Extract of Nicotiana tabacum as a potential control agent of Grapholita molesta (Lepidoptera: Tortricidae)

Abstract

Oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), is an important pest of stone and pome fruits. Growers usually depend on chemical insecticides to control this pest, but demand for more environmentally-friendly means of controlling pests is increasing. At least 91 plant extracts have been reported to be effective against other lepidopterans, but their acute toxicity against G. molesta has rarely been studied. Among these 91 materials, we assessed the residual toxicity of 32 extracts against first instar larvae (< 5 h old) of G. molesta in the laboratory. Nicotiana tabacum L., used at the concentration of 2 mg/ml, showed the highest corrected mortality (92.0%) with a lethal time (LT₅₀) value of 12.9 h. The extract was followed in its efficacy by Allium sativum L. (88.0%), Zanthoxylum piperitum (L.) De Candolle (70.0%), and Sapindus mukorossi Gaertner (65.0%), when mortality was assessed at 20 h after exposure. Against adult fruit moths (< 5 d old), N. tabacum also showed the highest corrected mortality among tested extracts, being 85 and 100% in adult females and males, respectively, at 168 h after exposure. However, there was no synergistic effect of the combined application of any of the top four extracts in either laboratory or greenhouse assays. Oviposition by G. molesta on peach twigs was reduced 85–90% when N. tabacum was applied at 4 ml/ twig compared to control (methanol), demonstrating that N. tabacum may have potential for use as a botanical insecticide against G. molesta.

Introduction

Oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), is a serious pest of fruit trees in the temperate regions, worldwide [1–4]. Its host range encompasses species within the family Rosaceae, mostly those from the genera Prunus and Pyrus [1]. Stone fruit peach [Prunus persica L. (Rosales: Rosaceae)] is considered the primary host of G. molesta whereas the pome fruits pear [Pyrus communis L. (Rosales: Rosaceae)] and apple [Malus domestica L. (Rosales: Rosaceae)] are considered secondary hosts [5].

Application of organophosphorus, carbamates, or synthetic pyrethroid pesticides is a common method for control of G. molesta in Korea [6, 7], but the development of insecticide resistance is a serious threat to the fruit industry [6], and G. molesta has developed resistance to 14 insecticides including 10 organophosphates [8]. As many of these insecticides are neurotoxins, they have some potential to be harmful to non-target organisms, including people and domestic animals [4]. To avoid such risks, new pest management tactics need to be developed for the management of G. molesta. Due to their less residual toxicity, lower development cost, and general safety to people, plant extracts have the potential to be effective alternatives for control of pest insects [9].

Secondary plant metabolites, such as polyphenols, terpenoids, alkaloids, steroids, lignans, essential oils, fatty acids, and sugars, are regarded as defense mechanisms against insect attack [10]. Some secondary metabolites inhibit insect development and reproduction, while others act as antifeedants, repellents, or fumigants [11–13]. Botanical insecticides degrade quickly, meaning their impact on beneficial or non-target organisms is less than that of conventional insecticides [14], thus would be more compatible with biological control agents than synthetic insecticides. Furthermore, botanical insecticides have also multiple modes of action, development of resistance in insects has been reported less frequently [15].

At least 91 plant extracts have been found effective against pest lepidopterans in studies published from 2000–2015 (Table 1). Some of these extracts have demonstrated a similar level of pest toxicity as synthetic insecticides. Extracts from goat weed (Ageratum conyzoides L.) and siam seed (Chromolaena odorata [L.]) controlled Plutella xylostella L. larvae, a rate similar to the synthetic insecticide emamectin benzoate [16]. Antifeedant activity was found for extracts of Chrysanthemum sp. and Achillea millefolium L. against Spodoptera littoralis (Boisduval) and Pieris rapae L., respectively [17, 18], and plant extracts have also been found to act as an oviposition deterrent; Reegan et al. [19] reported that a hexane extract of Limonia acidissima (L.) showed 100% oviposition deterrency for adults females of Culex quinquefasciatus Say and Aedes aegypti L.

Table 1. Plant extracts reported during 2000–2015 to show toxicity against lepidopteran insects.

Plant species	Plant parts	Solvent	Lepidopteran insects tested
			Species	Family
Abrus precatorius [38]	Seed	Ethanol	Galleria mellonella	Pyralidae
Achillea millefolium [18]	Leaf	Methanol	Pieris rapae	Pieridae
Acorus calamus [39]	Rhizome	Ether	Sitotroga cerealella	Gelechiidae
Ageratum conyzoides [16]	Leaf	Detergent	Plutella xylostella	Yponomeutidae
Allium cepa [40]	Fresh onion	Tween 20	Tuta absoluta	Gelechiidae
Allium sativum [40]	Fresh garlic	Tween 20	Tuta absoluta	Gelechiidae
Alpinia galanga [41]	Rhizome	Ethanol	Plutella xylostella	Yponomeutidae
Anona coriacea [42]	Leaf	Methanol	Spodoptera frugiperda	Noctuidae
Anona dioica [42]	Leaf	Methanol	Spodoptera frugiperda	Noctuidae
Anona muricata [43]	Leaf	Ethanol	Plutella xylostella	Yponomeutidae
Artemisia annua [18]	Leaf	Methanol	Pieris rapae	Pieridae
Artemisia vulgaris [44]	Whole plant	Methanol	Spodoptera littoralis	Noctuidae
Avicennia marina [45]	Aerial part	Hexane	Phthorimaea operculella	Gelechiidae
Azadirachta indica [46]	Seed	Water	Tuta absoluta	Gelechiidae
Bifora radiens [47]	Whole plant	Acetone	Thaumetopoea solitaria	Thaumetopoeidae
Cabralea canjerana [48]	Seed/ Fruit	Ethanol	Spodoptera frugiperda	Noctuidae
Capparis aegyptia [45]	Aerial part	Hexane	Phthorimaea operculella	Gelechiidae
Capsicum annum [49]	Leaf	Methyl. chloride	Spodoptera littoralis	Noctuidae
Capsicum frutescens [16]	Fruit	Detergent	Plutella xylostella	Yponomeutidae
Carica papaya [50]	Seed	Methanol	Spodoptera frugiperda	Noctuidae
Cassia sophera [16]	Leaf	Detergent	Plutella xylostella	Yponomeutidae
Chromolaena chaseae [51]	Leaf	Ethanol	Spodoptera frugiperda	Noctuidae
Chromolaena odorata [16]	Leaf	Detergent	Plutella xylostella	Yponomeutidae
Chrysanthemum grandiflorum [17]	Aerial part	Metanol	Spodoptera littoralis	Noctuidae
Chrysanthemum indicum [52]	Leaf	Water	Plecoptera reflexa	Noctuidae
Chrysanthemum macrotum [17]	Aerial part	Methanol	Spodoptera littoralis	Noctuidae
Chrysanthemum morifolium [53]	Leaf	Methanol	Trichoplusia ni	Noctuidae
Chrysanthemum segetum [17]	Aerial part	Methanol	Spodoptera littoralis	Noctuidae
Citrullus colosynthis [54]	Seed	Ammonium sulfate	Ectomyelois ceratoniae	Pyralidae
Citrus sinensis [55]	Leaf	Phenol	Phyllocnistis citrella	Gracillariidae
Cleome deoserifolia [44]	Aerial part	Ethanol	Phthorimaea operculella	Gelechiidae
Cleome spinosa [56]	leaves	Ethanol	Pieris rapae	Pieridae
Commiphora molmol [57]	Stem	Water	Spodoptera littoralis	Noctuidae
Croton urucurana [58]	Stem	Methanol	Anagasta kuehniella	Pyralidae
Cymbopogon martinii [59]	Whole part	Water	Euprosterna elaeasa	Limacodidae
Cyprus rotundus [41]	Tuber	Ethanol	Plutella xylostella	Yponomeutidae
Datura metel [60]	Leaf	Methanol	Helicoverpa armigera	Noctuidae
Delphinium consolida [44]	Whole plant	Methanol	Spodoptera littoralis	Noctuidae
Dimorphandra mollis [61]	Leaf	Ethanol	Sitotroga cerealella	Gelechiidae
Euphorbia lathyrus [62]	Seed	Ethanol	Spodoptera littoralis	Noctuidae
Fumaria officinalis [47]	Whole plant	Acetone	Thaumetopoea solitaria	Thaumetopoeidae
Ginkgo biloba [63]	Seed coat	Methanol	Spodoptera exigua	Noctuidae
Glycine max [64]	Leaf	Isooctane	Heliothis zea	Noctuidae
Gomphrena globosa [41]	Seed	Ethanol	Plutella xylostella	Yponomeutidae
Hordium sativum [38]	Seed	Ethanol	Galleria mellonella	Pyralidae
Hovenia dulcis [65]	Leaf	Water	Anticarsia gemmatalis	Erebidae
Humulus lupulus [47]	Whole plant	Methanol	Thaumetopoea solitaria	Thaumetopoeidae
Hymenoxys robusta [66]	Leaf	Methanol	Spodoptera exigua	Noctuidae
Ipomoea pauciflora [67]	Seed	Hexane	Spodoptera frugiperda	Noctuidae
Jatropha curcas [16]	Leaf	Detergent	Plutella xylostella	Yponomeutidae
Jatropha gossypifolia [68]	Leaf	Ethanol	Spodoptera frugiperda	Noctuidae
Laurus nobilis [38]	Seed	Ethanol	Galleria mellonella	Pyralidae
Lepidaploa lilacina [51]	Leaf	Ethanol	Spodoptera frugiperda	Noctuidae
Lychnophora ericoides [51]	Leaf	Ethanol	Spodoptera frugiperda	Noctuidae
Lychnophora ramosissima [51]	Leaf	Ethanol	Spodoptera frugiperda	Noctuidae
Melia azedarach [68]	Leaf	Ethanol	Spodoptera frugiperda	Noctuidae
Millettia ferruginea [69]	Seed	Water	Busseola fusca	Noctuidae
Momordica charantia [70]	Leaf	Methanol	Leucoptera coffeella	Lyonetiidae
Nerium indicum [71]	Seed	Water	Helicoverpa assulta	Noctuidae
Nicotiana tabacum [16]	Leaf	Detergent	Plutella xylostella	Yponomeutidae
Ocimum gratissimum [16]	Leaf	Detergent	Plutella xylostella	Yponomeutidae
Pachyrhizus erosus [72]	Seed	Methanol	Plutella xylostella	Yponomeutidae
Peganum harmala [73]	Leaf	Methanol	Spodoptera exigua	Noctuidae
Pelargonium zonale [40]	Leaf	Tween 20	Tuta absoluta	Gelechiidae
Petroselium sativum [38]	Seed	Ethanol	Galleria mellonella	Pyralidae
Peumus boldus [74]	Leaf	Water	Spodoptera frugiperda	Noctuidae
Piper amalago [75]	Leaf	Ethanol	Tuta absoluta	Gelechiidae
Piper glabratum [75]	Leaf	Ethanol	Tuta absoluta	Gelechiidae
Piper mikanianum [75]	Leaf	Ethanol	Tuta absoluta	Gelechiidae
Plantago lanceolata [70]	Leaf	Methanol	Leucoptera coffeella	Lyonetiidae
Plantago psyllium [38]	Seed	Ethanol	Galleria mellonella	Pyralidae
Pongamia pinnata [76]	Seed	Chloroform	Earias Vittella	Noctuidae
Psychotria goyazensis [77]	Leaf	Ethanol	Spodoptera frugiperda	Noctuidae
Psychotria prunifolia [61]	Leaf	Ethanol	Sitotroga cerealella	Gelechiidae
Quassia amara [78]	Wood	Methanol	Hypsipyla grandella	Pyralidae
Ricinus communis [79]	Leaf	Hexane	Spodoptera frugiperda	Noctuidae
Rhododendron molle [80]	Flower	Ethyl acetate	Hypsipyla grandella	Pyralidae
Ruta chalepensis [81]	Leaf	Hexane	Hypsipyla grandella	Pyralidae
Sapindus mukorossi [82]	Fruit	Water	Thysanoplusia orichalcea	Noctuidae
Siphoneugena densiflora [83]	Leaf	Methanol	Spodoptera frugiperda	Noctuidae
Synedrella nodiflora [19]	Leaf	Detergent	Plutella xylostella	Yponomeutidae
Tagetes erecta [84]	Leaf	Ethanol	Spodoptera frugiperda	Noctuidae
Tanacetum mucroniferum [44]	Whole plant	Methanol	Spodoptera littoralis	Noctuidae
Tanacetum zahlbruckneri [85]	Flower	Methanol	Spodoptera littoralis	Noctuidae
Tithonia diversifolia [61]	Leaf	Ethanol	Sitotroga cerealella	Gelechiidae
Trichilia pallens [86]	Twig	Water	Spodoptera frugiperda	Noctuidae
Trichilia pallida [86]	Twig	Water	Spodoptera frugiperda	Noctuidae
Trichogonia villosa [51]	Leaf	Ethanol	Spodoptera frugiperda	Noctuidae
Vernonia holosenicea [51]	Leaf	Ethanol	Spodoptera frugiperda	Noctuidae
Zanthoxylum limonella [87]	Bark	Ethyl acetate	Spodoptera litrura	Noctuidae
Zea diploperennis [88]	Leaf	Water	Spodoptera frugiperda	Noctuidae

As botanical insecticides are a potential alternative to conventional insecticides [9], the present study was conducted to assess the efficacy of various plant extracts against G. molesta. Among the 91 plant extracts reported in the literature, we could obtain only 32 plant extracts available and measured their acute toxicities against first instar larva and adults of G. molesta. We also evaluated the deterrent effect of these plant extracts on the oviposition of G. molesta females in the laboratory and under semi-field condition.

Materials and methods

Insect rearing procedures

Apples infested with oriental fruit moth were collected and kept in ventilated plastic containers (24.0 L × 17.0 W × 8.0 H cm) at 24.9 ± 0.1°C, 50.2 ± 1.3% RH, and a 16:8 h (L:D) photoperiod in an incubator (DS-11BPL, Dasol Scientific Co. Ltd, Hwaseong, Republic of Korea). When the larvae reached the fifth instar, they emerged from the apple and built their cocoons in the paper towel provided for pupation. Pupae were collected and held in breeding dishes (10.0 D × 4.0 H cm, 310102, SPL, Pocheon, Republic of Korea). When adult moths emerged, they were transferred into ventilated acrylic cylinders (25.5 H × 8.5 D cm), and provided with a piece of cotton soaked in 10% sugar solution as a food source. The acrylic cylinders were kept in a desiccator (36.0 L × 28.0 W × 25.0 H cm) and incubated at 25.6 ± 0.1°C and 91.2 ± 0.1% RH. When moths started to lay eggs on the wall, the cylinder was changed daily to collect freshly laid eggs. Acrylic cylinders bearing eggs on the walls were kept in a separate incubator at 25.6 ± 0.1°C and 91.2 ± 0.1% RH until egg hatch, after which first instar larvae were collected for the experiments or reuse in mass rearing.

Extract preparation

Methanol extracts of test plants were purchased from KPEB (Korea Plant Extract Bank, Cheongju, Republic of Korea) (Table 2). Extraction consisted of extraction, filtering and yield testing, concentration, drying, and storage (http://extract.kribb.re.kr).

Table 2. Thirty-two plant extracts evaluated in this study.

Plants (Reference number)	Extracted part	Family name	Plants (Reference number)	Extracted part	Family name
Gomphrena globosa L. (036–080)	Whole plant	Amaranthaceae	Ginkgo biloba L. (031–069)	Leaf-stem	Ginkgoaceae
Allium cepa L. (034-064)	Whole plant	Amaryllidaceae	Piper Kadzura Ohwi (001–223)	Leaf	Piperaceae
Allium sativum L. (033–033)	Whole plant	Amaryllidaceae	Plantago lanceolata L. (020-084)	Whole plant	Plantaginaceae
Artemisia annua L. (008–007)	Leaf	Amaryllidaceae	Cymbopogon tortilis J. Presl (010–002)	Whole plant	Poaceae
Nerium indicum L. (018–097)	Leaf	Apocynaceae	Delphinium maackianum Regel (012–093)	Whole plant	Ranunculaceae
Chrysanthemum boreale Makino (004–039)	Whole plant	Asteraceae	Hovenia dulcis Thunberg (015–094)	Stem-bark	Rhamnaceae
Chrysanthemum coronarium L. (034–061)	Whole plant	Asteraceae	Citrus unshiu Marc (018-017)	Leaf-stem	Rutaceae
Chrysanthemum indicum L. (011–005)	Whole plant	Asteraceae	Zanthoxylum piperitum (L.) De Candolle (011–088)	Leaf	Rutaceae
Chrysanthemum morifolium Ramat (032–009)	Whole plant	Asteraceae	Sapindus mukorossi Gaertner (021–040)	Leaf-stem	Sapindaceae
Tagetes erecta L. (035-092)	Whole plant	Asteraceae	Capsicum annum L. (026-010)	Leaf-stem	Solanaceae
Humulus japonicus Siebold & Zucc. (008–095)	Leaf-stem	Cannabaceae	Datura metel L. (037-098)	Aerial part	Solanaceae
Cleome spinosa Jacquin (033-098)	Aerial part	Cleomaceae	Nicotiana tabacum L. (036–022)	Leaf-stem	Solanaceae
Citrullus vulgaris Schrader (035–064)	Whole plant	Cucurbitaceae	Alnus japonica Thunberg (003–084)	Leaf	Betulaceae
Momordica charantia L. (034–065)	Whole plant	Cucurbitaceae	Arisaema takeshimense Nakai (001–136)	Leaf	Araceae
Rhododendron micranthum Turcz (003–023)	Leaf-stem	Ericaceae	Xylosma congestum (Lour.) Merrill (001–113)	Leaf	Flacourtiaceae
Ricinus communis L. (018–093)	Leaf	Euphorbeaceae	Acer takeshimense Nakai (001–128)	Leaf	Aceraceae

Laboratory bioassay

Evaluation of single plant extracts

Commercially produced plant extracts were diluted in our laboratory using methanol (99.5%, Daejung Chemicals and Metals Co. Ltd., Siheung, Republic of Korea) to make a 2 mg/ml stock solution. First instar (< 5 h old) larvae and adult male or female moths (3–5 d old) of G. molesta were used in our bioassays. Sex of adults used in bioassays was determined at the pupal stage by confirming the presence of an additional posterior abdominal segment in males [20]. Bioassays consisted of exposure of target life stage to an extract in scintillation glass vials (20 ml), to which 100 μl of each plant extract’s stock solution has been applied and allowed to air-dry, with rotation, for 2.5 h before the assay. This process allowed the methanol to fully evaporate, leaving the plant extract as a residue on the inner surface of the vial, after which five first instar (< 5 h old) larvae or adults were place in each vial. The vials were kept in the desiccators at 25.3 ± 0.03°C and 70.2 ± 0.8% RH for larvae and 25.2 ± 0.02°C and 70.5 ± 0.9% RH for adults in the incubator. Methanol was used as a negative control and the synthetic insecticide λ-cyhalothrin as a positive control. Mortality was observed every 4 and 24 h for larvae and adult, respectively, until death of all insects in the negative control. Bioassays were conducted with 30 larvae and 30 adults per treatment with six replications (5 insects/ replication).

Tests with mixed extracts

The synergistic effects of mixtures of pairs of plant extracts were determined by the co-toxicity coefficient (CTC) method in the laboratory [21, 22]. The mixture of two plant extracts, at a 1:1 ratio and concentration of 2 mg/ml, was applied to larvae and adults of G. molesta. Bioassays were conducted in glass scintillation vials similar to those described in the previous section.

Calculation of co-toxicity coefficients Sun and Johnson [21].

We calculated the co-toxicity coefficients of extract mixtures as per Sun and Johnson [21]: Co-toxicity coefficient (CTC) = (LT₅₀ of toxicant alone / LT₅₀ of toxicant in the mixture) × 100 (CTC = 100, similar action; CTC >100, synergistic action; CTC<100, antagonism).

Greenhouse bioassay

Plant extracts were also evaluated in greenhouse trials. Before the experiment, transparent film (O.H.P film, 210 mm × 297 mm, PP2910, 3M, Seoul, Republic of Korea) was put inside the acrylic cage used for adult moths as an oviposition substrate. Eggs of this film were then collected and used for experiments. After spraying 4 ml of a given plant extract (at a concentration of 2 mg/ml) on each twig of a potted peach tree, 25 eggs were attached to five twigs (5 eggs/twig) for each treatment. Tangle trap (Tanglefoot Company, Grand Rapids, Michigan, USA) was applied at the bottom of the twig to prevent hatched larva from escaping. After 7 d, twig infestation rates were determined.

Assessment of oviposition deterrence in laboratory assay

Oviposition deterrence effects of plant extracts were evaluated in the laboratory. Tests were carried out using peach tree twigs with five leaves each. At first, twigs (length of 10–12 cm) were put in conical flask (250 ml) filled with water to keep the twigs alive for about 7 d. Then, 4 ml of plant extracts were sprayed at a concentration of 2 mg/ml on the twigs, after which twigs were kept for 2.5 h to allow the plant extract to dry or 5 h to allow the positive control of λ-cyhalothrin to dry. Twigs in the conical flask were then placed on plastic trays and covered with ventilated acrylic cylinder cages (25.5 H × 8.5 D cm). Five mated female moths that had begun to lay eggs the previous day, together with five males, were released into each acrylic cylinder cage and held at 25.4 ± 0.1°C, 42.1 ± 0.4% RH, and a 16:8 h (L:D) photoperiod in the growth chamber. We then observed the number of eggs laid on each twig or on the wall of a cage every 24 h for up to five days. The experiments were replicated two times.

Assessment of oviposition deterrence in a greenhouse assay

The oviposition deterrence of plant extracts was also evaluated under greenhouse conditions. Four ml of each plant extract were sprayed onto potted peach plants at a concentration of 2 mg/ml and plants were then allowed to dry for 2.5 h. After fully drying, plants were covered with a pipe framed cage (47.0 L × 47.0 W × 115.0 L cm) screened with white-colored nylon fabric Then five female moths (mated and started oviposition one day before) and five males were released inside the cage. We then observed the number of eggs laid on each twig or on the wall of a cage every 24 h for up to five days. The experiments were replicated two times.

HPLC analysis

Instrumentation

An Agillent 1200 series (Agilent, Santa Clara, CA) HPLC system was equipped with bin pump (G1312A), degasser (G13796), column oven (250 × 4.6 mm and 5 μm particle size, Agilent, Santa Clara, CA), and diode array detector (G1315B). Agilent ChemStation software was used for data acquisition and system suitability calculations.

Chromatographic parameters

Reverse phase high performance liquid chromatography (RP-HPLC) was used for the analysis for N. tabacum and A. sativum extract according to the method described by Tanbwekar et al. [23] with a minor modification. In our study, column temperature was used at 25°C instead of 35°C. Column was used with flow rate of 1 ml/minute. Diode array detector in range of 200–800 nm was used for determining peak purity. Injection volume was 20 μl where phosphate buffer (pH 6.8; 10nm) with methanol (35.65% v/v) was used as mobile phase.

Statistical analysis

Larval mortality data were corrected using Abbott’s formula [24] and then were used to calculate the lethal median time (LT₅₀) using SAS 9.4 software [25]. Infestation of twigs in greenhouse and number of eggs laid on substrates in the oviposition deterrence experiment in the laboratory were analyzed using a Chi-square test with a post-hoc multiple comparison test analogous to Tukey’s test [26].

In the oviposition deterrence experiment in the greenhouse, the number of eggs was analyzed using single factor analysis of variance (ANOVA) and differences in the mean number of eggs were determined by Tukey’s test using Proc MIXED of SAS 9.4 [25]. Before analysis, normality and homogeneity were tested using a Kolmogorov-Smirnov test (P = 0.150) and a Levene test (P = 0.442).

Results

Laboratory bioassay

Evaluation of single plant extracts

Among the 32 plant extracts tested, Nicotiana tabacum L., Allium sativum L., and Zanthoxylum piperitum (L.) De Candolle showed the highest mortality on first instar larva (Table 3). The LT₅₀ values of N. tabacum, A. sativum, and Z. piperitum were 12.9 h (χ² = 9.99, df = 4, P = 0.041), 15.6 h (χ² = 4.02, df = 4, P = 0.403), and 16.1 h (χ² = 17.02, df = 4, P = 0.002), respectively. The LT₅₀ value of Sapindus mukorossi Gaertner was 17.5 h (χ² = 10.04, df = 5, P = 0.074), which was significantly higher than N. tabacum or A. sativum. Nicotiana tabacum showed highest corrected mortality of 92.0% followed by A. sativum (88.0%), Z. piperitum (70.4%), and S. mukorossi (65.2%) within 20 h (Fig 1). For the positive control, λ-cyhalothrin, 100% corrected mortality was found within 12 hours. On the basis of the LT₅₀ value, N. tabacum, A. sativum, Z. piperitum, and S. mukorossi were chosen as the four most effective plant extracts against first instar larvae of G. molesta, and these extracts were further evaluated in subsequent experiments.

Table 3. Statistical comparison of methanolic plant extracts (200μg/vial) against the 1st instar larva of Grapholita molesta by scintillation glass vial assay.

Treatment	LT50	95% C.I	Slope ± SE	χ2 (df)
λ-cyhalothrin	5.32a	4.92–5.72	6.21 ± 0.58	2.35
Nicotiana tabacum	12.92b	11.57–14.14	9.07 ± 1.09	9.99 (4)
Allium sativum	15.57c	15.03–16.09	11.16 ± 0.88	4.02 (4)
Zanthoxylum piperitum	16.09bcd	14.07–18.15	8.57 ± 1.40	17.02 (4)
Sapindus mukorossi	17.48d	16.32–18.62	9.74 ± 0.98	10.04 (5)
Tagetes erecta	17.95de	17.29–18.59	8.91 ± 0.64	8.24 (5)
Allium cepa	18.52de	17.94–19.09	11.30 ± 0.83	5.51 (5)
Citrullus vulgaris	18.70de	18.12–19.26	14.91 ± 1.15	6.52 (5)
Cymbopogon tortilis	19.07de	17.08–21.21	7.94 ± 1.19	20.49 (5)
Capsicum annum	19.09de	18.49–19.69	10.87 ± 0.80	8.16 (5)
Alnus japonica	19.09de	17.53–20.71	8.73 ± 1.09	14.41 (5)
Ricinus communis	19.36de	18.61–20.09	7.50 ± 0.50	8.66 (6)
Gomphrena globosa	19.50de	17.61–21.47	10.14 ± 1.61	23.04 (5)
Ginkgo biloba	19.78de	18.19–21.37	11.59 ± 1.65	18.63 (5)
Momordica charantia	20.55e	18.86–22.31	11.76 ± 1.84	20.45 (5)
Plantago lanceolata	20.90e	20.36–21.44	14.56 ± 1.15	6.25 (5)
Piper Kadzura	21.35e	19.87–22.91	13.38 ± 1.98	17.72 (5)
Cleome spinosa	21.50de	16.50–35.96	12.04 ± 4.16	103.07 (5)
Arisaema takeshimense	21.51de	17.56–27.97	9.16 ± 2.52	64.14 (5)
Delphinium maackianum	21.69e	20.15–23.28	9.16 ± 1.07	17.54 (6)
Chrysanthemum indicum	21.87e	19.05–25.61	10.72 ± 2.52	42.42 (5)
Chrysanthemum coronarium	22.25de	17.38–34.55	8.92 ± 2.84	80.31 (5)
Artemisia annua	22.67e	20.31–25.25	9.42 ± 1.64	37.51 (6)
Datura metel	22.77e	20.29–25.93	13.98 ± 3.27	41.16 (5)
Citrus unshiu	22.86e	21.39–24.36	12.84 ± 1.72	21.27 (6)
Xylosma congestum	23.09e	20.87–25.61	8.68 ± 1.39	30.74 (6)
Chrysanthemum boreale	23.17e	16.79–32.93	18.71 ± 6.76	94.56 (5)
Hovenia dulcis	24.02e	22.09–26.08	12.30 ± 2.03	31.59 (6)
Nerium indicum	24.15e	23.61–24.69	16.47 ± 1.28	4.80 (6)
Humulus japonicus	24.48e	22.91–26.28	27.58 ± 6.45	29.28 (5)
Acer takeshimense	25.02e	23.65–26.45	13.93 ± 1.88	18.30 (6)
Rhododendron micranthuma	-	-	-	-
Chrysanthemum morifoliuma	-	-	-	-

LT₅₀ values followed by different lower case letters are significantly different among treatments

^aLarvae died faster than control, so LT₅₀ was not calculated

Fig 1. Efficacy of different plant extracts against Grapholita molesta 1st instar larvae over time.

In the adult assay, 100 and 96.7% of adult males survived 24 and 48 h, respectively, but only 30.0% of adult males survived 144 h when held in vials treated with N. tabacum (Fig 2). Allium sativum and N. tabacum both caused higher mortality than S. mukorossi and methanol on adult males with LT₅₀ values of 107.5 (χ² = 3.08, df = 6, P = 0.799) and 109.9 h (χ² = 7.46, df = 5, P = 0.189), respectively (Table 4). In case of adult females, N. tabacum and A. sativum were also significantly more effective than other plant extracts, with LT₅₀ values of 131.9 (χ² = 14.39, df = 6, P = 0.026) and 158.3 h (χ² = 5.96, df = 7, P = 0.544), respectively (Table 4). Irrespective of treatments, adult male G. molesta adult died faster than females (Fig 2).

Fig 2. Survivorship of adult male and female of Grapholita molesta after exposure to single applications of plant extracts.

Table 4. Statistical comparison of tested methanolic plant extracts against adult Grapholita molesta.

Tested on	Treatment	LT50	95% C.I.	Slope ± SE	χ2 (df)
Male	λ-cyhalothrin	57.01a	53.11–61.29	14.90 ± 2.53	0.01 (2)
	Allium sativum	107.49b	99.15–115.55	7.03 ± 0.83	3.08 (6)
	Nicotiana tabacum	109.96bc	101.17–119.41	6.43 ± 0.85	7.46 (5)
	Zanthoxylum piperitum	126.35cd	116.72–135.85	6.05 ± 0.63	5.85 (8)
	Sapindus mukorossi	137.66de	130.26–144.81	10.99 ± 1.33	2.96 (7)
	Methanol	174.73f	166.86–182.33	12.28 ± 1.39	3.17 (9)
Female	λ-cyhalothrin	88.80a	81.91–95.44	8.53 ± 1.20	3.77 (4)
	Nicotiana tabacum	131.93b	115.23–150.72	8.63 ± 1.65	14.39 (6)
	Allium sativum	158.34bc	150.23–166.77	10.44 ± 1.33	5.96 (7)
	Sapindus mukorossi	201.46d	193.66–209.54	13.87 ± 1.65	7.67 (9)
	Zanthoxylum piperitum	209.58de	201.74–217.78	14.49 ± 1.83	5.33 (9)
	Methanol	215.49ef	207.77–223.23	15.15 ± 1.76	6.66 (10)

LT₅₀ values followed by different letters are significantly different among treatment.

Evaluation of mixed extracts

We also evaluated the effect of mixtures of plant extracts on first instar larvae (< 5 h old) and on both male and female adults (< 5 d old) of G. molesta. The first instar larvae of G. molesta died faster when treated with the mixture of N. tabacum+Z. piperitum, with corrected mortality of 90.5% at 20 h after treatment (Fig 3). The LT₅₀ value of the mixture of N. tabacum+Z. piperitum was 14.3 h (χ² = 11.32, df = 4, P = 0.023), but the co-toxicity coefficient value was 90.5 indicating that there was no synergistic effect of the mixture of N. tabacum+Z. piperitum. The lethal median time (LT₅₀) was 76.7 h (χ² = 2.87, df = 4, P = 0.579) for adult males, significantly different from the mixture of N. tabacum+A. sativum (Table 5) in which all adults died within 144 h (Fig 4). The co-toxicity coefficient value of N. tabacum+A. sativum was 140.1, indicating a synergistic effect of the mixture of these two extracts. However, in case of adult females, the LT₅₀ value was not significantly different between the mixture of N. tabacum+A. sativum and the mixture of A. sativum+S. mukorossi (Table 5). The mixture of N. tabacum+A. sativum showed 100% mortality within 144 h (Fig 4). The co-toxicity coefficient value of N. tabacum+A. sativum mixture was 107.5, indicating a synergistic effect of the mixture (Table 5), but, from the C. I. value, the mixture of N. tabacum+A. sativum was not significantly different from the single extract of N. tabacum. Here, we also found that adult males died faster than adult females in mixed extract treatment. From the above results, the mixture of N. tabacum+A. sativum would be the best choice for use against adult males, but the mixture of N. tabacum+A. sativum and N. tabacum by itself were both equally lethal to adult females.

Fig 3. Corrected mortality (%) of combinations of plant extracts against first instar larvae of Grapholita molesta.

Table 5. Statistical comparison of tested methanolic plant extracts (mixture) against Grapholita molesta.

Tested on	Treatment	LT50	95% C.I.	Slope ± SE	χ2 (df)	Co-toxicity coefficient
Larvaa, first instar	λ-cyhalothrin	5.32a	4.92–5.72	6.21 ± 0.58	2.35	-
	N. tabacum+Z. piperitum	14.27b	12.78–15.65	9.03 ± 1.17	11.32(4)	90.54
	N. tabacum+A. sativum	18.20c	16.52–19.90	8.31 ± 1.08	16.26(5)	70.99
	A. sativum+Z. piperitum	18.04c	17.47–18.60	11.40 ± 0.84	2.51(5)	86.31
	N. tabacum+S. mukorossi	18.99cd	17.83–20.10	12.44 ± 1.44	11.38(5)	68.04
	A. sativum+S. mukorossi	21.80cde	19.81–24.05	12.95 ± 2.45	28.37(5)	71.42
	Z. piperitum+S. mukorossi	21.65cdef	18.56–25.79	9.65 ± 2.34	43.92(5)	74.32
Adult, male	λ-cyhalothrin	54.87a	48.10–60.78	7.97 ± 1.54	1.67 (2)	-
	N. tabacum+A. sativum	76.70b	68.37–84.76	6.19 ± 0.91	2.87 (4)	140.14
	A. sativum+S. mukorossi	94.48c	86.63–101.98	8.35 ± 1.17	3.04 (5)	113.77
	N. tabacum+Z. piperitum	100.13cd	91.88–108.11	8.06 ± 1.14	1.84 (5)	109.82
	N. tabacum+S. mukorossi	122.87e	115.50–129.94	12.15 ± 1.72	0.74 (6)	89.49
	Z. piperitum+A. sativum	123.65e	114.92–132.41	8.58 ± 1.15	2.86 (6)	86.93
	Z. piperitum+S. mukorossi	135.43ef	127.90–142.82	12.69 ± 1.82	1.28 (6)	93.30
	Methanol	170.30g	161.87–178.65	12.45 ± 1.66	6.74 (8)	-
Adult, female	λ-cyhalothrin	86.03a	78.37–93.50	8.01 ± 1.18	1.79 (4)	-
	N. tabacum+A. sativum	122.69b	112.66–132.71	6.51 ± 0.80	6.51 (7)	107.53
	A. sativum+S. mukorossi	140.15bc	131.65–148.40	10.36 ± 1.36	1.71 (7)	112.98
	Z. piperitum+A. sativum	156.65cd	147.49–165.68	9.85 ± 1.21	3.55 (8)	101.08
	N. tabacum+Z. piperitum	175.50e	166.48–184.81	11.21 ± 1.46	4.49 (8)	75.17
	N. tabacum+S. mukorossi	187.83ef	178.40–197.59	10.81 ± 1.31	9.03 (9)	70.24
	Z. piperitum+S. mukorossi	231.07h	223.06–239.38	18.18 ± 2.40	7.66 (10)	90.70
	Methanol	200.61fg	191.94–209.00	13.99 ± 1.72	2.74 (10)	-

LT₅₀ values followed by different letters are significantly different among treatment

^aThe LT₅₀ value was calculated using corrected mortality

Fig 4. Survivorship of adult male and female of Grapholita molesta on mixed application of plant extracts.

Greenhouse bioassay

In the greenhouse bioassay, infestation levels of twigs were significantly reduced when twigs were sprayed with either N. tabacum or A. sativum (χ² = 30.74, df = 5, P < 0.001) compared to the negative control (Table 6). However, we found no significant differences among the plant extracts (χ² = 7.19, df = 3, P = 0.066).

Table 6. Efficacy evaluation of plant extracts on infestation rate of peach twigs in greenhouse.

Treatment	Hatchability (%)	Infestation rate
λ-cyhalothrin	88.0	0.09 (2/22)d
Nicotiana tabacum	88.0	0.27 (6/22)cd
Allium Sativum	84.0	0.38 (8/21)bdc
Zanthoxylum piperitum	88.0	0.45 (10/22)abcd
Sapindus mukorssi	84.0	0.67 (14/21)abc
Control	88.0	0.82 (18/22)a

Means within a column with different letters differ significantly (P < 0.05)

Oviposition deterrence in the laboratory

From the above experiments we found that N. tabacum, A. sativum, and the mixture of N. tabacum+A. sativum provided the best control of adult G. molesta, so, these treatments were compared in an oviposition deterrence test in the laboratory. Mated females laid only 29 eggs on the leaves treated with N. tabacum, significantly fewer than all other plant extracts, and an 85% reduction compared to the methanol control (χ² = 236.50, df = 4, P < 0.001) (Table 7). We found N. tabacum to be very effective in reducing oviposition, at levels similar to those provided by λ-cyhalothrin, for up to three days (Fig 5).

Table 7. Deterrent effect of plant extract on oviposition of G. molesta in laboratory.

Treatment	Total no. of eggs produced	% eggs on wall	% eggs on leaves
λ-cyhalothrin	267	97.75a	2.25a
Nicotiana tabacum	312	90.71b	9.29b
N. tabacum+A. sativum	319	67.71c	32.29c
Allium sativum	377	64.99c	35.01c
methanol	396	51.77d	48.23d

Means within a column with different letters differ significantly (P < 0.05)

Fig 5. Daily egg laying on cage walls and leaves up to five days.

Oviposition deterrence the greenhouse

The number of eggs laid by adult mated females was significantly lower for all plant extracts compared to the negative control (F = 9.82, df = 4, 9, P = 0.014), and the percentage of leaves with eggs and the total number of eggs laid were reduced in the N. tabacum treatment by 71 and 90%, respectively, compared to the methanol control (Table 8).

Table 8. Deterrent effect of plant extract on oviposition of G. molesta on greenhouse.

Treatment	No. of leaves/twig	Percent of twigs of which leaves with egg	Percent of leaves with egg	Total no. of eggs reproduced
λ-cyhalothtrin	9.36 (103/11)	0.00 (0/11)a	0.00 (0/103)a	0c
Nicotiana tabacum	8.56 (94/11)	36.36 (4/11)ab	8.51 (8/94)b	18b
Allium sativum	6.79 (95/14)	57.14 (8/14)b	15.79 (15/95)b	28b
N. tabacum+A. sativum	9.00 (117/13)	69.23 (9/13)b	19.67 (23/117)bc	42b
methanol	7.15 (93/13)	46.15 (6/13)b	29.03 (27/93)c	184a

Means within a column with different letters differ significantly (P < 0.05)

HPLC analysis

Nicotine the major compound of N. tabacum appeared 56.3% at RT 2.42 min with two unidentified minor compounds at RT 2.83 min (27.01%) and 3.77 min (10.13%) (Fig 6). From A. sativum, the major compound allicin appeared 100% at RT 3.19 min (Fig 7).

Fig 6. HPLC of methanol extract of Nicotiana tabacum.

Fig 7. HPLC of methanol extract of Allium sativum.

Discussion

The synthetic pesticide λ-cyhalothrin was more toxic than any of plant extracts to first instar larvae. Based on the comparison of plant extract LT₅₀ values to that of λ-cyhalothrin, we selected N. tabacum, A. sativum, Z. piperitum, and S. mukorossi as the most effective botanical extracts for control of first instar larvae of G. molesta. Although the highest mortality was observed in larval stage of G. molesta from N. tabacum treatment, for both adult males and females N. tabacum and A. sativum were equally effective in a subsequent assay. Nicotiana tabacum has several modes of action. It can be a nerve poison [27, 28], stomach poison, or repellent [29]. Baskaran and Narayanasamy [29] found N. tabacum to be effective against aphids, thrips, psyllids, tingids, beetles, sawflies, and lepidopterans. Evaluation of N. tabacum against G. molesta has been made here for the first time. In addition, N. tabacum is easy to apply in the field. Amoabeng et al. [16] ground N. tabacum leaves in tap water containing 0.1% Sunlight^® detergent solution and sieved them through fine linen for immediate application to a cabbage field. This preparation resulted in 93.0% reduction of Plutella xylostella larvae, while λ-cyhalothrin reduced the same population by only 51.0%. The best efficacy was recorded with the extract of N. tabacum against Cydia molesta Busch. (98.3%) and Anarsia lineatella Zell. (99.0%) [30]. Vandenborre et al. [27] found that a jasmonate-inducible lectin named NICTABA present in tobacco leaf is responsible for the larval mortality of lepidopteran insects. Nevertheless, a major active compound of N. tabacum was nicotine, which mimics acetylcholine and activates the nicotinic acetylcholine receptor causing an influx of sodium ions to flood the receptor [28]. Methanolic extracts of A. sativum have also caused mortality of 81.0% against Spodoptera litura [31]. A constituent of the A. sativum extract, alliin (derived from the amino acid cysteine) is converted by an enzyme to allicin, which is believed to act as an antifeedant, repellent, and insecticide [32].

We did not find any synergistic effects of N. tabacum and Z. piperitum on first instar larvae of G. molesta. However, the mixture of N. tabacum+A. sativum showed synergistic effects on adult males. The reason for this difference in the effectiveness of the mixture of N. tabacum+A. sativum between larvae and adults is unknown, but might be caused by differences in physiological structure. Similarly, Derbalah [33], who found that an extract of Bauhinia purpurea L. showed 83 and 80% mortality on adult and pupal stages of Trogoderma granarium Everts, respectively, but only 33.0% mortality on the larval stage. Interestingly, extracts of Caesalpinia gilliesii (Hook.) showed lower mortality on adult and pupal stages (43.0 and 43.0%, respectively), than on larvae (80%).

We found no synergistic effect of N. tabacum and Z. piperitum on the first instar larvae of G. molesta, and similarly Noosidum et al. [34] found no synergistic effect of the mixture of Litsea salicifolia Roxb. (0.1%) and Melaleuca leucadendron L. (0.3%) against adult females of Aedes aegypti (L.). However, the synergistic effects of mixtures of plant extracts have been reported in other studies. Alim et al. [35] found that a mixture of neem plus crown flower at a 1:1 ratio showed synergistic effects on Aleurodicus dispersus adults. Zibaee and Khorram [36] also found that essential oils of Eucalyptus globulus Labill. and Rosmarinus officinalis L. showed synergistic effects on Blattella germanica L.

Nicotiana tabacum extract was effective in deterring oviposition in both laboratory and greenhouse assays, which suggests it would be effective at reducing G. molesta populations in the field. Similarly, Amoabeng et al. [16] found that N. tabacum extract reduced 93.0% of a Plutella xylostella population in a cabbage field. In other work in Uganda, a crude extract of N. tabacum showed similar effectiveness to the synthetic insecticides against a bruchid beetle (Callosobruchus sp.) [37]. Nevertheless, plant extracts can be harmful to other beneficials: N. tabacum found to be harmful on Coccinella magnifica Redtenbacher and Episyrphus balteatus De Geer compared to tap water but less harmful than synthetic insecticides [16].

In conclusion, among the 32 tested plant extracts, N. tabacum extract showed highest toxicity against the first instar and adult of G. molesta, and oviposition was greatly reduced after the spray in both laboratory and greenhouse. Nevertheless, formulation should be improved as methanolic extracts in this study is not appropriate for organic farming. Based on these results, we are suggesting that the extract of N. tabacum can be a good botanical insecticide against G. molesta.

Supporting information

S1 File. Test of single plant extract on larva, test of single plant extract on adult, test of combination of extracts on larva, test of combination of extracts on adult, Greenhouse evaluation of plant extracts, Oviposition deterrency in laboratory, Oviposition deterrency in greenhouse.

(XLSX)

Data Availability

All relevant data are within the paper and its supporting Information files.

Funding Statement

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (IPET) through the Advanced Production Technology Development Program, funded by Ministry of Agriculture, Food, and Rural Affairs (MAFRA) (315007-03-2-HD050) to UTL.