is a blooming plant which belongs to the Heliantheae (Asteraceae) family. It is commonly known as congress grass, carrot weed, and broom bush in India. Similarly, in the USA the experts refer to it as feverfew and false ragweed [1
]. P. hysterophorus
is the product of natural hybridization between P. confertum
and P. bipinnatifidum
. P. hysterophorus
can thrive in hostile environments and suppresses the growth of other native species due to its allelopathic effects [2
]. The presence of P. hysterophorus
decreases the stability of grazing land establishment, thereby also reducing pasture production [3
]. P. hysterophorus
has emerged as the seventh most troubling weed globally [1
]. The popularity of this weed can be related to its infamous aggressive nature in the surroundings and crop fields [1
], and P. hysterophorus
infestations are increasing rapidly in many areas in India. In 1951, the Parthenium
was first discovered in Poona (Maharashtra State). In 1972, its habitat expanded to Kerala in the south and Kashmir in the north. Later, in 1979 it expanded and advanced up to Assam. Currently, it can be found all over the subcontinent. It infests about 5 million hectares in Karnataka state, making it the most dominant weed in the area [4
]. Particularly in India, Parthenium
now thrives even in regions with prohibitive climatic conditions [5
The dominant nature of this weed is due to its strong reproductive potential and its ability to grow at an exponential rate [1
]. A bitter glycoside parthenin and SQL (sesquiterpene lactones) are the major chemical constituents of P. hysterophorus
. All parts of P. hysterophorus
, including trichomes and pollen, contain SQL [6
]. Parthenin, ambrosin, and hymenin have been considered to be the main components of this weed responsible for its strong allelopathic effects on various crops [7
]. Apart from the loss of crop yield and plant biodiversity, P. hysterophorus
is also considered hazardous for human and animal health (e.g., it is responsible for dermatitis after contact with their leaves) [8
]. In humans, swelling and itching of the mouth and nose were observed when the body was exposed to its pollens. Further, it was also noted to cause asthma (allergic bronchitis) in the later stages [10
]. In the last few decades, the elimination of invasive plants has been done with the application of synthetic (chemical) herbicides (bromacil, chlorimuron ethyl, and buctril). These herbicides can cause environmental damage and also harm living organisms, including humans. In addition, the resistance of invasive herbs has been grown stronger with the misuse and abuse of chemical herbicides. Hence, the use of bio-herbicides represents a necessary advancement in weed control, in order to produce an environmentally and economically sustainable alternative. Bio-herbicides (allelopathy and allelochemicals) have been a challenge to synthetic (chemical) approaches [11
Allelopathy is a very realistic method to control weed spread. There has been increasing interest in research on plant allelopathy to control weeds in agroecosystems [12
]. The chemistry of allelochemicals affords control of the weeds directly or indirectly and has the potential to act as bio-herbicides [14
]. The discovery of new weed management technologies has become inevitable to overcome the constraints of synthetic herbicide. In the light of the above, allelopathy seems to be the most practical method of weed control as it fulfils the criteria of eco-friendliness and it is already cost effective in managing several weeds [2
is a perennial herb belonging to the Asteraceae family and is commonly known as wormwood [16
]. Absinthin, silica, thujone, anabsinthine, and tannic and resinous substances are among the main bioactive constituents of their leaves and flowers [16
]. In ethnobotany, A. absinthium
is used for its anthelmintic, antispasmodic, antiseptic, and febrifuge properties [17
spp. are also known for their allelopathic properties [18
] especially against other species that can become invasive in some areas, such as Convolvulus arvensis
and Portulaca oleracea
(guava) belongs to the Myrtaceae family and its leaves contain some bioactive compounds, like avicularin, quercetin, and guaijaverin [22
]. The guava leaves also contain potential allelopathic metabolites [23
], such as flavonoids, terpenoids, and cyanogenic acids [24
]. Only a few studies have tested the allelopathic effects of guava against other plant species [25
The present investigation was designed to test the allelopathic effect of aqueous leaf extracts of A. absinthium and P. guajava on seed germination, seedling growth, and some biochemical parameters of P. hysterophorus, thus exploring the possibility to use those extract as a bio-herbicide against this weed that is becoming extremely invasive in India.
2. Materials and Methods
The present work was performed in the School of Bioengineering and Biosciences Lovely Professional University, Phagwara, Punjab, India. For this experiment, P. guajava and A. absinthium were harvested in the wild from non-anthropic areas surrounding the University campus. Plants were harvested at their best balsamic period and before the flowering stage.
2.1. Aqueous Extract Preparation
After washing the leaves with tap water, the leaves were additionally washed with distilled water. The leaves were air dried for one month at room temperature and then ground to powder by using a mortar and pestle. The extracts were made by mixing 100 g of powdered leaves in 1000 mL of sterilized water and were kept at room temperature for 2 h. Leaf extracts were then filtered (Whatmann No. 1) and the crude extract was diluted to obtain different concentrations, i.e., 25%, 50%, and 75% (w/v) solutions. Treatments were cataloged as: P1 (25%), P2 (50%), P3 (75%), and P4 (100%) for P. guajava and A1 (25%), A2 (50%), A3 (75%), and A4 (100%) for A. absinthium. The control (CN) represents plants treated with distilled water. All the solutions were adjusted with sulfuric acid to pH 7.0 to avoid the confounding effect of different pH on plant performances.
2.2. Plant Material
The P. hysterophorus seeds were sown in mud pots measuring 10 cm in diameter and 10 cm in depth, filled with 60 g of top soil (sand/loam, 2:1). Freshly prepared concentrates of 25%, 50%, 75%, and 100% were sprayed on the surface of Parthenium seedlings in order to uniformly cover all the seedling surface.
After the first spray, two consecutively sprays were given on day 5 and 10. The control pots were also sprayed with distilled water. Every treatment was replicated three times. The seedlings were harvested following a month after sowing and were washed with tap water to clear any soil remaining on the roots. After that, the seedlings were examined for biophysical and biochemical parameters.
2.3. Pigment Analysis
2.3.1. Chlorophyll Content
A total of 1 g of fresh leaves was crushed using a mortar and pestle in 3 mL of 80% acetone. Then, the homogenized material was centrifuged at 10,000g
for 20 min at 4 °C (Eltek cooling centrifuge, Elektrocraft Pvt. Ltd., India). The supernatant absorbance was collected at 645 and 663 nm and pigments were quantified according to Arnon [26
] by using a UV-visible, double beam spectrophotometer (Systronics 2202, Systronics India Ltd., Ahmedabad, India).
2.3.2. Carotenoid Content
A total of 1 g of fresh leaves was homogenized using a mortar and pestle in 4 mL of 80% acetone. Then, the homogenized material was centrifuged at 10,000g
for 20 min at 4 °C. The supernatant absorbance was collected at 480 and 510 nm and carotenoid contents were quantified according to Maclachlan and Zalik [27
2.4. Malondialdehyde (MDA) Content
Membrane damage was assessed in terms of MDA by-product content, following the method of Heath and Packer [28
]. Leaves were extracted using 0.5 g of fresh material in 5 mL of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged at 5000g
for 10 min a 4 °C. Then, 1 mL of supernatant was mixed with 6 mL of 20% (w/v) TCA, containing 0.5% (w/v) of thiobarbituric acid. This mixture was heated at 95 °C for 30 min and then cooled in an ice bath. The absorbance of the supernatant was taken at 532 nm. Correction of non-specific absorbance was done by subtraction of absorbance taken at 600 nm.
2.5. Proline Content
The method used by Bates et al. [29
] was used for proline estimation. An aliquot of 0.5 g of fresh leaves was homogenized using a mortar and pestle in a sulfosalicylic acid solution (3% v/v), and centrifuged for 10 min at 10,000g
for 10 min at 4 °C. Then, 2 mL of ninhydrin and glacial acetic acid were added to 2 mL of the supernatant and the mixture was incubated in a boiling water bath for 1 h. Absorbance was taken at 520 nm.
2.6. Glycine-Betaine (GB) Content
The Grieve and Grattan [30
] method was used to measure GB content. A 1 g amount of fresh leaves was homogenized using a mortar and pestle in 10 mL of distilled water and then the extract was filtered using filter papers (Whatman N°1). After filtration, 1 mL of the supernatant was collected and 1 mL of 2 M HCl was added. Then, 0.5 mL of this mixture were taken and 0.2 mL of potassium tri-iodide solution was added. The mixture was cooled and shaken for 90 min in an ice bath. After that, 2 mL of chilled distilled water and 20 mL of 1-2 dichloromethane was added. Two layers appeared in the mixture. The upper layer was discarded and the absorbance of the organic layer was taken at 365 nm.
2.7. Antioxidant Enzymes
2.7.1. Catalase (CAT) Activity
The catalase activity was determined by using the method described by Aebi [31
]. The decomposition rate of hydrogen peroxide was followed by a decline in absorbance at 240 nm in a reaction mixture containing 1.2 mL of hydrogen peroxide (15 mM), 300 μL of enzyme extract, and 1.5 mL phosphate buffer (50 mM; pH 7.0).
2.7.2. Superoxide Dismutase (SOD) Activity
Superoxide dismutase activity was determined according to the method given by Kono [32
]. In the test cuvettes, a mixture of 1.3 mL of sodium carbonate buffer (50 mM, pH 10.2), 500 μL of 24 μM nitroblue tetrazolium (NBT), and 100 μL Triton X-100 (0.03% v/v) was prepared. The reaction started after adding 100 μL hydroxylamine hydrochloride. After two minutes, 70 μL of the enzyme extract was added and the rate of NBT reduction was recorded following the rise in absorbance at 540 nm.
2.7.3. Dehydroascorbate Reductase (DHAR) Activity
The DHAR activity was estimated according to the method of Dalton et al. [33
]. The mixture contained 1.5 mM reduced glutathione (reduced), 50 mM of phosphate buffer. 0.2 mM dehydroascorbate, and the crude extract. Then, the absorbance was taken at 265 nm. Using the extinction coefficient 14 mM−1
, the enzyme activity was determined.
2.7.4. Ascorbate Peroxidase (APX) Activity
An aliquot of 0.5 g of leaves was extracted in 3 mL of phosphate buffer (50 mM, pH 7.0) and centrifuged at 5000g
for 20 min. A mixture of 1.5 mL phosphate buffer (50 mM, pH 7.0), 300 μL ascorbate (0.05 mM), 600 μL H2
(1 mM), and 600 μL of plant extract was prepared and the reduction in absorbance was monitored at 290 nm [34
2.8. Total Phenolic Content
The total phenolic content was measured according to the method given by Singleton and Rossi [35
]. A total of 0.4 g of dried leaves was crushed using a mortar and pestle in 40 mL of 60% ethanol (v/v). After that, the extract was filtered using Whatman no. 1 filter paper and was diluted to 100 mL using 60% ethanol (v/v). Then, a 2.5 mL extract was taken and was diluted again with 25 mL of milli-Q water. In a 2 mL of sample extract, 10 mL of Folin–Ciocalteu reagent was added and the solution was mixed vigorously. Then, 2 mL of 75% sodium carbonate (w/v) solution was added after 5 min. The absorbance was taken at 765 nm.
2.9. Glutathione Content
The glutathione content in leaf samples was determined using the method described in Sedlak and Lindsay [36
]. Extraction was performed as described by Sedlak and Lindsay [36
]. Then, 100 mL of plant extract were added to 1 mL of Tris-HCl buffer, 4 mL of absolute methanol, and 50 μL of 5’-dithiobis-2-nitrobenzoic acid. The mixture was incubated for 50 min at room temperature and the absorbance of the supernatant was taken at 412 nm.
2.10. Ascorbic Acid Content
The method in Roe and Kuether [37
] was used to measure the ascorbic acid content. In 0.5 mL of plant extract, 0.5 mL of 50% TCA and 100 mg of charcoal were added. The mixture was mixed properly and then filtered with a Whatman no. 1 filter paper. Then, 0.4 mL of 2,4-dinitro phenyl hydrazine was added to the filtrate and the mixture was incubated for 3 h at 37 °C, followed by a cooling bath. Lastly, 1.6 mL of 65% H2
was mixed and kept at room temperature for 30 min. Sample absorbance was taken at 520 nm.
2.11. Statistical Analysis
All the experiments were carried out in triplicate and performed three times independently with consistent results. A representative dataset is reported herein. Data are expressed as the mean ± SD of replicates. All data were subjected to Bartlett’s test to assess homoscedasticity of data across populations. Differences between treatments, for each parameter under study, were then evaluated using one way-analysis of variance (ANOVA) and followed by Duncan’s test (p ≤ 0.05). Values of EC50 for growth parameters were calculated by fitting the data to a dose-response polynomial curve. The difference in EC50 between the two extracts was analyzed using Student’s t-test (p ≤ 0.05). All the statistical analyses were carried out using the SPSS16 software (SPSS INC., Chicago, IL, USA).
The biological control of weeds using allelopathic species or the use of allelochemicals isolated from plant extracts is preferred over both mechanical and chemical control in agriculture. Mechanical control results in high costs of management whereas the chemical control poses serious concerns for human health as well as environmental safety. Furthermore, intensive chemical control also promotes the development of herbicide-resistant weeds [3
The current study showed that the aqueous leaf extracts of A. absinthium
and P. guajava
were effective in limiting the seed germination and the growth of P. hysterophorus
, which is becoming invasive in India as well as in other areas of the world. The aqueous leaf extracts of A. absinthium
and P. guajava
had a critical impact on seed germination and seedling development (shoot and root length). As the concentration levels increased, these impacts likewise increased. To prove the interest in controlling this weed with other bio-herbicides, the phytotoxic effects of other allelopathic grasses, like Dichanthium annulatum
, Cenchrus pennisetiformis
, and Sorghum halepense
, have also been reported against Parthenium
The range of concentration both A. absinthium
and P. guajava
leaf extracts tested herein (25–100%) affected either the germination percentage or the root and shoot length of Parthenium
. Notably, besides a different chemical composition of leaf extract between the two species tested herein (not characterized in the present experiment), aqueous extracts obtained from both of the species exerted similar negative effect in terms of seed germination and root and shoot development of P. hysterophorus
when applied at the highest concentration level. A. absinthium
had a higher EC50 in terms of seed germination inhibition, whereas P. guajava
showed a significantly higher EC50 in terms of root inhibition. Therefore, these two extracts could be used together or individually at different stages during P. hysterophorus
infestations. According to other research [38
], the aqueous extracts of allelopathic grasses can be efficiently exploited to control the infestation of P. hysterophorus
in the field due to their capacity to affect different pathways of this weed.
Chlorophylls and carotenoids are key photosynthetic pigments for plants, and their content and functionality are essential to absorb and direct the light to photosystems [41
]. In this investigation, it was observed that the aqueous leaf extracts of A. absinthium
and P. guajava
reduced the level of chlorophylls and carotenoids, which suggests possible photosynthetic limitations exerted by both the extracts to Parthenium
leaves. An impaired photosynthetic process can generate a surplus of excitation energy burden in the chloroplast, thus leading, in turn, to an overproduction of harmful reactive oxygen species (ROS) [42
]. Increased levels of MDA by-products observed in Parthenium
seedlings support the production of free radicals and the occurrence of lipid peroxidation events [43
] induced by the treatment with A. absinthium
and P. guajava
Ascorbic acid (ASA) and glutathione (GSH) are considered to be key components of non-enzymatic cellular antioxidant defense system [44
]. For ASA to act as an antioxidant, it is necessary to preserve its reduced form by the activity of DHAR [44
]. In this study, it was observed that ASA content was remarkably reduced with increases in the concentrations of A. absinthium
and P. guajava
extracts. This occurred in concomitance with an enhancement in DHAR (compared to the control plants), which suggests that the regeneration of ASA in its fully reduced form was not enough to preserve the ASA pool. The content of glutathione was also drastically reduced by A. absinthium
and P. guajava
extracts application over the Parthenium
leaves. The stimulation of other non-enzymatic antioxidants, such as glycine-betaine and total phenolic compounds, might have further represented an attempt by Parthenium
plants to counteract the oxidative stress induced by the application of A. absinthium
and P. guajava
Besides DHAR, other antioxidant enzymes act as ROS scavengers in plant cell, including SOD, CAT, and APX [45
]. The present experiment revealed that the activity of all these enzymes was stimulated by the treatment of Parthenium
plants with A. absinthium
and P. guajava
extracts. Higher levels of MDA in cells subjected to these allelochemicals suggested that the antioxidant enzymatic system, although enhanced, did not completely eliminate the surplus of ROS and did not protect from ROS-triggered oxidative insult. A huge body of experimental evidence suggests that the induction of oxidative stress and enhancement of antioxidant battery are common responses exhibited by different plant species to allelochemicals (for a review see [46
]). Although the precise molecular target of ROS generated in plants responding to allelochemicals is not fully recognized, there is no doubt that several allelochemicals act as prooxidants.
Allelochemicals are well-known inhibitors of germination and plant growth and, in most cases, they lead to the modification of cell redox status [46
]. With the data provided by our dataset, we were not able to find the connection between the ROS triggered by A. absinthium
and P. guajava
application and the reduction in root and shoot development in Parthenium
plants. It seems, however, possible that the mechanical properties of the cell wall may be modified by enzymes, ROS, and their interaction [47
]. Among cell wall proteins, endoglucanases, xyloglucan endotransglycosylases, pectinases, pectin esterases, debranching enzymes, and non-enzymatic proteins, such as expansins, are responsible for cell wall extensibility [48
]. The second group of agents affecting cell wall extensibility are ROS, derived by spontaneous reaction or produced/consumed by cell wall associated proteins, such as APX, NADPH oxidase, and SOD [46
]. Therefore, both the direct effect of some ROS (principally •
OH and H2
OH which can be produced by Fenton reactions from NADPH oxidase-derived O2 •
− or by peroxidases supplied with O2
and NADH [49
]) and their inaction with the abovementioned cell wall protein might responsible for the reduction in plant growth. However, other possible effects exerted by both the tested extracts (e.g., hormonal interference, etc.) might have additionally contributed to the limited root and shoot growth inhibition of Parthenium
plants and this poses the bases for future research.