Two indigenous Berberis species from Spain were confirmed as alternate hosts of the yellow rust fungus Puccinia striiformis f.sp. tritici

Puccinia striiformis f.sp. tritici (Pst), which causes yellow (or stripe) rust on wheat, is a macrocyclic and heteroecious fungus. In this study, we investigated whether Berberis vulgaris subsp. seroi and B. vulgaris subsp. australis, which are indigenous in Spain, may serve as alternate hosts for Pst. Wheat leaves bearing telia of an isolate of Pst were harvested and used to inoculate plants of both barberry subspecies. Pycnia were observed on the adaxial side of the leaves from 10 days after inoculation (dai). Following successful fertilisation, aecia were observed on the abaxial side of the leaves from 16 dai. At 27 dai, barberry leaves bearing aecia were detached and used to inoculate susceptible wheat seedlings of cultivar ‘Morocco’. Uredinia were observed on wheat seedlings from 12 days after aeciospore exposure. Eighty-three single lesions were recovered from individual wheat leaves, of which 43 were genotyped using 19 Pst simple sequence repeat markers (SSR). In total, 19 multilocus genotypes (MLGs) were identified among the 43 progeny isolates. The SSR genotyping confirmed that all 43 isolates were derived from the parental isolate. Seven heterozygous SSR markers showed segregation among the progenies, whereas none of the 12 homozygous markers resulted in segregation. These results demonstrated that B. vulgaris subspp. seroi and australis can serve as alternate hosts for the yellow rust fungus, which may result in novel virulence combinations that can have a detrimental impact on wheat production. Although Pst has not been detected on these barberry species in nature, this study highlights the importance of rust surveillance in barberry areas where suitable conditions for completion of the sexual life cycle may be present.

Fluoroquinolone resistance in Achromobacter spp.: substitutions in QRDRs of GyrA, GyrB, ParC and ParE and implication of the RND efflux system AxyEF-OprN

Background Achromobacter are emerging pathogens in cystic fibrosis patients. Mechanisms of resistance to fluoroquinolones are unknown in clinical isolates. Among non-fermenting Gram-negative bacilli, fluoroquinolone resistance is mostly due to amino acid substitutions in localized regions of the targets (GyrA, GyrB, ParC and ParE) named QRDRs, but also to efflux. Objectives To explore quinolone resistance mechanisms in Achromobacter. Methods The putative QRDRs of GyrA, GyrB, ParC and ParE were sequenced in 62 clinical isolates, and in vitro one-step mutants obtained after exposure to fluoroquinolones. An in vitro mutant and its parental isolate were investigated by RNASeq and WGS. RT–qPCR and gene inactivation were used to explore the role of efflux systems overexpression. Results We detected seven substitutions in QRDRs (Q83L/S84P/D87N/D87G for GyrA, Q480P for GyrB, T395A/K525Q for ParE), all in nine of the 27 clinical isolates with ciprofloxacin MIC ≥16 mg/L, whereas none among the in vitro mutants. The RND efflux system AxyEF-OprN was overproduced (about 150-fold) in the in vitro mutant NCF-39-Bl6 versus its parental strain NCF-39 (ciprofloxacin MICs 64 and 1.5 mg/L, respectively). A substitution in AxyT (putative regulator of AxyEF-OprN) was detected in NCF-39-Bl6. Ciprofloxacin MIC in NCF-39-Bl6 dropped from 64 to 1.5 mg/L following gene inactivation of either axyT or axyF. Substitutions in AxyT associated with overexpression of AxyEF-OprN were also detected in seven clinical strains with ciprofloxacin MIC ≥16 mg/L. Conclusions Target alteration is not the primary mechanism involved in fluoroquinolone resistance in Achromobacter. The role of AxyEF-OprN overproduction was demonstrated in one in vitro mutant.

1456. Resistance Mechanisms of Tigecycline in Enterococcus faecalis

Background Enterococcus faecalis have been regarded as one of the leading causes of the nosocomial infections worldwide. Tigecycline (TGC) is considered as a choice of last resort for the treatment of infections caused by multidrug-resistant E. faecalis, however, the emergence of TGC non-susceptibility has posted the therapeutic challenge. Non-susceptibility in clinical strains could be due to resistance (MIC >0.5 mg/l) or heteroresistance. Therefore, this study aimed to understand the underlying molecular mechanisms of TGC resistance and heteroresistance in E. faecalis. Methods In vitro induction experiments were carried out under TGC pressure with two TGC- sensitive E. faecalis strains. Heteroresistance was evaluated by population analysis profiling (PAP) in 270 clinical TGC- sensitive E. faecalis strains. TGC susceptibility was determined by the agar dilution method. Resistance and heteroresistance mechanisms were investigated by identifying genetic mutations in tetracycline (Tet) target sites and susceptibility testing in the presence of the efflux protein inhibitors phenylalanine-arginine-β-naphthylamide (PaβN) and carbonyl cyanide m chlorophenylhydrazine (CCCP). Comparison of single nucleotide polymorphism in the whole genome between the parental isolate and two TGC-resistant strains were investigated by next-generation sequencing. Results No mutations in Tet target sites in seven TGC heteroresistant strains were present, whereas the mutations in Tet target sites of seven TGC-resistant E. faecalis were frequently found (Table 1). TGC MICs in heteroresistant strains were reduced by CCCP (Table 2). Whole genome sequencing revealed the same non-synonymous mutations and transcoding deletions in the exons of several genes encoding for various enzymes or transfer systems (Table 3). Table 1. The characteristics of the antimicrobial susceptibility, resistance mechanism of TGC-induced resistant isolates Table 2. Characteristics of clinical heteroresistant mother E. faecalis strains and heteroresistance-derived E. faecalis clones Table 3. List of mutation-related genes, amino acids and proteins by comparison of whole genome between the parental isolate and the TGC-induced resistant strains Conclusion Our data indicated that the main mechanism of TGC heteroresistance in E. faecalis might be associated with the efflux pumps. TGC resistance in E. faecalis was associated with mutations in the 16SrRNA site or 30S ribosome protein S10. The genetic mutations in several enzymes and transfer systems might also participate in the resistance development to TGC in E. faecalis. Disclosures All Authors: No reported disclosures

Study of Inheritance and Linkage of Virulence Genes in a Selfing Population of a Pakistani Dominant Race of Puccinia striiformis f. sp. tritici

Wheat stripe rust is a severe threat of almost all wheat-growing regions in the world. Being an obligate biotrophic fungus, Puccinia striiformis f. sp. tritici (PST) produces new virulent races that break the resistance of wheat varieties. In this study, 115 progeny isolates were generated through sexual reproduction on susceptible Himalayan Berberis pseudumbellata using a dominant Pakistani race (574232) of PST. The parental isolate and progeny isolates were characterized using 24 wheat Yr single-gene lines and ten simple sequence repeat (SSR) markers. From the one-hundred-and-fifteen progeny isolates, 25 virulence phenotypes (VPs) and 60 multilocus genotypes were identified. The parental and all progeny isolates were avirulent to Yr5, Yr10, Yr15, Yr24, Yr32, Yr43, YrSp, YrTr1, YrExp2, Yr26, and YrTye and virulent to Yr1, Yr2, Yr6, Yr7, Yr8, Yr9, Yr17, Yr25, Yr27, Yr28, YrA, Yr44, and Yr3. Based on the avirulence/virulence phenotypes, we found that VPs virulent to Yr1, Yr2, Yr9, Yr17, Yr47, and YrA were controlled by one dominant gene; those to YrSp, YrTr1, and Yr10 by two dominant genes; and those to YrExp2 by two complementary dominant genes. The results are useful in breeding stripe rust-resistant wheat varieties and understanding virulence diversity.

Inheritance of Virulence and Linkages of Virulence Genes in an Ethiopian Isolate of the Wheat Stripe Rust Pathogen (Puccinia striiformis f. sp. tritici) Determined Through Sexual Recombination on Berberis holstii (Retracted)

The authors of Siyoum et al. 103:2451-2459 (2019) retracted this article because it proved to contain errors in statistical analyses of the data and subsequent data interpretations. This article was retracted on 14 November 2019. Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most devastating wheat diseases in Ethiopia. To study virulence genetics of the pathogen, 117 progeny isolates were produced through sexual reproduction of an Ethiopian isolate of the stripe rust pathogen on Berberis holstii plants under controlled conditions. The parental and progeny isolates were characterized by phenotyping on wheat lines carrying single Yr genes for resistance and genotyped using 10 polymorphic simple sequence repeated (SSR) markers. The progeny isolates were classified into 37 virulence phenotypes and 75 multilocus genotypes. The parental isolate and progeny isolates were all avirulent to resistance genes Yr5, Yr10, Yr15, Yr24, Yr32, YrTr1, YrSP, and Yr76 but virulent to Yr1 and Yr2, indicating that the parental isolate was homozygous avirulent or homozygous virulent at these loci. The progeny isolates segregated for virulence to 12 Yr genes. Virulence phenotypes to Yr6, Yr28, Yr43, and Yr44 were controlled by a single dominant gene; those to Yr7, Yr9, Yr17, Yr27, Yr25, Yr31, and YrExp2 were each controlled by two dominant genes; and the virulence phenotype to Yr8 was controlled by two complementary dominant genes. A linkage map was constructed with seven SSR markers, and 16 virulence loci corresponding to 11 Yr resistance genes were mapped with some loci linked to each other. These results are useful in understanding host–pathogen interactions and selecting resistance genes to develop wheat cultivars with highly effective resistance to stripe rust.

Virulence and Simple Sequence Repeat Marker Segregation in a Puccinia striiformis f. sp. tritici Population Produced by Selfing a Chinese Isolate on Berberis shensiana

Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, frequently produces new races overcoming resistance in wheat cultivars. A recently identified race, V26 with virulence to Yr26 and many other stripe rust resistance genes, has a high potential to cause epidemics in China. In this study, teliospores from a single-urediniospore isolate of V26 (Pinglan 17-7) produced on the wheat line 92R137 (Yr26) were used to produce a sexual population through selfing by infecting Berberis shensiana plants under controlled conditions. One hundred and eighteen progeny isolates and the parental isolate were phenotyped for virulence/avirulence on 24 Yr gene lines of wheat. These progeny isolates were all avirulent to Yr5, Yr8, Yr15, and YrTr1 and virulent to Yr1, Yr2, Yr7, Yr9, Yr10, Yr17, Yr24, Yr25, Yr26, YrA, YrExp2, and YrV23, indicating that the parental isolate is homozygous avirulent or homozygous virulent at these loci. The progeny population segregated for avirulence to Yr6, Yr43, and YrSP at one locus (3 avirulent:1 virulent ratio); for virulence to Yr27 and Yr28 at one locus (3 virulent:1 avirulent); and for Yr4, Yr32, and Yr44 at two loci (15 virulent:1 avirulent). Among the eight segregating avirulence/virulence loci, association was found between virulence to Yr4 and Yr32, as well as between virulence to Yr6 and Yr43 based on χ2 tests. From 82 genotypically different progeny isolates, 24 pathotypes and 82 multilocus genotypes were identified. The results show that a highly diverse population can be produced from a single isolate by selfing on a barberry plant and sexually produced population can be used to genetically characterize virulence of the stripe rust pathogen.

Evolution of an Experimental Population of Phytophthora capsici in the Field

Populations of the vegetable pathogen Phytophthora capsici are often highly diverse, with limited gene flow between fields. To investigate the structure of a newly established, experimental population, an uninfested research field was inoculated with two single-zoospore isolates of P. capsici in September 2008. From 2009 through 2012, ≈50 isolates of P. capsici were collected from the field each year and genotyped using five microsatellite loci. The same two isolates were also crossed in the lab. High levels of diversity were detected in the research field, with 26 to 37 unique multilocus genotypes detected each year. Through 2012, genotypic diversity did not decline and no evidence of genetic drift was observed. However, during the 2011 and 2012 growing seasons, four new alleles not present in either parental isolate were observed in the field. Selfing (but not apomixis) was observed at low frequency among in vitro progeny. In addition, evidence for loss of heterozygosity was observed in half of the in vitro progeny. These results suggest that recombination, mutation, and loss of heterozygosity can affect the genetic structure observed in P. capsici populations.

Plasmid CDS5 Influences Infectivity and Virulence in a Mouse Model of Chlamydia trachomatis Urogenital Infection

ABSTRACTThe native plasmid of bothChlamydia muridarumandChlamydia trachomatishas been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol forChlamydia trachomatisthat for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate ofC. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 inin vivofitness and in induction of inflammatory responses. To our knowledge, these are the firstin vivoobservations ascribing infectivity and virulence to a specific plasmid gene.

Partial Disruption of Translational and Posttranslational Machinery Reshapes Growth Rates of Bartonella birtlesii

ABSTRACT Specialization of bacteria in a new niche is associated with genome repertoire changes, and speciation in bacterial specialists is associated with genome reduction. Here, we tested a signature-tagged mutant library of 3,456 Bartonella birtlesii clones to detect mutants that could grow rapidly in vitro. Overall, we found 124 mutants that grew faster than the parental wild-type strain in vitro. We sequenced the genomes of the four mutants with the most rapid growth (formed visible colonies in only 1 to 2 days compared with 5 days for the wild type) and compared them to the parental isolate genome. We found that the number of disrupted genes associated with translation in the 124 rapid-growth clones was significantly higher than the number of genes involved in translation in the full genome (P < 10−6). Analysis of transposon integration in the genome of the four most rapidly growing clones revealed that one clone lacked one of the two wild-type RNA ribosomal operons. Finally, one of the four clones did not induce bacteremia in our mouse model, whereas infection with the other three resulted in a significantly lower bacterial count in blood than that with the wild-type strain. IMPORTANCE Here, we show that specialization in a specific niche could be caused by the disruption of critical genes. Most of these genes were involved in translation, and we show that evolution of obligate parasitism bacteria was specifically associated with disruption of translation system-encoding genes.

Adaptation to Fungicides in Monilinia fructicola Isolates with Different Fungicide Resistance Phenotypes

The ability to develop fungicide resistance was assessed in Monilinia fructicola isolates with different fungicide sensitivity phenotypes by adapting mycelium and conidia to increasing concentrations of selective fungicides and UV mutagenesis. Results showed that adaptation to Quinone outside inhibitor (QoI) fungicide azoxystrobin and sterol demethylation inhibitor (DMI) fungicide propiconazole was more effective in conidial-transfer experiments compared to mycelial-transfer experiments. DMI-resistant (DMI-R) isolates adapted to significantly higher doses of azoxystrobin in both, mycelial- and conidial-transfer experiments compared to benzimidazole-resistant (BZI-R) and sensitive (S) isolates. Adaptation to propiconazole in conidial-transfer experiments was accelerated in BZI-R isolates when a stable, nonlethal dose of 50 μg/ml thiophanate-methyl was added to the selection medium. One of two azoxystrobin-resistant mutants from DMI-R isolates did not show any fitness penalties; the other isolate expired before further tests could be carried out. The viable mutant caused larger lesions on detached peach fruit sprayed with azoxystrobin compared to the parental isolate. The azoxystrobin sensitivity of the viable mutant returned to baseline levels after the mutant was transferred to unamended medium. However, azoxystrobin resistance recovered quicker in the mutant compared to the corresponding parental isolate after renewed subculturing on medium amended with 0.2 and 1 μg/ml azoxystrobin; only the mutant but not the parental isolate was able to adapt to 5 μg/ml azoxystrobin. In UV mutagenesis experiments, the DMI-R isolates produced significantly more mutants compared to S isolates. All of the UV-induced mutants showed stable fungicide resistance with little fitness penalty. This study indicates the potential for QoI fungicide resistance development in M. fructicola in the absence of a mutagen and provides evidence for increased mutability and predisposition to accelerated adaptation to azoxystrobin in M. fructicola isolates resistant to DMI fungicides.