Polymicrobial Multifunctional Approach for Enhancement of Crop 1

Productivity

Figure 3.2 Ectomycorrhizal and endomycorrhizal colonization of Acacia nilotica seed-

lings (Saravanan, 1998; Saravanan & Natrajan, 2000). a) Morphology of a root colonized
by the ectomycorrhizal fungus, Pisolithus tinctorius. b) Cross-section of a root colonized
by P. tinctorius. Thick mycelial covering (mantle) on the surface of the root (yellow
arrows) and intercellular hyphal network (“Hartig net”, shown by red arrows). 1.
epidermal cells;
2.cortical cells. c) Root surface showing germinated spores of Glomus mosseae (an AM
fungus) d) Stained arbuscules of G. mosseae in the root (arrows) e) Stained vesicles of
G. mosseae in the root (arrows). (For interpretation of the references to color in this
figure legend, the reader is referred to the online version of this book.)

plant to the fungi may also occur through the intraradical hyphae. After
colonization,AM fungi produce runner hyphae (extraradicular hyphae) that
grow from the plant root into the soil and take up P and micronutrients,
which are transferred to the plant. AM fungal hyphae, which grow from
the plant root, have a high surface-to-volume ratio, making their absorp-
tive ability greater than that of plant roots (Azcon, 2009; Bonfante & Anca,
2009). Also, these hyphae are finer than roots and can enter into pores of
the soil that are inaccessible to roots.The size and the relative proportion
of hyphae within the root and in the soil vary greatly between different
AM species (Abdel Latef, 2011; Dumas-Gaudot et al., 2004; Lumini,
Orgiazzi, Borriello, Bonfante, & Bianciotto, 2010). Furthermore, AM fungi
produce another morphologically distinct type of hyphae that grow from
the roots and colonize other host plants in the proximity.
The magnitude of bacteria/mycorrhizal fungus/plant symbioses depends
on a number of variables such as the extent of fungal colonization, C-
transfer
 2 C. Adinarayana Reddy and R. S.

from the plant, the amount of P in the soil, and the relative amount of P
transferred to the plant (Lee et al., 2012; Sanon et al., 2012;Veresoglou et al.,
2012; Zamioudis & Pieterse, 2012).The diversity of AM fungi in an ecosys-
tem influences the diversity and productivity of plants in that
environment (Adesemoye & Kloepper, 2009; Andreas & Martin, 2006;
Angeles, Ouyang, Aguirre, Lammers, & Song, 2009; Bago et al., 2003;
Finlay, 2008). Symbi- otic N-fixation between rhizobia (including
Sinorhizobium, Bradyrhizobium, Mesorhizobium, and Azorhizobium) and a
host plant involves signal molecules and pathways. This symbiosis may be
favorably influenced by simultaneous AM fungal colonization with the same
host plant (Bonfante & Anca, 2009; Bonfante & Requena, 2011; Harrison,
2005).
Signal molecules and pathways of AM fungi and the host plant for the
onset of symbiosis and the beneficial mutualism between the two have
been reviewed (Akiyama & Hayashi, 2006; Bonfante & Requena, 2011;
Harrison, 2005; Kiers et al., 2011). Signals are exchanged between plant
roots and AM fungi that are important for plant mycorrhizal symbiosis.
Recent evidence suggests that phytohormones produced by the plant
including strigolac- tones stimulate fungal metabolism as well as hyphal
branching (Bonfante & Requena, 2011; Castillo & Pawlowska, 2010; Dodd
& Ruiz-Lozano, 2012). The symbiotic signals (“Myc factors”) are active in
small doses and stimulate the growth of the root system, and the formation of
mycorrhiza association. Biotechnological processes for synthesizing large
amounts of these regulator molecules have been developed and will permit
the study of their effects on crops, with the objective of improving their
yield, without using chemical fertilizers (Bonfante & Requena, 2011;
Harrison, 2005).There are plant genes that are expressed by “Myc factors” at
the onset of AM-host plant symbiosis and the fungal genes involved in
structural and physiological alterations in the host plant have also been
identified (Dodd & Ruiz-Lozano, 2012; Hata et al., 2010). The physiological
functions of the host plant need to be modified so that the host plant can
provide the fungi with the required organic carbon compounds (through
root exudate) in exchange for water and nutrients pro- vided by the
fungus. Some common plant genes are expressed during the AM symbiosis
as well as N-fixation by rhizobium (Akiyama & Hayashi, 2006).

4.2. Ectomycorrhizae
Ectomycorrhizal fungi (EM fungi) are phylogenetically very diverse
and more than 2000 species of EM fungi worldwide have been identi-
fied, primarily from Basidiomycotina and Ascomycotina. These EM fungi
form characteristic mycorrhizal associations, almost entirely with woody
Polymicrobial Multifunctional Approach for Enhancement of Crop 3
Productivity

perennials, including Pinaceae, Betulaceae, Fagaceae, and Diperocarpaceae

in tropical, subtropical, and arid environments, and are regarded as key
organisms in nutrient and carbon cycles in forest ecosystems (Agerer, 2003;
Becerra et al., 2005; Jakucs, Kovacs, Agerer, Romsics, & Eros-Honti, 2005).
Unlike AM fungi, hyphae of EM fungi do not penetrate into the root cells
but are intercellular.The hyphae penetrate into the root cortex where they
form a hyphal network (“Hartig net”; see Fig. 3.2) in the intercellular space
through which minerals and nutrient materials are exchanged between the
fungus and the plant.The fungus forms a mantle of hyphae on the outside of
the plant root that extends into the surrounding soil (Anderson &
Cairney, 2007; Smith & Read, 2008).The structure of ectomycorrhizal
extramatrical mycelium (extraradical mycelium) varies considerably between
ectomycor- rhizal species, ranging from a weft of undifferentiated
mycelium around the root to a highly differentiated mycelium comprising
a foraging fungal front connected to roots via rhizomorphs (Bonfante &
Anca, 2009; Cairney, 2000). In angiosperm tree roots, the ectomycorrhizal
hyphae penetrate the epidermal layer and spread as hyphal network
(Hartig net) intercellularly (one cortical cell deep: Fig. 3.2, panels a and b),
but in the case of conifers and gymnosperms the hyphal penetration
reaches up to a depth of 3–4 cortical cells (Agueda, Parlade, de Miguel, &
Martinez-Pena, 2006; Lupatini, Bonnassis, Steffen, Oliveira, & Antoniolli,
2008; Massicotte, Melville, & Peterson, 2005). Similar to AM fungi,
ectomycorrhizae also exhibit syner- gistic interactions with other plant-
beneficial organisms such as symbiotic N2-fixers. For example,
ectomycorrhizal symbiosis enhanced the efficiency of inoculation of two
Bradyrhizobium strains on the growth of legumes (Andre et al., 2005). It
is also of interest that similar synergies were seen when AM fungus
(Glomus mosseae), EM fungus (Pisolithus tinctorius), and Bradyrhizobium
sp. were used together to inoculate Acacia nilotica, enhance- ment of N2
fixation, growth, and dry biomass were observed when all three organisms
were present (Saravanan and Natarajan, 1996, 2000). Moreover,
Bradyrhizobium sp. when co-inoculated with either the AM fungus or the
EM fungus, gave enhancement of N 2 fixatioin as compared to the control
with Bradyrhizobium sp. only (Fig. 3.3).
Ericoid and orchid mycorrhizas tend to be host-specific and colonize
only the plants in the family Ericaceae and Orchidaceae,respectively
(Bergero, Perotto, Girlanda,Vidano, & Luppi, 2000; Cairney, 2000;
Wilkinson, 2001). In ericoid mycorrhizas, colonization is simple.The fungus
develops inside epidermal cells, forming coils (hair-like roots enmeshed
in extensive weft of hyphae) that give rise to independent infection
units. Normally
 4 C. Adinarayana Reddy and R. S.

Figure 3.3 Potato dextrose agar (PDA) plate demonstrating the biocontrol property of
a PGPR bacterium (B1) against three fungal pathogens A, C, and F representing strains
of Alternaria, Curvularia, and Fusarium species, respectively. (For color version of this
figure, the reader is referred to the online version of this book.)

no sheath is formed. Fungi that form ericoid mycorrhizal associations are

all ascomycetes. These ericoid fungi play an important role in mobilizing
the organic nutrients in the soil and making them available to the plant
(Cairney, 2000; Smith & Read, 2008). In the ericoid type of mycorrhiza
(in Ericaceae tribes Ericeae,Vaccinieae, Rhododendreae, and Calluneae and
in related families), the fungus is endophytic. In the “arbutoid” type, found
in members of Ericaceae tribe Arbuteae and subfamilies Pyroloideae and
Monotropoideae, the association is ectendotrophic, i.e. the fungus grows
within and also ensheaths the root tissue.
Orchid mycorrhizal associations involve partially or completely
achlo- rophyllous plants (for some part of their life) and fungi of the
basidiomy- cete group (Cairney, 2000; Smith & Read, 2008). Fungal
symbionts of green orchids are highly effective saprophytes whereas
those of achloro- phyllous orchids are likely to form ectomycorrhizas on
autotrophic plants. Coils produced by orchid mycorrhizae occur mostly in
the inner layers of the root.
Polymicrobial Multifunctional Approach for Enhancement of Crop 5
Productivity

5. MICROBIAL INOCULANTS
To take advantage of the demonstrated beneficial effects of various
soil microbial groups in increasing plant growth and yields, many different
types of microbial inoculants (biofertilizers) have been in use for a long
time. Biofertilizers are defined as substances containing living microbes,
which when applied to seed, plant, or soil promote growth by the supply of
essential nutrients such as N, P, and other mineral nutrients. In this section,
the words “microbial inoculants” and “biofertilizer” are used
interchange- ably. There is a vast body of published research on the
construction of various microbial inoculants, their applications, and the
results obtained in greenhouse and field trials using these inoculants
(reviewed in Arora et al., 2011; Bashan, 1998; Dodd & Ruiz-Lozano,
2012; Kennedy et al., 2004; Malusa et al., 2012; Miransari, 2011). There is
growing interest in further research aimed at developing more effective,
economical, and environmen- tally friendly microbial inoculants.The obvious
reason behind this activity is the urgency to develop natural biofertilizers
to meet the food needs of the increasing human population and to
decrease our dependence on chemical fertilizers and pesticides.
The microbial inocula that have been in use mostly consisted of either
a single organism or a combination of beneficial organisms providing
one or more beneficial functions for obtaining increases in growth yields
of the crops tested. Primarily, one or more of the following three major
groups of microorganisms, considered beneficial to plant nutrition, were
included in constructing the bioinoculants/bioformulations: 1. AM and
ectomy- corrhizal fungi; 2. plant growth-promoting rhizobacteria
(PGPR), and
3. N2-fixing bacteria (Feldmann, Hutter, & Schneider, 2009; Fulton, 2011;
Ijdo, Cranenbrouck, & Declerck, 2011; Kennedy et al., 2004; Khan et al.,
2010; Malusa et al., 2012; Vessey, 2003). Since the late 1950s, mycorrhizal
fungi were utilized as biofertilizers to promote plant growth, because of their
abil- ity to increase the plant uptake of P, N, mineral nutrients, and water
(Feld- mann et al., 2009; Koide & Mosse, 2004; Miransari, 2011). In recent
years, research efforts in the field of microbial biofertilizers have steadily
increased and individual as well as various combinations of beneficial
microbes con- ferring one or multiple beneficial effects on plant growth
and yields have been described (Baldani & Baldani, 2005; Khalid et al.,
2009; Krishna, 2005; Peterson, 2004). Because of the extensive literature
on microbial inocu- lants, we have chosen to present here selected
representative examples of
 6 C. Adinarayana Reddy and R. S.

different microbial inoculants/formulations and some preliminary work

on polymicrobial inoculants.

5.1. Inocula, Carriers, and Applications

Inocula of various kinds (solid or liquid; bacterial or fungal; pure culture or
mixed culture inocula, etc.) have been in use for many years. One of the first
tasks to be completed in producing a microbial inoculant is to determine
the desired composition of organisms to be included in the formulation.
Once that is done, two other key aspects of the inoculant technology that
need to be addressed are the selection of a suitable carrier that provides
favorable microenvironment to keep the microbes in the formulation
viable until delivery to the field.
Mycorrhizal inocula are some of the most widely used biofertilizers
both in developed and developing countries. Much of the interest focused
on AM fungi because of the very wide range of economically important
food crops colonized by them ( Jeffries et al., 2003; Khan et al., 2010;
Peterson, Piche, & Plenchette, 1984). Preparation of inocula for AM
Fungi is quite involved because of their obligatory requirement for plant
tissue (Koide & Mosse, 2004). Commercial AMF inoculants are produced
mainly by grow- ing host plants in controlled greenhouse conditions, with
an inoculant containing different fungal structures (spores, hyphal
fragments, and mycor- rhizal root residues) from the roots of plants used
for propagation such as sorghum, maize, and onion. Substrates such as sand,
soil, or other materials such as zeolite and perlite are used to mass-produce
AM fungal inoculum in pots, bags, or beds (Adholeya,Tiwari, & Singh,
2005; Gianinazzi-Pearson, Séjalon-Delmas, Genre, Jeandroz, & Bonfante,
2007; Harrier & Watson, 2003; Ijdo et al., 2011).With this technique, it is
possible to reach high inoc- ulum densities (Feldmann et al., 2009). The
inoculum is diluted as needed prior to marketing using a carrier such as peat,
clay, and fly ash.
Methods for producing monoxenic cultures of AMF were developed in
the late 1980s utilizing split-plate cultures and Ri T-DNA transformed roots
of carrots under controlled conditions (St-Arnaud, Hamel,Vimard, Caron,
& Fortin, 1996). This method allows a higher efficiency than traditional
methods with the production on the average of 15,000 spores per Petri
dish 4–5 months after the beginning of the production cycle and has so
far been used mainly for physiological and genetic studies in the laboratory.
An improvement of this method resulted in production of about 65,000
spores in 7 months (Douds, 2002). A method for producing mycorrhizal
fungi on a larger commercial scale has also been described (Adholeya et
al., 2005).
Polymicrobial Multifunctional Approach for Enhancement of Crop 7
Productivity

Even so, the cost of producing the inoculums is somewhat high and may
not be practical for field applications.
Producing ectomycorrhizal fungi is relatively easy and can be
produced by fermentation using suitable media.The same is true for
producing inoc- ula of various trichoderma species. Inocula for most
bacteria used in plant growth formulations is usually prepared by growing
the organisms as aseptic liquid cultures, harvested, and the bacterial
suspension is diluted to give a
desired concentration of viable bacteria/ml (usually ≥108/ml). When mul-
tiple organisms are used in a given formulation, each organism is grown
individually and then mixed together to give a formulation with the right
amount of viable bacteria per ml (Reddy and Lalithakumari, 2009a).
Carriers for preparing inocula should be designed to provide a favorable
microenvironment for the PGPM to ensure their viability and adequate
shelf life of the inoculant formulation (preferably two months or more at
room temperature).A number of commercial inocula are sold as lyophilized
products that can be reconstituted with different liquid (or solid carriers).
The advantage with this is that the viability can be maintained for months
or years but a large-scale preparation is more involved, careful handling is
required, and the product may be relatively expensive. Maintaining viabil-
ity of the microbes after soil application is just as important as maintain-
ing viability during the storage period. Rhizosphere competence of the
microbes used in preparing the inoculant is an important requirement. A
desirable carrier should be easily available, stable, economical, ecofriendly,
easy to apply, and have good moisture-holding capacity and pH-buffering
capacity (Malusa et al., 2012). Carrier-type also determines the form of the
inoculant (solid or liquid). Common solid carriers include organic materials
such as peat, coal, clay, saw dust, wheat bran, peat supplemented with chitin-
containing materials, and inorganic materials such as vermiculite, perlite,
silicates, kaolin, and bentonite (see Adholeya et al., 2005; Kennedy et al.,
2004; Malusa et al., 2012; Fig. 3.4, panel d). In the case of solid inocula, the
size of the granules or beads used for immobilization of the microbe may
vary from 75 µm to 250 µm. Liquid inoculants can be broth cultures, sus-
pensions in solutions of humic acid, or suspensions in mineral or organic
oils or oil-in-water suspensions. Liquid or powder-type inoculants can be
used to coat the seeds, for root dipping at the time of transplantation of
seedlings, or apply directly into the furrow (or seed beds) or as a foliar spray.
Humic acid has been a popular carrier for a number of microbial inocu-
lant formulations for producing a stable inoculant at ambient temperature
(Reddy & Lalithakumari, 2009a, 2009b).
 8 C. Adinarayana Reddy and R. S.

Figure 3.4 Preparation of mycorrhizal inocula for enhancing cropproduction

(Saravanan, 1998; Saravanan & Natrajan, 2000). a) Spores of arbuscular mycorrhizae
(AM) garden soil for mass inoculum production. Numbers 1 to 5 show spores of
Glomus mosseae,
G. albidum, G. fasciculatum, G. macrocarpum, and G. dimorphicum, respectively. b)
Pot cultures of the five AM fungi mentioned in (a) above for production of spore
inocula.
c) Fruit body of ectomycorrhizal fungus, Pisolithus tinctorius. d) Mycelial inocula
(45-day-old) of the ectomycorrhizal fungus P. tinctorius. e) P. tinctorius immobilized
in sodium alginate beads (Mycobead). (For color version of this figure, the reader is
referred to the online version of this book.)

Humic acid is a principal component of humic substances, which are the

major organic constituents of soil, peat, and coal. It is primarily produced
by biodegradation of dead organic matter. Humic acids consist of a mixture
of weak aliphatic and aromatic organic acids that are not soluble in water
under acidic conditions but are soluble under alkaline conditions. Humic
acids play a vital role in soil fertility and plant nutrition. The benefits of
humic acids include: stimulation of beneficial microbial activity, better seed
germination, breaking up compacted soils allowing enhanced water pen-
etration and better root growth, increased fertilizer retention, enhanced P
uptake, chelation of micronutrients and increasing their bioavailability, and
addition of organic matter in organically deficient soils (see R. E. Petit, web
link: http://www.calciumproducts.com/articles/Dr.Pettit Humate.pdf ).
Peat has been commonly used as a carrier for PGPR, particularly for rhi-
zobial inoculants, due to its wide availability and a long history of field trials
(Babalola, 2010; Dutta & Podile, 2010; Siddiqui, 2006; Somers et al., 2004).
Polymicrobial Multifunctional Approach for Enhancement of Crop 9
Productivity

When added to peat, PGPR maintain activity and in some cases can continue
to multiply during the storage period, thus increasing their population size
(Adesemoye & Kloepper, 2009). However, a major drawback of peat is the
variability in its quality and composition (acidity), which affect the viability
of microbes. For example, adding N-fixing and P-solubilizing bacteria
increased the amount of N and P availability in the final product. Saw dust
was shown to be useful as a carrier for production of inocula containing
different strains of bacteria (reviewed in Arora et al., 2011; Babalola, 2010;
Miransari, 2011).
Organic polymers such as alginate (D-mannuronic acid and L-glucuronic
acid polymer), hydroxyethylcellulose, and carrageenan have also been sug-
gested as inoculum carriers. Different polymers entrap or encapsulate the
microorganisms, which are slowly released over a period of time. Alginate
appears to be a popular carrier in this group. It is the most common poly-
mer used for encapsulation of microbes (Fig. 3.4d). Formulations with
organic polymers as carriers offer a relatively long shelf life even at ambient
temperature (see Malusa et al., 2012 for details).
Application methods for the delivery of PGPM to crops in the field
are relatively limited. Farmers are not keen on purchasing specialized
equipment to be used for microbial-based products. Formulated inocula
should be read- ily applied using standard farming machinery to make it
appealing to farm- ers. Application of liquid inoculant is often more
convenient for the farmer because of less time required, ease of application,
and the equipment routinely used on a farm can be used. The development of
inexpensive and efficient technologies for the efficient delivery of inocula,
probably by modification of sprayers and sprinklers normally used for plant
irrigation, could facilitate use of PGPM formulations. Soil inoculation can be
done either with solid or liquid formulations. Normally, the carrier is mixed
with the inoculum in the factory, but it could be mixed by the farmer prior to
application, especially when liquid formulations are used. Depending on
the particular inoculant formulation, the inocula can be used for seed
coating, for dipping seedlings, direct application to the furrow, or as foliar
application.The use of fertilizers that were produced by mixing organic
matrices and insoluble phosphates with the addition of selected P-
solubilizing microorganisms can also be con- sidered for applying PGPM to
crops. Such fertilizers increase the availability of nutrients (particularly of P)
to plants and also improve the tolerance of the plant to pathogens (Khalid et
al., 2009; Malusa et al., 2012).
Using PGPM strains that form stable and effective biofilms could be a
strategy for producing commercially viable inoculant formulations (Malusa
et al., 2012; Seneviratne, Zavahir, Bandara, & Weerasekara, 2008).A
majority
 1 C. Adinarayana Reddy and R. S.

of plant-associated bacteria found on roots and in soil are found to form

biofilms (Ude, Arnold, Moon, Timms-Wilson, & Spiers, 2006). Bacterial,
fungal, and bacteria/fungal biofilms were suggested as possible inoculants.
This is a novel and interesting idea, but to what extent this approach would
be practiced remains to be seen.

5.2. Monoculture and Co-culture Inoculant Formulations

Rhizobial inoculants are extensively used around the world and the
ability of rhizobia to increase plant growth and yields, resulting in a lower
input of
chemical fertilizers, is well established.The plant growth-promoting ability of
rhizobia as inoculants varies with soil type, moisture, abundance and activity
of native rhizobia, yield potential of the crop, available N in the soil, crop
rota- tion, and other properties (Hilali, Przrost, Broughton, & Antoun,
2001). For example, an inoculant containing strains of R. leguminosarum bv.
trifolii, isolated from roots of wheat cultivated in rotation with clover from
loamy sand Rabat soils, gave a 24% increase (P < 0.1) in wheat dry biomass
and grain yields, while those isolated from the silty clay Merchouch soils
gave no appreciable increases in growth and yields. In trials conducted in
arid areas on legumes like mung bean (Vigna radiata), Bradyrhizobiuim
inoculation gave up to 10–25% yield benefits with normal rainfall (Adesemoye
et al., 2009; Bashan, 1998).
Field trials with P. fluorescens Pf1 showed that foliar application of this
organism at 7-d intervals consistently reduced the incidence of blister blight
(Exobasidium vexans) disease in tea (Camellia sinensis), almost comparable
in effectiveness with that of the chemical fungicide used. Also, tea yield
increased significantly compared to the untreated control
(Saravanakumar, Vijayakumar, Kumar, & Samiyappan, 2007). Ardakani,
Heydar, Khorasani, and Arjmandi (2010) showed that biocontrol efficacy of
strains of P. fluores- cens, using bentonite or peat as a carrier, was much
higher in protecting cot- ton seedlings against damping-off disease, as
compared to controls treated with the standard carboxin-thiram fungicide.
Bharathi, Vivekananthan, Harish, Ramanathan, and Samiyappan (2004)
evaluated the biocontrol effi- cacy of 13 PGPR strains of P. fluorescens
(Pf1) and B. subtilis against chilli fruit rot and die-back diseases caused by
Colletotrichum capsici, and found them to be in increasing the seed
germination and seedling vigor.
A number of studies showed that co-inoculation of two or more PGPR
organism(s) gives better productivity for a range of crops. For example,
legumes inoculated with Rhizobium and Azospirillum gave increase in
bio- mass, yield, and nitrogen content. Also, early and enhanced nodulation
by rhizobia co-inoculated with Azospirillum was attributable to an
 Polymicrobial Multifunctional Approach for Enhancement of Crop 11
Productivity

increased
1 C. Adinarayana Reddy and R. S.

secretion of root flavonoid substances that are involved in the activation

of the nodulation genes in Rhizobium (Dobbelaere et al., 2001). Stimulation
of nodulation following co-inoculation with Azospirillum may also be
due to increase in the production of lateral roots, root hair density and
branching, and differentiation of a greater number of epidermal cells into
root hairs, which are susceptible for infection by rhizobia. Considerable
increases in yield of grain and N, P, K content were seen when wheat was
coinoculated with A. brasilense and Sinorhizobium meliloti (Askary,
Mostajeran,Amooaghaei, & Mostajeran, 2009; Caballero-Mellado,
Carcano-Montiel, & Mascarûa- Esparza, 1992). Similar increases in N, P,
and K, and various micronutrients was found in Azospirillum-treated
maize, soybean, and rice (Caballero- Mellado et al., 1992; Naiman,
Latrónico, & Garcia de Salamone, 2009; Garcia de Salamone et al., 2010;
Bashan, 1998; Baldani & Baldani, 2005). More than a 100 crops and a
number of environmentally important plant species were shown to be
stimulated by Azospirillum (Bashan et al., 2004).
Trichoderma spp. and Pseudomonas spp. are well recognized as PGPR
organisms that stimulate plant growth by multifaceted action, but primar-
ily by their biocontrol and phosphate solubilization properties (Harman
et al., 2004).The mode of action of Trichoderma sp. is multifaceted including
antibiosis, parasitism, competition, and inducing systemic resistance (Harman
et al., 2004). Trichoderma harzianuim increased cucumber dry mass yield by
80% when compared to the control (Yedida et al., 2001). Pseudomonas
putida and T. atroviride were shown to improve both growth and fruit
yields when applied to mature healthy tomato plants grown under
hydroponic condi- tions; also, increase in the fresh weight of both the
shoot and the roots of tomato seedlings was observed (Gravel et al.,
2007). Multiple strains of Trichoderma viride and T. harzianum stimulate
growth of lettuce. Pseudomonas putida, known for it is inhibition of Fusarium
sp., was shown to increase root and shoot weight of corn (Myresiotis et al.,
2012).Also, P. putida and Pseudo- monas cepacia were shown to stimulate
growth and yield of winter wheat (de Freitas and Germida, 1991).
Pseudomonas chlororaphis, known as inducer of systemic resistance and
also as an effective biocontrol agent, produces phen- azine group of
antibiotics against Pythium aphanidermatum, a pathogen of hot pepper
seedlings. P. chlororaphis is also effective in eliminating soft-rot in leaves of
the tobacco plant caused by Erwinia carotovora (see Avis et al., 2008).
Inoculation of a mixture of mycorrhiza and PGPR in general gave
increased growth and yields of the crops tested, as compared to single organ-
ism inoculation (Belimov, Kojemiakor, Chuvarliyeva, 1995). AM fungi are
relatively nonspecific to host plant. Co-inoculation of AM fungi with one or
 Polymicrobial Multifunctional Approach for Enhancement of Crop 13
Productivity

more of the other PGPR organisms generally gives more consistent

results in enhancing growth and productivity for different crops (see
Adesemoye et al., 2008; Barea et al., 2005; Dobbelaere et al., 2003; Dutta &
Podile, 2010). For example, synergistic interactions between AM fungi along
with a PGPR organism such as Azospirillum, Azotobacter, Bacillus, or
Pseudomonas species, were found to be beneficial for enhancing plant growth
and yield for a num- ber of crops. However, the stimulatory effect of the
Azospirillum inocula on root growth did not significantly influence the
mycorrhization, regardless of the AM fungus involved, either in wheat or in
maize plants, in the presence of indigenous AM fungi or when maize
plants were artificially inoculated with G. mosseae and Glomus macrocarpum.
Positive effects of A. brasilense and AM fungal colonization on rice growth
and drought resistance have been reported (Ruiz-Sanchez et al., 2011).
Inoculation of a mixture of mycorrhiza and PGPR in general gave increased
growth and yields of the crops tested, as compared to single organism
inoculation (Belimov et al., 1995). Azospirillum- AM fungus combination
seems suitable for sustainable agriculture practices, since both types of
microorganisms are not only compatible with each other but give
synergistic benefits to plant productivity. Individual inoculation of B.
subtilis and A. brasilense Sp245 positively affected the growth and dry
weight of both shoots and roots of tomato plants, but the combination of the
two rhizobacteria had no synergistic or comparable effects on plant biomass.
In vitro tests and cellular analysis of root tips revealed growth inhibition of
the primary root, which is not related to a reduced persistence in the
rhizosphere of one or both bacteria (Dodd & Ruiz-Lozano, 2012).Vestberg et
al. (2004) used an inoculant containing one or more strains of G. mosseae,
B. subtilis,
P. fluorescens,T. harzianum, and Gliocladium catenalatum. Used either singly or in
dual mixtures in the presence or absence of the strawberry crown rot (caused
by Phytophthora cactorum) and red stele (caused by P. fragariae), the results on
decreasing disease incidence or increasing the yields have been mixed
with considerable variations in treatments.These results suggest that mixing
differ- ent microorganisms in the same inoculum/formulation can cause
interfer- ences and consequently give lower than expected performances.
Therefore, individual organisms in mixed inoculums should be carefully
chosen so that one can get synergistic increases in crop yields.

5.3. Polymicrobial Inoculant Formulations

The idea of developing a formulation containing a consortium of
beneficial microbes for increasing the yields of multiple field-grown
crops is not new (Antoun & Prevost, 2006; Kennedy et al., 2004), but
successful formulation
1 C. Adinarayana Reddy and R. S.

of this type are very few, if any. Dodd and Ruiz-Lozano (2012) suggested
that combining different classes of soil organisms within one inoculant
can take advantage of multiple plant growth mechanisms of such an
inoculant to enhance crop productivity. However, in many studies to date,
even when a mixture of PGPR and other organisms were used in
designing an inoculant, the focus in many cases was on one crop rather
than developing an inocu- lant which potentially gives positive increase
in yields for multiple crops. In developing a multifunctional
polymicrobial inoculant, one is expected to take into account existing
crop management systems and the compat- ibility of the inoculant with
routinely added soil organic amendments and agrochemicals. One of the
main limitations so far has been the absence of a single polymicrobial
inoculant formulation that would promote the growth of a range of crops
including legumes, cereals, and vegetable crops.
A multistrain inoculum containing three microbial strains isolated from
rice rhizosphere was used by Nguyen, Kennedy, and Roughley (2003)
as a biofertilizer for rice. This multistrain biofertilizer for rice contained
Klebsiella pneuminiae, P. fluorescens (or P. putida), and Citrobacter freundii;
P. fluorescens was selected for its N2-fixing ability, Klebsiella pneumoniae
for its P-solubilizing ability, and C. freundii, for its ability to produce toxic
substances that inhibited nearly 50% of the rice rhizosphere population
(apparently to facilitate colonization by the first two organisms).Application
of this biofertilizer to the rice crop allowed the reduction of urea applica-
tion by 50% as compared to the uninoculated control field, which actually
received twice the amount of urea fertilizer; also, 20–30% increase in rice
yield was observed. The combined effect of lowering the need for urea by
50% and increasing rice yield translates into considerable cost savings for
the former. Paikray and Malik (2012) reported the development of a micro-
bial formulation containing a consortium of “P. fluorescens, Pseudomonas
striata, Paenibacillus (Bacillus) polymyxa, B. subtilis,Azospirillum,
Rhizobium,Azotobacter,
T. harzianum, Trichoderma viride, Saccharomyces cerevisiae and Lactobacillus,
and nutrients”. This inoculum apparently showed a “wide variety of plant
growth-promoting properties including root and shoot length elongation,
early and high germination rate, high yield, decrease in soil pathogenic load
and increase soil micro and macronutrient status”, but details are lacking.
Polymicrobial formulations containing a diverse mixture of benefi-
cial rhizosphere microorganisms with multiple functionalities is attractive
because combining different classes of soil organisms can take advantage of
multiple plant growth-promoting mechanisms and could be applied to
mul- tiple crops (Avis et al., 2008; Gravel et al., 2007; Hayat et al., 2010;
Malusa
 Polymicrobial Multifunctional Approach for Enhancement of Crop 15
Productivity

et al., 2012; Vestberg et al., 2004). A key concept in constructing effective

polymicrobial multifunctional formulations is the selection and use of a
right combination of rhizosphere bacteria and fungi that are mutually
com- patible, have complementary functionalities, effectively colonize the
rhizo- sphere of the crop(s) of interest, and bring about a synergistic
promotion of growth and yield of crop(s) (Avis et al., 2008; Azcon, 2009;
Barea et al., 2005; Hata et al., 2010). It is also important to select a stable and
efficacious carrier that would provide a suitable microenvironment for
keeping the microbes in the inoculum viable and to develop relatively
simple and inex- pensive delivery methods. Considering the beneficial
effects of 1. PGPR;
2. AM fungi; and 3. symbiotic N 2-fixers, it is desirable to construct a poly-
microbial formulation that contains several microbial strains representing
each of these three functional groups and strongly promotes plant growth
and yields (Arora et al., 2011; Khalid et al., 2009; Malusa et al., 2012). It is
to be expected that a well-designed multifunctional formulations such as
the one described would be a welcome addition to the fast-growing
inoculant enterprises worldwide. Such an inoculant is also expected to be
eco-friendly and suitable for organic farming and other integrated
production systems, where synthetic fertilizer inputs are not allowed or
restricted by law. How- ever, construction of such complex formulations is
technically demanding. To the best of our knowledge, there is no single
inoculant on the market that satisfies the above mentioned criteria. Soilless
and protected crops such as hydroponics can also be benefitted by such an
inoculant because the predictability of the results with hydroponic
systems would be higher than in open fields where a number of
environmental variables may affect the outcome (Kennedy et al., 2004;
Malusa et al., 2012). Ongoing efforts in the authors’ lab to develop a
multifunctional polymicrobial formulation of the type described above are
briefly outlined below.
Several hundred bacterial strains were isolated from the root nodules
of various leguminous plants as well as from soil and rhizosphere samples
col- lected from diverse environmental sources (Reddy and Lalithakumari,
2009a, 2009b).A number of strains of Trichoderma species, perhaps the
most-studied fungi for their inhibition of plant pathogens by antibiosis,
competition, ISR, and parasitism (Harman et al., 2004), were isolated from
soil samples (representing cultivated and uncultivated agricultural soils)
obtained from geographically divergent regions. After screening a large
number of micro- bial strains for the desired functionalities mentioned above,
a polymicrobial formulation F2 containing phylogenetically diverse
consortium of over 20 microbial strains with complementary
functionalities was constructed using
1 C. Adinarayana Reddy and R. S.

humate (12%, pH 7.0) as the carrier.The microbial groups in F2 were iden-

tified using 16S rDNA sequencing, and these included strains representing
genera Rhizobium, Azorhizobium, Sinorhizobium, Bacillus, Pseudomonas, Steno-
trophomonas, Enterobacter, and seven strains representing multiple species of
genus Trichoderma (Reddy and Lalithakumari, 2009a, 2009b).
Greenhouse trials were done with various vegetables using F2 formu-
lation as the inoculants, and the results were compared to those obtained
with the controls without the added inoculant. These results are presented
in Table 3.3. Substantial statistically valid increases in yields were obtained
with all F2-treated plants as compared to the controls. Getting uniformly
higher yields in a broad spectrum of F2-treated crops was quite encour-
aging. In general, F2-treated plants showed early flowering and fruiting,
looked healthier, and good root nodulation was observed in legumes.
Representative results are shown in Fig. 3.5. The lowest yield (37.2%) was
observed with garden bean, while the highest yield (258%) was seen with
okra. Although these yields are quite impressive, these experiments were
done in controlled idealized conditions in a green house and the yields in
a field situation would not be expected to be as high because of a num-
ber of uncontrollable variables in field experiments. Some field trials have
been conducted using the F2 inoculant by BioSoil Enhancers Inc., under a
license from Michigan State University.When F2 formulation, later named
SumaGrow® (http://thegrowpros.com/the- science-of-sumagrow/), was
used in field experiments, the yields obtained were consistent with those
observed in the greenhouse experiments in that substantial increases were

Table 3.3 Green house evaluation of multifunctional polymicrobial formulation F2#

Yield (g)

Crops F2 Control % Increase

Garden Bean (Phaseolus vulgaris) 48.6* 23.5 106.8
Tomato (Lycopersicon esculentum) 1000* 380 163.1
Wonde bush beans (Phaseolus sp.) 72.9* 35.6 104.8
Pea (Pisum Sativum) 13.9* 7.52 84.8
Pea purple hull (Pisum arvense) 14.75* 10.75 37.2
Okra (Hibiscus esculentus) 138.7* 38.7 258.4
Peanut (Arachis hypogaea) 21.62* 6.48 233.6
Soybeans (Glycine max) 11.58* 5.1 127.1
Values represent four replications for each treatment.
#
F2 formulation contained over 20 microbes with multiple functional activities such as N-
fixation, phosphate solubilization, and biocontrol (see text).
*Significant, P = 0.022.
 Polymicrobial Multifunctional Approach for Enhancement of Crop 17
Productivity

observed in F2-treated crops as compared to controls; however, the extent

of increase in yields was less than that seen in green house experiments. For
example, results of double-blind field trials showed that F2-supplemented
crops gave 30%, 27%, 40%, 61%, and 75% increases in yields, respectively,
for corn, bell pepper, banana pepper, yellow squash, and tomato.
SumaGrow®- supplemented corn, with 50% of the normal NPK input,
gave 30–40% increase in corn yields as compared to un-inoculated field
with 100% NPK supplementation (http://thegrowpros.com/research/). In
other words, commercial chemical fertilizer input into SumaGrow®-
supplemented corn field can be reduced by 50% resulting in considerable
cost savings to the farmer and safer for the environment (data from BSEI).
These results are similar to the 30%–50% savings in fertilizer costs
observed with some bio- inoculants by previous investigators (reviewed
in Adesemoye et al., 2009; Kennedy et al., 2004; Siddiqui, 2006).To the
best of our knowledge, F2 for- mulation is one of the very few bioinoculant
products available today, that is specifically designed to contain a
consortium of microbial groups with

Figure 3.5 Green house experiments testing the efficacy of polymicrobial

multifunc- tional F2 formulation (see Text) with different plants and compared to the
respective controls. a) Tomato; b) Clover; d) Switch grass. In each panel, F2-treated
plants are given on the left side, while controls are given on the right side; c) Display of
root nodules in garden bean inoculated with F2. (For color version of this figure, the
reader is referred to the online version of this book.)
1 C. Adinarayana Reddy and R. S.

multiple complementary functions and is effective in substantially

increas- ing the productivity of a broad spectrum of legumes, cereals, and
vegetables (Reddy and Lalithakumari, 2009a, 2009b).

6. CONCLUSIONS
There is a growing worldwide awareness for the need to increase food
production to feed the rapidly expanding global human population. There
is also a growing consensus that the traditional approach of massive inputs of
chemical fertilizers and pesticides to increase crop yields is not sustainable.
Also, there is a considerable resistance in some areas of the world in using
genetically engineered crops for increasing food production. Therefore,
there is an obvious need for developing efficacious, environmental friendly,
affordable, and safe technologies using naturally occurring rhizosphere bac-
teria and fungi for constructing effective microbial inoculants to increase
the yields of food crops. Furthermore, successful implementation of this
approach would be sustainable, nonpolluting, efficacious, and preserves soil
health (Fig. 3.6).
Microbial inoculants have been in use for a long time. Symbiotic N2-
fixing rhozobia were perhaps the most widely used inoculants and are
still in common use. Microbial inoculants containing one or more of the
PGPR strains with or without N2-fixing rhozobia are also in wide

Figure 3.6 Field trials demonstrating the efficacy of F2 formulation (Sumagrow®),

in enhancing the growth of soybean (Glycine max). Left – sumagrow treated (T);
right – uninoculated control (UN). (Source: courtesy of BioSoil Enhancers Inc.). (For
color version of this figure, the reader is referred to the online version of this book.)
 Polymicrobial Multifunctional Approach for Enhancement of Crop 19
Productivity

use for enhancing crop productivity (see Table 3.2 for a selected list of
commercially available microbial inoculants). PGPR strains that are often
used (singly or in mixture) include species of genera Bacillus, Pseudomo-
nas, Azospirillum, Azotobacter, Trichoderma, and Glomus; species of other
genera are used but somewhat less frequently. There is a general consen-
sus now that these inoculants increase growth yields of various crops in
the 5–30% range. There is a continuing emphasis on developing inocu-
lants that increase yields by several different PGPR mechanisms with the
expectation that such inoculants might give better yields for a broader
spectrum of crops and may be more stable, affordable, and convenient to
use. Preliminary laboratory and field studies with such inoculants have
been encouraging.
Polymicrobial inoculants containing a large consortium of soil organ-
isms (such as the F2 formulatioin described here) with multiple
organisms representing each major PGP function such as N 2 fixation, P-
solubiliza- tion, biocontrol, ISR, and production of various plant growth-
promoting stimulants hold much promise for microbial enhancement of
food crop productivity. There are still many unknowns and this is a
productive area for intensified future research. Furthermore, in this
consortium approach, a single polymicrobial formulation with multiple
beneficial functions would have the ability to substantially increase
productivity of a broad spectrum of food crops in diverse geographic
regions. This would aid in less depen- dence on chemical fertilizers and
pesticides, reduction of costs of farm- ing, and protection against adverse
health and environmental consequences. Also, such polymicrobial
formulations consisting of microbes that naturally occur in nature, conserve
soil health by increasing the number of microbial groups in the rhizosphere
beneficial to crop productivity, and ensure better sustainable use of our
natural resources. Effective harnessing of the power of beneficial soil
organisms for alleviating human food needs appears feasible and the
expanding research involving microbial physiology/ecology, bio-
technology, genomics and proteomics (Tisserant et al, 2012; Wang, Ohara,
Nakayashiki, Tosa, & Mayama, 2005; Weidner, Puhler, & Kuster, 2003), and
applications of symbiotic N2-fixers and PGP bacteria/fungi should yield
rich dividends in the future.
ACKNOWLEDGMENTS
CAR sincerely acknowledges Dr Lalithakumari, J for her dedicated and invaluable work
on the bioinoculant project in my laboratory. Acknowledgments are also due to Dr Purna
Viswanathan for her help with the 16S rDNA sequencing work on the isolates.
2 C. Adinarayana Reddy and R. S.

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