Crop Science 43:1320-1324 (2003)
© 2003 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Hypersensitive Response to Striga Infection in Sorghum
A. Mohamed,
A. Ellicott,
T. L. Housley and
G. Ejeta*
Dep. of Agronomy, 1150 Lilly Hall of Life Sci., Purdue Univ., West-Lafayette, IN 47907-1150
* Corresponding author (gejeta{at}purdue.edu)
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ABSTRACT
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Availability of appropriate laboratory procedures that reveal the specific interactions between the parasitic striga (Striga spp.) and host genotypes in the early stages of infection facilitates characterization of the specific mechanisms of resistance. Our objective was to use an in vitro extended agar gel assay (EAGA) to characterize hypersensitive response (HR) to parasitic invasion of sorghum [Sorghum bicolor (L.) Moench] genotypes. The HR was characterized by expression of necrotic lesions at the haustorial attachment sites which discouraged further penetration of the parasite into host roots. We examined the HR reaction of seven cultivated, five wild, and 95 BC3F4 genotypes derived from a wild resistant (P47121) and two susceptible male sterile based populations (CK60 and KP9). The susceptible genotypes showed no necrosis. In contrast, resistant cultivars Framida and Dobbs, and a wild accession, P47121, showed necrosis in >67% of attached striga. In each of these lines, attached striga were discouraged from penetration and further development. P47121 had the highest level of necrosis (89.9%) and discouraged haustoria penetration (83.1%), followed by Framida (71.4, 56.7%) and Dobbs (67.8, 49.4%). However, resistant genotypes SRN-39, IS9830, and 555 with low striga germination distance did not exhibit any HR. Only Framida possessed both a low germination distance and high HR. Nine BC3F4 genotypes had a moderate to high HR. These results suggest that use of P47121 and other resistant genotypes would further enhance development of striga-resistant sorghum cultivars through directed introgression.
Abbreviations: ddH2O, double distilled water • EAGA, extended agar gel assay • HR, hypersensitive response
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INTRODUCTION
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STRIGA is an obligate parasitic weed that significantly constrains crop production in Africa and Asia. In some years, striga may infest >21 million ha of crop land in Africa (Sauerborn, 1991) with annual crop losses estimated at 
40% (Lagoke et al., 1991). Crop damage is most severe where drought and low soil fertility already limit productivity (Parker and Riches, 1993). Control of striga is complicated by its enormous seed reserve in the soil that can be triggered to germinate and damage crops even before the striga plant emerges above ground. Much of the striga endemic regions of Africa and India are inhabited by subsistence farmers who are unable to adopt expensive chemical control or use of modern cultural practices. Hence, development of high-yielding striga-resistant crop cultivars is perhaps the most feasible means for reducing crop losses (Ejeta and Butler, 1993).
Significant progress has been made in breeding for striga resistance in several crops (Singh and Emechebe, 1990; Ejeta and Butler, 1993; Kim et al., 1998). Appearance of striga on host plants in the field is the eventual expression of a series of interactive events between the parasite and its hosts. Empirical breeding for striga resistance in field crops has relied on selection of host plants that allow emergence of few parasitic plants and show little or no loss in productivity of the crop. Sorghum genotypes with good levels of striga resistance have been identified using this approach. However, the specific mechanisms of many of these resistance sources have not been determined. Nevertheless, there appears to be a general parallel between host–pathogen interactions in plant diseases and defense responses triggered during striga invasion. The major limitation to making a precise determination of these observations during the development of the parasite appears to be the lack of appropriate bioassays that reveal early interactions between the host and parasite.
One host-resistance mechanism, the HR, has been extensively studied in plant–pathogen interactions. Hypersensitive response has not been previously observed in sorghum, although some cultivated and wild sorghums have been reported to severely impede growth of the parasite following infection (Lane et al., 1991). In other plant–pathogen interactions, the HR generally refers to the appearance of a necrotic lesion around the site of attempted infection, followed closely by death of the affected host cells within hours of the attack (Agrios, 1988). The HR can be phenotypically diverse, ranging from a single cell response to large and spreading necrotic areas in a tissue accompanying parasitic colonization (Holub et al., 1994). Necrosis of the affected tissue has been shown, in some situations, to be directly related to the accumulation, oxidation, and polymerization of phenolic compounds (Nicholson et al., 1989). Our objective was to identify and characterize striga-resistant sorghum genotypes that express HR using an in vitro system, the EAGA, a modification of a procedure we have described earlier (Hess et al., 1992).
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MATERIALS AND METHODS
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Genetic Material
A collection of sorghum cultivars, wild accessions, and breeding lines from our sorghum breeding program was sampled for this study. Seven cultivated sorghum lines (SRN-39, Framida, IS9830, 555, Dobbs, IS4225, and Shan Qui Red) with known field reaction to striga infection, five wild sorghum accessions (P47121, P1885, P14539, P14529, and P12-26), and 95 BC3F4 progenies derived from a cross between P47121 and two male sterile based populations (CK60 and KP9) were evaluated for HR to striga invasion. All seeds were from plants grown at the Purdue University Agronomy Research Center, West Lafayette, IN.
Seeds of Striga asiatica (L.) Kuntze were obtained from the USDA/APHIS, Methods Development Center, Whiteville, NC, courtesy of Drs. Robert Eplee and Rebecca Norris. Striga seeds were stored and handled and experiments performed under quarantine restrictions in an approved quarantine laboratory located on the campus of Purdue University. Striga seeds were conditioned following a procedure described by Mohamed et al. (1998).
The Extended Agar Gel Assay
The EAGA is a modification of the agar gel assay described by Hess et al. (1992). In this assay, 150-mm Petri dishes with a thick agar layer were used to support growth of sorghum seedlings for a longer period than in the agar gel assay. Because seedlings in the EAGA could be grown for longer periods, effects of signal exchanges between sorghum roots and striga haustoria as well as host defense responses beyond germination could be observed. With the agar gel assay, we were only able to detect striga germination in response to exudates from host roots with no opportunity to observe post attachment parasitic reactions. The EAGA, on the other hand, allows observation of haustorial attachment and expression of HR events post-attachment following ethylene treatment, in addition to striga germination.
Sorghum seeds were soaked in 10 mL 50% sodium hypochloride (NaOCl) solution (500 mL L-1 commercial bleach) for 30 to 60 min and rinsed three times with double distilled water (ddH2O). They were then soaked in a 0.5% solution of N-[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarbomixide (Captan 50-W) for 24 h, washed three times in sterile ddH2O, transferred to Petri dishes that contained moist filter paper, and incubated in the dark at 28°C for 24 h. Only healthy germinated seeds were used for the EAGA.
About 1500 striga seeds (four drops of settled seeds) conditioned for 8 to 22 d were pipetted into a sterile 150-mm Petri dish. A 0.7% agar solution was autoclaved at 121°C and 103.5 x 103 Pa for 15 min, then cooled to 50°C for at least 1 h. The 50°C agar was poured into the Petri dish, containing conditioned striga seeds, which produced an even distribution of seeds. Four pregerminated sorghum seeds were placed at even intervals around the edges of each Petri dish so that the radicles, directed toward the center of the dish, just penetrated the gel. The Petri dishes were then covered and placed in an incubator at 28°C.
Three days following inoculation, each dish was observed for germination, parasitic attachment, and host root development, then treated with ethylene to remove any difference in host roots for inducing striga seed germination. Ethylene elicits germination of viable striga seeds including those not affected by host root exudates (Jackson and Parker, 1991). Ethylene treatment was done in a dosing chamber located in the biosafety cabinet to prevent ethylene contamination of the room. A 30-s burst inside the sealed chamber is sufficient to germinate viable striga seeds. Plates were left in this chamber for 2 d. After 48 h in the dosing chamber (5 d following inoculation), plates were vented in front of the biosafety cabinet to remove any residual ethylene before placing them under grow light for 12 h. Data were collected under the microscope where the entire length of the host seedling root was scanned under a magnification of at least 25x. The dishes were observed at 2, 5, and 7 d following ethylene dosing (5, 7, and 10 d after pouring the plates) for striga attachment, and number of attachment sites was recorded. Sites of attached striga were circled for future observation of necrosis and parasitic discouragement. A striga seedling was counted as having a haustorium only if hairlike projections (tubercles) were present on its radicle. The three most distant striga (with haustoria) from the host seedling root were identified and the shortest distance from the striga seedling to the primary host root was measured to the nearest 0.5 mm. The HR was observed when a clear necrotic lesion developed around the attachment site. The reaction normally started a few hours after attachment and the lesion became more intense in 24 h. Striga seedling discouragement was observed 3 d following the attachment. Whereas striga seedlings attached on susceptible host roots penetrated and developed, those on resistant roots were discouraged and never penetrated or developed beyond attachment.
Each experiment was conducted as a completely randomized design with three or four replications. Data were collected from 12 to 16 sorghum seedlings (four seedlings in each Petri dish, replicated three or four times) on total number of striga seeds that germinated, seedlings that attached, penetrated, caused necrosis, or those discouraged from further development. The percentage of striga seedlings with necrosis and with discouraged attachment relative to total attachments in each of four sorghum roots was analyzed statistically. Germination data were transformed to arcsine, examined by ANOVA, and back transformed. Analysis of variance was performed independently for experiments involving cultivated and wild sorghums using the program PROC-ANOVA in SAS (SAS Institute, 1990). Means in individual experiments were tested for significance using LSD.
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RESULTS AND DISCUSSION
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All susceptible sorghum cultivars had a normal association with striga (Fig. 1a) when observed under the conditions of the EAGA. Striga seedlings developed successfully without any sign of stress or damage. However, in some striga-resistant sorghum cultivars and wild sorghum genotypes, necrotic areas appeared at striga attachment sites on the root (Fig. 1b). These necrotic lesions most often started as red spots, which turned brown with time. The lesions were often large and some spread up to 2 mm from attachment site, but most remained localized. Attached striga at these necrotic sites often did not develop further (discouraged from penetration), and eventually died on the host.
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Fig. 1. Extended agar gel assay allows identification of postinfection host (H) response to Striga (S) in early stages of parasitic development. Susceptible genotypes support normal growth (a) whereas resistant genotypes exhibit a hypersensitive response (b) following haustorial attachment.
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Among cultivars, Framida and Dobbs showed considerable necrosis and the attached striga were discouraged from penetration (Table 1). No necrosis was observed on roots of susceptible genotypes. Some striga-resistant cultivars, including SRN-39, IS9830, and 555, also did not show necrotic lesions. These genotypes are resistant to striga because they are low producers of the striga germination signal, as indicated by germination distance (Table 1). Among the wild genotypes, P47121 {Sorghum verticilliflorum (Steud.) Stapf [= S. arundinaceum (Desv.) Stapf]} showed the highest levels of necrotic lesions and discouraged penetration (Table 1). Thus, sorghum genotypes were classified into two categories, those exhibiting HR and those with no necrosis. Susceptible genotypes induced striga germination and supported its normal growth and development. The EAGA was effective in demonstrating that different striga-resistant sorghum genotypes may possess different resistance mechanisms. The widely used cultivar Framida, with high field resistance, expressed both HR and a low germination stimulant production. Some genotypes with moderate field resistance expressed either low germination stimulant production (555, IS 9830) or a mild HR (Dobbs). The most widely used striga-resistant sorghum cultivar, SRN-39, did not exhibit HR in the EAGA.
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Table 1. Mean Striga seed germination, haustoria initiation, and percentage of Striga seedlings with necrosis and those discouraged from establishing on Sorghum cultivars with known field reaction. Each number is an average of 12 infected seedlings.
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The HR reaction of genotypes appeared to be graded and variable in intensity. A single infected sorghum root showed reddening at most but not all haustorial attachment sites. Some attachment sites appeared necrotic early and faded, with striga growing normally. Overall, however, genotypes possessing HR showed greatly reduced percentages of striga attachments and parasitic association relative to susceptible genotypes.
In a further evaluation of the two best genotypes for HR (P47121 and Framida) and examining a very large number of attachment sites (Table 2), we found that the wild genotype P47121 had a stronger necrotic reaction than Framida. The reaction in P47121 was almost twice as intense as in Framida, which showed the strongest HR among the cultivated types. Nearly 83% of the Striga attached to P47121 were discouraged from further development compared with 49% for Framida (Table 3). These results suggest that P47121 would be an excellent donor parent for introgressing HR gene(s) for striga resistance into sorghum cultivars.
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Table 2. Percentage of Striga seedlings with necrosis or those discouraged from establishing on wild sorghum accession P47121 and sorghum cultivar Framida, both with strongest reaction among the lines tested. Each number is an average of 16 infected seedlings.
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Table 3. Hypersensitive response reactions of BC3F4 progenies of (P47121 x CK60) and (P47121 x KP9) and their parents. Each number is an average of 12 infected seedlings.
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Of 95 BC3F4 progenies derived from the two populations involving P47121, nine had moderately high HR (Table 3). Before our screening with the EAGA, these progenies were selected for agronomic traits and not for HR. The parental populations CK60 and KP9 showed no necrosis and supported normal development of Striga seedlings when infected. Of the nine BC3F4 selections, CK32 and KP33 had HR similar to that of P47121 (Table 3). The number of attached striga seedlings discouraged from further penetration was also equally high on these two lines as it was on P47121.
In genotypes exhibiting HR, necrosis was observed as early as 3 d after infection (Fig. 2a). Discouragement of parasitic development was evident 7 d following infection, reaching a maximum in 12 d after infection (Fig. 2b). In general, sorghum genotypes that showed necrosis at attachment sites also showed parasitic discouragement (r2 = 0.85, P < 0.01) (Fig. 3). While parasitic discouragement can be noted, observation made on necrosis is more readily apparent and reliable since this symptom appears early when the health of the host tissue is of little concern. The HR to striga invasion is also readily observable with the paper roll assay (Mohamed et al., 2001).
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Fig. 2. Observation time for onset of necrosis (a) and Striga seedling discouragement (b) as detected with the extended agar gel assay.
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Fig. 3. Correlation between number of Striga seedlings attached to host seedling roots with clear necrosis at the haustorial attachment site and the number of attached striga seedlings that were discouraged from penetrating and establishing on sorghum roots.
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The HR observed in striga-resistant sorghum genotypes fits the classical definition of HR in plant–pathogen interactions, whereby death of host tissue occurs within a few hours of attempted parasitic ingress. Hypersensitive response in striga was expressed as a necrotic lesion at the attachment sites and served to discourage parasitic establishment. The HR as an active host defense response has been mostly studied in host–pathogen interactions. A similar phenomenon was observed in sunflower (Helianthus annuus L.) and chickpea (Cicer arietinum L.) infected with orobanche (Orobanche spp.) that involved phytoalexin synthesis (Wegmann et al., 1991). The HR component, phytoalexin, was reported to induce resistance in sunflower (cv. 81-14) and chickpea (cv. ILC 280) against orobanche. The toxic effect of the phytoalexin to orobanche was shown in submerged cell cultures where the attacked root responded by producing an HR expression of tissue necrosis followed by subsequent parasite death (Wegmann et al., 1991). Similarly, discouraged establishment of Striga on nonhost plants could be due solely to HR (Hood et al., 1998). The defense reaction in the nonhost was accompanied by browning or necrosis of cortical cells around the infection site, as well as accumulation of phenolic compounds (Gowda et al., 1999). The HR reaction we observed in sorghum appeared to be similar to those described as nonhost response to striga in these reports.
Though widely studied, the physiologic basis of HR in plant–pathogen interaction has not yet been clearly elucidated (Hammond-Kosack et al., 1996). Necrotic lesions around infection sites have been speculated to play a causal role in host plant resistance (Heath, 1980). On interaction with the parasite, the plant cell death reportedly deprives the parasite access to further nutrients. The role of the necrotic lesion may not be very evident, however, because some parasites can obtain nutrients from dead plant cells (Hammond-Kosack et al., 1996). On the other hand, cellular decomposition may lead to a release of toxic substances that are stored in the vacuole (Osbourn, 1996). Alternatively, the levels of induced phytoalexins which are usually rapidly turned over in plant cells may accumulate in inhibitory concentrations.
Hypersensitive response appears to be a potent mechanism of striga resistance in some sorghum cultivars. The intense expression of necrotic tissue at striga attachment sites of host roots followed with the eventual withering and death of parasitic seedlings suggest that HR expressed in the wild sorghum line P47121 and its derivatives CK32 and KP33 can be an excellent source of striga resistance in sorghum. Since HR expression is associated with discouraged attachment of the parasite, it could serve as a powerful striga resistance mechanism. For an in-depth understanding of the basis of HR in striga resistance, however, further investigation of the basic physiological mechanism needs to be pursued. Meanwhile, we are exploiting the simple inheritance of HR for effective pyramiding of genes for Striga resistance with multiple mechanisms through conventional breeding and introgression.
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ACKNOWLEDGMENTS
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This research was partially funded by USAID Grant DAN 254-G-00-002 through INTSORMIL, the International Sorghum and Millet Collaborative Research Support Program, and by the Rockefeller Foundation under Grant 2000 FS024 to Purdue University.
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NOTES
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This research was supported by the Rockefeller Foundation (Grant no. 2000FS024) and the Int. Sorghum and Millet (INTSORMIL) Collaborative Research Support Program (Grant no. LAG-g-00-96-90009-00). Purdue Agric. Res. Programs, Journal Paper no. 16 745.
Received for publication March 13, 2002.
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ABSTRACT
INTRODUCTION
