Pythium Sulcatum and P. Ultimum As Causal Agents of Cavity Spot Disease of Carrots in Egypt

Pythium sulcatum and P. ultimum as causal agents of cavity spot disease of carrots in Egypt

K. A. El-Tarabily1, M. A. Abouzeid2, G. E. ST. J. Hardy3, and K. Sivasithamparam4

1Department of Biology, Faculty of Science, United Arab Emirates University, Al-Ain, 17551, United Arab Emirates; 2Department of Microbiology, Faculty of Science, University of Ain Shams, Cairo, 11566, Egypt; 3School of Biological Sciences and Biotechnology, Division of Science and Engineering, Murdoch University, Murdoch, W.A., 6150, Australia (e-mail: [email protected]); 4Soil Science and Plant Nutrition, School of Earth and Geographical Sciences, Faculty of Natural and Agricultural Sciences, University of Western Australia, 35 Stirling Highway, Crawley, W.A., 6009, Australia. Received 30 January 2003, accepted 3 October 2003.

El-Tarabily, K. A., Abouzeid, M. A., Hardy, G. E. ST. J. and Sivasithamparam, K. 2004. Pythium sulcatum and P. ultimum as causal agents of cavity spot disease of carrots in Egypt. Can. J. Plant Sci. 84: 607–614. In a survey of carrot diseases of the Nile delta region of northern Egypt, Pythium sulcatum (Pratt and Mitchell) and P. ultimum (Trow) were isolated from 74 and 26%, respectively, of carrot roots showing cavity spot disease. In laboratory and glasshouse pathogenicity tests, P. sulcatum caused significantly (P < 0.05) more severe damage than P. ultimum. The greater level of pathogenicity of P. sulcatum was associated with its ability to produce a wider array of cell-wall-degrading enzymes with significantly (P < 0.05) higher enzymatic activities compared to P. ultimum. Polygalacturonase, pectin lyase, pectate lyase and cellulase were detected in cavity spot lesions induced by both P. sulcatum and P. ultimum. Pectin methylesterase was detected in tissues invaded by P. sulcatum and not by P. ultimum. This is the first record of cavity spot disease of carrots in Egypt and indicates P. sulcatum and P. ultimum as the causal agents of this disease in the region surveyed.

Key words: Carrots, cavity spot, cell-wall-degrading enzymes, Egypt, Pythium

El-Tarabily, K. A., Abouzeid, M. A., Hardy, G. E. ST. J. et Sivasithamparam, K. 2004. La cavité pythienne de la carotte causée par Pythium sulcatum et P. ultimum en Égypte. Can. J. Plant. Sci. 84: 607–614. Dans le cadre d’une enquête sur les maladies de la carotte dans la région du delta du Nil, dans le nord de l’Égypte, on a isolé Pythium sulcatum (Pratt et Mitchell) et P. ultimum (Trow) dans 74 % et 26 % des carottes atteintes de la cavité pythienne, respectivement. Lors des essais de pathogénicité en labo- ratoire et en serre, P. sulcatum a entraîné des dommages significativement (P< 0,05) plus importants que P. ulitimum. On associe la plus grande pathogénicite de P. Sulcatum au fait que le cryptogame produit un plus large éventail d’enzymes détruisant la paroi cellulosique et que l’activité de ces enzymes est significativement plus élevée (P < 0,05) que chez P. ultimum. On a retrouvé de la For personal use only. polygalacturonase, de la pectine lyase, de la pectate lyase et de la cellulase des deux pathogènes dans les lésions caverneuses. Les tissus envahis par P sulcatum contenaient aussi de la pectine méthylestérase, mais pas ceux attaqéus par P. ulitimum. Il s’agit de la première fois où l’on observe la cavité pythienne en Égypte et tout indique que cette maladie résulte de P. sulcatum et P. ultimum dans la région atteinte.

Mots clés: Carotte, cavité pythienne, enzymes attaquant la paroi cellulosique, Égypte, Pythium Can. J. Plant Sci. Downloaded from pubs.aic.ca by 134.115.68.21 on 07/24/12 Cavity spot causes severe losses in the marketable yield and Polyphenols accumulate in tissue around lesions. Secondary quality of carrots (Daucus carota L.) worldwide (Montfort fungi and bacteria may invade the wound site causing fur- and Rouxel 1988; Vivoda et al. 1991; Benard and Punja ther enlargement and darkening (Perry and Harrison 1979). 1995). Cavity spot is characterized by the formation of Cavity spot becomes apparent only after carrot roots are har- sunken, brown-black, circular to elliptical or irregularly vested and washed (Benard and Punja 1995). shaped lesions or pits 1–10 mm wide, sometimes surround- Several studies have been conducted to elucidate the possi- ed by a pale yellow halo on all root parts (Vivoda et al. ble cause(s) of cavity spot. It was described as a physiological 1991). Initially, the cavity spot lesions are depressed, disorder (Guba et al. 1961) potentially caused by a calcium approximately elliptical areas, with the long axis between 2 deficiency (Maynard et al. 1963). Infection by Rhizoctonia and 15 mm orientated across the breadth of the root (Perry solani (Kühn) (Mildenhall and Williams 1970), anaerobic pec- and Harrison 1979). Lesions enlarge as roots mature, the tolytic Clostridium species (Perry and Harrison 1979), soil cells of the outermost layers of the secondary phloem col- ammonification (Scaife et al. 1980), feeding injury by fungus lapse progressively and the periderm fractures, leaving a gnat larvae (Hafidh and Kelly 1982), aliphatic acids (Perry ragged edge. A wound periderm forms around the lesions, 1983), toxicity of ammonium ions (Goh and Ali 1983), high and suberin and lignin are deposited in the cell walls. soil alkalinity (Scaife et al. 1983), and environmental stress 607 608 CANADIAN JOURNAL OF PLANT SCIENCE

Fig. 1. In vitro pathogenicity test on mature carrot roots showing typical large lesions caused by Pythium sulcatum. The carrot on the top represents the non-inoculated control.

(high moisture, temperature > 28°C) (Soroker et al. 1984) have MATERIALS AND METHODS also been suggested as causes of cavity spot. Definitive evi- Isolation of Pythium spp. Associated with Cavity For personal use only. dence for the biological origin of this soil-borne disease was Spot Lesions provided by Groom and Perry (1985) and then by White Three months after planting, commercially grown and natu- (1988), who established that two slow-growing Pythium rally infected carrots with characteristic cavity spot lesions species, P. violae (Chesters and Hickman) and P. sulcatum were collected from 20 farms near Qalyob City (Qalyobia (Pratt and Mitchell), were the most important causal organisms province). This area is located 35 km north of Cairo and is in countries where cavity spot occurred. Several fast-growing Pythium species including P. ultimum (Trow) (Vivoda et al. in the major carrot-growing region in Egypt. 1991) and P. coloratum (Vaartaja) (El-Tarabily et al. 1996) Within 24 h of sampling, the carrots were cleaned under have also been demonstrated to cause cavity spot. running water for 4 h to remove soil particles and surface The hyphae of Pythium are known to penetrate the carrot microorganisms (El-Tarabily et al. 1996). Two hundred pieces (ca. 4-mm diameter) of diseased tissues were sliced

Can. J. Plant Sci. Downloaded from pubs.aic.ca by 134.115.68.21 on 07/24/12 roots through cracks produced by the outgrowth of the lat- eral roots or other wounds (Zamski and Peretz 1995). from 200 infected roots. The pieces were surface-disinfest- Digestion of the host cell wall and tissue maceration through ed by immersion in 70% ethyl alcohol for 3 min followed by the activities of cell-wall-degrading enzymes are crucial immersion in 1.05 % sodium hypochlorite for 4 min. aspects of fungal penetration and colonization of the carrot Surface-disinfested pieces were then rinsed 10 times (5 min tissues in cavity spot pathogenesis (Zamski and Peretz 1996; for each rinse) with sterile, distilled water to remove resid- Campion et al. 1997). ual hypochlorite, and then dried on sterile filter paper for 30 Recently, typical cavity spot lesions have been observed min in a laminar flow cabinet (El-Tarabily et al. 1996). in commercially grown carrots in Egypt; however, this dis- Tissues from the lesion margins were then excised asepti- ease has not been previously recorded here. The objectives cally and plated with the internal tissue facing down, on a of this study were to: (1) determine if Pythium species were modified selective medium (Jeffers and Martin 1986) for the associated with cavity spot symptoms in carrots grown in isolation of Pythium species. The medium consisted of Egypt; (2) confirm the pathogenicity of the most consistent- water agar (WA) amended with pimaricin 5 µg mL–1 ly obtained isolates, and (3) characterize activities of the (Sigma Chemical Co., St Louis, MO), rifampicin 10 µg cell-wall-degrading enzymes of the pathogenic Pythium mL–1 (Sigma), ampicillin (Sigma) 250 µg mL–1, and pen- species associated with cavity spot. tachloronitrobenzene 1000 µg mL–1 (134 mg of 75% a.i. EL-TARABILY ET AL. — CARROT CAVITY SPOT 609

Fig. 2. In vitro pathogenicity test on mature carrot roots showing small lesions caused by Pythium ultimum. The carrot on the top represents the non-inoculated control.

Terraclor, Uniroyal Australia Pty. Ltd. , Victoria, Australia). colonized PCA plugs under the same conditions served as The plates were incubated at 15 or 25°C in the dark for 7 d controls. After 7 d, the lesion diameters were measured

For personal use only. and examined daily. All Pythium isolates were maintained (length by width) for each inoculation point. An average on potato-carrot agar (PCA) (Plaats-Niterink 1981) slants, lesion diameter was determined from the three inoculation which were stored at 4°C and subcultured every 3 wk. The points on each root. isolates were also preserved in water, by transferring twenty 5-mm diameter plugs from Pythium-colonized PCA Identification of Pathogenic Pythium spp. plates into sterile distilled water in McCartney bottles. To measure the mycelial growth rate for each isolate, one These were stored at 15°C. 5-mm diameter plug was cut from the edge of a 6-d-old cul- ture grown at 25°C in the dark on PCA. This plug was trans- Laboratory Pathogenicity Test ferred to a 9-cm Petri dish containing 20 mL of PCA medium. Two hundred and twenty-three Pythium isolates obtained Growth rate (mm d–1) was assessed by measuring two, right

Can. J. Plant Sci. Downloaded from pubs.aic.ca by 134.115.68.21 on 07/24/12 from the cavity spot lesions were chosen for an in vitro angle diameters of the colony as described by Campion et al. screening test to evaluate their pathogenicity. All isolates (1997). According to the daily growth rate on PCA, all were cultured on PCA at 25°C in the dark for 8 d. Mature, Pythium isolates were initially clustered into either a slow- 3-mo-old carrot roots that were free of cavity spots were growing (10 mm d–1) or fast-growing (25 mm d–1) group. surface sterilized in 1.05% sodium hypochlorite for 2 min The slow- and fast-growing groups of Pythium caused and rinsed three times in sterile distilled water. The carrots lesions in the laboratory pathogenicity test, and as all iso- were placed in plastic trays lined with paper towels moist- lates within each group had similar cultural and morpholog- ened with sterile distilled water and inoculated by placing 5- ical characteristics, the three most pathogenic isolates mm diameter PCA plugs of an actively growing colony on representing the slow- (isolates 24, 89 and 134), and fast- the root surface. There were three inoculation positions on (isolates 5, 18 and 107) growing groups were chosen for fur- each root. Each tray contained four carrots and there were ther glasshouse pathogenicity test and assays of the cell- three replicate trays for each isolate. Trays were covered wall-degrading enzymes. with aluminum foil and incubated in the dark for 7 d at 25°C The influence of temperature on mycelial growth for each under humid conditions (Groom and Perry 1985). The roots Pythium isolate was determined using potato-carrot broth were sprayed daily with sterile distilled water to prevent (PCB). Erlenmeyer flasks (250 mL), each containing 50 mL drying of the inoculum and carrots. Roots exposed to non- of the broth, were inoculated with mycelial plugs (5-mm 610 CANADIAN JOURNAL OF PLANT SCIENCE

Table 1. Comparison of growth rates and morphological characteristics of Pythium sulcatum and P. ultimum isolated from carrot roots with cavity spot lesions in Egypt Characters Pythium sulcatum Pythium ultimum Daily growth rate on PCA at 25°C 10–12 mm d–1 25–27 mm d–1

Optimum temperature 24–28°C 25–30°C

Hyphal diameter 5–6.5 µm 7–9.5 µm

Appressoria Sausage shaped, 17 × 6 µm Not formed

Hyphal swellings Subglobose and peanut shaped Globose, terminal and intercalary, Terminal and intercalary, 20 × 27 µm in diameter 19–25 µm in diameter

Sporangia Filamentous and not differentiated from the Not formed vegetative hyphae

Zoospores Not produced Not produced

Oogonia Smooth, terminal and intercalary, subglobose, Smooth, terminal and intercalary, globose, 17–22 µm in diameter average 19 µm in diameter 15–22 µm in diameter average 18 µm in diameter

Antheridia 1–2, monoclinous and diclinous, often large, 2–3, monoclinous, sac-like and straight longitudinally appressed to and encircling the oogonium with slight folds and furrows

Oospores Aplerotic, globose, 13–21 µm in diameter Aplerotic, globose 12–20 µm in diameter average 18 µm in diameter average 19 µm in diameter

Oospore wall thickness 1.1–1.3 µm 2.7–3.2 µm

diameter) taken from the growing margins of 6-d-old cul- Colonized millet seeds, which had been autoclaved twice, tures of the Pythium isolates cultured on PCA plates. Liquid served as the control. cultures were grown in the dark at temperatures of 10–40°C at 2°C intervals. After 10 d, the mycelial mats were harvest- Soil Infestation

For personal use only. ed by filtration, washed in distilled water, dried for 24 h at Soil was collected from one of the fields where cavity spot 30°C, and then weighed. damaged carrots were found. Prior to use, the soil was Pythium isolates were identified by examining the mor- passed through a 1-cm mesh sieve and air dried. The char- phological characteristics of cultures growing on PCA fol- acteristics of the soil (El-Tarabily et al. 1997) were: pH 6.8 –1 lowing the key of Plaats-Niterink (1981). Sporangial (in 0.01 M CaCl2); electrical conductivity 1.74 dS m ; formation and zoospore discharge were induced using organic carbon 1.1%; with the following nutrients expressed Schmitthenner’s (1979) slush medium. Development of as mg kg–1 soil; bicarbonate extractable potassium and sexual reproductive structures were induced using grass- phosphorus 390 and 35, respectively; nitrate nitrogen 21, blade cultures (Plaats-Niterink 1981). The dimensions of ammonium nitrogen 74; sulfate 505, and iron 798. 300 of each of the asexual and sexual reproductive struc- Pathogenicity test was carried out with three isolates of

Can. J. Plant Sci. Downloaded from pubs.aic.ca by 134.115.68.21 on 07/24/12 tures were obtained from 4-d-old PCA cultures incubated at the slow-growing group (isolates number 24, 89 and 134) 25°C in the dark. All measurements were made using an and three isolates of the fast-growing group (isolates 5, 18 Olympus BH-2 microscope (Olympus Optical Co., Ltd, and 107). Each isolate was dispersed through the soil using Tokyo, Japan). 0.05 g millet seed inoculum g–1 air-dried soil by mixing in a cement mixer. This inoculum density had previously been Glasshouse Pathogenicity Test shown to be the optimum level required to cause cavity spot Inoculum Production under glasshouse conditions (El-Tarabily et al. 1996). Inoculum for each Pythium isolate was prepared by placing Colonized millet seeds were autoclaved and used as the con- 40 g of moist millet seed (Panicum miliaceum L.) into 250- trol. Free-draining pots (30-cm diameter) were filled with 8 mL Erlenmeyer flasks and autoclaving at 121°C for 30 min kg of soil. on 3 consecutive days (El-Tarabily et al. 1996). The sub- Carrot (cv. Nanco) seeds were surface-sterilized in 70% strate was then aseptically inoculated with eight agar plugs ethanol for 1 min and then in 1.05% sodium hypochlorite for (5-mm diameter) taken from the actively growing margins 2 min, and finally the seeds were washed five times with of the selected Pythium isolates grown on PCA. The flasks sterile, distilled water. Ten seeds were sown at a 0.5-cm were incubated at 25°C in the dark for 20 d and were peri- depth into each pot (El-Tarabily et al. 1996). After emer- odically shaken to ensure uniformity of colonization. gence (ca. 8 d), the seedling density was reduced to five per EL-TARABILY ET AL. — CARROT CAVITY SPOT 611

Fig. 3. Carrot roots showing typical cavity spot symptoms caused by Pythium sulcatum in the glasshouse pathogenicity test after 14 wk at 25 ± 2°C.

pot. Each treatment was replicated eight times with five Table 2. Effect of soil inoculation with Pythium sulcatum and P. ultimum plants in each replicate, in fully randomized blocks. on cavity spot development on carrot roots under glasshouse conditions The pots were placed in an evaporatively cooled Mean number glasshouse maintained at 25 ± 2°C. Pots were watered to Mean number of of lesions Disease container capacity daily and fertilized once weekly with Treatments lesions per root per cm2 of root indexy inorganic liquid fertilizer (Thrive®) (Arthur Yates & Co P. sulcatum 35.13* ± (1.27)z 2.91* ± (0.12) 4.46* ± (0.09) Limited, Milperra, NSW, Australia) at the manufacturer’s P. ultimum 6.71 ± (0.37) 0.28 ± (0.24) 1.82 ± (0.08) recommended rate. zValues in parentheses are the standard errors of the mean. y

For personal use only. Disease index rated on a scale of 1–5, where 1 = no visible cavity; 2 = up Disease Assessment to five cavities, each < 3 mm in diameter; 3 = up to 10 cavities < 3 mm in diameter; 4 = up to 20 cavities < 3 mm in diameter or up to two cavities > Carrots were harvested 14 wk after seeding, the leaves 10 mm in diameter; and 5 = more than 20 cavities < 3 mm, or up to 3 cav- removed and the roots washed, weighed (to determine yield) ities > 20 mm in diameter. and scored for cavity spots. Disease was evaluated as: (i) *Indicates significance at P < 0.05 between treatments in columns. number of cavity spot lesions per root; (ii) number of cavity spot lesions per cm2 of root and (iii) a disease index rated on a 1–5 scale (El-Tarabily et al. 1996). Details of the rating scale are presented in Table 2. To fulfil Koch’s postulates, 50 randomly selected cavity spot lesions were surface disin- 6.5 g–1 fresh weight of tissue). The homogenized tissue was centrifuged at 17 000 g for 20 min at 4°C and the supernatant Can. J. Plant Sci. Downloaded from pubs.aic.ca by 134.115.68.21 on 07/24/12 fested and the pathogens recovered as described above. was used to determine the activities of cell-wall-degrading Assay of Cell-wall-degrading Enzymes enzymes, including pectin lyase (Hultin and Levine 1963), The Pythium isolates were cultured on basal mineral agar pectate lyase (Nasuno and Starr 1967), polygalacturonase medium (Zamski and Peretz 1996). Agar blocks obtained (Wang and Veen 1970), pectin methylesterase and cellulase from the cultures were used to inoculate fresh carrots using (Zamski and Peretz 1996). The cell-wall-degrading enzymes the method described for the laboratory pathogenicity test. assayed in our study were based on the work of Zamski and Freshly inoculated carrot tissues were assayed for cell-wall- Peretz (1996) and Campion et al. (1997) on cell-wall-degrad- degrading enzymes produced by the pathogens. The control ing enzymes produced by the Pythium spp. responsible for consisted of carrots inoculated with noncolonized agar plugs. carrot cavity spot. There were ten replicates for each assay for After 7 d, tissues with lesions were removed with a 10-mm- each isolate. diameter cork borer. The tissues were dissected with a scalpel, ground with a mortar and pestle in liquid nitrogen and Statistical Analysis homogenized in an Omni mixer (OCI Instruments, Omni Data were statistically analyzed by the Student’s t test using Corporation International, Waterbury, CT) at 3000 rpm for 5 Superanova® (Abacus Concepts, Inc., Berkeley, CA) at min with wash buffer (3 mL of 60 mM phosphate buffer, pH P < 0.05. 612 CANADIAN JOURNAL OF PLANT SCIENCE

Table 3. Activities of cell-wall-degrading enzymes by Pythium sulcatum and P. ultimum 7 d after inoculation on carrot roots at 25°C Enzymatic activities P. sulcatum P. ultimum Polygalacturonase (enzyme units g–1 fresh weight h–1) 0.86* ± (0.02) z 0.13 ± (0.01) Pectin lyase (enzyme units g–1 fresh weight h–1) 1.84* ± (0.04) 0.47 ± (0.05) Pectin methylesterase acid equivalents (mmol h–1) 0.63* ± (0.03) 0.00 Pectate lyase (enzyme units g–1 fresh weight h–1) 3.85* ± (0.06) 1.34 ± (0.08) Cellulase (enzyme units g–1 fresh weight h–1) 0.44* ± (0.03) 0.14 ± (0.02) zValues in parentheses are the standard errors of the mean. *Indicates significance at P < 0.05 between treatments in rows.

RESULTS not shown), the results were combined for each species (Table 2). In all treatments, no disease symptoms were Isolation of Pythium spp. Associated with Cavity observed on the foliage and the non-infested controls Spot Lesions showed no cavity spot symptoms. Out of 200 surface-sterilized carrot pieces, 223 Pythium iso- Carrot yields (mean root weight) were not affected by lates were obtained. Of these, 165 (74%) were slow-grow- inoculation with P. sulcatum or P. ultimum (data not ing and 58 (26%) were fast-growing. shown). Both Pythium species were recovered from the cavity spot lesions. Laboratory Pathogenicity Test All 165 isolates of the slow-growing Pythium isolates pro- Assay of Cell-wall-degrading Enzymes duced large (> 30 mm) lesions, whilst only 44 of the 58 fast- Since there were no significant differences in the enzyme growing Pythium isolates produced lesions, which were all production profiles of the three isolates of P. sulcatum and small (< 15 mm). Lesions produced by the slow-growing the three isolates of P. ultimum, the results of the three iso- Pythium isolates were brownish-black with clearly defined lates were combined for each species. In the laboratory path- edges (Fig. 1) and, in all cases, the tissue beneath the perid- ogenicity test, polygalacturonase, pectin lyase, pectate lyase erm had turned black by 5 d after inoculation. The fast- and cellulase were detected in cavity spot lesions induced by growing Pythium isolates, produced lesions of a similar both P. sulcatum and P. ultimum (Table 3). P. sulcatum pro- colour (Fig. 2) and the periderm started to turn brown 7 d duced significantly (P < 0.05) higher enzymatic activities after inoculation. No lesions formed when non-colonized than P. ultimum. Pectin methylesterase was detected in tis- PCA plugs were applied to the root surfaces. sues invaded by P. sulcatum and not by P. ultimum.

Identification of Pathogenic Pythium spp. DISCUSSION

For personal use only. The slow- and fast-growing Pythium species were identified Pythium sulcatum and P. ultimum were the only Pythium as P. sulcatum and P. ultimum, respectively (Table 1). The spp. isolated from cavity spot lesions on carrots obtained identity of P. sulcatum (DAOM # 230748) was confirmed from commercial fields in Egypt. Both Pythium species by Dr. André Lévesque (National Fungal Identification caused cavity spot lesions on carrot roots in laboratory and Service, Agriculture and Agri-Food Canada, Ottawa, ON). glasshouse pathogenicity tests, although P. sulcatum was more aggressive than P. ultimum. P. ultimum produced only Glasshouse Pathogenicity Test a few cavity spots in the glasshouse trial. This is the first Typical cavity spot symptoms appeared on all carrots grown in report of cavity spot of carrots in Egypt and confirms the soil infested with P. sulcatum (Fig. 3), while soil infested with role of P. sulcatum and P. ultimum as the causal agents of P. ultimum produced only a few small cavities (Table 2). this disease.

Can. J. Plant Sci. Downloaded from pubs.aic.ca by 134.115.68.21 on 07/24/12 Carrots grown in soil infested with P. sulcatum showed signif- Pythium sulcatum is known to cause cavity spot in Japan icantly (P < 0.05) higher disease severity levels than those (Nagai et al. 1986), the Unite Kingdom (White 1988), the grown in soil infested with P. ultimum (Table 2). The disease Netherlands (Wagenvoort et al. 1989), Canada (Benard and symptoms observed in this trial were characteristic of cavity Punja 1995), and Australia (El-Tarabily et al. 1996; Davison spot with small, blackish-brown, spherical lesions surrounded and McKay 1998). Although P. ultimum was less pathogen- by a yellow halo. The lesions were generally less than 3 cm in ic in the present study, it did cause symptoms that would length and approximately 2–8 mm in depth, and were distrib- negatively affect the quality of carrot roots. P. ultimum has uted irregularly over the entire carrot root. The symptoms also been reported to cause cavity spot in the United observed on carrots growing in soils artificially infested with Kingdom (White 1986), the United States (Vivoda et al. either P. sulcatum or P. ultimum isolates were typical of the 1991) and Canada (Benard and Punja 1995). cavity spot lesions seen in the field, but were different from the Worldwide, many Pythium spp. have been implicated as lesions that developed on mature carrots in the laboratory path- the causal agents of cavity spot. This variation probably ogenicity test (Figs. 1 and 2). reflects the different environmental and soil conditions Since there were no significant (P > 0.05) differences in between countries (Guerin et al. 1994). For example, P. vio- the incidence and severity of symptoms among the three iso- lae, which has been reported to be the main causal agent of lates of P. sulcatum or the three isolates of P. ultimum (data cavity spot disease in Europe and North America (White EL-TARABILY ET AL. — CARROT CAVITY SPOT 613

1988; Vivoda et al. 1991; Benard and Punja 1995), was not Benard, D. and Punja, Z. K. 1995. Role of Pythium species in isolated from cavity spot lesions in the current survey. Its cavity spot development on carrots in British Columbia. Can. J. absence may be related to unsuitably high soil temperatures Plant Pathol. 17: 31–45. (>25°C) in the field at the time of sampling (Vivoda et al. Campion, C., Massiot, P. and Rouxel, F. 1997. Aggressiveness 1991) and/or due to the possible absence of the pathogen at and production of cell-wall degrading enzymes by Pythium violae, the test sites in Egypt. Pythium sulcatum and Pythium ultimum, responsible for cavity spot on carrots. Eur. J. Plant Pathol. 103: 725–735. The optimum temperature for P. sulcatum and P. ultimum Cherif, M., Benhamou, N. and Belanger, R. R. 1991. is reported to be within the range of 25–30°C (Plaats- Ultrastructural and cytochemical studies of fungal development Niterink 1981). In our study, the optimum temperature for and host reactions in cucumber plants infected by Pythium ulti- P. sulcatum and P. ultimum isolates were 24–28°C and mum. Physiol. Mol. Plant Pathol. 39: 353–375. 25–30°C, respectively, and falls within the range of the Davison, E. M. and McKay, A. G. 1998. Pythium spp. associated ambient temperatures recorded at the time of the field sur- with cavity spot of carrots in Western Australia. Australas. Plant vey (23–29°C). Both P. sulcatum and P. ultimum are Pathol. 27: 163–168. homothallic, and the oospores they produce are able to sur- El-Tarabily, K. A., Hardy, G. E. ST. J. and Sivasithamparam, vive in the environment prevalent in this region. K. 1996. Association of Pythium coloratum and Pythium sulcatum The enzymes that degrade cell wall polysaccharides are with cavity spot disease of carrots in Western Australia. Plant important in the development of the disease symptom char- Pathol. 45: 727–735. acteristics of Pythium species (Cherif et al. 1991; Zamski El-Tarabily, K. A., Hardy, G. E. ST. J. and Sivasithamparam, K. 1997. Effects of host age on development of cavity spot disease and Peretz 1996). In the present investigation, the greater of carrots caused by Pythium coloratum in Western Australia. pathogenicity of P. sulcatum was associated with its ability Aust. J. Bot. 45: 727–734. to produce a wider array of cell-wall-degrading enzymes Goh, K. and Ali, M. S. 1983. Effects of nitrogen fertilizers, calci- and at higher activities compared to P. ultimum. In Canada, um and water regime on the incidence of cavity spot in carrot. highly virulent isolates of P. violae and P. sulcatum were Fertil. Res. 4: 223–230. found to produce higher levels of pectolytic enzymes com- Groom, M. R. and Perry, D. A. 1985. Induction of ‘cavity spot- pared with moderately or weakly virulent isolates (Benard like’ lesions on roots of Daucus carota by Pythium violae. Trans. and Punja 1995). In a study in Israel, the degradation of car- Br. Mycol. Soc. 84: 755–757. rot cell walls by P. violae was associated with the in situ Guba, E. F., Young, R. E. and Ui, T. 1961. Cavity spot disease production of a wide spectrum of cell-wall-degrading of carrots and parsnip roots. Plant Dis. Rep. 45: 102–105. enzymes that specifically attacked cell wall polysaccharides Guerin, L., Mathilde, B. and Rouxel, F. 1994. Biochemical char- acterization of Pythium spp. involved in cavity spot of carrots in (Zamski and Peretz 1996). In their study, cellulase and poly- France. Ann. Appl. Biol. 125: 255–265. galacturonase showed highest levels of activity during the Hafidh, F. T. and Kelly, W. C. 1982. Cavity spot of carrots first day after inoculation. Pectin lyase, pectate lyase and caused by feeding of the fungus gnat larvae. J. Am. Soc. Hortic. pectin methylesterase gradually increased to their highest Sci. 107: 1177–1181. For personal use only. levels of activity 14 to 30 d after inoculation. This pattern of Hultin, H. O. and Levine, A. S. 1963. On the occurrence of mul- enzyme activities enables the penetration of P. violae tiple molecular forms of pectinesterase. Arch. Biochem. Biophys. through the walls of the carrot cells and the establishment of 101: 396–402. the hyphae (Zamski and Peretz 1996). P. sulcatum isolated Jeffers, S. N. and Martin, S. B. 1986. Comparison of two media from cavity spot on carrots in France have been reported to selective for Phytophthora and Pythium species. Plant Dis. 70: produce cell-wall-degrading enzymes in vitro in a non- 1038–1043. shaken chemically defined liquid medium (Campion et al. Maynard, D. N., Gersten, B., Young, R. E. and Vernell, H. F. 1997). Their strain of P. ultimum failed to produce pectin 1963. The influence of plant maturity and calcium level on the occurrence of carrot cavity spot. J. Am. Soc. Hortic. Sci 83: methylesterase or pectate lyase. However, we found that all 506–510. three strains of P. ultimum tested produced pectate lyase but Mildenhall, J. P. and Williams, P. H. 1970. Rhizoctonia crown rot

Can. J. Plant Sci. Downloaded from pubs.aic.ca by 134.115.68.21 on 07/24/12 not pectin methylesterase. These differences may be related and cavity spot of muck-grown carrots. Phytopathology 60: 887–889. to the geographic variation among strains of P. ultimum. Montfort, F. and Rouxel F. 1988. La maladie de la “tache” de la Although it is tempting to associate the activity of pectin carrote due á Pythium violae Chester et Hickman: Donnèes symp- methylesterase with the greater pathogenicity of P. sulcatum tomatologiques et ètiologiques. Agronomie 8: 701–706. observed in this study, caution must be exercised, as strong- Nagai, Y., Fukami, M., Murata, A. and Watanabe, T. 1986. ly pathogenic isolates of P. violae were found by Campion Brown-blotted root rot of carrots in Japan. (1) Occurrence, symp- et al. (1997) not to produce this enzyme. toms and isolations. Ann. Phytopathol. Soc. Jpn. 52: 278–286. The present investigation identified the pathogens associated Nasuno, S. and Starr, M. P. 1967. Polygalacturonic acid transe- with cavity spot disease of carrots in Egypt, and established a liminase of Xanthomonas campestris. Biochem. J. 104: 178–185. relationship between the aggressiveness of the pathogen and Perry, D. A. 1983. Effect of soil cultivation and anaerobiosis on cavity spot of carrots. Ann. Appl. Biol. 103: 541–547. the in situ production of cell-wall-degrading enzymes. Perry, D. A. and Harrison, J. G. 1979. Cavity spot of carrots. II. The effect of soil conditions and the role of pectolytic anaerobic ACKNOWLEDGMENTS bacteria. Ann. Appl. Biol. 93: 109–115. We thank Prof. Youssef. A. Youssef from the Microbiology Plaats-Niterink, A. J. Van der. 1981. Monograph of the genus Department, Faculty of Science, University of Ain-Shams, Pythium. Studies in Mycology Baarn. Centraalbureau voor Cairo, Egypt for valuable discussions. Schimmelcultures 21: 1–242. 614 CANADIAN JOURNAL OF PLANT SCIENCE

Scaife, M. A., Burton, A. K. and Turner, M. K. 1980. Cavity Wagenvoort, W. A., Blok, I., Mombarg, H. F. M. and spot of carrots—an association with soil ammonium. Commun. Veldhuizen, T. 1989. Cavity spot of carrots in relation to a Soil Sci. Plant Anal. 11: 621–628. Pythium sp. Gartenbauwissenschaft 54: 70–73. Scaife, M. A., Turner, M. K., Barnes, A. and Hunt, J. 1983. Wang, M. C. and Veen, N. T. 1970. Purification and characteri- Cavity spot of carrots—observance on a commercial crop. Ann. zation of endo polygalacturonase from Verticillium albo-atrum. Appl. Biol. 102: 567–575. Arch. Biochem. Biophys. 141: 749–757. Schmitthenner, A. F. 1979. Pythium species: isolation, biology White, J. G. 1986. The association of Pythium spp. with cavity and identification. Pages 33–36 in Advances in turfgrass patholo- spot and root dieback of carrots. Ann. Appl. Biol. 108: 265–273. gy. Proceedings of a Symposium on Turfgrass Diseases. White, J. G. 1988. Studies on the biology and control of cavity Minneapolis, MN. spot of carrots. Ann. Appl. Biol. 113: 259–268. Soroker, E., Bashan, Y. and Okon, Y. 1984. Reproducible induc- Zamski, E. and Peretz, I. 1995. Cavity spot of carrots: Interaction tion of cavity spot in carrots and physiological and microbial between the host and pathogen, related to the cell wall. Ann. Appl. changes occurring during cavity spot formation. Soil Biol. Biol. 127: 23–32. Biochem. 16: 541–548. Zamski, E. and Peretz, I. 1996. Cavity spot of carrots: II. Cell- Vivoda, E., Davis, R. M., Nuñez, J. J. and Guerard, J. P. 1991. wall-degrading enzymes secreted by Pythium and pathogen-related Factors affecting the development of cavity spot of carrot. Plant proteins produced by the root cells. Ann. Appl. Biol. 128: Dis. 75: 519–522. 195–207. For personal use only. Can. J. Plant Sci. Downloaded from pubs.aic.ca by 134.115.68.21 on 07/24/12