Gall structure and specificity in Bostrychia culture ... · 411 Rue des Moguerou, 29680 Roscoff...

Algae 2013, 28(1): 83-92http://dx.doi.org/10.4490/algae.2013.28.1.083

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Copyright © The Korean Society of Phycology 83 http://e-algae.kr pISSN: 1226-2617 eISSN: 2093-0860

Gall structure and specificity in Bostrychia culture isolates (Rhodomelaceae, Rhodophyta)

John A. West1,*, Curt M. Pueschel2, Tatyana A. Klochkova3, Gwang Hoon Kim3, Susan de Goër4 and Giuseppe C. Zuccarello5 1School of Botany, University of Melbourne, Parkville VIC 3010, Australia2Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY 13902-6000, USA 3Department of Biology, Kongju National University, Kongju 314-701, Korea411 Rue des Moguerou, 29680 Roscoff, France5School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, 6140, New Zealand

The descriptions of galls, or tumors, in red algae have been sparse. Kützing (1865) observed possible galls of Bostrychia

but only presented a drawing. Intensive culture observations of hundreds of specimens of the genus Bostrychia over

many years have revealed that galls appeared in only a small subset of our unialgal cultures of B. kelanensis, Bostrychia

moritziana / radicans, B. radicosa, B. simpliciuscula, and B. tenella and continued to be produced intermittently or con-

tinuously over many years in some cultures but were never seen in field specimens. Galls appeared as unorganized tissue

found primarily on males and bisexuals, but occasionally on females and tetrasporophytes. The gall cells usually were

less pigmented than neighboring tissue, but contained cells with fluorescent plastids and nuclei. The galls were not trans-

ferable to other potential hosts. Galls could be produced from gall-free tissue of cultures that originally had galls even

after transfer to new culture dishes. Electon microscopy of galls on one isolate (3895) showed that virus-like particles are

observed in some gall cells. It is possible that a virus is the causative agent of these galls.

Key Words: Bostrychia; galls; Rhodomelaceae; Rhodophyta; unialgal culture; virus-like particles

IntroductIon

Galls, or tumors, are unorganized tissue on otherwise

normal plants. Galls are usually associated with abnor-

mal cell division patterns and / or cell enlargement (Apt

1988, Scheffer 1997). Galls have not been reported ex-

tensively in red algae possibly because they are rare and

not considered important, but the deformation of tissue

could have fitness consequences for the host. While the

cause of all red algal galls is not known several causative

agents have been shown or suggested. Cyanobacteria

were reported to cause galls in Mazzaella laminarioides

(Bory de Saint-Vincent) Fredericq (as Iridaea laminarioi-

des Bory de Saint-Vincent) (Correa et al. 1993). Bacteria

were seen in the galls of the red algae Chondracanthus

teedei (Mertens ex Roth) Kützing (as Gigartina teedii

[Roth] Lamouroux) (Tsekos 1982), Grateloupia ameri-

cana Kawaguchi et Wang (as Prionitis lanceolata [Harvey]

Harvey), and Polyneuropsis stolonifera M. J. Wynne, D. L.

McBride & J. A. West (McBride et al. 1974). Subsequently

bacteria were proven to be the cause of galls on G. ameri-

cana (Apt and Gibor 1989, Ashen and Goff 1996, 1998,

Received November 19, 2012, Accepted February 1, 2013

*Corresponding Author

E-mail: [email protected]: +61-3-8344-8080

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://cre-ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Algae 2013, 28(1): 83-92

http://dx.doi.org/10.4490/algae.2013.28.1.083 84

rochrome DAPI (4′,6-diamidino-2-phenylindole, Sigma-

Aldrich, St. Louis, MO, USA) using a heat fixation method.

Algal thalli were dipped in 5 µg mL-1 DAPI solution in

seawater for 5 min, and then the cover slips were slightly

heated over a boiler for a few seconds. After staining, algal

thalli were mounted on slides in the DAPI solution and

were examined under a UV filter.

Micrographs were taken with Olympus DP50 digital

camera affixed to an Olympus BX50 microscope (Olym-

pus, Tokyo, Japan) using Viewfinder Lite and Studio Lite

computer programs (Better Light Inc., Placerville, CA,

USA) or with a Zeiss GFL bright field microscope (Carl

Zeiss AG, Oberkochen, Germany) using a Canon G3 cam-

era (Canon Inc., Tokyo, Japan) and Photoshop CS4 com-

puter program ( http://www.adobe.com/au/).

Transmission electron microscopy (TEM)

Galls were fixed in phosphate buffered saline (PBS) buf-

fer containing 2% glutaraldehyde at 4°C for 2 h. The glu-

taraldehyde was then rinsed out with PBS buffer and the

cells were postfixed with 2% osmium tetroxide at 4°C for

1.5 h. Thereafter, the cells were rinsed out with PBS buffer

and were dehydrated in a graded acetone series, embed-

ded in Spurr’s epoxy resin (Spurr 1969) and polymerized

overnight in a 70°C oven (Polysciences Inc., Warrington,

PA, USA). Sections stained with uranyl acetate and Reyn-

olds’s lead citrate (Reynolds 1963) were viewed and pho-

tographed on a Phillips Bio Twin Transmission Electron

Microscope (Phillips Electron Optics, Eindhoven, Nether-

lands). We were able to carry out TEM studies on only one

B. simpliciuscula isolate (3895).

rESuLtS

Refer to Table 1 for the time periods (years) in which

galls were seen in the various isolates. These galls were

predominantly found in isolates from Australia where

most isolates were obtained. Only a small percentage of

our isolates had galls and they were only observed in B.

simpliciuscula Harvey ex J. Agardh, B. moritziana / radi-

cans complex, B. radicosa (Itono) J. A. West, G. C. Zucca-

rello & M. H. Hommersand, B. kelanensis Grunow, and B.

tenella (Lamouroux) J. Agardh.

Bostrychia simpliciuscula

B. simpliciuscula is a polyphyletic species consisting

of three lineages (Zuccarello et al. 1999, Zuccarello and

2000, Ashen et al. 1999). A fungus possibly causes galls of

Catenella nipae Zanardini (Zuccarello 2008). The galls of

Prionitis and Catenella were originally described as red

algal parasites (Lobocolax deformans Howe and Catenel-

locolax leeuwenii Weber-van Bosse, respectively). Experi-

mental infection and formation of galls was not achieved

with fungi in Catenella nipae (Zuccarello 2008).

Galls have also been associated with viruses. Virus-

like particles (VLPs) were found in the galls of Gracilaria

epihippisora Hoyle (Apt and Gibor 1991). In this species

gall tissue was capable of autonomous growth but only in

an undifferentiated state. Pueschel (1995) also observed

VLPs in the filamentous red alga Acrochaetium savianum

(Meneghini) Nägeli (as Audouinella saviana).

We have studied Bostrychia over many years and it has

become useful in research on speciation, ecophysiology,

evolution and cell biology / video microscopy of repro-

duction (reviewed in Zuccarello and West 2011). A very

large collection of over 1,000 isolates of all the recognized

species of Bostrychia has been established (http://www.

botany.unimelb.edu.au/West). A small subset of these

isolates has produced undifferentiated tissue that persist-

ed in culture. Galls of Bostrychia were observed in 1865,

when Kützing illustrated many species of Bostrychia and

depicted a gall on B. cornifera Montagne (Kützing 1865,

Pl. 24) (Fig. 1A in this paper), later synonymized with B.

moritziana (Sonders ex Kützing) J. Agardh (King and Put-

tock 1989). Our observations on Bostrychia galls are pre-

sented below.

MAtErIALS And MEtHodS

Algal material and laboratory culture

Unialgal culture methods were described in West and

Zuccarello (1999) and West (2005). Culture isolates were

all maintained at 18-23°C, 12 : 12 LD daily photoperiods,

3-5 µmol photons m-2 s-1 cool white fluorescent or LED

lighting, MPM/2 culture medium (30‰ salinity). For fast-

er growth and reproduction cultures were placed in 10-15

µmol photons m-2 s-1 on rotary shaker (70 rpm).

Most isolates used for this research program are now

available at the Korean Marine Plants Collection, Chun-

gnam National University, 220 Gung-dong, Yuseong-gu,

Daejeon, Korea.

Bright field and fluorescence microscopy

Algal nuclei were stained with the DNA-specific fluo-

West et al. Galls of Bostrychia

85 http://e-algae.kr

Fig. 1. Morphology of galls in Bostrychia. (A) Drawing of B. cornifera (currently B. moritziana) with several ‘galls’ developing on a monosiphonous branch (arrows). Reproduced from Kützing (1865). (B-E) B. simpliciuscula microscopic images of galls developing on polysiphonous axis and laterals in different isolates (3895, 3931, and 3910). (D & E) Through-focus images of the same gall (3910). (F) B. simpliciuscula, fluorescent DAPI staining of the gall cells nuclei (blue color, DAPI-stained nuclei; red color, plastid autofluorescence; arrow points to the dead cells inside the gall). (G & H) B. simpliciuscula, transmission electron microscopy images of virus-like particles in gall tissues of isolate 3895. Scale bars represent: B-F, 50 µm; G & H, 200 nm.

A C

D E

B

F

HG

Algae 2013, 28(1): 83-92


Table 1. Bostrychia moritziana / radicans, B. kelanensis, B. radicosa, B. simpliciuscula, and B. tenella isolates observed. Rubisco spacer lineages of B. moritziana / radicans and B. simpliciuscula from Zuccarello and West (2003, 2006), Zuccarello et al. (1999) or during this study

Species Culture No.

Collection site / Date Phases in culture

Galls Lineage / Haplotype

B. moritziana / radicans

2746 Tooradin, Western Port Bay, VIC, AUS; Oct 25, 1986 T M F No 1/AU

2748 Tooradin, Western Port Bay, VIC, AUS; Oct 25, 1986 T M F M 1999 1/AU2749 Tooradin, Western Port Bay, VIC, AUS; Oct 25, 1986 T M F M 1989 1/AU3204 Beachwood, Natal, ZFA; Oct 4, 1991 T M F F 1997-2012 1/AU3571 Stuarts Point, NSW, AUS; Oct 2, 1995 T M F B M 2000-2005 1/AU3668 Clyde River, Nelligen, NSW, AUS; Dec 13, 1996 T M F B M B 2000-2008 1/AU3669 Clyde River, Nelligen, NSW, AUS; Dec 13, 1996 T M F M 1998-2004 1/AU3673 Clyde River, Nelligen, NSW, AUS; Dec 13, 1996 T M F B MBF 1998-2010 1/AU3675 Rhyll, Phillip Island, VIC, AUS; Dec 27, 1996 T M F M 2000-2003 1/AU3676 Rhyll, Phillip Island, VIC, AUS; Dec 27, 1996 T M F M 1998-2012 1/AU3679 Mooney Mooney, Hawkesbury River, NSW, AUS; Feb 11, 1997 T M F No 1/AU3680 Thorsby Creek, Newcastle, NSW, AUS; Feb 11, 1997 T M F M 2002, 2006 1/AU3813 Snowy River estuary, Malo, VIC, AUS; Dec 29, 1997 T M F M 2002, 2006 No data3879 Bridge crossing, Calliope R, Gladstone, QLD, AUS; Jan 25, 1998 T M F M 2002-2005 No data3905 Foster Beach, VIC, AUS; Dec 7, 1998 T M F M 2002 No data3913 Port Albert Wharf, N. Island, NZL; Dec 16, 1998 T M F M 2002-2008 No data3914 Helensville R. Reserve, N. Island, NZL; Dec 16, 1998 T M F M 2001-20023941 On Avicennia, Cowleds Landing, SA, AUS; Feb 10, 1999 M B M 2000,

B 2001, 20021/AU

3968 On Sonneratia, Nusa Lembongan, IDN; Apr 25, 1999 T M F M 2000-2006, 2012

1/AU

3026 Bahia Magdalena, Baja California Sur, MEX; Jan 7, 1990 T M F F 1995, 2000, 2002

5/A1

3492 Jetty at Port Aransas, TX, USA, coll. B. Baca; Sep 15, 1974 T M F T 2002 5/B3973 Nusa Lembongan, IDN; Apr 25, 1999 M B B 2002 6/NA3998 Tomkinson River, Arnhem Land, NT, AUS; Aug 23, 1999 T M F B T 2002 No data4124 Cedar Key, FL, USA; Sep 19, 2000 T M F F 2001-2009,

M T 20026/D

B. kelanensis 3810 Cossack, WA, AUS, Dec 9, 1997 T M F M 2012 No dataB. radicosa 4086 Tempusak (near Kota Belud), Sabah, MYS; Aug 13, 2000 T M F B B 2002 B. radicosa

haplotype4614 Okat Harbor, Kosrae, FSM; Feb 8, 2006 veg No B. radicosa

haplotype4621 Pacific Tree Lodge, Kosrae, FSM; Feb 8, 2006 veg No B. radicosa

haplotype4627 Fefan I. Chuuk, FSM; Feb 10, 2006 veg No B. radicosa

haplotype4663 Peniyak Village Weno I. Chuuk, FSM; Feb 11, 2006 veg No B. radicosa

haplotypeB. simplicius-

cula2747 Tooradin,Western Port Bay, VIC, AUS; Oct 25, 1986 T No H1

2853 Port Welshpool, VIC, AUS; Dec 22, 1987 T No H12914 Torrens Island, SA, AUS; Sep 22, 1988 T No H12915 Torrens Island, SA, AUS; Sep 22, 1988 T No H12933 Millers Landing, Wilsons Promontory, VIC, AUS; Dec 17, 1988 T No H13108 Williamstown, VIC, AUS; Dec 30, 1990 T M F No H13109 Sydney, New South Wales NSW, AUS; Jan 4, 1991 M 1999, 2002 H23110 Sydney, NSW, AUS; Jan 4, 1991 T M F B M 2003 H23111 Sydney, NSW, AUS; Jan 4, 1991 F 1995, 1998 H23114 Sydney, NSW, AUS; Jan 4, 1991 T M F B M 1998-1999,

2001-2003H2

3298 Moruya R., NSW, AUS; Jan 11, 1993 T No H13304 Merimbula, NSW, AUS; Jan 12, 1993 T M F B No No data3305 Bermagui R., NSW, AUS; Jan 12, 1993 T No H13306 Batemans Bay, NSW, AUS; Jan 12, 1993 T No H1



Species Culture No.

Collection site / Date Phases in culture

Galls Lineage / Haplotype

3308 Broughton Ck., NSW, AUS; Jan 11, 1993 T M F T 1995, M 1998-1999, 2002, F 1996, 1998, 2002

H2

3315 Tuross Lake, NSW, AUS; Jan 12, 1993 T No H1

3317 Narooma, NSW, AUS; Jan 12, 1993 T No H1

3319 Narooma, NSW, AUS; Jan 12, 1993 T M F B B 2002, M 2003 H1

3321 Wapengo Ck., NSW, AUS; Jan 11, 1993 T No H1

3322 Merimbula, NSW, AUS; Jan 12, 1993 F No H1

3330 Garden Island, SA, AUS; Jan 22, 1993 F No H1

3546 Brunswick Heads, NSW, AUS; Oct 23, 1995 T No H3

3562 Forster, NSW, AUS; Oct 23, 1995 T M F Only on M, at 17°C, not 23°C

H2

3576 Woolooware Bay, NSW, AUS; Dec 14, 1995 T No H1

3581 Williamstown, VIC, AUS; Feb 29, 1996 T M F B F 2002 H1

3612 Jawbone Reserve, Williamstown, VIC, AUS; Jun 21, 1996 T M F B B 2004 H1

3657 Sussex Inlet, NSW, AUS; Dec 14, 1996 T M F M 2004-2006, 2009

H1

3658 Sussex Inlet, NSW, AUS; Dec 14, 1996 T M F M 2001-2004, B 2003, 2005, 2009

H1

3663 Bermagui, NSW, AUS; Dec 13, 1996 T M F B B M 1999-2012 H1

3671 Merimbula, NSW, AUS; Dec 14, 1996 T M F B B M 2000-2001, 2003-2006

H1

3672 Sussex Inlet, NSW, AUS; Dec 14, 1996 T No H1

3677 Rhyll, Phillip I., VIC, AUS; Dec 27, 1996 T M F B B 2002 H1

3814 Corringle Beach, VIC, AUS; Jan 1, 1998 T M F No H1

3895 American R., Kangaroo I., SA, AUS; Sep 19, 1998 T M F M 2001-2012 H1

3897 Wynyard Wharf, TAS, AUS; Sep 13, 1998 T M F M 2003-2010 H1

3900 Batman Bridge, S. of George Town, TAS, AUS; Nov 17, 1998 T M F No H1

3910 Mimosa Rock National Park, NSW, AUS; Dec 16, 1998 M 2000-2010 No data

3912 Wapengo Lake, NSW, AUS; Dec 16, 1998 T No No data

3915 Kiama, Minnamura R., NSW, AUS; Dec 19, 1998 T No No data

3916 Kiama, Minnamura R., NSW, AUS; Dec 19, 1998 T No No data

3917 Sussex Inlet, NSW, AUS; Dec 19, 1998 T F No No data

3918 Sussex Inlet, NSW, AUS; Dec 19, 1998 T No No data

3929 Tumby Bay, SA, AUS; Feb 10, 1999 T B B 2001-2006 No data

3930 Tumby Bay, SA, AUS; Feb 10, 1999 M B M B 2000-2012 No data

3931 Tumby Bay, SA, AUS; Feb 10, 1999 T M F M 2003, 2006 No data

3932 Arno Bay, SA, AUS; Feb 10, 1999 T M F B B F 2002-2012 No data

3938 Blanche Harbour, SA, AUS; Feb 10, 1999 T M F No No data

3940 Blanche Harbour, SA, AUS; Feb 10, 1999 T M F B M 2001, B 2002-2012

No data

4042 Price, SA, AUS; Jan 13, 2000 F No No data

4045 Port Clinton, SA, AUS; Jan 10, 2000 T B B 2004-2012 No data

4046 Pt. Arthur, SA, AUS; Jan 10, 2000 T No No data

4203 Woolooware Bay, NSW, AUS; Oct 15, 2001 M No No data

4600 Lehn Mesi R., Pohnpei, FSM; Feb 4, 2006 NR No H3

4629 Fefan I. Chuuk, FSM; Feb 10, 2006 NR No H3

4636 Old Taliafak Bridge, GUM; Feb 12, 2006 NR No H3

B. tenella 2751 Initao, Misamis Oriental, PHI; Oct 25, 1986 T M F F 2003-2012 No data

VIC, Victoria; AUS, Australia; T, tetrasporophyte; M, male; F, female; ZFA, South Africa; NSW, New South Wales; B, bisexual; NZL, New Zealand; SA, South Australia; IDN, Indonesia; MEX, Mexico; NA, not available; WA, Western Australia; MYS, Malaysia; FSM, Micronesia; TAS, Tasmania; NR, no re-production; GUM, Guam; PHI, Philippines.

Table 1. continued

Algae 2013, 28(1): 83-92


West 2003, 2006, 2011) and galls were found on isolates

from three lineages (1, 5, and 6). Again the preponder-

ance of galls was found on isolates from Australia.

In 26 years of culture isolate 2748 produced galls only

once (May 1999) on the male spermatangial stichidia and

these were very similar in overall structure to those seen

in B. simpliciuscula. On the female of isolate 2749 galls

were noted only in 1989. By contrast, isolate 2747 from

the same locality never had galls. Isolate 3026 female

formed galls at irregular intervals in 1995, 2000, and 2002

whereas the tetrasporophyte and male showed no gall de-

velopment during 22 years of culture. Isolate 3492 (Texas,

USA) was isolated in 1974 and did not show galls until

July, 2002 and these developed primarily on cladohaptera

of tetrasporophytes.

Isolate 3204 female (South Africa) was collected in 1991

and had numerous galls on procarpic lateral branches

from 1997-2012 (Fig. 2A & B). Males and tetrasporophytes

had no galls. Isolate 4124 female (Florida, USA) was col-

lected in 2000 and developed galls with mostly colorless

living cells on the tips of vegetative laterals. Regeneration

of viable branches frequently occurred from gall tissue

(Fig. 2C & D). Galls were evident for almost 8 years (2001-

2009). Males and tetrasporophytes produced galls briefly

in 2002.

Galls were observed on 16 males, 4 females, 4 bisexu-

als, and 3 tetrasporophytes of all isolates in Table 1.

Bostrychia radicosa

Isolate 4086 (Sabah, Malaysia) was obtained in Au-

gust 2000 and showed gall formation on the nodes and

gametangial sectors of the bisexual gametophytes first in

May, 2002. Isolate 4178 (New Caledonia) males developed

galls in August, 2012 (Fig. 3A & B). Initially the gall cells

enlarged and retained fully pigmented chloroplasts (Fig.

3A) but as the galls matured pale hypertrophied cells with

enlarged vacuoles were evident (Fig. 3B). Other isolates of

B. radicosa from Thailand (4207) and Micronesia (4614,

4621, 4627, and 3662) did not develop galls.

Bostrychia kelanensis

In 31 isolates of B. kelanensis from Australia, Guam, In-

dia, Indonesia, Malaysia, and Micronesia only one male

(3810) from Western Australia developed galls. Grouped

cells divided and enlarged, projecting from tier cells (Fig.

3C) in spermatangial sectors of lateral branches. Eventu-

ally irregularly shaped masses developed numerous short

branch apices (Fig. 3D).

West 2006). Galls were found on 15 of 87 (17%) isolates of

lineages H1 and H2, exclusively with Australian isolates.

Galls were usually associated with viable spermatangial

stichidia, either on unisexual or bisexual plants, although

some were evident on the non-reproductive sectors. Tet-

rasporophytes and female gametophytes occasionally

had galls. This has been true over 26 years of culture for

some isolates. Galls never developed on young gameto-

phytes, only on reproductively mature gametophytes. On

isolate 3562 no galls formed on the tetrasporophytes, fe-

males or males grown at 23°C. However, galls developed

in the vicinity of spermatangial stichidia of males but not

on females or tetrasporophytes when grown at 17°C. Iso-

late 3562, having numerous galls, was placed in a culture

(on a shaker and in brighter light) for 3-6 weeks with the

male of another isolate (3108) having a long history with-

out galls. No galls developed on isolate 3108. This is only a

partial test of Koch’s postulates (http://en.wikipedia.org/

wiki/Koch’s postulates).

Galls varied in appearance and size. The initial stages

appeared as enlarged proliferating cells. Cell divisions

appeared random and very different from the polysipho-

nous tier cell division pattern of the normal host (Fig. 1B-

E). The cells in galls were often smaller than tier cells, of

various shapes and sizes and had enlarged vacuoles. Gall

cells were less pigmented than normal host tissue cells

(Fig. 1B). They appeared to have viable nuclei that often

were larger than tier-cell nuclei (Fig. 1F). In smaller galls

the cells all appeared viable but as the galls expanded

and the cell number increased some dead cells were seen

(arrow in Fig. 1F). In some larger galls new shoots arose

within the cell mass (no photo).

In many isolates galls were present intermittently, how-

ever, galls were continuously present on the male and bi-

sexual phases of 3663 (Fig. 1F) from 1999-2012 and on the

male phase of 3895 from 2001-2012 (Fig. 1B). Isolate 3932

was unusual because galls appeared on the bisexual or fe-

male thalli but not on males from 2002-2012.

Electron microscopic (TEM) observations. While TEM

fixation of cellular structure was difficult our micrographs

did show VLPs in gall cells of isolate 3895 (Fig. 1G & H).

These VLPs are of two distinct morphologies (staining dif-

ferently) approximately 70-75 nm in size and hexagonal

in shape.

Bostrychia moritziana / radicans

From 390 isolates of the B. moritziana / radicans spe-

cies-complex 24 (6%) produced galls. This species com-

plex consists of seven different lineages (Zuccarello and



Fig. 2. Galls on Bostrychia moritziana / radicans isolates 3204 and 4124. (A) Habit image of 3204 female with numerous white galls on branches bearing procarps. (B) Galls at tips of procarp bearing branches. Trichogynes (tr) visible projecting from gall at lower right. (C) 4124 female with gall at tip of vegetative lateral. Two regenerating shoots developed from gall cells. (D) 4124 female gall with mass of colorless cells, a few appear dead with collapsed protoplasts but most appear to be viable living cells. Healthy, branched shoot regenerated from gall tissue. Scale bars represent: A, 1 mm; B & D, 80 µm; C, 60 µm.

A

C

B

D

Algae 2013, 28(1): 83-92


Fig. 3. Galls on Bostrychia radicosa, isolate 4178 (A & B); B. kelanensis, isolate 3810 (C & D); B. tenella isolate 2751 (E-G). (A) B. radicosa, elongate spermatangia bearing lateral branches with two galls visible on left. Released spermatia near branch tips also visible, small developing gall with normally pigmented cells visible on far left. (B) B. radicosa, high magnification, enlarged cells lightly pigmented with enlarged vacuoles. (C) B. kelanensis, developing gall with normally pigmented cells projecting from branch. (D) B. kelanensis, older galls with extensive branch formation. (E) B. tenella, normal procarpic branches with abundant trichogynes (tr). (F) B. tenella, numerous galls on procarpic branches, trichogynes (tr). (G) B. tenella, high magnification of gall cells, tier and cortical cells with fully pigmented chloroplasts, trichogynes (tr). Scale bars represent: A & B, 50 µm; C & D, 77 µm; E & F, 100 µm; G, 25 µm.

A C D

B

E

G

F



also indicate that much of the transmission is vertical

within a single host. We did not test gall transmission to

progeny of gall bearing sexual parents.

Although not tested extensively three antibiotics (Peni-

cillin G, Ciprofloxacin, and Rifampin) were routinely add-

ed to various cultures without any effect on gall presence.

During antibiotic treatments various bacteria were also

clearly present on the hosts as well.

The greater susceptibility of males and bisexuals to gall

formation in B. simpliciuscula may be due to the numer-

ous spermatangia formed on male branches and the fre-

quent release of spermatia opening more surface areas to

attachment by viruses and bacteria.

We know very little about causative agents of the galls

seen in Bostrychia, the effects of environmental stressors

on gall formation, the transmission of the causative agent

or the effects of galls on the fitness of their hosts. The

study of potential pathogens of marine red algae should

be pursued more critically.

AcKnoWLEdGEMEntS

The Australian Research Council grants (A19917056

[1999-2001]; SG0935526 [1994]; S198122824 [1998];

S005005 [2000]), Australian Biological Resources Study

program (2002-2005), and Hermon Slade Foundation

(2005-2007) partially supported this work. Many thanks

to Ulf Karsten and Doug McBride for help with collecting

samples in various localities around Australia and to Alan

Critchley (South Africa), Rosario Braga (Brazil), and E. K.

Ganesan (Venezuela).

rEFErEncES

Apt, K. & Gibor, A. 1989. Development and induction of

bacteria-associated galls on Prionitis lanceolata (Rho-

dophyta). Dis. Aquat. Org. 6:151-156.

Apt, K. E. 1988. Galls and tumor-like growths on marine mac-

roalgae. Dis. Aquat. Org. 4:211-217.

Apt, K. E. & Gibor, A. 1991. The ultrastructure of galls on the

red alga Gracilaria epihippisora. J. Phycol. 27:409-413.

Ashen, J. B., Cohen, J. D. & Goff, L. J. 1999. GC-SIM-MS de-

tection and quantification of free indole-3-acetic acid

in bacterial galls on the marine alga Prionitis lanceolata

(Rhodophyta). J. Phycol. 35:493-500.

Ashen, J. B. & Goff, L. J. 1996. Molecular identification of a

bacterium associated with gall formation in the marine

red alga Prionitis lanceolata. J. Phycol. 32:286-297.

Bostrychia tenella

In 86 isolates of B. tenella from many different geo-

graphic regions only isolate 2751 (Philippines) developed

galls and that was only after 17 years in culture. Galls oc-

curred continuously from 2003-2012 on the procarp bear-

ing female, but never on the males or tetrasporophytes.

In normal females the lateral branches were uniform in

shape and heavily corticated, bearing numerous pro-

carps (Fig. 3E). Many gall structures appeared as irregu-

lar club-shaped enlargements around the procarps (Fig.

3F). The variably-shaped cells contained fully pigmented

chloroplasts (Fig. 3G). No branch shoot proliferation oc-

curred from these galls.

dIScuSSIon

While the presence of galls is infrequent in Bostrychia

culture isolates, their similar morphology and the pres-

ence of VLP in gall cells of B. simpliciuscula implicate vi-

ruses are the causative agent. Galls have been observed

previously in other red algal cells with VLP suggesting

them as a causative agent (Apt and Gibor 1991). Tumor-

ous growth is commonly caused by viruses in higher

plants (Francki et al. 1985, Scheffer 1997).Galls were not observed in laboratory culture on any

other Bostrychia species although this could be due to

limited sampling. We have many more isolates of the B.

moritziana / radicans (390), B. simpliciuscula (87), and B.

tenella (89) than of other species but it could be that these

species were more susceptible to the causative agent of

gall formation.

It is noteworthy that almost all species with galls lack

cortication except for well-corticated B. tenella in which

only one female (2751) had galls.

We have never observed galls on any Bostrychia spe-

cies in the field although we have examined thousands

of specimens, however Kützing (1865) observed and illus-

trated possible galls on field specimens.

The ability of galls to form from healthy tissue sepa-

rated from other gall tissue, suggests that the causative

agent (possibly a virus) may be latent in cells of some

Bostrychia isolates. Latent bacteria are known in higher

plants (Francki et al. 1985), and the stimulation of their

effects (e.g., cell proliferation) could be due to stressors

in the cells. This is seen in our experiments in which galls

were induced in low temperature conditions. The inabil-

ity of galls to be transmitted from one isolate to another

in Bostrychia and in Gracilaria (Apt and Gibor 1991) may

Algae 2013, 28(1): 83-92


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