Hybridizations Between formae speciales of Venturia inaequalis Pave the Way for a New Biocontrol Strategy to Manage Fungal Plant Pathogens.

Hybridization and adaptation to new hosts are important mechanisms of fungal disease emergence. Evaluating the risk of emergence of hybrids with enhanced virulence is then key to develop sustainable crop disease management. We evaluated this risk in Venturia inaequalis, the fungus responsible for the common and serious scab disease on Rosaceae hosts, including apple, pyracantha, and loquat. Field isolates from these three hosts and progenies obtained from five crosses between formae speciales isolates collected from pyracantha (f. sp. pyracantha) and apple (f. sp. pomi) were tested for their pathogenicity on the three hosts. We confirmed a strict host specificity between isolates from apple and pyracantha and showed that most isolates were able to cause disease on loquat. None of the 251 progeny obtained from five crosses between V. inaequalis f. sp. pyracantha and V. inaequalis f. sp. pomi could infect apple. If confirmed on more crosses, the inability of the hybrids to infect apple could lead to a novel biocontrol strategy based on a sexual hijacking of V. inaequalis f. sp. pomi by a massive introduction of V. inaequalis f. sp. pyracantha in apple orchards. This strategy, analogous to the sterile insect approach, could lead to the collapse of the population size of V. inaequalis and dramatically reduce the use of chemicals in orchards.

Threat to Asian wild apple trees posed by gene flow from domesticated apple trees and their "pestified" pathogens.

Secondary contact between crops and their wild relatives poses a threat to wild species, not only through gene flow between plants, but also through the dispersal of crop pathogens and genetic exchanges involving these pathogens, particularly those that have become more virulent by indirect selection on resistant crops, a phenomenon known as "pestification." Joint analyses of wild and domesticated hosts and their pathogens are essential to address this issue, but such analyses remain rare. We used population genetics approaches, demographic inference and pathogenicity tests on host-pathogen pairs of wild or domesticated apple trees from Central Asia and their main fungal pathogen, Venturia inaequalis, which itself has differentiated agricultural and wild-type populations. We confirmed the occurrence of gene flow from cultivated (Malus domestica) to wild (Malus sieversii) apple trees in Asian forests, potentially threatening the persistence of Asian wild apple trees. Pathogenicity tests demonstrated the pestification of V. inaequalis, the agricultural-type population being more virulent on both wild and domesticated trees. Single nucleotide polymorphism (SNP) markers and the demographic modelling of pathogen populations revealed hybridization following secondary contact between agricultural and wild-type fungal populations, and dispersal of the agricultural-type pathogen population in wild forests, increasing the threat of disease in the wild apple species. We detected an SNP potentially involved in pathogen pestification, generating an early stop codon in a gene encoding a small secreted protein in the agricultural-type fungal population. Our findings, based on joint analyses of paired host and pathogen data sets, highlight the threat posed by cultivating a crop near its centre of origin, in terms of pestified pathogen invasions in wild plant populations and introgression in the wild-type pathogen population.

Pyramiding Quantitative Resistance with a Major Resistance Gene in Apple: From Ephemeral to Enduring Effectiveness in Controlling Scab.

Genetic resistance is a useful strategy to control plant disease, but its effectiveness may be reduced over time due to the emergence of pathogens able to circumvent the defenses of the plant. However, the pyramiding of different resistance factors in the same plant can improve the effectiveness and durability of the resistance. To investigate the potential for this approach in apple to control scab disease we surveyed scab incidence in two experimental orchards located at a distance of more than 300 km planted with apple genotypes carrying quantitative resistance and major gene resistance alone or in combination. Our results showed that the effectiveness of pyramiding in controlling scab was dependent on the site and could not be completely explained by the effectiveness level of the resistances alone.

When virulence originates from nonagricultural hosts: evolutionary and epidemiological consequences of introgressions following secondary contacts in Venturia inaequalis.

In pathogens, introgressions through secondary contacts between divergent populations from agricultural and nonagricultural disease reservoirs are expected to have crucial evolutionary and epidemiological implications. Despite the importance of this question for disease management, experimental demonstrations of these implications remain scarce. Recently, we identified a virulent population of the apple scab pathogen Venturia inaequalis that migrated from nonagricultural hosts to European domestic apple orchards. Here, we investigated the occurrence of gene flow between agricultural and nonagricultural populations sampled in two orchards, and thereafter its consequences on the pathogenicity of hybrids. Population genetic structure and demographic inferences based on the genotypes of 104 strains revealed a high amount of gene flow between the two populations in one orchard. In this site, mating between populations was made possible by the presence of a common host. Our results revealed an invasion of the virulent trait in the agricultural population; a main direction of introgression in hybrids from the agricultural to nonagricultural genetic backgrounds; and a population of hybrids with transgressive traits. We demonstrate a secondary contact with gene flow between divergent populations of pathogens. Our findings highlight evolutionary and epidemiological changes in pathogens and have concrete implications for sustainable disease management.

Virulence Characterization of Venturia inaequalis Reference Isolates on the Differential Set of Malus Hosts.

A set of differential hosts has recently been identified for 17 apple scab resistance genes in an updated system for defining gene-for-gene (GfG) relationships in the Venturia inaequalis-Malus pathosystem. However, a set of reference isolates characterized for their complementary avirulence alleles is not yet available. In this paper, we report on improving the set of differential hosts for h(7) and propose the apple genotype LPG3-29 as carrying the single major resistance gene Rvi7. We characterized a reference set of 23 V. inaequalis isolates on 14 differential apple hosts carrying major resistance genes under controlled conditions. We identified isolates that were virulent on at least one of the following defined resistance gene hosts: h(1), h(2), h(3), h(4), h(5), h(6), h(7), h(8), h(9), h(10), and h(13). Sixteen different virulence patterns were observed. In general, the isolates carried one to three virulences, but some of them were more complex, with up to six virulences. This set of well-characterized isolates will be helpful for the identification of additional apple scab resistance genes in apple germplasm and the characterization of new GfG relationships to help improve our understanding of the host-pathogen interactions in the V. inaequalis-Malus pathosystem.

Differential selection pressures exerted by host resistance quantitative trait loci on a pathogen population: a case study in an apple × Venturia inaequalis pathosystem.

Understanding how pathogens evolve according to pressures exerted by their plant hosts is essential for the derivation of strategies aimed at the durable management of resistant cultivars. The spectrum of action of the resistance factors in the partially resistant cultivars is thought to be an important determinant of resistance durability. However, it has not yet been demonstrated whether the pressures exerted by quantitative resistance are different according to their spectrum of action. To investigate selection pressures exerted by apple genotypes harbouring various resistance quantitative trait loci (QTLs) on a mixed inoculum of the scab disease agent, Venturia inaequalis, we monitored V. inaequalis isolate proportions on diseased apple leaves of an F1 progeny using quantitative pyrosequencing technology and QTL mapping. Broad-spectrum resistances did not exert any differential selection pressures on the mixed inoculum, whereas narrow-spectrum resistances decreased the frequencies of some isolates in the mixture relative to the susceptible host genotypes. Our results suggest that the management of resistant cultivars should be different according to the spectrum of action of their resistance factors. The pyramiding of broad-spectrum factors or the use of a mixture of apple genotypes that carry narrow-spectrum resistance factors are two possible strategies for the minimization of resistance erosion.

Emergence of novel fungal pathogens by ecological speciation: importance of the reduced viability of immigrants.

Expanding global trade and the domestication of ecosystems have greatly accelerated the rate of emerging infectious fungal diseases, and host-shift speciation appears to be a major route for disease emergence. There is therefore an increased interest in identifying the factors that drive the evolution of reproductive isolation between populations adapting to different hosts. Here, we used genetic markers and cross-inoculations to assess the level of gene flow and investigate barriers responsible for reproductive isolation between two sympatric populations of Venturia inaequalis, the fungal pathogen causing apple scab disease, one of the fungal populations causing a recent emerging disease on resistant varieties. Our results showed the maintenance over several years of strong and stable differentiation between the two populations in the same orchards, suggesting ongoing ecological divergence following a host shift. We identified strong selection against immigrants (i.e. host specificity) from different host varieties as the strongest and likely most efficient barrier to gene flow between local and emerging populations. Cross-variety disease transmission events were indeed rare in the field and cross-inoculation tests confirmed high host specificity. Because the fungus mates within its host after successful infection and because pathogenicity-related loci prevent infection of nonhost trees, adaptation to specific hosts may alone maintain both genetic differentiation between and adaptive allelic combinations within sympatric populations parasitizing different apple varieties, thus acting as a 'magic trait'. Additional intrinsic and extrinsic postzygotic barriers might complete reproductive isolation and explain why the rare migrants and F1 hybrids detected do not lead to pervasive gene flow across years.

Identification and pathogenicity of Alternaria species associated with leaf blotch disease and premature defoliation in French apple orchards.

Leaf blotch caused by Alternaria spp. is a common disease in apple-producing regions. The disease is usually associated with one phylogenetic species and one species complex, Alternaria alternata and the Alternaria arborescens species complex (A. arborescens SC), respectively. Both taxa may include the Alternaria apple pathotype, a quarantine or regulated pathogen in several countries. The apple pathotype is characterized by the production of a host-selective toxin (HST) which is involved in pathogenicity towards the apple. A cluster of genes located on conditionally dispensable chromosomes (CDCs) is involved in the production of this HST (namely AMT in the case of the apple pathotype). Since 2016, leaf blotch and premature tree defoliation attributed to Alternaria spp. have been observed in apple-producing regions of central and south-eastern France. Our study aimed to identify the Alternaria species involved in apple tree defoliation and assess the presence of the apple pathotype in French orchards. From 2016 to 2018, 166 isolates were collected and identified by multi-locus sequence typing (MLST). This analysis revealed that all these French isolates belonged to either the A. arborescens SC or A. alternata. Specific PCR detection targeting three genes located on the CDC did not indicate the presence of the apple pathotype in France. Pathogenicity was assessed under laboratory conditions on detached leaves of Golden Delicious and Gala apple cultivars for a representative subset of 28 Alternaria isolates. All the tested isolates were pathogenic on detached leaves of cultivars Golden Delicious and Gala, but no differences were observed between the pathogenicity levels of A. arborescens SC and A. alternata. However, the results of our pathogenicity test suggest that cultivar Golden Delicious is more susceptible than Gala to Alternaria leaf blotch. Implications in the detection of the Alternaria apple pathotype and the taxonomic assignment of Alternaria isolates involved in Alternaria leaf blotch are discussed.

Evolution of the population structure of Venturia inaequalis, the apple scab fungus, associated with the domestication of its host.

Evaluating the impact of plant domestication on the population structure of the associated pathogens provides an opportunity to increase our understanding of how and why diseases emerge. Here, we investigated the evolution of the population structure of the apple scab fungus Venturia inaequalis in response to the domestication of its host. Inferences were drawn from multilocus microsatellite data obtained from samples collected on (i) the Central Asian Malus sieversii, the main progenitor of apple, (ii) the European crabapple, Malus sylvestris, a secondary progenitor of apple, and (iii) the cultivated apple, Malus x domestica, in orchards from Europe and Central Asia. Using clustering methods, we identified three distinct populations: (i) a large European population on domesticated and wild apples, (ii) a large Central Asian population on domesticated and wild apples in urban and agricultural areas, and (iii) a more geographically restricted population in M. sieversii forests growing in the eastern mountains of Kazakhstan. Unique allele richness and divergence time estimates supported a host-tracking co-evolutionary scenario in which this latter population represents a relict of the ancestral populations from which current populations found in human-managed habitats were derived. Our analyses indicated that the domestication of apple induced a significant change in the genetic differentiation of populations of V. inaequalis in its centre of origin, but had little impact on its population dynamics and mating system. We discuss how the structure of the apple-based agrosystem may have restricted changes in the population structure of the fungus in response to the domestication of its host.

Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturia pirina, Venturia aucupariae and Venturia asperata.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.

Quantitative Resistance to Plant Pathogens in Pyramiding Strategies for Durable Crop Protection.

Quantitative resistance has gained interest in plant breeding for pathogen control in low-input cropping systems. Although quantitative resistance frequently has only a partial effect and is difficult to select, it is considered more durable than major resistance (R) genes. With the exponential development of molecular markers over the past 20 years, resistance QTL have been more accurately detected and better integrated into breeding strategies for resistant varieties with increased potential for durability. This review summarizes current knowledge on the genetic inheritance, molecular basis, and durability of quantitative resistance. Based on this knowledge, we discuss how strategies that combine major R genes and QTL in crops can maintain the effectiveness of plant resistance to pathogens. Combining resistance QTL with complementary modes of action appears to be an interesting strategy for breeding effective and potentially durable resistance. Combining quantitative resistance with major R genes has proven to be a valuable approach for extending the effectiveness of major genes. In the plant genomics era, improved tools and methods are becoming available to better integrate quantitative resistance into breeding strategies. Nevertheless, optimal combinations of resistance loci will still have to be identified to preserve resistance effectiveness over time for durable crop protection.

Slow erosion of a quantitative apple resistance to Venturia inaequalis based on an isolate-specific Quantitative Trait Locus.

Quantitative plant resistance affects the aggressiveness of pathogens and is usually considered more durable than qualitative resistance. However, the efficiency of a quantitative resistance based on an isolate-specific Quantitative Trait Locus (QTL) is expected to decrease over time due to the selection of isolates with a high level of aggressiveness on resistant plants. To test this hypothesis, we surveyed scab incidence over an eight-year period in an orchard planted with susceptible and quantitatively resistant apple genotypes. We sampled 79 Venturia inaequalis isolates from this orchard at three dates and we tested their level of aggressiveness under controlled conditions. Isolates sampled on resistant genotypes triggered higher lesion density and exhibited a higher sporulation rate on apple carrying the resistance allele of the QTL T1 compared to isolates sampled on susceptible genotypes. Due to this ability to select aggressive isolates, we expected the QTL T1 to be non-durable. However, our results showed that the quantitative resistance based on the QTL T1 remained efficient in orchard over an eight-year period, with only a slow decrease in efficiency and no detectable increase of the aggressiveness of fungal isolates over time. We conclude that knowledge on the specificity of a QTL is not sufficient to evaluate its durability. Deciphering molecular mechanisms associated with resistance QTLs, genetic determinants of aggressiveness and putative trade-offs within pathogen populations is needed to help in understanding the erosion processes.

Erosion of quantitative host resistance in the apple×Venturia inaequalis pathosystem.

Theoretical approaches predict that host quantitative resistance selects for pathogens with a high level of pathogenicity, leading to erosion of the resistance. This process of erosion has, however, rarely been experimentally demonstrated. To investigate the erosion of apple quantitative resistance to scab disease, we surveyed scab incidence over time in a network of three orchards planted with susceptible and quantitatively resistant apple genotypes. We sampled Venturiainaequalis isolates from two of these orchards at the beginning of the experiment and we tested their quantitative components of pathogenicity (i.e., global disease severity, lesion density, lesion size, latent period) under controlled conditions. The disease severity produced by the isolates on the quantitatively resistant apple genotypes differed between the sites. Our study showed that quantitative resistance may be subject to erosion and even complete breakdown, depending on the site. We observed this evolution over time for apple genotypes that combine two broad-spectrum scab resistance QTLs, F11 and F17, showing a significant synergic effect of this combination in favour of resistance (i.e., favourable epistatic effect). We showed that isolates sampled in the orchard where the resistance was inefficient presented a similar level of pathogenicity on both apple genotypes with quantitative resistance and susceptible genotypes. As a consequence, our results revealed a case where the use of quantitative resistance may result in the emergence of a generalist pathogen population that has extended its pathogenicity range by performing similarly on susceptible and resistant genotypes. This emphasizes the need to develop quantitative resistances conducive to trade-offs within the pathogen populations concerned.

Evolution of pathogenicity traits in the apple scab fungal pathogen in response to the domestication of its host.

Understanding how pathogens emerge is essential to bring disease-causing agents under durable human control. Here, we used cross-pathogenicity tests to investigate the changes in life-history traits of the fungal pathogen Venturia inaequalis associated with host-tracking during the domestication of apple and subsequent host-range expansion on the wild European crabapple (Malus sylvestris). Pathogenicity of 40 isolates collected in wild and domesticated ecosystems was assessed on the domesticated apple, its Central Asian main progenitor (M. sieversii) and M. sylvestris. Isolates from wild habitats in the centre of origin of the crop were not pathogenic on the domesticated apple and less aggressive than other isolates on their host of origin. Isolates from the agro-ecosystem in Central Asia infected a higher proportion of plants with higher aggressiveness, on both the domesticated host and its progenitor. Isolates from the European crabapple were still able to cause disease on other species but were less aggressive and less frequently virulent on these hosts than their endemic populations. Our results suggest that the domestication of apple was associated with the acquisition of virulence in the pathogen following host-tracking. The spread of the disease in the agro-ecosystem would also have been accompanied by an increase in overall pathogenicity.

Revision of the nomenclature of the differential host-pathogen interactions of Venturia inaequalis and Malus.

The apple scab (Venturia inaequalis-Malus) pathosystem was one of the first systems for which Flor's concept of gene-for-gene (GfG) relationships between the host plant and the pathogen was demonstrated. There is a rich resource of host resistance genes present in Malus germplasm that could potentially be marshalled to confer durable resistance against this most important apple disease. A comprehensive understanding of the host-pathogen interactions occurring in this pathosystem is a prerequisite for effectively manipulating these host resistance factors. An accurate means of identification of specific resistance and consistent use of gene nomenclature is critical for this process. A set of universally available, differentially resistant hosts is described, which will be followed by a set of defined pathogen races at a later stage. We review pertinent aspects of the history of apple scab research, describe the current status and future directions of this research, and resolve some outstanding issues.