Although some immunocompetent patients have developed invasive aspergillosis, the vast majority of cases are seen in immunocompromised patients. COVID-19 infection has been proposed to cause immune dysfunction or suppression, which predisposes patients to fungal co-infections such as mucormycosis and aspergillosis.A 58-year-old woman was admitted to the hospital with confusion, dysarthria, and loss of consciousness. The patient had a 1-month prior history of severe COVID-19 infection. A computerized tomography (CT) scan and a magnetic resonance imaging (MRI) revealed an intraventricular lesion with perilesional edema and a significant midline shift, which was initially thought to be an intraventricular tumor. Following a posterior parietal craniotomy, the lesion was resected via a transcortical approach from the posterior parietal region to the right lateral ventricle. Histopathological findings confirmed intraventricular aspergillosis (IVA). The patient was treated with intravenous amphotericin B for two months and discharged with oral variconazole for 4 months.Covid-19 infections can result in- dissemination of fungal diseases such as aspergillosis. As a minor component of cerebral aspergillosis with a poor prognosis, intraventricular aspergillosis necessitates prompt treatment, which includes surgical resection and the administration of anti-fungal medications.Infection with COVID-19 causes immune dysfunction, which leads to fungal co-infection, including CNS aspergillosis. As a result, all COVID-19 patients who present with acute neurologic symptoms should have CNS aspergillosis considered in their differential diagnosis. •Infection with COVID-19 can cause immune dysfunction, which may lead to fungal co-infection, including CNS aspergillosis.•Workup of COVID-19 patients with acute neurologic symptoms should be broad and include fungal infectious.•Patients with CNS aspergillosis should receive prompt treatment because these cases have poor prognosis and can be fatal. Although uncommon in comparison to pulmonary aspergillosis, CNS aspergillosis is usually fatal if not treated promptly. They frequently occur within the brain parenchyma  or less Since the beginning of the ongoing global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19 infection has been shown to cause immune dysfunction or suppression, predisposing patients to bacterial and fungal co infections ,8. Altho2A 58-year-old woman presented to the emergency department our general hospital with a week of confusion, dysarthria, and stupor. The patient was hospitalized to the ICU ward and treated with corticosteroids among other medications for a serious COVID-19 infection she acquired a month earlier. Family and psychosocial history were negative. Physical examination revealed a patient who was ill, confused and disoriented, with a GCS of 9/15 (M5E2V2). A neurological examination left-sided hemiparesis and no papilledema. Motor examinations revealed that the upper and lower extremity muscle force of the left side were 2/5. Also, a positive plantar reflex was observed on the left foot. Her level of consciousness rapidly deteriorated a few hours after admission and neurosurgical consultation. The patient was immediately sent for a brain CT scan, which revealed a right hypodense intraventricular lesion with a midline shift. An MRI of the brain revealed a right intraventricular lesion that was isointense on T1&T2 weighted images. A large non-demarcated intraventricular lesion in the right lateral ventricle was observed, with perilesional edema, mass effect, and significant shift of the midline structures .Fig. 1PrThe neurosurgeon (A.K) performed a posterior parietal craniotomy was on the patient. Following the opening of the dura, a transcortical approach from the posterior parietal region to the right lateral ventricle was made. The lateral ventricle's ependymal layer was opened, and a soft, greyish-colored lesion with slight hemorrhage and local invasion into the ependyma was discovered. Using microscopic surgical techniques with suction and bipolar diathermy, a macroscopic gross total resection was performed. Histopathological examination revealed necrotic foci with numerous branching fungal hyphae with acute angle branching. This finding was consistent with a fungal infection caused by Aspergillus species .Fig. 2A:3Following surgery, the patient's condition and level of consciousness improved. Based on the histopathological reports, the patient was immediately started on intravenous (IV) amphotericin B 250 mg daily, which she received for two months before being switched to oral voriconazole 400 mg Bid for additional 4 months. Her postoperative recovery has been uneventful, and she has completely recovered from her symptoms. . For improved patient outcomes, additional treatment options such as intraventricular administration of amphotericin B have been described . Despite5COVID-19 infection causes immune dysfunction, and it is possible to co-infect with bacterial and fungal agents, including Aspergillus species. As a result, in COVID-19 patients who exhibit confusion, loss of consciousness, seizures, or any neurological symptoms, aspergillosis should be considered in the differential diagnosis. Early administration of an antifungal agent such as amphotericin B may significantly improve patient outcome and prevent further neurologic deterioration and death.Not commissioned, externally peer reviewed.None.Not required.Written Informed consent was obtained from the patient for publication of this case report and accompanying image. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.A.K, H.S: Conceived the idea.S.B.K, M.S: Data collection, writing the paper.A.K, S.B.K, S.A: Review and revised manuscript for intellectual content critically. All author approved the final version of the manuscript.1.Name of the registry:2.Unique Identifying number or registration ID:3.Hyperlink to your specific registration (must be publicly accessible and will be checked):Alireza Khoshnevisan.None.


The fluctuating climates, rising human population, and deteriorating arable lands necessitate sustainable crops to fulfil global food requirements. In the countryside, legumes with intriguing but enigmatic nitrogen-fixing abilities and thriving in harsh climatic conditions promise future food security. However, breaking the yield plateau and achieving higher genetic gain are the unsolved problems of legume improvement. Present study gives emphasis on 15 important legume crops, i.e., chickpea, pigeonpea, soybean, groundnut, lentil, common bean, faba bean, cowpea, lupin, pea, green gram, back gram, horse gram, moth bean, rice bean, and some forage legumes. We have given an overview of the world and India’s area, production, and productivity trends for all legume crops from 1961 to 2020. Our review article investigates the importance of gene pools and wild relatives in broadening the genetic base of legumes through pre-breeding and alien gene introgression. We have also discussed the importance of integrating genomics, phenomics, speed breeding, genetic engineering and genome editing tools in legume improvement programmes. Overall, legume breeding may undergo a paradigm shift once genomics and conventional breeding are integrated in the near future. Rising human population, fluctuating climates, and depleting arable land, coupled with lower productivity and post-harvest losses, pose a serious threat to global food security. Furthermore, surging pests and diseases challenge global researchers to develop stress resilient, high-yielding, nutritious crops to alleviate hunger and deprivation . LegumesLegumes have the capability to fix atmospheric nitrogen and are also believed to be involved in carbon sequestration and soil amelioration. Hence, they are considered an essential feature of sustainable agricultural production, particularly in arid regions . Their aPisum sativum L.), was the first experimental crop used by Gregor Johann Mendel (Father of Genetics) in his pioneering work to understand the basics of heredity and variation, which paved the way for establishing new branch of biological science, i.e., Genetics  over last two decades has been very slow compared to that achieved in cereals  Figure 1In addition, indeterminacy, plant morphology, environmental sensitivity, slow growth rate, and lack of management practises and government policies will further hinder legume improvement. Indeterminacy leads to non-synchronous flowering and maturity, which will affect the crossing and mechanical harvesting, respectively. Legume crops have poor photosynthetic efficiency, slow dry matter accumulation, reduced seedling vigour and canopy development, poor source-sink relationship, and rapid leaf senescence. Furthermore, they have reduced leaf area index (0.7-2.0) compared to cereal crops (3-6), which will reduce the gas exchange and photosynthetic efficiency. Legumes produce many flowers, but due to a lack of significant nutrient assimilation, most of them will fall before setting into fruit. For instance, about 70-90% and 80-95% of the produced flowers will drop in green gram and pigeon pea, respectively . Legume Given the importance of legume crops in a sustainable agricultural system and food security, we prepared a comprehensive review on challenges and strategies to overcome these challenges in legume breeding programmes. Furthermore, we discussed the advantages of integrating advanced genomics and phenomics approaches into distant hybridization to accelerate alien gene introgression and achieve higher genetic gain in these crops.Gene pool (GP) is a relationship among crop plants and related taxa that could be useful to breeders for crop improvement . Based oCicer arietinum L.) is the only domesticated species of the genus Cicer and was evolved from its immediate wild progenitor, C. reticulatum Ladiz. through natural selection. The genus Cicer consists of 46 species, of which 10 are annual and 36 are perennial in nature , GP-2 , and GP-3  M. Pop. and C. cuneatum Hochst. Ex Rich), respectively (Chickpea (n nature . Of the Anatolia . Among tectively .Cajanus cajan (L.) Millspaugh) is an important grain legume crop which includes nearly 13,200 cultivated and 555 wild accessions in the gene bank. Based on their crossability relationship with cultivated pigeonpea, all accessions were classified into three GPs. The C. cajana and C. cajanifolius (Haines) van der Maesen belongs to GP-1, whereas 10 species each belongs to GP-2 and GP-3, respectively (Pigeonpea (ectively .Glycine max (L.) Merr.) belongs to the genus Glycine, which has two subgenera, i.e., Glycine and Soja. The subgenus Glycine has 26 perennial species native to Australia  and its wild progenitor (G. soja Sieb.)  to GP-1 (G. max)  . Furtherertility . Majorit(G. max) .Arachis hypogaea L.) belongs to the genus Arachis, with 80 species. All groundnut species are divided into total nine taxonomic sections on the basis of their sexual compatibilities, morphological and cytogenetic features, and geographic distributions  as GP-3 . The genus Lens has seven closely related taxa and were categorized into four GPs  Hand.-Mazz and L. odemensis (Ladiz.) are crossable with cultivated lentils and may share the common gene pool (Wild species/sub-species of the genus four GPs . It has domensis . Furtherene pool .Phaseolus vulgaris L.) is the important legume crop with limited germplasm characterization. Over the course of its evolution and domestication, the common bean eventually formed two separate GPs, namely the Mesoamerican and the Andean  is an important legume crop. On the basis of seed weight, seed shape, and pod characteristics, faba bean has been divided into four groups, i.e., major, equine, minor, and paucijuga  belongs to the genus, Pisum, present in the tribe Fabeae, and is the oldest domesticated crop, with an estimated domestication of about 10,000 years ago  Asch. & Graebn., and P. abyssinicum A. Braun from their cultivated genepools (P. elatius) in the GP-1 , a member of GP-3  of these five sub-genera in Asia, Africa, and America. Domesticated cowpea (Vigna unguiculata (L.) Walp.) belongs to the section Catiang of sub-genus Vigna  and unguiculata (grain and vegetable cowpea) differ for their pod length and are cultivated in Asia and central Africa, respectively  R. Wilczek) and black gram (V. mungo (L.) Hepper) are the two most important legumes, which were believed to be originated from the single ancestor (V. radiata var. sublobata (Roxb.) Verdc and V. mungo var. silvestris Lukoki, Marechal & Otoul, respectively  Marechal)  Ohwi & H.Ohashi and V. stipulacea (Lam.) Kuntze with rice bean (V. umbellata (Thunb.) Ohwi & H.Ohashi)  Ohwi & H.Ohashi  , V. tenu.Ohashi) , and V. H.Ohashi . List ofAgricultural practises have domesticated hundreds of crop plants from their wild relatives with altered phenotype and genotype . During Favourable alleles stored in the wild repositories can be explored to break the yield plateau Figure 2http://www.nbpgr.ernet.in/Research_Projects/Base_Collection_in_NGB.aspx). A huge amount of chickpea germplasm  is conserved at various gene banks (https://www.croptrust.org/). Globally, ICRISAT, Hyderabad has the largest collections of cultivated chickpeas  and wild Cicer species (308 accessions from 18 species) from 60 countries. These reservoirs can be utilised in breeding programmes to boost the genetic gain in legumes. However, lack of appropriate germplasm characterization and pre-bred lines may limit their effective utilization in breeding programmes. Therefore, it has become indispensable to characterize the crop germplasm and develop the pre-breeding population.Huge germplasm collections of Chickpea , Groundnut , Pigeonpea , Soybean , Pea , Green gram , Common bean , Cowpea , Horse gram , and Rice bean  are stored in the National Bureau of Plant Genetic Resources (NBPGR), New Delhi (New combinations of genes resulting from spontaneous hybridization (between cultivars and their wild relatives) and trait introgression are considered as the major avenues for gene flow to expand the genetic diversity of cultivated crops . HoweverPre-breeding involves all the activities associated with identification of desirable traits/genes from unadapted germplasm  and transfer of these traits/genes into well-adapted, cultivar backgrounds . Pre-breeding offers a great opportunity to broaden the GP-1 by utilising genetic variability available in the wild species. This will ensure a steady flow of new and useful genetic variability into the breeding pipelines for the development of new cultivars with high levels of resistance and wider adaptability . Pre-breAs mentioned earlier, huge genetic variation is available in the wild relatives of legumes with several important traits. Despite their potential applications in crop improvement, legume breeders are reluctant to use wild species in their breeding programmes. This is owing to a lack of proper germplasm characterization, cross-incompatibility, and associated linkage drags Figure 3Chickpea is a cool-season, low-input demanding crop that ranks third in world production among the legumes . ChickpeCicer taxa. Furthermore, multi-parent populations such as MAGIC (Multiparent advanced generation inter-cross) and NAM (Nested Association Mapping) populations may enhance the allelic diversity and may also help in the inclusion of novel recombinants in GP-1  Desi and Kabuli ILs with enhanced resistance to wilt, foot rot, and root rot (C. arietinum and C. reticulatum (C. arietinum and C. echinospermum (C. reticulatum and C. echinospermum) were used to increase variability, transfer genes for several abiotic  and biotic  stresses, and also to exploit higher heterosis  Aba blight , cold  into JG-11 (foc2 and foc4) for Fusarium wilt resistance and pyramiding three genes, i.e., one for Ascochyta blight and two for Fusarium wilt resistance (foc1 and foc3), respectively   by transto JG-11 . This apectively . QTL-hot markers . Furtherchickpea . The Traultivars . A largedhal, seed husks used as cattle feed, dry stems used as fuel-wood, raw material to make huts and baskets, and soil mulch, and green leaves used for green manuring and medicinal purpose   . Seeds aitional) . Pigeonpitional) .Cajanus species are reservoirs of untouched diversity that can be explored to enhance the disease and insect pest resistance (C. scarabaeoides (L.) Thouars, a wild relative of pigeonpea has agronomically superior characteristics, including dwarfness, the number of fruiting branches and pods per plant, and resistance to pod borers and phytophthora stem blight (C. scarabaeoides have been successfully introgressed into the Pigeonpea (C. sericeus (Benth. ex Baker) Maesen for A1 CMS, C. scarabaeoides for A2 CMS, C. volubilis (Blanco) Blanco for A3 CMS, C. cajanifolius (Haines) Maesen for A4 CMS, C. cajan for A5 CMS, C. lineatus (Wight & Arn.) Maesen for A6 CMS, C. platycarpus (Benth.) Maesen for A7 CMS, C. reticulatus (Aiton) F.Muell for A8 CMS, and C. lanceolatus (W.Fitzg.) Maesen for A9 CMS  (G. canescens F.J.Herm. for powdery mildew (G. tomentella Hayata for nematode and leaf rust (G. latifolia (Benth.) Newell & T.Hymowitz for stem rot (G. formosana Hosok.) after screening 5,000 lines (2:3 and BC1F2 mapping populations (G. soja was identified on chromosome-14 and was found to increase the soybean yield by 9.4 per cent (G. tomentella (Kunitz trypsin inhibitor (kti) genes to develop trypsin inhibitor free lines (The wild accessions of us (YMV) , nematodus (YMV) , aphids us (YMV) , and brous (YMV)  It additus (YMV) . Other wy mildew , G. tomeeaf rust , and G. stem rot . These s00 lines . In addiulations . A yieldper cent . Genes fmentella . MABC haee lines .A. duranensis Krapov. & W.C.Greg. (AA) and A. ipaensis Krapov. & W.C.Greg. (BB)  . The kerrovement . FurtherA. cardenasii Krapov. & W.C.Greg. to cultivated peanuts  with A. hypogaea help to develop an IL  resistance to multiple diseases  and repeated use of the same genotypes in breeding programmes  into the cultivars  Grande) (Stemphylium blight (L. lamottei Czefr. and L. ervoides) (L. nigricans (M.Bieb.) Webb & Berthel. and L. ervoides) (L. ervoides) , high-micronutrient content  (L. odemensis)   (L. culinaris ssp. culinaris)  ( Grande) , resistarvoides) , resistarvoides) , architervoides) , extra eILWL401) , floweriemensis) , yield aentalis) , and tolgricans) , heat (Llinaris)  and salientalis) . Distantentalis) . Furtherentalis) .L. orientalis  , micronutrient content, and reduced generation time (80-100 days) (L. culinaris cv. Alpo and L. odemensis was constructed by using RNAseq methodology (Ascochyta blight resistance (on chromosome-6) were mapped in the interspecific crosses.Soil salinity affects the nodulation and nitrogen fixation in lentils. Collaborative research of ICAR, New Delhi  and ICARDA, Lebanon  identified the salt-tolerant accessions of -136670) . Success00 days) . Recentlhodology . SimilarPhaseolus is native to America, with 80 identified species (P. vulgaris L.), lima bean (P. lunatus L.), runner bean (P. coccineus L.), tepary bean (P. acutifolius A. Gray), and year bean (P. dumosus Macfad.), were domesticated for cultivation. Two of these species, P. vulgaris and P. lunatus, were independently domesticated at least twice in Mesoamerica and the Andes. This suggests that some domestication traits, such as determinacy, might be selected multiple times in common beans (Common bean is a popular legume crop that can be eaten as green pod vegetable or dried seeds (dry bean/Rajmash). Because of its medicinal properties, it can also be used to treat diabetes, heart problems, and bladder burn . The gen species . Among ton beans . Furtheron beans .Phaseolus species. For instance, P. acutifolius is known to possess relatively high levels of tolerance to most of the abiotic and biotic stresses  . In spitstresses . Therefoe traits .P. vulgaris and P. acutifolius was not successful without in vitro embryo culture  without the aid of embryo rescue to produce interspecific crosses between these two species  (P. coccineus cv. PI255956 to P. vulgaris cv. OR91G (PvPdh1 (Phaseolus vulgaris Pod dehiscence-1) located on chromosome-3, identified from the middle American domesticated beans was used to develop shattering tolerant common bean (Interspecific hybrids of  VRW-32) . In addiv. OR91G . Resistav. OR91G . Recentlmon bean .Faba bean is less resource consuming, cool season crop grown on marginal soils. It is also known as broad bean, bakala, horse bean, and tick bean . Middle-V. faba and V. narbonensis L. , L. albus L. (white lupin), L. luteus L. (yellow lupin), and L. mutabilis Sweet (Andean lupin) have been domesticated and are under cultivation (Lupin seeds have been used for thousands of years as food and feed. It has originated from the Old World (Mediterranean region), as well as from the New World (North America and Andean highlands) . Lupin itivation . There ativation .L. albus), thin seed coats and pod walls (L. angustifolius) (L. luteus), and high pod set . Genes for several agriculturally important traits, such as, vernalization independence (Ku and Julius), low-alkaloid content (iucundus), reduced pod shattering (tardus and lentus), soft seededness (mollis), white flower colour (leucospermus), and anthracnose resistance (Lanr1), have been identified and transferred into L. angustifolius (Crossing wild species with cultivated lupins is rarely successful . With huifolius) , reducedtifolius .iucundus and tardus, prevented the development of shattering resistant, alkaloid-free lupin lines  and pea cultivar, Alaska-81 to transfer the resistance genes for pea weevil into the cultivar background  from wild germplasm accessions. Introgressing these mutants into cultivar background may reduce the antinutritional factors. The P. elatius mutant has very low seed protease inhibitory activity, and this mutation has been successfully introgressed into cultivated pea , dry pea, or green manure crop and will also help in improving soil fertility by fixing atmospheric nitrogen. Peas are neglected by many farmers and will be grown on marginal lands, despite their importance in health benefits and sustainable crop production. Furthermore, due to limited genetic base coupled with changing climate conditions, wide range of biotic and abiotic stresses affects peas production. Novel genetic variations can be either exploited from the available wild relatives or can be created through mutagenesis . Distanta blight , P. satiium wilt , and Pis pinodes . Phenotya weevil . Geneticeritance . The resckground . In addi. fulvum . Howeverated pea . Furtherated pea .Vigna is an important genus in the Leguminaeceae family and it helps in restoring soil fertility by fixing atmospheric nitrogen. Cowpea (Vigna unguiculata (L.) Walp.) is a warm-season legume crop and is a rich source of nutrients and minerals. It can be used as a pot herb or as livestock feed. It has the ability to thrive well in drought-prone areas and in poor soils. Cowpea is a self-pollinated crop with a monophyletic origin. Several insect-pests, including pod borers, pod sucking bugs, flower bud thrips, and cowpea aphids, will greatly affect the cowpea yield  and V. sesquipedalis (L.) Fruwirth  A.Rich. as the resistance source to aphids, flower bud thrips, pod borers, and bruchids (symbiosis receptor-like kinase (SYMRK), through null mutants produced by CRISPR/Cas-9 technology  and abiotic factors  will cause severe yield reduction in these crops. Distant hybridization has allowed us to broaden the genetic base and offers the opportunity to deal with these problems.Green gram and black gram are the important short-duration legumes, and they can be intercropped with cereal crops to improve soil fertility . It is tears ago . Green gVigna, has useful genes for vigorous and erect growth, sturdy stems, broad leaves, and long and profuse pods with biotic and abiotic stress tolerance (V. radiata var. sublobata) (V. unguiculata subsp. dekindtiana (Harms) Verdc.), nematode (V. angularis (Willd.) Ohwi & H.Ohashi) (V. sublobata)  Benth., V. marina (Burm.) Merr., and V. vexillate) were identified (V. sublobata accessions were successfully introgressed using high-throughput genotyping and phenotyping tools (Wild relatives of olerance . Wild acblobata) , pod bug.Ohashi) , MYMD (mblobata) , and heaentified . Subsequng tools . Furtherng tools . Furtherng tools .V. aconitifolia) is largely cultivated as multi-purpose crop in arid regions of India. The prostrate, vining, and semi-trail growing patterns of moth bean help in reducing soil erosion. It can be used as a green manure crop and also helps to improve soil fertility by fixing atmospheric nitrogen. Moth bean has been considered as a vital source of amino acids, vitamins, and minerals (qVacBrc2.1) and minor (qVacBrc5.1) QTLs for seed resistance to Callosobruchus chinensis. Resistance genes for C. chinensis were further successfully introgressed into cultivars  is an underutilized legume that has gained attention due to its numerous applications, including profitability, diversity, and agricultural sustainability. It is also known as “climbing mountain bean,” “red bean,” or “Oriental bean.” It is used as a green manure crop as well as a cover crop. Rice bean is mostly grown for beans, although they serve as vegetable (green pod), fodder, and folk medicine. Despite these benefits, rice bean is grown in fewer areas owing to lack of ideal plant types, non-synchronous maturity, seed shattering, and insufficient marketing facilities (Rice bean (cilities . FurtherV. tenuicaulis)  , bruchidicaulis) . Interspmbellata . HoweverV. mungo . By emplmbellata . Furthermbellata .Macrotyloma uniflorum (Lam.) Verdc.) is an underutilised legume crop that originated in south-west India and it serves as a major source of vegetable protein for millions of rural residents in the Indian subcontinent  Verdc. and M. ciliatum (Willd.)Verdc. However, M. uniflorum, is the only cultivated species distributed in the Indian subcontinent   (M. africanum (Brenan ex R.Wilczek) Verdc. and M. axillare)  and lipid (10.85%) content in the horse gram (The genus ontinent . Wild spxillare) , increasDangwal) , and higxillare) . Introgrrse gram . Furtherrse gram .Fusarium spp., Pythium spp., root knot nematode, Anthracnose, collar rot, powdery mildew, Stemphylium leaf spot, Alfalfa leaf curl virus, and Alfalfa weevil)  ; (ii) fo weevil) ; and (ii weevil) . Forage Trifolium repens (L.). For instance, reproductive traits (such as flowering and seed yield) from T. nigrescens Viv.   .Medicago sativa L.) is possibly the world’s most important cultivated temperate perennial forage legume. M. arborea L. is another perennial forage legume, that serves as major source of genes for high level of heterosis (M. rugosa Desr. and M. scutellata (L.) Mill. into M. sativa (M. rugosa-M. truncatula Gaertn. and M. scutellate-M. truncatula were successfully produced by protoplast fusion to transfer the genomic fragments from the these species (through intergenomic recombination) into M. truncatula , selection intensity (I), and trait heritability (h2). Genetic variation is the fundamental requirement for crop improvement. However, domestication followed by intensive selection and utilization of same parental lines has significantly reduced the genetic diversity in the legume gene pool. Narrow genetic diversity of legumes can be widened either by unlocking the hidden variation present in the germplasm resources or by creating novel alleles or haplotypes not present in the crop germplasms  for specific traits of interest . Several2, RILs, BCs, DH, NILs, BILs, and immortalised F2), multi-parental (NAM and MAGIC), and unstructured populations (association panels), help to identify significant alleles (QTLs) or haplotypes. Multi-parent populations will combine allele richness and increase the mapping resolution by enhancing recombination frequency  (AhNFR1 and AhNFR5) and cowpea (SYMRK)  and genome editing can be effectively used to transfer favourable genes from unrelated species or produce novel genetic variations not present in the nature. Transgenesis has been successfully employed in broad range of legume species and it can potentially stack multiple genes for targeted traits . Mutatio century . CRISPR-C genes) . This ca (SYMRK) .Identified or created genetic variations  can be transferred into breeding populations by employing genomics-assisted breeding tools  . The MASHigh-throughput phenotyping platforms such as spectral reflectance provide an opportunity to utilize the vast amount of genomic data accumulating in databases. Furthermore, by combining phenomics and envirotyping with refined field trails and experimental designs, one can significantly improve heritability estimation and reduce spatial heterogeneity in the field, and, as a result, achieve higher genetic gain. However, decoding and using the information from such tremendous dataset generated from phenomics demands data science, particularly artificial intelligence and machine learning, to develop model-based breeding methods .via rapid generation advancement tools such as, single seed decent, shuttle breeding, doubled haploid  offers an avenue to broadening the genetic base of these crops. Taking into account the number of wild accessions available in gene banks, a rational sampling of target donors would have to be achieved. Reduced representation sequencing and high-throughput phenotyping offer the advantage of characterizing all germplasm lines stored in gene banks . FurtherGlycine (GP-3) have not yet been fully exploited. A. cardenasii has probably been one of the most widely used sources of useful genes in groundnut to date, even if crosses involving other species have also been used.One of the major lessons learned from wild species is that the valuable alleles present in CWRs can only be identified when they are incorporated into cultivar background. Alien gene introgression have offered us dividend results in chickpeas. Still, the chickpea hybridization programme needs meticulous planning and implementation for full utilization of wild resources to break the yield plateau. Wild species of pigeonpea offer the potential use in hybridization programmes for introgression of multiple traits, including resistance to pod borers and diseases. Exploitation of GP-3 and GP-4 has been attempted in soyabean, but it ended at the amphidiploid stage . The novThe wild lentil taxa are potential reservoirs of valuable genes controlling several traits. To increase and sustain lentil production and productivity, new gene sources need to be identified and transferred from different GPs. To maximize and sustain common bean production, high-yielding, superior quality cultivars that are less dependent on water, fertilizer, and pesticides should be developed. A tiered breeding approach involving alien gene introgression, gene deployment, and multi-trait improvement programmes would be the most appropriate strategy to accomplish these goals. Cross incompatibilities are the major barriers to improve the productivity of faba bean. However, modern breeding tools succeeded in transferring genes across incompatible species and opened opportunities to develop high-yielding faba bean with reduced anti-nutritional factors. Marker-based assessment of genetic diversity in geographically specified germplasm sets of cultivated lupin species might speed up the process of assigning different genotypes to “Old World” and “New World” farmed species. Transgressive segregants identified in distant hybridisation programmes enable the development of high yielding pea cultivars.Vigna species helps to discover novel alleles for yield improvement in different Vigna crops. The application of genomic tools to harness genetic diversity from wild relatives of cowpea has yet to be accessed. Introducing molecular genetics tools into cowpea distant hybridization helps in bypassing the incompatibility barriers and introgressing desirable alleles or haplotypes. Horse gram and rice bean are the climate-resilient future crops for dryland areas. Present and future challenges in the breeding of forage legumes demand for the exploration of genetic variation in breeding programmes. The most dependable and desirable way to use their wild relatives is to introgress desirable traits into modern cultivars. Forage yield, quality, and persistence are the most important economic traits and have been shown to be good candidates for the future forage breeding.Exploitation of wild In conclusion, concerted integrated strategies should be used to identify and introgress the valuable genes housed in the CWRs of legume crops. This will provide us with a generation of a multitude of novel pre-bred lines that can be utilised in mainstream breeding to fight today’s climatic vagaries.IS, PS, GS, HA and SG conceived the idea. Authors have contributed in writing individual crops: GS (Pigeonpea), AD (Groundnut), PU (Chickpea), PKS (Lentil), GN (Moth bean and Rice bean), LG (Forage legumes), DK (Soybean), PK (Lupin), AK (Common bean), AT (Black gram), SS (Green gram), GK (Faba bean), MDP (Pea and Cowpea), and PH (Horse gram). SG and GS prepared the figures. HA, SG, RK, SK and AD collected the data and finalized the tables. HA, PS, GS, and SG compiled and edited the manuscript. IS supervised the study and finalized the manuscript. All authors contributed to the article and approved the submitted version.The authors would like to thank everyone who contributed to improving the quality of the review article at various stages of writing manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Quercus robur) bark and heather  herb extracts against common bovine mastitis pathogens.Bovine mastitis has a negative impact on animals, and improper antibiotic use has caused an increase in bacterial resistance. Therefore, medicinal plants could serve as an alternative treatment for this condition. Polyphenols have potential as antibiotic agents. Oak bark has long been used as a medicine and has shown antibacterial effects. Moreover, research on heather plant demonstrated that it has antibacterial properties. This study aimed to assess the antibacterial effects of oak  and minimal bactericidal concentration (MBC) were determined for the most effective extracts.Dried oak bark and heather herb were used to prepare extracts using 30%, 50%, and 70% ethanol and acetone as solvents. Their polyphenol content was determined using the Folin–Ciocalteu method. Bovine mastitis-inducing clinical isolates of S. aureus and Streptococcus strains, but no statistically significant difference was observed in activity against E. coli. The disk diffusion test showed that the oak bark extracts obtained using acetone and ethanol at 30% yielded the best results. However, the 70% acetone oak extract alone affected all types of bacteria. Further antibacterial tests of 70% acetone and 30% ethanol oak extracts revealed that the lowest MIC and MBC scores were against S. aureus strains and E. coli reference cultures. Conversely, the heather herb extracts exhibited broader activity against all types of bacteria, although better results were observed against Gram-positive bacteria. There was also a negative correlation between solvent concentration and antibacterial effect . The highest inhibition zone scores and broadest spectrum were observed in samples prepared in 30% ethanol. There was no statistically significant correlation between the phenolic content of plants and their antibacterial effects.Oak bark extracts had variable antibacterial effects against Oak bark and heather extracts could be used as potential antibacterial agents against bovine mastitis pathogens. Bovine mastitis has been a problem for over 100 years in the dairy industry , 3. PropS. aureus was observed , E. coli [ID. V-2019-252], S. agalactiae [ID. V-2019-171], S. uberis [ID. V-2019-243], Serratia liquefaciens [ID. V-2019-251], and S. aureus [ID. V-2019-256]) were obtained from the Latvia University of Life Sciences and Technology, Research Laboratory of Biotechnology. The antibacterial susceptibility of the clinical isolates of bovine mastitis is described in S. aureus (ATCC 25923) and E. coli (ATCC 25922), were provided by Riga Stradins University, Biology and Microbiology Faculty.Six bovine mastitis clinical isolates   agar or Mueller-Hinton agar with 5% sheep blood (Oxoid) (MHBA). After inoculation, four sterile filter paper disks (5 mm in diameter) were placed on the agar surface and impregnated with 15 μL of heather herb or oak bark semi-solid extracts, which were prepared by evaporating the solvent and were quantitatively moved (dissolved with water up to 5 mL) to vials.The MH and MHBA agar plates were incubated under suitable conditions in a thermostat  for 18 h at 37°C. Twenty-four hours later, the antibacterial properties of the selected samples were analyzed by measuring the sterile area (diameter) around the disk.The MIC of extracts was determined using the broth microdilution method, which is the standard antibacterial susceptibility testing method. Bacterial suspensions of 0.5 McFarland density were used to obtain a final suspension with a bacterial concentration of 106 Colony-forming unit (CFU)/mL.6 CFU/mL). The 96-well plates were incubated in a thermostat (Memmert) for 18 h at 37°C. The MIC values were considered as the lowest concentration of the tested compound that completely inhibited bacterial growth in the microdilution wells, as detected by the unaided eye. To determine the MBC, 10 μL of a subcultured sample were inoculated on an MH plate from each microdilution well; one above the MIC value and three below the MIC value. The MH plates were incubated in a thermostat (Memmert) for 18 h at 37°C. The MBC was defined as the lowest concentration that kills all bacteria. Each sample was tested 4 times, and a solution of broth without extract was used as the control.The quantitative assay of the antibacterial activity was performed in 96-well plates . The dry extract was dissolved in 2 mL of dimethyl sulfoxide. The first extract solution was diluted 10-fold with Mueller-Hinton broth  and was then used for 2-fold serial dilutions of the compound solutions in a 50 μL volume of Mueller-Hinton broth . Each well was seeded with 50 μL of the bacterial suspension (10® Excel® 2019 MSO (16.0.10382.20010) 32-bit  and analyzed using The IBM® SPSS® Statistics Software . Simple descriptive statistics  were used to investigate the significance of differences between samples. Spearman’s correlation analysis was also used. In all cases, significance was set at p < 0.05.Data were tabulated using MicrosoftS. aureus and Streptococcus strains, whereas no significant difference was detected against E. coli. The largest inhibition zones were observed for oak bark extracts prepared using 30% acetone and 30% ethanol. However, the oak extract in 70% acetone alone affected all types of bacteria tested, including S. liquefaciens, indicating a broader antibacterial spectrum. The Mann–Whitney–Wilcoxon statistical test was applied to assess differences between the effects of the extracts on bacteria according to the cell wall type. Oak bark extracts prepared using 50% ethanol, 70% ethanol, 50% acetone, and 70% acetone showed statistically significant differences among Gram-positive types of bacteria  compared with Gram-negative bacteria (E. coli and S. liquefaciens) (p < 0.05). There was no difference in effectiveness between the extracts prepared using 30% ethanol and 30% acetone as a solvent in comparison according to cell wall type (p > 0.05). Overall, the extracts had better antibacterial effects against S. aureus strains. There was no significant correlation between the amount of polyphenols in the extracts and their antibacterial effects in either group (p < 0.05). Although there was a positive correlation between ethanol extract type and TPC, a higher ethanol concentration yielded a greater TPC , whereas no such correlation was observed in the acetone extract group (p > 0.05). For further analysis, we chose the oak bark extract prepared with 30% ethanol, for its highest scores, and the oak bark extract prepared using 70% acetone, for its broadest spectrum.The results of the agar diffusion test are provided in S. aureus strains. The antibacterial effectiveness of the ethanol extracts against the various bacterial strains was as follows (highest to lowest): S. aureus V256 > S. aureus ATCC > E. coli V4 > S. uberis V243 > S. liquefaciens V251 > S. agalactiae V171 > E. coli ATCC > E. coli V252. Spearman’s correlation statistical analysis showed a negative correlation between solvent concentration and antibacterial effect . The highest measurement scores for the inhibition zones (10.5–25.3 mm in diameter) and broadest spectrum were observed in 30% ethanol samples. The acetone-based extracts of heather herb exhibited the following effectiveness: S. aureus V256 > S. aureus ATCC > E. coli V4 > S. uberis V243 > S. liquefaciens V251 > S. agalactiae V171 > E. coli ATCC > E. coli V252. The best results were observed for the 30% acetone extracts. There was also a correlation between extract solvent concentration and antibacterial effect  lower concentration of acetone extract had better activity. Analysis of relation between extract solvent concentration and TPC showed correlation . Because the TPC was greater in extracts in which the solvent had more organic solvent, whereas a better antibacterial effect was observed at lower solvent concentrations, other extracted active substances might be responsible for the antibacterial effects observed here. Further investigation of the heather herb extracts composition is needed. The MIC and MBC tests were performed using heather herb 30% ethanol extracts.The analysis of the extracts of heather herb  revealedThe heather herb dry extract starting mass was 730.6 mg, that of the oak bark dry extract with ethanol was –493.5 mg, and that of the oak bark dry extract with acetone was –479.2 mg.S. aureus strains and E. coli ATCC. When comparing the two types of oak extracts, we found that both yielded similar results, although the ethanol extracts had lower MIC and MBC values against S. aureus. The MBC of the heather extracts ranged from 2.28 to 73.06 mg/mL. The lowest MIC and MBC values were observed for S. aureus strains, whereas the highest values were observed for Streptococcus and Serratia bacteria.The results of MIC and MBC for both extracts are shown in S. aureus, E. coli, and Streptococcus strains, whereas more types of heather herb extracts showed antibacterial activity against Serratia spp.In this study, six herbal extracts from two plants were tested. The analysis of the results showed that antibacterial effects varied between plant extracts and bacteria types. Oak bark extracts produced larger inhibition zones than did heather extracts. All concentrations of the oak bark extracts had effects on Schinopsis brasiliensis water extracts had better activity against S. aureus, whereas Caryocar brasiliense ethanol extracts had the highest activity against E. coli, possibly because of a more diverse chemical composition. Because the removal of tannins decreased the antibacterial activity, polyphenols may be responsible for this effect. A study of oak species showed that Q. robur had better antibacterial activities than other oak species. Its bark extract contained ellagic acid, gallic acid, protocatechuic acid, and vanillic acid, and antibacterial activities against Pseudomonas aeruginosa, Micrococcus flavus, and E. coli. The antibacterial activities of phenolic standards of the ellagic and caffeic acids were comparable and were higher than those of Q. robur and Quercus macrocarpa extracts, respectively [Differences in the chemical composition of the plant extracts and the type of the bacterial cell wall could affect the antibacterial effect. In the case of the oak bark extracts, their effectiveness varied to a great extent according to the bacterial cell wall structure, and generally, they presented better antibacterial effects against Gram-positive bacteria. Similarly, a study of mastitis-inducing bacteria also revealed lower effects against Gram-negative bacteria . Schinopectively . An analectively . ConsideS. aureus. The antibacterial effects of polyphenols have been described in many studies. Polyphenols, phenolic acids, and their mixtures in plant extracts have been shown to exhibit antibacterial activity against Gram-positive bacteria (S. aureus and resistant strains) [Candida albicans) [S. aureus and Staphylococcus epidermidis were lower than 8 mg/mL, whereas aqueous and alcoholic extracts showed higher values. The alcoholic heather extract yielded the highest MIC values in P. aeruginosa, E. coli, Klebsiella pneumoniae, and C. albicans [S. aureus and Staphylococcus hominis were 0.1 μg/mL. The hydroalcoholic extract contained quercetin, kaempferol, and myricetin derivatives [S. aureus, but no activity against E. coli strains [Agrobacterium tumefaciens, Erwinia spp., K. pneumoniae, and P. aeruginosa, but did not show activity against E. coli and Proteus spp. [Proteus vulgaris, whereas the activities on E. coli and Enterococcus faecalis strains were similar and lower. Compared with ethyl acetate and water extracts, the aqueous extract displayed the strongest effect against all tested bacterial strains, although the ethyl acetate extract had better activity against P. vulgaris [et al. [et al. [The heather herb extracts showed a correlation between polyphenolics and antibacterial effects, as well as better antibacterial effects against Gram-positive bacteria, especially strains) , 35. Antstrains) . A literlbicans) . Many stlbicans) , 37–40reivatives . A scree strains . The chevulgaris . The obs [et al.  and Bubo [et al. . Because [et al. , variousThe standardization of MIC levels for medicinal plants has been discussed by many authors –44. TherS. aureus mastitis isolated strain and E. coli reference strain. For heather herb, the best antibacterial effects were observed using the 30% ethanol extract against S. aureus strains, whereas the weakest effects were observed against Streptococcus and Serratia bacteria. In the case of oak bark ethanol extracts, there was a correlation between the yield of polyphenols and their antibacterial effects, whereas no such correlation was observed for the acetone extracts of oak bark or heather herb extracts. Thus, further investigation of the active substances of oak bark and heather herb extracts and their connection to antibacterial effects is necessary.Oak bark and heather herb are natural sources of polyphenolic compounds. The data generated in this study showed that the type of solvent affected the extracted amounts of polyphenols and the antibacterial effects of the extracts. The results of this study showed that oak bark and heather herb could be potentially used as antibacterials in the case of bovine mastitis, as well as for multi-antibiotic-resistant strains. For oak bark, the highest bacterial inhibition zone scores were observed for 30% ethanol extracts, whereas the broadest spectrum was detected for 70% acetone extracts. For both types of extracts, the lowest MIC and MBC values were noted against both DB and RK: Conceptualized the aim of the study and design. RS and IS: Designed and performed the experiments. RS: Analyzed the data, wrote and edited the manuscript. DB, IS, and RK: Supervised the study and corrected the manuscript. All authors have read and approved the final manuscript.


Cathaya Chun & Kuang as a new genus of Pinaceae. They included one fossil species, C.loehri (Engelh. & Kink.) Chun & Kuang and two extant species, C.argyrophylla Chun & Kuang and C.nanchuanensis Chun & Kuang. Under Art. 40.1 of the Shenzhen Code, they did not validly publish Cathaya because they did not designate a type species for the generic name. Four years later (Cathaya (in Chinese) where they cited the 1958 publication and included one species C.argyrophylla (with C.nanchuanensis in synonymy) under Cathaya. According to Art. 40.3, they validated both the generic name Cathaya and C.argyrophylla in 1962. Further examination of the type collection and botanical history of the discovery of C.argyrophylla revealed that the type collection Guang-Fu-Lin-Qu Exped. 00198 consists of 11 duplicates in theSouth China Institute of Botany (IBSC)and 9 duplicates in other herbaria  and that the intended type specimen (IBSC0000004) consists of two gatherings: the bark, reproductive shoot and seed cones collected in 1955, whereas pollen-bearing cones were collected in 1956. We thus lectotypify the name C.argyrophylla with the specimen Guang-Fu-Lin-Qu Exped. 00198 (IBSC0000004 excl. pollen-bearing cones).In 1958, Chun and Kuang described rs later , they ag Cathayaargyrophylla Chun & Kuang is the sole extant species of Cathaya Chun & Kuang (Pinaceae). The genus was widely distributed during the Tertiary  00198 (IBSC) as the type of C.argyrophylla. We examined the collection and found 11 duplicates of ‘Expeditionis Kwangfu Lingchu’) 00198 in IBSC. Under Art. 40.2 Note 1, these duplicates should be considered as syntypes (duplicates in other herbaria are considered isosyntypes). Of the 11 duplicates, IBSC0000004 .China. Guangxi, Longsheng Xian., alt. 1400 m, on sunny rocky slopes, 16 May 1955, Photos are in


MicroRNAs (miRNAs) regulate gene expression by binding to mRNAs, and thus reduce target gene expression levels and expression variability, also known as ‘noise’. Single-cell RNA sequencing (scRNA-seq) technology has been used to study miRNA and mRNA expression in single cells. To evaluate scRNA-seq as a tool for investigating miRNA regulation, we analyzed datasets with both mRNA and miRNA expression in single-cell format. We found that miRNAs slightly reduce the expression noise of target genes; however, this effect is easily masked by strong technical noise from scRNA-seq. We suggest improvements aimed at reducing technical noise, which can be implemented in experimental design and computational analysis prior to running scRNA-seq. Our study provides useful guidelines for experiments that evaluate the effect of miRNAs on mRNA expression from scRNA-seq. In Single-cell RNA sequencing (scRNA-seq) is a rapidly developing technology that enables direct profiling of gene expression in single-cell resolution. Quantification of cell-to-cell variation using scRNA-seq provides deep insights into expression level heterogeneity and stochastic gene expression . A derivDrawbacks of scRNA-seq data include the occurrence of stochastic noise, which is due to the sampling method, the small amount of starting material, and sequencing inefficiency . ApproacIn GSE81287 , and sinhttps://ftp.ncbi.nlm.nih.gov/geo/series/GSE81nnn/GSE81287/suppl/GSE81287_smartseq2_refseq_rpkms.txt.gz accessed on 1 December 2019). Genes expressed in more than five naive ESCs or five primed ESCs were retained in the analysis.The raw read count and RPKM matrices of mRNAs were downloaded from GEO . Samples sequenced with 51 bp reads (41 bp RNA fragments) were used to calculate miRNA expression. To maintain consistency, we normalized read count similarly as with the other dataset used in our analysis, GSE114071. The raw read count of each miRNA was first divided by the sum count of all miRNAs. Normalized data lower than 10e−4 were further set to this minimum level. Then, all the data were log2-transformed for downstream analysis. Differentially expressed miRNAs were provided in the supplementary file of Omid R Faridani’s paper [https://static-content.springer.com/esm/art%3A10.1038%2Fnbt.3701/MediaObjects/41587_2016_BFnbt3701_MOESM4_ESM.pdf accessed on 10 April 2022), determined using SCDE [The molecule count matrix of miRNAs was downloaded from GEO . The normalized expression values represented the fraction that each miRNA constituted the total miRNA content. The authors did not provide the read count for gene expression matrices, and only the RPKM matrix is available on GEO. DCA requires a read count matrix as input; therefore, we downloaded the raw mRNA sequencing data from SRA (https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA464059 accessed on 1 December 2019) and quantified the gene expression ourselves. Downloaded sra files were decompressed using fastq-dump v2.8.0 and then mapped to hg38 transcriptome using STAR v2.5.3a [The normalized miRNA expression matrix was downloaded from GEO ( v2.5.3a . AlignedAs the coefficient of variation (CV) and the mean of normalized counts are linearly related in the logarithmic scale , we fittDeep Count Autoencoder (DCA) was designed by Eraslan et al. to denoise scRNA-seq datasets . In the http://www.targetscan.org/cgi-bin/targetscan/data_download.vert72.cgi accessed on 10 July 2018) [http://www.mirdb.org/download.html accessed on 10 July 2018) [http://mirtarbase.mbc.nctu.edu.tw/php/download.php accessed on 10 July 2018, latest version at https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/download.php accessed on 17 October 2022) [The target genes of each miRNA were predicted using TargetScanHuman v7.2 (ly 2018) . TargetSly 2018)  and miRTer 2022) . miRDB pp value) was determined by the Kolmogorov–Smirnov (KS) test for different distributions of expression noises, and the Student’s t-test (two-tailed) for values in naive ESCs, primed ESCs, and K562 cells. All statistical analyses were performed using R v4.0.2.Statistical significance  and to cancer cells . These tPrevious studies have described a general tendency of decreasing the expression noise of mRNAs, when the mean expression level increases ,26. We oThe low capture rate of single-cell sequencing technology results in drop-out events, which are failures to detect expressed genes. These events produce “false” zero count observation and may lead to biased estimations of mRNA expression. A number of methods have been proposed to denoise single-cell gene expression data and reconstruct biological signals. We chose DCA  to furthThe effect of miRNA on target gene expression noise may vary according to expression level. Therefore, we grouped miRNAs according to their expression levels . Four groups were partitioned based on a gradient of the mean fraction in the logarithmic scale . We firsp < 0.05) [t-test, Benjamini FDR > 0.05). In fact, the distributions of mean expression and noise were nearly identical for each miRNA . The log fold change of mean expression or noise of some mRNAs was greatly increased or reduced. However, none of the mRNAs of the naive ESCs or primed ESCs showed an overall inclination to increase or reduce mean expression and noise  that provide both single-cell mRNA and single-cell miRNA profiling, to analyze miRNA regulation on the expression noise of target mRNAs. We endeavored to properly measure the expression noise of mRNAs by accounting for the effect of mean expression level, and used a denoising algorithm, DCA, to further reconstruct biological noise. We compared the expression noise of target mRNAs, between miRNAs with different expression levels in a group of cells, and between differentially expressed miRNAs in a number of cell types. Although some results indicated that the total noise of predicted target mRNAs is anti-correlated with the expression level of miRNAs, other results revealed that miRNA regulation is generally mild and its noise reduction effect on target mRNAs is not easily discerned.To date, very few studies have combined single-cell mRNA and single-cell miRNA profiling. In our experiments, we attempted to identify the technical noise that is part of the total noise, in order to estimate true biological variability. However, the datasets we used were not generated with this intention; hence, a large proportion of the technical noise could not be separated from the biological noise. Several methods have been developed to quantify technical noise and biological noise from scRNA-seq data. According to these methods, bulk-cell mixture controls or external spike-ins are required in the decomposition of total noise ,26,27. HWe propose several guidelines for future studies on the effect of miRNAs on gene expression with scRNA-seq. (i) The use of a UMI-based protocol rather than a read-based protocol in mRNA sequencing will greatly reduce amplification noise . (ii) Th


Enterobacter is a major nosocomial genus of Enterobacteriaceae responsible for a variety of nosocomial infections, particularly in prolonged hospitalized patients in the intensive care units. Since current antibiotics have failed treating colistin- and carbapenem-resistant Enterobacteriaceae, efforts are underway to find suitable alternative strategies. Therefore, this study conducted a reverse vaccinology (RV) to identify novel and putative immunogenic targets using core proteome of 20 different sequence types (STs) of clinical Enterobacter spp. Moreover, we introduced a structural-based approach for exploration of potential vaccine candidates against the Enterobacteriaceae family using their conserved domain analysis.Enterobacter spp. with a similarity of ≥ 50%. Nine proteins with a score of ≥ 20 considered as the shortlisted proteins based on the quartile scoring method, including three TonB-dependent receptors, WP_008500981.1, WP_058690971.1 and WP_058679571.1; one YjbH domain-containing protein, WP_110108068.1; three flagellar proteins, WP_088207510.1, WP_033145204.1 and WP_058679632.1; one spore-coat U domain-containing protein, WP_039266612.1; and one DD-metalloendopeptidase family protein, WP_025912449.1. In this study, proteins WP_058690971.1 and WP_110108068.1 were detected as the top candidates with regard to immune stimulation and interactions with TLRs. However, their efficacy is remaining to be evaluated experimentally.A number of 2616 core coding sequences (CDSs) were retrieved from 20 clinical strains of Enterobacteriaceae family. These proteins belong to the iron acquisition system and possess all criteria of suitable vaccine targets. Therefore, they need to be specifically paid attention for vaccine development against clinically important members of Enterobacteriaceae family.Our investigation introduced common ferrichrome porins with high sequence similarity as potential vaccine candidates against the  Enterobacter is a genus of common Gram-negative, facultatively anaerobic, rod-shaped, non-spore-forming, and motile bacteria belonging to the Enterobacteriaceae family. There are 22 highly similar species within this genus, comprising Enterobacter cloacae complex (ECC) [E. cloacae, E. hormaechei, E. kobei, and E. roggenkampii are frequently isolated from clinical specimens. E. hormaechei and E. kobei account for more than 70% of community-acquired infections. E. cloacae is responsible for 10% of post-surgical peritonitis and four to five percent of nosocomial sepsis, pneumonia cases, and UTIs [ex (ECC) , 2. Someex (ECC) . Among mand UTIs . These sand UTIs .Enterobacter spp. are often the second or the third most common nosocomial Enterobacteriaceae harboring carbapenemase enzymes [The treatment of ECC infections is problematic due to the increasing resistance to various antimicrobial agents . Recent  enzymes . Therefo enzymes , 9. Howein silico identification of novel putative vaccine candidates against ECC up to now. In 2020, three outer-membrane porin proteins, including LpfC, OmpA, and FimD as well as an arginine transporter were introduced using a subtractive study of E. cloacae reference proteome [et al., used bioinformatics to design a multi-chimeric vaccine against E. xiangfangensis using the ferrichrome porin (FhuA) and peptidoglycan-associated lipoprotein  [The ability of conventional methods to discover immunological compounds of microorganisms is limited . The appproteome . More rein  . Also, Pin  .Enterobacteriaceae.In the present study, we aimed to introduce novel putative immunogenic candidates against different sequence types (STs) of clinical ECC strains using their core proteome. In addition, we investigated consensus protein structures with promising immunogenicity against other clinically important E. cloacae complex causing human clinical infections isolated from various clinical samples , different geographical regions and different periods of time (from 2008 to 2020). Please see Enterobacter spp. belonging to seven species including E. cloacae, E. hormaechei, E. kobei, E. roggenkampii, E. cancerogenus, E. asburiae, and E. bugandensis, considering all the above criteria for our analysis. The complete genomes of selected ECC strains were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/). The genetic characteristics and clinical information of strains have been provided in The dataset included almost all https://pubmlst.org/) was used to determine strains’ STs based on the allele numbers of six housekeeping genes . PubMLST is a collection of integrated population sequence data, containing the provenance, phylogenetic and phenotypic information for over 100 different microbial species and genera [E. cloacae subsp. cloacae ATCC 13047 (accession number: NC_014121.1) was used as the reference strain. To elucidate their phylogenetic distances, a Neighbor Joining (NJ) dendrogram based on the core genome multi-locus sequence typing (cg-MLST) was performed using cano-wgMLST_BacCompare (http://baccompare.imst.nsysu.edu.tw). This server enables users to determine the evolutionary relationship of bacteria using a whole genome (wg-) MLST and canonical MLST [PubMLST  as a database to predict the subcellular localization of proteins [http://www.enzim.hu/hmmtop/) database which is an automatic server for predicting transmembrane helices and the topology of proteins [All proteins were uploaded to PSORTb (proteins . Only ceproteins .http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) using a cut-off value of ≥ 0.5. VaxiJen is a server for alignment-independent prediction of protective antigens [https://webs.iiitd.edu.in/raghava/algpred2/batch.html) with a cut-off value of ≥ 0.5. This web tool has been developed for prediction of amino acid residues with allergenic characteristics in a protein sequence [The antigenicity of the proteins was predicted with the VaxiJen online tool (antigens . Subsequsequence .https://blast.ncbi.nlm.nih.gov) with the defined threshold (coverage ≥ 30% and identity ≥ 25%) was used to assess the homology of selected proteins versus human proteome  [PSI-BLAST (d: 9606) , 24. PSIhttps://web.expasy.org/protparam/). This web tool computes various physical and chemical parameters for a particular protein [http://www.violinet.org/vaxign2) and VICMpred (http://www.imtech.res.in/raghava/vicmpred/) databases, respectively. Vaxign predicts adhesion probability of proteins using an optimized SPAAN. It has been approved that the SPAAN prediction has a sensitivity of 89% and specificity of 100% [The molecular weight and other physiochemical properties of the selected proteins such as theoretical pI, half-life, instability and hydropathy indexes were estimated using the ProtParam tool ( protein . The adh of 100% . VICMpre of 100% .http://www.cbs.dtu.dk/services/BepiPred/) was used to identify linear B-cell epitopes of all previously selected proteins with a threshold of ≥ 0.61. This tool uses a random forest algorithm trained on epitopes of antibody-antigen protein structures [http://tools.iedb.org/tepitool/) from the Immune Epitope Database (IEDB) resource, was used to predict human MHC II binding sites (T-cell epitopes) with a cut-off value of the top 5% of peptides. The ratio of MHC II binding sites was calculated for each protein . This database uses experimental data on antibody and T-cell epitopes studied in humans, non-human primates, and other animal species in relation to infectious diseases, autoimmunity, allergies, and transplantation [The BepiPred v2.0 tool (ructures . For eacantation .https://robetta.bakerlab) [https://swissmodel.expasy.org/qmean/) and the ProSA-web server (https://prosa.services.came.sbg.ac.at/prosa.php). The QMEAN server provides the quality estimation of protein structure models by ranking potentially unreliable regions within them [In this section, the tertiary structures of the putative immunogenic candidates were first predicted using the Robetta web tool (akerlab) . The quahin them . ProSA-whin them .http://tools.iedb.org/ellipro/) with a threshold value of ≥ 0.8 [Next, the conformational B-cell epitopes of selected proteins were identified using the IEDB analysis resource , B-cell epitopes ratio, T-cell epitopes ratio, and the number of conformational B-cell epitopes. The sum of all scores for each protein was considered in the final score. Proteins with a score of ≥ 20 were considered putative immunogenic proteins and underwent further analysis. The quartile method measures the dispersion of values by dividing their distribution into lower, median, and upper quartiles to form four intervals. The quartile method provides us a rational target selection by comparing proteins based on several unweighted criteria at the same time .https://kraken.iac.rm.cnr.it/C-IMMSIM/index.php). C-ImmSim utilizes a position-specific scoring matrices (PSSM) originated from machine learning techniques to simulate immunological reactions [https://life.bsc.es/pid/pydockweb/default/index). This is a web-server for the prediction of protein-protein rigid interactions [The immune-reactivity of the shortlisted proteins were simulated using C-ImmSim (eactions . C-ImmSieactions . The numractions .https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml), was used for prediction of taxonomic relationships of proteins and their functions based on their conserved domains [Enterobacteriaceae family using this web tool.The conserved domain database, CDD  and E. cloacae strain PIMB10EC27 (1776), respectively. In addition, E. asburiae strain A2563 (27 CDSs) and E. cancerogenus strain 1428 (362 CDSs) had the minimum and maximum numbers of unique CDSs, respectively. See During core proteome analysis, a number of 2616 core CDSs were found among 20 clinical strains of The pan/core gene profiling of ECC strains indicated that the number of pan gene families differs meaningfully among the strains. While, the distribution of core gene families was almost close. See Among 2616 core proteins, only 48 proteins were outer membrane or extracellular. The number of the transmembrane helices in all proteins were ≤ 1 and no proteins weighed >110 kDa. Among them, 42 proteins were antigenic and five were allergenic. Overall, 37 antigenic, non-allergen proteins with no similarity to human proteins were subtracted for comparative analysis. The flowchart summarizing step by step subtraction of vaccine candidates has been shown in Nine proteins with a score of ≥ 20 were considered as the shortlisted proteins based on eight properties, including adhesion probability, antigenicity index, hydropathy index, instability index, functional class , B-cell epitopes ratio, T-cell epitopes ratio, and the number of conformational B-cell epitopes. See Molecular weights of proteins ranged from 26.13 to 82.87 kDa. All of them were predicted to be stable. WP_033145204.1 has the lowest instability index (14.73). TonB-dependent siderophore receptors, FlgE and FliK were virulence factors while other remaining proteins were involved in cellular processes. WP_008500981.1 and WP_025912449.1 had the maximum and minimum number of linear B-cell epitopes, respectively. The B-cell epitope ratio ranged from 0.10 (for WP_058690971.1) to 0.51 (for WP_058679632.1). WP_058690971.1 and WP_025912449.1 had the highest and lowest number of T-cell epitopes, respectively. WP_088207510.1 had the maximum T-cell epitope ratio (0.15). The number of conformational B-cell epitopes ranged from two (for WP_025912449.1) to eight (for WP_058679571.1). Physicochemical properties, functional class, number of linear and conformational B-cell epitopes, MHC II binding sites of shortlisted proteins have been presented in Enterobacter spp. The results of immune simulation and molecular docking have been summarized in The results of the immune simulation showed that five proteins including WP_058690971.1, WP_110108068.1, WP_033145204.1, WP_058679632.1 and WP_025912449.1 could stimulate the production of IFN-γ, IgM and IgG1. See The results obtained from CDD demonstrated that nine shortlisted proteins have superfamilies with three main functions: iron uptake , exopolysaccharide production (WP_110108068.1) and flagellar assembly . WP_039266612.1 had a spore-coat U domain involved in spore coating. WP_025912449.1 had domains responsible for peptidoglycan hydrolase . The full results of the CDD search have been summarized in Enterobacteriaceae. We detected this ferrichrome porin in major clinically important Enterobacteriaceae including Escherichia coli (WP_000124438.1 and WP_000124388.1) Klebsiella pneumoniae (WP_004178624.1 and WP_012542816.1), Salmonella enterica (WP_000113211.1), Shigella flexneri (WP_011587185.1) and Citrobacter koseri (WP_012134008.1). All selected proteins scored ≥ 20 based on the quartile method. See Enterobacteriaceae ferrichrome porins have been presented in We identified the conserved domain of the ferrichrome outer membrane transporter superfamily in WP_058690971.1 (TonB-dependent siderophore receptor). This superfamily exists in ferrichrome porin FhuA of all members of Enterobacter spp. to screen several immunogenic candidates in the first place. The results of cg-MLST indicated a close phylogenetic relationship among selected strains on the core genomic level and the number of their core CDSs were also in the same range. Therefore, by excluding the accessory genetic content; the core proteome represents the most prevalent proteins with the high conservancy [This study utilized the core proteome of various servancy . KeepingAn ideal or promising immunogenic candidate has several criteria. It should be exposed to extracellular space to effectively elicit protective immune responses. Besides, it has to be highly antigenic without any allergenicity. It needs to be highly conserved among widely distributed strains. In addition, an ideal immunogenic candidate should play an important role in the pathogenesis of bacteria and ideally be expressed during bacterial infection . We usedKlebsiella spp. fulfilled all vaccine parameters [Acinetobacter baumannii, two TonB-dependent receptors (BauA and BfnH) showed a partial protective effect on animal models. Aghajani et al. indicated that the mortality rate and bacterial load of A. baumannii in immunized mice was lower compared to that of the control group [Salmonella enterica, an in vivo-induced antigen, elicited a significant immune-protection against the lethal wild-type challenge [Neisseria meningitidis conferred protection against serogroup B [In this study, we introduced nine putative immunogenic proteins as vaccine candidates against ECC, three of which were TonB-dependent proteins. In Gram-negative bacteria, these receptors are involved in the transport and uptake of large substrates such as iron siderophore complexes and vitamin B12 . TonB-derameters . In Acinol group . YncD ofhallenge . Furtherogroup B .Enterobacteriaceae family. In this study, the in silico investigation on ferrichrome porins in different members of Enterobacteriaceae demonstrates that they all have the criteria of being vaccine candidates. Despite diverse amino acid compositions of these proteins, our data showed that they are all similar to WP_058690971.1 with regard to physicochemical properties as well as the number of T-cell and conformational B-cell epitopes. The results of immune simulation and molecular docking for these proteins were also close to WP_058690971.1. See We realized that the TonB-dependent siderophore receptor (WP_058690971.1) has the conserved domain of the ferrichrome outer membrane transporter superfamily belonging to the in silico analysis, ligand-gated channel proteins FhuA, BfnH, PapC, DcaP, FatA and IutA were introduced as the best vaccine candidates against A. baumannii. Therefore, it seems that proteins with beta-barrel structure belonging to ferrichrome outer membrane transporter and ligand-gated channel superfamilies have potential for vaccine development against bacterial pathogens. Accordingly, structure-dependent exploration of vaccine candidates in bacterial genomes could serve as a method for in silico vaccine design itself. This method reduces our need for the comprehensive analysis of whole genomes to approach an appropriate vaccine candidate.From the other side, proteins belonging to the ferrichrome outer membrane transporter superfamily also have conserved domain of the ligand-gated channel superfamily. This domain was found in four of our candidates including WP_008500981.1, WP_058690971.1, WP_058679571.1 and WP_110108068.1, which had the strongest interaction with TLRs. Their beta-strands are connected with loops on their extracellular side which are required for substrate attachment and transport . It seemYersinia pestis and Haemophilus influenzae lacking NlpD showed less virulence in murine models and failed in growth, respectively [in silico study on Pseudomonas aeruginosa, Morganella morganii and Clostridioides difficile introduced flagellar assembly proteins as valuable vaccine candidates [Similarly, NlpD conserve domain seem to be an important structure in proteins with vaccine potential. NlpD of WP_025912449.1 has an important role in cell stability. ectively , 48 Protndidates , 50, 51.Enterobacteriaceae. Proteins belonging to the ferrichrome outer membrane transporter superfamily possess all criteria of suitable vaccine candidates. Therefore, they need to be seriously paid attention to eliminate clinically important Enterobacteriaceae by vaccine development. Based on our study, structural-based exploration of vaccine candidates can be considered as a fast and more convenient method for in silico vaccine development.This study introduced nine putative immunogenic candidates against clinical members of ECC using a pan/core-genomic analysis and RV approach. Considering the immune-simulation results, WP_058690971.1, TonB-dependent siderophore receptor, and WP_110108068, YjbH domain-containing protein, were the best immunogenic candidates against ECC with respect to TLRs interactions. However, experimental assays are remaining to determine the efficacy of these candidates. Our investigation introduced ferrichrome porins as ideal vaccine candidates against members of S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Puccinia triticina determined using PacBio high-fidelity (HiFi) sequencing. This gapless assembly contains two sets of chromosomes, showing that one contig represents one chromosome. Comparisons of homologous chromosomes between the phased haplotypes revealed that highly frequent small-scale sequence divergence shapes haplotypic variation. Genome analyses of Puccinia triticina along with other rusts revealed that recent transposable element bursts and extensive segmental gene duplications synergistically highlight the evolution of chromosome structures. Comparative analysis of chromosomes indicated that frequent chromosomal rearrangements may act as a major contributor to rapid radiation of Pucciniales. This study presents the first gapless, phased assembly for a dikaryotic rust fungus and provides insights into adaptive evolution and species radiation in Pucciniales.Chromosome evolution drives species evolution, speciation, and adaptive radiation. Accurate genome assembly is crucial to understanding chromosome evolution of species, such as dikaryotic fungi. Rust fungi  in dikaryons represent the largest group of plant pathogens, but the evolutionary process of adaptive radiation in Pucciniales remains poorly understood. Here, we report a gapless genome for the wheat leaf rust fungus IMPORTANCE Rust fungi  are the largest group of plant pathogens. Adaptive radiation is a predominant feature in Pucciniales evolution. Chromosome evolution plays an important role in adaptive evolution. Accurate chromosome-scale assembly is required to understand the role of chromosome evolution in Pucciniales. We took advantage of HiFi sequencing to construct a gapless, phased genome for Puccinia triticina. Further analyses revealed that the evolution of chromosome structures in rust lineage is shaped by the combination of transposable element bursts and segmental gene duplications. Chromosome comparisons of Puccinia triticina and other rusts suggested that frequent chromosomal arrangements may make remarkable contributions to high species diversity of rust fungi. Our results present the first gapless genome for Pucciniales and shed light on the feature of chromosome evolution in Pucciniales. Advances in long-read sequencing technologies have accelerated complete assemblies for complex genomes , 2. High7–de novo assembly using graph-binning algorithm and maintaining the contiguity of each haplotype, is a powerful tool to obtain phased assembly with high accuracy and contiguity. Other methods are also available in different projects  in dikaryons are the most speciose natural group of plant pathogens, and many members of this group are the causal agents of prevalent diseases in agriculture and ecology , 13. In Puccinia triticina is the most widely distributed wheat rust fungus . Whole-genome sequencing of Pt15 yielded 19.83-Gb HiFi reads with a mean length of 14.6 kb and 29.42-Gb Illumina reads (Table S2). hifiasm   analysisWe predicted a total of 25,429 protein-coding genes for haplotype A and haplotype B  . We usedaK/sK ratio; median, 0.4381; mean, 0.5314) of secreted protein genes was higher than that of BUSCO genes . The proportion of positively selected allelic genes encoding secreted proteins was higher than that of BUSCO genes, and the same was true for the proportion of allelic genes that underwent more relaxation of purifying selection . The distribution of TEs in these rust species was similar to that in our assembly . The repetitive sequences were almost evenly distributed on each chromosome (Table S3). The predominant transposable element (TE) type was LTR retrotransposons (LTR-RTs), which mainly included Gypsy and Copia elements . We alsoassembly , indicatPuccinia species and M. larici-populina. The gigabase-sized genome in A. psidii was attributed to the recent burst and the accumulation over a long period. Given that different TE families in A. psidii showed the burst at discrete time scales  or large-scale gene duplication events, consistent with the observations for sK distribution of paralogues in P. graminis, P. striiformis f. sp. tritici, P. coronata f. sp. avenae, A. psidii, and M. larici-populina . Most gene duplicates in P. triticina and other rust species were dispersed duplicates, which may result from extensive segmental duplications, given that segmental duplication events often occur in eukaryotic genomes with a high TE content . The results showed that gene duplicates were mainly enriched in pathways including metabolism, biosynthesis, and ABC transporters, which contribute to lifestyle adaptation (aK/sK ratio >1) and relaxed purifying selection (aK/sK >0.8) were significantly higher after P. triticina-P. graminis divergence than before P. triticina-P. graminis divergence , suggesting that rapid evolution of species-specific genes in P. triticina contributed to environmental adaptability.We performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of gene duplicates in aptation . The numvergence . SpeciesP. triticina chromosomes as the reference genome to perform comparative analysis with high-quality genomes of five other rust species. By mapping the P. graminis, P. striiformis f. sp. tritici, and P. coronata f. sp. avenae chromosome sequences onto the P. triticina genome, we found that overall synteny existed between genomes and genes of these closely related species, and we identified multiple chromosomal rearrangements on different chromosomes, most of which were large inversions . No significant large rearrangement was observed, largely due to the larger phylogenetic distance between M. larici-populina and P. triticina. A high degree of gene order divergence may be attributed to recurrent rearrangements from their ancestral chromosomes. When the supersized assembly in A. psidii was compared to the P. triticina chromosomes, multiple chromosomal rearrangements were detected regardless of the effect of large numbers of TEs  was relatively lower than the previous haplotypes of other  species , 22. ThiP. triticina and five other rust species, suggesting that TEs and segmental duplications synergistically contribute to genomic adaptation in the rust lineage.The dramatic expansions of TEs and gene families have shaped adaptive evolution of rust fungi , 36. We P. triticina chromosomes, we uncovered frequent chromosomal rearrangements in species from Pucciniaceae, Sphaerophragmiaceae, and Melampsoraceae. Given that these species have the larger phylogenetic distance across the rust lineage (n = 18) may be highly conserved in a wide range of species of the rust lineage, although multiple assembled chromosomes . Hi-C contact maps were produced with HiC-Pro 3.10 (Genome assembly was first obtained with HiFi reads using hifiasm 0.15 . A BLASTequences . Heterozequences . HERA (2equences  was usedPro 3.10  and HiCEPro 3.10 .TTAGGG or CCCTAA within 200 bp. Genome completeness was assessed using BUSCO 3.02 with genome mode (https://github.com/JanaSperschneider/GenomeAssemblyTools).Telomeres were identified by searching multiple repeats of ome mode  based onome mode  was usedome mode  with IllDe novo repeats were identified with RepeatModeler 2.0.2a and the option -LTRStruct (http://www.repeatmasker.org) (https://github.com/nextgenusfs/funannotate). Transcript and protein evidence included full-length cDNA from germinated urediniospores in our genome project and transcript sequences from Puccinia genomes (Table S6). Ab initio gene prediction tool AUGUSTUS (TRStruct . These rker.org)  were merAUGUSTUS  was traiAUGUSTUS  and GlimAUGUSTUS  were useAUGUSTUS .Gene annotation was evaluated using BUSCO 3.02 with protein mode . SecreteT) of LTR-RTs was calculated based on the following formula: T = K/2r, where K is the divergence rate between the 5′ and 3′ ends of LTRs and r is the fungal substitution rate of 1.05 × 10−9 nucleotides per site per year (K was estimated using R package ape (http://ape-package.ird.fr/) with the K80 model.Intact LTR-RTs were identified with LTR_retriever  based onper year . K was eThe ANI was calculated using fastANI with “–fragLen 1 Mb” . Whole-gsK distributions of paralogues and orthologues were generated using the wgd pipeline (https://github.com/arzwa/wgd)  . KEGG pazwa/wgd)  with a Per 2.3.3  was usedhttps://synvisio.github.io/#/).A whole-genome pairwise alignment of genome assemblies was generated using d-Genies . HomologaK), the number of substitutions per synonymous site (sK), and aK/sK values were estimated with the YN00 model in the PAML program (sK values of >5 in the analysis were excluded due to saturation.The protein sequences were aligned using MUSCLE . Protein program . Ks valuPRJNA848635 and PRJNA848634. All sequencing reads are available in the SRA database under BioProject number PRJNA848635.The genome sequence of Pt15 has been deposited in the NCBI database under BioProject numbers


Pinus bungeana, an endangered and native coniferous tree species in China, has considerable timber and horticulture value. However, little is known about needle diseases in P. bungeana. A needle blight of P. bungeana has been observed in Hebei Province, China. P. bungeana inoculated with mycelial plugs of fungal isolates presented symptoms similar to those observed under field conditions. Ten virulent fungal isolates were identified as a small-spored Alternaria species based on morphological observations. Maximum likelihood and Bayesian phylogenetic analyses carried out with multilocus sequence typing of eight regions  assigned the pathogen to Alternaria alternata. This is the first report of A. alternata causing needle blight on P. bungeana in China. Pinus bungeana Zucc. ex Endl.), a distinctive and evergreen coniferous tree species within the genus Pinus of the family Pinaceae, is mainly distributed across warm temperate areas and the north-subtropical and middle subtropical climatic zones [P. bungeana plays a key role in local forest ecosystems, with strong resistance to sulphur dioxide, ozone and soot pollution in nature [Bunge’s pine  and sequence analysis of rDNA and protein coding genes, such as nuclear internal transcribed-spacer regions (ITS), the mitochondrial ribosomal large subunit (mtLSU), the mitochondrial small subunit (mtSSU), translation elongation factor (TEF), beta-tubulin, endopolygalacturonase (endoPG) genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, RNA polymerase second largest subunit (RPB2), plasma membrane ATPase, Alternaria major allergen gene (Alt a 1), calmodulin (CAL) and the anonymous genomic regions OPA1-3, OPA2-1 and OPA10-2 [Alternaria [A. alternata from A. tenuissima [In the past, patterns . This apl traits . Therefo OPA10-2 ,21,22,23ternaria ,11. Furtnuissima ,25.Alternaria alternata (Fr.) Keissl, the type species for the genus Alternaria, is able to cause diseases in over 100 plants, including vegetables, fruits, herbs and ornamental trees [al trees ,27,28,29al trees . It is aal trees ,30,31. IP. bungeana have been reported, including needle cast, trunk rot, needle rusts and twig blight [P. bungeana. The aim of this study was to identify the pathogens that cause needle blight in Bunge’s pine using morphological and molecular phylogenetic approaches and lay a theoretical foundation for the control of this pathogen.A few diseases related to g blight ,33. HoweIn September 2020, leaf spot of Bunge’s pine was found in Hebei Province, China. Thirty symptomatic tissues, the margin between the lesioned and healthy pine leaves, were cut into 3 to 5 mm long pieces. These tissues were surface sterilised for 45 s in ethanol (75%), washed thrice in sterilised distilled water and blotted dry with sterile paper. Pieces were transferred to 2% potato dextrose agar (PDA) in Petri plates, supplemented with ampicillin at 100 μg/mL and incubated at 25 °C (±1 °C) in the dark for 4 days. The single-spore isolation technique was used to obtain purified fungal isolates . Single-P. bungeana needles under controlled conditions. Asymptomatic needles of P. bungeana were surface disinfected and air-dried. Then, one piercing wound was made on the mid-upper region of each needle with a sterile needle . The inoculation was performed by placing mycelial blocks (5 mm in length) from actively growing colony margins onto each stab wound. Needles inoculated with noncolonised PDA blocks were treated as negative controls. Each control and treatment, involving three needles per replicate, was placed into a Petri dish (9 cm) with moist sterile filter paper and sealed with plastic wrap to maintain a high relative humidity. Then, they were incubated at 25 °C in a growth chamber with a 12 h photoperiod. The whole experiment was carried out three times.All isolates were cultured on PDA and used for virulence tests on detached P. koraiensis Sieb. et Zucc.).Ten isolates that were confirmed to be pathogenic on the detached needles were selected to determine pathogenicity on potted Bunge’s pine. Bunge’s pine needles were disinfected with 75% ethanol and air dried. Then, wound inoculation was conducted on 2-year-old potted, healthy Bunge’s pine with a sterile needle. The blocks (3 mm in length) from colony margins with actively growing mycelia of 3-day-old isolates were placed on each wounded site. Blocks were removed 2 days post-inoculation. PDA discs with no mycelia were used as controls. Three potted plants were treated as one replicate, and three replicates were used. The inoculated plants were placed into a controlled-environment greenhouse. The size of the disease spot was recorded until representative symptoms appeared. The same procedure was carried out on 2-month-old seedlings of Korean pine  to observe the colony morphology . Micromotef1, endoPG, OPA10-2, Alt a1, SSU, LSU and gapdh genes were amplified with the primer pairs V9G/ITS4 [2O with a thermal cycler under the conditions listed in The CTAB method described by Damm et al.  was usedV9G/ITS4 ,39, EF1-V9G/ITS4 , PG3/PG2V9G/ITS4 , OPA10-2V9G/ITS4 , Alt-forV9G/ITS4 , NS1/NS4V9G/ITS4 ,43, and V9G/ITS4 , respectAlternaria spp. described by Woudenberg et al. [https://www.ncbi.nlm.nih.gov/ (accessed on 10 February 2021)). A. alternantherae (CBS 124392) was used as the outgroup. The alignments of nucleotide sequences were obtained by using Clustal W in BioEdit software [The reference sequences of 43 g et al.  selectedsoftware . Treatinsoftware .7 generations), with FigTREE v1.4.4 used to view the phylogenetic trees.Phylogenetic trees of combined genes were constructed with two independent optimality search criteria, Bayesian inference (BI) phylogenetic analysis and maximum likelihood (ML) analysis. The ML analysis was performed using IQ-TREE , choosinSymptoms appeared on Bunge’s pine needles and enlarged constantly. The colour of infected needles is off-white at the early stage and then turns to light brown gradually, with dark-brown spots appearing one by one B,C. At tTen isolates were pathogenic, and healthy needles exhibited symptoms similar to those in nature, while mock-inoculated control needles showed no symptoms . Light-bn = 50), typically obclavate, subglobose and ellipsoid, with 1–5 transverse septa and 1–3 longitudinal septa that slightly constricted near several septa. The conidia were yellow–brown and later turned black–brown . For these ten isolates, the PCR amplification and sequencing of each gene generated product sizes were about 1072, 942, 733, 619, 259, 516, 491 and 753 or 777 bp, respectively. The alignments (including the gaps) for eight genes were 1021, 849, 522, 579, 241, 473, 448 and 634 bp in size, respectively. The ten sequences of isolates along with sequences from 33 Alternaria strains were concatenated for the construction of a phylogenetic tree. The alignment of the eight-locus concatenated dataset consisted of 4767 characters, with 4356 constant characters, 245 parsimony-uninformative characters, and 166 parsimony-informative characters.A multilocus phylogenetic analysis was conducted on ten pathogenic isolates based on the sequences from eight genes: SSU, LSU, ITS, A. alternata CBS 121455 and CBS 121336. Two phylogenetic analyses revealed that all isolates with aggressiveness showed >95% similarity to the A. alternata isolates reported previously.ML and BI analyses generated basically the same tree topology, which demonstrated that the evolutionary relationships of the fungus isolates were statistically supported. A single tree with bootstrap proportions (BP) from ML and Bayesian posterior probabilities (BPP) from BI was generated . The phyA. alternata was confirmed to be the causal agent of needle blight on Bunge’s pine in China. This is the first report of A. alternata on P. bungeana.Because of its ability to assimilate harmful material in the needles, graceful appearance and fine timber, Bunge’s pine plays an essential role in ecology and the economy. Needle blight disease can not only worsen the pine appearance but also influence apical dominance. The loss of apical dominance reduces wood quality. Moreover, the death of trees can occur in severe cases. Generally, the diseases affecting Bunge’s pine damage the economy and ecology. Based on morphological characteristics and molecular identification with phylogenetic analysis of multiple gene sequences, Alternaria spp. are frequently misidentified due to morphological overlap with A. alternata [Alternaria species. Conidial morphology is dependent on culture conditions and conidium age [Several small-spore lternata . The dimlternata , but werlternata . This phdium age . The numAlternaria spp. were reclassified by Woundenberg et al. [Alternaria isolates, and nine gene regions  were selected to distinguish sect. Alternaria more effectively [Alternaria due to lineage sorting, recombination and horizontal transfer [Alternaria sections for low resolution of species delimitation in small-spored Alternaria [rpb2, endoPG, tef1, Alt a 1 and OPA10-2) was able to separate A. alternata from the A. arborescens species complex [rpb2) was excluded, while additional molecular markers  were included in this study as proposed by Woudenberg et al. [Due to morphological variability and minimal molecular variation, the taxa of g et al. . Whole-gectively . Phylogetransfer . Multilog et al. . The comg et al. ,17,20.Alternaria alternata was reported as a ubiquitous pathogen in the great majority of crops and some broad-leaved trees [A. alternata is the most important mycotoxin-producing genus as a result of the wide reports of TA, AME, AOH, ALT and ATX produced [A. alternata can not only colonise the phylloplane but also penetrate into living leaves [A. alternata was reported to be the dominant endophytic fungal taxon in the bark and needles of Chinese oil pine (Pinus tabulaeformis Carr.) and isolated from various plants [Raffaelea quercus-mongolicae [Pinus sylvestris, A. alternata was found to be a common primary or secondary saprotroph [A. alternata to colonise Bunge’s pine needles without wounding, which may be related to plant resistance or pathogenic activity. The result of unwounded inoculation indicated that wounding may play a significant role in the pathogenicity of A. alternata. In nature, needles are prone to chafing, which can induce laceration as a result of the wind. This may provide an opportunity for A. alternata to be virulent. In addition, the virulence of A. alternata may have been obtained horizontally from a recent common saprophytic ancestor [ed trees ,54,55,56produced . In addig leaves . Neverths plants . In addingolicae . When exprotroph . It is dancestor .A. alternata, as a pathogen of pine needles, has never been reported. Although the thicker epidermis and cuticle of needles make it more difficult for fungi to invade plants, it is noteworthy that wounds appearing on needles may lead to disease prevalence. Pathogenicity test results indicate that A. alternata has the ability to infect other Pinus species, and it is necessary to investigate the distribution and propagation of the disease caused by A. alternata. A. alternata may pose a great threat to ecology because the hosts that the pathogen can invade are increasing, especially in Pinus species. Studies on the pathogenicity mechanism of A. alternata and disease management should be conducted in the future.According to previous studies,