Australasian Plant Pathology, 2001, 30, 1–5

Effect of temperature and pH on the growth and sporulation of Phytophthora clandestina S. SimpfendorferAD, T. J. HardenB and G. M. MurrayC ACharles

Sturt University, School of Science and Technology, Wagga Wagga, NSW 2678, Australia; present address: NSW Agriculture, Tamworth Centre for Crop Improvement, Tamworth, NSW 2340, Australia. BCharles Sturt University, School of Wine and Food Sciences, Wagga Wagga, NSW 2678, Australia. CNSW Agriculture, Wagga Wagga Agricultural Institute, NSW 2650, Australia. DCorresponding author; email: [email protected]

Abstract. The effects of temperature and pH on the growth and sporangial production of isolates from each of the four known races of Phytophthora clandestina Taylor, Pascoe & Greenhalgh were investigated. Mycelial growth occurred at temperatures from 10 to 30°C and pH 3.5 to 9.0 with highest growth rates of all isolates being at 25°C with a pH of 6.0–6.5. Sporangial production was greatest between 20 and 25°C and pH 5.0–7.0 with all races. However, sporulation occurred over a temperature range from 10 to 30°C and from pH 4.0 to 9.0 with all isolates. There were no consistent differences between the four pathogenic races of P. clandestina in their relative growth rate or extent of sporangial production over a range of temperatures and pH values. Introduction Phytophthora clandestina Taylor, Pascoe & Greenhalgh causes an economically important taproot rot of subterranean clover (Trifolium subterraneum L.) in Victoria, Western Australia and New South Wales (Greenhalgh and Taylor 1985; Taylor et al. 1985a; Taylor and Greenhalgh 1987). The disease is associated with serious loss of seedlings in the autumn and early winter and is estimated to reduce annual dry matter production of subterranean clover by 47% on clay-loam soils and by 94% on light, clay-loam soils (Greenhalgh 1992). Taylor et al. (1985b) defined the optimum temperature for the radial growth of P. clandestina on agar as being 25°C, with a minimum to maximum temperature range for growth of 5°C–31°C. However, data currently do not exist for the optimum temperature for the production of sporangia in culture by P. clandestina. The pH of artificial media is also known to influence the growth rate of Phytophthora species. Wong et al. (1986c) determined that the optimum mycelial growth of P. clandestina at 25°C occurs at pH 6.0. They further demonstrated that P. clandestina has a relatively wide pH range (5.0–7.7) for its maximum mycelial growth at 25°C with the extent of growth being reduced by 65% under acid (pH 4) and by 32% under alkaline (pH 9) conditions when compared to growth at pH 6.0. However, both the pH and temperature studies were conducted on a single isolate of P. clandestina and when only one pathogenic race (race 0) of the fungus was known to exist. Currently, at least four pathogenic races of P. clandestina have been identified (Flett 1994). © Australasian Plant Pathology Society 2001

The effect of pH on the formation of sporangia in P. clandestina has not been investigated. However, sporangia of other Phytophthora species can form within a wide pH range from 4.0 to 9.0, although the optimum pH for sporangial formation varies both with the species and among isolates of the same species (Ribeiro 1983). This study defines the optimum temperature and pH for the growth and sporulation of isolates from all four of the currently identified pathogenic races of P. clandestina. Methods Fungal isolates Seven isolates of P. clandestina obtained from Dr Sze Flett, Department of Natural Resources and Environment, Victoria, were used in these studies. The isolates were obtained by standard soil baiting techniques (Eskdale and Murray 1991) from soil collected from New South Wales, Victoria and South Australia (Table 1). Effect of temperature on growth Growth was studied on a medium containing 200 mL L –1 V8 Juice (Campbell Soup Co., Camden, NJ, USA), 20 g L–1 agar and 2 g L–1 CaCO3. The pH of the medium was adjusted with 0.1 M NaOH or 0.1 M HCl to a pH of 6.0. The V8 agar was then autoclaved at 121°C for 15 min before dispensing 20 mL into sterile 90 mm plastic Petri dishes. Growth of seven isolates of P. clandestina (two each of races 0, 1 and 3; one of race 2) was measured at a range of temperatures from 5°C to 35°C at 5°C intervals. A single agar disc (7-mm-diameter) was cut from within the growing margin of 12-day-old cultures of each isolate and inverted centrally on a V8 agar plate. A total of five replicate plates was inoculated for each isolate and temperature combination. The edges of the plates were sealed with a single layer of Parafilm following inoculation and incubated at their respective temperatures in darkness. Mycelial growth was recorded daily by measuring two colony diameters at right angles and subtracting the width of the inoculation

10.1071/AP00054

0815-3191/01/010001

S. Simpfendorfer et al.

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Table 1.

Identification, origin, pathogenic race and culture number of isolates of Phytophthora clandestina

Isolate Pc8 Pc10 Pc12 Pc15 Pc3 Pc22 Pc24

RaceA

VPRI Culture No.B

0 0 1 1 2 3 3

19483 19487 18023 19493 18243 19509 19462

Origin Merrigum, Vic, 1993 Baddaginnie, Vic, 1993 Holbrook, NSW, 1991 Naracoorte, SA, 1992 Rutherglen Vic, 1992 Tongaboo, NSW, 1993 Tongaboo, NSW, 1993

ANumbers

represent the pathogenic race. lodged in the Victorian Plant Research Institute collection, Knoxfield.

BCultures

disc. These measurements were used to calculate radial growth rates (mm day–1) over a linear period of mycelial growth. Effect of temperature on sporangial production Agar discs, 7 mm in diameter, were cut from within the colony margin of 9-day-old cultures of each of seven P. clandestina isolates grown on V8 agar at 25°C. Five mycelial discs of an isolate were placed into 90 mm Petri dishes containing 20 mL of sterile deionised water (SDW) and incubated in the dark at a range of temperatures from 5 to 35°C at 5°C intervals. There were three replicate dishes for each combination of isolate and temperature. Sporangial formation at the mycelial fringe of each disc was observed daily at ×100 magnification using an inverted light microscope and the number of sporangia was recorded in four randomly selected fields of view for each disc after 7 days. Effect of pH on growth V8 agar was made up with the initial pH adjusted with 0.1 M NaOH or 0.1 M HCl to a range between 3.5 and 9.0. The V8 agar was then autoclaved at 121°C for 15 min and the pH checked with Duotest pH indicator paper (Macherey-Nagel, Germany). Autoclaving produced only minor changes (< 0.2 pH unit) in the final pH of the pH 7.5 to pH 9.0 media while media at pH 3.5 to 7.0 were not affected by sterilisation. Agar discs (7-mm-diameter) were cut with a cork borer from within the growing margin of 10-day-old colonies of the respective P. clandestina isolates on V8 agar and inverted centrally to inoculate 20 mL of the pH-adjusted medium in Petri dishes (90 mm diameter). The plates were sealed with Parafilm and incubated in darkness at 25°C. Five replicate plates of each P. clandestina isolate were prepared at each of the separate pH values. The radial growth rates (mm day–1) were determined as described previously. Effect of pH on sporangial production Agar discs (7-mm-diameter) were cut from within the colony margin of 9-day-old cultures of each P. clandestina isolate grown on pH 6.0 V8 agar at 25°C. Five mycelial discs from each isolate were submerged in 90 mm Petri dishes containing 20 mL of SDW adjusted with 0.1M NaOH or 0.1M HCl to provide pH values of 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0. Three replicate plates of each isolate and pH combination were prepared and incubated in the dark at 25°C. Sporangial formation was then determined after 7 days as described previously. Statistical analysis Results were analysed by analysis of variance with Genstat 5.1 (Lawes Agricultural Trust, Rothamsted Experimental Station).

Treatment means and standard errors of the means were calculated and means were compared with a least significant difference test (P < 0.05).

Results Effect of temperature on growth All seven isolates of P. clandestina had maximum mycelial growth rates at 25°C (Table 2). Mycelial growth of all isolates occurred at temperatures ranging from 10 to 30°C with no growth occurring at 5 and 35°C. Variation existed in the growth rates of isolates at the different temperatures tested. Pc12 and Pc22 had the greatest growth rates at 30°C whereas Pc15 and Pc24 were the fastest growing isolates at the lower temperatures of 10 and 15°C. Inherent differences were also evident in the growth rates of the seven isolates at the optimum growth temperature of 25°C. Isolates Pc12 and Pc22 were the fastest; Pc24, Pc15, Pc8 and Pc3 had medium growth rates, and Pc10 was the slowest growing isolate. Effect of temperature on sporangial production Maximum sporangial production by P. clandestina in SDW occurred when incubated at 20°C and 25°C (Table 3). All isolates produced sporangia within the temperature range 10 to 30°C. Only Pc12 had minor sporangial formation at 5°C, and none produced sporangia at 35°C. Considerable variation in the extent of sporangial production existed between the different isolates. Pc12 had the most prolific formation of sporangia at all temperatures except 15°C where the number of sporangia produced by it was not significantly different from those produced by Pc15 and Pc22. The isolates can be ranked in order of their extent of sporangial production at the optimum temperatures of 20°C and 25°C. In descending order of the proliferation of sporangial formation, Pc12 had the highest rate of production (51–56% greater than the next isolate at 20°C and 25°C, respectively); Pc8, Pc10, Pc15 and Pc22 all had a moderate level of sporangial production in SDW. Isolate Pc3 had a moderately low level of sporangial formation, whereas Pc24 had only sparse production of sporangia at all temperatures (Table 3). Effect of pH on growth The initial pH of the V8 agar significantly influenced the growth rate of P. clandestina with optimum mycelial growth of all seven isolates at 25°C occurring between pH 6.0 and 6.5 (Table 4). However, P. clandestina appears to have a relatively wide pH range for rapid growth between 5.5 and 7.0. With the exception of Pc12 at an initial medium pH of 3.5, all isolates grew at all pH values (3.5 to 9.0) examined in this study. However, only sparse mycelial growth occurred at the lowest pH of 3.5. P. clandestina appears to grow relatively better under alkaline than acid conditions at 25°C. Averaged across the seven P. clandestina isolates, mycelial growth was reduced by 80% under acid (pH 4.0) and only 58% under alkaline (pH 9.0) conditions when compared with growth on V8 agar with an initial pH of 6.0.

Growth and sporulation of Phytophthora clandestina

Table 2.

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Effect of temperature on the growth rate of seven isolates of Phytophthora clandestina on V8 agar Growth rate (mm day–1)

Temp. (°C)

Race 0

5 10

Pc8 0.0 0.36 eA

15 20 25 30 35

1.15 e 1.78 e 3.28 c 1.55 c 0.0

AMeans

Race 1

Race 2

Race 3

s.e.

Pc10 0.0

Pc12 0.0

Pc15 0.0

Pc3 0.0

Pc22 0.0

Pc24 0.0

—

0.44 de 1.36 d 2.21 c 2.70 e 1.36 d 0.0

0.46 d 1.49 c 2.00 d 4.55 a 3.40 a 0.0

1.03 b 1.96 b 3.15 a 3.30 c 1.59 c 0.0

0.50 d 1.44 cd 2.50 b 3.05 d 1.68 c 0.0

0.93 c 1.34 d 3.11 a 4.55 a 2.81 b 0.0

1.40 a 2.15 a 2.64 b 3.49 b 1.65 c 0.0

0.03 0.03 0.05 0.05 0.04 —

within rows followed by the same letter are not significantly different (P = 0.05).

Table 3.

Effect of temperature on the number of sporangia produced by seven isolates of Phytophthora clandestina in sterile deionised water

Temp.

No. of sporangia produced

(°C) 5 10 15 20 25 30 35

Race 0 Pc8 0.0 0.5 dA 4.1 b 15.5 b 15.1 c 1.5 c 0.0

Race 1

Race 2

Race 3

s.e.

Pc10 0.0

Pc12 0.0.4

Pc15 0.0

Pc3 0.0

Pc22 0.0

Pc24 0.0

—

0.5 d 2.1 c 12.4 d 13.7 d 0.7 d 0.0

5.3 a 10.2 a 24.1 a 27.2 a 8.0 a 0.0

1.4 c 10.1 a 14.2 c 17.4 b 0.4 d 0.0

0.4 d 1.1 d 7.6 e 9.1 e 0.4 d 0.0

3.2 b 10.2 a 15.9 b 14.5 cd 2.7 b 0.0

0.0 d 0.3 e 1.8 f 2.2 f 0.3 d 0.0

0.18 0.21 0.26 0.32 0.18 —

A Values

represent the mean number of sporangia in four randomly selected fields of view (×100 magnification). Means within rows followed by the same letter are not significantly different (P = 0.05).

Effect of pH on sporangial production Maximum sporangial production by the seven isolates of P. clandestina occurred over the pH range 5.0–7.0, although the optimum pH for sporangial formation varied between isolates (Table 5). All isolates had some sporangial formation under the extreme acid (pH 4.0) and alkaline (pH 9.0) conditions examined in this study. However, all isolates had significantly greater sporangial formation when mycelial agar discs were submerged in SDW within the pH range 5.0–7.0. Discussion This study defined the maximum, minimum and optimum pH and temperature ranges for both growth and sporangial production of P. clandestina under culture conditions. Such basic research on the biology of isolates from each of the four currently recognised pathogenic races of P. clandestina has not been previously reported. Optimum growth of P. clandestina was found to occur at 25°C whereas an incubation temperature between 20 and 25°C provided

optimum sporangial formation. Maximum mycelial growth and sporangial formation of all four pathogenic races of P. clandestina occurred over a fairly wide pH range from 5.5 to 7.0 for growth and from 5.0 to 7.0 for sporulation. This study contradicts earlier work by Purwantara et al. (1997), who found 20°C to be optimum for the mycelial growth of a single isolate from each of three pathogenic races (0, 1 and 2) of P. clandestina on lima-bean agar. No consistent differences were found between the different pathogenic races of P. clandestina in their relative growth or extent of sporangial production over a range of temperatures and pH values. There was also no trend in the geographical location from which P. clandestina cultures were originally isolated and their relative tolerance to either low or high temperatures for mycelial growth. Even isolates of P. clandestina from the same property had varying growth rates at the different temperatures. Pc22 and Pc24, which were both isolated from the same farm at Tongaboo, NSW in 1993 and are both race 3 cultures, had different growth curves over the various temperatures. Pc24 grew significantly better at the

S. Simpfendorfer et al.

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Table 4. Effect of pH on the growth rate of seven isolates of Phytophthora clandestina on V8 agar at 25°C Growth rate (mm day–1)

pH Race 0 3.5

Pc8 0.11 lA

4.0 4.5 5.0 5.25 5.5 5.75 6.0 6.25 6.5 6.75 7.0 7.5 8.0 8.5 9.0

0.81 k 2.03 i 2.99 f 3.23 de 3.26 cd 3.33 bcd 3.41 ab 3.46 a 3.40 ab 3.35 abc 3.11 e 2.99 f 2.73 g 2.25 h 1.30 j

Race 1

Race 2

Pc10

Pc12

Pc15

0.05 h 0.68 g 1.69 e 2.36 c 2.63 b 2.71 ab 2.76 a 2.78 a 2.76 a 2.81 a 2.78 a 2.64 a 2.41 c 2.23 d 1.80 e 1.41 f

0.00 k 0.45 j 2.20 i 3.40 e 3.99 c 4.13 b 4.40 a 4.45 a 4.43 a 4.34 a 3.96 c 3.80 d 3.41 e 3.04 f 2.71 g 2.33 h

0.05 m 0.23 l 0.85 k 1.55 i 2.15 g 3.24 b 3.30 ab 3.36 a 3.40 a 3.30 ab 3.10 c 2.85 d 2.58 e 2.29 f 1.91 h 1.38 j

Pc3 0.09 j 0.88 i 1.46 g 2.19 e 2.79 c 2.98 ab 3.03 a 3.06 a 3.01 ab 3.00 ab 2.90 bc 2.55 d 2.13 e 1.76 f 1.53 g 1.16 h

Race 3 Pc22

Pc24

0.21 k 1.24 j 2.31 h 4.06 e 4.39 c 4.46 bc 4.54 ab 4.59 a 4.56 ab 4.55 ab 4.48 abc 4.38 cd 4.26 d 3.50 f 2.85 g 1.79 i

0.05 j 0.80 i 1.93 g 2.78 e 3.25 cd 3.35 bc 3.55 a 3.56 a 3.63 a 3.55 a 3.43 b 3.28 cd 3.18 d 2.69 e 2.13 f 1.19 h

AMeans

within columns followed by the same letter are not significantly different (P = 0.05, s.e. = 0.039, lsd0.05 = 0.109).

Table 5.

Effect of pH on the formation of sporangia by seven isolates of Phytophthora clandestina in sterile deionised water

pH

Mean No. of sporangia Race 0 Pc8

4.0 5.0 6.0 7.0 8.0 9.0

4.2 eA 13.1 c 15.8 a 14.1 b 6.0 d 1.4 f

Race 1

Race 2

Race 3

Pc10

Pc12

Pc15

Pc3

Pc22

Pc24

3.8 c 11.5 b 12.6 a 13.0 a 4.5 c 1.2 d

9.7 e 28.4 a 26.1 b 24.2 c 16.0 d 4.6 f

4.7 e 14.3 c 17.1 a 15.9 b 9.9 d 2.9 f

2.3 e 10.4 a 9.1 b 8.1 c 4.4 d 1.3 f

6.6 d 12.1 c 14.5 b 16.4 a 12.2 c 3.4 e

0.4 d 3.0 a 2.2 b 2.2 b 1.3 c 0.3 d

AValues represent the mean number of sporangia in four randomly selected fields of view (×100 magnification). Means within columns followed by the same letter are not significantly different (P = 0.05, s.e. = 0.265, lsd = 0.736).

lower temperatures of 10°C and 15°C while Pc22 was faster growing at the higher temperatures of 20°C, 25°C and 30°C (Table 2). Phytophthora species produce sporangia during asexual reproduction and under adverse growth conditions (e.g. submersion of mycelia in SDW) as a means of dispersal (Taylor et al. 1985b). The induction of sporangial formation under controlled laboratory conditions is of practical importance, since large numbers of zoospores are often required both by plant pathologists as inoculum for infectivity

studies and by mycologists to enable detailed studies of these structures. Zoospores are readily released through the germination of sporangia following chilling in SDW at 4°C (Taylor et al. 1985b). The maximisation of sporangial production through the incubation of cultures at the optimal temperature (20–25°C) and pH (5.0–7.0) ranges therefore increases the concentration of zoospores following the germination of sporangia. Temperature and pH are also known to affect the survival of fungal pathogens in soil. Temperature affects the growth

Growth and sporulation of Phytophthora clandestina

rate of mycelium and the extent of sporangial formation in other Phytophthora species and thereby influences, at least to some extent, the rate of disease development (Duniway 1983). Although temperature effects on growth and sporulation cannot by themselves provide an accurate forecast of disease development, the temperature in a given climate must permit some growth of the fungal pathogen if the disease is to develop. Furthermore, the effects of temperature on root diseases incited by soilborne Phytophthora spp. sometimes parallel closely the effects of temperature on the growth rate of mycelium, even though other aspects of their disease cycles may be influenced somewhat differently by temperature (Duniway 1983). Wong et al. (1986d) demonstrated that the survival of P. clandestina in soil is affected by temperature and moisture. The conditions of high soil moisture at elevated temperatures of up to 25°C which favoured the survival of P. clandestina were similar to those which promote the parasitic growth of this fungus. The temperature requirements of all four pathogenic races of P. clandestina (range 10–30°C; optimum 25°C) as defined in this study agree with field observations in Western Australia where P. clandestina is readily detected after early rains in autumn (April–May) when soil temperatures range between 12 and 24°C (Wong et al. 1985). Propagules of P. clandestina survive in residues of subterranean clover (Wong et al. 1986b) and become active with the opening rains in autumn while the soil is still warm. Root disease then declines rapidly with the onset of winter conditions (Wong et al. 1986a). Thus, optimum growth and sporangial production in P. clandestina at 20−25°C are consistent with this seedling infection process. Acknowledgements An Australian Postgraduate Research Award provided funding for this project. References Duniway JM (1983) Role of physical factors in the development of Phytophthora diseases. In ‘Phytophthora: its biology, taxonomy, ecology and pathology’. (Eds DC Erwin, S Bartnicki-Garcia and PH Tsao) pp. 175–187. (American Phytopathological Society: St Paul, MN) Eskdale JW, Murray GM (1991) Baiting to detect root rot pathogens of subterranean clover. In Australasian Plant Pathology Society 8th

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Conference, 7–11 October 1991, Sydney University, Program and Abstracts, pp. 14. Australasian Plant Pathology Society: Sydney. Flett SP (1994) Studies on Phytophthora clandestina, the cause of taproot rot in subterranean clover. 1. Evidence for physiological specialisation in Phytophthora clandestina. Australian Journal of Experimental Agriculture 34, 1125–1129. Greenhalgh FC (1992) Studies on the etiology and significance of root diseases of subterranean clover, and the histopathology and control of Phytophthora root rot. PhD Thesis, La Trobe University, Victoria. Greenhalgh FC, Taylor PA (1985) Phytophthora clandestina, cause of severe taproot rot of subterranean clover in Victoria, Australia. Plant Disease 69, 1002–1004. Purwantara A, Flett SP, Keane PJ (1997) The effect of temperature on the growth of Phytophthora clandestina and infection of subterranean clover cultivars. Australasian Plant Pathology 26, 109–114. Ribeiro OK (1983) Physiology of asexual sporulation and spore germination in Phytophthora. In ‘Phytophthora: its biology, taxonomy, ecology and pathology’. (Eds DC Erwin, S BartnickiGarcia and PH Tsao) pp. 55–70. (American Phytopathological Society: St Paul, MN) Taylor PA, Greenhalgh FC (1987) Significance, causes and control of root rots of subterranean clover. In ‘Temperate pastures: their production, use and management’. (Eds JL Wheeler, CJ Pearson and GE Robards) pp. 249–251. Australian Wool Corporation/ Commonwealth Scientific and Industrial Research Organisation: Melbourne. Taylor PA, Barbetti MJ, Wong DH (1985a) Occurrence of Phytophthora clandestina in Western Australia. Plant Protection Quarterly 1, 57–58. Taylor PA, Pascoe IG, Greenhalgh FC (1985b) Phytophthora clandestina sp. nov. in roots of subterranean clover. Mycotaxon 22, 77–85. Wong DH, Barbetti MJ, Sivasithamparam K (1985) Fungi associated with root rot of subterranean clover in Western Australia. Australian Journal of Experimental Agriculture 25, 574–579. Wong DH, Barbetti MJ, Sivasithamparam K (1986a) Behaviour of Phytophthora clandestina propagules at a field site in Western Australia. Australian Journal of Soil Research 24, 485–491. Wong DH, Sivasithamparam K, Barbetti MJ (1986b) Soil behaviour of Phytophthora clandestina. Phytopathology 116, 67–73. Wong DH, Sivasithamparam K, Barbetti MJ (1986c) The influence of environmental factors on the growth and survival of Phytophthora clandestina. Canadian Journal of Microbiology 32, 553–556. Wong DH, Sivasithamparam K, Barbetti MJ (1986d) Influence of soil temperature, moisture and other fungal root pathogens on the pathogenicity of Phytophthora clandestina to subterranean clover. Transactions of the British Mycological Society 86, 479–482.

Received 17 March 2000, accepted 13 September 2000

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