Plant Physiol. (1986) 81, 1103-1109 0032-0889/86/81/1103/07$0 1.00/0 Changes in Cytokinin Concentrations in Xylem Extrudate following Infection of Eucalyptus marginata Donn ex Sm with Phytophthora cinnamomi Rands' Received for publication December 12, 1985 and in revised form March 25, 1986 DAVID M. CAHILL*, GRETNA M. WESTE, AND BRUCE R. GRANT School of Botany (D.M.C., G.M.W.) and Russell Grimwade School of Biochemistry (B.R.G.), University of Melbourne, Parkville 3052, Victoria, Australia ABSTRACI The concentrations of zeatin-type and isopentenyladenine-type cyto- kinins were reduced in the xylem extrudate collected from seedlings of Eucalyptus species following infection by Phytophthora cinnamomi Rands. The use of an enzyme-linked immunosorbent assay (ELISA) allowed the detection of these cytokinins over the range of 03 to 7 picomoles for the isopentenyladenine-type and 1 to 1000 picomoles for the zeatin-type. Isopentenyladenine-type cytokinins occurred in concen- trations less than 10% of the zeatin-type, but they could be readily detected and measured. This is the first report of their presence in xylem. The sensitivity of the assay allowed a short collection period (30 minutes) reducing any confusion with trauma-induced changes. Infection of the susceptible species Eucalyptus marginata Donn. ex Sm. resulted in significant reduction of zeatin-type cytokinins within 3 days of infection, and at 14 days postinfection the concentration of both cytokinin types was reduced to 26% of uninoculated controls. No reduction in cytokinins occurred with the field resistant Eucalyptus calophylla R. Br. It is suggested that failure of cytokinin transport from the root system may be responsible for the failure in water transport and symptoms of P. cinnamomi infection observed in infected susceptible eucalypts. The fungus Phytophthora cinnamomi Rands is a pathogen with a broad host range, and the diseases it causes are of economic importance throughout the temperate and tropical zone (36). While the primary symptom of infection by P. cinnamomi in a susceptible species is root rot, the organism also induces second- ary symptoms in the shoots of many perennial host plants which resemble droughting. These include wilting, chlorosis, micro- phylly, and shoot and bud death (dieback) (36). Studies of seedling eucalypts grown under controlled condi- tions showed that susceptible species died when as little as 8% of the root system was infected (9), indicating that loss of root tissue alone is unlikely to be the cause of either the secondary symptoms or of host death. Moreover, the same study demonstrated a significant reduction in hydraulic conductivity of the root system of a susceptible species 2 to 4 d after inoculation. This decline in root conductivity preceded reductions in leaf xylem water poten- 'Supported in part by a grant from the Reserve Bank Rural Credits Fund, UM/1282. 2Abbreviations: IgG, immunoglobulin G; ZR, trans-zeatin riboside; Z, zeatin; DiZ dihydrozeatin; IP, N6-isopentenyladenine; IPA, N6-isopen- tenyladenoside; ELISA, enzyme-linked immunosorbent assay. tial, leaf transpiration rate, and wilting in these plants. These changes were not observed in seedlings of field resistant species. Histological examination failed to show xylem blockage or extensive damage to the conducting system under these condi- tions (9) and there is no evidence of toxin production sufficient to account for the secondary symptoms (7). However, reductions in the concentration of cytokinin-like compounds in the tracheal fluid of crop plants infected with wilt by other fungal root pathogens, Verticillium spp., have been reported (16, 20, 24) with symptoms which also resemble drought. In this paper, we report that infection of susceptible eucalypts by P. cinnamomi also induces a major reduction in the concentration of cytokinins, and we suggest that this reduction may be sufficient to account for the secondary symptoms devel- oped on infection in susceptible species of this genus. A previous brief report of this work has appeared (8). MATERIALS AND METHODS Chemicals. trans-Zeatin riboside, zeatin, N6-isopentenylade- nine, N6-isopentenyladenosine, N6-furfurylaminopurine, N6- benzylamino-purine, p-nitrophenyl phosphate ('Sigma 104' grade), alkaline phosphatase conjugated IgG2 from rabbit, chicken egg albumin (ovalbumin), and dimethyldichlorosilane were purchased from the Sigma Chemical Company. Dihydro- zeatin, adenosine, and guanosine were a gift from F. Hassan (University of Melbourne), BSA Fraction V was from the Com- monwealth Serum Laboratories, Melbourne, Octan-l-ol was from Unilab Laboratory Reagents, and Freunds complete and incom- plete adjuvant were purchased from Difco. Phytophthora cinnamomi Cultures and Zoospore Production. The culture of P. cinnamomi (A2 strain) used was isolated from roots of Isopogon ceratophyllus R.Br. (25) and was maintained on 20%-V8 juice agar. Zoospores were produced axenically by the method of Byrt and Grant (5) and the concentration deter- mined with a haemacytometer prior to use as inoculum. Plant Materials and Growth Conditions. Eucalyptus marginata and Eucalyptus calophylla seeds were of known provenance (CSIRO Division of Forest Research, seedlot numbers 9899 and 8855, respectively). The seed coat of E. marginata was com- pletely removed and the seeds germinated on moistened filter paper in sterile Petri dishes at room temperature. After 1 to 2 weeks, the newly germinated seedlings were transferred to black plastic pots (15 cm high x 8 cm diameter) containing a 1:2 mixture of steam-sterilized sand (<2 mm) and vermiculite. E. calophylla seeds were planted directly into pots. Plants were grown in a temperature controlled glasshouse for 6 months (temperate range 20-28°C). The lower half of the root system was then removed, and plants were repotted into pots split 1103 https://plantphysiol.org Downloaded on December 1, 2020. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
Changes in Cytokinin Concentrations in Xylem Extrudatefollowing Infection of Eucalyptus marginata Donn ex Sm withPhytophthora cinnamomi Rands'
Received for publication December 12, 1985 and in revised form March 25, 1986
DAVID M. CAHILL*, GRETNA M. WESTE, AND BRUCE R. GRANTSchool ofBotany (D.M.C., G.M.W.) and Russell Grimwade School ofBiochemistry (B.R.G.), University ofMelbourne, Parkville 3052, Victoria, Australia
ABSTRACI
The concentrations of zeatin-type and isopentenyladenine-type cyto-kinins were reduced in the xylem extrudate collected from seedlings ofEucalyptus species following infection by Phytophthora cinnamomiRands. The use of an enzyme-linked immunosorbent assay (ELISA)allowed the detection of these cytokinins over the range of 03 to 7picomoles for the isopentenyladenine-type and 1 to 1000 picomoles forthe zeatin-type. Isopentenyladenine-type cytokinins occurred in concen-trations less than 10% of the zeatin-type, but they could be readilydetected and measured. This is the first report of their presence in xylem.The sensitivity of the assay allowed a short collection period (30 minutes)reducing any confusion with trauma-induced changes. Infection of thesusceptible species Eucalyptus marginata Donn. ex Sm. resulted insignificant reduction of zeatin-type cytokinins within 3 days of infection,and at 14 days postinfection the concentration of both cytokinin typeswas reduced to 26% of uninoculated controls. No reduction in cytokininsoccurred with the field resistant Eucalyptus calophylla R. Br. It issuggested that failure of cytokinin transport from the root system maybe responsible for the failure in water transport and symptoms of P.cinnamomi infection observed in infected susceptible eucalypts.
The fungus Phytophthora cinnamomi Rands is a pathogenwith a broad host range, and the diseases it causes are ofeconomicimportance throughout the temperate and tropical zone (36).While the primary symptom of infection by P. cinnamomi in asusceptible species is root rot, the organism also induces second-ary symptoms in the shoots ofmany perennial host plants whichresemble droughting. These include wilting, chlorosis, micro-phylly, and shoot and bud death (dieback) (36).
Studies of seedling eucalypts grown under controlled condi-tions showed that susceptible species died when as little as 8% ofthe root system was infected (9), indicating that loss ofroot tissuealone is unlikely to be the cause ofeither the secondary symptomsor of host death. Moreover, the same study demonstrated asignificant reduction in hydraulic conductivity ofthe root systemof a susceptible species 2 to 4 d after inoculation. This decline inroot conductivity preceded reductions in leaf xylem water poten-
'Supported in part by a grant from the Reserve Bank Rural CreditsFund, UM/1282.
tial, leaf transpiration rate, and wilting in these plants. Thesechanges were not observed in seedlings of field resistant species.
Histological examination failed to show xylem blockage orextensive damage to the conducting system under these condi-tions (9) and there is no evidence of toxin production sufficientto account for the secondary symptoms (7).However, reductions in the concentration of cytokinin-like
compounds in the tracheal fluid of crop plants infected with wiltby other fungal root pathogens, Verticillium spp., have beenreported (16, 20, 24) with symptoms which also resembledrought. In this paper, we report that infection of susceptibleeucalypts by P. cinnamomi also induces a major reduction in theconcentration of cytokinins, and we suggest that this reductionmay be sufficient to account for the secondary symptoms devel-oped on infection in susceptible species of this genus. A previousbrief report of this work has appeared (8).
MATERIALS AND METHODS
Chemicals. trans-Zeatin riboside, zeatin, N6-isopentenylade-nine, N6-isopentenyladenosine, N6-furfurylaminopurine, N6-benzylamino-purine, p-nitrophenyl phosphate ('Sigma 104'grade), alkaline phosphatase conjugated IgG2 from rabbit,chicken egg albumin (ovalbumin), and dimethyldichlorosilanewere purchased from the Sigma Chemical Company. Dihydro-zeatin, adenosine, and guanosine were a gift from F. Hassan(University of Melbourne), BSA Fraction V was from the Com-monwealth Serum Laboratories, Melbourne, Octan-l-ol was fromUnilab Laboratory Reagents, and Freunds complete and incom-plete adjuvant were purchased from Difco.
Phytophthora cinnamomi Cultures and Zoospore Production.The culture of P. cinnamomi (A2 strain) used was isolated fromroots of Isopogon ceratophyllus R.Br. (25) and was maintainedon 20%-V8 juice agar. Zoospores were produced axenically bythe method of Byrt and Grant (5) and the concentration deter-mined with a haemacytometer prior to use as inoculum.
Plant Materials and Growth Conditions. Eucalyptus marginataand Eucalyptus calophylla seeds were of known provenance(CSIRO Division of Forest Research, seedlot numbers 9899 and8855, respectively). The seed coat of E. marginata was com-pletely removed and the seeds germinated on moistened filterpaper in sterile Petri dishes at room temperature. After 1 to 2weeks, the newly germinated seedlings were transferred to blackplastic pots (15 cm high x 8 cm diameter) containing a 1:2mixture of steam-sterilized sand (<2 mm) and vermiculite. E.calophylla seeds were planted directly into pots. Plants weregrown in a temperature controlled glasshouse for 6 months(temperate range 20-28°C). The lower half of the root systemwas then removed, and plants were repotted into pots split
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longitudinally and taped so that the root systems remained easilyaccessible. The plants were transferred to a controlled environ-ment chamber in which the temperature was 24 ± 1°C (day andnight), and there was a 12:12 light regime with PAR of 700 ,uEm-2 s1'. Nutrients were supplied on alternate days using a half-strength eucalypt nutrient solution (17), and pots were flushedevery 2 weeks with distilled H20 to prevent salt accumulation.Treatment of Plants. After 2 to 3 weeks in the growth chamber,
40 plants of each species were selected on the basis of uniformityin size and vigor, and 10 plants were randomly assigned to eachtreatment. Five plants of each species were inoculated with P.cinnamomi and the other five served as controls. Root pruningand transfer to the growth chamber resulted in the productionof long new white roots which were accessible by carefullyremoving one-half of the split pot. Inoculation was made as
described previously (35). Three to six roots were inoculated perplant, and control plants were sham-inoculated using steriledistilled H20. Success of inoculation was tested by plating rootsonto P,oVP agar (31).Xylem Sap Collection. Extrudate (xylem sap extracted under
pressure) was collected 0, 3, 8, and 14 d after infection using aScholander pressure bomb (29). Plants were watered with dis-tilled H20 1 h before sap collection. Plants were removed fromthe split pots by immersion in half-strength nutrient solution (24± 1C) and the root system gently washed free of adhering sandand vermiculite. These steps were taken with care to avoiddamage to the root system. The root system was then placed ina cylindrical polyvinylchloride insert filled with half-strengthnutrient solution and inserted into the pressure bomb. The stemwas severed 3 cm above the developing lignotuber and thephloem and bark removed from the terminal 5 mm of stem.After sealing, the chamber pressure was increased by 0.2 MPa/min to a final pressure of 1 MPa. This pressure was maintainedfor 30 min and the extrudate collected at 5 min intervals from aparafilm (American Can Co., Greenwich, CT) funnel placedaround the protruding stem. The first drop of extrudate wasdiscarded. Extrudate was stored in 4 ml silanized glass vials (19)during the collection period and then frozen in liquid N2 andstored at -70°C until analysis. All manipulation of plants was
Extrudate collected from roots (30min)V
Samples sealed and frozen in liquid nitrogenand stored at -70°C
V
For analysis samples thawed andvolume recorded
V
Centrifuge 10,000 rpm, 5min toremove insolubles
Pass sample through Sep-pak
Elute cytokinins with methanol (3ml)
Eluate taken to dryness in vacuo <300C
Take up in 100 p l 10% DMSO PBS
Analyze for cytokinins using ELISA
FIG. 1. Method of collection and preparation of xylem extrudates fortesting in the ELISA.
200 plI of 100 ng/ml cytokinin-ovalbumin conjugate added toeach well of a microtitre tray and incubated for 3h
ITray is washed three times for 3min with phosphatebuffered saline containing 0.05% Tween 20 (pH7.2)
V250 pl of 0.25% ovalbumin added and
incubated for 1.5hv
Wash
VA mixture of 25 pI standard or sample plus 175 Vl ofantisera was added to each well and incubated for 3h
VWash
V200 pl of 1:2000 IgG alkaline phosphatasewas added and incubated 16h at 40C
VWashV
200 p I of 1 mg/ml p-nitrophenyl phosphatesubstrate in glycine buffer (pH 10.4) was added
VAbsorbance read at 405 nm after 30min
FIG. 2. The ELISA procedure.
*2-0
+.10
m
0
-2-0
So I0-025 0-1 0 25 1-0 2-5 10 25
ZR concentrotion (ng/well)FIG. 3. Logit transformed standard curve for zeatin riboside. A simi-
lar curve was produced for N6-isopentenyladenosine. Bars are SE (n =10).
carried out within the environment chamber.Diurnal Variation in Cytokinin Levels. To assess diurnal vari-
ation in cytokinin levels, 12 seedlings of E. calophylla were used.Extrudate was collected from two plants at both 0730 h and 0830h (lights on at 0800 h) and from four plants at 1300 h and 1630h (lights off 2000 h). Extrudate was stored at -70°C until ana-lyzed.Root Measurements. After extrudate had been collected, the
root systems were preserved in formalin-acetic acid-alcohol(FAA) (23). Root lengths were measured using a Comair rootlength scanner (Commonwealth Aircraft Corporation Limited,Australia) (27). Root tip number was determined using thetechnique of Richards and Rowe (26). The percentage of rootsystem infected was determined using the line intersect method(30).
Preparation of Cytokinin-Protein Conjugates. Conjugates ofZR and IPA to both BSA and ovalbumin were prepared by themodification of MacDonald et al. (18) of the Erlanger and Beiser
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CYTOKININ CHANGES FOLLOWING ROOT INFECTION BY P. CINNAMOMI
Table 1. Specificity ofZR and IPA AntiseraResults are expressed as % cross-reactivity and represent 50% inhibition value for ZR or IPA divided by the
50% inhibition value for the tested compound. Quantities of test compound required for 50% inhibition arealso given.
Compound Anti-ZR Anti-IPA
% ng % ng
ZR 100 2.5 0.26 3.9 x 104Z 45 4.5 0.031 3.2 x 105DiZ 31 8.2 0.017 5.9 x 105IPA 0.15 1.7 x 103 100 1 X 102IP 0.025 1.0 x 104 64 1.66 x 1026-Furfurylaminopurine 0.014 1.8 x 10' 9 1. X 1046-Benzylaminopurine 0.04 4 x 104 14 7.1 x 102Adenine 0 0Guanosine 0 0
FIG. 4. Logit transformations of dilutions of extrudate from controland infected roots of E. marginata and the standard curve for ZR. (x),ZR standards; (0, 0), dilutions of extrudate from 2 control root systems;(0, *), dilutions of extrudate from 2 infected root systems.
technique (11). Unconjugated cytokinin was removed using aSephadex G-l0 column eluted with degassed distilled H20. Theprotein concentration was determined by the method ofBradford(3) and adjusted to 8 mg/ml after concentration in an AmiconB 15 microconcentrator. Minimum coupling efficiency was esti-mated (10) and was found to be similar for ZR and IPA to bothproteins: 4 mol ZR: 1 mol protein and 7 mol IPA: 1 mol protein.Conjugates were stored at -70°C.
Immunization Schedule. Antisera were raised in New Zealandrabbits using intramuscular injection of 1 to 1.5 mg of conjugatein Freunds complete adjuvant initially, followed by two addi-tional injections of conjugate with incomplete adjuvant 1 weekapart. A boost injection of 0.5 to 1.0 mg of conjugate in incom-plete adjuvant was given 5 weeks later. Further booster injectionsdid not increase the titer significantly, and sufficient serum wasobtained from a single bleed from the marginal ear vein. Crudeserum was stored at -70°C.
Extraction and Assay of Cytokinins. The protocol for prepa-ration ofcytokinins from the xylem extrudate is shown in Figure1. In the purification step extrudates were purified before assayusing microparticulate Sep-Pak cartridges (Waters Associates)following the procedure of MacDonald et al. (19). The metha-nolic eluate was dried in vacuo at <30°C and taken up in 10%
101-
-u
01 1-O F
0
0.1 1.0 10Z R added (ng/well)
FIG. 5. Recovery of added ZR internal standards from dilutions ofextrudate from E. marginata. (x), standards with no extract; (0), 5 41concentrated extrudate/well of the microtiter tray; (Fl), 10 ul/well; (A),20 ul/well.
DMSO in 0.15 M PBS which contained: 8 g NaCl, 0.2 g KCI,1.15 g Na2HPO4, 0.2 g KH2PO4 per L (pH 7.2).The details of the ELISA protocol are shown in Figure 2. The
procedure for the indirect ELISA was developed based on themethods of Koenig and Paul (15). To reduce background ab-sorbance, conjugates of ZR and IPA were made to ovalbumin(chicken egg albumin) and these were bound to the microtitertrays as the antigen. Coating of trays with ovalbumin conjugateeffectively eliminated the binding of BSA-specific antibodieswhich are present in the sera. All additions except that of thestandards and samples were made with an eight channel multi-pipetter (Titertek, Flow Laboratories, Australia). Plates whichhad been incubated with 0.25% ovalbumin to block nonspecificbinding (step 3) could be stored for up to 6 months at -20°C,which eased the logistics of the assay. Absorbances were readwith a Biorad EIA Plate Reader (model No. 2550) fitted with a405 nm filter.
A
F
a I a I
0-025 0-1 0-25 1.0 2-5 10ulIofSamPle
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FIG. 6. Diurnal variation in concentrations of Z and IP cytokinins inE. calophvlla xylem extrudate during the light period. Bars are SE(n = 2 or 4). Arrows indicate beginning and end of collection period forother experiments. (0). IP cytokinins; (0). Z cytokinins.
Quantification. Absorbance values were converted to bindingvalues (B/Bo) which were then logit transformed (28). Standardcurves were fitted and calculations for samples made using a
Hewlett Packard General statistics package in conjunction witha program based on that of Brooker et al. (4).
RESULTS
Antiserum Properties. All immunized rabbits produced anti-sera to the cytokinin conjugates, but for each conjugate one
rabbit was chosen and its serum used throughout the experi-
ments. Serum titers for the conjugates differed considerably andreflected the difference in coupling ratios. The dilutions of serarequired to give 50% binding of antibody in the absence ofcompeting antigen were 1:50,000 and 1:15,000 for the IPA andZR antisera, respectively. Except at very low dilutions (1:100-1:900), preimmune sera did not bind.Standard curves relating concentration of cytokinin to binding
are typical (2, 18, 32).On logit transformation (Fig. 3) the curves are linearized giving
a usable measuring range of 10 pg to 2.5 ng (0.3-7 pmol) forIPA and 25 pg to 25 ng (1.0-1000 pmol) for ZR. Minimumdetection limits were 300 fmol (10 pg/25 ul) for IPA and 700fmol (25 pg/25 ,l) for ZR. The coefficient of variation fortriplicate samples within an assay was <4%.The cross-reactivity of antisera raised against ZR and IPA with
other cytokinins and structurally related compounds is shown inTable I and is in agreement with the results of others (12, 18, 32,33). Cross-reactivity of the ZR antiserum with the bases Z andDiZ (Z cytokinins) was appreciable as was the cross-reactivity ofIP with the IPA (IP cytokinins) antiserum. Hence, when calcu-lating amounts of cytokinins present in the extrudates, thesecompounds could contribute to the total. Data are thus given asZR or IPA equivalents.
Validation of the Assay. Experiments were performed to vali-date the assay on extrudate since eucalypt sap has not beenassayed by immunoassay before. Initial experiments showed thatdilutions of crude Eucalyptus extrudate did not always parallelthe standard curve. However, partial purification of the samplesusing Sep-Paks resulted in parallelism of sample dilutions andthe standard curve (Fig. 4). The recovery of added cytokininfrom this single step purification was found to be 90%. Parallel-ism between dilutions of unknowns and standard curves wasmaintained when dilutions were made with known amounts ofadded internal standard (Fig. 5).
Diurnal Variation of Cytokinin Concentrations. The collectionof extrudate from the 10 plants at each sampling time took 2.5to 3 h to complete. Even though this is a relatively short collectionperiod, diurnal variation in cytokinin content over this intervalcould influence the result. Figure 6 shows the quantities ofZ andIP cytokinins in the extrudate of E. calophylla at specific timesduring the 12:12 photoperiod day. There is increased synthesisof cytokinins during the day reaching a maximum between 0900and 1200 h, followed by a decrease. Increases in xylem cytokininlevels were observed in light and significant increases (P < 0.05)were detected within 1 to 1.5 h. However, the data show thatduring the time of collection which began at 0900 h, diurnalvariation would not have had a significant effect on the cytokininconcentrations in the extrudate.Changes in Cytokinins during the Experimental Period. Both
Z and IP cytokinins were detected in the xylem extrudate of E.marginata and E. calophylla. There was a general increase in
Table 11. Quiantities ofZ and IP Clvtokinins per Milliliter Xvlem Extrudate in Infected and Control Root Systems ofSeedlings ofE. marginata and E. calophvlla
Data are the mean of five replicates per treatment, ± SE; significance levels: a and c, P < 0.01; b and d, P < 0.05.
Z Cytokinins IP Cytokinins
Tilmeafter . inarginala E. calophylla E. marginata E. calophyllaControl Infected Control Infected Control Infected Control Infected
CYTOKININ CHANGES FOLLOWING ROOT INFECTION BY P. CINNAMOMI
Table III. Fresh Weight ofRoot Systems ofE. marginata and E. calophylla at Various Times afterInoculation
Root systems were severed below the lignotuber and blotted dry on absorbent paper before weighing. Valuesrepresent the mean ± SE (n = 5). Those values followed by the same letter are significantly different (P < 0.05).
Table IV. Fresh Weight ofShoots and Number ofLeaves per Shoot ofE. marginata and E. calophylla at Various Times after InoculationShoots included the lignotuber. Values represent the mean ± SE (n = 5). Those values followed by the same letter are significantly different (P <
0.05).E. marginata E. calophylla
Time Postinoculation Shoot weight Leaf number Shoot weight Leafnumber
Table V. Root Tip Numbers and Total Length ofRoot System for Control and Inoculated E. marginata and E. calophyllaRoot tip numbers and total root length were determined as described in "Materials and Methods." Measurements were not made on plants
sampled at 8 d after inoculation. Values represent the mean ± SE (n = 5). Values followed by an asterisk are significantly different from controls (P< 0.05).
Root Tip Numbers Total Root Length
Timenafter E. marginata E. calophylla E. marginata E. calophyllaInoculationControl Infected Control Infected Control Infected Control Infected
concentration of cytokinins during the 14-day experimental pe-riod (Table II). This paralleled the increase in root growth duringthis period (Table III). Levels of the Z cytokinins were consid-erably higher than the IP cytokinins from all samples. Extrudatenormally contained less than 100 pmol of Z. cytokinins andusually less than 10 pmol of the IP cytokinins.Changes following Infection. Three days after infection ofroots
of E. marginata by P. cinnamomi, there was a significant (P <0.01) reduction of Z and IP cytokinins compared with controlplants (Table II). By 14 d after infection, levels of both Z and IPcytokinins were reduced to 26% of controls.
In the field-resistant species E. calophylla, there was no changein either Z or IP cytokinins following infection, and concentra-tions of both cytokinins continued to increase during the exper-imental period. There was more variation between individualplants of E. calophylla, hence larger standard errors, but concen-trations of both Z and IP cytokinins were similar to that of theuninfected E. marginata.
Progress of Infection and Plant Growth. After inoculation,lesions developed in roots of E. marginata within 24 h andextended quickly. By 3 d after inoculation lesions had extendedthrough less than 2% of the root system, but by 14 d after
inoculation 20% of the root system was infected. In contrast,finite lesions were observed in roots of E. calophylla where thefungus became confined within 15 to 20 mm of the root tipwithin 3 d. All inoculated roots, whether from the susceptible orfield resistant species, contained P. cinnamomi. The root systemsof both species continued to grow actively and produce manylong white roots and large numbers of short translucent rootletswhether infected or not. Root systems of both eucalypts haddoubled in weight by 14 d.
Increase in weight was recorded as a measure of the growth ofroots (Table III) and shoots (Table IV) over the experimentalperiod. Shoots showed no symptoms of infection 14 d postino-culation. Shoot weights and leaf number had increased signifi-cantly in E. marginata particularly after the 8th d. Shoot weightand leaf number ofE. calophylla, a more slowly growing species,had not increased at 14 d after inoculation.Root Tip Number and Root Length. The number of root tips
of infected E. marginata were reduced by 40% compared tocontrols by 14 d postinoculation (Table V). E. calophylla rootsystems normally produced fewer root tips, but numbers weresimilar for both control and infected plants. Lengths of roots foran infected root system of E. marginata were reduced when
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compared to controls and mirrored the reduction in number ofroot tips.
DISCUSSION
Two groups of cytokinins, zeatin-type (Z, ZR, and DiZ) andN6-isopentenyladenine-type (IP and IPA) have been detected andquantified in xylem extrudate from 6-month-old seedlings of twoeucalypt species that differ in susceptibility to P. cinnamomi.This is the first record ofIP-type cytokinins in xylem sap althoughthey have been previously isolated from the phloem exudate ofa number of species (34). The removal of the phloem at thecollar and the discard of the first drop of exudate ensured thatall sap collected originated from the xylem. The ELISA techniqueenabled the detection and measurement of the low concentra-tions (pmol) ofcytokinins present in xylem sap and demonstratedtheir mass transport in xylem sap from root to shoot. Collectionofxylem material over the relatively short time of 30 min reducedthe chance of rapid cytokinin decomposition in sap isolated fromthe plant. The minimum injury and short collection period alsoreduced any error due to wound response, which may mask thechanges in endogenous phytohormones due to pathogen infec-tion.The picomolar levels of both the cytokinin types found in the
extrudates resemble those recorded from phloem exudate (34)and a soybean root pressure exudate (13).The role ofgrowth regulators in plant disease has until recently
been perceived as one in which altered levels produce growthabnormalities such as excessive growth, stunting, and tumors(21). Little regard has been given to the possibility that thealteration in levels of one or more of the phytohormones maybe crucial in disease development before other symptoms areobserved.Our results demonstrate that after infection by P. cinnamomi
the levels of both Z-type and IP-type cytokinins in the xylemextrudate of the susceptible E. marginata were drastically re-duced. A significant reduction in Z-type cytokinins was detectedas early as 3 d and of IP-type cytokinins 8 d after infection. Theseresults provide the first accurate evidence that particular cytoki-nins are affected by a root pathogen. Misaghi et al. (20) andPatrick et al. (24) also found reduced levels of cytokinin-likeactivity in tracheal fluid from Verticillium-infected cotton andtomato roots. However, this is a wilt pathogen that plugs thexylem.By contrast levels ofcytokinins in the roots ofthe field-resistant
species E. calophylla were not altered by infection. This agreeswith previous work where respiration, ion leakage, and roothydraulic conductivity were all changed by infection of thesusceptible E. marginata but not of E. calophylla (6, 7, 9).
P. cinnamomi preferentially penetrates and destroys the roottips and hence cytokinin synthesizing sites; it must be noted,however, that the magnitude of the reduction in cytokininsobserved is well in excess of the reduction in actively growingroot tips. At 3 d postinoculation, when root tip numbers ofinfected plants were similar to controls, a significant reductionin the levels of cytokinins was already detected.The secondary symptoms of P. cinnamomi invasion resemble
those due to water stress, and we have found significant reduc-tions in cytokinin concentrations which precede the reductionsin root hydraulic conductivity found by Dawson and Weste (9).Reductions in concentrations of cytokinins occur in waterstressed plants (14) and under these conditions there are largeincreases in the concentration of ABA in buds and leaves andthere is some evidence which suggests that there are also increasesin roots (1, 22). The alteration in balance between cytokininsand ABA may therefore be a major cause of the secondarysymptoms of the disease caused by P. cinnamomi. This would
explain the apparent contradiction of massive reduction in water
transport when only limited destruction of the root system hasoccurred.
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1108 CAHILL ET AL.
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