Relationship between Partial Resistance and Inheritance of Adult Plant Resistance Gene Lr 46 of Leaf Rust in Six Bread Wheat Varieties

Leaf rust caused by Puccinia triticina Eriks is one of the most important diseases of wheat (Triticum aestivum L.) in Egypt and worldwide. It causes severe losses in grain yield reaching about 23% or higher on susceptible cultivars under favorable environmental conditions for the pathogen [1]. Genetic resistance is the most economical and effective means of reducing yield losses to this disease [2]. Knowledge of the identity and diversity of leaf rust resistance genes in cultivars and commonly used germplasm in breeding programs can improve efficacy of developing new resistant cultivars. Near-isogenic lines (NILs) carrying Lr resistance genes were developed in the spring wheat cv. Thatcher through backcrossing [3,4].

At present, more than 80 genes and alleles of leaf rust resistance Lr genes have been identified and described. Among them 33 Lr genes were transferred from other species into Triticum aestivum L. [5-8]. Most of these resistance genes are race-specific and therefore several of these genes are ineffective due to the emergence of new virulent races soon after their deployment [9]. In contrast, slow-rusting resistance characterized by slow disease progress in the field despite a compatible host reaction [10]. Although slow-rusting resistance genes have small to intermediate effects when present alone, high levels of resistance have been achieved by combining four to five genes [11]. The leaf rust resistance gene Lr 46/Yr 29 confers slow-rusting resistance to leaf rust and stripe rust, which has also provide durable resistance in gene combination [12]. Another gene confers slow-rusting resistance to leaf rust and stripe rust is Lr 34/Yr 18 [13-15]. Lr 34/Yr 18 also confers resistance to powdery mildew (Blumeria graminis) [16], stem rust (Puccinia graminis tritici) [14] and barley yellow dwarf virus (Singh, 1993). Moreover, Lr 67/Yr 46 genes confer a slow-rusting leaf rust resistance transferred from accession PI250413 in Pakistan [17]. The leaf rust resistance gene Lr 67/Yr 46 also had an effect on stripe rust and was associated with leaf tip necrosis in the field [15,17,18].

Knowledge of the identity of the leaf rust resistance genes in released cultivars and germplasm is essential for the incorporation of new effective resistance genes into breeding programs and maintenance of a diversity of resistance genes in commonly grown cultivars. The study aimed to determine the level of adult plant resistance; partial resistance to leaf rust in six wheat varieties under field conditions. Also, to identify the adult plant resistance genes in tested varieties at adult plant stage.

This investigation aimed to determine partial resistance and identify adult plant resistance genes governing leaf rust resistance in six wheat varieties i.e Giza 168, Sakha 93, Gemmeiza 1, Gemmeiza 7, Gemmeiza 9 and Sids 1 under field conditions (Table 1). The selected monogenic lines were Lr 38, Lr 42, Lr 43, Lr 44, Lr 45, Lr 46, and Lr 47. The experiments of this study were carried out in the farm of Environmental Studies and Research Institute. Sadat City University, Sadat City.

Table 1: List of the six tested local wheat varieties, pedigree and year of release.

To estimate the level of partial resistance, the above mentioned wheat varieties were cultivated in three growing seasons i.e. 2011/12, 2012/13 and 2013/14 at Sadat City location. All experiments were carried out in a randomized complete block design, with three replicates. The tested wheat varieties were grown in 6 row plots, each row was 3 m long and 30 cm apart, where, the plot size was 3 m × 3 m=9 m². The experimental plots were surrounded by a rust spreader belt, planted with a mixture of the highly susceptible varieties to leaf rust i.e. Thatcher, Morocco and Triticum spelta saharensis to serve as a continuous spreader source for leaf rust urediniospores.

For identification of adult plant leaf rust resistance genes by genetic analysis, the parental varieties and Thatcher NILs were grown in plots (9 m²) contains rows of 3 m long and 30 cm apart during 2011/12 growing season in three successive dates at 15 days intervals to overcome differences in the time of flowering. The Thatcher NILs were used as male parents for crosses with each of the tested wheat cultivar to obtain F₁ seeds. The F₁ seeds were grown in the next following season (2012/13) in rows of 3 m long and 30 cm apart and spaced 30 cm in order to facilitate production of F₂ seeds. In 2013/14 growing season, parents and F₂ seeds were grown at Sadat City in plots; each plot of the parents and F₂ plants contains 9 rows of 3 m long and 30 cm between rows and seeds were 20 cm apart, therefore each row contained 15 plants and each plot contained 135 plants. Each of F₂ cross was evaluated at two plots contained 270 plants. All plots were surrounded by a spreader area in one meter width sown with a mixture of highly susceptible wheat cultivars i.e. Thatcher, Morocco and Triticum spelta saharensis.

Inoculation and disease assessment

Spreader plants were sprayed with water and dusting with spores powder mixture of the most prevalent and aggressive leaf rust pathotypes (one volume of fresh urediniospores mixture: 20 volume of talcum powder). Dusting was carried out in the early evening (at sunset) before dew point formation. The inoculation of all plants was carried out at booting stage according to the method of Tervet and Cassell [19]. To estimate the level of partial resistance, percentage leaf rust severity was recorded for the six wheat varieties using the modified Cobb’s scale described by Peterson et al. [20]. Rust severity data were scored after the appearance of the first symptoms (appear of the first pustule on any of the tested wheat varieties) at seven days intervals. The percentage final rust severity (FRS) was assessed according to Das et al. [21], as the disease severity (%) for each tested variety when the highly susceptible check variety (Morocco) was severely rusted and the disease rate reached the highest level of leaf rust severity.

Area under disease progress curve (AUDPC) was also calculated for each cultivar as a good reliable and more accurate estimator for rust resistance under field conditions. The values of AUDPC were calculated by using the following equation of Pandey et al. [22].

Where: D=Days between two consecutive recording (time intervals)

For identification of the adult plant resistance, data of leaf rust severity were recorded at the adult plant stage for parents and each of F₂ plant at milk stage when the susceptible wheat cultivars Thatcher, Morocco and Triticum spelta saharensis displayed a response between 80 S to 100 S using the modified Cobb,s scale of Peterson et al. [20]. Plant reaction was expressed in five infection types, Roelfs et al. [23]. The infection types were immune=(0), resistant=(R), moderately resistant=(MR), moderately susceptible=(MS) and susceptible=(S).

The F₂ plants of each cross were grouped into eight classes depending on their percentage of disease severity under field conditions. The disease severity classes were: 0-10; 11-20; 21-30; 31-40; 41 50; 51-60; 61- 70 and 71-80. Plants grouped in the first three classes were considered as resistant phenotype, while plants of the other classes (more than 30%) were considered as susceptible phenotype [24].

For identification of the adult plant leaf rust resistance genes in each cross, goodness of fit of the observed to the expected ratio of the phenotypic classes concerning the leaf rust severity and infection types, were determined by Chi square (χ2) analysis according to Steel and Torrie [25].

Evaluation of the tested wheat monogenic lines against leaf rust at adult plant stage

Data in Table 2 showed that, in 2011/12 growing season the wheat monogenic lines Lr 46 and Lr 47 were completely resistant and showed zero percent final rust severity compared with the other tested monogenic lines which showed final rust severity ranged from 5 MR to 20 MR.

Table 2: Response of seven wheat monogenic lines at adult plant stage to final rust severity grown at Sadat.

In 2012/13 growing season, the leaf rust resistance genes Lr 42 and Lr 45 were immune to leaf rust infection. Meanwhile, the other tested monogenic lines showed final rust severity ranged from Tr R to 20 MR.

In 2013/14 growing season, the wheat monogenic lines Lr 43, Lr 46 and Lr 47 were completely resistance and showed zero percent rust severity. Moreover, the other tested monogenic showed final rust severity ranged from Tr MR to 20 MR.

City during three successive growing seasons (2011/12- 2013/14)

Final rust severity includes two components: disease severity based on modified Cobb,s scale [20], where Tr=less than 5% and 5=5% up to 100=100% and host response based on scale described by Roelfs et al. [23]; where 0=immune (no visible infection), R=resistant (flecks and small uredinia with necrosis), MR=moderately resistant (large necrotic flecks and large uredinia), MS=moderately susceptible (moderate to large uredinia with chlorosis) and S=susceptible (large uredinia).

Evaluation of the tested wheat varieties under field conditions

Final rust severity (FRS%): Data of final rust severity in Table 3 recorded during the 2011/12 and 2012/13 growing season were found to be slightly higher compared with the third growing season 2013/14. Also, data showed that the final rust severity (%) of the tested wheat varieties was lower compared to the check variety Sids 1 during the three seasons at Sadat City. In 2011/12 growing season, the wheat varieties Gemmeiza 9 (10.00%), Giza 168 (16.67%) and Gemmeiza 7 (26.67%) showed the lowest values of final rust severity (did not exceed up to 30.00%) during this season. While, the wheat varieties Gemmeiza 1 (53.33%), Sakha 93 (66.67%) and Sids 1 (86.67%) showed the highest values of FRS (%).

Table 3: Percentage final rust severity and area under disease progress curve (AUDPC) of Puccinia triticina on six wheat varieties under field conditions at Sadat City location during 2011/12, 2012/13 and 2013/14 growing seasons.

Data of final rust severity (%) in the second season (2012/13) showed that the wheat varieties Giza 168 and Gemmeiza 9 (each with 13.33%) and Gemmeiza 7 (26.67) showed the lowest values of FRS (%) (Less than 30.00%). While the wheat varieties Gemmeiza 1 (50.00%), Sakha 93 (60.00%) and Sids 1 (80.00%) showed the highest values of FRS (%) during this season.

In 2013/14 growing season, the wheat varieties Gemmeiza 9, Giza 168 and Gemmeiza 7 showed the lowest values of FRS (%) i.e. 6.67%, 11.67% and 23.33%, respectively. While, the wheat varieties Gemmeiza 1, Sakha 93 and Sids 1 showed the highest values of FRS (%) i.e. 46.67%, 63.33% and 73.33%, respectively.

Data of mean FRS (%) during three seasons indicated that the wheat varieties Gemmeiza 9 (10.00%), Giza 168 (13.89%) and Gemmeiza 7 (25.56%) showed the highest resistance response with the lowest mean FRS (%). While, the wheat varieties Gemmeiza 1 (50.00%), Sakha 93 (63.33%) and Sids 1 (80.00%) were susceptible and showed the highest values of mean FRS (%).

Area under disease progress curve (AUDPC): Data presented in Table 3 showed the mean area under disease progress curve (AUDPC) estimated over the three seasons, the tested wheat varieties can be classified into two main groups depending on their values of AUDPC. The first group included the wheat varieties that displayed the highest levels of adult plant resistance under field conditions through the three growing seasons of the study.

These varieties were Gemmeiza 9, Giza 168 and Gemmeiza 7 which showed the lowest values of AUDPC (less than 300) i.e. 82.06, 111.22 and 225.56, respectively. Therefore, they have been classified as slowrusting or partially resistant varieties. The second group included the wheat varieties Gemmeiza 1 (521.11), Sakha 93 (703.89) and Sids 1 (886.67), which they showed the highest values of AUDPC to leaf rust infection. Also, they displayed the lowest levels of adult plant resistance and these varieties classified as fast-rusting varieties.

Identification of adult plant leaf rust resistance genes

Results presented in Table 4 showed that all of 233 F2 plants of the crosses between Lr 38 and the wheat variety Giza 168 revealed resistant reaction without any segregation under field conditions. This result indicated that, the wheat variety Giza 168 has the leaf rust resistance gene Lr 38. While, F₂ plant of the crosses between this gene and the wheat varieties Sakha 93, Gemmeiza 1, Gemmeiza 7, Gemmeiza 9 and Sids 1 segregated to (180 resistant: 47 susceptible), (174 R: 57 S), (166 R: 49 S), (177 R: 55 S) and (172 R: 63 S), respectively. These segregations fit the ratio (3 R: 1 S) indicated that the wheat varieties Sakha 93, Gemmeiza 1, Gemmeiza 7, Gemmeiza 9 and Sids 1 did not have this gene.

Table 4: Segregation and Chi square analysis of F₂ plants of the crosses between six bread wheat cultivars and seven leaf rust monogenic lines at adult stage under field conditions at Sadat City location during 2013/14 growing season.

F₂ plants of the crosses between the leaf rust resistance gene Lr 42 and the tested wheat varieties Giza 168, Sakha 93, Gemmeiza 1, Gemmeiza 7, Gemmeiza 9 and Sids 1 segregated to the ratios 175 R: 47 S, 190 R: 54 S, 153 R: 54 S, 171 R: 46 S, 166 R: 54 S and 161 R: 53 S, respectively. These ratios fitting 3 R: 1 S ratio indicating that these varieties don’t have gene Lr 42.

The F₂ plants of the crosses between Lr 44 and the wheat varieties Giza 168, Sakha 93, Gemmeiza 1, Gemmeiza 7, Gemmeiza 9 and Sids 1 segregated to the ratios 160 R: 52 S, 168 R: 59 S, 183 R: 65 S, 146 R: 55 S, 175 R: 48 S and 166 R: 43 S, respectively. These ratios fitting to 3 R: 1 S indicating that they don’t have this gene.

All of 215 and 218 F2 plants of the crosses between Lr 45 and the wheat varieties Giza 168 and Gemmeiza 9 were resistant and therefore expressed Lr 45 resistance but the wheat varieties Sakha 93, Gemmeiza 1, Gemmeiza 7, Gemmeiza 9 and Sids 1 segregated at 171 R: 61 S, 173 R: 47 S, 170 R: 65 S and 159 R: 49 S, respectively, fitting to 3 R: 1 S ratio and not therefore expressing this gene.

All of 233 and 231 F2 plants of the crosses between Lr 46 and the wheat varieties Giza 168 and Gemmeiza 9 were resistant and therefore expressed Lr 46 resistance but the wheat varieties Sakha 93, Gemmeiza 1, Gemmeiza 7, Gemmeiza 9 and Sids 1 segregated to 173 R: 67 S, 154 R: 53 S, 173 R: 47 S and 178 R: 55 S, respectively, which were a good fit to the ratio of 3 R: 1 S, but still indicating that these varieties did not express Lr 46 gene.

All of 218 and 225 F2 plants of the crosses between Lr 47 and the wheat varieties Giza 168 and Gemmeiza 9 were resistant expressed Lr 47 resistance but the wheat varieties Sakha 93, Gemmeiza 1, Gemmeiza 7, Gemmeiza 9 and Sids 1 segregated to 178 R: 66 S, 160 R: 52 S, 161 R: 56 S and 183 R: 49 S, respectively, fitting to the ratio 3 R: 1 S and not therefore expressing this gene.

Data in Table 5 indicated that, the wheat varieties Giza 168 and Gemmeiza 9 have the same three genes i.e. Lr 45, Lr 46 and Lr 47. While the other tested varieties Sakha 93, Gemmeiza 1, Gemmeiza 7 and Sids 1 do not carry any of the tested leaf rust resistance genes.

Table 5: Resistance genes for leaf rust identified in six bread wheat varieties at adult plant stage.

Partial resistance has been early recognized as more stable type of resistance in contrast to other forms of resistance [10,26].

In this study, the components of partial resistance against leaf rust determined in six wheat varieties i.e. Giza 168, Sakha 93, Gemmeiza 1, Gemmeiza 7, Gemmeiza 9 and Sids 1 under field conditions at Sadat City location during three growing seasons i.e. 2010/11, 2011/12 and 2012/13.

According to the percentage final rust severity (FRS), the tested wheat varieties could be classified into two main groups. The first one included partially resistant varieties i.e. Gemmeiza 9, Giza 168 and Gemmeiza 7 which showed low values of FRS (%) (less than 30%). While, the second group included the fast rusting varieties i.e. Gemmeiza 1, Sakha 93 and Sids 1 which showed the highest values of FRS (%) during the three growing seasons. Also, they displayed the lowest levels of adult plant resistance and these varieties classified as fast-rusting varieties. These results are previously supported by Bassiony, Nazim et al. and Herrera-Fossel et al. [1,27,28].

Area under disease progressive curve (AUDPC) is a good indicator of adult plant resistance under field condition [29]. Cultivars which had low AUDPC and terminal severity values thus may have good level of adult plant resistance [29]. Furthermore, AUDPC, in particular, is the result of all factors that influence disease development such as differences in environmental conditions, varieties and population of the pathogen [12,22,30,31]. According to the obtained results and depending on the values of AUDPC, the wheat varieties Gemmeiza 9, Giza 168 and Gemmeiza 7 showed the lowest values of AUDPC. Such results indicated that these varieties have good level of adult plant resistance under field conditions during the three growing seasons to leaf rust and can be used as resistance sources. Therefore, this group of varieties characterized as the partially or slow rusting resistant group. While, three wheat varieties Gemmeiza 1, Sakha 93 and Sids 1 showed the highest AUDPC values. These varieties classified as the highly susceptible or fast rusting varieties group, as reported by Nazim et al., Denissen, Singh et al. and Herrera-Fossel et al. [5,12,28,32].

To identify the gene (s) governing adult plant resistance to leaf rust in the wheat varieties; genetic analysis was conducted. This experiment included crossing of tested six wheat varieties i.e. Giza 168, Sakha 93, Gemmeiza 1, Gemmeiza 7, Gemmeiza 9 and Sids 1 with seven monogenic lines i.e. Lr 38, Lr 42, Lr 43, Lr 44, Lr 45, Lr 46, and Lr 47. The F₁ plants were selfed to obtain the F₂ plants. The F₂ plants were evaluated at adult plant stages and goodness of fit of the observed to the expected ratio of the phenotypic classes concerning the leaf rust severity and infection types, were determined by Chi square analysis according to Steel and Torrie [25].

From this study, the tested wheat varieties Giza 168 and Gemmeiza 9 have the adult plant resistance genes i.e. Lr 45, Lr 46 and Lr 47. While, the wheat varieties Sakha 93, Gemmeiza 1, Gemmeiza 7 and Sids 1 do not carry any of the tested wheat monogenic lines. These results in agreement with Ingal et al., Loladze et al. and Wang et al. [7,33,34]. Moreover, the data suggest that the two wheat varieties Giza 168 and Gemmeiza 9 possess genes that are responsible for partial resistance for Puccinia triticina. Meanwhile, these two wheat varieties Giza 168 and Gemmeiza 9 showed partial resistance to leaf rust; this mainly due to the presence of the partial leaf rust resistance gene Lr 46. Obtained results has been strongly supported by Sivasamy et al. [35] who found that five wheat lines carried leaf rust resistance gene Lr 46 and showed partial resistance to leaf rust.

Leaf rust resistance gene Lr 46 is a slow rusting gene. These genes do not provide the host plant with complete immunity against leaf rust (Puccinia triticina) races. The effect of Lr 46 delayed the infection process or reduces the development of symptoms caused by leaf rust races on adult plants; reduce colony size and lower disease severity [36]. Lr 46 was first described in 1998 by Singh et al. [37] in cultivar Pavon 76, and located on chromosome 1B. Also, Lr 46 linked with the stripe rust resistance gene Yr 29. The type of resistance conferred by Lr 46 is similar to that of Lr 34, although with a smaller effect [38].

Our results showed the presence of different levels of resistance in the tested wheat varieties to leaf rust at adult plant stage under field conditions. None of the tested varieties were immune. The wheat varieties Giza 168 and Gemmeiza 9 showed good levels of partial resistance and this resistance mainly due to the presence of the leaf rust partial resistance gene Lr 46. Our results do not exclude the presence of other genes for leaf rust resistance in the tested varieties.