Plants are generally more susceptible to disease in early stage than in late period and this could be due to the fact that there is an increase in resistance through time, with plants already resistance to a pathogen increasing their ability to overcome infection and colonization at a later growth stage (Develey-Riviere and Galiana, 2007). Many studies have been published on this phenomenon and reported for large number of crop plants.

So far different barley (Hordeum vulgaris L.) genotypes to Puccinia hordei were assessed in greenhouse tests at seedling growth stages and in the field at adult plant growth stages (Marcel et al., 2008; Yeo, 2008). For this barley leaf rust P. hordei isolate 1.2.1 was used to evaluate the level of partial resistance. Also, a number of quantitative trait loci (QTLs) were found to confer resistance to Puccinia triticina (Pt), Puccinia hordei-murini (Phm), and Puccinia hordei-secalini(Phs) when inoculated on three barley populations (L94xVada, Steptoe x Morex, and Oregon Wolfe Barley populations) (Bettgenhaeuser et al., 2014). From these, diversity of QTLs govern resistance in host system and also indicated that qualitative R genes are not involved in near nonhost resistance, however, genes conferring partial resistance to adapted pathogens may play role in nonhost resistance (Jafary et al., 2008; Niks, 2014).The effects of QTLs developed from those three barley populations, namely: Rphq2, Rphq11 and Rphq16 are plant stage dependent (Marcel et al., 2007), that they are effective only at seedling stage. Rphq3 on the other hand has a strong and consistent effect at both seedling and adult plant stages. Rnhq is a QTL for nonhost resistance, and was effective to Phm and Phs at seedling stage (Jafary et al., 2006).

Near-isogenic lines (NILs) differing with regard to disease resistance QTLs provide valuable material for a more detailed study into the genetic and molecular dissection of the mechanisms underlies the emergence of disease resistance during host development. Such NILs allows the evaluation of QTL in a nearly uniform genetic background, overcoming the difficulties of identifying QTL phenotypes (Marcel et al., 2007). QTL-NILs do not only provide a better estimate for the effect of single QTL alleles, but also provide a better insight into QTL x pathogen and QTL x environment interactions.

Therefore, this study used the NILs developed for partial resistance QTLs Rphq2, Rphq3, Rphq11, and Rphq16, and a nonhost resistance QTL, Rnhq with the objective to evaluate whether these QTLs show plant growth stage dependency on resistance to homologous and heterologous rust pathogen isolates. 

For inoculation of those plants at each stage, 1 mg (P. hordei 1.2.1) and 2 mg (heterologous rusts) of spores diluted 10 times with lycopodium spores were used as inoculum for each pot. Then, the inoculum was sprayed over the plants as uniformly as possible. The plants were then placed in a humidity chamber overnight (8 h) at 100% relative humidity in the dark at 18ºC to allow the spores to germinate. After incubation, the plants were transferred to a greenhouse compartment where the temperature was set at 14 ± 3°C with 30 to 70% relative humidity.

Data collection and analysis

Five to eight days after inoculation, when the infection flecks appear, observation zones containing proper density of flecks were delimited by marker. The observations started when the susceptible line showed the first mature pustules. The latency period (LP50) of three to five plants per QTL-NILs and two to three plants per parental line per stage in three replications and averages were considered to reflect the level of partial resistance for each QTL-NILs and donor lines. For all lines two leaves per pot per plant stages were scored.

For heterologous rusts, the frequency of visible infection sites (VIS; the number of both flecks and pustules per cm²) and infection frequency (IF; the number of pustules per cm²) following Jafary et al. (2006) were evaluated. Also, the latency period (LP) of the fungi on each plant was calculated. The data collected were analyzed using GenStat 12th edition statistical software. 

As shown in Table 2, for infection with P. triticina, effect of Rnhq-V showed significant effect on Su-Rnhq-V only at second leaf stage. In case of Phm at first leaf stage, the effect is not significant; however there was positive effect at second leaf stage. It had positive effect on RLP at first and second leaf stage for Phs. However, the effect seems to decrease at second and third leaf stages. In general, the effect of Rnhq-V on Su-Rnhq-V on RLP tends to decrease with increment in leaf layer in all three tested inappropriate rust species. Parental line ‘Vada’ showed no sporulating uredia on both P. triticina and Phm except very few on Phs, as a result RLP was not scored for this line on those two rust species (Table 3). 

Adequate standardization of plant age, inoculum density and quality, and environmental conditions are required to recognize true differences in susceptibility. In this research, the environmental conditions during plant growth prior to inoculation, during exposure of inoculated plants to dew, and during post-dew development were sufficiently defined and controlled to provide an acceptable level of variation in disease development attributable solely to environmental factors.

Near-isogenic lines (NILs) differing with regard to disease resistance QTLs provide valuable material for a more detailed study into the genetic and molecular dissection of the mechanisms underlying the emergence of disease resistance during host development. Such NILs allows the evaluation of QTL in a nearly uniform genetic background, overcoming the difficulties of identifying QTL phenotypes (Marcel et al., 2007). QTL-NILs do not only provide a better estimate for the effect of single QTL alleles, but also provide a better insight into QTL x pathogen and QTL x environment interactions.

In this study, three most effective QTL-NILs with SusPtrit background, Rphq2, Rphq3, and Rphq11 contributed in resistance to homologous rust were used to evaluate the effect of each QTL at different plant development stages.

As shown in Figure 3, Rphq2 on Su-Rphq2 had higher effect from first to third leaf layers as compared to Rphq3 on Su-Rphq3. Its effect starts to relatively decrease after fourth leaf layers in SusPtrit background NILs. Rphq2 is effective at seedling stage and gradually lose its effect as plant grows higher. The effect of Rphq3 on Su-Rphq3 consistently increases from first leaf layer to adult plant stage. This consistency in effect indicates that this QTL is stage independent. On the other hand, Rphq11 on Su-Rphq11 had no as such statistically significant difference from Rphq2 and Rphq3 on Su-Rphq2 and Su-Rphq3, respectively. It tended to have an effect intermediate between Rphq2 and Rphq3 (Figure 3).

In previous studies (Niks et al., 2000; Marcel et al., 2008) it was reported that Rphq2 had a strong effect in the seedling stage but almost no effect in adult plant stage, while Rphq3 was effective in seedling and adult plant stages indicating that Rphq3 is plant stage independent. Also it was reported (Yeo, 2008) that, Rphq11 was effective in seedling stage. However, in present study, it was observed that those QTLs which were effective at seedling stage were also effective across all plant stages with gradually decreasing effects as plants grew older. Previously, these QTLs were reported to be plant stage dependent because they were mapped at seedlings stage but not at adult stage. Here the QTL-NILs used for this study were only those in which QTLs of interest is in the plant material. So, that the QTLs which were reported to be plant stage dependent may not be as reported due to the fact that QTLs do function throughout the plant stage but its effect is smaller than other QTLs detected in a mapping population. However, the effect of Rphq3 is consistent in all leaf layers observed, indicating that this QTL is plant stage independent as reported in previous studies. As far as Rnhq is concerned, Su-Rnhq-V had positive effect in resistance at first leaf layer; however, its effect seems to decrease as plants grew older.

Generally, as reviewed in detail in Develey-Rivière and Galiana (2007), resistance acquisition during development has been reported for a large number of crops from both monocots and dicots (wheat, rice, maize, soybean, common bean, tomato, grapevine, tobacco). This resistance to diseases associated with major transitions happening during plant life cycle (Kus et al., 2002; Poethig, 2003; Rusterucci et al., 2005; Baurle and Dean, 2006), function of the maturity of tissue or organ (Zeier, 2005), acquired resistance with development (Panter et al., 2002; Rusterucci et al., 2005), the functional regulation of plant resistance (R) genes (Panter et al., 2002; McDowell et al., 2005), induction of defense mechanisms (Cameron and Zaton, 2004; Dong, 2004; Hugot et al., 2004; Xu et al., 2006). In this study, we observed the same phenomenon of resistance at different developmental stage of nearly isogenic barley lines. 

The authors have not declared any conflict of interests.

This work is part of M.Sc. thesis of the senior author. The study was financially supported by Netherlands Fellowship Program (NFP-AP).