Colonization of Muskmelon and Nonsusceptible Crops

Gordon, T. R., Okamoto, D., and Jacobson, D. J. 1989. Colonization of muskmelon and nonsusceptible crops by Fusarium oxysporum f. sp. melonis and other species of Fusarium. Phytopathology 79:4095-1100. The ability of Fusarium oxysporum f. sp. melonis to colonize roots abundance of the four fungi was similar on all seven crops; nonpathoof a susceptible muskmelon cultivar and six crop species rotated with genic strains of F. oxysporum were isolated most frequently, followed melons was quantified in the greenhouse and field. Root colonization by F. o. melonis. F. equiseti was the least frequently isolated of the four by F. solani, F. equiseti, and nonpathogenic strains of F. oxysporum fungi in greenhouse experiments, whereas F. solani was the least frequently also was recorded. Roots of alfalfa, cotton, Honey Dew muskmelon, isolated in the field. Differences in colonization frequencies among these sugar beet, tomato, and wheat were colonized by F. o. melonis. Crops fungi were not proportional to differences in their respective population with comparatively low populations of F. o. melonis on their roots also densities in soil. Nonpathogenic strains of F. oxysporum had the highest tended to have low populations of other Fusarium spp. The relative colonization frequency per colony-forming unit per gram of soil. Additional keywords: Fusarium wilt, soilborne pathogen. Fusarium oxysporum Schlecht. f. sp. melonis Leach & Currence MATERIALS AND METHODS emend. Snyd. & Hans., cause of Fusarium wilt of muskmelon (Cucumis melo L.), has been reported to colonize roots of corn Greenhouse studies in naturally infested soil. Colonization by and soybean grown in rotation with muskmelon (2). Colonization F. melonis was quantified on roots of wheat (Triticum aestivum of rotation crops also has been reported for other formae speciales L. 'Yecora Rojo'), alfalfa (Medicago sativa L. 'Moapa 69'), sugar of F. oxysporum (1,4,10,13,17,22). beet (Beta vulgaris L. 'SS-YI'), cotton (Gossypium hirsutum L. Root colonization of a nonsusceptible plant by a pathogenic 'SJ-2'), tomato (Lycopersicon esculentum Mill. 'UC 82'), strain of F. oxysporum implies the ability to compete with other muskmelon (C. melo L. 'Greenflesh Honey Dew'), and a western root-colonizing fungi. Nevertheless, it has been suggested that shipping-type muskmelon (C. melo L. 'PMR 45-SDJ') that is formae speciales of F. oxysporum are not well adapted to compete susceptible to Fusarium wilt. Greenflesh Honey Dew is susceptible with the less specialized fungi commonly found in agricultural in a root-dip assay (26) but does not develop Fusarium wilt in soils (5). Persistence of wilt pathogens is attributed to the the field. A Panhill clay loam soil, obtained from a commercial production of chlamydospores, rather than the pathogen's ability melon field naturally infested with race 2 of F. o. melonis, was to compete with nonpathogenic fungi (5). combined with sterile coarse sand (3:1, v/v) and blended in a One measure of a pathogen's ability to compete with other motorized mixing drum. Bulk density of the soil-sand mix was root-colonizing fungi is relative infection frequency on nonsusapproximately 1.2 times greater than the unamended soil. Each ceptible crops. Infection frequency commonly is estimated from crop was seeded in a pot containing approximately 300 g of soilcolonization frequency, that is, the number of sites on a given sand mix. The experiment included three replicates in a completely length of root that produce discrete colonies on an isolation randomized design. Each pot was kept in a clay saucer, which medium (9,11). Infection frequency can be determined for F. was filled with water whenever the soil surface became dry. oxysporum if the pathogen can be distinguished from Temperatures were 28 ±8 C during the day and 18 ±3 C at nonpathogenic strains. Smith and Snyder (22) reported that F. night. oxysporum f. sp. vasinfectum (Atk.) Snyd. & Hans. was Root samples were removed from each pot along with most morphologically distinct from nonpathogenic strains of F. of the soil 4 wk after seeding. Roots in the bottom 2 cm of oxysporum isolated from field soil from the San Joaquin Valley the pot were removed and not included in the sample assayed. of California. Similarly, we can distinguish F. o. melonis from Loosely adhering soil was removed with running tap water. nonpathogenic strains of F. oxysporum isolated from melon field Remaining soil was removed by three 30-mmn washes in 1% sodium soils in the San Joaquin Valley. hexametaphosphate, with a tap-water rinse between each wash. Fusarium wilt is a serious disease in major melon-growing areas After the final washing, roots were stored in sterile distilled water in the San Joaquin Valley (8), and there are no previous reports for up to 24 hr at 4 C before they were placed on Komada's concerning the ability of the pathogen to colonize rotational crops selective medium (14) amended with 1 ml/ L of tergitol NP-10 grown in this area. This study was undertaken to determine the (KM). Roots_• 1 mm in diameter that were not obviously damaged relative infection frequency of race 2 of F. o. melonis, F. equiseti or discolored were laid in parallel rows on the agar surface in (Corda) Sacc., F. solani (Mart.) Sacc., and nonpathogenic strains 9-cm-diameter plastic petri dishes. A minimum of 100 cm of roots of F. oxysporum on roots of six crop plants commonly grown was cultured per replicate (50 cm!/plate). Plates were incubated in rotation with susceptible muskmelon cultivars. A preliminary at room temperature (22 ±3 C) with 12 hr of light per day account of this work has been published (6). provided by two 34-W fluorescent tubes. Colonies emerging from plated roots were counted 6 or 7 days later. Species of Fusarium were identified on the basis of colony morphology on potato___________________________________________ dextrose agar (PDA) and spore morphology on carnation leaf © 1989 The American Phytopathological Society agar (CLA) (18). Vol. 79, No. 10, 1989 1095 Isolations were made from shoot tissue of the sampled plants. samples were taken from alfalfa (2-3 g/sample) and the largest Stem sections were taken from the cotyledonary node of all crops, from sugar beet (15-20 g/sample). Tissue pieces were suspended except wheat and sugar beet where shoot tissue was the first in sterile water and homogenized in a blender for 2-3 mm. stem node above the soil line and petioles of the first and second Dilutions of the resultant homogenate were spread over the surface true leaves, respectively. Tissue pieces were surface disinfested of KM. For both cotton and tomato, only the cortex, which by rinsing with tap water, followed by immersion for 30 sec in readily separated from the central stele, was assayed by this 70% ethanol and 90 sec in 0.5% sodium hypochlorite. Treated procedure. Sections of the stele from cotton and tomato taproots tissue pieces were placed directly on KM. were surface disinfested as previously described and placed directly Inoculum densities of F. o. melonis and other Fusarium spp. on KM. in the soil-sand mixture were estimated by soil dilution plating After the first washing of taproot samples, the sodium (16,25). Air-dried soil was homogenized in a blender, and 5or hexametaphosphate solution was centrifuged at 16,000 g for 15 10-g subsamples were suspended in 200 ml of 1% sodium min. The resultant pellet was dried at 35 C for 24 hr. Samples hexametaphosphate and agitated on a shaker for 30 min. The of 5-, 10-, or 25-mg of this material were resuspended in sterile suspension was diluted 1:4 (v/v) in 0.1% water agar, and 1.0water and spread over the surface of KM. ml samples were spread over the surface of plates containing Isolations also were made from shoot tissue of field-grown KM. plants. Samples were taken near the cotyledonary node, or, in Generation and characterization of benomyl-resistant strains, the case of sugar beets, from petioles of lower leaves. Shoot tissue Cornmeal agar (CMA) plates completely colonized by race 2 of was surface disinfested, as described above, and placed on KM. F. o. melonis were exposed to ultraviolet light (254 nm) for 30 Several weed species growing in or near field plots were sec at a distance of 6 cm; radiant energy at this distance was collected. Roots were washed and isolations were made as approximately 300 mW cm. Plates were overlaid with Puhalla's described for crop plants. A minimum of 50 cm of roots from minimal medium (21) amended with benomyl at 5 gg a.i./ml each weed species collected was examined for the presence of and maintained in darkness for at least 24 hr. After 5 to 10 days F. o. melonis. at 22 + 3 C. colonies on the agar surface were mass transferred Vegetative compatibility tests. Nitrate nonutilizing (Nit) to fresh plates of minimal medium amended with benomyl. mutants were selected from isolates of F. oxysporum by culturing Transfers of mycelium that continued to grow in the presence them on a medium containing chlorate as described by Puhalla of benomyl were mass transferred to water agar without benomyl, (21). Nit mutants were tested for vegetative compatibility with and these cultures were hyphal tipped to minimal medium tester strains of race 2 of F. o. melonis (12). amended with benomyl. Benomyl-resistant strains obtained by Data analysis. For each experiment, analysis of variance this procedure were tested for pathogenicity on susceptible (ANOVA) was used to test for significant differences in muskmelon cultivar Top Mark in a seedling root-dip assay (12). colonization of seven crops by F. o. melonis. Similar tests were Benomyl-resistant strains were recovered from diseased seedlings, done for F. equiseti, F. solani, and nonpathogenic F. oxysporum. A single-spore derivative of one of these strains, confirmed to ANOVA also was used to evaluate differences in colonization have wild-type morphology and growth rate on CMA, was frequency of four fungi on the same crop. Mean comparisons designated P2/19B and used in subsequent experiments, were made with Duncan's multiple range test. Within each Greenhouse studies in artificially infested soil. Benomylexperiment, colonization frequencies averaged over all seven crops resistant strain P2/19B of race 2 of F. o. melonis was grown and a crop X fungus interaction were evaluated by a two-way on CMA for 15 days at room temperatures under lights as ANOVA (23). When samples were taken from the same plot at previously described. Spores were washed from colonized CMA different times, data were analyzed by repeated measures ANOVA. plates with sterile water and added to a Yolo loam soil collected ANOVA and mean comparisons were performed on lognear Davis, CA. This soil, which had no detectable indigenous transformed data; nontransformed data are reported. Bartlett's population of F. o. melonis, was combined with sterile, coarse test was used to confirm homogeneity of variances. Statistical sand (3:1, v/v) before inoculum was added. After the spore computations were performed with NCSS versions 5.0 and 5.3 suspension was added, the soil-sand mixture was air dried and (J. L. Hintze, Kaysville, UT) on an IBM PC-AT. stored at room temperature (23 ± 3 C); it was assayed periodically over the next 6 wk by soil dilution plating. Inoculum density RESULTS of the soil-sand mix was adjusted to a final level of approximately 100 colony-forming units (cfu) of F. o. melonis per gram soil Identification of Fusarium spp. F. o. melonis, other isolates by adding noninfested soil and sand. The seven crops grown in of F. oxysporum, F. solani, and F. equiseti were identified by the naturally infested field soil also were grown in artificially colony morphology on KM. F. o. melonis was characterized by infested soil; temperatures were 32 ± 11 C during the day and dense, whitish aerial mycelium with a reddish-purple pigment that 21 ± 2 C at night. Root samples were harvested and processed diffused into the agar. Nonpathogenic strains of F. oxysporum as previously described. produced buff-colored colonies with very little mycelium and Field studies. To corroborate the results of our greenhouse abundant sporodochia. Differences in colony morphology also experiments, a preliminary field test was conducted to determine were apparent on PDA. On CLA, both F. o. melonis and other colonization frequencies of the four Fusarium spp. in a commercial isolates of F. oxysporum produced macroconidia characteristic field naturally infested with race 2 of F. o. melonis. Each crop of the species (18), with no clear distinction between pathogenic was seeded in three replicate plots, in a completely randomized and nonpathogenic strains. design; each replicate consisted of a single row 4.5 m long. Six To confirm the correlation between colony morphology and weeks after seeding, whole plant samples were collected, including pathogenicity, a total of 97 colonies of both morphological types as much of the root system as possible. Four randomly selected were isolated from roots and tested for virulence on seedlings plants were taken from each replicate, and roots were washed of susceptible muskmelon cultivar Top Mark in a greenhouse and placed on KM as described for greenhouse-grown plants. root-dip assay (12). Eighty-one isolates were identified as F. o. Also, stem sections were taken from each plant as described melonis on the basis of colony morphology, including a minimum previously, surface disinfested, and placed on KM. of 10 cultures isolated from each of the seven crops studied. All Plant samples also were collected at 10, 12, and 15 wk after 81 induced symptoms typical of Fusarium wilt, comparable to planting for all crops except wheat. As before, four plants were those on muskmelon seedlings inoculated with strain P2 of race taken from each replicate but isolations were made only from 2 of F. o. melonis (12). At least six of the 10 seedlings inoculated the taproots, which were washed in the same manner as the with a virulent strain eventually died. Water-treated controls previous root samples. Several cross sections, 2-3 cm long, were always were undamaged by the procedure. Sixteen colonies taken from each taproot. Sample weights varied with crop, identified as nonpathogenic strains of F. oxysporum did not reflecting differences in the mass of their taproots. The smallest induce symptoms on any of the inoculated muskmelon seedlings.