Spot blotch disease causes major yield losses to wheat crop grown in warm, humid regions world-wide. Although the disease was reported as early as 1914 (HCIO, NC12508), its importance was recognized only after the Green Revolution, when majority of semi-dwarf wheat cultivars were found to be susceptible to this disease. Thus, over the last four decades, spot blotch has been seen as a serious constraint in wheat production, not only in the Eastern Gangetic Plains (EGP) of north India, but also in Bangladesh, Nepal, Brazil and Africa (Table 1; Fig. 1). Disease severity is highest, when the crop’s late post-anthesis phase coincides with a period of high relative humidity and high temperature. Globally, an estimated 25 mha of wheat area is vulnerable to spot blotch (van Ginkel and Rajaram 1998), about 40% of which is grown in the Indian sub-continent as a part of a rice-wheat cropping system (Joshi et al. 2007a). Crop losses in the Indian subcontinent due to spot blotch have been estimated to be in the range of 15% to 25% (Dubin and van Ginkel 1991), but level of loss in individual fields can be much higher. The disease also reduces end-use quality of harvested wheat grains due to contamination with mycotoxins (deoxynivalenol, nivalenol, or zearalenone) produced by the pathogen, thus also causing concern for human and animal health. Keeping this in view, even a marginal reduction in disease severity would have a significant impact on farmer’s income.
In the past, the causative agent of spot blotch was believed to be a complex of the following three species: Helminthosporium sativum, Alternaria triticina and Pyrenophora tritici repentis (Maraite et al. 1998). However, later careful monitoring of the disease in the Eastern Gangetic Plain (EGP), carried out by sampling at various growth stages of the host suggested that the main pathogen is the hemibiotroph Bipolaris sorokiniana [syn. Drechslera prorokiniana (Sacc.) Shoem. Syn. Helminthosporium sativum, teleomorph Cochliobolus sativus; for taxonomy and nomenclature, see later], which is also the causal agent of common root rot, seedling blight, head blight, and black point of wheat and barley.
As a pathogen, B. sorokiniana first colonizes the older leaves at the base of wheat plant and then progresses to the upper part of the canopy (Joshi et al. 2002). Disease severity is affected by crop management, soil fertility, planting density, the growth stage of the plant and the weather conditions experienced during the later part of the growth (Joshi et al. 2007b). High relative humidity, which allows the canopy to remain wet for a prolonged period, is particularly favourable for infection and pathogen growth (Acharya et al. 2011). Outbreaks of spot blotch in Brazil were shown to be associated with periods, when the leaves remain wet for >18 h a day and the mean air temperature stays at >18°C (Reis 1991). In the Indian subcontinent, the disease spreads, when the temperature stays >26°C (Chaurasia et al. 2000), which explains why late-sown wheat is particularly vulnerable to the disease (Duveiller et al. 2005). In the Indian EGP, Nepal and Bangladesh, where wheat is typically planted after the harvest of preceding rice crop, loss in productivity is often due to a combination of spot blotch and terminal heat stress (Joshi et al. 2007c). The areas for occurrence of spot blotch globally are shown in Fig. 1. In this brief review, a summary of the current state of knowledge about wheat spot blotch caused by the anamorph, B. sorokiniana is presented. The emerging tools that could be used for breeding wheat cultivars exhibiting a high level of resistance to spot blotch are also discussed. The disease root rot, which is also caused by B sorokiniana will not be covered in this review.
NOMENCLATURE AND TAXONOMY OF B. SOROKINIANA AND C. SATIVUS
Bipolaris and Cochliobolus represent two of the three genera (the third genus being Curvularia), which form a complex, causing several diseases on a number of species from the family Poaceae with a worldwide distribution. The taxonomy of this complex has been a subject of discussion, such that frequent nomenclatural changes have been made and opinions continue to differ. A detailed description of “Bipolaris sorokiniana (Sacc.) Shoemaker” is available elsewhere (Sivanesan and Holliday1981; ¬Sivanesan 1990).
The pathogen that causes spot blotch was initially a part of the former genus Helminthosporium that was divided into three anamorphic genera: Bipolaris, Drechslera, and Exserohilum with their corresponding teleomorphs named as Cochliobolus, Pyrenophora, and Setosphaeria. Thus Bipolaris sorokiniana (Sacc.) is an anamorph representing the asexual state of Cochlibolus sativus (teleomorph), which represents the sexual state and occurs only rarely in nature. Analysis of gene sequences of rDNA ITS (internal transcribed spacer), GPDH (glyceraldehyde 3-phosphate dehydrogenase) gene, and the gene encoding the LSU (large subunit) of EF1-α (translation elongation factor 1-α) all suggest that B. sorokiniana and C. sativus may be the two stages of the same taxon (Manamgoda et al. 2012). It is also known that in ascomycetes, anamorph and teleomorph of the same taxon develop independently on different substrates, so that each phase is often collected in complete ignorance of the occurrence of the other form; this anomalous situation is not uncommon among fungal pathogens, and therefore according to the International Code of Botanical Nomenclature (ICBN), it is legal to have two separate binomials for two forms of the same pleomorphic fungus. This appears to be a useful option, but creates confusion in the minds of students and users, who would wonder how the same organism can have two names.
In the older literature, several synonyms of the anamorph B. sorokiniana have been used, which include the following: Helminthosporium sativum, H. sorokinianum, Drechslera sorokiniana, Drechslera prorokiniana (Sacc.) Shoem. teleomorph Cochliobolus sativus (Maraite et al., 1998). Initially, Shoemaker (1959) proposed the generic name Bipolaris for the Helminthosporium species with fusoid, straight, or curved conidia germinating by one germ tube from each end (bipolar germination). The fungus (anamorph B. sorokiniana) is distinguished from other members of the Bipolaris genus on the basis of morphological features of conidiophores. A key for distinguishing species of Bipolaris was given by Subramanian (1971), and an elaborate description of all species of Bipolaris has been provided by Manamgoda et al. (2012).
Infection, multiplication and transmission. Although, spot blotch is largely a seed-borne disease, primary infections can also be initiated from inoculum surviving on crop residues, alternate hosts (e.g. rice, barley and other grasses) or conidia in the soil. The fungus can survive on straw or in the soil for two months, after which its viability begins to decline (Chand et al. 2002; Pandey et al. 2005). It has been shown that the conidia found on wheat straw tend to aggregate into ‘clumps’ after storage for five months(Chand et al. 2002). Although sexual state of this fungus (Cochliobolus sativus) is known (see later), it is not a source for infection, leaving conidia as the major vehicle for dispersal and survival of the pathogen (Reis and Wünsche 1984).
The infection for spot blotch is initiated by a rapid (within four hours) adhesion of the conidial spores to the leaf surface, followed by their germination and formation of the germ tubes (Acharya et al. 2011). Within eight hours, the germ tubes swell sufficiently to produce an appressorium, from where infecting hyphae are developed (Jansson and Akesson 2003). The hyphae penetrate the host’s cuticle within 12 hours (Sahu et al. 2016) and multiply rapidly, spreading into the intercellular space within the mesophyll tissue of the leaf (Acharya et al. 2011). Conidia of a new generation are produced within 48 hours, and are carrie
d on single or clustered erect conidiophores that are 100-150 μm x 6-8 μm long. These conidia are olive-brown, oblong, tapered towards the ends and have a prominent basal scar. They measure 15-20 μm x 60-120 μm in size and have three to nine thick walled septa. Several cycles of conidia production are possible during the cropping season, which cause secondary infections involving dispersal through dew and rain (Acharya et al. 2011). The secondary infection of spot blotch is thus typically caused by the air-borne conidia (Duveiller et al. 2005) (Fig. 2).
The spot blotch pathogen is a hemi-biotrophic fungus, with initial biotrophic growth phase confined to individual epidermal cells, and the subsequent necrotrophic growth involving apoptosis of the host cells. Once the plant’s epidermal defense has been overcome, the speed of the pathogen’s spread within the host mesophyll tissue depends on the level of resistance expressed by the host. In case of seed-borne infection, the pathogen is able to respond rapidly to the germination of the host seed, and grows readily to reach the plumule and the tip of the coleoptile (Reis and Forcelini 1993). The subsequent systemic development of the pathogen within the host results in accelerated leaf senescence (Dehere and Oerke 1985). However, the symptoms like leaf spotting may not become apparent until the flag leaf has emerged (Duveiller et al. 2007).
Symptoms and diagnosis of the disease. The spot blotch symptoms typically appear on leaf, sheath, node and glumes as small light brown lesions mostly oval to oblong to somewhat elliptical in shape measuring 0.5 to 10 mm long and 3 to 5 mm wide. These lesions have brown margins and are often scattered throughout the leaves and gradually increase in size and coalesce to form larger necrotic patches (reaching several centimeters). The affected leaves soon appear dried, become chlorophyll deficient, and eventually die. Under most severe conditions, the pathogen also attacks the spikes and produces dark brown to black discoloration around the germinating point of seed, called ‘black point’. Severity of spot blotch is modulated by several abiotic factors such as soil fertility, moisture and moderate temperature.
The symptoms mentioned above may generally be used for diagnosis of the disease, but this may need verification by microscopic identification of the pathogen, which requires expertise and skill. This method is also time-consuming and is sometimes rather unreliable. Therefore, alternative methods for diagnosis using either antibody-based or DNA-based probes or molecular markers have also been used (Ward et al. 2004). As an example, a SCAR (sequence characterized amplified region) marker for PCR-based detection of B. sorokiniana in plant tissues and soil was developed by Aggarwal et al. (2011). This marker allowed the detection of the pathogen even when present in the latent form in inoculated wheat leaves, before the visual symptoms appear. This would help in early detection and timely action for disease management. RAPD markers were also used for identification of pathogen isolates belonging to different groups of spot blotch established on the basis of colour and shape of colonies (Pandey et al. 2008; for groups of spot blotch, see later). In a recent study from China, fungal isolates from volunteer wheat plants were identified to be B. sorokiniana, using three different DNA marker systems, which included internal transcribed spacer (ITS) of ribosomal DNA genes, β-tubulin gene, and the elongation factor (EF) gene (Sun et al. 2015).
Phenotyping the disease (approaches used to assess infection severity). The data on spot blotch infection in wheat crop can be recorded on a continuous scale using one of the following two available methods (Duveiller et al. 1998; Bashyal et al. 2010): a single digit scale with scores ranging from 0 (immune) to 9 (highly susceptible; Saari and Prescott 1975), or a double digit scale (00-99), where the first digit indicates the extent of the disease progression from ground level to the top of the canopy, while the second digit refers to the extent of leaf area showing disease symptoms (Eyal et al. 1987). The double-digit scale has been widely adopted for field-based evaluations, but has limited application when artificial inoculation is used, because the upper leaves are generally more heavily exposed to the inoculum. In a recent study, where 4925 wheat accessions from the NBPGR Genebank in India were screened for spot blotch (Kumar et al. 2016), following classification was used, based on double digit scale: (i) I- immune (00), (ii) R- resistant (<12), (iii) MR- moderately resistant (12 to 34), (iv) MS- moderately susceptible (35 to 56), (v) S- susceptible (57 to 78), (vi) HS- highly susceptible (>78). More recently, a sensor-based evaluation approach (involving a hand-held green seeker NDVI) has been developed to screen a large number of lines (Kumar et al. 2016).
Pathogen reproduction and the infection process. As mentioned earlier, B. sorokiniana (syn. Helminthsporium sativum) represents the asexual state of the spot blotch pathogen, its sexual state or the teleomorph being C. sativus. Therefore, it is obvious that the pathogen reproduces through asexual means mainly through conidia (primary source being seed, soil and plant debris; secondary source is air), although occurrence of sexuality and parasexual cycle has been reported in the sexual form of B. sorokiniana (C. sativus) and in several species of Helminthospoium (Nelson et al. 1960; Tinline 1962; Chand et al. 2003), which occur rather rarely. In one of these studies, nine of the 13 interspecific crosses involving eight different species of Helminthosporium, gave viable ascospore progenies; abundant perithecia were also available in four crosses, suggesting that sexuality does occur in related forms, and that there are pathogenicity genes, which segregate in these crosses. However, no isolates from wheat were involved in this study and no sexuality has been reported within or between B. sorokiniana isolates derived from wheat, which is therefore, described as an imperfect state or anamorph.
The existence of two distinct mating types (A, a) was suggested in an analysis of a large number of isolates of spot blotch (B. sorokiniana) collected from diverse locations (Wen and Lu 1991). The pathogen is generally heterothallic, and heterokaryons with multinucleate condition do occur, which is attributed either to the occurrence of mutations or to anastomoses that have been observed between strains (Bashyal et al. 2015). However, the occurrence of two mating types is insufficient evidence for sexual reproduction, unless actual mating or recombination is demonstrated, because if other requirements for mating are not available (e.g. compatibility between mating types and proper cultural conditions), no mating or recombination through a sexual or parasexual cycle will be possible. As a corollary, neither the geographic distribution nor the host preference of two different mating types can be used to explain the lack of detection of the sexual stage (Wen and Lu 1991). However, crosses have been made in isolates derived from barley and recombination has been studied, leading to the preparation of linkage maps for all the 15 chromosomes of B. sorokiniana = C. sativus (Zhong and Steffenson 2001).
Survival of the pathogen and the sources of inoculum. The survival of the pathogen on the host, and its availability as a source of inoculum depends on several factors. Firstly, a direct relationship between the melanin content and conidiogensis of B. sorokiniana has been reported, suggesting that melanin produced by the pathogen neutralizes antimicrobial activity of the host cells, thus contributing to propagule durability (Henson et al. 1999; Bashyal et al., 2010). Secondly, a number of species work as alternative hosts for the pathogen. Pandey et al. (2005) screened 22 grass species, and observed that each of a number of species could work as an alternative host for B. soroki
niana (see next section for details). When used for artificial inoculation, the isolates recovered from these species proved to be pathogenic on wheat. However, since their ability to infect wheat under natural conditions could not be confirmed, these alternative hosts may or may not be the likely source of natural inoculum in the EGP. Thirdly, the pathogen can also survive on crop debris (Chand et al. 2002), although the high rainfall associated with the monsoon season (June-September in the EGP) tends to induce water-logging in the paddy crop, so that the resulting anaerobic conditions in the soil are inimical to conidial survival. It was observed that the conidia isolated from soil during August onwards in India were not pathogenic (Pandey et al. 2005). In summary, the vast bulk of inoculum responsible for spot blotch is actually seed-borne (Pandey et al. 2005).
The host range of the pathogen. B. sorokiniana has a broad range of hosts. In addition to bread wheat, it is able to infect durum wheat, barley, triticale, rye, maize, pearl millet (Pennisetum typhoides), foxtail millet (Setaria italic), Guzmania species (Tillandsioideae), Panicum lacromanianum, Phleum pratense, along with a number of other wild grasses (Manamgoda et al. 2011). Phalaris minor is another potential alternative host (Singh et al. 1998; Hobbs and Morris 1996). In NE China, B. sorokiniana was found to be able to infect 29 crop species including several grasses(Acharya et al. 2011), while in the Yellow and Huai River plain region, at least 65 grass species were shown to support B. sorokiniana (Ma and He 1987). Under laboratory conditions, B. sorokiniana can easily infect the model plant, Arabidopsis thaliana (Zhao et al. 2005). A summary list of known alternative hosts is provided in Table 2, although it does not include all the species reported from China. However, the contribution of these species towards field inoculum for development of spot blotch in wheat crop has not been confirmed.
PATHOGEN VIRULENCE DIVERSITY, AND ITS CHARACTERIZATION
Diversity for virulence/pathogenicity. As early as 1926, it was noted that isolates of the pathogen (then referred to as Helminthosporium sativum) varied considerably with respect to their virulence on wheat and barley (Christensen 1926). At the morphological level, virulence is correlated with colony color and morphology (Chand et al. 2003; Pandey et al. 2008). Using the color of colonies and the nature of growth, the isolates were arranged into the following five groups: (i) black colonies with suppressed growth; (ii) brown/dull black colonies with suppressed growth; (iii) grey colonies with white spots, showing cotton-like growth; (iv) dull white/greenish black colonies with fluffy growth; and (v) white colonies with fluffy growth (Chand et al. 2003; Pandey et al. 2008). Isolates belonging to the first of these five groups (black colonies) are generally the most likely cause of large-scale outbreaks (Chand et al. 2003; Asad et al. 2009). In another study, the isolates from leaves were placed in five groups as above, but when isolates from seed were used, only the first three of the above five groups were available; more importantly, isolates of three major groups (black, white and mix; which appear as major groups) could be derived from the “mix” population in clonal studies (Pandey et al. 2008).
Variation in pathogenicity level of the same race under different conditions/locations has also been reported from Pakistan and Nepal (Asad et al. 2009; Mahto et al. 2002). A particular race may be relatively more virulent in locations, where wheat is continuously cultivated (El-Nashaar and Stack 1989). The level of virulence may also depend on hyphal fusion, nuclear migration or the occurrence of a multinuclear state (Chand et al. 2003; Pandey et al. 2008).
Pathotypes and virulence genes. It is generally recognized that a specific pathogen isolate for a disease carries a predominant virulence gene, which corresponds to a major resistance gene in the host (gene-for-gene relationship). Spot blotch pathogen is no exception to this rule, although an exact “gene-for-gene” relationship has not been established yet. It is also recognized that in a particular geographical region, the genes that need to be deployed for developing resistant cultivars should be selected according to the virulence genes present in the predominant race/pathotype, which occurs in that area. This demands that we should have information about the predominant pathotype and its population structure in a specific area. Information should also be available about the predominant virulence gene and frequencies of other virulence genes that the population may carry. Population structure of the pathogen will obviously differ in different areas with respect to different virulence genes, since frequencies of different virulence genes in a population keep on changing due to the pressure on the pathogen to survive. Often the most frequent virulence gene becomes ineffective in due course of time due to cultivation of resistant genotypes in an area. Unfortunately, this information is not available for wheat-spot blotch pathosystem, although some information for barley-spotblotch is available (see next).
Population differentiation in B. sorokiniana. As early as 1922, it was shown that like many other plant pathogens, specialization for virulence does occur in B. sorokinianaalso (Christensen 1922). However, this specialization of B. sorokinianais more apparent, when barley is the host, and is not so conspicuous, when wheat is the host. It has been observed that almost allB. sorokiniana isolates are virulent on wheat host and any isolatecould be used for screening wheat cultivars for resistance (Tinline 1988). This situation of wheat-B. sorokiniana pathosystem differs from wheat-Puccinia pathosystems, where specific races with virulence genes have been shown to interact with individual wheat cultivars with specific resistance genes. It has also been observed that unlike rusts, the B. sorokiniana isolatesdo not exhibit clear and unique differential virulence patterns on wheat and consist of strains that exhibit a continuum in aggressiveness with no specific host-pathogen interactions (Maraite et al. 1998; Duveiller and Garcia Altamirano 2000). Consequently, wheat differentials for classifying spot blotch isolates into pathotypes or races are not available. In a study involving analysis of infection responses (IRs) of 12 wheat cultivars inoculated with 206 B. sorokiniana isolates collected from 24 countries, it was observed that only 1 to 2% of the variance could be attributed to interactions between host and pathogen (Hetzler et al. 1991). In case of barley- spot blotch pathosystem, on the other hand, three, four or six pathotypes (0, 1, 2..) or eight virulence groups (VIGs; developed using coded triplet nomenclature system of Limpert and Müller 1994) were identified (Ghazvini and Tekauz 2007). A virulence gene VHv1 was also identified, which functions with barley host in a specific manner (Zhong et al. 2002), However, in a recent study involving bioinformatic analysis of whole genome sequence of the pathogen, a virulence gene ToxA showing virulence against the wheat host was also identified (see next).
Identification of a virulence genes Vta1 andToxA. The progeny consisting of more than 100 strains derived from the cross between two C. sativa isolates (ND90Pr and ND93-1) was used for study of virulence against both barley and wheat. In both cases, 1:1 ratio (virulent : non-virulent) was obtained suggesting that the two isolates used as parents differed for a single virulence gene. The gene in barley was designated as VHv1 and that for wheat was designated as Vta1 (Zhong et al. 2002). One would like to find out if these two genes are the same.
In another recent study, genomes of three Australian isolates of B. sorokiniana were sequenced and screened for known wheat pathogenicity genes, leading to identification of ToxA virulence gene in one of the three isolates, n
amely BRIP10943 (Macdonald et al. (2017). This gene was previously associated with wheat pathogens Parastagonospora nodorum and Pyrenophora tritici-repentis. Analysis of the regions flanking ToxA within B. sorokiniana genome suggested its occurrence within a 12kb genomic region that was nearly identical to the corresponding regions in P. nodorum and P. tritici-repentis.
A search was also made for the presence of ToxA gene in wheat isolates, and it was shown to be present in 12 of 35 isolates that were screened. Sequences of the ToxA genes in these isolates also revealed two haplotypes, which differ by a single non-synonymous nucleotide substitution. Pathogenicity assays suggested that a B. sorokiniana isolate harbouring ToxA gene, relative to a non-ToxA isolate, is more virulent on wheat lines carrying the sensitivity geneTsn1 that was identified earlier. The protein ToxA was already known to be a necrotrophic effector molecule produced by Stagonospora nodorum and Pyrenophora tritici-repentis (Tan et al. 2012) and the product of cloned gene Tsn1was earlier found to carry S/TPK and NBS-LRR domains that are characteristic features of disease resistance genes (Faris et al. 2010). Thus, a gene-for-gene relationship between ToxA as a avirulence gene and Tsn1 as a resistance gene can not be ruled out. It was also inferred that host-specific virulence genes can be horizontally acquired across multiple fungal species, thus increasing the virulence potential of the pathogen.
Biochemical characterization of the pathogen (including phytotoxins). At the biochemical level, virulence was shown to be negatively correlated with melanin level and positively correlated with the level of extracellular lytic enzymes like cellulase and pectinase in strains isolated from barley (Chand et al. 2014). Different species of Bipolaris also produce a range of phytotoxic compounds, which include prehelminthosporol, helminthosporol, helminthosporic acid and sorokinianin (Fig. 3). Prehelminthosporol (C15H22O2), which is believed to be the most abundant and active of these toxins, is a hydrophobic sesquiterpene of low thermal stability and poor water solubility (Carlson et al. 1991). Sorokinianin is also a sesquiterpene, and shares the same carbon skeleton as prehelminthosporol; it has been shown to have an inhibitory effect on germination of conidia (Nakajima et al. 1994). Jahani et al. (2006, 2014) identified the compound bipolaroxin, which produces yellow or necrotic lesions when placed on the leaf of a wheat plant. Above a level of 15 ng/mL bipolaroxin, all wheat cultivars produced a susceptible reaction. In a study involving 12 isolates of B. sorokiniana, the virulence of the pathogen was shown to be correlated with the level of toxin produced; the most virulent strain (B25) was shown to have the highest level of toxin and the least virulent strain (B7) was shown to have a relatively much lower concentration of the toxin, suggesting that virulence depends on the level of the toxin also (Jahani et al. 2006).
Molecular characterization of the pathogen (including molecular markers). Several approaches have been used for molecular characterization of the fungal isolates. These include molecular markers like AFLP, SSR, RFLP, etc, karyotyping using contour-clamped homogeneous electric field (CHEF) electrophoresis, and whole genome sequencing.
Molecular markers and linkage maps. Molecular markers have been used for the study of variability and diversity within B. sorokiniana. For instance, a number of universal rice primer (URP)-PCR based markers were found to be useful for the study of diversity and variability among different isolates of B. sorokiniana (Aggarwal et al. 2011; Mann et al. 2014). EST-SSRs and ribosomal DNA polymorphism involving sequence variation in the internal transcribed spacer (ITS) can also be utilized for characterization of isolates (Aggarwal et al. unpublished data).
More detailed characterization of B. sorokiniana isolates using molecular markers was possible using isolates from barley, where molecular maps for all the 15 chromosomes could be prepared using AFLP, SSR and other markers. The isolates representing two different mating types could also be crossed and progenies examined for recombination, which facilitated mapping. Linkage analysis gave 27 linkage groups, an individual chromosome having more than one linkage groups, so that the linkage groups did not match the number of chromosomes due to low density of markers (Zhong et al. 2002). The virulence gene VHv1and the mating locus MAT could also be mapped.
In B. sorokiniana isolates from barley, a correlation was also observed between the AFLP patterns and virulence, but not between AFLP pattern and geographic origin of isolates. To evaluate causes of genetic variation among isolates, the influence of mutation, migration and gene flow, and recombination within the B. sorokiniana population were discussed, suggesting genetic recombination between isolates possessing extreme levels of virulence in the population of the pathogen. Similar studies need to be conducted on isolates from wheat plants.
Genome sequencing (NRPS, PKS and SSP). Using whole genome sequencing, it was also possible to identify genes encoding different secondary metabolites and other proteins, which may be involved in virulence. The most important of the secondary metabolites that may be involved in providing virulence to the pathogen are non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). Small-secreted proteins (SSPs) are another class of molecules that may be involved in virulence. As many as 25 NRPSs, 18 PKSs and 167 SSPs were detected in C. sativus isolates through genome sequencing. Only two NPS genes that were present in VHv1 region of the B. sorokiniana genome were conserved in C. sativus and were believed to be responsible for virulence (Condon et al. 2013).
Karyotypes of B. sorokiniana. Karyotypes of 16 isolates collected from barley grown in different parts of the world, representing all the three pathotypes (0, 1, 2), were also studied using CHEF electrophoresis (Zhong and Steffenson 2007). Each of the 16 isolates (except two isolates from North Dakota) generally showed a unique banding pattern, which is a bit surprising, unless large-scale structural changes were involved in differentiation (normally the karyotypes within a species remains constant and unique). A similar study of isolates from wheat would resolve to what extent the strains infecting wheat and barley differ, and to what extent structural changes played a role in differentiation.
Single-copy DNA probes assigned to each of the 15 chromosomes also became available, so that Southern blots of CHEF-separated chromosomes could be hybridized with these probes to identify highly polymorphic chromosomes among isolates. Chromosomal rearrangements (translocations, deletions, duplications) were found in several isolates. DNA markers previously found linked to VHv1 were also used as probes in hybridizations with the CHEF blots. The results showed that the chromosome carrying VHv1 was larger than its counterpart without the gene in other isolates. This suggests that the genomic region carrying the virulence locus VHv1 is unique. This study provides useful information on genome structure and divergence, which is essential for advancing our understanding of the genetics and biology of C. sativus.
GENETICS OF RESISTANCE TO SPOT BLOTCH
Sources for resistance. Sources for resistance against spot blotch are available within the bread wheat gene pool, and also in related alien species from within the tribe Triticeae.
(i) Sources within the bread wheat genepool. Major known sources of resistance for wheat crop against spot blotch within the wheat gene pool are listed in Table 3.The major sources of resistance are available from Brazil, India, China, Mexico (CIMMYT), Nepal and Pakistan, although some of these entries from different countries may have the same origin/pedigrees (Mehta 1985;
Singh et al. 2015; van Ginkel and Rajaram 1998; Joshi et al. 2004; Alam et al. 1998; Bhatta et al. 1998). Resistant somaclonal variants were also reported in an independent study (Arun et al. 2003).
(ii) Resistance sources in alien species. During late 1980s, at CIMMYT, Thinopyrum curvifolium was used successfully as an alien donor for spot blotch resistance (Duveiller and Gilchrist 1994). A number of Aegilops and Triticum species that could also be used as resistance donors included the following: Aegilops triuncialis, Ae. speltoides, Ae. triaristata and Ae. cylindrica, T. dicoccoides (wild emmer), T. boeoticum, T. timopheevii, T. araraticum, T. persicum, T. sphaerococcum and T. urartu (Singh and Dhaliwal 1993; Smurova and Mikhailova 2007). Among the cultivated wheat, tetraploid durums tend to be more susceptible than bread wheat, suggesting that the genes for spot blotch resistance at the hexaploid level are probably available mainly within the D genome, although the genetic studies do not support such a hypothesis (see later).
Inheritance/genetics of spot blotch resistance. Inheritance for seedling/adult plant resistance to spot blotch has been studied using different approaches, which can be classified into two major categories. The studies in the first category used Mendelian approach involving crosses between resistant and susceptible genotypes followed by an analysis of segregation pattern, and the studies in the second category involved quantitative genetics approach using molecular markers. Molecular markers were used either for QTL interval mapping based on linkage involving use of mapping populations, or the genome wide association studies (GWAS) based on LD (linkage disequilibrium); each GWAS involved the use of an association panel consisting of a large number of genotypes. The results of these studies are summarized in Tables 5 and 6.
Mendelian approach. While following Mendelian approach, different workers used different sources of resistance, which could differ in their genetic make-up, and also with respect to the resistance genes they carry for the prevailing pathogen races. However, a lack of understanding of the population structure of the pathogen, and lack of identification of specific races of the pathogen, did not allow identification of avirulence genes in the pathogen and their corresponding resistance genes in the host, as has been possible for different rusts of wheat. Development of field-based epiphytotics was also not easy in all cases due to specific environmental conditions required for the development of the disease (high humidity and high temperature >26oC). A high level of genotype × environment interaction also contributed to the difficulty in inheritance studies. Despite these difficulties, a number of studies have been conducted, which involved crosses between resistant and susceptible genotypes, and suggested that resistance in different genotypes could be controlled by either dominant or recessive genes, which could also be involved in a variety of interactions including additive/duplicate/epistatic interactions. In one such study, two resistant cultivars (Chirya3, MS#7) were crossed each with the same susceptible cultivar (BL1473) and were also crossed with each other. The phenotypes observed in F1 and the segregation patterns observed inF2suggested that each resistant cultivar carried one resistant gene, and that the genes in two resistant cultivars differed and were duplicate in nature (giving 15:1 ratio in F2 generation; Neupane et al. 2007). In another study, one resistant cultivar (Chirya3) was separately involved in two crosses, each with a different susceptible cultivar (CS, WH147) and the results suggested that resistance in Chirya3 was governed by two recessive genes (hlbr1, hlbr2); these recessive genes were described to function in a complimentary manner, although apparently these were also duplicate in nature (1R:15S ratio in F2 generation; Ragiba et al. 2004a). Since Chirya3 was involved in both these studies, it is apparent that this genotype had separate sets of dominant and recessive genes for resistance, unless the same resistance genes were dominant in one combination and recessive in the other combination. One could perhaps conduct allelic tests to find out if same genes were involved; mapping of these genes on chromosomes could be another approach to find out if the dominant and recessive genes were really different. In another study, additive effects between two or three recessive genes (two in cultivars PBW343 and HS361, and three in RAJ3702) were inferred on the basis of the study of F1 and F2 generations derived from six crosses involving three cultivars (Bhushan et al. 2002). Similarly epistatic interactions among three dominant genes were inferred in another study (Sharma and Bhatta 1999; Neupane et al. 2007). Polygenic control with two or three genes providing good resistance was also reported in four moderately resistant cultivars (Velazquez 1994, MSc Thesis). In a comprehensive study, Joshi et al. (2004) investigated the segregating generations (F3, F4, F5 and F6) of three crosses between resistant (Acc. No. 8226, Mon/Ald, Suzhoe#8) and susceptible (Sonalika) parents. Resistance appeared to be under the control of three additive genes. Taken together, all these studies suggest a polygenic nature of resistance, which is also supported by studies involving QTL interval mapping and GWAS (see next paragraph).
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