1 Scope
This standard specifies the method for inspection and identification of Peronospora hyoscyami de Bary f. sp. tabacina (Adam) Skalichy in import plant quarantine.
This standard is applicable to inspection and identification of Peronospora hyoscyami de Bary f. sp. tabacina (Adam) Skalichy in import dry tobacco leaves and seeds and seedlings of nicotiana plants.
2 Principle
Peronospora hyoscyami de Bary f. sp. tabacina (Adam) Skalichy is an obligate parasite. After tobacco leaves are infected, nearly round spots with blurred edges and chlorosis will appear, and later pale brown necrotic spots that can fall off will form. Under suitable temperature and humidity conditions, light blue mold [namely, the sporangiophore and sporangium of Peronospora hyoscyami de Bary f. sp. tabacina (Adam) Skalichy] will be formed on the necrotic spot on the back of leaves; under certain conditions, oospore may be formed in diseased tissues. Parasitic specialization and morphological characteristics of pathogenic bacteria are the basis for the identification of Peronospora hyoscyami de Bary f. sp. tabacina (Adam) Skalichy.
3 Instruments and reagents
3.1 Handhold magnifier.
3.2 Magnifier with light source.
3.3 Binocular dissecting microscope (10×~50×).
3.4 Microscope (100×~1 000×).
3.5 Centrifuge (1 000r/min ~ 4 000r/min).
3.6 Stainless steel metal screen (aperture size of 60μm).
3.7 Oscillator (200r/min).
3.8 Phosphate buffer solution, 0.02mol/L (pH=7).
3.9 Aniline blue.
3.10 Phenol glycerin solution.
Preparation of phenol glycerin solution: mixture of 20mL of phenol crystal (dissolved by heating in water bath), 20mL of lactic acid, 40mL of glycerinum and 20mL of distilled water.
3.11 10% potassium hydroxide solution
Preparation of potassium hydroxide solution: taking 10g of potassium hydroxide and dissolving with 90mL of water to form 10% potassium hydroxide solution.
Foreword i
1 Scope
2 Principle
3 Instruments and reagents
4 Sampling
5 Inspection methods of tobacco leaves
6 Inspection methods for seeds and seedlings of nicotiana plant
7 Identification characteristics
8 Result judgment
Annex A (Informative) Oospore inspection by frozen homogenization method
