Weeds cause significant yield losses in crop production and influence the health of animals and humans, with some exotic weeds even leading to ecological crises. Weed control mainly relies on the application of chemical herbicides, but their adverse influences on the environment and food safety are a significant concern. Much effort has been put into using microbes as bioherbicides for weed control. As plant-associated bacteria (PAB), they are widely present in the rhizophere, inside crops or weeds, or as pathogens of weeds. Many species of PAB inhibit the seed germination and growth of weeds through the production of phytotoxic metabolites, auxins, hydrogen cyanide, etc. The performance of PAB herbicides is influenced by environmental factors, formulation type, surfactants, additives, application methods, and cropping measures, etc. These factors might explain the inconsistencies between field performance and in vitro screening results, but this remains to be clarified. Successful bioherbicides must be specific to the target weeds or the coinciding weeds. Detailed studies, regarding factors such as the formulation, application techniques, and combination with cultivation measures, should be carried out to maximize the performance of PAB-based bioherbicides. Weeds influence crops directly or indirectly by competing with them for nutrition, water, and light. Some weeds suppress the growth of crops through allelopathy . Many spThe use of cultivation techniques, mechanical operations, and chemical herbicides can reduce the yield losses caused by weeds. Weed control was a labor-intensive process before the invention of chemical herbicides. Although the discovery of chemical herbicides liberated the hands of farmers and significantly reduced yield loss caused by weeds, their adverse effects, such as the generation of residues in the environment and agricultural products and the development of resistance, have been well documented . A numbeXanthomonas campestris pv. poae JT-P482, Pseudomonas fluorescens D7, and P. fluorescens ACK55, have been used in the development of bioherbicides and applied for the control of annual bluegrass and brome, respectively [Plant-associated bacteria (PAB), including bacteria in the soil adjacent to the rhizosphere, rhizobacteria, endophytic bacteria, and phytopathogenic bacteria, co-evolve with crops and weeds. Many PAB show suppressive, herbicidal, or phytotoxic activity against weeds. The application of PAB displaying herbicidal activity leads to the colonization of target weeds by bacteria and the production of phytotoxins in weeds or the adjacent environment. Such herbicidal PAB have a high potential for use in the development of bioherbicides. Some PAB, such as ectively ,16,17.In this review, we summarized the progress in bioprospecting PAB for their herbicidal activities and developing bioherbicides with PAB as the active ingredients, with a focus on screening strategies, the diversity of herbicidal PAB, and their modes of action. We also discussed the factors affecting the performance of herbicidal PAB and the inconsistency between the activity levels determined by in vitro screening and field efficacy. Additionally, we predict the future of bioherbicides originating from PAB.Considering the diversity of microbes associated with weeds and crops, the screening for herbicidal or weed-suppressive bacteria among PAB must be rapid, robust, and cost-effective. Unlike chemical herbicides, which have a single mode of action, herbicidal PAB function through multiple modes of action. High-throughput screening with target enzymes or receptors is not suitable for herbicidal bacteria. At present, screening for herbicidal PAB is normally based on the seeds, seedlings, or tissue cultures of weeds.Euphorbia esula) for the screening of phytotoxic bacteria against Euphorbia sp. [8 CFU/mL bacterial suspension in a petri dish. To increase the throughput of the screening, a 24-well microplate screening system was set up with the callus tissues of leafy spurge [Euphorbia sp. were found to be phytotoxic to the callus tissues of E. esula, and the phytotoxicity was highly correlated with the activity experimentally determined using leafy spurge seedlings [As many herbicidal bacteria act through the production of phytotoxic metabolites, it is possible to use the calluses or cell cultures of weeds to screen for phytotoxic PAB. A rapid screening method was established with cell cultures and callus tissues of leafy spurge  is often selected because of its high sensitivity to the non-specific phytotoxic secondary metabolites produced by herbicidal microorganisms. A seed-germination assay with L. sativa was carried out to verify the phytotoxicity of deleterious rhizobacteria (DRB) isolated from the roots of leafy spurge with 30 μL of bacterial suspension per seed pre-germinated on 1% water agar [sativa could also be carried out in a box with germination paper [Orobanche sp. [Parthenium hysterophorus [Bromus tectorum [Avena fatua [Chenopodium album [The primary screening of the herbicidal activities of PAB can be achieved through the establishment of a screening procedure based on sensitive plants or weeds. Lettuce  was isolated from diseased P. annua with symptoms of bacterial wilt [annua appeared 7 to 10 days after the application of X. campestris culture; about 30% of the annual bluegrass wilted within 1 week, and 60–70% of the annual bluegrass plants were dead 3 weeks after the culture application [campestris pv. poae caused the systemic wilt of annual bluegrass when applied after mowing [campestris pv. poae to control annual bluegrass in Bermuda grass [9 cfu/mL after three applications. The host range of the bacterium was restricted to Poa sp. It is possible to use X. campestris pv. poa to control unwanted Poa sp. in crop fields or invasive Poa sp. in turf. The strains of X. campestris pv. poae from California and Michigan showed good control of P. annua ssp. annua and P. annua ssp. reptans in a growth chamber assay, but their effects in field tests decreased to 11 and 7%, respectively [campestris pv. poae for the control of P. annua. Nishino and Fujimori distinguished the isolates of X. campestris pv. poae collected in Japan into three groups [campestris pv. poae isolates, strain JT-P482 showed pathogenicity against annual bluegrass. The application of the JT-P482 culture to precut annual bluegrass caused over 75% weight loss due to plant wilting and death [campestris pv. poae JT-P482 was developed by Japan Tobacco Inc. as a bioherbicide with the commercial name Camperico® for the control of P. annua. Savage and Haygood also isolated many strains of X. campestris from asymptomatic plants, such as annual bluegrass, crabgrass, goosegrass, and other species of weeds collected in field and turf locations. Of these strains, MB245, 246, 249, 250, 253, and 260 were virulent against annual bluegrass, while MB251 targeted crabgrass [Bromus. Compared with MB218, turf grasses showed less susceptibility to MB245. With the reclassification of X. campestris, the xanthomonads that attack grasses were recategorized as X. translucens. Mitkowski found that X. translucens pv. poae, the casual agent of bacterial wilt in annual bluegrass, showed high specificity for P. annua and only weak pathogenicity against P. annua var. reptens and Poa trivialis cv. sabre [campestris pathovar PT1 isolated from P. trivialis (a common weed contaminant of creeping bentgrass seed stock) selectively controlled P. trivialis. The X. campestris PT1 treatment of turfgrass with transplanted P. trivialis plugs caused about 19.4% of the P. trivialis to wilt [Xanthomonas sp. from diseased common cocklebur (X. strumarium) leaves with angular-shaped spotting symptoms on the leaf margins and central leaf areas killed common cocklebur seedlings [Xanthomonas sp. isolates, LVA987 caused extensive blight in treated common cocklebur leaves 7–9 days after inoculation and the death of treated plants 10–12 days after treatment. The isolate LVA987 showed high pathogenicity against C. canadensis, A. artemisiifolia, A. trifida, and other Asteraceae plants. LVA987 was further characterized as X. campestris. The treatment with the LVA987 culture at 109 cfu/mL caused significant mortality and a reduction in growth in the rosette and bolting growth stages of C. canadensis [Selaginella moellendorffii through metagenome profiling and found that X. translucens was present in many diseased samples but not in the native club mosses [nt hosts . Many spial wilt ,37,38. Ilication . X. campda grass , and thee groups . Of the nd death , while nnd death ,44. The nd death . X. campv. sabre ,48. The b mosses .syringae, with a very wide spectra of hosts, was listed as No. 1 of the top 10 pathogenic bacteria in agriculture in a survey organized by Molecular Plant Pathology [syringae are of economic importance, causing bacterial speck in tomato (P. syringae pv. tomato), bleeding canker in horse-chestnut (P. syringae pv. aesculi), and bean halo blight (P. syringae pv. phaseolicola). Many pathovars also infect weeds or undesirable plants. P. syringae pv. phaseolicola infects kudzu (P. lobata). Younger kudzu seedlings treated with P. syringae pv. phaseolicola displayed water-soaked symtoms and disease severities were increased with multiple appliction, but the plants treated with P. syringae pv. phaseolicola showed a higher regrowth potential [syringae pv. tagetis is a pathogen of many plants, especially composite plants. Certain isolates of P. syringae pv. tagetis were found to infect marigold, sunflower, Jerusalem artichoke, and ragweed through wound inoculation [artemisiifolia) was also found to be a host of P. syringae pv. tagetis [syringae pv. tagetis infection is apical chlorosis, often accompanied by stunting. Early field trials showed that P. syringae pv. tagetis isolated from Canada thistle (Cirsium arvense) is more effective against annual weeds, such as A. artemisiifolia, C. canadensis, Lactuca serriola, and X. strumariaum [syringae pv. tagetis reduced the shoot growth of common ragweed and Canada thistle by 82 and 31%, respectively; the common ragweed treated with P. syringae pv. tagetis showed chlorosis in 60% of the leaf area [syringae pv. tagetis to control Canada thistle in the field [syringae pv. tagetis was also observed to infect Ambrosia grayi; almost 100% of the A. grayi became infected 6 weeks after a single dose of P. syringae pv. tagetis, with application weekly or every 3 weeks [syringae-CT99B016C, with differences to P. syringae pv. tagetis, was found to be pathogenic to Canada thistle, causing moderate to severe apical chlorosis and similar symptoms in Sonchus (moderate chlorosis in new growth areas) [oleraceus and S. asper  and T. officinale (dandelion). The metagenome profiling of diseased exotic club moss S. moellendorffii demonstrated the presence of P. syringae in many diseased moss samples [aeruginosa strain CB-4 isolated from infected corn leaves produced a herbicidal compound that strongly inhibited the growth of D. sanguinalis [P. athology . Many paotential . P. syriculation . Common  tagetis . A typicumariaum ,56,57. Peaf area . Tichichhe field . Although areas) . The str samples . P. aeruguinalis .Pseudomonas spp. and Xanthomonas spp., other phytopathogenic bacteria have also been studied for weed management. The pathogen that causes ginger  bacterial wilt, R. solanacearum, was investigated for the biocontrol of Kahili ginger (H. gardnerianum), an invasive weed in the tropical forests of Hawaii. The inoculation of R. solanacearum through stem injection or root cutting caused H. gardnerianum plants to exhibit irreversible chlorosis and severe wilting 3–4 weeks following inoculation. This inoculation also caused systemic infection in the rhizomes, which led to their death and decay [canadensis infected the healthy leaves of C. canadensis and inhibited the seed germination of rye grasses and Cichorium intybus by over 90% [andropogonis isolated from diseased chickweed showed herbicidal activity against weeds in the Caryophllaceae, Poaceae, and Fabaceae families, but the infection of B. andropogonis required wounds and infiltration under natural conditions [cereus XG1 isolated from watermelon showing symptoms of bacterial fruit rot exhibited herbicidal activity against D. sanguinalis [Besides nd decay . Bacteriover 90% . B. andrnditions . B. cereguinalis .Deleterious rhizobacteria (DRB) are predominantly saprophytic bacteria that aggressively colonize plant seeds, roots, and rhizospheres and readily metabolize organic substances released by plant tissues . DRB exifluorescens strain D7 isolated from the roots of winter wheat inhibited downy brome growth in laboratory bioassays [Bromus [tectorum by 42~64%. P. syringae strain 3366 from the rhizoplane of wheat and downy brome was active against downy brome in different assays [putida FH160, E. taylorae FH650, and X. maltophila FH131 were isolated from the rhizoplane of downy brome and other plants [trivialis X33d isolated from the rhizosphere of durum wheat (Triticum durum) seedlings strongly reduced the dry weight of the roots and shoots of B. diandrus and promoted the growth of many species of crops [Downy brome is a serious problem in wheat fields. P. ioassays  and undeioassays . The cel [Bromus , but wast assays . Three st assays , and oner plants ,78. FH16r plants . P. trivof crops , 80.fluorescens WH6 isolated from rhizosphere soil inhibited the germination of the seeds of grassy weeds, such as annual bluegrass (P. annua), without significantly affecting the growth of established grass seedlings and mature plants or the germination of the seeds of broadleaf plant species [fluorescens strains reduced the populations of the target invasive annual grasses in invaded pastures and grassland to nearly zero after 5 years of application and maintained control for 7 years after application [fluorescens strain BRG100, isolated from the rhizosphere of green foxtail (Setaria viridis), strongly inhibited the root elongation of green foxtail by 74% [rettgeri strain CPS67 and Pseudomonas isolate HWM11, strongly suppressed the growth of P. minor in pot assays [conjugatum in a soybean field inhibited the germination of seeds and the growth of P. conjugatum [kilonensis/brassicacearum strain G11, a cyanide producer isolated from Galium mollugo, strongly inhibited the growth of E. crus-galli, reducing the above-ground biomass by 95% and decreasing the root length [Chromobacterium sp. S-4 isolated from the rhizosphere of D. sanguinalis reduced the fresh weight of D. sanguinalis by 60% and caused chlorosis in the treated leaves 3 days after application [Pseudomonas sp. isolated from the rhizosphere of wheat caused up to 50.0% and 56.7% mortality in A. fatua and P. minor seedlings and reduced root length by up to 73.8% and 53.9%, respectively. Additionally, these consortium strains increased wheat shoot length, root length, fresh biomass, dry biomass, and leaf greenness by up to 41.6, 100, 79.9, 81.5, and 21.1%, respectively [Bacillus sp. X20, inhibited the germination of seeds of wild oat by 75% [fluorescens strain L2-19, S. maltophilia strain TFR1, and P. putida strain B1-7 isolated from the rhizosphere of green foxtail significantly reduced the growth of green foxtail [fluorescens G2-11 inhibited the germination of seeds and the root growth of S. viridis in silt loam and sandy loam [fluorescens strains, XJ3, XS18, and LRS12, inhibited the growth of annual bluegrass (P. annua), with shoot inhibition of 42–78% and root inhibition of 39–79% over 2-year field tests [fluorescens consistently inhibited the growth of the three weeds by 85 to 100% in the small-field studies. Field studies consistently showed a 50% reduction in downy brome, jointed goatgrass, and medusahead after three years of bacterial application. An almost complete suppression of these fall annual grass weeds was seen 5–7 years after a single application in fields containing desirable plants , perennial bunchgrasses, and native grasses).Many DRB also inhibit the growth of other monocotyledon weeds. P.  species . Some delication . P. fluol by 74% . DRB frot assays . An unidnjugatum . DRB isonjugatum . P. kilot length . The cellication . A consoectively . Li et at by 75% . P. fluo foxtail . P. fluondy loam . Kennedyld tests . Of the ld tests . Furtherfluorescens isolate LS102 and F. balustinum isolate LS105 inhibited the root growth of leafy spurge by 80 and 65%, respectively, in a hydroponic system [fluorescens LS102 and LS174 reduced the root weight and root carbohydrate content of leafy spurge by 20% [palmeri S. Wats.) strongly inhibited the plant’s seed germination, with Pseudomonas sp. strain TR10 reducing the seed germination rate to 5.26% and the seedling length by 92.85% in in vitro assays [fluorescens and A. delafieldii reduced the fresh and dry shoot weight of velvetleaf, while they showed no effects on the growth of corn [acuta, such as Xanthomoas sp, P. aeruginosa, P. fluorescens, B. subtilis, and B. cereus, significantly reduced the germination of seeds of S. acuta and inhibited the growth of roots [aeruginosa isolate KC1 from the rhizosphere of castor plants (R. communis) reduced the root and shoot length of seedlings of A. spinosus and P. oleracea without inhibitory effects on wheat [conyzoides in a soybean field strongly inhibited the germination of seeds and the growth of A. conyzoides [aeruginosa FS15, isolated from the soil adjacent to Chenopodium sp., completely inhibited the seed germination and root growth of C. arvensis and P. oleracea [Pseudomonas asplenii and P. syringae, significantly reduced the seedling dry weight and nitrogen fixation of white clover [fluorescens S61, showing the strong inhibition of Rhizobium leguminosarum, significantly inhibited the growth of white clover on a golf course [taylorae, isolated from weeds producing a high amount of indole acetic acid, inhibited the root growth of several species of weeds, including bindweed, velvetleaf, pigweed, green foxtail, and morning glory [fluorescens WSM3455 and WSM3456 and A. xylosoxidans WSM3457, isolated from weeds in a vineyard, specifically inhibited the growth of wild radish and caused some foliar symptoms, such as growth retardation, leaf chlorosis, and poor lateral root development [putida T42, P. fluorescens L9, P. fluorescens 7O0, P. aeruginosa O010, and P. alcaligenes W9, isolated from weed-infested wheat soil, significantly reduced the number of plants, root dry weight, seed production, and straw weight of broad-leaved dock, with maximum reductions of around 50%, while all the strains except strain O010 increased the wheat yield by 58~86% compared with the weedy control [conjugatum, A. conyzoides, Chromolaena odorata, and A. spinosus [conyzoides, the inhibition of seed germination and growth by isolates BL03 and BL07 was over 90%. B. flexus JMM24 isolated from the rhizosphere of L. aphaca, a common weed in mustard fields, caused a significant growth reduction in L. aphaca of up to 92%, while it increased the growth of mustard by up to 191% in terms of root dry weight and 119% in terms of shoot dry weight in pot assays [japonicum isolate GD3 and P. putida GD4 inhibited the root elongation of Ipomoea hederacea by about 26 and 90%, respectively, 7 days after inoculation [Leafy spurge is a serious problem in North America. Many DRB with phytotoxic activity have been isolated from the rhizosphere of leafy spurge . P. fluoc system . P. fluoe by 20% . Many DR of corn . DRB froof roots . The P. on wheat . The DRBnyzoides . P. aeruoleracea . Rhizobae clover . The DRBf course . The DRBng glory . The DRBelopment . P. puti control . Five unspinosus . For A. t assays . B. japoStriga sp. and Orobanche sp., infest many crops and cause significant losses in crop production. The DRB P. fluorescens strain QUBC3, isolated from the roots of Phalaris sp., strongly inhibited the radical growth of O. aegyptiaca and O. cernua [hermonthica, 15 P. fluorescens/P. putida isolates significantly inhibited the germination of S. hermonthica seeds and significantly reduced the emergence of shoots from treated maize [hermonthica-suppressive soil, belonging to the Bacillus, Streptomyces, and Rhizobium genera, caused seed decay in S. hermonthica [Certain parasitic weeds, such as . cernua . Of the ed maize . Severalmonthica .Vicia faba), the host of Orobanche sp., inhibited the seed germination of Orobanche sp. and the emergence of shoots. P. fluorescens strain Bf7-9, isolated from the rhizosphere of faba bean, positively influenced the growth of faba bean but reduced the pre-emergence of Orobanche crenata and Orobanche foetida by 71 and 81%, respectively, in root chamber assays. Soil drenching with Bf7-9 significantly reduced the number and dry weight of emerging shoots in O. crenata and O. foetida by 64 and 76% and 39 and 63%, respectively [Pseudonomuas marginalis strain Nc1-2 from the rhizosphere of faba bean promoted the growth of faba bean; meanwhile, it reduced the number emerging shoots in O. crenata [fluorescens also reduced the number of emerging shoots in O. crenata by 75% 2 months after soil treatment [subtilis and Bacillus pumilus reduced the number of emerging shoots and the fresh and dry weight of O. crenata 4 months after soil treatment [Bacillus circulans and Bacillus megaterium var. phosphaticum, isolated from the soil of a faba bean field, strongly reduced the germination and growth of O. crenata [megaterium var. phosphaticum with Rhizobium leguminosarum pv. viceae significantly reduced the seed germination of O. crenata. Faba bean bacterized with the combination of B. megaterium var. phosphaticum and Rhizobium leguminosarum pv. viceae reduced significantly the shoot emergence and dry weight of O. crenata in a field test [Bacillus velezensis JTB8-2, isolated from the rhizosphere soil of Egyptian O. aegyptiaca, promoted the growth and yield of tomato at a high application rate (3.2 L/105 m2), while it significantly reduced the number of emerging shoots, fresh weight, and biomass of O. aegyptiaca in pot and field experiments and the parasitic rate in the field [Some PGPR also show herbicidal activity against weeds. Bacteria associated with faba bean  suppressed little-seed canary grass, which improved wheat grain yield losses by 20.1 to 66.9% in pot assays and 34.3 to 64.3% in the field trial [0, and O010, were characterized as P. fluorescens, P. putida, P. aeruginosa, and P. fluorescens, respectively [Enterobacter sp. strain I-3, isolated from the soil of an agricultural field, significantly inhibited the seed germination and growth of Cyperus microiria. The culture of strain I-3 also reduced the seed germination of D. sanguinalis [Allelopathy was defined by Rice  as “the rthenium . Treatmedactylon . Abbas edactylon . Among tld trial . The fouectively . Enterobguinalis .Klebsiella pneumoniae strain YNA12 from evening primrose strongly inhibited seed germination in evening primrose and reduced the seedling length and biomass [Pseudomonas viridiflava strain CDTRc14, an endophyte isolated from Lepidium draba in a vineyard, inhibited the seed germination of L. draba and reduced the biomass and the root length of L. draba seedlings [Pseudomonas brassicacearum YC5480 from Artemisia sp. showed phytotoxicity to some monocotyl and dicotyl plants [conyzoides, Bidens biternata, and P. hysterophorus, with symptoms such as leaf curling, wilting, and burning, while some reduced the root and shoot growth [Endophytic bacteria have been isolated from diverse plants and show beneficial effects on the growth of host plants and the tolerance of host plants to biotic and abiotic stress. However, some endophytic bacteria show phytotoxic activity or produce herbicidal secondary metabolites. The endophytic bacterium  biomass . Pseudomeedlings ,124. A bl plants . Severalt growth .Serratia plymuthica A153 isolated from the roots of winter wheat selectively suppressed the growth of many species of dicotyledon weeds and crops but not the monocotyledon species of Poaceae [plymuthica A153 suppressed a range of weeds, such as C. album, Stellaria media, Polygonum convolvulus, and Galeopsis speciosa, etc. [Azospirillum brasilense L2 and L4, nitrogen-fixing bacteria isolated from the soil of a sorghum field, completely inhibited the seed germination of S. hermonthica, while strain L4 promoted the growth of sorghum [campestris pv. retroflexus, isolated from redroot pigweed, strongly inhibited the growth of Capsella bursa-pastoris, P. oleracea, and Amaranthus retroflexus, even at 30-times dilution, with reduction in shoot length and root length ranging from 52.6 to 74.8% and 72.1 to 83.6%, respectively [In this section, PAB that cannot be assigned to the above classifications are discussed.  Poaceae . In a fisa, etc. , but thi sorghum . The celectively .The possible modes of action of herbicidal PAB include the following mechanisms: the production of phytotoxic metabolites, the production of exopolysaccharides, the overproduction of auxins, and other unclear modes.syringae pv. tagetis produces tagetitoxins, which disrupt the biogenesis of chloroplasts, resulting in the chlorosis of newly developed leaves in treated plants [fluorescens WH6 produced 4-formylamino- oxyvinylglycine (FVG), an herbicidal germination-arrest factor (GAF) that irreversibly inhibited the seed germination of grassy weeds, such as annual bluegrass, without significantly affecting the growth of established seedlings and mature plants of annual bluegrass or the seed germination of broadleaf plant species [fluorescens, namely Pseudomonas synxantha, Pseudomonas chlororaphis, and Pantoea ananatis [fluorescens strain D7 was related to the production of a phytotoxic metabolite; at a concentration of 8%, the cell-free filtrate of strain D7 reduced the root elongation of downy brome by about 50% [aeruginosa H6 was attributed to the production of quinoline and its derivatives [fluorescens BRG100, strongly inhibited the root elongation of green foxtail by 67% [velezensis JTB8-2 inhibited the seed germination of O. aegyptiaca at 0.5~4.0 mM [campestris pv. retroflexus that contributed to its toxicity to A. retroflexus were cyclo-(Pro-Phe) and six organic acids [aeruginosa C1501, caused a large area of necrosis on the leaves of Amaranthus hybridus but was safe for sorghum [syringae strain 3366 controlled corn spurrey (Spergula arvensis L.) and fireweed (Epilobium angustifolium L.) seedlings at 10 ppm and reduced the root and shoot growth of cuttings of the perennial weeds birdsfoot trefoil (Lotus corniculatus L.) and silverleaf (Potentilla pacifica Howell) at 103 ppm [syrinagae strain 3366 included phenazine-1-carboxylic acid (PCA), 2-amino phenoxazone, and 2-amino phenol, of which PCA was the major phytotoxic metabolite. PCA inhibited downy brome root growth by 99% at 5.7 mg L−1 [putida GD4 was attributed to its production of phytotoxins [aeruginosa CB-4 strongly inhibited radicula and coleoptile elongation [hybridus and E. crus-galli caused by the cell-free filtrate of P. aeruginosa strain B2 isolated from the rhizosphere of wheat indicated the production of phytotoxic metabolites by the strain, and the isolated active compound completely inhibited the seed germination and seedling growth of A. hybridus and E. crus-galli at concentrations of 10 and 15 mg/L, respectively [The phytotoxicity of weed bacterial pathogens might be caused by the production of phytotoxins. P. d plants . Many DRd plants ,132. P.  species ,133,134.ananatis . The inhbout 50% , and thebout 50% . The actbout 50% . The herivatives . Pseudopl by 67% ,141. The5~4.0 mM . The phyic acids , which fic acids . 2-Hydro sorghum . The phy 103 ppm . The phy7 mg L−1 . These ptotoxins . The culongation . Leaf neectively .Bacillus clausii isolated from Dracocephalum tanguticum showed herbicidal activity against P. annua at 32 ppm [aphaca caused by the deleterious rhizobacteria of Brassica juncea could be attributed to the production of 5-aminoleveulinic acid [cereus 57-02, produced an unidentified phytotoxin, causing rot in the rootlets of lettuce seedlings at 57 mg/L [Lipopeptides from t 32 ppm . The gro 57 mg/L .fluorescens S241 increased the cyanide content in treated soil to inhibit the growth of bean and lettuce [Sida acuta by rhizobacteria could be attributed to the production of cyanide [aeruginosa PAO1 in the volatile-mediated killing of Arabidopsis thaliana [aeruginosa PAO1 resulted in a strong reduction in volatile-mediated phytotoxicity. The deleterious pseudomonads from the rhizosphere of weeds in an alfalfa field also produced hydrogen cyanide, which could explain its inhibition of the root growth of the weeds Axonopus affins (Chase) and Lens esculenta (Moench) [Cyanide has been associated with the phytotoxicity of many rhizobacteria against weeds. P.  lettuce . The inh cyanide . The stuthaliana . The dim(Moench) .Euphorbia spp. produced exopolysaccharides that significantly reduced the growth of leafy spurge calluses [Some rhizobacteria isolated form calluses . Althougtaylorae produced a high amount of auxins (72 mg/L IAA) and inhibited the root growth of field bindweed (C. arvensis) by 90.5% [japonicum might be related to its high production of IAA [Enterobacter sp. I-3 and the precursor of IAA significantly decreased leaf length, leaf width, and root length and increased the number of lateral roots in lettuce assays [Klebsiella pneumoniae YNA12 was partially attributed to its high production of IAA [Pseudomonas sp. [An appropriate concentration of auxins promotes the growth of plants, while a high concentration of auxins inhibits plant growth. PAB suppressed the growth of weeds through the high production of auxins, especially indole-3-acetic acid (IAA). E. by 90.5% . The inhn of IAA . The come assays . The inhn of IAA . The higonas sp. .Curtobacterium sp. MA01 suppressed the growth of petunia plants by causing oxidative stress and decreasing the amount of antioxidants. Curtobacterium sp. MA01 also decreased protein synthesis in the infected petunia plants [Enterobacter sp. I-3 controlled plant growth by inhibiting the gibberellin biosynthetic pathway and regulating abscisic acid and amino acid synthesis [putida NRBIC19 reduced the allelopathy between P. hysterophorus and neighboring plants by altering the soil microbial diversity nearby [SucB gene in strain NBRIC19 participated in the degradation of the toxic allelochemicals produced by P. hysterophorus [putida GD4 on the root surface of morning glory was about 100 times higher than that of the other investigated DRB, B. japonicum GD3, while both strains reduced the growth of morning glory, by 90 and 26%, respectively [a plants . Enterobynthesis . Certainynthesis . P. putiy nearby . The Sucrophorus . The supectively .fluorescens strains ACK55 and D7 inhibited the seed germination of B. tectorum in petri dish assays, but they showed no effects on B. tectorum during growth-chamber plant-soil assays and field tests [Up to now, very few PAB have been successfully developed as bioherbicides, but there are many reports of their unstable performance or even their lack of any effect in the field ,161,162.ld tests ,161. ThePseudomonas spp. and Xanthomonas spp. have a very wide range of hosts. P. syringae pv. tagetis is a common pathogen of sunflower, artichoke, and other crops. Ralstonia solanacearum is a common pathogen of Solanaceae plants, such as tomato, potato, pepper, and tobacco, etc. For the development of successful bioherbicides with bacterial pathogens of weeds, the host specificity is a key issue. Alongside the bacterial pathogens of weeds, all the other microbes must be evaluated for their host specificity. Microbes with a wide host range, especially those with pathogenicity to crops or desirable plants, must be avoided, or their application must be limited under certain ecological conditions.The host range of a weed-suppressive PAB must be studied before field assessments. Many bacterial pathogens not only infect unwanted weeds in the field, but also crops, especially crops in the same family as the targeted weeds. For example, syringae pv. tagetis isolated from common ragweed only infected ragweed, while other P. syringae pv. tagetis isolates from marigold, sunflowers, and Jerusalem artichoke caused leaf spots and apical chlorosis [annua) responded differently to treatment with X. campestris pv. poae at a concentration of 109 cfu mL−1. A single leaf-tip treatment controlled 80% of annual P. annua but only 60% of perennial P. annua [annua. In the screening for herbicidal DRB from crops and weeds, attention is always paid to the killing or growth inhibition effects in weeds. Some DRB also inhibit the growth of crops [hysterophorus inhibited the root growth of P. hysterophorus in in vitro assays and under glasshouse conditions, they also decreased the root growth, root dry mass, and shoot length of crop plants [Bacterial pathogens isolated from different weed hosts are indistinguishable by conventional biochemical tests, but they might show specificity to a certain host or to a broad spectrum of hosts. For example, when applied through spraying, the strains of P. hlorosis . DiffereP. annua . This miof crops . Althougp plants . Certainp plants . The saftrivialisX33d, showed greater suppression of the growth of B. diandrus and higher cell viability than talc–kaolin powder [putida and Acidovorax delafieldii with alginate maintained large populations of bacteria and produced a high concentration of cyanide in the soil, which reduced velvetleaf growth [fluorescens BRG100. Drying the pesta to 0.3 aw stabilized the population of BRG100 at 8.5 log10 cfu g−1 for 16 months. The starch types in the pesta formulation of P. fluorescens BRG100 also affected the disintegration of the pesta, with pea starch causing the fastest degradation [fluorescens and its colonization of plants was improved by encapsulating the bacterium in alginated beads by incorporating skim milk or skim milk with betonite clay [The formulation type affects the performance of herbicidal PAB in the field. A semolina–kaolin Pesta granules of P. n powder . The forf growth . Water aradation . The surite clay .syringae pv. tagetis suspension increased the infection of Canada thistle and common ragweed by P. syringae pv. tagetis, causing 100% disease incidence and greater disease severity [syringae pv. phaseolicola culture at 0.2% increased the infection of Kudzu leaves [Xanthomonas sp. LVA-987 [v/v) or Silwet 408 (0.2% v/v) to a P. syringae pv. tagetis (5 × 108 cfu mL−1) suspension resulted in 100% disease incidence and greater disease severity in Canada thistle [syringae pv. tagetis increased the population of P. syringae pv. tagetis in Canada thistle leaves and significantly reduced the shoot dry weight [Surfactants influence the infection of weeds by pathogenic bacteria. The nonionic organosilicone surfactant Silwet L-77 facilitated stomatal penetration by aqueous suspensions. The addition of 0.1% Silwet L-77 to a P. severity . The addu leaves . Silwet  LVA-987 . The add thistle . The addy weight .Bacillus lentus A05 and P. aeruginosa A08. They demonstrated that talc, bentonit, kaolin, and ground burned-rice husk were effective carriers for both bacteria, supporting populations of B. lentus A05 and P. aeruginosa A08 for up to 20 weeks and maintaining the stability of the two DRB’s biocontrol efficacy against A. conyzoides [fluorescens G2-11 in inhibiting the germination of seeds and the root growth of S. viridis [fluorescens BRG100 incorporating oat flour and 20% maltose increased the survival of bacteria and prolonged the shelf life of the product from 3 weeks to 32 weeks [trivialis X33d increased the stability of bacterial cells in the formulation [The carriers in a formulation also affect the performance of PAB-based bioherbicides. Rakian et al. evaluated the effects of using talc, bentonit, kaolin, and ground burnt rice husk as carriers on the shelf life of two DRB, nyzoides . The inc viridis . A pesta32 weeks . The addmulation .putida strain B1-7, P. fluorescens strain L2-19, and S. maltophilia strain TFR1 significantly inhibited green foxtail shoot growth in organically managed soil, which indicated that organic content is important for the growth-suppressive activity of DRB [fluorescens G2-11 significantly inhibited the seed germination and plant growth of S. viridis in fine sandy loam [syringae pv. phaseolicola to control kudzu might be late spring [syringae pv. tagetis to control Canada thistle for maximum disease incidence and disease severity, which might have been related to the precipitation levels during this time [fluorescens strain D7 showed greater suppression of the growth of downy brome in cool or moist conditions, which favor the seed germination and establishment of downy brome [syringae pv. tagetis to control A. grayi was found to be noon [campestris pv. poae JT-P482 to control annual bluegrass was determined to be fall or winter; whereas treatment in October and February through to April produced acceptable levels of fresh-weight loss the following spring, and treatment in October, November, and February reduced seedheads by more than 75%, April treatment did not prevent the spread of seeds [campestris pv. poae JT-P482 to effectively control annual bluegrass in glasshouse assays through heavy wilting or plant death [Environmental factors such as the temperature, humidity, and soil properties affect the performance and survival of weed-suppressive PAB. Soil properties, such as the soil type, organic matter content, and pH, affect the performance of DRB in the suppression of weed growth. P. y of DRB . P. fluondy loam . Temperae spring . Mid-Julhis time , and sucny brome . The opt be noon . The besof seeds . Temperant death .putida strain FH160, S. maltophilia strain FH131, and E. taylorae strain FH650 showed a more consistent colonization of the rhizosphere of downy brome through soil incorporation [syringae pv. tagetis caused 71% apical chlorosis in Canada thistle, while this figure was only 18–51% for other application methods [campestris pv. poae JT-P482, and shortening the interval between cutting and the application of JT-P482 increased the efficacy [Brassica and sweat clover, reduced the weed biomass by 90% in soybean fields and achieved a soybean yield that was higher than that of the weedy control and equivalent to or higher than that of conventionally grown soy [syringae pv. tagetis to small Canada thistle seedlings increased the efficacy of the treatment [fluorescens at limiting the spread of P. annua on football pitches [The application method also affects the weed-suppressive performance of herbicidal bacteria. In a comparison of different application methods, P. poration , but theporation . When ap methods . Backpacefficacy . The apprown soy . In genereatment ,59. Mowi pitches .fluorescens and Acidovorax delafieldii, it increased the production of HCN in root-free soil [−5 M L-tryptophan with E. taylorae increased the inhibition of the root growth of several species of weeds [syringae pv. tabaci used in biological weed control increased the survival of the bacteria; sucrose alone enhanced survival more than oil alone, but the beneficial effect of sucrose was reduced when it was combined with oil [The combination of certain simple chemicals with herbicidal PAB can potentiate their herbicidal activities. Although glycine alone had no effect on the growth of velvetleaf, when it was applied in combination with the cyanogenic rhizobacteria P. ree soil . The comof weeds . The addwith oil .Bacillus sp. isolates and Ochrobactrum quorumnocens RPTAtOch1, thwarted the control of angled onion with Pectobacterium carotovorum subsp. carotovorum because of its antagonism against P. carotovoraum subsp. carotovorum [Endophytes in weeds might counteract the efficacy of microbial weed-control agents. Some endophytic bacteria, such as otovorum .campestris pv. poae and P. fluorescens strains D7 and ACK55, have been developed and registered as bioherbicides [In this review, we focused on herbicidal PAB, considering their screening, diversity, modes of action, and performance. PAB are widely present in the soil adjacent to plants and inside plants (weeds and crops). A rapid screening process is a prerequisite for selecting highly effective herbicidal bacteria from the broad spectrum of PAB. Some herbicidal PAB, such as X. rbicides ,16,17. Trbicides ,161,162.pneumoniae and P. aeruginosa, are human pathogens. Although such human pathogens with herbicidal activities would not be approved as bioherbicides because of the strict registration regulations, it would be better to exclude them in the early stages of bioherbicide development. The application of herbicidal PAB with wide host ranges or crop pathogenicity, such as P. syringae and R. solanacearum, must be avoided or limited to specific areas to reduce their adverse effects on ecosystem balance and crop production. The application of herbicidal PAB for weed control might affect soil microbial communities, and more studies are required to clarify their effects on the entire soil microbiome of weeds and crops.In the development of bioherbicides based on PAB, we should consider their effects on crops and the environment and their safety for humans, as some herbicidal bacteria, such as K. As the fields of microbiology, multi-omics, and next-generation sequencing progress, more PAB will be found. Abundant PAB from crop and weed microbiomes have been isolated and characterized according to their herbicidal activities, especially those from the rhizosphere . As we k


Ratoon sugarcane is susceptible to chlorosis, characterized by chlorophyll loss, poor growth, and a multitude of nutritional deficiency mainly occurring at young stage. Chlorosis would significantly reduce the cane production. The molecular mechanism underlying this phenomenon remains unknown. We analyzed the transcriptome and metabolome of chlorotic and non-chlorotic sugarcane leaves of the same age from the same field to gain molecular insights into this phenomenon.The agronomic traits, such as plant height and the number of leaf, stalk node, and tillers declined in chlorotic sugarcane. Chlorotic leaves had substantially lower chlorophyll content than green leaves. A total of 11,776 differentially expressed genes (DEGs) were discovered in transcriptome analysis. In the KEGG enriched chlorophyll metabolism pathway, sixteen DEGs were found, eleven of which were down-regulated. Two photosynthesis pathways were also enriched with 32 genes downregulated and four genes up-regulated. Among the 81 enriched GO biological processes, there were four categories related to metal ion homeostasis and three related to metal ion transport. Approximately 400 metabolites were identified in metabolome analysis. The thirteen differentially expressed metabolites (DEMs) were all found down-regulated. The phenylpropanoid biosynthesis pathway was enriched in DEGs and DEMs, indicating a potentially vital role for phenylpropanoids in chlorosis.Chlorophyll production, metal ion metabolism, photosynthesis, and some metabolites in the phenylpropanoid biosynthesis pathway were considerably altered in chlorotic ratoon sugarcane leaves. Our finding revealed the relation between chlorosis and these pathways, which will help expand our mechanistic understanding of ratoon sugarcane chlorosis.The online version contains supplementary material available at 10.1186/s12870-022-03588-8. Sugarcane is a major food and energy crop globally. In China, sugarcane accounts for 90% of sugar production, contributing 6 to 8 billion RMB annually . In GuanAreca catechu (Arecaceae) [Areca catechu and cucumber [In plants, leaf chlorophyll loss causes chlorosis, and it could be due to accelerating chlorophyll catabolism or reduced chlorophyll production, or by both. Chlorophyll metabolism involves a series of enzymes, including HemA, HemB, chlH, chlM, por, and NOL . Iron deecaceae) . Low expecaceae) . Chloropecaceae) , 8. Low cucumber , 9. Chlocucumber , 11. Irocucumber , and itscucumber . Previoucucumber –16. Excecucumber , 3. Besicucumber . MechaniThe advanced omics technologies enable a comprehensive molecular analysis of complex biological problems such as pathogenesis and nutritional disorders . TranscrOur study aimed to describe the global response of sugarcane to ratoon chlorosis through transcriptome and metabolome analyses. Genes and metabolites related to chlorophyll metabolism, photosynthesis, metal ion homeostasis, and phenylopropanoid biosynthesis were analyzed. The findings of this present a new perspective of molecular regulatory mechanisms of chlorosis occurring in ratoon sugarcane.A locally adapted commercial sugarcane cultivar, GuiTang49 (GT49) (ROC22 x Ganzhe 14), developed by Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences, China . On ApriThe leaf samples from chlorosis with SPAD chlorophyll meter  reading less than ten were classified as chlorotic and used for further experiments. In contrast, leaf samples from the control group with a reading higher than 40 were grouped and used as control. Approximately 0.1 g of leaves were rinsed and ground in liquid nitrogen, and chlorophyll was extracted with 80% acetone for 24 h in dark. The content of chlorophyll was determined using a spectrophotometer [Five leaves were included in one sample, and each group contained five samples (5 replicates). The total RNA was extracted using Trizol  according to the manufacturer’s instructions. RNA quality was assessed by Agilent 2100 Bioanalyzer  and agarose gel electrophoresis. The mRNA, enriched by Oligo (dT) beads, was fragmented and reverse transcribed into cDNA first-strand with random primers. After synthesizing second-strand cDNA, the total cDNA was purified with a QiaQuick PCR extraction kit . Sequencing adapters were ligated to the cDNA. Size selection and PCR amplification were performed before sequencing. The sequencing was operated using Illumina HiSeq 2500 .p-value ≤ 0.05 were considered as DEGs . GO (gene ontology) and KEGG enrichment analysis was performed to depict the gene function and biological pathway of DEGs [Reads obtained from the sequencer were filtered before assembly using fastp . The hig of DEGs , 31.p-value ≤ 0.05 were considered to be significantly different between the chlorosis and control groups [The freeze-dry leaf samples for metabolome analysis were ground using a mixer mill . 100 mg of leaf powder was extracted overnight with 1.0 ml of aqueous methanol containing 0.1 mg/L lidocaine as internal standard. The supernatant was obtained by centrifugation and filtration. The metabolite compounds were analyzed by LC-ESI-MS/MS system . The chromatographic separations were performed using a Waters ACQUITY C18 column  under a flow rate of 0.4 ml/min at 40 °C. The mobile phase was water (0.04% acetic acid) as Phase A and acidified acetonitrile (0.04% acetic acid) as Phase B. The separation was run in a gradient condition: 95:5 Phase A/Phase B for the first 10 min, 5:95 Phase A/Phase B for the 11th to 12th min, 95:5 Phase A/Phase B for the 13th to 15th min. The effluent was detected with Sciex Triple Quad 6500 mass spectrometer  in a positive ion mode. The quantification of metabolites was performed according to multiple reaction monitoring methods . Analystl groups .ACAD gene was used as a reference gene to normalize the relative expression levels [The RNA used for transcriptome sequencing was also employed for qRT-PCR validation. The total RNA was digested by DNase, and reverse-transcribed into cDNA for qRT-PCR using PrimeScript RT reagent kit . The n levels . The qRTn levels . A totaln levels .The ratoon plantlets displayed distinct chlorosis symptoms compared with the green plants found in the same field at the same time Fig. A. The SPZea mays, Setaria italica, and Oryza sativa Japonica. 5745 unigenes matched the Nr database entries only, while KEGG, Swissprot, and COG had 218, 176, and 35 unigenes, respectively, and they were single matched  and 5484 (11.36%) were from Setaria italica and Oryza sativa Japonica Group, respectively , we identified a total of 11,776 differentially expressed genes (DEG) between chlorosis and control groups . The results showed the percentage of conserved orthologues of plants represented in our assembled sugarcane transcriptome. Completeness of all the unigenes resulted in a high rate of complete plant orthologues (75%). Of the 1440 orthologues searched in the BUSCO set of plants, 75% was complete, with the number being 1080. Of the 1080 complete BUSCO orthologues, 1060 were single-copy, while the other 20 orthologues were duplicates. The proportion of fragmented and missing BUSCOs was 14 and 11%, respectively  analysis was employed to emphasize the function of DEGs. GO enrichment analysis revealed that DEGs were most enriched in GO cell component categories of membrane, thylakoid and intrinsic component of membrane Fig. . In molep-value = 0.0224, Table SThe KEGG analysis indicated that DEGs involved in the chlorophyll metabolism pathway were enriched , Unidentified gene (unigene0019302), NHX2 (unigene0040569), DTX42 (unigene0056614), HMA1 (unigene0064621) and HCC1 (unigene0083098). Three genes, including TMN6 (unigene0036726), YS1 (unigene0055240) and IRO2 (unigene0085997), were only found in processes related to metal ion homeostasis.Among the 81 enriched GO biological processes, there were four categories related to metal ion homeostasis and three categories related to metal ion transport Fig. . In the p-value < 0.05 were classified as significantly differentially expressed metabolites (DEMs) between the chlorosis and control groups. All these metabolites, including six phenolic acids, three lipids, one alkaloid, one organic acid and one other compound, were down-regulated in the chlorosis group . The three compounds that belonged to the phenylpropanoid biosynthesis were coniferyl alcohol, ferulic acid and 5-O-caffeoyl shikimic acid.To determine the differences of metabolites in ratoon chlorosis sugarcane, we performed metabolome analysis using LC-ESI-MS/MS system. A total of 598 compounds were identified and quantified -PCR assays. The primary expression trend of the 20 genes was consistent Fig. A. The vaRatooning is a cultivation method in sugarcane production . Ratoon Chlorophyll is the primary source of pigment in plant leaves, which is correlated with the green color . The welA. thaliana, photosynthetic efficiency reduction was accompanied by a decrease in chlorophyll content [Leaf photosynthesis is positively correlated with chlorophyll content . In A. t content . In whea content . Transcr content . Likewis content . Lack of content . In chlo content . DEGs of content .Studies of ratoon sugarcane chlorosis in Guangxi suggest that the excessive Mn accumulation in parent stalks was the main reason for chlorosis . At the A. catechu L. [Arabidopsis [BGLU, a beta-glucosidase coding gene, was down-regulated in chlorosis sugarcane (Fig. In a previous study, the phenylpropanoid biosynthesis pathway was enriched in both transcriptome and metabolome analysis of chlorosis techu L. . Combinitechu L. . In chlotechu L. . In an itechu L. . Synthesbidopsis . We notiane Fig. . This reane Fig. .Here we studied transcriptome and metabolome responses of chlorotic and non-chlorotic sugarcane leaves. Our results provide more insights into chlorophyll synthesis, photosynthesis, metal ion metabolism, and phenylpropanoids biosynthesis in ratoon chlorosis sugarcane. Gene regulation dysfunction in metal ion homeostasis and transportation likely leads to Mn toxicity and Fe deficiency, which may cause ratoon chlorosis in sugarcane . The redAdditional file 1: Table S1. The primers for validation of transcriptome. The table included the twenty genes that selected for validation of transcriptome. The parameter of each primer, which included primer sequence, melting temperature, product length and so, were also listed. The FPKM value, GO and KEGG annotation of each gene was indicated.Additional file 2: Table S2. The expression and annotation of all the differential expression unigenes. This table included 11,776 unigenes. The read count, FPKM value, foldchange, KEGG and GO annotation were indicated.Additional file 3: Table S3. The GO enrichment analysis results of the differential expression unigenes.Additional file 4: Table S4. The KEGG pathway enrichment analysis results of the differential expression unigenes.Additional file 5: Table S5. The differential expression genes of Porphyrin and chlorophyll metabolism pathway.Additional file 6: Table S6. The differential expression genes of Photosynthesis and Photosynthesis - antenna proteins pathways.Additional file 7: Table S7. The differential expression genes related to metal ion homeostasis and transport process.Additional file 8: Table S8. The 598 metabolites identified in metabolome. The table included the quantification value, mass spectrum character, and compound identification information of each metabolites.Additional file 9: Table S9. The differential expression genes of Phenylpropanoid biosynthesis pathways.


Viola grypoceras via de novo assembly of Illumina data. The complete circular chloroplast genome is 158,357 bp long and contains four parts: a large single-copy (LSC) region of 86,764 bp, a small single-copy (SSC) region of 17,345 bp, and two inverted-repeat regions (IRa and IRb) of 27,124 bp each. Genome annotation predicted that this genome harbors 111 genes, comprising 77 protein-coding genes, 30 transfer RNA (tRNA) genes, and four ribosomal RNA (rRNA) genes. Phylogenetic analysis demonstrated that V. grypoceras shares a close systematic relationship with V. mirabilis and V. websteri by forming a basal clade in the genus Viola.We constructed and characterized the chloroplast genome of  Viola grypoceras Gray V. grypoceras plants have aerial stems and flowers with purplish to white petals with purple stripes. Plastid genomic information is available for only a few Viola species with aerial stems .The violet V. grypoceras. Fresh leaf samples were collected for DNA extraction on the banks of Gangjeong-cheon stream in Jeju Island, South Korea . A dried plant specimen was deposited in the Herbarium of the Nakdonggang National Institute of Biological Resources (NNH) under voucher number NNIBRVP90496 . Total genomic DNA was extracted from the leaf tissue using a DNeasy Plant Mini Kit . The isolated genomic DNA was used to construct a paired-end library (PE) with a mean insert size of 500 bp by Theragen Bio , followed by sequencing on an Illumina HiSeq 2500 platform. A total of 25.37 Gb of 150-bp PE reads were obtained by Illumina HiSeq NGS and assembled using CLC Genomics Workbench   of 27,124 bp each. We annotated 111 genes in the V. grypoceras chloroplast genome, which consists of 77 protein-coding genes, 30 transfer RNA (tRNA) genes, and four ribosomal RNA (rRNA) genes. The V. grypoceras chloroplast genome contains 20 introns, with one group I intron and 19 group II introns. The group I intron is present in the gene trnL-UAA  in algae (Jeong et al. Viola species. We inferred the evolutionary history of these species using the maximum-likelihood method and General Time Reversible model (Nei and Kumar We compared seven genes (Viola species formed three major clades in the phylogenetic tree, with 99–100% bootstrap support, using Passiflora miniata of the Passifloraceae and Salix bicolor of the Salicaceae as the outgroups (V. grypoceras belongs to the basal-most clade in the tree, along with V. mirabilis and V. websteri. Seven Viola species without aerial stems form a derived clade. Two caulescent species, V. raddeana and V. verecunda, were placed between the two clades. As shown in Viola species with aerial stems were placed in basal lineages of the genus Viola. The phylogenetic tree places V. grypoceras close to V. mirabilis. Further investigation of the caulescent Viola taxa at the interspecies and intraspecies levels would further our understanding of the relationships of Viola species.Twelve utgroups . V. gryp


These trials were also all scanned in an independent sample of functional MRI participants (n = 10), each with sufficient data to precisely define within-individual networks. Stable latent factors varied across trials and correlated with trial-level functional responses selectively across networks. One network linked to parahippocampal cortex, labeled Default Network A (DN-A), tracked scene construction, including for control trials that possessed minimal episodic memory demands. To the degree, a trial encouraged participants to construct a mental scene with imagery and awareness about spatial locations of objects or places, the response in DN-A increased. The juxtaposed Default Network B (DN-B) showed no such response but varied in relation to social processing demands. Another adjacent network, labeled Frontoparietal Network B (FPN-B), robustly correlated with trial difficulty. These results support that DN-A and DN-B are specialized networks differentially supporting information processing within spatial and social domains. Both networks are dissociable from a closely juxtaposed domain-general control network that tracks cognitive effort.Multiple large-scale networks populate human association cortex. Here, we explored the functional properties of these networks by exploiting trial-to-trial variation in component-processing demands. In two behavioral studies (NEW & NOTEWORTHY Tasks shown to differentially recruit parallel association networks are multifaceted, leaving open questions about network processes. Here, examining trial-to-trial network response properties in relation to trial traits reveals new insights into network functions. In particular, processes linked to scene construction selectively recruit a distributed network with links to parahippocampal and retrosplenial cortices, including during trials designed not to rely on the personal past. Adjacent networks show distinct patterns, providing novel evidence of functional specialization. Diverse higher-order functions, including autobiographical memory, spatial navigation, and social inference, have been attributed to a large monolithic network known as the default network DN; –7. This One challenge for understanding processes supported by the DN is that most prior studies rely on group-averaged data, which necessarily blurs anatomical details . Evidence from prior studies supports that assessing how people respond to complex stimuli can provide insight into processing demands . Participants could proceed after responding. After 10 s, they received a reminder to select a response. After answering the question, the participants were then presented with the response strategies scale RSS;  and aske1) spent less than 20 min on the survey, 2) reported being outside of our age range (18–28 yr old), 3) had no mouse clicks registered for multiple trial responses  showed clear stereotyped patterns of responding across trials, or 5) missed more than one check question. Participants were also flagged if they did not comment or write “None” (as requested) in a final feedback box, or if they missed any check questions . A single flag  did not result in exclusion if no patterned responding or other flags were noted.As is often observed in online experimentation, participant compliance varied. A series of quality control (QC) criteria were adopted to conservatively include only participants who fully engaged the task. The criteria were applied before any analysis of factors or assessments of reliability. Participants were excluded if they Assessment of response patterns was particularly vital to quality control. Thirteen strategy probe ratings for 11 trials (7 that appeared across multiple surveys and 4 unique to a survey) were visualized for each participant by two independent experimenters (LMD and OIA). Strategies were unlabeled during visualization to prevent experimenter bias. A participant was flagged for exclusion if strategy ratings did not differ across or within each trial, or if trials became uniform near the end of a survey. For example, a subset of participants chose a single value for all strategies within each trial , a subset selected values in a clearly stereotyped pattern , and a subset showed evidence of a drop off in performance . For participants flagged due to stereotyped responses, all trials were visualized as an additional check.F = 0.36, P = 0.90), supporting that a trial’s strategy pattern represented stable properties, rather than idiosyncratic aspects of the rating group. As the replication experiment will reveal, these conservative procedures yielded highly stable estimates of strategy use across independent groups of participants.As a final quality control check, ratings across strategies for the five trials consistently included across all participants were visualized to explore cohort differences. Strategy ratings for each trial showed similar patterns. A linear model testing for rating differences found no significant cohort effects  were recruited from the Boston area to complete 4 neuroimaging sessions each. All participants provided informed consent online; study protocols were approved by the Harvard University IRB. Each neuroimaging session featured a battery of tasks, including fixation and the episodic projection task. Full details of task acquisition and preprocessing parameters are provided in DiNicola et al. . Only paEach participant completed 11 runs of a passive fixation task, for intrinsic functional connectivity analysis , as well as 6 runs of an episodic projection task . Additional tasks were included in the neuroimaging battery, not discussed here  other task analyses and without examination of any of the trial-variation effects explored in this paper.During each run of the episodic projection task, participants responded to trials varying in self-relevance (Self vs. Non-Self) and temporal orientation . Crossing of these two dimensions (2 × 3) yielded 6 target conditions of 30 trials each . During each scan session, a rapid T1-weighted structural image was acquired, using a multi-echo magnetization prepared rapid acquisition gradient echo  were regressed from the T1-aligned BOLD fixation data. These data were then bandpass filtered at 0.01–0.10 Hz  maximum absolute motion greater than 1.8 mm, 2) slice-based signal-to-noise ratio less than or equal to 135 (as in Ref. 3) eyes closed during skipped task trials. Sixteen fixation runs were excluded across the participants, and one run was included for subject 6, in error, despite maximum motion higher than 1.8 mm. No episodic projection task runs were excluded . Networks were identified within the whole brain k-means outputs based on referential features  analyses were conducted on fixation data, within each individual, to precisely estimate whole brain network organization. a et al. . Briefly MATLAB v15b. Netwdividual . These ndividual . The netFor the episodic projection task, run-specific GLMs were created featuring separate regressors for every trial , after estimating average values for each network within individuals, we averaged trial values across individuals, producing a single network estimate for every unique trial. In this way, within-individual network definition allowed for estimates of network activity that were fully constructed within the idiosyncratic anatomy of each individual. At the same time, stable functional MRI estimates for each of the 180 individual trials were obtained because each trial estimate was the average across the 10 participants’ individualized networks.cor.test from stats v4.1.1 in R). Data from all trials were included, and Pearson’s correlation values were plotted to visualize patterns for each composite.Each of the 180 trials from our episodic projection task was paired with five mean strategy composite scores (from the online data) and six mean network activity values (from the neuroimaging data). We asked whether variation in our behavioral composite scores, across trials, related to variation in network activity. As a first step, we calculated correlations between trial-level behavioral composite scores and mean functional MRI BOLD response estimates from each of the six networks  for each composite was also calculated . A maximum explainable variance value was estimated as the product of the reliability, which we compared to our estimated R2 values.Regression models thus provided insight into how much variance in network activity was captured by our composite scores. For the strongest observed correlations, we also sought to contextualize these values as a percent of the explainable variance, the highest As will emerge in the results, initial regression models revealed evidence of intercorrelation between a composite score reflecting Difficulty and other composite scores. In post hoc analyses, we directly tested whether Difficulty impacted other composite-network relations by regressing the impact of Difficulty ratings from all other composite scores and plotting residual composite-network correlations.Finally, the analyses supported a double-dissociation between composite scores reflecting Scene Construction and Difficulty in relation to activity in brain networks DN-A and FPN-B. As a final stringent test of this discovery, we used the composite scores to create contrast maps based on trials with high and low values on each of these composites. For Difficulty, we identified the 10 trials with the highest and 10 trials with the lowest composite scores. For each individual, we then created a whole brain contrast map and overlaid the border of the individual’s FPN-B, to compare each individual’s contrast map to their specific FPN-B estimate. For Scene Construction, we used the same process, but only included trials originally considered controls . We selected the 10 control trials with the highest and 10 control trials with the lowest Scene Construction composite scores and then visualized resultant contrast maps in relation to each individual’s DN-A estimate.r > 0.80 except Specificity; see r = 0.94 across trials, r > 0.80 for 98% of trials; see Nearly every strategy probe showed high interexperiment reliability , adding to evidence that trials feature distinct rating combinations. Trials with high ratings for visual imagery , for example, were also likely to have high ratings for envisioning the physical locations of objects and places (“Loc_Obj_Places”). Trials with high ratings for considering others’ mental states (“Others_Feelings”) tended also to require imagining others’ personality traits . In exp 2, the correlation matrix showed similar structure (right), supporting the reliability of interstrategy correlations.To visualize relations among the 16 strategies, we created a correlation matrix, using data from all 180 trials. In rategies , left, atructure , right, exp 1, the greatest strategy differentiation appeared for a pair of correlated strategies . We cut the clustering dendrogram to preserve clusters at least as strong as this pair, which revealed five groupings (top). In exp 2, the independently estimated dendrogram revealed a similar structure. Using the same cut point criterion (above “Facts” and “Difficulty”) produced five strategy groupings that largely matched those from exp 1, which we heuristically labeled: (I) Difficulty, (II) Autobiographical, (III) Scene Construction, (IV) Others-Relevant, and (V) Self-Relevant.Using hierarchical clustering, we next identified five strategy groupings that could be combined into composite scores for subsequent network analysis. In roupings , top. Inexp 1 and exp 2 (“Loc_People” and “Relationships”), as well as those that showed weaker grouping across both experiments , were excluded from composite scores. Out of all strategy probes, “Specificity” also had the lowest interexperiment reliability (r = 0.66), and “Moral_Principles” had the lowest mean rating and SD  were used to create the strategy composite scores carried forward to analyses of the functional MRI data.Given those strategy ratings from r = 0.65; CI, P < 0.001). Overall, results from exp 1 and exp 2 provided evidence that trial-level ratings were stable . Conversely, DN-B showed a nonsignificant negative correlation to Scene Construction .Comparing Scene Construction scores, across trials, to DN-A activity revealed a strong positive correlation  revealed moderate intercorrelation between Difficulty, Autobiographical, and Self-Relevant scores  = 36.91, P < 0.001], and Scene Construction was the only significant predictor, accounting for most of the variance in DN-A response . For DN-B, the overall model was significant , but Scene Construction was not a significant predictor and accounted for almost no variance in DN-B response . These results supported a selective relation between Scene Construction scores and DN-A activity. Although DN-B is tightly juxtaposed to DN-A across the cortical mantle  was still a significant predictor, again accounting for most of the variance in DN-A response . For DN-B, this composite was not a significant predictor .As an additional consideration, the Scene Construction composite included strategies for using visual imagery and for considering the locations of objects or places. A third strategy, for considering the locations of people, clustered with this composite in Given that the episodic projection task was designed to target DN-A activity , we nextr = 0.33, CI, P < 0.05) and original control trials  tracked DN-A response (t(146) = −10.82, P < 0.001; see triangles in R2Scene Construction = 0.19, P < 0.001; R2Full Model = 0.32, Difficulty also P < 0.01).We, therefore, ran additional analyses restricted either to those original target trials or to control trials constructed not to include episodic projection demands . Correlation results revealed that Scene Construction scores for both the original target . Conversely, DN-A showed a weakly negative correlation to Difficulty scores , which were strongly associated with activity in the adjacent FPN-B . FPN-B, in turn showed no relation to Scene Construction , illustrating a functional double dissociation . For FPN-B, Difficulty was the only significant predictor and accounted for most of the model’s variance in network response .Multiple regression confirmed the double dissociation. In a model featuring Scene Construction, Difficulty, and Others-Relevant scores, Scene Construction accounted for the most variance in DN-A activity across trials and was the only significant predictor, as described above , as was reliability of the network activity values .R2 values to the product of these reliability scores, for each composite-network pair (as an estimate of explainable variance) suggested that Scene Construction scores accounted for ∼47% of the explainable reliable variance in DN-A activity, and Difficulty scores for ∼60% in FPN-B activity. Overall, the dissociation between DN-A and FPN-B adds to evidence that parallel association networks, with side-by-side regions across association cortex, can be robustly functionally dissociated . Effort does not account for Scene Construction’s relation to DN-A response (see top).Given evidence of a possible confounding effect of Difficulty on multiple composite-network relations, in post hoc analyses, we regressed the Difficulty composite scores from all other composites. Residual data continued to reveal a strong, selective relation between Scene Construction scores and DN-A activity , and the positive correlation between Scene Construction and Autobiographical composites strengthened. Scene Construction was still the strongest predictor of DN-A response within a model also featuring Others-Relevant, Autobiographical, and Self-Relevant composites . We unpacked these results in further post hoc testing .Autobiographical scores also showed a selective, positive correlation to DN-A activity after Difficulty regression . We initially expected a relation between Others-Relevant scores and DN-B response, based on evidence of DN-B recruitment for social functions  but only modestly predicted variance in DN-B activity overall (R2Full Model = 0.08). The positive relation to Others-Relevant scores accounted for similar variance to a negative relation to Scene Construction .Others-Relevant scores maintained a weak but selective positive relation to DN-B response after regression of Difficulty . No relation to RT was found for Scene Construction composite scores  and a weaker relation for Others-Relevant scores .For both Autobiographical and Self-Relevant composites, other observed network correlations did not survive regression of the Difficulty composite scores compare . These rThese findings indicate that our data could reduce to three clusters, related to Scene Construction, Others-Relevant, and trial Difficulty dimensions . Revisiting our initial composite characterization, both regressing and reverse-coding Difficulty composite strategies  preserved Scene Construction and Others-Relevant correlations, separable from an intercorrelated Self-Relevant cluster.The relation between Scene Construction scores and DN-A response and the separate relation between Difficulty scores and FPN-B response becomes even more compelling given these additional analyses. As an additional post hoc test, we next probed these two relations to assess whether maps based solely on trial groupings from the composite scores could yield selective network recruitment within individual participants.As a test of the discovery that Scene Construction and Difficulty scores differentiate activity between juxtaposed networks, we created within-individual, whole brain contrast maps using trials with high and low scores on the Scene Construction and Difficulty composites. Importantly, for Scene Construction, we restricted the analysis only to trials from the original control conditions. Thus, this contrast is completely orthogonal to the originally envisioned condition contrasts in DiNicola et al. .left column). Overlap was observed within midline regions, including in retrosplenial cortex and posterior parahippocampal cortex (PHC), previously linked to Scene Construction ratings . In most tested individuals,For Difficulty contrasts, each individual’s estimated FPN-B border was overlaid. Comparing the contrast, within individuals, to network borders again revealed evidence of overlap  and even across control trials , presenting a challenge to parsing differences. But among the control trials, subsets showed higher scores on either the Autobiographical or Scene Construction composite (middle). After visualizing the distribution of control trials, we selected a subset representing the 14 most extreme trials . Examining DN-A response across these trials revealed higher values for the Scene Construction subset .Scene Construction and Autobiographical scores were strongly correlated across all trials , three predictors were significant, including both Scene Construction probes  and Sequence_Events . Little variance in DN-A activity was accounted for by the Pers_Past_Exper probe , the only one specific to recollection of the past.What is more, unpacking the Autobiographical composite, one strategy probe measured reliance on memory  illustrated these results, with high correlations to both Scene Construction probes (before and after regression of Difficulty), and higher correlation to Sequence_Events than Pers_Past_Exper within the Autobiographical composite. A positive correlation to Specificity (not assigned to a composite during clustering) was also revealed. Collectively, these results add to evidence for DN-A’s role in constructing mental scenes, likely including specific details of dynamic events.Plotting correlation values between DN-A response and each individual strategy probe , top illProcesses linked to scene construction selectively recruited one specific distributed network, termed DN-A, that includes PHC, retrosplenial cortex, and multiple cortical association regions , DN-A showed no relation to Difficulty scores, and Difficulty-related networks did not track Scene Construction. DN-A can thus be functionally dissociated from parallel, and even interdigitated, networks within association cortex through a unique role in scene construction.These results build evidence in support of domain specialization for individually defined, distributed network DN-A. Specifically, our evidence suggests DN-A contributes to a previously hypothesized “construction system” , supportIn addition to network insights, the present work provided evidence that behavioral strategy ratings can tap into stable trial-level properties. If behavioral ratings varied by respondent group or by individual answers to trial questions, they would not inform independent neuroimaging data. But across two behavioral experiments, ratings of strategy probes showed striking stability. Even for individual trials, patterns were highly reliable, akin to trial-level fingerprints, and interexperiment reliability was high for nearly every strategy probe  and 2. WHere, neuroimaging data were also reliable , both frontoparietal networks (FPN-A and FPN-B) showed positive correlations to the Difficulty composite score. A question for future work concerns the precise roles of these networks. Distinct contributions to aspects of control and to network coordination have been proposed  or domain-general (FPN-B) processes .Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.No conflicts of interest, financial or otherwise, are declared by the authors.L.M.D., O.I.A., and R.L.B. conceived and designed research; L.M.D. and O.I.A. performed experiments; L.M.D., O.I.A., and R.L.B. analyzed data; L.M.D., O.I.A., and R.L.B. interpreted results of experiments; L.M.D. and R.L.B. prepared figures; L.M.D. and R.L.B. drafted manuscript; L.M.D., O.I.A., and R.L.B. edited and revised manuscript; L.M.D., O.I.A., and R.L.B. approved final version of manuscript.


Trypanosoma theileri, a non-pathogenic parasite of bovines, has a predicted surface protein architecture that likely aids survival in its mammalian host. Their surface proteins are encoded by genes which account for ∼10% of their genome. A non-pathogenic parasite of sheep, Trypanosoma melophagium, is transmitted by the sheep ked and is closely related to T. theileri. To explore host and vector specificity between these species, we sequenced the T. melophagium genome and transcriptome and an annotated draft genome was assembled. T. melophagium was compared to 43 kinetoplastid genomes, including T. theileri. T. melophagium and T. theileri have an AT biased genome, the greatest bias of publicly available trypanosomatids. This trend may result from selection acting to decrease the genomic nucleotide cost. The T. melophagium genome is 6.3Mb smaller than T. theileri and large families of proteins, characteristic of the predicted surface of T. theileri, were found to be absent or greatly reduced in T. melophagium. Instead, T. melophagium has modestly expanded protein families associated with the avoidance of complement-mediated lysis. We propose that the contrasting genomic features of these species is linked to their mode of transmission from their insect vector to their mammalian host.This article has an associated First Person interview with the first author of the paper. Summary: Comparing closely related non-pathogenic trypanosomes, we highlight differential investment in cell-surface protein encoding genes and predict this differential investment is associated with the life histories of their hosts and vectors. Trypanosomatidae are a family of single-celled eukaryotes of the class Kinetoplastea, which are characterised by a specialised mitochondrial genome, the kinetoplast. Trypanosomatidae are monoxenous (single host) or dixenous (two host) species. Dixenous trypanosomatids are obligate parasites of a broad diversity of animals and plants whilst monoxenous species are largely restricted to insects . HoweverTrypanosoma cruzi and Leishmania spp. cause important diseases in humans, Chagas disease and Leishmaniasis, respectively. Also, Trypanosoma brucei has been subjected to intense molecular and cytological study as two of its subspecies, T. b. gambiense and T. b. rhodesiense, are causative agents of Human African Trypanosomiases (HAT)  . In addielopment .T. brucei genome comprises 11 megabase chromosomes  , also known as leishmanolysin  . VSG vars (VSGs) . T. cruze genome  and, aloe genome , these ganolysin .Trypanosoma theileri and Trypanosoma melophagium, can be highly specific to their host and vector. These species represent an attractive model to study the basis of host and vector specificity. For the remainder of this manuscript, we refer to species that cause no overt pathogenicity in immunocompetent hosts as non-pathogenic. We recognise that ‘non-pathogenic’ species may still cause slight detriment to their hosts, which is often difficult to detect, especially when prevalence is high in the host population.Studies have largely focused on pathogenic dixenous trypanosomatids, which can hold broad host niches, capable of infecting multiple mammalian species . In contTrypanosoma theileri is a non-pathogenic bovine parasite which has a reported prevalence of 80% in cattle in the US and Europe when screened via culture-based methods  and one MSP family   . Lastly,ishmania . T. theima grayi , 2017, dic forms , 1931.Trypanosoma melophagium is a non-pathogenic trypanosome of the subgenus megatrypanum, transmitted between sheep via the sheep ked. This flightless insect vector has been eradicated from much of its original geographic distribution due to widespread pesticide use. However, where the sheep ked persists, it often carries T. melophagium , this variation being observed in its repeat and unique sequence  in comparison to other Trypanosoma isolates . The T. melophagium genome was annotated with 10,057 protein encoding genes, in comparison to 11,312 in T. theileri , contrasting with the kinetoplastid mean of 48.4%  and in universal single copy orthologs which are essential for every life-cycle stage of T. brucei . T. rangeli displays the highest level of selection pressure acting to increase translational efficiency . In comparison, T. congolense is more divergent from T. brucei (0.249) whilst the isolates within T. brucei  show less divergence than seen between T. theileri and T. melophagium.A species tree was generated as part of the orthologous protein clustering. Using genetic markers, previous studies have noted the similarity between theileri . Based o. brucei . T. graytheileri C. T. melT. theileri has a greater number of species-specific orthogroups than T. melophagium and 12.9% of its genes are assigned to one of these orthogroups, while T. melophagium has only 2.7% of its genes in a species specific orthogroup . To validate this, a blast search was performed using a relaxed cut off (1e-5) using the T. melophagium genome as the database and T. brucei TREU927/4 VSGs as the query. No hits were identified.Firstly, and as expected, T. theileri genome analysis, the genes and orthogroups from this study were annotated with the host interaction genes from T. theileri is predicted to contain 1,265 more genes than T. melophagium (T. theileri putative surface protein (TTPSP) revealed a large expansion in T. theileri  compared to T. melophagium (10 genes) which could equate to much of the disparity in genome size (T. theileri transcripts were subjected to a blastn search against a database consisting of the T. melophagium transcripts (1e-25 cut-off). The T. melophagium transcripts were derived from the genome annotation analysis. Only nine T. theileri TTPSPs aligned to T. melophagium.To enable comparison to the ophagium . Examinaome size . To confT. theileri genome analysis but were split between 66 orthogroups in this analysis  revealing their specific innovation in these related trypanosomatids.TTPSPs were split between four orthogroups in the original analysis   consisting of 11 proteins and annotated as MSP. Combined, these results suggest that the surface protein environment of T. melophagium and T. theileri are distinct and that genes encoding these proteins account for most of the discrepancy in the genome sizes.Following the TTPSPs, the largest expanded gene family in defences . This rodefences . OrthogrT. grayi, T. melophagium and T. theileri contain 42 clade-specific orthogroups including the MSP orthogroups OG0000590 and OG0008095. Both MSP orthogroups were expanded in T. theileri in comparison to T. grayi and T. melophagium. T. melophagium and T. theileri share 81 orthogroups specific to the two species. Twelve of the orthogroups are putative cell surface protein families, including five representatives from TTPSP and seven MSP orthogroups. Of the nine remaining annotated orthogroups, OG0008096 is the largest and contains trans-sialidsases. The orthogroup is expanded in T. theileri (n=17) compared to T. melophagium (n=5).T. theileri has 121 species specific orthogroups. Sixty-four of these were putative host interaction genes . Other annotated species-specific orthogroups consisted of an actin family (OG0018163) along with several families associated with protein binding, WD domain G-beta repeat (OG0018131) and a leucine rich repeat family (OG0018098).on genes . Two annT. theileri and T. melophagium, with 38 and eight genes, respectively. Of the four orthogroups containing trans-sialidase, two do not contain proteins from T. melophagium (OG0011818 and OG0015016). These two orthogroups contain proteins from T. cruzi and so are likely to have been lost by T. melophagium and T. grayi. In T. cruzi, trans-sialidases are involved in host immune evasion  that include 20 T.theileri invertase genes only have three members in T. melophagium and three members in T. grayi. This expansion might indicate an adaptation of T. theileri to its vector, the tabanid fly, which can feed on sugary flower nectar  contain only one gene from the plant parasite Phytomonas  were expanded in T. theileri (n=60) compared to T. melophagium (n=11).Other putative cell surface modifying moleculesT. brucei and T. theileri transcriptomes which revealed differences in the abundance of glycosomal enzyme mRNAs. Particularly, pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxykinase and malate dehydrogenase were found to be >10 fold more abundant in T. theileri than in T. brucei (T. melophagium (File S4) and found that T. melophagium has expanded the orthogroups associated with three glycolytic enzymes. These included pyruvate orthophosphate dikinase (OG0000570), phosphoenolpyruvate carboxykinase (OG0000120) and malate dehydrogenase (OG0000332), whilst T. melophagium has a reduced number of genes in the fumarate reductase orthogroup (OG0000078). Orthogroups associated with peroxisome targeting were found to be in equal numbers (OG0004108 PEX5 and OG0003998 – PEX7). All other glycolytic enzymes are present in equal numbers in the two species. Therefore, it is likely that the glycolysis pathway is conserved in T. melophagium.. brucei . We confT. brucei have identified genes which are associated with key stages of the T. brucei life cycle. These studies tracked genes associated with stumpy formation in the blood stream form (T. theileri. T. melophagium has expanded its orthogroups containing the kinases NRK (T. theileri and T. melophagium are missing metacaspase (MCA1) which is associated with the later stages of progression towards metacyclic forms in T. brucei . Therefore, a functional gene silencing pathway is likely to be present in T. melophagium, matching the prediction in T. theileri.All five core genes that represent the trypanosome RNAi machinery  were present in T. cruzi and T. vivax isolates, along with T. brucei Lister 427 2018 . T. melophagium and T. theileri have not expanded their retrotransposon repertoire. A similar pattern was observed for long terminal repeat (LTR) retrotransposon counts, which show a positive correlation with genome size .Retrotransposon counts highlighted an expansion in T. melophagium and T. theileri are closely related trypanosomes that have distinct hosts and vectors . Whole blood was collected into heparinized vacutainers and used within 2 days. 1 ml of blood was diluted with 5 volumes of a 50% mix of HMI9 supplemented with 20% fetal bovine serum (FBS) and Madin-Darby bovine kidney (MDBK) conditioned medium. All cultures were kept at 37°C and were examined microscopically every 3 days for 6 weeks. After propagation of T. melophagium by culturing of the blood sample, the specimens were transferred and co-cultured with fibroblast-like primary cells as feeder cells, isolated from the same blood sample. Due to the short lifespan of these primary cultured cells, T. melophagium was subsequently co-cultivated with MDBK cells and then progressively adapted to axenic conditions with a 50% mix of HMI9 and MDBK conditioned medium.T. melophagium using a MagAttract high molecular weight DNA kit, following the manufacturer's instructions (Qiagen) and cleaned via ethanol precipitation. The DNA was sequenced with Oxford Nanopore Technology's (ONT) MinION (R9.4.1), following the ONT Rapid Sequencing protocol. Base-calling was performed in high accuracy mode using Guppy (available at https://community.nanoporetech.com/) which produced 1.059 gigabases (Gb) of data. PycoQC was used to visualise the data (DNA was extracted from cultured the data . The samthe data .T. theileri sequencing data was downloaded from NCBI. 170 bp genomic reads were used (SRR13482812).Jellyfish and GenomeScope were used to provide a k-mer based estimate of the genome size and heterozygosity using the short DNA reads described above .https://github.com/nanoporetech/medaka) were performed. DNBseq reads were mapped to the Medaka polished assembly, and two iterations of Racon were performed. Short and long reads were aligned to the Racon polished assembly to complete two final iterations of polishing with Pilon (https://github.com/sujaikumar/assemblage) and BUSCO . These contigs were removed from the assembly. BUSCO and BlobTools were re-run on this trimmed assembly to assess the final assembly's completeness and coverage .Both sets of reads were mapped to the draft assembly. Each contig was subjected to a DIAMOND blastx search against the InterProScan database . The resT. theileri proteome and T. melophagium RNAseq evidence .Publicly available genomes, transcriptomes and proteomes were accessed from TriTrypDB  along wirom NCBI . The quan=992) and 3) single-copy universal orthologs, which are essential for every stage of the T. brucei life cycle (n=158). The last list of genes were identified by screening the universal single-copy orthologs for genes that had a significant reduction in transcript levels in every library of an RNAi phenotype screen (>1.5 log fold decrease) (The genomes from each of these kinetoplastid isolates were screened for transfer RNA genes using tRNAscan-SE . The outecrease) .http://transposonpsi.sourceforge.net) and LTR-harvest  retrotransposons using TransposonPSI . A similar search was performed to confirm the reduced TTPSP counts in T. melophagium. For this, the T. theileri transcripts were used to query a database made from the T. melophagium transcripts .To confirm the absence of VSGs in http://ggplot2.tidyverse.org) and ggrepel (available at http://github.com/slowkow/ggrepel).The cell surface orthogroups in this study were annotated with the orthogroups from