Microbial
pathogens and strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.)
____________________________________________________________________________________________
The potential of bacterial volatiles for crop protection against
phytophathogenic fungi
Laure Weisskopf1
1
Research Station Agroscope Reckenholz-Tänikon, Reckenholzstrasse 191, CH-8046 Zurich, Switzerland
The production of antifungal substances by bacteria has long been recognized and this knowledge is entering practical
life through the use of bacterial antagonists to protect crops against their fungal enemies. Recently, it has become clear
that in addition to diffusible substances, bacteria emit a wide range of volatile compounds into the atmosphere. Over the
last ten years, evidence has accumulated that these volatiles are not only able to promote plant growth, but also to
strongly inhibit fungal growth. As the demand for organic products and the need to render agriculture more sustainable
are rising, finding new environmentally friendly crop protection strategies is essential. In this perspective, the newly
discovered capacity of bacterial volatiles to efficiently repel phytopathogenic fungi in laboratory experiments holds great
promise. In this section, recent knowledge on the inhibiting potential of bacterial volatiles against fungi causing major
crop diseases is summarized, future research questions are highlighted and putative applications for environmentally
friendly crop protection are discussed.
Keywords antifungal volatiles, biocontrol, Plant-Growth Promoting Rhizobacteria
1. Volatile organic compounds – newly discovered players in bacteria-fungi
interactions
Bacteria and fungi are major inhabitants of soils, of the rhizosphere (the region of soil under the influence of the roots)
and of plant tissues. They engage in multifaceted interactions, whose outcome can be either beneficial or deleterious to
the plant, which they greatly depend upon to meet their carbon needs. Two well-known examples of plant-promoting
interactions between bacteria and fungi are the beneficial effects of so-called “mycorrhiza-helper bacteria” [1, 2] or on
the contrary the antagonistic activity of bacteria against phytopathogenic fungi. By antagonizing the plant’s enemies,
bacteria ensure continuous supply of root exudates while minimizing competition for those exudates by repelling
heterotrophic fungi. The mechanisms by which soil bacteria are able to inhibit crop disease-causing fungi are very
diverse and range from iron depletion of their fungal competitor through siderophore production [3] to degradation of
fungal virulence factors [4] or production of a large variety of antifungal compounds (reviewed in [5]). Another
sophisticated way of bacteria to help plants defend themselves against disease causing fungi is the induction of systemic
resistance (ISR), by which non-pathogenic rhizosphere bacteria can trigger a state of alert in the plant, which leads to
better defence against a wide range of different pathogenic organisms [6].
All the above-mentioned processes require close vicinity or even physical contact of the interacting partners, yet
bacteria are also able to inhibit phytopathogenic fungi from a distance. A classic example of such volatile-mediated
antifungal activity is cyanogenesis, i.e. the production of the respiratory poison hydrogen cyanide (HCN). HCN is
produced in various amounts by strains of different Pseudomonas species as well as by other bacteria such as
Chromobacterium violaceum [7]. In the biocontrol strain P. fluorescens CHA0, cyanogenesis was shown to be directly
involved in the biocontrol of Thielaviopsis-induced root rot of tobacco [8]. Until recently, HCN was the only volatile
molecule known to be active against fungi. However, in the last decade, increasing evidence has been brought about
that non cyanogenic bacteria are also capable of inhibiting fungi by emitting volatiles. In contrast to HCN, whose
practical application at least as a pure compound is limited due to its toxicity and to its lack of specificity, other, less
toxic volatiles produced by rhizosphere bacteria, which specifically inhibit pathogenic fungi might represent a
promising new strategy to control crop fungal diseases. Since the first discovery of production of antifungal volatiles by
non cyanogenic bacteria [9], many new reports have demonstrated widespread volatile-mediated antagonistic potential
in rhizosphere bacteria. These findings have been recently reviewed [10-12], mostly focussing on the emitter’s side, i.e.
the bacteria. This book chapter focusses on the target organisms and summarizes available knowledge on the potential
of bacterial volatiles to inhibit the fungal causing agents of major crop diseases. It also highlights present gaps of
knowledge and future research directions.
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2. The potential of bacterial volatile organic compounds (VOCs) for crop protection
against major fungal diseases
2.1. Production of antifungal volatiles by bacteria: a widespread phenomenon
Fungal diseases are responsible for tremendous losses in world-wide agriculture [13, 14]. Strikingly, most of the
research carried out so far on bacterial volatiles mostly dealt with the response of Rhizoctonia solani or Fusarium
species (Table 1). While these are of economical relevance, other fungi or fungi-like organisms pose also major threats
to global food security such as the causing agents of rice blast (Magnaporthe grisae), of potato late blight
(Phytophthora infestans), of powdery mildew in wheat (Blumeria graminis) as well as of the devastating rust (Puccinia
spp., Uromyces spp.) and smut (Ustilago spp.) diseases in cereals [14]. One explanation for this lack of knowledge on
many of those pathogens might be their obligate biotrophic lifestyle, which makes their cultivation in vitro very
challenging. While this has not prevented researchers to search for bacterial biocontrol agents against these pathogens,
the young field of volatile-mediated antagonism is still mostly dealing with readily cultivable fungi. Among those fungi
and oomycetes responsible for major crop diseases, those that have been tested for their reaction to bacterial volatiles
include species from the genera Alternaria, Botrytis, Colletotrichum, Fusarium, Phytophthora, Pythium, Rhizoctonia,
Sclerotinia and Verticillium. The results obtained when exposing those disease-causing agents to bacterial volatiles are
summarized in Table 1. So far, mostly Bacillus, Pseudomonas and Streptomyces species have been reported to
efficiently inhibit the growth of phytopathogenic fungi, while members of other genera (e.g. Burkholderia, Serratia,
Agrobacterium) have only sporadically been investigated (Table 1). Since very few comparative studies exist, in which
different bacterial species were analysed for VOCs-mediated antifungal activity [12, 15, 16], one cannot at present state
with certainty that Bacillus and Pseudomonas are relatively more active than other bacteria. Their overrepresentation in
the studies conducted so far might be a consequence of their being more readily isolated and cultivated than other
species, or their being intentionally selected for analysis of VOCs-mediated effects due to their effectiveness as
biocontrol agents, the latter relying at least partly on the production of non-volatile metabolites. Further studies
systematically comparing a broad diversity of bacterial species for VOCs-mediated antifungal activity should bring an
answer to the question of whether or not Pseudomonas and Bacillus species are more efficient producers of antifungal
volatiles than members of other genera.
Table 1 Overview of literature reporting inhibition of fungi and oomycetes by bacterial volatiles, with a focus on major crop
pathogens. n.a., not applicable; n.d., not detected (no volatile analysis); n.t., not tested (volatiles determined but not tested as pure
compounds). Ref., literature reference. Results on the effect of pure volatiles from plant origin (e.g. essential oils), but which are also
known to be produced by bacteria too [11], are also included.
Fungi
Effects
Bacteria
Volatiles
Ref.
Alternaria
alternata
Complete growth inhibition
n.a.
benzaldehyde
[17]
Mycelial growth inhibition
n.a.
1-hexanol, E-2hexenal and 2nonanone
[17]
Inhibition of mycelial and
germ tube growth
n.d.
nonanal, 2-nonenal,
2-nonanone, hexanal
[18]
Mycelial growth inhibition
Morphological distortions
Bacillus subtilis
n.d.
[19]
Mycelial growth inhibition
n.a.
Acetaldehyde
[20]
Mycelial growth inhibition
Pseudomonas corrugata
n.d. (not HCN)
[21]
Mycelial growth inhibition
Burkholderia ambifaria
[22]
Reduced growth
and pigmentation
Paenibacillus sp.
Bacillus sp.
dimethyl trisulfide, 2nonanone, 2undecanone
n.t.
Mycelial growth inhibition
Paenibacillus polymyxa
BMP-11
Alternaria
brassicae
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1-octen-3-ol,
benzothiazole,
citronellol
[23]
[24]
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Alternaria
brassicola
Alternaria
solani
Mycelial growth inhibition
Bacillus subtilis
n.t.
[25]
Mycelial growth inhibition
Paenibacillus sp.
Bacillus sp.
n.t.
[23]
Mycelial growth inhibition
Streptomyces
globisporus JK-1
n.d.
[26]
Alternaria sp.
Mycelial growth inhibition
Streptomyces
globisporus JK-1
n.d.
[26]
Botrytis cinerea
Complete growth inhibition
n.a.
benzaldehyde
[17]
Mycelial growth inhibition
n.a.
1-hexanol, E-2hexenal and 2nonanone
[17]
Mycelial growth inhibition
Bacillus subtilis
n.t.
[25]
Reduced disease on
blackberry and grape
n.a.
hexanal, 1-hexanol,
(E)-2-hexen-1-ol,
(Z)-6-nonenal, (E)-3nonen-2-one, methyl
salicylate, methyl
benzoate
[27]
Mycelial growth inhibition
n.a.
acetaldehyde
[20]
Inhibition of mycelial
growth and of spore
germination
Paenibacillus sp.
Bacillus sp.
n.t.
[23]
Complete growth inhibition,
inhibition of spore
germination and protoplast
retraction
Bacillus subtilis JA
n.t.
[28]
Mycelial growth inhibition
Disease reduction on
strawberries
Streptomyces platensis
n.t.
[29]
Mycelial growth inhibition
Paenibacillus polymyxa
BMP-11
1-octen-3-ol,
benzothiazole
[24]
Reduced disease on tobacco
plants (greenhouse)
Bacillus cereus
dimethyl disulfide
[30]
Complete growth inhibition
Reduced sporulation and
germination, reduced
infection on tomato fruits
Streptomyces
globisporus JK-1
n.d.
[26]
Mycelial growth inhibition
Photobacterium sp.
Salinivibrio costicola
Bacillus safensis
B. gibsonii
B. oceanisediminis
n.d.
[15]
Complete growth inhibition
n.a.
nonanal
[31]
Colletotrichum
accutatum
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Mycelial growth inhibition
n.a.
acetaldehyde
[20]
Inhibition of mycelial
growth and of pigmentation
Paenibacillus polymyxa
BMP-11
1-octen-3-ol,
benzothiazole,
citronellol
[24]
Mycelial growth inhibition
Bacillus sp.
Enhydrobacter
aerosaccus
n.d.
[32]
Colletotrichum
fragariae
Complete growth inhibition
n.a.
nonanal
[31]
Colletotrichum
gloeosporioides
Complete growth inhibition
n.a.
benzaldehyde
[17]
Mycelial growth inhibition
n.a.
1-hexanol, E-2hexenal and 2nonanone
[17]
Complete growth inhibition
n.a.
nonanal
[31]
Mycelial growth inhibition
Bacillus sp.
Enhydrobacter
aerosaccus
n.d.
[32]
Mycelial growth inhibition
Streptomyces
globisporus JK-1
n.d.
[26]
Fusarium
avenaceum
Mycelial growth inhibition
Bacillus subtilis
n.t.
[25]
Fusarium
culmorum
Mycelial growth inhibition
Bacillus subtilis
n.t.
[25]
Mycelial growth inhibition
Bacillus subtilis, Serratia
sp.
n.d.
[33]
Mycelial growth inhibition
Paenibacillus sp.
Bacillus sp.
n.t.
[23]
Mycelial growth inhibition
Streptomyces
globisporus JK-1
n.d.
[26]
Mycelial growth inhibition
P. fluorescens strains,
B. cereus, B. subtilis,
Erwinia herbicola
n.d.
[34]
Inhibition of mycelial
growth, of spore germination
and alteration of
morphology
Agrobacterium
radiobacter, Bacillus
cereus, Enterobacter
aerogenes, Escherichia
coli, Micrococcus luteus,
Nocardia corallina,
Proteus vulgaris,
Sarcina lutea,
Serratia marcescens
n.d.
[16]
Mycelial growth inhibition
Bacillus subtilis
n.d.
[19]
Inhibition of mycelial
growth and of pigmentation
Paenibacillus sp.
Bacillus sp.
n.t.
[23]
Colletotrichum
capsici
Fusarium
graminearum
Fusarium
oxysporum
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Fusarium solani
Mycelial growth inhibition
Herbaspirillum sp.
n.t.
[35]
Mycelial growth inhibition
Pseudomonas corrugata
n.d. (not HCN)
[21]
Mycelial growth inhibition
Paenibacillus polymyxa
BMP-11
1-octen-3-ol,
benzothiazole,
citronellol
[24]
Reduced pigmentation
Paenibacillus polymyxa
BMP-11
citronellol
[24]
Mycelial growth inhibition
Burkholderia gladioli pv.
agaricicola
n.t.
[36]
Inhibition of mycelial
growth and of spore
germination
Bacillus
amyloliquefaciens
benzenes, ketones
and aldehydes
[37]
Mycelial growth inhibition
Bacillus subtilis
n.t.
[25]
Mycelial growth inhibition
Agrobacterium
tumefaciens,
Pectobacterium
carotovorum 436R,
Pseudomonas
phaseolicola 796,
P. syringae 1142,
Xanthomonas campestris
pv vesicatoria 85-10
n.t.
[12]
Mycelial growth inhibition
Bacillus sp.
n.d.
[32]
Enhydrobacter
aerosaccus
Sclerotinia
sclerotiorum
Mycelial growth inhibition
Bacillus subtilis
n.t.
[25]
Inhibition of mycelial
growth and of germination
from ascospores and
sclerotia
Pseudomonas sp.
benzothiazole,
cyclohexanol, ndecanal, dimethyl
trisulfide, 2-ethyl 1hexanol, and nonanal
[38]
Mycelial growth inhibition
Bacillus subtilis,
Burkholderia cepacia,
Pseudomonas sp.
Serratia sp.
n.d.
[33]
Mycelial growth inhibition
Disease reduction on oilseed
rape
Mycelial growth inhibition
Streptomyces platensis
n.t.
[29]
Paenibacillus sp.
Bacillus sp.
n.t.
[23]
Agrobacterium
tumefaciens,
Pectobacterium
carotovorum 436R,
Pseudomonas
phaseolicola 796,
P. syringae 1142,
n.t.
[12]
Mycelial growth inhibition
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Verticillium
dahliae
Rhizoctonia
solani
Inhibition of spore
germination
Complete growth inhibition
Pseudomonas
chlororaphis PA23
Streptomyces
globisporus JK-1
n.d.
[39]
n.d.
[26]
Mycelial growth inhibition
Bacillus subtilis,
Pseudomonas sp.
Serratia sp.
n.d.
[33]
Mycelial growth inhibition
Paenibacillus sp.
Bacillus sp.
n.t.
[23]
Mycelial growth inhibition
Agrobacterium
tumefaciens,
Pectobacterium
carotovorum 436R,
Pseudomonas
phaseolicola 796,
P. syringae 1142,
Xanthomonas campestris
pv vesicatoria 85-10
n.t.
[12]
Inhibition of mycelial
growth and of sclerotia
formation
Mycelial growth inhibition
Serratia plymuthica
HRO-C48
n.t.
[40]
Streptomyces
globisporus JK-1
n.d.
[26]
Mycelial growth inhibition
Bacillus subtilis
n.t.
[25]
[41]
Mycelial growth inhibition
Bacillus subtilis,
Serratia sp.
n.d.
[33]
Mycelial growth inhibition
Pseudomonas sp.,
Serratia sp.,
Burkholderia cepacia
n.t.
[42]
Mycelial growth inhibition
Paenibacillus sp.
Bacillus sp.
n.t.
[23]
Mycelial growth inhibition
Disease inhibition on rice
Mycelial growth inhibition
Streptomyces platensis
n.t.
[29]
B. thuringiensis
n.t.
[43]
Serratia plymuthica
HRO-C48
Agrobacterium
tumefaciens,
Pectobacterium
carotovorum 436R,
Pseudomonas
phaseolicola 796,
P. syringae 1142,
Xanthomonas campestris
pv vesicatoria 85-10
n.t.
[40]
n.t.
[12]
n.d.
[44]
Mycelial growth inhibition
Mycelial growth inhibition
Mycelial growth inhibition
Bacillus strains
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Phytophthora
capsici
Mycelial growth inhibition
Paenibacillus polymyxa
BMP-11
1-octen-3-ol,
benzothiazole,
citronellol
n.d.
Mycelial growth inhibition
Streptomyces
globisporus JK-1
[26]
Mycelial growth inhibition
Burkholderia gladioli pv.
agaricicola
n.t.
[36]
Mycelial growth inhibition
Xanthomonas campestris
pv. vesicatoria 85-10
decan-2-one
[45]
Mycelial growth inhibition
Bacillus strains
n.d.
[46]
Mycelial growth inhibition
Burkholderia ambifaria
Dimethyl disulfide,
dimethyl trisulfide,
4- octanone, Smethylmethanethiosu
lphonate,
acetophenone,
Phenylpropan-1-one,
1-Phenyl-1,2propanedione, 2undecanone
[22]
Mycelial growth inhibition
Paenibacillus polymyxa
BMP-11
1-octen-3-ol,
benzothiazole,
citronellol
[24]
Mycelial growth inhibition
Bacillus sp.
n.d.
[32]
[24]
Enhydrobacter
aerosaccus
Pythium afertile
Mycelial growth inhibition
Bacillus subtilis
n.d.
[19]
Pythium
ultimum
Mycelial growth inhibition
Bacillus subtilis
n.t.
[25]
[41]
2.2. Specificity of the volatile-mediated interaction between bacteria and fungi
As can be seen from Table 1, the potential of bacterial volatiles for growth inhibition of fungi responsible for major
crop losses in today’s agriculture is abundantly documented. Most of the studies have focused on one particular
bacterial strain and its volatile-mediated effect on one target fungus of interest. However, few reports that address the
question of specificity are available, where the response of various target fungi to the volatiles of one or few bacterial
strains was compared. Liu and co-workers investigated the impact of four bacterial strains belonging to the Bacillus and
Paenibacillus genera on a wide variety of target fungi. They observed large differences in the susceptibility of fungi to
bacterial VOCs, with highest effects on Sclerotinia, followed by (decreasing order) Alternaria, Rhizoctonia,
Verticillium and Fusarium species [23]. This high sensitivity of Sclerotinia is corroborated by the observations of Li et
al., who observed complete inhibition of Sclerotinia as well as of Botrytis, while other tested fungi were only partially
inhibited by the bacterial VOCs [26]. Similar genus-specific variability in susceptibility was reported upon exposure to
VOCs from Bacillus subtilis: Fiddaman and Rossall observed already in the early nineties that B. subtilis VOCs caused
much greater inhibition of mycelial growth of S. sclerotiorum, R. solani or Botrytis cinerea than of P. ultimum or
Fusarium species [25] and Chaurasia et al. reported that the oomycete Pythium afertile was much more inhibited than
the ascomycetes Alternaria sp. or Fusarium sp. [19]. Beyond this genus specificity, different species within a genus can
react very differently, as was shown for F. graminearum, which was much less inhibited than F. oxysporum [23], and
even within a single species, susceptibility to bacterial VOCs might differ, as reported twice independently for R. solani
pathotypes [25, 46]. Beyond this taxon-specific variability, a general trend seems to emerge from the literature listed in
Table 1, namely the particular sensitivity of Sclerotinia sclerotiorum, of Botrytis cinerea, of Alternaria sp. or of
oomycetes and the relative tolerance of Fusarium species to bacterial VOCs [22, 33, 45]. This is also illustrated in Fig.
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1, where few strains isolated from the rhizosphere of white lupin [47] were tested for VOCs-mediated inhibition of
Fusarium poae and Microdochium majus. The two fungi reacted very differently to the VOCs from the same strains: M.
majus was strongly inhibited but F. poae was unaffected by the presence of bacterial VOCs.
On the other side of the
interaction, even closely related
strains might exhibit differential
VOCs-mediated effects on a given
target fungus, as previously shown
for plants [7, 48]. Few studies have
indeed
reported
differential
potential in emission of antifungal
volatiles in different bacterial
strains: Ann and co-workers
reported that only one out of six
Bacillus strains was able to inhibit
the growth of Phytophthora
capsici, while Colletotrichum and
Fusarium strains were inhibited by
all
six strains [32]. Similar strainFig. 1 Effect of volatiles from three Burkholderia strains on mycelial growth of
specific
effectiveness was observed
Microdochium majus (upper row) and Fusarium poae (lower row). 20 µl of overnight
by Berrada et al., where some
bacterial culture were spread on one half of the Petri dishes containing Luria-Bertani
bacilli caused up to 87% growth
medium. After 24h, a plug of fungal mycelium was placed in the middle of the other half
on malt extract agar. Plates were parafilmed and incubated for 10 days at room
inhibition in Botrytis cinerea,
temperature in the dark. Pictures: courtesy of Lukas Hunziker.
while other strains showed no
activity [15]. However, it should be
noted that the experimental setup used in this latter study did not allow univocal discrimination between effects due to
volatile compounds and those due to diffusible compounds.
2.3. The search for active molecules
Similar to the search for growth-promoting volatiles in plants [49], that for the active compounds responsible for the
VOCs-mediated antifungal activity of bacterial strains is very challenging. Beyond inorganic volatiles such as HCN or
ammonia (NH3) [21, 50], identifying organic compounds, which inhibit the growth of fungi when applied in
concentrations relevant to the biological situation has proven a hard task. In some studies, milligrams of the pure
volatiles are applied on a filter [24, 37], resulting in concentrations of orders of magnitudes higher than those occurring
upon exposure to the bacterial blend itself. When dose-response experiments are carried out, which has so far been
rather the exception than the rule, application of such high quantities of compounds often leads to growth inhibition,
while already a 10-fold dilution might prove ineffective [22]. Beyond the difficult adjustment of the concentration to be
tested, which is in great part due to the challenge of accurately quantifying the rate of bacterial volatile production, at
least three other challenges await the researcher on his quest for bioactive volatile discovery: the first one is the
relatively high proportion of unresolved peaks in the GC-MS profiles, which prevents the purchase or synthesis of the
possibly newly discovered molecules. The second lies in the application technology: most researchers apply the
volatiles to be tested once on the target organism, while bacterial emission results in slow, dynamic release over time, as
the fungus is growing. Lastly, we have learned from volatile research in the insect pheromone field that pure
compounds rarely account for the observed bioactivity, but that a mixture of different compounds is needed to achieve
activity. Testing synthetic mixtures is a very delicate enterprise, given the facts that the natural concentrations and
proportions can only be very approximately inferred from GC-MS chromatograms, that compounds usually differ in
their water affinity (some volatiles naturally occurring together might not be miscible when a synthetic mixture is
attempted) and that the mixture of single active volatiles might result in an inactive mixture, if it contains some
promoting and some inhibiting compounds. In view of these challenges, it is remarkable that at least some VOCs could
be identified as antifungal compounds, such as dimethyl di- or trisulfide, or other sulfur compounds, benzaldehyde and
long chain ketones like 2-undecanone or 2-nonanone (see Table 1). Beyond specific activity of given molecules, the
physical and chemical properties of the volatiles need to be considered, with special attention on pH changes. pH is one
of the most important environmental factors influencing microbial communities [51]. Overall, bacteria are generally
more sensitive to acid conditions and true fungi more to alkaline environments, whereas the preference of oomycetes is
so far unknown. Both HCN and NH3 would lead to drastic changes in pH in the media exposed to the bacterial volatiles,
which in turn might cause altered growth behaviour in target fungi. Interestingly, awareness of this phenomenon
appears to have been much stronger in earlier studies than in later ones, as exemplified by Moore-Landecker and
Stotzky, who analysed volatile-mediated pH changes upon growth of bacteria on different media. Significant
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alkalinisation was observed, yet the authors concluded that this was not the reason for growth reduction, since fungi
were growing normally on alkaline media in the absence of bacterial volatiles [16]. About 20 years later, Fiddaman and
Rossall also investigated pH changes but found only minor effects, which depended on the presence (acidification) or
absence (alkalization) of carbohydrates in the bacterial growth medium [25]. Here too, the authors concluded that pH
changes might not play a significant role in the overall antifungal activity of bacterial volatiles, at least in well-buffered
laboratory culture media. Whether or not volatile-mediated acidification or alkalinisation of the environment would play
a role in interactions occurring in nature remains to be elucidated.
2.4. From the Petri dish to the field: where are we now?
Despite the strong effects observed in the laboratory, few researchers have so far dared the step towards experimental
designs closer to the field situation and most of the results compiled in Table 1 originate from in vitro studies on fungi
and bacteria grown on rich culture media in sealed Petri dishes. Still, one of the pioneer studies on antifungal effects of
volatiles from non cyanogenic bacteria compared production of volatiles by Bacillus subtilis grown on rich medium
(Potato Dextrose Agar, PDA), with that of cells growing in sterilized sand, soil or directly on the roots of oil seed rape,
the target fungus Rhizoctonia solani being itself grown on PDA [25]. As expected, when no additional carbon supply
was added, production of antifungal volatiles was much reduced when bacteria were grown in sand, soil or plant roots
as compared to growth on PDA. However, when additional nutrients were supplied to the soil, the volatile-mediated
antifungal activity of B. subtilis was again very strong, suggesting that in specific niches, such as the rhizosphere of
plants exuding large amounts of carboxylates, sufficient antifungal volatile emission might occur to ensure efficient
antagonism of phytopathogenic fungi. Similar encouraging results were obtained with the inhibition of Sclerotinia
sclerotiorum by Pseudomonas strains growing in soil: mycelial growth and sclerotia formation were strongly inhibited
and failed to grow/germinate when transferred to fresh PDA plates. However, here too, the bacteria were first grown in
rich media and then inoculated with substantial amounts of these media to the soil, which still represents a rather
artificial system [38]. In few studies, the effectiveness of the antagonistic strains was tested in vivo, whereby
discrimination between volatile and non-volatile effects is difficult in such setups. Berrada and co-workers observed
that the strain showing best activity in vitro was also best protecting soil-grown plants, with long-lasting and complete
inhibition of Botrytis cinerea on tomato plants. The efficiency was strongly strain-specific, some bacilli causing up to
87% inhibition while others were ineffective [15]. Similar protection against B. cinerea in soil-grown tobacco was
achieved by inoculating B. cereus to the soil or by drenching the soil with dimethyl disulfide [30]. Even Fusarium
graminearum- induced head blight could be significantly alleviated by volatile-producing rhizosphere strains in potgrown wheat, whereby volatile effects could not be discriminated from non-volatile effects [34]. Until now, two studies
are available, which univocally show that production of antifungal volatiles can inhibit phytopathogenic fungi that grow
on or in their host plants: the first one eloquently demonstrated the potential of Streptomyces platensis to efficiently
inhibit three fungal disease causing agents, namely S. sclerotiorum in oilseed rape, R. solani in rice and B. cinerea on
strawberries [29]. These experiments were carried out in soil-grown plants but with an open Petri dish containing S.
platensis in a sealed growth chamber. More recently, Li and co-workers showed similar inhibition of B. cinerea on
tomato fruits upon exposure to the volatiles of Streptomyces globisporus [26]. Remarkably, the antifungal volatile
producing strain was grown on autoclaved wheat seeds, and not on rich medium in this latter study. While the abovementioned examples provide proof of concept of the transferability of in vitro experiments to soil-grown plants, the
road to practical field application remains very long. Apart from soil fumigation with active volatiles (such as dimethyl
disulfide), the best way to use bacterial producers of antifungal volatiles will most probably reside in inoculating the
strains themselves, thereby benefiting from their volatile-mediated activity as well as from other plant-beneficial
properties such as production of diffusible antifungal compounds or induction of systemic resistance. Therefore, in
addition to in vitro volatile-mediated activity, successful colonization of the rhizosphere as well as of the phyllosphere
will be an important asset for efficient plant protection against belowground as well as aboveground pathogens.
3. Open questions and future challenges
Based on the literature available on the volatile-mediated antifungal activity of bacteria, it is striking to see the
increasing number of papers published in the last couple of years, compared with relatively scarce documentation of the
phenomenon before 2007 (see also references from Table 1). This intensifying interest likely arises from the growing
needs for sustainable intensification of agriculture, from the toxicity of chemical fungicides or from the limitations of
the alternatives used so far in organic farming, e.g. the use of copper-based fungicides to fight oomycetes such as late
blight of potato and downy mildew of grapevine. As exemplified by drastic fungal growth reduction in laboratory
experiments but also by significant reduction of fungal diseases in pot-grown plants upon exposure to bacterial
volatiles, this newly discovered capacity of rhizobacteria to remotely inhibit fungal growth bears interesting application
potential and raises great hopes for the development of environmentally friendly crop protection measures. Yet, before
practical application of this knowledge can be envisaged, many important questions need to be answered.
Despite the drastic growth reduction occurring when challenging phytopathogenic fungi with the complex blend of
bacterial volatile emissions, researchers have so far struggled to pinpoint the active molecules responsible for the
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observed effects. In many of the studies listed in Table 1, the profiles of emitted volatiles were not analysed and when
they were, the identified molecules were rarely tested as pure compounds and hardly ever as synthetic mixtures.
Additional obstacles towards identification of candidate molecules is the use of irrelevant concentrations, leading to
false positives, or the oversimplification of experimental setups (i.e. filter papers with a one-time exposure to the
volatiles rather than a slow release as that originating from biological emission), leading to false negatives. Finally, the
high proportion of peaks in the GC-chromatograms, to which a high confidence match cannot be assigned, precludes
purchase and testing of putatively active compounds. Rapid progress in analytical techniques, in chemical database
coverage as well as increasing awareness of the synergetic effect of mixtures should lead to improved results in the
future.
One important point that has been almost completely overlooked so far is the elucidation of the mechanisms
underlying growth arrest in fungi exposed to bacterial volatiles. With the exception of HCN, a respiratory poison, which
obviously leads to growth arrest and death in an aerobic organism, the reasons for the described growth reduction,
hyphal distortion or inhibition of spore formation vs. germination are as of yet completely unclear. While identifying
the active molecules might provide indirect hints towards possible mechanisms of action, another way to elucidate those
mechanisms would be to monitor changes occurring at gene expression (transcriptomics) or protein (proteomics) level
upon exposure to bacterial volatiles. While such global studies have been carried out in plants exposed to bacterial
volatiles [52-54], no information is yet available on the physiological changes occurring in target fungi and oomycetes
when they “smell” bacteria, and which ultimately lead to growth arrest or death.
Finally, one of the biggest challenges will be to try to transfer the accumulating knowledge on volatile-mediated
inhibition of phytopathogenic fungi by bacterial volatiles to the field. In this respect, emission of antifungal volatiles
should be regarded as one of many components to successfully control disease-causing agents. Bacterial strains
showing a combination of efficient weapons in the battle against phytopathogenic fungi should be preferred to those
showing exclusively one or the other feature. Such antagonists would ideally be able to repel the plants’ fungal enemies
from a distance (through volatiles), but also through direct contact. They should also be rhizosphere and phyllosphere
competent and of course devoid of any virulence factor liable to endanger plant, animal or human health. Searching for
such a “perfect strain” might represent a long and strenuous quest, yet the perspective of contributing to the
development of new, sustainable strategies to fight off phytopathogenic fungi, a major cause of yield and quality losses
and starvation in many parts of the world, certainly makes this endeavour worth the effort.
Acknowledgements Excellent technical help of Lukas Hunziker is gratefully acknowledged.
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