Host growth stage is an important factor affecting development in many plant diseases. It is a common observation among growers of perennial ryegrass seed crops that stem rust becomes particularly severe around the time of anthesis, after flower stems have fully extended. This observation has led to a commonly-held assumption that the plants are especially susceptible to infection at and immediately after the time of flower stem extension.
The objectives of the study were to determine whether there are differences in susceptibility of perennial ryegrass plants to infection by the stem rust fungus that are correlated with tissue age and/or growth stage from tillering to flowering. In addition, I report here the very important influence of growth stage on the ability of the pathogen to spread from a single primary infection, producing many stem infections within an infected plant.
Perennial ryegrass (cultivar Morningstar) was grown outdoors in 1-gallon pots, 3 plants per pot, starting in September. For the experiments on growth stage and susceptibility, four different stages were induced by bringing plants from the outdoors into the warm, long-day greenhouse on four different dates. At the time of inoculation, the stages were: penultimate leaf (leaf below the flag leaf) visible, boot stage, head emerging, and anthesis beginning.
One tiller on each plant was selected for inoculation, and a standard amount of inoculum (rust spores) was applied to each tiller. Plants were placed overnight (15 h) in a chamber with conditions favorable for rust infection. The plants then were moved onto the greenhouse bench, and maintained until observed for results.
The number of pustules per plant organ (each leaf blade, leaf sheath, flower head and stem) on each inoculated tiller was determined 14 days, or approximately 1½ latent periods, after inoculation. Pustule number per inoculated plant area was calculated separately for each plant organ, based on the area present on the day of inoculation.
To test growth stage as a factor in average susceptibility of plants, a one-way analysis of variance (ANOVA) on whole-plant averages of infection severity (total number of pustules divided by total inoculated area for each pot) was used. For the evaluation of organ position as a factor in susceptibility, a one-way ANOVA was used within each developmental stage to test organ type and position as a factor affecting number of pustules per inoculated area.
Another experiment, to test the idea that the stem rust pathogen can spread to the elongating tiller stem from a single leaf sheath lesion, is described in the results section.
Growth stage and susceptibility. When perennial ryegrass plants were inoculated with urediniospores of the stem rust fungus, the average full-tiller susceptibility to infection (number of pustules divided by plant area inoculated) was not significantly different among the growth stages tested. That is, a given level of inoculum produced about the same number of infections per inoculated area of the tiller whether plants were in the stem extension stage, boot stage, head emergence, or fully-emerged head stage. Within each growth stage, however, there were statistically significant differences in disease severity (pustules per plant area) among plant organs of different positions on the tiller. For the youngest growth stage tested, the leaf blades were equally susceptible, whereas some of the lower (older) leaf sheaths were less susceptible. For the other growth stages, the most recently exposed plant organs were usually the ones with the most disease, although in some cases older organs also were as severely diseased as young organs. For tillers in the boot stage, the flag leaf sheath was particularly receptive to infection. When the head was emerging from the boot, the flag leaf sheath and the newly-exposed inflorescence were especially susceptible. After the inflorescence had emerged completely, and its subtending stem was newly exposed above the sheath of the flag leaf, this stem was significantly more receptive to infection than most other parts of the plant.
Within-plant spread of disease. Approximately 3 weeks after the inoculatIon date (1 week after the previously-described results were observed), elongated rust lesions appeared on the flower head and/or the flower stem on many of the tillers that had been inoculated at the boot or head-emerging stages. These elongated lesions appeared much later than most of the pustules, which had appeared about 9 days after inoculation. Furthermore, the late-appearing pustules appeared on flower heads and stems that had not yet been exposed at the time the initial inoculum was applied.
These observations suggested that the elongated diseased areas were the result of secondary spread within the plant, arising from primary infections on leaf sheaths enclosing the emerging flower heads. It appeared that primary infections on the flag leaf sheath penetrate its inner surface, where they provide inoculum for multiple, adjoining infections of the enclosed flower head as it moves upward past the inner surface of the primary infection site on the sheath.
To test the hypothesis of secondary spread on elongating tillers, individual tillers were each inoculated at a single site on the flag leaf sheath. About 2½ weeks later, elongated lesions occurred on flower heads or stems of most of the tillers that had a pustule on the sheath. There was no rust disease produced on the flower heads or stems of any tillers that lacked a lesion on the flag leaf sheath. Therefore, there was a clear association between lesions on the sheath and elongated lesions on the emerging flower. Additional measurements confirmed that the stem lesions arose from the sheath lesions.
The final length of rusted area on the flower stem results directly from the amount of stem elongation that occurs after the start of spread of infection from the overlying sheath. Under the conditions of these experiments, final lengths of secondary stem infections arising from each single sheath pustule ranged from 1½ to 6 inches.
Microscopic examination of the stem and enclosing sheath confirmed that the rust fungus penetrates the full thickness of the sheath, and produces sporulating pustules on both the outer and inner surfaces at about the same time. Within a day, rust spores released from the pustule on the inner sheath surface germinate on the surface of the enclosed stem and start new infections there.
The youngest leaves or stems of perennial ryegrass plants were generally more susceptible to rust infection than the older parts. However, in comparing average susceptibility over the whole plant (number of infections per inoculated plant area), there was no significant difference among growth stages from late tillering (prior to flag leaf emergence) to full extension of the inflorescence. Therefore, the increased level of rust commonly appearing near flowering is not due to a greater susceptibility of plants at this stage.
Whereas the effect of plant and tissue age on susceptibility has only limited bearing on stem rust epidemic development, the impact of growth stage on the process of within-plant spread is far more important. This within-plant spread permits a single primary infection on a leaf sheath to be multiplied manyfold through secondary infections on the enclosed, elongating flower head and stem. In addition, the newly expanded sheath and enclosed inflorescence are both young tissue and therefore particularly susceptible to infection. The result is a rapid surge in disease severity (and consequently additional inoculum for the rust epidemic) starting about one latent period (8-12 days) after head emergence, if infections have occurred on the flag leaf sheath. This increase is independent of airborne inoculum and, perhaps to some degree, weather conditions.
Although this study was performed on the flag leaf sheath and enclosed inflorescence, it is likely that the same process occurs on lower sheaths and the structures enclosed there as well. Given the great potential for secondary lesion elongation, within-plant spread may be the predominant mode of stem rust epidemic increase during tiller extension and early flowering in perennial ryegrass.
The existence of extensive, elongated areas of rust disease on the flower and stem of perennial ryegrass is a common observation, but the process by which they develop was previously unknown and generally assumed to be the result of numerous individual stem infections arising from airborne spores. We can now see that this phenomenon results from the ability of the pathogen to penetrate and sporulate on the inner surfaces of sheaths, thereby applying inoculum to an extensive length of particularly susceptible tissue as it elongates past the inoculation point.
The process of within-plant spread has important implications for disease management. The ability of fungicides to affect spore production at the inner face of the sheath, or to affect spore germination or penetration at the stem surface under the sheath, would be critical to their effectiveness. A study on this question is in progress. Also, it may be possible to select for host genetic resistance based on a plant's ability to restrict sporulation of the rust fungus on the inner surface of the sheath.
I thank Sheila Seguin for excellent technical assistance in this research.
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