Terpenes and phenolics in alcoholic extracts of pine needles exhibit biocontrol of weeds (Melilotus albus and Asphodelus tenuifolius) and insect-pest (Plutella xylostella)

2.1 Collection and processing of pine needles

Leaf needles of Pinus roxburghii were collected from Margala hills forest near Islamabad city, Pakistan (73° 02′ E longitude and 33° 36′ N latitude, 508 masl) during November 2018. Needles were air-dried for 2 weeks in shade between 15 and 25 °C. Dry needles were crushed by a grinder to make a fine powder, screened through a 2 mm size mesh sieve, and stored at 4 °C for further use in extraction.

2.2 Maceration of Pinus roxburghii needles

Powdered pine needles were treated separately with four different solvents, viz., cold water (20 °C), hot water (45 °C), methanol (20 °C) and ethanol (20 °C). Purpose to use various extractants was difference in their polarity as water with higher polarity (1.0) renders greater extraction yield, while solvents with lower polarity (<1.0, e.g., ethanol and methanol) produce less extraction yield (Ashraf et al., 2020). Same procedure was followed for all solvents extraction but in case of hot water extraction temperature was adjusted at 45 °C. Maceration with each solvent was performed in separate conical flask in the ratio of 1:5 (pine needle powder:solvent). Each solvent treatment had four sets to make solution through shaking for four different time durations (1, 3, 5 and 7 days) by covering with aluminum foil. After shaking, filtration was done using filter paper Whatman No. 1 and re-filtered three times. Resulting filtrate was concentrated through rotary evaporator, and the crude extract residues were stored at 4 °C in Eppendorf tubes.

2.3 Extract analysis

Biochemical compounds in the extract residues were measured through GC–MS (Agilant 5973–6890), operating at 70 eV in EI mode. Helium gas served as carrier @ 1 mL min⁻¹ along with HP-5MS capillary column (30 m, 0.25 mm, 0.25 µm). Initially, temperature of 50–100 °C was achieved @ 5 °C min⁻¹, later 100–250 °C @ 3 °C min⁻¹, and finally maintained at 260 °C for 20 min. The 2 µL aliquot was aspirated into the column at 200 °C, and the resolution for sample components was attained. Various components were identified via retention time comparison against that of authentic samples (Supleco; Bellefonte, USA) carried out through co-elution and MS analysis and database in the NIST library.

2.4 Weedicidal bioassays

Weedicidal bioassay was initiated by pre-testing of serial concentrations against seeds of Melilotus albus and Asphodelus tenuifolius weeds. Seeds were used after sterilizing in sodium hypochlorite (0.5%) solution for 2 min. Experiment on comparison of different solvent extracts obtained after 7 days incubation was performed with their 33%, 66% and 100% concentrations. First, the extracts were solubilized in Tween 20® (1%) and later diluted to required concentrations in distilled water. Filter papers were soaked separately with 2 mL methanol, ethanol, cold water and hot water extract solutions in the Petri plates being used for weed seeds germination test. Controls were maintained by moistening the filter paper with Tween 20® (1%) solution. In each Petri plate of the tested extract, 20 weed seeds were put for germination in the growth chamber at 24 °C. Germinated seeds were counted daily until 7th day after placement. Seeds germination was considered when growing plumule extended to 2 mm length. Germination (%) was calculated with the following formula: $$\mathit{Germin}ation\left(\%\right)=\frac{\mathit{Germin}atedseeds}{\mathit{Total}seeds}\times 100$$

2.5 Insect culture

Third-instar larvae of diamondback moth (DBM) were reared in Biocontrol Lab of the Entomology Department, PMAS Arid Agriculture University, Rawalpindi, Pakistan. Diamondback moth culture was maintained on young plants (45–50 days old) of cabbage (Asha cultivar) under 25 °C, 65 ± 5% RH and 16/8 h’ light/dark conditions following Qureshi et al. (2020).

2.6 Insecticidal bioassays

Pinus roxburghii needles extracts were tested via leaf dip bioassay following the Park et al. (2002) procedure against 3rd instar larvae of DBM. Three concentrations of extracts, viz., 1%, 3%, 5% were prepared from stock solutions of all solvents incubated for 1, 3, 5 and 7 days. Insecticidal bioassay was conducted by testing these serial concentrations along with respective controls (0%). Fresh cabbage (Asha cultivar) leaves were washed and then dried under room temperature. These leaves were dipped into respective concentrations of each extract for 10 s and allowed to dry at filter paper for 60 s, and then placed in Petri dishes having moist filter paper. Ten 3rd instar larvae of DBM were placed in each Petri dish and mortality data were recorded at time intervals of 24, 48, 72, 96 and 120 h. All extracts obtained at 1, 3 and 5 days were found less efficient as compared to day-7 extract. Therefore, concluding experiment was performed using different solvent extracts obtained at 7 days’ maceration with 5%, 10%, 15% and 20% concentrations along with respective controls (0%) by following the procedure described earlier. Mortality percentage was calculated using the formula as mentioned below: $$\mathit{Corrected}Mortality\left(\%\right)=\frac{P-Po}{100-Po}\times 100$$ here,

• P = Percent mortality in treatment.

• Po = Percent mortality in control.

2.7 Statistical analysis

Data related to extraction yield were analyzed by following the two-factor factorial arrangement under completely randomized design (CRD) with three replications. Weedicidal and insecticidal bioassays were analyzed through two-factor factorial CRD with six replications. Treatment means were compared using LSD test at 5% level of significance through Statstix 8.1 software. Graphical representations were made through MS-Excel software.

3.1 Extraction yield with different solvents

Water (hot and cold) as solvents brought a significant (P < 0.05) increase in crude extraction yield as compared to methanol and ethanol (Table 1). The highest average extraction yield was obtained through hot water (as solvent) followed non significantly by cold water, but significantly by both methanol and ethanol. In terms of incubation time, averagely there was non-significant difference from day-1 through day-7 except for hot water. Individually, with methanol the highest extraction yield was obtained at day-5 and the lowest at day-1. In case of ethanol, the highest extraction yield was obtained at day-3 and the smallest at day-7. Cold water as solvent rendered the highest crude extraction yield at day-3 and day-5 followed by day-1 and day-7 with a small difference. Hot water when used as solvent, greater extraction yield was obtained at day-7 with the lowest one at day-1. Interaction between solvents and incubation time was statistically significant (P < 0.05) with the highest value with hot water at day-7 and the lowest one with ethanol at day-1.

3.2 Biochemical composition of extracts

Compounds obtained from all the extracts after 1, 3, 5 and 7 days’ incubation are shown in Table 2. In cold water extract, the identified compounds at day-1 were; acids, amides, ketones, aldehydes, phenols and alcohols. At day-3 and day-5, greater contents of acids, siloxanes, amides, aldehydes, phenols and alcohols were achieved. From day-7, higher quantity of organic acids, siloxanes, amides, and phenolic compounds were obtained. Compounds identified from hot water extract at day-1 and day-3 were; acids, siloxanes, amides, ketones, phenols and alcohols. From day-5, compounds present in greater amount were acids, siloxanes, amides, and phenols while from day-7, acids, siloxanes and phenols were obtained. Compounds identified from the methanol extract of day-1 were; acids, siloxanes, terpenes, phenols, alcohols, ketones, aldehydes and alkanes. Similar compounds were recorded in day-3, 5 and day-7. Compounds obtained from ethanolic extract at day-1, 3, 5 and day-7 were; acids, siloxanes, terpenes, phenols, alcohols, aldehydes, alkanes and ketones. Overall, methanol and ethanol as solvents yielded higher quantity of phenols, terpenes, alcohols, alkanes, aldehydes and ketones as compared to cold- and hot water.

3.3 Efficacy of Pinus roxburghii needle extracts as bio-weedicide

Bioassays concerning weeds germination inhibition showed that all the tested needle extracts of Pinus roxburghii had variable weedicidal activities (Fig. 1). However, methanolic extract exhibited the highest germination inhibition (74%) followed by ethanolic extract (68%) both having significant difference with cold water extract (44%) and hot water extract (42%) for Melilotus albus at 100% concentrations. Each lower concentration of all the extracts exhibited significantly less germination inhibition if compared to one another. Trend of germination inhibition was as: 100% > 66% > 33% > 0% concentration. In case of Asphodelus tenuifolius, methanolic extract displayed the highest germination inhibition (65%) followed by ethanolic extract (64%), however, both differed significantly with cold water extract (34%) and hot water extract (33%) at 100% concentrations. Germination inhibition reduced gradually and significantly with extract concentrations of 66% and 33%, being minimal in control treatment (0% concentration). Current findings revealed that P. roxburghii needles possess active biochemical compounds which could serve as the potential source of bio-weedicides. Moreover, methanol and ethanol proved to be more efficient solvent to obtain the promising weedicidal results.

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Fig. 1

Effect of different extracts on seed germination inhibition of Melilotus albus and Asphodelus tenuifolius weeds.

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3.4 Efficacy of Pinus roxburghii needle extracts as bio-insecticide

Needle extracts of Pinus roxburghii exhibited promising insecticidal activity against Plutella xylostella or diamondback moth (DBM) 3rd instar larvae (Fig. 2). Data analysis depicted that efficacy of extracts was less in the beginning 24 h against 3rd instar larvae. There was positive correlation between extracts efficacy with time passed, and it was enhanced with increasing time, and reached the highest value at 72 h with methanolic extract. The highest larval mortality (92%) was recorded by methanolic extract followed by ethanolic extract (76%), cold water extract (66%) and hot water extract (56%) with 20% extract concentrations at an exposure duration of 120 h. In terms of time, the highest mortality was observed during 72 to 96 h in all the solvent extracts. Pinus roxburghii needles extract showed mortality to 3rd instar larvae of DBM slowly after 24 h of treatment but toxicity was increased sharply after 72 h. Higher concentration of each extract rendered significantly higher mortality rate as compared to its respective next lower concentration. So, the highest mortality rate was achieved by 20% and the lowest by 5% concentration. Pinus roxburghii extracts exhibited promising activity against the tested pest after 72 h. Current findings revealed that mortality was increased with the increase in concentration as well as exposure time of extract.

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Fig. 2

Effect of different extracts on Plutella xylostella mortality at 5, 10, 15 and 20% concentrations.

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4.1 Impact of solvents on extraction of biochemicals

Employing an appropriate solvent is one of the leading factors for efficient extraction of desired biochemical compounds. Crude extraction yield varies significantly with the polarity of the solvent used; whereas nature of biochemicals extracted varies due to affinity of functional groups of compounds with extractants. Polar solvents (e.g., methanol, ethanol) are more suitable for the extraction of hydrophilic compounds (Sasidharan et al., 2011). During this study, different solvents, viz., methanol, ethanol, hot and cold water were used to extract biochemical compounds from P. roxburghii needles. Among from these solvents, significantly higher extraction yield was obtained through hot water (as solvent) followed by cold water, methanol and ethanol. Greater extraction yield with water is due to its higher polarity (1.0); while solvents with lower polarity (<1.0, e.g., ethanol and methanol) produce less extraction yield (Ashraf et al., 2020). Similarly, cold- and hot water as solvent produced higher amounts of organic acids, siloxanes and amides. More efficient extraction of organic acids from sativum seeds with water has been reported recently by Ashraf et al. (2020). Water extraction also favored to obtain higher amount (7–10%) of tannins from P. roxburghii bark (Kumari et al., 2017). Under this study, methanol and ethanol as solvents produced greater amount of terpenes, phenols, and alcoholic compounds. From the viewpoint of natural product, concentration of several biochemicals although with lesser significance as biopesticide (organic acids and siloxanes) was greater with water. Compounds having greater efficiency as biocontrol agents (phenols and terpenes) were obtained in larger content through methanolic and ethanolic extraction.

Methanol is considered as one of the most effective organic solvents for the extraction of valuable phytochemicals from plant material (Parekh et al., 2006). It enables extraction of higher amount of phenols, terpenes and alcoholic compounds, which are known to be biocontrol agents (Bogolitsyn et al., 2019). Relatively more phenolic compounds can be extracted through methanol because it acts as a binary solvent (Anokwuru et al., 2011). Moreover, ethanol when used as solvent yielded higher concentration of phenolic compounds as compared to water (Koffi et al., 2010), which are in accordance with current findings. Thus, selection of proper solvent for the targeted group of plant secondary metabolites is quite important.

These findings revealed that extraction of different biochemical compounds from P. roxburghii needles depends upon the nature of solvent. Moreover, methanol and ethanol as solvents could be preferably used for extraction of phenolic compounds, terpenes, alcohols and ketones. Whereas, cold- and hot water extraction can be done preferably for organic acids, siloxanes and amides.

4.2 Biocontrol efficacy of different extracts

Several plant-based extracts are being used for weeds management. Pinus roxburghii needle extracts efficiently inhibited the germination of two obnoxious weeds (M. albus and A. tenuifolius). Methanolic extract showed the highest weed seeds germination inhibition followed by ethanol, cold water and hot water. Better performance of methanolic and ethanolic extract could be attributed to the presence of higher amount of terpenes, phenolic and alcoholic compounds in it as compared to that in cold- and hot water extracts. Pinus roxburghii bark and needles are good source of polyphenols and terpenes having potential to inhibit the weeds germination (Omezzine et al., 2011). Methanol and ethanol are efficient solvents to extract terpenes and phenolic compounds and had significant weedicidal impacts (Da-Silva et al., 2017). Terpenes, phenols and alcoholic compounds present in plant extracts exhibited greater weedicidal activities (Lim et al., 2017). Polyphenols and terpenes might be affecting the metabolic activities and seed hydration during the process of germination.

Needles extracts of P. roxburghii caused variable mortality percentage of the target insect-pest in bioassays. Methanolic extract was more effective to control 3rd instar larvae of Plutella xylostella L. when compared with other solvent based plant extracts. It could be attributed to the presence of higher quantity of phenolic compounds and terpenes. Pine needle extracts evaluated in this study have not been used before for insect-pest control as per our knowledge in any part of the world. These results suggest that P. roxburghii needles extract could serve as an effective option to insect-pest control. Different plant secondary metabolites and phytochemicals such as essential oils, terpenes, alkaloids, and phenolic compounds have shown pesticidal properties (Copping and Duke, 2007).

CRediT authorship contribution statement

Tajwar Alam: Conceptualization, Investigation, Writing – original draft. Ghulam Jilani: Conceptualization, Writing – original draft. Arshad Nawaz Chaudhry: Conceptualization, Writing – review & editing. Muhammad Sheeraz Ahmad: Conceptualization, Writing – review & editing. Rukhsanda Aziz: Writing – review & editing. Rizwan Ahmad: Writing – review & editing.