Pedro Talhinhas

Genetic and Morphological Characterization of Colletotrichum acutatum Causing Anthracnose of Lupins

ABSTRACT Anthracnose, caused by Colletotrichum sp., is a serious problem of lupins (Lupinus spp.) worldwide. Morphological characters and molecular markers were used to characterize 43 Colletotrichum isolates from lupins, 8 isolates from other hosts, and 18 reference isolates representing related Colletotrichum spp., to assess the pathogen diversity and resolve its taxonomy. All lupin Colletotrichum isolates tested positive with C. acutatum-specific polymerase chain reaction (PCR) and did not test positive with C. gloeosporioides-specific PCR. Spore shape and colony diameter as well as insensitivity to benomyl grouped the lupin anthracnose isolates closer to C. acutatum than to C. gloeosporioides. Analysis of internal transcribed spacer (ITS) sequences of 57 Colletotrichum isolates grouped all lupin isolates with C. acutatum and distinct from C. gloeosporioides. Further, tub2 and his4 sequences revealed groups concordant with ITS, reducing the excessive dependence on the latter. Arbitrarily primed-PCR and amplified fragment length polymorphism analyses revealed intraspecific subgroups, but neither was useful to decipher species level relationships. ITS, tub2, and his4 results strongly support designating lupin anthracnose pathogen as C. acutatum or its subspecies. Most Colletotrichum isolates from lupins from worldwide locations are genetically homogeneous and form a distinct subgroup within C. acutatum. Present results also underline the potential of the C. acutatum-specific PCR for routine pathogen diagnosis.

Molecular and Phenotypic Analyses Reveal Association of Diverse Colletotrichum acutatum Groups and a Low Level of C. gloeosporioides with Olive Anthracnose

ABSTRACT Anthracnose (Colletotrichum spp.) is an important disease causing major yield losses and poor oil quality in olives. The objectives were to determine the diversity and distribution pattern of Colletotrichum spp. populations prevalent in olives and their relatedness to anthracnose pathogens in other hosts, assess their pathogenic variability and host preference, and develop diagnostic tools. A total of 128 Colletotrichum spp. isolates representing all olive-growing areas in Portugal and a few isolates from other countries were characterized by molecular and phenotypic assays and compared with reference isolates. Arbitrarily primed PCR data, internal transcribed spacer of rRNA gene and β-tubulin 2 nucleotide sequences, colony characteristics, and benomyl sensitivity showed Colletotrichum acutatum to be dominant (>97%) with limited occurrence of Colletotrichum gloeosporioides (<3%). Among C. acutatum populations, five molecular groups, A2 to A6, were identified. A2 was widely prevalent (89%), coinciding with a high incidence of anthracnose and environmental conditions suitable to disease spread. A4 was dominant in a particular region, while other C. acutatum groups and C. gloeosporioides were sporadic in their occurrence, mostly related to marginal areas of olive cultivation. C. gloeosporioides, isolated from olive fruits with symptoms indistinguishable from those of C. acutatum, showed same virulence rating as the most virulent C. acutatum isolate from group A2. C. acutatum and C. gloeosporioides isolates tested in infected strawberry fruits and strawberry and lupin plants revealed their cross-infection potential. Diagnostic tools were developed from β-tubulin 2 sequences to enable rapid and reliable pathogen detection and differentiation of C. acutatum groups.

Genotypic and phenotypic diversity in Colletotrichum acutatum, a cosmopolitan pathogen causing anthracnose on a wide range of hosts

SUMMARY Colletotrichum acutatum causes anthracnose on a wide range of hosts including woody and herbaceous crops, ornamentals, fruits and conifers. Almond, citrus, lupin, olive and strawberry are some of the crops in which C. acutatum diseases are economically important. With the application of molecular markers and diagnostic PCR over the last 10-15 years, C. acutatum was identified as a major pathogen on a number of hosts instead of or along with C. gloeosporioides. C. acutatum displays high levels of genotypic and phenotypic diversity. The global populations of this cosmopolitan pathogen fit into at least eight distinct molecular groups, A1-A8, which show some degree of correlation with the morphological characteristics and varying patterns of host association and geographical distribution. The pathogen has complex epidemiology, exhibiting pathogenic and non-pathogenic lifestyles on target hosts, non-target crops and weeds. C. acutatum populations also show pathogenic variability and cross-infection potential in relation to a number of hosts. Molecular genetic tools are being developed to investigate the pathogenicity mechanisms of this key pathogen. This article mainly focuses on the global population diversity in C. acutatum, pathogen epidemiology and diagnosis, host colonization processes, and the development of tools for the identification and analysis of genes associated with pathogenicity. Background information on the pathogen origin, host range, disease symptoms and disease management strategies is also provided.

454-pyrosequencing of Coffea arabica leaves infected by the rust fungus Hemileia vastatrix reveals in planta-expressed pathogen-secreted proteins and plant functions in a late compatible plant-rust interaction

Coffee (Coffea arabica L.), one of the key export and cash crops in tropical and subtropical countries, suffers severe losses from the rust fungus Hemileia vastatrix. The transcriptome of H. vastatrix was analysed during a compatible interaction with coffee to obtain an exhaustive repertoire of the genes expressed during infection and to identify potential effector genes. Large-scale sequencing (454-GS-FLEX Titanium) of mixed coffee and rust cDNAs obtained from 21-day rust-infected leaves generated 352 146 sequences which assembled into 22 774 contigs. In the absence of any reference genomic sequences for Coffea or Hemileia, specific trinucleotide frequencies within expressed sequence tags (ESTs) and blast homology against a set of dicots and basidiomycete genomes were used to distinguish pathogen from plant sequences. About 30% (6763) of the contigs were assigned to H. vastatrix and 61% (13 951) to C. arabica. The majority (60%) of the rust sequences did not show homology to any genomic database, indicating that they were potential novel fungal genes. In silico analyses of the 6763 H. vastatrix contigs predicted 382 secreted proteins and identified homologues of the flax rust haustorially expressed secreted proteins (HESPs) and bean rust transferred protein 1 (RTP1). These rust candidate effectors showed conserved amino-acid domains and conserved patterns of cysteine positions suggestive of conserved functions during infection of host plants. Quantitative reverse transcription-polymerase chain reaction profiling of selected rust genes revealed dynamic expression patterns during the time course of infection of coffee leaves. This study provides the first valuable genomic resource for the agriculturally important plant pathogen H. vastatrix and the first comprehensive C. arabica EST dataset.

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.

Genome size analyses of Pucciniales reveal the largest fungal genomes.

Rust fungi (Basidiomycota, Pucciniales) are biotrophic plant pathogens which exhibit diverse complexities in their life cycles and host ranges. The completion of genome sequencing of a few rust fungi has revealed the occurrence of large genomes. Sequencing efforts for other rust fungi have been hampered by uncertainty concerning their genome sizes. Flow cytometry was recently applied to estimate the genome size of a few rust fungi, and confirmed the occurrence of large genomes in this order (averaging 225.3 Mbp, while the average for Basidiomycota was 49.9 Mbp and was 37.7 Mbp for all fungi). In this work, we have used an innovative and simple approach to simultaneously isolate nuclei from the rust and its host plant in order to estimate the genome size of 30 rust species by flow cytometry. Genome sizes varied over 10-fold, from 70 to 893 Mbp, with an average genome size value of 380.2 Mbp. Compared to the genome sizes of over 1800 fungi, Gymnosporangium confusum possesses the largest fungal genome ever reported (893.2 Mbp). Moreover, even the smallest rust genome determined in this study is larger than the vast majority of fungal genomes (94%). The average genome size of the Pucciniales is now of 305.5 Mbp, while the average Basidiomycota genome size has shifted to 70.4 Mbp and the average for all fungi reached 44.2 Mbp. Despite the fact that no correlation could be drawn between the genome sizes, the phylogenomics or the life cycle of rust fungi, it is interesting to note that rusts with Fabaceae hosts present genomes clearly larger than those with Poaceae hosts. Although this study comprises only a small fraction of the more than 7000 rust species described, it seems already evident that the Pucciniales represent a group where genome size expansion could be a common characteristic. This is in sharp contrast to sister taxa, placing this order in a relevant position in fungal genomics research.

Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestyles

Colletotrichum acutatum is a cosmopolitan pathogen causing economically important diseases known as anthracnose on a wide range of hosts. This fungus exhibits varied pathogenicity lifestyles and the tools essential to understand the molecular mechanisms are still being developed. The transformation methods currently available for this species for gene discovery and functional analysis involve protoplast transformation and are laborious and inefficient. We have developed a protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of C. acutatum. Using this protocol we were able to transform C. acutatum isolates belonging to different genetic groups and originating from different hosts. The transformation efficiency was up to 156 transformants per 104 conidia, with >70% transformants showing single location/single copy integration of T-DNA. Binary vector pBHt2-GFP was constructed, enabling green fluorescence protein tagging of C. acutatum strains, which will be a useful tool for epidemiology and histopathology studies. The ATMT protocol developed was used to identify putative pathogenicity mutants, suggesting the applicability of this technique for rapid generation of a large panel of insertional mutants of C. acutatum leading to the identification of the genes associated with the varied lifestyles.

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.

The distinctive population structure of Colletotrichum species associated with olive anthracnose in the Algarve region of Portugal reflects a host–pathogen diversity hot spot

Anthracnose (Colletotrichum spp.) is an important disease of olive fruits. Diversity and biogeographic relationships of the olive anthracnose pathogens in the Algarve (Portugal) were investigated, along with host association patterns and disease levels during 2004-2007, to test the hypothesis that this region is a host-pathogen diversity hot spot. Diverse Colletotrichum acutatum and Colletotrichum gloeosporioides populations were identified based on rRNA-internal transcribed spacer and partial beta-tubulin 2 gene sequences of 95 isolates. Spatial and temporal variations in the occurrence of the eight genetic entities of the pathogens were linked to olive biogeography. Disease occurrence patterns suggest that C. acutatum populations are more stable pathogens, while C. gloeosporioides populations appear to be more influenced by favourable conditions. Three unique C. acutatum populations were identified, but none of the eight populations were dominant, with the most frequent type representing only 27%. Thus, the population structure of olive anthracnose pathogens in the Algarve is distinct from other parts of Portugal and other world locations, where only one or two genetic entities are dominant. This pattern and level of genetic diversity in a restricted area, where oleaster (wild olive tree), ancient landraces and modern cultivars of olive occur in close proximity, suggests the Algarve as a centre of diversity of the anthracnose pathogens and corroborates recent work suggesting western Mediterranean as an important centre of olive diversity and domestication.

Magnaporthe oryzae Populations Adapted to Finger Millet and Rice Exhibit Distinctive Patterns of Genetic Diversity, Sexuality and Host Interaction

In this study, host-specific forms of the blast pathogen Magnaporthe oryzae in sub-Saharan Africa (SSA) were characterised from distinct cropping locations using a combination of molecular and biological assays. Finger millet blast populations in East Africa revealed a continuous genetic variation pattern and lack of clonal lineages, with a wide range of haplotypes. M. oryzae populations lacked the grasshopper (grh) element (96%) and appeared distinct to those in Asia. An overall near equal distribution (47–53%) of the mating types MAT1-1 and MAT1-2, high fertility status (84–89%) and the dominance of hermaphrodites (64%) suggest a strong sexual reproductive potential. Differences in pathogen aggressiveness and lack of cultivar incompatibility suggest the importance of quantitative resistance. Rice blast populations in West Africa showed a typical lineage-based structure. Among the nine lineages identified, three comprised ~90% of the isolates. Skewed distribution of the mating types MAT1-1 (29%) and MAT1-2 (71%) was accompanied by low fertility. Clear differences in cultivar compatibility within and between lineages suggest R gene-mediated interactions. Distinctive patterns of genetic diversity, sexual reproductive potential and pathogenicity suggest adaptive divergence of host-specific forms of M. oryzae populations linked to crop domestication and agricultural intensification.