Pleurotus ostreatus and Pleurotus djamor) collected from Rajaji National Park in Haridwar, India. For this purpose, mushroom samples were collected from selected locations  from June 2021 to July 2022 and subsequently analyzed for selected heavy metals using atomic absorption spectroscopy (AAS). Results showed that both Pleurotus spp. had significantly varying (p < 0.05) concentrations of heavy metals. However, P. ostreatus showed relatively higher concentration levels of these metals compared to P. djamor. The mean concentrations (mg/kg dry weight) of the Cd, Cr, Cu, Fe, Mn, and Zn in P. ostreatus and P. djamor were 0.10 and 0.08, 0.87 and 0.64, 16.19 and 14.77, 28.49 and 27.15, 9.93 and 8.73, and 18.15 and 15.76, respectively. As indicated by the multivariate analysis tools i.e., principal component analysis (PCA) and hierarchical cluster analysis (HCA), the locations near the residential, industrial, and transportation activities had higher concentration levels of heavy metals. Moreover, the health risk studies using the target hazard quotient (THQ < 1) showed no significant health risk as the consumption of both Pleurotus spp., except for at one location, had high-traffic activities. The findings of this study provide vital information about the occurrence of potentially toxic heavy metals in wild edible Pleurotus spp. in Rajaji National Park in Haridwar, India representing a safeguard for mushroom consumers.This study aimed at assessing the concentration of six heavy metals  in two wild edible oyster mushrooms ( Mushrooms are protein-rich and low-fat sources of food, making them nutritionally equivalent to meat for the rising vegan community around the world . AccordiTricholoma spp. to accumulate Cd concentrations with a bioconcentration factor (BCF) higher than 1 [Agaricus campestris, Boletus edulis, and Boletus reticulatus were found to be high accumulators of Cd and Hg, respectively [Agaricus bisporus mushrooms sold in Uttarakhand State local markets were assessed [Wild mushroom foraging is a cultural tradition of European countries, mainly in Central and Eastern Europe. Locals tend to forage collectively in the autumn–spring season, in which a diversity of delicious edible mushrooms flourish . Yet, thr than 1 . Howeverectively . Most ofectively . Recentlassessed . AuthorsPleurotus spp.) are mainly grown on lignocellulosic-rich materials or dead wood. These species are a great source of human protein, natural amino acids, and essential poly- and monounsaturated fatty acids [P. ostreatus grown on agro-industrial residues such as olive pruning residues and spent coffee grounds had the potential to accumulate some desirable and undesirable metals. However, the reported concentrations were within the same limits set by the European Commission (EC), World Health Organization (WHO), and United States Food and Drug Administration (FDA) [P. ostreatus, grown on spent mushroom substrate, supplemented with nano-amino additives outlined high Pb levels, (>0.3 mg/kg) exceeding limits set by the guidelines, which stipulate the need to reconsider consuming the produce regularly [P. ostreatus mushrooms were acknowledged to enclose higher concentrations of heavy metals compared to cultivated ones. In this context, this species collected from mine-polluted soils delineated Zn, Cd, and Cr concentrations higher than the safe limits, with concentrations in stalks higher than those in caps [P. djamor is well appreciated for its high antioxidants and protein content. This species has been reported to accumulate the highest amounts of heavy metals among Pleurotus spp., and mainly has the highest Pb concentrations [P. cirinopileatus > P. djamor > P. eryngii > P. ostreatus.Oyster mushroom species  . In the  in caps . Althougtrations . These a2). It is well known for its high biodiversity and is home to several rare plant and animal species [Pleurotus spp. and assesses their potential risk for human consumption. The findings of this study suggest a heavy-metal accumulation potential of wild edible Pleurotus spp. that could potentially be harmful to consumers.Rajaji National Park is a famous natural reserve located on both sides of the Ganges River, Uttarakhand (820 km species . This pa species . In thisP. ostreatus and P. djamor were obtained from ten (10) locations around the Chilla Forest Range of Rajaji National Park in Haridwar city, Uttarakhand, India. 2, respectively. The area receives an average annual rainfall of 2136.7 mm with August as the wettest month. The average temperature and humidity of the area are recorded as 23 °C and 68%, respectively [Pleurotus spp. The sampling locations are easily accessible to the local communities and do not need any special permission from the forest authorities. Therefore, wild mushrooms are commonly collected and consumed by the local people. The mushroom samples were collected from June 2021 to July 2022 following standard field sampling methods [P. ostreatus, n = 49; P. djamor, n = 39) for each species varied due to their availability. Herein, the Pleurotus spp. were morphologically identified using the field guide of Kumari et al. [https://www.mushroom.world/ and http://www.mycokey.com/ (accessed on 1 September 2022).For the present study, samples of two oyster mushroom species i.e., 3 and HClO4. The mixture was placed on a rotary shaker and self-digested for 12 h. After the completion of self-digestion, a 3% HNO3 solution was added to make the final volume of the contents 50 mL. Again, the flask was placed on a hot plate and digested for 1 h at 150 °C until 10 mL of residual volume was left. Finally, more HNO3 solution was added to make the liquid volume 50 mL and usable for heavy metal analysis using AAS. The analytical conditions of the AAS instrument such as detection limits, wavelength (nm), cathode lamp current (mA), slit width (0.2–0.7 mm), and flame (air-acetylene mixture) were adjusted based on the heavy metals as given in the previous study [The collected mushroom samples were oven dried at 60 °C until a constant weight was achieved. The dried samples were again converted into fine powder form using a mechanical grinder . Analysis of six heavy metals  was performed using atomic absorption spectroscopy . For this, 1 g of powdered mushroom sample was placed in a 150 mL-capacity conical flask and dissolved in 10 mL of a di-acid mixture having a 3:1 ratio of concentrated HNOus study ,25,26. TPleurotus spp. was analyzed using the target hazard quotient (THQ) method [In the present study, the health risk of heavy metals contents in two ) method ,27. The Ef is exposure frequency of heavy metals, Ed is exposure duration (365 days), Ir is the mushroom ingestion rate, HMc is the heavy-metal concentration in the mushroom sample (mg/kg), Bw is the average body weight (70 kg for adult and 16 kg for child), Cp is the consumption period , Rd is the reference doses  [−3 is the conversion factor, respectively. Moreover, the data were analyzed using principal component analysis (PCA) and hierarchical clustering (CL) to derive the relationship between heavy-metal concentration in Pleurotus spp. and their sampling locations (correlation matrix). CL is used to create clusters that have a predetermined ordering from top to bottom. Based on their similarity, nodes are compared with each other where larger groups are built by joining groups of nodes. The data were tested using a one-way analysis of variance (ANOVA) and a Tukey’s post hoc test. The level of statistical significance was adjusted as the probability (p) < 0.05.Moreover, the THQ sum of six heavy metals was used to present the cumulative health risk index (HRI) value. Following Equation (2) were used to calculate HRI: × 10−1) , and 10−Microsoft Excel 2019  and OriginPro 2022b  software packages were used for the data analysis and visualization.Pleurotus spp. collected from different locations of Rajaji National Park are shown in p < 0.05) heavy-metal contents among each species between selected locations. Both Pleurotus spp. showed the following mean heavy-metals compositional order: Fe > Zn > Cu > Mn > Cr > Cd. The contents of Cd ranged between 0.03 and 0.16, and between 0.05 and 0.11 for P. ostreatus and P. djamor, respectively, in all studied locations. Although, the mean Cd contents found in P. ostreatus and P. djamor were 0.10 and 0.08 mg/kg, respectively, noting no risk to human health; numerous locations  outlined Cd contents in P. ostreatus, approaching the safe limit set by Indian Standards (0.10 mg/kg) [P. djamor showed a value exceeding the safe limit only in Bijnor Canal Road (0.11 mg/kg). Moreover, the skewness test of heavy-metal contents in Pleurotus spp. showed both positive (0.05 to 1.76) and negative (–0.02 to –1.88) values, depicting random symmetric distribution throughout the sampling locations. Also, the Kurtosis tests showed both positive (0.23 to 1.07) and negative (–0.16 to –0.85) values, indicating that the data tend to have fewer outliers.The results of heavy-metals concentration in 0 mg/kg) . ConversP. ostreatus was acknowledged to have a high affinity to bioaccumulate Cd, especially when grown widely [Trichloma spp.) denoted high Cd contents in mushroom caps and stipes, with the former higher than the latter (Cd in caps > C in stipes) [In a previous study, n widely ; this is stipes) . In Cd-c stipes) . Further stipes) . Similar stipes) .P. ostreatus and P. djamor samples collected from all locations were far below the safe limits; [Annually, the Rajaji National Park witnesses spring water overflowing on the surface; this type of water often transports debris enclosing contaminants and other pollutants according to an earlier report from Penn State University , which m limits; ,29 being limits; .Pleurotus spp. and their sampling locations in and around Rajaji National Park, Haridwar, India. Results revealed that by using the PCA tool, the randomly distributed data was orthogonally transformed onto two principal components namely PC1 and PC2 for both Pleurotus spp. The standardized score plot given in P. ostreatus had eigenvalues of 16.50 and 6.90 covering the variances of 62.06 and 25.96%, respectively P. ostreatus, the axial vector length of Fe was highly correlated for Rishikesh Canal Road, Bheemgoda Barrage Forest, and Sapt Rishi Ghat, sampling sites while Zn and Cu dominated the Bijnor Canal Road location. Herein, the Mansa Devi Forest location showed the lowest heavy-metal contents For P. ostreatus, which suggested the safest location for P. ostreatus cultivation. P. ostreatus collected from different locations of Rajaji National Park, Haridwar, India. Hierarchal cluster analysis showed that Mn–Fe and Cd–Zn had the highest similarities in terms of analyzed heavy-metals concentration in P. ostreatus mushroom. Also, similarities were identified between different sampling locations such as Sureshwari Devi Forest–Chilla Forest Colony and Devpura Forest Colony–Rishikesh Canal Road, respectively.The multivariate analysis provides better insights into the understanding of randomly distributed data sets . In thisP. djamor samples were observed as 15.30 and 4.80 depicting 67.86 and 21.29% variance, respectively. As given in P. djamor samples. However, the similarities between sampling locations were not effectively established due to random distribution except for Sapt Rishi Ghat–Rishikesh Canal Road and Bheemgoda Barrage Forest–Chandi Devi Forest.On the other hand, the extracted eigenvalues of PC1 and PC2 for heavy metal contents in Agaricus bisporus mushroom collected from different locations in Uttarakhand State, India were analyzed using geospatial and multivariate statistical tools i.e., principal component analysis and cluster analysis. They found that both PCA and HCA helped identify the districts which had the highest heavy-metal contamination across the state. Similarly, Sarikurkcu et al. [Russula spp. from Turkey. They found that the variability of essential and non-essential heavy metals was successfully depicted using PCA and HCA. Moreover, Barea-Sepúlveda et al. [Macrolepiota procera mushrooms collected from different locations in Spain and Morocco. Thus, these reports suggested that PCA and HCA can be useful for understanding the heavy metal variabilities in mushroom samples.Multivariate statistical analyses are one of the most effective methods for generalizing randomly distributed large data sets. In a recent study by Kumar et al. , the conu et al.  also anaa et al.  also utiP. ostreatus and P. djamor, as well as their resultant HRIs. It was revealed that child THQs were always higher than the adult and that THQs of P. ostreatus were higher compared to those of P. djamor. A similar observation was recently acknowledged by Kumar et al. [Agaricus bisporus mushrooms collected from Uttarakhand state markets. The study of HRIs on Tricholoma spp. revealed that they were higher in mushroom caps than in mushroom stipes [Pleurotus spp. mushrooms; therefore, further studies should be put into action to detect any possible potential risk associated with a specific part of the edible mushrooms. The average child HRIs of P. ostreatus and P. djamor was 2.33-fold higher than the average adult HRIs in the same species. Moreover, child and adult HRIs were higher by 1.1–1.4-fold in P. ostreatus than in P. djamor, as shown in P. ostreatus, collected from Bijnor Canal Road (1.151), which was mostly attributed to the high Cd content.As wild mushroom consumption has increased in numerous regions of the world, especially in developing countries, the need to investigate any possible health risks has become indispensable. Therefore, this can be predicted by studying the target hazard quotient (THQ) and health risk index (HRI) in adult and child groups for a better insight into any possible toxicity on human health . Here, Tr et al.  regardinm stipes . ConversP. ostreatus from Bijnor Canal Road should be avoided as they may pose a serious effect on the development of their central nervous system leading to learning disabilities besides possible cancerogenic impacts [P. ostreatus, child HRIs’ in studied locations followed this decreasing order: Bijnor Canal Road > Rishikesh Canal Roa –Sapt Rishi Gat > BHEL Forest Colony > Devpura Forest Colony > Chandi Devi Forest > Mansa Devi Forest > Sureshwari Devi Forest > Bheemgoda Barrage Forest > Chilla Forest Colony; whereas, adult HRIs’ decreasing order was as follows: Bijnor Canal Road > Rishikesh Canal Road–Sapt Rishi Gat > BHEL Forest Colony > Devpura Forest Colony > Chandi Devi Forest > Mansa Devi Forest > Sureshwari Devi Forest > Bheemgoda Barrage Forest–Chilla Forest Colony.This region is currently heavily affected by the increasing activities of neighboring mines besides the uprising water canal pollution, although a restoration process took place in 2014. Accordingly, children’s intake of  impacts . For P. P. djamor, the following child HRIs trend was noted: Bijnor Canal Road > BHEL Forest Colony > Sapt Rishi Gat > Rishikesh Canal Road > Devpura Forest Colony > Bheemgoda Barrage Forest > Chandi Devi Forest > Mansa Devi Forest > Chilla Forest Colony > Sureshwari Devi Forest. Whereas, adult HRIs had the following decreasing order: Bijnor Canal Road > BHEL Forest Colony > Sapt Rishi Gat > Rishikesh Canal Road > Devpura Forest Colony > Chandi Devi Forest–Bheemgoda Barrage Forest > Mansa Devi Forest > Chilla Forest Colony > Sureshwari Devi Forest. The obtained findings simulate that HRI values were not only species-related, but also soil-related (mainly Cd contamination). Other recent studies outlined the strong inter-relationship between soil mercury (Hg) content and HRI values in wild edible mushrooms [Pleurotus spp.For ushrooms ,43,44. TP. ostreatus and P. djamor) collected from the Rajaji National Park, Haridwar, India showed the occurrence of selected heavy metals . Comparatively, the contents of six heavy metals were found at maximum in P. ostreatus followed by P. djamor. However, the concentration varied significantly (p < 0.05) based on the sampling locations. Overall, the decreasing order of mean heavy-metal concentration in both Pleurotus spp. was Fe > Zn > Cu > Mn > Cr > Cd. The findings revealed that the samples collected from locations close to the residential, industrial areas, and transportation activities showed relatively higher heavy-metal contents as compared to those collected from the deep forest areas as indicated by the multivariate analyses. Similarly, the health risk index studies also indicated safe consumption of Pleurotus spp. collected from all locations except for those near heavy traffic and industrial areas. Considering the importance of wild edible mushrooms in human nutrition, proper measures should be taken in terms of heavy-metal contamination and associated health risks. Further studies on the biomonitoring of heavy metals in other edible mushroom species are highly recommended.This study concluded that two oyster mushrooms (


Festuca L. species of uncertain origin and unclear taxonomy. A taxonomic study of seven diagnostic morphological traits was conducted on a representation of 22 species. Phylogenomic analyses were performed on a representation of these supraspecific taxa and all other Loliinae lineages using separate data from the entire plastome, nuclear rDNA 45S and 5S genes, and repetitive DNA elements. F. subgen. Mallopetalon falls within the fine-leaved (FL) Loliinae clade, whereas the remaining taxa are nested within the broad-leaved (BL) Loliinae clade forming two separate Mexico–Central–South American  lineages. MCSAI includes representatives of F. sect. Glabricarpae and F. subgen. Asperifolia plus F. superba, and MCSAII of F. subgen. Erosiflorae and F. sect. Ruprechtia plus F. argentina. MCSAII likely had a BL Leucopoa paternal ancestor, MCSAI and MCSAII a BL Meso-South American maternal ancestor, and Mallopetalon FL, American I–II ancestors. Plastome vs. nuclear topological discordances corroborated the hybrid allopolyploid origins of these taxa, some of which probably originated from Northern Hemisphere ancestors. The observed data indicate rapid reticulate radiations in the Central–South American subcontinent. Our systematic study supports the reclassification of some studied taxa in different supraspecific Festuca ranks.Allopolyploidy is considered a driver of diversity in subtribe Loliinae. We investigate the evolution and systematics of the poorly studied Mesoamerican and South American polyploid broad-leaved  Elymus L., Calamagrostis Adans. [Festuca L., [Poa L., [Despite considerable debate about the evolutionary fate of allopolyploids, alternatively viewed as drivers of biodiversity  or evolus Adans. ,17) and tuca L., ; Poa L.,[Poa L., ). Molecu[Poa L., , while t[Poa L., .Festuca and several closely-related genera nested within it [Festuca are indicated in italics and the phylogenetic lineages of Loliinae in plain text. Phylogenetic analyses have consistently inferred two main clades within the subtribe, the broad-leaved (BL) and fine-leaved (FL) Loliinae, characterized by distinct genomic and phenotypic features [Festuca contains approximately 600 species distributed worldwide, inhabiting cool seasonal regions of both hemispheres [Festuca’s main center of diversity is the Holarctic region, which harbors nearly 500 species, including all known diploid species of the genus and different polyploids, ranging from tetraploids to dodecaploids [Festuca occur in South America [Festuca species have been ranked into eleven subgenera according to the worldwide classification system of Alexeev [Festuca, which encompasses most of the fine-leaved taxa of both hemispheres, makes the bulk of the FL clade. It also includes the small subgenus Helleria E.B. Alexeev, also treated as a separate genus Hellerochloa Rauschert, and several other genera nested within [Festuca subgenera, except the FL Mallopetalon (Döll) E.B. Alexeev, contain species of the BL clade, some of which have been also treated as separate genera. Two of them are native to the Old World (Schedonorus (P. Beauv.) Peterm., Xanthochloa (Krivot.) Tzvelev), five to the New World  E.B. Alexeev, Subuliflorae E.B. Alexeev, Erosiflorae E.B. Alexeev, Mallopetalon) and two are native to both areas (Leucopoa (Griseb.) Hack., Drymanthele V.I. Krecz. & Bobrov). The BL clade also includes three additional separate genera nested within [Festuca subgenera have been classified in different sections and subsections based on morphological traits , Erosiflorae, Mallopetalon, Subulifolia (sect. Glabricarpae E.B. Alexeev), and F. argentina (Speg.) Parodi), endemic to Mexico, Central America or South America, some of which constitute the basal-most BL lineages but have uncertain taxonomic adscriptions and evolutionary circumscriptions [F. argentina, include tall fescues that show extravaginal (or mixed) innovations, flat leaves, and open and lax panicles. In a series of successive taxonomic studies, Alexeev described Festuca sect. Ruprechtia (type specimen F. amplissima Rupr.) [F. subgen. Asperifolia (type specimen F. lugens (E. Fourn.) Hitchc. ex Hern.-Xol) [F. sect. Glabricarpae  [F. subgen. Mallopetalon (type specimen F. fimbriata Nees) [F. subgen. Erosiflorae (type specimen F. quadridentata Kunth) [Festuca argentina, initially assigned to F. subgenus Festuca [F. subgen. Mallopetalon [Festuca ranks described by Alexeev were expanded with other close species described from Mesoamerica and South America by the same or later authors  and transferring taxa from F. sect. Glabricarpae  to it but without strong morphological or phylogenetic arguments.Although a large amount of biological and genomic resources has been generated for some economically important forage and grassland Schreb.; ), other riptions ,25,26,27a Rupr.) , F. subgrn.-Xol) , F. sectSwallen) , F. subgta Nees)  and F. sa Kunth)  based on Festuca , was alsopetalon ; howeveropetalon  and phylopetalon ,25 from  authors . StančíkFestuca ranks described by Alexeev plus F. argentina are of high interest as these polyploid taxa may constitute some of the ancestral lineages of the broad-leaved Loliinae [Despite the importance of previous taxonomic work, the broad-leaved species belonging to these groups have been little studied, and the morphological characters used to delimit their taxonomic ranks remain poorly understood. The high uncertainty about the taxonomic circumscriptions and the evolutionary placements of the five Mesoamerican–South American taxonomic Loliinae ,27. TherFestuca subgeneric and sectional ranks  plus an additional reproductive trait (monoecy vs. dioecy)   on the sF. subgen Erosiflorae sensu Alexeev [F. quadridentata, endemic from the Ecuadorean paramos, and in two other species distributed in the northern Andes, F. dichoclada Pilg. and F. horridula Pilg., incorporated into this subgenus by Alexeev [F. subgen. Erosiflorae to six new South American species of which two fulfilled all the main diagnostic characteristics proposed by Alexeev , one differed from them due to its shortly awned lemma (F. urubambana Stančík), another due to its partially nutant panicles and awned lemma (F. venezuelana), the fourth for its densely hairy ovary tip, shorter hilum and hyaline ligule with dentate apex (F. superba), and the fifth for its short ligule with ciliate apex, nutant panicles and awned lemma (F. steinbachii)  Kunth and F. steinbachii [F. sect. Glabricarpae the North Andean species F. dentiflora E.B. Alexeev ex Stančík & P.M. Peterson and F. woodii Stančík, which matched the sectional diagnostic features except for the sparsely hairy ovary tip of F. woodii  . Species Alexeev  differenD. Koch. . The spericarpae  are sepamma apex . Alexeevinbachii ,43. Staninbachii  added to. woodii . The spe Alexeev  departed.D. Koch . F. subg Alexeev  based sovary tip .Festuca lineages mentioned above. Festuca superba, a narrow endemic species from northwestern Argentina, was classified within the F. subgen. Erosiflorae by Stančík and Renvoize [Festuca argentina, endemic to Patagonia and the southern Andes, is the most phenotypically distinct species of all taxa analyzed. It has been attributed to fine-leaved F. subgen. Festuca by some authors [F. argentina shows unique traits, such as dioecy, a narrowly contracted lanceolate panicle and a sparsely hispid ovary tip  and seven species characterized only for a few loci  K. Richt., F. kingii (S. Watson) Cassidy, F. valdesii), along with genome skimming data on five species of the supraspecific Festuca taxa under study  and 21 species of other Loliinae lineages and two outgroups obtained in previous works [Phylogenomic analyses of a selection of 11 tic taxa , plus 23tic taxa . New genus works ,27 were F. asperella) to 32,808 (F. horridula) million Illumina pair-end (PE) reads (F. kingii) to 6532 bp (F. chiriquensis), with a total length of 6589 bp in the multiple sequence alignment (MSA) . This region showed a conserved structure along its aligned transcriptional unit, composed of the 5′-external transcribed spacer (ETS) (~715 bp), the 18S gene (1818 bp), the internal transcribed spacers and the 5.8S gene (ITS1-5.8S-ITS2) (577 bp), and the 25S gene (3392 bp), which had similar average lengths in the samples studied. The highly variable IGS region, studied for the first time in Loliinae, ranged from 977 (F. pratensis) to 1992 bp (F. gracillima Hook. f.), producing an MSA 2496 bp in length . The newly assembled sequences of the nuclear rDNA 5S region ranged from 298 bp  to 319 bp (F. gautieri). The 5S region consisted of a conserved 5S gene  and a 563 bp intergenic variable spacer (IGS) in the MSA . The newly assembled plastomes ranged from 131,438 bp (F. superba) to 133,638 bp (F. chiriquensis), matching the plastome length values obtained in previous studies [F. breviglumis and F. valdesii, which had lower read coverage (13×–26×). The MSA of the plastomes was 134,265 bp in length . Newly obtained sequences from each data set were deposited in GenBank under accession codes OP120917-OP120926 (35S), OP158132-OP158167 (IGS), OP142676-OP142686 (5S), SAMN30029287-SAMN30029296 (plastomes) (SRA data under BioProject PRJNA863311) (Genome skimming data from newly sequenced samples ranged from 5683 (E) reads . The seq studies ,27 for tA863311) .F. fimbriata  was nested within a strongly supported FL American I–American II clade, whereas the remaining species under study fell into two separate BL groups. Representative species of F. sect. Glabricarpae  and F. subgen. Asperifolia (F. asperella), together with F. venezuelana and F. superba, formed a robust Mexico–Central–South American I (MCSA I) clade, while the representative species of F. sect. Erosiflorae , F. sect. Ruprechtia  and F. argentina formed a Mexico–Central–South American II (MCSA II) group integrated into a robust clade that also included representatives of Leucopoa  and Subulatae-Hawaiian   was also nested within a robust FL American I–American II clade, whereas the other taxa fell within the BL clade. The robust MCSA I clade (Glabricarpae–Asperifolia–F. superba–F. venezuelana) was resolved as a sister to the also robust tropical–South African clade, although this relationship was weakly supported, and the strongly supported MCSA II clade (Erosiflorae–Ruprechtia–F. argentina) was resolved as a sister to a weakly supported Leucopoa clade, although this relationship was well supported  was also nested within the FL Loliinae clade but close to representative species of American Pampas, Subulatae-Hawaiian and Exaratae lineages and not to those of American II, American I and American–Neozeylandic lineages, which formed a nested group within the BL Loliinae clade  and MCSAII (pro parte) groups formed a clade, sister to another clade that included two species from the MSCAII group and representatives of the remaining BL lineages, with all these relationships showing full support. Within the MCSA superclade, Glabricarpae, Asperella and F. superba (MCSAI group) species were resolved as basal paraphyletic lineages, while F. venezuelana formed a fully supported clade with most elements of the more recently evolved and well supported MCSAII pro parte clade. The two species of the MCSAII group that departed from the MCSA superclade, F. horridula (Erosiflorae) and F. valdesii (Ruprechtia), formed a fully supported subclade together with American F. kingii (Leucopoa); this subclade, in turn, joined other Eurasian species of Leucopoa and of Subbulbosae in a fully supported lineage (Festuca ranks under study on its branches (The 35S maximum likelihood (ML) phylogenetic tree recovered the expected topology for the Loliinae as previously presented by Moreno-Aguilar et al. , consistCatalán) a. The  b. F. fimupported b. The 5Supported c. The 5Sae clade c. The plae clade d. In thi lineage d. To acc lineage  were thebranches .F. quadridentata) to 67.5% (F. chiriquensis)  showed the lowest percentage of repeatomes (38.8%) among the studied species, differing from the relatively high values shown by the American II and American I species , F. argentina and F. superba of Retand elements. F. fimbriata had a low coverage of Retand elements, as in some FL American II species , although unlike the American II and American I species, it showed a much lower coverage of Angela elements (F. kingii (Leucopoa) was resolved as the closest relative of this MCSA supergroup  fell within the expanded FL group in this repeatome-based network, nesting in an intermediate position between the American II and American I lineages  in the individual analysis of the newly sequenced samples a were coquensis) . Interes species  but beindinacea; ). LTR-Coelements a. A totaelements b. The MCpergroup b. F. fimlineages b. The red ex DC. ).Festuca lineages have been instrumental in unravelling the origins and systematics of the seven Loliinae groups studied  originated from ancestors of FL Loliinae, while species in the other six groups derived from ancestors of BL Loliinae  and American I  “fine-leaved” lineages [F. fimbriata, such as the possession of fimbriated lodicles and lemma apex , as shown in the nuclear, plastome and repeatome phylogenies  and F. sect. Glabricarpae  plus F. venezuelana and F. superba (F. tancitaroensis), while they differ in their erect (Asperifolia) vs. nutant (Glabricarpae) panicles  in the 35S, IGS, 5S (MCSAI) and repeatome-based nuclear phylogenies b, while  superba . Asperifpanicles . F. veneRenvoize  within Fa groups , althouga groups , with Asome tree d.F. subgen. Erosiflorae sensu Alexeev  and F. sect. Ruprechtia  plus F. argentina  in its non-rhizomatous caespitose habit, longer membranous ligule with a truncate and short ciliate apex and hispid ovary tip  and Subulatae-Hawaiian (F. molokaiensis) lineages (F. horridula) and one species of Ruprechtia  split from the MCSA superclade and fell within a separate BL lineage, nesting with the North American Leucopoa F. kingii in a strongly supported clade , probably derived from a Leucopoa ancestor, which likely acted as the paternal parent for most of these species  and F. valdesii (Ruprechtia) likely had both paternal and maternal Leucopoa-type parents (F. superba) are less clear. The nuclear topologies do not retrieve strongly supported relationships of these slightly older MCSA lineages with any of the remaining BL lineages  groups. This hypothesis agrees with the proposed DEC biogeographic models for colonizing ancestral BL Festuca lineages from the Northern Hemisphere to Mesoamerica and South America [Festuca ancestors [Festuca in these regions and throughout the southern hemisphere [Festuca [F. argentina)  . Similarilene L. ,54). Thilyploids . For theF. argentina, and MCSAI F. superba , which share muticous or mucronulate lemma apices and hairy ovary tips  Krivot., F. sibirica Hack. ex Boiss.) and their American descendants  .The morphological differences observed for the main diagnostic characters  of MCSAI superba b with reeviously ,46,48, m Festuca  discusseary tips , and cilrgentina . F. argelogenies b, supporlogenies . This telogenies , is shar. kingii b and var species ,56. As i America , the rarentina)  or within the BL clade , while the studied species of F. sects. Phaeochloa  and Muticae (F. modesta Nees) always nested within the BL clade [F. superba is presumably an allooctoploid, based on its perfectly paired bivalents observed at meiosis [F. subgen. Drymanthele but not to currently described sections of this rank, and because of its strong phylogenetic nesting within the Glabricarpae–Asperifolia clades of the MCSAI lineage, we propose to tentatively classify it within F. subgen. Drymanthele sensu lato without a sectional assignment until other close broad-leaved Meso-South American taxa are also phylogenomically studied.Renvoize . Howeverructions b. The movary tip , togethemanthele ,55. Howe Alexeev ,39,44,59BL clade . F. supe meiosis . Its hig meiosis . Based oFestuca by Linné [Festuca and fourteen close genera constitute the monophyletic subtribe Loliinae. Phylogenetic analysis has shown that fine-leaved F. subgen. Festuca species and some broad-leaved fescues  plus ten annual genera  Link, Narduroides Rouy, Podophorus Phil., Psilurus Trin., Vulpia C.C. Gmel., Wangenheimia Moench) make up the FL clade, while taxa of eight broad-leaved Festuca subgenera  plus three annual or perennial genera  form the BL clade ([Festuca) taxa is based on several diagnostic vegetative and reproductive morphoanatomical traits . We propose to apply the Festuca sensu lato classification scenario, which is based on a systematic evolutionary criterion that is nomenclaturally conservative and maintains a paraphyletic Festuca (with subgenera and sections) and other traditionally recognized genera. Our current study has demonstrated the applicability of our systematic approach in the group of studied broad-leaved MCSA and Mallopetalon species, for which their phylogenetic resolution does not always coincide with their taxonomic classification as a consequence of the high reticulation of the Loliinae but has helped to disentangle their hybrid allopolyploid evolutionary history.The systematics of Loliinae has undergone multiple classifications since the description of its main genus by Linné , resultiL clade  Parodi, Physis (Buenos Aires) 11: 498. 1935. Basionym: Poa argentina Speg., Revista de la Facultad de Agronomía y Veterinaria 3 (30–31): 584–585. 1897. Ind. loc.: “Argentina: Hab. ad margim orientalem Lago Argentino, anno 1884”. Type specimen: Lago Argentino, 1884, Sr. Tonini del Furia s.n. .Typus: Festuca argentina (Speg.) Parodi. It differs from the rest of the subgenera by the combination of its dioecy, caespitose habit, plicate leaves, tri-nerved lower glume, unawned lemma apex and sparsely hairy ovary tip. Etymology: Coironhuecu is based in the common Patagonian native name of F. argentina (Coirón huecú) due to its toxicity caused by its fungal endophytes.The subgenus is integrated only by Festuca species in the subgeneric and sectional taxa under study [Festuca species have a chromosome base number of x = 7; ploidy levels of the Meso and South American species studied  to the analysis. DNA was isolated from herbarium specimens or silica gel dried samples using a modified CTAB protocol [Total DNA sampling was performed on representative species of all Mesoamerican and South American supraspecific er study . We alsoprotocol  with ∼20protocol .F. pratensis plastome (JX871941) as a reference and standardized parameters . The plastomes of four samples with low number of PE reads  were assembled using a read-mapping strategy to, respectively, closely related Festuca plastomes using Geneious Prime 2022 (Assembled plastomes for most of the newly sequenced samples were obtained with Novoplasty v. 2.7.1  using thime 2022 . The plahttps://repeatexplorer-elixir.cerit-sc.cz accessed on 30 May 2022). Clustering was performed using default TAREAN tool settings  and an input of 500,000 PE reads per sample. 45S rDNA sequences were found in the TAREAN tandem reports of each sample. The 45S region was divided into its 35S and IGS regions using the Brachypodium distachyon (L.) P. Beauv. 45S sequence as reference  and the 5S sequences of two species  that could not be recovered by TAREAN were assembled employing a read-mapping strategy using, respectively, F. triflora J.F. Gmel. and F. pratensis as reference sequences in Geneious Prime 2022. Additional 35S and 5S sequences from other Loliinae lineages were retrieved from previous studies [The nuclear rDNA 45S region  region) of 27 of the 36 new Loliinae samples studied was extracted with the TAREAN tool of the Repeat Explorer2 (RE2) software ,66 throueference . The nuc studies ,27.automated1 parameter. Maximum likelihood (ML) phylogenetic trees were reconstructed for each separated data set with Iqtree imposing the best-fit nucleotide substitution model, according to the Bayesian Information Criterion (BIC), and estimating 1000 ultrafast bootstrap replicates (BS) for the branch support of the best tree [Entire plastomes and nuclear 35S, IGS and 5S sequences were aligned separately with MAFFT v. 7.031b . TrimAl est tree ,70,71. Test tree , imposinFestuca species were obtained from similarity graph-based clustering analysis of filtered PE reads using the Repeat Explorer pipeline of RE2 [The composition and proportion of repetitive elements of the studied e of RE2 . Previoue of RE2 , and refe of RE2 . Brieflye of RE2  using pae of RE2 .


Ehrlichia ruminantium, was first reported in dromedary camels in Kenya in 2016; investigations carried out at the time to determine the cause were inconclusive. In the present study, we screened sera from Kenyan camels collected before (2015) and after (2020) the 2016 disease outbreak for antibodies to Ehrlichia spp. using an E. ruminantium polyclonal competitive ELISA (PC-ELISA). Median antibody levels were significantly higher (p < 0.0001) amongst camels originating from areas where the heartwater-like disease was reported than from disease-free areas, for animals sampled in both 2015 and 2020. Overall median seropositivity was higher in camels sampled in 2015 than in 2020, which could have been due to higher mean age in the former group. Camels that were PCR-positive for Candidatus Ehrlichia regneryi had significantly lower (p = 0.03) median antibody levels than PCR-negative camels. Our results indicate that Kenyan camels are frequently exposed to E. ruminantium from an early age, E. ruminantium was unlikely to have been the sole cause of the outbreak of heartwater-like disease; and Ca. E. regneryi does not appreciably cross-react with E. ruminantium in the PC-ELISA. A disease with clinical and post-mortem presentation similar to those seen in heartwater, a tick-borne disease of domestic and wild ruminants caused by the intracellular bacterium  Camelus dromedarius) are important food-producing animals in northern Kenya. Their population has been steadily increasing year-on-year ). Continuous data sets were assessed for normality using the Shapiro–Wilk test and inspection of Q–Q plots. Effect of independent variables  on the dependent variable (PI) was analysed using Kruskal–Wallis test, Mann–Whitney U test, and Spearman’s correlation coefficient , ranged from a minimum of 2 months (0.17 years) to a maximum of 30 years. The camel samples were grouped into four age ranges  and the distribution of PI values obtained in the PC-ELISA is shown in p < 0.0001), and moderately significant (p = 0.0011) between the second and third groups. Thereafter, median PI showed a small but significant decline in camels aged between 16 and 30 years (p < 0.0001).Seropositivity in the PC-ELISA has previously been reported to increase with age ,24. The 30 years . ResultsE. ruminantium. Amongst the 636 camels for which sera were tested using the E. ruminantium PC-ELISA and for which sex was recorded, the median PI of the 161 male camels was 79%  and the median PI of the 475 female camels was 81%  (p = 0.16).There are no reports of differences between seropositivity of male and female animals for either the PC-ELISA or other assays for antibodies against  2–115%) . In a twp < 0.0001) (p < 0.0001) at 83%  than that of the 184 camels originating from areas where such disease was not reported  (n = 35) sampled in 2020 at sites reporting heartwater-like disease in camels also had a significantly higher median PI of 97  than sheep (n = 55) at sites not reporting disease  (p < 0.0001)  a. Second 0–107%) b. Sheep  0.0001) c. n = 237), the median PI of the 115 camels sampled in 2015  was significantly higher (p < 0.0001) than that of the 122 camels sampled in 2020  (n = 422), the median PI of the 258 camels sampled in 2015  was also significantly higher (p = 0.0002) than that of the 164 camels sampled in 2020   a. Amongse 0–99%) b. It shoe 0–99%) , which cEhrlichia spp. DNA. No E. ruminantium DNA was detected in any of the camels, but Ca. E. regneryi was detected in the blood of 40 out of 286 animals for which sera were available. To investigate whether E. ruminantium PC-ELISA positivity was associated with presence of Ca. E. regneryi in blood, indicating possible cross-reactivity between the two species, we compared PI of camels that were PCR-positive or PCR-negative for Ca. E. regneryi (n = 246) the PI ranged from 0–99%, median 78% (IQR 28). In a two-tailed Mann–Whitney test, there was a significant difference between the two groups, with higher mean PI in camels testing negative for Ca. E. regneryi (p = 0.03)  .Ehrlichia, using an ELISA developed for detection of exposure to E. ruminantium. Previously, reports of presence of Ehrlichia spp. in camels were based on diagnosis of suspected heartwater by microscopy and clinical signs [Ehrlichia in spleen tissue [groEL genes, this novel Ehrlichia was closely related to Ehrlichia minasensis , a pathogen of cattle harboured by Rhipicephalus microplus ticks [E. canis. The novel camel Ehrlichia, named Ca. E. regneryi, was placed in a separate branch from E. ruminantium within the Ehrlichia phylogeny [E. canis and distinct from Ca. E. regneryi [E. minasensis was not included in the phylogenetic analysis. In the first study carried out on Kenyan camels, 16S rRNA sequences with high similarity to Ca. E. regneryi and E. canis were detected in two sick animals during an investigation of heartwater-like disease in camels at two sites in northern Kenya [Ca. E. regneryi in 14.5% of healthy camels but failed to detect E. ruminantium or any other Ehrlichia spp. in camel blood. Our study represents the first attempt to screen sera from camels for antibodies to any species of al signs ,15 or mon tissue . Based ous ticks , and thehylogeny . A subseregneryi ; E. minarn Kenya . In our E. ruminantium. However, as far as we know, the specific anti-species conjugate required for the MAP1-B ELISA has not been developed for camels. The E. ruminantium PC-ELISA employed in the present study has the advantage that it can be applied to sera from any mammal, without the need for host species-specific reagents [E. ruminantium [E. ruminantium exposure in small ruminants in northern Cameroon [Ehrlichia spp. bacteria or Amblyomma spp. ticks have never been reported in or on domestic animals [Ehrlichia, which compared well with the cut-offs of up to 50 PI for E. ruminantium-negative, 51–70 PI for Ehrlichia-positive, 71–85 PI for sera positive for E. ruminantium or Ehrlichia ovis (sheep only) and 85 PI for E. ruminantium-positive sera [The indirect MAP1-B ELISA ,27 has breagents . A compainantium . The PC-inantium ,24 and aCameroon . We test animals ,36,37, tive sera ,18,20. Ehrlichia sp. Even with a more conservative cut-off point of 50 PI [E. ruminantium-specific cut-off of 85 PI [E. ruminantium, or an antigenically very closely-related bacterium. When applied to serum samples collected from camels during the present study and archived samples collected from camels in 2015, prior to the first report of heartwater-like disease in camels, the PC-ELISA PI values ranged from zero to >100%, but the vast majority  were >35, suggesting that most of the camels had been exposed to at least one of 50 PI , 86.9%% of 85 PI , 190 samE. ruminantium serosurveys did not distinguish between male and female animals. Using the MAP1-B ELISA, a survey of ranch cattle in Tanzania reported higher seroprevalence amongst females than males, although the difference was not statistically significant [E. ruminantium infection related to pregnancy [Bos taurus cattle in South Africa were particularly prone to develop the peracute form of heartwater [No difference was seen between PC-ELISA seropositivity levels of male and female camels; although three times as many females as males were tested, the overall sample size was sufficiently large to avoid bias. Most previous nificant . The onlregnancy ; heavilyartwater . E. ruminantium seropositivity and age was reported for goats (1–5 years old) or cattle (1–7+ years old) in Zimbabwe tested with the MAP1-B ELISA [Coxiella burnetii in camels up to 15 years of age, followed by a decrease in older animals, was reported from Tunisia [Anaplasma spp. in Egypt [C. burnetii in Algeria [The median seropositivity level of camels increased with age up to 16 years, with a subsequent decline in older animals. A similar pattern of seropositivity rates increasing with age was reported from cattle and small ruminants sampled in Ghana ,24, alth-B ELISA . As seen Tunisia . Howeverin Egypt , or C. b Algeria , althougE. ruminantium. Waning of antibodies to E. ruminantium following field exposure in cattle reported for the MAP1-B ELISA [E. ruminantium could only be investigated by experimental infection of known naïve camels with the pathogen and following antibody levels post recovery. Interestingly, the overall levels of seropositivity were higher in camels sampled prior to the first report of heartwater-like disease in camels than in camels sampled in 2020, regardless of whether or not they originated from an area where the disease was reported. It is unclear whether this was due to the higher average age of the 2015 cohort, or to some other factor. PI values were on average significantly higher for camels collected in, or originating from, areas of northern Kenya where heartwater-like disease had been reported in camels, than in areas where such disease had not been reported. This pattern was seen both in sera collected during the present study and in archived sera collected from camels in 2015, prior to the first report of the disease. However, from serology alone it was not possible to ascribe seropositivity specifically to exposure to -B ELISA  and the -B ELISA  could alEhrlichia spp. DNA in blood [E. ruminantium DNA, out of the 286 camels tested by PC-ELISA, 40 were positive for Ca. E. regneryi [E. ruminantium and Ca. E. regneryi. We found a negative correlation between Ca. E regneryi DNA in blood and PC-ELISA seropositivity in camels; low antibody levels (<55 PI) were seen in around a quarter of the PCR-positive animals, while only 10% of these animals had PI > 85. This suggested that the high antibody levels detected in sera from the majority of PCR-negative animals resulted from exposure to another Ehrlichia species. However, from the data presented here and in our previous study [Ehrlichia species was E. ruminantium. The camels sampled in Marsabit County in the present study were also tested for the presence of in blood  collecteregneryi . This prus study , we cannn = 40, representing 4/5 sites), with most exceeding 95 PI. In the latter areas, less than half of the sampled sheep (20/55) exhibited PI levels > 85. However, we found Amblyomma spp. ticks harbouring E. ruminantium in both areas of Marsabit County [Amblyomma gemma and Amblyomma lepidum ticks collected during our study, 436 were collected at the sites where heartwater-like disease had been reported in camels, while only 23 were collected from animals at sites where heartwater-like disease had not been reported in camels. Ticks in both sets contained E. ruminantium DNA at approximately similar rates (8.7% and 7.1%) [E. ruminantium to domestic ruminants [Amblyomma variegatum, the species most widely-associated with heartwater in West, Central, and East Africa [Amblyomma spp. in transmission of E. ruminantium to domestic ruminants and, potentially, camels in Kenya, remains unclear, indicating a need for further sampling and screening of both ticks and livestock.Although co-grazing sheep were only available for sampling at some of the sites, there was a much clearer distinction between seropositivity rates and PI levels between areas where heartwater-like disease had and had not been reported in camels. All sampled sheep in the former areas exhibited PI levels > 85  . Both spuminants . Amblyomt Africa . This tit Africa ,41,42 ant Africa . The rolE. ruminantium PC-ELISA revealed high rates and levels of seropositivity in Kenyan camels, particularly amongst animals sampled in areas where heartwater-like disease had been reported in camels. Co-grazing sheep in these areas also showed extremely high rates and levels of seropositivity. However, these results alone do not provide sufficient evidence to indicate whether or not camels become infected with E. ruminantium. Further experimental and field studies are needed to determine whether camels are susceptible to infection with E. ruminantium and, if so, whether clinical disease ensues and how it is manifest. High seropositivity could indicate that, under normal circumstances, camels are exposed to E. ruminantium and become infected but do not suffer disease. Heartwater-like disease in camels could occur when the animals are in poor condition or subject to extrinsic factors or systemic disturbances, as discussed previously [Ca. E. regneryi results either in heartwater-like disease or in high levels of serum antibodies detectable by the E. ruminantium PC-ELISA.The eviously ), althoueviously . Further


Rickettsia frequently infects ticks and causes tick-transmitted rickettsioses in areas of endemicity where ixodid ticks support host transmission during blood feeding. Ticks also serve as a reservoir for SFG Rickettsia. Among the members of SFG Rickettsia, R. rickettsii causes Rocky Mountain spotted fever (RMSF), the most lethal TBD in the United States. Cases of RMSF have been reported for over a century in association with several species of ticks in the United States. However, the isolation of R. rickettsii from ticks has decreased, and recent serological and epidemiological studies suggest that novel species of SFG Rickettsia are responsible for the increased number of cases of RMSF-like rickettsioses in the United States. Recent analyses of rickettsial genomes and advances in genetic and molecular studies of Rickettsia provided insights into the biology of Rickettsia with the identification of conserved and unique putative virulence genes involved in the rickettsial life cycle. Thus, understanding Rickettsia-host-tick interactions mediating successful disease transmission and pathogenesis for SFG rickettsiae remains an active area of research. This review summarizes recent advances in understanding how SFG Rickettsia species coopt and manipulate ticks and mammalian hosts to cause rickettsioses, with a particular emphasis on newly described or emerging SFG Rickettsia species.Ticks are hematophagous ectoparasites capable of transmitting multiple human pathogens. Environmental changes have supported the expansion of ticks into new geographical areas that have become the epicenters of tick-borne diseases (TBDs). The spotted fever group (SFG) of  Rickettsiales, Rickettsiaceae) are small (0.3- to 0.5- by 0.8- to 2.0-μm) Gram-negative bacteria with an obligate intracellular life cycle circulating between mammalian hosts and hematophagous arthropod vectors  in nature. Early studies using electron microscopy identified intracellular Rickettsia with a trilaminar cell membrane surrounded by a slime layer  with predictions of ~700 core genes, an ~30% G+C content, and a coding capacity of 69 to 84% . , 4. Rick disease . ComparaR. felis (TRG) strains have been identified, sequenced, and characterized for their diverse genetic makeup, pathogenicity, and vector adaptations (R. felis in booklouse (Liposcelis bostrychophila) is involved in the development of oocytes, maintained strictly via transovarial transmission, and is considered nonpathogenic in mammalian hosts , which has the smallest genome, causes the most severe and lethal disease (epidemic typhus), which has claimed countless lives over the last centuries  and limited access to molecular diagnostic tools in reference laboratories prohibit the prompt diagnosis and treatment of rickettsial infections. As a result, the actual incidence of tick-borne rickettsioses is predicted to be much higher, and the case fatality rate of SFG rickettsioses remains high in many parts of the world  , 36. Tican hosts . In Nort species –39. Thesttsioses . Delayedttsioses –42. Incrhe world –45. OverRickettsia, for instance, the duration of tick attachment, bacterial loads in tick saliva, and the transmission efficiency of Rickettsia. Under laboratory conditions, R. rickettsii transmission occurred as soon as 8 h after D. variabilis bites on guinea pigs, and the severity of clinical disease was dependent on the duration of tick attachment  in A. sculptum saliva dampened the proinflammatory immune responses of DCs infected with R. rickettsii  exposed to A. maculatum adult ticks and subsequently infected with an intradermal injection of 107R. parkeri cells suffered from persistent R. parkeri infection at the inoculation site (eschar) and systemic dissemination of R. parkeri, suggesting that tick feeding introduced immunomodulatory factors and enhanced the pathogenesis of R. parkeri rickettsiosis -mediated activation of DCs and natural killer (NK) cells important for innate rickettsial immunity and predisposes the animals to rickettsial infections (–R. sanguineus and A. maculatum) produce different classes and abundances of immunomodulatory molecules that pose various capacities to facilitate SFG Rickettsia survival and reduce the proinflammatory immune responses of DCs, NK cells, or other cellular components at the site of infection. Furthermore, additional studies are required to identify specific rickettsial factors that synergize with immunomodulatory factors in tick saliva, contributing to the enhanced transmission and pathogenesis of tick-borne rickettsiosis. For in-depth discussions, interested readers are directed to a recent review article highlighting the multifaceted and complex relationships between Rickettsia and arthropod vectors .–50.46–50–tachment . This sttachment , 50. Theley rats . Tick saley rats , 54. Besckettsia –55. Skinkettsia ––59. A reckettsii . The abuckettsia . Indian ttsiosis . Other ihe lungs . Howeverruitment . The R.  feeding . A pointfections –66. It r vectors .Rickettsia actively replicates at tick bite sites and produces epidermal and dermal necrotic lesions that are characterized as inoculation eschars within a few days of infection. Histopathological analyses often identify vasculitis and necrotic features associated with vascular thrombosis. Numerous clinical reports have documented the presence of multiple eschars on patients infected by several SFG rickettsial agents, such as R. parkeri, R. philipii, (previously known as Rickettsia sp. strain 364D), and R. africae  infected with R. rickettsii increased cell-associated interleukin-1α (IL-1α) production for the potential activation of IL-1 receptor 1 (IL-1R1) signaling in an intracrine and paracrine manner to coordinate the local inflammatory responses of endothelial cells and recruit professional phagocytes , and vascular cell adhesion molecule 1 (VCAM-1)  from endothelial cells, implying their roles in activating and recruiting neutrophils and monocytes to the sites of infection (77–R. conorii generated a marked release of IL-8 and MCP-1 (CCL2) in a dose- and TLR4-dependent manner, heat-inactivated R. africae caused rather modest responses in HUVECs (R. africae) subjects, Mediterranean spotted fever (MSF) patients displayed a marked elevation of serum IL-8, potentially contributing to the distinct clinical features of MSF  and IFN-γ induced the expression of two chemokines, CXCL9 and CXCL10, known to target CXCR3 expressed on T and NK cells  (R. conorii (R. conorii) in Italy and Spain displayed pronounced increases in serum levels of CXCL10  –76. Siminfection , 77–79. n HUVECs . In conts of MSF . Previous of MSF . It is ackettsia . R. conoections) . This ob conorii . Human pf CXCL10 . This waf CXCL10 .In vitro, R. rickettsii infection of HMEC-1 cells exploited microRNA-424 to actively modulate the expression of CX3CL1. While the exact mechanisms remain unknown, in vivo C3H/HeN mouse infection studies performed with R. conorii confirmed that the peak expression of CX3CL1 coincides with the recruitment of macrophages during the acute phase of systemic endothelial infection  patients displayed a significantly increased level of soluble CD40 ligand (sCD40L) in serum  as a transmembrane protein for the interaction with its receptor, CX3CR1, which is abundantly expressed on human innate immune cells capable of controlling early rickettsial infections, including NK cells, T cells, and monocytes/macrophages . Once clnfection . On the nfection , 89. Durnfection , 91. Whijaponica –95. Such africae –99. Of nractions , 101.Rickettsia species, such as R. rickettsii, R. conorii, R. parkeri, R. helvetica, and R. australis, have evolved to resist bactericidal mechanisms and establish a replicative niche within the cytosolic compartments of macrophages  and altered metabolic and lipid catabolic pathways favoring anti-inflammatory M2 responses , outer membrane protein B (OmpB), the most abundant and conserved protein required for the formation of protective surface- and capsule-like layers on Rickettsia, played a significant role in preventing the surface polyubiquitylation of OmpA and subsequent autophagy evasion , which upregulates rickettsicidal genes encoding guanylate-binding proteins (GBPs) and inducible nitric oxide synthase (iNOS)  and mouse (BMDM) macrophages also activated inflammasome responses and induced IL-1β and IL-18 secretion in a caspase 1- and TLR4-dependent manner  . R. austt manner , 114. Fuke cells . These srophages . At the rophages . Thus, sR. rickettsii and R. conorii were considered the major tick-borne Rickettsia species associated with human infections  in the Americas, Europe, and Africa  and characteristic eschars at the inoculation site of tick attachment  and gene rearrangements, partly due to the presence or absence of tra genes  in the Americas  Sca2 and RickA mediate actin-based motility in a time-dependent manner in tissue culture cells, (ii) Sca2 is required for R. parkeri pathogenesis and dissemination from the inoculation site to internal organs in Ifnar1−/−Ifngr1−/− mice, (iii) Sca4 associates with vinculin and mediates the cell-to-cell spread of R. parkeri, and (iv) OmpB (Sca5) blocks the ubiquitylation of rickettsial surface antigens to promote autophagy evasion in immune cells and contributes to eschar formation in Ifnar1−/−Ifngr1−/− mice  and a circular pRA plasmid , with 1,260 chromosomal and 11 plasmid open reading frames (ORFs) predicted  pathogenesis, survival in mammalian and arthropod hosts, and transmission remain unclear (R. africae causes ATBF and infects multiple species of Amblyomma (A. hebraeum and A. variegatum as the main tick vectors), Hyalomma, and Rhipicephalus ticks in areas of endemicity in sub-Saharan Africa (–R. africae have been reported for A. hebraeum (Amblyomma ticks (up to 100%) collected from cattle in south and central Mozambique are infected with R. africae  on cattle in Madagascar  and blood from cattle (22%)  and A. variegatum ticks on goats, sheep, and cattle in Kenya suggest that other ruminants may serve as alternative hosts for R. africae transmission and amplification in nature  , illustrates the importance of sustained surveillance for the expansion of ticks and the occurrence of associated tick-borne pathogens of veterinary and medical significance  , 152. Thulations –155. In demicity , 157. Reificance , 159. In to ATBF . Most R.patients –163.R. heilongjiangensis, first isolated in 1982 from Dysmicoccus sylvarum ticks in Suifenhe, a city in the Heilongjiang Province of China, is comprised of a 1.28-Mbp (32.3% G+C content) circular chromosome and no plasmid DNA  of R. heilongjiangensis are required to induce nonlethal acute disease when intraperitoneally injected into C3H/HeN or C57BL/6 mice, corroborating clinical observations of mild cases in patients infected with R. heilongjiangensis  in patients bitten by the reptile tick B. hydrosauri in Australia and Thai tick typhus in those exposed to Ixodes granulatus bites in Thailand  predicted to harbor 1,284 genes closely related to the genes present in R. rickettsii, R. conorii, and R. slovaca and no plasmid DNA  in southern Australia, where confirmed cases of FISF have been reported   , 183. Cler (ASF) , 183. Ofer (ASF) , 183. A reatment . Cases oal areas , 186. Whulations .R. amblyommatis GAT-30V consists of a single chromosome  and three circular pRM plasmids . Compared to pathogenic SFG Rickettsia species (R. rickettsii and R. conorii), the chromosomal DNA sequence of R. amblyommatis displayed high degrees of sequence identity, multiple genetic rearrangements, and regions undergoing gene decay   in D. variabilis is estimated to be less than 1% , also known as meat allergy, but the underlying cause remains unknown (–The genome of ne decay . Bioinfo to 64%) . In receetiology –191. On  than 1% . Seropretal host , 193. Hoularemia . In addi unknown –197.A. americanum tick who developed a macular rash at the tick bite site and had a positive PCR test for R. amblyommatis and the absence of Borrelia, Ehrlichia, Anaplasma, Babesia, Bartonella, and other pathogenic Rickettsia species  did not show any clinical illness but seroconverted by 14 days postinfection (6 cells of R. amblyommatis 9-CC-3-1 (isolated from Amblyomma cajennense) caused vascular inflammation in guinea pigs  and developing molecular tools for Rickettsia. Yet some deficiencies  continue to hinder investigative advances for this universal emerging pathogen, highlighting significant research opportunities for discovering novel molecular mechanisms associated with the obligate intracellular life cycle of SFG Rickettsia species.Several advances have been made within the last decade toward understanding basic


Bulbus Fritillariae (BF) is a kind of herbal medicine widely used in many countries including China, Japan, Korea, and so on. Among the known BF medicinal materials, Bulbus Fritillariae cirrhosae (BF cirrhosae) was reported to have the best curative effect. Due to the limited resources of BF cirrhosae, a lot of adulterants have emerged in the market, impairing the market order, resource development, and above all, clinical efficacy. Here, a novel nucleic acid amplification technique, Recombinase Assisted Loop-mediated isothermal DNA Amplification (RALA), was used to establish a real-time fluorescence isothermal molecular authentication method for five commonly used BF drugs. Moreover, this RALA-based assay can also be developed as a colorimetric detection method for on-site detection. Both real-time fluorescence and visual methods could detect as low as 0.1% genuine targets in the mixed samples. In summary, we report an isothermal detection system for five kinds of BF circulating in the market, providing a new choice for the molecular identification of BF drugs and showing promise in the laboratory testing as well as field identification of other herbal medicines. Workflow of RALA-based bulbus fritillariae identification.  Fritillaria. BF was reported to have the function of clearing away heat and moistening lungs, eliminating phlegm, and relieving cough  derives from the dried bulbs of various species of the genus ng cough . Howeverng cough , althougng cough . Among ang cough . Under sTraditional identification methods for BF drugs include morphologic detection, microscopical identification , physicaIn an effort to overcome the shortcomings of LAMP, an isothermal amplification method called RALA  was developed by our group . The priF. cirrhosa D. Don, F. unibracteata Hsiao et K. C. Hsia, F. delavayi Franch., F. taipaiensis P. Y. Li, F. przewalskii Maxim. and F. unibracteata Hsiao et K. C. Hsia var. wabuensis (S. Y. Tanget S. C. Yue) Z. D. Liu, S. Wang et S. C. Chen), BF ussuriensis (bulbus of F. ussuriensis Maxim.), BF pallidiflora , BF thunbergii (bulbus of F. thunbergii Miq.) and BF hupehensis (bulbus of F. hupehensis Hsiao et K. C. Hsia) were gifted by Chengdu Institute Food and Drug Control and authenticated by DNA barcoding using ITS as a marker gene. The DNA of all BF samples was extracted using the plant DNA isolation Kit  according to the manufacturer’s instructions. The concentration of DNA was determined by the ultra-micro ultraviolet spectrophotometer ND5000  and stored at 4°C. Finally, 1 μL DNA sample was directly added to the RALA system.BF cirrhosae 2SO4, 0.2 mM dNTPs, 3 U of EasyTaq polymerase, 0.2 μM each primer , 20 mM KCl, 2 mM MgSOh primer , and 1 μThe RALA primers included a pair of inner primers and one (or two) loop primers were designed according to the specific site of the ITS sequence or the P16 gene sequence. All oligonucleotides were synthesized and purified by BBI, and the purification level was PAGE. The RALA primer sequences of 5 kinds BF were shown in 4, 10 mM (NH4)2SO4, 0.1% Triton X-100, 0.8 M betaine, 0.8 mM dNTPs, 1.6 μM inner primers (FIP and BIP), 0.8 μM loop primers (LF and LB), 0.24 U/μL Bsm polymerase, 0.6 mM ATP, 6 ng/μL TthRecA, 0.2 × SYBR Green I, and 1 μL DNA sample. Each group of primers amplified five BF at the same time. The reaction mixture was placed in the PikoReal Real-Time PCR System  at 60°C for 80 min. The results could be analyzed through the real-time collected fluorescence signals and 2% agarose gel.The total volume of the RALA reaction mixture was 25 μL, including 20 mM Tris–HCl (pH 8.8), 60 mM KCl, 4 mM Mg SO–5 ng/μL) and the mixed templates which contained different percentages of BF cirrhosae in the mixed genome with maize .Taking BF cirrhosae as the target, the analytical sensitivity of RALA was investigated by using a 10-fold serial dilution of pure genomic DNA  under the background of white paper.Tth RecA binds with the inner primers FIP and BIP to form the nucleoprotein complex and open the target double-strand DNA (dsDNA) at the consensus region. Next, the primers anneal to the complementary strand and are extended by the DNA polymerase with strand displacement ability. After the initial extension and strand displacement of the inner primer, the dumbbell-like amplicon is generated and self-primed for further extension. Moreover, the new inner primers can anneal to the dumbbell-like amplicon to induce the next round of extension. Besides, the loop primers LF and LB were designed to anneal to the single-strand loop domain of the intermediated products, which can further improve the amplification rate of RALA. The whole amplification process can be completed at a constant temperature of 60°C and the products of RALA amplification were considerable stem-loop DNAs with inverted target repeats. For different analysis needs, both the real-time fluorescence and colorimetric reporting system were established to present the results .The genome sequence-based molecular detection leverages the specific gene fragment as the amplification target to accurately identify the medicinal plants of related species. After decades of practice, ITS sequence , rbcL seF. cirrhosa D. Don; (2) F. unibracteata Hsiao et K. C. Hsia; (3) F. przewalskii Maxim.; (4) F. delavayi Franch.; (5) F. taipaiensis P. Y. Li; (6) F. unibracteata Hsiao et K. C. Hsia var. wabuensis (S. Y. Tang et S. C. Yue) Z. D. Liu, S. Wang et S. C. Chen). Fortunately, the ITS sequences of the six source plants of BF cirrhosae are identical in large part, and there are multiple base sites different from the other BF (Bulbus Fritillariae cirrhosae is the bulbus of six species of Fritillariae: (1) other BF . To deveother BF  and evalother BF . As showother BF . In ordeR2 = 0.9702), indicating that this method has the potential to realize quantitative detection. To evaluate the sensitivity of the method in mixed samples, 25 ng of the mixed genome containing different ratios of BF cirrhosae genome  were used as the templates. After the specificity examination, we sought to evaluate the sensitivity of this method. The serially diluted genomic templates of BF cirrhosae were used to determine the minimum detectable quantity of BF by RALA. As presented in After the establishment of the real-time RALA assay for BF cirrhosae, we intended to extend its use to a simpler situation of field detection without the need for fluorescence monitoring. Many colorimetric dyes including calcein, phenol red, neutral red (N-red), and so on have been proved to be applicable in realizing on-site colorimetric isothermal detections . Among tF. ussuriensis Maxim.), BF pallidiflora , BF hupehensis (bulbus of F. hupehensis Hsiao et K. C. Hsia) and BF thunbergii (bulbus of F. thunbergii Miq.). The ITS sequences of these four plants were aligned and the specific primer sets for BF ussuriensis and BF pallidiflora were designed (KF934438.1) of BF thunbergii was selected as the candidate target. We designed PCR primers of the P16 gene (ON493814 and ON493815). By comparing the sequencing results, we obtained their specific sites and designed the RALA primers of BF thunbergii and BF hupehensis  the whole testing can be achieved at a constant temperature, which is less instrument-demanding and cost-efficient; (ii) the high sensitivity enables the method for qualitative and quantitative laboratory testing; (iii) the colorimetric detection could provide a highly useful method in field testing using a simpler heat blocker; (iv) the method could not only distinguish the genuine BF cirrhosae but also determine its possible adulterants, which may complement the isothermal toolbox for BF identification. Therefore, this RALA-based method offers a promising alternative to the molecular identification of BF. As a new isothermal amplification method, RALA combined the merits of recombinase polymerase amplification (RPA) and LAMP. It can realize similar amplification efficiency to LAMP, while requiring less primer sequence. The system of RALA is less complicated than RPA, thus decreasing the cost. Besides, the addition of recombinase could improve the detection specificity of the RALA assay. Moreover, the colorimetric assay developed here could be realized without uncapping operation, which eliminates the false positive caused by carry-over contamination. Despite the advantages above, the method faces some challenges. The first is the primer design of RALA still requires multiple primer sequences, though less complex than LAMP; the second is that RALA carries out under about 60°C, which means a temperature controller is needed, though it is much simpler than PCR-based apparatus. Once overcoming the challenges, we believe that the high practicality of the RALA-based method could be readily extended for the identification of other herbal medicines.ON493814 and ON493815.The original contributions presented in this study are publicly available. This data can be found here: NCBI, ZT and RC: conceptualization, supervision, and funding acquisition. YW: methodology, software, validation, investigation, experimental operation, data analysis, drafting, and revision. SD: methodology, experimental guidance, and revision. GC: experimental guidance. JD: investigation. FD: resources and investigation. XH and XC: investigation. All authors contributed to the article and approved the submitted version.


Mycotoxin contamination in food poses health hazards to humans. Current methods of controlling mycotoxins still have limitations and more effective approaches are needed. During the past decades of years, variable environmental factors have been tested for their influence on mycotoxin production leading to elucidation of a complex regulatory network involved in mycotoxin biosynthesis. These regulators are putative targets for screening molecules that could inhibit mycotoxin synthesis. Here, we summarize the regulatory mechanisms of hierarchical regulators, including pathway-specific regulators, global regulators and epigenetic regulators, on the production of the most critical mycotoxins . Future studies on regulation of mycotoxins will provide valuable knowledge for exploring novel methods to inhibit mycotoxin biosynthesis in a more efficient way. Aspergillus, Penicillium, Monascus and Fusarium genera, and represent a major threat to human and animal health  ..1, AFB2,The biosynthetic pathways of these mycotoxins have been characterized well and the reader is referred to other reviews for details on the biosynthetic pathways ,27,28,29Regulation of mycotoxin biosynthesis is a complex process with various environmental factors forming a hierarchial regulatory network, including pathway-specific regulators, global regulators and epigenetic modification  as summaThe genes involved in the biosynthesis of mycotoxin are typically arranged in a biosynthetic gene cluster (BGC), containing not only synthases and/or synthetases genes but also many tailoring enzymatic encoding genes . The cluA. flavus/A. parasiticus and A. nidulans. The AF/ST BGC includes ~30 genes and including two pathway-specific regulators AflR and AflS (previously named AflJ) CGA-3′ .,32.A. fl kingdom . AflS is kingdom . The exp5)CGA-3′ . In A. paffinity . The preCCSSG-3′ . Positio motif B . The abnflatoxin ,81.Penicillium, Aspergillus and Byssochlamys . Th. ThFUM gSimilar to carbon source, nitrogen source also affects production of all mycotoxins but not in a consistent manner. In some fungi, AreA, a GATA factor transcriptional regulator of nitrogen metabolism, has been deleted to explore impact on mycotoxin synthesis.aflR/aflS intergenic region by recognizing a GATA element which seems to prevent AflR binding  medium the ∆areA strain promoted AF biosynthesis when compared with the WT and OE::areA strains ,103aflR/ strains . As AreAP. expansum, cultures grown with organic nitrogen sources give better PAT yields than inorganic nitrogen sources. Peptone, glutamic acid and yeast extract are the best nitrogen sources for up-regulation of all pat gene expression and increase PAT production in P. expansum, while ammonium sulfate is the most unfavorable nitrogen source [In n source . But theMonascus M9 growth and CIT production than inorganic nitrogen [M. purpureus M3103 when grown with NH4Cl or NH4NO3 as the sole nitrogen source [Organic nitrogen is also a better source for nitrogen . Minimaln source .F. fujikuroi, AreA and a second nitrogen metabolism regulator, AreB, have been found to regulate TC biosynthesis and TC production  is the key TF in pH signal transduction in filamentous fungi, and recognizes 5′-GCCARG-3′ (R = G or A) in the target promoters . The Pace medium .A. flavus resulted in increased AF production by 10-fold [A. parasiticus, a putative PacC binding site (5′-GCCAAG-3′) was identified in the aflR promoter  (R = G or A) was found in the promoter regions of 9 pat genes, including its putative pathway-specific regulator patL  [In contrast to AF and PAT, CIT production was significantly increased when the pH value shift from acid to alkaline in A. niger . In P. e(pH 6~8) . No repoTRI gene transcription and TC production in F. graminearum. Neither TRI gene expression nor TC accumulation is detected when the pH is maintained at neutral or alkaline pH [TRI gene transcription and negatively regulates TC production. Overexpression of pacC in F. graminearum strongly repressed TRI gene expression and reduced TC accumulation at acidic pH [TRI genes, including the pathway-specific regulator TRI6  . Six FUM regions . HoweverA. nidulans [A. nidulans develops asexually in light and sexually in the dark, and VeA is involved in the shift from sexual to asexual spore formation. LaeA is constitutively present in the nucleus, while VeA and VelB appear to interact already in the cytoplasm then travel together into the nucleus by KapA  reduced expression of all FUM genes. Surprisingly, despite decreased expression of FUM genes, FM production in the ∆lae1 mutant was not significantly reduced compared with WT. However, the lae1 complemented strain produced 50% more FMs than WT [veA homologue was deleted in F. verticillioides, the production of FM was completely suppressed. VeA forms a complex with the velvet proteins VelB and VelC, and is necessary for the expression of the pathway-specific regulator gene FUM21 -like site  was shown to be involved in this regulation for both chemotypes. The NIV/FX chemotype has higher antioxidant capacities than DON/ADON chemotype in response to oxidative stress [Functional or non-functional r NIV/FX  [127,128ependent . A 0.5 me stress . Fgap1 , while other three isolates produced significantly less (<20%) FM [The effect of oxidative stress induced by H<20%) FM . This isLaeA investigations first suggested that epigenetic regulatory mechanisms were important for secondary metabolism synthesis . Since tsntB in A. nidulans and A. flavus changed the global levels of histone H3K9K14 acetylation, leading to the inhibition of ST and AF  magnetic field inhibits CIT contamination by reducing the expression of the pathway-specific regulator gene urpureus . Anotherurpureus . As most


Escherichia coli, toxigenic Clostridioides difficile, Campylobacter and Salmonella species. The review will discuss research gaps and propose future approaches to the translational development of plant-derived products for drug discovery purposes for the treatment and prevention of gastrointestinal infectious diseases.The rising burden of antimicrobial resistance and increasing infectious disease outbreaks, including the recent COVID-19 pandemic, has led to a growing demand for the development of natural products as a valuable source of leading medicinal compounds. There is a wide variety of active constituents found in plants, making them an excellent source of antimicrobial agents with therapeutic potential as alternatives or potentiators of antibiotics. The structural diversity of phytochemicals enables them to act through a variety of mechanisms, targeting multiple biochemical pathways, in contrast to traditional antimicrobials. Moreover, the bioactivity of the herbal extracts can be explained by various metabolites working in synergism, where hundreds to thousands of metabolites make up the extract. Although a vast amount of literature is available regarding the use of these herbal extracts against bacterial and viral infections, critical assessments of their quality are lacking. This review aims to explore the efficacy and antimicrobial effects of herbal extracts against clinically relevant gastrointestinal infections including pathogenic  Escherichia coli, Campylobacter, Enterococcus faecalis, Salmonella spp., Shigella, and Clostridioides difficile . In. InChoisIn general, antibacterial actions of action of plant-derived products include inhibition of bacterial cell wall synthesis, cell membrane destruction, inhibition of bacterial protein synthesis, DNA replication, and metabolic pathways, making them excellent candidates for future drug development ,15,23,24Escherichia coli (EHEC), specifically, E. coli O157:H7, is one of the most important serotypes among the different E. coli pathogenic groups, which include enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohaemorrhagic (EHEC), enteroinvasive (EIEC), diffusely adherent (DAEC), and enteroaggregative E. coli (EAEC) [Enterohemorrhagic i (EAEC) . It is ei (EAEC) . Plant-di (EAEC) .Zingiber officinale Roscoe) is one of the most used spices. Zingerone (vanillylacetone) is reported to be the active constituent responsible for the antidiarrhoeal efficacy of ginger [Rhus chinensis Mill , and Indian medicinal plant, Berberis aristata DC (Indian barberry or tree turmeric), were effective in preventing ETEC heat-labile enterotoxins from binding to their surfaces [Galla chinensis, was effective for treating LT-induced diarrhoea by causing inactivation of ETEC enterotoxins [Galla chinensis is gall caused by the Chinese aphid (family Pemphigidae) on the Rhus leaves of the family Anacardiaceous  [Galla chinensis inhibit intestinal bacterial growth in vitro including Clostridium perfringens, Clostridium paraputrificum, and E. coli without affecting the growth of the intestinal probiotics  [Galla chinensis was one of the active compounds to resist E. coli and protect mice from infection by Enterotoxigenic E. coli [Ginger  . Furtherbacilli) . It was  E. coli .Camellia sinensis tea leaves contain galloylated tea catechins, which have been observed to act at the level of the bacterial cell membrane level [E. coli cells by damaging the lipid bilayer membrane. These results suggest a strong effect of catechins from green tea on bacterial phospholipid synthesis [ne level ,52 inducne level . Epigallynthesis . Furtherynthesis .Syzygium aromaticum (L.) Merr. & L.M. Perry.), inhibits the production of two verotoxins (VT1 and VT2) in EHEC by modulating the transcription of stx 1 and stx 2, respectively [Stx1 and Stx2 gene expression levels were similarly reduced with sub-inhibitory concentrations of Curtisia dentata extract, trans-cinnamaldehyde (major component of cinnamon) and carvacrol (obtained from oregano oil) [Eugenol, the major component of clove (ectively . Stx1 anano oil) ,54. ThesE. coli, the leading cause of bacteria-mediated gastrointestinal infections, by reducing bacterial adhesion to the gastrointestinal mucosa [Vaccinium macrocarpon (cranberry) and show promising activity against bacterial virulence toxins and biofilm formation [Proanthocyanidins show antibacterial activity against l mucosa . These pormation ,56. Althormation ,57.Berberis vulgaris, exhibits antibacterial properties against a wide range of pathogenic bacteria either alone or in combination with antibiotics [Pseudomonas aeruginosa [An alkaloid, berberine, derived from the rhizomes of ibiotics ,59. Berbibiotics  to examiibiotics . Scanninibiotics . Furtheribiotics ,62. Inteibiotics . Throughruginosa .V. cholerae and E. coli has been extensively investigated. Berberine directly inhibited V. cholerae and E. coli enterotoxins in vitro [E.coli biofilm by berberine was associated with the suppression of quorum-sensing genes such as pfS, hflX, and fliA [E. coli heat-stable enterotoxins in a mouse model [E.coli by downregulating the capsule synthesis promoter (RcsF) and Undecaprenyl-diphosphatase (YbjG) [E. coli [E.coli by downregulating the capsule synthesis promoter (RcsF) and Undecaprenyl-diphosphatase (YbjG) [E. coli [The mechanism by which berberine treats diarrhoea caused by in vitro . The inhand fliA . In addise model . Even thse model . Berberise model . The absse model . Additiose model . In Gramse model  Berberine (YbjG) . Both are (YbjG) ,70. Cons[E. coli . Even th[E. coli . Berberi[E. coli . The abs[E. coli . Additio[E. coli . In Gram[E. coli  Berberine (YbjG) . Both are (YbjG) ,70. Cons[E. coli .C. difficile toxins A (TcdA) and toxin B (TcdB) and toxicity while maintaining normal gut flora growth [C. difficile-induced clinical symptoms were mitigated, causing a favourable shift in gut microbiota composition without negatively affecting gut microbiome diversity [C. difficile agent, but further clinical studies are needed.Aromatic essential oil constituents such as carvacrol (2-methyl-5-(1-methylethyl)-phenol) and cinnamaldehyde  possess broad antimicrobial activity that extends to food spoilage and pathogenic microorganisms, including drug-resistant and biofilm-forming microorganisms ,34. The a growth . As a reiversity . Based oAllium sativum L.) inhibits C. difficile toxin production (TcdA) along with the genes encoding TcdR, an RNA polymerase sigma factor needed for maximal expression of TcdA (4.7 mg/mL) [C. difficile toxins TcdA and TcdB by blocking either the toxin-binding site on toxin molecules or the host cell receptors (0.3 mg/mL) [C. difficile toxins TcdA and TcdB in vitro. Fresh onion bulb extract (12.5% v/v) significantly decreased toxin production and activity. The primary mechanism of action of this extract was the inhibition of C. difficile sporulation at different stages [C. difficile infection (CDI), berberine (100 mg/kg/day) combined with vancomycin was more effective than vancomycin alone. Treatment with berberine prevents CDI relapses and reduces mortality significantly [Several studies have shown that allicin in garlic juice (7 mg/mL) ,75. The 3 mg/mL) . In an et stages . In a moficantly .C. difficile [A novel lead compound, chromomycin A2/NP-00387, with potent anticlostridial activity in vitro and in vivo, conferred 100% protection to infected mice against clinical manifestations of CDI at a concentration of 0.03 μg/mL . The comifficile . It willScutellaria baicalensis Georgi. (Chinese skullcap) roots contain baicalin , a major flavone glycoside identified in the Chinese Pharmacopoeia as a medicinal herb [H. pylori in the murine stomach [Stx to inactivate the toxin and promote toxin oligomerization [E. coli O157:H7 and showed that baicalin reduced lethality in mice, but its exact mechanism of action remains unclear [nal herb . Researc stomach . Previourization . Moreove unclear .C. difficile. A recent study examined the effect of essential oil compounds on mixed human faecal microbiota. As a result of the study, thymol and geraniol at a concentration of around 100ppm may be effective in suppressing pathogens in the small intestine without damaging beneficial commensal bacteria of the colon [C. difficile in vitro [There are only a few reports available in which researchers have assessed essential oils ,79,80 anhe colon . SeveralCampylobacter jejuni that cause dysentery-like illness are more cytotoxic than other Campylobacter species [Campylobacter jejuni and Campylobacter coli, little is known about their effect on virulence factors of these bacteria [farnesiana, Artemisia ludoviciana, Opuntia ficus-indica, and Cynara scolymus. It has also been reported that Japanese green tea extracts show anti-Campylobacter jejuni activity [C. jejuni, with a strong antiadhesive effect in vitro, but not in vivo. Similarly, extracts of A. farnesiana, A. ludoviciana, C. scolmymus, and O. ficus-indica significantly reduced Campylobacter adhesion to Vero cells [It has become increasingly difficult to manage campylobacteriosis because of antibiotic-resistant strains of Campylobacter. Campylobacter has been reported to possess a wide variety of cytotoxic activities , but onl species . Althougbacteria . A prelibacteria . The fouactivity . A critiactivity ,87). In activity , okra exro cells . This inro cells . Furtherro cells ,15.The global healthcare system is heavily burdened by GIT disorders. Globally, diarrhoeal-inducing GIT disorders are one of the leading causes of death . Even thDue to the absence of significant pre-clinical and clinical research on the effectiveness of herbal medicine, it is important to gather whatever scientific data to support their use in treating different pathologies. A large proportion of herbal medicines are used in developing countries . There aAdansonia digitata) is eaten to treat diarrhoea and dysentery. According to a clinical study conducted in Senegal comparing Adansonia fruit with oral rehydration therapy, the two treatments did not differ significantly in terms of diarrhoea duration and weight gain [In India, raw baobab fruit (ght gain . It is pght gain .Psidium guajava (myrtaceae), originate from Central and South America where this tree forms part of the pantropical region. Additionally, the plant can be used to treat coughs, diabetes, dysentery, fevers, leukorrhea, rheumatism, toothaches, and wounds [P. guajava was shown to clear infection faster after 19 days in mice infected with Citrobacter rodentium [V. cholerae had intestinal ameliorative effects after a 4-h infection when treated with 250 mg/kg of ethanol extracts of P. guajava leaves [®, which contains P. guajava leaves with high flavonoid concentrations, on patients with infectious gastroenteritis. The duration of abdominal pain was significantly reduced by QG5® in these patients without any serious side effects [P. guajava extracts. According to one study, oral administration of 5000 mg/kg body weight of methanolic extract of P. guajava bark for 28 days did not cause toxicity in Wistar rats [E. coli was most prevalent. Clinical trials conducted on 100 gastroenteritis patients showed that berberine tannate was more effective than conventional antibiotics in treating diarrhoea [E. coli, Shigella spp., and Salmonella spp. were isolated from the patients [Guava is well-known for treating diarrhoea in tropical countries through ingesting teas, decoctions, or macerations . Guava ld wounds . The used wounds . Guava hodentium , and mica leaves . A rando effects . The littar rats , althougtar rats . Early ctar rats ,104. Amoiarrhoea . When beiarrhoea . Many dipatients .E. coli and V. cholerae diarrhoea during three consecutive 8-h periods after treatment. There have been no reported side effects associated with berberine [Bacillus cereus, E. coli, and V. cholerae, present at relatively high levels (12.5–469 μg/mL) [In a randomized controlled trial, berberine sulphate reduced mean stool volume in 165 adult patients with enterotoxigenic erberine . There aerberine , inhibiterberine ,109, enherberine , and its9 μg/mL) ,111.C. difficile antibiotic-associated diarrhoea, berberine cure rates were 84 and 92 percent, respectively [C. difficile toxin genes was reported by Shu et al. [In a study of 50 patients with CDI and non-ectively . A meta-ectively . The inhu et al.  to be a u et al. . Aside fu et al. ,116. Theu et al. ,118,119E. coli in all cases and was well tolerated with no reported adverse effects. In a placebo-controlled randomized trial conducted with 252 children, 150 mg of bismuth subsalicylate per kilogram of body weight per day decreased the duration of acute diarrhoea. C. jejuni, enteropathogenic E. coli, Salmonella spp., Shigella spp., and Vibrio spp. were found in stool samples collected at the beginning of the study [C. difficile, enterotoxigenic E. coli, S. enterica subsp. enterica serovar Typhimurium, and Shigella sonnei. Bismuth subsalicyclate was found to have MICs of 2, 8, 4–8, and 2–8 μg/mL, respectively [As part of a parallel, double-blind, randomized trial of 133 European tourists in West Africa and 112 American students in Mexico, bismuth subsalicylate significantly shortened travellers’ diarrhoea duration at both study sites . Bismuthhe study . There wectively . Despiteectively  The use ectively .Coptis spp., Berberis spp., and Hydrastis canadensis as capsules, tablets, and tinctures , Napiers, Edinburgh, UK; Berberine HCL 97% Capsules, British supplements, Milton Keynes, UK; and Hydrastis Canadensis capsules, Alfa herbal, Barcelona, Spain). In addition to supporting digestive function, these supplements are also used to treat gastrointestinal disorders, including diarrhoea [Several dietary supplements, herbal medicines, and over-the-counter pharmaceuticals are available on the international market that support and maintain GI health  . Variousiarrhoea ,110. Mosiarrhoea ,104. In iarrhoea ,123. In iarrhoea .Currently, omics approaches, which encompass genomics, transcriptomics, proteomics, and metabolomics, are becoming important in identifying and characterizing crucial gene–protein–metabolite networks, discovering new drug metabolites, identifying complete genomes, transcriptomes, and proteomes of medicinal plants, evaluating human cell responses to drugs and whole ethnobotanical plants for medicinal use, and facilitating large-scale production of plant-derived medicines ,133. A rThe discovery of plant biosynthetic pathways and gene clusters has increased dramatically over the past few decades, spurred by technological advances in sequencing and mass spectrometry technologies . Due to Aquilegia oxysepala extract on Staphylococcus aureus and its main chemical components , which are used for treating GI infections, were examined in comparison with nine antibiotics with known mechanisms of action [A. oxysepala targets are similar to those of protein synthesis inhibitors including lincolmensin, erythromycin, chloromycetin, streptomycin, and acheomycin, and its bioactive component maguoflorine accounted for most of the antibacterial activity. In this study, berberine showed similar effects to rifampicin and norfloxacin, both of which target nucleic acids [Metabolomics experiments offer a more efficient route to drug discovery than the classical approach in natural products research. In essence, metabolomics analyses all metabolites present in a living system qualitatively and quantitatively . Interesf action . As showic acids . These mCatharanthus roseus (L.) G. Don (Madagascar Periwinkle) revealed diverse iridoid-based monoterpene indole alkaloids [E. coli [Transcriptomics is an effective method for assembling genomic data from a variety of non-model therapeutic plants that lack a reference genome. Transcriptomic studies assist in understanding the molecular mechanisms involved in the formation of secondary metabolites and in probing pharmaceutically significant mechanisms . The tralkaloids . It was lkaloids  and thatlkaloids . There a[E. coli .A wide range of functions can be achieved with proteomics in medicinal plant research . Using pSalmonella enterica revealed that energy metabolism-related proteins and flagellar proteins were up-regulated with berberine treatment, indicating a possible antibacterial action at the molecular level [The proteomic analysis of ar level . Althougar level .These studies show how OMICS technologies have potential for identifying the mechanisms of action of bioactive compounds, as well as combating drug-resistant bacteria. Furthermore, these studies suggest that bacterial cell death cannot be explained solely by the direct interaction between bioactive compounds and their cellular targets ,152, witThe appeal of herbal medicine is multifactorial. It is widely believed by the general public that herbal medicines are safe to consume and are not associated with any significant side effects. Since these treatments are “natural”, they are thought to be safe as an alternative to conventional medicine. However, several side effects have been reportedly associated with herbal products used in the treatment of GI disorders. These are listed in Many previous studies concerning plant-derived antibacterial agents failed to standardize their methods to achieve reproducible and comparable results. The effectiveness of a plant’s antimicrobial properties is generally demonstrated through large-scale, well-designed clinical trials. It is therefore essential that clinical trials follow the guidelines of the U.S. FDA and the EMA when evaluating medicines indicated for treating bacterial infections ,181. PlaC. difficile, Enterobacter, E. coli, K. pneumonia, and Pseudomonas, are of intense interest. The potential synergy or potentiation of phytochemicals with other antibacterial agents has already been demonstrated in previous studies [As bacteria become increasingly resistant to commonly used antibiotics, new classes of plant-derived antimicrobials to treat infections caused by pathogens, such as  studies ,63,183.  studies .As part of their defence against microbial infections, plants use a variety of chemical strategies to reduce the selective pressure for developing antibiotic resistance. The activity of a compound can be significantly increased or decreased depending on its synergistic and antagonistic effects with other compounds found in plants. The complex chemical composition of plant extracts may also produce pharmacological effects, as is the case for traditional herbal medicines, whose efficacy is based on their complex composition, due to their synergistic or antagonistic interactions with a wide spectrum of phytochemicals ,185. DevThis review summarizes data on the clinical efficacy and antimicrobial properties of antibacterial agents of plant origin currently used to prevent and treat GI infections. A paradigm shift has occurred from focusing on conventional antibiotic therapy to alternative approaches due to new resistant bacterial strains. There is a wide variety of active constituents found in plants, making them an excellent source of antimicrobial agents with therapeutic potential as alternatives or potentiators of antibiotics. The identification of new and valuable antibacterial compounds from plants and the testing of their antibacterial properties are crucial for this purpose. Several factors may contribute to the effectiveness of herbal-derived compounds for fighting bacterial infections, two of which may be pivotal. First, from a phytochemical point of view, these compounds belong to various classes of plant secondary metabolites such as isoquinoline alkaloids, monoterpenoids, phenolic acids, and tannins, which may provide alternative antibacterial mechanisms to conventional antibiotics. Second, it is extremely likely that the utilization of unique traditional knowledge of herbal medicine will result in biocompatible, cost-effective, and promising solutions, ultimately leading to the discovery of new antibacterial agents. Although new plant-derived antibacterial compounds are increasingly being studied, few are still in clinical trials. However, most of them show antibacterial activity in vitro and in vivo. Most of these products act as direct growth inhibitors; however, certain products have antimicrobial properties due to their antitoxin properties . Inhibitory agents of biofilm formation may also be of interest. Plant-derived products may be used as biofilm inhibitors and synergistic agents in the future. It may be more fruitful to investigate combinations of plant compounds with conventional antibiotics. There may be a reason for this, since phytomedicines require complex combinational effects between their bioactive compounds to synergize their activities. Innovative approaches using high-throughput technologies (omics) and systems biology are crucial for effective proof-of-concept research and development of new types of plant-derived products that could be effective against antimicrobial resistance.