Plant growth-promoting properties of fynbos rhizobia and their diversity
Thesis (MSc)--Stellenbosch University, 2018.
ENGLISH ABSTRACT: Soil has a high diversity of microorganisms which differs from the bulk to the rhizosphere soil. Bulk soil often has a higher microbial diversity than the rhizosphere, whereas the rhizosphere has a higher microbial mass due the high levels of root exudates provided by the plant. Diazotrophs, free-living nitrogen fixing bacteria, play an important role in plant growth, particularly in the nutrient poor soil, a characteristic of fynbos soil. Aspalathus linearis (rooibos) and Cyclopia spp. (honeybush) are leguminous plants endemic to the fynbos region of South Africa. These plants thrive in the nutrient poor fynbos soils and have become popular as herbal teas. Leguminous plants in this region rely on rhizobia to fix nitrogen and supply plants with other important nutrients such as plant growth-promoting compounds in exchange for a carbon source. The aim of this study is to determine the diversity and function of diazotrophic bacteria and rhizobia associated with A. linearis and Cyclopia spp. plants. The aim was achieved by determining the diversity of the diazotrophic community in the bulk and rhizosphere soils of commercial and natural rooibos and honeybush plants. Terminal restriction fragment length polymorphism (T-RFLP) was used to determine the diversity of the nifH gene in soil microbial DNA. Real-time Quantitative Polymerase Chain Reaction (qPCR) was also used to quantify the nitrogen fixing bacteria in soil. The copy number of nifH in the bulk soil was lower than that in the rhizosphere. The results indicated no significant differences between the diazotrophic communities of commercial and natural plants. Rhizobia were isolated from rooibos and honeybush root nodules to determine the diversity of rhizobia that associate with A. linearis and Cyclopia plants. Sequence analysis of the rhizobial isolates was done on the 16S rRNA, recA, atpD housekeeping genes, as well as the nodA and nifH symbiosis genes. Burkholderia and Rhizobium species were isolated from the root nodules. Aspalathus linearis preferred to associate with α-proteobacteria, whereas Cyclopia spp. preferred Burkholderia (β-proteobacteria) species. Phylogenetic analysis showed that some of the isolates are closely related to isolates from other studies done on fynbos root nodules. Further studies have to be done on the isolates that are not closely related to known species to determine if they are novel species. In the final part of the study, the rhizobial isolates were screened for the production of plant growth-promoting properties. Isolates were plated on specialised media and tested for the production of ammonia, hydrogen cyanide, phosphatase, 1-aminocyclopropane-1-carboxylate deaminase, and indole acetic acid. Results indicate that rhizobia provide the host plant with more than just available nitrogen. Rhizobia also produce indole acetic acid, HCN, and phosphatase. Some isolates produced more plant growth-promoting compounds than others, and considerable variation between strains was observed. These overproducing isolates could be used in potting trials to determine their effect on plant growth. This study highlights the importance of symbiotic interactions between microorganisms and plants. Future studies should include sampling extensive areas, over different seasons, as well as screening, isolating, and identifying more rhizobia across various plant hosts.
AFRIKAANSE OPSOMMING: This study highlights the importance of symbiotic interactions between microorganisms and plants. Future studies should include sampling extensive areas, over different seasons, as well as screening, isolating, and identifying more rhizobia across various plant hosts. Risobakterieë is uit die wortelknoppies van rooibos- en heuningbosplante geїsoleer om die diversiteit van die risobakterieë wat met A. linearis en Cyclopia assosieer te bepaal. Volgorde bepalingsanalise van die 16S rRNA, recA, atpD huishouding-gene asook die nodA en nifH simbiose is gedoen om die isolate te identifiseer. Burkholderia en Rhizobium spesies is uit die wortelknoppies geїsoleer. Aspalathus linearis verkies om met α-proteobakterieë te assosieer, terwyl Cyclopia spp. verkies om met Burkholderia (β-proteobakterieë) te assosieer. Filogenetiese analise het getoon dat sommige isolate naby verwant is aan isolate wat tydens ander studies op fynbos-wortelknoppies geїsoleer is. Verdere studies moet nog op die isolate gedoen word wat nie naby verwant is aan bekende spesies nie om te bepaal of dit nuwe spesies is al dan nie. In die laaste gedeelte van die studie is die isolate gekeur vir die produksie van plantgroei bevorderende eienskappe. Isolate is op gespesialiseerde media uitgeplaat en vir die produksie van ammoniak, waterstofsianied, 1-aminosiklopropaan-1-karboksilaat deaminase en indool asynsuur getoets. Die resultate dui aan dat die risobakterieë meer as net stikstof aan die plant verskaf. Sommige isolate het meer plantgroei bevorderende verbindings geproduseer as ander en daar was aansienlike variasie tussen stamme. Die stamme wat hoë vlakke geproduseer het, kan in potplantproewe gebruik word om hulle effek op plantgroei te bepaal. Die studie lig die belang van simbiotiese interaksies tussen mikroörganismes en plante uit. Toekomstige studies kan uitgebreide areas van monsterneming, verskillende seisoene, keuring van meer isolate en die identifisering van meer risobia van verskillende plant-gashere insluit.