Helena Oliveira

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.

Cylindrocarpon root rot: multi-gene analysis reveals novel species within the Ilyonectria radicicola species complex

Ilyonectria radicicola and its Cylindrocarpon-like anamorph represent a species complex that is commonly associated with root rot disease symptoms on a range of hosts. During the course of this study, several species could be distinguished from I. radicicola sensu stricto based on morphological and culture characteristics. DNA sequence analysis of the partial β-tubulin, histone H3, translation elongation factor 1-α and nuclear ribosomal RNA-Internal Transcribed Spacer (nrRNA-ITS) genes were employed to provide further support for the morphological species resolved among 68 isolates associated with root rot disease symptoms. Of the various loci screened, nrRNA-ITS sequences were the least informative, while histone H3 sequences were the most informative, resolving the same number of species as the combined dataset across the four genes. Within the Ilyonectria radicicola species complex, 12 new taxa are delineated occurring on a diverse range of hosts, the most common being Cyclamen, Lilium, Panax, Pseudotsuga, Quercus and Vitis.

Multi-gene analysis and morphology reveal novel Ilyonectria species associated with black foot disease of grapevines

Black foot is an important disease of grapevines, which has in recent years been recorded with increased incidence and severity throughout the world, affecting grapevines both in nurseries and young vineyards. In the past the disease has been associated with infections by Ilyonectria macrodidyma, Ilyonectria liriodendri, Campylocarpon fasciculare, and Campylocarpon pseudofasciculare. Based on published data, a high level of genetic diversity was detected among isolates of I. macrodidyma. To resolve this issue, we employed a multigene analysis strategy (based on the β-tubulin, histone H3, translation elongation factor 1-α, and the internal transcribed spacers on both sides of the 5.8S nuclear ribosomal RNA gene) along with morphological characterisation to study a collection of 81 I. macrodidyma-like isolates from grapevine and other hosts. Morphological characters (particularly conidial size) and molecular data (highest resolution achieved with histone H3 nucleotide sequence) enabled the distinction of six monophyletic species within the I. macrodidyma complex, four of which (Ilyonectria alcacerensis, Ilyonectria estremocensis, Ilyonectria novozelandica, and Ilyonectria torresensis) are described here. This work forms part of an effort by the International Council on Grapevine Trunk Diseases to resolve the species associated with black foot disease, which we believe will clarify their taxonomy, and therefore help researchers to devise control strategies to reduce the devastating impact of this disease.

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.

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.

Black foot of grapevine: sensitivity of Cylindrocarpon destructans to fungicides

Black foot disease of grapevine is caused by Cylindrocarpon spp., with C. destructans being the main pathogen isolated from vine cuttings and young vineyards in Portugal. Few recommendations for black foot disease control are presently available, and they are not easy to implement within commercial nurseries. In this study, 14 fungicides were evaluated for their effect on the mycelial growth and conidium germination of four field isolates of C. destructans. Mycelial growth of the pathogen was inhibited by DMI fungicides, prochloraz (EC50 values <0.09 mg l-1) and to a lesser extent by difenoconazole (EC50 values <2.25 mg l-1), by the benzimidazole fungicide benomyl (EC50 values <0.35 mg l-1), and by the mixtures cyprodinil + fludioxonil and carbendazim + flusilazole, which gave EC50 values <0.75 mg l-1. Among these, only cyprodinil + fludioxonil (EC50 values <0.15 mg l-1), the strobilurin fungicides, azoxystrobin and trifloxystrobin (EC50 values <2.27 mg l-1) and the phenylsulphamide fungicide tolylfluanid (EC50 < 0.54 mg l-1) were effective in reducing conidium germination. Results from in vivo studies, carried out on potted grapevine plants (cultivar Castelao) showed that benomyl, tebuconazole and the mixtures carbendazim + flusilazole and cyprodinil + fludioxonil significantly (α=0.05) improved plant growth (plant height and number of roots) and decreased disease incidence compared with non-treated plants.

Virulence diversity of anthracnose pathogens (Colletotrichum acutatum and C. gloeosporioides species complexes) on eight olive cultivars commonly grown in Portugal

Olive anthracnose, caused by Colletotrichum acutatum and C. gloeosporioides species complexes, is a major disease affecting fruits at maturity, causing significant yield losses, and poor fruit and oil quality. Diverse genetic groups, particularly belonging to C. acutatum s.l. have been reported among the pathogens, with recent research proposing these genetic groups as distinct species. In this work, the virulence diversity of isolates representing different populations of C. acutatum s.l. and C. gloeosporioides s.s. was studied using a set of eight olive cultivars. Higher disease severity was produced by isolates belonging to groups A2 and A5 of C. acutatum s.l. (=C. nymphaeae and C. acutatum s.s., respectively) compared to C. gloeosporioides s.s. isolates as well as isolates of C. acutatum s.l. group A4 (=C. godetiae). Anthracnose severity was higher on the cultivars ‘Cobrançosa’, ‘Maçanilha de Tavira’ and ‘Galega Vulgar’ and lower in ‘Azeitoneira’, ‘Blanqueta’, ‘Negrinha de Freixo’ and ‘Picual’, but results indicate the occurrence of isolate × cultivar interactions. Differences in severity could be related to differences in conidia germination and appressoria formation, suggesting that early host-pathogen recognition events can in part explain disease severity under favourable environmental conditions. Overall results revealed the higher virulence and fitness levels of genotypes belonging to certain genetic groups within C. acutatum suggesting their ability to adapt to diverse agro-climatic conditions including specific hosts.

Characterization of Cylindrodendrum, Dactylonectria and Ilyonectria isolates associated with loquat decline in Spain, with description of Cylindrodendrum alicantinum sp. nov.

Thirty-one loquat orchards (Eriobotrya japonica ‘Algerie’) with plants exhibiting decline symptoms were surveyed between 2004 and 2007 in the province of Alicante, Spain. Twenty-eight representative isolates with Cylindrocarpon-like asexual morphs recovered from affected roots were included in this study, with the objective to characterize them by means of phenotypical characterization, DNA analysis and pathogenicity tests. Dactylonectria alcacerensis, D. torresensis and Ilyonectria robusta were identified based on morphological and cultural characteristics as well as DNA sequence data for part of histone H3, with D. torresensis the most frequent species. All of them are reported for the first time on loquat, and I. robusta is reported for the first time in Spain. In addition, one species is newly described, Cylindrodendrum alicantinum. Pathogenicity tests with representative isolates showed that these species were able to induce typical root rot disease symptoms, affecting plant development or even leading to plant death. This research demonstrates the association of species belonging to the genera Cylindrodendrum, Dactylonectria and Ilyonectria with root rot of loquat and loquat decline in the province of Alicante (eastern Spain). This information should be considered for the improvement of the current management strategies against these soil-borne pathogens when establishing new loquat plantations or introducing new susceptible fruit crops in the region.

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.