Introduction

Ray blight is a major disease of Asteraceae (Stevens 1907; Vaghefi et al. 2012), causing severe losses to the chrysanthemum (Chrysanthemum × morifolium) and pyrethrum (Tanacetum cinerariifolium) industries worldwide (Baker et al. 1949; Pethybridge et al. 2008b). The most conspicuous symptom of the disease in both hosts is discolouration and distortion of the flower buds and ray florets (Stevens 1907). Three morphologically similar but phylogenetically distinct species, Stagonosporopsis chrysanthemi, S. inoxydabilis and S. tanaceti have been found associated with ray blight of Asteraceae (Vaghefi et al. 2012), collectively referred to as ‘ray blight pathogens’.

Stagonosporopsis chrysanthemi (syn. Phoma ligulicola var. ligulicola) is considered a host-specific pathogen to chrysanthemum with a worldwide distribution (Boerema et al. 2004). Stagonosporopsis inoxydabilis (syn. P. ligulicola var. inoxydabilis) infects various Asteraceae genera including Tanacetum, Zinnia and Matricaria (Boerema et al. 2004; van der Aa et al. 1990). Both pathogens are important quarantine organisms in many European countries (EPPO 2015), however, due to historical confusion in their taxonomy (Vaghefi et al. 2012; Walker and Baker 1983), the reported host range and geographical distribution should be treated with caution (Rossi et al. 2014).

The taxonomy of the ray blight pathogens has undergone many changes, an extensive review of which is presented by Vaghefi et al. (2012). The pathogen causing ray blight of chrysanthemum was initially described as Ascochyta chrysanthemi (Stevens 1907), which was later reduced to a synonym of Phoma chrysanthemi (Garibaldi and Gullino 1971). This synonymy was later rejected by Walker and Baker (1983). In 1990, the pathogen was reclassified as Phoma ligulicola, and was divided into two varieties, namely var. ligulicola and var. inoxydabilis (Van der Aa et al. 1990). Studies on the host range of S. chrysanthemi and S. inoxydabilis (Chesters and Blakeman 1967; Peregrine and Watson 1964; van der Aa et al. 1990) were conducted prior to the separation of the two varieties of P. ligulicola, thus require further verification.

The two varieties of P. ligulicola were later elevated to species level and renamed as S. chrysanthemi and S. inoxydabilis (Vaghefi et al. 2012). A third species, S. tanaceti, was described as the ray blight of pyrethrum in Australia (Vaghefi et al. 2012). Stagonosporopsis tanaceti, also regarded as a quarantine pathogen in Europe (EPPO 2015), has only been reported in Australia associated with substantial crop losses in pyrethrum (Hay et al. 2015; Pethybridge and Wilson 1998; Pethybridge et al. 2007; Vaghefi et al. 2012). In glasshouse trials, S. tanaceti was also able to cause disease on Tagetes patula and Chrysanthemum carinatum (Pethybridge et al. 2008a).

Stagonosporopsis chrysanthemi and S. inoxydabilis are not yet known to occur in Australia, and may be regarded as a biosecurity threat to the Australian pyrethrum industry if able to infect and cause disease on pyrethrum. Both pathogens are homothallic (Chilvers et al. 2014; Vaghefi et al. 2015a) and the environmental conditions conducive to their sexual reproduction are frequently observed in Tasmanian pyrethrum production areas (Pethybridge et al. 2008b). Therefore, if an incursion were to occur, rapid establishment and dissemination of S. chrysanthemi and S. inoxydabilis in Australian pyrethrum fields is highly probable (Pethybridge et al. 2008b). Understanding the ability of the aforementioned species to infect pyrethrum is, therefore, a high priority. Moreover, ray blight of chrysanthemum was previously recorded in Australia (Oxenham 1963; Simmonds 1996); therefore, an endemic source of S. chrysanthemi may exist.

Despite the biosecurity importance of the ray blight pathogens in Europe and Australia, no reliable assay is available for their rapid detection and identification. Current quarantine measures for identification of S. chrysanthemi rely on visual inspection of the plant material, cultural and morphological identification of the pathogen and NaOH spot test (production of red pigments following NaOH application, indicating the presence of metabolite ‘E’; van der Aa et al. 1990) (EFSA PLH Panel 2013). However, visual inspection of plant material is not a reliable method of detection as S. chrysanthemi infection may be latent (Chesters and Blakeman 1966), and symptoms may be easily confused with other diseases and disorders (EFSA PLH Panel 2013). Differentiation of S. inoxydabilis and S. tanaceti based on morphology is also problematic and requires a high degree of expertise (Vaghefi et al. 2012). The need for a reliable molecular diagnostic assay for identification and differentiation of the three Stagonosporopsis species associated with ray blight of Asteraceae has been emphasised in several studies (EFSA PLH Panel 2013; Rossi et al. 2014; Vaghefi et al. 2012).

Of the small number of available sequences for Stagonosporopsis species (actin, β-tubulin, translation elongation factor 1-α, internal transcribed spacer and large subunit of ribosomal DNA), the actin sequence contained the highest variability and potential for species delineation (De Gruyter et al. 2012; Hyde et al. 2014; Vaghefi et al. 2012). Sequence information within the actin gene was used by De Gruyter et al. (2012) to develop TaqMan probes for the detection of S. andigena and S. crystalliniformis. However, our preliminary studies found that the actin sequence was not suitable for PCR-based differentiation of the three ray blight pathogens due to the limited number of consecutive nucleotide differences (unpublished data). The intergenic spacer (IGS) of the nuclear ribosomal DNA (nrDNA) is highly variable and well suited for discriminating closely related species, and has been used for development of highly sensitive diagnostic assays for multiple fungal pathogens (Chilvers et al. 2007; Liew et al. 1998; Muller et al. 2013; Sampietro et al. 2010; Suarez et al. 2005). In some species, however, the IGS sequence is variable at an intra-specific level (Appel and Gordon 1996; Dissanayake et al. 2009; Kawabe et al. 2005; Srinivasan et al. 2011); therefore, suitability of the IGS for diagnostic purposes and differentiation of the three ray blight pathogens requires validation.

The objectives of this study were to: i) investigate the ability of S. chrysanthemi and S. inoxydabilis to cause disease on pyrethrum; ii) determine the suitability of the IGS region for diagnostic purposes for discriminating the three Stagonosporopsis spp. associated with ray blight; and iii) develop a multiplex PCR assay for detection and identification of the target Stagonosporopsis spp. in a single reaction.

Materials and methods

Pathogenicity assay

Pyrethrum plants

An in vitro system was developed to establish pathogen-free pyrethrum plants in a quarantine incubator at the Australian Quarantine and Inspection Services (AQIS) approved premises at the Faculty of Veterinary and Agricultural Sciences, the University of Melbourne, Australia. Seed of variety ‘BR1’ were surface-sterilised by shaking in 1 % (ai) sodium hypochlorite for two minutes, and rinsing in sterile distilled water three times. Air-dried seed were placed on Potato Dextrose Agar (PDA) (5 seeds/Petri dish) and incubated for two weeks at 22 °C under a 12 h photoperiod. Seed were checked daily and contaminated seed removed. After two weeks, single pathogen-free seedlings were transferred to double-autoclaved peat pellets (Jiffy-7® pot; 3 × 3 cm pots containing 75 % sphagnum peat and 25 % coir fibre), and individually placed in transparent screw cap plastic containers under sterile conditions. Plantlets were incubated at 20 ± 2 °C with a 12 h photoperiod. One millilitre liquid fertiliser (Miracle-Gro MaxFeed, Australia) was added to the containers every month. Three-month-old plants were used for pathogenicity trials.

Fungal isolates and inoculum preparation

The ex-holotype strain of S. inoxydabilis (CBS 425.90) and reference strain of S. chrysanthemi (CBS 500.63; Boerema et al. 2004) were cultured on PDA and incubated at 22 °C under constant white light to induce sporulation. The ex-holotype strain of S. tanaceti (CBS 131484 = TAS1 = isolate PL1 in Jones (2009)), reported to be moderately aggressive on pyrethrum (Jones 2009), was cultured on V8 agar (200 mL V8 juice in 1 L sterile water, pH 6.25) and incubated at 22 °C under constant white light. Ten-day-old cultures were flooded with sterile distilled water and pycnidiospores were released by gently scraping with a sterile scalpel. The suspension was filtered through a sterile muslin cloth, and the spore concentration was adjusted to 104 spore mL−1 using a haemocytometer (Assistent®, Germany). Tween 20 was added to the inoculum at a concentration of 0.02 % (v/v).

Inoculation

To investigate the ability of S. chrysanthemi and S. inoxydabilis to infect and cause disease on pyrethrum, a replicated inoculation trial was performed on three-month-old pyrethrum plants in individual pots. The trial was conducted in a completely randomised design with four replicate plants inoculated with each of the S. chrysanthemi and S. inoxydabilis species. Four plants inoculated with S. tanaceti served as positive control, and four negative control plants were treated with sterile water containing 0.02 % (v/v) Tween 20. Depending on the size of the leaf, one to five drops (20 μL) of inoculum were placed on each leaf. After inoculation, all plants were incubated at 20 ± 2 °C under a 12 h photoperiod, and assessed for symptoms daily for three weeks. Days to symptom development was recorded for each plant, and isolations were carried out from the resultant necrotic lesions (three isolations per plant) to verify that symptoms were caused by the Stagonosporopsis spp. For this, infected tissue was excised and surface sterilised (0.4 % ai sodium hypochlorite), cultured on 2 % water agar, and incubated at room temperature in darkness for 3 to 4 days. Resultant fungal mycelia were transferred to V8 and PDA for species identification.

Species-specific PCR assay development

Fungal isolates and DNA extraction

Stagonosporopsis chrysanthemi, S. inoxydabilis and S. tanaceti isolates used for sequencing the IGS region are shown in Table 1. Additional fungal species used for testing the specificity of the developed assays are shown in Table 2. These included multiple pathogenic and saprophytic fungal species associated with pyrethrum in Australia (Pethybridge et al. 2003; Hay et al. 2015). Genomic DNA of each fungal species was extracted using a DNeasy Plant Mini Kit (Qiagen, Australia) and visualised on ethidium bromide-stained 1 % agarose gels. Genomic DNA of 18 Stagonosporopsis species, derived from ex-type or reference cultures according to Boerema et al. (2004), were obtained from Centraalbureau voor Schimmelcultures (CBS), The Netherlands. DNA of S. citrulli was kindly provided by Dr. M. T. Brewer, University of Georgia, Georgia, USA. Dilutions of 2 ng/μL were prepared for all DNA samples for use in PCR.

Table 1 Isolates used for sequencing the entire IGS region of Stagonosporopsis spp. associated with ray blight of Asteraceae. Ex-type strains are shown in bold and with an asterisk. Voucher strains according to Boerema et al. (2004) are in bold
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Table 2 Isolates used for diagnostic assay specificity validation. Ex-type strains are shown in bold and with an asterisk. Voucher strains according to Boerema et al. (2004) are in bold
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IGS amplification and sequencing

To assess the suitability of the IGS region of the nrDNA for marker development (lack of intra-specific variation), the entire region in seven S. tanaceti strains, isolated from various localities and years (Table 1), were sequenced by primer walking. The entire IGS region of S. tanaceti, S. chrysanthemi and S. inoxydabilis was amplified using the primers LR12R and invSR1R (Table 3) using the High-Fidelity Velocity PCR kit (Bioline, Australia). Fifty microliter PCR reactions contained Hi-Fi buffer including MgCl2 at a final concentration of 2 mM (Bioline), 0.1 μM of each primer, 0.25 mM dNTPs (Bioline), 1 U Velocity DNA polymerase (Bioline) and 10 ng DNA template. The PCR cycle was 3 min at 98 °C, followed by 35 cycles of 30 s at 98 °C, 30 s at the 67 °C, 1 min at 72 °C, and a final extension of 10 min at 72 °C. This yielded products of ~3 kb in all species. PCR products were purified using a PCR purification kit (Qiagen, Australia) and sequenced at the Australian Genome Research Facility (AGRF, Melbourne, Australia). For sequencing the 5′ and 3′ ends of the IGS, primers LR12R and invSR1R were used, which provided ~700 bp from each end. Several nested primers (Table 3) were subsequently designed using Primer3 (Rozen and Skaletsky 1999) for sequencing the internal region (Fig. 1).

Table 3 Primers used for sequencing the entire IGS region in Stagonosporopsis chrysanthemi, S. inoxydabilis and S. tanaceti
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Fig. 1
figure 1

Schematic presentation of the partial ribosomal DNA large subunit (LSU) and the entire Intergenic Spacer (IGS) sequence in Stagonosporopsis tanaceti, S. chrysanthemi and S. inoxydabilis. Position and orientation of primers used for amplifying and sequencing the IGS as well as the species-specific primers are indicated by arrows bearing the primer numbers specified in Table 3. The position and orientation of the repeat units are indicated by black, white and grey triangles in each species. Distances and lengths of repeat units are to scale. Approximate nucleotide positions are indicated by scale at the top of the figure. The repeat unit sequences are 5′ – GCR TTA GTA GGT TGB GRM R – 3′ (small black), 5′ – GCR TTA GTA GGT TGY GRC R – 3′(grey), 5′ – GTR GAG CCC CTA GCT TTG GCG RGT ACC GCC CAA AGG KTT TGR GRG GTC ASG G – 3′ (large black) and 5′ – GGG GGG TAG RCG CCY KRG CWT AGG GGC TCG ACY GCC TGY ACT AAG CGA GCA KAC CGC CTA GAG TTR GGG G – 3′ (white) in S. tanaceti; 5′ – GCG TTA GTA GGT TGS GGS R-3′ (black) and 5′ – GCR TTM GTR GGY TGY GRC R – 3′ (grey) in S. chrysanthemi; 5′ – GCG TTA GTA GGT TGG GGM R – 3′ (black) and 5′ – GCR TYA GYA RGT TGH RRY A – 5′ (grey) in S. inoxydabilis

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Qualitative PCR assay development

Sequenced fragments were visualised, trimmed and assembled using Geneious Pro v. 7.1.3 (http://www.geneious.com, Kearse et al. 2012). The entire IGS sequences of the three Stagonosporopsis species were aligned using MAFFT v. 7 (Katoh et al. 2013), and a variable region within the IGS, downstream of the 28S rDNA, was targeted for assay development. Primer3 (Rozen and Skaletsky 1999) was used to design one forward primer common to the three species (StagFd1) and three species-specific reverse primers (ScSR2 for S. chrysanthemi, SiSR3 for S. inoxydabilis and StSR03 for S. tanaceti) that amplified specific-sized amplicons when paired with the forward primer (Fig. 1). The designed primers were checked for specificity to NCBI GenBank database to minimise the chance of non-specific priming, and were synthesised by Sigma-Aldrich, New South Wales, Australia.

Each of the species-specific primer pairs were tested in separate conventional PCR reactions for amplification in a range of temperatures with variable primer and MgCl2 concentrations, and specificity against the DNA of the other two Stagonosporopsis species. PCR mixtures contained 10 ng genomic DNA, MangoTaq reaction buffer (Bioline), 1 or 2 mM MgCl2, 0.1 or 0.2 μM of each primer, 0.1 mM dNTPs (Bioline) and 0.7 unit MangoTaq DNA polymerase (Bioline). The PCR cycle included 3 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at a range of annealing temperatures (58 to 63 °C), zero or 30 s at 72 °C, and a final extension of 3 min at 72 °C. Each series of amplification reactions included sterile MilliQ water as a negative control as well as genomic DNA from the reference strains of S. chrysanthemi (CBS 500.63), S. inoxydabilis (CBS 425.90) and S. tanaceti (CBS 131484) as positive and negative controls. PCR reactions were conducted in a MyCycler thermal cycler (Bio-Rad, Australia) and the products were visualised on ethidium bromide-stained 1 % agarose gels. The optimised PCR assay for the three species-specific primer pairs compromised 15 μL reactions containing 10 ng genomic DNA, MangoTaq reaction buffer (Bioline), 1 mM MgCl2, 0.1 μM of each primer, 0.1 mM dNTPs (Bioline) and 0.7 unit MangoTaq DNA polymerase (Bioline). The PCR cycle included 3 min at 95 °C, followed by 35 cycles of a two-step amplification (30 s at 95 °C and 30 s at 60 °C), and a final extension of 3 min at 72 °C.

After ensuring the specificity of each primer pair for the target species at a range of temperatures, multiplex PCR reactions were carried out using a multiplex PCR kit (Qiagen, Australia). PCR mixtures included 0.8 or 1 × multiplex buffer (Qiagen), 0.3 or 0.6 μM of the forward primer (StagFd1), 0.1 or 0.2 μM of each reverse primer (StSR03, SiSR3 and ScSR2) and 10 ng genomic DNA. The PCR cycle included 5 min at 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s at a range of annealing temperatures (58 to 63 °C), 30 or 90 s at 72 °C, and a final extension of 5 min at 68 °C. PCR reactions were conducted in a MyCycler thermal cycler (Bio-Rad) and the products were visualised on ethidium bromide-stained 1 % agarose gels. The optimised multiplex PCR assay compromised 15 μL reactions of 0.8 × multiplex buffer (Qiagen), 0.3 μM of the forward primer (StagFd1), 0.1 μM of each reverse primer (ScSR2, SiSR3 and StSR03) and 10 ng genomic DNA. The PCR cycle was 5 min at 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s 60 °C, 30 s at 72 °C, and a final extension of 5 min at 68 °C.

The optimised multiplex PCR assay was further employed to verify the identity of 407 S. tanaceti isolates collected from commercial pyrethrum fields along the northern coast of Tasmania, Australia (Vaghefi et al. 2015b). Also, five additional S. chrysanthemi isolates and two additional S. inoxydabilis strains imported from international culture collections (Table 2) were screened using the multiplex PCR assay to ascertain the absence of intra-specific length variation in the PCR products.

Validation of the species-specific PCR assays

The conventional uniplex and multiplex PCR assays were tested for specificity, sensitivity, repeatability and reproducibility; the key performance criteria outlined by the OEPP/EPPO guideline (EPPO 2010) for validation of diagnostic assays. Specificity of each assay was tested against 10 ng genomic DNA from 21 Stagonosporopsis spp. as well as 14 fungal species associated with pyrethrum in Australia (Table 2), using PCR conditions described above, including 1 mM MgCl2, 0.1 μM of each primer, and an annealing temperature of 60 °C. To ensure that the DNA samples may be amplified by PCR, all samples were tested in an ITS PCR containing 10 ng genomic DNA, MangoTaq reaction buffer (Bioline), 2 mM MgCl2, 0.2 μM of each primer (V9G: 5′ – TTA CGT CCC TGC CCT TTG TA – 3′ and ITS4: 5′ – TCC TCC GCT TAT TGA TAT GC – 3′) (De Hoog and Gerrits van den Ende 1998; White et al. 1990), 0.1 mM of each dNTP (Bioline) and 0.5 U MangoTaq DNA polymerase (Bioline). Conditions for amplification included an initial denaturation step of 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 52 °C and 30 s at 72 °C, and a final denaturation step of 7 min at 72 °C. PCR reactions were visualised on ethidium bromide-stained 1 % agarose gels.

Sensitivity of the PCR assays was determined using two-fold serial dilutions of genomic DNA of S. chrysanthemi (CBS 500.63 and ICMP 2287), S. inoxydabilis (CBS 425.90) and S. tanaceti (CBS 131484 and DAR 70020) in sterile Type I water. The initial concentration of the DNA samples was determined using a Qubit fluorometer and a dsDNA BR Assay Kit (Life Technologies, Grand Island, NY, USA). Each of the PCR assays was conducted on a total of 8 ng to 4 fg genomic DNA to quantify the detection limit of each assay. A similar dilution series was prepared using a mixture of the DNA samples for testing the detection limit of the multiplex PCR assay.

Repeatability of the assays was tested by conducting the PCRs again under the same conditions and using the same reagents and PCR equipment as above. Reproducibility of the assays was investigated by performing the PCRs with minor variations, i.e., using different PCR reagents and equipment in a different laboratory. PCR reactions contained 10 ng genomic DNA, Standard PCR buffer (New England Biolabs Inc., Ipswich, MA, USA), 1 mM MgCl2 (New England Biolabs Inc.), 0.1 μM of each primer (synthesized by Integrated DNA Technologies Inc., CA, USA), 0.1 mM dNTPs (New England Biolabs Inc.), and 1 unit Taq polymerase (New England Biolabs Inc.). The PCR cycle was 3 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 60 °C, 30 s at 68 °C, and a final extension of 3 min at 68 °C. Each series of amplification reactions included sterile Type I water as a negative control. PCR reactions were conducted in a T100 thermal cycler (Bio-Rad, Hercules, CA, USA) and the products were visualised on a 1 % agarose gel amended with 0.5× (v/v) nucleic acid stain GelRed (Biotium Inc., Hayward, CA, USA). The multiplex assay was also performed as above, using 0.3 μM of the forward primer and 0.1 μM of each of the reverse primers.

Results

Pathogenicity assay

Stagonosporopsis chrysanthemi and S. inoxydabilis were pathogenic to pyrethrum and caused symptoms similar to S. tanaceti. Pyrethrum plants inoculated with S. inoxydabilis (CBS 425.90) and S. tanaceti (CBS 131484) developed symptoms after three to four days. The initial symptoms included small necrotic spots on the leaves. Plants inoculated with S. chrysanthemi (CBS 500.63) developed necrotic spots seven days after inoculation (Fig. 2). Subsequently, leaf spots expanded to larger lesions, which eventually killed the leaves and spread into the petioles. Two weeks after inoculation, plants started to develop necrosis at the base (crown) and white aerial mycelia were visible on most symptomatic leaves. All plants collapsed completely after three weeks. The three Stagonosporopsis species developed reproductive structures on diseased pyrethrum tissue (Fig. 3). Both pycnidia and pseudothecia were formed on plants inoculated with S. chrysanthemi and S. inoxydabilis while only pycnidia developed on the S. tanaceti-inoculated plants (Fig. 3). Isolation frequency of the Stagonosporopsis spp. from infected tissue was 100 %, confirming that the lesions were caused by the intended fungal species.

Fig. 2
figure 2

Pathogenicity of Stagonosporopsis chrysanthemi to pyrethrum in an in vitro inoculation trial; a necrotic spots on leaves one week after inoculation (wai), b necrotic lesions and dieback, c death of leaves and spread of necrosis into the petiole d necrosis in the crown area at 2 wai

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Fig. 3
figure 3

Pycnidia and pseudothecia development on pyrethrum plants inoculated with Stagonosporopsis spp.: a death of pyrethrum plants three weeks after inoculation with Stagonosporopsis chrysanthemi; b pseudothecia of S. chrysanthemi; c pseudothecia of S. inoxydabilis; and d pycnidia of S. tanaceti on pyrethrum petiole

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Species-specific PCR assay development

IGS amplification and sequencing

Sequencing of the entire IGS region resulted in a fragment of ~2.7 kb in Stagonosporopsis chrysanthemi and ~3.0 kb in S. tanaceti and S. inoxydabilis. The IGS sequences were identical among the seven isolates of S. tanaceti, and between the two S. chrysanthemi isolates. Alignment of the S. tanaceti IGS with S. chrysanthemi and S. inoxydabilis detected 78 % and 75 % sequence similarity, respectively. Sequences were deposited in the NCBI database (GenBank accession numbers KP161042 to KP161044).

Qualitative PCR assay development

The uniplex PCR assays amplified species-specific sized amplicons at a range of temperatures (Supplementary Fig. 1). Primers StagFd1 and StSR03 resulted in a ~ 400 bp band in S. tanaceti and no amplicon in either S. inoxydabilis or S. chrysanthemi over a range of annealing temperatures from 58 to 63 °C. Primers StagFd1 and SiSR3 amplified a band of ~630 bp in S. inoxydabilis and no band in either S. chrysanthemi or S. tanaceti over a range of annealing temperatures from 58 to 63 °C. Primers StagFd1 and ScSR2 resulted in a ~ 560 bp amplicon in S. chrysanthemi and no amplification in either S. inoxydabilis or S. tanaceti at 60 to 63 °C (Supplementary Fig. 1). The multiplex PCR assay produced single intense species-specific amplicons when DNA of only one species was included in the reaction; ~ 560 in S. chrysanthemi, ~ 630 bp in S. inoxydabilis and ~400 bp in S. tanaceti. All three amplicons were amplified when DNA of the three species were used in one reaction (Fig. 4).

Fig. 4
figure 4

Multiplex PCR assay enabling identification and differentiation of Stagonosporopsis tanaceti, S. chrysanthemi and S. inoxydabilis in one reaction. The first lane represents the 1Kb Plus DNA ladder (Life Technologies, Australia)

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Application of the multiplex PCR assay to screen 407 Stagonosporopsis isolates collected from pyrethrum fields in Tasmania detected a single ~400 bp amplicon for all the isolates, verifying their identity as S. tanaceti, and confirming lack of intra-specific length variation (data not shown). No intra-specific length variation was found among the amplicons from the six S. chrysanthemi or the three S. inoxydabilis isolates tested (Figs. 5 and 6).

Fig. 5
figure 5

Specificity test of the multiplex PCR assay using the DNA samples from pure cultures of 14 fungal species associated with pyrethrum in Australia. The first and last lanes in each gel are the 1Kb Plus DNA ladder (Life Technologies, Australia)

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Fig. 6
figure 6

Specificity test of the multiplex PCR assay against the DNA samples from pure cultures of Stagonosporopsis spp. The first and last lanes in each gel represent the 1Kb Plus DNA ladder (Life Technologies, Australia)

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Validation of the species-specific PCR assays

All PCR assays were specific to their target species. ITS amplification from the gDNA of the 14 fungal species associated with pyrethrum, as well as the imported DNA samples of the Stagonosporopsis spp., demonstrated that all the DNA samples were PCR-amplifiable (data not shown). No amplification resulted from the same DNA samples when used in the uniplex and multiplex species-specific assays (Figs. 5 and 6; Supplementary Fig. 2). Species-specific PCR assays using 2-fold dilution series of DNA samples from the target species detected DNA quantities as low as 4 fg (Supplementary Fig. 3). All assays were repeatable and reproducible, and the same results were obtained when repeating the assays under the same conditions, and when performing the assays in a different laboratory with changed PCR reagents, thermocycler and oligonucleotide supplier.

Discussion

Stagonosporopsis chrysanthemi and S. inoxydabilis were able to infect and cause disease on pyrethrum, with similar symptoms to those caused by S. tanaceti, resulting in plant death in only three weeks. This is the first report of S. chrysanthemi causing disease on pyrethrum. Due to quarantine restrictions, the pathogenicity trial was conducted in closed plastic containers in an incubator, creating a humid environment highly conducive to disease development, which may have accelerated the rate of symptom development and plant death. The timing of infection and severity of the symptoms may be different in a glasshouse assay or under field conditions. Further trials may provide more information on the timing and severity of infection in different hosts and environments.

A highly specific and sensitive multiplex PCR assay for the rapid and accurate detection and identification of the three quarantine Stagonosporopsis spp.; S. tanaceti, S. inoxydabilis and S. chrysanthemi, was developed from the IGS region of the nrDNA gene complex. Identification of Stagonosporopsis spp. based on morphological characteristics is time consuming, requires a high level of experience and expertise, and may not even be possible due to the high similarity of some phylogenetically-close Stagonosporopsis species as well as variability of morphological characters in vitro (EFSA PLH Panel 2013; Stewart et al. 2015; Vaghefi et al. 2012). This has resulted in the recent development of molecular methods for detection and identification of several important phytopathogenic Stagonosporopsis spp., using microsatellite loci and ITS and actin sequences (Brewer et al. 2015; De Gruyter et al. 2012; Pethybridge et al. 2004). The actin sequence could not be used for PCR-based differentiation of the three Stagonosporopsis spp. studied here due to the limited number of consecutive nucleotide differences, which resulted in cross-amplification at lower temperatures (data not shown). We targeted the IGS region of the nrDNA gene complex for species-specific marker development due to its high level of variability as well as high copy number, which enables development of highly specific and sensitive diagnostic assays (Chilvers et al. 2007; Liew et al. 1998; Muller et al. 2013; Sampietro et al. 2010; Suarez et al. 2005).

Although multiple copies of the IGS are known to evolve in a homogenous manner through concerted evolution (Nei and Rooney 2005), in some fungal species, intra-specific variation of the IGS has been reported (Appel and Gordon 1996; Dissanayake et al. 2009; Kawabe et al. 2005; Latha et al. 2003; Srinivasan et al. 2011). Sequencing the entire IGS region of seven S. tanaceti isolates and two S. chrysanthemi isolates detected no intra-specific nucleotide variation in these species. Another characteristic of the IGS sequence in Eukaryotes is the presence of repetitive elements (sub-repeat units) (Mirete et al. 2013; Pantou et al. 2003; Wang et al. 2012), which may differ in the copy number, resulting in the IGS size variability at an intra-specific (Jackson et al. 2000) or intra-genomic (Chang et al. 2008; Ganley and Scott 1998) level. Although the IGS sequences of S. chrysanthemi, S. inoxydabilis and S. tanaceti had a repetitive structure (Fig. 1), no intra-specific length variation was detected among the PCR amplicons from the six S. chrysanthemi isolates, three S. inoxydabilis isolates, or 407 S. tanaceti isolates tested in this study. This verified the suitability of the selected IGS region as a PCR target for detection and differentiation of the three ray blight pathogens.

The species-specific diagnostic PCR assays were highly specific, sensitive, repeatable and reproducible when tested against the DNA of pure fungal cultures. The multiplex PCR assay will provide a valuable tool for disease surveillance in Australian pyrethrum field, and may be used for screening of the Stagonosporopsis isolates collected from pyrethrum fields to enable timely detection of possible S. chrysanthemi or S.inoxydabilis incursions. Moreover, the S. tanaceti-specific assay may provide a rapid, reliable and sensitive method for pathogen detection in pyrethrum seed as infected seed is known to be a source of primary inoculum for ray blight epidemics in pyrethrum fields in Australia (Pethybridge et al. 2006). This application, however, requires in planta validation of the assay.

Once validated in planta, the developed assays may be further utilised for quarantine purposes in Australia, where S. chrysanthemi and S. inoxydabilis are not known to occur, or in Europe, where the three Stagonosporopsis spp. associated with ray blight of Asteraceae are listed as Level 2 quarantine pathogens (EPPO 2015). Current quarantine measures for importation of chrysanthemum propagative material to Australia and Europe are solely based on phytosanitary certificates, visual inspection of plant material and cultural and morphological identification of pathogen cultures (BICON 2016; EFSA PLH Panel 2013). The diagnostics assays may be incorporated into the AQIS or EPPO biosecurity processes to provide standard guidelines for rapid and accurate pathogen identification. This is of special significance for in planta detection of S. chrysanthemi or possible soil tests, as S. chrysanthemi is reported to be asymptomatically present in chrysanthemum cuttings (Baker et al. 1961; Chesters and Blakeman 1966; EFSA PLH Panel 2013) or as sclerotia in soil (Blakeman and Hornby 1966). Seedborne S. tanaceti inoculum infecting the germinating hypocotyls may also remain latent in pyrethrum crown tissue for weeks prior to the appearance of symptoms (P.W.J. Taylor, unpublished data). Sensitivity tests of the developed PCR assays for pathogen detection in symptomatic and asymptomatic plant tissue as well as infested seed or soil will further expand applications for biosecurity and quarantine purposes.