Genetic Analysis of Resistance to Whitefly Silvering in Cucurbita pepo L.

R. Bruce Carle, Susan Webb, and Annette Chandler

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

Additional index words. silverleaf disorder, squash, Bemisia agentifolii

Abstract. Inheritance of resistance to the whitefly induced silverleaf disorder was investigated in Cucurbita pepo L. using two different resistant lines (ZUC33-SLR/PMR and ZUC76-SLR) and three susceptible lines (Z153-PMR, YCN374-PMR, and YSN286-PMR). Eighteen F2 and 20 resistant BC populations were generated, exposed at the seedling stage for 6 hours to a whitefly colony, and scored for silverleaf symptoms at the 5th and 10th leaf stages. Chi-square analysis of segregation data suggested two to four recessive genes may be involved with the inheritance of resistance, depending on resistant source and susceptible line. Variation in the expressivity of resistance was observed and attributed to the level of whitefly infestation.

Squash silverleaf disorder (SSL) is a seri
ous physiological malady of Cucurbita caused by the feeding of the silverleaf whitefly, Bemisia agentifolii Bellows & Berring. It is characterized by the progressive silvering of the adaxial leaf surface, and in severe cases the bleaching of petioles, flowers, and fruit (Paris et al., 1987). SSL decreases the plant's photosynthetic capability (Jimenez et al., 1995; Yokomi et al., 1990) and reduces marketable yield (Costa et al., 1994).

SSL symptoms are induced by the feeding of whitefly nymphs during foliage and fruit development via an unknown mechanism. The silvered appearance is due to a separation of the upper epidermis from the underlying mesophyll cells during leaf expansion (Jimenez et al., 1995). The severity of symptoms is positively correlated with the level of nymphal infestation (Costa et al., 1993; Yokomi et al., 1990), however, as few as 3 whiteflies are sufficient to induce some silvering (Yokomi, 1995). In the absence of an economic threshold, growers routinely spray insecticides to control whiteflies as soon as any silvered foliage appears, increasing the cost of production and the environment's pesticide load.

Two different C. pepo breeding lines have been identified as having resistance to SSL, ZUC33- SLR/PMR, and ZUC76-SLR (McAuslane et al., 1995). Both lines remain green at high levels of

whitefly infestation. Their resistance results from the plant's tolerance of the effects of whitefly feeding rather than any modification of whitefly biology or behavior by the plant (Cardoza et al., 1998). The purpose of this study was to determine the inheritance of resistance conveyed by these lines.

Materials and methods

Plant material. Cucurbita pepo breeding lines, ZUC33-SLR/PMR, ZUC76-SLR, YSN286-PMR, ZUC153-PMR, and YCN374-PMR, developed at the University of Florida's Central Florida Research and Educational Center in Leesburg (UF-CFREC), were used to study the inheritance of SSL resistance. The SSL resistant lines ZUC33-SLR/PMR (R1) and ZUC76-SLR (R2) are unrelated zucchini inbreds developed from symptomless plants identified in a 1990 planting heavily invested with SSL. The three susceptible lines used show different severity of SSL symptoms. ZUC153-PMR (S1) is a susceptible zucchini, YCN374-PMR (S2) is a moderately susceptible yellow crookneck, and YSN286-PMR (S3) is a highly susceptible yellow straightneck.

Inheritance study. In Fall 1996, controlled hand-pollinated reciprocal crosses were made between R1 and S1 and between R2 and S1, S2, and S3 in the field at UF-CFREC research farm. The following winter in the UF-CFREC greenhouse, F2, susceptible backcross (S-BC) and reciprocal

 

Florida Agricultural Experiment Station journal series N-01604.

Cucurbitaceae '98


resistant backcross (R-BC) populations were generated. In the summer of 1997, six F2, six R-BC, and one S-BC for the R1 x S1 populations and four F2, four R-BC, and one S-BC for the R2 x S1, R2 x S2, and R2 x S3 populations were screened and scored for SSL resistance. Chi-square analyses were performed for F2, and R-BC populations, using one-, two-, and three-gene models for determining expected ratios.

Screening procedure. The UF-CFREC B. argentifolii colony is maintained on collards (Brassica oleracea acephala) in four isolation cages in a growth room at 24 to 26 °C, with a diurnal photoperiod of 16 h light and 8 h dark. The facility permits the screening of six planting flats (68 x 35 cm) at one time. Eight whitefly exposure cycles, each 6 h long, over a 6-day period were used. A split planting, 19 and 21 Apr. 1997, was made to accommodate screening logistics and to maintain an adequate B. argentifolii population.

For each segregating population, a flat of 72 seeds was sown (Speedling Planter Flat, 5 x 5 cm cells, soilless Vergro Plug Mix A). At full cotyledon expansion, seedlings were sprayed until run off with a 0.25% B-Nine solution (daminozide) to inhibit internode elongation. At the one- to two-leaf stage, seedlings were exposed for 6 h to the B. argentifolii colony. Flats were placed in the growth room and adult whiteflies were evenly distributed onto the seedlings by vigorously shaking six host collard plants from the isolation cages. At the end of each exposure cycle, before removal, adult whiteflies on the exposed plants were shaken onto the next group of flats. Additional whiteflies were deposited onto the new flats from two host collard plants. A control flat containing 36 susceptible and 36 resistant parents of the segregating populations being screened was included for each cycle. In addition, the last cycle included flats containing 24 plants of each parental line.

After whitefly exposure, the flats were placed in a greenhouse for a 3- to 5-day recovery period. During the recovery period, 10 first true leaves were sampled randomly from each flat. Three 18-mm discs were cut from each leaf, and the numbers of eggs and nymphs on the abaxial surfaces counted with the aid of a stereomicroscope. The egg and nymph counts for each plant were totaled

and averaged for each flat to obtain whitefly infestation scores, which ranged form 60 to 120 indicating a severe infestation throughout the exposure cycles.

Silverleaf rating. Plants surviving the rigors of treatment were transplanted to the field on 7 and 9 May 1997. Single rows (0.46 x 1.52 m spacing) on raised beds prepared with black plastic mulch and drip irrigation were used for each population. Plants were rated for leaf silvering at the 5th and 10th leaf stages using a 0 to 5 scale adapted from Paris et al. (1987): 0) no symptoms, 1) less than half of the leaf veins with parallel silvering, 2) more than half of the leaf veins with parallel silvering, 3) all leaf veins with parallel silvering, 4) all leaf veins with parallel silvering and some interveinal spaces silvered, and 5) entire leaf surface silvered. Severity of SSL symptoms exhibited by the controls were used as reference for scoring individual plants as resistant, intermediate or susceptible. Segregant plants rated 0 or 1 at the 10th leaf stage were considered resistant, 2 or 3 intermediate, and 4 or 5 susceptible.

Results and discussion

The severity of the whitefly infestation in all exposure cycles induced occasional light silvering among both resistant controls, with R1 noticeably more silvered then R2. All of the susceptible control populations silvered intensely with S3 showing the severest symptoms. No resistant progeny were found in any of the S-BC populations. Resistant progeny were recovered in all F2 populations and all but one R-BC population.

Inheritance data and chi-square analyses for F2 and R-BC populations derived from R1 and S1 are presented in Table 1. No intermediate types were observed among the F2 progeny. Four F2 populations conformed to a single recessive inheritance model for resistance and two populations fit a double recessive model. About 25% of the R-BC progeny were classified as intermediate, suggesting either distinct levels of R1 resistance are determined by different genotypes or a single genotype shows variable expressivity related to the level of whitefly infestation. R1-BC populations matched a number of models based on the interaction of two or three genes, however, a

Cucurbitaceae '98


consistent pattern of inheritance was not observed. Alternatively assuming variable expressivity, analyses with resistant and intermediate classes combined yielded four R1-BC populations fitting a single recessive model, and four populations fitting a double recessive model.

Inheritance data and chi-square analyses for F2 and R-BC populations generated from R2 and

S1 are presented in Table 2 and for populations derived with R2 and S2 are presented in Table 3. The pattern of inheritance among populations derived with R2 and S1 was similar to that seen for R1 and S1; F2 and R2- BC populations conformed to either single or double recessive models when the intermediate and resistant classes were combine for analysis. Populations generated from R2

Table 1. Whitefly silvering segregations in F2 and BC populations derived from crosses between ZUC33-SLR/PMR (R1) and ZUC153-PMR(S1).

Phenotypez

Cross S I R c2 P c2 P c2 P

F2 population 3S:1R+Iy 15S:1R+Iy 63S:1R+Iy

(R1 x S1)

#1 52 0 13 0.87 0.50­0.25 20.97 <0.005 143.66 <0.005

#2 53 0 12 1.48 0.25­0.10 16.54 <0.005 120.69 <0.005

#3 64 0 3 15.05 <0.005 0.36 0.75­0.50 3.70 0.10­0.05

(S1 x R1)

#1 57 0 11 2.82 0.10­0.05 11.44 <0.005 94.42 <0.005

#2 54 0 14 0.71 0.50­0.25 23.86 <0.005 160.03 <0.005

#3 60 0 4 12.00 <0.005 0.00 >0.995 9.14 <0.005

Combined 340 0 57 23.98 <0.005 44.55 <0.005 422.57 <0.005

Homogeneity 8.95 0.25­0.10 28.63 <0.005 109.07 <0.005

BC population 1S:1R+Ix 3S:1R+Ix 7S:1R+Ix

R1(R1 x S1)

#1 41 18 9 2.88 0.10­0.05 7.84 0.01­0.005 46.02 <0.005

#2 33 19 17 0.13 0.75­0.50 27.17 <0.005 99.30 <0.005

#3 24 9 6 2.08 0.25­0.10 3.77 0.10­0.05 24.03 <0.005

#4 46 15 8 7.67 0.01­0.005 2.56 0.25­0.10 27.38 <0.005

#5 41 12 7 8.07 <0.005 1.42 0.25­0.10 20.15 <0.005

#6 7 4 4 0.07 0.90­0.75 6.42 0.03­0.01 22.87 <0.005

15S:1R+Ix 3S:1R+Ix 7S:1R+Ix

(S1 x R1)R1

#1 50 18 0 15.06 <0.005 0.08 0.90­0.75 12.13 <0.005

#2 31 28 9 0.53 0.50­0.25 31.37 <0.005 109.21 <0.005

Combined 273 123 60 17.76 <0.005 55.68 <0.005 318.32 <0.005

Homogeneity 18.71 0.01­0.005 24.95 <0.005 42.78 <0.005

2S:1I:1Rw 4S:3I:1Rw 5S:2I:1Rw

R1(R1 x S1)

#1 41 18 9 5.26 0.10­0.05 3.68 0.25­0.10 0.14 0.95­0.90

#2 33 19 17 0.25 0.90­0.75 10.02 0.01­0.005 10.69 <0.005

#3 24 9 6 2.54 0.50­0.25 3.46 0.25­0.10 0.32 0.90­0.75

#4 46 15 8 9.09 0.03­0.01 8.45 0.03­0.01 0.53 0.90­0.75

#5 41 12 7 8.90 0.03­0.01 8.97 0.03­0.01 0.96 0.75­0.50

#6 7 4 4 0.07 0.97­0.95 2.91 0.25­0.10 3.03 0.25­0.10

(S1 x R1)R1

#1 50 18 0 24.59 <0.005 18.24 <0.005 9.88 0.01­005

#2 31 28 9 11.15 <0.005 0.54 0.90­0.75 10.26 <0.005

Combined 273 123 60 35.17 <0.005 22.51 <0.005 1.37 0.75­0.50

Homogeneity 26.67 <0.005 33.75 <0.005 34.44 <0.005

zPhenotypes: S = susceptible, I = intermediate, R = resistant.

yc2 df: individual = 1; combined = 1; homogeneity = 5.

xc2 df: individual = 1; combined = 1; homogeneity = 7.

wc2 df: individual = 2; combined = 2; homogeneity = 7.

Cucurbitaceae '98


and S2 had no intermediates, and the inheritance data fit predominately a single recessive model.

The severity of SSL symptoms has been shown

to be positively correlated with the level of nymphal infestation (Costa et al., 1993; Yokomi et al., 1990). Both R1 and R2 control plants screened with the F2

Table 2. Whitefly silvering segregations in F2 and BC populations derived from crosses between ZUC76-SLR (R2) and ZUC153-PMR(S1).

Phenotypez

Cross S I R c2 P c2 P c2 P

F2 population 3S:1R+Iy 15S:1R+Iy 63S:1R+Iy

(R2 x S1)F2

#1 51 3 11 0.42 0.75-0.50 25.93 <0.005 168.64 <0.005

#2 52 2 14 0.08 0.90-0.75 34.65 <0.005 213.34 <0.005

(S1 x R2)F2

#1 31 0 21 6.56 0.03-0.01 103.41 <0.005 509.54 <0.005

#2 35 5 26 16.99 <0.005 186.77 <0.005 884.73 <0.005

Combined 169 10 72 7.87 <0.005 299.00 <0.005 1579.08 <0.005

Homogeneity 16.17 <0.005 51.76 <0.005 197.17 <0.005

BC population 1S:1R+Ix 3S:1R+Ix 7S:1R+Ix

R2(R2 x S1)

#1 52 0 15 20.43 <0.005 0.24 0.75-0.50 5.99 0.03-0.01

#2 30 0 31 0.02 0.90-0.75 21.69 <0.005 81.89 <0.005

(S1 x R2)R2

#1 34 0 27 0.80 0.75-0.50 12.07 <0.005 56.26 <0.005

#2 47 0 22 9.06 <0.005 1.74 0.25-0.10 23.70 <0.005

Combined 162 0 95 17.92 <0.005 19.23 <0.005 139.54 <0.005

Homogeneity 12.39 0.01-0.005 16.52 <0.005 28.32 <0.005

zPhenotypes: S = susceptible, I = intermediate, R = resistant.

yc2 df: individual = 1; combined = 1; homogeneity = 3.

xc2 df: individual = 1; combined = 1; homogeneity = 3.

Table 3. Whitefly silvering segregations in F2 and BC populations derived from crosses between ZUC76-SLR (R2) and YCN374-PMR(S2).

Phenotypez

Cross S I R c2 P c2 P c2 P

F2 population 3S:1R+Iy 15S:1R+Iy 63S:1R+Iy

(R2 x S2)F2

#1 48 1 19 0.71 0.50­0.25 62.26 <0.005 342.89 <0.005

#2 47 0 19 0.51 0.50­0.25 57.22 <0.005 318.06 <0.005

(S2 x R2)F2

#1 50 0 15 0.13 0.75­0.50 31.41 <0.005 195.61 <0.005

#2 49 0 9 2.78 0.10­0.05 8.50 <0.005 73.43 <0.005

Combined 194 1 62 0.11 0.75­0.50 140.14 <0.005 850.57 <0.005

Homogeneity 3.62 0.50­0.25 11.60 0.01­0.005 44.17 <0.005

BC population 1S:1R+Ix 3S:1R+Ix 7S:1R+Ix

R2(R2 x S2)

#1 26 0 34 1.07 0.50­0.25 32.09 <0.005 107.01 <0.005

#2 46 0 20 10.24 <0.005 0.99 0.50­0.25 19.13 <0.005

(S2 x R2)R2

#1 26 0 34 1.07 0.50­0.25 32.09 <0.005 107.01 <0.005

#2 31 0 32 0.02 0.90­0.75 22.35 <0.005 84.46 <0.005

Combined 129 0 120 0.33 0.75­0.50 71.43 <0.005 290.03 <0.005

Homogeneity 12.07 0.01­0.005 16.09 <0.005 27.58 <0.005

zPhenotypes: S = susceptible, I = intermediate, R = resistant.

yc2 df: individual = 1; combined = 1; homogeneity = 3.

xc2 df: individual = 1; combined = 1; homogeneity = 3.

Cucurbitaceae '98


Table 4. Whitefly silvering segregations in F2 and BC populations derived from crosses between ZUC76-SLR (R2) and YSN286-PMR(S3).

Phenotypez

Cross S I R c2 P c2 P c2 P

F2 population 3S:1R+Iy 15S:1R+Iy 63S:1R+Iy

(R2 x S3 )F2

#1 63 2 2 12.94 <0.005 0.01 0.95­0.90 8.46 <0.005

#2 53 6 5 2.08 0.25­0.10 13.07 <0.005 101.59 <0.005

(S3 x R2)F2

#1 58 3 6 4.78 0.05­0.03 5.90 0.03­0.01 61.38 <0.005

#2 51 6 7 0.75 0.50­0.25 21.60 <0.005 146.29 <0.005

Combined 225 17 20 16.53 <0.005 27.71 <0.005 268.70 <0.005

Homogeneity 4.02 0.50­0.25 12.87 <0.005 49.01 <0.005

BC population 1S:1R+Ix 3S:1R+Ix 7S:1R+Ix

R2(R2 x S3)

#1 62 0 4 50.97 <0.005 12.63 <0.005 2.50 0.25­0.10

#2 49 0 0 49.00 <0.005 16.33 <0.005 7.00 0.01­0.005

(S3 x R2)R2

#1 57 0 3 48.60 <0.005 12.80 <0.005 3.09 0.10­0.05

#2 56 0 3 47.61 <0.005 12.48 <0.005 2.97 0.10­0.05

Combined 224 0 10 195.71 <0.005 53.61 <0.005 14.48 <0.005

Homogeneity 0.47 0.95­0.90 0.63 0.90­0.75 1.08 0.90­0.75

zPhenotypes: S = susceptible, I = intermediate, R = resistant.

yc2 df: individual = 1; combined = 1; homogeneity = 3.

xc2 df: individual = 1; combined = 1; homogeneity = 3.

and BC populations showed slight silvering, particularly the R1 plants exposed with the R1-BC populations. A reasonable explanation for inheritance of SSL resistance is that it is governed by two independent recessive genes present in both resistant parents. The fit of one and two-gene models to populations generated with S1 would result if S1 were homozygous dominant for one gene and heterozygous for the second gene. S2 would be homozygous dominant for one gene and homozygous recessive for the second gene. This difference in genotypes may explain the differences in susceptibility of S1 and S2, the former being more SSL sensitive.

This double recessive explanation is not entirely supported by the segregation data from the populations derived from R2 and S3 (Table 4). Although the F2 populations conform (two fitting a single recessive, one fitting a double recessive, and one nearly fitting either model), too few resistant progeny were recovered in the BC populations to match either a single or double recessive model. Three BC populations matched a triple recessive inheritance pattern. They also are suggestive of a four recessive gene model, although the population sizes are too small for a significant

test. No resistant progeny were recovered in the fourth BC population, which can be attributed to its smaller size if three or more recessive genes control SSL resistance. The S3 parent is highly susceptible to SSL, exhibiting more rapid and persistent silvering than S1 or S2. It is plausible that this higher level of susceptibility is conveyed by another gene or group of genes, but the disparity of fit between the F2 and BC populations remains unexplained.

The screening procedure utilized for the inheritance study was not ideal. The interaction between the whitefly and the plant is inherently variable. The expression of SSL symptoms has a large environmental component, with the age and health of both whitefly and plant being critical, as well as weather conditions and the presence of whitefly predators. The screening procedure did not insure that each plant received the same level of whitefly exposure, which was indicated by the slight variation in silvering among the R1 and R2 control plants. It was designed to economically screen large populations at a stringent level in order to reduce the number of false negative classifications, that is susceptible being classified as resistant. The stringency, however, potentially

Cucurbitaceae '98


Costa, H.S., M.W. Johnson, and D.E. Ullman, 1994. Row covers effect on sweetpotato whitefly (Homoptera: Aleyrodidae) densities, incidence of silverleaf, and crop yield in zucchini. J. Econ. Entomol. 87:1616­1621.

Costa, H.S., D.E. Ullman, M.W. Johnson, and B.E. Tabashnik. 1993. Squash silverleaf symptoms induced by immature, but not adult, Bemisia tabaci. Phytopathology 83:763­766.

Jimenez, D.R., R.K. Yokomi, R.T. Mayer, and J.P. Shapiro. 1995. Cytology and physiology of silverleaf whitefly-induced squash silverleaf. Physiol. Mol. Plant Pathol. 46:227­242.

McAuslane, M.J., S.E. Webb, and G.W. Elmstrom. 1996. Resistance in germplasm of Cucurbita pepo to silverleaf, a disorder associated with Bemisia argentifolii (Homoptera: Aleyrodidae). Fla. Entomol. 79:206­221.

Paris, H.S., H. Nerson, and Y. Buger. 1987. Leaf silvering of Cucurbita. Can. J. Plant Sci. 67:593­598.

Yokomi, R.K., K.A. Hoelmer, and L.S. Osborne. 1990. Relationships between the sweetpotato whitefly and the squash silverleaf disorder. Phytopathology 80:895­900.

Yokomi, R.K., D.R. Jimenez, L.S. Osborne, and J.P. Shapiro. 1995. Comparison of silverleaf whitefly-induced and chlormequat chloride-induce leaf silvering in Cucurbita pepo. Plant Dis. 79:950­955.

overwhelms the plant's resistance, and can not reliably reveal individuals with intermediate levels of resistance.

The inheritance study did determine that resistance is recessive with no major cytoplasmic effects involved. There was a strong indication that at least two independently inherited genes are involved with the resistant response. The different levels of SSL sensitivity manifested by the susceptible parents and the difference in inheritance observed among their derived progeny suggested other genes may also be involved with resistance. Certainly, the difference in silvering observed between R1 and R2 when overwhelmed with whiteflies supports this as well.

Literature cited

Cardoza, Y.J., H.J. McAuslane, and S.E. Webb. 1998. Mechanism of resistance of zucchini germplasm to squash silverleaf disorder induced by Bemisia argentifolii J. Econ. Entomol. (in press).

Cucurbitaceae '98