Multi-gene sequence analysis: a functional method in classifying phytoplasma causing little leaf disease on selected crops in the Philippines

Phytoplasmas are cell wall-less, phloem-inhabiting prokaryotes that belong to the Class Mollicutes and associated with more than 600 plant diseases worldwide. These are obligate parasites and unculturable in vitro, thus given the pathogenic nature, the mode of transmission is difficult to establish despite of reported phloem-feeding insect vectors (Bertaccini et al. 2014), dodders (Cuscuta and Cassytha spp.) and grafting. The geographical distribution and impact of the diseases depend on the host range and feeding behavior of insect vectors. Phytoplasmas cause 'yellows' disease, 'witches' broom (proliferation of shoots and branches), little leaf (leaf stunting), phyllody (greening of flower petals), suppressed flowering and shoot growth, loss of apical dominance of the shoots, phloem necrosis, branch dieback, and progressive loss of vitality. Field survey and collection conducted from 2021-2023 in provinces of Luzon and Mindanao islands, revealed the high incidence of little leaf in bitter gourd (Momordica charantia) and sponge gourd (Luffa aegyptiaca) Philippine bolo Gigantochloa levis (Blanco) Merrill. and even common weed species Passiflora biflora. Detection of phytoplasma from symptomatic plants were confirmed using the Polymerase Chain Reaction (PCR) and Loop-Mediated Isothermal Amplification (LAMP) assays. Based on gene sequences, target multigene i.e., chaperonin 60, protein transferase subunit A and 16Sr RNA were then classified under 'Condidatus' Phytoplosmo luffae. This high incidence of phytoplasma detected from other plant species may pose risk in terms of spatial and temporal distribution. As weeds may serve as an efficient alternate host, an in- depth understanding on the etiology of diseases caused by phytoplasma is much needed. Detection tool using the standard diagnostic protocols for phytoplasma set by the International Plant Protection Convention-International Standard for Phytosanitary Measures 27 (IPPC - ISPM 27) with the additional target genes were found useful in validating the presence of phytoplasma from infected samples.