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Do mutualistic organisms associated with wild Nicotiana benthamiana plants influence drought tolerance?

Dastogeer, Khondoker (2017) Do mutualistic organisms associated with wild Nicotiana benthamiana plants influence drought tolerance? PhD thesis, Murdoch University.

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In arid regions, such as those in north-western Australia, plants survive under water deficit, high temperatures, intense solar radiation and nutrient-impoverished soils. They employ various morphophysiological and biochemical adaptations including interaction with microbial symbionts. Seed from thirty-two accessions of four Nicotiana species (N. benthamiana, N. occidentalis, N. simulans, and N. umbratica) collected from wild plants from northern Australia, were grown and used to assess their responses to water stress. The original wild host plants were selected because they grow in an extremely hot environment where water supply is often unpredictable, and because they share a close genetic relationship to the international model plant N. benthamiana research accession 4 (RA-4). Under moderate water stress conditions, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight, root/shoot ratio, and relative water content of leaves was significantly affected. However, the degree to which the accessions were affected varied considerably. Some accessions of N. simulans, N. benthamiana and N. occidentalis were significantly more affected by water stress than others. There was significant inherent variation between accessions in leaf and shoot tip wilting times. Initial symptom expression (leaf wilting) was significantly delayed in two accessions of N. benthamiana and in one accession of N. umbratica. The least water stress tolerant lines, three accessions each of N. occidentalis and N. simulans exhibited advanced symptoms of water stress (shoot tip wilting) within 14-17 days of cessation of watering. This stage was significantly delayed in three accessions of N. benthamiana and two accessions each of N. occidentalis and N. simulans, which exhibited tip wilting after only 21-24 days. There were variations among the accessions of Nicotiana species on their tolerance to water stress. Plant responses to water stress could not be predicted from their phenotype under well-watered conditions.

We evaluated identity, host and tissue association, and geographical distribution of fungal endophytes isolated from above and below-ground tissues of wild plants of three indigenous Australian Nicotiana species. Isolation frequency and α-diversity were significantly higher for root endophyte assemblages than those of stem and leaf tissues. We recorded no differences in endophyte species richness or diversity as a function of sampling location, but did detect differences among different host genotypes and plant tissues. There was a significant pattern of community similarity associated with host genotypes but no consistent pattern of fungal community structuring associated with sampling location and tissue type, regardless of the community similarity measurements used.

We developed and evaluated two rapid screening methods to identify fungal endophytes that enhanced water deprivation stress tolerance in seedlings of N. benthamiana RA-4. Sixty-eight endophyte isolates taken from wild Nicotiana plants were co-cultivated with N. benthamiana RA-4 seedlings on either damp filter paper or on an agar medium before being subjected to water deprivation. The longevity of seedlings was compared under association with different fungal isolates and under the two screening methods. The filter paper method was faster and simpler than the agarbased method. Based on results, 17 isolates were selected for further testing under water deprivation conditions while growing in washed river sand in a glasshouse. Only two fungal isolates, one resembling Cladosporium cladosporioides (E-162) and a fungus not closely related to any described species (E-284), significantly enhanced seedling tolerance to moisture deprivation consistently in both in vitro and glasshouse-based tests. Although a strongly significant correlation was observed between any two screening methods, the results of the filter paper test was more strongly reflected (r = 0.757, p< 0.001) in results of the glasshouse-based test, indicating its relative suitability over the agar-based test. In another experiment, the same 17 isolates were inoculated to N. benthamiana plants growing in sand in a glasshouse under nutrient-limiting conditions to test their influence on growth promotion. Isolates resembling C. cladosporioides, Fusarium equiseti, and Thozetella sp. promoted seedling growth, evidenced by increased shoot length and higher biomass than non-inoculated control.

The two promising fungal endophytes identified from wild Nicotiana plants, E-162 (C. cladosporioides) and E-284 (an unidentified species) were inoculated to plants of N. benthamiana RA-4 to examine their metabolic response to endophyte colonisation under adequate water and water deficit conditions. We examined leaf metabolites using gas chromatography-mass spectrometry (GCMS) to compare levels of sugars, sugar alcohols, amino acids and other metabolites at various stages of plant growth and stress application. Ninety-three metabolites were detected in leaves, including 20 sugars, 13 sugar alcohols, 21 amino acids, 29 organic and fatty acids and ten other compounds. Endophyte colonization caused significant differential accumulation of 17-21 metabolites when the plants were grown under well-watered conditions. The presence of endophytes under water stress conditions caused differential accumulation of cytosine, diethylene glycol, galactinol, glycerol, heptadecanoate, mannose, oleic acid, proline, rhamnose, succinate, and urea. Accumulation of these metabolites suggests that fungal endophytes influence plants to accumulate certain metabolites under water-stress. Further, the two different endophytes tested caused slightly different accumulation patterns of some metabolites.

We evaluated how these two fungal endophytes as well as yellowtail flower mild mottle virus (genus Tobamovirus), influenced water stress tolerance in N. benthamiana RA-4 plants. The water stress tolerance of fungus-inoculated plants correlated with increased plant biomass, relative water content, soluble sugars, soluble proteins, proline content, increased activity of the antioxidant enzymes catalase, peroxidase and polyphenol oxidase, and decreased production of reactive oxygen species and electrical conductivity in plants under water stress. In addition, we found that there was significant differential upregulation of drought-related genes in the fungus-inoculated plants subjected to water stress. Plants inoculated with the virus exhibited a similar response to those plant inoculated with the fungi in terms of increasing plant osmolytes, antioxidant enzyme activity and gene expression. Although the fungus and virus infection similarly increased plant water stress tolerance by influencing plant physiology and gene expression, their presence together in the same plant did not have an additive effect, nor did they decrease water stress tolerance.

These findings suggest that both fungi and virus influence plant physiology and gene expression under water stress, and it suggests that there is potential to use endophytic fungi, and perhaps virus, to induce greater tolerance to water stress in agricultural production systems.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
Supervisor(s): Jones, Michael, Wylie, Steve and Li, Hua
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