Physiological and Molecular Plant Pathology 65 (2004) 181–185

www.elsevier.com/locate/pmpp

Biochemical changes in corn plants infected by the maize

bushy stunt phytoplasma
Ana Junqueira, Ivan Bedendo, Sérgio Pascholati*
Department of Entomology, Plant Pathology and zoology, Plant Pathology Session, Esalq-University of São Paulo,
Avenida Padua Dias, 11, ZC 13418-900 Piracicaba, SP, Brazil
Accepted 18 January 2005

Abstract
The objective of the present work was to study biochemical alterations in corn plants infected by the maize bushy stunt phytoplasma. A
resistant and a susceptible corn hybrid were used and changes in protein, phenolic compounds, chlorophyll and reducing sugars in infected
plants were evaluated. The experiments were carried out inside greenhouse, and the corn plants were inoculated by using infective Dalbulus
maidis leafhoppers 10 days after seeding. When symptoms started to appear, leaf samples were harvested at different times for the
biochemical analyses. The results showed an increase in all biochemical parameters evaluated for both hybrids, with exception for
the chlorophyll content that decreased. In a general way, the values observed for the level of the different compounds were higher in the
susceptible hybrid when compared to the resistant one. The increases in protein, reducing sugar and phenolic compound contents in
inoculated plants point out changes in host metabolism due to the phytoplasma, and the reduction in chlorophyll content shows that the
phytoplasma can interfere in photosynthesis and perhaps speedy senescence in the leaf tissue.
q 2005 Elsevier Ltd. All rights reserved.

Keywords: Biochemical changes; Metabolites; Stunt; Corn; Phytoplasma

1. Introduction starch were observed in leaves and decreases in root [15].

The diseases associated to phytoplasmas are important to
many different crops around the world [19]. Studies related
to biochemical and physiological changes in plants caused
by these microorganisms are rare. Some results have shown
that phytoplasma infection can change the level of some
compounds in plant tissues. Increases in phenolic compound
content were shown in apple and plum tissues infected with
phytoplasmas, but no changes in phenolic compounds were
observed in Catharanthus roseus plants infected with the
same phytoplasmas [20]. By using papaya plants, it was
demonstrated a higher carbohydrate content in diseased leaf
tissue, but a lower content in diseased stem and root [8].
Similar results were obtained with infected tobacco and
C. roseus plants in which increases in carbohydrate and
* Corresponding author. Tel.: C55 19 3429 4124; fax: C55 19 3434
4839.
E-mail address: sfpascho@esalq.usp.br (S. Pascholati).
0885-5765/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pmpp.2005.01.005
Higher hormone levels, as auxin in Sesamum sp [24] and
cytokinin in C. roseus [5] and Ribes houghtonianum [28]
were observed in those plants when infected with this
pathogen. In diseased C. roseus chlorophyll synthesis was
also altered during plant growth [4]. Recently, it was shown
that maize plants infected by mollicutes had lower protein
content than uninoculated plants [22].
The maize bushy stunt is an important disease in the
USA, Mexico, Brazil and Latin America [26,17]. In Brazil,
this disease has caused severe losses on susceptible hybrids
particularly on late-planted crops, e.g. when crops have
been planted in February and March [17]. Typical disease
symptoms are characterized by foliar chlorosis followed by
reddening, shortned internodes, ear proliferation and
stunting of plants [2]. The causal agent, a phytoplasma, is
a wall-less prokaryote, phloem inhabitant and naturally
transmitted by Dalbulus maidis leafhopper [6]. Up to now
there is no binomial classification for phytoplasmas, thus
according to the current classification the maize bushy stunt
phytoplasma is a representative of the group 16SrI, sub-
group B [12]. The diagnosis based upon symptoms
182 A. Junqueira et al. / Physiological and Molecular Plant Pathology 65 (2004) 181–185
generally need to be confirmed by phytoplasma detection by
PCR technique [6].
Thus, the objective of the present work was to study
some biochemical aspects related to protein, carbohydrate,
chlorophyll and phenolic compound contents in corn plants
infected by the maize bushy stunt phytoplasma.
2. Material and methods
The experiments were carried out using two corn hybrids
(Zea mays) XLX 520 (susceptible) and FT 9006 (resistant)
selected among nine hybrids previously studied [27]. The
criteria used were based upon symptom severity, plant
growth and grain yield. Symptoms in the resistant hybrid
were mild and grain production was higher in relation to the
susceptible one. Thus, symptoms in the susceptible hybrid
were characterized by severe foliar reddening, reduced plant
high, increased ear proliferation and higher stunting of
plants.
Pre-germinated seeds were sown inside plastic pots
(250 ml) having soil, organic matter and sand as substrate
(3:1:1; v/v/v). The material was kept under greenhouse
conditions and 10 days later the seedlings were inoculated
by using seven infective Dalbulus maidis leafhoppers
placed in contact with the leaves. After 4 days, the insects
and eggs were eliminated. Plants insect free were used as
control. Twenty days after inoculation each plant was
transferred to 5 l plastic bags having the same substrate
above and maintained in the greenhouse.
For biochemical analyses, disks (1.5 cm in diameter)
from leaves exhibiting the same physiological age and
situated at half of plant height, were obtained one day before
and 40, 50, 70 and 80 days after inoculation. Leaf samples,
represented by 0.3 g fresh tissue each, were used for protein,
carbohydrate, chlorophyll and phenolic compound determi-
nation. The experiment was a 2!2!5 factorial represented
by two corn hybrids (XLX 520 and FT 9006), two
treatments (inoculated and non-inoculated plants) and five
sampling times (0, 40, 50, 70 and 80 days). Triplicate
samples for the determinations were taken at each sampling
time and each of the above experiments was repeated at
least two times. The data were statistically analyzed by
using the ESTAT program (UNESP, Jaboticabal, SP,
Brazil).
The presence of the phytoplasma in the inoculated corn
hybrids and its absence in the control plants was demon-
strated by using the nested polymerase chain reaction
procedure [13]. Nucleic acid was extracted from fresh tissue
and four pairs of primers were used according to Gundersen
and Lee [7]: R16mF1-5 0 AAG ACG AGG ATA ACA GTT
GG 3 0 ; R16R1-5 0 TTC CCT CTT CTT GCG AAG TTA
GGC CAC CGA 3 0 ; R16F2n-5 0 GAA ACG ACT GCT AAG
ACT GG 3 0 ; R16R2-5 0 TGA CGG GCG GTG TGT ACA
AAC CCC G 3 0 . Leaf samples were harvested from both
hybrids when symptoms started to appear.
The amount of total proteins in the leaves was
determined by using the Bradford method [3], having
bovine serum albumin as standard. The results were
expressed as mg protein/gram fresh tissue.
The amount of reducing sugar content was estimated by
using p-hydroxybenzoic acid hydrazide [16], having
glucose as standard. Absorbance was read at 410 nm and
the results expressed as mg glucose/gram fresh tissue.
Total chlorophyll content in the leaves was determined
by using acetone [9]. The results were expressed as mg
chlorophyll/gram fresh tissue based upon the formulae: mg
total chlorophyll/gftZ20.2 (OD 645) C8.02 (OD 663).
(V/1000.W), where ODZoptical density at 645 and 663 nm,
VZfinal volume (ml) and WZtissue fresh weight (g) [1].
Total phenolic compound content was estimated in the
leaves by using hexane to remove chlorophyll and other
pigments, and the Folin-Ciocalteau reagent 2N (1:1; v/v).
Absorbance was determined at 750 nm and the results
expressed as chlorogenic acid equivalents/gram fresh
tissue [10].
3. Results and discussion
The symptoms in the plants and phytoplasma detection
started around 40 days after inoculation. PCR results always
showed phytoplasma presence in inoculated tissues of both
hybrids and its absence in control plants. By using the PCR
method it was not possible to quantify phytoplasma
concentration in the infected leaf tissue of resistant or
susceptible hybrids.
Chlorophyll content was significantly lower in inocu-
lated leaf tissue when compared to health leaf tissue from
the susceptible hybrid XLX 520 (Fig. 1). The content was
reduced around 36.1% in plants infected by the phytoplasma
and these plants also exhibited early leaf chlorosis. In the
resistant hybrid FT 9006, chlorophyll reduction was also
observed in inoculated plants but the difference in relation to
1.4
mg chlorophyll / g fresh tissue
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0 40 50 70 80
Days after inoculation
Fig. 1. Changes in chlorophyll in corn leaves of two hybrids (XLX 520-
susceptible; FT9006-resistant) in response to phytoplasma inoculation.
Plants 10 days-old were inoculated by using infected Dalbulus maidis
leafhoppers. Legend: (-%-) XLX520-control; (-&-) XLX520 inoculated;
(-:-) FT9006 control; (-C-) FT9006 inoculated.
 A. Junqueira et al. / Physiological and Molecular Plant Pathology 65 (2004) 181–185 183

the control plants was around 10%. Some researchers also 0.8

mg protein / g fresh tissue

reported chlorophyll reduction in infected plant tissues as
the reduction around 60% observed in coconut palms
affected by lethal yellowing [14]. Papaya plants exhibiting a
decline associated to a phytoplasma showed a significant
decrease in photosynthesis after the first symptoms appear
[8]. Although changes in photosynthetic rate were no
evaluated in the present work, it can be assumed that the
observed reduction in chlorophyll levels will probably
interfere with the photosynthetic capacity in the maize
leaves as stated for the papaya dieback-phytoplasma
pathosystem [8].
Regarding the amount of reducing sugar, inoculated corn
plants from both hybrids exhibited higher carbohydrate
content than the uninoculated plants (Fig. 2). During the
80 days period, differences were observed between the
hybrids since the inoculated tissues from the susceptible
hybrid XLX 520 and the resistant one FT 9006 exhibited
average increases around 46.2 and 16.3% in relation to the
health tissues, respectively. Increases in carbohydrate
content was also shown in coconut palms infected with
the lethal yellowing phytoplasma where sugar accumulation
occurred in leaves and a decrease was observed in roots
suggesting an inhibition in phloem transport [18]. Studies
carried out with infected tobacco and C. roseus plants also
showed that soluble carbohydrates and starch translocation
was severely altered since concentration of these substances
was higher in leaves and lower in roots [15]. In papaya
plants, sugar accumulation in leaves and its decrease in roots
were related to phloem block due to the pathogen or
metabolites produced by the host [8]. This hypothesis was
also considered for pear decline, where metabolites
produced by the pathogen and by the plant could make the
phloem not functional and interfere with metabolite
translocation between producer and consumer organs in
the plant [25]. In the papaya study, the carbohydrate
accumulation in the leaves occurred simultaneously or
8 chitinases and b-1, 3-glucanases. Increases in PR-proteins
mg glucose / g fresh tissue
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 20 40 60 80
Days after inoculation
Fig. 3. Changes in proteins in corn leaves of two hybrids (XLX 520-
susceptible; FT9006-resistant) in response to phytoplasma inoculation.
Plants 10 days-old were inoculated by using infected Dalbulus maidis
leafhoppers. Legend: (-%-) XLX520-control; (-&-) XLX520 inoculated;
(-:-) FT9006 control; (-C-) FT9006 inoculated.
slightly after a reduction in photosynthesis rate, and
probably lead to a reduction in pigments related to
photosynthesis [8]. The results obtained in the present
work where the inoculated corn plants exhibited high sugar
content and a reduction in chlorophyll and a possible
reduction in photosynthesis rate are in agreement to the
above report.
The protein content was higher in inoculated plants from
the resistant as well as the susceptible hybrids when
compared to non-inoculated ones (Fig. 3). In a general
way, protein increased during plant growth showing the
same pattern for both hybrids. For the susceptible hybrid
XLX 520 an average increase of 8.2% for protein was seen
in the inoculated plants while for the resistant hybrid the
increase was around 29.5%. Usually, plants infected by
pathogens show a high protein content that could be due to
the activation of the defense mechanisms from the host and
to the attack mechanisms from the pathogen [23]. Among
the proteins produced by the host plant there are those
related to the defense mechanisms and called pathogenesis-
related-proteins (PR-proteins) that include peroxidases,

7 in corn plants infected by the phytoplasma could contribute

6 to the increase in total protein [11]. Thus, the higher
5
accumulation of proteins in the resistant hybrid FT 9006
could explain in part its resistance level. On the other hand,
4
in the case of the hybrid XLX 520 the lower protein
3 accumulation could be related to its susceptibility. Different
2 results were obtained for corn plants inoculated with
1 mollicutes where a lower protein content was observed in
0 relation to the uninoculated ones [22]. The possible
0 20 40 60 80 explanation for this difference could be due to the fact
Days after inoculation that an extremely susceptible corn hybrid was used in the
Fig. 2. Changes in reducing sugars in corn leaves of two hybrids (XLX 520-
experiments.
susceptible; FT9006-resistant) in response to phytoplasma inoculation. Inoculated plants from both hybrids exhibited higher
Plants 10 days-old were inoculated by using infected Dalbulus maidis phenolic compound levels compared to non-inoculated
leafhoppers. Legend: (-%-) XLX520-control; (-&-) XLX520 inoculated; plants (Fig. 4). The phytoplasma present in the tissues
(-:-) FT9006 control; (-C-) FT9006 inoculated. caused an increase in phenolic compounds around 57.5%
184 A. Junqueira et al. / Physiological and Molecular Plant Pathology 65 (2004) 181–185
mg equivalent chlorogenic acid / g
0.18
0.16
0.14
0.12
fresh tissue
0.10
0.08
0.06
0.04
0.02
0.00
0 20 40
Days after inoculation
Fig. 4. Changes in phenolic compounds in corn leaves of two hybrids (XLX
520-susceptible; FT9006-resistant) in response to phytoplasma inoculation.
Plants 10 days-old were inoculated by using infected Dalbulus maidis
leafhoppers. Legend: (-%-) XLX520-control; (-&-) XLX520 inoculated;
(:-) FT9006 control; (-C-) FT9006 inoculated.
in the susceptible hybrid and 47.2% in the resistant one.
In plum and apple trees infected by phytoplasma a
significant increase in phenolic compounds was also seen
and it was related to defense basically involving the
inhibition of the pathogen inside the phloem vessels [20].
Phenolic compounds can be involved in the defense in
plants and their accumulation levels in response to infection
can be related to the pathosystem and the resistance level of
the host [21]. In the present work, it was observed that the
susceptible hybrid had a higher phenolic compound content
than the resistant one but it should be pointed out that
resistance is not only related to the presence of phenolic
compounds and also that those compounds have other
functions in plant tissue.
One of the typical symptoms caused by phytoplasma in
corn plants is the leaf redness. Anthocyanin accumulation, a
phenolic compound, is usually associated to the red color
exhibited by corn and sorghum plants infected by fungal
pathogens [21]. Thus, the redness in the corn leaves could
also be related to the phenolic compound accumulation that
occurred in plants of both hybrids infected with the
phytoplasma. In the present work, quantitative changes in
phenolic compound accumulation were determined but a
qualitative study is also necessary.
Finally, it was seen that for both corn hybrids, resistant or
susceptible to the bushy stunt phytoplasma, there was an
increase in the level of reducing sugars, proteins and
phenolic compounds, except in chlorophyll, showing that
metabolic changes occurs in the plants when inoculated with
the pathogen.
Acknowledgements
We want to thank Dr Patrı́cia Cia-Esalq/USP for artistic
assistance. IB and SP are grateful to CNPq for the
fellowship awarded.
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