Ann. appl. Biol. (1990), 117, 537-551 Printed in Great Britain 537

Phytuphthura cryptogea root rot of tomato in rockwool nutrient culture. 11. Effect of root zone temperature on infection,

sporulation and symptom development

By R. KENNEDY and G. F. PEGG Department of Horticulture, University of Reading,

Earley Gate, Reading RG6 2AU, UK

(Accepted 21 August 1990)

Summary The effect of root-zone temperature on Phytophthora cryptogea root rot was

studied in tomato cv. Counter grown under winter and summer conditions in rockwool culture. A nutrient temperature of 25 "C resulted in increased root initiation and growth, higher in winter-grown than in summer-grown plants. Rhizosphere zoospore populations were greatly reduced at 25 "C and above.

Growth of P. cryptogea in vitro was optimal between 20 "C and 25 "C and completely suppressed at 30 "C. Encystment was enhanced by increased temperatures above 20 "C. Zoospore release in vitro occurred in cultures maintained at constant temperatures in the absence of the normal chilling stimulus. Optimal release was at 10 "C; no zoospores were released at 30 "C.

Inoculated, winter-grown tomato plants maintained at 15 "C developed acute aerial symptoms and died after 21 days. Comparable plants grown at a root-zone temperature of 25°C remained symptomless for the 3-months duration of the experiment. Summer-grown infected plants at the higher root temperature wilted but did not die. Enhanced temperature was ineffective as a curative treatment in summer-grown plants with established infection.

Aerial symptoms of Phytophthora infection are seen as a function of the net amount of available healthy root. With high root zone temperatures this is determined by new root production and decreased inoculum and infection.

Key words : Phytophthora cryptogea, root rot, root zone temperature, control

Introduction Important root rot pathogens of tomato include the oomycetes Phytophthora cryptogea

Pethybr. & Lafferty, P. nicotianae B. de Haan var. parasitica (Dastur) Waterhouse and several Pythium spp. (Evans, 1979). Of these P . cryptogea is the most serious in commercial tomato crops. The presence of an infective motile zoospore stage makes these pathogens potentially the most damaging in soilless tomato culture. The importance of the motile stage in disease epidemics has long been recognised (Weste, Ruppin & Vithanage, 1976). Infection by motile and non-motile stages of the organism are important in plants with little or no propensity for new root initiation and regrowth (Pegg, 1986). Rockwool nutrient culture (RNC) has rapidly superceded other traditional alternatives including nutrient film technique (NFT) (Pegg & Jordan, 1990). Unlike NFT, there is no recycling of the nutrient and the risk of infection has been considered to be minimal. Severe outbreaks of Phytophthora cryptogea infection, however, have occurred in commercial tomato crops but the source of infection has not been established. The distribution of infection is often irregular, suggesting that plants or roots

0 1990 Association of Applied Biologists

538 R. KENNEDY AND G. F. PEGG

growing under sub-optimal conditions may be pre-disposed to infection. Factors affecting the establishment and spread of root pathogens in RNC have not been previously recorded.

Beneficial effects on yield of controlling tomato root-zone temperatures in NFT-grown tomatoes have been demonstrated by Hurd & Graves (1985) and Moss (1983). Increasing root temperatures led to an increase in final yield of 10%. Optimum temperatures for root growth usually in the range of 20 - 25 "C tend to be lower than for shoot growth (Marchener, 1986). Little has been published on the temperature relations of P . cryptogea, but the maxima for growth and sporulation in vitro are lower than for many other spp. (Waterhouse, 1963; Weste, 1983). Since the rockwool nutrient solution temperature can be regulated precisely, the possibility of exploiting differences in thermal tolerance between host and pathogen offers a potential non-chemical method of control. A previous paper has described the methods of analysis of tomato roots and infection in rockwool fibre (Pegg & Jordan, 1990). The present work describes the effect of temperature on the growth and sporulation of P . cryptogea and on the development of root rot and aerial symptoms in infected tomato plants in rockwool culture.

Materials and Methods

Growth of mycelium in vitro Cultures of P . cryptogea isolates BR 7 and Holt No 2 (MAFF, Coley Park, Reading) were

maintained on PDA at 20 "C in the dark. Discs of mycelium were taken from the perimeter of 21 days-old plate cultures using a 1 cm diameter cork borer. Erlenmeyer flasks containing 30 ml of sterilised V8 medium (1.25 mg CaCO,, 12.5 ml V8 juice, 112.5 ml glass distilled water) to which had been added 10 mg L p-sitosterol (Englander & Roth, 1980) were each inoculated with an agar mycelial disc. Flasks were incubated at 10, 15,20,25 and 30 "C (with eight-fold replication) and harvested after 4 days by filtering through Whatman No. 5 filter paper. Samples were dried at 100 "C for 24 h before weighing.

Zoosporangial production in vitro Cultures of P . cryptogea produced in V8 medium in 9 cm diameter plastic Petri dishes as

previously described were incubated at 15, 20, 25 and 30°C. For each temperature five replicate Petri dish cultures were counted at each of four sampling times (days) during the growth of the culture. Zoosporangial numbers were counted under an eye-piece grid from five random fields of view in aliquots from each plate at each temperature.

Production of zoospore inoculurn Cultures of P . cryptogea were maintained on PDA at 20°C in the dark. Zoospore

suspensions were prepared by transferring 1 cm discs of 14 - 21 days-old mycelium to flasks containing filtered V8 medium and incubating for 14 days in darkness. The resulting mycelial mat was washed x 4 with sterile distilled water (SDW) and chilled for 20 min at 5 "C prior to incubation at 20 "C for 2 h when zoospores were released. Zoospore concentrations were determined by haemocytometer counts from 1 ml aliquots of zoospore suspension, fixed and stained with 0.1 ml lactophenol-cotton blue. Zoospore concentrations were confirmed by serial dilutions of the original suspension in SDW plated on V8 juice agar.

Zoospore release and encystment in vitro Zoosporangial cultures produced as above were washed and chilled at 5 "C for 20 min to

induce zoospore release. Six replicate cultures were incubated in SDW at 15,20,25 and 30 "C. Zoospores released (encysted and non-encysted) were counted over 5 h in 10 random microscope fields using an eye-piece grid. Other zoosporangial cultures not subjected to a

EfSect of root temperature on Phytophthora cryptogea 539

chilling period were washed with SDW at 20°C and placed at 15, 20, 25 and 30°C immediately after washing. Zoospore release was measured by counting encysted and non- encysted zoospores in 10 random fields of view under an eye-piece grid at regular time periods during a 7 h incubation. The rate of zoospore encystment was counted in suspensions of 10 zoospores ml-1 SDW in 5 cm diameter Petri plates incubated for 7 h after release at temperatures between 5 "C and 30 "C.

Growth of tomato plants Tomato (Lycopersicon esculentum Mill.) cv. Counter, grown under low-light winter

conditions were placed in 10 x 10 x 6.5 cm Grodan growing blocks, watered with a nutrient solution (Anon., 1981) and maintained at pH 6 with a conductivity of 2.5 mS cm3. After the emergence of the first truss, plants were placed on 30 x 15 x 7.5 cm polyethylene-wrapped rockwool slabs (one plant per slab). In another experiment plants of the same cultivar were maintained entirely in 10 x 10 x 6.5 cm growing blocks during the winter growth season. Vegetative growth was restricted under low light conditions by increasing the conductivity of the nutrient solution to 6 mS cm3. Plants were grown in alternate rows in the same glasshouse, each row containing infected and non-infected plants. Conductivity and pH were monitored daily and maintained at 6.0 mS cm3 and 6.0 - 6.5 respectively.

Plants grown under high-light summer conditions were grown solely in growing blocks. Seedlings were grown at a conductivity of 2.5 mS cm3 and a pH of 6 . Conductivity was raised after transfer to the block, to 5.0 mS cm3 until the emergence of the third fruiting truss and thereafter maintained at 2.5 mS.

The growing and experimental protocol for summer and winter-grown tomatoes was as follows:

Procedure

Sowing in propagation block Transfer to growing block Transfer to growing slab Conductivity increased to

5 mS cm3 - 6 mS cm3 Conductivity reduced to

3 mS cm3 Plants inoculated

Controlled root-zone temperatures applied

Date of first fruit pick

Date r A

J

Winter Summer 9 Nov. 1987 13 Dec. 1987 18 Jan. 1988 -

30 Apr. 1988 18 May 1988

16 Dec. 1987 25 May 1988

30 Mar. 1988 25 Jun. 1988 25 Jul. 1988 16 Feb. 1988 &

1 Jun. 1988 18 Feb. 1988 &

16 Jun. 1988 3 Mar. 1988

27 Jul. 1988 1 Aug. 1988

Plant inoculation Winter-grown plants were inoculated at the 9th truss stage by applying 10 ml of a 1000

zoospores ml-I suspension uniformly to the surface of the growing block of each plant. Winter-grown plants of cv. Counter maintained entirely in rockwool blocks were inoculated at the 21st truss stage with 10 ml of a 5000 ml-' zoospore suspension as before. Plants sown in April and harvested in August (summer-grown) were inoculated at the 9th truss stage with 5 x lo-' zoospores in 10 ml. Higher or lower than ambient root-zone temperatures were applied 72 h after inoculation to winter and summer-grown plants. Root temperatures were applied to the winter-grown plants maintained in blocks 15 days after inoculation.

540 R. KENNEDY AND G. F. PEGG

Root-zone temperature Root-zone temperatures were controlled by heating the nutrient solution in a 100 litre plastic

tank with a coil through which hot water was circulated from a water bath heated by a Circotherm immersion heater. The water bath required a temperature setting of 30 "C to maintain the nutrient solution at 25°C. Root zone temperatures below ambient were maintained by chilling the nutrient solution using a chiller unit (GRANT CCl90). Heated or chilled nutrient solution was delivered to the plant in 1-5 cm diameter black alkathene pipes sheathed with 3 cm diameter polyurethane foam insulation. The final delivery of nutrients to the growing block was through non-insulated 5 mm diameter black polyethylene piping. Blocks and slabs were sited on 30 cm thick polystyrene sheets on a concrete floor and further insulated with 6 cm of glass fibre wrapping. Solution temperatures in the range 14 - 26 "C were maintained at k 2°C at the block surface. Excess nutrients were allowed to run to waste.

Glasshouse air temperature was maintained at 18 - 20 "C during the day and 14 - 16 "C at night for winter-grown crops. Plants grown during summer conditions were maintained at 20 - 25 "C during the day with night temperatures of 18 - 20 "C. Glasshouse heating, ventilator control and nutrient supply was controlled from sensors linked to a computerised control programme. Ambient air and root temperatures were monitored and recorded continuously.

Assessment of infection

Zoospore populations At 1 - 7 day intervals after inoculation, nutrient samples were removed in a 10 ml sterile

hypodermic syringe from the root-zones (growing blocks and/or slabs) of infected and non- infected plants. One ml aliquots of solution were plated on V8 medium selective agar containing a 10% mixture of benomyl, nystatin, PCNB, rifampicin and ampicillin (BNPRA) (Masago, Yoshikawa, Fukada & Nakanishi, 1977) and spread evenly over the surface of a 9 cm plate with an "L"-shaped sterile glass spreader. Colonies of P . cryptogea were counted from four replicate plates after 36 h incubation at 20 "C.

Root growth and infection At various intervals after inoculation a 3 cm diameter x 10 cm cylindrical core of mineral

wool and roots was removed from each of four replicate plant substrates at a point 4 cm below the surface of the growing block (in the median line). Cores were also removed from slabs 4 cm below the base of the growing block in the same line. Cores from each treatment were placed in 1.0 M H, PO4 for 45 min in a fume chamber (Pegg 8z Jordan, 1990) to dissolve the rockwool and binder and release intact roots. Dry weight of roots in each core were measured after washing and drying at 100 "C for 48 h. Other cores of the same dimensions were removed, dissected and the number of healthy and infected roots counted. Individual roots were recorded as infected if lesions caused by P . cryptogea were present on any part of a root in each sample. The presence and identity of the pathogen was confirmed by removing 1.0 g samples of root from each core and comminuting at 360 rpm in 10 ml of distilled water for 30 sec in a 25 ml chamber of a Silverson blender. The comminuted sample was diluted by 100 ml SDW, 1 ml of which was spread on a 9 cm BNPRA agar plate. Colony forming units (CFU) were expressed as means of counts of four root replicates per treatment made after 48 h incubation at 20°C in darkness. Dry weights of four replicates of infected roots from 10 cm x 3 cm diameter cores from growing blocks were made at 2 wk intervals from summer grown plants. The presence of P . crypfogea in 1.Og root samples was confirmed by root comminution as described before.

EfSect of root temperature on Phytophthora cryptogea 1

0 5 10 15 20 25 30

Temperature "C Fig. I . The effect of temperature on inycelial dry weight of P. cryprogru isolates -0--. isolate BR7 zoosporangial-producing, -.- Holt No . 2 . non-/oosporangial producing. Each point represents the means of eight replicate cultures on V8 medium at different temperatures. Vertical bars are the 95u'o confidence limits of the means.

t

T

541

Days

Fig. 2. The effect of temperature on zoosporangial production in virro 4-, 30°C; -a-, 25°C; - -. 20°C; - 0 -, 15°C. Figures are the means of five replicate cultures per temperature and fibe random microscope fields per replicate. Vertical bars are the 95% confidence limits of the means.

To assess the effect of zoosporangia or zoospores on CFU obtained from comminuates of root lesions, comparisons were made of CFU numbers obtained from homogenates of mycelia from zoosporangia-producing (BR7), and non-zoosporangial producing (Holt No 2), isolates of P. cryptogea. These had identical mycelial growth rates (Fig. 1). Ten replicate 1 cm

542 R . KENNEDY AND G. F. PEGG

mycelial discs, each from 6 and 14 days-old cultures of each isolate, grown on V8 medium at 20 "C were comminuted in 10 ml SDW for 30 s in a Silverson blender at 360 rpm. One ml aliquots from a one hundred-fold dilution were plated on BNPRA agar, and incubated at 20 "C. Identical numbers of CFU were obtained from each isolate for each culture age. It was concluded that colonies obtained from root homogenates represented mycelial colonisation with no significant contribution from sporangia or zoospores.

Statistical treatment of results All data expressed as percentages were analysed by analysis of variance following arcsin

angular transformation. Comparison between treatment means was made by a fixed range test citing the least significant difference (P = 0.05). The percentage values shown in Tables and Figures are actual percentage data.

Results

Eflect of temperature on growth and development of P. cryptogea in vitro Mycelial dry weights of P . cryptogea grown on aqueous (V8 medium) over the temperature

range 10 to 30 "C are shown in Fig. 1. Growth was optimal at 20 - 25 "C, falling rapidly at 30 "C. Maximal production of zoosporangia occurred at 20 "C as a peak at 8 days. At 15 "C a smaller but delayed peak occurred 4 days later. Few zoosporangia were formed at 25 "C and none at 30 "C (Fig. 2) .

Zoospore release in Phytophthora is normally associated in vitro with a chill shock and alternating temperatures. When sporangia were maintained at constant temperature,

t

" 1 2 3 4 5

Time (h)

Fig. 3. P. cryprogea zoospore release at different temperatures following a 5°C chill stimulus for 20 min. -0-, 30°C; -0-, 25°C; - -, 20°C; - 0 -, 15°C. Figures are the means of encysted and motile zoospores from six replicate cultures and ten random microscope fields per replicate. Vertical bars are the 95% confidence limits of the mean5.

Eflect of root temperature on Phytophthora cryptogea 543

0 I 2 3 4 5 6 7 8

Time (h)

Fig. 4. P . cryprogea zoospore release in cultures maintained at constant temperature in the absence of a chill stimulus -A-, 30°C. -0-, 25°C; -*-, 20°C; - 0--, 15°C; - -, 10°C. Figures are the means of encysted and motile zoospores from 6 replicate cultures and 10 random microscope fieldr per replicate. Vertical bars are the 95% confidence limits of' the means.

lullUwlllg a ,, L L11111 SL1111U1US 1U1 L W 111111, l l laXll l lal IIU111UL:IS UI IIIULIIL: allu IIUII-IIIULllL:

zoospores were produced at 15 "C and 20 "C. The release curve over 5 h was similar at each temperature. At 5 h there was a sharp decline in released zoospores between 15 "C and 30 "C (Fig. 3). When the experiment was repeated without a chill shock introduced before the imposition of the temperature regime, a similar pattern of release was obtained (Fig. 4) for 15, 20 and 25 "C. At 30 "C, however, zoosporangial release was completely suppressed. Maximum zoospore numbers were obtained at 10 "C after 2 h incubation.

The effect of temperature on encystment (Fig. 5) showed that between 5 "C and 20 "C, 70% to 90% of zoospores remained motile after 7 h. At 25 "C the rate of encystment was linear and by 6 h all motility had ceased. This was reduced to 3 h in cultures maintained at 30 "C.

Rhizosphere zoospore levels Zoospore levels in rhizosphere nutrient solutions obtained from growing blocks and slabs

are shown in Table 1.4 and B and in Fig. 6. Plants established during winter months had the 15 "C and 25 "C temperature regime applied, 72 h (Table 1A) and 15 days, (Fig. 6 ) after inoculation. Temperatures of 14, 18, 22 and 26 "C were established for summer-grown plants 72 h after inoculation (Table 1B).

In all cases, rhizosphere zoospore numbers (either encysted or non-motile) declined with increasing temperature. Numbers of zoospores were higher in summer than winter-grown root solutions. There was evidence from daily population samples of cycling zoospore production with periods ranging from 7 to 3 days (Fig. 6). Zoospore numbers declined sharply 24 h after a 10 degree temperature rise, consistent with in vitro data, and remained low.

544 R. KENNEDY AND G. F. PEGG

* 100 - *

*

A-A 0

I I I I I I 0 1 2 3 4 5 6 7

Time (h)

Fig. 5 . The effect of temperature on P. crypfogeu zoospore encystment. -0-, 30°C; - m-, 25°C; -A -, 20°C; - A -, 15"C.-O--, 5°C. * = Treatment means significantly different from all other treatments.

Table 1 . Zoospore numbers in the mineral wool rhizosphere solutions (nos ml-I, means of four plant replicates)

A . Winter-grown planis Days after inoculation

Temperature

15°C Block solution 14.8 25°C Block solution 0.5 Difference between means l4.3*

8 15 24 35

5.3 6.5 2.0 3.7 0.7 0.0 4.6* 5.8 2.0

15°C Slab solution 6.7 7.5 10.9 2.0 25°C Slab solution 0.3 4.3 0.5 1.3 Difference between means 6.4 3.2 10.4* 0.7 L S D. = 10.3 (P = 0.05)

B. Summer-grown plunts Days after inoculation

7 17 24 31 41 14°C Block solution 36.8 52.0 21.0 33.0 0.0 18°C Block solution 16.4 35.0 22.0 40.0 46.0 22°C Block solution 0.8 13.0 4.0 5.0 10.0 26°C Block solution 0.4 8.0 6.0 6.0 13.0 L S D . = 33.7 (f = 0.05)

Zoospore numbers in the mineral wool rhizosphere solution. Data represent colonies per ml of nutrient solution obtained from one ml aliquots plated on BNPRA agar and incubated for 36 h. Means of four plant replicates; A , from the block and slab solutions of winter-grown plants; B, from the growing block solutions of summer-grown plants. I AD. = results significantly different between any two mcans ( P : 0.05).

Efect of root temperature on Phytophthora cryptogea

Appearance of first wilt 15-25°C root symptoms zone temperatures

applied

T I t t

% W

8

2 40

0

545

1 30

Days from inoculation

Fig. 6 . Rhizosphere zoospore populations from winter-grown tomato plants maintained in rockwool growing blocks with root zone temperatures of 15°C - -, and 25°C. - 0 -. Vertical bars represent 95% confidence limits.

Root infection Roots of winter-grown plants grown at 15 "C showed 100% infection in both blocks and

slabs 21 days after inoculation (Table 2A). Plants with a root zone temperature at 25 "C had a significantly lower level of root infection. The percentage of roots infected at 25 "C in blocks and slabs decreased by 97 days after inoculation.

Table 2. Effect of root zone temperature on root infection

A . W'inler-gro wn plirnic Temperature Days after inoculation

21 51 91

150C Block 93'" 100 100 Slab 100 100 I00 Block 49* 84 5 5 * Slab 58* 100 12

25°C

B. Sur?rnier-grown plnn/y Temperature Days aftcr inoculation

9 23 45 62

14°C Block 18°C Block 22°C Block 26°C Block

13.6 100 100 100 16.9 88.1 100 100 2.5 92.4 96.9 100 6.3* 19.3* 90.1 91.2

a ) The number of roots showing any infection in a 3 cm diameter x 10 cm core of mineral wool fibre expres\ed as a percentage of the total number roots per core. * : Results statistically different from correspondins rreatmenl balues ( P = 0.05) based on arcs in transformed data. Means of four replicates,

546 R. KENNEDY AND G. F. PEGG

Table 3. Effect of root-zone temperature on root colonisation by P. cryptogea (Colonies g- ' fresh weight roots, means of 4 replicates)

A . Winter-gro wn plants Days after inoculation

Temperature 21 57 97

15°C Block 505 1625 287 25°C Block 310 425 175 Difference between means 195 1200* 112

15°C Slab 310 1450 262 25°C Slab 285 587 435 Difference between means 25 863* 163 ~ . s . D = 708 ( P = 0.05)

B. Summer-grown plants

Temperature 9 23 45 62

14°C Block 25 800 216 250 18°C Block 100 425 266 266 22°C Block 100 625 200 216 26°C Block 25 350 116 533 L.S.D. = 685 ( P = 0.05)

Days after inoculation

Data represent CFU obtained by comminuting 1.0 g root in 10 ml SDW in an homogeniser at 360 rpm for 30 5 . and plating 1.0 ml aliquots of a 1:lOO dilution o n BNPRA agar plates. L s D values are for differences between treatment means for each sample time.

The percentage infection of roots of summer-grown plants maintained with a root zone of 26 "C was significantly lower at 9 and 23 days after inoculation. Roots maintained at 14 "C were 100% infected at this time (Table 28).

Pathogen levels in 1.0 g comminuted samples from infected blocks and slabs of winter- grown tomatoes at 25 "C root-zone temperature were significantly lower 57 days after inoculation than at 15 "C (Table 3A). Numbers of colonies (1.0 g core samples) were generally higher from blocks than from slabs. No significant differences were found in numbers of Phytophthora colonies isolated from roots of summer-grown plants at any of the applied temperatures (Table 38).

Effect of root temperature on root growth The effects of nutrient at 15 "C and 25 "C on root growth of inoculated and uninoculated

winter-grown plants are shown in Table 4. Root biomass was largely confined to the growing block with no significant growth in the slab at either root temperature. Greater root growth of' winter-grown inoculated plants (Table 4) was recorded at 25 "C after 21 days. Root dry weight was significantly greater at 25 "C, than 15 "C, 97 days after inoculation. A similar pattern of root distribution was observed in uninoculated plants (Table 4). Root colonisation in slabs of uninoculated and inoculated winter-grown plants was poor (Table 4). There was no significant effect of a 25 "C root-zone temperature on root regrowth of plants where higher temperatures had been applied 15 days after inoculation. Dry weights of roots were 3.55 g at 25 "C (means of eight replicates) and 3.50 g (means of seven replicates) at 15 "C.

Root growth of summer-grown plants maintained at 26 "C for 25 days and longer after inoculation was higher than for those grown at lower temperatures (Table 5A) but failed to

Effect of root temperature on Phytophthora cryptogea 547

Table 4. Effect of infection and nutrient temperature on root growth in winter-grown tomato plants (Dry wt g . , means of 4 replicates)

Temperature 21 57 97 --- Inoculated Unlnoculated Inoculated Unlnoculated Inoculated Uninoculated

15°C Block 0.45 0.40 0.54 0.72 0.70 2.47 25°C Block 0.96 0.71 I .06 1.92 3.01 3.31 Difference between means 0.51 0.31 0.52 1.20 2.31* 0.84

15°C Slab 0.06 0.02 0.03 0.01 0.04 0. I4

Difference between means 0.03 0.10 0.00 0.02 0.12 0.04

I s D. for roots of inoculated plants = 0.72; I s 1) tor roots of uninoculated plants = I .25 ( P = 0.05). Data are the root dry weight in grams from blocks and slabs on three sampling dates after dissolution of the mineral wool and binder in 1.0 hl H,PO, for 45 min.

25°C Slab 0.03 0.12 0.03 0.03 0.16 0.10

Table 5. Effect of temperature on root dry weight of summer-grown tomato plants (Dry wt g. , means of 4 replicates)

A . Infected plants Days after inoculation Temperature

14°C Block 18°C Block 22°C Block 26°C Block

L.S.1). = 0.95 ( P = 0.05)

B. Control (healthy) plants

Days after inoculation

62

1 .S.D. = 1.57 (P = 0.05)

9 23 45

2.02 1.76 1.58 1.75 1.71 1.48 1.63 1.74 1.10 1.86 2.52 1.86

Temperature "C 14 18 22

2.08 2.92 3.01

62

1.33 0.94 I .28 1.86

26

2.98

Data are rhe root dry weighr in grams from blocks and slabs 011 three sampling dates after dissolution of the mineral wool and binder in 1.0 hl H,PO, for 45 rnin.

reach the 5% level of statistical significance. Root dry weights of uninoculated plants at 62 days (Table 5B) did not differ significantly.

Aerial symptoms The development of partial or acute root flaccidity in infected winter or summer-grown

plants was closely related to root infection. Winter-grown plants maintained at a root-zone temperature of 15 "C wilted and died 21 days after inoculation. At 25 "C only one plant developed wilt symptoms (Table 6 A ) ; the remaining plants continued growth and fruiting.

All summer-grown plants maintained with root-zone temperatures of 14 "C and 18 "C developed wilt symptoms and subsequently died 5 wk after inoculation. Some of the plants maintained at 26°C developed wilt symptoms 4 wk after inoculation, but did not die (Table 6B).

548 R. KENNEDY AND G. F. PEGG

Table 6. Effect o f root-zone temperatures on aerial symptoms induced by P. cryptogea

A . Winter-grown plunls Wk after inoculation

2 3 6 9 14

(Numbers of plants wilting out of 12) Temperature "C 15 5 12 I2 12 12 25 0 0 I 1 1

B. Summer-grown plants Wk after inoculation

I 2 3 4 5 6 8

(Numbers of plants wilting out of 14) Temperature "C 14°C 0 0 5 9 14 14 14 18°C 0 0 3 8 14 14 14 22°C 0 0 0 6 12 14 14 26°C 0 0 0 3 6 7 8

Figures refer to actual numberr of plants wilting out of I2 winter-grown, and 14 summer-grown plants at each nutrient temperature.

Discussion Increased temperature in the root environment affected disease development by P .

cryptogea in two ways: by enhancing root growth in the host plant and reducing inoculum production by the pathogen. In winter-grown plants where ambient air and substrate temperatures were reduced, high root temperatures increased root growth and initiation in infected and healthy control plants. Increased rates of root regrowth were reflected in decreased percentage root infection. Infected plants with little or no root regrowth at 15 "C developed severe wilt symptoms while the majority of infected plants with enhanced root regrowth at 25 "C remained symptomless over the 14 wk experimental period.

Summer-grown plants maintained at the high root temperature regime showed less root regrowth and higher levels of root infection than winter-grown ones. This reduced regrowth may have resulted from lower assimilate supply to the root as the carbon sink exerted by the fruit was strong at the time of inoculation (Pegg, 1986). However, the substrate volume was five times smaller than the slabs used for the winter experiment. Air temperatures of 20 - 25 "C prevailing during the summer experiment may also have depressed root initiation and growth. Eidsten & Gisler4d (1986) found root dry matter of curled parsley was lower in an ambient air temperature of 21 "C compared with 18 "C. Similarly root dry weight of winter-grown cucumber grown at 20 "C increased with increasing root-zone temperature and was higher than summer-grown plants with a mean air temperature of 25 "C (Newton, Sahraoui & Sherif, 1988). Winter-grown tomato plants inoculated in the pre-fruiting phase may have benefited from increased levels of assimilates to the roots by enhanced utilisation at the higher root temperature. High root-zone temperatures had no effect on root growth of plants showing aerial symptoms before application of the elevated temperature, by when photosynthesis and translocation were both reduced. Increased root regrowth may result in part from enhanced uptake of nutrients particularly of key elements such as phosphorus (Papadopoulos & Tiessen, 1987). Increasing root temperatures between 14 "C and 26 "C in NFT winter grown tomatoes led to enhanced uptake of nitrogen, calcium, magnesium and phosphorus (Adams, 1988).

Disease development was markedly influenced by the effect of temperature on pathogen growth, sporulation, zoospore release and encystment. High temperatures have been shown to be inhibitory to the growth and development of the pathogen in virro. McIntosh (1972)

Effect of root temperature on Phytophthora cryptogea 549

found that survival of P. cactorum zoospores in soil water declined with increasing temperatures. P. cinnamomi responded similarly (Malajczuk, Sanfelieu & Hossen, 1983) with an optimum at 20 "C. Zoospore cysts were more resistant and 7% of the population survived 43 days at temperatures from 20 "C to 30 "C. Zoospore numbers reached a peak between 14 and 18 days with clear evidence of subsequent recycling at a lower level. Unlike the soil environment, antagonism from rhizosphere organisms in rockwool nutrient solution would be expected to be greatly reduced or absent. The optimal temperature for zoosporangial production in uitro, 20 "C, agreed with that of Bumbieris (1978). Temperatures of 25 "C or 30 "C greatly reduced or prevented sporangial formation, a major factor in reduced secondary inoculum at 25 "C. Bumbieris (1979) showed that a low temperature stimulus enhanced zoosporangial production on P. cryptogea-infected pine root, prior to incubation at 25 "C. In non-sterile soil the optimum pre-incubation temperature was 5 "C but under sterile conditions it rose to 15 "C. Zoosporangial formation was affected by the depth of culture medium (Bumbieris, 1979). In rockwool, this would correspond to the solution filled free space in the fibre matrix. The higher O2 demand of roots at 25 "C could also adversely affect the development of zoosporangia.

The effects of temperature on zoospore release are in general agreement with those reported by other workers. Release of zoospores was stimulated by exposing sporangia to a 5 "C shock before incubating at the test temperature. At high temperatures (30 "C) non-chilled cultures caused failure of zoospore release compared to chilled cultures. Zoospore discharge has been reported to occur at 30 "C but stops abruptly at 33 "C (MacDonald & Duniway, 1978). The optimum temperature for mycelial growth of P. cryptogea isolate BR7 was in the range 20 - 25 "C similar to that reported by Brooks (1953) and Weste (1983). This suggests that lesion growth and mycelial spread of the pathogen may differ substantially at 15 "C and 25 "C. The continued rapid growth of lesions at high root temperatures may account for the development of root rot in the presence of low levels of secondary inoculum. Numbers of pathogen colonies derived from comminuted 1.0 g root samples from winter-grown plants at high root-zone temperatures decreased with time suggesting smaller lesions or death of the pathogen in a moribund root system. The differences in colony counts in comminuted samples from low temperature roots could not be accounted for by the presence of more abundant sporulation at this temperature. The comminution of mycelial samples from two isolates of P. cryptogea which differed in their production of zoosporangia gave colony counts on BNPRA amended agar which were not significantly different. Comminution may kill zoosporangia and zoospores, giving an underestimate of infective propagules.

The pattern of disease development by P . cryprogea on tomato roots is similar to that reported for other soilborne pathogens where disease development is most severe at temperatures unfavourable to the host (Johnson & Hartman, 1919; Dickson, 1923). The disease was dissimilar to root rot of avocado by P. cinnamomi which became more severe at temperatures favourable to host development (Zentmyer, 198 1).

Secondary zoospore production in the plant rhizosphere solution (Table 1) was greatly reduced at 25 "C compared with 15 "C in agreement with the in vitro results (Fig. 2). Thus while zoosporangia failed to develop after 12 days at 25 "C in vitro, the low counts of zoospores in the substrate solution at 25 "C persisting at 35 days and at 47 days at 26 "C in summer-grown plants reflect the difficulty of maintaining a uniform temperature in all parts of the system where nutrient channelling may lead to some variation between irrigations. Similarly, root exudates and the rhizosphere microflora may stimulate the overall production of zoosporangia at a given temperature compared with the in vitro experiment in sterile distilled water.

The suppression of aerial symptoms at high root-zone temperatures especially in winter- established tomato plants emphasises the relationship between root loss from infection and root gain when root, shoot sink strength priorities are changed (Pegg & Holderness, 1984;

550 R. KENNEDY AND G. F. PEGG

Pegg, 1986). Thus where infection is confined to a renewable resource such as feeder roots, rather than tap root and hypocotyl, the development of aerial symptoms is seen as a function of the available healthy root biomass. The size of this required to maintain the fruiting shoot will be further determined by the roots’ physiological efficiency. Where a treatment, such as high temperature, enhances root growth while simultaneously suppressing inoculum production and infection the net effect is a reduction or prevention of aerial symptoms. The less effective control by high temperatures in summer-grown plants may be explained a) by the reduced propensity for root regeneration and growth in a substrate volume of 650 cm3 compared with 4025cm3 for the winter experiment and b) the effect of massive root infection from a concentrated application of a high concentration of inoculum coinciding with little or no root regrowth due to a high fruit carbon demand.

The use of elevated root temperatures in winter-grown tomatoes in rockwool may offer a non-chemical method of controlling P. cryptogea-induced root rot. The effect of infection and root temperature on the growth and yield of the tomato plant will be described in a subsequent paper.

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(Received 20 November, 1990)