Abstract
The quantity and quality of oilseed production in rapeseed mustard are severely affected by biotic and abiotic stresses. Among these, the biotrophic fungus Erysiphe cruciferarum causes powdery mildew (PM) infection in Indian mustard cultivars, potentially reducing yield by up to 50% across affected regions in India. Considering recent developments in molecular plant pathology and their impact on sustainable management of challenging plant pathogens, this article reviews the current scenario for resistance and its mechanism to E. cruciferarum in Brassica cultivars. It also covers the complex molecular signaling pathways for resistance that are regulated by phytohormones along with differential gene expression, and effectors proteins in Brassica spp. The recent advancements in genomics have contributed to identification of resistance/susceptibility genes as well as quantitative trait loci (QTLs) involved in PM resistance. Furthermore, this review unfolds a comprehensive understanding of the genetic as well as genomic basis of resistance that can provide the valuable insights for breeding programs focused on developing PM-resistant rapeseed-mustard varieties. This review aims to provide the background on recent discoveries and future strategies on identification of resistance genes, aiding in the development of more resilient rapeseed-mustard crops and leading to significant improvements in crop protection and yield stability.
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Understanding the Powdery Mildew Pathogen and Rapeseed Mustard Interactions: Insights into Disease Resistance and Molecular Mechanisms to Enhance the Quality and Productivity of Oilseed Brassica Crops
Sayantani Chanda,a,# Laxmi Awasthi,a,# Rakhi Tomar,a Samridhi Mehta,a Ankita Kumari,a Shiv Shankar Sharma,a Prajjwal Rai,b Mahesh Rao,a Peru Nallathambi,c Prabhu Dayal Meena,d Archana Rai,e,* and Ashish Kumar Gupta a,*
The quantity and quality of oilseed production in rapeseed mustard are severely affected by biotic and abiotic stresses. Among these, the biotrophic fungus Erysiphe cruciferarum causes powdery mildew (PM) infection in Indian mustard cultivars, potentially reducing yield by up to 50% across affected regions in India. Considering recent developments in molecular plant pathology and their impact on sustainable management of challenging plant pathogens, this article reviews the current scenario for resistance and its mechanism to E. cruciferarum in Brassica cultivars. It also covers the complex molecular signaling pathways for resistance that are regulated by phytohormones along with differential gene expression, and effectors proteins in Brassica spp. The recent advancements in genomics have contributed to identification of resistance/susceptibility genes as well as quantitative trait loci (QTLs) involved in PM resistance. Furthermore, this review unfolds a comprehensive understanding of the genetic as well as genomic basis of resistance that can provide the valuable insights for breeding programs focused on developing PM-resistant rapeseed-mustard varieties. This review aims to provide the background on recent discoveries and future strategies on identification of resistance genes, aiding in the development of more resilient rapeseed-mustard crops and leading to significant improvements in crop protection and yield stability.
DOI: 10.15376/biores.20.4.Chanda
Keywords: Rapeseed mustard; Erysiphe cruciferarum; Powdery mildew; Yield loss; Host-pathogen interactions; Host resistance; Effectors genes; Genome editing and developing resilient rapeseed cultivars
Contact information: a: ICAR- National Institute for Plant Biotechnology, New Delhi-110 012, India; b: ICAR- Indian Agricultural Research Institute, New Delhi-110 012, India; c: ICAR-IARI Regional Station, Wellington, The Nilgiris Tamilnadu-643 231, India; d: ICAR-Directorate of Rapeseed-Mustard Research, Bharatpur, Rajasthan-321 303, India; e: Nuclear Agriculture & Biotechnology Bhabha Atomic Research Centre, Trombay, Mumbai – 400 085, India; #: These two authors have contributed equally to this work;
*Corresponding authors: 
raiarchu@barc.gov.in; ashish.pathology@gmail.com
INTRODUCTION
Rapeseed-mustard (Brassica spp.) stands as a cornerstone in global oilseed cultivation, notably contributing to the edible oil market alongside soybean and oil palm. However, a gap persists between the supply and demand of oilseeds, driven by the increasing global population. Moreover, the change in global climate scenario has posed a significant risk to Brassica cultivation resulting in an increased disease incidences and severity. A diverse range of plant pathogens infect Brassica crops. One such known fungal biotroph is E. cruciferarum Opiz ex L. Junell, which causes powdery mildew (PM) disease in this crop. There are about 700 species of PM pathogens capable of infecting approximately 10,000 plant species (Braun and Cook 2012). Within the last two decades, PM has become epidemic in rapeseed-mustard, affecting over 120 cruciferous host plant species in more than 25 countries (Mir et al. 2023). The disease is especially prevalent in regions with cool, dry conditions in leading rapeseed-mustard producing countries such as Canada, China, India, and the European Union (FAO 2022). In India, it spreads rapidly across major mustard-growing states such as Uttar Pradesh, Rajasthan, Gujarat, and Maharashtra (Dange et al. 2002; Mohitkar et al. 2012; Meena et al. 2018), thus posing a major bottleneck to the seed production, quality and profitability. Furthermore, the pathogen caused yield losses ranging between 20% and 40% across various regions of India, with severely affected fields experiencing losses of up to 50% (Kumar et al. 2016; Meena et al. 2018). These significant yield losses can translate into economic setbacks, affecting the livelihoods of farmers and the overall productivity of the agricultural sector.
Despite its significance, PM has received less research attention compared to other diseases such as Alternaria blight and white rust. Perhaps this is because it appears later in the crop growth stages and its damage is estimated frequently after harvest. While previous reports primarily have emphasized yield losses, PM affects overall productivity by reducing oil quality and altering the plant’s physiological processes, especially decreasing photosynthetic efficiency followed by overall plant vigour and seed yield. Due to the presence of powdery mass on leaf surfaces, significant reductions were observed in leaf gas exchange parameters like net photosynthetic rate and transpiration rate in the infected leaves of susceptible genotypes compared to resistant and moderate genotype groups. Chlorophyll fluorescence analysis revealed a decrease in the maximum quantum efficiency of photosystem-II in PM-infected leaves across the genotype groups in different crop plants (Sree et al. 2024; Saja et al. 2020). The net photosynthetic efficiency goes down in susceptible genotypes, which ultimately affects the productivity of the crop plants.
Striking research efforts by plant pathologists and breeders to combat PM in Brassica species have been bolstered by extensive work focused on elucidating the genetic level of resistance and developing resilient cultivars. Studies leveraging model plants, such as Arabidopsis thaliana, have given crucial insights for molecular defense mechanism, signalling pathways underlying resistance to PM and pathogen-host interactions. Genetic studies have identified candidate resistance genes (R genes) and quantitative traits loci (QTLs) imparting with PM resistance, facilitating marker-assisted selections and breeding programs targeted for improvement and development of resistant varieties for PM disease.
Moreover, the exploration of wild relatives of Brassica crops has unearthed promising genetic resources for enhancing resistance. These efforts are pivotal for the sustainable agriculture, aiming not only to minimize yield losses but also to reduce dependency on chemical pesticides, thereby promoting eco-friendly crop production practices. Cultural practices, such as crop rotation and debris management, help reduce disease pressure but they are not always sufficient alone. Breeding for resistance offers a sustainable solution, though it requires time and can be challenged by pathogen evolution. An integrated approach combining these strategies (cultural, genetic resistance, minimal use of pesticides) is the most effective for long-term PM disease management in Brassica crops. However, judicious exploration of such strategies will signify mustard cultivation with enhanced yield and quality. Hence, this review comprehends current knowledge and recent advancements in global rapeseed-mustard research, including Brassica genetics, the PM (E. cruciferarum) pathogen, associated seed yield and oil content losses, and management strategies. It further explores key aspects such as genetic diversity, host-pathogen interactions, resistance mechanisms, and breeding strategies, that are crucial for ensuring food and nutritional security, particularly in developing countries. Furthermore, it outlines future research directions aimed for elucidating the complexities of PM disease and accelerating the development of durable resistance in Brassica crops. By understanding host-pathogen interactions and pinpointing resistance-associated genes, the findings aid in the development of disease-resistant varieties through molecular breeding and biotechnological approaches.
Fig. 1. Image illustrating the characteristic powdery mildew disease symptoms on Brassica spp. leaf, stems, and pods.
ASPECTS OF THE PATHOGEN AND ITS CONTROL
Economic Significance of E. cruciferarum in Brassica Species
Powdery mildew disease is an epidemic and devastating disease in Indian mustard cultivars. Due to variable weather conditions in different agro-climatic regions, powdery mildew affects photosynthetic efficiency, leading to stunted growth, premature senescence, defoliation, and reduced biomass accumulation. This results in shrivelled seeds, reduced seed weight, and poor pod formation, ultimately lowering the oil content and collectively deteriorating overall crop productivity. Powdery mildew disrupts photosynthetic surfaces. This not only reduces the photosynthetic efficiency and yield, but it also causes a stress condition for the plant. Exposure to stress condition can alter metabolite profiling and the lipid biosynthesis pathways that ultimately change the fatty acid composition and reduce its seed nutritional quality (Baud and Lepiniec 2010). In B. juncea, profound yield losses were recorded due to E. cruciferarum infection, for example, in Gujarat, yield loss was reported at 24.1% (Dange et al. 2003), while in Haryana, the EC-126743 accession showed a yield loss of 17.4% and a drop in oil content of 6.5% (Saharan and Sheoran 1985). In Maharashtra, the Pusa Bold and Seeta cultivars experienced dramatic yield reduction ranging from 45% to 95% (Hare 1994). Another study from Maharashtra reported yield losses between 15% to 40% and oil content reduction up to 37.6% in cultivars Seeta, Pusa Bold, Bio-902, and TM-17 (Kohire et al. 2008). Kanzaria et al. (2013) estimated losses of 7.3%, 21.7%, 22.5%, and 21.5% in oil content, protein content, seed yield, and test weight, respectively, because of E. cruciferarum infection in Maharashtra and Gujarat, regions where the PM disease is highly prevalent in severe form. Powdery mildew has also been reported to cause yield losses exceeding 25% to 30% in Turkey, United States, Korea, France, Australia, and Poland where B. napus is grown widely (Sadowski et al. 2002; Mert-Turk 2008; Kaur et al. 2008; Khangura et al. 2011; Kim et al. 2013; Uloth et al. 2016; Meena et al. 2018). Brassica rapa cultivars such as Yellow Sarson in Canada and Europe have shown yield losses of 20% to 30% and oil content reductions of 10% to 20% (Smith and Johnson 2020). In B. oleracea, yield loss is often measured in terms of marketable quality rather than quantity, yet losses ranging from 20% to 50% in cultivars including Green Magic in California and Spain have been reported (Table 1; Saharan et al. 2005). These widespread and significant yield and oil content reductions around the world highlight the necessity for effective disease management programmes to mitigate the economic impact and yield losses of powdery mildew on Brassica crops.
Table 1. Impact of Powdery Mildew Severity on Seed Yield and Oil Quantity Losses in Brassica spp. under Indian and Global Perspectives
Genomic insights and Host Range of E. cruciferarum
Erysiphe cruciferarum is a fungal biotroph that is primarily responsible for causing powdery mildew (PM) in Brassica species including B. juncea, B. napus, B. nigra, B. oleracea, and B. rapa (Glawe 2008). The phenotypic and fundamental genomic information based on the conserved internal transcribed spacer (ITS) region of DNA has been made available for E. cruciferarum, but no whole genome assembly has been reported to date. However, the database is rich in genome of fungi causing PM disease in hosts other than Brassicaceae. The previous genome assemblies indicates that PM fungi are approximately four times larger in size compared to other ascomycetes, especially, the genome size of Golovinomyces orontii is approximately 160 Mb. (Saharan et al. 2023). Various E. cruciferarum isolates were identified through the amplification of ITS1 and ITS2 regions that flank the 5.8S rRNA using universal primers ITS1 (forward) and ITS4 (reverse), showing >99% nucleotide identity with GenBank sequences for E. cruciferarum. The entire ITS region of the DNA for PM isolate KUS-F24819 was sequenced after amplification with primers ITS5 and P3, and deposited under the accession no. KC862331 showing 100% identity with E. cruciferarum isolates from Arabidopsis thaliana, B. oleracea var. acephala, and B. rapa (Kim et al. 2013). Morphological, along with phylogenetic, analyses of various isolates were conducted using Erysiphaceae-specific primers such as PMITS1 and PMITS2 (Borges et al. 2023). Detection at the molecular level involved amplifying the ITS1 region with oligonucleotides EryF and EryR from infected plant tissues DNA (Attanayake et al. 2009; Pane et al. 2021). So far, the use of comparative genomic studies greatly facilitates the identification and characterization of genes linked to target traits in various species (Wan et al., 2008). However, additional research is needed to elucidate the genomics and genetic diversity of the PM pathogen in Indian mustard. In addition to cultivated Brassica species, the pathogen E. cruciferarum also infects a wide range of wild relatives across the Brassicaceae family worldwide (Table 2).
Table 2. Inventory of Different Brassica Crop’s Wild Relatives Infected by PM Pathogen Along with the Records of Various Locations Worldwide (Saharan et al. 2019)
Table 2. Inventory of Different Brassica Crop’s Wild Relatives Infected by PM Pathogen Along with the Records of Various Locations Worldwide (Saharan et al. 2019)
Furthermore, different Brassica genotypes exhibit varying degrees of resistance or susceptibility to E. cruciferarum. This variation is largely governed by the presence or absence of resistance (R) genes, differences in the speed and intensity of immune response activation, and hormonal regulation efficiency. For instance, certain B. juncea lines and wild relatives possess quantitative trait loci (QTLs) conferring partial to strong resistance, while commercial cultivars often lack these traits. Additionally, transcriptomic differences in defense gene expression further explain the differential responses, where resistant genotypes show a faster and stronger activation of PR genes, SA signaling, and ROS production compared to susceptible ones.
Mechanistic Insights Underlying Host-Pathogen Interactions in Brassica Species
The first line of defense: PTI response against PM infection
Inherently, plants have evolved a two-step defense mechanism that detect and deploy immune response appropriately: (1) Pattern recognition receptors or PRRs (Extracellular receptor-like kinases: RLKs and receptor-like proteins: RLPs) that detect PAMPs/DAMPs; (2) Intracellular receptors with nucleotide-binding and leucine-rich repeat domains (NLRs) that interact with pathogen specific secretory proteins called effectors (Krattinger and Keller 2016). The initial immune response to the pathogen attack is triggered when the receptors on the host cell’s surface, called the PRRs, recognize pathogen/damage-associated molecular patterns (PAMPs/DAMPs) (Jones and Dangl 2006), that are present on the pathogen’s surface. This recognition leads to PTI response, which then initiates further down-signaling in the form of MAPK cascade and other defense associated pathways. Previous research has shown that the chitin, in cell walls of fungal pathogen, triggered immune responses in Arabidopsis, as it was recognized by PRR receptors, such as LYK4/5 (lysine motif receptor-like kinases 4/5) and CERK1 (chitin elicitor receptor kinase1), which led to accumulation of defense protein and callose deposition (Cao et al. 2014). Moreover, mutations in CERK1 were also linked to increased susceptibility for PM, emphasizing its role in broad range disease resistance (Wan et al. 2008). Similarly, the recessive gene, ol-2, an analog to the MLO gene revealed in barley, facilitated histological resistance through the development of papillae. These papillae are composed of callose and other substances and form at the sites where the plant and powdery mildew interact, therefore halting fungal growth early and ensuring complete resistance (Bai et al. 2003, 2008). In Brassica species also, these mechanisms need to be worked out for the rapid screening and identification of durable resistant types.
R-gene base or vertical immunity (Effector-triggered immunity ETI)
Although PTI response provides strong defense against the infection, pathogens tend to constantly evolve novel ways to bypass PTI. One such strategy is secretion of specialized molecules called the effectors in the host cytoplasm. Effectors or the ‘avirulence’ factors are known to promote virulence and are highly diverse in nature (White et al. 2000). The ETI is marked by robust resistance responses, activated when pathogen effectors (avr proteins) are recognized by intracellular receptors present in the host cells, resulting in race-specific, major gene resistance. Such type of resistance follows gene-for-gene hypothesis according to which an effector gene causes avirulence in the host plant with its interactive resistance (R) gene. These R gene encode diverse intercellular receptors with two conserved domains, i.e., the nucleotide-binding site (NBS) and a region of leucine-rich repeats (LRRs). Apart from LRR or LRR-like domains, the R proteins typically possess toll/interleukin-1-receptor (TIR) domains, and serine/threonine kinases (S/TK). The R genes are usually categorized into two groups: those with a TIR domain towards N terminal are termed TNLs, while those with a CC (coiled-coil) domain towards N terminal are termed as CNLs. Additionally, R-genes with PM 8 (RPW8) domain have also been reported and are referred to as RNLs (Tirnaz et al. 2020). Usually, initiation of R-gene recognition system induces hypersensitive response (HR), which stops the pathogen from spreading to neighboring healthy cells by inducing localized cell death at the infection site, therefore, conferring systemic resistance (Keen et al. 1993). The RNL gene in Arabidopsis (viz., RPW8.1 and RPW8.2) has been reported to provide resistance by promoting accumulation of H2O2 and confined cell death through the SA-dependent pathway (Kim et al. 2014). Among this, RPW8.2 localize towards EHM (extra-haustorial membrane) to activate defense signaling, requiring specific protein interactions and transport mechanisms. The reports on mutations in RPW8.2 affect its function and resistance (Saharan and Krishnia 2001).
Role of phytohormones in PM resistance: Systemic acquired resistance (SAR)
For effectively deploying defense response against the pathogens, host plants have developed various signaling pathways during the initiation of the infection. These pathways are mostly activated in response to the changes in regulation of different phytohormones, especially SA, JA, and Et. While SA accumulates during the attack by biotrophic pathogens, JA and Et come together to stop the development of necrotrophic organisms. Previous studies have shown the role of SA in inducing systemic acquired resistance (SAR) in response to PM infection, which is caused by a biotrophic pathogen. Usually, SA accumulation leads to SAR response, resulting in a long-term resistance against multiple pathogens by activating a range of defense genes, particularly those encoding pathogenesis-related (PR) proteins (Park et al. 2007). Key studies have established the importance of SA signaling in the regulation of genes necessary for redox and calcium signaling, essential for activation of NPR1 gene (Chandran et al. 2009). This results in the increase of transcripts for genes involved in SA-production and PR genes. Mutations in SA signaling genes such as wrky18 and wrky40 can affect the plant’s PM disease response. Furthermore, mutation in EDR1 protein kinase affects the accumulation of defense-related transcripts, particularly those encoding TFs such as WRKY and AP2/ERF, thus increasing SA-dependent resistance against PM (Christiansen et al. 2011). Moreover, infected plants with the EDR1 mutation also revealed increased genes expression related to the endomembrane system and ROS generation. The EDR1 migrates from endoplasmic reticulum to the plant-fungal interaction site during PM disease, which suggests that it is a part of secretory pathway in the defense against PM (Christiansen et al. 2011; Wu et al. 2015). These results demonstrate complex roles that the secretory system and SA signaling play in Arabidopsis protection against E. cruciferarum. The PR-1 and PR-2 genes were significantly up-regulated in Raphanus alboglabra compared to B. napus, with the elicitor stimulating these genes upon PM infection (Alkooranee et al. 2015). These types of studies, thus, highlight the intricate interplay between SA signaling, transcription factors, and the secretory system in orchestrating effective defense mechanisms against powdery mildew, underscoring the complexity of plant immune responses.
Inheritance and Resistance Mechanisms in Brassica Species Against E. cruciferarum
Brassica species as potential genetic resistance sources for PM
Plant genetic resources are crucial components of agricultural biodiversity, essential for the development of novel varieties to feed the world’s burgeoning population. The genetic diversity within Brassica germplasm represents a valuable reservoir of resistance against PM disease, which can be harnessed effectively for the development of disease resilient cultivars. Therefore, extensive research has been conducted globally to explore the inherent potential for disease resistance in rapeseed-mustard against PM pathogen (Table 3). Assessment of 71 genotypes of B. juncea from China, India, and Australia revealing four genotypes, ‘JM06014’, ‘JM06015’, ‘JM06012’, and ‘JM06009’, provided PM resistance in natural field conditions (Singh et al. 2010). However, in contrast, all tested genotypes from China and India were found to be susceptible to PM disease. In another study, 200 genotypes were screened out of which 20 resistant, 9 moderately resistant, and 71 highly susceptible genotypes were selected (Singh et al. 2016). Similarly, 61 germplasm were tested under natural conditions, identifying eight immune and one highly resistant, with 36 moderately resistant genotypes (Chadar et al. 2020). One genotype ‘RDV 29’ was also reported as highly resistant along with 12 moderately resistant germplasm after screening of 1,020 Indian mustard accessions (Nanjundan et al. 2020). Apart from cultivated Brassica, wild relatives are also a rich reservoir of resistance against multiple diseases including PM; however, they are less explored. At present, eleven accessions of A. thaliana are reported to be resistant to PM disease. Therefore, more studies to explore PM resistance in wild relatives are necessary to fully exploit their genetic potential. Furthermore, there is a need for development of more robust screening techniques to categorize germplasm more accurately from immune to susceptible. The present screening methods involve phenotyping of the genotypes based on the observation of physical symptoms post PM inoculation through staple leaf method or conidial dusting with sterile brush (Bhosle et al. 2021) followed by categorization under the rating scale ranging from 0 (immune) to 9 (highly susceptible), as recommended by the AICRP-RM in India. This often has led to inaccuracies and discrepancies in the observations and inconsistency during the repeated experiments. Therefore, incorporation of molecular approaches like LAMP (Loop-Mediated Isothermal Amplification) and LFA (Lateral Flow Assay) assays can offer more rapid and accurate diagnosis of the pathogen and hence better characterization of the germplasm is possible based on the quantification of disease severity (Attaluri and Dharavath 2023).
Table 3. Genetic Resources for Resistance in Brassica Species against Powdery Mildew Pathogen
Inheritance pattern of PM resistance in Brassica species
Introgression or crossings have been developed widely to study the inheritance pattern of resistance in Brassica species for PM disease resistance (Alkooranee et al. 2015). Early breeding experiments crossing a susceptible parent from B. juncea with the resistant parent belonging to B. carinata (Varuna × PCC2; RH 30 × HC-1) demonstrated that one dominant gene provides wide resistance against multiple pathogens (PM, white rust and Alternaria blight disease) in B. carinata (Kumar et al. 2002). In a recent study, accessions of B. napus and other species were screened, and a B. carinata cv. ‘white flower’ was identified immune for PM under natural and in-vitro conditions (Gong et al. 2020). Hybridization of this B. carinata with the elite cultivar of B. napus ‘Zhongshuang11’ generated F1 hybrids that inherited cytoplasm from the resistant parent (B. carinata). The progenies were backcrossed to yield five and a single line from BC1F3 and BC2F2 generation, respectively, that showed highly resistance to moderate resistance against the PM disease. Moreover, these lines exhibited similar seed quality and morphological traits to ‘Zhongshuang11’, indicating successful introgression of resistance genes into B. napus. In India B. juncea, one accession, namely RDV29, was found to be completely resistant to the PM disease. The genetic analysis of populations obtained by crossing the resistant parent ‘RDV29’ with susceptible parent ‘RSEJ775’ was made. The screening and evaluation of F1 generation showed that the PM resistance in RDV29 is semi-dominant in nature and determined by two unlinked loci. The segregation ratios in F2 (9:6:1; resistant: susceptible: highly susceptible), susceptible backcross (1:2:1; partially resistant: susceptible: highly susceptible), and resistant backcross (all resistant) further confirmed this statement. Furthermore, it was also inferred from the data that the expression of the resistance depends on the gene dosage (Nanjundan et al. 2020). Similar findings were previously reported in A. thaliana, where resistance to PM was governed by a locus in 5 genotypes and by two distant loci in single germplasm. In some cases, resistance was encoded by semi-dominant alleles, while in others, susceptibility by dominant alleles (Adam and Somerville 1996). The locus RPW8 in A. thaliana consist of two resistance genes (RPW8.1 and RPW8.2) that are of dominant nature and confers broad range resistance to PM (Xiao et al. 2001). These reports collectively shed light on the complex and variable nature of resistance against the PM disease in Brassica crops, demonstrating that effective resistance can arise from single dominant genes, semi-dominant alleles, or multiple loci, thus offering insightful strategies for future breeding plans targeted at improving Brassica crops.
Mechanism of resistance against PM in Brassica spp.
Exposure of Brassica plants to E. cruciferarum triggers the initial defense mechanisms against pathogen infection and proliferation. These include physical barriers such as wax, cuticle, epidermal cell wall, stomata, leaf hairs, and thick-walled tissues, that prevent pathogen entry. In cruciferous plants, resistance to E. cruciferarum before penetration is mainly provided by the cuticle and waxes (Malinovsky et al. 2014). Microscopic and transcriptome analyses were performed for PM resistance in the progenies of inter-specific crosses between resistant B. carinata and susceptible parent B. napus and found formation of needle-like and few flaky particles on the leaf wax of progenies that showed resistance. Furthermore, the study also found elevated expression of genes involved in biosynthesis of wax (CER, KCS6, MAH1, and LACS2), which are necessary for modulating certain wax components in resistant genotypes (Zhang et al. 2022). Additionally, several cell wall integrity genes, such as PGIP1, PMR5, RWA2, PDCB1, C/VIF2, and PMEI9, were also observed to be upregulated. In contrast, low callose deposition in resistant progenies as compared to in susceptible ones was observed in the study, perhaps due to initiation of other structural responses deterring the pathogen entry in resistant genotypes. The histopathological and phenotypic studies revealed that necrosis as a result of PM disease was observed higher after infection in Camelina sativa and B. juncea as compared to Sinapis alba (Mir et al. 2023). This cell death was apprehended due to antioxidant enzyme activities in these species following infection by E. cruciferarum as corresponding to the cell death, the enzyme activity was observed to be relatively higher in C. sativa and B. juncea in comparison with S. alba. However, if the pathogen overcomes these initial defensive layers, it encounters a more systematic defense response through proven mechanisms viz., ETI and PTI. Similar mechanism of resistance was documented in other species of PM pathogen (Muthamilarasan and Prasad 2013). A form of cell death as HR (hypersensitive reactions) was reported in tomato powdery mildew interactions. This HR reaction is due to the interaction among pathogen avirulence (avr) factors and plant proteins (Nimchuk et al. 2003). There are two forms of HR responses in response to Oidium neolycopersici on tomato. The single cell HR reactions and fast HR reactions occur in the presence of two different genes, i.e., Ol-4 and Ol-6, respectively, which could happen if the epidermal cells are infected by the haustoria (Huang et al. 1998; Bai et al. 2005). Tomato plants carrying three Ol-genes (Ol-1,3, and 5) are associated with three accessions of Solanum habrochaites (Li et al. 2007). In Arabidopsis thaliana CPR5 mutants, hypersensitive response is accompanied by spatial and temporal overexpression of CEP1 in a specific manner, coinciding with the spontaneous cell death. However, it is noteworthy that while CEP1’s involvement in programmed cell death (PCD) is regulated by CPR5 and induced by E. cruciferarum, it is not essential for CPR5 mutants to show elevated amounts of resistance to PM (Misas-Villamil et al. 2016; Howing et al. 2017). The inherent powdery mildew resistance genes with crop plants encode different physiological responses in host cells and result in different levels of resistance. Similarly, Indian mustard cultivars also comprise various species and accessions from Brassica. Therefore, there are possibilities of different genes that could respond to such HR reactions, which need thorough investigations.
Biochemical indices associated with resistance against powdery mildew infections
Cruciferous plants generally synthesize biochemical molecules upon PM pathogen’s infection as defense response. In response to the pathogen penetration, it has been observed that the infected cells of Arabidopsis and other crucifer develop cell wall appositions (CWAs) that not only provide physical reinforcements but also act as a chemical antimicrobial barrier against E. cruciferarum (Hardham et al. 2007; Huckelhoven 2007). Post-penetration, the changes in biochemistry of the host plant come as part of SAR and ISR response, where the ISR pathway is arbitrated by NPR1 gene and is ultimately crucial for disease resistance and responds to changes in ethylene and jasmonic acid, while the SAR pathway is regulated by salicylic acid (SA) accumulation (Choudhary et al. 2007). This pathway usually regulates the PR proteins, such as PR1, PR2, and PR5, while the JA-ET pathway controls different defense genes, including PDF1.2 and PR3 in A. thaliana. Additionally, the plant cytochrome P450 gene also responds to the powdery mildew infection by encoding defense-related biochemical enzymes. In Arabidopsis, the mutants lacking the CYP83A1 gene were found to be involved in synthesizing plant chemicals that exhibit resistance to PM (Weis et al. 2013). It was then found that this gene (CYP83A1) aided in producing glucosinolates that are key molecules involved in host defense mechanisms (Bak et al. 2001). Glucosinolates have a key role in both deterring or attracting insects and combatting diseases such as powdery mildew (Bednarek and Osbourn 2009). Without sufficient glucosinolates, the tested pathogen struggles to infect Brassicaceae plants, reliant on these compounds for host recognition and penetration (Weis et al. 2014). Another tryptophan-derived compound called camalexin is also believed to contribute to enhanced PM resistance. Its production relies on several cytochrome P450 enzymes, viz. CYP71A13, CYP79B2, and CYP71B15 (associated with the camalexin and phytoalexin deficiency) (Nafisi et al. 2007; Schuhegger et al. 2007a, 2007b). Mutations within these genes usually hamper the camalexin production, therefore leading to reduced resistance (Saharan et al. 2019). These insights underscore the multifaceted nature of powdery mildew resistance in cruciferous plants, where diverse biochemical pathways and genetic components, including glucosinolates, camalexin, and PMR genes, collectively contribute to complex and dynamic defense responses against pathogen invasion.
Molecular events involved in resistance in Brassica species in response to E. cruciferarum
Although the histological or cellular reactions and the biochemical molecules are associated with some of the defense responses to PM infections, the genomic or molecular interactions are the ultimate end process for ascertaining the host resistance types. The evolutionary mechanism that helps maintain homologous R genes in B. napus, which provide broad spectrum resistance for PM pathogen, have been previously studied. In B. rapa, one gene and three genes in B. oleracea, have been identified as being similar to RPW8 (Li et al. 2016). Additionally, two loci RPW6 and RPW7 were located on chromosome 5 and 3, respectively, in A. thaliana genome that showed independent dominant inheritance against PM disease (Xiao et al. 1997). Most of the characterized R genes in A. thaliana for powdery mildew are C-terminal NB-LRRs, while there is a smaller number of N-terminal coiled-coil motif superfamily and transmembrane domains. Key genes include EDS1, EDR1, PAD4, EDS5, NPR1, EIN2, RAR1, COI1, SGT1b, PBS3, NDR1, and RPW8 (Xiao et al. 2005). In Arabidopsis, genetic screenings have also led to the identification of six recessive loci of PM resistant mutants (PMR1 to PMR6), with genes PMR2, PMR4/GSL5, PMR5, and PMR6 having been partially characterized in Arabidopsis (Vogel and Somerville 2000; Jacobs et al. 2003; Nishimura et al. 2003; Consonni et al. 2006). These mutants have shown increased resistance against PM disease. Furthermore, the susceptibility gene containing MLO locus was also screened and characterized in Brassica with respect to its sequence similarity with barley MLO and identified as a susceptibility gene (Consonni et al. 2006). Chandran et al. (2009) elucidated 67 transcription factors with altered expression at the PM infection site, revealing that MYB3R4 acts as a regulator for transcription, thus influencing host endo-reduplication at the infection site. This combination of homologous R genes, transcription factors, and resistance-related loci work together to provide robust defense against E. cruciferarum and other PM pathogens.
Non-host Resistance (NHR)
The most prevalent and important plant immunity, known as NHR, offers a broad- spectrum resistance to multiple pathogens (Mysore and Ryu 2004; Thordal-Christensen 2003). Such resistance prevents infections from pathogens that did not co-evolve alongside the host, mainly due to the lack of adapted fungal effectors or the presence of numerous resistance genes (Jones and Dangl 2006).
The NHR functions against fungal pathogens that are non-adapted like PM. The NHR has an independent, multi-component defense system, i.e., pre- and post-invasion immunities (Lipka et al. 2005; Wiermer et al. 2005; Stein et al. 2006; Meena et al. 2018). Arabidopsis NPR1 genes, which are important for systemic acquired resistance, are essential for the regulation of NHR (Chen et al. 2013; Zhong et al. 2015).
Previous studies on Arabidopsis have shown that it exhibits resistance to PMs, such as Blumeria graminis and E. pisi, that are not adapted to A. thaliana. These function through strong pre-invasion defenses that are mediated by PEN1 to PEN4 genes (Collins et al. 2003; Stein et al. 2006). The gene PEN1 codes a protein called syntaxin that prevents fungal penetration by forming ternary SNARE complexes (Assaad et al. 2004; Bhat et al. 2005; Kwon et al. 2008). PEN 2 and PEN 3 proteins also play roles in penetration resistance, with PEN 2 demonstrating myrosinase activity that can impede fungal infection (Lipka et al. 2005; Stein et al. 2006).
While the NHR response in pre-invasion stage is mediated by PEN (penetration gene), in the post-invasion stage, it is regulated by PM pathogen genes with increased disease susceptibility, phytoalexin-deficient, and genes associated with senescence (Saharan et al. 2019). The dual role of non-host resistance, where pre- and post-invasion immunity, regulated by various pathogen-related genes, collectively offer robust protection against non-adapted powdery mildew pathogens.
Genes Governing Intrinsic Resistance in Brassica Species and their Mechanisms Against E. cruciferarum
The resistance often has been controlled by Mendelian genes (Biffen 1905). Many research outcomes have accumulated the knowledge on different basis of plant resistance (Lucas 2011; Russel 2013). However, limited efforts have been made to explore this basis content for developing durable resistant genotypes. Recent advancements in genomic and transcript analysis techniques have significantly contributed to understanding the molecular pathway that contributes toward resistance against PM. Identified genes represent both SAR and NHR pathways and belong to R- gene (RPW8), SA signaling pathways (NPR1 and NPR2), calmodulin binding protein (MLO), transcription factors (WRKY70), etc. (Table 4).
A recent transcriptome analysis using RNA-seq data on two B. napus cultivars, displaying a contrasting range of resistance against powdery mildew disease, found that gene expressions involved during pectin modification and degradation were at elevated levels in resistant cultivar in comparison to susceptible. These genes included PM resistant5 (PMR5), polygalacturonase inhibitor1 (PGIP1), pectin methyl esterase inhibitor 9 (PMEI9), and reduced wall acetylation2 (RWA2) (Zhang et al. 2022). Studies conducted in Arabidopsis and other crops have recently confirmed that members of these gene families, such as PMR, PGIP, PMEI, and RWA, are strongly linked with susceptibility or resistance to pathogen (Engelsdorf et al. 2017; Lionetti et al. 2017). These studies highlight specific gene families and molecular pathways that play a key role in developing durable resistance against powdery mildew, emphasizing the potential for utilizing advanced genomic techniques to enhance resistance breeding strategies in Brassica species and other crops.
Table 4. Genes Along with their Functions Conferring Resistance in Crucifer’s Species Against Powdery Mildews
Major R Genes Identified for Brassica Powdery Mildew Resistance
Status of RPW8 gene in cultivated Brassica species
The major concern to ascertain durable resistance is whether resistance genes (R) after undergoing diploidization remain present or are lost in Brassica species. For instance, it was discovered that B. rapa and B. oleracea each have four homologs of the RPW8 gene. However, in B. napus (Bn), which is a cross between B. oleracea (Bo) and B. rapa (Br), seven homologs (RPW8) were observed. It was unclear whether these genes were lost in B. napus and through what evolutionary process (Li et al. 2016). The findings suggested that, although, BoHR homolog originating from B. oleracea remained largely unchanged, the BrHR homolog introduced from B. rapa displayed comparatively more variability within the genome of B. napus due to evolutionary processes such as gene loss, deletion, insertion, substitution, mutation, and intragenic recombination. Furthermore, the BnHR genes, although shared high sequence similarity with BoHR genes, the absence of homologs in B. napus accessions was explained through intragenic recombination involving two paralogs and two orthologs. Additionally, subcellular localization studies were done by fusing truncated BnHRa and BnHRb at the C-terminus, as well as full length BnHR with Yellow Fluorescent Protein (YFP). It was found that the BnHRb-YFP and BnHRa-YFP predominantly present at the extra-haustorial membrane encompassing the haustorium of PM pathogen. The study also revealed the role of these genes in induction of cell death leading to improved resistance against PM disease in Arabidopsis by ectopic expression studies (Li et al. 2016).
BjNPR1 gene
The non-expressor of PR genes family, having NPR1, NPR3, and NPR4, is extremely important in signaling pathway of the salicylic acid (SA) for plant defense. NPR1 acts as a positive regulator by activating (PR) genes upon SA accumulation during pathogen infection. Both NPR3 and NPR4 serve as negative regulators and SA receptors, controlling NPR1 activity by facilitating its degradation, ensuring a balanced immune response. The overexpressed BjNPR1 in transgenic B. juncea presented increased resistance to E. cruciferarum and A. brassicae, its involvement in broad-spectrum disease resistance is shown by its delayed symptom development and decreased disease severity (Ali et al. 2017).
Mildew Locus O (MLO) proteins
The MLO gene family plays a pivotal role in pathogen-host interactions and offering resistance to PM diseases. A transmembrane protein that the wild-type MLO gene produces is implicated in increasing powdery mildew susceptibility by aiding fungal entry and establishment within the host plant. This susceptibility pathway is disrupted by the loss-of-function mutations in MLO genes, which strengthens the plant defense against the pathogen. The significance of the MLO gene in improving plant resistance to PM has been highlighted by recent developments in gene editing technology (Shi et al. 2022). In Arabidopsis, studies have identified PMR2, which encodes MLO2, as a crucial regulator promoting compatibility between PM species (Consonni et al. 2006). MLO-2 aids in PM pathogen entry into plant cells, along with the close paralogs MLO6 and MLO12 (Panstruga 2005). These MLO genes are part of membrane proteins having a conserved role in plant defense (Devoto et al. 2003). Arabidopsis possesses a group of 15 MLO gene, among which MLO2, MLO6, and MLO12 are particularly influential in PM compatibility (Collins et al. 2003). The MLO proteins interact with calmodulin, influencing defense responses independently of signaling pathways such as SA or JA/ET (Bhat et al. 2005). The presence of MLO is crucial for E. cruciferarum infection, with pathogens utilizing MLO functions to inhibit host defense responses (Panstruga 2005). In rapeseed , mutating the BnMLO6 gene resulted in significant resistance to E. cruciferarum and Sclerotinia sclerotiorum (Shi et al. 2022). Moreover, resistant plants showed lower expression levels of the susceptibility genes MLO6 and MLO12, demonstrating their critical function in PM resistance (Zhang et al. 2022). Triple mutant devoid of AtMLO2, AtMLO6, and AtMLO12 exhibited nearly total resistance against E. cruciferarum infections (Consonni et al. 2006). Above-mentioned studies demonstrate the critical role of MLO genes in facilitating PM susceptibility and highlight the potential of gene-editing technologies, like CRISPR/Cas9, to disrupt MLO pathways and significantly enhance resistance to powdery mildew in various crops.