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Journal of Plant Pathology (2009), 91 (1), 141-146
Edizioni ETS Pisa, 2009
141
ENDOGENOUS CYTOKININ CONTENT IN COCONUT PALMS AFFECTED
BY LETHAL YELLOWING
M.L. Aguilar, F. Espadas, B. Maust and L. Sáenz
Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, A.C.
Calle 43 No. 130, Colonia Chuburná de Hidalgo, 97200 Merida, Yucatan, Mexico
Lethal yellowing (LY) is the most devastating phytoplasma disease affecting coconut palm (Cocos nucifera)
in the Americas. Based on symptoms, different pathogenicity models and factors have been proposed, including hormone unbalance and changes in endogenous cytokinins content. Symptomatology and PCR were used
to determine LY phytoplasma presence in coconut
palms from Yucatan, Mexico. To determine Z-type and
iP-type endogenous cytokinin content, young leaf and
primary and secondary root meristem tissue were analyzed by HPLC-ELISA. The content of six cytokinins,
three of the Z-type (Z, Z9G, ZR) and three of the iPtype (iP, iP9G and iPR) were determined in nine palms,
three of which were healthy, three were in disease stage
2; and three in stage 6. Both cytokinin types decreased
drastically compared with healthy controls in stage-2
leaf tissue: 13.3-fold in Z; 11 in Z9G; 17.8 in ZR; 17.7 in
iP; 382 in iP9G; and 17.5 in iPR. Decreases in stage-6
leaf tissue were more pronounced, so that Z9G, ZR, iP
and iP9G were not detected. Z-type cytokinins decreased also in primary root meristem from affected
palms at both stages (346 fold in Z; 3.9 in Z9G; and 6 in
ZR), but iP9G increased 7 times in stage-2 tissue and
iPR increased 26.45 times in stage-6 tissue. In stage-2,
secondary root meristem tissue, Z increased 20.78
times, iP increased 4 times and iPR 14 times. Lethal yellowing clearly affects endogenous Z-type and iP-type
cytokinin levels in coconut palm, which may lead to
some of the symptoms associated with this disease.
phloem. They cause diseases to several hundred plant
species (Agrios, 1978), among which lethal yellowing
(LY) (Plavsic-Banjac et al., 1972). This disease spreads
quickly, is fatal and affects many palm species in the
Americas, including coconut palm (Cocos nucifera)
(Harrison and Jones, 2004).
As LY progresses and becomes more severe, infected
coconut palms exhibit a variety of symptoms including
premature nut drop (stage 1), inflorescence necrosis
(stage 2 to 3), leaf chlorosis and senescence (stages 4 to
6) and spear leaf death (stage 7-8). Plant death (stage 9),
usually ensues within 3-6 months (McCoy et al., 1983).
To account for this complex symptomatology, different
pathogenicity models and factors have been proposed,
including toxins, impairment of phloem function, reduced nitrogen and carbon translocation, physiological
stress and hormone unbalance (Oropeza et al., 1997).
Symptoms such as stomatal closure, leaf yellowing and
senescence are similar to those linked with natural leaf
senescence and may be explained by a reduction in endogenous cytokinins. Cytokinins are plant growth regulators that promote stomatal opening (Dodd, 2003) and
delay senescence of whole plants (Noodén et al., 1990)
and excised plant parts (Singh et al., 1992). However,
there is evidence that plant pathogens such as viruses
can also alter endogenous cytokinin content; e.g. in tobacco (Whenham, 1989); potato (Dermastia et al., 1995;
Dermastia and Ravnikar, 1996) and beans (Clarke et al.,
1999). Therefore, the objective of the present study was
to determine the endogenous cytokinin content in LYaffected coconut palms.
Key words: Phytoplasma, Cocos nucifera, cytokinins,
ELISA, HPLC, PCR.
MATERIALS AND METHODS
SUMMARY
INTRODUCTION
Phytoplasmas (class Mollicutes) are minute prokaryotes lacking a cell wall, generally confined to the host
Corresponding author: L. Sáenz
Fax: +52. 999. 9813900
E-mail: [email protected]
Plant material. Samples from young leaves (+4, numbered from apex), primary and secondary root meristems, and trunk were collected from coconut palms (Cocos nucifera) growing along the north coast of Yucatan
state, Mexico, at San Crisanto (21°20’LN, 89°09’LW),
were weighed, dipped in liquid nitrogen and stored at 70ºC until lyophilized.
Chemicals and reagents. Cytokinins were purchased
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Cytokinins in LY-affected coconut palm
from Apex Organics (UK). Antibodies against ZR
(zeatin riboside) and iPR (iPR-isopentenyladenosine)
and alkaline phosphatase were supplied by Olchemim
(Czech Republic). Bovine serum albumin, ovoalbumin
and 4-nitrophenylphosphate were purchased from Fluka (USA); acid phosphatase from Sigma (USA); SepPak C18 cartridges from Waters Associated (USA); and
methanol for chromatography from Merck (Germany).
Detection of LY phytoplasma. Presence of LY phytoplasma in putatively infected plants was determined by
PCR according to Maust et al. (2003). Briefly, DNA was
extracted from ground trunk tissue with cetyltrimethyammonium bromide (CTAB) buffer (20 mM EDTA pH 8.0,
100 mM Tris-HCl pH 8.0, 2% CTAB, 1.4 M NaCl, 2mercaptoethanol in sterile distilled water) at 65°C for 1 h
and used as templates for PCR. Amplifications were done
in 25 µl reaction volumes, each containing a 1 µl sample of
DNA, 50 ng of each primer, 125 µM of each dNTP, 1 U
Taq DNA polymerase and standard PCR buffer containing 1.5 mM MgCl2. PCR was run for 35 cycles in a programmable Perkin Elmer GeneAmp PCR system 2400 using phytoplasma-universal rRNA primer pair P1/P7
(Deng and Hiruki, 1991; Smart et al., 1996), with the following parameters: denaturation for 60 sec at 94°C; annealing at 54°C for 50 sec; and extension at 72°C for 1
min 20 sec (10 min in the final cycle). The products of the
initial P1/P7-primed PCR were diluted to 1:40 with sterile
ultrapure water and reamplified for 30 cycles using nested
16S rRNA and the 503f/LY16Sr primer pair, as described
by Harrison et al. (1999). For nested PCR, the thermal cycling parameters were: denaturation for 30 sec at 94°C;
annealing at 60°C for 50 sec; and extension at 72°C for 1
min 20 sec (10 min in the final cycle). Aliquots (5 µl) of
each final reaction mixture were electrophoresed on 1%
agarose gels, stained with ethidium bromide, viewed and
photographed under an UV transilluminator.
Cytokinin extraction. Frozen tissues were placed in a
cold mortar, cooled in liquid nitrogen, ground to a fine
powder and extracted with 10 vol of ice-cold 90% (v/v)
aqueous ethanol. Extraction was continued on ice with
occasional stirring for 90 to 120 min, the solution was
centrifuged at 4000 rpm and the supernatant recovered.
Tissue samples were extracted again with 80% ethanol
(v/v, 20 ml) for 60 min, the solution centrifuged as
above, and the supernatant recovered. The two supernatants were combined and reduced to low volume (1
to 5 ml approx.) on a rotary evaporator under vacuum
at 35°C. This concentrated sample was passed through
a Sep-Pak C18 cartridge previously activated with
methanol, followed by 10 ml of 0.01 M triethylammonium acetate solution (TEAA).
Samples were placed on the Sep-Pak cartridge,
washed through with 10 ml TEAA, eluted with 10 ml
50% (v/v) aqueous methanol, reduced to low volume
Journal of Plant Pathology (2009), 91 (1), 141-146
on the rotary evaporator as before, and transferred to a
capped 1.5 ml polypropylene microcentrifuge tube.
Samples were stored at -20 °C until use.
HPLC and immunoassays. Cytokinins were extracted following the method of Jones et al. (1995) including
separation by reverse-phase HPLC and assay by ELISA.
A HPLC Beckman System Gold (USA) with diode array detector was used. Briefly, samples were applied to a
HPLC C18 ODS column (150 mm x 4.6 mm, 5 µm
spherical particles) (Beckman, USA), eluted in a gradient of 15 (v/v) to 40% (v/v) aqueous methanol containing 2 mM TEAA at pH 7 for 15 min at a flow rate of 1.5
ml min-1, followed by continued elution with 40%
methanol for a further 25 min. Before each assay, a set
of cytokinin standards was passed through the column
to establish retention times. All assays were done in duplicate for each run. Fractions (1.5 ml) were recovered
from the column effluent with a fraction collector. Individual column fractions were evaporated under vacuum
at 45°C using a Centrivap concentrator (Labconco,
USA). Dried HPLC fractions were resuspended in 350
µl deionized water and quantified by ELISA (50 µl per
assay), using two different polyclonal rabbit antibodies:
anti-Z9R antibodies to measure Z, ZR (zeatin riboside)
and Z9G (zeatin-9-glucoside) and anti-iP9R antibodies
to measure iP, iPR (isopentenyladenine riboside) and
iP9G (isopentenyladenine-9-glucoside).
In each assay, the experimental unknown was preceded by a calibration series containing cytokinin riboside at doses from 0.025 to 6.24 pmol per assay, doubling the concentration at each step. The amount of cytokinins present in the samples was estimated based on
the standard curves of each cross-reactive compound
and losses during sample purification. Recoveries were
generally greater than 60%. All assays were done in duplicate in each run.
Statistical analysis. Based on symptomatology and
PCR results, cytokinin content was determined on samples from 3 healthy palms; 3 stage-2 affected palms; and
3 stage-6 affected palms. Data represent the means ±
standard deviation (SD) of analysis results for the three
tissue types (young leaves, primary and secondary root
meristem) from palms in one of the three conditions.
Differences between the means were determined by an
analysis of variance (ANOVA) and the Student-Newman-Keuls test was used to determine the significance
of these differences.
RESULTS
LY phytoplasma detection in different tissues of infected coconut palms. DNA extracted from the trunks
of twenty-three palms was used as templates in a stan-
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Fig. 1. Agarose gel electrophoresis of PCR-amplified coconut trunk DNA using a nested PCR protocol with universal primers
P1/P7 and LY group-specific primers 503f/LY16Sr. The 930 bp band indicates the presence of phytoplasma DNA. M= 1 kb ladder; lanes 1 and 14, water control from first round re-amplified; lanes 2 and 15 positive control; lanes 3 to 13 DNA from symptomatic palms; lanes 16 to 23 DNA from symptomless palms.
dard PCR assay with the P1/P7 general primers.
Aliquots of these PCR reactions were used as templates
for nested PCR, using primers 503f and LY16Sr. Visible
bands matching the size of the 16S rRNA gene segment
of the LY phytoplasma (930 bp) were observed in DNA
extracts from six of eleven palms with overt symptoms
(Fig. 1). An amplicon was obtained from only one of the
eight symptomless palms. These PCR results and the
type of symptoms led to selection of nine palms for cytokinin analysis: three healthy, symptomless; three with
stage-2, LY symptoms (one necrotic inflorescence); and
three with stage-6, or advanced, symptoms (all leaves
yellow, spear leaf unaffected).
Endogenous cytokinins in the leaves. Healthy leaf
tissue had a higher iP cytokinin content [iP = 31.5;
iP9G = 393.53; iPR = 54.24 pmol g-1 dry weight (DW)]
than Z cytokinin content (Z = 7.37; Z9G = 1.56; ZR =
3.03 pmol g-1 DW), with iP9G having the highest overall concentration (Fig. 2). In contrast, the levels of both
cytokinin types decreased in stage-2 LY-affected leaf tissue, with generally larger decreases in the iP’s than in
the Z’s. In the Z-type, cytokinin decreases were 13.4fold in Z (7.37 to 0.55 pmol g-1DW), 11-fold in Z9G
(1.56 to 0.141 pmol g-1DW) and 17.8-fold in ZR (3.03
to 0.17 pmol g-1DW) (Figure 2A). Decreases in the iPtype cytokinins were 17.8-fold in iP (31.5 to 1.78 pmol
g-1DW), 382-fold in iP9G (393.53 to 1.03 pmol g-1DW)
and 17.5-fold in iPR (54.24 to 3.1 pmol g-1DW) (Figure
2B). In stage-6 LY-affected leaves the decreases in both
cytokinin types were much greater, to the point where
iP, iP9G, Z9G and ZR were not detected.
Endogenous cytokinins in primary roots. In contrast
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Journal of Plant Pathology (2009), 91 (1), 141-146
Fig. 2. Endogenous cytokinins in young leaves from healthy
coconut palms (control) and at stages 2 and 6 of lethal yellowing disease (LY). A. Z-type cytokinins. B. iP-type cytokinins.
Figures represent means ± standard deviation from three different palms in the same condition; different letters (a, b, c)
for the same cytokinin denote significant difference (P<0.05).
ND, not detectable.
Fig. 3. Endogenous cytokinins in primary root meristem from
healthy coconut palms (control) and at stages 2 and 6 of lethal
yellowing disease. A. Z-type cytokinins. B. iP-type cytokinins.
Figures represent means ± standard deviation from three different palms in the same condition; different letters (a, b, c)
for the same cytokinin denote significant difference (P<0.05).
to leaf tissue, primary root meristem of healthy coconut
palms exhibited higher Z cytokinin levels (Z = 66; Z9G
= 70; ZR = 31.78 pmol g-1DW) than iP levels (iP = 8.34;
iP9G = 2.7; iPR = 4.4 pmol g-1DW) (Fig. 3). In stage-2
LY-affected primary roots, Z-type levels clearly decreased, with a 346-fold decrease in Z (65.8 to 0.19
pmol g-1DW), 3.9-fold in Z9G (70.26 to 17.99 pmol g1DW) and 6.6-fold in ZR (31.78 to 4.8 pmol g-1DW)
(Figure 3A). Among the iP-type cytokinins, iP decreased 4.7 fold (8.34 to 1.78 pmol g-1DW), iP9G increased 7 fold (2.7 to 18.98 pmol g-1DW) and iPR remained at levels similar to those in healthy primary
roots (3.84 pmol g-1DW) (Fig. 3B). In stage-6 LY-affected primary roots, the Z types remained at levels similar
to those in stage 2, but iPR increased dramatically:
26.45 times higher than levels in healthy primary roots
(4.4 to 116.4 pmol g-1DW).
Endogenous cytokinins in secondary roots. Endogenous levels of both Z-type and iP-type cytokinins were
generally lower in the secondary root meristem than in
the primary roots: Z = 1.65; Z9G = 14.06; ZR = 16.8; iP
= 3.2; iP9G = 3.67; iPR = 0.96 pmol g-1DW (Fig. 4). In
stage-2 LY-affected secondary roots, Z9G and ZR levels
remained unchanged from healthy levels whereas Z levels increased 20.78 times (1.65 to 34.3 pmol g-1DW)
(Fig. 4A). For the iP types, the free base increased 4
fold (3.2 to 13.6 pmol g-1DW) and the iPR 14 fold (0.96
to 13.73 pmol g-1DW) (Fig. 4B). In stage-6 LY-affected
secondary roots, only Z9G levels changed, decreasing 4
fold (14.06 to 3.44 pmol g-1DW) to levels below those
in healthy secondary roots; all other Z and iP types remained at similar levels.
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Fig. 4. Endogenous cytokinins in secondary root meristem from
healthy coconut palms (control) and at stages 2 and 6 of lethal
yellowing disease. A. Z-type cytokinins. B. iP-type cytokinins.
Figures represent means ± standard deviation from three different palms in the same condition; different letters (a, b, c) for the
same cytokinin denote significant difference (P<0.05).
DISCUSSION
An extensive analysis of endogenous cytokinins in different tissues of healthy coconut palms has previously
been reported (Sáenz et al., 2003). The most abundant cytokinins were Z-type, followed by iP-type and DHZ-type.
Among the Z types, ZR or ZR5’P (zeatin ribotide) were
the most abundant, depending on the tissue, and among
the iP types iP9G was most abundant in most tissues.
In an effort to determine if LY affects the endogenous cytokinin content of infected coconut palms, the
present study extended analyses to the free base, riboside and glucoside, of the two main cytokinin types (Z
and iP). LY was found to decrease drastically the concentration of both cytokinin types in leaf tissue. In primary root meristem, Z types tended to decrease at both
infection stages, but iP9G and iPR tended to increase at
stage 2 and 6, respectively. In the meristem of the thick-
Aguilar et al.
145
er secondary roots, Z types increased at stage 2 to decrease at stage 6, while the iP type level tended to increase at stages 2 and 6.
Roots are known to be sites of endogenous cytokinin
synthesis (Letham, 1994), and the fact that the content
of these cytokinins increases in coconut palm when LY
begins may be taken as an indication that roots increase
cytokinin production for export to sites where they are
needed, as it was found in hybrid roses (Dieleman et al.,
1997). This would be in response to decreasing cytokinin level in other plant organs (e.g. leaves), which
may be a direct or indirect effect of the pathogen.
As mentioned, LY-induced leaf yellowing resembles
that characterizing natural leaf senescence and may be
explained as depending on reduction in endogenous cytokinins. Changes commonly associated with leaf senescence, such as decrease in photosynthetic capacity, pigment and protein content, have also been detected in
LY-infected palms (León et al., 1996). Senescence affects cytokinins also in soybean, in which xylem sap cytokinin levels decrease sharply with the onset of senescence (Noodén et al., 1990), and in tobacco leaves, in
which cytokinin, as well as the capacity to synthesize
them, decrease as leaf senescence progresses (Singh et
al., 1992). LY-affected palms also exhibit permanent
closure of stomata, which is believed to be a pivotal
change in LY mode of action (León et al., 1996). The
hypothesis of increased abscisic acid content as a possible cause for this has been tested, but no changes were
observed (Martínez et al., 2000). In any case, a decrease
in cytokinin content of the leaves promotes stomatal
closure (Davies and Zhang, 1991).
There are reports that endogenous cytokinin levels
and metabolism are affected in plants affected by other
pathogens, e.g. viruses. Infection of tobacco plants by
Tobacco mosaic virus (TMV) stimulates cytokinin catabolism, reduces free cytokinin concentration and increases cytokinin-O-glucoside concentration (Whenham,
1989). In potato plants infected by Potato virus Y
(PVY), the concentration of biologically active cytokinins as free bases and ribosides tends to shift toward terminal inactivation by 9-glucosylation after primary infection in the roots of susceptible cultivars (Dermastia et al., 1995). Furthermore, Dermastia and
Ravnikar (1996) identified a wide range of cytokinins in
tissue-cultured potato plants 8 weeks after PVY infection and reported an increase in total cytokinin content
in infected plants which they attributed to increases in
iP9G and ZR. In bean plants infected with White clover
mosaic virus (WCMV) concentrations of the free bases
and DZR decreased, while there was an increase of 9glucosides and nucleotides (Clarke et al., 1999).
Results of the present investigation have shown that
in LY-affected palms biologically active and free base
cytokinin levels decrease, along with inactive metabolites, such as 9-glucoside. This can be retained as an in-
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dication that LY affects the biosynthesis of these compounds which is the likely cause of symptom expression, thus beginning to unravel the mechanism of action
of this disease.
ACKNOWLEDGEMENTS
Partial funding was provided by CONACYT, Mexico
(grant 28883-N). The authors thank M. Narvaez and I.
Córdova for their assistance.
REFERENCES
Agrios G.N., 1978. Plant Pathology. Academic Press, New
York, NY, USA.
Clarke S.F., Mckenzie M.J., Burritt D.J., Guy P.L., Jameson
P.E., 1999. Influence of white clover mosaic potexvirus infection on the endogenous cytokinin content of bean.
Plant Physiology 120: 547-552.
Davies W.J., Zhang J., 1991. Root signal and the regulation of
growth and development of plants in drying soil. Annual Review of Plant Physiology and Molecular Biology 42: 55-76.
Deng S., Hiruki C., 1991. Amplification of 16S rRNA genes
from culturable and nonculturable mollicutes. Journal of
Microbiological Methods 14: 53-61.
Dermastia M., Ravnikar M., Kovac M., 1995. Increased cytokinin-9-glucosylation in roots of susceptible Solanum
tuberosum cultivar infected by potato virus YNTN. Molecular Plant-Microbe Interactions 8: 327-330.
Dermastia M., Ravnikar M., 1996. Altered cytokinin pattern
and enhanced tolerance to potato virus YNTN in the susceptible potato cultivar (Solanum tuberosum cv. Igor)
grown in vitro. Physiological and Molecular Plant Pathology
48: 65-71.
Dieleman J.A., Verstappen F.W.A., Perik P.R.J., Kuiper D.,
1997. Quantification of the export cytokinins from root to
shoots of Rosa hybrida and their degradation rate in the
shoot. Physiologia Plantarum 101: 342–352.
Dodd I.C., 2003. Hormonal interactions and stomatal responses. Journal of Plant Growth Regulation 22: 32-46.
Harrison N.A., Jones P., 2004. Lethal yellowing. In: Elliott
M.L., Broschat T.K., Uchida J.Y., Simone G.W. (eds).
Compendium of Ornamental Palm Diseases and Disorders. pp. 39-41. APS Press, St. Paul, MN, USA.
Harrison N.A., Cordova I., Richardson P., Dibonito R., 1999.
Detection and diagnosis of lethal yellowing. In: Oropeza
C., Verdeil J.L,. Ashburner G.R., Cardeña R., Santamaría
J.M. (eds). Current Advances in Coconut Biotechnology,
pp. 183-196. Kluwer Academic Publishers, Dordrecht, the
Netherlands.
Jones L.H., Hanke D.E., Eeuwens C.J., 1995. An evaluation
of the role of cytokinins in the development of abnormal
inflorescences in oil palm cultures (Elaeis guineensis Jacq)
Received July 29, 2008
Accepted November 24, 2008
Journal of Plant Pathology (2009), 91 (1), 141-146
regenerated from tissue culture. Journal of Plant Growth
Regulation 14: 135-142.
León R., Santamaría J.M., Alpizar L., Escamilla J.A., Oropeza
C., 1996. Physiological and biochemical changes in shoots
of coconut palms affected by lethal yellowing. New Phytologist 134: 227-234.
Letham D.S., 1994. Cytokinins as phytohormones-sites of
biosynthesis, translocation, and function of translocated
cytokinin. In: Mok D.W.S., Mok M.C. (eds). Cytokinins:
Chemistry, Activity and Function, pp. 57-80. CRC Press,
Boca Raton, FL, USA.
Martínez S., Cordova I., Maust B., Oropeza C., Santamaría J.,
2000. Is abscisic acid responsible for abnormal stomatal
closure in lethal yellowing of coconut palms? Journal of
Plant Physiology 156: 319-323.
Maust B.E., Espadas F., Talavera C., Aguilar M., Santamaria
J.M., Oropeza C., 2003. Changes in carbohydrate metabolism in coconut palms infected with lethal yellowing phytoplasma. Phytopathology 93: 976-981.
McCoy R.E., Howard F.W., Tsai J.H., Donselman H.M.,
Thomas D.L., Basham H.G., Atilano R.A., Eskafi F.M.,
Britt L., Collins M.E., 1983. Lethal yellowing of palms.
University of Florida Agricultural Experiment Station Bulletin No. 834. Gainesville, FL, USA.
Noodén L.D., Singh S., Letham D.S., 1990. Correlation of
xylem sap cytokinin levels with monocarpic senescence in
soybean. Plant Physiology 93: 33-39.
Oropeza C., Santamaría J.M., Ashburner G.R., 1997. A model
for the pathogenicity of lethal yellowing in coconut palms
(Cocos nucifera). In:. Eden-Green S.J., Ofori F. (eds). International Workshop on Lethal Yellowing-Like Disease of
Coconut pp. 109-115. Elmira, Ghana. Natural Resources
Institute, Chatham, UK.
Plavsic-Banjac B., Hunt P., Maramorosch K., 1972. Mycoplasma-like bodies associated with lethal yellowing disease of
coconut palms. Phytopathology 62: 298-299.
Sáenz L., Jones L.H., Oropeza C., Vláèil, D., Strnad M., 2003.
Endogenous isoprenoid and aromatic cytokinins in different plant parts of Cocos nucifera (L.) Plant Growth Regulation 39: 205-215.
Singh S., Letham D.S., Zhang X., Palni L.M.S., 1992. Cytokinin biochemistry in relation to leaf senescence. VI. Effect of nitrogenous nutrients on cytokinins levels and
senescence of tobacco leaves. Physiologia Plantarum 84:
262-268.
Smart C.D., Schneider B., Morrer R., Blomquist D.J., Guerra
L.J., Harrison N.J., Ahrens U., Lorenze K.H., Seemüller
E., Kirkpatrick B.C., 1996. Phytoplasma-specific PCR
primers based on sequences of the 16S-23S rRNA spacer
region. Applied and Environmental Microbiology 62: 29882993.
Whenham R.J., 1989. Effect of systemic tobacco mosaic virus
infection on endogenous cytokinin concentration in tobacco (Nicotiana tabacum) leaves: consequences for the control of resistance and symptom development. Physiological
and Molecular Plant Pathology 35: 85-95.