I would like to perform a field experiment testing the effect of beneficial endophytic bacteria on plants. I would like to deliver bacteria to the seeds in the form of powder inoculant, after mixing bacteria suspension with the carrier. I am not sure what materials are a suitable to use as a carrier; how I can prepare the carrier before bacteria can be mixed with it (grinding, sieving, sterilization, etc.) ; what volume ratio of carrier to bacteria suspension is optimal, what proportions of the inoculant vs..
You asked so many questions !! Anyway, The best method to prepare an in inoculant (either for free living or endophytic biofertilizers) is to use the peat moss material as a carrier for your inoculum. First, the peat moss is an acidic organic material, so you must neutralised this acidity via the application of calcium carbonate ~ 5% to adjust the pH to 7.0 +/- 0.2, then sieve the peat moss to pass through a sieve of 2 mm (eyes diameter). If you have activated charcoal, you can mix it with ~ 2-3 % (to increase the water holding capacity of the mixture). In the case you already have the carrier ready to be uploaded with your inoculum. Mix the carrier with the respected liquid inoculum by 60% of the determined water holding capacity -WHC ( the WHC of the carrier we are talking about is ~ 300 - 350 %). Now, to inoculate the plant seeds with the prepared inoculum, you must mix the seeds with a sticky material (e.g, liquid solution of Arabic gum ~10% to help the inoculum stick to the seeds, after adding the Arabic gum, spread the seeds on a clean plastic sheets in a shadow place to partially dry ). Finally, carefully mix the plant seeds with the prepared inoculum and then start cultivating your experiment. Regrading the shelf life of this inoculum, it could reach 3 months or little bit mor. You can keep the inoculum in perforated plastic bags each contains 1-2 Kg in a place away from the direct sun light and high temperature (20-25 oC)
Good luck
Thank you very much for the detailed guidance Abu-Bakr M. Gomaa! That is very helpful!
I am wondering also if there are any specifications on how much of the prepared inoculant should be mixed with e.g. 1kg of the seeds? Once I know how many seeds I will be planting, this would give me an idea of how much of the inoculating mixture I have to prepare.
I understand that the seeds can be coated with the sticky material e.g. day before planting, to let them dry down? But do you recommend to mix the sticky seeds with bacteria just before planting time, or can this be done also maybe a day earlier, so I can bring to the field one container of seeds already mixed with peat moss inoculant?
Thank you once more, I really appreciate all the advices from people with expertise in this area.
Thank you Prof. Hani Antoun!
This is interesting what you mentioned about producing freeze dried bacteria for inoculating the carrier, as this could be done in advance and allow me to spread the preparation of inoculant batches over longer period of time.
At Trent we have the equipment to freeze dry the liquid suspensions but I wonder how much of a dry bacteria pellet i would need to inoculate lets say 1 kg of peat moss/clay?
I need to prepare powder inoculant as the soybean seeds I am working with do not respond well on prolonged time of being submerged in bacteria liquid culture/control media - in case of some genotypes inoculation with liquid bacteria suspension drastically decreases germination rate.
You are welcome,
Regarding the proper quantity of the needed inoculum to be mixed with the seeds, it depends on the size of seeds; the small seed like clover, 1/2 kg of inoculum/ Kg seeds is enough (take into your consideration the moisture % of the perpared inoculum ~ 60%); for the big size seeds like soybean, fababean ....etc. the proportion is 1/1 (w/w). The point is the whole seeds should be well coated with the inoculum.
For the mixing process, it would be better to be done in a short period before cultivation, but it will make no great difference if it done 1 day before.
Best wishes
You may safely apply the method given in the reference with bacteria. Briefly: grow a lawn of bacteria/rhizobia on NA/YMA plate for 48-72 h; scrape bacterial growth and mix with 10g talc. For thorough mixing sieve it.
Singh PC, Nautiyal CS. A novel method to prepare concentrated conidial biomass formulation of Trichoderma harzianum for seed application.
J Appl Microbiol. 2012 Dec;113(6):1442-50. doi: 10.1111/j.1365-2672.2012.05426.x. Epub 2012 Sep 5.
Dear Anna, We generally select for heat stable ( sporebearing) microorganisms which we apply to the seed together with sterile 0.3% xanthan gum as a 'sticker'. After the first round assay in which we find out which microbes are effective by adding a n excess o f spores to the seed, we find out the lowest amount of cells or spores of the inoculant (cfu per seed or per g seed) is required for an effective plant response. We then determine for how long (days, months) the bacterial inoculant survives on the seed. If survival is poor you can use peat but will have to increase the titre of your inoculum.
In your case have you tested the viability of your culture after freeze drying? Do you use skim milk as a cryoprotectant durng freeze drying (because it may interfere with your plant assay). Please think of testing for viability after each step that you subject your culture to.
Best of luck
Chris
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