Cherry blossoms (Cerasus serrula) are native to the temperate zone around the Himalayas in the northern hemisphere, mainly distributed in the west and southwest of China, including Yunnan, Sichuan and Tibet. Cherry has high ornamental, edible and medicinal value. In August 2022, we observed that Cherry trees exhibited witches' broom and plexus bud in Kunming City, Yunan Province, China. The symptoms consisted of many small branches with little leaves at the top of branches, stipule lobation, and clustered adventitious buds that are tumor-like on the branches that usually cannot sprout normally. As disease intensity increased, the branches dried up from the top to the bottom till the death of the whole plant. We named this disease C. serrula witches’ broom disease (CsWB). We found CsWB in the areas of Panlong, Guandu, Xishan Districts in Kunming, where more than 17% of the plants we surveyed were infected. We collected 60 samples from across the three districts. These included 15 symptomatic and 5 asymptomatic plants per district. The lateral stem tissues were observed under a scanning electron microscope (Hitachi S-3000N). The nearly spherical bodies were found in the phloem cells of symptomatic plants. Total DNA extraction was conducted from 0.1 g tissue using the CTAB method (Porebski et al. 1997), ddH2O was used as the negative control, and Dodonaea viscose plants with witches’ broom symptoms were used as the positive control. The nested PCR was used to amplify the 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993) and PCR amplicon of 1.2 kb were amplified (GenBank accessions: OQ408098; OQ408099; OQ408100). The direct PCR specific to the ribosomal protein (rp) gene yielded amplicons of approximately 1.2 kb with primer pair rp(I)F1A and rp(I)R1A (Lee et a. 2003) (GenBank accessions: OQ410969; OQ410970; OQ410971). The fragment from 33 symptomatic samples was consistent with the positive control, and absent for asymptomatic samples, suggesting an association of phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of CsWB phytoplasma showed that it has a 99.76% similarity with Trema laevigata witches' broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99.75% identity with Cinnamomum camphora witches' broom phytoplasma (GenBank accession OP649594). An analysis with iPhyClassifier showed that the virtual RFLP pattern derived from the 16S rDNA sequence shares 99.3% similarity with that of the 'Ca. Phytoplasma asteris' reference strain (GenBank accession: M30790), and the virtual RFLP pattern derived from the fragment is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). Thus, CsWB phytoplasma is identified as ‘Ca. Phytoplasma asteris’-related strain belonging to sub-group 16SrI-B. The phylogenetic tree was constructed based on 16S rRNA gene and rp gene sequences by using MEGA version 6.0 (Tamura et al. 2013) with neighbor-joining (NJ) method and bootstrap support was estimated with 1000 replicates. The result indicated that the CsWB phytoplasma formed a subclade in 16SrI-B and rpI-B respectively. In addition, the clean 1-year-old C. serrula were tested positive for the phytoplasma using the nested PCR 30 days after being grafted with naturally infected twigs with CsWB symptoms. To the best of our knowledge, Cherry blossoms is a new host of ‘Ca. Phytoplasma asteris’-related strains in China. The newly emerged disease is a threat to the ornamental value of cherry blossoms and the production of wood quality.
‘ Candidatus Liberibacter asiaticus’ (CLas) is associated with the devastating citrus disease Huanglongbing (HLB). Young flushes are the center of the HLB pathosystem due to their roles in the psyllid life cycle and in the acquisition and transmission of CLas. However, the early events of CLas infection and how CLas modulates young flush physiology remain poorly understood. Here, transmission electron microscopy analysis showed that the mean diameter of the sieve pores decreased in young leaves of HLB-positive trees after CLas infection, consistent with CLas-triggered callose deposition. RNA-seq-based global expression analysis of young leaves of HLB-positive sweet orange with (CLas-Pos) and without (CLas-Neg) detectable CLas demonstrated a significant impact on gene expression in young leaves, including on the expression of genes involved in host immunity, stress response, and plant hormone biosynthesis and signaling. CLas-Pos and CLas-Neg expression data displayed distinct patterns. The number of upregulated genes was higher than that of the downregulated genes in CLas-Pos for plant−pathogen interactions, glutathione metabolism, peroxisome, and calcium signaling, which are commonly associated with pathogen infections, compared with the healthy control. On the contrary, the number of upregulated genes was lower than that of the downregulated genes in CLas-Neg for genes involved in plant−pathogen interactions and peroxisome biogenesis/metabolism. Additionally, a time-course quantitative reverse transcription-PCR-based expression analysis visualized the induced expression of companion cell-specific genes, phloem protein 2 genes, and sucrose transport genes in young flushes triggered by CLas. This study advances our understanding of early events during CLas infection of citrus young flushes.
‘Candidatus Liberibacter asiaticus’ is the bacterium associated with the citrus disease known as huanglongbing (HLB). This study evaluated the influence of ‘Ca. L. asiaticus’ infection on a number of key plant physiological variables concerning photosynthesis, cell integrity, reactive oxygen species scavengers’ activity, and osmoregulation of two different species of citrus—the pomelo Citrus maxima and the mandarin C. reticulata ‘Tankan’—relative to their measured ‘Ca. L. asiaticus’ infection load. Results indicated that all measured physiological variables except soluble sugar were affected by increased ‘Ca. L. asiaticus’ infection titers, wherein the variety C. maxima proved overall more resistant than C. reticulata. ‘Ca. L. asiaticus’ infection was linked in both plants to decrease in chlorophyll concentration, cell membrane permeability, and malondialdehyde, as well as increased free proline and starch contents. Chlorophyll fluorescence measurements taken 9 months after grafting the mandarin C. reticulata with ‘Ca. L. asiaticus’ scions revealed a significant decrease in the photosynthesis variables maximum photochemical quantum yield of photosystem II (PSII), effective photochemical quantum yield of PSII, and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae, whereas nonphotochemical fluorescence quenching increased significantly; C. maxima plants, on the other hand, did not show significant differences until the 12th month from infection exposure. The variables superoxide dismutase, catalase, peroxidase, and soluble protein initially increased and later decreased. In addition, progression of ‘Ca. L. asiaticus’ replication in both citrus species was accompanied by rapid changes in three reactive oxygen species scavenging enzymes in C. maxima, while the pattern was different in C. reticulata. We hypothesize that the observed interspecific differences in physiological change are related to their relative resistance against ‘Ca. L. asiaticus’ infection. These results provide a scaffold for better describing the pathogenesis, selecting the most resistant breeds, or even validating pertaining omics research; ultimately, these detailed observations can facilitate the diagnosis of ‘Ca. L. asiaticus’ infection.
‘Candidatus Liberibacter solanacearum’ (Lso) is the causal agent of zebra chip of potato (Solanum tuberosum), which can significantly reduce potato yield. In this study, a loop-mediated isothermal amplification (LAMP) method for the detection of Lso haplotypes A and B was developed and evaluated. Two sets of LAMP primers named LAMP-A and LAMP-B were designed and tested for specificity and sensitivity. Both LAMP-A and LAMP-B were specific to Lso in in silico analysis using the Primer-Blast tool. The LAMP-A and LAMP-B could only produce positive signals from DNA mixtures of Lso-infected tomato but not from the genomic DNA of 37 nontarget plant pathogens. The sensitivity of LAMP-A and LAMP-B on Lso haplotypes A and B were tested on gBlocks and genomic DNA from Lso-infected tomato. On the genomic DNA for LAMP-A, the lowest amount of template DNA for a positive LAMP reaction was 2 to 20 ng on four haplotype A strains and 20 to 80 ng on four haplotype B strains; for LAMP-B, the lowest amount of template DNA for a positive LAMP reaction was 0.02 to 2 ng on four haplotype B strains and 20 ng to no amplification on four haplotype A strains. On gBlocks for LAMP-A, the lowest number of copies for a positive LAMP reaction was 60 on haplotype A and 600 on haplotype B; for LAMP-B, the lowest number of copies for a positive LAMP reaction was 60 on haplotype B and 600 on haplotype A. Therefore, considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, the LAMP-A and LAMP-B primers can be used together to test the probable Lso-infected plant or psyllid samples to rapidly, accurately, and directly differentiate haplotypes A and B. We highly recommend this LAMP system to plant pathology practitioners and diagnostic labs for routine detection of Lso and confirmation of zebra chip disease on potato or tomato.
Two phloem-limited pathogens, 'Candidatus Arsenophonus phytopathogenicus' and 'Candidatus Phytoplasma solani', threaten sugar beet production in France, Switzerland and Germany. Previous studies of these pathogens in Germany had focused on its western and southern regions, leaving a knowledge gap about eastern Germany. Despite their importance, this study is the first to investigate phytoplasmas in sugar beet in Saxony-Anhalt, Germany. A phytoplasma strain related to 'Ca. P. solani' is found predominant in Saxony-Anhalt, unlike in France, where 'Ca. P. solani' has a minor role compared to 'Ca. A. phytopathogenicus'. The phytoplasma strain infecting sugar beet in Saxony-Anhalt was classified into a new subgroup designated as 16SrXII-P. The MLSA of non-ribosomal genes of the novel phytoplasma strain showed that it is significantly different from the reference and all previously reported 'Ca. P. solani' strains including strain from western Germany. Analyses of sugar beet samples from previous years confirmed the presence of the 16SrXII-P strain in sugar beet as early as 2020, and also in Bavaria in southern Germany. Based on 16S rDNA analysis, 'Ca. A. phytopathogenicus' in Saxony-Anhalt is identical to strains in sugar beet in other parts of Germany and France, as well as to a strain in potato from Germany. The presence and prevalence of two phytoplasmas in sugar beet in Germany, suggest that more attention should be directed towards understanding phytoplasma infection in sugar beet in this country.