Sugarcane White Leaf Phytoplasma Research Articles

Comparison of Three Genomic DNA Extraction Methods from Sugarcane for Detection of Sugarcane White Leaf Phytoplasma

Comparison of Three Genomic DNA Extraction Methods from Sugarcane for Detection of Sugarcane White Leaf Phytoplasma

Morphological and biochemical changes in asymptomatic and moderately symptomatic plants infected with sugarcane white leaf (SCWL) phytoplasma

Morphological and biochemical changes in asymptomatic and moderately symptomatic plants infected with sugarcane white leaf (SCWL) phytoplasma

Probing behavior of the leafhopper analyzed through DC electropenetrography and microscopy

Yamatotettix flavovittatus Matsumara is a new leafhopper species vector of sugarcane white leaf (SCWL) phytoplasma that causes sugarcane chlorosis symptoms. The effects of probing behavior of Y. flavovittatus on sugarcane and its implication for SCWL phytoplasma transmission are yet to be studied. In this research, we used DC electropenetrography (EPG) to define waveforms produced by adult and fifth-instar nymphal Y. flavovittatus on sugarcane and correlated them with salivary sheath termini (likely stylet tip locations) via light and scanning electron microscopy. The following six waveforms and associated activities are described: (NP) non-probing, (Yf1) stylet probing into epidermal cells, (Yf2) stylet probing through mesophyll/parenchyma, (Yf3) stylet contact with phloem and likely watery salivation, (Yf4) active ingestion of sap from phloem, probably sieve elements, and (Yf5) unknown stylet activity in multiple cell types. Study findings reveal that the Y. flavovittatus vector ingests sieve tube element more frequently and for longer durations than any other cell type, supporting that Y. flavovittatus is primarily a phloem feeder. Adult Y. flavovittatus show a longer total probing duration and produces a high density of puncture holes on sugarcane leaves. Moreover, probing behaviors revealed that adults typically ingest phloem sap more frequently and for longer durations than fifth-instar nymphs, enhancing sap ingestion. Furthermore, we propose that adults are more likely to acquire (during Yf4) and inoculate (during Yf3) higher amounts of phytoplasma than fifth-instar nymphs. This information on the penetration behavior of leafhopper Y. flavovittatus serves as a basis for advanced studies on the transmission mechanism of SCWL phytoplasma.

Complete genome sequence of "Candidatus Phytoplasma sacchari" obtained using a filter-based DNA enrichment method and Nanopore sequencing.

Phytoplasmas are phloem-limited plant pathogens, such as sugarcane white leaf (SCWL) phytoplasma, which are responsible for heavy economic losses to the sugarcane industry. Characterization of phytoplasmas has been limited because they cannot be cultured in vitro. However, with the advent of genome sequencing, different aspects of phytoplasmas are being investigated. In this study, we developed a DNA enrichment method for sugarcane white leaf (SCWL) phytoplasma, evaluated the effect of DNA enrichment via Illumina sequencing technologies, and utilized Illumina and Nanopore sequencing technologies to obtain the complete genome sequence of the "Candidatus Phytoplasma sacchari" isolate SCWL1 that is associated with sugarcane white leaf in China. Illumina sequencing analysis elucidated that only 1.21% of the sequencing reads from total leaf DNA were mapped to the SCWL1 genome, whereas 40.97% of the sequencing reads from the enriched DNA were mapped to the SCWL1 genome. The genome of isolate SCWL1 consists of a 538,951 bp and 2976 bp long circular chromosome and plasmid, respectively. We identified 459 protein-encoding genes, 2 complete 5S-23S-16S rRNA gene operons, 27 tRNA genes, and an incomplete potential mobile unit (PMU) in the circular chromosome. Phylogenetic analyses and average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values based on the sequenced genome revealed that SCWL phytoplasma and sugarcane grassy shoot (SCGS) phytoplasma belonged to the same phytoplasma species. This study provides a genomic DNA enrichment method for phytoplasma sequencing. Moreover, we report the first complete genome of a "Ca. Phytoplasma sacchari" isolate, thus contributing to future studies on the evolutionary relationships and pathogenic mechanisms of "Ca. Phytoplasma sacchari" isolates.

Detection of White Leaf Disease in Sugarcane Using Machine Learning Techniques over UAV Multispectral Images

Sugarcane white leaf phytoplasma (white leaf disease) in sugarcane crops is caused by a phytoplasma transmitted by leafhopper vectors. White leaf disease (WLD) occurs predominantly in some Asian countries and is a devastating global threat to sugarcane industries, especially Sri Lanka. Therefore, a feasible and an effective approach to precisely monitoring WLD infection is important, especially at the early pre-visual stage. This work presents the first approach on the preliminary detection of sugarcane WLD by using high-resolution multispectral sensors mounted on small unmanned aerial vehicles (UAVs) and supervised machine learning classifiers. The detection pipeline discussed in this paper was validated in a sugarcane field located in Gal-Oya Plantation, Hingurana, Sri Lanka. The pixelwise segmented samples were classified as ground, shadow, healthy plant, early symptom, and severe symptom. Four ML algorithms, namely XGBoost (XGB), random forest (RF), decision tree (DT), and K-nearest neighbors (KNN), were implemented along with different python libraries, vegetation indices (VIs), and five spectral bands to detect the WLD in the sugarcane field. The accuracy rate of 94% was attained in the XGB, RF, and KNN to detect WLD in the field. The top three vegetation indices (VIs) for separating healthy and infected sugarcane crops are modified soil-adjusted vegetation index (MSAVI), normalized difference vegetation index (NDVI), and excess green (ExG) in XGB, RF, and DT, while the best spectral band is red in XGB and RF and green in DT. The results revealed that this technology provides a dependable, more direct, cost-effective, and quick method for detecting WLD.

The Diversity, Distribution and Status of Phytoplasma Diseases in China

Phytoplasmas are important prokaryotic pathogenic bacteria without cell walls, which were formerly known as mycoplasma-like organisms, and belong to the Mollicutes class, Candidatus Phytoplasma genus. They are widely distributed in plants and insects, and can cause serious diseases in important food crops, vegetables, fruit trees, ornamental plants and trees, resulting in huge economic losses. To date, more than 100 phytoplasma diseases have been reported in China, which are distributed throughout the country. Jujube witches'-broom, paulownia witches'-broom, wheat blue dwarf, banana bunchy top, sugarcane white leaf, rice orange leaf and mulberry dwarf represent the phytoplasma diseases causing the most serious damage in China. New phytoplasma diseases and their strains are being reported continuously, indicating that phytoplasmas are more diverse than previously thought. Phytoplasmas are mainly transmitted by insect vectors, such as leafhopper and planthopper, and can also be spread by grafting or Cuscuta australis (known as dodder). Mixed infections of phytoplasmas and viruses, bacteria, and spiroplasmas have also become a serious problem in several crops and are responsible for more synergistic losses. With the continuous development and improvement of technology, molecular biological detection has become the main technique for phytoplasma detection and identification. Currently, research on phytoplasma diseases in China mainly focuses on pathogen identification and classification, and insect vector and host diversity; however, there is less focus on pathogenicity, comparative genomics, and effect factors. More research attention has been paid to wheat blue dwarf phytoplasma, paulownia witches'-broom phytoplasma, jujube witches'-broom phytoplasma, and sugarcane white leaf phytoplasma. Other phytoplasma diseases have been reported; however, there have been no in-depth studies. In this paper, the history and present situation of phytoplasma research, and the status, distribution, and diversity of phytoplasma diseases are summarized, and some possible research directions of phytoplasma in the future in China are proposed.

Sugarcane white leaf phytoplasma (white leaf of sugarcane)

sugarcane white leaf phytoplasma (white leaf of sugarcane)

Naked-Eye Colorimetric Loop-Mediated Isothermal Amplification for Rapid Screening of the Sugarcane White Leaf Phytoplasma

Naked-Eye Colorimetric Loop-Mediated Isothermal Amplification for Rapid Screening of the Sugarcane White Leaf Phytoplasma

The role of salicylic acid and benzothiadiazole in decreasing phytoplasma titer of sugarcane white leaf disease

The objective of this research was to study the effect of Benzothiadiazole (BTH) and Salicylic acid (SA) on the systemic acquired resistance (SAR) of sugarcane the phytoplasma associated with the sugarcane white leaf (SCWL) disease. The experiment was conducted on plants of the sugarcane variety Khon Kaen 3 (KK3) infected with SCWL phytoplasma using insect vectors. Biochemical changes related to the SAR such as SA and total phenolic compounds were followed according to 4 different timepoints: 7, 14, 21 and 28 days after inoculation. Together, phytoplasma were quantified by RT-qPCR using the secA gene of phytoplasma. According to our results, the spraying of BTH and SA tended to increase the amounts of SA, total phenolic compounds and a lower presence of phytoplasma in the plants in comparison with the inoculated control. Spraying BTH at a concentration of 2.4 mM and SA at a concentration of 2.4 mM exhibited the best efficiency to reduce the concentration of phytoplasma. According to RT-qPCR results, the inoculated plants sprayed with BTH displayed a significantly lower concentration of phytoplasma compared to the inoculated controls. Overall, our results indicated that the spray of BTH and SA could induce an efficient SAR response to the phytoplasma associated with the SCWL disease. We expect these results will give support to the development of new products for controlling white leaf disease in sugarcane.

Analysis of the transmission mode of sugarcane white leaf phytoplasma in Gengma, Yunnan Province, China

AbstractSugarcane white leaf (SCWL) is a destructive new sugarcane disease that has spread rapidly throughout low‐latitude plateau regions of Yunnan Province, China. To determine the transmission mode of SCWL phytoplasma in this region, the transmission mode of SCWL phytoplasma was analysed in the Gengma sugarcane area of Lincang City, Yunnan Province, where SCWL is severe. Five susceptible sugarcane varieties were grown with phytoplasma‐infected and pathogen‐free seedcane in 2018–2019, and then, systematic comparisons of newly emerging and ratoon crops were carried out. Comparisons using insect‐proof netting were also performed to confirm the role of insect vectors. The average rate of disease in newly emerging and ratoon crops grown with phytoplasma‐infected seedcane was 11.7%–72.9%, while in plants grown with pathogen‐free seedcane, 0% disease was observed. Moreover, the average rate in plants grown with pathogen‐free seedcane under insect‐proof netting was also 0%. These results suggest that the main transmission mode of SCWL phytoplasma was seedcane with phytoplasma in Gengma sugarcane area (SCWL serious occurrence area) of Lincang City, Yunnan Province. These findings provide a foundation for further studies of sugarcane phytoplasma disease, as well as a scientific basis for the effective prevention and control of SCWL in low‐latitude plateau regions in Yunnan Province.

Molecular identification of 16SrXI-B subgroup of phytoplasma related strain with yellow leaf disease of sugarcane (Saccharum officinarum) in India

In India, yellow leaf disease (YLD) is known to be caused by Sugarcane yellow leaf virus (SCYLV) and Sugarcane yellows phytoplasma (SCYP; 16SrI-B). Therefore, present study was conducted to confirm the association of specific phytoplasma with YLD in Co 0238 and CoS 510 sugarcane genotypes during 2019-2020 at Indian Institute of Sugarcane Research, Lucknow. The YLD affected genotypes were characterized by yellowing of leaf mid-ribs and leaf-lamina, leaf-tip necrosis, bunching of leaves at crown region and drying of plants with an incidence ranged from 28.26-35.81%. YLD symptomatic leaf samples were collected from the two genotypes and subjected to genomic DNA extraction by DNeasy Plant Mini Kit followed by nested PCR assay using universal phytoplasma primers (16S rDNA region). The nested PCR assays of samples from both the genotypes showed specific amplification. 16S rRNA gene sequence analyses of CoS 510 isolate (MN913611) revealed 100% identity with Sugarcane white leaf phytoplasma (SCWLP) belongs to 16SrXI-B subgroup. Similarly, virtual RFLP analyses revealed 1.00 similarity coefficient with 16SrXI-B subgroup. Thus, present study first time reports the association of SCWLP related strain with YLD of sugarcane in India. Phytoplasma infecting sugarcane are more diverse and needs systematic studies on their single or mixed infections in different varieties in India.

Tettigoniella viridis and Clovia conifer are potential insect vectors of sugarcane white leaf phytoplasma

Tettigoniella viridis and Clovia conifer are potential insect vectors of sugarcane white leaf phytoplasma

Geographical Distribution and Identification of Phytoplasma Strain Associated with Sugarcane White Leaf Disease in Sri Lanka

Sugarcane white leaf (SCWL) is one of the major sugarcane diseases in the Asian continent associated with phytoplasmas. It occurs in all sugarcane-growing areas of Sri Lanka, causing substantial economic losses. However, subgroup level identification has been not reported so far for the phytoplasma strains associated with this disease in Sri Lanka. In the present study, the geographical distribution of phytoplasma strains associated with SCWL in Sri Lanka, identification of their weed reservoirs and ribosomal subgroup analysis based on phytoplasma 16S rRNA gene were carried out. A total of 27 SCWL samples representing six main sugarcane-growing areas of Sri Lanka and six samples of two grass species (Cynodon dactylon and Brachiaria distachya) showing putative symptoms of phytoplasma were analyzed. A nested PCR product of 1.2 kb size was consistently amplified in all the symptomatic samples with primers amplifying the 16S rRNA gene. A higher level of sequence identity from 99.03 to 100% was shared among the 16S rRNA gene sequences of all tested SCWL phytoplasma strains. Both Bermudagrass white leaf (BGWL) and Brachiaria grass white leaf (BraWL) phytoplasma strains shared 97.5–98.1% identity of their 16S rRNA gene with SCWL strains. The 16S rRNA gene sequence comparisons and virtual RFLP analysis of SCWL, BGWL, and BraWL phytoplasma isolates allowed their affiliations with ‘Candidatus Phytoplasma sacchari’ (16SrXI-B subgroup) with SCWL and ‘Ca. P. cynodontis’ (16SrXIV-A subgroup) with the grasses samples. The phytoplasma subgroup 16SrXI-B was identified as the most widespread SCWL phytoplasma subgroup in commercial sugarcane varieties in Sri Lanka.

Alteration in the Stylet Probing Behavior and Host Preference of the Vector Matsumuratettix hiroglyphicus (Hemiptera: Cicadellidae) After Infection with Sugarcane White Leaf Phytoplasma.

The leafhopper (Matsumuratettix hiroglyphicus (Matsumura) (Hemiptera: Cicadellidae)) is a crucial insect vector of the phytoplasma associated with sugarcane white leaf (SCWL) disease. The aim of this study was to compare the stylet probing behaviors of M. hiroglyphicus on healthy sugarcane plants, asymptomatic, and symptomatic SCWL-infected sugarcane plants, using DC electropenetrography. We also used host-selection preference (free-choice) assays to identify the preferred types of host plants, and scanning electron microscopy to observe stylet puncture holes and salivary flanges after leafhopper probing. According to a quantitative analysis of M. hiroglyphicus stylet probing, mean durations per insect of both phloem ingestion (waveform D; the phytoplasma-acquisition behavior) and phloem salivation (waveform C; the phytoplasma-inoculation behavior) were significantly longer on both types of infected sugarcane than on healthy plants. These longer overall durations were mainly because the same number of significantly longer-duration C and D events was performed on infected sugarcane compared with healthy plants. On free-choice tested plants, M. hiroglyphicus displayed a significantly greater preference to settle on the infected plants (both types) than the healthy sugarcane. These results provide the first empirical evidence that acquiring the SCWL phytoplasma alters the host selection and stylet probing behaviors of its main vector (M. hiroglyphicus). Our study thus contributes to a better understanding of the interactions between the insect vector and SCWL phytoplasma-infected plants, and will aid in developing novel disease management tactics for sugarcane.

Development of loop mediated isothermal amplification assays for the detection of sugarcane white leaf disease

The Sugarcane white leaf (SCWL) phytoplasma from group 16SrXI is the causal agent of Sugarcane white leaf disease (SWLD) resulting in severe yield losses to the sugar industry. Although microscopy, serology based methods, dot blot DNA hybridization, and PCR have been developed as diagnostic tests, these techniques are labour intensive, costly and time-consuming and can only be undertaken in well-equipped laboratories. Therefore, the objective of the present study was to develop a loop-mediated isothermal amplification (LAMP) assay for SCWL as an alternative approach for quick and efficient detection of the sugarcane white leaf (SCWL) phytoplasma within 30 min. Three LAMP primer sets including a universal 16S rRNA based phytoplasma assay and two 16SrXI group-specific assays were used to detect the 16SrXI SCWL phytoplasma. Plant cytochrome oxidase (cox) LAMP primers that amplify a housekeeping gene in plants were used as controls. LAMP assays using a commercially available master mix and a real-time fluorometer allowed detection of the SCWL phytoplasma within 30 min at 63 °C via the visualization of an amplification plot on screen. The results obtained in this study revealed potential applicability of the LAMP-based assay with 16SrXI SCWL primers for in-field detection of SCWL in comparison with traditional methods that are labour-intensive and prone to cross-contamination during their implementation.

Survey of Incidence and Nested PCR Detection of Sugarcane White Leaf in Different Varieties.

Sugarcane white leaf (SCWL) is a devastating sugarcane (Saccharum officinarum) disease caused by a 16SrXI group phytoplasma, which is extremely harmful to sugarcane production. To determine the occurrence of SCWL in different varieties in 2018, we conducted a field survey and performed nested PCR detection of SCWL phytoplasma in cane-planting areas of Mangweng and Hepai in Gengma, Yunnan province, which are the areas most severely affected by SCWL in China. The results of the field survey showed that the symptomatic incidence of SCWL differed among varieties. The mean symptomatic incidence of SCWL on variety Yuetang60 was the highest (73.50%), and it was the lowest on Liucheng05-136 (13.67%). Using nested PCR, the SCWL phytoplasma was detected in symptomatic plants of all varieties more than 90% of the time; the SCWL phytoplasma was detected in 91 and 97% of symptomatic plants of Yingyu91-59 and Liucheng05-136 varieties, respectively. The SCWL phytoplasma was detected by PCR in 82% of the asymptomatic plant samples. The results of this study showed that field survey based on white leaf symptoms did not accurately reflect the actual occurrence of the SCWL phytoplasma.

First report of a ‘Candidatus Phytoplasma asteris‘‐related strain associated with Melia azedarach phyllody in India

Chinaberry tree (Melia azedarach), also known as bead tree and belonging to the family Meliaceae, is an important ethnomedicinal tree with a wide natural distribution (Ntalli et al., 3). Its roots, stem and leaves possess various medicinal properties and have applications in various types of medicine. Chinaberry trees showing phyllody-like symptoms and virescent flowers were observed in Narayangoan (Pune), Maharashtra, in April 2018. Samples of four plants with symptoms and four without symptoms (Figs. 1-2) were collected and tested to detect the presence of phytoplasmas and for further molecular characterisation. Total genomic DNA was isolated from petioles of both healthy and infected plants using a DNeasy Plant Mini Kit (Qiagen, Germany). PCR was performed using P1/P7 phytoplasma-specific universal primers followed by nested PCR with R16F2n/R16R2 primers (Deng & Hiruki, 1; Schneider et al., 4; Gundersen & Lee, 2). Bands of the expected size (c. 1.8 and 1.2 kb, respectively) were obtained in samples from all symptom-bearing plants but not in the symptomless ones. The 16Sr DNA sequence amplified from a representative sample from a symptomatic plant was deposited in GenBank (Accession No. MN830223). Sequence comparison by BLAST analysis showed the highest sequence identity with members of the 16SrI phytoplasma group (aster yellows), such as Cocos nucifera lethal wilt phytoplasma (KY814724), Sandal spike phytoplasma (MK627541), Phyllanthus emblica witches' broom phytoplasma (MK627538), Sugarcane white leaf phytoplasma (MK627531) and Santalum album phytoplasma (MG865436). Phylogenetic analysis (Fig. 3) using the neighbour-joining method (MEGA X software) showed that the Melia azedarach phytoplasma sequence clustered within group 16SrI, subgroup B. Virtual RFLP analysis using iPhyClassifier (Zhao et al., 5) further confirmed that the Melia azedarach phytoplasma sequence shares 99.8% identity with that of ‘Candidatus Phytoplasma asteris’ reference strain (M30790), and that the phytoplasma is a ‘Candidatus Phytoplasma asteris‘-related strain, belonging to the 16Sr group I, subgroup B. To our knowledge, this is the first report of ‘Candidatus Phytoplasma asteris’ (16SrI-B) affecting M. azedarach in India. This finding holds significant importance for future epidemiological studies since M. azedarach could be an alternate host for phytoplasma (16SrI-B group) which infects several important vegetable crops cultivated in the country.

A new phytoplasma strain associated with the sugarcane white leafdisease in Vietnam

Sugarcane white leaf (SCWL) disease is one of the most important diseases of sugarcane in Vietnam. The presence of phytoplasmas in phloem tubes of infected plants was confirmed by DAPI staining. A nested PCR assay using R16mF2/R16mR1 and R16F2n/R16R2 primer pairs amplified products with approximate length of 1,250 bp from the symptomatic sugarcane samples. The sequence analysis indicated that the phytoplasma strains from Khanh Hoa province shared 100% sequence identity with previously sequenced SCWL phytoplasma strains that were related to ‘Candidatus Phytoplasma oryzae’ (16SrXI). The virtual RFLP patterns of a 1,127 bp sequence obtained with the restriction enzymes BfaI, BstUI, EcoRI, HaeIII, HinfI, HpaI, RsaI and TaqI indicate that this SCWL-associated phytoplasma from Vietnam is a different strain from those in the 16SrXI described ribosomal subgroups. The two insect species, Matsumuratettix hiroglyphicus and Yamatotettix flavovittatus, identified as the vectors of SCWL in Thailand, were identified and found to be infected also in the SCWL symptomatic fields in Vietnam. The transmission experiments from SCWL-infected leafhoppers to healthy sugarcane and to insect feeding medium failed, suggesting that the infected planting material is the major transmission manner of SCWL disease in Vietnam.

Molecular Identification of Sugarcane White Leaf in Puer, Yunnan Province, China

To confirm the phytoplasma association in 13 sugarcane white leaf (SCWL) symptomatic samples collected from Puer, China, nested PCR assays with phytoplasma universal primer pairs was performed. A ~ 1240 bp product was obtained from 10 out of 13 symptomatic samples, and 16S rRNA gene sequencing results showed that all sequences of SCWL isolates were 100% identical to each other. BLASTn analysis revealed 100% sequence similarity with the 16S rRNA genes from SCWL phytoplasma strains LC7 and LC9 from Lincang, Yunnan province, China belonging to 16SrXI-B subgroup. Virtual restriction fragment length polymorphism also confirmed that Puer SCWL strains were member of the 16SrXI-B subgroup.

Characteristics of sugarcane white leaf phytoplasma transmission by the leafhopper Matsumuratettix hiroglyphicus

AbstractThe leafhopper Matsumuratettix hiroglyphicus (Matsumura) (Hemiptera: Cicadellidae) is the most important vector of sugarcane white leaf (SCWL) phytoplasma that significantly affects the sugarcane crop in Asia. Here, we aimed to study the characteristics of SCWL phytoplasma transmission by M. hiroglyphicus. To this end, the stylet penetration activities performed during the acquisition access period (AAP) and inoculation access period (IAP) were investigated by the direct current electrical penetration graph technique and confirmed by quantitative polymerase chain reaction (qPCR). Additionally, the latent period (LP) of SCWL phytoplasma in the vector was determined by qPCR and localised by fluorescent in situ hybridisation. The results indicated that the acquisition of SCWL phytoplasma occurred during phloem ingestion (waveform D), whereas its inoculation was associated with salivation into the phloem sieve element (waveform C). The minimum AAP was 15 min and the minimum duration of phloem ingestion was 2.35 min. The minimum LP of SCWL phytoplasma in the vector was at least 14 days; then, SCWL phytoplasma moved to the salivary glands of the insect, enabling the transmission of the pathogen to the host plants. The minimum IAP for a successful transmission of SCWL phytoplasma to the host plants was 11–12 min, with a minimum duration of salivation into phloem of 1.35 min. The female vectors had higher SCWL phytoplasma copy numbers than the male vectors, and displayed faster AAP, IAP, and LP. Overall, our findings provide important information related to the feeding behaviour of M. hiroglyphicus and its effect on the transmission of SCWL phytoplasma.