First Report of Pineapple Mealybug Wilt–Associated Viruses 3 and 5 Infecting Pineapple ( Ananas comosus ) in India

Mealybug wilt of pineapple (MWP), a severe disease first reported in Hawaii in 1910, threatens pineapple production globally, including the rapidly expanding cultivation of pineapple in India. MWP symptoms include leaf reddening and curling, and plant decline, and the disease is caused by several species of pineapple mealybug wilt–associated viruses (PMWaVs; genus Ampelovirus, family Closteroviridae) (Massé et al. 2024). Mixed infections of multiple PMWaV species are frequently linked to MWP, although it can be associated with infection by a single viral species (Dey et al. 2018). MWP was described in Indian plantations in the 1970s (Singh and Sastry 1974) and PMWaV-1 was detected subsequently in Indian pineapple germplasm (Sether et al. 2001; Larrea-Sarmiento et al. 2022). However, no other PMWaV species have been reported from India. In March 2025, leaf samples were collected from 12 pineapple plants, from five different locations in the Kolli Hills (Namakkal), exhibiting MWP symptoms such as leaf reddening and tip dieback (Figure 1). Total RNA was extracted using TRIzol reagent and used for RT-PCR assays targeting the RdRp and HSP70h genes of PMWaVs (Gambley et al. 2009; Massé et al. 2024). Mixed infections of PMWaV-1, PMWaV-3 and PMWaV-5 were detected in pineapple samples from three of the five locations of Kolli Hills. However, no PMWaVs were detected in samples from the other two locations, and PMWaV-2, -4, -6 and -7 were not detected in samples from any of the locations. The PCR amplicons were Sanger sequenced bi-directionally and the sequences submitted to GenBank (Accession Nos. PX644559 [PMWaV1], PX644560-PX644561 [PMWaV3] and PX644562-PX644563 [PMWaV5]). BLASTn analysis indicated that the Namakkal PMWaV-1, PMWaV-3 and PMWaV-5 isolates had 97% nucleotide identity with American isolate PMWaV-1/S2-1 (OP860292.1), 98.51% identity with an American reference isolate (NC_043406.1) and 84.64% with French isolate PMWaV-5-RE2016_L16-01 (OQ850040.1), respectively. This study is the first report of the association of PMWaV-3 and PMWaV-5 with MWP in India, PMWaV-1 having been reported previously. The co-infection of PMWaV-1, -3, and -5 is significant as these mixed infections appeared to be linked to the development of MWP symptoms. However, further research using high-throughput sequencing is recommended to thoroughly understand the MWP disease complex, as demonstrated in Reunion Island (Massé et al. 2024). This study is of major phytosanitary significance for the pineapple industry in India since vegetative propagation using infected suckers is a primary driver for spread of MWP. Key measures include establishing certified virus-free nursery programmes and implementing robust mealybug vector control. Integrating molecular diagnostics into agricultural extension services is crucial for effective disease surveillance and management. These steps are vital to safeguard the expanding pineapple industry from significant yield losses in India.