In this detailed genetic study we investigated the often-overlooked hypothesis that host genetic diversity and pathogen population size affects pathogen diversity. It is well-known that variation in the host phenotype affects virulence evolution in the pathogen [64]. However, studies that simultaneously evaluate host diversity and pathogen population size at neutral and selected loci in order to explore the link between fungal pathogen diversity and virulence appear to be absent. We predicted that pathogen neutral genetic diversity will increase with large pathogen N AND host diversity, whereas pathogen diversity under selection will increase with host diversity. This prediction was broadly supported, with the exception that pathogen neutral genetic diversity was found to increase with pathogen N. We also discovered strong evidence that pathogen migration from the diverse host (barley grass) is high, providing a mechanism for the effective transmission of virulence from weeds to the crop. Below we explore these findings and their implications in more detail.

Does host genetic diversity and/or pathogen N affect pathogen neutral genetic diversity and mode of reproduction?

Predicting whether pathogen neutral genetic diversity, and thus N e will change with host genetic diversity and/or host/pathogen N, is not necessarily straightforward. The number of alleles/genotypes maintained by mutation is expected to increase strongly with population size. Furthermore, irrespective of N, diverse host populations are predicted to select for diverse pathogen populations, likely via enhanced sexual reproduction in the pathogen, such as the case of Puccinia graminis on its alternate host [65, 66] and selection favouring novel mutations such as in rusts on wild oats in Australia [21, 22].

Whether or not host diversity overrides the importance of mutation in large populations, is an open question. The interplay between N and host diversity becomes particularly important when one considers weeds as a potential ancillary host of crop pathogens. This is because weeds are typically genetically more diverse than crops, but will often have smaller population sizes or sustain lower disease incidence. The possibility of variation in the degree of sexual reproduction between hosts may add a further layer of complexity.

Most fungi can reproduce both asexually and sexually, with fungal populations of a species with high rates of sexual reproduction typically being genetically more diverse than populations with less sexual reproduction. In fungal plant pathogens a key advantage of sexual reproduction is that it can combine virulences from two individuals into the same genetic background, thereby accelerating adaptation to novel combinations of resistance genes in the host. Meanwhile, the pathogen’s capacity for asexual reproduction can enable rapid propagation, while keeping well-adapted gene combinations together. In the present study of R. commune from barley, all neutral genetic diversity estimates based on the 14 SSR loci, including the number of MLGs, number of alleles, Nei’s gene diversity, Shannon’s information index and the scaled within population Shannon diversity, were higher than for populations from barley grass. This suggests lower levels of clonal reproduction in the pathogen on barley, in contrast to the expectation that pathogen neutral genetic diversity should be higher on the more diverse host. This is an unexpected finding since sex in fungi is often triggered by stress [67], which is predicted to be higher in non-cultivated host populations.

In an extension of genetic diversity estimates, the IMa2 N e estimate for the pathogen population from barley was approximately twice as large as that from barley grass, although we note some overlap of posterior density intervals. Nevertheless, the findings suggest that the larger population size (acreage) of barley is accompanied by an associated larger pathogen N with a more diverse pathogen population. Therefore, in our study system, host/pathogen N seems to be more important than host diversity for maintaining large N e and higher neutral genetic diversity of the pathogen. That is, higher pathogen neutral genetic diversity is correlated with a higher host/pathogen N, not higher host diversity, at least at loci evolving neutrally. This finding may reflect the reduced risk of rare allele loss in larger pathogen populations [68].

Although we predicted differences in the degree of pathogen sexual reproduction on barley and barley grass, both host-associated populations were in linkage disequilibrium, suggesting both are predominantly asexual. This finding is consistent with the observation that the sexual state has never been observed for R. commune. However, other lines of evidence suggest some sexual reproduction must occur in the pathogen, but its frequency appears to be insufficient to remove the strong signal of disequilibrium generated by recent founder events and bottlenecks [42].

Despite the common finding of disequilibrium in R. commune on both barley and barley grass, we found fewer MLGs and these were also distributed more evenly and at higher linkage disequilibrium in barley grass populations. This finding contradicts our prediction that the pathogen should reproduce sexually (have lower linkage disequilibrium) more frequently on barley grass than on barley. It is evident that studies that fail to combine careful and simultaneous consideration of both host genetic diversity and pathogen N may draw misleading conclusions.

Is there more pathogen migration from the weed to the crop?

A key factor for virulence evolution is the effectiveness of between-host transmission [6, 69–71]. It is therefore important to establish whether between-host transmission is occurring, in order to assess the importance of alternate hosts on pathogen evolution. Few studies have shown ongoing pathogen migration between wild and cultivated host plants [72, 73]. Our estimates of migration rates indicated there is no transmission barrier between the two hosts that would prevent newly evolved virulence types selected on barley grass from migrating to barley. In fact, the estimated migration rates of the pathogen from barley grass into the barley pathogen populations were significantly (P < 0.05) higher. These higher migration rates might be facilitated by a higher number of resistance genes in H. murinum [39], which block more migrant pathogen isolates from barley establishing on barley grass, than in the reverse migration. This further suggests that newly evolved virulence on the barley grass can be rapidly transmitted to barley-infecting populations, posing a significant threat to durability of disease resistance genes bred into barley cultivars.

Is genetic diversity under selection at an effector locus related to host genetic diversity?

The potential importance of frequency-dependent selection in maintaining genetic diversity within host-pathogen interactions is well recognized [74, 75]. However, while it is acknowledged in theory that weedy hosts could play a role in the evolution of plant pathogens [18, 24, 39, 76], investigations are often limited to simply quantifying the number of pathotypes present. Isolates from barley grass in this study exhibited slightly higher nip1 nucleotide diversity, more NIP1 amino acid types and harboured more virulent NIP1 types. Strong evidence for selection on NIP1 composition was also found in the higher frequency of ΔNIP (ΔNip1 = where nip1 is absent) in isolates from barley grass. Isolates with ΔNIP1 are able to escape host resistance conferred by Rrs1 [34, 36], indicating a high prevalence of Rrs1 in barley grass is selecting for infectious strains. Our results are therefore consistent with the expectation of frequency-dependent selection [74, 75] on the genetically more diverse weedy host driving increased diversity of the pathogen populations, despite smaller pathogen N.

Are pathogen populations from a genetically diverse host more aggressive/virulent than those from a monoculture crop?

More than 40 years ago, Ali and Boyd [77] suggested that weedy barley grass could have an important influence on the epidemiology of R. commune on cultivated barley. Since then, other studies have reported that isolates from barley grass were highly variable for pathogenicity [30, 39, 78] or display novel virulences [79]. Brown [78] found that in a comparison of 182 isolates from barley grass and 94 from barley, isolates from barley grass were significantly more virulent (could infect more cultivars) and had more pathotypes (19 in barley grass vs. 5 in cultivated barley isolates). Similarly, the present study found that despite evidence for host adaptation (isolates from barley tend to be more aggressive on barley, and the converse on barley grass), isolates from barley grass infected significantly more barley cultivars (91.1 % vs. 87.7 %; P = 0.011) and overall they were more aggressive than isolates from barley. Weeds, or genetically diverse hosts, as a source of virulence is often assumed but is rarely shown empirically, with a few notable exceptions [21, 22, 65, 73]. Combined with the evidence for R. commune migration between hosts as shown here and in [72], it is evident that weedy or wild hosts could play a major role in pathogen evolution.

Implications for pathogen evolution

Our study of barley/barley grass R. commune populations confirm that there is indeed a relationship among host and pathogen diversity and population sizes, but that the direction of the relationship can vary between neutral loci/traits to those under selection. In this case, increased pathogen diversity of effector alleles at nip1, but not alleles at neutral loci, was found on the genetically more diverse host, barley grass. This pattern of increased diversity at nip1 is consistent with an arms race between host and pathogen. Indeed, similar findings have been found for parasites [9, 80], bacteria [81] and rust pathogens on crops [16, 65] and wild hosts [10, 11], where pathogen race diversity is higher on diverse or alternate hosts where sexual reproduction occurs. An exception is found in the poplar rust pathogen Melampsora larici-populina with fewer pathotypes in wild than in cultivated populations (although some results were inconclusive), likely because of complex resistances bred into cultivated poplars [17, 82] and ongoing gene flow [73]. The contrasting findings that the neutral loci of the R. commune pathogen did not exhibit more diversity on the genetically more diverse host, suggest that pathogen N is a better predictor of the patterns of pathogen neutral genetic diversity. Similarly Zymoseptoria tritici shows evidence for faster genomic evolution and a higher effective population size on cultivated wheat compared to its sister species on wild grasses [19]. The discrepancy between effector alleles and neutral genetic diversity may also be attributed to the rapid rate at which virulence evolves on cultivated hosts due to the high selection pressure exerted by widely deployed resistance R-genes [20].

Practical implications for agriculture

Agricultural mechanisation has led to an ever increase in crop acreages with serious consequences for disease risk. In this study of R. commune, the large acreage of barley and associated large pathogen N was linked with increased pathogen neutral diversity, despite the lower genetic diversity of the host. It follows that host/pathogen population sizes need to be kept small to reduce the risk of accelerated adaptive evolution in the pathogen [20]. One strategy to achieve this is to employ various alternatives to crop monocultures such as intercropping (mixtures of different crop species) or the use of multiline cultivars or varietal mixtures (crop plants that differ in resistance specificities). Applying the strategy of host heterogeneity (eg. multilines or cultivar mixes) is known to negatively affect the pathogen’s ability to survive, reproduce and compete in several systems [83].

Ensuring host heterogeneity is also recommended to extend the life of resistant cultivars by retarding the rate of pathogen evolution [84, 85]. However, a potential risk of this strategy is that extensive use of host heterogeneity could lead to the development of complex pathogen races [86]. Indeed, in our case study of barley grass as a heterogeneous host, our findings of more pathotypes and higher virulence diversity compared with the pathogen on barley highlights this risk. To retard pathogen evolution, it is thus important to combine and rotate as many disease control and management strategies in space and time as possible [20, 23, 24, 87, 88].

Management of disease on crops must also include management of weedy ancillary hosts, which, as evident in this study, can harbour disproportionate supplies of virulent pathogen strains. However, it will be particularly challenging to manage ubiquitous weeds or wild hosts for this purpose. Applying herbicides adjacent to cultivated hosts is a feasible starting point, but may not prevent pathogen transmission from further a field. This is especially true for airborne pathogens or those where migration is assisted by human-mediated dispersal of infected plant material.