Nematode-infected (<i>Litylenchus crenatae</i> subsp. <i>mccannii</i>) bud scale cells showed variable levels of hypertrophy.

(A-C) Photomicrographs of control bud scales showing flat, rectangular-shaped cells. (D-F) Representative infected bud scale cells. Infected cells presented several levels of hypertrophy and a more disorganized distribution of the cells (F). (G) Different nematode stages (arrows) associated with abnormally large scale cells. (H) Example of nematodes (arrows) probing large scale cells. (I) Large numbers of eggs were often found in nematode infected scales. Scale bars = 50 μm.</p

Cell architecture of beech (<i>Fagus grandifolia</i>) leaves.

Photomicrographs of transverse sections of control and symptomatic beech leaf disease (BLD) leaves collected in early autumn. Sections were stained with a mixture of hematoxylin and eosin. (A) Representative control beech leaf. (B-C) Section of a mature control beech leaf showing differentiated cell layers as indicated in figure C. (D) Representative symptomatic BLD leaf. Dotted white, blue and orange squares represent sections of (G) asymptomatic, (H) green and (E-F) yellow banded areas of the same leaf used for histological analyses. (E-F) Sections of yellow symptomatic BLD areas showing extensive leaf thickness as a consequence of cell proliferation and cell hypertrophy. Nematode sections are stained (dark purple). (G) Section of a non-symptomatic area of a BLD leaf, which presents cell architecture similar to that of control leaves (B). (H) Section of a green interveinal area of a BLD leaf without nematodes. Note the additional cell layers in comparison to the control leaves (C). (I) Leaf thickness was measured from photomicrographs of control, asymptomatic (BLD_a) and symptomatic BLD areas without (BLD -n) and with (BLD +n) nematodes. For each condition a minimum of 50 measurements were made. (J-L) Cell surface (μm2) was measured for the upper epidermis (J), lower epidermis (K) and spongy mesophyll cells (L). Measures were made for control (n = 80 cells), asymptomatic (BLD_a, n = 100 cells) and symptomatic BLD areas without (BLD -n, n = 100 cells) and with (BLD +n, n = 100 cells) nematodes. Thick lines in the boxplot represent medians. Different letters denote statistical differences between cell sizes (P < 0.05, ANOVA with Tukey-Kramer test). Scale bars: 100 μm (A, B); 50 μm (C-H).</p

Measurement and weight of 50 buds collected from symptomatic beech leaf diseased trees in late February of 2023.

Measurement and weight of 50 buds collected from symptomatic beech leaf diseased trees in late February of 2023.</p

Nematode (<i>Litylenchus crenatae</i> subsp. <i>mccannii</i>) assessment of leaves and beech (<i>Fagus grandifolia</i>) buds in early spring.

(A) Representative branch showing sets of asymptomatic and symptomatic beech leaf disease (BLD) leaves, and poorly developing buds. (B-C) Detection of nematodes in symptomatic BLD leaves using acid fuchsin staining (B). Nematodes (arrows) were found migrating on the abaxial side of the leaf and penetrating the leaf tissues (C-C’). (D) Representative symptomatic BLD leaves dissected from poorly developing buds. (E) Percentage of asymptomatic and symptomatic BLD leaves associated with 50 poor-developing buds rated according to the phenotype scale presented in Fig 1. (F) Active nematodes and eggs extracted from a single, poorly developing bud. (G) Numbers of nematodes extracted from 50 poorly developing buds. Buds were classified from 0 to 4 based on their bud scale phenotype (see Fig 1). Scale bars: 5 mm (B); 1 mm (C, D, F); 500 μm (C´).</p

Histological analysis of shoot apex and leaf primordial tissues in non- and nematode-infected (<i>Litylenchus crenatae</i> subsp. <i>mccannii</i>) buds of beech (<i>Fagus grandifolia</i>).

Bright-field photomicrographs of bud scale cross-sections stained with a mixture of toluidine blue and basic fuchsin are shown. (A-B) Beech leaf shoot apex and leaf primordial cells marking nematodes within these tissues (arrows). (C-E) Examples of leaf primordial developing in buds infected with nematodes (arrows). Note the presence of different number of disorganized and abnormal cell layers (n > 6) in each leaf primordia. (F) Cross-section of leaf primordia from a non-infected bud showing six distinct layers of highly organized cells. Scale bars: 50 μm.</p

Measurement and weight of 50 poor developing beech buds collected in early spring of 2023.

Measurement and weight of 50 poor developing beech buds collected in early spring of 2023.</p

Sections of young developing leaves within nematode-infected (<i>Litylenchus crenatae</i> subsp. <i>mccannii</i>) beech buds (<i>Fagus grandifolia</i>) with altered interveinal leaf cell proliferation.

Morphological cell rearrangements of interveinal leaf cross-sections. (A) Representative section of control leaf interveinal areas with the typical six layers of cells. (B-D) Three interveinal areas of the same leaf infected by nematodes showing a variable and prominent increase in the number of cells layers. Outline drawings of some representative interveinal areas highlighting this upsurge in cell division associated with nematodes (B’-C’) compared to the control (A’). (E) Veinal areas of the same nematode-infected leaf displayed different levels of ectopic cell proliferation. (F) Differential cell proliferation within the same interveinal leaf area dissected from a nematode-infected bud. Scale bars: 20 μm.</p

Nematode quantification of buds collected from control and symptomatic beech leaf diseased trees in early autumn of 2021.

Nematode quantification of buds collected from control and symptomatic beech leaf diseased trees in early autumn of 2021.</p

Beech leaf disease nematode, <i>Litylenchus crenatae</i> subsp. <i>mccannii</i>, associated with naturally infected beech buds.

A total of 60 buds were collected randomly at the beginning of the fall from beech leaf disease symptomatic trees. (A) A representative set of beech buds ready to be processed for nematode extraction and morphological analysis. (B) Eggs in different developmental stages. (C-E) Juvenile stages. (F-G) Mature sexual adults. F: male; G: female. (H) Putative asexual female. Sclerotized, cuticularized anal openings were at times more difficult to observe in asexual females than in sexual, reproductive females. However, the strongly sclerotized stylet was always distinctly robust, and longer in sexually reproductive females than in asexual females [10.0 ± 0.0 (10.0–10.0) vs 7.4 ± 0.2 (7.0–7.5)], with a thicker, well-developed stylet and stylet knobs vs a thin stylet with weak, almost absent knobs. In addition, the location of the vulva on the body (V %) was higher in sexually reproductive females than in asexual females [80.1 ± 0.5 (79.6–81.0) vs 77.6 ±1.1 (75.0–79.1)]; and the tail length was smaller, with a mostly thorn-like terminus in sexually reproductive females [47.8 ± 8.9 (39.0–62.0) vs 59.9 ±7.1 (50.0–70.0)] with a long, pointed terminus in asexual females. The asexual females have longer esophageal glands, and longer, filiform tail. s: stylet; v: vulva. Scale bars: 20 μm. (PDF)</p

Histological analyses of non- and nematode-infected (<i>Litylenchus crenatae</i> subsp. <i>mccannii</i>) bud scales of beech (<i>Fagus grandifolia</i>).

Bright-field photomicrographs of bud scale cross-sections stained with a mixture of toluidine blue and basic fuchsin. (A) Representative image of non-infected bud scale composed of four layers of cells. Spherical hyaline structures are sections through leaf hair. (B-F) Representative photomicrographs of nematode-infected bud scale cells showing different developmental stages of hypertrophy. Non-infected bud scale cells can also be seen adjacent to hypertrophied cells, as shown in figures C-F. Arrows point to nematode sections associated with hypertrophied bud scale cells. (G) Bud scale cell surface (μm2) was measured in control (n = 30 cells) and nematode-infected (n = 45 cells) whole fresh bud scales. (H) Bud scale cell surface (μm2) was measure in control (n = 115 cells) and representative nematode-infected (n = 115 cells) fixed and bud scale cross-sections. (I) Nuclear surface (μm2) of bud scale cell sections of control (n = 60 nuclei) and nematode-infected (n = 60 nuclei) buds. Thick lines in the boxplot represent medians. Different letters denote statistical differences between control and diseased bud scale cells (P < 0.05, ANOVA with Tukey-Kramer test). Scale bars: 25 μm (A-C); 50 μm (D-F).</p