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Interrelated effects of mycorrhiza and free-living nitrogen fixers cascade up to aboveground herbivores

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Aboveground plant performance is strongly influenced by belowground microorganisms, some of which are pathogenic and have negative effects, while others, such as nitrogen-fixing bacteria and arbuscular mycorrhizal fungi, usually have positive effects. Recent research revealed that belowground interactions between plants and functionally distinct groups of microorganisms cascade up to aboveground plant associates such as herbivores and their natural enemies. However, while functionally distinct belowground microorganisms commonly co-occur in the rhizosphere, their combined effects, and relative contributions, respectively, on performance of aboveground plant-associated organisms are virtually unexplored. Here, we scrutinized and disentangled the effects of free-living nitrogen-fixing (diazotrophic) bacteria Azotobacter chroococcum (DB) and arbuscular mycorrhizal fungi Glomus mosseae (AMF) on host plant choice and reproduction of the herbivorous two-spotted spider mite Tetranychus urticae on common bean plants Phaseolus vulgaris. Additionally, we assessed plant growth, and AMF and DB occurrence and density as affected by each other. Both AMF alone and DB alone increased spider mite reproduction to similar levels, as compared to the control, and exerted additive effects under co-occurrence. These effects were similarly apparent in host plant choice, that is, the mites preferred leaves from plants with both AMF and DB to plants with AMF or DB to plants grown without AMF and DB. DB, which also act as AMF helper bacteria, enhanced root colonization by AMF, whereas AMF did not affect DB abundance. AMF but not DB increased growth of reproductive plant tissue and seed production, respectively. Both AMF and DB increased the biomass of vegetative aboveground plant tissue. Our study breaks new ground in multitrophic belowground–aboveground research by providing first insights into the fitness implications of plant-mediated interactions between interrelated belowground fungi–bacteria and aboveground herbivores.
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Interrelated effects of mycorrhiza and free-living nitrogen
fixers cascade up to aboveground herbivores
Botir Khaitov
1,2
, Jos
e David Pati~
no-Ruiz
1
, Tatiana Pina
1,3
& Peter Schausberger
1
1
Group of Arthropod Ecology and Behavior, Department of Crop Sciences, University of Natural Resources and Life Sciences, Peter Jordanstrasse
82,1190 Vienna, Austria
2
Division of Legume Crops, Department of Plant Sciences, Tashkent State Agrarian University, Universitetskaya street 2a, 370, Tashkent,
Uzbekistan
3
Departament de Ci
encies Agr
aries i del Medi Natural, Unitat Associada d’Entomologia UJI/IVIA, Universitat Jaume I, Campus del Riu Sec,12071,
Castell
o de la Plana, Spain
Keywords
Abovegroundbelowground interactions,
arbuscular mycorrhiza, fitness, multitrophic
interactions, nitrogen fixation, spider mites.
Correspondence
Peter Schausberger, Group of Arthropod
Ecology and Behavior, Department of Crop
Sciences, University of Natural Resources and
Life Sciences, Peter Jordanstrasse 82, 1190
Vienna, Austria.
Tel: 00 43 1 47654 3361;
Fax: 00 43 1 47654 3359;
E-mail: peter.schausberger@gmx.at
Funding Information
Eurasia Pacific Uninet (EPU 26/2013);
Erasmus Mundus CASIA.
Received: 10 July 2015; Revised: 15 July
2015; Accepted: 22 July 2015
Ecology and Evolution 2015; 5(17):
37563768
doi: 10.1002/ece3.1654
Abstract
Aboveground plant performance is strongly influenced by belowground
microorganisms, some of which are pathogenic and have negative effects, while
others, such as nitrogen-fixing bacteria and arbuscular mycorrhizal fungi, usu-
ally have positive effects. Recent research revealed that belowground interac-
tions between plants and functionally distinct groups of microorganisms
cascade up to aboveground plant associates such as herbivores and their natural
enemies. However, while functionally distinct belowground microorganisms
commonly co-occur in the rhizosphere, their combined effects, and relative
contributions, respectively, on performance of aboveground plant-associated
organisms are virtually unexplored. Here, we scrutinized and disentangled the
effects of free-living nitrogen-fixing (diazotrophic) bacteria Azotobacter
chroococcum (DB) and arbuscular mycorrhizal fungi Glomus mosseae (AMF) on
host plant choice and reproduction of the herbivorous two-spotted spider mite
Tetranychus urticae on common bean plants Phaseolus vulgaris. Additionally, we
assessed plant growth, and AMF and DB occurrence and density as affected by
each other. Both AMF alone and DB alone increased spider mite reproduction
to similar levels, as compared to the control, and exerted additive effects under
co-occurrence. These effects were similarly apparent in host plant choice, that
is, the mites preferred leaves from plants with both AMF and DB to plants with
AMF or DB to plants grown without AMF and DB. DB, which also act as AMF
helper bacteria, enhanced root colonization by AMF, whereas AMF did not
affect DB abundance. AMF but not DB increased growth of reproductive plant
tissue and seed production, respectively. Both AMF and DB increased the
biomass of vegetative aboveground plant tissue. Our study breaks new ground
in multitrophic belowgroundaboveground research by providing first insights
into the fitness implications of plant-mediated interactions between interrelated
belowground fungibacteria and aboveground herbivores.
Introduction
Plants are the prime links between the below- and above-
ground spheres, mediating interactions between below-
and aboveground living organisms that do not directly
interact with each other. Based on intensive research dur-
ing the past two decades, it is now generally acknowl-
edged that below- and aboveground plant-associated
processes are mutually dependent (Van der Putten et al.
2001; Bezemer and van Dam 2005; Rasmann and Turlings
2007; Erb et al. 2008; Koricheva et al. 2009; Heil 2011;
van Dam and Heil 2011; Schausberger et al. 2012).
Among others, aboveground plant performance is
strongly influenced by belowground microorganisms,
which may be either pathogens or mutualists, which in
turn affects herbivorous organisms inhabiting and feeding
3756 ª2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
on green plant parts (Van der Putten et al. 2001; Bezemer
and van Dam 2005; Van Dam and Heil 2011). Cases
in point are plants living belowground in concurrent,
usually mutualistic, symbiosis with functionally distinct
microorganisms such as nodulating rhizobial bacteria
(RB), and/or free-living nitrogen fixers, such as dia-
zotrophic bacteria (DB), and/or plant growth promoting
rhizobacteria, such as Pseudomonas sp., and/or arbuscular
mycorrhizal fungi (AMF) (Pineda et al. 2010, 2013). Con-
current associations with AMF and nitrogen fixers (RB or
DB) commonly act additively or synergistically on nutri-
ent uptake by the plants (e.g. El-Shanshoury et al. 1989;
Ibijbijen et al. 1996; Barea et al. 2005; Artursson et al.
2006; Sabannavar and Lakshman 2011). However, under
certain circumstances, also competition for nutrients,
space, or other resources between AMF and nitrogen fix-
ers may occur, resulting in subtractive effects as compared
to the predicted sum of each symbiont’s effect (Scheublin
and van der Heijden 2006).
Recent research provided insights into how belowground
interactions between plants and one functional type of
microorganism may cascade up to aboveground herbivores
(for plant growth promoting rhizobacteria: Tomczyk 2006;
Saravanakumar et al. 2008; for AMF: Hoffmann et al.
2009, 2011a,b; for RB: Kempel et al. 2009; Thamer et al.
2011; Dean et al. 2014) and their natural enemies (e.g.
Gange et al. 2003; Schausberger et al. 2012; Pineda et al.
2013). Also combined effects of different strains of a given
species of microorganism (e.g. Saravanakumar et al. 2008;
Roger et al. 2013) or of different, but functionally similar,
species of AMF (Gange et al. 2003) as well as microorgan-
ism community effects (Hol et al. 2010) have been looked
at. However, functionally distinct belowground microor-
ganisms commonly co-occur, but their combined effects
and relative contributions, respectively, on aboveground
herbivores and their natural enemies are virtually unex-
plored (Pangesti et al. 2013). Comparing bottom-up effects
of belowground plant associations with AMF, nitrogen fix-
ers and both in combination on arthropods living on
aboveground plant parts are an important ecological and
evolutionary issue in multitrophic research. This issue is
also relevant for applied ecology, for example, in agricul-
ture, forestry, or ecosystem restoration, because commer-
cially available, functionally distinct microorganisms are
often jointly used for promoting plant growth and health
(Sprent and Sprent 1990; Mrkovacki and Milic 2001; Wu
et al. 2005; Rathi et al. 2014).
We addressed this novel belowgroundaboveground
issue in a multitrophic system consisting of common
bean plants Phaseolus vulgaris L., two belowground
microorganisms, the arbuscular mycorrhizal fungus
Glomus mosseae Nicol. and Gerd. and free-living nitro-
gen-fixing bacteria Azotobacter chroococcum Beijerinck,
and an aboveground herbivore, the two-spotted spider
mite Tetranychus urticae Koch (Fig. 1). This below-
groundaboveground system is, for its easy experimental
accessibility and, except A. chroococcum, relatively well
understood (Hoffmann et al. 2009, 2011a,b; Schausberger
et al. 2012; Pati~
no-Ruiz and Schausberger 2014), includ-
ing the effects of nodulating rhizobia (Katayama et al.
2010). Tetranychus urticae is a globally distributed, poly-
phagous herbivore with >1000 recorded host plant species
(e.g. Bolland et al. 1998). The spider mites feed on their
host plants by piercing the parenchyma cells and sucking
out the cell contents. Their behavioral and life history
performance is enhanced by the AMF G. mosseae (e.g.
Hoffmann et al. 2009, 2011a,b; Pati~
no-Ruiz and Schaus-
berger 2014). Glomus mosseae is a widespread facultative
symbiont of vascular plants (Giovannetti et al. 1993). In
general, the AMF hyphae penetrate the roots and grow
intracellularly, penetrating individual cells and inside
forming arbuscules for exchange of resources with the
plant (Allen 1996). AMF may affect plant growth and
health by changing mineral nutrition, especially phospho-
rous (P) uptake (Smith and Read 1997; Clark and Zeto
2000), and consequently influences resistance and toler-
ance to biotic (Trotta et al. 1996; Azcon-Aguilar et al.
2002; Schausberger et al. 2012) and/or abiotic stressors
(Turnau and Haselwandter 2002). Azotobacter chroococ-
cum is an aerobic free-living soil bacterium, playing an
important role for natural nitrogen (N) availability. Azo-
tobacter sp.and other DB bind atmospheric N and release
N in the form of ammonium ions (NHþ
4), thereby mak-
ing N accessible to plants (e.g. Mrkovacki and Milic
2001). Azotobacter sp. are widely distributed in natural
and agricultural soils of temperate regions and more
Figure 1. Adult two-spotted spider mite female, Tetranychus urticae,
on bean leaf, Phaseolus vulgaris.
ª2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3757
B. Khaitov et al.Rhizosphere microbes drive leaf mites
abundant in the rhizosphere of plants, including of
legumes, than in soils unassociated with plants (Kole
et al. 1988; Rodelas et al. 1999; Mrkovacki and Milic
2001). Azotobacter sp. may also function as mycorrhiza
helper bacteria (MHB) by enhancing the environmental
conditions for AMF occurrence and establishment, for
example, by producing growth factors and/or inhibition
of competitors and antagonists and/or improving soil
conduciveness (e.g. Garbaye 1994; Frey-Klett et al. 2007).
The separate effects of AMF and RB on performance of
aboveground herbivores are highly species and context
dependent, and range from negative over neutral to posi-
tive, depending on whether the nutritional value or defen-
sive system of the host plant is more strongly affected
(Gehring and Whitham 2002; Kempel et al. 2009; Pineda
et al. 2010; Dean et al. 2009, 2014). In contrast, the
effects of free-living nitrogen fixers (DB) and the interre-
lated effects of DB and AMF, which commonly co-occur
(Frey-Klett et al. 2007), on aboveground herbivore perfor-
mance are unknown.
Using the multitrophic system of P. vulgaris,G. mosseae
(AMF), A. chroococcum (DB), and T. urticae, here we
examined (1) how the interactions between AMF and DB
affect the host plant choice and life history of the spider
mites feeding on aboveground plant parts, (2) how the
interactions between AMF and DB affect vegetative and
Figure 2. Cumulative number of offspring
produced by spider mite females feeding on
leaves from bean plants inoculated with
arbuscular mycorrhizal fungi (AMF+) and/or
diazotrophic bacteria (DB+) or left uninoculated
(AMF,DB) over 5 days; plant ages were 3
4 weeks (plant age 1) and 56 weeks (plant
age 2) postinoculation with the
microorganisms.
3758 ª2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
Rhizosphere microbes drive leaf mites B. Khaitov et al.
reproductive plant growth, and (3) how AMF and DB
affect each other’s occurrence, establishment, and growth
in the rhizosphere. We conducted two experiments. In the
first experiment, we assessed survival and reproduction of
the spider mites feeding on leaves of bean plants below-
ground associated with either G. mosseae alone (AMF+/
DB), or A. chroococcum alone (AMF/DB+), or both in
combination (AMF+/DB+) or neither of the two microor-
ganisms (AMF/DB). Additionally, we quantified vege-
tative and reproductive plant growth, as affected by the
four rhizosphere treatments, and AMF and DB occur-
rence. In the second experiment, we evaluated host plant
preference of adult spider mite females given a choice
between four leaves, one from each treatment AMF+/
DB, AMF/DB+, AMF+/DB+, and AMF/DB.
Materials and Methods
Plant growing and plant growth
measurements
To obtain plants and leaves for experiments, seeds of
P. vulgaris (var. Taylor’s Horticultural) were surface-ster-
ilized with a solution of 75 mL chloride +25 mL water
for 23 min, rinsed thoroughly with distilled water and
then put in perlite, previously autoclaved for 20 min at
121°C, inside 0.5 L pots for pregermination (20 seeds/
pot). Pots and saucers were disinfected with 75% ethanol
before use. After 810 days, the seedlings, then having the
cotyledons and primary leaves, were transplanted in
groups of three to 1 L pots filled with a 1 : 1 : 1 silicate
sand/expanded clay/soil substrate mixture, previously
autoclaved for 20 min at 121°C, and inoculated with
either AMF, or DB, or both AMF and DB, or left uninoc-
ulated. About 3 weeks after transplanting and inoculation,
the plants were ready for use in experiments. Plants were
grown under standardized conditions in a walk-in
environmental chamber (60 5% RH, 16:8 h L:D, and
23:18°C L:D). The relative placement of pots within the
environmental chamber was changed every 2 days to
exclude any inadvertent positioning effects. The plants
were watered in 23 days intervals with ~80100 mL per
pot of a reduced N and P fertilizer solution (22 mL/L
water). The ingredients of the fertilizer solution were
K
2
(SO
4
) 0.256 g/L, Mg SO
4
0.136 g/L, Fe
6
H
5
O
7
93H
2
O
50.0 g/L, Na
2
Bo
4
O
7
94H
2
O 1.3 g/L, MnSO
4
94H
2
O
1.5 g/L, ZnSO
4
97H
2
O 0.6 g/L, CuSO
4
95H
2
O 0.45 g/
L, Al
2
(SO
4
)
3
0.028 g/L, NiSO
4
97H
2
O 0.028 g/L, Co
(NO
3
)
2
96H
2
O 0.028 g/L, TiO
2
0.028 g/L, LiCi
2
0.014 g/L, SnCi
2
0.014 g/L, KJ 0.014 g/L, KBr 0.014 g/L,
and MoO
3
0.014 g/L.
After finishing the spider mite experiments and clip-
ping off leaves for experimental use, respectively, which
happened ~6 weeks after transplanting, all remaining
aboveground plant material and the roots were removed
from the pots for further analyses. The fresh weights of
pods, shoots, and roots were measured immediately after
removing the plants from the pots, and cleaning the
roots, respectively. During cleaning the roots, we also ver-
ified the absence of nodules, possibly emanating from
inadvertent soil contamination by RB. Subsequently, the
pods, shoots, and roots were dried at room temperature
(25 2°C and 3050% RH) for 14 days, and their dry
weights measured.
DB and AMF inoculation and quantification
To inoculate the growing substrate and roots, respectively,
with DB, we used a reduced DB solution, prepared by dis-
solving 2.5 mL of pure Azotovit
(obtained from Agro-
trader Agrarhandel, Austria) in 1 L of tap water. Azotovit
consists of pure water containing Azotobacter chroococcum
at a bacterial density of 5 910
9
/mL. For the AMF/DB+
and AMF+/DB+treatments, during plant transplanting the
roots were dipped in the DB solution and 40 mL of the
solution was poured into each pot. After 2 weeks, another
40 mL of DB solution was added to each pot.
To estimate the numbers of colony-forming units
(cfu) of A. chroococcum in the planting substrate, we
used an Azotobacter sp. specific culturing medium
(M372-500G Azotobacter Mannitol Agar; HiMedia Labo-
ratories, India). A total of 41.4 g Azotobacter sp. med-
ium (containing soil extract 5 g, mannitol 20 g, K
2
HPO
4
1 g, MgSO
4
0.2 g, NaCl 0.2 g, FeSO
4
traces, agar 15 g)
was suspended in 1 L distilled water, according to the
manufacturer’s specifications, heated to boiling to com-
pletely dissolve the medium, sterilized by autoclaving at
Table 1. Results of generalized estimating equations (GEE) for the
effects of arbuscular mycorrhizal fungi G. mosseae (AMF),
diazotrophic bacteria A. chroococcum (DB), and plant age (34 and 5
6 weeks postinoculation) on oviposition, activity and mortality of
two-spotted spider mites T. urticae feeding on leaves of common
bean P. vulgaris.
Source of
variation
Oviposition Activity Mortality
Wald ӽ
12
P Wald ӽ
12
P Wald ӽ
12
P
AMF 8.400 0.004 2.361 0.124 3.003 0.083
DB 9.470 0.002 0.040 0.841 1.015 0.314
Plant age 99.309 <0.001 18.259 <0.001 0.487 0.485
AMF*DB 0.139 0.710 0.207 0.649 0.979 0.322
AMF*plant
age
0.241 0.624 0.204 0.651 0.007 0.932
DB*plant
age
0.319 0.572 1.869 0.172 0.082 0.774
AMF, arbuscular mycorrhizal fungi; DB, diazotrophic bacteria.
ª2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3759
B. Khaitov et al.Rhizosphere microbes drive leaf mites
121°C for 15 min, and then poured into sterile Petri
dishes for solidification. The plate count technique was
used for determining the presence of A. chroococcum and
estimating its density: 1 g soil substrate was randomly
sampled from each pot and diluted to 10
5
in distilled
water. 1 ml of diluted soil sample was spread on the
surface of the agar plates using a sterile glass spreader.
All used devices such as tubes, dishes, and bottles were
previously autoclaved for 20 min at 121°C to prevent
any contamination. After 72 h incubation at 28°C,
A. chroococcum had formed large, moist colonies, which
were counted. All colonies turned dark brown after 5
7 days of incubation indicating A. chroococcum identity
(Banerjee et al. 2014).
To inoculate the plants with AMF, we used the Glomus
mosseae inoculum BEG 12 (http://www.kent.ac.uk/bio/
beg) (see Hoffmann et al. 2009). For treatments AMF+/
DB+and AMF+/DB,5gofG. mosseae inoculum was
added to each pot containing three plants. After the
experiments, the roots of all plants were checked for myc-
orrhizal colonization, which allowed assigning a specific
fungal colonization level to each pot used in experiments.
Plants were removed from pots and the substrate rinsed
off the roots with cold tap water. To assess the root
length colonized (RLC) by AMF, the roots were cleared
by boiling for 10 min in 10% KOH and stained by boil-
ing for 5 min in a 5% black ink (Sheaffer, Ft. Madison,
Iowa), household vinegar (equal to 5% acetic acid)
Figure 3. Activity (A) and mortality (B) of
spider mite females feeding on leaves from
bean plants inoculated with arbuscular
mycorrhizal fungi (AMF+) and/or diazotrophic
bacteria (DB+) or left uninoculated (AMF,
DB) over 5 days.
3760 ª2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
Rhizosphere microbes drive leaf mites B. Khaitov et al.
solution (Vierheilig et al. 1998). The RLC was estimated
according to Newman (1966) and the modified gridline
intersect method (Giovannetti and Mosse 1980).
Spider mite rearing and experiments
Two-spotted spider mites, T. urticae (Fig. 1), used in
experiments derived from a population reared on whole
bean plants P. vulgaris at room temperature. Plants used
for maintaining the stock population were grown in a
sand/clay mixture that did neither contain AMF nor DB.
To obtain mated spider mite females for experiments, spi-
der mite nymphs were withdrawn from the stock popula-
tion and placed in groups of 2030 individuals on leaf
arenas from either AMF/DB, AMF+/DB, AMF/
DB+, or AMF+/DB+plants until reaching adulthood.
Arenas consisted of squares (~2.5 92.5 cm) on detached
clean trifoliate leaflets placed upside down on moist filter
paper covering a water-soaked foam cube
(7 9795 cm) resting in a plastic box (10 910
96 cm) half-filled with water. Each leaf arena was
delimited by strips of moist tissue paper to prevent mite
escaping. The developmental progress of the spider mites
was monitored once per day until the mites had reached
adulthood and females were mated, respectively (Hoff-
mann et al. 2009). To assess oviposition, as influenced by
AMF and/or DB, the females were singly transferred from
the leaflets, on which they matured, to experimental leaf
arenas from either AMF/DB, AMF+/DB, AMF/
DB+, or AMF+/DB+plants (prepared as described above;
Figure 4. Residence (A; seven observations
per choice unit) and oviposition within 24 h (B)
of four spider mite females (one raised on
AMF/DB, AMF+/DB, AMF/DB, and
AMF+/DB+each) given a choice between four
interconnected leaves from bean plants
inoculated with arbuscular mycorrhizal fungi
(AMF+) and/or diazotrophic bacteria (DB+)or
left uninoculated (AMF,DB). Different
lower case letters inside bars indicate
significant differences between treatments
(Sidak for residence and LSD for oviposition
following GEE; P<0.05).
ª2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3761
B. Khaitov et al.Rhizosphere microbes drive leaf mites
Figure 5. Fresh and dry weight of pods (A),
shoots (B), and roots (C) of common bean
plants inoculated with arbuscular mycorrhizal
fungi (AMF+) and/or diazotrophic bacteria
(DB+) or left uninoculated (AMF,DB). Data
refer to three plants grown together in a pot.
3762 ª2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
Rhizosphere microbes drive leaf mites B. Khaitov et al.
females maturing on a given treatment were placed on a
leaf from the same treatment) and their survival, activity
(moving/stationary), and number of laid eggs assessed
once per day for five consecutive days. This experiment
was carried out in two series with six replicates per treat-
ment per series, hence performed at two plant ages (34
and 56 weeks after transplanting and inoculation).
To assess spider mite host plant choice, four leaflets, one
of each treatment (AMF/DB, AMF+/DB, AMF/DB+,
and AMF+/DB+), were arranged in the shape of a cross on
moist filter paper covering a water-soaked foam
(15 915 94 cm) resting in a plastic box (20 920
96 cm) half-filled with water, with all four leaf tips point-
ing to, and touching each other, in the center. The relative
position and sequence of the four leaflets was random and
switched between replicates. To enable free movement of
the mites, the four leaflets were smoothly connected by a
wax bridge. The wax bridge was created by dripping hot
wax from a nonfragrant candle onto the touching zone of
the leaf tips (Hoffmann et al. 2009). After cooling and
solidification of the wax, four mated spider mite females,
one from each treatment (AMF/DB, AMF+/DB,
AMF/DB+, and AMF+/DB+), were released in the middle
of the bridge and their residence observed every 30 min for
the first 3 h and then again after 24 h. After 24 h, the num-
ber of eggs present on each leaflet was recorded. Before
release, the four females were marked with different tiny
water-color dots on their dorsal sides to make them dis-
cernible. The choice experiment was replicated 22 times;
every choice unit and every female were used only once.
The leaf arenas used for raising the mites to adulthood
before the experiments and the experimental no-choice
and choice units were stored in an environmental cham-
ber at 25 1°C, 60 5% RH and 16:8 h L:D.
Statistical analyses
All statistical analyses were carried out using IBM SPSS
21 (IBM Corp. Armonk, NY). In the spider mite no-
choice experiment, we used separate generalized estimat-
ing equations (GEE) to analyze the influence of DB and
AMF presence/absence and plant age on daily egg produc-
tion (normal distribution, identity link) of the spider mite
females over 5 days, aggregated activity (moving/station-
ary; binomial distribution with logit link), and mortality
(yes/no; binomial distribution with logit link). Plant age
and observation day were auto-correlated (AR1) and used
as inner subject variables. In the spider mite choice exper-
iment, we used GEEs to analyze the residence (lumped
data of seven observations; Poisson distribution, log link)
and oviposition (Poisson distribution, log link) preference
of the mites for the leaflets from AMF/DB, AMF+/
DB, AMF/DB+, or AMF+/DB+plants (exchangeable
correlation structure between the four leaflets of each
replicate). Post hoc least significance difference (Sidak
and LSD) tests were used to compare treatment pairs.
The effects of DB and AMF presence/absence on the fresh
and dry weights (exchangeable correlation structure) of
pods, shoots, and roots were analyzed by separate GEEs
(normal distribution, identity link). RLC was compared
between AMF plants with and without DB, and DB den-
sity (bacteria counts) was compared between DB plants
with and without AMF, respectively, by generalized linear
models (normal distribution, identity link).
Results
In the spider mite no-choice experiment, both AMF and
DB increased offspring production by the spider mite
females, which also produced more offspring on leaves
from young than old plants (Fig. 2, Table 1). Neither
AMF nor DB affected spider mite activity, but the mites
were overall less active on young than old leaves. None of
the two-way interactions affected activity (Fig. 3A,
Table 1). AMF marginally significantly enhanced mite
survival, whereas DB, plant age, and the two-way interac-
tions did not have an effect (Fig. 3B, Table 1). In the spi-
der mite choice experiment, the site preference of the
spider mite females was influenced by plant inoculation
with AMF and/or DB (Fig. 4A; GEE; Wald ӽ
32
=22.59,
P<0.001) and ranked AMF+/DB+, AMF/DB+, AMF+/
DB, AMF/DB(Sidak; P<0.05). The oviposition
preference (Fig. 4B; GEE; Wald ӽ
32
=39.97, P<0.001)
followed the same ranking pattern, with slight differences
in pairwise treatment comparisons (LSD; P<0.05).
AMF but not DB increased pod weight (Fig. 5A,
Table 2); both AMF and DB increased shoot and root
weight (Fig. 5B,C, Table 2); for neither parameter, the
interaction of AMF and DB was significant (Table 2).
DB presence increased the RLC of AMF-inoculated
plants (Wald ӽ
12
=27.708, P<0.001), whereas AMF did
not affect the density of DB in the soil of DB-inoculated
plants (Wald ӽ
12
=0.040, P=0.841) (Fig. 6).
Table 2. Results of generalized estimating equations (GEE) for the
effects of arbuscular mycorrhizal fungi G. mosseae (AMF), and dia-
zotrophic bacteria A. chroococcum (DB), on weight of pods, shoots,
and roots of common bean plants P. vulgaris (three plants grown
together in a pot).
Source of
variation
Pods Shoots Roots
Wald ӽ
12
P Wald ӽ
12
P Wald ӽ
12
P
AMF 18.016 <0.001 4.248 0.039 7.946 0.005
DB 1.310 0.252 5.449 0.020 17.924 <0.001
AMF*DB 0.181 0.670 0.456 0.500 0.518 0.472
AMF, arbuscular mycorrhizal fungi; DB, diazotrophic bacteria.
ª2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3763
B. Khaitov et al.Rhizosphere microbes drive leaf mites
Discussion
The effects of single belowground microorganism species
on aboveground plant-associated herbivores are relatively
well understood, but virtually nothing is known about the
combined effects of functionally distinct belowground
microorganisms on the interactions between aboveground
plant parts, herbivores, and carnivores. Here, we scruti-
nized the interrelated effects of two primarily plant-mutu-
alistic belowground microorganisms, a mycorrhizal
fungus and a free-living nitrogen-fixing bacterium, on
herbivores feeding on aboveground plant parts. In detail,
we disentangled the effects of the DB A. chroococcum and
the AMF G. mosseae on host plant choice and life history
of the two-spotted spider mite T. urticae on common
bean plants P. vulgaris. In addition, we assessed plant per-
formance as affected by DB and AMF, and AMF perfor-
mance as affected by DB, which may act as AMF helper
bacteria (e.g. Garbaye 1994; Bhowmik and Singh 2004;
Behl et al. 2007; Frey-Klett et al. 2007). Both AMF and
DB alone increased spider mite reproduction to similar
levels and exerted additive effects under co-occurrence.
These effects were similarly apparent in host plant choice,
that is, the spider mites preferred and performed the best
on AMF+/DB+leaves, followed by AMF/DB+and
AMF+/DBleaves, and the worst on AMF/DBleaves.
DB enhanced root colonization by AMF, whereas the
density of DB was unaffected by the presence of AMF.
AMF but not DB enhanced reproductive plant growth,
that is, seed production. Both DB and AMF increased the
biomass of vegetative shoot and root tissue.
Although strongly context dependent (Kiers and Deni-
son 2008; Hoeksema et al. 2010), the most widespread
effects of mutualistic soil microorganisms on plants are
improvement of the nutritional status and thus promo-
tion of growth and/or enhancement of the defensive
system (Smith and Read 1997; Pineda et al. 2010; Friesen
et al. 2011). Accordingly, the net effect of a given
belowground microorganism on herbivore performance is
a trade-off between positive effects, due to improved
quality and/or quantity of the host plant, and negative
effects, due to strengthened constitutive and/or induced
resistance mechanisms (Bennett et al. 2006; Gehring and
Bennett 2009; Pineda et al. 2010; Schausberger et al.
2012; Pangesti et al. 2015). In our study, AMF and DB
alone increased vegetative plant tissue and, evident from
the enhanced life history of the spider mites, the nutri-
tional value of the plant tissue for the herbivores.
Together, the two microorganisms had additive effects on
both plant growth and spider mite life history and behav-
ior. AMF primarily enhances uptake of P and K by the
bean plants of our experimental system (Hoffmann et al.
2009) while DB primarily enhances uptake of N (Mrko-
vacki and Milic 2001). AMF additionally increased the
weight of bean pods, that is, enhanced growth of repro-
ductive tissue, reflecting the strong dependency of seed
formation on P availability (e.g. Sterner and Elser 2002).
The provision of additional P and N to plants, up to an
upper limit, profoundly affects plant interactions with her-
bivores, which are strongly P and N limited (Slansky and
Rodriguez 1987; Sterner and Elser 2002). Accordingly, vari-
ation in P and N content of plant tissue commonly affects
host plant choice and life history of herbivores (Coley et al.
2006) including spider mites (Wermelinger et al. 1985,
1991; Hoffmann et al. 2009). For example, N
2
fixation by
rhizobia increases the N content of host plant tissue (Sprent
and Sprent 1990), and thereby strongly determines host
plant quality to herbivores (Sch
adler et al. 2007; Chen et al.
Figure 6. Root length colonized (RLC) by
arbuscular mycorrhizal fungi (AMF) in presence
and absence of diazotrophic bacteria (DB) and
DB density in the soil in presence and absence
of AMF.
3764 ª2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
Rhizosphere microbes drive leaf mites B. Khaitov et al.
2008). However, in addition to primary plant compounds,
the concentration and/or composition of secondary
metabolites or morphological alterations may crucially
determine the outcome of plantherbivore interactions
(Kempel et al. 2009; Katayama et al. 2010). For example,
rhizobia can increase the production of alkaloids (Johnson
and Bentley 1991) or cyanogenic compounds (Thamer
et al. 2011), which are used for direct defense against herbi-
vores. Azotobacter sp. may induce morphological changes
such as thickening the leaf cell walls and leaf cuticulas (e.g.
Gonz
alez et al. 2011), making herbivore attacks more
difficult or energy consuming. Rhizobia or Pseudomonas
colonization may also change volatile emission of plants,
affecting the host plant choice of herbivorous beetles (Ball-
horn et al. 2013) and third trophic level natural enemies
such as parasitoids (Pineda et al. 2013). Therefore, the net
effect of mutualistic soil microorganisms on aboveground
herbivores depends on the trade-off between the enhance-
ment of nutritional quality versus defensive mechanisms
(Pozo and Azcon-Aguilar 2007; Kempel et al. 2010). The
P. vulgaris variety used in our study is poorly directly but
strongly indirectly defended (Hoffmann et al. 2009;
Schausberger et al. 2012). Accordingly, we argue that, in
our system, the net effects of AMF and DB on the herbi-
vores were largely positive due to enhancement of the
nutritional quality of their host plant. Improved uptake of
N and P and other elements conceivably enhanced the pro-
duction and availability of proteins and photosynthates
such as sugars in the leaf tissue, all of which are highly
important for mite reproduction (Slansky and Rodriguez
1987). However, involvement of third trophic level natural
enemies, and enhancement of indirect defense mechanisms,
such as the release of herbivore induced plant volatiles
(HIPVs), might turn the net effects into negative. For
example, Pangesti et al. (2015) observed that Pseudomonas
sp. changed aboveground volatiles to enhance recruitment
of parasitoids to herbivore-infested plants. Similarly,
Schausberger et al. (2012) showed that AMF increases the
attractiveness of HIPVs, induced by spider mites, to their
third trophic level natural enemies, predatory mites. The
combined effects of interacting soil mutualists of plants on
third trophic level natural enemies have not yet been
explored.
Regarding the mutual effects of the microorganisms,
our experiments revealed that the DB A. chroococcum,
which may act as AMF helper bacteria, increased the level
of root colonization by the AMF G. mosseae. In contrast,
AMF did not affect DB occurrence and density. For the
nodulating nitrogen fixer Frankia, Diem (1996) observed
exactly the opposite, that is, positive effects of mycorrhiza
on Frankia but lacking effects of Frankia on mycorrhiza.
Mycorrhiza helper bacteria (MHB) are a highly diverse
group of bacteria and include Azotobacter sp. (Garbaye
1991; O’Connor et al. 2002; Frey-Klett et al. 2007). MHB
may enhance formation and establishment of mycorrhiza,
the association between plant roots and AMF (Allen 1996;
Smith and Read 1997), via diverse mechanisms such as
producing substances/nutrients that stimulate AMF and/
or modify root exudates and/or stimulate the host to pro-
duce substances enhancing mycorrhiza formation (Fitter
and Garbaye 1994; Bansal et al. 2002).
Overall, our study provides a key example of the interre-
lated effects of two primarily plant-mutualistic microor-
ganisms, mycorrhizal fungi and free-living nitrogen-fixing
bacteria, on herbivores feeding on aboveground plant
parts. It breaks new ground in multitrophic belowground
aboveground research by providing first insights into the
implications of plant-mediated belowground fungibacte-
ria interactions on fitness of aboveground herbivores.
Acknowledgments
BK’s stay and work in Vienna was financially supported
by an Erasmus Mundus CASIA grant awarded to BK and
a grant from Eurasia Pacific Uninet (EPU 26/2013)
awarded to PS. TP’s stay and work in Vienna was sup-
ported by Universitat Jaume I (UJI Research Mobility
Grant, E-2013-05). We thank C. Gangl (Agrotrader
Agrarhandel, Austria) for providing Azotobacter sp. and
I.C. Christiansen, A. Walzer, and M. Seiter for comments
on a previous version of the manuscript.
Conflict of Interest
None declared.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Data S1. Raw data for oviposition, mortality, activity and
host plant choice of the spider mites, vegetative and
reproductive plant growth, root colonization by mycor-
rhiza and density of free-living nitrogen fixing bacteria in
the soil.
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Rhizosphere microbes drive leaf mites B. Khaitov et al.

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... The rhizosphere microbial community may consist of a huge diversity of pathogenic, neutral and/or mutualistic micro-organisms. Among the usually mutualistic symbionts, it is especially mycorrhizal fungi, rhizobia and plant growth-promoting rhizobacteria (PGPR) that may influence aboveground plant growth and defense against pathogens and herbivores (Hoffmann et al., 2009(Hoffmann et al., , 2011aHoffmann and Schausberger, 2012;Khaitov et al., 2015;Pangesti et al., 2015;Nunes and Kotanen, 2017;Friman et al., 2021b). Here, we studied plant-mediated interactions between free-living soil bacteria that are commonly considered PGPR and aboveground herbivorous spider mites on strawberry plants. ...
... Several studies have addressed indirect plant-mediated interactions between rhizobacteria and aboveground herbivores' performance (Zehnder et al., 1997;Khan et al., 2018). These interactions are highly system-and contextspecific and may range from positive (Khaitov et al., 2015) to neutral (Valenzuela-Soto et al., 2009) to negative (Zamioudis and Pieterse, 2012) effects on the herbivores, among others depending on the species of bacteria, plant and herbivore, the defensive array of the plant and the abiotic environmental conditions (Shah et al., 2021). Free-living rhizobacteria may increase the abundance of herbivores by enhancing the plant's nutritional state but may simultaneously enhance growth and biomass of vegetative shoot and root tissue (Khaitov et al., 2015). ...
... These interactions are highly system-and contextspecific and may range from positive (Khaitov et al., 2015) to neutral (Valenzuela-Soto et al., 2009) to negative (Zamioudis and Pieterse, 2012) effects on the herbivores, among others depending on the species of bacteria, plant and herbivore, the defensive array of the plant and the abiotic environmental conditions (Shah et al., 2021). Free-living rhizobacteria may increase the abundance of herbivores by enhancing the plant's nutritional state but may simultaneously enhance growth and biomass of vegetative shoot and root tissue (Khaitov et al., 2015). Thus, rhizobacteria may affect plant tolerance to herbivore infestation by enhanced regrowth following herbivore injury (Disi et al., 2019). ...
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Plants mediate interactions between below- and above-ground microbial and animal communities. Microbial communities of the rhizosphere commonly include mutualistic symbionts such as mycorrhizal fungi, rhizobia and free-living plant growth-promoting rhizobacteria (PGPR) that may influence plant growth and/or its defense system against aboveground pathogens and herbivores. Here, we scrutinized the effects of three PGPR, Azotobacter chroococcum, Azospirillum brasilense, and Pseudomonas brassicacearum, on life history and population dynamics of two-spotted spider mites, Tetranychus urticae, feeding on aboveground tissue of strawberry plants, and examined associated plant growth and physiology parameters. Our experiments suggest that these three species of free-living rhizobacteria strengthen the constitutive, and/or induce the direct, anti-herbivore defense system of strawberry plants. All three bacterial species exerted adverse effects on life history and population dynamics of T. urticae and positive effects on flowering and physiology of whole strawberry plants. Spider mites, in each life stage and in total, needed longer time to develop on PGPR-treated plants and had lower immature survival rates than those fed on chemically fertilized and untreated plants. Reduced age-specific fecundity, longer developmental time and lower age-specific survival rates of mites feeding on rhizobacteria treated plants reduced their intrinsic rate of increase as compared to mites feeding on chemically fertilized and control plants. The mean abundance was lower in spider mite populations feeding on PGPR-treated strawberries than in those feeding on chemically fertilized and untreated plants. We argue that the three studied PGPR systemically strengthened and/or induced resistance in above-ground plant parts and enhanced the level of biochemical anti-herbivore defense. This was probably achieved by inducing or upregulating the production of secondary plant metabolites, such as phenolics, flavonoids and anthocyanins, which were previously shown to be involved in induced systemic resistance of strawberry plants. Overall, our study emphasizes that PGPR treatment can be a favorable strawberry plant cultivation measure because providing essential nutrients needed for proper plant growth and at the same time decreasing the life history performance and population growth of the notorious herbivorous pest T. urticae.
... MHB may improve the formation and establishment of mycorrhiza, specifically the association between plant roots and AMF, through various mechanisms such as the production of AMF-stimulating substances/nutrients and/or the modification of root exudates and/or stimulating the plant host to produce substances that improve the formation of mycorrhiza. Plants, AMF and bacteria can therefore be considered tripartite associations leading to a consortium promoting plant growth [18]. The interaction between the micro-organisms in the rhizosphere influences plant growth and production and health directly or indirectly in agricultural and natural habitats by providing nutritional elements and resistance to biotic and abiotic stresses [19]. ...
... Microorganisms in bio-fertilizers can improve the growth of a plant through nitrogen fixation or the production of growth hormones [86]. Therefore a synergistic relationship promotes the growth and yield of plants because microorganisms allow the plants achieve greater absorption of phosphorus, nitrogen and other elements than those treated with a single inoculation and the control [18]. The enhancement of plant biomass by the co-inoculation of AMF and bacterial strain in comparison with AMF inoculation alone has already been reported [11,16,22]. ...
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The use of bio-fertilizers in agro-ecosystems is considered to have the potential to improve plant growth in extreme environments featuring water shortages. However, while arbuscular mycorrhizal fungi (AMF) and bacteria bio-fertilizers have been used in other plants to enhance stress tolerance, little is known about their symbiotic effect on sorghum ( Sorghum bicolor L.) growth under drought stress conditions. Therefore the aim of this study was to investigate the inoculation of sorghum with Nitroxin and Glomus mosseae and their interaction effects on the agro-physiological characteristics and grain yield of sorghum under drought stress conditions. Nitroxin is a bio-fertilizer that consists of a mixture of Azospirillum and Azotobacter bacteria. The results showed that co-inoculation of sorghum seeds with Nitroxin and AMF improved the chlorophyll (a, b and total) content, soluble proteins, water use efficiency) WUE(, relative water content (RWC), nitrogen (N) content in the plant, AMF spore density, proline content, grain yield, panicle length, the number of panicles per plant, grain number per panicle, 1000-grain weight and decreased the electrolyte leakage and water saturation deficit (WSD) in drought stress and non-stress conditions. Under drought stress conditions, there was a 27% increase in grain yield under the synergistic effects of bacteria and fungi compared to the non-application of these microorganisms. The results of this experiment show that Nitroxin and AMF bio-fertilizers can mitigate the negative effects of stress on plants in drought stress conditions by increasing the amount of photosynthetic pigments, soluble proteins and osmotic regulation and decreasing electrolyte leakage. We found that the combination of bacteria and AMF for sorghum growth and yield increment is a promising method to cope with the stress caused by drought.
... Our current knowledge of communications within the phytobiota is still largely based on interactions involving two or three components, and is frequently measured in controlled conditions. However, a further level of complexity in studying phytobiota relates to eukaryotic organisms that interact with plants, each of them harboring its own internal and external microbiota, influencing the interactions between plants and their microbial communities (Cusano et al., 2011;Khaitov et al., 2015). The intense communication among plant holobiont members and the observations that signals can be co-opted, modified, or even destroyed, by another member of the community reinforce the need for a system-level analysis of communication mechanisms to exploit phytobiota manipulation for crop improvement (Figure 1). ...
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Crop plants are associated with a wide diversity of microorganisms in all their parts. Crop microbiota influences plant phenotype, growth, yield and quality by contributing to plant resistance toward diseases, plant adaptation to abiotic stresses, and plant nutrition. The association between terrestrial plants and microbes developed at least 460 million years ago, as suggested by the fossil evidence of the earliest land plants, indicating the essential role of microbes for plants. Recent studies indicate that plants actively recruit beneficial microorganisms to facilitate their adaptation to environmental conditions. Cultivation methods and disease control measures can influence plant microbiome structure and functions. Both pesticide and biological control agent applications may alter the biodiversity inside the phytobiota and suppress beneficial functions. Nonetheless, to date, the effects of disease control measures on phytobiota and their possible side consequences on plant growth, crop productivity and quality remain a neglected field of study. The present work summarizes the known effects on phytobiota providing evidence about the role of plant microbial community in determining the overall efficacy of the applied control measure and suggests that future studies on plant disease control consider also the microbe-mediated effects on plant fitness.
... Alternatively, rhizosphere communities may affect the outcome of leaf interactions. For example, applying nematodes to the soil can reduce aphids on the leaves (Hol et al. 2013), and mycorrhizae and nitrogen-fixing bacteria in bean plants can lead to the attraction of mites (Khaitov et al. 2015). These results suggest that, after infections by phytopathogens, host plants recruit specific beneficial microbiota that enable them to resist and withstand diseases caused by these organisms (Berg et al. 2016). ...
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Background Utilizating the plant microbiome to enhance pathogen resistance in crop production is an emerging alternative to the use of chemical pesticides. However, the diversity and structure of the microbiota, and the assembly mechanisms of root-associated microbial communities of plants are still poorly understood. Results We invstigated the microbiota of the root endosphere and rhizosphere soils of the rice cultivar Nipponbare (NPB) and its Piz-t-transgenic line (NPB-Piz-t) when infected with the filamentous fungus Magnaporthe oryzae ( M. oryzae ) isolate KJ201, using 16S rRNA and internal transcribed spacer 1 (ITS1) amplicon sequencing. The rhizosphere soils showed higher bacterial and fungal richness and diversity than the endosphere except for fungal richness in the rhizosphere soils of the mock treatment. Bacteria richness and diversity increased in the endospheric communities of NPB and Piz-t under inoculation with KJ201 (referred to as ‘NPB-KJ201’ and ‘Piz-t-KJ201’, respectively) compared with the corresponding mock treatments, with the NPB-KJ201 showing the highest diversity in the four bacterial endocompartments. In contrast, fungal richness and diversity decreased in the endospheric communities of NPB-KJ201 and Piz-t-KJ201, relative to the corresponding mock treatments, with NPB-KJ201 and Piz-t-KJ201 having the lowest richness and diversity, respectively, across the four fungal endocompartments. Principal component analysis (PCA) indicated that the microbiota of Piz-t-KJ201 of root endophytes were mostly remarkablely distinct from that of NPB-KJ201. Co-occurrence network analysis revealed that the phyla Proteobacteria and Ascomycota were the key contributors to the bacterial and fungal communities, respectively. Furthermore, a comparative metabolic analysis showed that the contents of tryptophan metabolism and indole alkaloid biosynthesis were significantly lower in the Piz-t-KJ201 plants. Conclusions In this study, we compared the diversity, composition, and assembly of microbial communities associated with the rhizosphere soils and endosphere of Piz-t-KJ201 and NPB-KJ201. On the basis of the different compositions, diversities, and assemblies of the microbial communities among different compartments, we propose that the host genotype and inoculation pattern of M. oryzae played dominant roles in determining the microbial community assemblage. Further metabolomics analysis revealed that some metabolites may influence changes in bacterial communities. This study improves our understanding of the complex interactions between rice and M. oryzae , which could be useful in developing new strategies to improve rice resistance through the manipulation of soil microorganisms.
... Mycorrhizal helper bacteria may increase the plant root colonization with AMF by different mechanisms, such as producing the AMF stimulators substances and nutrients, enhance root exudates, and/or stimulate the host to generate substances improving mycorrhiza formation (Rigamonte, Pylro, and Duarte 2010). On the other hand, Azotobacter sp. may also assist AMF root colonization by providing the suitable environmental conditions for AMF activity via different ways, such as generating growth factors, deterrence of competitors and antagonists, and enhancing soil properties (Frey-Klett, Garbaye, and Tarkka 2007;Khaitov et al. 2015). ...
Article
Wheat is the main staple crop in Iran and thus of critical importance in human nutrition. This study was conducted to investigate the effect of individual and co-inoculation of azotobacter chroococcum bacteria (A. chroococcum) and a species (Rhizophagus irregularis) of arbuscular mycorrhizal fungi (AMF) on improving the nutritional quality of bread wheat grain. We performed a pot experiment, in which the effects of R. irregularis and A. chroococcum inoculation, and zinc (Zn) fertilization [0, 4.4, and 8.8 mg (kg)⁻¹] to cadmium (Cd)-spiked soil on increasing the Zn bioavailability and reducing the Cd concentration of wheat grains were tested. Inoculation with A. chroococcum increased the rate of root colonization by R. irregularis. Root colonization with AM fungus significantly reduced the grain Cd concentration especially after co-inoculation with A. chroococcum and soil application of ZnSO4 fertilizer. Mycorrhized plants, compared to non-mycorrhized plants had considerably higher grain concentrations of phosphorous (P) and nitrogen (N), which were affected by the application rate of ZnSO4 and co-inoculation with A. chroococcum. The response of grain Zn concentration to mycorrhization was dependent on the rate of Zn fertilization and inoculation with A. chorococum. Single inoculation with R. irregularis increased the concentration of phytic acid (PA) and also the molar ratio of PA:Zn in grains, which may have resulted in reduced Zn bioavailability. In contrast, double inoculation of plants with R. irregularis and A. chroococcum reduced the molar ratio of PA:Zn especially after application of ZnSO4. This study suggests that dual inoculation of wheat plants with A. chroococcum and R. irregularis together with Zn fertilization may enhance the nutritional quality of wheat grains.
... Plant communities are a key component of ecosystems as they are at the bottom of trophic networks playing also an important role in nutrient cycling [4]. However, plant survival may also depend on the associated microorganisms community [5], usually developing symbiotic relationships with them [6]. Soil microorganisms are responsible for multiple ecosystem functions, including litter decomposition, soil fertility, and water retention [7][8][9]. ...
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Soil microorganisms, together with water, play a key role in arid ecosystems, being responsible for the nutrient cycle, facilitating nutrient incorporation into plants, influencing plant drought tolerance, and enhancing their establishment. Therefore, their use for restoration practices is promising. We tested the potential of native strains of Actinobacteria from Monte Desert as growth promoters of native vegetation, isolating them from two substrates from their habitat (bare soil and leaf-cutting ant refuse dumps). Strains were inoculated into the soil where seedlings of three native plant species (Atriplex lampa, Grindelia chiloensis, Gutierrezia solbrigii) were growing. Seedlings were grown following a full factorial design experiment under greenhouse and field conditions comparing native Actinobacteria effects with a known growth-promoting strain, Streptomyces sp. (BCRU-MM40 GenBank accession number: FJ771041), and control treatments. Seedlings survived greenhouse condition but species survival and growth were different among treatments at field conditions, varying over time. The highest survival was observed in a native soil strain (S20) while the lowest in MM40. The low survival in MM40 and in the other treatments may be explained by the higher herbivory observed in those seedlings compared to control ones, suggesting a higher nutritional status in inoculated plants. Strains from refuse dumps were the best at enhancing seedling growth, while strains from soil were the best at maintaining their survival. Native Actinobacteria studied may increase plant species survival and growth by improving their nutritional status, suggesting their potential to facilitate vegetation establishment and, therefore, being good candidates for restoration practices. Furthermore, plant species respond differently to different strains, highlighting the importance of microorganism diversity for ecosystem functioning.
... Few studies have also documented that AMF 1 3 can modulate plant interactions with herbivores (Kempel et al. 2010), their natural enemies and pollinators (Pineda et al. 2010;Willis et al. 2013). Therefore, it is possible that AMF can have cascading effects on plant-insect interactions (Gehring and Bennett 2009;Khaitov et al. 2015), an area of research that warrants more attention. ...
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Beneficial plant–microbe interactions in the rhizosphere have been found to enhance plant growth and development. Arbuscular mycorrhizal fungi (AMF), a major group among these microbes, have been found to improve plant fitness through mycorrhizal symbiosis. Despite being well documented in various natural and domesticated study systems, few studies have examined whether AMF also has cascading effects on other traits, such as influencing insect community dynamics through attraction/repulsion of beneficial and harmful insects. To test this, we planted Sorghum-sudangrass (Sorghum x drummondii), a fast-growing annual grain/forage crop, either inoculated with commercial AMF mix or left as control in lab and field experiments. We hypothesized that AMF would enhance plant growth and influence the recruitment of insect herbivores and their natural enemies due to possible alterations in plant defense pathways. Our results suggest that while AMF-inoculated plants had significantly better germination, growth, and establishment; they also experienced a lower initial incidence of Spodoptera frugiperda, a major herbivore on Sorghum in the Lower Rio Grande Valley. In addition, our insect community trapping experiment revealed that AMF-inoculated plants attracted significantly more beneficial insects (predators and parasitoids) and a lower number of damaging herbivores. Taken together, our field and lab data show that AMF can not only positively influence plant growth traits but can also provide defenses against herbivores by selectively attracting beneficial insects and repelling herbivores, with implications for sustainable pest management strategies.
... A. chroococcum is known to produce plant growth-promoting substances in addition to nitrogen fixation. Further it is known to have synergistic interaction with AM fungi [32,33]. T. harzianum enhancing plant growth has been reported in several crops and has been attributed to the production of plant growthpromoting substances, thereby enhancing root growth and antimicrobial compounds deleterious to plant pathogens [34,35]. ...
Article
The response of teak (Tectona grandis L.f.) to the selected microbial consortium (Ambispora leptoticha, Azotobacter chroococcum, Trichoderma harzianum) was evaluated through large-scale nursery trials at three locations in Mandya district of Karnataka state, India. At each location, there were 500 inoculated and 500 uninoculated seedlings, totaling to the maintenance of 3000 seedlings at three locations. The growth performance was evaluated 180 days after planting. The increase in plant dry weight of inoculated seedlings was 97% compared to uninoculated plants. The seedlings inoculated with the microbial consortium under large-scale nursery trials were planted in the wasteland at three locations, and their growth was evaluated for nearly 6 years. The biovolume index of inoculated plants was 289% more than uninoculated plants 73 months after out-planting. It can be concluded that the selected microbial consortium is the best for inoculating teak in forest nurseries.
... Specifically, these bacteria (e.g. Azospirilla and Azotobacter) enhance plant N supply through production of plant growth promoting substances that act mainly, by increasing the ability of the root system to absorb more N from the soil (Dobbelaere et al. 2001;Khaitov et al. 2015). ...
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Arbuscular mycorrhizal fungi (AMF) and biochar applications have been found to enhance the nutrient cycling and agricultural crop production. This greenhouse study investigated the effects of AMF inoculation and biochar amendment at different level on the soil bacterial population, mycorrhizal root infection and nutrient uptake of cacao seedlings. Germinated twoweek old cacao (Theobroma cacao L.) seedlings were either uninoculated or inoculated with AMF during transfer from seed germination boxes into individual polybags filled with acidic red soil amended with increasing (3.75%, 7.5% and 15%) level of bamboo biochar (BB). After 15 months, AMF inoculation improved the N and P uptake of cacao seedlings by 65% and 90% over the control, respectively. Highest (25 mg plant-1) N uptake of the seedlings was observed in AMF+15% BB. P uptake of cacao seedlings was also improved by 7.5% BB (1.56 mg plant-1), 15% BB (1.90 mg plant-1) and AMF + 15% BB (1.89 mg plant-1). Biochar levels improved the percentage root infection in mycorrhizal (+AMF) seedlings from 79% to 83%. The population of nitrogen-fixing bacteria (NFB) and phosphate solubilizing bacteria (PSB) increased with biochar amendment, consistently at 15% level. Irrespective with biochar amendment, AMF only affected the PSB population. Moreover, PSB population has strong correlation with the P and N uptake of cacao seedlings while NFB population was significantly correlated only with P uptake. The bacterial population and nutrient uptake were not significantly correlated with the percentage mycorrhizal root infection. The significant effect of these treatments indicates a good effect in improving the growth performance of cacao seedlings.
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A field experiment was conducted during two consecutive growing seasons (2013 and 2014) to evaluate the effects of inoculations with Rhizobium and Azotobacter on the growth and yield of two chickpea (Cicer arietinum L.) varieties under saline (5.8 dS m�1) arid condition. The single treatment of either Rhizobium or Azotobacter exhibited to promote the growth of chickpea to some level, however, co-inoculation produced more effects and increased the shoot dry weight (30.3 and 26.4%), root dry weight (17.5 and 26.3%), nodule number (79.1 and 43.8 piece per plant), nitrogen content in roots (9.62 and 10.9%), in shoots (12.6 and 8.3%) and seed protein (7.1 and 4.3%) in both Flip06-102 and Uzbekistan-32 chickpea varieties compared to the control. Our studies showed that the highest yield response of 429 (27.9%) and 538 (23.9%) kg ha�1 over the control was revealed by the co-inoculation with Rhizobium and Azotobacter inoculants in Flip 06-102 and Uzbekistan-32, respectively. A new introduced Flip 06-102 chickpea variety was more salt tolerant and had higher root nodulation than the local Uzbekistan-32 chickpea variety. Nitrogen (N), phosphorus (P), and potassium (K) contents in the shoots and roots were significantly (p<0.05) higher in the co-inoculated treatment, while plant sodium (Naþ) concentration was the lowest for both chickpea varieties exhibiting co-inoculation alleviated the detrimental effects of salinity. Therefore, the co-inoculation of Rhizobium plus Azotobacter could be applied to improve the vegetative growth and yield of chickpea and to alleviate the effects of salt stress.
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Mycorrhizosphere includes the region around the mycorrhizal roots. The concept of mycorrhizosphere is based on the fact that mycorrhizae exert a strong influence on the microflora in the rhizosphere (Bansal and Mukerji 1994; Fitter and Garbaye, 1994; Garbaye, 1991; Mukerji et al. 1998; Paulitz and Linderman, 1991). As a result, there are taxonomic and functional differences in populations of bacteria, fungi and nematodes associated with rhizospheres of vesicular arbuscular mycorrhizal (VAM) and non-VAM plants. How, the symbiotic association with mycorrhizal fungi alters the rhizosphere microflora is one of the fundamental questions in mycorrhizal research.
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Mycorrhizal symbiosis should not be considered merely as a bipartite plant-fungus interaction, but should instead incorporate the associated organisms. These mycorrhiza-associated organisms are known to influence each other mutually, the outcome of which is described as the “mycorrhizosphere” (Foster and Marks 1966; Meyer and Linderman 1986; Frey-Klett and Garbaye 2005). The mycorrhizosphere comprises mycorrhizas, extramatrical mycelium and the associated microorganisms. In the same way the rhizospheres exert a pressure on microbial populations (Barea et al. 2005), the mycorrhizal roots and hyphae of mycorrhizal fungi (MF) shape the bacterial species composition due to root and hyphal exudation and turnover (Bowen 1993; Morgan et al. 2005). This “mycorrhizosphere effect” may lead to improved plant nutrition, growth and disease resistance (Linderman 1988; Frey-Klett et al. 2005). Determining the functional significance of the mycorrhizosphere organisms for plant productivity presents a major challenge for the future (Artursson et al. 2006).
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The efficiency of plant-growth-promoting rhizobacteria (PGPR), viz. Azospirillum sp., Azotobacter chroococcum, Pseudomonas fluorescens, Pseudomonas striata and yeast, viz. Saccharomyces cerevisiae was evaluated for maximization of Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe root colonization and spore number in the root zone of Rhodes grass (Chloris gayana Kunth). The pot culture experiment was carried out under polyhouse condition and observations were recorded at 45 days and 90 days of plant growth. The PGPR considerably enhanced mycorrhizal colonization compared to yeast, with Azospirillum sp. being the most efficient. They not only stimulated AM development, but also accelerated the root growth.
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The roots of most plants are colonized by symbiotic fungi to form mycorrhiza, which play a critical role in the capture of nutrients from the soil and therefore in plant nutrition. Mycorrhizal Symbiosis is recognized as the definitive work in this area. Since the last edition was published there have been major advances in the field, particularly in the area of molecular biology, and the new edition has been fully revised and updated to incorporate these exciting new developments. . Over 50% new material . Includes expanded color plate section . Covers all aspects of mycorrhiza . Presents new taxonomy . Discusses the impact of proteomics and genomics on research in this area.
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Biological control can be defined as the directed and precise management of biological resources, to protect plants against pathogens. The control of plant diseases by applying biocontrol techniques is a multifaced scientific area of great interest because of the need to reduce chemical inputs to agriculture and significantly enhance global sustainability. Some specific groups of microorganisms are able to protect the plant against pathogens by acting through several mechanisms. Among these organisms, arbuscular mycorrhizal fungi (AMF) are promising because of their ubiquity in natural and agricultural terrestrial ecosystems (Jeffries and Barea 2001).
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Nitrogen-fixing and phosphate-mobilizing bateria, as well as mycorrhizal fungi, can influence plant nutrition beneficially and thus be used as biofertilizers in agriculture. This paper briefly reviews the role of wheat genotypes in the interaction of wheat with soil microorganisms like phosphate solubilizing and nitrogen fixing bacteria, specifically Azotobacter sp., and with mycorrhizal fungi for the development of sustainable wheat crop production. The role of rhizosphere microorganisms and the mechanisms, factors affecting response of bioinoculants and the possibilities of breeding wheat genotypes responsive to these bioinoculants for sustainable wheat production in semi-arid tropics are discussed.
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A wide range of natural factors such as lightning-caused fires and geomorphic or palaeotectonic processes may affect the stability of natural ecosystems (Herrera et al. 1993). Additionally, human activities causing pollution of air, water and soil, and overuse of resources like grasslands or clear-cutting of forests have a strong impact on a wide range of ecosystems. They may become degraded to such an extent that spontaneous recovery is strongly limited, especially if the damaging agent is continuously present. In general, successful restoration requires the reconstruction of adequate biological, physico-chemical, hydrological and morphological conditions. Moreover, the presence of hazardous substances can necessitate chemical or bioremediated clean-up. A common reason for the failure of many restoration attempts is the neglect of the fact that the plant root systems are associated with a diverse community of active soil micro-organisms. It is well known that a functioning association between plants and rhizosphere micro-organisms can modify the substratum, facilitate plant establishment under hostile conditions, and counteract the stagnation of the succession.