Dear Sattar,

The following is the generally used method:

Symptoms of brown rot on apple and pear are easily identified and trained personnel can identify brown rot affected fruit. However, it is not possible to differentiate between the main brown rot fungi in the field using symptomology. Laboratory identification is essential to differentiate between the Monilinia spp. This is difficult, but can be achieved using morphological and cultural characters (Batra, 1979, Byrde and Willetts, 1977; Willetts et al.,1977; De Cal and Melgarejo, 1999) and ELISA (Hughes et al., 1998). However the culture identification should always be confirmed with molecular testing. PCR can also be undertaken directly from affected fruit (Hughes et al., 2000; Hughes, 2003).

Morphological and cultural methods

Determination of the three brown rot fungi using cultural methods is based on quantitative morphological characteristics (Leeuwen and van Kesteren, 1998). Such methods are

simple and low cost but require pure isolates, and are time consuming. Critical

morphological features include spore size, hyphal diameter, and germ tube length. Cultural features include colony growth rate, colony growth pattern and colony colour (Penrose et al., 1976; Mordue, 1979a; Mordue, 1979b; Mordue, 1979c; Lane, 2002). Many of these characters overlap between species meaning that identification has to be conducted under standardised conditions and starting with a pure culture. M. fructigena is difficult to distinguish from M. polystroma using the above criteria.

Isolation from plant material

Fruit rots are the most common symptoms of apple brown rot. Symptomatic fruit and stem cankers should be collected for isolation of the fungus.

Equipment Required

• Autoclave

• Class II Biohazard Cabinet or Laminar flow

• Bunsen burner

• Incubator at 25ºC

• Sterile petri dish

• Sterile scalpel and blades

Media

• Potato Dextrose Agar (PDA). Any commercial brand, follow manufactures instructions.

• Prune Extract Agar (PEA)

Prune Extract Agar (PEA) – to make 2L

• Prune extract 200 ml

• Sucrose 12 g

• Yeast extract 2.5 g

• Agar 32 g

• Water 1800 ml

1. Infuse 25 g finely sliced, pitted prunes in 450 ml water for 20 minutes in a beaker,

in a hot water bath, stirring frequently.

2. Remove from water bath and leave to cool for at least 5 minutes. Carefully filter warm liquid through two layers of white tissue paper. Pour filtrate into bottles / flasks and sterilise.

3. Once cool, store extract in the refrigerator.

4. Mix together the prune extract, sucrose, and yeast extract and make up to 2 l with

water. Pour in to 500 ml flasks containing 8 g agar. Mix together, seal and autoclave at 120°C for 10 minutes.

NOTE: Do not overcook otherwise the medium becomes cloudy and the agar will

hydrolyse and not gel properly.

Method

M. fructigena is easily cultured onto standard media such as prune extract agar (PEA) or potato dextrose agar (PDA) from fruit, shoots/branches. This can be achieved by inducing fruit to sporulate in a humidity chamber and transferring mass spores directly to a suitable culture medium or surface sterilising infected tissue and placing onto culture media.

The following procedures are commonly used for the isolation of Monilinia from infected

plant material:

Mass transfer of conidia:

Where sporulation of the fungus is present on infected plant material, directly transfer the spores from the sample to an agar plate with a sterile needle.

Direct isolation from infected tissue:

Samples are cleaned and then sterilised by soaking in 1 % sodium hypochlorite for 3 to 10

minutes (the thicker the tissue the longer the soaking) and rinsing in sterile water three times.

Suspected fruit samples can be surface sterilised by wiping the surface with cotton wool dipped in 96 % ethanol.

Small sections (2 to 3 mm3

) of the infected tissue are cut out with a sterile scalpel and

placed onto an appropriate culture medium. The sections should be cut out from the margin between the healthy and infected area.

Incubation:

Incubate the direct spore transfers or tissue isolations at 22ºC for five days in 12hr

light/12hr dark, then examine them for the presence of Monilinia spp.

4.1.2. Morphological identification

The three morphologically similar species of Monilinia can be identified using the synoptic key described by Lane (2002).

For more details, see attached file (Plant 3).

In addition, attached please find another two files: Plant 1 and Plant 2 which shed light on the isolation methods used.

Hoping this will be helpful,

Rafik

Thank you so nice Rafik Karaman for such a nice information.

Great job

HI:

You can just pickup some spores by sterilize needle and plate it on PDA petridish , incubate 25C for 4-5 day, after that pick the single isolates grow from single spore and plate it in new petridish.

Thanks

Dear Sattar Shah,

I have personally a good experience with a selective medium (acidified potato dextrose agar [pH 3.6] amended with fosetylaluminum [fosetyl-AL] at 500 μg/ml) (see attachment)

Best regards 

Vladimir

Vladimir Ostry ,Thank you so nice of you