Full Text: 1 Introduction Yellow or stripe rust of wheat, caused by Puccinia striiformis Westend. f. sp. tritici Erikss. (Pst) is one of the most economically important fungal disease of global spread (Chen et al., 2014). In India, stripe rust is a major biotic stress observed in cooler areas of the country and causing considerable yield loss during epidemic years (Hodson, 2011; Sharma & Saharan, 2011; Prashar et al., 2015; Khan et al., 2016). Under conducive weather conditions, stripe rust can cause yield loss up to 100%, but usually damage remains in the range of 10-70% depending upon crop stage, disease severity, and susceptibility of cultivar (Gangwar et al., 2019). Yield losses in wheat due to stripe rust are usually ascribed to reduction of kernel numbers per spike, shriveled grains, and reduced test weight due to decreased photosynthesis (Gangwar et al., 2019). Emergence of new pathotypes and subsequent breakdown of resistance is a major challenge in stripe rust management. In 1996, pathotype 46S119 which was virulent on widely used Pst resistance gene i. e. Yr9 was reported, followed by 78S84 having combined virulence on Yr9 and Yr27 was identified in 2001 (Prashar et al., 2007; Bhardwaj et al., 2019). Both pathotypes have predominantly distributed and responsible for most of the stripe rust epidemics in major-wheat growing regions of India (Prashar et al., 2015). Besides, 3 new Pst pathotypes viz., 110S119, 238S119 and 110S84 with additional virulence on Riebesel47/51, SuwonXOmar, Yr11, Yr14, and Yr24 were reported during 2013-14 (Gangwar et al., 2016; Gangwar et al., 2019). Thus, search for resistance to new Pst pathotypes in addition to existing virulent pathotypes is need of the hour.        Although, multiple applications of fungicides can save the crop from stripe rust, but development and deployment of resistant varieties is the most efficient and environmentally sustainable means of reducing the loss due to stripe rust (Chen, 2005). In India, as an emergent tool for managing wheat rusts under high disease incidence, the fungicides propiconazole 25% EC (tilt), tebuconazole 25% EC (folicur) and triadimefon 25% EC (Bayleton) are usually recommended at the rate of 0.1 percent (Bhardwaj et al., 2016). But use of resistant cultivars has remained preferable, economically viable, ecologically safe and effective method for managing wheat rusts epidemics. A rigorous screening of germplasm against the available population of rust pathogens leads to the identification of rust resistance sources. A dynamic rust resistance breeding programme should always think ahead of the pathogen through identification of sources of resistance to any new pathotype in addition to the prevalent ones. Current study related to identification of seedling and adult-plant resistance sources among the wheat germplasm collected from Northern hills are reported in the present communication. 2 Materials and Methods 2.1 Wheat genotypes The plant materials consisted of 464 germplasm accessions of cultivated bread wheat collected from Northern hills, India. 2.2 Stripe rust pathogen Four highly virulent pathotypes (pts.) of wheat stripe rust (Puccinia striformis f. sp. Tritici) namely, 78S84, 46S119, 110S119 and 238S119 were used in the present study. 2.3 Experimental setup The schematic diagram of experimental layout and flow of germplasm in different years and locations is presented in Figure 1. 2.4 Evaluation of wheat germplasm to stripe rust under natural field conditions Screening of 464 wheat germplasm to stripe rust was carried out at two locations, Hawalbagh (29⁰36’N, 79⁰40’E; 1250 m asl), Uttarakhand and Dalang Maidan (32⁰21’N, 77⁰14’E; 2347 m asl), Himachal Pradesh, India. These locations are considered as hot spots for stripe rust development. At these locations, stripe rust appears every year in severe form on susceptible varieties. At Hawalbagh, the wheat germplasm was evaluated in three regular cropping seasons (November to May) during 2013-2016. Whereas, at Dalang Maidan, the germplasm were evaluated in off-seasons (May to August) for two years (2014 and 2015). At both locations, the experiment was laid out in an augmented design consisting of 16 blocks. In each block, susceptible check (Agra Local) was repeated after every 29 test genotypes. Seeds of each genotype were sown by dibbling in two rows with 30 cm inter-row spacing and100 cm row length. The stripe rust infector’s, a homogeneous mixture of highly susceptible wheat lines such as NI5439, Avocet and Agra Local were used as stripe rust spreader in the field. Infector rows were settled surround the experimental plot to increase inocula load. The infection types (IT) and disease severity data were recorded using modified Cobb scale (Peterson et al., 1948) from boot to milk stages. The infection types were recorded as 0= no visible infection (immune); R= necrotic areas with or without uredia (resistant); MR= necrotic areas with small uredia (moderately resistant); MS=medium size uredia with no necrosis but some chlorosis (moderately susceptible); S= large sized uredia with no necrosis and chlorosis (susceptible); X= variable sized uredia with necrosis or chlorosis and fully susceptible (intermediate). Stripe rust severity was assessed as percentage of leaf area infected using modified Cobb scale. The response value of 0.0, 0.2, 0.4, 0.8 and 1.0 were assigned for 0, R, MR, MS and S, respectively. The coefficient of infection (CI) of each genotype was calculated by multiplying response value with the severity of infection (Pathan & Park, 2006). The average coefficient of infection (ACI) of a germplasm was determined by dividing the sum of CI values with total number of seasons (Stubbs et al., 1986). In the current study, sum of CI values was divided by five to derive ACI value of each genotype. Based on CI and ACI values, the germplasm was classified into different categories of rust resistance. Genotypes with CI/ACI values of 0, <5, <20, 21-40, 41-60 and >60 were considered as immune, highly resistant, high partial resistant, medium partial resistant, low partial resistant and susceptible, respectively (Sajid-Ali et al., 2009; Raghu et al., 2018). 2.5 Seedling resistance test (SRT) Highly field resistant genotypes with CI/ACI values of 0-5 (n=22) were evaluated at seedling-stage against four Pst pathotypes namely, 78S84, 46S119, 110S119 and 238S119 under glasshouse conditions. SRT was conducted at ICAR-Indian Institute of Wheat and Barley Research (IIWBR), Shimla, India, during 2016-17 cropping season. Aluminum trays containing sterilized mixture of fine loam and farmyard manure (3:1) were used for raising seedlings of genotypes. Each tray was sufficient to accommodate 18 genotypes along with a susceptible check. For each genotype, 5-6 seeds were sown in hills. One-week old seedlings were inoculated 10 mg spores of an individual Pst pathotype suspended in 1 ml light grade mineral oil (Soltrol 170; Chevron Phillips Chemicals Asia Pvt. Ltd., Singapore). Inoculated seedlings were incubated in dew chambers at 16±2°C for 48 hours. Subsequently, seedlings were transferred to glasshouse benches and incubated at 18±2°C with 60-75% relative humidity, illuminated at approximately 15,000 lux for 12 hours. The infection types (IT) were recorded at 16 days post-inoculation using standard procedure (Nayar et al., 1997). 2.6 Adult-plant stage resistance (APSR) Partial field resistant genotypes along with highly field resistant genotypes with CI/ACI values ranges between 0-60 (n=371), were tested for adult-plant stage resistance to stripe rust at Hawalbagh, ICAR-VPKAS, Almora, Uttarakhand, India, during 2016-17. The experiment was laid out in an augmented design. In each block, susceptible check Agra local was repeated after every 53 test genotypes. Seeds were sown in two-meter rows with 30-cm row to row distance during second fortnight of November 2016. Single row of infector was repeatedly planted after every entry. Additionally, the field was surrounded by infector rows to increase inocula load. From late January to early March, stripe rust epidemic was created artificially by inoculating infectors with a mixture of four Pst pathotypes. The Infector rows were first syringe inoculated with the mixed inocula of pathotypes followed by repeated sprays. Irrigations were carried out as required to maintain sufficient humidity for better rust infection. Disease severity and infection types were recorded five times at 15 days interval from boot to milk stage. The procedure for recording infection types and disease severity, and calculating coefficient of infection (CI) was same as described in natural field test. Area under the disease progress curve (AUDPC) was calculated using CI values (Gyawali et al., 2018). 2.7 Statistical analysis The CI values of wheat genotypes were subjected to ANOVA using PROC GLM of SAS (SAS Institute, 1988) statistical software package. The AUDPC of wheat genotypes was differentiated by Fisher’s least significant difference (LSD) (p=0.05) based on standard error of mean difference of repeated check Agral Local. The cut-off of resistance and susceptible genotype was determined by significant t test of susceptible checks and test genotypes at p<0.05(AUDPC=8.2) and LSD_0.05 (AUDPC=375.67). Therefore, genotypes with rust severity lower than the cut-off AUDPC 383.9 were considered resistance. 3 Results 3.1 Stripe rust resistance under natural field conditions The ANOVA for terminal stripe rust severity of 464 wheat germplasm evaluated under natural field conditions is given in the Table 1. Whereas, grouping of genotypes into different resistant and susceptible categories based on CI and ACI values are summarized in Table 2. The results of current study indicated that 22 (4.8%), 349 (75.2%) and 93 (20%) genotypes were highly resistant, partially resistant and susceptible across locations and seasons, respectively (Table 2). Of these 349 field partially resistant genotypes, 160 (34.5%), 104 (22.4%) and 85 (18.3%) genotypes showed high, medium and low degree of field partial resistance, respectively (Table 3). Location and season wise breakup of stripe rust resistance among 464 wheat germplasm under natural field conditions is shown in Figure 2. The susceptible check Agra Local showed stripe rust of 80S-100S with the ACI values of 96.0 under natural field conditions. Besides, the rust severity range of 80S-100S was observed in the infector’s rows indicating sufficient Pst inocula load in the test locations. 3.2 Seedling resistance test (SRT) The results of SRT of 22 highly field resistant genotypes are presented in Table 3. Out of 22 resistant genotypes tested, 14 genotypes namely, IC0138525, IC0469441, IC0469503, VHC-6161, IC0469464, IC0469440, VHC-6211, VHC-6162, VHC(BD)-79,  VHC-6202, IC0316103, VHC-6265, IC0281542 and VHC-6209 showed seedling resistance against all 4 Pst pathotypes (46S119, 78S84, 110S119 and 238S119). Further, two genotypes, IC0138518 and IC0138524 showed resistance at seedling stage against two Pst pathotypes, 46S119 and 78S84, and susceptible to 110S119 and 238S119. Another two genotypes, IC0138520 and VHC-6290 showed resistance to 78S84 and susceptible to 46S119, 238S119 and 110S119. Remaining 4 genotypes namely, IC0469478, VHC-6294, VHC-6372 and IC0138516 found susceptible to all 4 Pst pathotypes. 3.3 Adult-plant stage resistance The adult-plant stage resistance test of 371 genotypes (genotypes showed highly and partial resistance under natural field conditions) along with susceptible check Agra local is summarized in the figure 3. The cut-off AUDPC value 383.9 was determined by t test of susceptible checks and test genotypes at 0.05 probability [(AUDPC = 8.2 (p < .05) plus LSD_0.05 which was AUDPC = 375.67]. The genotypes with lesser than AUDPC 375.67 were considered resistant to stripe rust at adult-plant stage. Out of 371 genotypes tested, 36 showed APSR (Region-1 of figure 3)  and remaining 336 genotypes along with  susceptible check Agra local were found susceptible to APSR (Region-2 of figure 3). These 36 genotypes with APSR are also included 18 genotypes with seedling resistance against 4 Pst pathotypes indicating all stage resistance (ASR) in those genotypes (Table 3). Whereas, remaining 18 genotypes showed only APSR to Pst. However, the susceptible check Agra Local with corresponding AUDPC value of 3064.0 was placed in the region-2 indicating high susceptibility to stripe rust (Figure 3). 4 Discussion Shift in the pathotypic virulence and emergence of new Pst races are the major reasons for frequent outbreak of stripe rust in wheat (Prashar et al., 2015; Gangwar et al., 2016). This leads to frequent breakdown of known resistance and posing major challenges to rust resistance breeding programme (Prashar et al., 2007). Thus, it necessitates the identification and introgression of new sources of stripe rust resistance (Joshi et al., 2011). Kumar et al. (2016) evaluated entire collections of cultivated wheat germplasm (19,460) for rusts resistance and identified 45 new sources of resistance. Raghu et al. (2018) reported a seedling and adult plant resistance to stripe rust in Uttarakhand wheat landraces. It suggests that wheat germplasm from NW hills region possessing stripe rust resistance. Current study identified 22 genotypes (4.8%) showing high degree of stripe rust resistance over 5 seasons at two locations under natural field conditions (Table 2). In which five genotypes were found immune (ACI=0) and seventeen highly resistant (ACIPst pathotypes at seedling stage (Pal et al., 2018). Besides, 4 genotypes (IC0138518, IC0138524, IC0138520 and VHC-6290) were showed susceptibility to 2 new pathotypes (110S119 and 238S119) but found resistant to earlier reported pathotypes 46S119 and 78S84 at seedling stage (Table 3). This is a clear example of genotypes carrying both seedling resistance and adult plant resistance (APR). Badoni et al. (2017) reported genotypes carrying APR genes Lr34/Yr18/pm38 in combination with seedling resistance genes Yr5, Yr10 and Yr15 in wheat. Based on pathological studies, several studies reported similar kind of findings previously (Maccaferri et al., 2015;  Bulli et al., 2016; Kumar et al., 2016; Gyawali et al., 2018; Verma et al., 2018; Raghu et al., 2018). The seedling resistance expressed early in seedling-stage and it will remain effective at all post-seedling stages of plants (Chen, 2005; Lagudah, 2010). Often, it is governed by major genes result in a hypersensitive response associated with high levels of resistance (Maccaferri et al., 2015). So far, 76 officially named stripe rust resistant genes (Yr1 to Yr76) and 42 with temporary designations have been reported in wheat (McIntosh et al., 2016). Most of the reported Yr genes confer all-stage resistance (seedling resistance). In the current study, 18 genotypes showed seedling resistance, were also tested for adult-plant stage resistance against mixture of 4 Pst pathotypes (Table 3 and 4). Further, SRT and APSR tests in the current study revealed that, genotype IC0469478 possess only adult-plant stage resistance to stripe rusts (Table 3). In addition, 17 more new sources of adult-plant stage resistance to stripe rust were identified in this study (IC0279883, IC0281544, IC0281570, IC0282865, IC0282866, IC0310124, IC0310127, IC0313151, IC0316085, IC0316086, IC0316088, IC0316093, IC0356468, IC0393111, IC0398292 IC0398297 and IC0398310). Earlier, several new sources of adult-plant stage resistance to stripe rust were reported both in barley (Gyawali et al., 2018; Verma et al., 2018) and in wheat (Ali et al., 2008; Maccaferri et al., 2015;  Saleem et al., 2015; Bulli et al., 2016; Badoni et al., 2017; Kumar et al., 2016; Raghu et al., 2018). Unlike seedling resistance, adult-plant resistance (APR) typically expressed at adult-plant stages (post-seedling), is characterized by various degree of resistance (quantitative or partial resistance) and often shows race non-specific resistance (Lagudah, 2011). Although, varieties with seedling resistance are more attractive to the farmers, but quickly become susceptible due to emergence of new virulent pathotypes (Kolmer et al., 2009; Hubbard et al., 2015). Therefore, emphasis must be given for deployment of both seedling and adult plant resistance as a long term disease management strategies for stripe rust (Hulbert & Pumphrey, 2014). In the current study, we identified 18 novel sources of all-stage resistance and 18 adult-plant stage resistance among 464 germplasm of cultivated wheat of Northern hills, India. The present findings would prove to be a useful source for developing potential stripe rust resistant varieties. These genotypes could serve as potent donor for creating new utilizable variability in wheat against stripe rust under wheat improvement programme of India. Acknowledgement The authors convey their gratitude to different scientists and technical staff involved in wheat exploration trips and germplasm maintenance, the technical staffs of wheat program, ICAR-VPKAS and to the ICAR, New Delhi for providing financial support. Conflict of Interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise.