Browsing by Person "Ndambi Beninweck, Endah"

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Investigating the mode of action of the mycoherbicide component Fusarium oxysporum f.sp. strigae on Striga parasitizing sorghum and its implication for Striga control in Africa
(2011) Ndambi Beninweck, Endah; Cadisch, Georg
Amongst the factors that are a threat to food security in Africa, is the parasitic weed Striga hermonthica which affects mostly cereals that constitute the staple food for subsistence farmers, thus affecting the livelihood of millions of people. Popularly known as witchweed, attack due to S. hermonthica can completely destroy the yield of cereal crops. Efforts to combat Striga have had very limited success since farmers rarely adopt control methods due to the mismatch between technologies and farmers? socio-economic conditions. Being such a severe problem, an appropriated method for Striga management adapted for African farmers is very much needed. The use of soil-borne fungi for biocontrol is now being developed as an alternative to the use of chemicals considering the specificity of such fungi and the fact that most of the damage by Striga is done before its emergence. The fungus Fusarium oxysporum f.sp. strigae has been identified and shown to be effective and specific to S. hermonthica and S. asiatica but its mode of action is not yet well known. It is required that the mechanisms underlying the mycoparasitic process of this natural antagonistic agent be well understood before its use. Thus, studies on the effectiveness, specificity and timely colonization of Foxy 2 on S. hermonthica are necessary as well as studies on the effect of Foxy 2 in Striga-host plants which should demonstrate its non-pathogenicity to food crops. The objective of this study was therefore to investigate the mode of action of Foxy 2 in its target S. hermonthica and non-target Sorghum bicolor and also to examine the safety of the use of this mycoherbicide by evaluating its ability to produce toxins. In the first part of the thesis, the ability of Foxy 2 to colonize sorghum roots and possibly shoots was investigated using light and transmission electron microscopy. The efficacy of Foxy 2 to cause death of S. hermonthica seedlings attached to Foxy 2 colonized sorghum roots was also evaluated. Microscopic investigations revealed that the intensity of root colonization by Foxy 2 increased with time and Foxy 2 could survive and colonize the sorghum rhizodermis, root hairs and cortical parenchyma up to four weeks after sowing. This behaviour is well adapted for Striga control as it corresponds to the peak of Striga seedling attachment. Hyphae were completely absent from the sorghum root central cylinder even after four weeks and also absent from the sorghum shoots up to 11 weeks after sowing indicating the non-pathogenity of Foxy 2 to sorghum. Furthermore, Foxy 2 was effective in controlling S. hermonthica by causing disease in 95% and 86% of S. hermonthica seedlings when coated on seeds of tolerant and susceptible sorghum cultivars respectively. Therefore, Foxy 2 could be combined with the tolerant sorghum variety in an integrated approach against S. hermonthica and S. asiatica. The effect of Foxy 2 on various growth stages of S. hermonthica was investigated subsequently so as to understand the mechanisms of action of Foxy 2 within S. hermonthica in the real living complex between the mycoherbicide Foxy 2, the parasite S. hermonthica and its host sorghum. Light, scanning and transmission electron microscopy were used to evaluate the pattern of colonization and control of S. hermonthica seedlings and shoots by Foxy 2. Results showed that 26 days after sowing Foxy 2 coated sorghum seeds, all tissues of the young S. hermonthica seedlings attached to sorghum roots were completely degraded and destroyed by Foxy 2 including the haustorial intrusive cells, hyaline tissue, vessels, central xylem elements and Striga cortical parenchyma. Some S. hermonthica plants which attached to areas of the sorghum root which were not yet colonized by Foxy 2 (towards the root tips), were able to outgrow the fungus and emerged. In the emerged S. hermonthica shoots, hyphae had subsequently penetrated and colonized vessels clogging them over long distances and were identified up to the top of the plants. In some vessels there was an intensive blockage of the vessels by hyphae such that spaces or gaps were rare. Ultrathin sections showed that the diseased S. hermonthica shoots reacted to Foxy 2 invasion by forming an electron dense wall coating along the secondary vessel walls probably to prevent fungal digestion of the walls. The study thus identified two mechanisms by which Foxy 2 contributed to wilting and death of S. hermonthica which included complete digestion of underground S. hermonthica seedlings and hyphal clogging of vessels in emerged S. hermonthica plants which interfered with water conduction. In order to understand the reactions of sorghum towards the presence of Foxy 2 as part of the risk assessment to ensure the safe use of this biocontrol agent, the action of Foxy 2 and a known pathogenic Fusarium species, F. proliferation, were compared in the fourth chapter. Sorghum roots were also wounded to expose the vascular system so as to investigate whether removal of the endodermal barrier could give access to Foxy 2 into the vessels which could lead to digestion resulting in wilting of the sorghum plants. The colonization processes of the two Fusaria species were quite different at all stages of growth. While F. proliferatum degraded the endodermis, invaded the central cylinder and digested the xylem parenchyma two weeks after sowing, Foxy 2 was restricted to the cortex even up to four weeks after sowing. Hyphae of Foxy 2 filled the intercellular spaces at the outer endodermal wall but could not penetrate the endodermis. Sorghum roots were observed to react to Foxy 2 invasion by reinforcing the central cylinder as seen by an increase in blue auto fluorescence especially of the endodermis. Five days after wounding and inoculating sorghum roots, Foxy 2 hyphae invaded the central cylinder very close to the cut but were completely absent from the central cylinder at a distance of 3000 µm from the cut, meanwhile F. proliferatum hyphae had digested the cells of the central cylinder at this distance. This indicated that not only the endodermis was a barrier but there could also be a physiological barrier within the central cylinder of the sorghum root which did not allow further spread of Foxy 2. Hence, exposure of the vascular system did not serve as a route for the invasion of Foxy 2 which therefore implied that it could not cause wilting of the plant. In the last part of the thesis, S. hermonthica shoots were analyzed by HPLC-MS/MS to investigate the possible production of toxins by Foxy 2 to kill the plant. Amongst the toxins tested (beauvericin, fumonisins B1, B2, B3, C and P series, enniatins A, A1, B and B1, and moniliformin), only beauvericin (BEA) was detected to be produced by Foxy 2 in S. hermonthica shoots. The concentration of this toxin increased with increased infection e.g. 60 µg BEA/kg Striga shoot tissue (dry weight) were detected three weeks after emergence rising to 720 µg BEA/kg Striga shoot tissue after six weeks in the severely diseased S. hermonthica shoots. When beauvericin was applied on S. hermonthica shoots at concentrations of 50 µM, transmission electron microscopy showed that all cell types became necrotic. However, beauvericin as well as all the other toxins were not detected in sorghum grains harvested from sorghum plants which were hosts to the S. hermonthica plants and growing from Foxy 2 coated sorghum seeds. Given that some F. oxysporum strains were previously shown to be able to produce fumonisins which are among the toxins which have been reported to be of potential risks to human and animal health, a pure culture of Foxy 2 was evaluated for its fumonisin production ability. Results from real-time PCR using two specific primer pairs for the FUM1 gene (which is the key gene for fumonisin synthesis), were negative confirming that Foxy 2 was not able to produce fumonisins and might not be of major concern for human and animal health when used as a biocontrol agent in the field, therefore safe for use as a biocontrol agent. To conclude, Foxy 2 showed potential to control S. hermonthica by completely destroying young underground stages and clogging vessels in aboveground stages, as well as producing the toxin beauvericin, both actions contributing to wilting of the plants. Its non-pathogenicity to sorghum and its inability to produce fumonisins could be seen as factors which make it well suited as a biocontrol agent. Further research needs to be done to evaluate its efficacy under field conditions and the impact of naturally occurring soil microorganisms and abiotic conditions on performance of Foxy 2 so as to understand its interactions with the environment and to optimize its efficacy.