Potential suppression of broad-spectrum virulence of Blumeria graminis f. sp. tritici population to race-specific resistance genes in hexaploid wheat

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt), is one of the serious diseases that attack wheat plants during the growing season. The Bgt virulence was screened against 16 Egyptian wheat cultivars and 21 powdery mildew differential lines carrying different (Pm) resistance genes in two regions (Kafr-Elsheikh and El-Minya) in Egypt 2021–2022. Broad-spectrum virulence and high diversity were observed at both regions. Regional populations of Bgt in Egypt are independent of each other. Genes, Pm2a, Pm3a, Pm1b, Pm3b, Pm21, Pm34, Pm36, Pm37, Pm53, PmNCA6, PmMIAG12, and PmNCAG13 were nationally effective, while Pm4a, Pm4b, Pm6, and Pm8 appeared to be defeated throughout the country affected by broad-spectrum virulence. Field responses showed that only four cultivars, Misr-1, Misr-2, Sakha-95 and Shandweel-1 were resistant in both regions. A strong correlation was recorded between FDS and AUDPC which implies that wheat pathologists and breeders may be able to assess their lines by a single scoring at an appropriate time. Phenotypic and genotypic data proved that ineffective Pm8 was present in four susceptible cultivars, Sakha-93, Sakha-94, Gemmeiza-10, and Gemmeiza-11, while it was present in combination with effective Pm3a in two resistant cultivars, Sakha-95 and Shandweel-1. This suggests that cultivar susceptibility may be attributed to the existence of ineffective gene Pm8 potentially suppressed by effective Pm3a in hexaploid-resistant wheat. Pyramiding effective resistance genes particularly those that have a suppression effect like Pm3a may be a viable option to avoid the risk of broad-spectrum Bgt virulence at a regional scale.


Introduction
Bread wheat (Triticum aestivum L.) is one of the most important cereal crops over the world.Many pathogens attack bread and durum wheat during the growing season.Blumeria graminis (DC.)Speer f. sp.tritici (Em.Marchal) the causative agent of wheat powdery mildew, is recognized as one of the most serious threats of wheat which leads to high yield reduction.The yield loss of bread wheat because of powdery mildew could reach over 34% (El-Shamy et al. 2012).Furthermore, partial to total yield loss on susceptible wheat cultivars may occur when environmental conditions are favourable for powdery mildew (Zhang et al. 2015).Among all management strategies, the most effective way for controlling powdery mildew is breeding for resistant wheat cultivars (Zhang et al. 14).Two types of resistance could be used for this purpose, the first type has different terms i.e., race-specific resistance, effector-triggered immunity (ETI), qualitative resistance, and monogenic resistance (Kou and Wang 2010).The products of plant resistance initiated by monogenic resistance are recognized as specific pathogen effectors (Caplan et al. 2008).Relying on race-specific resistance genes does not guarantee a long-term of robust resistance as only one mutation is generally needed for a pathogen to change from avirulence to virulence.The wide cultivation of commercial cultivars, that rely upon monogenic resistance, for a long time might cause the resistance to be overcome by matching virulent pathotypes (Yu et al. 2001).
Polygenic resistance is another type of resistance which relies upon a quantitative resistance without the effect of race-specific reaction (Lindhout 2).This type of resistance is conferred by quantitative trait loci and is also termed polygenic resistance, partial resistance, quantitative resistance or race-nonspecific resistance (Kou and Wan 2010;Poland et al. 2009).Among 68 powdery mildew (Pm) resistance genes identified and mapped on specific chromosomes of the wheat genome, most are associated with qualitative traits (He et al. 2021).However, some of these genes were found to be associated with deleterious traits (Hurni et al. 2014).Hypersensitive (HR) non-associated polygenic resistance expresses more durability than monogenic resistance which associated with a hypersensitive response (Lindhout 2002).Hence, wheat breeders need to have enough information about the type of available resistance in commercial wheat cultivars and other breeding materials.Despite the importance of wheat powdery mildew, there is not a well-established Egyptian breeding program for introducing resistant wheat cultivars.Lacking information about the virulence of Bgt population and the available resistance genes in Egyptian wheat cultivars impedes the initiation of the successful breeding program.Only a few studies have investigated the virulence of the Egyptian Bgt population and the response of Egyptian wheat cultivars to the domestic Bgt pathotypes (El-Shamy et al. 2016;Abdelrhim et al. 2018;Draz et al. 2022).Also, the detection of some major resistance genes in commercial Egyptian wheat was studied by Emara et al. (2016) and Elsayed and Elkot (2020).The need for resistant Egyptian wheat cultivars is urgent, hence it is important to investigate the role of broad-spectrum virulence to race-specific genes in cultivar vulnerability and its potential suppression.This study is aimed to speculate which ineffective and effective resistance genes present in some Egyptian wheat cultivars by screening the response of Egyptian hexaploid wheat to particular Bgt pathotypes at seedling and adult plant stages across two geographically different regions.

Isolation and propagation of B. graminis f. sp. tritici
Samples of powdery mildew-infected wheat leaves carrying conidiospores of B. graminis f. sp.tritici were collected from wheat fields of two geographically isolated regions, Kafr-Elsheikh (31° 06′ 25.20" N, 30° 56′ 26.99" E) at North Egypt and El Minya (28° 06′ 35.57"N, 30° 45′ 1.08" E) at Middle Egypt during 2021-2022.Single pustule from infected wheat leaves were transferred using the spatula method on 10-day-old seedlings of the susceptible cultivar Jagalene, according to the inoculation method described by Browder (1972).The inoculated seedlings were kept in a greenhouse at 18 °C and 12 h of light until pustules developed.The purification was repeated twice to obtain pure Bgt isolates.The obtained pure isolates were propagated on Bgt susceptible hexaploid wheat cv Jagalene plants to obtain enough inoculum for virulence assay.

Characterization of Bgt races
A set of 21 wheat differential lines, each with a single Pm gene, plus Jagalene as a susceptible check were used for analysing the virulence of Bgt isolates (Table 1).The differential lines listed in Table 1 were provided by Dr. Christina Cowger's Lab at North Carolina State University.Each Bgt isolate was inoculated on 10-day-old seedlings of the differentials with five replicate pots.Seedling infection types were rated 10 days after inoculation following the 0-4 scale described by Shi et al. (1987), where ITs 0-2 refer to resistant response pattern (R) and their corresponding isolates are defined as avirulent (av), while ITs 3-4 refer to susceptible (S) and their corresponding isolates as virulent (v).

Virulence spectra and gene effectiveness
Virulence spectra of Bgt races were measured as the percentage of the virulence frequency on particular Pm genes to the total number of genes.Binary data generated from v/av patterns were used to construct a dendrogram.The effectiveness of Pm gene/cultivar was measured on the percentage of resistance frequency of a particular Pm gene/cultivar to the total number of Bgt races.

Virulence diversity and relatedness
Diversity index of virulence spectra for each Bgt race was measured on two indices, Simpsonʼs index (S) and Shannonʼs index (H) (Groth and Roelfs 1987) in the following ways: where n i = the number of virulences of the race i , and N = the sample size.
where P i = the frequency of virulence in a race i , and N = total number of virulences in the set race.
A virulence relatedness based on the simple match was generated using the PC software MVSP 3⋅1 (Kovach 1999).Cluster analysis was done using the unweighted pair group arithmetic mean (UPGMA) method.

Seedling evaluation of Egyptian wheat cultivars
The responses of 16 Egyptian bread wheat cultivars (Table 2) were evaluated against the obtained Bgt isolates.
In pots, a completely randomized design with five replicates was done for this test.Ten-day-old seedlings were inoculated separately with conidiospores of the obtained Bgt isolates.The inoculum preparation and the inoculation method were  (Zadoks et al. 1974), a field epidemic was initiated by inoculating spreader plants with a mixture of spores deriving from the obtained Bgt isolates.In which, the propagated conidiospores on a susceptible cultivar Jagalene described above (Browder 1972) were gently removed from the infected wheat plants and then dispersed over the spreader plants.All recommended agricultural practices for wheat crop in the commercial fields i.e., fertilization, irrigation, and other management were applied.When the susceptible cultivar "Jagalene" reached maximum severity (around GS-75), mildew scoring of adult plants was done using the 0-9 scale adopted by Saari and Prescott (1975).Mildew severity was assessed four times between GS45 and GS75 (Zadoks et al. 1974), with 10 days intervals according to Sharma and Duveiller (2007) using the following formula: where; D 1 indicates vertical disease progress on the plant and D 2 refers to severity measured as diseased leaf area.Final disease severity (FDS) was categorized based on the disease index, in which, 1-10% = Resistance (R), 11-30% = Moderately Resistance (MR), 31-50% = Moderately Susceptible (MS), 51-60% = Susceptible (S), 61% and above = Highly Susceptible (HS).The area under disease progress curve (AUDPC) was calculated according to Shaner and Finney (1977) as follows: in which Y = mildew severity (per unit) at the i th observation, X i = time (days) at the ith observation, and n = total number of observations.
The correlation coefficient (r) (Pearson and Hartley 1970) was analysed to determine the relationship between FDS and AUDPC.

Gene for gene hypothesis of Pm resistance genes
Pm genes were postulated in 16 Egyptian bread wheat cultivars according to the method described by Statler (1984).The tested wheat genotypes were inoculated separately at seedling stage (10-day-old) with the 12 Bgt races in a triplicate test.The inoculated seedlings were maintained in a greenhouse under favourable conditions.Response patterns for powdery mildew were evaluated 10 days after inoculation following the 0-4 scale described by Shi et al. (1987), where ITs 0-2 refer to resistant response pattern (R) and their corresponding isolates are defined as avirulent, while ITs 3-4 refer to susceptible (S) and their corresponding isolates as virulent.

Molecular verification of combined Pm genes
Molecular assay was carried out to verify the presence Pm genes postulated in Egyptian bread wheat cultivars.In this assay we selected two genes Pm8 and Pm3a based on virulence spectrum mediated by Bgt races along with their assembled presence.Pm8 gene was verified by using the closely linked Sequence-Tagged Site (STS) marker iag95 (primer sequence: fwd: AGC AAC CAA ACA CAC CCA TC, rev: ATA CTA CGA ACA CAC ACC CC) (Mohler et al. 2001), while the Pm3a was verified by using the closely linked simple-sequence repeat (SSR) marker PSP2999 (primer sequence: fwd: TCC CGC CAT GAG TCA ATC, rev: TTG GGA GAC ACA TTG GCC) (Bougot et al. 2002).
DNA was extracted from the tissue of wheat leaves using the Cetyl Trimethyl Ammo-nium Bromide (CTAB) protocol (Doyle and Doyle 1987).The PCR condition was performed using the Techne, PRO-GENE Thermocycler and optimized according to initial study of Mohler et al. (2001) and Bougot et al. (2002).PCR amplification products of 10 μl each sample were electrophoresed in 1.5% agarose gel stained with ethidium bromide at 100 V.A 100 bp DNA ladder H3 RTU, Nippon Genetics Europe GmbH, served in calibration.DNA bands were visualized using a UV-transilluminator (Herolab UVT 2020, Kurzwellig).

Assessment of genetic relatedness
Genetic relatedness of Egyptian wheat cultivars was assessed on Nei & Li's coefficient and generated using the PC software MVSP 3.1 ( Kovach 1999).Binary data generated from the presence/absence of resistance genes in Egyptian cultivars were used to construct a dendrogram.In which, presence was coded as 1, while absence as 0. Cluster analysis was done using the unweighted pair group arithmetic mean (UPGMA) method.

Statistical analysis
Data were pooled from repeated tests and subjected to analysis of variance using SPSS statistics computer program.Means were compared using LSD method (Steel and Torrie 1980) at 0.05 significance.

Virulence spectra and diversity
Data in Table 4 revealed the virulence spectra of Bgt races corresponding to 21 Pm genes.Broad-spectrum virulence was displayed with race Bgt11 derived from Kafr-Elsheikh region with a percent of 42.85%, followed by race Bgt4 (El-Minya) and Bgt10 (Kafr-Elsheikh) with a percent of 33.33% each.The race Bgt12 was the lowest virulent (4.76%), followed by the three races Bgt7 (13.04%),Bgt8 and Bgt9 (19.04% each) all derived from Kafr-Elsheikh region.All races displayed high diversity of virulence to Pm genes reached S = 0.875 and H = 2.079, except one race Bgt12 which had no diversity.The highest virulence diversity was recorded with race Bgt11 (S = 0.875 and H = 2.079), followed by Bgt4 and Bgt10 (S = 0.857 and H = 1.946).The

Cultivar effectiveness
The resistance responses of each Egyptian wheat cultivar to the Bgt races were used to assign the effectiveness (Fig. 4).Cultivar efficacy ranged from 8.33 to 83.33%.Sakha-95 and Shandweel-1 were the most effective cultivars (83.33%).

Discussion
Broad-spectrum virulence B. graminis f. sp.tritici to racespecific Pm resistance genes were screened in Egyptian and global wheat germplasm across two regions in Egypt during 2021-2022 where the disease is deep-rooted.The results indicated that some Pm genes i.e., Pm8 could become ineffective because of a persistent exposure to the pathogen population that is able to circumvent these genes in the long run.The virulence analysis of Bgt races corresponding to 21 Pm genes displayed broad-spectrum virulence and high diversity with race Bgt11 derived from Kafr-Elsheikh region, followed by race Bgt4 (El-Minya) and Bgt10 (Kafr-Elsheikh).A clustering pattern generated using virulence/avirulence profiles of the Bgt races revealed the highest virulence relatedness between two races Bgt1 and Bgt4, followed by two races Bgt3 and Bgt6 derived from El-Minya as well as between Bgt10 and Bgt11 from Kafr-Elsheikh.This indicates that regional populations in Egypt are independent of each other and no mixing of populations in both regions.Nine genes, Pm1b, Pm21, Pm34, Pm36, Pm37, Pm53, PmNCA6, PmMIAG12 and PmNCAG13 were nationally effective with 100% efficacy, followed by Pm2a (91.66%),Pm3a and Pm3b (83.33% each).At the other end of the efficacy spectrum, Pm4b, Pm6 (16.66% each), Pm4a and Pm8 appeared to be defeated throughout the country affected by broad-spectrum virulence of Bgt races.These results provide a clear understanding of the virulence of Bgt and the effectiveness of the genes where only a few studies have previously reported the virulence analysis of the Bgt population in Egypt (El-Shamy et al. 9; Abdelrhim et al. 2; Draz et al. 2022).
In the field trials, adult plant resistance was investigated based on infection types (ITs) and final disease severity (FDS).Although they are useful in characterizing specific resistance, they cannot be the only variable used when both partial resistance and effective race-specific resistance are present in a large set of cultivars.The AUDPC was also calculated to assess the partial resistance at adult plant stages.Partial resistance allows some epidemic development of the disease, but at a reduced level (Knudsen et al. 1986).The quantitative nature of the resistance means that it is more difficult to identify than a race-specific resistance, but it may be apparent as relatively low disease severity under high disease pressure (Yu et al. 2001;Das et al. 1993).Most Egyptian wheat cultivars showed susceptibility to the disease in both regions.Susceptibility at Kafr-Elsheikh was higher than that of El-Minya.Six cultivars, Misr-1, Misr-2, Misr-3, Sakha-95, Gemmiza-12 and Shandweel-1 were resistant at El-Minya, while five cultivars, Misr-1, Misr-2, Sakha-95, Giza-168 and Shandweel-1 were resistant at Kafr-Elsheikh.In contrast, most of Pm genes were resistant in both regions, with the exception of six genes, Pm4a, Pm4b, Pm6, Pm8, Pm25 and Pm35 at El-Minya region, and four genes, Pm1a, Pm4b, Pm6 and Pm8 at Kafr-Elsheikh.This suggests that most of the Egyptian wheat cultivars carries no effective Pm genes.It is noteworthy that Sakha-95 and Shandweel-1 exhibited resistance across both regions based on ITs and FDS.The AUDPC revealed four Egyptian cultivars, Misr-1, Misr-3, Sakha-95 and Shandweel-1 possessed a high level of partial resistance El-Minya, while only two Sakha-95 and Shandweel-1 had a high level of partial resistance.A strong correlation was recorded between FDS and AUDPC for powdery mildew estimated in the tested wheat genotypes including Egyptian cultivars and Pm resistance genes.This implies that wheat pathologists and breeders may be able to assess their lines by a single scoring at an appropriate time (Yu et al. 2001; Draz and Abd El-Kreem 1).In the field, inoculation increased disease severity compared with the levels on susceptible check genotypes, but the correlation between seedling resistance and adult partial resistance trait AUDPC was very high.This indicates, on one hand, that the inoculum used was representative of the local pathogen virulence spectrum, and on the other hand, that pathologists and breeders may be able to select resistant cultivars and lines without needing to perform inoculation trials, provided that check susceptible disease severity reaches a sufficiently high level (Yu et al. 2001).In the wheat mildew pathosystem, cultivars characterized by a susceptible reaction at the seedling stage and a resistant reaction at the adult plant stage are described to possess specific adult plant resistance which is controlled by major genes.By contrast, susceptible cultivars at the seedling stage but showing relatively resistance at the adult plant stage are described to possess adult plant partial resistance which is controlled by minor non-race-specific resistance genes, some behave oppositely and others correspond closely to seedling and adult stages (Bennett 1981;Das et al. 1993).
Relatively few Pm genes were widely effective against the Egyptian B. graminis f. sp.tritici population in our study.However, other genes still have efficacy in some regions, and may still be useful for gene-stacking or resistance gene pyramiding.This is the first study to determine race-specific resistance affected by the broad-spectrum virulence of B. graminis f. sp.tritici population in Egypt.We first discussed the genes that appear to be partially or completely ineffective in Egypt.Pm1a had been previously reported as completely ineffective in all sampled locations during 2013-2014 growing season (El-Shamy et al. 2016), however, our isolates from previously unsampled regions indicated that Pm1a is likely still effective in Kafr-Elsheikh in the North and El Minya in Middle Egypt.High virulence to Pm3a and Pm3b had been found in the United States (Parks et al. 2008) but low virulence in Western Europe (Clarkson and Slater 1999) and Morocco (Imani et al. 2002).In Egypt, we here categorized Pm3a as regionally effective.The Pm3a is one of the first loci reported for powdery mildew resistance in wheat, mapped to the short arm of chromosome 1AS (Feuillet et al. 1998).We found that Pm3a is also likely effective in the other previously unsampled regions of Kafr-Elsheikh and El Minya.Pm3b, Pm6, Pm8, and Pm17 were ineffective against Egyptian B. graminis f. sp.tritici isolates in our seedling assay.El-Shamy et al. (2016) stated that Pm6 and Pm17 could be a potential source of partial resistance at the adult wheat stage against Egyptian Bgt Population.Pm17 was reported as allelic to Pm8 and both genes derive from rye (Secale cereale) that explains the similarity in their response to Bgt population (Hsam and Zeller 1997;Niewoehner and Leath 1998;Draz et al. 2022).It has been reported that Pm8 resistant gene is overcome by several Bgt races in numerous countries; Atlantic coast, Morocco, Europe, Hungary, China, and Egypt (Hsam and Zeller 1997;Imani et al. 2002;Wang et al. 2005;Kom áromi and Vida 2009;Abdelrhim et al. 2018).Turning to the genes that appear to be nationally effective in Egypt, Pm1b is reported to be an effective resistance gene against Bgt population in Egypt (El-Shamy et al. 9).It was originally obtained from Triticum monococcum and transferred into wheat cv.MocZlatka (Hsam et al. 1998).In addition, Pm2a was effective against most Egyptian isolates (91.66).The absence of Pm2a in recent Egyptian wheat cultivars differentiates the efficacy of the gene among the regions as it lacks the selection pressure in some regions than others.In our study, Pm21 was completely effective against all B. graminis f. sp.tritici isolates from Egypt, and it should be useful in common wheat breeding programs there.Pm21 is important gene which is still effective in many regions including, Europe, China, Poland (Huang et al. 1997;Chen et al. 1995;Czembor et al. 2014;Liu et al. 2015).Transferred to common wheat from Haynaldia (= Dasypyrum) villosum, a wild wheat relative, via a translocation (6VS.6AL), this gene has been widely used in breeding (He et al. 2016) and was recently made accessible to breeders in a spring-habit background (Lukaszewski and Cowger 2017).
Response patterns of cultivars postulate the presence of Pm4a in Misr-1 and Misr-3, while Pm4b in Sids-12, Sids-12 and Sids-14.Pm8 was postulated to be present in six cultivars,  and Shandweel-1.The T1BL.1RS wheat-rye translocation chromosome cultivars, which contain chromatin from 'Petkus' rye, carry the resistance gene Pm8 (Hsam and Zeller 1997).Response pattern of Gemmeiza-12 suggests the existence Pm1a.A combined presence of Pm3a and Pm8 was postulated in two cultivars, Sakha-95 and Shandweel-1.The most frequent gene was Pm8, it was present in six cultivars, while the least one was Pm1a (one cultivar), followed by Pm3a and Pm4a (two cultivars each).Molecular data showed the combined presence of both Pm3a and Pm8 in both cultivars, Sakha-95 and Shandweel-1.The SSR marker PSP2999 developed 610-bp in both cultivars proving the existence of Pm3a, while the STS marker iag95 verified the presence of Pm8 in both cultivars with an amplified fragment of 1050-bp.This suggests that none of the evaluated bread wheat cultivars had effective Pm genes.The six wheat cultivars, Sakha-93, Sakha-94, Sakha-95, Gemmeiza-10, Gemmeiza-11 and Shandweel-1 showed a high frequency of resistance to B. graminis f. sp.tritici isolates collected from both regions.Cultivar effectiveness revealed that the resistant cultivars, Sakha-95 and Shandweel-1 were the most effective cultivars (83.33%), followed by .The least cultivar efficacy (8.33%) was recorded with Gemmeiza-9, followed by Giza-168 (16.66%), .This suggests that the susceptibility of Egyptian cultivars may be attributed to the presence of ineffective race-specific gene Pm8, since it was present in four susceptible cultivars Sakha-93, Sakha-94, Gemmeiza-10, Gemmeiza-11, with low effectiveness affecting broadspectrum virulence of Bgt races.Even though ineffective gene Pm8 was found in combination with effective gene Pm3a in two resistant cultivars Sakha-95 and Shandweel-1.This suggests that Pm8 may be suppressed by Pm3a in both cultivars Sakha-95 and Shandweel-1 (Hurni et al. 2014).

Conclusion
We demonstrate that hexaploid wheat susceptibility towards powdery mildew may not only be due to the lack of effective genes but may also be attributed to the presence of ineffective genes, such as Pm8 reported in current study.To our knowledge, this was the first time to demonstrate that Pm8, a race-specific gene affected by broad spectrum Bgt virulence was suppressed by effective gene Pm3a in Egyptian hexaploid wheat.Gene-stacking breeding process should be explained better.
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Fig. 2
Fig. 2 Effectiveness of powdery mildew (Pm) resistance genes against Bgt races collected from Egypt during 2021-2022.A cumulative line as a percentage of the total

Fig. 4 Fig
Fig. 4 Effectiveness of Egyptian wheat cultivars against Blumeria graminis f. sp.tritici races collected from Egypt during 2021-2022.A cumulative line as a percentage of the total

Table 1
Differential wheat lines used to analyse virulence spectra of Blumeria graminis f. sp.tritici in current study

Table 3
Characterization of 12 races of Blumeria graminis f.

Table 5
Adult plant responses of Egyptian wheat cultivars and Pm resistance genes to powdery mildew at two regions in Egypt during 2021-2022 final disease severity, AUDPC area under disease progress curve