First Report of ' Candidatus Phytoplasma asteris' (16SrI-B subgroup) associated with stunting and little leaves of guar (Cyamopsis tetragonoloba L.) in the world

Guar or cluster bean (Cyamopsis tetragonoloba L.) is a leguminous crop well-suited for cultivation in arid and semi-arid regions. India accounts for 90% of world's guar production. In September 2022, little leaf, and stunted plants (Fig. 1) were observed on 50 days-old guar plants with a disease incidence of less than 10%. To determine the cause, ten each of symptomatic (CyteJod1-10) and asymptomatic (CyteJod11-20) guar leaves were collected from the same field. Insects were also collected using the sweep-net method in polythene bags from in and around the guar fields. DNA extraction was done from leaf midribs of all the collected samples using the DNeasy Plant Mini Kit (Qiagen, Germany). Of the various insects collected(including aphids, whiteflies, thrips, mites, cutworms and leafhoppers) only the leaf hopper species are known to be putative vectors of phytoplasma (Ranebennur et al. 2022). Based on the morphological characters(De Long, 1931), the only leafhopper found within the field was Empoasca sp. which was further confirmed at a reference facility at the Division of Entomology, ICAR-IARI, New Delhi. Ten adults (Empoasca, C. tetragonoloba Jodhpur) were labelled (ECyteJod1-10) and used for DNA extraction using the Blood and Tissue kit (Qiagen, Germany).Nested polymerase chain reaction (Nested-PCR) was employed to determine phytoplasma species association using universal primers P1/P7 followed by R16F2n/R16R2 (Gundersen and Lee, 1996) and secA gene primers (secAfor1/secArev3 and secAfor2/secArev3) (Hodgetts et al., 2008). Out of the 10 symptomatic plants and 10 leaf hopper samples, 4 leaf hopper samples and all the symptomatic plants produced approximately 1.25 kb and 480 bp for the 16S rRNA and secA gene, respectively. The PCR products were cloned and sequenced. Since, all the 10 sequences were identical, only phytoplasma sequences of 2 isolates (CyteJod1& 2) were submitted to NCBI GenBank with accession numbers OQ058847-OQ058848 for 16S rRNA and OQ067519-OQ067520 for secA gene. Nucleotide BLAST analysis of 16S rRNA sequences revealed a 100% (1244/1244 nucleotides) identity with Ca. P. asteris (16SrI-B) obtained from leaf hopper (Tachycixius sp. Accession no. KF583778) from Lebanon, Italy. BLAST analysis of secA sequences revealed 100% (487/487 nucleotide) sequence similarity with 16SrI group of Ayapana triplinervis (water hemp plant) phytoplasma strain (OM142605) from India. The iPhyClassifier tool (Zhao et al., 2009) classified the 16S rRNA gene sequences into 16Sr group I, subgroup B, with a similarity coefficient of 1.0. Phylogenetic analysis done through MEGA11 (Tamura et al., 2021) showed that the guar phytoplasma strains clustered with 16SrI-B phytoplasma reference strains (Fig. 2a, 2b). The association of Empoasca sp. with 16SrI-B phytoplasma group has previously been reported in moth bean from Jodhpur, India (Singh et al., 2023). Previously, association of 16SrII-D subgroup with guar as host was reported from Haryana, India (Rao et al., 2019). This is a first report of guar disease caused by 16SrI-B subgroup phytoplasma worldwide. This has epidemiological significance and needs immediate attention since the pathogen may spread to other legume crops in similar geographical regions, posing a threat to agricultural systems where legumes are intercropped. The report highlights a new potential vulnerability for the guar and other pulses, emphasizing the need for vigilance and research to protect legume cultivation globally.