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Gall formation in Erythrina latissima
Originalarbeiten . Original Papers Department of Botany, University of Natal, Pietermaritzburg, South Africa
Gall Formation in Erythrina latissima J. VAN STADEN,
J. E.
DAVEY and A. R. A. NOEL
With 10 figures Received March 25,1976 . Accepted January 30,1977.
Summary The Chalcid galls that develop on the leaves of Erythrina latissima are morphologically consistent, suggesting that they either have an obligate fauna, or alternatively that one organism, probably a species of Eurytoma, dominates the whole system. Structurally the galls are as complex as Cynipid gaHs, consisting of four distinct zones. Of these the nutritive and protective zones are the most important as they provide the food for the developing larvae and protection for the pupae when they overwinter in the gall on the ground. It would appear as if the larvae within the galls exert a strong mobilizing effect and creates a strong sink which ensures the translocation of nutrients towards the galls. This is usually manifested in that the leaf greatly decreases in size with increased infestation. Key words: Gall development, Eurytoma wasps, Erythrina latissima.
Introduction The host-parasite relationship of plant galls has been the subject of numerous investigations and most of these studies have included a description of the morphology and anatomy of the galls (KOSTOFF and KENDALL, 1929; KUSTER, 1930, 1937; Ross, 1932; NOBLE, 1940; MANI, 1964). Limited attention has also been given to the problem of gall induction (BOYSEN-JENSEN, 1948; BOPp, 1963) but comparatively little research has been carried out on the general physiology of insect galls. In South Africa the genus Erythrina is represented by six species, all of which develop a similar type of large gall on their leaves (WOOD, 1912). As far as we are aware the causal organisms and the development of these galls have not previously been described, although a preliminary study of aspects of the fine structure of galls on E. raffra THUNB. was made by HOFFMANN and VILLIERS (1971). Z. Pflanzenphysiol. Ed. 84. S. 283-294. 1977.
284
J. VAN STADEN,
J.
E. DAVEY and A. R. A. NOEL
The development of galls of this type is generally considered to be initiated by chemical stimuli released by the larval instars of the parasite (COOK, 1923; BOYSENJENSEN, 1948; MARESQUELLE and MEYER, 1965). More recent publications have indicated that plant growth substances are involved in gall induction and development (MATSUBARA and NAKAHIRA, 1968; ENGELBRECHT et al., 1969; ENGELBRECHT, 1971; OHKAWA,1974).
Materials and Methods Leaves were gathered from indigenous specimens of Erythrina latissima E. MEY. growing in the environs of PietermaritZ!burg, Natal. In order to study the life cycle of the parasite and to obtain leaves and ga1ls at various stages of development, samples were taken throughout the leaf-bearing season (October to June). Preliminary scanning for stages of gall formation and localisation of the cercidozoa was facilitated by hand sections mounted in dilute FABIL (NoEL, 1964). Other material for light microscopy was fixed in F.A.A., embedded in wax and the sections stained with safranin land fast green ,using a conventional schedule. For scanning electron microscopy (SEM), the specimens were coated in a Hitachi high vacuum coating unit: first with 100 °A carbon and then with 200 °A gold-paladium (60 : 40). In order to reduce charging a small amount of colloidal silver was applied between specimen and stub. Leaf samples were prepared for transmission electron microscopy by fixation in cacodylate buffered glutaraldehyde (6 0/0, at pH 7.2) for 6 hours at 18°C. Post-fixation in 2 0/0 osmium tetroxide was followed by dehydration in ethanol and embedment in araldite. Sections were stained with uranyl acetate and lead citrate (REYNOLDS, 1963).
Observations
Structure of E. latissima leaf The large trifoliate leaves are strongly bifacial, which is reflected by the constant orientation of the galls. Whilst the young leaves are densely tomentose, at maturity only the prominent veins on the underside of the leaf, and the galls, retain a coarse tomentum. The adaxial epidermis has a relatively thick cuticle, while the abaxial epidermis, which is distinctly papilose, has thin, slightly cuticularized walls. The stomata are confined to the sides of the abaxial papillae. The mesophyll chlorenchyma is dissected into small blocks by the extreme reticulation of the vascular system and its associated mechanical tissue. The vertical stratification of the mesophyll is indistinct, although the presence of large sub-stomatal chambers ensures that the abaxial mesophyll is particularly well ventilated. As will be indicated, the distribution of galls is related to the pattern of leaf vascularisation. The vein islets are approximately 0.4 mm wide and about half of them have included, free vein endings. As a result, all the mesophyll cells are in particularly close proximity to a vascular supply. Z. PJlanzenphysiol. Bd. 84. S. 283-294. 1977.
Gall formation in Erythrina
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Fig. 1: Finger-like projections of mature galls as found on' the lower surface of E. latissima leaves. Bar 2 cm = 1 cm.
General biology of the galls
The appearance of the mature gall is shown in Figure 1. One or all three of the leaflets may carry a few to a great number of galls. The number of galls developed has a profound effect on overall leaf size reached, which decreases markedly as the infestation increases (Fig. 2). The mature galls are very conspicuous, solid finger-like structures, hanging for up to 20 mm below the lower surface of the leaf and projecting in a dome some 7 mm above the upper surface. The wasp that emerges from the gall during the summer and spring belongs to the Ettrytomidae (Chalcidoidae). Is probably of an undescribed species of Eurytoma. E. latissima is strongly deciduous and produces new leaves after flowering in September. The youngest leaves and buds do not appear to be readily parasitised, probably because of the very thick tomentum which is present at that stage although oviposition was observed on leaflets which were expanded to only 50 % of their mature area. At least under laboratory conditions, oviposition by the species of Eurytoma that emerge from the galls was observed to be carried out on both surfaces of the leaves. Approximately 20 % of the galls contain parasites which reach pupation by mid-summer (February), whilst the leaves are still green and on the tree. Both male (70010) and female (30010) Eurytoma adults (Fig. 3) emerge from the lower surface of these galls and oviposition takes place in the mature leaves. However, most of the pupae (± 80010) overwinter in senescent galls on fallen leaves on the ground. In spring (October), the proportion of males to females that emerge from the fallen leaves is greatly altered, namely 22010 males to 78010 females. These were observed to mate under laboratory conditions. The lifespan of the imago was found to be very short, from two to four days. Z. PJlanzenphysiol. Bd. 84. S. 283-294. 1977.
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VAN STADEN,
J.
E.
DAVEY
and A. R. A.
NOEL
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4cm
Fig. 2: Effect of insect infestation on the size of E. latissima leaves. Bar 1 cm = 4 em.
The size of the galls is to some extent related to the number of Eurytoma larvae they contain. At maturity most galls contain at least two larvae. However large complex galls which accomodate as many as eight larvae and reach a diameter of 20 mm, compared with the usual 4 mm, are not uncommon. The continued presence of living larvae is a prerequisite for the maintenance of the gall. When the larvae pupate, or if they are destroyed by parasites, the galls senesce rapidly. Otherwise, for example in abscised leaves in autumn, galls containing larvae remain as green islands on the senescing lamina. This effect persists for about eight days, after which the gall tissue also become senescent and dry up. When green leaves are stored under conditions that delay senescence, galls continue to grow and remain bright green while the leaf tissue dies. The outer tissues of the lower part of the gall dies first, but the dome may remain green and continue to grow from the inner core. Z. Pjlanzenphysiol. Bd. 84. S. 283-294. 1977.
Gall formation in Erythrina
Development and structure
287
of galls
The galls are first visible, on both surfaces of the leaf, as densely tomentose raised interveinal areas, two to three mm in diameter. In section, these young galls were seen to consist of achlorphyllous callus lying in the centre of the leaf mesophyll. Numerous crystals were clustered at the inner limit of chlorophyllous mesophyll surrounding the callus. Embedded within the callus, at the end of an ovipositor tract which terminated at either the upper or lower surface, was an egg, approximately 0.17 mm in diameter (Fig. 4). The parenchyma surrounding the egg continues to proliferate and eventually the egg became ~mbedded deep in callus. Young galls are frequently found in groups of two to three. As these galls develop, either directly
Fig. 3: Mature Eurytoma wasps found to emerge from galls on E. latissima leaves. X 20.
Fig. 4: Egg found in E. latissima leaf at the end of an ovipositor tract. X 170. Fig. 5: Smooth, some-shaped upper projections (arrow) of E. latissima galls. Bar 2 cm
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Pjlanzenphysiol. Bd. 84.
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Fig. 6: Diagrammatic presentation of a transverse section through a gall at the level of the larval cavity (LC). NZ = Nutritive zone, PZ = Protective zone, PL = Parenchyma layer, E = Epidermis, V = Vascular tissue.
under or on the side of a vem, they soon fill the vem islet and cease to exist as separate entities, producing multiple galls. Initially gall development is most noticeable immediately above the egg, towards the adaxial surface. Profuse periclinal cell division in this region is followed by rapid cell enlargement. As a result many large, callus-like cells are soon found immediately above the eggs. While the outermost two to three layers of these cells at first remain chlorophyllous, the innermost cells eventually become thick walled and lignified. This results in smooth, dome-shaped projections (Fig. 5) which consist mainly of mechanical tissue, being formed immediately above the developing larvae. The larval cavities are situated on about the same plane as the leaf, therefore this part of the gall would afford some heat protection to the parasite. The maturation of the lower, ridged, finger-like projection (Fig. 1) starts slightly later than that of the upper part, although the results are far more complex. Not only does a protective casing form around the larva, but provision is made for an adequate food supply by means of a well developed vascular system connected to that of the leaf, as well as a means by which adult wasps can escape from the gall. The complexity is increased by the fact that more than one larva is usually found within a gall, and that each is housed in an independent larval cavity, and has an independent escape route. Z. PJlanzenphysiol. Bd. 84. S. 283-294. 1977.
Gall formation in Erythrina
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Sections of the galls in the region of the larval cavities showed that as is common in Cynipid galls (COOK, 1923; KOSTOFF and KENDALL, 1929), four distinct zones can be distinguished (Fig. 6). The innermost or nutritive zone consists of two to three layers of thin walled parenchyma cells that surround the larval cavity (Fig. 7). Under the light microscope it would appear as if the walls of those cells lining larval cavities are thickened and spongy. Electron microscopy however revealed that this is a nacreous layer made up of the walls of compressed cells. These cells become depleted of their contents and compressed by the developing larva (Fig. 8). It is significant that the cells of the rest of the nutritive zone are undamaged. Surrounding the nutritive zone is a very pronounced zone of mechanical tissue with very thick secondary walls showing frequent plasmodesmata. (Fig. 9). Although the vascular bundles are connected to those of the leaf, they run parallel to the larval cavity and are not directed towards it as in other galls (DOCTERS VAN LEEUWEN, W. and ]., 1909). It is furthermore noticeable that in the vascular bundles xylem elements are very weakly developed. The larger vascular bundles occurring towards the outside of the protective zone are accompanied by sclerenchymatous or collenchymatous cells, supporting the pronounced ridges on the external surface of the gall. These ridges develop only when the galls are nearly mature. The sclerenchyma cells originate from columns of parenchymatous cells, forming septate fibres whose walls remain relatively thin, or become conspicuously thick and lamella ted. The third zone of gall tissue consists of parenchyma and extends from the protective zone up to the epidermis, the latter being regarded as the fourth zone (Fig. 6). Transverse sections towards the lower tip of the gall again reveal the presence of four distinct zones of cells as described above. No cavity is however present, and the middle core of this part of the gall is filled by extremely thin-walled, callus-like tissue. These parenchyma cells usually remain meristematic. Most of these cells contain chloroplasts that are abnormal in that they posses thylakoids that are highly disorganised. The disorganisation is very similar to those observed in «green islands» caused by powdery mildew on barley leaves (CAMP and WHITTINGHAM, 1975). Longitudinal sections show that this inner parenchymatous zone extends all the way from the larval cavity to the tip of the gall, where it terminates just behind a small epidermis-covered indentation. This parenchyma is extremely susceptible to water stress and collapse within 24 hours if the fully turgid leaves are removed from the tree. As a result of this cell collapse a schizogenous canal is formed from the larval cavity, which is then only separated from the atmosphere by a single layer of epidermal cells. The presence of this canal facilitates the escape of adult wasps from the gall in spring. This observation confirms the view of KOSTOFF and KENDALL (1929) that there is great variation in the structure and origin of gall exit canals, from the situations in which it corresponds to the ovipositor tract to those where it forms along a line of cell death and disintegration. Z. Pjlanzenphysiol. Bd. 84. S. 283-294. 1977.
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VAN STADEN,
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DAVEY
and A. R. A.
NOEL
NZ SJ1m
Z. PJlanzenphysiol. Rd. 84. S. 283-294. 1977.
Gall formation in Erythrina
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Host-parasite interaction
According to MARESQUELLE and MEYER (1965) galls are initiated only after emergence of the larva, the «unhatched» eggs having no cedogenic activity. There is however no doubt that E. latissima leaf galls develop at the earliest stages of parasite embryology, when it is represented only by a patch of cells on the surface of the yolk. Although the concept of «hatching» is inappropriate in this instance, nevertheless it must be supposed that the early nuclear divisions and cell formation in the egg are accompanied by intense metabolic activity. This may be related to the initiation, and the continued development of the gall up to its mature size. In this way it is possible to explain the occurrence of galls at all stages of development up to the mature size which contain only one or no eggs, although other specimens may contain various larval instars and pupae. By mid-January about 60 % of the galls contain larvae that are 2 X 0.5 mm in size and weigh approximately 0.5 mg. This figure rises to 95 Ofo by the middle of February.
Fig. 10: Scanning micrograph of a Eurytoma larva in the larval cavity in E. latissima galls. Bar 4 cm = 1 mm. Fig. 7: Light micrograph showing a section of a larva (L) in a larval cavity (LC). NZ Nutritive zone, PZ = Protective zone. X 25. Fig. 8: Electron micrograph showing larval cuticle (L) (Arrow) and cells of the nutritive zone (NZ). Bar 1.5 cm = 5 ,urn. Fig. 9: Electron micrograph showing thick secondary walls of the protective zone. Bar 1.5 cm = 5 ,urn.
Z. PJlanzenphysiol. Bd. 84. S. 283-294. 1977.
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VAN STADEN,
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DAVEY
and A. R. A.
NOEL
The later instars fit closely into the larval cavity (Fig. 10) and their cuticles are in contact with the spongy remnants of the inner nutritive layer of the gall (Fig. 8). The second larval instars are mouth less and, having a blind gut, no excrement is observed in the larval cavities. The cells of the nutritive layer are therefore not eaten. These observations suggest that these instars may continue to feed by absorbing food directly through their cuticles. This would mean that the larvae must create a very effective «sink» in order to mobilise sufficient nutrients for their development. In support of this it may be noted that the structure of the cells of the nutritive layer (Fig. 8) do not show signs of enrichment but rather various stages of cytoplasmic dissolution. In addition it has been shown (VAN STADEN, 1975) that larvae removed from mature galls in February contain high levels of cytokinins, compounds which are generally accepted to participate in the mobilization of food reserves (MOTHES et aI., 1959).
Discussion
The Chalcid galls on E. latissima leaves are structurally as complex as Cynipid galls (COOK, 1923; KOSTOFF and KENDALL, 1929; MAN I, 1964). They develop within the leaf mesophyll, in most cases immediately adjacent or on one of the more prominent veins. They can thus be classified as true mark galls (MANI, 1964). Although it cannot at present be definitely stated that only one insect is responsible for both the induction and maintenance of the galls, because of their morphological consistency, it seems that they either have an obligate fauna, or alternatively one organism dominates the whole system. The nutritive zone of E. latissima consists of large parenchyma cells that are evenly utilized by the larval instars over the whole inner surface area of the larval cavity. In some of the oak galls studied by KOSTOFF and KENDALL (1929) the corresponding zone showed an increased enrichment of protoplasm, and in some cases an accumulation of starch. No such observations were made in the present investigation. The larvae in E. latissima galls seem to utilize the contents of the nutritive cells by dissolution of the cell contents. This is carried out without damaging the cell walls. Emptied cells are compressed by the developing larva into a layer spongy in appearance. This spongy layer in all probability corresponds to the jelly-like material that KOSTOFF and KENDALL (1929) observed in the larval cavities of N euroterus batatus. In E. latissima galls a continued supply of water and nutrients is apparently reaching the cells of the nutritive zone by means of lateral transport from the vascular bundles in the outer periphery of the protective zone. These run parallel to the long axis of the gall but there are no branches extending directly through the protective zone or ending in the immediate vicinity of the larval cavity. The cells of the protective zone, which in spite of their heavily thickened walls, retain a functional protoplast and show numerous plasmodesmata, are well adapted for lateral translocation of nutrients. Z. P/lanzenphysiol. Bd. 84. S. 283-294. 1977.
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KOSTOFF and KENDALL (1929) suggested that «sclerenchyma» zone was a more suitable term than «protective» zone. Two reasons were given for this decision. Firstly it was pointed out that the cells in the material they examined were in fact sclereids. Secondly they considered this zone to be a product of plant reaction, where an interaction between foreign substances and the plant protective substances takes place. The present observations suggest that it would be more appropriate to retain the original term of protective zone because it is obvious that this layer does indeed protect the larvae and pupae during the period of overwintering. While in agreement with KOSTOFF and KENDALL (1929) that this layer does not serve to protect the plant from the parasite we cannot agree that in E. latissima this zone is one of interaction between plant and insect. If this argument is accepted it would imply that the plant is effectively capable of isolating the parasite. This is however not the case. As a result of stimulation by the insects the plant is forced to supply them with all their nutrient requirements, be it to its own detriment. The situation is somewhat analogous to that in seeds, the larvae fulfilling a similar function to the seed embryo (FELT, 1940). That this is so can be seen from the fact that galls in senescing leaves remain functional long after the leaf lamina is dead. The senescence of the galls resulting from the destruction of the larvae is also consistent with this and indicates that the plant itself cannot counter or eliminate the effect of the parasite. The presence of the larvae in the galls must create an effective sink to which nutrients and assimilates are translocated. Part of these nutrients, particularly that provided in excess of the larval requirement, can be used for the development of the protective layer. At present there is no evidence that the substances most probably responsible for gall induction and maintenance are foreign to the plant. This aspect will be discussed in a subsequent paper. Acknowledgements The authors gratefully acknowledge a running expenses grant from the Atomic Energy Board, Pretoria. The personnel of the Electron Microscope Unit of the University of Natal is thanked for its technical assistance.
References Bopp, M.: Wurzelhalsgalle und Zelldifferenzierung. Naturw. Rundsch. 16, 349-359 (1963). BOYSEN-JENSEN, P.: Formation of galls by Mikiola Jagi. Physiol. Plant. 1, 95-108 (1948). CAMP, R. R., and W. F. WHITTINGHAM: Fine structure of chloroplasts in «green islands» and in surrounding chlorotic areas of barley leaves infected by powdery mildew. Amer. J. Bot. 62, 403-409 (1975). COOK, M. T.: The origin and structure of plant galls. Science 26, 6-14 (1923). DOCTERS van LEEUWEN, W. and J.: Beitrage zur Kenntnis der Gallen von Java. Recueil des Travaux Botaniques Neerlandais 6, 67-98 (1909). ENGELBRECHT, L., U. ORBAN, and W. HEESE: Leaf-miner caterpillars and cytokinins in the «green islands» of autumn leaves. Nature, 223, 319-321 (1969). - - - Cytokinin activity in larval infected leaves. Biochem. Physiol. Pflanzen (BPP) 162, 9-27 (1971).
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FELT, E. P. : Plant galls and gall makers. Comstock, Ithaca New York, 1940. HOFFMANN, B., and T. A. VILLIERS: A preliminary investigation into the fine structure of an insect-produced gall on Erythrina ca//ra THUNB. Proc. S. A. Electron Mic. Soc. 1,
5-6 (1971).
KOSTOFF, D., and J. KENDALL: Studies on the structure and development of certain cynipid galls. BioI. Bull. 56, 402-443 (1929). KUSTER, E.: Anatomie der Gallen. Handbuch der Pflanzenanatomie. Band V. Borntraeger, Berlin, 1932. - On the histological structure of some Australian galls. Linn. Soc. N.5. Wales Proc.
52, 57-64 (1937). MANI, M. S.: The Ecology of Plant Galls. Dr. W. Junk Publishers, The Hague, 1964. MARESQUELLE, H. J., and J. MEYER: Physiologie et morphogenese de galles d'origine animale (zoocec·idies). In Encl. Plant Physiol. W. Ruhland Ed. Vol. 15(2),280-329 (1965). MATSUBARA,S., and R. NAKAHIRA:Cytokinin activity in an extract from the gall of Plasmodiophora-infected root of Brassica rapa L. Bot. Mag. (Tokyo) 80, 373-374 (1968). MOTHES, K., L. ENGELBRECHT, and o. KULAJEWA: Ober die Wirkung des Kinetins auf Stickstoffverteilung und EiweiEsynthese in isolierten Blattern. Flora (lena) 147, 445,
464 (1959). NOEL, A. R. A.: A staining and mounting combination for sections of plant tissues. Stain Tech. 30, 324-345 (1964). NOBLE, N. 5.: Trichiloga ster Acacia-longifoliae (FROGGATT) (Hymenopt., Chalcidoidae), a wasp causing galling of the flower-buds of Acacia longifolia WILLD., A. floribunda SIEBER and A. sophorae R. Br. Roy. Ent. Soc. London Trans. 90, 13-38 (1940). OHKA WA, M.: Isolation of zeatin from larvae of Dryocosmus euriphilus YASUMA TSU. Hort. Science 9, 458-459 (1974). REYNOLDS, E. S.: The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell. BioI. 17, 208-212 (1963). Ross, H.: Praktikum der Gallenkunde. Springer, Berlin, 1932. VAN STADEN, J.: Cytokinins from larvae in Erythrina latissima galls. Plant Science Letters
5,227-230 (1975). J. M.: Natal Plants Vol. 6. Bennett-Davis, Durban, 1912.
WOOD,
Prof. Dr. J. VAN STADEN, Department of Botany, University of Natal Pi?termaritzburg, South Africa.
z. P/lanzenphysiol.
Bd. 84.
s.
283-294. 1977.
Gall Formation in Erythrina latissima J. VAN STADEN,
J. E.
DAVEY and A. R. A. NOEL
With 10 figures Received March 25,1976 . Accepted January 30,1977.
Summary The Chalcid galls that develop on the leaves of Erythrina latissima are morphologically consistent, suggesting that they either have an obligate fauna, or alternatively that one organism, probably a species of Eurytoma, dominates the whole system. Structurally the galls are as complex as Cynipid gaHs, consisting of four distinct zones. Of these the nutritive and protective zones are the most important as they provide the food for the developing larvae and protection for the pupae when they overwinter in the gall on the ground. It would appear as if the larvae within the galls exert a strong mobilizing effect and creates a strong sink which ensures the translocation of nutrients towards the galls. This is usually manifested in that the leaf greatly decreases in size with increased infestation. Key words: Gall development, Eurytoma wasps, Erythrina latissima.
Introduction The host-parasite relationship of plant galls has been the subject of numerous investigations and most of these studies have included a description of the morphology and anatomy of the galls (KOSTOFF and KENDALL, 1929; KUSTER, 1930, 1937; Ross, 1932; NOBLE, 1940; MANI, 1964). Limited attention has also been given to the problem of gall induction (BOYSEN-JENSEN, 1948; BOPp, 1963) but comparatively little research has been carried out on the general physiology of insect galls. In South Africa the genus Erythrina is represented by six species, all of which develop a similar type of large gall on their leaves (WOOD, 1912). As far as we are aware the causal organisms and the development of these galls have not previously been described, although a preliminary study of aspects of the fine structure of galls on E. raffra THUNB. was made by HOFFMANN and VILLIERS (1971). Z. Pflanzenphysiol. Ed. 84. S. 283-294. 1977.
284
J. VAN STADEN,
J.
E. DAVEY and A. R. A. NOEL
The development of galls of this type is generally considered to be initiated by chemical stimuli released by the larval instars of the parasite (COOK, 1923; BOYSENJENSEN, 1948; MARESQUELLE and MEYER, 1965). More recent publications have indicated that plant growth substances are involved in gall induction and development (MATSUBARA and NAKAHIRA, 1968; ENGELBRECHT et al., 1969; ENGELBRECHT, 1971; OHKAWA,1974).
Materials and Methods Leaves were gathered from indigenous specimens of Erythrina latissima E. MEY. growing in the environs of PietermaritZ!burg, Natal. In order to study the life cycle of the parasite and to obtain leaves and ga1ls at various stages of development, samples were taken throughout the leaf-bearing season (October to June). Preliminary scanning for stages of gall formation and localisation of the cercidozoa was facilitated by hand sections mounted in dilute FABIL (NoEL, 1964). Other material for light microscopy was fixed in F.A.A., embedded in wax and the sections stained with safranin land fast green ,using a conventional schedule. For scanning electron microscopy (SEM), the specimens were coated in a Hitachi high vacuum coating unit: first with 100 °A carbon and then with 200 °A gold-paladium (60 : 40). In order to reduce charging a small amount of colloidal silver was applied between specimen and stub. Leaf samples were prepared for transmission electron microscopy by fixation in cacodylate buffered glutaraldehyde (6 0/0, at pH 7.2) for 6 hours at 18°C. Post-fixation in 2 0/0 osmium tetroxide was followed by dehydration in ethanol and embedment in araldite. Sections were stained with uranyl acetate and lead citrate (REYNOLDS, 1963).
Observations
Structure of E. latissima leaf The large trifoliate leaves are strongly bifacial, which is reflected by the constant orientation of the galls. Whilst the young leaves are densely tomentose, at maturity only the prominent veins on the underside of the leaf, and the galls, retain a coarse tomentum. The adaxial epidermis has a relatively thick cuticle, while the abaxial epidermis, which is distinctly papilose, has thin, slightly cuticularized walls. The stomata are confined to the sides of the abaxial papillae. The mesophyll chlorenchyma is dissected into small blocks by the extreme reticulation of the vascular system and its associated mechanical tissue. The vertical stratification of the mesophyll is indistinct, although the presence of large sub-stomatal chambers ensures that the abaxial mesophyll is particularly well ventilated. As will be indicated, the distribution of galls is related to the pattern of leaf vascularisation. The vein islets are approximately 0.4 mm wide and about half of them have included, free vein endings. As a result, all the mesophyll cells are in particularly close proximity to a vascular supply. Z. PJlanzenphysiol. Bd. 84. S. 283-294. 1977.
Gall formation in Erythrina
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Fig. 1: Finger-like projections of mature galls as found on' the lower surface of E. latissima leaves. Bar 2 cm = 1 cm.
General biology of the galls
The appearance of the mature gall is shown in Figure 1. One or all three of the leaflets may carry a few to a great number of galls. The number of galls developed has a profound effect on overall leaf size reached, which decreases markedly as the infestation increases (Fig. 2). The mature galls are very conspicuous, solid finger-like structures, hanging for up to 20 mm below the lower surface of the leaf and projecting in a dome some 7 mm above the upper surface. The wasp that emerges from the gall during the summer and spring belongs to the Ettrytomidae (Chalcidoidae). Is probably of an undescribed species of Eurytoma. E. latissima is strongly deciduous and produces new leaves after flowering in September. The youngest leaves and buds do not appear to be readily parasitised, probably because of the very thick tomentum which is present at that stage although oviposition was observed on leaflets which were expanded to only 50 % of their mature area. At least under laboratory conditions, oviposition by the species of Eurytoma that emerge from the galls was observed to be carried out on both surfaces of the leaves. Approximately 20 % of the galls contain parasites which reach pupation by mid-summer (February), whilst the leaves are still green and on the tree. Both male (70010) and female (30010) Eurytoma adults (Fig. 3) emerge from the lower surface of these galls and oviposition takes place in the mature leaves. However, most of the pupae (± 80010) overwinter in senescent galls on fallen leaves on the ground. In spring (October), the proportion of males to females that emerge from the fallen leaves is greatly altered, namely 22010 males to 78010 females. These were observed to mate under laboratory conditions. The lifespan of the imago was found to be very short, from two to four days. Z. PJlanzenphysiol. Bd. 84. S. 283-294. 1977.
286
J.
VAN STADEN,
J.
E.
DAVEY
and A. R. A.
NOEL
-
4cm
Fig. 2: Effect of insect infestation on the size of E. latissima leaves. Bar 1 cm = 4 em.
The size of the galls is to some extent related to the number of Eurytoma larvae they contain. At maturity most galls contain at least two larvae. However large complex galls which accomodate as many as eight larvae and reach a diameter of 20 mm, compared with the usual 4 mm, are not uncommon. The continued presence of living larvae is a prerequisite for the maintenance of the gall. When the larvae pupate, or if they are destroyed by parasites, the galls senesce rapidly. Otherwise, for example in abscised leaves in autumn, galls containing larvae remain as green islands on the senescing lamina. This effect persists for about eight days, after which the gall tissue also become senescent and dry up. When green leaves are stored under conditions that delay senescence, galls continue to grow and remain bright green while the leaf tissue dies. The outer tissues of the lower part of the gall dies first, but the dome may remain green and continue to grow from the inner core. Z. Pjlanzenphysiol. Bd. 84. S. 283-294. 1977.
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of galls
The galls are first visible, on both surfaces of the leaf, as densely tomentose raised interveinal areas, two to three mm in diameter. In section, these young galls were seen to consist of achlorphyllous callus lying in the centre of the leaf mesophyll. Numerous crystals were clustered at the inner limit of chlorophyllous mesophyll surrounding the callus. Embedded within the callus, at the end of an ovipositor tract which terminated at either the upper or lower surface, was an egg, approximately 0.17 mm in diameter (Fig. 4). The parenchyma surrounding the egg continues to proliferate and eventually the egg became ~mbedded deep in callus. Young galls are frequently found in groups of two to three. As these galls develop, either directly
Fig. 3: Mature Eurytoma wasps found to emerge from galls on E. latissima leaves. X 20.
Fig. 4: Egg found in E. latissima leaf at the end of an ovipositor tract. X 170. Fig. 5: Smooth, some-shaped upper projections (arrow) of E. latissima galls. Bar 2 cm
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Fig. 6: Diagrammatic presentation of a transverse section through a gall at the level of the larval cavity (LC). NZ = Nutritive zone, PZ = Protective zone, PL = Parenchyma layer, E = Epidermis, V = Vascular tissue.
under or on the side of a vem, they soon fill the vem islet and cease to exist as separate entities, producing multiple galls. Initially gall development is most noticeable immediately above the egg, towards the adaxial surface. Profuse periclinal cell division in this region is followed by rapid cell enlargement. As a result many large, callus-like cells are soon found immediately above the eggs. While the outermost two to three layers of these cells at first remain chlorophyllous, the innermost cells eventually become thick walled and lignified. This results in smooth, dome-shaped projections (Fig. 5) which consist mainly of mechanical tissue, being formed immediately above the developing larvae. The larval cavities are situated on about the same plane as the leaf, therefore this part of the gall would afford some heat protection to the parasite. The maturation of the lower, ridged, finger-like projection (Fig. 1) starts slightly later than that of the upper part, although the results are far more complex. Not only does a protective casing form around the larva, but provision is made for an adequate food supply by means of a well developed vascular system connected to that of the leaf, as well as a means by which adult wasps can escape from the gall. The complexity is increased by the fact that more than one larva is usually found within a gall, and that each is housed in an independent larval cavity, and has an independent escape route. Z. PJlanzenphysiol. Bd. 84. S. 283-294. 1977.
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Sections of the galls in the region of the larval cavities showed that as is common in Cynipid galls (COOK, 1923; KOSTOFF and KENDALL, 1929), four distinct zones can be distinguished (Fig. 6). The innermost or nutritive zone consists of two to three layers of thin walled parenchyma cells that surround the larval cavity (Fig. 7). Under the light microscope it would appear as if the walls of those cells lining larval cavities are thickened and spongy. Electron microscopy however revealed that this is a nacreous layer made up of the walls of compressed cells. These cells become depleted of their contents and compressed by the developing larva (Fig. 8). It is significant that the cells of the rest of the nutritive zone are undamaged. Surrounding the nutritive zone is a very pronounced zone of mechanical tissue with very thick secondary walls showing frequent plasmodesmata. (Fig. 9). Although the vascular bundles are connected to those of the leaf, they run parallel to the larval cavity and are not directed towards it as in other galls (DOCTERS VAN LEEUWEN, W. and ]., 1909). It is furthermore noticeable that in the vascular bundles xylem elements are very weakly developed. The larger vascular bundles occurring towards the outside of the protective zone are accompanied by sclerenchymatous or collenchymatous cells, supporting the pronounced ridges on the external surface of the gall. These ridges develop only when the galls are nearly mature. The sclerenchyma cells originate from columns of parenchymatous cells, forming septate fibres whose walls remain relatively thin, or become conspicuously thick and lamella ted. The third zone of gall tissue consists of parenchyma and extends from the protective zone up to the epidermis, the latter being regarded as the fourth zone (Fig. 6). Transverse sections towards the lower tip of the gall again reveal the presence of four distinct zones of cells as described above. No cavity is however present, and the middle core of this part of the gall is filled by extremely thin-walled, callus-like tissue. These parenchyma cells usually remain meristematic. Most of these cells contain chloroplasts that are abnormal in that they posses thylakoids that are highly disorganised. The disorganisation is very similar to those observed in «green islands» caused by powdery mildew on barley leaves (CAMP and WHITTINGHAM, 1975). Longitudinal sections show that this inner parenchymatous zone extends all the way from the larval cavity to the tip of the gall, where it terminates just behind a small epidermis-covered indentation. This parenchyma is extremely susceptible to water stress and collapse within 24 hours if the fully turgid leaves are removed from the tree. As a result of this cell collapse a schizogenous canal is formed from the larval cavity, which is then only separated from the atmosphere by a single layer of epidermal cells. The presence of this canal facilitates the escape of adult wasps from the gall in spring. This observation confirms the view of KOSTOFF and KENDALL (1929) that there is great variation in the structure and origin of gall exit canals, from the situations in which it corresponds to the ovipositor tract to those where it forms along a line of cell death and disintegration. Z. Pjlanzenphysiol. Bd. 84. S. 283-294. 1977.
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Host-parasite interaction
According to MARESQUELLE and MEYER (1965) galls are initiated only after emergence of the larva, the «unhatched» eggs having no cedogenic activity. There is however no doubt that E. latissima leaf galls develop at the earliest stages of parasite embryology, when it is represented only by a patch of cells on the surface of the yolk. Although the concept of «hatching» is inappropriate in this instance, nevertheless it must be supposed that the early nuclear divisions and cell formation in the egg are accompanied by intense metabolic activity. This may be related to the initiation, and the continued development of the gall up to its mature size. In this way it is possible to explain the occurrence of galls at all stages of development up to the mature size which contain only one or no eggs, although other specimens may contain various larval instars and pupae. By mid-January about 60 % of the galls contain larvae that are 2 X 0.5 mm in size and weigh approximately 0.5 mg. This figure rises to 95 Ofo by the middle of February.
Fig. 10: Scanning micrograph of a Eurytoma larva in the larval cavity in E. latissima galls. Bar 4 cm = 1 mm. Fig. 7: Light micrograph showing a section of a larva (L) in a larval cavity (LC). NZ Nutritive zone, PZ = Protective zone. X 25. Fig. 8: Electron micrograph showing larval cuticle (L) (Arrow) and cells of the nutritive zone (NZ). Bar 1.5 cm = 5 ,urn. Fig. 9: Electron micrograph showing thick secondary walls of the protective zone. Bar 1.5 cm = 5 ,urn.
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The later instars fit closely into the larval cavity (Fig. 10) and their cuticles are in contact with the spongy remnants of the inner nutritive layer of the gall (Fig. 8). The second larval instars are mouth less and, having a blind gut, no excrement is observed in the larval cavities. The cells of the nutritive layer are therefore not eaten. These observations suggest that these instars may continue to feed by absorbing food directly through their cuticles. This would mean that the larvae must create a very effective «sink» in order to mobilise sufficient nutrients for their development. In support of this it may be noted that the structure of the cells of the nutritive layer (Fig. 8) do not show signs of enrichment but rather various stages of cytoplasmic dissolution. In addition it has been shown (VAN STADEN, 1975) that larvae removed from mature galls in February contain high levels of cytokinins, compounds which are generally accepted to participate in the mobilization of food reserves (MOTHES et aI., 1959).
Discussion
The Chalcid galls on E. latissima leaves are structurally as complex as Cynipid galls (COOK, 1923; KOSTOFF and KENDALL, 1929; MAN I, 1964). They develop within the leaf mesophyll, in most cases immediately adjacent or on one of the more prominent veins. They can thus be classified as true mark galls (MANI, 1964). Although it cannot at present be definitely stated that only one insect is responsible for both the induction and maintenance of the galls, because of their morphological consistency, it seems that they either have an obligate fauna, or alternatively one organism dominates the whole system. The nutritive zone of E. latissima consists of large parenchyma cells that are evenly utilized by the larval instars over the whole inner surface area of the larval cavity. In some of the oak galls studied by KOSTOFF and KENDALL (1929) the corresponding zone showed an increased enrichment of protoplasm, and in some cases an accumulation of starch. No such observations were made in the present investigation. The larvae in E. latissima galls seem to utilize the contents of the nutritive cells by dissolution of the cell contents. This is carried out without damaging the cell walls. Emptied cells are compressed by the developing larva into a layer spongy in appearance. This spongy layer in all probability corresponds to the jelly-like material that KOSTOFF and KENDALL (1929) observed in the larval cavities of N euroterus batatus. In E. latissima galls a continued supply of water and nutrients is apparently reaching the cells of the nutritive zone by means of lateral transport from the vascular bundles in the outer periphery of the protective zone. These run parallel to the long axis of the gall but there are no branches extending directly through the protective zone or ending in the immediate vicinity of the larval cavity. The cells of the protective zone, which in spite of their heavily thickened walls, retain a functional protoplast and show numerous plasmodesmata, are well adapted for lateral translocation of nutrients. Z. P/lanzenphysiol. Bd. 84. S. 283-294. 1977.
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KOSTOFF and KENDALL (1929) suggested that «sclerenchyma» zone was a more suitable term than «protective» zone. Two reasons were given for this decision. Firstly it was pointed out that the cells in the material they examined were in fact sclereids. Secondly they considered this zone to be a product of plant reaction, where an interaction between foreign substances and the plant protective substances takes place. The present observations suggest that it would be more appropriate to retain the original term of protective zone because it is obvious that this layer does indeed protect the larvae and pupae during the period of overwintering. While in agreement with KOSTOFF and KENDALL (1929) that this layer does not serve to protect the plant from the parasite we cannot agree that in E. latissima this zone is one of interaction between plant and insect. If this argument is accepted it would imply that the plant is effectively capable of isolating the parasite. This is however not the case. As a result of stimulation by the insects the plant is forced to supply them with all their nutrient requirements, be it to its own detriment. The situation is somewhat analogous to that in seeds, the larvae fulfilling a similar function to the seed embryo (FELT, 1940). That this is so can be seen from the fact that galls in senescing leaves remain functional long after the leaf lamina is dead. The senescence of the galls resulting from the destruction of the larvae is also consistent with this and indicates that the plant itself cannot counter or eliminate the effect of the parasite. The presence of the larvae in the galls must create an effective sink to which nutrients and assimilates are translocated. Part of these nutrients, particularly that provided in excess of the larval requirement, can be used for the development of the protective layer. At present there is no evidence that the substances most probably responsible for gall induction and maintenance are foreign to the plant. This aspect will be discussed in a subsequent paper. Acknowledgements The authors gratefully acknowledge a running expenses grant from the Atomic Energy Board, Pretoria. The personnel of the Electron Microscope Unit of the University of Natal is thanked for its technical assistance.
References Bopp, M.: Wurzelhalsgalle und Zelldifferenzierung. Naturw. Rundsch. 16, 349-359 (1963). BOYSEN-JENSEN, P.: Formation of galls by Mikiola Jagi. Physiol. Plant. 1, 95-108 (1948). CAMP, R. R., and W. F. WHITTINGHAM: Fine structure of chloroplasts in «green islands» and in surrounding chlorotic areas of barley leaves infected by powdery mildew. Amer. J. Bot. 62, 403-409 (1975). COOK, M. T.: The origin and structure of plant galls. Science 26, 6-14 (1923). DOCTERS van LEEUWEN, W. and J.: Beitrage zur Kenntnis der Gallen von Java. Recueil des Travaux Botaniques Neerlandais 6, 67-98 (1909). ENGELBRECHT, L., U. ORBAN, and W. HEESE: Leaf-miner caterpillars and cytokinins in the «green islands» of autumn leaves. Nature, 223, 319-321 (1969). - - - Cytokinin activity in larval infected leaves. Biochem. Physiol. Pflanzen (BPP) 162, 9-27 (1971).
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FELT, E. P. : Plant galls and gall makers. Comstock, Ithaca New York, 1940. HOFFMANN, B., and T. A. VILLIERS: A preliminary investigation into the fine structure of an insect-produced gall on Erythrina ca//ra THUNB. Proc. S. A. Electron Mic. Soc. 1,
5-6 (1971).
KOSTOFF, D., and J. KENDALL: Studies on the structure and development of certain cynipid galls. BioI. Bull. 56, 402-443 (1929). KUSTER, E.: Anatomie der Gallen. Handbuch der Pflanzenanatomie. Band V. Borntraeger, Berlin, 1932. - On the histological structure of some Australian galls. Linn. Soc. N.5. Wales Proc.
52, 57-64 (1937). MANI, M. S.: The Ecology of Plant Galls. Dr. W. Junk Publishers, The Hague, 1964. MARESQUELLE, H. J., and J. MEYER: Physiologie et morphogenese de galles d'origine animale (zoocec·idies). In Encl. Plant Physiol. W. Ruhland Ed. Vol. 15(2),280-329 (1965). MATSUBARA,S., and R. NAKAHIRA:Cytokinin activity in an extract from the gall of Plasmodiophora-infected root of Brassica rapa L. Bot. Mag. (Tokyo) 80, 373-374 (1968). MOTHES, K., L. ENGELBRECHT, and o. KULAJEWA: Ober die Wirkung des Kinetins auf Stickstoffverteilung und EiweiEsynthese in isolierten Blattern. Flora (lena) 147, 445,
464 (1959). NOEL, A. R. A.: A staining and mounting combination for sections of plant tissues. Stain Tech. 30, 324-345 (1964). NOBLE, N. 5.: Trichiloga ster Acacia-longifoliae (FROGGATT) (Hymenopt., Chalcidoidae), a wasp causing galling of the flower-buds of Acacia longifolia WILLD., A. floribunda SIEBER and A. sophorae R. Br. Roy. Ent. Soc. London Trans. 90, 13-38 (1940). OHKA WA, M.: Isolation of zeatin from larvae of Dryocosmus euriphilus YASUMA TSU. Hort. Science 9, 458-459 (1974). REYNOLDS, E. S.: The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell. BioI. 17, 208-212 (1963). Ross, H.: Praktikum der Gallenkunde. Springer, Berlin, 1932. VAN STADEN, J.: Cytokinins from larvae in Erythrina latissima galls. Plant Science Letters
5,227-230 (1975). J. M.: Natal Plants Vol. 6. Bennett-Davis, Durban, 1912.
WOOD,
Prof. Dr. J. VAN STADEN, Department of Botany, University of Natal Pi?termaritzburg, South Africa.
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