Ultrastructure of Hirschmanniella diversa early-stage infection in browning rhizomes of Indian lotus

Indian lotus “Shina-shirobana” rhizomes were obtained from fields where H. diversa had not been detected. Nematode inoculum was obtained from infected fresh rootlets of the same lotus cultivar harvested from fields where rhizome browning caused by nematodes has been frequently found. Rootlets were chopped into approximately 1 to 2-cm pieces. Nematodes were collected from the rootlets using the Baermann funnel method with 72 hr of incubation at 24 ± 1°C.

Apices of young lotus rhizomes (1 cm length) were excised and placed in the center of 3.0-cm-diameter petri dishes containing 4.0 ml of 0.4% water agar, with the apex in contact with the bottom of the dish (Fig. 1A, B). Approximately 100 to 300 nematodes were poured around the rhizome tissue. Petri dishes were sealed with plastic tape to prevent the water agar from drying and maintained in darkness at 24±1°C. After 4.5, 24, and 72 hr, the petri dishes of young lotus rhizomes inoculated with nematodes were frozen rapidly in liquid nitrogen (Fig. 1C). After thawing, the inoculated lotus rhizome apices were observed with stereomicroscopy. Apices were fixed for light and electron microscopy (Fig. 1D).

Figure 1:

Inoculated (or uninoculated control) specimens were immersed in 2.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, for 12 to 72 hr at 4°C. Specimens were sectioned into 90-μm-thick slices with a microslicer (DTK-3000; Dosaka EM, Kyoto, Japan) and observed utilizing a light microscope.

Specimens inoculated with nematodes were immersed in 2.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, for 12 to 24 hr at 4°C. Postfixation with 1% osmium tetroxide was performed in the same buffer for 12 hr at 4°C. Specimens were dehydrated in a graded series of ethanol: 50, 70, 80, 90, and 100%. Thereafter, ethanol was exchanged with 100% t-butyl alcohol (2-methyl 2-propanol), and specimens were freeze-dried (ES-2030; Hitachi, Tokyo, Japan). The specimens were coated with approximately 8 nm of platinum by ion spatter (E-1010; Hitachi) and observed by field emission SEM (S-4700; Hitachi) at 25 kV. To observe the inner tissues of rhizomes that had been invaded by nematodes, specimens were vertically cut into two pieces with a razor blade. The pieces were coated with platinum and observed by SEM as described above.

Specimens inoculated with nematodes were immersed in 2.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, for 24 hr at 4°C and then postfixed in 1% osmium tetroxide in the same buffer at 4°C for 12 hr. The specimens were rinsed with distilled water for 10 min and dehydrated in an ethanol series: 50, 70, 80, 90, and 100%. Absolute 100% ethanol was then replaced with QY-1 (1-Butoxy-2, 3-epoxypropane), and specimens were embedded in Quetol 651 resin mixture (Nissin EM, Tokyo, Japan). For light microscopy, 0.5-μm-thick sections were cut using glass knives and stained with 1% toluidine blue in 1% sodium borate solution. For transmission electron microscopy, ultrathin sections were cut from resin blocks with an ultramicrotome (EM UC6; Leica, Vienna, Austria) using a diamond knife. Ultrathin sections were collected on 200 × 75mesh Formvar-coated grids, stained with saturated uranyl acetate in distilled water for 10 min, and then stained with lead citrate for 10 min. A Hitachi H-7650 transmission electron microscope was used to observe the interfaces between invading nematodes and host cells.

Nematodes began to move towards the apices of young rhizomes when nematode suspensions were poured into the surrounding water agar in petri dishes. After 4.5 hr of inoculation, many nematodes gathered around the surface of the apices and invaded them (Fig. 2). After thawing the agar, most of the nematodes remained attached to the apices when the agar around the nematodes was removed. At both 24 and 72 hr after inoculation, the number of nematodes invading the apices was much higher than at 4.5 hr after inoculation.

Figure 2:

We observed many invading nematodes in the epidermis and in a cavity where a few layers of cortical cells had disappeared at 24 hr after inoculation (Fig. 3A). Nematode invasion of some cortical cells was also observed. The host cell wall appeared to have collapsed in the cell invaded by the nematode (Fig. 3B). In total, 72 h after inoculation, nematodes had penetrated to a depth of about 1 mm from the surface (Fig. 3C). The host tissues surrounding the invading nematodes had disappeared and cavities were observed (Fig. 3D).

Figure 3:

Nematodes penetrating the epidermis were observed 4.5 hr after inoculation (Fig. 4A). We observed two patterns of penetration into the apex of the young rhizome: burrowing under the peeled part of the epidermis (Fig. 4B) and direct penetration of the epidermis through narrow indentations (Fig. 4C). In the latter case, several nematodes penetrated the epidermis from the same site. In total, 24 hr after inoculation, we observed several clusters of nematodes on the tissues where the epidermis was peeled off, accompanied by large indentations around the nematodes (Fig. 4D). The indentations were larger than those at 4.5 hr after inoculation, and neighboring indentations appeared to be connected (Fig. 4E). Most nematodes had invaded more deeply into the tissues than at 4.5 hr after inoculation (Fig. 4F).

Figure 4:

SEM images of cross sections of nematodes reached a depth of at most 1 mm by 24 hr after inoculation (Fig. 5). We observed cavities in the host tissues of up to 180 μm in diameter around the invading nematodes (Fig. 5B). Host cells at the sides of the cavity had disappeared, as if they had disintegrated (Fig. 5C).

Figure 5:

Artifacts generated by freezing in liquid nitrogen during sampling included the collapse of the cytoplasm and fragmentation of plasma membranes in uninoculated apices (Fig. 6A). However, artifacts were minimal on the cell walls and starch grains of host cells (Fig. 6A). By contrast, the epidermis and cortex around invading nematodes had degraded and were electron-dense and partly dissolved, suggesting that the nematodes had digested host tissues enzymatically (Fig. 6B, C). The area surrounding invading nematodes was clear (Fig. 6B, C).

Figure 6:

Cell walls facing the cavities were degraded and sometimes completely absent (Fig. 7A). Remaining cell walls and cell contents of the remaining portions were electron-dense (Fig. 7B). We observed fine granular materials around the invading nematode, which appeared to be denser closer to the nematode, suggesting that they were caused by secretions from the nematode (Fig. 7B, C). The outlines of the cell walls and cellular contents became indistinct at sites adjacent to the fine granular materials (Fig. 7C).

Figure 7: