Tolerance of Trifoliate Citrus Rootstock Hybrids to Candidatus Liberibacter Asiaticus
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Scientia Horticulturae 147 (2012) 71–80 Contents lists available at SciVerse ScienceDirect Scientia Horticulturae journa l homepage: www.elsevier.com/locate/scihorti Tolerance of trifoliate citrus rootstock hybrids to Candidatus Liberibacter asiaticus ∗ Ute Albrecht, Kim D. Bowman U.S. Horticultural Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, 2001 South Rock Road, Fort Pierce, FL 34945, USA a r t i c l e i n f o a b s t r a c t Article history: Candidatus Liberibacter asiaticus (Las) is the suspected causal agent of Huanglongbing (HLB) in Florida Received 22 May 2012 and other citrus producing countries around the world. Although little resistance to HLB is found within Received in revised form 29 August 2012 commercial citrus varieties, tolerance has been reported for some varieties that are commonly used as Accepted 31 August 2012 rootstocks. This study assesses the response of eight different rootstock varieties, which include the Cit- rus × Poncirus trifoliata hybrids Carrizo citrange, US-802, US-812, US-897, and US-942, Benecke trifoliate Keywords: orange, Volkamer lemon, and Cleopatra mandarin, to Las under controlled conditions in the greenhouse. Citrus disease The percentage of plants detected positive for Las did not exceed 7% in the genotypes Benecke, Carrizo, Citrus greening US-802, US-942, and Volkamer during the first 6 months after graft-inoculation (mai). Pruning resulted Disease resistance in much increased numbers of plants with detectable Las levels and induced disease symptoms in pre- Poncirus trifoliata Rootstock viously asymptomatic plants. At 12 mai, percentages of Las-positive plants were 91–96% for US-802, US-812, Cleopatra, and Volkamer, and 70–78% for US-897 and US-942. Lowest percentages (44–52%) were observed for Benecke and Carrizo. The number of Las genomes per g of leaf tissue in Las-positive plants 5 6 was considerably lower in Benecke and US-897 seedlings with 4.5 × 10 and 1.6 × 10 compared with 7 6 × Cleopatra (1.0 10 ), while numbers for the other genotypes ranged from 3.1–4.9 × 10 . At 12 mai, foliar disease symptoms, though variable in extent, were prominent in most genotypes, except US-897. Shoot masses were significantly reduced in Cleopatra, Benecke, US-802, and Volkamer seedlings in response to Las at this time. Continued observation until 18 mai found no significant growth reductions in Benecke seedlings. Our results suggest classifying Carrizo, US-897, and US-942 as tolerant, US-802, US-812, and Volkamer lemon as moderately tolerant, and Cleopatra mandarin as susceptible to Las. Despite irregular growth, low rates of infection and low Las numbers indicate some resistance of Benecke to Las. Addi- tional greenhouse experiments and field observations confirmed findings for US-802, US-897, US-942, and Cleopatra, although results for US-802 were more variable. © 2012 Published by Elsevier B.V. 1. Introduction China, and is now found in most citrus-growing areas worldwide, including Asia, Saudi Arabia, Africa, and the Americas (Bové, 2006; Candidatus Liberibacter spp. are the suspected causal agents Gottwald, 2010). The disease was found in Florida in 2005 (Halbert, of Huanglongbing (HLB), a destructive disease of citrus which 2005), 1 year after its first discovery in Sao Paula, Brazil (Teixeira threatens citrus production in Florida and other citrus-producing et al., 2005). countries. Three Candidatus Liberibacter spp. are associated with The Chinese name Huanglongbing refers to the appearance of HLB: Ca. L. asiaticus, Ca. L. africanus (Jagoueix et al., 1994) and yellow shoots in the canopy of affected trees, which are the result Ca. L. americanus (Teixeira et al., 2005). The ranking of Candida- of an asymmetric blotchy mottling of leaves or severe chloro- tus was assigned due to the difficulty of obtaining the pathogen sis, similar to zinc- or other nutrient deficiencies (McClean and in pure culture (Davis et al., 2008; Sechler et al., 2009). Liberib- Schwarz, 1970). Fruit of affected trees become misshapen, develop acters are gram-negative bacteria (Garnier et al., 1984) which are an undesirable taste and drop prematurely, thus generating sub- transmitted to the phloem by sap-sucking insects of the family Psyl- stantial losses to citrus growers in affected areas. Crop losses on lidae. In Florida and most other countries, HLB is associated with juice orange production in Florida during the past 5 years because Ca. L. asiaticus (Las) vectored by the Asian citrus psyllid Diapho- of HLB are estimated to be more than 20% (Hodges and Spreen, rina citri Kuwayama (Halbert and Manjunath, 2004; Halbert, 2005). 2011). HLB symptom development appears to be associated with HLB probably originated in India in the 1700s before spreading to phloem collapse and blockage of the translocation stream as a result of infection with liberibacters (Schneider, 1968; Achor et al., 2010). In addition, infection induces major changes in carbohydrate ∗ metabolism, phytohormone metabolism and other metabolic path- Corresponding author. Tel.: +1 772 462 5920; fax: +1 772 462 5986. E-mail address: [email protected] (K.D. Bowman). ways (Albrecht and Bowman, 2008, 2012; Kim et al., 2009; Fan et al., 0304-4238/$ – see front matter © 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.scienta.2012.08.036 72 U. Albrecht, K.D. Bowman / Scientia Horticulturae 147 (2012) 71–80 2010, 2011; Rosales and Burns, 2011). Current management strate- pieces from infected greenhouse-grown ‘Valencia’ orange plants, gies for HLB consist of planting disease-free trees, vector insect PCR-positive for Ca. L. asiaticus (Las) and symptomatic for HLB, onto control in combination with nutritional applications, and removal each plant. Twelve plants of each genotype were mock-inoculated of infected trees. with disease-free tissue pieces obtained from healthy greenhouse- HLB affects all citrus species and relatives, and commercial vari- grown ‘Valencia’ orange plants to be used as non-infected controls. eties such as sweet oranges (Citrus sinensis L.), mandarins (Citrus reticulata Blanco) and tangelos (hybrids of C. reticulata) are most 2.1.2. Experiment 2 susceptible (McClean and Schwarz, 1970; Nariani, 1981; Lopes Thirty-six greenhouse-grown 16-month-old seedlings of the and Frare, 2008; Miyakawa, 1980; Miyakawa and Zhao, 1990; genotypes Benecke trifoliate (P. trifoliata (L.) Raf.), Carrizo citrange Folimonova et al., 2009). Tolerance to HLB has been reported for × × (C. sinensis P. trifoliata), US-802, US-812 (C. reticulata ‘Sunki’ P. some cultivars commonly used as rootstocks, particularly trifoli- × trifoliata ‘Benecke’), US-897, US-942 (C. reticulata ‘Sunki’ P. trifoli- ate orange (Poncirus trifoliata L. Raf.) and some of its hybrids. In ata ‘Flying Dragon’) and Volkamer lemon (Citrus volkameriana Ten. South Africa, McClean and Schwarz (1970) did not find any well- & Pasq.) and 29 greenhouse-grown 16-month-old Cleopatra man- defined disease symptoms in P. trifoliata after graft-inoculation darin seedlings were used. Plants were inoculated in July 2010 as with diseased bud tissue. Similarly, Miyakawa (1980) found no dis- described above, but using 3 bark- or bud pieces per plant. Nine tinct HLB symptoms for P. trifoliata in Taiwan. However, moderate plants of each genotype were mock-inoculated with disease-free to severe disease symptoms were observed for trifoliate orange tissue pieces and 27 plants were inoculated with infected tissue. For and its hybrids Carrizo citrange and Troyer citrange (C. sinen- Cleopatra mandarin, 6 plants were mock-inoculated and 23 plants sis L. Osbeck × P. trifoliata) by Nariani (1981) in India. A study in were inoculated with infected tissue. Florida reported inconsistent results for greenhouse-grown P. tri- All inoculations were performed in groups containing one plant foliata seedlings after graft-inoculation with Las-infected budwood per genotype to ensure that different genotypes received tissue (Folimonova et al., 2009). In the same study, Carrizo citrange was pieces from the same source. classified as tolerant to HLB based on the absence of foliar dis- ease symptoms and healthy growth of infected plants. A recent 2.1.3. Experiment 3 study conducted in our laboratory identified the trifoliate hybrid Thirty 18-month-old greenhouse-grown plants with US-942 US-897 (C. reticulata × P. trifoliata) as tolerant to HLB (Albrecht and scion, generated from etiolated internode stem segments using Bowman, 2011). Transcriptional analysis of US-897 discovered a standard in vitro plant regeneration procedures (Edriss and Burger, number of genes possibly associated with tolerance to Las (Albrecht 1984; Zou et al., 2008) and micro-grafted onto decapitated epi- and Bowman, 2012). cotyls of seed-grown 3–5-week-old Volkamer lemon rootstock via The rootstock is an important component of commercially wedge-grafting, were used. Plants were inoculated in July 2010 as grown citrus trees, and may determine success or failure of a cit- described for experiment 2. Ten plants were mock-inoculated with rus operation (Castle, 2010). In addition to the desired effect on Las-free tissue pieces and 20 plants were inoculated with infected scion vigor, fruit size, fruit quality, and yield, rootstock selection is tissue. based on tolerance to different environmental conditions and resis- tance to pests and diseases. The USDA citrus breeding program has 2.1.4. Experiment 4 been developing and evaluating new citrus varieties since 1893 and Twenty-nine 18- to 24-month-old greenhouse-grown plants several new U.S. rootstock selections have been released in recent with US-812 scion, in vitro-generated and micro-grafted onto years (Bowman, 2001, 2007a,b, 2010). Although a field study con- Volkamer lemon as described for experiment 3, were used. Plants ducted in our laboratory found that rootstock did not induce high were inoculated in September 2010 as described for experiment 2. levels of resistance to HLB in the scion, differences in disease symp- Nine plants were inoculated with disease-free tissue pieces and 20 tom expression, canopy damage, stem growth, and fruit quality plants were inoculated with infected tissue.