TRPLSC-1256; No. of Pages 10

Review

Phytopathogen emergence in the genomics era Elisha Thynne*, Megan C. McDonald*, and Peter S. Solomon Plant Sciences Division, Research School of Biology, The Australian National University, Canberra, 2601, ACT, Australia

Phytopathogens are a global threat to plant agriculture and biodiversity. The genomics era has lead to an exponential rise in comparative gene and genome studies of both economically significant and insignificant microorganisms. In this review we highlight some recent comparisons and discuss how they identify shared genes or genomic regions associated with host virulence. The two major mechanisms of rapid genome adaptation – horizontal gene transfer and hybridisation – are reviewed and we consider how intra-specific pan-genome sequences encode alternative host specificity. We also discuss the power that access to expansive gene databases provides in aiding the study of phytopathogen emergence. These databases can rapidly enable the identification of an unknown pathogen and its origin, as well as genomic adaptations required for emergence. The study of phytopathogen emergence has never been easier Emerging plant pathogens represent a serious threat to agricultural industries, food security, and to the conservation of plant species across the world. Accordingly, the ability to quickly identify a new phytopathogen and understand how it has emerged is vitally important. A fascinating complex of events surround the emergence of a phytopathogen and the impact that humans have had in facilitating phytopathogen emergence (see Glossary) has been studied in detail; for example, through trade, farming practices, and climate change [1,2]. However, the exponential rise in abundance of genomic data has meant that, for the first time, researchers are now in a position to begin to understand the true extent of the genetic events involved in pathogen emergence, both recent and ancient. Sequenced genes and genomes for both pathogenic and non-pathogenic organisms have become ubiquitous, which allows in-depth comparative analyses to be performed (Figure 1). In this review we will address two specific genome adaptations relevant to phytopathogenicity that have been observed with comparative sequence analyses. Additionally, we describe how increased sequencing of both Corresponding author: Solomon, P.S. ([email protected]). Keywords: pathogen emergence; horizontal gene transfer; genome plasticity; hybridisation. * These authors contributed equally to this work. 1360-1385/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2015.01.009

pathogenic and non-pathogenic microorganisms, and from whole genomes to single loci, has not only imbued greater clarity to the study of phytopathogens and their emergence but has also opened our eyes as to the limitations of reference genomes. Unprecedented levels of access to genetic data have enabled rapid genomic comparisons. This means that a new phytopathogen can be quickly identified, its relationship to other microorganisms assessed, and an informed prediction of the mechanism of emergence and location of origin can be made. To demonstrate how the genomics era has assisted the study of phytopathogen emergence we provide two specific examples whose study has been greatly aided by genome sequences and the ability to share genetic data (i.e., genetic markers or genes). Initially we discuss Pseudomonas syringae and the emergence of ‘kiwifruit canker’ (Box 1). We also touch upon the devastating emerging disease, Magnaporthe oryzae ‘wheat blast’ (Box 2), whose study would be greatly aided by genome sequencing. We finally discuss how sharing of genetic data has assisted the study of Hymenoscyphus pseudoalbidus and the emergence of ‘ash dieback’ (Box 3).

Glossary Comparative genomics: studies performed to compare and contrast genes or genomes of multiple individuals. These studies allow comparisons of host specificity, virulence, or other phenotypes that can be associated with functional genome elements. Effector genes: genes that produce proteins or metabolites produced by a microorganism that enable successful infection and disease in a plant host. Avirulence genes, commonly referred to in agricultural pests, are a subclass of effectors. Horizontal gene transfer (HGT): a genome adaptation event whereby DNA is transferred between organisms (potentially from different species or kingdoms) in a manner different from common reproduction (i.e., sex). Interspecific hybridisation: the process of forming viable offspring between two genetically distinct species. For example, this can be achieved through sharing of alleles by distinct species via sexual reproduction or by the formation of a polyploid organism by transfer of whole-genome sequences. Intraspecific genomic plasticity: describes the dynamic nature of the genomes of some microorganism in which large regions (up to whole chromosomes) can be lost, gained, duplicated, or translocated to/from other isolates within the same species. Pan genome: refers to all possible genomic sequences that can belong to an organism or organisms within a pre-defined phylogenetic clade. Phytopathogen emergence: a process by which a microorganism is observed with increasing regularity to infect and cause disease in a new or previously unreported plant host. Phytopathogen emergence can be preceded by an adaptation event allowing a microorganism to infect a plant, or alternatively can be a result of pathogen movement into a naı¨ve host population. For the purposes of this review we will refer to adaptation events as those that have altered the genome of a microorganism to facilitate infection on a new host; for example, the gain of a virulence gene.

Trends in Plant Science xx (2015) 1–10

1

TRPLSC-1256; No. of Pages 10

Review

Trends in Plant Science xxx xxxx, Vol. xxx, No. x

The study of phytopathogen emergence has been greatly aided by the ability to obtain and compare genomic sequences against globally populated, expansive databases

(A)

(C)

(B)

(D)

? TRENDS in Plant Science

Figure 1. Understanding phytopathogen emergence in the genomics era. The ability to obtain the genomic sequence of a microorganism with relative ease has greatly improved our understanding of phytopathogen emergence. (A) A globally populated sequence database from all kingdoms of life. The true power of using genome analyses to study phytopathogen emergence comes from the ability to compare data against other sequences, thereby enabling rapid and accurate identification of a phytopathogen. Many studies in recent years have utilized genetic loci markers to resolve the boundaries of microorganism species, which in turn have populated databases with informative data. These data can be exploited for diagnostic purposes and for putative prediction of the centre of origin (Box 2). Similarly, the parental lineages of novel hybrid phytopathogen can be predicted using these resources. For an example of using genome sequences to observe a hybridisation event, see [35]. (B) Horizontal gene transfer (HGT) events can be predicted by comparisons to expansive sequence databases and tests of phylogenetic congruence. Access to large gene datasets for comparisons is therefore required because the standard method of HGT prediction is phylogenic incongruence. For example, the true phylogeny for our emergent phytopathogen is shown (below) to be incongruent with the phylogenetic tree for the horizontally transferred gene (above). For an example of a pipeline for HGT prediction in phytopathogens, see [32]. (C) Re-sequencing within pathogenic species reveals novel genetic information within the pan-genome. Often studies that sequence more than one isolate from a single species discover novel genetic data that determine host-specific virulence. Here three genome sequences of the same species show a novel insertion (or deletion) of a genome segment that contains genes. For an example of the identification of isolate specific genome regions that confer isolate-specific virulence genes see [50]. (D) Increasing levels of functional data assigned to gene sequences in public databases. Newly annotated genomes can be compared with functionally characterised genes. Here we depict the light-blue gene from bacteria and its homolog in plants known to interact with a receptor on in plant cells. For an example involving previously characterised plant expansins found within bacterial genomes see [18].

Observations of genomic adaptation and its impact upon phytopathogenicity For the purposes of this review we will not focus on the specific mechanisms of host resistance and susceptibility to phytopathogen effectors because this topic has been comprehensively reviewed [3]. Similarly, we will not focus on the beneficial outcomes of genome assemblies for important plants that are now available, especially complex genomes such as the hexaploid wheat genome (Triticum aestivum) [4]. Instead we will assess the impact that the ability to readily obtain genome sequences for a phytopathogen has had on improving our understanding of both the mechanisms of accelerated genome adaptation and the subsequent affect on the ability of a microorganism to cause plant disease. We will address two defined mechanisms of accelerated genome adaptation relevant to phytopathogenicity: horizontal gene transfer and inter-specific hybridisation. Horizontal gene transfer: sharing of phytopathogenicity-related genes between species and kingdoms One of the most publicized and sensationalistic mechanisms of accelerated genome adaptation studied has been horizontal gene transfer (HGT) [5,6]. Previously, the mechanisms of HGT, although well understood in prokaryotes [7], were hard to prove in eukaryotic organisms. One of the 2

primary reasons for the inability to accurately identify a HGT event was the lack of eukaryotic gene sequences available for comparison [5,8]. In more recent years this inability to successfully identify HGT has receded, and the breadth of HGT, intra and inter-kingdoms, has been explored; largely due to the ability to compare gene sequences against expansive, publicly-available databases [9– 22]. The importance of HGT on the emergence of phytopathogens has been shown to be significant for oomycetes and across the bacterial and fungal kingdoms. In bacteria, the relationship between horizontal transfer and genome adaptation has become well recognised. For example, the transfer of pathogenicity islands (PAIs) (regions of localised pathogenicity-related genes) between bacteria is well known [23,24], and recent genomic studies have reaffirmed this phenomenon and its impact on plant– pathogen interactions [25,26]. However, more recent widescale comparative analyses have revealed the horizontal transfer of specific pathogenicity genes not only between phytopathogens but also from plant hosts. Cross-kingdom HGT events were identified by searching public databases with a plant expansin homologue found in Bacillus subtilis, EXLX1 [18,27]. Subsequent phylogenetic analysis revealed the transfer of plant expansins into multiple microorganisms, including species of bacteria and fungi [18]. In plants, expansins are proteins involved in loosening cell walls [28]. It is speculated that these expansin homologues

TRPLSC-1256; No. of Pages 10

Review Box 1. Pseudomonas syringae and the emergence of ‘kiwifruit canker disease’ Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of the severe kiwifruit canker disease. The disease was first observed in 1984 in Italy and Korea. In 2008 a highly virulent strain of the phytopathogen was observed in Italy, and by 2010 had spread to New Zealand [75]. The emergence of kiwifruit canker disease by Psa has been studied extensively, aided by gene and genome sequences [49,76–78]. P. syringae are a globally spread bacterial species that have a wide range of plant hosts [33]. Much work has gone into understanding the lifecycle of these phytopathogens, including sequencing isolates of environmental reservoirs [68–80]. A recent study described the isolation of environmental strains related to P. syringae pv. tomato (Pto) that are able to cause disease on tomatoes [68]. These environmental strains also possessed an increased host range [more virulent than Pto on cauliflower (Brassica oleracea) and celery (Apium graveolens)] [68]. A mechanism of host-specific emergence was presented whereby less-specific, inter-recombining environmental samples may, if in contact with a suitable host, reproduce and reproduce clonally [68]. Clonal lineages observed in phylogenetic and comparative genomic studies involving Psa lend support to this theory [76,78]. Study of Psa emergence has been assisted by numerous genomic studies comparing multiple P. syringae genomes [49] and Psa genomes [77,78]. A multigene phylogeny of 40 isolates of Psa was presented [76]. These isolates, sampled from collections, included a range of samples from various countries and historical samples from the less-recent outbreaks [76]. Four clonal lineages of Psa were observed in this phylogeny. Interestingly, the New Zealand isolates included in this study were split between two clades, suggesting they belonged to two distinct lineages [76]. Similar results were described in a comparative analysis of 36 Psa genomes from New Zealand, and these showed the same separation into four defined clades as Chapman [76], including the two distinct New Zealand clades [78]. They concluded that there are at least four distinct lineages of Psa [78]. This scenario is concordant with the mode of P. syringae pathovar emergence from the environment, hypothesised in [68].

function by loosening cell walls to assist microorganism inhabitation of a plant and are, therefore, advantageous in plant–phytopathogen interactions [18]. The traditional weakness of using database analyses to identify HGT

Trends in Plant Science xxx xxxx, Vol. xxx, No. x

events is the subsequent lack of support from functional data (Table 1). However, in this example support for these claims was provided by a study showing that EXLX1 in B. subtilis was structurally similar to plant expansins. When knocked-out, the bacterium displayed reduced ability to colonise maize (Zea mays) roots [27]. Database analyses such as this are important because they characterise the spread of particular genes and their homologues across species and kingdoms. Such analyses are made possible by the deposition of the microorganism genes from genome sequencing projects into the US National Center for Biotechnology Information (NCBI) database (see Table 1 for other examples). Another prominent example of HGT from plants to fungi resulting in enhanced pathogenicity was described with the effector Ave1 found in Verticillum dahliae and Verticillium albo-atrum [21]. The presence of this gene in Verticillium spp. was functionally shown to increase virulence in tomatoes that lack the Ave1-recognition receptor, Ve1. Homology searches and phylogenetic analyses suggest that this effector has a plant origin [21]. Comparative genomics between race 1 and race 2 strains of V. dahliae showed that a 50 kb DNA region, containing the gene for Ave1, was only present in race 1 [21]. This large transfer of DNA within a chromosome would not have been observed without full genome sequencing. The ability to rapidly compare genomes of interest showing gain of genes and large regions of surrounding DNA, such as with Ave1, illustrates the relevance of HGT to the rapid adaptation of eukaryotic genomes and to facilitating phytopathogen emergence. Despite the significance of the above examples, there are still only a limited number of described HGT events from plants that have been functionally shown to increase phytopathogenicity. Inter-eukaryotic HGT events between microorganisms appear to be a more common occurrence. As demonstrated in bacteria with the HGT of PAIs, similar events have been demonstrated in phytopathogenic fungi with recent genome studies revealing the transfer of

Box 2. The emergence of wheat blast by Magnaporthe oryzae would be improved by access to a wheat blast isolate genome Magnaporthe oryzae is responsible for a devastating disease in rice (Sativa oryzae), known as rice blast [81]. Much research has gone into rice blast; there is a well-annotated reference genome ([82]; Magnaporthe Comparative Sequencing Project. Broad Institute of Harvard and MIT, http://www.broadinstitute.org/), re-sequenced isolates [53], and known effectors (including secondary metabolites and secreted proteins [83]). In 1985 M. oryzae was identified as the causal agent of a severe wheat (Triticum aestivum) disease in Brazil, and has since spread throughout Brazil and surrounding countries [84]. The study of wheat blast has thus far relied on this well-studied close relative. For example, a study used conserved sequences based on the reference M. oryzae strain 70-15 to identify glycosyl hydrolase (GH) 6 and 7 families, and subsequently the authors observed expression of these cellulases in the wheat blast isolate, BR48 in planta [85]. Knock-down of these homologues reduced virulence of this wheat-infecting M. oryzae isolate [85]. However, more could be achieved in terms of wheat-specific phytopathogenicity with access to a wheat blast genome. In a recent study the authors used microsatellites derived from rice blast in part to test the hypothesis that wheat blast emerged from local rice blast populations [86]. Rice populations were eliminated as the source population of wheat blast in view of

strong population differentiation and a lack of historical gene flow between the two host-infecting populations [86]. They propose that wheat blast may have emerged from a different grass (Poaceae)infecting population [86]. In fact, the source population of wheat infecting M. oryzae may derive from isolates infecting grass members of the subfamily Pooideae. This speculation is supported by observation of an independent host jump of a ryegrass (Lolium perenne)-infecting M. oryzae isolate onto wheat in Kansas [87]. Ryegrass-infecting isolates phylogenetically group closely to wheat blast isolates [88], and similarly wheat and ryegrass are both of the Poaceae subfamily, Pooideae (whereas rice is not). A comparative analysis utilising the genomes of wheat blast, ryegrass-infecting, and the ryegrass-origin wheat-infecting isolates could be performed to help test this hypothesis. Similarly, the wheat blast genome alone could be assembled to the M. oryzae reference genome to look for genes involved in host specificity. Although not a trivial endeavour, such a wide-scale analysis may highlight genomic region variation involved in altered host range. Synthesis of such a genome analysis with what is known about M. oryzae genome plasticity, and effectors and loci associated with wheat blast resistance, could be extremely fruitful in advancing our understanding of wheat blast emergence. 3

TRPLSC-1256; No. of Pages 10

Review

Box 3. Hymenoscyphus pseudoalbidus and the emergence of ‘ash dieback’ Ash dieback is a devastating disease of ash trees (Fraxinus excelsior and Fraxinus angustifolia) across Europe. First observed in Poland in the 1990s [89], this disease results from infection by the fungus Hymenoscyphus pseudoalbidus [90]. A pathogen profile that was recently compiled describes this phytopathogen and its recent emergence [89]. However, we seek to emphasize how the story of Ash dieback demonstrates the importance of public databases for quick and accurate identification of shared global diseases. In a recent phylogenetic study the authors utilized sequenced loci to distinctly delimit H. pseudoalbidus from the non-pathogenic H. albidus [90]. Previously, the relationship between the two species was unclear [90]. To perform their pivotal analysis, the authors sequenced several herbarium samples. Two samples from Switzerland (1978 and 1987) were reported to be H. pseudoalbidus, suggesting its presence in Europe before observation of the disease [90]. Multiple gene loci of H. pseudoalbidus from various herbariums and environmental samples have been sequenced, documenting the occurrence of the disease through Europe [91,92]. Subsequent to defining the boundaries of these species, H. pseudoalbidus was shown to cause local extinction of H. albidus [93]. A recent study sequenced the same loci from a phytopathogenic fungus found in Japan, Lambertella albida, and the authors determined this fungus to be in fact H. pseudoalbidus [94]. Greater genetic diversity was reported in Japanese isolates than in the European and further Asian isolates, and Hymenoscyphus spp. have subsequently been published, suggesting that this disease originated from Asia [94–96]. The genomes of H. pseudoalbidus and H. albidus have been sequenced. To date, however, they have only been used for the development of molecular markers [97,98]. In the future, they will undoubtedly be exploited to shed light on this emerging disease.

accessory chromosomes (ACs) [29,30]. One study showed evidence of accessory chromosome HGT, but also described controlled conditions in which transfer of an AC (chromosome 14) occurred between a Fusarium oxysporum strain pathogenic to tomato (Solanum lycopersicum) and F. oxysporum strains non-pathogenic to tomato [30]. The transfer of AC 14 from the tomato-pathogenic strain conferred the ability to infect tomato to the traditionally non-pathogenic strains [30]. These examples, as well as Ave1 above, highlight the power of genomic sequencing to capture the movement of large contiguous pieces of DNA. Although it is unknown how often these events occur, it is now clear that within such large regions pathogenicity genes can be present which are able to alter host specificity or trigger the emergence of a new disease [30,31]. Although we can observe the movement of large pieces of DNA, a glaring shortcoming of this analysis is the absence of any mechanisms that might underlie this movement (Box 4). More recent studies have shown that the scope of HGT between microbial eukaryotes can be much broader and often more subtle in effect. A recent analysis compared the cereal pathogen Fusarium pseudograminearum to other cereal and non-cereal pathogens [32]. In this study, multiple ancient transfer events were observed to have occurred during the evolution of this pathogen [32]. These events were subtler because they did not appear to enable sudden host specialisation (as were shown in the cases described in [30,31]). Instead, these HGT events were hypothesised to contribute to niche specialisation in cereals because the transferred genes were found in other wheat pathogens 4

Trends in Plant Science xxx xxxx, Vol. xxx, No. x

[32]. Similarly, a genome-wide phylogenetic analysis performed in oomycetes provided evidence that multiple HGT events were key to the evolution of oomycetes as phytopathogens [19]. Of the 34 putative HGT genes discussed, two were present in an animal-pathogen lineage, and the remainder were found in phytopathogen lineages of oomycetes. Accordingly, these genes were hypothesised to be involved in plant– pathogen interactions, for example, genes encoding enzymes that degrade plant sugars or plant structural components [19]. Both of these studies are exciting because they work towards defining specific genes required for specialisation on a given host (for another example, see [16]; Table 1). Unlike traditional targeted genome analyses that focus on the effector space of plant–pathogen interactions, these analyses provide a more unbiased approach, utilising sequences available in NCBI to identify HGT between unrelated organisms. Accordingly, they potentially provide an interesting insight into the minor genes that may well be important for successful plant colonisation. Inter-specific hybridisation can beget phytopathogen emergence Another mechanism of accelerated genome evolution linked to phytopathogen emergence is hybridisation. Signatures of hybridisation events include discordant gene sequences or polyploidy that can be easily observed and the parental origin speculated upon [33,34]. In this respect, the recent availability of genome sequences has rapidly accelerated our understanding of hybridisation and the role it plays in pathogen emergence. For example, the sexual exchange of diverged alleles has been shown to be capable of providing the offspring a selective advantage over the parents, or extending the host range of the phytopathogen. A recent study showed that Zymoseptoria pseudotritici, a fungal phytopathogen of various grass species sampled across the Middle East, emerged after a hybridisation event [35]. The sequenced genomes of five Z. pseudotritici were aligned and long (up to 100 kb) variable and non-variable regions observed [35]. The variable regions showed only two distinct haplotypes, suggesting two distinct parents [35]. Nevertheless, the parental species have not yet been discovered, and Z. pseudotritici is a prevalent grass phytopathogen in Iran [34,35], which supports the inference that hybridisation of distinct species can beget phytopathogen emergence [35]. Another fungal hybridisation event was described that resulted in an extended host range for the diploid hybrid, Verticillium longisporum. The approach involved phylogenetic comparisons with isolates of V. longisporum and other Verticillium spp. [36]. V. longisporum had been previously described as a hybrid [37], and sequence analysis has suggested that its parents were V. dahliae and an unknown Verticillium spp. [38]. However, the authors used multiple gene loci from 203 isolates to determine that there are three distinct lineages of V. longisporum that are likely the result of several independent hybridisation events between V. dahliae and two unknown Verticillium species [36]. These examples show that hybridisation can produce viable offspring with a new host range, which in the right environment can give rise to more widespread disease than either of its parental species.

Type of analysis

Donating organism

Comparative genomics and database search for effector gene Ave1

Verticillum darliae b

Unknown plant sp.

Comparative genomics between Fusarium spp. and functional validation assay

Fusarium oxysporum (non-pathogenic isolate)

Fusarium oxysporum f. sp. lycopersici

Reciprocal BLAST analysis between cereal pathogen proteins

Fusarium pseudograminearum b

Unknown bacterial sp. Unknown fungal sp.

Database search to predict inter-kingdom HGT in Pyrenophora spp. Comparative genomics of wilt: Verticillum spp. and F. oxysporum, and database search

Pyrenophora spp.

Unknown plant spp. Unknown bacterial spp.

Veritcillium spp b

Unknown bacterial sp.

Database search and phylogeny of oomycete proteomes

Oomycete spp

Database search for HGT into Colletotrichum spp.

Colletotrichum spp.

Database search of plant expansin homologue EXLX1

b

Unknown plant sp.

Unknown fungal spp.

Unknown bacterial spp.

Putative role in phytopathogenicity Loosening of cell walls to assist microorganism inhabitation of plants Unknown. However, increases virulence in tomato if the ve1 receptor not present Transfer of a dispensable chromosome (chromosome 14). This chromosomes contain host specific effectors (including Ave1) Functionally tested virulence factors; predicted to increase niche specialization, improving phytopathogenicity Predicted niche specialisation to increase phytopathogenicity Glucan glucosyltransferase virulence factor required for normal wilt disease progression Predicted niche specialisation to increase phytopathogenicity Predicted niche specialisation to increase phytopathogenicity

Inter-kingdom?

Targeted or unbiased approach Targeted approach

Refs

Yes

Functionally tested? Yes

Yes

Yes

Targeted approach

[21]

No

Yes

Targeted approach c

[30]

Yes and no

Yes

Unbiased approach

[32]

Yes

No

Unbiased approach d

[20]

Yes

Yes

Targeted approach c

[16]

Yes

No

Unbiased approach

[19]

Yes

No

Unbiased approach

[14]

a

[18]

The ability to quickly obtain and analyse genomes, and also to compare genes against expansive and globally populated databases, has enabled researchers to observe rapid genomic adaptations shown to be crucial for phytopathogen emergence (e.g., HGT). Presented here is a selection of HGT events into known phytopathogens (cited in this review) that have been observed since 2010 with the use of comparative genomics and/or database searches. The approaches and goals of each of the analyses vary. For example, some observe inter-kingdom HGT as opposed to intra-kingdom, and/or some utilise an unbiased approach to observe a range of HGT events as opposed to others that test the hypotheses that a particular gene or chromosome are horizontally acquired. However, although varied, they each provide insight into how HGT can impact upon the emergence of a microorganism as an effective phytopathogen. For an example of a pipeline that could be employed to observe for HGT in a recently emerged phytopathogen see Gardiner et al. [32].

b c

Although a single, or a specific selection of species has been the primary focus of acquisition via HGT, multiple secondary organisms were observed to have been affected.

Although the final approach was targeted, the initial analysis was an unbiased comparative genomics approach.

d

Although the analysis was mostly unbiased, limitations were placed on the kingdoms from which the donating organism could belong.

5

Trends in Plant Science xxx xxxx, Vol. xxx, No. x

Primary phytopathogen recipient Bacillus subtilis b

TRPLSC-1256; No. of Pages 10

Review

Table 1. Various strategies to explore the prevalence and impact of HGT on phytopathogenicitya

TRPLSC-1256; No. of Pages 10

Review

Trends in Plant Science xxx xxxx, Vol. xxx, No. x

Box 4. Outstanding questions Many of the examples we have presented in this review have raised interesting questions regarding the future directions of comparative genomic research. Below are three key unanswered questions that the genomics era has raised.  What mechanisms underlie horizontal gene transfer? Genome sequencing is very good at revealing HGT events between genetically distant organisms. The mechanism of this exchange, however, remains largely unknown. While some interesting mechanisms have been proposed (i.e., endosymbiosis, conidial anastomosis tubes), these deal mainly with the exchange of cytoplasm but not fusion of nuclei [99,100]. In the absence of obvious self-reproducing TEs, which where insertion mechanisms are known, the mechanism that allows movement of single genes between distantly related organisms remains a fascinating open question. In fungi the formation of conidial anastomosis tubes (CATs) has been proposed many times as a potential mechanism for HGT. Recent studies with Colletotrichum lindemuthianum have shown not only hyphal fusion but also heterokaryon formation [101]. Genome sequencing of these heterokaryons would be fascinating in terms of what signatures CAT heterokaryons leave in the merged nuclei.

Recent phylogenetic studies have also described the nature of hybridisation in shaping various populations of oomycetes [39–44]. For example, a study of the lineage of Andean, Solanum spp.-infecting Phytophthora andina, with the support of a multigene phylogeny, postulated that this species is a hybrid of Phytophthora infestans and an unknown species [40]. Furthermore, they speculate that the different Solanum spp. host ranges observed for P. andina to P. infestans are probably a result of the hybridisation event [40]. A comparison of 42 Phytophthora isolates from the environment in Australia and South Africa describes four hybrid strains among these isolates, reiterating the prevalence of hybridisation among oomycete populations [42]. However, as these samples were obtained from the environment and not from infected tissue, the impact of these hybridisation events on phytopathogenicity can only be speculated upon [42]. This above example [42] is limited because it only compared three nuclear and one mitochondrial loci. Definitive identification of hybrid species requires comparisons at the whole-genome level or across a wide distribution of unlinked loci. However, analyses such as the above example can have further practical applications. By broadening the genetic knowledge of phytopathogenic families, it may enable prediction of the parental origins of a hybrid species. Of the three hybrid species discussed above (Z. pseudotritici, V. longisporum, and P. andina) at least one of the parental species of each is as-yet undetermined [35,36,40]. Genetic sampling from sources beyond infected plant tissue, such as from herbariums or environmental samples, offers potential in answering such questions. Alternative host specificity is encoded within the pangenome It was thought that the increased availability of genomic sequences and genetic data would confirm the hypothesis that HGT was the primary mechanism of genomic adaptation leading to pathogen emergence [45]. Although true, comparative genome analyses have shown that intraspecific variation in gene content, outside the transfer of ACs, 6

 What role transposable elements play in pathogen emergence? Genome sequencing under current technologies is hampered by repetitive sequences. These limitations are likely to be lifted with the imminent release of single-molecule sequencing. Within some of the studies discussed in this manuscript, TEs are considered key genome elements that enabled phytopathogen emergence by generating novel sequences, and rearrangements of genes, within pathogen genomes.  How do we move beyond the ‘reference’ genome? Bacterial researchers have already coined the terms ‘open’ and ‘closed’ pan-genomes, which refer to organisms where increased genome sequences reveals significantly more novel gene sequences, or not, respectively [46,102,103]. A major limitation of the pan-genome concept is the difficulty in designing a fast and flexible coordinate system (analogous to a reference chromosome) to base genome comparisons upon. While a solution has recently been posed [104], it remains to be seen whether this can be more widely adopted by highly-plastic phytopathogen genomes. In the meantime fungal comparative genomics should be wary of comparisons limited to the reference genome alone. De novo assembly has improved dramatically in recent years [105], making this a valid option for examining genomic reads that do not assemble to the reference.

plays a large role in host specificity. From these re-sequencing studies the idea of the pan-genome has emerged as a means to capture all possible genetic information contained within breeding populations of a species [46]. Comparative genome analyses enable assessment of the variation between different species [16,32,47,48], and similar analyses comparing multiple isolates of the same species show occurrences of intra-species genome adaptation, whereby the genomes of re-sequenced isolates show high levels of presence/absence polymorphism at the gene level [49–53]. A comparison of 19 Pseudomonas syringae genomes describes genome structure and variation in the effector complement between isolates [49]. Large discrepancies were described in isolate gene content, whereby little more than two-thirds of the predicted genes per isolate were uniformly shared among all P. syringae genomes [49]. Furthermore, the 19 isolates compared had a diverse host range [including but not limited to tomato, barley (Hordeum vulgare), and sugarbeet (Beta vulgaris)]. This broad range was attributed to the significant variation in gene content observed. We elaborate on host-range variation with the example of an emergent Pseudomonas pathogen and discuss how genomic variability in environmental isolates can lead to rapid clonal expansion on a suitable plant host (Box 1). Similarly, a study compared the genomes of three pathovars of Xanthomonas axonopodis to investigate why the two isolates which were closely related at neutral loci had different host ranges [51]. Variation in chromosomal arrangement and plasmid content, as well as pathovar-specific effectors (PSEs), were observed [51]. It was hypothesised that these PSEs might be the factors responsible for the altered virulence and host patterns [51]. These studies highlight that the presence or absence of specific genes can alter host specificity. Importantly, these genes seem to be maintained in larger populations of the same species, thereby preserving the genetic history of the pathogen and distinguishing these presence/absence polymorphisms from HGT events. Similarly to the comparative BLAST analysis describing HGT among wheat pathogens

TRPLSC-1256; No. of Pages 10

Review [32], the genes displaying presence/absence polymorphisms within the bacterial species’ pan-genome also contribute to niche adaptation [46]. We detail another example of how re-sequencing the whole genomes of closely related M. oryzae isolates may provide insight into the niche adaptation and emergence of wheat blast (Box 2). Increased numbers of available genome sequences have also assisted in defining the pan-genome of phytopathogenic fungi. In fungi (particularly asexual species), the pangenome is more commonly referred to as isolate or lineage specific (LS) genome regions [50,54]. A recent study compared 11 genomes of V. dahliae, revealing significant chromosomal reorganisation between isolates and a large 4 Mb LS genome region that was shared or unique among only a subset of the eleven isolates [50]. The authors go on to show effector-like proteins found in isolates with unique LS regions are required for virulence for that strain, and they postulate that chromosome reorganisation in this asexual species, mediated by retrotransposons, is an adaptive mechanism to assist in increasing genetic variability in the absence of sexual reproduction [50]. In another example, three genomes were compared of the wheatinfecting fungal species, Pyrenophora tritici-repentis [52]. Two genomes were from pathogenic isolates and one from a non-pathogenic isolate [52]. In this study, an increased number of transposable elements (TEs) were observed within the pathogenic isolates compared to the non-pathogenic isolates, and TE-induced gene duplications, previously unobserved in fungi, were noted [52]. Increased repeat content of fungal genomes also makes it difficult to accurately assess genes that are truly missing from assemblies versus genes that are lost in repetitive DNA. We discuss further the challenges associated with studying pathogenic genomes enriched for TEs as well as the issues associated with relying on reference genomes [53,54] in Box 4. Unprecedented access to gene sequence data facilitates phytopathogen study The genomics era has resulted in a significant rise in the number of gene sequences available for comparison [55,56]. There is no doubt that the capability to quickly acquire and compare genomes has assisted our understanding of phytopathogen emergence. Extensive sequence-based analyses describing the biodiversity among microorganisms are efficacious for the study of phytopathogens and their emergence. Population genetic studies using gene sequence comparisons from an increased number of isolates have been employed to predict the origin of phytopathogen emergence, even in established pathogens [34,57,58]. The true power of these population studies, however, is the deposition of the gene loci sequenced (genes, SNPs, or microsatellite markers) into publicly-accessible databases. These sequence contributions are vital to the study of phytopathogens because they can be utilised by any researcher across the world to perform further comparative studies. With access to extensive databases or genome sequences, a new phytopathogen can be quickly identified and/or reclassified, compared to closely related species, and a potential origin predicted (Box 3). Furthermore, gene loci

Trends in Plant Science xxx xxxx, Vol. xxx, No. x

sequenced from the environment, instead of infected tissues, can be immensely useful for identifying close relatives of existing pathogens. Herbariums are now proving effective as sources of genetic material for studying changes in the diversity of plant populations and the populations of their phytopathogens over time (Box 3) [59–61]. They can also provide data about isolates that are scarce in nature. Sutton and Marasas (1976) described a fungal species Tiarosporella tritici found on wheat-straw in South Africa [62]. This is a species within the Botryosphaeriaceae family whose members include several significant phytopathogens [63]. To the best of our knowledge, this species has not since been documented from nature. Recently, however, T. tritici genes from herbarium samples were sequenced [64,65], allowing phylogenetic comparison to other members of the Botryosphaeriaceae family, thereby increasing our knowledge of this phytopathogenic family [64–67]. Genetic sampling of environmental sources, such as water or soil, can also be useful for determining the presence or spread of potential phytopathogens [41,42,68]. Likewise, sampling of microorganisms from healthy plant tissue reveals an alternative natural reservoir of novel phytopathogens. Greater interest in seemingly innocuous endophytes for natural products [69] and taxonomy/biodiversity [70,71] has highlighted the risk of spread of these essentially invisible organisms to naı¨ve host populations [63]. Under some conditions endophytes can resort to disease [72,73]. A study functionally demonstrated this shift with Diplodia mutila which is usually an endophyte of the palm, Iriartea deltoidea, but can become a pathogen in seedlings when exposed to increased light intensity [72]. Endophytes and potential environmental reservoirs represent an unseen risk and potential source of inoculum for spread and persistence of phytopathogens into new environments [63]. As an example, seemingly innocuous endophytes may themselves not cause disease but still pose an indirect threat of injecting new genetic diversity into allopatric populations. Accordingly, genetic sampling from a wide range of sources increases our knowledge of the biodiversity that is present, providing useful data for quickly identifying and preventing phytopathogen spread. Concluding remarks A review in 2011 summarised the findings of the comparative genome studies of phytopathogenic Xanthomonas spp. available at the time [74]. The authors stress the importance of continued genome sequencing for understanding the adaptations of microorganisms with the statement – ‘The power of comparative genomics for defining the adaptations that cause host and tissue specificity . . . is proportional to the number of genome sequences available’. We strongly believe in the validity of this statement, with the caveat that the power of this tool is limited by the question posed. There is simply too great of a volume of data contained within a genome to be able to reach any reasonable conclusions without a specific and targeted question. However, the deposition of these genome sequences populates public databases with valuable data that will enable other researchers with specific questions to utilise these databases for their own studies. 7

TRPLSC-1256; No. of Pages 10

Review The genomics era has enabled researchers to begin to appreciate and understand the range of genomic events that lead to the emergence of a microorganism as a phytopathogen. As more genomes are sequenced, this appreciation and understanding will continue to grow. However, perhaps the most exciting outcome of the genomics era is the ability to share and, in turn, exploit gene and genome sequences from across the planet. For the first time researchers are able to perform research as globalised as the phytopathogens they study. Origins of emergence can be predicted, and the integration of seemingly disparate data provides greater scope to the study of phytopathogens, for example, enabling observation of interkingdom gene transfers or collapsing the nomenclature of the same phytopathogens that are geographically separated. Future work should strive to unify these disparities in nomenclature, identify environmental sinks of potential phytopathogens, integrate genome-based diagnostic tools, and, importantly, be made publicly-available to push forward the greater communities understanding of the breadth of shared DNA sequences. Acknowledgements This study was supported by the Grains Research and Development Corporation (GRDC) (ANU00019). P.S.S is an Australia Research Council Future Fellow and E.T is a recipient of an Australian Postgraduate Award and a GRDC Grains Industry Research Scholarship. M.C.M acknowledges support by the Swiss National Science Foundation. We would like to thank our colleague Mariano Jordi Muria Gonzalez for his artistic contribution of the diseased leaf in Figure 1.

References 1 Anderson, P.K. et al. (2004) Emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers. Trends Ecol. Evol. 19, 535–544 2 Fisher, M.C. et al. (2012) Emerging fungal threats to animal, plant and ecosystem health. Nature 484, 186–194 3 Dodds, P.N. and Rathjen, J.P. (2010) Plant immunity: towards an integrated view of plant–pathogen interactions. Nat. Rev. Genet. 11, 539–548 4 Mayer, K.F. et al. (2014) A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science 345, 1251788 5 Keeling, P.J. and Palmer, J.D. (2008) Horizontal gene transfer in eukaryotic evolution. Nat. Rev. Genet. 9, 605–618 6 Raffaele, S. and Kamoun, S. (2012) Genome evolution in filamentous plant pathogens: why bigger can be better. Nat. Rev. Microbiol. 10, 417–430 7 Ochman, H. et al. (2000) Lateral gene transfer and the nature of bacterial innovation. Nature 405, 299–304 8 Andersson, J.O. (2005) Lateral gene transfer in eukaryotes. Cell. Mol. Life Sci. 62, 1182–1197 9 Kirsch, R. et al. (2014) Horizontal gene transfer and functional diversification of plant cell wall degrading polygalacturonases: key events in the evolution of herbivory in beetles. Insect Biochem. Mol. Biol. 52, 33–50 10 Slot, J.C. and Rokas, A. (2011) Horizontal transfer of a large and highly toxic secondary metabolic gene cluster between fungi. Curr. Biol. 21, 134–139 11 Yin, L-F. et al. (2014) Evolutionary analysis revealed the horizontal transfer of the Cyt b gene from Fungi to Chromista. Mol. Phylogenet. Evol. 76, 155–161 12 Gardiner, D.M. et al. (2013) Cross-kingdom gene transfer facilitates the evolution of virulence in fungal pathogens. Plant Sci. 210, 151–158 13 Gardiner, D.M. et al. (2014) Genomic analysis of Xanthomonas translucens pathogenic on wheat and barley reveals cross-kingdom gene transfer events and diverse protein delivery systems. PLoS ONE 9, e84995 8

Trends in Plant Science xxx xxxx, Vol. xxx, No. x

14 Jaramillo, V.D. et al. (2015) Identification of horizontally transferred genes in the genus Colletotrichum reveals a steady tempo of bacterial to fungal gene transfer. BMC Genomics 16, 2 15 Jaramillo, V.D.A. et al. (2013) Horizontal transfer of a subtilisin gene from plants into an ancestor of the plant pathogenic fungal genus Colletotrichum. PLoS ONE 8, e59078 16 Klosterman, S.J. et al. (2011) Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens. PLoS Pathog. 7, e1002137 17 Lawrence, D.P. et al. (2011) Interkingdom gene transfer of a hybrid NPS/PKS from bacteria to filamentous Ascomycota. PLoS ONE 6, e28231 18 Nikolaidis, N. et al. (2014) Plant expansins in bacteria and fungi: evolution by horizontal gene transfer and independent domain fusion. Mol. Biol. Evol. 31, 376–386 19 Richards, T.A. et al. (2011) Horizontal gene transfer facilitated the evolution of plant parasitic mechanisms in the oomycetes. Proc. Natl. Acad. Sci. U.S.A. 108, 15258–15263 20 Sun, B-F. et al. (2013) Multiple interkingdom horizontal gene transfers in Pyrenophora and closely related species and their contributions to phytopathogenic lifestyles. PLoS ONE 8, e60029 21 de Jonge, R. et al. (2012) Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing. Proc. Natl. Acad. Sci. U.S.A. 109, 5110–5115 22 Richards, T.A. et al. (2009) Phylogenomic analysis demonstrates a pattern of rare and ancient horizontal gene transfer between plants and fungi. Plant Cell Online 21, 1897–1911 23 Hacker, J. and Kaper, J.B. (2000) Pathogenicity islands and the evolution of microbes. Annu. Rev. Microbiol. 54, 641–679 24 Juhas, M. et al. (2009) Genomic islands: tools of bacterial horizontal gene transfer and evolution. FEMS Microbiol. Rev. 33, 376–393 25 Huguet-Tapia, J.C. and Loria, R. (2012) Draft genome sequence of Streptomyces acidiscabies 84–104, an emergent plant pathogen. J. Bacteriol. 194, 1847 26 Mann, R.A. et al. (2012) Comparative analysis of the Hrp pathogenicity island of Rubus-and Spiraeoideae-infecting Erwinia amylovora strains identifies the IT region as a remnant of an integrative conjugative element. Gene 504, 6–12 27 Kerff, F. et al. (2008) Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization. Proc. Natl. Acad. Sci. U.S.A. 105, 16876–16881 28 Cosgrove, D.J. (2000) Loosening of plant cell walls by expansins. Nature 407, 321–326 29 Hu, J. et al. (2012) Genomic characterization of the conditionally dispensable chromosome in Alternaria arborescens provides evidence for horizontal gene transfer. BMC Genomics 13, 171 30 Ma, L-J. et al. (2010) Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature 464, 367–373 31 Friesen, T.L. et al. (2006) Emergence of a new disease as a result of interspecific virulence gene transfer. Nat. Genet. 38, 953–956 32 Gardiner, D.M. et al. (2012) Comparative pathogenomics reveals horizontally acquired novel virulence genes in fungi infecting cereal hosts. PLoS Pathog. 8, e1002952 33 Giraud, T. et al. (2008) Speciation in fungi. Fungal Genet. Biol. 45, 791–802 34 Stukenbrock, E.H. et al. (2007) Origin and domestication of the fungal wheat pathogen Mycosphaerella graminicola via sympatric speciation. Mol. Biol. Evol. 24, 398–411 35 Stukenbrock, E.H. et al. (2012) Fusion of two divergent fungal individuals led to the recent emergence of a unique widespread pathogen species. Proc. Natl. Acad. Sci. U.S.A. 109, 10954–10959 36 Inderbitzin, P. et al. (2011) The ascomycete Verticillium longisporum is a hybrid and a plant pathogen with an expanded host range. PLoS ONE 6, e18260 37 Karapapa, V. et al. (1997) Morphological and molecular characterization of Verticillium longisporum comb. nov. pathogenic to oilseed rape. Mycol. Res. 101, 1281–1294 38 Collado-Romero, M. et al. (2010) DNA sequence analysis of conserved genes reveals hybridization events that increase genetic diversity in Verticillium dahliae. Fungal Biol. 114, 209–218 39 Bertier, L. et al. (2013) Host adaptation and speciation through hybridization and polyploidy in Phytophthora. PLoS ONE 8, e85385

TRPLSC-1256; No. of Pages 10

Review 40 Goss, E.M. et al. (2011) The plant pathogen Phytophthora andina emerged via hybridization of an unknown Phytophthora species and the Irish potato famine pathogen, P. Infestans. PLoS ONE 6, e24543 41 Hu¨berli, D. et al. (2013) Fishing for Phytophthora from Western Australia’s waterways: a distribution and diversity survey. Aust. Plant Pathol. 42, 251–260 42 Nagel, J.H. et al. (2013) Characterization of Phytophthora hybrids from ITS clade 6 associated with riparian ecosystems in South Africa and Australia. Fungal Biol. 117, 329–347 43 Nechwatal, J. and Lebecka, R. (2014) Genetic and phenotypic analyses of Pythium isolates from reed suggest the occurrence of a new species, P. phragmiticola, and its involvement in the generation of a natural hybrid. Mycoscience 55, 134–143 44 Oh, E. et al. (2013) Surveys of soil and water reveal a goldmine of Phytophthora diversity in south African natural ecosystems. IMA Fungus 4, 123 45 Oliver, R.P. and Solomon, P.S. (2008) Recent fungal diseases of crop plants: is lateral gene transfer a common theme? Mol. Plant Microbe Interact. 21, 287–293 46 Vernikos, G. et al. (2015) Ten years of pan-genome analyses. Curr. Opin. Microbiol. 23, 148–154 47 Adhikari, B.N. et al. (2013) Comparative genomics reveals insight into virulence strategies of plant pathogenic oomycetes. PLoS ONE 8, e75072 48 Ohm, R.A. et al. (2012) Diverse lifestyles and strategies of plant pathogenesis encoded in the genomes of eighteen Dothideomycetes fungi. PLoS Pathog. 8, e1003037 49 Baltrus, D.A. et al. (2011) Dynamic evolution of pathogenicity revealed by sequencing and comparative genomics of 19 Pseudomonas syringae isolates. PLoS Pathog. 7, e1002132 50 de Jonge, R. et al. (2013) Extensive chromosomal reshuffling drives evolution of virulence in an asexual pathogen. Genome Res. 23, 1271–1282 51 Jalan, N. et al. (2011) Comparative genomic analysis of Xanthomonas axonopodis pv. citrumelo F1, which causes citrus bacterial spot disease, and related strains provides insights into virulence and host specificity. J. Bacteriol. 193, 6342–6357 52 Manning, V.A. et al. (2013) Comparative genomics of a plant-pathogenic fungus, Pyrenophora tritici-repentis, reveals transduplication and the impact of repeat elements on pathogenicity and population divergence. G3: Genes Genomes Genet. 3, 41–63 53 Yoshida, K. et al. (2009) Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae. Plant Cell Online 21, 1573–1591 54 Syme, R.A. et al. (2013) Resequencing and comparative genomics of Stagonospora nodorum: sectional gene absence and effector discovery. G3: Genes Genomes Genet. 3, 959–969 55 Benson, D.A. et al. (2012) GenBank. Nucleic Acids Res. 40, D48–D53 56 Benson, D.A. et al. (2006) GenBank. Nucleic Acids Res. 34, D16–D20 57 Goss, E.M. et al. (2014) The Irish potato famine pathogen Phytophthora infestans originated in central Mexico rather than the Andes. Proc. Natl. Acad. Sci. U.S.A. 201401884 58 McDonald, M.C. et al. (2012) Phylogenetic and population genetic analyses of Phaeosphaeria nodorum and its close relatives indicate cryptic species and an origin in the Fertile Crescent. Fungal Genet. Biol. 49, 882–895 59 Ames, M. and Spooner, D.M. (2008) DNA from herbarium specimens settles a controversy about origins of the European potato. Am. J. Bot. 95, 252–257 60 Yoshida, K. et al. (2014) Mining herbaria for plant pathogen genomes: back to the future. PLoS Pathog. 10, e1004028 61 Yoshida, K. et al. (2013) The rise and fall of the Phytophthora infestans lineage that triggered the Irish potato famine. Elife 2, e00731 62 Sutton, B.C. and Marasas, W.F.O. (1976) Observations on Neottiosporina and Tiarosporella. Trans. Br. Mycol. Soc. 67, 69–76 63 Slippers, B. and Wingfield, M.J. (2007) Botryosphaeriaceae as endophytes and latent pathogens of woody plants: diversity, ecology and impact. Fungal Biol. Rev. 21, 90–106 64 Crous, P.W. et al. (2006) Phylogenetic lineages in the Botryosphaeriaceae. Stud. Mycol. 55, 235–253 65 Phillips, A. et al. (2013) The Botryosphaeriaceae: genera and species known from culture. Stud. Mycol. 76, 51–167 66 Jami, F. et al. (2012) Five new species of the Botryosphaeriaceae from Acacia Karroo in South Africa. Cryptogamie Mycol. 33, 245–266

Trends in Plant Science xxx xxxx, Vol. xxx, No. x

67 Jami, F. et al. (2014) Botryosphaeriaceae species overlap on four unrelated, native South African hosts. Fungal Biol. 118, 168–179 68 Monteil, C.L. et al. (2013) Nonagricultural reservoirs contribute to emergence and evolution of Pseudomonas syringae crop pathogens. New Phytol. 199, 800–811 69 Aly, A.H. et al. (2010) Fungal endophytes from higher plants: a prolific source of phytochemicals and other bioactive natural products. Fungal Divers. 41, 1–16 70 Jami, F. et al. (2013) Greater Botryosphaeriaceae diversity in healthy than associated diseased Acacia karroo tree tissues. Aust. Plant Pathol. 42, 421–430 71 Slippers, B. et al. (2013) Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework. Stud. Mycol. 76, 31–49 72 Alvarez-Loayza, P. et al. (2011) Light converts endosymbiotic fungus to pathogen, influencing seedling survival and niche-space filling of a common tropical tree, Iriartea deltoidea. PLoS ONE 6, e16386 73 Delaye, L. et al. (2013) Endophytes versus biotrophic and necrotrophic pathogens – are fungal lifestyles evolutionarily stable traits? Fungal Divers. 60, 125–135 74 Ryan, R.P. et al. (2011) Pathogenomics of Xanthomonas: understanding bacterium–plant interactions. Nat. Rev. Microbiol. 9, 344–355 75 Scortichini, M. et al. (2012) Pseudomonas syringae pv. actinidiae: a reemerging, multi-faceted, pandemic pathogen. Mol. Plant Pathol. 13, 631–640 76 Chapman, J. et al. (2012) Phylogenetic relationships among global populations of Pseudomonas syringae pv. actinidiae. Phytopathology 102, 1034–1044 77 Marcelletti, S. et al. (2011) Pseudomonas syringae pv. actinidiae draft genomes comparison reveal strain-specific features involved in adaptation and virulence to Actinidia species. PLoS ONE 6, e27297 78 McCann, H.C. et al. (2013) Genomic analysis of the kiwifruit pathogen Pseudomonas syringae pv. actinidiae provides insight into the origins of an emergent plant disease. PLoS Pathog. 9, e1003503 79 Morris, C. et al. (2010) Inferring the evolutionary history of the plant pathogen Pseudomonas syringae from its biogeography in headwaters of rivers in North America, Europe, and New Zealand. MBio 1, e00107–e00110 80 Morris, C.E. et al. (2008) The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle. ISME J. 2, 321–334 81 Dean, R. et al. (2012) The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 13, 414–430 82 Dean, R.A. et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434, 980–986 83 Valent, B. and Khang, C.H. (2010) Recent advances in rice blast effector research. Curr. Opin. Plant Biol. 13, 434–441 84 Kohli, M. et al. (2011) Pyricularia blast – a threat to wheat cultivation. Czech J. Genet. Plant Breed. 47, S130–S134 85 Van Vu, B. et al. (2012) Cellulases belonging to glycoside hydrolase families 6 and 7 contribute to the virulence of Magnaporthe oryzae. Mol. Plant Microbe Interact. 25, 1135–1141 86 Maciel, J.L.N. et al. (2014) Population structure and pathotype diversity of the wheat blast pathogen Magnaporthe oryzae 25 years after its emergence in Brazil. Phytopathology 104, 95–107 87 Pratt, K. (2012) UK researchers find important new disease. UKAgNews 24 April 88 Tosa, Y. et al. (2004) Genetic constitution and pathogenicity of Lolium isolates of Magnaporthe oryzae in comparison with host speciesspecific pathotypes of the blast fungus. Phytopathology 94, 454–462 89 Gross, A. et al. (2014) Hymenoscyphus pseudoalbidus, the causal agent of European ash dieback. Mol. Plant Pathol. 15, 5–21 90 Queloz, V. et al. (2011) Cryptic speciation in Hymenoscyphus albidus. For. Pathol. 41, 133–142 91 Chandelier, A. et al. (2011) First report of the ash dieback pathogen Hymenoscyphus pseudoalbidus (Anamorph Chalara fraxinea) on Fraxinus excelsior in Belgium. Plant Dis. 95, 220 92 Husson, C. et al. (2011) Chalara fraxinea is an invasive pathogen in France. Eur. J. Plant Pathol. 130, 311–324 93 McKinney, L.V. et al. (2012) Rapid invasion by an aggressive pathogenic fungus Hymenoscyphus pseudoalbidus replaces a native decomposer Hymenoscyphus albidus: a case of local cryptic extinction? Fungal Ecol. 5, 663–669 9

TRPLSC-1256; No. of Pages 10

Review 94 Zhao, Y-J. et al. (2013) Hymenoscyphus pseudoalbidus, the correct name for Lambertella albida reported from Japan. Mycotaxon 122, 25–41 95 Gross, A. et al. (2014) Population structure of the invasive forest pathogen Hymenoscyphus pseudoalbidus. Mol. Ecol. 23, 2943–2960 96 Zheng, H-D. and Zhuang, W-Y. (2013) Hymenoscyphus albidoides sp. nov. and H. pseudoalbidus from China. Mycol. Prog. 1–14 97 Gherghel, F. et al. (2014) The development of a species-specific test to detect Hymenoschyphus pseudoalbidus in ash tissues. For. Pathol. 44, 137–144 98 Gross, A. et al. (2012) A molecular toolkit for population genetic investigations of the ash dieback pathogen Hymenoscyphus pseudoalbidus. For. Pathol. 42, 252–264 99 Gabriela Roca, M. et al. (2005) Conidial anastomosis tubes in filamentous fungi. FEMS Microbiol. Lett. 249, 191–198

10

Trends in Plant Science xxx xxxx, Vol. xxx, No. x

100 Richards, T.A. et al. (2011) Gene transfer into the fungi. Fungal Biol. Rev. 25, 98–110 101 Ishikawa, F.H. et al. (2012) Heterokaryon incompatibility is suppressed following conidial anastomosis tube fusion in a fungal plant pathogen. PLoS ONE 7, e31175 102 Medini, D. et al. (2005) The microbial pan-genome. Curr. Opin. Genet. Dev. 15, 589–594 103 Tettelin, H. et al. (2005) Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial ‘pan-genome’. Proc. Natl. Acad. Sci. U.S.A. 102, 13950–13955 104 Nguyen, N. et al. (2015) Building a pan-genome reference for a population. J. Comput. Biol. 22, 1–15 105 Bankevich, A. et al. (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477