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Witches’ Broom Disease of Lime Contributes to Phytoplasma Epidemics and Attracts Insect Vectors

    Authors and Affiliations
    • A. M. Al-Subhi1
    • A. M. Al-Sadi1
    • R. A. Al-Yahyai1
    • Y. Chen2
    • T. Mathers2
    • Z. Orlovskis2
    • G. Moro2
    • S. Mugford2
    • K. S. Al-Hashmi1
    • S. A. Hogenhout2
    1. 1Department of Plant Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al Khod 123, Oman
    2. 2John Innes Centre, Department of Crop Genetics, Norwich NR4 7UH, United Kingdom

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    An insect-transmitted phytoplasma causing Witches’ Broom Disease of Lime (WBDL) is responsible for the drastic decline in lime production in several countries. However, it is unclear how WBDL phytoplasma (WBDLp) induces witches’ broom symptoms and if these symptoms contribute to the spread of phytoplasma. Here we show that the gene encoding SAP11 of WBDLp (SAP11WBDL) is present in all WBDLp isolates collected from diseased trees. SAP11WBDL interacts with acid lime (Citrus aurantifolia) TCP transcription factors, specifically members of the TB1/CYC class that have a role in suppressing axillary branching in plants. Sampling of WBDLp-infected lime trees revealed that WBDLp titers and SAP11WBDL expression levels were higher in symptomatic leaves compared with asymptomatic sections of the same trees. Moreover, the witches’ brooms were found to attract the vector leafhopper. Defense genes that have a role in plant defense responses to bacteria and insects are more downregulated in witches’ brooms compared with asymptomatic sections of trees. These findings suggest that witches’ broom-affected parts of the trees contribute to WBDL epidemics by supporting higher phytoplasma titers and attracting insect vectors.

    Phytoplasmas (genus ‘Candidatus phytoplasma’) and other members of the Class Mollicutes are small bacteria with single cell membranes and no cell wall and are thought to have evolved from a Gram-positive Clostridium-like ancestor (Bertaccini 2007; Hogenhout et al. 2008; Lee et al. 2000). The majority of mollicutes live in close association with eukaryotic hosts as symbionts or pathogens and have undergone severe genome reductions. Within the Class Mollicutes, members of Ca. Phytoplasmas form a monophyletic group that has common ancestry with Acholeplasma species (Harrison et al. 2011). All phytoplasmas characterized so far are intracellular pathogens of plants; these bacteria colonize the cytoplasm of phloem sieve cells of the plant vasculature and move systemically throughout the plant via migration through sieve plate pores (Bové and Garnier 2002). Phytoplasma are introduced into the sieve cells during feeding by phytoplasma-carrying sap-feeding insects of the order Hemiptera (Weintraub and Beanland 2006), grafts of phytoplasma-infected scions (Aryan et al. 2016; Kamińska et al. 2003), and parasitic plants, including dodder (Cascuta species; Garnier et al. 1991). Phytoplasma infect a wide variety of vascular plant species and occur worldwide (Bertaccini 2007; Hogenhout et al. 2008; Lee et al. 2000).

    Phytoplasma-infected plants often show characteristic symptoms, including witches’ brooms (stem proliferations from a single point), virescence (colored flower parts that remain green), phyllody (development of vegetative tissues instead of flowers), big buds (bulbous enlarged flower buds), sterility, chlorosis, stunting, changes in leaf morphology, and general decline of the plant (Bertaccini et al. 2014; Hogenhout et al. 2008; Lee et al. 2000). Phytoplasmas also invade and multiply within various tissues inside cells of insect vectors. Phytoplasma infections can lead to premature death of the insects, although insects that are efficient vectors of the phytoplasma often do not appear affected by the infection and sometimes benefit from carrying the phytoplasma through, for example, increased longevity and reproduction (Beanland et al. 2000; MacLean et al. 2014; Sugio et al. 2014).

    Phytoplasmas in the 16SrII phytoplasma group are classified into 21 subgroups (Yang et al. 2016) that include the 16SrII subgroup B ‘Candidatus. P. aurantifolia’ WBDLp (Bertaccini et al. 2014) and the 16SrII subgroup D phytoplasmas Alfalfa Witches’ Broom, Peanut Witches’ Broom, and Sweet Potato Little Leaf (SPLL; Al-Subhi et al. 2018). WBDL has a narrow host range restricted to some citrus crops, predominantly lime trees, in the Middle East and Brazil (Alhudaib et al. 2009; Garnier et al. 1991; Ghosh et al. 1999). WBDLp isolates from Oman, United Arab Emirates (UAE), and Iran show limited sequence variation (Al-Abadi et al. 2016; Al-Ghaithi 2017).

    Acid lime (Citrus aurantifolia [Christm] Swingle) is a major commercial crop in Oman and neighboring countries. Over the past four decades, acid lime production has dramatically declined because of Witches’ Broom Disease of Lime (WBDL) phytoplasma (Al-Sadi et al. 2017). This bacterial pathogen is transmitted by sap-feeding insects, predominantly by the leafhopper Hishimonus phycitis (Queiroz et al. 2016; Salehi et al. 2007). Trees infected with WBDL show characteristic witches’ brooms symptoms, which consist of dense proliferations of shoots with small light green to yellow leaves and reduced flower and fruit production. However, it is not yet clear if the phytoplasma benefits from inducing the witches’ brooms symptoms in its host plant. The symptoms become evident in one or few branches and spread throughout the tree over several years until the tree dies (Al-Yahyai et al. 2015). The disease was first reported in Oman during the 1970s (Waller and Bridge 1978). It has then been recorded in the UAE (Garnier et al. 1991), Iran (Bove et al. 2000), India (Ghosh et al. 1999), and Saudi Arabia (Alhudaib et al. 2009).

    Phytoplasmas have functional Sec-dependent secretion systems that translocate phytoplasma proteins, including virulence proteins (effectors), across the phytoplasma cell membrane into the cytoplasm of plant or insect cells (Kakizawa et al. 2004). The secreted proteins are released in the plant phloem and unload from the phloem to systemic plant tissues (Bai et al. 2009; Sugio et al. 2011a). Fifty-six candidate effector genes, named secreted aster yellows strain witches’ broom proteins (SAPs), were identified in the genome of aster yellows phytoplasma strain witches’ broom (AY-WB phytoplasma; Bai et al. 2009). Of these 56, two AY-WB effectors – SAP11 and SAP54 – have been characterized functionally. AY-WB SAP11 binds and destabilizes Arabidopsis thaliana TEOSINTE BRANCHED1 (TB1), CYCLOIDEA (CYC), and PROLIFERATING CELL FACTORS 1 and 2, known as TCP transcription factors, particularly class-II CINCINNATA and TB1/CYC TCPs, leading to A. thaliana leaf crinkling and shoot proliferations that resemble the witches’ brooms symptoms of phytoplasma-infected plants (Bai et al. 2009; Lu et al. 2014; Sugio et al. 2011a, 2014; Tan et al. 2016). SAP11 homologs of other phytoplasmas also bind TCPs (Janik et al. 2017; Tan et al. 2016) and TCPs are conserved among plant species (Navaud et al. 2007), suggesting that SAP11-mediated TCP destabilization leading to witches’ brooms may be a common phenomenon. SAP54 and homologs of this effector (also known as phyllogen) of phytoplasmas bind and destabilize plant MADS-box transcription factors leading to the development of leaf-like flowers that resemble those of phytoplasma-infected plants with phyllody symptoms (Kitazawa et al. 2017; MacLean et al. 2011; Maejima et al. 2014, 2015). Intriguingly, both SAP11 and SAP54 promote colonization of the leafhopper Macrosteles quadrilineatus on A. thaliana (MacLean et al. 2014; Orlovskis 2017; Orlovskis and Hogenhout 2016; Sugio et al. 2011a).

    A SAP11 homolog (henceforth referred to as SAP11WBDL in this study) was identified in the draft genome of a WBDLp isolate from acid lime tree (C. aurantifolia) from Oman (A. M. Al-Subhi, A. M. Al-Sadi, R. A. Al-Yahyai, and S. Hogenhout, unpublished) and in some Omani WBDLp isolates (Al-Ghaithi et al. 2018; Anabestani et al. 2017). However, a WBDLp-like phytoplasma identified in Brazil and that colonizes citrus trees does not induce obvious witches’ brooms symptoms, and a gene with sequence similarity to SAP11 effectors was not identified in the WBDLp-like phytoplasma of Brazil, although these citrus trees were more attractive to the insect vector (Queiroz et al. 2016). This raised the question whether witches’ broom symptoms play a role in insect vector attraction.

    In this study, we investigated the prevalence of witches’ broom symptoms and the presence of SAP11WBDL among Middle Eastern WBDLp isolates and how these symptoms may contribute to WBDL epidemics in the field. We detected SAP11WBDL in all WBDLp isolates collected from symptomatic trees, the majority of which showed witches’ broom symptoms. The SAP11WBDL sequence was conserved among the isolates and interacted with specific TCP transcription factors of A. thaliana and lime. Moreover, branches of lime trees in Omani orchards with witches’ broom symptoms had higher SAP11WBDL expression levels, higher WBDLp titers, and were more attractive to H. phycitis leafhoppers than non-symptomatic branches of the same trees. The symptomatic branches also had lower expression levels of plant defense genes. These data indicate that WBDLp induced witches’ broom symptoms that promote WBDL epidemics in the field.

    Materials and Methods

    Sampling of acid lime trees.

    A total of 40 lime trees, including both WBDLp-symptomatic (13) and asymptomatic plants (17) and trees with other symptoms (10), were sampled at various locations in North and South Oman, UAE, Saudi Arabia, Iran, and Brazil (Table 1) from 2013 to 2016. Usually, parts of the trees were symptomatic and only occasionally whole trees. Leaves of symptomatic trees (showing chlorosis, dieback, and/or yellowing or witches’ brooms) were sampled. For asymptomatic trees, young leaves from central areas of the trees were sampled at approximately 100 g per sample. The samples were snap-frozen in liquid nitrogen and shipped to the laboratory. Samples were ground to a powder in liquid nitrogen before storage at −80°C. Samples from acid lime trees that did not have WBDL symptoms (presumably non-infected trees) were included as negative controls (Table 1). DNA preparations of lime trees shown to be previously infected with WBDLp and alfalfa plants known to be infected with alfalfa witches’ broom disease (AlWD) phytoplasma were used as positive controls.

    Table 1. A list of 40 Witches’ Broom Disease of Lime (WBDL)-symptomatic, asymptomatic, and declining lime trees and two positive control samples used in this study with their geographical origins, and corresponding 16S rDNA, imp, tuf, and SAP11 genes and accession numbers of 19 WBDL phytoplasma strains of the 16Sr-II group deposited in the NCBI database

    WBDLp titers and relative expression levels of SAP11WBDL and selected acid lime defense genes were compared in symptomatic and non-symptomatic parts of six acid lime trees previously shown to be infected with WBDLp. These six trees were located at Sultan Qaboos University Agricultural Experiment Station in Al-Seeb, Oman. Two sample sets were collected from each infected acid lime tree, the first set from branches showing severe phytoplasma ‘Ca. P. aurantifolia’ symptoms (witches’ broom) and the second set from an asymptomatic branch. The 12 samples were stored at −80°C until further processing.

    DNA extraction and PCRs.

    Total DNA was extracted from the symptomatic and asymptomatic acid lime tree samples using the CTAB method described by Doyle and Doyle (1987) with some modifications as described by Al-Subhi et al. (2017).

    PCRs were conducted with specific primers for WBDLp phytoplasma including 16S rDNA, Immunodominant membrane protein (imp), elongation factor Tu (tuf), and SAP11 (Table 1; Supplementary Table S1). The WBDLp 16S rDNA gene was amplified with the universal primers’ pairs P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995) at a primer annealing temperature of 55°C and the nested primer pair R16F2n/R16R2 (Gundersen and Lee 1996) at a primer annealing temperature of 60°C. The WBDLp partial tuf gene was amplified using the primer pair TUF-II-F1/TUF-II-R1 as the direct PCR and the primer pair TUF-II-F2/TUF-II-R1 as the semi-nested PCR at primer annealing temperatures of 53°C (Al-Subhi et al. 2018). The full-length WBDLp imp gene was amplified with primer pairs IMP-II-F1/IMP-II-R1 and IMP-II-F2/IMP-II-R1, as the direct and the semi-nested PCR reaction, respectively, at annealing temperatures of 53°C (Al-Subhi et al. 2018). For amplification of the WBDLp (16SrII-B subgroup) SAP11 gene, the primer pairs SAP11-WBDL-F1/SAP11-WBDL-R1 (direct PCR) and SAP11-WBDL-F2/SAP11-WBDL-R2 (nested PCR) were used at annealing temperatures of 53°C (Al-Subhi et al. 2018). The SAP11 gene of AlfWD phytoplasma (16SrII-D subgroup) was amplified with primer pairs SAP11-IID-F1/SAP11-IID-R1 (direct PCR) and SAP11-IID-F2/SAP11-IID-R2 (nested PCR) at annealing temperatures of 53°C (Al-Subhi et al. 2018). PCR amplifications were carried out using “Ready-To-Go” PCR beads (GE Healthcare UK Limited, Buckinghamshire, UK) with 25 µl reaction volumes containing 50 ng of host genomic DNA and 10 pM of each primer. The PCR protocol followed was according to Al-Subhi et al. (2017).

    Sequencing and sequence analysis.

    One positive PCR product from each sample of the four genes was purified using a Qiaquick PCR Purification kit (Qiagen, Hilden, Germany) and Sanger sequenced at Macrogen Company (South Korea). Amplification products were sequenced from both directions using nested PCR primers of each gene (Supplementary Table S1). Sequences were visualized, assembled, and aligned using the software BioEdit (Hall 1999). Sequences were searched against the non-redundant nucleotide and protein databases of GenBank (NCBI, Bethesda, MD) using BLASTn or BLASTx (Wheeler and Bhagwat 2007). DNA sequences were deposited in GenBank (accession numbers in Table 1).

    Phylogenetic analyses.

    Sequences of WBDLp genes and other organisms (Table 1 and Supplementary Tables S3 to S5) were aligned using the tool CLUSTAL W (Thompson et al. 1994) and alignments were visually checked and corrected as appropriate, and phylogenies of the alignments were generated with the neighbor-joining method from the software MEGA 6 (Tamura et al. 2013) using the Kimura-2-parameter model (Kimura 1980) to estimate genetic distances. Bacillus subtilis 16S rRNA (AB042061) and tuf (GCA000789275) were used as outgroups to root the 16S rDNA and tuf phylogenetic trees.

    Yeast two-hybrid assays.

    Yeast two-hybrid (Y2H) assays were conducted with the GAL4 system (Invitrogen, Paisley, UK) following manufacturer’s instructions. SAP11 genes corresponding to the full-length SAP11 proteins without signal peptides were amplified from plant materials infected with WBDL, AY-WB (16SrI-A subgroup; Accession. No. GI:85057650), Sweet Potato Little Leaf (SPLL; 16rII-A subgroup; not published), and Maize Bushy Stunt Phytoplasma (MBSP; 16SrI-B subgroup; Accession. No. NZ_CP015149; Supplementary Table S1). A. thaliana TCP genes for TCP2 (GenBank accession no. NM_117950), TCP6 (NM_123468), TCP7 (NM_122234), TCP9 (NM_130131), BRC2 (NM_001334381), BRC1 (NM_112741), TCP13 (NM_111082), and TCP14 (NM_114630) were amplified with Gateway compatible primers (GenScript, Piscataway, NJ) and cloned into the pDONR207 entry vector using BP Clonase II (Invitrogen) as described in Sugio et al. (2011a). Sequences corresponding to the conserved TCP domains of Acid Lime TCP transcription factors (Supplementary Fig. S9) were cloned into pDONR207 entry vector using BP Clonase II (Invitrogen). The inserts were transferred from pDONR207 entry vectors into the destination vectors pDEST-GBKT7 (SAP11) or pDEST-GADT7 (TCPs) using LR Clonase II. Y2H assays were performed using the yeast (Saccharomyces cerevisiae) strain AH109 according to the manufacturer’s protocol (Clontech, Mountain View, CA). Selection of transformed yeast clones was conducted on leucine and tryptophan-lacking minimal synthetic dextrose (SD; SD/–LW) media. Interactions were analyzed on SD media lacking tryptophan, leucine, and histidine (SD/–LWH) with 20 mM of 3-amino-1,2,4-triazole (3AT; Sigma-Aldrich, Taufkirchen, Germany) or SD lacking leucine, tryptophan, adenine, and histidine (SD/–LWAH) with 20 mM of 3AT.

    RNA isolation and cDNA synthesis.

    Total RNA was extracted from symptomatic and asymptomatic branches of the six phytoplasma-infected acid lime trees that are included in this study using Tri-Reagent (Sigma-Aldrich, St. Louis, MO) in accordance with the manufacturer’s instructions. The purities and yields of the RNA extractions were determined with 1% agarose gel electrophoresis and a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Loughborough, Leicestershire, UK). One microgram of total RNA was treated using RQ1 DNase set (Promega, Madison, WI) to remove possible DNA contaminants before cDNA synthesis, then cDNA was synthesized from 1 µg of RQ1-DNase–treated RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) in accordance with the manufacturer’s instructions. The oligo-dT primer was used to synthesize cDNA to be utilized in acid lime host defense genes expression studies and the Random Hexamer primer (Thermo Fisher Scientific) was used to synthesize cDNA to be used in SAP11WBDL gene expression study.

    Real-time reverse transcription PCR analysis.

    The cDNA products were diluted 10-fold by distilled water and 0.5 µl of each diluted cDNA sample was used for 20 µl of real-time quantitative PCR reactions using SYBR Green JumpStart Taq Ready Mix (Sigma-Aldrich) with 0.5 µg of each gene-specific primer (Supplementary Tables S1 and S2). Two technical replicates of each sample were run in a CFX96 Real-Time System with a C1000 Thermal Cycler 298 (Bio-Rad, Watford, UK); cycling conditions were according to Orlovskis (2017).

    The coding sequence genome of Citrus sinensis and Citrus clementina were used to design the specific primers for the acid lime PR1, 13-lipooxygenase 2 (Lox2), isochorismate synthase1 (SID2), Myc2, 18S, actin, tubulin, elongation factor 1-alpha, and GAPDH genes (Supplementary Tables S1 and S2). The specific oligonucleotides for the conserved fragments of selected genes (Table 1) were designed using the program Primer3 (Rozen and Skaletsky 2000) and the PrimerQuest design tool. The relative expression level of target acid lime PR1, Lox2, SID2, and Myc2 genes were normalized to the expression levels of housekeeping genes, including 18S, actin, tubulin, elongation factor 1-alpha, and GAPDH. The relative expression level of SAP11WBDL was compared with those of the WBDLp housekeeping genes DnaB, ftsY, gyrA, pykF, and tuf (unpublished WBDLp phytoplasma draft genome; Supplementary Table S1). Relative gene expression levels were determined by the comparative Ct method (Schmittgen and Livak 2008). Phytoplasma titers were calculated from Ct values of WBDLp phytoplasma housekeeping genes normalized against the Ct values of the acid lime housekeeping genes. The expression stabilities of the selected reference genes were evaluated by the software program NormFinder (; Andersen et al. 2004; Latham 2010). The stability values of phytoplasma housekeeping genes and acid lime housekeeping genes were 0.079 and 0.002, respectively. Three biological replicates were analyzed for all gene expression level experiments.

    H. phycitis preference tests.

    The study was conducted by placing blue, transparent, and yellow color sticky traps in three sites in Oman, Al-Seeb (N: 23.600955, E: 58.164136) area from the Muscat governorate, Nizwa (N: 22.973664, E: 57.554600) from the Al-Dakhilia governorate, and Liwa (N: 24.493074, E: 56.560632) from the North Al-Batinah governorate. Two sticky traps of each type were hung on symptomatic and asymptomatic branches of each WBDL-infected acid lime tree at approximately 1.5 m above the ground. The yellow traps were hung on six lime trees in Al-Seeb, while the blue and transparent traps were each hung on three lime trees in Nizwa, two trees in Al-Seeb, and five trees in Liwa. H. phycitis insects (putative vectors of WBDL) trapped over 1-week periods were counted from each side of the sticky traps. The study was conducted over the period of 5 November to 13 November 2016.

    Statistical analysis.

    All statistical analysis was performed with a Kruskal–Wallis test (non-parametric test) using R program (R version 4.0.2.). The significant difference was analyzed at 95% confidence level.


    SAP11WBDL is detected in WBDLp-infected acid lime trees throughout the Middle East.

    The WBDLp was detected using PCR amplification of the 16S rRNA gene. Of the total of 40 trees sampled, 19 tested positive for WBDLp (all the 13 symptomatic trees and six out of the 21 asymptomatic trees). These were five out of eight trees in north Oman, two out of six trees in south Oman, three out of six trees in UAE, three out of 10 trees in Saudi Arabia, five out of five trees in Iran, and one out of five trees in Brazil (Table 1). Of the 19 trees in which WBDLp was detected, 13 trees displayed typical witches’ broom symptoms. The six trees out of 19 trees that were positive for WBDLp and that did not show symptoms were from south Oman (two trees), Saudi Arabia (three trees), and Brazil (one tree). WBDLp 16S rDNA, tuf, and imp genes were amplified and sequenced from all WBDLp-positive trees. The sequences of the WBDLp isolates collected herein were compared with those of a previous WBDLp isolate from Oman (Al-Subhi et al. 2018), a Brazil WBDLp isolate (Queiroz et al. 2016), an alfalfa witches’ broom isolate that belongs to another subgroup than WBDL, and available sequences from other phytoplasmas (Supplementary Figs. S1, S2, S3, S4, S5, S6, S7, and S8). These comparisons showed that the imp sequences were identical among the Middle Eastern isolates and that the Brazilian WBDLp isolate grouped with 16SrII-B subclade WBDL phytoplasmas (Supplementary Figs. S3 and S4). The 16S rDNA and tuf sequences of isolates collected from northern and southern Oman, UAE, Iran, Brazil and one of a Saudi Arabia isolate (KSA1) also grouped with 16SII-B WBDL phytoplasmas. The 16S rDNA sequences of the two WBDLp Saudi Arabia isolates (KSA2 and 3) from the Al-Ahsa region of Saudi Arabia grouped separately from other WBDLp isolates in the 16rII-B cluster (Supplementary Figs. S5 and S6). Moreover, the tuf genes of these three isolates grouped with 16SrII-D phytoplasmas (Supplementary Figs. S7 and S8). These data indicate that the WBDLp isolates from Al-Ahsa region of Saudi Arabia are distinct from those of other regions in the Middle East. Together these data indicate that the isolates collected from symptomatic lime trees in the Middle East in this study carry WBDL phytoplasma.

    The SAP11WBDL gene was amplified from 18 WBDLp positive trees in the Middle East but not from the WBDLp-infected citrus tree in Brazil, consistent with another report (Queiroz et al. 2016; Table 1). The SAP11WBDL sequences were identical among all Middle Eastern samples and to the SAP11WBDL sequence of previously reported Omani WBDLp of the 16SrII-B subgroup (KX358622; Al-Subhi et al. 2018; Table 1; Supplementary Figs. S1 and S2). Thus, the SAP11WBDL gene is commonly amplified in WBDLp phytoplasma isolates collected from lime trees across the Middle East.

    SAP11WBDL interacts with TCP transcription factors of A. thaliana and lime.

    To investigate if SAP11WBDL interacts with TCPs, Y2H assays of SAP11WBDL and other SAP11 homologs with class-II TCPs of A. thaliana and acid lime were conducted in the same way as shown above.

    SAP11AYWB interacts with class-II TCPs BRC1 (AtTCP18), BRC2 (AtTCP12), and AtTCP2 and AtTCP13 in the Y2H assays. Confirming other findings (Sugio et al. 2011a, 2014), SAP11MBSP interacted only with BRC1 and BRC2, SAP11SPLL with both BRC1 and BRC2, and SAP11WBDL only with BRC2 (Fig. 1A). Thus, SAP11WBDL interacts with one of the two A. thaliana CYC/TB1 TCPs that regulate stem branching (Chai et al. 2017; González-Grandío et al. 2013; Sugio et al. 2011a, 2014).

    Fig. 1.

    Fig. 1. SAP11s genes of Witches’ Broom Disease of Lime (WBDL) phytoplasma, Sweet Potato Little Leaf (SPLL) phytoplasma, Aster Yellows Strain Witches’ Broom (AY-WB) phytoplasma, and Maize Bushy Stunt Phytoplasma (MBSP) interact with A, Arabidopsis TCPs and B, acid lime TCP’s genes domain; the SAP11 genes were cloned into pDEST-GBKT7 as a binding domain of the GAL4 transcriptional activator (baits), Arabidopsis TCP’s were cloned into pDEST-GADT7 as an activation domain of GAL4 (prey); the transformed yeasts were selected and evaluated by plating on leucine and tryptophan-lacking minimal synthetic dextrose (SD; SD/-L-W), SD lacking tryptophan, leucine, and histidine containing 20 mM of 3AT (SD/-L-W-H), and SD lacking leucine, tryptophan, adenine, and histidine (SD/-L-W-A-H). EV, empty vector control. C, A phylogenetic tree of selected acid lime TCP domains, the Arabidopsis TCP domains, and the Zea mays TCP domains. Phylogenetic tree constructed by the neighbor-joining method with bootstrap values expressed as percentages of 1,000 replications.

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    Because the host range of WBDLp is mostly restricted to citrus, particularly lime, the citrus sequence data available in the Citrus Genome Database ( was mined for TCP sequences. Ten citrus TCP-like sequences were identified. Phylogenetic analyses of these sequences with TCPs from A. thaliana (Martín-Trillo and Cubas 2010) and maize (Zea mays L.; Chai et al. 2017) showed that acid lime TCP1-like, TCP12-like, and TCP18-like belong to the class-II CYC/TB1 clade, and the others to the class-II CINCINNATA-TCP or class-I PROLIFERATING CELL FACTORS clades (Fig. 1C).

    It has been shown that SAP11 binds with the conserved TCP domain of TCP transcription factors (Sugio et al. 2014). Therefore, the TCP domains of the TCP transcription factors were amplified from acid lime and cloned for Y2H analysis to assess binding of SAP11 homologs. The sequences of the lime TCPs domains were identical to those in the Citrus Genome Database (Supplementary Fig. S9). SAP11WBDL interacted strongly with the acid lime TCP1-like and TCP18-like CYC/TB1 TCPs, and weakly with the acid lime TCP12-like (Fig. 1B). SAP11SPLL interacted only with acid lime TCP1-like of the CYC/TB1 TCP clade, whereas SAP11AYWB did not, and both SAP11AYWB and SAP11MBSP interacted with the acid lime TCP12-like and TCP18-like TCPs (Fig. 1B). Thus, SAP11WBDL interacts with three acid lime CYC/TB1 TCPs. Because SAP11 effectors destabilize TCP transcription factors upon interaction and CYC/TB1 TCPs regulate stem branching in diverse plant species, including A. thaliana and maize (Sugio et al. 2011a, 2014), it is likely that SAP11WBDL plays a role in the induction of witches’ broom symptoms in WBDLp-infected acid lime.

    WBDLp has higher titers in witches’ brooms versus asymptomatic branches of acid lime trees.

    Next, we investigated WBDLp titers and SAP11 expression in witches’ brooms versus asymptomatic branches of six acid lime trees (Fig. 2A). Based on amplification levels of the 16S rDNA gene, WBDLp titers are higher in symptomatic versus asymptomatic branches of all six lime trees (Fig. 2B). To assess if WBDLp in the branches is alive and metabolically active, we also analyzed WBDLp transcript levels. Quantitative RT-PCRs of WBDLp genes for DnaB, FtsY, GyrA, PykF, and TUF, normalized using acid lime housekeeping genes, showed higher expression levels of the WBDLp genes in witches’ brooms than in non-symptomatic branches (Kruskal–Wallis, P = 2.968e-07; Fig. 2C). Moreover, relative to the WBDLp housekeeping genes, SAP11WBDL expression levels were higher in the witches’ brooms too (Fig. 2D). Therefore, WBDLp is more abundant and have upregulated SAP11WBDL expression levels in witches’ brooms versus asymptomatic parts of acid lime trees.

    Fig. 2.

    Fig. 2. Witches’ Broom Disease of Lime (WBDL) has higher titers in witches’ broom symptomatic (S) compared with asymptomatic (As) branches of WBDL-infected lime trees. A, A WBDL-infected acid lime tree with witches’ broom and asymptomatic branches. B, More WBDL 16S rDNA is amplified from witches’ broom S- compared with As-branches of six WBDL-infected acid lime trees (L1 to L6). The WBDL 1.8-kb 16S rDNA fragments (P1/P7 primers) were separated on a 1% (wt/vol) agarose gel. The notation “M” is a DNA ladder with fragment sizes indicated in kb at the left of the gels. C, WBDL gene expression levels are higher in witch’s broom S- versus As-branches of six WBDL-infected acid lime trees (L1 to L6). Relative titers (y-axis) of data points of the box plots were obtained from the average of the expression levels of each of the WBDLp dnaB, ftsY, gyrA, pykF, and tuf genes against the average expression levels of acid lime genes for 18S rRNA, tubulin, elongation factor 1-alpha, and GAPDH at n = 3 samples per tree. Kruskal–Wallis, PAS versus S = 2.968e-07. D, SAP11WBDL has higher expression levels in S- versus As-branches of six WBDL-infected acid lime trees (L1 to L6). The relative SAP11WBDL expression levels (y-axis) of the box plots were obtained by calculating the SAP11WBDL expression levels relative to the average expression levels of a WBDLp dnaB, ftsY, gyrA, pykF, and tuf genes at n = 3 samples per tree. Kruskal–Wallis statistical analysis: PAS versus S = 0.0001467.

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    The WBDLp insect vector H. phycitis prefers colonization of lime witches’ brooms.

    To investigate if the witches’ brooms attract the WBDL insect vector H. phycitis to lime trees, blue and transparent sticky traps were placed in symptomatic and asymptomatic branches of WBDL-infected acid lime trees in the al-Seeb, Nizwa, and Liwa orchards. Higher numbers of H. phycitis were captured on the blue and transparent sticky traps of witches’ brooms than those of asymptomatic branches (Kruskal–Wallis, P = 0.002287 for blue and P = 0.002143 for transparent sticky traps; Fig. 3A and B), suggesting that witches’ brooms are more attractive to the principal vector of WBDLp. The leafhopper preference for the witches’ broom parts was observed for lime trees in Al-Seeb, Nizwa, and Liwa cities in Oman (Fig. 3A and B). Interestingly, when yellow sticky traps were used in leafhopper choice tests, the leafhoppers did not prefer the witches’ broom symptomatic branches compared with asymptomatic ones of six acid lime trees in the Al-Seeb orchard (Fig. 5A to C). The witches’ broom symptomatic branches show denser and more yellow vegetation compared with asymptomatic plant parts (Fig. 5A). Indeed, the symptomatic branches have lower chlorophyll content and are more chlorotic (yellow) than asymptomatic branches (Kruskal–Wallis, P = 4.868e-08; Fig. 5B).

    Fig. 3.

    Fig. 3. The Witches’ Broom Disease of Lime (WBDL) leafhopper vector Hishimonus phycitis prefers witches’ broom (S) compared with asymptomatic (As) branches of acid lime trees. Choice experiments were done with A, blue and B, transparent sticky traps hung in witches’ broom S- and As-branches of acid lime trees. Data points in box plots of graphs show numbers of leafhopper insects found on the sticky traps (y-axis) in independent trees at n = 3 trees of farm 1 (Al-Dakhilia governorate), n = 2 trees of farm 2 (Muscat governorate), and n = 5 trees of farm 3 (North Al-Batinah governorate) for a period of 7 days. Data points of the three farms were combined for Kruskal–Wallis statistical analysis: A, PAs versus S = 0.002287; B, PAs versus S = 0.002143.

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    Jasmonate and salicylic acid synthesis gene expression levels are reduced in acid lime witches’ brooms.

    It was previously shown that plants with an impaired jasmonate (JA) signaling pathway are more attractive to leafhoppers, including those that vector phytoplasmas (Kallenbach et al. 2012; Sugio et al. 2011b). The expression level of the acid lime homolog of LOX2, which is involved in the synthesis of JA in plants (Chauvin et al. 2016), was downregulated in leaves of lime witches’ brooms compared with asymptomatic leaves (Fig. 4A, Kruskal–Wallis; P = 0.002287). In contrast, no expression differences were observed of the acid lime homolog of Myc2, which regulates the activation of JA-responsive genes of the signaling pathway (Liu et al. 2019), between symptomatic and asymptomatic branches (Fig. 4A; Kruskal-Wallis, P = 0.569). Similarly, for the salicylic acid (SA)-mediated plant defense pathway that also plays a role in plant defense to insects (Bruessow et al. 2010; Cowles et al. 2018), the lime homolog of isochorismate synthase1 (ICS1 or SID2), which is required for SA synthesis in plants, was downregulated in acid lime witches’ brooms versus asymptomatic ones (Fig. 4B; P = 0.008549), whereas the SA response gene for PR1 was not (Fig. 4B; P = 0.6351). Therefore, JA and SA synthesis, but not downstream signaling genes, may be downregulated in the witches’ brooms of acid lime trees.

    Fig. 4.

    Fig. 4. Genes involved in jasmonate and salicylic acid synthesis are downregulated in witches’ brooms (S) versus asymptomatic (As) branches of Witches’ Broom Disease of Lime (WBDL)-infected acid lime trees. Box plots of quantitative reverse transcription PCRs of the acid lime transcripts of A, jasmonate synthesis gene 13-lipooxygenase 2 (LOX2) and response gene Myc2 and B, response gene PR1 and salicylic acid synthesis gene SID2. Each data point represents the relative expression of two technical replicates per sample at n = 3 leaf samples per tree of six trees (L1 to L6) of the Muscat governorate (N: 23.600955, E: 58.164136). Kruskal–Wallis statistical analysis: A, PAs versus S LOX2 = 0.002287 and PAs versus S Myc2 = 0.569; B, PAs versus S PR1 = 0.6351 and PAs versus S SID2 = 0.008549.

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    Fig. 5.

    Fig. 5. The Hishimonus phycitis leafhoppers prefer visual cues from Witches’ Broom Disease of Lime (WBDL) symptomatic acid lime. A, Photos showing the differences in symptoms between the symptomatic (S) and the asymptomatic (As) branches; the S-branch shows witches’ broom and yellow color. B, Box plot represents chlorophyll content reading by SPAD-502 Plus Meter (Konica Minolta, Tokyo, Japan) of 10 leaves from each of the S- and As-branches for each of the six lime trees (L1 to L6) at the Muscat governorate (N: 23.600955, E: 58.164136). C, Yellow sticky traps hung in witches’ broom S- and As-branches of acid lime trees. Data points in box plots of graphs show numbers of insects found on the sticky traps (y-axis) in independent trees at n = six trees (L1 to L6) at the Muscat governorate (N: 23.600955, E: 58.164136) for a period of 7 days. Data points of the S-lime trees were combined for Kruskal–Wallis statistical analysis: B, PAs versus S = 4.868e-08; C, PAs versus S = 0.9352.

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    WBDL is a lethal disease of acid lime and is widespread in the Middle East (Al-Yahyai et al. 2012; Bove et al. 2000). This study provided new insights into mechanisms involved in WBDL disease progression and spread. Firstly, we show that SAP11WBDL is commonly present in WBDLp isolates of the Middle East and the majority of lime trees infected with these WBDLp isolates show sections with excessive axillary branching, termed witches’ brooms. We found that the witches’ brooms have higher WBDLp titers than sections of the same trees without these symptoms. The expression levels of a number of WBDLp metabolic genes are higher in witches’ brooms and the expression level of SAP11WBDL is particularly high. We also showed that SAP11WBDL interacts with acid lime TCP transcription factors. Given that SAP11 destabilizes TCPs (Chai et al. 2017; González-Grandío et al. 2013; Sugio et al. 2011a, 2014) and that TCPs have a conserved role in the regulation of axillary branching among plants (Chai et al. 2017; González-Grandío et al. 2013; Sugio et al. 2011a, 2014), our data suggest that SAP11WBDL is likely to play a role in the development of witches’ broom symptoms during WBDLp infection of acid lime. Moreover, the WBDL leafhopper vector H. phycitis (Orlovskis 2017; Orlovskis and Hogenhout 2016) is more attracted to the witches’ brooms. These data provide evidence that the witches’ broom symptoms correlate with WBDLp titer and attraction of leafhopper vectors on which WBDL relies for transmission.

    Phylogenetic analysis of the 16S rDNA, imp, and tuf gene of WBDLp isolates from Oman, UAE, Iran, and Brazil showed that all are placed in one subclade and clustered with 16SrII-B subgroup phytoplasma ‘Ca. P. aurantifolia’ with very strong bootstrap support, which is consistent with previous reports of WBDL phytoplasma (Al-Abadi et al. 2016; Al-Subhi et al. 2018; Silva et al. 2014; Garnier et al. 1991; Zreik et al. 1995) and suggest that the genetic diversity of WBDLp phytoplasma isolates across the large geographical distribution from Oman, UAE, and Iran is relatively low. Whereas the majority (72%) of lime trees positive for WBDLp and SAP11WBDL had witches’ brooms, 28% of the trees, including all Saudi Arabian and Brazilian trees, did not show these symptoms. Witches’ broom symptoms increase over time, and so some of the trees may have been recently infected and do not show witches’ broom symptoms yet. Moreover, phytoplasma isolates found in Brazilian and Saudi Arabian trees are different from the WBDLp isolates of Oman, UEA, and Iran. A SAP11WBDL homolog was not detected in the Brazilian WBDL-like phytoplasmas (Queiroz et al. 2016). In addition, the Saudi Arabian phytoplasma isolates grouped in different subclades from WBDLp within the 16SrII phytoplasma clade. The Saudi Arabian isolates could be a mixed infection of several 16SrII group phytoplasmas or a phytoplasma that carries genome sections of these groups, as interspecies recombination between phytoplasmas has been reported by Danet et al. (2011). Therefore, the SAP11 effectors in these isolates may not be expressed or regulated in a similar manner as in the other WBDLp isolates that show witches’ brooms.

    SAP11WBDL interacts with lime class-II CYC/TB1 TCP transcription factors, which have a conserved role as negative regulators of stem branching in diverse plants species, including monocots and dicots (Chai et al. 2017; González-Grandío and Cubas 2015; Martín-Trillo and Cubas 2010). SAP11 of diverse phytoplasmas destabilize TCPs (Janik et al. 2017; Mittelberger et al. 2017b; Orlovskis et al. 2017; Sugio et al. 2011a, 2014) and a reduction in class-II transcription factors of the CYC/TB1 TCP clade typically leads to increased branching, as is observed for the witches’ broom symptomatic branches of WBDL-infected acid lime trees. Moreover, the CYC/TB1 TCPs positively regulate chloroplast phytochrome and chlorophyll biosynthesis (Finlayson et al. 2010; González-Grandío et al. 2013; Wang and Wang 2015). Therefore, chlorosis and the downregulation of lime homologs of LOX2 and SID2, which are targeted to the chloroplasts (Bell et al. 1995; Straus et al. 2010), may be caused by the reduction of CYC/TB1 TCP abundance in witches’ brooms of WBDL-infected acid lime trees.

    WBDLp titers are high in witches’ broom symptomatic branches of the tree and these branches were also more attractive to WBDLp leafhopper vectors. WBDLp relies on these insect vectors for transmission and there is no other known path of spread of WBDLp to lime trees. Therefore, it is highly likely that the witches’ broom symptoms promote the spread of WBDL via leafhopper transmission; that is, the insects are more likely to acquire WBDLp from the witches’ broom than from asymptomatic sections of the trees and then transmit the pathogen to other trees. Moreover, progeny of these insects will also become WBDLp vectors (MacLean et al. 2014; Mittelberger et al. 2017a).

    The downregulation of LOX2 promotes egg laying of leafhoppers (Kallenbach et al. 2012; Sugio et al. 2011b). The simultaneous downregulation of SID2 may be counterproductive to the leafhopper because SA negatively interferes with the JA pathway and this provides an advantage for some herbivore insects (Bruessow et al. 2010) and protection of the insects from detrimental effects of bacterial pathogens (Hilfiker et al. 2014). However, given that phytoplasma is a bacterial pathogen, suppression of both the JA and SA defense pathways may be required to promote colonization by leafhopper and phytoplasma, respectively, of the witches’ brooms, and to provide more optimal conditions for WBDL epidemics. Another consideration is that the proliferation of branches and overall vegetation of witches’ brooms leads to an increase in vasculature and phloem sieve cells, which are colonized by phytoplasmas, thereby promoting WBDLp titers in symptomatic witches’ broom tissues. Given that the leafhopper vectors are phloem-feeding insects, this increase in vasculature is suggested to also promote insect feeding and acquisition of the bacteria by the insects. Therefore, various attributes of witches’ broom symptomatic sections of acid lime tree are likely to promote WBDL epidemics. Pruning of symptomatic acid lime branches is likely to be a good strategy in reducing the epidemics of WBDL.


    We thank Dr. Elham Kazerooni, Dr. Claudine Carvalho, Dr. Mohammed Al-Hamadi, and Dr. Khalid AL-Khoudther for their help in the collection of samples from Iran, Brazil, UAE, and Saudi Arabia, respectively, and Dr. Pecher for advice on the Y2H experiments.

    The author(s) declare no conflict of interest.

    Literature Cited

    Current address for Z. Orlovskis’s is Department of Plant Molecular Biology, University of Lausanne, Lausanne CH-1015, Switzerland.

    The author(s) declare no conflict of interest.

    Funding: This work was funded by Sultan Qaboos University under grant Nos. SR/17/01 and EG/12/03; and the Biotechnology and Biological Sciences Research Council under grant Nos. BB/K002848/1, BB/J0045531/1, and BB/P012574/1.