OTHERS

Fine structure of phloematic trypanosomatid–coconut treeinteraction

Maura da Cunha • Darlı Grativol Keller • Ivan Cunha Bustamante Filho •

Jorge Andre Sacramento de Magalhaes • Valdirene Moreira Gomes •

Wanderley de Souza • Jose Inacio Lacerda Moura • Jose Luiz Bezerra •

Flavio Costa Miguens

Received: 17 February 2009 / Accepted: 16 September 2009 / Published online: 15 December 2009

� The Phytopathological Society of Japan and Springer 2009

Abstract Phytomonas wilt or Hartrot is a fatal disease

of palm (Arecaceae) species including Cocos nucifera

(coconut) and is caused by a phloematic trypanosomatid, a

promastigote parasite that inhabits phloem sieve elements

of disease palms. In the present work, we described the

morphology of the interaction between a phloematic try-

panosomatid (Phytomonas staheli) and C. nucifera. Two

varieties and one ecotype of the adult coconut palm from

northeast and southeast Brazil were analyzed, totaling

34,000 plants. Coconut palm losses due to Hartrot varied

according to the variety or ecotype and geographic area.

Occurrence of Hartrot was insignificant in Rio de Janeiro

state (southeast), but in Bahia state (northeast) losses were

substantial when appropriate cultural practices were not

applied. Symptomatic and healthy palm tissues were ana-

lyzed by light and electron microscopy. Laboratory

diagnoses revealed the twisted promastigote form of phlo-

ematic trypanosomatids in the extracts of shoot apex, leaves,

stems and inflorescence in diseased plants, but not in the

healthy ones. No parasites were found in the roots. Although

the general anatomy of healthy and diseased palms was

similar, callose deposition in the sieve plates was revealed

by histochemistry and immunocytochemistry in the diseased

tissue. Plugging by the P-protein and plastid alterations was

also observed. Our observations strongly suggest that para-

site traffic between sieve elements took place, although their

cell bodies were larger than the sieve pores. Phloematic

trypanosomatid proliferation in the sieve tube elements

might interrupt the transport of phloem or/and consume

plant nutrients. In addition, an association between the

percentage of sieve elements colonized by pathogen in palm

tissues and disease severity was established.

Keywords Phytomonas staheli � Cocos nucifera �Wilt disease � Anatomy and ultrastructure �Histochemistry and immunocytochemistry

Introduction

Phytomonas, a flagellate protozoan belonging to the Try-

panosomatidae family, are heteroxenous promastigotes that

do not synthesize arginase in axenic culture. Their life

cycles take place in insects, as intermediate hosts, and in

plants, as definitive hosts (Camargo 1999; Camargo et al.

1987; Wallace et al. 1992). In 1909, the genus Phytomomas

was created by Donovan (1909) based on Lafont’s

description of protozoa in plants. The genus also has been

further grouped according to the location of the protozoa in

the plant: floricola (flower trypanosomatids), fruticola (fruit

trypanosomatids), laticicola (latex trypanosomatids) or

M. da Cunha � D. G. Keller � I. C. B. Filho �J. A. S. de Magalhaes � F. C. Miguens (&)

Laboratorio de Biologia Celular e Tecidual,

Universidade Estadual do Norte Fluminense Darcy Ribeiro

(UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil

e-mail: flavio.miguens@pq.cnpq.br

V. M. Gomes

Laboratorio de Bioquımica e Fisiologia de Microorganismos,

Universidade Estadual do Norte Fluminense (UENF),

Campos dos Goytacazes, Rio de Janeiro, Brazil

W. de Souza

Laboratorio de Ultraestrutura Celular Hertha Meyer,

Instituto de Biofısica Carlos Chagas Filho, Universidade Federal

do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil

J. I. L. Moura � J. L. Bezerra

Centro de Pesquisas do Cacau, Comissao Executiva do Plano da

Lavoura Cacaueira (CEPEC/CEPLAC), Itabuna, Bahia, Brazil

123

J Gen Plant Pathol (2010) 76:74–83

DOI 10.1007/s10327-009-0212-y

phloemicola (phloem trypanosomatids) (Vickerman and

Dollet 1992). Phloemicola or phloem-restricted trypano-

somatids (Dollet 2001) have always been associated with

diseases in coffee (Stahel 1931; Vermeulen 1963) and palm

(Camargo 1999; Dollet 2001) species. However, no

symptoms have been detected in laticiferous plants (da

Cunha et al. 2000; Dollet 1984), except in cassava, Man-

ihot esculenta, where they exert a clear pathogenic effect

(Kitajima et al. 1986). Fruticola and floricola flagellates are

never associated with diseases.

Recently, the molecular and serological properties of

phloem trypanosomatids associated with wilts of coconut

and oil palm in Latin America have been determined, and

phloemicola has been proposed to be a well-defined taxon

among all plant trypanosomatids (Dollet 2001; Sturm et al.

2007). Previously, phloem trypanosomatids were described

as a new species, Phytomonas staheli (McGhee and

McGhee 1979). It was found in sieve elements of diseased

coconut and oil palms, causing Hartrot or fatal wilt of

palms (Parthasarthy and van Slobbe 1978; Parthasarthy

et al. 1976) and Marchitez sorpressiva (Dollet and Lopez

1978; Dollet et al. 1977). Attalea funifera (Bezerra et al.

1983), Cocos nucifera (Bezerra and Figueiredo 1982),

Elaeis guineensis (Attias et al. 1987; Oliveira and Bezerra

1982; Oliveira et al. 1987) and Roystonea regia (Attias

et al. 1989) have also been reported to be infected by

phloem trypanosomatids in Brazil. Since 1984, insects of

the genus Lincus have been circumstantially described as a

vector of trypanosomatids in palms in Latin America

(Desmier de Chenon 1984; Louise et al. 1986; Perthuis

et al. 1985). Fatal wilt disease has been transmitted

experimentally by Lincus lobuliger (Hemiptera: Pent-

atomidae) from diseased coconut to healthy oil palm

(Resende et al. 1986). The southeast of Bahia state has

been considered as an endemic area (Moura et al. 2002).

The most pronounced effects of the wilt syndrome are

water stress and deficiencies in the plant transport systems

(Beckman 1987; Lucas 1998). Callose synthesis and

deposition, lignification, phenolic compounds, and P-pro-

tein accumulation in the sieve plate and pores have been

reported as general plant responses to stress (Beckman

1987; Eschrich 1975; Lucas 1998). Phloem plastids were

also reported to be involved in the defense mechanisms in

plants (Walsh and Melaragno 1981; Zucker 1983). The

mechanisms by which phloematic trypanosomatids causes

diseases in plants are not clear. However, there may be

phloem blockages or changes in phloem transport from the

accumulation of the parasites (Agrios 2005; Dollet 2001).

The first introduction of coconut trees in the western

hemisphere was in Cabo Verde and the Sao Tome Islands

by Vasco da Gama when he was returning from India and

East Africa in 1499 (Harries 1977, 1978). They were first

grown as a plantation crop in the 1840s in Ceylon (Child

1974). In Brazil, coconut was introduced in Sao Vicente

during Portuguese colonial times in the sixteenth century

and several ecotypes were reintroduced during the twenti-

eth century, mainly in the northeast of the country (Ferreira

et al. 1998). In 1996, there were 260,000 ha of coconut

palms in Brazil, and Phytomonas wilt, potential vectors and

susceptible palms shared the same geographic distribution.

Because coconut palm plantations are an important eco-

nomic activity in Brazil, infection of palms by phloematic

trypanosomatids can have serious economic consequences

because they can destroy the palm trees. In this work, we

analyzed the morphological aspects of the protozoa–host

interaction.

Materials and methods

Plant material and study sites

One healthy and five symptomatic 10-year-old coconut

trees, Cocos nucifera (var. Malayan Green Dwarf), and one

10-year-old symptomatic C. nucifera (var. West African

Tall) were collected from the germplasm bank at an

experimental station (Estacao Experimental Lemos Maia)

of the Cacao Research Center (CEPEC/CEPLAC). The

station is located close to the municipality of Una

(15�1800000S, 33�0500000W) in south-eastern Bahia state,

Brazil. The vegetation around the station is typical of the

Atlantic Rain Forest. The soil is clayey. The climate in this

region is warm, tropical humid with relatively high rainfall

and high atmospheric temperatures. The total annual pre-

cipitation was 2,045.8 mm, and the mean annual air tem-

perature was about 28�C in 2004. Twenty-one diseased and

one healthy C. nucifera ecotype Green Dwarf plants were

collected from commercial plantations in Quissama

(22�0602400S, 41�2802000W) and Conceicao de Macabu

(22�0500700S, 41�5200600W), municipalities located in

northern Rio de Janeiro state, Brazil. The vegetation around

the plantations is dominated by grasses. The soil is sandy in

Quissama and clayey in Conceicao de Macabu. The climate

in this region is warm, tropical subhumid with relatively

low rainfall and high atmospheric temperatures. The total

annual precipitation varied between 550 and 850 mm and

the mean annual air temperature was about 26�C in 2004.

Coconut trees were divided into three types according to

morphological symptoms. Palms considered to be in the

last stage of infection were called type 1, which presented

browning of the oldest leaves, usually starting from the

tips, yellowing of younger leaves, blackening of unopened

and open inflorescences, dropping of all nuts and putre-

faction of the shoot apex with a foul odour. In the inter-

mediate infection stage, type 2 plants presented yellowing

of older leaves, browning of unopened and open

J Gen Plant Pathol (2010) 76:74–83 75

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inflorescences, and nut drop, but no putrefaction of the

shoot apex. Plants of type 3, which had probably been

infected for less than 3 months, presented a gradual yel-

lowing and initial browning of the leaves, and initial

browning of unopened and open inflorescences. The per-

centage of losses from Phytomonas wilt were based on

field and laboratory diagnoses, for each variety or ecotype

and experimental area.

Light and electron microscopy

Extracts from roots, shoot apex, undifferentiated stem,

stem in the first stage of differentiation, leaf primordia,

young leaves, mature leaves, unopened inflorescences,

young and mature opened inflorescences were analyzed

with phase contrast microscopy and scanning electron

microscopy for laboratory diagnoses. Fragments of these

organs were also analyzed with light and electron micros-

copy. To determine the infection level, we counted the

number of infected and uninfected vascular bundles per

unit area (mm2) in 20 areas of stem meristem, stem, basal

petiole, inflorescence and root with the scanning electron

microscope.

For light microscopy, samples were fixed in formalin–

acetic acid–alcohol, dehydrated in ethanol and embedded

in paraffin (58–60�C). Sections (10–12 lm) were stained

with basic fuchsin-astrablau (Roeser 1962). Callose was

stained sky blue with 0.05% aniline blue (Johansen 1940).

Tannins were stained blue-green using 10% aqueous ferric

chloride (Johansen 1940). The samples were observed with

a Zeiss Axioplan photomicroscope (Carl Zeiss AG,

Oberkochen, Germany).

For electron microscopy, samples were fixed in 2.5%

glutaraldehyde, 2.0% formaldehyde in 0.05 M cacodylate

buffer, pH 7.4, for 2 h at room temperature. They were

post-fixed with 1.0% OsO4 in the same buffer. Then,

samples were rinsed in distilled water, stained en bloc in

0.5% aqueous uranyl acetate at room temperature for 2 h,

dehydrated in acetone and embedded in Epoxy resin

(Polybed�812, Polysciences, Inc., Warrington, PA, USA).

Sections were stained with uranyl acetate followed by lead

citrate and observed with a Zeiss EM 900 transmission

electron microscope at 80 kV accelerating voltage (Carl

Zeiss AG). For scanning electron microscopy, after post-

fixation, samples were dehydrated in acetone, dried with a

critical point dryer in CO2, sputter-coated with 20 nm gold

and observed with a Zeiss DSM 962 scanning electron

microscope (Carl Zeiss AG) at 25 kV accelerating voltage.

Immunocytochemistry

Antiserum against callose was prepared by immunization

of white New Zealand rabbits with laminarin (polymer of

b-1,3-glucan) from Laminaria digitata (Sigma, St. Louis,

MO, USA). Pre-immune serum was collected before

immunization. For immunocytochemical analysis, samples

were first fixed for 2 h at room temperature in a solution

containing 0.1% glutaraldehyde, and 2.0% formaldehyde in

0.1 M phosphate buffer (PB), pH 7.3, rinsed three times

with 0.1 M PB, pH 7.3, dehydrated in increasing concen-

trations of methanol (30–90%) and processed for LR Gold

(Polysciences, Inc.) embedding. Ultrathin sections were

treated following a previously described protocol (Pain

et al. 1996) with some modifications. A mixture of 10 mM

phosphate-buffered saline (PBS) and 0.15 M NaCl pH 7.5,

containing 1% bovine serum albumin (BSA) was used for

all rinsing steps and for diluting the reagents. Sections were

immunolabelled by immersing grids in drops (40 ll) of

solutions in the following sequence: (1) PBS ? BSA, for

30 min; (2) pre-immune serum in PBS ? BSA, for 20 min;

(3) anti-laminarin serum (1:100, v/v) in PBS ? BSA, for

2 h at room temperature; (4) five changes of buffer, for

10 min each; (5) goat anti-rabbit IgG antibody conjugated

with 5 nm or 10 nm colloidal gold (Sigma) (1:50, v/v) in

PBS ? BSA, for 2 h at room temperature; (6) five changes

of PBS ? BSA, for 10 min each; (7) five changes of

deionized water, for 10 min each. Sections were then air-

dried on a formvar coated grid, stained with uranyl acetate

and lead citrate and observed with a ZEISS 900 transmis-

sion electron microscope. Control sections were prepared

by replacing the primary anti serum with pre-immune

serum.

Results

Adult coconut tree losses due to Phytomonas wilt varied

depending on the variety or ecotype and geographic area.

In Rio de Janeiro state (*32,000 plants in experimental

area), Phytomonas wilt was rare, and the losses of the

Green Dwarf ecotype were \0.5%. In Bahia state at the

EELM, the Malayan Green Dwarf variety (2,000 plants)

had losses of *5% and a substantial loss ([30%) of the

West African Tall variety (1,000 plants in total). When

insecticides that control the vector(s) of the protozoa were

not sprayed, the entire crop of the Green Dwarf ecotype

(12,000 plants) was lost at a commercial farm near Una,

Bahia, Brazil.

Diseased specimens of C. nucifera had a gradient of

morphological symptoms, whereas healthy plants pre-

sented no morphological alterations. The shoot apex

putrefied in 100% of type 1 coconut palms and was related

to secondary infection. This symptom was not common in

type 2 plants and was absent in type 3. Laboratory diag-

noses using light and electron microscopy confirmed

morphological symptoms observed in the field. Phloematic

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trypanosomatids were observed in extracts of shoot apex,

stems, leaves, and inflorescences from diseased palms at all

stages of infection, but absent in root extracts. Yeasts and

bacteria were observed in type 1 and type 2 palms. Phlo-

ematic trypanosomatids presented twisted promastigote

morphology, between 10 and 18 lm long with 1.5–2.5 lm

diameter, in all organ extracts from diseased plants

(Fig. 1a). Parasites were not observed in organ extracts of

healthy palms (data not shown). In addition, as the disease

progressed, the percentage of infected sieve elements

increased in palm organs (Table 1); in most parts of their

organs, type 1 trees had the highest percentages of infected

sieve elements. However, highly infected sieve elements

were found next to the slightly infected sieve elements

(Figs. 1b, d, 2k).

Anatomically, the vascular bundles of healthy and dis-

eased plants did not differ. In all organs analyzed, bundles

in the differentiated vascular system were randomly dis-

persed in ground parenchyma. Strengthening tissue, form-

ing a bundle sheath with lignified fibers, surrounded the

vascular bundles. The xylem consisted of 1 or 2 elements

of metaxylem and 4–12 of protoxylem (Fig. 1b, c). The

phloem consisted of sieve elements, usually associated

with two companion cells and a variable number of

parenchyma cells (Fig. 1b, d). Tanniferous idioblasts were

observed at parenchyma tissues in healthy and diseased

plants (Fig. 1e).

Xylem and phloem presented a similar ultrastructure

in healthy and infected plants. However, peculiarities

were identified mainly in sieve elements of diseased

plants. Although plastids at the periphery of the sieve

elements were frequent in uninfected palms (Fig. 1f, g),

they were rare in diseased palms (Fig. 1b, d). Phloem

plastids had external and internal membranes, but thy-

lakoid membranes were not identified, and starch grains

were infrequent. Usually, crystalline-like, electron-dense

structures were found in plastid stroma (Fig. 1g). In

longitudinal sections of vascular bundles of severely

diseased type 1 palms, the parasites were also found in

vessel elements in the shoot apex. The abundance of

phloematic trypanosomatids in sieve elements varied

according to disease severity (Fig. 1h–k); the pathogen

occupied a greater volume of the vascular tissue as the

disease became more severe. Colonization level was

always high in stem meristem (palm heart) and undif-

ferentiated organs in the three types of diseased plants

(Fig. 1k).

In sieve elements, phloematic trypanosomatids

appeared as twisted promastigotes with all morphological

features typical of trypanosomatids, including sub-pellic-

ular microtubules, a flagellum attached to the cell body

through specialized junctions, a flagellar pocket, and

sometimes a paraflagellar rod (Fig. 1k). The flagellum had

a basic structure with a 9 ? 2 pattern of axonemal

microtubules. In addition, mitochondrion was usually

normal, but the kinetoplast had different levels of con-

densation (Figs. 1k, 2h).

In the lumen of healthy and diseased plants, filaments

of P-protein were dispersed throughout the sieve ele-

ments, but trapped at sieve pores. Transmission electron

microscopy showed that P-protein filaments were often

distributed close to parasites. Sometimes, parallel fibrils

of P-protein were observed in the sieve pore (Fig. 2a)

and perpendicularly oriented to the cell membrane

(Fig. 2b).

Sieve plates of the sieve elements were in the lateral

and terminal walls. Diameters of the pores in sieve plates

varied between 0.5 and 1.0 lm. Infected sieve elements

frequently presented fibrils in their lumen (Fig. 2a). Cal-

lose was deposited in sieve elements of healthy and dis-

eased plants. In healthy palms, callose deposition was

slightly stained with aniline blue as seen by light

microscopy (Fig. 2c) but was strongly stained in diseased

plants (Fig. 2d). With transmission electron microscopy,

callose appeared as an electron-lucent structure (Fig. 2a).

Fig. 1 Microghaphs of Cocos nucifera. a Extract of stem apical

meristem of var. Malayan Green Dwarf. Phloematic trypanosomatids

(solid arrow) presenting twisted cellular bodies. Starch grains (openarrows) and cellular debris (arrowheads) are in background. SEM.

b Longitudinal section of leaf primordia from var. West African Tall.

Five sieve elements (square) having different levels of colonization.

Vascular parenchyma (VP) and sclerenchyma (star) in formation

presenting typical anatomy. LM. c Transversal section of stem

meristem from var. Malayan Green Dwarf. Infected vascular bundle

(ellipse) of basal petiole from palm type 2 immature leaf. Vascular

bundle showing small parenchyma small cells, xylem (open arrow)

and phloem (white arrow). SEM. d Longitudinal section of primordial

leaf from ecotype Green Dwarf (type 2). The vascular bundle has five

infected sieve tube elements (asterisk). Note callose deposition in

sieve plate (open arrow) and companion cells (cc). LM. e Longitu-

dinal section of stem meristem from ecotype Green Dwarf (type 2).

A tanniferous idioblast with electron-dense tannin accumulation in

vacuole. TEM. f Transversal section of stem meristem from healthy

ecotype Green Dwarf. The vascular bundle has companion cells (cc)

and sieve elements. Phloem plastid (arrow) in sieve element. TEM.

g High magnification of sieve element from (f). Phloem plastid close

to cell wall has external and internal membranes, but no distinct

thylakoid membrane. Note crystal-like structures in stroma. TEM.

h Longitudinal section of basal petiole from ecotype Green Dwarf

(type 1). Phloematic trypanosomatids are highly colonizing the sieve

elements. SEM. i Longitudinal section of basal petiole from ecotype

Green Dwarf (type 2). Contiguous sieve element with intermediate

level of trypanosomatid colonization. SEM. j Longitudinal section of

basal petiole from ecotype Green Dwarf (type 3). Low colonization

by trypanosomatids is evident in sieve elements of type 3-plants.

SEM. k Transverse section of stem meristem from var. Malayan

Green Dwarf (type 3). Highly colonized sieve elements have a lateral

sieve plate with pores (black arrow). Phloematic trypanosomatids

have typical trypanosomatid morphology. Condensed kinetoplast

(black/white arrow) is the most evident structure. TEM. Scale bars in

a, b, d-f, h-j 10 lm; c 250 lm; g, k 5 lm

c

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More callose was deposited in infected sieve elements

(Fig. 2a, d, g–l) than in healthy plants. Callose deposition

appeared to be related to disease severity (Table 1).

Immunolabelling strongly suggested that electron-lucent

areas of the sieve plate resulted from callose deposition

(Fig. 2e, f).

Despite the fact that pathogen cell bodies were larger

than sieve pores, several images strongly suggested that the

78 J Gen Plant Pathol (2010) 76:74–83

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parasite moved between contiguous sieve tube elements

(Fig. 2g–l). Phloematic trypanosomatids were also found in

the sieve pores. Cell bodies spanning both sides of the sieve

pores were also identified (Fig. 2i–l). Apparently, sub-

pellicular microtubules (Fig. 2i) and the cytoplasm

(Fig. 2l) of the parasite cells were rearranged during tran-

sit. In addition, in lateral sieve plates (Fig. 2m) and end

sieve plates (Fig. 2n), parasite cell bodies were preferen-

tially oriented perpendicularly to sieve plates, and their

flagella were difficult to visualize, while those on free

protozoa were easily identified.

Discussion

Losses have been reported from phloematic trypanoso-

matids or P. staheli, mainly in coconut and oil palm in

Latin America (McCoy and Martinez-Lopes 1982; Ohler

1999; Waters 1978). Southeast Bahia state (northeast

Brazil) has been cited as endemic for Phytomonas wilt in

palms; there is an overlap of etiological agent, vectors and

host distribution, and a constant low rate of this disease is

expected (Moura et al. 2002). Yellow Dwarf varieties may

be more resistant than Green Dwarf varieties (Bezerra and

Figueiredo 1982). In the present study, devastation on a

commercial farm in Una was related to ecotype suscepti-

bility or deficient integrated disease management, and an

epidemic affecting the West African Tall variety was due

to crop management. In Rio de Janeiro state, fatal wilt

levels were insignificant, and the vector was not identified.

However, vector identification is fundamental for disease

control. Lincus lobuliger (Hemiptera: Pentatomidae) has

been implicated in protozoan transmission (Resende et al.

1986), and vectors of phloematic trypanosomatids have

Table 1 Disease symptoms and sieve elements colonization level by phloematic trypanosomatids and their association with levels of disease

(types 1–3) in all tested varieties and ecotypes of coconut trees (Cocos nucifera)

Symptoms Percentage of plants with symptoms

Type 1 Type 2 Type 3 Healthy

Externala

Nuts fall ????? ??? ?? –

Unopened inflorescence browning ????? ?? ? –

Young inflorescence browning ????? ??? ?? –

Mature inflorescence browning ????? ??? ?? –

Wilt of first leaf ????? ????? ????? –

Oldest leaf browning ????? ????? ????? ???

Older leaves browning ????? ??? ?? –

Younger leaves yellowing ???? ?? ? –

Shoot apex putrefaction ???? ?? – –

Internal symptoms

Tanniferous idioblastsMesophyll ? ? ? ?

P-protein/sieve pore ????? ???? ???? ?

Plastids/sieve elements 0 0–1 0–1 [1

Callose deposition/sieve plate ????? ??? ??? ?

Percentage with colonized vascular bundlesb

Stem meristem 98.4 ± 2.7 85.0 ± 6.0 72.4 ± 11.8 –

Stem 76.1 ± 4.9 67.0 ± 9.6 66.6 ± 8.2 –

Basal petioles: adult leaves 92.0 ± 6.4 90.8 ± 6.5 63.3 ± 9.6 –

Basal petioles: young leaves 51.3 ± 4.2 55.4 ± 9.0 30.3 ± 9.1 –

Unopened inflorescence 98.3 ± 2.6 89.8 ± 4.3 80.6 ± 9.4 –

Young inflorescence 97.0 ± 3.8 88.9 ± 5.3 76.0 ± 9.2 –

Mature inflorescence 52.5 ± 5.4 53.2 ± 7.9 43.1 ± 7.1 –

Primary and secondary roots – – – –

a ????? = 100%; 100% [ ???? C 75%; 75% [ ??? C 50%; 50% [ ?? C 25%; 25% [ ? [ 0%; – = 0%b Percentage (mean ± SD) of vascular bundles infected by phloematic trypanosomatids. n = 50 vascular bundles per structure

J Gen Plant Pathol (2010) 76:74–83 79

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been identified for the genera Lincus and Oclerus

(Camargo 1999; Dollet 2001).

Pathogenic species of the Trypanosomatidae have been

found in sieve elements of diseased coconut (Parthasarthy

and van Slobbe 1978; Parthasarthy et al. 1976), oil palms

(Dollet and Lopez 1978; Dollet et al. 1977) and other

plants from the Arecaceae family (Camargo 1999). Results

presented here establish a clear relationship between dis-

ease development and host morphological alterations and

also show the anatomy and ultrastructure of healthy and

diseased palms.

Phloem colonization is an important step in the pro-

gression of Phytomonas wilt in palms (Camargo 1999;

Dollet 1984; Wallace et al. 1992). The presence of the

parasite on both sides of sieve pores suggest that the larger

protozoa may pass through the smaller sieve pore by

deformation of flexible cell bodies; however, this question

about transport across the pore remains open. McGhee and

Cosgrove (1980) based on microscopic observations of the

swimming of several Trypanosomatidae genera show that

these flagella are capable of a considerable variety of

movements. Bending activity produces a sinusoidal wave,

which is not quite planar, so that the cell rotates at a low

frequency; the wave normally propagates from tip to base,

but when the flagellum touches an obstruction anywhere

along its length, the direction of propagation may be

reversed by changing the direction of movement. In the

present study, we found parasite flagella plugging sieve

pores of diseased tissue contrary to present knowledge of

the Trypanosomatidae species movement and flagellum

physiology.

The anatomy of healthy and diseased palms was very

similar, suggesting that trypanosomatids never caused a

destructive effect against host plant cells. Callose depo-

sition was the main difference. Callose is a linear b-1,3-

glucan with some 1,6- branches, and as a glucosidic

non-cellulosic polysaccharide, it differs from cellulose in

the type of linkage between the glucose residues

(Hopkins 1995). Usually, callose is detected by its

specific staining with aniline blue (Hong et al. 2001) and

by immunocytochemistry (Northcote et al. 1989). Callose

has been localized in the cell plate, plasmodesmata, root

hair, cotton-seed hair, and spiral thickenings even in

tracheids of healthy plants (Stone and Clarke 1992).

However, mature sieve-plate pores in monocotyledons

have little or no callose (Behnke 1971; Walsh and Evert

1975). The synthesis of callose can also be induced by

wounding, infection, and physiological stress (Kauss

1996; Stone and Clarke 1992). Callose is also deposited

in an apposition layer against the cell wall in diseased

tomato (Beckman et al. 1982). We found different

degrees of callose production during phloematic trypan-

osomatids infection. It is possible that a callosic apposi-

tion may seal the sieve plates, and that occlusions by

trypanosomatids and callose accumulation could func-

tionally disturb phloem transport in phloematic trypano-

somatid-infected coconut.

The sieve tubes of the phloem contain proteinaceous

structures, collectively called P-proteins (phloem pro-

teins), which accumulate in differentiating sieve ele-

ments and persist in translocating sieve elements. The

functional role of P-proteins remain unresolved. How-

ever, it seems likely that polymerized and unpolymer-

ized P-proteins exist in dynamic equilibrium within sieve

elements, where the concentration of each is responsive

to physiological changes within the vascular system

(Golecki et al. 1999). The P-protein is deposited initially

as ultrastructurally distinct polymorphous or crystalline

bodies during sieve element differentiation (reviewed in

Cronshaw 1975; Cronshaw and Sabnis 1990; Sabnis and

Sabnis 1995). Disruption of sieve elements during

wounding results in the accumulation of P-protein fila-

ments at the sieve plate, ostensibly blocking transloca-

tion via P-protein plugs (Hopkins 1995). In C. nucifera,

we identified two morphological kinds of P-like protein

in the sieve tube elements, and their accumulation at the

sieve plates was probably due to the infection by phlo-

ematic trypanosomatids and/or related vector feeding

that wounded sieve elements. P-protein plugs probably

affect long-distance movements of macromolecules in

phloem and impact the physiology of diseased coconut

trees.

In addition, phloem plastids have been reported to

explode in immediate response to a pressure release in the

sieve elements, which in turn frees substances that react

with the P-protein body material (Knoblauch and van Bel

1998). Starch and protein granules from broken plastids

could contribute to the plugging of the sieve tube elements

(Walsh and Melaragno 1981). Although the function of

plastid inclusions in monocotyledons is not clear, there is

evidence that they are involved in plugging mechanisms

(Eleftheriou 1990). Phloem plastids were highly visible in

uninfected vascular bundles of C. nucifera, but not in

infected ones. Our observations suggest that plastids frag-

ments in trypanosomatids-infected sieve elements of the

vascular bundle, also contribute P-protein that plugs the

phloem vessels in the presence of the protozoa. Injury to

the sieve elements by vectors could result in the same local

response.

In conclusion, our observations show that in symp-

tomatic coconut palms, at least 30% of sieve elements

of the phloem contain phloematic trypanosomatids or

80 J Gen Plant Pathol (2010) 76:74–83

123

P. staheli. In the terminal stage of disease, about 98% of

vascular bundles contain protozoa. In contrast, the path-

ogen was present in the vessel elements of the xylem only

in terminal stage of fatal wilt. These data strongly suggest

that the long-distance translocation of molecules in the

phloem is affected by the presence of phloematic try-

panosomatids, partially due to the plant response to the

infection.

J Gen Plant Pathol (2010) 76:74–83 81

123

Ackowledgements This research was supported by CNPq (Conse-

lho Nacional de Desenvolvimento Cientıfico e Tecnologico), FINEP

(Financiadora de Estudos e Projetos), FENORTE (Fundacao Estadual

do Norte Fluminense) and PRONEX (Programa de Nucleos de

Excelencia).

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