CYTOLOGICAL CHANGES INDUCED BY THE STEM ...

0 downloads 0 Views 1MB Size Report
solution of 0.005% aniline blue in 0.067 M phosphate buffer (Peterson et al., 1978) .... McMULLEN C. R., GARDNER W. S. and MYERS G. A., 1978 . Aberrant ...
I Nematol.

medii. (1980), 8: 153·163.

/stituto di Nemalologia agraria, C.N.R., Bari /stituto di Botanica dell'Universita degli Studi, Bari, Italy

CYTOLOGICAL CHANGES INDUCED BY THE STEM NEMATODE DITYLENCHUS DIPSACI IN STRAWBERRY LEAVES

by T. BLEVE-ZACHEO, G. ZACHEO, M. T. MELILLO, F. LAMBERTI and O. ARRIGONI (1)

The stem nematode, Ditylenchus dipsaci (KUhn) Filipjev, causes swellings and growth malformation on stems and leaves of various susceptible hosts in which the chlorophyllic parenchyma cells are hypertrophied or destroyed (Rhode, 1972). Watson et al. (1979) report that at the feeding site the nematode induces cell separation because of the dissolution of the middle lamella which according to Seinhorst (1957), is a process necessary for the development of the parasite. Hussey and Krusberg (1968, 1970) found that D. dipsaci attacks the embryonic leaves of shoot apices of resistant pea varieties by penetrating directly through the epidermis and induces formation of cavities in the parenchymatous mesophyll tissue only 48 hr after inoculation. They observed thickening of the cortical cell walls of the invaded tissues and an intense peroxidase activity, associated with the trophic activity of the nematode. Dropkin (1963), Krusberg (1960, 1967) and Riedel and May (1971) suggested that pectolytic enzymes are secreted by D. dipsaci within the plant tissue and that these are primarily responsible for cell separation during the early stages of the nematode infestation.

(l) The assistance of Mr. R. Lerario in preparing the illustrations is grate· fully acknowledged.

-

153 -

Campbell and Griffin (1973) observed « white flagging» associated with D. dipsaci in alfalfa leaves and noted a progressive reduction in the lamellar structure of the chloroplasts followed by a decrease in pigmentation. The object of the present investigation was to study the changes induced by D. dipsaci in strawberry (Fragaria vesca L.) leaves.

Materials and methods Fragments of curled leaves collected from a strawberry plantation heavily infested by D. dipsaci were fixed for 2 hr in 3% glutaraldheyde at 4u C, rinsed overnight in 0.1 M phosphate buffer at pH 7.2 and postfixed for 2 hr in 1 % osmium tetroxide. They were then dehydrated in a graded ethanol-propylene oxide series and embedded in Araldite. Sections 2 [Lm thick were stained in toluidine blue and observed under a light microscope. For the histochemical detection of callose, sections were immersed for 2 hr in NaOH methanol solution, which was changed repeatedly to eliminate the resin, stained with an aqueous solution of 0.005% aniline blue in 0.067 M phosphate buffer (Peterson et al., 1978) and examined with a Leitz Dialux 20 EB microscope with fluorescent light and exciter filter KP 490 and barrier filter TK 510/K 515. Ultrathin sections were stained for 1 hr in uranyl acetate and for 10 min in lead citrate (Reynolds, 1963) and examined at 80 KV in a JEM 100 B electron microscope.

Results Sections stained with toluidine blue revealed the presence of D. dipsaci in the lacunar tissue. The trophic activity of the nematode induced the dissolution of the middle lamellae of the cells, which separated from each other (Fig. 1 a) to form cavities surrounding a progressively degenerating area. The tissue formed by the parenchyma cells bordering the cavi ties became hypertrophied and the cell walls were remarkably thickened and sometimes interrupted. The cytoplasm of the cells was a granular mass apparently without any definite structure. These changes were

-

154-

more evident in the cells of the lacunar tissue and of the bundle sheaths and less evident in the cells of the palisade parenchyma (Fig. 1 b, c and d). Electron microscopy showed that the cell walls bordering the cavities consisted of a series o[ electron dense layers. Tissue stained with fluorescent aniline blue and examined under ultraviolet light did not reveal the presence of callose in these cells (Fig. 2 b). The structure of the cell layers adjacent to the cavity in the mesophyll tissue was less disorganized, but the cell walls showed some middle lamella dissolution (Fig. 2 a). Moreover, electron dense material and para mural bodies were present in the cytoplasm (Fig. 2 a, c), and the chloroplasts generally were swollen with disorganized lamellar systems and with gaps on their outer membranes. These chloroplasts contained several vesicles (Fig. 3 a and b). There were also chloroplasts in which it was almost impossible to identify the original structures which had been transformed into an amorphous matrix with numerous small vacuoles (Fig. 3 b and c). The accumulation of starch grains induced by the nematode transformed the original structure of the thylakoids and grana were displaced toward the outside of some chloroplasts (Fig. 3 d). These observations agree with those of Tanaka and Akai (1960) who demonstrated a decrease of the starch hydrolitic enzyme, [\-amylase, in the parasitized cells. The structure of the mitochondria was transformed into a dense matrix with few cristae. The membrane surrounding the cell nucleus became disconnected from the nuclear matrix, thus presenting irregular outline (Fig. 3 c). In the mesophyll cells the cytoplasm was transformed to a fibrous material, similar to that observed by Politis and Goodman (1978), (Fig. 4 a and b). Several paramural bodies (Fig. 4 c) and deposits of transparent electron material were present on the walls between plasmalemma and primary wall with interposed microtubules (Fig. 4 d). These deposits gradually extended into the cellular lumen and occupied a large part of it (Fig. 5 a). The aniline blue treatment revealed large deposits of callose which appeared as narrow dotted bands; sometimes the whole cell was filled with fluorescent material (Fig. 5 b). As the callose deposition progressed secondary walls were formed which protruded into the cytoplasm or into the intercellular spaces (Fig. 5 c and d).

-

155-

Fig. 1 - Crass sections of strawberry leaves attacked by Ditylenchus dipsaci stai ned with toluidi ne blue; a) scattered mcsophyll cells with numerous nematodes (arrows); b) mesophyll tissue with large cavities, nematodes and hypertrophied cells ( Hy); c and d) necrotic bundle sheath cells (nc), with des troyed cytoplasm and interrupted thickened walls ( IW).

Fig. 2 - b) Cell wall with electron dense layers assumed to consist o f s uberins (sb); a and c) cell wall with dark middle lamella in di ssolution a nd associated paramura l bodies (pb); note in tracellular space filled with an amorphous substance (as),

,I(.,

Fig. 3 - a and b) Chlol'Oplasts with gaps o n outer surface portions of cytoplasm (arrow) and connected to membranOLLS bodies (mb); b, c) plastids with disorgan ized grana in an a mo rphOliS matrix a nd numerous small vacuoles; mitochondria (m) with de nse matrix and scarce c ristae; roughed nucleus (rn) (note large space in the nuclear membrane): d) ab norma l plastid with large starch grai ns (sg),

I

,

Fi~. 4 - n, b) Large deposits of microfibrils in the cytoplas m and chloroplasts (my), Intermingled w ith paramura l bodies; c, d) differen t stages of membrane bound vesicles between plasmalemma and primary cell wall (mbv). Cytoplasm with numerous Goigi bodies (Gb) and po lysomes (p).

Fig. 5· a) Large deposition of callose on walls, plasmalemma ( pi); b) stained with a niline blue (ca); c) secondary wall ingrowth (cw) in the cytoplasm and d) on the outside of the intraceJluiar space.

Discussion In strawberry leaves attacked by D. dipsaci cavItIes developed in the tissues, due to the lytic action of the nematode, as reported by Dropkin (1963) and Krusberg (1960 and 1967). These authors, in fact, noticed activity of cellulytic and pectolytic enzymes in homogenates of the nematode and suggested that pectolytic enzymes secreted and injected by the nematode into the invaded tissues might be the main cause of the separation of the cells. The thickened walls of the cells bordering the cavities consist of electron dense layers, and is probably due to the presence of suberins as suggested by Lipetz (1970). In the leaves of virus-infected Gomphrena globosa suberin and lignin have been found to accumulate in the region of the virus local lesions (Appiano et al., 1977). The paramural bodies occur in tissue invaded by nematodes (Huang and Maggenti, 1969; Bleve-Zacheo et al., 1979), viruses bacteria and fungi (Wheeler, 1975; McMullen et al., 1978; Cohen and Loebenstein, 1975; Politis and Goodman, 1973; Politis and Goodman, 1978) and are characteristic of the host-parasite interaction. These organelles, which are associated with cell walls, are thought to be involved in \,·all synthesis. Arrigoni and Rossi (1963) and Esau et al. (1966) have demonstrated that paramural bodies most likely are concerned with cell \,·all growth. We suggest that the paramural bodies, through the extensi\·e callose deposition, could control new cell wall growth in the injured cell.

SUMMARY

Lltrastructural modifications induced by Ditylenchus dipsaci (Kuhn) Filipjev in stra\\·berry leaves show many similarities with those reported by other authors. The most remarkable changes in the cell concern the chloroplasts and the cell wall. Chloroplasts were modified with abnormal accumulation of starch grains. Cell walls were considerably thickened and the presence of callose was demonstrated histochemically. The early appearance of several paramural bodies supports the idea of an interrelationship between these organelks and callose biosynthesis.

-

161 -

RIASSUNTO

Alterazioni indotte da Ditylenchus dipsaci su fog lie di fragola. Le alterazioni ultrastrutturali indotte da Ditylenchus dipsaci (Kuhn) Filipjev su foglie di fragola sembrano, per molti versi, simili a quelle riportate da altri au tori. Le anomalie pili evidenti sono state riscontrate a carico dei cloroplasti e della parete cellulare. Nei cloroplasti, infatti, si rinviene un anormale accumulo di amido, mentre sulle pareti cellulari, notevolmente ispessite, si evidenzia, in seguito a trattamento con bleu di anilina, apposizione di callosio. La presenza di lomasomi, nei primi stadi dell'infezione, suggerisce l'ipotesi dell'esistenza di una relazione tra questi organelli e la biosintesi di callosio.

LITERATURE

CITED

ApPIANO A., PENNAZIO S., D'AGOSTINO G. and RIDOLFI P., 1977 . Fine structure of necrotic local lesions induced by tomato bushy stunt virus in Gomphrena globosa leaves. Physiol. Plant Pathol., 11: 327-332. ARRIGONI O. and ROSSI G., 1963 - I lomasomi: loro probabili rapporti con la crescita per distensione della parete cellulare. Giorn. Bot. !tal., 70: 476-481. BLEVE-ZACHEO T., ZACHEO G., LAMBERTI F. and ARRIGONI 0., 1979 - Cell wall protrusions and associated membranes in roots parasitized by Longidorus apulus. Nematologica, 25: 62-66. CAMPBELL W. F. and GRIFFIN G. D., 1973 - Fine structure analyses of stem nematode-induced white flagging in Medicago sativa. I. Nematol., 5: 123-126. COHEN J. and LOEBENSTEIN G., 1975 - An electron microscope study of starch lesions in cucumber cotyledons infected with tobacco mosaic virus. Phytopathology, 65: 32-39. DROPKIN V. H., 1963 - Cellulase in phytoparasitic nematodes. Nematologica, 9: 444-454.

ESAU K., CHEADLE V. I. and GILE R. H., 1966 - Cytology of differentiating tracheary elements. II Structures associated with cell surfaces. Amer. I. Bot., 53: 765-771.

HUANG C. S. and MAGGENTI A. R., 1969 - Wall modifications in developing giant cells of Vicia faba and Cucumis sativus induced by root-knot nematode, Meloidogyne javanica. Phytopathology, 59: 931-937. HUSSEY R. S. and KRUSBERG L. R., 1968 . Histopathology of resistant reactions in Alaska pea seedlings to two populations of Ditylenchus dipsaci. Phytopathology, 58: 1305-1310. HUSSEY R. S. and KRUSBERG L. R., 1970 - Histopathology and oxidative enzymes patterns in Wando peas infected with two populations of Ditylenchus dipsaci. Phytopathology, 60: 1818-1825. KRUSBERG L. R., 1960 - Hydrolytic and respiratory enzymes of species of Di· tylenchus. Phytopathology, 50: 9-22. KRUSBERG L. R., 1967 - Pectinase in Ditylenchus dipsaci. Nematologica, 13: 443-451. LIPETZ T., 1970 - Wound-healing in higher plants. Int. Rev. of Cytol., 27: 1-28. McMULLEN C. R., GARDNER W. S. and MYERS G. A., 1978 . Aberrant plastids in barley leaf tissue infected with barley stripe mosaic virus. Phytopathology, 68: 317-323. PETERSON R. L., HERSEY R. E. and BRISSON J. D., 1978 - Embedding softened herbarium material in Spurr's resin for histological studies. Stain Technology, 53: 1-9.

-

162-

POLITIS D. J. and WHEELER H., 1973 - Ultrastructural study of penetration of maize leaves by Colletotrichum gramineacola. Physiol. Plant Pathol., 3: 465-471. POLITIS D. J. and GOODMAN R. N., 1978 - Localized cell wall appositions: incompatibility response of tobacco leaf cells to Pseudomonas pisi. Phytopathology, 68: 309-316. REY:-..'OLDS E. S., 1963 - The use of lead citrate at high pH as an electron opaque stain in electron microscopy. 1. Cell. Biol., 17: 208-212. RIEDEL R. M. and MAl W. F., 1971 - A comparison of pectinases from Ditylenchus dipsaci and Allium cepa callus tissue. 1. Nematol., 3: 174-178. RHODE R. A., 1972 - Expression of resistance in plants to nematode. Ann. Rev. Phytopathol., 10: 233-252. SEI:-..'HORST J. W., 1957 - Ditylenchus: races, pathogenicity and ecology. Proc. S-19 Workshop Phytonem., Nashville: 1-7. TA:-..'AKA H. and AKAJ S., 1960 - On the mechanism of starch accumulation in tissue surrounding lesions in rice leaves due to the attack of Lochliobolus miyabeanus. II. On the activities of beta-amylase and invertase in tissues surrounding spots. (In Japanese, English summary). Ann. Phytopathol. Soc. lap., 25: 80-84. W.uso" A. K. and SHORTHOVSE T. D., 1979 - Gall formation on Cirsium arvense by Ditylenchus dipsaci. l. Nematol., 11: 16-22. WHEELER H., 1975 - Plant Pathogenesis. Springer Verlag, Berlin, Heidelberg, New York, 106 pp.

Accepted for publication on 6 May, 1980.

-

163-