Hello,

I used leaf discs for an experiment during my PhD and was able to keep the leaf for at least 10 days with the population intact. However, I was using Brassica rapa leaves with Myzus persicae. I did not invert the dish but simply put a light paper covering over the dishes. I used distilled water in the preparation and sterilised the equipment I used to cut. I have included an exert from the M & M of my thesis below, the whole thing can be found on my profile you are interested in seeing more, though think this is the most relevant part (it is 'Attack rate assay' where I use leaf discs). Hope any of this is of help to you. Feel free to contact with any questions. Hope the experiments go well,

Mandela

To prepare the leaf discs around 30ml of 1.5% agar solution was poured into a 7 cm Petri dish; this setup was similar to that used by Blande et al. (2004). A preliminary trial showed that the leaf in this media and condition was able to survive 10 days, at which point mummies would be clearly formed. Scissors used to cut the leaves were sterilised in 90% ethanol to reduce the chance of infection. As the agar cooled the leaf was gently embedded into the surface ensuring that the stem was in contact with the agar and could receive sufficient moisture. Caution was taken not to place the plant in before the agar was sufficiently cooled as this instantly kills the aphids or causes them to scatter.

Lipaphis erysimi (Kaltenbach) is a specialist crucifer feeding aphid. Mustard aphids are reared on potted mustard plant in a screen-house. Aphids are transferred with a brush to each bioassay chamber containing thin, surface sterilized leaf with 0.01% sodium hypochlorite. The leaf midrib is covered with sterilized moistened cotton. C. papaya leaves are used after washed thoroughly with sterilized water and place in a tray for 15 days for air-drying. The leaves are then ground in mortar and pestle to fine powder. Five gram leaf powder  to 100 ml acetic acid and left for 15 days to dissolve  completely. 20-40 aphids are employed  in bioassay in each chambers as control and tests with 5 replicates.

 Sometimes a minimal change from environment before - during bioassay realization is crucial for aphid survival. How many days go by before aphid die? Are neonate aphids? Are aphids eating Vicia faba on plant before staring the bioassay?

Hi,

I've done such experiment with Aphis gossypii on the leaves of cucumber. At first there were some troubles such as death of aphids. But the I used the protocol of IRAC online.I used 5 cm petri dishes, in 25 C and 65% rh chamber room. I thought that the problem is related to instability of leaves. If you can use another plants, or you must done on fresh plants ans leaves.

Thank you all for your replies!

I will try again with your advices.

To respond to Marta, aphids are reared on entire V. faba plants before the experiment. I use 10-days aphids and some of them die the next day whereas the leaf looks intact. In all the cases, I observed 50% mortality over the 3 fisrt days of the experiment.

I meant "at least 50% mortality"... there was a great variability. I should also mention that care was taken in aphid handling to not break their stylet.

you may want to check the papers by Cindy ten Broeke (https://www.researchgate.net/profile/Cindy_Ten_Broeke)

Thomas,

My group has worked on aphids (and other insects) doing insecticide resistance testing for some 30 years now. You would think it was easy to put an aphid on a leaf disc embedded in agar and spray it but not so.

If we are using an established method then my control mortality issues come from poor handing. Aphids are very fragile and people not used to working with them are too rough. An experienced staff of mine will kill almost none when transferring but someone new may have >50% control mortality. With practice most get their control mortality to acceptable levels but a few don't. So my first advice is just practice transferring, it is not as easy as most think. Practice transferring may well fix your problem.

Secondly, if the method is not established then that can certainly lead to excessive control mortality.  For instance, my standard bioassay method for Aphis gossypii has a 24 h hold with aphids on a leaf disc embedded in agar. If you extend the post spray hold to 48 h control mortality is rising. Extend it to 72 hours and control mortality is not acceptable. The test is not viable beyond 48 h.

So basically my standard method for Aphis gossypii will not work newer products like spirotetramat and pymetrozine that take several days to work. Keeping control mortality low with long post spray holds is very difficult.

Thomas,

Sorry I almost forgot. These methods can be very species specific. For instance, I first tested Myzus persicae using a  method developed by one of the entomologists at my research institute. Then Aphis gossypii became an issue so I needed to test those as well. Of course I tried Aphis gossypii with the method developed for Myzus persicae. The result? I killed all of them, 100% control mortality. I had to develop a new method to suit.

Well I work with A. pisum nymphs and using diet instead of leaf (several differences), Main problems (in my case): temperature (Small changes reduce aphid viability) and change from entire-plant to diet (reducing viability with increasing aphid age).

Hope going better this time!

Hi, Thomas. I have done aphid bioassay before. I tested the mortality of green peach aphid Myzus persicae and cotton aphid Aphid gossypii with entomopathogenic fungal spores by potter spray tower. I used 1% agar with distilled water (heat and cool to set) in 5.5cm diameter Petri dishes, placing chemical-free capsicum leaf discs on agar (before set). then introduced 10 aphid adults with a fine brush to each dish and covered with a vented creamer cup and placed in 22oC incubator for 24 hours, then removed adults and left 10-15 juvenile aphids  for the bioassay. This way, you have the same age aphids for the test. Please be very careful, do not touch the juvenile before and during the bioassay, they are very fragile!  I had no problem in regard to their survival in the Control. Hope this can help you. Good luck. 

I think you should check other parameters that can kill your aphids. The three main causes could be:

1.- the aphids died drowned in water drops

2.- the aphids died because the plants were treated with some insecticide because even if the leaf discs are in bad conditions (partially dehydrated and yellow) the aphids will survive

3.- the aphids have a infection of entomopathogenic fungus. Maybe the high HR inside the petri dish force the infection of spores on the aphids cuticle and after the infection them die. You can maintain some days the aphids on a wet ambient and check for the formation of spores.

The other possibility could be the high temperature of the agar when you place the leaves on it. Maybe you are cooking the discs and them it became non suitable for aphids feeding.

Good luck with you assais

So this kind of bioassay seems difficult to implement and its issue depends on numerous factors.

I will try again taking your advices into account.

Thank you all for the time you spent providing large explanations!

What was the aim of this bioassay?

The aim of the bioassay was to test the toxicity of entomopathogenic fungi towards aphids, both with spores and metabolites secreted in a liquid medium. Toxicity tests on entire plants require a lot of time in searching aphids, which sometimes get out of the system. The experiment is also limited in time as the plant continues to grow and becomes too large. So I wanted to try in Petri dishes but I had some difficulties to implement the bioassay as I explained above.

In my opinion the bioassays with leaf discs are interesting for short duration assays because if there last for a long time the discs are finally affected and the result could be distorted. I think the bioassays with entire plants are much more reliable. You can isolate the plant inside a bag of non-woven tissue as I used  in my publication, but if the plant continues to growth untreated leaves and stem will be available for aphids.

The other possibility is to use clip-cages to maintain the aphids confined in a selected area of the entire plant. You can also change the selected area periodically to ensure the leave is in perfect conditions.

Article New sources of resistance to lettuce aphids in Lactuca spp

I thought that I could change the leaf in the Petri dish in order to extend the duration of the bioassay as I read in some articles, but this might have induced more stress in aphids than benefits.

Your opinion is similar to that of Grant Herron and I feel you are right. Bioassays in Petri dishes appear eventually to be suitable to compare the virulence of fungal strains, but I have to manage with entire plants if I want to assess lethal doses.

Your suggestion about clip-cages looks nice. It will greatly reduce searching time.

Thank you!

I don't know what is the duration of your bioassay but maybe you can carry it out using entire leaves inside Petri dishes. I performed assays to asses lethal doses with a chemical insecticide using entire leaves with the petiole put inside a eppendorf full of water. Do you need to make the hole on the lid the most little as you could insert the petiole and moreover you can fix the leave with parafilm to avoid some leaks. It is also interesting to put a paper disc on the bottom of the dish to avoid drops.

I think it could be a good solution for you.

I recorded aphid mortality during five days. I already used entire leaves inside Petri dishes but these were inserted into agar. Maybe an eppendrof full of water will be more convenient. I will try it !

I have evaluated many JH and anti Jh analogue on Aphis craccivora  long back 25 years ago. We have designed a very cage mde up of hard plastic about half inch dia. One side was blocked with fine brass net and other side open. The leaf srface was treated in situ and known number of aphids of particular age/ stage was put in cage. The open end of this small cage (height half inch) was clipped on treated surface. The opposite side of leaf was covered with a small circular transparent polyvinyl sheet allowing photosynthesis.The aphids were handled with fine brush. U can easily handle even first instar larvae. In this way topically treated aphid population can be also be released in such cage. There is no question of drying of leaves etc. as we grow potted plants and put in lab under controlled condition.

 Dear Mandela

Did you use leaf discs or intact leaf itself? Kindly tell in detail, I am also doing the same but not getting fruitful results. If possible, could you send 1-2 pics of this experiments.

thanks

We clipped small cages on leaves grown in pots, constantly supplying the food and nutrition. The aphids could not escapes from cage. The living and dead specimens counted after 24 h interval or as desired

Hello Chet,

I cut around the edges of the leaf using sterilised scissors to better fit the Petri dish. I have managed to find only one photo of several leafs set-up in this way for my experiment (find attached). They are uncovered for the photo but I normally stored them with a piece of tissue paper resting over the top.

Hope you have luck with this experiment. If you have any more questions you are welcome to contact me.

Mandela

anyone can expain how to conduct the bioassay on whitfly, thrips, aphid, leaf hopper