Parental Isolates Research Articles

Unveiling the resistance risk and resistance mechanism of florylpicoxamid in Corynespora cassiicola from cucumber

Florylpicoxamid, a QiI fungicide, demonstrates broad-spectrum activity against a wide range of phytopathogenic organisms belonging to the phyla Basidiomycota and Ascomycota. Nevertheless, the potential for resistance and the underlying resistance mechanisms of Corynespora cassiicola against florylpicoxamid are still not fully understood. We determined the baseline sensitivity levels of 101C. cassiicola isolates to florylpicoxamid. The EC50 values varied from 0.01 to 1.18 μg/mL with an average of 0.50 μg/mL. Laboratory-induced fungicide adaptation of nine wild-type isolates generated seven C. cassiicola mutants exhibiting high level of resistance to florylpicoxamid, all originating from a single parental isolate. The mutants maintained their resistance even after undergoing ten successive cultivations on a medium devoid of fungicides. No cross-resistance was detected between florylpicoxamid and pyraclostrobin, fluopyram, prochloraz, or propineb. Five of the resistant mutants showed an improved compound fitness index (CFI) relative to their parental isolate, whereas the remainder displayed either a reduced or comparable CFI. All seven of the resistant mutants displayed an A37V substitution within the CcCytb protein, which was responsible for the resistance to florylpicoxamid, as validated through molecular docking analysis. Furthermore, an allele-specific PCR (AS-PCR) method for detecting the CcCytbA37V mutation was successfully established. In summarize, the findings of this study indicate a moderate risk of C. cassiicola developing resistance to florylpicoxamid, with the A37V substitution in CcCytb playing a key role in this resistance, detectable through the use of specific AS-PCR primers.

Advanced in the Production of L-Ascorbic Acid using Biotechnological Processes

Over the past decade there has been increasing demand to develop alternatives to the Reichstein process, a largely chemical synthesis by which the majority of vitamin C (L-ascorbic acid) is produced. Many studies improve the biotransformation of Reichstein intermediate, but no natural bacterial strain is capable of catalyzing the biosynthesis of intermediates in single step fermentation. The study aimed to Isolate and identify bacterial strains have the ability to produce Ascorbic acid and detect their ability to be cultivated on plant wastes as substrate. In this study a total of Seventy-five different bacterial colonies of acetic acid bacteria were isolated from eight different samples. The most potent isolate was identified by 16S rRNA and phylogenetic tree relationship showed that the isolated strain was novel and named Gluconobacter oxydans st SW with accession number OP429626. The optimal levels of different nutritional and cultural variables were reached. The data revealed that the yield of ascorbic acid was increased from 7.17 g/l to 10.348 g/l. The effect of low doses of gamma radiation was tested. For Gluconobacter oxydans st. SW, the highest ascorbic acid yield was obtained was 20.480 g/l at activation radiation dose 1.2 kGy compared with 10.34 g/l from the parent isolate at the optimum fermentation conditions. It was observed that the radiated Gluconobacter oxydans st SW can adapt to the waste’s hydrolysate more than the un-radiated one. The highest yield of ascorbic acid (21.2 g/l) was obtained from fermentation broth containing 70% supplemented hydrolsate and 30% synthetic fermentation broth.

Family member experience with restricted family presence in Canadian PICUs: an interpretive descriptive study

PurposeWe performed this study to explore family member experiences with restrictions to family presence during their child’s PICU admission, leveraging the unique context of the COVID-19 pandemic to aid in future ethical and informed decision-making.MethodsQualitative interpretive descriptive study with family members of Canadian PICU patients admitted from March 2020 to April 2021 who experienced restricted family presence (RFP) policies. Respondents were purposively sampled for demographic-based maximum variation.We generated themes through inductive thematic analysis of open-ended interviews.ResultsFourteen parental figures from five regions across Canada participated. We identified four themes associated with the lived experience and impact of the restrictions on family members of critically ill children: (1) challenges to fulfilling the parent role, (2) isolation from primary supports during a time of crisis, (3) navigating increased logistical difficulties, and (4) seeking compassionate approaches within the healthcare system.Conclusion: RFP policies created a range of barriers for family members of critically ill children. Healthcare organizations and teams may play a role in removing barriers through consistent and empathetic application of rules with consideration for the duality of the parent role in PICUs, providing important post-pandemic policy and practice implications.What is known:• Parental presence with critically ill children may improve health outcomes for patients and their families.• Restrictions to family presence in PICUs continue to occur internationally and, during COVID-19, resulted in parental isolation, anxiety, and increased stress. What is new:• By isolating family members and removing supports, COVID-19-related restrictions in Canadian PICUs challenged family members’ ability to fulfil their parent roles while meeting their own needs. • Families need empathetic, advocacy-based approaches from clinicians and healthcare systems to maintain trust and therapeutic relationships in a family-centered organization.

Factors Associated with Suicidal Behavior in Adolescents: An Umbrella Review Using the Socio-Ecological Model.

Adolescent suicide is a critical social issue with profound and lasting individual and collective consequences. This umbrella review examines factors associated with adolescent suicidal behavior through the socioecological framework of prevention and seeks to identify gaps in the existing literature. The review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and included a comprehensive search of the of the Web of Science, Scopus, and Cochrane databases in both English and Spanish, covering the period from 2018 to 2024, using terms related to adolescence and suicidal behavior. Out of 6,138 articles identified, 37 met the quality criteria and were selected for analysis. The studies highlighted individual risk factors such as age, gender, belonging to ethnic or gender minorities, emotional disorders, self-destructive behavior, emotional regulation, and experience of physical or emotional pain. Interpersonal factors such as parental relationships, sexual abuse, social isolation, peer pressure, and loneliness were also found. However, the review revealed a worrying lack of studies on societal and community factors and a paucity of research focusing on protective factors. The study highlights the need to include factors related to the physical and social environments that influence health and behavior in future research, as well as to enhance the resources and strengths of adolescents.

Resistance risk assessment of Rhizoctonia solani to four fungicides.

Hexaconazole, thifluzamide, difenoconazole and azoxystrobin are widely used fungicides for the control of Rhizoctonia solani in China. However, few studies have assessed the sensitivity and resistance risk of R. solani to these four fungicides. The sensitivities of 126 R. solani isolates to hexaconazole, thifluzamide, difenoconazole and azoxystrobin were determined, with average half maximal effective concentration (EC50) values of 0.0386, 0.0659, 0.663 and 1.508 μg mL-1, respectively. Field resistance monitoring of the four fungicides showed that the three isolates had moderate resistance to difenoconazole. Resistant mutants to the four fungicides were obtained by fungicide adaptation, and resistance could be stably inherited by most mutants. Compared with those of the parent isolates, the biological characteristics of hexaconazole-resistant mutants exhibited enhanced or similar compound fitness index (CFI), whereas most of the other mutants displayed reduced or comparable CFI. There was evidence of positive cross-resistance between hexaconazole and difenoconazole. In the presence of fungicides, the expression of the CYP51 genes in hexaconazole- and difenoconazole-resistant mutants significantly increased, the expression of SDH genes in thifluzamide-resistant mutants significantly decreased, and the expression of the Cyt b gene in azoxystrobin-resistant mutants did not significantly change. Based on these data, we speculated that R. solani had a low-to-medium resistance risk to four fungicides. The change of target gene expression may be one of the reasons for fungicide resistance in R. solani. This study provides a theoretical basis for monitoring resistance emergence and developing resistance management strategies to control R. solani. © 2024 Society of Chemical Industry.

Older Gay Men's Sexual Experiences as Boys with Men: An Empirical and Narrative Analysis.

A dominant view among researchers is that boys' sexual interactions with adult men are traumatizing. In contrast, many gay men recall childhood sexual experiences with adult males as positive. The current study tested for both of these outcomes by examining recalled boyhood sexual experiences of older gay men. Interviews were conducted in the 1970-1980s, and thus, before the public view became popular that child-adult sexual interactions must be traumatizing. Quantitative analyses suggested that gay men with boyhood sexual experiences with adult males (n = 7, mostly aged 11-16 at first experience with men aged 20s to 50s) were as well adjusted as those without these experiences (n = 10), and these experiences were usually viewed positively (71%). Narrative analyses indicated that adjustment problems in adulthood were not related to these early sexual experiences but to other factors such as parental abusiveness, societal intolerance, internalized homophobia, or social isolation.

Potential Mechanisms of Hexaconazole Resistance in Fusarium graminearum.

Fusarium head blight (FHB) caused by Fusarium graminearum is a serious fungal disease that can dramatically impact wheat production. At present, disease control is mainly achieved by the use of chemical fungicides. Hexaconazole (IUPAC name: 2(2,4-dichlorophenyl)-1-(1,2,4-triazol-1-yl)hexan-2-ol) is a widely used triazole fungicide, but the sensitivity of F. graminearum to this compound has yet to be established. The current study found that the EC50 values of 83 field isolates of F. graminearum ranged between 0.06 and 4.33 μg/ml, with an average EC50 value of 0.78 μg/ml. Assessment of four hexaconazole-resistant laboratory mutants of F. graminearum revealed that their mycelial growth and pathogenicity were reduced compared with their parental isolates and that asexual reproduction was reduced by resistance to hexaconazole. Meanwhile, the mutants appeared to be more sensitive to abiotic stress associated with SDS and H2O2, while their tolerance to high concentrations of Congo red, and Na+ and K+ increased. Molecular analysis revealed numerous point mutations in the FgCYP51 target genes that resulted in amino acid substitutions, including L92P and N123S in FgCYP51A, as well as M331V, F62L, Q252R, A412V, and V488A in FgCYP51B, and S28L, S256A, V307A, D287G, and R515I in FgCYP51C, three of which (S28L, S256A, and V307A) were conserved in all of the resistant mutants. Furthermore, the expression of the FgCYP51 genes in resistant strains was found to be significantly (P < 0.05) reduced compared with their sensitive parental isolates. Positive cross-resistance was found between hexaconazole and metconazole and flutriafol, as well as with the diarylamine fungicide fluazinam, but not with propiconazole, and the phenylpyrrole fungicide fludioxonil, or with tebuconazole, which actually exhibited negative cross-resistance. These results provide valuable insight into resistant mechanisms to triazole fungicides in F. graminearum, as well as the appropriate selection of fungicide combinations for the control of FHB to ensure optimal wheat production.

Process-induced protein aggregates influenced pea globulins’ structure formation upon heating

Pea protein ingredients play key role in formulations of plant-based foods. However, functional properties of pea ingredients are inconsistent depending on extraction process. Protein aggregation occurs simultaneously during protein extraction, thus examining the protein aggregated states as induced by processing is essential for better process design. This study investigated the influence of process-induced protein aggregated states on structure formation upon heating of pea protein ingredients. Combining rheological, spectroscopic, and microscopic techniques, the mechanisms underlining heat-induced structure formation have been unveiled from microscopic to macroscopic scales. The salt-extracted isolate (PPI*) where protein aggregation was minimized, developed mesh-like structure through intermolecular protein-protein interaction upon gelling similar to commercial protein concentrate (PPC). In turn, commercial isolate (PPI) as appeared as microscopic particles, formed gel through accumulation of protein particles with no structure development. The aggregated states of PPI* and PPI seemed to dictate vicilin and legumin purification by means of anion exchange chromatography. Purification process promoted intermolecular protein aggregate structures. However, these purified fractions regardless of parent isolates showed similar structure development as PPC and PPI* during gelling. Monitoring protein aggregation during extraction process can be a key to limit functional property variation in pea protein ingredients.

Resistant risk and resistance mechanism of florylpicoxamid in Colletotrichum gloeosporioides isolated from Chinese walnut

Colletotrichum gloeosporioides is the causal pathogen for the devastating walnuts anthracnose. A novel quinone inside inhibitor (QiI) fungicide florylpicoxamid has strong inhibitory efficacy against C. gloeosporioides. This study looked into the resistance risk and mechanism of C. gloeosporioides to florylpicoxamid. The basal level sensitivity of C. gloeosporioides isolates (n = 102) to florylpicoxamid was established with an average 50% mycelial growth inhibition concentration (EC50) value of 0.069 ± 0.035 μg/mL. Six stable florylpicoxamid-resistant mutants with resistance factors of >1000 were produced. The fitness of every mutant was much lower than that of their parental isolates. In general, the resistance risk of C. gloeosporioides to florylpicoxamid would be moderate. Molecular docking results revealed that the amino acid substitutions A37V, and S207L in CgCytb lead to a reduction in the binding affinity between florylpicoxamid and CgCytb, indicating that these two mutations (S207L and A37V in CgCytb) indeed confer florylpicoxamid resistance in C. gloeosporioides. These findings offer a fresh viewpoint on the mechanism underlying QiI fungicide resistance and could support the prudent application of florylpicoxamid in the future to combat walnut anthracnose.

Alteration in the Morphological and Transcriptomic Profiles of Acinetobacter baumannii after Exposure to Colistin.

Acinetobacter baumannii is often highly resistant to multiple antimicrobials, posing a risk of treatment failure, and colistin is a "last resort" for treatment of the bacterial infection. However, colistin resistance is easily developed when the bacteria are exposed to the drug, and a comprehensive analysis of colistin-mediated changes in colistin-susceptible and -resistant A. baumannii is needed. In this study, using an isogenic pair of colistin-susceptible and -resistant A. baumannii isolates, alterations in morphologic and transcriptomic characteristics associated with colistin resistance were revealed. Whole-genome sequencing showed that the resistant isolate harbored a PmrBL208F mutation conferring colistin resistance, and all other single-nucleotide alterations were located in intergenic regions. Using scanning electron microscopy, it was determined that the colistin-resistant mutant had a shorter cell length than the parental isolate, and filamented cells were found when both isolates were exposed to the inhibitory concentration of colistin. When the isolates were treated with inhibitory concentrations of colistin, more than 80% of the genes were upregulated, including genes associated with antioxidative stress response pathways. The results elucidate the morphological difference between the colistin-susceptible and -resistant isolates and different colistin-mediated responses in A. baumannii isolates depending on their susceptibility to this drug.

Genetic Alterations Associated with Colistin Resistance Development in Escherichia coli.

Background: The increased incidence of infections due to multidrug-resistant Gram-negative bacteria has led to the renewed interest in the use of 'forgotten' antibiotics such as colistin. In this work, we studied the chromosomal colistin resistance mechanisms among laboratory-induced colistin-resistant Escherichia coli isolates. Methods: Three colistin-susceptible (ColS) clinical isolates of E. coli assigning to ST131, ST405, and ST361 were exposed to successively increasing concentrations of colistin. The nucleotide sequences of pmrA, pmrB, pmrD, phoP, phoQ, and mgrB genes were determined. The fitness burden associated with colistin resistance acquisition was determined by measuring the in vitro growth rate. Results: Colistin resistance induction resulted in 16-64 times increase in colistin MICs in mutants (n = 8) compared with parental isolates. Analysis of chromosomal genes in colistin-resistant mutants compared with those of ColS ancestors revealed genetic alterations confined to PmrAB two-component system and included PmrA G53R/R81S/L105P and PmrB E121K/E121A/A159P/A159V/G302E changes. The PmrB E121 was found as a critical position for colistin resistance development being altered in three mutants with different ancestors. The acquired colistin-resistance phenotype was stable following 10 consecutive passages in the absence of selective pressure of colistin and it did not alter the susceptibility of mutants to other antimicrobial agents. All mutants exhibited growth rates similar to their respective ColS ancestors, except for one isolate, which revealed a significant growth defect. Conclusion: Our results revealed that colistin resistance in E. coli was more related to PmrAB alterations, which did not impose a fitness cost in most cases.

Variability in Chromosome 1 of Select Moroccan Pyrenophora teres f. teres Isolates Overcomes a Highly Effective Barley Chromosome 6H Source of Resistance.

Barley net form net blotch (NFNB) is a destructive foliar disease caused by Pyrenophora teres f. teres. Barley line CIho5791, which harbors the broadly effective chromosome 6H resistance gene Rpt5, displays dominant resistance to P. teres f. teres. To genetically characterize P. teres f. teres avirulence/virulence on the barley line CIho5791, we generated a P. teres f. teres mapping population using a cross between the Moroccan CIho5791-virulent isolate MorSM40-3 and the avirulent reference isolate 0-1. Full genome sequences were generated for 103 progenies. Saturated chromosome-level genetic maps were generated, and quantitative trait locus (QTL) mapping identified two major QTL associated with P. teres f. teres avirulence/virulence on CIho5791. The most significant QTL mapped to chromosome (Ch) 1, where the virulent allele was contributed by MorSM40-3. A second QTL mapped to Ch8; however, this virulent allele was contributed by the avirulent parent 0-1. The Ch1 and Ch8 loci accounted for 27 and 15% of the disease variation, respectively, and the avirulent allele at the Ch1 locus was epistatic over the virulent allele at the Ch8 locus. As a validation, we used a natural P. teres f. teres population in a genome-wide association study that identified the same Ch1 and Ch8 loci. We then generated a new reference quality genome assembly of parental isolate MorSM40-3 with annotation supported by deep transcriptome sequencing of infection time points. The annotation identified candidate genes predicted to encode small, secreted proteins, one or more of which are likely responsible for overcoming the CIho5791 resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.

Care, connection, and social distancing: The challenges of baby loss during the COVID-19 pandemic in Aotearoa New Zealand

ProblemThe COVID-19 pandemic hindered access to routine healthcare globally, prompting concerns about possible increases in pregnancy loss and perinatal death. BackgroundPUDDLES is an international collaboration exploring the impact of the COVID-19 pandemic on parents who experience pregnancy loss and perinatal death in seven countries, including Aotearoa New Zealand. AimTo explore parents’ experiences of access to healthcare services and support following baby loss during the COVID-19 pandemic in Aotearoa New Zealand. MethodsWe conducted in-depth, semi-structured interviews with 26 bereaved parents, including 20 birthing mothers, and six non-birthing parents (one mother and five fathers). Types of loss included 15 stillbirths, four late miscarriages, and one neonatal death. Participant ethnicities were broadly representative of Aotearoa New Zealand’s multi-ethnic society. Data were analysed using Template Analysis. FindingsAnalysis revealed five themes relating to pandemic impact on bereaved parent’s experiences. These were: ‘Distanced and Impersonal care’; ‘Navigating Hospital Rules’; Exclusion of Non-birthing Parents; ‘Hindered Access to Social Support’; and ‘Continuity of Relational Care’. DiscussionThe COVID-19 pandemic exacerbated isolation of bereaved parents through perceived impersonal care by healthcare professionals and restrictions on movement hindered access to social and cultural support. Compassionate bending of the rules by healthcare professionals and community postnatal visits by continuity of care midwives following the bereavement appeared to be mitigating factors. ConclusionSocial isolation is an added challenge for parents experiencing baby loss during a pandemic, which may be mitigated by flexible and compassionate care from healthcare professionals, especially continuity of care midwives.

Online Child Sexual Exploitation and Abuse of Children and Adolescents with Disabilities: A Systematic Review

Online child sexual exploitation and abuse (OCSEA) is a rising global problem affecting children and adolescents worldwide. Despite the escalating prevalence of OCSEA, there is limited research specifically focusing on children and adolescents with disabilities. To bridge this gap, this systematic review was conducted to identify the prevalence, nature and associated risk factors of OCSEA of children and adolescents with disabilities. Following the PRISMA Statement, this systematic review included scientific evidence from 12 academic databases and the gray literature published between 1993 and 2023. A total of 13 studies were extracted, and thematic analysis was conducted to analyze the data. The findings of this systematic review reveal the characteristics such as the gender, age and type of disabilities of OCSEA victims. Perpetrators use diverse techniques, including online grooming, manipulation, and cyber-threats, resulting in consequences such as mental health issues and social isolation of victims. The associated risk factors include lack of parental monitoring, social isolation, and low risk perception. The results of this research provide crucial insights into OCSEA of children and adolescents with disabilities, emphasizing the need for targeted interventions and further exploration in this understudied area.

Resistance risk assessment for benzovindiflupyr in Sclerotium rolfsii and transmission of resistance genes among population.

Sclerotium rolfsii is a destructive soil-borne fungal pathogen which is distributed worldwide. In previous study, the succinate dehydrogenase inhibitor (SDHI) fungicide benzovindiflupyr has been identified for its great antifungal activity against Sclerotium rolfsii. This study is aimed to investigate the resistance risk and mechanism of benzovindiflupyr in Sclerotium rolfsii. Eight stable benzovindiflupyr-resistant isolates were generated by fungicide adaptation. Although the obtained eight resistant isolates have a stronger pathogenicity than the parental sensitive isolate, they have a fitness penalty in the mycelial growth and sclerotia formation compared to the parental isolate. A positive cross-resistance existed in the resistant isolates between benzovindiflupyr and thifluzamide, carboxin, boscalid and isopyrazam. Three-point mutations, including SdhBN180D, SdhCQ68E and SdhDH103Y, were identified in the benzovindiflupyr-resistant isolates. However, molecular docking analysis indicated that only SdhDH103Y could influence the sensitivity of Sclerotium rolfsii to benzovindiflupyr. After mycelial co-incubation of resistant isolates and the sensitive isolate, resistance genes may be transmitted to the sensitive isolate. The in vivo efficacy of benzovindiflupyr and thifluzamide against benzovindiflupyr-resistant isolates was a little lower than that against the sensitive isolate but with no significant difference. The results suggested a low to medium resistance risk of Sclerotium rolfsii to benzovindiflupyr. However, once resistance occurs, it is possible to spread in the population of Sclerotium rolfsii. This study is helpful to understanding the risk and mechanism of resistance to benzovindiflupyr in multinucleate pathogens such as Sclerotium rolfsii. © 2024 Society of Chemical Industry.

Revealing inheritance of a Xinjiang isolate BGTB-1 of Puccinia striiformis f. sp. tritici and the shift of pathogenicity from avirulence to virulence at heterozygous AvrYr5 locus

In China, Xinjiang is a relatively independent epidemic region of wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, due to great genetic divergence of Xinjiang with other inland epidemic regions. In this region, race evolution was usually slower than inland populations. However, many new races have recently been found, and there needs to be more understanding of the virulence evolution of the Xinjiang population. So, in this study, a 65 sexual progenies, derived from a Xinjiang single-urediospore isolate BGTB-1 of P. striiformis f. sp. tritici by selfing on alternate host barberry (Berberis aggregata). It was phenotyped on the 25 single Yr lines, and genotyped by 19 kompetitive allele-specific PCR-single nucleotide polymorphism (KASP-SNP) markers. As a result, the 65 progenies were identified as 56 various virulence patterns (VPs), and neither of which was identical to the parental isolate, showed 100% virulence variation. Compared with the parental isolate, of all progenies, 39 (60.0%) had increased virulence, and 18 (27.7%) had decreased virulence. All progenies exhibited avirulence to Yr10, Yr15, Yr32, and YrTr1 loci, and avirulence and virulence segregation at the remaining 21 Yr resistance loci. The results showed avirulence to Yr5, Yr7, and Yr76 (A:V≍3:1) loci is controlled by a single dominant gene, and that to Yr6, Yr25, and Yr44 (A:V≍1:3) loci by a single recessive gene. However, avirulence to the remaining 15 resistant loci including Yr1, Yr2, Yr3, Yr4, Yr8, Yr9, Yr17, Yr26 (=Yr24), Yr28, Yr29, Yr43, YrSp, Yr27, YrA, and YrExp2, with various avirulence and virulence segregation ratios, is controlled by two genes with different gene effects, indicating complex genetic traits of the parental isolate. Totally, 65 dissimilar genotypes detected among progenies using overall molecular markers, by which a linkage map was constructed, with a genetic distance of 441.0 cM. Interestingly, although the parental isolate was avirulent to Yr5, but 17 progenies showed virulence, showing the change of pathogenicity from avirulence to virulence at this resistance locus. It was for the first time to report that progenies with virulence to Yr5 produced sexually from avirulent parental isolate at this resistance locus. To our knowledge, this study offers an insight into inheritance, sexual reproduction and virulence variation of P. striiformis f. sp. tritici in Xinjiang, facilitating us to understand evolution of the rust pathogen in this region and accounting for Xinjiang population distinguished form other inland populations. Additionally, it is necessary to further confirm the roles of sexual reproduction in the emergence of new races and affecting population genetic diversity of P. striiformis f. sp. tritici under natural conditions in Xinjiang.

Characteristics of fluopicolide-resistance mutants in Phytophthora nicotianae, the pathogen causing black shank disease in tobacco

Black shank, a devastating disease in tobacco production worldwide, is caused by the oomycete plant pathogen Phytophthora nicotianae. Fluopicolide is a pyridinylmethyl-benzamides fungicide with a unique mechanism of action and has been widely used for controlling a variety of oomycetes such as Plasmopara viticola, Phytophthora infestans, Pseudoperonospora cubensis, P. nicotianae and Bremia lactucae. However, the fluopicolide-resistance risk and molecular basis in P. nicotianae have not been reported. In this study, the sensitivity profile of 141 P. nicotianae strains to fluopicolide was determined, with a mean median effective concentration (EC50) value of 0.12 ± 0.06μg/mL. Five stable fluopicolide-resistant mutants of P. nicotianae were obtained by fungicide adaptation, and the compound fitness index of these resistant mutants were lower than that of their parental isolates. Additionally, cross-resistance tests indicated that the sensitivity of fluopicolide did not correlate with other oomycete fungicides, apart from fluopimomide. DNA sequencing revealed two point mutations, G765E and N769Y, in the PpVHA-a protein in the fluopicolide-resistant mutants. Transformation and expression of PpVHA-a genes carrying G765E and N769Y in the sensitive wild-type isolate confirmed that it was responsible for fluopicolide resistance. These results suggest that P. nicotianae has a low to medium resistance risk to fluopicolide in laboratory and that point mutations, G765E and N769Y, in PpVHA-a are associated with the observed fluopicolide resistance.

Exposure of Helicobacter pylori to clarithromycin in vitro resulting in the development of resistance and triggers metabolic reprogramming associated with virulence and pathogenicity.

In H. pylori infection, antibiotic-resistance is one of the most common causes of treatment failure. Bacterial metabolic activities, such as energy production, bacterial growth, cell wall construction, and cell-cell communication, all play important roles in antimicrobial resistance mechanisms. Identification of microbial metabolites may result in the discovery of novel antimicrobial therapeutic targets and treatments. The purpose of this work is to assess H. pylori metabolomic reprogramming in order to reveal the underlying mechanisms associated with the development of clarithromycin resistance. Previously, four H. pylori isolates were induced to become resistant to clarithromycin in vitro by incrementally increasing the concentrations of clarithromycin. Bacterial metabolites were extracted using the Bligh and Dyer technique and analyzed using metabolomic fingerprinting based on Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-Q-ToF-MS). The data was processed and analyzed using the MassHunter Qualitative Analysis and Mass Profiler Professional software. In parental sensitivity (S), breakpoint isolates (B), and induced resistance isolates (R) H. pylori isolates, 982 metabolites were found. Furthermore, based on accurate mass, isotope ratios, abundances, and spacing, 292 metabolites matched the metabolites in the Agilent METLIN precise Mass-Personal Metabolite Database and Library (AM-PCDL). Several metabolites associated with bacterial virulence, pathogenicity, survival, and proliferation (L-leucine, Pyridoxone [Vitamine B6], D-Mannitol, Sphingolipids, Indoleacrylic acid, Dulcitol, and D-Proline) were found to be elevated in generated resistant H. pylori isolates when compared to parental sensitive isolates. The elevated metabolites could be part of antibiotics resistance mechanisms. Understanding the fundamental metabolome changes in the course of progressing from clarithromycin-sensitive to breakpoint to resistant in H. pylori clinical isolates may be a promising strategy for discovering novel alternatives therapeutic targets.

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-∆I177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals.

The African swine fever virus (ASFV) mutant ASFV-G-∆I177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-∆I177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-∆I177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-∆I177L/∆EP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-∆I177L. Domestic pigs intramuscularly (IM) inoculated with either 102 or 106 HAD50 of ASFV-G-∆I177L/∆EP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-∆I177L/∆EP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 102 HAD50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-∆I177L/∆EP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-∆I177L/∆EP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-∆I177L/∆EP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-∆I177L, ASFV-G-∆MGF, or ASFV-G-∆9GL/∆UK. Therefore, ASFV-G-∆I177L/∆EP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.