Virus sampling design for pea plants inoculated with PSbMV.

<p>(A) Plants of the pea cultivar Vedette were mechanically inoculated with mixtures of two PSbMV variants 28 days after sowing on the two leaves I1 and I2. Twenty-two days post inoculation (dpi), corresponding to the anthesis of the first flower in the plant population, the three leaves L1 to L3 immediately above I2 were collected separately and analyzed. Sixty-one dpi, corresponding to the end of anthesis, the three leaves L4 to L6 immediately above L3 were collected separately and analyzed. Finally, all pods produced by the main stem of the plants were harvested at desiccation step, seeds were sown and seedlings were analyzed 22 days after sowing. (B) Different sets of plants were subjected to different sampling schemes. For plants numbered 20 to 49, samplings at 22 dpi and/or at 61 dpi were omitted.</p

Frequency of two PSbMV variants in pea leaves and seedlings in three sets of plants corresponding to three sampling designs.

<p>For plants 1 to 19, leaves were sampled at 22 and 61 dpi; for plants 20 to 39, leaves were sampled only at 61 days post inoculation (dpi) and for plants 40 to 49, no leaves were sampled. <i>n</i>: total number of leaves or seedlings analyzed. Seedlings were analyzed only for plants that produced nine seeds or more. The percentages of the DPD1-R variant in inocula 1 and 2 were 37.8% and 65.9%, respectively.</p><p>^aMean relative frequency (×100) and standard deviation (×100; between parentheses) of the DPD1-R specific marker in the viral population.</p><p>^bFrequency of healthy seedlings (i), of seedlings infected by DPD1 (ii) or DPD1-R (iii) only, and of seedlings co-infected by both PSbMV variants (iv).</p

<i>Ne</i> estimates for the systemic colonization of pea leaves by PSbMV between 22 and 61 days post inoculation (dpi).

<p>Estimates were obtained by two different methods and separately for two inocula corresponding to two different initial ratios of PSbMV variants. 95% confidence intervals estimated by bootstrapping among plants are indicated in brackets. The variable <i>f_i,j</i> is the relative frequency of virus variant 1 in plant <i>i</i> and leaf <i>j</i> (<i>j</i> in {1,2,3} for date 1 and <i>j</i> in {4,5,6} for date 2) (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003833#ppat.1003833.s001" target="_blank">Fig. S1</a> for details on its estimation).</p><p>^aFor inocula 1 and 2, <i>i</i>∈[1–10] and <i>i</i>∈[11–19], respectively.</p><p>^bFor inocula 1 and 2, <i>i</i>∈[20–29] and <i>i</i>∈[30–39], respectively.</p><p>^cThe variance and <i>F</i>_ST methods assume an increase of the variance of viral frequencies (respectively of the <i>F</i>ST statistics of viral populations) with time. “NA” (not available), indicates situations where these assumptions were not satisfied and, consequently, where genetic drift was negligible (<i>Ne</i> tends to infinity).</p

The 4 models of PSbMV vertical transmission.

<p>This figure illustrates the 4 sets of infection rules governing vertical transmission (<i>i.e.</i> seedling infection) and corresponding to the 4 models considered here (models M1, M2, M3 and M4). For each model, the rules leading to the 4 possible categories of seedling infection ((i) healthy, (ii) infected only by variant 2 (DPD1), (iii) infected only by variant 1 (DPD1-R) and (iv) infected by both PSbMV variants) are indicated and illustrated for values of <i>N¹</i> and <i>N²</i> ranging from 0 to 8 and <i>N^c</i> = 4. Let remember that where <i>N¹</i> (resp. <i>N²</i>) is the number of particles of type 1 (resp. 2) entering into the seed and <i>N^c</i> is a threshold for efficient seedling infection.</p

Models for virus vertical transmission.

<p><i>N¹</i> (respectively <i>N²</i>) is the number of PSbMV variant 1 (DPD1-R) (respectively variant 2 (DPD1)) particles entering a given seed of a given plant and <i>N^c</i> is a critical threshold for the infection of the seedling issued from this seed. Note that for <i>N^c</i> = 0 the models M1, M2 and M3 are identical to model M4.</p><p>^aWhen variant-variant interactions occur, two cases were distinguished depending on whether, or not, virus variants are interchangeable whatever their type. Variants are interchangeable for seedling infection if the contribution to seedling infection of a virus particle of one variant does not depend on the density of virus particles of the other variant.</p

Practical identifiability of virus seed transmission models.

<p>Correlation coefficients (and in brackets their standard deviations estimated with a bootstrapping method) between the true and estimated parameter values for the 4 models (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003833#ppat-1003833-t002" target="_blank">Table 2</a>) of virus seed transmission (over 100 simulated datasets).</p