Detection of Begomovirus in chilli and tomato plants using functionalized gold nanoparticles

Abstract

Begomoviruses are a major class of Geminiviruses that affects most dicotyledonous plants and causes heavy economic losses to farmers. Early detection of begomovirus is essential to control the spread of the disease and prevent loss. Many available detection methods like ELISA, immunosorbent electron microscopy, PCR or qPCR require expertise in handling sophisticated instruments, complex data interpretation and costlier chemicals, enzymes or antibodies. Hence there is a need for a simpler detection method, here we report the development of a visual detection method based on functionalized gold nanoparticles (AuNP assay). The assay was able to detect up to 500 ag/µl of begomoviral DNA (pTZCCPp3, a clone carrying partial coat protein gene) suspended in MilliQ water. Screening of chilli plants for begomoviral infection by PCR (Deng primers) and AuNP assay showed that AuNP assay (77.7%) was better than PCR (49.4%). The AuNP assay with clccpi1 probe was able to detect begomoviral infection in chilli, tomato, common bean, green gram and black gram plants which proved the utility and versatility of the AuNP assay. The specificity of the assay was demonstrated by testing with total DNA from different plants that are not affected by begomoviruses.

Figure 1

Schematic representation of the AuNP assay proposed in this work. Total DNA would be isolated from plant samples and screened for the presence of begomoviruses using the functionalized AuNPs.

Figure 2

Screening of plants for the presence of begomovirus by PCR using Deng primers (Deng et al., 1994). (a) Lanes 1 to 10: Chilli DNA samples C41 to C50; N—Negative; M—1 Kb DNA ladder. (b) Lanes 1 to 12: Tomato DNA samples isolated by Dellaporta method; P positive plasmid DNA; N negative; M—1 Kb DNA ladder.

Figure 3

Chilli leaf curl viral coat protein gene (partial) obtained in this study.

Figure 4

Microscopic and spectroscopic analysis of AuNPs. (a) Transmission Electron Micrograph (TEM) of AuNPs. (b) Energy Dispersive X-ray (EDX) spectroscopic analysis of synthesized AuNPs.

Figure 5

(a) Particle size analysis of functionalized AuNPs. (b) Zeta potential of functionalized AuNPs.

Figure 6

Schematic illustration of the principle of the AuNP assay for begomoviral detection. In the presence of viral DNA (target) the functionalized AuNPs retain red colour when NaCl was added as dsDNA (viral DNA + Probe DNA) prevents aggregation of the AuNPs. In the absence of viral DNA, functionalized AuNPs aggregate and turn purple due to the neutralization of electron cloud of AuNPs by the salt. S-thiol linkage; P-probe.

Figure 7

Detection of begomovirus in chilli samples using functionalized AuNPs. (a) With clccpi1 thiol probe; plant DNA (C71 to C80); positive (P)—begomovirus infected chilli DNA; N—healthy chilli DNA. (b) With chrppro1 thiol probe; plant DNA (C28 to C40); positive (P)—begomovirus infected chilli DNA; N—healthy chilli DNA. (c) Comparison of PCR and AuNP assay with different probes for chilli samples.

Figure 8

Detection of begomovirus in tomato plants using functionalized AuNPs. (a) With clccpi1 thiol probe, plant DNA (T27 to T38; T40 to T56 & T58), positive (P)—begomovirus infected tomato DNA, N—healthy tomato DNA. (b) With torppro1 thiol probe, plant DNA (To1 to To21), positive (P)—begomovirus infected tomato DNA, N—healthy tomato DNA. (c) Comparison of AuNP assay with different probes for tomato samples.

Figure 9

Comparison of the sensitivity of the AuNP assay and PCR (M13 primers) using different concentrations of plasmid (pTZCCPp3) diluted with Milli Q water. (a) AuNP assay with clccpi1 thiol probe was able to detect plasmid up to 500 ag/µl. (b) PCR was able to detect plasmid up to 10 ng/µl.

Figure 10

Specificity and versatility of AuNP assay for the probe—clccpi1 thiol. (a) Using unrelated plant DNA of three plants I. aspalathoides, P. amboinicus and C. roseus. (b) Using plants belonging to other family (Fabaceae)—P. vulgaris (common bean) that are infected by different begomoviruses. (c) PCR detection of begomovirus infection in bean plants (B1 to B4) using Deng primers. (d) Screening of begomovirus infected positive samples V. mungo (black gram) and V. radiata (mung bean) belonging to family Fabaceae were procured from TNAU. In 8c, the marker lane was from the same gel, refer supplementary figure (Supplementary file).