Cucurbit Genetics Cooperative Report 12:9-10 (article 2) 1989
D.C. Linde, J.M. Shively and B.B. Rhodes
Clemson University Edisto Research and Education Center, Blackville, SC 29817 (first and third authors); and Department of Biological Sciences, Clemson University, Clemson, SC 29632 (second author)
Anthracnose (causal agent = Colletotrichum lagenarium) is one of the most important diseases of cucumbers, and cucurbits in general. An in vitro or greenhouse screening aid for selecting anthracnose resistance could be valuable if it saved time and money. Toxins are one class of screening aids investigated increasingly for selecting host resistance.
The chlorotic halo sometimes observed around the necrotic lesion caused by C. lagenarium suggests that one or more toxins may be involved in its pathogenesis. On the basis that lipid toxins have been isolated from liquid cultures of C. nicotianae (1, 2) and C. capsici (3), we attempted to isolate lipid toxins from shake cultures of race 2 C. lagenarium.
The fungus was grown in modified (40 g/l) sucrose) Czapek Solution liquid medium (4L) for 2 weeks on a shaker run at 150 rpm. Standard partition chromatography methods with ethyl acetate were used to obtain acidic and neutral lipid fractions from the hyphae, culture broth, and culture pellet. Only the acidic and neutral lipid fractions from the culture broth were found to inhibit cucumber and, to a greater extent, lettuce seed germination. When the 2 fractions were combined in ethanol and spotted on punctured tobacco and cucumber leaves, large necrotic lesions with chlorotic halos similar to anthracnose lesions were observed. The control (ethanol only) produced a small, almost transparent lesion.
N-hexane washes of the acidic and neutral lipid fractions contained no detectable toxic activity with the lettuce seed germination assay. Thin layer chromatography was used to purify the toxic fractions. A total of 3 acidic and 1 neutral lipid toxin fractions were identified. Their mobilities in several solvent systems are shown in Table 1.
The fungus was grown again in liquid culture (4L), and the acidic and neutral lipid fractions were obtained using partition chromatography as above. The two fractions were combined (total weight = 0.13 g) and suspended in 1 ml ethanol. One u1 was used in a leaf puncture assay in the greenhouse on 7 cucumber genotypes with varying levels of resistance to race 2 C. lagenarium. The leaf puncture assay was also used of 16 F2 cucumber plants segregating for anthracnose resistance. The 7 genotypes and 16 respective F3 families were inoculated in the field with the same isolate. No relationship was found between the lesion size caused by the leaf puncture assay and the field disease rating for either the 7 genotypes or the 16 F2 plants and their respective F3 families.
The high concentration of lipids used in the leaf puncture assay may have precluded a proportional response to the toxin. An alternative hypothesis is that the putative toxin is only one element of the pathogen’s virulence.
Table 1. Rf values of the 4 toxic factions in 10 solvent systems on silica gel G thin layer chromatography.
Toxic Fraction y |
||||
Solvent systemz |
N1 |
Ala |
Alb |
A2 |
| Bz: EtAc: Ac 70 : 30: 1 |
0.250-0.0500 | 0.125-0.375 | 0.125-0.375 | 0.500-0.625 |
| Bz: MeOH 90:10 |
0.47 | – | – | – |
| Bz: EtAc 30:70 |
0.56 | – | – | – |
| Et2 100 |
0.50-0.60 | 0.00-0.10 | 0.30-0.50 | 0.00-0.10 |
| EtAc 100 |
0.50 | 0.00 | 0.00 | – |
| CHCl3: MeOH 90:10 |
0.58 | – | 0.00 | – |
| Acetone 100 |
– | 0.00-0.30 | 0.00 | – |
| EtOH 100 |
– | – | 0.10-0.30 | – |
| EtOH: MeOH | – | – | 0.70-0.80 | – |
| MeOH 100 |
– | – | 1.00 | – |
z BZ = benzene, EtAc = ethyl acetate, Ac = acetic acid, CHCl3 = chloroform, Et2 = diethyl ether, MeOH = methanol, EtOH = ethanol.
y‘N’ = neutral lipid toxic fraction, ‘A’ = acidic lipid toxic fraction.
Literature Cited
- Gohbara, M., S-B. Hyeon, A. Suzuki and S. Tamura. 1976. Isolation and structure elucidation of colletopyrone from Colletotrichum nicotianae. Agr. Biol. Chem. 40: 1453-1455.
- Gohbara, M., Y. Kosuge, S. Yamasaki, Y. Kimura, A. Suzuki and S. Tamura. 1978. Isolation, structures, and biological activities of colletotrichins, phytotoxic substances from Colletotrichum nicotinae. Agr. Biol. Chem. 42: 1037-1043.
- Grove, J.F., R.N. Speake and G. Ward. 1966. Metabolic products of Colletotrichum capsici: isolation and characterization of acetocolletotrichin and colletodiol. J. Chem. Soc. C: 230-237.