HORTSCIENCE 26(7):905-908. 1991. strains with resistance or tolerance to chlor- amben injury could be developed, then a production system that would incorporate Evaluations and Correlated Responses more effective chemical and cultural prac- tices for once-over mechanical harvest could for Resistance to Chloramben be implemented. Cucumbers have been shown to possess low phenotypic variability for re- Herbicide in Cucumber sponse to chloramben, most likely due to the effects of more than two genes (Miller et al., Jack E. Staub1 and Larry D. Knerr2 1973). Germplasm sources for resistance to Vegetable Crops Research, U.S. Department of Agriculture, Agricultural the methyl esterified form of chloramben Research Service, Horticulture Department, University of Wisconsin, (Miller et al., 1973) are no longer available. Therefore, a series of experiments were de- Madison, WI 53706 signed to: 1) identify sources of resistance

3 to commercially available non-esterified form Leslie A. Weston of chloramben (hereafter referred to as chlor- Horticulture Department, University of Kentucky, Lexington, KY 40546 amben) when applied under field conditions; 2) develop a laboratory procedure that could Additional index words. Cucumis sativus, recurrent selection, breeding evaluate germination response of seeds after Abstract. The U.S. cucumber germplasm collection (753 accessions) and U.S. adapted being challenged with chloramben; and 3) processing cucumber (Cucumis sativus L.) inbreds and hybrids were surveyed for re- determine whether a positive correlation ex- sponse to 6.7 kg ae/ha of chloramben. Nine plant introductions (PI 165952, 173892, ists between the laboratory germination pro- 179676, 275411, 277741, 279464, 279465, 436609, and 482464) were classified as tol- cedure and field emergence and/or seedling erant to chloramben, based on percentage and rate of field emergence and seedling performance. vigor. All adapted strains evaluated were susceptible to chloramben injury. The chlor- An initial screening of the U.S. cucumber amben-tolerant accessions (C0) were subjected to two cycles of recurrent half-sib family collection (753 accessions) and adapted inbred selection that resulted in 11 C2 families. These families, a susceptible adapted line (WI lines for tolerance to chloramben provided 2870), and the resistant PI 436609 were evaluated in the field (6.7 kg ae/ha) and tolerant parents for a base population (cycle laboratory (0.0, 0.01, and 0.0001 M) for response to chloramben challenge. Significant 1; C1). This population was subjected to two (P = 0.05) differences between families were observed for percentage emergence and cycles of half-sib recurrent selection for field phytotoxicity ratings. Correlations between emergence and phytotoxicity ratings at two tolerance to chloramben injury. The result- 2 dates were low (r = -0.32 and – 0.05). Significant (P = 0.05) interfamily differences ing C2 families and tolerant and resistant were also recorded for percentage germination, hypocotyl length, primary root length, strains were used to determine whether a lab- and number of lateral roots in the laboratory. Correlated responses between these oratory procedure would be correlated with growth variables were high (r2 = 0.78 to 0.84), but correlations between field and field observations. laboratory observations were low (r2 = -0.31 to 0.24). We hypothesize that the genetic Identification of sources of tolerance and response to chloramben challenge under laboratory conditions depends on the concen- population development. Field experiments tration of the chemical administered. Chemical name used: 3-amino-2, 5-dichloroben- were conducted at the Univ. of Wisconsin zoic acid (chloramben). Agricultural Research Station at Hancock on a Plainfield loamy sand (Typic Udipsam- Inadequate weed control is a major factor paraquat, and trifluralin. Bensulide and nap- ment; mixed mesic) in 1987 (screening) and that limits yield in commercial cucumber talam often give poor annual weed control 1989 (evaluation). In 1987, 25 seeds of each production. Average annual loss in value of (Knerr and Hopen, 1989; Nell, 1977; Ro- accession (753), and the U.S. Dept. of Ag- the pickling cucumber crop in the United manowski and Tanaka, 1965), while DCPA riculture processing cucumber inbred lines States due to weeds is estimated at causes severe crop damage when surface-ap- WI 1379, WI 1983, WI 2238, WI 2757, WI $17,359,000 (Weed Sci. Soc. Amer., 1984). plied before crop emergence (Monaco and 2870, WI 3888, WI 3733, and SMR 18, GY- Poor weed control is of particular concern in Miller, 1972). Trifluralin may result in crop 14, ‘Calypso’, ‘Clinton’, ‘Carolina’, ‘Expo’, culture using once-over mechanical harvest- injury and is limited to use in the southern and ‘Natsufuschinari’ were sown in twenty- ing since weeds adversely affect the opera- United States (Weed Control Manual, 1986). five 30-cm rows (replications) on 1.5-m cen- tion of mechanical harvesters, and high Moreover, it cannot be applied until the three- ters that were arranged in a randomized com- cucumber plant densities and rapid vine to four-leaf stage of crop development, plete-block design (RCBD). Chloramben growth, which impede cultivation, render thereby requiring mechanical control of weeds (75DF) was surface-applied preemergence at mechanical weed control impractical (Mon- until that time (Weed Control Manual, 1986). 6.7 kg ae/ha (three times the recommended aco and Miller, 1972; O’Sullivan, 1980). Glyphosate and paraquat are nonselective rate) to 20 replications (25 seeds per repli- Herbicides currently registered for cu- postemergence contact herbicides suitable only cation) with a tractor-mounted compressed- cumber production include bensulide, chlor- for use during seedbed preparation (Weed air-powered sprayer that delivered 190 amben, DCPA, glyphosate, naptalam, Control Manual, 1986). Chloramben is an liters·ha -1. The remaining five replications excellent grass and broadleaf herbicide. were left untreated as a control for visual Received for publication 13 Aug. 1990. The au- However, cucumber tolerance to chloramben observations and were not used in the analy- thors thank Justin Cruz, USDA apprentice, for his is marginal (Baker and Warren, 1962; Cole sis. Twelve hours after herbicide applica- invaluable help in performing the laboratory ex- and Jordan, 1974; Knerr and Hopen, 1989), tion, plots were irrigated with 13 mm of water periments. Mention of a trade name, proprietary requiring the addition of activated charcoal via overhead irrigation to move the herbi- product, or specific equipment does not constitute over the seed row as a safening agent (Union cides into the seed zone. After 4 days, plots a guarantee or warranty by the USDA and does Carbide Agr. Prod., 1989). Most growers were irrigated three times weekly to ensure not imply its approval to the exclusion of other are reluctant to use chloramben in this man- adequate moisture for germination and emer- products that may be suitable. The cost of pub- ner due to additional expenses and time in- gence. lishing this paper was defrayed in part by the pay- Seedlings in treated plots were compared ment of page charges. Under postal regulations, puts (S.F. Gorski, H.J. Hopen, T.J. Monaco, this paper therefore must be hereby marked ad- S. Weller, and B.H. Zandstra, personal to those in control plots 1 and 3 weeks after vertisement solely to indicate this fact. communications). Furthermore, close plant emergence, and visually rated for chloram- 1Research Horticulturist and Associate Professor. spacings required for mechanical harvest make ben injury on a five-point phytotoxicity scale 2Former Graduate Research Assistant. the use of activated charcoal impractical. [1 = no damage, 2 = slight injury (slight 3Assistant Professor. If commercially acceptable cucumber margin undulation or second leaf showing no

HORTSCIENCE, VOL. 26(7), JULY 1991 905 Table 1. Response of plant characters (emergence, germination, and hypocotyl and root growth) of cucumber strains to chloramben during field and laboratory evaluation.

zSeedlings in treated plots were compared to those in control plots 1 (designated 1) and 3 (designated 2) weeks after emergence, and rated for chloramben injury on a 5-point visual phytotoxicity scale [1 = no damage, 2 = slight injury (slight margin undulation or second leaf showing no damage), 3 = moderate injury (margins of leaves undulating or deformed, cotyledons or first true leaves chlorotic), 4 = severe injury (cotelydons fused, margins of first and/or second leaf undulating or deformed, cotyledons chlorotic, or seedlings without visible shoot apex), 5 = seedling death or no emergence]. yPlant introduction tolerant to chloramben at 6.7 kg ae/ha under field conditions. xInbred line susceptible to chloramben at 6.7 kg ae/ha under field conditions. damage), 3 = moderate injury (margins of highest ranking families were selected and either 5 ml of the water (control) or on 15 leaves undulating or deformed, cotyledons randomly mated to produce a C2 half-sib ml of the solidified Gelrite medium. Treat- or first true leaves chlorotic), 4 = severe population. Seeds of these families, WI 2870, ments were arranged in a RCBD and held at injury (cotelydons fused, margins of first and/ and ‘Calypso’ were produced by controlled 27C and 85% RH for 5 days, during which or second leaf undulating or deformed, cot- pollination under the same environmental time rate and percentage of germination and yledons chlorotic, or seedlings without vis- conditions (greenhouse; June through Sept. primary root and hypocotyl length were re- ible shoot apex), 5 = seedling death or no 1988) and were used in the development of corded daily. Seeds were considered ger- emergence]. Mean phytotoxicity ratings were an in vitro procedure for determining chlor- minated when radicles protruded 3 mm, and calculated for each accession and line after amben toxicity. root and hypocotyl measurements were taken each evaluation. Development of in vitro procedure. A se- from the emergence point to the apex of the Selection of tolerant plants was based on ries of experiments was designed to develop organ. initial injury (1 week post-application) and and evaluate a laboratory procedure for the Selected families and control strains ger- whether plants recovered after being chal- detection of chloramben injury. To deter- minated 100% after 3 days. When placed on lenged (3 weeks post-application). Plants were mine seed viability under optimum condi- media containing 0, 0.0001, or 0.00001 M selected that were rated 2 or lower after 1 tions, 10 seeds of the C2 selected families, of chloramben, seeds of ‘Calypso’ and the week and recovered after 3 weeks (subse- the tolerant PI 436609 (phytotoxicity rating heterogeneous bulk of the C2 families ger- quent new leaves showing slight or no dam- of 2.2) and the susceptible line WI 2870 minated 100%. The 0.01 and 0.001 M chlor- age). (phytotoxicity rating of 4) were placed in 9- amben treatments did not affect germination During initial field screening, PI 165952 cm petri plates on Whatman no. 1 filter pa- (100%), but did adversely affect primary root (Turkey), PI 173892 (India; ‘Khira’), PI per moistened with 5 ml of distilled water. and hypocotyl length. Seed placed in 0.1 M 179676 (India; ‘Kakri’), PI 275411 (Neth- Three replications of these families or lines chloramben did not germinate. erlands; ‘Lange Groene Broei’), PI 277741 were then arranged in a RCBD, placed in a The mean hypocotyl length of germinated (Netherlands; ‘Green Spot Super’), PI 279464 controlled environment (27C and 85% RH), ‘Calypso’ seeds (45.3 mm) was not signifi- (Japan; ‘Kara-Aonaga-Fushinari’), PI 279465 and rate and percentage of germination were cantly (P = 0.05) higher than bulked indi- (Japan; ‘Natsufushinari’), PI 436609 (Peo- recorded for 3 days. viduals (41.7 mm). The mean primary root ples Republic of China; ‘Tsin Sanz Yen A procedure for the detection of chlor- and hypocotyl lengths of germinated seed of 15919’), and PI 482464 (Zambia) received amben toxicity was developed by testing the bulked families and ‘Calypso’ grown on 0.01 a mean rating of 2.2 or lower (range = 1 to response of seed of selected families and and 0.001 M chloramben were significantly 3) and, therefore, were designated as tolerant ‘Calypso’ to chloramben in a water and a gel less than those grown on the other concen- to chloramben challenge at 6.7 kg ae/ha. All medium. For this dose-response experiment, trations examined, but were not different from other lines or hybrids tested received mean 100 seeds from four randomly selected half- each other. The mean root length of ‘Ca- ratings of 4.2 (range = 3 to 5) and were sib families were bulked to supply a heter- lypso’ germinated on 0.01 and 0.001 M classified as susceptible. ogeneous seed lot for testing. Chloramben chloramben was 6.2 and 6.9 mm, respec- Ten tolerant plants from each of these nine (DF75) was diluted in either distilled water tively, while root length of bulked families tolerant accessions were lifted from field plots or a Gelrite (Scott Laboratories, Fiskeville, was 7.4 and 7.6 mm, respectively. The mean and transplanted to a greenhouse where each R.I.)/MgCl 2 mixture (0.4 g Gelrite per 0.04 hypocotyl length of germinated ‘Calypso’ seed plant was randomly hand pollinated using g MgCl2 in 100 ml distilled water) to yield grown on 0.01 and 0.001 M chloramben was bulk pollen from four other tolerant plants to 0.1, 0.01, 0.001, 0.0001, and 0.00001 M 0.1 mm and 7.0 mm, respectively, and hy- produce half-sib families (C1). Seeds from solutions. Chloramben in the Gelrite/MgCl2 pocotyl length of the bulked families was 0.0 each fruit (Cl half-sib family) were sown in solution was dissolved by heating the mix- mm and 9.1 mm, respectively. 10 replications (25 seeds per replication), ture to a light boil. Three replicates of 25 Correlation between laboratory and field. challenged with chloramben as described seeds were placed in petri plates (9 cm) con- Based on results from the above experi- above, and 10 tolerant plants from the 11 taining the various chloramben treatments in ments, and the few half-sib family seed

906 HORTSCIENCE, VOL. 26(7), JULY 1991 Table 2. Correlations between field and laboratory response of 11 chloramben-tolerant cucumber ( +0.79) and number of lateral roots ( +0.84), -5 families to chloramben (6.7 kg ae/ha and 10 M, respectively). while root length and number of lateral roots were significantly correlated (+0.81; all correlations were significant at P = 0.05). If a laboratory screening procedure could be developed for the detection of plants tol- erant to chloramben injury under field con- ditions, then selection for chloramben tolerance could be conducted in the labora- tory. Laboratory selection would decrease zSeedlings in treated plots were compared to those in control plots 1 (1) and 3 (2) weeks after emergence, costs and time required for the development and rated for chloramben injury on a 5-point visual phytotoxicity scale [1 = no damage, 2 = slight of chloramben-tolerant germplasm. Corre- injury (slight margin undulation or second leaf showing no damage), 3 = moderate injury (margins of lated responses between field and laboratory leaves undulating or deformed, cotyledons or first true leaves chlorotic), 4 = severe injury (cotelydons data were low (Table 2). The lack of signif- fused, margins of first and/or second leaf undulating or deformed, cotyledons chlorotic, or seedlings icant positive correlations between the data without visible shoot apex), 5 = seedling death or no emergence. from the field and laboratory indicates that *,** Significant at P = 0.05 or 0.01, respectively. either these characters do not measure sim- ilar responses, or that perhaps herbicide available, 0, 0.01, and 0.0001 M chloram- of the tolerant control PI 436609. The mean challenge (particularly in the field) was not ben were chosen for evaluating correlated length of the primary root of families 1 and severe enough to provide identification of responses between field and laboratory tests. 2 was significantly longer than either control genotypic differences when they do exist.

Seeds of the 11 C2 families, PI 436609, and strain. Germinated seeds of families 1, 2, 5, The fact that the emergence of all strains WI 2870 (designated as susceptible) were 9, and 10 produced significantly more lateral tested was >90% in untreated plots and challenged by chloramben under field con- roots than the tolerant control, but the mean emergence and early growth of seedlings were ditions (6.72 kg ae/ha) and in the laboratory number of roots was not significantly larger significantly affected by chloramben treat- using the Gelrite medium procedure. The than that produced by the susceptible con- ment indicates that the herbicide challenge Gelrite medium was chosen to ensure that trol. in the field was measurable. It is known that chloramben would be evenly distributed and The mean germination percentage of se- soil moisture greatly affects chloramben ac- to prevent changes in concentration due to lected families and PI 436609 incubated on tivity (La Bonte and Hopen, 1985), and that evaporation. WI 2870 was chosen for the 0.0001 M chloramben was significantly higher immediate application of water after chlor- bioassay because of its greater sensitivity than than for WI 2870 (Table 1). The mean hy- amben application increases toxicity (Knerr, ‘Calypso’ (initial field screening and unpub- pocotyl elongation of seedlings of family 11 1987). In addition to controlled post-herbi- lished data). was lower than those of families 1, 2, 3, and cide application of water to treated plots in

In the field, 25 seeds of each C2 family 7 grown in 0.0001 M chloramben. Similarly, this experiment, »5 mm of rainfall was re- seed lot were sown in 60-cm-long plots ar- the mean primary root length of WI 2870 ceived in 1989 within 24 h after chloramben ranged in a RCBD with eight replications. was significantly less than those of families treatment. It is possible that some of the her- Chloramben was applied as previously dis- 3, 8, 10, and 11. The mean primary root bicide was driven below the seed, thus re- cussed, and percentage emergence and phy- length of PI 436609 was similar to all se- ducing the effective quantity of the chemical totoxicity ratings were taken 7 and 14 days lected families. While the number of lateral around the seed during germination and after planting. Laboratory treatments, con- roots recorded for WI 2870 was significantly emergence. This may explain the lack of dif- sisting of 25 seeds in 9-cm petri dishes, were less than for all families except 4 and 6, ferences among strains 3 weeks after emer- replicated three times, arranged in a RCBD lateral roots of families 2, 3, 4, 5, 6, and 7 gence. and held at 27C and 85% RH for 10 days, were shorter than those of PI 436609, which It is more likely that characteristics from at which time germination percentage, hy- was similar to those of other selected fami- the field and laboratory did not measure the pocotyl, primary root length, and number of lies. same physiological response and that the lateral roots were recorded. The radicles and cotyledons of seeds ger- germination environment can substantially Although the percent emergence of se- minated on medium containing 0.01 M modify the effects of the herbicide chal- lected families 2, 4, 5, 9, 10, 11 was sig- chloramben emerged, but hypocotyls did not lenge. For example, although the mean nificantly higher than the susceptible control elongate and lateral roots were not produced emergence percentage of families 9 and 10 (WI 2870), only seed of families 9 and 11 (Table 1). Germination percentage was rel- was similar in the field, the mean hypocotyl emerged more rapidly than the tolerant con- atively high (85% to 100%) among all fam- length (a potential measure of emergence) of trol (PI 436609; Table 1). Mean phytotox- ilies or lines tested, except for family 6 (69%), family 9 was similar to PI 436609. When icity ratings 1 week after emergence of for which it was significantly lower than for challenged in the laboratory with 0.0001 M families 5 and 10 were significantly lower all other families except 7 and 8 and WI and 0.01 M chloramben, no such differences than for PI 436609, and ratings of families 2870. Mean primary root length of WI 2870 were detected for these criteria. 5, 10, and 11 were significantly lower than was significantly less than that for PI 436609 The significantly high correlation between for WI 2870. Differences among families were and families 2, 5, 8, 9, 10, and 12. Primary hypocotyl length, primary root length, and also detected. Seedlings of family 10 were roots of family 2 were, on the average, sig- number of lateral roots suggests that one significantly more tolerant to chloramben than nificantly longer than those of family 6, which character provides indirect information about those of families 1, 2, 3, 4, 6, 7, and 9 at 1 was similar to WI 2870. the other measured traits. Therefore, during week. However, no differences were de- Data from selected families germinated on laboratory evaluation of progeny for chlor- tected among cucumber populations 3 weeks medium containing 0.0001 M chloramben amben tolerance it would be appropriate to after chloramben application. were used to calculate correlated responses monitor germination percentage and just one Under laboratory conditions, no differ- between and among characteristics recorded of the other traits measured, ences in germination percentage were de- in the field and laboratory. Although corre- Chloramben inhibits root development tected among the strains examined when lations between percentage emergence (PE) (Weed Sci. Soc. Amer., 1989); also, metab- grown on medium without chloramben. Al- and phytotoxicity ratings (PR-1 and PR-2) olism in certain plants appears to result in though the mean hypocotyl lengths of all were low ( –0.32 PE ´ PR-1, – 0.05 PE the formation of conjugates rather than deg- families except 4 and 6 were significantly ´ PR-2, + 0.37 PR-1 ´ PR-2), correla- radation products (Stoner and Wax, 1968). larger than those of the susceptible control, tions among some characteristics in the lab- Our data confirm these earlier results re- only the hypocotyls of families 1 and 2 were, oratory were relatively high. Hypocotyl length garding inhibition of root development. For on the average, significantly longer than those was highly correlated with root length instance, hypocotyl elongation of all strains

HORTSCIENCE, VOL. 26(7), JULY 1991 907 tested at 0.0001 M chloramben was de- Basis of variability in the cucumber for resis- Effects of seed preconditioning treatments on creased when compared to growth on media tance to chloramben methyl ester. Weed Sci. emergence of cucumber populations. Hort- without chloramben. Likewise, primary root 21:207-211. Science 21:1356-1359. length and the number of lateral roots were Monaco, T.J. and C.H. Miller. 1972. Herbicide Stoner, E.W. and L.M. Wax. 1968. Amiben me- significantly depressed with increasing con- activity in close-spaced, pickling cucumbers. tabolism and selectivity. Weed Sci. 16:283-288. Weed Sci. 20:545–548. Union Carbide Agricultural Products. 1989. Union centrations of chloramben in the growing Nell, C.J. 1977. Weed control in cucumbers in a Carbide 1989 chemical guide. Research Trian- medium. conventional planting and in a stale bed. Proc. gle Park, N.C. Differential response of cucumber strains Northeastern Weed Sci. Soc. 31:248-251. Weed Control Manual 1986 and Herbicide Guide. was noted with increasing concentrations of O’Sullivan, J. 1980. Irrigation, spacing, and ni- 1986. Ag. Consultant and Fieldman, a Meister chloramben. Mean primary root length of trogen effects on yield and quality of pickling Publ. p. 196-197. families 1 and 2 was significantly greater cucumbers grown for mechanical harvesting. Weed Science Society of America. 1984. Crop than of WI 2870 on medium without chlor- Can. J. Plant Sci. 60:923–928. losses due to weeds in Canada and the United Romanowski, R.R. and J.S. Tanaka. 1965. An States. Weed Sci. Soc. Amer., Champaign, Ill. amben. At 0.0001 M chloramben, the pri- mary root lengths of families 1 and 2 were evaluation of herbicides for use with cucumber p. 1–22. (Cucumis sativus) and watermelon (Citrullus Weed Science Society of America. 1989. Herbi- similar to those of WI 2870. Since the po- vulgaris) in Hawaii. Hawaii Agr. Expt. Sta. Prog. cide handbook of the Weed Society of America. tential effects of the growing environment, Rpt. 144. 6th ed. Weed Sci. Soc. Amer., Champaign, Ill. seed age, and seed handling procedures were Staub, J.E., J. Nienhuis, and R.L. Lower. 1986. p. 51-52, 192-193. eliminated in this experiment, this difference is probably due to genotype. The genetic re- sponse to chloramben challenge under lab- oratory conditions described here may depend on the concentration administered. The fact that the mean primary root length of family 2 individuals was significantly higher than that of WI 2870 individuals at 0.01 M chlor- amben lends support to this hypothesis. The physiological and biochemical processes that control germination and emer- gence in cucumber differ (Staub et al., 1986). The effect of chloramben on germination and primary root development appears to have a demonstrable genetic component. For in- stance, similar germination percentages were observed in family 8 and WI 2870 when grown on 0.01 M chloramben, but the pri- mary root extension of family 8 was signif- icantly greater than WI 2870. Likewise, the germination percentage of WI 2870 was higher (P = 0.10) than of family 6, but their primary root extension was similar. The fact that, when challenged with 0.0001 M chlor- amben, germination of family 4 individuals was 100% but their hypocotyl extension was not substantial lends support to this hypoth- esis. Development of efficient field and labo- ratory herbicide screening procedures may allow for development of germplasm with significant levels of herbicide tolerance. Such germplasm would be an important compo- nent of integrated pest management pro- grams to support low-input sustainable agriculture.

Literature Cited Baker, R.S. and G.F. Warren. 1962. Selective herbicidal action of amiben on cucumber and squash. Weeds 10:219–224. Cole, A.W. and T.N. Jordan. 1974. Seedling and yield response of cucumber to naptalam and chloramben. Weed Sci. 22:604-607. Knerr, L.D. 1987. Naptalam as a safener for cu- cumber (Cucumis sativus L.) against the phy- totoxic effects of chloramben. MS Thesis, Univ. of Wisconsin-Madison. p. 62–70. Knerr, L.D. and H.J. Hopen. 1989. Naptalam as a safener against chloramben in cucumber (Cu- cumis sativus L.). Weed Technol. 3:445-449. La Bonte, D.R. and H.J. Hopen. 1985. Apparent safening of chloramben for cucumbers by com- bination with naptalam. Proc. N. Central Weed Control Conf. 40:41. Miller, J.C., D. Penner, and L.R. Baker. 1973.

HORTSCIENCE, VOL. 26(7), JULY 1991