J Ind Microbiol Biotechnol (2009) 36:695–704

DOI 10.1007/s10295-009-0539-6

ORIGINAL PAPER

Microbial conversion and in vitro and in vivo antifungal

assessment of bioconverted docosahexaenoic acid (bDHA)
used against agricultural plant pathogenic fungi
Vivek K. Bajpai Æ Hak Ryul Kim Æ Ching Tsang Hou Æ
Sun Chul Kang

Received: 14 October 2008 / Accepted: 27 January 2009 / Published online: 4 March 2009
Ó Society for Industrial Microbiology 2009

Abstract Microbial modification of polyunsaturated fatty revealed a promising antifungal effect in vivo as compared
acids can often lead to special changes in their structure and to the positive control oligochitosan. Furthermore, elabo-
in biological potential. Therefore, the aim of this study was rative study of GC-MS analysis was conducted on
to develop potential antifungal agents through the microbial bioconverted oil extract of DHA to identify the transfor-
conversion of docosahexaenoic acid (DHA). Bioconverted mation products present in bDHA. The results of this study
oil extract of docosahexaenoic acid (bDHA), obtained from indicate that the oil extract of bDHA has potential value of
the microbial conversion of docosahexaenoic acid (DHA) industrial significance to control plant pathogenic fungi.
by Pseudomonas aeruginosa PR3, was assessed for its in
vitro and in vivo antifungal potential. Mycelial growth Keywords Docosahexaenoic acid
Bioconversion

inhibition of test plant pathogens, such as Botrytis cinerea, Antifungal activity
Plant pathogens

Colletotrichum capsici, Fusarium oxysporum, Fusarium Pseudomonas aeruginosa PR3
solani, Phytophthora capsici, Rhizoctonia solani and Scle-
rotinia sclerotiorum, was measured in vitro. bDHA
(5 ll disc-1) inhibited 55.30–65.90% fungal mycelium Introduction
radial growth of all the tested plant pathogens. Minimum
inhibitory concentrations (MICs) of bDHA against the Bioconversion is a ‘‘green’’ technology that converts
tested plant pathogens were found in the range of 125– fatty acids into entirely new chemical compounds with
500 lg ml-1. Also, bDHA had a strong detrimental effect antimicrobial, industrial or biomedical properties. The bio-
on spore germination for all the tested plant pathogens. conversion reactions by Pseudomonas aeruginosa PR3 have
Further, three plant pathogenic fungi, namely C. capsici, been cited extensively among microbial systems that pro-
F. oxysporum and P. capsici, were subjected to an in vivo duce mono-, di- and tri-hydroxy fatty acid derivatives from
antifungal screening. bDHA at higher concentrations unsaturated fatty acids [15]. Strain PR3, isolated from a
waste water stream on a pig farm in Morton, Illinois, USA,
was found to convert oleic acid to a novel compound, 7,10-
V. K. Bajpai
S. C. Kang (&)
dihydroxy-8(E)-octadecenoic acid (DOD), which inhibits
Department of Biotechnology, Daegu University,
Kyoungsan, Kyoungbook 712-714, Republic of Korea the laboratory growth of Candida albicans, a yeast that
e-mail: [email protected] sometimes causes thrush and other infections in humans [9].
This strain was also found to convert ricinoleic acid to
H. R. Kim
another novel compound, 7,10,12-trihydroxy-8(E)-octa-
Department of Animal Science and Biotechnology,
Kyoungpook National University, Daegu 702-701, decenoic acid (TOD), which inhibits the rice blast fungus,
Republic of Korea raising the prospect for a biological fungicide against this
pathogen [14]. These recently described compounds from
C. T. Hou
the microbial conversion of unsaturated fatty acids are
Department of Agriculture, Microbial Properties Research Unit,
National Centre for Agricultural Utilization Research potential value-added products that can inhibit or can be used
(USDA/MPRU/NCAUR), Peoria, IL, USA in the biocontrol of certain important plant pathogens.

123
696 J Ind Microbiol Biotechnol (2009) 36:695–704

A number of fatty acids have been shown to possess anti- Materials and methods
microbial activities. Recently, our research group reported
that bioconverted DHA showed strong antibacterial activity Microorganisms
against Staphylococcus aureus, Bacillus subtilis, Listeria
monocytogenes and Pseudomonas aeruginosa [25]. How- The bacterial strain Pseudomonas aeruginosa PR3 was
ever, information on the antifungal effect of unsaturated fatty provided by Dr. Ching Tsang Hou from United States
acids is scant, and especially so for information relating to Department of Agriculture, Microbial Properties Research
the plant pathogenic fungi. Unit, National Center for Agricultural Utilization Research
On the other hand, harvest losses due to fungal diseases (USDA/MPRU/NCAUR), Peoria, IL, and grown at 28°C
in the world crop production may amount to 12% or even aerobically at 200 rpm on screening medium containing
higher in developing countries [2, 24]. Research into the per liter 4 g dextrose, 2 g K2HPO4, 2 g (NH4)2 HPO4, 1 g
derivation of fungicides by microbial bioconversion of NH4NO3, 0.5 g yeast extract, 0.014 g ZnSO4, 0.01 g
unsaturated fatty acids is being intensified. It is evident that FeSO4
7H2O and 0.01 g MnSO4
7H2O [14].
microbially derived fungicides have enormous potential to The fungal cultures were obtained from the Korean
inspire and influence modern agro-chemical research [9, Agricultural Culture Collection (KACC, Suwon, South
14]. Furthermore, several studies reported that hydroxy Korea). Cultures of each fungal species were maintained on
fatty acids have antifungal activities [10, 12, 20]. A few potato dextrose agar (PDA) slants and stored at 4°C.
hydroxy fatty acids also exhibit cytotoxic activity against The fungal species used in the experiment were Botrytis
cancer cells [12, 25] and prostaglandin E-like activity [27]. cinerea KACC40573, Colletotrichum capsici KACC41078,
Recently, production of hydroxy fatty acids through bio- Fusarium oxysporum KACC41083, Fusarium solani
conversion by microorganisms has become a major focus KACC41092, Phytophthora capsici KACC40157, Rhizoc-
of research [8, 11]. tonia solani KACC40111 and Sclerotinia sclerotiorum
Omega-3 fatty acids are essential dietary cis-polyun- KACC41065.
saturated fatty acids (PUFAs). These PUFAs include
docosahexaenoic acid (C22: 6n - 3, DHA), eicosapenta- Fatty acid substrate and microbial conversion
enoic acid (C20: 5n - 3, EPA) and a-linolenic acid (C18:
3n - 3, LNA), associated with fish and marine vertebrates Docosahexaenoic acid (DHA) was purchased from Cay-
[16]. It has been well documented in our previous work that man Chemical Co. (Denver, CO). The purity of the fatty
one of the omega-3 fatty acids possessed a remarkable acid substrate was over 99%. Bioconversion was carried
antifungal effect to infections with certain plant pathogens out in screening medium [14]. A substrate concentration of
[4]. Therefore, in this study, microbial conversion of 0.5 ml of docosahexaenoic acid (DHA) in 50 ml culture
omega-3 fatty acids such as docosahexaenoic acid (DHA) medium or 1% (v/v) was added to 24-h-old culture of
has been exploited to produce new value-added hydroxy Pseudomonas aeruginosa PR3 and then continuously sha-
products to serve as a potential antifungal agent. ken for 2 days at 200 rpm in a Psycro Therm controlled
In addition to this, some of the plant pathogens focused environment shaker (New Brunswick Scientific, Edison,
on in this study, such as Botrytis cinerea, Colletotrichum NJ) at 28°C and for an additional 2–3 days to bring about
capsici, Fusarium oxysporum, Fusarium solani, Phyto- the conversion of the substrate fatty acid. At the end of
phthora capsici, Rhizoctonia solani and Sclerotinia conversion, the culture broth was acidified to pH 2.0 with
sclerotiorum, are more frequently reported as responsible 6 N HCl to kill the culture followed by immediate
for opportunistic plant fungal diseases associated with extraction twice with an equal volume of ethyl acetate and
marked industrial economic losses. Therefore, efforts have diethyl ether (9:1, v/v). The solvents used in this study
been focused on the significant evidence supporting the were obtained from Merck (Germany), and the purity of the
role of bioconverted oil extract of docosahexaenoic acid solvents was over 99.5%. Solvent evaporation from the
(bDHA) as a potential antifungal agent to control plant combined extract was achieved using a rotary evaporator
pathogenic fungi. In the present investigation, we report (EYELA N1000, Japan), and the bioconverted oil extracts
here the microbial conversion of docosahexaenoic acid of DHA were obtained [14].
(DHA) and, assessed in vitro and in vivo, the antifungal
efficacy of bioconverted oil extract of DHA as an industrial Gas chromatography-mass spectrometry (GC-MS)
potential against certain important plant pathogenic fungi analysis of bioconverted DHA
causing severe destruction to crop, vegetable and orna-
mental plants. Also, we analyzed the biotransformation The GC-MS analysis of bDHA was performed using a
products of bioconverted oil extract of DHA by GC-MS SHIMADZU GC-MS (GC-17A) equipped with a ZB-1 MS
analysis. fused silica capillary column (30 9 0.25 m i.d., film

123
J Ind Microbiol Biotechnol (2009) 36:695–704 697

thickness 0.25 lm). For GC-MS detection, an electron ion- In vitro antifungal susceptibility assessment assay
ization system with ionization energy of 70 eV was used.
Helium gas was used as the carrier gas at a constant flow rate The minimum inhibitory concentrations (MICs) of bio-
of 1 ml/min. Injector and MS transfer line temperature were converted oil extract of DHA (bDHA) were determined by
set at 220 and 290°C, respectively. The oven temperature twofold dilution method against B. cinerea, C. capsici,
was programmed from 50 to 150°C at 3°C min-1, then held F. oxysporum, F. solani, P. capsici and S. sclerotiorum
isothermal for 10 min and finally raised to 250°C at [22]. Four micro-liter oil samples of bDHA were dissolved
10°C min-1. Diluted samples (1/100, v/v, in methanol) of in 5% DMSO aseptically. This solution was serially diluted
1.0 ll were injected manually in the splitless mode. The with 5% DMSO and added to potato dextrose broth (PDB)
relative percentage of bDHA constituents was expressed as at final concentrations of 0, 62.5, 125, 250, 500 and
percentages by peak area normalization. 1,000 lg ml-1, respectively. Aliquots of 10-ll spore sus-
Identification of components of the biotransformation pensions (1.0 9 108 spores ml-1) of test strains were
products of bDHA was assigned by comparison of their inoculated in the test tubes in PDB medium and incubated
retention indices, relative to a series of n-alkane indices on for 2–7 days at 25°C. The standard reference drug, olig-
the ZB-1 capillary column and GC-MS spectra from the ochitosan, was used as positive control for the tested plant
Wiley 6.0 MS data and literature data [1]. pathogens, which was obtained from Sigma Chemicals (St.
Louis, MO). The minimum concentrations at which no
Preparation of fungal spore suspension visible growth was observed were defined as the MICs,
which were expressed in lg/ml. The MIC values represent
The fungi were grown on potato dextrose agar (PDA, Difco the results of three independent experiments.
laboratories, Detroit, MI) plates in dark at 25°C for
5–7 days, after which time spores were harvested from In vitro spore germination assay
sporulating colonies and suspended in sterile distilled water
containing 0.1% (v/v) Tween 20. The concentrations of For spore germination assay of the different fungi, including
spores in suspension were determined using a hematocy- Botrytis cinerea, Fusarium oxysporum, Sclerotinia sclero-
tometer and adjusted to 1.0 9 108 spores ml-1. tiorum, Colletotrichum capsici, Fusarium solani and
Phytophthora capsici [18], test samples (2 ll) were dis-
In vitro antifungal activity assessment assay solved in 5% DMSO to obtain 50, 100, 200, 300, 400 and
500 lg ml-1 concentrations of bioconverted oil extract of
The antifungal activity of bioconverted oil extract of DHA, where the final concentration of DMSO was 0.5%.
docosahexaenoic acid (bDHA) was assessed by disc dif- The samples were inoculated with spore suspension of each
fusion method using potato dextrose agar (PDA) in 9-cm fungal pathogen containing 1.0 9 108 spores ml-1. From
petri dishes [6]. Sterile Whatman paper discs of 6 mm this, aliquots of 10-ll spore suspension from each were
diameter were pierced in the agar, equidistant and near placed on separate glass slides in triplicate. Slides con-
the border, where 5-ll disc-1 oil extract of bDHA was taining the spores were incubated in a moisture chamber at
used separately. An agar plug of fungal inoculum 6 mm 25°C for 24 h. Each slide was then fixed in lactophenol-
in diameter was removed from a 10-day-old previous cotton blue and observed under the microscope for spore
culture of all the fungal strains tested and placed upside germination. The spore generated germ tubes were enu-
down in the center of the petri dishes. The plates were merated, and the percentage of spore germination was
incubated at 25°C for 5–7 days, until the growth in the calculated. The control (0.5% DMSO) was tested separately
control plates reached the edges of the plates. Controls for spore germination of different fungi.
were prepared in the same solvent (DMSO) employed to
dissolve the sample. Mycelial growth inhibition of each
fungal strain was calculated as the percentage of inhibi- In vivo antifungal activity assessment assay
tion of radial growth relative to the control. Three
replicate plates were used for each treatment. Based on the in vitro susceptibility, three plant diseases, leaf
The radial growth inhibition of treatment compared to spot of pepper caused by Colletotrichum capsici, tomato
control was calculated by percentage, using the following vascular wilt and necrosis caused by Fusarium oxysporum
formula: and leaf scorch of pepper caused by Phytophthora capsici,
were evaluated in vivo. The in vivo antifungal activity of
Inhibition (%Þ ¼ ½1  Radial growth of treatment (mm)= test samples was determined by a whole plant method in a
Radial growth of control (mm)]  100 greenhouse, as described previously [17].

123
698 J Ind Microbiol Biotechnol (2009) 36:695–704

In brief, for in vivo experimental design, the seeds of Percent inhibition (%Þ ¼ ½ðA  BÞ=A  100;
the tested plants were sown in separate pots in August
2007 in a greenhouse. The tested plants, possessing an where A and B represent the disease area on the untreated
average of four to six and six to nine leaves for pepper and treated plants, respectively.
and tomato plants, respectively, were kept under the
following greenhouse conditions: Day and night temper-
atures of 70–82°F and 62–64°F, optimum for tomato and Results
pepper plants, respectively, were maintained. This was
because, during cloudy weather, a temperature closer to GC-MS analysis of bDHA
the lower end of these ranges is preferred, while in sunny
weather, temperatures closer to the higher end are better GC-MS analyses of the bioconverted oil extract of DHA
for pepper and tomato plants. Below 60°F, nutrient defi- (bDHA) led to the identification of 44 different biotrans-
ciencies may occur because plants cannot absorb some formation products. The identified compounds according to
elements at cool temperatures, indicating a lack of their elution order on a ZB-1 capillary column were found
phosphorus uptake (even though there may be adequate to be carbonochloridic acid, methoxymethane, hydrazine,
phosphorus in the nutrient solution). Where day temper- L-prolin, oxalic acid, luprisol, propionaldehyde, caproal-
atures might exceed 85–90°F, cooling equipment is dehyde, propanoic acid, valeric acid, 1,1-dimethoxyoctane,
needed to maintain the regular growth of the plants. cyclooctane, sulfurous acid, butyric acid, 1,2,3,4-undec-
Therefore, ideally, the thermostat was located at blossom anetetrol, 2-propenoic acid, butanedioic acid, 2,2,3-
height of the greenhouse for good temperature control. triethyloxirane, acrolein, tetrahydropyrrolo, thaizole,
The optimum relative humidity for greenhouse-grown cyclohexanecarboxylic acid, diethyl carbinol, cyclohexa-
tomato and pepper plants was maintained at 65–75%. none, butanoic acid, decanoic acid, 4-pentenyl butyrate,
Lower humidity can decrease growth and stimulate flower 1-heptene, cyclohexanol, silane, 1-butyne, birnenoel, tri-
production, creating a large fruit load at the expense of a fluoroacetic acid, dodecane, pentalene-1,5-dione, 3-decen-
strong, healthy plant. The higher light intensity required 1-ol, cyclopentaneundecanoic acid, ethyl isohexanoate,
for pepper and tomato plants was also maintained. For 1-pentanol, propylphosphonic acid, 4-heptenoic acid, hex-
that, the greenhouse was equipped with spectral fil- 4-enoic acid, butyldimethylsilanol and 4-methylhexyl
ters that can alter the red and far-red light balance of acetate (Figs. 1, 2a–c). The major components in the oil
sunlight. detected were carbonochloridic acid (2.99%), meth-
Further, to prepare the test solutions at the concentration oxymethane (40.6%), caproaldehyde (7.85%), 2-propenoic
of 1,500 lg ml-1, 4 ll of bDHA was dissolved in 5% acid (8.13%) and cyclohexanone (2.07%).
dimethyl sulfoxide (DMSO) followed by diluting it with
water containing a surfactant Tween 20 (200 lg ml-1), In vitro antifungal activity
where the final concentrations of DMSO and Tween 20
were 0.5 and 0.1%, respectively. The initial concentration As shown in Table 1, the bioconverted oil extract of DHA
of the test solution was 1,500 lg ml-1; further test dilu- (bDHA) at 5 ll disc-1 exhibited moderate to high anti-
tions of 500 and 300 lg ml-1 of bDHA were employed. fungal activity against B. cinerea (55.30%), C. capsici
For applying the test samples of bDHA, 4 ml of each test (60.30%), F. oxysporum (65.90%), F. solani (62.90%),
sample solution was sprayed onto each pot at the same P. capsici (64.10%), R. solani (65.60%) and S. sclerotio-
time. The plants treated with different concentrations of rum (56.70%). F. oxysporum and R. solani were found to
bDHA were kept in a greenhouse for 1 day before being be the most inhibited plant pathogens by bDHA. The
inoculated by each pathogen. Further, 6 ml of fungal spore control (5% DMSO) did not inhibit the growth of any of
suspension (1.0 9 108 spores ml-1) of each fungal strain the plant pathogens tested.
was sprayed onto each pot. Controls were sprayed with
DMSO, Tween 20, DMSO ? Tween 20 solutions and In vitro antifungal susceptibility
water, where the final concentrations of DMSO and Tween
20 were 0.5 and 0.1%, respectively. Oligochitosan was The minimum inhibitory concentrations (MICs), defined
used as reference positive control. The area of lesions on as the lowest concentrations of bDHA that resulted in
treated plants was measured in millimeters using a vernier complete growth inhibition of B. cinerea, C. capsici,
caliper. All tests were conducted in three replicates. F. oxysporum, F. solani, P. capsici and S. sclerotiorum,
The effect of antifungal efficacy of the test samples on were found to be 500, 250, 125, 500, 250 and 250 lg ml-1,
each disease was evaluated after 14 days as a percentage of respectively (Table 1). C. capsici, F. oxysporum, P. capsici
inhibition calculated by the formula: and S. sclerotiorum were found more susceptible to bDHA

123
J Ind Microbiol Biotechnol (2009) 36:695–704 699

Fig. 1 GC-MS chromatogram of bioconverted oil extract of docosahexaenoic acid (bDHA)

as compared to B. cinerea and F. solani. DMSO did At the initial concentration of 1,500 lg ml-1, bDHA
not affect the growth of test fungal strains at the concen- exhibited 100% antifungal effect against leaf scorch of
tration used in this study. In this study, bDHA exhibited pepper caused by P. capsici, wilt/necrosis of tomato caused
a higher antifungal effect than that of standard oligochi- by F. oxysporum and leaf spot of pepper caused by
tosan in regard to the plant pathogenic fungi tested C. capsici. Further dilutions of bDHA applied to the plants
(Table 1). were 500 and 300 lg ml-1. Also at the concentration of
500 lg ml-1, a strong antifungal effect of bDHA was
In vitro spore germination inhibition observed against all the plant pathogenic fungi with 100%
antifungal effect. Whereas bDHA at the concentration
The results obtained for bDHA from the spore germination of 300 lg ml-1 had a moderate antifungal effect against
assay of each of the test fungi are shown in Fig. 3. DMSO C. capsici, F. oxysporum and P. capsici demonstrated
(0.5%, v/v) as a control did not inhibit the spore germi- respective percentages of inhibition of 40, 48 and 25%
nation of any of the plant pathogens tested. There was a (Table 2). It was observed that the antifungal effect of
significant inhibition of fungal spore germination by dif- bDHA was rapid and exhibited a remarkable antifungal
ferent concentrations of bDHA. A 100% inhibition of effect at higher concentration as compared to reference
fungal spore germination was observed in F. oxysporum standard oligochitosan; however, at low concentration a
and P. capsici at 500 lg ml-1 bDHA. bDHA also exhib- moderate antifungal effect was the characteristic feature of
ited a potent inhibitory effect on the spore germination of bDHA against the tested plant pathogens.
B. cinerea, C. capsici, F. solani and S. sclerotiorum in the
range of 50–90% at the concentrations ranging from 400 to
500 lg ml-1. Discussion

In vivo antifungal activity This study describes the qualitative and quantitative
antifungal effect of bioconverted oil extract of docosa-
In vivo antifungal activity of bioconverted oil extract of hexaenoic acid (bDHA). It appeared that bDHA at the
docosahexaenoic acid (bDHA) against the tested plant applied concentrations exhibited a wide range of antifungal
pathogens was assessed by the presence or absence of activity in vitro, affecting different plant pathogenic fungi.
disease area on the tested plants (Fig. 4). According to the Docosahexaenoic acid (DHA) as a negative control had no
results given in Table 2, the bDHA exhibited a wide range antifungal effect (data not shown). However, DHA has
of antifungal activity against all the plant pathogens tested, several potential applications in health promotion and
whereas docosahexaenoic acid (DHA) as a negative control disease prevention. A number of organizations have made
had no antifungal effect (data not shown). Also, the blind specific recommendations for the general population to
controls, such as DMSO (0.5%), Tween 20 (0.1%), increase its intake for nutrition. In response to and along
DMSO ? Tween 20 and water, did not inhibit any of the with these recommendations, DHA is being incorporated
fungal strains tested. into nontraditional food sources because of advances in the

123
700 J Ind Microbiol Biotechnol (2009) 36:695–704

Fig. 2 a Expanded GC-MS chromatogram (for 2–10 min) of bio- docosahexaenoic acid (bDHA). c Expanded GC-MS chromatogram
converted oil extract of docosahexaenoic acid (bDEA). b Expanded (for 20–30 min) of bioconverted oil extract of docosahexaenoic acid
GC-MS chromatogram (for 10–20 min) of bioconverted oil extract of (bDHA)

123
J Ind Microbiol Biotechnol (2009) 36:695–704 701

Table 1 Antifungal activity of bioconverted docosahexaenoic acid (bDHA) against the tested plant pathogens
Fungal pathogen bDHAa MIC (lg ml-1)
Radial growth (mm)b Radial growth inhibition (%)c bDHAd STDe

Botrytis cinerea (KACC40573) 20.35 ± 0.57 55.30 ± 0.01 500 1,640

Collectotrichum capsici (KACC41078) 17.95 ± 0.73 60.30 ± 0.02 250 [2,000
Fusarium oxysporum (KACC41083) 15.55 ± 0.55 65.90 ± 0.01 125 [2,000
Fusarium solani (KACC41092) 16.93 ± 0.68 62.90 ± 0.02 500 [2,000
Phytopthora capsici (KACC40157) 16.38 ± 0.33 64.10 ± 0.07 250 580
Rhizoctonia solani (KACC40111) 15.70 ± 0.67 65.60 ± 0.01 na na
Sclerotinia sclerotiorum (KACC41065) 19.65 ± 0.74 56.70 ± 0.01 250 1,500
Inhibition ratio (%) = 1 - [Radial growth of treatment (mm)/Radial growth of control (mm)] 9 100
DMSO (5%) as negative control (no antifungal effect); DHA and P. aeruginosa PR3 culture broth served as controls (no antifungal effect)
na Not applicable
Values are given as mean ± SD of three experiments
a
Bioconverted docosahexaenoic acid (tested volume 5 ll disc-1)
b
Radial growth of fungal pathogens
c
Radial growth inhibition percentage
d
Minimum inhibitory concentration
e
Oligochitosan (Standard positive control)

Fig. 3 Inhibitory effect of different concentrations (lg ml-1) of bioconverted docosahexaenoic acid (bDHA) on spore germination of tested
plant pathogens

technology to safely enrich the food supply in various inhibitory effect against all the plant pathogens tested.
industries of microbiology and biotechnology [28]. In Earlier in vivo studies on the analysis of the antifungal
addition, DHA has been found to show anti-inflammatory effect of various crude extracts showed that they had
effects, suppress interleuckin-1b, tumor necrosis factor-a varying degrees of antifungal effect against different plant
and interleukin-6 [26]. Over the last decades, an increasing pathogenic fungi [29]. Our study revealed similar results of
body of evidence has been accumulated on the beneficial antifungal effect of bDHA in vivo against the tested plant
effect of polyunsaturated fatty acids both in primary and pathogens under greenhouse conditions, and these results
secondary prevention of cardiovascular diseases [23]. Also were in agreement with our previous findings [4].
DHA reduces ambulatory blood pressure (BP) and heart Lee et al. reported that Piper longum-derived various
rate (HR) in hyperlipidemic men, which potentially crude extracts of methanol, chloroform and hexane exhib-
implicates its importance for human nutrition and the food ited a 100% antifungal effect against wheat leaf rust caused
industry [21]. Further, this research work also describes the by Puccinia recondita, 50% antifungal effect against
complex effect of bDHA on fungal spore germination and tomato late blight caused by Phytophthora infestans and
exhibited a wide range of antifungal activity. Regarding the 33% antifungal effect against rice blast disease caused by
in vivo antifungal effect, bDHA exhibited a remarkable Pyricularia grisea, respectively [17]. These results suggest

123
702 J Ind Microbiol Biotechnol (2009) 36:695–704

Fig. 4 In vivo antifungal

activity of bioconverted
docosahexaenoic acid (bDHA)
against the tested plant
pathogens. Arrows showing
antifungal effect at respective
concentrations of 1,500 and
500 lg ml-1 as 100% control
value, whereas at 300 lg ml-1,
arrows showing moderate
severity of the diseases caused
by Colletotrichum capsici,
Phytophthora capsici and
Fusarium oxysporum,
respectively

the availability of various crude extracts from different assumed that some of the minor components present in the
origins for trials in controlling plant fungal diseases under oil extract of bioconverted DHA might be responsible for
greenhouse conditions. Certain extracts from different ori- exerting this antifungal effect involving some type of syn-
gins act in many ways on various types of disease complex ergism with other active components of bDHA [19].
and may be applicable in the same way against agricultural Moreover, information on the antifungal effects of
plant pathogenic fungi as other agricultural chemicals. bDHA is scant, and these results show, for the first time,
bDHA can also be used as a leading factor in a wide range that bDHA possesses a substantial antifungal effect against
of activities against many phytopathogens, where these Botrytis cinerea (grey mold rot in grapes), Collectotrichum
pathogens have developed resistance against the specific capsici (leaf spot in pepper), Fusarium oxysporum (vas-
fungicides (benzimidazoles, dicarboximides, diethofuncar- cular wilt and necrosis in tomato), Fusarium solani (fruit
band and the sterol biosynthesis inhibitors) [7], resulting in rot in varied hosts), Phytophthora capsici (leaf scorch in
serious environmental damage. Among these pathogens, pepper), Rhizoctonia solani (stem canker pathogen on
wilt disease of cotton caused by F. oxysporum has been potato) and Sclerotinia sclerotiorum (water-soaked spots
reported to be very destructive to crops under field condi- and dry lesions on leaf, stalk and stem on bean and cab-
tions in China, where about 4 million hectares of cotton, bage). These pathogens are responsible for serious
accounting for 20% of the world’s total production, is economic losses in various parts of the world, and,
grown annually [5]. In our study, it has become clear that although control measures are available, they are of limited
bDHA possesses great potential to strongly inhibit the effectiveness [3]. As a result, work on alternative approa-
growth of F. oxysporum along with other plant pathogenic ches to control such pathogens is important. Thus, this
fungi tested. These activities could be attributed to the research would be worthy as an important applied micro-
presence of major biotransformation products of bDHA, bial approach to inhibit such severe plant pathogens. It
such as carbonochloridic acid, methoxymethane, caproal- would also be interesting to study the effects of biocon-
dehyde, 2-propenoic acid and cyclohexanone. It can also be verted docosahexaenoic acid (bDHA) on medically

123
J Ind Microbiol Biotechnol (2009) 36:695–704 703

Table 2 In vivo antifungal activity of bioconverted docosahexaenoic acid (bDHA) against the tested plant pathogens
Material Percentage of inhibition (%)a
Concentrationb LSPc LSHPd WNTe

bDHAf 1,500 100 ± 0.00 100 ± 0.00 100 ± 0.00

500 100 ± 0.00 100 ± 0.00 100 ± 0.00
300 40 ± 1.15 25 ± 1.52 48 ± 1.52
g
Oligochitosan 5% 62 ± 1.32 58 ± 1.20 84 ± 2.10
PCh – 0 0 0
DMSOi 0.5% 0 0 0
Tween 20j 0.1% 0 0 0
DMSO ? Tween 20k 0.5% ? 0.1% 0 0 0
Waterl 0.1% 0 0 0
Values are mean ± SEM of triplicate experiments
a
100%: Complete inhibition; 0%: no inhibition
b
lg ml-1
c
LSP leaf spot of pepper (Colletotrichum capsici)
d
LSHP leaf scorch of pepper (Phytopthora capsici)
e
WNT Wilt/necrosis of tomato (Fusarium oxysporum)
f
Bioconverted docosahexaenoic acid
g
Oligochitosan (positive control)
h
No treatment
i
Dimethyl sulfoxide (negative control)
j
Tween 20 (negative control)
k
Dimethyl sulfoxide ? Tween 20
l
Water

important fungi for development of new antifungal agents products present in bDHA and on the elucidation of their
for preventive treatment of serious fungal disease infec- antifungal mechanism.
tions in animals and human beings along with plant fungal
diseases.
In addition to this, microbial conversions of unsaturated References
fatty acids have yielded a diverse range of products that can
be used potentially as value-added products [13, 14]. To 1. Adam RP (2001) Identification of essential oil components by gas
enhance the potential of microbial conversion as an chromatography/quad-rupole mass spectroscopy. Allured Pub-
lishing Corporation, Carol stream
industrial approach to generate new antifungal agents, it is
2. Agrios GN (1997) Significance of plant diseases. In: Plant
necessary to improve the productivity and efficiency by pathology, 4th edn. Academic Press, San Diego, pp 25–37
developing better microbial strains and more efficient 3. Anon (2001) Crop protection compendium. CAB International,
bioconversion processes. In order to produce bioconverted Wallingford
4. Bajpai VK, Shin SY, Kim HR, Kang SC (2008) Anti-fungal
oil extracts of DHA in large quantity and to render the
action of bioconverted eicosapentaenoic acid (bEPA) against
bioprocess feasible and practical, we need to further plant pathogens. Ind Crops Prod 27(1):136–141. doi:10.1016/
improve the conversion rate and the process cost. j.indcrop.2007.07.014
In conclusion, our results suggest that the use of bDHA 5. Dong H, Zhang X, Choen Y, Zhoua Y, Li W, Li Z (2006) Dry
mycelium of Penicillium chrysogenum protects cotton plants
can be considered as an antifungal availability for trials in
against wilt diseases and increases yield under field conditions.
controlling plant safety standards. Therefore, bDHA can be Crop Prot 25:324–330. doi:10.1016/j.cropro.2005.05.003
a potential industrial product to control plant pathogenic 6. Duru ME, Cakir A, Kordali S, Zengin H, Harmandar M, Izumi S,
fungi causing severe destruction to crops and vegetables Hirata T (2003) Chemical composition and antifungal properties
of essential oils of three Pistacia species. Fitoterapia 74:170–176.
with potential applications in the agro- and/or food indus-
doi:10.1016/S0367-326X(02)00318-0
try. However, further studies should focus on the isolation 7. Elad Y (1991) Multiple resistance to benzimidazoles dicarboxi-
and structural determination of individual bioconverted mides and diethofencarb in field isolates of Botrytis cinerea in

123
704 J Ind Microbiol Biotechnol (2009) 36:695–704

Israel. Plant Pathol 41:41–46. doi:10.1111/j.1365-3059.1992. metabolites against the green mold pathogen (Penicillium digit-
tb02314.x atum Sacc.) of citrus fruit. Postharvest Biol Technol 48:113–121.
8. Hou CT (1995) Microbial oxidation of unsaturated fatty acids. doi:10.1016/j.postharvbio.2007.09.024
Adv Appl Microbiol 41:1–23. doi:10.1016/S0065-2164(08) 19. Marino M, Bersani C, Comi G (2001) Impedance measurements
70306-X to study the antimicrobial activity of essential oils from Lami-
9. Hou CT, Bagby MO (1991) Production of a new compound, 7,10- aceace and Compositae. Int J Food Microbiol 67:187–195. doi:
dihydroxy-8(E)-octadecenoic acid from oleic acid by Pseudo- 10.1016/S0168-1605(01)00447-0
monas sp. PR3. J Ind Microbiol 7:123–130. doi:10.1007/ 20. Masui H, Kondo T, Kojima M (1989) An antifungal compound,
BF01576074 9,12,13-trihydroxy-(E)-octadecenoic acid, from Colocasia an-
10. Hou CT, Forman RJ (2002) Growth inhibition of plant pathogenic tiquorum inoculated with Ceratocytis fimbriata. Phytochemistry
fungi by hydroxy fatty acids. J Ind Microbiol Biotechnol 24:275– 28:2613–2615. doi:10.1016/S0031-9422(00)98051-8
276. doi:10.1038/sj.jim.2900816 21. Mori TA, Bao DQ, Burke V, Puddey IB, Beilin LJ (1999)
11. Hou CT, Hosokawa M (2005) Value-added industrial products Docosahexaenoic acid but not eicosapentaenoic acid lowers
through biocatalysis: production of novel fatty acids. In: Hou CT ambulatory blood pressure and heart rate in humans. Hyperten-
(ed) Handbook of industrial biocatalysis. Taylor & Francis, New sion 34:253–260
York, pp 1–25 22. Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolke RH (1995)
12. Kato T, Yamaguchi Y, Abe N, Uyehera T, Nakai T, Yamnaka S, Manual of clinical microbiology. ASM, Washington
Harada N (1984) Unsaturated hydroxy fatty acids, the self- 23. Nannicini F, Sofi F (2006) Alpha-linolenic acid and cardiovas-
defensive substances in rice plant against blast disease. Chem cular diseases omega-3 fatty acids beyond eicosapentaenoic acid
Lett 25:409–412. doi:10.1246/cl.1984.409 and docosahexaenoic acid. Minerva Cardioangiol 54:431–442
13. Kim HR, Jang YS, Hou CT (2001) Effect of metal ions on the 24. Oreke EC, Dehne HW, Schonbeck F, Weber A (1994) Crop
production of isomeric 9, 10, 13 (9, 12, 13)-trihydroxy-11E(10E)- production and crop protection: estimated loses in major food and
octadecenoic acid from linoleic acid by Pseudomonas aeruginosa cash crops. Elsevier, Amsterdam
PR3. Enzyme Microb Technol 25:109–115 25. Shin SY, Bajpai VK, Kim HR, Kang SC (2007) Antibacterial
14. Kuo TM, Manthey LK, Hou CT (1998) Fatty acid bioconversions activity of bioconverted eicosapentaenoic (EPA) and docosa-
by Pseudomonas aeruginosa PR3. J Am Chem Soc 75:875–879. hexaenoic acid (DHA) against foodborne pathogenic bacteria. Int
doi:10.1007/s11746-998-0240-3 J Food Microbiol 113:233–236. doi:10.1016/j.ijfoodmicro.
15. Kuo TM, Kim H, Hou CT (2001) Production of a novel com- 2006.05.020
pound, 7, 10, 12-trihydroxy-8(E)-octadecenoic acid from 26. Simopoulos AP (2006) Evolutionary aspects of diet, the omega-6/
ricinoleic acid by Pseudomonas aeruginosa PR3. Curr Microbiol omega-3 ratio and genetic variation: nutritional implications for
43:198–203. doi:10.1007/s002840010287 chronic diseases. Biomed Pharmacother 60:502–507. doi:10.1016/
16. Leaf A, Kang JX, Xiao YF, Billman GE (2003) Clinical preven- j.biopha.2006.07.080
tion of sudden cardiac death by n - 3 polyunsaturated fatty acids 27. Wang JZ, Wang FB (1996) Studies on chemical constituents of
and mechanism of prevention of arrhythmias by n - 3 fish oils. Codonopsis pilosula. Nat Prod Res Dev 8:8–12
Circulation 107:2646–2652. doi:10.1161/01.CIR.0000069566. 28. Whelan J, Rust C (2006) Innovative dietary sources of n-3 fatty
78305.33 acids. Annu Rev Nutr 26:75–103. doi:10.1146/annurev.nutr.25.
17. Lee SE, Park BS, Kim MK, Choi WS, Kim HT, Cho KY, Lee SG, 050304.092605
Lee HS (2001) Antifungal activity of pipernonaline, a piperidine 29. Yoo JK, Ryu KH, Kwon JH, Ahn YJ (1998) Antifungal activities
alkaloid derived from long pepper, Piper longum L., against of oriental medicinal plant extracts against phytopathogenic
phytopathogenic fungi. Crop Prot 20:523–528. doi:10.1016/ fungi. Korean J Agric Chem Biotechnol 41(8):600–604
S0261-2194(00)00172-1
18. Leelasuphakul W, Hemmanee P, Chuenchitt S (2008) Growth
inhibitory properties of Bacillus subtilis strains and their

123