Taxonomy and biology of the lettuce anthracnose fungus

[ 61 9 ] Trans. Br . mycol. Soc. 86 (4), 619-628 (1986)

Printed in Grear Britain

TAXONOMY AND BIOLOGY OF THE LETTUCE ANTHRACNOSE FUNGUS By V. J. GALEA, T. V. PRICE S chool of Agriculture, La Trobe University, Bundoora ]08] Victoria, Australia AND B. C. SUTTON Commonwealth Mycological Institute, K ew, Surrey , U .K.

The morphology and development of the causal organism of lettuce anthracnose, Marsson ina panattoniana , was compared with Microdochium nivale, M. ory zae and M. stoveri. The species are congeneric and the new combination Microdochium panattonianum is proposed. Conidia from fresh lettuce or cultures differed significantly in size from those in herbarium material. Germination occurred at temperatures between 3-26 °C and pH 4-5'2. Germination decreased following exposures >9 h at 32°, 4 hat 34° and 1 h at 36°. Germination did not occur in the absence of free water, at -4 MPa water potential, or at conidial densities of 5100 mm ". Germ-tube growth was optimum at 20° and pH 5'2; at this temperature germination commenced after 4 h, the second germ-tube appeared after 20 h and sporulation at 44 h. Germ-tube growth was inhibited following 1 hat 32°, 34° and 36°. Colony growth was linear with time and was optimum at 15-20° with growth on PDA > MEA> TWA. Conidial production increased logarithmically 2-7 d after incubation at 20°. Lettuce anthracnose caused by Mars sonina panattoniana (Berl.) Magnus was first described from Italy by Berlese (1895). Appel & Laibach (1908) suggested the fungus be transferred from the genus as the conidia were not produced in an acervulus; more recently Moline & Pollack (1976) also came to this conclusion. The disease is recognized by necrotic spots on the leaves which drop off leading to a shot-hole appearance and the disease is also called 'ring spot '. Sunken elliptical lesions with whitish centres are also formed on the midrib . Since its discovery the disease has been found to be widespread in Europe, N , America and Australasia (Anon., 1976). The disease is more prevalent during late winter and early spring and in recent years epidemics have caused severe losses in California and Victoria where it is currently of economic importance. This paper reports on the taxonomy of the lettuce anthracnose fungus and its biolog y. MATERI ALS AND METHODS

M orphology

Con idia from lesions on fresh glasshouse infected lettuce, cv. Cos Verdi; herbarium specimens (T VP 471, Werribee South, Vic., 1983; TVP 479, Cranbourne, Vic., 1983; TVP 473, Keilor, Vic., 1983; TVP 470, Virginia, South Aust., 1983); isolates from lettuce (T VP 451, Werribee South, Vic. , 1981; TVP 471, Werribee South, Vic., 1983 ; TVP 480, Werribee South, Vic., 1984); and from

La ctuca serriola L. (T VP 472, Yea, Vic., 1983) grown for 10 d on Malt Extract Agar (M EA, Oxoid Ltd) at 15 °C were mounted in water, examined under phase contrast microscopy (x 400), photographed and measurements made of length and width of approximately 100 conidia. For SEM studies infected leaf tissue was fixed in glutaraldehyde (6 % in 0 '05 M phosphate buffer, pH 6'5) for 6 h, deh ydrated in an acetone series, critical point dried, mounted, coated with gold and examined in a Siemens Autoscan at 20 kV. Conidial germination Conidia were stained with lactophenol cotton blue prior to microscopic assessment of germination, Conidia were counted as germinated if the length of the germ-tube was greater than half the spore length. Measurements of germ-tubes were made from photographed specimens. Effe ct of temperature. Petri dishes (5 em) containing 10 ml tap water agar (T WA, 1'5 %) were inoculated at three sites with 5 Jll of a suspension of conidia (2' 2 x 105 mr '), incubated in the dark at 3'5,6,10, 15, 20, 23, 25, 26, 28, 30, 32, 34 and 37°, and assessed for germination 24 and 48 h later. Three replicate plates were also similarly inoculated with conidial suspensions (7'25 x 104 or 5,6 x 105 mr'), incubated at 32, 34 or 36° for varying periods after which they were transferred to 20° and assessed for germination.

620

The lettuce anthracnosefungus

Effect of relativ e humidity and fre e water. Greasefree microscope slides stored in alcohol were air dried in a laminar flow cabinet prior to inoculation with a suspension of conidia (1'36 x 10' ml") by means of a pressurized sprayer. The inoculated slides were placed on glass rods in square 10 cm Petri dishes containing sulphuric acid solutions adjusted to maintain relative humidities between 60 and 100 % (Weast & Astle, 1980). The Petri dishes were sealed with plastic film (W hatman), incubated at 20° and spores assessed for germination 24 and 48 h later. Other inoculated slides were either transferred immediately to Petri dishes maintained at 100 % r.h., or subjected to various drying and wetting regimes (T able 2) and incubated at 20° for 24 h. Each slide was assessed for germination of 100 conidia at three sites and each treatment was replicated three times . Effect ofwater potential. TWA plates were amended with NaCl (L ang, 1967) or KCl to give a range of water potentials from 0 to - 5 MPa at 20°. The plates were inoculated with 5 pI drops of a conidial suspension (4'6 x lOS mr '), incubated at 20° and assessed for germ ination. Each treatment was replicated three times . Effect of conidial density. Petri dishes (9 em) containing 20 ml TWA were inoculated at ten sites with 10 pI of conidial suspensions obtained from a 14-d culture on MEA grown at 15°. The conidial density on each plate following inoculation ranged between 1'5 x 10' and 1'5 x 104 mm " , Inoculated plates were incubated at 20° and the percentage germination measured at five sites on each plate . Effect of pH. Mixtures of phosphate buffer (0'2 M , NaH 2P0 4 , Na 2HP0 4) were used to prepare a pH range of 4'25, 5'2, 6'0, 7'1, 7'8 and 8'1 distilled water agar plates. The pH of each plate was rechecked with a flat electrode following pouring. Two plates of each pH were inoculated with two 5,u1 drops of a conidial suspension (4'5 x lOS ml" ) incubated at 20° and assessed for germination. Sequence offungal development in vitro

Petri dishes (9 ern) containing 4 ml TWA were inoculated at four sites with 5 pI of a suspension (5' 0 x 104 ml") of conidia. Two of the inoculated sites were immediately covered with a sterile cover slip. The plates were incubated in the dark at 10, 15 and 20°. At intervals the inoculated sites were examined microscopically under phase contrast and photographic records made of the sequence of development following incubation.

Effect of media on growth at different temperatures Petri dishes (9 em) containing 20 ml of TWA, MEA and Potato dextrose agar (PDA, Oxoid Ltd) were inoculated centrally with a droplet containing 2-5 conidia and incubated in darkness at 5, 10, 15, 20 and 25°. Three replicates were used for each treatment and the experiment was repeated twice. Two measurements of colon y diameter at right angles to each other were made weekly for 11 wk. Growth on Czapek Dox agar was not assessed as previous work by McKenna (1982) indicated that the growth rate and habit on TWA and Czapek Dox agar was identical. Rate of sporulation in culture Thirty agar slants containing 8 ml MEA in 28 ml McCartney bottles were inoculated with 10,u1of a conidial suspension (4'58 x 107 ml" ) which was spread evenly over the agar surface and incubated at 15°. At regular intervals three slant s were removed and conidia were detached by washing the slants with sterile water. The total number of conidia removed by washing was determined by haemoc ytorneter counts. RESUL TS

Taxonomy Mierodochium panattonianum (Berl.) Sutton, Galea & Price comb .nov. (Figs 1-7 ) Marsonia panattoniana Berl., R iv. Pat. Veg. 3: 342 (1895)· Marssonina panattoniana (Berl.) Magnus, H edwigia 45 : 9 1 (1906) . Marssoniaperforans Ell. & Ev., in Selby , Bull. Ohio Agri. Exp . St . 73 : 225 (1896). Didymaria perforans (Ell. & Ev. ) Dandeno, Rept Mich. Acad. Sci. 8: 47 (1906). A scochyta suberosa Rostrup ex N eergaard, Bot. Tidsskr. 44: 360 (1938). Mycelium inter- and intra-cellular, hyaline, branched, septate. Conidiomata epidermal, subcuticular or superficial, varying from single conidiophores to fascicles of several to small sporodochia which often become confluent and form on the surface of collapsed tissue , consisting of hyphal strands running parallel to the surface of the host and producing eustromatic tissue of loosely aggregated textura globulosa from which conidiophores or conidiogenous cells arise; individual cells of the strands may also produce conidiophores. Conidiophores supporting conidiogenous cells mayor may not be formed, 1-2 septate, hyaline , sparsely branched near the point of origin and bearing 1-4 conidiogenous cells which are

V.

J. Galea,

T. V. Price and B. C. Sutton

frequently produced at the same level, up to 10 flm long x 3' 5-4'0 flm wide. Conidiogenous cells hyaline, smooth, discrete or integrated on short conidiophores, 7'5-16'0 flm long, cylindrical to irregular or lageniform and then with a narrower conidiogenous region t : 5-2'0 flm wide and a broader base 2'5-4'0 flm wide, proliferating enteroblastically to produce additional conidia at successively higher levels and sometimes combined with occasional sympodial holoblastic proliferation. Conidia dry, forming effuse white to pink masses, holoblastic, hyaline, smooth, eguttulate or with several small guttules, fusiform, curved, medianly 1 (-2) eusep-

tate, obtuse at the apex with the upper cell wider, lower cell strongly tapered towards the truncate base, 12'5-15'5 x 2'5-3'5 flm. Specimens examined: on leaves of Lactuca sativa, Copenhagen, Denmark, C. Frisenette, 20 Aug. 1984, 1M1 276684, holotype of A. suberosa (C); Reading, Berks., U.K., E. W. Mason, July 1924, 1M1 22315; MAFF, Bristol, Avon, M. Cater, comm. 11 Dec. 1980, 1M1 254087; South Werribee, Victoria, Australia, T. V. Price 431, 12 Aug. 1981, 1M1 261874; on leaves of Laetuca scariola ( = L. serriola), Livland, Prov., Vidzeme, Riga, Latvia, U.S.S.R., J. Smarods, July 1941, F. Petrak Myc. gen. 663, 1M1 30563.

o:r Fig.

1.

621

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>:

Microdochium panattonianum, A, conidia; B, conidiophores and developing conidia.

~ ~_;""A

. !!!!!!Y~

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Figs 2-7. M. panattonianum. Scanning electron micrographs of lesions on Cos Verdi lettuce leaf surface. Scale bars = 10 tun. Fig. 2. A single conidiogenous cell emerging through the epidermis with conidia, produced at successive levels. Fig. 3. Conidiophore and conidium produced from a hyphal cell.

622

The lettuce anthracnose fungus

Fig. 4. Conidia produced holoblastically from conidiophores. Fig . 5. Conidiogenous cells and conidia emerging from a stoma. Fig. 6. Conidiomata on the leaf surface. Fig . 7. Conidiogenous cells with holoblastic conidia.

A reappraisal of conidiogenesis and conidiogenous cell growth was made by Minter, Kirk & Sutton (1982, 1983) and Minter, Sutton & Brady (1983) and the importance of describing the events surrounding conidial production in terms of seven recognizable stages was emphasized. By using these criteria to anal yse the situation in M . fragaria e (Lib.) Kleb. and M. panattoniana it is clear that the differences are comparatively small . In M.fragariae and other species of the genus conidia are holoblastic and proliferation of the conidiogeneous

cell, though enteroblastic and percurrent, is restricted to the formation of onl y 2-4 conidia (Sutton, 1980; Spiers & Hopcroft, 1983; Sutton & Webster, 1984). In M. panattoniana conidia are also holoblastic and proliferation of the conidiogeneous cell may be either enteroblastic and percurrent, holoblastic and sympodial or a combination of the two. The combination of percurrenr /sympodial conidiogeneous cell proliferation, dr y conidia and essentially sporodochial effuse confluent conidio-

V. J. Galea, T. V. Price and B. C. Sutton mata reinforces the conclusion by Moline & Pollack (1976) that M. panattoniana is incorrectly placed in Marssonina and places the fungus in the same genus as Fusarium nivale Ces. ex Berl, & Yogi. This species has undergone considerable taxonomic and nomenclatural revision recently. Based on a study of conidiogenesis, which was interpreted as annellidie, and a different ascomycete teleomorph (Monographella Petrak) Gams & Muller (1980) concluded that the species was incorrectly placed in Fusarium and proposed Gerlachia Gams & Muller to accommodate it. Subsequently Samuels & Hallett (1983) showed that in addition to percurrent proliferations the conidiogeneous cells also developed sympodially so they proposed taking up the earlier generic name Microdochium H. Sydow for such species. Gerlachia was reduced to the status of a taxonomic synonym. Arx (1984) finally removed the remaining species of Fusarium sect. Arachnites to Microdochium, a procedure which effectively tied up all the loose ends as far as the teleomorphs and anamorphs of species related to F. nivale are concerned. Comparison of the morphology and development of M. panattoniana with that of M. nivale (Ces. ex Bed. & Vogl.) Samuels & Hallett and M. oryzae (Hashioka & Yokogi) Samuels & Hallett in Gams & Muller (1980) and of M. stoveri (Booth) Samuels & Hallett (1983) shows that the species are congeneric.

Conidial size

The size of conidia from infected lettuce or from cultures were significantly greater than those from herbarium material (Table 1).

Table

1.

Conidial germination Effect of temperature. Germination occurred between 10° and 26° after 24 h incubation. It was inhibited at 3°,5° and 28° and did not occur at and above 30°. Germination occurred between 3° and 26° after 48 h incubation but there was no increase at higher temperatures (Fig. 8). Germ-tube growth was optimum at 20° and decreased linearly and more rapidly at higher than at lower temperatures (Fig. 9). Germination decreased rapidly following exposure for more than 9 h at 32°, 4 h at 34° and 1 h at 36° (Fig. 10) but a similar decline in germ-tube growth rate occurred after more than 1 h exposure to these temperatures. The effects of temperature and duration of exposure on germination and germ-tube lengths were best described by the equations: 1

and

respectively. Effect of relative humidity and free water. Germination at 100 % r.h. was very low (1-2 %) and conidia did not germinate at lower humidities. Germination increased in the presence of free water and drying for 24 h did not affect viability. Germination on

Conidial dimensions of M. panattonianumfrom various sources Length (flm)

Source of conidia Fresh Cos lettuce Cultures TVP 451 TVP 471 TVP 480 TVP 472 Herbarium material TVP 471 TVP 479 TVP 473 TVP 470

Width (flm)

Range

Mean

Range

10"3- 17"9

13"5

2"7-4'9

Mean 3'6

100

10"3-18'5 11'4-21'2 12"5-21'2 9'8- 17'9

14"0 14"0 14'9 14'7

2'2-4'9 2'7-5'4 2'7-4'9 1'2-4'9

3'6 3,6 3'4 3'5

100 100 100 100

8'7- 16'3 8'7-16'8 8'7-15'8 8'7- 16'3

12'0 12'0 11'4 12'1

2'2-4'3 2'2-3'8 2'2-3'8 1'6-3'8

2'9 3'0 2,8 2'6

88 93 97 75

LoS.D.

P < 0'°5 0'01

623

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0'15 0'19

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100

50

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30

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Temp erat ure (oC) I emp era Lure \ .... ) Fi g. 8

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10

20 30 Temp erature (OC) Temperat ure ( C) F ig. 9

F ig . 8. Effect of temperature on germinati on of M. panat tonianum conidia . After 24 h ( 0) ; 48 h ( e ). Fig. 9. E ffect of tem pe ra tu re on M . panattonianum germ-tube length after 24 h incuba tion .

glass slides was poor compared to germination on agar (T able 2).

Table 2. Effect offree water ongermina tion of conidia on glass slides afte r 24 h incubation at 2 0 °

Effect of wat er potential. Germination was slow at water potentials below - 1 MPa, with none at - 4 MPa. Germination at potentials between - 1 and - 3 MPa increased following incubation for

G ermination (% ± S.D.)

100

~ <:

60

1'4 ±I '7b 99·o± I '3c

48 h and this was more marked when KCl was used as the solute (F ig. 11). Germ-tube growth was also inhibited with decreasing water potential and this was also m ore rapid in the presence of NaC!.

"

<:

'§ o"

32' 0 ± 18·8a 40 '2 ± I s '8 a

M ean s foll owed by the same lett er are n ot signi fican tly different (P < O-D1) .

80

s

Slides wet , with distilled H,O Slides dried, th en rewet immediat ely with H 2 0 Slid es dried, then rewet after 24 h with H 2 0 Slides dry , at 100 % r.h . Tap wate r agar

40

20

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.....- "'T'"---,---'....--r-- """'T- - r - -......... ..., ) o 2 4 6 8 10 12 221 !2 Duration of expos ure (h) Duration of exposure (h) Fi g. 10. Effect of duration of exp osure to temperatures ab ove 30° on germination of M . panattonianum conidia aft er 24 h at 20°. 32° ( 0), 34° ( e ), 36° (.0:. ). LD,o = 9' 48 h, 32°; 4'02 h , 34° ; 2'99 h , 36°.

Effect of conidial density . Germination was sign ificantly (P < 0'001; l.s.d , = 3 '8 ) reduced at conidial den sities abo ve 1 ·6 x 10 3 mrn"? and did not occur at densities of 5 '1 x 10 3 mrn " after 24 h at 20° . Incubation for 48 h resulted in a slight but significant (P < 0 '001 ) increase at densities above 10 3 mm" (F ig. 12 ) . Effect of pH. Optimum germination occurred between pH 4 '25 and 5'2. Germination decreased rapidly ab ove pH 6 '0 and ceased at pH 8 ' 0 (Fi g. 13 ).

V. J. Galea, T. V. Price and B. C. Sutton 1O~ T

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-2 -3 -4 Wat er pote ntia l (MPa)

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Fig. 11. Effect of NaCI and KCl induced water stress on germination of M. panattonianum conidia at 20°. On NaCl, 24 h CO); on NaC!, 48 h Ce); on KCl, 24 h CL,); on KCl, 48 h CA.). LD.. = -2'12 MPa, NaCl, 24 h; -2'49 MPa, NaCl, 48 h; -2,61 MPa, KCl, 24 h; -3'89 MPa, KCl, 48 h.

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8

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7

Fig. 13. Effectof pH on germination of M. panattonianum conidia at 20°, 24 h CO), LD 50 = 6'45 ±0·80; 48 h, Ce), LD 50 = 6,80±0·81.

80

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100

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102

103

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Conidial den sity (mm- 2 )

Fig. 12. Effect of conidial density on germination of M. panattonianum conidia at 20°. After 24 h CO); 48 h ce). Incubation for 48 h did not result in any significant increase in germination. Optimum germ-tube growth occurred at pH 5'2 (Fig. 14).

Sequence of fungal development in vitro. The first germ-tube emerged after 4 hat 20° or 15° and after 8 h at 10°; a second germ-tube emerged after 20 h at 20°, 24 hat 15° and 44 hat 10°; branching of mycelia and sporulation occurred at 28 and 44 h

4

5

6

7

8

pH pH

Fig. 14. Effect of pH on germ-tube length of M. panattonianum conidia after 24 h at 20°, respectively at 20° whilst branching commenced at 32 h at 15° and 48 h at 10°.

Effect of media on growth at different temperatures The optimum temperature for growth was 15-20°. Growth on MEA> PDA > TWA. Growth was

626

The lettuce anthracnose fungus

Table 3. Growth rate of M. panattonianum on different media at different temperatures (0G) Growth rate (mrrr/ wk)

TWA

PDA

MEA

2'0

1'9 5,8 6,6

5'4 6'S 6,S l 'S

7' 0

0'3

slow and there was a linear relationship of growth with time (T able 3). Colonies on MEA and PDA had a pale flesh pink colour with a raised convoluted appearance. Colonies on TWA were white, sparse and spreading. The ability of the fungus to grow on TWA was tested further by inoculating plates of purified agar (Oxoid Ltd). The fungus again showed sparse and spreading hyphal growth and also sporulated on this medium. The hyphae also contained numerous bulbous cells (Fig, 15)·

Rate of sporulation in culture

Conidial production increased logarithmically from 3'3 x 104 conidia/slant to 2 '9 x 10 8 conidial slant 2-7 d after incubation reaching a maximum of 7 x 10 8 conidia/slant after 9 d.

Fig, lS . Hyphal growth of M . panattonianum on purified agar showing bulbous cells.

DISCUSSION

Marssonina panattoniana and its relat ionships have been the subject of much discussion and this has been reviewed by Moline & Pollack (1976). Most of th e confusion surrounds the interpretation of the conidiomata which have been variously described as hypha I (D andeno, 1906 ) or pycnidial (N eergaard, 1938 ), but the synonymy suggests that most workers have interpreted conidiomata as acervular (Brandes, 1918 ). It is evident from our stu dies that there is much variation in conidiomatal type but that the tissue bearing conidiophores in M. panattoniana is not produced exclu sively between the host's cuticle and upper epid ermal wall as in M. fragariae and does not remain discrete. Moline & Pollack (1976) also studied conidiogenesis in M. panattoniana and interpreted development as annellidic with percurrently proliferating conidiogenous cells. They distinguished M . panattoniana from other Marssonina species which, according to Arx (1970), produce conidia from what were termed phialides, and concluded that M. panattoniana should be removed from Marsson ina for th is reason. Such observation s have been confirmed but, in addition, it has been found that proliferation of the conidiogenous cells may be sympodial and holoblastic as well as percurrent and enteroblastic. These features place the species in Microdochium H . Sydow , a genus that now accommodates fungi like Fusarium nivale to which M . panattoniana is mo st closely related. The conidial dimensions from fresh and cultured specimens differed significantly, probably due to shrinkage by dr ying . Gams & Muller (1980) indicated that narrower spore measurements due to shrinkage also occur when lactic acid is used as a mountant. The conidial measurements in this study were somewhat shorter than those described by Berlese (1895), Dandeno (1906) and Brandes (19 18) but are within the ranges given by Pape (1929), Moline & Pollack (1976) and Marte & Capelli (1979) although it is not clear whether their measurements were made on conidia from fresh specimens and whether these were mounted in water . Conidial measurements from L . serriola have not previously been described; those obtained in this study were in the range obtained from L. sativa. M . panattonianum conidia were capable of germinating at low temperatures and this is in agreement with the infection at 2° reported by Moline & Pollack (1976) . The inhibition observed above 24 and 28° is in agreement with the results of Brandes (1918). The effects of temperature on germ-tube growth have not previously been reported and our results indicate that this is optimum at 20°. Although exposure at 30° for 24 h

V. J. Galea, T. V. Price and B. C. Sutton totally inhibited germination our results indicated that there was a relationship between temperature, time of exposure and germination and this would explain the failure by Brandes (1918) to obtain germination following exposure of conidia to 40° for 10 min. The average winter and spring temperatures in Werribee (July-october, Davies, 1981) and England (Stevenson, 1939) are within the range favourable for germination and growth of M. panattonianum and our results indicate that the prevalence of the disease during these months is largely due to temperature. The poor germination and growth above 26° also provides evidence as to why the disease is seen less frequently during the summer months, especially since ambient summer temperatures in Australia often reach and exceed 40°. The reduction in viability of conidia at high temperatures suggests that further studies are required to demonstrate how this fungus survives over summer since a teleomorph for M. panattonianum has not yet been found. Free conidia were able to survive air drying for 24 h without loss of viability. The necessity for free water for germination has not been previously reported. The availability of free water on lettuce leaf surfaces are, therefore, likely to influence both germination and infection. Increased infection has been reported to occur following overhead irrigation (Brandes, 1918), wet winters (Stevenson, 1939) and leaf wetness (Couch & Grogan, 1955), but was not linked to increased conidial germination. Free water on leaf surfaces is more likely to occur as dew during the low winter and spring temperatures; furthermore current lettuce cultivation practices in Victoria are based on fixed overhead sprinkle irrigation. The water requirements for germination of M. panattonianum conidia are, therefore, likely to occur throughout winter and spring. In spite of the presence of free water, conidial germination on glass slides was much reduced compared with germination on TWA. Brandes (1918) found that when conidia were mixed with liquid agar at 25° only those at the surface germinated and he suggested that this was due to an oxygen requirement. It is not known whether the reduction in germination on glass slides compared with that on agar is due to a requirement of oxygen and this needs further investigation. The effects of water stress on germination show a similar pattern to those reported for other fungi (Griffin, 1972, 1981; Cother & Griffin, 1974; Hocking & Pitt, 1979; Sung & Cook, 1981), with latent period increasing with decreasing water potential. Our results indicated that inhibition of germination and growth was greater with NaCI than KCI. Sung & Cook (1981) reported similar

effects of these salts on Fusarium roseum Link emend. Snyder & Hansen and thought they were due to increased uptake of K" at 20°. The increased germination when solutes other than NaCI is used at the same water potential has also been reported for other fungi (Cother & Griffin, 1974; Pitt & Hocking, 1977). Further work is required to elucidate the nature of the suppression by NaCI compared to KCI. A germination self inhibitor appears to be present at conidial densities above 1·6 x 103 mm -2. Although the nature of the inhibitor is not known our results are similar to those obtained with Glomerella cingulata (Stoneman) Spaulding & Schrenk (Lingappa & Lingappa, 1965), Penicillium griseofulvum Dierckx (Fletcher & Morton, 1970) and Rhynchosporium oryzae Hashioka & Yokogi ( = Microdochium oryzae) (Amu Singh & Sen Gupta, 1983). As the conidia were germinated on TWA it is unlikely that the inhibition was due to competition for nutrients. The optimum pH for germination and growth was much lower than that for M. oryzae (Amu Singh & Sen Gupta, 1983). The rate of development from germination to sporulation decreased with decreasing temperature. Our rates differ from those of Appel & Laibach (1908) and Brandes (1918). Their studies were conducted on nutrient-rich media and at higher temperatures and this could account for the differences observed. Our results on growth of M. panattonianum are in general agreement with Appel & Laibach (1908) and Moline & Pollack (1976), but our isolate had a faster growth rate at 20° than the latter. Media composition greatly affected growth rates and Yap (1969) has reported growth rates of 45 mm/mo on Corn meal agar. The growth habit on PDA was similar to that reported by Moline & Pollack (1976) and the growth on TWA appeared to be similar to the growth they obtained on Tochinai agar. M. panattonianum was capable of sporulating rapidly and profusely even on highly purified agar containing no nutrients. Its growth habit and pigmentation are, therefore, linked to the nature of the nutrients. This work was financed by a grant from the Reserve Bank of Australia, Rural Credits Development Fund. B. C. S. thanks the curator (Herb. C) for the loan of relevant herbarium material.

REFERENCES

AMU SINGH, S. & SEN GUPTA, P. K. (1983). Factors affecting germination, in vitro, of Rhynchosporium oryzae conidia. Transactions of the British Mycological Society 81, 66cH562.

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The lettuce anthracnose fungus

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(R eceived for publication 2] July 1985)