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Dep. of Agronomy, Lilly Hall of Life Sciences, 915 W. State Street, Purdue Univ., West Lafayette, IN 47907-2054
* Corresponding author (gejeta{at}purdue.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: DMBQ, 2,6-dimethoxy-1,4-benzoquinone • MGD, maximum germination distance • MHD, maximum haustorial distance
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Of the more than 40 species described in the genus Striga, only about a quarter parasitize crops. The rest grow on wild hosts in ecosystems evolving for an estimated 40 million years (Raynal-Roques, 1996). Striga hermonthica is the species broadly adapted to agricultural conditions and occurs almost exclusively on crop hosts. Striga asiatica, less broadly adapted but still a major crop pest, occurs commonly on wild grasses. Some argue that certain Striga spp. currently emerging as occasional weeds may eventually become noxious, perhaps even to the extent of S. asiatica and S. hermonthica (Raynal-Roques, 1996). Striga aspera may be the next to become a noxious weed since it is known to naturally hybridize with S. hermonthica (Kuiper et al., 1998).
This alarming possibility together with the severe devastation already wrought by the existing witchweed species may be slowed or even halted by the flow of striga resistance genes from the wild to an agricultural ecology. This flow to some extent has naturally occurred in Sorghum but will become less likely as modern agriculture continues to separate the crop from its wild companions. The selective advantages of wild sorghum that evolved under pressure from Striga spp. may be exploited in their cultivated relatives by deliberate introgression. As more is learned about the interactions between striga and its hosts, the search for specific opportunities to interfere with establishment of the parasitic association can be narrowed. Wild relatives of cereal hosts can be screened for the phenotypes corresponding to these opportunities. Although striga resistance in wild and related species has not been fully exploited, a few surveys of wild sorghums for striga resistance have been reported (Deodikar, 1951, Lane et al., 1994, Mohamed et al., 2003).
The sorghum host participates in the parasitic association with striga at many levels: exuding the stimulant for striga seed germination, providing the haustorial initiation signal, allowing penetration to its vasculature and producing assimilates and possibly other factors in forms usable by the parasite. Opportunities for genetic resistance in sorghum to striga may exist in each of these areas of cooperation. We have developed laboratory methods for observing specific stages of the sorghum x striga association and identify sorghum variants that do not cooperate in a manner that leads to parasitism.
In this study, we screened 55 wild sorghums and 20 cultivars for potential mechanisms of striga resistance using in vitro procedures developed in our laboratory. Our focus was on events observed early in the process of parasitic establishment, before striga attaches to sorghum. The objective of the study was to determine if new sources of preattachment striga resistance can be found within the primary gene pool of sorghum.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Laboratory Methods
Seed Conditioning
Striga asiatica seed was washed upon receipt with 1% (v/v) Tween-20 (polyoxyethylenesorbitan monolaurate), a surfactant, and several rinses of water to remove sand and debris. Cleaned weed seed was air dried and stored in a desiccator for at least two weeks before conditioning. Conditioning of striga began 10 to 14 d before infection in 0.5-g batches. Conditioning involved washing seed with 25 mL 75% (v/v) ethanol for 2 min in a sonicator. This was followed by three rinses in sterile water of 1 min each with sonication. The seed was then washed in a sonicator for 2 min with 25 mL Metricide 28 (Metrex, Inc., active ingredient: glutaraldehyde, 2.5%), a disinfectant, followed by three sterile water rinses. The seed was then washed with 25 mL 0.525% (v/v) sodium hypochlorite (bleach) for 2 min with sonication followed by three water rinses. Finally, surface sterilized seed was incubated at 28°C in 32 mL freshly prepared 50 µL mL–1 benomyl (Dragon, Inc., active ingredient: methyl 1-[butylcarbamoyl]-2-benzimidazolecarbamate, 50%), a systemic benzimidazole fungicide. The benomyl solution was changed after 2 d and subsequently every 3 to 4 d during the conditioning period.
Sorghum seed was deglumed and surfaced sterilized with 25 mL 1.3% (v/v) sodium hypochlorite solution for 1 h. Bleach was removed by several washes of sterile water. Sorghum seed was then treated overnight in 10 mL 5% (w/v) captan slurry (active ingredient: N-[trichloromethyl]thio-4-cyclohexene-1,1-dicarboimiide, 39%), a nonsystemic fungicide. After washing twice with sterile water, seed was transferred to sterile Petri plates containing filter paper thoroughly wetted with sterile water. Sorghum was germinated in covered plates in the dark overnight at 28°C.
Extended Agar Gel Assay
The assay described by Mohamed et al. (2003), with slight modifications, was used in all experiments. The extended agar gel assay involved the following steps. Germinated sorghum seeds were planted into 0.7% (w/v) agar containing conditioned S. asiatica seeds and incubated in darkness at 28°C for 3 d. At the end of the 3 d, plates were checked under a stereomicroscope through the bottom of the agar. Seedling vigor was estimated by a quick measurement of root length. Any seedling root that had not reached a length of 6 cm was not included in the results. Skipping the basal 2 cm of sorghum root, distances of the furthest three germinated striga to the primary sorghum root were measured to the nearest 0.5 mm. The extreme basal portion of the sorghum root was excluded because over this distance, the root usually had not yet reached the bottom surface of the agar where the weed seed was embedded. Maximum germination distance (MGD) for each accession was calculated by averaging three measurements around a healthy sorghum seedling. Three measurements were taken per seedling to minimize effects of local differences in striga seed distribution on the plates. If fewer than three germinated striga were present on the plate, a distance of 0 mm was used in calculating the MGD for the seedling on that plate.
After determining maximum striga germination distance, plates were treated with a 30 s burst of ethylene inside a sealed chamber. After 48 h in the ethylene chamber (5 d after plating), plates were vented and observed under a stereomicroscope. Striga seed batch germination potential in agar with ethylene was determined from plates into which no sorghum was planted. Germination counts were taken on these blank plates in 10 randomly selected 40x microscope fields (6-mm diam). To determine the maximum haustorial initiation distance (MHD), the radicles of germinated striga were examined for the presence of hair-like projections characteristic of haustoria. Ignoring the basal 2 cm of sorghum root, distances from haustoria to the sorghum root were measured to the nearest 0.5 mm. MHD for each seedling was calculated by averaging the three furthest measurements. If fewer than three germinated striga with haustoria were present on the plate, a distance of 0 mm was used in calculating the MHD for the seedling on that plate.
Experiment 1—Initial Screening by the Extended Agar Gel Assay.
Seventy-five entries including 55 wild sorghums and 20 cultivars were screened in five sets of three to four seedlings for each accession. Cultivars SRN39 (low stimulant line) and Shanqui Red (high germination stimulant line) were included as checks in each of the five sets. All seedlings of a particular entry were assayed within a single plate. Only the two checks were repeated between the five sets. One blank plate was used in each of the five sets to determine striga batch germination rate.
Experiment 2—Repeat Screen of Selected Accessions.
Ten wild accessions and two cultivar checks were tested by the extended agar gel assay. Representative wild sorghums with vigorous seedling characteristics from each of the groups listed in Table 1 were used in the second set of assays. These were compared with the same two cultivated sorghums checks, SRN39 and Shanqui Red, used in Exp. 1. Five replications were used to calculate the mean MGD and mean MHD of each.
Experiment 3—Further Comparison of Group 1 Type to Cultivars.
Sorghum bicolor drummondii PQ-434 and the two cultivar checks, SRN39 and Shanqui Red, were grown in the extended agar gel assay with 11 replications per entry. Measurements for MGD and MHD were taken as before at 3 and 5 d after plating, respectively. Additionally, the germination percentage of striga seed within 3 mm of the sorghum root was taken at 3 (before ethylene treatment) and 5 (after ethylene treatment) d by dividing the number of germinated weed seeds by the total weed seed count along the sorghum root length beginning 2 cm from the kernel at the basal end to the root apex. A mark was made on the plate at the sorghum root tip at 3 d so that the same total weed seed count could be used to calculate the percent germination over this length before and after ethylene treatment. Haustorial initiation percentage was calculated at 5 d by dividing the number of germinated striga seed having recognizable haustoria by the number of germinated weed seeds within the 3-mm sorghum root zone.
Statistics
Sorghum accessions were screened in a completely random design. Mean comparisons were performed by SAS version 8.2 (SAS Institute, 2001). Variance analysis was performed with PROC ANOVA to assess the difference between and within accessions. Means were compared through unequal variance t test as recommended for cases where the assumption of homogeneity is not met. Experiment-wise error was minimized by adjusting the critical probability for accepting differences at p < 0.05/n, where n is the number of pair-wise tests, as per Bonferroni.
In Exp. 1, 75 accessions were screened in five sets of 15 accessions each, including the two checks. For each trait, observations were made on three to four seedlings with three measurements were taken on each seedling, resulting in an averaged value for both MGD and MHD (Table 1). For both traits, the measure of variance among checks was used to assess the experimental error. Within each check, the level of significance for comparisons among the means was set up at p < 0.005.
In Exp. 2, variance analysis was performed to assess significant differences among accessions for MGD and MHD and means were compared through a t test with an adjusted 
value. Significant difference was observed at p < 0.0008.
In Exp. 3, the three entries were repeated eleven times in the experiment. The effect of ethylene treatment on germination was assessed through ANOVA and mean differences tested with a t test at an value of 0.008.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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10 mm is considered a high striga germination stimulator (Hess et al., 1992). Accessions were classified for their haustorial initiation capacity on the basis of their MHD values. A mean MHD of 2 mm was set as the threshold to distinguish high haustorial initiators from low ones in the groupings assigned in Table 1. This threshold mean MHD was chosen because it was the cut off for the lowest quartile of all measurements taken in Exp. 1. Entries are grouped in Table 1 according to the germination and haustorial initiation distances measured from the agar plates. A pair-wise comparison of means within each check, however, did not show significant differences between the sets, discarding the potential experimental error contributed by the sets. Accessions assigned to Group 1 are low germination stimulators (mean MGD < 10 mm) that are also low haustorial initiators (mean MHD < 2 mm). Group 2 accessions are low germination stimulators with high haustorial initiation activities (mean MHD 2 mm). Group 3 accessions are high germination stimulators (mean MGD 10 mm) with low haustorial initiation activity and Group 4 accessions showed both high germination stimulant and high haustorial initiation activity. Accessions were assigned to Group 5 if the mean of measured germination distances was >4 mm and standard deviation of the measurements was >50% of the mean. Wild sorghums of this group contained a mixture of high and low MGD seedlings, reflecting the heterogeneous nature of some accessions. The small sample size (3–4 seedlings) taken in the initial screen may have been too low to detect the heterogeneity of some wild accessions for the measured traits. Several wild sorghums displayed low germination stimulation of striga seed in agar (Table 1). The majority of wild accessions examined were high striga germination stimulators. The overall range of MGD values measured in Exp. 1 was greater among the wild accessions than among the cultivars. Fourteen putative low germination stimulators were identified among the wild sorghums. Thirteen of the 20 cultivars were low germination stimulators. One accession of S.b. drummondii (PQ-434) from Group 1 showed no germination of striga in agar before ethylene treatment. A comparison of the very low germination of striga near the roots of PQ-434 to that near a high stimulant cultivar CK60 is shown in Fig. 1 . After 3 d in agar with the growing roots of PQ-434, conditioned striga seed did not germinate (Fig. 1-a1), whereas those with CK60 showed high germination.
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The mean MHD values gathered from the checks in Exp. 2 did not significantly (p < 0.05) differ from the mean MHD values found in Exp. 1. However, MHD values were generally higher in Exp. 2 than those measured in Exp. 1 and ANOVA separated only the mean MHD of Group 1 type IS18803 (mean MHD = 0.6 mm) from the means of all other sorghums. The mean MHD of PQ-434 (1.1 mm) was not significantly (p < 0.0008) different than the mean MHD of SRN39 (2 mm) in this test.
In Exp. 3, the percentage of germinated striga with haustoria along the sorghum root was taken as an additional measure of haustorial initiation activity. PQ-434 was compared with the low stimulant cultivar SRN39 and high stimulant cultivar Shanqui Red. The mean MHD values measured in this experiment are compared in Fig. 4 . Although the differences between the mean values of PQ-434 and SRN39 are significant (p < 0.008), the greater distinguishing feature of the wild sorghum from the cultivars is the very low percentage of striga near its roots that form haustoria (Fig. 5) . By this measure, the striga-resistant cultivar SRN39 did not significantly differ from the striga-susceptible Shanqui Red in haustorial initiation activity. In contrast to the high correlation (Pearson r = 0.96, p < 0.0001) found in this experiment between MGD and near-root percent germination before ethylene treatment, MHD was only partly correlated with percentage of germinated striga that formed haustoria (r = 0.65). It is possible that screening for low haustorial initiation activity using only MHD might miss sorghums that produce a haustorial initiation signal but inhibit haustorial formation near their roots. Such sorghums may possess a very effective striga resistance mechanism.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Evidence of water-soluble inhibitors to striga germination in the root exudates of sorghum cultivars was reported by Weerasuriya et al. (1993). These unidentified inhibitors appeared to be present at low levels in both low and high stimulant producing cultivars. PQ-434 may produce higher levels of similar compounds or more effective ones. Although relatively fewer striga near the roots of PQ-434 germinated compared with weed seed near the roots of the checks or in agar with no sorghum after ethylene treatment, that some germination did occur suggests that whatever inhibition this wild sorghum has on striga germination is incomplete.
Host-derived cues of haustorial initiation are different from compounds that stimulate striga seed germination. Fewer compounds are demonstrably capable of cueing haustorial initiation. Compounds such as kinetin, simple phenolic compounds, and quinones like 2,6-dimethoxy-1,4-benzoquinone (DMBQ) are quite active haustorial initiators (Riopel and Timko, 1995), but their presence in host root exudates is only detectable when host roots are mechanically damaged (Riopel et al., 1990). It is hypothesized that striga aids in the release of DMBQ and other xenognostic quinones, catabolites of host cell wall peroxidases (Chang and Lynn, 1986).
A current model of haustorial induction in S. asiatica with sorghum describes how the process is assured to occur in close proximity to the sorghum root (Keyes et al., 2000). Localized production of hydrogen peroxide by the root tip of the, as yet, free-living striga provides the rate limiting cosubstrate for host cell wall peroxidases. The peroxidases use the H2O2 to oxidize the phenolic components of host cell walls such as syringic acid into xenognostic quinones such as DMBQ. Quinones and other reactive oxygen species are involved in defense mechanisms against pathogens (Lamb and Dixon, 1997). Striga might exploit these defense responses of the host to determine the proximity and viability of its roots. DMBQ then binds to a receptor site on striga and acts as part of a largely uncharacterized redox response circuit in which an electron is transferred through a semiquinone intermediate. The bound quinone is reduced and reoxidized as part of an electron transport chain that ultimately triggers the transition to the parasitic mode expressed through organogenesis of the haustorium. During a 6-h induction process, the xenognostic quinones must be in constant supply (Smith et al., 1990). Several messages were identified in molecular characterization of S. asiatica as expansins, compounds that allow expansion of cell walls during elongation. Three of these, SaExp1, SaExp2 and SaExp3, are intimately linked to the process of haustorial induction (O'Malley and Lynn, 2000).
The survey of the wild sorghum collection revealed accessions with a wide range of MHD values (Table 1), such as IS18874 and HD#758 with mean MHD > 4 mm and the low haustorial initiators PQ-434, IS14301, IS14313 and IS18803 with mean MHD < 0.5 mm. None of the known striga-resistant cultivars had such low MHD values as the latter group, but many exceeded the highest values measured among the wild sorghums assayed. In light of the xenognostic quinone model of haustorial initiation (Keyes et al., 2000), both high and low types are quite interesting. The high MHD values may reflect a varied cell wall composition in these sorghums that when digested with peroxidases release a more active or diffusible xenognosic compound than DMBQ. The low MHD sorghums may possess traits that interfere with H2O2 production, have lower levels of peroxidases, or altered root epidermal cell wall structure such that the xenognostic quinones are not produced. Alternatively, quinones released are inhibitory of the semiquinone redox response at the striga-binding site in a manner similar to those compounds described by Smith et al. (1996).
Low MHD sorghums on the other hand may have all the traits needed for the coordinated release of xenognostic quinones to stimulate haustorial formation in surrounding striga, but their roots may exude compounds like auxins, which are powerful inhibitors of haustorial induction (Keyes et al., 2000). The observation that sorghums with very low MHD usually have a low MGD suggests that some wider acting allelopathic compounds are being produced that inhibit both striga germination and haustorial initiation. Evidence gathered in Exp. 3 suggests that the inhibitory effects of PQ-434 on germination are not complete. It will be interesting to test this accession and others like it for inhibitory effects on haustorial initiation. It is unlikely that the association between very low MGD and MHD in these wild accessions is an artifact of the assay since many of the screened cultivars that showed similarly low MGD values in Exp. 1 gave high MHD measurements. Any haustorial initiation signal produced by these low MGD cultivars must have diffused through the agar to trigger haustorial formation by the time striga was germinated by applied ethylene.
Because the Carolina strain of Striga asiatica used in our assays is of a limited genetic base (Werth et al., 1984), predictions about the usefulness of the observed phenomenon in the wider solution to the striga problem are made guardedly. This collection of wild accessions has not yet been assayed in the presence of Striga hermonthica, which in much of Africa is a worse weed than S. asiatica. However, past selection of breeding materials for striga resistance in Africa has been aided by the agar gel assay using S. asiatica seeds (Hess et al., 1992; Haussmann et al., 2000). Low germination stimulant lines identified by measuring MGD with S. asiatica usually show similar values when assayed with S. hermonthica. However, there are notable exceptions when various populations of S. hermonthica seed are compared (Haussmann et al., 2001). The assays are useful in that they provide relatively quick measures of specific interactions and identify candidate entries for further testing. They can be used as a tool for combining mechanisms of resistance that act at specific points during the sorghum x striga association.
Heritability of the low haustorial initiation capacity has not been clearly established. Preliminary results from progeny of PQ-434 crossed with high germination stimulant lines indicate that the low haustorial initiation trait is simply inherited with dominant gene action (Mohamed, 2002). If the low MHD trait is transferable and can effectively prevent haustorial initiation in any Striga spp., then improved sorghums could be developed that would presumably avoid parasitic attack. More significantly, a low haustorial initiation trait could be combined with high germination stimulant production in a striga resistant sorghum that would also help to rid the surrounding soil of viable striga seed, eliminating the need to sacrifice a sorghum season to trap crops.
MHD, or percentage of haustoria formed in the extended agar gel assay, may be useful measures of a currently unexploited striga resistance mechanism of low haustorial initiation activity. The predictive value of MHD and percentage of haustorial initiation to actual striga resistance remains to be tested. It is evident from the survey of the twenty cultivars in Table 1 that other mechanisms beyond low germination stimulation and low haustorial initiation contribute to their striga resistance since some of those with high MGD and MHD values are reported to have field resistance. Potential post-attachment reactions in sorghum against striga have been reported including the hypersensitive (Mohamed et al., 2003) and incompatible (Arnaud et al., 1999; Grenier et al., 2001) responses. The former was identified in wild accession #47-121 from our collection (Mohamed et al., 2003). By combining genes conferring preattachment with postattachment mechanisms, aided by assays for each, sorghums with durable striga resistance can be developed. Wild sorghums may be sources of unique resistance traits lacking in cultivars since they have evolved under selective pressures imposed by Striga spp.
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication March 24, 2003.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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