Zucchini Squash Genotypes Resistant to Whitefly-induced Squash Silverleaf Disorder

Yasmin J. Cardoza and Heather J. McAuslane

Department of Entomology and Nematology, University of Florida, Gainesville, FL, 32611-0620

Susan E. Webb

Central Florida Research and Education Center, University of Florida, Leesburg, FL 34748-8232

Additional index words. Bemisia argentifolii, silverleaf whitefly, zucchini squash, host plant resistance, squash silverleaf disorder

Abstract. Squash silverleaf disorder (SSL) is a physiological disorder induced by feeding of immature Bemisia argentifolii Bellows and Perring which has had an economically important impact on the production of commercially grown Cucurbita species in Israel, the Caribbean, and the United States. Two zucchini squash breeding lines, ZUC76-SLR and ZUC33-SLR/PMR, found to show field SSL resistance in previous tests, were evaluated under greenhouse conditions using 'Zucchini Elite', a commercial zucchini variety susceptible to SSL, as a control. Our findings indicate that tolerance to whitefly feeding is the mechanism of resistance to SSL in ZUC76 SLR and ZUC33-SLR/PMR, additionally, six ZUC76-SLR sister lines were also evaluated for possible resistance to SSL when compared to the SSL-susceptible cultivar 'Zucchini Elite' and the highly SSL-resistant ZUC76-SLR. ZUC76-SLR sister lines tested included ZUC60-SLR, ZUC61-SLR, ZUC70-SLR, ZUC72-SLR, ZUC74-SLR, and ZUC78-SLR. We concluded that at the infestation level tested (100 pairs of whiteflies), five of the six ZUC76-SLR sister lines possess different levels of resistance to whitefly feeding.

The silverleaf whitefly (Bemisia argentifolii) is a devastating pest of cucurbit crops in many regions around the world including Israel (Paris et al., 1987), the United States (Maynard and Cantliffe, 1989), and the Caribbean (Segarra-Carmona et al., 1990). Heavy B. argentifolii infestations can cause wilting, stunting and loss of vigor in the affected plants. Also, heavy honeydew secretions can promote growth of sooty mold on leaf surfaces and thus reduce photosynthesis. Additionally, silverleaf whiteflies are associated with transmission of viral diseases (Bethke et al., 1991; Costa and Brown, 1991) and induction of physiological disorders such as squash silverleaf (SSL) (Maynard and Cantliffe, 1989). In the United States, B. argentifolii has become an important pest of cucurbits since squash silverleaf disorder (SSL) made its first appearance in 1987.

SSL disorder is one of many silverleaf whitefly-associated maladies. SSL is characterized by a developmentally progressive silvering of the adaxial leaf surface, and in severe cases, bleaching of the petioles, flowers, and fruit (Paris et al., 1987). SSL severely affects commercially grown Cucurbita species by reducing photosynthetic capabili

ties (Jimenez et al., 1995; Yokomi et al., 1990) and yield (Costa et al., 1994) in affected plants. Feeding of silverleaf whitefly immatures induces SSL, and severity of the symptoms is positively correlated with nymphal infestation (Costa et al., 1993b; Yokomi et al., 1990). However, the exact mechanism responsible for the onset of this disorder remains unknown and the only means by which SSL economic losses can be reduced is by managing B. argentifolii populations and by developing squash varieties resistant to these insects or to the disorder itself.

Control of B. argentifolii by insecticides is very difficult due to their habit of feeding on the underside of leaves, making them less exposed to chemical applications (Simmons and McCreight, 1996). Additionally, whiteflies have rapidly developed resistance to most commercially available products. This is due to heavy reliance upon insecticides for their management in past years (Prahbacker et al., 1985). For this reason, the identification and quantification of sources of resistance to either the silverleaf whitefly or SSL is an active area of investigation at the present time. Host plant resistance provides a durable and en

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vironmentally safe alternative to chemical control that can be incorporated into integrated pest management practices for whiteflies (de Ponti et al., 1990).

Most commercially grown Cucurbita species are susceptible to SSL (Maynard and Cantliffe, 1989; Simons et al., 1988). Cultivar groups of C. pepo differ, however, in their susceptibility to this disorder (Paris et al., 1993a, 1993b). Additionally, McAuslane et al. (1996) found that zucchini breeding lines ZUC76-SLR and ZUC33-SLR/PMR (referred to as A21-7 and Sunseed 3, respectively) showed resistance to SSL under field conditions.

The objectives of this study were to elucidate the resistance mechanisms to SSL in the resistant zucchini breeding lines, ZUC76-SLR and ZUC33-SLR/PMR and to screen ZUC76-SLR sister lines for possible resistance to SSL compared to the SSL-susceptible 'Zucchini Elite'.

Materials and methods

Plant and insect material. To determine the mechanisms of SSL resistance in the two zucchini breeding lines, tests were performed using 'Zucchini Elite', a variety susceptible to SSL, and zucchini breeding lines resistant to silverleaf disorder, ZUC76-SLR and ZUC33-SLR/PMR. Zucchini breeding lines were obtained from G. Elmstrom and R.B. Carle (Central Florida Research and Education Center, University of Florida, Leesburg). 'Zucchini Elite' was obtained from a commercial company (Harris Seeds, Rochester, N.Y.). For tests designed to screen ZUC76-SLR sister lines for SSL resistance, greenhouse experiments were conducted using 'Elite' zucchini, as a SSL-susceptible control, ZUC76-SLR, as a SSL-resistant control, and six ZUC76-SLR sister lines obtained from R. B. Carle. ZUC76-SLR sister lines tested included ZUC60-SLR, ZUC61-SLR, ZUC70-SLR, ZUC72-SLR, ZUC74-SLR, and ZUC78-SLR.

Individual seeds of each of the lines were sown in seedling-tray cells containing Metromix 200 (Grace Sierra, Milpitas, Calif.) and seedlings were allowed to develop to the first true-leaf stage in the greenhouse. Seedlings were then transplanted to terra-cotta clay pots (15.2 cm in diameter) containing a 1:3 (v: v) mixture of pasteurized sand and Peat-Lite soil mix (Conrad Fafard, Inc.,

Agawan, Mass.). Each plant was fertilized weekly, from transplantation until the end of the experiment, with 100 ml of a 3.38 g·L­1 solution of a soluble fertilizer (20N­20P­20K) (Peters, W.R. Grace, Fogelsville, Pa.).

Whiteflies (B. argentifolii) were obtained from a laboratory colony reared on cotton (Gossypium hirsutum L.) and collard (Brassica oleracea var. acephala L.) in a room maintained at 28 °C, 14 h light/10 h dark photoperiod, and ambient relative humidity. This colony was originally started in May 1994 with insects collected from hibiscus (Hibiscus rosa-sinesis L.) in an experimental greenhouse in Gainesville, Fla.

Experimental procedure. To determine the mechanisms of resistance in ZUC76-SLR and ZUC33-SLR/PMR, plants at the second true-leaf stage were infested with 0, 40, 80, or 160 whitefly pairs. At the time of infestation, each plant was covered with a 1-gal perforated plastic bag (26.4 ¥ 27.5 cm) (Ziploc, DowBrands L.P., Indianapolis, Ind.) to confine insects to a single plant. Whiteflies were aspirated from the laboratory colony into 7-dram plastic vials and then released into a vegetable bag. Whiteflies and bags were removed after 3 d and care was taken to remove all insects from plants. This study was arranged in a randomized complete block design with each level of infestation in each block. Three replicates were set up in the first trial, 4 in a second and an additional 3 in a third trial, for a total of 10 replicates.

Eggs were allowed to hatch and nymphs to develop until the red-eyed nymphal stage to induce SSL symptoms, after which time all leaves were removed and labeled according to block, infestation level, and position on the plant. Leaves were then taken into the laboratory where nymphs were counted and leaves were rated for SSL symptoms. Nymphs on the abaxial surface of the originally infested leaves were counted with the aid of a stereo microscope. Silverleaf symptoms were rated following the system used by Paris et al. (1987). In this system, a number from 0 to 5 is assigned to a leaf according to the severity of the symptoms as follows: 0) no silvering symptoms, 1) silvering in and parallel to less than half of the leaf veins, 2) silvering in all and parallel to more than half of the leaf veins, 3) silvering in all and

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parallel to all leaf veins, 4) all leaf veins and some spaces between veins silvered, and 5) entire upper leaf surface silvered.

Effects of genotype, trial, block nested within trial, and whitefly densities on number of nymphs, for all three genotypes, and on number of silvered leaves, maximal silverleaf rating, summed silverleaf rating, and average silverleaf rating, for 'Elite' and ZUC33-SLR/PMR, were tested using ANOVA in Proc GLM (SAS Institute, 1996). ZUC76-SLR was not included in this analysis because it did not show silvering at any of the whitefly infestation levels tested. Data for summed silverleaf rating were log (x + 1) transformed as needed to conform to assumptions of normality before analysis.

To screen for possible SSL resistance in ZUC76-SLR sister lines, plants at the second true-leaf stage were infested with 100 pairs of whiteflies. Whiteflies were removed after 2 d and care was taken to remove all insects from the plants. Eggs were allowed to hatch and nymphs to develop until the red-eyed nymphal stage to induce symptoms, then nymphs were counted and leaves were rated for silverleaf symptoms in a scale from 0 to 5 as described for the previous experiment. This study was arranged in a randomized block design with each genotype represented in each of 10 replicate blocks.

Results and discussion

Trial, block nested within trial, and genotype did not significantly affect number of nymphs produced from the initial whitefly infestations. Mean (sd) number of nymphs for the original whitefly infestation levels were 0.0 (0.0) for zero, 419.1 (83.5) for 40, 965.7 (229.9) for 80, and 2010.4 (318.9) for 160 whitefly pairs.

ZUC76-SLR did not show silverleaf symptoms at any of the whitefly infestation levels tested and thus was not included in the following analysis. The effect of trial was only found significant for the number of silvered leaves (F = 15.06; df = 2, 47; P = 0.0001) and summed silverleaf rating (F = 16.42; df = 1, 66; P = 0.0001).

Genotype was found to have a significant effect on the number of silvered leaves (F = 89.05; df = 1, 66; P = 0.0001), maximal silverleaf rating (F = 131.82; df = 1, 66; P = 0.0001), summed silverleaf rating (F = 122.31; df = 1, 66; P = 0.0001), and average silverleaf rating (F = 128.77; df = 1, 66; P = 0.0001).

Analysis of variance revealed a significant effect of whitefly density on the maximal silverleaf rating (F = 246.7; df = 3, 27; P = 0.0001; F = 47.61; df = 3, 21; P = 0.0001), average silverleaf rating (F = 401.5; df = 3, 27; P = 0.0001; F = 60.2; df = 3, 21; P = 0.0001), number of silvered leaves (F = 356.0; df = 3, 21; P = 0.0001; F = 72.7; df = 3, 21; P = 0.0001), and summed silverleaf rating (F = 367.3; df = 3, 21; P = 0.0001; F = 90.0; df = 3, 21; P = 0.0001), in 'Elite' and ZUC33-SLR/PMR, respectively (Table 1).

ZUC33-SLR/PMR and ZUC76-SLR were not as affected by insect feeding (i.e., they only exhibited slight silvering) as was 'Elite'. ZUC76-SLR did not show silverleaf symptoms at any of the levels of infestation tested in this study, even though the number of nymphs on each of the genotypes was not significantly different for any of the initial whitefly infestation levels. 'Elite' showed maximal silvering rates even at the lowest infestation level of 40 pairs of whiteflies. On the other hand, ZUC33-SLR/PMR showed slight silverleaf symptoms only at infestation levels above 80 pairs of whiteflies. Additionally, significant

Table 1. Number of silvered leaves, and maximal silverleaf rating, summed and mean silverleaf ratings [versus initial infesting whitefly densities (pairs) in two zucchini genotypes (± standard deviation)].

Silvered leaves Rating

Whitefly (no.) Maximal Summed Avg

density Elite ZUC33 Elite ZUC33 Elite ZUC33 Elite ZUC33

0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

40 4.0 ± 0.8z 0.1 ± 0.3 5.0 ± 0.0z 0.1 ± 0.3 16.0 ± 3.0z 0.1 ± 0.3 4.0 ± 0.4 z 0.1 ± 0.3

80 4.8 ± 0.8 z 2.1 ± 1.7 5.0 ± 0.0z 1.1 ± 0.9 18.8 ± 3.7z 3.0 ± 2.7 3.9 ± 0.5z 1.0 ± 0.7

160 5.5 ± 1.2 z 3.0 ± 0.9 4.9 ± 0.3z 1.9 ± 0.3 22.7 ± 3.9z 4.7 ± 1.9 4.2 ± 0.3z 1.5 ± 0.3

zMeans differed significantly between the two genotypes by Tukeys mean separation test at the 0.05 level.

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Table 2. Mean (± standard deviation) number of silvered leaves and silverleaf ratings for the susceptible control 'Elite' and three zucchini breeding lines affected by SSL symptoms.z

Parameter Elite ZUC61-SLR ZUC74-SLR ZUC78-SLR

Silvered leaves (no.) 4.9 ± 0.3 a 4.3 ± 0.5 a 2.1 ±0.9 b 2.3 ± 0.5 b


Maximal 5.0 ± 0.0 a 5.0 ± 0.0 a 0.9 ± 0.3 b 1.0 ± 0.0 b

Summed 19.8 ± 1.6 a 17.2 ± 1.5 b 2.1 ± 0.9 c 2.3 ± 0.5 c

Average 4.05 ± 0.3 a 4.03 ± 0.3 a 0.9 ± 0.3 b 1.0 ± 0.0 b

zData within a row followed by same letter are not significantly different by Tukeys mean separation test at the 0.05 level.

differences in maximal, summed and average silverleaf ratings between 'Elite' and ZUC33-SLR/PMR. 'Elite' had significantly more silvered leaves and greater maximal, summed and average silverleaf ratings than ZUC33-SLR/PMR in each of the nonzero whitefly infestation levels.

Since the number of nymphs infesting each of the genotypes were found statistically similar for a given initial whitefly infestation level, it can be concluded that resistance in ZUC76-SLR and ZUC33-SLR/PMR is not due to lack of oviposition preference by the adults. Furthermore, it can be said that resistance in the two breeding lines is not caused by the inability of immature whiteflies to develop on these plants thus, tolerance to whitefly feeding is the major mechanism of resistance to SSL in ZUC76-SLR and ZUC33-SLR/PMR zucchini breeding lines.

In the experiment conducted to evaluate SSL resistance in ZUC76-SLR, block did not have a significant effect on any of the variables tested. Additionally, the number of nymphs did not differ significantly among the genotypes tested. Plants averaged 1304 (±139.94) nymphs on the originally infested leaves. Some of the genotypes screened were, however, less susceptible to SSL. Three of the six sister lines, ZUC60-SLR, ZUC70-SLR, and ZUC72-SLR, evaluated in this study did not express any SSL symptoms, so only the remaining three, ZUC61-SLR, ZUC74-SLR, and ZUC78-SLR and the susceptible cultivar 'Elite' were included in the statistical analyses. Genotype had a significant effect on number of silvered leaves (F = 63.13; df = 3, 26; P = 0.0001), as well as on maximal (F = 2063.7; df = 3, 26; P = 0.0001), summed (F = 716.7; df = 3, 26; P = 0.0001), and average (F = 568.2; df = 3, 26; P = 0.0001) silverleaf ratings (Table 2). Number of silvered leaves and maximal and average

silverleaf ratings did not significantly differ between 'Elite' and ZUC61-SLR. 'Elite' had a significantly greater summed silverleaf rating than ZUC61-SLR. No differences in number of silvered leaves or silverleaf ratings were found between ZUC74-SLR and ZUC78-SLR. 'Elite' and ZUC61-SLR had a significantly higher number of silvered leaves and silverleaf ratings than ZUC74-SLR and ZUC78-SLR. Number of silvered leaves and silverleaf ratings were highest for 'Elite' and lowest for ZUC74-SLR.

Although SSL affects most commercially grown Cucurbita species (Maynard and Cantliffe, 1989; Segarra-Carmona et al., 1990; Simons et al., 1988), differences in SSL susceptibility have been reported for six groups of summer squash (Paris et al., 1993b, 1993c) and for two zucchini breeding lines (McAuslane et al., 1996). All zucchini genotypes used in this experiment were exposed to similar numbers of insects. ZUC60-SLR, ZUC70-SLR, and ZUC72-SLR maintained their normal leaf coloration throughout the entire experiment as did the SSL-tolerant control ZUC76-SLR. ZUC74-SLR and ZUC78-SLR were mildly affected by SSL showing only a maximal level of silvering of 1 in all replicates. On the other hand, ZUC61-SLR was as severely affected by SSL as was the susceptible control 'Elite'.

Since no statistical differences were observed in the number of nymphs infesting each genotype, it can be concluded that the ZUC76-SLR, ZUC33-SLR/PMR, and the ZUC76-SLR sister lines tested possess different levels of resistance to the effects of whitefly feeding. Additionally, it can be said that this resistance is not due to lack of oviposition attractiveness to adult whiteflies (antixenosis) or to negative qualities of the host that interfered with immature development (antibiosis).

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At the levels of infestation used in this experiment, resistance among the zucchini breeding lines ranged from nonexistent in ZUC61-SLR to mild in ZUC33-SLR/PMR, ZUC74-SLR, and ZUC78-SLR to complete in ZUC60-SLR, ZUC70-SLR, ZUC72-SLR, and ZUC76-SLR. The zucchini genotypes in this study did not seem to differ in their characteristics as hosts for immature stages. Thus, the resistance observed in these breeding lines might be due to the ability of the plant to tolerate nymphal feeding without silvering. Tolerance to plant pests is very desirable for integrated pest management programs because it does not exert selection pressure on the insect population and because it is compatible with other control tactics (Reese et al., 1994). Use of tolerant plants within an IPM program is very desirable because tolerant plants do not have any negative effects on natural enemies and they raise the economic injury level, reducing costly insecticide applications (Reese et al., 1994).

The introduction of B. argentifolii into the United States and its associated SSL has contributed to the urgency of developing active resistance breeding programs in Cucurbita. Crossing of genotypes with high degrees of resistance with genotypes of high commercial value may yield varieties that possess both of these desirable characteristics (de Ponti et al., 1990). Thus, identification of resistance sources to SSL in Cucurbita is crucial for the development of an active breeding program aimed at overcoming the economic losses caused by SSL. The resistant lines identified in this study need to be evaluated for consideration for future release as commercial varieties or/and their usefulness for future SSL resistance breeding programs. Finally, the variation in levels of resistance among the six closely related ZUC76-SLR sister lines may provide a good model to study their physical and genetic makeup to help understand the factors responsible for conferring their resistance to SSL.

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