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Research Paper Effects of volatile organic compounds from Streptomyces albulus NJZJSA2 on growth of two fungal pathogens Yuncheng Wu1,2,3, Jun Yuan1,2,3, Yaoyao E1,2,3, Waseem Raza1,2,3, Qirong Shen1,2,3 and Qiwei Huang1,2,3 1
2
3
National Engineering Research Center for Organic-based Fertilizers, Nanjing Agricultural University, Nanjing 210095, China Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing 210095, China
A Streptomyces albulus strain NJZJSA2 was isolated from the forest soil sample of Tzu-chin Mountain (Nanjing China) and identified based on its morphological and physiological properties and 16S rDNA gene sequence analysis. The strain S. albulus NJZJSA2 was evaluated for the production of antifungal volatile organic compounds (VOCs) against two fungal pathogens. Results showed that the VOCs generated by S. albulus NJZJSA2 inhibited mycelial growth of Sclerotinia sclerotiorum (SS) and Fusarium oxysporum (FO) by 100 and 56.3%, respectively. The germination of SS sclerotia and FO conidia was completely inhibited in the presence of VOCs produced by S. albulus NJZJSA2 in vitro. In soil, the VOCs delayed the germination of SS sclerotia and inhibited the germination of FO conidia for 45 days. The strain S. albulus NJZJSA2 was able to produce 13 VOCs based on GC/MS analyses. Among those, six compounds were purchased and used for the antifungal activity assay. Three relatively abundant VOCs, 4-methoxystyrene, 2pentylfuran, and anisole were proved to have antifungal activity. Microscopy analysis showed that the pathogen hyphae were shriveled and damaged after treatment with 4-methoxystyrene. These results suggest that the S. albulus strain NJZJSA2 produce VOCs that not only reduce the growth of SS and FO, but also significantly inhibit the SS sclerotia and FO conidia. The results are useful for the better understanding of biocontrol mechanisms by S. albulus strains and will help to improve the biological control efficiency of lethal plant diseases.
: Additional supporting information may be found in the online version of this article at the publisher's web-site. Keywords: Volatile organic compounds / Streptomyces albulus / Fusarium oxysporum / Sclerotinia sclerotiorum Received: November 27, 2014; accepted: February 21, 2015 DOI 10.1002/jobm.201400906
Introduction The widespread soil-borne pathogens, Fusarium oxysporum f. sp. cucumerinum J. H. Owen (FO) and Sclerotinia sclerotiorum (Lib.) De Bary (SS) are responsible for the Fusarium wilt of cucumber and sclerotinia stem rot of oilseed rape, respectively [1, 2]. These fungal pathogens persist in soil as dormant long-lived structures, chlamydospores, and Correspondence: Qiwei Huang, Jiangsu Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China E-mail: [email protected] Phone: þ86-025-84396212 Fax: þ86-025-84396824 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
sclerotia, which can survive for several years in the absence of host plants [3]. Traditionally, chemical controls can effectively protect plants from infectious pathogens [4]. However, sustainable agriculture requires that plant disease-control strategies become more ecologicalbased and less dependent on outside factors, such as synthetic chemicals. Biological control has been considered as an alternative safe method for controlling soilborne fungal pathogens [5, 6]. Among the biocontrol agents, actinomycetes have received considerable attention, particularly Streptomyces species. The efficacy of antagonistic Streptomyces spp. in the biological control of plant pathogenic fungi, such as
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Pyrenochaeta lycopersici [7], Sclerotiorum rolfsii [8], and Fusarium oxysporum [9], has been widely studied. Streptomyces species are well known for their ability to produce antibiotics and volatile organic compounds (VOCs) [10, 11]. The VOCs are low-molecular-weight compounds that easily evaporate at normal temperature and pressure, conferring the characteristic of diffusion through the atmosphere and soil [12]. The release of VOCs by soil microbes has been reported to inhibit the growth of fungal pathogens, promote plant growth, display nematicidal activity, and induce systemic resistance in crops [13, 14]. Previous studies about VOCs produced by Streptomyces spp. mainly focused on their effects on the environmental chemistry, such as off-odor in water pollution [15] or indoor air [16]. Recently, the studies of VOCs produced by Streptomyces spp. have focused on the control of plant diseases [17]. Wan et al. [18] reported that the VOCs produced by S. platensis F-1 demonstrated antifungal activity against Rhizoctonia solani, S. sclerotiorum, and Botrytis cinerea. Similarly, VOCs generated from Streptomyces globisporus JK-1 suppressed Penicillium italicum [19]. However, different organisms and different species release similar and different types of VOCs [11, 18, 19]. Therefore, it is important to investigate more and more biocontrol agents for the production of VOCs with novel structures to use those as biocontrol agents effectively. In this study, Streptomyces albulus strain NJZJSA2 was isolated from forest soil with a wide antifungal spectrum. S. albulus strains are known for their ability to produce e-polylysine, which exhibits antimicrobial activity against yeast, fungi, and Gram-positive and Gram-negative bacteria [20]. S. albulus strains have been reported as biocontrol agents against fruit and vegetable pathogens [21]. However, little information is currently available on the antifungal activity of VOCs produced by S. albulus strains against fungal pathogens. The goal of this study was to determine the effect of the VOCs produced by S. albulus NJZJSA on two fungal pathogens and to identify the antifungal VOCs released from S. albulus NJZJSA2.
Materials and methods Microorganisms and cultural conditions Two fungal pathogen strains Fusarium oxysporum f. sp. cucumerinum J. H. Owen and Sclerotinia sclerotiorum (Lib.) De Bary, which exhibited high virulence in cucumber (Cucumis sativus) and oilseed rape (Brassica napus), respectively, were used as the target fungi. Stock cultures were maintained on potato dextrose agar (PDA) ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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plates at 4 °C. Pre-cultures were established by transferring a stock agar plug containing mycelia of two pathogens onto PDA Petri dishes and incubating for 5 days at 25 °C for SS, and at 28 °C for FO. The S. albulus strain NJZJSA2 with broad inhibition spectrum (data not shown) was isolated from forest soil samples from the Tzu-chin Mountain (N32°030 , E118°510 , Nanjing, China,). For the S. albulus NJZJSA2, stock cultures were maintained on PDA at 4 °C, and a pre-culture (104 CFU ml1) was established by inoculating one agar plug (about 1 cm diameter) containing spores of S. albulus NJZJSA2 into 50 ml of Bennett medium [22] and incubated at 28 °C for 24 h in an orbital incubator at 170 rpm. Identification of strain NJZJSA2 To identify strain NJZJSA2, the physiological and biochemical characteristics were evaluated according to the International Streptomyces Project (ISP) [23]. The spore, shape, and chain morphology were observed using light microscopy. The assimilation of carbohydrates was studied using ISP9 medium containing different carbohydrates (1%, w/v) as the sole carbon source [24]. The 16S rRNA gene of S. albulus NJZJSA2 was amplified, sequenced [25], and BLAST searched against the NCBI database. The sequences of its close relatives were used to construct a neighbor-joining phylogenetic tree using MEGA 4.0. Culture preparation for the production of VOCs Autoclaved wheat seeds (autoclaved at 120 °C for 20 min) were used to prepare cultures of S. albulus NJZJSA2 for the production of VOCs. A 5 ml pre-culture of S. albulus NJZJSA2 as described above, was inoculated into the autoclaved wheat seeds (20 g wheat seeds, plus 40 ml distilled water) for two days before use. In soil tests, 5 ml pre-culture of S. albulus NJZJSA2 was centrifuged at 4000 rpm for 5 min and then resuspended in 5 ml of sterile distilled water before the inoculation of 20 g autoclaved soil. VOCs antagonistic effects on FO and SS growth The VOCs produced by S. albulus NJZJSA2 were tested according to the method of Fernando [26] with some modifications. The Petri plates containing 10 g of autoclaved wheat seeds inoculated with S. albulus NJZJSA2 were prepared as described above and covered with another Petri plate containing PDA inoculated with a 6 mm diameter plug of FO or SS. The two plates were then sealed with Parafilm to obtain a double-plate chamber. The average distance between autoclaved wheat seeds surface of lower plates and agar surface of upper plates was 1.5 cm. The plates containing FO
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were incubated at 28 °C for 84 h, and the plates containing SS were incubated at 25 °C for 60 h. The lower plates containing S. albulus NJZJSA2 were then removed and the upper plates containing fungal mycelia were re-covered with new Petri plates to eliminate the VOCs effect. The upper plates containing FO or SS were incubated for another 36 or 60 h. The control plates had only autoclaved wheat seeds. All treatments were performed in triplicate. The diameters of mycelia were measured at 12 h intervals during incubation. VOCs antagonistic effects on FO conidia and SS sclerotia germination The effects of VOCs on the germination of conidia of FO and sclerotia of SS were studied by different methods. The method used for the FO spore germination assay was as described above except that 100 ml of spore solution (106 CFU ml1) was spread on PDA instead of a FO agar plug. Sclerotia germination of SS was studied according to the method of Li [19]. Briefly, four small Petri dish bottoms (60 mm in diameter and 15 mm in height) were placed inside a larger Petri dish (150 mm in diameter and 30 mm in height). Three of the small Petri dish bottoms contained 5 g of autoclaved wheat seeds inoculated with S. albulus NJZJSA2 were prepared as described above, and the fourth dish contained six surface-sterilized SS sclerotia. Non-inoculated, autoclaved wheat seeds were used as a control. The larger dish was sealed with Parafilm and incubated at 28 °C. The FO spore germination was measured after 3 days of incubation, and the number of sclerotia germination were recorded after 10 days of incubation. Each treatment had three replicates. VOCs effects on FO conidia and SS sclerotia vitality in soil The effects of VOCs produced by S. albulus NJZJSA2 from autoclaved wheat seeds and soil were studied. The soil was collected from the rice field with the following properties: pH 7.29, organic carbon, 22.31 g/kg, total N, 2.55 g/kg, total P, 1.02 g/kg, and available K 272.89 mg/ kg. Four small Petri dish bottoms (60 mm in diameter and 15 mm in height) were placed inside a large Petri dish (150 mm in diameter and 30 mm in height). Three of the small Petri dish bottoms contained 10 g of autoclaved wheat seeds or soil inoculated with S. albulus NJZJSA2 were prepared as described above. The fourth dish contained 10 g of soil inoculated with 1 ml of FO spore solution (108 CFU ml1) or 10 surface-distilled sclerotia. The larger dish was sealed with Parafilm and incubated at 28 °C. Each treatment had three replicates. The surviving conidia and sclerotia were counted after 45 days. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Identification of VOCs by SPME-GC-MS The VOCs produced by S. albulus NJZJSA2 grown on autoclaved wheat seeds were collected using solid-phase micro-extraction (SPME) after four days incubation; the VOCs released from fresh autoclaved wheat seeds were used as control [13]. A SPME fiber coated with divinylbenzene-carboxene-PDMS (DCP, 50/30 mm) was used for extracting the VOCs, and the samples were analyzed using the Trace DSQ (Finnigan) GC-MS system. The SPME fibers were desorbed at 250 °C for 5 min with an RTX-5MS column (30 m, 0.25 mm inside diameter, 0.25 mm). The initial oven temperature of 40 °C was held for 2 min, increased at a rate of 4 °C min1 to 150 °C, and held for 1 min, further increased at a rate of 10 °C min1 to 250 °C, and held for 4 min. The mass spectrometer was operated in the electron ionization mode at 70 eV with a source temperature of 220 °C. A continuous scan from 50 to 500 m/z was used. Helium was used as the carrier gas at a constant flow rate of 1.0 ml min1. The mass spectra of VOCs were compared with those in the National Institute of Standards and Technology (NIST) database (Version 2.0). Effect of synthetic compounds on SS and FO growth The VOCs produced by S. albulus NJZJSA2 grown on autoclaved wheat seeds were analyzed by GC-MS, and 13 compounds were detected when compared with autoclaved wheat seeds only (Table 1). Six of the identified VOCs were selected to test for their antagonistic activity (Table 2). These pure compounds were purchased from Shenghe Chemical Reagent Co., Ltd. (Nanjing, China), Lingfeng chemical Reagent Co., Ltd. (Shanghai, China), and Aladdin Reagent Database, Inc. (Shanghai, China). The divided plate method was used to perform the antagonistic assay of the selected VOCs. Sterile filter papers (15 20 mm) containing 5, 10, 20, 40, 60, 80 ml of each VOC were placed on one side of divided plates, and FO or SS mycelia plugs were placed on the PDA media of the other side. The control divided plates had only sterile filter papers. The colony size in each treatment was recorded and the percentage inhibition of hyphae growth was calculated using the following equation: Percentage of inhibition ¼ [{(Control-treatment)/ Control} 100] [17]. Microscopic observation of SS and FO FO or SS mycelia plugs were inoculated on the PDA side of divided plates with cover slips inserted at an angle. Four concentration gradients (0, 10, 80, 200 ml/plate) of 4-methoxystyrene were added to another side of the divided plates after 2 days inoculation and kept incubating for another 2 days. To prepare the samples
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Table 1. Volatile organic compounds produced by S. albulus NJZJSA2. RT(min) 1.15 3.6 4.26 6.19 11.42 14.28 19.00 19.34 20.73 21.46 22.81 25.99 28.84
Possible compound
Relative peak area (%)
Toluene Styrene Anisole 2-Pentylfuran 4-Methoxystyrene 1H-Indene,1-ethylideneoctahydro-7a-methyl-,(1Z,3aa,7ab) Trans-1,10-Dimethyl-trans-9-decalol Tetradecane Naphthalene,decahydro-4a-methyl-1-methylene-7(1-methylethenyl)-,[4aR-(4aa,7a,8ab)] 1H-cyclopenta[1,3]cyclopropa[1,2]benzene,octahydro-7-methyl-3-methylene-4(1-methylethyl)- (3aS,3bR,4S,7R,7aR)Naphthalene, 1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)1H-Cycloprop[e]azulen-4-ol,decahydro-1,1,4,7-tetramethyl-, [1aR-(1aa,4b,4ab,7a,7ab,7b)] 2-Oxepanone, 7-hexyl-
0.04 3.55 10.7 11.83 32.15 0.99 7.97 4.06 3.2 13.63 2.51 2.57 6.78
The 13 compounds were identified by comparing with those in the NIST/EPA/NIH Mass Spectrometry Library with respect to the spectra in the Mainlib and/or Replib databases.
for scanning electron microscopy (SEM), the cover slips with mycelium were fixed with 2.5% glutaraldehyde and dehydrated with a graded series of ethanol washes followed by drying in a drier under CO2. Mounted specimens were coated for 2.5 min with 10 mA of goldpalladium and examined under a field emission SEM (S-4800, Hitachi, Japan) operating at 15 kV [5]. The FO or SS mycelia after being treated with 200 ml/plate of 4-methoxystyrene were examined by trypan blue staining according to the method of Weid et al. [27]. In brief, FO or SS mycelia was soaked with 5 ml trypan blue solution for 5 min, and then washed three times with
distilled water. Growth of mycelium was viewed with a fluorescent microscope (Nikon eclipse 80i).
Results Identification of S. albulus NJZJSA2 The newly isolated strain S. albulus NJZJSA2 was identified according to its morphological characteristic (Supporting Information Table S1), physiological properties (Supporting Information Table S2), and 16S rRNA gene sequence analysis (Supporting Information Fig. S1).
Table 2. Pure compounds purchased from reagent companies. Compound
Chemical structure
Source
Purity
Toluene
A
99.5
Styrene
B
99.0
Anisole
C
99.5
2-Pentylfuran
C
98.0
4-Methoxystyrene
C
98.0
Tetradecane
C
99.0
A: Shenghe Chemical Reagent Co., Ltd. (Nanjing, China), B: Lingfeng chemical Reagent Co., Ltd. (Shanghai, China), and C: Aladdin Reagent Database, Inc. (Shanghai, China). ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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The strain grew well on various media, produced abundant spores, and the aerial mycelium was white– gray. The morphological characteristics of S. albulus NJZJSA2 on different media are given in Supporting Information Table S1. The 16S rRNA gene sequence of S. albulus NJZJSA2 contained 1551 bp nucleotides (NCBI Accession Number KJ778868). The BLAST results indicated that S. albulus NJZJSA2 was branched with a member of Streptomyces strains and had the highest similarity of 99% with S. albulus AB024440 (Supporting Information Fig. S1). Most of the studied morphological and physiological characteristics of S. albulus NJZJSA2 were similar to S. albulus ATCC 31713 except for the production of indole and the negative result for the utilization of L-rhamnose and D-raffinose (Supporting Information Table S2). Thus, strain NZJSA2 was identified as S. albulus NZJSA2. Antagonistic effect of VOCs on SS and FO The effect of VOCs on the mycelia growth of two pathogenic fungi was evaluated. The VOCs produced by S. albulus NJZJSA2, grown on autoclaved wheat seeds inhibited the mycelia growth of FO and SS (Fig. 1A and B). The inhibition rates of the two fungi were increased with increasing incubation time. The greatest FO inhibition rate was approximately 56.3% after 84 h of incubation, and the inhibition rate of SS was 100% after 60 h of incubation (Fig. 2). The germination of SS sclerotia and
FO conidia was completely suppressed in the presence of VOCs produced by S. albulus NJZJSA2 (Fig. 1C and D). Interestingly, both fungi recovered their growth after the elimination of VOCs, but the growth speeds were slower compared to the control. These results indicate that the VOCs did not cause mycelium death but did inhibit the mycelia growth. Effect of VOCs on the vitality of FO conidia and SS sclerotia in soil Previous research has shown that the composition of the growth substrate directly affects the chemical composition and quantity of the VOCs [28, 29]. In this study, we used nutrient-rich media (wheat seeds) and nutrientpoor media (rice soil) to test the production of antifungal VOCs by S. ablulus NJZJSA2. After 45 days of incubation of S. albulus NJZJSA2 in the autoclaved wheat seeds or sterile soil, the survived FO conidia were 0.17 104 g1 and 1.43 106 g1, respectively. The survived FO conidia in the control autoclaved wheat seeds and sterile soil were 3.83 106 g1 and 2.53 106 g1, respectively. All of the SS sclerotia survived after exposure to the VOCs for 45 days, but the germination speed of different treatments was significantly different (Fig. 3). In the autoclaved wheat seeds, the germinated numbers of sclerotia were approximately 1.67 and approximately 6.67 in the control treatment after 72 h. As incubation proceeded, the germination of all sclerotia took an
Figure 1. S. albulus NJZJSA2 antifungal volatile activity in the dish chamber. Mycelia plug growth of FO (A) and SS (B) were inhibited in the presence of the S. albulus NJZJSA2 growth on autoclaved wheat seeds (right), compared to the control (left). Volatile organic compounds produced by S. albulus NJZJSA2 affected the germination of spores of FO (C) and sclerotia germination of SS (D) were inhibited in the presence of the S. albulus NJZJSA2 (left) as compared to the control (right). ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 2. Mycelia growth of SS (A) and FO (B) measured in the absence or presence of VOCs produced by S. albulus NJZJSA2. The dotted lines means that the VOCs inhibition effect were eliminated after 60 (A) and 84 h (B). The error bars indicate the standard deviation of the means calculated from three independent samples.
additional 48 h, while it only took another 12 h in the control treatment. In autoclaved soil, the germinated numbers of sclerotia were approximately 4.67 and approximately 7.00 in the control treatment after 72 h. As incubation continued, all sclerotia germination took another 24 h, while it only took another 12 h in the control treatment. GC/MS analysis of VOCs produced by strain NJZJSA2 Different organisms and different species release similar and different types of VOCs [18, 19]. Thirteen peaks were found significantly different when compared the GC/MS chromatogram of VOCs produced by
S. albulus NJZJSA2 grown on autoclaved wheat seeds with the GC/MS chromatogram of VOCs released from fresh autoclaved wheat seeds (Supporting Information Fig. S2). These peaks were identified as VOCs produced by S. albulus NJZJSA2. The thirteen VOCs produced by S. albulus NJZJSA2 belonged to aromatic hydrocarbons, alkyls, ethers, lipids, and alkenes (Table 1), which showed high similarity indices (90%) with the VOCs from the NIST library. Among those, only six VOCs could be purchased from the regent company (Table 2). Thus, these volatile compounds were selected for further individual testing of antifungal activity against FO and SS.
Figure 3. The survived numbers of sclerotia treated with VOCs produced by S. albulus NJZJSA2. AWS þ Str: VOCs produced by S. albulus NJZJSA2 grown on autoclaved wheat seeds. AWS: autoclaved wheat seeds only. SS þ Str: VOCs produced by S. albulus NJZJSA2 grown in sterile soil. SS: Sterile soil only. The error bars indicate the standard deviation of the mean calculated from three independent samples. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Effect of select volatile compounds against FO and SS The concentration of VOCs was an important factor while acting as antifungal agents [18]. All six compounds showed antifungal activity against FO (Fig. 4) and SS (Fig. 5). As incubation proceeded, the mycelia growth rate of FO and SS were slower in the presence of different concentration of the six pure VOCs than the control treatment. The growth rate was decreased when the concentration of the pure VOCs was increased. However, the inhibition rate of the two plant pathogen fungi was significantly different (Fig. 6). Among those, 4-methoxystyrene exhibited 91.32% inhibition of SS mycelia growth at 80 ml/plate, followed by 2-pentylfuran (76.87%), anisole (76.30%), styrene (50.86%), toluene (50.86%), tetradecane (12.71%). However, the most effective compound against FO, 4-methoxystyrene, exhibited only 45.90% inhibition of FO mycelia growth, followed by anisole (37.70%), 2-pentylfuran (24.59%), tetradecane (21.31%), styrene (14.75%), toluene (8.19%). Effects of 4-methoxystyrene on the ultrastructure of FO and SS Among the six selected VOCs, 4-methoxystyrene was the most effective VOC against both FO and SS. Thus, 4-methoxystyrene was chosen to observe its effect on the morphology of the growing FO or SS mycelia by SEM and optical microscope. The cell wall and plasmalemma of the hyphae from the untreated control were normal, regular, and homogenous (Fig. 7E and Fig. 8E). Slight changes were found on the hyphae of FO or SS after being treated with low concentrations of 4-methoxystyrene (Fig. 7F and Fig. 8F). In contrast, the hyphae treated with high concentrations of 4-methoxystyrene showed considerable changes in hyphal morphology. The apices of terminal hyphae of both FO and SS were shortened and abnormally swollen (Fig. 7C and D; Fig. 8C and D). The cell walls of both fungi were severely collapsed and shrunken (Fig. 7G and H; Fig. 8G and H). The shrunken degree of conidia of FO was also increased with the increasing concentration of 4-methoxystyrene (Fig. 7I–L). Optical microscope analysis revealed that most of FO or SS hyphae were deformed and the plasma membrane was strongly retracted (Fig. 9). Due to loss of cellular impermeability to trypan blue, some dead hyphae were visualized as dark blue hyphae owing to the incorporation of trypan blue dye (Fig. 9B and D). Discussion Streptomyces spp. have been identified as a promising resource for the biocontrol of plant diseases. They have the ability to produce active antifungal and antibacterial compounds that have been developed for agricultural ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
uses [30]. Streptomyces spp. are also known to produce strong odors, and previous studies have reported the antifungal activity of volatile emissions, that led us to investigate the antifungal potential of VOCs produced by newly isolated and identified S. albulus NJZJSA2. The results showed that the growth of FO and SS was inhibited in double-dish chamber assay. There was no direct contact between the fungus and S. albulus NJZJSA2, suggesting the presence of antifungal VOCs. The SS sclerotia and FO conidia are the primary long-term survival structures that remained viable for several years in soil and play important role in their disease cycles [31, 32]. Previous reports have shown that inhibiting the germination of resting structures could damage the disease cycles and protect plants [31]. Here, we found that the germination of SS sclerotia and FO conidia was completely inhibited when exposed to VOCs produced by S. albulus NJZJSA2. Unfortunately, both fungi recovered their growth after the elimination of VOCs. Our results were in agreement with the results of Li et al. [19], who reported that the mycelial plugs of P. italicum exposed to the antifungal VOCs for 5 days, showed growth after being transferred to fresh PDA plates. The information implied that VOCs produced by the strain S. albulus NJZJSA2 were responsible for the toxic effects on SS and FO, but could not kill them completely. The role of secondary metabolites is important for underground communication between living organisms [33]. The VOCs are ideal info-chemicals because they can act over a wide range of distances and their spheres of activity will extend from proximal interactions, due to aqueous diffusion, to greater distances via diffusion in the air, including soil pores [4]. Previous reports from Fernando et al. [26] showed the release of antifungal VOCs from soil amended with antagonistic bacterial. In this study, S. ablulus NJZJSA2 was also able to produce VOCs in sterile soil, which decreased the amount of surviving FO conidia and the vitality of SS sclerotia. After 45 days of treatment with VOCs, we observed that the numbers of FO conidia were significantly decreased and the SS sclerotia germination speed was slower as compared to the control (Fig. 3). It revealed that the continuous exposure of fungal resting structures to antifungal VOCs restrain their growth. However, the antifungal effect of VOCs produced from sterile soil was not as effective as observed from autoclaved wheat seeds. This might be because of the nutrient-poor conditions or the action of adsorbents in soil, which led to less release of VOCs [34]. These findings indicate the potential use of S. ablulus NJZJSA2 as a soil amendment for managing SS and FO soil-borne plant pathogens.
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Figure 4. Mycelia growth of Fusarium oxysporum in the presence of styrene (A), toluene (B), anisole (C), tetradecane (D), 2-pentylfuran (E), 4methoxystyrene (F), at different concentrations (5, 10, 20, 40, 60, 80 ml/plate).
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Figure 5. Mycelia growth of Sclerotinia sclerotiorum in the presence of styrene (A), toluene (B), anisole (C), tetradecane (D), 2-pentylfuran (E), 4-methoxystyrene (F), at different concentrations (5, 10, 20, 40, 60, 80 ml/plate).
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Figure 6. Effect of different pure volatile compounds with increasing concentrations on the percent inhibition of FO (A) and SS (B).
The antifungal effects of VOCs were closely related to the chemical nature of each component. Thus, the VOCs produced by S. ablulus NJZJSA2 were further identified by GC-MS. A total of 13 volatile substances of S. ablulus NJZJSA2 were identified in this study. The types and compositions of VOCs produced by S. ablulus NJZJSA2 were similar in part to those produced by other Streptomyces spp. [18, 19], which mainly included ketones, alkanes, alkenes, terpenes, and terpene derivatives. Trans-1, 10-dimethyl-trans-9-decalol (Geosmin), a tertiary alcohol with an earthy smell, was produced by numerous microorganisms, such as Penicillium spp., Aspergillus spp., Streptomyces spp., Noncyanobacteria, and Cyanobacteria [19]. We found two derivatives of naphthalene in this study, naphthalene-1, 2, 3, 4, 4a, 7hexahydro-1,6-dimethyl-4-(1- methylethyl)- has been reported to be one of the antimicrobial VOC in essential oils of wood or volatile constituents of propolis [19]. Among the identified VOCs, toluene, styrene, anisole, 2-pentylfuran, 4-methoxystyrene, and tetradecane were also produced by other microorganisms, including Muscodor albus [35], Hypoxylon sp. CI-4 [36], and B. megaterium [37]. But few reports studied their individual activity against fungal pathogen, especially against SS and FO. The anti-SS activity order of the six compounds was shown to be 4-methoxystyrene > anisole > 2-pentylfuran > toluene > styrene > tetradecane at the concentration level of 80 ml/plate. The anti-FO order of the six compounds was shown to be 4-methoxystyrene > anisole > 2-pentylfuran > tetradecane > styrene > toluene. The differences were probably due to the different sensitivity of these fungi toward the six compounds. According to our results, toluene and tetradecane were less effective ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
in inhibiting the growth of SS and FO, which is consistent with the report of Yuan et al. [13]. Huang et al. [38] reported that styrene could not inhibit mycelial growth of B. cinerea. However, it could inhibit mycelial growth of SS and FO in our study. Considering their antifungal activity and low production levels, toluene, tetradecane, and styrene were not promising candidates as effective antifungal VOCs produced by S. ablulus NJZJSA2. The compound 2-pentylfuran has been reported to exhibit plant growth regulatory properties [37] and nematicidal activity [39]. We found that 2-pentylfuran effectively inhibited the mycelial growth of SS and FO, that extended our new knowledge of the biological activity of 2-pentylfuran. Anisole released by C. crocatus was reported antifungal by Fitzgerald et al. [40]. Similarly, in this study, anisole was effective in inhibiting the growth of SS and FO. The 4-methoxystyrene was more toxic when compared with the other five tested VOCs. Huang et al. [39] reported that 4-methoxystyrene produced by B. megaterium YFM3.25 showed nematicidal activity. Considering the antifungal activity (Fig. 6) and the relative peak area (Table 1) of VOCs, 4-methoxystyrene, 2-pentylfuran, and anisole were potential candidates as effective antifungal VOCs produced by S. albulus NJZJSA2. The mycelia growth processes of SS and FO were suppressed in the presence of 4-methoxystyrene. The SEM micrographs showed that both SS and FO terminal hyphae were shortened and abnormally swollen when exposed to the high concentration of 4-methoxystyrene. The mycelia cell wall of both fungi was gradually shrunk, but completely intact. These results suggested that the action location of 4-methoxystyrene was not the
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Figure 7. Scanning electronic micrographs (SEM) of Fusarium oxysporum growing on PDA in the absence (A, E, I) or presence (B, F, J: 10 ml/ plate; C, G, K: 80 ml/plate; D, H, L: 200 ml/plate) of 4-methoxystyrene. A–D: terminal hyphae; E–H: hyphae; I–L: spores. Arrows indicate cell wall damaged by volatile substances.
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Figure 8. Scanning electronic micrographs (SEM) of Sclerotinia sclerotiorum growing on PDA in the absence (A, E) or presence (B, F: 10 ml/ plate; C, G: 80 ml/plate; D, H: 200 ml/plate) of 4-methoxystyrene. A–D: terminal hyphae; E–H: hyphae. Arrows indicate cell wall damaged by volatile substances.
Figure 9. Optical micrographs of hyphae stained by trypan blue. A and C: normal hyphae of Sclerotinia sclerotiorum and Fusarium oxysporum, respectively. B and D: hyphae of Sclerotinia sclerotiorum and Fusarium oxysporum treated with 4-methoxystyrene (200 ml/plate). Red arrows show hyphal deformation. Dark staining indicates dead hyphae. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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cell wall. However, a previous research reported that the VOCs from S. philanthi RM-1-138 damaged the fungal cell wall [17]. Different results were probably due to different components of the VOCs. Optical micrograph results indicated the loss of cellular impermeability to trypan blue after treated with 4-methoxystyrene at the concentration of 200 ml/plate. A previous study indicated that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue [41]. This indicated the cytoplasmic extrusions from the plasma membrane of fungal cells. This result was similar to the damage induced by the VOCs from S. globisporus JK-1 against P. italicum [19]. The antifungal mechanism of 4-methoxystyrene might be damaging the fungal cell membrane. In conclusion, we showed that the VOCs from S. albulus NJZJSA2 have efficacy against the two fungal pathogens, S. sclerotiorum and F. oxysporum in agar medium and in soil. The microscopic analysis revealed that the VOCs affected the pathogenic fungus by altering their hyphae and terminal hyphae morphology. The results are useful for the better understanding of the biocontrol mechanisms of S. albulus NJZJSA2 against S. sclerotiorum and F. oxysporum. The pot and field experiments are underway to evaluate the efficiency of VOCs produced by S. albulus NJZJSA2 under natural soil conditions to reduce the viability of S. sclerotiorum and F. oxysporum and the effect on soil microflora.
[4] Compant, S, Duffy, B, Nowak, J, Clement, C., et al., 2005. Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Appl. Environ. Microbiol., 71, 4951–4959. [5] Yuan, J., Ruan, Y., Wang, B., Zhang, J., et al., 2013. Plant growth-promoting rhizobacteria strain Bacillus amyloliquefaciens NJN-6-enriched bio-organic fertilizer suppressed fusarium wilt and promoted the growth of banana plants. J. Agric. Food Chem., 61, 3774–3780. [6] Ling, N., Xue, C., Huang, Q. W., Yang, X. M., et al. 2010. Development of a mode of application of bioorganic fertilizer for improving the biocontrol efficacy to Fusarium wilt. Biocontrol, 55, 673–683. [7] Bubici, G., Marsico, A., D'Amico, M., Amenduni, M., et al., 2013. Evaluation of Streptomyces spp. for the biological control of corky root of tomato. Appl. Soil Ecol., 72, 128–134. [8] Errakhi, R., Lebrihi, A., Barakate, M., 2009. In vitro and in vivo antagonism of actinomycetes isolated from Moroccan rhizospherical soils against Sclerotium rolfsii: a causal agent of root rot on sugar beet (Beta vulgaris L.). J. Appl. Microbiol., 107, 672–681. [9] Getha, K., Vikineswary, S., 2002. Antagonistic effects of Streptomyces violaceusniger strain G10 on Fusarium oxysporum f. sp. cubense race 4: indirect evidence for the role of antibiosis in the antagonistic process. J. Ind. Microbiol. Biotechnol., 28, 303–310. [10] Wilkins, K., Larsen, K., Simkus, M., 2000. Volatile metabolites from mold growth on building materials. Chemosphere, 41, 437–446. [11] Chater, K.F., Biro, S., Lee, K.J., Palmer, T., et al., 2010. The complex extracellular biology of Streptomyces. FEMS Microbiol. Rev., 34, 171–198.
Acknowledgments This research was financially supported by Jiangsu Collaborative Innovation Center, the Nature Science Foundation of China (31272255, C150705), Innovative Research Team Development Plan of the Ministry of Education of China (IRT1256), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the 111 project (B12009). Conflict of interest The authors have no conflict of interest.
[12] Morath, S.U., Hung, R., Bennett, J.W., 2012. Fungal volatile organic compounds: a review with emphasis on their biotechnological potential. Fungal Biol. Rev., 26, 73–83. [13] Yuan, J., Raza, W., Shen, Q., Huang, Q., 2012. Antifungal activity of Bacillus amyloliquefaciens NJN-6 volatile compounds against Fusarium oxysporum f. sp. cubense. Appl. Environ. Microbiol., 78, 5942–5944. [14] Raza, W., Faheem, M., Yousaf, S., Rajer, FU., Yameen, M., 2013. Volatile and non-volatile antifungal compounds produced by Trichoderma harzianum SQR-T037 suppressed the growth of Fusarium oxysporum f. sp. niveum. Sci. Lett., 1, 21–24. [15] Rosen, A., Safferman, R., Mashni, C., Romano, A., 1968. Identity of odorous substance produced by Streptomyces griseoluteus. Appl. Microbiol., 16, 178.
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Research Paper Effects of volatile organic compounds from Streptomyces albulus NJZJSA2 on growth of two fungal pathogens Yuncheng Wu1,2,3, Jun Yuan1,2,3, Yaoyao E1,2,3, Waseem Raza1,2,3, Qirong Shen1,2,3 and Qiwei Huang1,2,3 1
2
3
National Engineering Research Center for Organic-based Fertilizers, Nanjing Agricultural University, Nanjing 210095, China Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing 210095, China
A Streptomyces albulus strain NJZJSA2 was isolated from the forest soil sample of Tzu-chin Mountain (Nanjing China) and identified based on its morphological and physiological properties and 16S rDNA gene sequence analysis. The strain S. albulus NJZJSA2 was evaluated for the production of antifungal volatile organic compounds (VOCs) against two fungal pathogens. Results showed that the VOCs generated by S. albulus NJZJSA2 inhibited mycelial growth of Sclerotinia sclerotiorum (SS) and Fusarium oxysporum (FO) by 100 and 56.3%, respectively. The germination of SS sclerotia and FO conidia was completely inhibited in the presence of VOCs produced by S. albulus NJZJSA2 in vitro. In soil, the VOCs delayed the germination of SS sclerotia and inhibited the germination of FO conidia for 45 days. The strain S. albulus NJZJSA2 was able to produce 13 VOCs based on GC/MS analyses. Among those, six compounds were purchased and used for the antifungal activity assay. Three relatively abundant VOCs, 4-methoxystyrene, 2pentylfuran, and anisole were proved to have antifungal activity. Microscopy analysis showed that the pathogen hyphae were shriveled and damaged after treatment with 4-methoxystyrene. These results suggest that the S. albulus strain NJZJSA2 produce VOCs that not only reduce the growth of SS and FO, but also significantly inhibit the SS sclerotia and FO conidia. The results are useful for the better understanding of biocontrol mechanisms by S. albulus strains and will help to improve the biological control efficiency of lethal plant diseases.
: Additional supporting information may be found in the online version of this article at the publisher's web-site. Keywords: Volatile organic compounds / Streptomyces albulus / Fusarium oxysporum / Sclerotinia sclerotiorum Received: November 27, 2014; accepted: February 21, 2015 DOI 10.1002/jobm.201400906
Introduction The widespread soil-borne pathogens, Fusarium oxysporum f. sp. cucumerinum J. H. Owen (FO) and Sclerotinia sclerotiorum (Lib.) De Bary (SS) are responsible for the Fusarium wilt of cucumber and sclerotinia stem rot of oilseed rape, respectively [1, 2]. These fungal pathogens persist in soil as dormant long-lived structures, chlamydospores, and Correspondence: Qiwei Huang, Jiangsu Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China E-mail: [email protected] Phone: þ86-025-84396212 Fax: þ86-025-84396824 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
sclerotia, which can survive for several years in the absence of host plants [3]. Traditionally, chemical controls can effectively protect plants from infectious pathogens [4]. However, sustainable agriculture requires that plant disease-control strategies become more ecologicalbased and less dependent on outside factors, such as synthetic chemicals. Biological control has been considered as an alternative safe method for controlling soilborne fungal pathogens [5, 6]. Among the biocontrol agents, actinomycetes have received considerable attention, particularly Streptomyces species. The efficacy of antagonistic Streptomyces spp. in the biological control of plant pathogenic fungi, such as
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Pyrenochaeta lycopersici [7], Sclerotiorum rolfsii [8], and Fusarium oxysporum [9], has been widely studied. Streptomyces species are well known for their ability to produce antibiotics and volatile organic compounds (VOCs) [10, 11]. The VOCs are low-molecular-weight compounds that easily evaporate at normal temperature and pressure, conferring the characteristic of diffusion through the atmosphere and soil [12]. The release of VOCs by soil microbes has been reported to inhibit the growth of fungal pathogens, promote plant growth, display nematicidal activity, and induce systemic resistance in crops [13, 14]. Previous studies about VOCs produced by Streptomyces spp. mainly focused on their effects on the environmental chemistry, such as off-odor in water pollution [15] or indoor air [16]. Recently, the studies of VOCs produced by Streptomyces spp. have focused on the control of plant diseases [17]. Wan et al. [18] reported that the VOCs produced by S. platensis F-1 demonstrated antifungal activity against Rhizoctonia solani, S. sclerotiorum, and Botrytis cinerea. Similarly, VOCs generated from Streptomyces globisporus JK-1 suppressed Penicillium italicum [19]. However, different organisms and different species release similar and different types of VOCs [11, 18, 19]. Therefore, it is important to investigate more and more biocontrol agents for the production of VOCs with novel structures to use those as biocontrol agents effectively. In this study, Streptomyces albulus strain NJZJSA2 was isolated from forest soil with a wide antifungal spectrum. S. albulus strains are known for their ability to produce e-polylysine, which exhibits antimicrobial activity against yeast, fungi, and Gram-positive and Gram-negative bacteria [20]. S. albulus strains have been reported as biocontrol agents against fruit and vegetable pathogens [21]. However, little information is currently available on the antifungal activity of VOCs produced by S. albulus strains against fungal pathogens. The goal of this study was to determine the effect of the VOCs produced by S. albulus NJZJSA on two fungal pathogens and to identify the antifungal VOCs released from S. albulus NJZJSA2.
Materials and methods Microorganisms and cultural conditions Two fungal pathogen strains Fusarium oxysporum f. sp. cucumerinum J. H. Owen and Sclerotinia sclerotiorum (Lib.) De Bary, which exhibited high virulence in cucumber (Cucumis sativus) and oilseed rape (Brassica napus), respectively, were used as the target fungi. Stock cultures were maintained on potato dextrose agar (PDA) ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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plates at 4 °C. Pre-cultures were established by transferring a stock agar plug containing mycelia of two pathogens onto PDA Petri dishes and incubating for 5 days at 25 °C for SS, and at 28 °C for FO. The S. albulus strain NJZJSA2 with broad inhibition spectrum (data not shown) was isolated from forest soil samples from the Tzu-chin Mountain (N32°030 , E118°510 , Nanjing, China,). For the S. albulus NJZJSA2, stock cultures were maintained on PDA at 4 °C, and a pre-culture (104 CFU ml1) was established by inoculating one agar plug (about 1 cm diameter) containing spores of S. albulus NJZJSA2 into 50 ml of Bennett medium [22] and incubated at 28 °C for 24 h in an orbital incubator at 170 rpm. Identification of strain NJZJSA2 To identify strain NJZJSA2, the physiological and biochemical characteristics were evaluated according to the International Streptomyces Project (ISP) [23]. The spore, shape, and chain morphology were observed using light microscopy. The assimilation of carbohydrates was studied using ISP9 medium containing different carbohydrates (1%, w/v) as the sole carbon source [24]. The 16S rRNA gene of S. albulus NJZJSA2 was amplified, sequenced [25], and BLAST searched against the NCBI database. The sequences of its close relatives were used to construct a neighbor-joining phylogenetic tree using MEGA 4.0. Culture preparation for the production of VOCs Autoclaved wheat seeds (autoclaved at 120 °C for 20 min) were used to prepare cultures of S. albulus NJZJSA2 for the production of VOCs. A 5 ml pre-culture of S. albulus NJZJSA2 as described above, was inoculated into the autoclaved wheat seeds (20 g wheat seeds, plus 40 ml distilled water) for two days before use. In soil tests, 5 ml pre-culture of S. albulus NJZJSA2 was centrifuged at 4000 rpm for 5 min and then resuspended in 5 ml of sterile distilled water before the inoculation of 20 g autoclaved soil. VOCs antagonistic effects on FO and SS growth The VOCs produced by S. albulus NJZJSA2 were tested according to the method of Fernando [26] with some modifications. The Petri plates containing 10 g of autoclaved wheat seeds inoculated with S. albulus NJZJSA2 were prepared as described above and covered with another Petri plate containing PDA inoculated with a 6 mm diameter plug of FO or SS. The two plates were then sealed with Parafilm to obtain a double-plate chamber. The average distance between autoclaved wheat seeds surface of lower plates and agar surface of upper plates was 1.5 cm. The plates containing FO
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were incubated at 28 °C for 84 h, and the plates containing SS were incubated at 25 °C for 60 h. The lower plates containing S. albulus NJZJSA2 were then removed and the upper plates containing fungal mycelia were re-covered with new Petri plates to eliminate the VOCs effect. The upper plates containing FO or SS were incubated for another 36 or 60 h. The control plates had only autoclaved wheat seeds. All treatments were performed in triplicate. The diameters of mycelia were measured at 12 h intervals during incubation. VOCs antagonistic effects on FO conidia and SS sclerotia germination The effects of VOCs on the germination of conidia of FO and sclerotia of SS were studied by different methods. The method used for the FO spore germination assay was as described above except that 100 ml of spore solution (106 CFU ml1) was spread on PDA instead of a FO agar plug. Sclerotia germination of SS was studied according to the method of Li [19]. Briefly, four small Petri dish bottoms (60 mm in diameter and 15 mm in height) were placed inside a larger Petri dish (150 mm in diameter and 30 mm in height). Three of the small Petri dish bottoms contained 5 g of autoclaved wheat seeds inoculated with S. albulus NJZJSA2 were prepared as described above, and the fourth dish contained six surface-sterilized SS sclerotia. Non-inoculated, autoclaved wheat seeds were used as a control. The larger dish was sealed with Parafilm and incubated at 28 °C. The FO spore germination was measured after 3 days of incubation, and the number of sclerotia germination were recorded after 10 days of incubation. Each treatment had three replicates. VOCs effects on FO conidia and SS sclerotia vitality in soil The effects of VOCs produced by S. albulus NJZJSA2 from autoclaved wheat seeds and soil were studied. The soil was collected from the rice field with the following properties: pH 7.29, organic carbon, 22.31 g/kg, total N, 2.55 g/kg, total P, 1.02 g/kg, and available K 272.89 mg/ kg. Four small Petri dish bottoms (60 mm in diameter and 15 mm in height) were placed inside a large Petri dish (150 mm in diameter and 30 mm in height). Three of the small Petri dish bottoms contained 10 g of autoclaved wheat seeds or soil inoculated with S. albulus NJZJSA2 were prepared as described above. The fourth dish contained 10 g of soil inoculated with 1 ml of FO spore solution (108 CFU ml1) or 10 surface-distilled sclerotia. The larger dish was sealed with Parafilm and incubated at 28 °C. Each treatment had three replicates. The surviving conidia and sclerotia were counted after 45 days. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Identification of VOCs by SPME-GC-MS The VOCs produced by S. albulus NJZJSA2 grown on autoclaved wheat seeds were collected using solid-phase micro-extraction (SPME) after four days incubation; the VOCs released from fresh autoclaved wheat seeds were used as control [13]. A SPME fiber coated with divinylbenzene-carboxene-PDMS (DCP, 50/30 mm) was used for extracting the VOCs, and the samples were analyzed using the Trace DSQ (Finnigan) GC-MS system. The SPME fibers were desorbed at 250 °C for 5 min with an RTX-5MS column (30 m, 0.25 mm inside diameter, 0.25 mm). The initial oven temperature of 40 °C was held for 2 min, increased at a rate of 4 °C min1 to 150 °C, and held for 1 min, further increased at a rate of 10 °C min1 to 250 °C, and held for 4 min. The mass spectrometer was operated in the electron ionization mode at 70 eV with a source temperature of 220 °C. A continuous scan from 50 to 500 m/z was used. Helium was used as the carrier gas at a constant flow rate of 1.0 ml min1. The mass spectra of VOCs were compared with those in the National Institute of Standards and Technology (NIST) database (Version 2.0). Effect of synthetic compounds on SS and FO growth The VOCs produced by S. albulus NJZJSA2 grown on autoclaved wheat seeds were analyzed by GC-MS, and 13 compounds were detected when compared with autoclaved wheat seeds only (Table 1). Six of the identified VOCs were selected to test for their antagonistic activity (Table 2). These pure compounds were purchased from Shenghe Chemical Reagent Co., Ltd. (Nanjing, China), Lingfeng chemical Reagent Co., Ltd. (Shanghai, China), and Aladdin Reagent Database, Inc. (Shanghai, China). The divided plate method was used to perform the antagonistic assay of the selected VOCs. Sterile filter papers (15 20 mm) containing 5, 10, 20, 40, 60, 80 ml of each VOC were placed on one side of divided plates, and FO or SS mycelia plugs were placed on the PDA media of the other side. The control divided plates had only sterile filter papers. The colony size in each treatment was recorded and the percentage inhibition of hyphae growth was calculated using the following equation: Percentage of inhibition ¼ [{(Control-treatment)/ Control} 100] [17]. Microscopic observation of SS and FO FO or SS mycelia plugs were inoculated on the PDA side of divided plates with cover slips inserted at an angle. Four concentration gradients (0, 10, 80, 200 ml/plate) of 4-methoxystyrene were added to another side of the divided plates after 2 days inoculation and kept incubating for another 2 days. To prepare the samples
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Table 1. Volatile organic compounds produced by S. albulus NJZJSA2. RT(min) 1.15 3.6 4.26 6.19 11.42 14.28 19.00 19.34 20.73 21.46 22.81 25.99 28.84
Possible compound
Relative peak area (%)
Toluene Styrene Anisole 2-Pentylfuran 4-Methoxystyrene 1H-Indene,1-ethylideneoctahydro-7a-methyl-,(1Z,3aa,7ab) Trans-1,10-Dimethyl-trans-9-decalol Tetradecane Naphthalene,decahydro-4a-methyl-1-methylene-7(1-methylethenyl)-,[4aR-(4aa,7a,8ab)] 1H-cyclopenta[1,3]cyclopropa[1,2]benzene,octahydro-7-methyl-3-methylene-4(1-methylethyl)- (3aS,3bR,4S,7R,7aR)Naphthalene, 1,2,3,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)1H-Cycloprop[e]azulen-4-ol,decahydro-1,1,4,7-tetramethyl-, [1aR-(1aa,4b,4ab,7a,7ab,7b)] 2-Oxepanone, 7-hexyl-
0.04 3.55 10.7 11.83 32.15 0.99 7.97 4.06 3.2 13.63 2.51 2.57 6.78
The 13 compounds were identified by comparing with those in the NIST/EPA/NIH Mass Spectrometry Library with respect to the spectra in the Mainlib and/or Replib databases.
for scanning electron microscopy (SEM), the cover slips with mycelium were fixed with 2.5% glutaraldehyde and dehydrated with a graded series of ethanol washes followed by drying in a drier under CO2. Mounted specimens were coated for 2.5 min with 10 mA of goldpalladium and examined under a field emission SEM (S-4800, Hitachi, Japan) operating at 15 kV [5]. The FO or SS mycelia after being treated with 200 ml/plate of 4-methoxystyrene were examined by trypan blue staining according to the method of Weid et al. [27]. In brief, FO or SS mycelia was soaked with 5 ml trypan blue solution for 5 min, and then washed three times with
distilled water. Growth of mycelium was viewed with a fluorescent microscope (Nikon eclipse 80i).
Results Identification of S. albulus NJZJSA2 The newly isolated strain S. albulus NJZJSA2 was identified according to its morphological characteristic (Supporting Information Table S1), physiological properties (Supporting Information Table S2), and 16S rRNA gene sequence analysis (Supporting Information Fig. S1).
Table 2. Pure compounds purchased from reagent companies. Compound
Chemical structure
Source
Purity
Toluene
A
99.5
Styrene
B
99.0
Anisole
C
99.5
2-Pentylfuran
C
98.0
4-Methoxystyrene
C
98.0
Tetradecane
C
99.0
A: Shenghe Chemical Reagent Co., Ltd. (Nanjing, China), B: Lingfeng chemical Reagent Co., Ltd. (Shanghai, China), and C: Aladdin Reagent Database, Inc. (Shanghai, China). ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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The strain grew well on various media, produced abundant spores, and the aerial mycelium was white– gray. The morphological characteristics of S. albulus NJZJSA2 on different media are given in Supporting Information Table S1. The 16S rRNA gene sequence of S. albulus NJZJSA2 contained 1551 bp nucleotides (NCBI Accession Number KJ778868). The BLAST results indicated that S. albulus NJZJSA2 was branched with a member of Streptomyces strains and had the highest similarity of 99% with S. albulus AB024440 (Supporting Information Fig. S1). Most of the studied morphological and physiological characteristics of S. albulus NJZJSA2 were similar to S. albulus ATCC 31713 except for the production of indole and the negative result for the utilization of L-rhamnose and D-raffinose (Supporting Information Table S2). Thus, strain NZJSA2 was identified as S. albulus NZJSA2. Antagonistic effect of VOCs on SS and FO The effect of VOCs on the mycelia growth of two pathogenic fungi was evaluated. The VOCs produced by S. albulus NJZJSA2, grown on autoclaved wheat seeds inhibited the mycelia growth of FO and SS (Fig. 1A and B). The inhibition rates of the two fungi were increased with increasing incubation time. The greatest FO inhibition rate was approximately 56.3% after 84 h of incubation, and the inhibition rate of SS was 100% after 60 h of incubation (Fig. 2). The germination of SS sclerotia and
FO conidia was completely suppressed in the presence of VOCs produced by S. albulus NJZJSA2 (Fig. 1C and D). Interestingly, both fungi recovered their growth after the elimination of VOCs, but the growth speeds were slower compared to the control. These results indicate that the VOCs did not cause mycelium death but did inhibit the mycelia growth. Effect of VOCs on the vitality of FO conidia and SS sclerotia in soil Previous research has shown that the composition of the growth substrate directly affects the chemical composition and quantity of the VOCs [28, 29]. In this study, we used nutrient-rich media (wheat seeds) and nutrientpoor media (rice soil) to test the production of antifungal VOCs by S. ablulus NJZJSA2. After 45 days of incubation of S. albulus NJZJSA2 in the autoclaved wheat seeds or sterile soil, the survived FO conidia were 0.17 104 g1 and 1.43 106 g1, respectively. The survived FO conidia in the control autoclaved wheat seeds and sterile soil were 3.83 106 g1 and 2.53 106 g1, respectively. All of the SS sclerotia survived after exposure to the VOCs for 45 days, but the germination speed of different treatments was significantly different (Fig. 3). In the autoclaved wheat seeds, the germinated numbers of sclerotia were approximately 1.67 and approximately 6.67 in the control treatment after 72 h. As incubation proceeded, the germination of all sclerotia took an
Figure 1. S. albulus NJZJSA2 antifungal volatile activity in the dish chamber. Mycelia plug growth of FO (A) and SS (B) were inhibited in the presence of the S. albulus NJZJSA2 growth on autoclaved wheat seeds (right), compared to the control (left). Volatile organic compounds produced by S. albulus NJZJSA2 affected the germination of spores of FO (C) and sclerotia germination of SS (D) were inhibited in the presence of the S. albulus NJZJSA2 (left) as compared to the control (right). ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 2. Mycelia growth of SS (A) and FO (B) measured in the absence or presence of VOCs produced by S. albulus NJZJSA2. The dotted lines means that the VOCs inhibition effect were eliminated after 60 (A) and 84 h (B). The error bars indicate the standard deviation of the means calculated from three independent samples.
additional 48 h, while it only took another 12 h in the control treatment. In autoclaved soil, the germinated numbers of sclerotia were approximately 4.67 and approximately 7.00 in the control treatment after 72 h. As incubation continued, all sclerotia germination took another 24 h, while it only took another 12 h in the control treatment. GC/MS analysis of VOCs produced by strain NJZJSA2 Different organisms and different species release similar and different types of VOCs [18, 19]. Thirteen peaks were found significantly different when compared the GC/MS chromatogram of VOCs produced by
S. albulus NJZJSA2 grown on autoclaved wheat seeds with the GC/MS chromatogram of VOCs released from fresh autoclaved wheat seeds (Supporting Information Fig. S2). These peaks were identified as VOCs produced by S. albulus NJZJSA2. The thirteen VOCs produced by S. albulus NJZJSA2 belonged to aromatic hydrocarbons, alkyls, ethers, lipids, and alkenes (Table 1), which showed high similarity indices (90%) with the VOCs from the NIST library. Among those, only six VOCs could be purchased from the regent company (Table 2). Thus, these volatile compounds were selected for further individual testing of antifungal activity against FO and SS.
Figure 3. The survived numbers of sclerotia treated with VOCs produced by S. albulus NJZJSA2. AWS þ Str: VOCs produced by S. albulus NJZJSA2 grown on autoclaved wheat seeds. AWS: autoclaved wheat seeds only. SS þ Str: VOCs produced by S. albulus NJZJSA2 grown in sterile soil. SS: Sterile soil only. The error bars indicate the standard deviation of the mean calculated from three independent samples. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Effect of select volatile compounds against FO and SS The concentration of VOCs was an important factor while acting as antifungal agents [18]. All six compounds showed antifungal activity against FO (Fig. 4) and SS (Fig. 5). As incubation proceeded, the mycelia growth rate of FO and SS were slower in the presence of different concentration of the six pure VOCs than the control treatment. The growth rate was decreased when the concentration of the pure VOCs was increased. However, the inhibition rate of the two plant pathogen fungi was significantly different (Fig. 6). Among those, 4-methoxystyrene exhibited 91.32% inhibition of SS mycelia growth at 80 ml/plate, followed by 2-pentylfuran (76.87%), anisole (76.30%), styrene (50.86%), toluene (50.86%), tetradecane (12.71%). However, the most effective compound against FO, 4-methoxystyrene, exhibited only 45.90% inhibition of FO mycelia growth, followed by anisole (37.70%), 2-pentylfuran (24.59%), tetradecane (21.31%), styrene (14.75%), toluene (8.19%). Effects of 4-methoxystyrene on the ultrastructure of FO and SS Among the six selected VOCs, 4-methoxystyrene was the most effective VOC against both FO and SS. Thus, 4-methoxystyrene was chosen to observe its effect on the morphology of the growing FO or SS mycelia by SEM and optical microscope. The cell wall and plasmalemma of the hyphae from the untreated control were normal, regular, and homogenous (Fig. 7E and Fig. 8E). Slight changes were found on the hyphae of FO or SS after being treated with low concentrations of 4-methoxystyrene (Fig. 7F and Fig. 8F). In contrast, the hyphae treated with high concentrations of 4-methoxystyrene showed considerable changes in hyphal morphology. The apices of terminal hyphae of both FO and SS were shortened and abnormally swollen (Fig. 7C and D; Fig. 8C and D). The cell walls of both fungi were severely collapsed and shrunken (Fig. 7G and H; Fig. 8G and H). The shrunken degree of conidia of FO was also increased with the increasing concentration of 4-methoxystyrene (Fig. 7I–L). Optical microscope analysis revealed that most of FO or SS hyphae were deformed and the plasma membrane was strongly retracted (Fig. 9). Due to loss of cellular impermeability to trypan blue, some dead hyphae were visualized as dark blue hyphae owing to the incorporation of trypan blue dye (Fig. 9B and D). Discussion Streptomyces spp. have been identified as a promising resource for the biocontrol of plant diseases. They have the ability to produce active antifungal and antibacterial compounds that have been developed for agricultural ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
uses [30]. Streptomyces spp. are also known to produce strong odors, and previous studies have reported the antifungal activity of volatile emissions, that led us to investigate the antifungal potential of VOCs produced by newly isolated and identified S. albulus NJZJSA2. The results showed that the growth of FO and SS was inhibited in double-dish chamber assay. There was no direct contact between the fungus and S. albulus NJZJSA2, suggesting the presence of antifungal VOCs. The SS sclerotia and FO conidia are the primary long-term survival structures that remained viable for several years in soil and play important role in their disease cycles [31, 32]. Previous reports have shown that inhibiting the germination of resting structures could damage the disease cycles and protect plants [31]. Here, we found that the germination of SS sclerotia and FO conidia was completely inhibited when exposed to VOCs produced by S. albulus NJZJSA2. Unfortunately, both fungi recovered their growth after the elimination of VOCs. Our results were in agreement with the results of Li et al. [19], who reported that the mycelial plugs of P. italicum exposed to the antifungal VOCs for 5 days, showed growth after being transferred to fresh PDA plates. The information implied that VOCs produced by the strain S. albulus NJZJSA2 were responsible for the toxic effects on SS and FO, but could not kill them completely. The role of secondary metabolites is important for underground communication between living organisms [33]. The VOCs are ideal info-chemicals because they can act over a wide range of distances and their spheres of activity will extend from proximal interactions, due to aqueous diffusion, to greater distances via diffusion in the air, including soil pores [4]. Previous reports from Fernando et al. [26] showed the release of antifungal VOCs from soil amended with antagonistic bacterial. In this study, S. ablulus NJZJSA2 was also able to produce VOCs in sterile soil, which decreased the amount of surviving FO conidia and the vitality of SS sclerotia. After 45 days of treatment with VOCs, we observed that the numbers of FO conidia were significantly decreased and the SS sclerotia germination speed was slower as compared to the control (Fig. 3). It revealed that the continuous exposure of fungal resting structures to antifungal VOCs restrain their growth. However, the antifungal effect of VOCs produced from sterile soil was not as effective as observed from autoclaved wheat seeds. This might be because of the nutrient-poor conditions or the action of adsorbents in soil, which led to less release of VOCs [34]. These findings indicate the potential use of S. ablulus NJZJSA2 as a soil amendment for managing SS and FO soil-borne plant pathogens.
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Figure 4. Mycelia growth of Fusarium oxysporum in the presence of styrene (A), toluene (B), anisole (C), tetradecane (D), 2-pentylfuran (E), 4methoxystyrene (F), at different concentrations (5, 10, 20, 40, 60, 80 ml/plate).
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Figure 5. Mycelia growth of Sclerotinia sclerotiorum in the presence of styrene (A), toluene (B), anisole (C), tetradecane (D), 2-pentylfuran (E), 4-methoxystyrene (F), at different concentrations (5, 10, 20, 40, 60, 80 ml/plate).
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Figure 6. Effect of different pure volatile compounds with increasing concentrations on the percent inhibition of FO (A) and SS (B).
The antifungal effects of VOCs were closely related to the chemical nature of each component. Thus, the VOCs produced by S. ablulus NJZJSA2 were further identified by GC-MS. A total of 13 volatile substances of S. ablulus NJZJSA2 were identified in this study. The types and compositions of VOCs produced by S. ablulus NJZJSA2 were similar in part to those produced by other Streptomyces spp. [18, 19], which mainly included ketones, alkanes, alkenes, terpenes, and terpene derivatives. Trans-1, 10-dimethyl-trans-9-decalol (Geosmin), a tertiary alcohol with an earthy smell, was produced by numerous microorganisms, such as Penicillium spp., Aspergillus spp., Streptomyces spp., Noncyanobacteria, and Cyanobacteria [19]. We found two derivatives of naphthalene in this study, naphthalene-1, 2, 3, 4, 4a, 7hexahydro-1,6-dimethyl-4-(1- methylethyl)- has been reported to be one of the antimicrobial VOC in essential oils of wood or volatile constituents of propolis [19]. Among the identified VOCs, toluene, styrene, anisole, 2-pentylfuran, 4-methoxystyrene, and tetradecane were also produced by other microorganisms, including Muscodor albus [35], Hypoxylon sp. CI-4 [36], and B. megaterium [37]. But few reports studied their individual activity against fungal pathogen, especially against SS and FO. The anti-SS activity order of the six compounds was shown to be 4-methoxystyrene > anisole > 2-pentylfuran > toluene > styrene > tetradecane at the concentration level of 80 ml/plate. The anti-FO order of the six compounds was shown to be 4-methoxystyrene > anisole > 2-pentylfuran > tetradecane > styrene > toluene. The differences were probably due to the different sensitivity of these fungi toward the six compounds. According to our results, toluene and tetradecane were less effective ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
in inhibiting the growth of SS and FO, which is consistent with the report of Yuan et al. [13]. Huang et al. [38] reported that styrene could not inhibit mycelial growth of B. cinerea. However, it could inhibit mycelial growth of SS and FO in our study. Considering their antifungal activity and low production levels, toluene, tetradecane, and styrene were not promising candidates as effective antifungal VOCs produced by S. ablulus NJZJSA2. The compound 2-pentylfuran has been reported to exhibit plant growth regulatory properties [37] and nematicidal activity [39]. We found that 2-pentylfuran effectively inhibited the mycelial growth of SS and FO, that extended our new knowledge of the biological activity of 2-pentylfuran. Anisole released by C. crocatus was reported antifungal by Fitzgerald et al. [40]. Similarly, in this study, anisole was effective in inhibiting the growth of SS and FO. The 4-methoxystyrene was more toxic when compared with the other five tested VOCs. Huang et al. [39] reported that 4-methoxystyrene produced by B. megaterium YFM3.25 showed nematicidal activity. Considering the antifungal activity (Fig. 6) and the relative peak area (Table 1) of VOCs, 4-methoxystyrene, 2-pentylfuran, and anisole were potential candidates as effective antifungal VOCs produced by S. albulus NJZJSA2. The mycelia growth processes of SS and FO were suppressed in the presence of 4-methoxystyrene. The SEM micrographs showed that both SS and FO terminal hyphae were shortened and abnormally swollen when exposed to the high concentration of 4-methoxystyrene. The mycelia cell wall of both fungi was gradually shrunk, but completely intact. These results suggested that the action location of 4-methoxystyrene was not the
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Figure 7. Scanning electronic micrographs (SEM) of Fusarium oxysporum growing on PDA in the absence (A, E, I) or presence (B, F, J: 10 ml/ plate; C, G, K: 80 ml/plate; D, H, L: 200 ml/plate) of 4-methoxystyrene. A–D: terminal hyphae; E–H: hyphae; I–L: spores. Arrows indicate cell wall damaged by volatile substances.
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Figure 8. Scanning electronic micrographs (SEM) of Sclerotinia sclerotiorum growing on PDA in the absence (A, E) or presence (B, F: 10 ml/ plate; C, G: 80 ml/plate; D, H: 200 ml/plate) of 4-methoxystyrene. A–D: terminal hyphae; E–H: hyphae. Arrows indicate cell wall damaged by volatile substances.
Figure 9. Optical micrographs of hyphae stained by trypan blue. A and C: normal hyphae of Sclerotinia sclerotiorum and Fusarium oxysporum, respectively. B and D: hyphae of Sclerotinia sclerotiorum and Fusarium oxysporum treated with 4-methoxystyrene (200 ml/plate). Red arrows show hyphal deformation. Dark staining indicates dead hyphae. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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cell wall. However, a previous research reported that the VOCs from S. philanthi RM-1-138 damaged the fungal cell wall [17]. Different results were probably due to different components of the VOCs. Optical micrograph results indicated the loss of cellular impermeability to trypan blue after treated with 4-methoxystyrene at the concentration of 200 ml/plate. A previous study indicated that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue [41]. This indicated the cytoplasmic extrusions from the plasma membrane of fungal cells. This result was similar to the damage induced by the VOCs from S. globisporus JK-1 against P. italicum [19]. The antifungal mechanism of 4-methoxystyrene might be damaging the fungal cell membrane. In conclusion, we showed that the VOCs from S. albulus NJZJSA2 have efficacy against the two fungal pathogens, S. sclerotiorum and F. oxysporum in agar medium and in soil. The microscopic analysis revealed that the VOCs affected the pathogenic fungus by altering their hyphae and terminal hyphae morphology. The results are useful for the better understanding of the biocontrol mechanisms of S. albulus NJZJSA2 against S. sclerotiorum and F. oxysporum. The pot and field experiments are underway to evaluate the efficiency of VOCs produced by S. albulus NJZJSA2 under natural soil conditions to reduce the viability of S. sclerotiorum and F. oxysporum and the effect on soil microflora.
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Acknowledgments This research was financially supported by Jiangsu Collaborative Innovation Center, the Nature Science Foundation of China (31272255, C150705), Innovative Research Team Development Plan of the Ministry of Education of China (IRT1256), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the 111 project (B12009). Conflict of interest The authors have no conflict of interest.
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