Roles of Fenlong Technology in the Increase of Grain Yield and Construction of a Huge "Underground Reservoir"

Benhui WEI

AbstractUnder the premise of simulating the growth environment of algae in natural conditions, the responses of Microcystis aeruginosa to stress of Rabbosia japonica were studied. The results showed that the maximum specific growth rate of M. aeruginosa inhibited by water extract from R. japonica was -0.083 d-1, and the inhibition effects on the electrical conductivity and pH of M. aeruginosa solution were significant with long duration. When the concentration of water extract from R. japonica was low, the maximum inhibition rate of M.aeruginosa was lower than 20%. When the concentration was 500 or 1 000 mg/L, the inhibition rate was higher than 50% after 144 h. It was proved that the inhibition effect of high concentrations of R. japonica on M. aeruginosa was significant and stable, which can provide reference for the control of water bloom in aquaculture water.

Key wordsMicrocystis aeruginosa; Rabbosia japonica; Water bloom; Inhibition effect



Received: September 15, 2018Accepted: October 31, 2018

Supported by Natural Science Foundation of Tianjin City (17JCYBJC29800); Funding Plan Projects for Various Types of Talent in Tianjin Agricultural University (J01009030702).

Nan YNAG (1996-), female, P. R. China, master, devoted to research about exploitation and utilization of plant resources.

*Corresponding author. Email: njlnie@126.com.

In recent years, with the advancement of science and technology and the development of economy, a large amount of sewage, waste water, excess pesticides and fertilizer have produced and discharged into rivers and lakes, which has made the water eutrophication of main lakes in China increasingly serious and has brought increasing economic loss. Water eutrophication is the main factor causing cyanobacteria bloom. Cyanobacterial bloom will form a layer of bluegreen scented foam on the surface of water, which will hinder the exchange of air and the water. In addition, cyanotoxins will be released during the growth process of cyanobacteria (also called bluegreen algae) and can make water quality to be destroyed, which in turn seriously affects human health［1-3］. There are many species of bluegreen algae leading to water bloom, among which Microystis aeruginosa is the most common, and it is dominant in the bluegreen algae in China and the world. Therefore, research on the inhibition of M. aeruginosa growth has become the focus of researchers at home and abroad［4-6］.

Rabbosia japonica is a traditional Chinese herbal medicine of Rabdosia, Labiatae. It is a perennial erect herb with leaves opposite to each other, and the leaf margin is coarsely serrated, while the flowering and fruiting period is from June to September. It is mainly distributed in North China, Northeast China, Japan, North Korea and the former Soviet Union. The overground part of R. japonica can be used as medicine and is bitter and cold. It has effects of invigorating the stomach, invigorating the circulation of blood, clearing heat and detoxicating. It can be used to cure sore throat, a variety of inflammation, cancer, abdominal distension, cold and fever［7］. Moreover, R. japonica has the biological activity of protecting cardiac muscle, diminishing inflammation, anticancer and preventing the liver from being damaged by carbon tetrachloride. Glaucocalyxin A, one of highcontent components of R. japonica, can improve microcirculation and inhibit platelet aggregation［8］.

Different extracts of R. japonica have a certain inhibitory effect on prokaryotes such as Escherichia coli and Staphylococcus aureus. The antibacterial effect mainly comes from the synergistic effect of its own active ingredients such as glaucocalyxin A, diterpenoids, triterpenoids, sterols, etc［9］. Terpenoids can inhibit plant growth and affect cell membrane permeability［10］. In addition, R. japonica has inhibitory effects on algae and various microorganisms including marine fouling bacteria［11］. However, in an artificially simulated environment, the effect of R. japonica on algae has not been reported. In this study, in the artificially simulated natural growth environment of M. aeruginosa, the responses of M. aeruginosa to the inhibition of different concentrations of water extract from R. japonica were studied to analyze the ability of R. japonica to inhibit algae growth and provide scientific reference for the deep research of inhibition of algae growth by R. japonica.

Materials and Methods

Materials

The experimental species of M. aeruginosa was from the Institute of Hydrobiology, Chinese Academy of Sciences. R. japonica used in the experiment was bought from a Chinese medicine shop in Tianjin.

Main instruments

Main instruments included DDS307 conductivity meter (Shanghai Precision Scientific Instrument Co., Ltd.), CT6023 pentype acidity meter (Shenzhen Kodida Electronics Co., Ltd.), UH5300 Ultraviolet spectrophotometer (Beijing Puxi General Instrument Co., Ltd.), etc.

Methods

Domestication and culture of M. aeruginosa

M. aeruginosa was expanded in BG11 medium［12-13］. It was transferred under sterile conditions on a clean bench. It was cultured at room temperature and under natural light to allow cells of M. aeruginosa to enter the logarithmic phase. After the activation of M. aeruginosa, it was cultured at 30 ℃ in an artificial incubator for 7-10 d, with illumination intensity of 2 000 Lx and illumination ratio of 12∶12.

Preparation of water extract of R. japonica

Firstly, 0.025, 0.05, 0.1, 0.2, 0.5 and 1.0 g of R. japonica powder were weighed accurately and dissolved with water to 1 000 ml. After ultrasonic extraction was conducted for 30 min, they were stood at room temperature for 24 h, filtered by filter paper, and centrifuged for 10 min for use.

Setting of simulation environment

The soil sample that was collected from Jinghai previously and used to simulate the sediment in the growth environment of the algae was aired and sieved with a sieve with 40 meshes. 24 groups of 20 g of the sieved soil sample were put in culture flasks, to which a certain amount of the medium was added to stick the floating soil to the wall of the culture flasks. The culture flasks containing the soil sample were placed in a high temperature sterilization pot for wet sterilization and then were taken out for use after cooling. M. aeruginosa suspension was put in 21 sterilized glass jars (10 cm×10 cm×20 cm), and every 2 000 ml of the medium contained 20 ml of the algae solution and 20 g of the sterilized soil sample. A heating rod was placed in water to control water temperature at around 25 ℃. It was cultured for 5-10 d under natural light.

Test for inhibition of the algae

Firstly, 60 ml of water extract of R. japonica (25, 50, 100, 200, 500 and 1 000 mg/L) was added to the algae solution in the simulation environment. Meanwhile, 60 ml of distilled water was added to the algae solution, which was as the control group (CK). There were three parallel samples in each group. The absorbance, electrical conductivity and pH of the six test groups and the control group were detected after 0, 3, 6, 9, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240 and 264 h respectively. The glass jars were shaken twice a day, and the position was randomly changed. The experiment lasted for 11 d.

Determination methods

Density of algae cells was calculated with a blood counting chamber under a microscope (16×10 time)［13］. 

Specific growth rate of algae cells was calculated as follows:

μ= ln(Xi /X0)/( i-1)（1）

where μ was specific growth rate of algae cells; Xi was density of algae cells on the ith day after the water extract of R. japonica was added; X0 was initial density of algae cells before the water extract was added.

Inhibition rate of algae cells was calculated as follows:

IR = ( N0-Ns) /N0×100%（2）

where IR was inhibition rate of algae cells; N0 was density of algae cells in the control group; Ns was density of algae cells in the test groups［15］. 

In order to detect the electrical conductivity of the algae solution, the algae solution in each glass jar was stirred several times with a glass rod in the upper 1/3 position to make M. aeruginosa distribute evenly. The sensor of a conductivity meter was inserted into the algae solution in the upper 1/3 position, and readings on the sensor were recorded［16］. 

To measure the pH of the algae solution, the algae solution in each glass jar was stirred several times with a glass rod in the upper 1/3 position to make M. aeruginosa distribute evenly. Afterwards, the sensor of a pH meter was inserted into the algae solution in the upper 1/3 position, and readings on the sensor were recorded［16］.

Results and Analysis

Inhibition effect of different concentrations of water extract from R. japonica on M. aeruginosa

As shown in Fig. 1, when the concentration of the water extract increased from 25 to 200 mg/L, the cell density of M. aeruginosa reduced and was smaller than the control group, but there was no significant difference between the test groups and the control group. As the concentration of the water extract rose to 500 and 1 000 mg/L respectively, the cell density of M. aeruginosa decreased with the increase of treatment time. When treatment time was up to 264 h, the cell density was 3.646 when the concentration was 500 mg/L, significantly smaller than the control group. It showed that when the concentration of the water extract from R. japonica was higher than 500 mg/L, it would significantly inhibit the reproduction of M. aeruginosa.



Fig. 1Effects of different concentrations of water extract from R. japonica on the cell density of M. aeruginosa

Seen from Fig. 2, as the concentration of the water extract was 25-200 mg/L, the inhibition rate of algae cells changed slightly and was always smaller than 20%. After treatment time exceeded 216 h, the inhibition rate of algae cells began to decrease when the concentration of the water extract was 25, 100 and 200 mg/L. As the concentration of the water extract was 500 and 1 000 mg/L, the inhibition rate of algae cells rose with the increase of treatment time. That is, it increased from 50% to 60% with the increase of treatment time from 144 to 168 h, and it was above 60% when treatment time was 192, 216, 240 and 264 h. The results indicated that low concentrations of the water extract inhibited the reproduction of M. aeruginosa cells slightly, but the inhibition effect was obvious when the concentration was higher than 500 mg/L. The inhibition effect of water extract from R. japonica on M. aeruginosa showed clear timeeffect and dosageeffect relationship.

According to Table 1, the total degree of freedom was 20, and the degree of freedom among groups and in a group was 6 and 14 respectively. The calculation results showed that F=17.310, P=0.000<0.01, that is, there was 99% confidence that there was a significant difference between different concentrations of water extract from R. japonica in the inhibition effect of M. aeruginosa, thereby rejecting H0 and accepting H1. The inhibition effect of different concentrations of water extract from R. japonica on M. aeruginosa was significant at 0.01 level.



Fig. 2Inhibition rate of different concentrations of water extract from R. japonica on M. aeruginosa

Table1Analysis of variance of the inhibition effect of M. aeruginosa by different concentrations of R. japonica

Source of variationSum of squares (SS)Degree of freedom (df)Mean square (MS)FP value

Among groups1.53550.30719.0080

In a group0.194120.016

Total1.72917

Effects of different concentrations of water extract from R. japonica on the specific growth rate of M. aeruginosa in various treatment periods

Seen from Table 2, the specific growth rate of M. aeruginosa cells decreased with the increase of treatment time in the control group. The specific growth rate in the test groups was smaller than that of the control group, showing that the growth of M. aeruginosa was inhibited by different concentrations of water extract from R. japonica. When the concentration of the water extract was 25-200 mg/L, the specific growth rate was positive, indicating that the effect was not significant. As the concentration of the water extract was 500 mg/L, the specific growth rate became negative after 96 h, that is, the growth of M. aeruginosa cells was inhibited after 96 h. When the concentration of the water extract was 1 000 mg/L, the growth of algae cells was completely inhibited from the beginning. Seen from the changes of the specific growth rate, in the same concentration, the inhibition effect of the water extract increased with the increase of treatment time. In the simulation environment, with the increase of treatment time, the higher the concentration of water extract from R. japonica was, the lower the specific growth rate of M. aeruginosa, and the better the inhibition effect was.

Agricultural Biotechnology2019

Table 2Specific growth rate of M. aeruginosa inhibited by different concentrations of water extract from R. japonica

Time∥h

Specific growth rate∥d-1

0 mg/L 25 mg/L50 mg/L100 mg/L 200 mg/L500 mg/L1 000 mg/L

240.1880.1100.1180.1190.1070.069-0.047

480.1250.0980.0670.0710.0910.028-0.028

720.1010.0760.0540.0650.0760.004-0.026

960.0960.0680.0520.0620.061-0.011-0.029

1200.0870.0700.0570.0560.056-0.034-0.044

1440.0770.0620.0500.0550.057-0.05-0.046

1680.0700.0560.0440.0490.051-0.069-0.060

1920.0570.0450.0330.0390.041-0.065-0.064

2160.0520.0370.0320.0350.041-0.061-0.065

2400.0520.0420.0290.0430.047-0.054-0.074

2640.0490.0380.0280.0400.044-0.051-0.083

Effects of different concentrations of water extract from R. japonica on the electrical conductivity of M. aeruginosa solution in various treatment periods

As shown in Fig. 3, in the simulation environment, the electrical conductivity of M. aeruginosa solution in the control group fluctuated from 881 to 979 μs/cm within 216 h, but the change was not significant. With the increases in the concentration of water extract from R. japonica (25-200 mg/L) and treatment time, the electrical conductivity of M. aeruginosa solution rose, but there was no significant difference between the test groups and the control group. When the concentration of the water extract was 1 000 mg/L, the electrical conductivity of M. aeruginosa solution increased to 1 014 μs/cm after 120 h, significantly higher than that of the control group. It revealed that after the addition of high concentration of the water extract from R. japonica, the permeability of cell membranes of M. aeruginosa changed significantly, and their damage became more and more serious, so that the electrical conductivity of M. aeruginosa solution increased.



Fig. 3Effects of different concentrations of water extract from R. japonica on the electrical conductivity of M. aeruginosa solution

Effects of different concentrations of water extract from R. japonica on the pH of M. aeruginosa solution in various treatment periods

Seen from Fig. 4, in the simulation environment, the pH of M. aeruginosa solution in the control group was 8.3-10.2, showing an increasing trend. Meanwhile, the density of M. aeruginosa cells also increased gradually, showing that the higher the pH was, the higher the reproductive efficiency of M. aeruginosa. The pH of M. aeruginosa solution in the test groups (25-200 mg/L) was lower than that of the control group, but there was no significant change. When the concentration of water extract from R. japonica increased to 500 and 1 000 mg/L, the pH of M. aeruginosa solution was significantly lower than that of the control group, and decreased with the increase of treatment time after 192 h. When the concentration was 1 000 mg/L, the pH was lower than 8 after 24 h. The results were consistent with the result of effects of different concentrations of water extract from R. japonica on the specific growth rate of M. aeruginosa, which further proved that M. aeruginosa had a stronger response to the inhibition of high concentrations of water extract from R. japonica.



Fig. 4Effects of different concentrations of water extract from R. japonica on the pH of M. aeruginosa solution

Discussion

M. aeruginosa, which is the most common algae causing algal bloom, produces toxins and leads to absence of oxygen in water during the process of decomposition to destroy the normal food web and directly threaten human health and the survival of other animals and plants. Therefore, it is important to develop an environmentally friendly natural reagent used to inhibit algae growth. Studies have shown that the current natural environmental factors have more significant effects on the community structure of algae than human factors［17］. Environmental factors have synergistic effects on algae growth, and experimental conditions should be close to the environment during the process of research to obtain more reliable conclusions［18］. Therefore, temperature, light and soil conditions close to the environment were set in this experiment. It was found that in the artificial simulated natural environment, high concentrations of water extract from R. japonica could significantly inhibit the proliferation of M. aeruginosa cells.

M. aeruginosa is a prokaryote, and its structure is similar to that of E. coli and other bacteria. The inhibition effect of R. japonica on M. aeruginosa may be the result of pharmacological synergy of different types of chemical components［11］. The experimental results showed that when the concentration of the water extract exceeded 500 mg/L, the electrical conductivity of M. aeruginosa solution increased significantly. The reason is that the terpenoids in R. japonica might destroy the structure of M. aeruginosa cell membranes［10］, thereby inhibiting the proliferation of M. aeruginosa cells, which is consistent with the result of effect of different concentrations of water extract on the cell density of M. aeruginosa.

Most of the lakes in China are alkaline and the pH is high. Studies have shown that in the process of water bloom, the pH of water environment increases significantly with the growth of algae, and the pH of the water environment suitable for the growth of M. aeruginosa is 8-9 on average. Other studies real that high pH (about 11.0) can promote the growth and proliferation of algae cells［19-21］. As the concentration of the water extract was 1 000 mg/L, the pH of M. aeruginosa solution declined significantly after 24 h, lower than 8, while the pH of M. aeruginosa solution in the control group was always higher than 9. It showed that the environment with high pH was more suitable for the growth of M. aeruginosa, while the pH of water environment in the treatments containing water extract from R. japonica would be decreased to inhibit the growth of M. aeruginosa. The inhibition effect of water extract from R. japonica on M. aeruginosa showed clear timeeffect and dosageeffect relationship.

Conclusions

In the simulated natural growth environment, the responses of M. aeruginosa to the inhibition of different concentrations of water extract from R. japonica were studied. The results showed that low concentrations of water extract from R. japonica (25-200 mg/L) had no significant effect on the reproduction of M. aeruginosa. When the concentrations of water extract from R. japonica was higher than 500 mg/L, the cell density, specific growth rate and pH of M. aeruginosa decreased, while the inhibition rate and electrical conductivity of M. aeruginosa rose with the increase of treatment time and water extract concentration. According to the results of variance analysis, there was 99% confidence that the inhibition effect of different concentrations of water extract from R. japonica on M. aeruginosa was extremely significant. It provides reference for the application of R. japonica in water protection and the development of a new reagent used to inhibit algae growth in aquaculture water.

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