Elucidating the role of whitefly heat shock proteins and endosymbiont in transmission of cotton leaf curl virus
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Date
2020
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Punjab Agricultural University, Ludhiana
Abstract
Bemisia tabaci is a cryptic species complex, which poses a major threat to agricultural crops
worldwide. To unravel its genetic diversity and endosymbiont composition across Punjab, sampling of
whiteflies was done from fifteen different locations. Based on mitochondrial cytochrome oxidase I
(mtCOI) sequencing and phylogenetic analysis, two major B. tabaci groups i.e Asia II-1 and Asia-
1group were identified and the prevalence of endosymbionts within these groups was described. The
south western region of Punjab was dominated by AsiaII-1group, while Asia-1group was predominant
in the northern region. Infection status of endosymbionts in individual whitefly were analyzed using
specific primers corresponding to 16S/23S rDNA region of bacterial gene. The primary endosymbiont
Portiera aleyrodidarum and secondary endosymbiont Arsenophonus was present across all populations
of Punjab. The infection percent of Cardinium was 100 % in Asia II-1 group, while its presence in
Asia-1 group was variable across these locations. Presence of Rickettsia could not be associated with
any haplotype as its infection was varied within the same population. Wolbachia was restricted to only
Asia -1 group; however Hamiltonella was not detected from any of the tested populations. The present
study evidenced the predominance Arsenophonus in AsiaII-1 group, which might be implicated with
higher incidences of cotton leaf curl disease (CLCuD) in southwestern Punjab. The role of
Arsenophonus in transmission of cotton leaf curl virus (CLCuV) by whitefly was investigated after its
selective elimination with rifampicin and tetracycline @90μg/ml in sucrose diet. Virus transmission
assays showed that Arsenophonus-ve B. tabaci poorly transmits CLCuV in cotton plants compared to
control whiteflies. The functional role of Heat shock proteins, Knottin and Cyclophilin in transmission
of CLCuV was studied through knockdown of these genes in whitefly using RNAi. To have an
enhanced RNAi efficiency dsRNA (dsdsRNases) against gut nuclease gene (dsRNases) was co
administered with the target genes. Whiteflies were given feeding access to 400 ng/μl of dsRNA
against target gene along with dsdsRNase (400ng/μl) in sucrose diet for 48 h knockdown of Hsp and
knottin-1 resulted in increased viral load in the host vector and its high transmission ability in cotton
host plants compared to control whiteflies. On the contrary, cyclophilin knockdown led to decreased
viral load in vector and ultimately lower transmission of CLCuV in cotton plants compared to sucrose
fed whiteflies. Comparative study on fate of virus particle in two cotton species (Gossypium arboreum
and G. hirsutum) showing variable response to CLCuD revealed that the immunity in arboreum cotton
may possibly be due to the degradation of CLCuV particles after gaining entry in plant phloem through
vector feeding. The RT-PCR based detection assays (Syber green and Taq man) suggest that the virus
may be restricted to plant phloem for up to seven days and thereafter it degrades. No detection of virus
from cDNA synthesized from total RNA of inoculated leaf of arboreum compared to hirsutum also
suggest that there is a possibility that virus degrades or is trapped after gaining entry into the arboreum
cell. Bioformulation of Lecanicillium lecanii MTCC956 at middle and lower dose (10and 12g/liter)
was effective in reducing whitefly population on cotton under screenhouse conditions.