Obligate Pathogen Research Articles

Methanotrophy by a Mycobacterium species that dominates a cave microbial ecosystem.

So far, only members of the bacterial phyla Proteobacteria and Verrucomicrobia are known to grow methanotrophically under aerobic conditions. Here we report that this metabolic trait is also observed within the Actinobacteria. We enriched and cultivated a methanotrophic Mycobacterium from an extremely acidic biofilm growing on a cave wall at a gaseous chemocline interface between volcanic gases and the Earth's atmosphere. This Mycobacterium, for which we propose the name Candidatus Mycobacterium methanotrophicum, is closely related to well-known obligate pathogens such as M. tuberculosis and M. leprae. Genomic and proteomic analyses revealed that Candidatus M. methanotrophicum expresses a full suite of enzymes required for aerobic growth on methane, including a soluble methane monooxygenase that catalyses the hydroxylation of methane to methanol and enzymes involved in formaldehyde fixation via the ribulose monophosphate pathway. Growth experiments combined with stable isotope probing using 13C-labelled methane confirmed that Candidatus M. methanotrophicum can grow on methane as a sole carbon and energy source. A broader survey based on 16S metabarcoding suggests that species closely related to Candidatus M. methanotrophicum may be abundant in low-pH, high-methane environments.

Fine mapping and candidate gene analysis of CRA3.7 conferring clubroot resistance in Brassica rapa.

In this study, we fine-mapped a clubroot resistance gene CRA3.7 in Chinese cabbage and developed its closely linked marker syau-InDel3008 for marker-assisted selection in CR cultivars breeding. Chinese cabbage is an important leafy vegetable rich in many nutrients widely grown in China. Clubroot disease caused by an obligate biotrophic pathogen Plasmodiophora brassicae was rapidly spread and challenged to Chinese cabbage production. A clubroot resistance (CR) gene, CRA3.7, was mapped on chromosome A03 of Brassica rapa. A Chinese cabbage line 'CR510', which harbor homozygous resistance locus CRA3.7 was selected from a BC4F3 family. 'CR510' was crossed with a clubroot susceptible Chinese cabbage inbred line '59-1'. Total 51 recombinant plants were identified from an F2 population including 3000 individuals. These recombinants were selfed and the clubroot resistance of F2/3 families was evaluated. Finally, a clubroot resistance gene CRA3.7 was fine-mapped to an interval of approximately 386 kb between marker syau-InDel3024 and syau-InDel3008. According to the reference genome, total 54 genes including five encoding the TIR-NBS-LRR proteins was annotated in the fine-mapped region. Further, nine candidate's gene expression in parental lines at 7, 14 and 21 days after inoculation of P. brassicae were evaluated. Bra019376, Bra019401, Bra019403 and Bra019410 are highly expressed in 'CR510' than '59-1'. Gene sequence of Bra019410 from 'CR510' was cloned and identified different from CRa. Therefore, Bra019376, Bra019401, Bra019403 and Bra019410 are the most likely candidates for CRA3.7. Our research provides a valuable germplasm resource against P. brassicae Pb3 and CRA3.7 closely linked marker for marker-assisted selection in CR cultivars breeding.

Virulence characteristics of Blumeria graminis f. sp. tritici and its genetic diversity by EST-SSR analyses.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (an obligate biotrophic pathogen) is a worldwide threat to wheat production that occurs over a wide geographic area in China. For monitoring genetic variation and virulence structure of Blumeria graminis f. sp. tritici in Liaoning, Heilongjiang, and Sichuan in 2015, 31 wheat lines with known Powdery mildew resistance genes and 2 EST-SSR markers were used to characterize the virulence and genetic diversity. Results indicated that 90% of all isolates were virulent on Pm3c, Pm3e, Pm3f, Pm4a, Pm5, Pm6 (Timgalen), Pm7, Pm16, Pm19, and Pm1 + 2 + 9 and 62.6% to 89.9% of isolates were virulent on Pm3a, Pm3b, Pm3d, Pm4b, Pm6 (Coker747), Pm8, Pm17, Pm20, Pm23, Pm30, Pm4 + 8, Pm5 + 6, Pm4b + mli, Pm2 + mld, Pm4 + 2X, Pm2 + 6. The Pm13 and PmXBD genes were effective against most collected isolates from Liaoning and Heilongjiang Provinces. Only Pm21 exhibited an immune infection response to all isolates. Furthermore, closely related isolates within each region were distinguished by cluster analyses using EST-SSR representing some gene exchanges and genetic relationships between the flora in Northeast China (Liaoning, Heilongjiang) and Sichuan. Only 45% of the isolates tested show a clear correlation between EST-SSR genetic polymorphisms and the frequency of virulence gene data. However, the EST-SSR polymorphism of isolated genes did not correspond to the virulence diversity of isolates in the single-gene lineage identification of hosts.

N-Acyl Amides from Neisseria meningitidis and Their Role in Sphingosine Receptor Signaling.

Neisseria meningitidis is a Gram-negative opportunistic pathogen that is responsible for causing human diseases with high mortality, such as septicemia and meningitis. The molecular mechanisms N. meningitidis employ to manipulate the immune system, translocate the mucosal and blood-brain barriers, and exert virulence are largely unknown. Human-associated bacteria encode a variety of bioactive small molecules with growing evidence for N-acyl amides as being important signaling molecules. However, only a small fraction of these metabolites has been identified from the human microbiota thus far. Here, we heterologously expressed an N-acyltransferase encoded in the obligate human pathogen N. meningitidis and identified 30 N-acyl amides with representative members serving as agonists of the G-protein coupled receptor (GPCR) S1PR4. During this process, we also characterized two mammalian N-acyl amides derived from the bovine medium. Both groups of metabolites suppress anti-inflammatory interleukin-10 signaling in human macrophage cell types, but they also suppress the pro-inflammatory interleukin-17A+ population in TH 17-differentiated CD4+ T cells.

Complete Genome Sequence of Terrisporobacter glycolicus Strain WW3900, Isolated from Influent Wastewater at a Research Center with Multiple-Species Research Animal Facilities.

Terrisporobacter glycolicus is an emerging obligate anaerobic pathogen. We report the 3.9-Mbp genome sequence of T. glycolicus strain WW3900, which was isolated from wastewater at a research center with laboratory animal facilities. The genome sequence predicted a biosynthetic gene cluster encoding an S-adenosylmethionine enzyme and other synthetic genes associated with potential antimicrobial producers.

Evolution of Mycobacterium tuberculosis drug resistance in the genomic era.

Mycobacterium tuberculosis has acquired drug resistance to all drugs that have been used against it, including those only recently introduced into clinical practice. Compared to other bacteria, it has a well conserved genome due to its role as an obligate human pathogen that has adapted to a niche over five to ten thousand years. These features facilitate reconstruction and dating of M. tuberculosis phylogenies, giving key insights into how resistance has been acquired and spread globally. Resistance to each new drug has occurred within five to ten years of clinical use and has occurred even more rapidly with recently introduced drugs. In most cases, resistance-conferring mutations come with a fitness cost, but this can be overcome by compensatory mutations which restore fitness to that of wild-type bacteria. It is likely that M. tuberculosis acquires drug resistance while maintaining limited genomic variability due the generation of low frequency within-host variation, combined with ongoing purifying selection causing loss of variants without a clear fitness advantage. However, variants that do confer an advantage, such as drug resistance, can increase in prevalence amongst all bacteria within a host and become the dominant clone. These resistant strains can then be transmitted leading to primary drug resistant infection in a new host. As many countries move towards genomic methods for diagnosis of M. tuberculosis infection and drug resistance, it is important to be aware of the implications for the evolution of resistance. Currently, understanding of resistance-conferring mutations is incomplete, and some targeted genetic diagnostics create their own selective pressures. We discuss an example where a rifampicin resistance-conferring mutation which was not routinely covered by standard testing became dominant. Finally, resistance to new drugs such as bedaquiline and delamanid is caused by individually rare mutations occurring across a large mutational genomic target that have been detected over a short time, and do not provide statistical power for genotype-phenotype correlation – in contrast to longer-established drugs that form the backbone of drug-sensitive antituberculosis therapy. Therefore, we need a different approach to identify resistance-conferring mutations of new drugs before their resistance becomes widespread, abrogating their usefulness.

Comparative Proteomic Analysis of Potato Roots from Resistant and Susceptible Cultivars to Spongospora subterranea Zoospore Root Attachment In Vitro.

Potato (Solanum tuberosum L.) exhibits broad variations in cultivar resistance to tuber and root infections by the soilborne, obligate biotrophic pathogen Spongospora subterranea. Host resistance has been recognised as an important approach in potato disease management, whereas zoospore root attachment has been identified as an effective indicator for the host resistance to Spongospora root infection. However, the mechanism of host resistance to zoospore root attachment is currently not well understood. To identify the potential basis for host resistance to S. subterranea at the molecular level, twelve potato cultivars differing in host resistance to zoospore root attachment were used for comparative proteomic analysis. In total, 3723 proteins were quantified from root samples across the twelve cultivars using a data-independent acquisition mass spectrometry approach. Statistical analysis identified 454 proteins that were significantly more abundant in the resistant cultivars; 626 proteins were more abundant in the susceptible cultivars. In resistant cultivars, functional annotation of the proteomic data indicated that Gene Ontology terms related to the oxidative stress and metabolic processes were significantly over-represented. KEGG pathway analysis identified that the phenylpropanoid biosynthesis pathway was associated with the resistant cultivars, suggesting the potential role of lignin biosynthesis in the host resistance to S. subterranea. Several enzymes involved in pectin biosynthesis and remodelling, such as pectinesterase and pectin acetylesterase, were more abundant in the resistant cultivars. Further investigation of the potential role of root cell wall pectin revealed that the pectinase treatment of roots resulted in a significant reduction in zoospore root attachment in both resistant and susceptible cultivars. This study provides a comprehensive proteome-level overview of resistance to S. subterranea zoospore root attachment across twelve potato cultivars and has identified a potential role for cell wall pectin in regulating zoospore root attachment.

Biochemical profiling of Brassica juncea doubled haploid mutant genotypes under white rust disease stress

White rust, caused by oomycete pathogen Albugo candida, is a serious disease for the crops of Brassicaceae family. It is an obligate pathogen responsible for up to 60% yield loss with combined infection of leaves and inflorescence across the globe. Biochemical variations in host plants can alter the content of proteins, sugars, phenols, iron content etc. enables to understand the biochemistry of host-pathogen interaction. Therefore, the present study was conducted during 2017-2019 at Amity University, Noida, Uttar Pradesh to evaluate the biochemical response of Brassica juncea var. Varuna and its doubled haploid (DH) mutant genotypes infected with A. candida causing white rust at cotyledonary leaf stage. The seven mutant genotypes were tested for pathogenic response against five white rust isolates (WRI). In the challenge assay, mutant genotypes C66 and C69 showed resistance response as compared to susceptible host B. juncea var. Varuna against different isolates on the basis of disease severity. The biochemical parameters were found to be high in the uninoculated genotypes in comparison to infected cotyledons of B. juncea genotypes. The biochemical profile emphasizes significant increase in content of protein, sugar, and phenol from highest in genotype C66 positively correlating with resistance response against pathogen in comparison to other mutant genotype C69 and susceptible check Varuna. The results suggest that factors conditioning the response of mutant genotypes to WRI may differ or control in different ways and triggers novel defense signaling.

Micrografting: An Old Dog Plays New Tricks in Obligate Plant Pathogens

Micrografting, which was developed almost 50 years ago, has long been used for virus eradication, micropropagation, regeneration, rejuvenation, and graft compatibility. Recently, micrografting has been used for studies of long-distance trafficking and signaling of molecules between scions and rootstocks. The graft transmissiveness of obligate plant pathogens, such as viruses, viroids, and phytoplasmas, facilitated the use of micrografting to study biological indexing and pathogen transmission, pathogen-induced graft incompatibility, and screening for the pathogen resistance during the past 20 years. The present study provides comprehensive information on the latter subjects. Finally, prospects are proposed to direct further studies.

Evolution of pathogenicity in obligate fungal pathogens and allied genera.

Obligate fungal pathogens (ascomycetes and basidiomycetes) and oomycetes are known to cause diseases in cereal crop plants. They feed on living cells and most of them have learned to bypass the host immune machinery. This paper discusses some of the factors that are associated with pathogenicity drawing examples from ascomycetes, basidiomycetes and oomycetes, with respect to their manifestation in crop plants. The comparisons have revealed a striking similarity in the three groups suggesting convergent pathways that have arisen from three lineages independently leading to an obligate lifestyle. This review has been written with the intent, that new information on adaptation strategies of biotrophs, modifications in pathogenicity strategies and population dynamics will improve current strategies for breeding with stable resistance.

Bps polysaccharide of Bordetella pertussis resists antimicrobial peptides by functioning as a dual surface shield and decoy and converts Escherichia coli into a respiratory pathogen.

Infections and disease caused by the obligate human pathogen Bordetella pertussis (Bp) are increasing, despite widespread vaccinations. The current acellular pertussis vaccines remain ineffective against nasopharyngeal colonization, carriage, and transmission. In this work, we tested the hypothesis that Bordetella polysaccharide (Bps), a member of the poly-β-1,6-N-acetyl-D-glucosamine (PNAG/PGA) family of polysaccharides promotes respiratory tract colonization of Bp by resisting killing by antimicrobial peptides (AMPs). Genetic deletion of the bpsA-D locus, as well as treatment with the specific glycoside hydrolase Dispersin B, increased susceptibility to AMP-mediated killing. Bps was found to be both cell surface-associated and released during laboratory growth and mouse infections. Addition of bacterial supernatants containing Bps and purified Bps increased B. pertussis resistance to AMPs. By utilizing ELISA, immunoblot and flow cytometry assays, we show that Bps functions as a dual surface shield and decoy. Co-inoculation of C57BL/6J mice with a Bps-proficient strain enhanced respiratory tract survival of the Bps-deficient strain. In combination, the presented results highlight the critical role of Bps as a central driver of B. pertussis pathogenesis. Heterologous production of Bps in a non-pathogenic E. coli K12 strain increased AMP resistance in vitro, and augmented bacterial survival and pathology in the mouse respiratory tract. These studies can serve as a foundation for other PNAG/PGA polysaccharides and for the development of an effective Bp vaccine that includes Bps.

Biotrophic Fungal Pathogens: a Critical Overview.

Biotrophic fungi are one group of heterogeneous organisms and these fungi differ in their traits like mode of nutrition, types of reproduction, and dispersal systems. Generally, based on the nutritional mode, fungi are classified into three broad categories, viz. biotrophs, necrotrophs, and hemi-biotrophs. Biotrophs derive their nutrients and energy from living plant cells and survive within the interstitial space of the cells. Biotrophic fungi cause serious crop diseases but are highly challenging to investigate and develop a treatment strategy. Blumeria (Erysiphe) graminis, Uromyces fabae, Ustilago maydis, Cladosporium fulvum, Puccinia graminis, and Phytophthora infestans are some of the significant biotrophic fungi that affect mainly plants. One among the biotrophic fungus, Pneumocystis jirovecii (Taphrinomycotina subphylum of the Ascomycota) exclusively a human pathogen, can cause lung diseases such as "pneumocystis." Biotrophic fungus widely parasitizing Solanaceae family crops (Tomato and potato) has done massive damage to the crops and has led to economic impact worldwide. During infection and for nutrient absorption, biotrophs develops external appendages such as appressoria or haustoria. The hyphae or appressorium adheres to the plant cell wall and collapses the layers for their nutrient absorption. The pathogen also secretes effector molecules to escape from the plant defense mechanism. Later, plants activate their primary and secondary defense mechanisms; however, the pathogen induces virulence genes to escape the host immune responses. Obligate biotrophic fungi pathogenicity has not been fully understood at the molecular level because of the complex interaction, recognition, and signaling with the host. This review summarizes the mechanism of infection in the host, and immune response to emphasize the understanding of the biotrophic fungal biology and pathogenesis in crops. Thus, the detailed review will pave the way to design methods to overcome the resistance of biotrophic fungi and develop disease-free crops.

Reappraising host cellular factors involved in attachment and entry to develop antiviral strategies against porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a highly contagious disease that brings tremendous economic losses to the global swine industry. As an intracellular obligate pathogen, PRRSV infects specific host cells to complete its replication cycle. PRRSV attachment to and entry into host cells are the first steps to initiate the replication cycle and involve multiple host cellular factors. In this review, we recapitulated recent advances on host cellular factors involved in PRRSV attachment and entry, and reappraised their functions in these two stages, which will deepen the understanding of PRRSV infection and provide insights to develop promising antiviral strategies against the virus.

Species composition and identification of pathogens of real powdery mildew of pumpkin crops

When cultivating pumpkin crops in most agricultural regions of the world, one of the most aggressive diseases is real powdery mildew, the damage of plants in which, under optimal soil and climatic conditions for the pathogen, reaches 100%. In the open and protected ground, among the cultivated representatives of the Cucurbitaceae family, the most widespread and harmful are the species Sphaerotheca fuliginea (Schlecht ex Fr.) Poll and Erysiphe cichoracearum f. sp. cucurbitacearum (DC ex Merat). In regions where both phytopathogens are present, mixed infections may occur, making identification difficult, and, accordingly, further plant protection measures. The pathogens of this powdery mildew of pumpkin crops have different requirements for temperature and humidity. During the growing season, there may be a change in species composition when conditions change, for example, when a new season begins. Symptoms of the disease in the form of a white or yellowish powdery plaque in the form of rounded spots (mainly on the leaves) can manifest themselves at all stages of plant development, the susceptibility of which increases by the beginning of fruiting. According to biological characteristics, S. fuliginea and E. cichoracearum are obligate biotrophic pathogens, unable to exist independently without living photosynthetic tissue, except for the wintering (sexual) stage of teleomorphs in the form of globular kleistothecia. At this stage, it is not difficult to differentiate pathogens by the morphology of kleistothecium. When analyzing the plant material of melons with the naked eye, S. fuliginea and E. cichoracearum cause similar symptoms of damage, but by microscopy these organisms can be distinguished by a complex of morphological characteristics. Taxonomically significant features are: the location of the mycelium, the formation of conidia (single or chain), the presence or absence of noticeable fibrosin bodies, the features of the appressoria, the size and shape of the conidia, as well as the position and type of their sprout tubes. The works of Russian and foreign scientists have shown the possibility of determining the types and physiological races of pathogens using an indirect method: reactions to differentiator plants. To date, about 30 physiological races of the pathogen Podosphaera xanthii (Sphaerotheca fuliginea) and 2 races of E. cichoracearum are known. However, this method does not always correlate with the methods of molecular analysis. Determination of the species composition of powdery mildew pathogens is a starting point for the development of a further strategy for the cultivation of pumpkin crops and in breeding work for resistance to this disease.

Nontuberculous Mycobacteria as Sapronoses: A Review.

Mycobacteria are a unique group of microorganisms. They are characterised by exceptional adaptability and durability. They are capable of colonisation and survival even in very unfavourable conditions. In addition to the well-known obligate human pathogens, Mycobacterium tuberculosis and M. leprae, more than 200 other species have been described. Most of them form a natural part of the microflora of the external environment and thrive in aquatic and soil environments especially. For many of the mycobacterial species associated with human disease, their natural source has not yet been identified. From an ecological point of view, mycobacteria are saprophytes, and their application in human and animal diseases is opportunistic. Most cases of human disease from saprophytic mycobacteria occur in immunocompromised individuals. This adaptability and resilience to environmental pressures makes treatment of mycobacterial diseases (most often sapronoses and less often zoonoses) and permanent eradication of mycobacteria from the environment very difficult. Saprophytic mycobacterial diseases (sapronoses) are chronic and recurrent due to the fact of repeated endogenous or exogenous re-exposure. Therefore, knowledge regarding their occurrence in soil and dust would aid in the prevention of saprophytic mycobacterioses. In conjunction, their presence and ecological significance in the environment can be revealed.

Indications and contraindications for convalescent plasma therapy in COVID-19 — a mini-review

Introduction: Convalescent plasma (CP), containing neutralizing antibodies (NAbs), is considered to be potential COVID-19 therapy. Favorable effects of passive administration of NAbs were already demonstrated for other viruses, including SARS and MERS viruses belonging to the Coronaviridae family. Aim of the study: This review aims to present and systematize current indications and contraindications for COVID-19 CP therapy. Material and methods: A systematic review of the literature was conducted using Pubmed, Wiley Online Library, Science Direct, and Medline databases. Results: At present, there is no strong evidence from randomized clinical trials to create universal eligibility criteria for CP treatment. Based on conducted studies and expert opinions, it is considered reasonable to administer CP to patients with a severe course of infection or with a risk of such a course, in the early stages of infection, optimally during 72h from symptom onset. Regarding contraindications to CP therapy, to a large extent, they are the same as commonly known and accepted contraindications for transfusion of routinely obtained plasma. Additionally, CP donations are subjected to obligatory pathogen reduction (PR). The exposure to photosensitizing substances, remaining in the CP following PR, might be associated with adverse effects. Conclusions: CP transfusion should not be a stand-alone treatment but should be used in clinical trials as a complement to other available COVID-19 therapies if possible. Patients should be informed that the effectiveness of CP has not been sufficiently proven, and that therapy is dependent on the physician’s decision and the availability of CP.

Functional characterization of Melampsora larici‐populina Hog1‐type MAPK in response to osmotic stress

AbstractThe obligate biotrophic pathogen Melampsora larici‐populina, responsible for poplar foliar rust disease, causes annual epidemics and severe damages to poplar plantations worldwide. Flexible responses to external osmotic pressure changes are important for the growth and survival of pathogens. In a previous study, we suggested that the Hog1‐type MAPK MlpHog1 in M. larici‐populina may play a role in infectious growth and responses to various environmental stresses. In the present study, we analysed its biological characteristics. MlpHog1 displayed a conserved coding sequence pattern among five strains from different regions of China. MlpHog1 restored the Hog1‐orthologue mutant defects responding to hyperosmotic stress in both Saccharomyces cerevisiae and Magnaporthe oryzae. Transient expression in wheat protoplasts revealed that MlpHog1 is localized in the cytoplasm and nucleus. These results indicate that MlpHog1 plays a positive role in responding to osmotic stress in the poplar rust M. larici‐populina.

Et tu, Neisseria? Conflicts of Interest Between Neisseria Species.

Neisseria meningitidis and Neisseria gonorrhoeae are two obligate human pathogens that have evolved to be uniquely adapted to their host. The meningococcus is frequently carried asymptomatically in the nasopharynx, while gonococcal infection of the urogenital tract usually elicits a marked local inflammatory response. Other members of the Neisseria genus are abundant in the upper airway where they could engage in co-operative or competitive interactions with both these pathogens. Here, we briefly outline the potential sites of contact between Neisseria spp. in the body, with emphasis on the upper airway, and describe the growing yet circumstantial evidence for antagonism from carriage studies and human volunteer challenge models with Neisseria lactamica. Recent laboratory studies have characterized antagonistic mechanisms that enable competition between Neisseria species. Several of these mechanisms, including Multiple Adhesin family (Mafs), Two Partner Secretion Systems, and Type VI secretion system, involve direct contact between bacteria; the genetic organisation of these systems, and the domain structure of their effector molecules have striking similarities. Additionally, DNA from one species of Neisseria can be toxic to another species, following uptake. More research is needed to define the full repertoire of antagonistic mechanisms in Neisseria spp., their distribution in strains, their range of activity, and contribution to survival in vivo. Understanding the targets of effectors could reveal how antagonistic relationships between close relatives shape subsequent interactions between pathogens and their hosts.

GWAS Reveals a Novel Candidate Gene CmoAP2/ERF in Pumpkin (Cucurbita moschata) Involved in Resistance to Powdery Mildew.

Pumpkin (Cucurbita moschata Duchesne ex Poir.) is a multipurpose cash crop rich in antioxidants, minerals, and vitamins; the seeds are also a good source of quality oils. However, pumpkin is susceptible to the fungus Podosphaera xanthii, an obligate biotrophic pathogen, which usually causes powdery mildew (PM) on both sides of the leaves and reduces photosynthesis. The fruits of infected plants are often smaller than usual and unpalatable. This study identified a novel gene that involves PM resistance in pumpkins through a genome-wide association study (GWAS). The allelic variation identified in the CmoCh3G009850 gene encoding for AP2-like ethylene-responsive transcription factor (CmoAP2/ERF) was proven to be involved in PM resistance. Validation of the GWAS data revealed six single nucleotide polymorphism (SNP) variations in the CmoAP2/ERF coding sequence between the resistant (IT 274039 [PMR]) and the susceptible (IT 278592 [PMS]). A polymorphic marker (dCAPS) was developed based on the allelic diversity to differentiate these two haplotypes. Genetic analysis in the segregating population derived from PMS and PMR parents provided evidence for an incomplete dominant gene-mediated PM resistance. Further, the qRT-PCR assay validated the elevated expression of CmoAP2/ERF during PM infection in the PMR compared with PMS. These results highlighted the pivotal role of CmoAP2/ERF in conferring resistance to PM and identifies it as a valuable molecular entity for breeding resistant pumpkin cultivars.

Beyond Nuclear Ribosomal DNA Sequences: Evolution, Taxonomy, and Closest Known Saprobic Relatives of Powdery Mildew Fungi (Erysiphaceae) Inferred From Their First Comprehensive Genome-Scale Phylogenetic Analyses.

Powdery mildew fungi (Erysiphaceae), common obligate biotrophic pathogens of many plants, including important agricultural and horticultural crops, represent a monophyletic lineage within the Ascomycota. Within the Erysiphaceae, molecular phylogenetic relationships and DNA-based species and genera delimitations were up to now mostly based on nuclear ribosomal DNA (nrDNA) phylogenies. This is the first comprehensive genome-scale phylogenetic analysis of this group using 751 single-copy orthologous sequences extracted from 24 selected powdery mildew genomes and 14 additional genomes from Helotiales, the fungal order that includes the Erysiphaceae. Representative genomes of all powdery mildew species with publicly available whole-genome sequencing (WGS) data that were of sufficient quality were included in the analyses. The 24 powdery mildew genomes included in the analysis represented 17 species belonging to eight out of 19 genera recognized within the Erysiphaceae. The epiphytic genera, all but one represented by multiple genomes, belonged each to distinct, well-supported lineages. Three hemiendophytic genera, each represented by a single genome, together formed the hemiendophytic lineage. Out of the 14 other taxa from the Helotiales, Arachnopeziza araneosa, a saprobic species, was the only taxon that grouped together with the 24 genome-sequenced powdery mildew fungi in a monophyletic clade. The close phylogenetic relationship between the Erysiphaceae and Arachnopeziza was revealed earlier by a phylogenomic study of the Leotiomycetes. Further analyses of powdery mildew and Arachnopeziza genomes may discover signatures of the evolutionary processes that have led to obligate biotrophy from a saprobic way of life. A separate phylogeny was produced using the 18S, 5.8S, and 28S nrDNA sequences of the same set of powdery mildew specimens and compared to the genome-scale phylogeny. The nrDNA phylogeny was largely congruent to the phylogeny produced using 751 orthologs. This part of the study has revealed multiple contamination and other quality issues in some powdery mildew genomes. We recommend that the presence of 28S, internal transcribed spacer (ITS), and 18S nrDNA sequences in powdery mildew WGS datasets that are identical to those determined by Sanger sequencing should be used to assess the quality of assemblies, in addition to the commonly used Benchmarking Universal Single-Copy Orthologs (BUSCO) values.