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

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Abstract

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.
On-farm production of inoculum of indigenous arbuscular mycorrhizal fungi
and assessment of diluents of compost for inoculum production
q
David D. Douds Jr.
a,*
, Gerald Nagahashi
a
, Paul Reed Hepperly
b
a
US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA
b
The Rodale Institute, 611 Siegfriedale Rd., Kutztown, PA 19530, USA
article info
Article history:
Received 4 May 2009
Received in revised form 13 October 2009
Accepted 13 October 2009
Keywords:
Organic agriculture
Sustainable agriculture
Biofertilizer
abstract
On-farm production of arbuscular mycorrhizal [AM] fungus inoculum can be employed to make the ben-
efits of the symbiosis more available to vegetable farmers. Experiments were conducted to modify an
existing method for the production of inoculum in temperate climates to make it more readily adoptable
by farmers. Perlite, vermiculite, and peat based potting media were tested as diluents of yard clippings
compost for the media in which the inoculum was produced using bahiagrass (Paspalum notatum Flugge)
as host plant. All produced satisfactory concentrations of AM fungus propagules, though vermiculite
proved to be better than potting media (89 vs. 25 propagules cm
3
, respectively). Two methods were
tested for the growth of AM fungi indigenous to the farm: (1) adding field soil into the vermiculite and
compost mixture and (2) pre-colonizing the bahiagrass seedlings in media inoculated with field soil prior
to transplant into that mixture. Adding 100 cm
3
of field soil to the compost and vermiculite produced 465
compared to 137 propagules cm
3
for the pre-colonization method. The greater flexibility these modifi-
cations give will make it easier for farmers to produce inoculum of AM fungi on-the-farm.
Published by Elsevier Ltd.
1. Introduction
Arbuscular mycorrhizal [AM] fungi are native to agricultural
soils and form a mutualistic symbiosis with the majority of crop
plants. Among the benefits to the plant ascribed to the symbiosis
are enhanced: uptake of immobile nutrients, water relations, and
disease resistance (Smith and Read, 2008). These benefits make
utilization of the symbiosis attractive to sustainable agricultural
systems that are designed to minimize synthetic inputs of fertilizer
and pesticides, or in the case of organic agriculture, eliminate them
from the production system.
Inocula of AM fungi are available commercially in a variety of
forms ranging from high concentrations of AM fungus propagules
in carrier materials to potting media containing inoculum at low
concentrations. Effective AM fungus inoculum may also be grown
on-the-farm using a variety of methods. These techniques were
pioneered in Columbia (Dodd et al., 1990; Sieverding, 1991) and
India (Gaur, 1997; Gaur and Adholeya, 2002), but we have recently
developed a method suitable for temperate climates (Douds et al.,
2006). This method entails mixing compost with vermiculite to de-
crease the effective concentration of available nutrients in the
compost, notably of P (Douds et al., 2008a). High levels of P are
known to inhibit colonization of roots by AM fungi (Hepper,
1983). Bahiagrass (Paspalum notatum Flugge) seedlings, colonized
by specific AM fungi, are then transplanted into bags containing
the compost and vermiculite mixture. The AM fungi proliferate in
the media as the bahiagrass grows throughout the summer, and
the bags are weeded and watered as needed. The bahiagrass is then
frost killed. The AM fungi over winter in the media and the inocu-
lum is ready for use the following spring. Inoculum produced in
this fashion has increased the yield of potatoes (Douds et al.,
2007) and strawberries (Douds et al., 2008b).
Adoption of on-farm production of AM fungus inoculum in com-
post and vermiculite mixtures is hampered by two factors. First, the
lack of commercially available seedlings already colonized by se-
lected AM fungi means there is no starter inoculum of the form out-
lined in the procedure above. Second, the possibility of asbestos
contamination of certain sources of vermiculite may cause hesita-
tion in adoption of the method (US EPA, 2008). Experiments were
conducted to find an alternate starter inoculum to allow farmers
to propagate the AM fungi indigenous to their farms and test the
use of alternatives to vermiculite for dilution of the compost.
2. Methods
2.1. Propagation of indigenous AM fungi
Two methods were tested for the propagation of indigenous AM
fungi. The first entailed adding field soil to the compost and
0960-8524/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.biortech.2009.11.071
q
Mention of trade names or commercial products in this article is solely for the
purpose of providing specific information and does not imply recommendation or
endorsement by the US Department of Agriculture.
*Corresponding author. Tel.: +1 215 233 6421; fax: +1 215 233 6581.
E-mail address: david.douds@ars.usda.gov (D.D. Douds Jr.).
Bioresource Technology 101 (2010) 2326–2330
Contents lists available at ScienceDirect
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
vermiculite mixture into which nonmycorrhizal bahiagrass (P. not-
atum Flugge) seedlings were transplanted. The second tested the
inoculation of the bahiagrass seedlings with AM fungi prior to
transplant by a preliminary growth phase in potting media con-
taining field-collected soil.
The first experiment to propagate indigenous AM fungi was
conducted during the 2007 growing season at The Rodale Institute,
Kutztown, PA. Seven gallon (26.5 L) black plastic bags (‘‘Grow
bags”, Worm’s Way, Bloomington, IN 47404) were filled with
approximately 20 L of a 1:4 [v/v] mixture of compost and vermic-
ulite, respectively. The compost was produced from yard clippings
and leaves in windrows by the Lehigh Valley Compost Facility,
Allentown, PA. The compost was first sieved to pass through a
1 cm mesh, and heat pasteurized on two consecutive days for
approximately 8 h each. The latter was done to try to kill any AM
fungi in soil introduced into the compost during turning.
Field soil was collected from the top 10 cm from a field under
organic management at The Rodale Institute. A most probable
number bioassay (MPN) (Alexander, 1965) of this soil indicated
that there were 12 propagules cm
3
. Four levels of soil addition
were examined, each with three replicate bags. 0, 100, 200, or
400 cm
3
of this soil was mixed into the individual bags of compost
and vermiculite mixture just prior to transplanting three-month-
old nonmycorrhizal bahiagrass seedlings, at a rate of five per bag,
on June 20, 2007. The seedlings had been grown in a greenhouse
in 65 cm
3
conical plastic pots (RLC 4 ‘‘pine cell”, Stuewe and Sons,
Corvallis, OR 97333) in an autoclaved mixture of sand, soil, vermic-
ulite, and turface [SSVT] (Applied Industrial Materials, Corp., Deer-
field, IL, 60015) (1:0.75:1:0.75 v/v). For these experiments and all
others, bags were weeded and watered, as needed, throughout
the growing season.
The second experiment to propagate indigenous AM fungi was
conducted during the 2008 growing season. Bags of compost and
vermiculite mixture were prepared as above. However, in this
experiment the bahiagrass seedlings were grown for 3 months in
a mixture of field soil and SSVT (1:5 v/v). The field soil was col-
lected from the top 10 cm of the Farming Systems Trial at The Ro-
dale Institute. Clearing and staining of the root systems of five of
these seedlings withheld from transplant into the bags indicated
an average of 58 ± 5% root length colonized by AM fungi indige-
nous to that field soil (Phillips and Hayman, 1970). Seedlings were
transplanted into the bags on June 18, 2008, at a rate of five plants
per bag, fifteen bags total. Inoculum production with this modifica-
tion was compared to that in the standard method (Douds et al.,
2006). Identical bags of yard clippings compost and vermiculite
mixtures [1:4 v/v] were prepared and bahiagrass seedlings colo-
nized by Glomus mosseae (Nicol. and Gerd.) Gerdemann or Trappe
and Glomus claroideum Schenck and Smith, both originally isolated
from soils of The Rodale Institute, were utilized as nurse plants and
transplanted into the bags (five plants per bag, nine bags for each
AM fungus).
2.2. Alternate diluents for compost in the on-farm production of AM
fungus inoculum
Non-pasteurized yard clippings compost produced at the same
facility as above was sieved to pass through a 1 cm mesh. It was
then mixed 1:4 [v/v] with one of three materials: vermiculite
(coarse, The Schundler Co., Edison, NJ 98817), perlite (Pennsylvania
Perlite Corp., Bethlehem, PA 18018), or peat based horticultural
potting media (Berger BM6; Saint-Modeste, Quebec G0L 3W0). Six-
teen bags were filled with each mixture.
Bahiagrass seedlings were grown in the greenhouse in the SSVT
mixture inoculated with selected isolates of AM fungi. Plants were
inoculated by placing a one cm thick band of whole pot culture
inoculum in the middle of the soil column of the conical 65 cm
3
pots. Fungi used were: G. mosseae (Nicol. and Gerd.) Gerdemann
and Trappe, G. claroideum Schenck and Smith, both originally iso-
lated from soils of The Rodale Institute, Glomus intraradices
Schenck and Smith (DAOM 181602), and Glomus sp. isolated from
the Stoneleigh Estate, Villanova, PA. Initial colonization of the
plants inoculated with G. mosseae,G. claroideum,G. intraradices,
and Glomus sp. was 35 ± 4, 33 ± 1, 56 ± 5, and 47 ± 13 percent root
length colonized, respectively (n= 3 plants each). Plants were
transplanted into the bags, five per bag, on June 13, 2007. There
were four bags per AM fungus diluent treatment combination.
2.3. Data collection and analysis
Propagules of AM fungi in the bags were quantified in early
winter after freezes had killed the bahiagrass. Samples were col-
lected on January 9, 2008 for the alternate diluent and field soil
addition experiments and November 14, 2008 for the experiment
in which the bahiagrass seedlings were inoculated with field soil
while in the greenhouse. Media was collected from two to three
places within each bag, pooled, and stored in zip lock plastic bags
at 4 °C until analysis. Fifty cm
3
subsamples from each AM fun-
gus compost diluent treatment combination, and from each level
of field soil addition in the experiments conducted in 2007, were
pooled prior to analysis to yield just one MPN assay per treatment
combination for these experiments. Space and labor constraints
precluded conducting replicate MPN assays for that experiment
(Alexander, 1965). Three MPN bioassays were conducted each for
the comparison of inoculum production using seedlings grown in
the greenhouse with field soil inoculum vs. two inoculated AM fun-
gi (G. mosseae and G. claroideum). Most probable number bioassays
utilized successive tenfold dilutions from 10
1
to 10
5
using the
sterilized SSVT mixture and bahiagrass seedlings as the host plant.
Plants were grown in a controlled environment (day/night: 16/8 h,
25/18 °C, 60/70% R.H.) for four weeks.
Spores of AM fungi were quantified in 50 cm
3
subsamples of
media collected from each bag via wet sieving and centrifugation
(Jenkins, 1964; Gerdemann and Nicolson, 1963). Roots were col-
lected from the sample bags, rinsed, cleared and stained using try-
pan blue (Phillips and Hayman, 1970). Percentage root length
colonized by AM fungi was quantified using the gridline intersect
method using a dissecting microscope at 20magnification (New-
man, 1966).
Experimental units were arranged according to a completely
randomized design. Data were subjected to analysis of variance
after square root (X+ 1) (spore count and MPN) or arcsin (percent-
age root length colonized) transformation. Parameters for which
significant treatment effects were found were characterized fur-
ther using Tukey’s method of multiple comparisons to separate
means (
a
= 0.05).
3. Results
3.1. Propagation of indigenous isolates of AM fungi
Addition of field soil to the inoculum production system suc-
cessfully propagated indigenous AM fungi (Fig. 1). AM fungi pres-
ent in soil turned into the compost during its production
evidently survived the heat treatment to colonize and increase,
however survival was inconsistent with only 0.7% root length col-
onized in one bag and 19% in another, compared to an overall aver-
age above 60% root length colonized in the bags receiving field soil.
Colonization of roots in the bags receiving soil was significantly
greater than those not receiving soil, and not different among
themselves (Pr > f < 0.0001). Propagule production ranged from
21.5 cm
3
(0.4 10
6
per bag) in the uninoculated treatment to
D.D. Douds Jr. et al. / Bioresource Technology 101 (2010) 2326–2330 2327
465 propagules cm
3
(8.8 10
6
per bag) for the treatment receiv-
ing 100 cm
3
of soil. The latter represented an over 7000 fold in-
crease of propagules relative to the number contained in the
original soil.
Growth of bahiagrass seedlings in SSVT amended with field soil
prior to transplant into the on-farm inoculum production system
also successfully produced inoculum of AM fungi (Fig. 2). G. mos-
seae and G. claroideum together constituted a slight majority of
the spores produced in these bags (40 ± 7 and 27 ± 12 spores
50 cm
3
, respectively of an average of 132 ± 26 total spores
50 cm
3
). Spore production by these species in bags containing
plants preinoculated specifically with G. mosseae or G. claroideum
was much greater than that in the bags growing the indigenous
community (Table 1). However, quantification of the total amount
of propagules of AM fungi in the compost and vermiculite mixtures
indicated no statistically significant difference (Pr > f = 0.5990)
among the three groups (Fig. 2). Colonization of roots after frost
killed the bahiagrass was significantly greater in bags propagating
the indigenous AM fungus community than those specifically inoc-
ulated with G. mosseae (71 ± 3% vs. 59 ± 4% root length colonized,
respectively, Pr > f = 0.0456) and not different from those inocu-
lated with G. claroideum (65 ± 4% root length colonized).
3.2. Alternate diluents for compost
Inocula of all AM fungi tested were successfully produced using
perlite and horticultural potting media, in addition to the routine
use of vermiculite, as diluents of compost in the on-farm system
(Table 2). Populations of spores of the four fungi were not signifi-
cantly different among the three diluents. Colonization of roots
by G. mosseae was greater in perlite than in vermiculite, and colo-
nization by G. claroideum was greater in perlite and vermiculite
than in horticultural potting media. Colonization levels were not
significantly different across diluents for the other fungi tested.
Even though spore populations and colonization of roots were sim-
ilar across diluents, vermiculite tended to produce more propa-
gules than the other diluents as measured via MPN bioassay.
4. Discussion
Production of AM fungus inoculum on-the-farm potentially of-
fers several important advantages over commercially available
inocula. These have been detailed earlier (Douds et al., 2005,
2006) and include lower cost and potentially more taxonomic
diversity than commercially purchased inocula. The current modi-
fications allow for another benefit: the production of an inoculum
containing locally adapted isolates of AM fungi.
Adoption of on-farm production of AM fungus inoculum by
growers requires a greater degree of flexibility than that present
in the original method. The original method required that compost
be diluted with vermiculite and that the original starter inoculum
be in the form of purchased bahiagrass seedlings colonized by spe-
0100200400
0
20
40
60
80
100
0
100
200
300
400
500
Field soil added (cm3)
Colonization (% root length)
Propagules cm
-3
Fig. 1. Colonization of Paspalum notatum roots by AM fungi (line, means of three
observations ± SEM) and propagule production (bars, results of most probable
number bioassay using a pooled sample from three inoculum production bags).
Field soil was added to each inoculum production bag in the amounts indicated to
propagate the indigenous AM fungus community.
Field Soil G. mosseae G. claroideum
0
100
200
300
400
Inoculation treatment
Propagules cm-3
Fig. 2. Production of propagules of AM fungi as measured by most probable number
bioassay (means of three observations ± SEM). Paspalum notatum seedlings that
were transplanted into bags containing compost and vermiculite previously were
grown for 3 months in a potting mixture containing Glomus mosseae or Glomus
claroideum or field soil for the propagation of an indigenous AM fungus community.
Table 1
Populations of spores of two species of AM fungi resulting from growth of plants
specifically inoculated with those fungi or with field soil in which those fungi are
indigenous.
AM fungi Inoculation treatment Pr > f
G. mosseae G. claroideum Field soil
Spores 50 cm
3
Glomus mosseae 783 ± 219 40 ± 7 0.0001
Glomus claroideum 283 ± 70 27 ± 12 0.0001
n10 7 12
Table 2
Use of different diluents with compost for the on-farm production of AM fungus
inoculum.
a
AM fungus Perlite Potting
media
Vermiculite ANOVA
(Pr > f)
Spores 50 cm
3
Glomus mosseae 346 ± 71 512 ± 217 525 ± 55 0.5993
Glomus sp. 186 ± 41 123 ± 59 71 ± 10 0.2130
Glomus
intraradices
290 ± 77 650 ± 133 324 ± 97 0.0722
Glomus
claroideum
452 ± 135 1220 ± 506 946 ± 247 0.3048
Colonization (% root length)
Glomus mosseae 69.1± 6.4 56.4 ± 2.9 46.6 ± 0.8 0.0120
Glomus sp. 59.9 ± 4.7 60.9 ± 4.9 64.6 ± 5.2 0.7804
Glomus
intraradices
67.9 ± 6.1 65.8 ± 2.5 81.1 ± 2.5 0.0534
Glomus
claroideum
72.4 ± 32 58.3 ± 2.4 70.9 ± 2.2 0.0080
Propagules cm
3
Glomus mosseae 36.5 21.5 120.0
Glomus sp. 21.5 12.0 46.5
Glomus
intraradices
21.5 46.5 145.0
Glomus
claroideum
46.5 21.5 46.5
Mean 31.5ab 25.4b 89.5a 0.0224
a
Means of four observations for spore populations and colonization, results of
one pooled sample for propagules (most probable number bioassay).
2328 D.D. Douds Jr. et al. / Bioresource Technology 101 (2010) 2326–2330
cific isolates of AM fungi. These characteristics are restrictive, espe-
cially the latter. Experiments conducted here demonstrated that
these restrictions are readily overcome.
Inoculum of AM fungi was successfully produced in compost
mixed with vermiculite, perlite, or horticultural potting media
(Table 2). However, propagule numbers tended to be greater in the
vermiculite based media. Propagules of AM fungi consist of spores,
colonized root pieces with vesicles, and infective hyphal fragments.
The laminar sheets of vermiculite may provide an environment
conducive to the growth and persistence of AM fungus hyphae.
The similar spore populations and colonization of roots among
the three media amendments support this idea.
The production of inoculum using perlite as the diluent offers
an added benefit. Among the farmers targeted by the on-farm inoc-
ulum production method are vegetable farmers who grow their
own seedlings for later transplanting to the field. The goal is to
mix the inoculum into the horticultural potting media in which
the seedlings are grown. An inoculum consisting primarily of per-
lite stands out in greater contrast to the predominantly dark col-
ored potting media than does a vermiculite and compost based
inoculum. This aids in mixing the inoculum homogeneously
throughout the potting media.
Two methods were used successfully to produce inocula of
indigenous isolates of AM fungi. Inocula were produced both by
(1) mixing field soil into the compost and vermiculite mixture in
the production bags (Figs. 1 and 2) pre-colonizing the bahiagrass
seedlings during the preliminary greenhouse growth phase by
adding a small amount of field soil to the SSVT mix (Table 1,
Fig. 2). In addition to making the method more practical, there
are several reasons why the production and utilization of the indig-
enous community of AM fungi can be desirable. First, there is some
evidence that the indigenous community of AM fungi can be more
effective in promoting the growth of plants in its native soil than
introduced isolates (Sreenivassa, 1992; Oliveira et al., 2005). Sec-
ondly, functional diversity has been demonstrated among AM fun-
gi in characteristics such as glomalin production (Wright and
Upadhyaya, 1996), variability in growth response among plant spe-
cies (Pope et al., 1983), spatial exploration of the soil for P (Smith
et al., 2004), and patterns of colonization (Hart and Reader,
2002). This makes it beneficial to have a taxonomically diverse
inoculum, which was accomplished when the bahiagrass was inoc-
ulated with field soil.
Some degree of the diversity of the original community will be
lost, however, for a number of reasons. AM fungi are sometimes
patchy in distribution, and may not be present in the soil used to
inoculate the bahiagrass (St. John and Koske, 1988). In addition,
even though bahiagrass is a very good general host for AM fungi
(Struble and Skipper, 1985), use of the bahiagrass monoculture will
tend to select for those AM fungi among the community which
propagate best with that particular host (Hetrick and Bloom,
1986; Giovannetti et al., 1988). This is the primary reason why,
in the original method (Douds et al., 2006), different species of
AM fungi are propagated in separate bags rather than all together
in the same bag. This avoids competition among the isolates for
occupancy of the roots and, concomitantly, for the fixed carbon
needed for growth. The species diversity in the original method
comes from mixing inocula of various species together at the time
of harvest and utilization of the inoculum. Since AM fungus species
diversity and plant community diversity are correlated (van der
Heijden et al., 1998), the potential weakness in the propagation
of indigenous AM fungi via the on-farm methods developed here
could be combated by using several different host plants. This op-
tion has not been explored.
The combination of these modifications to the on-farm system,
e.g. the propagation of indigenous isolates of AM fungi in a perlite
plus compost mixture, was not tested explicitly. Since the starting
material in both systems, i.e. colonized bahiagrass seedlings in
SSVT mix, was the same and propagated AM fungi well in perlite,
vermiculite, or potting media plus compost mixtures, it should
not matter whether the AM fungi colonizing the bahiagrass was
a selected isolate or members of the indigenous community.
The naturally-occurring symbiosis between AM fungi and the
roots of most crop plants is a potentially powerful tool in sustain-
able agricultural systems. Agronomists and horticulturists now
have greater flexibility for the on-farm production of AM fungus
inoculum in temperateclimates.
Acknowledgements
We gratefully acknowledge the technical assistance of Joe Lee
and Stephanie Campbell. This work was supported in part by
USDA-CSREES Sustainable Agriculture Research and Education
Grant No. LNE03-172.
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... Various methods in use for the mass production of mycorrhizal inoculum include hydroponics, aeroponics, in vitro cultivation, substrate-based pot cultures, and on-farm production [7][8][9]. Among all these methods of AM fungus production, pot culture and on-farm production are the simplest and most widely used methods [10,11]. In general, an indigenous AM fungus inoculum that is locally suited to soil conditions performs best in providing plants and soil with numerous benefits [11]. ...
... Among all the available methods used in pot culture multiplication of AM fungi, amendments with organic substrates, such as vermicompost, peat-based materials, and agricultural wastes, and inorganic substrates, such as perlite and vermiculite, are most popular [7,8,10,11,16]. Douds et al. [8] suggested a mixture of compost from yard clippings and vermiculite in raised beds supplied with bahiagrass pre-colonized seedlings to produce a concerted AM fungus inoculum. Singh et al. [21] used vermicompost as the carrier material for delivering AM propagules and found that application not only enhanced the growth of target plants but also enhanced the AM fungal population in the rhizosphere of inoculated plants. ...
... Substrates with high OC and low P support AM fungal abundance [8,10,11]. Therefore, soybean hulls, kani, and burnt DOC ash were chosen. A similar approach was adopted by Douds et al. [10] in which to support the spore formation of Glomus mosseae, Glomus intraradices, and Gigaspora rosea, they selected a mixture of dairy manure and leaf composts having low phosphorus and high nitrogen content. ...
Article
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This study considered soybean processing mill waste (hulls) as an organic substrate for mass multiplication of indigenous arbuscular mycorrhizal (AM) fungi on sorghum and amaranthus as hosts. In the first experiment, from seven soybean processing mill wastes, three wastes were evaluated for their ability to multiply AM fungi on the two host plants. Among these wastes, hulls were found to be promising for the multiplication of AM fungi and were further examined in a second experiment in combination with vermicompost (VC), a mix of hulls plus vermicompost (SH + VC) amended with soil: sand mix (3:1 v/v) and a soil–sand mix used as a control (SS) in polybags containing the previous two host species. We found that SH blended with VC significantly improved AM fungus production in sorghum polybags assessed through microscopic (spore density in soil, colonization in roots) and biochemical parameters (AM signature lipids in soil: 16:1ω5cis neutral lipid fatty acid (NLFA); phospholipids fatty acid (PLFA) g⁻¹ soil; 16:1ω5cis ester lipid fatty acid (ELFA) g⁻¹ both in soil and roots; and glomalin content in soil. SH + VC contained significantly greater AM fungus populations than the other substrate combinations examined. Principal component analysis (PCA) also identified sorghum as a potential host supporting AM fungus populations particularly when grown under SH + VC conditions. Hence, the combination of soybean hulls and vermicompost was found to be a promising substrate for the mass production of AM fungi using sorghum as a host. These findings have important implications for developing AM fungus inoculum production strategies at the commercial scale.
... Arbuscular mycorrhizal fungi (AMF) are important components of soil that can be exploited to enhance the development of crops and contribute to the establishment of more sustainable agriculture. This approach would reduce, or even eliminate, the need for chemical fertilizers and pesticides in organic farming (Douds Jr et al., 2008. This is possible because plants colonized by AMF exploit higher volumes of soil (Smith and Read, 1997) and have higher nutrient uptake (Smith et al., 2011), as well as increased tolerance to drought, saline stress (Augé, 2001), and heavy metals (Rozpadek et al., 2014). ...
... Thus, they provide many additional benefits to acting as plant growth promoting agents (Azcón-Aguilar et al., 1997) and as a biological control against phytopathogens (Pozo et al., 2002;Moreira et al., 2016). Arbuscular mycorrhizal fungi are obligate biotrophic (Douds Jr et al., 2006, 2008, and complete their life cycles solely associated with the roots of living plants. Consequently, they cannot be multiplied separately in a defined culture medium (Douds Jr et al., 2006). ...
... To stimulate inocula production, studies have been developed to test the multiplication of spores under field conditions, called the on-farm method. These studies explore mycorrhizal colonization with fungal isolates that are environmentally adapted to local conditions, potentially representing a low-cost alternative for farmers (Douds Jr et al., 2006, 2008Schlemper and Stürmer, 2014). This method allows farmers and nursery workers to access inoculums with the most effective strains for their culture and their soil and climate conditions; furthermore, they can produce seedlings already mycorrhizal, with this benefit enhancing the establishment of seedlings in the field (Douds Jr et al., 2008). ...
Article
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The production of inoculum from arbuscular mycorrhizal fungi (AMF) at a large scale and low cost is essential for establishing methods to assist in producing pineapple plantlets with high nutritional and phytosanitary quality. However, this objective is difficult to accomplish because of the biotrophic nature of these fungi. The on-farm multiplication method for AMF inoculum presents a good alternative to supply the demand for the production of glomerospores. This study aimed to multiply and evaluate AMF inoculum originating from isolated species (including Rhizophagus clarus, Claroideoglomus etunicatum) versus native AMF from pineapple and coffee plantations multiplied by the on-farm method on the colonization in pineapple plantlets. Initially, inocula of R. clarus, C. etunicatum, and native AMF (pineapple and coffee) were multiplied by the on-farm method in Sorghum bicolor. After four months, the number of AMF spores and the percentage of viable spores at the layers of 0.00-0.05 and 0.05-0.10 m were evaluated. There were no differences in spore numbers in relation to the source of the inoculum (R. clarus, C. etunicatum, pineapple, and coffee) and evaluated layers, with an average number of 605 spores per 100 cm3 of soil. The percentage of viable spores was greater at the layer of 0.00-0.05 m (76.32 %) compared to the layer of 0.05-0.10 m (72.05 %), regardless of the inoculum source. The viability of the inoculum obtained from C. etunicatum was higher than that from the coffee crop (77.93 and 68.06 %, respectively). Subsequently, the spores were inoculated in pineapple plantlets to assess the rate of colonization. Pineapple plantlets inoculated with AMF had an average of colonization of 18 and 67.73 % after 50 and 180 days cultivation, respectively, with no significant difference being detected between treatments. Therefore, on-farm inoculum production was effective at multiplying the AMF of both isolates of R. clarus and C. etunicatum, as well as for commercial crops (pineapple and coffee), with spores having high viability. Arbuscular mycorrhizal fungi colonized pineapple plantlets independently of the inoculum utilized and favored its growth.
... The arbuscular mycorrhizal fungi (AMF -Glomeromycota) are obligate symbionts associated with plant roots (Parniske 2008). They have potential to increase the nutrients uptake and to stimulate plant growth (Douds Jr. et al. 2008.). Additionally, they have potential to reduce the effects of biotic and abiotic stress factors, improving soil quality. ...
... Regarding the farmers, it should be highlighted that the possibility of AMF inoculants production can be obtained through accessible procedures, such as the on-farm method (Douds Jr. et al. 2008), which favors the transplanting of colonized seedlings to the field. The on-farm method is an important and environmentally friendly social technology, because it favors not only the reduction of inputs, but also increases plant growth (Czerniak & Stümer 2014) and the farmers autonomy in the inoculant production. ...
Article
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Euterpe edulis Martius is one of the endangered species of the Brazilian Atlantic Forest which presents low germination rate and slow seedling growth. Arbuscular mycorrhizal fungi (AMF) are known by their symbiosis with plants, promoting an increase of water and nutrientes uptake. This study aimed at evaluating the effect of AMF inoculation on the initial growth (6 months) and nutrients uptake in E. edulis seedlings. Treatments consisted of the inoculation of pre-germinated seeds with AMF spores collected from three sites (forest, Juçara and crop), as well as a control with no inoculation. Seedlings growth, number of AMF spores in the substrate and uptake of the N, P, K, Ca and Mg macronutrients in plant tissues were analyzed. Inoculation with AMF improved the initial growth of seedlings, regardless of the source of inoculum used in the experiment, and the inoculation with material collected from rhizosphere increased the shoot and root dry biomass of seedlings by 43 % and 61 %, respectively. Inoculation with AMF provided a greater accumulation of all nutrients assessed in the shoot and root of seedlings, especially when spores were collected at the Juçara site. Inoculation with AMF is a promising strategy to improve the spread of this species. © 2016, Universidade Federal De Goias (UFG). All rights reserved.
... For each 50 L potting soil, organic substance � 65%, N+P2O5+K2O � 5%, pH = 5.5-6.5. We did not measure the characteristics of vermiculite because it is a well-known nutrient-poor substrate [36]. The resource amount was characterized by altering the percentage of nutrient-rich soil; i.e., a low resource amount contained 25% nutrient-rich soil and a high resource amount contained 50% nutrient-rich soil. ...
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Soil spatial heterogeneity involves nutrients being patchily distributed at a range of scales and is prevalent in natural habitats. However, little is known about the effect of soil spatial configurations at the small scale on plant foraging behavior and plant growth under different resource amounts. Here, we experimentally investigated how a stoloniferous species, Trifolium repens , responded to varied resource amounts and spatial configuration combinations. Plant foraging behavior (i.e., the orientation of the primary stolon, mean length of the primary stolon, foraging precision, and foraging scale) and plant growth (i.e., total biomass, root biomass, shoot biomass, and root/shoot) were compared among differently designed configurations of soil resources in different amounts. The relationships of foraging behavior and plant biomass were analyzed. The results showed that the effect of the spatial configuration of soil resources on Trifolium repens depended on the resource amount. Specifically, when the total resource amount was low, fragmented soil patches promoted root foraging and increased Trifolium repens plant biomass; however, when the total resource amount was high, the soil spatial configuration did not affect foraging behavior or plant growth. Our results also showed that plant growth was facilitated by root foraging scale to adapt to low resource amounts. We conclude that the spatial configuration of soil resources at small scales affects whole plant growth, which is mediated by a distinct foraging strategy. These findings contribute to a better understanding of how the growth strategy of clonal plants responds to heterogeneous environments caused by different resource amounts and its spatial configurations.
... The expensive technology of inoculum production comprises formation of single cultures of AMF. A cheaper method is the on-farm system (farmers on their own property can produce inocula) (Douds et al. 2008(Douds et al. , 2010. Both indigenous and introduced AMF can be included; however, native AMF can be more effi cient due to local adaptation to the environment (Sreenivassa 1992 ). Infective propagules of AMF (spores, hypha, and colonized roots) can be used as inocula (Sieverding 1991 ). ...
Chapter
The advances in plant cataloging and the increase of studies on mycorrhiza in South America (SA) have led to the compilation of information to better understand the native ecosystems and their constraints. Selected environments ranging from natural to anthropized ecosystems were analyzed according to their fungal-endophyte-associations and fungal-symbionts occurrence in relation to relevant physical-chemical properties of soils of the principal biomes in SA. Considering conservation units, no National Park is under continuous research in SA and few ones have been investigated for mycorrhizal symbioses. Ectomycorrhizas, with scant host-tree species in SA, are also investigated in Argentina and Chile forestry and mostly in exotic trees in Brazil. The study of the mycorrhizas and mycorrhizal fungi ecology and their response to global change, which is urgently recommended, is still incipient. Further, the publication revisions showed that Brazil, Argentina, Chile, Venezuela, and Ecuador are the countries with more published reports. Studies on mycorrhizas have developed largely; however, most of them were concerned with diversity and morphology, while the applications of mycorrhizas in environmental issues are still limited. The cooperative work between researchers from the Northern Hemisphere and SA could lead to greater advances on the quick and improved knowledge of the wonderful SA ecosystems and their mycorrhizas. This chapter revises and discusses the advances in mycorrhizal fungi understanding drawing on recent research.
... Trap plants are a group of plants known for their dependence on mycorrhiza and therefore used as routine hosts for the enrichment of AMF in pot culture for spore propagation. Recently, onfarm production of AMF inoculum has received considerable attention due to the cost-effectiveness and the use of indigenous micro-organisms (Douds et al. 2006(Douds et al. , 2008. The on-farm production helps to cut down the cost of processing steps involved in other methods like trap pot cultures. ...
Article
Aims: The propagation of pure cultures of AMF is an essential requirement for their large scale agricultural application and commercialization as biofertilizers. The present study aimed to propagate AMF using the single spore inoculation technique and compare their propagation ability with the known reference spores. Methods and results: Arbuscular mycorrhizal fungal spores were collected from the salt-affected Saemangeum reclaimed soil in South Korea. The technique involved inoculation of Sorghum-Sudan grass (Sorghum bicolor L.) seedlings with single, healthy spores on filter paper followed by the transfer of successfully colonized seedlings to 1 kg capacity pots containing sterilized soil. After the first plant cycle, the contents were transferred to 2.5 kg capacity pots containing sterilized soil. Among the 150 inoculants, only 27 seedlings were colonized by AMF spores. After 240 days, five inoculants among the 27 seedlings resulted in the production of over 500 spores. The 18S rDNA sequencing of spores revealed that the spores produced through single spore inoculation method belonged to Gigaspora margarita, Claroideoglomus lamellosum, and Funneliformis mosseae. Furthermore, indigenous spore Funneliformis mosseae M-1 reported a higher spore count than the reference spores. Conclusions: The AMF spores produced using single spore inoculation technique may serve as potential bio-inoculants with an advantage of being more readily adopted by farmers due to the lack of requirement of a skilled technique in spore propagation. Significance and impact of study: The results of the current study describes the feasible and cost effective method to mass produce AMF spores for large scale application. The AMF spores obtained from this method can effectively colonize plant roots and may be easily introduced to the new environment. This article is protected by copyright. All rights reserved.
... En la elección de los sustratos a evaluar se consideró la sensibilidad de las micorrizas a los niveles de fósforo, se seleccionaron dos sustratos con bajo contenido en P (<1 ppm). No se utilizó régimen de fertilización en las plántulas inoculadas por la relación inversa entre la disponibilidad de P y la colonización de hongos micorrízicos (21,22). .A ello se atribuye que el sustrato 3, con alto contenido de P, no mostró diferencia significativa entre las plantas inoculadas y sin inocular. ...
Article
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Micropropagation through in vitro plant cultivation allows large-scale production of identical individuals genetically to the starting material. Woody species have difficulties in the acclimatization stage due to their slowness in the development of physiological response to environmental changes. The ultimate success of in vitro propagation depends on the capacity of plants to adapt in the moment of transfering from the laboratory to the greenhouse conditions. One of the tools to offset losses during acclimatization is the use of arbuscular mycorrhizal fungi (AMF), which sets mutualistic symbiotic associations unspecific with 90 % of vascular plants. AMF, because of their action as agents of growth bioregulation as bio-fertilizers or biocontrollers have received special attention in handling and propagation of fruit plants. In this work the effects of inoculation with AMF at the start of acclimatization are presented to mycorrhization. Inoculation with one type of AMF over two rootstocks of apple was done in a clone of M9 and one rootstock of the Cornell-Geneva series (RN29 and Geneva®41 respectively) set in three different substrates. Seedlings inoculated with AMF when compared to the control, presented further expansion of their leaves, bigger diameter and greater height, all significantly different. Acclimatization period was reduced from 60 to 40 days. The incorporation of this type of technologies could generate a more sustainable management of plant production with less use of agrochemicals
Chapter
Beneficial soil-borne bacteria and fungi are central to the performance of most plants. Knowledge of beneficial microorganisms and the processes in topsoils that favour the association of beneficial organisms with plants allows us to better manage soils for higher productivity and environment sustainability. This review describes the main groups of symbiotic and free-living organisms and explores how they contribute to plant and soil health in managed and natural ecosystems. Many field studies have investigated the biodiversity, ecology and function of beneficial organisms in relation to root distribution in topsoils and land management practices. There is scant information however on whether beneficial bacteria and fungi can persist and enhance root function in subsoils. Opportunities for enhancing beneficial plant-microbe interactions in the subsoil deserve scrutiny particularly as crop productivity is becoming more dependent on subsoil moisture with declines in rainfall in many parts of the world.
Chapter
For a better understanding of natural, degraded areas and agro-ecosystems, the study of surface and deep soil responses to global change is required. To enhance the resilience of soil ecosystems, the examination and use of arbuscular mycorrhizas, was indicated since they are drivers of nutrient cycles participating in some ecosystem services, such as nutrient acquisition by plants. Currently, there is a holistic vision of AMF as multipurpose organisms with complex ecological functions in the soil. This chapter discusses advances on mycorrhizal fungi based on recent research from Southamerican countries. New reports on the occurrence of mycorrhizas in Amazonian dark earth, soils amended with vermicompost and biochar have resulted in a more detailed understanding of the soil biology from South America. Studies on mycorrhizas have developed largely; however, the understanding of mycorrhizas in anthropic environments are still incipient, and its limitations constitute a barrier for the contribution to sustainable cropping and forest systems. Few reports from South America showed that the addition of soil conditioners resulted in increases in plant cover and plant species richness. In this sense, the biochar/mycorrhizas interactions can be prioritized for sequestration of carbon in soils to contribute to climate change mitigation.
Chapter
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Numerous techniques have been developed in the past few decades for the mass production of arbuscular mycorrhizal (AM) fungi. The main obstacle behind the mass production techniques is the obligatory nature of these biotropic fungi and species level identification is not possible at early stage of development. Currently, in vitro cultivation methods such as hydroponic system and root organ culture have been widely used for mass production of AM fungi. But traditional method i.e. mass production in soil based media and living host is very popular and economical for the rapid production of these AM fungi. The aim of this review article is to highlight the recent and advanced methods used for mass production of AM fungi.
Article
When the influence of five host plants on spores produced by three mycorrhizal fungi was tested, the number of spores produced was highly variable within treatment. Sporulation of Glomus fasciculatum was significantly influenced by host plant but spore production by G. macrocarpum and G. mosseae was not. Spore production was not correlated with degree of root colonization but with plant dry weight, although differences in dry weight alone could not explain the influence of host plant on G. fasciculatum sporulation. Apparently, sporulation of G. fasciculatum is more heavily influenced by host plant than is sporulation of G. macrocarpum and G. mosseae. Different levels of host-fungus affinity may exist. When the efficiency (ability to colonize) of the inoculum produced by the three fungi on the five host plants was compared, few significant differences were evident even at the lowest dilution of 1.4 spores/42 g soil. Thus, the ability of spores to colonize their host may be relatively constant despite differences in spore numbers which have been produced.
Article
High N fertilization reduced percent infection by Glomus spp. ( Glomus fasciculatus (Thaxter Gerd. & Trappe and G. mossae (Nich. and Gerd.) & Trappe) endomycorrhizae on inoculated Podocarpus macrophyllus Thunb., Pittosporum tobira Thunb., and Rhododendron simsii Planch. plants. Inoculation with Glomus spp. benefited growth of the 3 woody plant species even at high levels of fertilization (1250 N, 1250 K and 230 Mg kg/ha·yr ⁻¹ ) although leaf nutrients levels showed little difference from noninoculated plants.
Article
Arbuscular mycorrhizae are the plant symbiosis the most widely spread on the planet. These fungi, grouped in the phylum Glomeromycota, are distributed over all terrestrial ecosystems and found associated with the majority of land plants. To the well-known positive impacts of arbuscular mycorrhizae on plant yields should be added several other benefits such as a better survival rate of colonized plants, the maintenance of plant biodiversity, the improvement of soil microflora, and the reduction in harmful effects of both biotic and abiotic environmental stresses. Given such a panoply of benefits to plants and their environment, one could believe that mycorrhizae represent a panacea for solving problems related to plant production and plant protection. In fact, the "plant - mycorrhizae - pathogen - environment" complex constitutes a standard condition to be maintained or to be recovered in order to ensure the sustainability of the environment. The potential of mycorrhizae as a biocontrol agent globally covers five known mechanisms of interaction. Three of them concern the direct effect of symbiosis on plants. They are: 1) plant growth stimulation through an increased nutritive contribution and, consequently, better plant health; 2) the morphological transformation of the root system; and 3) the induction or suppression of defense mechanisms, and this mainly at the enzymatic level. Another mechanism concerns the pathogen: 4) through a direct competition with mycorrhizal fungi linked with nutrient availability and infection sites. Finally, mycorrhizae indirectly influence the soil structure and quality through: 5) the modification of the soil microflora and an increase in organic matter.
A rapid automated method for total protein nitrogen has been developed, using the Technicon AutoAnalyzer with block digestor. Ammonia is determined by the automated ammoniasalicylate reaction at a rate of 40 samples/hr. A number of variations in digestion parameters have been evaluated. Hydrogen peroxide was used as a digestion accelerator. A salt-acid ratio of 1:1 and a block temperature of 425°C were chosen. The catalysts evaluated included HgO, CuS04, Se02, and Ti02/CuS04. Maximum nitrogen recovery in the shortest time (30 mill) was achieved with HgO as the catalyst. The results from multiple analyses of 10 experimental samples of different refractoriness with the block digestor method, using HgO or CuS04 as a catalyst, compared well with the results by the official AOAC Kjeldahl method. The accuracy, precision, economy, and saving of space offered by the Missouri-Technicon block digestor method make this an attractive alternative to classical Kjeldahl analysis for large numbers of total protein determinations in samples of different refractoriness. The AutoAnalyzer cartridge manifold is simple and reproducible in its performance, and a large dilution of the sample is made on dialysis which eliminates the matrix effects from the sample digests. At least 250 samples can be analyzed in 9 hr with 1 instrumentation setup.
Article
Three vegetable crops, namely coriander (Coriandum sativum), fenugreek (Trigonella foenumgraecum), and carrot (Daucus carota), were inoculated with vesicular-arbuscular mycorrhiza (VAM) fungi and grown in nutrient-deficient sandy-loam soils amended with organic matter. Under field conditions, shoot and root dry weights and total uptake of P and N were significantly increased in all the inoculated plants. The crops differed in the extent to which they were colonized by VAM fungi, the colonization being 76% in coriander, 63% in carrot, and 60% in fenugreek. Infection propagules were produced in greater numbers on coriander and fenugreek. The extent of increase in green yield following VAM inoculation was greater in plots amended with leaf compost in equal proportions (1:1) than in those amended with a higher proportion (2:1). The high levels of VAM colonization and large number of infectious propagules demonstrated the potential of these crops as substrates for inoculum production. The increased yields indicate the possibilities of using VAM to increase yield.
Article
Arbuscular mycorrhizal (AM) fungi colonize the roots of the majority of crop plants, forming a symbiosis that potentially enhances nutrient uptake, pest resistance, water relations, and soil aggregation. Inoculation with effective isolates of AM fungi is one way of ensuring the potential benefits of the symbiosis for plant production. Although inocula are available commercially, on-farm production of AM fungus inoculum would save farmers the associated processing and shipping costs. In addition, farmers could produce locally adapted isolates and generate a taxonomically diverse inoculum. On-farm inoculum production methods entail increasing inoculated isolates or indigenous AM fungi in fumigated or unfumigated field soil, respectively, or transplanting pre-colonized host plants into compost-based substrates. Subsequent delivery of the inoculum with seed to the planting hole in the field presents technological barriers that make these methods more viable in labor-intensive small farms. However, a readily available method for utilization of these inocula is mixing them into potting media for growth of vegetable seedlings for transplant to the field. Direct application of these inocula to the field and transplant of seedlings precolonized by these inocula have resulted in enhanced crop growth and yield.