Candidatus phytoplasma malaysianum (16SrXXXII) associated with Elaeocarpus sylvestris decline in South Korea

Abstract Phytoplasma has been newly detected in Elaeocarpus sylvestris, a tree species that grows on the island of Jeju, South Korea. Various symptoms of infection of E. sylvestris have appeared since 2013, including loss of leaf coloration, such as yellowing, darkening and branch dieback. We analyzed leaves and seeds of E. sylvestris sequences infected by phytoplasma, and found partial 16S rRNA sequences to be 99.5%, and secA genes 98.2% correlated with Malaysian periwinkle virescence group strain ELY-BN1, MaPV, MOP, MYD, and TtWB. Analyzing these sequences and through the virtual restriction fragment length polymorphism (virtual-RFLP), we report here the first occurrence of Elaeocarpus decline associated with a phytoplasma strain belonging to 16SXXXII groups, as well as the first detection of phytoplasma in the seeds of E. sylvestris in South Korea.


Introduction
Phytoplasmas are wall-less pleomorphic prokaryotes that were previously classified as mycoplasma-like organisms (MLOs) on account of their morphological similarity to mycoplasmas, first occurred in mulberry phloem with dwarf disease (Doi et al. 1967;McCoy et al. 1989;Bertaccini and Duduk 2009;Weintraub and Jones 2009;Pagliari and Musetti 2019). Phytoplasma chromosome size is about 600-880 kb, which is very small in different from other plant pathogens (Oshima et al. 2013).
Phytoplasma are pathogens that is the cause of over 1,000 diseases in different wild and cultivated plant species all over the world (Seemu€ uller et al. 2002;Bertaccini 2007;Hoshi et al. 2007;Kumari et al. 2019;Namba 2019). In South Korea, such diseases have included jujube witches' broom (Kim 1968), paulownia witches' broom (La 1968), mulberry dwarf (Chang and Kim 1971), and sumac witches' broom (Kim 1980) causing significant economic losses from a lot of trees affected throughout the country (Jung et al. 2012). Currently, including crops, about 64 species of host plants infected with phytoplasma have been reported in Korea. (Chung et al. 2011;Jung et al. 2012;Lee et al. 2017). Plants infected with phytoplasma also show many symptoms, including witches' broom, yellowing, phyllody, virescence, shortened internodes, and little leaf, as well as declining and vascular discoloration (Bertaccini and Duduk 2009;Kumari et al. 2019;Dermastia 2019). Meanwhile, with the development of molecular biology, PCR technique and sequence analysis on 16S ribosomal DNA, is in commonly used molecular diversity and taxonomy classification on phytoplasma (Lee et al. 1998;Bertaccini and Duduk 2009).
Elaeocarpus sylvestris var. ellipticus (Thunb.) H. Hara is an evergreen tree belonging to Elaeocarpaceae, grown in southern China, south central Japan, Taiwan, Vietnam, and on Jeju Island in South Korea and it reproduces mostly by seed germination (Kim and Kim 2014). However since 2013, E. sylvestris trees on Jeju have shown decline, while some trees have died on streets, in parks or in their natural habitat (Lee et al. 2017). Since the 1980s, similar disease have also been reported in Japan named ELY (Elaeocarpus yellows), and E. zollingeri trees have been infected with phytoplasma and are in danger of decimation (Ikeda and Hashimoto 1996;Narazaki and Tsuda 2008;Kawabe 2010;Odawar City Cultural Property Protection Committee 2013;Kawabe et al. 2011;Satoh et al. 2014;Iwabuchi et al. 2018).
Until the present study, no research on the decline of E. sylvestris trees from phytoplasma has been conducted in Korea to date, even though the number of areas with damaged trees is increasing every year. The purpose of this study is to identify the cause of the decline in E. sylvestris trees using PCR assays, base sequences, and virtual-RFLP. In addition, we will determine whether the phytoplasma is found in the seeds of tree with decline symptoms.

Diseased samples and extraction of DNA
Our investigation was conducted on E. sylvestris in Jeju island, we were collected 14 symptomatic samples which yellowing, darkening leaves and seeds near symptomatic leaves between April and September 2019 (Table 1). Samples were stored at 4 C, while to identify phytoplasma infection, DNA was extracted from 0.1 g of leaf midribs, petioles, and separating the flesh and seeds of the drupe, using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany).
Amplified PCR products were purified using a PCR Purification Kit (Bioneer, Seoul, South Korea) and clone used to transform Escherichia coli JM109 cells according to manufacturer's instructions (Qiagen, Valencia, CA, USA). The sequencing were performed by Macrogen Co. (Daejeon, korea). Sequence alignments were analyzed using GENETYX-WIN 4.0 (GENETYX, Tokoyo, Japan), and phylogenetic analysis was performed with MEGA ver. 10.0 software using the Neighbor-joining method, with the default values and 10,000 replications for bootstrap analysis.

Symptomatology
The most of diseased E. sylvestris trees were observed in various locations including streets, parks, around the apartment and natural habitats on Jeju. In some street trees, symptomatic trees appeared to decline symptoms collectively, more than 70% trees showed that decline symptom from April to October. Early symptoms were as the previous year's leaves began yellowing and darkening. A number of leaves fall sharply, the leafless branches becoming weak. After years of repetition of these symptoms the tree eventually dries up and dies over time (Figure 1).

Detection of phytoplasma and molecular diversity
Polymerase chain reaction with phytoplasma universal primers (P1/P7) amplified the expected DNA fragment of 1.8 kb and targeted 16S rRNA, The 16S-23S ITS region, and partial 23S rRNA. Nested PCR with R16F2n/R16R2 primers resulted in the predicted 1.2 kb amplicon in leaves, both flesh and seeds detected with phytoplasma ( Figure 2). The phytoplasma universal primers (SecAfor1/SecArev3) also amplified the expected size of 0.84 kb and targeted the partial secA gene (Figure 2). The nucleotide sequences of the 16S rRNA and secA genes of infected leaves and seeds on   Jeju showed a 100.0% identity starins ESDP-JJ1, JJ2, JJ3, JS1, and JS2. The pairwise alignment of the 16S rRNA gene sequence with available sequences in the NCBI database showed that the phytoplasma was more than 99.5% correlated with Malaysian periwinkle virescence group strain ELY-BN1, MaPV, MOP, MYD, and TtWB ( Figure 4, Table 2). The 16S rRNA and 16/23S ITS sequences of E. sylvestris decline phytoplasma  (ESDP) were deposited in GenBank accession numbers: OL689203, OL689204, OL689205, OL689206 and OL689207. The 16SrRNA phytoplasma sequences were insufficient for identification of phytoplasmas belonging to the same group. Pairwise alignment of the secA gene sequence with sequences obtained from the NCBI database revealed that the phytoplasma was more than 98.2% associated with Malaysian periwinkle virescence group ( Figure 5). The secA gene sequence of E. sylvestris decline phytoplasma (ESDP) were deposited in GenBank accession numbers: OL689198, OL689199, OL689200, OL689201 and OL689202. The virtual-RFLP patterns derived from the query 16S rDNA gene (F2nR2 fragment) of strain Elaeocarpus sylvestris decline phytoplasma (ESDP) is clearly different RFLP band pattern reported Malaysian periwinkle virescence (MaPV), Malayan Oil Palm (MOP), Malayan Yellow Dwarf (MYD) (Figure 3).

Discussion
The results of the pairwise alignment of the 16SrRNA and the secA gene sequence revealed that the phytoplasma in each was 99.5%, 98.2% linked to others with Candidatus phytoplasma malaysianum (16SrXXXII). In the virtual-RFLP results, it was different from the previously reported 16SrXXXII-A (MaPV), 16SrXXXII-B (MYD), 16SrXXXII-C (MOP) group (Nejat et al. 2013).
Among the 17 restriction enzymes, in BstUI enzyme case, 16S rDNA gene of strain ESDP were distinguished from 16SrXXXII-A (MaPV), 16SrXXXII-B (MYD), 16SrXXXII-C (MOP), and restriction enzyme BfaI can be distinguish 16SrXXXII-C (MYD). In addition, HhaI enzyme also distinguish 16SrXXXII-A (MaPV), 16SrXXXII-B (MOP), the other enzymes showed the same patterns. As a result of this, we propose that EDSP strain a new subgroup within the 16SrXXII group. Associated with Malaysian periwinkle virescence group (16SrXXXII) Strains including Malaysian periwinkle virescence (MaPV), Malaysian Oil Palm (MOP), Malayan Yellow Dwarf (MYD), Camptotheca acuminate witches'-broom (CAWB), Trema tomentosa witches'-broom (TtWB), and Elaeocarpus zollingeri yellows (ELY) (Nejat et al. 2009(Nejat et al. , 2013Iwabuchi et al. 2018;Yu et al. 2020), among these, E. zollingeri yellows (ELY) closely hosts related and symptoms are similar to E. sylvestris decline in Korea. PCR-RFLP and virtual RFLP analysis also showed that the korean and Japanese diseased Elaeocarpus displayed a band pattern that was similar to the analysis results with the restriction enzymes RsaI, HpaII, and MseI (Satoh et al. 2014). However, using the AluI enzyme, it was showed that different band pattern from E. sylvestris decline in Korea. Japanese studies of E. zollingeri yellows (ELY) reported phytoplasma infection by types other than Ca. phytoplasma malaysianum, including Ca. phytoplasma asteris (Kawabe et al. 2001. In our studies, however, only Ca. phytoplasma malaysianum was detected. Further research is needed on this. In Japan, insect vectors of ELY has not yet to be determined (Satoh et al. 2014;Iwabuchi et al. 2018). Likewise, it is still unknown whether Leafhoppers carry phytoplasma disease in Korea. Preventing the spread of phytoplasma disease requires identifying the insect hosts associated with it.
As for the reports about phytoplasma transmission in seeds, including apricot, peach, corn, and carrot (Necas et al. 2008;Ça glar et al. 2019;Carminati et al. 2019). Theoretically speaking, Phytoplasma transmission via seeds has not yet been reported in Korea (Shin 1980). However, seeds of diseased E. sylvestris, phytoplasma was detected by the PCR analysis performed in this study. This trees mostly reproduces by seed germination, but the phytoplasma was detected in the seeds. There are a risk of the spread of phytoplasma in the end.
Phytoplasma were present in trees displaying decline symptoms. The present study is the first confirmed report of Ca. phytoplasma malaysianum (16SrXXXII) in E. sylvestris leaves and seeds on Jeju Island in South Korea. In response to this, considering that various symptoms and signs of decline are evident, we provisionally propose to name it "Elaeocarpus sylvestris decline phytoplasma (ESDP)."

Conclusions
This study was conducted to discover phytoplasma in Elaeocarpus sylvestris growing in Jeju Island, various decline symptoms observed incluing yellowing, darkening and branch dieback. Trees infected with phytoplasma die progressing rapidly and a number of trees are being cut down. As a result of PCR using phytoplasma universial primer P1/P7, R16F2n/R2, and SecAfor1/SecArev3 phytoplasma detected in leaves and seeds. We analyzed that E. sylvestris decline sequences, and found partial 16S rRNA sequences to be 99.5%, and secA genes 98.2% similarities with Ca. phytoplasma malaysianum (16SrXXXII). As a result of virtual-RFLF analysis with 17 key restriction enzymes with 16S rRNA gene, ESDP strain was presumed a new subgroup within the 16SrXXXII group.

Disclosure statement
No potential conflict of interest was reported by the author(s).