Endophyte 3.1 (BWT)
ANSWER: Smaller is better, but the 2.5 cork borer may be the best to use for consistent results. The modified protocol is fine, but it is more difficult to keep the tissue sterile during the cork boring process. It is much easier to cut the disks first and then surface-sterilize them.
Experiment:
Magnolia alpha leaves were analyzed using ONLY the modified protocol: large pieces of leaf tissue were surface sterilized as usual, then leaf disks of four different sizes produced using sterile cork borers and plates. Care was taken that all the tissue pieces were dry before plating. Extra disks were used for photography and imageJ calculations for surface area to derive the actual radius and circumference of the pieces (Figure 11, Table 2).
Results:
Normalization to circumference gives very consistent numbers across the range of disk sizes (Table 5 and Figure 13).
It appears to be the ability of fungi to escape – and be near the edge of the tissue that is important. Smaller pieces also matter: smaller numbers of fungi emerging from each disk means less chance for inhibiting or obscuring the growth of other fungi.
Extrapolating the data (Figure 14), gives 0.089 fungi/mm2 if we made infinitely small disks.
Lessons learned:
The 2.5 mm borer may give the most consist results, small enough to have limited numbers of fungi, but large enough to give consistent results from surface sterilization.
The overall density of fungi appears to be low, ~0.1 per mm2.
The standard protocol is fine as long as care is taken to closely follow the protocol and to make sure the leaf pieces are dry before plating.
Table: Modified protocol, does size matter?
Figure: Does size matter with modified protocol.
Figure: Fungi/unit area vs. area of leaf disk
Extrapolating to 0 mm2 leaf disk gives ~0.1 fungi/mm2
