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Bioactive Constituents, Metabolites, and Functions

Differences in Active Defense Responses of Two Gossypium barbadense L. Cultivars Resistant to Fusarium oxysporum f. sp. vasinfectum Race 4 Lorraine S. Puckhaber, Xiuting Zheng, Alois A. Bell, Robert D. Stipanovic, Robert L. Nichols, Jinggao Liu, and Sara Duke J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05381 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 2, 2018

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Journal of Agricultural and Food Chemistry

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Differences in Active Defense Responses of Two Gossypium barbadense L

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Cultivars Resistant to Fusarium oxysporum f. sp. vasinfectum Race 4

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Lorraine S. Puckhaber,† Xiuting Zheng,† Alois A. Bell,† Robert D. Stipanovic,*† Robert L.

5

Nichols,§ Jinggao Liu,† Sara E. Duke†

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†U.

8

Research Center, 2765 F and B Road, College Station, Texas 77845

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§Cotton

S. Department of Agriculture, Agricultural Research Service, Southern Plains Agricultural

Incorporated, 6399 Weston Pkwy, Cary, North Carolina 27513, USA

10 11

* Corresponding Author: Fax: 979 260 9319; Phone: 979 823 5670

12

Email: [email protected] (R.D. Stipanovic)

13

ACS Paragon Plus Environment


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ABSTRACT: A highly virulent race 4 genotype of Fusarium oxysporum f. sp. vasinfectum

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(Fov) was identified for the first time in the western hemisphere in 2002 in cotton fields in the

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San Joaquin Valley of California. The Gossypium barbadense L. cotton cultivars 'Seabrook Sea

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Island 12B2' ('SBSI') and 'Pima S-6' are resistant to Fov race 4. Active defense responses were

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quantitated by monitoring the accumulation of antimicrobial terpenoids (i.e., phytoalexins) in

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inoculated stem stele tissue in these cultivars. The increase in the concentration of the most toxic

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phytoalexins was statistically faster after 24 h in 'SBSI' compared to 'Pima S-6'. The

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sesquiterpenoid hemigossylic acid lactone, which was observed for the first time in nature, also

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accumulated in diseased plants. Neither hemigossylic acid lactone nor the disesquiterpenoids

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gossypol, gossypol-6-methyl ether, and gossypol-6,6'-dimethyl ether showed toxicity to Fov.

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Segregation of F2 progeny from 'SBSI' x 'Pima S-6' crosses gave a few highly susceptible plants

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and a few highly resistant plants, indicating separate genes for resistance in the two cultivars.

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KEYWORDS: disease resistance; cotton; Gossypium barbadense; Fusarium wilt; Fusarium

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oxysporum f. sp. vasinfectum race 4 (VCG0114) isolate CA-9; Fov race 4; phytoalexins

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INTRODUCTION

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Fusarium oxysporum f. sp. vasinfectum race 4 (VCG0114) (Fov race 4) is a highly virulent

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fungal pathogen on cotton (Gossypium), particularly on G. barbadense cottons. Fov race 4 was

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first recognized in the western hemisphere in cotton fields in the San Joaquin Valley of

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California in 2002,1,2 and subsequently found in Texas in 2017.3 We are interested in identifying

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different mechanisms of resistance to Fov race 4 that can be utilized in a marker assisted

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selection breeding program. Among G. barbadense cultivars, the commercial cottons 'Pima S-6'

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and 'Pima S-7' are resistant and susceptible, respectively, to Fov race 4 in greenhouse and field

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studies.4,5

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In earlier work,6,7 we demonstrated that the resistant G. barbadense cultivar 'Seabrook

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Sea Island 12B2' ('SBSI') responds to the presence of the fungal wilt pathogen Verticillium

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dahliae faster than susceptible cottons, as shown by the rapid increase in the concentration of the

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sesquiterpenoid phytoalexins that are toxic to the pathogen. Because 'SBSI' is resistant to V.

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dahliae, and the mechanisms of resistance for 'SBSI' and 'Pima S-6' to Fov race 4 are unknown,

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we hypothesized that the same mechanism responsible for the resistance of 'SBSI' to V. dahliae

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might be responsible for resistance to Fov race 4, and may differ from the resistance mechanism

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in 'Pima S-6'. To test this hypothesis, we determined how quickly these two cottons respond to

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infection by determining the concentration of phytoalexins in stem stele tissue 24 and 48 h after

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inoculation with Fov race 4; the susceptible G. barbadense 'Pima S-7' was included as a control.

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Fov race 4 initially attacks the cotton root, and later invades stele tissue. We used a stem

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inoculation technique because the induced concentration of sesquiterpenoid phytoalexins, 1-4

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(Figure 1) in the root is masked by constitutive phytoalexins; conversely, healthy stele tissue is



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free of constitutive phytoalexins. Finally, 'SBSI' was crossed with 'Pima S-6' and segregation of

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resistance among F2 plants was determined and compared to the resistance of the parent cultivar.

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MATERIALS AND METHODS

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Phytoalexin responses. Cotton seed were germinated in a roll of damp germination

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paper for 1 d at 30 °C, followed by 1 d at 18 °C. The seedlings were transplanted into

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pasteurized soil in 16-oz plastic cups drilled for drainage, and then grown in an environmental

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growth chamber programmed for a 26 °C/13 h/light and 20 °C/11 h/dark cycle. After 42 d, when

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the plants had attained six true leaves, the stems were puncture inoculated with Fov race 4,

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isolate CA-9 (2-3 x 107 conidia/mL). Specifically, a 23-gauge needle fitted to a 1 mL syringe

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was used to apply a drop of inoculum to the stem surface 1 cm below and perpendicular to the

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cotyledonary petioles and then used to puncture through the drop into the stele tissue. A second

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inoculation was repeated 1 cm down and equidistant around the stem from the first application.

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The inoculated plants were replaced in the growth chamber and after 0, 24 or 48 h, stem stele

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was harvested from 10 plants of each cultivar. The stem internode between the cotyledonary

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node and the first true leaf node was removed from the plant and the bark stripped from the stele.

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The stem stele was then cross-sectioned into 1-2 mm pieces and transferred to a pre-weighed 5

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mL glass, screw-cap tube; tubes were reweighed to obtain the tissue weights and then 1.5 mL of

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acetone/1% aqueous ascorbic acid (9/1, v/v) extractant was added to each tube. The tubes were

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sealed and placed in a dark cold-room (2 °C). After 24 h, the samples were shaken and

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centrifuged at 3000 rpm for 5 min. Aliquots of the clear extracts were transferred to vials for

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HPLC analysis.


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Chromatographic analysis of extracts from inoculated plants. Plant extracts were

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analyzed using a 1200 HPLC equipped with a diode array detector (Agilent Technologies, Santa

78

Clara, CA); the column used was a 250 mm x 4.6 mm i.d., 5 µm, Hypersil MOS-1(Thermo

79

Scientific, Waltham, MA), which was maintained at 32 °C. The mobile phase was a gradient of

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methanol (solvent A) and water (solvent B) (both with 0.07% H3PO4) run at 1.25 mL/min. The

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gradient was as follows: 0.0 min, 20% A; 7.0 min, 70% A; 12.0 min, 80% A; 19.0 min, 90% A;

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19.2 min, 100% A; 23.0 min, 100% A; 26.0 min, 20% A; and 29.0 min, 20% A. The

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chromatogram signal was monitored at 235 ± 10 nm (referenced to 550 ± 50 nm), and spectra

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(210-600 nm) for detected peaks were stored. The injection volume was 50.00 µL. Ten extracts

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were placed on the HPLC instrument for analysis; the remaining vials were stored in the dark at

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-80 °C until the analysis of the preceding sample set was almost complete. Peak identities in the

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sample chromatograms were determined via retention time and spectroscopic comparisons with

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authentic compounds. The terpenoid concentrations in the stem stele were calculated from the

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relevant peak areas, curves of µg versus area from standard solutions of authentic compounds,

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and the stem stele weights. The experiment was replicated in two trials. The concentration

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results for each compound were analyzed separately. At time zero, no terpenoids were detected

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in the stem tissue of any plant; therefore, this data was excluded from analysis. Due to

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variability of terpenoid concentrations between cultivars, concentration data were transformed to

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ranks (within each trial) and these data were analyzed as a non-parametric Friedman two-way

95

ANOVA with cultivar and time (24 and 48 h) as fixed factors and trial as a random factor. For

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all compounds, there was no cultivar by time (h) interaction so each time point was analyzed

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separately as a Kruskal-Wallis ANOVA. Data were analyzed with the Mixed procedure in SAS

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ver. 9.4; least square mean comparisons were adjusted with Tukey-Kramer to control for Type-1



99 100 101

error. Data are presented in the units of the raw concentration data even though the statistical analyses were based on the ranked data. Isolation of hemigossylic acid lactone. Concentrated phytoalexin extracts were prepared

102

as described earlier.8 Purification procedures were conducted in minimum light. The crude

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extract was streaked on SilicAR TLC G254 plates (Mallinckrodt, St. Louis, MO) and developed

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with CHCl3/Me2CO/HCOOH (95/4/1), System 1; hexane/Et2O/HCOOH (70/30/1), System 2; or

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C6H6/MeOH (99/1), System 3. Hemigossylic acid lactone, 5 and other terpenoids were purified

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until they appeared as a single quenched spot under UV (254 nm). Hemigossypol, 3,

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hemigossypol-6-methyl ether, 4, gossypol, 6, and gossypol-6-methyl ether, 7 gave immediate red

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spots when sprayed with a 1:1 mixture of 10% phloroglucinol in EtOH and concentrated HCl.

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The compounds desoxyhemigossypol, 1 and desoxyhemigossypol-6-methyl ether, 2 turned red a

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few h after spraying while 5 did not react with phloroglucinol but appeared as a darker spot

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under 254 nm UV light. To obtain the individual compounds, the crude extract was streaked

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across the bottom of the TLC plate and developed with Systems 1 - 3 described above. TLC

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zones containing the terpenoids were scraped from the plates with a razor blade immediately

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after development. Terpenoids were eluted from the silica gel with Et2O and immediately

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reduced to dryness unless HCOOH was used in the developing solvent. If System 1 or 2 that

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contained HCOOH was used, the Et2O solution was first washed successively with NaHCO3,

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H2O, saturated NaCl, and finally dried over Na2SO4 as above. After filtering, the organic

118

solution was reduced to dryness in vacuo at 30 °C. 1H-NMR spectra were recorded on an FX-

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90Q (90 MHz) instrument (JEOL, Akishima, Japan) in CDCl3 with 7.28 δ used as an internal

120

standard. Low and high resolution mass spectra were determined by direct insertion probe on a

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CH7 (Varian Inc., Palo Alto, CA) and CEC-21-110 mass spectrometers (Consolidated


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Engineering Corporation, Pasadena, CA), respectively; EIMS m/z (M)+ (%) 258 (100), 244 (15),

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243 (96), and 229 (22); HREIMS m/z 258.085030. IR spectra were recorded on a 237B

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spectrometer (Perkin-Elmer, Waltham, MA); (KBr): 1755, 1640, 1628 cm-1. The melting point

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of the unknown (uncorrected) was determined on a Kofler hot stage.

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Toxicity of phytoalexins to Fov-11. As previously reported, a 96-well cluster plate

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bioassay was used to evaluate the toxicities of 5, 6, and mixtures of 6 and 7 as well as 8 to F.

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oxysporum f. sp. vasinfectum race 1 (VCG 01117) isolate 11 (Fov-11).9,10 The toxicity of the

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sesquiterpenoid phytoalexins 1-4 to Fov-11 have been reported;9 thus, the toxicity of 5, 6 and 7

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to Fov-11 was determined and compared to that reported for 2.9 The toxicity of 8 was also

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evaluated. The bioassay used a suspension of Fov-11 conidia in water (106 conidia/mL). This

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was mixed with a concentration series of terpenoids in buffered nutrient solution containing 2%

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DMSO to assist dissolving the terpenoids. The buffered nutrient solution was composed of 30

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mM NH4H2PO4, 115 mM D-glucose, 1.0 mM MgSO4.7H2O, 75 mM KH2PO4, and 75 mM

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Na2HPO4 (pH 6.3). The concentrations of terpenoids in the test mixtures spanned the ranges of

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5–20 µg/mL for 2, 5–30 µg/mL for 6, 5–35 µg/mL for mixtures of 6 and 7 (3:2 and 2:3 ratios of

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6 and 7) or 5–35 µg/mL for 8. Compound 5 was assayed only at 30 µg/mL due to the limited

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amount of pure compound.

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The test mixtures were added to a 96-well cluster plate along with a blank solution and a

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control suspension of conidia only with no terpenoid (200 µL per well; 6 wells per test mixture,

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blank or control). Immediately after plate preparation, the optical densities (OD) at 550 nm were

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read with an Emax Precision Microplate Reader (Molecular Devices, San Jose, CA) to obtain

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OD values due to compound absorption alone. The plates were incubated at 27 °C for 46 h in

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darkness, and the OD was measured. The mean blank OD value and the mean zero time



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compound OD values were subtracted from the mean after-incubation test mixture OD values;

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the mean blank OD value was also subtracted from the mean after-incubation control suspension

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OD. The fractional effect on the conidia growth for each terpenoid concentration was calculated

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by comparing the corrected mean test mixture OD values with the corrected mean control

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suspension OD. Where toxicity was observable, the data was analyzed in a multiple drug-effect

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program to obtain the effective terpenoid dose that reduced conidia growth by 50%.11

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In each bioassay, three different conidial suspensions were tested using the same

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terpenoid solution. These replications were used to calculate a mean and standard error for the

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fractional effect at each compound concentration. If the compound was sufficiently toxic, a

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mean ED50 with a standard error was calculated. Each terpenoid or terpenoid mixture, except 5,

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was bioassayed in at least two separate experiments; 5 was tested only once due to the limited

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amount of compound.

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Resistance determination in cultivars and F2 populations. 'SBSI' was crossed with

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'Pima S-6' in the greenhouse and the resulting F1 seed was planted; plants were self-pollinated to

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produce F2 seed. F2 seedlings prepared as described previously were planted into a pasteurized

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loam:sand (3:1) mix containing 2 g of ground oatmeal per 2 kg of soil. Immediately after

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transplanting, soils were infested with conidial suspensions of Fov race 4 isolate CA-9. Thus, 1

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mL of 2 x107 conidia/mL was injected with an 18-gauge needle at 6 locations in a hexagonal

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pattern located 1-2 cm away from the seedling. Inoculated plants were incubated at 23 °C/13

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h/light and 18 °C/11 h/dark cycle. After 60 d, plant height and shoot weight were measured to

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determine disease severity. The foliar disease index was calculated by dividing the number of

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symptomatic leaves on a plant by the total number of leaves and multiplying by 100.

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RESULTS AND DISCUSSION

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In agreement with greenhouse and field studies,4,5 in our growth chamber study 'Pima-S7'

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was susceptible and 'Pima S-6' was resistant to Fov race 4 isolate CA-9; 'SBSI' was also resistant.

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For this study, plants were inoculated with Fov race 4, placed in the growth chambers for 0, 24

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and 48 h, and stem sections were collected, cut into small pieces and extracted to provide

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samples for HPLC analysis. A typical HPLC trace showing separation of cotton terpenoids

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found in response to Fov race 4 is shown in Figure 2 and spectra of these compounds are shown

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in Figure 3. Terpenoid concentrations following inoculation are shown in Table 1. No terpenoids

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were detected at time 0 in any plant and are not shown. At 24 h after inoculation, the

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concentrations of all individual phytoalexins were significantly higher in 'SBSI' plants than in

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either resistant 'Pima S-6' or susceptible 'Pima S-7'. The toxicity of the phytoalexins to Fov are

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shown in Table 2.9 The concentrations of the most toxic phytoalexins 1 and 2 were significantly

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higher in 'SBSI’ than in either 'Pima S-6' or 'Pima S-7'. In addition, the combined concentrations

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of the most toxic phytoalexins at 24 h were significantly higher in 'SBSI' (Table 1). As expected,

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after 48 h the differences in phytoalexin concentrations changed among the resistant and

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susceptible cultivars. However, in 'SBSI' the combined concentration of 1 and 2 were still

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significantly higher compared to that in 'Pima S-6' or 'Pima S-7'. After 48 h, the concentration of

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1 in 'Pima S-7' was smaller but not significantly different than that in 'SBSI'. In contrast, the

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concentrations of phytoalexins in the resistant 'Pima S-6' were all significantly lower than in

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'SBSI'. This would be expected if the mechanism of resistance in 'Pima S-6' is not related to

189

early recognition of the pathogen.



190

In addition to the sesquiterpenoid phytoalexins, 1-4 we had previously identified,8,12 we

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found gossypol, 6, and its 6-methyl ether, 7. These two disesquiterpenoids have previously been

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reported in infected stem stele in 'SBSI'13 and in the G. hirsutum varieties 'Siokra 1-4' and 'Sicot

193

189.14 An unidentified compound, 5 also was observed in the chromatograms (Figure

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2). The unknown compound, isolated as white crystals, was subjected to EIMS analysis; the

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molecular weight of the parent ion [m/z 258 (100%)] was as expected for hemigossylic acid

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lactone, 5. The high resolution molecular weight measurement from the HREIMS (m/z

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258.089030) was consistent with that calculated for C15H14O4 (m/z 258.089190). Compound 5

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has been reported as a byproduct in the synthesis of 3.15 Our observed m.p. (228-232 °C) for 5

199

agreed with that reported by Wei et al. (220-222 °C), as did its 1H-NMR spectrum.15

200

The antimicrobial activity of the terpenoids was determined using methods published

201

previously.9,10 In these methods the terpenoids, 1-4 were tested for toxicity using a 96-well plate

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turbimetric bioassay to provide ED50 toxicity values. The declining order of toxicity of the

203

sesquiterpenoid phytoalexins to Fov-11 was 1 > 2 > 3 > 4 (Table 2).9

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The toxicity of 5, 6, and mixtures of 6 and 7 were evaluated in this study. To

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complement the study, 8 was also included in the assays. Additionally, 2 was included, and its

206

ED50 value obtained in this work (12.4 ± 3.0 µg/mL) was comparable with that of Zhang et al.

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(13.4 µg/mL).9 Fractional effects of 6 and 2 on conidia growth of Fov-11 are shown in Figure 4.

208

The decrease in the effect of 6 at concentrations greater than ca. 25 µg/mL is due to solubility

209

limitations of 6 in the bioassay medium. Compound 8 and mixtures of 6 and 7 showed no

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fungicidal activity at any concentration between 5 and 35 µg/mL and are not shown. Also, 5

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showed no fungicidal activity to conidia at 30 µg/mL.


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In our growth chamber studies, the reactions of the three G. barbadense cultivars to soil

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inoculations with Fov race 4, isolate CA-9, are shown in Table 3. These growth chamber studies

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agreed with results from field and greenhouse studies;4,5 that is, 'Pima S-7' was highly susceptible

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to the pathogen and 'Pima S-6' was highly resistant. 'SBSI' also was highly resistant. The

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resistant cultivars were not significantly different from their uninoculated controls in height or

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weight. The fungus was re-isolated from only 16% and 33% of the hypocotyls of 'SBSI' and

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'Pima S-6', respectively, compared to 100% from the 'Pima S-7' hypocotyls (Table 3).

219

The segregation of resistance to Fov race 4 among F2 progeny from a 'SBSI' x 'Pima S-6'

220

cross based on shoot weight and plant height is shown in Figure 5. If resistance in 'SBSI' and

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'Pima S-6' were due to separate dominant genes on different chromosomes, then four of the 60 F2

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plants theoretically should be homozygous recessive for both genes, and susceptible. In addition,

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four plants should be homozygous dominant, possibly with a higher level of resistance. In fact,

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one plant died and about 20 were more susceptible than either parent. Five plants were more

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resistant than either parent. Theoretically, 16 plants should have a single dominant gene from

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only one of the parents, which may account for the large number of moderately susceptible

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plants. These results support a two gene hypothesis and justify developing resistance from both

228

sources.

229 230

AUTHOR INFORMATION

231

Corresponding Author

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*E-mail: [email protected]. Tel: (979) 823-5670. Fax (979) 260-9319.

233

ORCID

234

Robert D. Stipanovic: 0000-0003-4455-6767



235

Funding

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This work was supported in part by Cotton Incorporated, Cary, North Carolina, USA [Grant #15-

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924].

238

Notes

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The authors declare no competing ﬁnancial interest.

240 241

ABBREVIATIONS USED

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Fov race 4, Fusarium oxysporum f. sp. vasinfectum race 4 (VCG0114); Fov-11, Fusarium

243

oxysporum f. sp. vasinfectum race 1 (VCG01117); PDA, potato dextrose agar.

244 245

Supporting Information. This material is available free of charge via the Internet at

246

http://pubs.acs.org.

247

Table S1. 1H-NMR data for 5 isolated from infected cotton stem stele tissue compared to

248

the synthetic compound prepared by Wei et al.15

249 250 251

REFERENCES (1) Davis, R. M.; Moore, N. Y.; Kochman, J. K. Characterization of a population of

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Fusarium oxysporum f. sp. vasinfectum causing wilt of cotton in Australia. Aust. J. Agric. Res.

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1996, 47, 1143–1156.

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(2) Kim, Y.; Hutmacher, R. B.; Davis, R. M. Characterization of California isolates of Fusarium oxysporum f. sp. vasinfectum. Plant Dis. 2005, 89, 366–372.

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(3) Halpern, H. C.; Bell, A. A.; Wagner, T. A.; Liu, J.; Nichols, R. L.; Olvey, O.; Woodward,

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J. E.; Sanago, S.; Jones, C. A.; Chan, T. C.; Brewer, M. T. First report of Fusarium wilt of cotton


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(4) Ulloa, M.; Hutmacher, R. B.; Davis, R. M.; Wright, S. D.; Percy, R.; Marsh, B. Breeding

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for Fusarium wilt race 4 resistance in cotton under field and greenhouse conditions. J. Cotton

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Sci. 2006, 10, 114–127.

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(5) Hutmacher, R. B.; Ulloa, M.; Wright, S. D.; Campbell, B. T.; Percy, R.; Wallace, T.;

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Bowman, D.; Jones, D.; Burke, J. Elite Upland cotton germplasm-pool assessment of Fusarium

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wilt resistance in California. Agron. J. 2013, 105, 1635–1644.

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(6) Bell, A. A.; Presley, J. T. Temperature effects upon resistance and phytoalexin synthesis in cotton inoculated with Verticillium albo-atrum. Phytopathology 1969, 59, 1141–1146. (7) Bianchini, G. M.; Stipanovic, R. D.; Bell, A. A. Induction of -cadinene synthase and

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sesquiterpenoid phytoalexins in cotton by Verticillium dahliae. J. Agric. Food Chem. 1999, 47,

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4403–4406.

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(8) Bell, A. A.; Stipanovic, R. D.; Howell, C. R.; Fryxell, P. A. Antimicrobial terpenoids of

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Gossypium: hemigossypol, 6-methoxyhemigossypol and 6-deoxyhemigossypol. Phytochemistry

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1975, 14, 225–231.

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(9) Zhang, J.; Mace, M. E.; Stipanovic, R. D.; Bell, A. A. Production and fungitoxity of the

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terpenoid phytoalexins in cotton inoculated with Fusarium oxysporum f. sp. vasinfectum. J.

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Phytopathol. 1993, 139, 247–252.

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(10) Puckhaber, L. S.; Stipanovic, R. D.; Bell, A. A. Kenaf phytoalexin: Toxicity of o-

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hibiscanone and its hydroquinone to the plant pathogen Verticillium dahliae and Fusarium

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oxysporum f. sp. vasinfectum. J. Agric. Food Chem. 1998, 46, 4744–4747.



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(11) Chou, T. C.; Talalay, P. Quantitative analysis of dose-effect relationship: The combined

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effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 1984, 22, 27–55.

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(12) Stipanovic, R. D.; Bell, A. A.; Howell, C. R. Naphthofuran precursors of

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sesquiterpenoid aldehydes in diseased Gossypium. Phytochemistry 1975, 14, 1809–1811.

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(13) Mace, M. E.; Bell, A. A.; Beckman, C. H. Histochemistry and identification of disease-

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induced terpenoid aldehydes in Verticillium-wilt-resistant and -susceptible cottons. Can. J. Bot.

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1976, 53, 2095–2099.

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(14) Hall, C.; Heath, R.; Guest, D. I. Rapid and intense accumulation of terpenoid

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phytoalexins in infected xylem tissues of cotton (Gossypium hirsutum) resistant to Fusarium

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oxysporum f. sp. vasinfectum. Physiol. Mol. Plant Pathol. 2011, 76, 182–188.

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(15) Wei, J.; Vander Jagt, D. L.; Toyer, R. E.; Deck, L. M. Synthesis of hemigossypol and its derivatives. Tetrahedron Lett. 2010, 51, 5757–5760.

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FIGURE CAPTIONS

295

Figure 1. Terpenoids, 1-7 produced by the cotton plant (Gossypium barbadense L.) in response

296

to pathogen infection and gossypol-6,6'-dimethyl ether, 8.

297

Figure 2. Partial HPLC trace showing the presence of hemigossypol, 3 (11.74 min),

298

desoxyhemigossypol, 1 (12.22 min), hemigossylic acid lactone, 5 (12.35 min), hemigossypol-6-

299

methyl ether, 4 (12.89 min), desoxyhemigossypol-6-methyl ether, 2 (13.05 min), gossypol, 6

300

(17.69 min), and gossypol-6-methyl ether, 7 (18.10 min) in an extract from 'Seabrook Sea Island

301

12B2' stem stele 48 h after stem-inoculation with Fusarium oxysporum f. sp. vasinfectum, race 4,

302

VCG0114, isolate CA-9. The complete chromatogram was 26 min overall.

303

Figure 3. HPLC diode array detector spectra for terpenoids found in extracts from 'Seabrook

304

Sea Island 12B2' stem stele 48 h after stem-inoculation with Fusarium oxysporum f. sp.

305

vasinfectum race 4 isolate CA-9. The spectra for gossypol-6-methyl ether, 7 and gossypol-6,6'-

306

dimethyl ether, 8 are very similar to that of gossypol and are not shown.

307

Figure 4. Fractional effects of desoxyhemigossypol-6-methyl ether, 2 and gossypol, 6 on the

308

conidial growth of Fusarium oxysporum f. sp. vasinfectum, race 1, VCG0117, isolate 11

309

determined via a 96-well plate turbimetric bioassay using minimal media with 2% DMSO. The

310

fractional effect is the comparison of the optical density of fungal suspension with phytoalexin

311

corrected for compound absorption versus suspension without phytoalexin.

312

Figure 5. Shoot weight and plant height of 59 F2 plants at 60 d after inoculation with Fusarium

313

oxysporum f. sp. vasinfectum, race 4, VCG 0114, isolate CA-9. Plants are arranged in order of

314

declining shoot weight. One plant died as a seedling and is not shown.



Table 1. Mean Concentrations (g/gm fresh stem stele) of Infection-induced Terpenoid Compounds 24 and 48 h after Inoculation with Fusarium oxysporum f. sp. vasinfectum Race 4, VCG0114, Isolate CA-9. Terpenoid a 1 2 3 4 5 1+2 (g/gm (g/gm (g/gm (g/gm (g/gm (g/gm Cultivar fresh stem fresh stem fresh stem fresh stem fresh stem fresh stem stele) stele) stele) stele) stele) stele) 24 h after inoculation 'SBSI' 6.90A 1.23A 3.01A 4.20A 'Pima S-6' 0.54B 0.04B 0.88B 0.02B 0B 0.58B 'Pima S-7' 0.89B 0.14 B 1.62B 0.02B 0B 1.03B 48 h after inoculation A A 'SBSI' 16.19 20.05 32.95A 11.22A 16.30A 36.24A 'Pima S-6' 7.49B 2.29B 9.65B 0.77B 1.40B 9.78B 'Pima S-7' 8.68A,B 2.99B 13.55B 1.14B 0.89B 11.67B a1 = desoxyhemigossypol; 2 = desoxyhemigossypol-6-methyl ether; 3 = hemigossypol; 4 = hemigossypol-6-methyl ether; 5 = hemigossylic acid lactone. bValues within columns with different superscripts differ significantly at P < 0.05 based on Kruskal-Wallis analysis of ranks within h; Tukey adjusted for multiple comparison. 1.81A,b

2.39A


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Table 2. ED50 of Cotton Phytoalexins to Fusarium oxysporum f. sp. vasinfectum Race 1,VCG01117, isolate 11 Conidia.a Compound ED50 (g/mL) Desoxyhemigossypol, 1 8.8 Desoxyhemigossypol-6-methyl Ether, 2 13.4 Hemigossypol, 3 29.3 Hemigossypol-6-methyl Ether, 4 >30 aZhang et al., 1993.



Table 3. Disease Severity of Three Gossypium barbadense L. Cultivars to Infection by Fusarium oxysporum f. sp. vasinfectum Race 4, VCG 0114, Isolate CA-9 at 47 d after Inoculation and Incubation at 23 °C/13 h Days and 18 °C/11 h Nights. Cultivar Foliar Disease Indexa Height Shoot Weight Re-isolation from Hypocotyl (%) (cm) (g) (%) b 'Pima S-6' 0 40.3 (2.3) 14.9 (0.1) 33 'Pima S-7' 100 7.7 (2.5) 0.1 (0.1) 100 'SBSI'c 0 54.7 (1.8) 18.7 (1.5) 17 aFoliar Disease Index = (No. Symptomatic Leaves / Total Number of Leaves) X 100. bStandard deviation; n=6 for 'Pima S-6' and 'SBSI' and 13 for 'Pima S-7'. c'SBSI' = 'Seabrook Sea Island 12B2'.


Page 18 of 24

Page 19 of 24


Figure 1

O CH

O HO

HO RO

OH

RO

6

6

Hemigossypol, 3 R=H Hemigossypol-6-methyl Ether, 4 R=CH3

Desoxyhemigossypol, 1 R=H Desoxyhemigossypol-6-methyl Ether, 2 R=CH3 O

O O HO

O CH

OH

HO

OH 6

HO

Hemigossylic Acid Lactone, 5

OH HC

R1O

6'

OR2

Gossypol, 6 R1, R2 = H Gossypol-6-methyl Ether, 7 R1 = H; R2 = CH3 Gossypol-6,6'-dimethyl Ether, 8 R1, R2 = CH3



Page 20 of 24

Figure 2

mAU 400

3

350 300

2

250 200

4 5

150

1

100

6

50

7

0 9

10

11

12

13

14

15


16

17

18

19 min

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Figure 3

228nm

224nm

Hemigossypol, 3

279nm

246nm

Hemigossypol-6-methyl ether, 4

267nm 372nm

352 nm 386nm

Desoxyhemigossypol, 1

246nm 224nm

224nm

304nm 294nm

223nm

Desoxyhemigossypol-6methyl ether, 2

305nm 293nm 337nm 236nm

Hemigossylic acid lactone, 5

Gossypol, 6

260nm 289nm

349nm

372nm

200 250 300 350 400 450 500 550 nm

200 250 300 350 400 450 500 550 nm



Page 22 of 24

Fractional Effect on Conidia

Figure 4

1.00 0.80 0.60 0.40

Desoxyhemigossypol-6-methyl ether, 2 Gossypol, 6

0.20 0.00 0.0

5.0

10.0

15.0

20.0

25.0

Compound Concentration (µg/mL)


30.0

35.0

80

80

70

70

60

60

50

50

40

40

30

30

20

20

Shoot Weight (g)

10

10

Plant Height (cm)

0

0 0

10

20 30 40 Plant Ranking No.

50

60

Figure 5


Plant Height (cm)


Shoot Weight (g)

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TOC


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