Methodological Guidelines for Accurate Detection of Viruses in Wild Plant Species

Abstract

Ecological understanding of disease risk, emergence, and dynamics and of the efficacy of control strategies relies heavily on efficient tools for microorganism identification and characterization. Misdetection, such as the misclassification of infected hosts as healthy, can strongly bias estimates of disease prevalence and lead to inaccurate conclusions. In natural plant ecosystems, interest in assessing microbial dynamics is increasing exponentially, but guidelines for detection of microorganisms in wild plants remain limited, particularly so for plant viruses. To address this gap, we explored issues and solutions associated with virus detection by serological and molecular methods in noncrop plant species as applied to the globally important Barley yellow dwarf virus PAV (Luteoviridae), which infects wild native plants as well as crops. With enzyme-linked immunosorbent assays (ELISA), we demonstrate how virus detection in a perennial wild plant species may be much greater in stems than in leaves, although leaves are most commonly sampled, and may also vary among tillers within an individual, thereby highlighting the importance of designing effective sampling strategies. With reverse transcription-PCR (RT-PCR), we demonstrate how inhibitors in tissues of perennial wild hosts can suppress virus detection but can be overcome with methods and products that improve isolation and amplification of nucleic acids. These examples demonstrate the paramount importance of testing and validating survey designs and virus detection methods for noncrop plant communities to ensure accurate ecological surveys and reliable assumptions about virus dynamics in wild hosts.

FIG 1

Ratio of absorbances (A_{405 sample}/A_{405 negative control}) obtained in ELISA for tested samples from Panicum virgatum plants. (a) Per-plant averages derived from 26 tissue samples per plant. (b) Per-tiller averages for each plant, derived for four mature green tillers (six tissue samples each) and one young tiller (two tissue samples). Empty circles, black circles, gray triangles, asterisks, and empty squares represent five different stems in each plant. (c) Averages per tiller class. Old NS (48 samples), mature green tiller with no symptoms of virus infection; Old S (48 samples), mature green tiller with symptoms; Young (8 samples), younger green tiller (all nonsymptomatic). (d) Averages derived for leaves (52 samples total) and stem portions of tillers (52 samples total). Error bars represent ±1 standard error of the means.

FIG 2

Agarose gel visualization of 832-bp BYDV-PAV PCR products from a switchgrass (Panicum virgatum) mixing experiment. Lane L, 1-kb Plus DNA ladder (Invitrogen). Samples coded with “T” prefixes were extracted with TRI Reagent, and those with “S” prefixes were extracted with a Sigma-Aldrich Spectrum Plant Total RNA kit. Suffix codes are as follows: N, negative (uninfected) tissue; O, infected oats; M, mix of infected oats and switchgrass (see Materials and Methods).

FIG 3

Proportion of samples (RNA extracts from uninfected Avena sativa, Koeleria macrantha, and Andropogon gerardii leaves each mixed 9:1 with RNA extract from BYDV-PAV-infected A. sativa leaves) for which BYDV-PAV was positively detected using the regular (a) and modified (b) RT-PCR protocols (i.e., including a 10-fold dilution of RNA extract prior to RT, the addition of a proteinaceous amplification facilitator, T4gp32 in both RT and PCR, and the addition of five amplification cycles to the PCR step). Black, gray, and white circles indicate the Plant RNA Reagent, TRIzol, and TRI-Reagent, respectively, used to extract RNA from uninfected leaves. Each data point illustrates the proportion of positively detected 9:1 mixed RNA samples out of 20 tested samples. Error bars represent ±1 standard error of the means.

FIG 4

Proportion of samples (RNA extract from uninfected plant tissue mixed 9:1 with RNA extract from BYDV-PAV-infected A. sativa leaves) that led to positive detection of BYDV-PAV using a regular RT-PCR protocol as a function of the average A₂₆₀/A₂₈₀ ratios obtained for RNA extracted from uninfected leaves from A. sativa (squares), K. macrantha (triangles), and A. gerardii (circles) plants and using Plant RNA Reagent (black), TRIzol (gray), and TRI Reagent (white) extraction reagent. Each data point illustrates the proportion of positively detected 9:1 mixed RNA samples out of 20 tested samples. Error bars represent ±1 standard error of the means.

FIG 5

Concentration of RNA as a function of the A₂₆₀/A₂₈₀ ratio of each of 180 RNA samples extracted from uninfected A. sativa (squares), K. macrantha (triangles), and A. gerardii (circles) plant tissues using Plant RNA Reagent (black), TRIzol (gray), and TRI Reagent (white) extraction reagent. Error bars represent ±1 standard error of the means.