Elevated Genetic Diversity in the Emerging Blueberry Pathogen <i>Exobasidium maculosum</i>

<div><p>Emerging diseases caused by fungi are increasing at an alarming rate. Exobasidium leaf and fruit spot of blueberry, caused by the fungus <i>Exobasidium maculosum</i>, is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA, severely reducing fruit quality in some plantings. The objectives of this study were to determine the genetic diversity of <i>E</i>. <i>maculosum</i> in the southeastern USA to elucidate the basis of disease emergence and to investigate if populations of <i>E</i>. <i>maculosum</i> are structured by geography, host species, or tissue type. We sequenced three conserved loci from 82 isolates collected from leaves and fruit of rabbiteye blueberry (<i>Vaccinium virgatum</i>), highbush blueberry (<i>V</i>. <i>corymbosum</i>), and southern highbush blueberry (<i>V</i>. <i>corymbosum</i> hybrids) from commercial fields in Georgia and North Carolina, USA, and 6 isolates from lowbush blueberry (<i>V</i>. <i>angustifolium</i>) from Maine, USA, and Nova Scotia, Canada. Populations of <i>E</i>. <i>maculosum</i> from the southeastern USA and from lowbush blueberry in Maine and Nova Scotia are distinct, but do not represent unique species. No difference in genetic structure was detected between different host tissues or among different host species within the southeastern USA; however, differentiation was detected between populations in Georgia and North Carolina. Overall, <i>E</i>. <i>maculosum</i> showed extreme genetic diversity within the conserved loci with 286 segregating sites among the 1,775 sequenced nucleotides and each isolate representing a unique multilocus haplotype. However, 94% of the nucleotide substitutions were silent, so despite the high number of mutations, selective constraints have limited changes to the amino acid sequences of the housekeeping genes. Overall, these results suggest that the emergence of Exobasidium leaf and fruit spot is not due to a recent introduction or host shift, or the recent evolution of aggressive genotypes of <i>E</i>. <i>maculosum</i>, but more likely as a result of an increasing host population or an environmental change.</p></div

Original <i>Vaccinium</i> host, tissue type, and geographic location for <i>Exobasidium maculosum</i> and <i>E</i>. <i>rostrupii</i> isolates.

<p>^a Isolates from lowbush blueberry marked by an asterisk (*) were previously identifed as <i>Exobasidium</i> sp. A (14). Isolates from Nova Scotia were collected by N. Nickerson and kindly provided by P. Hildebrand, Atlantic Food and Horticulture Research Centre, Kentville, Nova Scotia, Canada. The isolate from Maine was obtained from an infected leaf kindly provided by S. Annis, University of Maine, Orono, ME. Isolates of <i>E</i>. <i>rostrupii</i> from cranberry were obtained from infected leaves kindly provided by J. Polashock, USDA-ARS, Genetic Improvement of Fruits and Vegetables Laboratory, Chatsworth, NJ.</p><p>^b Species include: <i>V</i>. <i>virgatum</i> or rabbiteye blueberry (R), <i>V</i>. <i>corymbosum</i> hybrid or southern highbush blueberry (S), <i>V</i>. <i>corymbosum</i> or highbush blueberry (H), <i>V</i>. <i>angustifolium</i> or lowbush blueberry (L), and <i>V</i>. <i>macrocarpon</i> or cranberry (C). Cultivar is listed, if known.</p><p>Original <i>Vaccinium</i> host, tissue type, and geographic location for <i>Exobasidium maculosum</i> and <i>E</i>. <i>rostrupii</i> isolates.</p

Bayesian inferred phylogenies for isolates of <i>Exobasidium maculosum</i> for the loci A. ITS, B. <i>EF-1α</i>, C. <i>CAL</i>.

<p>Phylogenies were rooted with <i>E</i>. <i>rostrupii</i>. Bold nodes indicate bootstrap support values obtained by maximum likelihood and Bayesian posterior probabilities greater than 70 and 0.90, respectively. Isolate names are colored by geographic location: blue = Georgia; green = North Carolina; red = northeastern North America. The first column of boxes to the right of each phylogeny indicates isolates that were collected from leaf (white) or fruit (black) plant tissues. The second column indicates the host from which isolates were collected: violet = rabbiteye blueberry (<i>Vaccinium virgatum</i>); blue = southern highbush blueberry (<i>Vaccinium corymbosum</i> hybrid); red = highbush blueberry (<i>V</i>. <i>corymbosum</i>); yellow = lowbush blueberry (<i>V</i>. <i>angustifolium</i>). Isolates of <i>E</i>. <i>rostrupii</i> were collected from leaf spots on cranberry (<i>V</i>. <i>macrocarpon</i>).</p

Measures of genetic differentiation for populations of <i>Exobasidium maculosum</i> collected from blueberry.

<p>^a Geographic populations consist of isolates from Nova Scotia, Canada and Maine, USA (NE), isolates from the southeastern USA (SE), and isolates from Georgia (GA) and North Carolina (NC). Comparison of populations based on host species within SE include: <i>V</i>. <i>virgatum</i> or rabbiteye blueberry (R), <i>V</i>. <i>corymbosum</i> hybrid or southern highbush blueberry (S), <i>V</i>. <i>corymbosum</i> or highbush blueberry (H). Comparison of populations based on host tissue within SE include fruit spots and leaf spots</p><p>^<b>b</b><i>S</i>_nn and <i>K</i>_ST* calculated in DnaSP (28). Values that significantly differ from neutrality (<i>P</i> ≤ 0.05) based on 1000 permutations of the data are indicated</p><p>Measures of genetic differentiation for populations of <i>Exobasidium maculosum</i> collected from blueberry.</p

Changes in element concentrations by leaf position over time.

<p>Element concentrations in tobacco leaves inoculated with buffer (green circle, control plants) or <i>X. fastidiosa</i> cell suspension (red circle, infected treatment) were followed over time considering relative leaf position in the plant (1 = most basal leaf, 10 = most apical leaf). Data correspond to leaves in positions ≥#4. For the first column (initial), leaves were collected between 25–27 days post infection (dpi); for the second column (intermediate), samples were collected 39–47 dpi; and for the third column (final), samples were obtained 56–59 dpi. From top to bottom, rows of graphs correspond to concentrations of Ca and P expressed in mg per g of plant tissue. Values represent means and standard errors (n = 5) from one out of three experimental sets conducted. *Indicates significant difference (<i>p</i><0.05) between treatments at a specific leaf position according to one-way ANOVA or Kruskal-Wallis.</p

Canonical discriminate analysis of treatment and leaf position as classification variables.

<p>Concentrations of 12 elements analyzed were considered as independent variables, while experimental set and time points were considered as replicates. A) Leaves were separated according to relative position in the plant between leaves #1–3 (circles<b>)</b> which are older, directly-inoculated and leaves ≥ #4 (inverted triangles) that represent new growth after inoculation. B) Leaves at the same position were compared according to presence or absence of <i>X. fastidiosa</i>. Circle = position #4; inverted triangle<b> = </b>position #5; square<b> = </b>position #6; diamond<b> = </b>position #7; triangle<b> = </b>position #8. Phenotypic correlations for Can1 are driven significantly (<i>p</i><0.05) by Ca (r = −0.98), Mg (r = −0.89), Na (r = 0.85), K (r = 0.74), and Mn (r = −0.59) and for Can2 by Cu (r = 0.57) and S (r = −0.52). B, Fe, Mo, P, and Zn have no significant effect on discrimination among classes. Red symbols indicate leaves infected with <i>X. fastidiosa</i> and green symbols indicate non-infected leaves. The length of the axes is proportional to the accounted for percentage of the total multivariance.</p

Changes in total ionome of tobacco leaves infected with <i>Xylella fastidiosa</i>.

<p>Tobacco plants (<i>Nicotiana tabacum</i> ‘Petite Havana SR1’) growing in the greenhouse were inoculated with <i>X. fastidiosa</i> or buffer (control) and the ionome of each leaf was characterized by inductively coupled plasma optical emission spectroscopy (ICP-OES). Mean values of element concentrations (in mg/g of plant tissue) were obtained from leaves in positions ≥ #4. Mean relative percentage of change with corresponding standard errors (represented only towards 0 value) were calculated by comparing mean leaf concentrations across five time points and three experimental sets between leaves where <i>X. fastidiosa</i> was detected (‘infected’) against those were the bacterium was not detected (‘non-infected’) (n∼375/treatment). <i>P</i> values for main treatment effects are included for each element; those highlighted in bold with red bars are considered significant (<i>p</i><0.05).</p

Ionome comparisons of greenhouse tobacco and field-grown host plants.

1<p>Mean and standard error (SE) are calculated from all infected and non-infected leaves at all positions in greenhouse experiments.</p

Changes in element concentration of field-grown host plants after infection with <i>Xylella fastidiosa.</i>

1<p>n = number of pairs analyzed for each plant species. A pair consisted of a non-infected asymptomatic leaf and a symptomatic leaf infected (confirmed by <i>X. fastidiosa</i>-specific PCR) taken from the same or a neighboring plant.</p>2<p>Metal content determined by ICP-OES. Data represents average and standard error of percentage of changes in elemental composition of infected vs. non-infected leaves.</p>3<p>ND = not determined.</p

Infection of <i>Nicotiana tabacum</i> ‘Petite Havana SR1’ with <i>Xylella fastidiosa</i><b>.</b>

<p>A) Progression of symptom development in tobacco plants infected with <i>X. fastidiosa</i> Temecula. The percentage of symptomatic leaves showing marginal leaf scorch at a specific leaf position on the plant (increasing numbers from bottom to top of the plant) and at a specific time are represented in the graph. Sampling times numbered 1 through 5 in the graph correspond to 0, 22, 27, 38, 48, and 59 days post inoculation, respectively. Five tobacco plants were analyzed at each sampling time. The experiment was repeated three times and data in the graph corresponds to one representative experiment. B) Population of <i>X. fastidiosa</i> Temecula in petioles of infected tobacco plants. Bacterial populations were quantified by specific qPCR using sections of petioles as source material. Five tobacco plants were analyzed per sampling time at several days post-infection (dpi). Data represented in the graph correspond to means and standard errors of bacterial populations of all leaves at positions ≥#4 at each sampling time. The experiment was repeated three times and data in the graph corresponds to one representative experiment.</p