Status of Citrus aurantifolia Infected with Witches Broom Disease of Lime in Oman

R. Al-Yahyai1, a, I. Khan2, F. Al-Said1, A. Al-Sadi1, A. Al-Wahaibi1 and M. Deadman1 1 Department of Crop Sciences, College of Agricultural & Marine Sciences, Sultan Qaboos University, Oman 2 Department of Horticulture, Agriculture University, Faisalabad, Pakistan Keywords: citrus, arid regions, phytoplasma, Sultanate of Oman, Hishimonus phycitis Abstract Production of lime (Citrus aurantifolia) in Oman has been significantly reduced in recent years. The reduction in yield has been attributed to a combination of biotic and abiotic factors that adversely affected tree growth and productivity. Lime cultivation area has lost 50% of its acreage compared to 1990, mainly due to tree loss caused by Witches Broom Disease of Lime (WBDL) associated with Candidatus Phytoplasma aurantifolia. The disease that originated in the Sultanate and spread to neighboring countries has the potential to devastate lime production throughout the entire regions of western Asia and North Africa thus affecting fruit imports to Oman as well. Infection with WBDL has been worsened by increasingly stressful abiotic conditions, mainly frequent drought and soil and water salinity that ultimately led to the decline of lime production in the country. This decline subsequently resulted in loss of lime acreage and profitability and reduced income from largely traditional farming systems. The unsustainable lime production has eventually led to abandonment of many farms, to conversion of fruit farms to forage farms, or to a complete change of the land use into other commercial projects. While the causal agent of WBDL has long been identified, there have been no practical solutions to control the disease thus far and these challenges remain decades later. This review will address the current status of lime in Oman and propose some solutions to enhance productivity of infected trees. INTRODUCTION Oman is located in the south-eastern Arabian Peninsula, a region that is characterized by a semi-arid to arid climate. This area receives an average annual rainfall of about 100 mm, while ET exceeds rainfall throughout the year. However, topographic and climatic variations in the country allowed for the cultivation of various types of crops including temperate, subtropical and tropical fruit crops. The total area of fruit crops is 36,925 ha which constitute more than 50% of the total agricultural area of Oman. Tree fruits are an important component of the traditional and modern farming systems in the country that are often incorporated into date palm and banana plantations. Historically lime has occupied a large part of the cultivated land throughout Oman due to its popularity mostly as fresh fruit but also as dried fruit for the export market. Oman is acknowledged as a transit route for lime that later spread from Arabia to Africa and Europe and from there to the New World (Davies and Albrigo, 1994). Oman has remained the largest producer of lime in the Arabian Peninsula. Despite being grown throughout Oman, the largest area of fruit crop cultivation is in the coastal region of Al Batinah in northern Oman that has 62% of the Sultanates lime trees (Table 1). Other citrus species of economic value include traditional crops such as citron and sweet lime, as well as recently introduced sweet orange, grapefruit and lemon. LIME PRODUCTION IN OMAN Lime (Citrus aurantifolia) is a major fruit crop in Oman constituting 4% of all fruit crops grown in the country and the main citrus species cultivated in the country.
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alyahyai@squ.edu.om

Proc. XXVIIIth IHC IS on Citrus, Bananas and Other Trop. Fruits under Subtrop. Conditions Eds.: J.-N. Wnsche and L.G. Albrigo Acta Hort. 928, ISHS 2012

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Omani lime (locally known as loumy or limon) is also known as Acid, Indian, Mexican or Key lime (Hodgson, 1967). In addition to the Middle East, the lime is also produced in India, Pakistan, Florida and Mexico (Burke, 1967). Characteristically, it is a small-fruited type distinguishable from the seedless, large fruited Tahiti lime (Citrus latifolia) and lemons (Citrus limon). Lime is a traditional crop that is consumed fresh for its juice or dried. Sun-dried limes used to be a major export commodity perhaps second only to dates. In many Arab and Islamic countries dried-limes are still known as Omani-limes perhaps indicating its exporting source. In recent decades, however, production of lime (Citrus aurantifolia) in Oman has been significantly reduced (Fig.1). Loss of cultivated area is currently 50% of that in 1990 (Fig. 2). This has been mainly attributed to infection with Witches Broom Disease of Lime (WBDL) associated with Candidatus Phytoplasma aurantifolia that was initially reported in late70s and early 80s. However, lime remains one of the major fruit crops in Oman with a total of 341,285 lime trees in the country, 62.75% of them are grown in the Batinah region (Table 1). Oman produced 5,916 MT of limes in 2006 (FAO, 2007). WITCHES BROOM DISEASE OF LIME (WBDL) History of the Disease Witches broom disease of lime (WBDL) is a phytoplasma-caused disease that has been observed on Omani limes since late 1970s and early 1980s (Bove et al., 1988; Gamier et al., 1991). The disease resulted in severe damage to the lime industry in Oman when many trees were eradicated as an early control measure (Al-Sadi et al., 2004). Studies have shown that phytoplasma causing WBDL can also spread to other cultivated and wild plants in Oman. This graft-transmissible and presumably vector-borne pathogen can also be transmitted via the parasiting dodder plant (Cuscuta campestris). In the field severe symptoms appear on citranges and limes but not on lemon (C. limon), sweet lime (C. limettioides), sweet orange (C. sinensis), pummelo (C. grandis), sour orange (C. aurantium), grapefruit (C. paradisi) or rough lemon (C. jambhiri). Experimentally, sweet orange and sour orange were found tolerant or resistant to the phytoplasma causing WBDL but not Troyer citrange (Gamier et al., 1991). Research on WBDL Since the identification of WBDL as a major disease in Oman, several projects have been carried out to investigate the disease at the Ministry of Agriculture (MA) and Sultan Qaboos University (SQU) in collaboration with international organizations. Researchers at MA have identified the natural hosts and vectors of Candidatus Phytoplasma aurantifolia. Moghal et al. (1998) reported the list of hosts infected by the phytoplasma associated with WBDL in Oman to include several citrus species such as C. aurantifolia, C. limetta, C. medica, C. jambhiri, and C. limon. Citrange (Poncirus trifoliate x C. sinensis) and Rangpur lime (C. limonia) have developed symptoms of WBDL when graft-inoculated with Candidatus Phytoplasma aurantifolia lime shoots. The disease was also reported on periwinkle (Catharanthus roseus), Achyranthes aspera, Amaranthus graecizans and Suaeda fruticosa. Several resistant citrus species have been reported resistant to WBDL that included sweet lime, sweet orange, Tahiti lime, mandarins, grapefruit, sour orange, pummelo, and kumquat (Moghal et al., 1998). Polymerase Chain Reaction (PCR) studies conducted by MA on P. aurantifolia concluded that the leafhopper Hishimonus phycitis is the most likely vector for WBDL due to its abundance in infected limes (MAF, 2005, 2006b). Research has led to several milestones including: molecular identification of the causal agent, phytoplasma (Al-Saady and Khan, 2006); assessment of citrus cultivars that are susceptible to the disease (Moghal et al., 1998); screening of various rootstocks for tolerance to WBDL (Moghal et al., 1998); preliminary vegetative propagation techniques of lime and related citrus species (R. Al-Yahyai, unpublished data; MAF, 2006b); somatic

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hybridization of citrus to develop WBDL resistant lime cultivars (Khan and Grosser, 2004; Khan et al., 2004). Horticulture and Breeding The genus Citrus and its related genera are rich in diversity for rootstock and scion characteristics. There are several active breeding programs and germplasm collections of citrus in the world, including a USDA sponsored citrus germplasm repository at the University of California, Riverside, USA and a major inventory of breeding accessions developed and maintained at Lake Alfred, Florida. The FAO also has sponsored citrus germplasm collection, improvement and distribution programs for the genetic improvement of rootstock and scion cultivars as well as for disease management. Among countries in the region, Pakistan is one of the top citrus producing and exporting countries. A huge collection of citrus germplasm and breeding accessions is available in the Department of Horticulture, at the University of Agriculture, Faisalabad. Several accessions from this collection have been introduced to Oman as rootstocks for lime and are currently being evaluated. Most popular scions include Kinnow mandarin (Frost, 1935), Orobllanco and Melogold grapefruits, and a series of orange cultivars from Florida. Among rootstocks, the citranges have been a great success which include Troyer, Carrizo, C35, and C32 (Cameron and Soost, 1986). For rootstock as well as for the scion breeding programs, nucellar embryony has been presenting problems to the breeders. The breeders have developed biochemical and molecular markers to facilitate the identification of zygotic seedlings (Khan and Roose, 1988a, b). Somatic hybridization is not handicapped by the problems of polyembryony. It can be used to produce progenies within 2-3 years. For WBDL, molecular and serological probes are available which could be applied for early testing of resistance status of the somatic hybrids (Roistacher, 1991; Bove et al., 1988; Gamier et al., 1991; Kollar et al., 1990; Ko and Lin, 1994). Environmental Stresses The sultanate of Oman is located at 23 north of the equator in a region that is considered arid with average annual rainfall below 100 mm. Sporadic rainfall and frequent drought periods coupled with high evapotranspiration due to high temperatures have led to considerable loss in tree productivity due to water stress. In the Batinah region, where tree fruits are primarily grown, soil salinity has been continuously increasing leading to death of trees especially salinity-sensitive tree fruits. These soils are mostly sandy soils with low soil fertility that collectively resulted in reduced tree growth and yield. Several studies have demonstrated the effects of salinity and drought on vegetable and fruit crops. However, fruit tree growth, yield and physiological response remained mostly unknown. Moreover, the effects of various environmental stress factors on diseased trees such as disease progress, etiology and tree resistance are not well documented especially in tropical and subtropical regions such as Oman. Thus, there are no recommendations for effective and practical orchard management strategies of diseased trees such as irrigation, fertilizer application and pruning. Water and Soil Problems in Relation to Lime Production in Oman Drought and salinity are major abiotic stress factors that affect growth and productivity of trees. Plants require large amounts of water to produce their biomass ranging from several hundred to two thousand grams of water per one gram of dry matter produced (Hsiao, 1993). Thus plant water status is perhaps the most important factor that must be controlled to obtain high yields of good quality horticultural produce (Jones, 1990). Water deficits affect many physiological and developmental processes involved in fruit production, including growth (cell division and cell expansion), gas exchange (stomatal aperture, and photosynthetic and respiratory enzymes), and a wide range of biochemical and developmental processes (Jones et al., 1985). The response to these variables under stressful conditions varies between species and cultivars within the same 377

species, such as stress (German and Sardo, 1996). Mechanisms of salinity damage to tree fruits, such as Citrus spp., and its management have been studied extensively (Ferguson and Grattan, 2005; Boman et al., 2005; Kozlowski, 1997). The main damage is caused by specific ion toxicity due to accumulation of sodium chloride and/or boron in tree leaves and osmotic effects due to accumulation of salts in the soil (Ferguson and Grattan, 2005). Multiple stresses have greater influence on tree growth than a single stress factor. Research has demonstrated disease infection had greater influence on tree growth and yield when combined with salinity and water stress (Syvertsen and Levy, 2005). Blodgett et al. (1997) concluded that water stress levels that are typically found under field conditions increased disease development of Sphaeropsis sapinea on Red Pine. However, the effects of abiotic stress factors on witches broom disease of lime are unknown. Oman is located in a region that is classified as arid and semi-arid due to high evapotranspiration rate that exceeds annual rainfall of 100 mm however, that amount fluctuates greatly among and within years. Agriculture is entirely dependent on irrigation using scarce underground water resources. The amount of water reserves in Oman is estimated to be 1168 million m3 (Al-Gheilani and Al-Mulla, 2006), of which 92% is used for agriculture (Al Sulaiman and Al Wohaibi, 2006). The amount of consumption exceeds recharge by a deficit of 390 million cubic meters (Abdel Rahman and Abdel-Mjid, 1993). Despite the large amount of water being used for agriculture, tree fruits are often not adequately irrigated and show symptoms of water stress. This is due to the water being used primarily for forage crops and date palm plantations that consume large amounts of water. Soil and water salinity is another environmental factor reaching 35% of the total area of Oman (FAO, 1997) that led to decline in lime in Oman. In the early 1990s, 50% of the cultivated area in Batinah region was saline according to a survey conducted by the Ministry of Agriculture (MAF, 1993). The area affected by salinity has since expanded to include large areas of the coastal regions of Batinah, Sharqiah and Dhofar. Production of many fruit trees, including lime, has declined rapidly due to salinity stress. STRATEGIES TO DEAL WITH WBDL IN OMAN Recently MA has proposed a national project to revive citrus in Oman (MAF, 2006b). The project includes two major area: (1) Citrus disease indexing: disease diagnostics using ELISA and PCR techniques, development and usage of disease-free stock plants for propagating citrus, and field surveys for major citrus diseases. (2) Seedling production: aimed at production and propagation of healthy Omani lime using healthy mother-plants from southern Oman and propagation using tissue culture techniques. Ongoing research on lime at Sultan Qaboos University aimed at tackling the disease with short and long-term approaches. Short-term solutions are based on the concept of living with the disease through orchard and tree management aimed at enhancing fruit quality and yield, while long-term resistance or tolerance to WBDL is found. These research strategies aim to eventually provide practical solutions to tree fruit growers, thus enabling them to continue production from diseased trees while new, longterm solutions through resistant cultivars are being gradually evaluated and introduced. Literature Cited Abdel Rahman, H.A. and Abdel-Majid, I.S. 1993. Water Conservation in Oman. Water International 18:95-102. Al Gheilani, H.M. and Al-Mulla, Y.A. 2006. The water wealth in the Sultanate of Oman: Reality and ambitions. Proceedings of the International Conference on Economic Incentives and Water Demand Management. 18-22 March 2006. Muscat, Sultanate of Oman. Al Saady, N.A. and Khan, A.J. 2006. Phytoplasmas that infect plant species worldwide: Review. Physiol. Mol. Biol. Plants. 6:263-281. Al-Sadi A.M., Khan, I.A. and Deadman, M.L. 2004. Economic losses caused by witches broom disease of lime and some management aspects in Shinas area of Oman. 10th 378

International Citrus Congress, 15-20 February 2004, Morocco p.817-818. Al-Sulaiman, Z.K. and Al-Wohaibi, B.K. 2006. Water metering pilot project: Case study on water demand and management in the Sultanate of Oman. Proceedings of the International Conference on Economic Incentives and Water Demand Management. 18-22 March 2006. Muscat, Sultanate of Oman. Blodgett, J., Kruger, E. and Stanosz, G. 1997. Effects of Moderate Water Stress on Disease Development by Sphaeropsis sapinea on Red Pine. Phytopathology 87(4): 422-428. Boman, B., Zekri, M. and Stover, E. 2005. Managing salinity in Citrus. HortTechnology 15(1):108-113. Bove, J.M., Garnier, M., Mjeni, A.M. and Khayrallah, A. 1988. Witches broom disease of small fruited acid lime trees in Oman: First MLO disease of citrus. In: Proc. Int. Organ. Citrus Virol. 10:307-309. Burke, J.H. 1967. The commercial citrus regions of the world. p.40-189. In: W. Reuther, L.D. Batchelor and H.D. Webber (eds.), The Citrus Industry. Vol. I, University of California Press, Berkeley. Cameron, J.W. and Soost, R.K. 1986. C35 and C32: citrange rootstocks for citrus. HortScience 21:157-158. Davies, F.S. and Albrigo, L.G. 1994. Citrus. CABI International, Wiltshire, UK. p.1-2. FAO. 1997. Water Reports 9: Irrigation in the Near East Region in Figures. Food and Agriculture Organization, Rome, Italy. FAO. 2007. FAOSTAT-Food and Agriculture Organization of the United Nations. 1 Oct. 2008. <http://faostat.fao.org> Ferguson, L. and Grattan, S. 2005. How salinity damage citrus: Osmotic effects and specific ion toxicities. HortTechnology 15(1):95-99. Frost, H.B. 1935. Four new citrus varieties-the Kara, Kinnow and Wilking mandarins and Trovita orange. California Agriculture Experiment Station Bulletin No. 597:14. Gamier, M., Zreik, L. and Bove, J.M. 1991. Witches Broom, a lethal mycoplasma disease of lime trees in the Sultanate of Oman and the United Arab Emirates. Plant Disease 75(6):546-551. German, C. and Sardo, V. 1996. Relationship between net photosynthesis and transpiration rate in citrus trees. Proc. Intl. Soc. Citricult. 2:1117-1121. Hodgson, R.W. 1967. Horticultural Varieties of Citrus. p.431-592. In: W. Reuther, L.D. Batchelor and H.D. Webber (eds.), The Citrus Industry. Vol. I, university of California Press, Berkeley. Hsiao, T.C. 1993. Growth and productivity of crops in relation to water status. Acta Hort. 335:137-148. Jones, H.G. 1990. Physiological aspects of the control of water status in horticultural crops. HortScience 25:19-26. Jones, H.G., Lakso, A.N. and Syvertsen, J.P. 1985. Physiological control of water status in temperate and subtropical fruit trees. p.301-344. In: J. Janick (ed.), Hort. Rev. AVI Publ., Westport, Conn. Khan, I.A. and Grosser, J.W. 2004. Regeneration and characterization of somatic hybrid plants of Citrus sinensis (sweet orange) and Citrus micrantha, a progenitor species of lime. Euphytica 137(2):271-278. Khan, I.A. and Roose, M.L. 1988a. Frequency and characteristics of nucellar and zygotic seedlings in three cultivars of trifoliate orange. J. Amer. Soc. Hort. Sci. 113:105-110. Khan, I.A. and Roose, M.L. 1988b. Nucellar Embryony: Detection and Importance. Punjab Fruit J. 41:1-15. Khan, I.A., Lee, R.F., Grosser, J.W. and Hartung, J. 2004. Screening selected germplasm for resistance to witches broom disease of lime. 10th International Citrus Congress, 15-20 February 2004, Morocco p.814-816. Ko, K.C. and Lin, C.P. 1994. Development and application of cloned DNA probes for Mycoplasma like Organism associated with sweet potato witches-broom. Phytopathology 84:468-473. 379

Kollar, A., Seemuller, E., Bonnet, F., Saillard, C. and Bove, J.M. 1990. Isolation of the DNA of various plant pathogenic mycoplasma-like organisms from infected plants. Phytopathology 80:233-237. Kozlowski, T. 1997. Responses of woody plants to flooding and salinity. Tree Physiology Monograph 1:1-29. MAF. 1993. South Batinah Integrated Study. Directorate General of Agricultural Research, Ministry of Agriculture and Fisheries, Oman. MAF (Ministry of Agriculture & Fisheries). 2005. Summary of Results of the Agricultural and Livestock Research 2000-2003. Directorate General of Agriculture, Ministry of Agriculture & Fisheries, Muscat, Sultanate of Oman. MAF (Ministry of Agriculture & Fisheries). 2006a. Agricultural Census 2004/2005 Volume I. Directorate General of Planning and Investment Promotion, Ministry of Agriculture & Fisheries, Muscat, Sultanate of Oman. MAF (Ministry of Agriculture & Fisheries). 2006b. Summary of Results of the Agricultural and Livestock Research - 2005. Directorate General of Agriculture & Livestock Research, Ministry of Agriculture & Fisheries, Muscat, Sultanate of Oman. Moghal, S.S., Zidgali, A.D. and Moustafa, S.S. 1998. Natural host range and reaction of citrus species to witches broom disease of lime (WBDL) in Oman. Proc. IPM Con. SQU, Muscat, Sultanate of Oman. 23-35 Feb. 1998. Roistacher, C.N. 1991. Graft-Transmissible Diseases of Citrus. FAO Publication, Rome 286p. Syvertsen, J. and Levy, Y. 2005. Salinity interactions with other abiotic and biotic stresses in citrus. HortTechnology 15(1):100-103.

Tables

Table 1. Number of lime trees in various regions of Oman according to the Ministry of Agriculture Census (2004/05) (MAF, 2006a). Region Muscat Batinah Musandam Dhahirah Dakhliah Sharqiah Wusta Dhofar Total No. of lime trees 14,812 214,146 5,466 33,768 31,342 34,580 193 6,978 341,285 Percentage of total 4.34 62.75 1.60 9.89 9.18 10.13 0.06 2.04 100

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30,000 Production (MT) 25,000 20,000 15,000 10,000 5,000 0

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79

81

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87

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95

97

99

01

03

19

19

19

19

19

19

19

19

19

19

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Fig. 1. Lime production in Oman (1977-2006) (FAO, 2007).

100 Area Decline (%) since 1990 80 60 40 20 0

20 00 20 02 20 04 19 90 19 92 19 94 19 96 20 06 19 98

Fig. 2. Decline in lime cultivation areas of lime since 1990 (FAO, 2007).

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