Biological Activity and Synthesis of a Kenaf Phytoalexin Highly Active

Chapter 31

Downloaded by UNIV OF GUELPH LIBRARY on June 18, 2012 | http://pubs.acs.org Publication Date: May 14, 1998 | doi: 10.1021/bk-1998-0686.ch031

Biological Activity and Synthesis of a Kenaf Phytoalexin Highly Active Against Fungal Wilt Pathogens R. D. Stipanovic,L.S.Puckhaber, andA.A.Bell Agricultural Research Center, Southern Crops Research Laboratory, U.S. Department of Agriculture, College Station, TX 77845

A new potent phytoalexin has been isolated from kenaf (Hibiscus cannabinus) and identified as 3,8-dimethyl-1,2-naphthoquinone, which we call o-hibiscanone. o-Hibiscanone had ED values of 0.5 μg/ml and 1.1 μg/ml against conidia of Verticillium dahliae and Fusarium oxysporum f. sp. vasinfectum, respectively. In comparison, desoxyhemigossypol, the most potent phytoalexin in cotton xylem tissue, had ED values of 5.2 μg/ml and 8.8 μg/ml, respectively. V. dahliae detoxifies o-hibiscanone by converting it to the benign hydroquinone. Mansanone C, a phytoalexinfromelmwhich differs from o-hibiscanone in that the latter lacks the isopropyl group of the former, was less than one-half as toxic to V. dahliae. o-Hibiscanone is believed to be derivedfromδ-cadinene [2,3,4,6,7,8-hexahydro-1,6dimethyl-4-(1-methylethyl)naphthalene]. A biosynthetic pathway to account for the loss of the isopropyl group is proposed. 50

50

Losses to cotton production in the U.S. from Fusarium and Verticillium wilt were estimated to be in excess of $120M in 1996 (7) despite extensive efforts to increase resistance to these pathogens using traditional breeding techniques. New sources of resistance to these diseases are therefore needed. In 1966, Idessis tested the resistance offifteenplant species to twenty strains of Verticillium isolated from 17 plant species (2). Results of this test showed kenaf, camomile, alfalfa, and snapdragon were highly resistant to most isolates tested. Kenaf (Hibiscus cannabinus) is of particular interest because it resides in the same family as cotton (i.e. Malvaceae). In our own study, we found four varieties of kenaf hadfreshleaf weights two weeks after inoculation with the Verticillium dahliae strain V-76 (a strain virulent to cotton) which averaged 89% (median 87%) of the uninoculated 318

U.S. Government work. Published 1998 American Chemical Society

In Synthesis and Chemistry of Agrochemicals V; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

319 control. Under similar conditions, susceptible Acala 44 and resistant Acala Prema cultivars had fresh leaf weights two weeks after inoculation that averaged 8% and 59%, respectively, of the uninoculated controls (Table I). Table I. Effect of Verticillium dahliae Infection on Kenaf and Cotton Leaf Weight. Plant


Kenaf

Cotton

Cultivar

Leaf Weight**

Everglade 71

95 (13)

Guatamala 4

79 (7)

45-9

88 (4)

Everglade 41

95 (22)

Acala 44

8(8)

Acala Prema

59 (22)

a

Relative mean leaf weight at 2 weeks after inoculation at 27°C: infected leaf weight divided by average uninfected leaf weight times 100. Means and standard deviations for Everglade 71, Guatamala 4 and 45-9 are based on four leaf samples, for Everglade 41 on three leaf samples, and for Acala 44 and Acala Prema on eight leaf samples. b

The mechanism of resistance in kenaf is not known, although, in the case of cotton we have shown the phytoalexins play a critical role in the defense response (J). In order to examine the role of phytoalexins in the disease response in kenaf, we analyzed extracts from kenaf stems inoculated with V. dahliae. These extracts were spotted on TLC plates, developed in two dimensions, then over-sprayed with

HBQ o-Hibiscanone

Desoxyhemigossypol dHG

OCH Hibiscanal 3

Hemigossypol HG

Mansanone C


320 agar and with a spore suspension of V. dahliae. The spores were allowed to germinate over 2 days, and the plates were examined for clear zones which indicated antibiotic activity. Extracts from uninoculated plants were used as controls. Two compounds exhibiting antibiotic activity were isolated from the inoculated stems and identified as o-hibiscanone and hibiscanal. The characterization of these compounds is reported elsewhere (4). These compounds have certain structural characteristics in common with the phytoalexinsfromcotton [i.e. desoxyhemigossypol (dHG) and hemigossypol (HG)].


Fungitoxicity The fungitoxicity of ohibiscanone and hibiscanal to both V. dahliae and Fusarium oxysporum f. sp. vasinfectum have been determined using a direct turbimetric bioassay (5). Hibiscanal exhibit ED values of 25.8 jig/ml and 36.5 μg/ml to V. dahliae and F.o.v., respectively. o-Hibiscanone was significantly more toxic to these pathogens exhibiting ED values of 0.5 /xg/ml and 1.1 /xg/ml, to V. dahliae and F.o.v., respectively. dHG, the most toxic cotton phytoalexin (6), exhibited in the same bioassay ED values of 5.2 μg/ml and 8.8 ^g/ml (5) to V. dahliae and F.o.v., respectively. The toxicity of o-hibiscanone to mycelia of four isolates of V. dahliae has also been determined. The results of this experiment are shown in Table II and are compared to the toxicity of dHG as previously reported (6). These bioassays show o-hibiscanone is significantly more toxic to both V. dahliae and F.o.v. than the most effective phytoalexin produced by cotton. 50

50

50

Table II. Toxicity of desoxyhemigosssypol (dHG)* and 0-hibiscanone (HBQ) to mycelia of 4 isolates of Verticillium dahliae (cotton nondefoliating isolates PH and TS-2 and defoliating isolates V-44 and V-76). Percentage Live mycelia PH TS-2 V-44 V-76 dHG HBQ dHG HBQ dHG HBQ dHG HBQ ("g/ml) 20 0 0 0 0 42 0 15 64 22 3 3 56 0 6 10 6 100 100 100 6 100 3 33 5 25 36 61 0 100 100 100 100 100 100 100 100 Source: Adaptedfromref. 6. Mean percentage of wells in cluster dish with live mycelia; three replicate experiments of 12 samples per concentration. pH6.3 nutrient solutions with 1% DMSO for dHG and 2% DMSO for HBQ. Concentration c

a

b

c


321 Figure 1 compares the toxicity of o-Mbiscanone in the turbimetric bioassay to that of mansanone C. The latter is a phytoalexin isolated from elm (7,8). Mansanone C differs from o-hibiscanone in that the latter lacks the isopropyl group of the former. Loss of this isopropyl group increases the toxicity by more than five fold (ED mansanone C = 2.29 ^g/ml; ED o-hibiscanone = 0.44 ^g/ml). 50

50

Biotransformation of o-Hibiscanone by V. dahliae I3

o-Hibiscanone was synthesized as shown in Figure 2. By utilizing C-labeled methyl magnesium iodide, it was possible to prepare C-labeled o-hibiscanone. When o-hibiscanone was allowed to incubate with V. dahliae, the solution which was originally bright yellow turned colorless. A C-NMR study showed that the C-labeled methyl group which occurs at 23.6 ppm is gradually replaced over 140 minutes by a peak at 24.75 ppm. On mixing with air, the solution returns to its original color. The colorless conversion product was shown to be the hydroquinone by synthesis (i.e. chemical reduction of o-hibiscanone by sodium dithionite). o-Hibiscanone also is reduced slowly by glutathione. On standing overnight with glutathione (132 μg/ml), o-hibiscanone (40 μg/ml) was completely reduced to its hydroquinone. Under anaerobic conditions, this hydroquinone solution or a fresh solution of the quinone (40 μg/ml) and glutathione (132 μg/ml) were mixed with V. dahliae (V-76) conidia (10 conidia/ml) and allowed to stand for 0.2, 15, 30 and 60 minutes. After the indicated time, aliquots were diluted and pipetted onto individual PDA plates, and smeared to distribute the conidia evenly over the plate. After 5 days the number of colonies on each plate were counted. The bioassay was repeated three times. As shown in Table III, the hydroquinone was essentially non toxic to the pathogen under conditions in which the quinone was lethal.


13

I3

13

6

Table III. Comparison of the fungitoxicity of o-hibiscanone (HBQ) and its hydroquinone (HBQ-HQ) to conidia of Verticillium dahliae (V-76) exposed for the indicated time under anaerobic conditions. a

Time

Precentage of Live Conidia

minutes

HBQ

C

HBQ-HQ

0.2

10

100

15

1

100

30

0

100

0

100

60 a

b

6

Time of exposure of conidia (lxl 0 ) to compounds. Concentration of HBQ and HBQ-HQ were 40 μg/ml; media contained 132 μg/ml glutathione and 1.8% DMSO. approximate time required to mix chemicals with conidia and immediately dilute solution in preparation for transfer to PDA plates.


322


• o-Hibiscanone

I 1 . ' · · I I I I I I L. 1 2 3 4 Concentration of Compound (μ§/τη\)

—J

Figure 1. Toxicity of o-hibiscanone and Mansanone C to Verticillium dahliae (V-76)

13

Figure 2. Synthesis of o-hibiscanone (* indicates location of C-label).


323 HO

.CH

OPP

COOH

PPO


3

OPP

Mevalonic Acid Diphosphate

Farnesyl Diphosphate

Nerolidyl Diphosphate

δ-Cadinene

3-Hydroxy calamene

3-Hydroxy-a-calacorene

2,5-Dimethyl3 -hydroxy naphthalene 3,12-Dihydroxy- a-calacorene OH .OH

o-Hibiscanone Figure 3. Proposed biosynthesis of o-hibiscanone in kenaf.

Proposed Biosynthesis of o-Hibiscanone o-Hibiscanone is thought to be derivedfromδ-cadinene via the isoprenoid pathway. A proposed biosynthetic pathway is shown in Figure 3. Benedict and coworkers (9) have shown nerolidyl diphosphate is the precursor to δ-cadinene in cotton and Essenberg and coworkers (10) have shown the cadalenes are derived from δcadinene. Using GC/MS comparisons with synthetic compounds, we have identified 3-hydroxy calamene, 3-hydroxy-a-calacorene, and 2,5-dimethyl-3hydroxynaphthalene in extracts of cold shocked kenaf seedlings. 3,12-Dihydroxy-acalacorene is proposed as the intermediate between 3-hydroxy-ct-calacorene and 2,5dimethyl-3-hydroxynaphthalene. 3,12-Dihydroxy-a-calacorene is expected to spontaneously dehydrate with the concomitant loss of propene. Literature Cited 1.

Blasingame, D. In Beltwide Cotton Production Research Conference; P. Dugger; D. Richter, Eds.; National Cotton Council, Memphis, TN, 1996, p 227.


324 2. 3. 4. 5.


6. 7. 8. 9. D.; 10.

Idessis, V. F. In Cotton Wilt;M.V.Mukhamedzhanov, Ed.; Academy of Sciences of the Uzbek,USSR:1966; pp 61-64. Bell, Α. Α.; Stipanovic, R. D.; Mace, M. E.; Kohel, R. J. In Recent Advances in PhytochemistryVol.28; B. F. Ellis; G. W. Kuroki; H. A. Stafford, Eds.; Plenum Press, New York: 1994, pp 231-249. Bell, Α. Α.; Stipanovic, R. D.; Zhang, J.; Reibenspies, J.; Mace, M. E. Phytochemistry (submitted). Zhang, J.; Mace, M. E.; Stipanovic, R. D.; Bell, A. A. J. Phytopathology 1993, 139, 247-252. Mace, M. E.; Stipanovic, R. D.; Bell, A. A. Pest. Biochem. Physiol. 1990, 36, 79-82. Dumas, M. T.; Strunz, G. M.; Hubbes, M.; Jeng, R. S., Experientia 1983, 39, 1089-1090. Burden, R. S.; Kemp, M. S. Phytochemistry 1984, 23, 383-385. Alchanati, I.; Acreman-Patel, J. Α.; Liu, J.; Benedict, C. R.; Stipanovic, R. Bell, A. A. Phytochemistry (in press). Davis, Ε. M.; Tsuji, J.; Davis, G. D.; Piera, M. L.; Essenberg, M. Phytochemistry 1996, 41, 1047-1055.


Biological Activity and Synthesis of a Kenaf Phytoalexin Highly Active

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