First report of a ' Candidatus Phytoplasma australasiaticum'-related phytoplasma strain associated with shoot proliferation disease of variegated croton in Taiwan.

Codiaeum variegatum (family Euphorbiaceae) is a leaf ornamental commonly known as variegated croton, which is often found in gardens or grown as indoor plants. In December of 2022, two cutting-propagated variegated croton plants exhibiting abnormal shoot proliferation and little leaf symptoms (Fig. S1) were found in a nursery owned by a private breeder in Wanluan Township, Pingtung County, Taiwan. The plants were propagated from a single stock plant, which died during transplanting from a commercial nursery in Changzhi Township, Pingtung County. To determine the potential cause of such symptoms, leaf tissues were collected from the center of the two symptomatic plants. Their DNA were extracted with a Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics, Lebanon, NJ) and used for further testing. As controls, two symptomless stock plants were collected from the commercial nursery in Changzhi Township and used to produce cutting-propagated plants; leaf DNA was extracted from each plant as described above. The DNA samples were subjected to PCR testing using the phytoplasma-specific primer pair P1/P7 (Schneider et al. 1995), and only DNA from the symptomatic plants produced the expected 1.8-kb amplicon. The two phytoplasma isolates detected in the plants were designated as CvaA and CvaB. After sequencing and analyzing the data using the i PhyClassifier program (Zhao et al. 2009), both CvaA and CvaB were classified to subgroup 16SrII-A (GenBank accession no. L33765) with a similarity coefficient of 1.0. The near-full-length 16S rDNA fragments of the detected isolates (GenBank accession no. OR794242) were also identical (1,463/1,463 bp) to that of NCHU2014 (GenBank accession no. CP040925, bp 537768-539230), a reference ' Ca . Phytoplasma australasiaticum'-related strain (16SrII-A) found in Taiwan (Chang et al. 2015; Rodrigues Jardim et al. 2023). To validate the results, the DNA samples were also tested with 16SrII group-specific semi-nested PCR targeting the elongation factor Tu gene. The outer and inner primer pairs used were TUF-II-F1/TUF-II-R1 and TUF-II-F2/TUF-II-R1, respectively (Al-Subhi et al. 2017). An expected amplicon was detected in the symptomatic samples but not in the symptomless counterparts. The amplified fragments' sequences (GenBank accession no. OR634931) were identical to that of the elongation factor Tu gene of the ' Ca . Phytoplasma australasiaticum'-related strain NCHU2014 (989/989 bp; GenBank accession no. CP040925, bp 139344-140332). The protein translocase gene secY of the detected phytoplasma was also amplified and sequenced using semi-nested primers SecYF1(II), SecYF2(II) and SecYR1(II) (Lee et al. 2010). Again, the sequences of the detected isolates (GenBank accession no. OR862773) were identical to that of NCHU2014 (1,263/1,263; GenBank accession no. CP040925, bp 192846-194108). The quality of the DNA samples was confirmed by PCR targeting the plant host's 28S rDNA using primer pair 28KJ/28C (Cullings 1992) and all symptomatic and symptomless samples produced the target amplicon (0.7 kb). 16SrII phytoplasmas have been detected in different host plants in Taiwan (Chang et al. 2015). To our knowledge, this is the first record of this group of pathogens infecting variegated croton in Taiwan. Branch-inducing phytoplasma has been used to improve the ornamental values of poinsettias, another Euphorbiaceae species (Lee et al. 1997). Further testing is needed to determine whether the phytoplasma detected in this work could be used for similar purposes.