Resistance of wheat genotypes to Mycosphaerella graminicola isolates at seedling stage under greenhouse conditions

Abstract One of the most devastating foliar diseases of wheat worldwide is Septoria leaf blotch (STB), caused by Mycosphaerella graminicola (asexual stage/Anamorph: Septoria tritici) which has been recently intensified in some regions in Iran. In this study, 49 wheat genotypes and 20 wheat differential genotypes were evaluated for their reaction to infection by six isolates of M. graminicola collected from infected fields during 2016–2017 at seedling stage under greenhouse conditions. According to the analysis of variance (ANOVA) of leaf pycnidia coverage percentage, a significant difference (p < .01) was observed between M. graminicola isolates and wheat cultivars. The interaction between genotypes and isolates was also significant (p < .01) and the results indicated a specific interaction between genotypes and isolates. The results presented Dezful and West Azerbaijan isolates that were the most virulent with more pathogenesis on differential genotypes. Although 47 of the wheat genotypes were susceptible to all isolates, some genotypes, including Wc‐46,224 (Austria), Wc‐45,425 (Portugal), Wc‐45,565 (Turkey), P.S.No4 (Italy), Dehdasht, M3 Synthetic, KavKaz‐k4500, Arina, Flame, and Riband were resistant to all isolates. In addition, the isolates exhibited different virulence patterns on wheat genotypes. The results of this study revealed high virulence of M. graminicola isolates, and Iranian and foreign wheat genotypes, commonly used in the region, presented high susceptibility, and the resistance sources had been identified among genotypes that can be applied in the wheat breeding programs.

fungus (M.Graminicola) was first identified by Sanderson (1972) in New Zealand and later in Australia, Brazil, the Netherlands, the United Kingdom (Eyal et al., 1987), and Canada (Hoorne et al., 2002).
Wheat STB disease in Iran was first reported by Petrak which was then sporadically and negligibly on wheat (Dadrezaie et al., 2003).The disease has gradually become more important and expanded in Iran with beginning of cultivation of modified CYMMYT genotypes (Khelghatibana et al., 2004;Rajaie et al., 2004).The extension of STB will be much more intensive with the development of rust-resistant dwarf genotypes as well as with increased nitrogen fertilizer use and disease loss will be intensified if infection occurs prior to spike emergence (Eyal, 1999).The fungi caused the disease result in unexpected and very serious epidemics on susceptible genotypes in favorable environment, and significantly reduce yield quality and quantity, which in some cases exceeds up to 50% losses (Eyal et al., 1987).
Cultivating method control such as spraying fungicides and using resistant genotypes are recommended to control this disease.
Given the inefficiency of cultivating methods in the effective control of the disease, resistance of fungal isolates to fungicides, and the costs and pollution caused by the application of toxic chemicals, the use of resistant genotypes are considered one of the most cost-effective and best methods to control the disease (Eyal, 1999;Eyal et al., 1987).Physiologic specialization of the gene-for-gene was demonstrated in this pathosystem (Brading et al., 2002) several years ago and 22 resistance genes (Stb) were mapped to STB in different wheat genotypes (Arraiano et al., 2001(Arraiano et al., , 2003(Arraiano et al., , 2007;;Adhikari, Wallwork, & Goodwin, 2004;Brading et al., 2002;Chartrain, Berry, & Brown, 2005;Chartrain, Joaquim, et al., 2005;Chartrain, Brading, et al., 2005;Chartrain et al., 2009;Cowling, 2006;McCartney et al., 2003;Tabib Ghaffary et al., 2011, 2012;Yang et al., 2018).Previous studies proved that the growth of fungal biomass terminates in the resistant genotype with Stb genes after 12-15 days (Habibi et al., 2014).Specific resistance to M. graminicola single isolates was identified in bread wheat genotypes (Abrinbana et al., 2012) and local tetraploid wheat subspecies (Ghaneie et al., 2012).Unavailable information regarding pathogen virulence and specific resistance to different isolates makes it difficult to research and identify how genetically control resistance in resistant genotypes and their utilization in breeding programs for resistance to STB.A study of the genetic structure of M. graminicola using molecular markers revealed that high genetic differentiation and low levels of gene flow were observed among the populations of this fungus in Ardebil, Golestan, Khuzestan, Fars, and East Azerbaijan provinces (Abrinbana et al., 2010).The transmission of various genes, including virulence and avirulence genes, are restricted among the fungi populations in these areas under these circumstances.Accordingly, it is proposed that the response of the genotypes and lines used in each of these areas are examined with isolates of the same region to identify sources of effective resistance (Abrinbana et al., 2010).The purpose of this study is to aim to compare the resistance of various wheat genotypes and identify those with the highest level of resistance to STB for use in wheat breeding programs.
Seeds of 49 genotypes were received from the gene bank of Cereal Research Department of Seed and Plant Research Improvement Institute, Karaj, Iran.

| Collection of infected plant samples
The naturally infected leaves with Septoria leaf blotch from wheat fields in different regions of Iran were collected in 2016-2017 cropping season, the samples were then transferred to the laboratory, and subjected to fungal isolation, for which 1-2 disks per each leaf containing pycnidia were cut as sample.The samples were superficially disinfected with 2% sodium hypochlorite.The leaf disks were placed on wet filter paper in sterile petri dishes and kept at 20°C for 24 h.The oozes from the pycnidia ostiole were transferred onto potato-dextrose-agar (PDA; potato 200 g/L, dextrose 20 g/L, and agar 15 g/L) plates under a stereoscopic microscope with a sterile fine needle.The plates were then kept to allow colonies to grow.The samples were purified by drawing sketches on laboratory loop impregnated with spore suspension and the samples were then subcultured on PDA medium as pure fungal culture.
Seedlings were prepared by sowing wheat seeds with five to seven seeds in each plastic pot containing a mixture of peat moss and soil in a ratio of 1:1 in three replications.In order to prepare the suspension required for seedling inoculation, the liquid medium of yeast-glucose extract (YGM; 30 g/L glucose +10 g/L yeast extract + distilled water) was infected with segments of colony grown on PDA medium and placed on a shaker at 17°C for 5 days.The prepared spore suspension was centrifuged at 3000 rpm for 5 min and then the suspension of yeast spores deposited was prepared in sterile water with a concentration of 10 million spores per mL (10 6 Spor/mL).

| Zymoseptoria tritici isolates
Due to their specific interactions with some wheat genotypes (Tabib Ghaffary et al., 2012), six Z. tritici isolates (five isolates from Iran and one isolate from Algeria) were selected to be used in this study from different origins (Table 3).

| Evaluation of isolates' virulence using wheat cultivars in greenhouse
The virulence of six M. graminicola isolates was tested using 69 wheat cultivars and three control susceptible cultivars to Septoria leaf blotch disease (Bolani) in a completely randomized design with three replications in greenhouse conditions.Ten seeds of each genotype were sowed in 10-cm pots containing a mixture of field soil and peat moss in a ratio of 1:1, the seedlings were inoculated at the one-leaf stage (approximately 9 days after sowing when the first leaf completely expanded and the second leaf appeared) separately with fungal spore suspension with a concentration of 10 7 Spore/mL for each isolate by spraying until the spore suspension flowed from the leaf surface.analyzed 21 days after inoculation by measuring the percentage of leaf area with necrotic lesions bearing pycnidia (Kema, Verstappen, Todorova, & Waalwijk, 1996;Kema et al., 1996) and McCartney et al.
Analysis of variance of data obtained from measuring percentage of pycnidia coverage (PC) and necrosis level (NL) of leaves was performed after standardization using SPSS and Excel software, categorization of isolates and wheat cultivars based on average percentage of pycnidia coverage, and percentage of necrosis in leaf area using cluster analysis in the Ward and GGE Biplot approach.

| RE SULTS AND D ISCUSS I ON
Characteristics of wheat genotypes, differential genotypes, and Z.
tritici isolates used in this study are presented in Tables 1, 2, and 3, respectively.

| Response of wheat genotypes and comparison of virulence of M. graminicola isolates based on pycnidia coverage percentage
Results of analysis of variance (ANOVA) indicated significant differences (p < .01) between cultivars and isolates (Table 4).The analysis also revealed significant differences (p < .01) in interaction between genotypes and isolates, indicating a specific interaction between the studied genotypes and isolates.This may show the genetic differences between the host genotypes for resistance to the pathogen.
According to the results of this test, the mean of infection was 0% and the interactions that were not significantly different from the mean of 0% were considered as resistance.
Among the isolates, RM 5, RM 183, and RM 230 with 87.7% virulence on 43 genotypes and RM 251 with 79.5% virulence on 10 genotypes had the highest and lowest virulence isolates, respectively (Table 5).
As described by Brown et al. (2001), the isolate aggressiveness was measured for each wheat genotype on the basis of the mean disease severity by ignoring data for specific interactions.The most and the least aggressive isolates were RM230 and RM251, which presented the highest mean disease severity (40.1%) and the lowest mean disease severity (37%), respectively (Table 4).
To understand the pathogenicity and resistance in pathosystem of M. graminicola and wheat, it should be considered that wheat genotypes are continuously exposed to completely diverse populations of pathogens and the abundance of pathogenic isolates with specific pathogenicity, which are capable of adjusting and establishing different genotypes of wheat, and are gradually increasing.Investigation of pathogenicity differences of 56 M. graminicola isolates collected from seven provinces of Iran revealed that there are significant differences among the isolates in terms of invasive power (Bashiri et al., 2006).

| Z. tritici isolates' aggressiveness and Stb genes' efficacy against isolates
Different virulence patterns on the Stb differentials were observed in six isolates, indicating the effective resistance genes to a limited number of Z. tritici isolates in these genotypes.

| Cluster analysis
Cluster analysis results of the genotypes based on the mean pycnidia coverage percentage of leaf area grouped them into four separate clusters in response to isolates (Figure 1).
Cluster I containing five genotypes including four landraces 224,425,565, and P.S.No4) and one Iranian durum wheat cultivar (Dehdasht) showed high level of resistance to six isolates and mean disease severity data ranged from about 0% to 9.1% (Figure 1 and Table 5).
In cluster II, the isolate-specific resistance was identified in one  5).
In clusters III and IV, 39 genotypes (27 landraces and Iranian durum wheat cultivars, 10 bread wheat cultivars, and susceptible control) with low to high susceptible reactions were grouped with control showing the mean infection of 37.6% to about 85.5% (Figure 1 and Table 5).
The results of cluster analysis of differential genotypes also revealed that these genotypes can be divided into resistant and susceptible groups in response to M. graminicola isolates.
The second group containing five genotypes including Oasis, Te9111, Tadinia, Salamouni, and Veranopolis in which the isolate-specific resistance was identified in cluster I, respec-  2 and Table 5).
Clusters II and III contained control susceptible genotypes (Boolani, Taichung29, and Obelisk) and seven genotypes that indicated highly susceptible pattern to all isolates with a range of 44.2-66.3% (Figure 2 and Table 5).F I G U R E 1 Cluster analysis of 49 durum and hexaploid wheat genotypes based on their mean disease severities to each isolate.

| Relationships among studied Septoria isolates
The relationships among isolates can be seen in Figure 4.A close relationship was observed among isolates 1 and 3 with isolates 4, 5, and 6; in other words, they have more similarities in terms of virulence pattern.Isolate 2 is more distinguished from other isolates, so its virulence pattern is different from other isolates (Figure 4).

| Relationships between the superior wheat genotypes and Septoria isolates
The relationships of the identified superior genotypes were revealed based on mean disease severity data to the studied isolates in Figure 5. Therefore, genotypes 11, 33, 25, 9, and 7 were resistant genotypes with the highest resistance to all isolates.Genotype 42 Cluster analysis of wheat differential cultivars based on their mean disease severities to each isolate.

F I G U R E 3
The superior genotypes against Septoria tritici blotch caused by M. graminicola isolates.

| DISCUSS ION
Yield losses caused by wheat STB are increasing in recent decades, plant pathologists and wheat breeders have further focused on the disease and have studied its various aspects such as genetic diversity, virulence pathogen, and host resistance.The use of resistant genotypes not only provides the best and most effective way to control the disease economically and environmentally but also it can contribute to avoid the use of chemical-based fungicides (Eyal, 1999).
According to the results of studies conducted in Iran (Haghdel & Banihashemi, 2003;Khelghatibana et al., 2004;Kia et al., 2006) and the incidence of disease epidemics in some provinces of the country, most common wheat genotypes used in Iran appeared to be susceptible to this disease, and serious consideration to breeding resistant varieties of the disease requires study of virulence pathogenicity in different regions, evaluation of resistance of various wheat genotypes against isolates of infected areas, identification of resistant genotypes, and finally study of their genetic resistance.
The present study was conducted in this regard and single isolate of M. graminicola was used to evaluate the response of resistant genotypes.The results of this study revealed the presence of specific resistance in the studied wheat genotypes and the physiological specialization of the isolates, which are consistent with the findings of other studies (Eyal et al., 1985;Grieger et al., 2005;Kema, Annane, et al., 1996).
These results are consistent with the findings of Eyal et al. (1973) who first reported the presence of virulence differences in M. graminicola isolates and found virulent specific genes on some genotypes by assessing the virulence of 97 isolates on 35 wheat and triticale genotypes (Eyal et al., 1985).Razavi and Hughes (2003)  The mean total infection of the genotypes such as Aria, Oasis, TE9111, and Salamouni was low, due to the large number of isolatespecific resistance in these genotypes.Therefore, the studied genotypes presented no moderate resistance to the isolates.These results indicated that most of these genotypes are susceptible to wheat STB, which could justify the disease's outbreak and epidemic in this area.Although bread hexaploid wheat and tetraploid durum wheat are the most important hosts of M. graminicola, some studies have shown that host specificity at the host species level was also present among isolates of this pathogen, so isolates collected from durum wheat are nonpathogenic on bread wheat (Kema, Annane, et al., 1996;Van Ginkel & Schafren, 1988).In regions or countries where one of these wheat genotypes was predominantly cultivated, the fungal isolates of those areas were adjusted to the predominant wheat, and specialization at the host species level occurs as an important trait (Eyal, 1999).
A few number of isolates used in this study were different in the virulence pattern of isolates and probably reflect high genetic diversity in the fungal populations in the region.These results were similar to the findings of most of the research conducted in these areas (Abrinbana et al., 2012;Eyal et al., 1973;Eyal et al., 1985;Kema, Annane, et al., 1996;Makhdoomi et al., 2015;Mehrabi et al., 2015), these studies used different isolates through Relationships among studied Septoria isolates.

F I G U R E 5
Relationships between the superior wheat genotypes and Septoria isolates.
the world.There has been limited information available on the virulence diversity of M. graminicola isolates in a specific region or even a specific country to this date.However, in a study using isolates collected from Manitoba and Saskatchewan in Canada, the low virulence diversity of the isolates was reported for these fungi populations in the region (Grieger et al., 2005).Although disease scaling has been used as one of the methods of evaluation of wheat STB in some cases (Grieger et al., 2005;Mergoum et al., 2007;Rosielle, 1972), the expression of single-gene or vertical resistance to the disease has not been always decisive and qualitative and, in some cases, this type of resistance also occurs quantitatively.So, unlike some diseases such as wheat powdery mildew, resistance to STB has been assessed quantitatively, usually by measuring the percentage of host leaf area covered by lesions bearing pycnidia (Brown et al., 2001;Chartrain, Brading, Makepeace, & Brown, 2004).In this disease, the level of infection was continuous, ranging from complete immunized (without pycnidia coverage) to complete susceptible with 90% leaf area infection, which symptoms vary depending on the studied wheat isolates and genotypes (Chartrain, Brading, Makepeace, & Brown, 2004;Eyal, 1999).That was why the use of qualitative methods was not very accurate and it was recommended to evaluate the disease quantitatively by calculating the percentage of host leaf covered by pycnidia and identifying the specific isolate-host interaction in this pathosystem by statistical methods.Accordingly, in this study, the percentage of leaf area covered by lesions bearing pycnidia was considered as a criterion for disease evaluation, and racespecific resistance was determined by statistical method.A large number of specific interactions was identified by this method, in some of which the resistance was not decisive and qualitative as up to 5% infection was observed in some cases.
According to the results of this study, it seems that the isolates of M. graminicola had high genetic diversity and virulence, which makes it difficult to efficiently use resistant genotypes and breeding for resistance to wheat STB in some cases.Most of the common genotypes in the region, especially the durum wheat genotypes studied in this study, were susceptible to the disease, and some of them had resistance genes that were not effective against most isolates in the region.However, among these genotypes, resistance sources were identified that could be used in wheat breeding programs in the region.This requires the study of resistance genetics and identification of the genes that cause resistance in these genotypes.Furthermore, it is advisable to evaluate the response of other genotypes and lines to identify more effective resistance sources and to produce genotypes with wider spectrum resistance by pyramidization of the effective genes.Pyramidization of resistance genes may not be effective in the long run due to the high genetic diversity of the pathogenic population in this region and the potential for sexual reproduction of the fungus.Moderate resistance should be used along with single genes in order to achieve stable resistance and to prevent rapid breakage of resistance.The genotypes that had been studied in this research did not show a relative resistance, but genotypes with this resistance may be identified by studying other genotypes and lines, or genotypes with moderate resistance (Chartrain, Brading, Widdowson, & Brown, 2004) can be used in wheat breeding programs.Studies revealed that resistance follows the gene-for-gene model in the interaction between wheat genotypes and Z. tritici isolates (Brading et al., 2002;Kema et al., 2000;Kema et al., 2018).
In specific resistance, the avirulence gene (avir) of the pathogen is usually identified by the resistance gene (R) of the resistant genotype followed by induction of (HR) high resistance in the plant.Six isolates studied in this research were different in terms of high virulence to the studied genotypes.The RM 155 isolate was the most virulent isolate and should thus have fewer avirulence genes, on the contrary, the RM 251 isolate was the least virulent isolate and should have the most avirulence genes.
Long-term cultivation of genotypes on a large scale may cause selection on the pathogenic population, thereby causing infection by dominating the resistance gene and breaking up the resistance.
For example, resistance of genotypes containing Stb1 and Stb4 resistance genes in Argon was broken 5 years after its release due to evolution of the pathogen genotype (Adhikari et al., 2003;Chartrain, Brading, Makepeace, & Brown, 2004).
The research results revealed that the isolates had a different pathogenic pattern and were more virulence on most of the Stb genes, which were somewhat consistent with the findings of Abrinbana et al. ( 2012), Hosseinnezhad et al. (2014), andMazandarani et al. (2014) and Makhdoomi et al. (2015).Studies around the world have indicated that Stb genes were vulnerable to attack by STB isolates (Abrinbana et al., 2012;Adhikari et al., 2003;Chartrain, Brading, Makepeace, & Brown, 2004;Cowger et al., 2000).
The present study also indicated that most of these Stb genes were not effective against Iranian Z. tritici isolates.This genetic diversity suggests that Z. tritici may be able to adjust quickly to resistant genotypes.Therefore, new sources of resistance must be regularly introduced to manage the disease.Using genetic resistance has been the most cost-effective strategy for controlling this disease.Thus, identifying new sources of resistance and expanding wheat gene storage are essential for the management of this disease.According to the results of this study, Stb1,Stb2,Stb3,Stb4,Stb5,Stb6,Stb7,Stb8,Stb9,Stb11,Stb13,Stb14,and stb18 genes were ineffective against the studied isolates, so they cannot be appropriate sources of resistance toward Iranian isolates of Z. tritici.The Stb2, Stb5,Stb6,Stb7,Stb13,and Stb14 genes and Stb2,Stb3,Stb4,Stb5,Stb6,Stb7,Stb8,Stb9,Stb11,Stb13,Stb14,Stb16,Stb17,and  In the research of Kia et al. (2017), this genotype was resistant to two isolates.Simon et al. (2016) reported that the Flame indicated partial resistance to a higher number of fungal isolates.
This genotype revealed specific resistance against three isolates in a study by Hosseinnezhad et al. (2014).Abrinbana et al. (2012) suggested that Flame genotype had specific resistance against six isolates.According to Makhdoomi et al. (2015), Flame was resistant to three isolates.The Stb6, Stb7, and Stb11 genes have been identified in TE9111 genotype (Chartrain, Joaquim, et al., 2005).
In this study, this genotype was also resistant to RM 251 and RM 155 isolates.In study of Tabib Ghaffary et al. (2012), this genotype was susceptible to one isolate, and in the other research, TE9111 was resistant to five isolates (Mahboubi et al., 2020).According to the study of Abrinbana et al. (2012), Hosseinnezhad et al. (2014), and Makhdoomi et al. (2015), this genotype indicated resistance against four, six, and three isolates, respectively.Arina and Riband genotypes were the source of resistance to STB, and have Stb15 genes (Arraiano, 2007).Arina and Riband were resistant to all isolates in this study, but Riband had specific resistance against 12 isolates in the study of Hosseinnezhad et al. (2014) and was resistant to all isolates in a study by Makhdoomi et al. (2015).Arina genotype showed resistance to 6, 10, 18, 20, and 10 isolates in other researchers' studies (Mazandarani et al., 2014;Abrinbana et al., 2012;Hosseinnezhad et al., 2014;Mahboubi et al., 2020;Tabib Ghaffary et al., 2012).Czembor et al. (2011)  Salamouni was resistant to eight and four isolates in the study of Mahboubi et al. (2020) and Dalvand et al. (2017), respectively.Oasis showed resistance to Iranian isolates during 2013-2015 (Dalvand et al., 2016).Also, Veranopolis resisted 13 and four isolates (Tabib Ghaffary et al., 2012;Mahboubi et al., 2020) The results of this study revealed that the tested isolates were virulent on most of the resistance genes and only a few genes had effective resistance against the isolates.This result was in agreement with previous reports (Abrinbana et al., 2012;Makhdoomi et al., 2015;Mehrabi et al., 2015).Stb1,Stb2,Stb4,Stb6,Stb7,Stb11,Stb13,and Stb14 genes were resistant to one or more isolates and the rest of the genes did not show resistance.Among the differential genotypes, Kavkaz-K4500, Arina, Riband, Flame, and M3 genotypes had the highest resistance against of all the isolates.Therefore, they can be used in breeding programs to produce resistant genotypes to the disease.

| CON CLUS IONS
The wheat landraces can be used successfully in wheat breeding programs to produce cultivars resistant to plant diseases.The other interesting result in our experiments was the high resistance of some wheat lines to most isolates, e.g., Dehdasht line was resistant to all isolates while landraces such as Shotordandan, Chamran 2, and Behrang were semisensitive.Therefore, it is likely a novel resistance gene(s) to M. graminicola isolate in these lines.
In addition, most of these lines may have had several resistance genes with different effects, resulting in a high resistance to various isolates in general.
Iran has been considered as the center of diversity wheat and M. graminicola fungi (Stukenbrock et al., 2007) and the pathogen and host plant have been in continuous interaction and evolution for thousands of years, so the Iranian isolates of M. graminicola with high genetic diversity and various virulence spectrum can dominate most of the known Stb genes.Also, they can be used to investigate resistance of wheat genotypes toward STB in other important wheat cultivation areas of the country, especially in Golestan, Khuzestan, and Ardebil provinces.Moreover, it is required to continuously study genetic changes in the pathogenic fungal population and to identify genetic sources of resistance with effective resistance genes against pathogenic isolates.
landrace, two durum wheat cultivars, and two bread wheat cultivars including Wc-45,443 (France), Chamran2, and Shotordandan which were resistant to two isolates (RM 251 and RM 155), while Aria and Gonbad showed resistance responses to three isolates, respectively, RM 22, RM 183, and RM 230 and RM 251, RM 5, and RM 22 (Figure 1 and Table suggested that there is a significant difference in virulence and invasive strength among 90 isolates collected from a field.In this research, Wc-46,224, Wc-45,425, Wc-45,565, P.S.No4, Dehdasht, KavKaz-k4500, Arina, Riband, Flame, and M3 Synthetic were the most resistant genotypes.This indicates that Stb10, Stb12, Stb15, Stb16, and Stb17 are found to be in the ten resistant genotypes in combination or individually.
List of Durum and Hexaploid wheat genotypes used in this study.
TA B L E 1 List of wheat differentials used in this study and their resistance genes (Stb).
TA B L E 3List of origins of Zymoseptoria tritici isolates used in this study.
Results of combined variance analysis of wheat genotypes pycnidia and necrosis studied isolate of Septoria.
The results presented that Stb12,   Stb6, and Stb7), Arina (possessing Stb15 and Stb6), M3 synthetic (W-7976) (Stb16 and Stb17), Flame (possessing Stb6), and Riband (possessing Stb15) were resistant to all isolates.Salamouni possesses Stb13 and Stb14 resisted all isolates except RM 155.Oasis and Bulgaria possess TA B L E 4 ns, *, **: Nonsignificant, Significant at 5% and 1% probability levels, respectively.Stb1, Oasis showed resistance to four isolates (RM 251, RM 6, RM 22, and RM 183), while Bulgaria showed resistance responses to one (RM 251) isolate.Veranopolis (possessing Stb2 and Stb6) and TE9111 (possessing Stb11, Stb6, and Stb7) genotypes were resistant to two isolates ((RM 251, RM22) and (RM 251, RM 155)), respectively.Tadinia possesses Stb4 and Stb6 which were resistant to RM 251 isolate, and also Bulgaria 88 possesses Stb1 which was resistant to RM 251.The rest of the differential genotypes were susceptible to the isolates.Among Stb genes, Stb10, Stb12, Stb15, Stb16, and Stb17 genes were the most effective resistance genes that presented resistance to all isolates.The six isolates used in this study were different in aggressive and mean disease severity on 20 differential wheat genotypes.RM 155 and RM 230 highvirulence isolates were pathogenic on 14 differential genotypes and RM 251 isolates were the least virulent isolates and were pathogenic on nine differential genotypes.In terms of aggression, RM 155 isolate with the highest mean disease severity (44.1%) was the most aggressive isolate.The RM 251 isolate with the lowest mean disease severity (27.1%) had the lowest invasive power (or was the lowest aggressive isolate).

Analysis of GGE biplot of genotypes compared to isolates
Percentage of frequency of resistance and susceptible genotypes based on pycnidia coverage.
3.4.1 | The superior genotypes against Septoria tritici blotch caused by M. graminicola isolates In Figure 3, the superior genotypes are placed on the top of polyhedron relative to each isolate based on mean disease severity data.Superior genotypes, in terms of resistance components, measured TA B L E 5 and Veranopolis (carrying Stb2 and Stb6) genotypes have not been able to resist against RM 251, RM 5, RM 22, RM 183, and RM 230; RM 251, RM 5, RM 22, and RM 183; and RM 251 and RM 22 isolates, respectively.
Abrinbana et al. and Makhdoomi et al. (2015)tb6, Stb13, and Stb14 genes cannot be effective in Iranian's breeding programs.Tadinia (carrying Stb4 and Stb6) and Bulgaria 88 (carrying Stb1) were resistant to RM 251 isolate.In the research of Dalvand et al. (2017), Tadinia among the foreign lines presented partial resistance to isolates, and Bulgaria 88 showed susceptibility to all the isolates.In studies on Z. tritici isolates byAbrinbana et al. and Makhdoomi et al. (2015), these genotypes were only resistant toward one or two isolates.This suggested that the Stb1, Stb4, and Stb6 are ineffective against Iranian species, making them inefficient in wheat breeding programs.