Complete Genome Sequence of the plant pathogenic fungus Colletotrichum lupini.

Colletotrichum is a fungal genus (Ascomycota, Sordariomycetes, Glomerellaceae) that includes many economically important plant pathogens that cause devastating diseases of a wide range of plants. In this work, using a combination of long and short-read sequencing technologies, we sequenced the genome of Colletotrichum lupini RB221, isolated from white lupin (Lupinus albus) in France during a survey in 2014. The genome was assembled into eleven nuclear chromosomes and a mitochondrial genome with a total assembly size of 63.41Mb and 36.55 Kb, respectively. A total of 18324 protein encoding genes have been predicted, of which only 39 are specific to C. lupini. To the best of our knowledge this is the first genome of this species to be fully sequenced and to get publicly released. This resource will provide insight into pathogenicity factors and will help to get a better understanding of the evolution and genome structure of this important plant pathogen.

A qPCR Assay for the Fast Detection and Quantification of Colletotrichum lupini

White lupin (Lupinus albus) represents an important legume crop in Europe and other parts of the world due to its high protein content and potential for low-input agriculture. However, most cultivars are susceptible to anthracnose caused by Colletotrichum lupini, a seed- and air-borne fungal pathogen that causes severe yield losses. The aim of this work was to develop a C. lupini-specific quantitative real-time TaqMan PCR assay that allows for quick and reliable detection and quantification of the pathogen in infected seed and plant material. Quantification of C. lupini DNA in dry seeds allowed us to distinguish infected and certified (non-infected) seed batches with DNA loads corresponding to the disease score index and yield of the mother plants. Additionally, C. lupini DNA could be detected in infected lupin shoots and close to the infection site, thereby allowing us to study the disease cycle of this hemibiotrophic pathogen. This qPCR assay provides a useful diagnostic tool to determine anthracnose infection levels of white lupin seeds and will facilitate the use of seed health assessments as a strategy to reduce the primary infection source and spread of this disease.

Assessment of the differential gene expression in anthracnose treated seedlings of yellow lupin

Seedlings of yellow lupine treated with Colletotrichum lupini isolate were studied by the method of SRAP-analysis with the purpose to assess the differential expression of genes. As a result, the PCR fragment corresponding to tolerant seedlings was found. The genetic determinants found are likely involved in the control of the resistance (tolerance) of lupine plants to anthracnose.

Genetic diversity of Colletotrichum lupini and its virulence on white and Andean lupin

Lupin cultivation worldwide is threatened by anthracnose, a destructive disease caused by the seed- and air-borne fungal pathogen Colletotrichum lupini. In this study we explored the intraspecific diversity of 39 C. lupini isolates collected from different lupin cultivating regions around the world, and representative isolates were screened for their pathogenicity and virulence on white and Andean lupin. Multi-locus phylogeny and morphological characterizations showed intraspecific diversity to be greater than previously shown, distinguishing a total of six genetic groups and ten distinct morphotypes. Highest diversity was found across South America, indicating it as the center of origin of C. lupini. The isolates that correspond to the current pandemic belong to a genetic and morphological uniform group, were globally widespread, and showed high virulence on tested white and Andean lupin accessions. Isolates belonging to the other five genetic groups were mostly found locally and showed distinct virulence patterns. Two highly virulent strains were shown to overcome resistance of advanced white lupin breeding material. This stresses the need to be careful with international seed transports in order to prevent spread of currently confined but potentially highly virulent strains. This study improves our understanding of the diversity, phylogeography and pathogenicity of a member of one of the world’s top 10 plant pathogen genera, providing valuable information for breeding programs and future disease management.

Deciphering the Infectious Process of Colletotrichum lupini in Lupin through Transcriptomic and Proteomic Analysis

The fungal phytopathogen Colletotrichum lupini is responsible for lupin anthracnose, resulting in significant yield losses worldwide. The molecular mechanisms underlying this infectious process are yet to be elucidated. This study proposes to evaluate C. lupini gene expression and protein synthesis during lupin infection, using, respectively, an RNAseq-based transcriptomic approach and a mass spectrometry-based proteomic approach. Patterns of differentially-expressed genes in planta were evaluated from 24 to 84 hours post-inoculation, and compared to in vitro cultures. A total of 897 differentially-expressed genes were identified from C. lupini during interaction with white lupin, of which 520 genes were predicted to have a putative function, including carbohydrate active enzyme, effector, protease or transporter-encoding genes, commonly described as pathogenicity factors for other Colletotrichum species during plant infection, and 377 hypothetical proteins. Simultaneously, a total of 304 proteins produced during the interaction were identified and quantified by mass spectrometry. Taken together, the results highlight that the dynamics of symptoms, gene expression and protein synthesis shared similarities to those of hemibiotrophic pathogens. In addition, a few genes with unknown or poorly-described functions were found to be specifically associated with the early or late stages of infection, suggesting that they may be of importance for pathogenicity. This study, conducted for the first time on a species belonging to the Colletotrichum acutatum species complex, presents an opportunity to deepen functional analyses of the genes involved in the pathogenicity of Colletotrichum spp. during the onset of plant infection.

Phylogenetic Diversity and Effect of Temperature on Pathogenicity of Colletotrichum lupini

Although lupin anthracnose caused by Colletotrichum lupini is a significant threat for spring and winter lupin crops, it has been poorly studied so far. This study aimed at characterizing the (i) phylogenetic, (ii) morphological, and (iii) physiological diversity of collected isolates from anthracnose-affected lupins. The genetic identification of representative isolates (n = 71) revealed that they were all C. lupini species, further confirming that lupin anthracnose is caused by this species. However, multilocus sequencing on these isolates and 16 additional reference strains of C. lupini revealed a separation into two distinct genetic groups, both of them characterized by a very low genetic diversity. The diversity of morphological characteristics of a selected subset of C. lupini isolates was further evaluated. To the best of our knowledge, microsclerotia production observed for some isolates has never been reported so far within the Colletotrichum acutatum species complex. Finally, the modeling of growth responses of a subset of C. lupini strains revealed the capacity of some strains to grow in vitro at 5°C. This ability was also evidenced in planta, because C. lupini DNA was detectable in plants from 14 days postinoculation at 5°C onward, whereas symptoms began to appear a week later, although at a very low level. Since lupin crops are planted during winter or early spring, growth studies in vitro and in planta demonstrated the capability of the species to grow at temperatures ranging from 5 to 30°C, with an optimum close to 25°C. In this study, C. lupini-specific primers were also designed for real-time quantitative PCR on fungal DNA and allowed the detection of C. lupini in asymptomatic field samples. These results open perspectives to detect earlier and limit the development of this pathogen in lupin crops.

Molecular Detection of the Seed-Borne Pathogen Colletotrichum lupini Targeting the Hyper-Variable IGS Region of the Ribosomal Cluster

Lupins anthracnose is a destructive seed and airborne disease caused by Colletotrichum lupini, affecting stems and pods. Primary seed infections as low as 0.01–0.1% can cause very severe yield losses. One of the most effective management strategies is the development of a robust and sensitive seed detection assay to screen seed lots before planting. PCR-based detection systems exhibit higher levels of sensitivity than conventional techniques, but when applied to seed tests they require the extraction of PCR-quality DNA from target organisms in backgrounds of saprophytic organisms and inhibitory seed-derived compounds. To overcome these limitations, a new detection protocol for C. lupini based on a biological enrichment step followed by a PCR assay was developed. Several enrichment protocols were compared with Yeast Malt Broth amended with ampicillin, streptomycin, and lactic acid were the most efficient. A species-specific C. lupini primer pair was developed based on rDNA IGS sequences. The specificity was evaluated against 17 strains of C. lupini, 23 different Colletotrichum species, and 21 different organisms isolated from seeds of Lupinus albus cv. Multitalia, L. luteus cv. Mister, and L. angustifolius cv. Tango. The protocol described here enabled the detection of C. lupini in samples artificially infected with less than 1/10,000 infected seed.