DEVELOPMENT OF TETRAZYG ROOTSTOCKS TOLERANT OF THE DIAPREPES/PHYTOPHTHORA COMPLEX UNDER GREENHOUSE CONDITIONS

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1 include sanitation, optimal nutritional and irrigation programs and management of adjacent grove areas to reduce weevil hosts. Degree-days, and crawler monitoring can establish the need for scale control. Disease Management. Tree skirts can be pruned to 24 inches or higher above ground level to reduce brown rot of low-hanging fruit. Application of fungicides to control scab should depend on the intended market, as for fresh Temples and Murcotts, with no control generally necessary for processed fruit. When possible, sites with a past history of foot and root rot should be avoided, irrigation and drainage improved and resistant cultivars used. Melanose fungicide programs should be restricted to fresh fruit cultivars and spray applications reduced when fruit becomes resistant. Sampling for infected blossoms, especially in groves with a history of post-bloom fruit drop (PFD) and utilizing a weather forecasting model to schedule fungicide applications can reduce incidence of PFD. Implications for Florida Growers Surveys conducted in the late 1990s of groups with an expressed interest in ecolabels indicated approximately 60% of respondents ranked fresh fruit and vegetables as foods most in need of ecolabels, with only 10% ranking juice products as most in need (Hartman Group, 1997), suggesting a greater market potential for fresh than processed citrus. Conventional growers who are already committed to sustainable practices and who are already managing a range of production, harvesting, packing, processing, labor and environmental regulations but who do not seek organic certification might also market crops under ecolabeling programs that are less restrictive than organic programs. Since the fresh fruit export market frequently requires detailed documentation of production and related practices, growers may have an opportunity with ecolabeling programs to develop a more distinctive marketing label and to establish a more preferred status, especially with European and Japanese buyers. Innovative partnering between the University of Wisconsin, the USDA, the World Wildlife Fund, and Wisconsin fruit and vegetable growers associations could also be duplicated in Florida, creating novel funding, research, and marketing opportunities for Florida citrus and other horticultural crops. However, those in the ecolabeling movement stress that in the near future, synergy rather than competition among the various ecolabeling programs is needed, especially in terms of clear labeling, measurable standards, verification, and public education (Kane et al., 2000). Literature Cited Caldwell, D. J Ecolabeling and the regulatory framework a survey of domestic and international fora. policy/ecolab 1.htm Hartman Group The Hartman report, food and the environment: a consumer s perspective, Phase II. The Food Alliance, Portland, OR. Kane, D. B., B. Lydon, K. Richards, and M. Sligh Greener fields: signposts for successful eco-labels Rural Advancement Foundation International USA, Pittsboro, NC. Nogaj, R. J. and F. A. Nogaj A new ecolabel: Grown and Picked in the USA by Workers Paid a Living Wage (Abstr). Conference on Ecolabels and the Greening of the Food Market. Boston, MA. The Food Alliance Citrus evaluation criteria, Portland, Ore. Proc. Fla. State Hort. Soc. 116: DEVELOPMENT OF TETRAZYG ROOTSTOCKS TOLERANT OF THE DIAPREPES/PHYTOPHTHORA COMPLEX UNDER GREENHOUSE CONDITIONS JUDE W. GROSSER 1, JAMES H. GRAHAM, CLAY W. MCCOY, ANGEL HOYTE, HERBERTH M. RUBIO, DIANE B. BRIGHT AND JUAN L. CHANDLER University of Florida, IFAS Citrus Research and Education Center Horticultural Sciences/Plant Cell Genetics Department 700 Experiment Station Road Lake Alfred, FL Additional index words. citrus breeding, disease resistance, rootstock improvement, somatic hybrid, tetraploid The authors would like to thank Ed Stover and Scott Ciliento of the University of Florida, Indian River Research and Education Center, Ft. Pierce, FL, for their assistance with test soil collection. This research was supported by the Florida Agricultural Experiment Station and grants from the Florida Citrus Production Research Advisory Council and the IFAS/USDA Cooperative Agreement on Diaprepes research, and approved for publication as Journal Series No. N Corresponding author. Abstract. Diaprepes (Diaprepes abbreviatus L.) root weevil has become an increasingly important pest affecting Florida citrus production. No known citrus rootstock germplasm appears to be resistant to weevil larvae feeding. Mechanical wounds on tap and scaffold roots caused by weevil feeding create opportunities for invading fungi, especially ubiquitous Phytophthora spp. Our strategy for dealing with this problem is to develop complex rootstock hybrids that have the capacity to tolerate mechanical root damage caused by weevil feeding and then recovery by exhibiting vigorous root growth in challenging soils inoculated with both Phytophthora nicotianae and P. palmivora. Crosses were made of superior allotetraploid somatic hybrid rootstocks and resulting seed were planted in a high ph calcareous Winder depressional soil inoculated with both Phytophthora spp. in greenhouse flats. Vigorous healthy tetrazyg seedlings were selected and propagated by grafting to vigorous rootstocks and subsequently rooted cuttings. New mandarin + pummelo somatic hybrids were also included in the assays. Replicated Diaprepes no-choice feeding assays were conducted in conetainers, and hybrids selected for reduced root damage were replanted in the Winder /Phytophthora mix to assess recovery Proc. Fla. State Hort. Soc. 116:

2 potential. Several hybrids showed excellent capacity for complete recovery in this greenhouse test and are now being propagated for more extensive field evaluation. Citrus rootstock breeding and selection at the tetraploid level maximizes genetic diversity and selection efficiency, and shows great promise for generating new rootstocks that can tolerate the existence of Diaprepes and Phytophthora in clay loam soils. The Diaprepes root weevil (Diaprepes abbreviatus L.) was first detected in Orange County, Florida in Since this time it has spread primarily by movement of contaminated ornamental nursery stock into 22 counties including more than 12,000 ha of commercial citrus (Hall, 2000). The larvae feed and reproduce on all commercial citrus rootstocks, and advanced larval feeding causes severe mechanical damage to scaffold roots (Graham et al., 2003; Nigg et al., 2001). Wide intergeneric somatic citrus hybrids are also susceptible to feeding (Grosser and McCoy, 1996), which suggests that it will be difficult to identify citrus germplasm that is resistant to feeding. A few distant citrus relatives have been shown to have some resistance to weevil larval feeding, but they are sexually incompatible with citrus and not immediately amenable to variety improvement techniques (Bowman et al., 2001). Studies of infested citrus groves have more recently led to the discovery of a Phytophthora-Diaprepes complex, a complicated interaction of insect damage, invading fungi (Phytophthora nicotianae Breda de Hoan and P. palmivora (E. J. Butler) E. J. Butler), rootstock, and soil type (Graham et al. 1997, 2003; Rogers et al. 1996). At present, control strategy for Diaprepes is costly, and in some areas, marginally effective. In many areas infested groves are either in severe decline or out of production (Hall, 2000). An obvious research objective is therefore the development of improved rootstocks than can handle the Phytophthora/Diaprepes complex. A paradox regarding Phytophthora resistance has emerged that will make this objective more difficult to achieve. Historically, Phytophthora nicotianae has been a problem in Florida groves, particularly in flatwoods areas with heavy soils and/or poor drainage. Citrus improvement programs have focused on selecting hybrids that are resistant to P. nicotianae, using trifoliate orange as a source of resistance genes. Resistant trifoliate hybrids such as Swingle citrumelo have become popular but are not adapted to high ph, calcareous soils. More recently, in the presence of the Diaprepes/Phytopthora complex on challenging soils, trifoliate hybrids including Swingle citrumelo and Carrizo citrange are collapsing, and a second Phytophthora species, P. palmivora has been implicated (Bowman et al., 2002; Graham et al. 2003). It appears that the Phytophthora resistance genes from trifoliate orange are not effective against P. palmivora. However, P. nicotianae susceptible mandarin rootstocks, including Cleopatra and Sun Chu Sha, are showing resistance to P. palmivora (Graham et al., 2003). The challenge for rootstock improvement programs is to overcome this paradox by packaging resistance genes against both Phytophthora species into appropriate hybrids. Citrus rootstock improvement is a daunting task because a large number of traits must be packaged into any successful new rootstock. For Florida, these traits include resistance/tolerance to blight, citrus tristeza virus, Phytophthora spp., nematodes, Diaprepes, salinity, adaptation to challenging soils, tree size control, nucellar embryony for seed propagation, good nursery performance, and the ability to consistently produce high yields of quality fruit. During the past 20 years, we have built a successful somatic hybridization program at the CREC, including direct application to rootstock improvement (Grosser and Chandler, 2002; Grosser et al., 2000). More than 70 allotetraploid somatic hybrid combinations are currently being tested for rootstock potential, and a few hybrids look promising. Seed trees of many of the somatic hybrid rootstocks are now flowering, including several that appear to have commercial rootstock potential. This development opens the opportunity to conduct citrus breeding and selection at the tetraploid level. We have identified two somatic hybrids, sour orange + rangpur and Nova mandarin + Hirado Buntan pummelo (zygotic) that are performing well in field trials and produce a high percentage of zygotic seed. The latter hybrid is also outperforming all commercial and other somatic hybrid rootstocks in a field trial at a site with challenging soil and the Diaprepes/Phytopthora complex. In 1999, we began a unique tetraploid rootstock breeding program, using these two hybrids as females in crosses with other high-performance somatic hybrid rootstocks including sour orange + Carrizo, sour orange + Palestine sweet lime, and Cleopatra + trifoliate orange) as pollen parents. We have coined the term tetrazyg to identify zygotic tetraploid hybrids produced from crossing allotetraploid somatic hybrids. This novel approach offers an opportunity to maximize genetic diversity in tetraploid progeny. Our approach to date has been to germinate seed from such crosses directly in a high ph, calcareous Winder soil/phytophthora screen to identify superior tetraploid zygotic tetrazyg seedlings. Selected tetrazygs are then grafted with sweet orange containing a quick-decline isolate of citrus tristeza virus (CTV) to rapidly determine their resistance to CTV-induced quick-decline disease. At the same time, the selected hybrids are propagated by either rooted cuttings and/or topworking to provide clonal material for further evaluation, including the Phytophthora/Diaprepes greenhouse screening described herein. This overall approach is expected to shorten the time required to develop rootstocks that will allow profitable citriculture on flatwoods soils where the Phytophthora/Diaprepes complex exists. In this report, we outline our strategy and progress for dealing with this problem, namely to develop complex rootstock hybrids that have the capacity to tolerate mechanical damage caused by weevil feeding and then recovery by exhibiting vigorous root growth in challenging soils inoculated with both Phytophthora nicotianae and P. palmivora. Materials and Methods Plant Material. Test material included 50 previously selected tetrazygs from the year 2000 crosses of somatic hybrids; two mandarin + pummelo somatic hybrids (Amblycarpa mandarin + selected pummelo seedlings, Ling Ping Yau and Hirado Buntan B) and Cleopatra mandarin as a control. The tetrazygs were produced from crosses that included either Nova mandarin hybrid + Hirado Buntan (zygotic) pummelo or sour orange + rangpur somatic hybrids as females and either sour orange + Carrizo, sour orange + Palestine sweet lime, Cleopatra + Argentine trifoliate orange, Succari sweet orange + Hirado Buntan pummelo (zygotic), Cleopatra + sour orange, Cleopatra + rangpur, or Succari sweet orange + Argentine trifoliate orange somatic hybrids as pollen parents. Since seed trees of the plant material to be tested have not yet fruited, the selected tetrazygs and somatic hybrids were propagated by rooted cuttings. Two-node stem cuttings (bottom cut just below the node) were dipped directly in 100% IBA (indole-butyric acid) powder (Sigma) and 264 Proc. Fla. State Hort. Soc. 116: 2003.

3 placed in 48-welled conetainers and maintained on a mistbed until root systems were established. Nearly all cuttings rooted after 2-3 months on the mistbed. Following removal from the mistbed, cuttings were repotted in fresh commercial potting soil in larger containers (diameter = 5 cm) and enlarged until stem-diameter reached pencil-thickness. Conetainer Feeding Damage Assay. Five plants of each selection (one plant per cone) were inoculated with neonate larvae (1st instar from eggs of wild female weevils) of Diaprepes abbreviatus at five larvae per conetainer. Plants were watered and fertilized as needed. After 6 weeks, plants were removed from soil and their root systems were rated from 1-5 as follows: 1 = healthy roots, no feeding damage; 2 = visible root injury, minor damage; 3 = visible root injury, severe damage; 4 = severe girdling, no Phytophthora; 5 = severe girdling, evidence of Phytophthora (Table 1). A damage resistance index score was calculated Table 1. Diaprepes/Phytophthora complex - greenhouse testing of tetrazyg rootstocks. Conetainer challenge Recovery challenge Hybrid Average damage No. surviving plants Score Average damage No. surviving plants Score 2247x x x x x x S x x x x x Cleopatra mandarin x Amb B x x x x S x x x x x x x x Amb A x x x x x x x x x x S x x x x x x x x x x x x x x x x Proc. Fla. State Hort. Soc. 116:

4 for each rootstock candidate as follows: score = 5 (ave. root rating) (number of surviving plants) (Table 1). The higher the score, the less average physical damage to the root system. Numbers of surviving larvae per cone were also recorded. Recovery Assay. Fifteen tetrazygs, 1 somatic hybrid, and Cleopatra mandarin (control) were selected for the recovery (root regrowth) assay, based on performance in the feeding damage assay and plant source (Table 1). All plants of these groups scoring a 3 or lower were replanted in 4-inch deep flats containing a calcareous Winder soil (ph ) inoculated with both Phytophthora nicotianae and P. palmivora. The Winder soil was collected at the Indian River Research and Education Center (IRREC) in Fort Pierce, Fla. The Phytophthora inoculum was collected at the Kerr Center (Fort Pierce, Fla.) by collecting top soil, roots, and debri from below trees previously identified by J. H. Graham to have high Phytophthora inoculum counts. The two soils types were mixed thoroughly in a 3:1 ratio Winder/Phytophthora inoculum and used to fill the 4-inch deep plastic flats. Plants were watered and fertilized as needed. After 3 months, plants were removed from the soil and their root systems were rated from 1-5 as above. A recovery index score was calculated as follows: score = 5 (ave. root rating) (number of surviving plants) (Table 1). The higher the score, the better the recovery. All recovered plants were taken to the Kerr Center (Fort Pierce, Fla.) for field evaluation in a site challenged by the Phytophthora/Diaprepes complex. Results and Discussion The average root rating per candidate rootstock in the container and recovery challenges, the number of surviving plants per candidate in the two tests, and the index scores per candidate in each test are provided in Table 1. As expected, weevil larvae fed on all candidate rootstocks; however, it was Fig. 1. Upper left: plants of hybrid 2247x following weevil larvae feeding (hybrid with lower level of damage); upper-middle: plants of hybrid 2247x following larvae feeding (lower level of damage); plants of hybrid 2247x following larvae feeding (severe damage - none surviving); lower-left: damaged plants recovering in Winder soil/phytophthora mix; lower-right: two hybrid plants showing complete recovery in the Winder soil/ Phytophthora mix following Diaprepes damage. 266 Proc. Fla. State Hort. Soc. 116: 2003.

5 evident that the average amount of mechanical damage from feeding per candidate rootstock was quite variable (Table 1; Fig. 1). At least one live weevil larva was recovered from every cone (data not shown). The number of surviving plants per candidate rootstock ranged from 0 to 5. Only two of the hybrids had all five plants surviving, and nine hybrids had no survivors. The commercial rootstock Cleopatra mandarin was chosen as the standard in this study because it has been outperforming other popular commercial rootstocks in flatwoods groves affected by the Phytophthora/Diaprepes complex (Graham et al., 2003), including the two top Florida rootstocks, Swingle citrumelo and Carrizo citrange. Cleopatra did fairly well in the damage resistance assay, scoring in the upper 75%. However, Cleopatra mandarin was not able to recover from the damage sustained in the container challenge, as only one of four plants recovered. This agrees with a previous report by Graham et al. (2003) which showed that Cleopatra was unable to regenerate roots in the presence of Phytophthora. Nearly all of the hybrids included in the recovery challenge recovered much better than Cleopatra. For example, all five plants of hybrid 2247x completely recovered as shown by its maximum recovery index of 20 shown Table 1. This hybrid was the most vigorous of all the selections included in the study, and its high level of vigor may be responsible for this hybrid s superior ability to recover from weevilfeeding damage. All five plants of hybrid 2247x also recovered, even though this selection sustained more damage from the weevil feeding. Other hybrids combining minimal damage with good recovery potential were: 2247x , 2247x , 2247x , 2247x , 2247x , and 2247x All of these hybrids showed good vigor in the nursery as well, suggesting that vigor may be associated with recovery potential if there is adequate resistance to both Phytophthora species. The somatic hybrid B also suffered more weevil-feeding damage than Cleopatra, but showed very good recovery potential. The second mandarin + pummelo somatic hybrid included in the trial ( A) did not perform well. Mandarin + pummelo somatic hybrids are being produced in efforts to build a widely adapted CTV-quick decline resistant replacement rootstock for sour orange (Grosser and Chandler, 2002). Sour orange was recently shown to be a hybrid of pummelo and mandarin (Nicolosi et al., 2000). Concluding remarks. This study showed that although Diaprepes weevil larvae feed on all citrus rootstock candidates, the average level of damage varies considerably per rootstock candidate. Several tetrazyg hybrids selected for minimal feeding damage showed good root/plant recovery potential under greenhouse conditions in a challenging high ph, calcareous Winder depressional soil that was inoculated with both P. nicotianae and P. palmivora. A new mandarin + pummelo somatic hybrid also showed good root recovery potential. The hybrids performing well in this study should be tolerant to the Phytophthora/Diaprepes complex in a flatwoods grove situation, and plants are currently being propagated for replicated field trials to evaluate this. All of these hybrids have also been topworked to mature field trees to expedite their flowering and fruiting as necessary to determine if they can be propagated by seed. Breeding and selection at the tetraploid level maximizes genetic diversity in tetrazyg progeny and should accelerate the rootstock development process. Expanded crosses of this nature are being conducted annually, and we expect to identify additional new complex hybrids tolerant of the Phytophthora/Diaprepes complex. Literature Cited Bowman, K. D., J. P Shapiro, and S. L. Lapointe Sources of resistance to Diaprepes weevil in subfamily Aurantiodeae, Rutaceae. HortScience 36: Bowman, K. D., J. P. Albano, and J. H. Graham Greenhouse testing of rootstocks for resistance to Phytophthora species in flatwoods soils. Proc. Fla. State Hort. Soc. 115: Graham, J. H., C. W. McCoy, and J. S. Rogers The Phytophthora-Diaprepes weevil complex. Citrus Ind., August Graham, J. H, D. B. Bright, and C. W. McCoy Phytophthora-Diaprepes weevil complex: Phytophthora spp. relationship with citrus rootstocks. Plant Dis. 87: Grosser, J. W. and J. L. Chandler Somatic hybridization for citrus rootstock improvement, p In Proc. 7th Intl. Citrus Seminar Improvement. Estacao Experimental De Citricultura De Bebedouro, SP, Brazil. Grosser, J. W. and C. W. McCoy Feeding response of first instar larvae of Diaprepes abbreviatus to different novel intergeneric citrus somatic hybrids. Proc. Fla. State Hort. Soc. 109: Grosser, J. W., P. Ollitrault, and O. Olivares-Fuster Somatic hybridization in Citrus: an effective tool to facilitate variety improvement. In Vitro Cell. Dev. Biol. Plant 36: Hall, D. G History and importance of Diaprepes to Florida, p In S. H. Futch (ed.). Proc. Diaprepes Short Course. Univ. Fla. IFAS Coop. Ext Serv., Lake Alfred. Nicolosi, E., Z. N. Deng, A. Gentile, and S. La Malfa Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theor. Appl. Genet. 100: Nigg, H. N., S. E. Simpson, N. E. El-Gholl, and F. G. Gmitter, Jr Response of citrus rootstock seedlings to Diaprepes abbreviatus L. (Coleoptera: Curculionidae) larval feeding. Proc. Fla. State. Hort. Soc. 114: Rogers, S., J. H. Graham, and C. W. McCoy Insect-plant pathogen interactions: Preliminary studies of Diaprepes root weevil injuries and Phytophthora infections. Proc. Fla. State Hort. Soc. 109: Proc. Fla. State Hort. Soc. 116: