A dissertation submitted in partial fulfilment of the requirements of the degree of Master of Science in Environmental science School of Animal, Plant and Environmental Sciences University of Witwatersrand, Johannesburg. March 2017. / Plant growth promoting rhizobacteria (PGPR) promotes plant growth in a variety of modes of action and also suppresses several phytopathogens causing plant diseases. There is evidence that Pseudomonas strains are able to induce systemic resistance, thereby enhancing the defensive capacity of many plant species, and they do so without any negative impact on the environment. Currently, many agricultural systems rely more on the use of chemical pesticides to combat plants diseases. The chemicals have several negative impacts on both human health and the environment. Therefore, there is need to investigate the ability to fight plant pathogens of alternatives like the Pseudomonas spp that do not harm the environment. Several strains of this genus are yet to be tested to see if they induce systemic resistance. Previous studies showed that bio surfactants produced by Pseudomonas koreensis exhibited strong effect against oomycetes P. ultimum in tomato plants. Induced systemic resistance (ISR) potential of P. koreensis following exposure to viruses has not been fully demonstrated to date. This study sought to investigate whether this strain has an effect on viruses and if it is able to induce systemic resistance against viral pathogens. The study started by growing the model plant N. benthamiana. The second stage involved carrying out assays of tobacco mosaic virus (TMV) after inoculating this virus in three bio treatments: (i) seed treatment of N. benthamiana with P. koreensis (referred to as the early treatment), (ii) root treatment at the transplanting stage (late treatment) and (iii) the control. In bio treatments (i) seeds were first sterilized by dipping them into 70% alcohol for 3 minutes and 0.1 % HgCl2 for 1 minute and washing them with distilled water. Each seed was then soaked into 20ml of bacteria suspension for 30 minutes and in (ii) a litre of P. koreensis culture was then poured onto the roots of 36 N. benthamiana plants. The bacteria suspension was added at 107 colony forming units per gram of soil to each tray. It was observed that disease severity was lower in the P. koreensis plant treatments than for the control. Results of this investigation have shown that P. koreensis can induce systemic resistance in foliar parts when plant seeds or roots are inoculated with this strain. This was demonstrated by separation of plant growth promoting rhizobacteria (PGPR) bacteria and TMV. Seeds and roots were inoculated with bacteria while the leaves were inoculated with TMV. The early bio treatment had the lowest mean number of necrotic lesions, and exhibited the mildest effects from TMV compared to the late bio treatment and control. Plants in the late bio treatment were moderately affected while the control was severely affected (P˂0.0001) ˂0.05. The early and the late bio treatment both had higher leaf surface area than the control; (P˂0.0001) ˂0.05. The early bio treatment lost the fewest leaves, and the late bio treatment lost a moderate number while the control lost the highest number (P˂0.0001)˂0.05.The reduced symptoms exhibited by plants inoculated with P. koreensis is an indication that P. koreensis has anti-viral activity against TMV. It was concluded that P. koreensis can reduce plant‟s viral susceptibility and result in ISR. It is hence proposed that P. koreensis can be used as a biological control (bio control) agent against viruses. Key words: Tobacco Mosaic Virus (TMV), Pseudomonas koreensis (P. koreensis), induced systemic resistance (ISR) / LG2018

Thesis (M.S.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 89-99).

Thesis (Ph.D.)--University of Idaho, May 2006. / Major professor: Donald L. Crawford. Abstract. Includes bibliographical references. Also available online in PDF format.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 89-100). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 89-100). Also available on the Internet.

>Magister Scientiae - MSc / Approximately 50% of sorghum (Sorghum bicolour (L.) Moench) produced globally is used as human food, with 95% of its total consumption occurring in Africa. Unfortunately, sorghum crops are prone to pathogenic attack, notably leading to a reduction in production yields. Generally, chemical agents are used as fertilizers and/or biocides to increase crop production. However, these chemicals can have a detrimental environmental impact including the eutrophication of fresh water and marine ecosystems. Thus, there is increased interest in plant growth promoting rhizobacteria (PGPR), as an alternative to chemicals, to facilitate eco-friendly biological control of soil-borne pathogens. PGPRs colonize the plant root system (i.e rhizosphere and rhizoplane) and promote growth and production yields essentially via the biological control of plant pathogens and their role in the nutrient cycles (e.g N fixation). The aim of this study is to characterize the microbial communities associated with sorghum in South Africa, and to identify common bacteria which could further be developed and applied to improve sorghum growth and yield. Sorghum rhizospheric environments (rhizoplane and rhizosphere) were collected from three sites characterized by different agricultural practices (Free State, Limpopo and North West). Denaturing gradient gel electrophoresis (DGGE) and Terminal-restriction fragment length polymorphism (T-RFLP) were used to identify microbial community molecular fingerprints. Sorghum-associated microbial communities were found to be different in all rhizospheric soil samples which could be explained by differences in soil chemistry, agricultural practices and geographical location. The analyses also clearly demonstrated that the sorghum bacterial community structures were similar in the rhizoplane, indicating the strong influence that the sorghum plant has in determining the rhizoplane colonizers. The archaeal community structure from rhizoplane and rhizosphere in each sampling site were dissimilar, which could be explained by differences in soil type and/or agricultural practices. Both the T-RFLP and DGGE analyses revealed that Bacillus sp. were consistently associated with South African Sorghum, Arthrobacter sp. were detected in the rhizoplane, while Uncultured archaea were detected in the rhizoplane of sorghum. These microorganisms represent valuable targets for engineering to promote growth and yield in sorghum.

Wheat crops are known to be devastated by infections of soil-borne pathogens, especially the fungus <i>Gaeumannomyces graminis </i>var. <i>tritici</i> (Ggt) that causes ‘take-all’. Plant growth promoting rhizobacteria (PGPR) such as <i>Pseudomonas fluorescens</i> have received much attention as biocontrol agents against Ggt, mainly due to their ability to produce antibiotics. The polycetide secondary antimicrobial metabolite 2,4-diacetylphloroglucinol (DAPG) is produced by a number of fluorescent pseudomonad strains and is known to suppress Ggt. Another soil microbial group which have been under investigation for their biocontrol potential against Ggt, are arbuscular mycorrhizal (AM) fungi which have the potential to out-compete Ggt and improve host plant nutrition and vigour. In this thesis, I report results from experiments that investigate interactions among AM fungi, Ggt, and DAPG-producing bacteria. A central hypothesis is that carbon flow from plants and AM fungi stimulates DAPG production. I therefore focus on interactions among AM fungi, Ggt and bacteria <i>in vivo</i> with wheat plants and <i>in vitro</i> with only fungal exudates. The synergistic co-operation of pseudomonads and AM fungi against Ggt was demonstrated and the fungal exudates (from AM and Ggt) produced both <i>in vitro</i> and <i>in vivo</i> increased DAPG production by <i>P. fluorescens</i>. The ecology and functioning of beneficial AM fungi was found not to be influenced by the presence of either Ggt or DAPG, highlighting the potential sustainable suppression of “take all” in wheat rhizosphere.

Salinity is one of the most severe environmental factors that limits global crop yield. Enhanced phytoremediation using plant growth promoting rhizobacteria (PGPR) has proven to be an effective and environmentally responsible approach to remove salt from the surface soil and reclaim salt-impacted soil for crop production. PGPR enhanced phytoremediation systems (PEPS) were applied to two research sites, Cannington Manor North (CMN) and Cannington Manor South (CMS) in southern Saskatchewan. The sites were impacted by brine leakage during upstream oil and gas production. A salt mass balance study was performed based on data collected from these two sites. Both sites were planted in June. Soil samples were taken in June 2009 (beginning of the season), August (midseason) and October (end of the season). Soil salinity changes throughout the season were monitored by measuring soil electrical conductivity (EC). The average surface soil ECe decreased from 3.7 dS/m to 3.1 dS/m at CMN, and from 10.2 dS/m to 9.2 dS/m at CMS in 2009 season. Plant samples that were collected in August and October were analyzed for sodium and chloride concentrations. These values were then converted into predicted ECe changes for the soil to compare with the actual changes in soil ECe. Plant uptake of NaCl was calculated to account for 25.2% and 28.1% of the decrease in surface soil ECe at CMN and CMS, respectively. However, plant samples were washed prior to salt content analysis. A considerable amount of salt could have been lost during the washing process. Several plant samples from other salt-impacted sites in Saskatchewan and Alberta were selected to examine salt loss due to tissue washing. The salt ions lost by washing were determined to be 44.4% for Na+ and 63.8% for Cl-. After the adjustment of plant NaCl uptake data by the loss due to washing, plant accumulation of NaCl accounted for 59.9% of the decrease in surface soil ECe at CMN and 56.1% at CMS. When plant uptake of K+ and Ca2+ were also taken into consideration by a simulation study, the decrease in surface soil ECe that was caused by plant uptake of salt ions accounted for 107.5% at CMN and 117.5% at CMS. This indicated that plants can have a significant role in the remediation of salt-impacted soil. The effects of PGPR (Pseudomonas spp. UW4 and Pseudomonas corrugata CMH3) treatment on selected physiological indicators, such as proline, superoxide dismutase (SOD), membrane leakage and photosynthesis, were examined on annual ryegrass (Lolium multiflorum). Plants were grown under three saline conditions: non-saline topsoil, non-saline topsoil spiked with NaCl to 10 dS/m, and high saline soil collected from a salt-impacted site diluted with non-saline topsoil to reach 10 dS/m. The shoot fresh weight of plants grown in spiked salt soil decreased by 74% and in diluted salt soil by 44%, respectively, compared to control soil. Both types of salt soil increased SOD activities by approximately 50%, proline concentrations by 20 to 25 fold, and membrane leakage levels by 1.6 to 2.8 fold. Significant impairment of photosynthetic performances, as indicated by the decreases in the chlorophyll fluorescence parameters Fv/Fm, yield and qP, and a parallel increase in qN, was also observed using Pulse Amplitude Modulation (PAM) fluorometry for plants in diluted impacted soil. PGPR moderately increased fresh weight and SOD activity. Both UW4 and CMH3 significantly increased proline concentration and lowered membrane leakage relative to untreated plants. Therefore, PGPR improve plant performance under salt stress by elevating proline levels, which can act as a quencher of destructive reactive oxygen species. PGPR treatment also restored all the chlorophyll fluorescence parameters nearly to the non-stressed level, indicating protection of photosynthetic tissues of PGPR treated plants under salt stress. Overall, PEPS was successfully applied to the salt-impacted sites. Plant uptake of salt played a major role in the decrease of surface soil ECe. PGPR’s role in enhancing plant performance under salt stress was suggested by the elevated proline concentrations, the decreased membrane leakage levels and the restored photosynthetic activity.

Bacillus thuringiensis NEB17 is a plant growth promoting rhizobacterium that produces a compound that directly increases plant growth. The compound is a bacteriocin and we propose the name thuricin 17. Thuricin 17 is a novel peptide inhibiting the growth of Bacillus species/strains, displaying both bactericidal and static effects. Its molecular weight, estimated via SDS-PAGE and verified by MALDI-QTOF mass spectroscopy, is 3162 Da. The partial amino acid sequence was determined and is N-term---WTCWSCLVCAACSVELL, C-term-CAS. Heat and pH stability, production and susceptibility to proteolysis were conducted. Thuricin 17 is active in pH 1.00-9.25, stable above 60°C and produced in the late exponential growth phase. This is the first bacteriocin from a Bacillus PGPR and the first reported to increase plant growth. This work presents an original discovery regarding PGPR mechanisms.

Pseudomonas chlororaphis PA23 inhibits several root pathogens in both the greenhouse and field. A LysR-type transcriptional regulator (LTTR) called PtrA (Pseudomonas transcriptional regulator A) that is essential for Sclerotinia sclerotiorum antifungal activity was discovered through transposon mutagenesis. P. chlororaphis PA23 produces the antibiotics phenazine 1-carboxylic acid, 2-hydroxyphenazine and pyrrolnitrin, and several additional products that contribute to biocontrol. Phenotypic assays and proteomic analysis have revealed that production of these secondary metabolites are markedly reduced in a ptrA mutant. Most LTTRs regulate genes that are upstream of and divergently transcribed from the LTTR locus. A short chain dehydrogenase (scd) gene lies immediately upstream of ptrA in the opposite orientation. Characterization of an scd mutant, however, has revealed no significant changes in antifungal activity compared to wild-type PA23. Gene expression analysis of the ptrA mutant indicates that ptrA may exert its regulatory effects through the Gac-Rsm network, and may be controlling expression of the scd gene. Collectively these findings indicate that PtrA is an essential regulator of PA23 biocontrol and is connected to other regulators involved in fungal antagonism.