Subcuticular−Intracellular Hemibiotrophy of Colletotrichum lupini in Lupinus mutabilis

Anthracnose caused by Colletotrichum lupini is the most important disease affecting lupin cultivation worldwide. Lupinus mutabilis has been widely studied due to its high protein and oil content. However, it has proved to be sensitive to anthracnose, which limits the expansion of its cultivation. In this work, we seek to unveil the strategy that is used by C. lupini to infect and colonize L. mutabilis tissues using light and transmission electron microscopy (TEM). On petioles, pathogen penetration occurred from melanized appressoria, subcuticular intramural hyphae were seen 2 days after inoculation (dai), and the adjacent host cells remained intact. The switch to necrotrophy was observed 3 dai. At this time, the hyphae extended their colonization to the epidermal, cortex, and vascular cells. Wall degradation was more evident in the epidermal cells. TEM observations also revealed a loss of plasma membrane integrity and different levels of cytoplasm disorganization in the infected epidermal cells and in those of the first layers of the cortex. The disintegration of organelles occurred and was particularly visible in the chloroplasts. The necrotrophic phase culminated with the development of acervuli 6 dai. C. lupini used the same infection strategy on stems, but there was a delay in the penetration of host tissues and the appearance of the first symptoms.

Diversity and Agronomic Performance of Lupinus mutabilis Germplasm in European and Andean Environments.

The introduction of Lupinus mutabilis (Andean lupin) in Europe will provide a new source of protein and oil for plant-based diets and biomass for bio-based products, while contributing to the improvement of marginal soils. This study evaluates for the first time the phenotypic variability of a large panel of L. mutabilis accessions both in their native environment and over two cropping conditions in Europe (winter crop in the Mediterranean region and summer crop in North-Central Europe), paving the way for the selection of accessions adapted to specific environments. The panel of 225 accessions included both germplasm pools from the Andean region and breeding lines from Europe. Notably, we reported higher grain yield in Mediterranean winter-cropping conditions (18 g/plant) than in the native region (9 g/plant). Instead, North European summer-cropping conditions appear more suitable for biomass production (up to 2 kg/plant). The phenotypic evaluation of 16 agronomical traits revealed significant variation in the panel. Principal component analyses pointed out flowering time, yield, and architecture-related traits as the main factors explaining variation between accessions. The Peruvian material stands out among the top-yielding accessions in Europe, characterized by early lines with high grain yield (e.g., LIB065, LIB072, and LIB155). Bolivian and Ecuadorian materials appear more valuable for the selection of genotypes for Andean conditions and for biomass production in Europe. We also observed that flowering time in the different environments is influenced by temperature accumulation. Within the panel, it is possible to identify both early and late genotypes, characterized by different thermal thresholds (600°C-700°C and 1,000-1,200°C GDD, respectively). Indications on top-yielding and early/late accessions, heritability of morpho-physiological traits, and their associations with grain yield are reported and remain largely environmental specific, underlining the importance of selecting useful genetic resources for specific environments. Altogether, these results suggest that the studied panel holds the genetic potential for the adaptation of L. mutabilis to Europe and provide the basis for initiating a breeding program based on exploiting the variation described herein.

Response to Anthracnose in a Tarwi (Lupinus mutabilis) Collection Is Influenced by Anthocyanin Pigmentation.

Anthracnose, caused by Colletotrichum lupini, is a major limiting factor for lupin production. Tarwi or Andean Lupin (Lupinus mutabilis) is generally regarded as susceptible to anthracnose, but the high protein and oil content of its seeds raise interest in promoting its cultivation in Europe. In this study we evaluated the response to anthracnose of 10 tarwi accessions contrasting in anthocyanin pigmentation, by comparison to white lupin (Lupinus albus), using a contemporary Portuguese fungal isolate. A severity rating scale was optimized, including weighted parameters considering the type of symptoms and organs affected. All tarwi accessions were classified as susceptible, exhibiting sporulating necroses on the main stem from seven days after inoculation. Anthracnose severity was lower on anthocyanin-rich tarwi plants, with accession LM34/LIB209 standing out as the less susceptible. Accession I82/LIB201 better combines anthracnose response and yield. In global terms, disease severity was lower on white lupin than on tarwi. Although based on a limited collection, the results of the study show the existence of genetic variability among L. mutabilis towards anthracnose response relatable with anthocyanin pigmentation, providing insights for more detailed and thorough characterization of tarwi resistance to anthracnose.

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.

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 10(4) 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.

Molecular and phenotypic analyses reveal association of diverse Colletotrichum acutatum groups and a low level of C. gloeosporioides with olive anthracnose.

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 beta-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 beta-tubulin 2 sequences to enable rapid and reliable pathogen detection and differentiation of C. acutatum groups.

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.