Accumulation of the flavonoids betagarin and betavulgarin in beta vulgaris infected by the fungus cercospora beticola

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Reprinted from Physiological Plant Pathology (1977) 11, 297-303

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Accumulation of the flavonoids betagarin and betavulgarin

in

Beta vulgaris

infected by the fungus

Cercospora beticola

SusAN S. MARTIN

USDA, ARS, Crops Research Laboratory, Colorado State University, Fort Collins, CO 80523, U.S.A.

Physiological Plant Pathology (1977) 11, 297-303

Accumulation of the flavonoids betagarin and betavulgarin

in

Beta vulgaris

infected by the fungus

Cercospora beticola

SusAN S. MARTIN

USDA, ARS, Crops Research Laboratory, Colorado State University, Fort Collins, CO 80523, U.S.A.

(Accepted for publicatwn June 1977)

The flavanone betagarin and the isoflavone betavulgarin occur in necrotic lesions resulting from infection of sugarbeet (Beta vulgaris) leaves by Cercospora beticola. In whole infected leaves from cul ti vars of varied leaf spot susceptibility, there were significant differences among cultivars in content of both flavonoids, but only betavulgarin content was significantly cor-related with a visual rating of disease severity. In lesions, the content of both compounds differed significantly among cultivars, but only betavulgarin content differed with time after disease initiation. At 3 weeks after plants were inoculated with a suspension of fungal spores, lesion betagarin concentrations were 300 to 1050 µg/ml, depending on cultivar, and beta-vulgarin contents were from 50 to 200 µg/ml. When compared with data from in vitro bio-assays, these amounts appear potentially capable of limiting fungal growth. However, the correlation coefficient between visual rating of disease severity and compound contents per lesion was non-significant for each flavonoid.

INTRODUCTION

Betagarin (5,2' dimethoxy6, 7methylenedioxyflavanone) and betavulgarin (2' -hydroxy-5-methoxy-6, 7-methylenedioxyisoflavone) were first isolated and chemically characterized from leaves of sugarbeet (Beta vulgaris L.) infected by the fungus

Cercospora beticola Sacc. [ 4]. These compounds were tentatively identified as

phyto-alexins in the sugarbeet leaf spot disease caused by this fungus. Subsequently, Johnson et al. [8] presented data on the antifungal activity of the flavanone betagarin (B), the isoflavone betavulgarin (BV) and several other isoflavones. Against C.

beticola, the ED₆₀ (dose to reduce linear growth by 50% relative to control growth) for BV was approximately 65 µg/rnl (estimated graphically from data of Table 1, reference [ 8]). Betagarin had only slight antifungal activity against C. beticola,

inhibiting growth by 27% relative to the control at a betagarin concentration of 200 µg/rnl. In other bioassays of these compounds against C. beticola, BV had an ED₅₀

of about 100 µg/rnl and betagarin at 250 µg/rnl inhibited growth 22% [Ruppel & Martin, unpublished]. From its lesser antifungal activity betagarin appears less likely than betavulgarin as a potential phytoalexin, but because isoflavones are bio-synthesized via flavanones or the isomeric chalcones [5, 19], betagarin remains of interest as a possible betavulgarin precursor.

Although precise details of infection and symptom development in Cercospora leaf spot disease have not been known, geneticists have successfully developed relatively resistant sugarbeet cultivars, as judged by reduced severity of foliar symptoms. Johnson et al. [8] analyzed a single 0·5 g sample of dried, ground necrotic lesions from

298 S.S. Martin

each of six field-grown, Cercospora-infected sugarbeet cultivars of varied relative resistance, and found that lesions of infected resistant cultivars generally appeared to contain more betavulgarin than susceptible ones. No consistent association between betagarin content oflesions and resistance was apparent. As part of a broad study of sugarbeet Cercospora leaf spot, this paper reports an extensive examination of the presence of these compounds in infected sugarbeets.

MATERIALS AND METHODS

Anarysis

of

wlwle infected leaves

Nine sugarbeet cultivars were field-grown in a randomized complete block design with four replications. Each plot consisted of four 6· l m rows. An identical control experiment was planted 0·8 km away. At about 8 weeks of age, the treated experiment was inoculated with a spore suspension of C. beticola using techniques described previously [ 13]. Three samplings of control and diseased plants were made: (I) in mid-July, immediately before inoculation; (2) 25 days after inoculation, when disease symptoms were mild on inoculated plants; (3) 60 days after inoculation, when the epiphytotic was fully developed. At each sampling, visual disease ratings on a scale of 0 = no symptoms to IO = complete defoliation were made for each plot [ 13]. At each sampling, whole mature leaves were composited by replication, dried to constant weight and ground in a Wiley mill. A 5·0 g sample of dry ground leaf material was extracted twice with acetone and the filtrates combined and dried by rotary evapora-tion. The residue was taken up in 2·5 ml of chloroform-methanol (I : 1 v/v) and column chromatographed on a Sephadex LH-20 (3·8 x 80 cm) with the same solvent as eluant. Initial fractions containing mainly chlorophyll were discarded, and subsequent fractions were monitored by t.l.c. and u.v. Those containing the phyto-alexins were combined and rotary evaporated to dryness. Each sample was dissolved in l ·O ml dry acetone, sealed and stored at - 20 °C for later analysis. Final separation was by t.l.c. on 250 µm silica gel GF-254 in chloroform-diethyl ether (9 : I v/v). Betagarin and betavulgarin were readily identifiable on the t.l.c. plates in u.v. light, the first blue-fluorescing under long-wavelength (365 nm) excitation, at RF approx. 0·46, and the second at RF approx. 0·36 as a dark (absorbing) spot under short-wavelength (254 nm) excitation. Each spot was scraped from the plate, eluted in redistilled chloroform, evaporated to dryness and redissolved in ethanol of appropriate volume. The u.v. spectrum of each isolated compound was recorded from 220 to 350 nm on a B & L Spectronic 505 double-beam spectrophotometer, and quantitative calculations for betagarin and betavulgarin were made from published molar absorptions [4] at 280 and 256 nm, respectively. Minimum amounts quantitatively determinable were 15 and 6 µg/g dry wt for Band BV, respectively. Trace amounts, visible by t.l.c. but insufficient for spectral confirmation of identity and quantitative determination, were assigned a value of one-half the minimum determinable amount for calculation purposes.

Anarysis

of

lesions

Thirteen sugarbeet cultivars were planted in a randomized complete block design with two replications and inoculated in early July with a spore suspension of C. beticola.

From this design, six cultivars spanning the range of relative susceptibility and resistance to the disease were sampled at 3, 6 and 9 weeks after inoculation. Duplicate random samples were prepared from each replication by randomly punching out fifty 4 mm diameter discs, each including a necrotic lesion. Each sample was covered with 2 ml diethyl ether and sonicated for 15 min, then the ether was removed and the procedure repeated. Preliminary tests showed no further recovery of betagarin or betavulgarin could be attained by 6 h Soxhlet extraction of the residue with diethyl ether. Combined extracts were evaporated to dryness at room temperature and taken up in 0·2 ml dry acetone. Aliquots were separated by t.l.c. and B and BV determined as described above.

Bioassays for antifungal activity

Bioassays of betagarin and betavulgarin were conducted with C. beticola as the test fungus [Ruppel & Martin, unpublished]. Methods were similar to those outlined by Johnson et al. [8], except that 100 µl of acetone containing the appropriate amount of tested compound were added to each ml of medium, and the test duration was 120 h. Betavulgarin was tested in two trials at ten concentrations ranging from O (control) to 150 µg/ml of medium. Betagarin was tested in one trial at five concentrations from 0 to 250 µg/ml of medium. Inhibition of fungal growth was linear with betavulgarin concentration over the range tested, and was described by the regression equation y

=

0·44x+3·91 (r

=

0·96), where y

=

% inhibition of linear fungal growth and

x

=

µg betavulgarin per ml of medium. The relationship between betagarin content and fungal growth inhibition appeared logarithmic rather than linear, with fungal growth inhibited by 22% at 250 µg/ml, the highest concentration tested. Continued observation of the test plates after the test period ended indicated that inhibition of C. beticola growth by the tested compounds was not overcome, suggesting that the inhibition observed was not merely a delay in growth.

RESULTS AND DISCUSSION Ana(ysis of whole infected leaves

Only traces of B or BV were detected in leaves of diseased plants at the first two sampling periods, or in control plants at all three sampling dates. Only at the third sampling, 60 days after inoculation, when the disease was well developed in the inoculated plants, were the compounds present in measurable quantities in the whole leaf samples. Therefore, the following discussion refers to diseased plants sampled 60 days after inoculation.

The B and BV contents and visual disease rating for each cultivar are shown in Table 1. From previous field trials, the upper five cultivars of Table 1 were known to be relatively resistant, and the lower four relatively susceptible to the disease. Within each of these groups, the cultivars are listed top to bottom in approximate order of decreasing resistance to leaf spot. Disease ratings in Table 1 agree with these classifi-cations, and analysis of variance indicated significant differences in disease severity among cultivars (F

=

7·76**), or between the resistant and susceptible cultivar groups

(F

=

52·0**). Variability within cultivars was rather high for both disease rating and B and BV content. For chemical content, this may be due in part to variability

300 S.S. Martin TABLE 1

Visual disease ratings and content of betagarin and betavulgarin in C. beticola-infected sugarbeet

leaves. Disease rating based on visual estimation of symptom severity on a scale ef O

=

no symptoms to 10 = complete defoliation

µg/g dry weight Disease rating Betagarin Betavulgarin

Cultivar

xa

S.D.

x

S.D.

x

S.D. FC(504 x 502/2)-CMS x SP 6322-0 2·0 0·4 13 11 28 18 US201 2·4 0·5 242 88 55 19 FC 506x FC 701/2 2·1 0·6 104 54 54 20 652016sl-CMS x 661161H 3·0 0·7 99 28 29 12 SP 5822-0 3·1 1·6 55 30 35 22 USH9B 4·8 l · I 259 70 70 16 52-305 CMS x 52-334, F 1 5·4 1·8 306 137 66 14 R & G Pioneer 5·2 2·0 401 211 98 45 51-319x 52-334, F1 6·8 1·2 14 14 66 60

ax,

Mean; s.n., standard deviation.

among plants, and in part to varying symptom severity on leaves of a single plant, making it difficult to obtain a truly representative sample. Nevertheless, there were significant differences among cultivars in betagarin (F

=

8·53**), and the differ-ence among cultivars in betavulgarin content approached significance (F

=

2·27;

F_0.05

=

2·31).

The correlation coefficient between mean betagarin content and mean disease rating was not significant (r

=

0·39), whereas the correlation coefficient for betavulgarin and mean disease rating was significant (r

=

0·66*). The positive correlation observed between betavulgarin content and disease severity would appear to be the opposite of that expected of a phytoalexin. However, plants of susceptible cultivars had many more lesions or localized infection sites per leaf than resistant ones, leading us to examine accumulation of B and BV per lesion.

Anarysis of lesions

Preliminary studies showed that B and BV are localized in and immediately around

Cercospora-induced lesions, and neither was detectable in 50 leaf discs from uninfected

plants. An analysis of variance summary for Band BV content in composite 50-lesion samples from 6 cultivars at 3 post-inoculation sampling dates is shown in Table 2.

TABLE 2

Summarized ana(ysis of variance for betagarin and betavulgarin contents ef C. beticola-induced

sugarbeet leaf lesionsa

Source of variation Sampling date Cultivars Date x cultivar Residual Degrees of freedom 2 5 10 18 F-test value Betagarin Betavulgarin 1-70 6·70** 0·71 13·44** 10·01 ** 2·27

TABLE 3

Means of six sugarbeet cultivarsfor disease rating and betagarin and betavulgarin contents ofC. beti-cola-induced lesions at three post-inoculation dates"

Time after inoculation Disease Betagarin Betavulgarin (weeks) reading (µg/50 lesions) (µg/50 lesions)

3 l·O C 44-2a 8·9 b

6 2·8 b 33·9 a 14·5 a

9 4·5 a 31·4 a 5•! C

a Within columns, values followed by the same letter do not differ by Duncan's multiple range test (P

=

0·05).

There were significant differences among cultivars in content of both compounds, but only BV content differed with the sampling date (i.e. with degree of symptom development, which is progressive with time). The non-significant date x cultivar interaction for both compounds suggests that the disease developed in a consistent manner in all cultivars. Table 3 summarizes across cultivars the mean disease ratings and mean B and BV contents per 50 lesions at 3, 6 and 9 weeks post-inoculation.

Disease ratings at the three dates show the characteristic progressive development of

foliar symptoms. Mean amounts ofB and BV at the first sampling date, 3 weeks after

inoculation with C. beticola, correspond on a lesion volumetric basis to about 560 and

110 µg/ml, respectively, with individual cultivar means at this date ranging from 300 to 1050 µg/ml for betagarin content, and for betavulgarin from 50 to 200 µg/ml.

When bioassayed against C. beticola, betagarin at the highest concentration tested,

250 µg/ml, inhibited linear growth by 22% [Ruppel & Martin, unpublished],

whereas the ED50 for betavulgarin was about 65 µg/ml in one test [8] or about

100 µg/ml in two trials ofanother [Ruppel & Martin, unpublished]. The legitimacy

of extrapolation from in vitro bioassays to the in vivo state is always uncertain.

How-ever, the amounts of betagarin and betavulgarin present in and around necrotic

lesions 3 weeks after inoculation with C. beticola appear to have the potential, at least,

of limiting fungal growth. Several other flavonoids, particularly isoflavones, have shown antifungal or antibiotic activity, and some have been implicated as

phyto-alexins in other host-pathogen systems [2, 3, 8, 10, 14, 16-18]. These toxic effects of

isoflavonoids may be due to their structural similarities to steroids [7]. However, despite demonstration of the differential efficacy of particular flavonoid structures

against a given pathogen [12, 16], no clear relationship has yet emerged between

structure and antifungal effect against a range of pathogens. Also, isoflavonoid accumulation has been demonstrated in response to a variety of abiotic stimuli

[6, 9], suggesting that at least in some cases accumulation might be a general response

to wounding. However, Partridge & Keen [ 11] recently showed in soybean that

although phenylalanine ammonia-lyase and chalcone-flavanone isomerase, key enzymes located early in flavonoid biosynthesis pathways, were activated non-specifically in both resistant and susceptible cultivars by wounding or fungal inocu-lation, this could not explain the specific, rapid production of the isoflavonoid phytoalexin glyceollin in resistant plants.

The increase from no detectable quantities ofbetagarin and betavulgarin prior to infection to measurable amounts by the third week after inoculation could be due

302 S.S. Martin either to increased biosynthesis, the commonly assumed explanation for post-infectional increases in so-called secondary products, or to a blockage of a catabolic pathway, or both. Evidence of metabolic utilization or turnover of flavonoids has recently been summarized by Barz

[JJ.

As Stoessl et al. [ 15] have pointed out, the possibility of metabolic turnover of secondary products makes risky the assumption that the quantity measured under a given set of experimental conditions is equal to the quantity of the compound synthesized. In this case, of course, if turnover were occurring, total synthesized values for betagarin and betavulgarin would be even greater than the amounts found.

Mean BV content per 50 lesions decreased between the sixth and ninth weeks after inoculation. This cannot be interpreted as suggesting metabolic utilization by the plant, or possibly fungal degradation of BV, because it could be an artifact in these field studies in which the age of the lesions sampled could not be known, because sporulation, germination and initiation of new lesions occur continuously after the initial cycle. Greenhouse experiments in which sporulation can be prevented will permit determination in greater detail of the time course ofB and BV accumulation. Correlations were examined between disease rating and B or BV content per lesion at each of the three sampling periods. In every case, the correlation coefficients with disease rating for betagarin were positive but statistically non-significant, and those for BV were invariably negative but non-significant. BV in particular had a narrow range of values over the experiment, and this fact for any variable makes it almost impossible to detect correlation even if it is present. Furthermore, disease rating is a whole-plant or even whole-cultivar attribute, as it is a visual assessment of the severity of symptoms on an entire field plot. Thus a lack of significant correlation between disease rating and B or BV content does not necessarily negate a possible role for these compounds in disease resistance. For example, either or both could be involved in fungal limitation in the lesion, yet other factors could determine the numbers of lesions initiated, and therefore the overall disease susceptibility.

I thank Dr Garry A. Smith for supervision of field planting and maintenance of the experiments; Dr Earl G. Ruppel for supervision of the plant inoculations, disease readings and bioassays; Grace W. Maag for collection and preliminary workup of whole-leaf samples; and Vi Crockett for careful technical assistance.

This cooperative investigation of ARS, USDA, the Colorado State University Experiment Station and the Beet Sugar Development Foundation is published with the approval of the Director, Colorado State University Experiment Station, as Scientific Series Paper No. 2232.

Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.

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