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Selection of diploid and screening of polyploid guayule lines for Verticillium tolerance
INDUSTRIAL CROPS ANDPRODUCTS AN INTERNATIONAL JOURNAL
ELSEVIER
Industrial Crops and Products 4 (1995) 303-310
Selection of diploid and screening of polyploid guayule lines for Verticilhn tolerance D.T. Ray ap*,T.V. Orum b, D.M. Bigelow b, S.M. Alcorn b aDepartment of Plant Sciences, The University of Arizona, Tucson, AZ 85721, USA b Depafiment of Plant Pathology, The Universiry of Arizona, Tucson,AZ 85721, USA
Received 21 August 1995; accepted 20 September 1995
Abstract Guayule (Parthenium algentatum Gray) produces a high quality hypoallergenic latex, that is of interest for use in the medical industry. In order to meet the anticipated demand, guayule will need to be planted in mild climatic zones over a wide geographic area, and often following cotton (Gossypium sp.) or other crops susceptible to the root-invading fungus Vetiicillium dahliae Kleb. Since guayule has been shown previously to be susceptible to L! dahliae, the goals of this work were: (1) to develop a diploid guayule line that is tolerant to K dahliae through mass selection, and (2) to evaluate improved guayule lines for tirticillium tolerance. To achieve the first goal, a diploid guayule population was subjected to three cycles of selection, with only the plants with the greatest tolerance and the highest rubber yields used in the subsequent cycles of selection. Second, fifteen improved polyploid and three diploid lines were evaluated for tolerance to K dahliae under greenhouse conditions. Cuttings were found to be more tolerant than seedlings, and tolerance increased with age of the plant at the time of inoculation. The Verticillium screened diploids expressed greater tolerance than an unscreened diploid population in both greenhouse and field trials. Among the 18 improved lines, 13 of which had not been previously evaluated, a Vkticillium-screened diploid line (418-6) had the highest tolerance ranking, but was not significantly better than four unscreened polyploid lines. Continued evaluation of improved lines will help growers in making informed decisions about which lines to plant for their specific locations. The Verticillium-tolerantdiploid germplasm material developed in this study is available
to plant breeding programs. Keywords:
Parthenium
argentatum;
Ploidy levels; Verticillium wilt; Verticillium dahliae; Selection schemes
1. Introduction
Guayule (Parthenium argentahmz Gray) is a rubber producing, small, woody perennial shrub native to the Chihuahuan desert region of north-central Mexico and southwest Texas. Natural rubber is a commodity that accounts for an annual import * Corresponding author. Fax: 520 621-7186.
deficit of nearly $1 billion for the United States. Over 2,000 rubber producing species are known. However, only two, Hevea brasi1iensi.s (A. Juss.) Muell.-Arg. and guayule, have been exploited as commercial sources of natural rubber. Today, Hevea is the sole source of natural kbber for industry. Nevertheless, active research and development programs are underway to domesticate and commercialize guayule. Guayule is envisioned as a new
0926-6690/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved. SSDI 0926-6690(95)00046-l
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D. T. Ray et al. I Industrial Crops and Products 4 (1995) 303-310
or alternative crop for arid and semiarid regions of the southwestern United States, north-central Mexico, and regions with similar climates around the world (Ray, 1993; Thompson and Ray, 1989). In the twentieth century, three periods of interest in the domestication of guayule for the tire industry can be differentiated: (1) a program conducted by the Intercontinental Rubber Co. at the turn of the last century; (2) the Emergency Rubber Project (ERP) during World War II; and (3) the post ‘oil crisis’ research that followed the quadrupling of crude oil prices in the early 1970’s (Thompson and Ray, 1989). Major obstacles to guayule commercialization have been low rubber yields, high costs of growing and processing the shrub, and high capital investment for construction of processing plants (Estilai et al., 1992). It was hoped that co-products, such as resins, bagasse, and waxes, would generate the additional revenues necessary to make guayule a commercial success (Estilai and Ray, 1991; Thompson and Ray, 1989). However, the rubber industry has considered guayule as a crop to be used only during times of emergency, since they have their own Hevea plantations in Asia and Africa. Such lack of interest in guayule may change with the recent discovery that guayule rubber is hypoallergenic in contrast to Hevea latex rubber products, which can cause allergic reactions ranging from skin rashes to life-threatening anaphylactic shock (Cornish, 1995; Siler and Cornish, 1994). Hevea natural rubber products that have caused allergic reactions include gloves, condoms, catheters, and other medical devises. An estimated 6.5% of the general population in the United States and 7.7% of the general population in England produce antibodies to Hevea rubber proteins. Among health care workers, the estimates are higher and vary between 10 and 40% (0.5 to 2 million individuals) in the United States, alone. This specialty market can pay a premium for the hypoallergenic latex and thus lead to the commercialization of guayule, which was not economically feasible for the production of guayule rubber tires. Plant breeding has been, and will continue to be, one of the most effective methods of enhancing the production and quality of guayule
latex. Improvement in guayule, as in any crop, relies upon the propagation of superior strains of plants. Progress through selection is possible only if genetic variability is present in the available germplasm (Estilai and Ray, 1991; Thompson and Ray, 1989). Diploid guayule reproduces sexually, and polyploid plants reproduce predominately by apomixis (asexual reproduction by seed) (Thompson and Ray, 1989). By using sexual plants, traditional plant breeding schemes can be utilized, but diploid guayule plants generally yield lower in rubber and are more susceptible to Phytophthora clyptogea Pethybridge and Lafferty (Mihail et al., 1991) and K dahliae (Gerstel, 1950) than polyploids. Such problems with diploids have caused guayule breeding programs to emphasize selection within polyploid populations. Although progress has been slower than desired, improvement among polyploids through breeding has been realized (Estilai and Ray, 1991; Estilai et al., 1992; Gathman et al., 1992; Thompson and Ray, 1989). Progress among diploids remains a long-term goal (Ray, 1993.) During World War II, Vbticillium wilt was a significant problem in field plantings, and infected plants were of any age (Campbell et al., 1943; Presley, 1943). This was primarily a problem in the cooler areas of California, but was also observed in Arizona, New Mexico, and Texas (Presley and Westen, 1944). Schneider (1945, 1948) screened several guayule cultivars for susceptibility to K dahliae and determined the role of temperature and soil moisture in disease development. Cultivars were found with a range of susceptibility from severely affected to somewhat tolerant. The disease was exacerbated by frequent irrigations and cool temperatures. Gerstel (1950) noted that diploid plants were highly susceptible, and tetraploid plants were significantly more tolerant to l&ticilliumt Greenhouse studies have shown differences in tolerance to kticillium among related Parthenizun species and polyploid guayule germplasm (Cheo and Beaupre, 1981; Or-urn and Alcorn, 1986). Diploids have been used successfully in guayule breeding programs through crossing with polyploids (Ray, 1993). Although polyploids reproduce apomictically, meiosis is normal in pollen mother
D. I: Ray et al. /Industrial Crops and Products 4 (1995) 303-310
cells. Thus, they can be used as male parents in breeding schemes. Because guayule populations are very heterogeneous, with high levels of heterozygosity, when diploid plants (sexual) are used as female parents and polyploid plants used as males, every progeny plant resulting from these crosses has the potential to have a different genotype (Thompson and Ray, 1989). Crossings based on the I/erticiZZium-tolerant diploid line would not only allow for the release of new genetic diversity for yield components, but the progeny would receive genes for Vericillium tolerance. In an unpublished test (D.T. Ray, A.E. Thompson, and D.A. Dierig), unselected progeny from diploid x polyploid open-pollinated crosses yielded the highest biomass and fourth highest rubber yield among 26 lines. This breeding scheme shows great potential for increasing yields, releasing genetic variability and incorporating disease tolerance in guayule, and is the most likely avenue of application of the work reported here. The objectives of this work were (1) to develop a diploid guayule line through mass selection with tolerance to K dahliae for use in plant breeding programs, and (2) to evaluate newly available and improved guayule lines for PbticiZZium tolerance. 2. Materials and methods 2.1. Selection of verticillium-tolerant diploid guayule
Seed source: Seed, ‘36-Chromosome’ (PI 478663), was obtained from the National Seed Storage Laboratory, and grown in open-pollinated plots for increase by Dr. David Rubis of The University of Arizona. Seed from this increase plot formed the basis for the first cycle of selection (1980-1982). The second cycle of selection was completed in 1985, and the third cycle completed in 1990 (Table 1). Progenies of the second cycle of selection were used in comparative greenhouse and field tests. Seedlings were maintained in transplanting trays (one plant per cell) until about one month prior to inoculation when they were transplanted into loo-mm diameter pots. Inoculum: K dahliae cultures were grown on Czapeks agar (DIFCO, Detroit, MI) plates. The
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contents of three overgrown agar plates of the fungus blended in 500 ml of distilled water resulted in a suspension containing approximately lo6 to lo7 conidia/ml and lo4 to lo5 microsclerotia/ml of K dahliae. In the first generation and the first two rounds of the second generation of selections, isolate DX-2 was used. To reduce the possibility of strain specificity in the selection for tolerance, isolates 70-21, and 81-38a were used with DX-2 in the third round of the second generation of selection. A combination of eight isolates (Table 2) was used in the third generation of selection. Selection: Three cycles of selection were performed in the greenhouse and included three inoculations in each cycle: the first by soil drench (25 ml/pot) and the second and third by root dip (50 to 100 ml/plant). Plants were evaluated two to four months after each inoculation as follows: 0 = dead; 1 = almost all of the leaves and stem tissue dead, but with a few spots of green on the stem; 2 = unilateral chlorosis and necrosis on more than 3 leaves, with leaf twist and curl; 3 = unilateral chlorosis or necrosis on 3 or fewer leaves, with no leaf twist or curl; and 4 = no symptoms. Only plants rated 3 or 4 were selected for further evaluation. Plants selected after three inoculations were also rated for vascular discoloration. Four to six week old cotton (Gossypium hirsutum L.) plants (‘Delta Pine 90’) were inoculated with the guayule to confirm the pathogenic@ of the inoculum. Because seed was open-pollinated, plants surviving the selection screening were evaluated by standard cytological techniques to confirm the diploid chromosome number (Thompson and Ray, 1989) (Table 1). Surviving plants were also evaluated for rubber and resin content by the methodology described in Schloman et al. (1986). Only titiicillium-tolerant diploid plants with the rubber content above 5% (sampled at approximately 2 years of age) were used to produce seed for the next generation of screening (Table 1). 2.2. Evaluation of improved lines Greenhouse were conducted selected at the of the guayule
trials: Three greenhouse trials using rooted cuttings from plants end of the second generation diploid screening. In all cases,
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Table 1 Greenhouse screening of open-pollinated Selection cycle ;; 38
diploid guayule line ‘36chromosome’ (PI 478663) for Vericillium wilt tolerancea
Inoculation b, number of plants inoculated
Number of plants selected as
First c
Second d
Third d
Tolerant
Diploid
Source for next cycle
2393 863 346
816 400 76
281 253 40
68 66 24
33 65 24
6 12 24
a Seed was obtained from the national Seed Storage Laboratory, Fort Collins, CO, USA, and increased at The Arizona. b Only plants that were healthy or had mild symptoms after an inoculation were given a second or third inoculation; were inoculated at one time. c The first inoculation was by soil drench. d The second and third inoculations were by root dip. e First inoculation, December 1980-January 1982; second inoculation, March 1981-June 1982; third inoculation, November 1982. f First inoculation, July 1983-May 1984; second inoculation, October 1983-June 1984; third inoculation, December 1984. s First inoculation, March 1989; second inoculation, November 1989; third inoculation, February 1990.
Table 2 Isolates of Verticillium dahliae used in greenhouse screening for tolerance of guayule diploids and new breeding lines Isolate
Original host
Reaction on cotton a
DX-2 70-21 Sl-14a 81-14b 81-38a 85-47-4 b 86-34 87-31
Cotton Pepper Jojoba Jojoba Guayule Jojoba Okra Pistachio
Defoliating Not defoliating Defoliating Defoliating Defoliating Defoliating Defoliating Defoliating
a The K dahliae isolates were classified according to whether
or not they cause defoliation of cotton in greenhouse inoculation tests. All isolates infected cotton and caused typical symptoms of vascular discoloration and unilateral chlorosis of leaves. b In some inoculations, seven rather eight isolates were used with isolate 85-47-4 omitted.
cuttings were inoculated by root drench with 25 ml of inoculum consisting of a mixture of eight isolates of K dahliae (Table 2) prepared as described previously. The experiments were arranged in a randomized complete block design with a cutting from each selected diploid plant plus a cutting of ‘Cal 3’ (PI 478664, a diploid line selected for higher rubber yield, but unselected for Rtiicillium-tolerance, thus considered I/erticiZZium-susceptible), in each block. Environ-
University of not all plants
June 19811983-August
mental conditions in our greenhouse were variable enough to affect symptom expression. At the conclusion of each trial, plants were rated from 0 to 4 according to the previously described system. The first cutting experiment, evaluated on 15 December 1987, consisted of 21 cuttings from each of eight selected VWicillium-screened diploid plants. The second experiment, evaluated on 20 March 1989, consisted of 20 to 30 cuttings from each of six selected plants. The third cutting experiment, also evaluated on 20 March 1989, was a pair-wise comparison of cuttings from plant 418-4 with cuttings from Cal 3 plants. A greenhouse evaluation of diploid seedlings (as opposed to cuttings), from 12 selected second generation KHiciZZium-screened diploid plants, was concluded on 01 May 1989. Additionally, seedlings of eighteen (15 polyploid and 3 diploid) improved guayule lines were evaluated in four trials in the greenhouse for tolerance to K dahliae based on inoculations by soil drench using a mixture of seven isolates of the fungus (Table 2). Inoculation dates were 02 March, 06 April, 23 August and 11 November 1993, and these lines were scored on a scale of 0 to 4. Plants were 4,5,9 and 12 months old at the time of inoculation in trials 1 through 4, respectively. Field trial: A trial was conducted at The University of Arizona Agricultural Experiment Sta-
D. ‘I: Ray et al. I Industrial Crops and Products 4 (1995) 303-310
tion, Marana Agricultural Centre, in a field that had been maintained in cotton without rotation for over six years, and in which Vericillium wilt had been observed the previous year. Guayule tested were progenies from the second cycle of selection (VZrticilZium-tolerant) and Cal 3. Because lines had been tested previously in the greenhouse, symptom expression was defined. Guayule seeds from each line were planted into trays in the greenhouse, and transplanted into the field on 03 April 1991. Plants were arranged in plots 6 m long, and separated by 6 m blocks of cotton to monitor for the presence of Vericillium wilt in the field. K dahliae can be difficult to isolate from guayule, but is very easy to isolate from cotton. Therefore, isolations were made from the cotton interspersed with the guayule plantings to confirm the presence of K duhliue in the field during the course of the experiment. We also associated mortality in the guayule with I/erticillium wilt based on symptom expression, that was similar to plants inoculated in the greenhouse. Symptoms included twisting and curling of leaves, unilateral leaf necrosis, and vascular discoloration. Isolation of K duhliue from symptomatic guayule was successful less than 10% of the time. (Greenhouse experience suggests that there is a fairly narrow time interval following appearance of symptoms when it is relatively easy to isolate Kduhliue from guayule.) Statistical analyses: Statistical analyses were run on SAS PROC GLM program (SAS, 1985). Following a significant F-test (P 5 0.05), mean Verticillium tolerance ratings were compared using Duncan’s multiple range test.
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3. Results Three generations of selection of diploid germplasm for higher levels of k-ticillium tolerance were conducted between 1980 and 1990 (Table 1). Selection intensity was extremely high. In the first generation of selection, 34% of the seedlings survived the first round of inoculations, 12% of the original plants survived the second round of selection, and 3% survived a third round of inoculations. Of the 68 surviving plants, 33 (49%) were found to be diploid. After evaluating this 33 plants for rubber content, only 6 diploids (0.2% of the original population) were used as a seed source for the second generation of selection. The second round of selection yielded 253 plants (29%) surviving three rounds of inoculations (Table 1). Sixty-six plants were selected for J&ticillium tolerance and after evaluation for ploidy level, 65 were found to be diploid. Twelve plants (1.4%) were selected as the seed source for the third generation of selection. The third generation of selection yielded 24 plants (7%) from which seed are available for use in guayule breeding programs. The second generation of I/erticillium screened diploid plants showed improvement in tolerance over Cal 3 (the unselected diploid check) under greenhouse conditions (Table 3). Almost all (92%) of the Cal 3 plants showed either symptoms or death, with 38% of the selected diploids showing tolerance to the mixture of Kduhliue isolates. Under field conditions (Table 4), the Verticillium screened diploids performed even better (64%
Table 3 Comparison of Verricillium-screened diploid guayule plants with Cal 3, a Verkillium-susceptible All plants were inoculated by soil drench with a mixture of eight isolates of Verticillium dahliae
diploid line, in greenhouse trials;
Plants either dead or with severe symptoms (%)
Vert-screened Cal 3
diploids c
Experiment 1 a (15 Dec. 1987)
Experiment 2 a (20 Mar. 1989)
Experiment 3 a (20 Mar. 1989)
Experiment 4 b (1 Mav 1989)
32 57
10 55
5 39
62 92
a Plants propagated from cuttings. b Plants propagated from seed. c Plants tested were progeny from the second cycle of selection.
D.T. Ray et al. /Industrial Cmps and Products 4 (1995) 303-310
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Table 4 Comparison of Verricillium-screened diploid guaynle plants with Cal 3, a Krflicillium-susceptible guayule line, in a field with a history of tirticilliwn wilt disease of cotton Percent plants either dead Total number of plants evaluated a or with severe symptoms b (%) Vert-screened diploids ’ Cal 3
146 1.50
36 80
a Transplanted 03 April 1991. b Transplants evaluated on 20 November 1991. c Plants tested were progeny from the second cycle of selection.
tolerant), showing greater tolerance than those grown in the greenhouse. Sixteen improved breeding lines, in four separate inoculation experiments in the greenhouse (performed in 1993), showed statistically significant differences in I&-ticillium tolerance among lines (Table 5). These lines were tested against ‘N576’ and ‘11605’ (both polyploids) that are commonly used as unimproved controls in breeding experiments. Three of the 16 lines included in the evaluation were diploids, ‘AZR3’ an unscreened diploid line, and ‘214-9’ and ‘418-6’, two second generation Verticillium-screened diploid lines. Line 418-6 ranked highest in tolerance among all lines tested, but not significantly higher than N576. Line 214-9, on the other hand, ranked among the lowest in tolerance, and was not significantly different from the unscreened diploid line AZR3. Age of plants was important in screening. In the 1993 inoculation experiments, tolerance to K duhliue, averaged over all lines tested, increased with the age of the plants at the time of inoculation (Fig. 1). In greenhouse trials, Delta Pine 90 cotton plants inoculated with K duhliue as positive controls developed symptoms typical of Vericillium wilt on cotton by the time the guayule was evaluated for symptoms. Symptoms included unilateral chlorosis and necrosis of leaves, defoliation, and vascular discoloration. Over 95% of the inoculated cotton plants showed severe symptoms of the disease and K duhliue was consistently re-isolated from the inoculated cotton. In the field trial,
Table 5 Mean tolerance ratings for 18 guayule lines to Verticillium dahliae based on greenhouse inoculations by soil drench using a mixture of seven isolates of the fungus a Line
Ploidy level
Mean rating b
418-6 AZRl UC 104 N9-5 N576 11605 AZR2 016-3 UC 101 N7-5 016-l Cal 6 Cal 7 N6-5 AZR3 P3-1 214-9 UC 102
Diploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Diploid Polyploid Diploid Polyploid
2.2 ac 2.0 ab 2.0 ab 1.9 ab 1.8 abc 1.7 bc 1.4 cd 1.3 cde 1.3 cde 1.3 cdef 1.2 def 1.2 def 1.2 def 1.1 def 0.9 efg 0.8 fg 0.8 fg 0.6 g
a Mean ratings are based on the combined results of four experiments in which all lines were entered in a randomized complete block design, with between 16 and 20 blocks inoculated in each experiment. The four experiments were combined in an analysis of variance in which line effect is highly significant ( P=O.OOOl), but in which the interaction of line and experiment is not significant (P=O.l). The K dahliae isolates are listed in Table 2, with the exception of isolate 85-47-4, which was not used in this experiment. b 0 = dead; 1 = almost all leaves and stem tissue dead, but with a few spots of green on the stem; 2 = unilateral chlorosis and necrosis on more than three leaves, with leaf twist and curl; 3 = unilateral chlorosis or necrosis on 3 or fewer leaves, with no leaf twist or curl; 4 = no symptoms. c Means followed by the same letter are not significantly different with a probability level of 0.05 according to Duncan’s multiple range test.
K duhliue was consistently isolated from symptomatic naturally infected cotton in plots interspersed with the guayule plots.
4. Discussion Our results indicate that there is a wide range in symptom expression among guayule lines following infection by K duhliue, that corroborate the observations of Gerstel (1950) and Schneider (1945, 1948). Verticillium tolerance among the se-
D. I: Ray et al. I Industrial Crops and Products 4 (1995) 303-310
0.0. 0
3
6
9
12
15
Age (months) Fig. 1. Tolerance of guayule to Verticillium dahliae as a function of age at the time of inoculation. Approximately two months after inoculation by soil drench, plants were rated from 0 to 4, with 0 = death and 4 = no symptoms. Each dot represents the average tolerance rating of 16 to 20 plants from each of 18 guayule lines. Error bars represent 95% confidence intervals.
309
agricultural regions are known to have particular problems with WticiZZium wilt. In such locations, it is important not to plant guayule in fields where a susceptible crop (such as cotton) has been grown in monoculture for several years. However, if one must plant in such a field, it also would be prudent to use older transplants, since these appear to have some innate tolerance to this fungus. For guayule to be successful as a commercial crop, an ongoing breeding and selection program is important. The diploid material described here represents a good starting point for continuing selection for VericiZZium tolerance among new diploid lines. However, we believe that even with this I+tiiciZZium-tolerant diploid line, additional selections always should be from plants exposed to K dahliue either in the greenhouse or field. References
lected diploids was not complete, but improved. Variability in outcome is due to guayule’s complicated reproductive biology and heterogeneous population structure (Thompson and Ray, 1989), as well as temperature and soil moisture (Schneider, 1945, 1948). Diploid guayule reproduces predominately sexually, but is self-incompatible. Thus, out-crossing is enhanced, and selection is for heterozygosity. Since improvement for l&tic& Zium tolerance in the diploid germplasm has taken place, there has been genetic improvement. Yet, the environmental component for this trait is high. Greenhouse conditions (Table 3) generally exacerbated Verticillium symptoms, as compared to field conditions (Table 4), and tolerance increases with age of the plant (Fig. 1). There were also subtle variations in greenhouse conditions. During these studies, we found that plants in warmer spots in the greenhouse had less symptom expression, and this may have affected the screening by not yielding as high a level of tolerance as desired. Practical post-infection treatments to control Vhticillium are not available. Therefore, the best management approaches to controlling this disease are the use of tolerant varieties and cultural techniques, specifically the use of crop rotations with non-host species, and the avoidance of fields where this disease is known to be a problem. Some
Campbell, W.A., Leach, L.D., Presley, J.T. and Snyder, W.C., 1943. Some diseases of guayule in California. Plant Disease Reporter, 27: 63-66. Cheo, PC. and Beaupre, CM.&, 1981. Evaluation of Parthenium species for resistance to tirticillium dahliae. Plant Disease, 65: 658-661. Cornish, K., 1995. Hypoallergenic Natural Rubber Products from Parthenium argentatum (Gray) and Other Non-Hevea brasiliensis Species. U.S. Patent Office. 081423, 911. Estilai, A. and Ray, D.T., 1991. Breeding guayule. In: J.W. Whitworth and E.E. Whitehead (Editors), Guayule Natural Rubber. Office of Arid Lands, Univ. of Arizona, Tucson, AZ, pp. 47-92. Estilai, A., Ehdaie, B., Naqvi, H.H., Dierig, D.A., Ray, D.T. and Thompson, A.E., 1992. Rubber and resin yield performance of new guayule selections. Agron. J., 84: 420-424. Gathman, A.C., Ray, D.T. and Livingston, M., 1992. Comparison of three stability measures in guayule. Ind. Crops Prod. 1: 67-74. Gerstel, D.U., 1950. Is resistance to Verticillium wilt in guayule related to chromosome number? Agron. J., 42: 310-311. Mihail, J.D., Alcorn, S.M. and Whitworth, J.W., 1991. Plant health: the interactions of guayule, microorganisms, arthropods, and weeds. In: J.W. Whitworth and E.E. Whitehead (Eds.) Guayule Natural Rubber. Office of Arid Lands, Univ. of Arizona, Tucson, AZ, pp. 173-216. Orum, TV. and Alcom, S.M., 1986. Greenhouse evaluation of tolerance of triploid and tetraploid guayule lines to Verticillium dahliae. In: D.D. Fangmeier and S.M. Alcom (Eds.). Proceedings of the Fourth International Conference on Guayule Research and Development, Tucson, AZ. Guayule Rubber Society, pp. 329-336.
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Presley, J.T., 1943. Some diseases affecting cultivated guayule in the Southwest during 1942. Plant Disease Reporter, 27: 94-96. Presley, J.T and Weston, L., 1944. Fungi isolated from diseased guayule. Plant Disease Reporter, 32: 984-985. Ray, D.T., 1993. Guayule: a source of natural rubber. In: J. Janick and J.E. Simon (Eds.). New Crops. John Wiley and Sons, Inc., New York, NY, pp. 338-342. SAS, 1985. SAS/STAT Guide for Personal Computers, Version 6 Edition. SAS Institute Inc., Cary, NC, 378 pp. Schloman, W.W., Jr., Garrot, D.J., Jr. and Ray, D.T, 1986. Water stress and seasonal effects on rubber quality in
irrigated guayule. J. Agric. and Food Chem., 34: 383-385. Schneider, H., 1945. Surveys and observations of Verticillium wilt of guayule in California from 1943-45. Plant Disease Reporter, 29: 615-617. Schneider, H., 1948. Susceptibility of guayule to Verticillium wilt and influence of soil temperature and moisture on development of infection. J. Agric. Res., 76: 129-143. Siler, D.J. and Cornish, K., 1994. Hypoallergenicity of guayule rubber particle proteins compared to Hevea latex proteins. Ind. Crops Prod., 2: 307-313. Thompson, A.E. and Ray, D.T., 1989. Breeding guayule. Plant Breed Rev., 6: 93-165.
ELSEVIER
Industrial Crops and Products 4 (1995) 303-310
Selection of diploid and screening of polyploid guayule lines for Verticilhn tolerance D.T. Ray ap*,T.V. Orum b, D.M. Bigelow b, S.M. Alcorn b aDepartment of Plant Sciences, The University of Arizona, Tucson, AZ 85721, USA b Depafiment of Plant Pathology, The Universiry of Arizona, Tucson,AZ 85721, USA
Received 21 August 1995; accepted 20 September 1995
Abstract Guayule (Parthenium algentatum Gray) produces a high quality hypoallergenic latex, that is of interest for use in the medical industry. In order to meet the anticipated demand, guayule will need to be planted in mild climatic zones over a wide geographic area, and often following cotton (Gossypium sp.) or other crops susceptible to the root-invading fungus Vetiicillium dahliae Kleb. Since guayule has been shown previously to be susceptible to L! dahliae, the goals of this work were: (1) to develop a diploid guayule line that is tolerant to K dahliae through mass selection, and (2) to evaluate improved guayule lines for tirticillium tolerance. To achieve the first goal, a diploid guayule population was subjected to three cycles of selection, with only the plants with the greatest tolerance and the highest rubber yields used in the subsequent cycles of selection. Second, fifteen improved polyploid and three diploid lines were evaluated for tolerance to K dahliae under greenhouse conditions. Cuttings were found to be more tolerant than seedlings, and tolerance increased with age of the plant at the time of inoculation. The Verticillium screened diploids expressed greater tolerance than an unscreened diploid population in both greenhouse and field trials. Among the 18 improved lines, 13 of which had not been previously evaluated, a Vkticillium-screened diploid line (418-6) had the highest tolerance ranking, but was not significantly better than four unscreened polyploid lines. Continued evaluation of improved lines will help growers in making informed decisions about which lines to plant for their specific locations. The Verticillium-tolerantdiploid germplasm material developed in this study is available
to plant breeding programs. Keywords:
Parthenium
argentatum;
Ploidy levels; Verticillium wilt; Verticillium dahliae; Selection schemes
1. Introduction
Guayule (Parthenium argentahmz Gray) is a rubber producing, small, woody perennial shrub native to the Chihuahuan desert region of north-central Mexico and southwest Texas. Natural rubber is a commodity that accounts for an annual import * Corresponding author. Fax: 520 621-7186.
deficit of nearly $1 billion for the United States. Over 2,000 rubber producing species are known. However, only two, Hevea brasi1iensi.s (A. Juss.) Muell.-Arg. and guayule, have been exploited as commercial sources of natural rubber. Today, Hevea is the sole source of natural kbber for industry. Nevertheless, active research and development programs are underway to domesticate and commercialize guayule. Guayule is envisioned as a new
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or alternative crop for arid and semiarid regions of the southwestern United States, north-central Mexico, and regions with similar climates around the world (Ray, 1993; Thompson and Ray, 1989). In the twentieth century, three periods of interest in the domestication of guayule for the tire industry can be differentiated: (1) a program conducted by the Intercontinental Rubber Co. at the turn of the last century; (2) the Emergency Rubber Project (ERP) during World War II; and (3) the post ‘oil crisis’ research that followed the quadrupling of crude oil prices in the early 1970’s (Thompson and Ray, 1989). Major obstacles to guayule commercialization have been low rubber yields, high costs of growing and processing the shrub, and high capital investment for construction of processing plants (Estilai et al., 1992). It was hoped that co-products, such as resins, bagasse, and waxes, would generate the additional revenues necessary to make guayule a commercial success (Estilai and Ray, 1991; Thompson and Ray, 1989). However, the rubber industry has considered guayule as a crop to be used only during times of emergency, since they have their own Hevea plantations in Asia and Africa. Such lack of interest in guayule may change with the recent discovery that guayule rubber is hypoallergenic in contrast to Hevea latex rubber products, which can cause allergic reactions ranging from skin rashes to life-threatening anaphylactic shock (Cornish, 1995; Siler and Cornish, 1994). Hevea natural rubber products that have caused allergic reactions include gloves, condoms, catheters, and other medical devises. An estimated 6.5% of the general population in the United States and 7.7% of the general population in England produce antibodies to Hevea rubber proteins. Among health care workers, the estimates are higher and vary between 10 and 40% (0.5 to 2 million individuals) in the United States, alone. This specialty market can pay a premium for the hypoallergenic latex and thus lead to the commercialization of guayule, which was not economically feasible for the production of guayule rubber tires. Plant breeding has been, and will continue to be, one of the most effective methods of enhancing the production and quality of guayule
latex. Improvement in guayule, as in any crop, relies upon the propagation of superior strains of plants. Progress through selection is possible only if genetic variability is present in the available germplasm (Estilai and Ray, 1991; Thompson and Ray, 1989). Diploid guayule reproduces sexually, and polyploid plants reproduce predominately by apomixis (asexual reproduction by seed) (Thompson and Ray, 1989). By using sexual plants, traditional plant breeding schemes can be utilized, but diploid guayule plants generally yield lower in rubber and are more susceptible to Phytophthora clyptogea Pethybridge and Lafferty (Mihail et al., 1991) and K dahliae (Gerstel, 1950) than polyploids. Such problems with diploids have caused guayule breeding programs to emphasize selection within polyploid populations. Although progress has been slower than desired, improvement among polyploids through breeding has been realized (Estilai and Ray, 1991; Estilai et al., 1992; Gathman et al., 1992; Thompson and Ray, 1989). Progress among diploids remains a long-term goal (Ray, 1993.) During World War II, Vbticillium wilt was a significant problem in field plantings, and infected plants were of any age (Campbell et al., 1943; Presley, 1943). This was primarily a problem in the cooler areas of California, but was also observed in Arizona, New Mexico, and Texas (Presley and Westen, 1944). Schneider (1945, 1948) screened several guayule cultivars for susceptibility to K dahliae and determined the role of temperature and soil moisture in disease development. Cultivars were found with a range of susceptibility from severely affected to somewhat tolerant. The disease was exacerbated by frequent irrigations and cool temperatures. Gerstel (1950) noted that diploid plants were highly susceptible, and tetraploid plants were significantly more tolerant to l&ticilliumt Greenhouse studies have shown differences in tolerance to kticillium among related Parthenizun species and polyploid guayule germplasm (Cheo and Beaupre, 1981; Or-urn and Alcorn, 1986). Diploids have been used successfully in guayule breeding programs through crossing with polyploids (Ray, 1993). Although polyploids reproduce apomictically, meiosis is normal in pollen mother
D. I: Ray et al. /Industrial Crops and Products 4 (1995) 303-310
cells. Thus, they can be used as male parents in breeding schemes. Because guayule populations are very heterogeneous, with high levels of heterozygosity, when diploid plants (sexual) are used as female parents and polyploid plants used as males, every progeny plant resulting from these crosses has the potential to have a different genotype (Thompson and Ray, 1989). Crossings based on the I/erticiZZium-tolerant diploid line would not only allow for the release of new genetic diversity for yield components, but the progeny would receive genes for Vericillium tolerance. In an unpublished test (D.T. Ray, A.E. Thompson, and D.A. Dierig), unselected progeny from diploid x polyploid open-pollinated crosses yielded the highest biomass and fourth highest rubber yield among 26 lines. This breeding scheme shows great potential for increasing yields, releasing genetic variability and incorporating disease tolerance in guayule, and is the most likely avenue of application of the work reported here. The objectives of this work were (1) to develop a diploid guayule line through mass selection with tolerance to K dahliae for use in plant breeding programs, and (2) to evaluate newly available and improved guayule lines for PbticiZZium tolerance. 2. Materials and methods 2.1. Selection of verticillium-tolerant diploid guayule
Seed source: Seed, ‘36-Chromosome’ (PI 478663), was obtained from the National Seed Storage Laboratory, and grown in open-pollinated plots for increase by Dr. David Rubis of The University of Arizona. Seed from this increase plot formed the basis for the first cycle of selection (1980-1982). The second cycle of selection was completed in 1985, and the third cycle completed in 1990 (Table 1). Progenies of the second cycle of selection were used in comparative greenhouse and field tests. Seedlings were maintained in transplanting trays (one plant per cell) until about one month prior to inoculation when they were transplanted into loo-mm diameter pots. Inoculum: K dahliae cultures were grown on Czapeks agar (DIFCO, Detroit, MI) plates. The
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contents of three overgrown agar plates of the fungus blended in 500 ml of distilled water resulted in a suspension containing approximately lo6 to lo7 conidia/ml and lo4 to lo5 microsclerotia/ml of K dahliae. In the first generation and the first two rounds of the second generation of selections, isolate DX-2 was used. To reduce the possibility of strain specificity in the selection for tolerance, isolates 70-21, and 81-38a were used with DX-2 in the third round of the second generation of selection. A combination of eight isolates (Table 2) was used in the third generation of selection. Selection: Three cycles of selection were performed in the greenhouse and included three inoculations in each cycle: the first by soil drench (25 ml/pot) and the second and third by root dip (50 to 100 ml/plant). Plants were evaluated two to four months after each inoculation as follows: 0 = dead; 1 = almost all of the leaves and stem tissue dead, but with a few spots of green on the stem; 2 = unilateral chlorosis and necrosis on more than 3 leaves, with leaf twist and curl; 3 = unilateral chlorosis or necrosis on 3 or fewer leaves, with no leaf twist or curl; and 4 = no symptoms. Only plants rated 3 or 4 were selected for further evaluation. Plants selected after three inoculations were also rated for vascular discoloration. Four to six week old cotton (Gossypium hirsutum L.) plants (‘Delta Pine 90’) were inoculated with the guayule to confirm the pathogenic@ of the inoculum. Because seed was open-pollinated, plants surviving the selection screening were evaluated by standard cytological techniques to confirm the diploid chromosome number (Thompson and Ray, 1989) (Table 1). Surviving plants were also evaluated for rubber and resin content by the methodology described in Schloman et al. (1986). Only titiicillium-tolerant diploid plants with the rubber content above 5% (sampled at approximately 2 years of age) were used to produce seed for the next generation of screening (Table 1). 2.2. Evaluation of improved lines Greenhouse were conducted selected at the of the guayule
trials: Three greenhouse trials using rooted cuttings from plants end of the second generation diploid screening. In all cases,
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Table 1 Greenhouse screening of open-pollinated Selection cycle ;; 38
diploid guayule line ‘36chromosome’ (PI 478663) for Vericillium wilt tolerancea
Inoculation b, number of plants inoculated
Number of plants selected as
First c
Second d
Third d
Tolerant
Diploid
Source for next cycle
2393 863 346
816 400 76
281 253 40
68 66 24
33 65 24
6 12 24
a Seed was obtained from the national Seed Storage Laboratory, Fort Collins, CO, USA, and increased at The Arizona. b Only plants that were healthy or had mild symptoms after an inoculation were given a second or third inoculation; were inoculated at one time. c The first inoculation was by soil drench. d The second and third inoculations were by root dip. e First inoculation, December 1980-January 1982; second inoculation, March 1981-June 1982; third inoculation, November 1982. f First inoculation, July 1983-May 1984; second inoculation, October 1983-June 1984; third inoculation, December 1984. s First inoculation, March 1989; second inoculation, November 1989; third inoculation, February 1990.
Table 2 Isolates of Verticillium dahliae used in greenhouse screening for tolerance of guayule diploids and new breeding lines Isolate
Original host
Reaction on cotton a
DX-2 70-21 Sl-14a 81-14b 81-38a 85-47-4 b 86-34 87-31
Cotton Pepper Jojoba Jojoba Guayule Jojoba Okra Pistachio
Defoliating Not defoliating Defoliating Defoliating Defoliating Defoliating Defoliating Defoliating
a The K dahliae isolates were classified according to whether
or not they cause defoliation of cotton in greenhouse inoculation tests. All isolates infected cotton and caused typical symptoms of vascular discoloration and unilateral chlorosis of leaves. b In some inoculations, seven rather eight isolates were used with isolate 85-47-4 omitted.
cuttings were inoculated by root drench with 25 ml of inoculum consisting of a mixture of eight isolates of K dahliae (Table 2) prepared as described previously. The experiments were arranged in a randomized complete block design with a cutting from each selected diploid plant plus a cutting of ‘Cal 3’ (PI 478664, a diploid line selected for higher rubber yield, but unselected for Rtiicillium-tolerance, thus considered I/erticiZZium-susceptible), in each block. Environ-
University of not all plants
June 19811983-August
mental conditions in our greenhouse were variable enough to affect symptom expression. At the conclusion of each trial, plants were rated from 0 to 4 according to the previously described system. The first cutting experiment, evaluated on 15 December 1987, consisted of 21 cuttings from each of eight selected VWicillium-screened diploid plants. The second experiment, evaluated on 20 March 1989, consisted of 20 to 30 cuttings from each of six selected plants. The third cutting experiment, also evaluated on 20 March 1989, was a pair-wise comparison of cuttings from plant 418-4 with cuttings from Cal 3 plants. A greenhouse evaluation of diploid seedlings (as opposed to cuttings), from 12 selected second generation KHiciZZium-screened diploid plants, was concluded on 01 May 1989. Additionally, seedlings of eighteen (15 polyploid and 3 diploid) improved guayule lines were evaluated in four trials in the greenhouse for tolerance to K dahliae based on inoculations by soil drench using a mixture of seven isolates of the fungus (Table 2). Inoculation dates were 02 March, 06 April, 23 August and 11 November 1993, and these lines were scored on a scale of 0 to 4. Plants were 4,5,9 and 12 months old at the time of inoculation in trials 1 through 4, respectively. Field trial: A trial was conducted at The University of Arizona Agricultural Experiment Sta-
D. ‘I: Ray et al. I Industrial Crops and Products 4 (1995) 303-310
tion, Marana Agricultural Centre, in a field that had been maintained in cotton without rotation for over six years, and in which Vericillium wilt had been observed the previous year. Guayule tested were progenies from the second cycle of selection (VZrticilZium-tolerant) and Cal 3. Because lines had been tested previously in the greenhouse, symptom expression was defined. Guayule seeds from each line were planted into trays in the greenhouse, and transplanted into the field on 03 April 1991. Plants were arranged in plots 6 m long, and separated by 6 m blocks of cotton to monitor for the presence of Vericillium wilt in the field. K dahliae can be difficult to isolate from guayule, but is very easy to isolate from cotton. Therefore, isolations were made from the cotton interspersed with the guayule plantings to confirm the presence of K duhliue in the field during the course of the experiment. We also associated mortality in the guayule with I/erticillium wilt based on symptom expression, that was similar to plants inoculated in the greenhouse. Symptoms included twisting and curling of leaves, unilateral leaf necrosis, and vascular discoloration. Isolation of K duhliue from symptomatic guayule was successful less than 10% of the time. (Greenhouse experience suggests that there is a fairly narrow time interval following appearance of symptoms when it is relatively easy to isolate Kduhliue from guayule.) Statistical analyses: Statistical analyses were run on SAS PROC GLM program (SAS, 1985). Following a significant F-test (P 5 0.05), mean Verticillium tolerance ratings were compared using Duncan’s multiple range test.
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3. Results Three generations of selection of diploid germplasm for higher levels of k-ticillium tolerance were conducted between 1980 and 1990 (Table 1). Selection intensity was extremely high. In the first generation of selection, 34% of the seedlings survived the first round of inoculations, 12% of the original plants survived the second round of selection, and 3% survived a third round of inoculations. Of the 68 surviving plants, 33 (49%) were found to be diploid. After evaluating this 33 plants for rubber content, only 6 diploids (0.2% of the original population) were used as a seed source for the second generation of selection. The second round of selection yielded 253 plants (29%) surviving three rounds of inoculations (Table 1). Sixty-six plants were selected for J&ticillium tolerance and after evaluation for ploidy level, 65 were found to be diploid. Twelve plants (1.4%) were selected as the seed source for the third generation of selection. The third generation of selection yielded 24 plants (7%) from which seed are available for use in guayule breeding programs. The second generation of I/erticillium screened diploid plants showed improvement in tolerance over Cal 3 (the unselected diploid check) under greenhouse conditions (Table 3). Almost all (92%) of the Cal 3 plants showed either symptoms or death, with 38% of the selected diploids showing tolerance to the mixture of Kduhliue isolates. Under field conditions (Table 4), the Verticillium screened diploids performed even better (64%
Table 3 Comparison of Verricillium-screened diploid guayule plants with Cal 3, a Verkillium-susceptible All plants were inoculated by soil drench with a mixture of eight isolates of Verticillium dahliae
diploid line, in greenhouse trials;
Plants either dead or with severe symptoms (%)
Vert-screened Cal 3
diploids c
Experiment 1 a (15 Dec. 1987)
Experiment 2 a (20 Mar. 1989)
Experiment 3 a (20 Mar. 1989)
Experiment 4 b (1 Mav 1989)
32 57
10 55
5 39
62 92
a Plants propagated from cuttings. b Plants propagated from seed. c Plants tested were progeny from the second cycle of selection.
D.T. Ray et al. /Industrial Cmps and Products 4 (1995) 303-310
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Table 4 Comparison of Verricillium-screened diploid guaynle plants with Cal 3, a Krflicillium-susceptible guayule line, in a field with a history of tirticilliwn wilt disease of cotton Percent plants either dead Total number of plants evaluated a or with severe symptoms b (%) Vert-screened diploids ’ Cal 3
146 1.50
36 80
a Transplanted 03 April 1991. b Transplants evaluated on 20 November 1991. c Plants tested were progeny from the second cycle of selection.
tolerant), showing greater tolerance than those grown in the greenhouse. Sixteen improved breeding lines, in four separate inoculation experiments in the greenhouse (performed in 1993), showed statistically significant differences in I&-ticillium tolerance among lines (Table 5). These lines were tested against ‘N576’ and ‘11605’ (both polyploids) that are commonly used as unimproved controls in breeding experiments. Three of the 16 lines included in the evaluation were diploids, ‘AZR3’ an unscreened diploid line, and ‘214-9’ and ‘418-6’, two second generation Verticillium-screened diploid lines. Line 418-6 ranked highest in tolerance among all lines tested, but not significantly higher than N576. Line 214-9, on the other hand, ranked among the lowest in tolerance, and was not significantly different from the unscreened diploid line AZR3. Age of plants was important in screening. In the 1993 inoculation experiments, tolerance to K duhliue, averaged over all lines tested, increased with the age of the plants at the time of inoculation (Fig. 1). In greenhouse trials, Delta Pine 90 cotton plants inoculated with K duhliue as positive controls developed symptoms typical of Vericillium wilt on cotton by the time the guayule was evaluated for symptoms. Symptoms included unilateral chlorosis and necrosis of leaves, defoliation, and vascular discoloration. Over 95% of the inoculated cotton plants showed severe symptoms of the disease and K duhliue was consistently re-isolated from the inoculated cotton. In the field trial,
Table 5 Mean tolerance ratings for 18 guayule lines to Verticillium dahliae based on greenhouse inoculations by soil drench using a mixture of seven isolates of the fungus a Line
Ploidy level
Mean rating b
418-6 AZRl UC 104 N9-5 N576 11605 AZR2 016-3 UC 101 N7-5 016-l Cal 6 Cal 7 N6-5 AZR3 P3-1 214-9 UC 102
Diploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Polyploid Diploid Polyploid Diploid Polyploid
2.2 ac 2.0 ab 2.0 ab 1.9 ab 1.8 abc 1.7 bc 1.4 cd 1.3 cde 1.3 cde 1.3 cdef 1.2 def 1.2 def 1.2 def 1.1 def 0.9 efg 0.8 fg 0.8 fg 0.6 g
a Mean ratings are based on the combined results of four experiments in which all lines were entered in a randomized complete block design, with between 16 and 20 blocks inoculated in each experiment. The four experiments were combined in an analysis of variance in which line effect is highly significant ( P=O.OOOl), but in which the interaction of line and experiment is not significant (P=O.l). The K dahliae isolates are listed in Table 2, with the exception of isolate 85-47-4, which was not used in this experiment. b 0 = dead; 1 = almost all leaves and stem tissue dead, but with a few spots of green on the stem; 2 = unilateral chlorosis and necrosis on more than three leaves, with leaf twist and curl; 3 = unilateral chlorosis or necrosis on 3 or fewer leaves, with no leaf twist or curl; 4 = no symptoms. c Means followed by the same letter are not significantly different with a probability level of 0.05 according to Duncan’s multiple range test.
K duhliue was consistently isolated from symptomatic naturally infected cotton in plots interspersed with the guayule plots.
4. Discussion Our results indicate that there is a wide range in symptom expression among guayule lines following infection by K duhliue, that corroborate the observations of Gerstel (1950) and Schneider (1945, 1948). Verticillium tolerance among the se-
D. I: Ray et al. I Industrial Crops and Products 4 (1995) 303-310
0.0. 0
3
6
9
12
15
Age (months) Fig. 1. Tolerance of guayule to Verticillium dahliae as a function of age at the time of inoculation. Approximately two months after inoculation by soil drench, plants were rated from 0 to 4, with 0 = death and 4 = no symptoms. Each dot represents the average tolerance rating of 16 to 20 plants from each of 18 guayule lines. Error bars represent 95% confidence intervals.
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agricultural regions are known to have particular problems with WticiZZium wilt. In such locations, it is important not to plant guayule in fields where a susceptible crop (such as cotton) has been grown in monoculture for several years. However, if one must plant in such a field, it also would be prudent to use older transplants, since these appear to have some innate tolerance to this fungus. For guayule to be successful as a commercial crop, an ongoing breeding and selection program is important. The diploid material described here represents a good starting point for continuing selection for VericiZZium tolerance among new diploid lines. However, we believe that even with this I+tiiciZZium-tolerant diploid line, additional selections always should be from plants exposed to K dahliue either in the greenhouse or field. References
lected diploids was not complete, but improved. Variability in outcome is due to guayule’s complicated reproductive biology and heterogeneous population structure (Thompson and Ray, 1989), as well as temperature and soil moisture (Schneider, 1945, 1948). Diploid guayule reproduces predominately sexually, but is self-incompatible. Thus, out-crossing is enhanced, and selection is for heterozygosity. Since improvement for l&tic& Zium tolerance in the diploid germplasm has taken place, there has been genetic improvement. Yet, the environmental component for this trait is high. Greenhouse conditions (Table 3) generally exacerbated Verticillium symptoms, as compared to field conditions (Table 4), and tolerance increases with age of the plant (Fig. 1). There were also subtle variations in greenhouse conditions. During these studies, we found that plants in warmer spots in the greenhouse had less symptom expression, and this may have affected the screening by not yielding as high a level of tolerance as desired. Practical post-infection treatments to control Vhticillium are not available. Therefore, the best management approaches to controlling this disease are the use of tolerant varieties and cultural techniques, specifically the use of crop rotations with non-host species, and the avoidance of fields where this disease is known to be a problem. Some
Campbell, W.A., Leach, L.D., Presley, J.T. and Snyder, W.C., 1943. Some diseases of guayule in California. Plant Disease Reporter, 27: 63-66. Cheo, PC. and Beaupre, CM.&, 1981. Evaluation of Parthenium species for resistance to tirticillium dahliae. Plant Disease, 65: 658-661. Cornish, K., 1995. Hypoallergenic Natural Rubber Products from Parthenium argentatum (Gray) and Other Non-Hevea brasiliensis Species. U.S. Patent Office. 081423, 911. Estilai, A. and Ray, D.T., 1991. Breeding guayule. In: J.W. Whitworth and E.E. Whitehead (Editors), Guayule Natural Rubber. Office of Arid Lands, Univ. of Arizona, Tucson, AZ, pp. 47-92. Estilai, A., Ehdaie, B., Naqvi, H.H., Dierig, D.A., Ray, D.T. and Thompson, A.E., 1992. Rubber and resin yield performance of new guayule selections. Agron. J., 84: 420-424. Gathman, A.C., Ray, D.T. and Livingston, M., 1992. Comparison of three stability measures in guayule. Ind. Crops Prod. 1: 67-74. Gerstel, D.U., 1950. Is resistance to Verticillium wilt in guayule related to chromosome number? Agron. J., 42: 310-311. Mihail, J.D., Alcorn, S.M. and Whitworth, J.W., 1991. Plant health: the interactions of guayule, microorganisms, arthropods, and weeds. In: J.W. Whitworth and E.E. Whitehead (Eds.) Guayule Natural Rubber. Office of Arid Lands, Univ. of Arizona, Tucson, AZ, pp. 173-216. Orum, TV. and Alcom, S.M., 1986. Greenhouse evaluation of tolerance of triploid and tetraploid guayule lines to Verticillium dahliae. In: D.D. Fangmeier and S.M. Alcom (Eds.). Proceedings of the Fourth International Conference on Guayule Research and Development, Tucson, AZ. Guayule Rubber Society, pp. 329-336.
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Presley, J.T., 1943. Some diseases affecting cultivated guayule in the Southwest during 1942. Plant Disease Reporter, 27: 94-96. Presley, J.T and Weston, L., 1944. Fungi isolated from diseased guayule. Plant Disease Reporter, 32: 984-985. Ray, D.T., 1993. Guayule: a source of natural rubber. In: J. Janick and J.E. Simon (Eds.). New Crops. John Wiley and Sons, Inc., New York, NY, pp. 338-342. SAS, 1985. SAS/STAT Guide for Personal Computers, Version 6 Edition. SAS Institute Inc., Cary, NC, 378 pp. Schloman, W.W., Jr., Garrot, D.J., Jr. and Ray, D.T, 1986. Water stress and seasonal effects on rubber quality in
irrigated guayule. J. Agric. and Food Chem., 34: 383-385. Schneider, H., 1945. Surveys and observations of Verticillium wilt of guayule in California from 1943-45. Plant Disease Reporter, 29: 615-617. Schneider, H., 1948. Susceptibility of guayule to Verticillium wilt and influence of soil temperature and moisture on development of infection. J. Agric. Res., 76: 129-143. Siler, D.J. and Cornish, K., 1994. Hypoallergenicity of guayule rubber particle proteins compared to Hevea latex proteins. Ind. Crops Prod., 2: 307-313. Thompson, A.E. and Ray, D.T., 1989. Breeding guayule. Plant Breed Rev., 6: 93-165.