Burcucumber (Sicyos angulatus) is one of the first ecosystem disturbing plants designated by the Ministry of Environment, Republic of Korea in 1998 and is known to be distributed in one of the largest areas among ecosystem disturbing plants. S. angulatus is a broad-leaved annual species, member of Cucurbitaceae family and is originated from North America, and has been widely spread to Asia and Europe. It grows well in swamps, woodland floodplains, and river floodplains. Observation made in infested riverbanks in the Republic of Korea presents that huge seed germination could completely cover ground during the growing season (Kil et al., 2006). This monospecific cover is a threat to other species and to the whole ecosystem.
In Article 2 (7) of the Act on the Conservation and Utilization of Biodiversity, the Ministry of Environment defines 16 species of ecosystem disturbing plants have been designated so far through risk assessment (Kim, 2017). Since these ecosystem-disturbing plants are mainly around people’s lives (Moon et al., 2008), it can be said that the control of using commercial synthetic herbicides is practically limited. Until now, the means for the spread or removal of ecosystem disturbing plants in Korea mainly depend on physical methods such as disposal actions, but because of its strong self-sustaining and renewable power, the proliferation is accelerating to a level that only temporarily decreases the number of individuals (Choi, 2017). Such problems may tempt chemical control due to practical difficulties in controlling ecosystem-disturbing plants, but the use of synthetic herbicides is bound to be limited due to the habitat of ecosystem disturbing plants and safety problems that may occur when used. As an alternative solution, the discovery of natural herbicide-based materials and technology development are attracting attention. Natural herbicide materials are likely to exhibit different action characteristics from synthetic herbicides depending on their characteristics, and often have the advantage of securing safety for the environmental ecosystem (Christy et al., 1993; Dayan et al., 2009).
In Korea, research on the development of eco-friendly removal technologies for ecosystem disturbing plants is steadily underway. However, interest in eco-friendly herbicides with low phytotoxicity and biodegradability is increasing globally (Hoagland, 1990), and various studies using natural herbicides are being conducted. An example of secondary metabolites produced by soil bacteria is glufosinate-ammonium (Hoerlein, 1994), which is the first bacterial extract that was used for the discovery of bialaphos (Bayer et al., 2004; Duke et al., 1996). This bioherbicide still efficiently and widely being used. In addition, albucidin (Hahn et al., 2009), herbimycin (Li et al., 2003), herbicidin (Lee et al., 2013), have been studied as a material exhibiting herbicidal activity.
The soil bacteria KRA16-334 strain to be used in this study is a natural herbicide candidate material discovered by the Korea Research Institute of Chemical Technology. In this study, we tried to confirm the possibilities of using the culture filtrate of the KRA16-334 strain as an environmentally friendly agent derived from Streptomyces drozdowiczii against S. angulatus an ecological disturbance broadleaf plant.
Tested Streptomyces strain (KRA16-334)
The soil bacteria KRA16-334 strain was used in this study which was classified as Streptomyces drozdowiczii through sequencing analysis of 16s rRNA as a bacteria separated from the soil in Poongsan-ri, Boryeong-gun, Chungcheongnam-do. The KRA16-334 broth filtrate cultured using the M3 liquid medium as the basic medium was used, and the control activity for ecosystem disturbing broad-leaved S. angulatus was investigated. The KRA16-334 strain was cultured for 7 days at 27℃, stirring speed 170 rpm, and pH 7.0.
Soil application (inhibition of seed germination)
Seeds of S. angulatus (20 seeds per pot), were planted in pots. The pots (upper diameter: 10 cm, lower diameter: 8 cm, depth: 12 cm) containing commercial horticulture soil were used in the experiment. The soil was moistened after planting. Then the KRA16-334 broth filtrates were sprayed onto the soil surface at the application rates of x1/8, x1/4, x1/2, and x1 with a volume of 20 mL in each pot. Treated pots were placed in the greenhouse (25/15±℃ and 12/12 h light/dark). Plant fresh weight was measured 15 days after treatment (DAT).
In pre-applications, KRA16-334 reduced the germination rate of S. angulatus by 42 to 100% (Fig. 1). Despite some seeds still germinated at low concentrations, the roots and shoot were substantially shorter than untreated controls (data is not given). The control efficacy of KRA16-334 at x1/4 and x1/8 application rate were <60%. However, x1 dosage of the broth filtrate barely supressed seed germination of S. angulatus completely (100%).
Foliar application (inhibition of seedling growth)
The seeds of S. angulatus were sown separately into 105-hole seed propagation trays in a greenhouse. When emerged seedlings were at the first leaf stage, they were transplanted into pots (upper diameter: 10 cm, lower diameter: 8 cm, depth: 12 cm). The KRA16-334 samples were prepared at the same doses as in the pre-applications, but sprayed directly onto the foliage at the same carrier volume. Stock culture solutions were applied at second (3 mL), fourth (5 mL), and sixth (15 mL) leaf stages of the seedlings. Herbicide efficacy results were taken visually 5, 10, and 15 days after treatment. The fresh weight of S. angulatus plants was measured right after the third visual assessment. The greenhouse trials showed that the highest effectiveness (97-100%) of the filtrate application was at the second leaf stage of S. angulatus plants (Fig. 2 and 3). The control efficiency of KRA16-334 was declined as the growth stages of plants passed. At the late leaf stage, (six-leaf) control efficacy of the broth filtrate was much lower, showing the efficacy between 55 and 65% at x1/8 and x1/4 application rates respectively.
Field experiments were conducted in the experimental farm of Chungnam National University in Daejeon, Korea. Initially, seeds of S. angulatus were sown on 105-hole propagation trays and grown in a greenhouse. Then, the seedlings of S. angulatus (at around 2-3 growth stage) were transplanted into 2 m2 plots. The experimental design was set up as a randomized complete block with three replications. When seedlings reached 5-6 leaf stage KRA16-334 (Tween 20, 0.1%) broth filtrates were sprayed with a volume of 200 mL m-2 at the x1/4, x1/2, and x1 application rates. Treatments were applied using a backpack sprayer in 2,000 mL-1 carrier volume. During the three weeks, visual assessment results of herbicide efficacy were taken compared with untreated control at every 7 days. S. angulatus plants were harvested from the field and dried in the oven for 72 hours at 65℃ to estimate the effects of KRA16-334 broth filtrate on dry matter of plants.
On the second day, injury signs were witnessed on the leaves of treated weeds, and the marks were identical to contact activity as part of the plants became desiccated. Overall, the control efficacy of strain KRA16-334 to S. angulatus was over 85% 21 days after the treatment at the x1 application rate. Three weeks after the treatment, KRA16-334 could not control (<50%) S. angulatus at the application rate of x1/2 and x1/4 (Fig. 4). The relatively low rate of herbicidal activity was observed at the sixth leaf stage in both greenhouse and field conditions as seedling stem became harder.
Comparisons with commercial herbicides
In order to compare overall reaction speed, KRA16-334 at the x1 application rate with Tween 20 (0.1%), commercial herbicides, glufosinate ammonium (Basta, 1,440 g a.i. ha-1) and glyphosate (Keunsami, 3,280 g a.i ha-1) were applied on S. angulatus. S. angulatus was grown in 105-hole seed propagation trays in the greenhouse until the plants were at the second leaf stage. Each commercial herbicide was applied following the manufacturer’s recommendations. The herbicidal activity was determined by visual injury (0: no injury, 100: complete death) every day for two weeks.
The culture filtrate of KRA16-334 began to show symptoms 2-3 days after the treatment, showing the characteristics of discoloration of leaves and rapid stopping of growth, and was completely dead on the 7th day of the treatment. This was almost similar to glufosinate-ammonium. It is estimated that the herbicidal activity of KRA16-334 is in contact and rapidly destroys the membrane to kill the plant, as the activity of the transitional herbicides appears and progresses (Fig. 5). In general, S. angulatus reaction to the application of KRA16-334 was stronger than glyphosate but inferior than glufosinate-ammonium at the x1 rate application.
Based on these results, it can be concluded that the candidate soil bacteria KRA16-334 strain is expected to be used as a natural and eco-friendly herbicide against S. angulatus an-ecosystem disturbing plant in Korea. However, further studies such as culturing methods, agitations, or mutation of the broth filtrate will be required to increase herbicidal activity of the KRA16-334 strain in experimental trials.
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agricultural Machinery/Equipment Localization Technology Development Program, funded by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) (321056-05).
Umurzokov Mirjalol, https://orcid.org/0000-0001-8148-8041
Jung Sup-Choi, Korea Research Institute of Chemical Technology, Doctor of Philosophy
Ruziev Farrukh, Chungnam National University, Researcher
Young Sook Kim, Korea Research Institute of Chemical Technology, Doctor of Philosophy
Kwang Min Cho, https://orcid.org/0000-0003-0537-2164
Kee Woong Park, https://orcid.org/0000-0003-0053-9543