Reproductive systems are central to the evolutionary and ecological processes shaping population structure and adaptability. Rust fungi are a large group of obligate plant pathogens with complex life cycles and diverse reproductive modes. In the rust fungus Cronartium pini , which exists in both macrocyclic heteroecious and microcyclic autoecious forms, transitions between reproductive modes can be studied within a single species. Here, we used comparative genomics to analyse the structure, diversity, and organization of mating-type (MAT) loci across both forms of C. pini . We identified a canonical tetrapolar system in the heteroecious form, characterized by unlinked, multiallelic homeodomain (HD) and pheromone/receptor (P/R) loci, consistent with obligate outcrossing. In contrast, the autoecious form displayed distinct reproductive signatures, including MAT gene homozygosity in some samples—indicative of clonal reproduction—and MAT gene duplications in others, suggesting self-fertility or altered mating pathways. Three HD loci were detected, but only one exhibited high allelic diversity and was consistently present, indicating functional divergence. Expanded allelic diversity at the STE3.2 pheromone receptor further suggests plasticity at the P/R locus. Although allele sharing between life cycle forms was rare, isolated cases of shared MAT alleles suggest limited historical connectivity and possible trans-specific polymorphism. Together, our findings reveal flexibility in the mating system architecture of C. pini and suggest that transitions between sexual and asexual reproduction may be facilitated by retained, functionally ambiguous MAT structures. This work highlights C. pini as a powerful model for studying life cycle evolution and mating system transitions in rust fungi. ### Competing Interest Statement The authors have declared no competing interest. Carl Trygger Foundation, CTS 22:2086

BACKGROUNDThe hemibiotrophic fungus Zymoseptoria tritici causing Septoria tritici blotch (STB), is a devastating foliar pathogen of wheat worldwide. A common group of fungicides used to control STB are the demethylation inhibitors (DMIs). DMI fungicides restrict fungal growth by inhibiting the sterol 14-alpha-demethylase, a protein encoded by CYP51 gene and essential for maintaining fungal cell permeability. However, the adaptation of Z. tritici populations in response to intensive and prolonged DMI usage has resulted in a gradual shift towards reduced sensitivity to this group of fungicides. In this study, 311 isolates were collected pre-treatment from nine wheat-growing regions in Europe in 2019. These isolates were analysed by high-throughput amplicon-based sequencing of nine housekeeping genes and the CYP51 gene.RESULTSAnalyses based on housekeeping genes and the CYP51 gene revealed a lack of population structure in Z. tritici samples irrespective of geographical origin. Minimum spanning network (MSN) analysis showed clustering of multilocus genotypes (MLGs) based on CYP51 haplotypes, indicating an effect of selection due to DMI fungicide use. The majority of the haplotypes identified in this study have been reported previously. The diversity and frequencies of mutations varied across regions.CONCLUSIONUsing a high-throughput amplicon-sequencing approach, we found several mutations in the CYP51 gene combined in different haplotypes that are likely to cause fungicide resistance. These mutations occurred irrespective of genetic background or geographical origin. Overall, these results contribute to the development of effective and sustainable risk monitoring for DMI fungicide resistance. (c) 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Scots pine blister rust is a rust fungal pathogen that has become more noticeable in recent years and has increased in recorded incidence in Northern Scandinavia. This has prompted an initiation of resistance breeding to the fungus in the Northern Swedish pine breeding program. To accomplish this, it is necessary to evaluate the breeding potential for increased resistance and putative impact on other breeding objective traits that may have genetic correlations to resistance. To assess the potential of the trait to be included in the breeding program we examined half sibling families in four trials of which two had high incidence of Scots pine blister rust in a range of 25.4-42.1 %. We assessed vitality and height in one year and rust lesion frequency at two later time points. We found that rust resistance had a narrow sense heritability of 0.36-0.41, while vitality reached 0.17 and height 0.25. We found a high genetic correlation between sites in rust resistance and no genetic correlation between rust resistance and either vitality or height. This means that breeding for increased resistance in Scots pine can be done effectively without risking a negative impact on established breeding objective traits and that resistance is stable across environments.

Cronartium pini causes Scots pine blister rust. This rust fungus has two different forms without differentiation in morphology and internal transcribed spacer: the heteroecious form has a macrocyclic life cycle and infects pine and an alternate host; the autoecious form only infects pine. Epidemics caused by these two forms impose severe risk on the pine forest in Sweden, therefore knowledge of their distribution and diversity is needed for strategic disease management. We designed microsatellite markers with improved resolution based on the C. pini genome, developed a multiplex amplification system, and analyzed the C. pini population diversity and structure in Sweden using 396 isolates. The heteroecious and autoecious populations showed clear differences in diversity, linkage disequilibrium, and structure. The heteroecious isolates had unique multilocus genotypes. Autoecious isolates shared the same genotypes more frequently, especially three autoecious multilocus genotypes that were commonly found over a in northern Sweden. The genetic distances among autoecious isolates are closer than those among the heteroecious isolates. The results confirmed that heteroecious C. pini populations were sexual and autoecious C. pini populations were clonal. We further discussed the hypothesis that autoecious C. pini originated from self-fertilization, and frequent self-fertilization and infrequent mutation generate homozygous but diverse genotypes.

Cherry spruce rust causes huge yield losses in Norway spruce seed production in Fennoscandia. The causal agent, Thekopsora areolata , has three types of spores that disperse during spring: basidiospores are produced on basidia that grow out from teliospores in overwintered bird cherry leaf litter to infect new pistillate spruce cones, aeciospores are released from old diseased spruce cones to infect bird cherry leaves, and urediniospores are produced from new bird cherry leaves for reinfection. No study has examined the dispersal of T . areolata spores, including the basidiospores that cause primary infection in spruce cones. In this study, teliospores of T . areolata were germinated in the laboratory and the morphology of basidiospores was described. T . areolata spores were sampled in Ultuna, Sweden and Joutsa, Finland with 21 spore traps at each site. Peaks in aeciospores were observed from 11 to 25 May and from 2 to 8 June at the Finnish site, and from 4 to 18 May at the Swedish site. Urediniospores were first observed 2–3 weeks after the peaks in aeciospores and they were mainly distributed within 10 m from the bird cherry trees. Peaks of 1–2 weeks in basidiospore detection coincided with multiple rain events. The basidiospore peak overlapped with the spruce pollen peak in Finland but not in Sweden. The quantities of basidiospores from different spore traps within 100 m from the spore source had no gradient. Information on spatial and temporal spore release is important for making decisions on disease management strategies.

Thekopsora areolata infects pistillate cones of Picea spp. with monokaryotic basidiospores in the spring. Receptive monokaryotic hyphae in the cones are fertilized by monokaryotic spermatia in the summer, and dikaryotic aecia are produced in cones in late summer. Infected cones produce no fertile seeds, meaning the disease causes large reductions in seed production. To understand the seasonal variation of T. areolata genotypic diversity, 548 aecia from 55 infected cones were sampled from multiple seed orchards in 2015, 2019 and 2020. Cone bagging experiments were performed during two seasons to investigate the sexual reproduction of T. areolata. In addition to the published simple-sequence repeat (SSR) markers, we developed 10 new polymorphic SSR markers to improve the resolution of population genetic analysis. Aecia were genotyped with 18 SSR markers in total. In 2015, when disease incidence was high in the seed orchards, the T. areolata populations had high genotypic diversity (H = 4.69). In 2019 and 2020, when disease incidence was low, the T. areolata populations had lower genotypic diversity (H = 3.88 and 3.85) and several cones were dominated by a single multilocus genotype. The genotypic diversity of T. areolata in a recently established seed orchard was exceptionally low (H = 2.01). Seven bagged cones that were infected produced either aecial primordia or aecia with lower diversity than exposed cones. The results indicate that cross-fertilization is important for sexual reproduction and aecia formation of T. areolata, and genotypic diversity of T. areolata increased with higher disease prevalence.

Severe outbreaks of Scots pine blister rust, caused by Cronartium pini (Willd.) Jorst., have occurred in several regions in Europe and Asia for at least hundred years. The rust fungus has a complex biology and epidemiology with two different life-cycle forms and five different spore stages. This review summarizes research on: taxonomy and host species, geographical distribution and historic epidemics, life-cycle forms and spore stages, population structure, infection and lesion development, susceptibility of pine provenances, impact of environmental conditions, climate change effects and management. The focus is on conditions in Northern Europe.

BACKGROUND:Over the past decade, demethylation inhibitor (DMI) and succinate dehydrogenase inhibitor (SDHI) fungicides have been extensively used to control to septoria tritici blotch, caused by Zymoseptoria tritici on wheat. This has led to the development and selection of alterations in the target-site enzymes (CYP51 and SDH, respectively). RESULTS:Taking advantage of newly and previously developed qPCR assays, the frequency of key alterations associated with DMI (CYP51-S524T) and SDHI (SDHC-T79N/I, C-N86S and C-H152R) resistance was assessed in Z. tritici-infected wheat leaf samples collected from commercial crops (n = 140) across 14 European countries prior to fungicide application in the spring of 2019. This revealed the presence of a West to East gradient in the frequencies of the most common key alterations conferring azole (S524T) and SDHI resistance (T79N and N86S), with the highest frequencies measured in Ireland and Great Britain. These observations were corroborated by sequencing (CYP51 and SDH subunits) and sensitivity phenotyping (prothioconazole-desthio and fluxapyroxad) of Z. tritici isolates collected from a selection of field samples. Additional sampling made at the end of the 2019 season confirmed the continued increase in frequency of the targeted alterations. Investigations on historical leaf DNA samples originating from different European countries revealed that the frequency of all key alterations (except C-T79I) has been gradually increasing over the past decade. CONCLUSION:Whilst these alterations are quickly becoming dominant in Ireland and Great Britain, scope still exists to delay their selection throughout the wider European population, emphasizing the need for the implementation of fungicide antiresistance measures. © 2021 Society of Chemical Industry.

Epidemics of Scots pine blister rust, caused by Cronartium pini, have become an increasing problem in northern Finland and Sweden. The biology of the rust fungus is complex, with two different life cycle forms that cannot be morphologically distinguished, and it is unclear to what extent the two forms contribute to the epidemics. Genetic structure of fourteen populations of C. pini were investigated in Fennoscandia. Distinction between the two life cycle forms, a heteroecious and an autoecious one, was made by determining zygosity using microsatellite markers, and AFLP markers were developed to analyse population genetic relationships. The results showed that the two life cycle forms are clearly differentiated and occur in separate populations. Within the life cycle forms, geographic differentiation was evident, probably due to restricted gene flow as well as connection with different alternating hosts. The host-alternating form dominated in the epidemic regions in northern Fennoscandia. Implications for silvicultural practices are discussed. (C) 2021 The Authors. Published by Elsevier Ltd.

Fungicide resistance has become a challenging problem in management of Septoria tritici blotch (STB), caused by Zymoseptoria tritici, the most destructive disease of winter wheat throughout western and northern Europe. To ensure the continued effectiveness of those fungicides currently used, it is essential to monitor the development and spread of such resistance in field populations of the pathogen. Since resistance to the key families of fungicides used for STB control (demethyalation inhibitors or azoles, succinate dehydrogenase inhibitors or SDHIs and Quinone outside Inhibitors or QoIs) is conferred through target-site mutations, the potential exists to monitor resistance through the molecular detection of alterations in the target site genes. As more efficient fungicides were developed and applied, the pathogen has continuously adapted through accumulating multiple target-site alterations. In order to accurately monitor these changes in field populations, it is therefore becoming increasingly important to completely sequence the targeted genes. Here we report the development of a PacBio assay that facilitates the multiplex amplification and long-read sequencing of the target gene(s) for the azole (CYP51), SDHI (Sdh B, C, and D), and QoI (cytochrome b) fungicides. The assay was developed and optimised using three Irish Z. tritici collections established in spring 2017, which capture the range of fungicide resistance present in modern European populations of Z. tritici. The sequences obtained through the PacBio assay were validated using traditional Sanger sequencing and in vitro sensitivity screenings. To further exploit the long-read and high throughput potential of PacBio sequencing, an additional nine housekeeping genes (act, BTUB, cal, cyp, EF1, GAPDH, hsp80-1, PKC, TFC1) were sequenced and used to provide comprehensive Z. tritici strain genotyping.

The cherry spruce rust caused by Thekopsora areolata (Fr.) Magnus results in significant losses in spruce seed production in the forest industry. The pathogen is present in Asia and Europe but absent from North America where it has been considered as a potential threat and listed as a quarantine organism by the United States Department of Agriculture. A comprehensive list and in-depth information regarding the alternate hosts of this pathogen are important for conducting epidemiological studies and for optimal disease control. Prunus padus L. is the main alternate host reported for T. areolata. In this study, we investigated the susceptibility of domestic and exotic Prunus spp. and other potential alternate host-plant species native to Scandinavia to T. areolata infection through a field survey and aeciospore inoculation experiments in the greenhouse and laboratory. No new susceptible species were found. In Sweden, a new record of Prunus grayana Maxim. with low susceptibility to T. areolata was found. In addition, we updated the list of currently confirmed alternate hosts of T. areolata according to field observations and inoculation results. Prunus padus and Prunus serotina Ehrh., as well as their hybrids and subspecies of Prunus padus, are highly susceptible, while Prunus depressa Pursh, Prunus grayana, Prunus spinosa L., and Prunus tenella Batsch are considered slightly susceptible.

Abstract Rust fungi are obligate parasites, of plants, with complex and in many cases poorly known life cycles which may include host alteration and up to five spore types with haploid, diploid, and dikaryotic nuclear stages. This study supports that Thekopasora areolata, the causal agent of cherry‐spruce rust in Norway spruce, is a macrocyclic heteroecious fungus with all five spore stages which uses two host plants Prunus padus and Picea abies to complete its life cycle. High genotypic diversity without population structure was found, which suggests predominantly sexual reproduction, random mating and a high gene flow within and between the populations in Fennoscandia. There was no evidence for an autoecious life cycle resulting from aeciospore infection of pistillate cones that would explain the previously reported rust epidemics without the alternate host. However, within cones and scales identical multilocus genotypes were repeatedly sampled which can be explained by vegetative growth of the fertilized mycelia or repeated mating of mycelium by spermatia of the same genotype. The high genotypic diversity within cones and haplotype inference show that each pistillate cone is infected by several basidiospores. This study provides genetic evidence for high gene flow, sexual reproduction, and multiple infections of Norway spruce cone by the rust fungus T. areolata which expands the general understanding of the biology of rust fungi.

Demethylation inhibitor (DMI) and succinate dehydrogenase inhibitor (SDHI) fungicides are currently relied upon for the control of septoria tritici blotch (STB) in European wheat fields. However, multiple mutations have occurred over time in the genes encoding the targeted proteins that have led to a practical loss of fungicide efficacies. Among the different amino acid substitutions inZymoseptoria triticiassociated with resistance to these fungicides, S524T in CYP51 (DMI target) and H152R in SdhC (SDHI target) are regarded as conferring the highest resistance factors to DMI and SDHI, respectively. To facilitate further studies on the monitoring and selection of these substitutions inZ.triticipopulations, a multiplex allele-specific quantitative PCR (qPCR) assay allowing for estimation of both allele frequencies in bulk DNA matrices was developed. The assay was then used on complex DNA samples originating from a spore trap network set up in Belgium, Denmark, Sweden, and Ireland in 2017 and 2018, as well as on leaf samples with symptoms. The S524T allele was present in all field samples and its proportion was significantly higher in Ireland than in Belgium, whereas the proportion of H152R was only sporadically present in both countries. The frequency of S524T varied greatly in the airborne inoculum of all four countries; however, the H152R allele was never detected in the airborne inoculum. The method developed in this study can be readily adopted by other laboratories and used for multiple applications including resistance monitoring in field populations ofZ.tritici.

Summary Cherry spruce rust is a fungal disease of Norway spruce cones caused by Thekopsora areolata and responsible for significant losses in seed production in Sweden and Finland. Here, we report the first set of nine microsatellites, which will allow an effective genetic fingerprinting of T. areolata . The markers were isolated using the FIASCO method and were characterized using DNA from 49 single aecia sampled from spruce cones in three different seed orchards in Sweden. Eight of the nine markers were shown to be polymorphic among the aecia. The markers were unlinked and are therefore suitable for future population genetic studies.

Swedish forestry is sustained by planting about 200 million Norway spruce (Picea abies) seedlings every year. Most of these seedlings come from seed orchards, which have been established to transfer the genetic gain in growth and yield achieved through plant breeding since the 1940s. However, today there is a deficit of seeds coming from Swedish seed orchards mainly caused by irregular flowering, but also due to pest and pathogen infections such as Thekopsora areolata—the causal agent of cherry spruce rust, a fungus that significantly reduces seed production in Fennoscandia. Here, we aim to investigate the reproduction mode and population structure of the pathogen using a newly developed microsatellite marker and a hierarchical sampling strategy. Sampling was done at different locations: one in Norway, one in Finland and five in Sweden. For each location, one aecium per infected cone was analyzed. In addition, multiple aecia per scale and cone were sampled at two locations in Sweden. The results show an overall high genetic diversity in T. areolata at all hierarchical levels with no genetic structure, an indication of high gene flow and random mating. However, at the cone/scale level non-random mating was observed. These results suggest that T. areolata has long distance spore dispersal in Fennoscandia with common recombination events and vegetative spread in cones and scales.

Knowledge of putative insect species vectors of the coconut lethal yellowing disease (CLYD) in Mozambique is crucial to develop an effective disease management plan. Hemiptera specimens from the families Derbidae and Pentatomidae were collected in the Inhambane and Zambezia provinces of the coastal region of Mozambique in 2014, covering the two main growing seasons. Sequence analyses of the 16S rRNA gene were used for phytoplasma clustering. Polymerase chain reaction (PCR) amplification was performed employing three different primer sets specific for phytoplasma. BLAST sequence comparison and phylogenetic analysis of the 16S rDNA PCR products revealed that collected specimens of Derbidae Diostrombus mkurangai Wilson were carrying the CLYD phytoplasma. Virtual RFLP analyses of the obtained sequences confirmed this assigning the detected phytoplasmas to the 16SrXX-A subgroup, confirming that they are 'Candidatus Phytoplasma palmicola'-related strains. This is the first detection of a 'Candidatus Phytoplasma palmicola'-related strain in D. mkurangai.

BACKGROUND:Cereal rust fungi (Puccinia spp.) are among the most economically important plant pathogens. These fungi have a complex life cycle, including five spore stages and two hosts. They infect one grass host on which they reproduce clonally and cause the cereal rust diseases, while the alternate host is required for sexual reproduction. Although previous studies clearly demonstrate the importance of the alternate host in creating genetic diversity in cereal rust fungi, little is known about the amount of novel genotypes created in each successful completion of a sexual reproduction event.RESULTS:In this study, single sequence repeat markers were used to study the genotypic diversity within aecial clusters by genotyping individual aecial cups. Two common cereal rusts, Puccinia graminis causing stem rust and Puccinia coronata the causal agent of crown rust were investigated. We showed that under natural conditions, a single aecial cluster usually include several genotypes, either because a single pycnial cluster is fertilized by several different pycniospores, or because aecia within the cluster are derived from more than one fertilized adjoining pycnial cluster, or a combination of both.CONCLUSION:Our results imply that although sexual events in cereal rust fungi in most regions of the world are relatively rare, the events that occur may still significantly contribute to the genetic variation within the pathogen populations.