Methods of molecular identifications and lab. protocols
Growth rate/colony radius
We have found that basically growth rate/colony radius is a very useful, reliable -- and easy --character for definition of species. For example, there is no easier method of distinguishing T. harzianum from T. aggressivum or T. atroviride -- all morphologically similar and having globose, smooth conidia and occurring in mushroom houses -- than by growing the isolates at 35 C. Neither T. aggressivum nor T. atroviride will attain a colony radius of greater than 5 mm after 96 h whereas T. harzianum grows very well and sporulates at 35 C. Further, I think that all members of sect. Longibrachiatum grow well, and sporulate, at 37 C--which may be one reason why they are so commonly isolated from immunocompromised patients. This is important information that should be an integral part of species descriptions.
Growth rates are determined on PDA and SNA, a defined, low-sugar medium (Nirenberg, 1976). Vigorously growing colonies are established on cornmeal dextrose agar (CMD, Difco cornmeal agar + 2% (w/v) dextrose) at 20 C. After a few days when the colonies are visibly growing, but before conidial production, a 5 mm-diam plug is taken from the actively growing edge of the colony and inoculated onto freshly prepared medium. Vented 9 cm-diam plastic Petri dishes contain 20 mL of freshly made medium. The inoculum plug is placed mycelium-side-down approximately 1.5 cm from the edge of the Petri dish. The Petri dishes are incubated in darkness (except for the brief exposure to cool white fluorescent light when they are measured) at 15, 20, 25, 30 and 35 C. They are examined at 24-hourly intervals when the colony radius, measured from the edge of the inoculum plug, is recorded as was colony appearance, time of first appearance of green conidia, and any odor and yellow pigmentation in the medium or conidia. Each growth trial consists of a single Petri dish for each strain at each temperature. The growth trials are repeated three times at roughly weekly intervals, and the average radius is taken from the three independent measurements.
The growth trial is repeated three times independently because there is some variation within a single strain. Of course I include every strain that I have of a 'species,' and this tends to confirm a consistent growth rate. I don't put too much reliance on data derived from a single strain, but report results of a single strain if that is all that I have. If strains in a clade have widely different growth rates, I suspect that they are not conspecific and I have only seen this in clades that may have long branches, i.e. the members of the clade might not necessarily be taxonomically the same species. I measure the radius of a colony inoculated eccentrically on a Petri dish simply because most Trichoderma species would fill the 9 cm diam Petri dish well before enough measurements can be made. We use two media, a rich one (PDA) and a poor one (SNA) but the growth rates on the two media tend to be parallel, only occasionally does one see a different growth curve. PDA is useful for colony characters and pigmentation. SNA (and CMD) are good for giving a close approximation of what the Trichoderma looks like in nature, or at least better than PDA -- or malt extract for that matter -- does.
posted by Gary Samuels on 25 Oct, 2004