CHAPTER II

THE EFFECT OF TEMPERATURE AND CULTUREMEDIUM ON THE GROWTH AND SPORULATION OF

DRECHSLERA CATENARIA

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INTRODUCTION

Sporulation and growth of fungi are affected by light, temperature,

relative humidity and nutrient source. Light and temperature have been

described to affect the sporulation of many imperfect fungi (Carlisle,

1965; Leach, 1962a; Leach, 1962b; Leach, 1967; Marsh et aI, 1959).

Leach (1967) found Drechslera catenaria on PDA to sporulate best at

temperatures below 25C, but there was no sporulation of the fungus

without light regardless of temperature. Q. catenaria sporulated most

abundantly when near-ultraviolet was followed by darkness, though it

also sporulated under continuous light (Leach, 1967). Shoemaker (1962)

and Zeiders (1976) also reported a lack of sporulation of this fungus

without light.

However, nutrition has been implicated (Hawker, 1957) as perhaps

the most important single factor in the control of growth and reproduction

of fungi. Fungi formerly included in the genus Helminthosporium, have

been shown to grow and reproduce on potato-dextrose agar (Leach, 1967),

V-8 juice agar (Dienier, 1955; Zeiders, 1976), lactose casein hydrolysate

(Malca and Ullstrup, 1962) and various other media (Braverman and

Graham, 1960). Good growth and sporulation of Drechslera catenaria have

previously been reported on potato-sucrose agar (Drechsler, 1923),

potato-dextrose agar (Leach, 1967) and V-8 juice agar (Zeiders, 1976).

However, growth and sporulation of D. 'catertaria were variable on sucrose-

asparagine agar (Braverman and Graham, 1960).62

63

Originally there was some difficulty in inducing the Drechslera

catenaria isolate from bentgrass to sporulate adequately in culture for

preparation of inoculum for pathological studies (Larsen et aI, 1980).

The objective of this study was to determine favorable nutritional and

temperature conditions for good growth and sporulation of D. catenaria

in culture.

MATERIALS AND METHODS

Media composition. Three different media were used in this study:

lactose casein hydrolysate medium (LCH) (Malca and Ullstrup, 1962),

potato-dextrose agar (PDA) (Difco Laboratories, Detroit, MI 48201),

and 20% V-8 juice agar (V-8JA) (D~ener,1975). All three media were adjusted

to pH 6.0 with either 6N sodium hydroxide or 6N hydrochloric acid before

autoclaving using a Beckman Expandomatic SS-2 pH meter (Beckman

Instruments, Inc., Fullerton, CA 92634). Twenty ml of medium were

dispensed into each disposable polystyrene petri plate (100 x 15 em).

Fungal isolates used and 'procedure for seeding~ culture media.

A monoconidial isolate (DCL-2) and the mass isolate (DCL) of Drechslera

catenaria, previously described (Chapter I),were used in this study.

Test media were seeded with 8-rom diameter agar discs taken from ten

day LCH fungal cultures incubated at 20C and exposed to 12 hr daily

incandescent and fluorescent lighting (5.5 K lux). Each agar disc was

removed from an area approximately one cm from the margin of the colony,

inverted and placed in the center of a culture plate of appropriate

medium.

Treatment conditions. Seeded plates were incubated for 4, 7, 10

or 14 days in temperature-controlled environmental chambers at 15 ! 1,

20 ! 1, or 25 ! lC with alternating dark (12 hr) and light (12 hr)

periods. The sources of direct irradiation (5.5 K lux) in this study

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were unfilteredG.E. 40-W incandescent lamps and G.E. 40-W cool-white

fluorescent lamps at a distance of 20 cm from the cultures. Temperatures

were measured using a Tele-thermometer (~odel 42SC, Yellow Springs

Instrument Co., Inc., Yellow Springs, OR 45387) with a "Banjo-surface"

temperature probe (#408) and did 'not differ between the chamber air and

the air inside the agar-filled petri plates.

Data collection. Sporulation and mycelial growth were compared on

the three media at the three temperatures. :'For sporulation measurements,

fungal colonies in culture plates were flooded with 10 ml of 1% copper

sulfate-Tween 20 solution and brushed with a camel's hair brush to dis­

lodge conidia. After the conidial suspension was decanted, the cultures

were rinsed twice with 5 ml of the copper sulfate-Tween 20 solution to

remove remaining conidia. All three suspensions were combined and the

conidial concentration was determined using a Sedgwick-Rafter counting

cell (VWR Scientific, Columbus, OR 43215) in conjunction with a Roward

ocular microscopic disc (American Optical Corp., Buffalo, NY 14215).

Sporulation means are the averages of conidial counts of twenty ocular

fields.

Mycelial growth was expressed as an increase in mycelial dry

weight and colony area. The average diameter of a colony, the mean

of three random diameter measurements per colony, was used to calculate

the area of the nearly circular colony. For mycelial dry weight

determinations, colonies were removed from agar plates by immersing

the culture plate contents in 50 ml of hot distilled water (93C) for

10 minutes. The extracted colony was dried in a dry-air oven at 38C

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for 24 hr. Final pH was determined for the agar-distilled water

solution from each culture after ten day incubation at 25C.

Data were recorded for both fungal isolates at different harvest

dates after seeding. Data from the colonies included: area (cm2),

mycelial dry weight (mg/cm2

) and sporulation (conidia/mrn2

) as affected

by temperature and medium composition. The sporulation and area data

were taken from the same treatment plates. Dry weight data were from

different plates since a destructive technique was used for estimating

sporulation. This entire experiment was done three times with three

replications per treatment for the sporulation-colony area plates and

only one replication per treatment for the dry weight determination.

The treatment plates were completely randomized within the chambers

each time the experiment was done. Treatment means were analyzed

according to each harvest date alone and for all dates together for

each character recorded. Analysis was done using the Duncan's New

Multiple Range Test at the 5% level of significance.

RESULTS

Culture "characters. Colonies of both isolates at all temperatures

. and on all media were white four days after seeding the plates. The

two isolates did not differ in mycelial pigmentation under any conditions.

After seven days, the Q. catenaria colonies on LCH were olive-gray and

moderately appressed to the agar surface, whereas on PDA, colonies were

dark gray to black with very dense erumpent growth. Colonies on V-8JA

were light gray with concentric zones of light and dark growth. The

dark zones were closely appressed hyphae with abundant sporulation and

the lighter areas consisted of erumpent aerial hyphae with sparse

sporulation. The dark zones generally corresponded to regions of new

growth exposed to light. No zones were recorded until seven days after

seeding. After ten days, the hydrogen-ion concentration of the PDA

(6.3) and LCH (6.1) cultures remained nearly unchanged from the initial

pH of 6, but the pH of the V-8JA cultures increased significantly (8.3).

Final pH did not differ among isolates or environmental treatments.

The effects of temperature and culture medium on growth and

sporulation of the two isolates of D~ catenatia are presented separately

for each harvest date in tables 2.1-2.4. Means of treatments were

compared within each harvest date as well as compared with treatments

of other harvest dates. Although there were some significant main

effects, only significant interactions pertinent to the experimental

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objective are presented. When there were no differences between

isolates, treatment means for the isolates were averaged. There were

significant differences in sporulation between isolates at 7 days,

but there was no isolate effect at 4, 10 or 14 days.

Colonies incubatedfot'four days (Table 2.1). Initial mycelial

growth was significantly faster, that is greater area, on PDA at 25C

2(19.1 cm ) than at any other temperature-medium combination. There

were no significant differences in colony dry weight (mg/cm2

) among

treatments. Sporulation (conidia/rnm2

) was highest at 20C on LCH

(15.5) and PDA (15.7).

Colonies incubated for seven days (Table 2~2). After seven days,

the two isolates differed significantly in dry weight and sporulation

but not in colony area. Area was greatest at 25C on PDA and V-8JA,

with the colonies completely or nearly covering the entire medium

surfaces. Dry weight of DCL was highest on PDA at l5C (2.37). However,

dry weight of DCL-2 colonies was highest on LCH and PDA at 20C, but did

not differ significantly from colonies on PDA or V-8JA at l5C.

Sporulation was also higher for DCL than DCL-2 under optimum conditions.

Sporulation of DCL was highest at 20C on LCH (33.2) and on V-8JA

(31.2). DCL-2 colonies produced more conidia on LCH at 20C (26.5)

than under other conditions.

Colonies incubated for ten days (Table 2.3). Colonies had nearly

or completely covered the surfaces of PDA and V-8JA at the two highest

temperatures and on LCH at 25C. Mycelial dry weight was highest for

colonies on PDA at either 20C (2.64) or 25C (2.73). However, sporulation

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Table 2.1. Effect of temperature and culture medium on colony area,dry weight and sporulation of'Drechsleracatenaria fourdays after seeding. u

Area Dry w~ Sporulation2Temperature (oC) MediUrilv , , , '(cm2) w (JUg/em )x (conidia/nun ) y

15 LCH 5.5 EZ0.93 A 12.5 B

PDA 7.8 D 0.88 A 6.9 CV-8JA 6.8 DE 0.92 A 7.6 C

20 LCH 3.6 F 0.96 A 15.5 APDA 7.5 D 0.71 A 15.7 AV-8JA 8.6 D 0.90 A 7.5 C

25 LCH 13.1 C 0.76 A 7.3 CPDA 19.1 A 0.82 A 8.5 CV-8JA 15.0 B 0.68 A 4.1 D

u The experiment was conducted three times with each treatmentreplicated six times for area and sporulation and three times fordry wt. Means are averaged over the isolates DCL and DCL-2 sincethere was no isolate effect. All treatment plates had a daily12 hr exposure to fluorescent and incandescent light (5.5 Klux)and 12 hr dark period.

v Media used: LCH = lactose casein hydrolysate medium, PDA = potatodextrose agar and V-8JA = 20% V-8 juice agar. Each petri dishcontained 20 ml of a medium.

w Colony area calculated using mean of 3 random measurements of thecolony diameter.

x

y

Z

Dry weight of mycelia and conidia after steaming in water and drying.

Sporulation means calculated from average conidium counts of 20 fields.

Means followed by the same letter in the columns are not significantlydifferent (P = 0.05) according to the Duncan's New Multiple RangeTest.

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Table 2.2. Effect of temperature and culture medium on colony area, dryweight and sporulation of two isolates of Drechslera catenariaseven days ,after seeding.

u

(oC) 'MediUrilv - -Isolate'tVColony Area -Dry,Weight Sporulation

2Temperature Cem2rx ,- - 'u)'2)x (conidia/mm ) y~g em

15 LCH DCL 12.2 FZ0.93 EFGH 15.7 DEF

DCL-2 10.8 F 0.73 GH 12.6 FPDA DCL 18.1 E 2.37 A 14.7 DEF

DCL-2 15.8 EF 1.28 BCDE 13.9 EFV-8JA DCL 15.4 EF 1.54 BCD 21.2 C

DCL-2 18.1 E 1.23 CDEF 21.4 C

20 LCH DCL 19.3 E 0.76 FGH 33.2 ADCL-2 27.8 D 1.70 BC 26.5 B

PDA DCL 29.2 D 1.71 B .12.8 FDCL-2 29.9 D 1.63 BC 9.5 G

V-8JA DCL 30.9 D 1.62 BC 31.2 ADCL-2 27.4 D 1.15 DEFG 15.7 DEF

25 LCH DCL 43.8 B 0.89 EFGH 17.0 DDCL-2 36.7 C 0.52 H 16.1 DE

PDA DCL 56.7 A 1.73 B 14.2 DEFDCL-2 56.7 A 0.58 H 12.8 F

V-8JA DCL 52.4 A 0.92 EFGH 13.3 EFDCL-2 54.1 A 0.76 FGH 13.8 EF

u The experiment was conducted three times with each treatment replicatedthree times for area and sporulation and once for dry weight. Alltreatment plates had a daily 12 hr exposure to fluorescent andincandescent light (5.5 K lux) and 12 hr dark period.

v Media used: LCH = lactose casein hydrolysate medium, PDA = potatodextrose agar and V-8JA = 20% V-8 juice agar. Each petri dishcontained 20 ml of a medium.

w D. catenaria isolates: DCL = mass isolate, DCL-2 = monoconidialisolate.

x Colony area calculated using mean of 3 random measurements,of colonydiameter. Dry weight of mycelia and conidia after steaming and drying.

y

Z

Sporulation means calculated from average conidium counts of 20 fields.

Means followed by the same letter in columns are not significantlydifferent (P = 0.05) according to the Duncan's New Multiple RangeTest.

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Table 2.3. Effect of temperature and culture-medium on- colony area,dry weight and sporulation of'Drecnslera'catenaria tendays after seeding. u

Area Dry wt. SporulationTemperature COC) Mediumv Ccm2)w CmS/cm2)x -Cconidia/mm2)y

15 LCH 26.1 DZ

1.04 E 25.3 DEPDA 32.0 C 1.88 B 21.8 EV-8JA 29.1 CD 1.51 CD 30.2 D

20 LCH 43.5 B 1.24 DE 51.2 CPDA 56.7 A 2.64 A 22.3 EV-8JA 56.3 A 1.75 BC 19.5 E

25 LCH 56.7 A 1.35 D 70.4 APDA 56.7 A 2.73 A 57.6 BV-8JA 56.7 A 0.95 E 51.1 C

u The experiment was conducted three times with each treatmentreplicated six times for area and sporulation and three times fordry wt. Means are averaged over the isolates DCL and DCL-2 sincethere was no isolate effect. All treatment plates had a daily12 hr exposure to fluorescent and incandescent light C5.5 Klux)and 12 hr dark period.

v Media used: LCH = lactose casein hydrolysate mediurn t PDA = potatodextrose agar and V-8JA = 20% V-8 juice agar. Each petri dishcontained 20 ml of a medium.

w Colony area calculated using mean of 3 random measurements of thecolony diameter.

x Dry weight of mycelia and conidia after steaming in water and drying.

y

Z

Sporulation means calculated from average conidium counts of 20 fields.

Means followed by the same letter in the columns are not significantlydifferent (P = 0.05) according to the Duncan's New Multiple RangeTest.

72

was significantly higher at 25C on LCH (70.4) than on PDA (57.6) at

the same temperature. Sporulation was also very high on V-8JA at

25C (51.1) and on LCH at 20C (51.2).

Colonies "incubated for fourteertdays (Table"2~4). Colony area

had reached maximum dimensions at all temperatures on all culture media

after two weeks, except on LCH and V-8JA at l5C. Dry weight of the

colonies was significantly higher on PDA at 20C (4.84) than on PDA

at 25C (3.65), with both differing significantly from each other and

all other treatments. Sporulation of the fungus was highest (200.1) on

LCH incubated at 20C. This treatment combination was more conducive

to conidial production than any other treatment, with the next best

sporulation on LCH at l5C (131.2).

Since mycelial dry weight and sporulation both increased with

time, greatest sporulation and maximum growth were recorded at 14 days.

When data from each harvest date were compared to one another, dry

weight of the fungus was highest on PDA incubated at 20C after 14 days.

When comparing all harvest dates, sporulation was highest for both

DCL (241.6) and DCL-2 (158.6) on LCH at 20C after 14 days, the values

differing significantly.

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Table 2.4. Rffect of temperature and culture medium on colony area, dryweight and sporulation of Drechslera catenaria fourteen daysafter seeding. u

Area Dry.wt. SporulationTemperature (oC) Mediumv . Ccm2)w· . (mg/ cm2)X (coriidia/mm2) y

15 LCH 52.4 BZ 0.69 F 131.2 BPDA 56.7 A 2.21 C 111.1 CV-8JA 49.9 C 1.02 EF 88.3 D

20 LCH 56.7 A 1.75 CD 200.1 APDA 56.7 A 4.84 A 104.1 CV-8JA 56.7 A 1.43 DE 89.2 D

25 LCH 56.7 A 2.14 C 81.8 DPDA 56.7 A 3.65 B 56.4 EV-8JA 56.7 A 1.12 EF 46.2 E

u

v

w

x

y

Z

The experiment was conducted three times with each treatmentreplica.ted six times for area and sporulation and three times fordry wt. Means are averaged over the isolates DCL and DCL-2 sincethere was no isolate effect. All treatment plates had a daily12 hr exposure to fluorescent and incandescent light (5.5 Klux)and 12 hr dark period.

Media used: LCH = lactose casein hydrolysate medium, PDA = potatodextrose agar and V-8JA = 20% V-8 juice agar. Each petri dishcontained 20 ml of a medium.

Colony area calculated using mean of 3 random measurements of thecolony diameter.

Dry weight of mycelia and conidia after steaming in water and dry~ng.

SP?rulation means calculated from average conidium counts of 20 fields.

Means followed by the same letter in the columns are not significantlydifferent (P = 0.051 according to the Duncan's New Multiple RangeTest.

DISCUSSION

Vegetative growth and production of conidia of both isolates of

Drechslera catenaria were significantly affected by culture medium

and incubation temperature. Generally there was more rapid growth in

area at 25C and on potato-dextrose agar. Increase in mycelial dry

weight was greatest on PDA, but sporulation was generally highest on

lactose casein hydrolysate media. Both isolates of D. catenaria

sporulated by four days and continued to sporulate on all three media

at all three temperatures throughout the study. When the four harvest

times were averaged, isolates differed, with DCL generally having

higher dry weight than the monoconidial isolate (DCL-2).

Sporulation did not increase with increasing growth. Sporulation

was actually higher for those colonies having less growth. Linear

growth of fungi on agar is the least laborious method of estimating

growth, but a more accurate measurement of growth is determination of\

mycelial dry weight (Cochrane, 1958). Dry weight was higher on PDA

and V-8JA than on the LCH medium. Reproduction of fungi is thought to

be favored by depriving an established mycelium of one or more essential

nutrients or essential ones in inadequate concentrations (Cochrane,

1958). That is, fungal reproduction is most likely to occur when

vigorous mycelia exhaust their nutrients or are transferred to a medium

low in nutrients. This at times may be overridden by other factors.

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It is possible that the poor growth of Q~ catertatia, especially on LCH

medium, may be the result of inadequate nutrition causing higher

sporulation in these cultures, than those on the less defined PDA and

V-8JA media.

Most plant pathogens grow best on media at an initial pH of

5.0 to 6.5 (Cochrane, 1958). All three media used in this study were

adjusted to an initial pH of 6. However by the termination of treatments,

the pH of the V-8JA cultures had become quite alkaline (ca 8.0). This

change in pH was not observed in either the LCH or PDA cultures.

Growth and sporulation of Helminthosporium and Bipolaris species have

been shown to be better at neutrality or slightly lower (Tarr and

Kafi, 1968). Conidia produced at extreme hydrogen-ion concentrations

have also been reported to vary in dimensions (Tarr and Kafi, 1968;

Harding, 1975). Growth and sporulation of D. catenaria on V-8JA may

have been affected by this change in pH. Growth, sporulation and

conidial dimensions of D. catenaria should be reinvestigated using

V-8JA with a buffer system.

Light was necessary for sporulation of both isolates of

D. catenaria, regardless of temperature or culture medium. These

results are similar to those found by Leach (1967), Shoemaker (1962)

and Zeiders (1976). The quality of light necessary for sporulation,

however, was not determined. Sporulation of H. catenarium and other

fungi has been shown to be stimulated by near ultraviolet radiation

(Leach, 1967). Leach (1962b) suggested that since fluorescent lamps

emit a small but significant amount of near-UV, much of the literature

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reporting successful reproduction in visible light actually may have

resulted from stimulation by near-UV radiation. Bergquist et al (1972)

showed that incandescent and fluorescent lamps together give almost an

entire spectrum of light except the extremely low ranges of near-UV.

These wavelengths of light are not known to be excluded by any petri

djsh materials (Leach, 1971). Therefore, no wavelength of light could

be excluded from those thought to induce sporulation of D. catenaria.

The growth zones of Q. catenaria that formed on V-8JA could possibly

reflect an inductive reaction that is necessary for conidiogenesis,

as suggested by Leach (1967). Light is usually the primary environmental

factor causing zonation, that is, the formation of fruiting structures

in concentric rings on agar surfaces (Cochrane, 1958). Temperature

fluctuations may also induce those zonations, but in our study

temperature did not fluctuate. Regardless, Leach (1967) found

D. catenaria to be a "Constant Temperature Sporulator" that did not

need fluctuating temperatures for conidial production. The nutritional

and light requirements of the bentgrass isolate of Drechslera catenaria

in culture need to be further examined.

Although sporulation was highest after two weeks, a large number

of these conidia had already germinated or produced secondary

conidiophores from both apical and some intercalary cells. Therefore,

although harvesting from 14-day-old cultures results in higher numbers

of conidia, adequate numbers of conidia can be obtained from cultures

after only ten days. From this study, it appears that large numbers

of D. catenaria conidia can be induced to form on lactose casein

hydrolysate media by incubating cultures at 25C with exposure to

alternating light for ten days.

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