Overexpression of Water-Responsive Genes Promoted by Elevated CO2 Reduces ROS and Enhances Drought Tolerance in Coffea Species.

Drought is a major constraint to plant growth and productivity worldwide and will aggravate as water availability becomes scarcer. Although elevated air [CO2] might mitigate some of these effects in plants, the mechanisms underlying the involved responses are poorly understood in woody economically important crops such as Coffea. This study analyzed transcriptome changes in Coffea canephora cv. CL153 and C. arabica cv. Icatu exposed to moderate (MWD) or severe water deficits (SWD) and grown under ambient (aCO2) or elevated (eCO2) air [CO2]. We found that changes in expression levels and regulatory pathways were barely affected by MWD, while the SWD condition led to a down-regulation of most differentially expressed genes (DEGs). eCO2 attenuated the impacts of drought in the transcripts of both genotypes but mostly in Icatu, in agreement with physiological and metabolic studies. A predominance of protective and reactive oxygen species (ROS)-scavenging-related genes, directly or indirectly associated with ABA signaling pathways, was found in Coffea responses, including genes involved in water deprivation and desiccation, such as protein phosphatases in Icatu, and aspartic proteases and dehydrins in CL153, whose expression was validated by qRT-PCR. The existence of a complex post-transcriptional regulatory mechanism appears to occur in Coffea explaining some apparent discrepancies between transcriptomic, proteomic, and physiological data in these genotypes.

Population structure in Quercus suber L. revealed by nuclear microsatellite markers.

Quercus suber L. is a sclerophyllous tree species native to the western Mediterranean, a region that is considered highly vulnerable to increased temperatures and severe dry conditions due to environmental changes. Understanding the population structure and demographics of Q. suber is essential in order to anticipate whether populations at greater risk and the species as a whole have the genetic background and reproductive dynamics to enable rapid adaptation. The genetic diversity of Q. suber has been subject to different studies using both chloroplast and nuclear data, but population structure patterns remain unclear. Here, we perform genetic analyses on Q. suber using 13 nuclear microsatellite markers, and analysed 17 distinct locations across the entire range of the species. Structure analyses revealed that Q. suber may contain three major genetic clusters that likely result from isolation in refugia combined with posterior admixture and putative introgression from other Quercus species. Our results show a more complex structure scenario than previously inferred for Q. suber using nuclear markers and suggest that different southern populations contain high levels of genetic variation that may contribute to the resilience of Q. suber in a context of environmental change and adaptive pressure.

Worldwide Population Structure of the Coffee Rust Fungus Hemileia vastatrix Is Strongly Shaped by Local Adaptation and Breeding History.

The devastating disease coffee leaf rust, caused by Hemileia vastatrix, has been a major constraint to worldwide coffee production. Recently, H. vastatrix populations were shown to be structured into three divergent genetic lineages with marked host specialization (C1, C2, and C3). However, there is yet no overall understanding of the population dynamics and adaptation of the most widespread and epidemiological relevant H. vastatrix group (C3). We used restriction site-associated DNA sequencing to generate 13,804 single nucleotide polymorphisms (SNPs) across a worldwide collection of 99 H. vastatrix isolates. Phylogenetic analyses uncovered a well-supported structuring within C3, with three main subgroups (SGs; SGI, SGII, and SGIII), which seem to reflect the historical distribution, breeding, and exchange of coffee cultivars. SGI shows a ladder-like diversification pattern and occurs across all four continents sampled, SGII is mainly restricted to Africa, and SGIII is observed only in Timor, revealing a higher genetic differentiation. Outlier and association tests globally identified 112 SNPs under putative positive selection, which impacted population structure. In particular, 29 overlapping SNPs per se seemed to have an extremely strong effect on H. vastatrix population divergence. We also found exclusive and fixed alleles associated with the SGs supporting local adaptation. Functional annotation revealed that transposable elements may play a role in host adaptation. Our study provides a higher-resolution perspective on the evolutionary history of H. vastatrix on cultivated coffee, showing its strong ability to adapt and the strength of the selective force imposed by coffee hosts, which should be taken into account when designing strategies for pathogen dissemination control and selective breeding.

Biochemical and Molecular Profiling of Wild Edible Mushrooms from Huila, Angola.

The harvesting, processing, and sale of wild edible mushrooms (WEM) is a relevant economic activity in Angola and a good example of the use of non-wood forest products for food. Although there is deep traditional knowledge about the general properties of WEMs, a huge gap remains in detailed scientific knowledge. Thus, this study aimed to investigate the socio-economic importance of the species sold at local markets in Huila, Angola, from their molecular identification to the assessment of their nutritional, chemical, and bioactive profiles. From the eight WEM morphotypes studied, five were identified based on phenotypical and molecular approaches (four Russula spp., and Amanita loosei). The studied mushrooms proved to be a rich source of carbohydrates, proteins, and ashes, also presenting low amounts of fat. Chemical analyses further revealed mannitol as the main free sugar in all samples, and organic acids, namely, oxalic, quinic, malic, citric, and fumaric acids in low amounts. Additionally, the α-tocopherol isoform and monounsaturated fatty acids were predominant. Regarding phenolic acids, protocatechuic, p-hydroxybenzoic, p-coumaric, and cinnamic acids were detected in all mushroom hydroethanolic extracts, being responsible for their antioxidant, antibacterial, and antifungal activities. Our investigation contributes to the identification and knowledge of WEMs as important complementary food sources in Angola, some of which were reported for the first time, promoting their utilization as a basis of nutritional and functional ingredients, as being able to be part of a balanced diet and to be used in new bio-based formulations.

Using gradient Forest to predict climate response and adaptation in Cork oak.

Climate change is impacting locally adapted species such as the keystone tree species cork oak (Quercus suber L.). Quantifying the importance of environmental variables in explaining the species distribution can help build resilient populations in restoration projects and design forest management strategies. Using landscape genomics, we investigated the population structure and ecological adaptation of this tree species across the Mediterranean Basin. We applied genotyping by sequencing and derived 2,583 single nucleotide polymorphism markers genotyped from 81 individuals across 17 sites in the studied region. We implemented an approach based on the nearest neighbour haplotype 'coancestry' and uncovered a weak population structure along an east-west climatic gradient across the Mediterranean region. We identified genomic regions potentially involved in local adaptation and predicted differences in the genetic composition across the landscape under current and future climates. Variants associated with temperature and precipitation variables were detected, and we applied a nonlinear multivariate association method, gradient forest, to project these gene-environment relationships across space. The model allowed the identification of geographic areas within the western Mediterranean region most sensitive to climate change: south-western Iberia and northern Morocco. Our findings provide a preliminary assessment towards a potential management strategy for the conservation of cork oak in the Mediterranean Basin.

First Report of Dieback Caused by Neofusicoccum batangarum in Cashew in Guinea-Bissau.

Cashew (Anacardium occidentale L.) is a cash crop with a highly significant economic importance in West Africa, particularly in Guinea-Bissau (Monteiro et al. 2015, 2017). In October 2018, dieback-like symptoms such as wilt and necrosis of apical shoots were observed in 10 % of the cashew trees grown in a 100 plant-orchard in Bolama Island at Bijagós archipelago, Guinea-Bissau. Six symptomatic apical shoots from individual plants were collected for fungal isolation and identification. Tissue pieces (3 × 2 mm) from healthy to diseased margins were surface sterilized with 1 % sodium hypochlorite, washed twice with sterilized water, placed on potato dextrose agar (PDA, Difco® Laboratories) supplemented with potassium thiocyanate (50 µg/ml), and incubated at 24 ± 1 °C in the dark for 7 days. Four fungal colonies were isolated (67 %) and purified through hyphal tips removal, displaying rapid growth rate, and aerial mycelia that initially was white, turning later to dark greenish on PDA. Pycnidia produced on 1.5 % water agar and sterilized pine needles (± 25ºC; near-UV light) were solitary, covered by mycelium, obpyriform to ampulliform (152.5 ± 41.6 × 135.2 ± 30.8 µm, n = 30). Conidia were unicellular, hyaline, smooth, fusoid to ovoid, thin-walled, measuring 16.21 ± 1.52 × 5.84 ± 0.66 µm (n = 50, L/W 2.8). Such morphological features are characteristic of Neofusicoccum spp. (Phillips et al. 2013). For molecular identification, genomic DNA was extracted from a representative isolate GB160 and partial regions of ribosomal internal transcriber spacer (ITS) (ITS1/ITS4; White et al. 1990), translation elongation factor 1-α (EF1-α) (EF1-688F/EF1-1251R; Alves et al. 2008) and ß-tubulin (ß-tub) (Bt2a/Bt2b; Glass and Donaldson 1995) genes were amplified as previously described, respectively, with BSA (50 mg/ml). Amplicons were sequenced and deposited in GenBank (ITS, MN952993; EF1-α, MN952204; ß-tub, MN952208). BLAST analysis of ITS, EF1-α and ß-tub gene sequences showed 100 % identity with Neofusicoccum batangarum reference strain CBS124923 (FJ900608, FJ900654, FJ900635, respectively). Maximum-likelihood and Bayesian Inference analyses from the concatenated dataset placed GB160 isolate within the N. batangarum cluster (BS = 72 %; PP = 0.95). For pathogenicity assessment, 3-month-old cashew "Caju di Terra" plants (n = 8) grown in a greenhouse under controlled conditions were inoculated following a randomized block design as described by Lima et al. (2013). Briefly, 3 mm diam. stem tissue bark was removed and replaced with a 3 mm diameter PDA plug retrieved from the colony margin. Inoculation wound was covered with sterilized wet cotton and sealed with parafilm. Eight control plants were only treated with PDA plugs and the wound covered and sealed as described. After 15 days, all inoculated plants displayed similar symptoms to those observed in the field, and vascular lesions (10.8 ± 4.0 cm), whereas control plants remained symptomless. Koch's postulates were fulfilled by successful re-isolation of the pathogen from all inoculated stems and identification by morphology and gene sequencing. N. batangarum was identified associated with Anacardium spp. in Brazil (Netto et al. 2017) and recently reported as causing grapevine dieback in Brazil (Rêgo et al. 2020). To our knowledge, this is the first report of N. batangarum causing cashew dieback in Guinea-Bissau and West Africa. Occurrence of this disease may represent a significant impact for cashew production since this crop is the major agricultural commodity in Guinea-Bissau.

Pathological, Morphological, Cytogenomic, Biochemical and Molecular Data Support the Distinction between Colletotrichum cigarro comb. et stat. nov. and Colletotrichum kahawae.

The genus Colletotrichum has witnessed tremendous variations over the years in the number of species recognized, ranging from 11 to several hundreds. Host-specific fungal species, once the rule, are now the exception, with polyphagous behavior regarded as normal in this genus. The species Colletotrichum kahawae was created to accommodate the pathogens that have the unique ability to infect green developing coffee berries causing the devastating Coffee Berry Disease in Africa, but its close phylogenetic relationship to a polyphagous group of fungi in the C. gloeosporioides species complex led some researchers to regard these pathogens as members of a wider species. In this work we combine pathological, morphological, cytogenomic, biochemical, and molecular data of a comprehensive set of phylogenetically-related isolates to show that the Coffee Berry Disease pathogen forms a separate species, C. kahawae, and also to assign the closely related fungi, previously in C. kahawae subsp. cigarro, to a new species, C. cigarro comb. et stat. nov. This taxonomic clarification provides an opportunity to link phylogeny and functional biology, and additionally enables a much-needed tool for plant pathology and agronomy, associating exclusively C. kahawae to the Coffee Berry Disease pathogen.

Genome-Wide Signatures of Selection in Colletotrichum kahawae Reveal Candidate Genes Potentially Involved in Pathogenicity and Aggressiveness.

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.

Novel insights on colonization routes and evolutionary potential of Colletotrichum kahawae, a severe pathogen of Coffea arabica.

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.

Population genomic footprints of host adaptation, introgression and recombination in coffee leaf rust.

Coffee leaf rust, caused by Hemileia vastatrix (Hv), represents the biggest threat to coffee production worldwide and ranks amongst the most serious fungal diseases in history. Despite a recent series of outbreaks and emergence of hypervirulent strains, the population evolutionary history and potential of this pathogen remain poorly understood. To address this issue, we used restriction site-associated DNA sequencing (RADseq) to generate ∼19 000 single nucleotide polymorphisms (SNPs) across a worldwide collection of 37 Hv samples. Contrary to the long-standing idea that Hv represents a genetically unstructured and cosmopolitan species, our results reveal the existence of a cryptic species complex with marked host tropism. Using phylogenetic and pathological data, we show that one of these lineages (C3) infects almost exclusively the most economically valuable coffee species (tetraploids that include Coffea arabica and interspecific hybrids), whereas the other lineages (C1 and C2) are severely maladapted to these hosts, but successfully infect diploid coffee species. Population dynamic analyses suggest that the C3 group may be a recent 'domesticated' lineage that emerged via host shift from diploid coffee hosts. We also found evidence of recombination occurring within this group, which could explain the high pace of pathotype emergence despite the low genetic variation. Moreover, genomic footprints of introgression between the C3 and C2 groups were discovered and raise the possibility that virulence factors may be quickly exchanged between groups with different pathogenic abilities. This work advances our understanding of the evolutionary strategies used by plant pathogens in agro-ecosystems with direct and far-reaching implications for disease control.

A first insight into the involvement of phytohormones pathways in coffee resistance and susceptibility to Colletotrichum kahawae.

Understanding the molecular mechanisms underlying coffee-pathogen interactions are of key importance to aid disease resistance breeding efforts. In this work the expression of genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) pathways were studied in hypocotyls of two coffee varieties challenged with the hemibiotrophic fungus Colletotrichum kahawae, the causal agent of Coffee Berry Disease. Based on a cytological analysis, key time-points of the infection process were selected and qPCR was used to evaluate the expression of phytohormones biosynthesis, reception and responsive-related genes. The resistance to C. kahawae was characterized by restricted fungal growth associated with early accumulation of phenolic compounds in the cell walls and cytoplasmic contents, and deployment of hypersensitive reaction. Similar responses were detected in the susceptible variety, but in a significantly lower percentage of infection sites and with no apparent effect on disease development. Gene expression analysis suggests a more relevant involvement of JA and ET phytohormones than SA in this pathosystem. An earlier and stronger activation of the JA pathway observed in the resistant variety, when compared with the susceptible one, seems to be responsible for the successful activation of defense responses and inhibition of fungal growth. For the ET pathway, the down or non-regulation of ET receptors in the resistant variety, together with a moderate expression of the responsive-related gene ERF1, indicates that this phytohormone may be related with other functions besides the resistance response. However, in the susceptible variety, the stronger activation of ERF1 gene at the beginning of the necrotrophic phase, suggests the involvement of ET in tissue senescence. As far as we know, this is the first attempt to unveil the role of phytohormones in coffee-C. kahawae interactions, thus contributing to deepen our understanding on the complex mechanisms of plant signaling and defense.

The coffee leaf rust pathogen Hemileia vastatrix: one and a half centuries around the tropics.

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.

Evolutionary and Biogeographic Insights on the Macaronesian Beta-Patellifolia Species (Amaranthaceae) from a Time-Scaled Molecular Phylogeny.

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.

Comparative Validation of Conventional and RNA-Seq Data-Derived Reference Genes for qPCR Expression Studies of Colletotrichum kahawae.

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.

4Pipe4--A 454 data analysis pipeline for SNP detection in datasets with no reference sequence or strain information.

BACKGROUND: Next-generation sequencing datasets are becoming more frequent, and their use in population studies is becoming widespread. For non-model species, without a reference genome, it is possible from a panel of individuals to identify a set of SNPs that can be used for further population genotyping. However the lack of a reference genome to which the sequenced data could be compared makes the finding of SNPs more troublesome. Additionally when the data sources (strains) are not identified (e.g. in datasets of pooled individuals), the problem of finding reliable variation in these datasets can become much more difficult due to the lack of specialized software for this specific task. RESULTS: Here we describe 4Pipe4, a 454 data analysis pipeline particularly focused on SNP detection when no reference or strain information is available. It uses a command line interface to automatically call other programs, parse their outputs and summarize the results. The variation detection routine is built-in in the program itself. Despite being optimized for SNP mining in 454 EST data, it is flexible enough to automate the analysis of genomic data or even data from other NGS technologies. 4Pipe4 will output several HTML formatted reports with metrics on many of the most common assembly values, as well as on all the variation found. There is also a module available for finding putative SSRs in the analysed datasets. CONCLUSIONS: This program can be especially useful for researchers that have 454 datasets of a panel of pooled individuals and want to discover and characterize SNPs for subsequent individual genotyping with customized genotyping arrays. In comparison with other SNP detection approaches, 4Pipe4 showed the best validation ratio, retrieving a smaller number of SNPs but with a considerably lower false positive rate than other methods. 4Pipe4's source code is available at https://github.com/StuntsPT/4Pipe4.

Genomic Patterns of Positive Selection at the Origin of Rust Fungi.

Understanding the origin and evolution of pathogenicity and biotrophic life-style of rust fungi has remained a conundrum for decades. Research on the molecular mechanisms responsible for rust fungi evolution has been hampered by their biotrophic life-style until the sequencing of some rust fungi genomes. With the availability of multiple whole genomes and EST data for this group, it is now possible to employ genome-wide surveys and investigate how natural selection shaped their evolution. In this work, we employed a phylogenomics approach to search for positive selection and genes undergoing accelerated evolution at the origin of rust fungi on an assembly of single copy genes conserved across a broad range of basidiomycetes. Up to 985 genes were screened for positive selection on the phylogenetic branch leading to rusts, revealing a pervasive signal of positive selection throughout the data set with the proportion of positively selected genes ranging between 19.6-33.3%. Additionally, 30 genes were found to be under accelerated evolution at the origin of rust fungi, probably due to a mixture of positive selection and relaxation of purifying selection. Functional annotation of the positively selected genes revealed an enrichment in genes involved in the biosynthesis of secondary metabolites and several metabolism and transporter classes.

Tracking cashew economically important diseases in the West African region using metagenomics.

During the last decades, agricultural land-uses in West Africa were marked by dramatic shifts in the coverage of individual crops. Nowadays, cashew (Anacardium occidentale L.) is one of the most export-oriented horticulture crops, notably in Guinea-Bissau. Relying heavily on agriculture to increase their income, developing countries have been following a strong trend of moving on from traditional farming systems toward commercial production. Emerging infectious diseases, driven either by adaptation to local conditions or inadvertent importation of plant pathogens, are able to cause tremendous cashew production losses, with economic and social impact of which, in developing countries is often underestimated. Presently, plant genomics with metagenomics as an emergent tool, presents an enormous potential to better characterize diseases by providing extensive knowledge on plant pathogens at a large scale. In this perspective, we address metagenomics as a promising genomic tool to identify cashew fungal associated diseases as well as to discriminate the causal pathogens, aiming at obtaining tools to help design effective strategies for disease control and thus promote the sustainable production of cashew in West African Region.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Overview of the functional virulent genome of the coffee leaf rust pathogen Hemileia vastatrix with an emphasis on early stages of infection.

Hemileia vastatrix is the causal agent of coffee leaf rust, the most important disease of coffee Arabica. In this work, a 454-pyrosequencing transcriptome analysis of H. vastatrix germinating urediniospores (gU) and appressoria (Ap) was performed and compared to previously published in planta haustoria-rich (H) data. A total of 9234 transcripts were identified and annotated. Ca. 50% of these transcripts showed no significant homology to international databases. Only 784 sequences were shared by the three conditions, and 75% were exclusive of either gU (2146), Ap (1479) or H (3270). Relative transcript abundance and RT-qPCR analyses for a selection of genes indicated a particularly active metabolism, translational activity and production of new structures in the appressoria and intense signaling, transport, secretory activity and cellular multiplication in the germinating urediniospores, suggesting the onset of a plant-fungus dialogue as early as at the germ tube stage. Gene expression related to the production of carbohydrate-active enzymes and accumulation of glycerol in germinating urediniospores and appressoria suggests that combined lytic and physical mechanisms are involved in appressoria-mediated penetration. Besides contributing to the characterization of molecular processes leading to appressoria-mediated infection by rust fungi, these results point toward the identification of new H. vastatrix candidate virulence factors, with 516 genes predicted to encode secreted proteins.