White Leaf Disease Research Articles

Phytoplasma DNA Enrichment from Sugarcane White Leaves for Shotgun Sequencing Improvement.

Sugarcane white leaf (SCWL) disease, caused by Candidatus Phytoplasma sacchari, poses a significant threat to sugarcane cultivation. An obligate parasite, phytoplasma is difficult to culture in laboratory conditions, making the isolation of its DNA from the massive amount of plant host DNA extremely challenging. Yet, the appropriate amount and quality of plant microbiome-derived DNA are key for high-quality DNA sequencing data. Here, a simple, cost-effective, alternative method for DNA isolation was applied using a guanidine-HCl-hydroxylated silica (GuHCl-Silica)-based method and microbiome DNA enrichment based on size-selective low-molecular-weight (LMW) DNA by PEG/NaCl precipitation. qPCR analysis revealed a significant enrichment of phytoplasma DNA in the LMW fraction. Additionally, the NEBNext Microbiome DNA enrichment kit was utilized to further enrich microbial DNA, demonstrating a remarkable increase in the relative abundance of phytoplasma DNA to host DNA. Shotgun sequencing of the isolated DNA gave high-quality data on the metagenome assembly genome (MAG) of Ca. Phytoplasma sacchari SCWL with completeness at 95.85 and 215× coverage. The results indicate that this combined approach of PEG/NaCl size selection and microbiome enrichment is effective for obtaining high-quality genomic data from phytoplasma, surpassing previous methods in efficiency and resource utilization. This low-cost method not only enhances the recovery of microbiome DNA from plant hosts but also provides a robust framework for studying plant pathogens in complex plant models.

Transcriptomic Profiling of Sugarcane White Leaf (SCWL) Canes during Maturation Phase.

Sugarcane white leaf (SCWL) disease, caused by Candidatus Phytoplasma sacchari, results in the most damage to sugarcane plantations. Some SCWL canes can grow unnoticed through the maturation phase, subsequently resulting in an overall low sugar yield, or they can be used accidentally as seed canes. In this work, 12-month-old SCWL and asymptomatic canes growing in the same field were investigated. An abundance of phytoplasma in SCWL canes affected growth and sugar content as well as alterations of transcriptomic profiles corresponding to several pathways that responded to the infection. Suppression of photosynthesis, porphyrin and chlorophyll metabolism, coupled with an increase in the expression of chlorophyllase, contributed to the reduction in chlorophyll levels and photosynthesis. Blockage of sucrose transport plausibly occurred due to the expression of sugar transporters in leaves but suppression in stalks, resulting in low sugar content in canes. Increased expression of genes associated with MAPK cascades, plant hormone signaling transduction, callose plug formation, the phenylpropanoid pathway, and calcium cascades positively promoted defense mechanisms against phytoplasma colonization by an accumulation of lignin and calcium in response to plant immunity. Significant downregulation of CPK plausibly results in a reduction in antioxidant enzymes and likely facilitates pathogen invasion, while expression of sesquiterpene biosynthesis possibly attracts the insect vectors for transmission, thereby enabling the spread of phytoplasma. Moreover, downregulation of flavonoid biosynthesis potentially intensifies the symptoms of SCWL upon challenge by phytoplasma. These SCWL sugarcane transcriptomic profiles describe the first comprehensive sugarcane-phytoplasma interaction during the harvesting stage. Understanding molecular mechanisms will allow for sustainable management and the prevention of SCWL disease-a crucial benefit to the sugar industry.

Enhancing the Efficacy of Fungal Biocontrol Agents for Matsumuratettix hiroglyphicus, the Vector of Sugarcane White Leaf Disease, Through Serial Passaging

Enhancing the Efficacy of Fungal Biocontrol Agents for Matsumuratettix hiroglyphicus, the Vector of Sugarcane White Leaf Disease, Through Serial Passaging

Workshop on Innovative Strategies for White Leaf Disease Management in Sri Lanka

Workshop on Innovative Strategies for White Leaf Disease Management in Sri Lanka

Complete genome sequence of "Candidatus Phytoplasma cynodontis" GY2015, a plant pathogen associated with Bermuda grass white leaf disease in Taiwan.

The complete genome sequence of "Candidatus Phytoplasma cynodontis" strain GY2015, which consists of one 498,922-bp circular chromosome, is presented in this work. This uncultivated plant-pathogenic bacterium is associated with Bermuda grass white leaf disease in Taoyuan, Taiwan.

Nested PCR-Based Rapid Detection of Phytoplasma Leaf Wilt Disease of Coconut in Sri Lanka and Systemic Movement of the Pathogen

Phytoplasmas are associated with many plant diseases. In palms, lethal bronzing disease, Texas Phoenix palm decline, and coconut lethal yellowing decline are some of them. In Sri Lanka, coconut leaf wilt decline has been reported in the Weligama area of the Southern province, and the disease is called Weligama coconut leaf wilt disease (WCLWD). Unlike other phytoplasma diseases of palms, WCLWD shows slow disease progress. Pathogen detection entirely relies on nested polymerase chain reaction (PCR). However, inconsistencies in pathogen detection have been experienced, i.e., symptomatic plants often produce negative results. The objectives of this study were to reconsider the choice of primers and to determine the best sampling tissue types for consistent detection of the pathogen. Among the six universal primer combinations tested, P1/Tint nested with fU5/rU3 produced consistent results. BLASTn searches of the sequences showed 99-100% similarity to sugarcane white leaf disease (SWL) or grassy shoot (SGS) disease-causing phytoplasma. The optimized nested PCR protocol was successful, with the minimum success rating of 88% and 100% specificity. Midribs of milky white bud leaf samples were the best tissue type for rapid detection. Systemic movement of the pathogen and a tentative latent period were also reported. The findings are helpful in the early detection of the disease.

Enhancing the survival of sugarcane infected white leaf disease by using bacterial indoleacetic acid producers

Enhancing the survival of sugarcane infected white leaf disease by using bacterial indoleacetic acid producers

Detection of White Leaf Disease in Sugarcane Crops Using UAV-Derived RGB Imagery with Existing Deep Learning Models

White leaf disease (WLD) is an economically significant disease in the sugarcane industry. This work applied remote sensing techniques based on unmanned aerial vehicles (UAVs) and deep learning (DL) to detect WLD in sugarcane fields at the Gal-Oya Plantation, Sri Lanka. The established methodology to detect WLD consists of UAV red, green, and blue (RGB) image acquisition, the pre-processing of the dataset, labelling, DL model tuning, and prediction. This study evaluated the performance of the existing DL models such as YOLOv5, YOLOR, DETR, and Faster R-CNN to recognize WLD in sugarcane crops. The experimental results indicate that the YOLOv5 network outperformed the other selected models, achieving a precision, recall, mean average precision@0.50 (mAP@0.50), and mean average precision@0.95 (mAP@0.95) metrics of 95%, 92%, 93%, and 79%, respectively. In contrast, DETR exhibited the weakest detection performance, achieving metrics values of 77%, 69%, 77%, and 41% for precision, recall, mAP@0.50, and mAP@0.95, respectively. YOLOv5 is selected as the recommended architecture to detect WLD using the UAV data not only because of its performance, but this was also determined because of its size (14 MB), which was the smallest one among the selected models. The proposed methodology provides technical guidelines to researchers and farmers for conduct the accurate detection and treatment of WLD in the sugarcane fields.

Roles of β-Indole Acetic Acid (IAA) Producing Endophytic Bacteria on the Recovery of Plant Growth and Survival Ability of Sugarcane Infected White Leaf Disease (SWLD).

β-Indole acetic acid is produced in the rhizosphere by endophytic bacteria and promotes plant growth. Effects of bacterial IAA producers (BIPs) on plant growth and recovery of sugarcane seedlings infected with phytoplasma causing white leaf disease (SWLD) were examined. Fifty-five endophytic bacteria isolated from rice roots were collected from the Mekong River Delta, Vietnam. Seven isolates showed β-Indole acetic acid production in culture medium supplemented with tryptophan. Interestingly, two of them (BC17 and BTII2) produced the highest β-Indole acetic acid after 4days of culture. Based on 16S rRNA sequences and phylogenetic analysis, the BC17 and BTII2 isolates were identified as Delftia lacustris and Rahnella aquatilis, respectively. Plant growth induced by the BC17 and BTII2 isolates showed statistically significant differences in height, root length and fresh weight of rice seedlings compared with non-treatment as the control. Treatment of two bacterial isolates in SWLD infected sugarcane plants also showed differences in height of sugarcane seedlings, while gradual symptoms of exposure decreased plant mortality compared to non-treatment as the control. BIPs were shown to be efficient biofertilizer inoculants that promoted plant growth and also ameliorated damage caused by phytoplasma-associated diseases at the sugarcane seedling stage.

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.

Rice hoja blanca virus (white leaf disease of rice)

Rice hoja blanca virus (white leaf disease of rice)

Discovery of potential protein biomarkers associated with sugarcane white leaf disease susceptibility using a comparative proteomic approach.

Sugarcane white leaf disease (SCWLD) is caused by phytoplasma, a serious sugarcane phytoplasma pathogen, which causes significant decreases in crop yield and sugar quality. The identification of proteins involved in the defense mechanism against SCWLD phytoplasma may help towards the development of varieties resistant to SCWLD. We investigated the proteomes of four sugarcane varieties with different levels of susceptibility to SCWLD phytoplasma infection, namely K88-92 and K95-84 (high), KK3 (moderate), and UT1 (low) by quantitative label-free nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). A total of 248 proteins were identified and compared among the four sugarcane varieties. Two potential candidate protein biomarkers for reduced susceptibility to SCWLD phytoplasma were identified as proteins detected only in UT1. The functions of these proteins are associated with protein folding, metal ion binding, and oxidoreductase. The candidate biomarkers could be useful for further study of the sugarcane defense mechanism against SCWLD phytoplasma, and in molecular and conventional breeding strategies for variety improvement.

Role of leaf fibro-vascular bundle configuration on feeding of <em>Deltocephalus menoni</em> (Hemiptera: Cicadellidae) in the transmission of White Leaf Disease in sugarcane varieties

Purpose: Deltocephalus menoni is the vector of Sugarcane White Leaf Disease (WLD) and the causative agent of White Leaf Disease Phytoplasma (WLDP). WLDP is restricted to the phloem tissues facilitating its dispersal via sap containing phytoplasma during the vector feeding. Here, the configuration of the vascular bundle would be an important characteristic in plant’s resistance to vector feeding and thereby the disease spread. A study was conducted to determine the relationship between feeding of D. menoni and the configuration of leaf fibro-vascular bundles. Research Method: Feeding amount of D. menoni on sugarcane varieties, the configuration of leaf fibro-vascular bundle of leaf blade in gross cross-section and the WLD infection in the natural environment were measured. Variations and correlations between configuration of leaf fibro-vascular bundles between varieties, vector feeding and WLD infection were studied. Major anatomical characteristics of vascular bundles that contribute to feeding were also determined. Findings: The structure of the leaf fibro-vascular bundles varied with the sugarcane variety/ accession and the volume of feeding by D. menoni of sugarcane was correlated with the configuration of the leaf fibro vascular bundle. Distance from the epidermis to vascular parenchyma, distance from epidermis to vascular bundle and thickness of phloem fiber layer were negatively correlated with the feeding amount of D. menoni. The size of phloem tubes, the width of the vascular bundle and the total number of vascular bundles, were positively correlated. The level of WLD infection in natural environment was also correlated with the structure of leaf fibro vascular bundle. Originality/value: Configuration of leaf fibro vascular bundles could be used to predict feeding of D. menoni, level of WLD infection, which could be incorporated into selection and directional breeding of sugarcane against the vector.

Disease Transmission Ability and Pattern of Deltocephalus menoni (Hemiptera: Cicadellidae) Vector of Sugarcane White Leaf Disease

Sugarcane White Leaf Disease (WLD) is one of the most devastative phytoplasma disease which cause substantial losses to the sugar cane industry in Sri Lanka. Leaf hopper Deltocephalus menoni (Hemiptera: Cicadellidae) is the locally-identified vector of WLD. Disease transmission proficiency of the vector, is a key factor to consider in disease management. Therefore, the present study was conducted to find out the ability of males, females and nymphs of D. menoni in transmitting the WLD and to determine the level of persistency of D. menoni in WLD phytoplasma (WLDP) transmission. To determine the percentage WLD transmission by virulified D. menoni males, females, and nymphs were introduced to one-month-old healthy potted plants in insect proof field cages for a one week period. Test plants were maintained until WLD symptom development. In estimating the WLD transmission pattern of D. menoni, virulified one-day- old adult females were introduced to the healthy sugarcane plants allowing them to feed on healthy plant for 24h period. Then they were recollected and introduced to another set of healthy sugarcane plants. Above procedure was repeated in 24h frequency for 13 days. Test plants were maintained in insect proof field cages for three-month period. The Presence of WLDP in the tested-plants was confirmed by PCR with WLDP specific SPP1/SPP2 primers. Under local conditions, female and male adults, 2nd and 4th instar nymphs were capable of transmitting the WLDP to the healthy plants in a rate of 55.5, 44.5, 22.2 and 33.3% respectively. The transmission efficiency of females is comparatively highthan that of adult male insects. Adult female of D. menoni was capable to transmit the disease from just after acquisition of the WLDP up to maximum of 11 days. Results imply that D. menoni is incapable to propagate the WLDP within its body and circulative transmission dose not occurred within the insect. D. menoni is an efficient vector in secondary transmission of the WLD and it is a semi-persistent vector. Due to semi-persistent nature of the vector, the management practices are vital to prevent rapid spread of this disease in sugarcane plantations.

Varietal Screen for Susceptible stage of <em>Saccharum</em> hybrids for <em>Deltocephalus menoni</em> (Hemiptera: Cicadellidae), vector of sugarcane White Leaf Disease

<em>Deltocephalus menoni </em>is a sap-sucking insect causing severe losses to the cane sugar industry by feeding on the crop and acting as the vector of Sugarcane white leaf disease (WLD) in Sri Lanka. This study was conducted to determine the most preferred age of the sugarcane plant for optimum feeding of <em>D.</em> <em>menoni</em>. Sugarcane plants of the varieties SL 92 5588, SL 97 1442, SLC 2009 01, and SL 96 128 at six age categories from 1-6 months, were selected for the study. Female <em>D. menoni </em>adults fed on to measure the amount of feeding. Variation of the feeding of <em>D. menoni </em>on each variety with the plant age was estimated separately. The feeding of the <em>D. menoni </em>significantly varied with the age of the crop, and the highest feeding rate of 6.8 mm2, was recorded on four months old sugarcanes. The amount of secreted honeydew, measured as an indicator of the suitability of sugarcane for feeding of <em>D. menoni</em>, increased gradually from one month and reached the peak at four-month age. When the plant turned five-month-old, the amount of honeydew secretion reduced gradually, and the honeydew secretion significantly dropped at six months. A similar trend was observed in the amount of honeydew secreted by feeding on each variety, including the resistant check. The highest amount of feeding, 6.8, was recorded at four-month age, indicating four-month age of the sugarcane hybrids is the most vulnerable stage for <em>D. menoni</em> feeding, and the three to five-month period is the susceptible period for feeding.

Population Dynamics of Wolbachia in the Leafhopper Vector Yamatotettix flavovittatus (Hemiptera: Cicadellidae).

Wolbachia (Rickettsiales: Alphaproteobacteria) infections induce abnormalities in the reproductive system and affect various biological traits of the host insects. The density of Wolbachia is one of the major parameters that influence induced phenotypes and interactions with the hosts. Wolbachia occurs naturally in populations of the leafhopper Yamatotettix flavovittatus Matsumura (Hemiptera: Cicadellidae), which transmits phytoplasma that cause white leaf disease in sugarcane. However, the quantity and dynamics of Wolbachia in this leafhopper are not well understood. In the current study, we estimated the number of Wolbachia by absolute quantification of the copy number of wsp, which encodes the outer surface protein, using real-time quantitative polymerase chain reaction (PCR). This investigation was performed using natural populations and laboratory colonies from three lineages of leafhoppers (designated as UD, KK, and SK). There was no significant difference in the number of wsp copies in most of field-collected adults. During the immature developmental stages, there were differences in the dynamics of Wolbachia infection between the UD lineage and the other two lineages. However, the number of wsp copies increased in the early instar and plateaued in the later nymphal instars. Sex had no influence on the number of Wolbachia within the same lineages. The number of Wolbachia was relatively constant during the adult stage in the UD lineage but fluctuated in the other two lineages. In conclusion, the present data provide a framework for exploring the relationship between Wolbachia and the leafhopper and could facilitate future research into management strategies using Wolbachia.

Characterisation of phytoplasma associated with sugarcane white leaf disease in Uttar Pradesh, India

During the survey of six districts of Uttar Pradesh in 2013–2017, white leaf, grassy appearance, green grassy with no cane formation, leaf yellows, stunted growth were observed on sugarcane grown on farmer’s field. Two symptomatic from each surveyed location along with one non symptomatic canes were analysed through nested 16S rRNA primers (P1/P7 and R16RF2n/R16R2). All the symptomatic leaves were found positive and BLASTn search revealed highest 99% sequence identity with 16SrXI-B subgroups phytoplasma. The phylogenetic analysis further confirmed the BLSATn results and isolates revealed relationship with isolates of 16SrXI-B subgroup. The wide occurrence of phytoplasma disease on sugarcane is because of using infected propagation materials, therefore it is suggested to use hot water treated seed material and maintain the healthy seed nursery to avoid the further spread.

First report of a 'Candidatus Phytoplasma trifolii'-related strain (16SrVI-A) associated with white leaf disease in Spirea japonica

In August of 2018 phytoplasmas naturally infecting Spirea japonica were found in Şanlıurfa province, Turkey and they were identified by nested polymerase chain reaction (PCR), restriction fragment length polymorphism analysis (RFLP) and phylogeny based on sequence analysis. Plants exhibited typical white leaf disease symptoms with reduced leaf size. The phytoplasmas were found to be closely related to ‘Candidatus Phytoplasma trifolii’ (16SrVI-A). This paper has reported that Spirea japonica is a new host for ‘Ca. Phytoplasma trifolii’ which is associated with the white leaf disease in S. japonica.

Tri-Trophic Relationship among Sugarcane White Leaf Disease Phytoplasma (WLDP), &lt;em&gt;Deltocephalus menoni&lt;/em&gt; (Homoptera: Cicadellidae) and Sugarcane Plant in Secondary Transmission of the Disease

Purpose: Sugarcane White Leaf Disease (WLD) is one of the most devastative diseases in cane-sugar industry in Sri Lanka and the causative agent is a distinct phytoplasma (WLDP) strain within the 16SrXI. White Leaf Disease spreads through infected seed cane and insect vector; Deltocephalus menoni (Hemiptera: Cicadellidae). This paper presents the tri-trophic relationship of the plant, vector and pathogen in disease transmission. Research Method: Four-month-old sugarcane plants of the variety SL 96 128 and laboratory-reared populations of D. menoni were used for this study. Performance of D. menoni on healthy plants and WLD plants was examined in relation to the aggregation, amount of feeding, fecundity, number of nymphs emerged, nymphal period, survival of nymph and adult’s longevity. The plant architecture, leaf histological and physiological aspects were also studied. Findings: Except for nymphal period, all behavioural characteristics were significantly-high in WLDinfected plants in 10, 14, 30, 8, 12 and 17 percent, respectively. The nymphal period of D. menoni was significantly-reduced by 12.78 percent. Number of shoots (41.83 percent) and leaves (18.96 percent) were high and distance between two leaves tends to be reduced by 32.85 percent in diseased-plants altering plant architecture into a profuse and bushy nature. Sucking distance, thicknesses of lower epidermis and phloem fibre layer in leaves were significantly reduced in infected plants. Leaf N, P, K and moisture content increased (24.89, 34.78, 28.64 and 3.85percent) while leaf wax content was reduced by 4.93 percent. Originality/value: Disease infected plants are augmenting its vector; D. menoni by attracting, enhancing survival and population build-up which promotes the transmission of WLD phytoplasma itself. There by regular inspection and rogueing out of WLD infected clumps make a great impact on secondary transmission of WLD by D. menoni.

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.