Wild grass isolates of Magnaporthe (Syn. Pyricularia) spp. from Germany can cause blast disease on cereal crops

Pathogens that cause destructive crop diseases often infect wild host plants. However, surveys of major plant pathogens tend to be skewed towards cultivated crops and often neglect the wild hosts. Here, we report an emerging disease threat generated by the blast fungus Magnaporthe (Syn. Pyricularia) spp. in central Europe. We found that this notoriously devastating plant pathogen infects the wild grasses foxtail millet (Setaria spp.) and crabgrass (Digitaria spp.) in south-western Germany, a region previously deemed unfavorable for blast disease. Using phenotypic characterization and genomic analyses, we determined that the observed disease symptoms are associated with the Setaria spp.-infecting lineage of M. oryzae and its sister species Magnaporthe grisea. We showed that M. oryzae isolates can infect barley and wheat, thus highlighting the risk of pathogen spread to crops. In addition, M. oryzae isolates which co-occur in natural populations display compatible mating types and variable candidate effector gene content, which may enhance the pathogen’s adaptive potential. Our findings stress the risk of blast fungus infections expanding into European cereal crops through migration and host jumps. This underlines the importance for pathogen surveillance not only on cultivated crops, but also on wild host plants. Author Summary Wild plant species are often overlooked during pathogen virulence surveys. However, many of the diseases we observe in crops are a consequence of host jumps from wild to cultivated plant species. This is reminiscent of zoonotic diseases, where host jumps from wild animals to humans result in new disease outbreaks. Here, we report that the notoriously devastating blast fungus Magnaporthe (Syn. Pyricularia) spp. occurs on wild grasses in south-western Germany. This region, which is at the center of the European cereal belt, has so far been viewed as having unfavorable climatic conditions for the blast disease. The newly identified blast fungus isolates have the capacity to infect wheat and barley cultivars, highlighting the risk of the disease spreading to staple cereal crops. In addition, there is potential for sexual recombination in local populations, which may increase the evolutionary potential of the fungus and might facilitate host jumps to cereal crops. Our findings emphasize the urgent need for surveillance of plant diseases on both wild hosts and crops.


Introduction
Plant disease outbreaks are increasing at an alarming rate that is exacerbated by global trade and climate change, thereby threatening food security across the globe [1]. In addition, plant pathogens can jump from wild to cultivated host plants, resulting in new crop diseases [2]. This is similar to zoonotic diseases, where transmission from wild animal species to humans result in new disease outbreaks [3]. Although there are ongoing efforts to organize and increase global surveillance of plant diseases [4], these often neglect pathogens of wild hosts which have the potential to cause epidemics in crops [5]. As a consequence, our understanding of the distribution of potentially harmful pathogens reservoirs on wild hosts remains limited. This is particularly worrisome for crop pathogens with wide host ranges that can infect both cultivated crops and wild host plants.
One plant pathogen with a wide host range and a propensity to jump between hosts, is the ascomycete blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae). M. oryzae infects over fifty wild and cultivated grass species, including staple cereal crops such as rice (Oryza sativa), wheat (Triticum aestivum) and barley (Hordeum vulgare) [6]. In contrast, its sister species Magnaporthe grisea, is mainly associated with crabgrass (Digitaria spp.) infections [7,8].
M. oryzae is thought to be undergoing incipient speciation into genetically distinct lineages, however, some inter-lineage genetic exchange does occur [9]. To date, at least ten M. oryzae lineages predominantly associated with a single grass host genus have been described [10]. M.
oryzae is thought to have undergone host jumps multiple times from wild to cultivated grass species. The rice-infecting lineage, for example, likely originated after a host shift from the M. oryzae lineage infecting foxtail millet (Setaria spp.) [11], wheat blast emerged after a host jump from the M. oryzae lineage infecting ryegrass (Lolium spp.) [12], and a maize-infecting lineage has evolved after a host jump from barnyard grass (Echinochloa crus-galli) [13].
Blast fungus infections, especially on rice and wheat crops, have had dire economic and societal consequences. Wheat blast, for example, emerged in the mid 1980s and was initially restricted to South America, but has spread to South Asia (Bangladesh, 2016) and Africa (Zambia, 2018) in recent years, resulting in annual yield losses reaching up to 50% [14][15][16]. In Europe, blast outbreaks are largely restricted to southern countries due to more favorable environmental conditions for the fungus, which is especially well-documented for rice blast in the Mediterranean [17]. A phytosanitary risk assessment by the Julius Kühn Institute and the European and Mediterranean Plant Protection Organization (EPPO), last updated in 2019, stated "So far, the fungus M. oryzae is not present in Germany but it already occurs in other EU-Member States. (...) Presumably, M. oryzae will not be able to establish outdoors in Germany due to unsuitable climate conditions" (EPPO; 2019). So far, this major cereal producing region at the center of the European cereal belt has been spared from blast disease.
Climate change will likely result in newly emerging crop diseases across the European continent and could result in environmental conditions suitable for blast disease. Average near-surface temperatures in Europe have already increased by 1.9°C compared to pre-industrial levels (EEA; 2022), with temperatures expected to continue to rise in the next decades [18]. Climate change has resulted in shifts in the geographical range of crop pests and pathogens [19] which have been documented in a myriad of fungal pathogens [20]. For example, the Fusarium graminearum species complex, which causes Fusarium head blight disease in multiple cereal crops, is typically present in warm, humid conditions. However, in recent years it has become more common in Germany, where it poses a threat to maize production, likely enabled by increasing temperatures [21].
We surveyed grass species for blast disease symptoms in the summer of 2021 to better understand the diversity and distribution of the blast fungus in southern and central Europe. We observed typical symptoms of blast fungus infections on the wild host plants Setaria spp. and Digitaria spp. in south-western Germany, a region located in the center of the European cereal belt. Using phenotypic characterization and whole genome analyses, we placed the collected isolates in the general Magnaporthe phylogenetic framework and determined that the observed disease symptoms are associated with the Setaria spp.-infecting lineage of M. oryzae and its sister species M. grisea, which infects Digitaria spp. We found that the M. oryzae isolates can infect susceptible wheat (Triticum aestivum) and barley (Hordeum vulgare) cultivars under laboratory conditions. In addition, some co-occurring isolates carry opposite mating types and display virulence effector diversity, potentially increasing the adaptive capacity of the fungus.
Together, our findings highlight the risk that climate conditions in central Europe may become more conductive for blast diseases. Furthermore, blast fungus lineages infecting wild grasses may adapt to locally cultivated cereal crops, posing a risk to key agricultural sectors in the region. Ultimately, our results point towards a need for increased pathogen surveillance, not only on crops, but also on wild grass hosts.

Disease symptoms and morphological features suggest the presence of the blast fungus in Germany
We observed plants which displayed elliptical lesions with a necrotic center and dark edges, typical for blast disease on Setaria spp. (Fig 1A) and Digitaria spp. plants (Fig 1B) in south-western Germany in August 2021 (Fig 1C, 1D and Table S1). We collected aerial material of representative individuals from infected plants and transported them to the laboratory for further analysis. After processing of infected plant material, we cultured 18 single spore isolates from nine infected plant samples: 16 isolates from infected Setaria spp. and two isolates from infected Digitaria spp. While the colony morphology of the two Digitaria spp.-infecting isolates was similar, Setaria spp.-infecting isolates displayed diverse colony morphologies. Eight of these isolates formed entirely white, fluffy colonies, whereas eight developed a gray center indicative of sporulation or high pigmentation. We selected two Setaria spp.-infecting isolates with contrasting colony morphologies (GE10A2 and GE12B) and the two Digitaria spp.-infecting isolates (GE3 and GE16_2) for further investigation (Fig 1E and 1F).
Microscopic characterization confirmed typical blast fungus morphology, with two septate, spindle-shaped spores, that formed characteristic single-celled appressoria on the hydrophobic surface of cover slips (Fig 1G and 1H). Taken together, our results show that disease symptoms, colony, spore and appressorium morphology of the samples we collected in Germany are consistent with the blast fungus. To define the precise identity of the isolated fungi, we selected the four isolates described above for whole genome sequencing using long and short read sequencing technologies [22]. This resulted in four high quality genome assemblies (13-29 contigs) with a BUSCO completeness score 98.8-98.9% ( Table 1). Next, we mapped short reads of these four isolates, in addition to 413 publicly available M. grisea and M. oryzae samples (Table S2), to the reference assembly MG08 (isolate 70-15) [23]. We then performed variant calling and determined their genetic relationship by creating a genome-wide SNP-based Neighbor-Joining (NJ) tree (Fig S1A). For easier visualization, we subsampled 80 isolates representative for M. grisea and the different M. oryzae lineages (Fig 2).  (Fig S1B). To avoid a potential reference-bias, and complementary to the genome-wide SNP-based NJ trees, we used unmapped sequencing reads to estimate k-mer-based pairwise distances between the same subset of 80 representative M. oryzae and M.grisea isolates used to create NJ trees ( Table S2). The relationship between all isolates and their grouping into host-specific lineages was consistent in both analyses and corroborated the clustering of the German isolates based on genome-wide SNPs (Fig S2A). We repeated the reference-free, k-mer based classification using increased stringency (see Methods) which confirmed robustness of this method and the identity of German blast fungus isolates (Fig S2B). In summary, our genomic analysis confirmed the presence of two blast fungus species, M. oryzae and M. grisea, in Germany and highlights the robustness of the k-mer based, reference free method for pathogen identification.

German M. oryzae isolates can infect wheat and barley cultivars
Next, we investigated whether the blast fungus isolates we collected from wild grasses have the capacity to infect cereal crops, such as barley and wheat, which are extensively cultivated in central Europe. We performed leaf drop infection assays using the four sequenced isolates on the barley cultivars Nigrate and Golden Promise, and the wheat cultivars Fielder and Chinese Spring. As a positive control, we used the wheat-infecting blast fungus isolate BTMP-S13-1, which belongs to the highly virulent pandemic clonal wheat blast lineage ("B71 lineage") that caused major outbreaks after its introduction to Bangladesh in 2016 [14,15,[26][27][28][29]. The four tested German isolates showed differential virulence in these cultivars under laboratory conditions. While M. grisea isolates were largely avirulent on these plants, the Setaria spp.-infecting M. oryzae isolates caused disease symptoms in the form of progressing lesion development (Fig 3). Disease progression was most pronounced in the susceptible barley cultivars Nigrate and Golden Promise, but progressing lesions also formed occasionally on the wheat cultivars Fielder and Chinese Spring.   Fig S3). This suggests these isolates can complete their asexual cycle on wheat and barley cultivars under laboratory conditions. Taken together, our results suggest that the M. oryzae population present in Germany has the potential to cause disease on cereal crops.

Mating types and effector candidate genes vary across German blast fungus isolates
Sexual mating in the blast fungus requires the co-occurrence of compatible mating types [30].
To determine the potential for sexual reproduction in local blast fungus populations in Germany, we analyzed the mating types of the isolates we collected. We used a BLASTN sequence similarity-based approach using the previously described MAT1-1 and MAT1-2 mating type genes [31,32] (Fig 4). Notably, among the German isolates, six predicted effectors, including three predicted and one validated MAX effector, showed presence/absence variation between the two M. oryzae isolates infecting Setaria spp., GE10A2 and GE12B. In the Digitaria spp.-infecting isolates GE3 and GE16_2, two predicted effectors, including a predicted MAX effector, displayed presence/absence variation (Table S3). These variable effectors included the well-characterized AVR-Pita and AVR-Pia, which are associated with host-specialization in M.
oryzae [39,40]. In summary, candidate effector content varies even between the genetically similar German blast fungus isolates. should not be ignored. In the past, the blast fungus has undergone jumps between wild and cultivated host plants on multiple occasions [11][12][13]43]. In addition, host cross-infectivity in the field appears to be relatively common, where a single blast fungus lineage infects multiple host species [25]. Examples are the rice blast fungus isolate TH3 which was collected from barley in Thailand, or the wheat blast fungus isolate P29, which was collected from cheatgrass (Bromus tectorum) in Paraguay (Fig 2 and Table S2). Intermediate hosts, such as barley cultivars that are susceptible to most blast fungus lineages [44,45], may serve as "springboards" for rapid pathogen diversification under weak host selective pressure. This in turn may facilitate adaptation of the blast fungus to local cereal cultivars.

Mating type and effector gene variation may increase the adaptive potential of the blast fungus in Europe
The potential of plant pathogens to adapt to changing environmental conditions and novel host plants may be exacerbated if sexual recombination occurs in populations where fertile isolates of opposite mating types co-occur. Our analysis shows that opposite mating types co-occur in a local M. oryzae population infecting Setaria spp. in Germany (Fig 4). Sexual recombination between fertile blast fungus isolates is thought to occur in nature and can readily be achieved under laboratory conditions [9,28,36,46]. In addition, genomic analyses suggest that recombination occurs in some lineages, although pandemic clonal lineages of rice and wheat blast tend to dominate isolates obtained from these crops [9,25,28,29,36,47]. We recently showed that an isolate of the pandemic clonal wheat blast lineage ("B71 lineage") from Zambia can mate with an African finger miller isolate [28], further raising the possibility that recombination and hybridization between host-specialized lineages can drive the pathogen's evolutionary potential.
We observed presence/absence variation of predicted candidate effector genes across the four German blast fungus isolates we characterized. Differential virulence and adaptation to alternative host plants largely depends on the pathogen effector gene content and the immune receptor (resistance gene, or short, R-gene) repertoire of the host population [24,[38][39][40]48].

Two of the effector candidates that show presence/absence variation in German Setaria
spp.-infecting isolates are the well-characterized avirulence effectors AVR-Pita and AVR-Pia, which are recognized by host plants that carry the corresponding immune receptors Pi-ta and Pia, respectively [39,40]. Although the genotype and R-gene content of German Setaria spp.
hosts remain unknown, it is possible that R-gene diversity in these wild populations led to the observed variation of pathogen effector genes. Differences in blast disease susceptibility has been described for both cultivated (Setaria italica) and wild (Setaria viridis) Setaria spp.
accessions [49] and homologs of several blast fungus resistance genes, including Pia (syn. RGA4/RGA5) have been identified in Setaria spp. genomes [50,51]. In summary, our data suggests that the presence of compatible mating types and standing genetic variation, most notably of candidate effector gene content, might contribute to the adaptive potential of blast fungus infecting wild grass populations in Germany. it conceivable that, as temperatures rise, the blast fungus will spread further north into major cereal producing regions in Europe in the future [56]. How far north the blast fungus is established in Europe beyond the area we surveyed is unknown. This lack of knowledge is further exacerbated by the paucity of surveys on wild host species. We therefore urge the community to increase disease surveillance not only on crops, but also on wild hosts, as is routinely performed for zoonotic human diseases.

Conclusion
The climatic conditions in Germany and the wider central European cereal belt have so far been considered unfavorable for proliferation and spread of the blast fungus. It is unclear how widespread the fungus is in this region, especially since occurrences of the blast fungus on uncultivated wild plants often remain undetected. The sample location reported here is in the center of the southern-German Upper Rhine valley, one of the warmest areas in Germany [57].
It is thus possible that the spread of the blast disease is limited to this geographical region.
Nevertheless, our study exposes the blast fungus as an emerging threat to cereal crops grown in central Europe and highlights the importance for plant pathogen surveillance not just on crop plants but also on wild hosts. This is increasingly important in the face of changing pathogen dynamics as a consequence of climate change, and shifting global trade routes propelled by international conflicts.

Sample collection and phenotypic pathogen characterization
In

Visualization of worldwide blast fungus distribution
Maps were created with the R-package ggmap (v3.0) [58]. Locations of blast fungus samples plotted are those of the 417 M. oryzae and M. grisea samples which were used for SNP-based phylogenetic analyses (Table S2). In addition, locations of blast fungus reports from Russia [59] and Korea [60], as well as the location of reports of rice and wheat blast fungus from CABI, the Invasive Species Compendium, were plotted. If the exact coordinates of a sample were unknown, the coordinates of the county's geographical center were plotted ( Table S1).

Pathogen infection assays
The

Genome sequencing and assembly
We whole-genome sequenced two representative single spore isolates infecting Setaria spp.
(isolate ID GE12B and GE10A2) and two infecting Digitaria spp. (isolate ID GE3 and GE16_2) as described in [22]. For this purpose, we extracted high molecular weight DNA following the protocol described in [59]. Sequencing runs were performed by Future Genomics Technologies (Leiden, The Netherlands) using the PromethION sequencing platform (Oxford Nanopore Technologies, Oxford, UK). All samples were sequenced with a depth between 105X and 146X and a mean read length of >20kb (read N50 >30kb) (Tables S4 and S5). Long reads were assembled into contigs and corrected using Flye (v2.9-b1768) [60], and polished with long reads process was repeated with members of this lineage only (--max-missing 1.0 filtering resulted in 1'835,862 SNPs and --indep-pairwise 50 10 0.5 filtering resulted in 41,120 SNPs), and a NJ tree using 500 bootstraps was created (https://itol.embl.de/tree/14915521019236421655393224).

Reference-free clustering of blast fungus isolates based on K-mer sharing
To avoid potential biases due to differences in nucleotide divergence between the M. oryzae reference genome (rice blast fungus isolate  and each of the host-specific lineages, we leveraged the unmapped raw sequences of 80 representative Magnaporthe samples (Table S2) to estimate k-mer-based distances between isolates (https://github.com/smlatorreo/Magnaporthe-Germany). To eliminate possible adapter leftovers, we used AdapterRemoval (v2.3.1) [61] with default parameters. To calculate the k-mer-based pairwise distances we used Mash (v2.3) [68] with two different sets of parameters. The first consisted of sketch sizes of 100,000, k-mer sizes of 21 nucleotides, and a support of a minimum of 2 k-mer copies as a noise filter. The second settings consisted of sketch sizes of 1'000,000, k-mer sizes of 21 nucleotides, and a support of a minimum of 3 k-mer copies as a noise filter.

Determination of mating type and candidate effector gene repertoire
To determine the mating type of the four sequenced German isolates, we searched for sequence similarity to the MAT1-1 and MAT1-2 mating type locus idiomorphs [31] using BLASTN. As a control, we also tested isolates whose mating type have been previously determined [32]. In addition, to compare the repertoire of candidate effector genes present in the German blast fungus isolates and identify presence/absence variation between them, we extracted the DNA coding sequences of a set of previously identified, either experimentally validated or in silico predicted effectors [35]. The candidate effectors were filtered to exclude highly similar (≥ 90% sequence identity) sequences, resulting in a total of 178 effectors [36].
These effectors originate from isolates infecting a variety of host grasses. We searched for sequence similarity between these predicted effectors across eight M. oryzae and eight M.