Rhizoctonia solani and Fusarium oxysporum. The potential of beneficial rhizosphere microorganisms to control R. solani and F. oxysporum f.sp. capsici was evaluated in pepper plants. Paenibacillus polymyxa and Trichoderma longibrachiatum were isolated from rhizospheric soil samples of healthy pepper plants. In vitro, both isolates caused clear reductions in the radial growth of root rot and wilt pathogens. Scanning electron microscopy displayed lysis and abnormal shape of the pathogens in dual cultures with P. polymyxa and T. longibrachiatum. The incidence and severity of root rot and wilt diseases were significantly reduced in pepper plants treated with the growth-promoting fungi , P. polymyxa, or T. longibrachiatum in comparison to the control. Moreover, the induction treatments led to increased pepper growth compared with their control. The defense related gene (CaPR4) expression was shown to be significantly higher in the treated plants than in the control plants. In conclusion, the antagonistic isolates (P. polymyxa and T. longibrachiatum) and PGPF isolates have a clear impact on the prevention of root rot and wilt diseases in pepper plants incited by R. solani and F. oxysporum f.sp. capsici. The expression of the CaPR4 gene added to the evidence that PGPF isolates generate systemic resistance to pathogen infections.Pepper is vulnerable to soil-borne fungal pathogens such as  Capsicum annuum L.) is a commonly cultivated crop, from the family Solanaceae, in Egypt and all over the world. In agricultural production, soil-borne pathogens are the main problems causing reductions in crop yield and quality. The most dreaded diseases of vegetables are root rot, damping-off, charcoal rot, and wilt caused by Rhizoctonia solani, Alternaria solani, F. oxysporum f.sp. capsici, Sclerotium rolfsii, Macrophomina phaseolina, and Pythium spp. [Fusarium wilt is known to be one of the most important devastating and harmful diseases affecting pepper plants. Pre- and post-emergence damping-off, root rot, wire stem, seed decay, and hypocotyl or tap root with necrotic spots, all of these symptoms are caused by R. solani at multiple growth stages [F. oxysporum and R. solani were the most common isolates from root-rooted and wilted pepper plants. Pathogenicity tests on the isolated pathogens from the infected pepper plants demonstrated that plants were infected with F. oxysporum f.sp. capsici and R. solani [F. oxysporum f.sp. capsici showed damping-off and vascular wilt symptoms [Pepper  were effective in soil-borne disease control and enhancement of plant growth [F. moniliforme, F. oxysporum, F. solani, and F. merismoides reduced wilt incidence in tomato [Biological control could be a successful strategy for managing diseases and feasible pepper production. Biocontrol agents (BCAs) control plant pathogens through several mechanisms, such as fungistasis, antibiosis, hyperparasitism, induced systemic resistance, and modification of the rhizosphere . Plant gt growth ,5,6. Then tomato .Sclerotium cepivorum. Additionally, exalted expressions of defense-related genes, PR1 and PR2, have been described in plants treated with PGPF [CaChi2 gene was involved in resistance to F. oxysporum f.sp. capsici in chili plants. The objective of this research was to test the efficiency of certain bioagents in controlling pepper root rot and wilt diseases under greenhouse conditions. Additionally, the activities of defense enzymes and expression of defense genes were evaluated.Pathogen infection is associated with induced genes encoding pathogenesis-related (PR) proteins. Plants have evolved a variety of defensive mechanisms to restrict pathogen attack, including biochemical, physiological, molecular, and cellular processes and barriers, as well as inducible defense response ,6,7,9. Iith PGPF . The CaCFusarium equiseti GF19-1, F. equiseti GF18-3, and Phoma sp. GS8-3 were used against Bean yellow mosaic virus [R. solani and F. oxysporum f.sp. capsici.Root samples of diseased pepper plants were isolated and identified in our previous study . Samplesic virus . The supSeveral bioagents, isolated from the rhizosphere of healthy pepper roots, were gathered from different locations, i.e., Kafr El-Sheikh, El-Behira, North Sinai, and Alexandria governorates. The serial dilution method was utilized for isolation. Identification of bacterial isolates was carried out depending on cultural, morphological, and biochemical properties , while fR. solani (R) and F. oxysporum f.sp. capsici (F). Plates were concurrently inoculated by bacteria (four isolates per dish). Non-inoculated plates served as control. Plates were kept at 19–22 °C until the control treatment was fully covered with PDA. The diameter of inhibition zone was measured, and the relative power of antibiosis (RPA) was determined using the formula: RPA = Z/C [For antagonistic bacterial isolates, PDA plates were inoculated in the center with agar discs (5 mm) bearing mycelium of 7-days old cultures of PA = Z/C . Z: The For fungal antagonists, the dual culture technique was used for testing the antagonistic activity of fungal isolates. PDA plates were inoculated with discs (5 mm) of the pathogenic fungi (one-week old cultures), and on the opposite side, the discs of the antagonistic fungi were placed. Plates containing the pathogens alone have been considered as control. Plates were kept at 18–20 °C until the full growth in control plates. The degree of antagonisms was determined as elucidated by Bell et al. ,16.Paenibacillus polymyxa and Trichoderma longibrachiatum (the most effective bio-agents in the experiments compared with other bioagents in vitro) on the growth of F. oxysporum f.sp. capsici and R. solani [Scanning electron microscopy (SEM) has been utilized to evaluate the effects of . solani . The sam. solani  solution8 cfu/mL of P. polymyxa.Inoculum of the antagonistic isolate was prepared by growing on nutrient broth media in conical flasks (500 mL) at 28 °C for 5 days using shaking incubator (100 rpm). Cell suspension was diluted and adjusted to 10T. longibrachiatum, F. equiseti GF19-1, F. equiseti GF18-3, and Phoma sp. GS8-3 were prepared by growing them on potato dextrose broth medium (PDB) and incubated at 28–30 °C for 10 days. Spore suspensions of the isolates were counted and adjusted to 2 × 106 spore mL−1 [Inocula of the isolates F. oxysporum f.sp. capsici or R. solani, at the rate of 2% . Inoculum of each pathogen was mixed separately with soil. Infested pots were irrigated and kept for 5 days before transplanting [Pot experiments were carried out during two consecutive seasons for studying the effect of the five isolates on root-rot and wilt diseases of pepper. Plastic pots (24 cm in diameter) were filled with autoclave sterilized sandy loam soil infested with one of the two pathogens, a-Seed treatment8 cfu/mL) or fungal spore suspension (2 × 106 spore/mL) for 2 h before planting in seedling trays [Pepper seeds were treated by soaking in 10 mL of the bacterial suspension (10ng trays . Seeds wb-Seedling treatmentw/w). As a control treatment, autoclaved PDB was used instead of bioagent suspensions.A seedling tray was filled with potting soil and sown with sterilized pepper seeds (using 5% sodium hypochlorite). The 19-d-old seedlings, raised in the tray, were soaked in the suspension of each isolate for 2 h and transferred to small pots (5 cm in diameter) filled with the potting soil. Twenty days later, the seedlings were transplanted in pots artificially infested with the pathogen  were crushed into powder. Subsequently, the obtained powder was macerated for 30 s and homogenized with sodium phosphate buffer . The filtrates were centrifuged  after filtering the triturated tissues through four layers of cheese cloth. A sample of the clear supernatant was collected for enzyme extraction.−1 g−1) by spectrophotometric analysis [Peroxidase and polyphenol oxidase activities in the collected samples were estimated , with CaActin as an internal reference as described by Elsharkawy and El-Khateeb [Leaves were harvested at 2 days after pathogen inoculation. RNA Purification Kit  was used to extract RNA. The extracted RNA was converted to complementary DNA (cDNA) using revert Aid H minus reverse transcriptase. Real-time PCR with SYBR Green was used to measure the expression of mRNAs of the target gene .Trichoderma spp.  as well as 32 bacterial isolates were isolated from different rhizosphere samples of healthy pepper plants. The isolate T. longibrachiatum (T1) proved to have the highest antagonistic effect on the pathogens (F. oxysporum f.sp. capsici in Petri dish (P. polymyxa (B25) (R. solani. The highest inhibitory spectrum of the isolates was P. polymyxa (B25)  C. Six baxa (B25) D.Trichoderma isolate T1 was strongly related to the species longibrachiatum. It showed the highest sequence similarities with T. longibrachiatum (GenBank accession number OM666052) .The phylogenetic tree exhibited that the M666052) . On the  species . It can F. oxysporum f.sp. capsici . However, the most successful treatment on R. solani was GF18-3 as seedling treatment, which gave a disease severity percentage of 27.77% in both seasons. Likewise, the most useful treatment for disease incidence of Rhizoctonia root rot was GF18-3 as seedling treatment, which gave a disease incidence of 27.8%. Discoloration severity was estimated at the end of the experiment. Discoloration severity was reduced in the treatments with GF19-1 and GF18-3 as seedling treatments recording 14.8% in the second season /plant was verified due to biotic inducers. All treatments significantly improved growth parameters in plants infected with xysporum . The mos seasons .F. oxysporum. However, in case of R. solani, the highest activities of peroxidase, polyphenol oxidase and total phenols were achieved by the treatments GF19-1 and GF18-3 as seedling treatment. Control treatment (pathogen only) showed the lowest values.Significant differences were found in phenol, peroxidase and polyphenol oxidase . Applicap ≤ 0.05) increase of CaPR4 gene expression level in treated pepper plants compared to the control. Plants treated with GS8-3 showed the highest expression levels in the case of F. oxysporum and R. solani. In addition, plants infected with F. oxysporum and treated with GF18-3 resulted in the lowest up-regulation of CaPR4. However, under the infection with R. solani, treatment with GF19-1 presented the lowest gene expression  is a major commercial crop all over the world, with a huge societal and economic impact. It is one of Egypt’s most popular vegetable crops, cultivated either in open fields or in plastic greenhouses under a protected farming system. Pepper plants may be infected with various pathogens, including soil-borne pathogens [Fusarium, Macrophomina, Rhizoctonia, Verticilium, Pythium, and Sclerotinia frequently cause damping-off, root rot, and wilt diseases [Fusarium wilt of pepper, caused by Fusarium spp., has resulted in significant reductions in pepper production in many countries around the world [P. polymyxa and T. longibrachiatum were isolated and identified by morphological and molecular biology techniques. Similarly, P. putida strain F1 was successfully identified based on the sequence of 16S rDNA comparing the sequence similarities with the related bacterial species in Gene bank [F. oxysporum f.sp. capsici and R. solani. The results are in agreement with Sahii and Khalid [F. oxysporum was hampered as a response to the antagonistic effect of Trichoderma sp. The R. solani mycelial diameter, as well as infection with root rot and damping-off diseases, were substantially decreased by T. harzianum isolate [Pepper . Additionally, P. polymyxa NSY50 application on cucumber plants infected with F. oxysporum successfully decreased the incidence of Fusarium wilt [F. oxyporum and R. solani infections. The accumulation levels of defense enzymes and the transcription levels of PR1 and PR5 genes were increased in cucumber plants treated with T. atroviride (TRS25) and led to better resistance against R. solani [CaPR4 gene expression suggests that this gene is involved in systemic resistance to F. oxysporum and R. solani. JA and ET activated PR4, PR5, and PDF1.2 in a synergistic manner [P. simplicisimum GP17-2 in Arabidopsis and tobacco enhanced the expression of different pathogenesis-related genes [Plant growth promoting fungi (PGPF) is a kind of saprophyte that lives in the soil and promotes plant development. As a consequence of seed and seedling treatments, all of the evaluated biocontrol agents substantially decreased the severity of lication . Curiousf onions . In thisf onions  evaluateium wilt . In this. solani . The facc manner . Induceded genes .F. oxysporum and R. solani. PGPF has been shown to improve plant growth and disease control [Trichoderma isolates were considered proper biofertilizers, since they could improve the capacity of nutrients uptake in plants and the resistance toward plant pathogen [P. polymyxa NMA1017 promoted plant growth through nitrogen fixation and siderophore synthesis, which led to increased crop production [All treatments significantly enhanced growth characters in plants relative to the control infected with  control . Severalpathogen . Furtheroduction .F. oxysporum f.sp. capsici and R. solani, as well as increasing growth parameters in pepper plants. The activities of oxidative enzymes (Peroxidase and polyphenol oxidase), phenol contents, and the expression levels of CaPR4 were stimulated in the treated pepper plants, leading to induced resistance against F. oxysporum f.sp. capsici or R. solani. The ability to use these treatments to manage root rot and wilt diseases of pepper was already improved as a result of increased pepper growth.There is a growing request for safe biocontrol agents to improve management strategies of soil-borne diseases to reduce the poisonous effects of pesticides. The results indicated that the antagonistic isolates from healthy pepper plants and PGPF isolates can effectively control the pathogens,


West Nile virus (WNV), a single-stranded RNA virus belonging to the family of Flaviviridae, is an arbovirus transmitted to humans predominantly by mosquito bites. It exhibits a wide range of clinical findings ranging from asymptomatic presentation to severe several neurological disorders. WNV has afflicted several countries around the globe including Pakistan. News of yet another outbreak in the country by WNV is circulating again. Concerned authorities should act vigilantly before the endemic takes over completely and bring down the already bereaving healthcare of Pakistan. Dear Editor,Discovered and named after the West Nile district of Uganda, West Nile virus (WNV) is a positive-strand RNA virus replicating via RNA-dependent RNA polymerase cycle . It beloThe clinical manifestation following a WNV infection spans a wide range, with about 80% population remaining asymptomatic. Mild symptomatology includes systemic febrile illness, also known as West Nile Fever (WNF). The jeopardizing symptoms are seen in less than 1% population and develop when the virus invades the central nervous system (CNS), thereby leading to meningitis, encephalitis, or acute poliomyelitis-like syndrome . The sevThe virus has been endemic throughout the world including Israel, Africa, Asia, the Caribbean, Greece, Romania, Russia, and the United States of America (USA) . Among tTo ensure that the country does not undergo another healthcare hazard, all precautionary measurements must be employed. Government must allocate more funds to the healthcare department. Currently, Pakistan lacks testing services for WNV except for a few institutes . IncreasPublic awareness programs should be run throughout the country, educating masses on personal hygiene, use of mosquito repellants, and electrical devices. Database systems for proper reporting of any case must be well-developed and the cases should be followed-up. Lastly, research programs should be devised that will work on actively controlling the transmissions via investigating every possible route. As a lower-middle-income country, still recovering from coronavirus disease 2019 (COVID-19) blackout and dengue outbreaks, a potential threat of WNV can lead to a disastrous setback, thereby further depriving the healthcare economy of Pakistan. Hence it is imperative that the concerned authorities take immediate actions or WNV can be the next gigantic public health threat for the country.Not required.None.S·W and T.G.S conceived the idea, S·W, T.G.S, S·H.A, and R.M.U retrieved the data, did write up of letter, and finally, M.J.T and M.S.A reviewed and provided inputs. All authors approved the final version of the manuscript.1Name of the registry: Not required.2.Unique Identifying number or registration ID: N/A3.Hyperlink to your specific registration (must be publicly accessible and will be checked):Muhammad Sohaib Asghar.No financial support was provided relevant to his article.All authors report no conflict of interest relevant to his article.


Gadus macrocephalus (Pacific cod) is an economically important species on the northern coast of the Pacific. Although numerous studies on G. macrocephalus exist, there are few reports on its genomic data. Here, we used whole-genome sequencing data to elucidate the genomic characteristics and phylogenetic relationship of G. macrocephalus. From the 19-mer frequency distribution, the genome size was estimated to be 658.22 Mb. The heterozygosity, repetitive sequence content and GC content were approximately 0.62%, 27.50% and 44.73%, respectively. The draft genome sequences were initially assembled, yielding a total of 500,760 scaffolds (N50 = 3565 bp). A total of 789,860 microsatellite motifs were identified from the genomic data, and dinucleotide repeat was the most dominant simple sequence repeat motif. As a byproduct of whole-genome sequencing, the mitochondrial genome was assembled to investigate the evolutionary relationships between G. macrocephalus and its relatives. On the basis of 13 protein-coding gene sequences of the mitochondrial genome of Gadidae species, the maximum likelihood phylogenetic tree showed that complicated relationships and divergence times among Gadidae species. Demographic history analysis revealed changes in the G. macrocephalus population during the Pleistocene by using the pairwise sequentially Markovian coalescent model. These findings supplement the genomic data of G. macrocephalus, and make a valuable contribution to the whole-genome studies on G. macrocephalus. Gadus macrocephalus (Pacific cod) belongs to the order Gadiformes, Gadidae  has been widely applied in genomic studies of fish species ,12. GenoG. macrocephalus. The mitochondrial genome of G. macrocephalus was assembled. Moreover, phylogenetic relationships, microsatellite motifs and demographic history inference were also carried out. The genomic data and phylogenetic analysis will be significant for genetic research in the future.In this report, WGS data were employed to investigate the genomic characteristics of G. macrocephalus in the fishing port, Qingdao, Shandong Province, China, in December 2020. Then, the muscle tissue was quickly frozen in liquid nitrogen for 1 h before storage at −80°C. The DNeasy Blood and Tissue Kit  was used to extract total genomic DNA from the muscle tissue following the manufacturer’s instructions. For sequencing, DNA libraries were constructed with insert sizes of 350 bp, and paired-end sequencing was performed on the Illumina HiSeq 4000 platform. The sequence data were deposited in the Short Read Archive (SRA) database (http://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA793360.The experimental design, sequencing and data analysis pipeline were showed . We collG. macrocephalus genome, we used the clean data for K-mer analysis (K = 19) in Jellyfish software [K-mer analysis, we obtained the size, heterozygosity and repetitive sequence content of the genome. Genome size was estimated by using the equation Genome size = K-mer_num / peak_depth, where K-mer_num is the total number of best predicted K-mers and peak_depth is the expected value of the K-mer depth [K-mer errors via the formula Revised genome size = Genome size × (1 − Error rate) [To produce clean data, the raw data from WGS were filtered by Fastp 0.20.1 , removinsoftware . From ther depth . After tor rate) .N’ representing unknown sequences between two contiguous contigs [To obtain the genomic sequence, we used SOAPdenovo software  to assem contigs .G. macrocephalus genome, we used the Perl script MIcroSAtellite  to identify microsatellite motifs. The parameters were set as the detection of mono-, di-, tri-, tetra-, penta- and hexanucleotide SSR motifs with a minimum of 10, 6, 5, 5, 5 and 5 repeats, respectively [To search for simple sequence repeats (SSRs) in the ectively .G. macrocephalus in the NCBI GenBank database (accession number: NC036931) as the reference. Then, these contigs were used as a guide for structural annotation of the mitochondrial genome in MitoFish [To obtain the 13 protein-coding gene sequences of mitochondrial genome, the clean reads from Fastp software were used to construct the mitochondrial genome via MitoFinder v.1.4 . MitoFinMitoFish .Bregmaceros nectabanus, smallscale codlet) from the GenBank database. A phylogenetic tree was drawn by the maximum likelihood algorithm with 1000 replicates in MEGA 11 [http://www.timetree.org) [G. macrocephalus and Gadiculus thori (silvery pout) diverged 25.90 million years ago (Mya), (2) G. macrocephalus and Melanogrammus aeglefinus (haddock) diverged 10.90 Mya, and (3) G. macrocephalus and Boreogadus saida (polar cod) diverged 8.40 Mya.To investigate the phylogenetic relationships and divergence times of Gadidae species , we downloaded 13 protein-coding gene sequences of the mitochondrial genome of Gadidae species and an outgroup ( MEGA 11 . Additioree.org)  to deterG. macrocephalus, the whole genome of G. morhua  was retrieved from the NCBI database and used as a reference genome. Then, the clean reads of G. macrocephalus were aligned to the reference genome of G. morhua by using the bwa-mem algorithm in Burrows–Wheeler Aligner (BWA) [eN) using the pairwise sequentially Markovian coalescent (PSMC) model [g = 6 years and a mutation rate of μ = 8.29 × 10−9 site−1 year−1 (5.0 × 10−8 site−1 generation−1) [eN was estimated using 100 bootstrap replicates.To infer the demographic history of er (BWA) . We estiC) model . The PSMation−1) . By defaA total of 56.63 Gb raw data were generated by the Illumina HiSeq platform . After fK-mer analysis. As a result, the peak of the 19-mer frequency distribution was 69, and the total number of K-mers was 47,386,140,246 , and the identity of these two mitochondrial genome sequences reached 100%. These two mitochondrial genomes contained the same set of 37 genes, including 13 protein-coding genes, 22 tRNA genes and 2 rRNA genes. Among them, 8 tRNA genes  and ND6 were located in the light chain, and the others were located in the heavy chain. Besides, structural annotation of the mitochondrial genome indicated that it was composed of a coding region and a D-loop . A comparison of the lengths of the mitochondrial genomes of a D-loop . The D-lTheragra and Gadus displayed the closest genetic relationship and diverged 5.69 Mya. Furthermore, G. macrocephalus underwent genetic divergence, and G. morhua diverged 3.59 Mya. The sample of G. macrocephalus from China clustered with samples of G. macrocephalus from South Korea and Canada, and their divergence time was 0.07 Mya.A maximum likelihood phylogenetic tree was constructed based on the 13 protein-coding gene sequences of the mitochondrial genome of Gadidae species, and showed a bootstrap value > 90% (based on 1000 replicates). Divergence time analysis showed that Gadidae species shared a common ancestor approximately 25.90 Mya . And theeN  in the world and account for approximately 18% of the world’s total marine fish catch [G. morhua [M. aeglefinus [G. chalcogrammus  [Lota lota (burbot) [Gadus, the genomic data of G. morhua are relatively comprehensive [G. macrocephalus both at home and abroad.The order Gadiformes species include some of the most important commercial fish   and Lota(burbot) . Additioehensive ,32. HoweG. macrocephalus. The genome size of G. macrocephalus was relatively small. Similarly, the genome sizes of the other fishes of the order Gadiformes species were less than 1 Gb: G. morhua (830 Mb) [M. aeglefinus (653 Mb) [G. chalcogrammus (683.61 Mb) [Lota lota (576 Mb) [G. macrocephalus was similar to the repetitive sequence content of other Gadidae species, such as G. morhua (25.40%) [G. chalcogrammus (38.89%) [Lota lota (66.74%) [G. macrocephalus. For a genome assembly, if the heterozygosity is less than 1%, the genome is relatively effortless to assemble [G. macrocephalus, the slightly low proportion of heterozygosity reflected that the genome assembly is relatively easy. Overall, combined with the heterozygosity and repetitive sequence content being slightly low, the small genome will make genome assembly easier in subsequent studies.In the present study, WGS was applied to preliminarily reveal the genomic data of 830 Mb) , M. aegl(653 Mb) , G. chal Mb , M. (576 Mb) . The rep(25.40%)  and G. c(66.74%)  had high(66.74%) . Furtherassemble . TherefoG. macrocephalus were assembled. For the genome sequences of G. macrocephalus, the lengths of N50 contigs and scaffolds were short. A genome assembly would have high utility with an N50 contig size > 30 kb and an N50 scaffold size > 250 kb [K-mer (K = 19) possibly led to the low assembly quality [G. macrocephalus.This is the first time that the genome sequences of > 250 kb . Accordi quality . Conside quality ,12. The  quality  should bG. macrocephalus [G. macrocephalus genome was very high (1497.5 microsatellites per Mb), which was markedly higher than that reported in Sebastiscus albofasciatus (556.8 microsatellites per Mb) [Tor tambra (971.39 microsatellites per Mb) [Recently, SSRs, which are chromosome-specific and frequently inherited in a Mendelian codominant fashion, have become the most commonly used molecular markers . Many recephalus ,40. In t per Mb)  and Tor  per Mb) . To ensuG. macrocephalus to further decipher its taxonomic status and systematic evolution. The genome organization and gene composition of the mitochondrial genome of G. macrocephalus were consistent with the results of a previous study using fish-versatile PCR primers [G. macrocephalus, similar to patterns reported in most vertebrates [G. macrocephalus. Through the structural annotation of mitochondrial genome, we obtained the detailed information of genetic distribution and genomic structure, which could provide important raw materials for functional annotation and evolutionary analysis.Complete mitochondrial genome is a powerful and popular tool to unravel the higher-level relationships of teleosts . Hence,  primers . The AT tebrates . BesidesG. macrocephalus and G. morhua diverged 3.59 Mya, subsequently, G. macrocephalus from three different places diverged 0.07 Mya. Quaternary climate oscillations and geographical heterogeneity play important roles in determining species and genetic diversity distribution patterns [G. macrocephalus at the population level remains poorly understood. More efforts are still to be made in the further research on the divergence of this species.The phylogenetic tree showed that the phylogenetic relationships and divergence times among Gadidae species. 0.07–11.95 Mya, the diversity of seven lineages became increasingly abundant because climatic conditions were warmer and wetter . Importapatterns , but howG. macrocephalus, revealed that G. macrocephalus population also experienced bottleneck and expansion. Similarly, Canino et al. reported that G. macrocephalus expansions were evident by using the mismatch distributions and Bayesian skyline plots of mitochondrial genome, but the start of expansions appeared to predate the last glacial maximum  [G. macrocephalus.Demographic history can be used to clarify the impact of geological and climate changes on the current species distribution, and help to formulate reasonable and effective strategies to preserve endangered species . The prears ago) . This fiG. macrocephalus was performed based on WGS data. The present study uncovered genomic characteristics of G. macrocephalus, including small genome, slightly low heterozygosity and repetitive sequence content. SSRs were identified from the genomic data, which could provide novel insight into population genetics and germplasm resource conservation in the future. Besides, the evolutionary relationships and divergence times of Gadidae species were determined, and the demographic history of G. macrocephalus was reconstructed. To improve the assembly quality of G. macrocephalus genome, we suggest that high-quality genome sequences should be generated by using third-generation sequencing and Hi-C techniques in the future.In summary, the first whole-genome survey of


P. carotovorum group of isolates. Based on multilocus sequence typing (MLST) using genes dnaX, mdh, icdA, and proA, five representative isolates were identified as Pectobacterium carotovorum (Cheers F1 and Hippo F1), while two were identified as Pectobacterium versatile (Hippo F1) and Pectobacterium odoriferum (Hippo F1), respectively, indicating the presence of diverse Pectobacterium species even in combined infection in the same field. Among the obtained isolates, P. carotovorum was the most prevalent species (62.69%), while P. versatile and P. odoriferum were less represented . Multilocus sequence analysis (MLSA) performed with concatenated sequences of four housekeeping genes  and constructed a neighbor-joining phylogenetic tree enabled insight into the phylogenetic position of the Serbian cabbage Pectobacterium isolates. Bacterium P. odoriferum was found to be the most virulent species for cabbage, followed by P. versatile, while all three species had comparable virulence with respect to potato. The results obtained in this work provide a better understanding of the spreading routes and abundance of different Pectobacterium spp. in Serbia.The aim of this work was to identify and characterize the pectolytic bacteria responsible for the emergence of bacterial soft rot on two summer cabbage hybrids (Cheers F1 and Hippo F1) grown in the Futog locality , known for the five-century-long tradition of cabbage cultivation in Serbia. Symptoms manifesting as soft lesions on outer head leaves were observed during August 2021, while the inner tissues were macerated, featuring cream to black discoloration. As the affected tissue decomposed, it exuded a specific odor. Disease incidence ranged from 15% to 25%. A total of 67 isolates producing pits on crystal violet pectate (CVP) medium were characterized for their phenotypic and genotypic features. The pathogenicity was confirmed on cabbage heads. Findings yielded by the repetitive element palindromic-polymerase chain reaction (rep-PCR) technique confirmed interspecies diversity between cabbage isolates, as well as intraspecies genetic diversity within the  Brassica oleracea var. capitata L.) is one of the world’s most important vegetable crops, as its high yield and adaptability have facilitated worldwide distribution  corresponding to the interspersed repetitive sequence elements. Primer sequences of the used primers are listed in TM, Waltham, MA, USA), 1 µL of each (forward/reverse) primer (10 µM) and 1 µL of sample DNA. PCR amplifications for BOX-, ERIC-, and REP-PCR were performed under the conditions proposed by Louws et al.  and two single oligonucleotide primers [BOXA1R (BOX-PCR) and GTGs et al. , while ts et al.  was adopdnaX (dnaX-F/dnaX-R), icdA (icdA400F/icdA977R), mdh (mdh2/mdh4), and proA (proAF1/proAR1)), encoding DNA polymerase III subunit tau, isocitrate dehydrogenase, malate dehydrogenase, and gamma-glutamyl phosphate reductase, respectively. Primer sequences of the used primers are listed in dnaX, while those reported by Ma et al. [icdA and proA, and the strategy employed by Moleleki et al. [mdh. Presence of bands in the searched positions was checked on 1% agarose gel in relation to 200–10,000 bp SmartLadder MW-1700-10 (Eurogentec). PCR products were purified using the Qiagen QIAquick PCR Purification Kit before being sent to the Eurofins Genomics’ DNA sequencing service (Germany) for sequencing. The obtained sequences were manually checked for quality and were compared with the strains deposited in the National Center for Biotechnology Information (NCBI) database using the nucleotide BLAST (BLASTn) tool. All newly identified sequences were deposited to the NCBI database to obtain the accession numbers.DNA of the seven selected representative cabbage isolates  was amplified with the primers produced based on the partial sequences of four housekeeping genes , P. carotovorum , and P. versatile  isolated from different hosts  and countries  and retrieved from the NCBI database (dnaX (416 nt), icdA (479 nt), mdh (240 nt), and proA (504 nt). Before the tree construction, the sequences of all genes were aligned to the same size using the biological sequence alignment editor BioEdit 7.2 program and the ClustalW Multiple alignment tool. The tree was constructed in MEGA7 Software and was rooted with the Dickeya solani strain RNS 05.1.2A (Accession No. CP104920).The phylogenetic position of the seven representative isolates was determined in relation to 19 strains of three database . A neighThe virulence potential of the seven representative cabbage isolates  was assessed using cabbage heads (unknown cv.) and potato tubers (cv. Arizona). Prior to inoculation, cabbage heads and potato tubers were sterilized as previously described , and appiy = disease progress assessment at the ith observation, it = time (days) at the ith observation, and n = total number of observations. Cabbage heads and potato tubers were weighed, whereby the mean values of the initial and the final (5 and 7 d.a.i.) weights were used to calculate the disease progress curve (AUDPC) according to the following equation:p < 0.05 were considered statistically significant.All obtained values were statistically processed using Minitab 21 Statistical Software, whereby one-way analysis of variance (one-way ANOVA) was performed, and the resulting values were compared using Tukey’s honestly significant difference (HSD) test. Values having Pectobacterium-specific primer pair F0145/E2477  and Hippo F1 (33 isolates), forming characteristic cavities on CVP medium and small, whitish, irregularly shaped colonies on NA . All iso45/E2477 .The pathogenicity of all cabbage isolates was confirmed on cabbage heads by visually identifying irregularly shaped soft lesions (approximately 3−5 cm in diameter) around the inoculation points (holes) 1 d.a.i. The diameter of decomposing tissue enlarged daily, and the affected area spread from the outer leaves to the inner tissues, while causing tissue discoloration from cream to black. At 5 d.a.i., cabbage heads were almost completely macerated and exuded a specific odor. 5-, and REP-PCR banding patterns—are shown in 5 (I-VII), and REP (I–VI) (5-PCR generated seven (I-VII), while ERIC- and REP-PCR generated six distinct banding patterns (I-VI). Based on the BOX- and GTG5-PCR findings, isolates were divided into the same seven groups, namely I: Pc2021–Pc2821, II: Pc3121–Pc3921, III: Pc4221–Pc4921, IV: Pc5021–Pc5721, V: Pv1021–Pv1621 and Pv6121–Pv6621, VI: Po7021–Po7521 and Po9121–Po9621, and VII: Pc8021–Pc8721 (II), while REP-PCR indicated homogeneity of isolates Pc4221–Pc4921 and Pc5021–Pc5721 (DNA fingerprinting group III) , each representing one group on the UPGMA tree , were randomly selected for further characterization.The UPGMA trees showing genetic diversity among the 67 tested cabbage isolates—constructed based on the obtained BOX-, ERIC-, GTGP (I–VI) . The obt1–Pc8721 . Howeveroup III) . The disP. carotovorum , one representative cabbage isolate (Pv6321) was identified as P. versatile , and one isolate Po7521 was identified as P. odoriferum , with the percent identity in the 97.76–100%, 99.78–100%, and 99.57–100% range, respectively, depending on the sequenced gene , as shown in P. carotovorum was the most prevalent species (62.69%), while P. versatile and P. odoriferum were less represented . The sequences obtained for the seven representative cabbage isolates were deposited in the GenBank under the following accession numbers: OP729211-OP729217 (dnaX), OP729218-OP729224 (icdA), OP729225-OP729231 (mdh), and OP729232-OP729238 (proA).Based on the NCBI BLASTn analysis, five representative cabbage isolates  were identified as dnaX, icdA, mdh, and proA is presented in P. carotovorum, P. versatile, and P. odoriferum isolates/strains were separated into three clusters within the tree, each corresponding to one species. However, genetic heterogeneity  was observed within each species. Five of the seven tested cabbage P. carotovorum isolates examined in this study were separated into four groups  within the cluster. The remaining tested P. versatile isolate Pv6321 was the most closely related to the comparative P. versatile strain DSM 30169 isolated from cabbage in Germany, while the P. odoriferum isolate Po7521 was most similar to the type P. odoriferum strain CFBP 1878 isolated from chicory in France. D. solani strain RNS 05.1.2A was placed on a monophyletic tree branch as an outgroup.The NJ phylogenetic tree generated based on the concatenated sequences of genes The developed disease symptoms observed seven days after the spray-inoculation of cabbage heads and potato tubers with suspensions of the tested isolates are presented in The results of the AUDPC analysis of the tested isolates on cabbage heads and potato tubers are shown in P. odoriferum isolate Po7521 and P. carotovorum isolate Pc8321, respectively  virulence potential. Statistically significant differences between the initial (0 d.a.i.) and the final (5 and 7 d.a.i.) weights were observed after cabbage inoculation with the P. carotovorum isolates Pc2321 and Pc3821, as well as the P. odoriferum isolate Po7521. On the other hand, the weights measured 5 and 7 d.a.i. with the P. versatile isolate Pv6321 were comparable but differed significantly from the initial weight (0 d.a.i.).The AUDPC values pertaining to cabbage heads ranged from 4964.2 to 5990.46 for the ectively A. Based P. carotovorum isolates Pc2321 and Pc4821, respectively , one P. versatile Pv6321, and one P. odoriferum Po7521, the weights measured 5 and 7 d.a.i. were comparable, but differed significantly from the initial values (0 d.a.i.).The AUDPC values pertaining to potato tubers ranged from 249.92 to 342.15 for the ectively B. As theErwinia carotovora subsp. carotovora was identified in the Bačka region, to provide evidence on the presence and diversity of the three bacteria  causing soft rot in cabbage in Vojvodina (Serbia). The obtained results indicated presence of a combined infection in Field II, where all three identified species were confirmed on the cabbage hybrid Hippo F1. However, in Field I (hybrid Cheers F1), only P. carotovorum was detected. Analysis of the 67 cabbage isolates indicated that P. carotovorum was the most represented (62.69%), followed by P. versatile (19.40%) and P. odoriferum (17.91%). These findings are not surprising, given that P. carotovorum species is generally recognized as the main causal agent of soft rot in Brassicaceae plants [P. versatile and P. odoriferum on cabbage. In addition to P. carotovorum, P. versatile, and P. odoriferum, Chen et al. [P. aroidearum, P. brasiliense, and P. polaris on Chinese cabbage grown in different districts of Beijing (China). Presence of P. odoriferum was also reported on cabbage and Chinese cabbage in Central Poland, China, and Iran [P. odoriferum and P. versatile on cabbage does not imply its absence on this host, as it is at least partly due to their recent reclassification  within the genus Pectobacterium [To the best of our knowledge, this is the first study since the pioneering work of Arsenijević and Obradović published more than 20 years ago , in whice plants . This spe plants ,49,50,51n et al.  also repand Iran ,48,52. Hacterium .Pectobacterium includes heterogonous strains characterized by diverse biochemical, physiological, and genetic properties even within the same species [Pectobacterium spp. isolates tested as a part of the present study were genetically heterogeneous, forming seven (BOX- and GTG5-PCR) and six (ERIC- and REP-PCR) groups on the UPGMA tree, depending on the utilized primers. In other words, the rep-PCR results indicate interspecies genetic heterogeneity, as well as intraspecies heterogeneity within P. carotovorum isolates only, which were clustered in five (BOX- and GTG5-PCR) and four (ERIC- and REP-PCR) groups. The rep-PCR  analysis conducted by Alvarado et al. [P. carotovorum strains isolated from Chinese cabbage in Pernambuco state (north-east Brazil). Based on the rep-PCR with primers for BOX-, ERIC-, and REP-PCR, Loc et al. [Pectobacterium strains of the same species tend to group closely according to their respective taxonomic designations. Considering that authors of several extant studies singled out rep-PCR as discriminative enough to reveal subtle differences between different Pectobacterium spp., as well as those among the same species, as proposed by Zoledowska et al. [P. parmentieri strains from Poland, rep-PCR is a promising technique for the clarification of genetic diversity and discrimination of soft rot-causing Pectobacterium spp.It is known that the genus  species . Based oo et al.  also revc et al.  reached a et al.  for P. pdnaX, icdA, mdh, and proA) enabled appropriate identification of the tested cabbage isolates and a clear separation of each of the three identified species from one another. The existence of inter- and intra-species genetic heterogeneity between the three detected Pectobacterium species was again confirmed based on MLSA with concatenated sequences of the same four genes. According to Zeigler [dnaX gene is considered one of the best candidates for assigning bacterial strains to the species level. Sławiak et al. [dnaX for the identification of European potato Dickeya spp. strains. However, despite the demonstrated high resolution of the dnaX gene, the use of other protein-coding genes should not be discouraged due to the well-known claim that mutations, as the main engine of evolution, in Pectobacterium spp. mostly occur on these genes [icdA, mdh, and proA) were successfully used for the typing of Pectobacterium spp. in different combinations with other housekeeping genes  [In the present study, typing of four housekeeping genes  ,18,19,20S, etc.) .P. odoriferum isolate Po7521, followed by the P. versatile isolate Pv6321. On the other hand, the P. carotovorum isolates Pc2321, Pc3821, Pc4821, Pc5421, and Pc8321 were the least virulent, with a comparable virulence potential for cabbage. However, such statistical differences in aggressiveness among isolates/species were not detected on potato tubers. While these findings may be indicative of host−pathogen specificity in the case of bacteria P. odoriferum and P. versatile, more confident claims about such interactions would require more extensive studies performed on a larger number of known hosts. Bearing in mind the wide distribution, ubiquity, and polyphagous nature of P. carotovorum [P. odoriferum strains isolated from Chinese cabbage exhibited much higher virulence potential for Chinese cabbage compared to the tested P. carotovorum and P. brasiliensis strains (measured 24 h and 30 h post-inoculation), all obtained from the same host. These authors did not observe statistically significant differences in the virulence potential between P. carotovorum and P. brasiliensis strains. However, it remains to be established whether the differences between species would be sustained for the duration of the disease progression. Moreover, based on the results reported by Waleron et al. [P. carotovorum and P. odoriferum, or between P. carotovorum and P. brasiliensis, based on the analyses performed 3 d.a.i. In the virulence assessment assay performed as a part of this work, the lowest AUDPC values pertaining to cabbage heads  was observed for the otovorum , and theotovorum , P. odorn et al. , no statThis research significantly contributes to the current knowledge of the diversity of pectolytic bacteria affecting cabbage in Serbia, which has so far remained unexplored despite their great importance.


Sinosenecio B. Nordenstam (Asteraceae) currently comprises 44 species. To investigate the interspecific relationship, several chloroplast markers, including ndhC-trnV, rpl32-trnL, matK, and rbcL, are used to analyze the phylogeny of Sinosenecio. However, the chloroplast genomes of this genus have not been thoroughly investigated. We sequenced and assembled the Sinosenecio albonervius chloroplast genome for the first time. A detailed comparative analysis was performed in this study using the previously reported chloroplast genomes of three Sinosenecio species.Sinosenecio species exhibit a typical quadripartite structure. There are equal numbers of total genes, protein-coding genes and RNA genes among the annotated genomes. Per genome, 49–56 simple sequence repeats and 99 repeat sequences were identified. Thirty codons were identified as RSCU values greater than 1 in the chloroplast genome of S. albonervius based on 54 protein-coding genes, indicating that they showed biased usage. Among 18 protein-coding genes, 46 potential RNA editing sites were discovered. By comparing these chloroplast genomes' structures, inverted repeat regions and coding regions were more conserved than single-copy and non-coding regions. The junctions among inverted repeat and single-copy regions showed slight difference. Several hot spots of genomic divergence were detected, which can be used as new DNA barcodes for species identification. Phylogenetic analysis of the whole chloroplast genome showed that the four Sinosenecio species have close interspecific relationships.The results showed that the chloroplast genomes of four Sinosenecio albonervius was revealed in this study, which included a comparison of Sinosenecio chloroplast genome structure, variation, and phylogenetic analysis for related species. These will help future research on Sinosenecio taxonomy, identification, origin, and evolution to some extent.The complete chloroplast genome of The online version contains supplementary material available at 10.1186/s12864-022-08872-3. The sophisticated oxygenic photosynthesis performed by chloroplasts is the most remarkable function of modern plastids. As a photosynthetic organelle capable of supplying energy to green plants, chloroplasts play an important role in photosynthetic oxygen production and secondary metabolism and the biosynthesis of starch, fatty pigments, and amino acids. Chloroplasts and their complex signaling pathways provide a fine regulatory mechanism for plant development, metabolism, and environmental response, forming a major genetic system with the nucleus and mitochondria –3.Chloroplasts also have their independent genomes. Most chloroplast genomes of angiosperms are highly conserved and exhibits a typical quadripartite structure, usually with 110–130 genes, including a large single-copy region (LSC), a small single-copy region (SSC), and two inverted repeat regions (IRs), ranging in size from 120 to 160 kb . Due to Sinosenecio B. Nordenstam (1978) (Asteraceae) contains 44 species that are primarily found in central and southwestern China [Sinosenecio is divided into two species assemblages based on chromosome number and endothecial cell wall thickening patterns, namely the Sinosenecio s.s. group and the S. oldhamianus group [rn China –9. This us group –13. ThesndhC-trnV, rpl32-trnL, matK, and rbcL, were used to determine the relationship of Sinosenecio species. However, the chloroplast genomes of this genus have not been thoroughly investigated. Here, we sequenced and assembled the chloroplast genome of Sinosenecio albonervius Y. Liu & Q. E. Yang. Combined with reported three Sinosenecio species  B. Nord) chloroplast genomes, a detailed comparative analysis was carried out in this study.Previously, several chloroplast markers, including S. albonervius, which includes a pair of inverted repeat regions (IRs) of 24,848 bp separated by large single-copy region (LSC) of 83,355 bp and small single-copy regions (SSC) of 18,173 bp , 4 rRNAs , and 7 tRNAs . 16 genes  have a single intron, and 2 genes (ycf3 and clpP) contain two introns . Moreover, there is no significant difference in GC content between the analyzed genomes.We assembled a 151,224 bp closed circular chloroplast genome with a typical quadripartite structure from the sequencing data of S. albonervius chloroplast genome contained 53 simple sequence repeats (SSRs), including 26 mononucleotide repeats, seven dinucleotide repeats, eight trinucleotide repeats, and 12 tetranucleotide repeats . In contrast, S. albonervius has 26, as do S. jishouensis and S. baojingensis. Furthermore, we discovered repeat sequences (> 10 bp) in the chloroplast genomes , 21 reverse (R), 37 palindromic (P), and four complements (C) repeats, and the largest repeat is a palindromic repeat with a size of 48 bp.S. albonervius (Table The codon usage frequency and relative synonymous codon usage (RSCU) frequency were calculated using 54 protein-coding sequences from the chloroplast genome of S. albonervius (Table ndhB genes contain the most RNA editing sites (9 sites), while several genes  include only one editing site. C-T conversion occurred at the first (21.7%) and second codon positions (78.3%) of all RNA editing sites, indicating that the editing frequency of the third codon position was lower than that of the second or first codon positions. Furthermore, serine codons were edited more frequently than other amino acid codons, and the conversion from serine to leucine occurred the most frequently.A total of 46 potential RNA editing sites were found in 18 protein-coding genes from the chloroplast genome of us Table . The ndhSinosenecio species were compared and analyzed to determine the level of divergence, with S. oldhamianus as a reference  are found in following regions: trnK_UUU-rps16 (0.01583), ycf1 (0.01444), ccsA-ndhD (0.01333) and trnT_UGU-trnL_UAA (0.01306). However, these regions are primarily concentrated in LSC, implying that the LSC contains the most highly diverse regions.The chloroplast genomes of nce Fig. . IR regiies Fig. . The sizome Fig. . Pi valuSinosenecio of subtribe Tephroseridinae and the other two clades consist of eight species from subtribe Senecioninae. In the genus Sinosenecio, S. oldhamianus is the first to differentiate, followed by S. albonervius, and finally S. baojingensis and S. jishouensis. From the perspective of whole chloroplast genomes, Sinosenecio is phylogenetically close to Farfugium and Ligularia.An ML phylogenetic tree was constructed using the chloroplast genome sequence alignments of 14 Asteraceae species Fig. . All nodS. albonervius, and deposited it in Genbank (OL678114). Comparing the chloroplast genomes of S. albonervius and the other three Sinosenecio species revealed that their genomes have a uniformly typical quadripartite structure with the same numbers of total genes, protein-coding genes and RNA genes as well as consistent GC content. Meanwhile, they differ slightly in the size of the SC and IR regions, which reflects the high degree of conservativeness in angiosperms chloroplast genomes to some extent. 18 genes in S. albonervius contain introns that significantly affect RNA stability, regulation of gene expression, and alternative splicing [rps7 gene is unique to gymnosperms, while the loss of at least seventeen genes  was found to be common in angiosperms. However, it is noteworthy that the four Sinosenecio species retain the above seventeen genes that are easy to be deleted, and most of these genes are related to NADPH-quinone oxidoreduction [We assembled the complete chloroplast genome of nk OL6781. CompariSinosenecio species chloroplast genomes, while most of the SSRs appear in the SC regions, especially in the LSC region. We found that A or T mononucleotide repetition is the most primary repetitive type, and all mononucleotide repeats are composed of A and T. Such results are consistent with previous reports that A and T are the most abundant repeats in the most angiosperms chloroplast genome, and rarely contain tandem G or C repeats [S. albonervius chloroplast genomes, the largest of which is a 48-bp palindrome repeat. Repeat sequences are essential genetic resources that play a significant role in phylogenetic studies. Larger and more complex repeat sequences may significantly impact chloroplast genome rearrangement and sequence divergence [Simple sequence repeats (SSR) are tandem DNA repeats with short motifs found in plant nuclear, mitochondrial and chloroplast genomes, and exhibit polymorphism and a codominant inheritance pattern. These sequences have been widely used to speculate genetic variation among plant genotypes and as DNA markers in population genetic researches , 18. The repeats . Furthervergence –24.S. albonervius were all C-T conversions at the codon's second or third position (21.7 vs. 78.3%). According to previous research, the editing site is usually in the first or second base of codons, resulting in the hydrophilic amino acid being transformed into hydrophobic [Synonymous codons encode the same amino acids with different frequencies in many organisms, known as codon bias. The genetic code is usually conserved between organisms but differs in the frequency of codons usage for each amino acid. The selection for which codons are frequent and rare is generally consistent within each genome –28. In orophobic .Sinosenecio species are highly conserved, with high similarity and gene order conservancy. However, the IR and coding regions are more conserved than the SC and non-coding regions, supported by previous findings [Sinosenecio genomes. There were two models proposed to explain the extension of the IR regions. Small IR expansion and movement are due to gene conversion, while double-stranded DNA breaks and recombination cause major IR expansion [accD–pasI, trnK_UUU-rps16, ycf1, ccsA-ndhD, trnT_UGU-trnL_UAA) are mostly found in the LSC regions. Some of these regions, such as ycf1, ccsA-ndhD, and trnT_UGU-trnL_UAA, have been reported in previous studies on the chloroplast genome [We discovered that the chloroplast genomes of findings , 35. Thefindings . The lenxpansion , 38. Furxpansion . These ht genome . The IR t genome .Sinosenecio phylogenetic relationship, we assembled a dataset of chloroplast genome sequences. The interspecific relationship within Sinosenecio has been strongly supported by phylogenetic analysis, and this result is essentially consistent with their taxonomy. However, Sinosenecio is a large genus with 44 species, and only four species' chloroplast genome sequences were used in this analysis, making a more comprehensive comparison with phylogenetic results inferred from other chloroplast fragments  or nuclear genes impossible. In addition, according to Liu 2010, S. albonervius, S. baojingensis, S. jishouensis, and S. oldhamianus, based on chromosome number and patterns of endothecial cell wall thickenings, were considered to be partial members of S. oldhamianus group. This group is closely related to Nemosenecio (Kitam.) B. Nord of subtribe Tephroseridinae may represent a new genus or should be merged into Nemosenecio [Nemosenecio that we can use to illustrate this conclusion from the level of chloroplast genome at present. Therefore, more taxon sampling and a more rounded analysis of chloroplast genomes are necessary to deeply understand the Sinosenecio genetic relationship.The chloroplast genome sequences with sufficient variable loci have been successfully used for classification and phylogenetic studies . To deteosenecio , 44. StiS. albonervius was assembled and compared to other Sinosenecio species. Sinosenecio chloroplast genomes shared structural characteristics such as strict gene order, stable GC content, and relatively conservative IR and coding regions, while boundary region expansion and contraction influence genome size. Some codons encoding amino acids in S. albonervius have codon usage bias, which is critical for regulating gene expression. 46 RNA editing sites were detected based on 18 protein-coding genes showing that editing events often occurred in the first and second positions of the codon. Furthermore, the phylogenetic analysis strongly supported the interspecific relationship within Sinosenecio, and partial hotspot regions for this genus genome divergence can be used as new DNA barcodes in species identification. Our study provides valuable information for future research on taxonomy, identification, and systematic evolution in Sinosenecio.The complete chloroplast genome of S. albonervius leaves were collected from Hupingshan Natural Reserve in Hunan Province, China, and dried with silica gel. The voucher specimen was deposited at the herbarium of Jishou University. Plant Genomic DNA Kit DP305  was used to extract high-quality total DNA from the silica-dried leaf. Whole-genome sequencing was performed on the Illumina Hiseq platform by Guangdong Mercells Cell Biotechnology Co., Ltd. .Fresh S. albonervius by the program GetOrganelle [https://chlorobox.mpimp-golm.mpg.de/geseq.html) [S. jishouensis as a reference. Finally, the S. albonervius chloroplast genome was uploaded to NCBI (Genbank: OL678114). Furthermore, the chloroplast genome map of S. albonervius was drawn using the web link (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html) [The clean data were used to assemble the complete chloroplast genome sequence of rganelle , and thieq.html) . The obtaw.html) .https://webblast.ipk-gatersleben.de/misa/) [The simple sequence repeats (SSR) were detected by using MISA online tool (e/misa/) , and thee/misa/) . REPutere/misa/) , 50.Sinosenecio chloroplast genome sequences were studied using the IRscope online program (https://irscope.shinyapps.io/irapp/) [S. albonervius chloroplast genome was analyzed using CodonW in MEGA [http://prep.unl.edu/cgi-bin/cp-input.pl), with a cutoff value of 0.8 [The expansion and contraction of IR regions in /irapp/) . The cod in MEGA , and proe of 0.8 .Sinosenecio chloroplast genomes obtained from Genbank were compared with S. albonervius on the mVISTA online program using the Shuffle-Lagan model [S. oldhamianus as the reference.an model , with S.Sinosenecio complete chloroplast genome sequences were aligned using MAFFT [For the nucleotide diversity analysis, ng MAFFT . A sliding MAFFT , 56.Sinosenecio species and other ten Asteraceae species sequences, were downloaded from GenBank to clarify the phylogenetic position and relationship of S. albonervius with other related species. The genus Aster was selected as an out-group. All these sequences were aligned by using MAFFT, and RAxML-8.2.12 was used for maximum likelihood analysis on Cipres Portal  with the GTRGAMMA model, and 1000 bootstrap replicates [Thirteen complete chloroplast genome sequences, including three plicates .Additional file 1.


A drug- sensitive strain of E. coli missing the main multidrug efflux pump AcrB was constructed as a host and the yddA gene was knocked out in wild-type (WT) and drug-sensitive E. coliΔacrB to study the yddA function. Sensitivity to different substrates of WT E.coli, E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA strains was compared with minimal inhibitory concentration (MIC) assays and fluorescence tests. MIC assay and fluorescence test results showed that YddA protein was a multidrug efflux pump that exported multiple substrates. Three inhibitors, ortho-vanadate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and reserpine, were used in fluorescence tests. Ortho-vanadate and reserpine significantly inhibited the efflux and increased accumulation of ethidium bromide and norfloxacin, while CCCP had no significant effect on YddA-regulated efflux. The results indicated that YddA relies on energy released from ATP hydrolysis to transfer the substrates and YddA is an ABC-type multidrug exporter. Functional study of unknown ATP-binding cassette (ABC) superfamily transporters in the model organism E. coli is conducive to discovering new multidrug resistance-reversal targets and providing references for studying other ABC proteins of unknown function.A putative multidrug efflux gene,  Bacteria have several efflux pumps associated with drug resistance. By energy source and amino acid homology, drug efflux proteins of bacteria are usually divided into seven superfamilies: the ATP-binding cassette (ABC), the small multidrug resistance (SMR), the major facilitator (MFS), the multidrug and toxic compound extrusion (MATE), the resistance/nodulation/cell division (RND), the proteobacterial antimicrobial compound efflux (PACE) family and the p-aminobenzoyl-glutamate transporter (AbgT) family . Of thesE. coli K-12MG1655 strain genome was predicted to encode 69 ABC transporters. Eleven of the ABC transporters are presumed to be exporters [E. coli were reported to mainly include capsular polysaccharide transport pump KpsT [E. coli that have been reported included MsbA and MacA. MsbA is a multidrug efflux pump and MacAB is a single-drug efflux pump of macrolides[E. coli can deepen our understanding of the mechanism of multiple drug resistance. The E. coli K-12 genome has five predicted ABC family drug efflux pumps, YddA, YbhFSR, YhiH/YhhJ, YadGH and MdlAB, but relatively few reports of them are available. Amino acid sequence analysis showed that the N-terminus of YddA protein is a transmembrane domain formed by five transmembrane helix domains (TMDs). The C-terminus is a nucleotide-binding domain (NBD), and the NBD domain has Walker A, Walker B, signature, and other important motifs. However, few reports are available on the exact function of YddA protein. If YddA is an efflux pump of the ABC family, whether it can transfer a single substrate or recognizes and transfers multiple substrates at the same time is unclear. Further experiments are needed.ABC transporters exist in almost all organisms from microorganisms to humans , 5. ABC ump KpsT , hemolysump KpsT , 15, macump KpsT -19, multump KpsT , micromyump KpsT , and hemump KpsT . Drug efE. coli, E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA strains were cultured in LB liquid medium at 37°C. A drug-sensitive strain was constructed by knocking out the acrB gene in E. coli K-12, then the yddA gene was knocked out in WT E. coli and E. coliΔacrB. PCR amplification used pKD3 or pKD4 plasmids as templates. Target fragments and electrocompetent cells with pKD46 plasmid were added to a chamber and subjected to electric shock with a Bio- Rad electroporation system . Positive clones from plate were selected for PCR verification . Carbonylcyanide m-chlorophenylhydrazone (CCCP) and reserpine were from MedChemexpress . Norfloxacin disks were from Hangzhou Microbial Preparation (China). Gel extraction kits were from ThermoScientific. Wizard SV Gel and PCR Clean-UP System were from Promega . DH5α competent cells were from Tiangen Biochemical Technology Co., Ltd. (China). Competent cell preparation kits were from TaKaRa (Japan).Tetracycline, oxytetracycline, doxycycline, rifampicin, norfloxacin, cefminox, cefazolin, daunorubicin, doxorubicin, streptomycin, roxithromycin, deoxycholate, chloramphenicol, ofloxacin, acridine flavin, sodium cholate, ampicillin, quinine, Hoechst 33342, and ethidium bromide were from Coolaber (China). https://evomics.org/resources/software/bioinformatics-software/clustal-x/) was used for multiple sequence alignments of the YddA and other homologous proteins. Genetic distances were calculated and phylogenetic trees were constructed with Mega 7.0 software (https://www.megasoftware.net/) using the neighbor-joining method. A homology alignment of YddA with other proteins of the ABC family was conducted to identify conserved motifs using DNAStar software (https://www.dnastar.com/). Basic physicochemical properties of the protein were predicted using Protparam software (https://web.expasy.org/protparam/). The transmembrane domain was predicted using TMHMM2.0 software (http://www.cbs.dtu.dk/services/TMHMM/). ProtScale software  was used to analyze hydrophobicity.FASTA format sequences were obtained from the National Center for Biotechnology Information (NCBI) for all proteins. ClustalX software  broth, and after 1:1,000 dilution with MH broth, 100 μl was added to each well of black 96-well plates. Antibacterial drug solutions were separately added. The first to eleventh wells were treated with drug solution at 100 μl per well. In the twelfth well, cells were added as growth controls. Cells were cultured approximately 16-20 h at 37°C, and cell densities were determined using a spectrofluorometer . The lowest drug concentration that completely inhibited bacterial growth in wells was the MIC. MIC assays were according to Morita et al.  and Ohen [et al. . Three ret al. and Biswas et al. [E. coli and three mutant strains were tested for drug sensitivity with norfloxacin susceptibility discs. Cells were cultured in LB solid medium with norfloxacin susceptibility discs and grown at 37°C for 24 h. Diameters of inhibition zones were measured with Vernier calipers.According to the method of Traub s et al. -28, WT EEthidium bromide efflux and accumulation assays. Ethidium bromide (EB) is a substrate widely used with bacterial efflux pumps. It is a probe that can detect and quantify bacterial efflux activity. The difference between EB efflux and accumulation assays was that in efflux assays, EB was added to energy-starved cells for 5 min and then glucose (25 mM) was added. Immediately, EB efflux was detected with a spectrofluorometer  in real time, at 37ºC, with fluorescence emission recorded every 60 sec for 15 min.E. coli, E. coli ΔyddA, E. coli ΔacrB, and E. coli ΔacrBΔyddA strains. Cells were cultured in LB liquid medium to an OD600 of about 0.6 at 37°C. Cells were harvested and washed twice with 50 mM KPi buffer (pH 7.0) containing 5 mM MgSO4 and suspended in the same buffer, 0.5 mM 2,4- dinitrophenol was added to cell suspensions for 30 min to consume cellular energy. Cell suspensions were washed three times with 50 mM KPi (pH 7.0) containing 5 mM MgSO4 and 2.5 μM EB was preloaded into the energy-poor cells. Cultures were incubated for 5 min at 37°C, 25 mM glucose was added, and the EB efflux experiment was initiated. Excitation wavelength was 500 nm (9-nm slit), and emission wavelength was 580 nm (20-nm slit). The EB efflux tests were performed according to the protocol as described by Abdel-Motaal et al. [et al. [et al. [The EB efflux assay was performed on WT l et al. , Reuter E. coli, E. coliΔyddA, E. coliΔacrB, and E. coli ΔacrBΔyddA strains were grown in LB liquid medium supplemented with 0.5 mM 2,4-dinitrophenol. Cells were harvested at mid-logarithmic phase, washed with 50 mM KPi (pH 7.0) buffer containing 5 mM MgSO4 and 25 mM glucose, and suspended in the same buffer. EB (2.5 μM) and inhibitors  were added to cell suspensions. Cells were incubated for 10 min at 37°C, and fluorescence was recorded using a spectrofluorometer . Excitation and emission wavelengths were 500 nm and 580 nm.In accumulation assays, WT Norfloxacin accumulation and efflux assays. WT E. coli, E. coliΔyddA, E. coliΔacrB, and E. coliΔacrBΔyddA were grown in liquid LB medium supplemented with 40 mM potassium lactate to OD600 1.0. Cells were collected by centrifugation, washed with 0.1 M Tris-HCl buffer (pH 7.0), and resuspended in the same buffer to OD 600 1.0. After incubation at 25°C for 5 min, norfloxacin (16 μg/ml) was added to initiate the assay. One-milliliter samples were removed at timed intervals and immediately centrifuged at 12,000 ×g for 1 min at 4°C and then washed with the same buffer. Pellets were resuspended in 1 ml 100 mM glycine-HCl (pH 3.0). Where indicated, ortho-vanadate (1 mM), CCCP (40 μM) or reserpine (80 μM) was added to the assay mixtures. Cell suspensions were shaken vigorously at 37°C for 1 h to lyse cells, centrifuging at 12,000 ×g for 5 min at 4°C. The supernatants were diluted 2- fold with the same buffer and fluorescence values detected by a spectrofluorometer . The excitation and emission wavelengths were 277 and 448 nm for norfloxacin. The norfloxacin accumulation test was performed as described by Abdel-Motaal et al. [l et al. .Norfloxacin is a quinolone antibiotic. Quinolones have strong stable fluorescence in acidic media because they contain conjugated systems of chromophores or cosmophores. Therefore, to detect norfloxacin efflux, we referred to classic efflux detection methods for autofluorescent drugs such as tetracycline  or doxorE. coli, E. coliΔyddA, E. coliΔacrB, and E. coliΔacrBΔyddA strains were grown in liquid LB medium to OD600 0.8. Cells were resuspended and washed twice with 0.1 M Tris-HCl buffer (pH 7.0) and resuspended in 0.1 M Tris- HCl buffer (pH 3.0.) Norfloxacin (16 μg/ml) was added and cells incubated at 37°C for 5 min. Samples (200 μl) were put into black 96-well plates and glucose was added to cell suspensions (25 mM) to initiate norfloxacin efflux. Efflux of norfloxacin was continuously measured by a spectrofluorometer. The excitation and emission wavelengths were 277 and 448 nm.WT Inhibition of norfloxacin transport by MDR substrates. To determine if efflux of norfloxacin was inhibited by known MDR substrates, we used WT E. coli and mutant strains. The cells were grown to OD600 0.8. Collection of the cells was the same as for the norfloxacin accumulation assays. We mixed five concentrations of norfloxacin  with fixed concentrations of EB (or other MDR substrates) in Mg2+ buffer containing cells. Four concentrations of EB  were examined. This method was used to study the inhibitory kinetics of norfloxacin against different drugs [50 value was determined based on the concentration of the drug with a 50% inhibitory effect on 32 μg/ml norfloxacin efflux. Assays were conducted as described by Li et al. [Norfloxacin induction and qRT-PCR analysis. To explore whether norfloxacin can induce the transcription of yddA, WT E. coli and E. coliΔacrB strains were incubated in the presence of norfloxacin. Cells were cultured in 2 ml of MH broth medium with low concentrations of norfloxacin. After incubation at 37°C for 12 h, the concentration of selective norfloxacin was increased to continue the next induction. Finally, a series of induced mutants with different MICs of norfloxacin were obtained.-ΔΔCt method was used in the calculations. Three independent qPCR experiments were performed for each sample.Total RNA was extracted by using the conventional Trizol method. The first strand cDNA was synthesized with a Superscript III reverse transcription kit (Bioneer). The specific primers used for RT-PCR are shown in t-tests were used to analyze differences among groups. Data are expressed as mean ± standard deviation.Experiments were repeated at least three times. Student’s E. coli), LmrA (Lactococcus lactis), HorA (Lactobacillus brevis), and Sav1866 (Staphylococcus aureus) , P-gp (human) and Sav1866 (S. aureus), suggesting that YddA was a putative multidrug efflux pump belonging to the ABC family of E. coli  . This reh motifs . These m E. coli .yddA gene was amplified using genomic DNA of E. coli K-12 as template, and a 1,686 bp fragment was found by 1% agarose gel electrophoresis, consistent with the expected fragment size. The fragment was recovered and purified and ligated to the pMD18T vector. Single colonies picked from ampicillin-resistance plates were verified by PCR  When the yddA gene was knocked out in WT E. coli and E. coliΔacrB, three mutant strains showed increased sensitivity to rifampicin, norfloxacin, ofloxacin, tetracycline, oxytetracycline, doxycycline, cefazolin, EB and Hoechst 33342. (ii) When the yddA gene was knocked out in E. coliΔacrB, cells showed increased sensitivity to cefminox, daunorubicin and doxorubicin. E. coliΔyddA cells had the same results as WT E. coli. (iii) Three mutant strains showed increased sensitivity to quinine compared to WT E. coli. However, this inhibition was different from results in (i), with bacterial growth but densities lower than WT E. coli strain. (iv) No difference was seen between the three mutant strains and WT E. coli to roxithromycin, chloramphenicol, streptomycin, ampicillin, acridine flavin, sodium cholate, or deoxycholate  and other substrates such as EB and Hoechst 33342. WT ycholate . These rE. coliΔacrBΔyddA was significantly larger than for the E. coliΔacrB-sensitive strain, The single-knockout strain E. coliΔyddA had increased sensitivity to norfloxacin, as shown by a diameter increase of at least a 5-mm compared to the WT E.coli. Inhibition zones for different strains are in yddA gene was knocked out.Disk diffusion tests were used to verify results of norfloxacin. Strains showed results consistent with the MIC experiments. The inhibition zone of E. coli strain, the abilities of E. coliΔacrB, E. coliΔyddA, and E. coliΔacrBΔyddA to perform EB efflux were significantly lower, and the efflux ability of the E. coliΔacrBΔyddA double knockout was the lowest.As shown in ortho-vanadate 1 mM, CCCP 40 μM, reserpine 80 μM) were added to cell suspensions. Consistent with the results of efflux assays, when no inhibitor was added, levels of EB accumulation in the three knockout strains were significantly higher than in the control WT E. coli. Levels were higher in double knockout strains E.coliΔacrBΔyddA than in E. coliΔacrB strains , the concentration of ortho-vanadate (0.5-2 mM) or reserpine (20-160 μM) was increased gradually. Ortho-vanadate , MsbA (E. coli), MacB (E. coli), and BmrA (B. subtilis) [The hallmark of ABC transporters are similar topologies, two transmembrane domains (TMDs) and two cytoplasmic nucleotide-binding domains (NBDs). In bacteria, usually a TMD and an NBD fused to form a semi- transporter. The semi-transporter can form homodimers or heterodimers. The putative drug exporter YddA protein contains an N-terminal transmembrane domain and a C-terminal ATP binding domain to form a half- transporter. This structure is the same as the ABC family of multidrug efflux pumps LmrA (ubtilis) . These fE. coli genome [E. coli KAM3 to detect its drug sensitivity. The MIC was tested and no significant increase was seen after YddA overexpression. The methods for identifying ABC-type MDR transporters include knocking out candidate genes that will increase drug sensitivity, and overexpression of MDR transporters in sensitive strains that will increase drug resistance. In our study, acrB gene in the AcrAB system was knocked out, disrupting the integrity of the AcrAB transporter and destroying its function [E. coliΔacrB was constructed. We knocked out of the yddA gene in E. coliΔacrB and E. coli K-12 to study its function and we tested the sensitivity of the four strains to 20 drugs. The MIC assay confirmed that E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA cells showed increased sensitivity to rifampicin, norfloxacin, ofloxacin, tetracycline, oxytetracycline, doxycycline, cefazolin, EB, Hoechst 33342 and quinine. But there are three drugs, cefminox, daunorubicin and doxorubicinhat that show increased sensitivity only in double knockout strain E. coliΔacrBΔyddA. Resistance phenotypes are not easily observable due to the high redundancy of drug transporters with overlapping specificities in bacterial genomes, but studying the function of the target gene in sensitive strains helps. One special drug in the YddA- regulated export, quinine, behaves differently from other drugs in the MIC assay. When quinine was added, the mutant strains E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA were at a sub-suppressive level. But quinine could not inhibit YddA-regulated norfloxacin efflux. These two results seem contradictory, but they agreed with each other. At the same time, it provided us with a new idea that the efflux of quinine regulated by YddA transporter was not the same as the other substrates, therefore it is possible that the binding site of quinine to the YddA transporter is worse than other substrates.In 2001, Kunihiko Nishino and Akihito Yamaguchi analyzed all putative MDR proteins of the i genome , includifunction , and a dyddA and acrB genes with real-time PCR in norfloxacin-induced strains WT E. coli and E. coliΔacrB was studied. qRT-PCR analysis showed that the transcription of yddA was upregulated upon exposure to norfloxacin.The drug efflux transporters of ABC family are rarely expressed without performing certain functions; therefore, the expression of genes in the presence of a drug can be analyzed by qRT-PCR assay. If one or more drugs are capable of increasing the expression of a particular ABC gene, it is reasonable to assume that one or more drugs are efflux substrates. Similarly, comparing gene expression between drug-resistant strains and wild-type strains is also useful for identifying proteins associated with drug resistance. Therefore, we used the substrate norfloxacin of YddA transporter, and then tested the transcription levels of related genes in the drug-resistant strains and control strains. The relative expression levels of E. coliΔacrBΔyddA than E. coliΔacrB, while efflux capacity was significantly lower, indicating that yddA knockout reduced the efflux capacity and increased the accumulation of the drug. Three inhibitors, ortho-vanadate, reserpine, and CCCP were used in fluorescence experiments. Ortho-vanadate and reserpine significantly inhibited efflux and increased accumulation of EB, and norfloxacin inhibition was not seen with CCCP. This result showed that the energy source of YddA was ATP instead of proton-motive force. We used known MDR substrates to detect their effect on norfloxacin export. Several MDR substrates, EB, Hoechst 33342, rifampicin, cefminox, cefazolin, tetracycline, doxycycline, oxytetracycline, and chlortetracycline significantly inhibited norfloxacin efflux. Among these substrates, only CCCP and quinine did not inhibit norfloxacin efflux. These results indicated that YddA was a multidrug efflux pump belonging to the ABC family, with a wide range of substrates. Our study on the yddA gene is relatively preliminary. In theory, the YddA transporter can only extrude substrates to the periplasmic space instead of extracellular. Other auxiliary proteins are required to complete transportion, such as the membrane fusion protein (MFP) family proteins and the outer membrane channel proteins. Only two genes, yddA and yddB, were found in the operon where YddA is located. Bioinformatics analysis found amino acids 49-165 of the YddB protein contain a plug domain that belongs to TonB-dependent porin protein and YddB localizes to outer membrane. Some drug efflux transporters in bacterial can form tripartite efflux pumps, and these pumps typically consist of three essential components, an inner membrane transporter, a membrane fusion protein (MFP) and an outer membrane factor (OMF), such as MacAB-TolC system and AcrAB-TolC system from E. coli. In contrast, the pumps that are not organized in this manner and exist as single-component pumps in the inner membrane. These single-component pumps may take up drugs from the cytosol and function with porins or other types of protein channels to make the efflux process effective, such as Tet pumps. YddA gene is located in an operon with the outer membrane channel protein YddB but not with the MFP protein. Therefore, the YddA protein very likely forms single-component pumps with protein channels YddB to extrude substrates. YddA are similar to some known ABC multidrug transporters but have their own characteristics. The study of YddA protein will increase our understanding of the ABC family.We further detected the efflux and accumulation in cells of fluorescent substrates . Intracellular fluorescent substrate accumulation was significantly higher for E. coli. In our studies we found that YddA transporter had a wide range of drug substrates including tetracyclines , quinolones (norfloxacin and ofloxacin), cephalosporins (cefminox and cefazolin), anticancer antibiotics (daunorubicin and doxorubicin), antimalarial drugs (quinine), antituberculosis drugs (rifampicin) and other cationic substrates such as EB and Hoechst 33342. YddA is a prokaryotic homolog of P-gp similar to LmrA, MsbA and Sav1866.With the discovery of LmrA, the multidrug resistance of prokaryotes and eukaryotes merged. Prokaryotic multidrug efflux pumps of ABC such as LmrA, MsbA, and Sav1866 were consL. lactis), Sav1866 (S. aureus) and BmrA (B. subtilis), all of which are related to bacterial resistance. Therefore, the importance of ABC transporters in cell physiology and medicine cannot be underestimated. ABC transporters are now recognized as the most important of all protein families. Research on ABC proteins in various forms has now become a small industry. As in many areas, research on “advanced” eukaryotic systems cannot proceed quickly without fundamental advances in microbial research [ortho-vanadate and reserpine. Therefore, research on the function of YddA, a putative drug efflux pump of the ABC family of E. coli, a model organism of prokaryotes, may provide a useful reference for explaining medically important homologous proteins such as P-gp in humans and pathogenic microorganisms. Understanding the mechanisms by which eukaryotic and prokaryotic cells develop resistance is key to developing effective new drugs. In cells, the multidrug-resistant phenotype often results from multiple mechanisms, which may include multiple transporter systems. Therefore, more research on the function of hypothetical drug efflux pumps will deepen our understanding of the mechanism of MDR transporters. YddA is a multi-drug efflux transporter belonging to ABC superfamily, which can recognize and transport a variety of substrates. Functional studies of the yddA gene provide theoretical support for further understanding of the ABC family.Because multidrug ABC proteins cause serious public health problems, they are a main target of clinical research. In bacteria, several ABC-type multidrug transporters have been discovered, especially in gram-positive bacteria. These include LmrA (research . YddA ishttp://jmb.or.kr.Supplementary data for this paper are available on-line only at