Leer: Comprensión, Interpretación y Construcción de Sentido

Even in the same cultivar, the growth of endophyte-infected plants under drought conditions was variable. Some endophyte-infected grasses had higher, and some had lower, levels of dry biomass than the median dry biomass of endophyte-free grass under drought (Figure 4.3). This suggests that effects of endophyte on grass performance under drought are quite different. Thus we selected outliers with the most different dry biomass as the preliminary set of candidates for a transcriptomics study.

Outliers with lower biomass (slower growth rates) showed higher survival ability than those with higher biomass (faster growth rates) (Table 4.1). This was possibly because outliers with greater biomass use more water and are more likely to have hydraulic failure (failure to pump sufficient water from roots to leaves) than outliers with lower biomass. Alternatively, compared to outliers with high biomass, outliers

with low biomass may have invested more carbohydrate on production of osmolytes rather than growth, thus they may have better osmotic adjustment to reduce water loss under severe drought stress (Burg and Ferraris 2008; McDowell et al. 2008).

Most studies on endophyte impact on grass performance under drought emphasize the enhancement of grass growth (Cheplick 2007; Cheplick et al. 2000; Hesse et al. 2003; Kane 2011). However, given climate change and extreme drought conditions such as those experienced in New Zealand in 2013 (APNZ-News-Service 2013), survival ability may be more important than growth rate. Only grasses that survive under severe drought conditions can continue to grow. Thus within the set of outliers a smaller set was selected according to their survival ability for future study.

The photosynthetic efficiency parameter Fv/Fm is often used as a sensitive indicator for many types of plant stress, including drought (Nogues and Baker 2000; Posch and Bennett 2009). A Fv/Fm ratio between 0.79 to 0.84 is recognized as an optimal value for many plants (Kitajima and Butler 1975). Normally, the Fv/Fm ratio decreases when plant is under stress, or when the stress becomes severe. The more stress the plants suffers, the lower the Fv/Fm ratio is (Kitajima and Butler 1975). The decreased Fv/Fm ratios and LRWC (leaf relative water content) under drought confirmed that the grasses suffered water deficit stress (Figures 4.5 and 4.6). However, outlier 2 in Nine O One and Trophy cultivars maintained a significantly higher Fv/Fm ratio and LRWC than corresponding outlier 1 of these cultivars under severe drought condition. This suggested that outliers for both Nine O One and Trophy cultivars had distinctly different drought tolerance levels, and were suitable to be used in a transcriptomics study.

An important way that plants cope with drought stress is to reduce water loss by decreasing osmotic potential in their cells. This can be achieved by accumulating osmolytes such as proline, glucose, fructose, sucrose, fructans, mannitol etc (Burg and Ferraris 2008). Outlier 1 had significantly greater LOA than outlier 2 in both Nine O One and Trophy cultivars, indicating that osmotic adjustment might be one reason for

the different survival ability under severe drought stress between these two pairs of outliers (Table 4.1).

As mentioned in section 4.2.1, endophyte genotype affects its influence on grass performance under drought. Thus the species and genotypes of endophytes living in outliers were tested. Whilst real-time PCR confirmed all outliers were N. lolii (Table 4.3), SSR analysis revealed that the endophyte in Trophy outlier 1 had a different genotype from the other endophytes tested (Table 4.3), thus Trophy outliers were not suitable for a transcriptomics study.

Outliers of Nine O One cultivar were selected for future study, as (1) outlier 2 showed the ability to survive under severe drought, (2) the two outliers showed significantly different drought tolerance under relatively severe drought stress, (3) endophytes living in the two outliers were determined to be the same genotype based on real-time PCR and SSR assays.

4.2.3

Endophyte improved drought tolerance of both selected

ryegrass genotypes but to different extents

The endophyte effect on selected grasses in the same cultivar was studied by measuring several critical plant physiological parameters. The photosynthetic efficiency parameter Fv/Fm is often used as a sensitive indicator for many types of plant stress, including drought (Nogues and Baker 2000; Posch and Bennett 2009). Plants utilize light to produce ATP and NADPH (Baker 2008). Fv/Fm measurements compare the minimum fluorescence (dark-adapted leaf pre-photosynthetic fluorescent state) to maximum fluorescence (photosynthetic state in the presence of light). A Fv/Fm ratio between 0.79 to 0.84 is recognized as an optimal value for many plants (Kitajima and Butler 1975). Normally, the Fv/Fm ratio decreases when plant is under stress, or when the stress becomes severe (Kitajima and Butler 1975).

control conditions was around 0.81, indicating all four types of grasses were healthy (Figure 4.8). The decreased Fv/Fm ratios and LRWC (leaf relative water content) under drought confirmed that the grasses suffered water deficit stress (Figures 4.7 and 4.8). However the endophyte-containing plants DTE+ and DSE+ had higher Fv/Fm ratios, LRWC and TTN (total tiller number) (Figure 4.9) than DTE- and DSE- respectively under drought stress, implying that harboring endophytes relieved grass suffering from water stress. These results are consistent with previous observations of a positive effect of endophyte on grass drought tolerance (Elmi and West 1995; Hahn et al. 2008; Zhang and Nan 2007). However, a significant difference on Fv/Fm, LRWC and TTN was only observed between E+ and E- of DT genotype but not in DS genotype indicating that the extent of positive effect of endophyte on grass drought tolerance was variable between two grass genotypes. The interaction of host genotype and endophyte on grass drought tolerance was also found in others’ studies, where the authors found the effect of endophyte on grass drought tolerance can be either positive or negative depending on host genotype-endophyte combinations (Cheplick 2007; Kane 2011). However, no further comparison of positive genotype and negative genotype was reported in their papers.