Journal of Plant Diseases and Protection

https://doi.org/10.1007/s41348-017-0143-3 (0123456789().,-volV)(0123456789().,-volV)

ORIGINAL ARTICLE

Evaluating the efficacy of STB resistance genes to Iranian Zymoseptoria tritici isolates

1 1 1 Mohamad Dalvand • Mohamad Javad Soleimani Pari • Doustmorad Zafari

Received: 26 December 2016 / Accepted: 9 December 2017 Ó Deutsche Phytomedizinische Gesellschaft 2017

Abstract Zymoseptoria tritici (P. Crous; syn. graminicola, tritici), causal agent of Septoria tritici blotch (STB) disease, is one of the most important foliar diseases of in Iran and across the world. To identify resistance sources than can be relevant to breeding programs, it is necessary to determine the virulence factors of the . In this study, 26 differential wheat (carrying Stb1–18 genes) were inoculated as seedlings in a greenhouse with 10 individual isolates of Z. tritici collected from different regions of Iran under different environmental conditions. Iranian Z. tritici isolates showed new virulence patterns. Among wheat differentials, none of the cultivars were resistant to all the Iranian isolates used in this study. Oasis (carrying Stb1), Sulivan (carrying Stb2), and Bulgaria 88 (carrying Stb1 and Stb6) showed susceptibility to all the isolates. Stb5, Stb7, Stb13, and Stb14 possibly do not have a good resistance as most of the isolates were virulent to these genes. Therefore, these genes cannot be considered as effective sources of resistance to Z. tritici. Shafir (carrying Stb6) revealed a resistant reaction to most of the isolates. M3 (carrying Stb16 and Stb17) was susceptible to four Z. tritici isolates, and this is the first report of susceptibility reaction for this .

Keywords Differential cultivar Á Seedlings Á Resistant gene Á Zymoseptoria tritici Á Wheat

Introduction the agrochemical and breeding industry (Stammler et al. 2008; Torriani et al. 2009). Septoria tritici blotch (STB), caused by the ascomycete Despite agronomical practices and varietal resistance, Mycosphaerella graminicola with an amorphous the control of the disease relies mainly on chemical control Zymoseptoria tritici (old name: S. tritici) (Quaedvlieg et al. (McDonald and Mundt 2016). However, this method is 2011), is one of the most serious foliar diseases of wheat neither environmentally friendly nor it is entirely reliable. (Eyal et al. 1987). Therefore, resistance breeding in wheat to STB can provide The disease can affect both the quality and quantity of an effective, economic and environmentally safe strategy wheat thereby reducing yield losses for more than 40–50% for disease management (Brown et al. 2015; Ferjaoui et al. in areas with relatively high rainfall and moderate tem- 2015). peratures (Eyal 1981; Fones and Gurr 2015). Both qualitative and quantitative resistances against Z. In Iran, STB is a serious disease (Haghdel and Bani- tritici have been reported during the last few decades. hashemi 2005). Susceptibility of prevalent commercial Quantitative resistance is controlled by polygenic traits and bread and durum wheat cultivars to STB resulted in severe has incomplete resistance to Z. tritici. It has been widely in the major wheat-growing provinces of Iran reported in both seedling and adult plant stages. In contrast, (Abrinbana et al. 2012). Owing to the increasing impor- qualitative resistance confers complete or near-complete tance of STB in many regions of the world including Iran, resistance to particular isolates, as it has been reported for the disease is a main target as well as a serious concern of the Stb6 resistance gene in bread wheat (Brading et al. 2002). So far 21 resistance genes have been discovered and & Mohamad Dalvand mapped (Brown et al. 2015; Simo´n et al. 2012; Lenden- [email protected] mann et al. 2014).

1 Bu-Ali Sina University, Hamedan, Iran 123 Journal of Plant Diseases and Protection

Due to the rapid changes in the pathogen population, a concentration was determined by hemocytometer counts study of the virulence pattern of Z. tritici, and continuous and adjusted to 107 spores/mL. 0.15% Tween-20 was then identification of new sources of resistance to STB and their added to the medium prior to inoculation. incorporation in breeding programs is required for sus- tainable disease management. Plant material and virulence assays Seedling screens offer opportunities to identify the efficacy of resistance to a wide panel of isolates (Tabib The reactions of 26 differential cultivars (Table 2), each Ghaffary et al. 2011), and differential wheat genotypes carrying one or more Stb gene(s) (Stb1–Stb18), were carrying Stb genes can be used to determine the virulence evaluated for pathotyping of fungal isolates. This experi- diversity of Z. tritici populations (Czembor et al. 2011). ment was conducted at Dezful city in Khuzestan which is a This study was conducted in order to assess the efficacy tropical province in southern Iran. of STB resistance genes against Iranian isolates of Z. tritici Taichung 29 and Obelisk were used as international and to evaluate whether there is a pathogenic variation for susceptible cultivars, while Darab 2 was used as a national STB within Iranian Z. tritici isolates. susceptible cultivar. Cultivars were planted in a plastic pot with a diameter of 15.5 cm (three pots per isolate) filled with a mixture of Materials and methods sterile loam soil, peat, and perlite. The experimental design was completely randomized with three repeats. Seedlings Isolation, purification, and disease inoculum of each pot were inoculated at the third stage with preparation Table 2 Bread wheat differential genotypes with known Stb genes In this study, leaf samples infected with Iranian isolates of used in virulence pattern studies Z. tritici showing blotch symptoms were collected from No. Cultivar Gene different fields of Khuzestan, Lorestan, Ardabil, Markazi, and Golestan provinces that represent different environ- 1 Oasis Carrying Stb1 mental conditions (Table 1). Isolation of fungi was per- 2 Sulivan Carrying Stb1 formed as described by Eyal et al. (1987). First, pieces of 3 Bulgaria 88 Carrying Stb1 and Stb6 infected were attached to glass slides and placed in a 4 Veranopolis Carrying Stb2 and Stb6 Petri dish with wet filter paper. After 24 h, the oozing 5 Israel 493 Carrying Stb3 and Stb6 pycnidia is removed with a sterile needle and transferred to 6 Tadinia Carrying Stb4 and Stb6 a PDA plate. For inoculation preparation, one-liter flasks 7 Cs Synthetic Carrying Stb5 containing Yeast-glucose (YG) liquid medium (Yeast-Ex- 8 Flame Carrying Stb6 tract 10 g/L and glucose 10 g/L) were inoculated using a 9 Shafir Carrying Stb6 small piece of fungal colonies from the solid medium and 10 Estanzuela Federal Carrying Stb7 incubated in a shaker (Jal tajhiz, Karaj, Iran) adjusted at 11 M6 Synthetic Carrying Stb8 150 rpm and 15 °C. After a week, the inoculum 12 Courtot Carrying Stb9 13 Kavkaz-K4500 Carrying Stb6, Stb7, Stb10 and Stb12 14 TE9111 Carrying Stb6, Stb7 and Stb11 Table 1 Characteristics of Zymoseptoria tritici isolates used in this study 15 Obelisk Susceptible check 16 Taichung 29 Susceptible check Isolate Province Location 17 Salamouni Carrying Stb13 and Stb14 IR1 Khuzestan Dezful Bread wheat 18 Ariana Carrying Stb6 and Stb15 IR2 Khuzestan Shush Bread wheat 19 Riband Carrying Stb15 or another IR3 Markazi Arak Bread wheat 20 M3 Carrying Stb16 and Stb17 IR4 Khuzestan Dezful Bread wheat 21 Balance Carrying Stb6 and Stb18 IR5 Ardabil Moghan Bread wheat 22 Kulm Check IR6 Khuzestan Safiabad Triticale 23 3HD-126 Carrying Stb11 IR7 Khuzestan Safiabad Durum wheat 24 KM7 Carrying Stb16 IR8 Golestan Gorgan Bread wheat 25 KM41 Carrying Stb17 IR9 Khuzestan Shushtar Bread wheat 26 3HD-138 Carrying Stb18 IR10 Lorestan Delfan Bread wheat 27 Darab2 Local check

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20 ml of suspension using a hand sprayer. After spraying, Statistics (PAST) software version 1.74. The pycnidial the pots were covered with a clear polyethylene bag to coverage data (Table 3) of the wheat genotypes by each increase humidity and prevent cross-contamination. The isolate was subjected to a hierarchical cluster analysis pots were then kept for 72 h on a greenhouse bench at using the paired group algorithm, and the similarity was 21 ± 2/16 ± 2 °C (day/night) in a 16-hour day. The bags measured using the Euclidean distance method imple- were then removed, and the plants were kept in the mented in PAST software (Hammer et al. 2001). greenhouse under the same conditions (Weber 1922; Robert et al. 2005). The wheat responses were evaluated after 21 days of inoculation by visual estimation of the Results and discussion percentage of leaf area with necrotic lesions bearing pyc- nidia (Kema et al. 1996). In this study, pathogenicity patterns of Z. tritici isolates with different hosts and different geographical regions Data analysis were studied. Significant differences (p \ 0.01) were observed Individual and combined analyses of variance were per- between wheat genotypes in their response to different Z. formed. As fixed effects, the main effects of isolate, wheat tritici isolates. It seems that these differences were due to genotype, and their interactions have been included in this the highly significant variations (p \ 0.01) in the geno- analysis. SAS 9.1 for Windows was used for data analysis. type–isolate interaction (Table 4). The means were compared using Duncan’s multiple range Cluster analysis showed that Z. tritici isolates could be test. Cluster analysis was performed in Paleontological categorized into three major clusters (Fig. 1).

Table 3 Percentage of leaf area Genotype Isolate Mean of wheat genotypes covered by lesions bearing pycnidia of IR1 IR2 IR3 IR4 IR5 IR6 IR7 IR8 IR9 IR10 Zymoseptoria tritici isolates Oasis 20 50 50 30 50 70 50 70 20 30 36 Sulivan 50 50 50 50 70 50 50 70 20 50 41.667 Bulgaria 88 20 50 50 50 50 50 50 50 50 20 34.333 Veranopolis 50 30 30 50 30 30 10 0 20 0 20 Israel 493 20 20 20 30 50 50 0 0 20 0 15.667 Tadinia 5 0 0 20 10 30 10 0005.5 Cs Synthetic 30 20 30 30 50 70 10 0 20 10 21 Flame 30 20 0 30 30 50 000010.667 Shafir 0 0 0 30 0 50 00006 Estanzuela Federal 30 10 10 50 20 50 0 20 0 0 13 M6 Synthetic 10 0 20 10 10 40 00006.167 Courtot 20 10 0 50 10 70 20 0 10 0 14.333 Kavkaz-K4500 50 20 20 30 10 70 0 0 10 0 16.333 TE9111 10 50 10 10 40 50 0 0 20 0 12.5 Obelisk 50 30 50 30 50 30 20 50 50 30 35.333 Taichung 29 50 50 10 50 50 50 10 10 50 10 29 Salamouni 50 50 30 50 10 70 0 0 30 10 24 Ariana 20 20 0 10 10 70 00009.333 Riband 50 30 0 0 30 50 0 10 30 0 10.333 M3 300301000003008.333 Balance 20 10 10 10 00001005.333 Kulm 0 10 20 0000020104.667 3HD-126 20 20 30 10 0 50 50 0 50 10 22.333 KM7 30 0 20 50 50 10 0 20 50 5 17.5 KM41 50 0 30 000005008 3HD-138 0 0 0 20 50 50 0 20 50 30 15.733 Darab2 50 50 50 70 50 70 70 50 50 70 42.833

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Table 4 Combined analysis of Fixed effect df SS MS F value Pr [ F variance for percentage leaf area diseased by lesions bearing Isolate 9 43,703.7790 4855.9754 53.89 \0.0001** pycnidia of Zymoseptoria tritici isolates on wheat genotypes Genotype 26 104,961.6222 4036.9855 44.80 \0.0001** Genotype 9 isolate 234 108,275.5877 462.7162 5.14 \0.0001** df Degrees of freedom; SS sum of squares; MS mean squares **Significant at p B 0.01

Among the wheat differentials in this study, none of the cultivars were resistant to all used Iranian isolates. As shown in Fig. 2, the highest percentage of pathogenicity was observed on Darab2 (susceptible cultivar). Oasis (Carrying Stb1), Sulivan (carrying Stb2), and Bulgaria 88 (carrying Stb1 and Stb6) showed susceptibility to all the isolates. In studies on Z. tritici isolates in Iran, conducted by Abrinbana et al. (2012), Hosseinnezhad et al. (2014), and Makhdoomi et al. (2015), these cultivars were only resistant toward one or two isolates. This indicates that the Stb1 gene is not effective against Iranian species. There- fore, its use in wheat breeding programmes would not be efficient. Veranopolis cultivar that has Stb6 and Stb2 genes shows high partial resistance in field conditions (Brown et al. 2001; Chartrain et al. 2004). Among all Z. tritici isolates tested, this cultivar but also cvs CS Synthetic, Salamouni and Israel 493 showed resistance to only two isolates which make these cultivars not suitable sources of resistance to Fig. 1 Dendrogram resulting from l cluster analysis of 27 wheat STB in Iran. Similar results were obtained in studies by genotypes based on their mean disease severities to each isolate. The paired grouping method and Euclidean distance were used to generate Abrinbana et al. (2012) and Hosseinnezhad et al. (2014)on the dendrogram using Paleontological Statistics (past) software these fungal isolates in Iran, while Makhdoomi et al. version 1.74 (2015) introduced Salamouni as a resistant cultivar for Iran. According to the results, Stb5, Stb7, Stb13, and Stb14 All isolates from the Khuzestan province collected from possibly do not provide a good resistance and most of the bread wheat cultivars were similar in virulence pattern to isolates were virulent to these genes. Therefore, these isolates from the Ardabil province. However, the isolates genes cannot be considered as effective sources for resis- from Khuzestan that were collected on the triticale (IR6) tance to Z. tritici and used in breeding programs. and durum wheat (IR8) were categorized into different The only qualitative gene for STB resistance that has clusters. been shown to control a gene-for-gene relationship is Stb6, The isolates of the Lorestan, Golestan and Markazi at the distal end of the short arm of 3A provinces and one of the Khuzestan province isolates (Brading et al. 2002). In this study, Shafir is resistant to (collected on durum wheat) were categorized in the same most isolates, whereas Abrinbana et al. (2012) reported cluster. Shafir as a susceptible cultivar to the isolates that they had However, more than 90% of the area under wheat cul- studied. tivation is devoted to bread cultivars, albeit durum wheat is Two further genes including Stb16q and Stb17 were one of the most important crops in Iran (Mohammadi et al. discovered in the synthetic hexaploid line M3. Stb16q on 2015). Although durum and bread wheat cultivars are chromosome 3DL controlled the high variations in necrotic cultivated in close proximity with an irregular patchy dis- leaf area, leaf area bearing pycnidia, and latent period. The tribution in Iran, the isolates of Z. tritici do not exhibit host conferred resistance at the seedling stage to all Z. tritici species specificity (Ghaneie et al. 2012), but these results isolates was tested among the major reported Stb genes. Stb showed that in addition to the geography, the type of host 17 on 5AL was detected only at the adult plant stage, and it species(wheat or triticale) affects the virulence pattern. was less potent than Stb16q (Tabib Ghaffary et al. 2012; Brown et al. 2015). Hosseinnezhad et al. (2014) reported

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Fig. 2 The mean disease severity Zymoseptoria tritici on differential wheat cultivars (comparison of means by Duncan, 1% probability levels) that M3 was resistant to all Iranian isolates, but in our According to the results of this study, the virulence research, M3 cultivar was susceptible to the four Z. tritici pattern of Z. tritici is different in various regions and none isolates (Table 3). This is the first report of susceptibility of of the resistance sources to STB in Iran are effective this cultivar to STB. against all Iranian Z. tritici isolates. Therefore, it is nec- KM41 cultivar has the Stb17 resistance gene and was essary to design novel STB management and Stb gene used for the first time in Iran. Stb17 is the first gene for deployment strategies. adult plant resistance and interestingly, the results showed that this genotype had resistance against most of the iso- lates even at the seeding stage. This phenomenon needs Compliance with ethical standards further investigation on whether the related gene can be effective at the seedling stage or this cultivar has another Conflict of interest The authors declare that they have no conflict of resistance gene. interest. 3HD-138 cultivar (carrying the Stb 18 gene) was also applied for the first time in Iran. The Stb18 gene was first Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. reported in French cultivars and manifests its effect at the seedling stage. However, the impact of this resistance can Informed consent Informed consent was obtained from all individual prolong maturity of the wheat (Tabib Ghaffary et al. 2011). participants included in the study. Results showed this gene was only resistant toward three isolates. Induction of resistance against this disease is necessary. References Although chemical control can be a quick method for Abrinbana M, Mozafari J, Shams-bakhsh M, Mehrabi R (2012) disease control, it is costly and environmentally harmful. Resistance spectra of wheat genotypes and virulence patterns of Moreover, it induces resistance of toward Mycosphaerella graminicola isolates in Iran. Euphytica . The most economic method for controlling this 186(1):75–90 disease is to identify resistance sources. However, the vast Adhikari TB, Anderson JM, Goodwin SB (2003) Identification and molecular mapping of a gene in wheat conferring resistance to use of resistant cultivars induces extra pressure on patho- Mycosphaerella graminicola. Phytopathology 93:115–1164 gens and therefore breaks their resistance. There are Brading PA, Verstappen ECP, Kema GHJ, Brown JKM (2002) A numerous examples of this event, including the resistance gene-for-gene relationship between wheat and Mycosphaerella break of cultivars possessing Stb1 and Stb4 genes that last graminicola, the Septoria tritici blotch pathogen. Phytopathol- ogy 92:439–445 for only 5 years after the release of these cultivars in the Brown JKM, Kema GHJ, Forrer HR, Verstappen ECP, Arraiano LS, state of Oregon in the USA (Adhikari et al. 2003). Brading PA, Foster EM, Fried PM, Jenny E (2001) Resistance of

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