Anthracnose in white lupin : genetic diversity, virulence and host resistance

White lupin (Lupinus albus L.) is a grain legume that is known for its high protein content, nutritional quality, efficient nitrogen fixation and unique ability to form specialized cluster roots that support phosphorus uptake. Despite a severe production decline at the end of the past century, white lupin has seen a recent revival to sustain the demand for plant-based protein and reduce Europes dependency on imported soybean. A major problem in (white) lupin cultivation is anthracnose disease, caused by the globally dispersed, seed- and air-borne fungal pathogen Colletotrichum lupini. This PhD thesis aims to provide insights on how to sustainably control anthracnose disease in white lupin in order to support its re-introduction into European cultivation systems. It describes (i) the genetic diversity, phylogeography and virulence of C. lupini, (ii) the development of a high-throughput phenotyping protocol to reliably screen white lupin germplasm, (iii) a genome-wide association study identifying resistance candidate genes and (iv) the exploration of effective seed treatments to reduce the primary pathogen inoculum. Multi-locus phylogeny and morphological characterization of 39 C. lupini isolates showed that diversity is greater than previously reported, distinguishing a total of six genetic groups and ten distinct morphotypes. Highest diversity was found across the South American Andes, indicating it to be the center of origin of C. lupini. Results reveal that the current pandemic is caused by strains belonging to genetic group II, which are spread globally, and are genetically and morphologically uniform. Group II isolates were shown to be highly aggressive on tested white and Andean lupin accessions. Isolates belonging to the other five genetic groups were mostly found locally and showed distinct virulence patterns on both white and Andean lupin accessions. Despite its uniformity, it was shown that two highly virulent group II isolates from Chile could overcome resistance of elite white lupin breeding material, stressing the need to implement phytosanitary protocols for international seed transports. A reliable high-throughput phenotyping tool was developed to identify anthracnose resistance in white lupin germplasm and study pathogen-host interactions. Phenotyping under controlled conditions, performing stem wound inoculation on seedlings, showed to be applicable for high-throughput and its disease scores strongly correlated with two-year Swiss field disease assessments (r = 0.95) and yield (r = -0.64). Phenotyping a diverse set of 40 white lupin accessions revealed eight accessions with improved resistance to anthracnose, which can be incorporated into white lupin breeding programs. A genome-wide association study (GWAS) was done to dissect the genetic architecture for anthracnose resistance in white lupin. White lupin genotypes, collected from the center of domestication and traditional cultivation regions, were screened for anthracnose resistance and characterized through genotyping-by-sequencing (GBS). GWAS revealed two significant SNPs associated with anthracnose resistance on gene Lalb_Chr05_g0216161, encoding a RING zinc-finger E3 ubiquitin ligase potentially involved in plant immunity. Further validation experiments are now required to confirm involvement in plant resistance. Population analysis showed a remarkably fast linkage disequilibrium (LD) decay, weak population structure and grouping of commercial varieties with landraces, corresponding to the slow domestication history and scarce breeding efforts in white lupin. A total of eleven different seed treatments was tested in field trials in Switzerland between 2018 and 2021 to identify treatments that reduce C. lupini infection levels in white lupin. Treatments consisted of hot water, steam, electron, long term storage, vinegar, plant extracts and biological control agents (BCAs). The BCAs were tested under controlled conditions for potential antagonistic activity against C. lupini during white lupin infection prior to field trials. Results showed that long term storage and vinegar treatments can successfully reduce disease incidence and increase yield to levels similar to those observed for certified seeds. In order to sustainably and effectively control anthracnose disease in white lupin, an integrative approach, including modern breeding efforts, disease prevention strategies and mixed cropping systems, is recommended. Further research is required to increase our understanding on white lupin-C. lupini interaction and to identify genetic regions involved in resistance or virulence, respectively, which could greatly support white lupin breeding. This thesis provides the basis to further explore C. lupini population dynamics, virulence and host-speciation, white lupin resistance mechanisms and sustainable ways to control anthracnose disease in order to further facilitate successful white lupin cultivation.Die Weiße Lupine (Lupinus albus L.) ist eine Körnerleguminose mit hohem Proteingehalt, guter Nährstoffqualität, effizienter Stickstofffixierung und, aufgrund spezialisierter Proteoidwurzeln, erhöhter Phosphoraufnahme. Trotz eines starken Produktionsrückgangs Ende des 20. Jahrhunderts erlebt die Weiße Lupine jüngst einen Aufschwung aufgrund der Nachfrage nach pflanzlichem Eiweiß und Europas Abhängigkeit von Sojaimporten. Ein großes Problem im Anbau der (weißen) Lupine ist die Anthraknose-Krankheit, die durch den weltweit verbreiteten, samen- und luftbürtigen Pilzerreger Colletotrichum lupini verursacht wird. Ziel dieser Dissertation ist es, Erkenntnisse über Bekämpfungsstragien der Lupinenanthraknose zu gewinnen, um die Wiedereinführung dieser Kulturpflanze in europäische Anbausysteme zu unterstützen. Sie beschreibt (i) die genetische Vielfalt und Virulenz von C. lupini, (ii) die Entwicklung eines Hochdurchsatz-Phänotypisierungsprotokolls, (iii) eine genomweite Assoziationsstudie zur Identifizierung von Resistenzgen-Kandidaten und (iv) die Untersuchung wirksamer Saatgutbehandlungen zur Reduzierung des primären Pathogeninokulums. Charakterisierung von 39 C. lupini-Isolaten zeigen dass die Diversität größer ist als bisher berichtet, wobei insgesamt sechs genetische Gruppen und zehn Morphotypen unterschieden werden. Die größte Vielfalt wurde in den südamerikanischen Anden gefunden, was auf das Genzentrum von C. lupini hindeutet. Die Ergebnisse zeigen, dass die aktuelle Pandemie durch weltweit verbreitete und homogene Stämme der genetischen Gruppe II verursacht wird. Isolate der Gruppe II erwiesen sich als äußerst aggressiv gegenüber der Weißen und Anden-Lupine. Isolate der anderen fünf genetischen Gruppen wurden meist lokal gefunden und zeigten sowohl bei der Weißen als auch bei der Anden-Lupine unterschiedliche Virulenzmuster. Trotz ihrer Einheitlichkeit konnte gezeigt werden, dass zwei hochvirulente Isolate der Gruppe II aus Chile die Resistenz von fortgeschrittenem Zuchtmaterial der Weißen Lupine überwinden konnten. Dies unterstreicht die Notwendigkeit der Umsetzung von Pflanzenschutzprotokollen für internationale Saatguttransporte. Es wurde ein zuverlässiges Phänotypisierungsprotokoll mit hohem Durchsatz entwickelt, um Anthraknoseresistenz in genetischen Ressourcen der Weißen Lupine zu identifizieren. Die Phänotypisierung unter kontrollierten Bedingungen und mittels Wundinokulation des Stängels erwies sich als geeignet für den Hochdurchsatz, und die Krankheitsindizes korrelierten stark mit den Krankheitsbonituren (r = 0,95) und dem Ertrag (r = -0,64) eines zweijährigen Parzellenversuchs im Feld. Die Phänotypisierung von 40 Weiße Lupine-Akzessionen ergab acht Akzessionen mit verbesserter Anthraknose-Resistenz. Eine genomweite Assoziationsstudie (GWAS) wurde durchgeführt, um die genetische Architektur der Anthraknoseresistenz bei weißer Lupine zu entschlüsseln. Weiße Lupine Genotypen aus dem Domestikationszentrum und traditionellen Anbauregionen wurden mittels zuvor beschriebenem Phänotypisierungprotokoll auf Anthraknoseresistenz untersucht und mittels genotyping-by-sequencing (GBS) charakterisiert. GWAS ergab zwei signifikante SNPs, die mit Anthraknoseresistenz auf dem Gen Lalb_Chr05_g0216161 assoziiert sind. Dieses Gen kodiert für eine RING-Zink-Finger-E3-Ubiquitin-Ligase kodiert, die möglicherweise an der Pflanzenimmunität beteiligt ist. Die Populationsanalyse zeigte einen bemerkenswert schnellen Zerfall des Kopplungsungleichgewichts (LD), eine schwache Populationsstruktur und eine Gruppierung von kommerziellen Sorten mit Landsorten, was der langsamen Domestikations-geschichte und den geringen Züchtungsbemühungen bei der Weißen Lupine entspricht. Insgesamt elf verschiedene Saatgutbehandlungen wurden zwischen 2018 und 2021 in Schweizer Feldversuchen getestet. Die Behandlungen umfassten Heißwasser, Dampf, Elektronenbestrahlung, Langzeitlagerung, Essig, Pflanzenextrakte und biologische Bekämpfungsmittel (BCAs). Die Feldergebnisse zeigten, dass eine Langzeitlagerung und eine Behandlung mit Essig das Auftreten von Krankheiten erfolgreich reduzieren und den Ertrag auf ein ähnliches Niveau wie bei zertifiziertem Saatgut steigern kann. Zur nachhaltigen und wirksamen Bekämpfung der Anthraknose-Krankheit wird ein integrativer Ansatz empfohlen, der moderne Züchtungs-maßnahmen, Strategien zur Krankheitsvorbeugung und Mischkultur umfasst. Diese Arbeit bildet die Grundlage für die weitere Erforschung der Populationsdynamik von C. lupini, der Virulenz und der Wirtsspeziation, der Resistenzmechanismen der Weißen Lupine und nachhaltiger Möglichkeiten zur Bekämpfung der Anthraknosekrankheit, um den erfolgreichen Anbau der Weißen Lupine weiter zu erleichtern

Attack of the clones: Population genetics reveals clonality of Colletotrichum lupini, the causal agent of lupin anthracnose

Colletotrichum lupini, the causative agent of lupin anthracnose, affects lupin cultivation worldwide. Understanding its population structure and evolutionary potential is crucial to design successful disease management strategies. The objective of this study was to employ population genetics to investigate the diversity, evolutionary dynamics, and molecular basis of the interaction of this notorious lupin pathogen with its host. A collection of globally representative C. lupini isolates was genotyped through triple digest restriction site-associated DNA sequencing, resulting in a data set of unparalleled resolution. Phylogenetic and structural analysis could distinguish four independent lineages (I–IV). The strong population structure and high overall standardized index of association (r̅d) indicates that C. lupini reproduces clonally. Different morphologies and virulence patterns on white lupin (Lupinus albus) and Andean lupin (Lupinus mutabilis) were observed between and within clonal lineages. Isolates belonging to lineage II were shown to have a minichromosome that was also partly present in lineage III and IV, but not in lineage I isolates. Variation in the presence of this minichromosome could imply a role in host–pathogen interaction. All four lineages were present in the South American Andes region, which is suggested to be the centre of origin of this species. Only members of lineage II have been found outside South America since the 1990s, indicating it as the current pandemic population. As a seedborne pathogen, C. lupini has mainly spread through infected but symptomless seeds, stressing the importance of phytosanitary measures to prevent future outbreaks of strains that are yet confined to South America

Genome‑wide association study reveals white lupin candidate gene involved in anthracnose resistance

White lupin (Lupinus albus L.) is a re-emerging protein crop and promising alternative to soybean. Its cultivation, however, is severely threatened by anthracnose disease caused by the fungal pathogen Colletotrichum lupini. To dissect the genetic architecture for anthracnose resistance, genotyping-by-sequencing (GBS) was performed on white lupin accessions collected from the center of domestication and traditional cultivation regions. GBS resulted in 4,611 high-quality single-nucleotide polymorphisms (SNPs) for 181 accessions, which were combined with resistance data observed under controlled conditions to perform a genome-wide association study (GWAS). Obtained disease phenotypes were shown to highly correlate to overall three-year disease assessments under Swiss field conditions (r > 0.8). GWAS results identified two significant SNPs associated with anthracnose resistance on gene Lalb_Chr05_g0216161 encoding a RING zinc-finger E3 ubiquitin ligase which is potentially involved in plant immunity. Population analysis showed a remarkably fast linkage disequilibrium (LD) decay, weak population structure and grouping of commercial varieties with landraces, corresponding to the slow domestication history and scarcity of modern breeding efforts in white lupin. Together with 15 highly resistant accessions identified in the resistance assay, our findings show promise for further crop improvement. This study provides the basis for marker-assisted selection, genomic prediction and studies aimed at understanding anthracnose resistance mechanisms in white lupin and contributes to improving breeding programs worldwide

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

The potential of alternative seed treatments to control anthracnose disease in white lupin

White lupin (Lupinus albus L.) is a promising crop to meet the rising global demand for plant-based protein. The seed-borne pathogen Colletotrichum lupini, however, threatens lupin cultivation worldwide. Seed dressings using synthetic fungicides were shown effective to reduce infection levels, but their negative environmental impact and exclusion from organic production calls for more sustainable solutions. In this study, a total of eleven different alternative seed treatments were tested in field trials in Switzerland between 2018 and 2021. Treatment types consisted of: hot water, steam, electron, long-term storage, elevated partial pressure of oxygen (EPPO), vinegar, plant extracts and biological control agents (BCAs). The BCAs were tested for potential antagonistic activity against C. lupini during white lupin infection under controlled conditions prior to field trials. Long-term storage and vinegar treatments successfully reduced disease incidence and increased yield to levels similar to those observed for certified seeds, without significantly affecting germination rate. Although promising, effectiveness of these treatments needs further validation. Four BCAs showed significant disease reductions under controlled conditions. Besides lowering disease severity, two BCAs also reduced C. lupini DNA in stem tissue. These reductions, however, were not observed in the field, highlighting the importance of field validations. The treatments identified in this study provide a solid basis for the development of sustainable and effective seed protection strategies in white lupin to control C. lupini successfully.ISSN:0261-2194ISSN:1873-690