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An endophytic bacterium synthesizing homologous fragrant compounds as its host plant.

Gardenia jasminoides J. Ellis (Rubiaceae) is a popular ornamental plant with white and sweet fragrant flowers (1). For a long time, flowers of G jasminoides have universally been used for manufacturing of perfume (8). However, relying on the flower uniquely as material limits the yield of perfume due to the flowering once a year of the plants. Finding alternative resource from microorganisms for perfume production is considered.

Endophytic bacteria live in plant tissues without causing substantive harm to their hosts (10). Previous investigations indicated that endophytic bacteria exist in a variety of tissue types within a broad range of plants (16). It has been hypothesized and partially demonstrated that some endophytes can synthesize the same or homologous chemicals of their plant hosts (18). A typical example is the endophytic fungi from Taxus spp. could produce taxol (12). Although numerous studies concerning bacterial endophytes from different tissues of various plants have been reported11, rare attention was focused on that from flower. During a survey on the biodiversity of endophytic bacteria from fragrant flowers, ZZB08, a bacterial strain producing sweet flavor was isolated from flower of G jasminoides. Here, we identified the ZZB08 and characterized its volatile compounds comparing with the host flower by method of solid-phase micro-extraction (SPME) combined with GC/ MS.

MATERIALS AND METHODS

Flower sampling and isolation of endophytic bacteria

Fresh followers of G jasminoides were sampled in Kunming City in May, 2012. The samples were firstly washed thrice with sterile distilled water, then sterilized sequentially by 75% (v/v) ethanol and hypochlorite solution (3% available Cl-) for 30 sec, and finally washed thrice again with sterile distilled water. After surface sterilization, flower materials were homogenized in a mortar containing suitable sterile distilled water to obtain a concentration of [10.sup.-2]. After filtration to remove the tissue, a volume of 200 qL suspension was spread on a plate containing BPN ([L.sup.-1]: beef extract 3 g, peptone 10 g, NaCl 5 g, agar 20 g, water 1,000 ml, pH 7.2). Ten plates for each sample were preformed. After 48 h incubation at 37 [degrees]C, bacterial colonies were purified randomly, and those producing fragrant flavor were selected for further studies.

Bacterial identification

All physiological characteristics were tested with cells cultured using medium of Luria-Bertani (LB) with pH 7-8 at 28 [degrees]C. Gram staining was performed as described by Gregersen (4). Oxidase activity was detected using API oxidase reagent according to the manufacturer's instructions. Catalase activity was determined by assessing bubble production in 3 % (v/v) [H.sub.2][O.sub.2]. The oxidation of carbon source was performed using Biolog GEN III system (MicroPlate). Cell morphology and motility were observed by light microscopy (BH-2; Olympus) and transmission electron microscopy (H-7650; Hitachi). Growth at different pH (5-10, in increments of 1.0 pH units), temperatures (4, 10, 15, 20, 28, 37, 45, 50 and 55 [degrees]C) and sodium chloride regimes (0-12 % w/v, at intervals of 1%) was determined using LB agar plates incubated for up to 5 days.

Extraction of genomic DNA and amplification of the 16S rRNA gene were performed as described by Li et al. (13). The 16S rRNA gene sequences were aligned with representative bacterial sequences from the GenBank database by using ClustalX (20) and then manually adjusted. Distance matrices and phylogenetic trees were calculated by the Kimura two-parameter model (9) and neighbor-joining algorithms using the program MEGA (version 4) by bootstrap analysis of 1,000 replications (19). The tree was rooted using Thermoactinomyces vulgaris VTTE-062992 as outgroup.

Chemical analysis of bacterial culture and flower

Bacterium was cultured using BPN medium and their volatiles were extracted by SPME as described by Diaz et al. (3) with minor modification. Briefly, 75 iM fibers (Supelco, Bellefonte, PA, USA) used for SPME were first equilibrated with helium at 250 UC for 15 min. Extractions were performed inside 15 mL Supelco SPME vials filled with 9 ml bacterial culture and under the conditions of 40 UC for 1 h. Volatiles from BPN medium were used as control. For flower, the SPME fiber was inserted directly into the vial containing one fresh flower, and extraction was carried out at room temperature for 12 h. After extraction, the SPME fiber was directly inserted into the front inlet of GC-MS (MS, HP 5973, GC/ MS: Agilent Technologies, USA) and desorbed at 250 [degrees]C for 2 min. GC conditions were the same as Xu et al. (21). The volatile compounds were identified based on a comparison of the mass spectrum of the substance with GC/MS system data banks (Wiley 7 and NB S 75 k library).

RESULTS

Bacterial identification

Among the endophytic bacteria from flowers of G jasminoides, a strain designated ZZB08 producing sweet flavor on BPN medium was obtained. Cells of ZZB08 are motile, gram positive, bacilli with size of 0.71-1.0x1.88-4.27 im, occur singly and in short chains. Agar colonies are opaque, smooth, circular, entire, and 1.0-2.0 mm in diameter after growth at 28[degrees]C for 48 h. Growth occurs at 10-50[degrees]C, pH 6.0-9.0 and NaCl concentrations of 0-5 %. The optimal growth pH, temperature and NaCl concentration were 27-3 5[degrees]C, pH 7.0-8.0 and 0-2 %, respectively. Catalase and oxidase are positive. Growth is aerobic. Nitrate is reduced to nitrite. Starch and casein are hydrolyzed. Citrate is utilized, but propionate is not utilized. The 16S rDNA sequence of strain ZZB08 had been deposited in GenBank under the accession number JQ765433. Sequence comparison via BLAST searches against sequences from the GenBank, EMBL or DDBJ databases revealed that strain ZZB08 had a close relationship with members of the genus Bacillus, which was classified in the family Bacillaceae. The ZZB08 was most closely related to B. vallismortis with sequence similarity of 99.25%. Moreover, in a neighbour-joining phylogenetic tree based on 16S rDNA sequences, strain ZZB08 formed a stable clade with B. vallismortis (Fig. 1).

On the basis of morphologic, physiological, biochemical characteristics, and 16S rDNA sequence analysis, strain ZZB08 was identified as Bacillus vallismortis.

Comparison of the volatile compounds between flower and strain ZZB08

By method of SPME-GC/MS, 17 and 18 compounds with relative content (RT) more than 0.5% were respectively detected from flower and culture of ZZB08 (Table 1). These 35 chemical constituents covered a wide range of acids, alkenes, alcohols, aromatics, benzenes, esters, heterocyclics, ketones, monoterpenoids, monoterpene alcohols and sesquiterpenes. Major volatiles of the flower were linalool (RT = 35.74%), a-farnesene (11.43%), trans-b-ocimene (10.17%) and z-3-hexenyl tiglate (9.68%), in which their total RT values accounted to 67.02% of the all volatiles. While, the abundant compounds of the ZZB08 were 2-butanone, 3-hydroxy- (36.82%), acetone (9.12%), 2-thiophenecarboxylic acid, undec-10-enyl ester (8.39%) and linalool (5.28%). The four counted up to 59.61% of the volatiles produced by the bacterium.

Comparatively, five major volatile compounds of flower, linalool (RT = 35.74%), afarnesene (11.43%), trans-b-ocimene (10.17%), z-3-hexenyl tiglate (9.68%) and methyl tiglate (3.33%) were also found in culture of the ZZB08 but with lower concentrations than that in flower, in which their RT values were 5.28%, 3.54%, 2.33%, 2.15% and 0.86%, respectively. Additionally, those compounds of ketones and acids produced by ZZB08 were not found in the flower sample.

DISCUSSION

We identified a fragrant endophytic bacterium, B. vallismortis ZZB08, from fragrant flower of the plant G jasminoides. B. vallismortis was firstly isolated from soil in Death Valley Californial and as a novel taxon was reported (17). Strains of this species with different functions have previously been reported from other environments. B. vallismortis BIT-33 from seawater samples could produce cytotoxic compound and showed direct cytotoxic and apoptotic effects on colon cancer cells (7). B. vallismortis C89 from the South China Sea sponge Dysidea avara could produce compounds of neobacillamide A and bacillamide C (22). B. vallismortis BCCS 007 from the Maharla salt lake of Iran could produce lipase (5). B. vallismortis Ace02 from the traditional Korean condiment Chungkook-jang should be used for prevention of dental caries as well as being an effective thrombolytic agent. (Kim et al., 2007). B. vallismortis JY3A from the polluted soil could remove 90.5% of pyrene (14). As an endophyte, B. vallismortis ZZ185 was isolated from healthy stems of broadleaf holly Ilex latifolia, and exhibited a broad antifungal functions against phytopathogens by produced the compounds of bacillomycin mixtures (23). Endophytic bacteria have been found in virtually every plant and tissue types studied. However, endophytes colonizing plant reproductive organs have been rarely investigated, especially for the flower (2). To our knowledge, B. vallismortis ZZB08 was the first endophyte which could produce fragrant volatiles similar to that of its host.

As a famous fragrant plant, the volatile constituents from fresh flower of G. jasminoides had been investigated previously. By method of headspace-SPME-GC/MS, Liu and Gao (15) identified 54 volatile compounds from fresh flower of G jasminoides, in which farnesene (RT=64.87%), cis-ocimene (29.33%) and linalool (2.74%) were the major constituents. By same method, Chaichana et al. (1) identified 23 compounds from fresh flower of G. jasminoides, in which the major chemical constituents were linalool (38.23%), farnesene (21.40%), z-3-hexenyl tiglate (16.27%) and trans-bocimene (9.50%). By method of adsorption wireGC/MS, Huang et al. (6) reported that fresh flower of G. jasminoides could generate 86 volatile constituents with the major compounds of linalool (43.05%), b-myrcene (8.32%), methyl benzoate (7.61%) and p-xylene (7.17%). In this study, 17 were detected from G. jasminoides flower with major volatiles of linalool (with RT of 35.74%), afarnesene (11.43%), trans-b-ocimene (10.17%) and z-3-hexenyl tiglate (9.68%). The differences of volatile constituents and their relative contents among different investigations maybe related to the performance conditions used in headspaceSPME-GC/MS and the flower samples analyzed. However, based on the results above, linalool, farnesene, ocimene and z-3-hexenyl tiglate as the major constituents of G jasminoides flower should be confirmed.

In this study, we identified 18 volatiles from then endophytic bacterium B. vallismortis ZZB08. Of them, five major volatile compounds of flower, linalool, a-farnesene, trans-b-ocimene, z-3hexenyl tiglate and methyl tiglate, were also found in culture of the ZZB08. Though with lower concentrations of the major volatiles than that in the flower, the B. vallismortis ZZB08 showed the potential as the alternative of G jasminoides flower for perfume manufacturing in future.

ACKNOWLEDGMENTS

This work was funded jointly by projects from China National Tobacco Corporation (110201201009 BR-03), China Tobacco Yunnan Industrial Co. Lid. (2012JC08, 2011CP02), and Hongyun Honghe Tobacco (Group) Co. Lid. (HYHH2012HX05, HYHH2012HX06).

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(3.) Diaz, A., Vazquez, L., Ventura, F., Galceran, M.T. Estimation of measurement uncertainty for the determination of nonylphenol in water using solid-phase extraction and solid-phase microextraction procedures. Anal. Chim. Acta, 2004; 506: 71-80.

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(6.) Huang, Q.Q., Jiang, K.Z., Feng, J.Y, Chen, G.X. Analysis of volatile constituents of Gardenia flowers by GC/MS. Acta Botanica Yunnanica, 2004; 26(4): 471-474.

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(8.) Joulain, D. Flower scents from the pacific. Chem. Biodivers., 2008; 5: 896-909.

(9.) Kimura, M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol, 1980; 16: 111-120.

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(12.) Kumaran, R.S., Kim, H.J., Hur, B.K. Taxol promising fungal endophyte, Pestalotiopsis species isolated from Taxus cuspidate. J. Biosci. Bioeng., 2010; 110(5): 541-546.

(13.) Li, W.J., Xu, P., Schumann, P., Zhang, Y.Q., Pukall, R., Xu, L.H., Stackebrandt, E., Jiang, C.L. Georgenia ruanii sp. nov., a novel actinobacterium isolated from forest soil in Yunnan (China) and emended description of the genus Georgenia. Int. J. Syst. Evol. Microbiol., 2007; 57: 1424-1428.

(14.) Ling, J., Zhang, G., Sun, H., Fan, Y., Ju, J., Zhang, C. Isolation and characterization of a novel pyrene-degrading Bacillus vallismortis strain JY3A. Sci. Total. Environ., 2011; 409(10): 1994-2000.

(15.) Liu, B.Z., Gao, Y. Analysis of headspace constituents of Gardenia flower by GC/MS with solid-phase microextraction and dynamic headspace sampling. Chinese J. Chromatogr., 2000; 18(5): 452-455.

(16.) Lodewyck, C., Vangronsveld, J., Porteous, F., Moore, E.R.B., Taghavi, S., Mezgeay, M., Lelie, D.VD. Endophytic bacteria and their potential application. Crit. Rev. PlantSci., 2002; 86: 583-606.

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(21.) Xu, C.K., Mo, M.H., Zhang, L.M., Zhang, K.Q. Soil volatile fungistasis and volatile fungistatic compounds. Soil Biol. Biochem., 2004; 36: 1997-2004.

(22.) Yu, L.L., Li , Z.Y, Peng, C.S., Li, Z.Y., Guo, Y.W. Neobacillamide A, a novel thiazolecontaining alkaloid from the marine bacterium Bacillus vallismortis C89, associated with South China Sea Sponge Dysidea avara. Helv. Chim .Acta, 2009; 91(3): 607-612.

(23.) Zhao, Z.Z., Wang, Q.S., Wang, K.M., Brian, K., Liu, C.H., Gu, Y.C. Study of the antifungal activity of Bacillusvallismortis ZZ185 in vitro and identification of its antifungal components. Bioresource Technol., 2010; 101(1): 292-297.

Huang Ying [1], Dang Li Zhi [2], Chen Xing [2], Duan Yan Qing [2] * and Mo Ming He [1] *

Laboratory for Conservation and Utilization of Bio-Resources & Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, 650091, P R. China. China Tobacco Yunnan Industrial Co., Ltd., Kunming, 650231, P. R. China.

(Received: 16 April 2015; accepted: 02 July 2015)

* To whom all correspondence should be addressed. E-mail: xujin98@ynu.edu.cn

Caption: Fig. 1. Neighbour-joining tree derived from aligned 16S rDNA sequences, showing the position of strain ZZB08 in the genus Bacillus. Thermoactinomyces vulgaris was used as outgroup. Bar, 0.01 substitutions per nucleotide position
Table 1. Volatile components from flower of G jasminoides
and B. vallismortis ZZB08

S.     RT      Compound
No.    (min)

1      2.24    trans-1,4-hexadiene
2      2.56    cis-3-Hexenol
3      2.59    methyl tiglate
4      2.64    3-hexanol, 4-methyl-
5      3.57    toluene
6      4.82    trans-b-ocimene
7      5.35    acetone
8      6.03    3-hexen-1-ol
9      6.16    linalool
10     6.24    p-xylene
11     7.95    2,3-butanedione
12     9.03    oxetane,2,2-dimethyl-
13     11.25   b-myrcne
14     11.74   z-3-hexenyl tiglate
15     12.79   L-limonene
16     13.09   2-butanone,3-hydroxy-
17     13.42   cis-ocimene
18     14.82   trans-caryophyllene
19     15.63   methyl benzoate
20     16.07   2,3-butanediol
21     16.71   a-farnesene
22     18.04   butanoic acid,3-methyl-
23     18.55   hexanoic acid, 2-methyl-
24     19.28   1,3,8-p-menthatriene
25     21.36   2-heptanone
26     26.77   butanoic acid, 3-methoxy-
27     31.25   cis-3-hexenyl butyrate
28     34.23   2-furancarboxylic acid, nonyl ester
29     34.87   2-thiophenecarboxylic acid, undec-10-enyl ester
30     52.09   1H-indene-1,3(2H)-dione,2-hydroxy-2-(9-
                 methoxy-9H-fluoren-9-yl)-

S.     Group                  Formular
No.

1      alkenes                [C.sub.6][H.sub.10]
2      alcohols               [C.sub.6][H.sub.12]O
3      esters                 [C.sub.6][H.sub.10][O.sub.2]
4      alcohols               [C.sub.7][H.sub.16]O
5      benzenes               [C.sub.7][H.sub.8]
6      monoterpenes           [C.sub.10][H.sub.16]
7      ketones                [C.sub.3][H.sub.6]O
8      alcohols               [C.sub.6][H.sub.12]O
9      monoterpene alcohols   [C.sub.10][H.sub.18]O
10     benzenes               [C.sub.8][H.sub.10]
11     ketones                [C.sub.4][H.sub.6][O.sub.2]
12     heterocyclics          [C.sub.5][H.sub.10]O
13     monoterpenes           [C.sub.10][H.sub.16]
14     esters                 [C.sub.11][H.sub.18][O.sub.2]
15     monoterpenes           [C.sub.10][H.sub.16]
16     ketones                [C.sub.4][H.sub.8][O.sub.2]
17     monoterpenes           [C.sub.10][H.sub.16]
18     sesquiterpenes         [C.sub.15][H.sub.24]
19     aromatics              [C.sub.8][H.sub.8][O.sub.2]
20     alcohols               [C.sub.4][H.sub.10][O.sub.2]
21     sesquiterpene          [C.sub.15][H.sub.24]
22     acids                  [C.sub.5][H.sub.10][O.sub.2]
23     acids                  [C.sub.7][H.sub.14][O.sub.2]
24     monoterpenes           [C.sub.10][H.sub.10]
25     ketones                [C.sub.7][H.sub.14]O
26     acids                  [C.sub.5][H.sub.10][O.sub.3]
27     esters                 [C.sub.10][H.sub.18][O.sub.2]
28     esters                 [C.sub.14][H.sub.22][O.sub.3]
29     esters                 [C.sub.16][H.sub.24][O.sub.2]S
30     aromatics              [C.sub.23][H.sub.16][O.sub.4]

S.     Molecular   Relative content
No.    Weight      (>0.50%)

                   Flower   ZZB08

1      82          1.47     -
2      100         0.69     -
3      114         3.34     0.86
4      116         -        2.66
5      92          3.26     -
6      136         10.17    2.33
7      58          -        9.12
8      100         0.81     -
9      154         35.74    5.28
10     106         3.97     -
11     86          -        1.13
12     86          -        0.60
13     136         5.24     -
14     182         9.68     2.15
15     136         2.43     -
16     88          -        36.82
17     136         1.28     -
18     204         1.46     -
19     136         2.27     -
20     90          -        0.66
21     204         11.43    3.54
22     102         -        1.27
23     130         -        1.30
24     134         2.15     -
25     114         -        1.47
26     118         -        1.56
27     170         0.61
28     238         -        1.74
29     280         -        8.39
30     356         -        1.99

Note: - not identified; RT retention time
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Author:Ying, Huang; Zhi, Dang Li; Xing, Chen; Qing, Duan Yan; He, Mo Ming
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Mar 1, 2016
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