qPCR Assay as a Tool for Examining Cotton Resistance to the Virus Complex Causing CLCuD: Yield Loss Inversely Correlates with Betasatellite, Not Virus, DNA Titer

Abstract

Cotton leaf curl disease (CLCuD) is a significant constraint to the economies of Pakistan and India. The disease is caused by different begomoviruses (genus Begomovirus, family Geminiviridae) in association with a disease-specific betasatellite. However, another satellite-like molecule, alphasatellite, is occasionally found associated with this disease complex. A quantitative real-time PCR assay for the virus/satellite components causing CLCuD was used to investigate the performance of selected cotton varieties in the 2014-2015 National Coordinated Varietal Trials (NCVT) in Pakistan. The DNA levels of virus and satellites in cotton plants were determined for five cotton varieties across three geographic locations and compared with seed cotton yield (SCY) as a measure of the plant performance. The highest virus titer was detected in B-10 (0.972 ng·µg^-1) from Vehari and the lowest in B-3 (0.006 ng·µg^-1) from Faisalabad. Likewise, the highest alphasatellite titer was found in B-1 (0.055 ng·µg^-1) from Vehari and the lowest in B-1 and B-2 (0.001 ng·µg^-1) from Faisalabad. The highest betasatellite titer was found in B-23 (1.156 ng·µg^-1) from Faisalabad and the lowest in B-12 (0.072 ng·µg^-1) from Multan. Virus/satellite DNA levels, symptoms, and SCY were found to be highly variable between the varieties and between the locations. Nevertheless, statistical analysis of the results suggested that betasatellite DNA levels, rather than virus or alphasatellite DNA levels, were the important variable in plant performance, having an inverse relationship with SCY (-0.447). This quantitative assay will be useful in breeding programs for development of virus resistant plants and varietal trials, such as the NCVT, to select suitable varieties of cotton with mild (preferably no) symptoms and low (preferably no) virus/satellite. At present, no such molecular techniques are used in resistance breeding programs or varietal trials in Pakistan.

Keywords: cotton leaf curl Khokhran virus—Burewala strain (CLCuKoV-Bur); cotton leaf curl Multan betasatellite (CLCuMuB); cotton leaf curl disease (CLCuD); national coordinated varietal trials (NCVT); quantitative real-time PCR (qPCR); seed cotton yield (SCY).

Figure 1

Symptoms exhibited by the field collected samples for the varieties B-01, B-02, B-10, B-12, and B-23 of the 2014–2015 NCVT trials in the Central Cotton Research Institute (CCRI) Multan, National Institute for Agriculture and Biology (NIAB) Faisalabad (Fsd), and Cotton Research Station (CRS) Vehari. Plants B-12 at CCRI Multan, and B-01 and B-02 at NIAB Fsd were showing symptoms of vein darkening/swelling at Scale-1. Plants B-01, B-02, B-10, and B-23 at CCRI Multan were exhibiting Scale-2 symptoms, characterized by vein darkening/swelling, leaf curling, and enation. Plants B-10, B-12, and B-23 at NIAB Fsd, and B-01 at CRS Vehari were showing Scale-3 phenotype, which included vein darkening/swelling, leaf curling, leaf crumpling, enation, and stunting. Plants B-02, B-10, B-12, and B-23 at CRS Vehari were showing Scale-4 symptoms, characterized by vein darkening/swelling, leaf curling, leaf crumpling, enation, severe stunting, and extensive crumpling.

Figure 2

qPCR estimation of the virus, betasatellite, and alphasatellite titers (ng·µg⁻¹) in total DNA extracted from the NCVT trials cotton plants. The scatter chart displays the SCY of the varieties at each location as well as the calculated titer of begomovirus (A) betasatellite (B) and alphasatellite (C) with standard deviation (shown in vertical bars). Values preceded by the same letter are not significantly different at the 5% level using a two tailed t-test. The NCVT codes used for different varieties are B-1, B-2, B-10, B-12, and B-23; nonetheless, the prefix letter represents the location of the trial. The samples collected from CCRI Multan are prefixed with M, from NIAB Faisalabad with N, and from CRS Vehari with V. The healthy control (H.C) is a non-infected glasshouse-grown cotton plant. Values mentioned in the figure are mean of three independent replicates (n = 3) and values denoted by different letters (in small alphabets) differ significantly at 5% significance level.

Figure 3

Path coefficient analysis of the correlation of virus, betasatellite and alphasatellite with SCY and symptom severity at a significance level of 5%.