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In annual plants affected by inflorescence-invading smut pathogens, avoidance of infection is crucial, while in the event of infection, the existence of different degrees of tolerance could also affect the interaction dynamics. Two experiments were performed with Digitaria sanguinalis spikelets vacuum inoculated with ustilospores of Ustilago syntherismae. In the first experiment, they were sown in pots and mature plants were checked to detect internal hyphae. Observations revealed the presence of symptomless mycelium in a few plants. In the second experiment the spikelets, from two different lots, were grown in a chamber. The objective was to explore the importance of two factors in the degree of seedling infection, one genotypic – type of germination (TG; radicular or coleoptilar) and one environmental – 48 h dark treatment (DT) applied just after germination. Analysis of the infection frequency showed that all the main effects (seed lot origin, TG and DT) were significant, while interactions were not. For TG, the estimated least square mean infection percentages were 66% for radicular germination and 46% for coleoptilar germination. Darkness increased seedling infection by 25%. Differences between TG, DT and their relationship with mesocotyl length exposed to the germinating spores are discussed.
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Pathogens can affect host population dynamics through direct effects on the survival, growth and fecundity of individual plants (Burdon, 1987; Gilbert, 2002). In turn, co-evolutionary dynamics of host–pathogen interactions involve different rates of evolution depending on the strength of selection and the amount of genetic variation in both the resistance of the host and the virulence of the pathogen (Parker & Gilbert, 2004).
Systemic infections of grasses by sterilizing fungi, such as many smut diseases, can have severe effects on host plant abundance and distribution, depending largely on the particular life histories of the plant and the pathogen (Smith & Holt, 1997; Antonovics, 2009). Systemic smuts can destroy the plant inflorescences by producing a mass of spores, and so the dynamics of the interaction could be very different in plant populations depending on whether the species are capable of clonal growth or not. As Clay (1991) points out, in species capable of clonal growth, hosts susceptible to castration can persist and spread despite the loss of sexual reproduction, thus increasing the fitness of the pathogen, which releases spores that can infect new hosts; by preventing its host from reproducing sexually, the fungus maintains susceptible host genotypes and prevents the production of resistant progeny. Several authors have studied interactions between these two types of partners and have quantified the effects of sterilizing diseases on non-annual plant species populations (e.g. Morrison, 1996; García-Guzmán & Burdon, 1997; Piqueras, 1999). There are fewer studies on the characteristics of inflorescence sterilizing diseases in wild annual plants. The majority of them involve smut fungi that can infect more than one host species, and the two or more hosts occupy the same habitat; for example, Govinthasamy & Cavers (1995) studied the effects of Ustilago destruens, a smut pathogen of cultivated Panicum miliaceum, on the Canadian native grass Panicum dichotomiflorum, and the interactions between Ustilago bullata and Bromus tectorum and Bromus catharticus have been studied from different points of view (Mack & Pike, 1984; García-Guzmán et al., 1996; Meyer et al., 2010).
Ustilago syntherismae causes systemic infections of several species of Digitaria in most parts of the world (Farr & Rossman, 2012). Its effects were evaluated by Johnson & Baudoin (1997), who were interested in the potential of smut for biological control of Digitaria ciliaris. They achieved smutted plants by inoculating seeds and soil with spores, but not inoculating flowers. Large crabgrass (Digitaria sanguinalis) is another species of this genus grown in the Caucasus and Kashmir as a cereal in the past century, but currently known as a widely distributed summer annual weed (de Wet, 1992), even in the glyphosate-tolerant summer crops (e.g. Puricelli & Tuesca, 2005; Dewar, 2009; Mas et al., 2010).
In September 2004, smutted inflorescences of D. sanguinalis were observed in an uncropped edge of an arable field near Barcelona (Spain; Mas et al., 2006); the causal agent was identified as U. syntherismae (Vánky, 1994). Since 2004, the zone has been visited each year, and the plant and the pathogen populations still remain until the present time. No alternative host species has been detected in the area. The study looked at some aspects concerning the plant population demography, the effects of the systemic infection on plant fitness, as well as the phenology and morphology of smutted and non-smutted plants (Gallart et al., 2009; Verdú & Mas, 2013). The findings showed that: (i) the seedling emergence period did not limit the disease; (ii) field collected seeds show two distinguishable patterns of germination depending on the embryonic organ that first emerges, radicle or coleoptile; (iii) the disease is presumably monocyclic and spores from smutted inflorescences overwinter in soil; (iv) the relative percentage of smutted versus non-smutted plants varied between 20 and 60% from year to year; and (v) in addition to smutted plants and non-smutted seeded plants, an intermediate phenotype was observed, although poorly represented (1%): partially smutted plants that bear spores and seeds at the same time.
An annual summer species such as the grass D. sanguinalis has no mechanisms of asexual reproduction in temperate regions. If the only type of resistance to the inflorescence-sterilizing smut pathogen U. syntherismae was to avoid infection (and infected and uninfected plants were reproductively isolated), the local maintenance of the interaction from year to year would only be possible if the soil bank contained a stock of susceptible seeds and virulent ustilospores, as occurs in the interaction between the tropical annual grass Rottboellia cochinchinensis and the pathogen Sporisorium ophiuri (Smith et al., 1997). Nothing is known about the survival of the U. syntherismae ustilospores in the soil, but the available information about large crabgrass indicates that seed viability decreased to about 70% after almost 1 year of burial, with practically no seeds surviving after 3 years (Masin et al., 2006). Thus, as the U. syntherismae–D. sanguinalis interaction has been maintained for several seasons in the field, and the selection of the host by the pathogen is very strong, the existence of different levels of resistance within the plant population is expected. The abovementioned partially smutted plants can be considered to have quantitative resistance, but some kind of qualitative resistance could also be found. The literature indicates that qualitative plant resistance, which allows individuals to avoid infection, has a genetic basis governed by interaction loci, while quantitative resistance is governed by a range of traits with different types of inheritance and with possible complex interactions (see Parker & Gilbert, 2004 and Pariaud et al., 2009 for reviews).
In the D. sanguinalis–U. syntherismae interaction studied here, once the pathogen has gained entry to a host, further spread to another host does not occur until the following season. In turn, both partners have one generation per year, reaching the reproductive stage at the same time, and so the infection of plants by fungus is a crucial factor. How and when the infection process takes place is therefore probably one of the most important life history traits to study.
Recent reviews devoted to evolution of plants and their pathogens in natural habitats and across the agro-ecological interface have pointed out that progress in understanding host–pathogen evolutionary dynamics needs characterization of processes occurring at many spatiotemporal scales, including genes and cells, within host individuals, and within and among host and pathogen populations (Burdon & Thrall, 2008, 2009). Quantifying the consequences of the key life history features (such as infection, dispersion and reproductive system) should lead to predictions that can increase our knowledge of host–parasite interactions as well as contribute to the development of a broad conceptual framework for understanding the role of life history in host–pathogen evolution (Barrett et al., 2008). It must also be considered that the modifying influence of the environment on any basic host–pathogen interaction is a real and potent phenomenon (Burdon, 1987); environmental variation generates further complexity in life history interactions, and may further diversify resistance expression (Laine et al., 2011).
This work focuses on within-population variation in host resistance and is laid out in two experiments. The main purpose of the first experiment was to relate the visible levels of disease incidence in D. sanguinalis plants with the occurrence of internal growth of U. syntherismae hyphae. The objective of the second experiment was to find out if, in a very rich viable ustilospore environment, the pattern of seed germination (which is presumably a genetic trait), the amount of light that a seedling receives during emergence (which depends on the seed depth in soil and is therefore a microenvironment variable), and/or the interaction between them, are relatively more or less important in explaining the success of seedling infection.
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None of the mature control plants showed any external sign of disease, and none of the control seedlings observed (187) contained internal hyphae. This confirms that the pathogen is not present inside the seed and the infection must take place at the beginning of the seedling stage or later.
At the end of the first experiment, 265 adult plants were obtained from inoculated spikelets, and 134 (50·6%) of them produced seeds in inflorescences without any external sign of disease. Only two plants were partially diseased, bearing at the same time inflorescences with seeds and inflorescences fully transformed into sori. The rest, 48·7%, bore only ustilospores and thus fall into the category of completely smutted plants. The longitudinal sections of completely smutted plants revealed the presence of the internal mycelium of U. syntherismae in all the vegetative stem nodes, including those from the basal zone, where the position of the nodes and very thin internodes is poorly defined because of intravaginal tillering. Hyphae occurred in association with vascular bundles and also extended into the parenchyma. In the partially smutted plants, mycelium was observed at the base of the stem, in all the nodes of the smutted tillers and also in the first node of one seeded tiller. There were four non-smutted plants, all of them with inflorescences bearing spikelets, that bore smut mycelium at the stem bases. There was no external sign of infection, making this type of plant morphologically indistinguishable from non-infected plants.
The second experiment was conducted to facilitate physical contact between germinating seeds and germinating ustilospores, and thus allow evaluation of qualitative resistance, based on the assumption that inoculated seedlings that were not infected must be categorized as resistant. From the two assays, 39·7% of the individuals tested were resistant (Table 1).
Table 1. Mean percentage of infection recorded for Digitaria sanguinalis seedlings with different germination patterns and receiving different treatments, from spikelets inoculated with Ustilago syntherismae ustilospores
|Sources of variation||Percentage of infection|
|Seed origina||Pattern of germinationb||Darkness treatmentc||Assay 1|| n d ||Assay 2|| n d |
There were no significant differences in the germination behaviour between the control and the inoculated spikelets of each type (data not shown). The 09-field spikelets produced 72% (SE 6·7) of the radicular type, while for the 08-S1 spikelets the coleoptilar pattern reached 96% (SE 2·3). Verdú & Mas (2013), working with the same lots of spikelets, found percentages of 54% (SE 8·0) and 99% (SE 1·0), respectively. Table 1 shows the percentages of infection obtained after processing 886 seedlings germinated from inoculated spikelets, grouped according to the sources of variation considered. As was expected, with respect to the 08-S1 seed origin, the number of seedlings with a radicular pattern of germination was very small compared with those obtained in all the rest. For this reason the 08-S1 seedlings with a radicular pattern were removed from the generalized linear model analyses.
The percentage of infected seedlings was greater in the second assay than in the first, except 08-S1/radicular/darkness, which was the most poorly represented. This general difference could be attributable to the within-population variability of both plant and pathogen, probably amplified at the interaction level, together with other non-controlled sources of variation. For example, it is known that the 2 month period between the first assay and the second did not alter the seed germination behaviour, which is well characterized (Toole & Toole, 1941), but knowledge of the viability or the rate of germination of the ustilospores and their growing rates is scarce.
Despite the differences, within each assay the percentages of infection showed similar relative importance between the levels of seed origin, pattern of germination and darkness, and so interactions between the source of variation ‘assay’ and the other factors were not expected, especially if the 08-S1/radicular seedlings are removed from the analyses. Table 2 shows the results of the two generalized linear models performed to analyse the proportion of seedlings infected in the light of the raw results provided in Table 1. All the main effects considered were significant, while the interactions were not. The estimated least-squares means for each of the two levels of each main effect were significantly different at α = 0·05.
Table 2. Likelihood ratio statistics of different effects in two analyses of proportions of Digitaria sanguinalis seedlings infected with Ustilago syntherismae, and least-squares means of the significant (P < 0·05) sources. The first half of the table gives the results of observations from spikelets collected in the field in 2009 (09-field); the second half gives the results from seedlings with the coleoptilar pattern of germination only
|Effect||d.f.||Chi-square||P > chi-square||Levels||LS meana||Standard error|
|Darkness treatment (D)||1||28·11||<0·0001||Yes||0·6845||0·1435|
|Pattern of germination (G)||1||18·22||<0·0001||Coleoptilar||0·4630||0·1429|
|D × G||1||1·14||0·2856|| || || |
|Darkness treatment (D)||1||14·77||0·0001||Yes||0·7033||0·1479|
|Seed origin (S)||1||39·10||<0·0001||08-S1||0·7498||0·1453|
|D × S||1||0·03||0·8520|| || || |
The treatment of darkness was a significant source in both analyses (Table 2). First, the percentage of infection estimated with 09-field spikelets, which represents the fraction of the entire field plant population that bears seeds and showed no sign of disease, i.e. the fraction that contributes to the next generation, increased by 25% if seedlings spent 48 h in darkness compared to seedlings that were incubated in the general regime of 12 h light/12 h dark. Secondly, in seedlings with coleoptilar patterns of germination, from both 09-field spikelets and 08-S1 spikelets, the percentage of infection increased by 18% with the dark treatment. Regardless of seed origin and pattern of germination, the mesocotyl of seedlings that received dark treatment was about 10 mm longer on average than those incubated in the alternating light/dark regime, which had mesocotyls about 3 mm long (Table 3).
Table 3. Mean mesocotyl lengths of Digitaria sanguinalis seedlings with different germination patterns and receiving different darkness treatments
|Seed origina||Pattern of germinationb||Darkness treatmentc||Mean mesocotyl length (mm)||Confidence limitsd|| n |
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The absence of mycelia in the control seedlings indicates that U. syntherismae is an externally seedborne smut, confirming the suggestion of Johnson & Baudoin (1997). The spatial distribution of mycelium found inside the smut-infected plants has also been described in other wild pathosystems, such as the infection of the perennial grass Heteropogon contortus by Sorosporium caledonicum (Fullerton, 1975). The distribution of hyphae suggests that, once the fungus is inside the plant, colonization into certain inflorescences is mediated by the plant's ability to elongate the internodes or branch faster than the ability of the hyphae to colonize the developing buds. The importance that these presumed pathogen-induced changes may have in host development is apparent in the last finding of the first experiment: the symptomless presence of the pathogen. The same phenomenon has also been detected in other sterilizing diseases, e.g. U. bullata–B. tectorum (Meyer et al., 2010).
Excluding plants that escape infection because they are not in contact with ustilospores, four levels of within-population variation in host resistance can be distinguished in the light of the first experiment: (i) plants that avoid infection; (ii) infected plants that do not develop the disease; (iii) partially tolerant infected plants; and (iv) fully susceptible plants. With respect to the underlying genetic structure, it may be inaccurate to describe these degrees of resistance and tolerance as levels, in the qualitative sense, because there is probably a continuum between infected non-smutted and completely smutted phenotypes. However, it seems clear that in terms of plant and pathogen population dynamics, which is affected by some life history traits such as fecundity, the three levels of quantitative resistance (ii, iii and iv above) imply very different contributions to further generations: infected plants that do not develop the disease represent missed opportunities to pass on ustilospores; completely smutted plants are missed opportunities to pass on seeds; and finally, partially smutted plants can contribute to a greater or lesser extent to the next generation of both plant and pathogen. The field observations have shown that, within the group of partially smutted plants, there is a wide range of variation in the resource allocation to produce seeds and ustilospores (Gallart et al., 2009).
Pariaud et al. (2009), in a review focusing on crop plant–pathogen interactions, documented that the expression of quantitative resistance is not solely the result of the genetic basis, but is also dependent on environmental effects, mainly temperature, relative humidity and, particularly for biotrophic pathogens, plant physiological status. There are fewer published reports of wild interactions (García-Guzmán et al., 1996; Laine, 2007). The abovementioned studies, together with other previous ones, reinforce the idea that the phenotypic expression of all active disease resistance mechanisms (in relation to what happens once infection has occurred) may be markedly affected both by variations in environmental conditions and by ontogenetic changes in the host plants (Burdon, 1987). This study allows one to hypothesize that any environmental factor that influences stem growth and development, alone or in interaction with the genetic background, could lead to different phenotypes of quantitative resistance.
The second experiment focused on qualitative resistance, where there is much more information available in wild plant–pathogen interactions (reviewed in Kniskern & Rausher, 2001; Barrett et al., 2008; Burdon & Thrall, 2009; Laine et al., 2011). In emerging seedlings, the coleoptile stomata are natural openings that may provide a penetration opportunity for the infective hyphae, and the observations here suggest this possibility, as in several seedlings the maximum mycelium density was in the vicinity of the apical stem meristem, at the base of the coleoptile. However, there were also several infected seedlings with mycelium visible only in the mesocotylar zone, which indicates that the mesocotyl was a sensitive zone and that the fungus may have some chemical penetration mechanisms.
Thus, the results suggest that non-infected seedlings presumably triggered some resistance mechanisms, stimulated or not by physical contact with infective hyphae, which were not independent of the darkness treatment received, as this treatment was associated with mesocotyl elongation. In addition, the results also indicate that there was some genetic control of infection avoidance, because there were significant differences in mean percentages of seedling infection, both between radicular and coleoptilar patterns of germination of 09-field spikelets and between 09-field and 08-S1 material with the coleoptilar pattern of germination (Table 2). The difference in the mean mesocotyl length of seedlings receiving darkness treatment could be an explanation for the differences found between the infection percentage of 09-field and 08-S1 spikelets (Table 3). Seedlings with the radicular pattern of germination showed greater susceptibility than those with the coleoptilar pattern, whether they received the darkness treatment or not (Table 1). Two explanations, not mutually exclusive, can be made. On the one hand, it could be a case of the infection window; when a D. sanguinalis embryo begins germination in the radicular pattern there is a period of time of radicle growth in which the spikelet bracts are separated because the embryo is swollen. This period, which could allow fungal infection, does not occur if the seedling has a coleoptilar pattern of germination (Verdú & Mas, 2013), i.e. the interval of time in which the infection can take place is narrower. On the other hand, seedlings with a coleoptilar pattern of germination might be genetically able to spread more mechanisms of resistance than those with a radicular pattern of germination. How this could be inherited or what kind of genetic background could be responsible for it is currently unknown, because there are still many questions to answer about the degree of variability of both plant and pathogen populations.
The results clearly show that the infection process was mediated by the dark treatment, but this treatment did not interact with the differentiated phenotypes. In the 09-field spikelets, the estimated mean percentage of infection of seedlings with the radicular pattern of germination was 66%, compared to 46% of those with the coleoptilar pattern of germination (Table 2). The least infection occurred in seedlings with a coleoptilar pattern of germination without dark treatment (Table 1), while the greatest infection occurred in seedlings with a radicular pattern of germination that received the dark treatment. As c. 2/3 of this lot of spikelets exhibited the radicular pattern of germination and 1/3 the coleoptilar pattern, the results of this experiment indicate that, at the population level, mean infection of nearly 60% could be expected in the next generation if the environment were as rich in germinating ustilospores as that of the Petri dishes. However, given a mean range, infection could reach 70% if the seedlings were grown within the soil, in darkness, or it could remain as low as 50% if the spikelets were located on the soil surface; thus, the results highlight the importance, in terms of probability of infection, of the distance that a seedling has to cover from germination to emergence.
In the field, the percentage of infected seedlings would also be influenced by the proportion of germinated seeds that were able to emerge from different buried depths. In the field studied, due to the tillage system operations, spikelets and ustilospores can be found between the soil surface and a depth of 20 cm. Benvenuti et al. (2001), in a detailed study that observed extensive crabgrass emergence but also fatal germinations, noted that in the soil profile: (i) at a depth of 8 cm no germination occurred; (ii) at 12 cm the lack of germination ability was attributable to secondary dormancy in 85% of the spikelets; (iii) between 0 and 2 cm the percentage of germination did not vary and reached 60%, 35% at 4 cm, and 8% at 6 cm; (iv) the delay in germination was 1·5 days from 0 to 2 cm and 4·1 days from 2 to 4 cm; and (v) fatal germinations reached 18%. However, ustilospore germination could be more restricted by the higher summer temperatures and desiccation on or near the soil surface than at any of the depths used for the seed germination tests. Considering all these findings, together with the perturbation regime of the field, it seems that the advance of the disease has been restricted due to intrinsic causes, because c. 40% of the population is qualitatively resistant and a small percentage of the infected plants produced some seeds. However, there are also environmental causes, because the seedlings with the highest probability of emergence are also those with the lowest probability of being infected, as they are located nearest to the soil surface and show rapid development and poor mesocotyl elongation with respect to more deeply located seeds.
It is concluded that, in the D. sanguinalis–U. syntherismae interaction, how qualitative plant resistance and pathogen infectivity are inherited is far from being understood, as is how quantitative resistance and aggressiveness are environmentally mediated. Nevertheless, some important life history traits, particularly those related to seed germination and early seedling emergence, placed in the context of the microenvironment, provide an understanding of how the interaction is maintained at the local level.