Trans. Br. mycol. Soc. 80 (1) 121-125 (1983)

[ 121 ]

Printed In Great Britain

INHIBITORS OF AQUATIC AND AERO-AQUATICHYPHOMYCETES IN PINE AND OAK WOOD

By S. A. GUNASEKERA* AND J. WEBSTERDepartment of Biological Sciences, University of Exeter

Addition of pine and English oak wood powder to agar depressed the mycelial growth of nineaquatic and two aero-aquatic Hyphomycetes and spore germination of three aquaticHyphomycetes. Both powders were highly effective in depressing the growth of Aegeritacandida, Dactylella aquatica, Tetracladium marchalianum and T. setigerum, and spore germi-nation of Dactylella aquatica. They were least effective as inhibitors of Heliscus lugdunensisand Lemonniera aquatica. Inhibition is stronger with pine powder than with oak powder, andthe inhibitory effects were reduced or completely lost in leached powders.

Aquatic Hyphomycetes are abundant on leaves ofdeciduous trees and also occur on woody substratain rapidly-flowing streams (Webster & Descals,1981). It is well-known that deciduous tree litter isa better source of material of these fungi thanconiferous needles, and the same is true for woodysubstrata.

Willoughby & Archer (1973) found nine aquaticHyphomycetes on pre-sterilized twigs of oak(Quercus sp.) exposed for colonization in a stream.Among these, Heliscus lugdunensis, Anguillosporalongissima, Clavariopsis aquatica, Lemonniera aqu-atica, Alatospora acuminata, Tricladium splendensand Anguillospora crassa were considered as earlycolonizers, while Dactylella aquatica and Helico-dendron sp. were considered as late colonizers. Theyfound oak twigs to be the most satisfactory sub-strate for Alatospora acuminata and Articulosporatetracladia in comparison with others such as alder(Alnus glutinosa (L.) Gaertner), ash (Fraxinusexcelsior L.) and willow (Salix sp.). Fewer fungiwere recorded on wood blocks of Scots pine (Pinussylvestris L.) than on beech (Fagus sylvatica),submerged in a river (Kane, 1978). Among thecommon aquatic Hyphomycetes recorded wereHeliscus lugdunensis, Alatospora acuminata, Campo-sporium pellucidum and Campylospora chaetocladia.Contrary to observations by Ingold (1966), Barlo-cher, Kendrick &Michaelides (1978) and Barlocher& Oertli (1978a) found that aquatic Hyphomycetesgrow and sporulate on dead pine (P. resinosa, P.sylvestris and P. leucodermis) needles decaying instreams, but occur relatively infrequently. This lowfrequency of occurrence increased when theneedles were longitudinally halved or treated withsteam, alcohol, NaOH or hot HCI. Barlocher et al,

* Present address: Department of Botany,Universityof Kelaniya, Kelaniya, Sri Lanka.

(1978) suggested that increased frequency could bedue to the removal of the delaying effect of thecuticle on fungal colonization or to the improvementof needles as a growth substrate by leaching and/ordestruction of inhibitors. Untreated needle powder(Barlocher & Oertli, 1978a) and methanol, ethanoland petroleum ether extracts of needle powder(Barlocher, Oertli & Guggenheim, 1979) depressedthe growth of Clavariopsis aquatica, Anguillosporapseudolongissima, Lemonniera aquatica, Tetracla-dium marchalianum and Tricladium angulatum onmalt extract agar.

During a study of the effects of supplementarynitrate and phosphate on weight losses of pine andEnglish oak wood caused by several aquatic andaero-aquatic Hyphomycetes (Gunasekera, Webster& Legg, 1983), higher weight losses were recordedfrom oak than from pine. This led us to investigatewhether the woods of pine and oak containcompounds which inhibit mycelial growth andspore germination of aquatic and aero-aquaticHyphomycetes.

MATERIALS AND METHODS

Single-spore isolates of the following fungi wereinvestigated: aquatic Hyphomycetes - Dactylellaaquatica (Ingold) Ranzoni, Tetracladium marchalia-num de Wild., Tetracladium setigerum (Grove)Ingold, Tricladium giganteum Iqbal, Tricladiumsplendens Ingold, Clavariopsis aquatica Ingold,Anguillospora crassa Ingold, Heliscus lugdunensisSacco & Therry, Lemonniera aquatica Ingold;aero-aquatic Hyphomycetes - Aegerita candidaPers.: Fries and Helicodendron conglomeratumGlen-Bott.

Wood from air-dried pine (Pinus sylvestris L.)and English oak (Quercus robur L.) was ground to apowder in a micro-hammer mill with a 0'5 rom

122 Inhibitors of aquatic and aero-aquatic hyphomycetes

5·0 %

A

2·5 % 1·25 %

Non-leached pine

0·25 % MEA

Leached pine

Non-leached oak

B

5·0 % 2·5 % 1·25 %

0 '25 %M EA

Fig . 1. Growth of Helicodendron conglomeratum on 0'25 % malt extract agar with 5 %, 2'5 % and 1'25 %non-leached and leached Pine (A) and Oak (B) wood powder, 14 days at 20° after inoculation.

S. A. Gunasekera and J. Webster 123screen and dried at 60°C. During leaching, 25 g ofdry powder was placed in a 6 cm diam glasscylinder, one end of which was covered with severallayers of muslin. The cylinder was held verticallyand tap water allowed to run slowly through thepowder for 4 days, Finally, the powder was washedwith 500 em" distilled water, dried at 60° andre-weighed to estimate the weight loss due toleaching. Leached and non-leached powders weresterilized by autoclaving at 1 bar for 20 min.

For estimating mycelial growth, 1'0, 0'5 and0'25 g amounts of non-leached powders andresidues of these amounts of leached powders weremixed and stirred with 20 em" of hot 0'25 % maltextract agar (MEA) in 9 cm Petri dishes. Two Petridishes for each fungus were then inoculated withfour 4'5 rom disks cut from 2-3 week old coloniesgrown on 0'25 % MEA. Mean colony diameter wasassessed, usually after incubation for 14 days at 15°,based on eight values, obtained by measuring eachcolony in two predetermined diameters (Fig. 1)under a 6 x objective of a dissecting microscope. A.candida and H. lugdunensis, with fast growth rates,were measured at the end of 4 and 7 daysrespectively, and A. crassa, with a slow growth rate,was measured at the end of 21 days. Growth wasestimated as an increase in diameter of the colonyover the inoculum diameter and expressed as apercentage of the colony diameter in 0'25 % MEAcontrols. Inoculum disks which failed to showmycelial growth after the selected time intervalwere removed, inverted and replated on fresh0'25 % MEA, and viability estimated after 7-10days at 20°,

For spore germination tests, 1'0 g amounts ofleached and non-leached powders were mixed with15 ern" of molten 1 % tap water agar (TWA)contained in 9 cm Petri dishes . The solidifiedmedium was overlaid with ten sterile 19 romcellophane (Boots roasting wrap) disks. Afterincubation for 24 h at room temperature, 4-5 dropsof a spore suspension, prepared from pieces ofculture agitated in aerated sterile water (Webster &Towfik, 1972), were placed on each disk. After 16 hat 15°, disks were removed and mounted in 1 %cotton blue lacto-phenol and examined microsco-pically for germination. One hundred spores werecounted on ten replicate disks for each fungus.Controls were disks over TWA, and inhibition ofgermination was expressed as a percentage of thecontrol value.

RESUL TS AND DISCUSSION

The fungi can be divided into three groups withrespect to growth on media containing non-leachedoak and pine powder. Group I: D. aquatica, A.

candida, T . setigerum and T . marchalianum, wheregrowth is almost completely inhibited on both oakand pine; Group II : T. splendens, T.giganteum, C.aquatica, A . crassa and H. conglomeratum, whereinhibition is intermediate; and Group III : L.aquatica and H. lugdunensis, where growth inhibi-tion is minimal.

Non-leached pine powder produces significant(95% c.L., Student's t test) growth inhibition ingroups I and II fungi at all concentrations.Inhibition was complete in group I fungi; increasedwith concentration in group II fungi; and was low,showing no significant change with concentration,in group III fungi. Viability tests (Table 1) indicatethe fungicidal nature of non-leached pine powdertowards group I fungi. The effect on group I andII fungi was considerably reduced by leaching, anexception being A. candida which still showed a30 % inhibition. H. conglomeratum (Fig. 1A)showed a stimulation of growth after leaching.These results may perhaps explain why Kane(1978) observed only four aquatic Hyphomyceteson pine blocks immersed in a stream - one of thembeing H. lugdunensis which is least inhibited bynon-leached pine wood powder. It is of interest thatH . lugdunensis was first described from pine bark,and that we have made collections of the perithecialstate, Nectria lugdunensis, on Larix decidua in astream (Herb. Exr. 4028).

Non-leached oak powder also caused significantgrowth inhibition for all fungi tested at allconcentrations used - except for A. crassa; but isgenerally less inhibitory than non-leached pinepowder (T able 1). Leaching removes inhibitoryeffects from oak powder and results in growthstimulation in five of the fungi tested - the mostobvious being A . candida and H. conglomeratum(Fig . 1B) showing 35-50 % growth stimulation.These findings are consistent with the observationsof Willoughby & Archer (1973) that A. crassa, H .lugdunensis and L. aquatica are prominent earlycolonizers of sterilized oak twigs.

Spore germination in TWA supplemented withnon-leached and leached pine and oak woodpowders has only been briefly investigated here(Table 2). It is notable that the responses of thespores of the fungi tested, viz. T. splendens, C.aquatica and D. aquatica, show a close parallel tothe inhibition of mycelial growth. Inhibition ofspore germination and mycelial growth of D.aquatica by non-leached pine and oak powder, andthe loss of this effect on leaching, is of interestbecause it offers a possible explanation for thegeneral observation that D. aquatica favours wellleached and partly decomposed wood (includingoak twigs, Willoughby & Archer, 1973) .

Brown colouration was observed around colonies

124 Inhibitors of aquatic and aero-aquatic hyphomycetes

Table I , P ercentage g row th inhibition

Pine Oak

Powder (g) Non-leached: Leached : Non-leached : Leached :in 20 ern" inhibition Non-viable inhibition inhibition Non- viable inhibition

MEA (%) disks (max, 8) (%) (%) disks (max, 8) (%)Group I

Dactylella 0'25 100 8 9'6± I 9 76 '2±2'9 4'1±1 '4aquatica 0 '5 100 8 15'3±1 '4 100 0 7'8±2'4

1'0 100 8 16,o± 1 6 100 8 10'7±2' 1Aegerita 0 '25 ' 100 8 30 'O±4'O 77'4±2'3 -38'9 ±2 '7candida 0 '5 100 8 n8±3'5 100 a -36'6±3 2

1'0 100 8 47'5±3 '1 100 8 -34'O±3'3Tetracladium 0'25 100 8 7'O± I '5 91'3±2'7 5'4±I '1setigerum 0 '5 100 8 4'3±1'7 100 7 8'4 ±I '9

1'0 100 8 6'5±I '3 lOa 8 8'4±I '9Tetracladium 0 '25 96'7±4'9 6 o'o±5'3 97'O±2 '2 1 3'2± I'8marchalianum 0' 5 100 8 2'3±1'8 100 6 4'4±1'3

1'0 100 8 4'5±2'3 100 8 4'2±1'OGroup 2

Tricladium 0 '25 5h±1 '5 9'9±2'2 26'3 ±2 '7 4'1±2'3splendens 0'5 64'6±1 ,6 12'8±1 '7 48'5±2'6 1'6 ±3 '1

1'0 100 8 20'4 ±I 'O 62'2±1 ,6 8'4±3'6Tricladium 0'25 75'5±1 '9 6'4± 1,6 27'6± 10 5'4± 1,6giganteum 0'5 83,8±3 '1 12,8± 1,8 42'7±l 'l 4'3±I'1

1'0 100 8 15'3± I ,6 57' 5±I 'l l '1±1'6Claoariopsis 0 '25 88'6±I '6 1'2±2'O 71'7±2'4 -15 '3 ±2'3aquatica 0 '5 96'3±2'2 15'8±4'O 93'3 ± 2'1 9 '5±3'6

1'0 100 8 14'6±3'4 100 2 3'7 ±2'8A nguillospora O'Z5 52'2±2'4 10'O±l '6 7'5 ± 1'6 3'O±1 ,g

crassa 0 '5 71,o±2'7 7'5±I '7 4'8 ±2'O 7'2± I ,81'0 100 2 4'1 ± 1'3 24'5±26 5'2±I'7

Helicodendron 0 '25 68'4±4'Z -13 '6±2'7 13'z±2'5 -38'3±3'5conglomeratum 0'5 86'1 ± 2'6 -10'9±1'8 28'6±o'8 -5Z'5±2'6

1'0 100 -4'4±3'3 53'O±2'5 -47 '6±2'1Group 3

Heliscus 0'25 43 '5±I '2 6'o ± 1'4 48'7 ± 1'2 1'4±O'9lugdunensis 0 '5 49 '2±I '5 IO'3±I '6 69'9±1 '8 1'3± I '7

1'0 47 '2±2'9 IO'4±2'O 88'9±I '2 O'9 ±I '8Lemonniera 0 '25 49 '9±4'5 2'O±3'8 23'4±5'1 1'5±3'8aquatica 0'5 61'9±5'9 4' 1±3'O 43'2±5 '9 1'3± 4' 9

1'0 75,8±4'8 13'5±2'6 87'6±2'8 -16'4±4'5

± , 95 % Confidence limits, Student's t test; negative values indicate stimulation of growth ,

Table 2. Percentage inhibition of spore germination

Pine Oak

1'7 ± I 'S 16'6±4'7 4 'O±Z'5O'4 ± O'7 95'4±2'1 1'6 ±I '93'O±2'3 84'3±4'4 8,8±4'3

±, 95 % Confidence limits, Student's t test ,

Tricladium splendensClauariopsis aquaticaDactylella aquatica

TWAcontrols Non-leached Leached Non-leached

5'z± 3 '531'8 ±33 '868' 1± 13'6

Leached

o'3±O'3o

1'5± 1'0

S. A. Gunasekera and J. Webster 125of T . setigerum, D. aquatica and A . candida growingon plates containing non-leached oak powder, andaround colonies of H . lugdunensis growing on plateswith non-leached oak and pine powders. The extentand intensity of browning varied with the fungusand the concentration of powder used . Although D.aquatica showed browning on the control plates,the browning was intense and more extensive on theplates with non-leached oak powder. No browningwas observed on plates with leached oak andleached pine powder, except in the case of D.aquatica which showed the same degree ofbrowning as the controls. Factors that influencefungal growth on wood have been reviewed byKaarik (1974) ; among these, phenolics, which areknown to yield brown colours when oxidized, areconsidered to be the most important (Levin, 1976),and their presence in needle powders of Pinusleucodermis and Sequoia have been reported byBarlocher & Oertli (1978b). Other growth-inhibi-tory compounds reported from wood of Pinus spp.include pinosylvins (Scheffer & Cowling, 1966) anda range of other extraneous components (Erdtman,1952). Although the present observation pointstowards the probable involvement of phenolics ingrowth inhibition, we cannot make any statementregarding the nature of the inhibitorycompound(s).

We are thankful to R. A. Davey for completingthe experimental work on spore germination tests.One of us (S.A.G.) is grateful to the Association ofCommonwealth Universities for the award of aCommonwealth Fellowship, during the tenure ofwhich this work was done.

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(R eceived for publication 22 February 1982)