Diversifying the menu for crop powdery mildew resistance.

Powdery mildew, a potentially severe crop disease, can be controlled by mlo mutations, which suppress fungal proliferation but typically also reduce yield. Li et al. (2022) demonstrate that productivity can be restored by overexpressing a host sugar transporter, thus offering a new option for economically and environmentally benign disease control.

Harnessing landrace diversity empowers wheat breeding.

Harnessing genetic diversity in major staple crops through the development of new breeding capabilities is essential to ensure food security1. Here we examined the genetic and phenotypic diversity of the A. E. Watkins landrace collection2 of bread wheat (Triticum aestivum), a major global cereal, by whole-genome re-sequencing of 827 Watkins landraces and 208 modern cultivars and in-depth field evaluation spanning a decade. We found that modern cultivars are derived from two of the seven ancestral groups of wheat and maintain very long-range haplotype integrity. The remaining five groups represent untapped genetic sources, providing access to landrace-specific alleles and haplotypes for breeding. Linkage disequilibrium-based haplotypes and association genetics analyses link Watkins genomes to the thousands of identified high-resolution quantitative trait loci and significant marker-trait associations. Using these structured germplasm, genotyping and informatics resources, we revealed many Watkins-unique beneficial haplotypes that can confer superior traits in modern wheat. Furthermore, we assessed the phenotypic effects of 44,338 Watkins-unique haplotypes, introgressed from 143 prioritized quantitative trait loci in the context of modern cultivars, bridging the gap between landrace diversity and current breeding. This study establishes a framework for systematically utilizing genetic diversity in crop improvement to achieve sustainable food security.

The genotype of barley cultivars influences multiple aspects of their associated microbiota via differential root exudate secretion.

Plant-associated microbes play vital roles in promoting plant growth and health, with plants secreting root exudates into the rhizosphere to attract beneficial microbes. Exudate composition defines the nature of microbial recruitment, with different plant species attracting distinct microbiota to enable optimal adaptation to the soil environment. To more closely examine the relationship between plant genotype and microbial recruitment, we analysed the rhizosphere microbiomes of landrace (Chevallier) and modern (NFC Tipple) barley (Hordeum vulgare) cultivars. Distinct differences were observed between the plant-associated microbiomes of the 2 cultivars, with the plant-growth promoting rhizobacterial genus Pseudomonas substantially more abundant in the Tipple rhizosphere. Striking differences were also observed between the phenotypes of recruited Pseudomonas populations, alongside distinct genotypic clustering by cultivar. Cultivar-driven Pseudomonas selection was driven by root exudate composition, with the greater abundance of hexose sugars secreted from Tipple roots attracting microbes better adapted to growth on these metabolites and vice versa. Cultivar-driven selection also operates at the molecular level, with both gene expression and the abundance of ecologically relevant loci differing between Tipple and Chevallier Pseudomonas isolates. Finally, cultivar-driven selection is important for plant health, with both cultivars showing a distinct preference for microbes selected by their genetic siblings in rhizosphere transplantation assays.

Pathogen-induced biosynthetic pathways encode defense-related molecules in bread wheat.

Wheat is a widely grown food crop that suffers major yield losses due to attack by pests and pathogens. A better understanding of biotic stress responses in wheat is thus of major importance. The recently assembled bread wheat genome coupled with extensive transcriptomic resources provides unprecedented new opportunities to investigate responses to pathogen challenge. Here, we analyze gene coexpression networks to identify modules showing consistent induction in response to pathogen exposure. Within the top pathogen-induced modules, we identify multiple clusters of physically adjacent genes that correspond to six pathogen-induced biosynthetic pathways that share a common regulatory network. Functional analysis reveals that these pathways, all of which are encoded by biosynthetic gene clusters, produce various different classes of compounds­namely, flavonoids, diterpenes, and triterpenes, including the defense-related compound ellarinacin. Through comparative genomics, we also identify associations with the known rice phytoalexins momilactones, as well as with a defense-related gene cluster in the grass model plant Brachypodium distachyon. Our results significantly advance the understanding of chemical defenses in wheat and open up avenues for enhancing disease resistance in this agriculturally important crop. They also exemplify the power of transcriptional networks to discover the biosynthesis of chemical defenses in plants with large, complex genomes.

Trehalose and α-glucan mediate distinct abiotic stress responses in Pseudomonas aeruginosa.

An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PAO1 and define its role in abiotic stress protection. We show that trehalose metabolism in PAO1 is integrated with the biosynthesis of branched α-glucan (glycogen), with mutants in either biosynthetic pathway significantly compromised for survival on abiotic surfaces. While both trehalose and α-glucan are important for abiotic stress tolerance, we show they counter distinct stresses. Trehalose is important for the PAO1 osmotic stress response, with trehalose synthesis mutants displaying severely compromised growth in elevated salt conditions. However, trehalose does not contribute directly to the PAO1 desiccation response. Rather, desiccation tolerance is mediated directly by GlgE-derived α-glucan, with deletion of the glgE synthase gene compromising PAO1 survival in low humidity but having little effect on osmotic sensitivity. Desiccation tolerance is independent of trehalose concentration, marking a clear distinction between the roles of these two molecules in mediating responses to abiotic stress.

Naturally occurring circadian rhythm variation associated with clock gene loci in Swedish Arabidopsis accessions.

Circadian clocks have evolved to resonate with external day and night cycles. However, these entrainment signals are not consistent everywhere and vary with latitude, climate and seasonality. This leads to divergent selection for clocks which are locally adapted. To investigate the genetic basis for this circadian variation, we used a delayed fluorescence imaging assay to screen 191 naturally occurring Swedish Arabidopsis accessions for their circadian phenotypes. We demonstrate that the period length co-varies with both geography and population sub-structure. Several candidate loci linked to period, phase and relative amplitude error (RAE) were revealed by genome-wide association mapping and candidate genes were investigated using TDNA mutants. We show that natural variation in a single non-synonymous substitution within COR28 is associated with a long-period and late-flowering phenotype similar to that seen in TDNA knock-out mutants. COR28 is a known coordinator of flowering time, freezing tolerance and the circadian clock; all of which may form selective pressure gradients across Sweden. We demonstrate the effect of the COR28-58S SNP in increasing period length through a co-segregation analysis. Finally, we show that period phenotypic tails remain diverged under lower temperatures and follow a distinctive "arrow-shaped" trend indicative of selection for a cold-biased temperature compensation response.

Improving zinc accumulation in cereal endosperm using HvMTP1, a transition metal transporter.

Zinc (Zn) is essential for all life forms, including humans. It is estimated that around two billion people are deficient in their Zn intake. Human dietary Zn intake relies heavily on plants, which in many developing countries consists mainly of cereals. The inner part of cereal grain, the endosperm, is the part that is eaten after milling but contains only a quarter of the total grain Zn. Here, we present results demonstrating that endosperm Zn content can be enhanced through expression of a transporter responsible for vacuolar Zn accumulation in cereals. The barley (Hordeum vulgare) vacuolar Zn transporter HvMTP1 was expressed under the control of the endosperm-specific D-hordein promoter. Transformed plants exhibited no significant change in growth but had higher total grain Zn concentration, as measured by ICP-OES, compared to parental controls. Compared with Zn, transformants had smaller increases in concentrations of Cu and Mn but not Fe. Staining grain cross sections with the Zn-specific stain DTZ revealed a significant enhancement of Zn accumulation in the endosperm of two of three transformed lines, a result confirmed by ICP-OES in the endosperm of dissected grain. Synchrotron X-ray fluorescence analysis of longitudinal grain sections demonstrated a redistribution of grain Zn from aleurone to endosperm. We argue that this proof-of-principle study provides the basis of a strategy for biofortification of cereal endosperm with Zn.

The role of reactive oxygen in the development of Ramularia leaf spot disease in barley seedlings.

Background and Aims: Ramularia collo-cygni is an ascomycete fungus that colonizes barley primarily as a benign endophyte, although this interaction can become pathogenic, causing the disease Ramularia leaf spot (RLS). Factors, particularly reactive oxygen species, that resulted in the transition of the fungus from endophyte to necrotrophic parasite and the development of disease symptoms were investigated. Methods: Disease development in artificially inoculated seedlings of barley varieties varying in partial resistance to RLS was related to exposure to abiotic stress prior to inoculation. Histochemical and molecular analysis determined the effect of R. collo-cygni colonization on accumulation of reactive oxygen species and antioxidant gene expression. Development of RLS on barley lines defective in antioxidant enzymes and with altered redox status or non-functional chloroplasts was compared with the accumulation of fungal biomass to determine how these factors affect disease symptom expression. Key Results: Exposure to abiotic stress increased symptom development in all susceptible and most partially resistant barley varieties, in association with greater hydrogen peroxide (H2O2) levels in leaves. Decreased activity of the antioxidant enzymes superoxide dismutase and catalase in transgenic and mutant plants had no effect on the disease transition, whereas manipulation of H2O2 levels during asymptomatic growth of the fungus increased disease symptoms in most susceptible varieties but not in partially resistant plants. Barley mutants that undergo rapid loss of green leaf area when infected by R. collo-cygni or albino mutants with non-functional chloroplasts showed reduced development of RLS symptoms. Conclusions: These results imply that in seedlings the pathogenic transition of the normally endophytic fungus R. collo-cygni does not result from senescence as such, but rather is promoted by factors that result in changes to host reactive oxygen species. Barley varieties vary in the extent to which these factors promote RLS disease.

Increased yield stability of field-grown winter barley (Hordeum vulgare L.) varietal mixtures through ecological processes.

Crop variety mixtures have the potential to increase yield stability in highly variable and unpredictable environments, yet knowledge of the specific mechanisms underlying enhanced yield stability has been limited. Ecological processes in genetically diverse crops were investigated by conducting field trials with winter barley varieties (Hordeum vulgare), grown as monocultures or as three-way mixtures in fungicide treated and untreated plots at three sites. Mixtures achieved yields comparable to the best performing monocultures whilst enhancing yield stability despite being subject to multiple predicted and unpredicted abiotic and biotic stresses including brown rust (Puccinia hordei) and lodging. There was compensation through competitive release because the most competitive variety overyielded in mixtures thereby compensating for less competitive varieties. Facilitation was also identified as an important ecological process within mixtures by reducing lodging. This study indicates that crop varietal mixtures have the capacity to stabilise productivity even when environmental conditions and stresses are not predicted in advance. Varietal mixtures provide a means of increasing crop genetic diversity without the need for extensive breeding efforts. They may confer enhanced resilience to environmental stresses and thus be a desirable component of future cropping systems for sustainable arable farming.

Evolution of the EKA family of powdery mildew avirulence-effector genes from the ORF 1 of a LINE retrotransposon.

BACKGROUND: The Avrk1 and Avra10 avirulence (AVR) genes encode effectors that increase the pathogenicity of the fungus Blumeria graminis f.sp. hordei (Bgh), the powdery mildew pathogen, in susceptible barley plants. In resistant barley, MLK1 and MLA10 resistance proteins recognize the presence of AVRK1 and AVRA10, eliciting the hypersensitive response typical of gene for gene interactions. Avrk1 and Avra10 have more than 1350 homologues in Bgh genome, forming the EKA (Effectors homologous to Avr k 1 and Avr a 10) gene family. RESULTS: We tested the hypothesis that the EKA family originated from degenerate copies of Class I LINE retrotransposons by analysing the EKA family in the genome of Bgh isolate DH14 with bioinformatic tools specially developed for the analysis of Transposable Elements (TE) in genomes. The Class I LINE retrotransposon copies homologous to Avrk1 and Avra10 represent 6.5 % of the Bgh annotated genome and, among them, we identified 293 AVR/effector candidate genes. We also experimentally identified peptides that indicated the translation of several predicted proteins from EKA family members, which had higher relative abundance in haustoria than in hyphae. CONCLUSIONS: Our analyses indicate that Avrk1 and Avra10 have evolved from part of the ORF1 gene of Class I LINE retrotransposons. The co-option of Avra10 and Avrk1 as effectors from truncated copies of retrotransposons explains the huge number of homologues in Bgh genome that could act as dynamic reservoirs from which new effector genes may evolve. These data provide further evidence for recruitment of retrotransposons in the evolution of new biological functions.

Genetics of resistance to Zymoseptoria tritici and applications to wheat breeding.

This paper reviews current knowledge about genes for resistance to Septoria tritici blotch (STB) of wheat, caused by Zymoseptoria tritici (formerly Mycosphaerella graminicola). These genes can be placed into two classes, although a few may have characteristics of both classes. Qualitative resistance is controlled by genes which control large fractions of genetic variation, 21 of which have been discovered and mapped so far. Most of them have been shown to be genotype-specific, being effective against the minority of Z. tritici isolates which are avirulent, and Stb6 has been shown to control a gene-for-gene relationship. Most qualitative resistances are unlikely to be durable and some formerly effective genes have been overcome by the evolution of pathogen virulence. Quantitative resistance is generally controlled by genes with small-to-moderate effects on STB. They have generally weaker specificity than qualitative genes and have provided more durable resistance. 89 genome regions carrying quantitative trait loci (QTL) or meta-QTL have been identified to date. Some QTL have been mapped at or near loci of qualitative genes, especially Stb6, which is present in several sources of resistance. Another gene of particular interest is Stb16q, which has been effective against all Z. tritici isolates tested so far. In addition to resistance, the susceptibility of wheat cultivars to STB can also be reduced by disease escape traits, some of which may be undesirable in breeding. The fundamental requirements for breeding for STB-resistance are genetic diversity for resistance in wheat germplasm and a field trial site at which STB epidemics occur regularly and effective selection can be conducted for resistance combined with other desirable traits. If these are in place, knowledge of resistance genes can be applied to improving control of STB.

Differential effects of lesion mimic mutants in barley on disease development by facultative pathogens.

Lesion mimic mutants display spontaneous necrotic spots and chlorotic leaves as a result of mis-regulated cell death programmes. Typically these mutants have increased resistance to biotrophic pathogens but their response to facultative fungi that cause necrotrophic diseases is less well studied. The effect of altered cell death regulation on the development of disease caused by Ramularia collo-cygni, Fusarium culmorum and Oculimacula yallundae was explored using a collection of barley necrotic (nec) lesion mimic mutants. nec8 mutants displayed lower levels of all three diseases compared to nec9 mutants, which had increased R. collo-cygni but decreased F. culmorum disease symptoms. nec1 mutants reduced disease development caused by both R. collo-cygni and F. culmorum. The severity of the nec1-induced lesion mimic phenotype and F. culmorum symptom development was reduced by mutation of the negative cell death regulator MLO. The significant reduction in R. collo-cygni symptoms caused by nec1 was completely abolished in the presence of the mlo-5 allele and both symptoms and fungal biomass were greater than in the wild-type. These results indicate that physiological pathways involved in regulation of cell death interact with one another in their effects on different fungal pathogens.

Ramularia collo-cygni--An Emerging Pathogen of Barley Crops.

Ramularia collo-cygni is the biotic factor responsible for the disease Ramularia leaf spot (RLS) of barley (Hordeum vulgare). Despite having been described over 100 years ago and being considered a minor disease in some countries, the fungus is attracting interest in the scientific community as a result of the increasing number of recorded economically damaging disease epidemics. New reports of disease spread and fungal identification using molecular diagnostics have helped redefine RLS as a global disease. This review describes recent developments in our understanding of the biology and epidemiology of the fungus, outlines advances made in the field of the genetics of both the fungus and host, and summarizes the control strategies currently available.

A trade off between mlo resistance to powdery mildew and increased susceptibility of barley to a newly important disease, Ramularia leaf spot.

Ramularia leaf spot (RLS), caused by the fungus Ramularia collo-cygni, is a serious, recently emerged disease of barley in Europe and other temperate regions. This study investigated the trade off between strong resistance to powdery mildew conferred by mlo mutant alleles and increased susceptibility to RLS. In field trials and seedling tests, the presence of mlo alleles increased severity of RLS. Genetic analysis of a doubled-haploid population identified one quantitative trait locus for susceptibility to RLS, colocalizing with the mlo-11 allele for mildew resistance. The effect of mlo-11 on RLS severity was environmentally sensitive. Analysis of near-isogenic lines of different mlo mutations in various genetic backgrounds confirmed that mlo alleles increased RLS severity in seedlings and adult plants. For mlo resistance to mildew to be fully effective, the genes ROR1 and ROR2 are required. RLS symptoms were significantly reduced on mlo-5 ror double mutants but fungal DNA levels remained as high as in mlo-5 single mutants, implying that ror alleles modify the transition of the fungus from endophytism to necrotrophy. These results indicate that the widespread use of mlo resistance to control mildew may have inadvertently stimulated the emergence of RLS as a major disease of barley.

An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat.

BACKGROUND: CRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community. RESULTS: We identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately. CONCLUSION: Based on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency.

Multiple routes to fungicide resistance: Interaction of Cyp51 gene sequences, copy number and expression.

We examined the molecular basis of triazole resistance in Blumeria graminis f. sp. tritici (wheat mildew, Bgt), a model organism among powdery mildews. Four genetic models for responses to triazole fungicides were identified among US and UK isolates, involving multiple genetic mechanisms. Firstly, only two amino acid substitutions in CYP51B lanosterol demethylase, the target of triazoles, were associated with resistance, Y136F and S509T (homologous to Y137F and S524T in the reference fungus Zymoseptoria tritici). As sequence variation did not explain the wide range of resistance, we also investigated Cyp51B copy number and expression, the latter using both reverse transcription-quantitative PCR and RNA-seq. The second model for resistance involved higher copy number and expression in isolates with a resistance allele; thirdly, however, moderate resistance was associated with higher copy number of wild-type Cyp51B in some US isolates. A fourth mechanism was heteroallelism with multiple alleles of Cyp51B. UK isolates, with significantly higher mean resistance than their US counterparts, had higher mean copy number, a high frequency of the S509T substitution, which was absent from the United States, and in the most resistant isolates, heteroallelism involving both sensitivity residues Y136+S509 and resistance residues F136+T509. Some US isolates were heteroallelic for Y136+S509 and F136+S509, but this was not associated with higher resistance. The obligate biotrophy of Bgt may constrain the tertiary structure and thus the sequence of CYP51B, so other variation that increases resistance may have a selective advantage. We describe a process by which heteroallelism may be adaptive when Bgt is intermittently exposed to triazoles.

Stabilization of yield in plant genotype mixtures through compensation rather than complementation.

BACKGROUND AND AIMS: Plant genotypic mixtures have the potential to increase yield stability in variable, often unpredictable environments, yet knowledge of the specific mechanisms underlying enhanced yield stability remains limited. Field studies are constrained by environmental conditions which cannot be fully controlled and thus reproduced. A suitable model system would allow reproducible experiments on processes operating within crop genetic mixtures. METHODS: Phenotypically dissimilar genotypes of Arabidopsis thaliana were grown in monocultures and mixtures under high levels of competition for abiotic resources. Seed production, flowering time and rosette size were recorded. KEY RESULTS: Mixtures achieved high yield stability across environments through compensatory interactions. Compensation was greatest when plants were under high levels of heat and nutrient stress. Competitive ability and mixture performance were predictable from above-ground phenotypic traits even though below-ground competition appeared to be more intense. CONCLUSIONS: This study indicates that the mixing ability of plant genotypes can be predicted from their phenotypes expressed in a range of relevant environments, and implies that a phenotypic screen of genotypes could improve the selection of suitable components of genotypic mixtures in agriculture intended to be resilient to environmental stress.

Classification of wheat diseases using deep learning networks with field and glasshouse images.

Crop diseases can cause major yield losses, so the ability to detect and identify them in their early stages is important for disease control. Deep learning methods have shown promise in classifying multiple diseases; however, many studies do not use datasets that represent real field conditions, necessitating either further image processing or reducing their applicability. In this paper, we present a dataset of wheat images taken in real growth situations, including both field and glasshouse conditions, with five categories: healthy plants and four foliar diseases, yellow rust, brown rust, powdery mildew and Septoria leaf blotch. This dataset was used to train a deep learning model. The resulting model, named CerealConv, reached a 97.05% classification accuracy. When tested against trained pathologists on a subset of images from the larger dataset, the model delivered an accuracy score 2% higher than the best-performing pathologist. Image masks were used to show that the model was using the correct information to drive its classifications. These results show that deep learning networks are a viable tool for disease detection and classification in the field, and disease quantification is a logical next step.

Spatial heterogeneity, frequency-dependent selection and polymorphism in host-parasite interactions.

BACKGROUND: Genomic and pathology analysis has revealed enormous diversity in genes involved in disease, including those encoding host resistance and parasite effectors (also known in plant pathology as avirulence genes). It has been proposed that such variation may persist when an organism exists in a spatially structured metapopulation, following the geographic mosaic of coevolution. Here, we study gene-for-gene relationships governing the outcome of plant-parasite interactions in a spatially structured system and, in particular, investigate the population genetic processes which maintain balanced polymorphism in both species. RESULTS: Following previous theory on the effect of heterogeneous environments on maintenance of polymorphism, we analysed a model with two demes in which the demes have different environments and are coupled by gene flow. Environmental variation is manifested by different coefficients of natural selection, the costs to the host of resistance and to the parasite of virulence, the cost to the host of being diseased and the cost to an avirulent parasite of unsuccessfully attacking a resistant host. We show that migration generates negative direct frequency-dependent selection, a condition for maintenance of stable polymorphism in each deme. Balanced polymorphism occurs preferentially if there is heterogeneity for costs of resistance and virulence alleles among populations and to a lesser extent if there is variation in the cost to the host of being diseased. We show that the four fitness costs control the natural frequency of oscillation of host resistance and parasite avirulence alleles. If demes have different costs, their frequencies of oscillation differ and when coupled by gene flow, there is amplitude death of the oscillations in each deme. Numerical simulations show that for a multiple deme island model, costs of resistance and virulence need not to be present in each deme for stable polymorphism to occur. CONCLUSIONS: Our theoretical results confirm the importance of empirical studies for measuring the environmental heterogeneity for genetic costs of resistance and virulence alleles. We suggest that such studies should be developed to investigate the generality of this mechanism for the long-term maintenance of genetic diversity at host and parasite genes.

Succession of the fungal endophytic microbiome of wheat is dependent on tissue-specific interactions between host genotype and environment.

Fungi living inside plants affect many aspects of plant health, but little is known about how plant genotype influences the fungal endophytic microbiome. However, a deeper understanding of interactions between plant genotype and biotic and abiotic environment in shaping the plant microbiome is of significance for modern agriculture, with implications for disease management, breeding and the development of biocontrol agents. For this purpose, we analysed the fungal wheat microbiome from seed to plant to seeds and studied how different potential sources of inoculum contributed to shaping of the microbiome. We conducted a large-scale pot experiment with related wheat cultivars over one growth-season in two environments (indoors and outdoors) to disentangle the effects of host genotype, abiotic environment (temperature, humidity, precipitation) and fungi present in the seed stock, air and soil on the succession of the endophytic fungal communities in roots, flag leaves and seeds at harvest. The communities were studied with ITS1 metabarcoding and environmental climate factors were monitored during the experimental period. Host genotype, tissue type and abiotic factors influenced fungal communities significantly. The effect of host genotype was mostly limited to leaves and roots, and was location-independent. While there was a clear effect of plant genotype, the relatedness between cultivars was not reflected in the microbiome. For the phyllosphere microbiome, location-dependent weather conditions factors largely explained differences in abundance, diversity, and presence of genera containing pathogens, whereas the root communities were less affected by abiotic factors. Our findings suggest that airborne fungi are the primary inoculum source for fungal communities in aerial plant parts whereas vertical transmission is likely to be insignificant. In summary, our study demonstrates that host genotype, environment and presence of fungi in the environment shape the endophytic fungal community in wheat over a growing season.