Seedling and adult plant resistance to leaf rust in some Egyptian wheat genotypes

Leaf rust of wheat caused by Puccinia triticina Eriks. is one of the most widespread disease in Egypt. In this study, thirteen Egyptian wheat genotypes were evaluated for leaf rust resistance at seedling stage under greenhouse condition and adult plant stage under field conditions over three growing seasons that are, 2011/2012, 2012/2013 and 2013/2014 and three locations that are, Itay El-Baroud and Nubariya Agricultural Research Stations as well as the Farm of the Faculty of Agriculture, Minufiya University, Shibin El-Kom. The tested wheat genotypes were classified into three groups according to their resistance. The first group, race-specific resistant genotypes including Shandweel 1, Misr 1, Misr 2, Sids 12 and Sids 13, showed the lowest values of final rust severity (FRS %) and area under disease progress curve (AUDPC). The second group, slow-rusting or partially resistant genotypes including Sakha 94, Gemmeiza 9, Giza 168, Sakha 95, Gemmeiza 10 and Gemmeiza 11, displayed low level of FRS and AUDPC. The third one which includes Gemmeiza 7 and Sids 1, showed the highest values of FRS and AUDPC. Postulation of leaf rust resistance genes was differed between the tested genotypes.Results indicated that Sakha 95 and Sids 12 may have seven resistance genes. Moreover, Gemmeiza 10 may has five genes and Misr 1 may has three genes. While, Giza 168, Sids 1, Misr 2 and Shandweel 1 may have two genes. The wheat genotypes Gemmeiza 11 and Gemmeiza 12 may have only single gene. Also, all the tested wheat genotypes may contain some additional genes.In contrast, the wheat genotypes Sakha 94, Gemmeiza 7, Gemmeiza 9 and Sids 13 did not have any of the tested genes.


INTRODUCTION
Leaf rust caused by Puccinia triticina Eriks. is a widespread disease of wheat (Triticum aestivum L.) in Egypt and worldwide.Yield losses due to leaf rust disease may be more than 50% for some susceptible wheat genotypes (German et al., 2007).Breeding wheat genotypes with resistance to leaf rust is the most effective control method and environment friendly approach to reduce the yield losses (Winzeler et al., 2000).
At present, more than 80 genes and alleles of leaf rust resistance genes have been identified and described.Among them 33 Lr genes were transferred from other *Corresponding author.E-mail: walid_elorabey2014@hotmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License species into Triticum aestivum L. (Herrera-Foessel et al., 2011, 2012;Ingala et al., 2012;McIntosh et al., 2012).Most of the resistance genes are effective at seedling stage and remain effective through the adult plant stage.Some of the leaf rust resistance genes express resistance optimally in adult plants and are known as adult plant resistance (APR) genes, which depends on the genetics of the host pathogen interaction as well as favored environmental conditions.
Rust resistance in wheat has been based on the use of race specific resistance genes.But the short-lived nature of race specific hypersensitive resistance has created the necessity to search for the more durable type of resistance.Several researchers reported that some genotypes showed the ability to retard the rust development even though they had a susceptible reaction type (Caldwell et al., 1970;Singh et al., 1991).This type of resistance known as slow rusting resistance.
Avoiding major rust epidemics in the region is a complex challenge, given that fewer genotypes are being cultivated over large areas, and several of those genotypes are protected by the same resistance genes.To identify those genotypes with resistant sources that are the most fit for the cultivation in the more diseased areas of the country, genotype screening for leaf rust resistance is considered the best and the cheapest method.
The objectives of this research work are to determine the resistance of some Egyptian wheat genotypes at seedling and adult plant stages to leaf rust through postulating and identifying resistance genes in the tested wheat genotypes.

Seedling studies
Genotypes evaluation and resistance genes postulation of the tested wheat monogenic lines were carried out at seedling stage in the greenhouse of Wheat Diseases Res.Dept., Plant Pathology Res.Inst., ARC, Giza, Egypt.A total of 107 leaf rust samples were collected during three successive growing seasons.Forty samples were collected in 2010/2011, thirty eight samples in 2011/2012 and twenty nine samples in 2012/2013 (Table 1) from the wheat commercial fields and the trap nurseries grown in different locations of Egypt.These locations were Beheira (31 samples), Dakahlia (12), Gharbiya (10), Minufiya (12), Sharqiya (20), Domiata (3), Qalyubia (5) and Bani Swif (14).Sample (2 to 4 infected leaves) was kept at room temperature (18 to 24°C) overnight to be dried off then kept in glycine envelopes (8 × 15 cm) and stored with deiscator in the refrigerator at 2 to 5°C.The infected specimens were transferred through inoculation to the highly susceptible variety; Thatcher for isolation and purification.
The method of inoculation was carried out as described by Stakman et al. (1962), in which wheat seedling leaves (7 days old) were rubbed gently between moisted fingers with tap water, sprayed with water in the incubation chambers.Then inoculated by shaking or brushing rusted materials from collected samples over the plant leaves and sprayed gently again with water in order to form initial film of free water on the plants which is essential for spore germination and establishment of infection.The inoculated seedlings were incubated in humid chambers for 24 h to allow spore germination and cause infection.The inoculated plants were moved onto the benches in the greenhouse with daily temperature 10 to 25°C.After approximately 12 to 15 days, three single pustules were isolated separately from each sample for rust reproduction on seedlings of the highly susceptible wheat variety Thacher to obtain enough urediniospores for inoculation.
At the same time, seeds of the tested wheat genotypes i.e.Sakha 94, Sakha 95, Giza 168, Gemmeiza 7, Gemmeiza 9, Gemmeiza 10, Gemmeiza 11, Sids 1, Sids 12, Sids 13, Misr 1, Misr 2 and Shandweel 1 were sown in 6 cm square plastic pots.Five seeds of each tested wheat material were sown in each corner in a clockwise order.Seven days old seedlings of the tested wheat materials, when first leaf full emerged, were inoculated with single pustule isolates of P. triticina which was previously propagated.Inoculation was carried out by shaking with propagated urediniospores over the seedling leaves of the tested materials.The inoculated seedlings were transferred onto the greenhouse benches(18 to 20°C and 100% RH).

Seedling disease assessment
Infection type (IT) data for each tested wheat genotype were recorded 12 days after inoculation using standard infection type scoring scale 0 to 4 (Stakman et al., 1962).Genotypes which showed low infection types (scores = 0, 0; 1, and 2) were considered as resistant or low infection types (LITs).While, those with scores = 3 and 4 were susceptible or high infection types (HITs) (Stakman et al., 1962).

Virulence frequency
Virulence frequency was calculated as percentage of virulent isolates to the total number of the tested isolates.

Postulation of leaf rust resistance genes
Thirteen Egyptian wheat genotypesand 39 monogenic lines carrying single gene for leaf rust resistance were tested in seedling stage using 15pathotypes of leaf rust i.e.BBCHS, BCCMS, BJTJG, CJNBD, JFKTS, LCJKB, LMSDB, MHCQQ, NSHLQ, PBBFB, PTQTT, QHJHJ, RJCRQ, SCLKB and SKPSS.All plant materials were grown in plastic pots.Seedlings were inoculated by urediniospores of the selcted identified pathotypes during 2013/2014 growing season.The inoculated seedlings were incubated also as previously mentioned and transferred onto benches of the greenhouse.Both inoculation and incubation procedures were done according to the method described by Tervet  and Cassel (1951).Leaf rust disease infection type (IT) datawere recorded for the wheat testedmaterials as mentioned before using disease assessmentapproaches previously suggested by Stakman et al. (1962).

Field studies
Field work was carried out at three locations that are, Itay El-Baroud and Nubariya Agricultural Research Stations as well as the Farm of the Faculty of Agriculture, Minufiya University, Shibin El-Kom during 2011/2012, 2012/2013 and 2013/2014.Each of the previously mentioned thirteen wheat genotypes were planted in aplot (3 m X 3.5 m= 10.5 m 2 ) consisting of seven rows, and each row was 3 m long and 40 cm apart.Whole experimental plots were surrounded by spreader plants of one meter width sown with a mixture of the highly susceptible wheat genotypes to leaf rust that is, Thatcher and Morocco.The spreader plants were artificially inoculated with a mixture of urediniospores and talcum powder(1:20 v/v) of the most prevalent and aggressive fifteen leaf rust physiologic pathotypes previously mentioned.The methods of inoculation were described by Tervet and Cassel (1951).

Percentage of final rust severity (FRS %)
Percentage of leaf rust severity was recorded for the thirteen wheat genotypes using the modified Cobb's scale described by Peterson et al. (1948).Rust severity data were scored after the appearance of the first symptoms (appear of the first pustule on any of the tested wheat genotypes) at seven days intervals.The percentage final rust severity (FRS %) was assessed according to Das et al. (1993), as the percentage disease severity for each tested genotypes when the highly susceptible check genotype(Sids 1) was severely rusted and the disease rate reached the highest level of leaf rust severity.

Area under disease progress curve (AUDPC)
AUDPC was also calculated for each genotype under field conditions.The values of AUDPC were calculated by using the following equation of Pandey et al. (1989).
Where: D = Days between two consecutive recording (time intervals) Y1 + Yk = Sum of the first and last scores.

Statistical analysis
Least significant difference (LSD at 5 %) test was performed to determine the significant differences between means according to Steel and Torrie (1980).

Percentage frequency of virulence to the tested wheat genotypes
Wheat  1).

Postulation of leaf rust resistance genes (Lr , s)
Low and high infection types displayed by the thirteen tested wheat genotypes (Table 1) compared with the infection types of 39 known Lr genes (Table 2) against fifteen identified tested pathotypes of P. triticina under greenhouse condition.Data obtained in Tables 3 and 4 were summarized in Table 3

Percentage of final rust severity (FRS %)
Data in Table 5 showed that there are significant differences in FRS means among the tested wheat genotypes.Meanwhile, there are no significant differences in means of FRS of environments as well as the interaction between genotypes and environments.
The wheat genotype Shandweel 1 showed resistance infection type.While, the wheat genotypes Sids 12, Sids 13, Misr 1 and Misr 2 showed moderately resistance infection type and the reminder genotypes showed susceptible infection type at the three tested locations during the three seasons.

Area under disease progress curve (AUDPC)
Data in Table 7 estimated the mean values of AUDPC over the three years and three locations.There are significant differences in AUDPC means among the tested wheat genotype sat P = 0.05.Meanwhile, no significant differences in means of AUDPC of environments (years and locations) as well as the interaction between genotypes and environments.Moreover, the tested wheat genotypes can be classified into three groups.The first group race-specific resistant includes wheat genotypes of Shandweel 1 (4.67),Misr 1 (37.33),Sids 12 (44.46),Misr 2 (44.98) and Sids 13 (54.05).These genotypes displayed the highest levels of adult plant resistance and showed the lowest values of AUDPC.Also, they showed resistant and moderately resistant infection types.
The second group included the wheat genotypes Sakha 94, Gemmeiza 9, Giza 168, Sakha 95, Gemmeiza 10 and Gemmeiza 11, which they displayed acceptable levels of adult plant resistance.However, results showed low values of AUDPC for these genotypes that is, 57.04, 62.74, 80.24, 102.02, 137.28 and 171.63, respectively.Also, this group showed susceptible infection type.Therefore, they have been classified as slow-rusting or partially resistant genotypes.Whereas, the third group included Gemmeiza 7 (429.07)and Sids 1 (751.85),which they showed the highest values of AUDPC to leaf rust infection, Also, displayed the lowest levels of adult plant resistance and these genotype sclassified as fastrusting genotypes.Occurrence of virulence was stable on the wheat genotype Misr 1 during the three seasons of this study, while, virulence frequencies of Misr 2 and Sids 12 which were almost stable during the two growing seasons 2011/2012 and 2012/2013.Moreover, little changes in varietal resistance against the tested isolates in the growing seasons of study were recorded; this is might be due to the differences in the genetic make-up of the tested wheat genotypes (Negm et al., 2013).
Changing in race pathogenesis led the breeders to involving alternative forms of resistance that would be more durable such as slow rusting or partial resistance (Broers, 1989;Singh et al., 2000a, b).It has been demonstrated that durable rust resistance is more likely to be of adult plant type rather than of seedling infection type and is not linked with the genes producing hypersensitive reaction (McIntosh, 1992;Bariana et al., 2001).Durable rust resistance is a mechanism conferring resistance to a genotype for long period of time during its widespread cultivation in a favorable environment for a disease (Johnson, 1978(Johnson, , 1988)).This type of resistance is mainly associated with the minorgenes, which are also known as slow rusting genes.The concept of slow rusting in wheat was previously recommended by Caldwell (1968).
Many researchers have emphasized the need to identify and exploit durable resistance.Johnson and Law (1975) defined durable resistance as a resistance source that remained effective after widespread deployment over a considerable period.A general concept of a durable resistance source for cereal rusts is that it is polygenic, likely to express at adult plant stage, non-race-specific and produce non-hypersensitive response to infection.
The tested wheat genotypes were evaluated at adult plant stage at three locations that is, Itay El-Baroud and Nubariya Agricultural Research Stations as well as the Farm of the Faculty of Agriculture, Minufiya University, Shibin El-Kom during three successive growing seasons that is, 2011/2012, 2012/2013 and 2013/2014.Percentage of FRS was recorded for each of the tested genotypes.However, the wheat genotype Shandweel 1 was highly resistant and showed lowest percentage level of FRS and resistance infection type.Moreover, the wheat genotypes Sids 12, Sids 13, Misr 1 and Misr 2 were also highly resistant and showed low percentage level of FRS and moderately resistance infection type.Resistance to leaf rust in these wheat genotypes mainly due to race-specific resistance gene (s), which were showed infection type resistance (R) to moderately resistance (MR).German and Kolmer (1992) found that individual major genes for adult plant resistance to leaf rust can show enhancement effectiveness when combined in wheat genotypes.However, the wheat genotypes Sakha 94, Gemmeiza 9, Giza 168, Sakha 95, Gemmeiza 10 and Gemmeiza 11 showed low percentage levels of FRS (did not exceeded up to 20%) also, these genotypes showed susceptible infection type (S).These genotypes displayed an adequate level of partial resistance to leaf rust infection, in comparison with the two genotypes Gemmeiza 7 and Sids 1 (fast rusting or highly susceptible genotypes).These results are previously supported by Bassiony (1979) and Nazim et al. (1983Nazim et al. ( , 1990)).
AUDPC is a good indicator of adult plant resistance under field condition (Wang et al., 2005).They added that genotypes which had low AUDPC and terminal severity values may have good level of adult plant resistance (Wang et al., 2005).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 (Pandey et al., 1989;Lal Ahmed et al., 2004;Singh et al., 2005;Boulot, 2007).
According to the obtained results and depending on the mean values of AUDPC, the wheat genotypes Sakha 94, Gemmeiza 9, Giza 168, Sakha 95, Gemmeiza 10 and Gemmeiza 11 showed lowest values of AUDPC.These results indicated that such genotypeshave good level of adult plant resistance under field conditions in three locations through three growing seasons to leaf rust and can be used as resistance sources.Therefore, this group of genotypes characterized as partially or slow rusting resistant group.While, the two wheat genotypes Gemmeiza 7 and Sids 1 showed the highest AUDPC values.These genotypes classified as the highly susceptible or fast rusting genotypes group, similarly to those reported by Nazim et al. (1990); Denissen (1993) and Singh et al. (2005).

Conclusion
According to the data obtained, nineteen known leaf rust resistance genes and one or more unknown genes were postulated in the thirteen tested wheat genotypes.These findings should be useful in the Egyptian wheat breeding programs in order to improve leaf rust resistance.Also, the present study revealed that most of the tested wheat genotypes were having enough resistance, ranging from complete resistance to partial resistance.Also, most of the tested wheat genotypes exhibited better performance under high disease pressure shown by susceptible varieties.Further studies for testing stability of the tested wheat genotypes over growing seasons and locations against leaf rust along with other desirable characters must be studied.

Table 1 .
Percentage of virulence frequency of P. triticina isolates and level of varietal resistance to thirteen Egyptian wheat genotypes during three successive growing seasons at seedling stage.

Genotype 2011/2012 2012/2013 2013/2014 No. of virulent isolates No. of avirulent isolates Total No. of isolates Virulence frequency (%)* Level of varietal resistance (%)** No. of virulent isolates No. of avirulent isolates Total No. of isolates Virulence frequency (%) Level of varietal resistan ce (%) No. of virulent isolates No. of avirulent isolates Total No. of isolates
*Virulence frequency (%)= Percentage of P. triticina isolates virulent to each wheat variety to the total number of tested isolates;** Level of varietal resistance (%): Estimated as the percentage of virulent isolates to the total number of tested isolates;***Total No. of collected samples = 107.

Table 2 .
Infection types of thirteen Egyptian wheat genotypes against fifteen pathotypes of P. triticina under greenhouse condition during 2013/2014 growing season at seedling stage.

Table 3 .
Infection type of thirty nine monogenic lines (Lr , s) against fifteen pathotypes of P. triticina under greenhouse condition during 2013/2014 growing season at seedling stage.

Table 4 .
Leaf rust resistance genes (Lr , s) probably present in the thirteen Egyptian wheat genotypes.
* +? = Means that the concerned genotype may have additional gene (s)that were not detected using the tested Lr genes and isolates of the study.

Table 5 .
Number of postulated leaf rust resistance genes (Lr , s) and their percentage of frequency in thirteen Egyptian wheat genotypes at seedling stage under greenhouse condition during 2013/14 growing season.

Table 5 .
Contd.Gemmeiza 11, Gemmeiza 9, Giza 168, Gemmeiza 10 and Sakha 95 showed the lowest percentage values of FRS (less than 20.00%).While the wheat genotypes Gemmeiza 7 and Sids 1 showed the highest percentage values of FRS during this season at the three locations that is, Nubariya, Itay El-Baroud and Shibin El-Kom.

Table 6 .
Percentage of final rust severity (FRS) of P. triticina and infection type on thirteen wheat genotype sunder field conditions at three locations during three growing seasons.

Table 7 .
Area under disease progress curve (AUDPC) of Puccinia triticina on thirteenwheat genotypes under field conditions at three locations during three growing seasons.