Notes and brief articles

TAl, F. L. (1979). Sylloge Fungorum Sinicorum.viii +1527 pp. Beijing: Academia Sinica Science Press.

TAl, F. L. & WEI, C. T. (1933). Notes on Chinese fungi.III. Sinensia (Contributions from the MetropolitanMuseum of Natural History Academia Sinica) 4,83-128(abstract in Review of Applied Mycology 13,656, 1934).

TALVlA, P. (1965). The occurrence of plant diseases in1964. Maatalous ja koetoiminta Helsinki 19, 17-18(abstract in Review of Applied Mycology 45, 15, 1966).

WALKER, J. & BAKER, K. F. (1983). The correct binomialfor the chrysanthemum ray blight pathogen in relationto its geographical distribution. Transactions of theBritish Mycological Society So, 31-38.

WATER, J. K. (1981). Chrysanthemum white rust. EPPOBulletin 11,239-242.

INFLUENCE OF THE CONIDIAL MATRIX OF SPHAERELLOPSIS FILUM(DARLUCA FILUM) ON SPORE GERMINATION

BY ISABELLE LOUIS AND R. C. COOKE

Botany Department, University of Sheffield, Sheffield, SIO zTN

Spores of Sphaerellopsis filum in conidial matrix maintained a higher level of viability duringexposure to drying, freezing and u.v. irradiation than those from which the matrix had beenremoved by washing.

In numerous fungi, including many plant patho-gens, conidiogenesis within pycnidia or upon acer-vuli or sporodochia is accompanied by exopoly-saccharide and glycoprotein synthesis, the resultbeing a large spore mass immersed in a copious,water-soluble, mucilaginous matrix. The latterfacilitates escape of spores from the fructificationvia rain-splash which, in the case of pycnidia, maybe preceded by the extrusion of tendrils of spore-laden mucilage. In addition, there is some evidencethat matrix material persists around individualspores after their dispersal and that it may have arange of physiological roles. For example, it hasbeen shown to inhibit germination in Septorianodorum (Berk.) Berk., and to protect spores ofAscochyta, Colletotrichum and Phoma species fromthe damaging effects of adverse temperatures, lowrelative humidities and ultraviolet radiation(Uspenskaya & Reshetnikova, 1979; Griffiths &Peverett, 1980; Nicholson & Moraes, 1980).Hydrolases and invertase have been detected in thematrix of Colletotrichum graminicola (Ces.) Wils.,and cellulase, exopolygalacturonase and protease inColletotrichum orbiculare (Berk. & Mont.) Arx(Porter, 1969; Nicholson & Moraes, 1980; Berg-strom & Nicholson, 1981). Matrix enzymes mayplaya part in host penetration and the initial phasesof disease development.

Despite the possible importance of the matrix inspore biology few detailed studies of its functionhave so far been made and additional informationis obviously required. During a survey of inter-actions between the mycoparasite Sphaerellopsisfilum (Biv.-Bern.: Fr.) Sutton (= Darluca filum(Biv.-Bern. : Fr.) Cast.) and rust fungi it was noticed

that the spore matrix had a marked effect ongermination behaviour. This was investigatedfurther and the results are reported here.

A pycnidial isolate of S. filum (ATCC 317-76)was maintained at 25°C in the dark in 8'5 em diamPetri dishes containing 25 ml oatmeal agar. After6-8 weeks incubation spores plus spore matrix(unwashed spores), matrix-free spores (washedspores) and spore-free matrix were obtained asfollows. Mature pycnidia, characterized by theextrusion of spore masses through their ostioles,were located using a dissecting microscope.Pycnidia were then removed to a sterile specimentube with a fine, sterile scalpel and carefullysquashed to extract their spore-laden matrix. Thisunwashed spore preparation was then used im-mediately after appropriate dilution with steriledistilled water. In order to separate spores andmatrix, numbers of intact pycnidia (approximately0'3 g total fresh weight) were removed from theagar, weighed and then ground in a pestle andmortar in approximately 5 ml sterile distilled water.The resulting slurry was homogenized and largepycnidial fragments then removed by filtrationthrough two layers of sterile muslin. Filtrate wasfurther filtered using a 0'45 pm Millipore diskwhich retained the spores. This second filtrate wasconcentrated in vacuo at 25° using a rotaryevaporator to give approximately 1 ml of spore-freematrix preparation per 0'3 g fresh weight ofpycnidial starting material. This was then filter-sterilized by means of a 0'22 pm Millipore disk tobe used immediately. Spores removed during thesecond filtration were resuspended in 10 ml steriledistilled water, the suspension being agitated

Trans. Br. mycol. Soc. 81 (3) (1983) Printed in Great Britain

668 Notes and brief articles

100

Exposure period (days)

Fig. 1. Percentage germination of spores of S. filum onwater agar at 25° after various periods of exposure to40-&>% r.h. at 18-21°. Unwashed spores , -e-;washed spores, -0-; washed spores with spore-freematrix added before exposure , -.-. Variance in dataexpressed as 95% confidence limits.

~0:0.~

0:"§ 40...'"C)

30

20

10

a

further, more prolonged drying resulted in amarked improvement in their capacity to remainviable, although this did not match that ofunwashed spores. It was noted for washed sporesthat as the drying period increased there was a risein the number of heavily vacuolated and abnormallyswollen individuals observed during germinationtests . By contrast, unwashed spores retained theirnormal internal appearance and shape .

When frozen for up to two days at - 5° there waslittle difference between subsequent viability ofwashed and unwashed spores . However, althoughlonger freezing periods produced a loss of viabilityin both kinds of spore , the decline in that of washedspores was the greater (Fig. 2) . Exposure tofreezing resulted in an increase in the number ofempty or misshapen individuals observed during

vigorously for 2 min using a flask shaker, and wereagain removed from suspension using a 0"45 11mdisk. This process was repeated six times, thewashed spores being finally suspended in distilledwater at an appropriate density for immediate use.

The effect of drying was studied as follows.Samples of washed or unwashed spores (50 pi, 106

spores ml" ) were spread evenly on a series ofsterile, 22 mm square coverslips which were thenallowed to dry in a laminar flow cabinet at roomtemperature (18-21°) for 3 h. Covers lips bearingwashed spores then received either 50 pi of sterile,spore-free matrix preparation or the same volumeof distilled water. These coverslips were then driedfor a further 3 h as were those coverslips bearingunwashed spores. All coverslips were then placedin closed Petri dishes at room temperature with anuncontrolled r.h, ranging from 40 to 60%. Atintervals of 1, 2, 3 and 6 days triplicate coverslipsbearing unwashed, or matrix-treated or water-treated washed spores were taken and placedspore-side down on 2 % distilled water agar at 25°in the dark to allow germination to take place.Germination was assessed using 170 spores takenat random on each coverslip, spores beingconsidered to have germinated when germ-tubelength exceeded twice the spore diameter. In thisand subsequent experiments incubation periods of3-24 h were used in order to allow maximumgermination to occur . This was done to takeaccount of the effects of particular treatments on thevelocity of subsequent germination.

To study the effect of freezing, coverslips withdried coatings of washed or unwashed spores wereprepared as described above and then placed at- 5°. At intervals of 1, 2, 3, 4 and 7 days maximumgermination was assessed as before .

For studies on the effect of ultraviolet radiation,50 pi aliquots of spore suspension were spread overapproximately 22 mm square areas in a series ofplastic Petri dishes containing 2 % distilled wateragar. Excess surface liquid was then evaporated byleaving the dishes open in a laminar flow cabinet.The lids were replaced and the dishes exposed to350-450 om radiation from a Hanovia ultravioletlamp held at a distance of 10 em from the agarsurface . After exposure periods of 20,30,35,40,45,50, 60 and 75 min, one dish containing washedspores and one with unwashed spores were taken,incubated at 25° and maximum germinationassessed. This was done by removing three 0'5 cmdiam agar disks from each seeded area and scoring170 spores taken at random on each disk.

Drying had little effect on the viability ofunwashed spores but markedly reduced that ofwashed spores (Fig. 1). Treatment of dried films ofwashed spores with spore-free matrix prior to

Trans. Br . my col. Soc. 81 (3) (1983 ) Printed in Great Britain

100

~"~""§~ 40

30

20

10

Notes and brief articles 669

100

90

80

70

§ 60c"S!~ 50c"e....,

40o

30

20

10

Exposure period (min)

Fig. 3. Percentage germination of spores of S. filum onwater agar at 25° after various periods of exposure toultraviolet radiation. Unwashed spores,-e-; washedspores, -0-. Variance in data expressed as 95 %confidence limits.

oDays at - 5·

Fig. 2 . Percentage germination of spores of S. filum onwateragarat 25° after variousperiodsof freezing at - S°.Unwashed spores, -e-; washed spores, -0-.Variancein data expressedas 95 % confidence limits.

o 70 80

germination tests on both washed and unwashedspores. However, after four days at - 5° suchabnormalities were much more numerous amongwashed spores (28-35 %) than among unwashedspores (3-7 %). For the most part such spores failedto germinate.

Exposure to ultraviolet radiation reduced via-bility of both washed and unwashed spores and, inaddition, any post-irradiation germination required24 h for completion. Decline in viability was morerapid in washed spores, reaching zero after 40 minexposure (Fig. 3). At this point over 4 % ofunwashed spores were still viable and even after60 min nearly 2 % remained so.

These results provide additional evidence that,under laboratory conditions, the matrix is highlyeffective in protecting spores against the deleteriouseffects oflow temperature and water stress, and thatit also provides some protection against short-waveradiation. The mechanisms underlying this appar-ently multi-purpose role are far from clear, and itis indeed difficult to envisage those physico-chemi-cal characteristics of matrix material which wouldconfer upon it the combined properties of anti-freeze, anti-desiccant and radiation protectant. For

example, while it is conceivable that a coating ofdried matrix could reduce water loss, it would seemunlikely that it could provide much protectionagainst low-temperature coagulation of cytoplasm.Similarly, at least for the hyaline matrix of Si filum,its effectiveness as an extracellular radiation barriermight be doubted. Nevertheless, the findingsreported here are in accord with those few studieswhich have been carried out on other species(Uspenskaya & Reshetnikova, 1979; Griffiths &Peverett, 1980; Nicholson & Moraes, 1980).However, an additional function for the matrixsuggests itself here, and it may facilitate rapidrehydration of dried spores, so enhancing theirgerminability as well as maintaining their viability.Restoration of matrix to dehydrated matrix-freespores of S. filum just prior to germination testson agar significantly improves their germinationperformance over that of water-treated controls(Louis, unpubl.).

The question must now be asked as to whatextent laboratory behaviour of spores with andwithout matrix reflects ecological behaviour. A

Trans. Br. mycol, Soc. 81 (3) (1983) Printed in Great Britain

REFERENCES

BERGSTROM, G. C. & NICHOLSON, R. L. (1981). Invertasein the spore matrix of Colletotrichum graminicola.Phytopathologische Zeitschrift 102, 139-147.

CHUNG, H. S. & WILCOXSON, R. D. (1969). Effect ofconidial number and matrix on germination of conidiain Phoma medicaginis. Phytopathology 59, 44~442.

EL-SHATER, M. E. (1982). Aspects of the biology of therust mycoparasite Darlucafilum. PhD Thesis, Univer-sity of Sheffield.

GRIFFITHS, E. & PEVERETT, H. (1980). Effects of humidityand cirrhus extract on survival of Septoria nodorumspores. Transactions of the British Mycological Society75, 147-150.

NICHOLSON, R. L. & MORAES, W. B. C. (1980). Survivalof Colletotrichum graminicola: importance of the sporematrix. Phytopathology 70, 225-261-

PORTER, 1. M. (1969). Protease, cellulase, and differentiallocalization of endo- and exopolygalacturonase inconidia and conidial matrix of Colletotrichum orbiculate,Phytopathology 59, 1209-1213.

RAMBO, G. W. & BEAN, G. A. (1970). Survival andgrowth of the mycoparasite Darluca filum. Phytopatho-logy 60, 1436-1440.

USPENSKAYA, G. D. & REsHETNIKOVA, 1. A. (1979). Roleof pycnidial mucus and some ecological factors in thegermination ofconidia in the genera Ascochyta Lib. andPhoma Fr. Mikologia i Fitopathologia 14,334-337.

670 Notes and brief articles

major difficulty is that nothing is known concerning We wish to thank the University of Sheffield forthe amount and persistence of matrix carried by the award of a Research Studentship to Isabellespores during their natural dispersal. This will Louis during the tenure of which this work wasdepend on the mode of dispersal. Since the matrix carried out.is water-soluble, dispersal of wet spore masses viarain-splash can be expected to result in the matrixbecoming attenuated to a point where its effectsmay become negligible (Griffiths &Peverett, 1980).However, this is not always the case and in, forexample, Colletotrichum graminicola, spore massesproduced by acervuli may become dry, the resultantparticulate material then being wind-dispersed sothat considerable amounts of matrix accompanythe dispersed spores (Nicholson & Moraes, 1980).While the general ecological importance of matrixfor survival of dispersed spores remains uncertain,it may well be that maintenance of viability duringenvironmental stress in laboratory conditions isincidental to another, and quite different, ecologicalrole ofmatrix. With respect to the latter, matrix hasbeen shown to contain germination inhibitors, andthere seems to be no doubt that its major functionis to prevent germination either within thefructification or in spore masses liberated close toit (Chung & Wilcoxson, 1969; Rambo & Bean,1970; Griffiths & Peverett, 1980; EI-Shater, 1982).Any additional protective function is more likelyto be exercised within wet or dry spore massesawaiting rain or wind dispersal respectively.Ecological aspects of matrix function are now beinginvestigated further using a range of plant-pathogenic species.

Trans. Br. mycol. Soc. 81 (3) (1983) Printed in Great Britain