Association of ‘Candidatus Phytoplasma cynodontis’ with Bermuda grass white leaf disease and its new hosts in Qassim province, Saudi Arabia

ABSTRACT Typical symptoms of phytoplasma such as whitening of the leaves, shortening of the stolons on Bermuda grass, variegated leaves, yellows, stunting, little leaves and yellows on Giant reed, Cooba and sand olive shrub were observed in Qassim province, Saudi Arabia, during the autumn season of 2015. When tested for phytoplasma by universal primers P1/P7 followed by R16mF2/R16mR2, products of approximately 1400 bp (as expected) were amplified from 16 plants with symptoms but not from symptomless plants. Based on sequencing, phylogenetic analysis and virtual restriction fragment length polymorphism patterns of the 16S rDNA F2nR2 fragments of seven Qassim phytoplasma isolates, bermuda grass isolates 170, 175 and 177, giant reed isolate 180, sand olive isolates 181 and 182 and cooba isolate 185, the associated phytoplasma was identified as a member of ‘Candidatus Phytoplasma cynodontis’ which belong to the 16SrXIV-A subgroup. The 16S rDNA gene sequences of seven Qassim phytoplasma isolates exhibited over 99.2% identity with members of ‘Ca. Phytoplasma cynodontis’ group of phytoplasmas. This is the first report of characterization of ‘Ca. phytoplasma cynodonties’ (16SrXIV) associated with Cynodon dactylon in Saudi Arabia and its new hosts, Dodonaea angustifolia, Arundo donax and Acacia salicia.


Introduction
Bermuda grass (Cynodon dactylon), which is native to Africa, is normally grown as a turf grass or as forage for livestock, can be an invasive weed. It is considered to be the most widely used turf grasses in tropics and subtropics on athletic fields and golf course fairways. Bermuda grasses establish rapidly and spread by vegetative propagules by both stolons and rhizomes (Brosnan & Deputy 2008). Bermuda grass white leaf (BGWL), first described in Taiwan, is a destructive phytoplasmal disease of Bermuda grass caused by the phytoplasma group (16SrXIV) and characterized by whitening of the leaves and shortening of the stolons (Chen et al. 1972;Obura et al. 2010). BGWL disease is reported in many countries including Singapore (Koh et al. 2008), Italy (Marcone & Ragozzino 1997), Iran (Salehi et al. 2009), Cuba (Arocha et al. 2005), Australia Tran-Nguyen et al. 2000), Turkey (Çağlar et al. 2013), Sudan (Dafalla & Cousin 1988), Thailand (Sunpapao 2014), India (Rao et al. 2007;Snehi et al. 2008;Kumar et al. 2015), Malaysia (Nejat et al. 2009;Naderali et al. 2013), Myanmar (Win et al. 2013), Kenya, Tanzania and Uganda (Asudi et al. 2015), and Serbia and Albania (Mitrović et al. 2015). Phytoplasmas, wall-less prokaryotes, classified in the Class Mollicutes, Order Acholeplasmatales and genus Candidatus are uncultivable (Xiaodong et al. 2006). They infect a wide variety of plants such as fruit crops, timber, vegetables, grasses and other ornamental crops by inhabiting the phloem tissue resulting in economically significant epidemics throughout the world (Bekele et al. 2011). Highly conserved 16SrRNA based characterization is applied to identify and classify phytoplasmas. Since the concentration of phytoplasma in phloem tissue of some plants is very low, nested polymerase chain reaction (PCR) is used for amplifying DNA fragments from the first round amplification using internal primers (Gundersen & Lee 1996;Heinrich et al. 2001). Though, phytoplasma infection in Bermuda grass has been previously described (Marcone & Ragozzino 1997;Çağlar et al. 2013;Naderali et al. 2013;Win et al. 2013;Khanna et al. 2015;Mitrović et al. 2015) the information on Bermuda grass infection from Saudi Arabia was not available. However, typical phytoplasmal symptoms were observed in Qassim region of the country. Therefore, a study was carried out to characterize the phytoplasma infecting Bermuda grass in Qassim region using 16S ribosomal RNA.

Samples collection
During the autumn season of 2015, nine samples of Bermuda grass (C. dactylon) showing whitening of the leaves and shortening of the stolons were collected form three different places from Qassim region (University campus, Al Safra and Mulayda); three samples of sand olive shrub (Dodonaea angustifolia) with stunting, little leaves and yellows; two samples of small evergreen tree, cooba (Acacia salicia) with yellows and two samples of Giant reed (Arundo donax) displaying variegated leaves were collected from Faculty of Agriculture and Veterinary Medicine farm. All samples were stored at 4°C until processed for DNA extraction. Asymptomatic samples of the same species (C. dactylon, D. angustifolia, A. salicia and A. donax) were also collected for use as negative controls.

DNA extraction
Total nucleic acids were extracted from fresh leaves of all symptomatic and asymptomatic samples. Hundred milligram of each sample were powdered in liquid nitrogen and transferred to a 1.5-ml Eppendorf tube for subsequent processing of DNA extraction using i-genomic plant DNA extraction Mini Kit (iNtRON Biotechnology Inc., Cat. No. 17371, Korea). The DNA was eluted in 100 μl of elution buffer and were kept at −20°C to use as DNA template in PCR assays.

Amplification of phytoplasma 16S rDNA gene fragments
The universal phytoplasma primer pair P1/P7 in first round (Deng & Hiruki 1991;Schneider et al. 1995) were used to amplification of 1.8 kbp DNA fragment comprising the nearly full length 16S rDNA. The pair primers, R16mF2 (5`-CATG-CAAGTCGAACGGA-3`) and R16mR2 (5`-CTTAACCC-CAATCATCGA-3`) were used in second round nested PCR assays which amplify an internal DNA fragment of 1400 bp from the 16S rDNA gene (Baric & Dalla Via 2004). PCR assay was performed in a thermal cycler (SwiftTM MaxPro Thermal Cycler, ESCO healthcare). The PCR reaction mixture (20 μl in the first PCR round) contained 1 µl (50 ng) nucleic acid as template, 1 µl of each primer (10 pmol), (4 µl) of 5× FIREPol ® Master Mix (Solis BioDyne, Estonia) and 13 µl of Nuclease free water (Promega, USA), and the volume of 40 µl was used in the second round of PCR with primers R16mF2/R16mR2 with double the amount of the 5× FIREPol ® Master Mix (Solis BioDyne, Estonia), 1 µl of each primer   (10 pmol), 1 µl of the primary PCR product and 29 µl of Nuclease free water (Promega). The PCR program with P1/ P7 primers was followed with initial desaturation at 94°C for 3 min, 34 cycles with denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min and followed by a final extension step at 72°C for 7 min. The same PCR program was used in the second round with primers R16mF2/ R16mR2 except the annealing temperature of 46°C. The amplified PCR products were analyzed by electrophoresis through a 1.5% agarose gel, stained with ethidium bromide, and DNA bands were visualized using a UV transilluminator (G:BOX F3 system, Syngene).

Sequencing PCR products and phylogenetic analysis
Seven internal amplified fragments of 16Sr DNA by the primer pairs R16mF2/R16mR2 were sequenced in both orientations (Macrogen Inc., Korea). The sequences obtained from Qassim 'Candidatus Phytoplasma cynodontis ' isolates, 170, 175, 177, 180, 181, 182 and 185 were deposited in the GenBank databases under accession numbers, LT220876, LT220879, LT220880, LT220881, LT220882, LT220883 and LT220884, respectively. The obtained sequences were gathered and edited using GAP4 program (Bonfield et al. 1995). Multiple sequence alignments and nucleotide sequence similarity were carried out using Clus-talW (Thompson et al. 1994). The 16S rDNA gene sequences of seven Qassim phytoplasmas isolated from Bermuda grass (isolate 170, 175 and 177), giant reed (isolate 180), sand olive (isolates 181 and 182) and cooba (isolate 185) plants in this study were compiled in FASTA format and compared with each other and with 11 other reference phytoplasmas which belong to different 16S rDNA subgroups and with 12 'Ca. phytoplasma cynodontis' strains reported from different countries using Clus-talW (Thompson et al. 1994). Phylogenetic tree was constructed using the neighbor-joining phylogenetic method implemented in MEGA4 program (Tamura et al. 2007) with 1000 bootstrap replications. Acholeplasma laidlawii was used as outgroup.

PCR amplification
PCR was carried out using P1/P7 primers in the first round followed by nested PCR with R16mF2 and R16mR2 primers in the second PCR round. All tested symptomatic samples of C. dactylon, A. donax, A. salicia and D. angustifolia gave DNA fragments of approximately 1400 bp. No amplification was obtained from symptomless plants ( Figure 5).

Virtual restriction enzyme digestions
Virtual restriction fragment length polymorphism (RFLP) patterns of the 16S rDNA F2nR2 fragments of seven phytoplasma Qassim isolates, three C. dactylon isolates (170, 175 and 177), two isolates of D. angustifolia (181 and 182), one isolate of both A. donax and A. salicia (180 and 185) using iPhyClassifier (Zhao et al. 2009) revealed that pattern similar to that of 'Ca. Phytoplasma cynodontis' which belongs to phytoplasma 16SrVIX group and subgroup A (GenBank accession: AJ550984) with a similarity coefficient of 0.97 ( Figure 6). BGWL phytoplasma was previously identified as 16SrXIV-A subgroup based on virtual RFLP in India (Khanna et al. 2015). Additionally, the virtual RFLP analysis was used successfully to (i) reveal the genetic diversity among other phytoplasmas such as cactus witches' broom phytoplasma strains infecting Opuntia species in China (Cai et al. 2008), (ii) grouped potato purple top phytoplasma strains to four different phytoplasma groups (16SrI, 16SrII, 16SrIII and 16SrXIII) in Mexico (Santos-Cervantes et al. 2010), (iii) identified the Indian arecanut palm yellow leaf disease phytoplasma as a member of 16SrXI-B subgroup (Ramaswamy et al. 2013) and (iv) classified Iranian cucumber phyllody (CuP) and squash phyllody (SqP) phytoplasmas to different phytoplasma subgroups 16SrII-M and 16SrII-D, respectively (Salehi et al. 2015).

Nucleotide sequence identities and phylogenetic analysis
Sequence analysis conducted on seven Qassim phytoplasma isolates, which compared to other 33 phytoplasma strains from different countries revealed that all Qassim phytoplasma isolates have identities ranging from 99% to 99.8% with each other and they are closely related to 'Ca. Phytoplasma cynodontis' (16S XV-A subgroup) isolates reported from Italy, Albania and Myanmar with high similarities from 99.2% to 99.7% (Table 1). Also, they shared identities of more than 98.9% with three Serbian and Chinese isolates (KJ000024, KF383981, KP019339 and EU377477). However, the lowest identities of 98.1% were seen with two Indian isolates (EU032485 and GQ403690) ( Table 1). The phylogenetic tree confirmed the result obtained from virtual RFLP (Figure 7). Therefore, all Qassim phytoplasma isolates and other 'Ca. Phytoplasma cynodontis' isolates were placed in one clade and were separated from 'Ca. Phytoplasma phytoplasmas' members with bootstrap of 100%. The 'Ca. Phytoplasma cynodontis' clade was regionally divided into four subclades (A, B, C and D). Hence, where the seven Qassim phytoplasma isolates were clustered together in subclade A with bootstrap 95%, the Italian and Albanian isolates formed a subclade B. Further, isolates from Myanmar, China and India were found in subclade C, whereas Serbian isolates were grouped in subclade D. The results presented in this study are in concurrence with previous work which showed that the BGWL phytoplasmas from different geographical regions are identical (Marcone & Ragozzino 1997;Wongkaew et al. 1997;Lee et al. 1998;Tran-Nguyen et al. 2000). As the 16S rDNA sequence identity is greater than 97.5% among Qassim isolates and other members of 'Ca. Phytoplasma cynodontis' (16SrXIV) which is enough for defining the status of novel 'Candidatius' phytoplasma species, the Qassim phytoplasma isolates associated with C. dactylon, D. angustifolia, A. donax and A. salicia plants should be considered as members of 'Ca. Phytoplasma cynodontis' group. However, another prospective analysis based on RFLP and other molecular markers are needed for better differentiation among Qassim phytoplasma isolates to confirm their taxonomic position. To the best of our knowledge, this is the first report of the characterization of 'Ca. Phytoplasma cynodonties' (16SrVIX) associated with C. dactylon, two D. angustifolia, A. donax and A. salicia plants in Saudi Arabia.