Fungal Diversity

Why are Halophytophthora species well adapted to mangrovehabitats?

KM. Leafiol*, KB.G. Jones2 and L.L.P. Vrijmoed2

IAquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, 5021Hoilo, Philippines; * e-mail: edleano@aqd.seafdec.org.ph2Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue,Kowloon, Hong Kong S.A.R., P.R. China

Leafio, E.M., lones E.B.G. and Vrijmoed, L.L.P. (2000). Why are Halophytophthora specieswell adapted to mangrove habitats? In: Aquatic Mycology across the Millennium (eds K.D.Hyde, W.H. Ho and S.B. Pointing). Fungal Diversity 5: 131-151.

Halophytophthora species are commonly isolated from fallen mangrove leaves from early tolate stages of decay. In this study we show that these organisms are well adapted to mangrovehabitats as they have a wide tolerance to varying levels of pH, salinity and temperature. Theyalso produce, abundant zoospores, and are chemotactically attracted to decaying mangroveleaves, and can readily attach to suitable substrata. In general, the four tested isolates (H.vesicula, H. avicennae, H. kandeliae and H. bahamensis) grew at pH 6-9, with maximumgrowth recorded at neutral pH. Vegetative growth and sporulation were observed over a widerange of salinities (from freshwater to marine) and temperatures, although optimumrequirements varied from species to species. Zoospores of Halophytophthora spp. werechemotactically attracted to mangrove leaf-extracts and some other compounds that arecommon to the surrounding environment. The zoospores attached and germinated on bothartificial (glass coverslips and polycarbonate membranes) and natural (mangrove leaves)substrata. Scanning electron micrographs show that newly attached zoospores, cysts, andgerminating cystospores of H. vesicula produced fibrillar adhesive mucilage on both types ofsubstrata. The growing germ tube/hypha also produced mucilage for attachment as was evidentby debris sticking to their tips. More adhesive mucilage was produced by encysted andgerminating cystospores on natural as compared to artificial substrata. Cystospores andgermlings of H. vesicula and H. avicennae were also found to attach firmly to a perspex disceven after being subjected to a high shear stress of 3.19 Newton per square meter (Nm'2).Enzyme treatment and staining of attached cystospores indicate that the adhesive produced iscomposed of acidic polysaccharide with a-I,4 linkages, and with either sulphate or phosphatefunctional groups. Once the cystospores were attached to the substratum, they could not bereadily dislodged, and successful germination and colonization followed.

Key words: Halophytophthora spp., physiological growth requirements, spore attachment,zoospore chemotaxis.

Introduction

Mangroves are highly productive ecosystems which are not only self-

131

sustainingl but export sufficient nutrients to support large trophic webs (Ongl1995). The bulk of the energy input is derived from leaf litter, which isprimarily decomposed by fungi and straminipilous (zoosporic) organisms. Mostmangrove habitats, however, are subject to varying environmental conditionsresulting in constant fluctuations of some water parameters (e.g. salinity andtemperature). Thus, they can only support populations of organisms with a widetolerance to varying salinity and temperature levels (Leafio et al., 1998a).

Among the initial coIonizers of fallen mangrove leaves, the straminipilousHalophytophthora species are reported to be among the most frequent species(Newell et al., 1987; Nakagiri et al., 1989; Newell and Fell, 1992; Tan and Pek,1997). Halophytophthora species known to colonize fallen senescent mangroveleaves are listed in Table 1. Their abundance is attributed to the efficient

production of zoospores (Nakagiri et al., 1989) and the ability to competeagainst higher fungi in colonizing fallen mangrove leaves (Newell and Fell,1997). Their success may largely depend on the homing ability of theswimming zoospores. The homing mechanism of zoospores of straminipilousorganisms are well documented for parasitic species (e.g. Phytophthora andPythium species) (AlIen and Harvey, 1974; Halsall, 1976; Morris et al., 1995;Tyler et al., 1996), but no reports are available for saprobic species. This studywas therefore initiated to evaluate: the physiological requirements for growthand sporulation of selected Halophytophthora spp.; and, the homing responseof zoospores that could lead to succesful colonization of the substrata.

Materials and methodsTest Isolates

Four Halophytophthora species were used in this study namely: H. vesicula(LEM-031) (Leafio et al., 1998a); H. avicennae1(ATCC 64709); H. kandeliae(ATCC 66503); and, H. bahamensis (ATCC 64761).

Physiological studiesPhysiological studies with respect to pH and to the combined effects of

salinity and temperature are described by Leafio et al. (1998a).

Zoospore chemotaxisZoospore chemotaxis was evaluated as described in Leafio et al. (1998b).

The test compounds used included L-amino acids, saccharides, plant extractsand ethanol.

I Supplied as H. avicennae from ATCC, but suggested as another strain of H. vesicula (S.Newell, pers. comm.)

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Fungal Diversity

Table 1. Records of Halophytophthora species colonizing fallen/decaying mangrove leaves(modified from Leafio et al., 1998a).

1,6, 10

1, 13

13

1, 13

1,7,12,13

3,6

1,5, 10

2,9,11,13

References·

1,2,3,5,6,7,9,10,11,12, 13

7, 10

8, 13

A. germinans

B. gymnorrhyza, R. stylosa, R. mangle

R. stylosa, B. gymnorrhyza, Avicenniamarina (Forsk.) Viehr.

Mangrove leaf sourceRhizophora mangle L., R. stylosa Griff., R.apiculata BL, R. mucronata Lam, Burgieragymnorrhyza Lamrk., Sonneratia albaSmith, Avicennia germinans (L.) Steam,Conocarpus erectus L., Lagunculariaracemosa (L.) Gaertn., Kandelia candel (L.)Druce

R. mangle, L. racemosa

R. stylosa, B. gymnorrhyza

R. mangle, R. stylosa, B. gymnorrhyza

K. candel, Avicennia alba Blume, A.germinans, R. stylosa, B. gymnorrhyzaA. germinans

FungiHalophytophthora vesicula

(Anastasiou and Church!.)H.H.Ho and Jong

H. spinosa var. spinosa (Felland Master) H.H.Ho andJong

H. spinosa var. lobata (Fell and B. gymnorrhyza, R. stylosa, R. apiculataMaster) H.H.Ho and Jong

H. kandeliae H.H. Ho, H.S.Chang and S.Y. Hsieh

H. masteri Nakagiri and S.Y.Newell

H. exoprolifera H.H. Ho,Nakagiri and S.Y.NewellH. epistomium (Fell and Master) R. mangle, R. stylosa, B. gymnorrhyza

H.H. Ho and JongH. bahamensis (Fell and

Master) H.H.Ho and JongH. batamanensis (Garretson­

Comell and Simpson) H.H.Ho and Jong

H. polymorphica (Garretson­Comell and Simpson) H.H.Ho and Jong

H. mycoparasitica (Fell andMaster) H.H. Ho and Jong

"I = Fell and Master, 1975; 2 = Newell et al., 1987; 3 = Ho et al., 1991; 4 = Ho et al., 1992; 5= Newell, 1992; 6 = Newell and Fell, 1992; 7 = Nakagiri, 1993; 8 = Wilkens and Field, 1993; 9= Nakagiri et al., 1994; 10 = Newell and Fell, 1994; 11 = Newell and Fell, 1995; 12 = Tan andPek, 1997; 13 = Nakagiri, 1998.

Attachment and germinationScanning electron microscopyA zoospore suspension (ca. 104 cells mL-1) was inoculated onto

polycarbonate membranes and glass coverslips and the zoospores were allowedto attach and germinate for 30-60 mins at room temperature. Discs (1 cm diam.)from green and senescent leaves of Kandelia candel were submerged in azoospore suspension for 30-60 mins to allow attachment and germination.

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Sample preparation for scanning electron microscopy followed the procedure ofDurso et al. (1993) with modifications. Fixation was in 2.5% gluteraldehyde in0.2 M cacodylate buffer (pH 7.5) for 2 h at 4 C. Fixed samples were washed incacodylate buffer (3 times) then post-fixed with 1% osmium tetroxide overnightat 4 C. The specimens were washed in distilled water for 15 mins (3 times) toremove excess osmium tetroxide and dehydrated in a graded ethanol seriesfrom 5 to 20% (at 5% intervals; 20 mins for each step), then from 30 to 90% (at10% intervals), to 95%, and followed by three changes of 100% ethanol (15mins for each dehydration step). The ethanol was replaced with acetone at 2: I,1:1, and 1:2 (ethanol:acetone) series followed by 3 changes in pure acetone (15mins for each step). Dehydrated samples were critical point dried using liquidC02 as the drying agent, mounted on aluminum stubs using carbon tape, andcoated with gold/palladium using JEOL JFC-II00E ion sputter. Specimenswere examined at 20 kV using a Leica Stereoscan scanning electronmIcroscope.

Strength of spore attachmentStrength of spore attachment was evaluated using the Fowler cell adhesion

measurement module following the procedure of Au (1993). The effects ofdifferent carbohydrate-digesting enzymes (a-amylase, a-mannosidase) andproteolytic enzymes (a-chymotrypsin, papain) on detachment rates of attachedcystospores were also evaluated.

Cytochemical stainingAttached and germinating cystospores (on glass coverslips) were fixed

following the procedure of Daniel et al. (1987). Toluidine blue and Alcian blue8GX were used for cytochemical staining (Chayen and Bitensky, 1991).

Statistical analysesData (where appropriate) were analyzed using one-way or two-way

analysis of variance (ANOV A). Significant differences among treatment meanswere compared using the Student Newman-Keul's test (Zar, 1996).

Results and discussionPhysiological studies

The fundamental niche of any organism is determined by the range ofenvironmental conditions under which it can survive and reproduce (Cooke andWhipps, 1993). In the aquatic environment, there is a range of chemical,physical, and biological factors, which control the distribution, activities, andabundance of fungal inhabitants. Three important physico-chemical factors

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Fungal Diversity

Table 2. Vegetative growth of Halophytophthora spp. in PYGS 1 broth at different pH levels(salinity = 20%0; temperature = 25 C).

(6.9)(8.0)(8.6)

pH Biomass (mg dry weight)'!'H. vesicula H. avicennae H. kandeliae H. bahamensis

4 (4.02)L ng ng ng ng5 (5.10) ng ng ng ng6 (6.02) 6.6 ± 1.2 b (6.08) 3 9.2 ± 2.1 b (5.96) 5.5 ± 2.6 • (6.02) ng7(7.12) 51.1±4.5n (5.10) 15.0±2.0·b (3.9) 15.0±3.6n (5.0) 7.1±3.6n8(8.07) 47.0±2.8' (6.92) 19.2±1.3' (5.6) 12.6±5.0' (7.9) 5.1±1.0'9 (8.88) ng ng 4.0 ± 0.5' (8.7) 5.0 ± 1.6 •

J PYGS = peptone-yeast extract-glucose-seawater (refer to Leafio et al., 1998a);2 pH after autoclaving; 3 pH after incubation; ng = no growth.l' after 3 days incubation (H. kandeliae) and 5 days incubation (H. avicennae and H.bahamensis).

Note: Column means (± SEM) with the same letter superscripts are not significantly different(P>0.05). Bold values indicate maximum growth.

include pH, salinity, and temperature (Dix and Webster, 1995). Results obtainedfrom this study reveal that the saprobic Halophytophthora spp. are well adaptedto the mangrove environment with their wide tolerance to varying pH (Table 2),salinity (Table 3) and temperature (Table 4) levels for both vegetative growthand sporulation.

Hydrogen ion concentration (pH) has been shown to affect zoosporeactivity of straminipilous organisms, spores of aquatic fungi, their mycelialgrowth, enzyme activities, and reproduction (Dix and Webster, 1995). The pHof a body of water is not constant, and can vary over wide limits due tophotosynthetic activity of algae and other macrophytes in the water (Dix andWebster, 1995), especially in lotic, lentic and estuarine environments.Laboratory results from this study showed that all the test isolates grew best atneutral to basic pH (pH 7-8). In Mai Po mangrove, where H. vesicula wasisolated, water pH ranges from 6.9-7.6 (Sadaba, 1996), which conforms withthe pH requirements of the test isolates. Other studies (lones and Harrison,1976; Bahnweg and Bland, 1980; Kitancharoen et al., 1996), on both marineand freshwater straminipilous organisms, also indicate that pH requirements foroptimum growth correlates with the pH ranges in their natural habitats.

Most physiological studies on marine fungi have centered on the individualeffects of salinity and temperature. In the natural environment, however,fluctuations of these parameters occur concurrently; thus combined effects ofsalinity and temperature on growth and sporulation of Halophytophthora spp.were evaluated in this study. All the test isolates showed wide tolerance tosalinity both for vegetative growth and sporulation (Table 3). Similar resultshave been reported for other Halophytophthora spp. isolated from fallen leaves

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Table 3. Vegetative growth and sporulation of Halophytophthora spp. at different salinity levels(~- range; _ optimum).

H. masteri3

I this study; L Nakagiri, 1993; j Nakagiri and at., 1994.

Test isolates

Vegetative growthH. vesicula I

H avicennae I

H. kandeliae I

H. bahamensis I

H. spinosa var lobata 2

H. exoprolifera 2

H. tartarea 3

H. masteri3

SporulationH. vesicula I

H. avicennae I

H. kandeliae I

H. bahamensis I

H. spinosa var lobata 2

H. exoprolifera 2

H. tartarea 3

o 10Salinity (%0)

20 30 40

-? (>20)

--

50 60

in mangrove habitats (Table 3) (Nakagiri, 1993; Nakagiri et al., 1994). Studieson marine parasitic straminipilous organisms show a contrary situation with thesaprobic Halophytophthora spp., where growth and sporulation were limited toseawater salinities which also limits the type of hosts that they can infect (Bianet al., 1979; Hatai et al., 1992; Kitancharoen et al., 1994; Kitancharoen andHatai, 1995).

136

; I

Fungal Diversity

Table 4. Vegetative growth and sporulation of Halophytophthora spp at different temperaturelevels (--range, - optimum).

Test isolates

Vegetative growthH. vesicula I

H avicennae I

H. kandeliae I

H. bahamensis I

H. spinosa var lobata 2

10 15Temperature (C)

20 25

-

30

-

-

35

H. exoprolifera 2

H. tartarea 3

H. masteri3

SporulationH. vesicula I

H. avicennae I

H. kandeliae I

H. bahamensis I

------------- ..•------------

-

H. spinosa var lobata 2

H. exoprolifera 2

H. tartarea 3

H. masteri3

I this study; L N~kagiri (1993); j Nakagiri et al. (1994).

--

-

The wide tolerance of these organisms to salinity variations might explaintheir distribution and high frequency of occurrence in aquatic habitats,particularly mangroves. Mangroves vary considerably in the salinity of theseawater, ranging from brackish (0-20%0, e.g. Mai Po mangrove, Hong Kong),to fully saline (35%0), to hypersaline water (44%0, e.g. Sinai mangrove, Egypt)(Leafio et al., 1998a). In some mangrove habitats in particular, the constantfluctuations in both salinity and temperature levels can only support

137

populations of organisms with wide salinity and temperature tolerance levels(Leafio et al., 1998a). Wide tolerance to temperature (ranging from 10 to 35 C)was also observed in all isolates tested in this study (Table 4). These results arein agreement with other studies on freshwater, estuarine, and marinestraminipilous organisms, both parasitic and saprobic species (Hatai et al.,1990; Nakagiri, 1993; Nakagiri et al., 1994; Willoughby, 1994; Yuasa andHatai, 1994; Kitancharoenetal., 1996; Sinmuketal., 1996).

Zoospore chemotaxisAside from the physiological requirements for growth and reproduction of

straminipilous organisms, many other factors also affect their abundance anddistribution in the aquatic environment. A unique feature of these organisms istheir ability to locate a suitable substratum or host for colonization/infectionthrough zoospore taxis. Among the various forms of taxes, chemotaxis wasreported to be the most important (Carlile, 1993) especially in an aquaticenvironment. In this study, zoospores of Halophytophthora spp. were found tobe chemotactically attracted to mangrove leaf extracts and to a wide range ofcompounds that could be present in their natural environment (Tables 5, 6).Strongest chemotactic responses were observed for pectin and ethanol onzoospores of H. vesicula, and pectin and different amino acids (except arginine)for zoospores of H. avicennae (Table 5).

Scanning electron micrographs of the zoospores of H. vesicula showed thatthey accumulated on the stomatal openings of senescent Kandelia candelleaf(Fig. 6). Stomatal openings might be a source of diffusates leaching fromsenescent cells that can chemotactically attract free-swimming zoospores in theaquatic environment. Newell and Fell (1992), however, reported thatchemotaxis of zoospores toward the leaves was only of localized significanceand that water flow was the dominant vector for Halophytophthora zoosporesin colonizing mangrove leaves. But once very near the leaves, zoospores canswim towards leaf leachates and find stomatal openings for subsequentcolonization, as shown in this study.

Chemical attractants and host-derived diffusates, that have been reported toinduce chemotactic responses of zoospores of straminipilous organisms, can beconsidered important factors in signaling between organisms, between oneanother, and their environment (Jansson and Thiman, 1992). Many studiesreport the importance of chemotaxis among parasitic oomycetes as a pre­requisite for infection of susceptible host plants (Khew and Zentmyer, 1973;Halsall, 1976; Donaldson and Deacon, 1993; Morris et al., 1995; Tyler et al.,1996) or animals (Thomas and Peterson, 1990). However, very few reports areavailable on zoospore chemotaxis of saprobic oomycetes (Muehlstein et al.,

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Fungal Diversity

Table 5. Chemotactic response of Halophytophthora spp. zoospores to plant extracts, aminoacids and ethanol.

Test attractants Chemotactic ratio (chemotactic responseT)H. vesiculaL H. avicennae H. kandeliae H. bahamensis

Leaf extractsK. candel

6.7 ± 2.5(H)1.1 ± 0.0(+)9.9 ± 0.4(H)t 1.9 ± 0.4(+)A. ilicifolius

3.6 ± 0.8(H)2.3±0.1(+)6.2 ± 0.4(++) 3.0±0.1(+)V8-juice

1.4±0.3(+)0.9 ± 0.1(-)6.9 ± 1.9(H) 1.7 ± 0.2(+)Amino acids Glutamic acid

5.9 ±0.9(H)8.6 ± 1.0(H)t4.4 ± 0.6(H) 6.2 ± 1.4(++)Asparagine

8.3 ± 0.6(H)10.2 ± 2.0(+H)t 2.7 ± 0.4(+)2.9±0.2(+)Arginine

1.0 ± 0.0(-)1.0 ± 0.1(-)1.0 ± 0.3(-)3.3 ±0.9(++)Histidine

0.9± 0.2(-)11.9± 1.4(+H)t 1.7 ± 0.3(+)1.8 ± 0.5(+)Methionine

0.8 ± 0.0(-)15.6±3.0(+H)t 0.9±0.1(-)3.8 ± 1.0(++)Ethanol

27.7 ± 5.9 (H+)t 2.0 ± 0.2(+)8.5 ± 1.0(++)t 7.7 ± 2.3(++)tChemotactic ratio = mean score on test attractant:mean score on control (Halsall, 1976); 1(_)= negative; (+) = weak; (++) = moderate; (+++) = strong;2 data from Leafio et al., 1998b;t significantly different (P<0.05) from the rest of the test attractants;Concentrations: leaf extracts = lOOmg mL'l; amino acids = lOOmM; ethanol = 20%.

Table 6. Chemotactic response of Halophytophthora spp. zoospores to saccharides.

Test attractants Chemotactic ratio (chemotactic responseT)H. vesiculaL H. avicennae H. kandeliae H. bahamensis

, ) 1.1 ± 0.0(+)0.6 ± 0.1

, )5.3 ± 1.0(H) 0.6±0.1

, )1.9 ± 0.3(+)0.6 ± 0.1

, +)T

1.2 ± 0.2(+)0.8 ± 0.1

, )1.1 ± 0.2(+)0.5 ± 0.1

1988), mainly because of their lesser immediate economic importancecompared to the parasitic species (Leafio et al., 1998a). However, with theincreasing interest in the ecological importance of saprobic straminipilousorganisms, there is a need to understand the different mechanisms involved in

139

-------------------------------------- ..-------.------

Figs. 1-2. Scanning electron micrographs of germinating cystospores of Halophytophthoravesicula attached on glass coverslip (Fig. I) and polycarbonate membrane (Fig. 2). Note: debris(d) attached on the cystospore wall (Fig. I); fibrillar mucilage (Mu) on the cyst wall and nearthe area of germ tube (gt) emergence (Fig. 2). Bars = 5 ~m.

140

Figs. 3-5. Scanning electron micrographs of germinating cystospores of Halophytophthoravesicula attached on green leaf of Kandelia candel. Note: higher amounts of fibrillar mucilage(Mu) produced near the area of germ tube emergence (Figs. 3, 5); large amount of debris (d)sticking on the hyphal tips (Figs. 4, 5). Bars: 3 = 5 Jlm; 4-5 = 2 Jlm.

141

Figs. 6-8. Scanning electron micrographs of newly attached and germinating cytospores ofHalophytophthora vesicula on senescent Kandelia candel leaf. Note: accumulation ofcystospores on the stomatal openings (S) (Fig. 6); extensive amounts of fibrillar mucilage (Mu)produced by the newly attached (Fig. 7) and germinating (Fig. 8) cytospores (gt = germ tube).Bars: 6 = 50 ~m; 7-8 = 5 ~m.

142

Fungal Diversity

the colonization of different substrata. Results from this study indicate that theability of Halophytophthora zoospores to locate a potential food source, givesthem an advantage in colonizing suitable substrata over other fungi ororganisms that produce non-motile spores. Thus, they are found to be one of themost common initial coIonizers of fallen leaves in many mangrove habitats(Nakagiri et al., 1989; Newell and Fell, 1995; Tan and Pek, 1997; Nakagiri,1998). Moreover, mangrove habitats undoubtedly support a high potentialinoculum, with a steady supply of leaves from mangrove plants. These leaves,being colonized by Halophytophthora spp. producing a significant number ofzoospores, will ensure a high zoospore inoculum to account for the rapidcolonization reported.

Attachment and germinationAfter finding a suitable substratum, zoospores encyst and attach, prior to

germination. Initiation of zoospore encystment can be regarded as a stimulus­mediated secretory process (lones et al., 1991). Zoospore encystment occurs byrecognition of a host surface component (Deacon, 1997) which can involveboth subtle and complex interactions (Kerwin et al., 1991). Encystment mayalso be triggered by various compounds/nutrients that are present in the aquaticenvironment and which are usually released by hosts' tissues through excretionor upon degradation.

Attachment of fungal spores to host cell surfaces is one of the early steps inthe penetration and colonization of the host cell (Hamer et al., 1988; Tunlid etal., 1992) and precipitates a variety of biological events that could influencefungal survival (Hazen and Glee, 1995). Attachment involves an active processof secretion of adhesive materials, which in some cases are highly specific torecognition of, and binding to, a particular host species (Nicholson and Epstein,1991). Among straminipilous organisms, most studies on spore attachment dealwith parasitic species. Detailed studies on zoospore attachment of plant and fishpathogenic oomycetes provided evidence on the production of adhesivemucilage during encystment, attachment, and germination (Hardham andGubler, 1990; Bartnicki-Garcia and Wang, 1993; Durso et al., 1993). This studyis the first detailed report on the attachment of zoospores of saprobicoomycetes.

Zoospores of Halophytophthora species, in this study, were also found toattach on a variety of surfaces, both artificial (glass and polycarbonatemembranes) and natural (mangrove leaves) (Figs. 1-7). This feature indicatestheir non-specificity in attachment which may be explained by their saprobicnature. Most parasitic species selectively attach to specific or limited hosts(Longman and Callow, 1987; Mitchell and Deacon, 1987; Manocha and Chen,

143

1990; Kerwin et al., 1991) which usually involve recognition of the specificsurface topography of the host or binding between complementary moleculeson both host and parasite cell surface (Douglas, 1987; Manocha and Chen,1990). Other parasitic species, however, also showed non-specificity withregards to zoospore attachment (Sing and Bartnicki-Garcia, 1975; Gubler et al.,1989).

In this study, attached cystospores of H. vesicula produced fibrillaradhesive mucilage on both artificial and natural substrata, but little mucilagewas evident when cystospore attached to a glass surface (Fig. 1), while onpolycarbonate membranes moderate amounts of adhesive mucilage wereproduced (Fig. 2). Almost the same amount of mucilage was produced by theattached cystospores on green leaves of K. candel (Figs. 3, 5). Large amounts ofdebris were also found attached to the hyphal tips (Fig. 4) indicating the activeproduction of mucilage by the growing hyphae.

The most profuse production of mucilage was observed on attached andgerminating cystopsores on senescent K. candelleaves (Figs. 7, 8). The higheramounts of adhesive produced on this substratum indicate the adaptation ofHalophytophthora zoospores to securely attach on fallen mangrove leaves. Inall cases, the mucilage produced was fibrillar in nature and extended a veryshort distance from the cyst wall, with greater amounts near the area of germtube emergence. This implies that zoospores of these organisms have pre­formed adhesives which are immediately released upon encystment andattachment.

Firm attachment of the settled cystospores is also an advantage incolonizing new substrata. Although several studies indicated that cystospores ofstraminipilous organisms can attach firmly on surfaces through the productionof adhesives (Sing and Bartnicki-Garcia, 1975; Gubler and Hardham, 1988;Gubler et al., 1989; Durso et al., 1993), this study is the first report to evaluatethe strength of attachment of settled and germinating cystospores. Encysted andgerminating cystospores of Halophytophthora spp. attach firmly to perspexeven when subj ected to shear stresses of up to 3.19 Newton per square meter(Nm·2) (Fig. 9).

The strength of attachment of attached cystopsores was significantlylowered when cystospores were treated with the carbohydrate-digesting enzyme

ex-amylase (Fig. 10). The other carbohydrate-digesting enzyme, ex-mannosidase,and the two proteolytic enzymes (ex-chymotrypsin and papain) did not affect thedetachment rates of attached cystospores of the test isolates. Characterization ofthe mucilage by light microscope cytochemical staining (Table 7) revealed thatthe adhesive is comprised of a polysaccharide with ex-1,4 linkages, and witheither sulphate or phosphate functional groups. Adhesives produced by most

144

Fungal Diversity

Table 7. Cytochemical staining reactions for the adhesive secreted by encysted and germinatingcystospores of Halophytophthora spp.

Stains H. vesicula H. avicennaeToluidine blue

pH 5.6 + +pH 4.2 + +pH 1.0 + si +

A1cian Blue 8GX

pH 5.8 + +pH 0.5 + +0.25 M MgCI2 si + +0.5 M MgCI2 si + si +1.0 M MgCh si + si +2.0 M MgCI2 + ++ = positive reaction; si + = slight positive reaction.

H. kandeliae

+si ++

++++++

straminipilous organisms are mainly made up of glycoprotein (Gubler andHardham, 1988; Gubler et al., 1989; Donaldson and Deacon, 1992; Deacon andDonaldson, 1993), or protein alone (Tunlid et al., 1991). Polysaccharides, onthe other hand, were also reported as the main components of adhesivesproduced by aquatic hyphomycetes (Read et al., 1992; Au et al., 1996, 1997),which are responsible for secure, rapid, spore attachment, often in turbulentwater conditions, making them more competitive in the colonization ofsubstrata (Au et al., 1996). Thus, the nature of the adhesive produced bysaprobic Halophytophthora spp. also allows them to effectively attach andcolonize suitable substrata in mangrove habitats.

ConclusionsHalophytophthora spp. are well adapted to environmental conditions in

mangrove habitats. Thus, they are reported to play a significant role in themicrobial degradation of fallen mangrove leaves, which is an essential part inthe overall trophic activities in the mangrove ecosystem.

The zoospores play a very important role in the colonization of substrata inthe aquatic environment. The presence of suitable substrata, together with thediffusates being leached-out, can significantly contribute to the chemotacticresponse of zoospores towards the source, thus aiding to the location offavourable environments for growth and reproduction. After locating suitablesubstrata, they can firmly attach on the surface through the production ofadhesive mucilage which can withstand the water conditions (e.g. wave action)of the aquatic environment. Once attached, successful germination andcolonization will take place.

145

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Shear Force (Nm-2)

Shear Force (Nm-2)

Fig. 9. Attachment (%) of Halophytophthora vesicula (A) and Halophytophthora avicennae (B)zoospores on Perpex disc at different shear stress using the Fowler cell adhesion measurementmodule. Attachment time = 30 mins.

a•aaa

Time of exposure (h)

~

~ be e

\f t t

tt b,b-+- -:B

o 2 4 6 8 10 12

100

90i 80~ 70

~ 60= 50'"

E 40..c:

E 30.:;: 20

10

o

Time of exposure (h)

A

o 2 4 6 8 ]0 ]2

]00

90

80

70

60

50

40

30

20

10

o

In general, the success of Halophytophthora spp. in colonizing fallenmangrove leaves (Fig. 11) can be attributed to their wide growth tolerance tovarying levels of pH, salinity and temperature (Nakagiri, 1993; Nakagiri et al.,1994; Leano et al., 1998a), abundant zoospore production (Nakagiri et al.,1989; Leano et al., 1998a), their chemotactic ability to locate suitable substrata

Fig. 10. Attachment (%) of Halophytophthora vesicula (A) and Halophytophthora avicennae(B) zoospores after treatment with a-amylase at different exposure time using the Fowler celladhesion measurement module. Means (for each shear stress) with the same letter are notsignificantly different. • = control (3.19 Nm-2); • = 3.19 Nm-2; .A.= 1.59 Nm'2.

146

Wide toleranceto

fluctuatingenvironmental

paramenters:

-pH

- Salinity

- Temperature

Chemotaxisaids substrate

detection

Fungal Diversity

Availability ofdifferent

attractants from

decaying leaves

Abundant supplyof mangrove

leaves all yearround

Wave action oftidal water

Fig. 11. Success of Halophytophtora species in mangrove habitats.

(Leafio et al., 1998b), and their ability to securely attach on mangrove leavesfor rapid germination and colonization.

AcknowledgementsThe authors wish to thank the City University of Hong Kong (CityU) for the award of

research studentship to E.M. Leafio, to Larni Angelie Espada (ATandT, U.S.A.) for statisticaladvice, to Doris Au (CityU) and TK. Cheung (University of Hong Kong) for their assistance inscanning electron microscopy, and to the Royal Society (U.K.) and CityU for the award of theKan Tong Po Visiting Professorship to E.B.G. Jones.

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(Received 30 October 1999, accepted 27 June 2000)

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