Keywords

1 Introduction

Wheat is one of the most important sources of food. At global level, approximately 95% of wheat cultivated is bread wheat (T. aestivum, hexaploid), while the remaining 5% being durum wheat (T. durum., tetraploid) and few other less important types (Shewry 2009). Wheat yield has been constantly under threat due to biotic and abiotic factors. In the event of abiotic stress, wheat cultivars are inclined by stress because of humidity, salt, temperature, and micronutrient. Continuous experience to high temperatures in rain-fed areas leads to drought stress and osmotic stress and higher salt concentrations in soil created by rapid evaporation of water, while the major biotic stress in wheat is due to diseases such as rusts, powdery mildew, karnal bunt, loose smut, blast, etc. Among the diseases, powdery mildew (PM), caused by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt), is one of the most serious threats limiting wheat production in several regions of the world including India. PM reduces the photosynthetic leaf area and the available nutrients, thereby reducing the yield significantly (by up to 25%). Breeding and developing PM-resistant cultivars is commonly viewed as the most efficient, powerful, and ecologically friendly technique to manage the disease. PM management using resistance genes enhances the stability and durability of the cultivars. Such durable resistance is exceedingly beneficial to farmers as it leads to increase in yield along with reduced cost of cultivation and is environmentally pleasant (Shah et al. 2017).

2 Disease

Powdery mildew is a wind-borne disease favored by the presence of disease in the preceding season. Disease infection can start during early crop growth when conditions are cool and wet. As the temperature rises and the humidity falls, the incidence and severity tend to diminish. The disease is preferred by mild temperatures (10–22 °C) (Beest et al. 2008), and 100% relative humidity (RH) favors the conidium germination. Prolonged cloudy weather fastens the disease development. During winter, spores survive in the host tissue after infection and may come from earlier infections within the field or from fields farther away. The disease is most common in dense early sown crops with high nitrogen fertility and rapid plant growth. Good soil moisture with potassium deficiency promotes canopy humidity which in turn favors the pathogen infection. Warm weather with alternate dry and wet conditions with wind may lead to epidemics during which even a resistant variety can become susceptible (Cunfer 2002).

2.1 Causal Organism

Powdery mildew is a fungal leaf disease caused by Blumeria graminis (DC.) E.O. Speer f. sp. tritici Em. Marchal (Bgt) = Erysiphe graminis DC. Ex Merat f. sp. tritici Em. Marchal. The pathogen is an obligate fungus which is host specific. Blumeria has been previously treated as a species of Erysiphe. But it differs from all species of Erysiphe because its anamorph possesses unique features such as digitate haustoria, secondary mycelium with bristle-like hyphae, and bulbous swellings of the conidiophores and unique structure of the ascocarps (Braun 1987). As a biotrophic parasite, B. graminis has evolved to specialize on particular hosts of Poaceae family. The wind-borne polycyclic pathogen greatly reduces yield and grain quality in wheat varieties that are susceptible. Blumeria is a true ascomycete fungus, forming the order of Erysiphales with only one family, the Erysiphaceae.

2.2 Geographical Distribution

PM of cereals is globally distributed. The disease is more common in regions with frequent rain and relatively cool temperature (Kashyap et al. 2021; Bennett 1984). Powdery mildew has been reported in several countries like the United Kingdom, Russia, Germany, Japan, Africa, and all parts of West Asia (Bennett 1984). Powdery mildew is a rampant disease in the cooler regions of China, Japan, and Central Asia, in North and East Africa, in northern Europe, and in eastern North America (Roelfs 1977; Saari and Wilcoxson 1974). In warmer, humid regions with mild winters such as parts of South America and the southeastern United States, the disease tends to be severe. But in regions where rain is frequent and heavy, the occurrence of powdery mildew is usually less as the spores are washed away from the leaves (Merchan and Kranz 1986). In India, PM is increasingly becoming problematic particularly in the northern and southern hill zone and some parts of north western plain zone.

2.3 Symptoms

Powdery mildew, caused by Blumeria graminis f. sp. tritici (syn. Erysiphe graminis), attacks wheat exclusively. The disease is characterized by white cottony powdery patches of fungal mycelium and conidia on the surface of the leaf. Initially, the symptoms appear as yellow flecks on the leaves. As the growing season progresses, the fluffy white fungal colonies can also affect the stem and earheads. Young plants having shorter tillers can become easily susceptible as they remain lower in the canopy. In severe condition it also appears on the awns. At maturity, the fluffy white colonies turn tan or brown in color. Symptoms usually progress from the lower to the upper leaves. As the infection gets older, small, black sexual fruiting bodies called chasmothecia (previously named cleistothecia) appear as distinct black dots on the maturing plants. When severe, the individual colonies often merge together and eventually cover most part of the stem and leaf surface. Moderate to severe infections can result in the death of the leaf tissues. Crop infected with powdery mildew usually appears yellow when seen from far due to the early death of leaves (Cunfer 2002).

2.4 Epidemiology

Powdery mildew severity occurs during periods of rapid crop growth, i.e., when plants begin to joint. The disease is first observed during tillering but does not continue after ear emergence. The sexual fruiting bodies known as chasmothecia produced during the late spring are highly resistant to extreme temperature and are thus an important source of inoculum for the following season. The chasmothecia survives on the wheat straw or as mycelium on infected wheat. During humid weather conditions, chasmothecia release ascospores which can germinate and infect plants under cool, humid conditions. Conidia are easily discharged from the lesions and are disseminated over long distances by wind and rain. An optimal temperature of 20°C is conducive for the production of conidia, and it declines rapidly above and below that temperature (Ward and Manners 1974). Fresh pustules with conidia are produced every 7–10 days at 97–100% relative humidity (Esmail and Draz 2017; Piarulli et al. 2012). The cycle is repeated for the continuous production of spores. As per the reports of Friedrich and Boyle (1993), relative humidity below 92% reduces the germ tube growth and appressorium production. Wheat powdery mildew infection and development tend to diminish when the temperatures rise above 25 °C and as humidity declines. Frequent and heavy rains slow the development of established pustules as the conidia are washed away by the rain water (Merchan and Kranz 1986).

2.5 Disease Cycle

The mildew fungus has multiple, rapid life cycles in a growing season. The fungus survives on the stubble as chasmothecia. Windborne conidia cause initial infections on the leaf surface which leads to secondary infections. The life cycle of powdery mildew includes both sexual (between seasons) and asexual (within season) stages particularly adapted to specific host habitats (McDonald and Linde 2002). Also, powdery mildew propagates very efficiently on wild plants and forms a huge reservoir of deeply rooted parasites that could serve as a source of inoculums to initiate epidemics in the fields (Dinoor 1974).

2.6 Yield Losses due to Powdery Mildew

Heavy economic losses around the globe have been reported to be due to powdery mildew diseases of wheat (Alam et al. 2012; Chen 2005). Powdery mildew can cause serious crop damage under severe conditions. Generally, the yield losses range from 12 to 34% (Griffey et al. 1993; Conner et al. 2003). But greatest yield losses up to 50% may occur if the disease occurs on the flag leaf during the heading and grain filling stage (Griffey et al. 1993; Leath and Bowen 1989). Based on the time of disease, epidemic onset, and its severity, the yield losses can reach up to 60% (Oerke et al. 1994). The pathogen reduces photosynthesis, decreases leaf assimilation index, and negatively affects grain yield components in wheat crop (Bowen et al. 1991; Henry and Kettlewell 1996; Samobor et al. 2005). At later growth stages, heavily colonized leaves can be killed prematurely which can significantly reduce yield up to 25% by reducing photosynthetic leaf area and crop available nutrients. In the colonized plants, the infection increases the metabolism of the attacked plants producing smaller shriveled grains. Also, during the attack of powdery mildew, the ability of the plant to resist other pathogens is decreased (Paulech 1995). Even low level of powdery mildew infection leads to the production of a greater number of nonproductive tillers which leads to reduced yield. Severe infection of powdery mildew can also cause delayed maturity, which increases the chances of reinfection and can also cause crop lodging through weakened stems. The earlier the infection, the larger is the potential yield loss.

In India, powdery mildew disease of wheat has caused serious consequences especially in parts of North Western Plain Zone, Northern Hill Zone, and Southern Hill Zone (Singh et al. 2009). However, sporadic incidence of powdery mildew has been reported from Rajasthan, Maharashtra, and Karnataka (Arya and Ghemawat 1953; Gadore and Patwardhan 1965; Patil et al. 1969). The disease was reported for the first time from Bombay (Maharashtra) (Gadore and Patwardhan 1965) and from Karnataka (Patil et al. 1969). From time to time, disease has been observed in severe form in U.P., Punjab, Haryana, Rajasthan, and Delhi (Swaminathan et al. 1971). In India, accurate data regarding the losses caused by mildew are not available since the disease mainly occurs in hills where wheat area is very limited.

2.7 Powdery Mildew Disease Assessment

Precise assessment of wheat powdery mildew is necessary for identifying the resistant and susceptible plants. Assessment of powdery mildew severity is done both in the seedling and adult plant stages using a visual scoring scale. Seedlings are generally assessed in the glasshouse under controlled conditions. The infection types (IT) of 10–15 days post-inoculation (dpi) of wheat leaves were scored using the 0–4 scale (Zhang et al. 2010). According to this scale, the scoring of “0,” “0;”, “1,” “2,” “3,” and “4” indicate “no visible symptoms,” “necrotic flecks without sporulation,” “highly resistant,” “resistant,” “susceptible,” and “highly susceptible,” infection types, respectively. In field conditions, powdery mildew is generally assessed using a 0–9 scale (Saari and Prescott 1975), based on the progression of symptoms. This scale is divided into three classes of infection types (ITs). 0–3 is considered as resistant, ITs 4–6 is considered as intermediate, and ITs 7–9 is considered as susceptible. The adult plant scoring was done once per season when the powdery mildew symptoms fully developed around GS-75 (Zadoks et al. 1974) and the most susceptible cultivars reached maximum severity (Table 12.1).

Table 12.1 Adult plant scale for powdery mildew disease scoring in wheat
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3 Disease Management

Many strategies are involved to control PM in wheat. However, genetic/host resistance is the most economical, reliable, sustainable, and environmentally safest way to control the PM disease.

3.1 Genetic Resistance to Powdery Mildew

Genetic resistance is among the most useful means to control powdery mildew (Xin et al. 2012; Summers and Brown 2012). Crops are diverse in their defense capacity against pathogens, and the genetic status of both host and pathogen determines the outcome of the interaction. The resistance depends on the interaction between the host and the pathogen. In general, there are two types of resistance to powdery mildew, i.e., quantitative resistance (horizontal or polygenic) and qualitative resistance (complete, vertical, and race-specific).

Each cell of the plants has innate immune system with systemic signaling capability from the site of infection (Jones and Dangl 2006). Upon infection, pathogens produce elicitors which are called pathogen-/microbe-associated molecular patterns (PAMP/MAMP), which includes peptides, metabolites, cell wall components, enzymes, and toxins (Dodds and Rathjen 2010; Giraldo and Valent 2012). These elicitors suppress the plant’s defense mechanisms. Post-infection, the host produces certain signal molecules known as damage-associated molecular patterns (DAMP) (Boller and Felix 2009). These elicitors or PAMP/MAMP/DAMP are recognized by the specific receptors (PRRs) in the plasma membrane (Frescatada-Rosa et al. 2015). As a primary level of defense response, the PAMP/MAMP triggers downstream genes resulting in no symptoms or hypersensitive response, generally referred to as the PAMP/pattern-triggered immunity (PTI) or non-host resistance (Baxter et al. 2014; Dodds and Rathjen 2010).

Certain pathogens produce race-specific intracellular elicitors known as effectors which are produced by specific avirulence (AVR) genes (Boller and Felix 2009). These effectors are recognized by plant-produced specific receptors (R proteins), encoded by R genes (Du et al. 2015; Sarris et al. 2015; Jones and Dangl 2006). These effectors suppress other PAMPs and also the host resistance genes to become more virulent (Lo Presti et al. 2015). As a secondary level of defense response, the effectors trigger downstream genes resulting in race-specific hypersensitive response to contain the pathogen, generally referred to as the effector triggered immunity (ETI) or qualitative resistance or vertical resistance (Boller and Felix 2009; Giraldo and Valent 2012).

In contrast, a weaker immune response, PTI and ETI response along with lack of hypersensitive response due to reduced or non-functionality of genes producing effectors and PAMP/PRR proteins and production of enzymes and toxins by pathogens, facilitating the pathogen to advance further is considered as incomplete resistance or quantitative resistance (Kim and Hwang 2015; Waszczak et al. 2015).

3.1.1 Race-Specific Resistance

Race-specific resistance or qualitative resistance has proved to be an integral part of crop breeding for resistance in wheat for many decades (Lillemo et al. 2010; Shamanin et al. 2019). This type of resistance is usually linked to immunity and constitutes resistance at all stages. Both PAMP/pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are to be considered as qualitative resistance, where the plant immune response is either a complete resistance with hypersensitive response or susceptibility. Race-specific resistance or vertical resistance to powdery mildew is controlled by major genes that is effective for only few Bgt isolates but is ineffective for others. Race-specific resistance is mainly via a hypersensitive foliar reaction directly involving single major R genes, which follows a gene-for-gene model (Bennett 1984; Hsam and Zeller 2002). More than 70 formally designated powdery mildew resistance genes (Pm) have been cataloged thus far (McIntosh et al. 2014). Major genes are expressed in seedlings and throughout the life stages of wheat. The widely used powdery mildew resistance gene Pm6, derived from T. timopheevi (Kuckuck 1970), is best expressed from the three-leaf stage onward (Jorgensen and Jensen 1972), and it is moderately effective.

Cultivars with race-specific resistance genes generally provide immunity or near-immunity to disease. This exerts a selection pressure on the pathogen that often leads to the rapid build-up of isolates with matching virulence genes (McDonald and Linde 2002). The R genes encode a class of resistance proteins which are the nucleotide-binding site leucine-rich repeat (NLR)-type receptors (Jones and Dangl 2006). He et al. (2018) reported that the powdery mildew resistance gene Pm21 encodes a typical NLR protein, yet it confers a broad-spectrum resistance at both seedling and adult plant stages to PM. To date, 11 mildew resistance genes, viz., Pm3 (Srichumpa et al. 2004), Pm38 (Krattinger et al. 2009), Pm8 (Hurni et al. 2012), Pm46 (Moore et al. 2015), Pm2 (Sánchez-Martín et al. 2016), Pm21 (Cao et al. 2011), Pm17 (Singh et al. 2018), Pm60 (Zou et al. 2018), Pm5 (Xie et al. 2020), Pm24 (Lu et al. 2020), and Pm41 (Li et al. 2020a, b) all encoding the nucleotide binding sites and leucine-rich repeat (NBS-LRR) proteins have been cloned.

Change in the virulence frequency is mainly influenced by the resistance genes present in the cultivars grown in a particular area. This has led to the rapid increase in virulent strains within the pathogen population, and consequently many of the major genes were overcome by new virulent pathotypes in a short span of time. For instance, a widely used gene Pm17 was overcome by new virulent pathotypes (Persaud et al. 1994). Recent ineffectiveness of some genes such as Pm17, Pm3a, and Pm4a in Eastern and mid-Atlantic regions of the USA (Cowger et al. 2009) and Pm8 in China (Wang et al. 2005) has urged the breeding community to further enrich the resistance sources to PM by identification and mobilization of new genes (Wallwork 2009).

Though many resistance genes have been identified from wheat and wheat-related species, most of them are race-specific and can be easily be overcome by new Bgt isolates (Li et al. 2014). Development of powdery mildew ceases when the day time temperature exceeds 26 °C, and thus even a moderate level of host resistance is adequate. In areas prone to severe epidemics, a common approach to control powdery mildew is to adopt host resistance by deploying one or more Pm genes in a better genetic background. Another strategy is to deploy non-race-specific quantitative resistance conferring more durable broad-spectrum resistance.

3.1.2 Non-race-Specific Resistance

Another type of resistance to powdery mildew is called non-race-specific resistance or quantitative or adult plant resistance (APR) or horizontal resistance which is governed by genes which is expressed in adult plants but not in seedlings. APR is quantitative in nature, and Keller et al. (1999) reported nearly 18 QTLs to govern powdery mildew resistance. APR is non-race-specific and is also referred as “slow mildewing” (Shaner 1973) and “partial resistance” (Hautea et al. 1987). APR is more durable than race-specific resistance to powdery mildew. Non-race-specific or quantitative resistance is commonly effective at the post-seedling stage. The resistance conferred by these genes is the result of the small effects of many genes and does not lead to complete absence of infection; instead, it reduces the fungal sporulation and duration (Burdon et al. 2014; Li et al. 2014).

Poland et al. (2009) reported that the molecular mechanism underlying quantitative resistance is that there is a multigenic basis, but the recent evidence suggests a likely diversity of mechanisms, some overlapping with race-specific resistance. Depending on the number and effects of genes controlling resistance, it is possible to distinguish quantitative resistance from race-specific resistance. According to Cowger et al. (2012), APR to powdery mildew has been identified in numerous widely cultivated cultivars which have been effective for more decades.

Hsam et al. (2003) reported that Pm12, Pm16, and Pm20 genes confer most effective defense response against powdery mildew in the adult plant stage. Majority of the APR genes have been mapped in the winter wheat. However, some of the APR genes such as Pm38 located at the Lr34/Yr18 locus and Pm39 located at the Lr46/Yr29 locus have been identified in spring wheat sources. Majority of the QTLs governing APR to powdery mildew have been reported on the chromosomes 1B, 2A, 2B, 2D, 3A, 3B, 4A, 4D, 5A, 5D, 6A, 7B, and 7D.

It is difficult to recognize plants with both race-specific resistance and APR to powdery mildew based on phenotype variation. With the advent of the molecular markers closely associated with genes such as Pm1 (Hu et al. 1997), Pm2 and Pm3 (Ma et al. 1994), Pm4 (Hartl et al. 1999), Pm12 (Jia et al. 1996), Pm12 (Cenci et al. 1999), Pm21 (Qi et al. 1996), and Pm25 (Shi et al. 1998) revealed that these genes have been reported with both race-specific resistance and APR to powdery mildew.

Several quantitative trait loci (QTLs) governing APR to wheat powdery mildew have been mapped near loci where the defeated major genes such as Pm4, Pm5, and Pm6 have been located (Keller et al. 1999; Liu et al. 2001). Keller et al. (1999) observed that the resistance conferred by such QTLs may be the result of the residual effects of defeated major genes and partial resistance may be the result of alternate alleles present at the R gene loci. Thind et al. (2017) reported that some genes involved in the quantitative resistance such as adult plant resistance (APR) gene also encode an NLR protein.

Humid and high rainfall conditions generally favor the rapid spread of powdery mildew throughout the plant canopy until senescence. Powdery mildew infection in the early plant growth stages reduces the tillering, whereas infection in the flowering stages reduces the losses in grain yield and quality (Everts et al. 2001). Thus control of powdery mildew in late plant growth stages is necessary for maximum protection of grain yields (Bowen et al. 1991; Griffey et al. 1993). Recently, additional sources of APR to powdery mildew have been identified, characterized, and validated. In order to develop durable and effective resistance to powdery mildew in wheat, it is necessary to combine QTLs having additive and complementary effects and that express during different growth stages.

However, the effective use of APR to powdery mildew resistance was largely limited due to lack of knowledge of effective sources of resistance, the quantitative nature, and precise tools for selection. This approach is cost-effective and time-consuming. Development of large mapping population segregating for the quantitative trait is required. Also the linkage analysis is restricted to loci in genomic regions containing polymorphisms between the two parental lines (Tanksley 1993).

3.2 Sources of Resistance to Powdery Mildew

Transferring PM-resistant genes to hexaploid wheat cultivars has been considered as an effective way to contain the disease. Most of the powdery mildew resistance genes actually come from wild relatives of wheat such as Triticum monococcum, T. urartu, T. turgidum ssp. dicoccoides, Aegilops speltoides, Ae. tauschii, etc. (Hsam and Zeller 2002; Jiang et al. 1994). Within the so far identified Pm genes derived from alien species or sparsely cultivated subspecies, 22 are assigned on the B genome, while only 14 are assigned on the A genome and 8 on the D genome (Tang et al. 2018).

3.2.1 Diploid Sources

Diploid Ae. tauschii Coss (2n = 2x = 14, DD), a distant relative of wheat, has proved to be a valuable source of powdery mildew resistance (Gill et al. 1986; Cox et al. 1992). Two genes, namely, Pm2 and Pm19, were transferred from A. tauschii into common wheat (Hsam and Zeller 2002). Ae. speltoides and Ae. longissima are both diploid species which were the donor of Pm1d, Pm12, Pm32, and Pm12 (Hsam and Zeller 2002; Hsam et al. 2003; Cenci et al. 1999). Pm29 gene was introduced from Ae. ovate to hexaploid wheat (Hsam et al. 2003). Similarly, Pm57 was identified on chromosome 2BL in Ae. searsii (Liu et al. 2017). More distantly related species which includes Ae. caudate, Ae. ovate, Ae umbellulate, Ae triuncialis, and Ae variabilis are also reported as valuable sources for PM resistance (Chen et al. 1995; He et al. 2009).

3.2.2 Tetraploid Sources

Tetraploid T. carthlicum (2n = 4x = 28, AABB genomes) was the source of Pm4b and Pm33 (Hsam and Zeller 2002; Zhu et al. 2005). Tetraploid species such as T. timopheevii and T. araraticum constitute the secondary gene pool. T. timopheevii and its wild form, T. araraticum (2n = 4x = 28, AAGG), contributed Pm6, Pm27, and Pm37 (Mains 1934; Järve et al. 2000; Hsam and Zeller 2002; Murphy et al. 2002; Perugini et al. 2008). Though tetraploid T. durum (2n = 4x = 28, AABB) is a less valuable source of resistance to powdery mildew (Mains 1934; Hsam and Zeller 2002), it contributed Pm3h (Zeller and Hsam 1998).

For common wheat, its progenitor wild emmer is also a rich donor of adaptive diversity to various diseases and can be exploited for trait improvement (Huang et al. 2016). Many confirmed Pm genes originate from wild species and primitive forms including wild emmer. Incorporating Pm genes into commercial cultivars is made possible as wild emmer is easily crossable with both hexaploid common wheat and tetraploid durum wheat (Rong et al. 2000; Elkot et al. 2015). Tetraploid wild emmer wheat (T. dicoccoides) (2n = 4x = 28, AABB) is the progenitor of common tetraploid and hexaploid wheats (Liu et al. 2002). Some of the powdery mildew resistance genes such as Pm16, Pm30, Pm31, Pm36, Pm41, Pm42, Pm49, and Pm50 have been transferred from wild emmer into common hexaploid wheat (Piarulli et al. 2012; Mohler et al. 2012). Cultivated species, T. dicoccum, is the source of resistance gene, Pm5, which is a recessive gene for powdery mildew (Mclntosh 1973).

3.2.3 Hexaploid Sources

Hsam and Zeller (2002) reported that old wheat cultivars, landraces, and related species were screened for resistance to powdery mildew early in the 1930s. PM genes were identified in many different, widely distributed wheat cultivars and landraces cultivated for thousands of years under extreme environments which are more genetically polymorphic in disease resistance and widely adapted to abiotic stresses (Talas et al. 2011; Li et al. 2016). Landraces can be readily crossed for the desired traits into new cultivars in comparison to distant relatives. Globally, many spring and winter wheat genotypes having seedling and adult plant resistance to powdery mildew have been identified and utilized in breeding programs. A total of 22 resistance alleles at 10 loci including Pm1, Pm2, Pm3 (3a, 3b, 3c, 3d, 3e, and 3f), Pm9, Pm18, Pm22, and Pm45 were identified in T. aestivum indicating the presence of more PM genes in cultivated wheat (Hsam and Zeller 2002). So far, nearly 33 designated genes have been identified from T. aestivum.

3.2.4 Other Sources

Li et al. (2018) and Chen et al. (2012) have reported that rye (Secale cereale L.) and Haynaldia villosa (H. villosa, syn. Dasypyrum villosum) had been used as a source of powdery mildew resistance genes. Genes for resistance to powdery mildew such as Pm8 and Pm17 have been successfully transferred into commercial wheat cultivars from rye (Jiang et al. 1994; Kim et al. 2004).

Pm8 is one of the most widely used genes in wheat breeding (Ren et al. 1997). This gene has played a major role in reducing the wheat yield loss due to powdery mildew infection. Pm8 was originally transferred from the “Petkus” rye into hexaploid wheat. Lutz et al. (1992) reported the emergence of new Bgt isolates that overcame the resistance of Pm8 during the 1990s. But the use of Pm8 in wheat breeding programs continued, especially in the twenty-first century, because the wheat-rye 1BL/1RS translocation carried other agronomic traits such as wide adaptability and high yield potential together with multiple disease resistance (Luo et al. 2009; El-Shamy et al. 2016). Globally, Pm8 has played an important role in wheat breeding and has been effective against the powdery mildew pathogen (Hurni et al. 2012). Pm17 is another resistance gene located on the short arm of the 1R chromosome in rye. Another gene Pm17 is also from rye identified in 1AL/1RS wheat-rye translocations (Friebe et al. 1994).

Chen and Liu (1982) reported another potential source of alien wheat powdery mildew resistance gene, Pm21 derived from H. villosa during the early 1980s. Pm21 showed a broad spectrum of resistance against most of the isolates of Bgt (Liu et al. 2015) and has remained effective for more than 40 years. Also, the resistance gene Pm51 was identified on chromosome 2BL in D. villosa (Zhan et al. 2014).

Oettler et al. (2005) reported that the hexaploid triticale (× Triticosecale Wittmack, AABBRR, 2n = 6x = 42), synthesized artificially by combining the genomes of Triticum turgidum (AABB, 2n = 4x = 28) and S. cereale (RR, 2n = 2x = 14), is an excellent source of powdery mildew resistance. Easy transfer of rye chromosomes into common wheat is possible as the rye components in triticale have been adapted to the wheat nucleus and cytoplasm (Ma and Gustafson 2008). As direct cross between wheat and rye requires precise embryo rescue techniques (Oettler et al. 2005), triticale serves as an alternative source for transferring the resistance contained in the rye chromosome to the hexaploid wheat. Hybridization of several triticale lines resulted in the development of triticale cultivar that varied in the rye genomes. As it combines the broad stress tolerance of different triticale lines, it can be effectively used to improve the powdery mildew and rust resistance of wheat in a short time.

Other species with potentially useful powdery mildew resistance genes are Ae. markgrafii, Ae. umbelluata, Ae. variabilis, Ae. triuncialis, and Ae. mutica, as well as the perennial subspecies of Triticeae, such as Elymus, Leymus, Elytrigia, and Thinopyrum (Jiang et al. 1994; Eser 1998; Hsam and Zeller 2002; Luo et al. 2009). Therefore, the identification of new and effective alien genes from wild relatives of wheat and their further translocation into crops may be a significant contribution to develop durable resistance to a broad spectrum of pathogen (Tester and Langridge 2010). Developing more resilient cultivars thereby solves the problem of low resistance of crops to powdery mildew of cereals and grasses and other fungal diseases (Pietrusińska et al. 2019).

According to Ma et al. (2018), the significance of breeding for powdery mildew resistance depends not only on its effectiveness for disease control but also on the agronomic performance of its donor (Zhao et al. 2012). Thus the identification of novel genes from elite wheat germplasm is a smart outlook for the rapid genetic improvement of resistance.

3.3 Effectiveness of Powdery Mildew Genes in Resistance Breeding

Cultivation of disease-resistant cultivars/varieties is an efficient method for commercial breeding, and disease control by the introgression of resistance genes enhances the durability of the variety. Host resistance is more likely to be durable when two or more resistance genes are pyramided in a single wheat variety. Information about the genetic diversity and distribution of Pm genes in a set of wheat varieties is required for the pyramiding of resistance genes.

Until now, 68 Pm genes/alleles (Pm1–Pm68) (Pm8 is allelic to Pm17, Pm18 = Pm1c, Pm22 = Pm1e, Pm23 = Pm4c, Pm31 = Pm21) have been identified in 60 loci from common wheat and its wild relatives (Li et al. 2019; McIntosh et al. 2019) (Table 12.2). Genes encoding resistance to powdery mildew have multi-allelic sites, where selected PM genes that respond differently to Bgt isolates are located at the same locus in different genotypes. Such genes include Pm1 (Pm1a-1e), Pm2 (Pm2a-2c, PmX3986-2, PmWFJ, PmD57-5D, PmLX66, and others), Pm3 (Pm3a-3j), Pm4 (Pm4a-4e), Pm5 (Pm5a-Pm5e), and Pm24 (Pm24a-Pm24b) (Ma et al. 2016; McIntosh et al. 2017). However, only a few PM genes have been successfully utilized in developing powdery mildew-resistant wheat cultivars such as Pm2, Pm4, Pm6, Pm21, and Pm30 which confer resistance against the pathogen isolates (Huang et al. 2012; Li et al. 2011; Liu et al. 2008).

Table 12.2 Genes associated with powdery mildew resistance, source, and their chromosomal location
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Pm3, existing in seven functionally distinct alleles (Pm3a to Pm3g), is the first wheat powdery mildew resistance gene to be cloned (Srichumpa et al. 2005; Yahiaoui et al. 2006). Pm3 alleles being the largest allelic series provide additional diversity for resistance toward different Bgt isolates while enriching the genetic basis for PM resistance breeding in wheat. The Pm4 locus is one of the most widely recognized loci of genetic resistance to powdery mildew, and Pm4a resistance allele has been used in breeding for several decades (Li et al. 2017). Further studies on the function characterization of this gene might provide an insight into the effectiveness of this gene toward the newly emerging Bgt pathotypes.

Pm6 is another important gene transferred from T. timopheevi, and the resistance conferred by Pm6 exhibits moderate resistance at the seedling stage and high resistance at the adult plant stage in a distinctly developmental stage-dependent manner (DDSDM) (Bennett 1984). Purnhauser et al. (2011) reported that Pm6 resistance has been found to be enhanced especially when used in combination with Pm2. The fine mapping and further cloning of Pm6 will facilitate not only the better utilization of Pm6 in wheat breeding but also a better understanding of the molecular mechanism of DDSDM resistance in plants.

Another popular gene Pm8 derived from the 1RS chromosome of rye has made a significant contribution to powdery mildew resistance in wheat since the 1990s. But the linked secalin glycopeptide in 1RS resulted in a poor flour quality (Friebe et al. 1989; Lee et al. 1995) making it unsuitable for breeding program. Other effective PM resistance genes that continue to be resistant include Pm1c, Pm12, and Mlxbd. But they have not been exploited in the present breeding program due to the poor agronomic traits associated with either alien chromosome segments or un-adapted genetic backgrounds (Duan et al. 1998; Qiu and Zhang 2004). Also, the PM genes derived from landraces usually have been reported to be linked with poor agronomic performance and require several backcrosses to eliminate the associated linkage drag (Xu et al. 2015; Li et al. 2020a, b). Thus, breeding for PM resistance depends not only on the effectiveness of the gene but also on the agronomic performance of its donor source (Zhao et al. 2012; Ma et al. 2018).

Though most of the resistant genes continue to be resistant, it could not be exploited due to poor agronomic traits associated with the alien translocations. For instance, Pm16 has been reported to give broad-spectrum resistance to wheat PM, but the linkage drag associated with this gene caused physiological deficiencies of yield leading to 15% yield loss (Summers and Brown 2012) which is the major constraint in the deployment of this gene.

Other factors such as the number of Bgt isolates virulent to particular gene should be considered while evaluating the putative resistance genes in bread wheat. Pm2a, which is believed not to be highly effective against Bgt isolates, remained effective in some parts of the world (Miedaner and Flath 2007); thus, this allele offers a novel source to be pyramided with other PM resistance genes in future breeding programs. Similarly, the recently identified Pm41 gene was reported to be highly resistant to Bgt isolates, and it is as a valuable and never exploited powdery mildew resistance gene. Thus, Pm41 could make an important contribution to wheat breeding through gene stacking.

An increased effort is required to explore new powdery mildew resistance genes and to improve the agronomic traits with currently identified genes. Though conventional wheat breeding has been remarkably successful, it is generally subjective, inefficient, and unable to achieve stable improvement (Gupta et al. 2010). Marker-assisted selection can successfully provide a valuable complement to conventional breeding (Gupta et al. 2010). With the advent of closely linked molecular markers, marker-assisted selection (MAS) can facilitate the elimination of adverse genes and accelerate breeding progress (Jiang 2015).

3.4 Molecular Markers Linked to Powdery Mildew Resistance Gene

Molecular markers that are tightly linked to the resistance genes can be used to identify the resistance of the wheat varieties in early generations (Gupta et al. 2010). Molecular markers tightly linked to the QTLs and R genes greatly facilitate in marker-assisted breeding and pyramiding of QTLs (Tucker et al. 2006). Molecular markers aid in the selection of resistant lines during the early growth stages and could be evaluated for high yield with high heritability, and they can easily be found by the genetic linkage of the desirable gene group on the chromosome associated with disease resistance (Hua et al. 2009). Molecular mapping allows for the accurate detection of molecular markers that are closely linked to the resistance to powdery mildew pathogen Bgt (Yao et al. 2007). Development and application of molecular markers in crop genetics are impactful in parental selection, genetic diversity estimation, reducing linkage drag, etc. and therefore of paramount importance in genetic mapping and gene discovery (Rasheed et al. 2017).

Different molecular techniques have been used to characterize and manipulate resistance genes and to dissect different types of resistance. RFLPs were the first molecular markers that developed and used in genetic analysis, initially in humans (Botstein et al. 1980), since the early 1980s, RFLPs have been used successfully for a wide range of plant species. The RFLP was also one of the first methods used for genetic typing and also known as genetic fingerprinting or profiling or testing. Despite that the RFLP have many benefits, it is still a slow and more tedious process to screen the mapping of genes compared to some of the newer DNA analysis techniques. In this manner, RFLPs have restricted application in wheat improvement programs. More number of powdery mildew resistance genes are identified by RFLP markers in wheat, such as Pm1, Pm2, Pm3b, Pm4a (Ma et al. 1994), Pm2 (Mohler and Jahoor 1996), Pm6 (Tao et al. 2000), Pm12 (Jia et al. 1996), Pm12 (Cenci et al. 1999), Pm17 (Hsam et al. 2000), Pm29 (Zeller et al. 2002), etc.

In the most recent decade, the RAPD procedure dependent on the polymerase chain reaction (PCR) has been one of the most commonly used procedures for detection of resistance genes. The RAPD analysis gives a speedy and proficient screen for DNA grouping-based polymorphism at uncountable loci. The significant favorable position of RAPD incorporates that it doesn’t need pre-sequencing of DNA. It is inexpensive and informal to use. Numerous powdery mildew resistance genes marked with RAPD markers, such as Pm1 (Hu et al. 1997), Pm1, Pm2, Pm3, Pm3a, Pm3b, Pm3c, Pm4a, Pm12 (Shi 1997), Pm25 (Shi et al. 1998), Pm12 (Cenci et al. 1999), etc.

Microsatellites or simple sequence repeated (SSR) loci, and simple sequence length polymorphisms (SSLPs), are co-dominant marker which found mostly in eukaryotes and to a lesser extent in prokaryotes more suitable for screening large populations than RFLPs. They are tandemly repeated (normally 5–20 times) in the genome with a minimum repeat length of 12 base-pairs. In recent times, several powdery mildew resistance genes are identified and mapped using SSR markers such as Pm27 (Järve et al. 2000), Pm30 (Liu et al. 2002), Pm33 (Zhu et al. 2005), Pm34 (Miranda et al. 2006), Pm43 (He et al. 2009), and Pm45 (Ma et al. 2011), etc.

3.5 Marker-Assisted Gene Pyramiding

Marker-assisted gene pyramiding entails introducing more than one resistance gene in order to increase durability and broad-spectrum resistance. Successful accumulation/stacking of multiple powdery mildew resistance genes is possible by rational application of marker-assisted gene pyramiding. Liu et al. (2008) successfully pyramided three powdery mildew resistance gene combinations, Pm2 + Pm4a, Pm2 + Pm21, and Pm4a + Pm21, into elite wheat cultivar ‘Yang047’. Plants deployed with single Pm2 gene had lower resistance, while those with Pm4a showed moderate resistance to powdery mildew. Notably, plants with Pm2 + Pm4a showed enhanced resistance than those with single Pm2 or Pm4a alone. Elkot et al. (2015) transferred two powdery mildew resistance genes PmTb7A.1 and PmTb7A.2 from T. boeticum into T. aestivum via T. durum (three-way cross) through marker-assisted backcross. Pietrusińska et al. (2011) pyramided Pm21 gene along with two leaf rust resistance genes into T. aestivum. Zheng et al. (2020) stacked powdery mildew resistance gene, yellow rust resistance (Yr26), and high-quality glutenin subunits Dx5 + Dy10 into the dwarf mutant wheat cultivar. Using the gene-linked markers, among 60 Chinese wheat cultivars, 24 cultivars are detected to carry Pm4b gene in combination with other Pm resistance genes (Pm2 + Pm4b + Pm8) in cultivars Xinxuan 2039, Lankao 008, and Zhengmai 366, while Yumai 368 was detected with (Pm2 + Pm4b + Pm6) multiple genes (Mwale et al. 2017).

Zhang et al. (2002) identified 11 wheat lines stacked with multiple genes Pm4b, Pm12, and Pm21 that showed high degree of resistance toward Bgt than single gene. Zhou et al. (2005) developed a resistant line (VPM1/7∗Bainong 3217 F4) against Bgt which had promising agronomic traits in addition to powdery mildew resistance. Later, Wu et al. (2018) characterized the stability of PM resistance gene, Pm4b, in wheat line VPM1/7∗Bainong 3217 F4 using 46 Bgt isolates and also developed 4 SNP and 3 SSR markers using BSR-Seq technique. Koller et al. (2018) pyramided transgenic lines with Pm3 allelic series and identified enhance powdery mildew resistance in field conditions compared to the parental lines transformed with single Pm3 alleles. Pyramiding of quantitative trait loci (QTLs) can be an alternate effective approach for developing durable resistance to powdery mildew in wheat. Simeone et al. (2020) mapped 18 QTL for APR powdery mildew resistance from 1AS, 2BS, 3BL, 4BL, and 7BS and 3 QTL for SR on 3BS chromosome region. Bai et al. (2012) identified QTLs responsible for adult plant resistance (APR) to powdery mildew in superior Chinese cultivars Bainong 64 and Lumai 21 and pyramided the same four QTLs and three QTLs, respectively, which showed high degree of resistance against Bgt. Strategy of pyramiding different PM resistance genes offers better protection for wheat against powdery mildew and provides a way of utilizing resistance gene resources for breeding new types of resistance lines and cultivars, which will have significance not only in breeding practice but also in theoretical research.

4 Advance in Powdery Mildew Resistance Breeding

Expanding the genetic tools by using genetic modification (GM) and genome editing is one of the foremost ways to enhance disease resistance. Genetic improvement for wheat breeding through GM enables easy transfer of resistance genes from one species to another compared to conventional crossing and also overcomes sexual incompatibilities. Obtaining stable inheritance of traits is often challenging due to the polyploid nature of common wheat. High-throughput genotyping platforms have established their potential role within the estimation of genetic diversity, construction of the high-density genetic maps, dissecting polygenic traits, and better understanding their interactions through GWAS (genome-wide association studies), QTL mapping, isolation of R genes, etc.

Wheat genome sequence (17 Gb) in a good quality is the hindrance of exploring the relationship between genotype and phenotype as it is 40 times bigger than rice (0.43 Gb) and 126 times bigger than Arabidopsis thaliana (0.125 Gb) (Brande and Moscou Matthew 2014). This poses a research barrier over the years. Until then, various technologies have been developed to reduce the complexity of genomes before sequencing. High-throughput genotyping platforms such as DArT-Seq, SNPs, GBS markers, and population-specific tGBS (targeted genotyping-by-sequencing) have accelerated the precise mapping of genomic regions sustaining rust and powdery mildew resistance (Qureshi et al. 2018; Nsabiyera et al. 2020).

4.1 Genome-Wide Association Studies (GWAS)

Genome-wide association studies (GWAS) is primarily focused on quickly screening germplasm collection for genes of interest and to capture historical recombination to obtain high-resolution mapping at a selected locus (Babu et al. 2020). As an advantage over mapping in bi-parental populations, this approach makes use of already existing natural populations, accounting for more genetic diversity at a given locus among the varied individuals. It relies on the recombination events that occur throughout the evolutionary process of germplasm (Yao et al. 2019). Four powdery mildew resistance QTLs have been identified using SNP genotyping-based genetic linkage analysis (Jia et al. 2018). Simeone et al. (2020) evaluated 221 accessions of wild and cultivated genotypes belonging to seven T. turgidum subspecies against Bgt; among them three QTL for SR (QPm.mgb-3BL.3, QPm.mgb-5AL.2, QPm.mgb-7BS.2) were mapped on chromosome assumed to be a new source conferring resistance to wheat powdery mildew.

Focused Identification of Germplasm Strategy (FIGS) is a rational method that provides information of specific plant traits along with the geographic and agro-climatic information. Using the FIGS strategy, Bhullar et al. (2009) identified 7 alleles for PM resistance from 1320 bread wheat landraces out of large collection of 16,089 accessions. Similarly, Vikas et al. (2020) exploited FIGS strategy to identify collection of accessions which had the maximum probability of gene of interest from a collection of 19,460 accessions of wheat.

4.2 Exome Capture

A high-throughput next-generation sequencing (NGS) technology can be used to assess genome-wide diversity in a single step. High-density SNP genotyping arrays and NGS have aided in the molecular detection of powdery mildew resistance genes/QTLs in wheat (Chao et al. 2019). Exome capture is an alternate genomic approach to SNP arrays also known as whole exome sequencing (WES) (King et al. 2015). The bread wheat exome constitutes only 1–2% of the total genome size of the targeted sequencing of the protein-coding portion of the genome. Such sequence can be specifically accessed by “exome capture.” Similar to Southern blotting, exome capture tolerates a high mismatch, thereby allowing efficient capture of diverged homologous sequence space in tetraploid and hexaploid wheat sequences (Saintenac et al. 2011; Henry et al. 2014). Wendler et al. (2014) studied the targeted genome complexity reduction strategy focused on exome sequencing, resulted in the discovery of markers in the cultivated and wild relatives of barley and in wheat (Allen et al. 2012). Genetic diversity and variations in wheat and barley genomes have been extensively explored using exome capture assays (Mascher et al. 2012). Ingvardsen et al. (2019) used a TILLING population for which the captured exome sequence of >1500 lines is available to obtain Mlo-based powdery mildew mutations in tetraploid wheat “Kronos.” The most common R gene encoded products such as nucleotide-binding leucine-rich repeat domain-containing (NB-LRR) proteins are confined to a smaller fraction of a plant exome. A typical plant genome is populated by several hundred R genes of the NB-LRR class (Meyers et al. 2003), and thus exome capture is an efficient method to identify and explore the plant exomes in mutant populations (Henry et al. 2014; King et al. 2015).

As the wheat genome (17 Gb in size) is too large to work, exome capture and sequencing is one of the approaches which greatly reduces sequencing volume and is highly cost efficient. It also covers the entire coding regions and reveals sufficient mapping information (Mo et al. 2018).

4.3 Genotype-by-Sequencing

Genotype-by-sequencing (GBS), an alternative genotypic approach, was presented by Elshire et al. (2011). In contrast to exome capture arrays, it does not rely on a fixed set of SNPs and its reference genome, whereas GBS involves genome complex reduction strategy followed by restriction enzyme-based sequencing allowing marker discovery focused on population-specific and genome wide studies. GBS-based labeling genomic regions conferring resistance genes in germplasm collections enables the discovery of resistance genes absent in reference genomes (Sanchez-Martin and Keller 2019). Exome capture or SNP-based arrays relying on reference genomes will miss out germplasm collection-specific resistance genes, while in this regard GBS acts an excellent genomics-assisted breeding approach for de novo platform. Cheng et al. (2020) studied the diversity of known powdery mildew resistance gene loci among Chinese wheat germplasm against the whole genome using genotyping-by-sequencing (GBS).

4.4 TILLING

Polyploid wheat tolerates a high mutational load compared with diploid species (Uauy et al. 2017). TILLING (Targeting Induced local Lesions in Genomes) reverse genetics methodology that integrates chemical mutagenesis with a high-throughput detection of single nucleotide mutations of target of interest in mutagenized populations. McCallum and coworkers were the first to introduce Targeting Induced Local Lesions in Genomes (TILLING), 20 years ago (McCallum et al. 2000). Acevedo-Garcia et al. (2016) reported the advantage of the non-transgenic TILLING technology to select partial loss-of-function alleles of TaMlo, the orthologue of the barley Mlo (mildew resistance locus o) gene which is known to confer durable broad-spectrum powdery mildew resistance.

Eco-tilling (Ecotype-Targeting Induced Local Lesions IN Genome) is a modified procedure of TILLING, and it relies on the enzymatic cleavage of hetero-duplexed DNA with single-strand-specific nuclease followed by detection through Li-Cor genotypes (Gokidi et al. 2017). It can be used to identify polymorphisms from within a naturally occurring population of crop plants (Comai et al. 2004). It can be used to characterize phylogenetic diversity, and the technique can help to identify important alleles within cereal crops. Eco-tilling is a relatively underexplored method now possible for the characterization of wheat disease resistance (Bhullar et al. 2009).

4.5 Mlo Proteins

The mildew resistance locus o (MLO)-based resistance trait was first characterized in barley where a loss of function mutation in an MLO gene conferred broad resistance against Bgt pathotypes which was later reported in wheat (Acevedo-Garcia et al. 2016). In 1942, Jorgensen was first to demonstrate and exploit the MLO-susceptible gene in barley toward Blumeria graminis f. sp. hordei (Bgh) (Jorgensen 1992). On resistant mlo mutant plants, Blumeria graminis pathogenesis is terminated at penetration stage, and consequently, fungal sporelings do not form haustoria inside host cells (Aist et al. 1987).

Freialdenhoven et al. (1996) identified two genes responsible for MLO resistance in barley, Ror1 and Ror2. Thenceforth, MLO genes have been reported in rice (OsMLO3) and TaMlo-A1, B1, and D1 (Konishi et al. 2010) in Triticum aestivum, located on chromosomes 5AL, 4BL, and 4DL (Elliott et al. 2002).

In wheat, in contrast to barley, no mlo mutants in natural condition were reported (Acevedo-Garcia et al. 2016). Mlo genes are largely conserved eukaryotic gene family among the plant kingdom, with comparative studies showing that wheat and barley reflect conserved similarity in genome structure (Kang et al. 2020). Similarly, coevolution of host-specific pathogens Bgt and Bgh occurred displaying gene collinearity (Mayer et al. 2011; Oberhaensli et al. 2011). The functional characterization of barley Mlo genes should be able to assist exploration of wheat mlo-based resistance because TaMlo (MLO in Barley Orthologue of Wheat) shows about 88% similarity to that of barley (Elliott et al. 2002).

In hexaploid wheat, three (TaMlo homologs) orthologs of the barley Mlo gene were located on ABD genome (Elliott et al. 2002). As of now eight bread wheat MLO members were identified (Konishi et al. 2010). Rakszegi et al. (2010) identified Tamlo mutants in spring bread wheat cv. Cadenza by TILLING approach using ethyl methane sulfonate (EMS). In hexaploid wheat, TILLING-derived Tamlo (TaMlo-A1, TaMlo-B1, and TaMlo-D1) missense mutants provided partial protection against Bgt while enhanced the resistance toward parental type (Acevedo-Garcia et al. 2016). Recently, Ingvardsen et al. (2019) tested mlo mutants in tetraploid durum wheat (Triticum turgidum var. durum). TILLING partial loss-of-function mutants of “susceptibility genes” showed mild pleiotropy (Huckelhoven et al. 2012). Gruner et al. (2020) observed no undesired pleiotropic phenotypes such as early signs of leaf senescence or spontaneous callose deposits in leaf mesophyll cells in Tamlo triple mutants in contrary to the mlo mutants of barley.

4.6 TALENS-Derived Bgt Resistance

In hexaploid bread wheat, the transgenic approach mediated by TALEN (transcription activator like effector nucleases) leads to complete resistance against the pathotypes of Bgt (Wang et al. 2014). TALEN (transcription activator-like effector nuclease) genome editing technology was used to generate transgenic winter wheat plants containing simultaneous knockout lesions in the three TaMlo homologs (Acevedo-Garcia et al. 2016). Gruner et al. (2020) reported that TALEN-derived triple mutant was completely resistant toward Bgt (5% host cell entry) and was highly heritable, whereas the respective susceptible parental wild-type line, cv. KN199, had an entry rate (71%) and cv. Cadenza (78%). Study also showed that TALENS derived TaMlo mutants when subjected to study the infection phenotypes against pathogens Zymoseptoria tritici, and Magnaporthe oryzae pv. Triticum (MoT), respective mutants were highly susceptible to the pathogens and showed high degree of resistance towards Bgt.

4.7 CRISPER

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats)-mediated knockout facilitates enhanced powdery mildew resistance in wheat (Wang et al. 2014). In wheat, the first successful experiment using the CRISPR/Cas9 system was editing of TaMLO, a powdery mildew resistance locus (Wang et al. 2014). In the above study, all six copies of TaMlo were simultaneously mutated, and the edited plants exhibited resistance toward the powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt). The Cas9-mediated gene was used to generate and induce variation in hexaploid wheat. CRISPR/Cas9 technology is less expensive, more versatile, and easier to design and has thus largely overtaken the other genome editing technologies (Hilscher et al. 2017).

4.8 Taedr1-Basal Resistance for Bgt

EDR1 (enhanced disease resistance 1) is another type of negative regulator apart from MLO which plays a negative role in defense mechanism against powdery mildew. Dangl and Jones (2001) suggested basal resistance toward pathogen infection as quantitative disease resistance. It is highly conserved in many species of the plant kingdom (Frye et al. 2001). Mutation of EDR1 was found to confer resistance to PM but more of a basal resistance (Huckelhoven 2005). Zhang et al. (2017) found that EDR1 would be a source for enhancing powdery mildew resistance in wheat. Resistance in EDR1 is accompanied by the accumulation of callose marginal growth reduction and mildew-induced mesophyll cell death (Frye and Innes 1998).

Zhang et al. (2017) exploited CRISPR/Cas9 technology and generated Taedr1 wheat plants by targeting all three homologs of wheat. The Taedr1 mutants were resistant to Bgt but without mildew-induced cell death. Wang et al. (2014) speculated that Taedr1 plants might confer resistance to other wheat pathogens also. Thus, the targeted knockout of negative regulators and/or susceptibility genes via genome editing represents a powerful approach for plant disease resistance breeding.

4.9 Candidate Gene Approach in Wheat

In plant kingdom, the major classes of disease resistance genes include NLR proteins and protein kinases (PKs). The candidate gene approach has emerged as a promising method of merging QTL analysis with the extensive data available on the cloning and characterization of genes involved in plant defense (Faris et al. 1998). Hitherto, more than 100 Bgt QTLs have been mapped in homologous chromosome groups from different mapping studies, with some of them being positioned in the same marker intervals (Kang et al. 2020). Infection of Bgt in wheat may be suppressed by host immune responses leading through the massive secretion of small virulence proteins called effectors (Bourras et al. 2019). Wheat plant genome encodes hundreds of cloned R genes that code for NB-LRR proteins which directly or indirectly recognize these effectors from pathogens to activate defense responses.

To date, seven R genes, namely, Pm3 allelic series (Brunner et al. 2010), Pm8 (Hurni et al. 2012), Pm2 (Sánchez-Martín et al. 2016), Pm17 (Singh et al. 2018), Pm21 (Cao et al. 2011; He et al. 2018; Xing et al. 2018), Pm60 (Zou et al. 2018), and Pm1a (Hewitt et al. 2021), for powdery mildew resistance have been cloned. These genes encode NB-LRR immune receptors that recognize pathogen effectors and activate effector-triggered immunity (ETI) with known resistance for powdery mildew specificities. R-gene enrichment sequencing (AgRenSeq) approach is reference-free which was successfully demonstrated to clone R genes from alien sources of domesticated crops which expedite the discovery of new NLR genes and counteracts the pathogen profile when most pathogenic strains are used (Martin et al. 2019). These cloned genes confer high level of resistance against Bgt usually culminating in the induction of a type of programmed cell death known as the hypersensitive response (HR) (Collier and Moffett 2009). However, Pm38/Yr18/Lr34/Sr57 and Pm46/Yr46/Lr67/Sr55 also code for NLR proteins, but they provide partial resistance to powdery mildew and rust diseases in adult plants. These genes encode an ATP binding cassette (ABC transporter) and an altered hexose transporter, respectively.

4.10 R Gene Cassettes

Stacking of PM genes is an important strategy to extend the life span of race-specific resistance genes (Li et al. 2014; Burdon et al. 2014). Taking benefit from effector-assisted breeding, stacking of multiple R gene-based resistance provides robust and broad-spectrum disease resistance (Martin et al. 2019). Molecular stacking is an effective alternative to conventional gene stacking through marker-assisted selection. Through molecular stacking, multiple R gene cassettes can be assembled on to one plasmid and then introduced as a cluster at a single genetic locus through plant transformation (Que et al. 2010) in routine breeding (Ainley et al. 2012). But the length of the DNA insert delimits the number of genes to be inserted into the vector (Que et al. 2010).

4.11 Virus-Induced Gene Silencing

The resistance action of candidate genes can be studied by the complementary functional assay—virus-induced gene silencing (VIGS) (Lee et al. 2012). VIGS offers a fast and rapid transient assay for silencing of gene expression. The most widely used vectors for VIGS in wheat are those derived from barley stripe mosaic virus (BSMV), a plant virus with a tripartite RNA genome (RNAα, RNAβ, and RNAγ) that readily spreads throughout tissues following mechanical rub-inoculation onto the leaves.

Bhullar et al. (2009) used combined strategy of VIGS and transient transformation assay to assign the function of previously undescribed Pm3 alleles. Moreover, through VIGS, silencing of the TaMlo homologs leads to powdery mildew resistance in wheat (Varallyay et al. 2012). Zhang et al. (2017) reported the knockdown of TaEDR1 (negative regulator of MLO protein) in mutant lines with VIGS and observed that these lines were showing enhanced resistance to powdery mildew. Xing et al. (2018) used barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) for targeting NBS domain and the LRR domain to evaluate the function of NLR1-V gene in Pm21. Later, he and his coworkers identified and investigated two candidate genes, viz., DvRGA1 and DvRGA2 (CC-NBC-LRR proteins), for Pm21-mediated resistance in wheat variety Yangmai 18 using BSMV-VIGS. Results suggested that silencing of DvRGA2 allowed abundant development of Bgt colonies with disease symptoms and fungal sporulation on the leaves, rather than DvRGA1 gene. Thus it was identified that DvRGA2 is associated with Pm21 resistance (He et al. 2018). Zou et al. (2018) validated the function of Pm60-NB LRR gene through VIGS and transient expression assays. VIGS was also useful in wheat genotypes that were difficult to transform and in those for which mutant/TILLING populations were unavailable. It can be also be used for simultaneous silencing of all homologs without the need for further genetic crosses.

5 Challenges in Breeding for Powdery Mildew Resistance

For the past few decades, developing disease-resistant wheat varieties mainly relied on the conventional breeding. Increased yield with desirable agronomic characters is the main prioritization of the modern wheat breeding community. This has led to the loss of genetic diversity for disease resistance. Resistant genes from alien sources of wheat were transferred to commercial cultivars through conventional breeding approaches. However, introduction of alien segments always was associated with a linkage drag. The deleterious effect associated with the alien gene may indirectly affect the yielding components of the host plant. Thus the potential linkage drag limits the transfer of PM genes to wheat. Also it might take several years for fixing a resistant gene in a particular wheat background through conventional breeding methods (Cowling 2012). Thus isolation of resistance genes from alien sources and recombination between alien chromosomes and wheat chromosomes are largely limited (Lukaszewski 2000; Mago et al. 2002; Qi et al. 2007).

These limitations have been largely overcome by combining genetic, cytogenetic, and molecular methods together. Recently, more number of molecular markers linked to the resistance genes has been identified. Precise physical mapping of powdery mildew resistance genes is very crucial for the identification of candidate genes (Kang et al. 2020). Locating the inserted alien segments in the wheat genome is also important for the successful development of resistant cultivar (Dundas et al. 2007). Genetic and cytogenetic methods need to be put into practice to promote recombination and minimize the adverse effects brought by the alien chromatin.

Precise genotyping and phenotyping are necessary for the identification of novel PM-resistant genes. Changing climatic conditions often influence the phenotypic data. In case of quantitative resistance, response of the host is very difficult to be scored compared to qualitative resistance. Time of scoring and nature of phenotypic expression are very crucial in assessing accurate level of severity. Visual scoring for the estimation of disease severity is always subjective and error prone particularly when large populations are screened (Poland and Nelson 2011).

Transgenic-based technologies are also influenced by the environmental factors as there might be biological and physiological interaction of genetic factors with the transgene expression (Ueda et al. 2006). Also, there are limitations in broad-spectrum application of molecular techniques in developing mildew-resistant lines as it is more expensive. Certain uncontrolled transgene insertion might also be associated with detrimental effects on plant growth and development (Kang et al. 2012). Yet, this approach involves the direct transfer of the functional genes eliminating the linkage drag (Jacobsen and Schouten 2007) and thus offers a long-term solution to global agricultural challenges. Identified trait-linked SNPs can be converted into allele-specific PCR assays which can be breeder friendly. Using R gene cassettes, despite the advantages of molecular stacking, the number of genes that can be introduced through molecular stacking is often restrained by the limit in the length of the DNA insert that can be put into a vector (Que et al. 2010). This limitation can be overcome if DNA fragments can be sequentially inserted at the same genomic target. Recent breakthroughs in genome editing technologies in plants enable such targeted insertion of DNA fragments in diverse crop species (Kumar et al. 2016; Voytas 2012).

6 Conclusions and Future Prospects

Significant breakthrough in the development of powdery mildew-resistant wheat varieties was achieved after the de novo sequencing of whole wheat genome. Marker-based approach is an effective method in the selection of target genes. Also, these markers might make it possible to identify novel genes for the development of powdery mildew-resistant wheat varieties. In future, strategies that combine conventional and molecular approaches for easy and rapid characterization of useful germplasm might be necessary for categorizing resistant genes.

Discovering and introducing novel sources of resistance to powdery mildew either from common wheat background or other wheat-related species should be the major objective of the wheat breeding. Chromosome micromanipulation and microinjection techniques are new and effective technology to be utilized in the future for the introduction of resistance to mildew into common wheat.

Generally, selection of lines with quantitative genes is difficult as extensive tests on selected mildew population are required and there are no proper methods to select the appropriate plant. The process is time-consuming and laborious. Thus a novel rapid selection technique for phenotyping seedling and adult plants is the need of the hour. Additionally, information regarding the genetic diversity and distribution of PM genes in hexaploid wheat is necessary for the enriching the resistance basis to PM in wheat. Interspecific and wide crosses will still continue as an effective strategy for developing resistant lines to mildew.