10. Heinselman, M. L. 1963. Forest sites, bog processes, and peatland New York.types in the glacial Lake Agassiz region, Minnesota. Ecol. Monogr. 16. Molina, R., and Palmer, J. G. 1982. Isolations, maintenance, and33:327-374. pure culture manipulation of ectomycorrhizal fungi. Pages 115-129

11. Heinselman, M. L. 1970. Landscape evolution, peatland types, and in: Methods and Principles of Mycorrhizal Research. N. C. Schenck,the environment in the Lake Agassiz Peatlands Natural Area, ed. American Phytopathological Society, St. Paul, MN.Minnesota. Ecol. Monogr. 40:235-261. 17. Mueller, G. M. 1982. The genus Laccaria in North America excluding

12. Marx, D. H. 1969. The influence of ectotrophic mycorrhizal fungi Mexico. Ph.D. thesis. University of Tennessee, Knoxville. 341 pp.on the resistance of pine roots to pathogenic infections. I. Antagonism 18. Nguyen, T. T., and Niederpreum, D. J. 1984. Hyphal interactionsof mycorrhizal fungi to root pathogenic fungi and soil bacteria, in Schizophyllum commune: The di-mon mating. Pages 73-125 in:Phytopathology 59:153-163. The Ecology and Physiology of the Fungal Mycelium. Symp. Br.

13. Mason, P. 1974. The genetics of mycorrhizal associations between Mycol. Soc., Bath University, April 1983. D. H. Jennings and A. D. M.Amanita muscaria and Betula verrucosa. Pages 567-574 in: The Rayner, eds. Cambridge University Press, Cambridge, England.Development and Function of Roots. J. G. Torrey and D. T. Clarkson, 19. Raper, J. R. 1966. The Genetics of Sexuality of Higher Fungi. Ronaldeds. Academic Press, New York. Press, New York. 283 pp.

14. Mason, P. A., Wilson, J., Last, F. T., and Walker, C. 1983. The 20. Raper, J. R., and Hoffman, R. M. 1974. Schizophyllum commune.concept of succession in relation to the spread of sheathing mycorrhizal Pages 597-626 in: Handbook of Genetics. I. R. C. King, ed. Plenumfungi on inoculated tree seedlings growing in unsterile soils. Plant Press, New York.Soil 71:247-256. 21. Swanson, D. K. 1988. Properties of peatlands in relation to

15. Melin, E. 1962. Physiological aspects of mycorrhizae of forest trees. environmental factors in Minnesota. Ph.D. thesis. University ofPages 247-263 in: Tree Growth. T. T. Kozlowski, ed. Ronald Press, Minnesota, St. Paul. 220 pp.

Physiology and Biochemistry

Effects of Dihydrofusarubin and Isomarticin from Fusarium solanion Carbohydrate Status and Metabolism of Rough Lemon Seedlings

S. Nemec, D. Phelps, and R. Baker

First and second authors, plant pathologist and research associate, respectively, U.S. Department of Agriculture, Agricultural ResearchService, U.S. Horticultural Research Laboratory, Orlando, FL 32803; third author, chemist, U.S. Department of Agriculture,Agricultural Research Service, U.S. Citrus and Subtropical Products Laboratory, Winter Haven, FL 33880.

We wish to recognize the support of Mr. Joe Pagano, research technician, Orlando, FL, on this project.Accepted for publication 16 February 1989.

ABSTRACT

Nemec, S., Phelps, D., and Baker, R. 1989. Effects of dihydrofusarubin and isomarticin from Fusarium solani on carbohydrate status and metabolismof rough lemon seedlings. Phytopathology 79:700-705.

Rough lemon citrus seedlings inoculated with Fusarium solani and b and ATPase activity, and no differences were present in the treatedseedlings suspended with their roots in solutions of the F. solani-produced versus the control plants. In general, naphthoquinone-treated plants, asnaphthoquinones dihydrofusarubin (DHF, 100 mg L-') and isomarticin well as inoculated plants, accumulated more minerals in leaves than did(50 mg L-') developed similar wilt symptoms. Only those seedlings treated control plants. Respiration increased in DHF-treated plants both in awith DHF developed chlorosis symptoms. Total soluble and reducing greenhouse and in a growth chamber. This respiration response was studiedsugars and starch were reduced in inoculated plants, but only starch was in growth chamber experiments with the inhibitors dinitrophenol, sodiumconsistently reduced in naphthoquinone-treated plants. Total protein did azide, salicylhydroxamic acid, and sodium fluoride. These studiesnot differ between inoculated plants, naphthoquinone-treated plants, and suggested that respiration is stimulated via mitochondrial oxidases.their controls. DHF-treated plants were evaluated for chlorophylls a andAdditional keywords: Citrus limon, carbohydrates, phytotoxins.

Fusarium so/ani (Mart.) Appel & Wr. emend. Snyd. & Hans. Six naphthoquinones inhibited anaerobic decarboxylation ofis an opportunistic pathogen of citrus causing fibrous (23) and pyruvate in vitro and the decarboxylation of cs-ketoglutaric acidscaffold root rot symptoms (6,15,21) and cankers on trunks and (17). Of these six, the marticins inhibited glutamine synthetase,branches (22). The most common symptom on a wide range of diminished the semipermeability of pea leaf tissue, and disruptedhosts is a cortical rot (25). Although F. solani is not generally chloroplast membranes of algae (17). In recent studies, bothconsidered a vascular pathogen (25), it readily invades the xylem isomarticin and dihydrofusarubin (DHF) affected the semi-of citrus stems from inoculated root systems (23). F. so/ani permeability of citrus leaf tissue, reduced water uptake in intactelaborates a large number of phytotoxic naphthoquinone citrus seedlings, and stimulated vessel plugging in seedlingspigments (4), and some isolates have been shown to produce maintained in solution cultures of the toxins (24). However, onlytrichothecenes (1 1) and fusaric acid (9). Limited information is DHF caused veinal chlorosis in citrus leaves.available on the effects of naphthoquinones on plant metabolism. This study was undertaken to examine further the effects of

F. solani on the metabolic activities of citrus. These studies wereconducted with citrus inoculated with F. solani or treated withDHF and isomarticin, two naphthoquinones produced by this

This article is in the public domain and not copyrightable. It may be treely fungus. The production of large-scale quantities of certain of thesereprinted with customary crediting of the source. The American naphthoquinones (4) has made it possible to study the effectsPhytopathological Society, 1989. of these toxins in whole plant systems.

700 PHYTOPATHOLOGY

MATERIALS AND METHODS filtered through Whatman No. I filter paper. The resulting glucosewas determined by the glucose oxidase procedure (Sigma Tech.

Plant production and naphthoquinone preparation. Rough Bull. 635, Sigma Chemical Co.)lemon (Citrus limon (L.) Burm. f.) seedlings were grown from Protein analysis. Soluble proteins were determined in samplesseed to 30-40 cm tall in steam-pasteurized Astatula fine sand of the same leaf tissue analyzed for carbohydrates. One-half gramsubsoil (hyperthermic, uncoated typic quartzipsamments) in flats of dry leaf tissue was ground in a mortar with liquid N2 to ain a greenhouse. Seedlings were carefully removed, and, after their fine powder. An extraction buffer, 50 mM Tris-HC1, pH 8.4,roots were washed, they were transferred to 14-L cylindrical containing 1 mM EDTA, 30 mM MgCI2, 5 mM 2-mercapto-polypropylene containers containing solutions of the naphtho- ethanol, and 2% polyvinylpyrrolidone (PVP), was added to thequinones. powder and the mixture reground in the mortar. The mixture

The containers were fitted with covers perforated with eight was centrifuged at 3,620 g for 30 min. The pellet was resuspended4.5-cm-diameter holes. The seedlings were supported, one per in 3 ml of 0.1 N NaOH and incubated at 60 C for 1 hr. Thehole, with soft foam plugs, and their roots were immersed in mixture was recentrifuged at 2,000 rpm for 15 min in a tabletopthe solutions aerated with an air pump. centrifuge and the supernatant analyzed for soluble proteins by

The naphthoquinones, isomarticin and DHF (Rel- the Lowry procedure (19).[3R,4aR, lOaR]-5, 10-dioxo-3,4,4a,5, 10, lOa-hexahydro-7- Chlorophyll assay. Chlorophyll was extracted from leaves ofmethoxy-3-methyl-3,6,9-trihydroxy-l-H-naphtho [2,3-c]pyran), rough lemon rootstock in a separate test in which the seedlingswere produced by F. solani in a sucrose-mineral salts liquid were incubated in 100 mg of DHF per liter. Three hydroponicmedium (5), and purified as previously described (5,28). containers were prepared with solutions of DHF, and three wereConcentrations of 50 mg of isomarticin per liter and 100 mg controls. Seven days after the test was established, five to sevenof DHF per liter were used per container in these studies. The leaves of each plant in each container were cut, pooled, andnaphthoquinones were first solubilized in 125 ml of 95% ethanol. weighed. Chlorophyll was isolated from 5 g of leaf tissue fromEach toxin solution was diluted to 12 L with deionized water each container replicate, according to a modification of thein each container; the final ethanol concentration was 1% for procedure by Goldberg and Brakke (13). The leaves were cuteach rate. The control was a 1% ethanol solution. into small pieces with a pair of scissors and ground in a mortar

Plants were maintained under greenhouse conditions and with a pestle in 80% acetone three times. Between each grinding,observed daily for up to 7 days. Plant wilt ratings based on a the acetone extract was filtered through Whatman No. 50 filterscale of 0-5 were made the final day of each test. Zero represented paper. The residue in the mortar after the third grinding wasno wilt and 5 indicated severe wilt. no longer green. All combined filtrates were diluted to 200 ml

Inoculum production and greenhouse inoculation methods. A with 80% acetone. Aliquots of each sample were diluted 10-foldknown pathogenic isolate of F. solani, isolated by Nemec from with 80% acetone and read at both 663 nm (chlorophyll a) andthe stele of root-rot diseased fibrous roots on blight-diseased trees 645 nm (chlorophyll b) versus 80% acetone. The concentrationsin Florida, was selected for this study. Inoculum was produced of chlorophyll a and b were converted to milligrams of chlorophyllby first growing the culture on Fries-medium slants (30) for 4 per gram of leaf tissue (2).days and then transferring an aqueous suspension of conidia from Chloroplast isolation and ATPase determination. Leaves (10 g)the culture to a liquid Fries medium. After being shaken for from the same hydroponic containers used for chlorophyll4 to 5 days, the mycelial colonies were collected by filtration analyses were cut into small pieces with a pair of scissors andthrough Whatman No. 1 filter paper and were resuspended in placed in a nylon bag. Ten milliliters of isolation medium (0.25 Mwater. Seedling root systems were dipped in this suspension for sucrose, 10 mM 3-(N-morpholino)propane-sulfonic acid, in 5%about 2 min. The plants were then potted in steam-pasteurized PVP, pH 7.2) was added to the bag in a mortar, and the contentsAstatula fine sand in 15-cm clay pots and placed on a greenhouse of the bag were ground with a pestle for a minute. The extractbench. Wilt was rated 7 days after inoculation as described above, was decanted and centrifuged at 3,000 g for 10 min. The resultantWilt was a consequence of extensive fibrous root rot caused by green pellet was suspended in isolation medium and stored atthe fungus common in tests of this type (23). -70 C until used.

Carbohydrate analysis. Leaf tissue for carbohydrate analysis ATPase activity was determined in the chloroplast fractionswas collected from rough lemon seedlings incubated for 5 days from the above procedure. Ca-ATP was used as the ATPasein DHF, from seedlings incubated for 7 days in isomarticin, and substrate. The Ca-ATPase activity was measured according tofrom seedlings 7 days after inoculation with F. solani. In the the procedure of Lien and Racker (18), using the phosphate assaytoxin studies, leaves were composited from each replicate of Taussky and Shor (29).hydroponic tank, three tanks per treatment. Three control tanks Leaf mineral analysis. Leaf tissue from plants treated withwere set up with each test. Leaves were composited from four isomarticin (50 mg L-') and DHF (100 mg L-') was collectedplants per replicate from each of four replicates in the inoculations 7 days after exposure of plants to the toxins. Leaves were analyzedwith F. solani. Four control replications were processed in the for N, P, K, Ca, Mg, 5, and B. Nitrogen was determined bysame way. digesting leaves in H2SO 4 and measuring released ammonia N

Leaf tissue was dried at 90 C for 90 min, then at 70 C for colorimetrically with basic phenol and NaOCl solution (7).22 hr. The tissue was ground in a Wiley mill to a 40-mesh particle Phosphorus, K, Ca, Mg, 5, and B were extracted from leaf tissuesize. One-half gram samples were extracted with 76% ethanol digested by nitric-perchloric acid (wet-ashed). Phosphorus wasfor 6 hr in a Soxhlet apparatus. The ethanol extract was determined colorimetrically with ammonium molybdate-ascorbicevaporated to near dryness in a rotary evaporator, diluted with acid solutions. Sulfur was determined turbidimetrically withwater, and then filtered through glass wool on a Whatman No. barium chloride, and B was determined colorimetrically with1 filter paper disk. The filtrate was mixed with a 1:1 mixture curcumin solution. The other elements were determined by atomicof Dowex-5O and Dowex-1 cation and anion resins (one volume absorption (3). All analyses were done by Agroservices Inter-of resin mixture per one volume of filtrate), shaken for 1 hr, national of Orange City, FL.and then filtered through Whatman No. 1 filter paper. The filtrate Metabolism. Oxygen uptake was determined first in leaves fromwas analyzed for total soluble sugars by the anthrone method DHF-treated and control plants maintained for 192 hr in a growth(16) and reducing sugars by the Somogyi-Nelson method (16). chamber (34 C day, 12 hr; 30 C night, 12 hr; 300 1.E m- sec-')

The residue from the extraction in the Soxhlet was analyzed and in plants maintained in a greenhouse (32-36.3 C, day; 20 Cfor starch by the method of Carter et al (8). The residue was minimum, night; 450 #.E m-2 sec-I). In this test, oxygen uptakeoven-dried to remove ethanol and then brought to a simmer in was determined at 48-hr intervals after seedlings were incubated10 ml of distilled water, cooled, and amended with 10 ml of in 100 mg of DHF and control solutions per liter in the growth0.5% amyloglucosidase in 0.1 M citrate buffer, pH 4.5. This chamber and greenhouse. Forty 5-mm-diameter leaf disks permixture was incubated at 55 C for 2 hr with shaking and then treatment replicate (five disks per plant from eight plants) were

Vol. 79, No. 6, 1989 701

placed in a Gilson Differential Respirometer flask and dark and DHF, respectively. No wilt developed in control plants. Onlyrespiration determined. The flasks were suspended in a 30 C water the isomarticin treatment resulted in a decrease in total solublebath with the shaker set at 100 rpm. The center well of the flask sugars compared to that of the controls (Table 1). Starch alsocontained 0.2 ml of 10% KOH and a filter paper disk. Oxygen decreased in plants treated with both toxins. In the isomarticinuptake was calculated as mean microliters per milligram of dry treatment, reducing sugars were 31.1 mg g-' and total proteinleaf weight per hour from three replicates per treatment. 291 mg g-' compared with control values of 33.3 and 295 mg

The effects of dinitrophenol (DNP), an uncoupler of oxidative respectively; these comparisons were not significantly different.phosphorylation, sodium azide, salicylhydroxamic acid (SHAM), In the DHF treatment, reducing sugars were 28.1 mg g- andand NaF were evaluated in other tests on oxygen uptake, using total protein 268 mg g-1 relative to control values of 21.4 andleaf disks from the control and from treatments with DHF at 250 mg g-', respectively, comparisons that were also not100 mg L-'. Forty leaf disks per treatment were infiltrated with significantly different.5 mM DNP, 10 mM sodium azide, 10 mM SHAM, or 20 mM Although DHF caused a wilt rating of 4.3 compared to noNaF by laying the disks stomate side down on filter paper wilt in treated plants, changes in chlorophyll a or b betweenimpregnated with the compound solubilized in 67 mM potassium treatments were too small to be significant. Chlorophyll a in thephosphate buffer, pH 6.7. The leaf disks were exposed for 3 hr DHF treatment was 5.1 mg g-' versus 5.7 in the control, andat 27 C in closed petri plates sealed with Parafilm. Plants in chlorophyll b was 2.2 mg g-' in the DHF treatment versus 2.3this study were maintained in a growth chamber set to the same in the control. The a/b ratio, however, was significantly lowerenvironmental parameters as described in the first metabolism in treated plants (Table 1). ATPase in the DHF treatment (6.8test. Data were reported as mean microliters of 02 per milligram prm hr-' mg-' of chlorophyll) was not significantly different fromof dry leaf weight from two replicates per treatment. Oxygen actiVity in the control (4.7 pm hr-' mg-1 of chlorophyll).uptake data were collected at 15-min intervals for 60 min. Oxygen Inoculated plants contained significantly less total soluble anduptake slopes generated by each treatment were analyzed by linear reducing sugars and less starch compared to controls, but totalregression and beta values of each slope separated by Duncan's protein did not differ between the treatments (Table 2).multiple range test at P < 0.05. In general, naphthoquinone-treated plants, as well as inoculated

plants, accumulated more minerals in leaves than did the controls.RESULTS All minerals except S accumulated in leaves of plants treated

with DHF, while N, Ca, and Mg accumulated in leaves ofPlant wilt occurred in seedlings treated with both naphtho- isomarticin-treated plants (Table 3).

quinones, with optimum expression occurring in 7 days. Plant Respiration rates of DHF-treated plants in both the greenhousewilt was scored as 2.8 and 3.0 for plants treated with isomarticin and the growth chamber increased significantly during the first

48 hr compared to those of controls, and they subsided morerapidly with time in DHF-treated plants maintained in the growth

TABLE 1. Total soluble sugar, starch, and chlorophyll a/b ratio in leaves chamber than in those maintained in the greenhouse (Fig. 1).of rough lemon rootstock seedlings incubated in solutions of isomarticin Respiration increased in greenhouse control plants after 96 hr,and dihydrofusarubin' and by the end of the test respiration in this treatment did not

Totalsolublesugar'bc Starchb,c Chorophyllb TABLE 2. Plant wilt, total soluble and reducing sugars, starch, and total

Treatment (mg/g) (mg/g) a/b protein in leaves of rough lemon seedlings inoculated with Fusariumsolania

Isomarticin (50 mg L-') 54.3* 27.8*** --Control 86.7 285.8 - Total TotalDihydrofusarubin, Plant soluble reducing Total

(100 mg L-1) 60.5ns 67.5*** 2.3 wiltb'c sugarc sugarc Starch proteinControl 53.4 742.2 2.5* Treatment (0-5) (mg/g) (mg/g) (mg/g) (mg/g)

Inoculated 3.0 51.3* 22.1"* 70.9*** 285flS'All data are means from three hydroponic tanks per treatment, each Cnol 0.0 84.5 35.7 30.1 29containing eight seedlings. Sugar and starch values are reported on a Control ____ ________________dry weight basis. Treatment means compared with control, by Student's aAll sugar and protein values are reported on a dry weight basis. Treatmentt test: * - P < 0.05; *** - P <_ 0.001; ns - not significant, - indicates means compared with control, by Student's t test: * - P < 0.05; ** -no test was made. P•< 0.01' *** - P<_ 0.001; ns - not significant.

bDihydrofusarubin: leaf samples taken 5 days after exposure to toxin bwilt rated on a scale of 0-5: 0 = no wilt, 5 = severe wilt and leaffor sugar and starch analysis and 7 days after exposure to toxin for desiccation. Leaf wilt and leaf samples taken 7 days after inoculation.chlorophyll analysis. CAll data are means of four samples per treatment; four plants were pooled

cisomarticin: leaf samples taken 7 days after exposure to toxin, to comprise each sample.

TABLE 3. Mineral analyses of leaves from rough lemon rootstock seedlings incubated in solutions of isomarticin and dihydrofusarubin and ofleaves of seedlings inoculated with Fusarium solani

Percent composition, dry wta B

Treatment N P K Ca Mg S (ppm)

Isomarticinb(50 mg [7') 3.27 0.25 1.31 2.61 0.26 0.20 58

Control 3.04* 0.22ns 1.28ns 2.21 * 0.22* 0.18ns 52fls

Dihydrofusarubin(100 mg L-) 4.06 0.32 1.66 2.36 0.24 0.22 70

Control 3.27** 0.24"* 1.23"** 2.06* 0.18* 0.18ns 53*

Inoculated 3.84 0.22 1.31 2.48 0.21 0.20 74Control 3.21ns 0.15ns 1.12ns 2.04"** 0.20ns 0.13* 70flS

"AlI toxin data are means from three hydroponic tanks per treatment, each containing eight seedlings. Data from inoculation test are means offour samples per treatment; four pooled plants comprised each sample. Treatment means compared with control, by Student's t test: * -- P _< 0.05;•**- P < 0.01' ** P < 0.001; ns -not significant.

hlsomarticin: leaf samples taken 7 days after exposure to toxin; dihydrofusarubin: leaf samples taken 5 days after treatment.

702 PHYTOPATHOLOGY

2.6. - differ significantly from respiration in the DHF-treatedgreenhouse-maintained plants (Fig. 1). Lowering daytime growth-

2.6 chamber air temperature from 34 to 23 C reduced symptomdevelopment in toxin-treated plants (data not shown).

X 2.4 DNP significantly enhanced respiration in control plants and>- had a slight but significant stimulatory effect on respiration ofo 2.2 DHF-treated seedlings (Fig. 2A). Beta values (data not shown)

of all slopes in Figure 2A were significantly different from onekm 2 another. Beta values of all slopes in Figure 2B were also signifi-cantly different from one another, indicating that sodium azideL 1.6 (cytochrome c oxidase inhibitor) inhibited respiration in bothcontrol and DHF-treated tissue. Beta values for slopes generatedo 1.6 ' by the control and DHF-treated tissue were not significantly

r_ j 0 GREENHOUSE CONTROL different in Figure 2C, but sodium fluoride significantly inhibited" 1.4 A GREENHOUSE - DHF respiration independent of treatment. Beta values for slopes0 GROWTH CHAMBER CONTROL generated by the control and DHF-treated tissue were not1.2 0 GROWTH CHAMBER - OHF significantly different in Figure 2D, but sodium azide in

i I I i I combination with SHAM (alternate pathway inhibitor)0 48 96 144 192 significantly inhibited respiration in all tissue and also repressed

any DHF-related stimulation of respiration.HOURS

Fig. 1. Respiration of rough lemon seedlings treated with 100 mg of DISCUSSIONdihydrofusarubin per liter and of controls in a greenhouse and a growthchamber for 192 hr. The production of large-scale quantities of certain naphtho-

3.6 2

3.2 -- , CONTROL+DNP 1.8 CONTROL+NaN3e--- DHF+DNP 0- COHF+NaTO H--• 2.6 - * CONTROL+DHF . 1.6 - - CONTROL3C E3 CONTROL 1C CONTROLC 2.4 ccS0- 1.2

2 1.6

1& 0.8~ 1.2 0.6

0.8 0.4

0.4 a b0 0

2.8 CONTROL +NaN +SHAM2 - CONTROL+NaF e- OHF +NaN 3 +S.AMDHF+NaF -*- CONTROL+DHF-0- CONTROL+DHF 2.4 -9--CONTROL

1.t2 1.1 100.2 -

R-4 0.8 .

0.4 •

C 0.4

d000I 15 30 45 600 15 30 45 600 53456MINUTES MINUTES

Fig. 2. Effects of inhibitors on respiration of rough lemon seedling leaf disks from plants maintained in solutions of 100 mg of dihydrofusarubin(DHF) per liter and in control solutions. Oxygen uptake values were recorded at 15-mmn intervals for 60 min. A, Effect of dinitrophenol (5 mM)on respiration of rough lemon leaf tissue from plants treated with DHF and from control plants. B, Respiration of leaf disks from DHF-treatedplants and control plants, both treated with sodium azide (10 mM). C2, Respiration of leaf disks from DHF-treated plants and control plants,both inoculated with NaF (20 mM). D, Respiration of leaf disks from DHF-treated plants and control plants, both incubated with 10 mM sodiumazide and 10 mM salicylhydroxamic acid (SHAM).

Vol. 79, No. 6, 1989 703

quinones has made it possible to evaluate effects of these toxins may be the responsible agents. A continuation of metabolic studss

on whole plant systems (4). The first expression by the plant with inoculated plants may be a means to clarify their involvement

of toxin uptake in these studies was a significant increase in in pathogenesis. Other toxins reported from F. solani, such as

respiration (Fig. IA). This coincides with the first irreversible fusaric acid (S. Nemec, unpublished data) and trichothecenes

wilt symptoms that appear 24-48 hr after toxin treatment (24). (Osamu Kawamura, personal communication) have not been

Increases in respiration usually are characteristic of most diseased detected in cultures of F. solani from citrus tissue.

plant tissues (31) and are nonspecific reactions in plants (14).Increased respiration caused by DHF partially depleted starch LITERATURE CITEDreserves, which were also depleted in plants in the isomarticinand inoculation treatments. 1. Aldridge, W. M., and Street, B. W. 1971. The relation between the

Total protein was unaffected in DHF- and isomarticin-treated specific binding of trimethyltin and triethyltin to mitochondria and

seedlings as well as in inoculated plants. The general early increase their effects on various mitochondrial functions. Biochem. J. 124:221-234.

in protein levels that occurs in fungus-infected plants (14) did 2 Arnon, D. I. 1949. Copper enzymes in isolated chioroplasts. Poly-

not develop in this study, and plant health had not deteriorated phenoloxidase in Beta vulgaris. Plant Physiol. 24:1-15.

enough for the marked decreases in protein apparent in late stages 3. Baker, D. E., and Sulr, N. H. 1982. Atomic absorption and flame

of disease development caused by fungus pathogens (14). Both emission spectrometry. Pages 13-27 in: Methods of Soil Analysis.DHF and isomarticin caused small but significant increases in Part 2, Chemical and Microbiological Properties, 2nd ed. No. 9,

leaf N, an effect seen in fungus-infected plants (14); in this study Agronomy. A. L. Page, ed. American Society of Agronomy, Madison,

such increases appeared unrelated to protein synthesis. WI.

These toxins are known to exert an effect on chloroplasts. 4. Baker, R. A., Tatum, J. H., and Gaffney, K. M. 1983. Large-scale

Isomarticin disrupted chloroplast structure in Spyrogyra (17) and recovery of naphthazarins from culture filtrates of Fusarium solani.

in citrus mesophyll cells (D. Achor, personal communication). (Abstr.) 83rd Annual Meeting, p. 245. American Society of

Veinal chlorosis, a symptom caused by DHF (24), occurred on 5. Baker, R. H., Tatum, J. H., and Nemec, 5. 1981. Toxin production

plants treated in DHF in this study but was not related to a by Fusarium solani from fibrous roots of blight-diseased citrus.general chloroplast degradation in this tissue (D. Achor, personal Phytopathology 71:951-954.communication). Furthermore, no changes in chlorophylls a and 6. Bender, G. S., and Menge, J. A. 1984. Dry root disease of citrus-

b were detected between treatments in our study. However, in Predisposing roots to infection by Fusarium solani. (Abstr.)

another study, reduction in photosynthesis without a change in Phytopathology 74:816.

leaf chlorophyll content did occur in cotton affected by 7. Bremner, J. M., and Mulvaney, C. S. 1982. Nitrogen-total. Pages

Verticillium wilt (20). DHF effected no change in ATPase. Othei 595-624 in: Methods of Soil Analysis. Part 2, Chemical and

possible changes in chloroplast functions were not investigated. Microbiological properties, 2nd ed. No. 9, Agronomy. A. L. Page,l c g ed. American Society of Agronomy, Madison, WI.

Isomarticin is known to interfere with respiration by inhibiting 8. Carter, J. L., Garrard, L. A., and West, S. H. 1973. Effect of gibberellicthe anaerobic decarboxylation of pyruvate in vitro and the acid on starch degrading enzymes in leaves of Digitaria decumbens.oxidative decarboxylation of a-ketoglutarate (17). This toxin was Phytochemistry 12:251-254.the most toxic of the naphthoquinones tested on peas (10,17). 9. Claydon, N., Grove, J. F., and Pople, M. 1977. Fusaric acid from

At a lower dosage than that of DHF, isomarticin was equally Fusarium solani. Phytochemistry 16:603.

effective in altering the physiological parameters reported here, 10. Dorn, S. 1974. Zur Rolle von Isomarticin, einem Toxin von Fusarium

as well as water relations of citrus in another study (24). martii var. pisi in der Pathogenese der Stengel- und Wurgelfaule an

Isomarticin may have been the preferred toxin for evaluating Erbsen. Phytopathol. Z. 81:193-239.

inhibition of respiration in citrus, but unfortunately our fungal 11. El-Banna, A. A., Scott, P. M., Lau, P., Sakuma, T., Platt, H. W.,and Campbell, V. 1984. Formation of trichothecenes by Fusarium

cultures did not produce enough of the compound for this type solani var. coeruleum and Fusarium sambucinum in potatoes. Appl.

of test. Environ. Microbiol. 47:1169-1171.Experiments with DHF in combination with sodium azide, 12. Emanuel, E. L., Carver, M. A., Solani, G. C., and Griffiths, D. E.

sodium azide and SHAM, and NaF were conducted, in which 1984. Differential inhibition of FoF 1-ATPase-catalysed reactions in

DHF was expected to have a maximal effect on respiration (Fig. 1). bovine-heart submitochondrial particles by organotin compounds.

The studies suggest that the cause of the stimulation of respiration Biochim. Biophys. Acta 766:209-214.

is to be found in the pathways using the mitochondrial terminal 13. Goldberg, K., and Brakke, M. K. 1987. Concentration of maize

oxidases (Fig. 2A and D) and not in the pentose phosphate chlorotic mottle virus increased in mixed infections with maize dwarf

pathway (Fig. 2C). Sites in the affected pathways at which the mosaic virus, strains B. Phytopathology 77:162-167.stimulathwa(ion occs hves y tobe afectermined .pa hwaysatio the 14. Goodman, R. N., Kirtly, Z., and Wood, K. R. 1986. The Biochemistrystimulation occurs have yet to be determined. Inhibition of the and Physiology of Plant Disease. University of Missouri Press,

mitochondrial cytochrome c oxidase alone did not significantly Columbia.affect the DHF-related stimulation of respiration (Fig. 2B). 15. Graham, J. H., Timmer, L. W., and Young, R. H. 1983. Necrosis

The one effect of DHF on respiration that is obvious is the of major roots in relation to citrus blight. Plant Dis. 67:1273-1276.

ability of DHF both to stimulate respiration and at the same 16. Hodge, J. E., and Hofreiter, B. T. 1964. Determination of reducing

time to retard or block the stimulation of respiration by DNP sugars and carbohydrates. Pages 380-393 in: Methods in Carbohydrate

(Fig. 2A). Such an effect has been reported in studies on organotin Chemistry, Vol. 4. R. L. Whistler, ed. Academic Press, New York.

compounds in mitochondria (1). The proposed site of interaction 17. Kern, H. 1978. The naphthazarins of Fusarium. Annu. Phytopathol.

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in mitochondria. 20. Mathre, D. E. 1968. Photosynthetic activities of cotton plants infected

It is not known to what extent these toxins are produced by with Verticillium albo-atrum. Phytopathology 58:137-141.

F. solani in naturally infected roots. The fungus is systemic in 21. Nemec, 5. 1984. Characteristics of Fusarium solani-infected pioneer

xylem of fibrous roots and wood of scaffold roots on field trees roots on blight-diseased and healthy citrus. Proc. Fla. Soil Crop Sci.

and husmaysynhesze hes toins n tis isse. onvntinal 22.Soc. 43:177-183.andthu ma sytheizethee txin inthi tisueCoveniona 2. Nemec, 5. 1987. Fusarium solani association with branch and trunk

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F. solani are active in pathogenesis in citrus, the naphthoquinones Proc. Fla. State Hortic. Soc. 93:36-41.

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