First report of a ‘Candidatus Phytoplasma sacchari’-related strain associated with yellowing and decline of Silver Bluestem in Texas, U.S.A.

Authors: RIOS, DANIELLE - Texas A&M University; UECKERT, JAKE - Texas A&M University; ONG, KEVIN - Texas A&M University; BARILLAS, JOSE - Texas A&M University; Costanzo, Stefano

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/27/2024
Publication Date: 4/30/2024
Citation: Rios, D., Ueckert, J., Ong, K., Barillas, J.R., Costanzo, S. 2024. First report of a ‘Candidatus Phytoplasma sacchari’-related strain associated with yellowing and decline of Silver Bluestem in Texas, U.S.A.. Plant Disease. https://doi.org/10.1094/PDIS-03-24-0524-PDN.
DOI: https://doi.org/10.1094/PDIS-03-24-0524-PDN

Interpretive Summary: The discovery of bacteria known as phytoplasmas infecting silver bluestem, a crucial warm-season grass native to Texas, presents a significant ecological concern. These organisms infest the tissue that is responsible for transporting nutrients throughout the plant. Over time, their infection alters the normal functioning of the plant leading to disease symptoms such as abnormal plant development, yellowing and browning of leaves and general decline. This previously unrecognized phytoplasma strain, named Texas Silver Bluestem-2 (TXSB-2), was identified in 2022 from silver bluestem plants in a grassland near Pipe Creek, Texas, marking the first reported instance of such infection in this grass species and in the Americas. The research conducted by an ARS scientist revealed molecular traits of TXSB-2 that can distinguish it from previously described phytoplasma species. This phytoplasma strain was determined to be related to a known species 'Candidatus Phytoplasma sacchari', the phytoplasma associated with sugarcane grassy shoot disease, which is one of the most destructive diseases of sugarcane. This phytoplasma is known to be prevalent in various sugarcane-growing regions, particularly in the Indian subcontinent and South-East Asia but never reported in the United States. This discovery holds implications for sugarcane producers, research scientists, and plant disease management professionals, emphasizing the need for understanding the insects involved in the transmission of this phytoplasma strain to plants and the genetic diversity of this bacterial pathogen.

Technical Abstract: 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. 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, 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, and two additional housekeeping genes (tuf and secA) were amplified as previously described. 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). 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, 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. The same phytoplasma was recently confirmed infecting sorghum in India. 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.