Vítor Várzea

Application of the Apn2/MAT locus to improve the systematics of the Colletotrichum gloeosporioides complex: an example from coffee (Coffea spp.) hosts

To improve phylogenetic resolution of the Colletotrichum gloeosporioides species complex we developed and tested the performance of a new set of primers for the Apn2/MAT locus with a case study of 22 isolates. These were isolated mainly from coffee plants and represent six divergent and well characterized species within the C. gloeosporioides complex. Following previous studies on this locus, we have generated sequence data from an expanded region (> 4600 bp), revealing increased phylogenetic informativeness when compared to other commonly used markers such as ITS, β-tub2 and GS. Within the Apn2/MAT locus the ApMAT marker alone was almost as informative in terms of phylogenetic resolution as a seven-gene concatenated dataset. Our results further revealed that gene-tree discordance may come to be a common issue in resolving evolutionary relationships in the C. gloeosporioides complex, highlighting the importance of multilocus approaches. The use of state-of-the-art data analysis techniques and a highly informative dataset as employed here may abate this issue and hopefully assist in disentangling the C. gloeosporioides complex.

Host‐jump drives rapid and recent ecological speciation of the emergent fungal pathogen Colletotrichum kahawae

Ecological speciation through host‐shift has been proposed as a major route for the appearance of novel fungal pathogens. The growing awareness of their negative impact on global economies and public health created an enormous interest in identifying the factors that are most likely to promote their emergence in nature. In this work, a combination of pathological, molecular and geographical data was used to investigate the recent emergence of the fungus Colletotrichum kahawae. C. kahawae emerged as a specialist pathogen causing coffee berry disease in Coffea arabica, owing to its unparalleled adaptation of infecting green coffee berries. Contrary to current hypotheses, our results suggest that a recent host‐jump underlay the speciation of C. kahawae from a generalist group of fungi seemingly harmless to coffee berries. We posit that immigrant inviability and a predominantly asexual behaviour could have been instrumental in driving speciation by creating pleiotropic interactions between local adaptation and reproductive patterns. Moreover, we estimate that C. kahawae began its diversification at <2200 bp leaving a very short time frame since the divergence from its sibling lineage (c. 5600 bp), during which a severe drop in C. kahawae’s effective population size occurred. This further supports a scenario of recent introduction and subsequent adaptation to C. arabica. Phylogeographical data revealed low levels of genetic polymorphism but provided the first geographically consistent population structure of C. kahawae, inferring the Angolan population as the most ancestral and the East African populations as the most recently derived. Altogether, these results highlight the significant role of host specialization and asexuality in the emergence of fungal pathogens through ecological speciation.

Validation of reference genes for normalization of qPCR gene expression data from Coffea spp. hypocotyls inoculated with Colletotrichum kahawae

BackgroundCoffee production in Africa represents a significant share of the total export revenues and influences the lives of millions of people, yet severe socio-economic repercussions are annually felt in result of the overall losses caused by the coffee berry disease (CBD). This quarantine disease is caused by the fungus Colletotrichum kahawae Waller and Bridge, which remains one of the most devastating threats to Coffea arabica production in Africa at high altitude, and its dispersal to Latin America and Asia represents a serious concern. Understanding the molecular genetic basis of coffee resistance to this disease is of high priority to support breeding strategies. Selection and validation of suitable reference genes presenting stable expression in the system studied is the first step to engage studies of gene expression profiling.ResultsIn this study, a set of ten genes (S24, 14-3-3, RPL7, GAPDH, UBQ9, VATP16, SAND, UQCC, IDE and β-Tub9) was evaluated to identify reference genes during the first hours of interaction (12, 48 and 72 hpi) between resistant and susceptible coffee genotypes and C. kahawae. Three analyses were done for the selection of these genes considering the entire dataset and the two genotypes (resistant and susceptible), separately. The three statistical methods applied GeNorm, NormFinder, and BestKeeper, allowed identifying IDE as one of the most stable genes for all datasets analysed, and in contrast GADPH and UBQ9 as the least stable ones. In addition, the expression of two defense-related transcripts, encoding for a receptor like kinase and a pathogenesis related protein 10, were used to validate the reference genes selected.ConclusionTaken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the interaction between Coffea spp. and C. kahawae.

Cellular and molecular analyses of coffee resistance to Hemileia vastatrix and nonhost resistance to Uromyces vignae in the resistance-donor genotype HDT832/2

In Arabica coffee breeding, some of the most used sources of resistance to leaf rust (Hemileia vastatrix) are natural Coffea arabica x canephora hybrids (“Híbrido de Timor”). To decipher the cellular and molecular nature of that resistance, leaves of genotype HDT832/2, were challenged with H. vastatrix race II, and monitored using light microscopy and RT-qPCR expression analysis of genes involved in plant immunity mechanisms (receptor-like kinase, WRKY transcription factor 1, phenylalanine ammonia-lyase, chalcone synthase, 13-lipoxygenase, glycosyltransferase, pathogenesis related PR1b and PR10). These were compared to the nonhost resistance responses of HDT832/2 to the infection by the cowpea rust fungus (Uromyces vignae). H. vastatrix ceased growth more frequently after stomata penetration, forming few haustoria, inducing a hypersensitive-like response, phenol accumulation and haustorium encasement with callose. U. vignae could enter stomata but failed to form haustoria, while inducing hypersensitive-like responses and phenol accumulation. In host and nonhost interactions, activation of genes involved in signalling coincided with the differentiation of appressoria, and cellular responses (hypersensitive-like responses and accumulation of phenolic compounds) were recorded from the full appressorium or penetration hypha stages onwards. Similarly, a gene related to the JA pathway was first activated at the penetration hypha stage for both interactions, while genes related to the SA pathway were only activated in the host interaction, the latter being the single clear difference between host and nonhost interactions. The cellular and molecular resistance responses of HDT832/2 to these rust fungi suggest that common immunity components are shared between host and nonhost resistance, which may explain the longer durability of this resistance.

Additional physiological races of coffee leaf rust (Hemileia vastatrix) identified in Kenya

Coffee leaf rust (CLR), caused by the fungus Hemileia vastatrix, is among the most important diseases affecting coffee all over the world. In Kenya, it is currently the second most important disease, and breeding coffee to obtain new resistant cultivars has been a priority. Over time, new rust pathogenic races able to infect hitherto resistant coffee genotypes have been registered. To date, 49 races of the pathogen have been characterized all over the world. The most recent races to be characterized are able to infect derivatives of Timor Hybrid (HDT), which is a major source of resistance in breeding programs. This work aimed to identify new races of the pathogen in Kenya, emphasizing infected leaves sampled from CLR resistant varieties and breeding lines collected from two sites (Ruiru and Koru). Twenty-four samples were characterized, out of which 22 samples corresponded to new races of the pathogen. A total of six new races (III, XVII, XXIII, XXXVI, XLI and XLII) were characterized, revealing three new virulence genes (v1, v7, v8) and possibly a fourth virulence gene, the v9. This finding represents a serious threat to coffee production and also a challenge to coffee breeding programs that are in progress in Kenya.

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.

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.

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

Colletotrichum kahawae Waller and Bridge is a highly aggressive and specialized fungal pathogen of coffee, causing the devastating Coffee Berry Disease (CBD), particularly at high altitudes. The disease arises from the unique ability of the pathogen to infect green developing coffee berries. This pathogen is currently confined to the African continent in all countries that grow Arabica coffee (Coffea arabica L.), leading to up to 80% yield losses, if no control measures are applied (Silva et al., 2006; Vossen and Walyaro, 2009; Hindorf and Omondi, 2011). For such huge economic impact, it is ranked as a quarantine pathogen and even as a biological weapon (Australia Group, 2014). Consequently, the pathogens potential dispersal to other Arabica coffee cultivation regions is greatly feared, particularly to those at high altitude also found in Latin America and Asia. Recently, this recognized species was brought down to a subspecific level (C. kahawae subsp. kahawae) based on molecular data (Weir et al., 2012), clustering together with a generalist and cosmopolitan group of Colletotrichum isolates unable to cause CBD (C. kahawae subsp. ciggaro). Since then a growing number of studies have reported the identification of C. kahawae in various hosts and regions of the world (Liu et al., 2013; Afanador-Kafuri et al., 2014; Mosca et al., 2014; Schena et al., 2014; Ismail et al., 2015; Garibaldi et al., 2016a,b; Perrone et al., 2016). Although these reports are referring to C. kahawae subsp. ciggaro, some of them could not distinguish the pathogen at the subspecific level, and this is leading to a wave of confusion of whether the long accepted species C. kahawae, the CBD pathogen, has escaped from Africa and extended its host range. Given the extreme impact that this situation may trigger and the subsequent biosecurity implications, there is a practical need to completely distinguish these pathogens taxonomically as to avoid the risk of misidentification, and caution should be taken on assigning/reassigning taxonomic ranking and nomenclature. Here we consider the evidences sustaining and contradicting the classification proposed by Weir et al. (2012), and discuss the risks and practical implications of changing the CBD pathogens species status in a plant pathology context.

Aggressiveness profiling of the coffee pathogen Colletotrichum kahawae

A. Vieira, I. Diniz* , A. Loureiro, A. P. Pereira, M. C. Silva, V. V arzea and D. Batista CIFC – Centro de Investigac ~ ao das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, 2784-505 Oeiras; CoBiG – Computational Biology and Population Genomics Group, cE3c – Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa; and LEAF – Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, 1349-017 Lisboa, Portugal