Abstract

The wheat leaf rust fungus tends to grow deep into the mesophyll of the host plant leaf. A wheat leaf rust isolate was inoculated on two wheat cultivars on the adaxial or on the abaxial side of the first seedling leaf. After incubation, some leaves were fixed adaxial side up and others abaxial side up. Latency period and infection frequency were measured on both sides of each leaf. Pustules tended to appear earlier (shorter latency period) and were more frequent (higher infection frequency) in the side of the leaf that faced down, which could be either the adaxial or the abaxial side. We conclude that P. triticina tends to grow downwards into the leaf (negative phototropism or positive geotropism) especially when the adaxial leaf side is facing down.

Introduction

Leaf rust, caused by P. triticina, is an important disease in most wheat growing areas (Saari & Prescott, 1985). In compatible interactions the rust colony grows into the leaf mesophyll tissue. The colony finally develops a uredium (also called pustule) when disrupting the leaf epidermis and releasing new uredospores. Inoculations and records on number or size of pustules are usually performed on the adaxial leaf side (Parlevliet, 1975; Jacobs & Buurlage, 1990). Sometimes, especially in the seedling stage, more uredia appear earlier on the abaxial than on the adaxial side whereas on other occasions the opposite occurs.

The purpose of this experiment is to study whether growth of the leaf rust fungus is oriented towards the down facing leaf side or towards the abaxial side of the leaf.

Materials and Methods

This study was performed on the susceptible cv. Thatcher and the partially resistant cv. Akabozu. The isolate of P. triticina used in the experiment was ´Flamingo´ (Wageningen University, The Netherlands).

Experiments were carried out at the seedling stage. Twenty seedlings were raised per tray (37x39x5 cm). Two replications were performed. Eleven days after sowing, first leaves were fixed with pins in a horizontal position and five different treatments were applied, four seedlings per treatment:
- Standard, inoculation on adaxial side. After incubation pins were removed allowing leaves to recover their natural attitude.
- Inoculation on the adaxial side. After incubation leaves stayed adaxial side up for five days.
- Inoculation on the adaxial side. After incubation leaves were turned and stayed abaxial side up for five days.
- Inoculation on the abaxial side. After incubation leaves were turned and stayed adaxial side up for five days.
- Inoculation on the abaxial side. After incubation leaves stayed abaxial side up for five days.

Inoculation was done by dusting three milligrams of spores, diluted 10 times with Lycopodium spores, on the leaves in a settling tower. Deposition resulted in, approximately, 100 spores/cm². For the incubation plants stayed, for 10 hours, in a mist chamber at 18ºC with 100 % relative humidity. Then, depending on the treatment, leaves were turned, and trays were transferred to a climate glasshouse compartment (20ºC, 14 hours of photoperiod) where natural light was supplemented with sodium lamps.

Latency period and infection frequency were recorded both on adaxial and abaxial side of each leaf. Latency period was taken as the time period from the beginning of incubation until the moment that 50 % of the uredia had appeared (Rubiales & Niks, 1995). Latency period was determined by daily counting the number of uredia visible in a marked area (on both sides the same area) until the number no longer increased. Infection frequency was determined on the marked area of the leaves. The final number of uredia was used to calculate the number of uredia per cm².

Statistical analysis was done comparing latency period and infection frequency between adaxial and abaxial side on each treatment separately for either cultivar. Arc sin transformed data were analysed by means of a paired-sample comparison (two-tailed Student's T, 0.05).

Results

Over all treatments within each cultivar differences in latency period between adaxial and abaxial leaf sides were very small (Table 1). However the infection frequency on the adaxial side was, on average, greater than on the abaxial one. In both cultivars the latency period was shorter, and the infection frequency higher on the down facing side, irrespective whether that was also the inoculated side or whether it was morphologically the adaxial or abaxial side. When leaves were left to recover their semi vertical position (in the treatment where leaves were not fixed) the latency period tended to be longer and the infection frequency shorter in the side that was facing up (data not shown). With respect to the inoculated side almost no differences were found. However, on Akabozu, latency period was significantly longer on the inoculated side.

Referring to the different treatments (Table 2) the tendency was, in general, similar both in Thatcher and Akabozu. Latency period was clearly longer on the up-facing side throughout all treatments. It is remarkable that when the adaxial side was facing up the infection frequency on both sides were similar (only lower on Akabozu when the adaxial side was inoculated) whereas when the abaxial side was facing up the infection frequency was clearly lower on that side. Another difference between Thatcher and Akabozu was that in treatments in which the inoculated side faced down, there were no differences in latency period between leaf sides on Akabozu.

Discussion

Within the first 24 hours after inoculation, the mycelium of P. triticina grows vertically into the mesophyll layers of the leaf (Niks, 1986). The present data indicate that leaf rust mycelium does not necessarily need to cross the leaf tissue towards the opposite epidermis. Whether leaves are inoculated on the adaxial or abaxial side, the mycelium will grow preferably and faster towards the down facing epidermis, and form a uredium there. Our data do not provide clues whether the orientation is due to negative phototropism or to positive geotropism. In plants, phototropism is a well know phenomenon. For instance the tip of a shoot in any plant grows towards a source of light. Phototropism has been studied extensively, for the biochemical compounds involved (auxines and photoreceptors) and the spectrum of visible light that acts as signal and for the expressed genes, e.g. in Arabidopsis thaliana (Liscum, 2002). Negative geotropism of germ tubes has been reported in axenic cultures of Cronartium spp. (pine rust) (Diner & Mott, 1982; Moricca & Ragazzi, 1996). Koch & Hoppe (1987) found that uredospores of soybean rust (Phakopsora pachyrhizi) always germinated at the shadowed side and germ tubes had a negative phototropism when developing. All studies concerned spore germination or germ tube growth but nothing was found about phototropism of the infection hyphae. Further experiments are needed to find out whether the growth of P. triticina is due to negative phototropism or positive geotropism.

The substomatal vesicle of P. triticina develops a primary infection hypha that grows deeper into the mesophyll. However, in other rusts, such as barley leaf rust (P. hordei) or rye leaf rust (P. recondita) the substomatal vesicle develops two primary infection hyphae that spread horizontally parallel to the epidermis (Niks, 1986). It may be that the presently reported orientation of P. triticina does not apply to rusts that develop hyphae that run parallel to the epidermis. Indeed, in P. hordei experiments it is not a regular observation that pustules predominantly appear in the down facing epidermis.

When leaves stayed long abaxial side up uredia came earlier on the opposite side and the infection frequency was higher on the adaxial side. However, when leaves stayed long adaxial side up uredia came earlier on the down facing abaxial side, but the infection frequency was similar on both surfaces. It seems that colonies indeed grow downwards, but they show a certain preference for spreading on the adaxial side rather than on the abaxial one. This is perhaps due to the different foliar anatomy of the wheat leaf between adaxial and abaxial side. The advantage for the rust fungus to be oriented primarily to the down facing leaf surface and to develop in the adaxial side remains unknown.

Acknowledgemets

The authors acknowledge Anton Vels for his technical assistance. This work has been supported by a postdoctoral fellowship granted by the Secretaria de Estado de Educacion y Universidades of Spain and cofinanced by the European Social Fund.

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