RESUMO
Symptoms of suspected phytoplasma infection were observed in cauliflower (Brassica oleracea var. botrytis) (cultivar NS60N) at Integrated Farming System Research Station, Trivandrum, Kerala, India (08o28'28"N, 76o57'47"E) in April-2021. The disease incidence was recorded up to 10% in different fields. The disease manifested as stunting, phyllody, floral malformation and flattening of stem (Fig.1A,B). Ten symptomatic and five asymptomatic plants were assayed for the presence of phytoplasma using nested PCR assays performed with P1/P7 and R16F2n/R16R2 primer pairs for 16S rRNA gene and SecAfor1/ SecArev3 and SecAfor2/ SecArev3 for secA gene (Deng and Hiruki 1991; Gundersen and Lee 1996; Hodgetts et al. 2008). The expected amplicons of ~1.25 kb and ~480 bp were consistently amplified in all the symptomatic cauliflower samples with the phytoplasma specific universal 16S rRNA and secA gene specific primers. Nested PCR products (~1.2 kb and 480 bp) amplified from cauliflower was cloned in EcoRI restriction sites of pGEM-T Easy vector (Promega, USA). The cloned nested PCR products were directly sequenced (16S rRNA gene: Acc. Nos. MZ196223, MZ196224; secA gene: MZ215721, MZ215722) in both forward and reverse directions which showed 99.77% sequence identity with Candidatus Phytoplasma cynodontis reference strain (Acc. No. AJ550984). Further analyses of the 16S rRNA and secA genes based phylogenetic tree (Fig. 2A and B) and the iPhyClassifier-based virtual RFLP analysis of 16Sr RNA gene study demonstrated that the phytoplasma-associated with cauliflower phyllody & flat stem disease (CaPP) belonged to 16SrXIV-A subgroup with a similarity coefficient of 1.0. No amplicon was observed from any of the asymptomatic cauliflower plants with the specific tested primers of both the genes. Earlier association of 16SrXV-A subgroup (Candidatus Phytoplasma brasiliense) and 16Sr III-J subgroup in Brazil (Canale and Badendo, 2013; Rappussi et al. 2012), 16SrII-A (Candidatus Phytoplasma aurantifolia) subgroup in China (Cai et al. 2016) and 16SrVI-A (Candidatus Phytoplasma trifolii) subgroup in Iran (Salehi 2007) were reported in cauliflower. Another species of cabbage, Brassica oleracea var. capitata L. was reported as host of Ca. P. trifloii (16Sr VI-D subgroup) from north India (Gopala et al. 2018). To our knowledge, this is the first report of a 'Candidatus Phytoplasma cynodontis', 16SrXIV-A subgroup related phytoplasma strain associated with cauliflower phyllody and flat stem in the world. The results described in this report confirm that the 16SrXIV-A phytoplasma, a widely distributed strain associated with sugarcane, wheat, grasses, sapota and many ornamentals in India (Rao 2021), has also infected cauliflower. This is not only the first instance of cauliflower phyllody disease found in India, but also the first instance of CaPP disease caused by 16SrXIV-A subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as cauliflower is the one of the most common vegetable crop grown all over India.
RESUMO
Nano-PCR is a potential tool for the early detection of plant viruses. In the current study, different concentrations of silver nanoparticles (20 nm) and magnesium oxide nanoparticles (50 nm) were included in the PCR mixture to improve the sensitivity of PCR for the detection of tomato leaf curl virus. The inclusion of nanoparticles in single or combination in PCR mixture has resulted in improvement of PCR sensitivity. Four-fold improvement was exhibited by the inclusion of 3 ng/µL silver nanoparticles, whereas the combination of silver and magnesium oxide nanoparticles (3 ng/µL and 200 ng/µL, respectively), resulted in a 4.5-fold improvement. The inclusion of 200 ng/µL of magnesium oxide nanoparticles in the PCR mixture exhibited a 7.6-fold increase in PCR sensitivity. Replacement of magnesium chloride with a combination of silver and magnesium oxide nanoparticles (3 ng/µL and 275 ng/µL, respectively) resulted in a 12-fold increase. A 13-fold improvement in PCR sensitivity was observed by the replacement of magnesium chloride in PCR buffer with 275 ng/µL of magnesium oxide nanoparticles. This could also produce detectable amplicon in PCR with a minimum of 25 cycles, resulting in a 26.5% reduction in the duration of PCR. This is the first report on the use of magnesium oxide nanoparticles in PCR for the early detection and better management of tomato leaf curl virus. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03842-2.