Aspects of sexual reproduction in Mycosphaerella species on wheat and barley : genetic studies on specificity, mapping, and fungicide resistance

Mycosphaerella species are haploid ascomycetes that cause major economic losses in crops that include cereals, citrus fruits, and bananas, among others. Two organisms in this genus are Mycosphaerella graminicola (Fuckel) .I. Schröt (anamorph Sepioria tritici) and Septoriapasserinii. M graminicola is the causal agent of septoria tritici blotch of both bread wheat and durum wheat species, and S. passerinii causes septoria speck!ed 1eaf blotch of barley. M. graminicola is a heterothaliic fungus with a very active sexual cycle, while no sexual cycle has been reported for S. passerinii.This thesis inciudes studies on mating and genetics of both M. graminicola and S. passerinii. Chapter 1 gives an introduction to these pathogens and an overview of the research topics. In Chapter 2. we studied the possibility of in planta generation of sexual progeny of the fungal wheat pathogen M graminicola when one of the parents was avirulent on a resistant host. We found that avirulent isolates are able to survive and even increase in biomass after inoculation onto resistant wheat cultivars and can complete sexual cycles on resistant cultivars to yield viable ascospores as long as the other parent is virulent. To our knowledge, this is the first time such a phenomenon has been described, and the possibility to generate such crosses opened the door for studies in Chapters 3 and 4.Chapter 3 describes the construction of two high-density genetic linkage maps of M graminicola using Diversity Arrays Technology (DArT) and the integration of these into a core map with common markers due to a common parental isolate. One of the maps was constructed based on segregations of progeny of two bread wheat-derived isolates, IPO323 and IPO94269, and the other was constructed from segregations of progeny of IPO323 and the durum wheat-derived isolate IPO95052. In total, 1,144 markers made up the integrated core map. Analyses from this study revealed that progeny had translocations, diploid and partial diploid linkage groups, and loss of entire linkage groups.Although M. graminicola causes disease on both bread wheat and durum wheat, isolates within the population show clear distinctions in either virulence on bread wheat or on durum wheat (host specificity)- In Chapter 4, we studied the genetic basis of host specificity in M. graminicola using 163 progeny from crosses between the Dutch bread wheat-derived 1PO323 and the Algerian durum wheat-derived TPO95052. Phenotyping of progeny was performed on a set of seven differential cultivars, and progeny crossed on either bread wheat or durum wheat could infect cultivars of bread wheat, durum wheat, both, or neither. These results were used to map nine quantitative trait loci (QTLs) on seven linkage groups in the high-densiry genetic linkage map from Chapter 3. One of these loci was previously mapped for cultivar specificity of IPO323in bread wheat, and the same locus was now mapped for host specificity of IPO323 to durum wheat. Our results show that the reported host specificity is probably the result of combinations of a number of independently inherited avirulence factors.In addition to avirulence genes, fungi can inherit other traits for survival. One such heritable trait is a point mutation in the mitochondrial genome that conveys resistance to strobilurin fungicides. Chapter 5 describes a study on the inheritance of strobilurin resistance. Resistant and sensitive isolates of M. graminicola were crossed on wheat seedlings that were both untreated and preventively treated with various concentrations of azoxystrobin (Amistar™), and progeny were analyzed to determine the rate of inheritance of the aforementioned mutation. Preventive rates from 3.125-200% Amistar™ resulted in completely resistant progeny populations despite the fact that the segregation of nuclear genes confirmed regular meiotic behavior. We conclude that sensitive isolates overcome the disruption of mitochondrial respiration and participate in sexual reproduction even under high fungicide pressure and that fungicide stress induces or results in preferential mating in M. graminicola.The barley pathogen S. passerinii clusters closely to M. graminicola in phylogenetic studies based on ITS sequences, and a high degree of genetic variation among isolates is found in nature. However, no teleomorph has been reported for this S. passerinii, and hence, it was considered to be asexual. Nevertheless, mating type idiomorphs were recently detected and isolated. In Chapter 6, we studied the possibility of a Mycosphaerella teleomorph associated with S. passerinii. Isolates with opposite mating types were co-inoculated onto barley cultivars, and leaves were monitored for the discharge of ascospores. Characterization of a segregating population by both molecular and phenotypic analyses confirmed that we successfully generated the hitherto unknown Mycosphaerella teleomorph of S. passerinii.Finally, the results of this thesis are discussed in a broader perspective in Chapter 7 in relation to epidemiology, co-evolution, and durability of resistance in the wheat-M. graminicola pathosystem. The proven ability of avirulent isolates of M. graminicola to generate sexual progeny on resistant cultivars represents a new dynamic in population genetics that has not'previously been considered in epidemiology. Resu!ts from this thesis emphasize the complex ways in which the sexual cycle contributes to the overall success of M. graminicola on wheat.