Springer 2005
Plant Growth Regulation (2005) 47:149–154 DOI 10.1007/s10725-005-3217-3
Growth inhibitors of lettuce seedlings from Bacillus cereus EJ-121 Lam Hoang, Gil-Jae Joo, Won-Chan Kim, So-Young Jeon, Sun-Ha Choi, Joung-Woong Kim, In-Koo Rhee, Jong-Moon Hur and Kyung-Sik Song* School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Deagu 702-701, Korea; *Author for correspondence (e-mail:
[email protected]; phone: +82-53-950-5715; fax: +82-53-956-5715) Received 14 March 2005; accepted in revised form 14 September 2005
Key words: 2-Aminobenzoic acid, Bacillus cereus EJ-121, Growth inhibitors, Lettuce, Sodium vanillate
Abstract The ethyl acetate extract of the Bacillus sp. EJ-121 culture broth exhibited growth inhibitory activity on a lettuce (Lactuca sativa L.) seedlings assay. Bacillus sp. EJ-121 was identified as Bacillus cereus by the morphological characteristic and nucleotide sequence of the 16S rDNA. The bioassay-guided fractionation of the ethyl acetate extract led to the isolation of two compounds. Their structures were deduced by spectroscopic methods and determined as sodium vanillate (1) and 2-aminobenzoic acid (2). Both compounds 1 and 2 inhibited more than 90% of root length at 50 ppm (0.26 and 0.36 mM, respectively) while they had a limited effect on shoot growth at the same concentration level. Roots and shoots of lettuce seedlings showed severe deterioration at 100 ppm. In order to study the fundamental structure–activity relationship, several structurally related benzoic acid derivatives were also assayed. The existence of a polar carboxyl moiety seemed to be responsible for the stronger activity.
Introduction The net effect of plant–microbe interaction on plant growth could be positive, neutral or negative in the rhizosphere of plants, where it is usually occupied by both deleterious rhizobacteria and plant growth promoting rhizobacteria (Nehl et al. 1996). The rhizobacteria, which inhibit the plant growth, are also described as allelopathic bacteria that directly or indirectly release the allelochemicals into the rhizosphere (Barazani and Friedman 1999). Such organic substances produced by bacteria which commonly found in soil are called plant growth regulators. Plant growth inhibitory activity is one of its main functions besides promoting plant growth substances.
The compounds from microbes are considered as a source of lead compounds for plant growth regulators, which can be used as fertilizer, a lodging-preventer, and herbicide (Bernart et al. 1990; Kanbe et al. 1993). Some species of plant growthpromoting bacteria may produce more than one substance such as three different phytotoxins, geldanamycin, nigericin, and hydanthocidin which were isolated from Streptomyces hygroscopicus (Barazani and Friedman 1999). Plant growth regulators such as hydoxysuochrin and sulochrin were isolated from culture filtrate of the fungus Aureobasidium sp., having an inhibitory activity effect on the growth of tea pollen tubes (Shimada et al. 2001). Penicillic acid isolated from a fungus (genus and species unknown) inhibited
150 root elongation of rice seedlings (Sassa et al. 1971). Efforts to introduce natural allelochemicals as plant growth-regulating agents in agriculture have yielded two commercial herbicide, phosphinothricin, a product of Streptomyces viridochromogenes and bialaphos from S. hygroscopicus (Barazani and Friedman 1999). Kremer and Souissi (2001) proposed that cyanide production by rhizobacteria suppress weed seedling growth. In our continuing search for plant growth regulators from microbes, two strains were isolated as potent inhibitors of plant growth out of over 2900 rhizosphere microorganisms obtained from the soils of the 500 domestic fields. One was the Bacillus subtilis IJ-31 which produced indole 3propionic acid and indole 3-acetic acid, which inhibited the growth of soybean seedlings (Lee et al. 2003). The other strain was EJ-121 which exhibited growth inhibitory activity on lettuce. In this report, identification of the EJ-121 strain, and the isolation and structure determination of the active compounds are discussed regarding their inhibitory activities on lettuce seedlings.
Materials and methods
from various farmland and was identified to the species level by its morphological, physiological, and biochemical characteristics and by its 16S rDNA sequencing. Chromosomal DNA of the EJ-121 was prepared according to the method of Sambrook et al. (1989) with some modifications and the relevant 16S rDNA was amplified with the primer set forward 27F (5¢-aga gtt tga tcc tgg ctc ag 3¢) and reverse 1492r (5¢-tac ctt gtt acg act t-3¢) by the method described by Dunbar et al. (2000). The sequence of the 16S rDNA was analyzed by the courtesy of CoreBioSystem Co., Ltd., Korea. The homology search of the determined 16S rDNA sequence was performed with the Ribosomal Database Project II (RDP, http://rdp.cme.msu.edu/ classifier/classifier.jsp).
Fermentation One percent of the seed culture was inoculated to a 50 l of production medium containing 2% peptone, 0.25% glucose, 0.5% NaCl, and 0.25% K2HPO4 (pH 7.0). The production culture was carried out at 30 C for 3 days under the condition of 30 rpm and aeration of 10 v.v.m. (volume of air per volume of medium per minute).
General Extraction and isolation Silica gel used for column chromatography was purchased from Merck (Kieselgel 60, Art. 7734, USA). TLC plate was a precoated Kieselgel 60 F254 (Art. 5715, Merck). HPLC (Waters, USA) was performed with an Apollo C18 5l column (10 · 250 mm) from Alltech (USA). 1H NMR (400 MHz) spectra were recorded on the Unity Inova (Varian, USA). Chemical shifts were given in d ppm from TMS. Authentic compounds were purchased from Sigma (USA).
Isolation and identification of rhizobacteria One gram of soil was suspended in 9 ml of saline (0.85% NaCl), and the suspension was diluted to 10 410 6. Aliquots (100 ll) were smeared on TSA agar media (Difco Co., USA) and then incubated at 30 C for 24 h. The isolated colonies were incubated on the same medium at 30 C for 24 h. Strain EJ-121 was one of 2900 isolates obtained
The cultures broth of B. cereus EJ-121 (8 l) was centrifuged at 12,000 rpm, 4 C for 5 min and the supernatant was consecutively partitioned with the same volume of dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and butanol. The most active EtOAc soluble fraction (1.38 g) was chromatographed on a silica gel column (3 · 50 cm, CH2Cl2– MeOH–Acetic acid=300:1:1% fi 1:1:1%) to yield 7 fractions (fr.). By the HPLC (Apollo C18 5l 10 · 250 mm column, 42% MeOH, PDA detector, flow rate 2 ml/min) separation, compounds 1 (0.6 mg) and 2 (1.4 mg) were obtained from fr. 2 (40.0 mg).
Bioassay Lettuce (Lactuca sativa L.) seeds were germinated in a Petri dish (20 · 150 mm) lined with a filter paper moistened with deionized water (6 ml) in a
151 growth chamber under continuous white fluorescent light at 24 C and 78% relative humidity for 24 h. Seedlings that had 2.0 mm long roots were selected and transferred to a Petri dish (15 · 90 mm) containing 2 ml of deionized water with or without various concentrations of the test compounds. They were further incubated in a growth chamber under the same conditions described above for an additional 96 h. At the end of the incubation, the length of the roots and shoots were measured, and the results were compared with those of a water-treated control.
Structure determination Compound 1
The EtOAc soluble fraction showed significant inhibitory activity on the lettuce growth at 1000 ppm while the dichloromethane, butanol, and water soluble fractions were relatively weak. Two active compounds were isolated by silica gel column chromatography and HPLC of the EtOAc soluble fraction from the culture broth of the B. cereus EJ-121. Compound 1 was analyzed by MS and 1H NMR. The EI-MS showed the fragment ion at m/z 167 [M+ - Na]. In 1H NMR, d7.55 (1H, d, J=2.4 Hz, H-2), 7.54 (1H, dd, J=8.0, 2.0 Hz, H-6), and 6.81 (1H, d, J = 8.0 Hz, H-5) indicated the typical signals of 1.3.4-tri-substituted benzene. From these data, 1 was postulated as a salt form of vanillic acid. Table 1. Homology search of 16S rDNA from strain EJ-121.
+
1
EI-MS: m/z 167 [M -Na], H NMR (400 MHz, methanol-d4); d7.55 (1H, d, J=2.4 Hz, H-2), 7.54 (1H, dd, J=8.0, 2.0 Hz, H-6), 6.81 (1H, d, J=8.0 Hz, H-5).
Compound 2 1
H NMR (400 MHz, methanol-d4); d7.80 (1H, dd, J = 7.8, 2.4 Hz, H-6), 7.21 (1H, dt, J = 7.8, 2.4 Hz, H-4), 6.72 (1H, dd, J = 7.8, 2.4 Hz, H-3), 6.56 (1H, dt, J = 7.8, 2.4 Hz, H-5). Each compound was re-confirmed by the cochromatography with authentic samples (tR’s; 19.35 min for compound 1 and 29.10 min for compound 2). Authentic 2-aminobenzoic acid was purchased from Sigma (USA) and sodium vanillate was synthesized by adding 1 ml of 0.1 N NaHCO3 to the 1 ml of methanolic solution containing 16.8 mg of vanillic acid (Sigma, USA).
Strain
Homology Accession number
Bacillus cereus 16S rDNA gene, ACTT10702 Bacillus cereus 16S rDNA gene, TA32–5 Bacillus cereus 16S rDNA gene, RC607 Bacillus cereus str. CH70–2 Bacillus cereus 16S rDNA gene, CH70–2 Bacillus megaterium str. MK64–1 Bacillus megaterium 16S rDNA gene, MK64–1
1.000
BCY15053
1.000
BCY15050
1.000
BCY15048
0.978 0.978
Y15046 BCY15046
0.833 0.833
Y15052 BMY150520
Results and discussion The rhizobacteria were isolated from the various farmlands to provide a pool of bacteria from which to screen for bacteria produced plant growth regulators. The isolate, which was the most effective at inhibiting lettuce root elongation, was selected for further study. The nucleotide sequence of the 16S rDNA of strain EJ-121 exhibited 100% of homology with Bacillus cereus (Table 1). The strain was designated as Bacillus cereus EJ-121.
Figure 1. The structures of (1) sodium vanillate, (2) 2-aminobenzoic acid, and related compounds: (3) isovanillic acid; (4) methyl salicylate; (5) salicylaldehyde; (6) salicylamide; (7) salicylic acid; (8) vanillic acid; (9) salicylhydroxamic acid.
152 By comparing its 1H NMR data with an authentic sample, 1 was identified as sodium vanillate. Proton NMR data of compound 2 showed the characteristic signals of 1.2-disubstituted benzene: d7.80 (1H, dd, J=7.8, 2.4 Hz, H-6), 7.21 (1H, dt, J=7.8, 2.4 Hz, H-4), 6.72 (1H, dd, J=7.8, 2.4 Hz, H-3), and 6.56 (1H, dt, J=7.8, 2.4 Hz, H-5). These 1H NMR data of compound 2 was identical to those of authentic 2-aminobenzoic acid (anthranilic acid) (Figures 1–3). Plant growth inhibitory activities of isolated sodium vanillate and 2-aminobenzoic acid were measured using the root and shoot growth of lettuce. As shown in Figure 2, compounds 1 and 2 inhibited the growth of lettuce roots and shoots in a dose-dependent manner. At 10 ppm, compounds 1 (0.05 mM) and 2 (0.04 mM) inhibited root elongation by 68.4% and 69.2%, respectively. Shoot growth, however, was not inhibited as much as the root growth at the same concentration. Both compounds 1 and 2 inhibited lettuce seedlings root elongation by more than 90% at 50 ppm (0.26 and 0.36 mM, respectively) (Table 2). The calculated I40 values (concentration required for 40% inhibition) of 1 and 2 were 1.10 and 1.23 ppm (6 · 10 3 and 9 · 10 3 mM) for root growth, and 3.61 and 4.14 ppm (1.9 · 10 3 and 3.0 · 10 3 mM) for shoot growth, respectively. The roots and shoots of lettuce seedlings showed
severe deterioration at 100 ppm [0.53 mM (1) and 0.73 mM (2)]. In order to study structure–activity relationships, the inhibitory activities of structurally related compounds such as isovanillic acid (3), methyl salicylate (4), salicylaldehyde (5), salicylamide (6), salicylic acid (7), vanillic acid (8), and salicylhydroxamic acid (9) were compared. Compounds 4 and 5, whose polar carboxylic acid groups were exchanged with non-polar groups (ester and aldehyde, respectively) were not effective regarding the growth of lettuce seedlings, suggesting that the polar carboxylic moiety, including amide, was responsible for the stronger activity. Except for 4 and 5, all the samples inhibited over 90% of root growth at 1 mM. Among them, 7 and 8 were the most effective. Their effects on the shoot growth (about 49.4– 57.2%) were not so much as the root growth at the same concentration. Searching for plant growth regulators suitable for developing new herbicides and for new lead compounds has been investigated. Kimura et al. (2002) reported that compounds isolated from Myxotrichum stipitatum had plant growth regulatory activities: myxostiolide inhibited the pollen tube growth; myxostiol accelerated, and clavatoic acid inhibited the root growth of lettuce, respectively. Vanillic acid has been isolated from plant
Figure 2. Inhibitory activities of compounds 1 and 2 on the roots and shoots growth of lettuce. 1, compound 1; 2, compound 2. Mean ± SE values of duplicate experiments with 30 seedlings each. : 5 ppm [0.03 (1), 0.04 mM (2)], : 10 ppm [0.05 (1), 0.07 mM (2)], : 50 ppm [0.26 (1), 0.36 mM(2)].
153
Figure 3. Inhibitory activities of related compounds on root and shoot growth of lettuce. (3) isovanillic acid; (4) methyl salicylate; (5) salicylaldehyde; (6) salicylamide; (7) salicylic acid; (8) vanillic acid; (9) salicylhydroxamic acid. Mean ± SE values of duplicate experiments with 30 seedlings each. : 0.05 mM, : 0.1 mM, : 0.25 mM, : 0.5 mM, : 1 mM.
Table 2. I40 values of related compounds on growth of lettuce. I40 values * Samples 1. 2. 3. 4. 5. 6. 7. 8. 9.
Sodium vanillate 2-aminobenzoic acid Isovanillic acid Methyl salicylate Salicylaldehyde Salicylamide Salicylic acid Vanillic acid Salicylhydroxamic acid
Root growth
Shoot growth
0.006 0.009 0.23 >1.0 >1.0 0.22 0.22 0.22 0.24
0.02 0.03 0.41 >1.0 >1.0 0.45 0.40 0.41 0.47
*Concentration required for 40% inhibition measured in mM.
resource such as stem–leaf of Welsh onion, and exudates of germinating watermelon seeds as a major component of allelopathic substance(s). It inhibited the growth of radical and shoot growth of lettuce, chrysanthemum, and tomato (Choi et al. 1998; Kushima et al. 1998). Anthranilic acid (2-aminobenzoic acid) and vanillic acid obtained from groundnut shell extracts inhibited Alternaria alternata spore germination at 1.9 mM (Kalaichelvan and Mahadevan 1988). A mixture of 2aminobenzoic acid and 4-acetamidophenol promoted root and sprout growth of cucumber (Socha 1989). It, however, is the first time that vanillic and 2-aminobenzoic acids were isolated from rhizobacteria as lettuce seedling growth inhibitors. The compounds isolated are expected to be useful as lead compounds for a lodging-preventer or as herbicide.
Acknowledgements This work was supported by a grant from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.
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