Silverleaf whitefly-resistant common beans: an investigation of antibiosis and/or antixenosis

: The silverleaf whitefly Bemisia tabaci biotype B is one of the most important sucking pests of common bean, which causes severe damages and requires frequently synthetic insecticides spraying to protect crop productivity. The use of common bean cultivars resistant to whitefly attack is an important strategy within an integrated pest management (IPM) program. The biological development of B. tabaci confined to 17 bean genotypes was evaluated in greenhouse trials to verify the occurrence of antibiosis. Whitefly adults were released on plants of these genotypes to oviposit, afterward the incubation period of eggs, nymphal period, complete development period (egg–adult), and viability of the silverleaf whitefly nymphs were recorded. As main results, genotype CHIP 300 prolonged the developmental period from egg to adult (~10 days) and BRS Estilo, Arcelina 4, IPR Garça, Tybatã, CHIP 300, IPR Eldorado, H96A102-1-1-1-52, SCS-202-Guará and CHIB 06 caused nymphal mortality, suggesting high levels of antibiosis and/or antixenosis. These genotypes may be helpful in common bean breeding programs aimed at developing commercial cultivars resistant to B. tabaci biotype B.


INTRODUCTION
Common bean (Phaseolus vulgaris L., Fabaceae) has great socioeconomic importance in Brazil, being widely consumed as a source of proteins and minerals for people´s diet (Carvalho et al. 2014), and besides that, it is a source of income for thousands of agricultural producers, especially family farmers (FAO 2015). The occurrence of insect pests and diseases of common bean are one of the main causes for the reduction of bean productivity (Moraes et al. 2006;Costa et al. 2018).
The silverleaf whitefly, Bemisia tabaci (Gennadius 1889) (Hemiptera: Aleyrodidae) biotype B, is one of the main sucking pests that colonize common bean crops (Musa and Ren 2005;Boykin et al. 2018). Bemisia tabaci is considered a complex of cryptic species that are morphologically indistinguishable, with a total of 43 identified species (De Barro et al. 2011;Tay et al. 2017). Some authors considered biotype B as Middle East-Asia Minor 1 (MEAM1) (Dinsdale et al. 2010;De Barro et al. 2011). However, the authors of the present study chose to use the older nomenclature that is still adopted by many researchers.
Bemisia tabaci biotype B can cause severe direct and indirect damage to the plants. The direct injury is due to feeding of nymphs and adults, which suck the phloem sap and inject toxins, impairing the vegetative and reproductive development of the plants (Villas Bôas 2005). Due to the large volume of honeydew excreted during feeding, there is usually an increase in the incidence of sooty mold (Capnodium sp.), with negative impacts on photosynthesis and crop productivity (Musa and T. L. B. Santos et al. washed coarse sand, and autoclaved organic matter (tanned manure), in a ratio of 1:1:1 (v/v/v), and commercial substrate (Plantamax, Joinville, SC, Brazil) in a ratio of 3:1 (v/v). The plants were housed in a greenhouse, free from insect infestation, and received the fertilization recommended for the crop (Fancelli 2010). When they reached the phenological stage V3-V4 (Valle et al. 2012), the plants were used in the trials.

Colony of Bemisia tabaci biotype B
The initial population of B. tabaci biotype B was obtained from IAC and maintained in a greenhouse (2.5 × 2.5 × 2 m) closed on the sides and on the ceiling with glass and an anti-aphid screen. For the maintenance of the insects, cabbage plants [Brassica oleracea (L.) var. acephala] were grown in plastic pots (2.5 L), and were irrigated and replaced as needed, in order to maintain their nutritional quality and also the vigor of populations of B. tabaci biotype B. This population was also characterized was characterized before the start of the research to confirm the biotype (Walsh et al. 1991;Simon et al. 1994;De Barro et al. 2003).

Bioassays
The biological performance of B. tabaci biotype B on the different genotypes was evaluated to verify the possible expression of antibiosis-resistance. Accordingly, the leaves (of the middle third) of the genotypes were individualized with cages made of "fabric voile" tissue (15 × 15 cm), which were fixed to the petioles of the leaflets with a satin ribbon. With the aid of a buccal aspirator, 150 pairs of whiteflies were collected from the breeding stock and released inside the cages, where they remained for 24 h, in order to obtain the eggs on the previously selected leaves. After this period, the cages were removed as well as the insects from the plants.
Under a stereoscopic microscope (40× magnification), the abaxial face of the leaflets was examined and an area containing 30 viable eggs per leaflet was delimited with Glitter (Acrilex, São Bernardo do Campo, SP, Brazil). Egg surplus was removed using cotton swabs (Cotonetes, Johnson & Johnson, São Paulo, SP, Brazil) (Cruz et al. 2014). Three pots per genotype were used and each leaflet (2 per plant) represented one replicate, totaling six replicates per genotype (n = 180), arranged under a randomized design.
The insects were daily observed to evaluate the following biological parameters: incubation period, duration of instars, total nymphal period, development period from egg to adult, mortality of nymphal instars, and nymphal viability.

Statistical analysis
The normality of residuals and homogeneity of variances were verified using the Shapiro-Wilk and Levene tests, respectively. When the assumptions were satisfied, the data were subjected to the analysis of variance using the F test and, when there was a difference between the treatments, the means were compared using the Fisher LSD test (p > 0.05). For all analyses, the statistical package PROC MIXED-SAS, version 9.2 (SAS Institute 2008) was used.

RESULTS
The duration of the embryonic development of B. tabaci biotype B in the different genotypes of common bean varied from 8.40 to 10.57 days, with the highest mean values observed in the genotypes SCS Predileto, BRS Notável, SCS 202 Guará, CHIP 300, and Arcelina 4 ( Table 2). The genotypes Tybatã, Pérola, IPR Eldorado, and IPR Garça had the shortest incubation periods.
There was a large variation in the total nymphal period of B. tabaci biotype B, with emphasis on the CHIP 300 genotype (24.91 days), which extended the nymphal period by approximately nine days in comparison to the standard susceptible genotype (Pérola) ( Table 2), differing from all other genotypes. In CHIP 348 (21.84 days) and H96A102-1-1-1-52 (21.60 days), intermediate prolongation was observed compared with the standard genotype, differing from most genotypes ( Table 2). The largest prolongation of the egg-adult period was observed in the CHIP 300 genotype (34.22 days), which differed from all other genotypes (Fig. 1). In turn, the susceptible standard genotype (Pérola) had the shortest egg-adult developmental period (24.36 days) (Fig. 1).

DISCUSSION
Reportedly, phytophagous insects consume large amounts of tissues and suck sap of various plant structures, despite the immense variation in the amount of nutrients, as well as the existence of innumerable physical and chemical barriers developed by plants to inhibit or prevent the attack (Bernays and Chapman 1994). However, the acceptance or rejection of host plants by insects depends on defense responses used by plants. Thus, successful colonization of pest insects depends on the presence or absence of various secondary metabolites associated with the possible host plant (Douglas 2018).
In general, insects attempting to colonize plants with antibiosis resistance, that is, plants with the capacity to affect their biology, present reductions in size and weight, diverse deformities, and prolongations in the lifecycle phases and, consequently, high mortality rates (Painter 1951). In the present study, the incubation period of the whitefly eggs in some of the evaluated genotypes was observed; in similar studies, the incubation period of the eggs of B. tabaci biotype B in bean genotypes ranged from 8.00 to 11.00 days (Torres et al. 2012;Peixoto and Boiça Júnior 2014). In the present study, the Tybatã and Pérola genotypes had the shortest incubation periods (8.40 and 8.66 days, respectively), consistent with the study by Peixoto and Boiça Júnior (2014), who observed that the incubation periods for these two genotypes ranged from 8.03 to 8.59 days. The change in the incubation period may be associated with biochemical causes related to common bean genotypes, or even to environmental factors (Smith 2005). Some authors suggest that the pedicel, besides fixing eggs on the plants, works absorbing water and even other compounds present on the plant, interfering on the whitefly embryonic development on common bean (Gameel 1974, Byrne andBellows Junior 1991). In another study with B. tabaci biotype B in common bean, authors suggest that prolonged incubation periods might be influenced by low temperatures and humidity (Oriani and Lara 2000). However, in the present study these factors have not been evaluated and may be considered in further studies.
Genotype CHIP 300 (24.91 days) prolonged the nymphal period of the whitefly by approximately nine days compared with the susceptibility-standard genotype, Pérola (15.73 days). Such an extension may be associated with the presence of morphological factors, such as waxiness and/or trichomes (Glas et al. 2012), or even by chemical factors associated with resistance (Bernays and Chapman 1994;Smith 2005;Douglas 2018). The high prolongation of the instars from insect pests in the genotypes CHIP 300, CHIP 348, and H96A102-1-1-1-52 suggests the occurrence of antibiosis and/or antixenosis type resistance in the respective genotypes (Painter 1951).
The largest prolongation from egg to adult was observed in the genotype CHIP 300 (34.22 days), which required approximately 10 days more to complete the cycle compared with the susceptible Pérola genotype (24.36 days). This may be due to a lower nutritional adequacy or the presence of antibiotic factors in this genotype, which are aspects that must be better investigated in future studies. Based on the available literature, the duration of the egg to adult period varies between bean genotypes, and there are reports of periods between 16.20 and 41.00 days (Oriani and Lara 2000;Torres et al. 2012). Some references reported a large variation in the development period of the whitefly on cowpea genotypes (Cruz et al. 2014), cabbage (Villas Bôas et al. 1997, cotton (Prado et al. 2015), soybean (Cruz and Baldin 2016), tomato (Baldin et al. 2005), squash varieties (Baldin and Benduzzi 2010), melon (Baldin et al. 2012) and, more recently, peppers, where a duration of 30.25 days was observed (Pantoja et al. 2018), similar to the present study.
In a study with the genotypes Iapar 81 and IPR Eldorado, high nymphal viability of whitefly was verified, with rates of 88.70 and 69.30%, respectively (Silva et al. 2014), in contrast to the present study, where viability in the two genotypes did not exceed 45%. These divergences are probably associated with the methodological or climatic differences (uninformed temperature) employed in the study of these authors. Other studies reporting low rates of nymphal viability of whitefly to antibiosis were also described in different hosts, such as cowpea (45.50 to 89.10%) (Cruz et al. 2014), soybean (68.60% to 89, 60%) (Cruz et al. 2016), zucchini (36.10 to 100%) (Baldin and Beneduzzi 2010), and peppers (0 to 25%) (Pantoja et al. 2018). As they negatively affect the performance of the insect's immature stages, the plants with antibiosis commonly cause high rates of nymphal or larval mortality, compromising the emergence of adults (Painter 1951;Panda and Khush 1995;Smith 2005), as verified in some genotypes in the present study.
The genotypes that demonstrated high levels of mortality in the different nymphal stages were CHIP 300, BRS Estilo, Arcelina 4, IPR Garça, and Tybatã. The mortality increased from the second instar, probably owing to increased insect feeding activity. Although the five genotypes showed a significant deleterious effect on the whitefly nymphs, it was observed in the CHIP 300 genotype that the effects were more intense, with common findings of individuals with deformities ( Fig. 4 b), and difficulties in the molting processes (Fig. 4 d) and adult emergence (Fig. 4 f), compared with the same-stage individuals confined to the susceptible Pérola genotype (Fig. 4 a, c, e). The images (Fig. 4 b, d, f) indicate antibiosis as a mechanism of resistance in these genotypes. However, new studies are required to further elucidate the possible role of chemical resistance factors such as enzyme inhibitors or antibiotic compounds (alkaloids, flavonoids, and terpenoids) (Kubo and Hanke 1986) involved in the resistance of these materials.
Although the studies that characterized the expression of antibiosis in bean genotypes to whitefly are scarce, some authors have suggested that secondary compounds could be responsible for the negative effects on the biology of B. tabaci biotype B, as seeing in a study carried out with the Arcelina bean genotype 4 cultivated during the dry season, the nymphal mortality index of 80% of B. tabaci biotype B was verified (Oriani and Lara 2000). In the study, the authors suggested the presence of the arcelin protein as a possible resistance factor in the tested genotype, which may be responsible for the high mortality of insect nymphs. In soybean crop, it has been suggested that flavonoid-bearing genotypes may also negatively affect the biology of the whitefly (Vieira et al. 2016). In cotton genotypes, the presence of higher levels of gossypol in certain materials was negatively correlated with the biology of B. tabaci biotype B (Guo et al. 2013).

CONCLUSION
Considering all the results obtained in the biological performance tests of B. tabaci biotype B in common bean genotypes, it was verified that the CHIP 300 genotype caused a greater prolongation of the egg to adult period of B. tabaci biotype B, indicating the expression of antibiosis and/or antixenosis. In addition, CHIP 300 and the genotypes BRS Estilo, Arcelina 4, IPR Garça, Tybatã, IPR Eldorado, H96A102-1-1-1-52, and CHIB 06 negatively affected the development (viability) of the whitefly, indicating high levels of resistance through antibiosis and/or antixenosis. However, the resistance factors associated with these materials must be better investigated by characterizing the biochemical composition of these genotypes. Thus, these genotypes may constitute important sources of resistance to B. tabaci biotype B for breeding programs to obtain resistant cultivars.