J Gen Plant Pathol (2007) 73:342–348 DOI 10.1007/s10327-007-0031-y
FUNGAL DISEASES
Selective media for Fusarium oxysporum Norio Nishimura
Received: 30 October 2006 / Accepted: 7 March 2007 / Published online: 7 September 2007 The Phytopathological Society of Japan and Springer 2007
Abstract Selective media without pentachloronitrobenzene were developed for quantitative assays of Fusarium oxysporum in soils. Media Fo-G1 and Fo-G2 were effective for naturally infested soils, Fo-W1 and Fo-W2 for wildtype isolates in soils containing a nitrate-nonutilizing (nit) mutant, and Fo-N1 and Fo-N2 for nit mutants. Selective media were made using ammonium citrate dibasic, L-sorbose, econazole nitrate, 25% iminoctadine triacetate solution and 50% tolclofos-methyl wettable powder for soil dilutions of 100-fold or more (Fo-G1, FoW1 and Fo-N1) and 10-fold (Fo-G2, Fo-W2 and Fo-N2). Potassium chlorate was added to Fo-N1 and Fo-N2. The efficacy for selectively isolating F. oxysporum was confirmed using six soils naturally infested with one of six formae speciales of F. oxysporum and with soil dilutions containing conidia of wild-type strains or nit mutants from the six formae speciales. On Fo-G1 and Fo-G2, most colonies of F. oxysporum were compact and round with purplish or reddish pigment in the reverse. Cylindrocarpon sp. formed colonies as large as those of F. oxysporum but were distinguishable by their colony morphology. Other contaminants such as F. solani, F. moniliforme, and Trichoderma were suppressed by medium ingredients and colonies of F. oxysporum. On FoW1 and Fo-W2, colony morphology of F. oxysporum and contaminants corresponded to that on Fo-G1 and Fo-G2, although F. oxysporum failed to produce the pigment. On Fo-N1 and Fo-N2, nit mutants formed clear colonies from 100- and 10-fold soil dilutions, respectively, and contaminants seldom formed large colonies.
N. Nishimura (&) National Agricultural Research Center for Kyushu Okinawa Region, 1823 Mii, Kurume, Fukuoka 839-8503, Japan e-mail:
[email protected]
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Keywords Selective medium Fusarium oxysporum Nitrate-nonutilizing mutant
Introduction Analysis of the population dynamics of Fusarium oxysporum Schlechtendahl in soil provides critical information on the epidemiology of the disease and its control. Therefore, many selective media have been devised for the quantitative detection of wild-type isolates and nitratenonutilizing (nit) mutants of F. oxysporum from soil. For naturally infested soils, examples are modified peptonepentachloronitrobenzene (PCNB) medium (Papavizas 1967) and Komada medium (Komada 1975); for wild-type isolates in soils containing a nit mutant, GMBP (Takehara et al. 2003); and for nit mutants, FMMCPA (Hadar et al. 1989), modified FMMCPA (Komada et al. 1995) and CGMBP (Takehara et al. 2003). These media all contain PCNB as a wettable powder to suppress contaminants such as zygomycetes. However, the registration of PCNB products as agricultural chemicals was cancelled by 2000 in Japan, and consequently their production was ceased. In addition, experiments with artificially infested soils revealed that Fusarium wilt of spinach and Fusarium yellows of celery occurred at about 10 colony-forming units (CFU)/g dry soil (Naiki and Morita 1983; Elmer and Lacy 1987) and root rot of butterhead lettuce at 1 CFU/g dry soil (Nishimura 2002), indicating that these diseases could develop with fewer CFU than the detection threshold of the media. If the density of F. oxysporum in soil was 10 CFU/g dry soil, the dilution rate of the soil should be as low as 10-fold. Therefore, to increase sensitivity for detecting wild-type isolates and nit mutants of F. oxysporum in light of the
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unavailability of PCNB products, I developed new selective media without PCNB. Because we have no selective media for detecting F. oxysporum from 100- or 10-fold dilutions of soil, the threshold of the media was evaluated with naturally infested soils and oven-dried or raw soil dilutions containing conidia. This paper describes the development of new selective media for F. oxysporum and their sensitivity in detection.
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F. oxysporum and contaminants form large or irregular colonies merging with each other, which made colony count difficult. To suppress their excessive growth, the amount of econazole nitrate, 25% iminoctadine triacetate solution and 50% tolclofos-methyl wettable powder was increased. Consequently, Fo-G2, Fo-W2 and Fo-N2 were developed.
Media Materials and methods General Selective media were modified on the basis of CGMBP and GMBP, which allowed the growth of contaminants such as F. solani, F. moniliforme and Trichoderma. (1) Because iminoctadine inhibited the growth of Trichoderma but not F. oxysporum (K. Tanabe, Nippon Soda Co., personal communication), 25% iminoctadine triacetate solution (Wako Pure Chemical Industries) was selected. (2) Miconazole nitrate was replaced by econazole nitrate because of its greater suppression of F. moniliforme and Cylindrocarpon sp. (3) L-Asparagine was replaced with ammonium citrate dibasic because of its stronger suppressiveness of F. solani. (4) Trial media with these three compounds and without PCNB allowed the growth of Rhizopus and Mucor, thus L-sorbose was used as a carbon source because it suppresses mucoraceous fungi (Awuah and Lorbeer 1986). (5) Because Syncepharastrum and an unidentified zygomycetes sparsely grew and formed conidia on trial media containing L-sorbose, 50% tolclofos-methyl wettable powder (Rizolex wettable powder, Sumitomo Chemical Co.) was used to inhibit their formation of conidia. (6) When trial media containing potassium chlorate were autoclaved, the vapor from the autoclave irritated the throat. In addition, when L-sorbose and the iminoctadine triacetate solution were added before autoclaving, the former stained the medium and the latter caused precipitation at high concentrations. Therefore, the potassium chlorate, L-sorbose, iminoctadine triacetate and tolclofos-methyl were added after autoclaving. As a result, a selective medium named Fo-G1 was developed for naturally infested soils, Fo-W1 for wild-type isolates and Fo-N1 for nit mutants. Fo-G1 and Fo-W1 were useful for soils diluted 100-fold or more. Fo-N1 with 5 mg/ l of econazole nitrate was useful for soils diluted 100-fold, but often failed to sustain growth of some nit mutants from the agar solution. Therefore, Fo-N1 containing 2 mg/l of econazole nitrate was developed for isolation from soil diluted more than 100-fold (Table 1). When soil dilutions of 10-fold were spread on plates of Fo-G1, Fo-W1 and Fo-N1, nutrition from soil made
Fo-G1, the basic medium, contained KH2PO4, 1 g; KCl, 0.5 g; MgSO47H2O, 0.5 g; ammonium citrate dibasic, 2 g; econazole nitrate, 5 mg (dissolved in 0.5 ml of dimethyl sulfoxide); H3BO3, 0.5 g; trace element solution, 0.2 ml; chloramphenicol, 0.25 g; and agar, 20 g per 1 l of distilled water. After autoclaving for 15 min at 121C, L-sorbose, 20 g; 25% iminoctadine triacetate solution, 0.05 ml; and 50% tolclofos-methyl wettable powder, 1 mg (suspended in a small amount of distilled water) were added. After cooling down to about 60C, the medium was adjusted to pH 3.7–3.9 with 10% H3PO4 using a pH meter, and 15 ml aliquots were dispensed in 9-cm plates. Plates were covered with lids and kept in the darkness for 3–7 days to dry the surface of the medium. The trace element solution contained citric acid, 5 g; FeSO47H2O, 5 g; ZnSO47H2O, 1 g; CuSO45H2O, 0.5 g; MnSO45H2O, 0.5 g; Na2MoO42H2O, 0.05 g per 95 ml of distilled water. Ingredients in other selective media are shown in Table 1. Potato dextrose agar containing 0.25 g/l of chloramphenicol (PDA+c) was used for evaluating sensitivity of the selective media during recovery tests of conidia from agar solutions and oven-dried soil dilutions.
Soils and fungal strains Six soils naturally infested either with F. oxysporum f. sp. lactucae race 1, f. sp. colocasiae, f. sp. fragariae, f. sp. cucumerinum, f. sp. apii or f. sp. lycopersici race 2 were used to evaluate the isolation efficacy of the media from different soils (Table 2). They were collected between 1998 and 2000. After sieving through a 2-mm mesh, a portion of each soil was stored at 9C and used by 2002. The rest was oven-dried at 105C for 12 h. Twelve F. oxysporum strains were used for artificial infestation (Table 2). Strains Focu-1S and Foly2-A1S were isolated from the soils. Other pathogens were selected from stock cultures. Nit mutants were isolated by the method of Correll et al. (1987). Conidia were obtained from 8-day-old potato-sucrose agar cultures as follows: small agar blocks with mycelia were cut from the center of cultures, soaked in distilled water, and filtered through a double-layered
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Table 1 Amount of nitrogen sources, trace element solution and antifungal ingredients in each selective medium Ingredienta
Selective medium Fo-G1
Fo-G2
Fo-W1
Fo-W2
Fo-N1
Fo-N2
Ammonium citrate dibasic (g/l)
2
2
–
–
2
2
NaNO3 (g/l)
–
–
2
2
2
2
Econazole nitrate (mg/l)
5
10
5
10
5 or 2b
10
Trace element solution (ml/l)
0.2
–
0.2
–
0.2
–
Potassium chlorate (g/l)
–
–
–
–
10
10
Iminoctagine triacetate 25% solution (ml/l)
0.05
0.4
0.05
0.4
0.05
0.4
Tolclofos-methyl 50% wettable powder (mg/l)
1
3
1
3
1
3
a
Other ingredients were the same as those of Fo-G1 in the text
b
5 mg/l of econazole nitrate for soils diluted 100-fold, 2 mg/l for soil diluted more than 100-fold
Table 2 Soils, pathogenic Fusarium oxysorum present, and strains used as inoculum source Naturally infested soil (location)a
Strains introduced as conidia to dilutions of each soil Wild-typeb
Nit mutantc
Haplic andosols with F. o. f. sp. lactucae race 1 (Nagano)
Fol-96-7S (lactucae race 3)
Fol-nit3 (lactucae race 3)
Pachic andosols with F. o. f. sp. colocasiae (Miyazaki)
Fo-25-2S (colocasiae)
Foc-nit3 (colocasiae)
Yellow soil with F. o. f. sp. fragariae (Fukuoka)
Fof-96-2S (fragariae)
Fof-nit3 (fragariae)
Yellow soil with F. o. f. sp. cucumerinum (Oita)
Focu-1S (cucumerinum)
Focu-nit3 (cucumerinum)
Gray lowland soil with F. o. f. sp. apii (Fukuoka)
Foa-2S (apii)
Foa-nit3 (apii)
Gray lowland soil with F. o. f. sp. lycopersici race 2 (Saga)
Foly2-A1S (lycopersici race 2)
Foly3-nit3 (lycopersici race 3)
a
F. o. showed F. oxysporum
b
Introduced to dilutions of oven-dried soils
c
Introduced to dilutions of the naturally-infested soils and their oven-dried counterparts
paper towel. The density of conidia in each filtrate was measured with a hemacytometer and adjusted to 8,000 conidia/ml. These conidial inocula were added to 0.05% agar solution and soil dilutions to obtain 20 colonies per plate.
Isolation from naturally infested soils One-hundred-fold dilutions of naturally infested soils were made by adding 1 g of raw soil to 99 ml of 0.05% agar solution in a 200-ml Erlenmeyer flask, and 0.5-ml of the dilution was spread on each plate, containing Fo-G1 or FoW1. Because the density of F. oxysporum in raw soils was too high for Fo-G2 and Fo-W2, raw soils were mixed with oven-dried counterparts at 1:9 (v/v), and then 10 g of the soils were suspended to 90 ml of the agar solution, followed by spreading 0.5-ml portions on plates of Fo-G2 and Fo-W2. In the case of nit mutants, conidial inocula each were added to the 100- and 10-fold dilutions of a naturally infested soil that had the same forma specialis as that of inoculum (Table 2), and 0.5- and 1-ml samples were spread on plates of Fo-N1 and Fo-N2. Experiments were repeated 2–4 times, with five plates per sample.
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Recovery from agar solution and oven-dried soil dilutions containing conidia Oven-dried soils were diluted on a fresh mass basis (20% moisture content), i.e., the 100- and 10-fold dilutions were made by adding 0.8 and 8 g of each oven-dried soil to 99.2 and 92 ml of the agar solution, respectively. Conidial inocula each were added to the agar solution and to the dilutions of an oven-dried soil for which the forma specialis corresponded to that of inoculum (Table 2), then 0.5ml portions were spread on plates of selective media and PDA+c. Experiments were repeated 2–4 times with five plates per sample. As for nit mutants, recovery tests from naturally infested soil dilutions were simultaneously carried out with the same conidial inocula.
Suspension, incubation and sensitivity of F. oxysporum recovery Soil dilution and agar solution containing conidia were reciprocally shaken for 20 min at about 120 cycles/min. Aliquots were pipetted from each sample agitated on a magnetic stirrer and spread on agar plates placed on a
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b Fig. 1 a Colonies of Fusarium oxysporum and contaminants on plates of Fo-G1 medium. A 0.5-ml sample of the 100-fold dilution of naturally infested soils was spread on plates. Upper row, surface view; lower row, reverse view. Left to right, soils naturally infested with F. oxysporum f. sp. lactucae race 1, f. sp. apii and f. sp. lycopersici race 2. Abbreviations to left of colonies: O, F. oxysporum; Z, small, reddish-purple colonies; S, F. solani; C, Cylindrocarpon sp.; P, Penicillium; and A, Aspergillus. b The surface of colonies of Fusarium oxysporum f. sp. apii (left) and Cylindrocarpon sp. (right) on Fo-G1. c Colonies of Fusarium oxysporum and contaminants on Fo-W1. A 0.5-ml sample of a 100-fold dilution of a soil naturally infested with F. oxysporum f. sp. apii was spread on plates. Upper, surface view; lower, reverse view. Letters to the left of colonies were the same as those on Fig. 1. d The surface of colonies of Fusarium oxysporum f. sp. apii (left) and Cylindrocarpon sp. (right) on Fo-W1. e Colonies of a nit mutant of F. oxysporum f. sp. apii on Fo-N1 and Fo-N2. Left, Fo-N1 plates that received 0.5 ml of 100-fold dilution. Right, Fo-N2 plates that received 1 ml of 10-fold dilution. Upper row, surface view; lower row, reverse view. Abbreviations: N, nit mutant; S, F. solani; C, Cylindrocarpon sp.; and P, Penicillium
soils, including formae speciales lactucae race 1 (Fig. 1 left), apii (Fig. 1 middle), colocasiae and fragariae, were compact with a reddish surface and reddish-purple reverse. Most colonies from the soil infested with f. sp. lycopersici race 2 were compact but purplish-red with little aerial hyphae (Fig. 1, right). They had the same characteristics as pathogenic strain Foly2-A1S. Colonies from the soil infested with f. sp. cucumerinum tended to be large. Although small, reddish-purple colonies, some of which were F. oxysporum, were observed (Fig. 1), most small colonies seldom formed typical microconidia of F. oxysporum on PDA. Therefore, they were not identified to species. The following contaminants were found on Fo-G1 (Fig. 1). F. solani formed small colonies when F. oxysporum colonies were numerous. The colony surface and the reverse were white, yellowish-orange or brown. Colonies of F. moniliforme were few and initially small. Cylindrocarpon sp. formed colonies similar to F. oxysporum with a light brown or reddish-brown reverse (Fig. 1, middle). However, the colony surface of Cylindrocarpon sp. was coarser than that of F. oxysporum (Fig. 1b). This fungus was abundant in the soil infested with F. oxysporum f. sp. apii but was lower than the detection limits in other soils. Paecylomyces sp. in the soil infested with F. oxysporum f. sp. fragariae also formed relatively large colonies; however, its colonies were dark green and infrequent. Penicillium formed small colonies, while Aspergillus sometimes produced large ones (Fig. 1, left). As for Fo-G2 with the 10-fold dilutions of soils, colony morphologies of F. oxysporum and contaminants looked similar to those on Fo-G1.
turntable with a glass spreader. The number of colonies was counted after incubation at 25C for 7–9 days. Sensitivity of F. oxysporum recovery was calculated as (average number of colonies on a selective medium/average number of colonies on PDA+c) · 100.
Results Colony morphology on Fo-G1 and Fo-G2 Colonies of F. oxysporum predominantly developed from six naturally infested soils on Fo-G1 (Fig. 1a). They showed variation in size and color. Colonies from four
Table 3 Recovery of nit mutant conidia on PDA+c, Fo-N1, and Fo-N2 from agar solution and dilutions of oven-dried and raw soils Nit Mutanta
Number of colonies per plate and sensitivity of F. oxysporum recovery Agar solution
Dilution rate of soil and the amount of sample per plate 100-fold, 0.5 ml
10-fold, 0.5 ml
Oven-dried soil PDA+c
b
Fo-N1
c
%
e
PDA+c
Fo-N1
Raw soil d
%
Fo-N1
d
Oven-dried soil PDA+c
Fo-N2
Raw soil %
Fo-N2
Fol-nit3
13 ± 3
12 ± 4
92
16 ± 4
19 ± 1
119
17 ± 2
25 ± 1
22 ± 4
88
22 ± 5
Foc-nit3
12 ± 1
12 ± 4
100
17 ± 1
19 ± 2
112
19 ± 1
20 ± 2
21 ± 2
105
19 ± 0
Fof-nit3
16 ± 2
13 ± 0
81
14 ± 4
14 ± 7
100
15 ± 6
20 ± 1
22 ± 3
110
21 ± 1
Focu-nit3
10 ± 3
11 ± 4
110
19 ± 2
19 ± 1
100
19 ± 0
16 ± 2
16 ± 1
100
16 ± 6
Foa-nit3
11 ± 2
10 ± 3
91
14 ± 4
14 ± 6
100
15 ± 5
15 ± 5
18 ± 6
120
22 ± 6
Foly3-nit3
11 ± 1
10 ± 4
91
21 ± 8
22 ± 4
105
20 ± 1
23 ± 3
83
24 ± 8
a
Conidia were added to agar solution and soil dilutions to obtain 20 colonies per plate
b
PDA supplemented with 0.25 g/l of chloramphenicol
c
Fo-N1containing 2 mg/l of econazole nitrate
d
Fo-N1containing 5 mg/l of econazole nitrate
e
(Average number of colonies on Fo-N1 or Fo-N2/average number of colonies on PDA+c) · 100
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Colony morphology on Fo-W1 and Fo-W2
Discussion
The 100-fold dilutions of the soil suspensions were also spread on plates of Fo-W1. Colonies of F. oxysporum and the contaminants on plates corresponded to those on Fo-G1 (Figs. 1c, 1d). Similar results were obtained with Fo-W2. However, F. oxysporum colonies failed to produce the pigment as with Fo-G1 in the reverse. The surface of colonies was white, except that a few colonies from the soil infested with f. sp. lactucae race 1 were bluish-purple.
F. oxysporum formed compact colonies on the new selective media without PCNB. When the colonies were abundant, the growth of most colonies of F. solani, F. moniliforme and Trichoderma were suppressed. These characteristics of the new media made it possible to isolate F. oxysporum from the 100- or 10-fold dilution of soil. The characteristics of each medium and comments on practical use are described next. Fo-G1 and Fo-G2 allow the growth of nit mutants that must be distinguished from the wild type after isolating both of them from the soil. Therefore, these two media are suitable for the isolation of the wild type from naturally infested soils. The density of F. oxysporum in the field is usually presumed to be more than 200 CFU/g (Chuang and Ko 1981). If 0.5-ml portions of the 100-fold dilutions that are spread on plates of Fo-G1 produced one colony of F. oxysporum per plate, the density in the soil is presumably ca. 250 CFU/g dry soils; therefore, Fo-G1 should be usable for most soils. When the population densities of both F. oxysporum and fast-growing contaminants such as Cylindrocarpon are low, the detection limit of F. oxysporum would be lowered further by using 10-fold dilutions and Fo-G2. On these media, both the pathogenic strains of F. oxysporum that were used as conidial inocula and those that were isolated from naturally infested soils produced relatively large, distinguishable colonies. On the other hand, nonpathogenic F. oxysporum was isolated not only from relatively large colonies but also from small ones identified with a z in Fig. 1. However, most of the small colonies were not identified to species. Because the density of nonpathogenic F. oxysporum cannot be measured, we need to investigate the small colonies further. Fo-W1 and Fo-W2 are used to isolate wild-type strains from soils that have nit mutants because these media do not allow colony formation of nit mutants. Nonpathogenic F. oxysporum has often been introduced to disinfested soils as biocontrol agents to suppress pathogenic F. oxysporum (e.g., Tezuka and Makino 1991; Ichikawa et al. 1998). In such experiments, it was possible to investigate population dynamics of both pathogenic and nonpathogenic forms when these media were combined with Fo-N1 or Fo-N2 (Nishimura 2002). Furthermore, a strain of F. oxysporum f. sp. lactucae race 3 produced a bluish-purple pigment specifically on Fo-W1, which was used to distinguish between the pathogenic strain and others (Nishimura 2003). As for small colonies, because they also appear on these media, further investigation is necessary. Fo-N1 and Fo-N2 allowed the growth of nit mutants in soil dilutions of 100- and 10-fold, respectively, but strongly suppressed colony formation of wild-type isolates of F. oxysporum and contaminants. Detection limit of Fo-N2
Colony morphology on Fo-N1 and Fo-N2 Nit mutants developed clear colonies on Fo-N1 containing 5 mg/l of econazole nitrate from the 100-fold dilutions of raw soils. Contaminants, including other Fusarium spp., were strongly suppressed. Similar results were obtained with Fo-N2, when either a 0.5- or 1-ml portion of the 10fold dilutions was applied. Contaminants such as Cylindrocarpon sp. and F. solani seldom formed large, definite colonies on Fo-N2. An example of the soil infested with F. oxysporum f. sp. apii was shown in Fig. 1e. Nit mutants whose parent isolates produced the pigment formed clear colonies with a reddish-purple pigment on Fo-N1 and Fo-N2, except that a mutant of F. oxysporum f. sp. lycopersici race 3 failed to produce the pigment on Fo-N2.
Recovery from agar solution and oven-dried soil dilutions containing conidia The sensitivity of the selection for F. oxysporum was evaluated by comparing the recovery of the fungal inocula (conidia) on a selective medium relative to its recovery on PDA+c. The number of colonies of the six wild-type isolates was almost equivalent on PDA+c and Fo-G1 or Fo-G2, when conidia were suspended in the agar solution and in the 100- or 10-fold dilutions of ovendried soils. The sensitivity of F. oxysporum recovery ranged from 83 to 120% for six isolates (data not shown). Similar results were obtained with Fo-W1 and Fo-W2 under the same regime, and their recovery sensitivities ranged from 84 to 113% (data not shown). In recovery tests of Fo-N series media, Fo-N1 containing 2 and 5 mg/ l of econazole nitrate and Fo-N2 were used for the agar solution, the 100-fold dilutions and the 10-fold ones, respectively. Their sensitivities ranged from 81 to 120%; moreover, when the same conidial inocula were added to soil dilutions of raw soils, the numbers of colonies were almost equivalent to those from oven-dried soil dilutions (Table 3).
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was presumed to be about 10 CFU/g soil, because it was possible to dispense 1 ml of the 10-fold dilution on a plate. Thus, these media are useful for precisely examining the population dynamics of the pathogen. However, it should be noted that some nit mutants were unable to form colonies on Fo-N1 with 5-mg/l econazole nitrate when conidia were suspended in agar solution. When the dilution of soil exceeds 100-fold, Fo-N1 containing 2-mg/l econazole nitrate should be used. Alternatively, if oven-dried soil is added to the soil dilutions at the rate of 1:99, Fo-N1 containing 5-mg/l econazole nitrate can be used. The growth of F. oxysporum and contaminants on the media was influenced by soil nutrition and antifungal ingredients. Therefore, it is important to use these selective media according to the dilution rate of the soil and to dispense at least 0.5 ml of sample on a plate. Variability among F. oxysporum strains should also be noted; e.g., certain strains of F. oxysporum formed small colonies on Fo-G1, Fo-G2, Fo-W1 and Fo-W2, and the nit mutant, Foly3-nit3, of F. oxysporum f. sp. lycopersici race 3 lacked pigmentation on Fo-N2. Because some strains and formae speciales were strongly influenced by the antifungal ingredients, it is necessary to check in advance how a target F. oxysporum strain grows from soil dilutions on the selective media. Acknowledgments The author is grateful to Dr. N. Matsumoto, National Institute for Agro-Environmental Sciences, for critical reading of the manuscript and to Dr. K. Tanabe, Nippon Soda Co., Ltd., for the offer of the information on iminoctadine triacetate.
References Awuah RT, Lorbeer JW (1986) A sorbose-based selective medium for enumerating propagules of Fusarium oxysporum f. sp. apii race 2 in organic soil. Phytopathology 76:1202–1205
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J Gen Plant Pathol (2007) 73:342–348 Chuang TY, Ko WH (1981) Propagule size: its relation to population density of microorganisms in soil. Soil Biol Biochem 13:185– 190 Correll JC, Klittich CJR, Leslie JF (1987) Nitrate nonutilizing mutants of Fusarium oxysporum and their use in vegetative compatibility tests. Phytopathology 77:1640–1646 Elmer WH, Lacy ML (1987) Effects of inoculum densities of Fusarium oxysporum f. sp. apii in organic soil on disease expression in celery. Plant Dis 71:1086–1089 Hadar E, Katan J, Katan T (1989) The use of nitrate-nonutilizing mutants and a selective medium for studies of pathogenic strains of Fusarium oxysporum. Plant Dis 73:800–803 Ichikawa T, Makino T, Doi M (1998) Biological control of Fusarium root rot of butterhead lettuce in soil culture by application of nonpathogenic Fusarium (in Japanese). Proc Kansai Pl Prot 40:119–120 Komada H (1975) Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Rev Plant Protec Res 8:114–125 Komada H, Ueda S, Yamamoto H (1995) Improvement on selectivity of nitrate non-utilizing mutant selective medium for Fusarium (in Japanese). Plant Protect 49:163–166 Naiki T, Morita Y (1983) The population of spinach wilt fungus, Fusarium oxysporum f. sp. spinaciae, and the wilt incidence in soil. Ann Phytopath Soc Jpn 49:539–544 Nishimura N (2002) Control of root rot of butter head lettuce by soil disinfection and nonpathogenic Fusarium oxysporum (abstract in Japanese). Jpn J Phytopathol 68:203–204 Nishimura N (2003) Vegetative compatibility groups in Fusarium oxysporum f. sp. lactucae race 3 causing butter-head lettuce root rot in Fukuoka (in Japanese with English abstract). Kyushu Pl Prot Res 49:37–40 Papavizas GC (1967) Evaluation of various media and antimicrobial agents for isolation of Fusarium from soil. Phytopathology 57:848–852 Takehara T, Kuniyasu K, Mori M, Hagiwara H (2003) Use of a nitrate-nonutilizing mutant and selective media to examine population dynamics of Fusarium oxysporum f. sp. spinaciae in soil. Phytopathology 93:1173–1181 Tezuka N, Makino T (1991) Biological control of Fusarium wilt of strawberry by nonpathogenic Fusarium oxysporum isolated from strawberry (in Japanese with English abstract). Ann Phytopath Soc Jpn 57:506–511