http://orcid.org/0000-0002-3893-9545
Figures
Abstract
The western tarnished plant bug, Lygus hesperus Knight (Hemiptera: Miridae) and the whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) are key hemipteran pests of numerous crop plants throughout the western United States and Mexico. Management in the U.S. currently relies on only a few insecticides and is threatened by the evolution of resistance. New chemistries or alternative management strategies are needed to reduce selection pressure on current insecticides and enhance control. Here, we investigated the bio-insecticidal toxicity of the French marigold, Tagetes patula Linnaeus (Asterales: Asteraceae), against both L. hesperus and B. tabaci. Assays indicated significantly reduced survival of both pest species on T. patula plants, and in diet incorporation assays containing aqueous and methanolic marigold foliar extracts. Mortality was concentration-dependent, indicating the presence of one or more extractable toxicants. These data suggest that T. patula plants have insecticidal constituents that might be identified and developed as novel alternatives to conventional chemical treatments.
Citation: Fabrick JA, Yool AJ, Spurgeon DW (2020) Insecticidal activity of marigold Tagetes patula plants and foliar extracts against the hemipteran pests, Lygus hesperus and Bemisia tabaci. PLoS ONE 15(5): e0233511. https://doi.org/10.1371/journal.pone.0233511
Editor: Nicolas Desneux, Institut Sophia Agrobiotech, FRANCE
Received: November 4, 2019; Accepted: May 6, 2020; Published: May 19, 2020
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the manuscript.
Funding: This research was supported by the U.S. Department of Agriculture, Agricultural Research Service, National Program 304 – Crop Protection & Quarantine (#2020-22620-022-00D to JF and DS), Bayer CropScience AG Grants4Targets Grant (#2016-01-51 to JF), and the National Cotton Council (#2020-22620-021-16J to JF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: This research was supported in part by the Bayer CropScience AG Grants4Targets Grant Program (#2016-01-51). This does not alter our adherence to PLoS ONE policies on sharing data and materials.
1. Introduction
Many plants exhibit resistance to insect attack that typically arises from one or more mechanisms; tolerance, non-preference, or antibiosis [1–4]. Antibiosis (where plant defenses affect pest biology) is often facilitated by phytotoxins, some of which have been developed as botanical pesticides. Pyrethrum, nicotine, neem oil, essential oils, and rotenone are examples of phytochemicals used as commercial botanical pesticides [5–7]. Several more recently investigated plant extracts and essential oils also show promising activity as bio-insecticides [8–12] or repellents [13,14].
Several species of marigold (Tagetes spp.) are known to contain phytochemicals with pesticidal activity. For example, numerous studies have shown insecticidal activity associated with Tagetes erecta L. (African marigold), T. minuta L. (Mexican marigold), or T. patula L. (French marigold) against mosquitoes [15–20], sand flies [21], a leaf hopper [22], grain/seed beetles [23–26], termites [27], human head lice [28], bed bug [29], an aphid [30], and several caterpillars [31,32]. These marigold species have also shown activity as acaricides [33,34] and nematocides [35–37].
Numerous insecticidal compounds have been isolated from essential oils and root extracts of Tagetes spp. [30,38]. Monoterpenoids, carotenoids, and flavonoids are the major biocidal constituents of volatile oils from aerial plant parts of Tagetes spp. [30,38]. Photoactive thiophenes, present primarily in roots and flowers, are also biologically active against several insect species [19,23,39–41]. Unfortunately, many of these active compounds have limited practical use because they are volatile and have poor persistence under field conditions [5].
The western tarnished plant bug, Lygus hesperus Knight (Hemiptera: Miridae) and the whitefly, Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) are two major pests of cotton and other crops throughout the western United States [42–44]. Tarnished plant bug management typically relies on multiple applications of conventional insecticides of various chemical classes [45–46]. Whitefly control in the western U.S. is primarily dependent on insect growth regulators (IGRs) and neonicotinoids [46–47], although broad-spectrum insecticides are also used [46]. In Arizona, an Integrated Pest Management program has been implemented against both lygus and whitefly [44]. However, success of this program is dependent on the continued effectiveness of only a few insecticides [48]. Because these few chemicals have been widely used for many years and because both pest species have evolved resistance to numerous other insecticides [48–52], new control tactics are needed. Hence, the discovery of new compounds or complex mixtures of bioactive compounds with novel insecticidal modes of action are needed to reduce such selection pressure, and novel botanical insecticides could fill such pest management niches [9]. Here, we demonstrate that the marigold, T. patula, and its crude extracts, have toxicological activity against two important hemipteran pest species, revealing that future benefits might include use of foliar extracts for direct use as botanical insecticides as well as a resource for future isolation and development of beneficial compounds.
2. Materials and methods
2.1 Insects
Adult L. hesperus were obtained from a laboratory-reared colony maintained on common bean pods (Phaseolus vulgaris L.) and raw sunflower seeds (Helianthus annuus L.) as shown previously [53]. The colony was initiated from adults collected from alfalfa (Medicago sativa L.) on the University of Arizona Maricopa Agricultural Center farm, Maricopa, AZ. Because L. hesperus reared for more than three generations exhibit effects of laboratory selection [53], we examined only insects collected less than three generations from the field. Adults were collected from the colony within 24 h after adult eclosion.
Adult B. tabaci were from a Middle East-Asia Minor One (MEAM1) laboratory colony maintained at the U.S. Arid Land Agricultural Research Center, Maricopa, AZ. Whiteflies were continuously reared on Brassica oleracea L. within a 35 x 35 x 85-cm mesh cage in a greenhouse maintained between 21-32°C with ambient photoperiod [54]. To synchronize ages of whiteflies for bioassays, uninfested broccoli plants were placed into the cage harboring our whitefly lab colony. After 24-48 h the infested plants were removed from the cage and adult whiteflies were removed by shaking plants and/or gently brushing with paint brush. The plants with newly oviposited eggs were held in new cage within greenhouse for 1-2 weeks until initiation of adult whitefly emergence. Adult whiteflies were again removed by shaking/brushing. Then, newly emerged adult whiteflies were collected by aspiration within 24 h and used immediately in diet and on-plant tests.
2.2 Plant propagation
Seeds from the marigold, T. patula L. (Livingston Seed Co., Columbus, OH), were sown in pots containing filtered Sunshine Mix-1 (Sun Gro Horticulture, Agawam, MA) soil:sand mixture [9:5 soil:sand (v/v)]. Plants were grown in a greenhouse at 25 ± 8°C with 15-50% relative humidity (R.H.). Flowering marigold plants were harvested for leaf extractions or used for on-plant assays 3-5 months after seedling emergence. For on-plant assays, common bean (P. vulgaris L.) seed (Ferry-Morse, Norton, MA) and cotton (Gossypium hirsutum L.) seed (Bollgard II, Monsanto, St. Louis, MO) were sown and grown in the 9:5 soil:sand potting mix in a greenhouse using the conditions described above.
2.3 On-plant bioassays
On-plant tests of L. hesperus survival were conducted in the greenhouse at 25 ± 8°C with a natural daylength of 10-12 h. Adults less than 24 h-old were collected, chilled on ice, and sorted into cohorts, each containing 20 males and 20 females. The 40 adults were simultaneously released into 37 x 37 x 61-cm plexiglass cages with mesh sides and a hole in the bottom that accepted a 3.8-liter pot (Fig 1A). Tap water was provided ad libitum in each cage via 30-mL vials sealed with foam plugs. Each of the three experimental repetitions included three each of T. patula test plants and three P. vulgaris control plants (six plants per repetition). Survival of the L. hesperus adults was recorded by counting the live and dead insects every 24 h for 13 d.
For on-plant assays (A), live adults were released into plexiglass cages with either marigold or control (common bean) plant and survival was assessed daily for 13 d. For incorporation assays, marigold extracts were mixed into diets for L. hesperus (B and C) or B. tabaci (D and E) and survival was recorded daily for up to 3 (B. tabaci) or 14 d (L. hesperus).
For B. tabaci on-plant assays, age-synchronized adults (<24-h old) of indeterminate sex were aspirated into 30-mL plastic vials and released into plexiglass cages (described above) containing test plants within a plant growth chamber (Conviron E8, Controlled Environments, Winnipeg, MB, Canada) at 26.0 ± 1.0°C, 40–60% R.H. under a 14:10 (L:D) h photoperiod. A total of 55-60 whiteflies were released into each cage. Survival of adults and numbers of eggs were counted every 24 h for 3 d. Each of the five experimental repetitions consisted of two each of the T. patula and two G. hirsutum plants per repetition.
2.4 Plant extractions
For aqueous extractions, 10 g of fresh marigold leaves were removed from mature plants, rinsed under tap water, cut into pieces, and placed into a mortar containing 30 mL of ultrapure water. Leaf material was crushed with a pestle for 5 min. After adding 20 mL of ultrapure water to rinse the pestle, the slurry was transferred to Erlenmeyer flask and total volume was brought to 150 mL with ultrapure water. The flask was covered with Parafilm M (Pachiney Plastic Packaging, Chicago, IL) and the contents were mixed with a magnetic stirrer for 2-3 hr. Equal amounts of the slurry were poured into each of four 50-mL capped centrifuge tubes and centrifuged at 10,000 x g for 10 min. Supernatants were vacuum filtered through Whatman #2 filter paper, then evaporated to dryness at 40°C on a heated stir plate. The dried residue was resuspended in 12 mL of ultrapure water and transferred to a pre-weighed centrifuge tube. Aqueous extracts were lyophilized using a benchtop lyophilizer (FreeZone 6 Liter, Labconco, Kansas City, MO). The dry extract was weighed and stored at 4°C until used in feeding bioassays.
For methanol extractions, 10 g of fresh marigold leaves were crushed with mortar and pestle in 30 mL of methanol (Fisher Scientific, Waltham, MA). The slurry was transferred to an Erlenmeyer flask, brought to 150 mL with methanol, covered, and stirred for 2-3 hr. The slurry was centrifuged and filtered as indicated above. Filtered supernatants were evaporated to dryness overnight at 30°C on a heated stir plate. The dried residue was resuspended in 12 mL methanol, transferred to a pre-weighed centrifuge tube, and dried in a fume hood under an air stream. Once dry, tubes were weighed and stored at 4°C.
2.5 Diet incorporation bioassays
To test marigold extracts for toxicity against L. hesperus and B. tabaci, we incorporated increasing concentrations of either aqueous or methanolic extracts into the food and observed mortality over time. For L. hesperus, aliquots of liquid diet [55] were mixed with dried soluble marigold extract [0 to 25000 parts per million (ppm)], or with methanol extract (0 to 10000 ppm) dissolved in 1% Tween 80 (Sigma, St. Louis, MO) (Fig 1B and 1C). Three-mL aliquots of diet mixtures were placed into inverted lids of 30-mL plastic vials and covered with tightly stretched Parafilm M. A brush was used to apply 15% (w/v) sucrose to the Parafilm to entice feeding by the insects. To account for possible buffer effects in the methanol extracts, 0 ppm doses contained the same volume of 1% Tween 80 used in extract samples. We repeated the assays three times, with each treatment replicated three times in each assay. Repetitions of the assays were conducted on different days and used fresh extract preparations each time. Lids containing the diets were each placed into the center of a 355-mL paper cup and 10 L. hesperus were released into each container (3 replicates x 5 doses x 3 repetitions x 10 adults per cup = 450 L. hesperus per test extract). Cups were covered with fine mesh secured with a rubber band. Feeding cups were held in a growth chamber at 26.0 ± 1.0°C, 40–60% R.H. under a 14:10 (L:D) h photoperiod. Survival and mortality was recorded by direct visualization and counting of live and dead insects every 24 h for up to 14 d.
For B. tabaci diet-incorporation bioassays, 50 adults (<24-h-old) were aspirated from B. oleracea into 30-mL plastic vials (Fig 1D), previously rubbed with a dye- and fragrance-free dryer sheet (Essential EverydayTM, SuperValu Inc., Eden Prairie, MN) to decrease static electricity. Each vial of insects was covered with stretched Parafilm M. Whitefly diet consisted of filter-sterilized 5% (w/v) DifcoTM yeast extract (Thermo Fisher Scientific, Waltham, MA) in 15% (w/v) sucrose with 1% Tween 80. Serial dilutions of marigold aqueous or methanol extracts (0 to 50,000 ppm) were incorporated into whitefly diet. Diet aliquots (0.3 mL) containing marigold extract were dispensed on top of the Parafilm stretched over each vial containing the test whiteflies (Fig 1E). Test vials were held in a growth chamber at 28.0 ± 1.0°C, 30% R.H. under a 14:10 (L:D) h photoperiod. Whitefly mortality was recorded at 0, 24, 48, and 72 h. Each repetition of the experiment consisted of three replicates for each dose of marigold extract (3 replicates x 5 doses x 3 repetitions x 50 adults per vial = 2250 B. tabaci per test extract).
2.6 Data analysis
For statistical analyses of on-plant survival assays and whitefly oviposition, we used Prism 7 for Mac OS X to plot and perform Analysis of Covariance (ANCOVA) to test whether the dose-response slopes obtained by linear regression differed among plant species. For diet-incorporation assays, survival analysis was performed using the LIFETEST procedure of SAS [56] and the Wilcoxon statistic. Multiplicity-adjusted comparisons among the survival functions of different doses within each extract were made using the ADJUST=SIMULATE option of the STRATA statement.
3. Results
3.1 Marigold toxicity to Lygus hesperus
When L. hesperus adults were held on intact plants, mortality occurred more rapidly on marigolds compared with the common bean control (Fig 2; ANCOVA F = 6.23; df = 1,22; P = 0.021). The rate of mortality (%/d) was higher for those on marigold (slope ± SE, 3.38 ± 0.209; intercept ± SE, 9.05 ± 1.66; R² = 0.960; F = 261.6; df = 1,11; P < 0.001) than for those on common bean (slope ± SE, 2.82 ± 0.080; intercept ± SE, 0.448 ± 0.634; R² = 0.991; F = 1245; df = 1,11; P < 0.001).
Lines indicate the linear regressions and bars represent standard errors.
3.2 Marigold toxicity and reduced oviposition in Bemisia tabaci
The difference in B. tabaci on-plant mortality (%/h) between marigold and cotton plants was also pronounced (Fig 3A; F = 35.51; df = 1,56; P < 0.001). Mortality of B. tabaci on marigold occurred relatively rapidly (slope ± SE, 1.21 ± 0.193; intercept ± SE, -7.83 ± 9.99; R2 = 0.584; F = 39.30; df = 1,28; P < 0.001) whereas little mortality was observed on cotton (slope ± SE, 0.045 ± 0.031; intercept ± SE, 1.64 ± 1.59; R2 = 0.073; F = 2.20; df = 1,28; P = 0.149). Correspondingly, oviposition was reduced on marigold relative to cotton (Fig 3B; F = 34.24; df = 1,56; P < 0.001). On cotton, B. tabaci laid 8.00 ± 1.20 eggs per h (R² = 0.614; df = 1,28; P < 0.001), while on marigold the rate was 0.51 ± 0.44 per h (R² = 0.046; df = 1,28; P = 0.258).
Mortality (A) and oviposition (B) of adult Bemisia tabaci on Tagetes patula L. (marigold) and Gossypium hirsutum L. (cotton). Lines indicate the linear regressions and bars represent standard errors.
3.3 Toxicity of marigold foliar extracts to Lygus hesperus and Bemisia tabaci
Tests of aqueous foliar extracts from T. patula L. for toxicity against L. hesperus indicated a significant dose-mortality response (χ2 = 195.6, df = 4, P < 0.001; Fig 4A and Table 1). Although differences among the L. hesperus survival functions were indicated among doses of the methanol extracts (χ2 = 72.92, df = 4, P < 0.001), multiple comparisons showed only few doses that were significantly different (Fig 4B and Table 2). Furthermore, the mortality responses did not indicate a consistent dose-dependence. It was notable that the highest mortality was associated with the lowest concentration of the methanolic extract.
Survival functions of adult Lygus hesperus (A and B) and Bemisia tabaci (C and D) fed on Tagetes patula (marigold) foliar extracts incorporated into artificial diet. Aqueous (A and C) and methanol (B and D) extracts were fed for up to 14 d for L. hesperus and 72 h for B. tabaci, respectively.
Adult B. tabaci exhibited dose-dependent toxicity when fed both aqueous (χ2 = 5512, df = 4, P < 0.001; Fig 4C and Table 3) and methanolic marigold extracts (χ2 = 760.7, df = 4, P < 0.001; Fig 4D and Table 4), although differences among doses appeared less marked for the methanol extracts compared with the aqueous extracts.
4. Discussion
Concerns for the impact of pesticides on human health and the environment, as well as the constant evolution of pesticide resistance, necessitates a renewed interest in the discovery and use of novel non-synthetic bio-pesticides [6,57,58]. Many plants, including those from the Asteraceae family to which Tagetes spp. belong, contain antibiosis, antifeedant, or repellant compounds [5–6] with potential as pest management resources. Due to the substantial delay between discovery and commercialization of new bio-pesticides, only a few of the many promising plant-based compounds have come to market [5,7]. The dearth of commercialized botanical insecticides is due to regulatory limitations in the approval of products, inconsistency in the performance or delivery of active compounds, and limited availability of resources to pursue commercialization [5]. Limitations in resources often involve barriers to economical production or formulation of the active compounds. Here, our approach was to test both T. patula L. whole plants and minimally-processed leaf extracts for inherent activity against two arthropod pests endemic to the western U.S. and elsewhere. Our findings suggest that marigolds are a relatively untapped resource for the future identification, development, and commercialization of bioactive pesticidal compounds and/or for implementation of minimally-processed, whole plant-based insecticidal materials that might by-pass some of the delays in commercial pesticide development.
Because whiteflies and mirid bugs are global pests of many important crops, there is interest in using repellent plants in companion plantings [59–60]. Examples exist that demonstrate potential benefits of repellent companion crops for management of whiteflies [61,62], and efficacy of marigolds as companion plants has been reported for several insect pests [63–68]. In the southwestern U.S., while cotton is a suitable host, both pests prefer other plants [69–70], which lends itself to the use of cultural control strategies. For example, alfalfa (Medicago sativa L.) planted adjacent to cotton can act as a “trap crop” to reduce lygus damage [71–73]. Cotton can also be intercropped with plants that repel or deter L. hesperus and B. tabaci [59–60]. Studies show the potential management benefits of such companion plants for these pests at a relatively small scale [61,62]. However, the biotic mechanisms that underlie such repellency are often not well understood [74] and the use of this management tactic requires establishment and maintenance of the repellent plants. Furthermore, although Conboy et al. (2019) [62] showed that T. patula L. can serve as a repellent companion plant against the whitefly Trialeurodes vaporariorum Westwood on tomatoes in greenhouses, it is unknown what, if any, benefits would be achieved in lesser-value crops and/or in open field settings. Hence, additional research is needed to assess marigolds as companion plants for their effectiveness, feasibility, and economics for different crops under real-world conditions. Alternatively, extracts of bioactive compounds from plants such as T. patula may be developed as novel insecticides [75,76].
Our results indicate that T. patula possesses intrinsic insecticidal activity against two important hemipteran pests, L. hesperus and B. tabaci. We observed significantly reduced oviposition by B. tabaci on T. patula plants and significantly higher mortality for both L. hesperus and B. tabaci adults on marigold compared with control plants. These results could reflect altered behavior (e.g., repellency or feeding deterrence) rather than direct intoxication. However, we also observed dose-dependent mortality in diet-incorporation bioassays for both L. hesperus and B. tabaci adults. Differences between the dose-dependency of aqueous extracts, compared with methanolic extracts, may also provide useful insights. The clear dose-dependency of mortality associated with the aqueous extracts suggest minimal if any feeding deterrence. In contrast, dose-dependency (or inverse dose-dependency for L. hesperus) observed for the methanolic extracts suggest a methanol-soluble constituent (likely the toxicant) may have deterred feeding by the insects.
Although it was difficult to ascertain whether all insects within the replicates fed equivalently on the diet, several lines of evidence suggest the effect of the marigold extracts was manifested through toxicity rather than starvation. Feeding by both L. hesperus and B. tabaci on artificial diets containing marigold extracts was qualitatively indicated by 1) the aggregation of adult insects on provided diet, 2) direct observation of insects probing the diet through the Parafilm, 3) conspicuous reductions in the volumes of artificial diet with increased time of exposure, and 4) presence of dark green or brown excrement (the color of incorporated marigold extracts) within the assay containers (J.A.F., personal observation). Because processing of the aqueous and methanol extracts involved several drying steps at elevated temperature, most of the volatile organic compounds were likely removed and therefore not responsible for the responses we observed. Hence, we suggest that the foliage of T. patula likely contain compounds that are toxic against both L. hesperus and B. tabaci adults which are not innately repellant. Furthermore, our preliminary results indicate a direct impact on L. hesperus and B. tabaci, as the marigold extracts appeared to alter water transport and/or osmotic balance by targeting insect aquaporin proteins when expressed in a heterologous system (J.A.F. and A.J.Y., unpublished data).
Our results indicate T. patula possesses insecticidal constituents that may be useful as novel insecticides against mirid or whitefly pests. Before commercial implementation, residual traces of marigold phytochemicals would need to be evaluated for potential off-target effects against natural enemy and other beneficial arthropod communities [77] as well as unintended health impacts in human consumers. However, marigold extracts have been used for medicinal and dietary purposes by humans since ancient times. As reviewed by Gupta and Vasudeva (2012) [78], species of Tagetes have served as traditional medicinal herbs in Mexico and India for treating a variety of ailments. Aqueous and alcoholic extracts of the leaves have been used for example as antimalarials, anti-inflammatory agents, diuretics, treatments for gastrointestinal disorders in humans, and nematicides and insecticides in pests. Hence, further investigation is needed to identify specific toxicants and their modes of action in order to assess their potential value as alternatives to conventional pesticides.
Acknowledgments
We thank B. Hefner and C. Hoban for technical assistance. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U. S. Department of Agriculture. USDA is an equal opportunity provider and employer.
References
- 1.
Painter RH. Insect resistance in crop plants. The University Press of Kansas; 1951.
- 2. Koch KG, Chapman K, Louis J, Heng-Moss T, Sarath, G. Plant tolerance: a unique approach to control hemipteran pests. Front Plant Sci. 2016;7:1363. pmid:27679643
- 3. Mitchell C, Brennan RM, Graham J, Karley AJ. Plant defense against herbivorous pests: exploiting resistance and tolerance traits for sustainable crop protection. Front Plant Sci. 2016;7:1132. pmid:27524994
- 4. Stenberg JA, Muola A. How Should Plant Resistance to Herbivores Be Measured? Front Plant Sci. 2017;8:663. pmid:28491077
- 5. Isman MB. Botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annu Rev Entomol. 2006;51:45–66. pmid:16332203
- 6. Regnault-Roger C, Vincent C, Arnason JT. Essential oils in insect control: low-risk products in a high-stakes world. Annu Rev Entomol. 2012;57:405–24. pmid:21942843
- 7. Campos EVR, Proença PLF, Oliveira JL, Bakshi M, Abhilash PC, Fraceto LF. Use of botanical insecticides for sustainable agriculture: Future perspectives. Ecol Indic. 2019;105:483–95.
- 8. Campolo O, Malacrinò A, Zappalà L, Laudani F, Chiera E, Serra D, et al. Fumigant bioactivity of five Citrus essential oils against Tribolium confusum. Phytoparasitica. 2014;42:223–33.
- 9. Isman MB. A renaissance for botanical insecticides? Pest Manag Sci. 2015;71:1587–90. pmid:26251334
- 10. Campolo O, Romeo FV, Algeri GM, Laudani F, Malacrinò A, Timpanaro N, et al. Larvicidal effects of four citrus peel essential oils against the arbovirus vector Aedes albopictus (Diptera: Culicidae). J Econ Entomol. 2015;109(1):360–5. pmid:26357845
- 11. Pavela R, Benelli G. Essential oils as ecofriendly biopesticides? Challenges and constraints. Trends Plant Sci. 2016;21(12):1000–7. pmid:27789158
- 12. Campolo O, Cherif A, Ricupero M, Siscaro G, Grissa-Lebdi K, Russo A, et al. Citrus peel essential oil nanoformulations to control the tomato borer, Tuta absoluta: chemical properties and biological activity. Sci Rep. 2017;7:13036. pmid:29026207
- 13. Conti B, Canale A, Cioni PL, Flamini G, Rifici A. Hyptis suaveolens and Hyptis spicigera (Lamiaceae) essential oils: qualitative analysis, contact toxicity and repellent activity against Sitophilus granarius (L.) (Coleoptera: Dryophthoridae). J Pest Sci. 2011;84(2):219–28.
- 14. Krčmar S, Gvozdić V. 2016 Field studies of the efficacy of some commercially available essential oils against horse flies (Diptera: Tabanidae). Entomol Gen. 2016;36(2):97–105.
- 15. Maradufu A, Lubega R, Dorn F. Isolation of (5E)-ocimenone, a mosquito larvicide from Tagetes minuta. Lloydia. 1978;41(2):181–3.
- 16. Green MM, Singer JM, Sutherland DJ, Hibben CR. Larvicidal activity of Tagetes minuta (marigold) toward Aedes aegypti. J Am Mosq Control Assoc. 1991;7(2):282–6. pmid:1895085
- 17. Sharma M, Saxena RC. Phytotoxicological evaluation of Tagetes erectes on aquatic stages of Anopheles stephensi. Indian J Malariol. 1994;31(1):21–6. pmid:7958125
- 18. Perich MJ, Wells C, Bertsch W, Tredway KE. Toxicity of extracts from three Tagetes against adults and larvae of yellow fever mosquito and Anopheles stephensi (Diptera: Culicidae). J Med Entomol. 1994;31(6):833–7. pmid:7815394
- 19. Perich MJ, Wells C, Bertsch W, Tredway KE. Isolation of the insecticidal components of Tagetes minuta (Compositae) against mosquito larvae and adults. J Am Mosq Control Assoc. 1995;11(3):307–10. pmid:8551298
- 20. Hadjiakhoondi A, Vatandoost H, Khanavi M, Abaee MR, Karami M. Biochemical investigation of different extracts and larvicidal activity of Tagetes minuta L. on Anopheles stephensi larvae. Iran J Pharm Sci. 2005;1(2):81–4.
- 21. Dinesh DS, Kumari S, Kumar V, Das P. The potentiality of botanicals and their products as an alternative to chemical insecticides to sandflies (Diptera: Psychodidae): a review. J Vector Borne Dis. 2014;51(1):1–7. pmid:24717195
- 22. Morallo-Rejesus B, Eroles LC. Two insecticidal principles from marigold (Tagetes spp.) roots. Philipp Ent. 1978;4:87–97.
- 23. Weaver DK, Wells D, Dunkel FV, Bertsch W, Sing SE, Sriharan S. Insecticidal activity of floral, foliar, and root extracts of Tagetes minuta (Asterales: Asteraceae) against adult Mexican bean weevils (Coleoptera: Bruchidae). J Econ Entomol. 1994;87(6):1718–25.
- 24. Weaver DK, Zetileh LJ, Wells CD, Baker JE, Bertsch W, Throne JE. Toxicity of fractionated and degraded Mexican marigold floral extract to adult Sitophilus zeamais (Coleoptera: Curculionidae). J Econ Entomol. 1997;90(6):1678–83.
- 25. Kéïta SM, Vincent C, Schmit J-P, Ramaswamy S, Bélanger A. Effect of various essential oils on Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). J Stored Prod Res. 2000;36(4):355–64. pmid:10880813
- 26. Boeke SJ, Baumgart IR, van Loon JJA, van Huis A, Dicke M, Kossou DK. Toxicity and repellence of African plants traditionally used for the protection of stored cowpea against Callosobruchus maculatus. J Stored Prod Res. 2004;40(4):423–38.
- 27. Elango G, Abdul Rahuman A, Kamaraj C, Bagavan A, Abduz Zahir A, Santhoshkumar T, et al. Efficacy of medicinal plant extracts against Formosan subterranean termite, Coptotermes formosanus. Ind Crops Prod. 2012;36(1):524–30.
- 28. Cestari IM, Sarti SJ, Waib CM, Branco AC. Evaluation of the potential insecticide activity of Tagetes minuta (Asteraceae) essential oil against the head lice Pediculus humanus capitis (Phthiraptera: Pediculidae). Neotrop Entomol. 2004;33(6):805–7.
- 29. Politi FAS, Nascimento JD, da Silva AA, Moro IJ, Garcia ML, Guido RVC, et al. Insecticidal activity of an essential oil of Tagetes patula L. (Asteraceae) on common bed bug Cimex lectularius L. and molecular docking of major compounds at the catalytic site of ClAChE1. Parasitol Res. 2017;116(1):415–24. pmid:27838836
- 30. Ravikumar P. Chemical examination and insecticidal properties of Tagetes erecta and Tagetes patula. Asian J Bio Sci. 2010;5(1):29–31.
- 31. Leger C, Riga E. Evaluation of marigolds and entomopathogenic nematodes for control of the cabbage maggot Delia radicum. J Sustain Agr. 2009;33(2):128–41.
- 32. Aldana-Llanos L, Salinas-Sánchez DO, Valdés-Estrada ME, Gutiérrez-Ochoa M, Rodríguez Flores EY, Navarro-García VM. Biological activity of dose extracts of Tagetes erecta L. on Spodoptera frugiperda (J. E. Smith). Southwest Entomol. 2012;37(1):31–8.
- 33. Andreotti R, Garcia MV, Cunha RC, Barros JC. Protective action of Tagetes minuta (Asteraceae) essential oil in the control of Rhipicephalus microplus (Canestrini, 1887) (Acari: Ixodidae) in a cattle pen trial. Vet Parasitol. 2013;197(1-2):341–5. pmid:23778081
- 34. Politi FAS, Souza-Moreira TM, Rodrigues ER, Queiroz GM, Figueira GM, Januário AH, et al. Chemical characterization and acaricide potential of essential oil from aerial parts of Tagetes patula L. (Asteraceae) against engorged adult females of Rhipicephalus sanguineus (Latreille, 1806). Parasitol Res. 2013;112(6):2261–8. pmid:23508761
- 35. Hooks CRR, Wang K-H, Ploeg A, McSorley R. Using marigold (Tagetes spp.) as a cover crop to protect crops from plant-parasitic nematodes. Appl Soil Ecol. 2010;46(3):307–20.
- 36. Marahatta SP, Wang KH, Sipes BS, Hooks CRR. Effects of Tagetes patula on active and inactive stages of root-knot nematodes. J Nematol. 2012;44(1):26–30. pmid:23482862
- 37. Palacios-Landín J, Mendoza-de Gives P, Salinas-Sánchez DO, López-Arellano ME, Liébano-Hernández E, Hernández-Velázquez VM, et al. In vitro and in vivo nematocidal activity of Allium sativum and Tagetes erecta extracts against Haemonchus contortus. Turkiye Parazitol Derg. 2015;39(4):260–4. pmid:26809911
- 38.
Rodríguez E, Mabry TJ. Tageteae - chemical review. In Heywood VH, Harborne JB, Turner BL, editors. The biology and chemistry of the Compositae vol 2. London, New York: Academic Press; 1977. p. 785–97.
- 39. Arnason T, Swain T, Wat C-K, Graham EA, Partington S, Towers GHN, et al. Mosquito larvicidal activity of polyacetylenes from species in the Asteraceae. Biochem Syst Ecol. 1981;9(1):63–8.
- 40. Vasudevan P, Kashyap S, Sharma S. Tagetes: a multipurpose plant. Bioresour Technol. 1997;62:29–35.
- 41. Gil A, Ghersa CM, Perelman S. Root thiophenes in Tagetes minuta L. accessions from Argentina: genetic and environmental contribution to changes in concentration and composition. Biochem Syst Ecol. 2002;30(1):1–13.
- 42. Scott DR. An annotated listing of host plants of Lygus hesperus Knight. Bull Entomol Soc Am. 1977;23(1):19–22.
- 43.
Wheeler AG. Biology of the plant bugs (Hemiptera: Miridae): pests, predators, opportunists. Ithaca, NY: Comstock Publishing Associates, Cornell University Press; 2001.
- 44. Naranjo SE, Ellsworth PC. Fifty years of the integrated control concept: moving the model and implementation forward in Arizona. Pest Manage Sci. 2009;65(12):1267–86.
- 45.
Greene JK, Bundy CS, Roberts PM, Leonard BR. Identification and management of common boll-feeding bugs in cotton. 2006 [cited 2019 Nov 1]. Available from: https://www.cottoninc.com/wp-content/uploads/2015/12/Boll-Feeding-Cotton-Bugs-Mgmt.pdf.
- 46.
Godfrey LD, Goodell PB, Natwick ET, Haviland DR, Barlow VM. UC IPM Pest Management Guidelines: Cotton UC ANR Publication 3444; 2019 [cited 2019 Nov 1]. Available from: http://ipm.ucanr.edu/PMG/r114301611.html.
- 47.
Ellsworth PC, Naranjo SE. Whitefly management with insect growth regulators and the influence of lygus controls. Cotton: A College of Agriculture Report No. AZ1123, College of Agriculture, University of Arizona, Tucson, AZ; 1999 [cited 2019 Nov 1]. Available from: https://repository.arizona.edu/handle/10150/197272.
- 48. Palumbo JC, Horowitz AR, Prabhaker N. Insecticidal control and resistance management for Bemisia tabaci. Crop Prot. 2001;20(9):739–65.
- 49. Snodgrass GL, Gore J, Abel CA, Jackson R. Acephate resistance in populations of the tarnished plant bug (Heteroptera: Miridae) from the Mississippi River Delta. J Econ Entomol. 2009;102(2):699–707. pmid:19449652
- 50. Parys KA, Luttrell RG, Snodgrass GL, Portilla MR. Patterns of tarnished plant bug (Hemiptera: Miridae) resistance to pyrethroid insecticides in the Lower Mississippi Delta for 2008–2015: Linkage to pyrethroid use and cotton insect management. J Insect Sci. 2018;18(2):29;1-9.
- 51. Dennehy TJ, DeGain B, Harpold VS, Zaborac M, Morin S, Fabrick JA, et al. Extraordinary resistance to insecticides reveals exotic Q biotype of Bemisia tabaci in the New World. J Econ Entomol. 2010;103(6):2174–86. pmid:21309242
- 52. Ma W, Li X, Dennehy TJ, Lei C, Wang M, DeGain BA, et al. Pyriproxyfen resistance of Bemisia tabaci (Homoptera: Aleyrodidae) biotype b: Metabolic mechanism. J Econ Entomol. 2010;103(1):158–65. pmid:20214381
- 53. Spurgeon DW. Physiological consequences of laboratory rearing of Lygus hesperus (Hemiptera: Miridae). Environ Entomol. 2012;41(2):415–9. pmid:22507017
- 54. Van Ekert E, Chauvigné F, Finn RN, Mathew LG, Hull JJ, Cerdà J, et al. Molecular and functional characterization of Bemisia tabaci aquaporins reveals the water channel diversity of hemipteran insects. Insect Biochem Mol Biol. 2016;77:39–51. pmid:27491441
- 55. Debolt JW. Meridic diet for rearing successive generations of Lygus hesperus. Ann Entomol Soc Am. 1982;75(2):119–22.
- 56.
Institute SAS. SAS release ed. 9.4. SAS Institute, Cary, NC. 2012.
- 57. Dayan FE, Cantrell CL, Duke SO. Natural products in crop protection. Bioorg Med Chem. 2009;17(12):4022–34. pmid:19216080
- 58. Biondi A, Guedes RNC, Wan FH, Desneux N. Ecology, worldwide spread, and management of the invasive South American tomato pinworm, Tuta absoluta: Past, present, and future. Ann Rev Entomol. 2018;63, 239–58.
- 59. Emilie D, Mallent M, Menut C, Chandre F, Martin T. Behavioral response of Bemisia tabaci (Hemiptera: Aleyrodidae) to 20 plant extracts. J Econ Entomol. 2015;108(4):1890–901. pmid:26470332
- 60. Du W, Han X, Wang Y, Qin Y. A primary screening and applying of plant volatiles as repellents to control whitefly Bemisia tabaci (Gennadius) on tomato. Sci Rep. 2016;6:22140. pmid:26907368
- 61. Legaspi JC. A preliminary investigation of giant red mustard (Brassica juncea) as a deterrent of silverleaf whitefly oviposition. J Entomol Sci. 2010;45(3):262–71.
- 62. Conboy NJA, McDaniel T, Ormerod A, George D, Gatehouse AMR, Wharton E, et al. Companion planting with French marigolds protects tomato plants from glasshouse whiteflies through the emission of airborne limonene. PLoS ONE 2019;14(3):e0213071. pmid:30822326
- 63. Hokkanen HMT. Biological and agrotechnical control of the rape blossom beetle Meligethes aeneus (Coleoptera: Nitidulidae). Acta Entomol Fenn. 1989;53:25–30.
- 64. Srinivasan K, Krishna Moorthy PN, Raviprasad TN. African marigold as a trap crop for the management of the fruit borer Helicoverpa armigera on tomato. Int J Pest Manage. 1994;40(1):56–63.
- 65. Hussain B, Bilal S. Marigold as a trap crop against tomato fruit borer (Lepidoptera: Noctuidae). Int J Agric Res. 2007;2(2):185–8.
- 66. Jankowska B, Poniedziałek M, Jędrszczyk E. Effect of intercropping white cabbage with French marigold (Tagetes patula nana L.) and pot marigold (Calendula officinalis L.) on the colonization of plants by pest insects. Folia Hortic. 2009;21(1):95–103.
- 67. Ben-Issa R, Gomez L, Gautier H. Companion plants for aphid pest management. Insects 2017;8(4):112.
- 68. Kumar V, Mahla MK, Lal J, Singh B. Effect of abiotic factors on the seasonal incidence of fruit borer, Helicoverpa armigera (Hub.) on tomato with and without marigold as a trap crop. J Entomol Zool Stud. 2017;5(2):803–7.
- 69. Barman AK, Parajulee MN, Carroll SC. Relative preference of Lygus hesperus (Hemiptera: Miridae) to selected host plants in the field. Insect Sci. 2010;17(6):542–48.
- 70. Jiao X, Xie W, Wang S, Wu Q, Zhou L, Pan H, et al. Host preference and nymph performance of B and Q putative species of Bemisia tabaci on three host plants. J Pest Sci. 2012;85(4):423–30.
- 71. Stern VM, Van den Bosch R, Leigh TF. Strip cutting alfalfa for lygus bug control. Calif Agric. 1964;18(4):4–6.
- 72. Stern VM, Mueller A, Sevacherian V, Way M. Lygus bug control in cotton through alfalfa interplanting. Calif Agric. 1969;23(2):8–10.
- 73. Sevacherian V, Stern VM. Movements of lygus bugs between alfalfa and cotton. Environ Entomol. 1975;4(1):163–5.
- 74. Barbosa P, Hines J, Kaplan I, Martinson H, Szczepaniec A, Szendrei Z. Associational resistance and associational susceptibility: having right or wrong neighbors. Annu Rev Ecol Evol Syst. 2009;40(1):1–20.
- 75. Flores G, Hilje L, Mora GA, Carballo M. Antifeedant activity of botanical crude extracts and their fractions on Bemisia tabaci (Homoptera: Aleyrodidae) adults: II. Sechium pittieri (Cucurbitaceae). Intl J Trop Biol. 2008;56(4):2115–29.
- 76. Hammad EAF, Zeaiter A, Saliba N, Talhouk S. Bioactivity of indigenous medicinal plants against the cotton whitefly, Bemisia tabaci. J Insect Sci. 2014;14:105. pmid:25204756
- 77. Desneux N, Decourtye A, Delpuech JM. The sublethal effects of pesticides on beneficial arthropods. Annu Rev Entomol. 2007;52:81–106. pmid:16842032
- 78. Gupta P, Vasudeva N. Marigold a potential ornamental plant drug. Hamdard Med. 2012;55(1):45–59.