Research Article Received: 12 February 2019 Revised: 14 June 2019 Accepted article published: 25 June 2019 Published online in Wiley Online Library: 27 July 2019 (wileyonlinelibrary.com) DOI 10.1002/ps.5531 Dwarf-cashew resistance to whitefly (Aleurodicus cocois) linked to morphological and histochemical characteristics of leaves Elaine SS Goiana,a Nivia S Dias-Pini,a* Celli R Muniz,a Arlete A Soares,b James C Alves,b Francisco C Vidal-Netoa and Cherre S Bezerra Da Silvac Abstract BACKGROUND: The cashew whitefly (CW), Aleurodicus cocois, is an important pest of cashew in Brazil. The use of resistant plants may be an effective strategy for the control of this pest. In a preliminary assay, we found that dwarf-cashew clones show different levels of resistance to CW. Here, we hypothesized that such resistance is associated with morphological characteristics of cashew leaves and their content of phenolic compounds. RESULTS: We determined (i) the attractiveness and suitability for oviposition of five dwarf-cashew clones towards CW, (ii) the leaf morphology and chemistry of those clones, and (iii) the relationship between leaf characteristics and resistance to CW. In greenhouse multiple-choice assays, PRO143/7 and CCP76 showed, respectively, the lowest and highest counts of both CW adults and eggs. Scanning electron microscopy (SEM) analysis revealed that PRO143/7 and EMBRAPA51 have, respectively, the highest and lowest numbers of leaf glandular trichomes. We found a negative correlation between number of trichomes in the abaxial surface of cashew leaves and CW oviposition. In addition, confocal microscopy analysis and histochemical tests with ferrous sulfate indicated a higher accumulation of phenolic compounds in the resistant clone PRO143/7 relative to the other clones. Dwarf-cashew clones did not significantly differ based on the number of leaf epicuticular striations, and the thickness of both leaf lamina and the epidermal layer. CONCLUSION: The resistance of dwarf-cashew plants to CW is associated with an elevated number of trichomes and accumu- lation of high levels of phenolics in leaves. Additionally, the contribution of epicuticular striation density and thickness of leaf lamina/epidermal layer are insignificant. © 2019 Society of Chemical Industry Keywords: Anacardium occidentale; leaf glandular trichomes; leaf phenolic compounds; leaf lamina; leaf surface; Aleyrodidae; Antixeno- sis; deterrence 1 INTRODUCTION genotypes expressing traits associated with insect resistance 7,8 The cashew whitefly (CW), Aleurodicus cocois Curtis (Hemiptera: could be valuable for breeding and IPM programs. Aleyrodidae), is a sap-sucking insect that causes direct damage to Plant resistance to whiteflies has been associated with a number crops by puncturing leaves during feeding, and indirect damage of factors, including increased epidermal thickness, type and size by promoting the proliferation of sooty molds and viruses.1 CW is of trichomes, production of secondary metabolites, and color of 9–12 13 a major pest of dwarf-cashew (Anacardium occidentale L.) and is the leaves. According to Aoyama and Labinas, enhanced widespread in all growing areas of northeastern Brazil, occurring in epidermal thickness confers rigidity to leaves owing to increased the form of episodic but intensive outbreaks that cause significant numbers of overlapping cellulose microfibrils incrusted with lignin. losses in productivity.2,3 Sap-sucking insects need to insert their mandibular stylets deep Despite the agricultural losses caused by CW, there is a scarcity into plant tissues in order to access the vascular bundles, and of cashew-specific pesticides registered in the Brazilian Ministry a thickened leaf epidermis hinders this process by increasing of Agriculture to control this pest. Under these circumstances, the adoption of an integrated pest management (IPM) strategy involving resistant cashew genotypes is an appealing option. ∗ Correspondence to: NS Dias-Pini, Laboratório de Entomologia, Embrapa The advantages of such an approach include the reduction of Agroindústria Tropical, Rua Doutora Sara Mesquita 2270, Fortaleza, CE, 60511-110, Brazil. E-mail: [email protected] pest populations to levels that do not cause economic damage, positive impacts on the environment, compatibility with other a Laboratório de Entomologia, Embrapa Agroindústria Tropical, Fortaleza, Brazil pest control methods, cost-effectiveness, and lasting results.4,5 Despite the plethora of cashew clones that are commercially b Laboratório de Morfologia e Anatomia Vegetal, Universidade Federal do Ceará, Fortaleza, Brazil available in Brazil,6 little is known about their resistance to CW 464 and respective mechanisms of resistance. Identification of cashew c Laboratório de Entomologia, Embrapa Algodão, Campina Grande, Brazil Pest Manag Sci 2020; 76: 464–471 www.soci.org © 2019 Society of Chemical Industry Mechanisms of dwarf-cashew resistance to whitefly www.soci.org energy demand.14 Moreover, an increase in the number of tector counted. Seedlings were used as an exception to adult plants in trichomes on the leaf surface may act as a physical barrier by this assay because it required plant confinement in cages, which limiting the mobility of nymphs and larvae,15–17 while the release of would not be feasible with adult plants. However, adult 3-year-old secondary compounds from glandular trichomes may affect food plants were used in the next three microscopy experiments. preference, oviposition, and survival.18,19 Here, we hypothesized that the resistance of cashew plants 2.3 Scanning electron microscopy to CW is associated with morphological characteristics of leaves Morphological analyses were carried out by scanning electron and their content of phenolic compounds. We determined (i) microscopy (SEM) using a Tescan (São Bernardo do Campo, Brazil) the relative attractiveness and suitability for oviposition of five Vega 3 microscope under an acceleration voltage of 15 kV. The dwarf-cashew clones towards CW, (ii) the leaf morphology and experimental design was completely randomized with three chemistry of those clones, and (iii) the relationship between leaf replicates each involving five leaf fragments removed from three characteristics and resistance to CW. different individuals of each dwarf-cashew clone. The leaves were collected from 3-year-old adult plants in the experimental station 2 MATERIAL AND METHODS of Embrapa Agroindústria Tropical (Pacajus, CE, Brazil). Plant mate- 2.1 Plant material rial was treated with Karnovsky fixative for 48 h, washed three The dwarf-cashew clones CCP76, BRS226, EMBRAPA51, BRS274 times (10 min each) with phosphate buffer, post-fixed with 1% and PRO143/7, originating from the germplasm bank of Embrapa osmium tetroxide for 1 h, washed in distilled water, dehydrated by Agroindústria Tropical (Fortaleza, CE, Brazil), were selected for treatment with ethanol solutions of increasing concentration (20, investigation of the characteristics associated with resistance to A. 30, 40, 50, 60, 70, 80, 90%) (15 min each) and finally washed three cocois. This selection was based on preliminary experiments where times (15 min each) in 100% ethanol. Samples were submitted the infestation rates of A. cocois on 25 dwarf-cashew genotypes to critical point drying, mounted on stubs, sputter coated with was assessed in the field. The clones that stood out as the most platinum, and examined by SEM. For each sample, we determined 2 susceptible or resistant to A. cocois were selected.20 the number of glandular trichomes in 1.05 mm areas of abaxial surface, the number of epicuticular striations between stomata, the thickness of the leaf lamina (μm), and the thicknesses of the 2.2 Attraction and oviposition assays epidermal layers on the abaxial and adaxial surfaces (μm). The objective of this multiple-choice assay was to correlate leaf characteristics with attraction and oviposition preferences of CW females. The experimental design was completely randomized 2.4 Confocal microscopy with five replicates, each involving one dwarf-cashew seedling Histochemical analyses of leaves of the selected dwarf-cashew featuring mature leaves (120 days old) selected from each of the clones were performed in order to detect the presence of phenols. studied clones. The plants were distributed randomly inside a In the first assay, six leaf fragments were removed from each of 1.0 m cube screen cage and spaced at least 15 cm apart in order the clones and submitted to paradermic and longitudinal sec- to prevent contact between the leaves. One hundred adult female tioning. Samples were separated into two groups, one of which whiteflies (20 insects per plant) were released into the cage and was retained as non-treated control and the other was treated the attraction of insects to plants was evaluated after 24 h by with Neu’s reagent [2-aminoethyl diphenylborinate (10 g L−1)and counting, with the aid of a mirror, the number of insects present polyethylene glycol 4000 (50 g L−1) in 95% ethanol], which binds on the abaxial surfaces of the leaves. The insects were carefully to specific chemical groups and enhances the fluorescence of removed in tubes. Subsequently, the eggs laid on leaves were flavonoids.21 Treated and non-treated samples were mounted Figure 1. Resistance of dwarf-cashew clones to cashew whitefly (CW), Aleurodicus cocois, in greenhouse, multiple-choice tests. (A) Number of CW adults on leaves. (B) Number of CW eggs on leaves. N = 5. Adults: F = 3.47, P = 0.0262. Eggs: F = 3.70, P = 0.0207. Boxes with different letters significantly 4,20 4,20 465 differ from each other according to Tukey’s test ( = 0.05). Pest Manag Sci 2020; 76: 464–471 © 2019 Society of Chemical Industry wileyonlinelibrary.com/journal/ps www.soci.org ESS Goiana et al. Figure 2. Morphological characteristics (mean ± SEM) of leaves of five dwarf-cashew clones with different levels of resistance to the cashew whitefly, Aleurodicuscocois, from scanning electron micrographs. (A) Number of glandular trichomes on the abaxial leaf surface. (B) Number of epicuticular striations between stomata. N = 3 plants, each donating five leaf fragments for analysis. Trichomes: F4,10 = 18.11, P = 0.0001. Striations: F4,10 = 6.21, P = 0.0089.