Abstract: Bacillus subtilis G8, isolated from soil in China, produced antifungal volatile organic compounds (VOCs). Bioassay in sealed dishes revealed that these volatiles significantly inhibited the mycelial growth and completely prevented the pigment production of all tested soil-borne plant pathogens (43-93% inhibition, respectively) and effectively controlled the overwintered sclerotoid germination of Sclerotinia sclerotiorum. Such effective antifungal VOCs were extracted using Solid Phase Microextraction (SPME) and given further identification by Gas Chromatography-Mass Spectrometry (GC-MS) technique. The detected volatile compounds included alkyls, alcohols, esters, ketones, acid, amine, oxime, phenols and heterocyclic compounds. Present results demonstrate that soil bacteria are rich resources of bioactive volatiles and may play an important role in reducing disease levels.
INTRODUCTION
Soil-borne disease is one of the most serious diseases of vegetables and crops in the world. Several strategies such as crop rotations, breeding resistant varieties and applying chemical fungicides may be useful, however, these cultural practices alone are rarely adequate and resistant cultivars are effective only against certain specific fungi species. Chemical fungicides, though usually more effective than other strategies, have caused significant environmental problems and fungicide-resistance. These years, biocontrol, for example, the application of antagonistic fungi and bacteria (Paulitz and Bélanger, 2001; Minuto and Spadaro, 2006), seems to arouse our great interest because it is eco-friendly, safe and may provide long-term protection to the crop (Fernando et al., 2005).
Recently, volatile compounds produced by fungi and some bacteria have been demonstrated with the potential antifungal or nematicidal nature by several studies (Alstrom, 2001; Wheatley, 2002; Xu et al., 2004; Zou et al., 2007; Gu et al., 2007) and the application of antifungal volatiles from fungi had been carried out in greenhouse (Mercier and Manker, 2005; Koitabashi, 2005). However, little is known about the effective volatiles produced by B. subtilis in the broad-spectrum control of soilborne plant diseases. In this study, eight pathogenic fungi that can colonize a wide range of host plants were chosen for bioassay and the method of SPME-GC/MS was expatiated for the extraction and identification of bacterial VOCs.
MATERIALS AND METHODS
Bacterial Culture
Bacillus subtilis G8 was isolated from the soil
in greenhouse in China and maintained in Nutrient Broth (NB) supplemented
with 20% (v/v) glycerol at -20 °C before use. For bioassay and analysis
of volatile compounds, bacterium was inoculated into 25 mL TSB-YE (Bacto-tryptone
15 g L-1, soya peptone 5 g L-1, yeast extract 6.5
g L-1, NaCl 5 g L-1, pH 7.2) in 50 mL sample vial.
The vial was rapidly sealed with parafilm (Menasha) and cultured at 30 °C
under agitation (180rpm) in the dark up to an OD600 of 1.0-1.5.
Pathogenic Fungi and Storage Conditions
Eight pathogenic fungi that can colonize a wide range of host plants
and have consistent high virulence, including S. sclerotiorum,
Botrytis cinerea, Alternaria brassicae, Alternaria
solani, A. citrullina, Fusarium oxysporum, Cercospora
kikuchii Chupp, Rhizoctonia solani, were used for the bioassay
in sealed dishes (Fig. 1). For short-term maintenance,
fungi were cultured on PDA plates at 22~25 °C under darkness. For long-term
storage, tiny pieces of an actively growing colony were brought into Potato
Dextrose Agar (PDA) slants at 4 °C.
Antifungal Activities of Bacterial Volatiles Against Mycelial Growth
Briefly, in sealed dishes (Fernando et al., 2005), 200 μL
bacterial cultures (108 cfu mL-1) described above
were spread onto the bottom dish of a sterile Petri-dish containing TSA-YE
(Bacto-tryptone 15 g L-1, Soya peptone 5 g L-1,
yeast extract 6.5 g L-1, NaCl 5 g L-1, agar 15 g
L-1, pH 7.2). A 5mm mycelial plug was taken from the margin
of the colony and placed in the centre of a second bottom dish containing
fresh PDA. The bacterial dish was immediately inverted over the fungi
dish and the dishes were rapidly sealed with parafilm. The dishes were
incubated at 25 °C in the dark (Fig. 2). Volatiles
from TSB-YE and indoor sterile air instead of bacterial volatiles serve
as controls. The diameter (mm) of the fungal colony was measured in a
crisscross fashion when the radial mycelium of the controls extended to
3/4 plate. There were four replicates for each treatment and the experiments
were repeated twice.
Inhibition Effects of Volatiles on Sclerotoid Germination
The adult sclerotia of S. sclerotiorum were collected to a
sterile Petri plate and stored at -4 °C for two months before use.
Briefly, the sclerotia with similar size and quality was selected and
placed in the centre of a petri dish (90 mm diam) containing fresh PDA.
Then 200 μL of bacterial cultures were spread onto another petri
dish containing TSA-YE. The treatments with bacterial volatiles in sealed
dishes were carried out as described above. The diameter of radial mycelial
around germinated sclerotia was determined every 24 h until seven days.
The control dishes had no bacterium. There were four replicates for each
treatment and the experiments were repeated twice.
| Fig. 1: | Radial mycelial growth of different pathogenic fungi exposed to bacterial volatiles in sealed dishes. The growth of radial mycelium was determined and the percent inhibition compared to the controls (indoor controls and medium controls) was calculated. Error bars indicate±SD |
| Fig. 2: | Antifungal activities of bacterial volatiles against Alternaria solani in sealed dishes. Volatiles produced by G8 on the TSA-YE dish presented visible inhibition effects on the mycelial growth and the pigment production of A. solani (B), whereas the mycelium grew normally and abundant of pigment was produced in the control dishes in absence of bacterial volatiles (A) |
Volatile Organic Compounds Extraction
Bacterium was cultured as described above. Briefly, three SPME fibers
(100 μm PDMS, 65 μm PDMS/DVB, 50/30 μm CAR/DVB/PDMS, purchased
from SUPELCO) were chosen to extract volatiles and conditioned with helium
at 250 °C for 2 h before use, respectively. The sample vial was clamped
inside a thermostatic water bath and placed on a hot stirrer. Samples
were equilibrated at 40 °C for 30 min. The SPME needle was allowed
to pierce through the parafilm and the fiber was exposed to the headspace
of the sample vial for 40 min. The VOCs from 25 mL TSB-YE medium was used
as controls. After extraction, the SPME fiber was directly inserted into
the front inlet of the gas chromatography.
GC/MS Parameters and Analysis
SPME fibers were desorbed at 210 °C for 1 min in the injection
port of GC/MS-QP2010 (70 ev; SHIMADZU) equipped with a Rtx-5 Capillary
column (30 m length, 0.25 mm i.d., 0.25 μm film thickness, Restek).
GC/MS runs were 32 min and the fibers were conditioned at 210 °C for
10 min before re-used. The injection port was operated in split mode at
a ratio of 5:1. The carrier gas was He. The initial oven temperature was
40 °C, held for 2 min, ramped at 6 °C min-1 to 180 °C
and ramped at 10 °C min-1 to 250 °C and held for 3 min.
The temperature of the transfer line and ion trap were 200 and 230 °C,
respectively. The ions were detected in the range 30-350 m/z. The mass
spectra of the unknown compounds were compared with those in the NIST05
Library.
RESULTS AND DISCUSSION
Volatiles Produced by G8 Displayed a Broad-Spectrum Antifungal
Activity
It is obvious that all the mycelial growth in treatment dishes full
of antifungal volatiles was significantly restricted, as compared to those
in the two control dishes without bacterial volatiles (Fig.
1). However, it seemed that there was species-specificity among different
fungi. Volatiles from G8 inhibited mostly the growth of S.
sclerotiorum, B. cinerea and C. kikuchii Chupp. (>75%
inhibition, p>0.05), whereas the inhibition against A. brassicae
and R. solani was lesser than 46% inhibition (p>0.05). This
result implied the antifungal potential and the possibility of bacterial
volatiles in soil plant diseases. The mycelium treated with volatiles
from TSB-YE medium (medium controls) had the same growth rate with that
in control dishes full of indoor sterile air (indoor controls, Fig.
1), which indicated that volatiles from TSB-YE had no antifungal activity.
Antifungal Effects of Volatiles on Fungal Pigments
Previously, the pigments of pathogenic fungi, such as melanin, were
testified nearly interrelated with fungal pathogenicity and could endow
fungi some special recovery function, such as anti-radiation, anti-oxidation,
scavenging free radical, etc. (Cao and Yang, 2006; Souad et al.,
2002). Some fungicides, such as tricyclazole and carpropamid, killed fungi
mostly by inhibiting the production of melanin. In this study, we found
that volatiles from G8 strain exhibited visible inhibition
to the pigments of all tested pathogenic fungi, including B. cinerea,
A. brassicae, A. solani, A. citrullina, F. oxysporum,
C. kikuchii Chupp., R. solani. A case in point was shown
in Fig. 2. Therefore, it seems that these volatiles would
be possible to play a significant role in reducing the pathogenic fungal
infection ability. That could also be a positive support for bacterial
volatiles in reducing disease level.
Inhibition Effects of Volatiles on Sclerotoid Germination
When transferred to fresh PDA culture medium, the overwintered sclerotia
of S. sclerotiorum in control dishes gradually germinated within
24~48 h and their radial mycelium could overgrow the whole PDA dishes
within four days after treatment (Fig. 3). However, the
sclerotia in treatment plates full of antifungal volatiles could not germinate
at all even if cultured on fresh PDA for seven days. The prevention of
overwintering sclerotoid germination is the key in the control of S.
sclerotiorum, which would reduce the apothecial formation, further
decimate ascospore infection and would consequently reduce plant diseases
(Abawi and Grogan, 1979). Therefore, if these volatiles could be applied
in greenhouses, the diseases caused by S. sclerotiorum would be
first suppressed effectively.
Determination and Analysis of Antifungal VOCs from G8
Eventually, a 50/30 μm CAR/DVB/PDMS fiber was chosen to
extract the VOCs from G8, because of the most peak numbers
and peak area of extracted compounds (Fig. 4). Totally,
thirty organic compounds were determined by SPME-GC/MS, including alkyls,
alcohols, esters, ketones, organic acid, amine, oxime, phenols as well
as some heterocyclic compounds (Fig. 4, Table
1).
Mass spectra were obtained using the scan modus (total ion count, 30-350 m/z). The confirmation of compound identity was done by comparison of the retention time and mass spectra with those in the NIST05 library (similarity index >80, Fig. 5).
Here we selectively focused on these small organic molecules (molecular mass <300) that characteristically have a high vapour pressure and easily volatilize.
| Fig. 3: | Inhibition effects of bacterial volatiles against sclerotoid germination of S. sclerotiorum in sealed dishes. The sclerotia exposed to the antifungal volatiles from G8 could not germinate at all (B) even if cultured on fresh PDA for seven days (The diam of radial mycelium was 0 mm). However, the sclerotia in control dishes germinated normally and the mean diam of the radial mycelium had reached 21 mm (A) after cultured for four days |
| Fig. 4: | GC profiles of bacterial volatile. These headspace volatiles were extracted by SPME and analyzed by a GC/MS-QP2010 from Shimadzu. The determined peaks of organic compounds were orderly numbered from 1 to 30 |
| Fig. 5: | GC-MS analysis of 3-Octanone. The mass spectrum of determined target and the compound 3-Octanone with 95% similar index searched by NIST05 Library was shown as A and B, respectively |
Such VOCs are ideal infochemicals because they can act over a wide range of distances and their spheres of activity will extend from proximal interactions to greater distances via diffusion in air, including in soil pores (Wheatley, 2002).
It seems that volatiles produced by G8 contain more than one kind of bioactive compounds and the different compounds may determine the different antagonistic natures of bacterial volatiles. Gu et al. (2007) found that phenol, 2-octanol, 2-nonanone and 2-undecanone displayed 100% nematicidal activities to both free-living nematode Panagrellus redivivus and pinewood nematode Bursaphelenchus xylophilus. Among those, phenol is known for its toxic effects on cells and has been used as an antiseptic in clinical applications for a long time. Besides, benzothiazole, cyclohexanol, 2-ethyl-1-Hexanol and nonanol could completely inhibit the mycelial growth of S. sclerotiorum (Fernando et al., 2005), but some ketones like 2-udecanone and 2-tridecanone had no inhibition. In our study, such effective compounds were also determined, whereas the antifungal natures of other tested novel compounds (such as oxime and morpholine) need to be further studied.
| Table 1: | VOCs from B. subtilis G8 determined by SPME-GC/MS method |
The analysis for these novel structures could give us a revelation that some of the bacterial volatiles could be used as main skeleton for developing novel fungistatic or nematicidal agents by further chemical modifications.
The B. subtilis G8 strain cultured in the TSB-YE medium with rich nutrition produced different bioactive compounds, therefore, such antagonistic volatiles-producing bacterium may have the potential to be effective biocontrol agents against soil-borne pathogens (such as fungi and nematodes) and less likely to select for resistance than synthetic fungicides composed of a single compound. However, the application of bacterial volatiles in the control of pathogens in greenhouse was under way.
In addition, the method used in this study may be useful for the extraction and identification of volatile metabolites produced by other microorganism (bacteria or fungi etc).
ACKNOWLEDGMENTS
This research was supported by National Key Technology R and D Program of China (2006BAD08A03) and by Shandong Agricultural University Postdoctor Foundation.