Cucurbit Genetics Cooperative Report 12:9-10 (article 2) 1989
Toxins: Potential Screening Aid for Selecting Anthracnose
Resistance in Cucumbers
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.
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|
Solvent systemz |
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|
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Bz: EtAc: Ac
70 : 30: 1 |
|
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|
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Bz: MeOH
90:10 |
|
|
|
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Bz: EtAc
30:70 |
|
|
|
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Et2
100 |
|
|
|
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EtAc
100 |
|
|
|
|
CHCl3: MeOH
90:10 |
|
|
|
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Acetone
100 |
|
|
|
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EtOH
100 |
|
|
|
|
EtOH: MeOH |
|
|
|
|
MeOH
100 |
|
|
|
|
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.
