Browsing by Subject "Striga"

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    Impacts of the fungal bio-control agent Fusarium oxysporum f.sp. strigae on plant beneficial microbial communities in the maize rhizosphere
    (2016) Musyoki, Mary Kamaa; Cadisch, Georg
    Striga hermonthica causes severe yield reduction in cereal crop production in Sub-Saharan Africa. Intergrated Striga management has been proposed as one of the best options to control striga. Along this line, the use of biocontrol agent (BCA) Fusarium oxysporum f.sp. strigae (Foxy-2) has been proven as an effective and environmental friendly management strategy. It is well established that a prerequisite for a successful BCA is sufficient risk assessment analysis. Towards this direction, Foxy-2 was assessed for its potential non-target impacts on the abundance, community structure of bacterial and archaeal nitrifying prokaryotes as well as enzymatic activities of proteolytic bacteria. Maize rhizosphere soils treated with or without Foxy-2, Striga and high quality organic residues (i.e., Tithonia diversifolia) as N source were evaluated by quantitative polymerase chain reaction (qPCR) and terminal restriction fragment length polymorphism (TRFLP). It was observed that Foxy-2 had a promoting effect on archaeal abundance under controlled conditions in sandy soils. Furthermore, crop growth stage, seasonality and soil type had a strong effect on abundance and community structure of nitrifying prokaryotes over Foxy-2 inoculation. In addition proteolytic enzymatic activities analysis showed that Foxy-2 did not affect their activities. Correlation analysis also showed that abundance and community structure on nitrifying communities positively correlated with extractable organic carbon, extractable organic nitrogen and soil pH, while proteolytic enzymatic activities correlated with extractable organic nitrogen and soil ammonium. It was concluded that Foxy-2 is compatible with nitrifying prokaryotes and proteolytic enzymatic activities.
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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.
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    The biocontrol agent Fusarium oxysporum f. sp. strigae - Monitoring its environmental fate and impact on indigenous fungal communities in the rhizosphere of maize
    (2016) Zimmermann, Judith; Cadisch, Georg
    The fungal biocontrol agent (BCA) Fusarium oxysporum f. sp. strigae (Fos) has proven to be effective in the suppression of the parasitic weed Striga hermonthica, which causes substantial yield losses in cereals in Sub-Saharan Africa. A prerequisite for widespread implementation of the biocontrol technology is the official registration of the BCA Fos by country authorities in Sub-Saharan Africa. The FAO and OECD institutions established international registration regulations to ensure the environmental safety of microbial BCAs. The present thesis aimed on assessing the potential of the BCA Fos to meet these registration requirements and was, therefore, based on the following two major objectives: (1) A specific DNA-based monitoring tool for Fos was developed which allows following its population kinetics in soils as driven by contrasting environmental impacts, such as soil type, plant growth stage and seasonality. (2) Risk assessment studies were conducted to assess potential side effects of Fos inoculation on non-target soil microorganisms.
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    Understanding the role of plant growth promoting bacteria on sorghum growth and biotic suppression of striga infestation
    (2014) Mounde, Lenard Gichana; Sauerborn, Joachim
    Witchweeds (Striga sp.) are parasitic weeds of great agricultural significance, parasitizing the roots of their hosts. Striga, like all other root parasitic weeds, drain essential organic and inorganic resources from their hosts leading to poor crop development and low yield. In Africa, about 50 million ha in over 30 countries are infested by Striga spp. causing grain loss of cereals. Estimated yield losses of maize, sorghum, millets and upland rice are between 30 and 90%. The parasite, therefore, is ranked as the leading biotic constraint to cereal production in the continent. Plant growth promoting rhizobacteria (PGPR) are promising components for integrated solutions to agro-environmental problems because inoculants possess the capacity to promote crop growth and reduce the population of deleterious microbes in the rhizosphere. Although there are numerous studies on crop growth promotion and biological control of diseases, weeds, nematodes and parasitic weeds using PGPR, little is known about the potential of some Bacillus subtilis, B. amyloliquefaciens and Burkholderia phytofirmans strains in sorghum growth promotion and resistance against Striga infection. The main objective of the study was to assess the effect of B. subtilis Bsn5, B. subtilis GBO3, B. amyloliquefaciens FZB42 and Burkholderia phytofirmans PsJN on growth promotion of sorghum crop and suppression of Striga development, thus providing a basic understanding on the sorghum-PGPR-Striga interaction. This study opens with an elaborate review of the state-of-the-art knowledge on the tripartite interactions between Striga, sorghum and different species of PGPR. Prior to this, bipartite relationship between sorghum and Striga, PGPR-sorghum and PGPR-Striga are reviewed with a focus on understanding Striga impact on sorghum, sorghum defence responses to infection, plant growth and disease suppression benefits by PGPR on sorghum, and the effect of PGPR on Striga development. Knowledge gaps in both bipartite and tripartite relationships are described, and future research recommendations given. A key recommendation from the review is to conduct experiments under controlled environmental conditions using Bacillus subtilis, B. amyloliquefaciens and Burkhoderia phytofirmans strains in order to understand their relationship with sorghum and Striga at bipartite and tripartite levels. Petri dish bioassays and root chamber experiments under controlled conditions were conducted at the Institute of Plant Production and Agroecology in the Tropics and Subtropics, University of Hohenheim between 2012 and 2014. B. subtilis Bsn5, B. subtilis GBO3, B. amyloliquefaciens FZB42 and Burkholderia phytofirmans strain PsJN inocula and their corresponding cell culture supernatants were evaluated for their growth promotion potential on sorghum and suppressiveness on Striga development. Sorghum root exudates and synthetic stimulant GR24 were used to induce Striga seed germination. Bacillus subtilis Bsn5 supernatant, which showed the greatest inhibitory activity on Striga germination and radicle elongation, was separated by ethyl acetate into lipophilic and hydrophilic phases. The purpose of this extraction was to try and identify the polarity of the inhibitor. Protein composition by mass spectrometry (MS) was also done on the supernatant with a view of establishing the presence of peptides because peptides have been associated with Orobancheceae germination and radicle inhibition in previous studies. In addition, determination of plant growth hormones in bacteria supernatants was also conducted using Radio-Immuno-Assay (RIA) in order to relate PGPR hormone production and sorghum growth enhancement. Burkholderia phytofirmans PsJN significantly (<0.05) induced a higher vigor index (VI) on sorghum seedlings (>18,000) compared to other PGPR and control treatments. The lowest VI (7626) was recorded in seeds inoculated with Bacillus amyloliquefaciens FZB42. Complete Striga germination inhibition (0% germination) occurred in seeds exposed to all PGPR inocula suspended while the highest germination (>60%) occurred in control treatments (10% Luria Bertani (LB) + GR24 and sterile distilled water (SDW) + GR24). The effect of bacterial supernatants on the germination percentage and radicle length of Striga seeds was also significantly (<0.05) different among treatments. The least germination (7.4 %) was observed in Bacillus subtilis Bsn5 + GR24 while the highest (66 %) was observed in SDW + GR24 control. Bacillus subtilis Bsn5 supernatant produced the lowest mean radicle lengths (0.1 mm) while the highest radicle lengths were observed in SDW + GR24 (2.2 mm). Therefore, Bacillus subtilis Bsn5 supernatant was selected for further investigation of compounds causing inhibition of Striga germination and preventing radicle elongation. The supernatant was separated into hydrophilic and hydrophobic fractions using ethyl acetate. Each fraction was then prepared in 1%, 25%, 50%, 75% and 100% concentrations before being evaluated for their inhibitory activity in Striga germination and radicle elongation. The highest germination percentage (63%) and radical length (2.9 mm) was observed in SDW + GR24 control treatment. The ethyl acetate (lipophilic) fraction at both 100% and 1% concentration + GR24 produced a germination percentage of >40% which was similar to 10% LB + GR24 and ethyl acetate + GR24 controls. There was complete inhibition of Striga seed germination after exposure to either Bacillus subtilis Bsn5 supernatant + GR24 or 100% hydrophilic fraction of the supernatant + GR24. However, at 25% and 1% concentration + GR24, Striga germination percentage increased to 34% and 49%, respectively. Light microscopy examination of Striga radicles exposed to Bacillus subtilis Bsn5 supernatant + GR24 revealed that stunting of the radicles was due to reduction in cell sizes at the radicle elongation zone. Extended agar gel assays (EAGA) experiments showed a similar trend of results with B. subtilis Bsn5 showing the highest inhibitory activity on Striga germination and radicle elongation compared to other PGPR and control treatments. Results from root chamber experiments demonstrated significant (p<0.05) differences in biomass production between Striga-free and Striga-infected sorghum. Total biomass yield in uninoculated Striga-free plants was 40% higher than uninoculated Striga-infected sorghum plants. Bacillus amyloliquefaciens FZB42, B. subtilis GBO3 and Burkholderia phytofirmans PsJN inoculated Striga-free sorghum showed a 75%; 142% and 158% increase in biomass yield, respectively, compared to uninoculated Striga-free sorghum. There were no significant differences in biomass yield observed between inoculated and uninoculated Striga-infected plants. All PGPR supernatants and 10% LB media showed production of phytohormones cytokinin, IAA, GAs and ABA. Cytokinin content in PGPR supernatants was significantly (>0.05) higher than blank 10% LB control media. There was a significant negative correlation (r= -0.96) between IAA and cytokinins. However, there was no significant positive correlation between any phytohormone and sorghum plant height, SPAD values, biomass production, Striga germination, attachment and tubercle death. Finally, this study shows that Bacillus subtilis Bsn5, B. subtilis GBO3, B. amyloliquefaciens FZB42 and Burkholderia phytofirmans PsJN might accelerate sorghum growth and suppress key stages of Striga development under laboratory conditions. Greenhouse and field experiments are recommended to better understand these interactions under natural conditions where other biotic and abiotic factors come into play. These findings could contribute to a better understanding of sorghum and beneficial bacteria interactions and provide novel information of the long-term effects of a PGPR on sorghum development, opening new avenues for Striga control and sustainable, ecofriendly sorghum production.