Australasian Plant Pathol. (2016) 45:561–570 DOI 10.1007/s13313-016-0446-z REVIEW Revisiting Stagonosporopsis species associated with chrysanthemum and pyrethrum ray blight Niloofar Vaghefi 1 & Sarah J. Pethybridge 1 & Frank S. Hay 1 & Rebecca Ford 2 & Marc E. Nicolas 3 & Paul W. J. Taylor 3 Received: 12 July 2016 / Accepted: 19 September 2016 / Published online: 29 September 2016 # Australasian Plant Pathology Society Inc. 2016 Abstract Ray blight is a destructive disease of Asteraceae affecting chrysanthemum and pyrethrum industries worldwide. Three morphologically similar but phylogenetically distinct species of the family Didymellaceae; Stagonosporopsis chrysanthemi, S. inoxydabilis and S. tanaceti are associated with the disease. Despite their close evolutionary relationship and cross host pathogenicity, these species have marked differences in their biology and epidemiology. Stagonosporopsis chrysanthemi and S. inoxydabilis are both homothallic with a MAT locus that carries both mating type genes. Ascomata play a major role in survival and dispersal of S. chrysanthemi, contributing to the onset of ray blight epidemics on chrysanthemum. However, S. tanaceti is either asexual or heterothallic due to the presence of only MAT1-2 idiomorph in its genome. Morphological similarity of the species causing ray blight on various Asteraceae, multiple changes in their taxonomy and historical confusion with other Phoma-like species have resulted in lack of clarity in their taxonomy, host range and global distribution. Host specificity studies and reports on global distribution of S. chrysanthemi published before its separation from S. inoxydabilis should be treated with caution. A recently developed species-specific multiplex PCR assay provides a rapid and robust tool to study the global distribution of these important quarantine pathogens. When global * Niloofar Vaghefi nv232@cornell.edu 1 School of Integrative Plant Science, Plant Pathology & Plant-Microbe Biology Section, Cornell University, Geneva, NY 14456, USA 2 School of Natural Sciences, Griffith University, Nathan, QLD 4111, Australia 3 Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Melbourne, VIC 3010, Australia populations from cultivated and/or wild Asteraceae are available, population genetic analyses may aid in understanding the origin, evolutionary history and global migration patterns of the Stagonosporopsis spp. associated with ray blight of Asteraceae. Keywords Ascochyta chrysanthemi . Phoma ligulicola . inoxydabilis . Stagonosporopsis tanaceti . Tanacetum cinerariifolium Introduction Ray blight is a destructive disease of Asteraceae worldwide, affecting all above-ground parts with the most conspicuous symptom being necroses of the ray florets (Stevens 1907). Reported on many Asteraceae species, ray blight is of economic importance to chrysanthemum (Chrysanthemum × morifolium) and pyrethrum (Tanacetum cinerariifolium) industries (Baker et al. 1949; Pethybridge et al. 2008b). On chrysanthemum, ray blight develops rapidly under favourable environmental conditions on potted plants, stock beds and cuttings grown in the field and under protected cultivation, and is capable of causing complete loss of susceptible varieties (Engelhard 1984; Baker et al. 1949). Moreover, asymptomatic infected flower cuttings may develop symptoms during transport or storage and become unmarketable (Baker et al. 1949). Ray blight has also been reported in many commercial pyrethrum production areas such as Kenya and Tanzania (Peregrine and Watson 1964; Robinson 1963), however, it is most damaging in Australia (Pethybridge et al. 2008b). Dieback of pyrethrum leaves and stems caused by ray blight results in substantial reduction in the number of flowers and, therefore, pyrethrin yield (Pethybridge and Hay 2001; Pethybridge et al. 2005b, 2007a). 562 Three morphologically similar but phylogenetically dist i n c t s p e c i e s ; S t a g o n o s p o ro p s i s c h r y s a n t h e m i , S. inoxydabilis and S. tanaceti are associated with ray blight of Asteraceae worldwide (Vaghefi et al. 2012), collectively referred to hereafter as ‘ray blight pathogens’. Stagonosporopsis chrysanthemi is associated with ray blight of chrysanthemum worldwide (Baker et al. 1949; EPPO 2016). Stagonosporopsis inoxydabilis is reported to infect various members of the Asteraceae (Van der Aa et al. 1990) but little information exists on its distribution and economic importance. Stagonosporopsis tanaceti is the cause of ray blight of pyrethrum in Australia and poses a major threat to the Australian pyrethrum industry. All of these species are important quarantine pathogens in many parts of the world. Stagonosporopsis chrysanthemi and S. inoxydabilis are level 2 quarantine pathogens in Europe (EPPO 2016), and are also considered a biosecurity threat to the Australian pyrethrum industry since both species are able to infect pyrethrum (Vaghefi et al. 2016). Stagonosporopsis tanaceti has to date only been detected in Australia and is therefore listed as a quarantine pathogen by European and Mediterranean Plant Protection Organization (EPPO 2016). Since the first report of ray blight on chrysanthemum, and the description of the causal agent as Ascochyta chrysanthemi (Stevens 1907), multiple taxonomic changes have been made and additional fungal species have been described as associated with ray blight symptoms on various Asteraceae. This has led to substantial confusion surrounding the taxonomy, host range, and global distribution of the pathogens associated with this disease. Considering the quarantine importance of these species, there is a need to better understand their biology, host-specificity, and global distribution (EFSA PLH Panel 2013; Rossi et al. 2014). The objective of this paper is to review current knowledge on the biology, host-specificity and global distribution of these important quarantine species, and identify areas of uncertainty and confusion that require further research. Symptoms Ray blight causes tissue necrosis, which distorts and discolours flowers (Pethybridge and Wilson 1998; Stevens 1907). Necrotic regions may extend 20–30 mm down the flower stem, blackening and weakening the tissue and causing the flower head to droop, resulting in the Bshepherd’s crook^ appearance of the infected buds and flowers (Fig. 1; Pethybridge and Wilson 1998). Infection during early stages of blossom development may result in asymmetrical growth resulting in necrotic and deformed buds. Leaf infection results in irregular brownish-black lesions of up to 3 cm which, under favourable environmental conditions, rapidly coalesce and cause leaf necrosis. Leaf lesions may extend into the petiole N. Vaghefi et al. and flower stem resulting in stem girdling. This results in drooping of the terminal shoot, chlorotic leaves, and stunted plants (Fox 1998; Horst and Nelson 1997; Pethybridge and Wilson 1998; Stevens 1907). On chrysanthemum, the major loss from ray blight is from infection of flowers and cuttings, which are highly susceptible (Baker et al. 1949, 1961; Stevens 1907). On mature chrysanthemum plants, stem girdling may result in the abnormal appearance of the corresponding non-infected shoots, leaf spotting, wilting and necrosis of the non-infected plant parts (McCoy and Dimock 1970). The appearance of symptoms on non-infected plant parts was proposed to result from the production of phytotoxins such as chrysanthone (Albinati et al. 1989; Arnone et al. 1990; Schadler and Bateman 1974, 1975). This was because injection of sterile culture filtrates of S. chrysanthemi into healthy plants resulted in development of similar symptoms (McCoy and Dimock 1970). Phytotoxic compounds have not yet been reported in the pyrethrumS. tanaceti pathosystem. Root infection has also been reported in the chrysanthemum-S. chryanthemi pathosystem (Chesters and Blakeman 1966; Baker et al. 1961) but not in S. tanaceti infection of pyrethrum. Taxonomic history of the causal agents Upon the first description of ray blight on chrysanthemum in 1904, the causal agent was described as Ascochyta chrysanthemi F. Stevens (1907). Sexual structures (perithecia) were reported in 1949, and the teleomorph was described as Mycosphaerella ligulicola K.F. Baker, Dimock & L.H. Davis (Baker et al. 1949), from the Latin ligua; ray flower, and colo; to inhabit. At that time, Baker et al. (1949) noted that a similar fungus, Sphaerella chrysanthemi Tassi, had previously been isolated from C. marginatum in Italy (Tassi 1900). However, the description of Sphaerella chrysanthemi was too ambiguous for Baker et al. (1949) to determine its relationship with M. ligulicola. Baker et al. (1949) did not examine the type specimen of Sphaerella chrysanthemi, and stated that M. ligulicola was probably a distinct pathogen since ray blight had not been reported outside the USA (Baker et al. 1949). Mycosphaerella ligulicola was later re-classified as Didymella ligulicola (Baker, Dimock & Davis) Von Arx (Müller and Von Arx 1962), based on the presence of non-fasciculate asci that arose between pseudoparaphyses (in pseudothecia) and contained ascospores constricted at the septum (Müller and Von Arx 1962). In 1963, ray blight of chrysanthemum was reported in Italy to be caused by D. ligulicola, and Sphaerella chrysanthemi was proposed to belong to a different species (Gambogi 1963). Upon re-examination of Sphaerella chrysanthemi specimens, Garibaldi and Gullino (1971), although unable to find asci, renamed the species as Didymella chrysanthemi (Tassi) Garibaldi & Gullino, on the basis of empty ascocarps and loose Stagonosporopsis spp. associated with chrysanthemum and pyrethrum 563 Fig. 1 Stagonosporopsis tanaceti (CBS 131484). a drooping flower head; b pycnidia on pyrethrum seed; c close-up of a pycnidium, showing darkened ostiolar area; d colony sporulating on V8 agar; e colony sporulating on oatmeal agar; f chlamydospores in chain; g conidia. (Scale bars = 20 μm) spores, and listed D. ligulicola as a synonym. They also referred to A. chrysanthemi as a synonym for Phoma chrysanthemi Voglino (1902), a pathogen of C. indicum in Italy. The synonymy proposed by Garibaldi and Gullino (1971) was later adopted extensively in the literature, assuming identity between M. ligulicola (anamorph A. chrysanthemi) described in the USA, and D. chrysanthemi (anamorph P. chrysanthemi) reported in Europe (Boerema and Bollen 1975; EPPO 1980, 1982; Punithalingam 1980; Richardson 1979; Van Steekelenburg 1978; Vlasveld 1977). Interestingly, Sphaerella chrysanthemi was later transferred to Mycosphaerella chrysanthemi (Tassi) Tomilin (Tomilin 1979), suggesting that Tassi’s Sphaerella chrysanthemi were not identical to the pathogen causing chrysanthemum ray blight, which was a Didymella species. Walker and Baker (1983) subsequently examined specimens from Tassi’s collection and found several ascocarps with mature asci and ascospores, and provided a detailed description which confirmed re-classification of Sphaerella chrysanthemi to Mycosphaerella chrysanthemi (Tassi) Tomilin. Walker and Baker (1983) were unable to locate type specimens of Phoma chrysanthemi Voglino, and stated that, based on the original description of P. chrysanthemi (Voglino 1902), there was no firm evidence that A. chrysanthemi Stevens and P. chrysanthemi Voglino were the same species. Therefore, they proposed the continued use of the binomials Didymella ligulicola and Ascochyta chrysanthemi for the pathogen causing ray blight on chrysanthemum (Walker and Baker 1983). Based on the introduction of new morphological criteria for differentiation of Ascochyta and Phoma (Boerema and Bollen 1975), A. chrysanthemi was renamed as Phoma ligulicola, and subsequently divided into two varieties based on a macrochemical reaction; the NaOH spot test (Van der Aa et al. 1990). Phoma ligulicola var. ligulicola, the type variety isolated from C. morifolium, produced a colourless antibiotic metabolite ‘E’ that upon application of NaOH oxidised to red pigment β on malt extract agar. The second variety did not produce metabolite ‘E’ and therefore no oxidisation occurred upon NaOH application, hence the name inoxydabilis. Van der Aa et al. (1990) proposed that Phoma ligulicola var. ligulicola was a specific pathogen of florists’ chrysanthemum described by Stevens (1907) in USA, while var. inoxydabilis was the cause of ray blight on various wild and cultivated Asteraceae in Europe. Subsequent molecular and morphological studies of Phoma and related genera resulted in re-classification of D. ligulicola as Stagonosporopsis ligulicola (Aveskamp et al. 2010). The genus Stagonosporopsis was originally separated from Ascochyta by Diedicke (1912) based on occasional formation of multi-septate (Stagonospora-like) conidia. In the phylogenetic reassessment of Didymellaceae (Aveskamp et al. 2010) based on the sequences of the large subunit (LSU) and Internal Transcribed Spacer (ITS) of the nrDNA, and beta-tubulin region, multiple Phoma species, including P. ligulicola, were recovered in a highly supported clade with the interpretive types of the genus Stagonosporopsis; S. actaeae (Boerema et al. 1997, 2004) and S. hortensis (Boerema and Verhoeven 1979; Clements and Shear 1931), and, thus, were reclassified as Stagonosporopsis (Aveskamp et al. 2010). The binomial Stagonosporopsis ligulicola was later changed by Vaghefi et al. (2012) due to preference of the epithet chrysanthemi over ligulicola. The epithet ligulicola had been introduced by Baker et al. (1949) to avoid confusion with Sphaerella chrysanthemi Tassi (1900). Later, when A. chrysanthemi was transferred to the genus Phoma, Van der Aa et al. (1990) maintained the epithet ligulicola to avoid homonymy with Phoma chrysanthemi Voglino (1902). Therefore, after transferring the species to Stagonosporopsis, the older epithet, ie, chrysanthemi, should have been re-proposed over ligulicola (Vaghefi et al. 2012). 564 Current taxonomic position of the ray blight pathogens Stagonosporopsis chrysanthemi is the current name for the pathogen described as the cause of ray blight of florists’ chrysanthemum by Stevens (1907) (Vaghefi et al. 2012). Based on a five-locus phylogeny including LSU, ITS, translation elongation factor 1-α, beta-tubulin and actin sequences, combined with morphological studies, Stagonosporopsis ligulicola var. inoxydabilis was elevated to species level as S. inoxydabilis. It differs from S. chrysanthemi by slower growth rate, less conidial variability and lack of pigment production after NaOH application (Vaghefi et al. 2012). Stagonosporopsis tanaceti was also described as the cause of ray blight of pyrethrum in Australia, which differs from S. chrysanthemi by absence of ascomata in culture, slower growth rate, wider and less variable conidia and no production of pigments upon NaOH application. Stagonosporopsis tanaceti shows morphological similarity to S. inoxydabilis but can be differentiated by faster growth rate, larger conidia, presence of chlamydospores, and lack of ascomata in culture (Vaghefi et al. 2012). Subsequent studies revealed that S. tanaceti has a different reproductive system to S. inoxydabilis and S. chrysanthemi, supporting its description as a distinct species (Vaghefi et al. 2015a). Mycosphaerella chrysanthemi (Tassi) Tomilin (Basionym: Sphaerella chrysanthemi Tassi; syn. Didymella chrysanthemi (Tassi) Garibaldi and Gullino) and Phomopsis chrysanthemi (Voglino) M.E.A. Costa and Sousa da Câmara (Basionym: Phoma chrysanthemi Voglino) are most likely different from the three Stagonosporopsis species currently known as the cause of ray blight on Asteraceae. The pathogenicity of Mycosphaerella chrysanthemi on chrysanthemum has not yet been determined and it may have been present on chrysanthemum foliage as a saprophyte (Walker and Baker 1983). Van der Aa et al. (1990) proposed that Phoma chrysanthemi Voglino (1902) may refer to S. inoxydabilis (P. ligulicola var. inoxydabilis) as S. chrysanthemi was not recorded in Italy until the 1960s (Gambogi 1963). However, Voglino’s description of the symptoms caused by P. chrysanthemi did not mention flower necrosis which is the hallmark symptom of the disease (Walker and Baker 1983). Moreover, the anamorphteleomorph relationship between M. chrysanthemi and P. chrysanthemi, proposed by Garibaldi and Gullino (1971) and later adopted by Boerema and Bollen (1975), has not yet been unequivocally established. Other Stagonosporopsis spp. reported from Asteraceae have included S. artemisiicola on Artemisia spp., S. dennisii on Solidago spp., S. dorenboschii on Callistephus sp., S. heliopsidis on Heliopsis spp. and Ambrosia artemisiifolia; and S. rudbeckiae on Rudbeckia spp. Little information exists on the pathogenicity of these species on their respective hosts, and their association with ray blight-type symptoms is unknown (Vaghefi et al. 2012). N. Vaghefi et al. Diagnostics Reliable detection and differentiation of the three Stagonosporopsis species associated with ray blight of Asteraceae based on only morphological and cultural characterisation is time consuming, requires a high level of experience and expertise, and may be impossible due to variability of morphological characters in culture (EFSA PLH Panel 2013; Vaghefi et al. 2012). Visual inspection of plant material for disease symptoms is also not a reliable method of diagnosis because S. chrysanthemi and S. tanaceti may be latently present within asymptomatic plant material (Chesters and Blakeman 1966; PWJ Taylor unpublished) or the symptoms may be confused with other diseases and disorders (EFSA PLH Panel 2013). Due to the inefficiency of using morphology for species identification, molecular markers have been developed for reliable identification of the ray blight pathogens. Although a DNA-based diagnostic assay based on the ITS sequence of S. chrysanthemi and S. tanaceti, was highly sensitive, it was not able to differentiate the two species (Pethybridge et al. 2004). Rapid and reliable identification of the Stagonosporopsis species associated with ray blight of Asteraceae can be achieved through multi-locus sequence typing (Aveskamp et al. 2010; Vaghefi et al. 2012) and also with a species-specific multiplex PCR assay developed by Vaghefi et al. (2016). This assay is based on a four-primer PCR that targets the intergenic spacer of the nrDNA of the ray blight pathogens, producing species-specific amplicons of ~ 560 in S. chrysanthemi, ~ 630 bp in S. inoxydabilis and ~ 400 bp in S. tanaceti, which can be easily differentiated on an agarose gel. Primer StagFd1 (5′-TGC ARA GTA CAM GGC AGA GG-3′) is common to the three ray blight pathogens, while ScSR2 (5′- CCA TTG ATT AAC GAT ACC TCG AC -3′), SiSR3 (5′- GGC ACG CAC AAT AAA GAG TG -3′) and StSR03 (5′- TAC CCT CAC CTT TAG GGG GAA T -3′) are specific to S. chrysanthemi, S. inoxydabilis and S. tanaceti, respectively. The high specificity of this assay h as be en c on f i r m ed by t e s t i ng ag a i ns t 2 1 o t h er Stagonosporopsis spp. and also 14 pathogenic and saprophytic fungal species associated with pyrethrum in Australia (Vaghefi et al. 2016). To date, the multiplex PCR assay has only been used for pathogen identification using DNA from pure fungal cultures. Once validated in planta, this assay may have the potential for pathogen detection in seed, symptomatic or asymptomatic plant material. Origin and global distribution Ray blight was originally detected in 1904 on florists’ chrysanthemum (Chrysanthemum × morifolium hybrids, syn. Chrysanthemum morifolium, C. grandiflorum, Dendranthema morifolium, D. grandiflorum), in North Stagonosporopsis spp. associated with chrysanthemum and pyrethrum Carolina, USA (Stevens 1907), and for many years remained restricted to North and South Carolina (Baker et al. 1961). The disease is believed to have spread within and outside of the USA following the expansion of the chrysanthemum industry, through global shipping of cuttings and flowers (Baker et al. 1949, 1961). The first reports of ray blight of chrysanthemum outside the USA were in Japan (Fujioka 1952) and New South Wales, Australia (Anonymous 1955). Ray blight was reported in England in 1959 on chrysanthemum plants that had been grown from cuttings imported from the USA (Baker et al. 1961), and later throughout Europe. An overview of the initial international spread of ray blight is given by Baker et al. (1961). Ray blight symptoms on pyrethrum were first reported in Tanzania (Peregrine and Watson 1964), and later in Kenya (Robinson 1963) and Papua New Guinea (Shaw 1984). However, these reports pre-date the division of the causal agent into two varieties (Van der Aa et al. 1990). Therefore, it is unclear if these reports relate to S. inoxydabilis (syn. P. ligulicola var. inoxydabilis) or S. chrysanthemi (syn. P. ligulicola var. ligulicola). Stagonosporopsis tanaceti has thus far only been reported in Australia and associated with substantial crop loss on pyrethrum (Hay et al. 2015; Pethybridge and Wilson 1998). Its distribution within Australia is not known, however, population genetic studies using microsatellite markers detected a probable second introduction of S. tanaceti into Tasmanian pyrethrum fields in 2012, which suggested the presence of an unknown wild source of S. tanaceti in Australia (Vaghefi et al. 2015b). Based on the phylogenetic affinity of the three ray blight pathogens, it is plausible that S. tanaceti may have originated outside of Australia, and was introduced to Australia either on propagative material imported from India in the 1980s or earlier, such as pyrethrum plants imported from Japan, UK and the USA in the 1930s (Bhat and Menary 1984) or Austria in 1890s (Von Mueller 1895). Stagonosporopsis chrysanthemi may have been indigenous to Japan (Van der Aa et al. 1990; De Gruyter et al. 2002; Boerema et al. 2004) as East Asia is known as the centre of origin for Chrysanthemum (Da Silva 2003). Although many fungal species originate from their hosts’ centre of origin (McDonald et al. 2012), the centre of origin of the host and pathogen do not necessarily correspond (Pérez et al. 2008; Zaffarano et al. 2006). An important step in understanding the origin and global migration paths of the ray blight pathogens is to elucidate their global distribution, which may be achieved using speciesspecific molecular markers (Vaghefi et al. 2016). Global populations of the ray blight pathogens from both cultivated and wild Asteraceae would enable comparative population genetic studies to be conducted to better understand their origins and evolutionary histories. 565 Host range Peregrine and Watson’s (1964) report of ray blight on pyrethrum (T. cinerariifolium) in Tanzania was the first report of the disease on a host other than florists’ chrysanthemum. Subsequent assays by Chesters and Blakeman (1967) reported that soilborne inoculum of the ray blight pathogen of chrysanthemum was mildly pathogenic to globe artichoke (Cynara scolymus), rudbeckia (Rudbeckia hirta), zinnia (Zinnia elegans), sunflower (Helianthus annuus) and dahlia (Dahlia variabilis), while highly pathogenic to lettuce (Lactuca sativa var. crispa). These findings should be interpreted with caution as the study was conducted prior to the separation of the species into different varieties (Van der Aa et al. 1990); therefore, the identities of the species used in this study is uncertain. Stagonosporopsis chrysanthemi (syn. Phoma ligulicola var. ligulicola) has been long assumed to be a specific pathogen of florists’ chrysanthemum (Boerema et al. 2004; Van der Aa et al. 1990). However, recent studies showed that S. chrysanthemi was able to infect pyrethrum (Vaghefi et al. 2016), albeit under in vitro conditions. Stagonosporopsis inoxydabilis was reported to infect various wild and cultivated Asteraceae including T. cinerariifolium, T. parthenium, Z. elegans and Matricaria sp. (Boerema et al. 2004; Van der Aa et al. 1990). Stagonosporopsis tanaceti caused disease on Tagetes patula and C. carinatum in glasshouse trials but, in contrast to S. inoxydabilis, failed to infect Z. elegans (Pethybridge et al. 2008a). Role of sexual reproduction in disease cycles Stagonosporopsis chrysanthemi and S. inoxydabilis are capable of sexual reproduction. Their homothallic nature was established based on single-conidium isolates producing pseudothecia (Fig. 2) (Van der Aa et al. 1990; Baker et al. 1949), and was later confirmed by detection of the two alternate mating type genes (MAT1-1-1 and MAT1-2-1) in their genome (Chilvers et al. 2014; Vaghefi et al. 2015a). Stagonosporopsis chrysanthemi has been reported to produce ascomata only in vivo while S. inoxydabilis form ascomata in both culture and in vivo (Van der Aa et al. 1990). This has been used as a criterion for species recognition. For example, it was suggested that ray blight of wild chrysanthemum, C. boreale, reported by Kim et al. (2001), was likely caused by S. inoxydabilis since the pathogen produced pseudothecia in culture (EFSA PLH Panel 2013). However, in our experience, some S. chrysanthemi strains produced pseudothecia in culture as well as in vivo (unpublished data); therefore, formation of pseudothecia in culture cannot be used as a criterion for species recognition. In the S. chrysanthemi-chrysanthemum pathosystem, the sexual form plays an important role in ray blight epidemiology since 566 N. Vaghefi et al. Fig. 2 Stagonosporopsis chrysanthemi. a close-up of a pycnidium, showing darkened ostiolar area (CBS 500.63); b pseudothecium on pyrethrum petiole (CBS 500.63), with darkened ostiolar area; c conidia (CBS 500.63); d-e asci and ascospores (DSMZ 62547); f ascospores (CBS 500.63) (ascospore sheath visible in f). (Scale bars = 20 μm) pseudothecia are the major overwintering structures (McCoy and Dimock 1973). Ascospores are also responsible for long-distance dispersal, as their active discharge helps them pass through the laminar boundary layer of the air surrounding the plant for transport by wind currents (McCoy 1973; McCoy and Dimock 1973). Stagonosporopsis tanaceti, on the other hand, was suggested to be heterothallic based on the discovery of only one mating type gene (MAT1-1-1) in its de novo assembled genome (DDBJ/EMBL/Gen Ban k acc ession nu mber JUDZ00000000; Wingfield et al. 2015) and the sole presence of MAT1-1 isolates in the S. tanaceti population in Australia (Vaghefi et al. 2015a). However, a heterothallic mode of reproduction for S. tanaceti cannot be unequivocally confirmed as isolates with the alternate mating type gene (MAT1-2-1) have not yet been identified but may be present in low numbers. Alternatively, S. tanaceti could well be an asexual species that has lost the MAT1-2-1 gene. Availability of a global population of S. tanaceti may help elucidate the mating system through potential discovery of the second mating type. Disease cycle and epidemiology Stagonosporopsis chrysanthemi In the S. chrysanthemi-chrysanthemum pathosystem, ascospores are considered the principal primary inoculum that initiates infections throughout the growing season. Ascospores emerge from pseudothecia; the overwintering structures that survive low winter temperatures in plant debris, and discharge windborne ascospores throughout the growing season (Baker et al. 1949). Ascospores are also dispersed over long distances and can infect neighbouring fields (Blakeman and Hadley 1968). The optimum temperature for pseudothecia formation is 21 °C, and no pseudothecia formation occurs at temperatures more than 24 °C (McCoy 1973; McCoy and Dimock 1973). Arid conditions are more favourable for development of pseudothecia and ascospores while high relative humidity favours production of pycnidia and splash-dispersed pycnidiospores (Baker et al. 1949; McCoy et al. 1972). Pycnidia can also develop under very dry conditions (eg 18 weeks at 6 % relative humidity) within a wide range of temperatures (Blakeman and Hadley 1968). Conidia exude in gelatinous drops (cirri) and are dispersed by rain splash, irrigation water or by the cloth covers that are often pulled over chrysanthemum plants to regulate day length. Under high relative humidity, conidia infect the petals within 6 h at a wide range of temperatures (3–30 °C) but the optimum temperature is 26 °C (McCoy and Dimock 1972). Germinating conidia penetrate the host directly through or between the epidermal cells. A branched mycelium rapidly grows intra- and intercellularly through the tissue, causing necrotic lesions (Baker et al. 1949; Blakeman and Dickinson 1967). Pycnidiospores are discharged throughout the growing season, resulting in multiple infection cycles and polycyclic epidemic progression. Although pseudothecia are considered the predominant overwintering structures, pycnidia produced on plant debris were also reported to survive winters with a minimum temperature of -29 °C, and produce viable pycnidiospores in spring (Baker et al. 1949). In addition to ascospores, another important means of longdistance spread of S. chrysanthemi is shipment of infested plant cuttings, as the pathogen is capable of surviving epiphytically on roots of chrysanthemum cuttings and 12 other plant species (Chesters and Blakeman 1966). Stagonosporopsis spp. associated with chrysanthemum and pyrethrum Stagonosporopsis chrysanthemi has been reported to produce loose sclerotia that survive in soil for at least 30 weeks over which infectivity declines (Blakeman and Hornby 1966). However, the EFSA PLH Panel (2013) noted that sclerotia or other thick-walled survival structures such as chlamydospores were not reported in earlier literature from studies conducted within the USA (Stevens 1907; Baker et al. 1949, 1961), or in the morphological description of S. chrysanthemi (Boerema et al. 2004; De Gruyter et al. 2002; Van der Aa et al. 1990). Therefore, the description of these structures in some European publications should be treated with caution due to potential confusion with the related species, S. inoxydabilis or M. chrysanthemi. 567 Chlamydospores (Fig. 1) and loose sclerotia are formed in culture and in vivo (Pethybridge and Wilson 1998; Vaghefi et al. 2012) but their role in the disease cycle is unknown and their survival in soil or plant debris has not been investigated (Pethybridge et al. 2008b). No sexual form has been found in Australian pyrethrum fields and the role of a sexual stage in the disease cycle is not yet known (Pethybridge and Wilson 1998; Jones 2009). The presence of only one mating type in Australian pyrethrum fields, combined with the evidence of linkage disequilibrium, high level of clonality and lack of recombinant multi-locus genotypes in population genetic studies (Pethybridge et al. 2012; Vaghefi et al. 2015b), also suggested that sexual reproduction was absent in the Australian pyrethrum fields. Stagonosporopsis tanaceti The epidemiology of S. tanaceti in Australian pyrethrum fields is markedly different to the S. chrysanthemi-chrysanthemum pathosystem, due to different reproductive systems as well as differences between the chrysanthemum and pyrethrum production systems. Pyrethrum production in Australia is semi-perennial and fields are established by seed in late winter and early spring (July to September). Flowering stems develop in the subsequent spring, and flowers are harvested thereafter in summer (15–18 months after planting December and January). Plants continue growing after the first harvest, become semidormant in winter, and produce flowering stems again in the spring and flowers that are harvested annually for 3–5 years (Pethybridge et al. 2008b). Infected seed is considered to be the primary source of inoculum when pyrethrum fields are established. This is supported by spatiotemporal analyses and multivariate modelling of epidemics as well as the high incidence of ray blight infection in pyrethrum seed lots (Pethybridge et al. 2005a, 2006, 2011). Stagonosporopsis tanaceti infects the outer and inner layers of the seed coat, and is transmitted to the developing embryo and cotyledon during germination. Depending on the inoculum intensity as well as environmental conditions, this may result in seedling death or growth of symptomatic seedlings. Also, the pathogen may transition into a quiescent phase in the developing seedling, and cause symptoms under favourable conditions (Bhuiyan et al. 2016). Pycnidia develop on the infected plant tissue and produce rain splash dispersed pycnidiospores, which cause multiple secondary infections of leaves, stems and flowers throughout the growing season (Pethybridge et al. 2005a, 2008b). Under humid conditions, pycnidiospores germinate and penetrate into the epidermal cells directly within 12 h, resulting in pin point necrotic spots after 24 h, and lesions subsequently develop by intra- and intercellular hyphal colonisation (Bhuiyan et al. 2015). Stagonosporoposis tanaceti pycnidia survive the mild Australian winters on diseased plants and contribute to disease initiation in subsequent years (Pethybridge et al. 2011). Disease management Stagonosporopsis chrysanthemi-chrysanthemum pathosystem Methods used to control ray blight of chrysanthemum include propagation of disease-free material, soil treatment by steam or chemicals, fungicide application, and spacing of plants to improve ventilation. Also, surface watering instead of overhead irrigation and removal, burying or burning of infected material and plant debris have also been used as cultural control methods (Fox 1998; Horst and Nelson 1997). M a n y f u n g i c i d e s a r e e ff e c t i v e i n c o n t r o l l i n g S. chrysanthemi, including benomyl, dicarboximide derivatives, propiconazole, iprodione and thiophanate-methyl (Baker et al. 1949; Crane et al. 1970; EFSA PLH Panel 2013; Engelhard 1984; Punithalingam 1980). Some S. chrysanthemi strains have developed resistance to benomyl and thiophanate-methyl (Punithalingam 1980). Little information is available on the effects of more recent fungicides on S. chrysanthemi. The utility of fungicides applied regularly targeting the control of other fungal diseases of chrysanthemum such as white rust (EFSA PLH Panel 2013; Fides 2006), resulting in only sporadic outbreaks of ray blight on chrysanthemum. Resistance of Chrysanthemum × morifolium cultivars to ray blight was reported (Engelhard 1984; Green and Engelhard 1974), but there is little information on the levels of resistance within current commercial cultivars (EFSA PLH Panel 2013). Stagonosporopsis tanaceti-pyrethrum pathosystem Extensive research has been conducted towards the development of management strategies to minimise crop loss from ray blight in Australian pyrethrum fields. Sampling strategies have been developed to accurately assess disease incidence and severity (Pethybridge et al. 2007a, b, 2008c). Epidemiological studies 568 (Pethybridge et al. 2005a, 2006, 2009, 2011, 2013) have helped to better understand the spatiotemporal dynamics of outbreaks. Identification of environmental and host risk factors favouring epidemics (Pethybridge and Hay 2001; Pethybridge et al. 2009), combined with fungicide efficacy trials (Pethybridge et al. 2005a, 2008d, 2010, 2013; Jones et al. 2007; Hay et al. 2015), have also assisted in the development of holistic management plans that have increased pyrethrin yield up to 80 % (Pethybridge et al. 2005b, 2008d, 2010, 2013). Currently, ray blight management in pyrethrum depends heavily on the use of fungicides, as no sources of resistance have been identified (Pethybridge et al. 2008b). Seed are treated with fungicides prior to planting to reduce seed borne inoculum (Pethybridge et al. 2006). Fields may receive up to 11 applications of fungicides in the first 18 months before the first harvest for management of ray blight and multiple other fungal diseases. This includes autumn and winter fungicide applications (three times in two-week intervals) as well as prophylactic use of fungicides in spring, when new leaves and young flowering stems are developing (Pethybridge et al. 2010, 2013). Reduced plant density and use of anecdotally less susceptible pyrethrum cultivars are other options that should be considered for disease management, especially in those fields with high risk sitespecific factors (Pethybridge et al. 2009). Concluding remarks Currently, only three morphologically similar and phylogenetically closely-related Stagonosporopsis species; S. chrysanthemi, S. inoxydabilis and S. tanaceti are known to be associated with ray blight symptoms on Asteraceae. Further research is required on other Stagonosporopsis or Phoma-like species isolated from various Asteraceae to establish pathogenicity on respective hosts and association with ray blight-type symptoms. Although recent molecular studies have helped elucidate the taxonomy and biology of the three Stagonosporopsis spp. associated with ray blight of Asteraceae (Vaghefi et al. 2012, 2015a, b), their origin, global distribution and host range is yet to be fully understood. The recently developed molecular diagnostic assay (Vaghefi et al. 2016) provides a rapid and robust tool for investigating the global distribution of these important quarantine pathogens. Understanding the geographical distribution of the ray blight pathogens is of paramount importance to the Australian pyrethrum industry. Multiple reports of chrysanthemum ray blight suggest that S. chrysanthemi and/or S. inoxydabilis may be present in Australia (Anonymous 1955; Oxenham 1963; Simmonds 1996). This requires further investigation, as the aforementioned species are capable of infecting pyrethrum (Vaghefi et al. 2016) and thus may pose a biosecurity threat to the Australian N. Vaghefi et al. pyrethrum industry. Supporting evidence suggesting the presence of an unknown, wild source of S. tanaceti was also obtained from population genetics studies using microsatellite markers (Vaghefi et al. 2015b). The presence of a permanent or wild host for S. tanaceti may increase the effective population size of the pathogen, which may enhance its variability and persistence by reducing the severity of bottlenecks and decreasing genetic drift (Barrett et al. 2008). Also, this has severe consequences for the Australian pyrethrum industry in terms of introducing new genetic diversity and/or a potential second mating type (MAT1-2-1). Regular surveillance and monitoring of pyrethrum fields is hence required to detect potential emergence of the alternate mating type or additional Stagonosporopsis spp. Since the MAT genes are determinants of sexual reproduction, they are believed to play a pivotal role in speciation events. In some species complexes, co-occurrence of species on the same hosts is argued to be the cause and result of sympatric speciation due to recombination events at the MAT locus (Woudenberg et al. 2012). Recombination (fusion or deletion) of MAT genes can cause reproductive barriers between isolates, and eventually, result in the establishment of new species (Arzanlou et al. 2010; Woudenberg et al. 2012). Due to the phylogenetic affinity of S. tanaceti, S. chrysanthemi and S. inoxydabilis, and their ability to coinfect the same hosts, a similar scenario for these species may be possible. This is supported by the presence of a small fragment with high similarity to S. chrysanthemi and S. inoxydabilis MAT1-2-1 ORF adjacent to the MAT1-1-1 ORF in S. tanaceti (Vaghefi et al. 2015a). This fragment could be a remnant of a functional MAT1-2-1 ORF in a hypothetical homothallic ancestor with a MAT locus similar to that of S. chrysanthemi and S. inoxydabilis. Two possibilities may have occurred, a deletion in the MAT1-2-1 ORF of the hypothetical ancestor may have left the MAT locus dysfunctional, resulting in an asexual S. tanaceti; or separation of the MAT11-1 and MAT1-2-1 ORFs may have given rise to a heterothallic S. tanaceti. Global populations of the ray blight pathogens from both cultivated and wild Asteraceae will help elucidate the reproductive system of S. tanaceti, and will also enable comparative population genetic studies to better understand their origins and evolutionary histories. Acknowledgments We thank Mr Tim Groom, Botanical Resources Australia – Agricultural Services Pty. Ltd., for constructive discussions. This project was supported by Botanical Resources Australia – Agricultural Services Pty. Ltd. 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