Dear S. Rehman

For collection of Entomopathogenic fungi, please find areas that have humiditidy, and slight raining after several hours. In dry weather it is alittle difficult to find. 

From soil you can use dilution method, from diseased insects in humid areas its very easy to isolate and for identification, i think the ITS method is very easy.

I hope it could helps you.

sehroon

 Dear Sehroon Khan 

Thank you. I have used dead insects - beetle and mealybug for isolating Entomopathogens. i got one fungi look like Hirsutella under microscopic observation and colony morphology. Now i am trying to get pure single colony.

Can you describe or help anyway actually what does 'ITS' meant? i searched using ITS word to understand actually whats the ITS protocol for isolation. Is it its primer for fungal sequencing?

Best wishes.

S. Rehman

Dear Shuvrah Rehman,

for isolation of entomopathogenic fungi from soil or litter you can also use th "insect bait method" which was first described by Zimmermann in 1986. Please find attached pdf file of N. Meyling concerning this method and other methods used for isolation of epf from soil.

Best wishes,

Cezary Tkaczuk

Dear S. Rehman,

You can use the insect bait method (Goettel et al., 2000) to isolate Beauveria. I may also suggest that you have a look at Microbial-Based Biopesticides: Methods and Protocols (Methods in Molecular Biology) by Travis R. Glare (Editor), Maria E. Moran-Diez (Editor). The hardcover of this book is due to be released 12 Sep 2016. 

Dear Shuvrah Rehman,

I agree with Cezary Tkaczuk for the isolation of entomopathogenic fungi.  

I use the insect bait method also for the isolation of entomopathogenic nematodes.

Best wishes,

Giuseppe Mazza

Hello Suvrah 

I agreed with the other colleagues about insect bait method , you can use either galeria melonella or Tenebrio mollitor as baits . Otherwise for isolate Beauveria bassiana there is a simple medium you can use with Bengale rose coloration , is in any handbook of micology or manual that you can find it , later I can look in the files and send it to you , but is a very common media.. Keep trying !!! When I started research many years ago I did not have available many things and also dodine ,,, you will manage it .. !!Regards, claudia

Hello Shuvrah,

I come back to direct extraction of EPFs from soil samples since you already got a lot of advice regarding bait methods. We use a proptocol similar to the one described in Kessler et al 2003, see attachment. We use the medium of Strasser et al, 1996 to isolate Beauveria and Metarhizium, see attachment. Dodine is an important ingredient, but you don't need to order the pure substance. You can use commercial products containing dodine, e.g. Syllit from Arysta Life Science, I am quite sure that some of these products are available worldwide (don't forget to adjust concentrations accordingly!)

All the best

Giselher

Dear Shuvrah,

After I collect the samples, I normally dissect their abdomen and plate pieces from inside on PDA. Inside their abdomen, especially during early stages of development, there are cells called hyphal-bodies, which will develop into hyphae after few days after the host's death. Another Method I use is directly from ascospores, but since you're interested in species that normally just produce the anamorphic state, I don't know if this method would work for conidia as well.

You can further confirm by SSU and LSU BLAST. I say those genes because they are both the easier ones to obtain by PCR in my experience (rather than ITS for example).

Good luck!

PS: Remember to avoid plating by accident, even small pieces, of the host cuticle. It carries lots of contaminants

The ideal time to isolate entomopathogenic fungi could be in the days after the rainy days as high humidity and temperature conditions (25-30 degrees) , insect favor infection by entomopathogenic fungi. To find them in the field will depend on the insect which you want to isolate , could be on the undersides of the leaves. To find out if they are infected by fungi in live insects , they could have a different rest behavior, such as slow movements , in addition to the appearance of spots on your body. In dead insects you could observe mycosis (which is the covered body of mycelium) or spores on the insect . In the soil you could be isolated by the method of " insect trap " with larvae of Galleria mellonella.

Dear Shuvrah, 

i have a simple insect pathology manual which may help to collect and isolate entomopathogens. I will be glad to share it with you. 

yours, 

Wesam

Generally one should collect alive insects rather than  dead. Maintain them individually on diet or food plant parts. Monitor your collection daily for the appearance of any disease disorder. Suspected samples can be examined separately(smear or culture media)  according to the type of disease agent ( virus, fungi or bacteria). Identification of disease agent are made according to appropriate references online. Beside fresh sources of diseased insects, some insects could be followed up until death and examined in the same way. Needless to say that knowledge of microbiology is required at least by collaboration. 

To isolate entomopathogenic agents is necessary to find in nature. For example: insects, soil, waste broth. This material must undergo a culture medium and to grow it must be isolated and tested for its ability entomopathogenic. This step is made through biological toxicity tests.

Please check the publication  Biocontrol 46:105-124, (2001)

The approach I take uses all of the methods described by Salah and Christos and then some like insect baiting. Detailed step by step procedures are presented by several authors in:  Lacey, L. A. (Ed.) 2012. Manual of Techniques in Invertebrate Pathology, 2nd edition. Academic Press/Elsevier, San Diego. 484 pp. It's available Through Elsevier and Amazon. 

GOOD RECEPTION

basically we can get it from insect's cadaver, soil and plant either, I guess when i saw ur case for the simple one we can isolate it from insect' cadaver afterward u can go to next step which is get it from soil or u can use dead's insect as a trap.

Well goodluck for your work

indeed, it has already made in isolation from insects covered corpses of mycelium we found dead on land (grasshoppers, moth potato: Phthorimaea operculella ..)

good luck in your work.

t is very easy , if you get larvae Galleria mellonella and take it to the soils where you want to isolate entomopathogenic fungi The Soil should have field moisture and leaves them there , checking the larvae every three days until the larvae found colonized by fungi

Dear all colleagues

Thank you all

Dear Sahir-Halouane Fatma 

your sending file unfortunately not downloading. May I request you to send again?

Dear Shuvrah : sorry , in fact what I told you was that I agreed with the other colleagues , that you can try insect bait with soil , you can use Tenebrio mollitor larvae or Galeria melonella , this technique is very usefull and also for Beauveria bassiana you can try selective médium with Bengala rose , is a very common médium that you can read in any mycology handbook .. this Works too and is not expensive. keep trying and don ´t give up !! Good Luck and success for you. regards, Claudia

Dear Shuvrah, we got better results using CTC medium as compared to Strasser medium (see the PDF). I also attach two Word files. One is a brief introduction to selective media and insect baiting, the other a 'cook-book' for some (semi)selective media one can use for EPF isolation. Good luck with your research!

Dear Shuvrah Rehman 

for get an entomopathogenic fungi pure and can be use to biological control you most be use a selective media by add a material (CTAB) Cetyle tri methyl amino bromide, another way use an insect wax moth Galleria sp. especially to get Beauveria sp.

Dear Shuvrah,

Isolating entomopathogenic fungi from insect is hard. those dead insect with visible fungi covered their body will just consider as infected by entomopathogenic fungi. 

alternatively, u can isolate entomopathogenic fungi from soil. there are two methods selective medium and insect baiting with soil using Galeria melonella  or Tenebrio mollitor (mealworm) larvae. mealworm is used for the fishing bait. U can refer to meyling 2007. Methods for isolation of entomopathogenic fungi from the soil environment. make sure the soil not  too dry or too wet. 

Selective medium such as CTAB, rose bengal can inhibit saprophytic fungi. I discovered new medium for isolate entomopathogenic fungi, u can refer it once the paper is published later. 

Dear Shuvrah Rehman,

Sorry for being late,entomopathogens include:bacteria,fungi and protozoa.

I would like to inform me what do you want to isolate ?

And  i will send you the technique of choise.

Best Regards

Most of the fungi I have isolated were from infected insects from a wide variety of habitats. Some of the techniques I've used are in the attached chapter from the Manual of Techniques in Invertebrate Pathology, Lacey (Ed.), 2012.

Dear Hussein Kaoud

Thanks for being cordial. I am interested about Fungal entomopahtogens. I hope you can help me.

Best Regards

Hello Dear

Different techniques were bee apllicated by specialist for this purpose. I aplied good methods for isolating Beauveria and Metarhizium. You can read them in my publications. I think they are useful for you. Thanks

Hello Dear

I study the pathogenicity of entomopathogenic fungi for control Oryctes and Oryzaephilus with novel technique. Please see them in my publication. I hope that will be useful. Thanks

Thank you 

Masoud Latifian.

Regards

Shuvrah Rehman

Dear Shuvrah Rehman

Use selective media, it will facilitate you the isolation from soil or insect cadaves

Good luck

Please find attached a detailed review of the isolation of entomopathogenic bacteria and fungi. Here the majority of the queries related to the medium and bait-insects are covered. https://www.mdpi.com/2076-2607/9/1/16

You can refer to my article on isolation of Beauveria.

Mwamburi, L.A. 2016. Isolation and Assessment of Stability of Six Formulations of Entomopathogenic Beauveria bassiana. In: Glare, T.R. Moran-Diez, M.E (Eds.), Microbial-Based Biopesticides: Methods and Protocols. Series: Methods in Molecular Biology, Springer, Humana Press Inc. USA. Volume 1477 Chapter 4. pp 85-91.

Hi Sharif

You can use larvae for mealworm "Tenebrio molitor " as a bait to get Metarhizium