Análisis microbiológico

Four Epichloë festucae var. lolii strains were represented in the plant material used for this research: AR1, AR37 and two common-toxic endophyte strains [AR93 in clone- plants (Table 2.3) and an unknown common-toxic strain in plants of the A12421 accession (Table 2.4)]. Although the exact molecular nature of the two common-toxic strains may have been slightly different, they were considered to have an equivalent alkaloid profile (personal communication, David Hume). Both were therefore referred to as ‘CT strain’ in this thesis. The genetic identity of the endophytes in all mature plants used over the course of this thesis was confirmed on the basal (1 cm) piece of two tillers per plant, by one of two methods:

(i) Simple sequence repeat (SSR, also known as ‘microsatellites’) analyses were performed by the team of the molecular marker laboratory of the Forage Science group of AgResearch (Palmerston North, New Zealand) on one set of clone- plants. Thereby, selected fungal DNA segments were amplified and analysed by electrophoresis according to a standard protocol (Card et al., 2014a), using the two markers ans25 and egs02 to discriminate between the strains.

(ii) High resolution melting (HRM) analyses were performed by SlipStream Automation (Palmerston North, New Zealand) on all mature E+ _{clone-plants and} source plants. Strain identification by HRM is also based on differences in selected DNA segments but uses small differences in the dissociation behaviour of well-known, polymorphic DNA sequences when exposed to increasing temperatures to discriminate between endophyte strains (Applied Biosystems, 2009). Although this method is not able to discriminate between all known endophyte strains, it is able to reliably distinguish AR1, AR37 and CT strains (personal communication, Mike Cook, SlipStream Automation).

Epichloë festucae var. lolii is maternally transmitted, i.e. from a plant to its seeds (Popay & Hume, 2011). Transfer by pollen or by physical contact during normal plant maintenance work has not been observed to date (Christensen & Voisey, 2007). Artificial inoculation attempts on mature plants generally fail (Liu et al., 2011a). Empirical evidence suggests that only targeted laboratory work on axillary buds has ever succeeded in establishing endophytic symbionts into mature, endophyte-free tillers and that even then, the success rate was very low (Simpson et al., 1997). An endophyte-free (E-_{) plant}

could, therefore, be expected to remain so under standard handling procedures. Endophyte-infected (E+_{) plants, however, may occasionally produce E}- _{tillers when the} meristematic zone of an axillary bud has not been colonised by the fungus before its separation from the meristematic zone of its mother plant (Christensen & Voisey, 2007). Endophyte presence needed, therefore, to be confirmed in every tiller used for experimental work. Two methods described in Simpson et al. (2012) were used for that in this thesis: (i) direct microscopy (also known as ‘staining’) and (ii) immunodetection or immunoblotting (‘blot’, ‘blotting’). Direct microscopy supplied immediate results and did not require sacrificing the tiller of interest. The protocol for this method was to first remove all dirt and necrotic leaf sheaths from the tiller (Figure 2.7a), then peel off the outermost of the living sheaths and cut off an approx. 0.5-cm-long piece of the lower leaf sheath with a sharp scalpel (Figure 2.7b). That piece was then opened by a vertical cut in the middle and transferred to a glass slide, in a drop of aniline blue solution (50% glycerol, 25% lactic acid, 24.95% water, 0.05% aniline blue) with the adaxial epidermis facing upwards (Figure 2.7c). A cover slip was placed over it before the slide was held over a naked flame until the aniline solution started to boil, enabling the stain to better penetrate the tissues (Figure 2.7d). Once the slide had cooled, it was examined for blue-stained fungal hyphae with a compound microscope (Carl Zeiss AG, Oberkochen, Germany), at magnifications of 100 to 400x (Figure 2.7f). Endophytes were easily distinguished from saprophytes and other fungi when using this method, by the characteristic, cell-parallel and rarely branched arrangement of their hyphae. This method did not detect endophytes which had colonised the tiller meristem but had failed to establish in leaves, however.

Figure 2.7. Detecting endophyte presence by direct microscopy. (a) A green leaf is removed from a tiller; (b) Ca. 5 mm of the inner epidermis or (c) Ca. 5 mm of the full leaf sheath is cut off and spread in a drop of aniline blue, inner side upwards; (d, e) The slide is covered and the solution heated over a flame for the stain to better penetrate the tissues; and (f) Stained hyphae as observed at 400× magnification. © Mike Christensen.

(f)

(a) (b)

(c)

Immunodetection was a more practicable method when large numbers of tillers from a same plant had to be screened, but could not be performed without killing the screened tiller. The protocol for this method was to rid the tiller to test of dirt and necrotic material, severe it at the very base of the shoot, get a clean cross section of the shoot by a second cut with a sharp, clean scalpel a few mm above the first cut and press this cross- section onto a nitrocellulose membrane (‘NCM’, AmershamTM _ProtranTM _{0.45 NC,} Global Science & Technology Ltd, Auckland, New Zealand). Plant and fungal (if the tiller was E+_{) proteins were deposited on the membrane by this process. The NCM was} then developed as described by Simpson et al. (2012) in the following steps:

1) The remaining surfaces on the NCM were blocked by immersion in a milk protein blocking solution (BS) [2.42 g Tris(hydroxymethyl)methylamine, 2.92 g NaCl, 5 g non-fat milk powder, 10 mL of 1M HCl, made up to 1 L with RO water (i.e. water filtered by a reverse osmosis process) and adjusted to pH 7.5]. The NCM was shaken for ≥ 2 h in this solution on an orbital shaker at room temperature. 2) The old BS was decanted off the NCM and the NCM rinsed twice with fresh BS

before being shaken for 15 minutes in a solution of primary antibody (rabbit anti- endophyte; produced at AgResearch in collaboration with the Massey University Small Animal Production Unit) and BS (1:1000 dilution) and left incubating in it overnight at 4°C.

3) The NCM was rinsed twice in fresh BS to remove any unbound primary antibody. 4) The NCM was shaken at room temperature for 15 minutes in a solution of

secondary antibody (goat anti-rabbit IgG-AP, sc-2034, Santa Cruz Biotechnology, Dallas, TX, U.S.A.) and BS (1:4000), and left incubating in this solution for 5 h at 4°C.

5) Excess antibodies were removed by decanting and rinsing the NCM twice in BS. 6) Two separate chromogen solutions were prepared in amounts adjusted to the NCM surface area to be developed. For any 10 cm2 _{NCM, 20 mg Fast Red TR} and 12.5 mg of naphthol AS-MX phosphate (F-2768 and Sigma N4875, respectively, both products of Sigma-Aldrich New Zealand Ltd, Auckland, New Zealand) were dissolved in 12.5 ml of Tris buffer each [24.2 g

Tris(hydroxymethyl)methylamine in 1 L RO water, adjusted to pH 8.2]. The NCM was then immersed into a combination of these two chromogen solutions and shaken at room temperature for ca. 15 minutes until tillers known as E+ _(or positive reference tiller blots added to the NCM for this purpose) had developed a bright red colour (Figure 2.8)

7) Finally, the NCM was rinsed three times in ROwater.

Immunodetection occasionally resulted in falsely positive (E+_{) records, e.g. when} the NCM was old and/or got being contaminated with soil during the handling (David Hume, personal communication) or when fungal pathogens or saprophytes were present in the tiller and left fungal protein on the NCM. Endophytes present in the base of the tiller, but having failed to colonise the leaves were revealed too, however.

Figure 2.8. Revealing endophyte presence by immunodetection, blotting results. (a) Developed immunoblot, dry; (b) Developed immunoblot, humidified for better discrimination of negative (pale pink) and positive results (bright red).