Biological Control 65 (2013) 200206

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Biological Control
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Antimicrobial effects of volatiles produced by two antagonistic Bacillus strains on the anthracnose pathogen in postharvest mangos
Min Zheng a, Jingying Shi b, Jian Shi c, Qingguo Wang a,, Yanhua Li d
a

Post-harvest Laboratory, College of Food Science and Engineering, Shandong Agricultural University, Taian, Shandong Province 271018, China Food Engineering Department, College of Food Science and Engineering, Shandong Agricultural University, Taian, Shandong Province 271018, China c Shandong Key Laboratory of Agricultural Microbiology, College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018, China d Shandong YingYangYuan Food Technology Co. Ltd., Jinan, Shandong Province 251400, China
b

h i g h l i g h t s
" Volatiles produced by two Bacillus

g r a p h i c a l a b s t r a c t
Mycelia growth inhibition of Colletotrichum gloeosporioides exposed to VOCs from biological control agents TB09, TB30, TB52, and TB72, in vitro (left) and in vivo (right).

strains inhibited anthracnose pathogen. " Mycelial growth of Colletotrichum gloeosporioides was suppressed. " Anthracnose on mango fruit was reduced. " Four volatiles can inhibit mycelia growth in vitro completely.

a r t i c l e

i n f o

a b s t r a c t
Four bacterial strains of Bacillus spp. which were antagonistic to the mango anthracnose pathogen were isolated and screened. Among them, TB09 and TB72 were identied by 16S rDNA sequence as Bacillus pumilus and Bacillus thuringiensis, respectively. In vitro, the anthracnose fungus showed 88.87% and 80.07% of mycelia growth inhibitions in presence of B. pumilus and B. thuringiensis, respectively and in vivo, the inhibitions of the disease were 94.28% and 87.06%, respectively. Based on the Gas ChromatographyMass Spectrometer (GCMS) analysis, 11 volatile compounds produced by B. pumilus and B. thuringiensis were identied. Among them, ve volatiles showed better inhibition effects on the pathogens. 2-nonanone, b-benzeneethanamine, 2-decanone completely inhibited mycelia growth in vitro at a concentration of 100 lL L1, and thymol inhibited growth at concentrations of 50 mg L1 and 100 mg L1. The inhibition rate of 40 lL L1 articial mixture of 5 volatiles was 98.75% in the plate test. The results showed that the two screened antagonistic bacteria, and some of their produced volatiles and articial mixtures could be promising control methods for anthracnose in harvested mango fruit. 2013 Elsevier Inc. All rights reserved.

Article history: Received 18 October 2012 Accepted 11 February 2013 Available online 28 February 2013 Keywords: Volatile compounds Bacillus pumilus Bacillus thuringiensis Bio-fumigation Colletotrichum gloeosporioides Mango fruit

1. Introduction Mango is valued for its color and enjoyed for its taste and nutritional value in dietary ber, vitamin C and especially the high content of b-carotene and other carotenoid sources of pro-vitamin A.
Corresponding author.
E-mail address: wqgyyy@126.com (Q. Wang). 1049-9644/$ - see front matter 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2013.02.004

Anthracnose, caused by Colletotrichum gloeosporioides (Penz.), is the most prevalent postharvest disease affecting the marketability mango. At present, the control of anthracnose disease in mangos remains reliant on fungicides including prochloraz amines, azoxystrobin and thiabendazole (Dodd et al., 1991; Donkin and Oosthuyse, 1996; Sundravadana et al., 2007). These fungicides effectively suppress the development of anthracnose symptoms, but selection of resistant forms of the pathogen is often associated

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with long term use in the absence of an integrated resistance management plan. Indiscriminant fungicide use may cause environmental pollution, including known ecotoxicity to sh, and health risks because of their suspected carcinogenic properties. Some alternative or complimentary physicochemical approaches to disease suppression that have received research attention include storage under CAP (controlled atmosphere package), hot water immersion (Benitez et al., 2006), hot dry air or chitosan coatings (Abd-AllA and Wafaa, 2010), or UV-C irradiation, as well various sequential or co-treatment combinations. Apart from hot water immersion, alternative methods, despite promising research outcomes, have yet to demonstrate sufcient efcacy to be adopted into practical application. The use of antagonistic microorganisms may be an alternative to fungicides for the control of postharvest diseases. Antagonistic bacteria such as Bacillus licheniformis (Govender et al., 2005), Pseudomonas uorescens (Vivekananthan et al., 2004) and Bacillus spp. (Jager et al., 2001) or fungi such as Trichoderma harzianum or yeast such as Saccharomyces cerevisiae (Vivekananthan et al., 2004) and Rhodotorula minuta (Patinovera et al., 2005) have been found effective for the control of the anthracnose of mango under controlled research conditions. Recently, volatile organic compounds (VOCs) produced by microorganisms have been evaluated as a new approach for postharvest disease control. For instance, the endophytic fungus Muscodor albus (Strobel et al., 2001) has been studied for controlling fungal diseases of the table grapes, peaches, apples and lemons (Mercier and Jimenez, 2004; Mercier and Smilanick, 2005; Mercier et al., 2010); Geobacillus sp. (M-7), a thermophilic soil bacterium, can produce VOCs which have the biological activity against a series of fungal pathogens, such as Aspergillus fumigatus, Geotrichum candidum, Sclerotinia sclerotiorum, Trichoderma viride, Botrytis cinerea, and Verticillium dahliae (Ren et al., 2010). Other examples of biological control agents include, Bacillus strains for black spot of citrus (Arrebola et al. 2010),ce:italic>Streptomyces globispourous against B. cinerea on tomato fruit (Li et al., 2012), and Candida intermedia for postharvest diseases of strawberry (Huang et al., 2011). However, limited research information is available related to the efcacy of biofumigation volatiles for the control of mango anthracnose pathogen. The objective of this study was to select effective antifungal volatile-producing strains from various rhizosphere microbes as the potential biological agents, evaluate the effects of mycofumigation produced by these strains on C. gloeosporioides in vitro and in vivo. A solid phase micro-extraction syringe was used to trap volatiles, and identied them by the GCMS process. The key volatiles that could be used to suppress pathogens were identied. 2. Materials and methods 2.1. Microorganisms C. gloeosporioides accession 36326 was provided by the Agricultural Culture Collection of China. The pure culture isolate was grown on potato dextrose agar (PDA) at 28 C for up to 7 days prior to use. Bacterial strains were obtained from the rhizosphere soil associated with the Shandong YingYangYuan Food Technology Co. Ltd., Jinan, China. For use in experiments, the strains were grown on nutrient agar (NA) incubated at 37 C for 12 days. A suspension was created by gently scraping the bacterial lawn from NA and diluting with water to approximately 106 cfu mL1, as assessed with McFarland Turbidity Standards. 2.2. Screening for bacteria producing volatile antimicrobials All of the bacteria isolated from soil were tested for antagonistic activity in two-compartment plastic plates (Kai et al., 2007). For

the fumigation assay against C. gloeosporioides, the PDA medium was poured into one side of the plates (92 16 mm; Sarstedt AG, Nrnbrecht, Germany) and the NA medium was poured into the opposing sector. Each isolated bacteria (50 lL; 1 106 cfu mL1) was innoculated on the NA medium side of the plate and incubated for 1 day at 37 C. Subsequently, an agar plug (U 5 mm) of C. gloeosporioides was placed on the PDA sector. Sterile distilled water was used as the control factor instead of the isolated bacterial. The plates were sealed with paralm, incubated for 5 days at 28 C and the diameter of C. gloeosporioides expansion was measured. Each experiment was performed with three replications and the experiment was repeated three times. 2.3. In vivo tests of disease control by volatile producing bacteria Freshly harvested, mature and healthy mangos with approximate size and color ($30 g per fruit, $4 cm across) were used in this experiment. The fruits were free from visible defects and rot. Before each trial, the mangos were washed with water and their surface sterilized in 2% sodium hypochlorite for 10 s, then rinsed under running sterile water for 1 min and air-dried. Each fruit was wounded with a 5 mm diameter hole-puncher and injected with 5 mm diameter mycelia plugs of C. gloeosporioides or 50 lL C. gloeosporioides conidial suspension (105 conidia mL1) in the wound area. To study the antagonistic activity of volatiles on mycelia growth or conidial germination, ten-inoculated fruits were arranged in a 6.5 L container as one replicate of each antagonist strain. Then, 20 lL suspensions of the presumptive bacterial antagonists (1 106 cfu mL1) were inoculated separately on NA for 24 h at 37 C. Also, six cultures were placed in the container with no direct contact with the mangos. For the control groups, sterile water of the same volume replaced the antagonist cultures. After 7 days at 25 C, the diameters of decayed spots were measured and the lesion area was calculated to contrast treatment effects and antagonistic activity. Three replications of each treatment were performed and the experiment was repeated three times. 2.4. Taxonomic characterization of antagonistic isolates TB09 and TB072 The isolated bacteria with presumptive volatile antifungal activity were identied based on morphological phenotype and 16S rDNA sequence analysis. The sequences for the primers are 27F: (50 -AGAGTTTGATCCTGGTCAGAACGCT-30 ) and 1492R: (50 TACGGCTACCT TGTTACGACTTCACCCC-30 ). Bacterial DNA was isolated from each strain using the established CTAB method as described in Jin et al. (2011). The resulting PCR products were puried and used directly for sequencing (Invitrogen, Shanghai, China). Homology search comparisons were carried out using the database available at NCBI BLAST (http://www.ncbi.nlm.nih.gov/). 2.5. Analysis of volatile compounds produced by the antagonistic bacteria 20 lL suspensions of the presumptive bacterial antagonists (1 106 cfu mL1) were inoculated separately on NA in sample vials. Volatiles from the Bacillus strains were collected and analyzed after 7 or 14 days of incubation. The NA medium without the antagonistic bacteria was set up as a control. The volatiles were trapped by the solid phase micro-extraction syringe (SPME) (Supelco Inc., PA-USA). SPME bers coated with 65 lm (PDMS/DVB) was used. Adsorption was allowed to continue for 45 min at room temperature. After that, samples were immediately analyzed by GCMS (GC/MS-QP2010 Plus, Shimadzu Co., Kyoto, Japan). The injector was operated in split-less mode for all chromatographic runs, and helium gas was used as the carrier. The injector

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temperature was kept at 250 C and the detector at 280 C. The oven temperature was programmed as follows: 40 C for 5 min then increased to 250 C at a speed of 20 C min1. Mass spectra data of the volatile compounds were compared with data in the NIST08 Mass Spectral Library (Software Version2.0). 2.6. Assay of identied volatile compounds on mycelia growth in vitro Different amounts of individual volatile compounds (technical grade) were used to study the effect on radial growth of hyphae. An agar plug (U 5 mm) was placed on the PDA and a Standard amount of individual volatile compounds were added to another plate. Then the two lidless plates were sealed with paralm. Equivalent amounts of sterile distilled water were used as the control. After 7 days incubation at 28 C, the colony diameter was assessed. For treatments in which the C. gloeosporioides agar plug was completely inhibited, the original plug was transferred to the fresh PDA in the absence of volatiles to assess whether the inhibition was fungistatic or fungicidal. Following an additional ve days on fresh media the diameter of outgrowth was measured. Each experiment was performed in three replications and the experiment was repeated three times. 2.7. Antifungal assay of articial mixture of identied volatile compounds A constructed mixture was made by using authentic standard chemicals based on the volatile prole determined from live cultures. The proportion of each positively identied compound was calculated from the relative peak areas (2-nonanone: b-benzeneethanamine: 2-methylpyrazine: 2-decanone: thymol = 1:2:10:10:1; V/V). A 5 mm diameter agar plug with C. gloeosporioides was placed on PDA, and on the other plate, different amounts of the constructed mixture were added. Then the two lidless plates were sealed with paralm. After 7 days incubation at 28 C, the colony diameter was assessed. Three replications of each treatment were performed and the experiment was repeated three times. 2.8. Statistical analysis The data was subjected to analyses of variance (ANOVA) using SPSS17.0 Windows Software (SPSS Inc., Chicago, USA). Least significant differences (LSD) were calculated to compare the results at 0.05 level. 3. Results 3.1. Screening for bacterial antagonist producing volatile antimicrobial Two hundred and six strains of bacteria were isolated from the soil using repeated plate streaking (Fig. 1). Preliminary screening showed that 4 isolates had readily observed inhibitory activity towards C. gloeosporioides mycelia growth, identied as TB09, TB30, TB52 and TB72. The diameters of mycelia growth were inhibited over 80% by TB09 and TB72 and reduced over 30% in the presence of volatiles released from TB30 or TB52 (Table 1). 3.2. In vivo tests of the volatile compounds produced by bacteria The development and expansion of disease symptoms induced by the anthracnose pathogen was inhibited effectively by volatile compounds synthesized by four strains identied in in vitro screens, TB09, TB30, TB52 and TB72 (Fig. 2). For the inoculated control fruit, the lesion area extended to 9.39 mm2 after 7 days incubation at room temperature, whereas for the inoculated fruits

Fig. 1. Mycelia growth of Colletotrichum gloeosporioides exposed to volatile compounds from TB09, TB30, TB52, TB72 and control in vitro.

Table 1 Inhibition on mycelia growth of Colletotrichum gloeosporioides in plates by volatile compounds produced by various bacteria. Bacteria isolate TB09 TB30 TB52 TB72 Control Mycelia growth (cm) 5.40 0.79 26.07 0.31 31.73 0.31 9.67 0.42 48.50 0.66 a c d b e Inhibition rate (SD) (%) 88.87 1.70 46.25 0.94 34.57 1.50 80.07 1.09 d c b a

Means in the same column with different letters are signicantly different (P = 0.05).

exposed to volatiles from TB09, TB30, TB52 and TB72, the lesion areas were limited to 0.54, 1.18, 1.42 or 1.16 mm2. These correspond to relative inhibition incidences of 94.3, 87.4, 84.9 and 87.6 for each isolate, respectively (Table 2). In the test of spore suspension inoculations, the VOCs of four antagonistic strains also signicantly inhibited the growth of anthracnose pathogen in fruit tissue, but the inhibition values of isolates TB09 and TB72 were greater than TB30 and TB52. 3.3. Identication of bacteria TB09 and TB072 The two bacterial antagonistic strains TB09 and TB72 were identied by morphological observation, physiological test and DNA sequence analysis. The results showed that TB09 was most similar to Bacillus pumilus strain AY876289 (98.01%); similarity overlap included Bacillus altitudinis strain AJ831842 (96.22%) as second comparison and Bacillus stratosphericus strain AJ831841 (94.58%) as third comparison. Based on colony appearance criteria, the identity of TB09 was best assigned as a B. pumilus strain. Likewise, the results obtained from sequence comparison indicated TB72 was most similar to Bacillus thuringiensis strain D16281 (99.50%) and, to a lesser extent, Bacillus cereus strain HE716871 (85.63%). Thus TB72 was identied as a B. thuringiensis strain. 3.4. Analysis of the volatile compounds produced by bacteria The volatile proles analyzed by GCMS showed that TB09 produced more volatile compounds (VOCs) than TB72 and the control (Fig. 3). Diethyl phthalate and heptadecane existed in all the samples and a peak corresponding to diethyl phthalate represented the main fraction in all assays (Table 3). 2-decanone and 2-methylpyrazine were the secondary predominant substances

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Fig. 2. Mycelia growth of Colletotrichum gloeosporioides exposed to volatile compounds from TB09, TB30, TB52, TB72 and control in vivo. (A) The fruits inoculated mycelia plugs. (B) The fruits inoculated with spore suspensions.

Table 2 In vivo tests of the volatile compounds produced by antagonistic bacteria. Treatment Inoculate mycelia plugs Rot area a (mm2) TB09 TB30 TB52 TB72 Control 0.54 0.06 1.18 0.09 1.42 0.06 1.16 0.14 9.39 0.47 a b c b d Control value b (%) 94.28 0.55 87.41 0.96 84.85 0.82 87.64 1.60 c b a b Inoculate spore suspensions Rot area (mm2) 0.75 0.03 2.91 0.12 3.53 0.18 1.03 0.07 8.02 0.40 a c d b e Control value (%) 90.81 0.55 63.67 0.65 55.81 4.15 87.06 1.04 d b a c

Means in the same column with different letters are signicantly different (P = 0.05). a Rot area (mm2) = p (rotted symptom radius)2. b Control value (%) = 100 {rot area of untreated fruit rot area of treated fruit} rot area of untreated fruit.

Fig. 3. Chromatogram corresponding to volatile organic compounds produced by TB09 (A), TB72 (B) and Control (C) on PDA. The remainder peaks are from the bers itself or the atmosphere. (1) 2-methylpyrazine (2) 2-nonanone (3) 2-decanone (4) Thymol (5) BHT (6) Diethyl phthalate (7) b-benzeneethanamine (8) Heptadecane (9) Gentisic acid (10) Heneicosane (11) n-hexadecanoic acid.

produced by TB09 and TB72, respectively. 2-nonanone, 2-decanone, gentisic acid and n-hexadecanoic acid were volatiles uniquely produced by TB09, while TB72 was differentiated by peaks identied as 2-methylpyrazine and thymol.

3.5. Inhibitory effect of identied volatile compounds on the mycelia growth in vitro Among the eleven volatile compounds mentioned in Table 3, seven of these technical grade volatile compounds are available in a liquid state and another four occur in a solid state at normal room temperature. 2-nonanone, b-benzeneethanamine and 2-decanone

completely inhibited the growth of mycelia in vitro at the tested concentration of 100 lL L1 (Table 4). Thymol completely inhibited the growth of mycelia at concentrations of 50 mg L1 and 100 mg L1 (Table 5). 2-methylpyrazine showed strong inhibitory activity against the growth of mycelia at a concentration of 100 lL L1, while the remaining compounds showed weak or no inhibitory activity. The C. gloeosporioides agar plug from the completely inhibited test plate could not initiate outgrowth to the fresh PDA following transfer even in the absence of volatiles for ve days (data not shown). 3.6. Effect of a constructed mixture of Bacillus volatile compounds on mycelia growth of C. gloeosporioides Following 7 days incubation exposure at 28 C, the growth inhibitions of C. gloeosporioides mycelia were 77.06% and 98.75% in the

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Table 3 GC/MS volatile prole of the two antagonists Bacillus strains and control. Possible compound Control Diethyl phthalate Heptadecane Bacillus TB09 2-Nonanone 2-Decanone BHT Diethyl phthalate b-Benzeneethanamine Heptadecane Gentisic acid Heneicosane n-Hexadecanoic acid Bacillus TB72 2-methylpyrazine Thymol BHT Diethyl phthalate b-Benzeneethanamine Heptadecane Heneicosane 98% 95% 89% 90% 96% 98% 89% 94% 72% 83% 70% 95% 82% 94% 98% 89% 96% 84% 13.17 14.25 5.69 7.26 12.17 13.16 13.85 14.25 14.83 15.57 17.02 2.41 9.31 12.17 13.16 13.85 14.25 15.57 10.81 1.28 0.64 9.37 4.30 32.09 4.53 2.91 0.16 1.11 1.36 11.01 0.62 5.07 62.12 6.45 2.47 11.71 Similarity Retention time (min) Relative peak area (%)

The compounds generated by Bacillus strains were tentatively identied by mass spectra comparison to those in the NIST Mass Spectral Library (probability based match > 80%).

Table 4 Effect of liquid state volatile compounds on the mycelia growth of C. gloeosporioides after 7 days in vitro. Volatile compound Mycelia growth (cm) at different concentrations of volatiles 0 l L L 1 Heneicosane 2-Nonanone 2-Decanone 2-Methylpyrazine Heptadecane Diethyl phthalate b-Benzeneethanamine 8.59 0.02 8.59 0.02 8.59 0.02 8.59 0.02 8.59 0.02 8.59 0.02 8.59 0.02 d d d d b d d 1 lL L1 7.94 0.18 7.98 0.00 7.39 0.10 7.46 0.20 8.38 0.11 6.17 0.22 5.77 0.01 c c c c ab c c 10 lL L1 7.44 0.06 6.04 0.09 5.54 0.08 5.44 0.08 8.39 0.04 5.71 0.03 5.56 0.08 b b b b ab b b 100 lL L1 5.65 0.10 0a 0a 1.45 0.38 8.22 0.08 4.82 0.13 0a a

a a a

Table 5 Effect of solid state volatile compounds on the mycelia growth of C. gloeosporioides after 7 days in vitro. Volatile compound Different concentrations of volatiles 0 mg L1 BHT Gentisic acid Thymol n-Hexadecanoic acid 8.59 0.02 8.59 0.02 8.59 0.02 8.59 0.02 c c b c 50 mg L1 6.20 0.16 b 7.51 0.12 b 0a 6.40 0.18 b 100 mg L1 5.19 0.08 a 7.09 0.16 a 0a 5.20 0.13 a

Table 6 Mycelia growth inhibition of C. gloeosporioides exposed to different concentrations of the articial mixture of volatile compounds identied in Bacillus. Different concentrations (lL L1) 0 20 40 60 80 100 Mycelia growth(cm) 7.06 0.80 c 1.63 0.28 b 0.09 0.02 a 0a 0a 0a Inhibition rate (SD) (%) 77.06 2.16 a 98.75 0.29 b 100 b 100 b 100 b

presence 20 or 40 lL, respectively (Table 6) of a constructed mixture of primary volatile compounds.

Inhibition rate was calculated as percentage growth inhibition as compared to the control in the absence of the articial mixture. Means in the same column with different letters are signicantly different (P = 0.05).

4. Discussion In this study, the antifungal volatile-producing strains TB09 and TB72 were selected from a broader screen of candidate soil isolates and emerged as the most promising biological control agents for postharvest control of anthracnose on mango fruit. Based on the sequence analysis of their 16S rDNA region after PCR amplication and other criteria, TB09 and TB72 were grouped with B. thuringiensis and B. pumilus, respectively. B. thuringiensis and B. pumilus are the most important Bacillus species in industrial biotechnology (Raddadi et al., 2012). The B. pumilus M-38 strain had been evaluated in a Petri plate against three potato tuber pathogens: Fusarium culmorum, Fusarium oxysporum and Fusarium sambucinum. The inhibition zone values of the B. pumilus M-38 strain ranged from 24 to 33 mm (Kotan et al., 2009). B. pumilus has also been tested for control of gray mold on apples caused by Botrytis mali (Jamalizadeh et al., 2010). B. thuringiensis has been suggested to be an important alternative for future use to reduce fungicide application rates to control postharvest diseases (Lucon et al., 2010). In previous research, some Bacillus strains, such as Bacillus subtilis, have played an important role in bio-control of postharvest fungal

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diseases by the production of antibiotics (Toure et al., 2004). However, little is known about volatiles produced by Bacillus spp. in controlling postharvest disease of fruit. The volatiles produced by B. subtilis or Bacillus amyloliquefaciens showed signicant inhibition of decay incidence of citrus diseases in vitro and in vivo (Arrebola et al., 2010). Volatiles generated by the B. subtilis JA strain signicantly inhibited B. cinerea (Chen et al., 2008). In this study, volatile substances produced by B. thuringiensis and B. pumilus had signicant inhibitory effects on mycelia growth of C. gloeosporioides in vitro and in vivo. In the in vivo test, the fruit inoculated with mycelia plugs and inoculated with spore suspensions were affected differently in presence of the antagonistic bacteria TB30 or TB52. However for TB09 and TB72, the results were similar. Thus outcome is consistent with in vitro test in which TB30 and TB52 affect the mycelia growth much more than spore inhibition. On the other hand, results also showed that the bacterial isolates TB09 and TB72 could have stable and efcient capabilities to control C. gloeosporioides and merit further testing under simulated distribution conditions for the prevention of anthracnose during transportation and storage periods. The antimicrobial mechanism of the VOCs is another important consideration to effectively design a biofumigation program. In a previous study, transmission electron microscopy of fumigated and untreated B. cinerea showed excessive vesiculation or thickened cell walls in exposed conidia and increased vesiculation or strong retraction of plasma membrane in exposed hyphae (Li et al., 2012). These results provide a better understanding of the volatiles mode of action. In previous studies, all VOCs produced by microorganisms could generally be chemically grouped into esters, alcohols, alkenes, alkanes, alkynes, organic acids, ketones, terpenoids, aldehydes and disuldes (Corcuff et al., 2011; Dilantha et al., 2005; Wan et al., 2008). The VOCs with highly inhibitory capability towards conidial germination and mycelia growth included phenylethyl alcohol and caryophyllene. 2-nonanone has been identied from the volatiles produced by C. intermedia strain to control postharvest disease of strawberry (Huang et al., 2011) and thymol was applied as the essential oil to inhibit fruit rot fungi (Ippolito et al., 2012). Our results demonstrated that in addition to 2-nonanone and thymol, other bioactive volatile compounds including 2-decanone, 2-methylpyrazine and b-benzeneethanamine produced by the two isolated strains also have antimicrobial activity. These VOCs didnt exist in the control treatment and so the results indicated that these VOCs were the derived from bacterial metabolites and not from the plastic ware and culture medium. The C. gloeosporioides agar plugs fumigated by these three compounds could not germinate even after they were transferred to the fresh PDA. This result suggested that these volatiles might be lethal to anthracnose pathogen. It is noteworthy that these compounds have been widely used in elds for such use as avor additives and pharmaceuticals for human consumption. For example, 2-nonanone is naturally produced in volatiles which are released by red raspberries and strawberries (Vaughn et al., 1993) and thymol has been used in oat our as an antimicrobial agent (Sandoval et al., 2011). As a result of the widespread use of these volatiles in the food industry, they have come to be considered a safe alternative to control mango anthracnose disease. The effects of the mixed compounds showed that the higher the amount of the constructed mixture, the greater the inhibitory activity against the anthracnose fungi. The inhibition rate could reach as high as 98.75% if the concentration of the articial mixture was 40 lL L1. The potential use of the articial mixture to control the anthracnose pathogen in mango is being tested. Future research will be needed on designing agriculturally acceptable and practical ways for efcient use of the antifungal volatiles to keep mango quality during the postharvest storage, distribution and marketing period.

Acknowledgments We acknowledge Dr. Chao Wang (Department of Horticulture Science and Engineering, Shandong Agricultural University) for the analysis of GC/MS.

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