Chu, Chia-Ching

Little ironweed (Cyanthillium cinereum; syn. Vernonia cinerea) is an herbaceous plant in the Asteraceae family (Trang et al. 2024). The plant is distributed in Asia, Australia and Africa, and has been used as a traditional medicine (Trang et al. 2024). In January 2026, little ironweed plants exhibiting phyllody symptoms (Fig. S1) were observed in a grass-covered area (measuring 3 m × 6 m) in Qishan District, Kaohsiung, Taiwan. Around 8% (22/285 plants) of the population showed symptoms. Six symptomatic and four asymptomatic plants were collected. Flower-like tissues from symptomatic plants were collected for DNA extraction with a Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics). For asymptomatic plants, capitula were used instead. One DNA sample was extracted for each plant. The samples were first tested with primers specific to the plant’s 26S rDNA (28KJ/28C; Cullings 1992), and all of them produced the target amplicon (~0.69 kb), indicating sufficient quality for polymerase chain reaction (PCR). Nested PCR targeting phytoplasmas’ 16S rDNA was conducted with outer and inner primer pairs P1/P7 and fU5/rU3 (Lorenz et al. 1995). In all PCR assays, negative controls (nuclease-free water) were included. All samples from symptomatic plants amplified the outer and inner amplicons (~1.81 kb and ~0.88 kb), whereas DNA from the asymptomatic plants did not. After assembling sequences of the outer and inner fragments for each symptomatic sample, identical 16S rDNA sequences were found. A representative sequence was deposited in GenBank (1,459 bp; accession no. PZ193724), and BLASTn search against GenBank revealed that it was identical to that of a reference phytoplasma strain belonging to subgroup 16SrII-A (NCHU2014; accession no. CP040925; 100% identity, 100% query coverage; Rodrigues Jardim et al. 2023). A 1,248-bp fragment within the obtained sequence was further analyzed using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi), and its virtual RFLP pattern was the same as 16SrII-A’s reference pattern (similarity coefficient = 1.00; accession no. L33765). The detected phytoplasma’s identity was further confirmed by amplification and sequencing of the elongation factor Tu (tuf) and protein translocase (secY) genes. Fragments containing tuf and secY were respectively amplified by primer sets TUF-II-F1/TUF-II-F2/TUF-II-R1 (for semi-nested PCR; Al-Subhi et al. 2017) and SecYF1(II)/SecYR1(II) (for PCR; Lee et al. 2010). The tuf sequences were obtained from the inner amplicon, whereas sequences of secY were obtained by sequencing followed by primer walking (accession nos. PZ377067 and PZ377068). The detected phytoplasma and the reference strain NCHU2014 (‘Ca. Phytoplasma australasiaticum’-related strain; 16SrII-A; accession no. CP040925) shared identical sequences for both tuf (986 bp; partial) and secY (1,263 bp; complete) fragments. This is the first report of little ironweed infected by a 16SrII-A phytoplasma in Taiwan; this specific host-pathogen association has only been reported in Hainan Province of China (Yu et al. 2024). Because 16SrII-A phytoplasma can infect economic crops in Taiwan (Wu et al. 2026) and little ironweed is widely distributed in the island, it is possible that the weed may represent a potential reservoir host. Thus, monitoring and eradication of symptomatic little ironweed is advisable in crop production areas.

ABSTRACT Ponkan mandarin ( Citrus reticulata cv. Ponkan) is an important fruit crop planted across different countries. One of its most devastating diseases is citrus huanglongbing (HLB), which is associated with infection by the bacterium Candidatus Liberibacter asiaticus ( C Las). Although studies on HLB in Ponkan mandarin have been conducted, symptom heterogeneity and its association with C Las titer under field conditions remain underexplored. We examined the association between symptom variation and C Las infection status in a Ponkan mandarin grove by categorizing symptoms at both the leaf and canopy (tree) level. Chlorotic leaves were sampled and classified into different categories, including leaves exhibiting interveinal chlorosis (Cat. A), those showing vein yellowing (Cat. B), and blotchy mottled leaves (Cat. C). Polymerase chain reaction (PCR) and quantitative PCR (qPCR) assays targeting C Las revealed that Cat. A leaves, which resembled nutrient-deficient leaves, had higher C Las detection rates and a larger proportion of samples with high C Las titers. Canopy-level assessment of symptom diversity within each tree also revealed that plants bearing Cat. A leaves were more likely to carry C Las and have high pathogen loads. The likelihood was also greater in trees with higher leaf symptom diversity. Additional prophage typing of the detected C Las strains revealed that leaf symptom variations were independent of prophage type difference. Nutrient analyses revealed reduced magnesium, manganese, and zinc contents in Cat. A leaves, indicating that leaves prone to C Las infection also exhibited nutrient deficiencies. These findings highlight the complexity of potential cultivar-specific associations between C Las and citrus under field conditions. IMPORTANCE Although it is well-established that HLB in Ponkan mandarin is associated with C Las infection, the relationship between leaf symptom variation in this cultivar and the presence and titer of C Las under complex field conditions remains unclear. By examining symptom heterogeneity in natural field conditions at both the leaf and canopy level, this study demonstrated that HLB leaf symptom variation in naturally infected Ponkan mandarin corresponds to differences in C Las presence and titer at both levels. Notably, although blotchy mottling is considered the typical leaf symptom of HLB, we found that C Las detection rates and titers tended to be greater in leaves showing nutrient deficiency-like symptoms, which contrasts with patterns in other citrus cultivars. Recognizing inconsistencies among cultivar-specific patterns is important for interpreting HLB symptoms, devising sampling strategies, and exploring uncharacterized factors affecting symptom expression under field conditions.

ABSTRACT Psyllids (Psylloidea) are host-specific, phloem-feeding insects that are associated with the transmission of destructive plant diseases caused by the bacterial genus “ Candidatus Liberibacter”. While psyllid-liberibacter interactions have been studied in great detail, most research mainly focused on species associated with important crop diseases. Therefore, a more general understanding of the associations between these two groups of organisms remains limited. The present study investigated the occurrence of infection by “ Ca . Liberibacter” across undercharacterized psyllid lineages in Taiwan. Samples of 46 psyllid species, representing six of the seven known Psylloidea families from host plants spanning 22 families, were obtained. The presence of “ Ca . Liberibacter” in adult psyllid DNA samples was determined by PCRs targeting 16S rDNA of “ Ca . Liberibacter”. PCR tests amplifying mitochondrial 16S DNA of the psyllids were also carried out for quality confirmation. The assays detected “ Ca . Liberibacter” in five of the psyllid species tested, namely Calophya nigridorsalis , Homotoma radiata , Cacopsylla tobirae , Epipsylla albolineata , and Trioza quadrimaculata . BLASTn searches and phylogenetic analysis on near-full-length 16S rDNA sequences identified the strain detected in C. tobirae as “ Ca . Liberibacter europaeus” and showed that E. albolineata carried a strain closely related to a “ Ca . Liberibacter” strain found in Bhutan, while the remaining detected strains could potentially be novel “ Ca . Liberibacter” species. The findings from this study showed that a large diversity of “ Ca . Liberibacter” could exist among underexplored psyllid and plant species, opening new avenues for investigating the ecology of “ Ca . Liberibacter”. IMPORTANCE Bacteria of the genus “ Candidatus Liberibacter” can cause some of the most devastating plant diseases. Gaining a broader perspective on the diversity of associations between these bacteria and their psyllid vectors is crucial for both fundamental and applicative purposes. By taking advantage of the biological diversity in Taiwan, the present study conducted one of the broadest surveys on the presence of “ Ca . Liberibacter” in psyllids, in terms of the diversities of the psyllids examined. The data from this work indicated that previously unknown “ Ca . Liberibacter” genotypes and perhaps even novel species may be more prevalent among psyllid species than previously known. These findings highlight the potential importance of exploring psyllid-liberibacter associations from a broader ecological perspective.