EXPLORING BIOLOGICAL CONTROL AND TRANSGENIC WEED MANAGEMENT APPROACHES AGAINST INFESTATION BY STRIGA HERMONTHICA IN MAIZE

Abstract

Striga hermonthica and low soil phosphorus (P) are major contributors to food insecurity in sub Saharan Africa (SSA). Infestation of cereal fields by S. hermonthica results in about 30% to 90% yield loss hence emerging as a major constraint in cereal production in SSA. Such great loss is attributed to two aspects of S. hermonthica lifecycle namely elevated seed fecundity and its remarkable synchronized lifecycle to that of its host. It is also estimated that crop yield is limited by P availability in 30% to 40% arable land. This is because although the total content of phosphorus in the lithosphere is high, its partial bioavailability is a major limiting factor in crop production. Furthermore, there exists a tripartite relationship among maize, S. hermonthica and low P levels, whereby low soil P levels prompt maize roots to produce exudates rich in volatile S. hermonthica seed germination stimulants known as strigolactones. The strigolactones are produced as a signal to initiate arbuscular mycorrhiza colonization. Unfortunately these chemical cues are hijacked and used by Striga seeds as germination stimulant and hence signal the presence of a susceptible host. To date, there is no integrated, sustainable and effective S. hermonthica control strategy that has been widely adopted by small-scale farmers in Africa. This can however be provided by simultaneously addressing poor P access by maize as well as biological control of germinated S. hermonthica or piled seed banks in the soil. This therefore calls for the integration of biotechnologies that will shatter the tripartite relationship among low soil P level, stimulation of Arbuscular Mycorrhizal Fungi (AMF) colonization and S. hermonthica seed germination. The aim of this study was to simultaneously explore biocontrol options by bio-prospecting the effectiveness of culturable microbes against S. hermonthica as well as enhance P availability to maize by genetic transformation of ecologically adapted maize genotypes with P efficient Purple Acid Phosphatase (PAP) genes from Lupinus albus (LaPAP) and Medicago truncatula (MtPAP). To explore the biocontrol frontier, bacterial and fungal isolates from Striga suppressive soils were assayed for their ability to produce extra cellular enzymes and antibiotic compounds as well as their ability to induce S. hermonthica seed decay and later genotyped using 16S rRNA and 18S rRNA genes, respectively. In order to develop transgenic maize plants expressing target PAP genes, a regeneration protocol with an assortment of callus induction and callus maturation/shoot induction media were evaluated. Further, the transformability of target maize varieties was assessed via histochemical analysis of β-glucuronidase (GUS) reporter gene. Finally, Agrobacterium tumefaciens-mediated transformation of the maize varieties over-expressing PAP gene cassette was achieved and transgenic lines evaluated using S. hermonthica-host plant infection assays in vitro and in potted experiments. The morphometric analysis of bacterial and fungal descriptors identified bacterial isolates that displayed array of enzymatic and antibiosis properties and also that had ability to cause Striga seed decay. For instance isolate SM5ISS (KY041696) with 99% genetic affiliation to Bacillus recorded high antibiosis (8cm) and extra cellular enzymatic values (2.5±0.03) and also recorded the highest number of S. hermonthica xv seed decay (45±0.23%). This bioprospection study summarily identified candidate isolates that caused S. hermonthica seed decay. The regeneration study revealed that Namba nane, KSTP'94 and CML144 varieties recorded a regeneration frequency of 26.1±1.11%, 32.1±1.28% and 35.4±1.24%, respectively, while their corresponding GUS transformability efficiency values were 0.8±0.03%, 1.4±0.19% and 2.1±0.20%, respectively. Transformation of Namba nane with LaPAP and MtPAP gene construct recorded a transformation efficiency of 0.33±0.03% and 0.36±0.04%, respectively, while the corresponding values for LaPAP and MtPAP gene constructs in KSTP'94 were 0.69±0.05% and 0.37±0.03%, respectively. Transformation of CML144 with LaPAP and MtPAP gene construct recorded a transformation efficiency of 0.65±0.03% and 0.34±0.03%, respectively. These results demonstrated that the target maize germplasm was transformable. Over-expression of LaPAP and MtPAP in the selected maize genotypes resulted in low number of S. hermonthica colonizing transgenic maize in comparison to wild type maize. For instance, in Namba nane the average number of Striga plants colonizing individual wild maize plant in both rhizotron and bucket experiments were 9 and 4 while the corresponding numbers for LaPAP and MtPAP transgenic were 4, 1 and 5, 2, respectively. For KSTP'94 the average number of Striga plants colonizing individual wild maize plants in both rhizotron and bucket experiments were 4 and 3 while the corresponding numbers for LaPAP and MtPAP transgenic was 3, 1 and 3, 1, respectively. In the case of CML144 the average number of Striga plants colonizing individual wild maize plant in both rhizotron and bucket experiments were 12 and 7 while the corresponding numbers for LaPAP and MtPAP transgenic plants was 6, 2 and 8, 3, respectively. Analysis of the ability of root exudate to induce S. hermonthica seed germination was higher in wild type than transgenic maize. For instance, the average number of Striga seeds stimulated to germinate in Namba nane under treatments; wilt-type, LaPAP and MtPAP was 7, 4 and 6, respectively. In KSTP'94, the average number of Striga seeds stimulated to germinate in Namba nane under treatments; wilt-type, LaPAP and MtPAP was 5, 2 and 3 respectively. Lastly, in CML144 the average number of Striga seeds stimulated to germinate in Namba nane under treatments; wilt-type, LaPAP and MtPAP was 5, 2 and 3, respectively. Summarily, this study identified microbes that were potent against S. hermonthica and proposes their use in reduction of S. hermonthica seed bank in infested soils. Further, it was demonstrated that indeed overexpression of PAP genes in maize results in less S. hermonthica infestation. The use of the two approaches is therefore recommended in an integrated S. hermonthica management package that would be able to impede the parasite in infested and low P soils especially in western Kenya.