Trying to Enhance the Growth of Sweet Clover With Glomus I Cultures From Soil.

I was growing a natural nitrogen fixing fungi called Glomus I, for short. I took sterilized potting soil soaked it in water for a few days, and decanted the soil mix into Jugs of water and soil medium. Di-ammonium phosphate, sodium chloride, potassium chloride, and small amounts of Tween 20 were added to the mix.

The samples were filtered 5x to get rid of any impurities, which means soil and other stuff that could "interfere" with a pressurized sprayer for crops.

The fungi, when phosphorus, nitrogen, and potassium with a very small amount of sodium are present, convert NH4+ ions into NO2- ions, thus eventually forming NO3 ions. This is the nitrification process with fungi. Certain bacteria can do this, but these fungi are more efficient and probably modified to handle more salt.

Here are some requirements for this project to work correctly. Please see below for more information.

1. Special Premix Potting Soil. Amazon.
2. Tap water. H20.
3. Potassium chloride salt. KCl.
4. Diammonium Phosphate salt. (NH4)2HPO4.
5. Chicken bone broth includes protein and Sodium chloride. NaCl.
6. Tween 20 (Preservative at 0.1% v/v (Volume per volume.).
7. Bitterex powder. Denatonium benzoate.
8. 2 L beakers.
9. Cups graduated marking of 20 ml to 200 ml.
10. 2 L plastic bottles with Lids (Medium for fungi).
11. A dark room (Glomus I does not like light that much.)
12. Funnels and filter paper or paper towels can work too.
13. Wild sweet clover outside. A few centimeters in length for control and testing.
14. Pressurized 1 L spray bottle (Amazon has it for 40 dollars Canadian.)
15. Sodium sulfate crystals. Some carbon dioxide is trapped in the crystals, making the filtered solution slightly acidic due to carbonic acid (H2CO3).
16. Pop a popsicle stick to stir the soil with tap water.

I discovered this fungus by adding a very high salt medium, 5g of salt (Sodium chloride) in soil with 1000 ml of water. We had 500 grams of soil. I first thought the white hairs were mold, but they did not turn green, so it was not mold! It was a nitrogen-fixing fungus.

I allowed it to soak in soil and water for 2 weeks before doing anything. Decanting and filtering 5x was necessary.

I added to the soil about 3 grams of diammonium phosphate, 1 gram of potassium chloride, and 8 grams of chicken broth powder. The salt (NaCl was 80 mg per L) and the potassium chloride was 1000 mg per L. Tween 20, around 1 ml max was added to the soil with tap water.

2 grams of bitterex is then added to the soil. This is allowed to sit for 2 weeks.

A clean jug that could hold 1.6 L was used with clamps and rods to hold the funnel. A cotton paper towel, one piece, was used to filter the sample once.

I had 4 extra 1.6 L bottles or jugs with caps I recycled cranberry juice for this (optional). The soil and water (most of the soil was added to the funnel once, and the sample was then filtered super finely with 5 filter papers (cotton towels work well for this).

Once filtered five times, the samples are tested with NO2, NO3 strip papers. As the picture above suggest it NO2 was 250 ppm and N03 was 50 ppm. Quite high eventually most of the NO2 converts by exposure to air to NO3.

N02- +1/2O2 >>>>> NO3-.

We measure out 200 ml of sample and add 800 ml of water to dilute it by 1:4. We should have a very slightly yellow color due to the Tween 20.

We have taken the liquid and tested the pH because a small amount of sodium sulfate trapped some carbon dioxide (CO2), we have a slightly acidic medium for the water samples with fungi. Ph was tested and measured at 6.

We let the solution of soil sit in a 2 L beaker for 2 weeks then we decant into a 2 L jug made of plastic with cheese cloth or filter paper. We do this five times and add tween 20 about 1 ml of it to 1 L sample.

We take 200 ml of sample and add 800 ml of water to give a 1 L solution with low concentrations of salt and bitterex. The bitterex repels animals from eating the crops.

We spray the pressurized material (Fungi plus salts and Tween 20) on the roots of the plant at night to prevent phototoxic effects on sweet clover that may harm it. We do this once a day to see what will happen to the clover. The fungi will eventually convert NH3 ions into NO3 ions, but is this sufficient to increase growth alone?

I had some weeds that I tried spraying 5 g of Potassium Nitrate in 500 ml, which gave 10 grams of KNO3 in water. It ended up wilting and damaging the plants when exposed to 100 ml for 3 days. There is a limit of how much diluted salt you can add to plants without damaging or killing the plant.

3 grams (Diammonium Phosphate) + 80 mg Sodium chloride + 1 g Potassium chloride per Liter of sample. This equals 4080 mg or 4.08 g salts divided by 1/5 (200 ml/1000 ml spray sample. This gives a diluted salt sample of 816 mg of salt total or 0.816 g salt.

Tween 20 at 1 ml per 1000 ml was also added. The fungi were in a very low concentration when diluted by 1:5, which may be an issue in future tests.

I am not sure of the final results. Did the fungi convert Ammonium ions into NO3 ions? How many ammonium ions were present in the spray total? 3 grams per L of di-ammonium phosphate times 0.20 (200 ml/1000 ml sample gives 0.6 grams of Salt (diammonium phosphate).

(NH4)2HPO4 + H20 >>>>> 2NH4(aq) + HPO4-1 (aq).

2:1 ratio, so 1.2 grams NH4 present with some sodium, phosphate, potassium, and nitrogen (NH4 and NO3) present should cause some increase in the roots and increase in sweet clover growth.

A total of 20 ml of spray was used on the sweet clover. Per day. 1.2 grams NH4+ times 20 ml/1000 ml equals

0.024 grams or 24 mg of Ammonium ions.

I think this was too weak to grow the clover directly; however, this amount of ammonia and sunlight could have enhanced the growth of sweet clover. Certain plants use this fungus naturally, including clover, hot peppers, tomatoes, potatoes, and many other plants.

The plant sprayed was 5 cm it went to 20 cm in 1 week, the control went to 5 cm to 10 cm in length. Again I dont know if this the direct effect.

I am not sure if that tiny amount of ammonium ions had a direct effect on the sweet clover, but researchers use this fungus on plants with great success. [1] Scaling the solution of Glomus I up is difficult for a small lab like mine, but I wanted to try this for myself.

Other researchers have inoculated a similar fungus into grapes, and the results were amazing. Please see [2] for more details. The grape plant grew faster than normal and was healthier with the Glomi culture and a different culture, but this explains if done correctly, it can have good results.

If we can find an alternative to Bitterex or maybe buy some cultures of Glomus I cheaply, I could test this fungus on different controls in the future! For now this is an interesting project. The thing is Bitterex should not be added to crops you may eat (Potatoes, Tomatoes, Grapes, Peppers, etc).

This is because it would make the crop taste very horrible and bitter, so we need an alternative. I haven't found one yet. Maybe coffee in small amounts may make it undesirable for the animals and break down naturally?

https://myco-grape.krs.hr/en/2021/09/ [1].

https://www.sciencedirect.com/science/article/abs/pii/S2589014X25001422 [2].