Plant Disease

Huanglongbing (HLB) is a citrus infectious disease caused by ‘Candidatus Liberibacter’ spp. Recently, it has begun to spread rapidly worldwide, causing significant losses to the citrus industry. Early diagnosis of HLB relies on quantitative real-time PCR assays. However, the PCR inhibitors found in the nucleic acid extracted from plant materials pose challenges for PCR assays because they may result in false-negative results. Internal standard (IS) can be introduced to establish a single-tube duplex PCR for monitoring the influence of the PCR inhibitor, but it also brings the risk of false-negative results because the amplification of IS may compete with the target. To solve this problem, we proposed a mutation-enhanced single-tube duplex PCR (mSTD-PCR) containing IS with mutant-type primers. By introducing the 3′-terminal mutation in the primer of IS to weaken its amplification reaction and its inhibition of ‘Candidatus Liberibacter asiaticus’ (CLas) detection, the sensitivity and quantitative accuracy of CLas detection will not be affected by IS. In evaluating the sensitivity of CLas detection using simulation samples, the mSTD-PCR showed consistent sensitivity at 25 copies per test compared with the single-plex CLas assay. The detection result of 30 leaves and 30 root samples showed that the mSTD-PCR could recognize false-negative results caused by the PCR inhibitors and reduce workload by 48% compared with the single-plex CLas assay. Generally, the proposed mSTD-PCR provides a reliable, efficient, inhibitor-monitorable, quantitative screening method for accurately controlling HLB and a universal method for establishing a PCR assay for various pathogens.

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.

‘Candidatus Liberibacter’ spp. are the most prevalent microorganisms in the citrus plant, associated with citrus huanglongbing, which are transmitted by psyllid vectors. In Colombia, the vector Diaphorina citri Kuwayama has been reported in different regions, but ‘Ca. Liberibacter asiaticus’ (CLas) has only been detected in insect vectors, not in citrus host plants. To identify the presence and quantify the pathogen in citrus tissues, we employed a combined strategy that involved three techniques based on polymerase chain reaction (PCR). First, we used endpoint PCR with specific primers for CLas (OI1/OI2c) to confirm the infection. Second, we used qPCR with specific primers CIT295a/CIT298 designed on 16S rDNA gene regions to quantify the pathogen load. Finally, we used droplet digital PCR (ddPCR) to determine the copy number of the pathogen in citrus tissues using the β-subunit of the ribonucleotide reductase gene (nrdB) that is specific to CLas. We identified the presence of CLas in citrus plants for the first time in Colombia and quantified its titer in the plant tissue. We employed ddPCR and qPCR to provide crucial information for the country’s disease management, control strategies, and general crop health. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.

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).

Recurrent epiphytotics of X-disease, caused by ‘Candidatus Phytoplasma pruni’, have inflicted significant losses on commercial cherry and peach production across North America in the last century. During this period, there have been multiple studies reporting different disease phenotypes, and more recently, identifying different strains through sequencing core genes, but the symptoms have not, to date, been linked with genotype. Therefore, in this study we collected and assessed differing disease phenotypes from multiple U.S. states and conducted multi-locus sequence analysis on these strains. We identified a total of five lineages associated with the induction of X-disease on commercial Prunus species and two lineages that were associated with wild P. virginiana. Despite a century of interstate plant movement, there were regional trends in terms of lineages present, and lineage-specific symptoms were observed on P. avium, P. cerasus, and P. virginiana, but not on P. persica. Cumulatively, these data have allowed us to define ‘true’ X-disease-inducing strains of concern to the stone fruit industry across North America, as well as potential sources of infection that exist in the extra-orchard environment.

Chia (Salvia hispanica L., Lamiaceae) is an important commercial and medicinal crop recently popularized in India and widely cultivated in Karnataka (Joy et al., 2022). During the field survey of chia crop diseases, characteristic virescence like symptoms were observed at Main Agricultural Research Station, UAS, Raichur as well as at Mysuru and HD Kote region. The incidence was ranged from 2 – 4 per cent in an area of 30 hectares. Typical symptoms associated with chia are malformed shoot and/or inflorescence axis with reduced floral parts with greenish florets. The stem axis become thick, flattened, leaves are reduced towards terminal region. A total of five phytoplasma suspected samples and five suspected healthy samples were used for identification purpose. The Plant Genomic DNA Miniprep Kit (Sigma Aldrich, USA) was used to extract the DNA from five symptomatic and five asymptomatic samples and the DNA was used as template to amplify the phytoplasma-specific 16S rDNA gene using P1/P7 primers (Deng and Hiruki, 1991; Schneider et al., 1995) followed by nested PCR using R16F2n/R16R2 primers (Gundersen and Lee 1996). The expected 1.25-kb amplicon was detected from the suspected symptomatic samples. Nested PCR products were purified and sequenced from both the directions using ABIX370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The analysis revealed that all five sequences shared 100 per cent identity with Candidatus Phytoplasma aurantifolia (OM649850, ON975012) and Tomato big bud phytoplasma (EF193359). The in-silico RFLP pattern of F2n/R2 primed region of 16S rDNA gene analyzed by using iPhyClassifier (Zhao et al. 2009) revealed that the sequence shared 98.72 per cent nucleotide sequence similarity with coefficient value of 1.00 to the reference strain RFLP pattern of 16Sr group II, subgroup D (witches'-broom disease of lime; U15442). Based on 16SrDNA sequences and in-silico RFLP analysis, the phytoplasma associated with the chia virescence was identified as a member of 16SrII-D group. Further, SecA gene was also amplified from the samples using SecAfor1/SecArev3 primer pair (Hodgetts et al., 2008). All samples produced ~400 bp products and sequenced as detailed above. Sequence analysis by nBLAST revealed 100 per cent similarity to Ca. P. australasia (MW020545) and Ca. P. aurantifolia isolate Idukki Kerala 1 (MK726369) both representing 16SrII-D group phytoplasma. The representative sequence (16Sr: PP359693, PP359694; secA:PP386558, PP386559) were deposited in GenBank. Chia virescence phytoplasma belonging to Ca. phytoplasma australasia has not been reported anywhere. The phytopathological studies associated with chia crop are very limited. Joy et al. (2022) reported the occurrence of foot rot disease caused by Athelia rolfsii. Several hosts are recorded to be associated with 16SrII D phytoplasma which includes china aster, eggplant and crotalaria (Mahadevakumar et al., 2017, Yadav et al., 2016a, b). Now the wide occurrence of the phytoplasma in the area might have transmitted by vectors. The occurrence of virescence is of great importance as it affects the overall yield which reduces the market value. To our knowledge, this is the first report of a group 16SrII-D phytoplasma associated with chia virescence in India.

Citrus greening disease was first reported in Saudi Arabia during the 1970s when characteristic foliar and fruit symptoms were observed in commercial citrus groves. However, ‘Candidatus Liberibacter asiaticus’ (CLas) was not detected in symptomatic trees until 1981 to 1984 when CLas-like cells were observed by transmission electron microscopy in leaves collected from symptomatic citrus groves in southwestern Saudi Arabia. Despite the anticipated establishment of the CLas-Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama) pathosystem, CLas presence has not been verified in suspect trees nor have ACP infestations been documented. Given the recent expansion of citrus production in Saudi Arabia, a systematic country-wide survey was carried out to determine the potential CLas distribution in the 13 citrus-growing regions of the country. Citrus trees were surveyed for the presence of CLas-psyllid vector(s) and characteristic disease symptoms in commercial and urban citrus trees. Adult psyllids collected from infested citrus trees were identified as ACP based on morphological characteristics. Real-time quantitative PCR amplification (qPCR) of the CLas β-subunit of the ribonucleotide reductase (RNR) gene from citrus leaf and fruit samples and/or ACP adults revealed that trees were positive for CLas detection in 10 of the 13 survey regions; however, CLas was undetectable in ACP adults. Phylogenetic and single nucleotide polymorphism (SNP) analyses of a PCR-amplified, cloned fragment of the CLas 16S rRNA gene (∼1.1 kbp) indicated Saudi Arabian isolates were most closely related to Florida, U.S.A., isolates. Analysis of climate variables indicated that the distribution of the ACP-CLas pathosystem observed in Saudi Arabia was consistent with published predictions of terrains most likely to support establishment.