Diversity Of Isolates Research Articles

Efficacy of tigecycline alone or in combination for experimental infections by KPC carbapenemase-producing Klebsiella pneumoniae

Although in vitro data suggest that tigecycline is active against Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp), experimental and clinical data are limited. We studied the effect of tigecycline alone or in combination for experimental infections by KPC-Kp. A total of 540 male C57BL/6 mice were infected with three genetically diverse KPC-Kp isolates susceptible to tigecycline with meropenem minimum inhibitory concentrations (MICs) of 4, 16 and 256 μg/mL, respectively. Mice were randomly treated with water for injection, tigecycline, meropenem and colistin alone, and double or triple combinations of tigecycline, colistin and meropenem. Mouse survival was recorded for 14 days. In separate experiments, mice were sacrificed 6 h and 24 h after bacterial challenge for quantitative culture of tissues and serological analysis. Time-kill curves were performed. Tigecycline, colistin and meropenem concentrations were measured in tissues and serum by high-performance liquid chromatography (HPLC). Survival was significantly prolonged when mice were treated with tigecycline alone and tigecycline-containing regimens compared with control mice and mice treated with tigecycline-sparing regimens. Tigecycline-sparing regimens were active only against the isolate with a meropenem MIC of 4 μg/mL. Mortality was associated with progression to multiple organ failure. Tigecycline and tigecycline-containing regimens achieved a rapid decrease of bacterial loads both in tissues and in vitro. Tigecycline concentrations in tissues were negatively correlated with tissue bacterial load. Tigecycline alone or in combination with meropenem and/or colistin achieves effective treatment of experimental KPC-Kp infections irrespective of the meropenem MIC.

Evaluation of Lesions and Viral Antigen Distribution in Domestic Pigs Inoculated Intranasally with African Swine Fever Virus Ken05/Tk1 (Genotype X).

The understanding of the pathogenic mechanisms and the clinicopathological forms caused by currently circulating African swine fever virus (ASFV) isolates is incomplete. So far, most of the studies have been focused on isolates classified within genotypes I and II, the only genotypes that have circulated outside of Africa. However, less is known about the clinical presentations and lesions induced by isolates belonging to the other twenty-two genotypes. Therefore, the early clinicopathological identification of disease outbreaks caused by isolates belonging to, as yet, not well-characterised ASFV genotypes may be compromised, which might cause a delay in the implementation of control measures to halt the virus spread. To improve the pathological characterisation of disease caused by diverse isolates, we have refined the macroscopic and histopathological evaluation protocols to standardise the scoring of lesions. Domestic pigs were inoculated intranasally with different doses (high, medium and low) of ASFV isolate Ken05/Tk1 (genotype X). To complement previous studies, the distribution and severity of macroscopic and histopathological lesions, along with the amount and distribution of viral antigen in tissues, were characterised by applying the new scoring protocols. The intranasal inoculation of domestic pigs with high doses of the Ken05/Tk1 isolate induced acute forms of ASF in most of the animals. Inoculation with medium doses mainly induced acute forms of disease. A less severe but longer clinical course, typical of subacute forms, characterised by the presence of more widespread and severe haemorrhages and oedema, was observed in one pig inoculated with the medium dose. The severity of vascular lesions (haemorrhages and oedema) induced by high and medium doses was not associated with the amount of virus antigen detected in tissues, therefore these might be attributed to indirect mechanisms not evaluated in the present study. The absence of clinical signs, lesions and detectable levels of virus genome or antigen in blood from the animals inoculated with the lowest dose ruled out the existence of possible asymptomatic carriers or persistently infected pigs, at least for the 21 days period of the study. The results corroborate the moderate virulence of the Ken05/Tk1 isolate, as well as its capacity to induce both the acute and, occasionally, subacute forms of ASF when high and medium doses were administered intranasally.

Completion of Maize Stripe Virus Genome Sequence and Analysis of Diverse Isolates.

Maize stripe virus is a pathogen of corn and sorghum in subtropical and tropical regions worldwide. We used high-throughput sequencing to obtain the complete nucleotide sequence for the reference genome of maize stripe virus and to sequence the genomes of ten additional isolates collected from the United States or Papua New Guinea. Genetically, maize stripe virus is most closely related to rice stripe virus. We completed and characterized the RNA1 sequence for maize stripe virus, which revealed a large open reading frame encoding a putative protein with ovarian tumor-like cysteine protease, endonuclease, and RNA-dependent RNA polymerase domains. Phylogenetic and amino acid identity analyses among geographically diverse isolates revealed evidence for reassortment in RNA3 that was correlated with the absence of RNA5. This study yielded a complete and updated genetic description of the tenuivirus maize stripe virus and provided insight into potential mechanisms underpinning its diversity.

Heterothallism among spatiotemporally diverse Colletotrichum lindemuthianum isolates and its implication in common bean anthracnose resistance breeding in the Northwestern Himalayan region

Heterothallism among spatiotemporally diverse Colletotrichum lindemuthianum isolates and its implication in common bean anthracnose resistance breeding in the Northwestern Himalayan region

High frequency of fungicide resistance-associated mutations in the wheat yellow rust pathogen Puccinia striiformis f. sp. tritici.

Reliance on fungicides to manage disease creates selection pressure for the evolution of resistance in fungal and oomycete pathogens. Rust fungi (Pucciniales) are major pathogens of cereals and other crops and have been classified as low-risk for developing resistance to fungicides; no case of field failure of fungicides in a cereal rust disease has yet been recorded. Recently, the Asian soybean rust pathogen, Phakopsora pachyrhizi evolved resistance to several fungicide classes, prompting us to screen a large sample of the globally widespread wheat yellow rust pathogen, Puccinia striiformis f. sp. tritici (Pst), for mutations associated with fungicide resistance. We evaluated 363 Pst isolates from Europe, the USA, Ethiopia, Chile, China and New Zealand for mutations in the target genes of demethylase inhibitor (DMI; Cyp51) and succinate dehydrogenase inhibitor (SDHI; SdhB, SdhC and SdhD) fungicides. A high proportion of Pst isolates carrying a Y134F DMI resistance-associated substitution in the Cyp51 gene was found among those from China and New Zealand. A set of geographically diverse Pst isolates was also found to display a substitution in SdhC (I85V) that is homologous to that reported recently in P. pachyrhizi and linked to SDHI resistance. The identification of resistance-associated alleles confirms that cereal rusts are not immune to fungicide resistance and that selection for resistance evolution is operating at high levels in certain locations. It highlights the need to adopt fungicide resistance management practices and to monitor cereal rust species for development of resistance. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

GABA Production by Human Intestinal Bacteroides spp.: Prevalence, Regulation, and Role in Acid Stress Tolerance.

The high neuroactive potential of metabolites produced by gut microbes has gained traction over the last few years, with metagenomic-based studies suggesting an important role of microbiota-derived γ-aminobutyric acid (GABA) in modulating mental health. Emerging evidence has revealed the presence of the glutamate decarboxylase (GAD)-encoding gene, a key enzyme to produce GABA, in the prominent human intestinal genus Bacteroides. Here, we investigated GABA production by Bacteroides in culture and metabolic assays combined with comparative genomics and phylogenetics. A total of 961 Bacteroides genomes were analyzed in silico and 17 metabolically and genetically diverse human intestinal isolates representing 11 species were screened in vitro. Using the model organism Bacteroides thetaiotaomicron DSM 2079, we determined GABA production kinetics, its impact on milieu pH, and we assessed its role in mitigating acid-induced cellular damage. We showed that the GAD-system consists of at least four highly conserved genes encoding a GAD, a glutaminase, a glutamate/GABA antiporter, and a potassium channel. We demonstrated a high prevalence of the GAD-system among Bacteroides with 90% of all Bacteroides genomes (96% in human gut isolates only) harboring all genes of the GAD-system and 16 intestinal Bacteroides strains producing GABA in vitro (ranging from 0.09 to 60.84 mM). We identified glutamate and glutamine as precursors of GABA production, showed that the production is regulated by pH, and that the GAD-system acts as a protective mechanism against acid stress in Bacteroides, mitigating cell death and preserving metabolic activity. Our data also indicate that the GAD-system might represent the only amino acid-dependent acid tolerance system in Bacteroides. Altogether, our results suggest an important contribution of Bacteroides in the regulation of the GABAergic system in the human gut.

Genetic changes are introduced by repeated exposure of Salmonella spiked in low water activity and high fat matrix to heat

WGS is used to define if isolates are “in” or “out” of an outbreak and/or microbial root cause investigation. No threshold of genetic differences is fixed and the conclusions on similarity between isolates are mainly based on the knowledge generated from previous outbreak investigations and reported mutation rates. Mutation rates in Salmonella when exposed to food processing conditions are lacking. Thus, in this study, the ability of heat and dry stress to cause genetic changes in two Salmonella serotypes frequently isolated from low moisture foods was investigated. S. enterica serovars S. Agona ATCC 51,957 and S. Mbandaka NCTC 7892 (ATCC 51,958) were repeatedly exposed to heat (90 °C for 5 min) in a low water activity and high fat matrix. No increased fitness of the strains was observed after 10 repeated heat treatments. However, genetic changes were introduced and the number of genetic differences increased with every heat treatment cycle. The genetic changes appeared randomly in the genome and were responsible for a population of diverse isolates with 0 to 28 allelic differences (0 to 38 SNPs) between them. This knowledge is key to interpret WGS results for source tracking investigations as part of a root cause analysis in a contamination event as isolates are exposed to stress conditions.

Epidemiological typing of ST80 vancomycin-resistant Enterococcus faecium: core genome multilocus sequence typing versus single nucleotide polymorphism-based typing

Vancomycin-resistant Enterococcus faecium (VRE) belonging to sequence type 80 (ST80) have been successfully disseminating in hospital settings. This study aimed to explore whole-genome sequencing (WGS)-based approaches in epidemiological typing of VRE by investigating a collection of ST80 strains. In total, 41 VRE ST80 isolates in three suspected transmission chains were subjected to WGS. The genetic relationship of the isolates was analysed by core genome multilocus sequence typing (cgMLST) and single nucleotide polymorphism (SNP)-based typing. The published genome sequence of a predominant ST80 clone in Copenhagen was also included in the analyses. Group 1 isolates (n=10) were recovered in the same geographic area, including two isolates from an outbreak of vanA genotype. Group 2 isolates (n=4) were detected from another outbreak. Group 3 consisted of 3 non-outbreak isolates and 24 representative isolates from an outbreak of vanB genotype. Our findings indicated ≥24 allelic difference and >16 SNPs as the cut-off for excluding VRE strains from an outbreak. cgMLST identified >200 differing alleles between genomes of the Copenhagen clone and outbreak strains in this study. Our findings also demonstrated that SNP analysis was not suitable for typing samples from different groups, even with the same ST, owing to lack of an optimal reference genome. cgMLST and SNPs provided comparable epidemiological discrimination for isolates with a suspected transmission chain. For diverse isolates, SNP analysis could be suboptimal. An approach applying cgMLST as the first-line typing method and SNPs as a complementary tool is suggested.

Genetic Diversity and Mating Type Distribution of Pseudocercospora fijiensis on Banana in Uganda and Tanzania.

Black Sigatoka, caused by Pseudocercospora fijiensis, is a major foliar disease of banana and plantain worldwide. There are few available data regarding the genetic diversity and population structure of the pathogen in East Africa, which are needed to design effective and durable disease management strategies. We genotyped 319 single-spore isolates of P. fijiensis collected from seven regions in Uganda and Tanzania and five isolates from Nigeria using 16 simple sequence repeat markers and mating type-specific primers. Isolates from each country and region within the country were treated as populations and subpopulations, respectively. A total of 296 multilocus genotypes (MLGs) were recovered, representing a clonal fraction of 7%. Subpopulations had a moderate level of genetic diversity (Hexp = 0.12 to 0.31; mean, 0.29). Mating type distribution did not deviate from equilibrium (MAT1-1: MAT1-2, 1:1 ratio) in Uganda; however, in Tanzania the mating types were not in equilibrium (4:1 ratio). The index of association tests (IA and r̄d) showed that all populations were at linkage equilibrium (P > 0.05), thus supporting the hypothesis of random association of alleles. These findings are consistent with a pathogen that reproduces both clonally and sexually. Low and insignificant levels of population differentiation were detected, with 90% of the variation occurring among isolates within subpopulations. The high intrapopulation variation has implications in breeding for resistance to P. fijiensis because isolates differing in aggressiveness and virulence are likely to exist over small spatial scales. Diverse isolates will be required for resistance screening to ensure selection of banana cultivars with durable resistance to Sigatoka in East Africa.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Staphylococcus aureus in Intensive Pig Production in South Africa: Antibiotic Resistance, Virulence Determinants, and Clonality.

Although Staphylococcus aureus is a major threat to the veterinary, agricultural, and public health sectors because of its zoonotic potential, studies on its molecular characterisation in intensive animal production are rare. We phenotypically and genotypically characterised antibiotic-resistant S. aureus in intensive pig production in South Africa, using the farm-to-fork approach. Samples (n = 461) were collected from the farm, transport vehicles, and the abattoir using the World Health Organisation on Integrated Surveillance of Antimicrobial Resistance (WHO-AGISAR) sampling protocol. Bacteria were isolated using selective media and identified using biochemical tests and polymerase chain reaction (PCR). Phenotypic resistance was determined using the disk diffusion method. Selected resistance and virulence genes were investigated using PCR. Clonality among the isolates was determined using the repetitive element sequence-PCR. In all, 333 presumptive staphylococcal isolates were obtained, with 141/333 (42.3%) identified as staphylococci biochemically. Ninety-seven (97; 68.8%) were confirmed as S. aureus using PCR, 52.6% of which were identified as methicillin-resistant S. aureus (MRSA) through the mecA gene. All the 97 S. aureus isolates (100%) were resistant to at least one of the antibiotics tested, with the highest resistance observed against erythromycin and clindamycin (84.50% each), and the lowest observed against amikacin (2.10%); 82.47% (80/97) were multidrug-resistant with an average multiple antibiotic resistance index of 0.50. Most of the phenotypically resistant isolates carried at least one of the corresponding resistance genes tested, ermC being the most detected. hla was the most detected virulence gene (38.14%) and etb was the least (1.03%). Genetic fingerprinting revealed diverse MRSA isolates along the farm-to-fork continuum, the major REP types consisting of isolates from different sources suggesting a potential transmission along the continuum. Resistance to antibiotics used as growth promoters was evidenced by the high prevalence of MDR isolates with elevated multiple antibiotic resistance indices >0.2, specifically at the farm, indicating exposure to high antibiotic use environments, necessitating antibiotic stewardship and proper infection control measures in pig husbandry and intensive pig production.

Characterizing the Genetic Architecture of Nonhost Resistance in Barley Using Pathogenically Diverse Puccinia Isolates

Barley is an intermediate or near nonhost to many cereal rust pathogens that infect grasses, making it a highly suitable model to understand the evolution and genetic basis of nonhost resistance (NHR) in plants. To characterize the genetic architecture of NHR in barley, we used the Oregon Wolfe Barley doubled haploid and Morex × SusPtrit recombinant inbred line mapping populations. To elicit a wide array of NHR responses, we tested 492 barley accessions and both mapping populations with pathogenically diverse cereal rust isolates representing distinct formae speciales adapted to Avena, Hordeum, Triticum, and Lolium spp.: P. coronata f. sp. avenae (oat crown rust pathogen) and P. coronata f. sp. lolii (ryegrass crown rust pathogen), P. graminis f. sp. avenae (oat stem rust pathogen) and P. graminis f. sp. lolii (the ryegrass stem rust pathogen), and P. striiformis f. sp. tritici (wheat stripe rust pathogen) and P. striiformis f. sp. pseudo-hordei (barley grass stripe rust pathogen). With the exception of P. coronata f. sp. lolii and P. coronata f. sp. avenae, susceptibility and segregation for NHR was observed in the barley accessions and both mapping populations. Quantitative trait loci (QTLs) for NHR were mapped on all seven chromosomes. NHR in barley to the heterologous rusts tested was attributable to a combination of QTLs with either or both overlapping and distinct specificities. Across both mapping populations, broadly effective NHR loci were also identified that likely play a role in host specialization.

Diversity of Culturable Bacteria Isolated from Highland Barley Cultivation Soil in Qamdo, Tibet Autonomous Region.

The soil bacterial communities have been widely investigated. However, there has been little study of the bacteria in Qinghai-Tibet Plateau, especially about the culturable bacteria in highland barley cultivation soil. Here, a total of 830 individual strains were obtained at 4°C and 25°C from a highland barley cultivation soil in Qamdo, Tibet Autonomous Region, using fifteen kinds of media. Seventy-seven species were obtained, which belonged to 42 genera and four phyla; the predominant phylum was Actinobacteria (68.82%), followed by Proteobacteria (15.59%), Firmicutes (14.29%), and Bacteroidetes (1.30%). The predominant genus was Streptomyces (22.08%, 17 species), followed by Bacillus (6.49%, five species), Micromonospora (5.19%, four species), Microbacterium (5.19%, four species), and Kribbella (3.90%, three species). The most diverse isolates belonged to a high G+C Gram-positive group; in particular, the Streptomyces genus is a dominant genus in the high G+C Gram-positive group. There were 62 species and 33 genera bacteria isolated at 25°C (80.52%), 23 species, and 18 genera bacteria isolated at 4°C (29.87%). Meanwhile, only eight species and six genera bacteria could be isolated at 25°C and 4°C. Of the 77 species, six isolates related to six genera might be novel taxa. The results showed abundant bacterial species diversity in the soil sample from the Qamdo, Tibet Autonomous Region.

Aggressiveness of Diverse French Aphanomyces euteiches Isolates on Pea Near Isogenic Lines Differing in Resistance Quantitative Trait Loci.

Aphanomyces root rot is a major disease in many pea growing regions worldwide. Development of resistant varieties is necessary to manage the disease. Near isogenic lines (NILs) carrying resistance alleles at main quantitative trait loci (QTLs) were developed by marker-assisted backcrossing. This study aimed to evaluate the aggressiveness of diverse French isolates of Aphanomyces euteiches on NILs carrying different resistance QTLs. Forty-three A. euteiches isolates from different French pea growing regions were tested for aggressiveness on eight NILs carrying single or combinations of resistance QTLs and two susceptible or resistant control lines, in controlled conditions. Three clusters of isolates, unrelated to geographical origin, were identified, including 37, 56, and 7% of isolates with high, moderate, and low average levels of aggressiveness, respectively. Three groups of pea lines were also identified. The first group consisted of a pea resistant control line, moderately to highly resistant to all of the isolates. The second group included five NILs carrying a major-effect resistance allele at QTL Ae-Ps7.6, with a medium to broad range of effects on the isolates. The third group consisted of three NILs carrying minor-effect resistance alleles, with a narrow range of effects on the isolates. The results suggest that highly aggressive isolates occur naturally, which may be selected by future partially resistant pea varieties carrying QTLs and increase the risk of erosion of QTL effect. QTL pyramiding strategies for a higher level and a broader range of effect of quantitative resistance on A. euteiches populations will be required for breeding for durable pea resistant varieties.

Genomes of Diverse Isolates of Prochlorococcus High-Light-Adapted Clade II in the Western Pacific Ocean

Genomes of Diverse Isolates of Prochlorococcus High-Light-Adapted Clade II in the Western Pacific Ocean

Limiting protease production plays a key role in the pathogenesis of the divergent clinical isolates of Staphylococcus aureus LAC and UAMS-1

ABSTRACT Using the USA300, methicillin-resistant Staphylococcus aureus strain LAC, we previously examined the impact of regulatory mutations implicated in biofilm formation on protease production and virulence in a murine sepsis model. Here we examined the impact of these mutations in the USA200, methicillin-sensitive strain UAMS-1. Mutation of agr, mgrA, rot, sarA and sigB attenuated the virulence of UAMS-1. A common characteristic of codY, rot, sigB, and sarA mutants was increased protease production, with mutation of rot having the least impact followed by mutation of codY, sigB and sarA, respectively. Protein A was undetectable in conditioned medium from all four mutants, while extracellular nuclease was only present in the proteolytically cleaved NucA form. The abundance of high molecular weight proteins was reduced in all four mutants. Biofilm formation was reduced in codY, sarA and sigB mutants, but not in the rot mutant. Eliminating protease production partially reversed these phenotypes and enhanced biofilm formation. This was also true in LAC codY, rot, sarA and sigB mutants. Eliminating protease production enhanced the virulence of LAC and UAMS-1 sarA, sigB and rot mutants in a murine sepsis model but did not significantly impact the virulence of the codY mutant in either strain. Nevertheless, these results demonstrate that repressing protease production plays an important role in defining critical phenotypes in diverse clinical isolates of S. aureus and that Rot, SigB and SarA play critical roles in this regard.

The Barley HvWRKY6 Transcription Factor Is Required for Resistance Against Pyrenophora teres f. teres.

Barley is an important cereal crop worldwide because of its use in the brewing and distilling industry. However, adequate supplies of quality malting barley are threatened by global climate change due to drought in some regions and excess precipitation in others, which facilitates epidemics caused by fungal pathogens. The disease net form net blotch caused by the necrotrophic fungal pathogen Pyrenophora teres f. teres (Ptt) has emerged as a global threat to barley production and diverse populations of Ptt have shown a capacity to overcome deployed genetic resistances. The barley line CI5791 exhibits remarkably effective resistance to diverse Ptt isolates from around the world that maps to two major QTL on chromosomes 3H and 6H. To identify genes involved in this effective resistance, CI5791 seed were γ-irradiated and two mutants, designated CI5791-γ3 and CI5791-γ8, with compromised Ptt resistance were identified from an M2 population. Phenotyping of CI5791-γ3 and -γ8 × Heartland F2 populations showed three resistant to one susceptible segregation ratios and CI5791-γ3 × -γ8 F1 individuals were susceptible, thus these independent mutants are in a single allelic gene. Thirty-four homozygous mutant (susceptible) CI5791-γ3 × Heartland F2 individuals, representing 68 recombinant gametes, were genotyped via PCR genotype by sequencing. The data were used for single marker regression mapping placing the mutation on chromosome 3H within an approximate 75 cM interval encompassing the 3H CI5791 resistance QTL. Sequencing of the mutants and wild-type (WT) CI5791 genomic DNA following exome capture identified independent mutations of the HvWRKY6 transcription factor located on chromosome 3H at ∼50.7 cM, within the genetically delimited region. Post transcriptional gene silencing of HvWRKY6 in barley line CI5791 resulted in Ptt susceptibility, confirming that it functions in NFNB resistance, validating it as the gene underlying the mutant phenotypes. Allele analysis and transcript regulation of HvWRKY6 from resistant and susceptible lines revealed sequence identity and upregulation upon pathogen challenge in all genotypes analyzed, suggesting a conserved transcription factor is involved in the defense against the necrotrophic pathogen. We hypothesize that HvWRKY6 functions as a conserved signaling component of defense mechanisms that restricts Ptt growth in barley.

Unexpectedly high mutation rate of a deep-sea hyperthermophilic anaerobic archaeon.

Deep-sea hydrothermal vents resemble the early Earth, and thus the dominant Thermococcaceae inhabitants, which occupy an evolutionarily basal position of the archaeal tree and take an obligate anaerobic hyperthermophilic free-living lifestyle, are likely excellent models to study the evolution of early life. Here, we determined that unbiased mutation rate of a representative species, Thermococcus eurythermalis, exceeded that of all known free-living prokaryotes by 1-2 orders of magnitude, and thus rejected the long-standing hypothesis that low mutation rates were selectively favored in hyperthermophiles. We further sequenced multiple and diverse isolates of this species and calculated that T. eurythermalis has a lower effective population size than other free-living prokaryotes by 1-2 orders of magnitude. These data collectively indicate that the high mutation rate of this species is not selectively favored but instead driven by random genetic drift. The availability of these unusual data also helps explore mechanisms underlying microbial genome size evolution. We showed that genome size is negatively correlated with mutation rate and positively correlated with effective population size across 30 bacterial and archaeal lineages, suggesting that increased mutation rate and random genetic drift are likely two important mechanisms driving microbial genome reduction. Future determinations of the unbiased mutation rate of more representative lineages with highly reduced genomes such as Prochlorococcus and Pelagibacterales that dominate marine microbial communities are essential to test these hypotheses.

Relatedness of type IV pilin PilA amongst geographically diverse Moraxella bovoculi isolated from cattle with infectious bovine keratoconjunctivitis.

Introduction Moraxella bovoculi is frequently isolated from the eyes of cattle with infectious bovine keratoconjunctivitis (IBK; pinkeye). As with M. bovis, which has been causally linked to IBK, M. bovoculi expresses an RTX (repeats in the structural toxin) cytotoxin that is related to M. bovis cytotoxin. Pilin, another pathogenic factor in M. bovis , is required for corneal attachment. Seven antigenically distinct pilin serogroups have been described in M. bovis .Hypothesis/Gap StatementMultiple different serogroups exist amongst type IV pilin encoded by M. bovis , however, it is not known whether M. bovoculi exhibits a similar degree of diversity in type IV pilin that it encodes.Aim This study was done to characterize a structural pilin (PilA) encoded by M. bovoculi isolated from cases of IBK to determine if diversity exists amongst PilA sequences.Methodology Ninety-four isolates of M. bovoculi collected between 2002 and 2017 from 23 counties throughout California and from five counties in four other Western states were evaluated.Results DNA sequencing and determination of deduced amino acid sequences revealed ten (designated groups A through J) unique PilA sequences that were ~96.1–99.3 % identical. Pilin groups A and C matched previously reported putative PilA sequences from M. bovoculi isolated from IBK-affected cattle in the USA (Virginia, Nebraska, and Kansas) and Asia (Kazakhstan). The ten pilin sequences identified were only ~74–76 % identical to deduced amino acid sequences of putative pilin proteins identified from the previously reported whole-genome sequences of M. bovoculi derived from deep nasopharyngeal swabs of IBK-asymptomatic cattle.ConclusionsCompared to the diversity reported between structural pilin proteins amongst different serogroups of M. bovis , M. bovoculi PilA from geographically diverse isolates derived from IBK-affected cattle are more conserved.

Broad spectrum blast resistance alleles in newly developed Malaysian rice (Oryza sativa L.) genotypes

Rice blast, caused by Magnaporthe oryzae, is a destructive disease. A total of 23 rice genotypes, were screened using a set of 51 simple sequence repeats SSR/ InDel markers and 51 single nucleotide polymorphic (SNP) markers to evaluate the sequence variation. Six newly identified Malaysian isolates of M. oryzae were used for blast screening. The resistant genotypes, consisting of Pongsu Seribu 2, O. rufipogon accessions, Tetep and IR64 had a resistant disease score of 2/3 showing minimal symptoms of blast. In contrast, Mahsuri and MR264 were the most susceptible and had a disease score of 5. In total, 11 sequence variations and two putative haplotype blocks, were observed in the SNP data representing Pi54 and Pita. The SNPs for Pita and Pi54 revealed the presence of resistant alleles that generated reproducible and aggressive calls based on the Sequenom data, but when screened with SSR and INDel markers showed poor correspondence to phenotype effects. Therefore, SNPs for Pi54 and Pita were found to be the most reliable for marker-assisted selection. The blast-resistant alleles identified in this study indicate that they are involved in the defence response against diverse isolates and play a role in putative broad spectrum resistance.

Nodulation Potential and Phenotypic Diversity of Rhizobia Nodulating Cowpea Isolated from Major Growing Areas of Ethiopia

Rhizobia are specific to a particular legume and need to be characterized by different methods. In Ethiopia, very few efforts have been made to evaluate the phenotypic characteristics and diversity of cowpea nodulating rhizobia. This study aimed to assess the nodulation potential and phenotypic diversity of rhizobia nodulating cowpea isolated from major growing areas of Ethiopia. Thirty soil samples were collected from different cowpea growing areas of Ethiopia of which 28 (93.3%) samples induced nodulation in the greenhouse. A total of 28 isolates obtained from nodulated plants were evaluated using presumptive tests and subjected to different phenotypic characteristics. On YEMA containing CR, the isolates produced colony colors that were whitish to pale pink. All isolates showed no growth on peptone glucose agar and the isolates were capable to grow on ketolactose medium but did not turn the media color to yellow. All the isolates were also confirmed to be gram-negative, rod-shaped, and non-spore-forming. The colony texture study revealed that 78.57% of the isolates had large mucoid colony whereas 21.43% of the isolates exhibited large watery colony texture with diameters ranging between 1 and 5mm. Depending on the colony color on YEMA containing bromothymol blue, 22 (78.57%) isolates were slow-growing whereas 6 (21.43%) isolates were fast-growing rhizobia. The cluster analysis based on phenotypic characteristics revealed a grouping of the isolates into two clusters at a similarity level of 75% and six further sub-clusters at a similarity level of about 91% indicating that there are variations and diversity among the isolates. Overall, this study revealed great variations among the cowpea nodulating rhizobial isolates and suggests that the soils of Ethiopia harbor populations of phenotypically diverse isolates. A further test to confirm these findings using genetic characterization and inclusion of reference strain is highly recommended.