Seedling and Adult Plant Resistance in the Ethiopian Bread Wheat Landraces to Stripe Rust Disease

High yielding farmers’ bread wheat cultivars are threatened by emerging race(s) of stripe (yellow) rust caused by Puccinia striiformis f.sp. tritici (Pst) in the highlands of Ethiopia. In depletion of rust resistance in commercial cultivars, researchers often look for new sources from close relatives and landraces. The objective of this study was to determine stripe rust resistance in selected Ethiopian bread wheat landraces obtained from the Ethiopian Institute of Biodiversity (IBCE). In 2017, a total of 152 accessions were exposed to the prevailing stripe rust races in hot spot areas (Kulumsa and Meraro) in Arsi zone of Oromia region. In the second year (2018), only promising landraces (57) were evaluated both at seedling and adult plant growth stages. The seedling test was conducted in the greenhouse at Kulumsa research center using three (PstS2 (v32), (PstS11) and (PstS11 v25) Pst races. In field evaluations, terminal severity (TRS), coefficient of infection (CI), area under disease progress curve (AUDPC), disease progress rate (DPR) and head infection (HI) were considred. High disease pressure was noted with 100% severity on susceptible entries at both locations and seasons. Highly significant (P<0.001) differences were noted among the landraces for all disease parameters indicated above. Of the 152 landraces, 57(38%) exhibited lower or equal disease reaction compared to the resistant check(Enkoy) across locations. Overall, 18 accessions showed resistance to the prevailing Pst races both at seedling stage and field conditions whereas14 exhibited susceptible /intermediate reaction at seedling stage, but had lower disease reaction under field conditions. This study has identified potential sources of overall and adult plant resistance in the Ethiopian bread wheat landraces to the prevailing Pst races. The authors recommend further studies to determine the diversity and/or novelity of resistance genes in selected accessions. Future wheat improvement should focus on utilization of these genetic resources to minimize the re-current outbreak of rust diseases.


Introduction
Wheat is a traditional crop cultivated by small scale farmers under rainfed conditions in Ethiopia. The country is the largest wheat producers in Sub-Saharan Africa (SSA). The crop ranks fourth in area coverage next to tef, maize and sorghum, respectively (CSA, 2018). The area under wheat production is estimated to be about 1.7 million hectares of land with average productivity of 2.7 tons/ha. Wheat production is constrained by several biotic, abiotic and socio-economic factors in the country. The Ethiopian highlands are suitable for the wheat plant as well as for perpetuations wheat rust diseases (Hulluka et al., 1991). Stripe (yellow) rust caused by P. striiformisf. sp. tritici. Westend (Pst) is the most threatening wheat disease in the highlands of Ethiopia (Badebo et al., 2008). The disease was first reported in the early 1940's, but it gained importance with the commercialization of wheat in the early 1980s (Gebremariam, 1991). Recurrent stripe rust epidemics has occurred since then; however, the 2010 epidemics was the most damaging. Most of the commercial bread wheat cultivars, including Kubsa and Galama scummbed to the new Yr27+ virulent race and caused 70 to 100% yield loss in major wheat producing areas of the country (Abeyoet al., 2013).
including hand planting and harvesting (Feuillet et al., 2008). The main reasons for farmers to maintain these historic wheat landraces could be due to their specific adaptability and preferred quality for home use. Wheat landraces contribute important traits including disease resistance (Cavanagh et al., 2013;Lopes et al., 2015), tolerance to abiotic stresses (Olmstead and Rhode, 2002), and protein content and gluten quality (Zhanget al., 2012). Wheat landraces have been successfully used by wheat breeders to improve agronomic traits, particularly disease and pest resistance (Nevo and Payne, 1987).
In Ethiopia, Vavilov identified six wheat "species" in his early expedition to Ethiopia in the late 1920s, but mainly bread wheat (Triticum aestivum) and durum wheat (T.turgidum var. durum) are cultivated nowadays. Durum wheat is an indigenious crop while bread wheat is considred to be of recent introductions to the country (Gebre Mariam, 1991). However, the recent archaeo-botanical studies identified bread wheat at Aksum, northern Ethiopia dating to the mid-sixth to early seventh centuries (Boardman, 1999). This suggest that bread wheat might have a longer history in Ethiopia than was previously thought. Most of the wheat landraces have been replaced by high yielding semi dwarf bread wheat varieties, but still grown by farmers mainly in the northern part of Ethiopia. A large number of bread wheat collections are preserved in the IBCE (Demissie and Habtemariam, 1991). However, bread wheat landraces have not been well exploited in modern wheat breeding in Ethiopia (Broers and De Haan, 1994). So far, only limited bread wheat landraces have been characterized for their resistance to rust diseases (Badebo and Tessema, 2012;Muleta et al., 2017).
In this study a set of Ethiopian bread wheat landraces from IBCE have been characterized for their resistance to the prevailing Pst races under field and greenhouse conditions.

Description of the Study Areas
The field study was conducted at Kulumsa and Meraro research stations in Arsi Zone of Oromia Region, Ethiopia. The greenhouse experiment was conducted at Kulumsa Agricultural Research Center. Meraro and Kulumsa are known to be hot spot areas for wheat stripe rust, although the disease pressure increases with altitude. Kulumsa represents mid highlands (2200 m.a.s.l.) and located at 39 ᴼ 09'11''E l and 08 ᴼ 01'10''N. It has average maximum 22.8 ᴼ C and minimum 10.5 ᴼC temperatures and receives 832 mm rainfall annually. Meraro represents extreme highlands (2960 m.a.s.l.) and located at 39 ᴼ 14'56''E and 07 ᴼ 24' 27''N. Meraro receives 1196 mm annual rainfall with maximum and minimum temperatures of 18.1 ᴼ C and 5.7 ᴼ C, respectively.

Planting Materials
A total of 152 bread wheat landrace accessions plus 3 checks were used in this study. The accessions and checks were obtained from the institute of biodiversity of Ethiopia (IBCE) and the wheat breeding program at Kulumsa research center, respectively. In 2017, all of the accessions were tested under field conditions whereas only selected entries were exposed to the prevailing races of Pst in the field and greenhouse during 2018.

Field Experimental Design and Testing Procedures
The experiment was laid out in augmented design where the three check varieties; Digelu (susceptible), Pavon-76 (moderately resistant) and Enkoy (resistant)were replicated in each block. Each plot consisted of 2 rows of 1 m length with 20 cm between rows. To ensure uniform spread of inoculum and sufficient disease development, infector plants consisted of mixtures of different susceptible bread wheat varieties (Morocco, Digelu and Kubsa) bordered the plots in all direction. Fertilizer application and other agronomic practices were applied according to the recommendations for each location.

Field Disease Assessments
Different epidemiological parameters which include terminal rust severity (TRS), coefficient of infection (CI), area under disease progress curve (AUDPC) and disease progress rate (DPR) were used to determine stripe rust resistance in the wheat landarces.

Disease Severity
Disease severity notes were taken five times on plot bases starting from the onset of rust within 10 days intervals. Stripe rust severity was estimated visually as a proportion leaf area affected by stripe rust using the modified Cobb'sscale (Roelfs et al., 1992) and the host plant response (infection type) was noted according to Peterson et al. (1948). The CI was calculated by multiplying the level of disease severity and the constant value of infection type. The constant values for infection types were used based on; R = 0.2, MR = 0.4, M= 0.6, MS = 0.8, S = 1 (Stubbs et al., 1986). Head infection was noted using 0-5 scale and then converted to percentages for analysis:0 = no infection, 1 = 20%, 3 = 60%, 4 = 80% and 5 = 100% severity.

Area under Disease Progress Curve (AUDPC)
AUDPC is an indicator of disease expression over time (Van der plank 1963), and it was calculated for each experimental unit based on the formula by Wilcoxson et al. (1975): AUDPC 0.5 xi x t ti , Where, xi = the average coefficient of infection of i ͭ ͪ record, x i+1 = the average coefficient of infection of i+1 th record and t i+1 -t i = Number of days between the i th record and i+1 th record, and n = number of observations.

Disease Progress Rate
Disease progress rate was estimated from the logistic model ln (Y/(1-Y)) (Van der Plank, 1963) and gompertz model: -ln [-ln(Y)] (Berger, 1981). Where y = the percent of severity divided by 100; t = time measured in days.
The model with higher coefficients of determination (R 2 ) was considered.In this study, Gompertz model was used for all assessment dates because of the higher values of coefficient of determination (R 2 ) compared to the logistic model.

Field Data Analysis
Data on different epidemiological parameters were generated for each test material. The data have been transformed using √ 0.5 before analysis. A standard analysis of variance was conducted to identify significance differences among the wheat landarces for each disease parameter. Correlation analysis was done among disease parameters and TKW using Minitab Software (2017). The data were analyzed using SPAD and SPSS software (SPAD 2002;SPSS 2005).

Seedling Test Procedures
A total of 57 accessions were selected for seedling resistance tests based on their field performances in 2017. The experiment was conducted according to standard procedures (Badebo et al., 2008;Hovmøller et al., 2017) in the greenhouse at Kulumsa research center in 2018. Five to six seeds of each entry plus three checks were grown in 7x7x6 cm plastic pots filled with compost, soil and sand at a ratio of 1:2:1 (v/v/v) under spore-free greenhouse compartment under supplemental light. The first l of seven to eight days old seedlings were inoculated with spores of three (PstS2 (v32), (PstS11) and (PstS11 v25) individual Pst isolates suspended in light weight mineral oil (Soltrol 170) using atomizer. Inoculation was carried in an enclosed cage that was rinsed with water subsequently to avoid spore contamination. Inoculated seedlings were allowed to dry for 20 minutes and then incubated in a dew chamber for about 24 hrs at 9-10 ᴼC and 100% relative humidity. Seedlings were kept in semi open plastic cubicles in a greenhouse compartment at 18-22 ᴼC and 70 to 80% relative humidity. The seedlings were supplemented with 12 hrs light using florescent lamps. The experiment was repeated based on the infection type on susceptable variety (Morocco). Seedlings were evaluated 16-20 days after inoculation using a 0-9 scale (McNeal et al., 1971). Entries with ITs 0-6 were considred as resistant and 7-9 susceptible.

Field Reaction of Landraces to Stripe Rust
Stripe rust disease pressure was high in the study areas during 2017 and 2018. The disease severity went up to 100% on susceptible entries accross locations and seasons. There were significant (p<0.001) differences among the test entries for terminal severity (TS), coefficent of infection (CI), area under disease progress curve (AUDPC), infection rate (DPR) and head infection (HI) as shown in Table 1. The landraces exhibited various reaction to stripe rust on wheat leaves and heads (spikes). The disease pressure was more at Meraro (2900 masl) than at Kulumsa (2200 masl). Key: DF= digree of freedom, CI= coefficient of infection, TRS=terminal severity, AUDPC=area under disease progress curve, DPR= disease progress rate, HI= head (spike) infection

Terminal Rust Severity on Wheat Leaves (TRS)
In 2017, the mean severity scores on leaves of resistant, moderately resistant and susceptible checks were, 9.6, 40 and 78% at Kulumsa and 20%, 59% and 94% at Meraro, respectively whereas the mean severity on spikes for the respective checks were 18%, 82% and 62% at Meraro (Table 3). The frequency distribution of bread wheat landrace accession under different disease severity (TRS) classes at Kulumsa and Meraro during 2017 is depicted in Figure 1.The frequency of susceptible entries were more at Meraro than at Kulumsa. A total of 62 (40.8 %) landraces at kulumsa and 54 (35.5 %) and 62 (40 .8%) at Meraro exhibited low (0-20%) severity on leaves and heads, respectively whereas 33 (21.7 %) landraces at Kulumsa and 12 (7.9 %) and 13 (8.6 %) at Meraro, respectively had moderate TRS (21-40%. A total of 29(19.1%) and 28 (18.4 %) exhibited immune reaction to stripe rust on leaves and heads respectively across locations (data not shown). Spike infection on moderately resistance check (Pavon-76) was higher than on the susceptible and late maturing check (Digelu). This could be due to spike morphology and some agronomic traits such as days to heading and maturity. Early headed spikes are more infected by stripe rust than the late ones (data not shown). There is no susbstantial information if resistance on leaves and spikes are controlled by the same or different genes; however, some morphological and agronomic traits were identified to be associated with spike infection (Allan and Pritchett, 1972) On highly infected wheat genotypes, the grains were shriveled and resulted in losses in yield, quality and TKW (data not shown). This result is consistent with the findings of Purdy and Allan (1966) who reported the negative effect of spike infection on yield and yield components. Trace to low levels of spike infection on the moderately resistant and resistant varieties did not affect TKW, yield and yield components. Spike infection of wheat by stripe rust at higher altitudes result in 100% losses (Badebo et al., 2008). The present study identified considerable variations among the Ethiopian bread wheat landraces in TRS at both locations and seasons. The higher disease severity level was observed at Meraro compared to Kulumsa. This may be attributed mainly due to more favourable environmental conditions such as cool temperature and high humidity at Meraro than at Kulumsa. In this study, 55(96.5%) and 31(54%) accessions exhibited high level of slow rusting resistance at Kulumsa and Meraro, respectively.
Wheat lines with a low terminal disease severity under high disease pressure might have more additive genes that render adult plant resistance (Singh et al., 2005). This kind of resistance is possibly polygenic controlled by more than one gene (Dehghani and Moghaddam, 2004). Ali et al. (2007) proposed that lines with CI values of 0-20, 21-40, and 41-60 were regarded as possessing high, moderate, and low levels of slow rusting resistance, respectively. According to thier classification, a total of 55 (94.5%) and 3 (5.3%) accessions at Kulumsa and 32 (56%) and 22 (38.6%) at Meraro could be categorized as high and moderate level of slow rusting resistance, respectively (data no shown). Resistance in these accessions might be controlled by several minor genes which give longlasting resistance.

Area under Disease Progress Curve
The distribution of bread wheat landraces under AUDPC values across locations in 2017 and 2018 seasons is shown in Figure 3. In 2017, of the total number of bread wheat landraces, 51 (33.6%) and 22(14.5%) showed lower AUDPC values than the resistant (Enkoy) and moderately resistant (Pavon-76) checks at Kulumsa and 50(32.9%) and 26(17.1%) at Meraro, respectively. The landrace accession with lower AUDPC values than the resistant check (Enkoy) are considred to posess slow rusting resistance. AUDPC is a good indicator of adult plant resistance under field condition (Wang et al., 2005). It is directly related with grain yield loss (Jeger, 2004;SubbaRaoet al., 2008) and provides critical information for designing effective disease management practices for lines with different types and levels of resistance.

Disease Progress Rate (DPR)
The  Of the total 57 bread wheat landraces, 16(28%) and 2(5.3%) showed no progress in disease severity in 2018. However, 40(70.2%) and 12(21.1%) had lower DPR value per unit time than the resistant and moderately resistant checks at Kulumsa and 42(73.7%), 13(22.8%) at Meraro, respectively (data not shown). Disease progress rate in terms of disease spread was lower in resistant varieties compared to the susceptible ones. In general, a low AUDPC value in an accession might not ensure low in DPR, because the later depends on the differences in the initial and final disease severity. Once the pathogen reaches its maximum infection level, the DPR value may decrease or remain constant as disease progresses, because the green leaf area is not available for spread of the pathogen (Ali et al.,2009). The Ethiopian bread wheat landrace accessions with low DPR value per unit time could be potential sources to be exploited in breeding program.

Correlation among Disease Parameters and Thousand Kernel Weight (TKW)
Highly significant (P<0.001) and positive correlations observed among the disease parameters across locations and seasons (Table 2). There was high correlations between terminal rust severity with AUDPC and disease progress rate at Kulumsa (r = 0.976 and 0.949) and Meraro (r = 0.980 and 0.945), respectively. Relatively lower correlations were noted between AUDPC and DPR compared to the other slow rusting parameters at Kulumsa (r =0.880) and Meraro (r = 0.896). Despite the severity and AUDPC increase, the rate of infection could be slowed down over time because as the epdemics progresses less plant tissue could be available for further infection of the pathogen (Freedman and Mackenzie, 1992). The TKW has negatively correlated at P<0.001 with all the disease paramters at both locations.  Highly significant and positive correlation observed between spike (head) infection and the major disease paramters; however, the correlation of HI with TKW was negative. The positive correlations among the disease parameters observed in this study is in agreement with the results of other researchers in cereal rust pathosystems (Safavi, 2012). All disease parameters were highly correlated in the present study and all affected TKW. Qamaret al. (2007) andSafaviet al. (2013) reported higher selection gains of slow rusting resistance using low terminal rating, AUDPC and CI under field condition.
Figure5. Frequency distribution of resistant and susceptible bread wheat landrace accesions to three stripe rust races at seedling stage

Characterization of Stripe Rust Resistance in Bread Wheat Landraces
A total of 57 bread wheat landraces evaluated both at seedling and adult plant growth stages and of these, 32 (56.1%) exhibited lower values for all disease paramters under field conditions (  Wheat genotypes could be susceptible at seedling tests but exhibit moderate resistance to moderate susceptible reaction at adult plant stage and these lines with show slow rusting resistance parameters at adult plant stage could have durable resistance (Singh et al. 2005). This kind of resistance can be kept for a long time even if pathogen changes its genotype, it is controlled by more than one gene (Dehghani and Moghaddam 2004). Generally, the seedling resistance genes are also active during the adult plant stage and they are classified into race-specific resistance types (Lagudah, 2010).

Conclusion
Most of the commercial bread wheat cultivars in Ethiopia scumb to new races of stripe rust in hot spot areas in Ethiopia. In the depletion of resistance in the cultivated wheat, new sources are sought from landraces. In this study, a total of 152 bread wheat landrace accessions from IBCE have been tested to stripe rust during 2017 whereas only selected 57 accessions were exposed to the prevailing Pst races both in the field and at seedling stage in the greenhouse 2018. Overall, 32 accesions exhibited resistance in the field across locations and seasons. Of these, 18 and 14 accesions showed resistant and susceptible reaction to the three PSt races at seedling stage, respectively. This study has shown that Ethiopian bread wheat landraces are potential sources of overall (seedling) and adult plant resistances (APR) to stripe rust. A large number of bread wheat collections are preserved in the Institute of Biodiversity (IBCE). To cope up with the ever threatening races of the pathogen, future wheat improvement should focus on exploiting these gene pool in the breeding program.