Abstract

Natural allelochemicals are considered as a source of algaecides. To uncover the anti-algal activity of Cinnamomum camphora fallen leaves and promote their usage as algaecides, the composition of their water and methanol extracts was analyzed, and the inhibitory effects of extracts on the growth of Microcystis aeruginosa and Chlamydomonas reinhardtii, and chlorophyll (Chl) content and photosynthetic abilities in C. reinhardtii were investigated. Twenty-five compounds were detected in the water extracts, mainly including terpenoids, esters, alcohols, and ketones. Compared to water extracts, there were more compounds and higher concentration in methanol extracts. Both water and methanol extracts inhibited the growth of the two algae, and 15 mg·ml−1 methanol extracts killed the algal cells after 48 h. The levels of Chl a and Chl b, as well as maximum quantum yield of photosystem II photochemistry (Fv/Fm) in C. reinhardtii cells reduced gradually with increasing the concentration of extracts, while the maximum quantum yield of non-photochemical de-excitation (φDO) increased gradually. At the same concentration, methanol extracts showed stronger inhibitory effects than water extracts, due to their higher number of compounds and higher concentration. Therefore, C. camphora fallen leaves have a potential value as an algaecide.

INTRODUCTION

With the increasing input of nutrients mainly nitrogen (N) and phosphorus (P) to waters, eutrophication becomes more serious (Cloern 2001) and promotes the excessive growth of cyanobacteria and green algae, and even blooms. Algae blooms can lower the water quality and cause a series of ecological problems (Dodds et al. 2009; Qin 2009). Algae release an abundance of volatile organic compounds (VOCs), which frequently lead to an unpleasant, earthy–musty odor in the water. Geosmin and 2-methyl borneol are considered as the main compounds to cause the odor (Fujise et al. 2010). In addition, these VOCs can inhibit other algal growth by inducing photosynthetic pigment degradation and inhibiting photosynthesis (Zhao et al. 2016; Xu et al. 2017; Ye et al. 2018). Besides VOCs, algae can also produce lots of toxins, including microcystin, hepatotoxins, neurotoxins, neosaxitoxins, anatoxin-a, etc. (Codd 2000; Frangópulos et al. 2004). Previous studies have reported that algal toxins showed inhibitory effects on the growth of other algae (Sanna et al. 2004; Li & Li 2012), aquatic plants (Pflugmacher 2002), zooplankton (Abrantes et al. 2006), and even fishes (Guzmán-Guillén et al. 2013). Moreover, they are a potential hazard to human health through the usage of the water for drinking and recreation (Hoeger et al. 2007).

For the benefit of ecosystems and human health, extensive methods have been developed to control the growth of undesired algae, including the usage of yellow loess (Choi et al. 1998), biquaternary ammonium salt (Liu et al. 2004), TiO2 (Kim & Lee 2005), copper sulfate (Costas & Lopez-Rodas 2006; Song & Wang 2015), sediment capping (Huang et al. 2011), phosphorus inactivation (Lürling & van Oosterhout 2013), and also biomanipulation such as with viruses (Garry et al. 1998) and bacteria (Park et al. 1998; Cai et al. 2011). During the experiments, these methods seem to be efficient in controlling the algal growth, but they may bring potential disaster to the environment (Jeong et al. 2000; Song & Wang 2015) and have high financial costs (Kim & Lee 2005; Huang et al. 2011; Lürling & van Oosterhout 2013) once they are used in the field.

Plant allelochemicals are natural compounds, which effectively inhibit neighbor plant growth and can be degraded in nature (Zuo et al. 2011; Zhang et al. 2012). They have been considered as a source of potential agents for algaecides (Zhou et al. 2008; Ni et al. 2012; Pęczuła 2013). Some plants have been found to have inhibitory effects on algal growth, e.g., extracts from Rhizoma coptidis and Semen arecae on Alexandrium tamarense (Zhou et al. 2007), garlic solution on A. tamarense, A. satoanum, A. catenella and Scrippsiella trochoidea (Zhou et al. 2008), and grape extracts on Chlamydomonas reinhardtii (Zuo et al. 2015). When Microcystis aeruginosa cells were treated with the extracts from Artemisia annua (Ni et al. 2012) and Iris wilsonii (Chen et al. 2012), a remarkable inhibition was found on the cell growth, and the anti-algal activity compounds are artemisinin in A. annua, and phenolics and tannin in I. wilsonii. Although lots of plants have inhibitory effects on algae, the usage of plant wastes from agricultural production as algaecides was more economically favorable and environmentally friendly, such as rice hulls (Chung et al. 2007), barley straws (Grover et al. 2007; Pęczuła 2013), and grape pruning wastes (Zuo et al. 2015).

Cinnamomum camphora (L.) Presl is an evergreen landscaping and forestation tree species, drops old leaves in May to June, and is widely planted in the south of China. This species synthesizes abundant secondary metabolites, mainly terpenoids, which can repel herbivore attack and resist fungal infection (Frizzo et al. 2000; Chen & Dai 2012; Yang et al. 2014), indicating that the plants may have the inhibitory effects on algae. To develop effective algaecides using C. camphora wastes, the components of the extracts from C. camphora fallen leaves were analyzed, and the inhibitory effects of the extracts on the growth of typical species of cyanobacteria (M. aeruginosa) and green algae (C. reinhardtii) were investigated. Meanwhile, the variation of photosynthetic pigment and photosynthetic abilities in C. reinhardtii were measured to uncover the inhibitory mechanism of C. camphora extracts on photosynthesis.

MATERIAL AND METHODS

Cell cultures

M. aeruginosa FACHB-912 provided by Freshwater Algae Culture Collection at the Institute of Hydrobiology, China, and C. reinhardtii strain CC-125 wild type mt+ [137c] from Dr E. H. Harris (Duke University, Durham, NC, USA) were grown in BG-11 (Rippka et al. 1979) and tris-acetate-phosphate (TAP) (Gorman & Levine 1965) medium, respectively. They were kept in 16 h light (30 μmol·m−2·s−1)/8 h dark, with temperature at 25 °C. They were used for experiments when their density reached the mid-logarithmic phase. The cell density was determined by using the blood cell counting plate, with each value being the means of six repeats.

Preparation of C. camphora fallen leaf extracts

Fallen leaves from C. camphora were collected in Zhejiang A & F University (30°15′ N, 119°43′ E) in May to June. The leaves were dried using a drying oven at 60 °C, and smashed with a pulverizer. The pulverized materials of 10 g were extracted with 100 ml distilled water and 50% methanol, respectively, at 25 °C for 48 h. The water- and methanol-extracted solution was centrifuged at 5,000 r·min−1 for 6 min, and then its concentration was 100 mg·ml−1.

Treatments with fallen leaf extracts

The fallen leaf extracts were added into the BG-11 medium to treat M. aeruginosa (6 × 106 cells·ml−1), and into TAP medium to treat C. reinhardtii (2 × 106 cells·ml−1), with the concentration of 1, 5, 10 and 15 mg·ml−1, respectively. The medium was added into the same amount of distilled water or 50% methanol as the control for water extract treatment and methanol extract treatment, respectively. The live cell density of the two algae were determined by using neutral red staining method (Wang et al. 2007) after 24 h and 48 h treatment, and the chlorophyll (Chl) content per 106 cells and Chl fluorescence parameters per 106 cells in C. reinhardtii were determined after 48 h.

Determination of Chl content

C. reinhardtii cells of 5 ml were collected by centrifugation and the pellets were resuspended in 3 ml 80% acetone. After removal of insoluble materials by centrifugation, the Chl content was determined following Arnon's method (Arnon 1949).

Measurement of photosynthetic efficiency

According to our previous method (Zuo et al. 2012), 10 ml C. reinhardtii culture was collected by centrifugation and resuspended in 10 μl of the same culture medium. The resuspended cells were pipetted on a piece of filter paper to form 0.5 cm2 spots. After incubation in darkness for 15 min, their Chl fluorescence was measured by a non-modulation Chl-fluorescence analyzer (Yaxin-1161, Yaxinliyi Science and Technology Ltd Co., Beijing, China) following the procedure of Strasser et al. (1995). Maximum quantum yield of photosystem II (PSII) photochemistry (Fv/Fm) and maximum quantum yield of non-photochemical de-excitation (φDO) were calculated using the formula given by Strasser et al. (1995).

Analysis of fallen leaf extracts

The methanol extracts of 20 ml were distilled to 8 ml at 50 °C (lower than the boiling point of methanol at 64.5 °C) using a rotary evaporator to remove the methanol. The distilled extracts were supplemented to 20 ml using distilled water to keep the extract concentration at 100 mg·ml−1. Six millilitres of removed methanol extracts and water extracts were separately extracted with 1 ml ethyl acetate, and analyzed by gas chromatography mass spectrometry (GC-MS). The GC (7890B, Agilent Technologies Company, CA, USA) was run with a 30 m × 0.25 mm × 0.25 μm HP-5MS capillary column. The temperature of the column was programmed to increase from 50 °C to 180 °C at a rate of 20 °C·min–1 and kept for 4 min. Then it was increased to 220 °C at a rate of 10 °C·min–1 and kept for 15 min. The MS (5977B, Agilent Technologies Company, CA, USA) was run under the following conditions: electron ionization mode of ionization energy at 70 eV and source temperature at 230 °C, mass range between m/z 28 and m/z 450, interface temperature at 250 °C and quadrupoles temperature at 150 °C. The qualitative and quantitative analyses of the GC/MS data were obtained from NIST/EPA/NIH Mass Spectral Library (NIST 14) (National Institute of Standards and Technology, Gaithersburg, USA). D-Limonene, eucalyptol, terpinene, linalool, camphor and E-nerolidol were used as the standard samples to calculate the concentration of the corresponding compounds in the extracts. The concentration of sesquiterpenoids (C15) was calculated referring to E-nerolidol, while the concentration of monoterpenoids (C10) and other compounds was calculated referring to camphor.

Calculations and statistical analyses

The response index (RI) was calculated according to the method described by Williamson & Richardson (1988).  
formula
where C and T are control response and treatment response, respectively. The positive values of RI indicate stimulation by the treatments relative to the controls, while negative values indicate inhibition.

Statistical analyses of one-way analysis of variance and drawing figures were performed with Origin 8.0 (Origin Lab, USA).

RESULTS

Composition of C. camphora fallen leaf extracts

There were 25 compounds in the water extracts from C. camphora fallen leaves, mainly including terpenoids, esters, alcohols, and ketones. Among these components, linalool, camphor and 6-epi-shyobunol were the main compounds, with the concentration of 510.6, 666.7 and 161.3 μmol·l−1, respectively. Compared to water extracts, 12 new compounds were detected in the methanol extracts, including ethyl linalool (66.2 µmol·l−1), coumaran (47.0 µmol·l−1), Z-9-tetradecenal (28.0 µmol·l−1), spathulenol (115.9 µmol·l−1), α-santalol (119.7 µmol·l−1), isolongifolol (343.6 µmol·l−1), palustrol (47.2 µmol·l−1), isoaromadendrene epoxide (33.1 µmol·l−1), β-santalol (75.3 µmol·l−1), platambin (53.1 µmol·l−1), methyl linolenate (36.3 µmol·l−1), and octadecanol acetate (43.2 µmol·l−1). Not only compound types, but the concentration of most of the compounds in methanol extracts was higher than that in water extracts, and their total concentration was three-fold that in water extracts (Table 1).

Table 1

The main compounds in C. camphora fallen leaf extracts

Retention time (min) Compounds Formula Concentration (μmol·l−1)
 
Water extracts Methanol extracts 
4.515 3-Methyl-2-pentanone C6H124.3 ± 0.4 34.0 ± 3.8 
4.756 Isobutyl acetate C6H12O2 11.0 ± 0.7 77.8 ± 7.1 
5.029 Mesityl oxide C6H1017.8 ± 2.0 59.6 ± 6.8 
5.237 Butyl acetate C6H12O2 3.4 ± 0.6 19.4 ± 1.9 
7.530 D-Limonene C10H16 10.7 ± 7.2 165.6 ± 37.2 
7.569 Eucalyptol C10H1890.6 ± 4.6 146.3 ± 13.2 
7.928 (S)-Linalool oxide C10H18O2 31.6 ± 4.7 24.5 ± 1.8 
8.162 Linalool C10H18510.6 ± 64.1 395.8 ± 35.1 
8.589 Camphor C10H16666.7 ± 54.8 985.4 ± 73.0 
8.746 δ-Terpineol C10H1859.7 ± 6.2 91.0 ± 3.7 
8.854 Hotrienol C10H18O2 99.2 ± 3.3 87.8 ± 14.4 
8.922 α-Terpineol C10H18101.7 ± 12.9 186.6 ± 14.0 
9.479 2,6-Dimethyl-1,7-octadien-3,6-diol C10H18O2 2.6 ± 0.3 86.1 ± 12.0 
10.017 Ethyl linalool C12H22a 66.2 ± 12.4 
10.702 Coumaran C15H24 a 47.0 ± 12.5 
11.180 Z-9-Tetradecenal C12H22a 28.0 ± 9.3 
11.350 Epiglobulol C15H2610.7 ± 0.7 9.6 ± 1.2 
11.850 E-Nerolidol C15H267.0 ± 0.5 1,096.1 ± 74.6 
12.345 Cis-Lanceol C15H2434.7 ± 11.8 87.4 ± 17.0 
12.213 Spathulenol C15H24a 115.9 ± 6.8 
12.310 Caryophyllene oxide C15H246.5 ± 1.1 116.9 ± 20.0 
12.428 Bihydro-β-ionone C13H2236.5 ± 7.2 197.0 ± 12.2 
13.218 6-Epi-shyobunol C15H26161.3 ± 9.6 646.4 ± 21.2 
13.997 α-Santalol C15H24a 119.7 ± 14.4 
14.449 Isolongifolol C15H26a 343.6 ± 43.1 
14.757 Dehydrosaussurea lactone C15H20O2 8.3 ± 4.2 93.8 ± 23.2 
14.994 Palustrol C15H26a 47.2 ± 11.4 
15.435 (E)-Atlantone C15H2210.5 ± 2.9 121.5 ± 20.6 
15.536 Saussurea lactone C15H22O2 1.2 ± 0.5 15.9 ± 0.8 
15.644 Cedrenol C15H2411.4 ± 7.7 30.3 ± 1.7 
15.705 Proximadiol C15H28O2 20.2 ± 1.2 a 
16.021 Isoaromadendrene epoxide C15H24a 33.1 ± 1.3 
16.283 β-Santalol C17H26O2 a 75.3 ± 8.5 
16.390 Platambin C15H26O2 a 53.1 ± 7.1 
18.070 1,8-Bimethyl-8,9-epoxy-4-isopropyl-spiro[4.5]decan-7-one C15H24O2 18.0 ± 1.4 93.7 ± 16.6 
19.459 Methyl linolenate C19H32O2 a 36.3 ± 3.0 
21.285 Octadecanol acetate C20H40O2 a 43.2 ± 10.5 
Retention time (min) Compounds Formula Concentration (μmol·l−1)
 
Water extracts Methanol extracts 
4.515 3-Methyl-2-pentanone C6H124.3 ± 0.4 34.0 ± 3.8 
4.756 Isobutyl acetate C6H12O2 11.0 ± 0.7 77.8 ± 7.1 
5.029 Mesityl oxide C6H1017.8 ± 2.0 59.6 ± 6.8 
5.237 Butyl acetate C6H12O2 3.4 ± 0.6 19.4 ± 1.9 
7.530 D-Limonene C10H16 10.7 ± 7.2 165.6 ± 37.2 
7.569 Eucalyptol C10H1890.6 ± 4.6 146.3 ± 13.2 
7.928 (S)-Linalool oxide C10H18O2 31.6 ± 4.7 24.5 ± 1.8 
8.162 Linalool C10H18510.6 ± 64.1 395.8 ± 35.1 
8.589 Camphor C10H16666.7 ± 54.8 985.4 ± 73.0 
8.746 δ-Terpineol C10H1859.7 ± 6.2 91.0 ± 3.7 
8.854 Hotrienol C10H18O2 99.2 ± 3.3 87.8 ± 14.4 
8.922 α-Terpineol C10H18101.7 ± 12.9 186.6 ± 14.0 
9.479 2,6-Dimethyl-1,7-octadien-3,6-diol C10H18O2 2.6 ± 0.3 86.1 ± 12.0 
10.017 Ethyl linalool C12H22a 66.2 ± 12.4 
10.702 Coumaran C15H24 a 47.0 ± 12.5 
11.180 Z-9-Tetradecenal C12H22a 28.0 ± 9.3 
11.350 Epiglobulol C15H2610.7 ± 0.7 9.6 ± 1.2 
11.850 E-Nerolidol C15H267.0 ± 0.5 1,096.1 ± 74.6 
12.345 Cis-Lanceol C15H2434.7 ± 11.8 87.4 ± 17.0 
12.213 Spathulenol C15H24a 115.9 ± 6.8 
12.310 Caryophyllene oxide C15H246.5 ± 1.1 116.9 ± 20.0 
12.428 Bihydro-β-ionone C13H2236.5 ± 7.2 197.0 ± 12.2 
13.218 6-Epi-shyobunol C15H26161.3 ± 9.6 646.4 ± 21.2 
13.997 α-Santalol C15H24a 119.7 ± 14.4 
14.449 Isolongifolol C15H26a 343.6 ± 43.1 
14.757 Dehydrosaussurea lactone C15H20O2 8.3 ± 4.2 93.8 ± 23.2 
14.994 Palustrol C15H26a 47.2 ± 11.4 
15.435 (E)-Atlantone C15H2210.5 ± 2.9 121.5 ± 20.6 
15.536 Saussurea lactone C15H22O2 1.2 ± 0.5 15.9 ± 0.8 
15.644 Cedrenol C15H2411.4 ± 7.7 30.3 ± 1.7 
15.705 Proximadiol C15H28O2 20.2 ± 1.2 a 
16.021 Isoaromadendrene epoxide C15H24a 33.1 ± 1.3 
16.283 β-Santalol C17H26O2 a 75.3 ± 8.5 
16.390 Platambin C15H26O2 a 53.1 ± 7.1 
18.070 1,8-Bimethyl-8,9-epoxy-4-isopropyl-spiro[4.5]decan-7-one C15H24O2 18.0 ± 1.4 93.7 ± 16.6 
19.459 Methyl linolenate C19H32O2 a 36.3 ± 3.0 
21.285 Octadecanol acetate C20H40O2 a 43.2 ± 10.5 

Data are means of three independent experiments ± standard deviation.

aNo compound was found.

Effects of C. camphora extracts on algal cell multiplication

When M. aeruginosa cells were treated with water extracts from C. camphora fallen leaves at 1, 5, 10 and 15 mg·ml−1 for 24 h, the cell multiplication was markedly inhibited, with the RI of −0.22 (P < 0.01), −0.36 (P < 0.01), −0.39 (P < 0.01) and −0.43 (P < 0.01), respectively. After 48 h, the inhibitory effects alleviated, but significant (P < 0.01) inhibition was also detected in the treatment at 5, 10 and 15 mg·ml−1 (Figure 1(a)). Compared to water extracts, methanol extracts showed stronger inhibition at the same concentration. In the treatment with methanol extracts at 15 mg·ml−1, the cells were killed completely (Figure 1(b)).

Figure 1

Effects of water (a) and methanol (b) extracts on M. aeruginosa cell multiplication. **Compared to the control, the significant difference at P < 0.01 level. Data are means of four replicates ± standard error.

Figure 1

Effects of water (a) and methanol (b) extracts on M. aeruginosa cell multiplication. **Compared to the control, the significant difference at P < 0.01 level. Data are means of four replicates ± standard error.

When C. reinhardtii cells were treated with C. camphora water and methanol extracts, the cell multiplication was inhibited significantly, and the inhibition enhanced with prolonging the treatment time. Similar to M. aeruginosa, C. reinhardtii cells were also killed completely in the treatment with methanol extracts at 15 mg·ml−1 (Figure 2).

Figure 2

Effects of water (a) and methanol (b) extracts on C. reinhardtii cell multiplication. **Compared to the control, the significant difference at P < 0.01 level. Data are means of four replicates ± standard error.

Figure 2

Effects of water (a) and methanol (b) extracts on C. reinhardtii cell multiplication. **Compared to the control, the significant difference at P < 0.01 level. Data are means of four replicates ± standard error.

Impacts of C. camphora extracts on Chl levels

The content of Chl a in C. reinhardtii cells reduced in the treatment with C. camphora water extracts, and the RI was −0.04, −0.09 (P < 0.05), −0.32 (P < 0.01) and −0.50 (P < 0.01), respectively, at 1, 5, 10 and 15 mg·ml−1. At the same concentration, methanol extracts showed stronger impacts on the content of Chl a in contrast to water extracts (Figure 3(a)). Similar reduction was also found in the content of Chl b (Figure 3(b)).

Figure 3

Effects of C. camphora extracts on the levels of chlorophyll a (a) and chlorophyll b (b) in C. reinhardtii cells. *Compared to the control, the significant difference at P < 0.05 level. **Compared to the control, the significant difference at P < 0.01 level. The cells were killed completely in the treatment with methanol extracts at 15 mg·ml−1, so the chlorophyll content was not measured. Data are means of four replicates ± standard error.

Figure 3

Effects of C. camphora extracts on the levels of chlorophyll a (a) and chlorophyll b (b) in C. reinhardtii cells. *Compared to the control, the significant difference at P < 0.05 level. **Compared to the control, the significant difference at P < 0.01 level. The cells were killed completely in the treatment with methanol extracts at 15 mg·ml−1, so the chlorophyll content was not measured. Data are means of four replicates ± standard error.

Effects of C. camphora extracts on Chl fluorescence parameters

C. camphora extracts remarkably reduced the Fv/Fm in C. reinhardtii cells, with the RI of −0.14 (P < 0.01), −0.35 (P < 0.01), −0.51 (P < 0.01) and −0.65 (P < 0.01), respectively, in water extracts at 1, 5, 10 and 15 mg·ml−1, and −0.34 (P < 0.01), −0.55 (P < 0.01) and −0.72 (P < 0.01), respectively, in methanol extracts at 1, 5 and 10 mg·ml−1 (Figure 4(a)). However, C. camphora extracts significantly (P < 0.01) promoted the increase of φDO in C. reinhardtii cells, and methanol extracts showed stronger effects compared to water extracts (Figure 4(b)).

Figure 4

Effects of C. camphora extracts on Fv/Fm (a) and φDO (b) in C. reinhardtii cells. **Compared to the control, the significant difference at P < 0.01 level. The cells were killed completely in the treatment with methanol extracts at 15 mg·ml−1, so the Fv/Fm and φDO were not measured. Data are means of four replicates ± standard error.

Figure 4

Effects of C. camphora extracts on Fv/Fm (a) and φDO (b) in C. reinhardtii cells. **Compared to the control, the significant difference at P < 0.01 level. The cells were killed completely in the treatment with methanol extracts at 15 mg·ml−1, so the Fv/Fm and φDO were not measured. Data are means of four replicates ± standard error.

DISCUSSION

An abundance of terpenoids that comprise terpenes and their derivatives were detected in the extracts from Cinnamomum genus (Kaul et al. 2003; Lee 2009; Ragasa et al. 2013; Monteiro et al. 2017; Taha & Eldahshan 2017; Tomazoni et al. 2017; Wei et al. 2017; Yuan et al. 2017). For C. camphora, the main compounds in the extracts from fresh leaves were terpenoids, especially oxygenated monoterpenes, and linalool and camphor were the two compounds with higher amount (Hamidpour et al. 2013; Yang et al. 2014; Tomazoni et al. 2017). In addition, in our previous studies, monoterpenes and oxygenated monoterpenes were the main VOCs released from the plants, and the emission amount of oxygenated monoterpenes was higher than that of monoterpenes, especially at high temperature (Zuo et al. 2017a). Similarly, amounts of terpenoids were found in the extracts from C. camphora fallen leaves, mainly including monoterpenoids and sesquiterpenoids, and the oxygenated terpenes were the main components (Table 1).

Among the extracts of water, methanol, ethyl acetate, hexane and dichloro-methane from fenugreek seeds, the methanol and ethyl acetate extracts showed the highest content of extracted compounds, indicating that organic compounds were more likely to be dissolved by methanol and ethyl acetate (Belguith-Hadriche et al. 2013). When grape pruning wastes (stems and leaves) were extracted by water and methanol, more compounds, mainly including terpenoids and phenolic compounds, were detected from the methanol extracts (Zuo et al. 2015). Similar results were also found in the extracts from C. camphora fallen leaves (Table 1).

When M. aeruginosa was treated with the extracts from A. annua, Conyza canadensis and Erigeron annuus, the cell growth was inhibited markedly, and their main components, terpenoids, may be the main anti-algal active ingredients (Ni et al. 2011). When M. aeruginosa cells were exposed to artemisinin, a terpenoid identified from A. annua, their soluble protein content decreased obviously, and superoxide dismutase activity and abscisic acid content increased remarkably (Ni et al. 2012). C. reinhardtii cell growth and photosynthesis were inhibited after the cells were treated with grape extracts with terpenoids and phenolic compounds as the main components (Zuo et al. 2015). Meanwhile, abundant VOCs including lots of terpenoids released from M. flos-aquae cells under non-N condition can inhibit Chlorella vulgaris cell growth (Xu et al. 2017), and from M. aeruginosa under non-P condition can inhibit C. reinhardtii cell growth (Ye et al. 2018). In this study, the inhibitory effects of C. camphora extracts on the growth of M. aeruginosa and C. reinhardtii should be caused by the terpenoid components (Figure 1), and the methanol extracts showed stronger effects due to their higher number of compounds and higher concentration (Table 1).

Previous studies have reported that linalool and camphor can inhibit the growth of several microorganisms, such as Campylobacter spp. (Duarte et al. 2016), Candida albicans (Zore et al. 2011), Schistosoma japonicum (Yang et al. 2014), Microsporum canis and M. gypseum (Silva et al. 2017), Escherichia coli, Staphylococcus aureus and C. albicans (Cutillas et al. 2017), by affecting membrane integrity and arrest of cell cycle (Zore et al. 2011). Meanwhile, linalool enhanced antifeedant activity against agricultural pests (Rani et al. 2014) and showed antitumor activity (Miyashita & Sadzuka 2013), and camphor treated several diseases, such as infection, inflammation, congestion, and pain (Hamidpour et al. 2013). The two compounds showed higher concentration in C. camphora extracts, which may be the main compounds to inhibit algal growth. In exposure to eucalyptol and limonene, C. vulgaris and C. reinhardtii cell growth reduced, due to the degradation of photosynthetic pigments and decrease of PSII efficiency (Zhao et al. 2016; Zhou et al. 2016; Xu et al. 2017). The two compounds existed in C. camphora extracts, and should play inhibitory roles on the growth of M. aeruginosa and C. reinhardtii. In addition, there were other terpenoids in C. camphora extracts, which might also contribute to the inhibitory effects.

In algae, Chl is an essential photosynthetic pigment and functions by capturing light and transducing it to biochemical energy during photosynthesis. Its content in C. reinhardtii cells declined after the cells were exposed to grape extracts (Zuo et al. 2015). When C. vulgaris and C. reinhardtii were treated with eucalyptol and limonene, their Chl including divinyl-pheophytin a, divinyl-Chl a, divinyl-Chl b, monovinyl-protochlorophyllide a, monovinyl-Chl b, and monovinyl-Chl a were degraded, and the degradation enhanced with increasing the compound concentration (Zhao et al. 2016; Zhou et al. 2016). This indicated that terpenoids can induce photosynthetic pigment degradation, which may be the reason for the decline of Chl levels in C. reinhardtii cells treated with C. camphora extracts (Figure 2).

Photosynthesis is the most fundamental biological process supporting algal growth and nutrient uptake. The characteristics of Chl fluorescence provide an insight into PSII photochemical efficiency and the damage level of the photosynthetic apparatus to a certain extent (Maxwell & Johnson 2000). Terpenoids or plant extracts containing amounts of terpenoids can block the PSII quantum production and electron transport in algae, and promote the absorbed solar energy dissipating as heat (Ni et al. 2012; Yang et al. 2012; Zuo et al. 2015; Zhao et al. 2016; Zhou et al. 2016; Zuo et al. 2017b; Xu et al. 2017). Similarly, the extracts from C. camphora fallen leaves inhibited the Fv/Fm in C. reinhardtii cells, and increased φDO (Figure 3).

CONCLUSIONS

Water and methanol extracts from C. camphora fallen leaves can inhibit M. aeruginosa and C. reinhardtii cell growth by inducing Chl degradation and reducing photosynthesis, and methanol extracts showed stronger inhibitory effects than water extracts at the same concentration, due to their greater number of components and higher concentration. This indicates that C. camphora fallen leaves have a potential value as an algaecide, and the algaecide is suitably extracted by methanol.

ACKNOWLEDGEMENTS

Authors Zumulati Yakefu and Wulan Huannixi contributed equally to this work. This research was supported by the Natural Science Foundation of Zhejiang Province (No. LY17C160004), the National Students' Innovation and Entrepreneurship Training Program (No. 102-2013200053), the Student Research Training Program in Zhejiang A & F University (No. 2013200038), the National Natural Science Foundation of China (No. 31300364), and the Personnel Startup Project of the Scientific Research and Development Foundation of Zhejiang A & F University (No. 2013FR069).

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Author notes

These authors contributed equally to this work.