The allelochemicals accounting for the allelopathic effects of Myriophyllum spicatum on the cyanobacterium Microcystis aeruginosa
Contributions of five previously identified allelochemicals, (+) catechin, eugeniin, and ellagic, gallic and pyrogallic acid, in the allelopathic effects of Myriophyllum spicatum on the cyanobacterium Microcystis aeruginosa were investigated. Release rates from M. spicatum were determined, and on the basis of the results, artificial culture solutions of M. spicatum were prepared. By comparing the inhibitory effects of artificial and real culture solutions of M. spicatum on M. aeruginosa, these polyphenols were found to contribute, on average, to 50% of the allelopathic effects of M. spicatum.
Contributions of five polyphenols, (+)-catechin, eugeniin, and gallic, ellagic and pyrogallic acid, in the allelopathic effect of Myriophyllum spicatum on the cyanobacterium Microcystis aeruginosa were investigated.
Allelopathy, cyanobacteria, growth inhibition, polyphenol, Myriophyllum spicatum.
Myriophyllum spicatum is a submerged aquatic plant known to cause allelopathic growth inhibition of cyanobacteria (Gross et al. 1996; Nakai et al. 1999). We confirmed allelopathic growth inhibition of the cyanobacterium Microcystis aeruginosa by assaying culture solutions of M. spicatum (CSMs) obtained by culturing 100 g-wet/L of M. spicatum in 20-times-diluted Gorham’s medium for 3 and 1 days (Nakai et al. 1999). In a study on M. spicatum exudates, Gross and Sütfeld (1994) found a galloyl ester and derivatives of ellagic acid (EA) in a solid-phase extract of CSM, and eugeniin (EUG; tellimagrandin II), EA and gallic acid (GA) in a M. spicatum extract; EUG and EA have also been detected in a solid-phase CSM extract (Gross et al., 1996). In a more recent study, (+)-catechin (CAT) and pyrogallic acid (PA), in addition to EA and GA, were found in both a CSM and M. spicatum itself (Nakai et al., 2000).
By comparing the inhibitory effects of CSMs with those of artificial culture solutions of M. spicatum (ACSMs) prepared by adding CAT, EA, GA, and PA to a cyanobacterial medium on the basis of estimated release rates, i.e. 0.024 μg/g-wet/d for CAT, 0.33 μg/g-wet/d for EA, 0.33 μg/g-wet/d for GA, and 0.17 μg/g-wet/d for PA, Nakai et al. (2001) revealed that, on average, these 4 polyphenols contribute to approximately 17% of the allelopathic effect on M. aeruginosa. Gross (1999) found that EUG is the cause of the anti-cyanobacterial allelopathic effect of M. spicatum due to the high content of EUG observed in the plant itself (10-15 mg/g-dry); however, Smolders et al. (2000) showed that of 16 submerged aquatic plants, M .spicatum contained the highest phenolic content (84 mg/g-dry). These results allowed us to surmise that phenols such as the five polyphenols reported above might account for the allelopathic effect of M. spicatum on the cyanobacteria M. aeruginosa.
To help understand the allelopathic mechanisms of M. spicatum on cyanobacteria, this research was carried out to determine the contribution of CAT, EUG, EA, GA and PA in the allelopathic effects of M. spicatum on M. aeruginosa.
M. spicatum and M. aeruginosa
M. aeruginosa (NIES-87) was obtained from the microbial collection of the National Institute for Environmental Studies (NIES), Japan, and M. spicatum was collected from Asakawa River, Tokyo, Japan. M. aeruginosa was cultivated in triplicate using modified C (CB) medium at 25 °C under a light intensity of 5000 lux (Watanabe & Satake 1991). Its growth was monitored by determining the turbidity with a T-2600 DA turbidity meter .
Contributions of the 5 polyphenols to the allelopathic effect of M. spicatum on the growth of M. aeruginosa were determined by comparing the inhibitory effects of ACSMs with those of CSMs obtained from equivalent concentrations of M. spicatum. The inhibitory effects of the ACSMs were investigated using a quasi-continuous addition method, which was used to evaluate the inhibitory effects of the CSMs (Nakai et al. 1999, 2001). Under the quasi-continuous addition method, CSMs added on day 0 of M. aeruginosa cultivation were prepared from 100 g-wet/L M. spicatum cultured for 3 days, while those added on days 1-5 were prepared from 100 g-wet/L M. spicatum cultured for 1 day (Nakai et al. 1999). Therefore, we prepared ACSMs by adding the amounts of the five polyphenols released from 100 g-wet/L M. spicatum for 3 days or 1 day to the CB medium, as summarized in Table 1. The inhibitory effects of the five polyphenols on M. aeruginosa were investigated by comparing the growth in the ACSMs with that in the control experiment.
Table 1. Polyphenol concentrations [μg/L] of an artificial culture solution of M. spicatum (ACSM) prepared according to the release rates from 100 g-wet/L of M. spicatum.
Time when added in the quasi-continuous addition method
*Data reported by Nakai et al. (2001)
Calculation of release rate of EUG
The releases of CAT, EA, GA and PA from M. spicatum were already reported (Nakai et al. 2001). Therefore, determined here is the release rate of EUG (a) by the same manner for CAT, EA, GA and PA on the basis of the mass balance of EUG in the CSM as expressed by Equation (1) assuming that microbial degradation of EUG did not occur.
d(CV)/dt = aW – kEUGCV
where C = the apparent EUG concentration in the CSM (µg/L), V = the volume of the CSM (L), W = the wet weight of M. spicatum in the CSM (g-wet), a = the release rate of EUG from M. spicatum (µg/g-wet/d), and kEUG = the experimentally obtained autoxidation rate coefficient of EUG (1/d). kEUG and C were obtained experimentally, as described below. Note, autoxidation rate of EUG was expressed as a function of pH, as described later.
Determination of the autoxidation rate coefficient (kEUG)
The autoxidation rate coefficient of EUG, kEUG, over a pH range of about 6.4-7.4 was measured using pH-buffered Gorham’s medium diluted 20-fold. Briefly, after adjusting the pH, CAT, EA, GA and PA were respectively added to 50 ml of medium. Next, over 6 hours, a 100-µl sample of the medium was analyzed by high performance liquid chromatography (HPLC) equipped with an electrochemical detector (HP1049A, Hewlett Packard) on an ODS column (TSK-gel ODS 80Ts, 250 × 4.6 mm, TOSOH) using a previously reported elution profile (Nakai et al. 2000).
Measurement of the apparent EUG concentration (C)
M. spicatum was cultured at 100 g-wet/L for 3 days in Gorham’s medium diluted 20-fold (Zehnder & Gorham 1999). Apparent concentrations of EUG in the medium and its pH were measured using HPLC and a pH meter over a 3-day cultivation period.
Autoxidation rate coefficient (kEUG)
Autoxidation of EUG in the medium followed pseudo-first order kinetics, similar to that of CAT, EA, GA, and PA (Nakai et al. 2001). The autoxidation rate coefficient, kEUG, was pH dependent and could be
expressed as a function of pH as shown in Figure 1. At the used range of pH, the autoxidation rate of EUG was almost same as that of GA (kGA = 0.028[pH] – 0.15), but higher than that of CAT (kCAT = 0.011[pH] – 0.05) and EA (kEA = 0.0057[pH] – 0.033). PA was the most rapidly autoxidized as expressed by the equation; kPA = 0.11[pH] – 0.60 (Nakai et al. 2001).
Figure 1. Relationship between pH and the autoxidation rate coefficient of EUG.
Calculation of the release rate of EUG (a)
The apparent concentrations of EUG in the CSM were in the range of 150 µg/L, while pH varied from 6.5 to 7.3 (Figure 2). Note that the apparent concentration of EUG was much higher than those of CAT, EA, GA, and PA (data not shown).
Figure 2. EUG concentration and pH in the CSM with time.
In calculating the release rate of EUG from M. spicatum, we made 2 assumptions: (1) that a and the pH on each day were constant, and (2) that the pH represented the average pH measured each day. Thus, the daily kEUG was obtained by applying the average pH to the equation presented in Figure 1; the release rate of EUG, a, was then calculated from equation 1. The amount of EUG released from 100 g-wet/L of M. spicatum during the 3-day cultivation period was then estimated (Nakai et al. 2001). Although the M. spicatum samples used were collected in the same season, estimated amounts of EUG released varied widely; the maximum value was about 10 times that of the minimum. Due to this wide variation, we used the logarithmic mean value (0.77 μg/g-wet/d) for the basis of the EUG concentrations used in the ACSMs (Table 1). Note that the value was higher than that of other four polyphenols; CAT (0.024 μg/g-wet/d), EA (0.33 μg/g-wet/d), GA (0.33 μg/g-wet/d), and PA (0.17 μg/g-wet/d).
Growth inhibition of M. aeruginosa by ACSMs
Figure 3 compares the growth inhibition of M. aeruginosa by the ACSMs prepared based on concentrations of polyphenols in 100 g-wet/L with that by CSMs. In this study, the ACSMs inhibited growth of M. aeruginosa by 50% compared to the control; however, in our previous research, the ACSMs without EUG did not show such a strong effect (Nakai et al. 2001). This result confirms the significant contribution of EUG in the allelopathic effect of M. spicatum on M. aeruginosa. However, comparison of the inhibitory effects of the CSMs and ACSMs indicated that the inhibitory effects of the ACSMs were 1/2 as strong as those of the CSMs. These results indicate that, on average, the 5 polyphenols contribute to 50% of the allelopathic effect of M. spicatum.
Figure 3. Comparison of the inhibitory effects of ACSMs and CSMs after 7 days incubation of M. aeruginosa. *Data obtained from Nakai et al., 2001.
Previous research conducted to understand the allelopathic effects of M. spicatum has focused on phenolic and polyphenolic compounds, such as hydrolyzable tannins and their derivatives (Gross and Sütfeld 1994; Gross et al. 1996; Nakai et al. 2000 and 2001). The present study indicates that unknown allelochemicals still exist. We recently succeeded in identifying the saturated/unsaturated fatty acids in CSMs (Nakai et al. in press). In the future, to fully understand the mechanism of allelopathy, the possible contribution of other compounds such as fatty acids in the allelopathic effect of M. spicatum must be verified.
Of the five phenols examined, eugeniin was the most abundant in culture solutions of M. spicatum. After determination of the release rates from M. spicatum, artificial culture solutions were prepared and assayed for M. aeruginosa growth. These polyphenols were found to contribute, on average, to 50% of the allelopathic effects of M. spicatum on M. aeruginosa.
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