- Email: [email protected]
Phytotoxicity of fusaric acid and analogs to cotton
Toxicon 57 (2011) 176–178
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
Phytotoxicity of fusaric acid and analogs to cotton R.D. Stipanovic*, L.S. Puckhaber, J. Liu, A.A. Bell USDA, Agricultural Research Service, Southern Plains Agricultural Research Center, 2765 F and B Road, College Station, TX 77845, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 July 2010 Received in revised form 5 October 2010 Accepted 8 October 2010 Available online 16 October 2010
We developed a cotton cotyledonary leaf bioassay to test the phytotoxicity of fusaric acid (5-butylpicolinic acid), picolinic acid and related analogs. The compounds were dissolved in aqueous Tween 80, and 20 mL of the test solution was placed at three positions on the leaf, and a needle was used to puncture the leaf through each drop; the results were evaluated after 48 h. In contrast to previous studies, we found the carboxylic acid group is essential for phytotoxicity. Nicotinic acid was considerably less phytotoxic than picolinic acid and conversion of picolinic acid to the amide or N-oxide decreased phytotoxicity. Increasing the alkyl chain length at the 5-position on picolinic acid from two up to five carbons atoms increased phytotoxicity. Fusaric acid methyl ester, the most phytotoxic compound tested, is a naturally occurring compound; as such it has potential as a herbicide in organic farming. Published by Elsevier Ltd.
Keywords: Phytotoxin Fusarium oxysporum f. sp. vasinfectum Fusarium nygamai Fusaric acid Methyl fusarate Cotton 3-Butylpyridine
Fusaric acid has been isolated from 11 Fusarium species including the cotton pathogen, Fusarium oxysporum f. sp. vasinfectum. Although fusaric acid shows low toxicity to animals, it is classified as a wilt-inducing toxin in many plants. Capasso et al. (1996) reported that fusaric acid and its methyl ester were produced by Fusarium nygamai, and proposed that these compounds could be used as bioherbicides in controlling witchweed (Striga hermonthica). Subsequently, Vischetti and Esposito (1999) extended these studies and found that fusaric acid methyl ester is degraded to 3-butylpyridine in the soil. In other work, a series of papers reported that the cotton plant converts fusaric acid into 3-butylpyridine, aka vivotoxin, and this compound is 100fold more toxic than fusaric acid to cotton (Gossypium hirsutum) (Bekker et al., 1971; Becker et al., 1972; Shilina et al., 1973). These results led us to consider that fusaric acid and its analogues might provide base compounds for developing new herbicides. Furthermore, because these compounds occur in nature they might be useful in organic farming. * Corresponding author. Tel.: þ1 979 260 9232; fax: þ1 979 260 9319. E-mail address: [email protected] (R.D. Stipanovic). 0041-0101/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.toxicon.2010.10.006
Our interest in cotton production prompted us to develop a cotton cotyledonary leaf assay to evaluate the phytotoxicity of a series of fusaric acid analogues including 3-butylpyridine. Fusaric acid may be chemically defined as a substituted pyridine. The structural components added to this primary nucleus are the position of the carboxylic acid group, derivatives of the carboxylic acid, and variations in the length of the alkyl side chain and its location on the pyridine ring. Of the large number of possible combinations, we chose to investigate the phytotoxicity of the compounds shown in Fig. 1. Our bioassay utilized acid delinted Coker-312 cottonseed that were placed in moist paper rolls and allowed to germinate in the dark at 30 C for two days. Individual seedlings were then selected for uniformity and transplanted into sterilized greenhouse planting mix in 450 mL plastic cups in a growth chamber with a 13-h, 28 C day and 11-h, 22 C night cycle. At the beginning of the day cycle only incandescent lights were activated, followed by onehalf of the florescent lamps after 15 min and all fluorescent lamps after an additional 15 min. Lights were turned off in the reverse order prior to darkness. Solutions of chemicals (8, 4, 2, 1, and 0.5 mM) were prepared in 0.1% Tween 80 and
R.D. Stipanovic et al. / Toxicon 57 (2011) 176–178
R2
R3 N
N
R1
Fusaric Acid; R1 = COOH; R2 = n-Butyl; R3 = H 1) R1, R3 = H; R2 = Methyl 2) R1, R3 = H; R2 = Ethyl 3) R1, R3 = H; R2 = n-Butyl 4) R1 = COOH; R2, R3 = H 5) R1 = CONH2; R2, R3 = H 7) R1 = COH; R2, R3 = H 8) R1 = CN; R2, R3 = H 9) R1, R2 = H; R3 = COOH 10) R1 = n-Butyl; R2 = H; R3 = COOH 11) R1 = COOCH3; R2 =n-Butyl; R3 = H 12) R1 = COOH; R2 = CH2 HO H ;R3 = H CH2 CH
COOH
O 6
CH3
13) R1 = R1 = COOCH3; R2 = CH2 HO H ;R3 = H CH2 CH CH3 14) R1 = COOH; R2 = Ethyl; R3 = H 15) R1 = COOH; R2 = n-Propyl; R3 = H 16) R1 = COOH; R2 = n-Pentyl; R3 = H 17) R1 = COOH; R2 = n-Heptyl; R3 = H Fig. 1. Structures of fusaric acid and analogues.
water. Test solutions were stored in the refrigerator until used. Plants were inoculated with the test solutions on the morning on the 7th day after planting. To introduce the chemical, drops (20 mL each) of test solutions were placed at three different positions on a cotyledonary leaf, and a needle was used to puncture the leaf through each drop. Forty-eight hours after inoculation, cotyledonary leaves attached to the plant were evaluated for necrosis by three individuals and then photographed to provide a permanent record for later referral. The degree of necrosis was scored
177
on a scale of 0–5 with 0 being no effect and 5 being severe necrosis and leaf curling around the area of inoculation (see Supplementary material). Some of the chemicals tested were purchased including: fusaric acid, 3-methylpyridine (1), 3-ethylpyridine (2), 3-butylpyridine (3), nicotinic acid (9), picolinic acid N-oxide (6) [Aldrich Chemical, Milwaukee, WI]; 5-propylpicolinic acid (15), and 5-pentylpicolinic acid (16) [Princeton Gold Collection I, Monmouth Junction, NJ]; 2-cyanopyridine (8), and picolinic acid (4) [Alfa Aesar, Ward Hill, MA]; picolinaldehyde (7) [Acros Organics, Morris Plains, NJ]; and 5-ethyl picolinic acid (14) [Ryan Scientific, Mt. Pleasant, SC]. Several of the compounds were synthesized using published methods: fusarinolic acid (12), 5-butylnicotinic acid (10) and 5-heptylpicolinic acid (17) [Song and Yee (2001)]; picolinamide (5) (Pavlik, 2005); and the methyl esters of fusaric and fusarinolic acids (11) and (13) (Hasimoto et al., 1981). We used picolinic acid as a model compound to test the effects of pyridine ring substitutions on toxicity (Table 1). The picolinic acid showed some phytotoxicity, but significantly less than fusaric acid. Conversion of the acid group to an amide or reduction to an aldehyde reduced the activity below that of picolinic acid. Shifting the carboxylic acid group from position 2 to 3 (i.e., nicotinic acid) also reduced activity, as did conversion of picolinic acid to its N-oxide. Addition of alkyl groups to the 5-position of picolinic acid led to a gradual increase in phytotoxicity as the length of the alkyl group increased. Thus, phytotoxicity increased in the order of picolinic acid < 5-ethylpicolinic acid < 5propylpicolinic acid < 5-butylpicolinic acid (i.e., fusaric acid) < 5-pentylpicolinic acid. However, 5-heptylpicolinic acid was less toxic than 5-pentylpicolinic acid. Interestingly, fusarinolic acid with the more hydrophilic side chain was less toxic than fusaric acid. Methyl fusarate, a phytotoxin found in F. nygamai (Vischetti and Esposito, 1999),
Table 1 Relative toxicity ratings (concentration mean score) [0 to 5 (S.D.)]a of fusaric acid, derivatives and analogs. Assayed by adding 20 mL of mM solution (0.1% Tween 80 in water) in three drops to the drops to the surface of a 7-day-old cotyledon and piercing through each drop with a needle then rating the necrosis after two days. Comp. #
Compound name
Concentration (mM)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Fusaric acid 3-Methylpyridine 3-Ethylpyridine 3-Butylpyridine Picolinic acid Picolinamide Picolinic acid N-oxide Picolinaldehyde 2-Cyanopyridine Nicotinic acid 5-Butylnicotinic acid Methyl fusarate Fusarinolic acid Methyl fusarinolate 5-Ethylpicolinic acid 5-Propylpicolinic acid 5-Pentylpicolinic acid 5-Heptylpicolinic acid
1.0 0 0 0 0.1 0 0 0 0 0 0 2.8 0.2 0.2 0.2 0.3 1.6 0.7
0.5
a b
1.0 (0.0)
(0.1)
(0.3) (0.3) (0.3) (0.1) (0.3) (0.4) (0.3)
1.4 0 0 0 0.3 0 0 0 0 0 0 3.2 0.3 1.0 0.9 1.1 2.3 1.5
2.0 (0.5)
(0.4)
(0.2) (0.3) (0.7) (0.1) (0.3) (0.2) (0.4)
Visual rating: 0 ¼ no necrosis; 5 ¼ severe necrosis (See Supplementary material). Significant (p < 0.0001) herbicidal effect observed with increasing concentration.
3.0 0 0 0 0.6 0.2 0 0 0 0 0.2 4.0 1.3 1.6 1.5 1.2 3.4 2.6
4.0 (0.3)
(0.6) (0.3)
(0.3) (0.6) (0.5) (0.5) (0.2) (0.3) (0.1) (0.6)
3.8 0 0 0 1.5 0 0.1 0 0 0 1.3 4.6 1.8 2.3 2.5 2.9 4.2 3.3
8.0 (0.6)
(0.9) (0.1)
(0.7) (0.4) (1.3) (0.4) (0.2) (0.8) (0.2) (0.3)
4.4 (0.3)b 0 0 0 2.8 (0.6)b 0 (0.1) 0.9 (0.2) 0 0 0.6 (0.2) 1.6 (0.3)b 5.0 (0.0)b 3.1 (0.6)b 3.1 (0.2)b 3.7 (0.1)b 4.5 (0.4)b 4.5 (0.1)b 4.1 (0.2)b
178
R.D. Stipanovic et al. / Toxicon 57 (2011) 176–178
was more active than fusaric acid, whereas fusarinolic acid and methyl fusarinolate exhibited similar toxicity. In contrast to previous reports (Bekker et al., 1971; Becker et al., 1972; Shilina et al., 1973), the carboxylic acid group or its methyl ester were found to be essential for toxicity and the position of the carboxylic acid group also affected activity such that moving the carboxylic acid from the 2-position (i.e., picolinic acid) to the 3-position (i.e., nicotinic acid) resulted in reduced phytotoxicity. In the case of fusaric acid, conversion of the carboxylic acid group to the methyl ester provided a more toxic compound. Of the compounds tested, fusaric acid and its methyl ester were among the most phytotoxic compounds showing severe wilting at 8.0 mM and detectable necrosis at the point of piercing at 0.5 mM. Thus, it would appear that evolutionary pressures within F. oxysporum and F. nygamai have combined to produce effective phytotoxins from a basic pyridine carboxylic acid building block. Acknowledgements We thank Cotton Incorporated for supporting this research. We thank Sara Duke for helpful statistical analysis. Conflict of interest statement The authors have no conflict of interest.
Appendix. Supplementary material Supplementary material associated with this article can be found in the online version, at doi:10.1016/j.toxicon. 2010.10.006. References Becker, S.E., Pushkareva, I.D., Poletaeva, V.F., Shilina, S.G., Yasakova, E.I., 1972. Nature and biosynthesis of Fusarium wilt toxin, its mechanism of action, and its transformation in the cotton plant. Bodenkultur 23, 256–271. Bekker, E.E., Dovletmuradov, K.D., Pushkareva, I.D., Poletaeva, V.F., Shilina, S.G., Yasakova, E.I., 1971. Nature and biosynthesis of the toxin of the causative agents of fusariosis wilt, the mechanism of its actions, and its possible transformation within the cotton plant. Izv. Akad. Nauk SSSR, Ser. Biol. 5, 749–754. Capasso, R., Evidenet, A., Cutignano, A., Vurro, M., Zonno, M.C., Bottalico, A., 1996. Fusaric and 9,10-dehydrofusaric acids and their methyl esters from Fusarium nygamai. Phytochemistry 41, 1035–1039. Hasimoto, N., Aoyama, T., Shiori, T., 1981. New methods and reagents in organic synthesis.14. A simple efficient preparation of methyl esters with trimethylsilyldiazomethane (TMSCHN2) and its application to gas chromatographic analysis of fatty acids. Chem. Pharm. Bull. 29, 1475–1478. Pavlik, J.W., 2005. Synthesis and spectroscopic properties of some dideuterated cyanopyridines. J. Heterocycl. Chem. 42, 73–76. Shilina, S.G., Bekker, Z.E., Goshaev, M.G., 1973. Isolation and comparative characterization of vivotoxin from wilt-infected cotton plants and of fusaric acid. Ekol.-Fiziol. Metody Bor’be Fuzarioznym Viltom Khlop 2, 219–230. Song, J.J., Yee, N.K., 2001. A concise synthesis of fusaric acid and (S)(þ)-fusarinolic acid. J. Org. Chem. 66, 605–608. Vischetti, C., Esposito, A.,1999. Degradation and transformation of a potential natural herbicide in three soils. J. Agri. Food Chem. 47, 3901–3904.
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
Phytotoxicity of fusaric acid and analogs to cotton R.D. Stipanovic*, L.S. Puckhaber, J. Liu, A.A. Bell USDA, Agricultural Research Service, Southern Plains Agricultural Research Center, 2765 F and B Road, College Station, TX 77845, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 July 2010 Received in revised form 5 October 2010 Accepted 8 October 2010 Available online 16 October 2010
We developed a cotton cotyledonary leaf bioassay to test the phytotoxicity of fusaric acid (5-butylpicolinic acid), picolinic acid and related analogs. The compounds were dissolved in aqueous Tween 80, and 20 mL of the test solution was placed at three positions on the leaf, and a needle was used to puncture the leaf through each drop; the results were evaluated after 48 h. In contrast to previous studies, we found the carboxylic acid group is essential for phytotoxicity. Nicotinic acid was considerably less phytotoxic than picolinic acid and conversion of picolinic acid to the amide or N-oxide decreased phytotoxicity. Increasing the alkyl chain length at the 5-position on picolinic acid from two up to five carbons atoms increased phytotoxicity. Fusaric acid methyl ester, the most phytotoxic compound tested, is a naturally occurring compound; as such it has potential as a herbicide in organic farming. Published by Elsevier Ltd.
Keywords: Phytotoxin Fusarium oxysporum f. sp. vasinfectum Fusarium nygamai Fusaric acid Methyl fusarate Cotton 3-Butylpyridine
Fusaric acid has been isolated from 11 Fusarium species including the cotton pathogen, Fusarium oxysporum f. sp. vasinfectum. Although fusaric acid shows low toxicity to animals, it is classified as a wilt-inducing toxin in many plants. Capasso et al. (1996) reported that fusaric acid and its methyl ester were produced by Fusarium nygamai, and proposed that these compounds could be used as bioherbicides in controlling witchweed (Striga hermonthica). Subsequently, Vischetti and Esposito (1999) extended these studies and found that fusaric acid methyl ester is degraded to 3-butylpyridine in the soil. In other work, a series of papers reported that the cotton plant converts fusaric acid into 3-butylpyridine, aka vivotoxin, and this compound is 100fold more toxic than fusaric acid to cotton (Gossypium hirsutum) (Bekker et al., 1971; Becker et al., 1972; Shilina et al., 1973). These results led us to consider that fusaric acid and its analogues might provide base compounds for developing new herbicides. Furthermore, because these compounds occur in nature they might be useful in organic farming. * Corresponding author. Tel.: þ1 979 260 9232; fax: þ1 979 260 9319. E-mail address: [email protected] (R.D. Stipanovic). 0041-0101/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.toxicon.2010.10.006
Our interest in cotton production prompted us to develop a cotton cotyledonary leaf assay to evaluate the phytotoxicity of a series of fusaric acid analogues including 3-butylpyridine. Fusaric acid may be chemically defined as a substituted pyridine. The structural components added to this primary nucleus are the position of the carboxylic acid group, derivatives of the carboxylic acid, and variations in the length of the alkyl side chain and its location on the pyridine ring. Of the large number of possible combinations, we chose to investigate the phytotoxicity of the compounds shown in Fig. 1. Our bioassay utilized acid delinted Coker-312 cottonseed that were placed in moist paper rolls and allowed to germinate in the dark at 30 C for two days. Individual seedlings were then selected for uniformity and transplanted into sterilized greenhouse planting mix in 450 mL plastic cups in a growth chamber with a 13-h, 28 C day and 11-h, 22 C night cycle. At the beginning of the day cycle only incandescent lights were activated, followed by onehalf of the florescent lamps after 15 min and all fluorescent lamps after an additional 15 min. Lights were turned off in the reverse order prior to darkness. Solutions of chemicals (8, 4, 2, 1, and 0.5 mM) were prepared in 0.1% Tween 80 and
R.D. Stipanovic et al. / Toxicon 57 (2011) 176–178
R2
R3 N
N
R1
Fusaric Acid; R1 = COOH; R2 = n-Butyl; R3 = H 1) R1, R3 = H; R2 = Methyl 2) R1, R3 = H; R2 = Ethyl 3) R1, R3 = H; R2 = n-Butyl 4) R1 = COOH; R2, R3 = H 5) R1 = CONH2; R2, R3 = H 7) R1 = COH; R2, R3 = H 8) R1 = CN; R2, R3 = H 9) R1, R2 = H; R3 = COOH 10) R1 = n-Butyl; R2 = H; R3 = COOH 11) R1 = COOCH3; R2 =n-Butyl; R3 = H 12) R1 = COOH; R2 = CH2 HO H ;R3 = H CH2 CH
COOH
O 6
CH3
13) R1 = R1 = COOCH3; R2 = CH2 HO H ;R3 = H CH2 CH CH3 14) R1 = COOH; R2 = Ethyl; R3 = H 15) R1 = COOH; R2 = n-Propyl; R3 = H 16) R1 = COOH; R2 = n-Pentyl; R3 = H 17) R1 = COOH; R2 = n-Heptyl; R3 = H Fig. 1. Structures of fusaric acid and analogues.
water. Test solutions were stored in the refrigerator until used. Plants were inoculated with the test solutions on the morning on the 7th day after planting. To introduce the chemical, drops (20 mL each) of test solutions were placed at three different positions on a cotyledonary leaf, and a needle was used to puncture the leaf through each drop. Forty-eight hours after inoculation, cotyledonary leaves attached to the plant were evaluated for necrosis by three individuals and then photographed to provide a permanent record for later referral. The degree of necrosis was scored
177
on a scale of 0–5 with 0 being no effect and 5 being severe necrosis and leaf curling around the area of inoculation (see Supplementary material). Some of the chemicals tested were purchased including: fusaric acid, 3-methylpyridine (1), 3-ethylpyridine (2), 3-butylpyridine (3), nicotinic acid (9), picolinic acid N-oxide (6) [Aldrich Chemical, Milwaukee, WI]; 5-propylpicolinic acid (15), and 5-pentylpicolinic acid (16) [Princeton Gold Collection I, Monmouth Junction, NJ]; 2-cyanopyridine (8), and picolinic acid (4) [Alfa Aesar, Ward Hill, MA]; picolinaldehyde (7) [Acros Organics, Morris Plains, NJ]; and 5-ethyl picolinic acid (14) [Ryan Scientific, Mt. Pleasant, SC]. Several of the compounds were synthesized using published methods: fusarinolic acid (12), 5-butylnicotinic acid (10) and 5-heptylpicolinic acid (17) [Song and Yee (2001)]; picolinamide (5) (Pavlik, 2005); and the methyl esters of fusaric and fusarinolic acids (11) and (13) (Hasimoto et al., 1981). We used picolinic acid as a model compound to test the effects of pyridine ring substitutions on toxicity (Table 1). The picolinic acid showed some phytotoxicity, but significantly less than fusaric acid. Conversion of the acid group to an amide or reduction to an aldehyde reduced the activity below that of picolinic acid. Shifting the carboxylic acid group from position 2 to 3 (i.e., nicotinic acid) also reduced activity, as did conversion of picolinic acid to its N-oxide. Addition of alkyl groups to the 5-position of picolinic acid led to a gradual increase in phytotoxicity as the length of the alkyl group increased. Thus, phytotoxicity increased in the order of picolinic acid < 5-ethylpicolinic acid < 5propylpicolinic acid < 5-butylpicolinic acid (i.e., fusaric acid) < 5-pentylpicolinic acid. However, 5-heptylpicolinic acid was less toxic than 5-pentylpicolinic acid. Interestingly, fusarinolic acid with the more hydrophilic side chain was less toxic than fusaric acid. Methyl fusarate, a phytotoxin found in F. nygamai (Vischetti and Esposito, 1999),
Table 1 Relative toxicity ratings (concentration mean score) [0 to 5 (S.D.)]a of fusaric acid, derivatives and analogs. Assayed by adding 20 mL of mM solution (0.1% Tween 80 in water) in three drops to the drops to the surface of a 7-day-old cotyledon and piercing through each drop with a needle then rating the necrosis after two days. Comp. #
Compound name
Concentration (mM)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Fusaric acid 3-Methylpyridine 3-Ethylpyridine 3-Butylpyridine Picolinic acid Picolinamide Picolinic acid N-oxide Picolinaldehyde 2-Cyanopyridine Nicotinic acid 5-Butylnicotinic acid Methyl fusarate Fusarinolic acid Methyl fusarinolate 5-Ethylpicolinic acid 5-Propylpicolinic acid 5-Pentylpicolinic acid 5-Heptylpicolinic acid
1.0 0 0 0 0.1 0 0 0 0 0 0 2.8 0.2 0.2 0.2 0.3 1.6 0.7
0.5
a b
1.0 (0.0)
(0.1)
(0.3) (0.3) (0.3) (0.1) (0.3) (0.4) (0.3)
1.4 0 0 0 0.3 0 0 0 0 0 0 3.2 0.3 1.0 0.9 1.1 2.3 1.5
2.0 (0.5)
(0.4)
(0.2) (0.3) (0.7) (0.1) (0.3) (0.2) (0.4)
Visual rating: 0 ¼ no necrosis; 5 ¼ severe necrosis (See Supplementary material). Significant (p < 0.0001) herbicidal effect observed with increasing concentration.
3.0 0 0 0 0.6 0.2 0 0 0 0 0.2 4.0 1.3 1.6 1.5 1.2 3.4 2.6
4.0 (0.3)
(0.6) (0.3)
(0.3) (0.6) (0.5) (0.5) (0.2) (0.3) (0.1) (0.6)
3.8 0 0 0 1.5 0 0.1 0 0 0 1.3 4.6 1.8 2.3 2.5 2.9 4.2 3.3
8.0 (0.6)
(0.9) (0.1)
(0.7) (0.4) (1.3) (0.4) (0.2) (0.8) (0.2) (0.3)
4.4 (0.3)b 0 0 0 2.8 (0.6)b 0 (0.1) 0.9 (0.2) 0 0 0.6 (0.2) 1.6 (0.3)b 5.0 (0.0)b 3.1 (0.6)b 3.1 (0.2)b 3.7 (0.1)b 4.5 (0.4)b 4.5 (0.1)b 4.1 (0.2)b
178
R.D. Stipanovic et al. / Toxicon 57 (2011) 176–178
was more active than fusaric acid, whereas fusarinolic acid and methyl fusarinolate exhibited similar toxicity. In contrast to previous reports (Bekker et al., 1971; Becker et al., 1972; Shilina et al., 1973), the carboxylic acid group or its methyl ester were found to be essential for toxicity and the position of the carboxylic acid group also affected activity such that moving the carboxylic acid from the 2-position (i.e., picolinic acid) to the 3-position (i.e., nicotinic acid) resulted in reduced phytotoxicity. In the case of fusaric acid, conversion of the carboxylic acid group to the methyl ester provided a more toxic compound. Of the compounds tested, fusaric acid and its methyl ester were among the most phytotoxic compounds showing severe wilting at 8.0 mM and detectable necrosis at the point of piercing at 0.5 mM. Thus, it would appear that evolutionary pressures within F. oxysporum and F. nygamai have combined to produce effective phytotoxins from a basic pyridine carboxylic acid building block. Acknowledgements We thank Cotton Incorporated for supporting this research. We thank Sara Duke for helpful statistical analysis. Conflict of interest statement The authors have no conflict of interest.
Appendix. Supplementary material Supplementary material associated with this article can be found in the online version, at doi:10.1016/j.toxicon. 2010.10.006. References Becker, S.E., Pushkareva, I.D., Poletaeva, V.F., Shilina, S.G., Yasakova, E.I., 1972. Nature and biosynthesis of Fusarium wilt toxin, its mechanism of action, and its transformation in the cotton plant. Bodenkultur 23, 256–271. Bekker, E.E., Dovletmuradov, K.D., Pushkareva, I.D., Poletaeva, V.F., Shilina, S.G., Yasakova, E.I., 1971. Nature and biosynthesis of the toxin of the causative agents of fusariosis wilt, the mechanism of its actions, and its possible transformation within the cotton plant. Izv. Akad. Nauk SSSR, Ser. Biol. 5, 749–754. Capasso, R., Evidenet, A., Cutignano, A., Vurro, M., Zonno, M.C., Bottalico, A., 1996. Fusaric and 9,10-dehydrofusaric acids and their methyl esters from Fusarium nygamai. Phytochemistry 41, 1035–1039. Hasimoto, N., Aoyama, T., Shiori, T., 1981. New methods and reagents in organic synthesis.14. A simple efficient preparation of methyl esters with trimethylsilyldiazomethane (TMSCHN2) and its application to gas chromatographic analysis of fatty acids. Chem. Pharm. Bull. 29, 1475–1478. Pavlik, J.W., 2005. Synthesis and spectroscopic properties of some dideuterated cyanopyridines. J. Heterocycl. Chem. 42, 73–76. Shilina, S.G., Bekker, Z.E., Goshaev, M.G., 1973. Isolation and comparative characterization of vivotoxin from wilt-infected cotton plants and of fusaric acid. Ekol.-Fiziol. Metody Bor’be Fuzarioznym Viltom Khlop 2, 219–230. Song, J.J., Yee, N.K., 2001. A concise synthesis of fusaric acid and (S)(þ)-fusarinolic acid. J. Org. Chem. 66, 605–608. Vischetti, C., Esposito, A.,1999. Degradation and transformation of a potential natural herbicide in three soils. J. Agri. Food Chem. 47, 3901–3904.