Epidemiology of Pythium Sulcatum Associated with Brown-Blotted Root Rot of Carrots

日植病報 62: 130-133 (1996)

Ann. Phytopathol. Soc. Jpn. 62: 130-133 (1996)

Epidemiology of Pythium sulcatum Associated with Brown-blotted Root Rot of Carrots

Koji KAGEYAMA*, Misako TACHI*, Masakazu UMETSU* and Mitsuro HYAKUMACHI*

Abstract

Pythium sulcatum was a predominant species isolated from carrot lesions showing brown-blotted root rot, and induced the similar symptoms to those naturally occurring in carrot fields with artificial inoculation. The fungus was also isolated from asymptomatic storage and absorbing roots from seedling to harvest stage, especially during spring cropping season. The infection sites of P. sulcatum was the upper part of asymptomatic storage roots in which the blotted lesion might appear. P. ultimum, P. sylvaticum, P. coloratum and P. spinosum were also obtained from asymptomatic roots and residues of root or leaf, but these species showed slight pathogenicity. The absorbing root residue was found to be the primary infection source of P. sulcatum. P. sulcatum was widespread in the intensive carrot-cultivated area.

(Received September 20, 1995; Accepted December 27, 1995)

Key words: carrot, Pythium sulcatum, brown-blotted root rot, epidemiology.

lesions were cut into ca. 5mm blocks, directly plated on INTRODUCTION the Pythium selective medium4) (cornmeal agar (CMA) amended with 5mg/l pimaricin, 100mg/l pentachloro- Brown-blotted root rot of carrot (Daucus carota L.) nitrobenzene and 100mg/l agrimycin), and incubated at first occurred in a main carrot-producing area of Chiba 25•Ž in the dark for 3-7 days. Prefecture9), and later in Kakamigahara of Gifu Prefec- Carrot materials together with storage and absorbing ture in which carrots have been cropped twice in a year. roots were monthly collected during two cropping sea-

The disease is characterized by small water-soaked sons of winter (1992) and spring (1993) from fields with brown spots, referred to blots, on storage root. The history of severe outbreak of brown-blotted root rot. lesions at harvest time are 2-3mm long, and not quite The residues of absorbing root and leaf were collected

noticeable. The lesions extend up to 20mm or more after harvest in 1992. The specimens were washed under during transportation to commercial markets and there running tap water, and cut randomly into 2-5mm pieces. sometimes cause a big loss showing severe soft decay. After removing the excess water on filter papers, 50 to Therefore, the disease is recognized as a post-harvest 100 pieces of storage and absorbing roots and 40 to 80

disease, although the symptoms appear at harvest time. pieces of root or leaf residues at each sampling time Nagai et al.9) and Watanabe et al.13) demonstrated that were placed on the selective medium. brown-blotted root rot was caused by Pythium sulcatum To determine the infection sites, 15 samples of Praat & Mitchell. However, little is known on the asymptomatic storage roots at harvest stage were col- ecology of the fungus. lected from two fields. A root was cut from top to The purpose of this study is to clarify: (i) time and bottom into two stripes of ca. 1cm wide and placed on sites of infection, (ii) primary inoculum source in the the selective medium, facing the periderm of the root to field, (iii) pathogenicity of several Pythium species iso- the agar. lated from carrot fields, and (iv) the distribution of P. Pythium colonies were transferred to CMA slants, sulcatum. when developed. Isolates were identified based on their morphological characteristics on both CMA and pieces

MATERIALS AND METHODS of colonized grass in water14) according to the key of Plaats-Niterink10) and the original description of P.

Isolation from plant materials. Carrots show- sulcatum11). To verify the identification of P. sylvaticum ing brown-blotted root rot were collected in spring Campbell & Hendrix, the isolates were paired with the cropping season in 1992. The root specimens with the type cultures5) and incubated at 25•Ž for a week or more

* Faculty of Agriculture , Gifu University, Gifu 501-11, Japan 岐阜大学農学部 Ann. Phytopathol. Soc. Jpn. 62 (2). April, 1996 131 to determine their heterothallic nature. sulcatum was predominantly obtained from storage and Pathogenicity tests. Carrot storage roots ob- absorbing roots from seedling to harvest stage. How- tained from the market were washed, halved vertically, ever, P. ultimum and P. coloratura were rarely isolated and then placed in large petri dishes or plastic boxes from storage or absorbing roots. P. sylvaticum were with moistened filter paper. Pythium isolates were more obtained in this season than in winter but the grown on potato carrot agar10). The 5mm agar disks frequency was low. with mycelial mat were taken from the edge of each P. sulcatum were mainly isolated from the part of 0- isolates of Pythium species tested, and placed in two 10cm from the top of the asymptomatic storage roots rows consisting of five disks on the halved storage roots. collected in both fields (Table 2). The other Pytlhium Five 5mm agar disks without mycelia were served as species were obtained from the same parts of the roots controls between the rows. The inoculated storage roots in a field but rarely isolated from all part of the roots in were incubated at 15•Ž and 25•Ž. All experiments were the other field. repeated three times. After 4 to 7 days of incubation, the Isolation from residues roots were examined for the appearance of lesions on P, sulcatum, P. ultimum, P. sylvaticum, P. coloratum the inoculated site. and P. spinosum Sawada were isolated from absorbing Isolation from soil. In 1993, soils from nine car- root or leaf residues of carrots (Table 3). P. sulcatum rot and one turnip fields were collected to quantify P. was obtained from root residues collected in Oigi field. sulcatum. Three samples (ca. 250g) from each field were P. ultimum was a predominant species, especially in leaf mixed to make a composite sample. Ten to five mm blocks of carrot storage roots were placed in the test Table 2. Frequency of isolation of Pythium species soil in 9cm diameter petri dish. After 3-days incubation from various parts of carrot roots without at 20•Ž in the dark, the blocks were recovered from the visible lesions at harvesting stage collected soil, washed under tap water for about 30min, and then from two fieldsa) dried and placed on the selective medium.

RESULTS

Isolation from roots Pythium spp. were isolated from 20 out of 50 lesions showing brown-blotted root rot. Nineteen of 20 isolates were P. sulcatum (95%), while the other was P. sylvaticum (5%). In periodical isolation, four species of Pythium, namely P. coloratum Vaartaja, P. ultimum Trow, P. sulcatum and P. sylvaticum, were obtained from asymptomatic storage and absorbing roots (Table 1). In winter cropping season P. sulcatum was predominantly isolated from storage roots in November and December and from absorbing roots in November. P. ultimum were consistently but not frequently isolated from storage a) Fifteen roots collected from each field were placed on and absorbing roots, while P. sylvaticum and P. cobra- the selective medium. turn were rarely isolated. In spring cropping season, P. b) P. sub, P. sulcatum; P. spp, the other Pythium spp.

Table 1. Frequency of isolation of Pythium species from carrot roots without the symptoms of brown-blotted root rot

a) P. sul, P. sulcatum; P. ult, P. ultimum; P. syl, P. sylvaticum; P. col, P. coloratum. Fifty to a hundred pieces of asymptomatic carrot roots were plated on the selective medium. 132 日本栢物病理学会報第62巻第2号平成8年4月

Table 3. Frequency of isolation of Pythium species from carrot plant residues after harvest in two different locations with histories of serious brown-blotted root rot

a) P. sul, P. sulcatum; P. ult, P. ultimum; P. syl, P. sylvaticum; P. col, P. coloratura; P. spi, P. spinosum. Fifty to a hundred pieces were plated on the selective medium.

Table 4. Pathogenicity of Pythium species to sliced Table 5. Frequency of isolation of Pythium sulcatum storage roots of carrots from ten fields using baiting methoda)

a) Thirty 5mm blocks of carrot storage root, after in-

cubating in the test soil at 20•Ž for 3 days, were placed

on the selective medium.

a) The values are the percentage of lesions formed on b) P. sul, P. sulcatum; P. ult, P. ultimum; Other, the other

carrot root by inoculating separately with 5mm disks unidentified Pythium spp. The values are the percent-

taken from agar cultures of different Pythium spp. ages of carrot blocks from which Pythium spp. were b) Mycelia were covered with the surface of roots, isolated.

although no symptom appeared. c) Carrots have been intensively cultivated in the fields

designated as No.1 to 8. The field No.9 is a carrot field

adjacent to main carrot cropping area and has no residues. P. sylvaticum and P. coloratura were infre- history of monoculture of carrot. quently isolated from root and leaf residues. P. spinosum which had been obtained from neither storage nor absorbing roots was isolated from leaf residues. DISCUSSION Pathogenicity P. sulcatum frequently induced the similar symptoms Five species of Pythium were isolated from storage to brown-blotted root rot naturally occurring in carrot and absorbing roots, and root and leaf residues of car- fields at both 15•Ž and 25•Ž (Table 4). The isolates of the rots. P. sulcatum was the predominant species isolated other Pythium species showed moderate pathogenicity from diseased roots and induced the similar symptoms at 25•Ž, but the symptoms were different from brown- to the brown-blotted rot naturally occurred in carrot blotted rot, showing water-soaked lesions without hol- fields with artificial inoculation. Therefore, this fungus is low and brown color. At 15•Ž, those isolates rarely considered to be a principal pathogen. P. ultimum has induced lesions except for one isolate of P. coloratum, been described to cause cavity spot12) and root dieback7) which did not induce typical symptoms. of carrots, and was frequently isolated from asympto- Distribution of P. sulcatum matic absorbing and storage roots and leaf residues in P. sulcatum was frequently isolated from the soil of all the present study. However, White15) and Howard et al.3) fields where carrots have been cultivated intensively, suggested that P. ultimum was not associated with root and the isolation ranged from 3.3 to 30.0% by bait disease of carrot. In the present study, P. ultimum was method (Table 5). The fungus was not isolated in the not seemed to cause brown-blotted root rot, since this fields apart from the main carrot cropping area (Fields fungus was not isolated from the lesions and showed low No.9 and No.10). Both P. ultimum and the other virulence and different symptoms with artificial inocula- Pythium spp. were obtained only from three out of 10 tion. Our results also suggested that P. sylvaticum, P. fields, and the frequency of isolation was low. coloratum and P. spinosum are not the primary cause of Ann. Phytopathol. Soc. Jpn. 62 (2). April, 1996 133

carrot root disease since they induced slight symptoms, 2. Groom, M.R. and Perry, D.A. (1985). Induction of •ecav- although isolated from carrot roots3,6-8,12,15,16). ity spot-like•f lesions on roots of Daucus carota by P. sulcatum was first isolated from brown rot of Pythium violae. Trans. Br. Mycol. Soc. 84: 855-757. carrot root in Wisconsin and Florida of the United 3. Howard, R.J., Pratt, R.G. and Williams, P.H. (1978). Pathogenicity to carrots of Pythium species from States8), and was described as a new species by Pratt et organic soils of North America. Phytopathology 68: the time, the fungus has been reported as a 1293-1296. causal agent of carrot root disease, such as root 4. Kageyama, K. and Ui, T. (1980). A selective medium dieback3,6) and cavity spot15,16). P. sulcatum was isolated for isolation of Pythium spp. Ann. Phytopathol. Soc. Jpn. from asymptomatic storage and absorbing roots at 46: 542-544. seedling stage in the field where brown-blotted root rot 5. Kageyama, K., Ui, T. and Uchino, H. (1991). Pythium of carrot had occurred. The fungus was also obtained sylvaticum isolates from bean and sugarbeet, and their from storage roots of harvest stage without symptoms, sexual behavior. Trans. Mycol. Soc. Jpn. 32: 283-289. and the infection sites were the same as the parts in 6. Kalu, N.N., Sutton, J.C. and Vaataja, O. (1976). which the blotted lesions might appear in storage roots. Pythium spp. associated with root dieback of carrot in

The results suggested that P. sulcatum could infect Ontario. Can. J. Plant Sci. 56: 555-561. latently the carrot roots from the early growing stage to 7. Liddell, C.M., Davis, R.M., Nunez, J.J. and Guerard, J.P. harvest time. The disease may promptly develop under (1989). Association of Pythium spp. with carrot root a favorable environmental condition such as high humid- dieback in the San Joaquin Valley of California. Plant ity. Dis. 73: 246-249. The symptoms of brown-blotted root rot appear on 8. Mildenhall, J.P., Pratt, R.G., Williams, P.H. and Mit- storage roots. Our study demonstrates that absorbing chell, J.E. (1971). Pythium brown root and forking of muck-grown carrots. Plant Dis. Rep. 55: 536-540. root residues are the possible primary source of 9. Nagai, Y., Fukami, M., Murata, A. and Watanabe, T. inoculum for the next year cropping, because P. sul- (1986). Brown-blotted root rot of carrots in Japan (1) catum was isolated from absorbing roots and their Occurrence, symptoms and isolation. Ann. Phytopathol. residues. Soc. Jpn. 52: 278-286. P. sulcatum could not be isolated from soil, using 10. Plaats-Niterink, A.J. van der (1981). Monograph of the direct isolation method of Ali-Shtayeh et al.1) This genus Pythium Stud. Mycol. 21: 1-242. species grows slower (12mm/day at 25•Ž on potato- 11. Pratt, R.G. and Mitchell, J.E. (1973). A new species of carrot agar) compared to the faster-growing (25-31mm/ Pythium from Wisconsin and Florida isolated from day) species, namely P. sylvaticum, P. ultimum and P. carrots. Can. J. Bot. 51: 333-339. spinosum. P. violae, which causes carrot cavity 12. Vivoda, E., Davis, R.M., Nunez, J.J. and Guerard, J.P. spot2,12,15), is also slow-growing and has been detected (1991). Factors affecting the development of cavity indirectly by trapping but not directly from soil12). spot of carrot. Plant Dis. 75: 519. Baiting method was found to be useful for isolation of P. 13. Watanabe, T., Nagai, Y. and Fukami, M. (1986). sulcatum from soil as well as P. violae. Brown-blotted root rot of carrots in Japan (2) Culture P. sulcatum was obtained only from fields with history and identification. Ann. Phytopathol. Soc. Jpn. 52: 287- 291. of intensive monoculture of carrot, but not from the 14. Waterhouse, G.E. (1967). Key to Pythium Pringsheim. fields without the history apart from the main carrot Commonwealth Mycol. Inst. Mycol. Paper 109: 1-15. cropping area. Nagai et al.9) suggested that the host of 15. White, J.G. (1986). The association of Pythium spp. P. sulcatum was limited to carrot among the eight crops with cavity spot and root dieback of carrots. Ann. appl. tested. The population of P. sulcatum may increase Biol. 108: 265-273. significantly due to continuous monoculture of carrots. 16. White, J.G. (1988). Studies on the biology and control We conclude that P. sulcatum infects both storage and of cavity spot of carrots. Ann. appl. Biol. 113: 259-268. absorbing roots of carrots without causing any symptom from the early growing stage. Monoculture of carrot 和文摘要 might lead to severe yield loss since the primary inoculum is present in absorbing root residues in the field 景山幸二・多地美砂子・梅津雅一・百町満朗:ニンジンのしみ soil. Further investigation on the mechanism of latent 腐病に関与するPythium sulcatumの伝染環 infection and lesion development are necessary for a Pythium sulcatumは,しみ腐れの症状を示しているニンジン greater understanding of brown-blotted root rot of car- から分離される優占種であり,人工接種により同じ病徴を示し rot. た。本菌は,特に春作で苗の段階から収穫期まで無病徴の貯蔵根および吸収根から分離された。また,無病徴の貯蔵根における感 Literature cited 染部位は上部に限られており,実際に病徴が現れる部位と一致していた。P. sylvaticum, P. ultimum, P. coloratumおよびP. 1. Ali-Shtayeh, M.S., Lim-Ho, C.L. and Dick, M.W. (1986). spinosumもニンジンの根および根や葉の残渣から分離された An improved method and medium for quantitative esti- が,病原性はほとんどなかった。吸収根の残渣は,P. sulcatum mates of populations of Pythium species from soil. の第一次伝染源であることが認められた。また,本菌はニンジン Trans. Br. Mycol. Soc. 86: 39-47. を連作している圃場に広く分布していた。