[ 185 ]

Trans. Br. mycol. Soc. 65 (2), 185-200 (1975)Printed in Great Br itain

EFFECT OF SALINITY ON THE REPRODUCTIONOF TERRESTRIAL AND MARINE FUNGI

By P. J. BYRNE AND E. B. GARETH JONES

Department <if Biological Sciences, Portsmouth Polytechnic,Portsmouth, PO I 2D r

(W ith 7 Text-figures)

The effect of salinity on the sporulation of twenty selected fungi, whichincluded terrestrial (10) , fresh-water (2) and marine (8) speci es, was investi­gated using a variety of techniques, e.g. growth on agar plates, shake culture,a range of substrates and perfusion chambers. Sporangiospores of terrestrialmucoraceous fungi were produced over the whole range of salinities testedbut zygospore production was inhibited at higher saliniti es. Terrestrialascomy cetes showed limited tolerance to high salinity and this may beaffected by the available growth factors present. Marine ascomycetes have awide tolerance to low-salinity conditions and may be less conditioned by theavailable substra te. The sporulation of the majority of the fungi imperfecti,irrespective of their ecological origin, exhibited a broad tol erance to high­and low-salinity conditions. Ex ceptions were the appendaged marine hypho­mycetes which are intolerant of extreme low-salinity conditions, and theappendaged fresh-wate r hyphomycetes which were inhibited by high-salinityconditions. In Chaetomium globosum Kunze ex Fr. th e effect of salinity onperithecial development depends on wh en the sea-water concentration is addedto the perfusion medium. The significance of these observations in relation toth e ecologica l distribution ofaquatic fungi is discussed, and what is meant by theterm •marine fungi' briefly consider ed.

A number ofworkers have investigated in a preliminary way the fruitingof higher marine fun gi under laboratory conditions (Meyers & Reynolds,1959a, b; Meyers & Simms, 1965, 1967 ; Meyers & Hoyo, 1966; Meyers& Scott, 1967; Kirk, 1967) . However, few of these have considered theeffects of sea-water dilutions on the sporulation of marine fungi. This maybe important when considering the ecological distribution ofaquatic fungi.

M eyers & Reynolds (I959a) and Meyers & Hoyo (1966) have shownthat the deuteromycetes Humicola alopallonella Meyers & Moore, Tricho­cladium achraspora (Meyers & Moore) Dixon, Varicosporina ramulosa Meyers& Kohlm. and <,alerion maritimum (Linder) Anastasiou sporulate morereadily in £h11 sea water than in distilled water, while Monodictys pelagica(Johnson) Jones (= Piricauda arcticoceanorum) produced abundant conidiain distilled water but none in sea water. However, this species is relativelycommon on wood removed from the sea (Jones, 1968). Kirk (1967) hasshown that clinical and marine isolates ofAllescheria boydii Shear were ableto produce mature cleistothecia and conidia over the whole range ofsalinities (0- 100 % ). Similar results were obtained by Meyers & Simms(1967) for Halosphaeria mediosetigera Cribb & Cribb and Torpedosporaradiata Meyers, but not with Luluiorthia floridana Meyers.

186 Transactions British Mycological Society

Table I. Fungi and growth media employed

Terrestrial Medium Fresh water Medium

Phycomycetes Hyphomycetes C.M.A.Mucor hiemalis Wehmer Y.G.A. Heliscus lugdunensisAbsidia glaucaHagem Y.G.A. Sacco Tetracladium setigerum C.M.A.

(Grove) Ingold & TherryFungi imperfecti Marine

Aspergillus nigervan Tiegh. Fungi imperfectiWardo"!yces anomala Brooks Y.G.A. Asteromyces cruciatus (F. & Mml, Y.G.A.& Hansford Moreau) ex. Hennebert

Doratomyces stemonitis Corda C.M.A. Dendryphiella salina (Suth.) Y.G.A.Ascomycetes Pugh & Nicot

Neurospora crassa Shear & Y.G.A. Tetracladium setigerum (Grove) C.M.A.Dodge Ingold

Chaetomium globosum Kunze C.M.A. Ascomycetesex Fr. Ceriosporopsis halima Linder Wood

Gelasinospora retispora Cain Y.G.A. Corollospora maritima WoodSordaria fimicola (Rob.) Y.G.A. Wedermann

Ces. & de Not. Halosphaeria hamata (Hohnk) WoodKohlm,

H. appendiculata Linder WoodLulworthia sp. WoodMicrothelia maritima (Linder) WoodKohlm,

Little is known of the salinity tolerance of terrestrial and fresh-waterfungi (Siepmann, 19590, b).

The process of reproduction is of prime importance for the continuedgenetic viability of the species, and also ensures the continued propagationof healthy mycelium with the ability to seek out and exploit fresh sub­strates. The physiological and morphological changes which occur duringthe development of reproductive structures may be sensitive to many andvaried environmental conditions, which may not influence so severely thegrowth of vegetative mycelium.

Since reproduction may play such a major role in controlling thedistribution offungi within certain environmental extremes, it is importantthat the effects of the physical factors extant in the environment beinvestigated in the laboratory. Studies on the effect of salinity on thereproduction of fungi have been greatly neglected, more emphasis beingplaced on growth requirements in terms of nutrients and temperature.

This paper examines the salinity tolerance of a range of terrestrial,fresh-water and marine fungi in an attempt to understand the ecologicaldistribution of these fungi.

MATERIALS AND METHODS

Sea water was collected at the mouth of Langstone harbour on a risingtide, filtered, and stored in carboys until required. Glass-distilled waterwas employed for sea-water dilutions.

Wood extracts were obtained by finely powdering IO g of beech (Fagussylvatica L.) and Scots pine (Pinus sylvestris L.) wood chips in a Glen­Creston microhamrner mill (0'5 mm mesh) and extracted in 250 ml ofdistilled or sea water for 24 h and then made up to 1'0 I with the appro­priate water for the experimental studies.

Salinity and reproduction. P.J. Byrne and E. B. G.Jones 187The fungi used are listed in Table I. Fresh isolates of the fresh-water and

marine species were made prior to the experiments. Two media wereemployed (Table I) : V.G.A. (glucose 10 g; yeast extract 3 g; agar 20 g)and C.M.A. (cornmeal 2 g; agar 1 5 g) and made up in 1 '0 I of sea wateror distilled water of the appropriate salinity . All media were sterilized byautoclaving at 12I

o for 15 min.All fungi were grown at 20

0 in a temperature-controlled room. Liquidcultures were placed on a three-tier orbital shaker and shaken at 120 eve]min over a stroke of 1'5 in.

ProceduresReproduction in agar-plate culture

Media were made up with a range ofsalinities from sea water to distilledwater at 10 % dilution intervals, and 20 ml aliquots aseptically dispensedinto sterile Petri dishes. Plates were inoculated, with a 3 mm diam plugtaken from the periphery on an actively growing culture on an agar plate.Five replicates were used at each salinity.

In the case of Mucor hiemalis plates were mated with 3 mm disks fromboth + and - (minus) strains approximately 40 mm apart and zygo­spores produced.

The methods employed in the quantitative and qualitative assessmentof reproduction were dictated by the group of fungi under investigation.The following techniques were used for replicate plates at each salinity.

Sporangiospores of phycomycetes. Each plate was flooded with 10 ml of0'02 % Tween 80 in distilled water, six glass balls (4'5 mm) introducedand the plates shaken vigorously for 30 sec to release the spores. Theresultant spore suspension was decanted, a further 5 ml Tween solutionadded, the plate reshaken and the second suspension added to the first.The spore suspension was diluted with Tween 80 solution until a suitablespore concentration for accurate counting was achieved. A haemocyto­meter was used for counting and the number ofspores per em" of mycelialcolony calculated.

Zygospores ofphycomycetes. Four 3 mm diameter agar plugs were removedat random from each plate, the number of zygospores on each counted,and the number of zygospores per em- calculated.

Ascomycetes. Each plate was examined daily for young perithecia. Whenpresent these were squashed in lactophenol and examined for the firstappearance of asci and ascospores.

To determine the number and size of perithecia, four 3 or 6 mm plugs(depending upon the density of perithecial production) were removedfrom each plate. The number of perithecia present were counted and thesize of each measured. The results are expressed as the number of peri­thecia per em- and as the average diameter (}lm) of the perithecia inrelation to salinity.

Fungi impeifecti. Two 3 mm agar plugs were removed at random fromeach plate and placed in 50 ml centrifuge tubes containing 20 ml 0'02 %solution Tween 80 in distilled water, and six glass beads were introduced.The tube was shaken vigorously to obtain a homogeneous spore suspensionand a check made to ensure that no spores remained attached to conidio-

13 MYC 65

188 Transactions British Mycological Society

Cellulose-t----+t-substrateEvaporation

vessel

Petri-dish lid

Petri-dish base-'-':~~~lJ~~

Nutrient solution

Cotton wool bung --...-.t:;

100 ml Erlenmeyer flask

Evaporation wick

Fig. I. Perfusion apparatus.

phores. The concentration ofspores was counted and expressed as numberof spores per em? of mycelial colony.

In the case of Doratomyces stemonitis, the number of synnemata per em­was calculated by direct counts of 3 mm agar plugs removed at randomfrom the plate.

For the aquatic hyphomycetes Heliscus lugdunensis and Tetracladiumsetigerum, 55 Petri dishes, each containing 20 ml distilled water C.M.A.were prepared. The plates were inoculated and incubated at 10° for 3-4weeks. Eleven sets of five replicate plates were flooded with sterile 10 mlaliquots of a range of sea-water dilutions and incubated for 3-4 days untilconidia were produced on the submerged mycelium. Six glass beads wereintroduced and plates were agitated to release the conidia. The sporesuspension was decanted and the spore concentration determined asdescribed above.

Reproduction ofmarine ascomycetes

A range of sea-water dilutions was prepared containing o· I gil yeastextract, and 20 ml aliquots dispensed into 100 ml Erlenmeyer flasks. Asterile strip of balsa wood (70 x 7 x 1'5 mm) was introduced into eachflask, and all flasks were then inoculated with mycelium of the fungus to betested. Flasks were incubated on a shaker for 2-3 weeks or until abundantmycelial growth had developed on the wood. They were then maintainedas standing cultures and examined at frequent intervals for the presence ofperithecia, asci and ascospores.

Perfusion technique for reproduction studies ofChaetomium globosum

This study was initiated so as to determine the site of inhibition ofreproduction at high salinities in Ch. globosum.

The apparatus employed was adapted after Eggins, Malik & Sharp(1968) and is illustrated in Fig. I. The substrate in the Petri dish was purewood cellulose Solka floc strips 40 x 10 mm with a tapered wick at each

Salinity and reproduction. P. J. Byrne and E. B. G. Jones 189

(.6) in

80x

1:6 -<)

<i>

'"'I:>.

0-, ~

000

'-A4 >.N....'"',.D

E;:>Z

2

80

and zygospores (e) at

40 60Sea water (%)

Fig. 3and zygospores

20

....~E 2;:>

Z

o 8x1:o~

~ 6P..

.900::

'"o 4P..en'­o

3 x1:o~~oP..

2 (3

~'­o

o100

•

80

x

IA A\ ~

j 0 U--'---'---'-..... I ~o 20 40 60

Sea water (%)

Fig. 2

Fig. 2. Mucor hiemalis, Production of sporangiospores (e)relation to salinity at 20

0•

Fig. 3. Absidia glauca. Production of sporangiospores (6)varying salinities at 200

•

3~-

end. The whole assembly was autoclaved and a small metal cup glued tothe base of each Petri dish. The cup was filled with sterile distilled waterto maintain a high humidity within the assembly.

For the salinity test, perfusion sets were made up with a range of sea­water concentrations and containing I gil yeast extract. The substrate wasinoculated with one drop of a macerate of mycelium and spores andduplicate sets at each salinity incubated at 20° in a growth room. Plateswere examined for reproductive structures each day.

In the salinity transfer experiments, using the perfusion technique, gosets were prepared each with I gil yeast extract in distilled water (per­fusing nutrient). These were inoculated and incubated as described above.At 12 h and/or 24 h intervals, five perfusion sets were removed and thedistilled water nutrient replaced with 60, 70, 80, go and 100 % sea water(containing yeast extract) respectively. Several drops of the new perfusatewere also added to the substrate to effect an immediate change-over of theionic environment. These sets were then incubated as before.

The perfusion technique proved an excellent method for the exactmanipulation of ions in the growth medium.

RESULTS

Salinity markedly affects the reproduction of M. hiemalis (Fig. 2).Sporangiospore production fell from 3 x 106 in distilled water agar to i ofthis number on 100% sea-water agar (S.W.A.). Zygospores were notproduced at salinities above 60 % S.W., with maximum production on10% S.W.A.

190 Transactions British Mycological Society

2

20%6 100

75 E-3oj

'u<l)

~50 ~

'-0...<l)

0:;E

25 ""is

Perithecia Asci

Fig. 4

Ascospores 0 20 40 60 80 100Sea water (%l

Fig. 5

Fig. 4. Neurospora crassa. Time taken to produce reproductive structures at varyingsalinities.

Fig. 5. Neurospora crassa, Effect of salinity on the number (_) and size (e) of peritheciaat 20°.

The reproduction ofA. glauca (Fig. 3), a homothallic species, exhibited adifferent response to that of M. hiemalis in that sporangiospore productionwas stimulated with increasing salinities up to go % S.W. A second peakin sporangiospore production was observed at 60-70 % S.W., an inherentfeature since the same pattern occurred in a repeat experiment. As withM. hiemalis, zygospore production was greatest on distilled water and 10 %S.W., but unlike that species, they were produced over the whole salinityrange, although increased salinities caused a reduction in zygosporeformation.

Results of the effect of salinity on the fruiting offour terrestrial ascomy­cetes are presented in Figs. 4 and 5 and Table 2. In no case were ascosporesproduced at salinities above 60 % S.W. and in Neurospora crassa it was aslow as 20 % S.W. These results show that the time required for the pro­duction of perithecia increased with increasing salinities, for example forSordaria fimicola two days in distilled water agar but eight days on go %S.W.A. Increasing the salinity also had a pronounced effect on the num­bers and average size of the perithecia produced. For example, in S.fimicola 3'5 x 102 perithecia per em> were produced on distilled water agar,11'5 x 102 on 30% S.W.A. but only 0'1 x 102 on 90% S.W.A., the latterlacking asci and ascospores. The average diameter of the perithecia ofS. fimicola decreased from 200-2 10 p,m (on distilled water agar and 10%

S.W.A.) to 45 p,m on go % S.W.A.The salinity at which the maximum number ofperithecia were formed

varied from 10 to 40 % S.W., but in each case the largest were pro­duced in distilled water media (Fig. 5). Thus, in general low salinities

Table 2. The effect oj salinity on the reproduction of three terrestrial ascomycetesSea water (%)

A

DW 10 20 30 40 50 60 70 80 90 100

Chaetomium globosum PAS (9) PAS (9) PAS (9) PAS (9) PAS (9) PA (II) P (IS) P (17)No, of peritheeia per em" 29 75 76 48 45 5 3 I

Gelasinospora retisopora PAS (2) PAS (2) PAS (2) PAS (2) PAS (2) PAS (2) P (g) P (g)No, of perithecia per em" x 103 0'7 0'9 I'g 1'9 2,8 1'7 0'2 0'1Average size of peri thecia (!tm) 130 130 115 100 70 60 50 45Sordaria fimicola PAS (2) PAS (2) PAS (2) PAS (2) PAS (g) PAS (g) PAS (4) PA(5) P (7) P (8)No, of perithecia per em" x 10" ss 6'5 8'0 11'5 4'5 4'5 S'O 1'5 0'2 0'1Average size of perithecia (!tm) 200 210 160 185 ISS 160 60 65 50 45

P, perithecia; A, asci; S, spore; -, no fruiting; (), Days taken to form perithecia,

Table 3. The effect of glucose concentrations and various carbon sources on the time taken (days)to develop reproductive structures of Sordaria fimicola

Glucose concentration (gil)> Scots pine Beechwood

0 0'1 1'0 5'0 20'0 Cellulose Maltose Fructose extract extractr--"----., r--"----., r--"----., ,-----.A---o. ~ ,-----.A---o. ~ ~ ~ ~

Salinity P A S P A 5 P A 5 P A 5 P A 5 P A 5 P A 5 P A S P A S P A SDW 3 6 7 3 6 7 3 5 6 3 6 7 3 5 6 5 8 9 s 7 8 6 9 II 8 10 13 6 II IS

Sea water (%)10 3 5 6 3 5 6 3 5 6 3 5 6 3 5 6 4 7 8 2 6 7 5 8 10 5 8 10 5 9 II20 3 5 6 3 5 6 s 5 6 3 6 7 3 5 6 4 8 9 s 6 7 6 9 II 8 5 9 II

3° 4 7 8 3 5 6 3 5 6 3 6 7 s 7 7 5 8 9 3 7 8 6 10 12 12 6 10 1240 4 8 9 3 6 7 3 6 7 3 7 8 4 7 8 4 8 9 8 II 13 850 5 9 10 4 7 8 4 7 8 4 8 10 4 7 8 5 10 12 10 Ig 15 II60 6 II Ig 5 10 12 5 9 10 5 10 12 5 8 10 670 8 Ig 6 12 6 12 6 II 5 II 880 8 8 8 790 9 9 9 8

100

A, asci; P, perithecia; 5, ascospores,

~l::l

T abl e 4- The e.fJect of salinity on the sporulation of some f ungi imperfecti ~

~("'>

Sea water (%) .....(5-

DW 60 80 ~10 20 3° 4° 5° 7° 90 100 ."Fr esh water e;;,

Heliscus lugdunensis (x 103) * 2'5 2'4 0'7 0,6~-Tetracladium setigerum ( X 103) * 1,6 0,8

Terrestr ial ~Aspergillus niger ( X 107)* JO'4 15"5 16,8 19'0 17'1 14'7 13'1 12'3 11'7 11'3 JO'5

~Doratomyces stemonitis 45'0 94'0 133'0 225'0 218'0 123'0 J08'o 94'0 42'0 28'0 15'0Synnemata per em"

("'>Stachybotrys atra (x J06)* 2'3 2'7 2,6 2'5 3'2 2'9 3'5 3'5 4'7 3'9 4'7 <:::lWardomyces anomala (x J06)* 16,6 12'2 9'5 6'1 4'3 4,6 3,8 3'4 4'1 3'8 4'5 ~

Marine()tj~-Asteromyces cruciatus (x J06)* 0'6 1'2 " 3 "3 2'0 2' 1 2'9 3'0 2,6 2'9 2,6 l::l

Dendryphiella salina (x JO')* 12'1 9'9 g'6 11'9 11'4 12'7 11'3 10'9 11 ' 2 12'5 12'5 ........

* Number conidia per ern"; -, no spor ula tion, ~~.Q

Salinity and reproduction. P. J. Byrne and E. B. G. Jones 193maintained relatively few large perithecia, at the optimum salinity alarge number of medium-sized perithecia, while at higher salinities a fewsmall perithecia were produced (Table 2).

In S.fimicola, many of the perithecia formed on 60 % S.W.A. containednormal and malformed asci. The malformed asci failed to produce sporesand they were shorter and broader than normal asci. Malformation ofasci reached a further stage on 70 % S.W.A., the lateral swelling was moreevident, giving the asci a globose appearance. There was no evidence ofspore formation and an apical pore to the ascus was lacking (Byrne, 1971).Malformed asci were not observed in the other ascomycetes tested.

Results presented in Table 3 show that increasing glucose concentra­tions from o-I to 20'0 g had little effect on the time taken to produceindividual fruiting structures. However, the replacement of glucose withcellulose, fructose, Scots pine extracts and beech extracts had a pro­nounced effect on the tolerance of S. fimicola to salinity. Carbohydratesource had an effect on the time taken to produce fruiting structures atdifferent salinities. On maltose at 50 % S.\"'., the time taken to produceperithecia and mature spores was 5 and 12 days respectively, while 10 and15 days were required to develop the same structures on fructose at thatsalinity. Ascospores were produced on 30 % and 10 % S.W. respectivelywhen grown on cellulose and Scots pine extracts. This observation may besignificant when considering the colonization of different substrates in thesea by fungi.

The sporulation of the two fresh-water hyphomycetes (H. lugdunensis,T. setigerum) was severely inhibited at low salinities (30% and 10% S.W.respectively) but no malformation of the conidia was observed (Table 4).The four terrestrial hyphomycetes (Table 4) produced conidia over thewhole range of salinities. In Wardomyces anomala, spore production fell withincreased salinity, while in Stachybotrys atra Corda spore production in­creased. A. niger and Doratomyces stemonitis exhibited optimum sporeproduction at 30 % S.W. Sporulation in Asteromyces cruciatus increased withincreasing salinities while in D. salina sporulation was little affected bysalinity (Table 4).

Of the six marine ascomycetes which produced ascocarps on balsa woodin pure culture, five iCeriosporopsis halima, Corollospora maritima, Halo­sphaeria hamata, Lulworthia sp. and Microthelia maritima) produced maturefruiting structures over the whole range of salinities. Halosphaeria appendi­culata also produced perithecia over the whole range of salinities, but failedto develop asci and ascospores in distilled water media.

Results for the reproduction of Ch. globosum on cellulose perfused with arange of sea-water dilutions are presented in Table 5. Reproduction wasmore tolerant to increasing salinities under these conditions as comparedto the agar plate cultures. Perithecia containing mature ascospores wereproduced at salinities up to 60 % S.W. compared with a 40 % S.W. limiton Y.G.A. plates (Table 2). At 70% S.W., a few immature peritheciadeveloped with no evidence of ascus formation.

Table 6 presents the results for the reproduction of Ch. globosum, wherethe perfusing nutrient was changed from distilled water (DWPN) to 60,70, 80, 90 and 100 % SWPN at 12 hand 24 h intervals. Exchanging

194 Transactions British Mycological Society

Table 5. The effect if salinity on thefruiting of Chaetomium globosumonperfused cellulose

+(4) ++(8) ++(10) +

+++

Seawater (%) ...

PeritheciaAsciAscospores

DW.'" 10 20 30 40 50 60 70

+ + + + ++ + + ++ + + +

'" Time in days to develop reproductive structures.

Table 6. The effect ifexchanging thedistilledwaterperfusate with various salinitiesat increasing incubation times on the reproduction if Chaetomium globosum

Sea water (%)A

Days

I

2

33!44!55t6789

10

60,....----"---.PAS

+ + ++ + ++ + ++ + ++ + ++ + ++ + ++ + ++ + ++ + ++ + ++ + ++ + +

7°,....----"---.PAS

++++++++ + ++ + ++ + ++ + ++ + ++ + +

80r---"---.PAS

+++++++ ++ ++ + +

90,....----"---.PAS

+++++++ ++ ++ + +

100r---"---.PAS

+++++++ ++ ++ + +

DWPN with 60 % SWPN had no effect on the subsequent maturation ofperithecia, as sporulation occurred at this salinity (Table 5).

In 70% SWPN nutrient, perithecia are formed but lack asci and asco­spores. Similar results were obtained when DWPN was exchanged for70% SWPN up to and including the fifth day of incubation. However,after 5! days, 70% SWPN as the perfusing nutrient allowed the furthermaturation of perithecia, even though asci were not formed until theeighth day of incubation. Thus the salinity-sensitive stage in the develop­ment of the perithecia had passed and the further development of asciand spores was then insensitive to 70% SWPN.

Exchanging DWPN with 80, go and 100 %SWPN caused the inhibitionof reproduction at the stage reached on the particular day of transfer;that is, no further maturation of a perithecium occurred once subjected tothese high salinities.

DISCUSSION

The results show that the terrestrial ascomycetes, mucoraceous fungiand fresh-water hyphomycetes tested were all affected to varying degreesby increased salinities. The most severely affected were H. lugdunensis andT. setigerum and also the four ascomycetes, in which the time taken toproduce perithecia increased with increasing salinities. Thus, increasing

Salinity and reproduction. P. J. Byrne and E. B. G. Jones 195salinity not only totally inhibits reproduction at the higher values, butalso causes a slowing down of the reproductive processes. Salinity had apronounced effect on the number and average size of peri thecia producedin culture. The salinity at which the maximum number of perithecia wereformed varied from 10 to 30 % S.W., but in each case the lar gest peritheciaoccurred on distilled water media, with a gradual decrease in size withincreasing salinities. Siepmann (195g a) reported similar results forRosellinia medullaris Ces, & de Not. In M. hiemalis and A. glauca zygosporeproduction was inhibited at all salinities above 10 % S.W . This was alsotrue ofsporangiospore production in M . hiemalis, but for A. glauca optimumsporangiospore production was on 30% S.W. with a second peak at 60 %S.W.

In S. fimicola, salinity-induced ascus malformation occurred. At 60 %and 70 % S.W. the asci became shortened and more globose with evidenceof wall thickening. At 60 % S.W. there was also inhibition of spore forma­tion. In some cases, fragmentation of the cytoplasm prior to spore forma­tion was only partial, and it is possible that high salinities may influencemeiotic and mitotic nuclear divisions. However, Meyers & Hoyo (lg66)have shown that variation of salinities can affect the conidial morphologyof the marine hyphomycete Varicosporina ramulosa. At salinities below Ig %S.W. the conidial cells became thick-walled and formed cysts. Chlamydo­spores, usually absent at salinities above 20 % S.W., were also formed at lowsalinities. Salt-induced morphological and cytological changes have beenreported for Endomycopsis fibuliger (Lindn.) Dekker (Takada & Matsuda,1965; Takada & Kasakawa, Ig66a, b) when concentrations of up to 1'0 M

NaCl caused yeast cells to become more spherical and ovoid, while at thesame time the ratio of yeast to filamentous cells increased. Harrison &Jones (lg71, Ig74) have shown similar responses to salinity in some fresh­water phycomycetes.

Preliminary results indicate that a reduction in the amount of availablecarbohydrate has the effect of slowing down the rate of reproduction,while reproductive tolerance to salinity is reduced on certain carbo­hydrates (cellu lose) when compared with others (maltose, fructose).Similar results have been observed by Harrison & Jones (1971) forSaprolegnia parasitica Coker. This aspect may be very important whenconsidering the ecological distribution of aquatic fungi. The apparentexclusion of terrestrial species from lignocellulose materials in the highersaline portions of an estuary may be due to combinations of factors, in­cluding salinity, temperature and the nature of the substrate.

The use of a perfusion technique enabled the effect of salinity on thedevelopment of Ch. globosum to be studied. Salinities of up to 60 % S.W.allowed normal development of the sex initials with subsequent develop­ment of ascogenous hyphae and vegetative cells to form the ascocarpwall (Whiteside, Ig62a, b; Cooke, Ig6ga, b). The addition of 70% S.W.allowed the development of the sex initials, but prevented the develop­ment of ascogenous hyphae. However, the vegetative stalk cells were un­affected and allowed the subsequent formation of the ascocarp wall. It isalso evident that having passed a critical stage in the formation of asco­genous hyphae, at about the sixth day of incubation, further development

Transactions British Mycological Society

Sea water (%) 0

Vegetative growth

50o

100

Reproduction

50o

100

Spore germination

50 100

Terrestrial, freshwaterMucor hiemalis •••••••

Absidia glauca

Chaetomium globosumGelasinospora retispora

Sordariafimico/aPenicillium no/alum

AspergillusnigerStachybotrys atraHe/isms /ugdunensisTetracladium setigerum

MarineCoro/lospora maritimaLulworthiasp.AsteromycescruciatusZalerion maritimumDendryphiella sa/ina

Fig. 6. Physiological responses of selected fungi to salinity. *, No results available.

was unaffected by 70 % S.W., with the perithecia producing maturespores after 10 days incubation. This phenomenon was observed only at70 % S.W., with salinities of 80, 90 and 100 % S.W. each causing theabrupt cessation ofperithecia I development. These observations emphasizethe extreme sensitivity of developmental stages in the reproduction ofterrestrial ascomycetes to conditions of increasing salinity.

The marine ascomycetes and fungi imperfecti and the terrestrialhyphomycetes tested all produced spores over the whole range of salini­ties with the exception of H. appendiculata. In W. anomala spore productionwas inhibited at the higher salinities, while Asteromyces cruciatus showed asimilar trend at lower salinities.

The effect of salinity on the vegetative growth (Byrne & Jones, inpreparation), reproduction and germination (Byrne & Jones, 1975) of arange of fungi are summarized in Fig. 6. This shows that while thevegetative growth of the terrestrial fungi (ascomycetes, mucoraceae andfungi imperfecti) exhibited a broad tolerance to increasing salinities, sexualreproduction and spore germination is markedly affected by high salinities.The salinity at which complete inhibition occurred varied between fungi.Asexual reproduction was less severely affected by high salinities, exceptin the case of the aquatic hyphomycetes, when complete inhibitionoccurred between 20 % and 40 % S.W. The marine fungi tested exhibiteda broad tolerance to decreasing salinities, with vegetative growth the mostmarkedly affected.

In this study, salinities up to 100 % S.W. only have been tested. It isknown that many marine fungi can tolerate salinities in excess of full seawater, e.g. higher fungi (Barghoorn & Linder, 1944) and Thraustochy-

Salinity and reproduction. P. J. Byrne and E. B. G. Jones 197

Vegetative growth

oSea water (%) 0 50 100

Mucoraceae

Terrestrialascomycetes

Terrestrialfungi imperfccti

Aquatichyphomycetes

Marineascomycetes

Marinefungi imperfecti

EcologicalReproduction Spore germination observations

0 050 100 50 100 50 100

Fig. 7. Physiolog ical and ecological respons es to salinity of variou s fungal groups.1illJ , R esults abstracted from th e literature.

triales (Bremer, 1974). This aspect may be important when consideringtheir ecological distribution. For example, Thraustochytrium sp. have beenisolated from concentrated brine pans (Scholz, 1958) and the highermarine fungi have been found growing on sand grains (Koch, 1974).

Fig. 7 summarizes the salinity tolerance and the known ecological dis­tribution of various groups of fungi. The ecological data is a summary ofthe observations of Byrne & Eaton (1972), who studied the colonization(by fungi) of test blocks submerged at different salinities under laboratoryconditions. There is good correlation between the physiological responsesof terrestrial and marine fungi to increasing salinities and the observeddistribution of these fungi under natural conditions. Mucoraceous fungihave rarely been isolated from marine or estuarine substrates (Borut &Johnson, 1962; Steele, 1967). Physiological work suggests that sporegermination may be severely inhibited by increasing salinities, which mayaccount for their absence from the marine environment. In addition,mucoraceous fungi thrive on substrates rich in soluble sugar compounds(Webster, 1970), but the substrates found in the marine environment(e.g. wood, cordage, plant remains) are low in soluble carbohydratecontent and may not prove favourable for the growth and reproductionof these fungi.

Terrestrial ascomycetes are rarely found in the sea (Jones, 1962) but arepresent at low salinities in estuaries (Shearer, 1972). The effects ofsalinitiesabove 60-70% S.W. on the reproduction of these fungi could account fortheir absence in the higher salinity regions of an estuary or in the sea.

The spore germination of terrestrial fungi imperfecti was markedlyaffected by increased salinities. With the exception of Phoma spp. and

198 Transactions British Mycological SocietyS. atra, few terrestrial hyphomycetes have been isolated from sea water(Steele, 1967 ). This could be due to the lack of suitable substrates or aninability to grow and reproduce in full sea water. In the case of theaquatic hyphomycetes, reproduction and spore germination were affectedby high salinities. These results are in agreement with the ecologicalobservations ofJones & Oliver (1964) and Shearer (1972) .

The marine ascomycetes and fungi irnperfecti show a wide tolerance tovariations in salinity. However, few lignicolous marine fungi have beenfound growing on timber submerged in fresh water or at low salinities(Jones & Oliver, 1964; Eaton & Jones, 1971 a, b; Byrne & Eaton, 1972;Irvine, Eaton & Jones, 1972; Jones, 1972; Shearer, 1972; Byrne & Jones,1974) ·

Various attempts have been made to define the term 'marine fungus'(Moore & Meyers, 1959; Ritchie, 1960; Kohlmeyer & Kohlmeyer, 1964).Physiological observations show that few higher marine fungi have anabsolute requirement for sodium chloride at concentrations found in seawater (Jones &Jennings, 1965). However, some of the lower marine fungido have such a requirement for sodium chloride (Vishniac, 1955, 1958;Johnson, 1960; Goldstein, 1963 a-c; Alderman & Jones, 1971). The workpresented in these series of papers indicates that the reduced vegetativegrowth, reproduction and spore germination of terrestrial and fresh-waterfungi under saline conditions may be factors in maintaining the fungusflora of the sea distinct from that ofnon-marine habitats. A marine fungusis one which is capable of producing successive generations by sexualand/or asexual means in natural oceanic waters, or on intertidal substrates.However, recent ecological observations suggest that there may be a groupof fungi which are restricted to brackish water (Tubaki & Ito, 1973;Shearer, 1972; Jones et al. 1972).

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(Acceptedfor publication 21 December 1974)