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Morphological and anatomical changes associated with gall formation on the leaves of Cussonia spicata
Morphological and anatomical changes associated with gall formation on the leaves of Cussonia spicata C. Malan, J. van Staden, J. Gouws and P.J. Robbertse Research Institute for Grain Crops , Potchefstroom; Department of Botany, University of Natal , Pietermaritzburg; Department of Botany, University of the North, Pietersburg and Department of Botany, University of Pretoria, Pretoria
The simple tubular galls which develop on the leaves of Cussonia spicata are apparently caused by a new species of psylla , of the genus Trioza. As a result of feeding by the in sect, most of the leaf cells in the vicinity of the feeding sites resumed meristematic activity and divided . As a consequence, the normal leaf structure became disorganized and the very distinctive chlorotic galls developed. During gall development the chloroplasts decreased in number and size and the grana degenerated . Associated with this degeneration was the appearance of phytoferritin. These changes could be correlated with lower levels of endogenous cytokinins in the gall tissue . S . Atr. J . Bot. 1982, 1: 58- 63
Die eenvoudige buisagtige galle wat op die blare van Cussonia spicata ontwikkel word blykbaar veroorsaak deur ' n nuwe onbeskryfde spesie van psylla van die genus Trioza . As gevolg van die insek se voeding raak meeste van die selle in die omgewing van die voedingsposisie meristematies en verdeel. Dit het tot gevolg dat die normale blaarstruktuur ongeorganiseerd raak en die karakteristieke chlorotiese galle ontwikkel. Soos die galle ontwikkel het , het die chloroplaste in die selle minder en kleiner geword en hulle grana het gedegenereer. Gedurende hierdie degenerasie is fitoferritien in die chloroplaste waargeneem. Hierdie verandering het saamgeval met laer hoeveelhede natuurlike sitokiniene in die galweefsel. S.·Afr. Tydskr. Pla ntk. 1982, 1: 58-63
Keywords: Galls , C. spicata, psylla, chloratic, phytoferritin , cytokinins
C. Ma la n* Research Institu te fo r Gra in Crops, Pri vate Bag X1251, Potchefstroom 2520, Republic of South Afri ca J. van Staden Department of Botany, Uni versit y of Natal, Pieterma ri tzbu rg 3200, Republ ic of South A fri ca J . Gouws Department of Botany, Uni versity of th e North , Private Bag X5090, Pietersburg 0700 , Republic o f South Afri ca and P .J . Robbertse Department of Botany, University of Pretoria, Pretoria 0002 , Republic of South Africa *To whom corres pondence should be addressed Accepted 8 February 1982
Introduction The leaves of Cussonia spicata frequently become distorted and abnormal as a result of the development of tubular, pocket-like galls on their abaxial surfaces . The galls, as well as the tissue immediately around their openings are chlorotic, and contrast sharply with the dark green appearance of the unaffected leaf lamina (Van Staden, Gilliland & Bandu 1981) _ While the host-parasite relationships of plant galls have been the subject of many investigations (Kostoff & Kendall 1929; Kuster 1932; Mani 1964), the biology of the Cussonia leaf gall system has apparently not been described. Material and Methods Uninfested leaves of Cussonia spicata Thunb., as well as leaves with galls at different stages of development, were collected during the summer months (December February) from plants growing in their natural habitat. Material for light microscopy was fixed in FAA, embedded in wax and the sections stained with safranin and aniline blue as described by Johansen (1940). For scanning electron microscopy (SEM), material was dried in a critical point drier, coated with gold, and investigated with a JSM T 200 scanning electron microscope. Samples for transmission electron microscopy (TEM) were prepared by fixation in sodium cacodylate buffered gluteraldehyde (4% at pH 7 ,2) for 24 h . Post-fixation in 207o osmium tetroxide was followed by dehydration in ethanol and embedment in Spurr's low viscosity resin or in Araldite. Sections were stained with uranyl acetate and lead citrate (Reynolds 1963) _ Uninfested leaf laminae and galls were analysed for cytokinins according to the method of Van Staden & Davey (1978) . Twenty grams of fresh plant material was homogenized in 80% ethanol and the homogenate extracted at 5° C for 24 h . After filtration the ethanolic extracts were subjected to Dowex 50 purification and the adsorbed cytokinins removed from the cation exchange resin with 5 mol dm - J NH 4 0H. The ammonia was removed under vacuum in an evaporator and the residue was redissolved in 3 ml 80% ethanol. This extract was striploaded onto Whatman no . 1 chromatography paper and separated with iso-propanol:25% ammonium hydroxide: water (10: I: I v/ v) . The chromatograms were dried thoroughly and divided into 10 equal Rf strips, which were assayed for cytokinin activity using the soybean callus bio-
59
S. Afr. J. Bot. 1982, 1(3)
Figure 1 Galls on C. spica/a leaves.
assay (Miller 1965). All extractions and bioassays were carried out in duplicate and the results presented are the means of the two separate bioassays.
ment and maintenance. Investigations showed that female adult psylla attach their eggs to young emerging leaves of C. spicata. The eggs which are much smaller than the structures shown in Figure 3 are secured by the insertion of a projection of the outer egg membrane into the epidermis or hypodermis of either the ad- or abaxial surface of the leaf (Figure 4). A similar type of attachment has been reported for Cardiaspina densitexta which parasitizes Eucalyptus fasciculosa (White 1970).
Results and Discussion General biology of the insect and morphology of the galls In summer, simple tubular, pocket-like galls which are highly chlorotic (Figure I & 2) develop from the abaxial leaf surfaces of C. spicata. As egg-like structures were observed in the galls (Figure 3) it was essential to establish whether or not these structures are involved in gall develop-
After hatching of the eggs, almost all the nymphs move to the abaxial leaf surface where they commence feeding by penetrating the vascular tissue with their stylets (Fig-
Figure 2 Chlorotic pocket-like galls (g) on the leaves of C. spica/a.
Figure 3 Egg-like structure (e) found in C. spica/a leaf galls.
60
S.-Afr. Tydskr. Plantk . 1982, 1(3)
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Figure 4 Photomicrograph showing the attachment of psylla egg (e) to the leaf surface by means of a projection (P) of the outer membrane of the egg.
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Figure 6 Photomicrograph showing the palisade (p) and spongy mesophyll (s) of a healthy leaf.
Figure 5 Photomicrograph showing a nymph stylet (s) penetrating the leaf vascular ti ssue.
ure 5). This is the stage when gall formation is initiated, indicating that the large egg-like structures which occur in the galls (Figure 3) are not involved in gall development. If the nymphs, which belong to a new undescribed species of psylla (Homoptera: Psyllidae) from the genus Trioza (Hollis: British Museum, pers. comm., 1981), are removed or die, gall development ceases. With time the gall tissue and parts of the surrounding leaf tissue become chlorotic. Frequently however, chlorosis is masked by the accumulation of a purple-red pigment in the galls. The accumulation of anthocyanin pigments is a common occurrence in plant tissues which have been invaded by foreign organisms (Hanchey-Bauer 1978). The dome-shaped, pocket-like galls that develop on the leaves of C. spicata probably serve a shelters for the psylla nymphs during feeding . Adult psylla emerge from the fifth nymphal stage. Anatomy and physiology of the galls In the normal state the leaves of C. spicata have a welldeveloped palisade and spongy mesophyll (Figure 6). Within the galls however, there is a more or less uniform
Figure 7 Photomicrograph of gall tissue showing the disorderly arrangement of the cells . Note the large number of cells which have divided .
mass of ill-defined parenchyma cells (Figure 7). Localized areas of the gall tissue show meristematic activity, the nuclei being in an active state with distended nucleoli. Newly formed cell walls occur frequently, indicating that active cell division was taking place in those areas where the insects fed (Figure 8). Some of the cells in the galls show extreme disturbance of the membrane system with myelin bodies being present (Figure 9). In the cells of the uninfested leaves, and the uninfested parts of the leaves where galls were present, the chloroplasts in the palisade and spongy mesophyllline the cells for maximum exposure to light. These chloroplasts have a very dense stroma with numerous thylakoid lamellae
61
S. Afr. J. Bot. 1982, 1(3)
Figure 10 Chloroplast structure in uninfested normal cells. Figure 8 Electron micrograph of gall tissue showing newly formed cell wall (arrow).
Figure 9 Electron micrograph of myelin bodies in the gall tissue.
arranged in stacks to form large grana. A few plastoglobuli are present (Figure 10). In contrast, the chloroplasts in the gall tissue are much less numerous, and they are also smaller and swollen. The overall number of thylakoid lamellae is greatly reduced and these have lost their regular arrangement, leaving an appearance of distortion. The stroma is still dense and the inner membrane of the chloroplast envelope develops many peripheral vesicles which extend into the chloroplast. Such vesicles usually develop when leaves are under stress (Laetsch 1974). Within these stressed chloroplasts crystal-like structures could be observed (Figure 11). With time the chloroplast structure deteriorated gradually (Figure 12) and the crystal-like structures became more prominent. Under high magnification the crystal-like particles could be seen to have a structure similar to that which is normally associated with phytoferritin (Cronshaw eta!. 1966; Robards & Humpherson 1967; Amelunxen et at 1970; Maramorosch & Hirumi 1973). They seem to be made up of four or five opaque subunits arranged around a central translucent core, and surrounded by a light area (Figure 13). Phytoferritin is an iron complex which occurs abundantly in the chloroplasts of diseas-
Figure 11 Chloroplast in gall tissue. Thylakoid lamellae are reduced greatly and crystal-like (c) structures can be seen.
ed plants where the grana are deteriorating (Cronshaw et at. 1966; Maramorosch & Hirumi 1973). With time the chloroplasts disintegrated completely (Figure 14). Aphids and psylla are the two main vectors for virus transfer to plants. The question arose as to whether the crystal-like structures observed in the chloroplasts were viruses rather than phytoferritin. This question is of considerable importance as viruses (Kuriger & Agrios 1977), and other pathogens such as fungi (Patrick eta!. 1977) and mycoplasmas (Gaborjanyi & Sziraki 1978) cause leaf chlorosis, which is associated with lower levels of endogenous cytokinins in the infested tissues. If the observed crystal-like particles were viruses it would be expected that the degree of infestation would have included other organelles and would not be confined to the plastids only as was found in this investigation. Of particular significance is the fact that the degeneration of chloroplasts, chlorophyll breakdown, and thus the appearance of phytoferritin could be retarded by the application of synthetic cytokinins (Mlodzianowski & Kwintkiewicz 1973; Gailhofer & Thaler 1975). This suggests that there may be a close relationship between cytokinin levels
62
S.-Afr. Tydskr. Plantk . 1982, 1(3)
Figure 12 Crystal-like structures (arrow) which appear in the chloroplasts of the gall cells.
Figure 13 Electron micrograph of the crystal-like structures found in the chloroplasts of gall tissue.
Figure 14 Electron micrograph of gall tissue showing the disintegrated parts of a chloroplast (ch).
and the appearance of phytoferritin in chloroplasts. Two physiological phenomena, viz. cell division and chlorophyll breakdown, both of which are influenced by cytokinins (Van Staden & Davey 1979), were affected by the parasitic psylla. As a result it was considered necessary to investigate the levels of endogenous cytokinins in the healthy leaf lamina and in the chlorotic leaf galls. Higher levels of endogenous cytokinins, which cochromatographed with zeatin and ribosylzeatin, were detected in the non-infested leaf lamina than in the chlorotic gall tissue (Figure 15). Psylla feed on C. spicata leaves by inserting their stylets into the vascular tissue from which they then withdraw compounds normally transported to or from the leaves . As a result of this feeding the chlorotic leaf galls develop.
At present it is very difficult to decide whether the induction of cell division, and chlorophyll loss are induced by compounds inserted into the leaf tissue by the insect, or whether these phenomena are due to the creation of a hormonal imbalance resulting from the removal of endogenous hormones from the areas where the insects feed. Lower levels of endogenous cytokinins were detected in the chlorotic leaf galls indicating that these hormones were apparently removed from the parasitized leaf tissue. The lower level of cytokinin could be correlated with the deterioration of the chloroplast structure and the appearance of phytoferritin. In the galls which occur on the leaves of Erythrina latissima low levels of cytekinin were also detected in the meristematic and slightly chlorotic gall tissue (Van Staden & Davey 1978). This situation was ap-
63
S. Afr. J. Bot. 1982, I (3)
References I
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Figure 15 Soybean callus bioassays of extracts from the equivalent of 20 g of healthy leaf tissue (A) and 20 g of gall tissue (B). Dowex 50 purified extracts were separated on paper using iso-propanol:25!tfo NH 40H :water (10: I: I v/ v) Z = zeatin; ZR = ribosylzeatin. Shaded areas represent activity significantly different from the controls at the I !tfo level.
parently created by the larvae of the Eurytoma wasp which are found inside the galls and which accumulate large amounts of cytokinin (Van Staden 1975). In the leaves of C. spicata the same situation is apparently created by the continuous feeding of the psylla nymphs and the removal of vascular transported cytokinins. These cytokinins which can occur within chloroplasts themselves (Davey & Van Staden 1981), are apparently necessary for the prevention of chlorophyll breakdown. In terms of the Cussonia system it is necessary to reconcile the lower level of endogenous cytokinin which was present in the meristematic gall tissue, with the generally accepted view that these hormones are necessary for cell division. Usually, and probably incorrectly, the cell division process is associated with high levels of endogenous cytokinins (Van Staden & Davey 1979). It is, however, well established that this process is inhibited or retarded by both high and low concentrations of cytokinin. In the case of zeatin and ribosylzeatin the optimum response is in the vicinity of 0,2-2,0 ng dm - J (Davey & Van Staden 1967). As cell division was induced in the gall regions where lower levels of endogenous cytokinins were detected, it would appear as if the cytokinin level could have been reduced to one which was stimulatory to this process. In mature leaves where cell division does not occur, high levels of endogenous cytokinin were detected (Van Staden 1976, 1977; Van Staden & Davey 1978). This indicates that high cytokinin levels need not necessarily be associated with increased meristematic activity. At present, however, it is not known whether or not cell-division inducing compounds are introduced into the leaves of C. spicata by the psylla during feeding. Acknowledgements The Electron Microscope Units of the University of Natal, Pietermaritzburg and the University of Pretoria are thanked for their assistance. J. van Staden thanks the C.S.I.R. Pretoria for financial support.
AMELU NXEN, F., THALER, I. & HARSDORFF, M. 1970. Die Struktur der Phytoferritinkristalle von Phajus grandifolius. Z . Pflanzenphysiol. 63: 199-210. CRONSHAW, J., HOEFERT, L . & ESAU, K. 1966. Ultrastructural features of Beta leaves infected with beet yellow virus. J. Cell. Bioi. 31: 429 - 443 . DAVEY, J.E. & VAN STADEN, J. 1976. Cytokinin translocation. Changes in zeatin and zeatin-riboside levels in the root exudate of tomato plants during their development. Planta 130: 69- 72. DAVEY, J.E. & VAN STADEN, J. 1981. Cytokinins in spinach chloroplasts. Ann. Bot. 48: 243-246. GABORJANYI, R. & SZIRAKI , I. 1978. Cytokinins in white clover plants infected by clover phyllody mycoplasma. A cta Phytopath. Acad. Soi. Hungaricae 13: 33-40. GAILHOFER, M. & THALER, I. 1975. Einfluss von 6-Benzylaminopurin auf die Feinstruktur von Mitochondrien und Plastiden in vitro kultivierter Protokorme von Cy mbidium Phyton (Austria) 17: 159-165 . HANCHEY-BAUER, P. 1978. Physiological plant pathology. In: Plant Physiology, eds, SALISBURY, F.B. and ROSS , C.W. 2nd edn. 399 - 402. Wadsworth , Belmont , California. JOHANSEN, D.A. 1940. Plant Microtechnique. McGraw-Hill, New York. KOSTOFF, D. & KENDALL, J. 1929. Studies on the structure and development of certain cynipid galls. Bioi. Bull. 56: 402-443. KURIGER, W.E. & AGRIOS, G.N. 1977. Cytokinin levels and kinetin-virus interactions in tobacco ringspot virus-infected cowpea plants. Phytopathology, 67 : 604 - 609. KUSTER, E . 1932. Anatomie der Ga llen. Handbuch der P flanzenanatomie. Band V. Borntraeger, Berlin. LAETSCH, W.M. 1974. The C 4 syndrome: a structural analysis. Ann. Rev. Plant Physiol. 25 : 27- 52. MAN!, M.S. 1964. The Ecology of Plant Galls. Dr. W . J unk Publishers, The Hague. MARAMOROSCH, K. & HIRUMI , H. 1973. Phytoferritin accumulations in leaves of diseased coconut palms. Protoplasma 78 : 175 - 180. MILLER, C.O. 1965. Evidence for the natural occurrence of zeatin and derivatives: Compounds from maize which promote cell division. Proc. Nat/ A cad. Soi. USA . 54: 1052- 1054. MLODZIANOWSKI, F. & KWINTKlEWICZ, M. 1973. Th e inhibition of kohlrabi chloroplast degeneration by kinetin. Proloplasma 76: 211 - 226. PATRICK, T .W., HALL, R. & FLETCHER, R.A . 1977. Cytokinin levels in healthy and Verticillium-in fected tomato plants. Can. J. Bot. 55: 377 - 382. REYNOLDS, E .S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. Bioi. 17: 208-212. ROBARDS, A .W. & HUMPHERSON, P .G . 1967. Phytoferritin in plastids of the cambial zone of willow. Planta 76: 169 - 178. VAN STADEN, J. 1975. C ytokinins from larvae in Ery thrina /atissima galls. Pl. Sci. Letters 5: 227 - 230. VAN STADEN, 1. 1976. Seasonal changes in the cytokinin content of Ginkgo biloba leaves. Physiol. Plant. 38: I- 5. VAN STADEN, J. 1977. Seasonal changes in the cytokinin content of the leaves of Salix babylonica. Physiol. Plant. 40: 296 - 299. VAN STADEN, J. & DAVEY, J.E. 1978. Endogenous cytokinins in the laminae and galls of Erythrina latissima leaves. Bot. Gaz. 139: 36 - 41. VAN STADEN, J. & DAVEY, J .E. 1979. The synthesis, transport a nd metabolism of endogenous cytokinins. Plant Cell & Environ. 2: 93- 106. VAN STADEN, J., GILLILAND, M.G . & BANDU, V. 1981. Structure of Cussonia spica/a leaf galls. Proc. Electron M icrosc. Soc. SouthAf r. 11:171-172. WHITE, T.C.R. 1970. Some aspects of the life history, host selection, dispersal, and oviposition of adult Cardiaspina densitexta (Homoptera: Psyllidae). Aust. J. Zoo/. 18: 105 - 11 7.
The simple tubular galls which develop on the leaves of Cussonia spicata are apparently caused by a new species of psylla , of the genus Trioza. As a result of feeding by the in sect, most of the leaf cells in the vicinity of the feeding sites resumed meristematic activity and divided . As a consequence, the normal leaf structure became disorganized and the very distinctive chlorotic galls developed. During gall development the chloroplasts decreased in number and size and the grana degenerated . Associated with this degeneration was the appearance of phytoferritin. These changes could be correlated with lower levels of endogenous cytokinins in the gall tissue . S . Atr. J . Bot. 1982, 1: 58- 63
Die eenvoudige buisagtige galle wat op die blare van Cussonia spicata ontwikkel word blykbaar veroorsaak deur ' n nuwe onbeskryfde spesie van psylla van die genus Trioza . As gevolg van die insek se voeding raak meeste van die selle in die omgewing van die voedingsposisie meristematies en verdeel. Dit het tot gevolg dat die normale blaarstruktuur ongeorganiseerd raak en die karakteristieke chlorotiese galle ontwikkel. Soos die galle ontwikkel het , het die chloroplaste in die selle minder en kleiner geword en hulle grana het gedegenereer. Gedurende hierdie degenerasie is fitoferritien in die chloroplaste waargeneem. Hierdie verandering het saamgeval met laer hoeveelhede natuurlike sitokiniene in die galweefsel. S.·Afr. Tydskr. Pla ntk. 1982, 1: 58-63
Keywords: Galls , C. spicata, psylla, chloratic, phytoferritin , cytokinins
C. Ma la n* Research Institu te fo r Gra in Crops, Pri vate Bag X1251, Potchefstroom 2520, Republic of South Afri ca J. van Staden Department of Botany, Uni versit y of Natal, Pieterma ri tzbu rg 3200, Republ ic of South A fri ca J . Gouws Department of Botany, Uni versity of th e North , Private Bag X5090, Pietersburg 0700 , Republic o f South Afri ca and P .J . Robbertse Department of Botany, University of Pretoria, Pretoria 0002 , Republic of South Africa *To whom corres pondence should be addressed Accepted 8 February 1982
Introduction The leaves of Cussonia spicata frequently become distorted and abnormal as a result of the development of tubular, pocket-like galls on their abaxial surfaces . The galls, as well as the tissue immediately around their openings are chlorotic, and contrast sharply with the dark green appearance of the unaffected leaf lamina (Van Staden, Gilliland & Bandu 1981) _ While the host-parasite relationships of plant galls have been the subject of many investigations (Kostoff & Kendall 1929; Kuster 1932; Mani 1964), the biology of the Cussonia leaf gall system has apparently not been described. Material and Methods Uninfested leaves of Cussonia spicata Thunb., as well as leaves with galls at different stages of development, were collected during the summer months (December February) from plants growing in their natural habitat. Material for light microscopy was fixed in FAA, embedded in wax and the sections stained with safranin and aniline blue as described by Johansen (1940). For scanning electron microscopy (SEM), material was dried in a critical point drier, coated with gold, and investigated with a JSM T 200 scanning electron microscope. Samples for transmission electron microscopy (TEM) were prepared by fixation in sodium cacodylate buffered gluteraldehyde (4% at pH 7 ,2) for 24 h . Post-fixation in 207o osmium tetroxide was followed by dehydration in ethanol and embedment in Spurr's low viscosity resin or in Araldite. Sections were stained with uranyl acetate and lead citrate (Reynolds 1963) _ Uninfested leaf laminae and galls were analysed for cytokinins according to the method of Van Staden & Davey (1978) . Twenty grams of fresh plant material was homogenized in 80% ethanol and the homogenate extracted at 5° C for 24 h . After filtration the ethanolic extracts were subjected to Dowex 50 purification and the adsorbed cytokinins removed from the cation exchange resin with 5 mol dm - J NH 4 0H. The ammonia was removed under vacuum in an evaporator and the residue was redissolved in 3 ml 80% ethanol. This extract was striploaded onto Whatman no . 1 chromatography paper and separated with iso-propanol:25% ammonium hydroxide: water (10: I: I v/ v) . The chromatograms were dried thoroughly and divided into 10 equal Rf strips, which were assayed for cytokinin activity using the soybean callus bio-
59
S. Afr. J. Bot. 1982, 1(3)
Figure 1 Galls on C. spica/a leaves.
assay (Miller 1965). All extractions and bioassays were carried out in duplicate and the results presented are the means of the two separate bioassays.
ment and maintenance. Investigations showed that female adult psylla attach their eggs to young emerging leaves of C. spicata. The eggs which are much smaller than the structures shown in Figure 3 are secured by the insertion of a projection of the outer egg membrane into the epidermis or hypodermis of either the ad- or abaxial surface of the leaf (Figure 4). A similar type of attachment has been reported for Cardiaspina densitexta which parasitizes Eucalyptus fasciculosa (White 1970).
Results and Discussion General biology of the insect and morphology of the galls In summer, simple tubular, pocket-like galls which are highly chlorotic (Figure I & 2) develop from the abaxial leaf surfaces of C. spicata. As egg-like structures were observed in the galls (Figure 3) it was essential to establish whether or not these structures are involved in gall develop-
After hatching of the eggs, almost all the nymphs move to the abaxial leaf surface where they commence feeding by penetrating the vascular tissue with their stylets (Fig-
Figure 2 Chlorotic pocket-like galls (g) on the leaves of C. spica/a.
Figure 3 Egg-like structure (e) found in C. spica/a leaf galls.
60
S.-Afr. Tydskr. Plantk . 1982, 1(3)
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Figure 4 Photomicrograph showing the attachment of psylla egg (e) to the leaf surface by means of a projection (P) of the outer membrane of the egg.
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Figure 6 Photomicrograph showing the palisade (p) and spongy mesophyll (s) of a healthy leaf.
Figure 5 Photomicrograph showing a nymph stylet (s) penetrating the leaf vascular ti ssue.
ure 5). This is the stage when gall formation is initiated, indicating that the large egg-like structures which occur in the galls (Figure 3) are not involved in gall development. If the nymphs, which belong to a new undescribed species of psylla (Homoptera: Psyllidae) from the genus Trioza (Hollis: British Museum, pers. comm., 1981), are removed or die, gall development ceases. With time the gall tissue and parts of the surrounding leaf tissue become chlorotic. Frequently however, chlorosis is masked by the accumulation of a purple-red pigment in the galls. The accumulation of anthocyanin pigments is a common occurrence in plant tissues which have been invaded by foreign organisms (Hanchey-Bauer 1978). The dome-shaped, pocket-like galls that develop on the leaves of C. spicata probably serve a shelters for the psylla nymphs during feeding . Adult psylla emerge from the fifth nymphal stage. Anatomy and physiology of the galls In the normal state the leaves of C. spicata have a welldeveloped palisade and spongy mesophyll (Figure 6). Within the galls however, there is a more or less uniform
Figure 7 Photomicrograph of gall tissue showing the disorderly arrangement of the cells . Note the large number of cells which have divided .
mass of ill-defined parenchyma cells (Figure 7). Localized areas of the gall tissue show meristematic activity, the nuclei being in an active state with distended nucleoli. Newly formed cell walls occur frequently, indicating that active cell division was taking place in those areas where the insects fed (Figure 8). Some of the cells in the galls show extreme disturbance of the membrane system with myelin bodies being present (Figure 9). In the cells of the uninfested leaves, and the uninfested parts of the leaves where galls were present, the chloroplasts in the palisade and spongy mesophyllline the cells for maximum exposure to light. These chloroplasts have a very dense stroma with numerous thylakoid lamellae
61
S. Afr. J. Bot. 1982, 1(3)
Figure 10 Chloroplast structure in uninfested normal cells. Figure 8 Electron micrograph of gall tissue showing newly formed cell wall (arrow).
Figure 9 Electron micrograph of myelin bodies in the gall tissue.
arranged in stacks to form large grana. A few plastoglobuli are present (Figure 10). In contrast, the chloroplasts in the gall tissue are much less numerous, and they are also smaller and swollen. The overall number of thylakoid lamellae is greatly reduced and these have lost their regular arrangement, leaving an appearance of distortion. The stroma is still dense and the inner membrane of the chloroplast envelope develops many peripheral vesicles which extend into the chloroplast. Such vesicles usually develop when leaves are under stress (Laetsch 1974). Within these stressed chloroplasts crystal-like structures could be observed (Figure 11). With time the chloroplast structure deteriorated gradually (Figure 12) and the crystal-like structures became more prominent. Under high magnification the crystal-like particles could be seen to have a structure similar to that which is normally associated with phytoferritin (Cronshaw eta!. 1966; Robards & Humpherson 1967; Amelunxen et at 1970; Maramorosch & Hirumi 1973). They seem to be made up of four or five opaque subunits arranged around a central translucent core, and surrounded by a light area (Figure 13). Phytoferritin is an iron complex which occurs abundantly in the chloroplasts of diseas-
Figure 11 Chloroplast in gall tissue. Thylakoid lamellae are reduced greatly and crystal-like (c) structures can be seen.
ed plants where the grana are deteriorating (Cronshaw et at. 1966; Maramorosch & Hirumi 1973). With time the chloroplasts disintegrated completely (Figure 14). Aphids and psylla are the two main vectors for virus transfer to plants. The question arose as to whether the crystal-like structures observed in the chloroplasts were viruses rather than phytoferritin. This question is of considerable importance as viruses (Kuriger & Agrios 1977), and other pathogens such as fungi (Patrick eta!. 1977) and mycoplasmas (Gaborjanyi & Sziraki 1978) cause leaf chlorosis, which is associated with lower levels of endogenous cytokinins in the infested tissues. If the observed crystal-like particles were viruses it would be expected that the degree of infestation would have included other organelles and would not be confined to the plastids only as was found in this investigation. Of particular significance is the fact that the degeneration of chloroplasts, chlorophyll breakdown, and thus the appearance of phytoferritin could be retarded by the application of synthetic cytokinins (Mlodzianowski & Kwintkiewicz 1973; Gailhofer & Thaler 1975). This suggests that there may be a close relationship between cytokinin levels
62
S.-Afr. Tydskr. Plantk . 1982, 1(3)
Figure 12 Crystal-like structures (arrow) which appear in the chloroplasts of the gall cells.
Figure 13 Electron micrograph of the crystal-like structures found in the chloroplasts of gall tissue.
Figure 14 Electron micrograph of gall tissue showing the disintegrated parts of a chloroplast (ch).
and the appearance of phytoferritin in chloroplasts. Two physiological phenomena, viz. cell division and chlorophyll breakdown, both of which are influenced by cytokinins (Van Staden & Davey 1979), were affected by the parasitic psylla. As a result it was considered necessary to investigate the levels of endogenous cytokinins in the healthy leaf lamina and in the chlorotic leaf galls. Higher levels of endogenous cytokinins, which cochromatographed with zeatin and ribosylzeatin, were detected in the non-infested leaf lamina than in the chlorotic gall tissue (Figure 15). Psylla feed on C. spicata leaves by inserting their stylets into the vascular tissue from which they then withdraw compounds normally transported to or from the leaves . As a result of this feeding the chlorotic leaf galls develop.
At present it is very difficult to decide whether the induction of cell division, and chlorophyll loss are induced by compounds inserted into the leaf tissue by the insect, or whether these phenomena are due to the creation of a hormonal imbalance resulting from the removal of endogenous hormones from the areas where the insects feed. Lower levels of endogenous cytokinins were detected in the chlorotic leaf galls indicating that these hormones were apparently removed from the parasitized leaf tissue. The lower level of cytokinin could be correlated with the deterioration of the chloroplast structure and the appearance of phytoferritin. In the galls which occur on the leaves of Erythrina latissima low levels of cytekinin were also detected in the meristematic and slightly chlorotic gall tissue (Van Staden & Davey 1978). This situation was ap-
63
S. Afr. J. Bot. 1982, I (3)
References I
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Figure 15 Soybean callus bioassays of extracts from the equivalent of 20 g of healthy leaf tissue (A) and 20 g of gall tissue (B). Dowex 50 purified extracts were separated on paper using iso-propanol:25!tfo NH 40H :water (10: I: I v/ v) Z = zeatin; ZR = ribosylzeatin. Shaded areas represent activity significantly different from the controls at the I !tfo level.
parently created by the larvae of the Eurytoma wasp which are found inside the galls and which accumulate large amounts of cytokinin (Van Staden 1975). In the leaves of C. spicata the same situation is apparently created by the continuous feeding of the psylla nymphs and the removal of vascular transported cytokinins. These cytokinins which can occur within chloroplasts themselves (Davey & Van Staden 1981), are apparently necessary for the prevention of chlorophyll breakdown. In terms of the Cussonia system it is necessary to reconcile the lower level of endogenous cytokinin which was present in the meristematic gall tissue, with the generally accepted view that these hormones are necessary for cell division. Usually, and probably incorrectly, the cell division process is associated with high levels of endogenous cytokinins (Van Staden & Davey 1979). It is, however, well established that this process is inhibited or retarded by both high and low concentrations of cytokinin. In the case of zeatin and ribosylzeatin the optimum response is in the vicinity of 0,2-2,0 ng dm - J (Davey & Van Staden 1967). As cell division was induced in the gall regions where lower levels of endogenous cytokinins were detected, it would appear as if the cytokinin level could have been reduced to one which was stimulatory to this process. In mature leaves where cell division does not occur, high levels of endogenous cytokinin were detected (Van Staden 1976, 1977; Van Staden & Davey 1978). This indicates that high cytokinin levels need not necessarily be associated with increased meristematic activity. At present, however, it is not known whether or not cell-division inducing compounds are introduced into the leaves of C. spicata by the psylla during feeding. Acknowledgements The Electron Microscope Units of the University of Natal, Pietermaritzburg and the University of Pretoria are thanked for their assistance. J. van Staden thanks the C.S.I.R. Pretoria for financial support.
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