First report of 16SrII-V phytoplasma associated with cowpea (Vigna unguiculata) phyllody in China

Abstract Cowpea plants exhibiting virescence, phyllody and little leaf symptoms were observed in the fields of Zhanjiang City, Guangdong province of China in October 2020. Total DNA was extracted from symptomatic cowpea leaves. Using phytoplasma universal primer pairs (R16mF2/R16mR1, P1/P7) and a secA primer pair (secAfor1/secArev3), expected PCR fragments of approximately 1400, 1800 and 800 bp were amplified from all symptomatic cowpea leaves. MUSCLE analysis revealed that the representative 16S rRNA gene sequence of cowpea phyllody phytoplasma (CPP) shared 98.67% nucleotide identity with the reference strain WBDL of ‘Ca. Phytoplasma aurantifolia’ (GenBank accession number U15442); and 100% nucleotide identity with 16SrII-V phytoplasmas associated with ‘Praxelis clematidea’ phyllody (GenBank accession number KY568717); ‘Desmodium triflorum’ little leaf (GenBank accession number MT452308); and ‘Ixeris chinensis’, ‘Emilia sonchifolia’, and ‘Desmodium ovalifolium’ witches’ broom (GenBank accession number MT416114, MT420682 and MK956144, respectively). Phylogenetic analyses based on the 16S rRNA and secA gene sequences revealed that the CPP strain clustered with the 16SrII-V subgroup and 16SrII group, respectively. Virtual restriction fragment length polymorphism analysis of the 16S rRNA sequence revealed that CPP was associated with the 16SrII-V subgroup. To our knowledge, this is the first report of the16SrII-V subgroup of phytoplasmas associated with cowpea phyllody disease.