Selaginella, which is the sole genus of the Selaginellaceae family, have an important place in evolutionary history, along with ferns, as such plants are valuable resources for deciphering plant evolution. In this study, we sequenced and assembled the plastid genome (plastome) sequences of two Selaginella tamariscina individuals, as well as Selaginella stauntoniana and Selaginella involvens. Unlike the inverted repeat (IR) structures typically found in plant plastomes, Selaginella species had direct repeat (DR) structures, which were confirmed by Oxford Nanopore long-read sequence assembly. Comparative analyses of 19 lycophytes, including two Huperzia and one Isoetes species, revealed unique phylogenetic relationships between Selaginella species and related lycophytes, reflected by structural rearrangements involving two rounds of large inversions that resulted in dynamic changes between IR and DR blocks in the plastome sequence. Furthermore, we present other uncommon characteristics, including a small genome size, drastic reductions in gene and intron numbers, a high GC content, and extensive RNA editing. Although the 16 Selaginella species examined may not fully represent the genus, our findings suggest that Selaginella plastomes have undergone unique evolutionary events yielding genomic features unparalleled in other lycophytes, ferns, or seed plants.The early vascular plants in the genus  A and IRB) [ndh genes in distantly related fern lineages [Chloroplasts, representing the most typical form of plastids, are semiautonomous cellular organelles found in photosynthetic plants and algae that contain their own genomes. Plastid genomes (plastomes) are typically 120–160 kb long, with a quadripartite architecture comprising one long single-copy (LSC) region and a short single-copy (SSC) region separated by two inverted repeats (IRand IRB) . Althougand IRB) ,5, IR exand IRB)  and fernand IRB) , and thelineages  and in slineages ,11 and alineages ,14,15.Selaginella [Selaginella genus contains over 700 species distributed in a diverse range of habitats, including deserts, tropical rain forests, and alpine and arctic regions.Pteridophytes are free-sporing vascular plants comprising two classes—Lycopodiopsida (lycophytes) and Polypodiopsida (ferns)—which form distinct evolutionary lineages in the tracheophyte phylogenetic tree . Lycopodaginella ,20,21. TchlB, chlL, and chlN in Psilotum SW and Tmesipteris [ndh genes and reduction of the small single-copy length in Schizaeceae [Analyses of plastome sequences have revealed that considerable genomic changes have occurred in some lineages of pteridophyte species. Comparative analyses with moss plastomes revealed five inversions in the fern plastomes . One ~3.sipteris ,24 and tizaeceae . Therefoizaeceae ,22,25,26Huperzia lucidula [Huperzia species  [Isoetes flaccida  [Selaginella species  [H. lucidula with those of bryophytes and seed plants revealed a 30 kb inversion in H. lucidula and seed plants [Anthoceros formosae), and four lycophytes  and identified an inversion between the plastomes of I. flaccida and Selaginella species and a microinversion between H. lucidula and I. flaccida. Furthermore, the gene ycf2 is present in Huperzia, but has been deleted in Isoetes [H. lucidula and bryophytes, but the plastome of S. uncinata is considerably different from those of bryophytes, which were derived from a unique inversion event, transpositions, and many gene losses [Since the release of the first plastome sequence for the lycophyte lucidula , completodiales) ,29, Isoeoëtales) , and Selellales) ,34,35,36d plants . Karol e Isoetes . Tsuji eSelaginella plastomes is the direction of the repeat blocks. Although the near or complete loss of IRs has been reported in some plant lineages of conifers and Fabaceae [A and IRB in most plastomes [A and DRB) in most plastomes in the genus Selaginella [Selaginella species are a genus-wide GC bias and the high occurrence of RNA editing. An analysis of the 3507 plastome sequences from algae to seed plants at the National Center for Biotechnology Information (NCBI) as of November 2019 [Selaginella plastomes was 52.8%, ranging from 51.0% in S. moellendorffii to 54.8% in S. uncinata [Selaginella plastid genes. Whereas about 200–500 sites of C-to-U RNA editing have been detected in flowering plant plastomes, more than 3400 C-to-U RNA editing events were discovered in S. uncinata plastomes [Selaginella genus [One notable feature of Fabaceae ,5, the tlastomes ,38. Receaginella ,34,35,36ber 2019  revealedlastomes . This exla genus .Selaginella species—Selaginella tamariscina, Selaginella stauntoniana, and Selaginella involvens—via whole-genome sequencing (WGS) using both the Illumina sequencing platform and the Oxford Nanopore long-read platform and assembled their complete plastomes. We performed a genus-wide comparison of genomic features, including GC contents, structural changes in the genome, and gene losses. This analysis uncovered remarkably dynamic features of the plastomes of plants of the Selaginella genus in the Division Lycopodiophyta.In the current study, we sequenced the plastomes of three Selaginella species, including S. tamariscina, S. stauntoniana, and S. involvens  in the Lycopodiopsida, which were obtained from NCBI  instead of the inverted repeats (IRs) found in most plastomes. This DR structure was confirmed by PCR amplification using primer combinations designed based on the junction sites for the hypothetical DR and IR structures  genes than those of non-Selaginella species (S. hainanensis had the highest number of plastome genes (103 genes), whereas S. tamariscina had the fewest (75 genes). Huperzia and Isoetes species contained almost all plastid genes. All 19 lycophyte species analyzed carried two copies of the four ribosomal RNA (rRNA) genes located in the repeat regions.The number of plastome genes differed significantly among lycophyte species, pointing to the frequent occurrence of gene loss events. In particular, the plastomes of  species . Within Isoetes and Huperzia species contained most plastid genes, except for a few that were lost or pseudogenized. In Selaginella species, many genes were absent or pseudogenized, specifically tRNA genes, ribosomal protein genes, and ndh genes. While non-Selaginella species had an average of 29.7 tRNA genes, Selaginella species averaged 11.6 tRNA genes, i.e., less than half the number in non-Selaginella species. The S. stauntoniana and S. tamariscina plastomes each contained only six tRNA genes. There were also many losses of protein-coding genes, such as the loss or pseudogenization of most ndh genes in S. lepidophylla, Selaginella lyallii, Selaginella indica, S. stauntoniana, S. tamariscina, Selaginella vardei, and Selaginella bisulcata, as well as losses of many ribosomal subunit genes throughout the genus. Other genes lost in Selaginella species included genes related to lipid biosynthesis (accD), translation initiation (infA), and other miscellaneous functions .Selaginella species and two lineages within the Selaginella genus, all divided by two main divergence events  and insertions/deletions (InDels) to analyze the intraspecies diversity in the S. tamariscina collections. The collections from regions within Korea showed relatively little variation, harboring up to 1 SNP and 15 InDels. However, a comparison of SNP and InDel counts in collections from Korea vs. China revealed much more variation. Plastomes of Korean collections showed approximately 1218 SNPs when compared to those of two Chinese collections: China2018 and China2019. Furthermore, between the two Chinese collections [chlB and chlN) that induced frameshifts resulting in premature stop codons. While China2019 retained all three chl genes, the other four collections contained the remains of chlB and chlN as pseudogenes that are likely nonfunctional. A BEAST phylogenetic tree of five S. tamariscina individuals with S. stauntoniana, which is the closest relative, as the outgroup showed that SNU2014, SNU2018, Korea2020, and China2018 diverged from each other around 90 thousand years ago, but diverged from China2019 around 900 thousand years ago  in the plastomes of the 19 lycophytes . Of the Selaginella species had considerably higher GC ratios than non-Selaginella species . There was no correlation between the orientation of the repeats and the GC content. Overall, Selaginella species lost more intron sequences compared to non-Selaginella lycophytes, and the intron pattern was shared in each lineage.Analysis of the GC contents of the plastomes revealed that the  species . The GC S. tamariscina, S. stauntoniana, and S. involvens, respectively, started with ACG instead of ATG. The annotation of the three plastomes was hindered by the frequent absence of authentic ATG start codons for protein-coding genes, as many genes contained ACG nucleotides instead of ATGs at their start sites. Stop codons were also missing in some genes. After a final manual curation of the gene annotations, we determined that 76.8%, 77.2%, and 53.3% of protein-coding genes in S. tamariscina plastome. We mapped raw RNA sequencing data from S. tamariscina onto the complete plastome sequence of the same species to determine whether the potential RNA editing sites were indeed edited in the mRNA sequences. We calculated the ratios of reads aligned to the start and stop codons in the unedited (C nucleotide) vs. edited (T nucleotide) forms using CLC find variations (ver. 4.3.0). In the S. tamariscina plastome sequence, 43 of the 56 genes contained potential editing sites for start codons, and four genes contained potential editing sites for stop codons  had a higher ratio of RNA-edited reads with the T nucleotide (switched from the U nucleotide during RNA sequencing) than unedited reads with the C nucleotide.To validate our hypothesis about RNA editing in these sequences, we conducted a transcriptome analysis of the p codons . The coeSelaginella species had a higher rate of abnormal start codons , while the Huperzia species had very low percentages (<10%), and I. flaccida was on the lower end of the range for Selaginella species (Selaginella sanguinolenta had the highest percentage (41.79%), and the levels for most other Selaginella species were similar; the exceptions were S. lyallii, S. kraussiana, S. lepidophylla, S. stauntoniana, and S. tamariscina, with percentages < 10%. Some codons (marked as ‘others’) did not fall into either category, as they lacked authentic start or stop codons, but did not contain codons that were potential targets for RNA editing . In stop codons, the proportions of potential RNA editing sites ranged from 4.71% to 5.81% for H. serrata and H. lucidula, respectively, while those for Selaginella species ranged from 0% for S. kraussiana to 41.79% for S. sanguinolenta  to 42.65% (S. involvens), averaging 35.73%. These values are higher than those in non-Selaginella species (12.73–18.72%)  [Selaginella species contrasts with the SSC contraction in Schizaeceae ferns, in which the SSC was reduced by as much as 2255 bp to contain only two genes in Schizaea elegans [Viviania, Hypseocharis, and Pelargonium, but expanded in the genera Melianthus, Francoa, and California [Selaginella genus.Overall, our results support previous findings that the plastome sequences of d plants . As of F–18.72%) , as welllifornia . The oveIsoetes and Huperzia. However, the Selaginella genus contains both inverted and direct repeats. A large inversion occurred just after the divergence of Selaginella from other lycophyte genera, which gave rise to DRB in the Selaginella plastomes. DRs have also been reported in S. tamariscina [S. vardei, and S. indica [I. flaccida with S. vardei, proposing that this inversion might have occurred between 142.5 and 281.5 mya during the Late Triassic [Selaginella genus, except for the three species of S. lepidophylla, S. hainanensis, and S. uncinata. Further inversions involving DRB occurred independently in S. lepidophylla and in S. hainanensis and S. uncinata, in both cases restoring IRB and lengthened LSCs. S. lepidophylla split from its sister species S. indica and S. vardei approximately 25.64 mya. Therefore, the inversion in S. lepidophylla must not be older than 25.64 million years old. S. hainanensis and S. uncinata diverged from each other approximately 1.03 mya, and from their sister species S. bisulcata and S. pennata approximately 4.51 mya. Therefore, this inversion must have occurred between 4.51 and 1.03 mya. The grouping of Selaginella species is generally in agreement with the reported phylogeny of Selaginellaceae [Selaginella species are relatively younger than those from previous reports [rbcL [Canonical plastomes are arranged in a quadripartite structure composed of an LSC and SSC region separated by two inverted repeats . This isariscina , S. vard. indica . Zhang eTriassic , which iellaceae ,21. Howe reports . These dts [rbcL , leadingts [rbcL , which iSelaginella species, only one instance of DRs has been reported, in the red alga Porphyra purpurea [Selaginella species.IRs are an interesting feature of circular plastomes. Although one missing IR has been reported in eudicots in Fabaceae ,42 and ipurpurea . IR and purpurea . Aside fpurpurea . The synpurpurea . AlternaSelaginella species showed evidence of severe gene losses that resulted in their overall small plastomes. The most noticeable losses involved ndh genes, encoding for NADH dehydrogenase that function in electron transfer during photosynthesis. ndh genes were lost or pseudogenized in several Selaginella species. The ndh genes are also absent or pseudogenized in some species of green algae and almost all gymnosperms [ndh genes is compensated for by the nucleus-encoded ndh genes in gymnosperms [ndh genes in S. tamariscina [Selaginella species adapted to dry environments have lost their ndh genes, suggesting that this gene loss might be related to their adaptation to these habitats [ndh genes was recent, as revealed by the pseudogenization of all ndh genes in S. bisulcata, whereas its sister species S. pennata retained the complete set: These two species only diverged 3.61 mya  is thought to have driven most plastomes to become AT biased [S. uncinata and S. moellendorffii plastomes, Smith proposed that unbiased mutation/gene conversion equilibrated the nucleotide composition to ~50% GC contents in Selaginella plastomes. Alternatively, he suggested that RNA editing has influenced the GC contents in these plastomes, as numerous GC-rich codons were changed into AT-rich codons through RNA editing [Unlike nuclear genomes, organellar genomes are AT biased, with an average GC content of 36.45 ± 2.81% and 36.87 ± 8.23% detected in 11,542 mitochondrial genomes and plastomes, respectively . TherefoT biased ,57. Base editing .Selaginella plastomes are subject to extensive RNA editing. C-to-U RNA editing is a post-transcriptional modification mechanism that grants gene diversity. There are 30–50 in plant plastomes, but S. uncinata contains 3415 RNA editing sites [Selaginella plastomes. The frequency of RNA editing at the start codons of plastid genes was >50% in some Selaginella species. Furthermore, S. tamariscina RNA sequencing reads confirmed these editing sites. This high number of editing sites coincides with the exceptionally high number of pentatricopeptide repeat (PPR) protein gene families (>800) in the S. moellendorffii nuclear genome, representing proteins essential for plastid RNA editing [S. moellendorffii [Isoetes engelmannii [S. tamariscina plastomes, the number of total events observed could be even greater than that in S. uncinata [ng sites . Accordi editing ,58. Hechndorffii . Grewe eelmannii , indicatuncinata .S. tamariscina (SNU2014) individual was collected from Baegunsan, Gwangyang-si, Jeollanam-do, Korea. A second S. tamariscina individual (SNU2018) was collected from Jeju Island. S. stauntoniana from Danyang-gun, Chungcheongbuk-do, Korea, and S. involvens from Jeju Island were provided by Hantaek Botanical Garden, Yongin, Gyeonggi-do, 17183, Republic of Korea.One Selaginella species and three non-Selaginella lycophytes were obtained from NCBI (Table 1). To analyze the intraspecies diversity in S. tamariscina, two plastome sequences obtained in the current study (SNU2014 and SNU2018) were used, along with assembled and annotated sequences for another Korean collection reported by Park et al. collected from Wolsong-ri, Jijeong-myeon, Wonju-si, Korea [The complete plastome sequences of 13 i, Korea  and two i, Korea ,35.S. tamariscina (SNU2014), S. stauntoniana, and S. involvens. The DNA quality and concentration were assessed by gel agarose electrophoresis and UV-spectrophotometry . The extracted DNA was sequenced on the Illumina NextSeq platform, generating 7.68, 2.2, and 2.0 Gb of raw data from S. tamariscina (SNU2014), S. stauntoniana, and S. involvens, respectively.Leaves were collected from the plants and ground in liquid nitrogen with a mortar and pestle. A modified cetyltrimethylammonium bromide (CTAB) method  was usedThe plastomes of the three species were assembled using the de novo assembly of Low-Coverage Whole genome sequence (dnaLCW) approach with the CLC genome assembler program  ,63. In sS. tamariscina individual (SNU2018), total genomic DNA was extracted using an Exgene Plant SV midi kit . The DNA quality and concentration were assessed via agarose gel electrophoresis and UV-spectrophotometry. The extracted DNA was sequenced on a MinION sequencer from Oxford Nanopore Technologies. Library preparation was performed with a Ligation Sequencing Kit (SQK-LSK108) following the manufacturer’s instructions. One flow cell was run for 48 h. In total, 10 Gb of raw sequencing reads was filtered by removing adaptor sequences using Porechop  and corrected with Canu using default parameters [https://github.com/ruanjue/smartdenovo). The complete plastome sequence assembled using Illumina sequencing data was used as a reference to extract plastome-related contigs assembled by Nanopore sequencing using BLASTN.For the second rameters . The Canhttps://chlorobox.mpimp-golm.mpg.de/geseq.html) with complete plastome references of other species in the Selaginella genus and non-Selaginella species registered in NCBI. The annotated sequences were manually curated for details such as start and stop codons and correct coding frames using Artemis [https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) for sequence homology. Furthermore, the aligned sequence was searched in the Conserved domain search provided by NCBI for complete gene structures. Genes that have sequence homology detected by TBLASTN but have incomplete gene structures due to the lack of sequences of internal stop codons were assigned as pseudogenes. Genes that were not searched at all through TBLASTN were marked as missing genes. This validation step for the pseudogene/missing gene assignment was conducted for other already published sequences used in this study.The plastomes assembled in the previous steps were annotated using GeSeq . Primers were used in different combinations to amplify the junction sites of the plastome. Hypothetical direct repeat junctions were amplified by primer combinations 1/2, 3/4, 5/2, and 3/6, while hypothetical inverted repeat junctions were amplified by different primer combinations of 1/3 and 2/4 for the first repeat block (IRB) (To confirm the direct repeat structure of ck (IRB) . Taq polymerase, 2.5 µL of 10× reaction buffer, 0.2 mM dNTPs , 10 ng genomic DNA, and 10 pmol of each primer. The PCR was carried out in a thermocycler with the following parameters: 5 min at 94 °C; 28 cycles of 20 s at 94 °C, 20 s at 62 °C, and 20 s at 72 °C; and 7 min at 72 °C for the final extension step. The PCR products were visualized by electrophoresis using a 1% agarose gel with a 100 bp ladder.PCR amplification was performed in a 25 µL volume containing 1U http://www.cbs.dtu.dk/services/FeatureExtract/). Existing genes and pseudogenes were marked. Among the extracted CDS, 45 genes that were common to all 19 species were combined into one long fasta sequence for each species. These sequences were aligned using MAFFT . The aligned sequences were trimmed using Gblocks [Isoetes and Selaginella families reported by Wikstrom and Kenrick [CDS, tRNA, and rRNA sequences for all 19 species of interest were extracted using FeatureExtract 1.2 L  by following the manufacturer’s instructions. The RNA quality and concentration were assessed using the Agilent Bioanalyzer  and Nanodrop spectrophotometer  with parameters RIN ≥ 7, 28S:18S > 1, and OD260/280 ≥ 2.https://github.com/trinityrnaseq; [http://weizhongli-lab.org/cd-hit/; [For RNA sequencing, the cDNA libraries were constructed using TruSeq Stranded mRNA  and samples were processed with next-generation sequencing (NGS) procedures on an Illumina Hiseq3000 with a paired-end method. The NGS reads were filtered by the threshold of >20% ‘N’ bases, ≤Q20 in Phred quality, and a length > 50 bp. De novo assembly was performed using TRINITY  and CD-cd-hit/; ).Genes in the plastomes of all 19 species were surveyed to check for the presence of intron regions. Potential C-to-U RNA editing sites were surveyed in the start and stop codons of the CDS from the plastomes of ten lycophyte species. Start codons with an ACG sequence instead of ATG and stop codons with CAA, CAG, and CGA sequences instead of TAA, TAG, and TGA were counted as potential RNA editing sites. Potential RNA editing sites were marked for all ten species and analyzed.S. tamariscina_2 were mapped onto the reference genome produced from the assembly of S. tamariscina (SNU2014) sequencing data. The start and stop codon positions of the CDS were obtained, and CLC find variations software (ver. 4.3.0) was used to examine the distribution of unedited mRNA reads with the C nucleotide and edited mRNA reads with the T nucleotide (previously U in RNA that changed to T during the process of RNA sequencing).To determine whether the potential RNA editing sites had gone through the editing process, raw RNA sequencing data from


Staphylococcus pseudintermedius group (SIG) is an emerging threat in veterinary medicine, particularly methicillin-resistant (MRSP) isolates, which are frequently associated with multidrug resistance. Reliable identification of SIG members is critical to establish correct antimicrobial treatments. However, information on the molecular epidemiology and antimicrobial resistance patterns of MRSP in some regions is still limited. This study aimed to assess the antimicrobial resistance of SIG isolates recovered from animals at the Veterinary Teaching Hospital of Complutense University of Madrid (Spain) during a 10-year period (2007–2016). A total of 139 selected Staphylococcus isolates were subjected to species-level identification by different bioanalytical techniques  and subsequent antimicrobial susceptibility testing. Methicillin-resistant isolates (n = 20) were subjected to whole genome sequencing for further characterization of their antibiotic resistance determinants. Our results showed that there was a good correlation between PCR and MALDI-TOF identification, whereas VITEK showed very divergent results, thus confirming MALDI-TOF as a good alternative for species-level identification of coagulase-positive staphylococci. Notably, S. pseudintermedius, including the epidemic MRSP genotype ST71, was the only SIG species found among canine isolates. In addition, we found a high prevalence of multidrug resistance and resistance to fluoroquinolones, cephalosporins and macrolides. Finally, diverse genes associated with antibiotic resistance were detected among MRSP isolates, although the genetic basis of some of the resistant phenotypes (particularly to fluoroquinolones) could not be determined. In conclusion, our study reveals the circulation of MRSP in the veterinary setting in Spain, thus highlighting the emerging threat posed by this bacterial group and the need for further epidemiological surveillance.The  Staphylococcus aureus is a well-recognized pathogen that is responsible for a plethora of clinical manifestations  in humans and animals worldwide. Coagulase-positive Staphylococcus (CoPS) isolates other than S. aureus, including the members of the Staphylococcus intermedius group  are emerging as a serious threat in veterinary medicine, especially in small animals. In particular, S. pseudintermedius is now considered the main CoPS species recovered from household pets [S. pseudintermedius is also part of the normal microbiome of small animals and is considered an opportunistic pathogen that is implicated in a variety of infections  [S. pseudintermedius may pose a serious but often neglected zoonotic risk [old pets . S. pseus, etc.) . Additiotic risk . S. aureus (MRSA) strains have become a major cause of concern in animal and human medicine. Similarly, decreased antimicrobial susceptibility has been detected in S. pseudintermedius isolates from companion animals. Strains carrying methicillin resistance genes  are of special importance in animal and human pathology [Methicillin-resistant athology . Detaileathology . MRSP ofathology . This maathology . In addiStaphylococcus species [S. intermedius and S. pseudintermedius is essential to clarify important aspects of their epidemiology [Correct species-level identification of the staphylococci is a critical step for the establishment of adequate antimicrobial treatments, and is key for successful clinical outcomes. In this sense, oxacillin minimum inhibitory concentration (MIC) breakpoints depend on the  species , which eemiology . Currentemiology ,7, incluemiology .S. pseudintermedius isolates recovered from animals at the Veterinary Teaching Hospital of Complutense University in Madrid  over a period of ten years (2007–2016). Isolates were identified to the species level, and phenotypically and genotypically characterized. In recent years, the clinical relevance, molecular epidemiology and antimicrobial resistance patterns of MRSP have been assessed in different countries ,9. HowevS. pseudintermedius (n = 117) identification by PCR and MALDI-TOF (κ = 0.528). In contrast, concordance between PCR and VITEK was much lower (κ = 0.010). Considering MALDI-TOF as the gold standard technique for S. aureus identification, VITEK identified correctly all isolates but one, which was assigned to S. intermedius. However, VITEK misidentified 13 S. pseudintermedius and one S. schleiferi isolates as S. aureus. All three S. schleferi isolates were identified by PCR and MALDI-TOF. In general, the correlation between PCR and MALDI-TOF results was good while VITEK showed very divergent results.Most Staphylococcus isolates analyzed in this study were recovered from dogs , but isolates also came from other sources including horses , cats and mice , and lion, sheep and swine . Isolates were collected from ear exudates , body fluids  or other origins . The results of the different CoPS identification techniques used in this study are summarized in S. pseudintermedius and 9 S. aureus) showed resistance exclusively to penicillin, the most frequent combination of antimicrobial resistance was tetracycline/penicillin , and 66 isolates  were multidrug resistant (MDR), i.e. resistant to at least three distinct classes of antimicrobial agents. The trend in the proportion of AMR-CoPS isolates over time was assessed by comparing the number of resistant isolates at three different periods covering a similar number of isolates: 2007-2010 (n = 44), 2011-2014 (n = 44) and 2015-2016 (n = 51). Among all of the tested antimicrobial agents, ciprofloxacin (P = 0.013), norfloxacin (P = 0.02), ofloxacin (P = 0.013) and trimethoprim-sulfamethoxazole (P = 0.029) showed increasing resistance rates over time (Cochran–Armitage Test)  . P > 0.05, multiple proportions Z-test), except for gentamicin (P ˂ 0.05), for which 27.6% isolates from ear exudates but only 10.5% of isolates from body fluids were resistant to this antibiotic.The association between AMR and the clinical origin of the isolates  was not significant , which was resistant to fluoroquinolones . S. pseudintermedius showed significantly higher resistance rates to kanamycin (P = 0.006), tetracycline (P = 0.001), trimethoprim-sulfamethoxazole (P = 0.003), clindamycin (P = 0.04) and chloramphenicol (P = 0.013) than S. aureus. However, S. aureus isolates were significantly more resistant to amikacin (P = 0.009) than S. pseudintermedius.The three S. pseudintermedius and 3 S. aureus isolates) were subjected to molecular analysis. All the strains were isolated from dogs, except S. aureus strain 94S, which was isolated from a pig’s lung. There was a significant association between MDR and methicillin resistance for S. pseudintermedius isolates . MRSP and MSSP isolates showed resistance against the same antibiotics except in the case of amikacin, which was only detected in one MRSP isolate. Eighteen isolates (90%) were found to possess the methicillin resistance-encoding mecA gene (this gene was not found in the genomes of S. pseudointermedius isolates 125S and 134S) (mec) analysis of the 18 mecA-containing isolates and found that most S. pseudointermedius isolates belonged to the SCCmec III type (3A) , whereas two isolates belonged to the SCCmec V type (13.3%). Likewise, three different sequence types (STs) were detected among MRSP isolates: ST71 , ST196  and ST261 . Interestingly, there was a perfect correlation between SCCmec types and STs, as all SCCmec III (3A) isolates belonged to ST71, whereas MRSP isolates of the SCCmec V (2B) and SCCmec V types belonged to STs 261 and 196, respectively. Regarding the MLST results of MRSA isolates, one of the isolates (94S) belonged to ST398, which is the ST typically associated with the livestock-associated MRSA (LA-MRSA) lineage, whereas the other two MRSA isolates belonged to two other different lineages, namely, community-associated MRSA (CA-MRSA) lineage , and hospital-associated MRSA (HA-MRSA) lineage . A total of 20 CoPS isolates resistant to oxacillin and with a positive result in the ALERE PBP2a test (17 nd 134S) . We perfblaZ. The presence of genes encoding resistance to macrolides (clarithromycin and erythromycin) were detected in 18 MRCoPS isolates (90%) including 15 (75%) methylase gene erm(B), and 2 (10%) methylase gene erm(C). In addition, the ABC transporter-encoding gene msrA and modifying enzyme mphC gene were found in the same MRSA isolate, while erm(B) or erm(C) genes were not detected. Macrolide resistance-encoding genes were not detected in two MRSP. Moreover, detection of lincosamide nucleotidyl transferase-encoding gene, lnu(A) and linA genes were detected in only one MRSA isolate. Membrane-associated efflux pump genes tet(K) and ribosome binding site gene tet(M) were detected in five (25%) and three (15%) MRCoPS isolates. One MRSA isolate simultaneously carried tet(K) and tet(M) genes.The molecular AMR profiles of 20 MRCoPS are summarized in aph(3´). In at least 90% of isolates, two aminoglycoside-streptothricin resistance-encoding genes were simultaneously detected with ant(6) + aph(3’) + aad(6) + SAT-4 being the most frequent combination (15 out of 20) among MRCoPS in our study. The cat(pc221) chloramphenicol acetyltransferase gene was detected in the four chloramphenicol-resistant MRSP isolates. Finally, 80% of the isolates (16 MRSP) carried the trimethoprim resistance gene dfr(G).Results for aminoglycoside-streptothricin resistance-encoding genes are summarized in Staphylococcus pseudintermedius has been identified as the most important SIG pathogen in dogs [S. pseudintermedius is key to the implementation of control measures for successful treatment and disease management [S. pseudintermedius may help to clarify the current clinical role of this species in veterinary medicine [ in dogs , althoug in dogs  and huma in dogs . Further in dogs . In thisnagement . Additiomedicine .S. pseudintermedius was the only SIG species found among canine isolates, which agrees with the results of previous studies [S. pseudintermedius is frequently misidentified as S. intermedius by phenotypic methods [S. pseudintermedius isolates from our strain collection were misidentified as S. aureus and 85 as S. intermedius by the VITEK system. A possible explanation for these discrepancies could be the lack of β-galactosidase test in the VITEK panel, which is frequently used to differentiate between S. pseudintermedius and S. aureus [S. pseudintermedius isolates can be misidentified as S. intermedius based on mannitol fermentation tests [nuc-based multiplex PCR assay described by Sasaki et al [S. pseudintermedius were reported [S. pseudintermedius isolates included in this study were misidentified as S. intermedius by PCR. In this study,  studies . Differe methods . Indeed,. aureus . Similaron tests . Previouki et al . The suiki et al , althougreported . AccordimecA gene. This contrasts with the results of previous studies that found a higher genetic diversity for S. pseudintermedius  belonged to the same sequence type (ST71), which is perfectly correlated with the SECCmec III(3A) type in those isolates containing the isolates . The higreported . MoreoveS. pseudintermedius isolates had the greatest levels of resistance to penicillin . The resistance rates to fluoroquinolones detected in the present study (around 20%) are in accordance with previous reports [ reports . S. aureus and S. pseudintermedius have emerged in the last decade. The relevance of MRSP in veterinary clinics is related to their frequent multidrug resistance, even to all licensed drugs available for dogs [The methicillin resistance of our selected isolates was confirmed by a PBP2a-based culture colony test, which has been recognized as a suitable diagnostic test to detect beta-lactam-resistant SIG isolates . Methicifor dogs . In addifor dogs . In partfor dogs ). In confor dogs .β-lactams, macrolides, lincosamide and aminoglycosides [Different studies have highlighted the emergence of multidrug-resistant CoPS , which wycosides ,11. In oycosides ,31. Howeycosides , who fouS. pseudintermedius may contribute to these discrepancies [Most antibiotic resistance genes detected in the genome of our collection of MRSP isolates have been reported by other authors and are presumed to be acquired via transposon transfer from other Gram-positive bacteria . Howeverepancies .mecA gene in the genome of S. pseudintermedius isolates has been previously reported [tet(K) gene was the most frequent gene in the set of MRSP isolates included in this study, which agrees with the results obtained by other authors  [tet(M) was only present in the ST196 and ST261 isolates (one isolate each) that we found, and tet(L), previously found in MSSP isolates from Spanish dogs [The presence of the reported . In our n et al. ). It has [tet(K) , which wish dogs , was notcat(PC221)-carrying plasmid was the most frequent mechanism of chloramphenicol resistance identified in our collection of S. pseudintermedius isolates [blaZ gene. Furthermore, the aph(3’) gene, which encodes an amynoglycoside-modyfying enzyme was the most frequent aminoglycoside resistance encoding gene found in isolates from dogs, thus agreeing with previous reports [ant(6), the kanamycin resistance-encoding gene aph(3’), and the streptothricin acetyltransferase encoding gene sat-4 were simultaneously detected in 65% of our CoPS isolates, which is a lower frequency than in previous reports from Spain [erm(B) as the most frequent marker of macrolide/lincosamide resistance [dfr(G) was detected in most trimethoprim-resistant MSRA and MRSP isolates as previously found [As in other studies, the catPC22-carrying reports . Remarkaom Spain  and othecatPC22-carryingsistance . The dihly found ,13. S. pseudintermedius, which may be due to the lack of harmonized strategies for antimicrobial susceptibility testing and the interpretation of resistance, as previously suggested by Moodley et al. [There is a notably high degree of heterogeneity in antibiotic resistance results across different studies focused on y et al. . Our stuStaphylococcus spp. was identified in 842 samples (15.4%) according to VITEK results . Of these, 139 isolates  were randomly selected for the present study for species-level identification by different bioanalytical techniques and subsequent antimicrobial susceptibility testing (as detailed below). A total of 5468 clinical samples were subjected to bacteriological analysis at the Veterinary Teaching Hospital of Complutense University of Madrid  from 2007 to 2016. After VITEK identification, all studied isolates were subjected to MALDI-TOF mass spectrometry analysis, following the procedures detailed in Perez-Sancho et al. . Brieflynuc) gene [S. aureus isolates were only identified by MALDI-TOF MS, as this technique achieves reliable identification of this Staphylococcus species [Additionally, SIG isolates were also identified by a multiplex PCR based on thermonuclease-encoding (uc) gene , which iuc) gene ,37. On t species ,39. Staphylococcus aureus ATCC 25923 was included as the quality control strain in all these analyses.Antibiotic susceptibility was assessed by the disk diffusion method following the Clinical and Laboratory Standard Institute (CLSI) guidelines ,41,42. Mhttps://github.com/tseemann/mlst) against the PubMLST database [mec was performed using SCCmecFinder (https://cge.cbs.dtu.dk/services/SCCmecFinder/). The resistomes inferred from the draft genomes were analyzed by BLASTN [https://github.com/tseemann/abricate) searches against the ResFinder [All isolates resistant to oxacillin and with a positive result in the ALERE PBP2a test were subjected to whole genome sequencing (WGS) for further characterization of their antibiotic resistance determinants. Bacterial strains were sequenced on a MiSeq (Illumina) platform as previously described . Brieflydatabase . Searchiy BLASTN  and ABRiesFinder  and CARDesFinder . The preesFinder .Staphylococcus species (S. aureus and S. pseudintermedius) and AMR frequencies was assessed using Fisher’s exact test. The relationship between the origin of the sample  and AMR were also evaluated using Fisher’s exact and multiple proportions Z-test with Bonferroni adjustment for multiple comparisons, respectively. The association between the resistance rates to the different antibiotics tested was determined by Cohen’s kappa coefficient (κ). The Cochran–Armitage trend test was used to determine the increase in different AMR over time. Statistical tests were performed using SPSS 25 software , except the Cochran–Armitage trend test, which was performed with the WinPepi package [P < 0.05.The association between  package . StatistIn conclusion, the results of this study confirm the value of MALDI-TOF technologies for species-level identification of SIG isolates of animal origin. Furthermore, we observed a high prevalence of MDR and resistance to fluoroquinolones, cephalosporins and macrolides, and detected epidemic MRSP genotypes such as ST71 among isolates from dogs. Finally, we found a variety of genes that have been related to antibiotic-resistant phenotypes in the genome of MRSP isolates. All in all, these results highlight the emerging threat posed by MRSP and the need for continuous surveillance of this bacterial group for antimicrobial resistance in the veterinary setting.


Currently, population aging presents a new challenge for all the countries in the worldwide. It is a natural and inevitable process, always associated with physiological and biological decline . Longer A group of 368 older persons aged 60 yr and above were successfully included by accidental non-probabilistic sampling through face-to-face interview.P-value <0.05 was statistically significant.Study protocol was explained and informed consent was obtained. Exclusion criteria were elders with dementia or speech impairments. Pearson's chi-square test is used for analysing data provided by SPSS software . P<0.05). Similar findings were reported elsewhere . Women were more likely to complain about irritable bowel, constipation, bloating, swollen belly, tight clothing and asthenia (%. Women


Morchella spp.) are popular edible fungi with significant economic and scientific value. However, white mold disease, caused by Paecilomyces penicillatus, can reduce morel yield by up to 80% in the main cultivation area in China. Paecilomyces is a polyphyletic genus and the exact phylogenetic placement of P. penicillatus is currently still unclear. Here, we obtained the first high-quality genome sequence of P. penicillatus generated through the single-molecule real-time (SMRT) sequencing platform. The assembled draft genome of P. penicillatus was 40.2 Mb, had an N50 value of 2.6 Mb and encoded 9454 genes. Phylogenetic analysis of single-copy orthologous genes revealed that P. penicillatus is in Hypocreales and closely related to Hypocreaceae, which includes several genera exhibiting a mycoparasitic lifestyle. CAZymes analysis demonstrated that P. penicillatus encodes a large number of fungal cell wall degradation enzymes. We identified many gene clusters involved in the production of secondary metabolites known to exhibit antifungal, antibacterial, or insecticidal activities. We further demonstrated through dual culture assays that P. penicillatus secretes certain soluble compounds that are inhibitory to the mycelial growth of Morchella sextelata. This study provides insights into the correct phylogenetic placement of P. penicillatus and the molecular mechanisms that underlie P. penicillatus pathogenesis.Morels ( Morchella spp.) are valuable edible fungi that belong within the order Pezizales. There is a long history of morel consumption in Asia, Europe, and North America [Fusarium incarnatum—F. equiseti species complex and pileus rot disease caused by Diploöspora longispora [Paecilomyces penicillatus, is another major disease affecting morels. This disease was first reported in Sichuan, China in 2013 [P. penicillatus infection in one location can quickly spread to neighboring morel cultivation areas and can account for up to 80% of morel yield losses. Currently, no commercial fungicide has been registered to control for morel white mold disease, and the management of this disease relies primarily on the removal of the symptomatic fruiting bodies. The molecular basis of P. penicillatus pathogenicity is, to the best of our knowledge, unknown. This makes it difficult to develop tailored solutions to control this disease.Morels  sequences and 5.8S rDNA sequences [P. penicillatus was first isolated from rotten mushrooms by Samson in 1974 and was originally placed in Trichocomaceae, order Eurotiales, class Eurotiomycetes based on the morphological characteristics of this pathogen [P. penicillatus within the order Hypocreales of Sordariomycetes, and revealed an uncertain affinity with Clavicipitaceae [P. penicillatus genomic resources, a comprehensive phylogenetic analysis based on single-copy orthologous genes is not available.equences . P. penipathogen . Howeverpitaceae . In addipitaceae ,13,14. Ppitaceae ,16,17. DP. penicillatus remain unclear. Though not known from P. penicillatus, lysis of the host cell wall is a common mechanism of mycoparasitism [Cladobotryum dendroides and Cladobotryum protrusum, which can cause cobweb disease, as well as Hypomyces perniciosus, the causative agent of wet bubble disease [Trichoderma spp. have been used as model systems to study the mechanisms of mycoparasitism [Trichoderma harzianum, the causative agent of green mold disease on white button mushroom, produces various fungal cell wall-degradation enzymes that are being used in biotechnology and in biological control [Trichoderma atroviride and Trichoderma virens indicated that they contain a large numbers of genes encoding fungal cell wall-degradation enzymes compared to non-mycoparasitic fungi [The molecular mechanisms of mycoparasitism in rasitism . Many my disease ,15,16. Irasitism . For ins control ,21,22. Gic fungi .P. penicillatus using the single-molecule real-time (SMRT) sequencing platform [P. penicillatus. Based on carbohydrate-active enzymes (CAZymes) analysis, secondary metabolite analysis, and virulence factor analysis, we demonstrated the putative molecular mechanisms of mycoparasitism of P. penicillatus. This study and the genomic resources made available herein provide a basis for the future functional characterization of the pathogenesis-related genes in P. penicillatus as well as the development of morel white mold disease management strategies.In this study, we obtained the first genome sequence of platform . ThroughP. penicillatus strain CCMJ2836 was provided by the Engineering Research Center of the Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University , which was isolated from white mold-diseased morel fruiting bodies in Sichuan Province, China. The mycelial plugs of strain CCMJ2836 were grown on potato glucose agar (PDA) medium overlaid with cellophane sheets for 10 d at 25 °C under a light/dark photoperiod (12/12 h).The P. penicillatus CCMJ2836 genomic DNA was extracted from the mycelia using NuClean Plant Genomic DNA Kits  following the manufacturer’s instructions. The 20 kb-gDNA library was prepared and sequenced on two SMRT cells with a PacBio Sequel system. Raw data were de novo assembled via SMARTdenovo (https://github.com/ruanjue/smartdenovo). Genome assembly completeness was assessed with both the Core Eukaryotic Genes Mapping Approach (CEGMA) [http://sourceforge.net/projects/glean-gene) through both extrinsic and ab initio algorirhms. (CEGMA)  and Benc (CEGMA) ). Protei (CEGMA)   referencing the following databases: InterPro, TrEMBL, National Center for Biotechnology Information non-redundant (NCBI-nr), Swiss-Prot [http://www.repeatmasker.org) and Tandem Repeat Finder [Annotations of the predicted genes were obtained through BLASTp  with protein sequences from the representative species of Eurotiomycetes , Ophiocordyceps unilateralis [Stachybotrys chartarum [Neurospora crassa [Schizosaccharomyces pombe [Schizosaccharomyces pombe was included in this analysis as an outgroup taxon.Reciprocal BLASTp searches were conducted  and Sorobertsii , T. atroroviride , T. harzarzianum , Trichoda reesei , T. vire. virens , C. dendndroides , C. protrotrusum , H. pernniciosus , Cordyceilitaris , Fusariuminearum , and Nieateralis , Stachybhartarum , and onea crassa ) were ines pombe  in Schizes pombe , and thees pombe  with 100P. penicillatus genes were searched against the CAZy database (http://www.cazy.org/) [e-value ≤ 1 × 10−5, identity ≥ 40% and coverage ≥ 40%), and were further grouped into auxiliary activities (AAs), carbohydrate-binding modules (CBMs), carbohydrate esterases (CEs), glycoside hydrolases (GHs), glycosyl transferases (GTs), and polysaccharide lyases (PLs). Modular analysis of GH family 18 was carried out with InterProScan (http://www.ebi.ac.uk/Tools/InterProScan/). Signal peptides of GH family 18 were predicted using the SignalP 5.0 server .The deduced amino acid sequences of the zy.org/)  with BLAhttps://fungismash.secondarymetabolites.org/) with the following built-in features activated: ActiveSiteFinder, ClusterBlast, Cluster Pfam analysis, KnownClusterBlast, Pfam-based GO term annotation, and SubClusterBlast. The cutoff similarity value for a known cluster was 40%. Genes within the clusters were further searched against the Pathogen-Host Interactions database (PHI-base) and the database of fungal virulence factor (DFVF) by BLASTp  to predict the putative involvement of the gene clusters in the pathogenesis of P. penicillatus. Pathogenic genes were identified using the PHI (http://www.phi-base.org/) [http://sysbio.unl.edu/DFVF/Download.php) [Gene clusters encoding secondary metabolite enzymes were analyzed with AntiSMASH fungal version 3.0 (se.org/)  and DFVFoad.php)  databaseP. penicillatus strain CCMJ2836 and M. sextelata strain GB-Ch-102 were inoculated on HIMEDIA corn meal peptone yeast agar (CMA) and incubated at room temperature for 5 d. P. penicillatus agar discs were inoculated on one side of regular or two-compartment CMA dishes. After 6 d, M. sextelata agar discs were inoculated on the other side of the petri dish at about 4 cm from the P. penicillatus agar disc and then incubated for two more days. Each confrontation culture was replicated 10 times and incubated at room temperature. Cultures were photographed with an Epson Perfection V700 Photo scanner.Cultures of P. penicillatus CCMJ2836 strain was sequenced on the PacBio SMRT Sequel platform using two SMRT cells. A total of 4,266 Mb clean data (~105 × coverage) was generated  content , Swiss-Prot (63.48%), KEGG (62.07%), COG (44.16%), TrEMBL (95.65%), InterPro (70.85%), and GO (49.55%). Overall, 96.34% genes had homologs in at least one database  were the most abundant representing 5.64% of the genome , which includes all the enzyme families known to function in carbohydrate metabolism [P. penicillatus is the GH family 18. The second and the third most abundant CAZyme families are the GH family 16 and the AA family 3, respectively . The drastic differences in the number and sequence divergence of sgC in these phylogenetically closely-related mycoparasitic organisms suggests a strong evolutionary pressure on this protein family.The GH family 18 contains all the enzymes that have been previously shown to contribute to the degradation and replenishment of both intrinsic and extrinsic fungal chitin  Figure . GH famionducted . InteresTrichoderma spp. and other representative fungal pathogens of plants or animals, P. penicillatus and Trichoderma spp. encode the highest number of GH family 18 proteins among the fungal organisms included in this study [P. penicillatus and Trichoderma spp. in comparison to the other filamentous fungi. Similarly, P. penicillatus contains the highest number of GH family 75, which could be involved in the decomposition of chitinous carbohydrates [P. penicillatus produces a plethora of chitinases and β--glucanases in resemblance to the well-characterized mycoparasitic Trichoderma spp.Beside chitinases, β--glucanases are another enzyme family that decomposes the fungal cell wall and belongs to GH family17, 55, 64, and 81 . Compareis study . For GH hydrates . Our resP. penicillatus. Among them, seven gene clusters had significant hits to known secondary metabolite biosynthesis gene clusters (P. penicillatus genes in these gene clusters showed significant sequence similarity to genes in the Pathogen-Host Interactions database (PHI-base) and/or the database of fungal virulence factor (DFVF), indicative of possible involvement of these gene clusters in the pathogenesis of P. penicillatus. Specifically, Squalestatin 1 (40% similarity) is a potent inhibitor of squalene synthase, an essential enzyme for sterol biosynthesis [Phoma sp. and several ascomycetes [Aureobasidium pullulans [Aspergillus nidulans and A. niger [P. penicillatus also contains a gene cluster that has a 40% similarity to that of a gene involved in the biosynthesis of leucinostatin, a compound with antitrypanosomal properties. Interestingly, most Paecilomyces spp. identified thus far are entomophagous [Paecilomyces sp. isolated from soil [P. penicillatus with 100% similarity, including dimethylcoproge [P. penicillatus on morel mycelia and fruiting bodies, or whether they coordinate or contribute to interspecies competition against other microbes living in the same environmental niche.We identified 74 putative secondary metabolite biosynthesis gene clusters in clusters , includiynthesis . Squalesynthesis  and was omycetes ,66. AbT1ullulans . AbA hasA. niger ,68. The ophagous , and leurom soil . In addilcoproge , monascolcoproge , and 6-mlcoproge . It remaP. penicillatus may produce certain inhibitory compounds that can affect the hyphal growth of morels. To test this possibility, we conducted dual culture assays where agar plugs of both P. penicillatus and M. sextelata were inoculated onto the same petri dish. Indeed, M. sextelata had minimum growth in the direction towards the colony of P. penicillatus, and extensive growth of M. sextelata was observed in the direction away from P. penicillatus (P. penicillatus does produce host-inhibitory substances in a contact-independent manner. In an attempt to further investigate whether the substances are volatile or soluble, we also conducted confrontation assays on enclosed two-compartment petri dishes, where P. penicillatus and M. sextelata were inoculated on the center of each compartments. We expected to observe a similar inhibitory effect of P. penicillatus on M. sextelata in the case of volatile compounds being produced by P. penicillatus. However, M. sextelata grew rapidly in all directions at nearly equal speed in these assays (P. penicillatus exhibits the antagonistic activity against M. sextelata through extracellular fungal cell wall-degradation enzymes or mycotoxins, as predicted in this study.Based on the above analyses, we hypothesized that cillatus A, suggese assays B. This iP. penicillatus, the causative agent of the morel white mold disease. The genome of P. penicillatus is ~40.20 Mb in size with an N50 value of 2.59 Mbp and 9454 coding genes. The phylogenetic analysis based on the single-copy orthologous genes demonstrated that P. penicillatus is within Hypocreales and is closely related to Hypocreaceae, a family that is known to include many mycoparasitic species. CAZymes analysis further illustrated that the genome of P. penicillatus has a large number of fungal cell wall degradation enzymes. We identified a plethora of gene clusters that are known to be involved in the biosynthesis of cytotoxic secondary compounds that may function coordinatively during the pathogenesis of P. penicillatus. We also showed that P. penicillatus can produce contact-independent soluble host-inhibitory compounds. Taken together, we provide strong evidence in support of the phylogenetic placement of P. penicillatus within Hypocreales and provide a basis for the future functional characterization of the fungal cell wall-degradation enzymes and secondary metabolites produced by this mycoparasitic fungus.In this study, we present the first whole-genome sequence of


Odontonema tubaeforme) plants exhibiting leaf curling and chlorosis. The molecular features of the viral genomic RNA and proteins resemble those of ampeloviruses. Based on sequence comparisons and phylogenetic analysis, we propose a new species in the genus Ampelovirus, which we have tentatively named Firespike leafroll-associated virus (FLRaV). Bioassays showed that the virus is mechanically transmissible to Nicotiana benthamiana. In addition, a full-length cDNA clone of FLRaV could successfully infect N. benthamiana via agroinfiltration.A novel RNA virus was identified in firespike ( Ampelovirus (family Closteroviridae) are plant RNA viruses that infect mainly fruit crops in orchards and stock nurseries, causing serious reductions in fruit yield and quality worldwide . A . A Ampeloviruses b 12]. O. OAmpelooviruses c 13]. O. OAmpeloc region . Putativc region d 15]. O. OAmpeloectively ,23.Ampelovirus species showed a range of 22.5–50.6% sequence identity  of inoculated plants  The virus contains a large, single-stranded, positive-sense RNA genome  ThA genome ; (iii) Pelovirus . In addielovirus , suggestAmpelovirus through phylogenetic analysis, the FLRaV virus genome possesses some features similar to members of subgroup I. For example, unlike members of subgroup II, the FLRaV virus genome has a small ORF positioned between the RGB and the QGB, and two additional ORFs located at the 3′ end of the genome. To the best of our knowledge, it is the first identified ampelovirus that possesses features of both subgroups. By analogy to similarly located ORFs of other members of the family Closteroviridae , FLRaV P25 and P37 might be involved in the suppression of host RNA interference [Although clustered in the subgroup II of rference ,31,32. TClosterovirus is mechanically transmissible to the model plant N. benthamiana [Ampelovirus was successfully transmitted to N. benthamiana, although the transmission rates were very low and the best transmission rates were obtained using transgenic N. benthamiana plants expressing HC-Pro from the turnip mosaic virus [N. benthamiana. It should be noted that reads with similarity to cucumber mosaic virus (CMV) and columnea latent viroid were identified in the BLASTx analysis as well, and firespike is a common host of CMV [N. benthamiana without inducing visible symptoms, similar to the full-length cDNA clone of GLRaV-3 [N. benthamiana.It has been shown that GLRaV-2 in the genus thamiana ,34, but ic virus . Here, wt of CMV . Further GLRaV-3 . Future N. benthamiana. This experimental system can be used for further analysis of ampelovirus-host interactions.By using unbiased high-throughput sequencing and conventional molecular biology tools, a new ampelovirus, tentatively named FLRaV, was identified. Full-length cDNA of FLRaV was infectious to a convenient laboratory plant host


Tricholoma matsutake; Agaricales, Tricholomataceae) is an ectomycorrhizal fungus that produces a commercially valuable, edible mushrooms. Attempts to artificially cultivate T. matsutake has so far been unsuccessful. One method used to induce T. matsutake to produce fruiting bodies of in the wild is shiro  transplantation. In vitro ectomycorrhization of T. matsutake with seedlings of Pinus densiflora has been successful, but field trials showed limited production of fruiting bodies. Few studies have been done to test what happens after transplantation in the wild, whether T. matsutake persists on the pine seedling roots or gets replaced by other fungi. Here, we investigated the composition and the interaction of the root fungal microbiome of P. densiflora seedlings inoculated with T. matsutake over a 3 year period after field transplantation, using high-throughput sequencing. We found a decline of T. matsutake colonization on pine roots and succession of mycorrhizal fungi as P. densiflora seedlings grew. Early on, roots were colonized by fast-growing, saprotrophic Ascomycota, then later replaced by early stage ectomycorrhiza such as Wilcoxina. At the end, more competitive Suillus species dominated the host roots. Most of the major OTUs had negative or neutral correlation with T. matsutake, but several saprotrophic/plant pathogenic/mycoparasitic species in genera Fusarium, Oidiodendron, and Trichoderma had positive correlation with T. matsutake. Four keystone species were identified during succession; two species  had a positive correlation with T. matsutake, while the other two had a negative correlation . These findings have important implications for further studies on the artificial cultivation of T. matsutake.The pine mushroom ( Inocybe, Rhizopogon, or Suillus) require small amount of carbon from hosts and are usually found in pine seedling in disturbed area  produces edible fruiting bodies during symbiosis with members of Pinaceae, especially Pinus densiflora  to uninfected pine trees  there is a significant change in root fungal communities during transplantation and seedling growth, and (ii) there are some fungi with positive or negative correlation with T. matsutake that affect the survival of T. matsutake on pine seedling roots.Advances in high-throughput sequencing have greatly contributed to our understanding the diversity and function of fungi in various environments , and havTricholoma matsutake strain KBFERI 20T05 (GenBank accession no. AF367417) was cultured in K-liquid media . Uncontaminated seedlings were transferred to culture vessels inoculated with T. matsutake in a sterilized culture room, then co-cultured for 3 months in a clean room illuminated with a fluorescent lamp . Then, P. densiflora seedlings were moved to a greenhouse filled with autoclaved soil from a nearby pine forest. Sixteen pine seedlings were sampled at 6 different post T. matsutake-inoculation periods: 3 months , 10, 17, 24, 31, and 38 months . In total, 96 seedling roots were harvested.This experiment was conducted at Gyeongsangbuk-do Forest Environment Research Institute in Gyeongju, South Korea. id media , and traT. matsutake , and stored at−80°C prior to DNA extraction. We did a preliminary morphological examination of roots to confirm the presence of atsutake . Seedlinatsutake . We conf primers .TM PCR SV kit , a unique identifier sequence was attached to each PCR products with a second round PCR following the Nextera XT index kit protocol . Second PCR products were purified as above. Concentration of each amplicon library were measured using a NanoDrop2000 . Amplicon libraries were pooled in equimolar quantities and sequenced using Illumina MiSeq platform at Macrogen .The fungal internal transcribed spacer 2 (ITS2) region was amplified with primers ITS3 and ITS4  with IllAfter sequencing, the raw data were processed using the Quantitative Insights Into Microbial Ecology v.1.8.0. (QIIME) pipeline . Fastq-jpost hoc test adjusted using the Benjamini and Hochberg method  were calculated in QIIME. Statistical analysis was performed in R software . Sequencing data were deposited in NCBI Sequence Read Archive (SRA) under Project ID PRJNA638021.To test for correlations between species, Sparse Correlations for Compositional data (SparCC)  network included . The netincluded . Clusterincluded . For theP. densiflora seedlings, from 63 OTUs found in M03 (mean = 7.88 OTUs per sample) to 487 OTUs in M38 (mean = 155.88 OTUs per sample)  remained with a Good’s coverage of 0.998–0.999. Based on taxonomic level, the OTUs represented 8 phyla, 28 classes, 89 orders, 188 families, and 327 genera. The number of OTUs significantly increased with the age of  sample) . Chao1 r periods .P. densiflora seedlings over time, based on the adonis analysis  and Cladosporium sp.  in M03, Pseudogymnoascus pannorum  and Oidiodendron echinulatum  in M10, and Wilcoxina mikolae  in M17. After M24, the most abundant OTU was Suillus granulatus  followed by W. mikolae  in M24, S. luteus in M31 , and W. mikolae (10.22%) in M38  and Basidiomycota (35.516%). Abundance of the next most abundant phylum, Mortierellomycota, was low at less than 0.1% . The abu) in M38 .P. densiflora seedlings, SparCC analysis was performed. Tricholoma matsutake (OTU 1) and 35 major OTUs with relative abundances >0.5%, accounting for 83.4% of total sequence reads, were clustered into four groups and one isolated OTU (T. matsutake (O. echinulatum (ericoid mycorrhiza). Based on our selection criteria, four OTUs were identified as keystone species during fungal succession of pine seedling roots: Cylindrocarpon pauciseptatum (OTU 15), Suillus granulatus (OTU 3), Fusarium oxysporum (OTU 8), and Fusarium sp. (OTU 37). All of the keystone taxa belonged to the same cluster. Among these keystone taxa, Cylindrocarpon pauciseptatum (OTU 15) and S. granulatus (OTU 3) showed significant negative correlation with T. matsutake, while Fusarium oxysporum (OTU 8) and Fusarium sp. (OTU 37) showed a positive correlation with T. matsutake  increased, while pathotrophs decreased. The presence of saprotrophic fungi is commonly reported in roots  and endophytic fungi , which are considered common fungi in an early successional stage , and Fusarium oxysporum  showed a significantly positive correlation with T. matsutake. As Suillus granulatus is a strong competitor and crucial in the establishment of pine seedlings  and also available on request to the corresponding author.The datasets generated for this study can be found in the NCBI Sequence Read Archive (SRA) under BioProject ID S-YO, MP, and YL contributed to conceiving and designing the experiments. KP and S-YO performed the experiments and analyzed the data. KP, S-YO, and SY wrote the manuscript with revisions from JF, MP, and YL. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Trichoderma lixii MUT3171, isolated from a historically PAH-contaminated soil in Italy, can grow on phenanthrene, as a sole carbon source. Here, we report the draft genome sequence of T. lixii MUT3171 obtained with high-throughput sequencing method. The genome of T. lixii MUT3171 was compared with other 14 Trichoderma genomes, highlighting both shared and unique features that can shed a light on the biotransformation of PAHs. Moreover, the genes potentially involved in the production of important biosurfactants and bioactive molecules have been investigated. The gene repertoire of T. lixii MUT3171 indicates a high degrading potential and provides hints on putative survival strategies in a polluted environment.Mono- and polycyclic aromatic hydrocarbons (PAHs) are widespread and recalcitrant pollutants that threaten both environmental and human health. By exploiting the powerful enzymatic machinery of fungi, mycoremediation in contaminated sites aims at removing a wide range of pollutants in a cost-efficient and environmentally friendly manner. Next-generation sequencing (NGS) techniques are powerful tools for understanding the molecular basis of biotransformation of PAHs by selected fungal strains, allowing genome mining to identify genetic features of biotechnological value.  Trichoderma  gathers successful colonizers of very diverse environments and is found wherever decaying plant material is available. Species from this genus are mostly known as biocontrol agents, capable of protecting plants from pathogens [Trichoderma spp. have also become of biotechnological interest due to their diversified enzymatic arsenal that facilitates their adaptability to a wide range of substrates [Trichoderma spp. into efficient and competitive generalists, through the constant interaction with different organisms [Trichoderma spp. as efficient phytosaprotrophs was likely acquired ancestrally through mycoparasitism of fungal phytopathogens [Trichoderma spp. to grow even in highly polluted environments [The fungal genus athogens  by produathogens . Trichodbstrates . These frganisms . For exaathogens . These wronments , a capacTrichoderma strains [Several environments are subjected to a variety of pollution sources, largely due to improper human activities. Mycoremediation in contaminated sites aims at removing hazardous pollutants in a cost-efficient and environmentally friendly manner. Increasing evidence revealed that the success of filamentous fungi in mycoremediation is aided by their ability to make pollutants more bioavailable, through the production of biosurfactants ,8. Howev strains ,10. The  strains ,12.Trichoderma lixii MUT3171, isolated from a highly polluted environment. Through the comparison with other sequenced Trichoderma and by investigating the peculiar genetic traits of T. lixii MUT3171, we bring perspectives on its hypothetical application in mycoremediation, and present a novel genomic resource, as no other T. lixii genomic sequences are currently available in public databases.Here, we investigate the genome of Trichoderma lixii MUT3171 was isolated from a site with a long history of industrial exploitation in Fidenza, Italy (N44.866126 W10.074016): the main contaminants were polycyclic aromatic hydrocarbons (PAHs), benzene, toluene, three xylene isomers (BTEX), and alkanes  of the Department of Life Sciences and Systems Biology, University of Torino, Torino . The soil dilution plate method was used to isolate microorganisms on a selective medium . Trichoderma lixii MUT3171 was isolated in the presence of phenanthrene at 200 mg/L. Mycelium for DNA extraction was grown on 2% malt extract agar at 25 °C for 7 days.The strain is deposited at Mycotheca Universitatis Taurinensis  and translation elongation factor 1-alpha  gene sequences obtained for T. lixii MUT3171 are available at GenBank NCBI under the accession numbers MF305834 and MT435114, respectively.The identification at species level was carried out through morphological and molecular analyses. The internal transcribed spacer region  (ABTS) (1 mM). In the presence of laccase activity, the colorless medium turns dark green. Plates were incubated at 24 °C for 7 days.The ability of Trichoderma lixii MUT3171 was inoculated in a modified mineral salt medium: soybean oil (4% w/w) was added to stimulate the surfactants producing metabolism. The flasks were incubated at 24 °C and 120 rpm. The culture broth was separated from the mycelium by centrifugation  and aliquots of the supernatant were analyzed. Positive controls were set up with Tween 80, whereas negative controls were performed using deionized water.For biosurfactants production, a qualitative drop collapse test and oil dispersion assay were performed as described by Boudour and Miller-Maier , and by T. lixii MUT3171 using the QIAamp DNA Microbiome Kit . DNA quality and concentration were assessed with Nanodrop 2000 , and 1 ng DNA was used for library preparation. The paired-end genomic libraries (2 × 250 bp) were built using the MiSeq v. 3 reagents (600 cycles) and sequenced at EUROFINS GENOMA Group .Total DNA was extracted from mycelial samples of Trichoderma asperellum (NC_037075.1), Trichoderma gamsii (NC_030218.1), Trichoderma hamatum (NC_036144.1) and Trichoderma reesei (NC_003388.1) as reference sequences.Reads quality was assessed with fastQC v.0.11.9 , and adarRNA) and transfer-RNA (tRNA) were predicted with RNAmmer v.1.2 [Nuclear genome statistics were calculated using QUAST v. 4.5 . RepeatMer v.1.2  and tRNAer v.1.2 , respectFusarium graminearum as training species. Proteome completeness was calculated with BUSCO v. 3.0.1 [Trichoderma  [https://www.geneious.com/download).De novo gene prediction was performed with Augustus v. 2.5.5  using Fuv. 3.0.1  using thv. 3.0.1 . Brieflyv. 3.0.1  and Targv. 3.0.1 , while pv. 3.0.1  and by Sv. 3.0.1 , respectv. 3.0.1 . CAZymesv. 3.0.1  and the v. 3.0.1 . For thev. 3.0.1 , targetise (CDD)  and visuSecondary metabolites gene clusters identification and characterization have been performed with antiSMASH v. 5.1.1  and BIG-ITS and TEF1-alpha indicated that Trichoderma lixii MUT3171 is closely related to Trichoderma lixii (based on BLASTn results with MF305834 and MT435114 as query).The results of colony morphology and phylogenetic analyses based on T. lixii MUT3171 nuclear genome is approximately 41 Mbp and possesses 11,923 protein-coding genes (T. lixii MUT 3171 (GenBank accession: MT495248) is 29.79 kbp in length with GC% 27.42. The mitochondrial assembly contains the gene for the ribosomal protein S3 (rps3), 14 core genes that encode proteins involved in oxidative phosphorylation and electron transport, and a highly degraded copy of the 3′ fragment of the NAD4 gene. Moreover, mitochondrial genes include the small and the large subunit of rRNA, as well as 25 tRNA genes. The gene order is conserved among other Trichoderma mitochondrial sequences .The final assembly for ng genes . The cirrRNAs, 44 8S rRNAs (of which 16 organized in clusters), 182 tRNAs, and five pseudogenes. Around 28% of the nuclear genome is repeated. The nuclear genome encodes for 472 secreted proteins, of which around 40% are smaller than 300 amino acids, making them candidate secreted effectors. More details on nuclear genome characteristics are provided in The nuclear genome contains single copies of 18S and 28S T. lixii MUT3171 by characterizing the enzyme-encoding genes that may participate in the cleavage of PAHs and other pollutants found in the sampling site. The genome of T. lixii MUT3171 was compared with those of other Trichoderma strains deposited in NCBI (to January 2020). We established an orthology relationship between the gene products of 14 species from the genus (including T. lixii MUT3171), i.e., we identified oxidoreductases, CAZymes, and biosurfactants, that probably derive from a common ancestor within the species analyzed in this study [Trichoderma harzianum complex , numbers of putative detoxifying enzymes are highly comparable, whereas at the genus level they are not. Multicopper oxidases . Biochemical evidence of intracellular laccases in Trichoderma is completely missing. Only thanks to molecular phylogenetic analyses, their occurrence in T. harzianum and T. reesei genome have been demonstrated [Trichoderma atroviride, T. harzianum and T. asperellum [Trichoderma strains are barely capable of producing extracellular laccases constitutively. As found for Trichoderma camerunense [Trichoderma koningiopsis [T. lixii MUT3171 showed a minimal laccase activity: the oxidation of ABTS in solid plate test was marginally appreciable . However, laccase gene transcription is highly sensitive to xenobiotics-related stress. The production of laccases in Trichoderma can be stimulated by the presence of aromatic compounds as alachlor [We have investigated the degradative potential of is study . Figure is study . Our anaoxidases a seem tonstrated . Extraceperellum ,47,48. Rerunense  and Tricngiopsis , T. lixialachlor , guaiacoalachlor , and pyralachlor , as a dialachlor .T. lixii MUT3171 genome  oxidoreductases [T. lixii MUT3171 genome is comparable to that of the other Trichoderma species examined in this study.The entire degradation pathway may involve other enzymes such as dioxygenases ,53 and c1 genome b. Our annt class c, being observed d. In thiductases , includiTrichoderma genomes possess a large number of glutathione S-transferases and glycosyltransferases  was observed.The knowledge of biosurfactants production by fungi is still very scarce, although these compounds represent a key factor in the PAHs transformation by enhancing the PAHs bioavailability into the soil . Only a erunense  and T. r. reesei . A classrfactant . Likewiso-ulmins h, whose T. lixii MUT3171 and other sequenced Trichoderma species in terms of degrading enzyme composition, we focused on other genetic traits that may guarantee the survival of T. lixii MUT3171 in petroleum-polluted environments. Three gene sequences  from T. lixii MUT3171 possess a PF13532 domain that indicates their involvement in the repair of DNA following alkylation damages. These enzymes, such as AlkB, mostly received attention in bacteria inhabiting petroleum-contaminated soils [T. harzianum homolog (GCF03025095.1), the phylogenetic reconstruction highlights a stronger differentiation between g11653.t1 and g7464.t1 , a poorly studied wood saprotroph [T. lixii MUT3171 protein include only Dothideomycetes, with Lepidopterella palustris, and Glonium stellatum, both wood decayers [Aureobasidium pullulans. However, little is known about the degrading potential of fungal quinoprotein alcohol dehydrogenases. Few reports, largely based on bacteria, have provided evidence of a specific role in phenanthrene degradation [Notably, the genome of choderma . For exachoderma a with a protroph . Indeed,decayers , togetheradation .T. lixii MUT3171 , polyketide synthases (PKS) and their hybrids (hybrid NRPS-PKS). In T. lixii MUT3171, we identified 23 PKS, 19 NRPS, and 8 NRPS-PKS hybrids, often co-localized on the genome with P450 monooxygenases, FAD-binding proteins, general substrates transporters, and multicopper oxidases. Nine siderophores were predicted as well, indicating that T. lixii MUT3171 has a relevant potential in iron sequestration, a process associated with microbial competition, resistance to oxidative stress, and development under limiting conditions [T. lixii MUT3171 cannot be further annotated as their composition is not similar to any known biosynthetic gene cluster. Nevertheless, we were able to infer a distance-based classification of two NRPS, three PKS, and one NRPS-PKS. Through multiple sequence alignments, the BIG-SCAPE analysis calculated the distances between the analyzed sequences and those present in the MIBiG repository [NRPS genes of T. lixii MUT3171 . While the production of cyclosporin A is known in T. harzianum [Trichoderma species, although phylogenetic results and lifestyle relationships between Escovopsis and Trichoderma [We identified candidate genes for the production of secondary metabolites and bioactive molecules in clusters , and thenditions . Most ofpository  of known MUT3171  had at larzianum , melinacchoderma  may provPKS clusters (T. lixii MUT3171 resemble those for the production of naphthopyrone in Aspergillus nidulans FGSC A4 (BGC0000107), betaenone in Phoma betae (BGC0001264 and BGC0001280), monoascorubrin in Talaromyces marneffei (BGC0000099) and stipitatic acid in Talaromyces stipitatus ATCC 10500 (BGC0000154). Naphthopyrones are pigments that protect Ascomycetes from a wide range of predators [Trichoderma. The candidate biosynthetic gene cluster for the production of the antimalarial drug stipitatic acid appears incomplete in T. lixii MUT3171, missing an essential cytochrome P450 and a FAD-binding protein [PKS genes (g7562.t1 in T. lixii MUT3171) are nearly identical.Among the clusters , three gredators , but the protein . The gen protein , containhybrid NRPS-PKS , a major facilitator transporter (g9544.t1) and a zinc-binding dehydrogenase (g9538.t1). Evidence that phomasetin and equisetin may be antibiotics with inhibitory activity against HIV-1 integrase has been provided [The detection of a NRPS-PKS  in T. lichoderma . Setins provided .T. lixii MUT3171 highlighted peculiar genetic traits that could explain its ability to inhabit extremely polluted environments and to grow on phenanthrene as the sole carbon source. Trichoderma lixii MUT3171 shares a similar degrading potential with other Trichoderma species, being capable of producing a large number of multicopper oxidases, monooxygenases, and biosurfactants. The genetic features of T. lixii MUT3171 and the comparison to the aforementioned Trichoderma species lead to some conclusions that may be drawn. It is possible to speculate that the T. lixii MUT3171 genome has not undergone simplification or loss of functions because of the extreme lifestyle in a polluted environment, at least in term of gene number, secreted proteins repertoire and content of CAZymes as glycosyltransferases, that are iconic in the Trichoderma genus [T. lixii MUT3171 to survive in a highly polluted environment may not only depend on its degradative enzymes. Indeed, genome mining revealed several traits that are unique to T. lixii MUT317, such as the presence of specialized mechanisms for DNA repair, the protection from protein unfolding and even the tolerance to heavy metals. Given the lack of homology with other Trichoderma sequences, these genes might be the result of horizontal gene transfer, a widespread phenomenon in the Trichoderma genus [Trichoderma lixii MUT3171 encodes for 58 secondary metabolites gene clusters that probably guarantee its competitiveness and survival. These genetic features are studied not only because they can provide adaptation to an extremophilic lifestyle, but also due to their biotechnological potential and economical value. Our results indicated the involvement of iron in the fungal tolerance to pollutants, as it is crucial for the functioning of DNA repair enzymes (2Fe-OG dioxygenases) and antioxidant enzymes that contain iron-sulfur clusters. This observation is also confirmed by the presence in T. lixii MUT3171 genome of 9 genes encoding for siderophores. This finding opens the possibility of using direct iron supplementation, or bioaugmentation using mixed microbial consortia to increase iron availability and enhance the fungal performance [In this work, the genome mining of ma genus . Moreovema genus . Trichodformance .T. lixii MUT3171, making it a good candidate for the treatment of PAH-contaminated soils.These observations emphasize the perspective of testing degradative ability of


Leuconostoc lactis is a glycopeptide-resistant, gram-positive, facultative anaerobic coccus isolated from dairy products, whereas Staphylococcus nepalensis is coagulase-negative coccus that has not been identified as human pathogen. We report an instructive case of L. lactis and S. nepalensis bacteremia in a 71-year-old man who experienced Boerhaave syndrome after a meal. Leuconostoc lactis is an intrinsically glycopeptide-resistant but ampicillin-susceptible, gram-positive, facultative anaerobic coccus  and an anaerobic bottle (13.3 h to culture) and S. nepalensis in 1 anaerobic bottle (24.3 h to culture). The 2 bacteria were indications of true bacteremia; therefore, we escalated ampicillin/sulbactam  with a MICrofast7J panel and determined the susceptibility of L. lactis according to Clinical and Laboratory Standards Institute (CLSI) guidelines , and red and white wine. We cultured samples from these products on blood agar medium; colonies of L. lactis, confirmed by MALDI-TOF mass spectrometry, were derived from cheese samples  scan showed collapse of the lower esophagus wall and expansion of the mediastinum; medical staff diagnosed a spontaneous esophageal rupture and performed emergency surgery. Surgical findings demonstrated a 5 cm perforation of the lower esophagus with no rupture to the thoracic and abdominal cavity. The final diagnosis included Boerhaave syndrome, esophageal hiatus hernia, and mediastinitis. Two sets of blood culture taken on day 1 were positive for gram-positive cocci, which we identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry as . lactis  Table 1 dmission  Table 2. samples .L. lactis bacteremia have been reported , rather than S. nepalensis, from the dry-cured ham colonies, we could not conclusively demonstrate the entry of S. nepalensis to the bloodstream. Moreover, contamination with S. nepalensis was possible; only 1 anaerobic bottle of blood culture taken at admission was positive. However, because S. nepalensis is not normally found in the human microbial flora but is a part of the predominant flora in dry-cured ham together with S. equorum and S. xylosus (S. nepalensis bacteremia diagnosis was correct.L. lactis into the human bloodstream and show results implying that L. lactis can be a pathogen of bacteremia, as previous reports have shown (,S. nepalensis bacteremia; further investigation is needed for confirmation.In conclusion, we demonstrate the point of entry for Leuconostoc lactis and Staphylococcus nepalensis bacteremia in a patient with spontaneous esophageal rupture. Additional information about


Spirodela polyrhiza (greater duckweed). The reduced anatomy of S. polyrhiza did not lead to a reduction in LOX family genes. The 13-LOX subfamily, with seven genes, predominates, while the 9-LOX subfamily is reduced to two genes, an opposite trend from known LOX families of other plant species. As the 13-LOX subfamily is associated with the synthesis of JA/MeJA, its predominance in the Spirodela genome raises the possibility of a higher requirement for the hormone in the aquatic plant. JA-/MeJA-based feedback regulation during culture aging as well as the induction of LOX gene family members within 6 h of salt exposure are demonstrated.Lipoxygenases (LOXs) (EC 1.13.11.12) catalyze the oxygenation of fatty acids and produce oxylipins, including the plant hormone jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA). Little information is available about the LOX gene family in aquatic plants. We identified a novel LOX gene family comprising nine LOX genes in the aquatic plant  Duckweeds can flower, but normally propagate vegetatively, with several species doubling their biomass almost daily under optimal conditions in nature or the laboratory   . A cross92545.1) . LOX genequences  were dowequences . Putativ/hmmer/) ,63. Moreescribed ,40,45. Sescribed . The prehttp://www.ebi.ac.uk/Tools/msa) [S. polyrhiza 7498  . A phylools/msa) ,67. Thisiza 7498 . An evolhttp://meme-suite.org/tools/meme/). Searching for the classical 6X histidine signature motif was done manually for each LOX protein and displayed using WebLogo3 (http://weblogo.threeplusone.com/) [https://www.ebi.ac.uk/Tools/psa/emboss_stretcher/).The MEME suite  was applne.com/) . The proS. polyrhiza from the phytozome database (http://www.phytozome.net/) and analyzed for intron-exon and intron phase distribution patterns (http://gsds.cbi.pku.edu.cn/). Tandemly duplicated gene pairs were identified as described [https://www.ebi.ac.uk/Tools/msa/clustalo/). More than 90% sequence similarity among genes was considered as segmental duplication [Genomic DNA and coding DNA sequences corresponding to each identified tomato LOX gene were retrieved for escribed . Similarlication , while t260/280 ratio of 1.8–2.0 were then electrophoresed on agarose gels to ensure the presence of intact rRNA bands. Methods for cDNA synthesis and qRT-PCR were essentially as described previously [CT−∆∆ method [S. polyrhiza actin (Spipo17G0011400) and 18S rRNA (Spipo23G0000600) genes were used as standard housekeeping genes to normalize the expression of target genes [S. polyrhiza 7498 genome for specific hits. Moreover, each primer pair was tested for its specificity to yield a single amplicon on 1.2% agarose gel. Primers used in this study are listed in Total RNA was extracted from 100 mg of each sample using the plant RNeasy kit according to manufacturer’s instructions . RNA samples with an Aeviously ,72. An iT method . The S. et genes . qRT-PCRS. polyrhiza 7498 LOX gene family in response to MeJA was carried out using a slight modification of an previously published method [Transcript regulation of the d method . MeJA  suite was used for statistical analysis. ANOVA was performed for significant differences in LOX gene expression. For the MeJA experiment and salt treatment, significant differences were calculated against non-treated control samples at each time point, and categorized at * s before .Spirodela polyrhiza, was identified and characterized by comparative bioinformatics, domain-scan analysis, sequence/phylogenetic analysis, and transcript abundance quantification. Lemna-specific LOX genes were also identified. Jasmonate-mediated changes in expression levels of the Spirodela LOX genes were seen and described. Salt stress identified a different trend of LOX gene expression in the aquatic Spirodela plants as compared with land plants.Lipoxygenases (LOXs) catalyze synthesis of a group of compounds collectively called oxylipins. Little is known about this family in aquatic plants. In this study, the LOX gene family of the greater duckweed,