clade with the phytoplasmas belonging to 16SrII group, with 98% and 100% bootstrap value separately. To our knowledge, this is the first report that Ipomoea obscura can be infected by phytoplasmas belonging to 16SrII-A subgroup in China. This report adds to the host range of 'Ca. Phytoplasma aurantifolia', documenting the symptoms on I. obscura which will assist in monitoring and control of the associated pathogen.

First Report of Aspergillus niger causing rot of Morchella sextelata in China.

As an important edible mushroom, morel mushroom (Morchella spp.) has been widely spread and cultivated in China. However, between 2022 and 2023, a rot disease with a natural incidence of 28% occurred in morel mushroom farms in the Qingpu district of Shanghai (N30°97', E121°06'), China. High temperatures (>20℃) and high humidity (>70%) provide conditions conducive to the spread of this disease. First, a small white mold-like symptoms appeared on the surface or the pinnacle of pileus. The tissues in the infected parts stop growing and developing.Then the lesion developed to encircle the pileus and spread gradually to the stipe, seriously affecting its yield and quality. The infected tissue of morel fruiting body at the edge of the lesions was isolated and cultivated on potato dextrose agar (PDA) at 28℃ in the dark. After 3 days, monospore cultures formed black cottony colonies. In order to reliably identify, isolates were transferred to Czapek Yeast Autolysate agar (CYA) (Samson et al, 2014). On CYA fungal colonies consisted of a white mycelium, covered by a layer of black conidiophores. Scanning electron microscope analysis revealed that mature mycelia produced conidiophores ended with numerous metula and phialides. The phialides showed the number of conidia bearing rounded spores, which coincides with previous research(Silva et al, 2020). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS), beta-microtubulin (BenA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) gene of the isolate were amplified by PCR (White et al. 1990; Glass et al. 1995; Hong et al. 2005; Liu et al. 1999). The resulting sequence was deposited in GenBank with accession OQ931346.1, OR393310, OR393311, and OR393312, respectively. PCR results and morphological observations indicated the isolated strain was a pure culture and the strain was designated as MOR02. Comparison results indicated that the sequences with accession numbers KF305756.1, MK450794.1, HQ285594.1, and HQ285594.1 have high identity with the molecular sequences of A. niger MOR02, which is 99%, 98%, 98%, 99%, respectively. Phylogenetic analysis with ITS and RBP2 genes of the isolated strain and 9 Aapergillus spp. strains were performed using MEGAX software with Neighbor-Joining (NJ) method. Based on the results of growth habits, morphological observations, and phylogenetic analysis, the pathogen was identified as A. niger. A spore suspension of the A. niger strain MOR02 (1 x107 conidia/mL) was inoculated back to healthy morel mushrooms. Five healthy fruit bodies of M. sextelata were injected, and another five healthy morels were treated with potato dextrose broth（PDB） medium as controls. Morels were incubated for 7 days at 20℃ and 85% to 90% relative humidity. The pathogen successfully infected the morel showing a similar white mold-like lesion as the natural occurrence disease. The controls remained healthy without any symptoms. The pathogen was reisolated from the affected lesions and identified as A. niger MOR02 based on its morphological characteristics and phylogenetic marker genes. To our knowledge, this is the first report of A. niger causing rot disease of M. sextelata. This study confirms that A. niger is the pathogenic fungus causing morel rot on the Qingpu farm in Shanghai. The disease occurs under conditions of high humidity and high-temperature conditions. Better production management is the most important to prevent the disease.