Summary

Control and Disposal of Invasive Japanese Knotweed Reynoutria japonica Houtt. Using Microwave Treatment

Published: November 15, 2024
doi:

Summary

This protocol provides a method for microwave control of Japanese knotweed in the field and disposal of dug-up rhizomes in laboratory conditions. The advantages and disadvantages of both methods are discussed. Future research directions are also suggested to optimize the use of microwaves for controlling Japanese knotweed.

Abstract

The study aims to assess the effectiveness of microwave treatment (MWT) at a frequency of 2.45 GHz and a power of 800 W to control Japanese knotweed (Reynoutria japonica Houtt.) using a self-propelled device that was built in the in-house facility. The MWT was applied in the field population of knotweed in July 2022. First, plants were mechanically moved from the area of 1 m2, and next, the cut shoots around 4 cm high were microwave-treated for 25 min, 20 min, and 15 min. The control treatments were: 1) only cut plants and 2) rhizomes dug out to 30 cm deep. The effectiveness of the microwave treatments was observed for the next 11 months by counting the number of newly grown shoots. The results showed that a 25 min MWT was 100% effective in Japanese knotweed loss of vitality, while a 15 min MWT microwave treatment stimulated plant growth by around 50%, compared to controls. Rhizomes were dug out in a separate in vitro experiment for laboratory testing. The rhizomes were categorized by thickness and subjected to a 60 s MWT using a commercial microwave, after which their temperature and vitality were assessed. The temperature of rhizomes following MWT depended on their thickness. Those rhizomes that warmed to temperatures above 42 °C were effectively destroyed. Summing up, the time plants are exposed to microwaves plays a major role in the effectiveness of this method. The longer the exposure to MWT, the better control. The thinner the rhizomes, the more effective the in vitro MWT rhizomes disposal.

Introduction

Japanese knotweed (Reynoutria japonica Houtt.) is one of the seven invasive plant species that threaten the natural environment in Poland1. This plant, outside its original range, exhibits a wide spectrum of habitats from anthropogenic habitats, including railway embankments, roadsides, parks, cemeteries, home gardens, various types of urban and post-industrial wastelands to natural ones, e.g., forest edges, riverbanks, thickets. It can also sometimes be found in agricultural areas. It copes well with various types of soils with various pH, from acidic to slightly alkaline2,3. It exhibits high tolerance to high temperature, salinity, periodic flooding, and drought2. It is also very resistant to soil contamination, including sulfur compounds4. Knotweed seriously threatens nature and contributes to the decline in plant species richness. They effectively compete with native species, preventing their regeneration through rapid growth and limiting light access5. They affect other plants alleopathically and cause changes in the physical and chemical properties of the soil. In addition, they negatively affect the human economy by limiting visibility along roads, destroying flood protection, reducing the attractiveness of investment and tourist areas, and causing economic losses associated with their control6,7.

Many attempts have been made to control Japanese knotweed, mainly using synthetic herbicides such as glyphosate or 2,4-D8. However, due to unfavorable environmental effects, this method is not recommended for most sites occupied by knotweed. On the other hand, mechanical methods involve regular mowing of plants, which are ineffective due to the deep system of rhizomes from which new shoots emerge9. An interesting solution is to use dense nets that limit the growth of knotweed, but this method also has limitations due to possible damage to the net or shoots growing outside its area. Therefore, lateral methods of controlling this species are sought. One such method may be using microwaves10.

Microwaves are electromagnetic waves with frequencies from 0.3 GHz to 300 GHz and wavelengths from 1 m to 0.001 m. Microwave radiation is invisible to the human eye. The electromagnetic spectrum of a microwave oven falls within the range of infrared radiation and radio frequencies11. Of the wide range of microwave frequencies, only a few are intended for medical or industrial applications. Federal Communications Commission regulations specify the use of specific microwave frequencies. Microwaves are transmitted through electrically neutral materials such as paper, glass, ceramics, and most plastics and are reflected by metals. In the absorbing material, they cause heat to be generated12. The electromagnetic field at microwave frequencies mainly provides energy to living organisms within its range. The thermal effect consists of increasing the body temperature due to the body's absorption of some energy. The appropriate frequency, field intensity, and the organism's ability to thermoregulate are required to increase the temperature of the tissue. It also depends on the time of exposure and the type of tissue. When a critical tissue heat level is exceeded, protein denaturation occurs13.

Microwave radiation has been used in natural sciences for many years. It is used, for example, for heating air in greenhouses14, disinfecting soil15,16,17, and drying fruits and vegetables18,19,20. Microwaves can also destroy insect pests of crop plants21,22,23 or weeds at a seedling stage24. Recent studies also indicate the high effectiveness of the microwave method in combating invasive Sosnowsky's hogweed10,25.

The device HOGWEED was built in the Faculty of Forestry of the University of Agriculture in Krakow26. It has its drive and moves on a caterpillar chassis, which can be used in areas with difficult access. Such a drive system does not damage the ground because the rubber tracks exert low unit pressures on the terrain. A radio remote control remotely controls the vehicle. The device was constructed to study the effect of microwaves on invasive weeds in natural ecosystems.

The study aims to determine the effectiveness of microwave radiation with a wave of 2.45 GHz, a power of 800 W, and an assumed operating time (from 15-25 min) for controlling the growth of Japanese knotweed plants (Reynoutria japonica Houtt.) in the field using the HOGWEED device. The study also aims to determine the disposal of rhizomes in laboratory conditions using a commercial microwave device. Disposal is important in the safe management of invasive plant waste so that it does not threaten environmental safety.

Protocol

We conducted the field experiment using a field population of invasive Japanese knotweed (Reynoutria japonica Houtt.) localized in Kraków (50.04 N, 19.63 E) under the written agreement with and supervision of the Kraków Municipal Greenery Board, which manages this area.

1. Field control of Japanese knotweed using a specialized device emitting microwaves

  1. Build the microwave emitter using a magnetron, which generates waves at a frequency of 2.45 GHz and a power of 800 W. Keep the aperture area of the horn antenna at 0.024254 m2 (134 mm x 181 mm) and the microwave power density at 32.8 kW/m2. Make the waveguide and antenna of four 1 mm thick brass sheets and join them with soft solder. Ensure the inner side of the sheet plate is silver coated to increase the conductivity of the metal surface26.
  2. Conduct microwave control of Japanese knotweed during its intensive growth period when plants are around 0.5-1.0 m high.
  3. Count the number of above-ground knotweed shoots per 1 m². Cut all above-ground parts of the plants using a hand mower approximately 4 cm above the ground surface.
  4. Mechanically remove dry leaves from the surface using a leaf blower to prevent burning during microwave treatment.
  5. Record the temperature of the prepared surface before treatment with the thermal camera.
  6. Place the microwave emitter on the prepared surface on the shoots in its center and push it slightly to adhere tightly to the ground. Emit microwaves by pressing a button on the machine and carry out the treatment for 25 min, 20 min, and 15 min for a surface of 268 mm x 362 mm dimensions.
  7. Record the temperature of the treated surface with the thermal camera.
  8. For controls, use surfaces on which the above-ground parts are only mechanically cut off using a hand mower at around 4 cm above the ground (control 1 – mowed), and the rhizomes are dug out to an approximate depth of 30 cm (control 2 – dig out). To help dig out the rhizomes, use a mobile compressor with a narrow-stream nozzle first and then pull out the visible rhizomes with the help of a metal cutter.
  9. Check the growth of plants in the research area monthly and compare it with both control areas during the next several months until the month of intensive growth of plants, e.g., from July to May. Manually count and document photographically the number of new knotweed shoots.

2. In vitro disposal of Japanese knotweed rhizomes using microwaves

  1. As a microwave source, use a commercial chamber device with a frequency of 2.45 GHz and a power of 800 W, with an electrically controlled capacity of 28 L.
  2. Dig out Japanese knotweed rhizomes from a depth of up to 30 cm and cut them into 28 cm sections using shears.
  3. Divide rhizome into three thickness classes, using a caliper to measure the largest diameter of the rhizome. Give the measurement result in centimeters to two decimal places. Use a drawing ruler to calculate the result in centimeters to one decimal place. Class I up to 1.00 cm; Class II 1.01-2.00 cm; Class III above 2.01 cm.
  4. Choose ten representative rhizomes per thickness class. Weigh the rhizomes' fresh mass with a balance. Express the results in g to two decimal places.
  5. Place the rhizomes in a microwave oven and microwave them for 60 s. Immediately after microwave treatment, take a thermogram with the thermal imaging camera to determine the temperature to which a given rhizome had heated up.
  6. Weigh the microwaved rhizomes again after microwave treatment when they cool down to room temperature.
  7. Take an additional eight rhizomes to determine their moisture and dry mass. Weigh rhizomes before placing them in the laboratory dryer at 105 °C for 2 days. After that time, weigh them again.
  8. Determine the temperature of the rhizomes based on the thermograms of the thermal imaging camera. Determine the average, maximum, and minimum temperature of a marked area or section. In this experiment, each rhizome was divided into 5 equally spaced points-ellipses of an area of around 2 cm that did not extend beyond the outline of the rhizome. Then, calculate each rhizome's average, maximum, and minimum temperature from the 5 points.
  9. Place the microwaved rhizomes individually on trays lined with sterile cotton wool. Ensure one tray contains rhizomes of the same thickness class. Use separate trays for the control rhizomes.
  10. Water the trays with tap water. Cover with colorless food foil to reduce water loss. Place the trays in a shaded area, monitor them, and top with water when needed. Perform monitoring until new shoots are observed or a visible decay of tissues occurs, e.g., for 14 days.
  11. For the rhizomes that survive and develop new shoots, perform additional analysis of their temperature along the entire rhizome length.

Representative Results

Field control of Japanese knotweed using a specialized device emitting microwaves
The average number of shoots per 1 m2 of the microwave-treated area was 27. Figure 1 shows the average number of shoots per 1 m2 that grew after microwave treatment for 11 months after microwave exposure. No new knotweed shoots appeared in areas treated with microwaves for 25 min. Compared to the control area, a decrease in the number of above-ground shoots was noted in the plot, where the microwave radiation time was 20 min. Over 11 months, the number of shoots was 20 pieces (pcs.)/m2. In turn, microwave treatment for 15 min contributed to an increase in the number of sprouting shoots. In September and October 2022, 50 pcs./m2 were noted, and about 40 pcs./m2 in the place where the rhizomes were removed (mechanical control). In May 2023, the number of above-ground shoots on the surface with removed rhizomes was 30 pcs./m2, similar to the control (26 pcs/m2). On the other hand, on the surface that was irradiated for 15 min in May 2023, 70 pcs/m2 were recorded. Therefore, the stimulating effect on the production of an increased number of above-ground shoots of knotweed after 15 min of microwave treatment was more noticeable in the long-term studies than immediately after the treatment.

Figure 1
Figure 1: Count of Japanese knotweed shoots. The number of Japanese knotweed (Reynoutria japonica) shoots 11 months after microwave treatment (MWT). MWT time: 0 (control, mowed), 15 min, 20 min, and 25 min. Dig out – rhizomes that were dug out. Please click here to view a larger version of this figure.

In vitro disposal of Japanese knotweed rhizomes using microwaves
After microwave treatment for 60 s, all rhizomes lost mass (Figure 2). The rhizomes in the first thickness class, i.e., the thinnest (17.9%), lost the most mass. That is almost 16% more than rhizomes in the third thickness class (the thickest). The rhizomes from all thickness classes subjected to microwave treatment had a lower dry mass (on average 30.49%) than the rhizomes dried immediately after digging (34.7%). The rhizomes from the first thickness class (up to 1 cm) had the lowest dry mass after drying. Their dry mass was almost 6% lower than the rhizomes from the second and third classes and over 8% lower than those previously dried at 105 °C.

Figure 2
Figure 2: Weight measurement for Japanese knotweed. Percentage of mass in Japanese knotweed (Reynoutria japonica) rhizomes after microwave treatment for 60 s and percentage of dry mass (105 °C) to control rhizomes. The data is classified based on the thickness class of rhizomes. I – first thickness class, II – second thickness class, III – third thickness class. Number of samples n=10; bars show standard error. Please click here to view a larger version of this figure.

After microwave treatment of the rhizomes for 60 s, the highest temperature was observed in the rhizomes from the first thickness class and the lowest in the rhizomes from the third thickness class (Figure 3). The thinner the rhizomes, the higher the temperature after microwave treatment. In thickness class I, the minimum temperature of the rhizomes was 55 °C. In thickness class II, it was 43 °C; in class III, it was almost 39 °C. The maximum temperature of the rhizome in thickness class III was 83 °C, which was lower than the average temperature of rhizomes in thickness class I (97 °C).

Figure 3
Figure 3: Temperature measurement for Japanese knotweed. The temperature of Japanese knotweed (Reynoutria japonica) rhizome after 60 s of microwave treatment depending on rhizome thickness, I – first thickness class, II – second thickness class, III – third thickness class, Tavg – average temperature, Tmax – maximum temperature, Tmin – minimum temperature. Number of samples n=10; bars show standard error. Please click here to view a larger version of this figure.

After 14 days of microwave treatment, the control rhizomes that were not subjected to microwave radiation or drying showed 100% survival (new shoots sprouted from the rhizomes). The rhizomes dried at 105 °C were dead. The use of microwave radiation showed that 60 s is enough to completely dispose of living rhizomes with a diameter of less than 2 cm. Above this limit, the efficiency of microwave treatment was 80%. Analyzing the results of changes in rhizome temperature, it was noticed that new shoots sprouted from those rhizome fragments that had a temperature after microwave treatment below 42 °C (Figure 4).

Figure 4
Figure 4: Japanese knotweed (Reynoutria japonica) rhizome with two new shoots. (A) Temperature graph of the rhizome along its entire length, (B) thermogram with the line function, (C) Rhizome with growing shoots, (D) thermogram of the rhizome with the ellipse functions marked, (E) results of measurements of the temperature ellipse function in the form of a table. The red blocks show two spots on the rhizome (C) with the new shoots and reference temperature (A) of these spots directly after microwave treatment. Ellipses refer to the fragments of the rhizome (C) where detailed temperature measurements were taken (E). Please click here to view a larger version of this figure.

It was also observed that rhizomes secreted drops of a sticky substance with a yellowish color (Figure 5A) and an intense, unpleasant smell shortly after microwave treatment. In addition, dead rhizomes were covered with a white coating 14 days after the microwave treatment (Figure 5B), probably due to mycelium growth.

Figure 5
Figure 5: Examples of Japanese knotweed (Reynoutria japonica) rhizomes. (A) Japanese knotweed immediately after microwave treatment with visible drops of sticky, yellowish secretion, (B) dead Japanese knotweed (Reynoutria japonica) rhizome covered probably with a white mycelium. Please click here to view a larger version of this figure.

Discussion

The effectiveness of Japanese knotweed (Reynoutria japonica Houtt.) control using the constructed device and knotweed rhizomes disposal using a commercial microwave oven were demonstrated. Both devices emitted microwaves at a frequency of 2.45 GHz and a power of 800 W.

It was observed that the longer the exposure of knotweed plants to microwave radiation, the lower and later their regeneration. In Japanese knotweed, 25 min microwave treatment effectively destroyed 100% of plants in the ground (in the research area). Concerning the control area (only mowed), 20 min microwave treatment caused a reduction and halt in the growth of above-ground parts of plants. Digging out rhizomes (mechanical control) and 15 min microwave treatment increased the number of sprouting shoots compared to the control-only mowed area. Studies on the disinfection of nursery substrate show that increasing exposure time to microwave radiation energy causes an increase in the depth of heating17. The system of Japanese knotweed rhizomes can reach up to 3 m into the soil27. Therefore, the appropriate selection of the duration of the emitted energy is very important and is a key critical step of this protocol. The same microwave device was used to control another invasive species – Japanese hogweed. The results showed that 10 min and 15 min of microwave treatment effectively destroyed 100% of Japanese hogweed plants in the leaf rosette and flowering stages, respectively10. The Japanese hogweed plant has a root system that can reach a depth of 2 m28, which is 1 m shallower than the Japanese knotweed rhizome. For these reasons, in this study, the microwave treatment time of the soil with Japanese knotweed rhizomes was assumed to be over 15 min.

The stimulating effect of microwaves was observed in vitro on sequoia plants irradiated with low-power microwaves29. Long-term signals with a power density of 10 mW/m2 and a frequency of 915 Mhz caused significant morphological modifications in Phaseolus vulgaris30. A slight increase in temperature to over 30 °C combined with microwaves at a frequency of 9.3 GHz stimulated plant growth31. It also contributed to a better distribution of saccharides in seeds or plants and a reduction in the content of non-structural carbohydrates.

The effect of microwave treatment can also be influenced by biological and environmental factors, especially soil moisture and initial temperature, which is another critical factor of this protocol. Studies show that in two identical soils with the same moisture content and subjected to the same microwave radiation energy, the soil with a higher initial temperature will heat up faster, e.g., due to solar radiation32. The effect of the microwave field also depends on the degree of hydration of the object being tested. Long-term exposure contributes to the evaporation and even boiling of water in the tissues, which directly causes the organism's death33. Seeds heat up and are destroyed faster when their water content exceeds the soil moisture16. The soil in this protocol had an average moisture content of 17.1%, as measured by the dry-weight method34. The collected rhizomes had an average water content of over 65% (34.7% dry mass). Therefore, they differed in moisture content from the soil almost four times, which could have influenced the faster heating of the rhizomes.

The difference in rhizome heating after microwave treatment was also noticeable between the three classes of rhizome thickness in the in vitro protocol. The thinnest rhizomes reached the highest temperature, and the thickest ones the lowest. Microwaves were used to control various organisms, and the effectiveness of this method was noticeable when a given body was heated to a critical level. This is usually the temperature at which protein denaturation occurs, i.e., around 42 °C17. In the case of knotweed rhizomes, it was also of crucial importance. Rhizomes that reached the minimum temperature above 42 °C along their entire length were disposed of. Those rhizomes that did not warm up to this temperature in some parts survived and sprouted. Results showed that 20% of rhizomes with a diameter above 2 cm survived, and none below this diameter survived. The dry mass of the thinnest rhizomes with a diameter of up to 1 cm was 26.6%, and rhizomes with a diameter of over 1 cm had almost 6% higher dry mass after drying. When the plant's outer part has a high polar liquid content, it causes strong field attenuation and heat concentration in the near-surface parts35. However, due to conduction or convection, the temperatures of the rest of the body are equalized. Therefore, due to being older and having uneven cross-section moisture, thicker rhizomes may not heat up in deeper parts to the temperature at which protein denaturation occurs.

High tissue temperature during microwave treatment caused the secretion and accumulation of a sticky substance on the surface of the rhizomes. The microwaved rhizomes also had a lower average dry mass (30.5%) than the control-dried ones (34.7%), which may indicate the disintegration of cells and tissues. Tissue disintegration and the secretion of juices were observed during microwave treatment of Sosnowsky's hogweed (Heracleum sosnowskyi Manden). It was noted that this treatment causes protein denaturation, changes in amino acid metabolism, and glyoxylate and galactose pathways. In addition, these juices were rich in sugars and numerous amino acids10. studies on Withania somnifera showed that exposure of plants to 900 MHz electromagnetic waves for 72 h causes an increase in the accumulation of harmful phenolic compounds by 32.1%, flavonoids by 14.9%, and a decrease in the activity of DPPH radical scavenging by 56.3%36. Therefore, the substance released during microwave treatment of Japanese knotweed rhizomes should be further tested to check its composition.

In summary, using the microwave method to remove knotweed plants in natural conditions is possible and promising. However, the method requires optimization, especially in terms of the effect of temperature and humidity of the soil and rhizomes on the effectiveness of the microwave treatment and optimizing the microwave time.

In the case of the disposal of knotweed rhizomes, the method used is very promising, especially concerning young rhizomes with a diameter of less than 2 cm. Just 60 s of microwave exposure is enough to prevent the rhizomes from regenerating. The limit temperature for the regeneration and proper control of microwaved knotweed rhizomes is 42 °C. For these reasons, the in vitro disposal technique of Japanese knotweed rhizomes has the potential for broader applications.

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

This research was funded by the Ministry of Science and Higher Education of the Republic of Poland.

Materials

AXIS BTA2100d AXIS Sp. z o.o. balance
CompAir C50 LECTURA GmbH Verlag mobile compressor 
FLIR E60  FLIR Systems, Inc. thermal imaging camera 
FLIR Tools  FLIR Systems, Inc. software to analyse the temperature from the thermogram
HDL_ANT version 3b4 program PC Software by W1GHZ software
Heraus UT 6120  Heraeus  laboratory drier

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Słowiński, K., Grygierzec, B., Tabor, S., Bucior, S., Synowiec, A. Control and Disposal of Invasive Japanese Knotweed Reynoutria japonica Houtt. Using Microwave Treatment. J. Vis. Exp. (213), e67660, doi:10.3791/67660 (2024).

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