Garlic, a widely cultivated global vegetable crop, is threatened by the underground pest Bradysia odoriphaga in China. Previous reports indicated that garlic essential oil, of which the dominant components are sulfides or thiosulfinates, exhibits insecticidal activity against pests. However, it is unclear whether the resistance of garlic to B. odoriphaga is related to thiosulfinates. Here, we compared the resistance of 10 garlic cultivars at various growth stages to B. odoriphaga by field investigation and indoor life-table data collection. Furthermore, the relationship between thiosulfinates content and resistance, as well as the toxicity of garlic oil and allicin against B. odoriphaga larvae was determined. Field surveys demonstrated that the garlic cultivars Qixian and Cangshan possessed the highest resistance, while Siliuban and Yishui were the most sensitive. When reared on Qixian, B. odoriphaga larval survival and fecundity declined by 26.2% and 17.7% respectively, but the development time was prolonged by 2.8 d compared with Siliuban. A positive correlation was detected between thiosulfinates content and resistance. Furthermore, garlic oil and allicin exhibited strong insecticidal activity. We screened out 2 pest-resistant cultivars, for which thiosulfinate content was highest. Additionally, the insecticidal activity displayed by sulfides and allcin suggests their potential for exploitation as botanical insecticides.
Plant resistance to insects plays an important role in the long-term coevolution of insect phytophagy1. It relies on physical barriers, chemical defenses and induced direct or indirect plant defenses2. Phytochemicals play a vital role in the chemical defense against herbivores, which causes adverse impacts on the development and fecundity of insects alone or synergistically3. Recently, various phytochemicals such as glucosinolates, cyanogenic glucosides, alkaloids, phenolics and proteinase inhibitors have been proven to play obvious roles in plant resistance to insects4, 5. Extensive research on plant resistance, especially with regards to phytochemicals, provides new impetus for Integrated Pest Management (IPM)6.
Garlic, Allium sativum (Liliaceae: Allium), is an important commercial crop widely grown around the world, especially in Asia and North Africa7, and China is the largest global exporter of garlic8. It is also one of the most commonly used ingredients as a flavor enhancer for sausage. Additionally, garlic is appreciated for its medicinal properties, and various garlic preparations are widely used as health care products9,10,11. A number of studies showed that sulfur-containing organic compounds, such as thiosulfinates, played important role in these function12. The most important active substance, allicin (2-propene-1-sulfinothioic acid S-2-propenyl ester), formed during lysis of alliin by alliinase, as antibacterial, antiviral, antifungal and antiprotozoal properties, and also has beneficial effects on the cardiovascular and immune systems12,13,14,15. Clinical trials confirmed that the allicin extracted from garlic can inhibit the proliferation of cultured cancer cells15. In addition, allicin has been documented to reduce Campylobacter jejuni colonization in broilers when added to the drinking water16. Allicin was even found to be capable of inhibiting DNA and protein synthesis in Salmonella enterica and had an immediate effect on RNA synthesis17. More research is needed to determine the efficacy of allicin in various industrial areas.
Root maggot is a devastating soil pest that feeds on garlic bulbs. In China, where the technology of film mulched planting has been promoted, the chive maggot Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae) has become the most significant pest, replacing the onion fly Delia antiqua, which was the primary root maggot pest in the past18. Bradysia odoriphaga feeds on 7 plant families and more than 30 plants species, especially Chinese chive (Allium tuberosum.), garlic (Allium sativum), welsh onion (Allium fistulosum) and onion (Allium cepa)19, 20. Bradysia odoriphaga larvae tend to aggregate in fields and directly damage plants by feeding on the root and corm tissues in the growth medium, resulting in weak or withered plants. Since the larvae primarily damage the underground portions of plants, they are difficult to prevent or control. One of the most prevalent management practices against B. odoriphaga is the application of insecticides, such as organophosphates, carbamates and neonicotinoids21. However, insecticide use is increasingly restricted due to environmental pollution and human health concerns, as well as the development of pesticide resistance22. Therefore, breeding insect-resistant cultivars and seeking environmentally friendly insecticides are important channels for the effective control of pests. China is the largest cultivator of garlic and harbors abundant germplasms, including more than 370 cultivars23, providing the invaluable resources required for the exploitation and utilization of cultivars.
Previous research found that the development of B. odoriphaga was adversely affected when reared on garlic compared with Chinese chives; the most favorable host24, 25. A component analysis of the ethanol extract of liliaceous hosts showed that sulfides, the major components of the pungent odor of garlic11, 26, play an important role in host plant resistance to B. odoriphaga 24. Some reports confirmed that garlic extracts exhibited insecticidal activity against storage pests, such as Tribolium castaneum (Herbst)27 and Sitotroga cerealella (Lepidoptera: Gelechiidae)28. Diallyl disulfide and diallyl trisulfide, the stable polysulphide breakdown sulfides of allicin (an unstable thiosulfinate), are two major components in garlic essential oil29,30,31. Some studies investigated that thiosulfinates contents differed among garlic cultivars resulted the different sulphide in garlic32. However, whether the resistance of garlic cultivars to B. odoriphaga differs and could be related to thiosulfinates, or whether sulfides exhibit insecticidal activity against B. odoriphaga, has scarcely been studied.
In the current study, we investigated the differences in pest-resistance among garlic cultivars by field investigation. Then, we demonstrated life table for B. odoriphaga reared on 10 garlic cultivars using the age-stage, two-sex life table method, which is able to portray the actual dynamics of the population more accurately than the traditional life table by comprehensively analyzing the information between different developmental stages and different sexes33, 34. This work also investigated the correlation between garlic cultivar resistance to the root maggot and thiosulfinates content in garlic. Additionally, the toxicity of garlic essential and allicin against B. odoriphaga were determined.
Quantity of B. odoriphaga occurring in the fields of different garlic cultivars
We recorded the degree of damage to the garlic bulbs caused by B. odoriphaga larvae for the different cultivars at the mature stage (May 20th, 2015). Figure 1 presents the significant differences in B. odoriphaga damage among the 10 cultivars (n = 50; df = 9, 40; F = 8.265; P < 0.001). The larval quantity on Siliuban and Yishui garlic cultivars was 1,302 and 1,194 larvae per 100 garlic plants respectively, constituting the most susceptible cultivars for root maggots. By contrast, the quantities of root maggots on Qixian and Cangshan cultivars were 558 and 684 larvae per 100 garlic plants respectively, constituting the most insect-resistant cultivars. The resistance level of the remaining cultivars was in between, with the quantity of root maggots recorded ranging between 774 and 1,002 larvae per 100 garlic plants.
Life table of B. odoriphaga reared on 10 cultivars garlic at the mature stage
Development and reproduction
Bradysia odoriphaga reared on the 10 garlic cultivars successfully reached the adult stage from eggs. Development, survival and fecundity parameters of B. odoriphaga reared on different garlic cultivars were noticeably different (Table 1). Obvious overlaps were observed in these curves (Fig. 2), showing the different development rates among individuals. The development time of larval stages for B. odoriphaga reared on the 10 cultivars was the most significantly different variable during the generation period. Particularly, we found that B. odoriphaga larvae grew slower when reared on the Qixian cultivar than on the others, and the larval developmental time was 22.29 d. The larval development times when reared on the Yishui (18.78 d), Zajiao (19.29 d), Sichuan (19.43 d), Nanfang (19.52 d), Zhongmu (19.84 d), Jinxiang (20.60 d) and Cangshan (21.03 d) cultivars were shorter than when reared on the Qixian cultivar. Additionally, the larval development time when reared on Siliuban cultivar was dramatically shorter than any of the others (18.71 d). There were no differences in the development times of the egg and pupa stages. The development time of eggs ranged between 3.25 d and 3.42 d across all 10 garlic cultivars, while the pupal stage was between 3.36 d and 3.49 d.
Differences in fecundity and adult longevity were observed across the different cultivars (Table 1). The mean fecundity of females was 93.71 eggs on Qixian cultivar, which was significantly lower than the other cultivars. The mean fecundity when reared on Cangshan cultivar was 96.27 eggs; the second lowest. Those reared on Yishui and Siliuban cultivars exhibited higher fecundity than on the other cultivars, with 113.81 and 118.67 eggs respectively. The fecundity when reared on other cultivars ranged between 102.59 and 110.58 eggs. With regards to Qixian cultivar, the adult longevity was observed to be the shortest at 3.62 d (females) and 3.96 d (males) respectively. Conversely, the longest adult longevity was observed on Yishui (3.93 d, females) and Nanfang cultivars (4.39 d, males). Male adults emerged earlier than female adults and had longer lifespans.
The s xj curves indicate the probability that a newly hatched egg will survive to age x and develop to stage j (Fig. 2). The occurrence of death at the larval stage was an important factor causing the mortality differences of B. odoriphaga reared on different garlic cultivars (Table 1). Figure 2 suggests that the young larval stage is critical, being associated with a rapidly decreasing survival rate. The larval mortality of B. odoriphaga reared on Qixian cultivar was higher 56.06%, which was higher in comparison with those reared on the Cangshan, Nanfang, Xinxiang, Zajiao, Sichuan and Jinxiang cultivars (47.37%, 44.27%, 43.51%, 42.75% and 40.46% respectively). The mortality when reared on Zhongmu garlic and Yishui cultivars was lower at 32.06% and 30.30% respectively. The mortality when reared on Siliuban cultivar was the lowest at 29.77%. There were no significant differences between egg and pupal stage mortality (Supplementary Tables S1).
The population parameters were calculated using the bootstrap method presented in Table 2. The intrinsic rate of increase (r), the finite rate of increase (λ) and the net reproductive rate (R0) of B. ordoriphaga reared on Qixian and Cangshan cultivars were 0.082 d−1, 1.085 d−1, 14.91 d eggs and 0.097 d−1, 1.102 d−1, 21.24 d eggs respectively; significantly lower than the values obtained from other garlic cultivars. However, the values obtained from Siliuban (0.128, 1.137 and 41.67) and Yishui (0.124, 0.132 and 37.74) cultivars were higher than the others. Similarly, the mean generation time (T) obtained from Siliuban and Yishui cultivar (29.05 and 29.19 d) was shorter than that of Qixian cultivar (32.95 d). The population parameters of B. ordoriphaga reared on other cultivars were middle-ranked, such as an intrinsic rate of increase r = 0.109–0.118, finite rate of increase λ = 1.115–1.125 and the net reproductive rate R0 = 27.85–35.03.
Survival, fecundity and developmental time of B. ordoriphaga reared on garlic at different growth stages
As for the larvae reared at different growth stages, the larval development time, fecundity and larval mortality of B. ordoriphaga differed significantly (Fig. 3). For example, for B. ordoriphaga reared on the Qixian cultivar we found that the larval development time at the mature stage (22.29 d) was dramatically longer than the seeding stage (20.02 days; P < 0.001) and growth stage (19.21d, P < 0.001), while the seeding and growth stage were significantly different (P < 0.001). The larval mortality at the mature stage (56.06%) was higher than at the seeding stage (38.21%; P < 0.001) and growth stage (29.30%; P < 0.001). The fecundity at the mature stage was 93.71 eggs for each female, which was significantly lower than the seeding stage (106.73 eggs for each female; P = 0.016) and growth stage (109.21 eggs for each female; P = 0.021). B. odoriphaga had a more adverse reaction when reared on the Qixian cultivar at the mature stage, followed by the seeding stage and growth stage, followed by the seeding stage and growth stage. These parameters showed a similar trend when B. odoriphaga was reared on the Cangshan cultivar.
Correlation between thiosulfinates and insect resistance
The garlic were significantly different among the cultivars at the mature stage, and varied widely between the growth stages (Table 3). At the mature stage, the thiosulfinates contents in the Qixian, Cangshan and Nanfang cultivars were relatively high, at 30.95, 27.32 and 25.42 μmol/g respectively. Conversely, those in the Xinxiang, Sichuan, Zajiao, Jinxiang and Zhongmu cultivars were between 19.54 and 23.67 μmol/g, constituting in between values. However, the thiosulfinates contents in Yishui and Siliuban garlic were observed at a minimum level, at 18.27 and 17.90 μmol/g respectively. In addition, the thiosulfinates content at the mature stage in Qixian garlic was highest (30.95 μmol/g), followed by the seeding stage (23.75 μmol/g), while the lowest was recorded at the growth stage (19.90 μmol/g). Thiosulfinates contents in the other cultivars showed a trend similar to the Qixian cultivar, being the highest at the mature stage, at moderate levels in the seeding stage and the lowest in the growth stage, with the exception of the Sichuan, Zhongmu, Yishui and Siliuban varieties. The thiosulfinates contents of these 4 garlic cultivars exhibited no differences among the growth stages.
Correlation between thiosulfinates and insect resistance
We analyzed the correlation between thiosulfinates content and the resistance performance to B. ordoriphaga (larval development time, fecundity and larval mortality) using linear-regression analysis. We found that thiosulfinates contents were significantly correlated with the insect-resistance of garlic to B. ordoriphaga (Fig. 4). Larval development time (df = 1, 28, F = 71.680, P < 0.001) and larval mortality (df = 1, 28; F = 139.135, P < 0.001) were positively linearly correlated with thiosulfinates content, while fecundity (df = 1, 28, F = 88.695, P < 0.001) showed a clear negative linear correlation. In other words, when thiosulfinates contents increased, larval development time was prolonged and the mortality increased, but fecundity decreased.
Toxicity of synthetic garlic oil, allicin and 4 insecticides to the fourth instar of B. odoriphaga
The toxicity of the tested insecticides against the fourth instar developmental stage of B. odoriphaga is provided in Table 4. In terms of rapid effect (treated for 48 h), the botanical insecticides (synthetic garlic oil and allicin) showed unremarkable insecticidal activity when compared with Phoxim (LC50 = 1.751 mg/L) and Emamectin benzoate (LC50 = 40.594 mg/L). Synthetic garlic oil (LC50 = 65.574 mg/L) showed stronger insecticidal activity. In terms of chronic effects (treated for 96 h), synthetic garlic oil (LC50 = 24.706 mg/L) showed the most potent insecticidal activity, followed by allicin (LC50 = 50.372 mg/L). The toxicities of the garlic compounds were all determined to stronger than Sophocarpidine (LC50 = 86.925 mg/L) and Azadirachtin (LC50 = 72.079 mg/L).
Three-to-four generations of B. odoriphaga per year are produced in the garlic fields of Shandong province in China. April to June is suitable for its growth, resulting in severe damage during the mature stage of garlic development18. In this study, the resistance of 10 garlic cultivars to B. odoriphaga was investigated for the first time. The result showed that the Qixian and Cangshan garlic cultivars exhibited the highest resistance to B. odoriphaga, while Yishui and Siliuban were the most susceptible (Fig. 1). Wang23 also reported that the Zhongmu, Cangshan and Jinxiang cultivars showed obvious resistance to Delia antiqua, however, we found that Zhongmu garlic was sensitive to B. odoriphaga. These differences may be due to regional variations or difference in planting conditions.
In general, the stronger the resistance of plant to a pest is, the longer the developmental duration of the insect lasts, resulting in lower survival and fecundity35. Previous research has indicated that although B. odoriphaga possesses a wide range of hosts, the different hosts exert various effects on B. odoriphaga 24, 25. Xue24 found that the survival and fecundity of B. odoriphaga declined when reared on garlic compared with Chinese chives and other liliaceous vegetables. In this study, our results revealed that the population growth rate of B. odoriphaga was influenced differently by the various garlic cultivars as observed by the differences in larval developmental time, larval mortality and fecundity, and consequently, the differences in the population parameters (r, R0, and T). When reared on the insect-resistant cultivars Qixian and Cangshan, the larval development time of B. odoriphaga was prolonged and the survival and fecundity decreased compared with those reared on insect-susceptible cultivars Siliuban and Yishui (Table 1); exhibiting major differences in resistance performance. Our findings are in agreement with many studies on the effects of host plants on the development, survival and reproduction of insects35, 36. The larval mortality was highest across the entire larval lifecycle, especially during the young stage (Fig. 2). This suggested that larvae in the young stage were the most vulnerable to external negative factors. Thus, this represents the ideal stage for controlling the insect population. Meanwhile, the different growth stages of garlic were found to differ significantly with respect to B. odoriphaga resistance, and the results indicated that garlic at the mature stage exhibited the highest resistance followed by the seeding stage, while the growth stage was the lowest (Fig. 3). Therefore, we recommend Qixian and Cangshan garlic as B. odoriphaga-resistant cultivars, and cultivation on a large scale might efficiently reduce the costs of B. odoriphaga control; a hypothesis which we verified under field conditions. The various effects on the development and fecundity of phytophagous insects resulting from host plants may also be attributed to nutrition and phytochemical differences in the different host plants37. Thus, an analysis of the sap composition of the garlic cultivars will help to clarify the factors affecting the population growth parameters of B. odoriphaga. Xue24 reported that B. odoriphaga reared on garlic cloves possessed lower survival and fecundity compared with those reared on garlic sprouts, and speculated that the differences in nutrition and phytochemicals led to this result.
Phytochemicals play an important role in plant insect-resistance6, 38. Xue24 reported that sulfides constituted the critical component for the resistance of Allium plants against B. odoriphaga. Wang23 analyzed the differences in resistance to D. antiqua among 34 garlic cultivars in the field, and found that the cultivar with high allicin content exhibited strong resistance. In addition, allicin accounted for about 60–90% of thiosulfinates in garlic11, 26. Our biochemical measurements confirmed that the thiosulfinates content in garlic was significantly correlated with resistance to B. odoriphaga. The cultivar with higher thiosulfinate content possessed higher insect-resistance, which confirms the results of Wang23. The difference in thiosulfinates content among various growth stages can also explain the differences in resistance mentioned above. This correlation may result from the insecticidal activity of sulfides or garlic essential oil as reported in previous studies.
Many plant essential oils possess potential insecticidal activity39, 40. As the majority of these chemicals degrade rapidly and have little or no harmful effects on the environment or humans41, 42, some of them including Azadirachtin and Sophocarpidine have been applied as insecticides. Garlic essential oil is known to possess repellent and insecticidal activity against arthropod pests43,44,45,46. Prowse45 also found that garlic oil possessed insecticidal efficacy against 2 target dipteran pests, Delia radicum (L.) and Musca domestica (L.). Additionally, garlic oil showed significant acaricidal effects, and exhibited lethal effects on Tetranychus urticae (Tetranychidae), reducing the fecundity of the treated mites46. Results in this study confirmed that garlic essential oil and allicin possessed good insecticidal activity against B. odoriphaga larvae, and their LC50 toxicity values were lower than that of the botanical insecticides Azadirachtin and Matrine. Additionally, allicin, an unstable thiosulfinate, decomposed into various sulphides easily at normal condition, and diallyl disulphide is the main breakdown products. Sulfides are known to be the dominant component of garlic oil extractions, comprising a mixture of diallyl disulfide, diallyl trisulfide, diallyl sulphide and other sulfides11. Our laboratory found that sulfides accounted for 99.9% of the garlic essential oil, while diallyl disulfide, diallyl sulfide and diallyl trisulfide accounted for 68.59%, 20.18% and 11.22% respectively. Elucidation of the pesticidal mechanism of garlic essential oil and their constituents is helpful in providing necessary information regarding the exploitation of the most appropriate formulation and delivery means. Moreover, according to the results of Yang27, diallyl trisulfide acts as a fumigant against the stored product pest Tribolium castaneum (Herbst). Our results confirmed that allicin exhibited a little low insecticidal activity against B. odoriphaga larvae compared with the garlic oil. We also speculated that sulphides, the decomposition products of thiosulfinate possess better insecticidal activity, and which sulphide, diallyl disulphide or diallyl trisulfide, playing dominant role needed further study.
Planting insect-resistant cultivars may reduce damage from pests; however, this method does not eliminate all damage. To ensure crop quality and yield, insecticides are frequently used in the field. Although many plant essential oils are known to possess insecticidal or repellent activity against insect pests, the control of B. odoriphaga to date has depended on synthetic chemicals. We have now confirmed that garlic essential oil exhibits strong insecticidal activity against B. ordoriphaga. Furthermore, Park43 discovered that garlic essential oil possessed high insecticidal activity against Lycoriella ingenua (Diptera: Sciaridae), a proximal species of B. ordoriphaga, and diallyl disulfide exhibited fumigant activity. These results all indicate that garlic essential oil or sulfides have the potential to act as novel botanical insecticides against dipteran pests. In addition, we also found that B. odoriphaga adults had positive taxis to low allicin concentration and phobotaxis to high concentration47. Thus, the insecticidal activity may be related to garlic oil concentration. Moreover, many studies have reported the expense of using botanical insecticides for pest control compared with chemical insecticides, as larger doses are required due to their weak potency. We found that a mixture of botanical insecticide (Azadirachtin) and chemical insecticide (Clothianidin) could control pests at a lower cost48. However, it is not clear whether garlic essential oil could be mixed with chemical insecticide for the control of B. odoriphaga. Further work needs to be conducted to confirm the dosage and methods of garlic essential oil in the control of B. odoriphaga.
Ten garlic cultivars, namely Qixian (originally from Qixian, Henan), Cangshanpuke (originally from Cangshan, Shandong), Zajiao (originally from Laiwu, Shandong), Xinxiang (originally from Xinxiang, Henan), Jinxiang (originally from Jinxiang, Shandong), Sichuan (originally from Chengdu, Sichuan), Yishui (originally from Linyi, Shandong), Nanfang (originally from Guiyang, Guizhou), Zhongmu (originally from Zhongmu, Henan) and Siliuban (originally from Cangshan, Shandong), were obtained from the College of Horticulture, Shandong Agricultural University (Tai’an, China). These 10 garlic cultivars comprise the main cultivated cultivars, and many previous studies had have investigated their diversity23, 32, 49, 50. All garlic cultivars used in this study were planted in the experimental field of Shandong Agricultural University on October 5th 2014, and harvested on June 5th 2015. Before covering with plastic film, 330 g/L of the herbicide pendimethalin (EC) was applied. The 10 cultivars were planted randomly, and the planting area of every cultivar measured approximately 120 m2 (length 20 m; width 6 m) with 5 duplicates. During planting, the plants were sufficiently watered and no insecticides were applied.
B. odoriphaga colonies
Bradysia odoriphaga colonies were originally obtained from a Chinese chive field in Tai’an, Shangdong, China in April 2013. Insect colonies were maintained in the Shandong Provincial Key Laboratory of Applied Microbiology and reared on an artificial diet for more than 10 generations. According to the rearing method described by Xue24, eggs, larvae and pupae were reared in culture dishes (Φ = 9 cm) covered with wet filter paper, and newly emerged adults were placed in pairs in individual oviposition containers (plastic cups of 3 cm diameter × 1.5 cm height) containing a piece of 30 mm filter paper moistened with deionized water. To facilitate egg collection, the artificial diet was cut into 1 cm-long pieces and placed in the rearing cages to allow females to oviposit. The B. odoriphaga colonies were maintained in growth cabinets at 25 ± 1 °C, 12 h: 12 h L: D, 75% ± 5% RH.
Insecticides and synthetic garlic oil
Sophocarpidine (5% technical concentrate), emamectin benzoate (88.3%), azadirachtin (5%) and phoxim (95.5%) were supplied by the Key Laboratory of Pesticide Toxicology and Application Technique, Shandong Agricultural University, Tai’an. Alternatively, synthetic garlic oil (EO; containing 3 major constituents, 68.59% of diallyl disulfide, 11.22% of diallyl trisulfide, and 20.18% of diallyl sulphide: Supplementary Tables S2 and Fig. S1) and allicin (95.8%, CFN90201) were purchased from the Wuhan ChemFaces Limited. L-cysteine and 5,5′-dithiobis (2-nitrobenzoic acid; DTNB) were purchased from Sigma-Aldrich. Acetone and Tween 80 were purchased from Tianjin Caratton Industry Limited.
Investigation of the occurrence of B. odoriphaga in the garlic field
Using a chess board sampling technique, on May 20th 2015 we randomly selected 10 points in every cultivar of the garlic field with every 1 point being represented by 5 plants, and recorded the number of B. odoriphaga larvae on every garlic bulb.
Life table study
The garlic plants exhibiting no injuries across the different growth stages (the seeding stage, November 2014; the growth stage, April 2015; and the mature stage, May 2015) were used as test plants. The B. odoriphaga colonies were reared on the garlic bulbs. Prior to the life table study, B. odoriphaga individuals were reared on these 10 garlic cultivars for a single generation. The eggs spawned by the adults reared on the different cultivars were gathered as the test insects. A total of 150 eggs (15 eggs from each female) were used for the life table study with each garlic cultivar. These eggs and hatched larvae were reared on the same cultivar sequentially. Every garlic cultivar bulb was cut into thin slices (approx. 3 mm) and placed in a separate petri dish. The eggs mentioned above were placed around the garlic thin slices. The eggs were observed daily and the hatching rates were recorded. Each day, newly hatched larvae were transferred into a new culture dish separately for supply of one of the diets. The survival of B. odoriphaga was recorded daily and fresh supplies of the diets were provided to avoid fungal growth until all adults perished. Deionized water was replenished daily to keep the filter paper moist. Just after the transformation from larvae into pupae, the pupae were moved to new petri dishes. After the emergence of adults, male and female insects were paired and placed in individual oviposition plastic containers. Adults were checked daily and the number of eggs of each individual was recorded until death.
Physiological determination of thiosulfinate content in garlic
The garlic cloves were ground with a mortar and mixed with water (25 mL per 0.5 g). The suspension was shaken for 15 min at 25 °C and filtered through gauze. The undissolved material was removed by centrifugation at 300 × g for 4 min. The thiosulfinate contents were determined using the method described by Han in 199526. The reaction mixture included 0.5 mL garlic extract and 4.5 mL cysteine solution (200 μM, Hepes buffer pH 7.5). After 10 min, every 1 mL reaction mixture was incubated in a cuvette with 4 mL DTNB (1.5 mM, phosphate buffer pH 7.5). The residual concentration of cysteine in the mixture was determined by measuring the amount of 2-nitro-5-thiobenzoate (NTB) formed after reaction with DTNB and the molar extinction coefficient (1 cm light path) of 14,150 at 412 nm.
Bioassays were conducted on the newly emerged fourth instar larvae of B. odoriphaga using a standard contact and stomach bioassay method (insecticide-impregnated filter method). Two pieces of filter paper (Φ = 9 cm) were immersed into the tested solution and then one was flattened in the culture dish. Fresh Chinese chive cauloids (0.5 cm) were cut and dipped into the test solution for 10 s with gentle agitation, and air dried at room temperature. Twenty B. odoriphaga larvae were placed around the Chinese chive cauloid placed on the filter paper, and the remaining piece of filter paper was placed on top of the tested larvae. Every treatment included 100 larvae for 5 replications, using a pure water treatment as the control. Serial dilutions (mg/L) of the active ingredient diluted with 0.1% Tween 80 solution were prepared. The test larvae were reared on the Chinese chive pseudo stems mentioned above and maintained in growth cabinets at 25 ± 1 °C, 12 h: 12 h L:D, 75% ± 5% r. h. Larval survival was checked and recorded following treatment for 48 h or 96 h.
Raw data of all individuals in the life table were analyzed according to the age-stage two-sex life table theory33. The developmental time, longevity, fecundity and population parameters and their mean values and standard errors were estimated using the bootstrap method included in the computer program TWOSEX-MS Chart33, 34. Differences in B. odoriphaga populations among the cultivars were compared using the paired test included in the software.
We tested the other variables for homogeneity of group variances using Levene’s test and normality using the Kolmogorov-Smirnov test prior to statistical analysis. For the analysis, the quantity of B. odoriphaga on each garlic cultivar was regarded as the dependent variable, while the garlic cultivars were regarded as the independent variables in one-way ANOVA followed by Tukey’s HSD multiple comparisons. With regards to the thiosulfinates content analysis at the same growth stages, the content in each garlic cultivar was regarded as the dependent variable, while the garlic cultivars were regarded as independent variables using one-way ANOVA followed by Tukey’s HSD multiple comparisons. Conversely, when analyzing the thiosulfinates content in the same garlic cultivar, the content at each growth stage was regarded as the dependent variable, while the growth stages were regarded as independent variables in the above-mentioned method. In the correlation analysis, thiosulfinates content in each cultivar and each growth stage of garlic was regarded as the independent variable, and B. odoriphaga larval development time, mortality, and fecundity reared on each cultivar were regarded as the dependent variables in the regression analysis followed by linear regression. In the bioassays, the number of dead B. odoriphaga larvae when treated with each insecticide dose (including the 0 mg kg−1 group, i.e., the control group) was regarded as the response frequency, while the doses were regarded as the covariate, and the total tested number of larvae in each treatment was regarded as the total observed in the regression analysis, which was followed by Probit. All analyses were performed with PASW Statistics 18.0.0 (2009; SPSS Inc. Quarry Bay, HK). Figures were constructed using SigmaPlot 12.0.
Panda, N. & Khush, G. S. Host plant resistance to insects. (Wiley, 1989).
Wu, J. & Baldwin, I. T. New insights into plant responses to the attack from insect herbivores. Annu. Rev. Genet. 44, 1–24 (2010).
Hogenhout, S. A. & Bos, J. I. Effector proteins that modulate plant–insect interactions. Curr. Opin. Plant. Biol. 14, 422–428 (2011).
Sadasivam, S., Thayumanavan, B., Sadasivam, S. & Thayumanavan, B. Molecular host plant resistance to pests. Molecular Host Plant Resistance to Pests (2003).
Mason, C. M. & Donovan, L. A. Does investment in leaf defenses drive changes in leaf economic strategy? A focus on whole-plant ontogeny. Oecologia. 177, 1053–1066 (2015).
Smith, C. M. & Clement, S. L. Molecular bases of plant resistance to arthropods. Annu. Rev. Entomol. 57, 309–328 (2012).
Etoh, T., Simon, P. W., Rabinowitch, H. D. & Currah, L. Diversity, fertility and seed production of garlic. Genetics 1887, 101–118 (2002).
Ding, C. The reality and countermeasure of garlic export trade in China. China Veget. 4, 1–5 (2005).
Göksel, S., Abdullah, S. & Yeğen, B. Ç. Role of garlic in the prevention of ischemia-reperfusion injury. Mol. Nutr. Food Res. 51, 1345–1352 (2007).
Sfaxi, I. H. et al. Inhibitory effects of a manganese superoxide dismutase isolated from garlic (Allium sativum L.) on in vitro tumoral cell growth. Biotech. Progr. 25, 257–264 (2009).
Vergel, S. J. N., Bustos, R. A., Rodríguez, C. D. & Cantor, R. F. Laboratory and greenhouse evaluation of the entomopathogenic fungi and garlic–pepper extract on the predatory mites, Phytoseiulus persimilis and Neoseiulus californicus and their effect on the spider mite Tetranychus urticae. Bio. Control. 57, 143–149 (2011).
Lawson, L. D. & Hughes, B. G. Characterization of the formation of allicin and other thiosulfinates from garlic. Planta Medica 58, 345–350 (1992).
Lawson, L. D. & Gardner, C. D. Composition, stability, and bioavailability of garlic products used in a clinical trial. J. Agr. Food Chem. 53, 6254–6261 (2005).
Harris, J. C., Plummer, S., Turner, M. P. & Lloyd, D. The microaerophilic flagellate Giardia intestinalis: Allium sativum (garlic) is an effective antigiardial. Microbiology. 146, 3119–3127 (2000).
Ban, J. O. et al. Inhibition of cell growth and induction of apoptosis via inactivation of NF-kappaB by a sulfurcompound isolated from garlic in human colon cancer cells. J. Pharmacol. Sci. 104, 374–383 (2007).
Robyn, J. & Rasschaert, G. Is allicin able to reduce Campylobacter jejuni colonization in broilers when added to drinking water? Poultry Sci. 92, 1408–1418 (2013).
Feldberg, R. S. et al. In vitro mechanism of inhibition of bacterial cell growth by allicin. Antimicrob Agents and Ch 32, 1763–1768 (1988).
Zhang, Q. C., Xue, M., Wang, Z., Zhang, Y. X. & Li, X. X. Toxicity and effects on the growth, development and reproduction of neonicotinoid insecticides to Delia antique (Meigen). Acta Phytophy Sin. 38, 159–165 (2011).
Yang, J. K. & Zhang, X. M. Notes on the fragrant onion gnats with descriptions of two new species of Bradysia odoriphaga (Diptera: Sciaridae). J. China Agr. Univ. 10, 153–158 (1985).
Feng, H. Q. & Zheng, F. Q. Studies of the occurrence and control of Bradysia odoriphaga Yang et Zhang. J. Shandong Agr. Univ. 18, 71–80 (1987).
Li, X. X., Ma, X. D., Xue, M., Zhao, H. P. & Li, Z. X. Toxic effects of clothianidin and other five kinds of insecticides to Bradysia odoriphaga. Acta Phytophylacica Sinica. 41, 225–229 (2014).
Wang, H. X., Sha, Y., Sun, J. T. & Sun, J. Z. A report of food poisoning by consuming of organophosphate pesticide contaminated vegetable. Occupat. Health 22, 512–513 (2006).
Wang, H. P. et al. Identification of resistance of Allium sativum germplasm resources to Delia antique. J. Plant Genet. Res. 11, 578–582 (2010).
Xue, M., Pang, Y. H., Wang, C. X. & Qiang, L. I. Biological effect of liliaceous host plants on Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae). Acta Entomol Sinica. 48, 914–921 (2005).
Zhang, P., Liu, F., Mu, W., Wang, Q. & Li, H. Comparison of Bradysia odoriphaga Yang and Zhang reared on artificial diet and different host plants based on an age-stage, two-sex life table. Phytoparasitica. 43, 107–120 (2015).
Han, J., Lawson, L., Han, G. & Han, P. Spectrophotometric method for quantitative determination of allicin and total garlic Thiosulfinates. Anal. Biochem. 225, 157–160 (1995).
Yang, F. L., Zhu, F. & Lei, C. L. Garlic essential oil and its major component as fumigants for controlling Tribolium castaneum (Herbst) in chambers filled with stored grain. J. Pest Sci. 83, 311–317 (2010).
Yang, F. L., Zhu, F. & Lei, C. L. Insecticidal activities of garlic substances against adults of grain moth, Sitotroga cerealella (Lepidoptera: Gelechiidae). Insect Sci. 19, 205–212 (2012).
Park, I. K. & Shin, S. C. Fumigant activity of plant essential oils and components from garlic (Allium sativum) and Clove Bud (Eugenia caryophyllata) oils against the Japanese termite (Reticulitermes speratus Kolbe). J. Agr. Food Chem. 53, 4388–4392 (2005).
Park, I. K. et al. Nematicidal activity of plant essential oils and components from garlic (Allium sativum) and cinnamon (Cinnamomum verum) oils against the pine wood nematode (Bursaphelenchus xylophilus). Nematology. 7, 767–774 (2005).
Kimbaris, A. C., Kioulos, E., Koliopoulos, G., Polissiou, M. G. & Michaelakis, A. Coactivity of sulfide ingredients: a new perspective of the larvicidal activity of garlic essential oil against mosquitoes. Pest. Manag. Sci. 65, 249–254 (2009).
Yang, F. J., Liu, S. Q. & Wang, X. F. Effect of different cultivars and trace fertilizers on allicin content in bulb of garlic. Shandong Agr. Sci. 4, 22–25 (2004).
Chi, H. Life-Table Analysis incorporating both sexes and variable development rates among individuals. Environ. Entomol. 17, 26–34 (1988).
Chi, H. & Su, H. Y. Age-stage, two-sex life tables of Aphidius gifuensis (Ashmead) (Hymenoptera: Braconidae) and its host Myzus persicae (Sulzer) (Homoptera: Aphididae) with mathematical proof of the relationship between female fecundity and the net reproductive rate. Environ. Entomol. 35, 10–21 (2006).
Lin, L. & Ren, S. X. Development and reproduction of Bemisia tabaci (Homoptera: Aleyrodidae) on three bean species. Insect Sci. 12, 25–30 (2005).
Özgökçe, M. S. & Atlıhan, R. Biological features and life table parameters of the mealy plum aphid Hyalopterus pruni, on different apricot cultivars. Phytoparasitica 33, 7–14 (2005).
Serdar, S. & Raymond, Y. Effect of temperature and host on development of Brachycaudus schwartzi (Homoptera: Aphididae). Ann Entomol Soc Am. 95, 597–602 (2002).
Kainulainen, P., Nissinen, A., Piirainen, A., Tiilikkala, K. & Holopainen, J. K. Essential oil composition in leaves of carrot varieties and preference of specialist and generalists sucking insect herbivores. Agric. For. Entomol. 4, 211–216 (2002).
Rameshwarsingh, R. Mechanism of action of insecticidal secondary metabolites of plant origin. Crop Prot. 29, 913–920 (2010).
Choi, W. I., Lee, E. H., Choi, B. R., Park, H. M. & Ahn, Y. J. Toxicity of plant essential oils to Trialeurodes vaporariorum (Homoptera: Aleyrodidae). J. Econ. Entomol. 96, 1479–1484 (2003).
Rice, P. J. & Coats, J. R. Insecticidal properties of several monoterpenoids to the house fly (Diptera: Muscidae), red flour beetle (Coleoptera: Tenebrionidae), and southern corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 87, 1172–1179 (1994).
Sinzogan, A. A. C., Kossou, D. K., Atachi, P. & Van Huis, A. Participatory evaluation of synthetic and botanical pesticide mixtures for cotton bollworm control. Int. J. Trop. Insect Sci. 26, 246–255 (2007).
Park, I. K., Kim, J. N., Lee, Y. S., Lee, S. G. & Ahn, Y. J. Toxicity of plant essential oils and their components against Lycoriella ingenua (Diptera: Sciaridae). J. Econo. Entomol. 101, 139–144 (2008).
Isman, M. B. Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Ann. Rev. Entomol. 51, 45–66 (2006).
Prowse, G. M., Galloway, T. S. & Andrew, F. Insecticidal activity of garlic juice in two dipteran pests. Agr Forest Entomol. 8, 1–6 (2006).
Attia, S. et al. Effective concentrations of garlic distillate (Allium sativum) for the control of Tetranychus urticae (Tetranychidae). J. Appl. Entomol. 136, 302–312 (2012).
Xue, M., Yuan, L. & Xu, M. L. Olfactory response of Bradysia odoriphaga adult to volatile substance and toxicity comparison among different pesticides. Chinese J. Pestic. Sci. 4, 50–56 (2002).
Zhu, G. D. et al. Toxicity of botanic insecticide to Bradysia odoriphaga larva and application value assessment. North. Hortic. 22, 135–139 (2016).
Chen, S. X. et al. Cluster analysis and evaluation of GarlicAllium sativum L. Germplasm based on principal components. J. Plant Genet. Resour. 13, 429–434 (2012).
Gao, Y. Y. et al. Studies on characteristics for growth of product organs of different garlic cultivars. J. Changjiang Veg. 17, 41–45 (2016).
This research was funded by “International Commonweal Scientific Research Special Fund-Research and Demonstration of Crop Root Maggot Control Technology (No. 201303027)”. We thank LetPub for its linguistic assistance during the preparation of this manuscript.
The authors declare that they have no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
About this article
Cite this article
Zhu, G., Luo, Y., Xue, M. et al. Resistance of Garlic Cultivars to Bradysia odoriphaga and Its Correlation with Garlic Thiosulfinates. Sci Rep 7, 3249 (2017) doi:10.1038/s41598-017-03617-9
Green synthesis of garlic oil nanoemulsion using ultrasonication technique and its mechanism of antifungal action against Penicillium italicum
Ultrasonics Sonochemistry (2020)
Efficient and stereoselective synthesis of thiosulfinates from disulfide using cumene hydroperoxide in the presence of titanium-based catalyst systems
Synthetic Communications (2019)
Effect of the nitrogen-fixing bacterium Pseudomonas protegens CHA0-ΔretS-nif on garlic growth under different field conditions
Industrial Crops and Products (2019)
Effects of Feeding on Different Host Plants and Diets on Bradysia odoriphaga Population Parameters and Tolerance to Heat and Insecticides
Journal of Economic Entomology (2017)