---
kind: "section"
citation: "7 C.F.R. § 201.58d"
title: "7"
number: "201.58d"
heading: "Fungal endophyte test."
url: "https://uscodex.org/cfr/7/201.58d"
---

# §201.58d. Fungal endophyte test.


A fungal endophyte test may be used to determine the amount of fungal endophyte (Acremonium spp.) in certain grasses.

- (a) Method of preparation of aniline blue stain for use in testing grass seed and plant material for the presence of fungal endophyte:
  - (1) Prepare a 1 percent aqueous aniline blue solution by dissolving 1 gram aniline blue in 100 ml distilled water.
  - (2) Prepare the endophyte staining solution of one part of 1 percent aniline blue solution with 2 parts of 85 percent lactic acid (C3 H6 O3).
  - (3) **Use stain as-is or dilute with water if staining is too dark.**
- (b) **Procedure for determining levels of fungal endophyte in grass seed—**
  - (1) Take a sub-sample of seed (1 gram is sufficient) from the pure seed portion of the kind under consideration.
  - (2) Digest seed at room temperature for 12-16 hours in a 5 percent sodium hydroxide (NaOH) solution or other temperature/time combination resulting in adequate seed softening.
  - (3) **Rinse thoroughly in running tap water.**
  - (4) **De-glume seeds and place on a microscope slide in a drop of endophyte staining solution.** Slightly crush the seeds. Use caution to prevent carryover hyphae of fungal endophyte from one seed to another.
  - (5) **Place coverglass on seed and apply gentle pressure.**
  - (6) Examine with compound microscope at 100-400x magnification, scoring a seed as positive if any identifiable hyphae are present.
  - (7) Various sample sizes may be used for this test. Precision changes with sample size; therefore, the test results must include the sample size tested.
- (c) Procedure for determining levels of fungal endophyte in seedlings from seed samples suspected to contain fungal endophyte:
  - (1) **Select seeds at random and germinate.**
  - (2) **Examine seedlings from the sample germinated after growing for a minimum of 48 days.**
  - (3) **Remove the outermost sheath from the seedling.** Tissue should have no obvious discoloration from saprophytes and should have as little chlorophyll as possible.
  - (4) **Isolate a longitudinal section of leaf sheath approximately 3-5 mm in width.**
  - (5) **Place the section on a microscope slide with the epidermis side down.**
  - (6) Stain immediately with the endophyte staining solution as prepared in [paragraph (a)](#a) (2) and (3) of this section. Allow dye to remain at least 15 seconds but no more than one minute.
  - (7) **Blot off the excess dye with tissue paper.** Sections should remain on the slide, but may adhere to the tissue paper; if so, remove and place in proper position on the slide.
  - (8) **Place a coverglass on the sections and flood with water.**
  - (9) **Proceed with evaluation as described in paragraph (b) (6) and (7) of this section.**

## Notes

### Amendments

[59 FR 64515, Dec. 14, 1994]

### Authority

Authority: 7 U.S.C. 1592.

### Amendments

[59 FR 64515, Dec. 14, 1994]