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First Report of a New Citrus Decline Disease (CDD) in Association with Double and Single Infection by ‘Candidatus Liberibacter asiaticus’ and ‘Candidatus Phytoplasma aurantifolia’ Related Strains in Iran

    Authors and Affiliations
    • H. Alizadeh , Department of Plant Protection, Faculty of Agriculture, University of Jiroft, Iran
    • F. Quaglino , Department of Agricultural and Environmental Sciences, University of Milan, Italy
    • M. Azadvar , Plant Protection Department, South Kerman Agricultural and Natural Resources Research and Education Center, AREEO, Jiroft, Iran
    • S. Kumar , Department of Agricultural and Environmental Sciences, University of Milan, Italy, and Dr. Rajendra Prasad Central Agricultural University, Bihar, India
    • A. Alizadeh
    • F. Bolboli , Department of Plant Protection, Faculty of Agriculture, University of Jiroft, Iran
    • P. Casati
    • P. A. Bianco , Department of Agricultural and Environmental Sciences, University of Milan, Italy.

      Since 2010, a new emerging citrus decline disease (CDD) widely spread in the southern Kerman region of Iran, killing around 10% of cultivated citrus species trees. CDD can be observed in at least 6-year-old sweet orange, grapefruit, and mandarin plants grafted on Bakraee (Golein et al. 2012), a local citrus variety largely employed as rootstock. Early CDD symptoms include pale green leaves, no production of fresh sprouts, and general retardation of growth. Late symptoms include evident tree decline along with reduction and decay of the root system. Due to CDD epidemiological behavior, typically observed in infectious diseases, and CDD symptoms resembling those of other citrus diseases associated with infection by obligate bacterial parasites, we verified the possible presence of liberibacters and phytoplasmas in CDD-affected citrus trees. During field surveys, carried out in March 2017 in southern Kerman, one leaf and root sample was collected from each of six citrus (Citrus sinensis [L.] Osbeck) trees showing early (three trees) and late (three trees) CDD symptoms, and from each of three symptomless citrus trees. Extracted total nucleic acids from these 18 samples were used as templates in PCR reactions carried out to amplify 16S rRNA gene of liberibacters (primers OI1/OI2c) (Jagoueix et al. 1996) and phytoplasmas (primers P1/P7 and F2n/R2) (Lee et al. 1998). Both 16S rDNA fragments were amplified in four symptomatic trees, indicating a coinfection; only OI1/OI2c or F2n/R2 fragment was amplified in both other symptomatic plants, indicating single infection. No amplification was observed in symptomless citrus trees. Liberibacters were identified exclusively in roots, while phytoplasmas in both roots and leaves. Five OI1/OI2c and six F2n/R2 amplicons were directly sequenced and analyzed. Based on sequence identity, CDD-associated liberibacter strains exhibit two 16S rDNA sequence variants (KY990821 and KY990822) attributed to the species ‘Ca. Liberibacter asiaticus’ (99.9 to 100% identity with strains GFB-Selangor [EU224393] and H36YPENINSULAR [JQ867409]). Species affiliation of these five CDD-associated liberibacter strains was confirmed by sequence analyses of ribosomal protein (rplJ) gene, amplified with primers A2/J5 (Hocquellet et al. 1999). In fact, such liberibacter strains exhibit two rplJ sequence variants (KY990823 and KY990824), showing 99 to 100% identity with ‘Ca. L. asiaticus’ strains HSP1 (KP210470) and A4 (CP010804). CDD-associated phytoplasma strains exhibit six 16S rDNA sequence variants (KY990815–20) attributed to the species ‘Ca. Phytoplasma aurantifolia’, showing 99.4 to 99.7% identity with the reference strain WBDL (U15442) and carrying species-specific signature sequence. Furthermore, iPhyClassifier analyses of F2n/R2 fragments allowed the attribution of such phytoplasma strains to subgroups 16SrII-B, -C, and a new putative 16SrII subgroup. Phylogenetic analyses confirmed the species affiliation of CDD-associated liberibacter and phytoplasma strains. Previous studies reported citrus diseases (e.g., Huanglongbing) associated with single or double infection by ‘Ca. L. asiaticus’ and genetically distinct phytoplasmas (Lou et al. 2014; Teixeira et al. 2008). This study reinforces this evidence, firstly reporting the association of a new citrus decline disease with single and double infection by ‘Ca. L. asiaticus’ and ‘Ca. P. aurantifolia.’ Further investigation will be needed to know the accurate etiology and epidemiology of CDD in order to determine its potential impact.