Pepper genotypes

Fifteen landrace genotypes of C. annuum, including the cultivated Huero, Dulce, X´catic, Verde and the semiwilds Maax, Blanco, Pozol, Picopaloma, Miraparriba, Pijadegato, Crespo, Chawa, Sucurre, Amaxito and Simojovel were collected in Southern Mexico (Chiapas, Campeche, Yucatan). Seeds were extracted manually from dried fruits collected in the field, home gardens or local rural markets. The commercial genotype Jalapeño was included as a control for susceptibility to B. tabaci.

Planting and seedling management

Seeds were immersed for 48 h in 250 mg L−1 gibberellic acid (Plant Health Care, Mexico) under constant oxygenation by using an aquarium air pump (Elite®, USA) to homogenize germination. Seeds were sown in 200 square plug polystyrene trays (45 × 70 cm) in a sphagnum peat moss substrate (Cosmocel, Mexico). The substrate was maintained at 60 % moisture. The seedlings were fertilized with 19N:19P:19K (Poly-feed triple 19, Haifa, Mexico) at 2 g L-1 dissolved in the irrigation water. Thirty-five days after sowing, the seedlings were individually transplanted in 2 L pots containing substrate composed of red soil, peat moss and fine gravel in a weight proportion of 50, 30 and 20 %, respectively. Plants in pots were fertilized with 4.5 g L-1 of the previously described fertilizer. Plants were kept into a greenhouse at the Instituto Tecnológico de Conkal, where the experiments were carried out, for 10 days after transplanting plants (45 days after sowing).

Colony of Bemisia tabaci

Adult whiteflies (B. tabaci) were originally collected from habanero pepper (Capsicum chinense Jacq.) agroecosystems in Conkal, Yucatan, Mexico. The stock colony was reared on habanero pepper into a greenhouse at the Instituto Tecnológico de Conkal, Yucatan, Mexico. To have homogeneous cohorts of B. tabaci for the experiments,a separated colony was maintained and reproduced on healthy plants of habanero pepper in entomological cages (1.2 × 1.2 × 1.2 m) made of aluminum and anti- aphid mesh and placed under the same greenhouse as the stock colony (Ballina-Gomez et al., 2013). Adults from homogeneous cohorts (1-2 days old) were collected with a small mouth operated aspirator made of a 50 mL glass jar with two openings on the screwable cap, one opening was connected to 1-cm diameter rubber tube and the other one was connected to a 5-mm diameter rigid plastic tube.

The free ending of the rigid plastic tube was placed on leaves where B. tabaci adults were settled, while a gently suction was applied with the mouth to the free ending of the rubber tube. Adults were kept in the glass jar for no more than 5 min before being transferred to the entomological cages for the bioassay on oviposition preference or being transferred to a fridge at 4-5 ºC where they were briefly chilled (1-2 minutes) to immobilize. Immobilized adults were observed under the stereomicroscope. Females were separated based on body size and abdomen shape. Adult females were then immediately transferred to the clip cages with the aid of a fine camel-hair brush for the experiments on nymphal mortality.

Bioassay of oviposition preference

For oviposition preference, free-choice bioassay was performed as described by ^{Ballina-Gómez et al. (2013)}. Healthy plants (45 days after sowing) were kept in entomological cages as previously described for whitefly rearing. Ten cages were set up for this experiment. A single cage contained 32 randomly distributed plants (two plants of each genotype). In total, 20 plants per pepper genotype were evaluated. A plant represented a replicate. Infestation of plants set in the entomological cages was carried out with unsexed adults. Individuals were released from the glass jar placed in the center of the cage. Each cage received 640 adults (average rate of 20 individuals per plant). Oviposition was evaluated 2 d after adult release in two leaves of the upper third section of plants. The number of eggs per leaf was recorded using a stereoscopic microscope at 40X (Leica Co., Jalisco, Mexico). After egg counting, leaf area (cm2) was individually measured with an area meter (LI3100, LiCOR, Lincoln, Nebraska, USA). Environmental conditions inside the cages were 18-35 °C of temperature, 65-95 % of relative humidity and natural photoperiod of 13:11 h (light:dark).

Evaluation of nymphal mortality

To evaluate nymphal mortality, the clip cage procedure was used to obtain a homogeneous cohort of nymphs as described by ^{Ballina-Gómez et al. (2013)} and ^{Firdaus et al. (2011)}. Briefly, 20 adult females were confined in a section (1 cm2) of a fully expanded young leaf of a plant, after 24 h, the adults were removed. Some eggs were also removed to left groups of 25 eggs per leaf section. The leaf section with the eggs was enclosed again with the clip cage to prevent migration of first-instar nymphs. After 15 d after the eggs were laid, leaves were cut and nymphal mortality was evaluated by observing the leaf sections with nymphs in a stereoscopic microscope at 40X (BME; Leica, Jalisco, Mexico). Nymphs were considered dead if they were blackish, deformed or dry (^{Ballina-Gomez et al., 2013}). Twenty leaves (one per plant) were evaluated per genotype. A leaf represented a replicate. A second assay of nymphal mortality was conducted with only four genotypes, including two that produced high mortality and two that produced low mortality of B. tabaci nymphs. The environmental conditions for the assays of nymphal mortality were the same as described for the oviposition preference study.

Evaluation of morphological leaf traits

Evaluations were performed in fully expanded young leaves of the upper stratum of plants. For all traits, a leaf represented a replicate. Each trait was assessed in a separated group of plants. Trichome density was evaluated in two leaves per plant. Forty leaves were assessed per genotype. The classification system consisted of five levels or classes of trichome density (^{Matos et al., 2011}), where the approximate average of trichomes in 5 cm2 of leaf section was 0 for class 1, 8.7 for class 2, 73.6 for class 3, 211.7 for class 4 and 900.2 for class 5. Leaf area was measured with a leaf area meter (LI3100C, LI-COR, Lincoln, NE, USA) in two leaves per plant. Twenty plants (40 leaves) were assessed per genotype. Leaf toughness was determined using a TA Plus Penetrometer (Texture Technologies, Hamilton, MA, USA). Leaf toughness was reported as the force required to punch a 4- cm² leaf section; the values were given in kg cm-2 s^-1. One leaf per plant was used. Twenty plants were assessed per genotype.

Chemical composition of leaves

After performing the second assay of nymphal mortality on four pepper genotypes with differential responses on nymphal mortality, four leaves of the middle and upper strata per plant were cut and pooled, which made a group of 80 leaves in total per genotype. Then, four groups of 20 leaves were formed, washed with distilled water containing 5 % neutral detergent (Extran MA02 5 %) and rinsed with distilled water. Each group of 20 leaves was placed in a paper bag and dried at 45 ºC for 72 h. Four samples of 3g of dried leaves were taken per genotype. Each sample represented a replicate. The nitrogen content of samples was determined by the Kjeldahl method. The content of P and K was determined by the wet digestion method. Quantification of mineral elements was carried out by atomic absorption spectroscopy. Total phenolic content (g equivalent of gallic acid per g of dry mass) was determined by the Folin-Ciocalteau method and total flavonoid content (g equivalent of quercetin per g of dry mass) by the aluminum chloride colorimetric method as described by ^{Hossain et al. (2011)}.

Experimental design and data analysis

The experiment was set under a completely randomized design. The pepper genotypes represented the treatments. All data were checked for normality and homoscedasticity. Results for oviposition, nymphal mortality, leaf area and leaf toughness in the 16 genotypes were analyzed by one-way analysis of variance and the Scott-Knott multiple comparison test. This test consists of a cluster analysis when a large amount of means is compared (^{Scott and Knott, 1974}). Results of trichome density were analyzed by the nonparametric Kruskal-Wallis test. To determine the relationship between physical leaf traits with oviposition preference or nymphal mortality, an analysis of correlation was performed.

Data of the second assay of nymphal mortality and chemical composition of leaves were analyzed by analysis of variance and Tukey´s multiple comparison tests. All data analyses were carried out in InfoStat software (InfoStat, Cordoba, Argentina). Differences were considered significant if P ≤ 0.05.

Oviposition preference and nymphal mortality

Table 1 Response of Capsicum annuum genotypes to Bemisia tabaci and physical characteristics of leaves 

^{Mean values ±}standard error with different letter within the same column are significantly different (Scott-Knott test, P ≤ 0.05). Values of trichome density are in categorical data, from a five-classes scale described by Matos et al. (2011).

Results showed significant differences in oviposition preference of B. tabaci among C. annuum genotypes (P= 0.0042). The density of whitefly eggs on the genotypes Jalapeño, Pijadegato, Crespo, X´catic, Chawa, Verde Sucurre, Amaxito and Simojovel (range of 0.19 to 1.7 eggs cm-2) was significantly lower than that of the genotypes Huero, Maax, Blanco, Pozol and Pico Paloma, with 4.3, 3.6, 3.3, 3.1 and 3.1 eggs cm-2 on their leaves, respectively (Table 1).

Nymphal mortality was significantly different among genotypes (P = 0.001). Nymphal mortality was higher on genotypes Pijadegato (45.4 %), Simojovel (51.6 %) and Amaxito (59.9 %) than on the other genotypes (Table 1).

Table 2. Pearson correlation coefficients (r) between physical leaf traits and host response to Bemisia tabaci.

Table 2 Pearson correlation coefficients r between physical leaf traits and host response to Bemisia tabaci  

Physical leaf traits

Physical leaf traits (trichome density, leaf area and leaf toughness) varied among C. annuum genotypes. Leaf area showed significant differences among genotypes (P = 0.001). High density of trichomes (class 4) was observed in leaves of the genotypes Picopaloma, Chawa and Simojovel, whereas the lower density of trichome (class 1) was observed in leaves of Huero, Pozol and Dulce (Table 1). The highest leaf area was observed in the genotypes Sucurre (9.87 cm²), Huero (7.69 cm²) and Pijadegato (7.48 cm2), and the lowest leaf area was recorded in Simojovel (1.44 cm2) (Table 1). Leaf toughness was higher (P = 0.0001) in the genotypes Maax (0.92 kg cm-2 s^-1), Picopaloma (0.89 kg cm^-2 s^-1), Crespo (0.88 kg cm^-2 s^-1), X´catic (0.89 kg cm^-2 s^-1), Chawa (0.96 kg cm^-2 s^-1), Verde (0.89 kg cm^-2 s^-1), Amaxito (0.95 kg cm^-2 s^-1) and Simojovel (0.89 kg cm-2 s-1) relative to that of the other genotypes (Table 1).

Slight negative correlation was observed between trichome density and B. tabaci egg density; likewise, between leaf toughness and egg density; however, these correlations were not significant (P > 0.05) (Table 2).

Chemical composition of leaves

The second assessment of nymphal mortality on four genotypes with differential response to B. tabaci showed that nymphal mortality on Simojovel (85.1 ± 3.9 %) and Amaxito (98.3 ± 2.3 %) was significantly higher than that on X´catik (11.8 ± 4.1 %) and Jalapeño (25.7 ± 6.5 %). Analysis of mineral elements in leaves of these genotypes showed that the content of N in Simojovel was significantly higher than that in Amaxito. In contrast, no differences were observed in content of P and K among genotypes (Table 3). Total phenolics and flavonoids in leaves were significantly different among genotypes. The genotype X´catic had the highest content of total phenolics, whereas the content of flavonoids was significantly higher in leaves of Jalapeño compared to that in Amaxito (Table 4).

Table 3 Mean content (± standard error) of N, P and K in leaves of Capsicum annuum genotypes. 

^.Mean values with different letter within the same column are significantly different (Tukey, P ≤ 0.05).

Table 4. Mean content (± standard error) of total phenolics and flavonoids in leaves of Capsicum annuum genotypes. 

^†For the calculation of total phenolics, the contents are presented as mg equivalent of gallic acid per g of leaf dry mass.

^††For the calculation of total flavonoids, the contents are presented as mg equivalent of quercetin per g of leaf dry mass. Mean values with different letter within the same column are significantly different (Tukey, P ≤ 0.05).