ICAR-National Bureau of Plant Genetic Resources, New Delhi-110012, India
*Corresponding author: Sundeep Kumar (E-mail: sundeep@nbpgr.ernet.in)
The importance of wheat as staple food is well known as it is most popular, widely grown cereal crop of the world (Breiman and Gaur, 1995). Due to fast growing population of the world especially in the developing countries the demand of wheat is keep on increasing (Rajaram, 2000; Nagarajan, 2005). The present global wheat demand is about 640 Million tonnes and to satisfy the demand of growing population in 2020 AD, we need to produce between 840 to 1050 million tonnes (Rosegrant et al., 1995; Kronsted, 1998). In India, it is expected that we need to produce around 109 million tonnes to feed India in 2020 (Sharma et al., 2002, Joshi et al., 2007; Nagarajan, 2005).
It has been learnt from past experiences that there are number of constraints in the successful wheat production and in the way to make dream true to feed the world growing population in coming years. Apart from abiotic stresses like drought, heat and salinity etc (Kumar et al., 2013), there are number of biotic stresses which are challenging successful wheat production across the world (Joshi et al., 2007). The major biotic stresses in wheat are rusts (leaf, stem and stripe), spot blotch, Fusarium leaf blight, powdery mildew, karnal bunt etc (Joshi et al., 2007; Kumar et al., 2012). However, leaf rust (Puccinia recondita) is one of the most devastating and persisting disease thus, considered as economically important disease in many parts of the world (Singh and Rajaram, 1992). Although, this disease was reported about a century back and recognized for its devastating nature by Dr. K.C. Mehta in 1930, this disease gained importance after green revolution as only few high yielding semi-dwarf wheat cultivars were grown at large scale which favored leaf rust pathogen to have epidemics and caused significant yield losses in many parts of the world.
Even today leaf rust is causing significant yield losses varying between 15 to 60 per cent (McIntosh, 1998) losses vary from region to region and year to year. However, losses been experience for almost every year since 1968. For last 40 to 50 years numbers of breeding programs have been implemented across the world at national and international level with the aim to minimize the yield losses following conventional or non-conventional breeding approaches. However, it is still considered as big challenge before wheat breeders. The best approach to save the yield losses occurring due to this disease is to follow durable disease resistant program in commercially adopted cultivars which are otherwise good agronomic traits and quality but susceptible to leaf rust. According to Chen et al. (2013), resistant cultivars are the cheapest, most reliable and environment friendly way to control rust disease.
Numbers of research programs are currently being progressing to develop durable leaf rust resistance cultivars at international level by CIMMYT, Mexico and at national level in different countries around the world. A number of genes like Lr9, Lr19 and Lr24, which are effective against most of the pathotypes of leaf rust, are available in the improved genotypes. But sometimes, these resistant genes lack durability. Thus, the short lived nature of race-specific hypersensitive response has created the necessity to search for more durable type of resistance. Therefore, the most efficient and best strategy to control the rusts, lies in combining genes irrespective of whether the genes are minor or major (Sawhney, 1995).
So far 67 leaf rust resistant genes have been reported conferring resistance to leaf rust. These genes alone or in combination provide a satisfactory level of resistance against leaf rust. However, due to increasing threat of rust and short lined nature of most of the resistant genes conifers vertical resistance. Now scientists are looking forward for other attractive approaches like slow rusting (Shaner et al., 1978; Singh et al., 1995; Sareen et al., 2012). Effect of slow rusting gene come in the form of longer latent period low susceptibility or infection frequency smaller uredial size reduce duration and quantity of spore production that can effect disease progress in the field (Wilcoxson, 1981; Navi et al., 1989; Sareen et al., 2012). Some varieties have the ability to retard rust development even those they have susceptible reaction type (Cardwell et al., 1970; Singh et al., 1991). The purpose of slow rusting or partial resistant is to achieve durable resistant and provide a sustainable approach of disease control (Johnson, 1988; Ittu, 2000; Sareen et al., 2012). Numbers of slow rusting gene have been identified so far. However, the known slow rusting genes which present in number of CIMMYT release germplasm line or cultivars are lr34 and lr46 present in combination with other minor gene (Bai et al., 1999).
Despite of the several advantages, very little efforts have been made to utilize slow rusting in the control of rust epidemics. Impact of slow rust resistant genes alone or in combination with other resistance genes to achieve durable resistance/near immunity is yet to be done. Genes responsible for slow rusting can be brought together with the help of molecular markers. Further, available wheat germplasm need to be characterized for slow rusting to make them enable to utilize them in breeding durable leaf rust resistant cultivars.
Problematic areas
Leaf rust pathogen is widely spread and presented rust. Almost every corner of the world wherever wheat is grown, presence of leaf rust is confirmed The favourable conditions for fast spreading of leaf rust is cloudy weather and low temperature around 15 to 25 0C. Different and distinct virulent pathotypes of Puccinia triticinia have been reported in various geographical regions of the world by Kolmer (1989, 1992) and eastern and western Canada by Leonard et al. (1992), in United State and Europe by Park and Felsenstein (1998). Singh and Rajaram (1992), classified different parts of the world for stripe, leaf and stem rusts based on the losses as major, minor and local problem (Table 1).
Members of leaf rust pathogen Puccinia recondita attack wide number of grasses all those they risk hardly any green rich and pathotypes are specialized to a particular host plant. Puccinia triticinia is primarily pathotypes of wheat its ancestors/wild relatives and triticale. The main source of disease spread is mycelium or uredinia which survive on volunteer plants during off season. Uredinia spores spread from one place to another place by the wind (Hirst and Hurst, 1967; Watson and Sousa, 1983) and may introduce a new virulent race in new area but effect can be easily visualized on susceptible cultivars. Rust pathogens are biotrophic in nature and required living plant cell to survive.
Symptoms
Symptoms appear in the form of small brown pustules develop on the leaf blades in a random manner as scattered distribution. Brown circular uredinia normally appear on the upper leaf surface, but in case of severe epidemics, sheath infections can also be seen. Uredinia can be seen in small clusters or may be irregularly scattered all over the lamina surface. They are bigger in size than the uredia of yellow rust fungus. Older uredia of leaf rust cannot be distinguished by colour from those of the yellow rust except for their irregular arrangement.
Pathotypes of leaf rust
Like resistant gene number of pathogens have been reported so far which have given differential response against durum and bread wheat cultivars. The pathotypes 77 and 104 have been observed most virulent to the bread wheat cultivars while, other pathotypes are relatively more virulent in durum wheat (Mishra et al., 2001). Other leaf rust races due to their low level of virulent against leaf rust resistant genes. However, these races observed more virulent to durum wheat compare to bread wheat. Other pathotypes which were virulent in bread wheat were 77-1 and 77-7 while in case of durum wheat the pathotypes 12-4, 100-6, 100-8 and 100-62 need to be included for evaluating leaf rust resistant. According to Dutta et al. (2006), 121R, 63-1 is one of the most frequent and virulent leaf rust pathotypes.
Control measures
There are different ways to control rust epidemics. The current available chemicals may be effective, but are difficult to use in developing countries due to cost, lack of timeliness and distribution problems. Use of resistant varieties is the best way to control rust epidemics as introduction of new rusts resistance genes reduces inoculums drastically. Resistance to rust can be broadly divided into different classes.
(1) Resistance to rust controlled by major gene
(i) Highly effective but lack durability. Many countries lack flexibility to undertake rapid cultivar changes following resistance failure.
(ii) Genes such as Lr9 and Lr19 have become ineffective in some areas.
(iii) Genes such as Lr21, Lr22a and a few others remain widely effective, but are likely to become ineffective if widely deployed.
(iv) A major gene break-down may result in a rapid change in pathotype profile.
(2) Resistance governed by slow rusting genes
(i) Slow rusting has been identified in a wide range of germplasm.
(ii) Associated with high infection types at low frequency and non-hypersensitivity.
(iii) Oligogenic in nature.
(iv) Genes governing slow rusting are additive in nature.
(v) Best way to select a germplasm line for slow rusting is terminal disease rating prior to maturity.?Susceptibility leads to early leaf death.
(3) Genetic diversity in resistance can also provides kind of insurance against expected yield losses.
(4) Multilines and cultivar mixtures.
a. May have a role in regions where rust development is late and rapid.
b. A mixed line enforces host genetic diversity independently of area.
In the case of leaf rust breeders’ choice is to improve the wheat for general resistance (slow rusting) based on historically proven stable genes. This nonspecific resistance can be further strengthening by accumulating several minor genes and then combining them with different specific genes to provide a certain degree of additional genetic diversity. The additional advantages of slow leaf rusting genes are their effectiveness against other diseases such as stripe rust and powdery mildew. For achieving effective durable resistance in germplasm, highly diverse sources of resistance for rusts and other diseases are being intentionally used in the crossing program. The major sources are germplasm from national programs, germplasm received from other gene banks, advanced lines and CIMMYT’s wide crossing program.
The best example of leaf rust resistance stabilization by using genes derived from many sources is Brazilian cultivar Frontana (Singh and Rajaram, 1992). No major epidemic has been observed for almost 20 years in Brazil. Four partial resistance genes, including Lr34, give a slow rusting response and have been the reason for no epidemics in the developing world during the past 15 years. Most of the cultivars in these countries carry these minor genes. About 60 percent of the CIMMYT germplasm carry one to four of these partial resistance genes. Lr34 is linked to Yr18 as well as to a morphological marker leaf tip necrosis, which makes the gene particularly attractive for breeders (Singh, 1992). Breeders continue to look for new sources of partial resistance for developing new lines with improved resistance.
A study has shown that most of the lines having durable resistance to leaf rust have at least 10-12 slow rusting genes. In most of the cases either Lr34 or Lr46 or both are present in addition to other resistant genes (Singh et al., 1998). These genes provide durable and non-specific adult plant resistance to the plants than that of race specific genes (Singh et al., 1995). Sometimes these resistant genes have pleiotropic effect or combined with other resistant genes and provide multiple diseases resistant to the plants. For example, Lr34 is linked with Yr18, Lr46 with Yr29 which are also slow rusting genes for strip rust. Hence, there is need of in depth characterization of slow rusting mechanism to achieve durable leaf rust resistance. In order to obtain cultivars with good level of protection under high disease pressure several such slow rusting complexes need to be combined in a cultivar. Wheat breeders can use slow rusting genes as an additional weapon to fight against leaf rust in comparison to race specific gene. The availability of molecular markers greatly facilitated the molecular breeders to introgress the desirable genes into the genotypes efficiently in lesser time. Any addition of a minor gene in the background of major leaf rust genes always adds some value (Singh et al., 1995) (Table 2).
In wheat, slow rust mechanism is recognized by low infection type. However, due to their minor effect in resistance mechanism it is difficult to establish their presence in a cultivar unless some tightly linked markers are not available. The genetic nature of this type of resistant is usually complex due to additive interaction of few to several genes having minor to intermediate effect.
Durable resistance is resistance that is long lasting and a pathogen cannot easily overcome it, possibly because of a lack of genetic flexibility and/or because the required adaptation(s). The best example of durable resistance is non-host resistance, which for most non-host plant-pathogen combinations, has lasted for recorded history. The primary focus of any disease resistance breeding program is to work on achieving durable resistance (Johnson, 1988), which often involves race-nonspecific, slow-rusting (Caldwell, 1968) mechanisms and can be identified easily at adult-plant stage. Race-specific resistance, provides low infection type at seedling stage and most of the time short lived in nature due to emergence of new virulence in the pathogen population in response to host selectivity in the field (Kilpatrick, 1975). The basic requirements for breeding for the development of race non specific durable resistant cultivars are as follows.
(i) A relevant and reliable rust nursery is prerequisite.
(ii) Appropriate host genotype is required for infection rows to ensure the presence of relevant pathotypes.
(iii) Crossing strategies should address the desired type of resistance and the major genes that are likely to be effective.
(iv) Selection must focus on the desired type and level of resistance.
(v) The international centres have a role in advising relevant host:?pathogen knowledge to assist NARs in local selection programs.
(vi) Molecular and other markers should be employed where possible.
The concept of general (race-nonspecific) resistance was advocated for breeding stem rust resistance in wheat by Borlaug (1972), leaf rust resistance by Caldwell (1968) and yellow rust resistance by Johnson (1988). The wide-scale application of such a concept in breeding for leaf rust resistance, commonly known as slow rusting, has been intensively used in CIMMYT in bread wheat improvement for almost 30 years after green revolution (Marasas et al., 2002). Due to advancement in science, it is now better understood the genetic basis of durable resistance to rust diseases, and this knowledge is being applied in rust breeding program. However, breeding for durable resistance based on minor additive genes has been challenging and often slow, due to following reasons:
1) Lacking genotypes having sufficient number of minor genes.
2) A source genotype may be poorly adapted.
3) There may be confounding effects from the segregation of both major and minor genes in the population.
4) Crossing and selection schemes and population sizes are more suitable for selecting major genes than minor genes. Lacking reliable molecular markers for several minor genes and their use in Marker assisted selection (MAS). However, such germplasm carrying combinations of minor genes are very useful in transferring these genes to adapted local cultivars.
Molecular markers for slow rusting, minor genes
Availability for tightly linked DNA markers in the future can be useful in maintaining and diversifying the combinations of additive slow rusting resistance genes in the wheat germplasm and cultivars. In the last 10-20 years, tremendous progress has been made in the field of molecular markers that have contributed significantly towards identification of resistance genes to various leaf rust races (McIntosh et al., 2011). A number of SSR markers are known for their linkage with various leaf rust resistance genes which are helping breeders to select the desirable genotypes through marker assisted selection (Gultyaeva et al., 2009). Over 100 genes of resistance to rust fungi: Puccinia recondita f. sp. tritici, (67 Lr – leaf rust genes), P. striiformis (48 Yr – yellow rust genes) and P. graminis f. sp. tritici (49 Sr – stripe rust genes) have been identified in wheat (Triticum aestivum L.) and its wild relatives. However, only 21 Lr, 10 Sr and 10 Yr genes have been mapped so far that can be used in marker assisted selection (MAS) (Table 3).
The actual use of molecular markers in breeding is to characterize and select the parents to be used in specific crosses as the field screening is very reliable and cost-effective. However, if such genes need to be incorporated in adapted cultivar that contains an effective race-specific resistance gene, then markers are the only option and will be used despite the more cost.
Advances in identifying molecular markers for slow rusting, minor genes
Leaf rust resistance genes, namely Lr1, Lr9, Lr10, Lr13, Lr19, Lr23, Lr24, Lr25, Lr27, Lr28, Lr29, Lr31, Lr34, Lr35, Lr37 and Lr47 have been mapped on chromosomes, using different molecular markers. The first molecular STS marker was developed by Schachermayr et al. (1994) for the Lr9 gene derived from Aegilops umbellulata.
William et al. (2003a) used AFLP markers to map Lr46 on the distal end of 1BL. The authors also found that Lr46 was tightly linked or pleiotropic to a stripe rust resistance gene designated Yr29. The tight linkage of a slow rusting gene to a stripe rust resistance gene was also found for the pair Lr34/Yr18. Suenaga et al. (2003) determined that the microsatellite locus Xwmc44 is located 5.6cM proximal to the putative QTL for Lr46. Sivasamy et al. (2014) revealed the phenotypic and molecular confirmation of durable adult plant leaf rust resistance (APR) genes Lr34+, Lr46+ and Lr67+ linked to leaf tip necrosis (LTN) in select registered Indian wheat (T. aestivum) genetic stocks.
Significant advances have been made in characterizing genes that confer resistance to biotic stresses in several crops, thanks to the use of molecular markers. Although, molecular markers have been successfully applied to characterize race-specific resistance genes, finding markers closely associated with durable resistance genes is comparatively more challenging. Because several minor genes are needed in a cultivar to achieve adequate protection under high disease pressure, the mapping populations generated often segregate for a number of these genes as quantitative trait loci (QTLs). Usually populations of doubled haploid (DH) or nearly homozygous recombinant inbred lines (RILs) are used for mapping in wheat. Initial efforts aimed at identifying QTLs for durable rust resistance were documented by Nelson et al. (1997), involving linkage mapping using the ITMI reference population, and by William et al. (1997) using bulked segregant analysis (Michelmore et al., 1991) in a CIMMYT spring wheat cross. PCR-based markers such as microsatellites (SSRs) and amplified fragment length polymorphisms (AFLPs) has made it possible to have more success in characterizing adult resistance genes for a number of diseases including leaf rust and yellow rust. CIMMYT has used full linkage mapping as well as bulked segregant analysis with multiple populations to determine the number and genomic locations of genes conferring durable resistance and to identify molecular markers with strong linkages with resistance alleles.
Known slow rusting resistance genes for leaf rust and yellow rust include Lr34/Yr18, Lr46/Yr29, and Yr30. Nelson et al. (1997) using RFLPs first associated molecular markers with Lr34/Yr18 on the ITMI reference mapping population. The ITMI population exhibited considerable polymorphism as it is derived from the cross of the spring wheat cultivar ‘Opata 85’ and synthetic hexaploid wheat. However, these markers did not show polymorphism in several other mapping populations involving spring wheat cultivars at CIMMYT. The availability of a large number of SSR markers with adequate coverage of the wheat genome and other marker systems such as AFLPs has helped to associate markers with genes such as Lr34/Yr18 (Suenaga et al., 2003). These have subsequently been validated in other populations (William et al., 2003b; Schnubusch et al., 2003).
Some examples of slow rusting genes are
(i) Lr34. 7DS. Association with Yr18, Bdv1, Pm? Many genotypes. Markers available.
(ii) Lr46. 1BL. Pavon 76. Markers available.
(iii) QLr.sfr.1BS. Forno.
(iv) QLr.ocu-2B. CI 13227
(v) QLr.ocu-7BL. CI 13227
Future prospects of slow rusting in rust control
Due to drastic change in climate condition, new leaf rust races are emerging very fast which has made most of the existing varieties susceptible to leaf rust. Resistant controlled by major genes in short linked and not in durable nature hence we have to depend upon the alternative tools like slow rusting to achieve durable resistant in near future to feed the growing population and sustainability in production. The optimal use of markers linked with slow rusting genes and the genetic engineering might be of great use in any development of leaf rust resistant cultivars. Use of tightly linked morphological markers is of great use in rust breeding. The association of adult plant resistance to leaf rust (Puccinia triticina) in some Indian bread wheat (Triticum aestivum) accessions with the morphological marker leaf tip necrosis has been established by Kumar et al. (2014).
So development of Bt genes in maize and cotton has given new dimensions to this field and same thing can be done for leaf rust in wheat. Some other known experiment are coat protein (Cp) gene of rice a strip virus introduced in to rice (Hayakawa et al., 1992) and few thaumatin like protein gene introduce in to wheat (Chen et al., 1999) have provided enhanced level of resistant against plant pathogens. So far genetically modified wheat has not been grown commercially. However, genetically modified crops are continuously kept on expanding day by day. Hence it is required clone resistant genes conferring durable resistant and their use in developing disease resistant transgenic wheat (Horvath et al., 2003). Thus technology can improve the effectiveness of individual slow rusting genes and can exploit their effect by combining them with other resistant genes. However, transformations techniques need to be further improve. The current use of biotechnology in this regards can be done is the form of molecular markers linked with various slow rusting genes/minor genes. This will definitely improve over ability to select gene combinations required to enhance the durable of resistance.
However, still their gap has molecular markers are available only for 21 leaf rust resistant genes while, 67 leaf rust resistant genes have been reported so far (Bossolini et al., 2006). There is need to initiate work to identify marker for rest of the resistance genes so that they can also be utilized in developing resistant cultivars following marker assisted selection approaches.
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