Molecular identification of phytoplasmas in fasciated cacti and succulent species and associated hormonal perturbation

Abstract

Fasciation, a frequent phenomenon in Cactaceae, has been attributed to various causes. The present study reports on phytoplasma-induced fasciation in Euphorbia coerulescens (Euphorbiaceae), Orbea gigantea (Asclepiadaceae), Opuntia cylindrica (Cactaceae), and Senecio stapeliiformis (Asteraceae). DNA was extracted from symptomless and fasciated tissues and amplified by nested PCR using universal primers P1/P7 followed by R16F2n/R16R2 produced amplicons of 1.2 Kb. The nucleotide sequence analyses of the amplicons indicated that fasciated plants were infected by phytoplasma. Phylogenetic analysis placed the cacti fasciation phytoplasmas in 16SrII group. The hormonal content of symptomless and fasciated tissues including indole-3-acetic acid (IAA), kinetin (Kin), N⁶-benzyladenine (BA), abscisic acid (ABA), and gibberellic acid (GA₃) was quantified by high performance liquid chromatography (HPLC). The results indicated that fasciation in O. gigantea was correlated with the accumulation of Kin and IAA increasing five and two times, respectively, as compared to symptomless tissue. However, there was no consistent pattern of hormones in other fasciated species (E. coerulescens, O. cylindrica, and S. stapeliiformis), suggesting that different plant species might have different mechanism to develop fasciation associated with phytoplasma infection.

Keywords:

• cactus
• cristation
• fasciation
• phytoplasma

Introduction

Fasciation, cristation, or abnormal cohesion of organs is a widespread phenomenon that may randomly affect vascular plants both in vitro and in vivo. It occurs in the morphology of plant organs and typically involves broadening of the shoot apical meristem, flattening of the stem, and changes in leaf arrangement (Iliev & Kitin 2011). It has been noted that many biotic and abiotic causes lead to the development of fasciated forms including fungal and nematode infections (Stange et al. 1996) and mineral deficiency; zinc deficiency is known to cause fasciation (Rance et al. 1982), temperature fluctuation (Binggeli 1990), chemical mutagens (Soroka & Lyakh 2009), and mutations (El-Banna et al. 2013). Fasciation is a frequent phenomenon in Cactaceae and in many other succulents’ family like in Euphorbiaceae, Crassulaceae, and Asclepiadaceae. A pure fasciation is also completely different than monstruosity, where axils (areoles) are multiple-activated and the apical meristem loses dominance. All column-like and globular cactican be touched by this anomaly growth form which remains, however, rare. According to origin, fasciations are classified as physiological or genetic, but comparatively little is known on their epigenetic or genetic nature at the molecular level (Iliev & Kitin 2011). Previous studies on fasciation of cacti and succulent plants focused mainly on the causative factors of fasciation (Snyder & Weber 1966; Boke & Ross 1978; Werner 1988; Bourque & Pierson 1998). Recently, tissue anatomy, protein pattern, and RAPD polymorphisms in fasciated cacti and succulent species indicated that occurrence of fasciation is an epigenetic mutation of tissues (El-Banna et al. 2013).

Phytoplasmas are diverse and cell wall-less bacteria associated with several hundreds plant species worldwide (McCoy et al. 1989; Lee et al. 2000). Symptoms of yellowing, witches’ broom, phyllody, virescence, dwarfing, and irregular growth of leaves are associated with several phytoplasma-caused diseases (Lee et al. 2000; Duduk & Bertaccini 2006). Phytoplasma has been reported to infect many plants in Egypt such as Catharanthus roseus (Omar et al. 2008), eggplants, tomato plants, and squash (Omar & Foissac 2012). These infected plants exhibited stunting, leaf yellows, and flower development abnormalities (Omar & Foissac 2012). Phytoplasmas can be detected by PCR assays using specific and universal primers designed on the basis of 16S rRNA gene (Deng & Hiruki 1990; Ahrens & Seemüller 1992; Davis & Lee 1993). The extremely conserved 16S rRNA gene is the most widely used for phylogenetic studies of prokaryotes (Martini et al. 2007). Phylogenetic analysis of 16S rRNA gene sequence revealed the phylogenetic status of phytoplasma and classified them as the class Mollicutes (Lee et al. 2000).

Fasciated mutants are evaluated in plant breeding programs for their ornamental characteristics and are widely cultivated by commercial growers (Krusmann 1995; Van Gelderen & Van Hoey Smith 1997; Dirr 1998). Some fasciations are perceived as real living sculptures and are sought after by horticulturists and collectors. Although, fasciated plants are very attractive when potted, and on the one hand, have ornamental values (Vallicelli 2010), on the other hand, they can be phytoplasma-host and cause infection to many food crops (Omar et al. 2008). The objective of this study was to detect and categorize the phytoplasma in Euphorbia coerulescens (Euphorbiaceae), Orbea (Stapelia) gigantea (Asclepiadaceae, Leach 1975), Opuntia cylindrica (Cactaceae), and Senecio stapeliiformi (Asteraceae), using 16S rRNA gene sequence. We also determined different hormones content in symptomless versus fasciated tissues in order to improve the understanding of the physiological mechanism of fasciation associated with phytoplasma.

Materials and methods

Plant materials

Fasciated and symptomless stem segments from the same plant of E. coerulescens, O. gigantea, O. cylindrica, and S. stapeliiformis were used in this study (Figure 1). The samples were kept at −80°C for further studies. The plants were taken from a local nursery located in El-Gharbia governorate and grown in 20-cm-diameter clay pots containing a mixture of clay:sand (1:1, v/v). Plants were irrigated once a week during the summer season and once a month during the winter season. The plants were grown under 50% shade net and a compound fertilizer (19:19:19; N:P₂O₅:K₂O) at 1 g L⁻¹ was applied once a month.

Figure 1. Morphological features of fasciated (left-side) versus symptomless (right-side) stems of E. coerulescens (A), O. cylindrica (B), O. gigantea (C), and S. stapelliformis (D).

DNA extraction

Total DNA of four cacti plants (E. coerulescens, O. gigantea, O. cylindrica, and S. stapeliiformi) having fasciation symptoms and other four asymptomatic were extracted from 1 g of fresh phloem-rich tissue areas of cladodes, which initially were mashed in liquid nitrogen with a mortar and pestle, using i-genomic plant DNA extraction Mini Kit (iNtRON Biotechnology, Inc., Cat. No. 17371) according to the manufacturer's instructions.

Condition, amplification of PCR, and 16S rDNA gene

A 1.8 kb product covering the partial sequence of the 16S rDNA gene, the intergenic spacer region, and the 5′-end region of the 23S rRNA gene regions of the phytoplasma genome was amplified using the universal primer pair P1/P7 (Deng & Hiruki 1991; Schneider et al. 1995). The first amplification were diluted with sterile distilled water (1:10), and 1 µl of each dilution was used as DNA template for amplification by nested PCR using primer pair R16F2n (5′-GAA ACG ACT GCT AAG ACT GG-3′) and R16R2 (5′-TGA CGG GCG GTG TGT ACA AAC CCC G-3′), which was used for amplification of the 1200 bp fragment of the phytoplasma16S rDNA (Lee et al. 1993). PCR was performed for 34 cycles, which were conducted in an automated thermal cycler (C1000^TM Thermal Cycler, Bio-RAD).The following parameters were used: the primary denaturation step at 94°C for 3 min, 34 cycles with denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min, followed by a final extension step at 72°C for 7 min. Each PCR mixture (25 µl) was as follows: (1 µl) of 25 ng nucleic acid, 1 µl of each primer (10 pmol), (12.5 µl) of GoTag®Colorless Master Mix (Promega Corporation, USA), and 9.5 µl of Nuclease free water (Promega). Fifteen microliter of all PCR products was analyzed by electrophoresis through a 1% agarose gel, stained with ethidium bromide, and DNA bands were visualized using an ultraviolet transilluminator.

The nested PCR products of four cacti samples were sent for sequencing directly. The sequence was in two directions with primers R16F2 and R16R2 in a sequenciator ABI. ISM 3700 (Applied Biosystems, USA). Raw sequence chromatograms were assembled and edited using GAP4 (Bonfield et al. 1995) to correct ambiguous bases or remove low-quality stretches from the termini of the sequences. Similarities to known sequences were detected using the BLASTN algorithms (Altschul et al. 1997) against the GenBank database at http://www.ncbi.nlm.nih.gov/blast. Multiple alignments were performed using ClustalW (Thompson et al. 1994), and the phylogenetic analyses were conducted with MEGA4 using the neighbor-joining method (Tamura et al. 2007). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al. 2004). Partial 16SrDNA gene sequences of four phytoplasma strains obtained from E. coerulescens fasciation phytoplasma, O. gigantean fasciation phytoplasma, O. cylindrica fasciation phytoplasma, and S. stapeliiformis fasciation phytoplasma have been deposited in the EMBL/GenBank/DDBJ databases under accession numbers, HG421070, HG421073, HG421071, and HG4210702, respectively. The 16S rDNA gene sequences of different groups of phytoplasmas used for comparisons were retrieved from GenBank (www.ncbi.nlm.nih.gov).

HPLC hormones estimation

The analysis of hormones was performed by high performance liquid chromatography (HPLC) according to Shindy and Smith (1975) with modifications. In different samples, 5 g fresh weight was ground and soaked in 80% (v/v) aqueous methanol and allowed to extract for 72 h. Then they were filtered through Whatman no. 42 paper. The filter paper and the residue were returned to the flask with a fresh volume of methanol and filtered again. The procedure was repeated once more, and the combined extracts were evaporated to the aqueous phase using a rotary evaporator. The aqueous phase was partitioned into two with equal volume (15 ml each). One partition was adjusted to pH 8.6 with 1% NaOH and extracted thrice with ethyl acetate. The combined ethyl acetate fraction was evaporated to dryness, dissolved in 1 ml of HPLC methanol, and used for quantification of basic hormones such as BA and Kin at 269 nm. The other partition of aqueous phase was adjusted to pH 2.8 with 1% HCl, extracted thrice with ethyl acetate. It was evaporated to dryness, dissolved in 1 ml of HPLC methanol, and used for quantification of acidic hormones such as IAA, ABA, and GA₃ at 254 nm. The extract was filtered through a membrane filter (0.45 µm) before injecting (10 µl) into a HPLC system (Waters 746, HPLC 510 pump; Tunable absorbance detector, Waters Associates, Millford, MA, USA) equipped with µ bonda pack C18 300 mm × 3.9 mm column. A mobile phase consisting of methanol HPLC containing 2% of glacial acetic acid was used at a flow rate of 1.0 ml min⁻¹. The concentrations of hormones in sample were computed from the response ratio of target compound and the appropriate internal Sigma standard (IAA – 12273; GA3 – 48880; ABA – A1049; Kin – 48130; BA – 13151).

Statistical analysis

Data were presented as means ± SE. Mean separation was by Duncan's multiple range test (DMRT) at 5% level.

Results and discussion

Morphological features

Morphological differences were clearly visible in fasciated plants compared with symptomless plants (Figure 1). The symptomless form of E. coerulescens had regular shape, while the cristated form had irregular shapes. The symptomless form of O. cylindrica was regular and has a cylindrical shape, but the fasciated form has spherical and irregular shape. Dense and small spines were found in the fasciated form compared to the symptomless form. Symptomless form of O. gigantea was elongated and has sharp spines, while fasciated forms had curled spherical shape with protrusions. The symptomless form of S. stapeliiformis was elongated and had polygonal shape, while the fasciated form was flat. Fasciation is typically characterized by the development of a flattened organs and plant parts, most commonly on stem and inflorescences. CLAVATA1 mutant of Arabidopsis plants have enlarged apical vegetative and floral meristems, leading to fasciation, altered phylotaxis, and extra floral organs and whorls (Clark et al. 1993). Similarly, fasciation in pea is characterized by abnormal enlargement of the stem apical meristem leading to distortions in shoot structure (Sinjushin & Gostimsky 2006, 2008). Fasciated shoots of Betula pendula induced in vitro as well as Prunus avium and Fraxinus excelsior formed flattened stems with densely arranged lanceolate leaves that were dramatically increased in size (Kitin et al. 2005; Mitras et al. 2009; Iliev & Kitin 2011).

The fasciation is thought to be genetically determined in dicot species (Karakaya et al. 2002), while in Lilium henryi, it was associated with the presence of Rotylenchus and ditylenchus nematode species (Stumm-Tegethoff & Linskens 1985). In Arabidopsis plant, two genes have been identified to cause fasciation (Reboredo & Silvares 2007). Also, fasciation has been experimentally produced by X-rays and chemical mutagens (Reboredo & Silvares 2007).

DNA amplification

The PCR amplification using primer pair P1/P7 specific of phytoplasma 16S rRNA from four symptomatic cacti plants (E. coerulescens, O. gigantea, O. cylindrica, and S. stapeliiformi) did not show any bands as the first PCR (data not shown). The same primers failed to produce any PCR products when the DNA from the four symptomless plants was used as control. The nested PCR amplification using primer pair R16F2n/R16R2 generated DNA fragments of 1.2 kb of the four strains. They were observed by electrophoresis and corresponded to the amplification of the 16S rRNA gene according with the DNA positive control. No band was observed in the negative control (data not shown).

Sequences and phylogenetic analysis

Four 16S rDNA (R16F2n/ R16R2 nested PCR) products were sequenced and compared with other 16S rDNA of groups and subgroups reported in GenBank as well as each other. The four strains were given the following names: E. coerulescens fasciation phytoplasma (EcFP), O. gigantea fasciation phytoplasma (OgFP), O. cylindrica fasciation phytoplasma (OcFP), and S. stapeliiformis fasciation phytoplasma (SsFP). The results revealed that the four cacti isolates are not identical even though they shared identities that ranged from 99.4 to 99.9, and they also shared high identities from 99.5 to 99.9 with Papaya yellow crinkle phytoplasma (White et al. 1998), eggplant phyllody phytoplasma (Omar & Foissac 2012) – which was previously classified as 16SrII-D subgroup – and Peanut witches-broom phytoplasma of 16SrII-A (Gundersen et al. 1994), and more than 99.2% with Euphorbia heterophylla phytoplasma (not classified); however, they have identities that ranged from 98.6 to 99.4 with Cactus witches-broom phytoplasma different subgroups according to Cai et al. (2008). Lower identities of 94.1–95.3 were with the Euphorbia dendroides fasciation phytoplasma, Euphorbia linifolia fasciation phytoplasma, and Euphorbia pulcherrima phytoplasma; hence, they belong to different 16Sr subgroups. The phylogenetic analysis of the four strains of phytoplasma showed that they clustered together with 16Sr II subgroup members. E. coerulescens fasciation phytoplasma isolate (EcFP), S. stapeliiformis fasciation phytoplasma isolate (SsFP), and O. cylindrica fasciation phytoplasma isolate (OcFP) were more closely related to Papaya yellow crinkle phytoplasma and Eggplant phyllody phytoplasma, while O. gigantea fasciation phytoplasma isolate (OgFP) stood out of this subclade (Figure 2). Phytoplasmas belonging to Peanut witches’ broom group (16SrII) and Aster yellows group (16SrI) were detected in Opuntia species showing witches’ broom symptoms (Wei et al. 2007; Cai et al. 2008). Phytoplasma representing different groups have been reported to be associated with stem fasciation in different plant species in Lilium martagon (16SrI-C) by Bertaccini et al. (2005), while in Petunia spp., the phytoplasma belongs to the AY group (Chung & Huh 2008); in Senna surattensis, it belongs to 16SrXII and 16SrV (Wu et al. 2012), in Cichorium intybus, it belongs to 16SrV-B (Li et al. 2012); in Crotalaria spectabilis, it belongs to the 16SrI-B subgroup (Kumar et al. 2010); and in Asparagus officinalis (16SrI-B) in Czech Republic (Fránová & Petrzik 2010). The 16SrII-D phytoplasmas are considered to be a serious problem in many crops (chickpea, sesame, mungbean, faba bean, tomato, eggplant, squash, papaya, and pale purple coneflower), in many countries, causing phyllody and virescence symptoms (Akhtar et al. 2010; Alfaro-Fernández et al. 2012; Omar & Foissac 2012; Pallavi et al. 2012) and stem fasciation in Linum usitatissimum (16SrII-D) in Pakistan (Akhtar et al. 2013).

Figure 2. The phylogenetic tree constructed using the neighbor-joining method, showing the phylogenetic relationships for the partial 16S-rDNA nucleotide sequences for four Egyptian (E. coerulescens, O. gigantea, O. cylindrica, and S. stapeliiformis) fasciation phytoplasma (marked in red), compared with representative from other 16S-rDNA phytoplasma groups. GenBank accession numbers for the published sequences are shown beside the name of isolates. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. Phylogenetic analyses were conducted in MEGA4.

Hormonal content in symptomless versus fasciated tissues

Hormonal content of symptomless and fasciated tissues of E. coerulescens, O. cylindrica, O. gigantea, and S. stapiliiformis is shown in Figure 3. Fasciated tissues of E. coerulescens had lower content of BA, Kin, IAA, GA₃, and ABA than that of symptomless tissues. In O. gigantea, Kin and IAA contents were significantly increased in fasciated tissues as compared to symptomless tissue. However, fasciated tissues had low content of GA₃ and ABA as compared to symptomless tissue. BA content showed no significant difference between fasciated and symptomless tissues. Phytoplasma-associated disorders are classified among the auxonic diseases of plants. The phytoplasma symptoms such as growth aberrations indicate perturbations in plant hormonal balance. It has been reported that in phytoplasma-infected micropropagated periwinkle shoots, the level of IAA increased 10 times compared with the control (Pertot et al. 1998). The development of green flowers, a symptom of phytoplasma diseases, was correlated with the accumulation of high levels of cytokinin in the floral parts (Davey et al. 1981). Nicolaison and Horvath (2008) studied gene expression in E. pulcherrima infected with a branch-inducing phytoplasma. They identified four differentially expressed genes after phytoplasma infection. Three of them (histidine-containing phosphotransmitter, an α-expansin, and a protein with similarity to the gibberellin-regulated GAST-1) showing high homology with genes involved in the biosynthesis or the perception of plant hormones or other growth regulators, such as cytokinin and gibberellins, were shown to be upregulated.

Figure 3. Contents of BA, Kin, IAA, GA₃, and ABA in symptomless versus fasciated tissues of E. coerulescens, O. cylindrica, O. gigantea, and S. stapelliiformis. Values are means ± SE (n = 3). Different letters above bars indicate significant differences (P < 0.05) among means.

The circumferences of fasciated tissues are several times greater than those of the symptomless ones due to increases in rates of cell growth and cell division (Iliev & Kitin 2011). These fasciation characteristics indicate perturbations in plant hormonal balance. There is evidence for a hormonal basis in pathological fasciation caused by Rhodococcus fascians, which has a plasmid-borne virulence gene fas-1 encoding an isopentenyl transferase that produces cytokinin (Crespi et al. 1994; Stange et al. 1996). Changes in hormone metabolism also correlated with fasciation in hybrid Populus transformed with the Agrobacterium rhizogenes rolC gene, and fasciated tissues had a high level of free cytokinins (Nilsson et al. 1996). It has also been shown that plants of Helianthus annuus with fasciated stems (STF) had higher endogenous free IAA levels (Fambrini et al. 2006). The observed phenotype and the higher levels of auxin detected suggest that the STF gene is necessary for the proper initiation of primordia and for the establishment of a phyllotactic pattern through control of both shoot apical meristem arrangement and hormonal homeostasis (Fambrini et al. 2006). Furthermore, the increased hormonal levels point to a possible hormonal control mechanism of the development of fasciated shoot apical meristem phenotypes. A possible genetic mechanism which operates through a hormonal imbalance restricted to the meristem and its immediate vicinity in fasciated stems has been suggested (Boke & Ross 1978). Previous reports demonstrated that exogenously applied cytokinins induced fasciation in Kalanchoe blossfeldiana (Varga et al. 1988), B. pendula (Iliev 1996), Mammillaria elongata (Papafotiou et al. 2001), and P. avium (Kitin et al. 2005). Also, it has been reported that fasciation in cotton is conditioned by GA₃ and genotype (Nadjimov et al. 1999).

Conclusion

The present study confirmed the presence of four new phytoplasma strains in cacti and succulent species. Phytoplasmas have been identified as members of 16SrII group. Our results also indicated that fasciation in O. gigantea was correlated with the accumulation of Kin and IAA which increased five and two times, respectively, as compared to symptomless tissue. However, there was no consistent pattern of hormones in other fasciated species suggesting that different plant species might have different mechanism to develop fasciation associated with phytoplasma infection.