Determination of levels of resistance in Pakistani bread wheat cultivars against stripe rust ( Puccinia striiformis ) under field conditions

The bread wheat is an important crop of Pakistan. The average yield of wheat in the country is much behind the potential yield of the improved wheat cultivars. The low yield of wheat is the consequence of many factors including rust diseases that cause yield along with quality losses. Most of breeding programs in the country lack facilities for screening against rust diseases. Usually, selection is done with inadequate rust epidemic or improper pathotypes. As a result, only a few of released cultivars exhibit desired resistance against the prevailing rust races/pathotypes. A study was conducted from 2007 to 2011 at National Agricultural Research Center, Islamabad, Pakistan to provide information on the level of resistance in 113 released Pakistani bread wheat cultivars against stripe rust. The results indicated that most of the cultivars exhibited compatible reaction with prevailing pathotypes/races of stripe rust fungus at Islamabad. Only six cultivars including, Tatara, Faisalabad 08, Bahawalpur 94, Sassui, Chakwal 86 and Fakhr-e-Sarhad demonstrated hypersensitive type of resistance against naturally prevailing stripe rust races/pathotypes. However, year x cultivar interaction was observed in many cultivars against stripe rust races. Among the cultivars that showed compatible type of reactions, four were highly partial (non-hypersensitive) resistant (disease severity (DS) < 5% of Morocco), nineteen were partially (non-hypersensitive) resistant (DS < 20% of Morocco), forty-three were moderately partial (non-hypersensitive) resistant (DS < 50% of Morocco) and forty-one were moderately to highly susceptible. Virulence was observed against resistant genes YrA, Yr2, Yr4 Yr6, Yr7, Yr9 and Yr27.


INTRODUCTION
Being the staple food of people of Pakistan, wheat (Triticum aestivum L.) has economical importance.It is grown on an area of 9.05 million ha with an average yield of 2657 kg ha -1 (Anonymous, 2010).The average yield of wheat in Pakistan is resting much behind the potential *Corresponding author.E-mail: maqsoodqmr@gmail.com.yield of the improved wheat cultivars which is 7000 to 8000 kg ha -1 . This low national average yield can be attributed to many factors (Asif and Kamran, 2011;Ahmed et al., 2012) but plant diseases are considered to be the main hindrance.
Wheat is affected by many diseases that cause yield and quality losses.The rust is one of the most important diseases that pose constant threat to sustainable production of wheat.There are three types of rusts viz, stripe rust (Puccinia striiformis West.f. sp.tritici Eriks.and E. Henn.), leaf rust (Puccinia triticina Eriks) and stem rust (Puccinia graminis Pers.f. sp.tritici Eriks.and E. Henn).Mostly, semi-dwarf spring wheat cultivars are grown over most of the area in the country since green revolution in 1960s.These cultivars are resistant to leaf rust and provide an ample control on stem rust (Singh et al., 2004).However, the emergence of a new race of P. graminis "Ug99" in Uganda in 1998 (Pretorius et al., 2000) and its migration to East Africa, Yemen, Sudan and Iran signals a serious threat for sustainability of wheat production in the country.In addition, the evolution of two Ug99 variant TTKST (Wanyera et al,. 2006) and TTSSK (Jin et al., 2008a;Jin et al., 2008b) in Kenya during 2006 and 2007 with virulence to resistance genes Sr24 and Sr36, respectively, further intensifies this threat.
Stripe rust is principally a significant disease of wheat throughout world.It usually infects wheat crops in winter or early spring or at cooler area.Wet conditions and lower temperature (6 to 23°C) favors germination and development of stripe rust epidemics (Cortazar, 1985).P. striiformis outbreak can be adequately restricted by using fungicides at proper time.However, the fungicides application is uneconomic and environment unfriendly.
The incorporation of resistance genes in commercial cultivars is found to be the most inexpensive, efficient, user and environmentally safe method to manage (Ittu, 2000).Recently, over 70 genes for stripe rust resistance have been detected and many have been characterized and incorporated into commercial wheat cultivars (McIntosh et al., 1995(McIntosh et al., , 2003(McIntosh et al., , 2004(McIntosh et al., , 2005(McIntosh et al., , 2008)).A number of them have been mapped (Uauy et al., 2005).Most of them are race-specific showing hypersensitive type of reaction.The utilization of race-specific type of resistance (hypersensitive resistance) has dominated wheat breeding since discovery of genetics of resistance by Biffen (1905), pathogen specialization by Stakman and Levine (1944) and gene-for-gene hypothesis by Flor (1956).A single or a few race specific genes (Yr) confer this type of resistances and they are simple to incorporate into improved germplasm.
This resistance is ephemeral in nature because new virulent races of the pathogen, P. striiformis f sp.tritici, are continuously evolving (Stubbs, 1985) through mutation or migration.Resistance based on single, major, race-specific gene often causes "boom and bust cycles" in wheat production within 5 years after release of a wheat line as a commercial cultivar (Kilpatrick, 1975).
There are some alternatives to resolve this problem such as continual deployment of genetic diversity for resistance in new cultivars (McIntosh, 1992;William et al., 2003), gene pyramiding and use of durable resistance.The race-nonspecific genes mostly express non-hypersensitive type of resistance also called partial resistance at adult plant stage (Van der Plank, 1963;Caldwell, 1968) that is considered to be more durable and the genes that are being incorporated should not be negatively correlated with the agronomic performance.
The developed countries have created well established procedures for selection of resistance against (Parlevliet, 1985).Thus, resistance should be highly rust disease (Roelfs et al., 1992).This increases the chance of discerning resistant parents and progenies.In Pakistan, selection for resistance is usually practiced with inadequate rust epidemic or improper pathotypes.In addition, insufficient information regarding the genetic diversity for virulence in extremely specialized stripe rust population, coupled with quick occurrence of new virulent races have complicated breeding for rust resistance.As a result, only a few of released cultivars exhibit desired resistance against the rust races/pathotypes prevailing in the country (Singh et al., 2004).Thus, there is a need to maintain existing level of resistance and developing new and improved source of resistance by designing a realistic breeding strategy.Therefore, the present study was conducted to improve existing information regarding the levels of resistance in Pakistani bread wheat cultivars against stripe rust (P.striiformis) under field conditions.

MATERIALS AND METHODS
A research was conducted from 2007 to 2011 at National Agricultural Research Center (NARC), Islamabad, Pakistan to determine the level of resistance in Pakistan old and currently growing spring bread wheat (T.aestivum L.) cultivars against stripe rust (P.striiformis) at adult plant stage under natural infection in field conditions.Host genotype consists of 113 commercial spring bread wheat cultivars that have been released in Pakistan for general cultivation since 1930s.These cultivars were provided by Wheat program NARC, Islamabad.Table 3 shows the cultivars that were used in this research, along with their parentage, year of release and presumed Yr genes for resistance.The experiment was multi-year and in each year, the sowing was performed on the mid of November.The experiment comprises of single replication, each entry consisted of six rows of 5 m length, and rows were spaced at 25 cm.Two rows of the susceptible wheat check cultivar Morocco were planted as spreader at perpendicular against each sub-plot to provide maximum opportunity for establishment of rust infection.The environmental conditions were fairly conducive for natural epidemic buildup of stripe rust during all years under study except in 2008 (Table 1, and Figures 1 and 2).The field was regularly visited after the first appearance of stripe rust on susceptible check Morocco.The data was observed three times at tillering stage, jointing stage and flowering stage during February and March.However, data here in this study have been recorded from flag leaf at flowering stage.The field was kept under careful observation from February to the end of April.Percentageof infection of leaf area (diseases severity) was assessed by using modified Cobb scale (Peterson et al., 1948).Infection type (IT) was recorded twice at the time of tillering and grain filling stage.IT was evaluated according to a scale (Table 2) suggested by McNeal et al. (1971).Level of resistance was calculated by comparing relative disease severity (DS) of each of cultivar with that of susceptible check Morocco.The relative DS was calculated by the formula as follows: (Average DS of the cultivar/average DS of Morocco)  100 IT of 7 or higher were considered as compatible (high IT), whereas ITs lower than 7 were regarded as hypersensitive resistant or incompatible (low IT).Non-hypersensitive/partial resistance can only be determined on the genotypes that exhibit a compatible (IT 7 or more).Thus, cultivars that showed a compatible IT and the relative DS < 5% of susceptible check "Morocco" were considered as highly partial resistant, the cultivars with relative

RESULTS
Generally, months of February and March have favorable weather in Islamabad for natural outbreak of stripe rust.
There is always higher rainfall, higher relative humidity and optimum temperature for germination and establishment of colonies of stripe rust fungus during these months.The observation recorded on the IT showed that most of the cultivars had a compatible (7 or above) type of host pathogen-relationship (Table 3).Only six cultivars viz, Tatara, Faisalabad 08, Bahawalpur 94, Sassui, Chakwal 86 and Feakhir-e-Sarhad demonstrated hypersensitive type of resistance against naturally prevailing stripe rust races/pathotypes.This result showed that virulence exists for the most Yr genes in Pakistani old and current bread wheat cultivars.However, data on DS showed that there was a wide range of difference among the cultivars for susceptibility or partial resistance.Likewise, a year specific response of different cultivars was observed in both DS and IT against prevailing stripe rust races/pathotypes.The most interesting result was that a lower IT and DS was recorded on very old cultivars and the cultivars that had unknown Yr genes.For example, the very old cultivars C 518, C 591, C 228, C 217, C 250, C 271 and C 273 showed a very high hypersensitive resistance to moderately susceptibility (IT, 0 to 7).The DS observed on these cultivars was ranged from 0 to 20 MS.These cultivars had been released before 1958 and yet they have not been studied for gene postulation.Perhaps, these cultivars carry some genes for both hypersensitive resistance and non-hypersensitive resistance.Pressure of virulent races/pathotypes for these race-specific genes might not be very common at Islamabad because they are not under cultivation so far.
Among the cultivars that showed compatible type of reactions, four were highly partial resistant (DS < 5% of Morocco), nineteen were partially resistant (DS < 20% of Morocco), forty-three were moderately partial resistant (DS < 50% of Morocco) and forty-one were moderately to highly susceptible.3).Qamar et al. 5891

DISCUSSION
The majority of Pakistani bread wheat cultivars were protected against stripe rust by incorporating the Yr genes, YrA, Yr2, Yr4 Yr6, Yr7, Yr18, Yr9, Yr22 and Yr27; however, the genes, Yr6, Yr7 and Yr9 are occurring more frequently either in combination with other Yr genes or alone (Table 3).Except Yr18, all these genes confer a hypersensitive type (race-specific) of resistance.In this research, the virulence for all these major genes was observed against prevailing races/pathotypes of stripe rust resistance (  2010) observed that the spring wheat cultivar WL 711 (Yr2) showed a reaction type of 0 to 80 S, Tandojam 83 (Yr6+) 0 to 100S, Zarghoon (Yr6+) 0 to 70S, Pak 81 (Yr9, Yr7) 0 to 70S, Blue Silver (Yr6+YrA) 0 to 60S, Zardana (Yr7) 0 to 80S, Inqalab 91 (Yr6, Yr7, and Yr27), 30 to 50S.Ahmad (2004) had reported that four major stripe rust outbreaks have occurred in the country during 1973, 1978, 1995 and 2003 that caused yield losses up to 35, 55, 37.5 and 20%, respectively.These losses occurred due to widespread cultivation of cultivars Pirsabak-85, Pak-81 and Inqalab-91 (Khan and Mumtaz, 2004).The resistance in these cultivars was conferred by the resistance genes Yr7, Yr9 and Yr27 (Table 1).In a study, Mirza et al. (2004)      They also claimed that these cultivars were highly susceptible at the seedling stage and partially resistance at the adult plant stage.In this study, they also found that among sixty tested cultivars by molecular marker, 12% showed bands for Yr18 in polymerase chain reaction (PCR) product.
On the basis of this study, we can conclude that there is considerable genetic diversity among Pakistan wheat cultivars for partial resistance against stripe rust.This type of resistance is always expressed at later stage of development.The genes responsible for this type of resistance are race non-specific, temperature sensitive and usually active at relatively high temperature (Roelfs et al., 1992).These genes usually slow down the progress of disease (Parlevliet and Van Ommeren, 1975) due to pre-haustorial abortion of infection units and post-haustorial growth retardation (Jacobs, 1990;Rubiales and Niks, 1995).They do not provide complete resistance to the host genotype.The resistance conferred by these genes is affected by temperature (Cortazar;1985;Qayoum and Line, 1985;Milus and line, 1986a;Milus and Line, 1986b;Uauy et at., 2005;Dereje and Fininsa, 2007) genetic background of the cultivar in which they have been deployed (Rapilly, 1979), number of the resistance genes present in a cultivar (Singh et al., 2000) and some time aggressiveness of stripe rust races (Niks and Lindhout, 1995;Todorova, 2000;Milus et al., 2006;Milus et al., 2009).Thus, it can be inferred that most of Pakistan cultivars are protected with major genes against stripe rust have cruelly been attacked by the relevant races and granted susceptibility to them.However, a considerable genetic diversity for partial (non-hypersensitive) resistance against stripe rust is also present among them.

Table 1 .
Meteorological data recorded at Water Resource Research Institute, Field Station, NARC, Islamabad.
Figure 1a.Average maximum temperature for 4 months over 5 years.

Table 2 .
The scale used for estimation of stripe rust infection type (IT) in wheat.
> 50% of Morocco were considered as susceptible.

Table 3 .
Parentage, year of release (YR) and presumed Yr genes for resistance in different wheat cultivars and disease severity (DS), relative (100) DS (RDS) tocheck cultivar Morocco and infection type (IT) expressed by them against stripe rust under natural conditions over 4 years.