Preliminary Study on the Effects of Different Nutrient Solutions on the Parasitism of Cuscuta grandis to Mikania micrantha

Haifan WANG Ruijie ZHANG Yuanjun XU Jie GUO

Abstract [Objectives] This study was conducted to investigate the effects of different nutrient solutions on the parasitism of Cuscuta grandis to Mikania micrantha.

[Methods]The growth-promoting effect of C. reflexa parasitizing M. micrantha was studied through the use of different concentrations of plant growth regulators （naphthalene acetic acid， gibberellic acid） and compounds containing trace elements （manganese sulfate， copper sulfate， zinc nitrate， boric acid）.

[Results] Mixing naphthalene acetic acid with a concentration of 0.01 ml/L and gibberellic acid with a concentration of 1 mg/L at a ratio of 1∶3 and then adding an equal volume of compounds containing trace elements including manganese sulfate with a concentration of 0.1 mg/L， copper sulfate with a concentration of 0.1 mg/L， zinc nitrate with a concentration of 0.1 mg/L and boric acid with a concentration of 2 mg/L， the average stem length of the breeding group increased significantly， about 2.47 cm， showing a significant difference from the CK. The survival rate of the experimental group using the plant growth regulators and the trace elements was 66.67%， and the survival rate of the control group was 56.67%.

[Conclusions]This study can fill the gap in the study of seed morphology of C. reflexa， and is also of great significance for the identification and quarantine of C. reflexa.

Key words Breeding group; Plant growth regulator; Trace elements; Survival rate

Received： April 3， 2021  Accepted： June 5， 2021

Supported by Yunnan Forestry Technological College Fund [（KY（YB）201925）]; Special Fund for Scientific Research in Forestry Public Welfare Industry （201204518）.

Haifan WANG （1987-）， male， P. R. China， master， devoted to research about forestry science.

*Corresponding author.

Mikania micrantha is a perennial vine. It climbs and entangles arbors and shrubs in its suitable area， and presses on the top of plant canopies， hindering the photosynthesis of host plants and leading to the death of hosts. It is one of the most dangerous and harmful plants in the world[1-2]. In China， M. micrantha mainly harms natural secondary forests and plantations， especially local forest stands with small canopy closure below 6-8 m[3]. After a large number of experimental studies by scientific researchers， it has been found that the parasitism of Cuscuta reflexa on M. micrantha can effectively control M. micrantha[4]， that is to say， C. reflexa can become a treasure as a harmful plant， which can not only fill the gap in the study of the seed morphology of C. reflexa， but also has very important significance for the identification and quarantine of C. reflexa[5].

Materials and Methods

Experimental materials

Ruili City， Dehong Prefecture， Yunnan Province was selected as the experimental site. Plant growth regulators and trace elements suitable for the rapid growth of C. reflexa were selected through indoor and outdoor experiments， and the plant growth regulators and trace elements produced by C. reflexa parasitizing M. micrantha were used to improve its rate of successful parasitism[4，6]. Meanwhile， the most suitable parameters for C. reflexa to entwine and parasitize M. micrantha were analyzed through the outdoor experiments， so as to achieve the purpose of using C. reflexa for controlling M. micrantha[7-8].

Experimental design

Indoor experiments

The C. reflexa plants used in the following experiments were randomly taken in the field. They had basically the same stem thickness， and intact stem tips free of damage. The experimental tools were penicillin vials， graduated cylinders， beakers， plastic droppers， glass rods and water.

Indoor experiment No.1： Effects of different growth regulators on the growth of C. reflexa

Objective： Six kinds of plant growth regulators were used to cultivate C. reflexa， and the effects of compounds containing trace elements on the growth and survival rate of C. reflexa were observed.

Experimental agents： The plant growth regulators were naphthalene acetic acid， benzyl aminopurine， gibberellic acid， ethephon， indole acetic acid， indole butyric acid.

Agent preparation： The solutions were prepared according to the experimental requirements （Table 1）， and each group in the experiment was set with five replicates.

Cultivation of C. reflexa： C. reflexa was cultured in sterilized penicillin bottles， and five replicates were done for each concentration.

Recording and analysis of experimental data： The length and stem thickness of C. reflexa were measured and recorded every 2 d. After 8 d， the length and thickness of C. reflexa were compared to select the most suitable solution for the growth of C. reflexa.

Indoor experiment No.2： Cultivation of C. reflexa with compounds containing trace elements

Objective： The compounds containing trace elements were prepared into solutions to cultivate C. reflexa， and the effects of the compounds containing trace elements on the growth and survival rate of C. reflexa were observed.

Experimental agents： Naphthalene acetic acid with a concentration of A2 （0.01 ml/L） and gibberellic acid with a concentration of C1 （1 mg/L）; compounds containing trace elements， manganese sulfate， copper sulfate， zinc nitrate， and boric acid.

Agent preparation： According to the experimental requirements， the solutions of the following concentrations were prepared respectively （Table 2）. In the experiment， each group was repeated 5 times.

Cultivation of C. reflexa： C. reflexa was cultured in sterilized penicillin bottles.

Recording of experimental data： The length and stem thickness of C. reflexa were measured and recorded every 2 d. After 8 d， the length and thickness of C. reflexa were compared， and the optimal dilutions for the growth of C. reflexa were screened out.

Indoor experiment No.2： Cultivation of C. reflexa by mixing trace element compounds and plant growth regulators

Objective： The plant growth regulators and compounds selected in Experiment 1 and Experiment 2 were mixed together to cultivate C. reflexa and observe the growth status of C. reflexa.

Experimental agents： Naphthalene acetic acid， gibberellic acid， manganese sulfate， copper sulfate， zinc nitrate， boric acid.

Agent preparation： According to the experimental requirements， solutions were prepared as follows： naphthalene acetic acid with the concentration of A2 （0.01 mg/L）， gibberellic acid with the concentration of C1 （1 mg/L）， manganese sulfate with the concentration of I2 （0.1 mg/L）， copper sulfate with the concentration of J2 （0.1 mg/L）， zinc nitrate with the concentration of K2 （0.1 mg/L）， and boric acid with the concentration of R3 （2 mg/L）.

Cultivation of C. reflexa： The prepared naphthalene acetic acid and gibberellic acid were mixed in volume ratios of 1∶ 1∶ 1∶3， 2∶ and 3∶ respectively. The mixtures were added into medical penicillin vials， which were then added with the same volume of manganese sulfate， copper sulfate， zinc nitrate and boric acid prepared at desired concentrations. C. reflexa was inserted into the vials and cultured.

Observation and recording of data： The thickness and length of the stem growth of C. reflexa were measured every 5 d， and the data were recorded in the table.

Outdoor experiment

Experimental objective

The plant growth regulators and compounds containing trace elements screened out in the laboratory were used in the experiment of C. reflexa parasitizing M. micrantha in the experimental site to demonstrate the practical value.

Experimental equipment and agents

Experimental materials： Centrifuge tubes， packaging tape， bamboo sticks， measuring cylinders， droppers， rubber head droppers， beakers， glass rods， domestic water.

Experimental agents： naphthalene acetic acid， gibberellic acid， manganese sulfate， copper sulfate， zinc nitrate， boric acid.

Sample plot selection

A place with flat terrain， sufficient light and coverage of M. micrantha of 90% was selected. Two plots of the same size （5 m×5 m） were selected as the sample plots， and one of the plots was used as the control group.

Parasitizing experiment

C. reflexa plants with a stem length of 15 cm were collected， and entwisted on 30 M. micranthas plants in plots one and two， respectively. The number of windings was three. After winding， they were fixed with packing tape.

Preparation and use of agents

The mixed solution of naphthalene acetic acid with a concentration of 0.01 ml/L， gibberellic acid with a concentration of 1 mg/L， manganese sulfate with a concentration of 0.1 mg/L， copper sulfate with a concentration of 0.1 mg/L， and zinc nitrate with a concentration of 0.1 mg/L and boric acid with a concentration of 2 mg/L， was prepared， 5 ml each. The prepared solution was added in centrifuge tubes， which were inserted into the soil， and the other end of the stem tip of C. reflexa parasitizing on M. micrantha in the experimental group was put in the liquid in a centrifuge tube （each C. reflexa was inserted in a centrifuge tube）.

Observation and recording of data

After 8 d， we went to the experimental site to count the number of suckers produced in the sample plot 1 and sample plot  and measure and record the length of the stem of C. reflexa.

Results and Discussion

Analysis of the results of different plant growth regulators on the growth of C. reflexa

From the data in Table 3， it can be concluded that various plant growth regulators had little effects on the stem thickness of C. reflexa， and mainly had greater impacts on the stem length and survival rate of C. reflexa. However， when cultivating C. reflexa with ethylene （ethephon）， it not only could not promote the lateral growth of C. reflexa， but also made the survival rate of C. reflexa close to 0， so ethylene （ethephon） was not suitable for the growth of C. reflexa as a plant growth regulator.

Through the analysis of variance， it could be concluded that among the five plant growth regulators， naphthalene acetic acid and gibberellic acid had the greatest impacts on the stem length of C. reflexa （Table 4-5）， and the stem length increased by 12.76 and 13.5 cm， respectively. The survival rates were 90% and 80%， respectively， which were also relatively high.

Haifan WANG et al. Preliminary Study on the Effects of Different Nutrient Solutions on the Parasitism of Cuscuta grandis to Mikania micrantha

Result analysis of compounds containing trace elements on the growth of C. reflexa

The analysis of variance between the control group and the trace element-containing compounds （manganese sulfate， copper sulfate， zinc nitrate， and boric acid） gave P<0.05， that is， the control group and the four trace element-containing compounds were significantly different. It showed that the compounds containing trace elements， manganese sulfate， copper sulfate， zinc nitrate， and boric acid， could promote the growth of the stem length of C. reflexa （Table 6）.

Analysis on the results of plant growth regulators and trace element compounds on C. reflexa

The data in Table 7 represent mean ± standard error， and the data were analyzed by LSD multiple comparison analysis; different lowercase letters indicate that the stem lengths of C. reflexa cultivated at different ratios of naphthaleneacetic acid and gibberellic acid were significantly different （P<0.05 means the difference is significant， and P>0.05 means the difference is not significant）; and different capital letters indicate a significant difference in the survival rate. Specifically， CK was significantly different from all of O O O3， O4 and O5; O1 was significantly different from O O4 and O5; O1 and O3 were not significantly different; O2 was significantly different from O1 and O3; and O O4 and O5 were not significantly different.   Therefore， O1 and O3 showed the best effect. And because the survival rate of O3 was 100%， it could be seen that mixing plant growth regulators and compounds containing trace elements in different proportions caused differences in the cultivation of C. reflexa. O3， that is， mixing naphthaleneacetic acid and gibberellic acid at a ratio of 1∶3 with the same volume of trace element compounds， achieved the best effect， which was reflected in the stem length increased the most， reaching 13.4 and the survival rate which was also higher.

Conclusions and Discussion

Indoor experiments

Through the analysis of the effects of different plant growth regulators on C. reflexa， the growth of the stem length of C. reflexa was the best at the concentration of naphthalene acetic acid of A2 （0.01 mg/L）， the concentration of benzyl aminopurine of B1 （0.2 mg/L），  the concentration of gibberellic acid of C1 （1 mg/L）， the concentration of indole acetic acid of G1 （2 mg/L） and the concentration of indole butyric acid of H1 （2 mg/L）， and the average increases in the stem length were 12.76， 12.38， 13.56，12.40 and 12.38 cm， respectively.

The results of the experiment on the effects of compounds containing trace elements on the growth of C. reflexa showed that the growth of the stem length of C. reflexa was the best at the concentration of manganese sulfate of I2 （0.1 mg/L）， the concentration of copper sulfate of J2 （0.1 mg/L）， the concentration of zinc nitrate of K2 （0.1 mg/L）， and the concentration of boric acid of R3 （2 mg/L）， and the average increases in the stem length were 12.0 11.58， 11.24 and 12.02 cm， respectively.

Analysis of variance and multiple comparisons screened out that the naphthalene acetic acid at the concentration of A2 （0.01 mg/L） and the gibberellic acid at the concentration of C1 （1 mg/L） among the plant growth regulators were the best for the stem length growth and survival rate of C. reflexa. The average increase in the stem length of C. reflexa in naphthalene acetic acid at the concentration of A2 （0.01 mg/L） was 12.76 cm， and the survival rate was 90%. Gibberellic acid with a concentration of C1 （1 mg/L） increased the average stem length of C. reflexa by 13.56 cm， and the survival rate was 80%.

When cultivating C. reflexa in plant growth regulators naphthalene acetic acid （0.01 mg/L） and gibberellic acid （1 mg/L） at certain proportions， mixed with the same volume of manganese sulfate （0.1 mg/L）， copper sulfate （0.1 mg/L）， zinc nitrate （0.1 mg/L）， boric acid （2 mg/L）， mixing naphthalene acetic acid and gibberellic acid at a ratio of 1∶3 with an equal volume of trace element compounds achieved the best effect， as the stem length grew the most， reaching 13.42 cm， and the survival rate was as high as 100%.

Outdoor experiments

The experimental results showed that mixing naphthalene acetic acid with a concentration of 0.01 mg/L and gibberellic acid with a concentration of 1 mg/L at a ratio of 1∶3 and then adding an equal volume of compounds containing trace elements including manganese sulfate with a concentration of 0.1 mg/L， copper sulfate with a concentration of 0.1 mg/ L， zinc nitrate

with a concentration of 0.1 mg/L and boric acid with a concentration of 2 mg/ L， the average stem length of the breeding group increased significantly， about 2.47 cm. The survival rate of the experimental group using the plant growth regulators and the trace elements was 56.67%， and the survival rate of the control group was 66.67%.

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