Jam.12946

REVIEW ARTICLE
A review: what is the spermosphere and how can it be
studied?
S. Schiltz, I. Gaillard, N. Pawlicki-Jullian, B. Thiombiano, F. Mesnard and E. Gontier
Biologie des Plantes et Innovation (BIOPI), Universit
e de Picardie Jules Verne, Amiens, France
Keywords
analytical techniques, germination, microbial
communities, seed exudates, spermosphere.
Correspondence
S
everine Schiltz, Biologie des Plantes et Inno-
vation (BIOPI), Universit
e de Picardie Jules
Verne, EA3900 ^
Ilot des Poulies, 33 rue de
Saint Leu, 80039 Amiens Cedex, France.
E-mail: severine.schiltz@u-picardie.fr
2015/1013: received 19 May 2015, revised
27 July 2015 and accepted 15 August 2015
doi:10.1111/jam.12946
Summary
The spermosphere is the zone surrounding seeds where interactions between
the soil, microbial communities and germinating seeds take place. The concept
of the spermosphere is usually only applied during germination sensu stricto.
Despite the transient nature of this very small zone of soil around the
germinating seed, the microbial activities which occur there may have long-
lasting impacts on plants. The spermosphere is indirectly characterized by
either (i) seed exudates, which could be inhibitors or stimulators of micro-
organism growth or (ii) the composition of the microbiome on and around
the germinating seeds. The microbial communities present in the
spermosphere directly reflect that of the germination medium or are host-
dependent and influenced quantitatively and qualitatively by host exudates.
Despite its strong impact on the future development of plants, the
spermosphere remains little studied. This can be explained by the technical
difficulties related to characterizing this concept due to its short duration,
small size and biomass, and the number and complexity of the interactions
that take place. However, recent technical methods, such as metabolite
profiling, combining phenotypic methods with DNA- and RNA-based
methods, could be used to investigate seed exudates, microbial communities
and their interactions with the soil environment.
Introduction
Seed germination is a crucial step for plant development
and agricultural production. The process of germination
starts with the uptake of water by the dry seed and is
completed when the radicle tip is visible. Uptake of water
by a dry seed is triphasic with a rapid initial uptake
(phase I, i.e. imbibition), followed by a plateau phase
(phase II) (Bewley et al. 2013). The third phase is charac-
terized by (i) a resumption of water uptake, (ii) endo-
sperm rupture and (iii) an elongation of the embryo axes
leading to radicle protrusion, which marks the end of
germination sensu stricto (Weibrecht et al. 2011). How-
ever, the third phase and water uptake continue during
the transition to seedling growth.
Upon first contact with water, the seed rapidly swells
and changes in size and shape (Robert et al. 2008). The
influx of water into cells of dry seeds during phase I
results in temporary structural disruptions, particularly to
membranes, which lead to an immediate and rapid leak-
age of solutes and low molecular weight metabolites into
the surrounding imbibition solution (Koizumi et al.
2008). In mucilaginous seeds, mucilage composed pri-
marily of pectins and hemicelluloses (Western 2012) is
released quickly from the seed surface during imbibition
(Windsor et al. 2000) and is then degraded to CO2 and
soluble sugars in the presence of soil microbial communi-
ties (Yang et al. 2012). This degradation increases the soil
microbial biomass and promotes early seedling growth.
This specific zone of interactions surrounding seeds,
between the soil, seed-borne microbial communities and
germinated seeds, was first fully studied and named the
spermosphere by Verona (1958). Nelson (2004) defined it
as ‘the zone of microbial interaction around the seed that
is under the influence of seed carbon deposition’. Associ-
ations developing on and around germinating seeds are
of major interest as they mark the first point of contact
between plants, pathogens and soil micro-organisms, with
Journal of Applied Microbiology 119, 1467--1481 © 2015 The Society for Applied Microbiology 1467
Journal of Applied Microbiology ISSN 1364-5072

either beneficial or deleterious future effects on plant
growth, development and health (Nehl et al. 1996; Singh
et al. 2011). Despite the transient nature of the spermo-
sphere, associations initiated within it have long-lasting
impacts on plants because they are crucial for the future
implementation of the plant rhizosphere, which signifi-
cantly influences plant growth and crop yield. The rhizo-
sphere, and especially plant growth-promoting
rhizobacteria (PGPR), have been widely studied because
they are a ‘green’ alternative to chemical fertilizers for the
promotion of sustainable agriculture (Avis et al. 2008;
Babalola 2010; Bhattacharyya and Jha 2012). PGPR are
economical, not harmful to the environment and can
easily be detected and isolated. Despite the importance of
the spermosphere in the establishment and development
of the rhizosphere, few studies have focused on the early
stages of the implementation of the interactions between
seeds and microbial communities during seed germina-
tion. The greatest deficiencies in the knowledge of the
spermosphere could be explained by the large number of
molecules exuded by seeds, their small quantity and their
short transient exudation into the environment as well as
the extreme complexity of soil microbial communities
and thus their complex interactions with seeds. However,
with the recent development of high-tech analytical tech-
niques, it is now possible to identify and quantify seed
exudates precisely as well as to determine the micro-or-
ganisms involved in the first steps of interactions with
seeds. The objective of this review is to examine the cur-
rent knowledge about the environment in the immediate
vicinity of germinating seeds: the spermosphere (Fig. 1).
In the first part of this review, the spermosphere is
defined in space and time and the different elements that
compose it, as well as their evolution in space and time,
What is the spermosphere ?
How can the spermosphere be characterised ?
where interactions between soil, microbial communities and germinating
seeds take place, with long-lasting imapacts on plants.
Seed
Radicule
- “ A small zone” from 2 to 12 mm around seeds,
- A short-lived concept only applied during seed germination sensu stricto,
By compounds exuded by seeds By microbial communities
They have two origins
Host-dependent
Soil-dependent
Nature of microbes
Eubacteria (Gram-negative and positive)
Analytical methods to identify microbial
communities
Phenotypic methods (plating methods, CLPP,
PLFA-FAME, biosensors)
DNA and RNA methods (DGGE, molecular
markers, 16S rRNA)
Fluorescence in situ hybridization
Stable isotope probing
Multi-omics approaches
True fungi (like Ascomycota)
Fungi-like organisms (Stramenopiles like
During 3 peaks of exudation
At initiation of seed imbibition
Few hours after imbibition
At radicle emergence
Nature of seed exudates
Carbohydrates, organic acids, alcohols fatty
acids, amino acids, proteins, secondary
metabolites and inorganic ions
The composition of seed exudates varies with
temperature
Analytical methods to identify seed exudates
Chromatographic techniques (TLC, HPLC, ion-
exclusion-partition, ion exchange)
Metabolic profiling approch (GC- and LC-MS,
NMR)
palnt species, soil type, soil pH, soil moisture and
Oomycota)
Figure 1 Schematic representation of the
spermosphere. During germination, seeds
passively exude a variety of compounds ( )
into the environment. The composition and
quantity of these exudates are highly variable
and contribute to creating specific micro-
environments in the vicinity of the seeds.
Specifically adapted micro-organisms (●)
colonize this zone and set up beneficial or
detrimental interactions with seeds. These
early relationships play a major role in future
plant health and growth.
Journal of Applied Microbiology 119, 1467--1481 © 2015 The Society for Applied Microbiology
1468
The spermosphere S. Schiltz et al.

are described. In the second part, the review focuses on
the technical approaches developed to study the spermo-
sphere. To conclude, some strategies are proposed to
understand better the functioning of the spermosphere to
manipulate it for agronomic purposes.
How can the spermosphere be defined?
The spermosphere is the region of soil directly under the
influence of seeds and the critical interface between plants
and microbes where beneficial and detrimental interac-
tions occur. The spermosphere has only a temporary exis-
tence during germination sensu stricto: before
germination, there is no interaction between the mature
dry seed and the soil, while after the emergence of the
radicle, the soil environment surrounding the seed is
defined as the rhizosphere.
In space
For many years, the zone of seed influence was estimated
by the germination response of micro-organism propag-
ules at various distances from the seed (Stanghellini and
Hancock 1971; Short and Lacy 1974; Short and Wyllie
1978). These authors showed that spore germination was
recorded between 2 and 12 mm from the seed surface.
The size of the spermosphere is variable. It depends on
the methodology used: the type of seeds and micro-or-
ganisms and the conditions of the study. Generally, a
higher soil moisture facilitates the diffusion of seed exu-
dates through the soil and so extends the area of the
spermosphere (Short and Lacy 1974). These authors also
showed that, at 50% soil moisture, the maximum dis-
tance from the seed at which Fusarium solani chlamy-
dospores germinated decreased with increasing
temperature. However, the zone of influence of seeds is
not uniformly distributed in the soil around them and
varies according to soil texture, moisture and temperature
(Stanghellini and Hancock 1971; Short and Lacy 1974),
plant species and genotypes (Short and Lacy 1974, 1976;
Roberts et al. 2009).
Soil is a highly heterogeneous environment formed by
numerous microenvironments where biotic (predation,
competition and root growth) and abiotic (pH, tempera-
ture, water tension and nutrient availability) factors
impose stresses on micro-organisms (Van Veen et al.
1997). These many microenvironments can profoundly
affect the distribution, physiology and survival of micro-
bial communities (Roberts and Kobayashi 2011). More-
over, seeds release an extremely diverse range of organic
and inorganic compounds into the soil, which has a con-
siderable effect on its physical and chemical properties
and significantly influences the nutritional environment
encountered by indigenous soil microflora (Badri and
Vivanco 2009; Hartmann et al. 2009). The lack of unifor-
mity in exudation across the seed surface leads to a
heterogeneous spatial distribution of microbial communi-
ties on and around seeds. The density of bacteria
increases with proximity to the future radicle emergence
and is greatest at cracks (Short and Lacy 1974; Tombolini
et al. 1999; Nelson 2004; Ofek et al. 2011). Generally, the
zone immediately behind the root tip is considered a
major site of exudation, as are the points of secondary
root emergence (Badri and Vivanco 2009). The spermo-
sphere, like the rhizosphere, can be thought of as a gradi-
ent system where diffusible compounds released from the
seed influence micro-organisms in regions of soil that
extend radially at millimetre distances from the plant
(Toal et al. 2000).
In time
In addition to varying in quantity and in quality, the
compounds exuded by seeds are not continuously
released following imbibition; in fact three peaks of exu-
dation have been revealed (Nelson 2004). The first peak
is concomitant with the initiation of seed imbibition fol-
lowed by a second peak a few hours later (Simon and
Raja Harun 1972). Windstam and Nelson (2008) showed
that significant levels of fatty acids and sugars were
released by both corn and cucumber seeds 15 min after
the initiation of imbibition but they did not reveal the
exudation peak within 6 h after imbibition. Temperature
affects the pattern of exudation but does not significantly
affect the amount exuded (Short and Lacy 1976). These
authors showed that the majority of carbohydrates were
exuded during the first 18 h of incubation at 22 or 30°C
and persisted for about 48 h at 10°C. The first two peaks
of exudation result from the rupture of membranes due
to rapid water uptake by the seed during the first phase
of germination. Then, the integrity of the membranes is
restored and exudation ceases. The third peak occurs
when the radicle emerges during the last phase of germi-
nation. The distribution of nutrients in the spermosphere
at a given point in space will also vary over time due to
the growth and maturation of the root and the develop-
ment of attendant soil microbial communities. Thus, the
spermosphere is spatially and temporally heterogeneous
on both micro- and macroscales with regard to the con-
centration of individual nutrients and their availability
(Roberts and Kobayashi 2011).
By the exudate composition
When seeds imbibe, a variety of compounds are passively
exuded from them into the spermosphere. The
S. Schiltz et al. The spermosphere

composition of these seed exudates varies with plant spe-
cies and with other factors such as soil type, soil pH, soil
moisture, temperature, available nutrients in the soil and
the presence of micro-organisms. Compared to the analo-
gous rhizosphere around roots, relatively little research
has been done on the spermosphere. Exudates released
from germinating seeds are usually ‘normal products’ of
seed metabolism and they generally consist of carbohy-
drates, amino acids, flavonoids, sterols and salts (Vancura
and Hovadik 1965; Rovira 1969; Gamliel and Katan 1992;
Nelson 2004) (Table 1). Volatile compounds such as
aldehydes, alcohols, ethylene, CO2 and volatile carboxylic
acids are also released from germinating seeds (Stotzky
and Schenck 1976; Vancura and Stotzky 1976; Gorecki
et al. 1985). Most of the carbohydrates exuded by seeds
are simple sugars such as sucrose, glucose, fructose and
maltose, but the ratios of these sugars are strongly depen-
dent on the developmental stage (Lugtenberg et al. 1999).
Molecules exuded by seeds during germination have an
effect on the immediate biotic and abiotic environment.
The release of organic acids into the spermosphere
changes the surrounding soil pH and alters the solubility
of inorganic phosphorous compounds, increasing phos-
phorous availability (Hinsinger 2001). Organic acids also
form metal chelate complexes, which can increase iron
solubility in low-pH (iron-limiting) conditions and
reduce aluminium toxicity (Jones 1998). It has been
hypothesized that other micronutrients, such as zinc and
copper, may be solubilized and mobilized by organic
acids, but little research has been done to investigate this
hypothesis (Jones et al. 2003). Seed exudates are crucial
to initiate the establishment of rhizobacteria, which can
affect plant development and health (Nelson 2004). Rapid
utilization of seed exudates can grant an advantage to
some biocontrol bacteria such as Pseudomonas over seed
pathogens like Pythimum ultimum (Fukui et al. 1994).
Seed exudates may be involved in the attraction of rhizo-
sphere micro-organisms and may modulate important
bacterial properties that confer the ability to adhere and
grow competitively in the seed vicinity. For example, seed
exudates collected from two varieties of soybean induced
a chemotactic response, supported active cell division and
induced biofilm formation of Bacillus amyloliquefaciens
BNM 339. However, root exudates did not have the same
effect (Yaryura et al. 2008). It is still unclear which differ-
ences between seed and root exudate compositions lead
to different responses in bacteria.
By the origin of microbial communities and their
presence in the rhizosphere
At present, there are two points of view on the origin of
microbial communities in the spermosphere. The first
suggests that the seed’s acquisition of its microbiome,
prior to extension of the primary root, results from a
passive encounter between microbes conveyed by the soil
solution and the germinating seed (Buyer et al. 1999;
Green et al. 2006; Ofek et al. 2011). In this theory, the
seeds do not influence the composition of the microbial
communities, which depend exclusively on the medium.
Buyer et al. (1999) found that seeds of different plant
species shared highly similar spermosphere bacterial com-
munities when planted in the same soil, although the
amounts and composition of the seed exudates differed
greatly. In this case, the microbial communities of the
spermosphere directly reflected that of the germinated
Table 1 Compounds exuded by seeds during germination
Compounds Analytical methods
Carbohydrates: Arabinose, Fructose, Galactose, Glucose, Maltose,
Mannose, Lactose, Raffinose, Rhamnose, Ribose, Sorbose, Sucrose, Xylose
GCMS, NMR (Da Silva Lima et al. 2014)
HPLC (Lugtenberg et al. 1999)
Anion exchange chromatography (Casey et al. 1998)
Ion-exchange chromatography (Kamilova et al. 2005)
Amino acids: Alanine, Glutamic acid, Glutamine, Glycine, Homoserine,
Leucine/Isoleucine, Methionine, Phenylalanine, Pyroglutamic acid, Serine,
Threonine, Tryptophan, Tyrosine, Valine
GCMS, NMR (Da Silva Lima et al. 2014)
Thin layer chromatography (Smith 1969)
RP-HPLC (Kamilova et al. 2005)
Organic acids: Acetic acid, Citric acid, Formic acid, Malonic acid,
Oxalic acid, Succinic acid
GCMS (Da Silva Lima et al. 2014)
Secondary metabolites: Phenolic derivatives, Steroids, Terpenoids GCMS (Da Silva Lima et al. 2014)
Alcohols: 1-Hexanol, 2-ethyl Glycerol, Mannitol GCMS (Da Silva Lima et al. 2014)
Fatty acids: Hexadecanoic acid, Octadecanoic acid isomers, Tridecanoic acid GCMS (Ruttledge and Nelson 1997; Da Silva Lima et al. 2014)
Proteins: Chitinases, Cysteine-rich protein, Galactosidases, Glycosyl hydrolases Electrophoresis and Immunoblotting (Terras et al. 1995)
Electrophoresis and LC-ESI-MS/MS (Scarafoni et al. 2013)
Inorganic ions: Cl, K, F, N, P, S Flame emission, atomic absorption spectrometry, Anion exchange
chromatography (Kato et al. 1997; Casey et al. 1998)
1470

medium. Then, the microbial community evolved under
the influence of root growth and a disparity between seed
and root microbial communities was observed: many
seed-colonizing populations were not detected in root
samples (Normander and Prosser 2000; Green et al. 2006;
Ofek et al. 2011). Unlike seeds, roots determine the com-
position, size and spatial distribution of bacterial
communities.
The second point of view argues that spermosphere-
colonizing bacteria are host-dependent and that their
levels of metabolic activity are based largely on both the
quantitative and qualitative composition of host exudate
released during seed germination (Roberts et al. 2009). In
this second theory, microbial communities of the sper-
mosphere depend on the seeds and their exudates. For
example, germination of sporangia of Pythium spo-
rangium was higher in the presence of seed exudates of
plants such as carrot, corn, lettuce, pea, radish and wheat
than in the presence of cotton, cucumber, sunflower, and
tomato seed exudates (Kageyama and Nelson 2003).
Simon et al. (2001) reported that population densities of
cultivable bacteria on tomato seeds were affected by the
genotype. Similarly, significant differences in the number
and species of endophytic bacteria were observed among
the seeds of four offspring hybrid maize and their paren-
tal lines (Liu et al. 2012). In this case, seed exudate
seemed to have pronounced selective and promoting
effects on specific microbial populations.
Both theories on the origins of microbial communities
may coexist. Indeed, the colonization of seeds by micro-
bial populations occurs a few hours after sowing by the
populations present in and on the seeds (Nelson 2004)
and by the populations detected in the initial potting
mixes (Buyer et al. 1999; Green et al. 2006; Ofek et al.
2011). Bacteria and fungi are the main microbial com-
munities found in the spermosphere (F€
urnkranz et al.
2012). Analysis of the bacterial community composition
revealed a prevalence and diversity of Bacteroidetes
(Green et al. 2006). The investigation of interactions
between fungal and bacterial community structures
showed a significant concordance between these two
microbial communities (Singh et al. 2009). The evolution
of the microbial community structure (MCS) during the
stages of seed to root transformation showed that devel-
oping roots may select specific groups of organisms as
those that proliferate in the spermosphere appear to dif-
fer from those colonizing the rhizosphere (Nelson 2004).
On the contrary, Green et al. (2006) observed the persis-
tence of some of the seed-associated bacteria on root
surfaces. However, plant species had a limited impact on
the microbial community compared to the biotic and
abiotic conditions around germinating seeds, as well as
the quantity and quality of compounds exuded by seeds
and their temporal release. Singh et al. (2009) showed
that moisture greatly influenced bacterial communities,
while soil nitrogen and carbon had a strong impact on
the fungal community. Microbial communities present
on or adjacent to seeds constitute the first inoculum of
the spermosphere. Then, compounds exuded by the ger-
minating seeds and the pedoclimatic conditions shape
the structure of the microbial communities that can be
similar or different from the initial microbial communi-
ties.
By the beneficial effects on seed germination
Recent development and research has described the vari-
ous mechanisms used by micro-organisms, especially
PGPR (plant growth-promoting bacteria), to enhance
plant growth (for reviews, see Ahemad and Kibret 2014;
P
erez-Monta~
no et al. 2014). However, to date, very few
studies have elucidated the mechanisms specifically
involved in the spermosphere. This review will only focus
on the beneficial micro-organisms naturally present or
introduce by inoculation in the spermosphere to increase
their beneficial effects on seed germination, as these are
of interest for future agronomic applications (Table 2).
Beneficial micro-organisms present in the spermosphere
enhance seed germination, i.e. its percentage and speed,
and the vigour of seedlings. This capacity could be attrib-
uted to the excretion of phytohormones by bacteria into
the culture medium at a concentration sufficient to pro-
duce morphological and physiological changes in seed tis-
sues (Dodd et al. 2010). Beneficial micro-organisms
could also enhance seed germination under osmotic
stress, salt or suboptimal temperatures by inducing plant
physiological protection against oxidative damage (Mas-
touri et al. 2010). Biocontrol micro-organisms are also
known to control seed-borne diseases. For example, the
production of antifungal metabolites with a broad spec-
trum of activity coupled with a strong ability to colonize
the spermosphere makes Pseudomonas chlororaphis
MA342 an effective biocontrol agent of cereal seed-borne
diseases (Johnsson et al. 1998). Competition for plant-
derived metabolites is also an efficient means of biocon-
trol against plant pathogens. Van Dijk and Nelson (2000)
have demonstrated that Enterobacter cloacae can limit the
availability of fatty acids to P. ultimum. A competitive
relationship is established in favour of Ent. cloacae, which
suppresses the germination of P. ultimum sporangia and
subsequent plant infection. By enhancing seed germina-
tion and controlling seed-borne diseases, development of
beneficial microbial communities in the spermosphere are
of the highest interest as they positively influence plant
growth and crop yield and may be an alternative to the
use of phytochemicals.

How can the spermosphere be studied?
The spermosphere was first studied indirectly. In the ear-
lier works, its study and characterization were achieved
through germination and/or the growth of various
micro-organisms in the immediate environment of the
seeds (Stanghellini and Hancock 1971; Short and Lacy
1974, 1976). More recently, these methods are still used
to assess the effect of seed treatment containing micro-or-
ganisms on improving seed emergence (Bennett et al.
Table 2 Some examples of beneficial micro-organisms and their effects on plant growth after the process of enrichment of the spermosphere by
seed inoculation
Micro-organisms Effect compared with control
Bacteria
Azospirillum brasilense Az39 and
Bradyrhizobium japonicum E109
(alone and in combination)
Promote seed germination, nodule formation and early development of Zea mays L. and
Glycine max L. (Cass
an et al. 2009)
Azotobacter chroococcum 12, Azospirillum
lipoferum OF, Pseudomonas fluorescens
169 and Bacillus subtilis FzB24
(alone or in combination)
Increase the percentage of seed germination, speed of germination, mean germination time
of Crataegus pseudoheterophylla (Fatemeh et al. 2014)
Bacillus pumilus CNPSo 2481 Promote seed germination and increase the root volume in Z. mays L. (Szilagyi-Zecchin
et al. 2014)
Enterobacter asburiae CNPSo 2480 Promote seed germination and increase the root volume in Z. mays L. (Szilagyi-Zecchin
et al. 2014)
Enterobacter cloacae Control Pythium ultimum damping-off on seeds of carrot, cotton, cucumber, lettuce, radish,
tomato and wheat (Kageyama and Nelson 2003)
Methylobacterium sp. NC4 (proteobacteria) Increase seed germination of Triticum aestivum (Meena et al. 2012)
Pseudomonas aeruginosa Control pre- and postemergence Colletotrichum truncatum damping-off and enhance seed
germination on soybean (Begum et al. 2010)
Pseudomonas chlororaphis MA342 Control seed-borne diseases (Drechslera graminea in barley, Drechslera teres in barley,
Drechslera avenea in oats, Ustilago hordei in barley, Tilletia caries in wheat) (Johnsson
et al. 1998)
Pseudomonas fluorescens Increase seed germination of chickpea, pea, soybean, lentil (Mishra et al. 2013) and
groundnut (Bhatia et al. 2008)
Control disease by inhibiting mycelia growth of Sclerotium rolfsii, Rhizoctonia solani,
Fusarium oxysporum f. sp. ciceri, Macrophomina phaseolina, Sclerotinia sclerotiorum
(Bhatia et al. 2008; Mishra et al. 2013)
Is a plant growth promoter (Meschke and Schrempf 2010)
Streptomyces lividans Suppress Verticillium dahliae proliferation when they are co-inoculated on Arabidopsis
thaliana seeds and resulting plants have a healthy appearance including an intact root
system (Meschke and Schrempf 2010)
Fungus
Ceratobasidium (Some clades isolated from
Ionopsis utricularioides)
Stimulate seed germination in Vanilla (Porras-Alfaro and Bayman 2007)
Clonostachys rosea Improve emergence time of carrot seeds (Bennett et al. 2009), increase control of seed-borne
fungal pathogens (Jensen et al. 2004)
Fusarium verticilloides (GM-1) Increase G. max L. seed germination and plant growth under salinity stress conditions
(Radhakrishnan et al. 2013)
Trichoderma harzianum Increase seed germination of several plants (chickpea, pea, soybean, lentil and tomato)
exposed to biotic stress (Sclerotium rolfsii, Rhizoctonia solani, Fusarium oxysporum f. sp.
ciceri, Macrophomina phaseolina, Sclerotinia sclerotiorum (Mishra et al. 2013) and Pythimum
ultimum (Mastouri et al. 2010) and to abiotic stresses (osmotic, salinity, chilling or heat stress)
(Mastouri et al. 2010)
Trichoderma harzianum and Trichoderma
virens (alone or in combination)
Control Pythium aphanidermatum damping-off on Cucumis sativus seeds (Pill et al. 2009) and
on Beta vulgaris L. seeds (Pill et al. 2011)
Control Colletotrichum truncatum damping-off on soybean seeds (Begum et al. 2010)
Others
Mixed formulation Trichoderma
harzianum PBAT-43 and Pseudomonas
fluorescens PBAP-27
Increase seed germination from 255 to 7211% and disease control from 4768 to 7600% in
different crops (chickpea, pea, soybean and lentil) (Mishra et al. 2013)
1472

2009) and to protect against damping-off during seed
germination (Pill et al. 2009). Two major experimental
approaches can be identified to study the spermosphere
and to understand the interactions between seeds, micro-
organisms and the environment. The first is to identify,
quantify and elucidate the role of molecules exuded by
seeds during seed germination. The second is to charac-
terize the composition of the microbiome on and around
germinating seeds. These two approaches are also com-
bined to obtain an integrated functioning of all the
constituents of the spermosphere.
Seed exudate characterization
Direct quantitative measurement of seed exudates in nat-
ural soils has proved almost impossible because sugars
and amino acids are quickly taken up and metabolized by
indigenous soil microflora. The study of the spermo-
sphere via the study of seed exudates has most often been
performed in in vitro and sterile conditions. Seeds were
surface sterilized and the exudates were collected from
germinating seeds immersed in water (Short and Lacy
1976; Kageyama and Nelson 2003; Windstam and Nelson
2008). The exudates could then be tested as inhibitory or
stimulatory of microbial activity. Some authors prefer not
to sterilize seeds prior to the preparation of exudates to
avoid washes with methanol or hypochlorite, which could
potentially remove components from the exudate (Casey
et al. 1998).
Plant seed exudates generally consist of carbohydrates,
amino acids, vitamins, organic acids and other miscella-
neous compounds. However, their concentrations are
always very low. Different chromatographic techniques
may be performed to analyse these molecules. Thin layer
chromatography procedures (Smith 1969; Begonia and
Kremer 1999) were used for the determination of sugars
and organic acids. Organic acid compounds from tomato
root exudates were identified by HPLC (Tan et al. 2013).
Ion-exclusion-partition and ion-exchange chromato-
graphic techniques were also used to profile the compo-
nents of seed exudates (Casey et al. 1998; Kamilova et al.
2005). A metabolite profiling approach (Shu et al. 2008;
Haichar et al. 2014; Wolfender et al. 2015) based on gas
chromatography-mass spectrometry (GC-MS) could be
investigated to analyse seed exudates.
Seed microbial community characterization
The spermosphere can also be characterized by the com-
position of the microbiome on and around germinating
seeds (Buyer et al. 1999; Green et al. 2006; Ofek et al.
2011). However, the characterization of the composition
and functioning of the spermosphere in natural soil is
extremely complex due to the diversity of the microbial
populations and the interactions between these commu-
nities, the seed and the environment. To simplify the
experimental model to better understand the mechanisms
involved in these interactions, seeds could be sterilized
and then inoculated with some specific bacterial strains
in a sterilized or controlled inert medium. For example,
the influence of host seeds on the metabolic activity of
Ent. cloacae during colonization of pea and cucumber
seeds (Roberts et al. 2009) was examined in control cul-
ture medium. Similarly, Simon et al. (2001) tested the
influence of host seeds on the growth of two micro-or-
ganisms, Bacillus and Pseudomonas strains, by using
tomato lines from a recombinant inbred line. Given the
strong influence of the pedoclimatic conditions and the
seed itself (genotype, species, stage of development) on
the form, structure and working of the spermosphere, it
is essential to carry out its study in well-controlled and
well-characterized experimental conditions to have repro-
ducible and explicable results.
To date, no specific method has been employed to
study the microbial community in the spermosphere;
they are the same as those employed for analysing the
rhizosphere or the bulk soil. However, there are clearly
additional difficulties for microbial analysis in the sper-
mosphere: short time duration (72 h) and spatial size
(10 mm). Each method presents its own advantages and
drawbacks so a multiphasic approach is increasingly fol-
lowed to better understand microbial life—growth, meta-
bolic activities, interactions with seeds and roots—in the
spermosphere/rhizosphere. In this review, the methods
are presented according to their main principle. Table 3
summarizes their main advantages and drawbacks.
The first group consists of phenotypic methods, based
on cultivation techniques. These techniques, used for a
long time, enable the MCS to be estimated. For example,
Chen et al. (2007) used community level physiological
profiles (CLPP) and PhosphoLipid fatty acid (PLFA)
methods to assess the effect of soil moisture and plant
species (white clover and rye-grass) on the MCS. Accord-
ing to Ramsey et al. (2006), PLFA provides the most
powerful technique to show changes in the MCS com-
pared to CLPP and PCR-based methods. However, these
techniques, particularly standard plating methods, are less
frequently used alone due to media and cultivation con-
ditions that prevent the growth of most microbial com-
munities. Other phenotypic methods are based on the
detection of a specific phenotypic characteristic of the
biological agent to be identified. In the spermosphere/rhi-
zosphere, this kind of method is suitable for monitoring
a particular microbial group such as inoculated bacteria
(PGPR, biocontrol agent). This specific detection, quan-
tification and location can be achieved by fluorescence or

Table
3
The
main
methods
used
to
characterize
the
microbial
community
structure
of
the
spermosphere
Objectives
of
the
methods
Principles
Advantages
Drawbacks
References
Phenotypic
methods
based
on:
(1)
Cultivation
techniques
Estimate
the
overall
microbial
community
and
give
an
insight
into
the
microbial
diversity
Standard
plating
on
selective
media
CCPP
(community
level
physiological
profiles)
using
BIOLOG
system
PLFA
(PhosphoLipid
fatty
acid)
profiles
Simple
and
inexpensive
Can
be
combined
with
recent
methods
Nondestructive
(except
PLFA)
Unable
to
grow
and
isolate
all
the
microbial
communities
Not
suitable
to
differentiate
inoculated
bacteria
from
native
microbes
Singh
et
al.
(2004);
S
€
o
derberg
et
al.
(2004);
Ramsey
et
al.
(2006);
Chen
et
al.
(2007);
Ahmad
et
al.
(2011)
(2)
Specific
monitoring
with
the
use
of
biosensors
Quantify
a
specific
microbial
group
and
characterize
nutrient
mobilization
or
uptake
Reporter
gene
(Gfp
or
enzymes
like
lux)
inserted
in
studied
micro-organisms
(biosensors).
Optical
detection
by
microscopy
Monitoring
of
specific
microbial
groups
Study
of
nutrient
uptake
In
situ
localization
by
microscopy
Limited
number
of
reliable
reporter
genes
Behaviour
of
biosensor
may
be
different
from
the
wild
type
Strong
influence
of
environmental
conditions
on
biosensor
efficiency
Potential
loss
of
reporter
gene
during
growth
in
soil
For
ex
situ
experiments,
large
amount
of
soil
is
needed
Boldt
et
al.
(2004);
Roberts
et
al.
(2009);
Ahmad
et
al.
(2011);
Marschner
et
al.
(2011)
DNA-
and
RNA-
based
methods
Characterize
community
composition,
determine
the
relative
abundance
and
species
diversity
and
analyse
the
interactions
between
plant,
soil
and
microbial
community
Use
of
DNA
probes
or
selective
markers
or
16S
rRNA
Amplification
of
targeted
genes
(PCR,
RT-PCR)
Separation
of
amplified
genes
by
DGGE
(denaturing
gradient
gel
electrophoresis),
T-RFLP
(terminal
restriction
fragment
analysis)
Ease
of
DNA
extraction
from
soil
Relative
low
cost
Large
number
of
samples
Large
number
of
primers
available
Quantification
of
genes
by
q-PCR
Destructive
sampling
Difficulties
in
sampling
of
rhizosphere/spermosphere
soil
(soil
adhered
to
seed
or
root)
Effect
of
soil
composition
(water,
organic
content)
and
texture
on
sampling
Large
amount
of
soil
is
needed
Low
concentration
of
RNA
in
soil
for
accurate
detection
No
detection
in
situ
in
soil
Normander
and
Prosser
(2000);
Tiquia
et
al.
(2002);
Green
et
al.
(2006);
Kang
and
Mills
(2006);
Xu
et
al.
(2009);
Ahmad
et
al.
(2011);
Marschner
et
al.
(2011);
Bouasria
et
al.
(2012);
F
€
u
rnkranz
et
al.
(2012);
Liu
et
al.
(2012);
Mehrabi-Koushki
et
al.
(2012)
Fluorescence
in
situ
hybridization
(FISH)
Detect,
quantify
and
localize
micro-organisms
in
situ
Oligonucleotide
probes
or
antibodies
are
labelled
with
florescent
marker
Detection
in
situ
using
confocal
microscopy
Nondestructive
method
Detection
in
situ
Use
in
combination
with
micro-radiography
Auto-fluorescence
of
micro-organisms
themselves
and
of
material
surrounding
bacteria
in
environmental
samples
Lack
of
specificity
of
the
oligonucleotide
probe
False
negative
results
Diem
et
al.
(1978);
Moter
and
G
€
o
bel
(2000);
Simon
et
al.
(2001);
Singh
et
al.
(2004);
Watt
et
al.
(2006);
Ahmad
et
al.
(2011);
Marschner
et
al.
(2011)
(Continued
)
1474

bioluminescence with the incorporation of an exogenous
reporter gene encoding for a protein like Gfp or an
enzyme like lux responsible for bioluminescence. The
quantification and visualization of the modified micro-
organisms, named biosensors, are performed by optical
detection methods such as fluorescence microscopy, spec-
trofluorometry or flow cytometry (Roberts et al. 2009;
Ahmad et al. 2011). This method can also be used for to
characterize nutrient availability at specific microsites
after extraction from soil or in situ. With this aim, the
reporter genes are linked to specific promoters involved
in nutrient uptake or nutrient mobilization (Marschner
et al. 2011). However, biosensors also present several
limitations (Table 3).
The second group consists of DNA- and RNA-based
methods, which are more recent and widely used. Molec-
ular detection techniques can characterize community
composition and determine the relative abundance and
species diversity. In fact, they can find micro-organisms
in natural environments with the use of DNA probes or
selective markers (antibiotic resistance genes, chro-
mogenic markers). DNA or RNA is extracted from the
soil followed by amplification of targeted genes with PCR
or RT-PCR (Hirsh et al. 2010). The amplified genes can
be separated by various fingerprinting methods such as
denaturing gradient gel electrophoresis (DGGE), which is
often chosen, capillary electrophoresis (CE) or terminal
restriction fragment analysis. For example, the effect of
compost amendment and growth stages on the bacterial
community compositions of seed and root surfaces of
cucumber was studied by using 16S rRNA genes ampli-
fied and separated by PCR-DGGE (Green et al. 2006).
Differential Display RT-PCR was developed to investigate
the gene expression of a selected Trichoderma species dur-
ing colonization of tomato-germinating seeds and roots
(Mehrabi-Koushki et al. 2012). To overcome the risk of
similar molecular markers in native strains, methods
based on the detection of polymorphism in the genome
involving a PCR step were tested: amplified fragment
length polymorphism, single strand conformation poly-
morphism (SSCP) and random amplified polymorphic
DNA (Ahmad et al. 2011; Marschner et al. 2011). For
example, the diversity of bacterial and fungal rhizosphere
communities of four species of mountain grasslands was
characterized using CE-SSCP molecular profiling (Bouas-
ria et al. 2012). Recent advances in molecular methods
and sequencing tools like the bacterial artificial chromo-
some (BAC) library, which contains large fragments of
labelled DNA in vectors, give further understanding at
the metagenomic level (Singh et al. 2004). For example,
16S rRNA libraries were used to investigate endophytic
bacterial communities in seeds of hybrid maize in
comparison with their parental lines (Liu et al. 2012).
Table
3
(Continued
)
Objectives
of
the
methods
Principles
Advantages
Drawbacks
References
Stable
isotope
probing
(SIP)
Enable
functional
activity
to
be
linked
to
microbial
community
structure
and
study
plant-microbial
interactions
Track
the
incorporation
of
heavy
stable
isotopes
from
specific
substrates
into
phylogenetically
informative
biomarkers
(PLFA,
DNA,
RNA
and
proteins)
associated
with
microbes
that
assimilate
the
substrate
Construction
of
a
bacterial
artificial
chromosome
(BAC)
library
Direct
link
between
microbial
metabolic
capability
and
phylogenetic
and
metagenomic
information
within
a
community
context
Very
limited
availability
High
cost
of
labelled
substrates
Low-throughput
techniques
Wellington
et
al.
(2003);
Singh
et
al.
(2004);
Treonis
et
al.
(2004);
Whiteley
et
al.
(2006);
Uhlik
et
al.
(2013);
Abram
(2015)
Multi-omic
approaches
Investigate
microbial
community
structure,
function,
activity
and
interactions
in
situ
and
understand
how
microbial
communities
respond
to
environmental
changes
Each
level
of
information
(DNA,
RNA,
proteins
and
metabolites)
is
investigated
by
genomics,
transcriptomics,
proteomics,
metabolomics
and
microbiomics
with
in
situ
environmental
characteristics
High-throughput
technique
Identify
and
isolate
novel
micro-organisms
Integration
with
SIP
Inability
to
link
specific
functions
to
individual
populations
Incompleteness
of
genetic
databases
R
€
o
ling
et
al.
(2010);
Uhlik
et
al.
(2013);
Abram
(2015)

The third group of methods is based on fluorescence
in situ hybridization (FISH), which enables the phyloge-
netic identification of uncultured bacteria in natural envi-
ronments using fluorescent group-specific phylogenetic
probes and fluorescence microscopy (Moter and G€
obel
2000). This technique enables the quantification and
localization in situ of individual microbial cells. Simon
et al. (2001) evaluated the percentage of Pseudomonas
species relative to total bacteria in the spermosphere of
seeds of tomatoes using FISH while Watt et al. (2006)
showed by FISH that the root caps were the zone of the
wheat rhizosphere most colonized by bacteria. FISH can
also be combined with micro-radiography to study the
uptake of carbon into micro-organisms (Singh et al.
2004; Wagner et al. 2006).
The fourth group of methods is stable isotope probing
(SIP). This recent technology enables functional activity to
be linked to the structure of microbial communities (Singh
et al. 2004; Uhlik et al. 2013; Abram 2015). A stable isotope
atom from a particular substrate is tracked in components
of microbial cells that provide functional information such
as lipids (Treonis et al. 2004), proteins (Uhlik et al. 2013),
DNA or RNA (Wellington et al. 2003; Whiteley et al.
2006). For example, 13
C-enriched DNA obtained by the
SIP technique contains the entire genome of each active
microbe of the community. Consequently, a BAC library
can be achieved by cloning large fragments of the labelled
DNA into vectors for optimal microbial community analy-
sis (Singh et al. 2004). Protein-SIP is also a very promising
technique as it may provide a more substantial access to
real microbial activity as proteins are the most explicit indi-
cators of metabolic activity (Uhlik et al. 2013). The recent
integration of SIP with metagenomics has also enabled a
more comprehensive understanding of the functional com-
munity dynamics of entire microbial systems (R€
oling et al.
2010; Uhlik et al. 2013; Abram 2015).
The fifth group of methods is constituted of multi-
omics approaches. More recently, microbial communities
are considered metaorganisms and each level of biological
information (DNA, RNA, proteins and metabolites) is
investigated along with in situ environmental characteris-
tics. Multi-omics approaches, i.e. metagenomics, meta-
transcriptomics, metaproteomics, metabolomics and SIP-
omics, can be employed to investigate collectively MCS,
potential, function, activity and interactions (R€
oling et al.
2010; Abram 2015). Microbiomics, which refers to the
application of omics technologies to the microbial com-
munities associated with the plant spermosphere, endo-
sphere, rhizosphere and phyllosphere, is a promising
approach to study the spermosphere as a biological sys-
tem including seed, soil, micro-organisms and their inter-
actions. This systems-biology approach enables the
conception of networks that could manage, organize and
integrate the huge amount of multilevel molecular data.
For example, these approaches have been developed on
the genus Brassica to characterize their associated micro-
biome (Witzel et al. 2015) and to understand the com-
plex interactions between plants and arbuscular
mychorrhizal fungi, which are ubiquitous symbionts of
plant roots (Salvioli and Bonfante 2013).
No single method will elucidate all the interactions
between plants and microbes as both culture-based and
culture-independent approaches have their own advan-
tages and limitations. A combination of different tech-
nologies may answer some specific questions. For
example, combining FISH with microautoradiography
could provide information about specific substrate-utiliz-
ing microbes and the presence of particular functional
genes in these organisms (Singh et al. 2004). This
approach could be further improved by the use of isotope
arrays, which, in addition to deciphering the function of
individual populations in a microbial community, can
also reveal key physiological interactions between the dif-
ferent members of these communities (Wagner et al.
2006). However, the cultivation-based techniques are still
a crucial means to verify and investigate the physiology
and genetics of individual contaminant-degrading micro-
organisms, to facilitate bioaugmentation and to enable
the improved annotation of metagenomics databases
(Uhlik et al. 2013).
Conclusion and perspectives
Despite its great importance for the development of
healthy and productive plants, the spermosphere remains
little studied compared to works on seed germination
and the rhizosphere. The nature of the compounds
exuded by germinating seeds has been well characterized
but a general framework of their time of exudation is still
not available. This lack of a general outline could be
explained not only by the large number of exuded com-
pounds (more than 140 have been identified in the sper-
mosphere (Nelson 2004) and in the rhizosphere (Badri
and Vivanco 2009) but also by the complexity of the
metabolic pathways they follow after seed exudation.
These compounds could be degraded in the environment
and/or used by the microbial communities. The strong
influence of the species, the genotype and pedoclimatic
conditions are also a limitation for the precise timing of
the release of these compounds. The fine characterization
of the microbial communities of the spermosphere has
recently been facilitated by the development of high-
throughput molecular biology techniques. However, few
studies have examined the functioning of the spermo-
sphere, especially the mutual interactions between exuded
seed compounds and soil micro-organisms.
1476

Considering the high complexity of the spermosphere
and its functioning, many studies have been carried out
in simplified conditions. Seeds have been sterilized as well
as the medium culture and the influence of a limited
number of micro-organisms has been characterized. The
use of stable labelled isotopes coupled to the characteriza-
tion of the metabolome surrounding the germinating
seeds would constitute an original way to study the sper-
mosphere dynamically throughout the process of germi-
nation. Metabolites of interest would be labelled with
stable isotopes during the seed filling period. Then, these
seeds containing some labelled metabolites would be ger-
minated in a defined environment. Compounds exuded
by these seeds could then be discriminated by isotopic
labelling from those molecules already present in the
environment which were not labelled. Analytical tech-
niques such as GC-MS, LC-MS and NMR would enable
both the identification and precise quantification of these
compounds. The labelling of metabolic fluxes during the
early steps of seed germination would give highly repro-
ducible results and allow an hourly collection of exudates
during seed germination. Nevertheless, this method is
time-consuming to implement and requires, as a first
step, the production of mature seeds with compounds of
interest labelled with stable isotopes. Metabolomic analy-
ses of seed exudates would provide a better understand-
ing of the interactions between seeds, micro-organisms,
and the environment during the early stages of seed
development. Despite efforts made to enhance the germi-
nating capacity and vigour of seedlings through bacteriza-
tion, very few results have led to efficient and large-scale
applications at the agronomic level. The fact that bacteria
from the seed are rapidly replaced by soil bacteria could
be due to the low competitivity of the natural seed inocu-
lum. The inoculation of seed with one PGPR species
could lead to positive enhancement but only for a short
period of time. Thus, why not try inoculating the seeds
with a composite inoculum, potentially including aerobes
and microaerophiles or anaerobic bacteria? Then, to
maintain microenvironments compatible with micro-or-
ganisms that need oxygen and others that do not, a
heterogeneous coating would probably be necessary. An
artificial seed coat made with polysaccharides and includ-
ing soil particles, clays and/or zeolites could be envisaged.
Such a strategy could be inspired by synthetic seed tech-
nologies developed in the late 1990s. The density of bac-
teria should probably be as high as possible. Then, which
microbial consortium should be added? Where and how
could its viable constituents be found? Most probably,
the answer lies in the primo-rhizosphere of seedlings
showing high growth and vigour in a natural environ-
ment. This more systemic way of proceeding would prob-
ably be more efficient than a prima facie targeted strategy
based on the inoculation of seeds with only one selected
PGPR micro-organism. These are only hypotheses based
on the limited success of seed-bacterization to date.
Much more work remains to be done. A more systemic
approach, including the omics capacities, may contribute
to further progress in knowledge of the spermosphere.
Spermosphere biocontrol for the improvement of rhizo-
sphere functioning could then contribute to more sus-
tainable agriculture practices.
Acknowledgements
This work was supported by a grant from the CoMet
project from the Conseil R
egional de Picardie.
Conflict of Interest
No conflict of interest declared.
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