Sugarcane Grassy Shoot Phytoplasma Research Articles

First Report of a 'Candidatus Phytoplasma sacchari'-Related Strain Associated with Yellowing and Decline of Silver Bluestem in Texas, U.S.A.

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.

A Fractional Order Approach in the Eco-epidemiological Model of Sugarcane Grassy Shoot Disease Using Controls

A Fractional Order Approach in the Eco-epidemiological Model of Sugarcane Grassy Shoot Disease Using Controls

Micro structuring and host tissue patterning of sugarcane grassy shoot phytoplasma through scanning electron microscopy

Micro structuring and host tissue patterning of sugarcane grassy shoot phytoplasma through scanning electron microscopy

Natural infection of Ca. Phytoplasma sacchari causing sugarcane grassy shoot disease in Sorghum bicolor

Natural infection of Ca. Phytoplasma sacchari causing sugarcane grassy shoot disease in Sorghum bicolor

Production of sugarcane grassy shoot disease free setts by using hot water treatment

Production of sugarcane grassy shoot disease free setts by using hot water treatment

Sugarcane grassy shoot phytoplasma (grassy shoot of sugarcane)

Sugarcane grassy shoot phytoplasma (grassy shoot of sugarcane)

'Candidatus Phytoplasma sacchari', a novel taxon - associated with Sugarcane Grassy Shoot (SCGS) disease.

Sugarcane Grassy Shoot (SCGS) disease is known to be related to Rice Yellow Dwarf (RYD) phytoplasmas (16SrXI-B group) which are found predominantly in sugarcane growing areas of the Indian subcontinent and South-East Asia. The 16S rRNA gene sequences of SCGS phytoplasma strains belonging to the 16SrXI-B group share 98.07 % similarity with 'Ca. Phytoplasma cynodontis' strain BGWL-C1 followed by 97.65 % similarity with 'Ca. P. oryzae' strain RYD-J. Being placed distinctly away from both the phylogenetically related species, the taxonomic identity of SCGS phytoplasma is unclear and confusing. We attempted to resolve the phylogenetic positions of SCGS phytoplasma based on the phylogenetic analysis of 16S rRNA gene (>1500 bp), nine housekeeping genes (>3500 aa), core genome phylogeny (>10 000 aa) and OGRI values. The draft genome sequences of SCGS phytoplasma (strain SCGS) and Bermuda Grass White leaf (BGWL) phytoplasma (strain LW01), closely related to 'Ca. P. cynodontis', were obtained. The SCGS genome was comprised of 29 scaffolds corresponding to 505 173 bp while LW01 assembly contained 21 scaffolds corresponding to 483 935 bp with the fold coverages over 330× and completeness over 90 % for both the genomes. The G+C content of SCGS was 19.86 % while that of LW01 was 20.46 %. The orthoANI values for the strain SCGS against strains LW01 was 79.42 %, and dDDH values were 22. Overall analysis reveals that SCGS phytoplasma forms a distant clade in RYD group of phytoplasmas. Based on phylogenetic analyses and OGRI values obtained from the genome sequences, a novel taxon 'Candidatus Phytoplasma sacchari' is proposed.

Mixed Infection of Sugarcane Yellow Leaf Virus and Grassy Shoot Phytoplasma in Yellow Leaf Affected Indian Sugarcane Cultivars.

Sugarcane is an important sugar crop contributes more than 80% of world sugar production. Mosaic, leaf fleck, and yellow leaf (YL) are the major viral diseases affecting sugarcane, amongst YL occurrence is widely reported in all the sugarcane growing countries. It is caused by Sugarcane yellow leaf virus (SCYLV) and detailed works were done on complete genome characterization, transmission, and management. However, in countries like Egypt, South Africa, Cuba, Mauritius and Hawaii, the disease was reported to the cause of sugarcane yellow leaf phytoplasma (SCYP) and/or SCYLV as single/combined infections. Hence, we have investigated in detail to identify the exact Candidatus phytoplasma taxon associated in Indian cultivars affected with YL. The sequencing results and the restriction fragment length polymorphism pattern of the PCR products using the universal phytoplasma primers confirmed presence of sugarcane grassy shoot (SCGS) phytoplasma (16SrXI group) in the YL-affected plants. Mixed infection of SCYLV and SCGS phytoplasma was estimated as 32.8% in YL affected plants. Evolutionary genetic relationship between SCYP and SCGS phytoplasma representatively taken from different countries showed that SCYP from South Africa and Cuba were diverged from others and had a highest similarity with SCGS phytoplasma. Although we wanted to identify SCYP from YL affected Indian sugarcane cultivars, the study clearly indicated a clear absence of SCYP in YL affected plants and we found SCYLV as the primary cause for the disease.

Draft Genome Sequences of Two Phytoplasma Strains Associated with Sugarcane Grassy Shoot (SCGS) and Bermuda Grass White Leaf (BGWL) Diseases

We performed whole-genome sequencing of two phytoplasmas associated with sugarcane grassy shoot (SCGS) and Bermuda grass white leaf diseases. These are the first draft genomes of SCGS phytoplasma (strain SCGS) and 'Candidatus Phytoplasma cynodontis' (strain LW01) and may help to delineate these phytoplasmas at a finer taxonomic level.

Emerging incidence and diversity analysis of sugarcane grassy shoot phytoplasma in western Uttar Pradesh, India

Emerging incidence and diversity analysis of sugarcane grassy shoot phytoplasma in western Uttar Pradesh, India

Grassy shoot: The destructive disease of sugarcane

Sugarcane is one of the important commercial cash crops grown widely for sugar production. Red rot, smut, wilt, grassy shoot and yellow leaf are the major diseases of sugarcane worldwide; however the sugarcane grassy shoot (SCGS) is considered the most destructive one due to the associated total loss of millable canes production. Although the crop is cultivated in tropical and sub-tropical regions across the globe, widespread occurrence of the disease is mostly reported only from South and South East Asian countries. The disease was first reported in India in 1949. Based on its symptomatology, it is known as grassy shoot in India, Pakistan, and white leaf in Thailand, Vietnam, Myanmar, China and known by both names in Sri Lanka. The past seven decades witnessed several research advancements in symptomatology recognition, diagnosis and management of this disease. The nested PCR based diagnostic approach initiatives taken during 1990s followed by the in silico virtual RFLP analyses development in the period of 2007–2009 revolutionized the molecular characterization of the pathogen based on the variability analyses which resulted in the description of reports of many phytoplasma strains infecting sugarcane. Several phloem sap feeding leafhoppers such as Deltocephalus vulgaris, Cofana unimaculata, Exitianus indicus and Maiestas portica in India, Matsumuratettix hiroglyphicus and Yamatotettix flavovittatus in Thailand and D. menoni in Sri Lanka were reported as insect vectors during the last ten years. The recent use of next generation sequencing technologies helped to sequence whole genome of the SCGS phytoplasma. Currently, heat therapy and pathogen elimination through meristem tissue culture based seedling production are the only available management techniques to prevent the disease spread into new areas. Despite the fast pace of research in pathogen detection and genomics, the research on disease management under field conditions still remain grey and pose challenges to sugarcane pathologists.

First Report of Sugarcane Grassy Shoot Phytoplasma (16SrXI) Associated with Little Leaf Disease of Chrysopogon zizanioides from India

First Report of Sugarcane Grassy Shoot Phytoplasma (16SrXI) Associated with Little Leaf Disease of <i>Chrysopogon zizanioides</i> from India

Incidence, symptomatology and transmission of sugarcane grassy shootdisease in Assam, India

A roving survey was conducted during 2015 and 2016 to study the incidence, symptomatology and transmission of sugarcane grassy shoot (SCGS) disease in the sugarcane growing areas of four districts of Assam, viz. Biswanath, Golaghat, Sonitpur, and Nagoan. Major symptoms observed in the fields were the production of narrow and chlorotic leaves ranging from pale yellow to completely white color, the development of chlorotic streaks in leaves, unproductive tiller proliferation and the bushy appearance of the infected stool, with thin and non millable canes with short internodes. The average disease incidence ranged from 10-22%. The transmission of the disease was attempted by different methods. Out of five predominant leafhopper species (Cofana unimaculata, Exitianus indicus, Hishimonus phycitis, Nephotettix nigropictus, Orosius albicinctus) collected and identified from the sugarcane fields, in all the four districts, only E. indicus was successful in transmitting the SCGS disease. The disease was also transmitted by the setts collected from sugarcane grassy shoot symptomatic canes and by dodder.

FIRST REPORT OF A 16SRXI GROUP PHYTOPLASMA ('CANDIDATUS PHYTOPLASMA ORYZAE') ASSOCIATED WITH CYPERUS spp. WHITE LEAF DISEASE IN IRAN

In 2014, Cyperus spp. (Cyperaceae) white leaf (CWL) disease was observed in lime orchards in Minab (Hormozgan province, Iran). Main disease symptoms were whitening of aerial plant parts and stunting. To investigate the phytoplasma presence, total DNAs extracted from five symptomatic and five symptomless Cyperus spp. plants were tested by nested PCR using primers P1/P7 followed by R16F2n/R16R2 (Lee et al., 1998). Amplicons of ca. 1.8 and 1.2 kb, were obtained from samples of symptomatic but not symptomless plants. All amplicons were cloned and sequenced. The obtained 16S rDNA sequences showed 100% identity with each other and a representative of the same sequences deposited in GenBank (accession No. MF136620). BLAST search using the full length 16S rRNA gene sequence revealed that the CWL sequence showed 99% identity with sugarcane white leaf and sugarcane grassy shoot phytoplasmas (AB646271 and KF908792, respectively), representatives of 16SrXI phytoplasma group. Computer-simulated restriction analysis using iPhyClassifier, showed that the RFLP profile of CWL phytoplasma was very similar to the reference pattern of the 16SrXI-B subgroup (X76432). Phylogenetic analysis using Mega software version 7 confirmed that the CWL phytoplasma clustered with16SrXI group phytoplasmas closest to 16SrXI-B subgroup. To our knowledge this is the first report of a 16SrXI group phytoplasma in Iran. Rice and sugarcane plants are two economically important hosts of 16SrXI group-related strains (Arocha and Jones, 2010), thus the CWL-associated phytoplasma could act as a source of inoculum for infecting rice and sugarcane crops in Iran.

Identification and Characterization of 16SriX and 16SrXI Groups of Phytoplasmas Associated with Leaf Yellows and Declining Disease of Garlic and Onion in India

Leaf yellows and declining symptoms were observed on garlic and onion plants at Pune and Nasik region of Maharashtra, and Gorakhpur region of Uttar Pradesh in March-April 2016. The disease incidence varied from 12% to 30% in different surveyed regions. After nucleic acid extraction and nested PCR analysis using phytoplasma specific primer pairs (P1/P7 followed by 3F/3R) of a 16S ribosomal gene an expected DNA fragments of 1.3 kb were amplified in two symptomatic onion samples (from Gorakhpur and Narayangaon) and one symptomatic garlic sample (from Pune). Pair wise sequence comparison of 16S rRNA gene of the onion leaf yellows phytoplasma (GKP strain) showed 100% sequence identity with a strain of phytoplasma associated with sugarcane grassy shoot disease (KP746932) and 99% sequence identity with phytoplasma strain associated with Jasminum sambac little leaf disease (KF728950) and Ca. P. oryzae (AB052873) belonging to rice yellow dwarf (16SrXI) group. However, another strain of onion leaf yellows phytoplasma (Narayangaon strain) and garlic leaf yellows and declining phytoplasma (Pune strain) showed 99% sequence identity with phytoplasma strains of grapevine yellows (KX011516), eggplant big bud (JX464672) and sesame phyllody (JX464672) belonging to Ca. P. phoenicium (16SrIX) group. Phylogenetic analysis of 16Sr RNA gene sequences of phytoplasma strains from onion and garlic in the present study confirmed their close relationships with phytoplasma strains of 16SrXI and 16SrIX groups. This is the first report of association of 16SrXI and -IX groups of phytoplasma causing leaf yellows and declining disease of onion and garlic in India.

Use of secA Gene for Characterization of Phytoplasmas Associated with Sugarcane Grassy Shoot Disease in India

Surveys of commercial sugarcane varieties were conducted to the phytoplasma disease incidence in eight major sugarcane growing states of India (Uttarakhand, Uttar Pradesh, Maharashtra, Bihar, Assam, Chhattisgarh, Haryana and Tamil Nadu) during 2014–2015. Leaves from 24 symptomatic sugarcane plants of eight varieties showing grassy shoot and chlorosis symptoms, and of 8 non-symptomatic plants were collected and analyzed for phytoplasma presence using 16S rRNA and secA gene-specific primers. Amplification of 1.8- and 1.2-kb products using nested primers (P1/P7 and R16F2n/R16R2) of 16S rRNA gene and 880- and 480-bp products using secA gene-specific primer pairs (SecAfor1/SecArev3 and SecAfor2/SecArev3) was obtained for all the 24 symptomatic sugarcane samples. Pairwise sequence comparison, phylogenetic and in silico RFLP analysis of partial 16S rRNA and secA gene sequences of eight strains of sugarcane grassy shoot phytoplasma representative of the eight states confirmed the association of ‘Candidatus phytoplasma oryzae’-related strains (16SrXI-B) with symptomatic sugarcane varieties. The study confirmed that secA gene-specific primers could be employed for molecular characterization of phytoplasmas associated with sugarcane grassy shoot phytoplasmas belonging to 16SrXI group.

New subgroup 16SrXI-F phytoplasma strain associated with sugarcane grassy shoot (SCGS) disease in India.

A novel phytoplasma subgroup strain SC-Phy385 (GenBank accession number HF586648) was found associated with Sugarcane Grassy Shoot (SCGS) disease in India. Sugarcane (Saccharum officinarum L.) leaf samples with 'grassy shoot' symptoms were collected from the western region of Maharashtra state. The analysis based on restriction fragment length polymorphism (RFLP) profiles and 16S rRNA gene sequences indicated that most of the phytoplasma strains belong to the 16SrXI-B group. However, SCGS phytoplasma strain SC-Phy385 showed unique RFLP profiles with a similarity coefficient of 0.87 using online interactive phytoplasma classification tool, iPhyClassifier. The actual and virtual RFLP profiles of SC-Phy385 using amplified products of the 16S rRNA partial gene and its multiple sequence alignment with other RYD group phytoplasma sequences revealed that this strain was different from phytoplasma groups described earlier, representing new subgroup 16SrXI-F. The 16S rRNA gene-based phylogenetic analysis of reported RYD and BGWL group phytoplasmas showed that they are a genetically diverse group of phytoplasmas.

New Efficient Natural Leafhopper Vectors of Sugarcane Grassy Shoot Phytoplasma in India

Sugarcane grassy shoot (SCGS) disease is associated with the presence of 16SrXI group phytoplasmas that are transmitted by leafhoppers; limited studies have been performed in India toward its natural transmission. To determine the insect vectors that transmit the disease in nature, leafhopper species from SCGS-infected fields at Shahjahanpur, Central Uttar Pradesh, India, were collected and analyzed for phytoplasma presence using nested polymerase chain reaction with phytoplasma-specific primers. An ~1.2-kb amplified DNA fragment was detected in nested PCR from the three major leafhopper species, viz. Maiestas portica (Melichar), Exitianus indicus (Ross) and Cofana unimaculata (Signoret), and the symptomatic sugarcane leaves of variety CoS 07250. BLASTn analysis of ~1.2-kb 16S rDNA partial sequences obtained from symptomatic sugarcane plants and these leafhoppers revealed 99–100 % sequence identities among themselves and 99 % identity with other reported strains of ‘Candidatus Phytoplasma oryzae’ (16SrXI group). Phylogenetic analysis of 16S rRNA sequences of SCGS, M. portica, C. unimaculata and E. indicus phytoplasma strains also indicated the closest phylogenetic relationship with those of ‘Ca. P. oryzae’ group. Transmission tests and population sampling study further confirmed that M. portica and C. unimaculata were vectors of the SCGS phytoplasma from diseased to healthy sugarcane plants. The identification of new vectors of SCGS phytoplasma suggested that these leafhopper species may be responsible for secondary spread of SCGS phytoplasma.

Detection of sugarcane grassy shoot phytoplasma (16SrXI-B subgroup) inPyrilla perpusillaWalker in Uttar Pradesh, India

Heavy infestation of Pyrilla perpusilla was recorded in sugarcane fields on varieties Co0238, CoS767, CoS07250 infected with sugarcane grassy shoot (SCGS) disease showing white leaf symptoms at Shahjahanpur district of Uttar Pradesh. To confirm the possibility that P. perpusilla could carry SCGS phytoplasmas DNA was extracted from specimens collected from symptomatic sugarcane fields and from SCGS symptomatic leaf samples. PCR assay was performed with phytoplasma specific universal P1/P7 primers and nested PCR assay with 3for/3rev primers, which yielded about 1.3 kb amplicons in both the P. perpusilla and white leaf symptomatic samples. Phytoplasma amplicons from three samples each from P. perpusilla and SCGS samples were sequenced. After alignment, the obtained sequence were analyzed through pair wise comparison and showed 100% identity among themselves and 99% with strains of 16SrXI group. RFLP analysis assigned both the phytoplasma strains from P. perpusilla and SCGS to the 16SrXI-B subgroup. The study confirmed that P. perpusilla carry the same phytoplasmadetectedinSCGSplants thereforeitmayplay roleasvectorintransmissionof thesephytoplasmasinsugarcanecrops.

ABC Transporter from Sugarcane Grassy Shoot Phytoplasma: Gene Sequencing and Sequence Characterization

The present study focuses on the sequencing and characterization of genes coding for ABC transporter in sugarcane grassy shoot (SCGS) phytoplasma. Amplification of ABC transporter genes from SCGS phytoplasma was obtained using three sets of degenerated primer pairs. Sequenced fragments were assembled and trimmed to 2362 bp corresponding to full-length evbG gene and a partial evbH gene of transporter system. The evbG gene was characterized through in silico approaches. Six transmembrane helices and two different conserved domains were identified. The first domain consists of 283 amino acids, is located from 15aa to 298aa, and is annotated as transporter domain. The second domain is 234aa long and laid between amino acid position 331 and 565; it is characterized by a conserved nucleotide phosphate-binding motif. Comparative modeling techniques were used to generate three-dimensional structure of the evbG protein of SCGS phytoplasma which showed 31 helices, 19 strands, and 43 turns. This is the first report of sequencing and characterization of ABC type transporter genes from sugarcane grassy shoot phytoplasma.