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Somatic Hybridization in the Uredinales


Robert F. Park1 and Colin R. Wellings1,2
1 Plant Breeding Institute, The University of Sydney, Sydney, New South Wales 2570, Australia; email: robert.park@sydney.edu.au 2 New South Wales Department of Primary Industries, Orange, New South Wales 2800, Australia; email: colin.wellings@sydney.edu.au

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Annu. Rev. Phytopathol. 2012. 50:21939 The Annual Review of Phytopathology is online at phyto.annualreviews.org This articles doi: 10.1146/annurev-phyto-072910-095405 Copyright c 2012 by Annual Reviews. All rights reserved 0066-4286/12/0908-0219$20.00

Keywords
durable resistance, heterokaryosis, Melampsora, pathotype, Puccinia, rust, Ug99

Abstract
Rust fungi are cosmopolitan in distribution and parasitize a wide range of plants, including economically important crop species such as wheat. Detailed regional, national, and continental surveys of pathogenic variability in wheat-attacking rust pathogens over periods of up to 90 years have shown that in the absence of sexual recombination, genetic diversity is generated by periodic introduction of exotic isolates, single-step mutation, and somatic hybridization. Laboratory studies have provided evidence for somatic hybridization between many rust species and formae speciales, and there is evidence for the process in nature within and between rust species on Linum, poplar, Senecio, wheat, and several grass species. Although the mechanisms involved in somatic hybridization are not well understood, they are thought to involve the fusion of dikaryotic vegetative hyphae, nuclear exchange, and possibly exchange of whole chromosomes between nuclei or parasexuality via the fusion of the two haploid nuclei, followed by mitotic crossing over and vegetative haploidization. In three cases, hybrid isolates rendered resistant plant genotypes susceptible because of new combinations of virulence. Implications for resistance breeding and future prospects in understanding the process are discussed.

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INTRODUCTION
Rust: the disease that results from the interaction between a rust fungus and a host plant; referring to the fungus only as rust is incorrect Common host: a plant species infected by more than one rust species or more than one formae speciales within a species

The Uredinales (phylum Basidiomycota, class Teliomycetes, order Uredinales), commonly known as the rust fungi, are plant pathogens that are typied by the orange-brown-red rust color of one or more spore stages. Destructive rust epidemics in cereal crops have impacted on the development of human civilization, with early accounts of rust epidemics coming from the Bible and from Greek and Roman literatures (60). Rust fungi are cosmopolitan in distribution and parasitize a wide range of plants, including ferns and conifers, and most families of dicotyledon and monocotyledon angiosperms. They are of great biological interest because of their obligate parasitism in nature and often complex life cycles, which can involve two phylogenetically distant hosts. Many are serious pathogens of important plant species and hence are of great economic importance. This is particularly so for the three rust species that infect common wheat: Puccinia graminis f. sp. tritici [Pgt; causal agent of stem (black) rust], Puccinia striiformis f. sp. tritici [Pst; causal agent of stripe (yellow) rust], and Puccinia triticina [causal agent of leaf (brown) rust]. Estimates of losses in wheat due to stem rust in Australia, for example, include AU23 million in 1889 (56), AU2 million in 1916 (97), AU7 million in 1947 (16), and AUD$200300 million in 1973 (104). In the United States, losses to stem rust in 1904 were estimated at USD$10 million and in 1916 at USD$181 million (17). Murray & Brennan (62) estimated average annual losses to the Australian wheat industry due to Pst at AUD$127 million and further estimated losses of AUD$994 million if current control measures were not used. The considerable success in breeding new wheat cultivars with genetic resistance to these pathogens owes a great deal to ongoing surveys and studies of rust pathogen virulence dynamics. This success has been greatest when pathogen surveys have been closely aligned with prebreeding and breeding efforts targeting the incorporation of genetic resistance.

The role of sexual recombination in generating genetic variability in rust fungi is well documented. According to Roelfs (81) and Peterson et al. (76), the eradication of barberry in the United States from 1918 to 1980, on which Pgt undergoes sexual recombination, had a large impact on reducing genetic diversity and increasing the stability of races of Pgt collected from wheat in the Great Plains. Intraspecic and interspecic hybridization events are now recognized as important contributors to variation, with molecular phylogenetic analyses providing evidence of this phenomenon in a range of fungi (85). Two examples of interspecic hybridization in the Uredinales involve rust pathogens of trees. In a study of white pine blister rust caused by Cronartium ribicola, Joly et al. (39) genotyped aecial isolates collected from three widely separated stands of Pinus exilis using 12 codominant polymerase chain reaction based markers. Tests revealed heterozygosity and novel alleles at all loci in up to 29% of the isolates from each site over two years. Subsequent studies established that this was due to the presence of one allele from C. ribicola and one from Cronartium comandre, the comandra blister rust pathogen, which infects Pinus contorta. Aeciospore morphology of these isolates was intermediate between C. ribicola and C. comandre, supporting the hypothesis that they arose from hybridization between the two rust pathogens on the common host P. exilis. Host range and virulence studies were not conducted (39). Newcombe et al. (66) described Melampsora X columbiana, believed to be a hybrid between the poplar-infecting rust pathogens Melampsora medusae and Melampsora occidentalis. The former species was described originally from Populus deltoides and forms its aecial state on eastern larch (Larix laricina), whereas the latter was described originally from Populus trichocarpa and alternates on Douglas r (Pseudotsuga menziesii ). Uredinia and telia of M. X columbiana were intermediate in morphology between M. medusae and M. occidentalis, and isolates often contained internal transcribed spacer (ITS) sequences from both parental species. A particular concern was the observation that the hybrid

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rust appeared to have a broader host range than either M. medusae or M. occidentalis (65). In addition to sexual recombination, long-term surveys of pathogenicity, especially in the cereal rust pathogens, have shown the importance of mutation as a source of variation. The detection of Pgt races virulent for the stem rustresistant wheat cultivars Eureka (carrying resistance gene Sr6 ), Gabo (Sr11), Spica (Sr17), and Festival (Sr9b) soon after release in Australia was attributed to independent mutational changes that resulted in the pathogen acquiring virulence for each resistance gene (99, 102). Although the precise molecular basis of mutation to virulence in Pgt remains unknown, it is generally accepted that it does occur in nature and that it is often responsible for the boom and bust cycle in which rust-resistant cultivars increase in popularity (boom), only to succumb to a new virulent pathogen genotype (bust) (58). Evidence for the asexual exchange of genetic material in rust fungi was obtained soon after the detailed studies of parasexual recombination in Aspergillus nidulans by Pontecorvo and Roper in 1952 (77). This process is often referred to as somatic hybridization, and in the rust fungi it is thought to involve the fusion of dikaryotic vegetative hyphae, nuclear exchange, and possibly exchange of whole chromosomes between nuclei. Alternatively, parasexuality via the fusion of the two haploid nuclei, followed by mitotic crossing over and vegetative haploidization of the diploid nuclei, may also occur. This review details previous studies of rust fungi in which somatic hybridization has been either demonstrated or implicated under controlled laboratory conditions and in nature. It discusses the potential importance of the process in gene transfer within and between rust taxa and implications for resistance breeding strategies. The utility of new marker platforms and genome sequencing in studying somatic hybridization in the rust fungi is also discussed.

SPECIES, FORMAE SPECIALES, AND PATHOTYPES


Approximately 7,000 rust species are currently accepted as valid taxa, most of which belong

to 12 of the (approximately) 164 accepted genera. There are also 139 generic names that are regarded as either synonyms or of uncertain status. The largest genera are Puccinia and Uromyces, both within the family Pucciniaceae, which account for 3,0004,000 and 600700 species, respectively. Critical taxonomic assessments of these two genera, including studies based on DNA sequencing, suggest strongly that they are not monophyletic (53, 94). Within species of rust fungi, it is common to nd variants that, although morphologically indistinguishable, are adapted to parasitizing different host species. Jakob Eriksson, a Swedish plant pathologist, was the rst to coin the term forma specialis (f. sp.) to designate these variants (3). The forma specialis is an informal rank that is not regulated by The International Code of Botanical Nomenclature and is usually named according to the host with which the dikaryon is most commonly associated. Three of the many f. spp. that have been characterized in the stem rust pathogen P. graminis are: Pgt (on wheat, Triticum aestivum), P. graminis f. sp. avenae (Pga; on oats, Avena sativa), and P. graminis f. sp. secalis (Pgs; on cereal rye, Secale cereale). Despite the host specicity displayed by these f. spp., there are some host species, or genotypes of host species, that are susceptible to two or more f. spp., and these are referred to as common hosts. For example, some genotypes of wheat are common hosts for Pgt and Pgs. In studies using one of these common host genotypes to investigate the genetic basis of resistance in wheat to Pgs, several wheat cultivars that possessed no or few effective genes for resistance to Australian isolates of Pgt were found to carry one [cultivar (cv.) Yalta], two (cv. Mona), three (cv. Pusa), or four (cv. Mentana) genes for resistance to a standard eld-collected isolate of Pgs (83, 84). In P. graminis, the monokaryons of related f. spp. generally share the same alternate host. The alternate host of Pgt, Pgs, and Pga is Berberis vulgaris, and although viable sexual crosses have been made between some of these f. spp., progeny in general have a lower range of virulence than the parental isolates and often grow
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Pathogenicity: the pathogen character determined by the interaction with a host, the contrasting phenotypes of which are avirulence and virulence Alternate host: one or the other of the different hosts infected by heteroecious rust fungi

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Physiologic race (pathotype, strain): a group of isolates that share common pathogenic attributes, which may or may not be genetically identical

poorly. Attempts to infect barberry with basidiospores derived from an F1 hybrid between Pgt and Pga resulted in few pycnia, which did not produce nectar, and attempts to produce aecia failed (37). Interestingly, urediniospores and teliospores were occasionally observed in old pycnial infections, indicating severe disruption of the genetic control of the normal life cycle of P. graminis in the Pgt X Pga hybrid. The same disruption was also observed in the F1 of a cross between Pgs and Pga. Johnson (37) also found that in successful crosses between what were considered closely related f. spp., the tness of the F1 progeny was lower than that of the parent on a given host. It was concluded that sterility and reduced tness in hybrids limits the significance of hybrids between f. spp. of P. graminis in nature. The existence of physiologic races within a forma specialis or species was rst reported by Stakman & Piemeisel (91) in Pgt. They dened races by their ability (virulence) or inability (avirulence) to infect 12 wheat genotypes, known as differentials because of their abilities to differentiate between the races. This discovery, along with that of Mendelian inheritance of stripe rust resistance in the durum wheat cultivar Rivet by Bifn (10) and of Mendelian inheritance of pathogenicity to specic cultivars in Pgt by Newton et al. (69), formed the foundation upon which the gene-for-gene hypothesis was formulated (27). Races (pathotypes) of cereal rust pathogens are now identied usually on the basis of virulence/avirulence on specic resistance genes rather than differential stocks that may carry several resistance genes. On this basis, the gene-for-gene hypothesis allows the prediction of pathogen genotype from a compatible (virulent) or incompatible (avirulent) phenotype.

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of urediniospores to airborne dispersal, has imposed considerable technical demands on studies of somatic hybridization under laboratory conditions. Of these, contamination between isolates is potentially a major source of experimental error (9). Although most of these studies have used a host genotype that is susceptible to the rust isolates under examination, at least one has used a host genotype resistant to one of the isolates under investigation (63). Studies of somatic hybridization under controlled conditions have targeted cereal rust pathogens, often using virulence for known resistance genes and/or urediniospore color as markers to identify and select putative hybrid genotypes. Mutations in urediniospore color are often detected in greenhouse studies with rust pathogens (107), and urediniospore color mutants, usually orange or yellow in contrast to the wild-type, red-colored urediniospores, have been found for many of the cereal rust fungi. In Pgt, urediniospore color is determined by two independent factors, one for spore wall color and one for cytoplasm color, each inherited in a 3:1 ratio (69). Urediniospores appear a normal red-brown when both factors are present in the dominant condition and colorless when both factors are homozygous recessive. When the factor for spore wall color is dominant and the factor for color of cytoplasm is homozygous recessive, urediniospores appear gray-brown. Urediniospores are orange when the factor for cytoplasm color is dominant and the factor for spore wall color is homozygous recessive.

Melampsora lini
Laboratory studies of somatic variation in Melampsora lini were based on isolates recovered from mixtures of four F1 hybrids derived from crossing races 22 and 1 (28, 29). Although race 22 was virulent on 14 of 16 differentials used to characterize races of M. lini, race 1 was virulent on only one (Bison). Unlike most other studies of somatic hybridization under controlled conditions, the genotypes of the four F1 hybrids used with respect to pathogenicity

STUDIES OF SOMATIC HYBRIDIZATION UNDER CONTROLLED CONDITIONS


The need to grow rust fungi on living plants because of their slow and fastidious growth in axenic culture, combined with the adaptation
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for each of the 16 differentials were known. Previous knowledge of the inheritance of avirulence for each differential coupled with genetic studies indicated that race 22 (and therefore the haploid nucleus contributed to the F1 by this race) was homozygous for virulence on all 16 differential genotypes. In contrast, race 1 (and therefore the haploid nucleus contributed to the F1 by this race) was heterozygous for pathogenicity on four differentials. The four F1 hybrids were mixed and used to coinfect Bison, susceptible to both parents, and the resulting inoculum screened on four host genotypes susceptible to race 22 and resistant to race 1 and the F1 hybrids. In the rst study (28), a total of 129 isolates virulent for one of the four host genotypes were recovered, 118 of which were typed as race 22. Asexual nuclear exchange of race 22 + and mating-type nuclei between F1 hybrids was proposed as the simplest explanation for this result. All 11 nonrace 22 variants isolated differed from the F1 hybrids with the addition of a single virulence, and it was considered that these most likely arose from single-step mutation. Similar results were obtained in the second study, and no evidence was obtained to suggest cytoplasmic inuences, heterokaryosis, or parasexual processes were involved.

were isolated. The authors concluded that although the former two races may have resulted from nuclear exchange following hyphal anastamosis, this could not explain the latter nine races. It was suggested that these may have arisen as a result of a multinucleate condition in the parental isolate, or alternatively if the parental isolate was dikaryotic, then somatic recombination following nuclear fusion and mitotic crossing over may have occurred.

Heterokaryosis: the capacity of haploid nuclei to form various associations within vegetative cells

Puccinia graminis
Early published studies of somatic hybridization in rust fungi under controlled conditions involved mixing races of the wheat stem rust pathogen Pgt. Nelson et al. (64) studied mixtures of races 38 and 56 and recovered a variant that was fully virulent on Khapli Emmer, a feature not seen previously among North American isolates of Pgt. Subculturing of this isolate over 25 uredinial generations was accompanied by a gradual loss of virulence on Khapli, and further characterization led to the identication of two new races that showed pathogenic features intermediate between the parental races 38 and 56. Urediniospores and hyphal cells with three to four nuclei were identied, and it was suggested that the instability and subsequent generation of new races observed may have resulted from dissociation of these nuclei. Later studies involving mixtures of red-brown race 11 (avirulent on Vernal Emmer) and gray-brown race 121 (virulent on Vernal Emmer) demonstrated that factors controlling urediniospore color could also be transmitted via somatic hybridization (63). Several orange-spored isolates virulent on Vernal Emmer were recovered, and although some of these were stable, many reverted back to the parental color. Cytological studies revealed the presence of trinucleate urediniospores at a low frequency (less than 1%) in all orange isolates, and the reversion to parental color was again interpreted as trinucleate isolates reverting to a dikaryotic state via dissociation of nuclei. These studies also demonstrated that it was possible to recover recombinant isolates
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Puccinia coronata f. sp. avenae


Crown rust, caused by Puccinia coronata f. sp. avenae (Pca), is the most widespread and damaging disease of oat (87). Surveys of Pca have shown high levels of pathogenic variation in both the presence (e.g., in the United States; 87) and absence (e.g., in Australia; R.F. Park, unpublished results) of the alternate host Rhamnus. Barto s et al. (9) made four mixtures of two Canadian races of Pca, 228 and 393, from which they recovered isolates that differed in pathogenicity from the two parental races. Two nonparental races were recovered, one of which (race 214) had not been detected previously in Canada. In additional studies in which single spore cultures of two races (393 and 229) were established, a further nine nonparental races

from inoculating a host genotype that was not susceptible to both parental isolates. Watson (101) and Watson & Luig (105) undertook studies of mixtures of races NR-1 and 111. Race NR-1 had orange urediniospores and was considered to be homozygous for many recessive alleles for virulence, and race 111 had red urediniospores and was considered to have several contrasting heterozygous genes. Both races were avirulent on Acme, a trait which at that time was uncommon in North America. In both studies, isolates virulent on Arnautka, Mindum, and Spelmar, all now known to carry Sr9d (60), were found. These results were difcult to explain because previous studies (38) had established that virulence for Sr9d was dominant, indicating that both parents should have been homozygous avirulent for this gene. Selfing of race 111 on barberry by Watson & Luig (105), however, resulted in progeny both virulent and avirulent on the three Sr9d genotypes, and so the occurrence of virulent progeny was suggested to be due to segregation of genes for pathogenicity other than those reported by Johnson (37). In addition, 11 putative hybrid isolates were identied in the later study (105), indicating that processes beyond simple nuclear exchange were involved in generating somatic hybrid isolates. Similar ndings were reported by Bridgmon (11), who isolated at least 15 races from mixtures of three parental races, and Ellingboe (24), who used six mixtures of two races, two of which yielded one and two races. The latter could be attributed to simple nuclear exchange. A further four of these two race mixtures yielded between four and 15 hybrid races, indicating more complex processes. Among the 15 recombinant isolates identied by Bridgmon (11), two were of particular interest because they were avirulent for Einkorn, despite both parental isolates being virulent. This result was inconsistent with what would be expected from a sexual intercross between isolates virulent for Einkorn, because virulence for this cultivar was shown in previous studies to be recessive (38). According to Watson (101), dikaryons most likely to exchange nuclei would appear to be those arising from wide crosses such as
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intervarietal (interf. spp.) crosses of P. graminis. Interestingly, Watson (103) concluded that the isolate of race 111 used in earlier experiments most likely arose as a sexual hybrid derived from an intercross between Pgt and Pgs. Watson & Luig (106) mixed a red-spored Australian isolate of Pgs with an orange-spored NR-2 of Pgt used in earlier studies (101, 105) and isolated two hybrid isolates that were intermediate in their pathogenicity between the two parental isolates. Of particular interest was one isolate that was virulent on the wheat cultivar Yalta (Sr11), but avirulent on the highly susceptible cultivar Morocco. In subsequent studies, Watson & Luig (107) mixed urediniospores of three recombinant isolates obtained previously: M-9-a and M-10-a (derived from mixing Pgt and Pgs) and M-2 (derived from mixing two isolates of Pgt) (106). The three mixtures used in these experiments in essence involved mixing isolates of either Pgt X Pgs hybrid by Pgt (M-9-a by M-2; M-1-a by M2) and Pgt X Pgs hybrid by Pgt X Pgs hybrid (M-9-a by M-10-a). A total of 10 derivative hybrid isolates were identied. The ranges of pathogenicity and urediniospore color observed among these isolates were again suggestive of processes other than simple nuclear exchange, and it was suggested that the expression of pathogenicity could be affected as nuclei move into different cytoplasm (107). In a third study (49), eld isolates of Pgt (race 210 Yellow) and Pgs obtained from Queensland were mixed, and from this two isolates that gave intermediate infection types on the wheat cultivars Morocco and Transfer were recovered.

Puccinia striiformis f. sp. tritici


The emergence of races in regions where Pst populations have been shown to be clonal has led to the common conclusion that mutation is the major factor generating race variability (4, 18, 19, 109). The overriding observation from these studies implicating single mutation events has been the near-isogenic virulence/avirulence relationships between emerging and preexisting races (109, 110).

Wellings

In contrast, detailed studies of Pst populations in countries such as China (22, 55) and Pakistan (5) have demonstrated complex pathogenic variability beyond single-step race relationships. The evidence from these studies implicates recombination events for virulence/avirulence phenotypes among races observed. The report of Berberis as the sexual stage host for Pst by Jin et al. (36) and the predicted distribution of these hosts in these regions suggests that sexuality in Pst is inuencing the emergence of races in nature. Although this has yet to be demonstrated in the eld, supporting evidence includes a study that showed greater telial production and therefore greater potential for sexuality in Pst populations shown to have recombinant race phenotypes (1). Somatic recombination is an alternative mechanism that could also generate increased levels of race variability that may not be explained by single-step mutation. Laboratory studies of somatic recombination in Pst have involved the use of isolates contrasting in urediniospore color and race phenotype. Little & Manners (42) provided the initial evidence for heterokaryosis in Pst, where new races were concluded to have arisen from mixed races through hyphal fusion and the reassortment of heterokaryotic nuclei. Further histological observations supported these conclusions (43). Several independent studies observed new recombinant races carrying virulence for Yr1 (31, 115). Cytological studies demonstrated the absence of diploid nuclei in somatic hyphae, which precludes the possibility of recombination of virulence in a parasexual cycle (43, 114). The phenomenon of somatic hybridization in Pst presumes the occurrence of heterothallism between coinfecting races in order to allow anastamosis and hyphal fusion events necessary to give rise to heterokaryosis. McGinnis (57) examined the cytological status of a range of macro- and microcyclic rust fungi and noted a correlation between chromosome number and sexuality. Goddard (30) observed six chromosomes in mitotic cells of Pst and concluded on the basis of McGinnis work that this was further evidence for the potential

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role of heterothallism. In contrast, Wright & Lennard (114) reported three chromosomes in cytological studies of basidiospores. Newton (67) speculated that double-stranded RNA (dsRNA) mycoviruses, which are ubiquitous but variable in many rust pathogens, including Pst (21), may have a role in determining heterothallism. Drawing on work from other fungi, they suggested that dsRNA mycoviruses may produce specic toxins that function to govern hyphal compatibility/incompatibility between isolates and f. spp. Newton et al. (68) attempted to hybridize Pst and P. striiformis f. sp. hordei (Psh; pathogen causing barley stripe rust) on the common host Fong Tien barley. White-colored urediniospore variants in Psh and virulence for Yr1 in Pst were potential indicators of hybridization events, and although several putative isolates were recovered, the evidence suggested that these were either contaminants or spontaneous mutants. On this basis, Newton (67) suggested that observed contrasts in dsRNA between Pst and Psh may have encoded different toxins and so caused incompatibility between infection hyphae. The hypothesis and the role of dsRNA in Pst remain unconrmed.

Microcyclic: lacking the aecial and uredinial states; only the pycnial, telial, and basidial states are formed during the life cycle

Puccinia triticina
Several attempts have been made to induce somatic hybridization in the leaf rust pathogen of wheat, P. triticina (formerly Puccinia recondita), under controlled conditions. Barto s et al. (9) were unable to detect any evidence of recombination among six mixtures of two races with contrasting virulence and urediniospore color. The absence of variation in these experiments was of particular interest given that considerable variation and evidence of somatic hybridization was detected in parallel experiments on a similar scale with Pca by the same researchers. Vakili & Caldwell (93) established mixed infections of two races of P. triticina that differed in color and virulence (avirulent red race 2 and the virulent yellow race 122) and inoculated ve wheat cultivars (Malakof, Carina, Brevit,
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Autoecious: requiring only one host species or group of closely related host species to complete the life cycle Macrocyclic: having all ve spore types produced by rust fungi in the life cycle

Webster, and Loros) that were immune to the former and susceptible to the latter. A total of 156 red pustules were found on the ve cultivars, and from these they recovered 33 races, 17 of which had not been previously detected. In contrast, these results could not be repeated using the same two isolates in later studies by Barr et al. (6). Although a low frequency of variant pustules was found in mixtures of other races of P. triticina, the authors concluded that this was likely due to contamination. Based on previous studies in which red urediniospore wall color was shown to be dominant over yellow, 14 red isolates known to be heterozygous for this trait were mixed in various combinations with the aim of generating distinctive yellow hybrid isolates. This approach provided a much more critical assessment of somatic recombination, and despite using 20 mixtures, no such hybrids were identied (6). The failure to detect recombinant isolates in all of these studies was interpreted by the authors as evidence against nuclear reassociation, parasexuality, and somatic meiosis as mechanisms of vegetative recombination in P. triticina. Sharma & Prasada (86) created separate mixtures of four normal-colored P. triticina isolates and a yellow urediniospore mutant, race 107 CM. Although two of the mixtures produced negative results, one yielded seven and another six putative recombinant isolates, of which ve had not been previously recorded from India. The apparent lack of recombinant isolates in two of the mixtures was interpreted by the authors as indicating that production of somatic hybrid isolates depends upon the pathogen genotypes used and also the host genotypes used to screen for and identify putative recombinant isolates.

such as pathogenicity or morphology, may be impossible. In regions where sexual recombination is absent or extremely rare, such as occurs with the wheat rust pathogens in Australia, identifying isolates of hybrid origin is more straightforward and has been based on the detection of genotypes with new pathogenicity traits, combinations of rare pathogenicity features, and/or pathogenic features intermediate between existing pathogen genotypes.

Melampsora lini
Barrett et al. (7) examined variation in 39 isolates of M. lini collected from Linum marginale, an endemic wild herbaceous plant, from locations across its geographic distribution in southern temperate areas of Australia. Two genetically and geographically divergent pathogen lineages were identied among the isolates using molecular markers. Nine microsatellite markers revealed that one of the lineages (AA) displayed low heterozygosity and that the second (AB) was highly heterozygous. Furthermore, all of the microsatellite loci that were polymorphic in lineage AB generated an allele shared by both lineages and for which AA was typically homozygous. Lineage AB isolates were consistently heterozygous for a second allele that was not present in isolates from lineage AA. It was suggested that lineage AB was derived via genetic exchange between one or more isolates from lineage AA and a second unidentied lineage. Although M. lini is autoecious and macrocyclic, the actual mechanism involved in generating the hybrid was not discussed. In a later study of 275 isolates that included genotyping the avirulence loci AvrP123 and AvrP4, several isolates from lineage AB with identical AFLP genotypes, but different Avr gene alleles, were identied (8). The authors concluded that although multiple hybridization events between identical AFLP genotypes may have given rise to these genotypes, it was equally probable that somatic exchange via hyphal fusion among genetically different individuals had occurred.

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EVIDENCE OF SOMATIC HYBRIDIZATION IN NATURE


In regions where sexual recombination occurs, discriminating somatic hybrids from sexual recombinants based on simple features,

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Melampsora medusae and Melampsora larici-populina


The rst poplars to be grown in New Zealand were introduced from 1840 to 1850 and were used for erosion control, shelter, and timber production. The two rust diseases M. medusae and Melampsora larici-populina were rst detected in New Zealand in March 1973 and are believed to have been wind-borne from Australia, where they were rst detected in the previous year (88). Over the following years, M. larici-populina became the dominant species; M. medusae was detected sporadically at low levels, and in fact was not detected at all between 1984 and 1990 (89). Because of the susceptibility to both rusts of three poplar cultivars that accounted for approximately 90% of poplar plantings at the time of introduction, new resistant hybrid selections were identied and released. In 1991, several hybrids that were resistant to both pathogens became heavily rusted. Microscopic and pathology studies demonstrated that the rust responsible had most likely arisen from hybridization between M. medusae and M. larici-populina. The new rust pathogen, with the proposed named Melampsora medusae-populina, shared morphological features of both parents, but in terms of telial host range, was more similar to M. medusae (89). Although the hybrid rust pathogen produced basidiospores, no evidence was obtained to show that it had overwintered, and it was assumed that it had died out because of lack of a suitable telial or aecial host (89). The hybrid was, however, detected again in 1998 (88). It was concluded that the hybrid was less likely to have arisen from sexual recombination on the basis of the inability of isolates of M. medusae from New Zealand to infect the alternate host Larix decidua.

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(46, 48). On the basis of pathogenic features, it was concluded that standard race 126 had an exotic origin. Standard race 21 was rst detected in Australia in 1954, and by the following year, it had increased rapidly to dominate Pgt populations in southern New South Wales, Victoria, and Tasmania (105). Like standard race 126, standard race 21 was considered to have had an exotic origin (44). Pgt pathotype 34-2,11, rst detected in Australia in northern New South Wales in 1957, combined certain pathogenic features of isolates typied by standard races 126 and 21 (103). On this basis, it was regarded as the product of somatic hybridization between an isolate from each group (44, 51). This was supported by subsequent studies in which isozymic variability was examined, which showed that the eight isozyme phenotypes of pathotype 34-2,11 and nine presumed derivative pathotypes were identical to those of representative pathotypes from standard race 21, with the exception of the enzyme GOT (glutamate oxalate transaminase), which resembled that of standard race 126 (15). Pathotype 34-2,11, along with several pathotypes that were considered to be mutational derivatives from 34-2,11 with virulence for genes such as Sr9e, Sr17, and Sr36, were common during the late 1950s and throughout the 1960s (48). Of these, only pt 34-2,4,5,11 was signicant in agriculture because it combined virulence for several resistance genes present in the cultivar Mendos (Sr7a, Sr11, Sr17, Sr36) (103).

Puccinia graminis f. sp. tritici and Puccinia graminis f. sp. secalis


Elymus scaber (formerly Agropyron scabrum), common wheat grass, is a perennial grass species that is endemic to many parts of Australia and is an important host to Pgt in northeastern Australia (79). Prior to the detection of rye stem rust (Pgs) in Australia in 1957 (100), all isolates of P. graminis recovered from E. scaber were identied as Pgt or, on one occasion only, Pga (9799). In 1963, an isolate of P. graminis resembling the somatic
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Puccinia graminis f. sp. tritici


Pgt standard race 126 was rst detected in Australia in 1925, after which it increased rapidly in frequency, and from 1929 until 1941 it dominated the Pgt population in Australia

hybrids generated in mixing experiments with Pgt and Pgs under controlled conditions by Watson & Luig (106) was isolated from E. scaber in Queensland (49). Detailed studies of 107 similar stem rust isolates collected between 1967 and 1971 allowed the identication of 27 strains (races) based on differences in pathogenicity on 21 wheat cultivars and two rye cultivars. These isolates were considered to have arisen from somatic hybridization between Pgt and Pgs, most likely on E. scaber, although possible common hosts also include Hordeum leporinum and cultivated barley, both of which are infected by Pgt, Pgs, and the scabrum stem rust in Australia (49). Burdon et al. (14) obtained evidence in support of the hybrid origin of the scabrum stem rust based on studies of isozyme variability among 11 isolates of Pgs, 20 of Pgt, and 14 of the scabrum stem rust. All scabrum isolates had a leucine amino peptidase phenotype identical to the Pgs isolates and an NADH diaphorase phenotype identical to that of the Pgt isolates. Phenotypes for the isozymes esterase and glutamate oxalo-transaminase also supported a hybrid origin for all scabrum isolates. These results were consistent with a mechanism of whole nuclear exchange between fused somatic hyphae. Isolates of the hybrid scabrum rust continue to be isolated from samples of stem-rusted barley and E. scaber submitted to the University of Sydney for race analysis (51, 71, 72; R.F. Park, unpublished results). Because E. scaber can be infected by Pgs, Pgt, or the hybrid scabrum rust, isolates from this grass are applied to genotypes of wheat, rye, and barley. Isolates are identied as Pgs when the differential genotype Black Winter rye is susceptible, and infection types X, 2, and 0 are produced on the wheats W2691, Little Club, and Yalta, respectively. Where isolates that are avirulent on Black Winter rye (1, 2) produce infection types 2 = or 3+ on Yalta, and are virulent on W2691 and Little Club, they are considered to be Pgt. Collections that cannot be categorized as Pgs or Pgt are classied as scabrum rust. The ability to identify the scabrum isolates in this way is not completely accurate. For example, studies of variability at the IGS-1 locus suggest that the pathotype
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116-4,5, identied in 1964 as a pathotype of Pgt (isolate accession number 640726; collected from Milguy, northern New South Wales), arose most likely via a hybridization event between Pgt and Pgs ( J.P. Silk, J.J. Burdon, and R.F. Park, unpublished results).

Puccinia triticina
Only one compelling example of somatic hybridization in nature between isolates of P. triticina has been reported. P. triticina has been present in Australia since at least European colonization, and in the absence of the alternate host Thalictrum, it comprises a large asexually reproducing population. Pathotype 64(6),(7),(10),11 was rst detected in Australia in northern New South Wales in 1990 (73). Greenhouse tests of pathogenicity on wheat and triticale genotypes indicated that it was similar to pathotypes of P. triticina considered to belong to two clonal lineages tracing back to two founding pathotypes of independent exotic origins. The rst founding pathotype, 1042,3,(6),(7),11, was detected initially in Victoria in 1984 and was considered an exotic incursion because it differed from isolates of P. triticina endemic at that time in pathogenicity and in the presence of a unique Pgm2 c allele (74). Pathotype 104-2,3,(6),(7),11 spread quickly throughout Australia and underwent mutation to produce a lineage comprising many derivative pathotypes (74). The second founding pathotype, 53-1,(6),(7),10,11, was rst detected in Australia in 1984 after being initially detected in New Zealand in 1981 (47). This pathotype possessed several pathogenic features not seen previously in Australasia, including virulence for Lr13, and it carried two unique isozyme alleles, Pgm2 a and Got b. It remained largely conned to northern New South Wales and Queensland, and reached epidemic levels on several cultivars carrying Lr13 in the latter state during 1988. Although pathotype 64-(6),(7),(10),11 was similar to pathotypes within the 1042,3,(6),(7),11 and 53-1,(6),(7),10,11 lineages, it differed from them in at least three pathogenic features, indicating that it had not arisen by

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simple mutation. Detailed comparative studies indicated that pathotype 64-(6),(7),(10),11 combined several pathogenic and isozymic features that prior to its detection were known only in pathotypes 104-2,3,(6),(7),11 and 53-1,(6),(7),10,11, strongly suggesting that it had arisen via somatic hybridization between them. Of particular interest was that the hybrid carried the Got b allele, previously found only in pathotype 53-1,(6),(7),10,11, and combined the two PGM2 alleles Pgm2 a and Pgm2 c, each previously known only from the two putative parental lineages. The hypothesis that pathotype 64-(6),(7),(10),11 arose via somatic hybridization was further supported by random amplication of polymorphic DNA (RAPD) phenotypes, which showed similarity with multiple isolates of the two putative parental genotypes. These studies also identied three RAPD bands found only in isolates of pathotype 53-1,(6),(7),10,11 and one band found only in isolates within the pathotype 104-2,3,(6),(7),11 lineage, all four of which were present in two isolates of the putative hybrid pathotype. Comparative greenhouse tests on adult plants established that the hybrid pathotype 64-(6),(7),(10),11 combined virulence for Lr1 with partial virulence for gene Lr13. These studies further demonstrated susceptibility of the hybrid wheat cultivars Meteor and Pulsar, both of which carried the resistance genes Lr1 and Lr13 in the heterozygous state because of their hybrid status. Although pathotype 64-(6),(7),(10),11 was isolated infrequently from 1990 to 2000 and has not been isolated since, it composed 16% of the isolates pathotyped from northeastern Australia in 1993 (32 isolates from a total of 205) and was commonly recovered from hybrid wheat cultivars, including Meteor and Pulsar. It was concluded that the susceptibility of these cultivars to pathotype 64-(6),(7),(10),11 most likely related to partial virulence for Lr13 in the rust and heterozygosity for Lr13 in the hosts. Incomplete dominance of host resistance and pathogen avirulence have been demonstrated for several corresponding gene pairs in the

P. triticina:Triticum aestivum host:pathogen system (40). Prior to the detection of pathotype 64-(6),(7),(10),11, combined virulence for Lr1 and Lr13 had not been detected in Australia.

Puccinia Species on Senecio


Puccinia lagenophorae was rst described in 1884 in Australia from Lagenophora billardieri and is believed to have spread from there to France, where it was rst recorded in 1960 on Senecio vulgaris. It spread quickly throughout Europe and was subsequently recorded in countries such as Egypt, Israel, Argentina, the United States, and Canada. Detailed studies by Morin et al. (61) of an aecial rust pathogen, Aecidium sp., on reweed (Senecio madagascariensis) in South Africa, provided evidence for a putative natural hybrid rust pathogen derived from P. lagenophorae and a second unidentied rust. Sequence comparisons of the ITS region and of the -tubulin1 gene from seven South African isolates of Aecidium sp. with isolates of P. lagenophorae from Australia, Europe, the United States, and Canada, and of several other rust species, indicated that all South African isolates belonged to the P. lagenophorae complex and had not been recorded previously in South Africa. Restriction and sequence analyses of the ITS region and of the -tubulin1 gene from the isolates revealed further that although one South African isolate carried single copies of each, two divergent copies of both genes occurred in the remaining six South African isolates. The presence of two different copies of each gene region in these isolates and the presence of only one in the Australian isolates were viewed as evidence that the South African isolates arose from hybridization between P. lagenophorae and a second rust pathogen, likely on S. madagascariensis in South Africa. Although the results of ITS sequencing suggested that the second parent is related to a member of a complex typied by Phytolacca dioicae, this was not supported by the results of the -tubulin1 sequencing, and hence the identity of the second parent could not be established.
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Heteroecious: requiring two different host species for completion of the life cycle

Based on the rare occurrence of pycnia in P. lagenophorae, somatic hybridization was suggested as the most likely process by which the hybrid rust arose. No obvious differences were noted between the hybrid rust and P. lagenophorae either in aeciospore morphology or in infection processes. The evolutionary signicance of the hybrid remains unknown; however, it was suggested that the more common occurrence of the hybrid rust among the seven random isolates studied could indicate greater tness (61).

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POSSIBLE MECHANISMS INVOLVED IN GENETIC REASSORTMENT


Although studies have provided evidence of nuclear reassortment and parasexuality in rust fungi, the actual mechanisms involved are not known. The parasexual cycle, rst found in Aspergillus nidulans, involves hyphal anastamosis, heterokaryosis via nuclear reassortment, fusion of two unlike haploid nuclei (diploidization), multiplication of the diploid nucleus and mitotic crossing over, and vegetative haploidization of the diploid nuclei (77, 92). Various studies of rust fungi have provided evidence of some of these mechanisms.

that grew together were prevented from pycniospore exchange. Of these, 110 formed aecia, indicating that the two haploid components of each mating type were able to diploidize each other only by means of hyphal fusion. Rodenhiser & Hurd-Karrer (80) observed orange-brown round bodies called fusion bodies at the tips of some germ tubes when urediniospores of several Puccinia species were germinated on agar containing mineral nutrients and dextrose. Hyphal networks with fusion bodies on the strands were also occasionally observed on wheat leaves inoculated with P. triticina urediniospores. Detailed studies on the formation of fusion bodies in P. triticina by Wang & McCallum (96) demonstrated that they were essential for germ-tube anastamosis, and furthermore, in some cases both nuclei in each germ tube migrated into the fusion body so that all four nuclei came into close proximity.

Nuclear Exchange
Craigie (20) was the rst to show that rust dikaryons are also heterokaryons. The importance of nuclear status in the rust fungi is clear in heteroecious species, in which heterokaryosis conditions pathogenicity on one host group and homokaryosis conditions pathogenicity on a completely different host (alternate) group. Wherever the vegetative mycelium of a rust fungus is composed of a dikaryon involving two different nuclei, it is by denition heterokaryotic. In experiments under controlled conditions, evidence for somatic hybridization has not, however, been obtained within single heterokayotic isolates (24), suggesting that associations of certain types of nuclei may be involved in disturbances leading to somatic hybridization (75). Several studies targeting mixtures of rust isolates found derivative isolates with three or more nuclei. As already noted, Nelson (63) found a low frequency of trinucleate urediniospores among orange-spored putative hybrid isolates and concluded that an observed reversion to parental spore color was due to instability in these isolates and reversion to a dikaryotic

Anastamosis
The fusion of vegetative structures (germ tubes, substomatal vesicles, appressoria, hyphae), often referred to as anastamosis, has been observed in a range of rust fungi, including P. triticina (2, 96) and P. graminis (64, 54, 111). In studies of the latter, anastamosis among germ tubes was observed in Pgt both on articial media and on wheat leaves as well as in mineral oil in mixtures involving Pgt and Pgs, Pgt and Pga, and Pgt and P. graminis f. sp. poae. Hermansen (35) similarly observed fusion between Pga and P. graminis f. sp. phlei-pratensis. Evidence for hyphal fusion in the monokaryotic stage was obtained in studies of coalescing haploid pustules of Puccinia helianthi on sunower (12). In these studies, 288 pairs of pycnial pustules
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state via dissociation of nuclei. Yang et al. (117) reported the presence of multinucleate isolates of P. striiformis f. sp. tritici at various rates from different locations in Gansu Province in China. Subculturing eight isolates with a multinucleate frequency higher than 1% on a susceptible genotype through several generations led to a decline in frequency of the multinucleate state.

Parasexuality
If the generation of somatic hybrid isolates resulted only from simple nuclear reassortment, the maximum number of new genotypes possible would be two if the nuclei in a dikaryon carry different mating type loci. The recovery of more than two recombinant genotypes in several studies (e.g., 11, 101) therefore suggests that the processes involved in genetic reassortment are more complex than simple nuclear exchange. Parasexuality after nuclear exchange requires the formation of a somatic diploid phase via karyogamy. Brown (13) observed the production of uninucleate hyphae from binucleate (dikaryotic) hyphae in systemic infections of Lactuca pulchella by the perennial rust Puccinia minussensis, which may have been due to either dissociation of the nuclei into homokaryons or nuclear fusion. In studies of the axenic culture of Pgt, Maclean et al. (52) isolated a monokaryotic isolate from the Australian Pgt race 126-Anz-6,7 that was pathogenic on wheat over six generations. Subsequent studies by Williams (112) established that this and a uredinial monokaryotic strain established from it (isolate 71868) were diploid on the basis of comparisons of nuclear size that showed the nucleus of the monkaryotic isolate had a diameter one-third greater and a volume about twice that of the nucleus of the dikaryotic parental isolate. Comparisons of the two isolates using cytouorometry indicated that the nucleus in the monokaryon had twice the DNA content of that in the single haploid nucleus of the parental dikaryon (113). Extensive experiments with the diploid uredinial isolate 71868 failed to detect evidence of dissociation into dikaryons (103).
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Hartley & Williams (34) proposed a means by which the appearance of many hybrid genotypes could arise from controlled pairings of rust isolates following simple nuclear exchange without parasexuality. These authors suggested that if a reciprocal transfer of whole, homologous chromosomes were to occur between the nuclei of a single dikaryon, the overall genetic constitution of the cell would not be altered but the genotype of each nucleus would change. In such a situation, it could be possible for any heterozygous isolate of a rust pathogen to carry a number of different haploid nuclei within the one phenotype, and if two such isolates (or possibly even a single isolate) were to undergo simple nuclear reassortment, many new phenotypes could be generated.

Sexual versus Somatic Hybridization


Pontecorvo (77) considered a genetic system based on the parasexual cycle to have all the elements of one based on sexual reproduction plus some novel aspects. This potentially allows storage within a population of a large amount of genetic diversity from processes such as mutation and provides for the reassortment of this diversity ready for the sieve of natural (or articial) selection. In the rust fungi, the model proposed by Hartley & Williams (34) provides a mechanism through which considerable genetic diversity could be generated and stored in rust pathogen populations. The driving force behind the extensive barberry eradication program in the United States from 1918 to 1980 was the role of this host in sexual recombination and the generation of new combinations of virulence in P. graminis, principally Pgt because of the importance of wheat. Despite the technical challenges of studying sexual recombination in many rust species, such as germinating dormant teliospores, studies have shown limited interfertility between f. spp. of rusts such as P. graminis. For example, intercrosses have been established between Pgt and Pgs (32, 90) and Pgt and P. graminis f. sp. agrostidis (90). In general, the progeny of these crosses had host ranges wider than the parents,
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but they were less virulent on each of the hosts infected by the parental f. spp. Green (32) speculated that these features resembled the ancestral form of P. graminis from which these f. spp. evolved and furthermore stated that although not potentially dangerous to cereal crops, such hybrids could be of evolutionary signicance. The existence of species of grass and of Berberis that are susceptible to more than one f. sp. of P. graminis, and the apparent rarity of hybrids between these taxa in nature, led Anikster (3) to propose that there must be barriers preventing such intercrossing and that if a hybrid is created either by sexual or by somatic recombination, it apparently cannot compete with the parental forms. Although probably rare, processes including interspecic matings and parasexuality may facilitate gene introgression or large genomic rearrangement and restructuring (85). Hybridization is now considered an important mechanism for rapid evolution and speciation in fungi (70).

the noncropping phase. Although there is no evidence that this has occurred, adaptation to the wild barley grass species Hordeum leporinum and H. glaucum, presumably by stepwise mutation for virulence on one or more resistance genes in these grass species, has been shown within the wheat stripe rust pathogen Pst in Australia (C.R. Wellings, unpublished results).

Resistance Breeding
The ability of rust fungi to undergo genomic reassortment via somatic hybridization has implications for the development of new cultivars with rust resistance. The presumed somatic hybrid rusts M. medusae-populina on poplars in New Zealand, Pgt pathotype 34-2,4,5,11 on wheat in Australia, and P. triticina pathotype 64(6),(7),(10),11 on wheat in Australia rendered resistant genotypes of each crop susceptible because of new combinations of virulence. In the case of P. triticina, the susceptibility of several hybrid wheat cultivars carrying Lr13 in a heterozygous form to the hybrid pathotype was believed to have resulted from partial dominance of avirulence and resistance in the pathogen and host, respectively. Studies of somatic hybridization between Pgt and Pgs under controlled conditions by Watson & Luig (106) showed that it was possible to generate a hybrid isolate with virulence on Vernal Emmer (Sr9e). These authors considered that there seemed no reason why such interf. spp. crosses would not produce hybrids capable of rendering other rust resistance genes ineffective. In later studies of the genetic basis of stem rust resistance in wheat, Sanghi & Luig (84) demonstrated that the resistance gene Sr11, although effective at that time to eld isolates of Pgt, was ineffective to isolate 57241 of Pgs and to sexual and somatic hybrids between Pgs and Pgt. Studies by Luig & Watson (50) demonstrated virulence for the rye-derived resistance gene Sr27 in eld isolate 69090 of Pgs, an articially generated somatic hybrid between Pgt and Pgs 69090, and for 5 out of 13 eld-collected Pgt by Pgs scabrum isolates. Studies of variability at the IGS-1 locus among

IMPLICATIONS FOR RUST CONTROL Rust Pathogen Epidemiology


It is interesting to consider that although sexual hybrids between Pgt and Pgs generated under controlled conditions are less virulent on the hosts of parental rusts, somatic hybrids between them have formed in nature in Australia and have survived (49). Assuming that cerealattacking rust pathogens evolved from grass rust pathogens (41), specialization of hybrids between Pgt and Pgs on E. scaber could be seen as a step towards the more primitive state of P. graminis (49). However, such adaptation, including possible mutation to virulence for resistance genes in E. scaber following hybridization, would confer increased survival ability to the hybrids, especially if, like E. scaber, the host is a perennial and grows during the whole year (49). Subsequent adaptation of such hybrids to cereals, via processes such as mutation or further hybridization, could result in cereal-attacking isolates with increased ability to survive during
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Australian isolates of Pgt provided evidence that Pgt pathotype 116-4,5, identied in 1964 (isolate accession number 640726), originated not from simple mutation but possibly a hybridizational event between Pgt and Pgs ( J.P. Silk, J.J. Burdon, and R.F. Park, unpublished results). This pathotype is virulent on several rust resistance genes in wheat and based on pathogenicity alone would be regarded as a pathotype of Pgt. These studies show clearly that isolates derived via somatic hybridization have the capacity to recombine virulence for resistance genes and render cultivars susceptible. The ability of Pgt and Pgs to hybridize in nature is of particular interest, given that several rust resistance genes have been transferred from cereal rye to hexaploid wheat (e.g., Lr25, Lr26, Sr27, Sr31, Sr50, Yr9) (60). However, virulences that have been detected for most of these genes, including virulence for Sr31 in the important Pgt race Ug99 (78, 95) and for two rye-derived stem rust resistance genes in triticale (59), are believed to have arisen by mutation in isolates of Pgt rather than hybridization between Pgs and Pgt. The concept of combining multiple resistance genes to bestow greater durability to diseases such as rusts was proposed by researchers more than 50 years ago and is based on the diminishing probability of a pathogen genotype acquiring virulence matching increasing numbers of resistance genes (e.g., 108). Cloning disease resistance genes from plants and transforming constructs combining multiple cloned genes as a means of developing durable resistance holds great promise in the quest for durable resistance (25). The ability of pathogens to acquire new virulence combinations via processes such as somatic hybridization should not, however, be ignored. In addition to simple hybridization, there also seems no reason why hybrid isolates could not undergo further hybridization and possibly backcrossing. Evidence for this was obtained in the presumed sexual hybrid M. X columbiana by Newcombe et al. (65), who were able to show considerable pathogenic variability among hybrid isolates and interpreted this as evidence

that the F1 hybrids may have undergone backcrossing to either parent.

CONCLUDING REMARKS
Controlled studies of basidiomycete fungi, such as the cultivated mushroom Agaricus bisporus, have shown a high frequency of somatic recombination (116). It has been suggested that this process may be less common in basidiomycetes, such as rusts, in which the vegetative heterokaryon comprises dikaryotic hyphal compartments, than in basidiomycetes like A. bisporus (116) and Heterobasidion annosum (33), in which multinucleate hyphal compartments are found. Although most published studies of attempts to induce somatic hybridization in rust fungi under controlled conditions have provided evidence for this process, few clear examples exist of the process in nature. This could indicate either that it is rare, that it is common but survival of the hybrid isolate is rare (103), and/or that our ability to discriminate somatic hybrids from isolates generated by other mechanisms is poor. The identication of 27 races of the hybrid scabrum stem rust by Luig & Watson (49) is of interest. Presumably, all of these races arose between 1957, when Pgs was introduced into Australia (100), and 1971. Whether these isolates developed from multiple hybridization events or only one followed by parasexuality is not known, although the nonrandom distribution of the locations from which the isolates were collected was seen as evidence that the different races had arisen from different hybridization events. The application of isozyme markers has already provided some insight into somatic hybridization in rust fungi, but this has been more from the perspective of conrming the hybrid nature of suspected hybrid isolates. These tests have also provided evidence contradicting results based on pathogenicity. For example, the Australian Pgt pathotype 34-4,7 (isolate 57096), which was collected only once in 1957, was considered by Luig (45) to have originated from somatic hybridization between Pgt and
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Pgs. Isozyme analyses by Burdon et al. (15) demonstrated that this pathotype possesses distinctive isozyme phenotypes for both GOT and LAP not found in Australian isolates of Pgs or Pgt, suggesting it more likely had its origins outside Australia. The more recent use of DNA marker technology, such as RAPDs (73), AFLP (8), microsatellite markers (7), and restriction and sequence analyses of specic loci (61), has provided much greater power in resolving the origin of rust isolates. Newer technologies such as next-generation sequencing and genotyping by

sequencing (26) hold great promise in advancing our understanding of the role of somatic hybridization in generating genetic variability in rust pathogens. Whole-genome sequences are now available for several rust species, including M. larici-populina (23), which may provide a powerful tool to look more critically at the mechanism that gave rise to the hybrid M. medusae-populina. The existence of comprehensive historical collections of viable rust isolates maintained in liquid nitrogen in several laboratories provides a unique resource that can now be tapped using these new technologies.

SUMMARY POINTS 1. Understanding rust pathogen variability has contributed signicantly to the development of new plant cultivars with genetic resistance to these pathogens. 2. Long-term surveys of rust pathogens have shown that in the absence of sexual recombination, genetic diversity in rust fungi is generated by periodic introduction of exotic isolates, single-step mutation, and somatic hybridization. 3. Although the mechanisms involved in somatic hybridization remain unknown, some studies have provided convincing evidence that it involves more than simple nuclear exchange. It is thought to involve the fusion of dikaryotic vegetative hyphae, nuclear exchange, and possibly exchange of whole chromosomes between nuclei or parasexuality via the fusion of the two haploid nuclei, followed by mitotic crossing over and vegetative haploidization. 4. Laboratory studies have provided clear evidence for asexual exchange of genetic material between many rust species and between f. spp. of the stem rust pathogen P. graminis. 5. There is evidence for somatic hybridization in nature within and between rust species on Linum, poplar, Senecio, wheat, and several grass species. 6. A lack of genetic markers has made it difcult to determine the frequency of this process in nature, although the studies that have been conducted suggest that it is rare. 7. In three cases, isolates believed to have arisen via somatic hybridization have rendered resistant plant genotypes susceptible because of new combinations of virulence. Although apparently rare, somatic hybridization can therefore be important in generating new and signicant pathogen genotypes.

FUTURE ISSUES 1. The recent demonstration of Berberis as the alternate host of the stripe rust pathogen Puccinia striiformis, after 100 years of searching (36), shows clearly we still have much to learn about the biology of rust fungi.

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2. Although DNA-based marker technologies have already contributed to understanding the hybrid origin of some rust isolates, advances in high-throughput sequencing now provide new tools that will enable a critical appraisal of the frequency and importance of somatic hybridization in nature. 3. The future use of transgenic technologies in moving resistance genes between sexually incompatible plant species should take into consideration the ability of pathogens to undergo rare but potentially large genome changes via processes such as somatic hybridization.

DISCLOSURE STATEMENT
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The authors are not aware of any afliations, memberships, funding, or nancial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS
The authors would like to pay particular tribute to the long-term work of Prof. W.L. Waterhouse, Prof. I.A. Watson, and Dr. N.H. Luig in monitoring pathogenic variability in P. graminis in Australasia. We also thank the Australian Grains Research and Development Corporation and predecessors for long-term nancial support of cereal rust research at the University of Sydney. LITERATURE CITED
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Contents
An Ideal Job Kurt J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 Arthur Kelman: Tribute and Remembrance Luis Sequeira p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 15 Stagonospora nodorum: From Pathology to Genomics and Host Resistance Richard P. Oliver, Timothy L. Friesen, Justin D. Faris, and Peter S. Solomon p p p p p p p p p p 23 Apple Replant Disease: Role of Microbial Ecology in Cause and Control Mark Mazzola and Luisa M. Manici p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 45 Pathogenomics of the Ralstonia solanacearum Species Complex St ephane Genin and Timothy P. Denny p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 67 The Genomics of Obligate (and Nonobligate) Biotrophs Pietro D. Spanu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 91 Genome-Enabled Perspectives on the Composition, Evolution, and Expression of Virulence Determinants in Bacterial Plant Pathogens Magdalen Lindeberg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 111 Suppressive Composts: Microbial Ecology Links Between Abiotic Environments and Healthy Plants Yitzhak Hadar and Kalliope K. Papadopoulou p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 133 Plant Defense Compounds: Systems Approaches to Metabolic Analysis Daniel J. Kliebenstein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 155 Role of Nematode Peptides and Other Small Molecules in Plant Parasitism Melissa G. Mitchum, Xiaohong Wang, Jianying Wang, and Eric L. Davis p p p p p p p p p p p p 175 New Grower-Friendly Methods for Plant Pathogen Monitoring Solke H. De Boer and Mar a M. L opez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 197 Somatic Hybridization in the Uredinales Robert F. Park and Colin R. Wellings p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 219

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Interrelationships of Food Safety and Plant Pathology: The Life Cycle of Human Pathogens on Plants Jeri D. Barak and Brenda K. Schroeder p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 241 Plant Immunity to Necrotrophs Tesfaye Mengiste p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 267 Mechanisms and Evolution of Virulence in Oomycetes Rays H.Y. Jiang and Brett M. Tyler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 295 Variation and Selection of Quantitative Traits in Plant Pathogens Christian Lannou p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 319
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Gall Midges (Hessian Flies) as Plant Pathogens Jeff J. Stuart, Ming-Shun Chen, Richard Shukle, and Marion O. Harris p p p p p p p p p p p p p p 339 Phytophthora Beyond Agriculture Everett M. Hansen, Paul W. Reeser, and Wendy Sutton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 359 Landscape Epidemiology of Emerging Infectious Diseases in Natural and Human-Altered Ecosystems Ross K. Meentemeyer, Sarah E. Haas, and Tom as V aclav k p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 379 Diversity and Natural Functions of Antibiotics Produced by Benecial and Plant Pathogenic Bacteria Jos M. Raaijmakers and Mark Mazzola p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 403 The Role of Secretion Systems and Small Molecules in Soft-Rot Enterobacteriaceae Pathogenicity Amy Charkowski, Carlos Blanco, Guy Condemine, Dominique Expert, Thierry Franza, Christopher Hayes, Nicole Hugouvieux-Cotte-Pattat, Emilia L opez Solanilla, David Low, Lucy Moleleki, Minna Pirhonen, Andrew Pitman, Nicole Perna, Sylvie Reverchon, Pablo Rodr guez Palenzuela, Michael San Francisco, Ian Toth, Shinji Tsuyumu, Jacquie van der Waals, Jan van der Wolf, Fr ed erique Van Gijsegem, Ching-Hong Yang, and Iris Yedidia p p p p p p p p p p p p p p p p p p p p p p 425 Receptor Kinase Signaling Pathways in Plant-Microbe Interactions Meritxell Antol n-Llovera, Martina K. Ried, Andreas Binder, and Martin Parniske p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 451 Fire Blight: Applied Genomic Insights of the Pathogen and Host Mickael Malnoy, Stefan Martens, John L. Norelli, Marie-Anne Barny, George W. Sundin, Theo H.M. Smits, and Brion Duffy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 475 Errata An online log of corrections to Annual Review of Phytopathology articles may be found at http://phyto.annualreviews.org/
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