standard protocol for anthracnose screening of...

Protocol for Screening Anthracnose Resistance in Pepper of 8

Protocol for Screening Anthracnose

Resistance in Pepper

INTRODUCTION

PART A. Isolation of Colletotrichum spp from field sample

PART B. Long-term storage of Colletotrichum spp.

PART C. Propagation of Colletotrichum spp. for inoculation

PART D. Inoculation of pepper lines in the laboratory with Colletotrichum spp

PART E. Evaluation of pepper lines inoculated with Colletotrichum spp


Introduction Anthracnose of pepper (Capsicum spp.) is caused by Colletotrichum spp. and is a major disease of pre-and post-harvest decay of fruits during the warm and wet seasons in tropical and subtropical areas (AVRDC,1997). Reports from Korea were that they found two species of anthracnose fungi as C. gloeosporioides infects both green and red ripe fruits whereas C. capsici infects mainly red ripe fruits (Kim, 1989). In Taiwan, C. acutatum was consistently the most aggressive but they reported that some species of pepper were also infected with C. chinense, C. gloeosporioides and C. capsici. (Black,1997). In Thailand, C. gloeosporioides and C. capsici are the most widespread, reducing marketable fruit yield in the wet season. At the moment, no commercial varieties are available that are resistant to anthracnose fruit rot disease. General information Symptoms: Typical symptoms first appear on mature fruit as small water-soaked, sunken lesions that rapidly expand. The lesions may increase to 3-4 cm in diameter on large podded fruit. Fully expanded lesions are sunken and range from dark red to light tan with varying amounts of visible dark stomatic fungal tissue. Pale buff to salmon spore masses occur scattered or in concentric rings on the lesions. The fungus over winters on and in pepper seed, and in residue from diseased plant. Disease is promoted by wet conditions and high relative humidity. Conditions: The disease has a wide geographic distribution occurring wherever pepper is grown under overhead irrigation or rainfall conditions. Immature fruits are infected, but generally symptoms are not expressed until they become fully mature and undergo the final color change. The pathogen can be seed-born in pepper, persists in crop debris, and have a wide host range. Control: Clean seed and crop rotation are important elements in disease management. Application of fungicides can reduce disease.


A. Isolation of Colletotrichum spp from field samples

1. Collect the infected fruits from the field. 2. Surface sterile the infected fruit with 1% NaClO for 3 min. Wash two

times in sterile distilled water and dry with tissue paper. 3. Placed the samples in a moist chamber and incubate at room

temperature for 24-48 hr. 4. Pick up the conidial mass by pasture pipette and streak on 4% WA;

incubate at room temperature for 24 hr. 5. Cut a single spore by using a pasture pipette under the microscope

and transfer to PDA. 6. Incubate at room temperature for seven days.

Figure 1. Anthracnose symptom on pepper fruit in the field.

Figure 2. Spores of Colletrotichum capsici (left) and Colletotrichum gloeosporioides (right).


B. Long-term storage of Colletotrichum spp.

1. Culture plate with sterile filter paper on PDA.

2. Conidia and mycelium were collected on membrane filters and dried on silica gel blue.

3. Cut the dried filter paper and place in a small vial. Store at 4°C.


C. Propagation of Colletotrichum spp. for inoculation

1. Grow the pathogen from stock culture (stored on filter paper) on PDA (10mg/l streptomycin; 200 ml PDA). Incubate at room temperature for seven days.

2. Add 0.5-1 ml. of sterile distilled water into an eppendorf tube, then, gently scrape the conidial mass with a loop and add it

to the eppendorf tube. 3. Mixed the conidial suspension with a vortex. 4. Add 1% Tween20 to the inoculum and vortex.

5. Adjust the concentration to approximately about 5x106 conidia/ml using a hemocytometer

D. Inoculation of pepper line in the laboratory with Colletotrichum spp Plant preparation

1. Selected mature green fruit, they should be non-infected fruit and uniform in size. Use a known variety that is the most tolerant as a resistance check and a known variety that is susceptible as a susceptible control.

Capsicum chinense (left) and Capsicum annuum (right)


2. Use a sterile knife to cut the fruit stem

3. Surface sterilize with 1% Sodium Hypochlorite pH 7 for five minutes.

4. Wash twice in sterile distilled water and then dry with tissue paper.


Inoculation method (Detached fruit method)

1. Make two or three pore holes (0.5 mm deep) on the fruit samples (depends on size of fruit) using a needle and wipe the wound with tissue paper.

2. Inoculate by injecting 2 µl of conidial suspension.


Incubation method Place the fruit samples in plastic bags to keep them moist. Cover the plastic bags and incubate them at 30ºc, near 100% RH.

D. Evaluation of pepper lines inoculated with Colletotrichum spp Lesions are measured in millimeter (mm.) at 4 DAI. Separate to disease rating scale

Rating Scale: 1 = No visible lesion 2 = Small lesion < 0.3 mm 3 = Medium lesion 0.31 – 1.0 mm 4 = Large lesion 1.01 – 2.0 mm 5 = A large expending lesion > 2.0 mm

D. Media PDA (per litre) MgSO4.7H20 0.3 g K2HPO4 2.0 g Yeast extract 4.0 g Casein hydrolysate 8.0 g Sucrose 10 g Agar 15 g Autoclave.

standard protocol for anthracnose screening of...

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