Ann. appl. Biol. (1978), 90,293-302 With 5 plates Printed in Great Britain

A wilt disease of coconuts from Trinidad associated with Phytomonas sp., a sieve tube-restricted protozoan flagellate

293

BY HENRY WATERS Coconut Industry Board, P.O. Box 204, Kingston 10, Jamaica

(Accepted 24 May 1978)

SUMMARY

A wilt disease of coconut palms in Trinidad has been responsible for the death of more than 15 000 palms in the last 2 years. Symptoms of the disease include: loss of nuts, premature browning of successively younger leaves, necrosis of open and unopened inflorescences and of the young unexpanded leaves at the centre of the crown; rotting of the growing point, stem apex and roots.

Samples from each of 10 diseased palms contained flagellates which were classified as trypanosomatid protozoans of the genus Phytomonas. The protozoans, which by squeezing could be expressed easily from tissues for observation by phase contrast light microscopy, were 1-1.5 ym wide and 27 ym long including the flagellum which was approximately 7 Fm long.

Sections of inflorescence rachillae from diseased palms revealed that the phytomonads were restricted to the sieve tubes. Phytomonas was always associated with diseased palms but it was never found in healthy palms.

A brief description of the ultrastructure of the presumed pathogen of wilt disease is also given.

I N T R O D U C T I O N

Since 1975 a wilt disease of unknown aetiology has killed approximately 15 000 coconut palms (COCOS nucifera L.) in the Cedros area of Trinidad as well as killing an undetermined number of palms in at least four other coconut growing areas of the island. The disease is therefore a serious threat to the cultivation of coconuts and the associated industries in Trinidad. This report describes the symptomatology of the disease and a sieve element inhabiting protozoan flagellate which was found to be associated with diseased palms.

M A T E R I A L S A N D M E T H O D S

Symptomatology. Nine palms of different ages from four coconut growing areas (Text-fig. 1) were felled, dissected and the symptoms recorded. Eight of these palms were diseased and one was apparently healthy (Table 1). Samples were taken from these palms for the direct detection of flagellates by light microscopy.

Direct examination offlugellutes. Flagellates from living material or material fixed in buffered glutaraldehyde/paraformaldehyde were observed by phase contrast light microscopy in the sap droplet expressed from tissues by squeezing a sample approximately 1 a 5 x 0.5 x 0.5 cm with a pair of pliers.

Electron and light microscopy of sectioned tissues. Inflorescence samples were collected from two diseased and two healthy palms growing at St. Annes estate in the Cedros area (Text-fig. 1). The samples from diseased palms were taken from apparently healthy tissue of the rachilla of

294 H. W A T E R S

I To co

Text-fig. 1 . Map of Trinidad showing the sampling sites (m). Palms exhibiting symptoms have been observed at Moruga. The widespread distribution of the wilt disease is evident. The main coconut-growing areas are within the broken lines.

unopened inflorescences approximately 5 cm below a black distal necrosis. Control samples from healthy palms were taken from comparable sites. Samples were fixed in Trinidad for 3 h at approximately 30 OC in a buffer mixture (pH 7.2) containing 0.05 M sodium cacodylate, 0.15 M sucrose and 2 m M calcium chloride to which glutaraldehyde and paraformaldehyde were added to give final concentrations of 3% each. Following primary fixation and two lhr-rinses in the buffer mixture (i.e. without glutaraldehyde or paraformaldehyde) the samples were sent in buffer by airmail to Jamaica where post fixation was carried out for 2 h in 2% w/v osmium tetroxide in the buffer mixture at approximately 21 OC. Dehydration through an ascending acetone series preceeded embedding in Spurr’s resin (Spurr, 1969). Sections were cut with diamond knives and mounted supported by formvar on 50 mesh grids. Contrast was enhanced with 20% w/v methanolic uranyl acetate and lead citrate (Venable & Coggershall, 1965). Observations were made using an AEI 80 1A electronmicroscope.

For light microscopy the material embedded in Spurr’s resin was sectioned at 2-4 pm with dry glass knives on an LKB UltratomeIII after which the sections were stained either with Paragon stain (Gurr) or 1% w/v toluidine blue 0 in 1% w/v aqueous di-sodium tetraborate and made permanent with Permount (Fisher Scientific).

Table 1. Palms sampled for symptomatology and direct examination of Phytomonas

Palm no. Condition Est. age yr Stem height m Collected at

20-25 15-20 20-25

2-3 2-3

10-15 1.5 2-3

15-20

11 8

1 1 1 1 4.5 O* 1 6-5

Cedros Cedros Manzanilla Arima Arima Cedros Toco Toco Cedros

* Seedling palm; H, healthy; D, diseased.

A wilt disease of coconuts and an associated protozoan 295

RESULTS

Symptomatology of the wilt disease A healthy coconut palm generally produces one leaf and sheds one leaf each month. Thus in

a healthy palm the oldest leaf is the only one showing signs of advanced senescence which takes the form of browning and desiccation. The disease first becomes evident as a more or less simultaneous browning of the oldest two or three leaves and perhaps the shedding of medium sized nuts. Symptoms of the disease include the loss of nuts, browning of successively younger leaves, necrosis of open and unopened inflorescences and of the immature leaves of the spear', a rot of the growing point, stem apex and roots. Table 2 lists the sampled palms ranked in order of advancing symptom progression for eight diagnostic characters. The time from the first diagnosis of disease to complete death is between 8 and 12 wk depending upon the age and condition of the palm, the more vigorously growing palms dying more rapidly. Palms of all ages (Table 1) are susceptible as are the tall and dwarf varieties cultivated in Trinidad. The length of any incubation period is not known.

Table 2. Range of symptom expression shown by the one healthy and eight diseased coconut palms which were sampled. Palms are ranked in order of advancing disease symptoms

No. Necrosis of Rot of No. partly No. 7- -7

Palm brown brown green Nut open unopen stem Root no. leaves leaves leaves fall infl. i d . spear G.P. apex death

0 4

13 - *

* -

8 5 8 9

26 12 I 2 0 0 0 0 0

0 0 + 0

prebearing prebearing

+ +

+ + seedling

prebearing + +

0 0 0 0

+ 0 + +

+ + + +

+ + + + +

0 0 0 0 0 0 0 0 + 0

0 + +

- ** + + + +

0 0 + +

Palm numbers refer to Table 1; *the number of brown leaves could not be established because they were removed as they appeared as part of the estate management; ** seedling palm; G.P., growing point; +, present; 0, absent.

Palms in the early stages of disease (Table 2 , palms 2 , 3,4) have leaves which may be grouped into three classes i.e. those that are green and apparently healthy, those that are partially brown and those that are completely brown, dead and desiccated. As the disease progresses all leaves eventually turn brown (Table 2, palm 9). Leaf browning follows a characteristic pattern. The distal pinnae first turn yellow/bronze and then soon turn brown and become desiccated. This is repeated for all pinnae as the browning extends proximally. Unlike normal senescence where the leaves are shed, diseased palms tend to retain many of the dead leaves which fall against the stem forming a skirt of brown leaves (Plate 1, fig. 1). Before collapsing against the stem, the rachis of a dead leaf often breaks leaving the distal part hanging down (Plate 1, fig. 1). No rachis break was recorded in mature green leaves.

Complete nut loss is usual in bearing palms with well advanced symptoms. Nut fall begins with medium aged green nuts, usually when the affected palm has two or three brown leaves. An internal browning of the husk and a blackening of the developing shell of medium sized nuts

The spear is defined as the group of exposed leaves at the centre of the crown (usually 3 or 4 in a bearing palm) in which the pinnae are unexpanded.

296 H . W A T E R S

was noted in palms 2 and 3 (Table 2). These observations were made on nuts deliberately removed from inflorescences to avoid confusion with decay or bruising following natural nut fall.

Inflorescences of bearing palms showed symptoms both on the female flowers and the rachillae 1-2 months after the opening of the spathes (Plate 1, fig. 2). The female flowers or buttons (fertilised female flowers) were grey/brown and desiccated. Often they were shed leaving the dry calyx attached to the rachillae (Plate 1, fig. 2). Rachillae were either distally necrosed and desiccated or missing (Plate 1, fig. 2). Some inflorescences failed to open fully and in such instances the entire inflorescence, when fresh, showed a dark brownlblack necrosis (Plate 1, figs 3,4). Where aborted opening of inflorescences was observed on palms in which the inflorescence had been partially open for 4 or 5 months (e.g. Table 2, palm 6), the inflorescences were greylbrown and desiccated. No necrosis of the spathes of these inflorescences exserted from the leaf axils was observed.

In bearing palms, differences between the symptoms expressed on the immature unopened inflorescences were related to the stage of disease development. In palm 2 (Table 2) there was no necrosis of the unopened inflorescences. Where the disease was more advanced but green leaves were still present within the crown (e.g. Table 2, palm 3), unopened inflorescences were either distally or completely necrosed (Plate 2, figs 1, 2). At this stage there was no necrosis of the spathes. In palms where symptoms were more advanced (e.g. Table 2, palm 6), the necrosis extended to include the spathe. The nature of the necrosis itself also differed in relation to the overall stage of disease development. At the intermediate stage the necrosis was dark brown or black and odourless. When the spathes of these inflorescences were opened a clear fluid ran out but the necrosed tissues were firm. Where the symptoms were more advanced and the necrosis included the spathe, the rot was light brown, soft, wet and foul smelling. In neither of these instances was the contiguous leaf tissue rotten or necrosed.

The developing inflorescences of pre-bearing palms (Table 2, palms 4, 5, 8) were also rotten within the crown. The rot included the spathes and was light brown, soft, wet and foul smelling. Like the rot of unopened inflorescences from bearing palms with advanced symptoms, this rot did not extend to adjacent leaf tissues.

Necrosis of the spear was invariably associated with the disease. This began on the pinnae margins of spear leaves as small patches of pale greylbrown papery necrosis which subsequently enlarged and coalesced until the whole spear was necrosed. The onset of necrosis was associated with the development of a wet brown rot in the bases and petioles of affected spears at a site enclosed by older leaf bases and only 10 to 30 cm above the growing point. When all the leaves comprising a spear were necrosed, it fell from its normally erect position within the crown. At this stage the spear could be pulled from the bud and the tissues so exposed were rotten, soft, pale yellow, wet and foul smelling.

A foul smelling, ochre and liquid rot which destroyed the growing point and the associated immature stem tissues (Plate 2, cf. figs 3,4) was recorded in half the palms examined (Table 2). In four of the palms this rot extended down into the more mature tissues of the stem apex; 60 cm in palm 9 (Table 2). The rotting of the stem was variable. For example in palm 6 (Table 2) it was dark brown, wet and peripheral whereas in palm 9 (Table 2) it was pale brown, wet and central.

Roots were not extensively studied but it was clear that root death was associated with this disease. Primary roots, recently emerged from the bole, showed apical or complete necrosis. Excavated secondary and tertiary roots also showed rotting and desiccation.

Sequence of symptom expression The first symptom which may be recognised is the browning of the oldest leaves and perhaps

the loss of some of the medium sized nuts. A rapid progression of symptoms takes place with spear necrosis, additional mature leaves turning brown and the appearance of the ‘skirt’ of dead

A wilt disease of coconuts and an associated protozoan 291

leaves. At this stage early symptoms can be expected in the inflorescences. Nut fall increases with younger and older nuts being lost. When all the leaves in the crown are brown, the spear symptoms will be well advanced and it will probably be hanging down within the crown. The unopened inflorescences and their spathes will also be rotten and the growing point will have been destroyed. Only a very few old nuts may now be present. In the final stages, all nuts will have been lost and a rot of the stem apex will have begun. Finally, what remains of the crown falls from the stem. This can happen in one of two ways. The stem may fold over 60-80 cm below the crown; alternatively the crown falls from the stem leaving it standing like a telegraph pole.

The protozoan flagellate and its association with diseased palms Using the squeezing technique for the light microscopic detection of flagellates, the eight

palms with disease symptoms each provided at least one tissue sample which contained flagellates (Table 3). None of the samples from six different tissues (Table 3) from the healthy palm examined contained flagellates. Each of these negative records was the result of at least five replicate extractions. It is evident that even in the earliest stages of symptom expression, diseased palms have been extensively invaded by the presumed pathogen (e.g. Table 2, palm 2, where the un-necrosed and therefore apparently healthy tissues of the bud and unopened inflorescences contained flagellates (Table 3)).

Flagellates were present in sectioned inflorescence samples from two diseased palms (at a stage similar to that described for palm 3, Table 2) but none were found in comparable samples from healthy palms (Table 4). The organisms were invariably restricted to the mature sieve elements. They were not present in other phloem cells, the xylem or the contiguous ground tissue (Plate 3, fig. 1). The flagellates were orientated with their longitudinal axes parallel to the

Table 3. Incidence offlagellates in the sampled coconut palms

Sample site tested

Soft tissue of the bud c. 10 cm directly below the growing point Rachis or petiole of spear leaves Roots Unopened inflorescences with necrosis Unopened inflorescences without necrosis Opened inflorescences Stem base, centre, c. 50 cm above ground level Stem base, peripheral 5-8 cm, 50 cm above ground level Stem apex at centre At least one tissue sample positive for flagellates

Palm no.* 1 2 3 4 5 6 7 8 9

o + + + - - + - - o + + - + + + + - o o - - - - - - - 0 - + - - - - _ _ o + o - - - - - - o - + - - o - - - - - + - - + - - + - _ + + - + - - - - - + - - + - - -

o + + + + + + + + * Palm numbers refer to those given in Table 1; +, flagellates present; 0, flagellates absent from at least five

replicate preparations; -, no data available.

Table 4. Numbers of sieve elements containing Phytomonas in healthy and diseased inflorescence samples

H:althy palms Diseased palms A B A B

Mature sieve elements 0 0 1955 1076 containing Phytomonas Mature sieve elements 615 1295 256 137 without Phytomonas % affected sieve elements 0 0 88.4 88.7

29 8 H . W A T E R S

longitudinal axis of the sieve element (Plate 3, figs 1, 2). The organism varied in their numbers per infected cell but often they were densely packed (Plate 3, figs 1,2; Plate 4).

Flagellates isolated from tissues by squeezing had a promastigote morphology (Hoare & Wallace, 1966) which is shown in Plate 3, figs 3-5. The body was finely tapered posteriorly and blunter anteriorly where a single robust flagellum was inserted within a flagellar reservoir (Plate 3, figs 4, 5). Organisms were 1-1.5 pm across at the widest point and approximately 27 pm long, including the flagellum which was approximately 7 pm long. Many of the flagellates expressed from fresh or fixed tissues were twisted (Plate 3, fig. 4); in preparations made of living flagellates the twisted forms were as active as the non-twisted forms. Swimming activity was rarely observed, the organisms remaining more or less stationary, flexing the body so that the anterior was brought round towards the posterior.

Ultrastructure of the protozoan flagellate The individual organisms were surrounded by a continuous pellicular membrane which lined

the reservoir and covered the flagellum (Plate 5 , figs 2 , 5 , 6). A single row of subpellicular microtubules lay immediately below this membrane (Plate 4; Plate 5, figs 5 , 6). These microtubules lay obliquely to the longitudinal axis of the organisms (Plate 5, figs 6, 7). The largest number (30-40) of microtubules was observed at the widest point of the body; the numbers fall off both anteriorly and posteriorly to as few as 12 implying that the microtubules are unequal in length.

The anterior flagellar reservoir was asymmetrical, the lip being longer on one side than the other (Plate 5 , fig. 1). No subpellicular microtubules lined the reservoir except for a discrete group of four short microtubules which was opposite microtubule doublets 3 and 4 of the flagellar axoneme (Plate 5 , fig. 5) . Distally they appeared to be unequal in length because in some sections of the flagellar reservoir their number was reduced to two. Their proximal origin could not be determined.

The single flagellum arose from the floor of the reservoir and for much of its length carried a paraxial rod (Plate 5, figs 1 to 3). This flagellar appendage was composed of an amorphous weakly electron dense material and it was always opposite the shorter side of the flagellar reservoir and coincided with microtubule doublets 5 and 6 of the axoneme (Plate 5 , figs 1, 5 ) . The paraxial rod was absent from the flagellum both proximally and distally. It originated approximately 0.5 pm above the floor of the reservoir and terminated approximately 1.0 pm from the distal tip of the flagellum (Plate 5, figs 1 , 2). Where the paraxial rod was present, the flagellum measured 250 x 175 nm in diameter; elsewhere the flagellum was more or less circular in section, measuring approximately 175 nm in diameter.

Within the cytoplasm of the organism, the root of the flagellum was associated with a kinetoplast (Plate 5 , fig. 1) which was composed of a reticulate network of electron dense DNA- like material. The ribosome-rich cytoplasm also contained a single nucleus as well as diffuse membrane-bound structures (Plate 4) probably homologous with the ‘dense bodies’ described by Paulin & McGhee (1 97 1) from Phytomonas elmassiani (Migone).

The promastigote morphology, its presence within plant sieve tubes, and the ultrastructural features of the flagellate allow it to be classified as a trypanosomatid of the genus Phytomonas.

D I S C U S S I O N

Trypanosomatid protozoa of the genus Phytomonas (Donovan, 1909) are found in the latex expressed from many species of laticiferous plants, especially those belonging to the Euphorbiaceae and Asclepiadaceae (Nieschulz. 193 1). P. dauidi (Lafont) is associated with the Euphorbiaceae and P. elmassiani with the Asclepiadaceae. It has been demonstrated (McGhee & Hanson, 1964) that Oncapeltus fasciatus (Dall.) is the vector of P. efmassiani which may be

A wilt disease of coconuts and an associated protozoan 299

transmitted to a wide range of Asclepiadaceae but not Euphorbiaceae (Hanson, McGhee & Blake, 1966). It is not known whether the apparent failure of P. elmassiani to infect euphorbiaceous hosts is because of a deficiency in the vector or the inability of the phytomonad to survive in the laticifers of the Euphorbiaceae (Hanson, McGhee & Blake, 1966). Within a population of susceptible plant species flagellosis rarely occurs in more than 20% of plants (McGhee & McGhee, 1971). In the laticiferous host the protozoan does not exert any overt phytopathological effect; it is not possible to differentiate between infected and uninfected plants by macroscopic observation of the intact plant.

Important differences are immediately apparent in the hodparasite relationship when non- laticiferous plants contain flagellates. The phytomonads are apparently restricted to the mature phloem sieve tubes and in the few known instances cause diseases which result in the death of host plants. The records of flagellosis of non-laticiferous plants derive from South America. In that continent, Coffee (Coflea spp.) (Stahel 1931a; 1931b; 1933; Van Emden, 1962; Vermuelen, 1963; 1968), coconut (Parthasararthy, van Slobbe & Soudant, 1976), African oil palm (EIaeis guineensis L.) (Dollet, Giannotti & Olagnier, 1977) and maripa palm (Maxmiliana maripa Drude) (Parthasarathy, 1977), are affected. Suriname is the only country so far known where all the species referred to are affected by phytomonads.

Stahel ( 1 93 l a ) was first to describe a flagellate (P. lepfovasorum Stahel) associated with a disease of coffee previously described as phloem necrosis (Stahel, 1920). This disease causes multiplication and concomitant disorganisation of the phloem, the sieve elements of which are characteristically shortened and smaller in diameter than normal. P. Zeptovasorum can be transferred from diseased to healthy plants, thereby inducing disease, by grafting roots from diseased to healthy trees (Stahel, 193 l a ; Van Emden, 1962; Vermuelen, 1963; 1968).

In the three known palm hosts there is no reported disorganisation of the phloem in the tissues examined and none was observed during this study. Demonstration of pathogenicity is not possible using grafting in the palms and as yet the vector of the phytomonad is unknown. The data presented here do not demonstrate pathogenicity of Phytomonas in coconuts affected by a wilt disease in Trinidad. However, the consistent association of the flagellate with diseased tissues even in the earliest stages of this disease and its absence from healthy tissues is powerful circumstantial evidence for associating the phytomonad with this disease. Apart from the results presented, the author has carried out an extensive ultrastructural investigation of another coconut disease (lethal yellowing) and Phytomonas has never been observed in any diseased or healthy tissue examined.

No attempt at classification of the coconut Trypanosomatid beyond the genus Phytomonas has been made because the taxonomy of the genus is only poorly understood. Many of the determinations in the past have been based upon size and gross morphology. It has been demonstrated (Hanson et al. 1966) that size is not a reliable taxonomic character. In controlled transmission experiments where six Asclepiadaceae host species were infected with P. elmassiatii using 0. fasciatus as the vector, the organisms recovered from the individual species were sufficiently different in size to suggest that at least two species were involved instead of one (Hanson et al. 1966). Clearly the size of organisms is influenced by the host as is the morphology. P. elmassiani when introduced to species of the Apocynaceae presents a choano- flagellate morphology rather than the usual promastigote form (McGhee & Hanson, 1971). The determination of a species for the presumed pathogen of coconut wilt disease therefore requires further study.

The ultrastructure of P. elmassiani has been described (Paulin & McGhee, 1971) and the ultrastructure of the phytomonad from coconut wilt disease tissue is little different except that the paraxial rod does not extend to the distal extreme of the flagellum and the flagellar reservoir is markedly more asymmetrical. Amastigote forms were never observed in any fixed or live material examined in the present study but they were common in preparations of P . elmassiani (Paulin & McGhee, 197 1).

300 H . W A T E R S

The symptomatology of the coconut wilt disease in Trinidad is similar to hartrot of coconuts in Suriname and lethal yellowing disease (Personal observations, unpublished). Hartrot has now been associated with a phytomonad (Parthsarathy eC al. 1976) and lethal yellowing is considered to be caused by a mycoplasma-like organism (Beakbane, Slater & Posnette, 1972; Plavsic- Banjac, Hunt & Maramorosch, 1972). It is not known whether hartrot and wilt disease are coidentical though this seems likely. Lethal yellowing has a different aetiology despite having similar symptoms.

To the commercial grower the wilt disease represents a serious threat because all coconut varieties appear to be equally susceptible. Maas (1 9 7 1) has stated that no substantial coconut industry exists in Suriname because of the ravages of hartrot although otherwise conditions are favourable for its cultivation.

This work was undertaken at the request of the Trinidad and Tobago Ministry of Agriculture and was carried out as part of the Lethal Yellowing Research Scheme which is funded by the United Kingdom Ministry of Overseas Development, the Government of Jamaica and the Coconut Industry Board, Jamaica. The financial support of these four organisations is acknowledged. The author is grateful to Dr R. M. Barrow of the Red Ring Research Division of the Trinidad and Tobago Ministry of Agriculture for bringing the disease to his attention and for supplying facilities for its study during a visit to Trinidad and Tobago. Thanks are also due to Dr Peter Hunt of the Botany Department, University of the West Indies for his constructive criticism of the manuscript and Ir W. G. van Slobbe for his help in providing information concerning hartrot in Suriname.

R E F E R E N C E S

BEAKBANE, A. B., SLATER, c. H. w. ~r POSNETTE, A. F. (1972). Mycoplasmas in the phloem of coconut, COCOS nucifera L. with lethal yellowing disease. Journal of Horticultural Science 47,265.

DOLLET, M., GIANNOTTI, M. & OLAGNIER, M. (1977). Observation de protozoaires flagellks dans les tubes cribles de Palmiers a huile malades. Comptes rendus hebdomadaire des sbances de I’Acadbmie des sciences 284,643-645.

DONOVAN, c. (1909). Kala Azar in Madras. Lancet 177, 1495. HANSON, w. L., MCGHEE, R. B. ~r BLAKE, J. D. (1966). Experimental infection of various latex plants of the

family Asclepiadaceae with Phytomonas elmassiani. Journal of Protozoology 13, 324-327. HOARE, c. A. L WALLACE, F. G. (1966). Developmental stages of Trypanosomatid flagellates: a new

terminology. Nature, London 212, 1385-1 386. MAAS, P. w. TH. (1 97 1). A coconut abnormality of unknown etiology in Surinam. F A 0 Plant Protection

Bulletin 19, 8C-85. MCGHEE, R. B. L HANSON, w. L. (1964). Comparison of the life cycle of Leptomonas oncopelti and

Phytomonas elmassiani. Journal of Protozoology 11,555-562. MCGHEE, R. B. B HANSON, w. L. (1971). Changes in structures of Phytomonas elmassiani in experimental

infections of Apocynaceae, a presumably foreign host. Journal of Protozoology 18,8&8 1. MCGHEE, R. B. 8r MCGHEE, A. H. (1971). The relation of migration of Oncopeltus fasciatus to distribution

of Phytomonas elmassiani in the eastern United States. Journal of Protozoology 18, 344-352. NIESCHULZ, 0. (193 1). Die parasitischen Protozoen der PAanzen. In Handbuch der Pathogenen Protozoen

by S. von Prowarek & W. Noller, 1799-1813. Leipzig, J. A. Barth. PARTHASARATHY, M. v., VAN SLOBBE, w. G. & SOUDANT, c. (1976). Trypanosomatid flagellate in the

phloem of diseased coconut palms. Science, New York 192, 1346-1348. PARTHASARATHY, M. v. (1977). “Hartrot” - Status report of the disease and research efforts.

Contribution to the 3rd meeting of the International Council on Lethal Yellowing. West Palm Beach, Florida.

PAULIN, J. J. ~r MCGHEE, R. B. (1971). An ultrastructural study of the trypanosomatid Phytomonas elmassiani from the milkweed Asclepias syrica. Journal of Parasitology 57, 1279-1287.

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PLAVSIC-BANJAC, B., HUNT, P. & MARAMOROSCH, K. (1 972). Mycoplasma-like bodies associated with lethal yellowing disease of coconut palms. Phytopathology 62, 298-299.

SPURR. A. R. (1969). A low-viscosity epoxy resin embedding medium for electronmicroscopy. Journal of Ultrastructure Research 26, 3 1-43.

STAHEL, G. (1920). De Zeefvatenziekte (Phloeemnecrose) van de Liberiakoffie in Surinam. Departement van den Landbouw (Paramaribo, Suriname) Bulletin no. 40,26 pp.

STAHEL, G . (1 93 la). Zur Kenntnis der Siebrohrenkrankheit (Phloemnekrose) des Kaffeebaumes in Surinam. I. Mikroskopische Untersuchungen und Infektionsversuche. Phytopathologische Zeitschrgt

STAHEL, G. (193 Ib). Zur Kenntnis der Siebrohrenkrankheit (Phloemnekrose) des Kaffeebaumes in

STAHEL, G . (1933). Zur Kenntnis der Siebrohrenkrankheit (Phloemnekrose) des Kaffeebaumes in

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E X P L A N A T I O N O F P L A T E S

Plate 1 Fig. 1. Diseased coconut palm showing leaves with rachis break (R), inflorescences without mature nuts after nut fall (I) and the 'skirt' of persistent dead leaves (L).

Fig. 2. Inflorescence, 2-3 months after opening showing desiccated immature nuts (buttons) (D) which can be compared with the more or less normal buttons (B). In some instances the buttons have been shed leaving dry calyxes (C) attached to the rachillae which are withered (W). Scale bar represents 10 cm. Fig. 3. Inflorescence showing aborted opening of the spathe (S). Scale bar represents 10 cm. Fig. 4. Detail from fig. 3 to show the desiccated and necrosed inflorescence with its withered rachillae (W). (S), spathe. Scale bar represents 10 cm.

Plate 2 Fig. 1. Immature inflorescence the spathe ( S ) of which was deliberately split open to show the distal necrosis (N) of the rachillae and the white apparently healthy tissue (H). Scale bar represents 10 cm. Fig. 2. Inflorescence, one month older than that in fig. 1. The spathe (S) was deliberately opened to show the more or less complete necrosis of the rachillae (N). Scale bar represents 10 cm. Fig. 3. Stem apex of a healthy coconut palm split to show the radial longitudinal surface with the soft white tissues of the spear leaf bases (SL), the growing point (G) and the leaf bases of mature leaves (LB). Scale bar marked in cm. Fig. 4. Stem apex of a diseased palm split as fig. 3. When compared with fig. 3 the destruction of the growing point (G) is apparent. The base of the spear has also been destroyed (SL). Rotting of the more mature stem tissues is also shown, the effect of which has been to ret the tissue leaving the fibrous sheathes of the vascular bundles (V). Scale bar marked in cm.

Plate 3 Fig. 1. Transverse section of part of a vascular bundle from an immature inflorescence rachilla of a diseased palm. A single metaxylem vessel is shown (X) as well as some cells with darkly staining contents that are presumed to be tanniferous cells (T). Numerous mature sieve elements are present the majority of which contain flagellates. Cf. sieve elements S', S" and S"' which illustrate the differences in flagellate density which may be observed in any one section. Scale bar represents 10 pm. Fig. 2. Longitudinal section of sieve elements (S) containing flagellates confirming the predominantly longitudinal orientation of the organisms. Scale bar represents 10 pm.

302 H . W A T E R S

Fig. 3. Flagellates as they appear in an expressed sap droplet, by phase contrast microscopy. Scale bar represents 10 ym. Fig. 4. Single phytomonad to show the promastigote morphology and the short robust flagellum (F). The organism illustrated is a twisted form. Scale bar represents 5 pm. Fig. 5 . Electronmicrograph of a negatively stained preparation of a phytomonad. The apical flagellar reservoir (R) and the flagellum (F) are shown. Scale bar represents 5 ym.

Plate 4

Electronmicrograph of a transverse section of a single mature sieve element showing more or less complete occlusion of the cell lumen by flagellates. Transverse sections of flagella are indicated by darts. Sections through the bodies of organisms show nuclei (N), kinetoplasts (K), dense bodies (D) and single rows of subpellicular microtubules (M). Scale bar represents 1 ym.

Plate 5 Fig. 1 . Approximately median longitudinal section of the flagellar reservoir (R) showing the assymetry of the reservoir, the kinetoplast (K) and the flagellum with its paraxial rod (P). Note that the paraxial rod coincides with the shorter side of the flagellar reservoir. Scale bar represents 250 nm. Fig. 2. Longitudinal section of the distal apex of the flagellum note that the paraxial rod (P) does not continue to the tip. (U), pellicular membrane. Scale bar represents 100 nm.

Fig. 3. Transverse section of a flagellum showing the paraxial rod (P) associated with microtubule doublets 5 and 6 of the axoneme. Scale bar represents 100 nm. Fig. 4. Flagellum sectioned transversely and distal to the paraxial rod, demonstrating the classical 9 + 2 arrangement of microtubules. Scale bar represents 100 nm. Fig. 5. Transverse section through the flagellar reservoir (R) showing the pellicular membrane (U), the single row of subpellicular microtubules (M) and the discrete group of four microtubules (bracketed) opposite microtubule doublets 3 and 4 of the flagellar axoneme. The section of the flagellum shows a 9 + 1 organisation of microtubules because the plane of sectioning is at the level of the axosome. Scale bar represents 100 nm. Fig. 6. Transverse section of Phytomonas in the posterior region of the body showing the pellicular membrane (U) and the subpellicular microtubules (M) some of which are sectioned obliquely indicating that the microtubules lie obliquely to the longitudinal axis of the organism. Scale bar represents 250 nm. Fig. 7. Longitudinal section of two phytomonads. The plane of sectioning has passed through the subpellicular microtubules (M) and their obliquity is apparent. Scale bar represents 250 nm.