Study on the impact of MikaniamicranthaKunthon soil chemistry,crop yields, andforest canopy in an Indo-Burma biodiversity hotspot region

1. INTRODUCTION

Invasive plants have a significant negative impact on crop productivity, biodiversity, and the environment[1-3]. Global food safety and agricultural stability are affected by several interrelated and tightly linked factors, including population growth, poverty, environmental deterioration, and the use of synthetic herbicides [4]. Crop yields are negatively impacted by weed competition for resources including light, nutrients, space, water, and allelopathy [5-7]. The financial burden and unsustainable nature of traditional weed management practices jeopardized farmers livelihoods, which strained the general public and policymakers [8-9].

            MikaniamicranthaKunth. (“mile-a-minute” weed)is an extremely fast-growing perennial vine belonging to the Asteraceae family and genus Eupatorium in the Compositae family[10] (Figure 1a). It is native to the sub-tropical regions of South, Central, and North America [11]. M. micrantha was known as the top 100 noxious invadersby the International Union for Conservation of Nature (IUCN)[12-13]. The flowers of M. micrantha produced crown hair seeds that can proliferate over a long distance and hence difficult to control [14] (Fig. 1a).Invasive spread of M. micrantharesults in a notable decrease in the output ofagricultural crops and hence endanger food security[15]. The harmful effects of M. micranthaare primarily caused by allelopathic effect (release of allelochemicals/secondary metabolites) which modulated the soil chemistry, microbial diversity, agricultural yield, and biodiversity in a manner to sustain its invasive spread[16-18].In terrestrial ecosystems,M. micrantha climbs and cover trees and rapidly causes the death of trees and shrubs, thereby severely impacting the native biodiversity[11]. This abrupt spread of M. micranthacan perturb the normal photosynthetic metabolism of native plants, enforcing their senescence and expanding the canopy gaps (Fig. 1b). In this respect, past studies also observed that M. micrantha compete with other neighboring plants/edible crops for nutrients and other abiotic resources by blocking the sunlight [45, 2119-20]. This above-canopy spread of M. micranthasignificantly lowers the growth and productivity of a number of crops and has the ability to choke, suffocate, penetrate crowns, and pull over plants[21].

            In this study, we investigate the allelopathic effects of M. micrantha on a selected food crop (i.e., Pisum sativum L.) of wide dietary intake and nutritional value.P. sativum(locally known as green pea) isanutritious legume crop that is high in protein, vitamins, minerals, and health-promoting beneficial compounds[22-23].Pea has been used for experiments for decades pertaining to a plethora of cytogenetics, and ecological studies as it is susceptible to broad leaf herbicides, can be used year-round, andperforms well in bioassays. Inthis respect, the analysis of major allelochemicals present on the plant parts, allelopathy, physicochemical characterization of soil, and their linkage with the habitat attributes of M. micrantha (such as light intensity, canopy gap analysis, and leaf area index (LAI)) was studied which is a rather novel integrated approach in its invasion ecology. Henceforth, this article aimed to evaluate the allelopathic potential of M. micrantha by the (i) Phytotoxic effects of the leafextract on the growth ofP. sativum (ii) Total phenolic content (TPC) and tannins present ininvasive plant parts,and(iii) Physicochemical characterization of soil

Description of invasive alien plan and study area(a)Mikaniamicrantha plant during flowering stagewith abrupt spread covering the shrubs/tree vegetation (b). M.micranthapenetrates the tree crown and covers the canopy which reduces the sunlight reaching the forest floor which may ultimately lead to their senescence and hence perturb the dense canopy (photo courtesy R.B. Syngkli)

• MATERIALS AND METHODS
  • Study Site

The research was carried out in Aizawl district, Mizoram, North East India (‘23.87 ºN 92.89 ºE’)at an altitude of ‘800 meters to 1200 meters’ which is underlying in an Indo-Burma global biodiversity hotspot (Figure 1c). The area receives 2350 mm of rain falls annually. Summer temperatures range from 20 to 35 °C, while winter temperatures range from 9 to 21 °C. The research was conducted during May, 2023toApril, 2024.Laboratory work was done at Mizoram University, Tanhril, located 846 meters above sea level in Mizoram, India (23.73784167ºN 92.66741944ºE). The ambient light of the site ranged 2248.33-3152.33 LUX during the study period.

• Preparation of leaves aqueous extracts of M. micrantha and its effects on the growth of test crops

Aqueous leaf extract of M. micrantha was prepared by following the method of Ismail and Chong[24]. Fresh leaves of M. micrantha were collected from the invaded region, and faced with several anthropogenic disturbances. The leaves were washed thoroughly with clean water to remove dust and dirt and chopped into small pieces to keep them in a conical flask. Further, 100 ml distilled water was added into the flask and agitated in a rotary shaker (Rotary Flask Shaker, Scientech, Science Enterprises, Delhi, India) at 140-150 RPM for 8 h. The aqueous extract was then filtered through 2 layers of filter papers (Whatman No. 41) and maintained it eventually into 4 concentrations (i.e., 50 gL^-1, 37.5 gL^-1, 25 gL^-1, and 12.5 gL^-1). Besides, these concentrations of aqueous extracts, a control (distilled water) was used to compare the results. The petri dishes (90 x15 mm) were covered with filter paper which acted as medium to absorb the leaf extracts. Ten seeds of P. sativum were kept in petri dishes. Further, 5ml of different extract concentrations and control were added in petri dishes and covered with filter papers. All experiments along with the control were done in duplicate for better comparative analysis. The petri dishes were covered and incubated Biochemical Oxygen Demand (BOD) Incubator, at 30º C for 4 days.Finally, the seed germination percentage (%), radicle, and plumule length of the test crops were measured to compare the effects of M. micrantha-based leaf extracts in comparison to the control.

• Sample extractions

Allelochemicals of M. micranthawere extracted from the plant parts(i.e., leaves and flowers) using a traditional cold maceration solvent extraction method, which was slightly modified as per Handa et al. [25]. An extraction was conducted using a sample-to-solvent ratio of 10:100 (w/v). A 250 mL stoppered Erlenmeyer flask with 100 mL of 95% methanol was used to macerate 10 grams (10 g) of the air-dried soil samples. The mixtures were let to stand at room temperature while being stirred frequently which lasted for 72 hours. Following filtration using Whatman No. 1 filter paper (125 mm), the solvent was evaporated using a rotary evaporator (BUCHI Rotavapour R-300). After evaporation, a crude extract of leaves and flowerswas obtained.

• Quantification of allelochemicals

Total phenolic content (TPC)

The Folin-Ciocalteu colorimetric method byAinsworth and Gillespie [26] was applied to determine the total phenolic content. 0.5 mL of aliquot of 10, 20, 40, 80, and 100 μg/mL gallic acid solutions were mixed with 5 mL of 10% Folin-Ciocalteu and kept for 5 mins. 4 mL of 7% Sodium Carbonate (Na₂CO₃)wasadded, and the mixture was allowed to incubate at dark room temperature for 60 mins. The optical density was measured using a spectrophotometer at 765 nm. The same procedure was used for all plant parts extract. The stock solution for plant extract was prepared using 20 ml of methanol and 20 mg of the crude extract.TPC was calculated using gallic acid’s standard calibration curve and represented as μg/mLgallic acid equivalent (GAE) of the dry extract.

Tannins

 The Folin-Ciocalteumethod by Lahare et al. [27] was used to determine the total tannin content of the crude extract. The stock solution was prepared using 20 ml of methanol and 20 mg of the crude extract.7.5 ml of distilled water wasused to dissolve 0.1 ml of crude extract. The mixture was mixed with 0.5 ml of Folin reagent, 1 ml of 35% (Na₂CO₃), and diluted with 10 ml of distilled water. The mixture is allowed to stand at room temperature for half an hour after being thoroughly mixed. Using a spectrophotometer, the optical densities of the sample and standard solutions were determined at 725 nm. The tannin concentration was measured in terms of gallic acid equivalents (GAE) per gram of extracts, with gallic acid concentrations (10, 20, 40, 80, and 100 μg/ml)being selected as standard.

• Physicochemical characterizationof soil on M. micrantha invaded site

Soil temperature were measured onsite with a portable soil analysis kit. For other parameters, the soil was collected from the rhizosphere (15cm depth)and surrounding of M. micranthausing a shovel and kept in a clean plastic tray.Unwanted debris such as roots, stones, leaves, and plant materials were removed. Soil pH was analyzed by ‘potentiometric method’ with a 5:1 ratio of water to the soil using a pH glass electrode (µ pH system 361, Systronics). The moisture content was determined by the ‘gravimetric method’ [28].The soil was air-dried and sieved by 2 mm sieve. Water holding capacity was analyzedby standard methods.Soil organic carbon (SOC) was estimated by following ‘Walkley and Black’s’[29]‘rapid dichromate oxidation method’.The estimation of available soil Nitrogen was done by ‘Kjeldahl’s Digestion Method[28]. Available Phosphorus was estimated by using the ‘Bray method’. The available potassium will be determined by a standard method using a flame photometer.

• Canopy openness, leaf area index, and light intensity of invaded site

The canopy openness of M. micrantha infested site was analyzed by hemispherical photography and gap light analyzer (GLA) software method, as devised by Sooraj et al. [30]. The hemispherical photographs were taken in a skyward direction perpendicular to the ground using a fish eye lens (Skyvik Sign One 10mm Fisheye Lens) mounted on a smartphone (Samsung Galaxy M51). Hemispherical photos were taken in triplicates from 16 random areas (named asG1, G2, G3, ……., and G10) on a cloudy day and select 1 best photograph to act as an input to GLA software for canopy gap analysis (Figure 2). Coordinates and elevation of the site were taken using GPS (Garmin GPSMAP 64s). The magnetic declinationfor the configuration settings of GLA was taken from the National Oceanic and Atmospheric Administration (NOAA) website[31]. The light intensity and LAI of the area were measured using a plant canopy analyzer (Integration of handheld and Software from Kaizen Imperial and Quantum Sensors from Apogee Instruments, USA) from 16 random spots under the forest canopy. The data of the plant canopy analyzer (PCA) were recorded and stored in the handheld device attached to the sensors which were later extracted using PCA software. Herein, plant canopy analyzer data and hemispherical photographs were collected to facilitate the study on the habitat ecology and further elucidate the role of canopy gaps in influencing the invasive spread of M. micrantha, which might be tightly linked with its allelochemical attributes.

Hemispherical photographs used for forest canopy openness estimation. The monochromatic images are processed (working image) whereas the colour images are the original images (registered image) used to analyze canopy openness on Gap light analyzer software.

• RESULTS AND DISCUSSION

3.1 Impacts on the growth of Pisum sativum

The aqueous leaf extract of M. micranthahas negative inhibitory effects on the growth of P. sativum.In this respect,50gL^-1the extract showed the lowest inhibition, whereas, 25gL^-1 showed the highest inhibition (26.7% reduction) in terms of seeds germination (Fig. 3a). The biomass of the seedlings was reduced by 33.4% at 25gL^-1but the reduction was recorded only up-to the extent of 1.01% at higher concentration of50gL^-1 (Fig. 3b). The radicle length also showed inhibitory effect at all concentration with the highest inhibition being recorded at 47.51% at 25gL^-1 (Fig. 3c). Similarly, the length of plumule were also reduced at all concentrations with the highest reduction observed at 25gL^-1 (31.97% reduction).The aqueous leafextract of M. micranthahas therefore exhibited a significant effect on Pea plant growth parameters at 25gL^-1and 37.5gL^-1. Past studies such as those of Sahu and Devkota[32] also corroborated that M. micrantha aqueous leafextract inhibits the germination and seedling growth of Oryza sativaL. and RaphanussativusL.Similarly, Wu et al. [33] also found that M. micrantha inhibits the germination and seedling growth of 26 species which comprised native, naturalized exotic species, legumes, and non-legumes species.Further, it has also been observed that volatile organics of the leaves and flowers of M. micrantha also inhibit the germination rates of LactucasativaL., Chrysanthemum coronariumL., BidenspilosaL., and Abutilon theophrastiMedik.[34].

c

d

3.2 Allelochemicals analysis

The TPC values recorded in the leaves (15.13 μg)and flowers (29.97 μg) ascertained the presence of phenolics as the most ubiquitous allelochemic compound. Similarly, tannins were also estimated in the leaves (15.84 μg) and flowers (35.67 μg), demonstrating the abundance of secondary metabolites in plant parts of M. micrantha. Interestingly, TPC and tannins were recorded higher concentrations in floral extract when compared with the leaves.The presence of these allelochemic compounds confirms the inhibitory effect of M. micrantha aqueous extract on P. sativum, thereby, validating the NWH.Past studies havealso reported that M. micranthacontainsstructurally diverse allelochemicals that have a significant bioactivity (Ríos et al., 2014; Dong et al., 2017; Ishak et al., 2018; Sumantri et al., 2020)[35-38]. Thus, present reports on allelochemic potential of M. Micrantha was well corroborated by past studies and pave the way for further glocal studies in sustainable management of invasive alien plants.

3.3 Physicochemical characterization of soil

TheM. micrantha invaded site remarkably altered the soil physicochemical parameters when comparedwith the control (Table 2). The soil temperature at the highly invaded site was recorded 25.3°C, which reflects the ability of M. micranthato withstand high temperatures, ascribed to its phenotypic plasticity [39-40]. Similarly, soil moisture content and water holding capacity (17.88%, 87.47%) were also estimated higher at the invaded site.Therefore, it is quite evident that environmental variables can influence the invasion success ofplant invaders in concert with their biological traits[41]. In this respect, abiotic environmental factors such as water is one of the key determinants in the spread of invasive alien plants [42-43].Especially, Mikania prefers habitatswithhigh water content in the soil such as wetlandsand other aquatic ecosystems [19].Likewise, Yue et al. [41]also found that M. micranthahas highergrowth characteristics and population in wet habitatsthan dry habitats.Similarly, available nitrogen (229.97 kg/ha) was also higher at the invaded site when compared with the control. However, pH (5.18), SOC (1.74%), available phosphorus (16.85 kg/ha), and available potassium (339.73 kg/ha) were lower at the invaded site as compared to the control site.Importantly, past studies also elucidated that M. micrantha alters the nutrients in the soil by mobilizing the activities of microbial enzymes which inturn affect the functional metabolism of microbial nitrogen and phosphorus[18].Previous studies demonstrated that other invasive alien species from Asteraceaelike Chromolaena odorata(L.) R.M. King & H. Robprefer to grow on soil with low phosphorus content [44-45]. Agricultural fieldsrich in nutrient content are therefore prone to infestation of invasive alien plants, which reduce agricultural yield to threaten food security and socioeconomic or farmer’s livelihood[46-47].

3.4 Forest canopy openness, leaf area index, and photosynthetically active radiation (Incoming and diffused)

The forest canopy openness analysis was to determine the overall canopy/forest cover of the study site invaded by M. micrantha.Sites where M. micrantha was absent were denoted by ‘0’while‘1’was assigned for sites that marked their presence (Table 3). The site comprises of forest area, human settlements, and other anthropogenic activities such as quarry, agricultural activities, small industries, and rock grinding mills. The canopy openness of the site ranged from 10.71 % (lowest) to 98.73 % (highest) with the average value being 35.32 % (Table 3).In this respect, the average LAI was estimated 1.55 at the invaded site wherein 5.3 was the highest and 0.05 was the lowest recorded value(Table 3).The incoming average PAR was 396.48 µ mol m^-2 s^-1 and diffused PAR was 183.40 µ mol m^-2 s^-1. Diffused PAR is the sunlight that reachs the forest floor which is already absorbed and reflected by the forest canopy.The canopy openness was observed higher in the area where M. micranthawas present as the dominant invasive plant.Past studies in this Indo-Burma biodiversity hotspot region also noted thathigh canopy openness, high light availability, and low LAI indicate the high disturbance status of the novel introduced site [48]. Huang et al. [44] found that light intensity escalates the growth of invasive alien plants like C. odorata,therefore, an open canopy area with abundant light is prevalent to M. micrantha. When there is less availability of understory light, M. micrantha climbs the forest canopy for sunlight (Fig. 1b).

• CONCLUSION

This study revealed that M. micrantha aqueous extracts have negative inhibitory effects on thegermination and growth of P. sativumat different concentrations.The results weresupported by the presence of TPC and tannins on the leaves and flower extracts of M. micrantha which were tightly regulated by invasive plants induced modulation of soil physico-chemical attributes.The high canopy openness, light availability (diffused PAR), and low LAI indicated that M. micrantha tends to grow and spread abruptly at sites faced with high anthropogenic disturbance.These disturbances facilitated the spread of M. micranthawhich was inextricably linked with its allelopathic potential and soil chemistry, as clearly demonstrated in results of present bioassay and ecological investigation.To this end, areaswith less canopy cover have low LAI which allows solar radiation to reach the forest floor. Therefore, it further facilitates the spread of M. micrantha and other invasive alien species which have allelopathic impacts on surrounding plants and crops. In

present bioassay study, P. sativumgrowth parameters were affected by M. micranthadue to imposed competition forthe availability of PAR, soil nutrients, and allelopathy.Thus, establishment of explicit interrelated framework of abiotic parameters such as PAR, LAI, and canopy gaps need to be studies to elucidate the allelochemic potential of M. micrantha on edible crops like pea.Sustenance of pristine or undisturbed sites with low canopy openness and less anthropogenic disturbances can be more resistant tothe invasion of M. micrantha.

ACKNOWLEDGEMENT

The authors are also thankful to the Ministry of Tribal Affairs, Governmentof India for financial assistance in theform of a National Fellowship for ST,Department of Biotechnology (DBT‐BT/PR24917/NER/95/907/2017),and department of Science and Technology (DST‐ Nexus Project) vide research project no. DST/TMD/EWO/WTI/2K19/EWFH/2019 (C), for financial assistance.Authors are grateful to the Department of Environmental Science, Mizoram University, Aizawl, Mizoram, India, for providing the laboratory facilities.

DECLARATION

The authors announce that they have no conflict of interest and follow the ethical norms established by our respective institutions.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

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