Disease: Aphanomyces root rot (black root)
Host: Sugar beet (Beta vulgaris)
Symptoms and signs (14)
Seedlings. Optimal conditions for Aphanomyces damping-off and root rot include wet and
warm (optimal: 20-30 °C) soil conditions; A. cochlioides seldom infects
at soil temperatures less than 15 °C (3). Symptoms include brown, water-soaked
tissue that can extend up to, and include, the cotyledons (Fig. 1). The
infected hypocotyl and root rapidly turn black and shrink to a dark, slender
thread (Fig. 2). Infected seedlings occur in patches ranging in size from a
square meter to extreme cases, where entire fields of 2- to 5-week-old plants
are destroyed. Disease frequently occurs in portions of fields that tend to
remain wet –- near drainage ditches, low spots, and in compacted soils. Aphanomyces-infested
seedlings that survive when soil becomes dry produce adult roots that are
malformed and scarred in appearance (Fig. 3).
Older Roots. In wet seasons, Aphanomyces root rot occurs from the seedling stage until harvest. Root rot can develop in plants that were infected as seedlings or from new infections on sound older roots. Aboveground symptoms include undersized plants with yellowed lower leaves that wilt during hot sunny days (Fig. 4) and recover overnight and on cool, cloudy days. Below ground, a brown to black rot develops at junctures of lateral roots and/or at the root tip (Fig. 5). Infected roots often are severely stunted (Fig. 6). Roots also may have rotted, tasseled root tips (Fig. 7). Severely infected plants die. When foliage is mechanically removed from infected beets at harvest, rotted roots are easily dislodged or are too small to be harvested. Older roots that recover from seedling infections (Fig. 3), or that are infected later in the season, have reduced yield and sucrose content, and higher levels of impurities (non-sucrose constituents, which makes sucrose extractions difficult and expensive).
A. cochlioides causes disease on other commercial crops within B.
vulgaris (table beet, mangel, chard), Spinacia oleracea (spinach),
and several wild species of Beta, including B. maritima and B.
patellaris (6). Over 30 species in several families (Aizoaceae,
Amaranthaceae, Caryophyllaceae, Chenopodiaceae, Hydrophyllaceae, Linaceae,
Papaveraceae, Portulacaceae, Solanaceae) became infected when artificially
inoculated in the greenhouse, but it is unknown if they are hosts in the field.
Cultures of A. cochlioides have been isolated from lambsquarter (Chenopodium
album), carpetweed (Mollugo verticillata), bouncing bet (Saponaria
ocymoides), and New Zealand spinach (Tetragonia tetragonioides) grown
in naturally infested soil; the cultures were pathogenic to sugar beet. For
further information, see Papavizas and Ayers (6).
Aphanomyces root rot generally occurs in regions where sugar beet crops are
grown including North America, Europe, and Chile (6).
A. cochlioides is difficult to isolate from plants unless tissue is recently infected. Firm tissue near the margin of the lesion (hypocotyls for seedlings) should be selected, washed in tap water to remove soil, immersed in 0.5% NaOCl for 15 sec, rinsed twice in sterile distilled water (SDW), and blotted dry. Tissue segments then are placed on Metalaxyl-Benomyl-Vancomycin medium (MBV, see Appendix), which is semi-selective for Aphanomyces species and incubated at 22-24 °C (8). Cultures should be observed daily over 5 days and when characteristic mycelium is observed, it should be transferred to fresh MBV and then to cornmeal agar (CMA), potato-dextrose agar (PDA), or oatmeal agar (OMA). To minimize problems with bacterial contamination, CMA may be supplemented with rifampicin (50 mg/L) and penicillin G (50 mg); A. cochlioides will not grow on CMA supplemented with streptomycin. Or, PDA may be supplemented with streptomycin sulfate (100 mg), neomycin (50 mg), and ampicillin (50 mg).
To quickly detect A. cochlioides and other common seedling pathogens (Pythium spp., Rhizoctonia solani), prepare newly infected tissues as described above. Then, float tissue in 5 ml of SDW for 24-48 hr at 21 ± 1 °C and examine microscopically for the presence of grape-like clusters of primary zoospores at the tips of hyphae-like zoosporangia (Fig. 8). It may be necessary to observe tissue daily for a few more days before zoosporangia are observed. Pythium and R. solani also produce characteristic structures in water culture (3).
If A. cochlioides is not isolated from tissues with suspicious symptoms, or if it is suspected as occurring in a particular field, a soil "baiting" technique can prove useful. Seed of a susceptible sugar beet cultivar treated with fungicide (Apron + Thiram, 0.625 + 2.5 g a.i./kg, respectively) is sown into soil and incubated at 18-20 °C. When seedlings emerge, the temperature is increased to 25-30 °C and soil is watered daily so it remains moist. Dying seedlings are removed daily and assayed in water culture to confirm infection by A. cochlioides, as described above. Soil also can be indexed to determine root rot potential (7, 15).
To prepare pure cultures, zoospore suspensions are obtained from plant material (as described above) or from mycelium (see section Pathogenicity Tests). Zoospore suspensions are drawn into capillary pipettes, transferred to the surface of a thick layer of agar, and then the entire agar disk is inverted with a spatula (9). The next day, the bottom of the plate is examined (X200) and colonies are marked that are developing from single zoospores (they should be isolated from neighboring zoospores). Within 48-96 hr, mycelia that have grown to the agar surface are ready to hyphal-tip and transfer to fresh plates of CMA, PDA, or OMA.
Pure cultures also can be obtained by diluting zoospores in sterile water and then spreading a small quantity (0.5–1.0 ml) of the suspension over the surface of a 2-3 day-old plate of culture media (10 ml/9-cm diameter petri dish). After 18 to 24 hr, germinating zoospores and a small portion of surrounding agar can be removed and transferred to CMA, PDA, or OMA.
Pathogen identification is primarily based on host specificity to B. vulgaris but microscopic features also are helpful (6, 12). Hyphae (3-9 mm diameter) are nonseptate, hyaline, with granular cytoplasm and the colony forms a delicate, sparse, arachnoid, wandering growth on and within media (this latter trait distinguishes it from Pythium, which has a more strongly directional growth). Main hyphae commonly branch in a Y-shaped junction and side branches are often short with a pointed apex. Zoosporangia (up to 3-4 mm in length) are delimited from hyphal branches and are sinuous, irregular in diameter, and involve large segments of the vegetative thallus. Primary zoospores encyst at the orifice upon emergence from the zoosporangium (Fig. 8); secondary zoospores are reniform and laterally biflagellate. Oogonia are borne terminally on short lateral branches, are subspherical (20-29 mm diameter), with a wall (1-2 mm thick) that is smooth at the outer surface and sinuous on the inner surface. Antheridia (6-10 mm diameter x 9-18 mm length) are of diclinous origin, club-shaped with apical prolongations, and separated from the stalk by a septum; 1 to 5 antheridia (usually 3-4) wrap around the oogonium. Oospores are hyaline to yellow (16-24 mm diameter) with granular contents, a large central reserve oil globule about 12 mm in diameter, and a smaller conspicuous refractive body. Oospore walls are 1.5-2 mm thick.
Recently, Malvick et al. (4) characterized pathogenic and genotypic traits of A. euteiches and A. cochlioides.
A. cochlioides is easily stored in sterile water. Bacterial-free cultures are grown on water agar. Six pieces of agar (5 mm x 5 mm) are cut from the advancing edge of an actively growing colony and placed in a 20-ml glass vial containing 15 ml SDW; cultures will die if stored in plastic vials. Caps are lightly screwed on the vials, and then are stored in the dark at room temperature. Cultures can be stored for 6 to 9 months (or longer if the water does not dry out) and are replenished by culturing them on CMA and then repeating the process for storage.
Other storage methods (7) include preservation on slants of CMA, PDA, or OMA at 12 °C, with hyphal transfers every 3-4 mo (will not survive at 4 °C). A. cochlioides also can be stored by covering cultures on agar slants with mineral oil. Oospores can be stored in artificially infested soil (see section on oospore production).
Both zoospores and oospores are used as inoculum in pathogenicity tests. Inoculation of plants with ground mycelium is not recommended because inoculum cannot be quantified. Soil with a large proportion of peat, including some potting mixes, appear to limit disease severity (11). In seedling tests, a root rot index (0-100 scale) often is calculated from a disease severity assessment (7, 15), whereas for older roots, a root rot index (0-7 scale) is used (15).
Zoospore production. For rapid production of limited amounts of inoculum, five 5-mm diameter disks are transferred from the margin of an advancing culture growing on CMA or PDA to a 9-cm diameter petri dish. Then, 15-20 ml of deionized water is added and plates are incubated at 22-28 °C. Primary zoospores are extruded from sporangia within 24-48 hr (too many disks will interfere with zoospore production) and secondary zoospores will be released from primary zoospores 4-8 hr later; zoospores may undergo repeated encystment and emergence.
A procedure developed by Mitchell and Yang (5) and modified by Malvick (W-L Research, Inc., Evansville, Wisconsin, personal communication) is useful for large-scale production of zoospores. A. cochlioides is grown in a sterilized broth containing Sigma type IV peptone (20 g/L) and glucose (5 g/L) in deionized water; 30 ml is dispensed into 125 ml flasks or 40 ml into 250 ml flasks. Two days after inoculation, the broth is decanted; 50 ml of 50% Mineral Salts Solution (MSS, see Appendix) is added to the mycelial mat; the suspension is mixed vigorously and then left undisturbed for 1.5 hr. The solution is discarded from the mycelial mat and the process repeated. After the second wash solution is removed, 30 ml of full-strength MSS is added to mycelium in the flask, swirled vigorously, and then left undisturbed overnight at 21-22 °C for production of zoospores. Zoospore density is determined by placing an aliquot of the suspension in a test tube and placing it in a vortex mixer for 30 sec so zoospores encyst (glass containers are preferred since zoospores and cysts tend to stick to certain plastics); zoospore cysts then are counted with a hemacytometer. Zoospore production varies among isolates; high-producing isolates should yield about 105/ml. Zoospores are extremely sensitive to low concentrations of organic and inorganic ions, so all glassware must be meticulously cleaned and rinsed with glass-distilled deionized water (avoid distilled water passed through copper tubes).
For pathogenicity tests, A. cochlioides requires a warm, water-saturated environment for infection of roots. Sugar beet seedlings are inoculated by dispensing zoospores onto the soil surface at various times after emergence or they can be applied around roots of older plants. Schneider (10) found that application of 1 x 105 - 2 x 105 zoospores/10-cm pot of 14-day-old seedlings identified resistant sugar beet cultivars. Zoospore suspensions should be handled with care for pathogenicity tests since zoospores tend to encyst when physically agitated.
Oospore production. Homogenized Oatmeal Broth is ideal for abundant production of oospores and ease of preparation (11, see Appendix). Oospores are counted and added to a vermiculite carrier. Oospore inoculum in vermiculite remains infective for more than 1 yr when stored at 4 or –9 °C. Severity of disease may progressively decline with storage, so preliminary testing of inoculum before use is advised. Oospores also are produced abundantly on the same agar media recommended for mycelial growth.
Oospore inoculum can be mixed into steamed or raw soil to the desired concentration. Seed is sown directly into soil, following procedures outlined for baiting A. cochlioides from soil (see section on Pathogen Isolation). A concentration of 10 or 100 oospores/g dry weight of field soil resulted in 40 and 87% loss of seedling stand 4 weeks after planting, respectively (1). Oospore densities of 250-350 per gram of soil also have been used for studies on sugar beet seedlings (7).
The most comprehensive taxonomic treatise of the genus Aphanomyces is a 1961 monograph by Scott (12). Additional plant pathogenic species have subsequently been described (2, 13).
1. Brantner, J.R., and Windels, C.E. 1999. Aphanomyces damping-off of susceptible and partially resistant sugar beet cultivars over a range of oospore concentrations. (Abstr.) Phytopathology 89:S104.
2. Ichitani, T., Kodama, T., Horimoto, K., and Ikeda, A. 1986. Aphanomyces iridis sp. nov. causing Aphanomyces basal rot of Dutch iris in Japan, Ann. Phytopathol. Soc. Jpn. 52:590- 598.
3. Leach, L.D. 1986. Seedling diseases. Pages 4-8 in: Compendium of Beet Diseases and Insects. E.D. Whitney and J.E. Duffus (eds.). APS Press. The American Phytopathological Society, St. Paul, MN. 76 pp.
4. Malvick, D.K., Grau, C.R., and Percich, J.A. 1998. Characterization of Aphanomyces euteiches strains based on pathogenicity tests and random amplified polymorphic DNA analyses. Mycol. Res. 102:465-475.
5. Mitchell, J.E., and Yang, C.Y. 1966. Factors affecting the growth and development of Aphanomyces euteiches. Phytopathology 56:917-922.
6. Papavizas, G.C., and Ayers, W.A. 1974. Aphanomyces species and their root diseases in pea and sugarbeet. U.S. Dep. Agric., Agric. Res. Serv. Tech. Bull. 1485. Washington, D.C. 158 pp.
7. Parke, J.L., and Grau, C.R. 1992. Aphanomyces. Pages 27-30 in: Methods for Research on Soilborne Phytopathogenic Fungi. L.L. Singleton, J.D. Mihail, and C.M. Rush (eds.). APS Press, The American Phytopathological Society, St. Paul, MN. 265 pp.
8. Pfender, W.F., Delwiche, P.A., Grau, C.R., and Hagedorn, D.J. 1984. A medium to enhance recovery of Aphanomyces from infected plant tissue. Plant Disease 68:845-847.
9. Schmitthenner, A.F., and Hilty, J.W. 1962. A modified dilution technique for obtaining single-spore isolates of fungi from contaminated material. Phytopathology 52:582-583.
10. Schneider, C.L. 1954. Methods of inoculating sugar beets with Aphanomyces cochlioides Drechs. Proc. Am. Soc. Sugar Beet Technol. 8:247-251.
11. Schneider, C.L. 1978. Use of oospore inoculum of Aphanomyces cochlioides to initiate blackroot disease in sugarbeet seedlings. J. Am. Soc. Sugar Beet Technol. 20:55-62.
12. Scott, W.W. 1961. A monograph of the genus Aphanomyces. VA Agric. Exp. Stn. Tech. Bull. 151. 95 pp.
13. Singh, S.L., and Pavgi, M.S. 1977. Aphanomyces root rot of cauliflower. Mycopathologia 61:167-172.
14. Windels, C.E., and Lamey, H.A. 1998. Identification and control of seedling diseases, root rot, and rhizomania on sugarbeet. University Minnesota Ext. Serv. and North Dakota State University Ext. Serv. PP-1142, BU-7192-S. 20 pp.
15. Windels, C.E., and Nabben-Schindler, D.J. 1996. Limitations of a greenhouse assay for determining potential of Aphanomyces root rot in sugarbeet fields. J. Sugar Beet Res. 33:1-13.
Metalaxyl-Benomyl-Vancomycin (8). For isolation of Aphanomyces species from infected plant tissue:
Autoclave the above ingredients and add the following when agar cools to 45-50 °C:
Keep plates in the dark at room temperature. Transfer colonies
to fresh MBV as soon as possible because they are sensitive to contamination.
Colonies may then be transferred to PDA or CMA.
Mineral Salts Solution (5; modified by Malvick, W-L Research, Inc., Evansville, Wisconsin, personal communication). A rinsing solution for production of zoospores by Aphanomyces cochlioides and A. euteiches:
Homogenized Oatmeal Broth (11). For production of oospores of Aphanomyces cochlioides:
Comminute rolled oats in 300 ml distilled water in a blender for 5 min, add water to bring total to 1 L, heat to 50 °C and adjust pH to 6.6 before autoclaving. Inoculate with 5 mm disks of A. cochlioides grown on CMA or PDA for not more than 5 days. Incubate 30 days (or longer if necessary) at room temperature in the dark. Comminute mycelial mats in water in a blender for 5 min. Determine oospore concentration, dilute with water to desired concentration, add to vermiculite at a rate of 2 parts fine grade (0.5-2 mm diameter) or 2.5 parts medium grade (2-5 mm diameter) to 1 part oospore suspension (v:v). Mix the vermiculite and oospore suspension, then air-dry the inoculum for 14 days and store at 4 °C.