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Symptomatic expression of tristezainfected citrus plants in Pakistan

a

Yasir Iftikhar , S.M. Mughal , M.M. Khan , M.A. Khan , M.A.

e

Nawaz & Z. Hussain

Department of Plant Pathology, University College of

Agriculture, University of Sargodha, Sargodha, Pakistan
b

Department of Plant Pathology, PMAS University of Arid

Agriculture, Rawalpindi, Pakistan
c

Institute of Horticultural Sciences, University of Agriculture,

Faisalabad, Pakistan
d

Department of Plant Pathology, University of Agriculture,

Faisalabad, Pakistan
e

Department of Horticulture, University College of Agriculture,

University of Sargodha, Sargodha, Pakistan
Version of record first published: 30 Oct 2012.

To cite this article: Yasir Iftikhar, S.M. Mughal, M.M. Khan, M.A. Khan, M.A. Nawaz & Z. Hussain
(2012): Symptomatic expression of tristeza-infected citrus plants in Pakistan, Archives Of
Phytopathology And Plant Protection, DOI:10.1080/03235408.2012.734718
To link to this article: http://dx.doi.org/10.1080/03235408.2012.734718

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Archives of Phytopathology and Plant Protection

iFirst article 2012, 17

Symptomatic expression of tristeza-infected citrus plants in Pakistan

Yasir Iftikhara*, S.M. Mughalb, M.M. Khanc, M.A. Khand, M.A. Nawaze and
Z. Hussaine

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Department of Plant Pathology, University College of Agriculture, University of Sargodha,

Sargodha, Pakistan; bDepartment of Plant Pathology, PMAS University of Arid Agriculture,
Rawalpindi, Pakistan; cInstitute of Horticultural Sciences, University of Agriculture, Faisalabad,
Pakistan; dDepartment of Plant Pathology, University of Agriculture, Faisalabad, Pakistan;
e
Department of Horticulture, University College of Agriculture, University of Sargodha,
Sargodha, Pakistan
(Received 4 July 2012; nal version received 23 September 2012)
Variable symptoms were recorded during a survey in the citrus trees infected or
suspected to be infected with citrus tristeza virus based on ELISA tests. Sweet
orange manifested main symptoms like bud union crease, pin holing, bark
cracking, incompatibility, yellowing and dropping of leaves. Pin holing was
particularly prevalent in Sweet orange (Citrus sinensis Osbeck), Kinnow (Citrus
reticulata Blanco) and Grapefruit (Citrus paradisii Mcaf.), while incompatibility
was only in Sweet orange and Grapefruit.
Keywords: citrus; CTV; ELISA; symptoms

Introduction
Citrus is attacked by a large number of diseases caused by dierent pathogens,
like fungi, bacteria, nematodes, viruses, viroids and prokaryotes, but virus and
virus-like diseases are the major threat in the citrus orchards. Tristeza disease in
citrus, caused by Citrus tristeza closterovirus (CTV) is one of the most serious
virus diseases prevalent and damaging the citrus orchards. It has already been
reported in Pakistan (Catara et al. 1988; Abbas et al. 2008). Dierent isolates of
CTV have been reported to produce a wide range of symptoms ranging from
mild and severe condition to unapparent and non-perceptible damage (Miao and
Skaria, 2002).
Symptom expression of CTV in citrus hosts is highly variable and depends
upon host species, rootstock and scion combination, virulence of CTV isolates and
soil or environmental conditions. Characteristics symptoms of CTV are vein
clearing in acid Lime, seedling yellows, decline, stunting and leaf corking and stem
pitting on dierent citrus hosts like Sweet orange, Grapefruit, grafted on Sour
orange (Citrus aurantium L.) rootstock (Brlansky et al. 2003; EPPO Bulletin 2004;
Lbida et al. 2005). Among the citrus varieties, Sweet orange ultimately leads
towards decline when propagated on susceptible rootstock such as Sour orange
(Niblett et al. 2000).

*Corresponding author. Email: yasiriftikhar@uos.edu.pk

ISSN 0323-5408 print/ISSN 1477-2906 online
2012 Taylor & Francis
http://dx.doi.org/10.1080/03235408.2012.734718
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Y. Iftikhar et al.

Two major syndromes due to CTV called decline and stem pitting have
already been studied extensively by the scientists. Sweet orange on tolerant
rootstocks remains asymptomatic (Garnsey et al. 1998). Infected plants exhibit
smalling and cupping of leaves, yellow ecks along the veins and stem pitting. In
case of Sweet orange plants grafted on Sour orange rootstock show dieback,
characteristic over growth at bud union, honey combing and resulting in sudden
wilt and decline (Wallace and Drake 1951). Almost all citrus species including
hybrids are liable to be infected by CTV. Symptoms produced in the CTVinfected plants are variable as described earlier and virology in Pakistan has been
neglected since a long time (Iftikhar et al. 2009), therefore a survey was initiated
to study the symptoms expression of CTV-infected plants in the main citrus
growing areas of Punjab and Khyber Pakhtonkhwah (Former N.W.F.P) provinces
of Pakistan.
Materials and methods
Survey of citrus growing areas
Major citrus growing areas of Punjab (Faisalabad, Sahiwal, Toba Tek Singh,
Sargodha, Bhalwal) and Khyber Pakhtonkhwah (Haripur, Marda, Peshawar) were
extensively surveyed to observe the symptoms of CTV-infected trees. The samples
from infected trees or trees to be suspected from CTV were collected and the trees
were marked to conrm through enzyme-linked immunosorbent assay (ELISA).
Anatomical changes like stunting, necrosis of cambium, pin holing/stem pitting,
bark cracking and bud union crease have been studied during preliminary surveys.
After conrmation, the anatomical changes/features were recorded in a tabulated
form for each variety.
Collection of samples
Leaves, small portion of twig bark and stem portion from bud union were collected
from the infected or suspected trees of CTV infection, in sterile polythene bags for
further process. The samples were brought to the laboratory and chopped into small
pieces and their sap was extracted separately in the extraction buer for ELISA test.
Enzyme-linked immunosorbent assay
DAS-ELISA (Clark and Adams 1977) was used to analyse the samples. ELISA kit
(1102 CTV; Adgia, USA) with polyclonal antibodies were used. ELISA ve trees
were marked for further investigations. ELISA plates were coated with coating
buer, diluted with polyclonal antibodies (IgG). Micro-plates were incubated at 48C
for overnight. After incubation plates were washed with washing buer (phosphate
buer saline Tween) three times with ve min intervals. ELISA plates were charged
with Antigen (Extract) (200 ml/well) followed by incubation and washing as earlier.
Enzyme conjugate (200 ml) was added to each well and kept the plates for incubation
and washing. Finally, the substrate (p-nitrophenyl phosphate) in substrate buer at 1
mg/ml were added per well. The plate was incubated at room temperature
(25 + 28C) for about 30 min to 1 h for the colour development. Colour reaction
at 405 nm was measured with a microplate reader and 3M sodium hydroxide was
added as a stopping reaction.

Archives of Phytopathology and Plant Protection

Transmission of CTV
Mechanical and insect transmissions were attempted on dierent citrus species,
including acid lime (Citrus aurantifolia) as an indicator plant for CTV. Carborandum
powder (600 mesh as an abrasive) was used for the mechanical transmission. Plants
were observed for symptom development and were subjected to ELISA for the
conrmation of CTV. Cotton aphid, Aphis gossypii (Glov.) and whitey, Bemisia
tabaci (Genn.) were used for insect transmission in the absence of brown citrus aphid
in Pakistan.

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Results and discussion

The results in Table 1 and Figure 1 shows that dierent eld symptoms of CTV were
observed on the basis of initiation of cambium necrosis, revealed the presence of
inverse pitting/stem pitting and incompatibility between rootstock and scion
followed by the conrmation through ELISA. In addition, examination of suspected
trees in the eld also showed some more symptoms such as cracking of bark,
gummosis on the branches, yellowing and dropping of leaves and pin holing in the
stem portion when the bark from the bud union was removed. Small numerous holes
were present in the stem portion which helped in eld diagnosis.
Another probable and possible symptom, i.e. bud union crease was also found in
Sweet orange. ELISA positive samples (trees) of Sweet orange also showed small
numerous pits in the stem portion from the bud union, bud union crease,
incompatibility between the scion and rootstock, gummosis on the upper branches,
bark cracking, yellowing of leaves and leaf dropping were pronounced in Sweet
orange. Kinnow showed only pin holing, bark cracking, yellowing of leaves and leaf
dropping, while there was no bud union crease, gummosis and incompatibility
between the rootstock and scion in Kinnow.
The samples of Grapefruit with CTV infection showed the pin holing, bud union
crease, incompatibility and leaf dropping only (Table 1). Dierent symptoms
observed during eld surveys are shown in Figure 1(ae). During the transmission
trials, acid lime shows mild leaf ecking on mechanical transmission whereas insect
transmission was failed due to unknown reasons, although mechanical transmission
is very dicult to produce symptoms. Cotton aphid and the whitey were used, as
the brown citrus aphid is fortunately not prevailing in Pakistan. ELISA test also
conrmed the presence of CTV in the samples of acid lime after the transmission
trials (Tables 2 and 3).
Symptomology of CTV infection is one of the primary and conventional schemes
in the identication of CTV, inspite of the fact that highly sensitive and rapid
Table 1.

Anatomical changes in the citrus varieties infected with CTV.

Citrus species

ELISA reaction

PH

BUC

Incom.

BC

GUM

YOL

LD

Sweet orange
Kinnow
Grapefruit

ve
ve
ve

7
7

PH, pin holing; BUC, bud union crease; Incom., incompatibility of scion and rootstock; BC, bark cracking;
GUM, gummosis; YOL, yellowing of leaves; LD, leaf dropping. Response: highly present,
moderately present, 7 not present. Response depends upon the prevalence of symptoms on the citrus trees
and conrmation by ELISA.

Y. Iftikhar et al.

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Figure 1. (a) Peg like structure on the stem portion; (b) pin holing and bud union crease in
the stem bark; (c) bark cracking; (d) incompatibility; (e) gummosis on the upper branches.

Archives of Phytopathology and Plant Protection

Table 2.

Mechanical inoculation of CTV on citrus species.

Mechanical transmission

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Insect transmission

Citrus species

Inoculated

Infected

% Infection

Infected

% Infection

Kinnow
Mosambi
Acid lime
Grapefruit
Eureka lemon
Rough lemon
Citrumello

10
10
10
10
10
10
10

0
0
7
0
0
0
0

0
0
70
0
0
0
0

0
0
0
0
0
0
0

0
0
0
0
0
0
0

Table 3.

ELISA test of infected acid lime with CTV.

Samples
CTV infected
Healthy
Positive control
Negative control
Buer

No. of wells
charged

Colour

OD values
at 405 nm

7
2
2
2
2

Light yellow to moderate yellow

Negative
Moderate yellow
Negative
Negative

0.4960.565
0.252
0.385
0.212
0.198

techniques have been developed. Similarly, response of the citrus species to CTV has
already also been reported by Mughal (2004). The reaction of citrus species against
CTV also depends upon strains of CTV, scion and rootstock combination and other
factors like soil conditions. The symptomatic expression can be highly variable with
respect to CTV strains which range from mild to severe. The characteristics
symptoms of CTV have also been elaborated by many plant pathologists (Brlansky
et al. 2003; Lbida et al. 2005). Symptomology of CTV infected plant were studied
under the local environmental conditions which may in combination with virus
strain inuence the symptoms expression of the CTV infected plants. Variable
response of dierent citrus species against CTV was reported by Price (1970) and
Miao and Skaria (2002). Vein clearing and vein corking symptoms may be dierent
on dierential hosts (Brlansky et al. 2002). Sometime presence of CTV strains may
confuse the symptoms expression due to synergistic eect of more than one strain
(Garnsey et al. 1987; Hilf 2004). Host infected with CTV infection can be
asymptomatic or unapparent on the tolerant rootstock but infection can
consequently and conspicuously cause other abnormalities in phloem and xylem
development, stunting, reduced vigour, loss in fruit size and quality but tree does not
die (Garnsey et al. 1998). Symptoms produced by dierent CTV infection can be
identied and dierentiated through indicator plant as Mexican or acid lime in the
greenhouse, but some strains notably strain T519 do not produce symptoms on the
indicator plant (Roistacher 1991; Halbert et al. 2004).
Our results obtained are also in close conformity with Wallace and Drake (1951)
who studied the symptoms expression of Sweet orange plant grafted on Sour orange
rootstock showing sudden wilt, decline and characteristic over growth bud union
and honey combing. Some isolates induce few scattered pits leaving no remarkable
deleterious eect on tree vigour but some isolates can cause extensive heavy pits

Y. Iftikhar et al.

under the bark and with this type of pitting, trees may continue to grow without any
aect on the tree vigour with small fruit size. But in case of more severe strains of
CTV-SP, the tree growth is markedly reduced; bark and twigs may become thicker
and brittle. Leaves turn chlorotic with dieback symptoms. Phloem necrosis also
causes the graft incompatibility in citrus and other fruit trees (Bos 1999).
Environmental conditions along with strains present in conventional cultivars may
aect the symptomatic and asymptomatic infection behaviour.

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Conclusion
It can be concluded with ndings presented that tristeza badly aects the citrus trees.
Symptoms can be variable depending on the environmental conditions and root
stock used in the region. Results would be helpful for the initial diagnosis of disease
in citrus trees so that citrus researchers and growers could make a strategy for
identication and management of this devastating disease.

Acknowledgements
We are thankful to the Ministry of Science and Technology (MoST), Government of Pakistan,
for providing nancial support. We are also thankful to the sta of the Citrus Sanitation
Laboratory, Institute of Horticultural Sciences, University of Agriculture, Faisalabad,
Pakistan, for providing assistance in the collection and preparation of samples.

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