Induction of Dothistroma blight symptoms with dothistromin

Physiological Plant Pathology (1981) 19, 49-55

Induction of Dothistroma blight symptoms with dothistromin LOUIS SHAIN

Department of Plant Pathology, University qf Kentucky, Lexington, Kentuc/9J 10516, U.S.A.

and

ROBERT

A.

FRANICH

Forest Research Institute, New ZealandForest Service, PrivateBag, Rotorua, New Zealand (Acceptedfor publication March 1981)

Dothistroma blight of Pinus radiata and other pine species, caused by Dothistroma pini, is characterized by red bands through necrotic lesions. The red color in these lesions is due to the accumulation of dothistromin, a difuranoanthraquinone, during the infection process, This compound is the product of the pathogen and was obtained from culture extracts. Evidence is presented supporting the hypothesis that dothistromin plays a significant role in pathogenesis and should be considered a toxin. Typical necrotic and red-band symptoms were induced by introducing 10 to 100 ng of dothistromin in acetone into puncture wounds in pine needles. Lesion length and ethylene production were used to assess dothistromin-induced injury, Natural lesions contained 1 to 10 j.lg of dothistromin, I,e. substantially greater amounts than those required for induction of artificial lesions. Cited histological studies of natural lesions demonstrated that host tissue was killed in advance of the pathogen-suggesting toxic action. Symptoms of artificially induced lesions developed within hours and, in some cases, continued to expand during a 72 h test period. When acetone alone was introduced into similar puncture wounds, only small necrotic flecks developed. Development of both natural and artificially-induced lesions was favored by high light intensity. The capability to induce lesions artificially with dothistromin may prove useful in the search for resistance and in the clarification of resistance mechanisms.

INTRODUCTION

Control of Dothistroma blight, caused by Dothistroma pini Hulbary, in New Zealand is based upon the aerial application of copper oxychloride to radiata pine (Pinus radiata D. Don) plantations during the susceptible age of 2 to 15 years. In 1979, 60000 ha were sprayed [10J at a cost of $10 to $20 ha -1. A supplementary or alternative means of control is selection and breeding for resistance. The chances of success for this means of control could be enhanced by an understanding of the mechanisms of disease resistance. Dothistroma blight is characterized by red bands through necrotic lesions (Plate 1). The red color in these lesions is due to the accumulation of dothistromin, a difuranoanthraquinone (Fig. 1) [1]. This compound is the product of the pathogen and has been obtained from culture extracts of the fungus [1, 8]. Although dothistromin has been shown to inhibit the growth of Chlorella pyrenoidosa [1, 9J and Bacillus mcgaterium [9], toxicity to pine-needle tissue has not been demonstrated. The role of dothistromin in the development of blight symptoms, therefore, is an unresolved but pertinent question whose solution has both basic and practical 004-8-4059/81/040049+09 $02.00/0 © 1981 Academic Press Inc. (London) Limited

50

L. Shain and R. A. Franich

o

o

OH

FIG. 1. Molecular formula of dothistromin [1].

implications, A capability to induce lesions artificially with dothistromin could be useful in the search for resistance and in the clarification of resistance mechanisms. On the basis of these considerations, additional studies were undertaken to determine if dothistromin is causally related to symptom development. A preliminary report on this work was presented earlier [12]. MATERIALS AND METHODS

Production of dothistromin Cultures of D. pini were grown in autoclaved 10% malt extract at 20±1 °0 on a rotary shaker. Ten, 14-day-old cultures were homogenized and extracted with ethyl acetate. Dothistrornin was obtained from concentrated ethyl acetate extracts by preparative t.l.c, using ethyl acetate: chloroform: formic acid (50: 50 : 4). Dothistromin was the major red band at c. R F 0·5. Introduction of dothistromin intopine needles A variety of techniques were used to introduce dothistromin into pine needles. These included uptake of buffered, aqueous, saturated solutions (c. 30 p.p.m.) by the transpiration stream of detached needles and vacuum infiltration of needles with aqueous solutions. Aqueous treatments did not result in typical necrosis, therefore some organic solvents were tried. Needles were punctured with a hypodermic needle (26 gauge) previously dipped into dothistrornin dissolved in: acetone, ethyl acetate, a-pinene hexane and terpineol. All of these (curiously including the last 3 which are constituents of pine oleoresin), with the exception of acetone, caused substantial necrosis by themselves when introduced into puncture wounds (Plate 2). In subsequent studies, therefore, acetone was used as the carrier to introduce dothistromin into wounds made with a hypodermic needle. Incubation conditions In preliminary studies, blight symptoms were not induced after dothistromin, dissolved in acetone, was introduced into puncture wounds in detached pine needles incubated indoors in low light. Symptoms, however, were successfully induced in similarly treated needles of a P. radiata seedling maintained outdoors in full sunlight. Furthermore, previous work demonstrated a positive correlation between light

Induction of Dothistroma blight symptoms

51

intensity and D. pini infection [7]. A study was conducted, therefore, to determine the effect of light and darkness on dothistromin-induced symptom development. Pine needles were punctured 4 times at 2 em intervals starting at their base with a hypodermic needle that was dipped before each puncture into acetone or dothistromin dissolved in acetone (5 mg rnl r-). Needles were placed individually into test tubes containing 0·2 ml distilled water and sealed with septa. Half the needles were maintained in about one-third full sunlight (760±SO uE m -2 s-1) at 25±2 °0 for 3 days while the other half was maintained in the darkness at the same temperature for the first 2 days and then transferred to light for the 3rd day. Needles from the same fascicle were distributed among treatments. Lesion development was assessed as described below for 3 days at 24 h intervals.

l)osage resjbonse Three concentrations of dothistromin in acetone, 1,5, 10 mg ml r" as well as acetone alone, were introduced separately into 4 puncture wounds at 2 em intervals in each of 3 needles. Each needle of fascicles containing 4 needles thus received one of the above treatments. Needles were placed into test tubes and incubated in light as described above. Ethylene production, a common response of plants to injury and infection [13J, and lesion length were used to assess dothistromin-induced injury. Qy.antijication ofdothistromin in pine needles Reversed-phase t.l.c. combined with densitometer scans of developed chromatograms were used to quantify the amount of dothistromin in artificially induced or natural lesions. Lesions were cut into pieces c. 0·5 mm long and placed into 1 ml of ethyl acetate containing 1 drop of formic acid. After 10 h at room temperature, the extract was evaporated to dryness, redissolved in 25 ul ethyl acetate and spotted on silica gel GF 254 which was previously developed in 5% paraffin oil in hexane. Dothistromin standards of 20, 50, and 100 ng also were spotted on each t.l.c. plate prior to its development in methanol: water: formic acid (67 : 33 : 4). Chromatograms were irradiated in a densitometer at A.x 436 nm with a blocking filter Aem > 500 nm. Dothistromin fluoresced orange at c. R F 0·7 and was quantified by a log Xlog plot of fluorescence weight -1. RESULTS

Symptoms of Dothistroma blight were induced when dothistromin in acetone was introduced into puncture wounds in pine needles which were then incubated under high light intensity (Plates 3 to 5). Necrosis was evident within hours of such treatment and, in some clones, continued to expand during a 72 h test period (Table 1). Symptoms did not develop around acetone-treated puncture wounds (Plates 2 and 4) nor on needles with dothistrornin-treated puncture wounds that were incubated in darkness up to 48 h (Plate 3). Symptoms, however, did develop after dothistrornintreated needles incubated in darkness were exposed to high light intensity. Differences in ethylene production by acetone or dothistromin-treated needles usually were greater at 48 than at 24 h after introduction of test solutions. Although greater amounts of ethylene were produced by needles incubated in light, significantly more ethylene was produced by dothistromin than acetone-treated needles incubated in

L. Shain and R. A, Franich

52 TABLE

Effects

if light and darkness on ethylene production Pinus radiata after introduction

24 Ethylene production" (nl g-l dry wt h- 1 )

Lesion length" (mm)

1

and lesion development by detached needles

rifdothistrotnin intopuncture wounds

Time (h) 48 Ethylene production Lesion (nl g-t dry length wt h- 1 ) (mm)

72 Ethylene production (nl g-l dry wt h- 1 )

if

Lesion length (mm)

Treatment Acetone" Dothistromin"

26·1 a 24·9 ab

Acetone Dothistromin

3-6d

Light" 12·4 c nil 29·1 a 1·8 yz

nil 1·5 xy

9·3 c nil 27·7 a 2·3 z Light" 13·0 bc nil 18·7 abc 1·2 x

Dark nil nil

6·9 e

2·7 d 7·1 e

nil nil

" Pine needles were punctured 4 times at 2 ern intervals with a 26 gauge hypodermic needle that was previously dipped into acetone releasing c. 10 nl into each wound. b Pine needles punctured as above with a hypodermic needle previously dipped into a solution of dothistromin in acetone (5 mg ml- l ) releasing c. 10 nl containing 50 ng dothistromin into each wound. c Light intensity was c. one third full sunlight (760±50 IlE m- 2 S-l). d Average amount of ethylene produced by each of 3 pine needles. Averages followed by the same letter are not significantly different. L.S.D. (P = 0,05) = 12·02 and 2·71 for light and dark-treated needles, respectively. • Average length of 12 lesions (4 on each of 3 needles). Averages followed by the same letter are not significantly different L.S.D. (P = 0,05) = 0·51. The "nil" reflects minimal necrosis of c. 0·5 rom surrounding acetone-treated wounds.

5

2.5

,

s:

~

;a

2.0

e -e lOt 15

:s

r1 o

I

/ s

10

Dothlat,omln cencentrctton (mg mJ- 1 ocetone l

FIG. 2. Ethylene production (6) and lesion length (0) 48 h after puncturing needles of

Pinus radiate with a hypodermic needle previously dipped into acetone or acetone containing 3 concentrations of dothistromin. About 10 nl was released into each of 4 puncture wounds per needle. Ehtylene production was for final 2411 period. Vertical lines delimit standard errors of means. Minimal necrosis surrounding acetone-treated wounds was assigned a length 0[0·5 mm.

Induction of Dothistroma blight symptoms

53

darkness. This implies that the host was responding to dothistromin in darkness (Table I) even though gross symptoms were not evident (Plate 3). The effect of increasing concentrations of dothistromin on ethylene production and lesion length is summarized in Fig. 2. Ethylene production increased with increasing concentration of dothistromin until saturation was reached at 5 mg ml -1. Lesion length on the other hand, increased linearly at concentrations up to 10 mg ml-1 . The amount of dothistromin actually introduced into needle punctures was determined quantitatively by extracting 20 wounds made with a hypodermic needle dipped into dothistromin at 5 mg ml-1 • These extractions were made promptly (within minutes) after wounding to minimize the possible effect of dothistromin breakdown in pine-needle tissue. The average amount of dothistromin extracted from these wounds was 50·25±11·05 ng (P = 0'05). It follows, therefore, that the volume of solution deposited into each puncture wound was c. 10 n1. The amount of dothistromin detected in natural lesions, on the other hand, was I to 10 ug. Evidence was obtained to suggest that sensitivity to dothistromin is related to blight susceptibility in trees of susceptible age. Six clones of P. radiata, maintained at a physiol.ogical age of 6 years by hedging [11] were selected from a heavily infected area containing more than 100 clones. Three of these dones were rated "field resistant" while the others were highly susceptible. Lesion size 48 h after introduction of dothistromin (5 mg ml -1) was significantly greater (P = 0·05) in needles of susceptible clones (x = 2·9 mm) than in resistant clones (x = 1·5 mm) (Plate 5). Lesion size, furthermore, was largely stabilized at 24 h in "field resistant" needles whereas it continued to expand during a 72 h test period in susceptible needles. Ratios of ethylene production by dothistromin-treated needles to their acetonetreated controls at 48 h tended to be higher in susceptible clones (x = 7·1 : 1) than in resistant clones (x = g'6 : 1) but these differences were not significant. DISCUSSION The fungal metabolite dothistromin can induce symptoms of Dothistroma blight at concentrations substantially less than those detected in naturally occurring lesions. The comparatively rapid production of dothistromin in culture, i.e., 6 to 10 days [8, 9J, suggests that it is not a staling product of old cultures. Development of both natural [7] and artificially-induced lesions is favored by high light intensity. Histological studies [6J, furthermore, demonstrated that host tissue was killed in advance of hyphal penetration, suggesting the diffusion of a toxic entity from hyphae to uninfected tissue. These results suggest strongly that dothistromin plays a significant role in pathogenesis and should be considered a toxin. Isolates of D. pini lacking the capability to produce dothistromin, unfortunately, were not available for inoculation studies to further test this hypothesis. Another point of evidence favoring the toxin hypothesis is that in a limited trial of clonal material of susceptible age, susceptibility was correlated with sensitivity to dothistromin, In other limited tests, however, a correlation between decreased sensitivity to dothistromin and mature-tree resistance was not obtained. This could reflect a continuing sensitivity of mature trees to dothistromin from the juvenile stage and their later acquisition of other mechanism(s) which retard needle penetration, such as those proposed elsewhere [4, 5].

54

L. Shain and R. A. Franich

Ethylene production by host foliage has been used successfully to assay another fungal toxin. A dosage response by susceptible tissue occurred with toxin (victorin) dilutions of 10 -0 to 10 -7. Resistant and susceptible tissue, furthermore, could be distinguished readily by the amount of ethylene produced in response to a given amount of toxin, i.e. ethylene production was positively correlated with tissue damage [13J. Although these trends were similar in the present study the range of effective dosages and the magnitude of response were quite limited by comparison. Perhaps incubation conditions can be altered to optimize the effects of dothistromin on ethylene production. Lesion length, in any case, may provide an adequate means for toxin assay (Fig. 2, Plate 4). Light appears to be a major factor in the development of natural and artificiallyinduced lesions in this as well as other diseases [2,3]. The results of this study suggest that necrosis occurs only when there is an interaction between dothistromin and photosynthetically-active tissue. The reported effect of dothistromin on C. pyrenoidosa and B. megaterium was inhibition of RNA synthesis which, among other things, would eventually affect chlorophyll synthesis. The mode of action of dothistromin in pine needle tissue, as well as critical studies on the effect of light quality and quantity on symptom development, await future investigations. Rapid induction ofarti:ficiallesions may be used in other studies of host-resistance mechanisms. For example, studies were initiated to determine if dothistromin catabolism or phytoalexin production differ in resistant and susceptible tissue with increasing time after lesion induction. Such studies may facilitate screening for resistance to Dothistroma blight. Journal Paper No. 80~1l-288 of the Kentucky Agricultural Experiment Station, Lexington" Kentucky 40546. This research was conducted at the Forest Research Institute, Rotorua, New Zealand while the first author was on sabbatical leave from the University of Kentucky. He acknowledges with gratitude a senior research fellowship awarded by the New Zealand National Research Advisory Council. The statistical assistance of Ian Andrew is acknowledged gratefully. REFERENCES ]. BASSETT, C., BUOHANAN, M., GALLAGHER, R. T. & HODGES, R. (1970). A toxic difuroanthraquinone from Dothisiroma pini. Chemistry andIndustry 52, 1659-1660. 2. CAMPBELL, G. R. & DEVERALL,:B.]. (1930). The effects oflight and a photosynthetic inhibitor on the expression of the Lr20 gene for resistance to leaf rust in wheat. Physiological Plant Pathology 16,415-423. 3. COHEN, Y. & ROTEM, J. (1970). The relationship of sporulation to photosynthesis in some obligatory and facultative parasites. Phytopathology 60, 1600-1604. 4. FRANICH, R. A., GAD GIL, P. D. & SHAIN, L. (1981). Fungistatic effects offoliar epicuticular resin acids during infection of Pinus radiata by Duthistroma pini. Physiological Plant Pathology 19 (submitted). 5. FRANrcH, R. A., WELLS, L. G. & BARNETT,]. R. (1977). Variation with tree age of needle cuticle topography and stomatal structure in Pinus radiata. Annals if Bata,!)' 41, 621-626. 6. GADGIL, P. D. (1967). Infection of Pinusradiata needles by Dothistroma pini. New ZealandJournal of Botany 5, 498-503. 7. GADGIL, P. D. & HOLDEN, G. (1976). Effect of light intensity on infection of Pinus radiate by Dothistroma pini. New ZealandJournal ifForestry Science 6, 67-71. 8. GALLAGHER, R. T. & HODGES, R. (1972). The chemistry ofdothistromin, a difuroanthraquinone from Dothistroma pini. Australian Journal if Chemistry 25, 2399-2407.

PLATE 1. Needles of Pinus radiate infected with Dotbistroma pini, Notice red bands through necrotic lesions. PLATE 2. The effect of some organic solvents on needles of P. radiata. Each needle was wounded 4 times at 2 em intervals with a hypodermic needle previously dipped into (left to right) terpineol, l-pinene, hexane, ethyl acetate or acetone. PLATE 3. The effect of light and darkness on dothistromin-Induced lesion development. Needles of P. radiata from the same fasicle were wounded 4 times at 2 em intervals with a hypodermic needle previously dipped into an acetonic solution of dothistromin (5 mg ml " "). Needles were incubated for 48 h in light (760 ±50 u.E rn -2 s-1), left, or darkness, right. PLATE 4. Lesions on a needle of P. radiata caused by D. pini or induced by different concentrations of dothistromin. Artificial lesions were induced 48 h previously by introducing dothistromin in acetone at 1,5, or 10 mg ml ! (D) starting at the needle base, respectively into puncture wounds. Acetone (A) alone was introduced into the puncture wound between the latter and the natural lesion (N) at the- needle tip. r

PLATE 5. Lesions induced 48 h previously by introducing dothistrornin in acetone (5 mg ml- 1) into puncture wounds in needles from 6 clones of P. radiata at a physiological age of 6 years. The 3 needles on the right were from clones rated field resistant (lesion x = 1·5 mm) whereas those on the left were rated susceptible (lesion ;Xi = 2·9 mm),

ffacing page 54]

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A. M ., B AIT, R. D. & P RITC HARD, G. G. (1976). In hibition of R NA synthesis in Chlorella jJJrtnoidoslt and Bacillus megaterillm by the pin e-blight toxin , do thistrornin. J ournal of General Microbiology 95, 268- 276. 10. KERSHA W , D.]., GAD GIL, P. D ., LEGG AT , c.j., RAY,]. W. & VANDE Il.PAS, ]. B. ( 1979). Handbook .for the Assessment and Control of Dothistroma Needle B light. New Zeal and Forest Service, R otorua, 11. LIB BY, W . J., BROWN, A. G. & F WL1)IN G, ]. M . (1972). Effects of hedging r a di ata pine on pro d uction, rooting and early gro wth of cut tings. New Z ealandJounul1 cf Forestry Science 2, 263-283. 12. SHAIN, L. & FRANICH, R. A . (1980). Induction of dothistrom a blig h t sym p toms with do thistrornin. Phytopathology 70 (in p ress) (Abstr.) . 13. S H AIN, L. & WHEELER , H . (1975). Production of ethy lene by oat s res istant a nd suscep tible to victor in, Phytopathology 65, 88-89. 9.

HARVEY,