Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

The techniques presented here are useful for investigating the endophytic fungi community associated with different plant organs. Endophytic fungi isolation allows their identification, and is valuable in other techniques such symbiotic germination. To begin, prepare eight to 10 centimeter PDA culture medium Petri dishes supplemented with three milliliters per liter of antibiotic.

Check for contamination by incubating the culture medium Petri dishes at 36 degrees Celsius for 24 hours before use. On the next day, superficially disinfest healthy mychoheterotrophic plant fragments using a standard procedure and place them in a sterile Petri dish. To evaluate the efficacy of superficial disinfestation of samples, inoculate a few drops of distilled water used during the last wash of the samples on PDA.

Next, using a flamed scalpel and forceps, section the samples into 0.2 centimeter thick pieces. Section the samples to amplify the surface area to be in contact with the medium. Place five fragments of the subterranean organs in the antibiotic supplemented PDA plate as far apart as possible without touching the dish edges.

Seal the Petri dishes with cling film, and store them in the dark at 25 to 27 degrees Celsius for five days. A day before subculturing, prepare five centimeter Petri dishes using five to seven grams per liter AA medium, and incubate the plates at 36 degrees Celsius for 24 hours. To purify fungal isolates, differentiate the PDA plate colonies by color, growth pattern, texture, and margin format.

Then delimit the colony margins on the bottom side of the dish. Also identify the colonies with a code. Using the tip of an autoclaved wooden toothpick, recover a tiny amount of mycelium from the margins of the identified fungal colony and striate the AA plate, producing three striae at one centimeter from one another and the dish edges.

Label the AA plate with the appropriate code using sticker paper and pencil. Seal the plates before incubating them in the dark at 25 to 27 degrees Celsius for three days. After incubation, meticulously observe the plates to identify fine hyphae forming individual colonies.

Delimit the area of individual colonies using a permanent marker. Cut a portion of the medium containing the colony using an autoclaved toothpick, and transfer the cut volume to the center of a new PDA Petri plate without antibiotics. After labeling the Petri dishes with the codes of the isolates, seal them with cling film, and maintain them in the dark at 25 to 27 degrees Celsius for seven to 14 days.

To begin the preservation of the fungal isolates with Castellani's method, add 0.5 milliliters of distilled water to the autoclaved two milliliter microcentrifuge tube. Using an autoclaved toothpick, cut 0.5 by 0.5 centimeter cuboid medium from the mycelium margins of already grown purified isolates. Place four to six cuboids in the microcentrifuge tubes with distilled water.

Store the tubes in the dark at 25 degrees Celsius for as long as needed. When required, recuperate a cuboid and place it in the center of a new PDA dish to grow a stored isolate. Begin the tease mount method by placing a drop of a Toluidine blue-O stain on a clean glass slide.

Different stains can be used in such a technique. Using an autoclaved toothpick, carefully remove some hyphae from the grown isolate, and place them in the drop of stain. Place a cover slip and analyze it under a light microscope.

For the adhesive tape mount method, place a drop of a lactophenol cotton blue stain on a clean glass slide. Cut a strip of transparent adhesive tape in a size that fits the glass slide and the central stain drop well. Direct the sticky surface of the adhesive strip to the mycelium surface, and try to collect some hyphae without pressing or collecting too many of them.

Then stick the tape to the glass slide, ensuring the stain is in contact with the collected hyphae. Place a drop of water above the tape and place a cover slip before analyzing the slide under a light microscope. The growth of small groups of filamentous fungi mycelia emerging from the interior of the fragments was observed after five days of inoculation of the mychoheterotrophic plant organ fragment on the PDA medium.

During seven to 14 days of inoculation of the purified isolate, the centrifugal growth of pure fungal isolate colony was observed on PDA, forming a sole circular mycelium. Possible contaminations were easily identified, compromising the colony homogeneity concerning its growth form aspect, color, and pigment production in the medium. A colony isolated from the fusiform roots of the mychoheterotrophic orchid, Wullschlaegelia aphylla, was opaque with no diffusible pigments or exudates in the medium.

The colony was whitish to gray on the upper side, and brownish on the lower side. The mycelia were aerial and abundant, and the margins were irregular and aerial. The colony had a velvety texture, and a wrinkled topography without macroscopic structures.

The structures which were not easily visible in the nonstained mycelium were exhibited after staining the fungal mycelium with lactophenol cotton blue and Toluidine blue-O, facilitating their identification. The fungal isolation methods presented here may also be applied to green plants for identifying the fungal endophytes. The isolated fungi can be used in symbiotic germination trials.