African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 3, Num. 2, 1995, pp. 217-222

Integrating biotechnological approaches for the control of Striga

R.K. VOGLER^1, G. EJETA^1 and L.G. BUTLER^2

^1 Department of Agronomy, ^2 Department of Biochemistry, Purdue University, West Lafayette. IN 47907, USA.

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ABSTRACT

Our research addresses the Striga problem as a series of interactions between the parasite and its hosts, with potential for intervention. The working hypothesis is that the intricate relations between Striga and its host can be interrupted leading to development of control strategies. Using this approach, key compounds involved in Striga germination, and an effective bioassay for isolating sorghum cultivars that are resistant to Striga has been developed. This assay has been useful in establishing the mode of inheritance for resistance against Striga, and has been used to transfer the gene for low stimulant production into more productive sorghum cultivars, and to map this gene in the molecular linkage map of sorghum currently under construction. In January 1995, eight high yielding Striga resistant food-grain sorghum cultivars were released for wide use in Striga-endemic areas of Africa. Plans are underway for a pilot project between Purdue University, World Vision Relief and Development Inc., and the United States Agency for International Development (USAID) to increase and distribute seed of these cultivars to farmers in ten African countries.

Key Words: Bioassay, Striga germination, stimulant production, sorghum, resistance

RESUME

Notre recherche examine le probleme de Striga dans des series d'interactions entre le parasite et ses hotes afin de pouvoir intervenir l'hypothese de travail consiste a interrompre les relations entre le Striga et son hote pour developper des strategies de controle. Avec cette approche, des composantes impliquees dans la germination du Striga et la technique de bioassay permettant d'isoler les varietes resistances ont ete mises au point. Le bioassay s'est avere utile dans l'etablissement du mode d'heritabilite de la resistance au Striga et le transfert du gene pour la faible stimulation de la production dans les cultivars hautement productifs du sorgho ainsi que dans le positionnement de ce gene dans le lien moleculaire en construction en ce moment. Enjanvier 1995, 8 cultivars de sorgho a graines de haut rendement et resistants au Striga etaient diffuses pour une large utilisation dans les zones d'Afrique endemiques au striga. Un projet pilote est en train d'etre monte entre l'Universite de Purdue, le World Vision Relief and Development Inc, et I'USAID pour multiplier et distribuer les semences de ces cultivars aux agriculteurs de 10 pays africains.

Mots Cles: Bioassay, Germination de striga, stimulant de la production, sorgho, resistance

INTRODUCTION

Control measures developed through conventional research have been of limited value to subsistence farmers in combating the menace of Striga in the semi-arid tropics (Ogborn, 1987 ). The low efficacy of control measures, such as chemical fertilizers or herbicides, or cultural practices. may be due, in part, to lack of access to an already overpriced market of production inputs (Eplee and Norris, 1987) and also, in part, to the limited understanding of the host-parasite interaction. The specific mechanisms of physiological, biochemical, and genetic adaptations of the parasite to its hosts have eluded researchers to date. This is why we have focused our research on the fundamental understanding of the signals involved in parasitism (Ejeta et al., 1991; Ejeta and Butler, 1993).

Striga spp. are obligate parasites whose interactions with hosts are vital to their survival. By interrupting this interaction, one can develop control strategies. But first. there is need to fully understand the basic biology of the host-parasite interactions. Past and present research has shown that successful parasitism depends on a series of chemical signals produced by the host roots. It has been observed, too, that host plants vary in their capacity to produce these signals and that host resistance is often based on the ability or inability to produce and exchange these signals with the potential parasite.

Although it is known that host resistance is complex, and often involves physiological and physical mechanisms. we are attempting to unravel host-resistance by breaking it into component parts based on the signals exchanged at each stage of the Striga life cycle.

APPROACH

Unlike other Striga research programmes, ours is focused on developing control strategies primarily based on host-plant resistance. To this end, integratedbiotechnological approaches combining techniques in biochemistry, tissue culture, geneucs and plant breeding, and molecular biology have been employed and are briefly described, below.

Biochemistry. Germination stimulants are produced by roots of host and non-host plants. Very small quantities are needed to induce germination ofconditioned Striga seeds (Hsiao et al., 1981).

Cook et al. (1966) first identified strigol from the root exudates of cotton. a non-host plant. The first germination stimulant isolated from a host plant (sorghum) was sorgoleone (Chang et al., 1986). which was unstable in its active form and of low water solubility. There is lack of correlation between sorgoleone production and field resistance by sorghum cultivars (Hess et al., 1992). We also developed a simple and rapid bioassay for stimulant production, the agar gel assay, which is used to screen individual host seedlings for their capacity to germinate conditioned Striga seeds embedded in a water agar. The assay is sensitive to water soluble stimulants, and it corresponds with the reported field resistance against Striga for the cultivars tested (Hess et al., 1992).

Hauck et al. (19921 were the first to isolate and identify a major water soluble germination stimulant lot Striga, sorgolactone, a strigol analog, from sorghum root exudate. The same group also identified the major stimulant from cowpea as another strigol analog, alectrol (Muller et al., 19921. Using HPLC and mass spectrometry. Siame et al. (1993) recently identified strigol as the major stimulant produced by.maize and proso millet, and as a minor stimulant in sorghum root exudates.

Ethylene biosynthesis and germination in Striga asiatica seeds. Ethylene has long been known for its role in fruit ripening and as a plant growth regulator. Recent reports revealed that endogenous ethylene plays a key role in the response of Striga hermothica (Del.) Benth. to germination stimulants (Jackson and Parker, 1991; Logan and Stewart, 1991). Babiker et al. (1993b) used gas chromatography with an activated alumina column and a flame ionization detector to determine ethylene production (Wang and Woodson, 1989) in strigol-induced germination of Striga asiatica (L.) Kuntze seeds. Their results confirmed the same response in S. asiatica (Babiker et al., 199 3b) as found in S. hermonthica. Strigol had an increasing ability to stimulate ethylene production and the subsequent germination of S. asiatica seeds with increasing conditioning over an eight day period. Babiker et al. (1993b) concluded that conditioning results in the partial release of the ethylene biosynthetic pathway by triggering the activation and/or synthesis of the ethylene forming enzyme (EFE), which in turn converts the immediate precursor, ACC (1-amino cyclopropane-l-carboxylic acid) to ethylene. A germination stimulant is also needed to trigger the conversion of ACC to ethylene.

In a related experiment, Babiker etal. (1993a) observed that cytokinins increase the capacity of S. asiatica seed to convert ACC to ethylene and also the germination of seeds. Based on previous reports that cytokinins enhance ACC oxidase activity in plant seeds and vegetative parts, and that auxin-cytokinin combinations enhanced ethylene production, Babiker et al. (1994) also demonstrated the cytokinin-like activity of thidiazuron, a substituted urea commercially available as a cotton defoliant, that when combined with several auxins elicits ethylene production. Not only was ethylene production increased. but germination of S. asiatica seeds also increased when compared with controls. It was concluded that ethylene biosynthesis and action are crucial in the germination of S. asiatica seeds; and that dormancy in Striga seeds is associated with the low capacity of the seeds to convert ACC to ethylene.

Tissue culture. Cai et al. (1993) and Butler et al. (1994) cultured S. asiatica in vitro and revealed that a host signal is required for the further development of Striga after germination and haustorial attachment. Only on host-conditioned media (prepared by culturing excised sorghum roots or sorghum plants, or by addition of sorghum root exudates, root or shoot extracts) did they observe the development of parasitic-type S. astatica seedlings. On control media, containing exogenous hormones but lacking any host components, non-parasitic-type Striga seedlings developed. A differential response in Striga secdling growth to root and shoot extracts from Striga susceptible and Striga resistant sorghum cultivars was also observed. Striga seedlings grew well on media conditioned with extracts from the susceptible cultivar IS 4225 compared to the weak growth observed on media conditioned with extracts from the resistant cultivar SRN39.

Cytokinins and auxins modulate development of S. asiatica. Previous reports revealed that auxins and cytokinins have synergistic effects on ethylene production and morphogenic regulation in several plants (Fellman et al., 1987). Babiker et al. (1994) and Butler et al. (1994) observed hormone- dependent morphology of S. asiatica seedlings grown in vitro. Exogenous plant hormones prevented shoot formation and delayed shoot appearance. The auxin 2,4-D, a commercial herbicide, resulted in callus formation with no further differentiation of S. asiatica. Only nonparasitic type Striga seedlings developed.

Response of sorghum plants to S. asiatica extracts. It has long been thought that host plants may use antibiosis as a mechanism against Striga parasitization (Saunders, 1933). Recent experiments in our laboratory have shown that Striga may also be transmitting toxins to its hosts. Sorghum calli were exposed to varying concentrations of S. asiatica extracts (from leaf and stem tissue) under conditions in which most of the cells died (Cai et al., unpublished). A few surviving ceils were rescued and grown in toxinfree media. These new clones will be tested for field resistance/tolerance to Striga infestation. In another experiment, 10-day old sorghum seedlings of both field susceptible and field resistant cultivars to aqueous extracts or S. hermonthica (from shoots) and S. asiatica (from shoots or unemerged seedlings) were exposed (Bell-Lelong etal., 1993). The aqueous Striga extracts exhibited allelopathic activity (chlorosis, wilting, and leaf curling) against the sorghum seedlings.

Genetics and plant breeding. Field screening for Striga resistant cultivars is often unreliable and slow because of the inconsistent nature of Striga infestation both within the same field and among different fields across years. Stable and durable sources of Striga resistance are also rare. Based on extensive testing across several years, Hess and Ejeta (1992) determined that the sorghum cultivar SRN39 is a superior source of field resistance against Striga which is recessive anct simply inherited. i.e., controlled by one to two genes. SRN39 was officially released in 1991 for commercial cultivation by farmers in Striga endemic areas of Sudan.

Inheritance of low production stimulation for a Striga seed germination factor in the sorghum cultivar SRN39. Striga resistance in some sorghum varieties has been reported to be due to low stimulant production. Screening for stimulant production was tedious until 1990 when the agar gel assay was developed (Hess et al., 1992). The assay is simple and rapid, as well as nondestructive. It screens host plant seedlings for their capacity to germinate conditioned Striga seeds, thus it could be used to distinguish low and high stimulating genotypes. The assay also correlated well with the reported field resistance of the cultivars tested. Using this assay, it was detennined that Striga resistance in SRN39 was due primarily to low production of germination stimulants, and that this is inherited as a single recessive gene (Vogler, 1992). This study examined three diverse crosses involving low x high stimulant producing genotypes, where SRN39 was the low parental type.

Conventional breeding of Striga resistant sorghum cultivars. Conventional plant breeding for Striga resistance has traditionally involved field evaluation of germpIasm under artifictal or natural infestation. However, field screening is usually Iimited to replicated evaluation of small numbers of entries, and the procedure does not lend itself to screening large numbers of entries because of the difficulty of establishing uniform infestations and the complexities of the hostparasite-environment interactions arising from such an evaluation. In general, progress in breeding for broad, durable resistance against Striga has been limited because of 1) species specific resistance against Striga, 2) intraspecific or physiological variants ofStriga, 3) the paucity of resistance genes in crop germplasm, and 4) the lack of rapid and effective screening techniques (Ejeta et at., 1991, 1993).

The development of the agar gel assay (Hess et al., 1992), however, has made possible the rapid screening and cataloguing of a large amount of germplasm and breeding lines based on one mechanism of resistance, low stimulant production. Moreover, because this assay has demonstrated correspondence with field resistance against Striga (Hess et al., 1992; Vogler, 1992), our sorghum breeding efforts for Striga resistance have focused, since 1990, on the development of lines with low stimulant production. Sorghum cultivar SRN39 has been used extensively in our breeding programme as the source of this resistance mechanism.

Molecular biology. Molecular markers can be powerful tours for learning the genetics of certain traits, even compIex traits like Striga resistance. A DNA marker identifies or "marks" the chromosomal segment in its vicinity and can be followed through genetic manipulations (Stuber, 1989). RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers are being used to construct a genetic map of a sorghum population derived from a diverse cross between SRN39, a zera zeta fi'om Africa, and Shanqui Red, a kaoliang from China (Melake Berhan et al., 1993).

Location ofa RAPD molecular marker linked to a germination stimulant production. Weerasuriya (1994) helped to further saturate the RFLP map with RAPD markers and. using the agar gel assay to screen the above mentioned population, he mapped the gene for germination stimulant production to one end of a RAPD derived linkage group. The map distance between this gene and the closest marker, SSR903b, an inter-simple-sequence- repeat (I-SSR) primer, was 13.5 cM. A maize RFLP, Pi200275, mapped 46 cM from the locus for stimulant production.

ACKNOWLEDGMENTS

The research on Striga is partially supporteel by USAID Grant No. DAN 1254-G-00-0021-00 through INTSORMIL, the International Sorghum and Millet CRSP, by Program Support Grant No. DSAN-XII-G-0124, by Grant No. GA AS 8905 from the Rockeleller Foundation, and by the McKnight Foundation.

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