Wertheim, F., Extension Educator, Agriculture/Horticulture, University of Maine Cooperative Extension
David Douds, Research Scientist, USDA-ARS
David Handley, Extension Professor, Small Fruit and Vegetable Specialist, University of Maine Cooperative Extension
Mark Hutton, Extension Professor, Vegetable Specialist, University of Maine Cooperative Extension

ABSTRACT

University of Maine Cooperative Extension faculty collaborated with a local organic grower and the USDA-ARS to evaluate the potential of arbuscular mycorrhizal [AM] fungi to boost yields in field grown leeks using both commercially-available AM fungus inocula and a “farm raised” inoculum.  The farm-raised inoculum was produced in bags of compost and vermiculite using bahiagrass as nurse plant.  The study was conducted in 2010 at two farm sites, and at one farm in 2011.  Data collected in 2010 indicated that leeks treated with the farm-raised inoculum were 32% heavier than controls while those treated with the commercial inoculum MycoGrow™ Soluble were not significantly different from the control.  In 2011, leeks treated with farm-raised inocula yielded 12% higher than controls though the result was not statistically significant. Of the two commercial inocula used in 2011, MycoApply® Endo yielded 30% higher while MycoGrow™ was no different from the control.  These results indicate that growers need to test various commercial sources of AM fungus inoculum to find one that works for their conditions and that the farm-raised inoculum may be used instead of commercial inoculum.

Introduction

Arbuscular mycorrhizal [AM] fungi are naturally occurring soil fungi that form a mutualistic symbiosis with the majority of crop plants.  Among the benefits to the plant ascribed to the symbiosis are enhanced nutrient uptake, water relations, and disease resistance (Smith and Read, 2008).  The primary benefit is enhanced uptake of immobile soil nutrients, i.e. P, Zn, and Cu.  The extra-radical hyphae of the AM fungi function, in effect, as extensions of the root system to increase the volume of soil explored for these nutrients.  The fungi benefit through the receipt of photosynthetically fixed carbon from the host plant.  These fungi appear to be totally dependent upon colonization of host roots for the procurement of sugars for carbon needed for growth and reproduction, and as such are called “obligate symbionts.” 

 Inoculation with AM fungi has been shown to enhance plant growth and yield in greenhouse and field studies.  Though these fungi are indigenous to agricultural soils, crop growth can benefit from inoculation with effective isolates of AM fungi.  Two strategies for use of inoculum are: 1) delivery of inoculum to the planting hole when seedlings are outplanted or when seeds are sown, and 2) inoculation of potting media to produce AM fungus colonized seedlings prior to outplanting.  The former strategy can be labor intensive and requires utilization of a sufficient number of propagules of the AM fungi to be competitive with, or to significantly supplement, the indigenous population.  The second option is normally regarded as more effective, and has the advantage of producing plants ready to utilize the symbiosis immediately upon outplanting.  This strategy is available to vegetable growers who produce their own seedlings for transplant to the field.

Inocula of AM fungi are available commercially in a variety of formulations ranging from preinoculated potting media to liquid concentrates into which roots can be dipped prior to transplant.  A number of research articles are available comparing their ability to produce AM fungus colonization of roots (Corkidi et al., 2004; Tarbell and Koske, 2007; Wiseman et al., 2009). 

Another option for farmers is to grow their own inoculum on-the-farm.  Methods for the on-farm production of AM fungus inoculum were first developed in the tropics and propagated either indigenous or introduced isolates of AM fungi in beds of fumigated soil (Sieverding, 1991; Gaur, 1997; Gaur et al., 2000).  Another method was developed for temperate climates (Douds et al., 2006; 2008a; 2010).  Inoculum produced via this method has been demonstrated to enhance the yield of potatoes, strawberries, and peppers (Douds et al., 2007; 2008b; 2012a).  In addition, inoculation of leek seedlings with inoculum of AM fungi produced on-farm enhanced yield over 2.5 fold over uninoculated controls in soil that had been repeatedly cultivated to produce a stale weed seedbed (Douds et al., 2012b).

Use of the mycorrhizal symbiosis, and indeed all natural ways to enhance plant nutrient availability, should be essential in organic production systems that are prohibited from application of synthetic fertilizers and pesticides.  Despite the potential for increased yields, decreased inputs, and increased revenue, inocula of AM fungi are not being used extensively in vegetable crop production.  Therefore, continued demonstrations of its utility and dissemination of results are warranted.  Here we report results of two years of field experiments in which inocula of AM fungi, purchased commercially or produced on-farm, were utilized to grow leeks on two organic farms in Maine.  Leeks were chosen for this study for their low pest profile, market value, and ease of harvest and data collection. 

 

Methods

University of Maine Cooperative Extension agriculture research faculty collaborated with a local organic grower in 2010 and 2011 to evaluate the potential of AM fungi to boost yields in field-grown leeks (Allium porrum L. cv. Tadorna) using both commercially available and “farm-raised” inocula.  The study was conducted at both the Highmoor Farm Experimental Horticulture Research Station in Monmouth, ME and at Wolf Pine Farm, an organic CSA farm located in Alfred, ME.

AM fungus inocula

One commercially available inoculum was utilized in 2010, MycoGrow™ Soluble (Fungi Perfecti LLC, PO Box 7634, Olympia, WA 98507), and two in 2011: MycoGrow™ Soluble and MycoApply® Endo (Mycorrhizal Applications Inc., PO Box 1029, Grants Pass, OR 97528).  MycoGrow™ is a water soluble inoculum containing nine species of AM fungi (29,275 propagules lb^-1(64,405 kg^-1)) plus other beneficial fungi and bacteria.  MycoApply® contains four AM fungus species and has 60,000 propagules lb^-1 (132,000 kg^-1).

AM fungus inoculum was also produced on-farm according to the method developed by researchers of the USDA-ARS in collaboration with the Rodale Institute (Douds et al., 2006).  Briefly, a 1:4 [v/v] mixture of compost and vermiculite was placed into plastic bags (7 gallon “Gro Bags”, Sunleaves Garden Products, Bloomington, IN 47404, USA).   Bahiagrass (Paspalum notatumFlugge) seedlings, previously grown for two months in conical plastic pots (66 cm³, RLC-4 pine cell, Stuewe & Sons, Corvallis, OR 97333 USA) were transplanted into these bags in early June of 2009 and 2010.  The bahiagrass seedlings were pre-colonized with one of the following AM fungi: Glomus mosseae, Glomus etunicatum, Glomus claroideum, and Glomus sp, all originally isolated from the soils of the Rodale Institute Experimental Farm, Kutztown, PA.  Five seedlings were transplanted into each bag, and AM fungi were segregated into different bags.  Bags were weeded and watered as needed throughout the growing season, during which the bahiagrass roots and associated AM fungi proliferated in the compost and vermiculite mixture.  The bahiagrass winter killed, and the bags over wintered outdoors.  The following spring, the compost and vermiculite mixtures from the different bags were recovered and mixed.  Most probable number bioassays estimated the AM fungus propagule density of the spring-harvested inocula to be 15.5 cm^-3 in 2010 and 25 cm^-3 in 2011 (Alexander, 1965).

 

Plant production and experimental treatments

Leek seeds were sown into 72 cell trays containing organic potting media (Living Acres NP Mix, 251 Weeks Mills Road, New Sharon, Maine, 04955) (0.4-0.5-0.3-1-0.2 (N-P₂O₅-K₂O-Ca-S)).  Seedlings then were transplanted at the cotyledon stage into 4 inch square pots containing 370 cm³ of a 9:1 [v/v] mixture of Living Acres NP Mix and:

Treatment 1: control mix (1:4 [v/v] compost:vermiculite, i.e. no AM fungi).

Treatment 2: farm-grown inoculum.

Treatment 3: MycoGrow™ inoculum: (same addition as control followed by an initial watering with 2.5 g MycoGrow™ (4 L)^-1 water, as directed).

Treatment 4: (2011 only): same addition as control, plus MycoApply® powder at a rate of 66.7g kg^-1 potting mix.

This yielded 574 and 925 propagules pot^-1 in treatment 2 for 2010 and 2011, respectively, and a calculated 14 and 925 propagules pot^-1 for MycoGrow™ and MycoApply®, treatments 3 and 4, respectively.  All plants were watered as needed and no supplemental fertilizer was added.  A subsample of five plants from each treatment was withheld from outplanting in 2010 and assayed for percentage root length colonized by AM fungi.  Roots were cleared in 10% KOH and stained with trypan blue (Phillips and Hayman, 1970) and colonization was quantified via the gridline intersect method (Giovannetti and Mosse, 1980) using a dissecting microscope at 20X magnification.  Plants were not sampled prior to outplanting for the 2011 season.

Experimental design, harvest, and analysis

The experiment was conducted in a randomized split block design.  The leeks were transplanted into the field on July 21, 2010 at both Wolf Pine Organic CSA, and Highmoor Farm Experiment Station; and in the 2011 study on July 27, 2011 at Highmoor Farm Experiment Station.  Fields at both farms had been in mixed vegetable production prior to the experiment.  Plants were grown in double rows on plastic-mulched beds, with 3 ft (0.91 m) between rows and 12 in (30.5 cm) between plants.  In 2010, there were six blocks per farm, each block containing three beds, each with 15 plants from one randomly assigned AM fungus inoculation treatment.  In 2011, there were five replicate blocks at the one farm, each block containing four beds (due to the additional commercially-available AM fungus inoculum) each with 15 plants from one randomly assigned AM fungus inoculation treatment. 

Leeks were harvested on November 15, 2010 and November 12, 2011.  The roots were washed and the tops trimmed.  The individual weight of marketable leeks was recorded for each plot.  The data were analyzed using Stastix9 ™ (Analytical Software, Tallahassee, FL) pooling data from both farms in 2010.

Figure 3.  Microscopic image of non-mycorrhizal roots of leek.  Roots were first treated with KOH, stained with trypan blue, and photographed under the dissecting microscope.  Note the very lightly staining cortex and darker vascular cylinder.

 

Discussion

Both farm raised and commercially produced inocula of AM fungi have the potential to increase the growth and yield of leek.  It is evident from the results reported here that commercially produced inocula are not equally effective in a given situation.  Effectiveness of the commercially available inocula used appeared simply to be directly proportional to the number of propagules of AM fungi delivered to the plant while in the greenhouse.  Though both were added according to manufacturer’s instructions, there was a 58 fold difference in the number of propagules of AM fungi applied per plant.  However, a variety of cultural and environmental factors influence both the formation of mycorrhizas in plant and AM fungus interactions and the result of that interaction in terms of the potential for a growth response.

Formation of mycorrhizas in the greenhouse environment is affected by several factors.  One important factor is the available P level in the potting media/nutrient regime (Jasper et al., 1979; Meikle and Amaranthus, 2008; Shukula et al., 2012).  High levels of available P will inhibit colonization of roots by AM fungi.  Mycotrophic status of the host plant is another factor.  Some crop plant families, eg. the Brassicaceae, do not become colonized by AM fungi (Smith and Read, 2008).  In addition, plants differ in terms of the level of available P which inhibits colonization by AM fungi (Douds, 2009).  The degree of colonization, as well as the resulting growth response, achieved in a particular plant and fungus pairing also depends upon the identity of the partners (Blaszkowski, 1993).  There exist optimal host plant and fungus interactions both from the perspective of the plant (potential for a growth response) and from the fungus (potential for growth and spore production).  For these and other reasons, a taxonomically diverse inoculum is generally considered preferable.  All inocula used in the experiments reported here had four or more AM fungus species.

Several conditions in the field also affect the outcome of inoculation with AM fungi.  The most commonly studied factor is the soil available P level: increasing P decreases the probability of a plant growth response to inoculation with AM fungi.  The soil P level above which a response is unlikely varies from 50 to 140 µg P g^-1 soil and is affected by the interaction of host plant and soil type (Amijee et al., 1989; Thingstrup et al., 1998).  The other factor which influences the outcome of the inoculation is the size of the population of AM fungi indigenous to the soil in the field (Sieverding, 1991).  The larger, healthier, and more diverse the native population the less likely an inoculation will produce a growth response.  However, research has demonstrated that seedlings outplanted to the field can benefit from inoculation during their greenhouse growth phase even in the presence of high P and a healthy background population of AM fungi (eg. Sorensen et al., 2008).

The lack of colonization by the commercial inoculum suggests the inoculum may have been mishandled prior to use resulting in death or at least decreased viability of the fungi.  One issue in the use of these inocula is that “no clear criteria have been set for the quality control of commercial inoculum” (IJdo et al., 2011).  Indeed, in published studies comparing numerous commercial inocula, few produced satisfactory levels of colonization at their recommended rates of application, and some produced no colonization (Corkidi et al., 2004, 2005, Tarbell and Koske, 2007; Wiseman et al., 2009).

Inoculation with AM fungi has the potential to enhance crop nutrient uptake, disease resistance, and water relations.  Optimal utilization of this, as well as other symbioses and natural sources of nutrients, is essential in agricultural systems in which synthetic chemical inputs are restricted.  Technologies for the on-farm production of inoculum of AM fungi have been shown to reliably produce a viable, infective inoculum.  Inocula of AM fungi are also available commercially; however several of these should be purchased and tested for effectiveness under prevailing conditions to ensure chances of growth promotion.

 

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