First Report of Association of a ‘ <i>Candidatus</i> Phytoplasma trifolii’-Related Strain with Little Leaf and Declining Disease of Pulasan ( <i>Nephelium mutabile</i> Blume) in Kerala, India

Pulasan (<i>Nephelium mutabile</i> Blume; family Sapindaceae) is a tropical fruit known for its high orilagin, ascorbic acid and thiamine content, known for its potent antioxidant and anti-inflammatory properties. Native to Southeast Asia, pulasan is extensively cultivated in Malaysia, Thailand, Indonesia, Sri Lanka, India and Philippines (Afzaal et al., 2023). In India, pulasan is cultivated in tropics, where it is cherished as a homestead crop. A recent survey conducted in September 2024 at Pangode village (8°45'23.22"N 76°58'48.68"E), Thiruvananthapuram, Kerala, showed little leaf symptoms along with drying of side branches along with stunted growth in pulasan trees (variety P15). Only two pulasan trees were recorded with symptoms out of 20 trees examined in the campus. To investigate the molecular identity of the phytoplasma, leaf samples from two symptomatic and asymptomatic trees were collected and subjected to total DNA extraction by DNA extraction kit (Qiazen). The extracted DNA was amplified using nested PCR assays with universal primers P1/Tint (Smart et al., 1996) followed by 3Fwd/3Rev (Manimekalai et al., 2010) specific for the 16S rRNA gene and tuf gene-specific primer pairs fTufl/rTufl (Schneider et al., 1997). The PCR assays yielded amplicons of ~1.3 kb and ~1.1 kb for the 16S rRNA and tuf genes, respectively, from all the symptomatic leaf samples, whereas no amplification was observed from any asymptomatic samples. One sequence of each gene of pulasan phytoplasma strain (PK-1) were deposited into the GenBank database [(Acc Nos. PQ865399 (16S rRNA gene) and PQ877893 (tuf gene)]. BLASTn analysis of the both gene sequences of the PK strains revealed a 99.62% sequence identity with eggplant little leaf (Acc. No. MW273757; 16S rRNA gene) and 100% sequence similarity with <i>Cucurbita pepo</i> phytoplasma (Acc. No. KX358589; tuf gene) belongs to the clover proliferation phytoplasma subgroup. Phylogenetic analysis using MEGA 11 employing the Maximum likelihood method, demonstrated that the PK phytoplasma strains clustered within the 16SrVI-D subgroup-related strains. The virtual RFLP pattern generated for the 16S rRNA gene of PK phytoplasma strain through pDRAW analysis was found identical to the reference pattern of 16VI-D subgroup (Periwinkle little leaf phytoplasma; GenBank Acc. No. AF228053). Sequence comparison analysis confirmed the presence of a '<i>Candidatus</i> Phytoplasma trifolii'-related strain associated with pulasan showing little leaf disease and stunting in India. While, phytoplasma associations have been reported in various fruit species globally (Fiore et al., 2018; Mitra et al., 2023), this study represents the first documented case of a phytoplasma disease infecting pulasan worldwide. Notably, '<i>Ca.</i> P. trifolii' (16SrVI-D subgroup) has previously been identified associated with several economically important agricultural and horticultural crops worldwide (Zhao and Wei, 2020; Rao, 2021). The results of this study revealed that pulasan trees serve as an alternative natural reservoir for the '<i>Candidatus</i> Phytoplasma trifolii' (16SrVI-D) phytoplasma strain, may potentially facilitate its dissemination to other crops through insect vectors. Considering the significant phytosanitary concerns, epidemiological factors and economic impact of pulasan in Southeast Asia for its global export potential, the detection of phytoplasma-associated disease in pulasan is noteworthy as it not only poses a potential threat to cultivation and productivity but may also have broader implications for regional trade and biosecurity protocols.