Reed fescue and lolium perenne endophytic fungus real-time fluorescent PCR detecting method

A technology of endophytic fungi and reed-like fescue, applied in biochemical equipment and methods, biological testing, fluorescence/phosphorescence, etc., can solve the problems of slow growth, time-consuming, difficult identification, etc., and achieve strong specificity and high sensitivity Effect

Inactive Publication Date: 2007-11-14
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After isolation and cultivation, the morphological characteristics of endophytic fungi can be used for identification, but it takes a long time. The isolation and cultivation of N.coenophialum and N.lolii by Liu Yueting et al. Fungi are difficult to reproduce, or grow slowly, or do not produce sporulation or have weak sporulation ability (Fisher, PJ, Sutton, BC, Pctrini, LE, Pctrini, O, 1994. Fungal endophytes from Opuntia stricta a first report. Nova Hedwigia, 59:195~200), all these bring difficulties to the identification

Method used

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  • Reed fescue and lolium perenne endophytic fungus real-time fluorescent PCR detecting method
  • Reed fescue and lolium perenne endophytic fungus real-time fluorescent PCR detecting method
  • Reed fescue and lolium perenne endophytic fungus real-time fluorescent PCR detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the collection and cultivation of relative species of endophytic fungi in pastures and the isolation and cultivation of N.coenophialum and N.lolii in imported grass species;

[0038] The strains involved in the experiment include N.coenophialum and N.lolii purchased from the American Culture Bank and their related species N.huerfanum, N.chisosum, N.aotearoae, N.sp., and N.coenophialum isolated and cultured in our laboratory There are 14 strains of 6 species and N.lolii, see Table 1.

[0039] Table 1: Test strains

[0040] strain number

species name

Host

host source

TJN1

TJN2

TJN3

TJN4

TJN5

TJN6

TJN7

TJN8

TJN9

TJN10

TJN11

TJN12

TJN13

TJN14

N.coenophialum

N.coenophialum

N.coenophialum

N.coenophialum

N.coenophialum

N.lolii

N.lolii

N.lolii

N.lolii

N...

Embodiment 2

[0045] Example 2: Aniline Blue Staining Microscopic Examination of Imported Grass Seeds Carrying N.coenophialum and N.lolii

[0046] The seeds involved in the experiment include reed fescue and perennial ryegrass seeds imported from Tianjin port in recent years, each of 4 varieties, as shown in Table 2.

[0047] Table 2: Seeds tested

[0048] Numbering

species name

Variety

Carrier rate

plant origin

1

2

3

4

5

6

7

8

F. arundinacea

F. arundinacea

F. arundinacea

F. arundinacea

L.perenne

L.perenne

L.perenne

L.perenne

Ky31

Rebel sentry

Southeast

Bargena II

Gator

Victorian

MD warrior

Turf

62.5％

63％

53％

0

89.5％

33.3％

68.6％

0

U.S

U.S

U.S

U.S

U.S

new Zealand

Australia

U.S

[0049] 1. Solution preparation

[0050] 5% NaOH solution: NaOH 25 g, an...

Embodiment 3

[0056] Example 3: Mycelial genomic DNA and genomic DNA extraction of N. coenophialum and N. lolii from single seeds

[0057] The genomic DNA of mycelium and the genomic DNA of N.coenophialum and N.lolii in single seeds were extracted using Shanghai Sangon genomic DNA purification kit (No. SK1252).

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Abstract

This invention involves a method of N.coenophialum and N.lolii which uses real-time fluorescence PCR technology to detect tall fescue and perennial ryegrass endophytic fungi, namely. It belongs to grass fungus detection area of the original illness. This invention relates to two Taqman probes and primers: a set is universal primers and probe for the grass fungus Neotyphodium in monochrome fluorescence PCR detection. The other set is specific primers and probes for forage and N.loli N.coenophialum of the two-color fluorescent PCR detection. The invention establishes a simple, rapid, specific, sensitive real-time fluorescence PCR detection method, and its sensitivity reaches a grass seeds. Detection time of the conventional method of can be at least cur from one month to 7-8 hours.

Description

technical field [0001] The invention relates to a method for detecting endophytic fungi parasitizing in the grass seeds of Poaceae forage grass reed fescue (Festuca arundinacea Schreb.) and perennial ryegrass (Lolium perenne L.) using real-time fluorescence PCR (real-time fluorescence PCR) technology, The two endophytes detected were the reed fescue endophyte (Neotyphodium coenophialum) and the perennial ryegrass endophyte (N.lolii). It belongs to the field of forage endophytic fungi detection. Background technique [0002] Reed fescue and perennial ryegrass are important grasses in the world of grasses, with high nutritional value, they are favored by all kinds of livestock, and are widely used in the establishment of grazing grasslands. Among the developed animal husbandry countries, for example, the planting area of ​​perennial ryegrass artificial grassland in New Zealand is 7.6 million hectares, and the artificial grassland area of ​​reed fescue in the United States and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/48C12Q1/68
Inventor 黄国明廖芳刘跃庭崔铁军罗加凤刘勇
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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