Abstract: Background and Objective: Resistance through reduced strigolactones is one of the sustainable ways of managing Striga asiatica. To verify the existence of reduced strigolactone production in sorghum genotypes, an agar jel assay was carried out on seven sorghum bicolor lines and one Sorghum arundinaceaum sourced in Zimbabwe. Methodology: In the first experiment, pre-germinated Striga seeds were pipetted into a Petri dish with drying agar jel and pre-germinated sorghum seedlings were grown across the Petri dish. The eight sorghum genotypes were also grown in a sand culture and the number of Striga that attached were recorded. Results: The results indicated that sorghum genotypes varied significantly (p<0.05) with respect to maximum germination distance (Mgd) with wild sorghum and SC sila having the largest mgds indicating that they produced the largest quantities of strigolactones. The genotypes Mukadziusaende had the highest tiller numbers while SC sila had the lowest. Striga counts were highest on Wild Sorghum, Ruzangwaya and Hlubi. There was a negative correlation between mgd and tiller number showing that the highest strigolactone producers had low tiller numbers. A correlation coefficient of 0.564 between mgd and Striga counts showed that as Strigolactones increase Striga counts also increase. Conclusion: It can therefore be concluded that resistance through reduced strigolactones was found in the sorghum genotype Mukadziusaende. The direct relationship between mgd means that tiller number can be used to select for reduced strigolactone production in the field.
INTRODUCTION
The average Sorghum productivity in the Sub-saharan Africa subcontinent is low as a result of a myriad of production constraints of which Striga asiatica is a major obstacle. Striga asiatica is an obligate hemi-parasite that attach to the roots of several crop species leading to severe yield loss1. This Striga spp. is geographically the most widespread species with large populations having been reported throughout sub-saharan Africa, south east China and the Indian subcontinent while smaller isolated populations have been reported in Arabia, Indonesia, Philippines, north and east Carolina (USA) and Australia2. The S. asiatica is the predominant species towards the east African coast and southern Africa3. According to Bouwmeester et al.4 these parasites especially the Striga spp. infests about two thirds of the 70 million ha used for cereal production in Africa. Jamil et al.5 asserts that about 20-80% yield losses or even complete crop failure can occur due to parasitism.
The root parasitic weed has developed the ability to germinate only when they are exposed to germination stimulants released from the host roots, thus synchronising their life cycle to those of their potential hosts only germinating when a suitable host seed is in proximity to the Striga seed6. Fernadez-Aparacio et al.7 reported that the synchrony is vital for parasitic weed survival because they have an absolute requirement for nutritional support from the host.
According to Bouwmeester et al.4 and Akiyama and Hayashi8, the first critical step in the life cycle of Striga, the germination of its seed, is regulated by strigolactones. The dependence on strigolactones could be exploited for Striga management through selecting for low strigolactones producing cultivars. The seeds of these parasitic plants will only germinate after perceiving a germination stimulant of their host9. After radicle emergence, the haustoria attaches and penetrates the host roots10. Cardoso et al.11 reported that once germination has been triggered, the radicle protrudes from the testa, elongates towards the root and develops haustorium, an organ that can attach to and penetrate roots of the host plant. The parasitic plant grows underground for 4-7 weeks prior to emergence and utilises host water, nutrients and photosynthates12. Much of the damage will have occurred by the time the Striga emerges above the ground.
Jamil et al.12 found significant variation among NERICA rice cultivars and their parents for strigolactones production and Striga germination. Production of low germination stimulants results in low numbers of Striga asiatica attachments thereby producing a resistant phenotype. Studies done in Sorghum have also shown that genotypes with low production of germination stimulant have demonstrated to be resistant to Striga in the field13-15. Since the root parasites affect the crop from the time they attach to the root, the development of new control strategies should focus on the initial steps in host parasite interaction16.
Ejeta et al.17, Wilson et al.18, Gurney et al.19 and Gurney et al.20 demonstrated that the near relatives of cereals could provide new sources of tolerance and or resistance to the parasite infection and may provide the way forward for the control of Striga spp. According to Doggett21,22 and De Wet23 the cultivated Sorghums of today primarily originated in Africa from the wild Sorghum bicolor spp. Arundinaceaum. Southern Africa has more sorghum landraces and whilst the quest to find a land race that produce the least strigolactones is still on, there is need to look at the wild Sorghum and the vast number of Sorghum landraces that are under cultivation in the African savannah. Given the high genetic diversity of the Sorghum spp. including wild Sorghum, there is need to quantify strigolactones in most of these cultivated lines as they are grown in Striga infested fields. According to Ejeta and Butler 24 different Sorghum genotypes differed by as much as a billion fold in the amount of germination stimulants they produce. Jamil et al.5 asserts that strigolactones have a triple role which is underground communication between the plant and AM fungi and parasitic plants and the regulation of tillering. According to Umehara et al.25 and Lopez-Raez et al.26 strigolactones inhibit tillering in plants and therefore the ability to tiller can be used as a selection criteria for reduced strigolactones production. Therefore, the objective of this study was to quantify strigolactones in Sorghum lines using agar jel analysis to determine Sorghum lines that produce the least strigolactones and therefore confers the highest resistance. The research also aimed at correlating the maximum germination distance (MGD) and tillering and Striga asiatica counts.
MATERIALS AND METHODS
Experiment 1: Agar jel assays
Germplasm and chemicals: Seeds of 8 Sorghum bicolor varieties were obtained from the gene bank, Harare research Station in Zimbabwe. The seeds of Sorghum arunaceaum were obtained from Gwebi agricultural college fields which is 27 km west of the city of Harare.
Experimental design
Surface sterilisation and Sorghum seed germination: Sorghum seeds were soaked in 1% sodium hypochlorite solution for 60 min and rinsed in double deionised water. The seeds were soaked in an aqueous solution of 10% captan overnight. Seeds were rinsed with deionized water three times and then incubated in moist filter paper at 27°C. At 48 h germinating seeds were placed in agar plates as outlined by Hess et al.14. The Sorghum genotypes whose strigolactones were quantified are shown in Table 1.
Surface sterilisation and conditioning of Striga seed: Striga asiatica seeds were placed in 30 mL sample bottles and rinsed 3 times by adding 3-5 drops of the detergent tween 20 into 10 mL of distilled water. Sonication was done using an ultra sonic cleaner 3 min during the first rinse27. The Striga seeds were incubated at 27°C for 3 days prior to transferring them into the fresh sterile flasks containing 15 mL of 0.001% aqueous benomyl solution. The sample bottles were re-incubated at 25°C for 35 days before they were ready for use in the agar jel assay.
Assay set up: Pre-conditioned Striga seeds were pipetted into Petri dishes. Water agar was then poured over the seed. The roots of the germinating Sorghum seeds were placed in the solidifying agar with the root tip pointing across the plate. The plates were incubated in the dark for 5 days. The maximum germination distances (mgd): distance between the host root and the furthest germinated Striga seed were used as indicators of the quantities of strigolactones produced.
Sand culture: About 1 g of S. asiatica seeds were weighed for every treatment and mixed thoroughly with 2 kg of washed river sand. The sand was sterilized before the start of the experiment by heating in an oven at 120°C for 48 h to kill any Striga weed seeds that could be in the sand. The method was adapted from Jamil et al.5. Plastic pots of dimensions 18 cm diameter and 20 cm height were used.
| Table 1: | Sorghum genotypes used in strigolactone quantification |
Experimental design: There were 8 cultivars (Table 1) and the experiment was replicated four times and laid down as a completely randomized design.
Planting and data collection: Five Sorghum seeds were planted in pots at a depth of 0.5 cm the seedlings were thinned to one plant per pot at 2 WACE. The Sorghum were allowed to grow in pots for 15 weeks and the Striga that emerged were counted. The number of tiller in the Sorghum plants were also counted. The data was analyzed according to the model:
where, Yij is the measured parameter e.g., tiller number, μ is general mean, Ti effect of the treatment and eij is the error term. Correlation of the measured parameters were determined using SPSS.
RESULTS
Maximum germination distance (MGD): The Sorghum cultivars differed strongly (p = 0.001) with regard to maximum germination distance which is indicative of the strigolactones quantity produced (Fig. 1) at 120 h of incubation time at 30°C. The minimum germination distance was 1.225 cm and it was for Mukadziusaende whilst the maximum was 2.775 cm for SC Sila (Fig. 1). The higher the maximum genetic distance the higher the quantity of strigolactones production.
Tillering: Sorghum varied strongly (p<0.01) in tillering and the average number of tillers varied from 1.5 for SC Sila to 4.5 for Mukadziusaende (Fig. 2).
| Fig. 1: | Maximum germination differences for various Sorghum cultivars |
| Fig. 2: | Effect of Sorghum cultivars on tiller numbers |
| Fig. 3: | Effect of Sorghum cultivars on Striga counts |
The cultivars that had the highest number of tillers were Mukadziusaende and wild Sorghum while the lowest were SC Sila and Zambia (Fig. 2).
Striga counts: The Sorghum cultivars varied strongly in eliciting Striga germination (counts) (p<0.01) the cultivars that had the highest number of Striga counts were Ruzangwaya and Hlubi while the lowest were Mukadziusaende and Chiredhi (Fig. 3).
Correlations between maximum germination distance, tillering and Striga counts: A correlation coefficient between germination distance and tiller numbers shows a negative relationship with a correlation coefficient of -0.191. When the maximum germination distance increases the tiller number decreases. The correlation coefficient between germination distance and Striga counts has a value of 0.564 which shows a relatively strong relationship between the two variables. As mgd increases, Striga counts also increase (p<0.001).
DISCUSSION
The objective of this study was to determine the Sorghum genotype that had produced the lowest strigolactones and the correlate strigolactones production to tillering. The Sorghum genotypes with lowest maximum germination distance were Mukadziusaende, Chiredhi and Isifumbathe whilst wild Sorghum and SC Sila had the biggest maximum germination distance. All the genotypes were susceptible to Striga asiatica. This is based on the stipulation that genotypes with a germination distance of less than 1 cm are resistant while those with more than 1 cm are susceptible to Striga asiatica14. However, the level of susceptibility differs with the genotypes.
These results are consistent with previous observations in cereals like rice5. The results confirm the existence of large genetic variation among the Sorghum cultivars. According to Jamil et al.5 up to about 500-fold difference exist in the amounts of strigolactones exuded by rice Germplasm. Ejeta and Butler24 confirmed the same results in Sorghum and reported differences as much as a billion fold in the amounts of stimulants produced in Sorghum.
Tillering varied among Sorghum genotypes with Mukadziusaende having the highest tiller number whilst the lowest was SC Sila (Fig. 2). In Sorghum, tillering has been proposed to be under genetic and environmental control and according to Kim et al.28 tillering has not been comprehensively addressed by the carbohydrate supply and demand framework. In this study the Sorghum genotypes were subjected to the same environmental conditions hence the environmental influences were eliminated. Therefore the differences suggested that hybrids may also differ in their propensity to tiller which is independent of the carbon supply demand28. The differences could be due to differences in hormonal signaling as the plants were in the same environment. According to Umehara et al.25 the hormone strigolactones reduced tillering in plants so plants that produce less strigolactones have profuse tillering compared to those that produce more. The results of this study support the propensity to tiller hypothesis as the genotypes were grown in the same conditions.
Taken together the results of this study suggested that elevated levels of strigolactones contribute to the inhibition of tiller buds. Therefore tiller growth is promoted when strigolactones are decreasing in the plant. The same results were supported by Umehara et al.25 and Dun et al.29. The implications of this study are that when high tillering Sorghum cultivars are introduced Striga can be managed.
CONCLUSION
The genotype Mukadziusaende had the least mgd and more tiller numbers and also was attached to less Striga asiatica plants. Tiller numbers can be used to select for reduced strigolactones production in the field since they is an inverse relationship between tillers and strigolactones. Tillering potential of Sorghum becomes a marker of the plant’s susceptibility to Striga infection. The selection of high tillering cultivars could be helpful strategy to reduce Sorghum loss to Striga. Therefore, farmers in Zimbabwe can utilize tiller number to select for moderately resistant Sorghum lines.
SIGNIFICANCE STATEMENT
Sorghum landraces vary with respect to strigolactones production Strigolactones quantities are inversely linked to tillering. The quantity of strigolactones produced is directly related to the number of Striga attachments in Sorghum Farmers and researchers can use tiller numbers to select for low strigolactones producers.
ACKNOWLEDGMENTS
Funding from the Research and Post Graduate Centre of the Bindura University of Science Education and the Research Council of Zimbabwe (RCZ) are acknowledged. The Zimbabwe Gene bank for the provision of varieties and the Seed Company of Zimbabwe for the variety SC Sila. Striga seed was sourced from Henderson Research Station (The Weed Research team) under the Department of Research and Specialist Services, Harare Research Station, Zimbabwe.