Host susceptibility and fungal virulence in the wheat-Parastagonospora nodorum interactions

doi:10.21203/rs.3.rs-3198290/v1

Download PDF

Research Article

Host susceptibility and fungal virulence in the wheat-Parastagonospora nodorum interactions

https://doi.org/10.21203/rs.3.rs-3198290/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

The fungus Parastagonospora nodorum is responsible for substantial economic losses of wheat worldwide. P. nodorum secretes multiple proteinaceous host-specific toxins known as necrotrophic effectors (NE) that induce compatible interactions with hosts possessing the corresponding dominant susceptibility (S) genes. In this study, the reaction of 40 wheat genotypes including 19 Iranian cultivars, 16 world cultivars and five differential genotypes against 33 P. nodorum isolates was investigated. In addition, the occurrence of known NEs and their corresponding S genes was assessed. Wheat genotypes were classified into six groups based on their variable reaction towards P. nodorum isolates. The S gene Tsn1 was present in 70% of highly susceptible cultivars and was completely absent in all resistant cultivars. The cultivars possessing all three S genes Tsn1, Snn1 and Snn3 were susceptible to all P. nodorum isolates. S gene Snn3 was detected in all Iranian wheat cultivars whereas the proportion of this gene in world cultivars was 62.5%. SnTox1-Snn1 was the most abundant NE-S gene frequency (100%) in Iranian P. nodorum isolates and Iranian cultivars. Furthermore, SnToxA was produced in Pichia pastoris and infiltration of SnToxA resulted in necrosis response exclusively on wheat cultivars harboring the Tsn1 sensitivity gene indicating that toxin infiltration can be used as a quick and accurate assay in breeding programs. Overall, these findings extended our substantial knowledge about P. nodorum-wheat interactions and their underlying molecular basis in Iran, a country located in the Fertile Crescent where both wheat and its pathogens coevolved.

Wheat glume blotch

Pathogenesis pattern

Necrotrophic effector

Sensitivity gene

Toxin infiltration

The ascomycete fungus Parastagonospora nodorum is the causal agent of stagonospora nodorum blotch (SNB) or glume blotch, one of the most devastating wheat diseases worldwide which causes significant yield loss up to 50% by reduction of thousand-kernel-weight (Duba et al., 2018). The symptoms of SNB appear on all aerial parts of the plant, including leaves, pods, stems, spikes, and awns. Initial symptoms develop as small brown spots surrounded by a yellow halo progressing to brown lens-shaped necrotic lesions. After tissue degradation, pycnidia may appear as dark brown to black dots on the surface of infected tissues (Solomon et al., 2006; Mehra et al., 2018).

P. nodorum is a model necrotrophic fungal pathogen that kills plant cells by producing proteinaceous toxins and cell wall-degrading enzymes. Multiple host-specific proteinaceous toxins known as necrotrophic effectors (NEs) are secreted by P. nodorum and interact with the corresponding plant sensitivity (S) gene products in a so-called inverse gene-for-gene manner, leading to host cell death and disease development (Oliver et al., 2012; McDonald et al., 2013). P. nodorum benefit from cell death by hijacking host immunity responses including PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) through the production of NEs (Shi et al., 2016). It has been suggested that NEs exploit the plant defense response in wheat lines possessing S genes causing susceptibility rather than resistance (Shi et al., 2016; McCombe et al., 2022; Peters Haugrud et al., 2022). So far, nine such interactions between P. nodorum NE and wheat S genes have been identified; SnToxA-Tsn1, SnTox1-Snn1, SnTox267-Snn2, SnTox3-Snn3-B1, SnTox3-Snn3-D1, SnTox4-Snn4, SnTox5-Snn5, SnTox267-Snn6, and SnTox267-Snn7. However, only five NE genes (SnToxA, SnTox1, SnTox3, SnTox5, and SnTox267) and three wheat S genes (Tsn1, Snn1, and Snn3-D1) have been cloned and studied at the molecular level to date (Peters Haugrud et al., 2022).

Global P. nodorum populations have been examined for their genetic diversity with both microsatellites (also called simple sequence repeats or SSRs) and NE loci, showing that Iranian isolates of P. nodorum harbor the most genetic diversity at neutral microsatellites loci and the highest number of NE alleles for SnToxA, SnTox1 and SnTox3 (Ghaderi et al., 2020). Iran is located in the Fertile Crescent. It is believed that wheat alongside with its pathogens co-evolved in the Fertile Crescent as a center of their origins (Stukenbrock et al., 2007), but to date our knowledge on the phenotypic and molecular basis of wheat-P. nodorum interactions in this region is limited. In this study, we investigated the virulence pattern of 33 different isolates of P. nodorum on 40 diverse wheat genotypes, including 19 Iranian cultivars, 16 world commercial cultivars, and a set of five SNB differential lines. The frequency of NE genes SnToxA, SnTox1, SnTox3, and SnTox5 in P. nodorum isolates as well as the proportion of S genes Tsn1, Snn1, and Snn3 in wheat genotypes were determined. Finally, a NE infiltration assay was used with SnToxA as a tool for the marker-assisted selection of wheat cultivars in breeding programs.

Plant materials

A total of 40 wheat genotypes, including 19 popular Iranian cultivars (Baharan, Barat, Chamran 2, Dena, Ehsan, Gonbad, Hana, Heidari, Khalil, Mehregan, Narin, Ofogh, Rakhshan, Sarang, Shabrang, Shavoor, Shoosh, Sirvan, Talaei); 16 world commercial wheat cultivars (3HD-126, 4B-160, Arina, Bulgaria, Coulter, Erik, ES-Federal, Katepwa, Kavkaz-k4500, KM7, Kulm, M-3, ND 495, Obelisk, Tadinia and Veranopolis); and a set of five SNB differential lines (Table 1) carrying known susceptibility genes were evaluated for their response to SNB.

Table 1

Wheat differential lines with known SNB sensitivity genes (dominant *SnnN*) used as a control in this study
Differential cultivar	Sensitivity/ non sensitivity genes
BG220	Snn3-B1
BG261	Tsn1
BR34	No sensitivity gene
Chinese Spring (CS)	Snn1
Grandin	Tsn1, Snn3-B1

Fungal materials

Spikes and leaves showing typical SNB symptoms (undetermined wheat cultivars) were collected from the main wheat-growing regions in Iran. Small pieces of necrotic tissues having pycnidia were surface sterilized for one minute in 70% ethanol solution, followed by rinsing in sterile distilled water and dried between two sheets of sterile filter paper. Infected segments were fixed on microscopic slides and kept in a humid chamber until the exudation of cirrhi containing pycnidiospores. The pycnidiospores were harvested using a sterilized needle and transferred onto V8-PDA (V8 juice, 200 ml; potato, 200 g; dextrose, 20 g; CaCO₃, 3 g; agar, 18 g per liter) and YSA (yeast extract, 10 g; sucrose, 10 g; CaCO₃, 3 g; agar, 18 g per liter) plates amended with 50 µg/ml chloramphenicol. Plates were incubated at 22°C for four to five days. Hyphal tips were then subcultured on new media to assess the morphological properties (Alllingham & Jackson, 1981). To stimulate pycnidia development, two special sporulation media were used; Czapek-Dox V8 agar (NaNO₃, 2 g; KCl, 0.5 g; MgSO₄, 0.5 g; FeSO₄, 0.01 g; K2HPO₄, 1 g; sucrose, 30 g; CaCO₃, 3 g; V8 juice, 200 ml; agar, 15 g per liter) and WSA (wheat germ powder, 40 g; sucrose, 20 g; CaCO3, 3 g; agar, 18 g per liter). About 2–3 cm pieces of autoclaved wheat straws were placed on the surface of each medium and isolates were cultured by transferring mycelial plugs grown on YSA onto the center of Czapek-Dox V8 and WSA plates containing wheat straw. The plates were incubated at 10°C under alternating 12-h NUV photoperiod/ 12-h darkness for three weeks (Douaiher et al., 2004). Pycnidiospores were stained using lactophenol cotton blue solution to assess morphological properties. Thirty-two isolates of P. nodorum, including eight isolates from each province were selected for pathogenicity assay and detection of NE genes (Table 2). P. nodorum reference isolate SN15 was obtained from Prof. Richard Oliver, Curtin University, Western Australia.

Table 2

Isolates of *Parastagonospora nodorum* used in this study
Isolate code	Collection region (Province/Town)
Pn-FN1	Fars/ Noor Abad
Pn-FN2	Fars/ Noor Abad
Pn-FN3	Fars/ Noor Abad
Pn-FN4	Fars/ Noor Abad
Pn-FN5	Fars/ Noor Abad
Pn-FN6	Fars/ Noor Abad
Pn-FN7	Fars/ Noor Abad
Pn-FN8	Fars/ Noor Abad
Pn-KG1	Kohgiluyeh Boyer-Ahmad/ Gachsaran
Pn-KG2	Kohgiluyeh Boyer-Ahmad/ Gachsaran
Pn-KG3	Kohgiluyeh Boyer-Ahmad/ Gachsaran
Pn-KG4	Kohgiluyeh Boyer-Ahmad/ Gachsaran
Pn-KG5	Kohgiluyeh Boyer-Ahmad/ Gachsaran
Pn-KG6	Kohgiluyeh Boyer-Ahmad/ Gachsaran
Pn-KG7	Kohgiluyeh Boyer-Ahmad/ Gachsaran
Pn-KG8	Kohgiluyeh Boyer-Ahmad/ Gachsaran
Pn-KhD1	Khuzestan / Dezful
Pn-KhD2	Khuzestan / Dezful
Pn-KhD3	Khuzestan / Dezful
Pn-KhD4	Khuzestan / Dezful
Pn-KhD5	Khuzestan / Dezful
Pn-KhD6	Khuzestan / Dezful
Pn-KhD7	Khuzestan / Dezful
Pn-KhD8	Khuzestan / Dezful
Pn-GAl1	Golestan/ Ali Abad
Pn-GAl2	Golestan/ Ali Abad
Pn-GAl3	Golestan/ Ali Abad
Pn-GAl4	Golestan/ Ali Abad
Pn-GAl5	Golestan/ Ali Abad
Pn-GAl6	Golestan/ Ali Abad
Pn-GAl7	Golestan/ Ali Abad
Pn-GAl8	Golestan/ Ali Abad
SN15	Australian Centre for Necrotrophic Fungal Pathogens (ACNFP), Curtin University, Western Australia

Inoculation and pathogenicity assay

Inoculation experiments were conducted in a factorial randomized complete block design with six replications under greenhouse conditions as described previously with some modifications (Liu et al., 2004). To do so, 32 Iranian isolates of P. nodorum plus reference isolate SN15 were grown on WSA-containing wheat straws as described above for further propagation to use as inoculum. Seeds of 40 different wheat cultivars were sown in eight centimeters diameter pots containing soil, leaf mold and peat moss in a ratio of 1: 1: 1 in six replications and kept under greenhouse conditions at an average temperature of 24°C until the three-leaf stage (about 14 days after sowing). The emerging first and third formed leaves were clipped off and the second leaf was subjected to the pathogenicity assay. To prepare P. nodorum inoculum, the pycnidiospores were harvested from the surface of the culture medium using a sterile scalpel and passed through a miracloth using a stream of sterile distilled water. Spore concentration was adjusted to 10⁶ spore/ ml^− 1 using a hemocytometer and supplemented with 0.15% Tween-20 as a surfactant. Seedlings were inoculated using a hand-held sprayer until runoff. Also, the same amount of sterile water containing 0.15% Tween-20 was sprayed on wheat genotypes as a negative control. To provide high relative humidity (RH), the plants were covered with transparent polyethylene bags for 48 hours in darkness. Afterward, the plastic bags were removed, and pots were placed on greenhouse benches covered with transparent plastic sheets under 85% RH, 25–27°C, and 16-h light/ 8-h dark conditions. Disease severity (DS) on the second leaf was visually scored seven days post inoculation (dpi) based on the percentage of leaf area covered with lesions. To improve the accuracy in recording data, infected leaves were photographed and subjected to digital image analysis using Color Analysis software for Android. To conduct Koch’s postulates, all 33 isolates of P. nodorum were re-isolated from randomly selected infected leaves using the method mentioned above and were morphologically characterized.

Statistical and cluster analysis

Data were subjected to the arcsin square root-transformation method to secure normal distribution. Statistical and cluster analysis of data were performed using SAS software v9.4. The General Linear Model (GLM) was employed based on the percentage of wheat leaf area infected by P. nodorum isolates under a factorial experiment in a randomized complete block design with six replications. Individual and combined analysis of variance for fixed effects isolate, genotype and their interaction were conducted, and the mean percentage of infected leaf area was compared by calculating the t-test least significant differences (LSD) value. Also, the data were subjected to a hierarchical cluster analysis using Ward’s method and Euclidean distance.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from both wheat leaves and P. nodorum mycelia using the CTAB method (Murray & Thompson, 1980). To confirm the morphological identification of P. nodorum, ITS4/5 primers were utilized for amplification of the internal transcribed spacer region (ITS) of the ribosomal DNA (White et al., 1990). For the detection of four necrotrophic effector genes (SnToxA, SnTox1, SnTox3, and SnTox5), and three wheat susceptibility genes (Tsn1, Snn1, and Snn3) specific primers were designed using Primer 3 online software (https://primer3.ut.ee/) (Untergasser et al., 2012) (Table S1) and employed in a multiplex PCR reaction. PCR amplifications were performed in a 15 µl reaction containing 30–40 ng of genomic DNA, 1x Taq DNA Polymerase, 2x Master Mix RED (Ampliqon, Cat. No. A190301), and 0.5 µM of each primer under the following conditions: initial denaturation at 95°C for 3 min, 35 cycles of denaturation at 95°C for 45 s, annealing at 57°C for 45 s, and extension at 72°C for 50 s with a final extension at 72°C for 10 min. PCR products were resolved on horizontal 1.2% agarose gel in 1x TBE buffer and visualized by ethidium bromide staining. To clarify the detection of necrotrophic/ sensitivity genes, one amplicon from each sample was sequenced by Pishgaman Co, Tehran, Iran. The sequences from this study were deposited in the NCBI GenBank database.

Heterologous expression of SnToxA in Pichia pastoris and toxin infiltration

For heterologous expression of SnToxA, the yeast strain Pichia pastoris X33 and the vector pGAPZa were used. The vector pGAPZa was first modified into a Gateway® compatible cloning for BP reaction. To do so, the donor vector pDONR™207 was used as a template to amplify the entire length of the ccdB gene along with two flanking regions of attP1 and attP2 with primer pairs ccdB_F_KpnI/ ccdB_R_SacII containing KpnI and SacII restriction sites, respectively (Table S1). Afterward, the resulting PCR product was double digested with KpnI/SacII restriction enzymes and inserted into KpnI/SacII digested pGAPZa vector using T4 DNA ligase yielding pRM147 according to the user manual (BioLabs inc., Cat No. M0202S). To generate an entry clone harboring SnToxA, the full length of the coding region of SnToxA cDNA was amplified using primer pairs attB1_ToxA_F/attB2r_ToxA_R (Table S1) to introduce the flanking recombination sites of attB1 and attB2 to each side of PCR amplicon and then inserted into the pRM147 (derived from pGAPZa) through BP reaction based on manufacturers instruction (Invitrogen, Carlsbad, CA, Cat. No. 11789020). Five micrograms of the expression construct were used for yeast transformation. The preparation and transformation of competent P. pastoris cells were done according to the user manual (Invitrogen, Carlsbad, CA, Cat. No. V205-20). Positive clones of P. pastoris were cultured in 100 ml of YPD liquid medium (1% yeast extract; 2% peptone, and 2% dextrose) amended with 100 µg/ml Zeocin™ and placed on a shaking incubator at 25°C and 150 rpm for seven days. P. pastoris cells were centrifuged at 7000 g for 5 min and the supernatant was used for toxin infiltration assays. The toxin was injected into the underside of wheat second leaves using a syringe without a needle. The reaction of wheat genotypes to the toxin infiltrates was assessed 72-h after injection. To ensure that the necrosis occurred only by toxin injection, YPD medium, as well as sterile distilled water, were also injected as negative controls.

Isolation and characterization of P. nodorum isolates from infected wheat

Ninety-two wheat samples presenting typical SNB symptoms were collected from the main wheat-growing regions in Iran (Fars, Kohgiluyeh Boyer-Ahmad, Khuzestan and Golestan provinces) in order to isolate P. nodorum (Fig. 1a). In total, thirty-two P. nodorum strains were successfully isolated. The fungal colonies grown on YSA medium were grayish-white with a smooth border that after 10 days had a diameter of about four centimeters (Fig. 1b). Colonies of P. nodorum isolates on Czapek-Dox V8 agar are shown in Supplementary (Fig. S1). The measurement of the fungal colony diameter at 10 days post-culturing did not reveal any significant variations based on the geographical origin of the isolates. Nevertheless, the isolates demonstrated superior growth when cultured on the WSA medium (Figs. 1b and 1c). Both Czapek-Dox V8 agar and WSA containing wheat straw were appropriate media to stimulate sporulation. After about three weeks, pycnidia appeared throughout the wheat straws, which contain orange pycnidiospores outflowing in pinkish cirri (Fig. 1d). Pycnidiospores were characterized by 1–3 septates, were cylindrical in shape with curved ends, light-colored, and had a diameter of 13–20 × 2.5–4.5 µm (Fig. 1e). Koch’s postulate was validated for all 32 isolates and SN15 control (data not shown). Finally, sequencing of the ITS rDNA region of all 32 isolates showed 100% identity with P. nodorum reference sequence. Given the morphological characteristics and molecular data, all 32 isolates were identified as P. nodorum.

Virulence pattern of P. nodorum isolates

The virulence pattern of 32 Iranian isolates of P. nodorum plus reference isolate SN15 was investigated using pathogenicity assay on 40 different wheat cultivars including 19 Iranian cultivars, 16 world commercial and five SNB differential lines in a factorial randomized complete block design. The first symptoms of SNB were observed at two dpi as small yellow spots. Symptoms at seven dpi ranged from small necrotic spots to spindle-shaped brown lesions surrounded with yellow halos and, in some cases, bearing of pycnidia scattered as black dots on the leaf surface at 10 dpi (Fig. 2). The symptoms were quantified based on the percentage of leaf area infected by the pathogen.

Analysis of variance showed that the effect of isolate, cultivar, and their interaction was significant (P < 0.0001), representing both isolate-specific and cultivar-specific responses in P. nodorum-wheat interaction (Table 3). There was a significant difference among isolates of P. nodorum in virulence against wheat genotypes. The means of DS data were compared using LSD_5%, showing that Pn-FN8 was the most virulent isolate with a mean DS of 75.66% on Shoosh genotype and a total mean DS of 41.07% through all wheat genotypes. The least virulent isolate was Pn-GAl5 with a mean DS of 1.00% on genotype BR34 and a total mean DS of 12.12% through all wheat genotypes (Table S2). In total, the highest mean of percentage leaf area infected was observed among isolates from Fars (Pn-FN) (mean DS = 33.12), compared to isolates from Kohgiluyeh Boyer-Ahmad (Pn-KG) (mean DS = 29.66), Khuzestan (Pn-KhD) (mean DS = 26.20) and Golestan (Pn-GAl) (mean DS = 15.76) provinces, respectively (Fig. 3). Moreover, the range between the minimum and maximum of the mean DS, which represents the distribution of mean DS for each sampling site, was as follows: Fars: 1.33% (Pn-FN4) − 75.66% (Pn-FN8), Kohgiluyeh Boyer-Ahmad: 1% (Pn-KG3) − 69.33% (Pn-KG4), Khuzestan: 1.66% (Pn-KhD3) − 64.66% (Pn-KhD2), Golestan: 1% (Pn-GAl3-Pn-GAl8) − 41.83% (Pn-GAl8) (Fig. 3 and Table S2).

Table 3

Analysis of variance for percentage of leaf area infected by 33 isolates of *Parastagonospora nodorum* on 40 wheat cultivars
Source	DF	Type III SS	Mean Square	F Value	Pr > F
Replication	5	99.233	19.847	5.09	0.0001
Cultivar	39	1470711.787	37710.559	9679.81	< .0001
Isolate	32	458158.520	14317.454	3675.10	< .0001
Cultivar*Isolate	1248	339770.379	272.252	69.88	< .0001
DF: Degree of freedom; SS: Sum of square

Despite the significant differences in virulence observed among isolates from different provinces (Fig. 3), certain genotypes displayed remarkable resistance to all the isolates. Notably, the cultivar BR34 demonstrated the highest level of resistance. The majority of Iranian cultivars were more susceptible than the world commercial cultivars (Fig. 4). The mean DS value on Iranian wheat cultivars was 36.87% while the mean DS value on all world wheat cultivars was 14.81%. Also, Iranian cultivars with a range of mean DS 9.66% (Heidari) − 75.66% (Shoosh) were more diverse than the combination of world and SNB differential lines with a range of mean DS 1% (BR34) − 51.16% (Grandin) (Fig. 4 and Table S2).

A high frequency of necrotrophic effector genes is observed among isolates of P. nodorum

The observed difference in virulence is likely due to the differential presence of NE genes in the different isolates and populations. A multiplex PCR was thus developed to detect NE genes SnToxA, SnTox1, SnTox3, and SnTox5 in P. nodorum isolates. Expected fragments of 806 bp, 465 bp, 693 bp, and 366 bp were amplified in P. nodorum isolates carrying NE genes SnToxA, SnTox1, SnTox3, and SnTox5, respectively (Fig. S2 and Table 4). Sequencing some of these fragments confirmed the specific amplification of each NE gene. The reference isolate SN15 contains three NE genes, SnToxA, SnTox1 and SnTox3, but not SnTox5, as previously reported. NE gene SnTox5 was identified and cloned in isolate Sn2000 (Kariyawasam et al., 2021). Two NE genes, SnTox1 and SnTox5, were detected in all Iranian isolates (Table 4). SnToxA and SnTox3 were detected in 59% and 69% of the isolates, respectively (Table 4). SnToxA was absent in the majority of isolates from KG province (gene detected in three out of eight isolates), while it was present in the majority of isolates in other provinces (Table 4 and Fig. S3). Four out of eight isolates from KG province possessed SnTox3 (Table 4). Only four isolates missed both SnToxA and SnTox3 (Pn-KG2, Pn-KG4, Pn-KG6 and Pn-GAl6). However, it is worth noting that these isolates were not classified as lowly virulent isolates, indicating the possibility of their possession of additional NE genes that play a significant role in virulence.

Table 4

Necrotrophic effector genes detected within *Parastagonospora nodorum* isolates collected from major wheat-growing regions in Iran
P. nodorum isolate	SnToxA	SnTox1	SnTox3	SnTox5
Pn-FN1	+	+	-	+
Pn-FN2	-	+ (MW454816)	+	+
Pn-FN3	+	+	-	+
Pn-FN4	+	+	-	+
Pn-FN5	+	+	+	+
Pn-FN6	-	+	+	+
Pn-FN7	+	+	+	+
Pn-FN8	+	+	+	+
Pn-KG1	+	+	+	+
Pn-KG2	-	+	-	+
Pn-KG3	-	+	+	+
Pn-KG4	-	+	-	+
Pn-KG5	+	+	-	+ (OQ709799)
Pn-KG6	-	+	-	+
Pn-KG7	+	+	+	+
Pn-KG8	-	+	+	+
Pn-KhD1	-	+	+	+
Pn-KhD2	+	+	+	+
Pn-KhD3	-	+	+	+
Pn-KhD4	-	+	+	+
Pn-KhD5	+	+	+	+
Pn-KhD6	+	+	+	+
Pn-KhD7	+	+	+	+
Pn-KhD8	+ (MW454818)	+	-	+
Pn-GAl1	-	+	+	+
Pn-GAl2	+	+	+	+
Pn-GAl3	+	+	+ (MW454817)	+
Pn-GAl4	+	+	+	+
Pn-GAl5	-	+	+	+
Pn-GAl6	-	+	-	+
Pn-GAl7	+	+	-	+
Pn-GAl8	+	+	+	+
SN15	+	+	+	-
plus (+) indicates positive detection of given necrotrophic effector genes in each isolate. NCBI accession numbers are presented in parentheses.

Wheat genotypes were clustered into two major susceptible and resistant groups

The reaction of 40 wheat genotypes towards 33 different P. nodorum isolates was assessed under greenhouse-controlled conditions. The DS values were used in cluster analysis that grouped the genotypes into two major groups, susceptible and resistant ones (Fig. 5). In addition, a quantitative scale was used to categorize the reaction of wheat cultivars towards P. nodorum isolates into six groups: 1 ≤ DS ≥ 5 highly resistant; 6 ≤ DS ≥ 10 resistant; 11 ≤ DS ≥ 15 moderately resistant; 16 ≤ DS ≥ 25 moderately susceptible; 26 ≤ DS ≥ 40 susceptible and DS > 41 highly susceptible. The susceptible cluster consisted of 28 wheat genotypes with the mean DS ranging from 19.55% (Katepwa) to 50.26% (Hana). This cluster had three subclasses including highly susceptible (DS: 38.60–50.26%; comprising the reference Grandin cultivar that carries Tsn1 and Snn3 S genes), susceptible (DS: 28.28–36.18%; comprising the reference BG220 and Chinese Spring that carry the Snn3 and Snn1 genes, respectively) and moderately susceptible (DS: 19.55–25.08%; comprising the reference BG261 that carries the Tsn1 gene). The resistant cluster contained 12 wheat genotypes with the mean DS ranging between 2.28% (BR34) and 13.84% (ES-Federal). This cluster consisted of three subclusters including highly resistant (DS: 2.28%; only the reference BR34 cultivar that does not carry S genes), resistant (DS: 7.12–10.88%), and moderately resistant (DS: 12.46–13.84%). Consistent with the virulence pattern (Fig. 3), all Iranian cultivars belong to the three susceptible subclusters.

A high frequency of susceptibility genes was observed among different wheat genotypes

Multiplex PCR enabled the identification of three susceptible genes Tsn1, Snn1, and Snn3 across the wheat genotypes, amplifying 380 bp, 532 bp, and 657 bp fragments, respectively which were confirmed by sequencing (Fig. S4 and Table 5). Cultivars Barat, Chamran 2, Gonbad, Katepwa, Kulm, Mehregan, Ofogh, Sarang, Shavoor, Shoosh, and Sirvan had all three S genes while genotypes 3HD-126, 4B-160, BG220, BG261, Chinese Spring, Coulter, KM7, ND495, and Vernapolis carried a single S gene. The expected dominant S genes were identified in differential lines BG261 (Tsn1), Grandin (Tsn1, Snn3-B1), Chinese Spring (Snn1), BG220 (Snn3-B1) which were used as the control. None of the S genes was identified in BR34 reported as a differential line carrying recessive tsn1 and snn3 genes, consistent with being a highly resistant control cultivar in this study. The proportion of the combination with all three S genes is presented in Supplementary (Fig. S5). A higher number of Iranian wheat cultivars were found to carry all three S genes (Tsn1 + Snn1 + Snn3+), whereas a larger proportion of world cultivars tended to possess Snn1 and Snn3 S genes (Tsn1- Snn1 + Snn3+). The total proportion of all S genes Tsn1, Snn1, and Snn3 in Iranian wheat cultivars and world wheat cultivars was 84.21% and 58.33%, respectively. The proportion of Tsn1 in Iranian wheat cultivars and world wheat cultivars was 52.63% and 25%, respectively. S gene Snn1 was identified in all Iranian wheat cultivars while 87.5% of world cultivars had the Snn1 gene. S gene Snn3 was detected in all Iranian wheat cultivars whereas the proportion of this gene in world cultivars was 62.5%.

Table 5

Susceptibility genes detected within Iranian, world commercial and *Parastagonospora nodorum* differential wheat genotypes
Wheat cultivar	Tsn1	Snn1	Snn3
BG220 (Snn3-B1 DL)	-	-	+
BG261(Tsn1 DL)	+	-	-
BR34 (No S gene, DL)	-	-	-
Chinese Spring (CS) (Snn1 DL)	-	+	-
Grandin (Tsn1, Snn3-B1 DL)	+	-	+
Baharan	-	+	+
Barat	+	+	+
Chamran 2	+	+	+
Dena	-	+	+
Ehsan	-	+	+
Gonbad	+	+	+
Hana	-	+	+
Heidari	-	+	+
Khalil	-	+	+
Mehregan	+	+	+
Narin	-	+	+
Ofogh	+ (MW454820)	+	+
Rakhshan	-	+	+
Sarang	+	+	+
Shabrang	-	+	+
Shavoor	+	+	+
Shoosh	+	+	+
Sirvan	+	+	+
Talaei	+	+	+
3HD-126	-	+	-
4B-160	-	+ (MW454819)	-
Arina	-	+	+
Bulgaria	-	+	+
Coulter	+	-	-
Erik	-	+	+
ES-Federal	-	+	+
Katepwa	+	+	+
Kavkaz-k4500	-	+	+
KM7	-	+	-
Kulm	+	+	+
M-3	-	+	+
ND495	+	-	-
Obelisk	-	+	+
Tadinia	-	+	+
Veranopolis	-	+	-
plus (+) indicates positive detection of given susceptibility genes in each genotype. NCBI accession numbers are presented in parentheses. DL: Differential line

Infiltration of SnToxA caused necrosis response on wheat cultivars harboring Tsn1 sensitivity gene

To assess the response of 40 wheat genotypes towards NE SnToxA and verify the presence of the Tsn1 gene, an infiltration assay was conducted. The cultivars displayed either complete sensitivity or complete insensitivity, with no intermediate reactions observed. Only wheat cultivars carrying the dominant Tsn1 allele showed a necrotic response to SnToxA infiltration. Water-soaked necrotic lesions appeared 72-h after SnToxA infiltrate injection (Fig. 6).

Since Iran is located in the Fertile Crescent where wheat was initially domesticated more than 12,000 years ago (Blixt, 2009), it is suggested that wheats and their pathogens are in a coevolutionary arms race (Stukenbrock et al., 2007). Our understanding of the interactions between wheat and P. nodorum in natural populations derived from Iran remains limited to date. Hence, the objective of this study was to assess the virulence profile of 33 isolates of P. nodorum on a diverse panel of wheat genotypes, and to analyze the molecular patterns of NEs and S genes in P. nodorum and wheat genotypes, respectively. In a previous study, the genetic structure of P. nodorum was assessed using microsatellite markers, revealing that Iranian isolates of P. nodorum exhibited the highest genetic diversity among 693 isolates collected from five continents worldwide. These findings provided supporting evidence for the hypothesis that P. nodorum has undergone co-evolution with its host in the Fertile Crescent (Ghaderi et al., 2020). This is in line with our current findings, as we have demonstrated that most Iranian isolates exhibited higher DS values compared to the reference isolate SN15. Fars (Pn-FN) isolates had the highest DS value, while Golestan (Pn-GAl) isolates had the lowest DS value compared to other isolates derived from other provinces. Previus study also showed that Fars and Golestan provinces had the highest and lowest genetic diversity, respectively among the Iranian population of P. nodorum (Ghaderi et al., 2020). In addition to their low genetic diversity and virulence, the Golestan isolates exhibited distinct differentiation from other populations. This can be attributed to the geographical separation of Golestan, which is over 800 km distant from the other three collection sites. In addition, physical barriers, such as mountains and deserts, act as differentiating factors between this population and the other populations examined in this study. In contrast to the Golestan population, there was no significant difference in the mean DS value between the Fars and Kohgiluyeh Boyer-Ahmad populations. This lack of difference could potentially be attributed to the presence of gene flow between these two populations. The findings of a previous population structure study also supported the presence of gene flow between the Fars and Kohgiluyeh Boyer-Ahmad populations (Ghaderi et al., 2020).

Our results may also suggest that high genetic diversity is positively correlated with increased virulence. It is reported that a diverse gene pool accelerates the generation of appropriate recombinants compensating for the lack of virulence (Phan et al., 2020). With higher genetic diversity there is a higher chance of selection for highly virulent isolates. In contrast, populations with low genetic diversity tend to be vulnerable to the introduction of new cultivars as they are unable to quickly recombine (Phan et al., 2020). Active sexual reproduction during the growing season enables P. nodorum to evolve quickly leading to the appearance of new virulent pathotypes occupying new ecological niches. Hence, the previously observed high genetic diversity in Fars province (Ghaderi et al., 2020), along with the increased virulence of isolates found in this study, suggests that the Fars population likely engages in more active sexual reproduction compared to the Golestan isolates. However, further comprehensive investigations are required in the future to address this aspect in greater detail.

Pathogenicity assay showed that all Iranian wheat cultivars fall into highly susceptible (47.37%), susceptible (31.58%) and moderately susceptible (21.06%) cultivars to SNB. SnTox1-Snn1 was the most abundant NE-S gene frequency (100%) in Iranian P. nodorum isolates and Iranian wheat cultivars. Contrary to the most NEs, the C-terminal chitin-binding region of NE SnTox1 acts as a PAMP-type stimulator and interacts with the cell surface pattern recognition receptor Snn1 (Liu et al., 2012) triggering PTI (rather than ETI) response in the host, which is one of the general defense mechanisms (Liu et al., 2016; Faris & Friesen, 2020). Therefore, considering the dual function of SnTox1 both as a necrotrophic effector stimulating the PTI in the plant and its role in protecting the fungus against wheat chitinases (Liu et al., 2016), the high-frequency of SnTox1 in the Iranian P. nodorum population is logical, which is comparable with its relatively high frequency (85%) in a large global P. nodorum populations as determined previously (Liu et al., 2012; Faris & Friesen, 2020). Considering the protective role of SnTox1 in disease development, which operates independently of its function in inducing cell death (Liu et al., 2016; McDonald et al., 2022), it was expected to observe a high frequency (100%) of SnTox1 presence across all disease severity (DS) categories (Table 6).

Table 6

The proportion of SNB susceptibility (S) and necrotrophic effector (NE) genes under different disease severity (DS) classification
S /NE gene	DS > 41	26 ≤ DS ≥ 40	16 ≤ DS ≥ 25	11 ≤ DS ≥ 15	6 ≤ DS ≥ 10	1 ≤ DS ≥ 5
Tsn1	70	50	50	0	0	0
Snn1	90	80	75	100	100	0
Snn3	100	70	75	100	57	0
SnToxA	100	69	50	50	-	-
SnTox1	100	100	100	100	-	-
SnTox3	100	69	67	75	-	-
SnTox5	100	94	100	100	-	-

S gene SnToxA is the main determinant in causing SNB, tan spot, and spot blotch diseases in plant lines harboring Tsn1. In the absence of Tsn1, the severity of the disease reduces significantly even in the presence of other S genes (Peters Haugrud et al., 2022; McCombe et al., 2022; Liu et al., 2012) which is consistent with the results here. S gene Tsn1 was present in 70% of highly susceptible cultivars. Tsn1 frequency decreased to 50% of susceptible and moderately susceptible cultivars and was completely absent in moderately resistant, resistant and highly resistant cultivars (Table 6).

The mean DS (36.87%) of Iranian wheat cultivars (regarded as susceptible) was correlated with the presence of all S genes Tsn1, Snn1 and Snn3 (84.21%). Additionally, the mean disease severity (DS) of world cultivars (14.81%), categorized as moderately resistant, corresponded to the low proportion (58.33%) of having all three S genes. Those cultivars possessing all three S genes Tsn1, Snn1 and Snn3 were all categorized as highly susceptible, susceptible and moderately susceptible. Also, the frequency of S genes in highly susceptible cultivars (86.66%) was much higher than the frequency of these genes in resistant cultivars (52%). Furthermore, an increased number of NE gene SnToxA was positively correlated to virulence. In DS > 41, the frequency of SnToxA was 100% decreasing gradually to 50% of P. nodorum samples in 16 ≤ DS ≥ 25 and 11 ≤ DS ≥ 15 (Table 6). Despite the absence of certain NEs in some isolates of P. nodorum, they exhibited high virulence, indicating the potential existence of other unidentified NEs and additional disease determinants. This finding further confirms the complex nature of the P. nodorum-wheat interaction (Oliver et al., 2009). It was suggested that susceptible cultivars carrying multiple S genes could rapidly select pathogen isolates having the corresponding NE genes (McDonald et al., 2013). Therefore, the frequency of the NEs in the pathogen population could be directly related to the frequency of the S genes in its host. In the absence of host S genes, the NE will be neutral and subjected to genetic drift (McDonald et al., 2013). Although the overall higher occurrence of both NEs and S genes in Iranian P. nodorum isolates and wheat genotypes supports this hypothesis, drawing a definitive conclusion requires monitoring a large population size of P. nodorum and host genotypes over multiple years.

Wheat cultivars were categorized into six groups (highly resistant, resistant, moderately resistant, moderately susceptible and highly susceptible) based on their variable reaction towards P. nodorum isolates. Most previous studies rated wheat plants for their reaction to SNB by a qualitative 0–5 scale (Liu et al., 2004; Károlyiné Cséplő et al., 2013; Singh et al., 2006; Feng et al., 2004). The quantitative scale implemented in this study can be compared to the previous qualitative scale, where values 0, 1, 2, 3, 4, and 5 correspond to immune, highly resistant, resistant, moderately susceptible, susceptible, and highly susceptible, respectively (Singh et al., 2006; Feng et al., 2004). Therefore, based on the quantitative scale defined here, Iranian wheat cultivars with a mean DS value of 36.87% were categorized as susceptible while commercial world cultivars with a mean DS value of 14.81% were classified as moderately resistant cultivars to SNB. The findings regarding the response of differential and world commercial cultivars to SNB were in line with qualitative assays conducted by previous researchers where BR34 was classified as highly resistant, Erik, M-3, Vernapolis, and Arina as resistant, ND 495 and Katepwa as moderately susceptible, 4B-160 and Coulter as susceptible, and Grandin as highly susceptible cultivar to SNB (Liu et al., 2004; Singh et al., 2006; Károlyiné Cséplő et al., 2013; Tommasini et al., 2007). Hence, the utilization of the quantitative scale as a substitutional assay for assessing the response of wheat cultivars to P. nodorum provides a more precise and reliable approach, which can serve as a valuable parameter in breeding programs.

NE infiltration represents one of the simplest and quickest strategies for evaluating the sensitivity reaction of host plants. Response to toxins occurs quickly, which provides accurate and reproducible results by minimizing the environmental effects on symptom development facilitating the early detection of resistant and susceptible cultivars (Sharma, 2012; Waters et al., 2011). As an example, the sensitivity of 60 bread wheat cultivars from Western Australian was assessed through NE SnTox3 infiltration, which served as an expedited approach for breeding purposes (Waters et al., 2011). The screening of wheat cultivars using purified toxins allows breeders to promptly identify and eliminate susceptible genotypes in the early stages of breeding cycles (Oliver et al., 2009). As mentioned earlier, the interaction between SnToxA and Tsn1 is an important factor in the development of SNB. Therefore, in order to achieve a high level of resistance against both SNB and tan spot, breeders should prioritize the selection of genotypes that lack the Tsn1 gene (Peters Haugrud et al., 2023). Considering the coexistence of SNB and tan spot, two major diseases in wheat (Phan et al., 2020), the application of NE SnToxA holds potential for marker-assisted selection in screening wheat cultivars against SNB, tan spot, and spot blotch diseases. The developed SnToxA cloning and infiltration assay demonstrated the capability to identify Tsn1-resistant cultivars within a 72-hour timeframe, enabling early detection and selection.

Genetic analysis of wheat lines revealed that the resistance to SNB can be inherited and controlled in a quantitative trait (Liu et al., 2004; Friesen et al., 2008). Most quantitative trait loci have minor effects posing a challenge for their utilization in breeding programs (Solomon et al., 2006; Liu et al., 2004). Hence, adopting new screening strategies developed based on the mechanisms of interaction between P. nodorum and wheat is essential. These methods, including NE infiltration assays, glasshouse assays, and PCR-based disease susceptibility screening (Waters et al., 2011), effectively contribute to the selection of non-susceptible wheat cultivars, thereby enhancing our capability to improve wheat breeding programs. Identifying the presence of NE genes alerts breeders to avoid using corresponding sensitivity genes in the host. In this study, we have demonstrated the effectiveness of NE infiltration coupled with multiplex PCR as a rapid and efficient method for detecting the S gene in the host and NE genes in P. nodorum. This offers valuable directions and tools that can significantly enhance breeding programs aimed at developing wheat cultivars with increased resistance against SNB.

In conclusion, this study sheds light on the interactions between the fungus P. nodorum and wheat genotypes, providing valuable insights into the molecular basis of these interactions. The research demonstrated the significant impact of necrotrophic effectors (NEs) secreted by P. nodorum on wheat, leading to substantial economic losses worldwide. By investigating 40 wheat genotypes against 33 P. nodorum isolates, the researchers identified six distinct groups based on their reactions to the pathogen. The presence and absence of specific dominant susceptibility (S) genes in different wheat cultivars played a crucial role in determining their susceptibility to P. nodorum isolates. Notably, the S gene Tsn1 was found in highly susceptible cultivars, while all resistant cultivars lacked this gene. The presence of all three S genes (Tsn1, Snn1, and Snn3) in a cultivar rendered it susceptible to all P. nodorum isolates. Furthermore, the study highlights the prevalence of NE-S gene frequencies, with SnTox1-Snn1 being the most abundant in Iranian P. nodorum isolates and Iranian wheat cultivars. The detection of S gene Snn3 in all Iranian wheat cultivars and its presence in 62.5% of world cultivars signifies its importance in the susceptibility of these wheat varieties.

Overall, the study contributes significantly to our understanding of the P. nodorum-wheat interactions, especially in the specific context of Iran's agricultural landscape, which is part of the Fertile Crescent—a region where both wheat and its pathogens have coevolved. The insights gained from this research could have practical implications for wheat breeders and plant pathologists, offering valuable information to develop more resilient and disease-resistant wheat varieties, ultimately mitigating economic losses caused by P. nodorum.

Acknowledgments

This research was financially supported by Isfahan University of Technology (IUT) and Iran National Science Foundation (INSF) with project No. 99007240. Thanks to Dr. F. Ghaderi for providing part of P. nodorum isolates and B. Najafipour and Dr. Y. Norouzi for guidance with statistical analysis. Also, we thank Prof. R. P. Oliver, Dr. K. C. Tan and Dr. E. Furuki for providing P. nodorum isolate SN15.

Data availability

The sequences were deposited in the GenBank and accession numbers are given in Tables 4 and 5. The data that support the findings of this study are available from the first author upon request.

Conflict of interest

All authors declare that they have no conflicts of interest.

Alllingham, E. & Jackson, L. (1981). Variation in pathogenicity, virulence, and aggressiveness of Septoria nodorum in Florida. Phytopathology, 71, 1080-1085.
Blixt, E. (2009) On Phaeosphaeria nodorum in wheat. Ph.D. Dissertation. Swedish University of Agricultural Sciences., Uppsala.
Douaiher, M., Halama, P. & Janex-Favre, M. (2004). The ontogeny of Stagonospora nodorum pycnidia in culture. SYDOWIA-HORN, 56, 39-50.
Duba, A., Goriewa-Duba, K. & Wachowska, U. (2018). A Review of the interactions between wheat and wheat pathogens: Zymoseptoria tritici, Fusarium spp. and Parastagonospora nodorum. International journal of molecular sciences, 19, 1138.
Faris, J. D. & Friesen, T. L. (2020). Plant genes hijacked by necrotrophic fungal pathogens. Current Opinion in Plant Biology, 56, 74-80.
Feng, J., Ma, H. & Hughes, G. R. (2004). Genetics of resistance to Stagonospora nodorum blotch of hexaploid wheat. Crop Science, 44, 2043-2048.
Friesen, T. L., Faris, J. D., Solomon, P. S. & Oliver, R. P. (2008). Host‐specific toxins: effectors of necrotrophic pathogenicity. Cellular Microbiology, 10, 1421-1428.
Ghaderi, F., Sharifnabi, B., Javan‐Nikkhah, M., Brunner, P. C. & McDonald, B. A. (2020). SnToxA, SnTox1, and SnTox3 originated in Parastagonospora nodorum in the Fertile Crescent. Plant Pathology, 69, 1482-1491.
Kariyawasam, G. K., Richards, J. K., Wyatt, N. A., Running, K. L., Xu, S. S., Liu, Z., et al. (2022). The Parastagonospora nodorum necrotrophic effector SnTox5 targets the wheat gene Snn5 and facilitates entry into the leaf mesophyll. New Phytologist, 233, 409-426.
Károlyiné Cséplő, M., Csősz, L., Gal, M., Veisz, O. B. & Vida, G. (2013). Seedling resistance to stagonospora nodorum blotch in wheat genotypes. Czech Journal of Genetics and Plant Breeding, 49, 77-85.
Liu, Z., Faris, J. D., Oliver, R. P., Tan, K.-C., Solomon, P. S., McDonald, M. C., et al. (2009). SnTox3 acts in effector triggered susceptibility to induce disease on wheat carrying the Snn3 gene. PLoS Pathogens, 5, e1000581.
Liu, Z., Friesen, T., Rasmussen, J., Ali, S., Meinhardt, S. & Faris, J. (2004). Quantitative trait loci analysis and mapping of seedling resistance to Stagonospora nodorum leaf blotch in wheat. Phytopathology, 94, 1061-1067.
Liu, Z., Gao, Y., Kim, Y. M., Faris, J. D., Shelver, W. L., de Wit, P. J., et al. (2016). SnTox1, a Parastagonospora nodorum necrotrophic effector, is a dual‐function protein that facilitates infection while protecting from wheat‐produced chitinases. New Phytologist, 211, 1052-1064.
Liu, Z., Zhang, Z., Faris, J. D., Oliver, R. P., Syme, R., McDonald, M. C., et al. (2012). The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1. PLoS Pathogens, 8, e1002467.
McCombe, C. L., Greenwood, J. R., Solomon, P. S. & Williams, S. J. (2022). Molecular plant immunity against biotrophic, hemibiotrophic, and necrotrophic fungi. Essays in Biochemistry, 66, 581-593.
McDonald, M. C., Oliver, R. P., Friesen, T. L., Brunner, P. C. & McDonald, B. A. (2013). Global diversity and distribution of three necrotrophic effectors in Phaeosphaeria nodorum and related species. New Phytologist, 199, 241-251.
McDonald, M. C., Williams, S. J. & Solomon, P. S. (2022). The role of tox effector proteins in the Parastagonospora nodorum–wheat interaction. In: Plant Relationships: Fungal-Plant Interactions. Springer, pp. 53-63.
Mehra, L., Adhikari, U., Cowger, C. & Ojiambo, P. S. (2018). Septoria nodorum blotch of wheat. PeerJ Preprints.
Mehrabi, R., Karami, N. & Torabi, M. (2015). The presence of the susceptibility gene, Tsn1, in some wheat lines and their reactions to ToxA infiltration produced by Pyrenophora tritici-repentis. Iranian Journal of Plant Protection Science, 46.
Murray, M. & Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8, 4321-4326.
Oliver, R. P., Friesen, T. L., Faris, J. D. & Solomon, P. S. (2012). Stagonospora nodorum: from pathology to genomics and host resistance. Annual Review of Phytopathology, 50, 23-43.
Oliver, R. P., Rybak, K., Solomon, P. S. & Ferguson-Hunt, M. (2009). Prevalence of ToxA-sensitive alleles of the wheat gene Tsn1 in Australian and Chinese wheat cultivars. Crop and Pasture Science, 60, 348-352.
Peters Haugrud, A. R., Shi, G., Seneviratne, S., Running, K. L., Zhang, Z., Singh, G., et al. (2023). Genome-wide association mapping of resistance to the foliar diseases septoria nodorum blotch and tan spot in a global winter wheat collection.
Peters Haugrud, A. R., Zhang, Z., Friesen, T. L. & Faris, J. D. (2022). Genetics of resistance to septoria nodorum blotch in wheat. Theoretical and Applied Genetics, 1-23.
Phan, H. T., Jones, D. A., Rybak, K., Dodhia, K. N., Lopez-Ruiz, F. J., Valade, R., et al. (2020). Low amplitude boom-and-bust cycles define the septoria nodorum blotch interaction. Frontiers in plant science, 10, 1785.
Sharma, I. (2012) Disease resistance in wheat. Cabi.
Shi, G., Zhang, Z., Friesen, T. L., Raats, D., Fahima, T., Brueggeman, R. S., et al. (2016). The hijacking of a receptor kinase–driven pathway by a wheat fungal pathogen leads to disease. Science advances, 2, e1600822.
Singh, P., Mergoum, M., Ali, S., Adhikari, T., Elias, E. & Hughes, G. (2006). Identification of new sources of resistance to tan spot, Stagonospora nodorum blotch, and Septoria tritici blotch of wheat. Crop Science, 46, 2047-2053.
Solomon, P. S., Lowe, R. G., TAN, K. C., Waters, O. D. & Oliver, R. P. (2006). Stagonospora nodorum: cause of stagonospora nodorum blotch of wheat. Molecular Plant Pathology, 7, 147-156.
Stukenbrock, E. H., Banke, S., Javan-Nikkhah, M. & McDonald, B. A. (2007). Origin and domestication of the fungal wheat pathogen Mycosphaerella graminicola via sympatric speciation. Molecular Biology and Evolution, 24, 398-411.
Tommasini, L., Schnurbusch, T., Fossati, D., Mascher, F. & Keller, B. (2007). Association mapping of Stagonospora nodorum blotch resistance in modern European winter wheat varieties. Theoretical and Applied Genetics, 115, 697-708.
Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., et al. (2012). Primer3 new capabilities and interfaces. Nucleic Acids Research, 40, e115-e115.
Waters, O. D., Lichtenzveig, J., Rybak, K., Friesen, T. L. & Oliver, R. P. (2011). Prevalence and importance of sensitivity to the Stagonospora nodorum necrotrophic effector SnTox3 in current Western Australian wheat cultivars. Crop and Pasture Science, 62, 556-562.
White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications, 18, 315-322.

Supportinginformation.docx
Table S1 Primers used for the detection of necrotrophic effector and susceptibility genes and cloning SnToxAinto pRM147 Fig. S1 Colonies of 33 isolates of Parastagonospora nodorum 10 days after culturing on Czapek-Dox V8 agar; 1-8: Pn-FN1-8, 9-16: Pn-KG1-8, 17-24: Pn-KhD1-8, 25-32: Pn-GAl1-8, 33: SN15 Fig. S2 PCR pattern obtained by multiplex PCR reaction to detect necrotrophic effector genes SnToxA, SnTox1, SnTox3and SnTox5 (amplicons of 806 bp, 465 bp, 693 bp and 366 bp, respectively) in Parastagonospora nodorum isolates; (1) Pn-FN1, (2) Pn-FN2, (3) Pn-FN5, (4) Pn-FN6, (5) Pn-FN7, (6) Pn-KG1, (7) Pn-KG3, (8) Pn-KG7, (9) PN-KG8, (10) Pn-KhD3, (11) Pn-KhD4, (12) Pn-KhD5, (13) Pn-KhD7, (14) Pn-KhD8, (15) Pn-GAl1, (16) Pn-GAl2, (17) Pn-GAl3, (18) Pn-GAl5, (19) Pn-GAl7, (20) Pn-GAl8, (21) SN15, (22) water control(M) 100 bp DNA marker Fig. S3 The frequency of all possible combinations for four necrotrophic effector genes (SnToxA, SnTox1, SnTox3 and SnTox5) in Parastagonospora nodorum isolates collected from different provinces. Plus (+) and minus (-) indicates the positive and negative presence of given NE genes in each combination, respectively Fig. S4 PCR pattern obtained by multiplex PCR reaction to detect SNB sensitivity genes Tsn1, Snn1 and Snn3 (amplicons of 380 bp, 532 bp, and 657 bp, respectively) in wheat cultivars; (1) Gonbad, (2) Barat, (3) Arina, (4) Dena, (5) Chamran 2, (6) Kulm, (7) Ofogh, (8) Mehregan, (9) Hana , (10) Heidari, (11) Tadinia, (12) Vernapolis, (13) 3HD-126, (14) Shabrang, (15) BG261, (16) Rakhshan, (17) Obelisk, (18) Shavoor, (19) KM7, (20) Sarang, (21) Ehsan, (22) Chinese Spring (M) 100 bp DNA marker Fig. S5 The frequency of all possible combinations for three susceptibility genes (Tsn1, Snn1 and Snn3) in Iranian and world wheat genotypes. Plus (+) and minus (-) indicates the positive presence and negative presence of given S genes in each combination, respectively
TABLES2.xlsx
Table S2 The average percentage of leaf area infected in pathogenicity assay of 33 different Parastagonospora nodorum isolates on 40 wheat genotypes

Download PDF

Version 1

posted

You are reading this latest preprint version

Host susceptibility and fungal virulence in the wheat-Parastagonospora nodorum interactions

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Results

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1