Agron. Sustain. Dev.
c INRA, EDP Sciences, 2010
DOI: 10.1051/agro/2010018
Review article
Available online at:
www.agronomy-journal.org
for Sustainable Development
Pathogenic and beneficial microorganisms in soilless cultures
J. Vallance1,2,3, F. D´eniel1,2, G. Le Floch1,2, L.Gu´erin-Dubrana3, D. Blancard3, P. Rey3*
1 Université Européenne de Bretagne, France
2 Université de Brest, EA3882 Laboratoire Universitaire de Biodiversité et Écologie Microbienne, IFR148 ScInBioS, ESMISAB,
Technopôle Brest-Iroise, 29280 Plouzané, France
3 UMR Santé Végétale 1065, INRA, ENITA de Bordeaux, Université de Bordeaux, 33175 Gradignan, France
(Accepted 29 March 2010)
Abstract – Soilless cultures were originally developed to control soilborne diseases. Soilless cultures provide several advantages for growers
such as greater production of crops, reduced energy consumption, better control of growth and independence of soil quality. However, diseases
specific to hydroponics have been reported. For instance, zoospore-producing microorganisms such as Pythium and Phytophthora spp. are
particularly well adapted to aquatic environments. Their growth in soilless substrates is favoured by the recirculation of the nutrient solution.
These pathogenic microorganisms are usually controlled by disinfection methods but such methods are only effective as a preventive measure.
Contrary to biofiltration, active treatments such as UV, heat and ozonisation have the disadvantage of eliminating not only the harmful
microorganisms but also the beneficial indigenous microorganisms. Here, we review microbial populations that colonise ecological niches
of hydroponic greenhouse systems. Three topics are discussed: (1) the general microflora; (2) the pathogenic microflora that are typical to
hydroponic systems; and (3) the non-pathogenic and possibly beneficial microflora, and their use in the control of plant diseases in soilless
greenhouse systems. Technical, economic and environmental concerns are forcing the adoption of new sustainable methods such as the use
of microbial antagonists. Thus, increased attention is now focused on the role of natural microflora in suppressing certain diseases. Managing
disease suppression in hydroponics represents a promising way of controlling pathogens. Three main strategies can be used: (1) increasing the
level of suppressiveness by the addition of antagonistic microorganisms; (2) using a mix of microorganisms with complementary ecological
traits and antagonistic abilities, combined with disinfection techniques; and (3) amending substrates to favour the development of a suppressive
microflora. Increasing our knowledge on beneficial microflora, their ecology and treatments that influence their composition will help to
commercialise new, ready-to-use substrates microbiologically optimised to protect plants in sustainable management systems.
antagonistic agents / biological control / microbial ecology / disinfection methods / hydroponics / recirculating solutions / root rots /
suppressive microflora / wilting / zoosporic pathogens
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Ecology of the microflora in soilless systems . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1 Influence of the kind of substrate on microflora . . . . . . . . . . . . . . . . . 2
2.2 Root system and nutrient solution microflora . . . . . . . . . . . . . . . . . . . 3
2.3 Influence of the rhizosphere on the microbial communities . . . . . . 4
2.4 Evolution of microbial communities. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3 Unique disease problems in soilless cultures . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.1 Infections by zoosporic oomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.2 Complex of pathogens on necrotic roots . . . . . . . . . . . . . . . . . . . . . . . . 5
3.3 Symptomless and minor pathogen infections on roots . . . . . . . . . . . 5
3.4 Other potentially pathogenic microorganisms in soilless cultures. 5
4 Effect of disinfection techniques on the microflora of soilless systems. 6
4.1 Active methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.2 Passive method: slow filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5 Disease suppression in soilless systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
* Corresponding author:
[email protected]
Article published by EDP Sciences
2 J. Vallance et al.
6 Management of the soilless microflora for disease suppression . . . . 8
6.1 Increasing the level of suppressiveness by the addition
of antagonistic microorganisms . . . . . . . . . . . . . . . . . . 8
6.2 Use of a mixed culture of antagonistic microorganisms
with disinfection techniques . . . . . . . . . . . . . . . . . . . 9
6.3 Nutritional amendments . . . . . . . . . . . . . . . . . . . . . . 9
7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1. INTRODUCTION
Soilless cultures are used worldwide. Depending on the
country, growers use a variety of complex technologies, all of
which offer advantages making them appropriate alternatives
to traditional soil culture (Fig. 1A). In cases where the soil is
polluted by chemical residues or contaminated by pathogens
which colonise and persist in the soil for years or when excessive
salinity causes water problems, soilless cultures can be
an alternative. The main advantage of soilless cultures is that
plants grow in a controlled environment. For instance, nutrient
solution supply, electrical conductivity, pH and temperature
are monitored and regulated by the grower. It provides an
ideal environment for growth and development of plants and
a greater yield is frequently obtained than with traditional cultural
methods. The majority of greenhouse crops are grown
using artificial substrates (Fig. 1B), which improves control
of water, aeration, nutrition and root distribution. These systems
were originally developed as open systems and excess
nutrient solution was disposed of outside the greenhouse. In
recent years, closed hydroponic systems have been developed
to minimise pollution. In a closed system, the nutrient solution
is recovered, replenished with nutrients and water, depending
on plant uptake, and the pH adjusted before resupplying to the
plants.
Microbial contamination of the root system in these culture
systems can arise from many sources: plant material, growth
media, and water from lakes, rivers and wells (Stanghellini
and Rasmussen, 1994). Root colonisation by fungi and bacteria
is favoured by at least three factors: (i) genetically uniform
host plants, (ii) environmental conditions, i.e. suitable
temperature and moisture regime, and (iii) rapid dispersal of
root-colonising agents throughout the cultural system via the
recycled nutrient solution.
The activity of microorganisms, however, may be
pathogenic or protective, so two scenarios are possible. (1)
One of the reasons for developing soilless culture was to prevent
root diseases caused by soil-pathogenic microorganisms.
Although a decrease in the diversity of root-infectingmicroorganisms
has been reported, root diseases still occur frequently
in hydroponics and disease outbreaks are sometimes greater
than in soil (Stanghellini and Rasmussen, 1994). Some minor
infections have become threats in soilless culture, indicating
that unique diseases are observed with this method of
plant cultivation. (2) The role of natural microflora in suppressing
certain diseases was demonstrated by comparing systems
with and without their original microflora (Postma et al.,
2000; Minuto et al., 2007). Indeed, it has been shown that the
natural microflora can suppress diseases (Berger et al., 1996;
Chen et al., 1998) and that a high density of bacteria in the
rhizosphere can limit pathogenic attacks on roots (Tu et al.,
1999). From these observations ensued the hypothesis that indigenous
bacteria were involved in disease biosuppression.
In this study we focused on the microbial communities
colonising the root systems of plants growing in soilless cultures
and highlighted the specificity of microbes in this type of
cultivation system. Three topics were reviewed: (i) the general
microflora; (ii) the pathogenic microflora of typical diseases
related to hydroponics; and (iii) the non-pathogenic and possibly
beneficial microflora and their use in the control of plant
diseases in soilless greenhouse systems.
2. ECOLOGY OF THE MICROFLORA
IN SOILLESS SYSTEMS
Soon after the start of a soilless culture, a microflora rapidly
colonises three ecological niches: the substrate, the nutrient
solutions and the rhizosphere of the cultivated plants. The density
and diversity of this microflora are affected by the type of
substrate (organic or inorganic), the nutrients in the solutions
and the age and cultivar of the plant species.
Cultural methods have been used to characterise this
microflora, but in recent years other methods based on
sole-carbon-source utilisation (Khalil and Alsanius, 2001;
Khalil et al., 2001b), phospholipid fatty acid profiling
(Waechter-Kristensen, 1996; Khalil and Alsanius, 2001;
Khalil et al., 2001a, b) and molecular fingerprinting (Postma
et al., 2000; Calvo-Bado et al., 2003, 2006) have provided
structural and functional analysis of the soilless microflora.
Recent studies on microflora have provided key information
on the microbial diversity and dynamics of soilless systems.
2.1. Influence of the kind of substrate on microflora
In soilless cultures a microflora rapidly develops soon after
the start of the culture via the plants and the water supply,
even though inorganic substrates contain few microbes. Once
plants are introduced into greenhouses, extensive colonisation
of rockwool substrates by bacteria and fungi rapidly occurs
(Price, 1980; Carlile and Wilson, 1991). Inorganic substrates
are mainly colonised by bacteria while organic substrates are
colonised by fungi (Koohakan et al., 2004). In the case of crops
of tomatoes, for instance, bacteria including fluorescent pseudomonads
were higher in rockwool than in peat substrates and
Pathogenic and beneficial microorganisms in soilless cultures 3
[A]
[C]
[D]
[E]
[F]
Figure 1. Tomato soilless culture and the main associated fungal pathogens. Suspended substrate in a tomato soilless culture (A), rockwool
containing healthy and altered roots (B), Phytophthora cryptogea sporangia on the surface of a necrosed root (C), Pythium aphanidermatum
oospores (round with a thick wall) in the root cortex cells (D), macroconidia of Fusarium oxysporum f. sp. radicis-lycopersici with chlamydospores
in formation (E), Colletotrichum coccodes acervulus with black seta (F).
the reverse was observed for fungi, actinomycetes and Trichoderma
spp. (Khalil and Alsanius, 2001). This might be due
to the presence of available organic compounds within the
organic substrates which may modify the microbial equilibrium
through reduced competition (Koohakan et al., 2004).
The level of conduciveness to the diseases caused by a given
pathogenic agent might be determined by the nature (structure,
composition) of the growth substrate of the crop. For instance,
rockwool is more conducive to Pythium root rot and
crown rot in cucumber culture than coir dust, pumice and perlite
(van der Gaag andWever, 2005). Temperature and oxygen
concentration did not explain the differences between the media
but the higher incidence of disease on rockwool was associated
with a much greater water content than in the three others.
When the height of the rockwool slabs was increased, the
percentage of diseased plants decreased. These results indicated
that water content plays a major role in the development
of root and stem rot and that the type and height of substrate
are important tools for decreasing yield losses.
2.2. Root system and nutrient solution microflora
Microorganisms multiply rapidly on roots and in nutrient
solutions. Large populations of heterotrophic bacteria (105–
106 cfumL−1) developed in the circulating nutrient solutions
20 h after planting tomatoes (Berkelmann et al., 1994). The
number of bacteria on young tomato roots can be as high
as 1010 cfu g−1 of fresh roots (Waechter-Kristensen et al.,
1994). However, there are differences between microbial communities
colonising roots and nutrient solutions; more fungi
and bacteria were detected on roots than in nutrient solutions
4 J. Vallance et al.
(Koohakan et al., 2004). Besides the densities, the structure
and the diversity of bacterial communities, as assessed by
a molecular fingerprint method (Single-Strand Conformation
Polymorphism, SSCP), were also different on roots and nutrient
solutions (Renault, 2007).
The cultural systems (inorganic and organic media, deep
flow technique and nutrient film technique) favoured in different
ways the growth of unique indigenous microorganisms
(Koohakan et al., 2004). Fungi and Fusarium spp. were found
to colonise preferentially roots grown in a coconut-fibre system
(organic medium) compared with a rockwool system (inorganic
medium). Pythium spp. were mainly detected in nutrient
solutions and on roots from the nutrient film technique
system. Among the non-specific bacterial genera, aerobic bacteria
seemed predominant on roots and in nutrient solutions,
with only slight differences between the four systems (inorganic
and organic media, deep flow technique and nutrient
film technique).Whatever the system, fluorescent pseudomonads
were frequently detected on roots and in nutrient solutions,
which was consistent with previous findings showing
that 40% of the cultivable bacteria belonged to the genus Pseudomonas,
known to contain potentially antagonistic agents
toward pathogens (Berkelmann et al., 1994). Similar results
were obtained in the recycled nutrient solution during a sixmonth
experiment in a soilless tomato greenhouse (Déniel
et al., 2004). These findings might be explained by the fact
that the temperature, high nitrogen content and oxygen concentration
of the nutrient solutions offer an optimal growth environment
for this genus.
2.3. Influence of the rhizosphere on the microbial
communities
There is a clear relationship between cultivated plants and
the establishment of the rhizosphere microflora. In closed
hydroponic systems, it results from the release of organic
compounds by the roots (Waechter-Kristensen et al., 1997).
Passive or active leakage of carbon sources from plant roots
differs in quantity and quality depending on plant species,
plant cultivar and environmental factors such as light, climate,
nutrient source, pH, humidity, etc. Whatever the hydroponic
habitat, the diversity of microorganisms depends on their ability
to metabolise the available carbon sources. Although a nutrient
film technique system is much simpler than a soil-based
culture system, SSCP analyses showed the bacterial diversity
of the rhizoplane to be as high as that of the rhizosphere in
soil (Chave et al., 2008). However, further studies comparing
the microorganisms colonising soil and soilless cultures are
needed to draw any conclusion.
2.4. Evolution of microbial communities
As mentioned above, biological processes in the rhizosphere
are strongly affected by plant root exudates that attract
specific microbial populations and stimulate their growth and
evolution. Based on viable counts, aerobic bacteria colonising
the rhizosphere of four types of soilless tomato production
systems (inorganic substrate: rockwool; organic substrate:
coconut-fibre; deep flow technique, nutrient film technique)
were found to become stable at 1010 cfu g−1 (of fresh roots)
in all systems investigated, contrary to fungi, that tended to
increase throughout the experiment (Koohakan et al., 2004).
However, changes in the composition of the microflora have
been demonstrated by molecular and biochemical analyses.
For instance, Khalil et al. (2001b) highlighted the differences
between the microflora of two supposedly identical hydroponic
cultivations by comparing sole-carbon-source utilisation
(SCSU) patterns and phospholipid fatty acid profiles (PLFA).
In tomato soilless cultures, Renault et al. (2008) also observed
a temporal shift over a cropping season in the bacterial
composition both in the nutrient solution and on the roots.
Indeed, community-level physiological profiles (CLPPs) indicated
that bacterial metabolism in nutrient solutions progressively
shifted from carbohydrates towards the degradation of
specific amino acids and carboxylic acids.
There is no consensus about whether shifts in the rhizosphere
microflora can result from pathogenic attacks. Indeed,
changes in the microbial communities of the rhizosphere
could be a consequence of both root damage caused
by pathogens such as P. ultimum and secondary colonisation
due to the resulting nutrient leakage (Naseby et al., 2000;
Hagn et al., 2008). On the other hand, it has been reported
that the microbial communities established early on the roots
of tomatoes grown in soilless systems were robust and resistant
to the effect(s) of the introduction of oomycete pathogens
or of switching from a recirculating to a run-to-waste nutrient
supply (Calvo-Bado et al., 2006). However, this assumption,
arising from experiments conducted over only 1.5 months, is
contradicted by the observation of changes in the microbial
communities of tomato plants grown hydroponically over the
6-month experiments of Vallance et al. (2009). SSCP analyses
of three different DNA regions indicated increases in the complexity
and size of the fungal microflora as the cropping season
progressed. Nevertheless, both studies suggest that there
are no substantial changes in the genetic structure of the indigenous
rhizospheric fungal community after root inoculation
with the non-pathogenic oomycete P. oligandrum or the
pathogenic oomycetes Pythium group F, P. aphanidermatum
and P. cryptogea.
3. UNIQUE DISEASE PROBLEMS IN SOILLESS
CULTURES
3.1. Infections by zoosporic oomycetes
Among the pathogenic microorganisms frequently detected
in hydroponic cultures, those producing zoospores, i.e.
Pythium spp. and Phytophthora spp., are particularly well
adapted to these cultivation systems (Favrin et al., 1988; Rafin
and Tirilly, 1995) (Figs. 1C, 1D). As zoospores can swim,
recycling facilitates rapid dissemination and subsequent root
Pathogenic and beneficial microorganisms in soilless cultures 5
infection of the whole culture (McPherson et al., 1995). Disease
epidemics can occur, particularly in periods of stress, because
of high temperatures and the low concentrations of dissolved
oxygen in the nutrient solution (Gold and Stanghellini
1985; Stanghellini and Rasmussen 1994; Chérif et al., 1997).
Highly pathogenic Pythium species, i.e. Pythium ultimum,
P. irregulare and P. aphanidermatum (Blancard et al., 1992;
Jenkins and Averre, 1983; Linde et al., 1994), caused root rot
and wilting.
In Brittany (France), two stages in root infection by
Pythium spp. in commercial tomato greenhouses were observed
by Rey et al. (2001). The first is generally from the start
of the winter crop (February) to June. A small population of
Pythium spp. is frequently detected. The population then dramatically
increases between July-August and the end of the
cropping season (October–November); this increase is sometimes
associated with root necrosis and root rot, but generally
infections are limited to root necroses and are even symptomless.
This pattern was particularly observed in greenhouses
with organic (peat) and, to a lesser extent, inorganic substrates
(rockwool). With a nutrient film technique system, Pythium
spp. invasion was earlier and more severe than in other cultures,
but with no amplification of symptoms.
A DNA macroarray for the detection and identification of
more than 100 Pythium species was developed to assess the
number and diversity of Pythium species on a single root sample
(Tambong et al., 2006). This technology has the advantage
of combining DNA amplification with the screening capability
of DNA arrays, resulting in a high degree of sensitivity
and multiple species specificity. The results of the DNA array
tests confirmed that the substrate was almost free of oomycetes
at the start of plant culture. P. dissotocum (or Pythium group
F) was spontaneously detected on roots throughout the growing
period but other Pythium species (P. intermedium, P. ultimum
and P. sylvaticum) were sporadically detected (Le Floch
et al., 2007). The relative predominance of P. dissotocum (or
Pythium group F) and the low diversity of Pythium species
confirm the results of previous studies conducted in soilless
cultures (Herrero et al., 2003; Moorman et al., 2002; Moulin
et al., 1994; Rafin and Tirilly, 1995; Rey et al., 1997).
3.2. Complex of pathogens on necrotic roots
A variety of fungal complexes and oomycetes are responsible
for root necroses. A three-year experiment in tomato soilless
cultures in France revealed that the distribution of the
fungi and of the oomycetes was region-dependent (Blancard,
unpubl. data). In the South-West, between two and five fungi
and oomycetes were frequently found on roots, whereas in
the five other regions (Brittany, the Eastern Pyrenees, Nantes
region, Orleans region, the South-East), up to three different
microorganisms were isolated from the samples. Some fungi,
including Fusarium oxysporum f. sp. radicis lycopersici, and
oomycetes, such as Pythium species, were found in all the
greenhouses investigated in the six French regions (Fig. 1E).
Other fungi, i.e. Colletotrichum coccodes, Rhizoctonia solani
and Thielaviopsis basicola, or oomycetes such as Phythophthora
spp. were only found on roots in some of the greenhouses
(Fig. 1F).
3.3. Symptomless and minor pathogen infections
on roots
Asymptomatic root colonisation in hydroponic cultures can
be correlated with yield loss (Rey et al., 1997; Stanghellini and
Kronland, 1986). Pythium dissotocum caused yield reductions
of up to 54% in hydroponically grown lettuce although there
was no visible damage (Stanghellini and Kronland, 1986).
Such infections might be more common in soilless greenhouse
systems than originally thought, because of the lack of
root symptoms (Favrin et al., 1988). Immunoenzymatic staining
procedures showed that Pythium spp. were the most frequent
fungal invaders in asymptomatic roots of hydroponically
grown tomato plants. Pythium spp. represented around 40% of
the colonised segments as opposed to 12% for the other fungi.
Pythium group F accounted for 75 to 90% of all the Pythium
isolates from the loose or dense mycelia of Pythium spp. on
the root epidermis (Rafin and Tirilly, 1995; Rey et al., 1997).
Certain strains produce large numbers of zoospores (Rafin,
1993), possibly facilitating the spread and the development of
Pythium group F in soilless cultures. When plants were grown
under optimal conditions Pythium group F-infected roots were
symptomless. However, roots looked generally macroscopically
healthy but the oomycete caused limited changes in the
root cortex (Rey et al., 1998) and produced metabolites that
may facilitate Pythium group F infections (Rey et al., 2001).
Moreover, due to high Pythium group F populations over the
cropping season, limited but repeated damage to root cortexes
could lead to slight yield reductions (Rey et al., 1997). Severe
damage, such as root rot, only occurs when plants are
placed under physiological stress conditions, i.e. lack of oxygen
in nutrient solutions (Chérif et al., 1997). The nature of
Pythium group F is still unclear. The taxonomic position of this
oomycete has only become clearer in recent years with the increased
interest in Pythium group F. Van der Plaats-Niterink
(1981) used the term group F because oomycetes of this
group only produce non-inflated filamentous sporangia on traditional
culture media and sexual structures are not observed.
However, after molecular characterisation of Pythium group
F isolates by ribosomal and intermicrosatellite-DNA regions
analysis, Vasseur et al. (2005) suggested that Pythium group F
isolates could be P. dissotocum-like isolates unable to form
sexual structures on traditional media. Moreover, Lévesque
and De Cock (2004) suggested that Pythium group F could
be related to P. dissotocum because of the complete homology
of ITS sequences.
3.4. Other potentially pathogenic microorganisms
in soilless cultures
The pathogenicity of a few microorganisms (Humicola
sp., Olpidium brassicae and Plectosporium tabacinum)
(Figs. 2A–2E) needs to be determined because some root
6 J. Vallance et al.
[C]
[D]
[E]
[A]
Figure 2. In situ and in vitro appearance of three fungi sometimes associated with root rot in tomato soilless culture but whose aggressiveness
has never been proven on this Solanaceae. Olpidium brassicae resting spores aligned in several root cells (A), Plectosporium tabacinum
bicellular conidia within root cortex cells (B), phialides of Plectosporium tabacinum (C), aleuriospores (dark brown) of Humicola sp. (D),
phialides of Humicola sp. perpendicular to the mycelium; they form chains of conidia (E).
microorganisms of minor importance in soils have become
of major economic importance in hydroponic cultures
(Stanghellini & Rasmussen, 1994). Hydroponics, for instance,
have favoured the development of Phytophthora cryptogea on
lettuce, whereas, in the field, no attacks by this fungus have
been reported. Plectosporium tabacinum (formerly Fusarium
tabacinum), frequently isolated from soilless tomato cultures
in France (Blancard, unpublished data) is a possible pathogen.
It is detected on a variety of soil-grown plants, i.e. melon
(Soran and Ozel, 1985), sunflower (Mirza et al., 1995) and
basil (Minuto et al., 1997). Matta (1978) and Pascoe et al.
(1984) reported that it caused necrotic lesions on young leaves
in tomato plants and El-Gindy (1991) noticed necrosis and
root rot in plantlets. Such symptoms have never been observed
on tomato plants grown hydroponically. However, considering
the pathogenic potential of P. tabacinum and its frequency
in greenhouses, its pathogenicity in hydroponics needs to be
assessed. Another example is Humicola fuscoatra. Gruyter
et al. (1992) reported the association of H. fuscoatra with
corky root symptoms in wilted glasshouse tomatoes. However,
Menzies et al. (1998) pointed out that Humicola fuscoatra
colonised roots, but did not cause necrosis and was, therefore,
not pathogenic in tomato plants. These findings highlight
the difficulties in distinguishing minor pathogens from other
fungi, as both frequently colonise roots in soilless cultures.
4. EFFECT OF DISINFECTION TECHNIQUES
ON THE MICROFLORA OF SOILLESS
SYSTEMS
Closed hydroponic systems increase the risk of pathogen
attack by using water contaminated with pathogenic microorganisms
(McPherson et al., 1995; van Os, 1999). Therefore,
Pathogenic and beneficial microorganisms in soilless cultures 7
prevention of these infections has become a major challenge
in the last decade (Runia, 1995; Ehret et al., 2001).
4.1. Active methods
The so-called “active” methods disinfect the nutrient solutions
and are very effective (Ehret et al., 2001; Goldberg
et al., 1992; Rey et al., 2001; Runia, 1995; Steinberg et al.,
1994); for example, UV radiation and heat treatment can eliminate
up to 99% of the microflora colonising the flowing solutions.
UV irradiation of recirculating solution was effective
in controlling Pythium spp.-induced root rot in tomato and
cucumber plants (Postma et al., 2001; Zhang and Tu, 2000).
Tirilly et al. (1997) reported a delay in Pythium root infection
in soilless culture with this method; however, in several
cases there was no difference in root colonisation from nondisinfected
greenhouses. Moreover, re-contamination of the
disinfected nutrient solution nullified the effect of disinfection
(Déniel et al., 1999). Such drastic treatments create a microbiological
vacuum in which microbial pioneers spread more easily
because of the lack of competition (Paulitz and Bélanger,
2001; Postma, 2004). The microbial differences in solutions
treated with UV and slow filtration often disappeared once
they flowed through the rockwool slabs containing plant roots
(van Os et al., 2004). Chlorination is effective in disinfecting
water in storage tanks and reduces and delays root colonisation
by Pythium spp. (Déniel et al., in press). However, this
treatment has the disadvantage of eliminating not only harmful
but also beneficial indigenous microorganisms; a weakness
of “active” methods of disinfection. Zhang and Tu (2000) imputed
the lack of control of P. aphanidermatum on tomato
roots to the reduction of bacterial communities caused by UV
radiation.
4.2. Passive method: slow filtration
The traditional technique of slow filtration, used for more
than 100 years for water disinfection (Graham and Collins,
1996; Ellis, 1985), has been adapted for horticulture over the
last decade (Ehret et al., 2001). Water flows slowly through
a bed of substrate, i.e. sand, rockwool or pozzolana; mechanical
and biological factors are thought to be responsible for
the efficacy of the system (Ellis, 1985; Weber-Shirk and Dick,
1997). Experiments to improve slow filtration efficacy have
focused on the determination of flow rates through the filter
unit as well as on the nature and the optimal depth of
substrates in filter tubes (Wohanka et al., 1999). Further investigations
showed that the formation of bacterial microcolonies
or biofilms on substrates enhanced efficiency. Indeed,
after sterilising a filtering column, a dramatic loss in Xanthomonas
campestris pv. pelargonii elimination has been reported
(Brand and Wohanka, 2001). Pseudomonas was the
predominant genus (50%) from the cultivable bacteria colonising
the filtering media, especially the top layers of sand filters,
and 10% of isolates were identified as Bacillus (Brand, 2000;
Calvo-Bado et al., 2003). The Bacillus and Pseudomonas genera
were recently reported to account for 42 to 86% of the total
cultivable bacterial flora in a biocenosis film of pozzolana
grains used as filtering medium (Déniel et al., 2004).
Pathogens eliminated efficiently by this technique include
zoosporic fungi, i.e. Phytophthora spp., bacteria, i.e. Xanthomonas
campestris, nematodes and even viruses (Ehret
et al., 2001; van Os et al., 1999). During a 3-year experiment
in a commercial greenhouse, Déniel et al. (2006) reported
that a biofilter eliminated more fungi than bacteria under
tomato production conditions. The efficiency of elimination
of pathogenic fungi was genus-dependent. Pythium spp. were
more effectively eliminated (99%) than Fusarium oxysporum
(92.7 to 99.3%). The high percentage of Pythium spp. elimination
was correlated with low root colonisation by these
pathogens. Effluents of filtering columns have been shown to
be colonised by a considerable natural bacterial microflora
(102–104 cfumL−1) (Déniel et al., 2004, 2006; Renault, 2007).
Moreover, molecular fingerprinting analyses of the total microflora
(denaturing gradient gel electrophoresis, DGGE, and
SSCP) pointed out clear changes in bacterial communities after
the passage of the nutrient solution through the filter unit
(Postma et al., 1999; Renault, 2007). Thus, slow filtration preserved
part of the natural microflora, because it is harmless
to specific groups of bacteria which are assumed to preserve
microbial ecosystems in the plant rhizosphere. Furthermore,
resident bacteria of nutrient solutions were shown to reduce
Pythium root rot in closed soilless systems (Tu et al., 1999).
The potential benefit of microflora in soilless cultures thus has
to be taken into account.
5. DISEASE SUPPRESSION IN SOILLESS
SYSTEMS
Pathogen-suppressive soils have been defined as “soils in
which (i) the pathogen does not establish or persist; (ii) establishes
but causes little or no damage; or (iii) establishes
and causes disease for a while but thereafter the disease
is less important, although the pathogen may persist in the
soil” (Borneman and Becker, 2007). Soils suppressive to several
pathogens have been widely described and investigated
(Alabouvette et al., 1979; Jager et al., 1979; Lifshitz et al.,
1984; Garibaldi et al., 1989; Whipps and Lumsden, 1991),
while the first studies of suppressiveness in soilless systems
were by McPherson et al. (1995) and Tu et al. (1999).
Both studies demonstrated the potential of the indigenous microflora
to inhibit root diseases in hydroponic cultures. In
soilless cultures, the term “suppressiveness” referred to the
cases where (i) the pathogen does not establish or persist;
or (ii) establishes but causes little or no damage. McPherson
et al. (1995) described the spread of Phytophthora cryptogea
in tomato nutrient film technique systems. In closed systems,
the pathogen caused less damage than in the parallel
run-to-waste ones; they therefore suggested that the potentially
beneficial microflora colonising the recycled nutrient solution
were responsible for disease suppression. They also suggested
that the method of disinfection, i.e. “active” or “passive”
8 J. Vallance et al.
(by total or partial elimination of the microflora) could be important
in the maintenance of the disease suppression. Tu et al.
(1999) also showed that Pythium root rot disease was less severe
in closed rockwool systems than in open culture due to
the greater numbers of bacteria in closed systems. They found
a strong correlation between the resident bacteria and the biosuppression
of Pythium.
The presence of microflora suppressing Pythium aphanidermatum
in cucumber rockwool substrate has been reported
and some of the microorganisms involved in the suppressiveness
identified (Postma et al., 2000, 2004, 2005). Pythium
damage was lower in non-autoclaved than in autoclaved rockwool;
the disease incidence was reduced by 50 to 100%.
Suppressiveness could be restored in sterilised rockwool substrates
by re-introducing the original microflora through contact
with untreated rockwool or through the nutrient solution
taken from untreated slabs. These results indicate that disease
suppression is of biological origin and is transferable. Experiments
on the microbial communities of rockwool showed a
positive association between disease suppressiveness and the
composition and diversity of bacteria and culturable filamentous
actinomycetes. Actinomycetes may prevent the colonisation
of dead root fragments by Pythium zoospores, whilst
bacteria may secrete antibiotics, surfactants, etc. preventing
colonisation of fresh root fragments.
Suppression of Fusarium oxysporum f. sp. radicis lycopersici
has also been demonstrated. The incidence of Fusarium
oxysporum f. sp. radicis lycopersici on tomato seedlings was
significantly reduced with recycled, non-disinfected rockwool
compared with new rockwool (Minuto et al., 2007); and in
tomato soilless culture, by the re-use of perlite and perlite-peat
substrates (Clematis et al., 2008). The indigenous microorganisms
colonising these recycled substrates were considered responsible
for the suppressive effects.
How the suppressive microflora becomes established is relatively
unknown, but it has been suggested that pathogens
themselves might influence suppressiveness. For instance, a
study showed that P. ultimum induced shifts in cucumber indigenous
microflora, favouring groups known to include potential
biocontrol agents (Hagn et al., 2008). However, knowledge
of structural and functional interactions and synergisms
between the microorganisms of the suppressive microflora is
limited and the influence of the plant and the pathogens on the
whole system needs further investigation (Weller et al., 2002;
Burdon et al., 2006).
6. MANAGEMENT OF THE SOILLESS
MICROFLORA FOR DISEASE SUPPRESSION
Factors influencing disease suppression such as the activity
of the total microflora, the diversity of the microbial
communities and the presence of specific antagonists are not
fully understood (Postma, 2004). Nevertheless, managing disease
suppression in hydroponics represents a promising way
of controlling pathogens. Three main strategies can be used:
(i) increasing the level of suppressiveness by the addition of
antagonistic microorganisms; (ii) using a mixed culture of microorganismswith
complementary ecological traits and antagonistic
abilities combined with disinfection techniques; and
(iii) amending substrates to favour the development of the suppressive
microflora.
6.1. Increasing the level of suppressiveness
by the addition of antagonistic microorganisms
Environmental conditions in greenhouses are controlled
and can be optimised to suit antagonistic agents. The biological
vacuum and the limited volume of the matrix of the
soilless substrates are thought to facilitate the introduction, establishment
and interaction of the biocontrol agent with the
root environment (Paulitz and Bélanger, 2001; Postma, 2004).
Thus, representatives of a range of bacterial (Pseudomonas,
Burkholderia, Bacillus, Serratia, Actinomycetes), fungal (Trichoderma,
Penicillium, Gliocladium, non-pathogenic Fusarium)
and oomycete (Pythium) groups have been tested as biocontrol
agents in soilless cropping systems. The antagonistic
activities of these microorganisms can be divided into several
categories: competition for nutrients and space, parasitism, antibiosis
and systemic induced resistance (Garbeva et al., 2004;
Alabouvette et al., 2006; Lemanceau et al., 2006). Nevertheless,
biocontrol of root diseases is often inefficient and only a
few antagonists are available commercially.
The lack of efficiency is due to unsuitable methods of
selection of antagonistic microorganisms. Results from in
vitro studies did not always correlate with the antagonistic
activity of the biocontrol agent once they were introduced
into greenhouses (Fravel, 2005; Alabouvette et al., 2006;
Georgakopoulos et al., 2002). These results also demonstrated
the importance of the medium used for doing the in vitro tests;
it has to be as close as possible to the environment into which
the antagonistswill be introduced. Even then, the colonisation,
survival and antagonistic activity of the biocontrol agent may
be insufficient and/or inconsistent at the infection site because
the antagonist is not adapted to the soilless environment. The
use of microorganisms selected from the indigenous suppressive
microflora and not from a suppressive soil or a different
crop might solve this problem: the microorganisms would be
better adapted to the soilless crop environment and the ecological
niche where their interaction with the pathogens will take
place.
For example, the pathogenic fungi or oomycetes most frequently
involved in root diseases in soilless cultures are those
producing zoospores, such as Pythium spp. and Phytophthora
spp., making them particularly well adapted to the aquatic environment
of hydroponics. The use of an antagonist belonging
to the same taxonomic group (i.e. oomycetes), with the same
life cycle and similar properties, is of particular interest. An
example of such an antagonist is the oomycete P. oligandrum
(Rey et al., 2008; Vallance et al., 2009); it has been widely
reported as an effective biocontrol agent (Foley et al., 1986;
Jones and Deacon, 1995; Benhamou et al., 1997; Rey et al.,
1998, 2005; Wulff et al., 1998). The beneficial effects of P.
oligandrum are due to its potential to colonise roots without
Pathogenic and beneficial microorganisms in soilless cultures 9
damaging the host plant cells and to survive in the rhizosphere.
P. oligandrum biocontrol in the rhizosphere is a complex process
including direct control of pathogens by mycoparasitism,
antibiosis or competition for nutrients and space; and/or indirect
control via the plant, i.e. induction of resistance and
growth promotion (Le Floch et al., 2005; Rey et al., 2008).
Persistent root colonisation by P. oligandrum strains may be
associated with an increase in tomato yield in soilless cultures
(Le Floch et al., 2003), a transient increase (Le Floch et al.,
2007) or not (Vallance et al., 2009).
When root colonisation by P. oligandrum is assessed, results
from molecular (DNA macroarray and real-time PCR)
and culture-dependent methods may be contradictory. Indeed,
in the experiment of Le Floch et al. (2007), P. oligandrum
was detected throughout the growing season (6 months) with
molecular methods, but only for three months with plate
counting on semi-selective media. These findings have important
implications for biocontrol strategies aimed at protecting
plants. Indeed, two different strategies could be envisaged:
(i) based on cultural data, P. oligandrum inoculation on roots
should be repeated three months after the first application; or
(ii) conversely, based on molecular results, reinoculation is unnecessary
because P. oligandrum is still present. In conclusion,
the second strategy probably represents the true pattern of root
colonisation by the antagonist, because detection by DNA array
and real-time PCR is more accurate. Appropriate methods
should therefore be used to detect the antagonistic agent(s) in
assessment of biocontrol.
A strategy for increasing suppressiveness and therefore
making biocontrol more successful might be to associate
several antagonistic agents with complementary and/or synergistic
modes of action against one or several pathogens
(Spadaro et al., 2005). This is the case in naturally suppressive
soils, where suppression is the result of complex
interactions between several microorganisms acting together.
Known examples are soils suppressive to Fusarium
wilts where non-pathogenic Fusarium and fluorescent Pseudomonas
were identified as the main antagonists (Alabouvette
and Lemanceau, 1999). The non-pathogenic Fusarium competes
for carbon sources while bacterial antagonists produce
siderophores competing for iron. In soilless cropping systems,
the association of the non-pathogenic Fusarium strain Fo47
and fluorescent Pseudomonas strain C7R12 controlled fusarium
diseases better than single inoculations of each antagonistic
microorganism (Eparvier et al., 1991). Another strategy
was to combine inoculation of Lysobacter enzymogenes with
chitosan. Chitosan enhanced the biocontrol efficacy of L. enzymogenes
in the control of P. apahidermatum in cucumber
soilless greenhouse systems. Chitosan either served as a C and
N source for the antagonist, induced antagonistic gene expression,
or both (Postma et al., 2009).
6.2. Use of a mixed culture of antagonistic
microorganisms with disinfection techniques
A more complex strategy consists of combining nutrient solution
disinfection methods with biocontrol agents to colonise
and protect the roots from pathogenic attack. One of the first
experiments of this type combined slow filtration and P. oligandrum
inoculation on roots in a tomato soilless greenhouse
system (Rey et al., 1999). Then, the association of slow sand
filtration and antagonistic strains of Fusarium spp. and Trichoderma
spp. isolated from a gerbera rhizosphere was successfully
tested (Garibaldi et al., 2003). A similar experiment also
reported that slow filtration and antagonistic fungi (Fusarium
spp. and Trichoderma spp.) operated synergistically to significantly
reduce the incidence of P. cryptogea root rot in gerbera
crops (Grasso et al., 2003). Another strategy with slow
filtration is to enhance efficiency by biological activation of
the filtering columns with bacteria with suppressive traits, i.e.
antagonistic activities, or siderophore and auxin production
(Déniel et al., 2004). These bacteria, i.e. Bacillus and Pseudomonas
strains, were isolated from a mature tomato hydroponic
slow filtration unit and then inoculated into a new filter
(Renault et al., 2007). Further investigations showed that the
six-month period for the control filter to reach maximum efficiency
against F. oxysporum was shortened in the bacteriaamended
filter; in addition, filtration was highly efficient from
the first month. Fast colonisation of pozzolana grains by selected
bacteria and their subsequent interaction with F. oxysporum
is probably responsible for filter efficiency. Pseudomonas
spp. are supposed to act by competing for nutrients and Bacillus
spp. by antibiosis and/or direct parasitism (Déniel et al.,
2004). However, after nine months of operation, bacteria from
the genera Pseudomonas and Bacillus, used to inoculate the
filters, were not recovered in significant numbers from substrates
in these filtering columns (Renault, 2007). Therefore,
although early bacterial inoculations promote filter efficacy
and induce a significant shift in microbial communities, the
inoculated bacteria do not colonise the filtering substrates for
long periods.
6.3. Nutritional amendments
Although physico-chemical factors influence the prevalence
of Pythium diseases in certain substrates (van der Gaag
and Wever, 2005), the main factor regulating disease suppression
in hydroponic cultures is the microflora. The rhizosphere
competency of potential biocontrol agents is often limited
due to a lack of available organic nutrients in soilless
growth media. Indeed, the main source of nutrients for the microflora
on inorganic substrates is the plant roots, i.e. exudates,
mucigel, sloughed root cells, etc. In conventional agriculture
many other sources are available: organic amendments such
as compost can be used as fertilisers or to improve the physical
structure of the soil. Composted organic amendments are
also substrates capable of suppressing plant diseases caused by
a wide range of pathogens and pests, including bacteria, fungi
and nematode species (Hoitink and Boehm, 1999; Alabouvette
et al., 2006; Termorshuizen et al., 2006). Therefore, to maintain
a critical threshold population of antagonistic microorganisms
in soilless substrates, two approaches (similar to those in
conventional cultures) could be considered: (a) the use of a different
organicmaterial, i.e. compost, as an alternative substrate
10 J. Vallance et al.
for greenhouse production, and (b) the introduction of a food
base for the biocontrol agent to sustain its antagonistic activity
without stimulating that of the pathogen.
(a) Composted organic amendments have been tested as
alternative substrates to peat in soilless systems to preserve
peat bogs. Two different types of citrus compost and their
water extracts were investigated as partial peat substitutes
for melon seedlings in greenhouse nurseries. Compared with
peat, both composts (containing plant nutrients and auxin- and
cytokinin-like compounds) enhanced the plant growth; biocontrol
of Fusarium oxysporum was also achieved due to the
biotic component. Water extracts had no effect on plant yield
but their biocontrol ability was similar to that of their solid
matrices (Bernal-Vicente et al., 2008). Another study showed
that the suppressiveness of compost is related to the ability
of its microflora to degrade organic compounds. The microbial
communities associated with three substrates with varying
capacities of Fusarium wilt suppression were characterised:
peat (conducive to wilt), cork (moderately suppressive) and
grape marc (very suppressive). The nature and composition
of the plant growth medium determined the microbial communities:
in suppressive media, the microflora preferentially
metabolised less easily biodegradable compounds such as carboxylic
acids, amino acids, amines, phenolic compounds and
polymers; while the microflora of peat used mostly sugars
(Borrero et al., 2006).
(b) As the availability of nutrients is a limiting factor for the
growth of the microbial communities in various plant habitats,
the use of nutritional amendments has been studied to
selectively increase the communities’ size and the biocontrol
efficacy of a target biocontrol agent. The feasibility of
selective enhancement and maintenance of desired populations
of naturally-occurring biocontrol agents such as Pseudomonas
putida by amending the nutrient solution with a
nitrogen stabiliser, N-Serve, has been demonstrated. Both
active and inert ingredients in N-Serve were involved in the
suppression of root disease of pepper and cucumber caused
by Phytophthora capsici and P. aphanidermatum. Xylene and
1,2,4-trimethylbenzene, the constituents of the inert fraction
of N-Serve, served as carbon sources for the selective enhancement
of the pseudomonad populations, and nitrapyin,
the active ingredient, reduced the vegetative growth of both
pathogens (Pagliaccia et al., 2007, 2008).
7. CONCLUSION
The last three decades have convincingly shown that, in
soilless culture, the initial goal of growing plants free of
soilborne microorganism attacks was not realistic. Diseases
specific to this type of cultivation have been frequently reported;
indeed, the elimination of the soil did not remove
the pathogenic issue but has simply moved it. For instance,
in comparison with soil, some diseases are only observed or
have taken on a greater importance in soilless cultures. In that
context, control methods have to be adapted to soilless greenhouses.
One of the main options that has gradually emerged
in recent years has been the use of non-pathogenicmicroflora.
This assumption was based on the finding that if hydroponics
is a “solution” for the development and spread of pathogenic
zoosporic fungi and oomycetes, much evidence indicates that
it can also be one for the management of the plant protective
microflora. The development of sustainable control methods
such as classical biological control but also new kinds of experiments,
i.e. the re-use of substrates (with their suppressive
microflora) or the use of suppressive ready-to-use substrates,
is a must for soilless cultures. As numerous environmental parameters
are controlled, managing the microflora is much easier
in soilless culture than on soil. It will be a testing ground on
which the results could be used for transfer to more complex
systems such as soil.
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