Breeding Mycorrhizae

[The Supreme Weeder]

Ok, so the organic section looks in need of more topics. It would sure be easier if rather than trawling the internet for information, we can find what we need here. With that in mind I'll be trying to collate resources I've found on t'interweb and bring them here. I can't state to its veracity with any degree of certainty but I guess it'll give people a starting point for reading up and trialling different ideas.

Today I'll start with breeding mycorrhizae. I've found a few different sources for information. Some are contradictory, but if this interests you it might be worth a look.

Taken from a post on thcfarmer by Samoan who got this from elsewhere...

I found that it is a lot of info on the web on how to use mycorrhizae, but not on how to breed it ourselves. I guess this is because many companies need to make money from a product that is extremely easy to breed, and needed for increased growth. Never buy mycorrhizae again. $60 a bottle is expensive so lets do it.

1. Purchase whatever Mycorrhizae brand that you like best. Make sure you get the good stuff for this is what you'll be breeding from here on out.



2. Get any bucket or tub to place some soil in. I use a 30 gallon tote because I will be using it throughout my grow and bloom cycle. I also have a few extra totes to increase my fungi yield.



3. Place the soil in the tote or totes you are using and make sure it is moist but not too damp. Water it as if your germinating seedlings. Use organic soil because chemicals tend to kill fungi, so this goes the same for watering.


4. Add oatmeal to the soil mix for the fungi to feed off of and mix it through the soil evenly.


5. Add sugars to your soil mix for the fungi to feed off of also. It isn't any sense in adding carboload or bloom boosters to the mix because it is expensive. The fungi feed off plain sugars such as grain sugars and molasses. Make sure its mixed throughout the soil.


6. Now for the water: water the mix throughout but not too much. Do not use city water because the chlorine products in it will kill off fungi. Instead use rain water or melted snow to water with. But do not use snow if your gonna use the soil, because melted snow slows plant growth. Distilled water is great but gets costly.


7. Now we can add the mycorrhizae that we spent so much money on to the mix. Take powder mycorrhizae and sprinkle some over the top or take liquid mycorrhizae and spray it over the top of the soil mix with a spray bottle.


8. Place a heating pad under the soil container and keep it on high if the tote is large and medium if the tote is small. Theres no need to buy a very expensive heat mat.


9. Make sure to cover the container because fungi need a dark place to grow. Lights will kill them if kept on them for long periods.

Wait a few days and you will see the fungi colonizing. It will look white and fuzzy and you will think mold Nooooooooooo, but this is what you are after. The more fungi you add to the soil the faster it will colonize, but much isn't needed at one time. They will pretty much multiply from any point.



Keep feeding them sugar and oatmeal if you want. Some individuals plant a plant to let the fungi sync with then very little feeding is needed. It's great to throw a store bought potatoe in the soil for the fungi to work with. Also add some bennies to the soil over time. Give the fungi a few days to grow and colonize before adding bacteria, for bacteria eats fungi in its early stage.


When you want to start another tub of mycorrhizae fungi just take some fungi or soil and transfer it to a new soil container, and allow it to multiply.

Try not to break up the colony as much as possible but if so, dont worry they will do there thing in no time. I mix my fungi throughout the soil so I can pull out one big cake to add to my dwc system.

If watering try not to damage the colony. Try to gently spray unchlorinated water to the top of the soil. If adding more fungi: do the same.

Now never run out of your favorite brand again!

[The Supreme Weeder]

One issue I see with this method is that I believe Mycorrhizae doesnt survive long without being attached to roots. The Rodale institute has a method using Bahia grass, and Ive seen another method using leeks and replanting them after taking some of the root mass to inoculte another plant.

[The Supreme Weeder]

Part 1 of a Rodale institute piece on mycorrhizae

HOW TO INNOCULATE ARBUSCULAR MYCORRHIZAL FUNGI ON THE FARM, PART 1
by Molly Lohman, Christine Ziegler-Ulsh and David Douds

Dec 08, 2010

CATEGORIES
Soil Health
CORE CONCEPTS
Soil Health


For the last 8 years, The Rodale Institute has partnered with the USDA-ARS to pioneer an On-Farm AM Fungus Inoculum Production System; a system which would make the economic and environmental benefits of mycorrhizae available to more farmers. Over the years, our trials have focused on developing a low-cost procedure and identifying features to maximize propagule production. Here we summarize what we’ve learned from those years and share everything you need to know to start your own on-farm system.

AM fungi basics
Arbuscular mycorrhizal (AM) fungi are the most important mycorrhizae in agricultural ecosystems due to the fact that they colonize the majority of crop plants. Known as “obligate symbionts,” AM fungi must associate with plant roots to survive; it is this association that begins a mutually beneficial between the fungi and the plant. In return for sugars from a plant, the long, thread-like structures of fungi, the hyphae, act as an extension of a plant’s root system and increase a plant’s access to immobile nutrients including phosphorus (P), zinc and copper. While plant root hairs extend 1-2 mm into the soil, the mycorrhiza’s hyphae explore a greater volume of soil and can extend up to 15 cm from the plant’s roots. The relationship between mycorrhizae and crop plants often enhances plant growth and yield, but even when no growth enhancement occurs, the majority of P uptake can be attributed to mycorrhizae. Mycorrhizae have also been credited with increasing a plant’s disease resistance, improving a plant’s ability to grow under drought conditions, and improving soil structure.

While some standard agricultural practices including frequent tillage and heavy phosphorus fertilization negatively impact mycorrhizae, many sustainable farming practices can be used to bolster native mycorrhizal fungus populations. Even soils that have been intensively managed for an extended period of time contain populations of mycorrhizae that can be augmented by using cover crops, developing a diverse crop rotation, and growing crops that form a symbiosis with AM fungi.

AM fungi inoculum
Inoculation with mycorrhizal fungi offers another opportunity to take advantage of the benefits of mycorrhizae. Mycorrhizal spores, pieces of colonized crop roots, and viable mycorrhizal hyphae function as active propagules of AM fungi that can be used as inoculum to “infect” other plants with AM fungi.

Under certain circumstances, using inoculum may be preferable to using management practices to bolster AM fungi populations. Previous fungicide use and other extreme cases may require inoculation to reintroduce AM fungi to the severely degraded soil microbial community. In less severe circumstances, inoculum may be used to produce pre-colonized seedlings that can take advantage of mycorrhizae’s benefits from their first day in the field. Past research shows that this competitive advantage can have a positive impact on yields. In a 2008 study, strawberry plants inoculated with AM fungi prior to outplanting produced 17% more fruit than uninoculated controls (Douds et al. 2008). One study with peppers observed a 14 to 23% yield increase from inoculated plants grown in compost-amended soil and a 34% increase one year from inoculated plants treated with chemical fertilizers (Douds and Reider 2003). Yield increases have also been observed for a variety of other crops including tomatoes, potatoes, onion, peanuts, watermelon, garlic and celery.

While commercially produced inoculum is available, it comes at a cost to farmers. The price of commercial inoculum reflects the costs of current production methods including greenhouse or lab space as well as the labor and time associated with isolating AM fungi from the original medium and/or mixing the spores with a carrier substrate. These costs, as well as shipping and handling, are all passed on to the farmer (Douds 2010).

On-farm production of inoculum
The goal of our research was to develop an on-farm inoculum production system that generated a potent, effective, species rich inoculum that was inexpensive to produce. By avoiding the associated costs of commercially produced inoculum, on-farm production makes the economic and environmental benefits of AM fungi available to a larger number of farmers.

Although significant yield increases vary by year, crop, and cultivar, our system was designed with routine use in mind. With yearly use, farmers will be poised to profit when mycorrhizae ease the conditions that depress yields, but in years when inoculum does not impact yields, the inexpensive system will not be an economic burden. We are targeting vegetable farmers who produce their own seedlings on site for later outplanting to the field, so adapting their current systems to produce colonized seedlings is simple. Although large scale, direct application of inoculum to fields is possible, the return on investment for crops such as field corn and soybean is too low to make inoculum application economically attractive.

The on-farm system starts by planting “host plant” seedlings into black plastic bags filled with a mix of compost, vermiculite and local field soil. AM fungi present in the field soil colonize the root of the host plants and over the growing season, the mycorrhizae proliferate as the host plants grow. When the host is frost killed, the mycorrhizae will naturally overwinter in the compost and vermiculite mixture, and the inoculum will be ready for use in the spring. Most aspects of the system, from the type of compost used to the dilution rate, have been examined through experimentation to pinpoint the best methods to maximize production. The result is a system that has successfully propagated all AM fungi tested and can produce hundreds of propagules per cubic centimeter. In one trial, 465 propagules cm-3 were produced, a 7000 fold increase relative to the concentration in the field soil that was initially added to the bag.

Here we walk through the process step by step and analyze the details that will make your on-farm system a success. Citations are provided to direct you to articles for further reading (see the end of this article for all reference information).

Choosing a host plant
The most important factor in choosing a host plant is selecting a plant that supports mycorrhiza growth. Crops such as spinach, sugar beet, lupine and members of the mustard family do not form a symbiosis with AM fungi. A dependable host for the majority of AM fungi species, bahiagrass (Paspalum notatum Flugge) has been used extensively. Additionally, to prevent the spread of pathogens, the host plant should be from a different family than the inoculated crop. Due to the fact that the inoculum system targets vegetable producers, bahiagrass, a member of the grass family, is an ideal general host. Finally, as a tropical plant, bahiagrass will be frost killed and will not become a weed pest in the field.

As of yet, bahiagrass seedlings are not commercially available. In order to use bahiagrass as a host plant, farmers must establish their own seedlings. We simply germinate bahiagrass seeds in either vermiculite or seed starter and transplant the seedlings into conical plastic pots filled with a sand and soil mixture. Taller than the typical greenhouse flat, these conical pots produce seedlings with a long root system. When outplanted into the bags of diluted compost, the long root ball will come into contact with propagules deep in the bag more quickly than would seedlings produced in shallower pots. The sand and soil mixture we use in the conical pots is a 1:3 soil:sand (volume basis) mix that uses sterilized field soil and coarse swimming pool sand. If the bahiagrass seedlings are grown in a typical greenhouse potting medium, we have found that they become iron deficient.

The process of germinating bahiagrass and establishing seedlings is usually started in the greenhouse four months before the last frost date so the plants can be transplanted as soon as possible after the frost. (For a printable timeline and information on where find these materials see our Quick and Easy Guide to On-Farm Mycorrhizae Inoculum Production.)

Finding the right mix of media
When the host seedlings are ready to be planted outside, it is important to transplant them into an appropriate medium. The nutrient availability of the medium has a significant impact on the number of mycorrhizal fungus propagules produced. Plants grown in high nutrient situations, especially high phosphorus, can limit the colonization of their roots and therefore, the proliferation of the fungus. The possibility of using pure soil, pure compost or soil diluted with vermiculite have all been dismissed due to the low colonization and/or low AM fungus spore production observed using these media.

Select dilutions of compost, however, have been successful in producing mycorrhiza inoculum. As a nutrient dense medium, compost supplies all the nutrients needed for bahiagrass growth as well as a broad array of microbes that benefit soil heath and suppress plant disease. However, due to compost’s high P concentration, it must be diluted with a nutrient poor substrate such as vermiculite, perlite or peat. Another benefit of this dilution is the resulting light weight medium that can be easily recovered and utilized.

Our research shows that the optimal compost dilution rate varies with the species of AM fungus and the type of compost used. Experiments conducted in 2003 and 2004 suggested general dilution levels that supported overall mycorrhiza growth better than others (Douds et al., 2008). Composts that were high in N, low in P and have moderate K levels (a yard clippings compost and a dairy manure and leaf compost in this study) had a high probability of success at dilutions of 1:2 to 1:4 [compost:diluent] based on volume. Composts that were high in P, low in N and had moderately high K levels (a controlled microbial compost) were most successful at dilutions of 1:19 or 1:49. In general, we have regular success with dilution ratios between 1:3 and 1:9 and we will typically use a 1:4 volume based dilution with any yard clippings compost produced by municipal composting facilities.

We have also experimented with the type of diluent and quantified the effect of perlite, vermiculite and peat based horticultural potting media on inoculum production (Douds et al., 2010). Inoculum can be successfully produced with all of the media amendments; spore production by all AM fungi studied was not significantly different among mixes using different diluents. However, a most probable number bioassay, a technique used to determine the density of infectious propagules, showed that vermiculite did tend to produce more overall propagules than peat based potting media. We hypothesize that the laminar sheets of the vermiculite may be an ideal environment for the growth and persistence of mycorrhizal hyphae. The similar spore populations and root colonization between the three diluents support this theory.

This dilute compost mix is used to fill seven gallon plastic bags three-quarters full. Field soil will be mixed into each bag as an inoculum starter, as discussed below, and the bahiagrass seedlings will be planted into this mix. The total amount of media needed depends on the number of plants that will be inoculated the following spring. Our printable Quick and Easy Guide to On-Farm Mycorrhizae Inoculum Production has instructions for making 200 or 400 ft3 of inoculated greenhouse potting medium, which can be used as a baseline to calculate the raw material needs for inoculum production of all scales.

The inoculum starter
While we most often propagate isolates of a specific AM fungus species for research, one of our first trials highlighted the possibility of using the on-farm method to propagate indigenous mycorrhizal fungi. In this early trial, contaminant fungi were found in what should have been a single species inoculum. These contaminant fungi most likely came from soil that was mixed into the compost during turning the previous year. This inadvertent propagation of native AM fungi illustrated that the on-farm method could be used to create an inoculum that was not only cheaper than single-species commercial inoculum, but also had the added benefit of containing a diverse group of locally-adapted mycorrhizal fungi that could be used to boost a farm’s native populations (Douds et al. 2005).

Research has shown that using a multi-species inoculum with local isolates is important for a number of reasons. Many AM fungi species are unique in characteristics such as their patterns of colonization, spatial exploration of the soil for P, ability to produce a growth response between plant species, and glomalin production. Typically absent from commercial inoculants, but present in healthy soil, Gigaspora spp. are significant producers of glomalin, which aids in soil aggregation. Additionally, some research suggests that the indigenous AM fungi are more effective in promoting plant growth in their local soil than introduced species.

To obtain a locally adapted and taxonomically diverse inoculum, field soil can be mixed into the dilute compost mix as a source of native AM fungi (Douds et al. 2010). To ensure a diverse sampling of mycorrhizae, soil should be collected from a natural area of the farm such as a wood lot or fence row. Collecting soil from these areas is preferable since it should contain a diverse and healthy mycorrhizal fungus population that has not been impacted by agricultural practices. Collecting soil from a production field is also an option, but the field should not have been used in the past two years to grow the crop that will be inoculated. This precaution is to avoid introducing pathogens to the inoculum. Additionally, due to the fact that mycorrhizal fungi can be distributed irregularly, pooling four to five samples is suggested. Most mycorrhizae are found in the top 10 cm of soil, so samples do not need to include soil deeper than this layer.

Once a pooled sample has been collected, sieve out any rocks or roots. Add 100 cm3 of soil from the pooled sample to each seven-gallon bag filled with medium, mix well, and transplant four to five bahiagrass seedlings into each bag. This step should be performed as soon as possible after the last frost in order to maximize the host plant’s growing time.

Harvesting inoculum
Throughout the season the bags only require 5 to 10 minutes of work per week; the bags should be watered as necessary and weeded to avoid introducing weed seed into the inoculum. As the host plant grows, the mycorrhizae will proliferate. At the end of the season, senescence of the host will prompt the mycorrhizae to sporulate. As occurs naturally in the field, the spores will overwinter in the compost and vermiculite mixture and the inoculum will be ready for use in the spring.

Spores, pieces of colonized roots and viable mycorrhizal hyphae all function as infectious propagules, and all three are produced using the on-farm system. Hyphae and colonized roots will be abundant from the previous season’s growth, while spore density can vary based on the species of AM fungi and how close the medium came to an ideal dilution. In past studies, the typical 1:4 dilution of yard clippings compost:vermiculite produced an average of 30 spores cm-3 (Douds et al. 2006). Combined with the number of colonized root pieces and hyphae, the on-farm system produces hundreds of propagules cm-3 using the 1:4 dilution; a quantity that exceeds the target “mass production” inoculum density of 80-100 propagules cm-3 (Douds et al. 2005).

While the spores and hyphae are mixed in with the compost and vermiculite medium, the host plant’s roots must be chopped up in order to take advantage of the mycorrhizal vesicles inside. Produced by most AM fungi, vesicles are spore-like, globular organs that contain energy reserves. In the spring, the mycorrhiza can regrow from these vesicles. Using the on-farm system, bahiagrass typically have 70-80% of their root length colonized by mycorrhizal fungi. Therefore, even small root pieces contain AM fungi and can be mixed into the medium to increase the number of infectious propagules.

We have found that the best way to harvest the spores and viable hyphae part of the inoculum is to cut off dead leaves, remove the root ball from the bags, and shake off the medium into a large bin. The root system can then be cut into pieces with scissors and mixed into the inoculum. The inoculum is now ready to be mixed with potting media in the greenhouse.

Producing colonized seedlings for outplanting
When deciding how much inoculum should be added to potting mix, the potency of the inoculum and the volume of the cells in which seedlings will be grown needs to be considered. When seven cooperating farms used the on-farm system to produce inoculum for three years, the average production was 82 ± 20 propagules cm-3. However, in a 2006 study, we observed an average of 503 and 240 propagules cm-3 for the 1:4 and 1:9 dilutions of yard clippings compost and vermiculite, respectively (Douds et al. 2006). In either case, only several cubic centimeters would be needed per cell to supply the target 100-200 propagules per plant.

Although in theory only a small amount of inoculum is needed in the potting soil, we suggest using a lower dilution rate than necessary due to the difficulty of obtaining a completely heterogeneous mixture. One study tested a 1:9 and 1:19 inoculum:potting media dilution (volume basis) using an inoculum with 120 propagules cm-3 (Douds 2009). Eight different cultivars of both peppers and tomatoes were planted into the mix and grown in 50 cell flats (70 cm3 per cell). After four weeks the tomatoes averaged 30.5% and 12.9% root length colonization for the 1:9 and 1:19 dilutions, respectively. AM fungi colonization of the peppers averaged 14.8% and 8.0%. All of these degrees of colonization are sufficient to impact growth or yield. The cell size will ultimately determine which dilution should be used. To ensure sufficient propagules per cell, a 1:9 dilution should be used in cells that are 50 cm3 or smaller, a 1:19 dilution can be used in larger cells.

Adapting greenhouse practices
Given the symbiosis’ sensitivity to phosphorus levels, it is important to ensure that the greenhouse fertilization regime is modified for the inoculated seedlings. Phosphorus availability controls the level of root colonization directly by impacting AM fungus hyphae growth and indirectly through the effect of plant tissue P concentration on exudation of signalling molecules from roots. Research clearly shows an inverse relationship between P availability and mycorrhizal fungus colonization. The challenge has been to identify a greenhouse potting mix and fertilization regime that will produce seedlings with satisfactory levels of colonization that are also of a size competitive with plants grown in high P conditions.

For conventional growers, we suggest a standard potting mix supplemented with a balanced nutrient solution low in P (Douds 2009). A phosphorus addition of 3 ppm or less is suggested no more than three times a week. Fertilization three times per week with solutions containing 31 ppm P resulted in effectively no colonization by AM fungi, yet gave little or no increase in shoot weight compared to pepper and tomato seedlings receiving significantly less P.

The situation for organic growers is more challenging than for conventional growers due to the difficulty in precisely controlling P levels. This part of the project is still a work in progress. For now, we suggest using an unaltered potting mix for organic growers. In one study, we used an organically approved potting mix (NP mix from Living Acres, New Sharon, ME) with an NPK analysis of 0.4-0.5-0.3. The manufacturer recommended no nutrient addition to seedlings grown in this medium in the greenhouse. The unamended medium was compared to treatments including the medium with vermiculite and supplemental fertilization. The colonization of the plants grown in the unamended medium was better than in some conventionally managed greenhouses, so we recommend the unamended potting mix as a place to start. If your potting medium requires additional fertilization, use a low P solution such as fish hydrolysate.

For organic growers that mix their own potting medium, it is important to consider the nutrient availability of the mixture. If the medium recipe includes compost, it may be important to cut back on the compost to limit P availability. The complication here, of course, is the concomitant reduction in availability of other nutrients as well. This season’s greenhouse trial with leeks, peppers, and tomatoes included 10% compost and 50% compost treatments. The media treatments was amended with two fertilization regimes, once a week or three times a week with a hydrolyzed fish fertilizer (12-0.5-1). The low colonization observed in all of the organic treatments illustrated that P levels were too high, even in the 10% compost medium with infrequent fertilization. However, in another trial, the 10% compost-based potting mix produced corn seedlings with 19% root length colonization after 19 days. This was considered a satisfactory level of colonization. While these different colonization levels illustrate the varying responses of different crops, it also illustrates the need to continue searching for an ideal farm-produced potting medium recipe that has the ideal nutrient levels to promote colonization.

Getting your system going
Our research has looked at inoculum’s effect on a variety of crops in organic and conventional systems and in high P soils. In each situation, yield increases due to inoculation can be variable from year to year, and even between cultivars. However, when the symbiosis offsets conditions that depress yields, the resulting gain can be significant.

On-farm inoculum production makes it easy to harness the benefits of AM fungi. Our low-cost on-farm system is easy to integrate into any farming system due to the low hands-on time required. Simply plan ahead to make your on-farm system a success; remember, a full season is required to produce the inoculum.

REFERENCES
Douds, D. D., and C. Reider. 2003. Inoculation with mycorrhizal fungi increases the yield of green peppers in a high P soil. Biological Agriculture and Horticulture 21: 91–102.
Douds, Jr., D. D., G. Nagahashi, P. E. Pfeffer, W. M. Kayser, and C. Reider. 2005. On-farm production and utilization of mycorrhizal fungus inoculum. Canadian Journal of Plant Science 85: 15–21
Douds, Jr., D. D., G. Nagahashi, P. E. Pfeffer, C. Reider, and W. M. Kayser. 2006. On-farm production of AM fungus inoculum in mixtures of compost and vermiculite. Bioresource Technology 97: 809–818.
Douds, D. D., G. Nagahashi, C. Reider, and P. Hepperly. 2008. Choosing a mixture ratio for the on-farm production of AM fungus inoculum in mixtures of compost and vermiculite. Compost Science and Utilization 16: 52–60.
Douds, D. D., G. Nagahashi, J. E. Shenk, and K. Demchack. 2008. Inoculation of strawberries with AM fungi produced on-farm increased yield. Biological Agriculture and Horticulture 26: 209–219.
Douds, D. D., G. Nagahashi, and P. R. Hepperly. 2010. On-farm production of inoculum of indigenous arbuscular mycorrhizal fungi and assessment of diluents of compost for inoculum production. Bioresource Technology 101: 2326–2330.
Douds, D. D. and C. Ziegler. 2003. Improve your soil, increase your yields, and reduce your expenses with AM fungi. The Rodale Institute

[The Supreme Weeder]

HOW TO INNOCULATE ARBUSCULAR MYCORRHIZAL FUNGI ON THE FARM, PART 2
by David Douds

Feb 22, 2017

Arbuscular mycorrhizal [AM] fungi are naturally-occurring fungi that are found in most, if not all, soils. They colonize roots of most crop plants to form a mutualistic (beneficial to both partners) symbiosis. Notable among crop plants that do not form this symbiosis are members of the mustard family (eg. rapeseed, broccoli), spinach, and sugar beet. The AM fungi assist plants in the uptake of mineral nutrients from the soil and enhance resistance to disease and drought. The primary benefit to plants is the enhanced uptake of mineral nutrients such as phosphorus, zinc, and copper that are “immobile” in the soil solution; that is, they do not dissolve well in the water in the soil and hence do not move toward roots as plants withdraw water from the soil. In the absence of mycorrhizal fungi, these nutrients can be taken up from a distance of only 1-2 mm away from the root. The thread-like strands (“hyphae”) of the AM fungi that are external to the root extend 8 cm or more away from the root, drastically increasing the volume of soil from which these immobile nutrients can be extracted.

Given these benefits, better utilization of the AM symbiosis is attractive to farmers, especially organic farmers who seek to minimize off-farm inputs. Farmers have two options for this: 1) adopt farm management practices that increase the functioning of their native populations of AM fungi or 2) inoculate with AM fungi. Economic and practical constraints make option 1 the likely route for row crop farmers. Vegetable farmers who grow their own seedlings for outplanting to the field can efficiently and economically use inocula of AM fungi.

Inoculum of AM fungi are commercially available, but farmers can grow inoculum of the AM fungi indigenous to their farms using a simple and reliable method developed through a collaboration by the USDA and researchers of the Rodale Institute.

The Method
General principles
AM fungi are “obligate symbionts,” meaning they need to colonize a host plant to grow and complete their life cycles. This means that, to produce inoculum of these fungi, one needs to supply access to plants, in particular to living plant roots. Here, we will use bahiagrass (Paspalum notatum) seedlings. Bahiagrass is a good host for AM fungi and is not winter hardy in temperate climates. Therefore it will not become a weed pest in the inoculum. Other Graminaceous species should work as host plant, but one will want to be sure to prohibit seed production.
One therefore needs to supply the things the plant will need to grow: nutrients, water, and sunlight. Nutrients are supplied via compost. The media in which the plants are grown, and therefore in which the inoculum is produced, is a mixture of 1:4 (volume basis) of compost and vermiculite. Locate the inoculum production system in full sunlight and water as needed.
Lastly, one needs a “starter inoculum.” The original design of this method (see resource no. 1, below) entailed initial production of the bahiagrass seedlings in potting mixture inoculated with individual species of AM fungi. The hope was that after demonstration of the success of the method, commercial sources would make available the “pre-colonized” bahiagrass plants. This did not happen, thus the need to explore another source of starter inoculum.
Starter inoculum
Using field soil from your farm as starter inoculum has two advantages: it is free and the AM fungus community already in your field soil is adapted to your climate and soil conditions. Two ways to incorporate field soil into the inoculum production procedure are 1) to add it to the potting mix in which the bahiagrass seedlings are grown prior to transplant into the inoculum production system or 2) to mix the soil into the compost and vermiculite mixture. These were both studied in earlier work (see resource no. 2, below). Option 1 is unattractive because it requires collecting soil when fields may be frozen. The current project was initiated because inoculum production with Option 2 has been inconsistent.

Overview of the on-farm inoculum production procedure
The target date for the setup of the inoculum production system is just after the threat of spring frost has passed in your area (see Figure 1). Begin producing your bahiagrass seedlings three months prior to that. They take one week or more to germinate and are initially slow growers. Then, after the threat of frost has passed, fill seven-gallon (26.5 L) plastic bags (“Grow Bags,” Sunleaves Garden Products, Bloomington, IN 47404) two-thirds full with a 1:4 (v/v) mixture of screened (6.4 mm mesh) compost and vermiculite, respectively. Next, mix 200 cm3 (approx. one cup) of sieved field soil (more on that below). Transplant your bahiagrass seedlings into the bags, 5 per bag. Weed and water the bags as needed throughout the growing season (see Figure 2). There will likely be some weed seeds germinating from the compost, and the goal is to remove these winter-hardy plants from the bags. The bahiagrass is then winter-killed, and the bags overwinter in place, outdoors. The following spring, the compost and vermiculite mixtures from bags are harvested and mixed into horticultural potting media (1:9 inoculum:media, volume basis) for use in the greenhouse to produce mycorrhizal vegetable seedlings.

This Experiment
There is no easy way to check the level of the population of AM fungi in the soil you have chosen to use as starter inoculum, especially since you are collecting it just prior to use. Therefore, the objective of this experiment was to test soils from a variety of farm habitats as starter inoculum for the on-farm production of inoculum of AM fungi with the goal of making a general recommendation of where to collect soil on your farm.

Soil was collected on June 15, 2016 from the following fields at the Rodale Institute:
1. pasture
2. wooded fence row
3. field last planted to soybean, followed by a winter wheat cover crop
4. field last planted to wheat, followed by over wintering hairy vetch
5. field last planted to corn, followed by a rye cover crop

8‑10 samples were collected per site, each to a depth of 6 inches, which were then pooled. The soils were sieved to pass a 2 mm mesh, and 200 cm3 (approx. one cup) was mixed into the top 1/4 or so of the compost vermiculite mixture of a bag. Five bags were prepared for each soil source. In addition, one set of 5 bags received no soil (as a control), and another set of 20 bags received bahiagrass seedlings pre-colonized by one of four AM fungi (5 bags per AM fungus). Each bag received 5 bahiagrass plants.

Data collection
Inoculum production bags were sampled on November 15, 2016 after the bahiagrass had winter-killed. Roots were analyzed for percentage root length colonized by AM fungi. Samples of the compost and vermiculite mixture from three bags of each treatment/ soil source, as well as three samples of a mixture of the 4 inoculated AM fungus species, were utilized to conduct Most Probable Number [MPN] bioassays to determine the number of propagules of AM fungi per cm3 of mixture.

Briefly, we conducted MPN assays by diluting one volume of inoculum with 9 equal volumes of a sterilized soil and sand based potting mixture to create a one-tenth dilution of the original. This was used to fill 5 small plant pots. A subsample of the one-tenth dilution mixture was again mixed with 9 volumes of sterilized mix to produce a one-hundredth dilution, and on through the one-ten-thousandth dilution, the goal being to dilute the original inoculum “to extinction” (that is, until AM fungi are no longer present).

Each pot then received a bahiagrass seedling. Plants grew for 4 weeks, after which the entire root systems of each pot were collected and examined for colonization by AM fungi. They were scored simply plus / minus for presence or absence of colonization. Statistical tables were consulted to yield the most probable number of propagules of AM-fungi-per-cm3 of inoculum. Since results of MPN assays can show much variability from one replicate to another, the percentage root length colonized by AM fungi of roots in the one-hundredth dilution also was determined as a measure of the amount of viable AM fungi in that dilution. MPN bioassays also were conducted on the original soil samples at the time of collection in June.

Results
Farm habitat from which soil was collected for use as starter inoculum had a significant effect upon inoculum production. Soil from the soybean/wheat and corn/rye plots had significantly greater density of AM fungus propagules at the time of collection (June, 2016) than soil from the wooded fence row (Figure 3), the other sites being intermediate. Colonization of bahiagrass roots within the inoculum production bags, sampled at the end of the growing season, was not significantly different among treatments, and ranged from 39% of root length in the non-inoculated controls to 54% in the soybean/wheat treatment. This indicated contamination of the control bags via either roots or soil mixed into the compost and/or colonization of the bahiagrass up from the soil, through the weed barrier fabric upon which the bags rested (see Figure 2).

Quantifying the inoculum produced at the end of the growing season via the MPN bioassays produced similar results whether propagules or colonization of roots were measured (Figure 4). The best inoculum production occurred in the bags that received the pre-colonized seedlings (the “standard method”) or soil from the soybean/wheat or corn/rye fields. Soils from the pasture, wooded fence row, and the wheat/vetch plots produced inoculum levels that were not statistically different from those of the control.

Conclusion
Though this, like all field experiments, should be repeated, the results indicate that soil collected in late spring/early summer from fields used to grow a row crop the previous year, followed by an overwintering cover crop of a cereal grain, serve as the best starter inoculum for the on-farm production of AM fungi.


Figure 1. On-farm production of AM fungus inoculum. Follow the steps from 1 (sowing bahiagrass seeds 3 months prior to the average date of the last frost) through 8 (use of the inoculum the subsequent spring). Parenthetical suggestions, in green, are for locations with short growing seasons to ensure enough time for bahiagrass growth and AM fungus proliferation.

Figure 2. On-farm inoculum production bags at the Rodale Institute showing growth of bahiagrass toward the end of the growing season. The bags rest on weed barrier fabric.

Figure 3. Propagules of AM fungi per cm3 of field soil collected from 5 different locations on the Rodale Institute farm on June 15, 2016. Results of 3 MPN bioassays each.

Figure 4. Percentage root length colonized by AM fungi in the one hundredth dilution (top panel) and total AM fungal propagules (bottom panel) present in the Most Probable Number bioassay conducted on the inoculum produced on farm. Means of 3 groups of 5 plants (top) or 3 MPN bioassays (bottom).
David Douds is a research microbiologist at USDA Agricultural Research Service in Wyndmoor PA.

For questions about this material, please contact the author by emailing david.douds@ars.usda.gov.

To read about early work on propagating indigenous AM fungi, and on the use of perlite and horticultural potting media as substitutes for vermiculite in the growth media in the bags, please see Douds, D.D., G. Nagahashi and P.R. Hepperly. 2010. Production of inoculum of indigenous AM fungi and options for diluents of compost for on-farm production of AM fungi. Bioresource Technology 101: 2326-2330.

[KD]

I always end up with extra mycotrex left over from my grow kit so I might give this a go if I remember one day. Very interesting read. Lots of stuff like this about now, Im loving it :)

[The Supreme Weeder]

I always end up with extra mycotrex left over from my grow kit so I might give this a go if I remember one day. Very interesting read. Lots of stuff like this about now, Im loving it :)

More things to try than hours in the day buddy. The thought occurs to me though that in a perpetual grow; the mycorrhizae will colonise new plants as long the new plants are in before the old ones are cropped and/or the roots of the cropped plants are left in the bed to degrade.

[KD]

I always end up with extra mycotrex left over from my grow kit so I might give this a go if I remember one day. Very interesting read. Lots of stuff like this about now, Im loving it :)

More things to try than hours in the day buddy. The thought occurs to me though that in a perpetual grow; the mycorrhizae will colonize new plants as long the new plants are in before the old ones are cropped and/or the roots of the cropped plants are left in the bed to degrade.

Now you say that I've read I can reuse my soil up to 4 times with a splash of added myco and another tab or 2. I'm scared of wasting a plant on it if it messes it up though. Might try this with the old soil of the next plant I do

[The Supreme Weeder]

Always worth a go pal

[KD]

Always worth a go pal

I suppose it's the only way I'll know for sure innit. I could actually whack an auto in it on the roof to see what happens. It'll be nice and warm again in about 3 months :)