Accumulation of the flavonoids betagarin and betavulgarin in Beta vulgaris infected by the fungus Cercospora beticola

Physkdogical Plant Pathology (1977)

l&297-303

Accumulation of the flavonoids betagarin and betavulgarin in Bela vulgaris infected by the fungus Cercospora beficola SUSAN

S.

MARTIN

USDA, AR&‘, Crops ResearchLaborakny, Colorado S&ateUnioersify, Fort Collins, CO 80523, U.S.A. (Acceptedfor publication June 1977)

The flavanone betagarin and the isoflavone betavulgarin occur in necrotic lesions resulting from infection of sugarbeet (Beta vulgaris) leaves by Cmcospmo beticola. In whole infected leaves from cultivars of varied leaf spot susceptibility, there were significant differencea among cultivars in content of both flavonoids, but only betavulgarin content was significantly correlated 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 pg/ml, depending on cultivar, and betavulgarin contents were from 50 to 200 &ml. When compared with data Tom in z&o bioassays, these amounts appear potentially capable of limiting fkngal growth. However, the correlation coefficient between visual rating of disease severity and compound contents per lesion was non-sign&ant for each flavonoid.

INTRODUCTION Betagarin (5,2’-dimethoxy-6,7-methylenedioxyflavanone) 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 Cercosporabeticola Sacc. [4]. These compounds were tentatively identified as phytoalexins 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. beticofu, the ED,, (dose to reduce linear growth by 50% relative to control growth) for BV was approximately 65 pg/ml (estimated graphically from data of Table 1, reference [S]). Betagarin had only slight antifungal activity against C. beticofu, inhibiting growth by 27% relative to the control at a betagarin concentration of 200 pg/ml. In other bioassays of these compounds against C. b&oh, BV had an ED,, of about 100 pg/ml and betagarin at 250 pg/ml 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 biosynthesized via flavanones or the isomeric chalcones [S, 191, betagarin remains of interest as a possible betavulgarin precursor. Although precise details of infection and symptom development in Cercosporaleaf 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. [S] analyzed a single O-5 g sample of dried, ground necrotic lesions from

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298

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 of lesions 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

Analysis of whole infected leaves Nine sugarbeet cultivars were field-grown in a randomized complete block design with four replications. Each plot consisted of four 6.1 m rows. An identical control experiment was planted O-8 km away. At about 8 weeks of age, the treated experiment was inoculated with a spore suspension of C. betkola using techniques described previously [13]. Three samplings of control and diseased plants were made : (1) 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 10 = complete defoliation were made for each plot [13]. At each sampling, whole mature leaves were cornposited by replication, dried to constant weight and ground in a Wiley mill. A 5-O g sample of dry ground leaf material was extracted twice with acetone and the filtrates combined and dried by rotary evaporation. 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.1.c. and U.V. Those containing the phytoalexins were combined and rotary evaporated to dryness. Each sample was dissolved in I.0 ml dry acetone, sealed and stored at - 20 “C for later analysis. Final separation was by t.1.c. on 250 q silica gel GF-254 in chlorofoxm+liethyl ether (9 : 1 v/v). Betagarin and betavulgarin were readily identifiable on the t.1.c. plates in U.V. light, the first blue-fluorescing under long-wavelength (365 nm) excitation, at R, approx. 046, and the second at RF approx. O-36 as a dark (absorbing) spot under shortwavelength (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 mn, respectively. Minimum amounts quantitatively determinable were 15 and 6 pg/g dry wt for B and BV, respectively. Trace amounts, visible by t.1.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. Analysis 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.

Phytoalexins

in Beta vulgaris

2gg

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.1.c. and B and BV determined as described above. Bioassaysfor antij&ngal activity Bioassays of betagarin and betavulgarin were conducted with C. beticola as the test fungus [Ruppel h Martin, unpublished]. Methods were similar to those outlined by Johnson et al. [S], except that 100 + 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 0 (control) to 150 pg/ml of medium. Betagarin was tested in one trial at five concentrations from 0 to 250 w/ml of medium. Inhibition of fungal growth was linear with betavulgarin concentration over the range tested, and was described by the regression equation y = 044x+3*91 (r = O-96), where y = y0 inhibition of linear fungal growth and x = pg 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 pg/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

Analysis 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 classifications, and analysis of variance indicated sign&ant differences in disease severity among cultivars (F = 7*76**), or between the resistant and susceptible cultivar groups (F = 52-O**). 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

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800

Vkual dkwse ratings and content of betagarin and bdavulgarin in C. b&cola-infscbd sugarbeat kaves. LXseaserating based on ohal &math of sp@om sevtri~ on a scok of 0 = no p#oms to 10 = complcb &foliation Disease rating t3ultivar

3

S.D.

FC(504 x 502/2)-CMS x SP 6322-O us 201 FC 506 x FC 701/2 652016sl-CMS x 661161H SP 5822-O US H9B 52-305 CMS x 52-334, F, R & G Pioneer 51-319 x 52-334, F,

2.0 2.4 2.1 3.0 3.1 4.8 5.4 5.2 6.8

0.4 0.5 0.6 0.7 1.6 1.1 l-8 2.0 1.2

a Z, Mean;

S.D.)

Betagarin if

Betavulgarin

S.D.

C8

S.D.

11 88 2:: 104 99 2”: 55 30 259 70 306 137 401 211 14 14

28 55 54 29 35 70 66 98 66

18 19 20 12 22 16 14 45 60

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 difference among cultivars in betavulgarin content approached significance (F = 2.27; F 0’06 = 2.31). The correlation coefficient between mean betagarin content and mean disease rating was not significant (r = O-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.

Analysis 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 B and BV content in composite 50-lesion samples from 6 cultkars at 3 post-inoculation sampling dates is shown in Table 2.

SUmmaztd

analysis

TABLE 2 of variancefor &tag& and bekzuulgorin contentsof C. beticola-induced sugorbeet kaf W

Source of variation

Degrees of fkedom

Samphg date Cultivan Date x cultivar Residual a Significance at the 1 o/0 level of probability

2 5 10 18

F-test value Betagarin Betavulgarin 1.70 6.70** 0.71 -

13*44+* 10*01** 2.27 -

is indicated by double asterisk.

Phytoalexins

301

in Beta vulgaris TABIS

3

Meansof sixsugarb6at cultivars fM diseas.9 ratingandbetagarinandb#tamlg& contents of c. beticola-inah& Time

after inoculation (weeks)

Disease reading

followed

by tbe same

da@P

3ktagarin (w/50 lesions)

l*Oc 2.8b 4.5 a

9 a Within wlumns, values range test (P = 0.05).

lesi0n.s a# three partiffoculorion

44.2 a 33-9 a 31.4 a letter

do not differ

Ektavulgarin (&Xl lesions) 8.9 b 14.5 a 5-l c by Duncan’s

multiple

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 of B 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 pg/ml, respectively, with individual cultivar means at this date ranging from 300 to 1050 pg/ml for betagarin content, and for betavulgarin from 50 to 200 kg/ml. When bioassayed against C. beticola, betagarin at the highest concentration tested, 250 pg/ml, inhibited linear growth by 22% [Ruppel & Martin, unpublished], whereas the ED,, for betavulgarin was about 65 pg/ml in one test [8] or about 100 tJ.g/ml in two trials of another [Ruppel & Martin, unpublished]. The legitimacy of extrapolation from in vitro bioassays to the in viva state is always uncertain. However, the amounts of betagarin and betavulgarin present in and around necrotic lesions 3 weeks after inoculation with C. beticofa 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 phytoalexins in other host-pathogen systems [Z, 3, 8, 10, 14, I&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 [I,?, 161, no clear relationship has yet emerged between structure and antifiungal effect against a range of pathogens. Also, isoflavonoid accumulation has been demonstrated in response to a variety of abiotic stimuli [S, 91, 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 nonspecifically in both resistant and susceptible cultivars by wounding or fungal inoculation, this could not explain the specific, rapid production of the isofiavonoid phytoalexin glyceollin in resistant plants. The increase from no detectable quantities of betagarin and betavulgarin prior to infection to measurable amounts by the third week after inoculation could be due

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302

either to increased biosynthesis, the commonly assumed explanation for postinfectional 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 [I]. 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 of B 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-sign&ant. 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. REFERENCES 1. BARZ, W. (1975). Abbau von Flavonoiden und Isoflavonoiden-ein Deutschm Botaniwhm Gesellschaft 88, 7 l-81. 2. BREDENBERO, J. B. (1961). Identification of an antifungal factor Suomcn KemistiMti 34B, 23.

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Fortschritts a’sr Chsmis