In the interactions between fungal endophytes and their hosts, the host may benefit through protection against pathogens and pests, growth promotion and tolerance to abiotic stresses. Non-pathogenic Fusarium oxysporum endophytes of banana have been shown to reduce the damage caused by the Cosmopolitus sordidus and the burrowing nematode Radopholus similis. The mode of protection against the burrowing nematode involves induced resistance, but the molecular basis of this resistance yet to be demonstrated. It has further been reported that protection of the host by multiple endophytes can lead to better control of target pests, probably because of the multiple modes of action involved. This phenomenon, however, has not been fully demonstrated for F. oxysporum endophytes of banana. This study aimed to investigate the molecular and biochemical basis of endophyte protection of East African Highland bananas (EAHB) against C. sordidus and R. similis. Expression of banana defence-related genes following endophyte inoculation and R. similis challenge varied greatly between the nematode-susceptible cv Nabusa and the nematode-tolerant cv Kayinja. In cv Nabusa, only the peroxidase (POX) and lectin genes were responsive to endophyte colonization of roots, or R. similis challenge. POX and lectin activities were significantly down-regulated 2 and 33 days after endophyte inoculation (dai), respectively. In cv Kayinja, endophyte colonization resulted in transient up-regulation of POX and a down-regulation of endochitinase (PR-3), lectin, pectin acetylesterase (PAE), phenylalanine ammonia-lyase (PAL) and PIR7A (peroxidase). Similar to systemic acquired resistance, PR-1 and catalase activities were up-regulated in the cv Kayinja 33 dai. Genes involved in signal transduction, cell wall strengthening, jasmonic acid pathway and defence molecule transport were differentially expressed in endophyte-inoculated plants. The expression profiles of four defence-related genes following endophyte inoculation and R. similis challenge were studied using quantitative real-time PCR. ABC transporter, Β-1,3-glucan synthase, coronatine insensitive 1 (COI1) and lipoxygenase (LOX) were up-regulated following endophyte inoculation. Β-1,3-glucan synthase and COI1 were highly up-regulated following R. similis challenge of endophyte-inoculated plants of the susceptible cv Nabusa, while COI1 and LOX were highly up-regulated following nematode challenge of endophyte-inoculated plants of the tolerant cv Kayinja. However ABC transporter gene activity was not up-regulated following nematode challenge of plants of both cultivars. UP-regulation of phenylpropanoid pathway enzymes PAL, POX and PPO has been observed in roots following colonization by both pathogenic and non-pathogenic fungi. In the current study, endophyte inoculation resulted in down-regulation of PAL activity in both a susceptible (cv Nabusa) and tolerant (cv Yangambi) banana. In cv Nabusa, endophyte inoculation primed PAL activity for up-regulation 30 days post nematode challenge (dpnc). However, in cv Yangambi PAL activity was up-regulated 7 dpnc irrespective of endophyte inoculation. Endophyte inoculation transiently up-regulated POX in cv Nabusa, but activity reduced to the levels in the controls 30 dai. Similar to PAL, R. similis challenge of endophyte-inoculated plants of Nabusa caused significant up-regulation of POX 7 dpnc. Nematode challenge of control plants of cv Yangambi resulted in a non-significant up-regulation of POX compared with non-challenged controls, but a significant up-regulation compared to all endophyte-inoculated plants. PPO activity was transiently up-regulated in cv Nabusa and down-regulated in cv Yangambi 7 dai. For all treatments, PPO activity was significantly reduced between 7 dai and 120 dai (60 dpnc). Fusarium oxysporum endophyte isolates Emb2.4o and V5w2 were successfully marked with benomyl- and chlorate resistance and transformed with fluorescent protein genes, while Eny1.31i, Eny7.11o and V4w5 were marked with benomyl resistance only. Most mutants and fluorescent protein transformants maintained resistance to the selective chemical on PDA and after plant colonization. Benomyl- and chlorate-resistant mutants were successfully used to determine actual plant colonization percentages by inoculated endophytes. Similarly, GFP transformants were successfully used to ascertain the pattern of endophytic root colonization in vivo. In plants dually inoculated with isolates Emb2.4o BR 8 and V5w2 CHR 9, both isolates were recovered from roots and rhizomes 4 weeks after inoculation, but isolate V5w2 CHR 9 proved a better colonizer of the two tissue types. Root colonization by isolate V5w2 CHR 9 was boosted when inoculated dually with Emb2.4o BR 8, while that by Emb2.4o BR 8 was reduced in the presence of V5w2 CHR 9. Where growth advantages were observed for dually inoculated plants, it occurred where plants were challenged with R. similis. In the absence of pests, control plants showed better growth than endophyte-inoculated plants. On the other hand, weevil challenge of control plants resulted in significant reductions in plant height, number of live roots and root fresh weight. Dual endophyte inoculation resulted in a significant reduction in R. similis populations in nematode only challenged plants, compared with plants inoculated with Emb2.4o BR 8 singly and control plants challenged with the nematode. In one replicate banana weevil damage to the outer and inner pseudostem base, and the inner rhizome were significantly reduced for dually-inoculated plants. Copyright / Thesis (PhD)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

East African highland bananas (Musa spp., AAA-EA genome group) are a major staple and income-generating fruit crop in the highlands of eastern and central Africa, grown across the countries of the Great Lakes region (i.e. Rwanda, Burundi, Uganda, eastern Democratic Republic of Congo and North-West Tanzania). Despite its importance, farmers and researchers are reporting that yields are declining, most notably in areas with low soil fertility. Although numerous studies have been conducted on yield constraints of bananas in the East African highland region, there is virtually no understanding of the impact of plant density management on the yields of these low-input banana systems. The productivity and profitability of various plant densities was studied in contrasting agro-ecological sites of Rwanda (Ruhengeri, Rusizi, Karongi, Butare, Ruhango, Kibungo and Bugesera), that differed distinctly in terms of altitude (1400-1960 m a.s.l), temperature (17-20°C), annual rainfall (950-1400 mm yr-1) and soil types (Nitisols, Ferralsols, Acrisols and Andosols). Under those cropping systems, the plant density is one management factor that resource poor farmers have some control over. An on-farm survey was conducted in all sites to determine the influence of climatic and edaphic factors on variations in on-farm plant density practices and bunch mass. In addition, three researcher-managed banana density experiments were conducted in contrasting agroecological sites (Kibungo low rainfall with medium soil fertility, Rubona high rainfall with low soil fertility and Ruhengeri high rainfall with high soil fertility) to (i) investigate the influence of plant density on the vegetative growth and yield parameters of AAA-EA bananas for typical highland agro-ecological zones, (ii) to assess the effect of plant density on nutrient deficiencies and imbalances, and (iii) to assess the magnitude and variability of nutrient depletion in the smallholder banana systems that are characterized by low external input use. Three different local EA highland banana varieties (i.e. “Ingaju”-cooking type, “Injagi”- cooking type, “Intuntu”-beer type) were each planted at five different plant densities (plants ha-1) of 1428, 2500, 3333, 4444 and 5000. Agronomic data (growth and yield traits) were collected over two cropping cycles (plant and ratoon crops). Soil, plant and climate data were also collected. Approaches such as compositional nutrient diagnosis (CND), boundary line functions and yield gap analysis were used to quantify the contribution of each identified yield limiting factor to yield gap. A first order magnitude of nutrient depletion was determined using partial nutrient balance calculations. Plant density positively correlated with water supply (i.e. difference between rainfall and evapotranspirative demand of bananas), with highest plant densities (>1500 mats ha-1) found in high rainfall areas (>1200 mm yr-1) with water surplus (218-508 mm yr-1) and lowest plant densities (1000-1400 mats ha-1) found in lower rainfall areas (1000-1200 mm yr-1) with water deficit (from -223 to -119 mm yr-1). Bunch masses were significantly higher at the lowest plant densities (18.1-20.8 kg fresh mass plant-1) when compared to the highest plant densities (14.7-15.5 kg). Lower soil and banana leaf nutrient contents were observed on weathered soils (Acrisols) and were associated with smaller bunch mass in comparison to fertile soils (Andosols, Nitisols). Farmers tended to reduce mat densities (i) if they wanted to intercrop, and (ii) to increase bunch mass to adapt to market preferences for large bunches. The plant densities generally recommended by extension bodies (3 × 3 or 2 × 3 m; i.e. 1111 and 1666 mats ha-1, respectively) are seldom practiced by farmers, nor do they seem to be very appropriate from an agronomic or economic perspective. Per hectare bunch and above ground biomass yields increased with increasing plant density, but maximum yield strongly depended on agro-ecological site. Bunch yields of beer bananas continued to increase with density, but maximum yields for the cooking cultivars were observed at 4444 plants ha-1 at Kibungo and Rubona, whereas yields continued to increase linearly beyond this level at Ruhengeri. Relationships between bunch yield, the total above ground dry matter yields and soil chemical properties suggest that nutrient deficiencies were larger at Kibungo (i.e. notably K) and Rubona (i.e. K, P, Ca, Mg) when compared with Ruhengeri. With increasing densities, leaf area index (LAI) continues to increase up to a value of 4 with 95% of photosynthetic active radiation (PAR) intercepted by the crop canopy. This suggests that further density and LAI increases would probably have little additional positive effect on total per hectare production. Compositional nutrient diagnosis (CND) indices showed that K, Mg and P were the most deficient elements in areas with low inherent soil fertility (Kibungo and Rubona) compared with relatively fertile areas (Ruhengeri). The boundary line functions and yield gap analysis also confirmed that K was the most limiting factor, contributing to an expected yield gap of 55.3% at Kibungo, while P and Mg contributed to a 35% yield gap at Rubona. An increase in plant density resulted in an increase in average yield gap from 45.6 % to 70.2% at Kibungo, whilst average yield gap decreased significantly from 47.5% to 30.2% at Rubona, and 76.6 to 53.7% at Ruhengeri. Nutrient uptake increased with plant density. Partial N and K balances (kg ha-1 yr-1) were estimated to be strongly negative at Rubona and Ruhengeri, while Ca and Mg were positive at Kibungo and Ruhengeri, but negative at Rubona. The results of this study indicate that, generally, soil fertility is a more limiting factor than water, but both CND norms and boundary line analysis showed that expected yield gaps seem to be high for plant density due to low inherent soil fertility. Partial nutrient balances provide a first order magnitude of nutrient depletion. Nutrient mining is significant, particularly for K. The current extraction rates will not allow farmers to sustain their yields, and options should be developed to improve the productivity of EAH banana cropping systems. The limited availability of manure and inorganic fertilizers is a real threat to the food and income security role that banana production plays in smallholder systems. In summary, the results from this study suggest the optimal density for bananas depends on water availability, soil fertility and cultivar. The agronomic optimal plant density is lower (< 4444 plants ha-1) in low rainfall (< 1000 mm yr-1) and less fertile areas, but seems to be higher (> 5000 plants ha-1) in areas with high fertility, which receive high rainfall (> 1300 mm yr-1). Improved plant density management can serve as an important entry point for resource poor farmers to maximize yield potential of EAH bananas in the various production zones. Blanket density recommendations do not make sense. While farmers can significantly improve their banana production, increased densities will put significant additional stress on limited nutrient resources, and region-specific integrated soil fertility recommendations should be developed and adopted to ensure sustained improvements of banana production and smallholder livelihoods. / Thesis (PhD)--University of Pretoria, 2013. / gm2014 / Plant Production and Soil Science / unrestricted