Comparison of Models for Quantification of Tomato Brown Rugose Fruit Virus Based on a Bioassay Using a Local Lesion Host

Abstract

Considering the availability of serological and molecular biological methods, the bioassay has been paled into insignificance, although it is the only experimental method that can be used to demonstrate the infectivity of a virus. We compared goodness-of-fit and predictability power of five models for the quantification of tomato brown rugose fruit virus (ToBRFV) based on local lesion assays: the Kleczkowski model, Furumoto and Mickey models I and II, the Gokhale and Bald model (growth curve model), and the modified Poisson model. For this purpose, mechanical inoculations onto Nicotiana tabacum L. cv. Xanthi nc and N. glutionosa L. with defined virus concentrations were first performed with half-leaf randomization in a Latin square design. Subsequently, models were implemented using Python software and fitted to the number of local lesions. All models could fit to the data for quantifying ToBRFV based on local lesions, among which the modified Poisson model had the best prediction of virus concentration in spike samples based on local lesions, although data of individual indicator plants showed variations. More accurate modeling was obtained from the test plant N. glutinosa than from N. tabacum cv. Xanthi nc. The position of the half-leaves on the test plants had no significant effect on the number of local lesions.

Applicability of Different Methods for Quantifying Virucidal Efficacy Using MENNO Florades and Tomato Brown Rugose Fruit Virus as an Example

“…The number of local lesions after 5–7 dpi (days post inoculation) on the half-leaf units of N. glutinosa plants is shown in Table 2 . Both the Kleczkowski model [ 54 , 56 ] and the growth curve model [ 49 , 54 ] were fitted to the local lesion number induced by ToBRFV-suspensions with virus concentrations of 128 ng/mL up to 2 mg/mL with the least error, meaning a very good fit ( Table 2 ). Both models fitted best with transformed means of the number of local lesions observed for inoculation of highest and middle concentrations of ToBRFV particles (i.e., 2, 0.016, and 0.0032 mg/mL) ( Table 2 ).…”

“…These equations were used for the quantification of the virus load [ 54 ]. Using the estimated model parameters fitted to the known dilution series, we constructed the inverse function of the Kleczkowski model and the growth curve model, respectively: …”

“…Moreover, it provided sufficient friction as a mechanical force to release dried virus particles from these surfaces. It has been shown that the different models could quantify the ToBRFV titer in the plant sap and virus suspensions [ 54 ]. Based on the data presented in Table 2 , bioassays could estimate a higher concentration of ToBRFV (e.g., 2 mg/mL), and its lower detection limit was equal to or lower than that of qELISA.…”

“…It has been shown that 4% MENNO Florades has very light or no phytotoxicity to the plants. It has been shown that a higher concentration than 2 mg/mL of ToBRFV can cause advanced necrosis on the inoculated leaves of N. glutinosa [ 54 ] due to aggregation of adjacent lesions. MENNO Florades (BVL registration number 044407-00) remains the only authorized plant protectant within the EU to be used for the disinfection of agricultural and horticultural surfaces/objects against harmful plant pathogens.…”

“…There is a relation between the number of local lesions on the host plants and the titer of a virus in the inoculum [ 47 ]. Several mathematical models have been introduced to correlate virus concentration in the suspension and the number of local lesions induced in bioassay [ 48 , 49 , 50 , 51 , 52 , 53 , 54 ]. Neither qELISA nor RT-qPCR provides the number of infectious particles in a sample, as only protein or nucleic acid, respectively, is quantified.…”