Harnessing Koelreuteria paniculata Seed Extracts and Oil for Sustainable Woolly Apple Aphid Control

Abstract

The woolly apple aphid—WAA (Eriosoma lanigerum Hausmann, 1802) poses a significant threat to intensive apple production. Given the limitations of conventional synthetic pesticides, there is an urgent need for effective and sustainable pest management strategies. Botanical extracts derived from plants with insecticidal properties mitigating aphid populations without adverse environmental impacts are scarce where WAA is concerned. Thus, the pertinent study aimed to investigate the aphicidal potential of Koelreuteria paniculata seed ethanolic extract (derived from the seed coat) and mechanically extracted oil (derived from the seed itself). At concentrations of 2.5% and 5%, both solutions expressed undeniable insecticidal potential, providing absolute (100%; oil) or significant (86–100%; ethanolic extract) mortality rates both in vivo and in vitro. Predominant phenolic compounds determined in the ethanolic extract were gallic and protocatechuic acids and three derivates—p-coumaric, quercetin, and luteolin acid derivates—contributing to more than 90% of the total phenolic content, while phenolic compounds were not detected in the oil, indicating activity of different active compounds. Although deriving from different seed parts and distinct extraction methods, both ethanolic extract and oil exhibited significant aphicidal effects against WAA. The integration of botanical extracts from invasive species into pest management practices supports ecological balance and sustainable agricultural productivity, fostering a healthier environment and more resilient agricultural systems.

1. Introduction

With an annual production of 95.84 million tons in 2022 [1] and a gross production value of USD 84.93 billion [2], apple holds the number one position in the production and trade of temperate fruit species, followed by pears and peaches [3]. Due to scientific and technological advancements, today, intensive apple production entails incorporating high-yield varieties with trellis and irrigation systems, hail protection, soil cultivation within and between tree rows to eliminate competition for water and nutrients (weeds), as well as high usage of mineral and organic fertilizers, along with pesticides [4,5]. Nevertheless, in Europe, some indications suggest that the potential for further increases in apple production has been exhausted [6]. One such indicator is the high degree of susceptibility of high-value apple cultivars to a wide range of pests and pathogens [7]. To effectively control the spread of pests and diseases and maintain marketable yields, farmers rely heavily on regular pesticide applications, often applying from 10 to 18 sprays per growing season [5,8]. Amongst numerous diseases and pests, the woolly apple aphid—WAA (Eriosoma lanigerum Hausmann, 1802., Hemiptera: Aphididae)—is a significant and widespread pest affecting apple trees (Malus domestica Borkh.). Originating from North America, its primary host is Ulmus americana L. [9], while apple trees are among its many usual hosts (fruit and ornamental species from genera Malus, Cotoneaster, Pyracantha, Cydonia, Crataegus, Pyrus, and Sorbus). WAA forms dense colonies covered with a white, waxy, filamentous secretion on trunks, larger branches, new shoots, and roots of apple trees (Figure 1a,b). They can induce hypertrophic gall formation on the roots and major branches. Severe infestations can impair tree growth and vigor, destroy buds, reduce yield, and diminish fruit quality [10].

Both airborne and soil-dwelling populations of WAA pose a significant economic risk to apple production and are a formidable management challenge [11]. The conventional control of WAA, given that it inhabits the roots and rootstock shoots, is to use low-vigor resistant rootstocks combined with intensive pruning. Pruning alone is not a sufficient agro-technical measure to affect WAA control, but it is an appropriate supplementary measure to rootstock selection [12]. Considering that the widely planted standard dwarfing rootstocks M9 and M26 show relatively high WAA infection susceptibility [13,14], a biological control relying upon natural antagonists is recommended [15]. Currently, the most common chemical control method for WAA in Europe is the application of Movento ^® SC 100 (Bayer CropScience Deutschland GmbH, Monheim, Germany—active ingredient: spirotetramat). However, urgent alternative control strategies for WAA are necessary, as the approval for this active ingredient is set to expire by July 2024 [16].

With the imminent loss of registration and environmental issues associated with residues, the focus is turning towards more sustainable and biologically based control strategies. As a potential alternative to synthetic pesticides, biopesticides are products derived from microbes, animals, and plants [17]. Such products are usually less harmful to the environment, target specific organisms, are effective in small quantities, decompose quickly, and are to a lesser extent prone to resistance development. According to Swapan et al. [18], biopesticides are also associated with lower production of greenhouse gases, thus contributing to the mitigation of climate change. The reduced GHG emissions and carbon footprint associated with biopesticide production, as opposed to synthetic pesticides, are primarily related to the natural sources of raw materials and less energy-intensive production processes. Additionally, biopesticides can be produced locally, minimizing the negative impact of transporting pesticides over long distances to consumers. To be accepted by producers, biopesticides’ effectiveness must at least match the effectiveness of synthetic pesticides. Sustainable agriculture employing biopesticides must be simultaneously socially acceptable, economically productive, and environmentally responsible which are the main pillars of the Sustainable Development Goals (SDGs) [19,20]. To foster sustainable agriculture, the EU has set a goal to cut the overall use and risk of synthetic pesticides. This initiative supports the targets of a circular economy [21], Zero Pollution Action Plan [22], Farm to Fork Strategy [23], biodiversity strategy [24] and bioeconomy strategy [25].

Biopesticides, botanicals, or phytochemicals, are derived from different plant tissues or organs and are largely applied in the form of extracts or essential oils. Biopesticides used in the past and now commercialized have included plant extracts and oils from neem, cotton, tobacco, garlic, wild oregano, euphorbia, citrus, and pepper [26]. Comparing neem oil and chlorpyriphos effectiveness, Kacho et al. [27] showed that there were no significant differences in WAA population density reduction, while Kumar and Gupta [28] additionally proved that neem oil was both effective in controlling WAA and safe for parasitoids compared to the same conventional insecticide. Furthermore, some piperine-based ester derivatives have shown potential in E. lanigerum suppression [29].

However, all the plants mentioned are considered industrial, edible, and/or medicinal, putting a question mark on the competitiveness between their current use and biopesticide feedstock, while a large group of unemployed and underutilized species remains unexplored. While plants have evolved a diverse array of chemical defense mechanisms to protect against biotic and abiotic stressors, invasive alien species (IAS) have mechanisms that are enhanced compared with native species. IAS generally exhibit a greater tolerance to climatic variability, are highly competitive, and thus are more broadly distributed compared to native species. These characteristics enable IAS to adapt more readily to new environments, establish stable populations, and provide constant feedstock that can be used as green solutions in combat with various pest issues. IAS-derived extracts proven to have a biopesticidal effect include Ailanthus altissima Mill., Simaroubaceae, and Morus alba L., Moraceae against Lymantria dispar L. and Lepidoptera: Erebidae under laboratory conditions [30]; Elaeagnus angustifolia L., Elaeagnaceae against Drosophila melanogaster Meig. and Diptera: Drosophilidae, tested in vitro [31]; Fallopia japonica (Houtt.) Ronse Decr., Polygonaceae, and Artemisia vulgaris L., Asteraceae against Botrytis cinerea Pers., Sclerotiniaceae, Fusarium oxysporum f. sp. radicis-lycopersici W.C. Snyder and H.N. Hansen, Nectriaceae, Rhizoctonia solani J.G. Kühn, Ceratobasidiaceae, and Sclerotinia minor Jagger, Sclerotiniaceae, showing antifungal activity both in vitro and in vivo [32]; and Robinia pseudoacacia L., Fabaceae as an aphicide in oilseed rape production, tested both in laboratory and field settings [33].

Regarding Koelreuteria paniculata Laxm. (Sapindaceae) specifically, there are only two reports on its biopesticidal activity [34,35]. Nevertheless, its plant parts, especially seeds, could serve as a feedstock for biopesticide production, concomitantly reducing invasiveness (by generative regeneration diminishing) and increasing alternative, sustainable pesticides’ application [36]. Since horticultural and medicinal crops are known sources of biopesticidal solutions [17], this novel approach could avoid harvesting of useful medicinal and edible plants grown on arable land and promote sustainable use of plant invaders already established on various unfavorable sites and soils.

Aligning with the globally defined goals, as well as providing a toxin-free environment, pertinent research strives towards the application of botanical pesticides, contributing to several global actions. Due to the confirmed K. paniculata invasiveness in the continental climate, the absence of its evaluation as a biopesticide as well as WAA’s detrimental influence on apple production, the objectives of the proposed research were to (i) assess the effects of K. paniculata (seed extract and oil) on WAA mortality using both in vivo and in vitro methods, (ii) to determine the chemical composition of K. paniculata, particularly the phenolic compounds, using ethanol and mechanical extraction methods, and (iii) to match potential biopesticide development with globally defined SDGs and the European Green Deal, as suggested by Fetting [37].

2. Materials and Methods

2.1. Orchard Location and Description

The trial was performed in an apple orchard situated in the Bačko Dobro Polje, Vojvodina Province, Serbia (N45.5125538, E19.6813725) on plain terrain 78 m above sea level. Given that Vojvodina Province is characterized by a mild climate (temperate–continental), with an average annual rainfall of 600–700 mm [38] and the chernozem soil type, the area is ideal for various grain, vegetable, and fruit crop production [39]. An apple orchard was established in the spring of 2019 on a 3-hectare area with “Gala” as a leading variety and “Granny Smith” as a pollinator variety, both on the M9 rootstock (T337), 0.8 m apart within the row and with 3.2 m-wide laneways. The entire orchard area is covered by a hail-protecting net and a drip irrigation system. The system is powered by a well, located at a depth of 82 m.

2.2. Koelreuteria Paniculata Seed Collection and Processing

Koelreuteria paniculata is an ornamental species of specific appearance and amenity tree properties. Though commonly cultivated in Europe and the United States, K. paniculata, the Chinese golden rain tree, is native to eastern Asia, including China and South Korea. However, in continental climate conditions outside its origin, the Chinese golden rain tree exhibits significant invasive potential with abundant seed production and germination success.

Seeds were collected in October 2023 from an adult solitary tree approximately 7 m tall and wide, described in detail in [40], where the specimen was used for biodiesel studies. The selected tree showed no signs of dry, damaged, or broken branches, dieback, or mechanical damage, and exhibited notable health and vitality. Seeds collected from all four sides of the crown and mixed in one sample of 2 kg were air-dried at room temperature for 7 days. During this period, total moisture loss was 25 g (1.25%). Due to the extremely hard and water-impermeable seed coat [41], a core sample of 200 g of dried seeds randomly selected with the split-plot method was used for subsequent extraction. Dried seeds (200 g) were extracted with 97% ethanol (1000 mL) as a solvent by simple soaking for four weeks at room temperature until the first change in color. Seeds were then extracted for an additional 2 weeks. This extract was considered an initial botanical preparation (IBP). The volume of 20 mL of ethanolic extract was evaporated by a rotary vacuum evaporator (IKA RV 10, Staufen, Germany) to obtain the dry extract. All results of phytochemical analysis are expressed as grams of dry weight (DW).

In addition to ethanolic extracts, oil obtained from the remaining 2 kg of the harvested seeds also served as a presumed feedstock for biopesticide. Oil extraction from K. paniculata was performed by mechanical pressing with a screw press without previous treatment of the seeds, except for drying to optimum humidity, presumably when seed coats showed no further evaporation, indicated by a seed coat color that remained black grayish instead of glossy black [42].

2.3. Phenolic Content Characterization

The concentration of polyphenols was measured utilizing high-performance liquid chromatography (HPLC) and a photodiode array (PDA) on a Nexera X2 instrument manufactured by Shimadzu (Kyoto, Japan). The system was equipped with an SPD-M20A diode array detector, LC-30AD binary pump (for organic and aqueous phase), SIL-30AC autosampler, CTO-20AC thermostated column compartment, and communication bus module (CBM-20A) and controlled by LabSolutions version 5.71 SP2 software (Shimadzu Corporation, Kyoto, Japan). The compounds were separated on a 40 °C thermostated Luna C18(2) column (150 mm × 2 mm i.d., 3 μm) and C18 guard column (4 mm × 2 mm i.d, 3 μm), both from Phenomenex (Torrance, CA, USA). Polyphenol constituents were determined using gallic acid and protocatechuic acid standards and by tentative identification using spectral data (p-coumaric acid, quercetin and luteolin derivatives). Gallic acid (98%) was purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and protocatechuic acid (99%) was purchased from HWI Analytic GmbH (Neumarkt, Germany). The calibration curve for the measured polyphenols was obtained by creating standard solutions with varying concentrations (1, 10, 20, 40, 60, 80, and 100 μg/mL). Ethanolic extract (1 mL) was filtered (through a 0.22 μm filter) and transferred to a vial without dilution. The volume of the injection and the rate at which it was delivered were 5 μL and 0.25 mL/min, respectively. Samples of oil were subjected to liquid–liquid extraction in order to obtain ethanolic extracts, which underwent the same procedure. The solvent gradient for the binary mobile phase that was employed was: 0–5 min, 5%; 5–10 min, 20%; 10–17 min 20%; 17–30 min, 25%; 30–35 min, 30%; 35–45 min, 70%; 45–50 min, 70%; 50–55 min, 5%; 55–60 min, 5% (solvent B). The mobile phase consisted of a 1% (v/v) aqueous solution of formic acid (A) and a 1% solution of acidified methanol (B). The volume of the injection and the rate at which it was delivered were 5 μL and 0.25 mL/min, respectively. The autosampler was maintained at a temperature of 15 °C. The column pressure was 20 MPa. Chromatographic data were collected in a wavelength range of 190 to 650 nm, with a reference wavelength of 600/50 nm. All analyses were performed in triplicate and the results expressed as a mean value of three measurements. Obtained results are expressed as the concentration of polyphenols in the formulation (μg/mL) and as quantity of polyphenols in the dry extract (mg/g of dry weight—DW). Derivatives were expressed using gallic acid equivalents. Limits of quantification and detection (LOQ and LOD) were also provided to support the data.

Regarding the mechanically pressed oil, liquid–liquid extraction from the obtained oil was performed using ethanol for further polyphenol determination.

2.4. Aphid Mortality Assays

The solution used for aphicidal assays was created by diluting 2.5 and 5 mL of the extract (labeled 2.5% and 5% IBP, respectively) into 100 mL of distilled water. Similarly to this, extracted oil was diluted with distilled water to concentrations of 2.5% and 5% (labeled 2.5% oil and 5% oil) with one drop of non-toxic emulsifying agent, Tween 80 addition to facilitate the water and oil homogenization and slow the separation. Since the WAA-colonized trees were present in a patchy manner throughout the orchard, treatments were applied within rows where 15 infected trees were in a continuous line, applying each of the five treatments in three replications. Blocks of 15 new trees were targeted for each of the three presented dates. Firstly, in vivo, trees characterized by both trunk- and branch-infected wounds were marked and chosen for spraying. Subsequently, for the in vivo studies, bark slices (15 × 15 mm, Figure 2) were collected from the trunks of the same 15 infected trees. On the same days, in vivo orchard applications were followed by immediate in vitro testing by transferring collected bark slices containing wingless aphids to laboratory conditions and petri dishes at the Faculty of Agriculture, Novi Sad, Serbia (Horticultural Plants Breeding Laboratory). Once established in the laboratory conditions, wingless WAA adults were allowed to acclimate and activate before the treatments, so as to transfer and spray only the live ones. Even though the effect was visible immediately, sprayed aphids were counted and mortality confirmed after 10 min, 1 h, 3 h, and after 24 h, both in vivo and in vitro.

Within each 15 tree blocks and petri dishes containing bark slices from those trees, 3 were sprayed with distilled water as a control, 3 were sprayed with 2.5% IBP, 3 with 5% IBP, 3 with 2.5% oil, and finally 3 were sprayed with 5% oil.

All treatments were applied with the onset of optimal temperatures (more than 10 °C during a couple of days) in the spring of 2024—24 February, 16 March, and 1 April—upon determination of WAA activity. The extracts were sprayed into complete bark wounds located on the trunk and branches in vivo and onto apple bark slices in vitro. Bark slices and bark wounds treated with distilled water were considered as controls. In both in vivo and in vitro trials, spraying was applied in the form of a mist and at a distance of 30 cm from bark or petri dishes. The volume sprayed into the wounds and onto the bark slices was approximately the same: 0.5 mL per wound over the broadest area (100–150 cm²) and on the petri dish of the same area. Applied volumes were measured and calculated as difference in volume before and after a single treatment in a graduated cylinder. When calculated for orchard conditions, the amount was 0.13–0.14 L per tree, corresponding to a standard insecticide application of 0.13 L per tree with a working pressure of 11.5.

2.5. Statistical Analysis

To compare the effect of the treatments, the mortality rate is expressed as a percentage and was analyzed using ANOVA followed by Tukey’s HSD when p < 0.05 for both in vivo and in vitro trials. The data were transformed by arcsine transformation, but for simplicity’s sake, percentages are shown in the tables. Also, the results of Tukey’s test for both percentages and transformed data revealed the same homogeneous groups, fortifying data presentation decisions.

The data obtained were transformed in Microsoft Excel 2007 (Microsoft, Redmond, WA, USA) and statistically processed using STATISTICA 14 software (Tibco, Palo Alto, CA, USA).

3. Results

3.1. In Vivo Efficacy of K. paniculata Seed Extract and Oil in Suppression of Wingless WAA Adults

In general, the number of live and active wingless WAA adults in vivo varied from 49 to 112 in the trunk wounds and 24 to 58 in the branches, due to the different wound shapes, observation dates, and pest developmental phases (

Table 1. Live and active wingless woolly apple aphid (WAA) adults counted on the trunk and branch wounds on three dates in the winter–spring of 2024.

Treatment	Number of Live Wingless WAA Adults	Apple Trunk	Apple Branch	Apple Trunk	Apple Branch	Apple Trunk	Apple Branch
		24 February		16 March		1 April
2.5% IBP *	Before	79	58	61	37	49	28
	After	6	5	3	4	5	4
	Mortality %	92 b **	91 b	95 b	90 c	90 c	86 c
5% IBP	Before	67	52	53	41	68	37
	After	4	0	2	2	3	2
	Mortality %	94 b	100 a	96 b	95 b	96 b	96 b
2.5% Oil	Before	102	36	97	58	84	43
	After	0	0	0	0	0	0
	Mortality %	100 a	100 a	100 a	100 a	100 a	100 a
5% Oil	Before	87	48	81	24	112	25
	After	0	0	0	0	0	0
	Mortality %	100 a	100 a	100 a	100 a	100 a	100 a
Distilled water	Before	92	39	96	28	54	31
	After	92	39	96	28	54	31
	Mortality %	0	0	0	0	0	0

* Initial botanical preparation (IBP) diluted by transferring 2.5 mL (labeled 2.5% IBP) and 5 mL (labeled 5% IBP) of the obtained solution and 97.5 and 95 mL of distilled water, respectively, to the bottle of 100 mL. ** Mean values designated with the same letter were not significantly different according to Tukey’s HSD test (p ≤ 0.05). Control treatment was excluded from the analysis.

). All these properties among the investigated trees and dates were very variable and are thus presented as “case observations” (examples are given in Figure 3a,b and Figure 4a,b), rather than averages from three days. Since there was no difference in the counting after 10 min, 1 h, 3 h, and 24 h upon the treatments’ applications on the same days, those data are presented only once.

Both oil concentrations (2.5% and 5%) proved to have absolute aphicidal effect, with no adult alive minutes after the spraying, while the WAA mortality upon seed extract application was between 90% and 96% in the trunk wounds and between 86% and 100% in the branch wounds, with the higher percentages associated with the higher IBP concentration.

More precisely, the first treatment (2.5% IBP) resulted in 3–6 live adults of up to 79 and 5% IBP resulted in 0–4 adults of up to 68 counted WAAs. Even though the number of wingless WAA adults in the given wounds reached 102 and 112 in the treatments with 2.5% and 5% oil, respectively, the mortality was absolute. On the contrary, the mortality rate was zero in the control treatments with distilled water. Tukey’s honest significant difference test for given plant parts and date of observation further proved the differences in WAA suppression by K. paniculata seed extract and oil. Applied treatments—extract, oil, and water—were generally separated into significantly different homogeneous groups and designated with different letters, without any transitional components between either of the neighboring two (a–b or b–c) based on Tukey’s HSD test.

3.2. In Vitro Efficacy of K. paniculata Seed Extract and Oil in Suppression of Wingless WAA Adults

To assess only the seed extracts and oil effect, live adult aphids from uniform bark slices were transferred to petri dishes and fully exposed to the same treatments as in the in vivo conditions in triplicate from the same three trees chosen in the orchard. In general, the number of live and active wingless WAA adults from the 1.5 cm² bark slices varied from 11 to 38. Again, since there was no difference in the counting after 10 min, 1 h, 3 h, and 24 h following treatment application on the same days, those data are presented only once. Since the spraying was applied in the form of a mist and at a distance of 30 cm from the petri dishes, mimicking the orchard conditions, two cases of live adult aphids after the treatment were recorded, for 2.5% and 5% extract, on 16 March. In both cases, live aphids seemed unsprayed and emerged from the dead ones. In all other petri dishes, no wingless WAA adults were counted following treatment (100% mortality rate), while no dead WAA was found (0% mortality rate) when sprayed with distilled water (

Table 2. The average number of live and active wingless woolly apple aphid (WAA) adults counted in the petri dishes on three dates in the winter–spring of 2024.

Treatment	Number of Live Wingless WAA Adults	24 February	16 March	1 April
2.5% IBP *	Before	19	12	11
	After	0	1	0
	Mortality %	100 a **	92 c	100 a
5% IBP	Before	21	25	20
	After	0	1	0
	Mortality %	100 a	96 b	100 a
2.5% Oil	Before	36	30	17
	After	0	0	0
	Mortality %	100 a	100 a	100 a
5% Oil	Before	29	20	38
	After	0	0	0
	Mortality %	100 a	100 a	100 a
Distilled water	Before	22	12	15
	After	22	12	15
	Mortality %	0	0	0

* Initial botanical preparation (IBP) diluted by transferring 2.5 mL (labeled 2.5% IBP) and 5 mL (labeled 5% IBP) of the obtained solution and 97.5 and 95 mL of distilled water, respectively, to the bottle of 100 mL. ** Mean values designated with the same letter were not significantly different according to Tukey’s HSD test (p ≤ 0.05). Control treatment was excluded from the analysis.

).

When treated with distilled water, live adults remained active and tended to group during the moisture presence (Figure 5). Once the water had evaporated, aphids continued to move separately. In the case of seed extract and oil, in a 1-minute time span, no treated aphids showed any signs of activity. Similar to the in vivo trial, Tukey’s honest significant difference test showed statistically significant differences in WAA suppression by K. paniculata seed extract and oil. Applied treatments—extract, oil, and water—generally separated into significantly different homogeneous groups without any transitional components between either of the neighboring two (a–b or b–c) based on Tukey’s HSD test.

3.3. Phenolic Content of Seed Ethanolic Extract and Oil

The concentration of polyphenols measured by high-performance liquid chromatography (HPLC) in the initial botanical preparation (IBP) showed an abundance of phenolic compounds (Figure 6). Interestingly, none of the phenolic compounds was present in the mechanically extracted cold-pressed oil (Supplement Figure S1).

Although the total phenolic content amounted to 65.4 μg/mL or 27.9 mg/g DW, five compounds were distinguished in the sample, contributing to the 59.09 μg/mL and 25.3 mg/g DW (more than 90% total phenolic content). Two phenolic acids, gallic and protocatechuic, and three derivatives—p-coumaric acid derivative, quercetin derivative, and luteolin derivative—achieved maximum peaks (Figure 6). Regarding the phenolic acids, IBP was more abundant in protocatechuic acid (5.901 mg/g DW) than in gallic acid (1.823 mg/g DW), while amongst the polyphenol derivatives, the p-coumaric acid derivative and quercetin derivative were significantly present, with 8.925 and 7.593 mg/g DW, while luteolin derivative showed only 1.011 mg/g DW (

Table 3. Phenolic profile of the initial botanical preparation from Koelreuteria paniculata seeds.

Compound	Abbr.	c (μg/mL)	c (mg/g DW)	Equation	R2	LOQ	LOD
Gallic acid	GalA	4.2661	1.823	y = 55033x + 7774	0.999	5.25	1.73
Protocatechuic acid	PCA	13.8079	5.901	y = 92394x − 44370	0.999	10.1	3.33
p-Coumaric acid derivative	p-CoumA	20.8849	8.925	y = 178713x − 45820	0.999	7.04	2.32
Quercetin derivative	Que	17.7682	7.593	y = 107707x − 26982	0.999	7.21	2.38
Luteolin derivative	Lut	2.3668	1.011	y = 111276x − 88071	0.999	8.05	2.66
	Sum	59.0939	25.253

DW (dry weight), LOQ (limit of quantification), LOD (limit of detection).

).

3.4. Matching Potential Biopesticide Development from IAS with UN Sustainable Development Goals (SDGs) and European Green Deal (EGD)

To assess the significance of biopesticide development from threatening IAS seeds, an analysis of broad possible environmental, economic, and social effects was conducted, including both direct and indirect possible influences. Overall, development of biopesticide from IAS can contribute directly to the globally defined SDGs: SDG3—Good Health and Well-Being; SDG11—Sustainable Cities and Communities; SDG12—Responsible Consumption and Production; SDG13—Climate Action; SDG15—Life on Land; and SDG17—Partnerships for the Goals. Concomitantly, indirect effects are expected to appear on SDG2—Zero Hunger, SDG5—Gender Equality, SDG6—Clean Water and Sanitation, SDG7—Affordable and Clean Energy, SDG8—Decent Work and Economic Growth, SDG9—Industry, Innovation, and Infrastructure, and SDG14—Life below Water (

Table 4. Matching biopesticide production from invasive alien species with the Sustainable Development Goals (SDGs) and European Green Deal (GD).

	SDG	Direct Link	Indirect Link	GD Matching
1	No Poverty		✓
2	Zero Hunger		✓	GD7 *
3	Good Health and Well-Being	✓		GD7
4	Quality Education
5	Gender Equality		✓
6	Clean Water and Sanitation		✓
7	Affordable and Clean Energy		✓	GD2
8	Decent Work and Economic Growth		✓
9	Industry, Innovation, and Infrastructure		✓
10	Reduced Inequalities
11	Sustainable Cities and Communities	✓		GD7
12	Responsible Consumption and Production	✓		GD5
13	Climate Action	✓		GD1
14	Life below Water		✓
15	Life on Land	✓		GD6
16	Peace, Justice, and Strong Institutions
17	Partnerships for the Goals	✓

* Green Deal main goals: GD1—increasing the EU’s climate ambition for 2030 and 2050; GD2—supplying clean, affordable, secure energy; GD3—mobilizing industry for a clean and circular economy; GD4—building and renovating in an energy- and resource-efficient way; GD5—a zero pollution ambition for a toxin-free environment; GD6—preserving and restoring ecosystems and biodiversity; GD7—Farm to Fork: a fair, healthy and environmentally friendly food system; GD8—accelerating the shift to sustainable and smart mobility. Sign ‘✓’ indicates possible direct or indirect contribution of biopesticide development to specific SDG.

).

It can be noted from Table 4 that biopesticide acquisition from invasive alien plants specifically aligns with GD5 (zero pollution ambition for a toxin-free environment), GD6 (reserving and restoring ecosystems and biodiversity), and GD7 (Farm to Fork: a fair, healthy, and environmentally friendly food system).

4. Discussion

Apple is the second-most traded fruit in the world after bananas [43], with China being a top producer, followed by the USA, Poland, and Turkey. Holding such a high position, Turkish researchers have forecast an increasing trend for apple production with 3,706,954 to 3,987,050 tons from 2020 to 2025 solely within their nation [44], expecting the same trend in global apple production due to the abundance of varieties and rootstocks, as well as novel precision agricultural tools and management practices. With the recent crisis caused by COVID-19 as well as numerous environmental and climate-related issues, both urban and rural fruit production increased, with the same demand: to provide healthy food based on integrated or organic practices [45]. Three main fruit production systems are in place globally at the moment—conventional, organic, and integrated [46]—with the last approach softening the edges of both conventional and organic production. Regarding the WAA-resistant apple rootstocks suitable for organic and integrated production, there are only a few progenies carrying the long-utilized Er1 and Er2 genes, deriving from “Northern Spy” and “Robusta 5,” respectively, or from the newly determined “Aotea 1” (Malus sieboldii genotype) [13]. To meet the expanding apple market demand, integrated pest management and pure organic production require minimization or exclusion of synthetic pesticides and fertilizers, with a concomitant increase in organic fertilizers [47] and acceptable biobased pesticide application [48]. The search for biologically acceptable pesticides has become pronounced for WAA control, due to the abandonment and prohibition of previously utilized chemicals—chlorpyrifos and imidan—which do not meet new legislative requirements.

Addressing the issues above, this study assayed and determined that seed extract and oil derived from an extremely invasive ornamental species, K. paniculata, had an almost immediate aphicidal effect on wingless WAA adults in both in vivo and in vitro orchard and laboratory assays. Slightly higher in vivo statistically significant differences in wingless WAA adult mortality immediately upon treatment application might be due to the basic properties of those botanical preparations. Specifically, the ethanolic extracts were light and easily evaporated, whilst the oil preparation was heavier, and although it was diluted with water, it did to some extent affix itself to the applied body or area. In addition, when applied in vivo, not all wingless WAA adults were definitely covered by the spraying, since the colony had a tridimensional form, compared to the aphids transferred to the petri dishes in a two-dimension plate and full exposure. Furthermore, since in both cases of adult WAA in vitro survival, where live aphids seemed unsprayed and emerged from the dead ones, it can be assumed that the botanical preparation and extracted K. paniculata oil have a contact aphicidal effect, as does the neem oil [49]. Similarly, Ebrahimi et al. [50] showed that aldehydes, phenols, and monocyclic terpenes of Azadirachta indica, Eucalyptus camaldulensis, and Laurus nobilis showed contact aphicidal effects, and the effect of Melaleuca alternifolia oil was proved to be even greater against Myzus persicae than the previously reported effect of neem, eucalyptus, and laurel oil [51].

Regarding the natural active compounds, Czerniewicz et al. [52] concluded that mixtures of phenolic acids extracted from the edible and medicinal species Juglans regia, Mentha piperita, Sambucus nigra, and Hypericum perforatum exhibited insecticidal activity against peach–potato aphid and bird cherry–oat aphid. Assessing the phenolic profile, our study showed that the major phenolic compounds (more than 90%) in the K. paniculata ethanolic extract numbered five: two phenolic acids (gallic and protocatechuic) and three derivatives (p-coumaric acid derivative, quercetin derivative, and luteolin derivative). A high concentration of gallic and caffeic acid in the hazel leaves was responsible for their resistance to filbert aphid [53], while Ozay et al. [54] suggest that pistachio hull extract, rich in gallic and protocatechuic acid, has great potential as a biopesticide feedstock. Özbek et al. [55] also determined that pistachio hull extract was rich in gallic acid, p-hydroxybenzoic acid, protocatechuic acid, syringic acid, p-coumaric acid, quercetin, and caffeic acid, some of which dominated in our extract in much greater amounts (more than five-fold when expressed in mg/g).

Investigating the insecticidal effect of gallic acid on the larvae of Spodoptera litura and its parasitoid Bracon hebetor, Punia et al. [56] showed high efficacy, while Dolma et al. [57] determined the same for Aphis craccivora Koch, a leguminous crop aphid. Surveying waste side products during the processing of acerola, Marques et al. [58] determined that phenolic compounds including gallic acid, epigallocatechin gallate, catechin, p-coumaric acid, salicylic acid, and quercetin resulted in the mortality of the fall armyworm—Spodoptera frugiperda (J.E. Smith). Another side product, olive mill wastewater, abundant in gallic and protocatechuic acid [59], showed great potential in controlling the pests Euphyllura olivina and Aphis citricola [60], as well as Ceratitis capitata [61]. Furthermore, gallic acid proved to be among other microalgal substances with larvicidal or insecticidal activity [62]. The p-coumaric acid derivate was predominant among the derivates in our study. Previous studies linked this acid with grain aphid suppression [63,64] and stalk borer control [65]. Considered together, gallic, protocatechuic, and p-coumaric acid, present in our study, were also major constituents responsible for aphicidal activity against Aphis spiraecola Patch [66].

Regarding WAA specifically, back in 1975, phenolics were linked with apple resistance to WAA [67], while in 2012, Ateyyat et al. [68] showed that the flavonoids quercetin dehydrate, rutin hydrate, and naringin were active as aphicides against WAA, with mortality of wingless adults higher than that obtained against apterous adults. Concomitantly, the three tested flavonoids did not affect the parasitoids of WAA compared with the detrimental effect of commercial insecticide. Although not specifically targeted in our study, during the in vivo application, ladybugs, spiders, and honeybees were present in the spraying area, but showed no signs of detrimental effect immediately or hours after application. In another study, Naga et al. [69] determined only a relative toxicity of phenolics against E. lanigerum, comparing them to the conventional insecticide imidacloprid. Although our initial assumption was also that the extremely hard and water-impermeable seed coat would not allow sufficient active compound extraction or efficacy (thus 20% concentrated initial solution was prepared), immediate high WAA mortality with both 2.5% and 5% IBP indicates that these concentrations could be further reduced and applied at larger-scale orchard levels, increasing the cost-effectiveness of ethanolic solution.

Since oil characterization showed no presence of phenolic compounds, other chemically active substances might be employed in the K. paniculata oil aphicidal effect against WAA. Our previous study on the seed oil from the very same K. paniculata specimen [40] proved the presence of various monounsaturated fatty acids derivates with a significant portion of eicosenoic acid (43.3–47.2%) and oleic acid (28.4–25.3%), followed by linoleic (8.28–11.0%) and palmitic acid (4.50–7.99%). Eicosenoic fatty acid was the primary compound in the Rhizophora mucronata (Lam.) leaf extracts, expressing both larvicidal and repellent activity against three different mosquitoes [70]. In a similar study, oleic acid proved to be the most responsible for the biopesticidal activity of cassava seed oil against the Bihar hairy caterpillar (Spilarctia obliqua) and cowpea aphid (Aphis craccivora), with 93.3% mortality in the larval population [71]. Furthermore, moringa seed oil, abundant in arachidic, behenic, oleic, and palmitic acid (similar to K. paniculata), proved effective in the suppression of Tetrancus urticae (Koch) and the cotton mealybug Phenacoccus solenopsis [72]. Notable fatty acids—palmitic, oleic, linoleic, and eicosenoic—present in the K. paniculata seed oil from our study were also determined in Ulva rigida extracts, with pronounced antibacterial and antifungal properties [73]. In some older studies performed in 2004–2006, “Savona” and “Bioshower” insecticidal soaps composed of 100% fatty acids were utilized against peach and apple aphids [74,75], fortifying our assumption that the active ingredients in the oil are fatty acids.

Reaching a toxin-free environment requires the sustainable use of agricultural waste as bioproduct feedstock, such as biopesticides. This approach aligns with the core principles of zero waste, which aims to eliminate waste and protect our “green” planet through “3R” (reduce, reuse, recycle) or “5R” (refuse, reduce, reuse, recycle, repair), fully integrating the circular economy approach [76]. These environmentally friendly, green solutions or NBSs are alternatives to conventional pesticides, and their implementation in current agricultural practices directly increases the rate of environmental toxicity reduction [77,78]. Sowińska-Świerkosz and García [79] highlight one of the key characteristics of nature-inspired solutions: their high effectiveness, as well as their economic and resource efficiency in addressing problems, compared to the traditional gray actions that have been present and applied so far. Since the European Commission proposes five questions to determine whether an activity or action can be characterized as an NBS [80], our and future similar studies should address those demands:

• Do nature-based solutions utilize nature and natural processes?
• Do they provide/enhance social benefits?
• Do they provide/enhance economic benefits?
• Do they provide/enhance environmental benefits?
• Do they ensure net benefits for biodiversity?

Considering that existing K. paniculata mature trees are present in both urban and rural green areas, the species can be regarded as an NBS derived from nature, while its intrinsic chemical compounds are a result of natural biochemical processes (Question 1). Furthermore, this solution can enhance both integrated and organic fruit production suitable for traditional field and modern urban apple pest control, bringing social innovation, citizens’ engagement, and participatory development of new products and practices (Question 2). Taken altogether, biopesticide acquisition from IAS enhances environmental benefits on multiple levels (preserves vegetative parts contributing to oxygen production and decreases the generative potential from seed dispersal), addressing Question 4, and thus directly contributing to biodiversity stabilization, respecting “do no harm” to natural predators and native flora while reducing invasiveness and the detrimental effect of WAA (Question 5).

5. Conclusions

With increasing demands for safe and healthy fruits produced according to sustainable principles, this study aimed to assess the in vivo and in vitro aphicidal effect of Koelreuteria paniculata seed ethanolic extract and oil against WAA (Eriosoma lanigerum). Although underexplored, the chosen invasive alien species proved to possess high aphicidal activity against wingless adults with applied concentrations of 2.5% and 5% of both seed ethanolic extracts and oil. The mortality of in vivo live aphids upon the seed extract application was between 96% and 100% in the trunk wounds and between 86% and 100% in the branch wounds, with the higher percentages associated with the higher concentration. On the contrary, in vitro application mainly resulted in 100% mortality, with only two cases of wingless WAA adult aphid survival. Oil demonstrated a complete aphicidal effect, with no adult aphids surviving minutes after spraying in vivo or in vitro.

Even though notable mortality was observed upon application, different chemical compounds seem to be responsible for such high effectiveness. Initial ethanolic extract was abundant in gallic and protocatechuic acids, and three derivates—p-coumaric acid, quercetin and luteolin derivatives—contributed more than 90% of total phenolic content and reached the maximum peaks. Since oil characterization showed an absolute absence of phenolic compounds, other chemically active substances, such as fatty acids previously proved to be associated with this species, might be crucial for the K. paniculata oil aphicidal effect against WAA. Due to the extremely high mortality from the 2.5% and 5% concentrations of the oil and initial botanical preparation, future steps shall include the investigation of further diluted concentrations’ effect on WAA mortality.

Future research shall be devoted to a complete economic analysis and undoubtedly to recommend the solutions obtained as suitable for sustainable apple production contributing to biological control.