Abstract

Effect of incubation time, 9.5 versus 17.5 hr in the dew chamber, was determined on barley lines inoculated with the inappropriate wheat leaf rust fungus, Puccinia triticina. On the susceptible reference wheat line 8860, both incubation times gave a similar infection frequency, but on the barley lines, the longer incubation period resulted in a one order of magnitude higher infection frequency than under the more natural night length of 9.5 hr. We presume that the pre-haustorial resistance is not well expressed when, at the time of haustorium formation, the plants are not exposed to daylight. The practical implication of our results is that experiments to screen for quantitative resistance to rust pathogens, should be incubated at natural night lengths, and not at the 17 to 24 hr as practised at many labs.

Introduction

Rust fungi are among the largest and economically most important plant pathogen groups in agriculture. They have many important features in common, such as a strictly biotroph life style. Rust fungi produce haustoria or intracellular hyphae that invaginate the plasmalemma of host cells in order to take up nutrients from the living plant cell. In most crops, resistance to rust pathogens occurs, and these are of various types (Niks and Rubiales, 2002). The most widely occurring and applied type of resistance is the hypersensitivity resistance, which is based on a gene-for-gene interaction. Typically, the hypersensitivity reaction is elicited after the pathogen has started to form its haustoria in the plant cell. Therefore, this resistance is also called post-haustorial. The disadvantage of hypersensitivity resistance is its limited durability. Therefore, alternative types of resistance have been investigated, that are, hopefully, less easy to overcome by the pathogen. Partial resistance may be such an alternative. This resistance slows down the epidemic build-up in a quantitative way, and is not based on a hypersensitivity reaction. In several plant-pathosystems, partial resistance has been demonstrated to be inherited quantitatively, and to be based on high rates of failed haustorium formation (Niks, 1986; Niks and Rubiales, 2002). Therefore, this resistance is considered pre-haustorial.

In many plant-pathosystems, partial resistance and hypersensitivity resistance occur side by side, and inherit independently. Pre- and post-haustorial types of resistance also occur against powdery mildews, but again the post-haustorial resistance is typically not durable, whereas the quantitatively inherited pre-haustorial resistance may be durable (Niks and Rubiales, 2002).

Interestingly, plants that are inoculated with an inappropriate rust or powdery mildew fungus, typically display a combination of hypersensitive post-haustorial and non-hypersensitive pre-haustorial reaction (Hoogkamp et al., 1998; Niks and Rubiales, 2002). The genes responsible for the pre-haustorial resistance of barley to the inappropriate leaf rust fungi of wheat (P. triticina) and of wall barley (P. hordei-murini) may also contribute to the partial resistance of barley to the barley leaf rust fungus P. hordei (Zhang et al., 1994; Hoogkamp et al., 1998).

At the Department of Plant Breeding, research on partial resistance dates back to the early 1970s, when Parlevliet started his work on barley - barley leaf rust (Parlevliet, 1975). This work consisted of field trials, but also of numerous greenhouse trials, in which he estimated the various components of the partial resistance, like latency period and infection frequency (Neervoort and Parlevliet, 1978). The work was complemented by studies on the histology of partial resistance (Niks 1982, 1986), and on the mechanisms of resistance of barley to inappropriate rust fungi (Zhang et al., 1994; Hoogkamp et al., 1998).

Since rust spores need water-saturated air and darkness to germinate, inoculated plants should be placed in a dark dew chamber. At the Department of Plant Breeding, a greenhouse compartment served as dew chamber. A humidifier was used to create high air humidity, and natural night ensured darkness for about 9 to 15 hours, depending on the season.

Since the beginning of 2000, experiments were conducted in improved greenhouse facilities, but we have found that our infection experiments did not give the same results as before. Hypersensitive resistance appeared to be reliably expressed, as before, but partial resistance and resistance to inappropriate rust fungi was expressed at lower levels than previously found. On seedlings of Vada, Parlevliet (e.g. 1975), Zhang et al. (1994) and Qi et al. (1998) measured a latency period of P. hordei about 25% longer than on the susceptible control line L94, whereas in the new facilities this difference was reduced to only about 10%. Second, we found in the past, that the wheat leaf rust P. triticina could infect seedlings of barley line L94 to some extent. The number of pustules of this inappropriate rust fungus was much less than 50% compared to that on a susceptible wheat control line (Zhang et al. 1994). In the new facilities, L94 was often almost as susceptible to the wheat leaf rust as the wheat control line. A third discrepancy was, that in the past, fewer than 3% of accessions in a barley collection were (somewhat) susceptible to the wheat leaf rust fungus, i.e. allowed at least 10 pustules to develop per seedling leaf, (Niks et al., 1996), whereas a recent screening in the new facilities showed that about 50% of the barley accessions were susceptible to that rust (unpublished data).

One of the features of the new facilities was the presence of a conditioned dew chamber. Temperature in this dew chamber was kept constant at 18 °C, and inoculated plants were placed in complete darkness for about 17 h, which is considerably longer than natural night length.

We set out to compare the effect of night length during incubation on the level of resistance to wheat leaf rust measured on barley seedlings.

Material and methods

Seven barley lines were selected that had been included also in the experiment to establish the level of susceptibility of barley to the wheat leaf rust (Niks et al., 1996). The same lines had also been tested in the new facilities, and most of them appeared more susceptible there (Niks, unpublished data). The susceptible wheat line 8860 was added as susceptible reference. These eight lines were sown in three sets, each to be incubated under different conditions. Each set fitted in a 37 x 39 cm plant box, containing three seedlings per line. At 12 days after sowing, the first seedling leaves were fixed in horizontal position on the soil of their box. Urediospores of wheat leaf rust isolate "Flamingo" were taken from the collection stored in a – 80 °C freezer, and thawed rapidly in a water bath of about 40 °C. Twelve milligrams of spores were thoroughly mixed with Lycopodium powder, to increase the volume about 10 times. This mixture was weighed, and divided into three equal parts. This ensured administration of the same amount of inoculum to each box. The inoculum was applied in a settling tower.

One box was incubated in the dark dew chamber at 18 °C, from 15:00 to 8:30 the next morning (17.5 hr). The second box was returned to the greenhouse compartment after inoculation, and transferred to the dew chamber at 23:00 (9.5 hr incubation), the third box was placed in a greenhouse compartment in which a humidifier was switched on from 22:30 until 8:00 (effective incubation about 9.5 hr, ~ 17 °C). The latter treatment mimicked as much as possible the conditions prevailing in the old facilities.

After incubation, the pins were removed to allow the leaves to resume any position. On the 10th day after inoculation the number of pustules per seedling leaf was counted. On densely infected barley lines and control wheat, the infection frequency was determined by placing a metal plate with a 1 cm² (2 x 0.5 cm) window over the leaf surface, and counting the pustules visible in the window. On lines with low amounts of pustules in all treatments, all pustules on each leaf were counted.

Results

The infection frequency on wheat line 8860 was similar in all three boxes (Table 1). Within the boxes, the barley lines varied in the number of pustules, due to the genetic variation of barley for resistance to this inappropriate rust fungus. It was obvious that the barley seedlings in the box incubated for 17.5 h had much higher numbers of pustules of wheat leaf rust than those in the two boxes incubated for about 9.5 h (Table 1; Figure 1).

On four lines (Volla, Varunda, Freegold and Valeta) hardly any pustule developed after incubation for 9.5 h. They were virtually immune. In the 17.5 h incubation treatment these lines not only carried about 1 to 12 pustules, but also showed pale flecks, indicating that the infection units developed further than in the 9.5 h incubated boxes (Fig 1). On other lines, the infection frequency was about 4 times (L100) to 16 times (L94) higher in the 17.5 h incubated box than in the 9.5 h incubated boxes.

There was no obvious difference observed in the amount or appearance of infections between the compartment and dew chamber at 9.5 h incubation.

The apparent lack of effect of incubation duration on the susceptible control indicates that development of the fungus, once it completed germination, is unaffected by daylight, but genes for resistance in barley are clearly more effective at 9.5 h incubation than at 17.5 h incubation.

Discussion

In our studies on resistance of barley and wheat to their leaf rust pathogens, we focus on quantitative aspects of the resistance. After we had moved to new facilities, the resistance was less pronounced than before. The experiment presented here, strongly indicates that the lower expression of the resistance in the new facilities was mainly due to the longer incubation duration.

Of course, the condition of darkness and high humidity should last long enough to allow germination and appressorium formation by the pathogen. Probably eight hours should suffice. The subsequent stages of the infection process may occur during daylight.

In the past, we found that the barley leaf rust fungus forms its first haustoria in cells of susceptible barley about at noon on the day after the germination had started, i.e. about 14 to 16 hours after inoculation (Niks, unpublished data). The rate of the development of the fungus depends on temperature.

We presume that at incubation at 18 °C for 17.5 h, the first attempts to form haustoria takes place when plants are still in the condition of darkness and high humidity. In our treatment of 9.5 h incubation, the germination started 8 h later, and attempts of first haustorium formation should occur at daylight. The results suggest that at 9.5 h incubation, the prehaustorial resistance is more effective. So, blocking haustorium formation by the plant cells is more effective when attempts take place at daylight than when they take place in darkness. This is true for resistance to an inappropriate leaf rust species, like the wheat leaf rust fungus in the present study, but has also been confirmed for partial resistance against barley leaf rust (data to be published later).

The results suggest that the prehaustorial resistance is more effective when photosynthesis takes place, and hence, that the prehaustorial resistance is an energy requiring process. It is interesting, that Parlevliet (1975) reported that in ageing flag leaves, partial resistance was less prominently expressed. Removal of spikes at the heading stage retarded the senescence process, and increased the level of partial resistance displayed by the plants (Parlevliet and Kuiper, 1983). He presumed that the spikes act as sinks of carbohydrates, leaving fewer resources for partial resistance to be expressed in the flag leaves. Also treatments like heat shocks and protein synthesis inhibitors are known to reduce the effectiveness of pre-haustorial resistance (Heath, 1979) indicating that this resistance requires metabolic activity of the plant.

Greenhouse and climate room experiments on rust fungi usually rely on dew chamber incubation, and at many labs, duration of incubation is 20 h or even longer. The important implication of the present experiment is, that such a long incubation duration will lead to underestimation of quantitative forms of resistance. It remains to be determined whether the effect of incubation duration is a peculiarity of the barley - leaf rust system, of applies also to other plant-pathosystems, maybe even including powdery mildews.

References

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