Publications

3561

Huanglongbing (HLB), which is associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), is a devastating disease that affects citrus trees worldwide. Because of the pervasiveness of the bacteria and psyllid vector, the disease is considered endemic in Florida. Although the effects of CLas on tree growth and physiology have been investigated for decades, most studies compared infected and noninfected trees under greenhouse conditions. This study used newly planted field-grown ‘Valencia’ sweet orange (Citrus sinensis) trees on two different rootstocks to monitor the distribution and accumulation of CLas in aboveground and belowground tissues following natural psyllid colonization and assess tree physiological responses and biomass reductions under HLB-endemic conditions. Trees were transplanted into the field with individual protective covers (IPCs), which are used to exclude psyllids and prevent infection. Openings were cut in the IPCs of half of the trees; to promote infection, these IPCs were temporarily removed during the main vegetative flushing period when psyllid populations were high. All trees that were exposed to psyllids became infected and displayed the symptoms typically associated with HLB. Throughout the study, higher levels of CLas were detected in the leaves compared with those in the fibrous roots. Trees that were not exposed to psyllids remained noninfected and healthy. After 18 months, a subset of trees was excavated to assess biomass differences between infected and noninfected trees. Infected trees had root system reductions of 37% and shoot system reductions of 20%, thereby significantly reducing the belowground-to-aboveground biomass ratio. Fibrous root loss was 49% and more severe than the loss of the rest of the root tissue. This study is the first to demonstrate the full extent of damage caused by CLas infection under natural HLB-endemic conditions. The results confirm previous observations that suggested fibrous root loss as one of the major consequences of infection and colonization with CLas. They also reinforce the benefits of using IPCs to prevent infection of young citrus trees during the first years of growth in the field.

Silver bluestem [Bothriochloa laguroides (DC.) Herter] is a warm-season grass native to Texas. This perennial grass plays a crucial role in maintaining ecological balance and supporting wildlife in the region. In September 2022, while investigating the ecological impact of invasive grass species on a grassland located near Pipe Creek (TX), B. laguroides plants were observed showing symptoms that included yellowing of the blades and occasionally brown discoloration of the midveins and stems (Fig. S1). Disease incidence was estimated as 2% of silver bluestem plants in the 2 hectares surveyed. To investigate the possibility of a phytoplasma association with the symptoms, four symptomatic and four asymptomatic leaf samples were collected for further study. Total DNA was extracted from leaf midribs using a DNeasy Plant Mini Kit (Qiagen). The DNA extracts were tested using a phytoplasma-specific quantitative PCR assay (Hodgetts et al. 2009), which identified two out of the four symptomatic B. laguroides samples as positive for phytoplasmas. A semi-nested PCR assay for amplification of the 16S rRNA gene fragment was then performed on these samples with primers P1/16S-SR followed by P1A/16S-SR (Deng, and Hiruki 1991; Lee et al. 2004), and two additional housekeeping genes (tuf and secA) were amplified as previously described (Makarova et al. 2012; Hodgetts et al. 2008; Bekele et al. 2011). All amplicons of the expected size, 1.5 kb (16S rRNA), 0.4 kb (tuf) and 0.6 kb (secA), were purified and bi-directionally sequenced using primers from each gene second round PCR amplification. Analysis of the sequences derived from the three gene fragments revealed no variation between the two plant samples and confirmed they originated from a phytoplasma, termed strain TXSB-2 (Texas Silver Bluestem). Sequences from a single B. laguroides plant DNA extract were deposited in GenBank with accession numbers OR711913 (16S rRNA), OR709687 (tuf) and OR709688 (secA). A BLAST search of the 16S rRNA gene sequence from TXSB-2 against the NCBI nucleotide database, showed 99.58% sequence identity with an unclassified phytoplasma clone 139-1 from a leafhopper collected in Australia (MW281491) (Fig. S2). The partial nucleotide sequence of the tuf and secA genes showed 90.60% and 89.78% similarity, respectively, to the corresponding genes in ‘Ca. P. sacchari’ strain SCWL1 (CP115156) associated with sugarcane in China. The iPhyClassifier, an interactive online tool for phytoplasma identification and classification (Zhao et al. 2009), was used to determine the ‘Candidatus Phytoplasma’ species affiliation and group/subgroup classification status of this phytoplasma strain. The result showed that the TXSB-2 16S rDNA shared 98.94% sequence identity with that of the 'Ca. P. sacchari' reference strain (GenBank accession: MN889545), indicating TXSB-2 is a 'Ca. P. sacchari'-related strain. The result from virtual restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA F2nR2 fragment revealed that TXSB-2 possessed a collective RFLP pattern that is distinct from the reference patterns of all established phytoplasma ribosomal subgroups and is proposed as the representative strain of a new subgroup designated as 16SrXI-H. ‘Candidatus Phytoplasma sacchari’ has been reported associated with sugarcane grassy shoot disease, which is considered among the most damaging diseases of sugarcane across parts of Southeast Asia and India (Kirdat et al. 2021). The same phytoplasma was recently confirmed infecting sorghum in India (Nithya et al. 2024). To our knowledge, this is the first report of a ‘Ca. P. sacchari’-related strain infecting B. laguroides in the United States. Moreover, B. laguroides is a new host for strains related to ‘Ca. P. sacchari’. Further investigation is required to elucidate the prevalence of this disease in the area, its natural vectors, and the potential consequences arising from this novel phytoplasma strain within its ecosystem in Texas.

ABSTRACT The genome of “ Candidatus Xenohaliotis californiensis” was assembled from shotgun metagenomic sequencing of experimentally infected white abalone. Ninety-one percent genome completeness was achieved with low contamination. Sequencing this genome provides the opportunity to track pathogen evolution over time, conduct gene expression experiments, and study dynamics between this pathogen and its phage.

Ipomoea biflora L., commonly known as morning glory, is an herbaceous vine plant in the Convolvulaceae family and is widespread at low elevations in Taiwan and other East Asian countries. In September 2023, six I. biflora plants exhibiting small leaves, leaf yellowing, and shoot proliferation were observed in a vacant lot in Taiwan Agricultural Research Institute (TARI), Wufeng District, Taichung, Taiwan, representing 100% disease incidence in the area. All the symptomatic morning glory climbed onto Murraya paniculata L. (common jasmine orange) which however showed no similar symptoms. The total DNA (two samples for each plant) from leaf tissues of three symptomatic morning glory plants, two asymptomatic morning glory plants, and one asymptomatic common jasmine orange was isolated by the CTAB method (Fulton et al. 1995) and used for PCR with the universal primers, P1 (Deng and Hiruki 1991)/P7 (Schneider et al. 1995), to amplify a fragment containing partial 16S rDNA. Expected 1.8-kb bands were amplified from DNA extracted from all symptomatic plants, whereas no PCR product was detected from that of the asymptomatic I.biflora and M. paniculata plants. Six PCR products were cloned and sequenced in the Biotechnology Center DNA-sequencing facility at National Chung Hsing University, and one representative sequence was selected and deposited in GenBank. BLAST analysis revealed that the obtained 16S rDNA sequence (PP230905) shared 99.92% identity with the following phytoplasma strains: rapeseed phyllody phytoplasma (CP055264), plumbago auriculata leaf yellowing phytoplasma (MN239503), and aster yellows phytoplasma (MK992774), which all belong to the 16SrI subgroup. The query 16S rDNA sequence shares 99.84% identity with that of the ‘Candidatus Phytoplasma asteris’ reference strain (M30790), suggesting that the phytoplasma is a ‘Ca. Phytoplasma asteris’-related strain. A virtual restriction fragment length polymorphism (RFLP) analysis was conducted using iPhyClassifier tool (Zhao et al. 2009), and the pattern derived from the 16S rDNA fragment of the I. biflora phytoplasma was identical (similarity coefficient 1.00) to the reference pattern of 16SrI, subgroup B (onion yellows phytoplasma OY-M; AP006628). Six total DNA samples from symptomatic plants were used as templates to amplify 842 bp secA sequences with SecAfor1 and SecArev3 primers (Hodgetts et al. 2008), and one representative sequence was deposited in GenBank. The partial secA sequence (PP263636) showed 98.22% identity with that of Trema levigatum witches’-broom phytoplasma (MW032212) that also belongs to the 16SrI group (Wan et al. 2021). Phylogenetic analysis of both 16S rDNA and secA confirmed I. biflora phytoplasma as 16SrI, subgroup B. Taken together, we concluded that the morning glory phytoplasma in this study was a ‘Ca. Phytoplasma asteris’-related strain belonging to the 16SrI group. To the best of our knowledge, this is the first report of a phytoplasma-infected I. biflora in Taiwan, suggesting morning glory as a new natural host of 16SrI phytoplasmas, alongside other plants like roselle and citrus (Tseng et al. 2014; Feng et al. 2015).