CHAPTER II THE EFFECT OF TEMPERATURE AND CULTURE MEDIUM ON THE GROWTH AND SPORULATION OF DRECHSLERA CATENARIA

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1 CHAPTER II THE EFFECT OF TEMPERATURE AND CULTURE MEDIUM ON THE GROWTH AND SPORULATION OF DRECHSLERA CATENARIA 61 \

2 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 sucroseasparagine agar (Braverman and Graham, 1960). 62

3 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.

4 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 64

5 were unfilteredg.e. 40-W incandescent lamps and G.E. 40-W cool-white 65 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 dislodge 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

6 for 24 hr. Final ph was determined for the agar-distilled water 66 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 (cm 2 ), mycelial dry weight (mg/cm 2 ) and sporulation (conidia/mrn 2 ) 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.

7 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 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 67

8 68 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/cm 2 ) among treatments. Sporulation (conidia/rnm 2 ) 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

9 69 Table 2.1. Effect of temperature and culture medium on colony area, dry weight and sporulation of'drechsleracatenaria four days after seeding. u Area Dry w~ Sporulation 2 Temperature (oc) MediUril v,,, '(cm2) w (JUg/em )x (conidia/nun ) y 15 LCH 5.5 E Z 0.93 A 12.5 B PDA 7.8 D 0.88 A 6.9 C V-8JA 6.8 DE 0.92 A 7.6 C 20 LCH 3.6 F 0.96 A 15.5 A PDA 7.5 D 0.71 A 15.7 A V-8JA 8.6 D 0.90 A 7.5 C 25 LCH 13.1 C 0.76 A 7.3 C PDA 19.1 A 0.82 A 8.5 C V-8JA 15.0 B 0.68 A 4.1 D u v w x y The experiment was conducted three times with each treatment replicated six times for area and sporulation and three times for dry wt. Means are averaged over the isolates DCL and DCL-2 since there was no isolate effect. All treatment plates had a daily 12 hr exposure to fluorescent and incandescent light (5.5 Klux) and 12 hr dark period. Media used: dextrose agar and V-8JA = 20% V-8 juice agar. Each petri dish contained 20 ml of a medium. LCH = lactose casein hydrolysate medium, PDA = potato Colony area calculated using mean of 3 random measurements of the colony diameter. Dry weight of mycelia and conidia after steaming in water and drying. Sporulation means calculated from average conidium counts of 20 fields. Z Means followed by the same letter in the columns are not significantly different (P = 0.05) according to the Duncan's New Multiple Range Test.

10 Table 2.2. Effect of temperature and culture medium on colony area, dry weight and sporulation of two isolates of Drechslera catenaria seven days,after seeding. u 70 (oc) 'MediUril v - -Isolate'tV Colony Area -Dry,Weight Sporulation 2 Temperature Cem2rx,- -'u)'2)x (conidia/mm ) y ~g em 15 LCH DCL 12.2 F Z 0.93 EFGH 15.7 DEF DCL F 0.73 GH 12.6 F PDA DCL 18.1 E 2.37 A 14.7 DEF DCL EF 1.28 BCDE 13.9 EF V-8JA DCL 15.4 EF 1.54 BCD 21.2 C DCL E 1.23 CDEF 21.4 C 20 LCH DCL 19.3 E 0.76 FGH 33.2 A DCL D 1.70 BC 26.5 B PDA DCL 29.2 D 1.71 B.12.8 F DCL D 1.63 BC 9.5 G V-8JA DCL 30.9 D 1.62 BC 31.2 A DCL D 1.15 DEFG 15.7 DEF 25 LCH DCL 43.8 B 0.89 EFGH 17.0 D DCL C 0.52 H 16.1 DE PDA DCL 56.7 A 1.73 B 14.2 DEF DCL A 0.58 H 12.8 F V-8JA DCL 52.4 A 0.92 EFGH 13.3 EF DCL A 0.76 FGH 13.8 EF u v The experiment was conducted three times with each treatment replicated three times for area and sporulation and once for dry weight. All treatment plates had a daily 12 hr exposure to fluorescent and incandescent light (5.5 K lux) and 12 hr dark period. Media used: dextrose agar and V-8JA = 20% V-8 juice agar. contained 20 ml of a medium. LCH = lactose casein hydrolysate medium, PDA = potato Each petri dish w D. catenaria isolates: DCL = mass isolate, DCL-2 = monoconidial isolate. x y Colony area calculated using mean of 3 random measurements,of colony diameter. Dry weight of mycelia and conidia after steaming and drying. Sporulation means calculated from average conidium counts of 20 fields. Z Means followed by the same letter in columns are not significantly different (P = 0.05) according to the Duncan's New Multiple Range Test.

11 71 Table 2.3. Effect of temperature and culture-medium on- colony area, dry weight and sporulation of'drecnslera'catenaria ten days after seeding. u Area Dry wt. Sporulation Temperature COC) Medium v Ccm2)w CmS/cm 2 )x -Cconidia/mm 2 )y 15 LCH 26.1 D Z 1.04 E 25.3 DE PDA 32.0 C 1.88 B 21.8 E V-8JA 29.1 CD 1.51 CD 30.2 D 20 LCH 43.5 B 1.24 DE 51.2 C PDA 56.7 A 2.64 A 22.3 E V-8JA 56.3 A 1.75 BC 19.5 E 25 LCH 56.7 A 1.35 D 70.4 A PDA 56.7 A 2.73 A 57.6 B V-8JA 56.7 A 0.95 E 51.1 C u v w x y The experiment was conducted three times with each treatment replicated six times for area and sporulation and three times for dry wt. Means are averaged over the isolates DCL and DCL-2 since there was no isolate effect. All treatment plates had a daily 12 hr exposure to fluorescent and incandescent light C5.5 Klux) and 12 hr dark period. Media used: LCH = lactose casein hydrolysate mediurn t PDA = potato dextrose agar and V-8JA = 20% V-8 juice agar. contained 20 ml of a medium. Each petri dish Colony area calculated using mean of 3 random measurements of the colony diameter. Dry weight of mycelia and conidia after steaming in water and drying. Sporulation means calculated from average conidium counts of 20 fields. Z Means followed by the same letter in the columns are not significantly different (P = 0.05) according to the Duncan's New Multiple Range Test.

12 was significantly higher at 25C on LCH (70.4) than on PDA (57.6) at 72 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. LCH incubated at 20C. Sporulation of the fungus was highest (200.1) on 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.

13 73 Table 2.4. Rffect of temperature and culture medium on colony area, dry weight and sporulation of Drechslera catenaria fourteen days after seeding.u Area Dry.wt. Sporulation Temperature (oc) Medium v. Ccm2)w. (mg/ cm2)x (coriidia/mm2) y 15 LCH 52.4 B Z 0.69 F B PDA 56.7 A 2.21 C C V-8JA 49.9 C 1.02 EF 88.3 D 20 LCH 56.7 A 1.75 CD A PDA 56.7 A 4.84 A C V-8JA 56.7 A 1.43 DE 89.2 D 25 LCH 56.7 A 2.14 C 81.8 D PDA 56.7 A 3.65 B 56.4 E V-8JA 56.7 A 1.12 EF 46.2 E u v w x y The experiment was conducted three times with each treatment replica.ted six times for area and sporulation and three times for dry wt. Means are averaged over the isolates DCL and DCL-2 since there was no isolate effect. All treatment plates had a daily 12 hr exposure to fluorescent and incandescent light (5.5 Klux) and 12 hr dark period. Media used: dextrose agar and V-8JA = 20% V-8 juice agar. contained 20 ml of a medium. LCH = lactose casein hydrolysate medium, PDA = potato Each petri dish Colony area calculated using mean of 3 random measurements of the colony 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. Z Means followed by the same letter in the columns are not significantly different (P = according to the Duncan's New Multiple Range Test.

14 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. was actually higher for those colonies having less growth. Sporulation 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. 74

15 It is possible that the poor growth of Q~ 75 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

16 76 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

17 of D. catenaria conidia can be induced to form on lactose casein 77 hydrolysate media by incubating cultures at 25C with exposure to alternating light for ten days.