March 26, 2008

Choosing a Mixture Ratio for the On-Farm Production of AM Fungus Inoculum In Mixtures of Compost and Vermiculite

By Douds, David D Jr Nagahashi, Gerald; Reider, Carolyn; Hepperly, Paul R

Arbuscular mycorrhizal [AM] fungi are potentially important tools in sustainable agriculture due to their roles in crop nutrient uptake, disease resistance, and water relations and in stabilizing soil aggregates. Inocula of these fungi can be effectively produced on-farm in mixtures of compost and vermiculite with a suitable plant host, such as bahiagrass (Paspalum notatum Flugge). Success of this method, however, depends upon utilizing the optimal compost and vermiculite mixture ratio. Experiments were conducted over two years utilizing a complete factorial design with three composts, four mixture ratios, and three AM fungi with the objective of producing regression equations to predict optimal mixture ratios using routine measures of compost nutrient analyses as independent variables. Growth of colonized P. notatum in yard clippings and dairy manure + leaf composts; which were high in N, low in P, with moderate K levels; produced more spores of AM fungi at mixture ratios of 1:2 to 1:4 [v/v compost: vermiculite] relative to higher dilutions. Dilution ratios of 1:19 and 1:49 were best for controlled microbial compost, which was high in P, low in N, and moderately high in K. Simple equations were developed which predict the optimal fraction of compost in the mixture for each of the three AM fungi studied (Glomus intraradices, Glomus mosseae, and Gigaspora rosea). Percent N, P, and K and N:P ratio were the significant independent variables. These equations allow a farmer to choose a mixture ratio for the on-farm propagation of AM fungi knowing only the nutrient analysis of the compost to be used. Introduction

Arbuscular mycorrhizal [AM] fungi are obligate symbiotic soil fungi that colonize the roots of most crop plants. The resulting mutualistic symbiosis (the "mycorrhiza") is more efficient in the uptake of immobile soil nutrients such as P, Zn, and Cu; than an uncolonized root. The extraradical phase of the fungus functions, in effect, as an extension of the root, exploring a greater volume of soil for nutrients. The symbiosis can result in enhanced plant growth and yield, especially under nutrient poor conditions. Even when no growth enhancement occurs, the majority of P uptake by the plant can be attributed to the functioning of the mycorrhiza (Smith et al. 2004; Li et al. 2006; Schnepf & Roose 2006).

The symbiosis has been shown to impart other benefits to both the plant and the ecosystem. For instance, colonization of plants by AM fungi increased water stress resistance and disease resistance in experimental situations (Auge 2001; Dalpe 2005). The diversity of the AM fungus community in the soil can be an important regulator of the diversity of the associated plant community (Stampe & Daehler 2003; Johnson et al. 2005). In addition, AM fungi secrete the glycoprotein glomalin which is believed to play important roles in stabilization of soil aggregates and in soil carbon sequestration (Wuest et al. 2005; Purin et al. 2006; Rillig & Mummey 2006).

Given these benefits, the optimal utilization of the AM symbiosis is desirable for farmers, in particular those interested in minimizing or eliminating input of synthetic fertilizers and pesticides. One way to better utilize this symbiosis is to inoculate with effective isolates or strains of AM fungi. This is efficiently done by vegetable farmers who grow their own seedlings in on-farm greenhouses for later transplant to the field, allowing them to take advantage of the benefits of transplanting into the field a seedling that is already colonized by AM fungi. Inocula are commercially available and some come ready mixed within horticultural potting media. An alternative, however, is for the farmer to grow AM fungus inocula on the farm.

On-farm inoculum production systems have been developed in the tropics using beds of fumigated soil in which either the indigenous AM fungus community or introduced isolates are propagated using one or a series of host plants (Sieverding 1991; Gaur 1997; Gaur et al. 2000). We have developed a method for the onfarm production of AM fungus inoculum in temperate climates which requires no input of synthetic chemicals or fumigation and its merits relative to other systems have been discussed (Douds et al. 2005, 2006). Bahiagrass seedlings, colonized by AM fungi, are transplanted in late spring into raised bed enclosures containing a mixture of compost and vermiculite. The plants are tended over the growing season (weeded and watered as needed) and the AM fungi proliferate as roots spread throughout the enclosure. Frost then kills the bahiagrass. The AM fungi naturally over winter in the media and the inocula are ready for use the following spring. This system has successfully propagated all AM fungi tested and produced hundreds of propagules cm^sup -3^ in a 1:4 [v/v] mixture of yard clippings compost and vermiculite (Douds et al. 2005).

Maximal production of inoculum in this system requires the proper dilution of compost with a nutrient poor substrate such as vermiculite (Douds et al. 2006). Colonization of roots by AM fungi, and hence, growth of the fungus, is inhibited by high nutrient levels, notably of available P (Abbott et al. 1984; Schubert & Hayman 1986; Douds & Schenck 1990). Composts are high in nutrients and can be applied to soils en lieu of chemical fertilizers to meet the NPK requirements of crops (Reider et al. 2000). Growth of colonized bahiagrass plants in enclosures full of dairy manure plus leaf compost resulted in neither spread of the fungus to new roots nor production of spores (Douds, unpublishable results). Therefore, the objective of this study was to develop a prediction formula whereby, given the chemical analysis of a compost, one could calculate the proper volume of vermiculite to add to the compost for the on-farm production of AM fungus inoculum.

Materials and Methods

Two experiments were conducted at The Rodale Institute Experimental Farm in Kutztown, PA over the 2003 (Experiment I) and 2004 (Experiment II) growing seasons. Each utilized a complete factorial experimental design with three factors: 1) compost (three types), 2) mixture ratio with vermiculite (4 levels), and 3) AM fungus (3 types); with three replications per treatment combination.

Production of AM Fungus Colonized Plants

Bahiagrass (Paspalum notatum Flugge) seedlings were transplanted into 66 cm conical plastic pots (RLC 4 'Pine Cell,' Stuewe and Sons, Corvallis, Oregon 97333) containing a 0.75:1:1:0.75 [v/v/v/v] mixture of field soil, sand, vermiculite, and calcined clay ('Turface/ Applied Industrial Materials Corp., Deerfield, IL 60015) and grown in a greenhouse for 3-5 months prior to use in the experiment. Inoculation treatments in these experiments included three of the following (see below): Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe originally isolated from the Fanning Systems Trial at The Rodale Institute Experimental Farm, Glomus intraradices Schenck & Smith (DAOM 181602); Gigaspora rosea Nicol. & Schenck (DAOM 194757); and uninoculated control plants. Plants typically were inoculated by placing a band of pot culture inoculum, from a 6- 8 month-old culture of P. notatum grown in the same potting mix, in the center of the column of media mix in each conical pot. The exception was G. intraradices. In this instance, inoculum was produced in vitro according to the split plate technique of St- Arnaud et al. (1996). Media containing spores, hyphae, and colonized Ri TDNA transformed carrot roots from Petri dishes was blended in a Waring blender in 10 mM sodium citrate (pH=6.0) at high speed for 45 sec to dissolve the gelling agent (Doner & Becard 1991). Fungal tissues and root pieces were collected on a 40pm sieve, rinsed, and pipetted onto the soil-based media in the center of the column of the conical pots. Plants received Hoagland's nutrient solution minus P (Hoagland & Arnon 1938) biweekly while in the greenhouse.

Experiment I

Experiment I was initiated on May 29, 2003. Twelve enclosures (1 m ? 1 m; 0.3 m tall) were constructed with silt fence walls supported by wooden stakes. Each had a weed barrier cloth floor and was divided into nine 0.11 m sections using black plastic sheeting. Each enclosure was filled to a depth of 20 cm with a unique compost and vermiculite dilution treatment. Three composts were used: yard clippings compost [YCC], dairy manure + leaf compost [DMLC], and controlled microbial compost [CMC]. The YCC was produced in windrows by the Lehigh County Compost Facility, Allentown, PA. The DMLC was made on site with a 1:4 [v/v] mixture of manure and leaves, respectively. The manure component contained shredded newspaper bedding. The CMC was purchased, and consisted of animal manure, bedding, clay loam, rock powder inoculant, and finished compost. The mixture had been composted for 10-14 weeks with frequent aeration. Analyses of the composts indicated that CMC had the highest level of P and the lowest C:N ratio (Table 1). Each compost was diluted with vermiculite at the following ratios (v/v, compost: vermiculite): 1:2, 1:4,1:9, and 1:49, yielding 12 such treatment combinations (one per enclosure). Each of three sections of all enclosures then received seven plants colonized by G. mosseae and three received plants colonized by G. rosea. The remaining three sections of each enclosure received seven plants which were uninoculated. TABLE 1.

Summary of chemical characteristics of composts

A subsample of plants was withheld from the experiment and used to quantify the status of mycorrhizas at time zero. Roots were cleared and stained (Phillips & Hayman 1970) and assayed for percentage root length colonized by AM fungi via the gridline intersect method (Newman 1966). The roots and potting media from plants at the time of initiation of the experiment also were used to conduct most probable number (MPN) bioassays to estimate the number of AM fungus propagules introduced to the enclosure sections (Alexander 1965). The samples were diluted 10" through 10" with the soil, sand, vermiculite, and calcined clay potting mixture, and potted in conical plastic pots with bahiagrass seedlings. Plants were grown for 4 weeks in a controlled environment growth chamber (day/night: 16/8 h, 25/18[degrees]C, 60/70% R.H.). Entire root systems then were recovered, stained as above, and examined to detect presence or absence of AM fungi. Plants colonized by G. mosseae had 23.2 +- 2.2% (n=3) root length colonized and the rooting volume of seven of these were estimated to contain 20000 propagules. Plants colonized by G. rosea had 43.6 +- 1.1% (n=3) of their root length colonized and seven of these were estimated to contain 2000 propagules. Uninoculated plants were not colonized and rhizosphere soils produced no colonization of test plants in the MPN bioassay.

Leaf (from G. mosseae sections only) and root samples were collected from the enclosures on August 5. Care was taken not to sample from the rhizosphere of the original seedlings' rooting volume. Roots were assayed for percentage root length colonized as above. Leaves were dried in a forced draft oven (2 d, 80[degrees]C) and analyzed for N and P via the methods of Wall and Gehrke (1975) and Murphy & Riley (1962), respectively, after digestion with H^sub 2^SO^sub 2^ and H^sub 2^O^sub 2^. Above ground biomass was harvested from the sections with uninoculated plants only on November 20 to assess the effect of the compost dilutions upon plant growth. Inoculated plants were not sampled for biomass production because the presence of the intact senescent shoot may contribute to the over wintering survival of the inoculum. Shoots were weighed after drying to a constant weight in a solar assisted oven. Compost and vermiculite mixtures and roots were sampled finally on December 4, after death of the bahiagrass, for quantification of colonization of roots and inoculum production. Colonization of roots was assayed as above. AM fungus spore production was quantified under a dissecting microscope after isolation of spores from 50 cm samples of mixture, one from each enclosure section, by wet sieving and centrifugation (Gerdemann & Nicolson 1963; Jenkins 1964).

Experiment II

Experiment II was initiated on May 21, 2004 with the same experimental design as the first, but with several important modifications. First, the dilution ratios were modified based upon the findings of Experiment I. The YCC and DMLC were diluted 1:1, 1:2,1:4, and 1:9 [v/v] with vermiculite and the CMC was diluted 1:9, 1:19, 1:49, and 1:99. Inoculation treatments included G. mosseae and G. rosea as before, but G. intraradices was used as the third inoculation treatment. In addition, seven gallon black plastic bags ('Grow Bags,' Worm's Way, Bloomington, IN 47404) were used instead of the silt fence enclosures (five colonized seedlings per bag). Each was filled with 18.2 L of compost and vermiculite mixture. Three bags were filled for each compost X dilution X AM fungus treatment combination. Plants colonized by G. mosseae, G. intraradices, and G. rosea had 25.2 +- 1.5, 59.5 +- 4.1, and 29.3 +- 3.7% (n=5) of their root length, respectively, colonized at the time of transplanting into the bags. MPN bioassays were not conducted at that time.

Leaves of bahiagrass were sampled from plants colonized by G. rosea only on August 16 and analyzed for N and P as above. Total above ground biomass was collected on November 5 only for plants colonized by G. intraradices and processed as above. Differences among AM fungus treatments were not expected, and the primary reason for nutrient and biomass sampling was to examine the effects of compost x dilution ratio combination upon plant growth. Compost and vermiculite mixtures were collected from each bag on December 2, after cold temperatures had killed the bahiagrass. These samples were used to quantify spore production, final percentage root length colonized by AM fungi, and propagule concentration as in Experiment I. Though G. intraradices produces spores primarily within plant roots, its spores were fairly abundant in the media under these conditions at the time of sampling, i.e. after plant senescence. Only these spores were quantified.

Statistical Analyses

Data were analyzed via analysis of variance after SQRT(x+1) or arcsin transformation for spore counts and percentage root length colonized data, respectively, via SAS (SAS Institute, Cary, North Carolina 227513, USA). Each compost treatment was analyzed separately in Experiment II because the dilution ratios were not the same across all composts. Measurements for which significant treatment effects were found were characterized further using Tukey's Method of Multiple Comparisons (a =0.05).

Equations to predict the optimal compost: vermiculite ratio were developed in a two step process using linear regression via SAS. First, equations of the form y= a+a x+a x2 or y= a+a x+a x2+a x3 were developed where y= spore population, x= fraction of compost in the growth mixture, and a and a are the intercept and slope regression coefficients, respectively, of the quadratic or cubic equations. These equations were generated for each AM fungus x compost combination of each experiment, i.e. each line in Figures 1, 2, and 3. They were then solved to yield the fraction of compost giving the maximal sporulation within the range of compost and vermiculite mixtures studied. Finally, those optimal compost fractions for a given AM fungus were used as the dependent variable for regression against compost nutrient analysis results as the independent variables (Table 1) using the FORWARD selection procedure of SAS linear regression. The default F statistic of 0.5 for entry to the model was used. This generated three equations, one for each AM fungus studied, predicting the optimal fraction of compost as a function of one or more characters of compost chemistry.


Plant Growth and Nutrient Concentrations

Growth and nutrient concentration of bahiagrass shoots in Experiment I were affected by both compost type and dilution (Table 2). Biomass was significantly greater for plants grown in the CMC mixtures, followed by YCC then DMLC. Above ground biomass production was insensitive to dilution of the compost until the 1:49 dilution, which produced significantly less biomass than the other dilutions. Nitrogen concentration in bahiagrass leaves was unaffected by dilution, but was higher when plants were grown in YCC and CMC than in DMLC. Phosphorus concentration in shoots was significantly lower in the 1:49 dilution ratio than in other dilutions, and higher in plants grown with DMLC than the other compost mixtures.


Nutrient concentration in leaves and total above ground biomass of bahiagrass grown in three composts amended with vermiculite, Experiment I. Tissue was collected for N and P analyses on August 5,2003, after 10 weeks of growth and for biomass on November 20, 2003.1

Dilution did not affect P or N concentrations in leaves of plants grown with DMLC, P concentration in CMC, or N concentration in YCC (Table 3) in Experiment II. Plants grown in the most concentrated YCC and CMC treatments had significantly greater P and N concentrations, respectively, than other dilutions of those composts. Dilution significantly affected above ground biomass accumulation in all compost treatments. Biomass production in the most concentrated treatments was 2.5 to 4 fold greater than that found in the highest dilutions (Table 3).


Nutrient concentration in leaves and total above ground biomass of bahiagrass grown in three composts amended with vermiculite, Experiment II. Tissue was collected for nutrient analysis August 16,2004, after 10 weeks of growth, and November 5,2004 for biomass1.

AM Fungus Colonization of Roots

Most AM fungi spread to colonize roots of bahiagrass growing out from the original rhizospheres of the transplants at the 10 week sampling in Experiment I (Table 4). Glomus mosseae tended to be more aggressive than G. rosea, but CMC compost diluted 1:9 or less with vermiculite slowed its spread at 10 weeks and, indeed, that of both AM fungi for the remainder of the growing season (Table 5). Roots were sampled only at the end of the production period in Experiment II. Dilution ratio had no effect upon colonization of roots grown in YCC (Table 6). Plants inoculated with G. mosseae had on average 59% root length colonized, G. rosea: 64%, and G. intraradices: 74%. Dilution ratio affected the colonization of plants inoculated with Glomus species in DMLC and CMC however. Roots tended to be more colonized at the 1:2 through 1:9 [compost: vermiculite, v/v] dilutions of DMLC. Colonization of roots by G. intraradices was not influenced by dilution ratio in CMC, but colonization of plants inoculated by the other fungi increased as the compost became more dilute.


Colonization of bahiagrass roots by AM fungi after 10 weeks of growth. Plants were grown in three composts diluted to various levels with vermiculite, Experiment I1.


Colonization of bahiagrass roots by AM fungi after frost killing of the plant. Plants were grown in three composts diluted to various levels with vermiculite, Experiment I1. TABLE 6.

Colonization of bahiagrass roots by AM fungi after frost killing of the plant. Plants were grown in three composts diluted to various levels with vermiculite, Experiment II1.

Sporulation of the Fungi

Spore populations in the compost and vermictilite mixtures in Experiment I were affected by both compost type and dilution with vermiculite (Figure 1). Sporulation of G. rosea increased with increasing proportion of compost in the mixture with YCC and DMLC (Pr>F = 0.0330 and 0.0001, respectively) and with decreasing proportion of compost with CMC (Pr>F = 0.0001). A similar pattern was shown by G. mosseae. Maximum sporulation of G. mosseae occurred at the 1:4 (v/v compost : vermiculite) dilution with YCC and DMLC but at the 1:49 (v/v compost : vermiculite) dilution with CMC.

Compost dilution ratios were modified in Experiment II based upon the results of Experiment I. A 1:1 [v/v] mixture was initiated with YCC and DMLC, and the 1:49 dilution was dropped, to attempt to find the peak response for G. rosea in these composts. Likewise, a 1:99 [v/v] mixture was included with CMC, and the 1:2 dilution was dropped, in an attempt to find the optimal response of sporulation in this compost. Sporulation in Experiment [Eth] followed the general patterns established in Experiment I. Glomus mosseae sporulated best in both DMLC and YCC at the 1:4 (compost: vermiculite [v/v]) dilution and the 1:49 dilution in CMC (Figures 2b and 3). We did not see a consistent pattern of sporulation by G. rosea in YCC and DMLC. It sporulated best in YCC at the 1:4 and 1:1 dilutions (Pr>F= 0.0019) and in DMLC in the 1:9 dilution (Pr>F= 0.0005) (Figure 2a). As in Experiment I, G. rosea sporulated significantly better in CMC at the 1:49 dilution (Pr>F= 0.0043) (Figure 3). Extraradical spores of G. intraradices were more abundant in YCC and DMLC at the 1:4 and 1:9 dilutions, respectively (Pr>F= 0.0006 and 0.0001). Spores of G. intraradices were more abundant in CMC compost at the 1:19 dilution (Figure 3).

FIGURE 1. Spore production by Gigaspora rosea (A) and Glomus mosseae (B) in mixtures of compost and vermiculite, Experiment I. YCC= yard clippings compost, CMC= controlled microbial compost, DMLC= dairy manure + leaf compost. Means of 3 observations +- SEM.

FIGURE 2. Spore production by Gigaspora rosea (A), Glomus mosseae (B) and Glomus intraradices (C) in mixtures of compost and vermiculite, Experiment II. YCC= yard clippings compost, DMLC= dairy manure + leaf compost. Means of 3 observations +- SEM.

FIGURE 3. Spore production by Glomus intraradices, Glomus mosseae, and Gigaspora rosea in mixtures of CMC (controlled microbial compost) and vermiculite. Means of 3 observations +- SEM.

Predictive Equations

Equations were generated via a two step process using linear regression (see Methods). Analyses showed %N, %K to be the best predictors of fraction of compost in the optimal mixture for growth of G. mosseae, %P for G. intraradices, and %N and N:P ratio for G. rosea (Table 7).


Equations to predict optimal fraction of compost in compost: vermiculite mixtures for the production of AM fungus spores1.


Inocula of three AM fungi were successfully produced in three different composts, each diluted to four concentrations with varying amounts of vermiculite. The optimal dilution ratio, compost: vermiculite [v/v], varied with both AM fungus grown and type of compost used.

Phosphorus availability has long been known to affect the development of arbuscular mycorrhizas (Mosse 1973; Menge et al. 1978; Jasper et al. 1979). High levels of available P limit colonization of roots, sporulation, and hyphae production by AM fungi (Douds & Schenck 1990; de Miranda & Harris 1994). AM fungi differ in their susceptibility to P levels in the soil. Glomus intraradices, one of the fungi studied here, is considered to be P tolerant (Sylvia & Schenck 1983; Alkan et al. 2006). Indeed, its ability to colonize roots of bahiagrass was largely unaffected by compost dilution, and it produced over 50% colonized root length at the most concentrated mixture of the high P CMC (Table 6). One mechanism whereby colonization is inhibited is when roots grown in P- sufficient conditions exude less of the hyphal branching signal to the rhizosphere (Nagahashi et al. 1996). This lessens the fungus' ability to find the root and form appressoria.

Nitrogen addition also affects AM fungi. Some studies indicate N addition can be detrimental (Johnson et al. 1980) or stimulatory (Hepper 1983; Furlan & Bernier-Cardou 1989) to the formation of mycorrhizas. The proper balance between N and P can regulate the formation of mycorrhizas (Sylvia & Neal 1990) and contribute to optimal functioning of the symbiosis from an inoculum production perspective (Douds & Schenck 1990). A low level of available P encourages colonization of the roots and formation of mycorrhizas. Sufficient availability of N is important for shoot growth and photosynthesis, the source of the fixed carbon the obligately symbiotic AM fungi require for growth and sporulation. Hepper (1983) found a positive correlation between colonization of lettuce by G. mosseae, as measured by glucosamine content, and N:P ratio of the applied nutrient solution.

Successful production of inocula by this method depends upon the proper dilution of nutrient rich compost with vermiculite. Vermiculite serves as a relatively inert ingredient and diluent. Proper dilution is governed by the nutrient levels of each compost, which in turn is dependent upon the starting materials and method of composting. Therefore, these experiments provided the framework for developing predictive formulae to aid in choosing a dilution. Equations predicting the optimal fraction of compost in the final mixture were developed (Table 7). These equations are quite simple and the agreement between predicted and observed values is very high.

The AM fungi studied exhibited different optimal compost dilution levels and different final predictive equations. This may be considered a negative factor since the fungi grown at a given site may not be the three for which the equations were developed here. Developing predictive equations for all of the 150+ species of AM fungi described to date is not possible. Also, the data support general recommendations based upon the nutrient concentrations of the compost used. Composts high in N, low in P, with moderate K levels, such as YCC and DMLC may be used at dilutions of 1:2 to 1:4 [v/v compost: vermiculite] for propagation of AM fungi with a high probability of success. Composts high in P, low in N, and moderately high in K, such as the CMC used here, should be diluted to 1:19 or 1:49.


This work was supported in part by a grant from the Northeast Sustainable Agriculture Research and Education program of USDA- CSREES (no. LNE-03179). We would like to thank S. Campbell and J. Lee for technical assistance and J.A. Fortin for the original split plate cultures of G. intraradices.

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.


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David D. Douds, Jr.1, Gerald Nagahashi1, Carolyn Reider2 and Paul R. Hepperly2

1. U.S. Department of Agriculture, Agricultural Research Service,

Eastern Regional Research Center, Wyndmoor, Pennsylvania

2. The Rodale Institute, Kutztown, Pennsylvania

Copyright J.G. Press Inc. Winter 2008

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