experimental

Project 2) Can we culture any endophytes from Magnolia leaf tissue?

12 days of incubation and overgrowth

Endophyte 1.0 (Fall semester- early Spring 2014)

ANSWER:  YES! We see some morphotypes consistently emerging from Magnolia leaf tissue.

Experiment:

Our first experiments were exploratory in nature. We adapted published techniques to conditions in our lab (Bascom-Slack et al., 2012 and supplementary material; Appendix). We explored various ways of consistently producing equivalent size pieces of tissue inspired by Gamboa et al., 2002. Undergraduates were highly involved with designing these experiments, generating the written protocols for use in the lab and producing the data.

Initially, we used scissors to hand-cut square pieces of Magnolia alpha leaf tissue, following Gamboa et al., 2002. The major vein was avoided, as were areas of damage or discoloration.

Pieces were cut into 2 cm2, 1 cm2 and 0.5 cm2 squares, surface-sterilized with ethanol and bleach, and cultured on PDA or MEA plates (see Supplemental Material from Bascom-Slack et al., 2011, Figure 1). Five to fourteen days later, endophytes emerged from the tissue and were scored (Figures 2-4)

The biggest problem was consistently making the pieces the same size.

Figure 5: Magnolia alpha leaf pieces on culture media, 2 cm2, 1 cm2 and two 0.5 cm2 tissue (counterclockwise)
Figure 1: Magnolia alpha leaf pieces on culture media, 2 cm2, 1 cm2 and two 0.5 cm2 tissue (counterclockwise)
Figure 2: Endophytes emerge from leaf tissue after 3-4 days of incubation
Figure 2: Endophytes emerge from leaf tissue after 3-4 days of incubation
Figure 3: 7 days of incubation, two major morphotypes are evident: Pinky and Ghosty. Bacterial contamination is also evident!
Figure 3: 7 days of incubation, two major morphotypes are evident: Pinky and Ghosty. Bacterial contamination is also evident!
Figure 7: 7 days of incubation, two major morphotypes are evident: Pinky and Ghosty. Bacterial contamination is also evident!
Figure 4: 7 days of incubation, two major morphotypes are evident: Pinky and Ghosty. Bacterial contamination is also evident!

Endophytes were easily identified emerging from the leaf tissue. We have observed three major fungi by morphotype, two of which can be seen in the Figures 6-8 above

Pinky is fairly prevalent, emerges early and is easy to identify (although we have noticed distinct differences in morphology when grown on different media!). (see figure below)

Ghosty also is fairly prevalent and emerges early. There are likely to be a few different species of this white fast growing group.

Slowop is a very opaque, white slow-growing fungi that emerges later in culturing and may be the most prevalent endophyte we have cultured.

There are a number of minor morphotypes (orange, green, gray/green, blue) that are much rarer.

The protocol seemed robust and worked well in a number of different hands. We detected slightly less than one endophyte per disk, although ~16% had no fungi emerging at all (Table 1). Pinky may be under-represented on the north side – but we need better numbers.

A few ideas to come from these experiments:

We need a better way to produce consistently sized pieces efficiently.

Future experiments are done in “quad” petri dishes to isolate leaf tissue and emergent fungi from each other.

Table 1 Endophytes scored from Magnolia alpha, all sizes pooled

# leaf pieces (pooling all sizes) Total # fungi Fungi/leaf piece % disks with no fungi % disks with >1 fungi # pink % pink
South side leaves     425      375      0.88      18     82      48    12.8
North side leaves      144      133      0.92      14      86       3       2.3