Antifungal Activity of Diketopiperazines and Stilbenes Against Plant Pathogenic Fungi In Vitro S. Nishanth Kumar & Bala Nambisan Received: 15 July 2013 / Accepted: 30 September 2013 / Published online: 12 October 2013 # Springer Science+Business Media New York 2013 Abstract The present study aimed to investigate antifungal activity of a stilbene and diketopiperazine compounds against plant pathogenic fungi, including Phytophthora capsici, P. colocasiae, Botrytis cinerea and Colletotrichum gloeosporioides. Minimal inhi- bition concentrations (MIC) and minimal fungicidal concentrations (MFC) of stilbenes and diketopiperazines for each fungus were determined using microplate method. Best activity was recorded by stilbenes against P. capsici and P. colocasiae. All four test compounds were effective in inhibiting different stages of the life cycle of test fungi. Stilbenes were more effective than diketopiperazines in inhibiting mycelial growth and inhibiting different stages of the life cycle of P. capsici and P. colocasiae. Rupture of released zoospores induced by stilbenes was reduced by addition of 100 mM glucose. The effects of stilbenes on mycelial growth and zoospore release, but not zoospore rupture, were reduced largely when pH value was above 7. In addition, stilbenes were investigated for its antifungal stability against Phytophthora sp. The results showed that stilbenes maintained strong fungistatic activity over a wide pH range (pH 4–9) and temperature range (70–120 °C). The compound stilbenes exhibited strong and stable broad-spectrum antifungal activity, and had a significant fungi- cidal effect on fungal cells. Results from prebiocontrol evaluations performed to date are probably useful in the search for alternative approaches to controlling serious plant pathogens. Keywords Stilbenes . Plant pathogen . Phytophthora sp . Life cycle Introduction Modern environmentally sound plant disease management constantly requires new low- toxicity, anti-polluting antifungal agents that differ from the fungicides currently developed in their mode of action and chemical properties [1]. Moreover, fungal plant pathogens readily develop resistance against existing systemic fungicides, resulting in great difficulty in practi- cally controlling key plant diseases in agriculture. Plants are constantly exposed and threatened by a variety of pathogenic microorganisms present in their environments. Diseases caused by Appl Biochem Biotechnol (2014) 172:741–754 DOI 10.1007/s12010-013-0567-6 S. N. Kumar : B. Nambisan (*) Division of Crop Protection/Division of Crop Utilization, Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram 695017, India e-mail: [email protected]
Transcript
Antifungal Activity of Diketopiperazines and StilbenesAgainst Plant Pathogenic Fungi In Vitro
S. Nishanth Kumar & Bala Nambisan
Received: 15 July 2013 /Accepted: 30 September 2013 /Published online: 12 October 2013# Springer Science+Business Media New York 2013
Abstract The present study aimed to investigate antifungal activity of a stilbene anddiketopiperazine compounds against plant pathogenic fungi, including Phytophthoracapsici, P. colocasiae, Botrytis cinerea and Colletotrichum gloeosporioides. Minimal inhi-bition concentrations (MIC) and minimal fungicidal concentrations (MFC) of stilbenes anddiketopiperazines for each fungus were determined using microplate method. Best activitywas recorded by stilbenes against P. capsici and P. colocasiae. All four test compounds wereeffective in inhibiting different stages of the life cycle of test fungi. Stilbenes were moreeffective than diketopiperazines in inhibiting mycelial growth and inhibiting different stagesof the life cycle of P. capsici and P. colocasiae. Rupture of released zoospores induced bystilbenes was reduced by addition of 100 mM glucose. The effects of stilbenes on mycelialgrowth and zoospore release, but not zoospore rupture, were reduced largely when pH valuewas above 7. In addition, stilbenes were investigated for its antifungal stability againstPhytophthora sp. The results showed that stilbenes maintained strong fungistatic activityover a wide pH range (pH 4–9) and temperature range (70–120 °C). The compound stilbenesexhibited strong and stable broad-spectrum antifungal activity, and had a significant fungi-cidal effect on fungal cells. Results from prebiocontrol evaluations performed to date areprobably useful in the search for alternative approaches to controlling serious plantpathogens.
Modern environmentally sound plant disease management constantly requires new low-toxicity, anti-polluting antifungal agents that differ from the fungicides currently developed intheir mode of action and chemical properties [1]. Moreover, fungal plant pathogens readilydevelop resistance against existing systemic fungicides, resulting in great difficulty in practi-cally controlling key plant diseases in agriculture. Plants are constantly exposed and threatenedby a variety of pathogenic microorganisms present in their environments. Diseases caused by
S. N. Kumar : B. Nambisan (*)Division of Crop Protection/Division of Crop Utilization, Central Tuber Crops Research Institute,Sreekariyam, Thiruvananthapuram 695017, Indiae-mail: [email protected]
plant pathogenic fungi significantly contribute to the overall loss in crop yield worldwide [2, 3].Moreover, fungi cause enormous economic loss in agriculture and the food industry bydestroying crops in the field and during storage [4]. In an effort to combat diseases, plantshave devised various mechanisms to fend off microbial invaders. However, despite theexistence of defense mechanisms, plants are exposed to attack by plant pathogenic fungi.Synthetic fungicides are the most common and effective method of disease control. But,concerns occurred by some chemical pesticides with potentially harmful effects for humanhealth and environment lead us to minimize these inputs [5].
For many years, a variety of different synthetic chemicals has been used as antifungalagents to inhibit the growth of various plant pathogenic fungi. However, there are a series ofproblems for the effective use of these chemicals in areas where the fungi have developedresistance [6, 7]. Thus, there is a growing interest on the research on the possible use ofnatural products such as microbial secondary metabolites, which may be less damaging forpest and disease control. Microbial metabolites exhibit a number of chemical and biologicaladvantages as fungicides. Microorganisms can synthesize secondary metabolites of versatilechemical structures with diverse biological activities that exceed the scope of syntheticorganic chemistry [8]. Therefore, finding new compounds that could be biodegradable andsafe to human health will be essential for the control of plant diseases. In recent years, therehas been a growing interest in the potential use of microbial metabolites as agrochemicals.Microbial metabolites are expected to overcome the problems associated with resis-tance of pests, and are generally more biodegradable and more environment friendlythan synthetic compounds [9]. Such products are relatively broad-spectrum,bioefficacious, economical, biodegradable and environmentally safe and can be idealcandidates for use as agrochemicals [10].
Phytophthora is a plant pathogen with about 50 species causing wide variety of diseaseon large number of host plants [11]. It causes root, stem and fruit rots resulting in severereductions in crop yield. Phytophthora blight, caused by the oomycete pathogenPhytophthora capsici, is one of the most important fungal diseases of pepper plants and iswidely distributed in worldwide [12–14]. Leaf blight of taro, caused by P. colocasiae is themost destructive disease of Colocasiae. It has become a limiting factor for taro production inall taro-growing countries causing yield loss of 25–30 % [15].
We have recently reported the antimicrobial stilbenes and diketopiperazines from Bacillussp. N strain associated with entomopathogenic nematode [16, 17]. This paper describes theantifungal activity of stilbenes and diketopiperazines against phytopathogens and morespecifically to P. capsici and P. colocasiae in vitro.
Materials and Methods
Media
Microbiological media (potato dextrose agar [PDA] and broth) were purchased from Hi-Media Laboratories Limited, Mumbai, India. All other reagents were of analytical grade, andthe other chemicals used in this study were of the highest purity.
Stilbenes, Diketopiperazines and Standard Antibiotics
The test diketopiperazines [cyclo-(L-Pro–L-Leu), cyclo-(D-Pro–L-Leu) and cyclo-(D-Pro–L-Tyr)and stilbene compounds [3,4′,5-trihydroxystilbene and 3,5-dihydroxy-4-isopropylstilbene]
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(Table 1) were isolated and purified from the cell free culture filtrate (Tryptic soyabroth) of a Bacillus sp. N strain associated with a novel EPN, Rhabditis (Oscheius)sp. and chemical structure of the compounds were established on the basis ofextensive spectral analyses [16, 17]. The standard antibiotics oligochitosan was pur-chased from Sigma Aldrich.
Test Fungus
P. capsici and P. colocasiae were kindly provided by Dr. M.L. Jeeva (principal scientist,Department ofMolecular Plant Pathology, Central Tuber Crops Research Institute, Trivandrum,India). Botrytis cinerea MTCC 359 and Colletotrichum gloeosporioides MTCC 2151 werepurchased from Microbial Type Culture Collection Centre, IMTECH, Chandigarh, India. Allfour fungal isolates were maintained on PDA (potato infusion from 200 and 20 g/l dextrose and15 g/l agar) in the dark at 25 °C.
Table 1 List of cyclic dipeptides and stilbenes used in this study
S. No compound Antimicrobial class Structure
1 Cyclo-(L-Pro-L-Leu) Cyclic dipeptide
2 Cyclo-(D-Pro-L-Leu) Cyclic dipeptide
3 Cyclo-(D-Pro-L-Tyr) Cyclic dipeptide
4 3,4',5-trihydroxystilbene Poly phenol
5 3,5-dihydroxy-4-isopropylstilbene Poly phenol
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Preparation of Spore Suspension and Test Samples
The spore suspensions of test pathogens obtained from 7-day-old cultures were prepared insterile distilled water. A haemocytometer was used to obtain a homogenous spore suspen-sion of 1×108 spores/ml. To prepare the stock solutions of test compounds, the compounds(2 mg/ml) were dissolved in dimethyl sulfoxide (DMSO). Samples with known weightswere further diluted with 5 % of the respective solvents used to prepare test samples, wherethe final concentration of the solvent was 0.5 % (v/v).
Determination of Minimum Inhibitory and Minimum Fungicidal Concentrations
A microplate method, as previously described [18], was used with slight modifications todetermine minimal inhibitory concentration (MIC) values of test compounds and antibiotic.Test compounds were serially diluted, ranging from 2,000 to 1 μg/ml whereas antibiotic wasserially diluted, ranging from 500 to 1 μg/ml. Serially diluted test compounds and antibioticwere mixed with 100 μl of the fungal spore suspension (2×106 spores/ml in fresh PDB). Themicroplates were incubated for 2–3 days at 27 °C with daily monitoring. All experimentswere done in triplicate. The MIC readings were performed spectrophotometrically with amicroplate reader at 600 nm. MICs values were calculated by comparing growth in controlwells and the blank, which consisted of uninoculated plates. The MIC of the compounds wasdefined as the lowest concentration of compounds that caused growth inhibition of morethan 90 % at 48 h, as compared to the control.
The in vitro minimum fungicidal activity (MFC) was determined as described by Espinel-Ingroff et al. [18]. After 72 h of incubation, 20 μl was subcultured from each well that showedno visible growth (growth inhibition of over 98 %), from the last positive well (growth similarto that for the growth control well), and from the growth control (extract-free medium) ontoPDA plates. The plates were incubated at 27 °C until growth was seen in the growth controlsubculture. The minimum fungicidal concentration was regarded as the lowest concentration ofthe test compound that did not yield any fungal growth on the PDA plates used.
Screening for Antifungal Activity
A 6-mm-diameter plug of each fungus was transferred to the center PDA plates and incubated at28 °C in the dark until the fungal colony was approximately 25 mm from the edge of the plate.The fungal bioassays were performed based on the paper disk diffusion method [19]. Eachpaper disk (6 mm diameter) was impregnated with MIC concentration of the test compounds.The loaded paper disks were placed on the PDA medium about 15 mm from the margin of thegrowing colony and 10 mm from the edge of the plate. The plates were incubated at 28 °C andthe diameter of zone of inhibition (distance between the center of the paper disk and the end ofclear zone) was recorded. The disk loaded by 50 μl solvent was considered a control. Theexperiments were performed in three replicates and repeated twice.
Effect of Stilbenes and Diketopiperazines on Each Life Stage of P. capsici and P. colocasiae
Mycelial Dry Weight
For the mycelial dry weight test, mycelia disks (6 mm in diameter) of P. capsici and P.colocasiae grown on PDA plates were cut from the margins of the colony and wereinoculated into PDB (potato dextrose broth) in a flask containing different concentrations
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of stilbenes and diketopiperazines (0, 25, 50, 100, 150, 200, 300, 400, 500 μg/ml). Flaskswere placed in a shaker at 25 °C, 120 rpm for 5 days. The mycelia were harvested at the endof the fifth day, filtered through pre-weighed filter paper, and followed by drying to aconstant weight at 80 °C for about 24 h in an oven, after which the yield of mycelial biomasswas measured. All the runs were replicated twice and the averaged values are presented inthe present work.
Sporulation
For the sporulation test, three mycelia disks (6 mm in diameter) were cut from the edge of theactively growing culture and were immersed in 25 ml sterile Petri solution (Ca(NO3)2: 0.4 g;KH2PO4: 0.5 g; Mg(NO3)2: 0.15 g; CaCl2: 0.06 g; distilled water: 1,000 ml) containing 0, 1, 2,4, 8, 16, 32, 64, 128 and 252 μg/ml stilbenes and diketopiperazines in plates. After 3 days ofincubation in the dark at 25 °C, the zoosporangia along the margins of each mycelial disk wereobserved with a light microscope (Carl Zeiss stereomicroscope, Stemi 200C, Germany).
Zoospore Release
The effect of stilbenes and diketopiperazines on zoospore release was tested in Eppendorftubes. Zoosporangia suspension (2×104 zoosporangia/ml) was obtained by the method ofZheng [20]. A total of 1 mg of the stilbenes and diketopiperazines solution was added to thetube containing 2 ml of the zoosporangia suspension to make the final concentrations ofstilbenes and diketopiperazines of 0, 25, 50, 100, 150, 200, 400 and 500 μg/ml. After theincubation at 25 °C for 30 min, approximately 100 zoosporangia were observed with thelight microscope, and the inhibition percentages of zoospore release of P. capsici and P.colacasia were calculated. The experiments were repeated twice with three replicates.
Cystospore Germination
For the determination of the effect of stilbenes and diketopiperazines on the cystosporegermination of P. capsici and P. colacasia, zoospore suspension (1×105 zoospore/ml) wasobtained by the method of Ozgonen and Erkilic [21]. Stilbenes and diketopiperazines wereadded to the suspension from the start of the incubation at 25 °C. Approximately 100cystospores were observed with a light microscope (microscope (Carl Zeiss stereomicro-scope, Stemi 200C, Germany) and the percentages of cystospore germination were calcu-lated. All experiments were repeated twice with three replicates.
Effect of ATP and Glucose on Cystospore Germination by Stilbenes
ATP (an energy supplier) or glucose (an osmotic stabilizer) was added to the zoosporangia orzoospore suspension to gain more information on mode of action of stilbenes using themethod described by Mitani et al. [22]. Briefly, ATP in Tris–HCl buffer (pH 7.3), or glucosein distilled water, was added to the zoosporangia or zoospore suspension at 10 min beforeaddition of stilbenes to give final concentrations of 20 and 100 mM, respectively.
Influence of pH on Antifungal Activity of Stilbenes
To evaluate the antifungal activity of stilbenes at different pH values closer to those in realsoil systems, the influence of pH on stilbenes activity was assayed by using P. capsici and P.
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colocasiae. The pH of PDB solutions containing test compounds (MIC concentration) wasadjusted to 4 to 9, with 5 N NaOH or 5 N HCl aseptically. All pH values were measured witha pH meter (Eutech pH 700; Thermo Scientific, Singapore). Each medium with different pHvalues was inoculated with 1 % volume of the fungal suspensions. The cultivation wascarried out on a test tube shaker at 28 °C for 72 h, while compounds free solutions withdifferent pH values were used as each control. The effect of pH on antifungal activity ofstilbenes was measured as the inhibitory rate of fungal growth. Triplicate set of experimentwere performed.
Effect of Temperature on Antifungal Stability of Stilbenes
Generally, antifungal activity of the antibiotics was associated with their stability, which wasusually influenced by temperature. Stilbenes were treated under different temperatures from−70 °C to 160 °C for 15, 30 and 60 min, respectively, by the MIC concentration. Fungalsuspensions were added to each medium and then cultivated at 28 °C for 72 h. Fungalgrowth was evaluated specific to the control fungi. The measurement of the inhibitory ratefor P. capsici and P. colocasiae was used to reflect the effect of temperature on antifungalstability of stilbenes and diketopiperazines. Each experiment was repeated three times.
Statistical Analysis
Analysis of variance for individual parameters was performed by Duncan’s multiple rangetests on the basis of mean values to determine the significance at P<0.05.
Results
MIC and MFC
MIC and MFC values were established for stilbenes and diketopiperazines and are shown inTable 2. Out of four phytopathogen tested, P. capsici and P. colocasiae were most sensitivefungi to stilbenes and diketopiperazines. P. capsici was most sensitive to stilbenes anddiketopiperazines with MIC values ranged from 2 to 64 μg/ml, followed by P. colocasiaewith MIC values ranged from 4 to 125 μg/ml. Best activity was recorded by 3,5-dihydroxy-4-isopropylstilbene against all test fungi, and a best MIC value of 2 μg/ml was recorded
Table 2 MIC and MFC values of diketopiperazines and stilbenes against fungi
Compounds P. capsici P. colocasiae B. cinerea C. gloeosporioides
against P. capsici followed by P. colocasiae. 3,4′,5-Trihydroxystilbene also recorded similarpattern of activity and a best MIC value of 8 μg/ml was recorded against P. capsici. Bothstilbenes and diketopiperazines recorded higher antifungal activity than the standard fungi-cide oligochitosan against P. capsici and P. colocasiae. The result of disk diffusion assayconducted by the MIC concentration of stilbenes and diketopiperazines is presented inTable 3. Best activity was recorded by 3,5-dihydroxy-4-isopropylstilbene against P.colocasiae (26 mm), followed by P. capsici (24 mm).
Effect of Stilbenes and Diketopiperazines on Each Life Stage of P. capsici and P. colocasiae
A significant inhibition of diketopiperazines and stilbenes on the mycelial dry weight of P.capsici and P. colocasiae was observed (Fig. 1). Best activity was recorded by 3,5-dihy-droxy-4-isopropylstilbene against P. colocasiae and inhibited the mycelial dry weight over90 % at 25 μg/ml followed by P. capsici (50 μg/ml) (Table 4). 3,4′,5-Trihydroxystilbene alsorecorded a similar pattern of activity.
Strong inhibition of diketopiperazines and stilbenes on different stages in the life cycle ofP. capsici and P. colocasiae was observed. The MIC values of diketopiperazines, stilbenesand oligochitosan for sporangium formation, zoospore release, and cystospore germinationare shown in Table 4. The inhibition of zoosporangia production was the most sensitivestage to the test compounds, and the two stilbenes recorded the most significant activity. Thebest MIC values was recorded against P. capsici and P. colocasiae by 3,5-dihydroxy-4-isopropylstilbene on zoosporangia formation (1 μg/ml) (Table 4). A very similar pattern ofactivity was recorded by 3,4′,5-trihydroxystilbene. After incubation for 24 h, there wasapproximately 123 (±11) zoosporangia formed around margin of one mycelial disk in steriledistilled water. In the presence of 2 μg/ml 3,5-dihydroxy-4-isopropylstilbene, there wasnearly no new mycelium around original mycelial disks, and formation of sporangium wascompletely inhibited.
Incubation with diketopiperazines and stilbenes could lead to the rupture of zoosporesand the best activity was recorded against P. capsici by 3,5-dihydroxy-4-isopropylstilbene.After 60 min incubation with 5 μg/ml 3,5-dihydroxy-4-isopropylstilbene, 97 % zoosporeswere ruptured (Table 4).
After 1 h of incubation, cystospore germination in the control was ca. 85 %, while 3,5-dihydroxy-4-isopropylstilbene and 3,4′,5-trihydroxystilbene at 5 μg/ml inhibited cystosporegermination of P. capsici completely. The MIC value of 3,5-dihydroxy-4-isopropylstilbeneagainst cystospore germination of P. colocasiae was also 5 μg/ml (Table 4).
Table 3 Antimicrobial activity of diketopiperazines and stilbenes
Zone of inhibition (diam., in mm)
Compounds P. capsici P. colocasiae B. cinerea C. gloeosporioides
Effect of ATP and Glucose on Cystospore Germination
Two stilbene compounds recorded significant activity against P. capsici and P.colocasiae and was selected for the further studies to know the effect of ATP andglucose on cystospore release and germination. After incubation for 4 h, zoosporerelease in sterile distilled water was ca. 97 %. 3,5-Dihydroxy-4-isopropylstilbene at50 μg/ml exhibited 80 % and 82 % inhibition on zoospore release against P. capsiciand P. colocasiae and this effect reduced markedly to 30 % and 33 % when 20 μMATP in pH 7.3 Tris–HCl buffer was applied 10 min before stilbenes (Fig. 2a).However, Tris–HCl buffer applied alone was able to reduce this effect to 22 % and19 % for P. capsici and P. colocasiae. At a higher concentration of 100 μg/mlinhibition of 3,5-dihydroxy-4-isopropylstilbene was less reduced by Tris–HCl bufferwith and without ATP, where as 3,4′,5-trihydroxystilbene recorded no inhibition onzoospore release (Fig. 2b).
Glucose with the final concentration of 100 mM protected zoospore from rupture whenconcentrations of 3,5-dihydroxy-4-isopropylstilbene were 50 μg/ml or below (Fig. 3a).While on the contrary, presence of 20 mM ATP at pH 7.3 in Tris–HCl buffer showed noeffect on 3,5-dihydroxy-4-isopropylstilbene induced zoospores rupture. After 22 h incuba-tion, cystospore germination in control was ca. 88 % while 3,5-dihydroxy-4-isopropylstilbene at 150 μg/ml inhibited 97 % and 93 % cystospore germination for P.capsici and P. colocasiae. But 3,4′,5-trihydroxystilbene inhibited only 30 % cystosporegermination (Fig. 3b).
Influence of pH on Stilbenes’ Activity
Figure 4 shows the estimation of the influence of pH on the inhibitory activity of stilbenesagainst P. capsici and P. colocasiae. The greatest inhibitory efficacy of stilbene 1 occurredbetween pH 7 and 8, and fungal growth inhibition rate was >90 %, whereas stilbene 2recorded the greatest inhibitory activity between pH 6 and 7 (Fig. 3). For stilbene 1, thelowest activity was recorded at pH 4. On the whole, stilbenes showed strong antifungalactivity over wide pH range from 5 to 8.
Fig. 1 Inhibition of compounds on the mycelial dry: a P. capsici and b P. colocasiae
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Tab
le4
Effectof
diketopiperazinesandstilb
enes
ondifferentstages
inthelifecycleof
P.capsiciandP.
colocasiae
Testcompo
unds
Stagesin
thelifecycleof
P.capsiciandP.
colocasiae
Mycelialdryweigh
t(μg/ml)
Zoo
sporangiaprod
uctio
n(μg/ml)
Zoo
sporerupture(μg/ml)
Cystosporegerm
ination(μg/ml)
P.capsici
P.colocasiae
P.capsici
P.colocasiae
P.capsici
P.colocasiae
P.capsici
P.colocasiae
Cyclo-(L-Pro–L-Leu)
200
400
1632
150
150
100
50
Cyclo-(D-Pro–L-Leu)
400
500
88
100
200
5010
0
Cyclo-(D-Pro–L-Tyr)
500
300
816
200
100
100
50
3,4′,5-Trihydrox
ystilbene
100
501
210
255
10
3,5-Dihydroxy-4-isopropylstilb
ene
5025
11
525
55
Olig
ochitosan
100
200
816
100
150
5050
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Temperature Effect on Antifungal Stability of Stilbenes
The evaluation of the temperature effect on antifungal stability of stilbenes was shown inTable 5. After treatment under different temperatures ranging from −70 °C to 120 °C, theinhibitory rates of the stilbenes against P. capsici and P. colocasiae was maintained at over70 %. The greatest inhibition efficacy of stilbene 1 appeared at 20 °C, whereas for stilbene 2the greatest inhibition was at 30 °C When treated at 160 °C for >15 min (i.e., for 30 and60 min), the inhibitory effect of stilbenes was completely lost, but at 160 °C for 15 min,approximately 40 % activity remained.
Discussion
Although a great amount of research has been undertaken to overcome the damage causedby plant pathogens, a major difficulty encountered is the lack of effective antifungals forcontrolling against some severe diseases [23]. In recent years, there has been a growinginterest in the potential use of microbial metabolites as agrochemicals. Microbial metabolitesare expected to overcome the problems associated with resistance of pests, and are generallymore biodegradable and more environment friendly than their synthetic counterparts [9, 24].
Fig. 2 Effect of ATP on zoospore release inhibition by oligochitosan. pH 7.3 Tris–HCl buffer with or without20 mM ATP was added 10 min before addition of stilbenes. Inhibition of stilbenes on zoospore release wasmeasured after 4 h. a 3,5-Dihydroxy-4-isopropylstilbene and b 3,4′,5-trihydroxystilbene
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Previously, we reported the antifungal activity of three cyclic dipeptides and two stilbenesisolated from the cell free culture filtrate of Bacillus sp. N strain associated with rhabditidentomopathogenic nematode against plant and human pathogenic fungi. Diketopiperazinespossess diverse biological activities such as antifungal [25] and antibacterial [26] activities.In the present study, we have used three cyclic dipeptides namely cyclo-(L-Pro–L-Leu),
Fig. 3 Effect of glucose on zoospore rupture caused by stilbenes. Glucose (100 mM) in sterile distilled waterwas added 10 min before addition of stilbenes. Zoospore rupture was measured after 60 min incubation. a 3,5-Dihydroxy-4-isopropylstilbene and b 3,4′,5-trihydroxystilbene
Fig. 4 Influence of pH on antifungal activity of stilbenes
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cyclo-(D-Pro–L-Leu) and cyclo-(D-Pro–L-Tyr). Cyclo(Leu–Pro) peptides having antifungalproperty have been isolated from Lactobacillus plantarum [27]. Rhee [28] also reported theantifungal activity of cyclo(L-pro–L-Leu) against many plant pathogenic fungi and highestactivity of this compound was recorded against rice blast fungus, Pyricularia oryzae. Astrong antifungal activity of 3,5-dihydroxy-4-isopropylstilbene against Aspergillus flavus,Candida tropicalis, etc., was reported [29].
In the present study, we describe the antifungal activity of three cyclic dipeptides and twostilbenes against four plant pathogenic fungi. All compounds recorded significant antifungalactivity plant pathogenic fungi tested. Out of five compounds tested, two stilbene com-pounds recorded significant activity against P. capsici and P. colocasiae.
Two stilbenes at low concentrations inhibited different stages in the life cycle of P. capsiciand P. colocasiae including zoosporangia production, zoospore release, cystospore germi-nation, and induced the rupture of released zoospore. It is well known that both zoosporerelease and zoospore rupture of Phytophthora are connected to impairment of the energygeneration system [22] and osmotic pressure [30]. Addition of ATP (an energy supplier) or
Table 5 Inhibitory activity of the stilbenes treated under different temperatures
Temperature (°C) Time (min) Inhibitory rate (%, x ± SD)
glucose (an osmotic stabilizer) prior to treatment with respiration inhibitor was able toreduce the inhibition on zoospore release and rupture of zoospore, respectively [22]. It isnotable that addition of ATP was not able to reduce the inhibition of stilbenes on zoosporerelease (Fig. 2) and zoospore rupture (Fig. 3). On the contrary, zoospore rupture was greatlyreduced by the addition of 100 mM glucose prior to treatment with 25–50 μg/ml stilbenes(Fig. 3). Similar results were previously reported on different fungi treated with chitosan[31]. These observations indicate that, similar to chitosan, stilbenes may act on the cellmembrane by upsetting osmotic pressure.
Many fungitoxic compounds that exhibits strong activity against many pathogenic fungihas been reported previously [32]. However, the compounds produced by EPN associatedbacteria have not been extensively used in agriculture because of their limited antifungalspectrum and instability. In our experiments, cyclic dipeptides and stilbenes exhibited broad-spectrum antifungal activity against plant pathogenic fungi because no visible fungal growthwas observed at the compound concentrations >100 μg/ml. Comparing with other antibi-otics, such broad-spectrum antifungal compounds like cyclic dipeptides and stilbenes hasnot been previously reported against the pathogen we tested. This indicated that stilbenescould be used as an effective candidate for the development of a new type of agriculturalantibiotics. In addition, it showed strong fungistatic activity against some important humanopportunistic pathogens, such as C. albicans [17]. In addition, another outstanding feature ofstilbenes was its antifungal stability. It showed a good biological control performance over awide pH range and possessed excellent heat resistance properties. Particularly under normalconditions (pH from 5 to 8, temperature from 0 to 60 °C), the inhibitory efficacy againstplant pathogenic fungi was optimal. Even under the extreme conditions (pH 4 or 9,>100 °C), stilbenes could also maintain relative higher antifungal activity. This excellentadaptability to the environment could satisfy the need for biocontrol of the fungal diseasesunder various conditions in agriculture.
Phytophthora spp. are dangerous filamentous fungi and have various host range [33]. Atpresent, no proper treatment of such infections has been satisfactorily established. In ourstudy, the stilbenes showed excellent fungal growth suppression ability againstPhytophthora sp. Although we did not determine the MFC values for them at the highestcompound concentration (1,000 μg/ml), a scheme for controlling these fungal diseases wasproposed according to their growth inhibition curves. Stilbenes treatment was dose-dependent; when its antifungal effect ended, inhibited fungi would continue growingbecause of the chalamydospores’ germination. So we can add this compound at regularintervals to control the fungal diseases caused by Phytophthora sp.
Conclusion
Crop protection using industrial fungicides is a powerful tool for effective control ofmany plant pathogenic fungi. However, extensive use of such fungicides is nowrecognized, worldwide, to be associated with a range of environmental risks. Thefindings of the present study indicate that stilbenes produced by Bacillus sp. is apromising antifungal compound for practical application in the agricultural biocontrolbecause of its strong, broad and stable inhibitory activity against plant pathogenicfungi. Thus, further study will need to focus on (1) more detailed identification ofantifungal mechanism against plant pathogenic fungi; (2) the optimization of fermen-tation for stilbenes because productivity of the compound also plays a major role forbiocontrol; this work is currently in progress.
Appl Biochem Biotechnol (2014) 172:741–754 753
Acknowledgments The authors are grateful to the Indian Council Medical Research (ICMR), Governmentof India for funding. We thank the Director, CTCRI, for providing the facilities for this work.
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