EVALUATION OF LEVELS OF NON-HYPERSENSITIVE RESISTANCE IN DIFFERENT SPRING WHEAT CULTIVARS TO LEAF RUST

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1 Sarhad J. Agric. Vol.25, No.2, 2009 EVALUATION OF LEVELS OF NON-HYPERSENSITIVE RESISTANCE IN DIFFERENT SPRING WHEAT CULTIVARS TO LEAF RUST MAQSOOD QAMAR*, M. YAQUB MUJAHID*, M. ANWAR KHAN*, RIENT E. NIKS** and M. ASIF* * National Agricultural Research Center, Islamabad, Pakistan ** Plant Breeding Institute, Wagenigen University, The Netherlands ABSTRACT Levels and components of non-hypersensitive resistance in wheat (Triticum aestivum L.) to leaf rust (Puccinia triticina) were studied at seedling and adult plant stage in green house and field conditions during 2001 at Wageningen University, The Netherlands. In seedling stage, level of non-hypersensitive resistance or partial resistance was assessed from latency period (LP), infection frequency (IF), percentage of early aborted infection units without plant cell necrosis (EA-N %) and colony diameter of infection units of single-pustule isolates of Puccinia triticina INRA on 12 spring wheat cultivars/lines. In adult plant stage, non-hypersensitive resistance was evaluated from LP and IF of single-pustule isolate of P. triticina on 8 spring wheat cultivars and 4 lines, while in a field experimental area under disease progress curve (AUDPC) was calculated to assess the level of non-hypersensitive resistance in 8 spring wheat cultivars and two lines. In both seedling and adult plant stage, host genotypes varied for all the parameters studied, indicating a variety of genes with minor effect for non-hypersensitive resistance, since non-hypersensitive resistance is considered to be quantitatively inherited. In partially resistant genotypes the fungus showed a longer LP, lower IF, an increase in EA-N% and reduced colony diameter than on highly susceptible genotypes. Differences between genotypes for the level of nonhypersensitive resistance were larger in adult plant stage than in seedling stage. Also variations among the cultivars for area under disease progress curve (AUDPC) were greater than for components of nonhypersensitive resistance in green house experiments. A long LP was associated with a low IF, increased EA- N% and reduced colony diameter of infection units, indicating that the mechanism responsible for longer LP, lower IF, higher EA-N% and reduced colony diameter might be controlled by the same genes or closely linked genes. Non-hypersensitive resistance measured in seedling stage for LP and IF showed no correlation with the non-hypersensitive resistance measured in adult plant stage. Adult plant stage IF and colony diameter of fungus isolates significantly associated with AUDPC assessed in the field, however a long LP and high IF at seedling stage, a high IF and increased EA-N % were non-significantly associated with low AUDPC. Apparently, nonhypersensitive resistance measured in field cannot completely be explained from the component analysis data in the green house. Key Words: Durable resistance, Hypersensitive resistance, Non-hypersensitive resistance, Partial resistance, Puccinia triticina, Triticum aestivum, Wheat leaf rust Citation: Qamar, M., M.Y. Mujahid, M.A. Khan, R.E. Niks and M. Asif Evaluation of levels of nonhypersensitive resistance in different spring wheat cultivars to leaf rust. Sarhad J. Agric. 25(2): INTRODUCTION More than 50 leaf rust resistance genes and QTLs have been described in wheat (Triticum aestivum) (Blaszczyk, et al. 2004). Many of them are race-specific genes showing hypersensitive resistance (HR), and several are currently being used by breeders to develop new cultivars. However, the resistance provided by these genes can be short-lived as new races of the pathogen, Puccinia triticina, are continuously evolving and acquire virulence against these genes. Resistance based on single, major, race specific gene often become ineffective within five years after release a of wheat line as a commercial cultivar (Kilpatrick, 1975). For example, leaf rust resistance of the spring wheat cultivar MacKellar, was overcome by a newly emerged pathotype 10-1, 3, 9,10,11,12 (Bariana et al. 2007) after a few years of its release. Similarly, in Australia, recently introduced stripe rust (Puccinia striiformis f. sp. tritici) pathotype 134E16A+ evolved to produce to variants, one with added virulence for Yr10 and other Yr17 during (Wellings, 2007). Currently, more attention is given to other forms of resistance, such as non-hypersensitive resistance (NHR), which are assumed to be more durable than HR. A resistance is considered to be durable if it has been observed in a cultivar grown on large scale for long time in different environments, favourable for the pathogens, without loosing its effectiveness (Johnson, 1984). Partial resistance is also called Non-hypersensitive as narrated by Parlevliet, et al. (1980). Theoretically, it was defined by Van der Plank (1963) and applied to the wheat leaf rust by Caldwell (1968). The term Non-hypersensitive resistance is denoted to the reduced rate of epidemic buildup of a pathogen on a

2 Maqsood Qamar, at al. Evaluation of non-persensitive resistance in spring wheat to leaf rust 234 susceptible host (Parlevliet and Van Ommeren, 1975). Though Non-hypersensitive resistance is partitioned into longer latency period, lower infection frequency (IF) and smaller spore production, but Lalentcy period (LP) is being the most important component (Shaner and Finney, 1980, Teng et al., 1977). Histologically, NHR in wheat to leaf rust was characterised by a reduced rate of fungal growth, resulting in smaller colonies with late and less sporulation as compared to highly suceptible cultivars (Jacobs, 1989). A small group of leaf rust resistance genes are known as "slow rusting genes (NHR)", such as Lr34 (Singh, and Huerta-Espino ) and Lr46 (Martínez, et al. 2001). They provide durable and non-specific adult plant resistance but their effect is more reduced than that of race-specific genes. Lr34 is pleiotropic or completely linked toyr18, which confers NHR to stripe rust. A similar tight linkage or pleiotropic effect was observed between Lr46 and Yr29, also a non-hypersensitive resistance (NHR) gene for stripe rust. In order to obtain cultivars with good levels of protection under high disease pressure, several of these slow rusting gene complexes need to be combined. Wheat breeders can use non-hypersensitive resistant genes as a complement to race-specific genes. Singh et al. (2000) identified spring wheat lines with high NHR levels, reaching up to immunity level for leaf rust, through compiling genes with additive effect for non-hypersensitive resistance. Polycyclic tests (field tests) can be used to determine the exact levels of NHR. Because of difficulties in the field tests, however, monocyclic tests or histological studies can also be used by evaluating one or more individual components. Little information is available on the relationship between components measured in green house and epidemic parameters assessed in the field. Therefore, this study was initiated to determine the level of non-hypersensitive resistance in some spring wheat cultivars/lines at different development stages and prediction of level of PR in field by components analysed in greenhouse. MATERIALS AND METHODS Levels of non-hypersensitive resistance in spring wheat to leaf rust were evaluated in Laboratory of plant breeding, Wageningen University the Netherlands during 2001 by using different spring wheat genotypes. The study was comprised some monocyclic tests (at seedling stage and adult plant stage) in green house and a polycyclic test at field. Non-hypersensitivity resistant cultivars BH1146 and Akabozu as well as susceptible cultivar Skalavatis Bearded, Skalavatis Unbearded, Thatcher, Lalbahadur were used as reference cultivars. Cultivars from the list of recommended cultivars in the Netherlands (Baldus, Anemos and Minaret) were studied to determine levels of non-hypersensitive resistance. Near isogenic lines (NILs) Lalbahadur Lr34 Lalbahadur Lr46, Thatcher Lr12 and Thatcher Lr34 were included to study the effect of Lr genes. Akabozu could not be included in adult plant stage experiment due to its very late maturing characteristics. A newly emerged French isolate of P. triticina namely, INRA, provided by Dr Gouyot used for all experiments in green house and field. This isolate was purified by single pustule isolation and multiplied on young seedlings of Pavon 76, a wheat cultivar. As this isolate was virulent to hypersensitivity resistance genes Lr1, Lr10 and Lr13 (Niks, personnel communication) in Pavon 76, so, Pavon 76 was used for its multiplication. Seedling Stage Test Ten days after sowing, primary leaves of six seedlings of each cultivar/lines raised in wooden boxes measuring 30 x 45 cm 2 were inoculated at the upper surface in a settling tower. Urediospores mixed with lycopodium spores (1:10, vol. / vol.) were 4 mg per box. In each box a greased glass slide was placed for later determination of inoculation density. The spore density per cm 2 was measured and statistically analysed by using F-test. The average inoculation density was about 231/cm 2. The inoculated seedlings were incubated under darkness for 19 hours at 100% relative humidity. After incubation process, the seedlings were transferred to a green house chamber. Four seedlings per cultivar were used to estimate LP, IF and IT while two were used for histological test. LP was estimated by using the method of Parlevliet (1975). Infection frequency was recorded using a metal strip with an opening measuring 2 x 0.5 cm 2 (Parlevliet and Kuiper, 1977). Infection type was determined by using 0-9 rating scale (McNeal et al. 1971). Four days after inoculation, about 3 cm long pieces of central parts of two leaves per cultivar were collected for histological study. The leaf segments were boiled in lacto-phenol / ethanol (2:1) and stained in 0.1% Uvitex following the procedure of Rohringer et al. (1977). The leaf segments mounted in glycerol were observed for percentage of established colonies, percentage of early aborted young infection units without plant cell necrosis (EA-N %), percentage of early aborted infection units with plant cell necrosis (EA+N %) and mature colony diameter under an auto-fluorescence microscope at 100x. All the infection units (mycelial structures originating from one urediospore that had formed at least one haustorium mother cell); number of haustorium mother cells and presence of necrosis (as indicated by plant cell auto-florescence at infection sites) were recorded. The infection units having less than six haustorium mother cells were considered as early aborted

3 Sarhad J. Agric. Vol.25, No.2, and vice versa for established colonies. Ten established colonies per leaf segment were measured with an eyepiece micrometer for measuring colony diameter. Adult Plant Stage Test All the cultivars were sown in pots of 15 cm diameter in a green house at several consecutive sowing dates for getting plants at the same developmental stage (fully expended young flag leaves) for inoculation. Per cultivar three pots were used and each pot was considered as a replication. The plants were inoculated by dusting urediospores mixed with lycopodium spores one milligram per pot. Under complete darkness, the inoculated plants were kept overnight (19 hours) in a high humidity chamber (100% relative humidity) and then transferred to green house. Latency period, infection frequency and infection type were recorded on the flag leaf. Three flag leaves per pot were used to measure LP and IF. Observations started five days (120 hours) after inoculation to estimate LP. IF and IT were recorded after completion of sporulation. LP, IF and IT were measured on the central part of leaves by using same method as in seedlings test. Field Experiment All cultivars were planted during the month of May in the field using randomized complete block design (RCBD) with 3 replicates having plot size of 1.25m x m. The plants of susceptible wheat line 8860 in pots, inoculated with isolate INRA were used as sporulating spreader. The spreader pots were kept there for two weeks for effective infection. The experimental plot was irrigated uniformly at regular intervals to enhance disease severity. Scoring for the disease severity was started two weeks after inoculation when the susceptible check cultivar, Thatcher, showed approximately 30 pustules on upper three leaves per tiller. Twenty tillers on each genotype within each replication were sampled randomly and numbers of pustules on upper three leaves were counted. Observations were recorded five times with an interval of four days. Area under the disease progress curve (AUDPC), which is a better indicator of disease expression over time (Van der Plank, 1963; Chaurasia et al. 1999), was calculated by the formula given by Joshi et al., (2002) and Joshi and Chand (2002). a AUDPC = [{ Y i + Y (i + 1) }/2 ] x {t (i + 1) - t i }, i = 1. Where, a = total number of observation days, ti = day i (time) expressed as number of days after sowing, t (i + 1) t i = Time (days) between two disease observation dates and Y i = Number of pustules at time t i. Data were subjected to statistical analysis using analysis of variance (Steel and Torrie, 1981) to determine the level of significant difference between genotypes. Duncan s Multiple Range Test (P = 0.05) was used to compare the genotypic means. Pearson s correlation coefficient was used to describe the association between different components of non-hypersensitive resistance and correlation between levels of non-hypersensitive resistance measured at different plant development stages. RESULTS AND DISCUSSION Seedling Stage Test Most important component of non-hypersensitive resistance is latency period (LP) which can only be determined on the genotypes that exhibit a compatible infection type (IT = 7 or high). All cultivars / lines studied in this study showed a high IT (7-9) to P. triticina and EA+N% remained lower than 5%, which clearly indicates absence of hypersensitive reaction in this material. Analysis of variance showed that at seedling stage (primary leaf), the variation among cultivars/lines was highly significant for LP, IF and colony diameter where as it was significant for EA-N% (Table I). Variation among cultivars was continuous and narrow indicating effect of more than one minor gene (Table I). The longest average LP (on Akabozu) was only about 12% longer than the shortest LP (on Skalavatis Unbearded). Skalavatis Bearded displayed significantly longer LP than Skalavatis Unbearded. Lalbahadur and its isolines did not differ significantly. Average LP of the rust fungus on any of Dutch cultivars (Minaret, Baldus and Anemos) was not as longer as on Akabozu. However, they showed lower susceptibility than reference cultivar Skalavatis Unbearded. The lines with Lr34 gene, Thatcher Lr34 and Lalbahadur Lr34 showed the same level of non-hypersensitive resistance. The lowest IF was observed on partially resistant cultivar, Thatcher Lr34 (48 pustules cm -2 ) and Akabozu (48.8 pustules cm -2 ) whereas the highest IF on Lalbahadur and Lalbahadur Lr46 (107 pustules cm -2 ). The three Dutch cultivars did not differ significantly from each other for IF (Table I). The IF on Dutch cultivars

4 Maqsood Qamar, at al. Evaluation of non-persensitive resistance in spring wheat to leaf rust 236 except Baldus was similar to that in reference cultivar Akabozu. There was also no difference between Skalavatis Bearded and Unbearded. Lalbahadur and its isolines also showed similar level of susceptibility. Difference among the cultivars/lines for EA-N% was significant in the seedling stage experiment (Table I). The extremes were Skalavatis Bearded (2.4%) and Thatcher Lr34 (12.8%). Variation between cultivars for colony diameter of wheat leaf rust was continuous (Table I). The extremes were Skalavatis Bearded (0.818 mm) and BH1146 (0.567 mm). There was no difference among Dutch cultivars for colony diameter but the average colony diameter of the leaf rust was significantly smaller in Dutch cultivars than in susceptible cultivar, Skalavatis Bearded. There was non-significant difference between Lalbahadur and its isolines for colony diameter, indicating that Lr34 and Lr46 are less effective at seedling stage. Adult Plant Stage Test Highly significant difference among cultivars/lines were noticed for latency period (LP) and infection frequency (IF) at adult plant stage (on flag leaf), partially resistance cultivar BH 1146 showed the longest LP whereas the susceptible check cultivar, Thatcher showed the shortest LP (Table II). The lowest IF of leaf rust pustule was observed on Lalbahadur Lr 34while the IF frequency was the highest on flag leaves of Lalbahadur (Table II). Field Experiment Area under disease progress curve (AUDPC) for ten-spring wheat cultivars to one isolate of P. triticina (INRA) was calculated in a polycyclic experiment in the field. Variation between cultivars for AUDPC was highly significant (Table III). Differences among cultivars for AUDPC were higher in the field than for single components in the green house. Values registered for this parameter varied between 1.9 for Akabozu and 48.8 for Thatcher. Akabozu was the most resistant cultivar followed by partially resistant cultivar BH1146 and Thatcher Lr34. The most susceptible was Thatcher followed by Thatcher Lr12 and Skalavatis Bearded. The variations among cultivars/lines for LP against leaf rust at seedling stage as well as at adult plant stage were continuous and significant (Table I-II). These results suggest that the cultivars/lines varied for LP against wheat leaf rust. Ranges here for LP were comparable to the ranges observed in winter wheat (Ohm and Shaner, 1976; Shaner et al., 1978), in spring wheat (Broers and Jacobs, 1989; Rubiales and Niks, 1995) and in barley/barley leaf rust (Parlevliet, 1975; Parlevliet and Kuiper 1977 and Niks et al., 2000). It is persuasive to speculate about the implication of continuity of the variation. Possibly a variety of LP-prolonging genes, with minor effects may exist in the spring wheat germplam. This is in agreement with the result of Broers and Jacobs (1989) who reported that the LP-prolonging genes probably have unequal effect and the wheat cultivars Akabozu, Westphal 12A and BH1146 possessed 2, 3 and 3-4 genes respectively. The results of this research were also in agreement with the result of Singh et al. (2000). They believed that the resistance in some partially resistant spring wheat lines might involve a combination of about 4-5 genes in addition to Lr34. Relatively, LP was longer in adult plant stage than in seedling stage. Similar results were found in spring wheat (Broers, 1989a; Rubiales and Niks, 1995), in winter wheat (Ohm and Shaner, 1976; Pretorius et al., 1988) and in barely/barley leaf rust (Parlevliet, 1975). The increase in resistance with increase in growth stage might be due to: a) morphological and physiological differences in leaf tissues and b) LP-prolonging genes may not be expressed in seedling stage (Qi et al., 1998) or LP-prolonging genes may better express in adult plant stage. Since seedling stage and adult plant stage experiments were not conducted at the same time, so increase in adult plant LP also has been caused by differences in environmental conditions. Generally, differences between susceptible and partially resistant host genotypes were more pronounced in adult plant stage than in seedling for LP. It was observed that there was non-significant difference between Lalbahadur and its isolines as well as between Thatcher and Thatcher Lr34 in primary leaf (Table I) but they differed significantly in flag leaf (Table II). These results indicate that the Lr34 and Lr46 genes are more active in flag leaf than in primary leaf. This result is in agreement with the observation by Rubiales and Niks (1995) on Lr34 gene. Continuous and significant variation was found for IF among the spring wheat cultivars/lines in seedling stage (Table I) as well as in adult plant stage (Table II). These research findings are quite in agreement with conclusions drawn by Broers (1989a) in spring wheat, Ohm and Shaner (1976) and Shaner et al. (1978) in winter wheat and Parlevliet (1975) and Parlevliet and Kuiper (1977). Ittu (2000) and Todorova (2000) also found significant host plant effect for IF to wheat leaf rust at seedling stage.

5 Sarhad J. Agric. Vol.25, No.2, Significant host genotype effect was observed for EA-N% and colony diameter (Table I) on primary leaf in seedling stage. Jacobs (1990) and Rubiales and Niks (1995) also observed significant differences among spring wheat genotypes for percentage of early aborted structure without plant cell necrosis and colony size. In this study, percentage of early aborted infection units of isolate "INRA" without plant cell necrosis was ranged from 2.4 (Skalavatis Bearded) to 12% (Thatcher Lr34). In barley to leaf rust, Niks (1982) reported that percentage of early aborted infection units without plant cell necrosis was ranged from 9 (C-120) to 59% (Vada). It seems that early abortion of infection units has greater effect on low IF in barley/barley leaf rust than in wheat to leaf rust. Thatcher Lr34 Akabozu, BH1146 and Lalbahadur Lr34 showed higher percentage of early aborted infection units without plant cell necrosis (EA-N %) and reduced colony diameter. These findings in percentage of early aborted infection units run parallel with those found for LP in primary leaf (Table I). These results suggest that non-hypersensitive resistance leads to early abortion of infection units, reduction in haustorial formation, reduced colony size and a long LP. Such effects were reported for non-hypersensitive resistance by Jacobs (1989, 1990), Rubiales and Niks (1995) and by Lee and Shaner (1984), although the latter did not find early aborted infection units. The values obtained for AUDPC (Table III) allowed the discrimination of wheat genotypes, in terms of disease progress, from very slow (1.9 in late maturing partially resistant cultivar Akabozu) to very fast (48.8 in early maturing susceptible cultivar Thatcher). This result suggests that cultivars characterized by a slower development of disease (AUDPC <10.9), could be considered non-hypersensitive resistant to wheat leaf rust. Based on this data it is suggested that Akabozu, BH1146, Thatcher Lr34 could be considered non-hypersensitive type. Broers (1989b) and Rubiales and Niks (1995) also reported the high level of resistance in Akabozu, BH1146 and Thatcher Lr34. Accordingly, Dutch cultivars, Baldus and Anemos could be considered partially resistant. This result is in agreement with that in the list of recommended cultivars in the Netherlands (Anonymous, 2001) where Baldus was ranked as high, Anemos as medium and Minaret as rather poor resistant to leaf rust. However, in this study, late maturing cultivars had a lower ADUPC than early maturing cultivars. It could be possible that very early cultivars Thatcher and its isolines were assessed more susceptible than they were and very late cultivar Akabozu more resistant than it was. Variation among cultivars for AUDPC was greater in the field study than for single components in green house experiments (monocyclic tests), as was observed for the barley/barely leaf rust system (Parlevliet, 1975). More clear differences among the cultivars for AUDPC measured in field could be explained by cumulative effect of AUDPC, as AUDPC is an impression of increase of the infection in time. It is also possible that relatively lower temperature outside green house caused longer LP on partially resistant wheat genotypes in field than in green house. Table I. Mean LP, IF, EA-N, EA+N colony diameter on primary leaf of 12 spring wheat cultivars at seedling stage to an isolate of P. triticina (INRA) Cultivar* LP (hours) IF (pustules cm -2 ) A-N% EA+N % Colony Diameter (mm) Skalavatis Unbearded 126 c x 78.7 bcd 4.0ab bcd Skalavatis Bearded 137 b 70.7 cde 2.4a a Lalbahadur Lr b ab 5.1ab cde Minaret 140 b 62.0 de 4.7ab abc Lalbahadur 140 b a 3.3a cde Lalbahadur Lr b 89.0 abc 7.0abc e Baldus 142 b 78.4 bcd 3.1a bcd Thatcher 142 b 75.0 bcd 5.3ab ab Anemos 144 ab 67.5 cde 7.1abc bcd Thatcher Lr ab 48.0 e 12.8c de BH a 52.5 de 8.5abc e Akabozu 153 a 48.8 e 9.7bc bcd Mean P Value for cultivar effect * Cultivars have been written in ascending order on the basis of average LP. x Numbers within a column followed by a letter in common are not significantly different (Duncan multiple range test, P 0.05).

6 Maqsood Qamar, at al. Evaluation of non-persensitive resistance in spring wheat to leaf rust 238 Table II. Relative portion (%) of mean latency period (LP) and mean Infection frequency (IF) to check cultivar Thatcher on flag leaf of 12 spring wheat cultivars/isogenic lines at adult plant stage to one isolate of P. triticina (INRA) Cultivar* LP% IF% Thatcher c x 100 cd Minaret c 153 b Baldus c 124 c Thatcher c 74d e Lalbahadur c 104 cd Skalavatis Bearded c 121 c Skalavatis Unbearded c 120 c Anemos c 74 de Lalbahadur b 57 e Lalbahadur b 194 a Thatcher ab 101 cd BH a 64 e Mean LSD (P = 0.05) Level of significance * Cultivars have been written in ascending order on the basis of average LP. x Numbers within a column followed by a letter in common are not significantly different (Duncan multiple range test, P 0.05). Table III. Areas under disease progress curve on ten spring wheat cultivars/lines to one isolate of P. riticina (INRA) and relative proportion (%) to check cultivar Thatcher and stage of development at first assessment date Cultivars AUDPC % Development stage a Akabozu 1.9a x 3.9 Late tillering BH ab % flag leaf emergence Thatcher Lr34 5.6bc % heading Baldus 8.7cd % flag leaf emergence (1% heading) Anemos 10.9d % flag leaf emergence (5% heading) Minaret 18.3e % flag leaf emergence (1% heading) Skalavatis (UB) y 23.2f % heading Skalavatis Bearded 31.6g % flag leaf emergence Thatcher Lr g % heading Thatcher 48.8h % heading Level of Sig x Numbers followed by the same letters are not significantly different (Duncan multiple range test, P = 0.05). a Development stage at the time of first assessment date y Skalavatis Unbearded Correlation between Components Both in primary leaf and in flag leaf (Table IV) a longer LP of wheat leaf rust fungus was significantly associated with a low IF. This association was stronger in flag leaf than in primary leaf, suggesting that the components at flag leaf are better indicators of selection for non-hypersensitive resistance than the components at primary leaf. The correlation (r = -0.80; P<0.01) between longer LP at flag leaf and reduced colony diameter was also highly significant and negative. These findings propound that the mechanism responsible for longer LP, lower IF and reduced colony size might be controlled by different genes. The results obtained here are in agreement with the observations reported by Rubiales and Niks (1995) in spring wheat and Lee and Shaner (1984) in winter wheat leaf rust and in barley leaf rust (Parlevliet, 1986), where LP, colony size and IF were seem to be coded by the same genes or closely linked genes. Similarly, LP and colony size and colony size and IF, respectively, seem to be coded by the same genes or closely linked genes. Broers (1989a and 1989b) did not agree with this idea and suggested that LP, IF and colony size might be inherited independently.

7 Sarhad J. Agric. Vol.25, No.2, Table IV Pearson s correlation coefficient between different components of non-hypersensitive resistance Seedling stage IF Adult Plant stage LP Adult Plant stage IF EA-N % Colony diameter Seedling stage LP (0.05) (0.03) (0.33) Seedling stage IF (0.02) (0.92) Adult Plant stage LP (0.002) 0.65 (0.03) (0.003) Adult Plant stage IF (0.09) 0.40 (0.22) EA-N % (0.07) Values in parenthesis represent probability (P-Values) Correlation between LP and percentage of early aborted infection units without plant cell necrosis (EA- N%) both at seedling stage and adult plant stage were positive and significant (r = 0.63; P < 0.03) (Table IV). This result suggests that same genes might be coded for longer LP and a higher percentage of early aborted infection units. These results also agree with those obtained by Jacobs (1990) and Rubiales and Niks (1995) who found that a longer LP was significantly associated with a reduced percentage of early aborted infection units. Similarly, the correlation between a lower IF at seedling stage and a higher percentage of early aborted infection units was significant (r = -0.65, P 0.02). This result is agreed with that reported by Jacobs (1990) in wheat to leaf rust. This indicates that non-hypersensitive resistance not only leads to a postponed sporulation of fungus but also seems to be responsible for reduced colony size, early abortion of infection units and lower IF of wheat leaf rust in partially resistant genotypes. Correlation between different Plant Development Stages Non-significant positive correlation coefficients between seedling LP and adult plant stage LP was observed. Similarly, the association between seedling stage IF and adult plant stage IF was negative and nonsignificant (Table V). This result indicates that components measured in seedling stage could not fully explain the adult plant effects. This is true especially for the cultivars like Lalbahadur and its isolines, Dutch cultivars Minaret, Baldus and Anemos and Thatcher and its isolines. These cultivars did not differ significantly at seedling stage from Thatcher for LP (Table I). In adult plant stage, Thatcher Lr34, Lalbahadur Lr34 and Lalbahadur Lr46 differed significantly from Thatcher (Table II). Parlevliet (1987) and Broers (1989a) also believed that non-hypersensitive resistance at seedling stage is a poor indicator for non-hypersensitive resistance than at adult plant stage. So seedling stage tests are advisable only as rough screening tests for components of non-hypersensitive resistance. Qi et al. (1998) observed a moderate correlation between seedling stage LP and adult plant stage LP in barley/barley leaf rust. They identified six QTLs for Non-hypersensitive resistance. Three QTLs were effective in seedling stage and five were effective in adult plant stage. Table V Pearson s correlation coefficient between developments Adult Plant stage LP Seedling stage LP 0.51 (0.11) Seedling stage IF (0.47) Values in parenthesis represent probability (P-Values) Adult Plant stage IF (0.28) 0.38 (0.25) Association between level of non-hypersensitive resistance measured in green house both at seedling stage and adult plant stage and level of non-hypersensitive resistance measured in the field (AUDPC), expressed in Pearson's correlation coefficient was moderate (Table VI). A large colony size of infection unit at seedling stage was highly significantly correlated (r = 0.76, P 0.05) with a greater AUDPC in the field. Correlation between AUDPC and flag leaf LP was significant (r = 0.65, P 0.01). A longer LP at seedling stage and higher percentage of early aborted infection units without plant cell necrosis were non-significantly associated with a low AUDPC in the field. Correlation between AUDPC and primary leaf IF as well as flag leaf IF were nonsignificant. This suggests that flag leaf LP and colony size of infection units in green house can predict AUDPC in field to some extent. These findings here agree to some extent with those observed by Parlevliet (1987) and Broers (1989a), who claimed that LP is a better indicator of non-hypersensitive resistance in the field. IF and colony size (spore production per pustule) are also good indicators but they are more difficult to evaluate accurately.

8 Maqsood Qamar, at al. Evaluation of non-persensitive resistance in spring wheat to leaf rust 240 So these values suggest that individual components measured in green house could not exactly predict AUDPC measured in field conditions. These findings here agree with those observed by Broers (1989a), who claimed that individual components could not fully explain the variation found for the epidemiological parameters in the field. Moreover the performance of NHR in the field is a combine effect of components determined. Table VI Pearson s correlation coefficient between AUDPC and components of PR measured in green house Components AUDPC Probability Primary leaf LP Flag leaf LP -0.65* 0.05 Primary leaf IF Flag leaf IF EA-N% Colony diameter 0.76** 0.01 * Significant at P = 0.05 ** Significant at P = 0.01 In general, it seems that the PR as measured in field cannot be fully completely explained from the component analysis data. This discrepancy can be explained by differences in earliness, variation in environmental conditions, especially temperature, difference in incubation period and variance in initial inoculum density. It is concluded that variations among the host genotype for level of non-hypersensitive resistance will be greater in adult plant stage than that of seedling stage. A long LP was linked with a low IF, increased EA-N% and reduced diameter of infection units which indicates the mechanism responsible for longer LP, lower IF, higher EA-N% and reduced EA-N% might be controlled by same genes or closely linked genes. Similarly, nonhypersensitive resistance as measured in field cannot be fully completely explained from the component analyzed data. However, the green house tests for individual components can be performed for screening purpose. REFERENCES Anonymous List of recommended cultivars in the Netherlands. Ministry of Agric. Govt. of The Netherlands. pp Broers, L.H.M. 1989a. Influence of development stage and host genotype on three components of partial resistance in wheat to wheat leaf rust. Euphytica. 44: Broers, L.H.M. 1989b. Partial resistance to wheat leaf rust in 18 spring wheat cultivars. Euphytica. 44: Broers, L.H.M. and T. Jacobs The inheritance of host plant effect on latency period of wheat leaf rust in spring wheat. II: Number of segregating factors and evidence for transgressive segregation in F3 and F5 generation. Euphytica. 44: Bariana, H.S., G.N. Brown, U.K. Bansal, H. Miah, G.E. Standen and M. Lu Breeding triple rust resistant wheat cultivars for Australia using conventional and marker-assisted selection technologies. Aust. J. Agric. Res. 58: Blaszczyk, L., H. Goyeau, X.Q. Huang, M. Doder, L. Stepien and J. Chelkowski Identifying leaf rust resistance genes and mapping gene Lr37 on the microsatellite map of wheat. Cell. Mol. Biol. Lett.9: pp Caldwell, R.M Breeding for general and/or specific plant disease resistance. In: Proc. 3 rd Int l wheat Genetics Symp. Canberra (Australia). pp Chaurasia, S., A.K. Joshi, R. Dhari and R. Chand Resistance to foliar blight of wheat: A search. Genet. Res. Crop Evol. 46: Ittu, M Components of Partial Resistance to Leaf Rust in Wheat. Acta. Phytopathologica et Entomologica Hungarica. 35: Jacobs, T Haustorium formation and cell wall oppositions in susceptible and partially resistant wheat and barley seedlings infected with leaf rust. J. Phytopath. 127: Jacobs, T Abortion of infection structures of wheat leaf rust in susceptible and partially resistant wheat genotypes. Euphytica. 45: Johson, R A critical analysis of durable resistance. Annual Review of Plant Path. 22: Joshi, A.K. and R. Chand Variation and inheritance of leaf angle and its relationship with resistance to spot blotch in wheat (Triticum aestivum). Euphytica. 123: Joshi, A.K., R. Chand and B. Arun Relationship of plant height and days to maturity with resistance to spot blotch in wheat (Triticum aestivum). Euphytica. 124: Kilpatrick, R.A New wheat cultivars and longevity of rust resistance U.S. Agric. Res. Serv. North-east Reg. ARS-NE NE-64. Lee, T.S. and G. Shaner Infection process of Puccinia recondita in Slow- and fast-rusting wheat cultivars. Phytopath. 12:

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10 Maqsood Qamar, at al. Evaluation of non-persensitive resistance in spring wheat to leaf rust 242