Shailender Kumar

Characterization of a 16SrI-B Subgroup Phytoplasma Associated with Sugarcane Leaf Yellows Disease in India

Leaf mid-rib yellowing along with yellowing of adjoining lamina and development of irregular yellow patches on leaf lamina was observed on two sugarcane varieties (CoLk 94184, CoSe 92423) during 2013–2014 crop season. Phytoplasma association with leaf yellows and leaf lamina with yellow patches symptomatic samples was confirmed through PCR and nested PCR assays using phytoplasma specific primers P1/P6 followed by R16F2n/R16R2. Sequencing and BLASTn analysis of ~1.2xa0kb product of 16Sr RNA gene fragments of the two phytoplasmas shared 99 and 100xa0% sequence identity with strains of ‘Candidatus Phytoplasma asteris’ (16SrI). Further characterization through in silico restriction enzyme digestion of 16Sr DNA using iPhyClassifier online tool assigned both the strains into subgroup 16SrI-B and phylogenetic analyses confirmed that both the phytoplasmas were closely related to 16SrI group. This is the first report of association of 16SrI-B subgroup phytoplasma with sugarcane leaf yellows disease in India.

Identification of a novel phytoplasma (16Sr XIV-A subgroup) associated with little leaf and witches’ broom of Cannabis sativa L. ssp. sativa and C. sativa L. ssp. indica in India

Symptoms of yellowing, little leaf and witches’ broom on Cannabis sativa L. ssp. sativa and C. sativa L. ssp. indica plants were recorded in and around sugarcane fields at Shahjahanpur, Uttar Pradesh, India, during May–June 2013. Symptomatic plants yielded phytoplasma DNA bands of ~1.2xa0kb from nested PCR assays using universal phytoplasma primers P1/P6 and R16F2n/R16R2. BLASTn search analysis of both C. sativa ssp. phytoplasma isolates showed that the 16S rDNA sequences shared a 99xa0% of sequence identity between each other and with those of the phytoplasma strains of 16SrXIV group (‘Candidatus Phytoplasma cynodontis’). Phylogenetic analysis of the 16SrDNA sequences of both phytoplasma isolates revealed their closest relationship with members of the 16SrXIV group. This supports the association of phytoplasma strains of the 16Sr XIV group with Cannabis little leaf and witches’ broom diseases (Can LL-WB). Actual and virtual RFLP analysis of the 16S rDNA sequences of Can LL-WB phytoplasma isolates confirmed that they belonged to the 16Sr XIV-A subgroup. This is the first world report of a 16SXIV-A subgroup phytoplasma infection in C. sativa sp. sativa and C. sativa sp. indica plants.

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. Maiestasportica (Melichar), Exitianusindicus (Ross) and Cofanaunimaculata (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 99–100xa0% sequence identities among themselves and 99xa0% 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.

Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars

Recombinase polymerase amplification (RPA) is a rapid, isothermal amplification method with high specificity and sensitivity. In this study, an assay was developed and evaluated for the detection of banana bunchy top virus (BBTV) in infected banana plants. Three oligonucleotide primer pairs were designed from the replicase initiator protein gene sequences of BBTV to function both in RPA as well as in polymerase chain reaction (PCR). A total of 133 symptomatic as well as asymptomatic banana leaf samples from various cultivars were collected from the different regions of India and evaluated for BBTV infection using the RPA assay. BBTV was efficiently detected using crude leaf sap in RPA and the results obtained were consistent with PCR-based detection using purified DNA as template. To our knowledge, this is the first report of reliable diagnosis of BBTV infection by RPA using crude leaf sap as a template.

Exitianus indicus (Distant): a putative vector for 'Candidatus Phytoplasma cynodontis' in India

Chlorotic little leaves, witches’ broom and shortened stolons/rhizomes on Cynodon dactylon was observed in and around sugarcane fields at Sugarcane Research Institute, Shahjahanpur, Uttar Pradesh, India during August–September 2013. The association of phytoplasmas with symptomatic C. dactylon was confirmed by direct and nested PCR amplification of phytoplasma 16Sr gene. Out of the different prevalent species of leafhoppers on Bermuda grass, only Exitianus indicus tested positive for phytoplasma presence. Phylogenetic analysis of 16Sr gene sequence suggested that the identified phytoplasmas from C. dactylon and E. indicus in the present study were 99% similar and belonged to 16SrXIV group. The study suggests that, E. indicus may be a putative vector for ‘Candidatus Phytoplasma cynodontis’ and may play a role in transmitting 16SrXIV group phytoplasmas in nature.

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.

First Report of Mild Mosaic in Ground Orchid, Phaius tankervilleae, in India Associated with Infection of Calanthe mild mosaic virus

Phaius tankervilleae is a species of ground orchid widespread in the Indian subcontinent, China, Japan, Malaysia, Philippines, USA (Florida and Hawaii), Australia and is grown commercially in India. During a December 2016 survey in Sikkim and Darjeeling hills, several plants of P. tankervilleae were observed with mild mosaic and streak symptoms in the germplasm collection of National Research Centre for Orchids, Pakyong, Sikkim. Negatively stained preparations of symptomatic leaf samples showed flexuous filamentous particles measuring about 800 nm in length and 12 nm in width under the electron microscope. To further characterize this virus, total RNA was extracted from symptomatic leaf tissues of P. tankervilleae, using an RNeasy plant mini kit (Qiagen, Valencia, CA). RT-PCR results showed the absence of Cymbidium mosaic virus and Odontoglossum ringspot virus in the sample. RT-PCR was further performed using potyvirus generic primers (Van der Vlugt et al., 1999) that target the partial coat protein (CP) ...

Molecular characterization confirming phytoplasma association with spear rot disease of oil palm in India

Oil palm is grown in 0.179 million ha of 11 states of India. Among the diseases reported, spear rot disease (SRD), endemic to the plantations in the state of Kerala and with sporadic incidence in Sullia district of Karnataka, is the most important. Characteristic symptoms recorded were chlorosis of the youngest whorl of fronds, rotting of spear and reduction in the frond size. The affected palms succumb within five years from manifestation of symptoms. Phytoplasma presence was earlier reported based on phytoplasma detection in affected palms by electron microscopy observations, dodder transmission of the agent from diseased to healthy palms, and symptom reduction after oxytetracycline treatments. In the present study, molecular characterization by using universal phytoplasma specific primers based on 16S rRNA gene sequence confirmed the phytoplasma association with the disease. Sequence comparison and phylogenetic analysis revealed the association of 16SrXI group phytoplasmas with spear rot disease, first report in oil palm in the world.

Molecular confirmation and interrelationship of phytoplasmas associated with diseases of palms in South India

Suspected phytoplasma symptoms of yellowing of inner whorl of leaves and rotting of spear in oil palm, yellowing of outer whorl of leaves in arecanut and flaccidity, yellowing and necrosis of crown leaves in coconut were observed in Kerala and Karnataka states of India. Phytoplasma presence in symptomatic oil palm, arecanut and coconut was confirmed by nested PCR assays with phytoplasma specific universal primers. 16Sr DNA sequence analysis and phylogenetic relationship studies confirmed the presence of 16SrXI group phytoplasmas in spear rot disease of oil palm from Kollam and Wayanad districts and in yellow leaf disease of arecanut from Wayanad district of Kerala. However, 16SrXIV group phytoplasmas were identified from root (wilt) disease of coconut from Kollam district of Kerala and yellow leaf disease of arecanut from Sullia of Dakshina Kannada district of Karnataka. This is the first report of association of 16SrXIV group phytoplasma with root (wilt) disease of coconut and yellow leaf disease of arecanut. These studies indicated the possible involvement of more than one group of phytoplasmas (16SrXI and 16SrXIV) in palm diseases in South India.