Ana Vieira

Streptococcus pyogenes genotype emm1 is a successful, globally distributed epidemic clone that is regarded as inherently virulent. An emm1 sublineage, M1_UK, that produces increased levels of SpeA toxin was associated with increased scarlet fever and invasive infections in England in 2015/2016. Defined by 27 SNPs in the core genome, M1_UK is now dominant in England. To more fully characterize M1_UK, we undertook comparative transcriptomic and proteomic analyses of M1_UK and contemporary non-M1_UK emm1 strains (M1_global). Just seven genes were differentially expressed by M1_UK compared with contemporary M1_global strains. In addition to speA, five genes in the operon that includes glycerol dehydrogenase were upregulated in M1_UK (gldA, mipB/talC, pflD, and phosphotransferase system IIC and IIB components), while aquaporin (glpF2) was downregulated. M1_UK strains have a stop codon in gldA. Deletion of gldA in M1_global abrogated glycerol dehydrogenase activity, and recapitulated upregulation of gene expression within the operon that includes gldA, consistent with a feedback effect. Phylogenetic analysis identified two intermediate emm1 sublineages in England comprising 13/27 (M1_13SNPs) and 23/27 SNPs (M1_23SNPs), respectively, that had failed to expand in the population. Proteomic analysis of invasive strains from the four phylogenetic emm1 groups highlighted sublineage-specific changes in carbohydrate metabolism, protein synthesis and protein processing; upregulation of SpeA was not observed in chemically defined medium. In rich broth, however, expression of SpeA was upregulated ~10-fold in both M1_23SNPs and M1_UK sublineages, compared with M1_13SNPs and M1_global. We conclude that stepwise accumulation of SNPs led to the emergence of M1_UK. While increased expression of SpeA is a key indicator of M1_UK and undoubtedly important, M1_UK strains have outcompeted M1_23SNPs and other emm types that produce similar or more superantigen toxin. We speculate that an accumulation of adaptive SNPs has contributed to a wider fitness advantage in M1_UK on an inherently successful emm1 streptococcal background.

Antimicrobial resistance in enteric or urinary Escherichia coli is a risk factor for invasive E. coli infections. Due to widespread trimethoprim resistance amongst urinary E. coli and increased bacteraemia incidence, a national recommendation to prescribe nitrofurantoin for uncomplicated urinary tract infection was made in 2014. Nitrofurantoin resistance is reported in E. coli isolates in the UK, however, mechanisms underpinning nitrofurantoin resistance in these isolates remain unknown. This study aimed to identify the genetic basis of nitrofurantoin resistance in urinary E. coli isolates collected from north west London and then elucidate resistance-associated genetic alterations in available UK E. coli genomes. As a result, an algorithm was developed to predict nitrofurantoin susceptibility. Deleterious mutations and gene-inactivating insertion sequences in chromosomal nitroreductase genes nfsA and/or nfsB were identified in genomes of nine confirmed nitrofurantoin-resistant urinary E. coli isolates and additional 11 E. coli isolates that were highlighted by the prediction algorithm and subsequently validated to be nitrofurantoin-resistant. Eight categories of allelic changes in nfsA, nfsB, and the associated gene ribE were detected in 12412 E. coli genomes from the UK. Evolutionary analysis of these three genes revealed homoplasic mutations and explained the previously reported order of stepwise mutations. The mobile gene complex oqxAB, which is associated with reduced nitrofurantoin susceptibility, was identified in only one of the 12412 genomes. In conclusion, mutations and insertion sequences in nfsA and nfsB were leading causes of nitrofurantoin resistance in UK E. coli . As nitrofurantoin exposure increases in human populations, the prevalence of nitrofurantoin resistance in carriage E. coli isolates and those from urinary and bloodstream infections should be monitored.

Phylogenetic relatedness is a key diversity measure for the analysis and understanding of how species and communities evolve across time and space. Understanding the nonrandom loss of species with respect to phylogeny is also essential for better-informed conservation decisions. However, several factors are known to influence phylogenetic reconstruction and, ultimately, phylogenetic diversity metrics. In this study, we empirically tested how some of these factors (topological constraint, taxon sampling, genetic markers and calibration) affect phylogenetic resolution and uncertainty. We built a densely sampled, species-level phylogenetic tree for spiders, combining Sanger sequencing of species from local communities of two biogeographical regions (Iberian Peninsula and Macaronesia) with a taxon-rich backbone matrix of Genbank sequences and a topological constraint derived from recent phylogenomic studies. The resulting tree constitutes the most complete spider phylogeny to date, both in terms of terminals and background information, and may serve as a standard reference for the analysis of phylogenetic diversity patterns at the community level. We then used this tree to investigate how partial data affect phylogenetic reconstruction, phylogenetic diversity estimates and their rankings, and, ultimately, the ecological processes inferred for each community. We found that the incorporation of a single slowly evolving marker (28S) to the DNA barcode sequences from local communities, had the highest impact on tree topology, closely followed by the use of a backbone matrix. The increase in missing data resulting from combining partial sequences from local communities only had a moderate impact on the resulting trees, similar to the difference observed when using topological constraints. Our study further revealed substantial differences in both the phylogenetic structure and diversity rankings of the analyzed communities estimated from the different phylogenetic treatments, especially when using non-ultrametric trees (phylograms) instead of time-stamped trees (chronograms). Finally, we provide some recommendations on reconstructing phylogenetic trees to infer phylogenetic diversity within ecological studies.

Plants and their pathogens are engaged in continuous evolutionary battles, with pathogens evolving to circumvent plant defense mechanisms and plants responding through enhanced protection to prevent or mitigate damage induced by pathogen attack. Managed ecosystems are composed of genetically identical populations of crop plants with few changes from year to year. These environments are highly conducive to the emergence and dissemination of pathogens and they exert selective pressure for both qualitative virulence factors responsible for fungal pathogenicity, and quantitative traits linked to pathogen fitness, such as aggressiveness. In this study, we used a comparative genome-wide approach to investigate the genomic basis underlying the pathogenicity and aggressiveness of the fungal coffee pathogen Colletotrichum kahawae infecting green coffee berries. The pathogenicity was investigated by comparing genomic variation between C. kahawae and its non-pathogenic sibling species, while the aggressiveness was studied by a genome-wide association approach with groups of isolates with different phenotypic profiles. High genetic differentiation was observed between C. kahawae and the most closely related species with 5,560 diagnostic SNPs identified, in which a significant enrichment of non-synonymous mutations was detected. Functional annotation of these non-synonymous mutations revealed a significant enrichment mainly in two gene ontology categories, “oxidation–reduction process” and “integral component of membrane.” Finally, the annotation of several genes potentially under-selection revealed that C. kahawae’s pathogenicity may be a complex biological process, in which important biological functions, such as, detoxification and transport, regulation of host and pathogen gene expression, and signaling are involved. On the other hand, the genome-wide association analyses for aggressiveness were able to identify 10 SNPs and 15 SNPs of small effect in single and multi-association analysis, respectively, from which 7 were common, giving in total 18 SNPs potentially associated. The annotation of these genomic regions allowed the identification of four candidate genes encoding F-box domain-containing, nitrosoguanidine resistance, Fungal specific transcription factor domain-containing and C6 transcription factor that could be associated with aggressiveness. This study shed light, for the first time, on the genetic mechanisms of C. kahawae host specialization.

Colletotrichum kahawae is a specialized plant pathogen of arabica coffee in Africa, able to infect green berries. The economic impact of this pathogen means there is an urgent need to better understand its pathogenic lifestyle, in particular its aggressiveness. In this study, several quantitative traits including disease severity, latent period and incubation period were measured to concomitantly assess the aggressiveness of 26 C. kahawae isolates. The results show that the area under disease progression curve is the most informative variable, particularly when joined together with the index disease intensity 10 days after inoculation and latency period, while the incubation period is not a reliable trait to distinguish aggressiveness levels in C. kahawae . This study also confirms the suitability of hypocotyls and detached green berries to perform C. kahawae aggressiveness assays, revealing that hypocotyls are a more reproducible testing material. Based on isolate profiles, three aggressiveness classes were established (high, moderate and low). A cytological analysis of representative isolates from each class showed that aggressiveness can be related to the development of post‐penetration stages, rather than conidia germination and appressoria differentiation. This study provides, for the first time, the best metrics to evaluate C. kahawae aggressiveness, characterizing the profile of a broad range of isolates, and defining a set of parameters that can be used to classify new isolates. Furthermore, the collected information will contribute to the improvement of coffee breeding programmes, through the selection of tester isolates for prescreening of resistant coffee materials, and offers the opportunity to engage on future genotype–phenotype studies.

Summary: Pathogenic fungi are emerging at an increasing rate on a wide range of host plants, leading to tremendous threats to the global economy and food safety. Several plant pathogens have been considered to be invasive species, rendering large‐scale population genomic analyses crucial to better understand their demographic history and evolutionary potential. Colletotrichum kahawae ( Ck ) is a highly aggressive and specialized pathogen, causing coffee berry disease in Arabica coffee in Africa. This pathogen leads to severe production losses and its dissemination out of Africa is greatly feared. To address this issue, a population genomic approach using thousands of single nucleotide polymorphisms (SNPs) spaced throughout the genome was used to unveil its demographic history and evolutionary potential. The current study confirms that Ck is a true clonal pathogen, perfectly adapted to green coffee berries, with three completely differentiated populations (Angolan, Cameroonian and East African). Two independent clonal lineages were found within the Angolan population as opposed to the remaining single clonal populations. The most probable colonization scenario suggests that this pathogen emerged in Angola and immediately dispersed to East Africa, where these two populations began to differentiate, followed by the introduction in Cameroon from an Angolan population. However, the differentiation between the two Angolan clonal lineages masks the mechanism for the emergence of the Cameroonian population. Our results suggest that Ck is completely differentiated from the ancestral lineage, has a low evolutionary potential and a low dispersion ability, with human transport the most likely scenario for its potential dispersion, which makes the fulfilment of the quarantine measures and management practices implemented crucial.

Summary: Taxonomy and History: Hemileia vastatrix Berk. and Broome (Basidiomycota, Pucciniales) was described in 1869 in eastern Africa and Ceylon as the agent of coffee leaf rust and has spread to all coffee cultivation areas worldwide. Major disease outbreaks in Asia, Africa and America caused and continue to cause severe yield losses, making this the most important disease of Arabica coffee, a cash crop for many tropical and sub‐tropical countries. Life cycle and Disease symptoms: Hemileia vastatrix is a hemicyclic fungus with the urediniosporic life cycle as its most important (if not only) source of inoculum. Chlorotic spots are the first macroscopic symptoms, preceding the differentiation of suprastomatal, bouquet‐shaped, orange‐coloured uredinia. The disease can cause yield losses of up to 35% and have a polyetic epidemiological impact on subsequent years. Disease control: Although the use of fungicides is one of the preferred immediate control measures, the use of resistant cultivars is considered to be the most effective and durable disease control strategy. The discovery of ‘Híbrido de Timor’ provided sources of resistance that have been used in several breeding programmes and that have been proven to be effective and durable, as some have been in use for more than 30 years. Genetic diversity and Molecular pathogenicity: Although exhibiting limited genetic polymorphism, the very large genome of H. vastatrix ( c . 797 Mbp) conceals great pathological diversity, with more than 50 physiological races. Gene expression studies have revealed a very precocious activation of signalling pathways and production of putative effectors, suggesting that the plant–fungus dialogue starts as early as at the germ tube stage, and have provided clues for the identification of avr genes.

Legitimacy and Implications of Reducing Colletotrichum kahawae to Subspecies in Plant Pathology

The Western Mediterranean Region and Macaronesian Islands are one of the top biodiversity hotspots of Europe, containing a significant native genetic diversity of global value among the Crop Wild Relatives (CWR). Sugar beet is the primary crop of the genus Beta (subfamily Betoideae, Amaranthaceae) and despite the great economic importance of this genus, and of the close relative Patellifolia species, a reconstruction of their evolutionary history is still lacking. We analyzed nrDNA (ITS) and cpDNA gene (matK, trnH-psbA, trnL intron, rbcL) sequences to: (i) investigate the phylogenetic relationships within the Betoideae subfamily, and (ii) elucidate the historical biogeography of wild beet species in the Western Mediterranean Region, including the Macaronesian Islands. The results support the Betoideae as a monophyletic group (excluding the Acroglochin genus) and provide a detailed inference of relationships within this subfamily, revealing: (i) a deep genetic differentiation between Beta and Patellifolia species, which may have occurred in Late Oligocene; and (ii) the occurrence of a West-East genetic divergence within Beta, indicating that the Mediterranean species probably differentiated by the end of the Miocene. This was interpreted as a signature of species radiation induced by dramatic habitat changes during the Messinian Salinity Crisis (MSC, 5.96–5.33 Mya). Moreover, colonization events during the Pleistocene also played a role in shaping the current diversity patterns among and within the Macaronesian Islands. The origin and number of these events could not be revealed due to insufficient phylogenetic resolution, suggesting that the diversification was quite recent in these archipelagos, and unravelling potential complex biogeographic patterns with hybridization and gene flow playing an important role. Finally, three evolutionary lineages were identified corresponding to major gene pools of sugar beet wild relatives, which provide useful information for establishing in situ and ex situ conservation priorities in the hotspot area of the Macaronesian Islands.

Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica—C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the model-based method used by NormFinder software. For C. arabica—C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern, developmental stage and host interaction.