Browsing by Subject "Biological control"

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Molecular perspectives on the ecologically inconsistent effectiveness of the mycoherbicide Fusarium oxysporum f. sp. strigae against Striga hermonthica
(2022) Anteyi, Williams Oyifioda; Rasche, Frank
Cereals are a major staple that is crucial for food security in sub-Saharan Africa (SSA). Sadly, the obligate hemiparasitic witchweed, Striga spp., especially Striga hermonthica (Delile) Benth., is a major biotic constraint to cereal production in SSA, causing enormous crop yield losses estimated at US$10 billion annually. Fusarium oxysporum f. sp. strigae (Fos) is the most renowned fungal biological control agent (BCA) for specifically and significantly tackling S. hermonthica under agricultural systems. Field surveys, however, have revealed the inconsistent effectiveness of Fos isolates against S. hermonthica in differing zones of SSA (i.e., West Africa, East Africa). This daunting phenomenon is a critical challenge that affects Fos reliability and deters its use for S. hermonthica management. The inconsistent effectiveness of Fos against S. hermonthica was presumably ascribed to the interactions that occur between the differing location-specific ecological factors of the pathosystem i.e., abiotic (climate, moisture, or soil physico-chemistry) or biotic (S. hermonthica, Fos isolate, or the plant microbiome). Without doubt, the diversity of a host or pathogen is a primary determinant of the innate susceptibility or virulence of the host or pathogen, respectively. In terms of S. hermonthica diversity, genomic variation of individuals, or regional genetic variation of the sampling zone, were the two major forces suspected. However, the important determiner out of the two forces was unknown. Besides, despite the suppression/death that Fos causes to S. hermonthica, the physiological damage S. hermonthica initiates to an infested cereal crop is mostly irreversible. Hence, in examining strategies for circumventing the main problem of Fos inconsistent effectiveness against S. hermonthica, and the physiological consequences of S. hermonthica on the host cereal crop, the integration of other (non-Fos inoculum) BCA were suggested as possible means for improving the efficiency of S. hermonthica biocontrol. For example, by utilizing a bioherbicide cocktail of Fos and plant growth promoting rhizobacteria (PGPR), or Striga seed germination-inhibiting fungal toxins. Apart from the popular reputation of PGPR in enhancing crop health and growth, certain PGPR strains (especially Bacillus subtilis isolate GB03) have been earlier reported for their highly-promising potential of antagonizing S. hermonthica development. Similarly, certain fungal extracellular metabolites (exometabolites), especially of Fusarium origin, were reported to completely inhibit S. hermonthica seed germination in vitro at very low concentrations (≤ 1 mM). Unfortunately, knowledge of the microbe (Fos)–microbe (PGPR) interaction, their localization and ecological niche, for enabling their expected synergistic impact of simultaneously suppressing S. hermonthica and enhancing the Striga-infected cereal crop biomass, was unknown. Also, it was unknown if highly potent/efficient Striga seed germination-inhibiting fungal exometabolites will consistently suppress S. hermonthica in planta. Thus, in the context of genetic diversity in S. hermonthica, the PhD study focused on gaining (molecular) insights into the inconsistent effectiveness of Fos against S. hermonthica; including the examination of some strategies for improving S. hermonthica biocontrol efficiency, precisely by integrating PGPR, or Striga seed germination-inhibiting Fusarium exometabolites, into a S. hermonthica biocontrol system. The first research examined the molecular genetic basis, underlying the variable susceptibility of S. hermonthica populations sampled from differing zones of SSA (West Africa, East Africa) to contrasting Fos isolates (Foxy-2, FK3). Regardless of sampling zone, the S. hermonthica populations displayed divergent susceptibility patterns to the Fos isolates i.e., a S. hermonthica class was susceptible to both Foxy-2 and FK3, while the other class was susceptible to either Foxy-2 or FK3. This manifestation correlated with nucleotide mutations at certain loci. Thus, genomic variation in S. hermonthica is a superior determinant of the inconsistent effectiveness of Fos isolates, rather than the S. hermonthica sampling zone. The second research examined the impact of coinoculating Fos and a PGPR (B. subtilis isolate GB03) into a S. hermonthica-sorghum parasitic system. Notwithstanding the colocalization of Fos and GB03 in common ecological niches of diseased S. hermonthica shoot (mainly in flavonoid-rich regions), GB03 thwarted Fos suppressive activity against S. hermonthica. Interestingly, a novel, alternative Fos entry route into S. hermonthica (through the trichome) was discovered. The coinoculation of Fos and GB03 presented no added advantage for S. hermonthica control. Finally, the third research screened a set of highly phytotoxic Fusarium exometabolites against S. hermonthica seed germination (in vitro) and incidence (in planta). This was to identify the most potent/efficient Fusarium exometabolite for S. hermonthica biocontrol. Among the tested exometabolites, diacetoxyscirpenol (DAS) was the most potent/efficient to completely suppress S. hermonthica both in vitro and in planta. Fos, however, did not produce DAS, due to underexpression of key genes necessary for Fusarium trichothecene biosynthesis. In conclusion, owing to the obligate outcrossing mating system in S. hermonthica, genomic variation is an inevitable phenomenon. This, therefore, plays a crucial role in the variable susceptibility of S. hermonthica to Fos. The newly discovered Fos (direct) entry route into S. hermonthica (trichome entry), elucidates a novel paradigm to the infection mechanism occurring under the S. hermonthica (host)–Fos (pathogen) interaction, in addition to the previously reported indirect, rhizosphere-transmission. Thus, this novel phyllosphere-transmission, paves the way for further research that exploit this alternative Fos infection route for better S. hermonthica biocontrol. Lastly, considering the potency and broadscale efficacy against diverse S. hermonthica populations, the exometabolite DAS could serve as a new agent for a more efficient S. hermonthica biocontrol. Though, further examination of its specific mode of action against the target weed (S. hermonthica), as opposed to non-target organisms, is required.
Molecular systematics of selected Diadegma species (Hymenoptera: Ichneumonidae: Campoplegine) important in biological control
(2006) Wagener, Barbara; Zebitz, Claus P. W.
The genus Diadegma (Hymenoptera: Ichneumonidae: Campopleginae) represents a large group of parasitoids with 201 species worldwide. Adult Diadegma females parasitise larvae of various lepidopteran species and some species, in particular Diadegma insulare (Cresson) and D. semiclausum (Hellén), have gained economic importance as biological control agents of Plutella xylostella (Linnaeus). A low parasitism rate of <15 % of the parasitoid complex (Diadegma sp., Oomyces sokolowskii (Kurdjumov) and Diaplazon laetatorius (Fabricius)) in unsprayed cabbage and kale fields infested with P. xylostella in eastern and southern Africa was the starting point for the development of a biological control project for P. xylostella which was implemented by the International Centre of Insect Physiology and Ecology (ICIPE), Kenya. One of the objectives of the biocontrol project was to examine the taxonomic status of Diadegma species associated with P. xylostella in eastern and southern Africa and the exotic parasitoid D. semiclausum imported to Kenya from Taiwan (Asian Vegetable Research and Development Centre, AVRDC) by cross breeding experiments and molecular methods. Thus, two different molecular regions, a fragment of the mitochondrial cytochrome c oxidase subunit (COI) and the second internal transcribed spacer (ITS2) of ribosomal DNA were amplified utilising polymerase chain reaction (PCR) and digested afterwards with several restriction enzymes (PCR-Restriction Fragment Length Polymorphism-RFLP). In the due course of the study examinations of several Diadegma species attacking P. xylostella were undertaken with the PCR-RFLP method developed previously for the African Diadegma. This molecular method could solve some taxonomic difficulties of the genus Diadegma. Sequence analyses were used to investigate the phylogenetic relationship of nine Diadegma species (D. blackburni (Cameron), D. insulare, D. leontiniae (Brèthes), D. chrysostictos (Gmelin), D. armillata (Gravenhorst), D. fenestrale (Holmgren), D. mollipla (Holmgren), D. semiclausum, D. rapi (Cameron)) and the phylogenetic relationship of the genus Diadegma within the superfamily Ichneumonoidea. Cross breeding experiments were carried out between two populations of D. mollipla from eastern and southern Africa. No significant differences in the total number of progeny per female and the number of male offspring were obtained, whereas the female progeny showed significant differences. Hybrid females resulting from both reciprocal crosses were reproductively compatible with males of both parental lines, which indicated that no genetic incompatibility was apparent between the two D. mollipla populations. In contrast, crosses between D. mollipla and D. semiclausum resulted only in the occurrence of male offspring, which is typical for unfertilised progeny in Diadegma. The laboratory cultures of D. mollipla and D. semiclausum were highly male biased. Inbreeding, where homozygosity is much higher, is leading to a higher diploid male production. Diploid males can easily be detected by isoenzyme variations as a genetic marker. Heterozygote females/males of D. semiclausum and D. mollipla were identified by phosphoglucomutase (PGM) electrophoretic banding patterns. Crosses between a mother (heterozygote, diploid) and her son (homozygote, haploid) resulted in one diploid male in D. mollipla and none in D. semiclausum. Information about diploid males in D. semiclausum detected with PGM has already been published and different methodologies might be the reason why in D. semiclausum no diploid male was detected. Therefore the present analyses with PGM as molecular marker should be seen as a preliminary study.
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.
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.