Dec 12, 1985 - In GF Pegg, ed, International Verticillium. Symposium. ... O'BRIEN TP, ME MCCULLY 1981 The Study of Plant Structure. Principles and.
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 cytokinins 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 concentrations 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 secondary symptoms in the shoots of many perennial host plants which resemble droughting. These include wilting, chlorosis, microphylly, and shoot and bud death (dieback) (36). Studies of seedling eucalypts grown under controlled conditions 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-isopentenyladenoside; 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 conditions (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 developed 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-isopentenyladenine, N6-isopentenyladenosine, N6-furfurylaminopurine, N6benzylamino-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. Dihydrozeatin, adenosine, and guanosine were a gift from F. Hassan (University of Melbourne), BSA Fraction V was from the Commonwealth Serum Laboratories, Melbourne, Octan-l-ol was from Unilab Laboratory Reagents, and Freunds complete and incomplete 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 determined 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 completely 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 (
