ROLE OF ALLELOCHEMICALS AND TRICHOME DENSITY IN THE RESISTANCE OF TOMATO TO WHITEFLY

In Brazil, the tomato is cultivated in almost all geographic regions even though its yield is significantly reduced by pests and diseases. Among these pests and diseases, whitefly is a major problem that causes direct and indirect damage due to its role in transmission of geminiviruses. The aim of the present study was to compare the degree of resistance to the silverleaf whitefly of tomato plant lines rich in acyl sugars (AS), zingiberene (ZGB), and 2-tridecanone (2-TD) and to establish whether selection for strains with higher densities of glandular trichomes promotes sufficient resistance to this insect pest. The investigated lines were developed by performing back-crossings in tomato (Solanum lycopersicum) from the interspecific crossing of S. lycopersicum x S. habrochaites var. glabratum ‘PI 134417’. Lines rich in AS, ZGB, and 2-TD were tested for their levels of resistance to B. tabaci biotype B. Two tomato plant lines (TOM-584 and TOM-679) with normal AS levels and one wild specimen (PI 134417) with high 2-TD content were used as controls. TOM-687, ZGB-703, and TOM-622 were less preferred by B. tabaci for oviposition, and nymphs exhibited a poorer survival rate on those plant lines than on the susceptible controls, TOM-584 and TOM-679. Allelochemicalrich lines exhibited pest resistance, whereas the controls did not, and the allelochemical-rich lines did not differ significantly from each other in the level of resistance. The results show that the development of tomato plant lines rich in 2-TD, ZGB and AS is an effective option for indirect selection for resistance to B. tabaci biotype B. All lines selected based on high trichome density except for BPX365F-751-05-01-03 exhibited fewer nymphs than the susceptible controls, indicating that selection for more glandular trichomes was generally effective in promoting increased resistance to the silverleaf white flay.


INTRODUCTION
In Brazil, the tomato (Solanum lycopersicum L., syn.Lycopersicon esculentum Mill.) crop is cultivated in virtually every geographic area and is one of the most important crops.Yields are significantly reduced by pests and diseases (FERNANDES et al., 2009).
The silverleaf whitefly, Bemisia tabaci (GENNADIUS, 1889) biotype B (Hemiptera: Aleyrodidae) (= Bemisia argentifolii), is commonly present in plantations, where it causes direct (yield reduction) and indirect damage due to the transmission of geminiviruses.This pest is usually managed by applying insecticides, which may cause damage to humans and the environment and may lead to the development of insecticide resistance (BACCI et al., 2007;SILVA et al., 2009).
Brazilian tomato plant breeding programs aimed at developing pest-resistant cultivars have adopted the strategy of incorporating resistance alleles from wild specimens containing resistance-related allelochemicals into commercial cultivars (MALUF et al., 2007).Maluf et al. (2010) assessed the AS-mediated resistance of the tomato plant to arthropod pests and found that hybrids with intermediate AS contents exhibited satisfactory resistance to Bemisia tabaci biotype B and Tuta absoluta (Lepidoptera: Gelechiidae), similar to homozygous lines with high AS contents.Silva et al. (2009) compared the degrees of resistance to B. tabaci biotype B and Tetranychus urticae (Thysanoptera: Tetranychidae) exhibited by tomato plant hybrids resulting from the crossing of lines with high ZGB and AS contents with those of the parental lines and commercial controls.These authors found that both allelochemicals were effective in conferring pest resistance in both homozygotes and heterozygotes.Maluf et al. (2007) assessed the correlation between glandular trichome density and resistance to the mite T. urticae and found that a high density of glandular trichomes negatively affects the average distance traversed by mites.Therefore, plants with high numbers of trichomes may be selected to develop genotypes resistant to this pest.Aragão, Dantas and Benites (2000) reported that the allelochemical 2-TD has potential for the introgression of resistance to arthropod pests, which can be utilized by selecting genotypes coding for a high density of glandular trichomes.
Therefore, improved tomato plant lines rich in AS, ZGB, and 2-TD were studied regarding their degree of resistance to the silverleaf whitefly (B.tabaci biotype B) and aimed to establish whether glandular trichome density promotes satisfactory degrees of resistance to that pest.

MATERIALS AND METHODS
The experiments were conducted in a greenhouse at the Experimental Vegetable Station at HortiAgro Sementes Ltd., Palmital Farm, Ijaci County, MG, and at the Olericulture Unit of the Universidade Federal de Lavras -UFLA, Lavras County, MG.
For the purpose of infestation by B. tabaci biotype B, as previously identified and used by Silva et al. (2009) and Maluf et al. (2010), silverleaf whiteflies were grown in a protected greenhouse with a 12-m 2 domed protective structure covered by transparent plastic.Adult insects were randomly collected at several tomato cv.Santa Clara plantations in the Lavras-MG area and transferred to the protected greenhouse.The tomato cultivar Santa Clara was used as a substrate for oviposition and, later, as food for the nymphs.Twenty days after transplantation, when several cv.Santa Clara plants exhibited signs of damage caused by infestation (wilting, leaf drop, small fruits), the various investigated genotypes were transported to the protected greenhouse that had been previously infested by a population of B. tabaci biotype B. Six days after infestation, oviposition was assessed by counting eggs; four leaflets from the upper third of the fourth leaf were sampled using a binocular stereoscopic microscope under 20X to 80X magnification.Twenty-three days after infestation, the same sampled leaflets were assessed for nymph development to identify the last instar or pupal stage, also using a binocular stereoscopic microscope.
Once satisfied the assumptions of ANOVA , a fully randomized design was used Analysis of variance was applied to the results of the test of resistance to silverleaf whitefly, and the averages of the genotypes were compared using Tukey's test (p  0.05) as implemented in the SAS software (STATISTICAL ANALYSIS SYSTEM INSTITUTE-SAS, 1989).Selected contrasts between groups of genotypes with different allelochemical contents were calculated to characterize possible differences in the degree of pest resistance as a function of allelochemical content.Finally, the pearson' correlation between the number of eggs of B. tabaci biotype B and the glandular trichome density of the tomato plant leaflets was assessed (this density was measured in the previous generation by MALUF et al., 2007).The amount of trichomes is similar to that found by Maluf et al. (2007) because we used the same materials, and this analysis merely required counting the trichomes present in the sample, although sampling error may have occurred.

RESULTS AND DISCUSSION
Oviposition by adults and number nymphs of B. tabaci biotype B were affected by the presence of the allelochemicals 2-TD, ZGB, and AS in the assessed tomato plant genotypes (oviposition: F=83,12, Pr>F 0,0001; nymphs: F=105,77, Pr>F 0,0001) (Table 1).Lines TOM-622, TOM-687, and ZGB-703 were less preferred by insect pest for oviposition and interfered with the survival of nymphs more than the susceptible controls TOM-584 and TOM-679.
None of the assessed genotypes was as resistant to the silverleaf whitefly, based on the average number of eggs and nymphs present, as the wild specimen PI 134417 (Table 1).This indicates that this genotype, whose leaflets are rich in methyl-ketones (2-TD in particular), allelochemicals might not be the only factor contributing to resistance to this pest.Similar results were found by Maluf et al. (2007) and Oriani, Vendramim and Vasconcelos (2011).The genotypes corresponding to the F7 and F5 generations, except for genotype BPX-365F-899-07-04-02, were less preferred by the silverleaf whitefly than the control TOM-584, particularly based on the average number of eggs on the leaflets (Table 1).Nombela and Muñiz (2010) found the satisfactory resistance of tomato plants to B. tabaci biotype B to be mediated by the isolation and cloning of the gene Mi-1.This preference for non genotypes under study, may be associated with gene activity Mi-1.
The contrast estimates between the groups of assessed genotypes (Table 2) show that genotype PI 134417 was less preferred for oviposition and nymph survival than genotypes with low allelochemical contents (contrast C1), indicating the probable effect of the allelochemicals and of high glandular trichome density on the resistance to the pest.
Contrasts C2, C3, and C4 indicated that the allelochemicals 2-TD, AS, and ZGB at the levels exhibited by lines TOM-622, TOM-687, and ZGB-703, respectively, were broadly effective in the induction of a lower preference for oviposition and lower number nymphs compared to the lines with low contents of these allelochemicals.
Similar results as indirect selection were found by Oliveira et al. (2012), selected genotypes with the highest trichome density and found levels of resistance to the moth Tuta absoluta that were much higher than those exhibited by susceptible controls, and treatment BPX-367D-238-02 was particularly effective.Maluf et al. (2007) found similar results of indirect selection for resistance to the red spider mite T. urticae.This finding indicates that a correlation exists between trichome density and silverleaf whitefly oviposition; that is, the higher the glandular trichome density, the lower the level of oviposition (Tables 3 and 4).Oriani, Vendramim and Vasconcelos (2011) found genotypes with very high antixenosis with regard to oviposition associated with the presence of type IV glandular trichomes.According to Aragão, Dantas and Benitas (2000), who identified and quantified foliar trichomes in tomato specimens, leaflets with the highest concentrations of 2-TD were associated with the highest glandular trichome densities.However, that correlation was not perfect because the measurement of glandular trichome density is associated with sampling error and does not consider possible differences in the concentration of allelochemicals in each trichome.
Table 3 -Mean density (nº/cm 2 ) of glandular trichomes on the abaxial side and adaxial side and the total glandular density on both sides of the plants obtained during the tomato genotype selection for the highest density of trichomes performed by Maluf et al. (2007).Analogous results with the allelochemicals AS and ZGB were found by Silva et al. (2009), in which AS-rich (TOM-688 and TOM-689) and ZGB-rich (ZGB-703 and ZGB-704) lines exhibited greater resistance to the silverleaf whitefly.However, Silva et al. (2009) observed that ZGB was significantly more efficient than AS in reducing number nymphs, indicating that ZGB may bestow a higher degree of antixenosis than AS.
Large differences in oviposition and reduced nymph survival were not observed in the present study between the AS-rich (TOM-687), 2-TD-rich (TOM-622), and ZGB-rich (ZGB-703) lines (Table 1), except for contrast C6 (Table 2); this finding appears to indicate that 2-TD induced less preference for oviposition and number nymphs than AS.

Table 1 -
Number of eggs and nymphs of B. tabaci biotype B on different tomato genotypes.

Table 2 -
Contrasts of interest estimates between groups of genotypes with different levels of allelochemicals.*, * Significant (p  0.01) and (p  0.05), respectively, based on the F test. *