Yu, Shao-Shuai

Peony (Paeonia suffruticosa Andr.), belonging to family Paeoniaceae, is an important medicinal and ornamental plant. During August of each year from 2016 to 2023, peony plants at Heze city were found to exhibit leaf yellows symptoms. The incidence rate of the symptomatic plant was recorded from 10% to 30% in four peony gardens with about 200 acres. Total DNA was extracted from 0.10 g fresh plant leaf tissues from 24 symptomatic and 8 asymptomatic samples using rapid plant genomic DNA isolation kit (Aidlab Biotechnology, Beijing, China). The extracted DNA was amplified by nested polymerase chain reaction using universal primers R16mF2/R16mR1 followed by R16F2/R16R2 (Lee et al., 1993; Gundersen and Lee, 1996) specific for the 16S rRNA gene and new designed tuf gene specific primers JWB-tuforfF1 (5′-ATGGCTGAAATATTTTCAAGAG-3′) and JWB-tuforfR1 (5′-TTATTCTATGATTTTAATAACAG-3′) followed by JWB-tuforfF2 (5′-ATGTAAACGTAGGAACTATTGG-3′) and JWB-tuforfR2 (5′- TCCGATAGTTCTTCCACCTTCAC-3′). Amplicons of about 1.25 kb and 1.02 kb (16S rRNA gene and tuf gene, respectively) were obtained in 8 symptomatic samples from four peony gardens. However, no amplification was obtained in any of the asymptomatic samples. The representative amplicons of 16S rRNA and tuf genes of three positive samples (Heze-9, -18 and -27) were cloned into a zero background pLB-simple vector (Tiangen Biotechnology, Beijing, China) and sequenced by Taihe Biotechnology, Beijing, China. Sequences obtained in the study were deposited in NCBI GenBank with accession numbers PP504882, PP504883 and PP504884 for the 16S rRNA gene as well as PP530237, PP530238 and PP530239 for the tuf gene. The phytoplasma strain under the study was described as peony yellows (PeY) phytoplasma, PeY-Heze strain. Alignment analysis by DNAMAN software showed that three 16S rRNA gene sequences obtained in the study shared 99.36% to 99.60% sequence identity and three tuf gene sequences obtained in the study were identical. BLAST analysis of the 16S rRNA gene sequences of the PeY-Heze phytoplasma strains showed 99.60%-99.84% sequence identity with ‘Candidatus Phytoplasma ziziphi’ (GenBank accession: CP025121). And tuf sequences of the strains showed 100% similarity with ‘Ca. P. ziziphi’ (CP025121). Interestingly, the virtual RFLP patterns derived from three 16Sr RNA gene sequences obtained in the study by iPhyClassifier (Zhao et al., 2009) were different from the reference patterns of all previously established 16Sr groups/subgroups. The most similar are the reference pattern of the 16Sr group VII, subgroup E (AY741531), with a similarity coefficient of 0.72, which is less than 0.85. These phytoplasma strains may represent a new 16Sr group. Phylogenetic analysis based on 16S rRNA genes using MEGA 7.0 by neighbor-joining (NJ) method with 1000 bootstrap value indicated that PeY-Heze strains clustered into one clade with the phytoplasma strains of ‘Ca. P. ziziphi’ with 68% bootstrap value. Although there are several reports available on ‘Ca. P. solani’ infecting peony in Shandong Province, China (Gao et al., 2013). To our knowledge, this is the first report of ‘Ca. P. ziziphi’-related strains infecting peony in China. The findings in this study will be beneficial to the detection, quarantine, and prevention of peony yellows phytoplasmas in China.

Areca catechu palm is an important cash plant in Hainan Island of China and also in the tropical regions of the world. A. catechu palm yellow leaf (AcYL) disease caused by phytoplasmas is a devastating disease for plant production. In the study, the phytoplasmas associated with the AcYL disease were identified and characterized based on their conserved genes, and genetic variation and phylogenetic relationship of the phytoplasma strains in the 16SrXXXII group were demonstrated. The results indicated that A. catechu palm plants showing yellow leaf symptoms were infected by ‘Candidatus Phytoplasma malaysianum’-related strains belonging to the 16SrXXXII-D subgroup. BLAST and multiple sequence alignment analysis based on 16S rRNA and secA genes showed that the AcYL phytoplasmas shared 100% sequence identity and 100% homology with the ‘Ca. P. malaysianum’-related strains. Phylogenetic analysis indicated that the AcYL phytoplasmas and ‘Ca. P. malaysianum’-related strains belonging to the 16SrXXXII group clustered into one clade with a 100% bootstrap value. Based on computer-simulated digestions, six kinds of restriction fragment length polymorphism patterns within the 16SrXXXII group were obtained, and a novel subgroup in the 16Sr group was recommended to propose and describe the relevant strains in this 16Sr subgroup. To our knowledge, this is the first study to report that A. catechu palm showing yellow leaf symptoms was infected by ‘Ca. P. malaysianum’-related strains belonging to the 16SrXXXII group. A novel 16Sr subgroup, 16SrXXXII-F, was proposed based on the systematical analysis of genetic variation of all phytoplasmas within the 16SrXXXII group. The findings of this study will support references for monitoring the epidemiology and developing effective prevention strategies for AcYL disease.

The pathogens associated with citrus Huanglongbing symptoms, including yellowing and mottled leaves in Citrus maxima, an important economic crop on Hainan Island of China, were identified and characterized. In the study, detection, genetic variation and phylogenetic relationship analysis of the pathogens were performed based on 16S rRNA and β-operon gene fragments specific to phytoplasma and Candidatus Liberibacter asiaticus. The results indicated that the pathogens—such as phytoplasma strains of CmPII-hn belonging to the 16SrII-V subgroup and CmPXXXII-hn belonging to the 16SrXXXII-D subgroup, as well as Candidatus Liberibacter asiaticus strains CmLas-hn—were identified in the diseased plant samples, with numbers of 12, 2 and 6 out of 54, respectively. Among them, mixed infection with the 16SrII-V subgroup phytoplasma and Candidatus Liberibacter asiaticus was found in the study, accounting for 7.4% (four samples). The phytoplasma strains of CmPII-hn—Tephrosia purpurea witches’ broom, Melochia corchorifolia witches’ broom and Emilia sonchifolia witches’ broom—were clustered into one clade belonging to the 16SrII-V subgroup, with a 99% bootstrap value. The phytoplasma strains of CmPXXXII-hn and Trema tomentosa witches’ broom belonging to 16SrXXXII-D, and the other 16SrXXXII subgroup strains were clustered into one clade belonging to the 16SrXXXII group with a 99% bootstrap value. There were 16 variable loci in the 16S rRNA gene sequences of the tested 16SrXXXII group phytoplasma strains, of which two bases had an insertion/deletion. The strains of Candidatus Liberibacter asiaticus, identified in the study and the strains that had been deposited in GenBank, were in one independent cluster with a 99% bootstrap value. To our knowledge, this is the first report showing that Citrus maxima can be infected by 16SrII-V and16SrXXXII-D subgroup phytoplasmas in China. Moreover, this is also the first report in which the plants are co-infected by 16SrII-V subgroup phytoplasmas and Candidatus Liberibacter asiaticus. More comprehensive and detailed identification and characterization of the pathogens associated with the diseased symptoms in Citrus maxima on the island in China would be beneficial for epidemic monitoring and for the effective prevention and control of related plant diseases.