Long-term sustainability of sugarcane production through the use of diazotrophic and plant growth promoting rhizobacteria

Hypotheses General hypothesis Growth and yield of sugar cane (S. officinarum L.) can be made more sustainable by incorporating practices that favour beneficial bacteriological components such as plant growth promoting rhizobacteria and/or endophytic diazotrophic bacteria. Specific hypotheses Plant growth promoting rhizobacteria and endophytic diazotrophs can be isolated from sugar cane grown in Mauritius. These bacteria can be re-inoculated into different cultivars of sugar cane. Successful inoculation of PGPR can lead to growth promotion compared to non-inoculated controls. The use of endophytic diazotrophs can complement the N nutrition of sugar cane. Appropriate inoculant technology is developed to facilitate and implement practical and low cost biofertilization . Objectives Based on these hypotheses, the following objectives were formulated: Overall objective To gain insight into how the sugar cane crop might benefit from biological nitrogen fixation (BNF), eventually leading to a reduction in the amount of N fertilizer used at field level with the simultaneous environmental advantage arising there from. In addition, the potential of plant growth promoting rhizobacteria (PGPR) to improve growth and development and subsequently yield would be studied. Specific objectives From sugar cane grown in Mauritius, to collect, identify and characterize a number of local isolates of endophytic and plant growth promoting rhizobacteria of three genera: (i) Gluconacetobacter (ii) Herbaspirillum (iii) Azospirillum To preserve the isolates and deposit them in International Collections. To verify whether the isolates can fix N2 in vitro. To verify whether the isolates can produce IAA and solubilize phosphate in vitro. To bulk the bacteria and develop practical inoculation methodologies for pot and field trials. To monitor colonization/ establishment of inoculated bacteria in sugar cane by appropriate methods. To quantify N2-fixation in vitro using bacterial cultures and in planta after inoculation. To monitor and compare growth and development of inoculated and non-inoculated cultivars. To evaluate the effect of pesticides on PGPRs and endophytic diazotrophs to gauge their robustness under field conditions. Thesis outline In Chapter 1, the status of the sugar cane industry worldwide is discussed with particular reference to the dismantling of subsidies and preferential trade agreements that have redefined the economics of sugar cane production. The historical link between sugar cane and Mauritius is discussed together with the country’s vulnerability in the new economic order. The potential of plant growth promoting rhizobacteria and endophytic diazotrophs to reduce costs of production is addressed. In Chapter 2, island wide surveys and samplings are conducted in commercial and non-commercial fields of Mauritius and Rodrigues in order to isolate Gluconacetobacter diazotrophicus from various sugar cane tissues such as leaf, leaf sheath, stem, roots, and trash. The isolates collected are characterized phenotypically and at the molecular level to gauge their diversity and they are compared to type strains. The ability of the isolates to fix N2, produce IAA and solubilize phosphate as well as their resistance / susceptibility to antibiotics and pesticides is evaluated. In Chapter 3, various detection methods for Gluconacetobacter are compared. The MPN technique using semi-solid LGIP medium is compared to serological and molecular techniques. Antisera raised against G. diazotrophicus are used in serological techniques such as ELISA and dot immunobinding assay. Molecular techniques such as PCR and IC-PCR are devised to lower the detection thresholds. In Chapter 4, various strategies are evaluated in order to improve the success rate of inoculation. Several inoculation methods including leaf spray, root immersion and decapitation are tested. Furthermore, various sources of the plant material are evaluated including vegetative cuttings, progenies from crosses and micropropagated plants. A range of cultivars is also tested with the aim of finding the right compatible reaction between host and microbe. In Chapter 5, two widely planted cultivars M 1176/77 and R 570 are put to the test in a semi-controlled field experiment using a specially-built concrete tank filled with a sub-soil of low N status. The inoculum used was the famous G. diazotrophicus Pal 5 and it was compared to a mixture of local isolates. Inoculation was carried out initially through roots at planting in virgin cane and subsequently by decapitation of the young regrowth in first ratoon. The N regime was lowered to 50% of the recommended dose and complemented by inoculation with bacteria. Growth and development as well as yield were monitored over a period of three years. N2 fixation was evaluated by the natural abundance technique. In Chapter 6, island-wide surveys and samplings were carried out to collect as many isolates of Azospirillum as possible from commercial and non-commercial fields of Mauritius and Rodrigues. Isolation was attempted from roots, rhizoplane and trash of sugar cane. The isolates were compared phenotypically and at the molecular level to gauge their diversity. Collected isolates were eventually used in glasshouse and field trials. In Chapter 7, the effect of inoculation with Azospirillum sp on two different cultivars R 570 and M 1176/77, with and without drought stress, is monitored. Growth as well as root development is monitored over a period of 138 days following inoculation. The interaction between cultivar, water regime and inoculation is analyzed to further understand the complexity of the microbe-plant association under sub-optimal conditions of drought stress. In Chapter 8, the effect of inoculation of two cultivars R 570 and M 1176/77 with Azospirillum sp. is compared to the effect of inoculation with synthetic IAA. Both treatments are compared to an untreated control to gauge the benefits that the crop could derive from inoculation. The effects of inoculation are monitored by analyzing parameters such as bud germination, presence of roots at the node, root fresh and dry weights, number of roots/node, number of lateral roots/node and length of roots/node. In chapter 9, a field experiment is set up to determine the best Azospirillum inoculation method. Three cultivars namely R 570, M 1400/86 and M 1176/77 are inoculated by three different inoculation techniques namely dipping, soaking and drenching. Growth and yield are monitored over a period of 2 years for plant cane and first ratoon to determine any beneficial effect of inoculation. In Chapter 10, island-wide surveys are conducted to establish and quantify the presence of Herbaspirillum spp in commercial and non-commercial fields of Mauritius and Rodrigues. Herbaspirillum is isolated from symptomatic as well as symptomless plants to confirm the endophytic nature of the bacterium. The plant tissues assayed are leaf, leaf sheath, root, stem and trash. The isolates collected are characterized by phenotypic, physiological as well as molecular means. The ability of the isolates to fix N2, produce IAA and solubilize phosphate is examined. In Chapter 11, a field experiment is conducted to evaluate the potential benefits that can be derived from sugar cane inoculation with Herbaspirillum rubrisubalbicans. Five cultivars are inoculated by an injection technique through the leaf tissues. Growth as well as yield is monitored over a two-year period in plant cane and first ratoon. Nitrogen fixation is determined by the natural abundance technique. In Chapter 12, the major conclusions of this research work are drawn and recommendations and suggestions are made to improve research in the field of biological nitrogen fixation and plant growth promoting rhizobacteria. In appendix E, an alternative method to the widely used and criticized Acetylene Reduction Assay to measure N2 fixation in vivo is evaluated. The flow-through gas exchange system indirectly estimates N2 fixation by monitoring the amount of hydrogen gas produced. This method has been used to compare the rate of N2 fixation of the local Gluconacetobacter isolates. Various experiments were conducted to evaluate the effect of oxygen, temperature, light and nitrate on the efficiency of the nitrogenase enzyme. In vivo measurements of N2 fixation were carried out for lablab and sugar cane. In appendix F, a range of substrates is examined for their abilities to sustain growth and multiplication of diazotrophic and plant growth promoting rhizobacteria. The aim is to determine the potential of these substrates as carriers in the development of appropriate and cheap inoculant technology for use with sugar cane.status: accepte