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Crop Protection 26 (2007) 5965

Effect of citrus leaf-miner damage, mechanical damage and

inoculum concentration on severity of symptoms of

Asiatic citrus canker in Tahiti lime

R.S.C. Christiano, M. Dalla Pria, W.C. Jesus Junior, J.R.P. Parra,L. Amorim, A. Bergamin Filho

Department of Entomology, Phytopathology and Zoology, ESALQ, University of Sao Paulo, Piracicaba, SP, CEP 13418-900, Brazil

Received 29 July 2005; received in revised form 9 January 2006; accepted 29 March 2006

Abstract

Citriculture in Sao Paulo State, Brazil, is threatened by Asiatic citrus canker (Xanthomonas axonopodis pv. citri). The introduction of

the Asian citrus leaf miner (Phyllocnistis citrella [CLM]) has resulted in an increase in the number of disease foci and has changed the

spatial pattern of citrus canker symptomatic trees from strong aggregation to intermediate aggregation and random patterns. We

evaluated the effect of inoculum concentration (101, 102, 104 and 106 colony forming units (cfu) ml1) on infection of leaves of Tahiti lime

(Citrus latifolia) with three distinct treatments: (1) intact leaves, (2) mechanically wounded leaves, and (3) leaves exhibiting injuries from

CLM at the egg stage, first instar, third instar, and pupal stages. The minimum inoculum concentration to cause symptom development

in intact leaves was 104 cfu ml1; in mechanically wounded leaves and in leaves with CLM injury at the egg stage and first instar stage,

102 cfuml1; in leaves with CLM injuries at the third instar and pupa stage, 10 1 cfuml1. The injuries from the third instar and pupa

stages resulted in greater disease severity than other treatments at all inoculum concentrations (five times higher than in the intact leaf).

Disease severity in leaves with mechanical wounding and with CLM injuries caused at the egg and first instar stages did not differ fromintact leaves.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Xanthomonas axonopodis pv. citri; Phyllocnistis citrella; Stomatal penetration; Mechanical wound

1. Introduction

Brazil has the largest citrus production areas in the

world. Most of the citrus production is concentrated in Sao

Paulo State, accounting for about 94% of Brazilians

production of Tahiti lime (Citrus latifolia Tanaka). The

fruits are sold primarily fresh in both internal and exportmarkets (FNP Consultoria & Agroinformativos, 2005).

One of the most severe citrus diseases in Sao Paulo State is

Asiatic citrus canker, caused by Xanthomonas axonopodis

pv. citri (Hasse) Vaut. (Xac). The pathogen was identified

in Sao Paulo as early as 1957 (Rosetti, 1977) and causes

erumpent lesions on fruit, leaves, and young stems.

Defoliation, dieback and premature fruit drop can occur

when the disease is severe, reducing fruit quality and yield

(Goto, 1992). However, the most significant impact of the

disease is through imposition of quarantine restrictions,

which limit and disrupt trade (Gottwald et al., 2001). Xac

penetrates through stomatal openings and wounds made

by thorns, sand, and human or insect activity. The

expansion rate of leaf lesions from infection throughstomata is about 1 mm month1 for 68 months, but

infection through mechanical wound results in rapid lesion

growth for 30 d, after which expansion decreases to near

zero (Graham et al., 1990).

Citrus canker has been kept under relative control in Sao

Paulo through an eradication program. However, from

1997 a change in disease behavior was observed. The

number of disease foci increased drastically from 25, in

1995, to 4180, in 1999 (Fundecitrus, 2005). The spatial

pattern of the diseased trees, earlier characterized as

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Corresponding author.

E-mail address: [email protected] (R.S.C. Christiano).
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strongly aggregated, changed to an intermediate aggrega-

tion and even random pattern (Bergamin Filho et al.,

2001). This change was associated with the introduction, in

1996, of Phyllocnistis citrella Stainton (Lepidoptera:

Gracillariidae), the Asian citrus leaf miner (CLM).

CLM has seven development stages (phases): egg, first

instar, second instar, third instar, fourth instar, pupa, andadult (Chagas and Parra, 2000). CLM targets fresh flush

and an adult CLM lays eggs near the leaf mid-rib, and on

hatching the first instar larva burrows into the epidermal

cell layer of the leaf, forming galleries (mines) and exposing

the leaf mesophyll. Sohi and Sandhu (1968) observed that

leaves with CLM injuries had higher canker incidence than

non-infested leaves. Normally, only one miner develops per

leaf, however under high infestation conditions, two to

three miners/leaf can occur. Exposure of leaf mesophyll

during feeding allows direct penetration of Xac into the leaf

tissue. Also, CLM larvae may become contaminated with

bacteria, disseminating them through the feeding galleries

(Gottwald and Garnsey, 1991; Gottwald and Graham,

1992; Gottwald et al., 1993, 1997).

The objective of the experiments described in this paper

was to compare the effect of different inoculum concentra-

tions of Xac on development of symptoms of citrus canker

on leaves of Tahiti lime that were either undamaged

(bacteria entering through stomata), mechanically

wounded, or infested with CLM at different stages of

development.

2. Material and methods

Tahiti lime plants were obtained by budding ontoPoncirus trifoliata in plastic tubes (200 150 mm). Grow-

ing the plants in the tubes was necessary to conduct the

experiment in a closed room to conform to phytosanitary

regulations. Plants 2025 cm height were pruned to

standardize flushing.

The effect of different leaf conditions on infection by Xac

were tested: (1) undamaged leaves (penetration through

stomata), (2) mechanical wounding, and (3) injury caused

by CLM. All treatments were applied to plants with young

leaves in new flush (leaves less than 75% expanded).

Mechanical wounding was achieved by puncturing leaves

with a histological needle at six equidistant points; CLM

infested Tahiti lime plants were obtained by placing the

plants in egg-laying colonies of P. citrella inside entomo-

logical cages, as described by Chagas and Parra (2000) and

removing them at the (1) egg stage, (2) first instar stage, (3)

third instar stage or (4) the pupa stage.

Plants with intact leaves, mechanically wounded leaves

and CLM-injured leaves were spray inoculated with a

suspension of Xac at concentrations of 100, 101, 102, 104

and 106 colony forming units (cfu) ml1. The Xac isolate

(IBSBF 1421, from Dr. Julio Rodrigues Neto, Intituto

Biolo gico, Sao Paulo) was grown on nutrient agar for 48 h

at about 28 1C before suspending in sterile distilled water.

The inoculum was diluted to the desired concentration

using a colorimeter. After inoculation, plants were placed

in wetted acrylic boxes to maintain surface moisture and

high humidity for 72 h at 28 1C and 12 h photophase. After

that, plants were kept at 28 1C and 12 h photophase. There

were a total of 30 treatments (six leaf pretreatments five

inoculum concentrations), each replicated 8 times (one plant/

replicate), in a completely randomized design. The experi-ment was repeated once.

Citrus canker incidence (% of diseased plants) was

assessed every 2 d for all treatments. At 30 d after

inoculation, the 104 and 106 cfu ml1 treatments were

sampled (one leaf collected from each infected plant 8

leaves per treatment). This procedure was carried out at

50 d after inoculation for the 101 and 102 cfu ml1 treat-

ments. Different sample times were dictated by the fact that

symptoms developed very slowly at low inoculum con-

centrations (Goodman, 1982). These leaves were scanned

and the disease severity (% leaf area infected) of each leaf

image measured by image analysis, using QUANT v.1.0

software (Vale et al., 2001). Where leaves were mechani-

cally wounded or damaged by CLM, only the lesions

immediately associated with the trauma were assessed.

Lesions on undamaged parts of the leaf were not counted

to demonstrate the isolated effect of injury on infection and

subsequent symptom development.

Disease incidence progress curves were plotted against

time. Incubation period (time between inoculation and

appearance of symptoms) was defined as: 50% of the

plants in a particular treatment group showed lesions. This

allowed calculation of the mean incubation period in days

for each treatment.

Area under the disease progress curves (AUDPC) foreach treatment was calculated by trapezoidal integration:

AUDPC Xn1i1

Xi Xi1

2

ti1 ti ,

where n is number of assessments; X, disease incidence (%);

and (ti+1 ti), time interval between two consecutive

assessments. In order to allow comparison between

treatments that were assessed during different periods of

time, the AUDPC integral variable was divided by its

respective observation period (tn t1), and marked with an

asterisk (*). Thus, AUDPC* is the standardized area under

the citrus canker incidence progress curve and can be

interpreted as the mean incidence of the disease while the

experiment was conducted. AUDCP* data of treatments at

each inoculum concentration were compared by ANOVA,

and a post hoc means separation was performed using

Tukeys HSD test at the 5% level of probability (po0:05)

using SAS v8.2 PROC GLM (SAS, Inc., 2001).

A nonlinear exponential model was fitted to the disease

severity data using STATISTICA software (StatSoft, Inc.,

2001). The appropriateness of the temporal models was

evaluated by correlation of observed versus predicted

values, standard residual plots, and coefficient of determi-

nation of regression. Disease severity data for treatments at

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R.S.C. Christiano et al. / Crop Protection 26 (2007) 596560


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each inoculum concentration were analyzed using SAS

with the PROC GLM CONTRAST command (SAS, Inc.,

2001) and 1% level of probability (po0:01).

3. Results

The results shown represent mean data from two runs ofthe experiment. Citrus canker lesions were observed in all

treatments, except 100 cfu ml1. The progress curves for

disease incidence for leaf treatments and inoculation

concentrations show that disease developed in all combina-

tions (Fig. 1). Inoculum concentrations of 104 and

106 cfu ml1 resulted in 100% incidence in all leaf treat-

ments, while at 102 cfuml1 only the CLM (pupal stage)

resulted in 100% infection. Mechanical wounding, CLM

(egg stage, first and third instar) had similar results

(5063% infection), and intact leaves had an incidence of

37.5%. At 101 cfuml1, the CLM (pupal stage) resulted in

the highest incidence (87%), followed by CLM (3rd instar)

(63%) and mechanical wounding (45%). Other treatments

showed 12% to 38% disease incidence.

The mean incubation period (MIP) ranged from 6 to

3 0 d (Fig. 1). The lowest MIP (6d) was observed at

inoculum concentrations of 106 cfu ml1 with leaves

infested with CLM (pupal stage and third instar), followed

by mechanical wounding (8 d). Intact leaves and CLM

infested leaves (egg stage and first instar) ranged from 11 to

13 d. At 101 and 102 cfu ml1, intact leaves, CLM (egg stage

and first instar) gave the highest MIPs (up to 30 d). In

general, treatments with mechanical wounds and CLM

infestation (third instar and pupal stages) had lower MIPs,

while the intact leaf and CLM infestation (egg and first

instar stages) had higher MIPs. With all treatments MIP

increased as inoculum concentration decreased.

AUDPC* increased with increasing concentration as the

number of diseased plants (incidence) increased and

incubation period decreased (see Fig. 2). For all inoculumconcentrations, AUDPC* of the CLM (pupal stage)

treatment was the greatest (po0:05), followed by CLM

(third instar stage) and mechanical wounding. Intact

leaves, CLM (egg stage and first instar) treatments had

lower, similar AUDPC*s for most concentrations.

Disease severity increased exponentially with the loga-

rithm of the inoculum concentration (Fig. 3). At

106 cfu ml1 intact leaves had a severity of 1.5%, mechani-

cally wounded leaves 0.9%, and CLM infestation for the

egg stage 2.2%, for the first instar 2.41%, for the third

instar 7.4% and for the pupal stage 8.8% (Fig. 3). More

severe CLM injury resulted in exponentially increased

disease severity (Fig. 4) with similar rates of disease

increase for 102, 104 and 106 cfu ml1. Contrast analysis

showed that at all concentrations disease severity in leaves

with CLM (third instar and pupal stages) was higher

(po0:01) than all other treatments (Table 1). Only at

106 cfu ml1 was disease severity resulting from CLM

(pupal stage) greater than severity resulting from CLM

(third instar, po0:01). Intact leaves, mechanical wounding,

CLM (egg and first instar stages) were not statistically

different, except at a concentration of 106 cfu ml1, when

CLM (egg and first instar stages) were more severely

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Fig. 1. Effect of leaf treatment and inoculum concentration on the progress of disease incidence (% diseased plants) of Asiatic citrus canker in Tahiti lime.

Leaf treatments: intact leaf (IL), mechanically wounded (MW) and infested with various stages of citrus leaf miner: egg stage (ES), first instar (1I), third

instar (3I) and pupal stages (PS). Inoculum concentrations of Xanthomonas axonopodis pv citriare: 101 (1), 102 (2), 104 (4) and 106 (6)cfuml1. Each point

represents the mean of two repetitions of the experiment.

R.S.C. Christiano et al. / Crop Protection 26 (2007) 5965 61


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infected than the mechanically wounded leaves. Disease

severities associated with CLM (third instar and pupa

stages) were 5 and 6 times higher, respectively, in relation

to intact leaves at all inoculum concentrations tested.

4. Discussion

The ability of Xac to cause infection with subsequent

symptom development after inoculation at very low

concentration (101

cfu ml1

) demonstrates the efficiencyof the infection process for this pathogen. These data agree

with the observations of Gottwald and Graham (1992),

who demonstrated that infection can result from as few as

one to two bacterial cells inoculated into the sub-stomatal

chamber. The minimum inoculum concentration needed to

cause infection with Xac in more than 50% of plants varied

depending on the damage status of the leaf. A concentra-

tion of 104 cfu ml1 resulted in 450 incidence when intact

leaves were inoculated, yet only 102 cfu ml1 were needed

when the leaves were mechanically wounded or injured

with CLM (at the egg and first instar stages), which agrees

with the observations of Goto (1992), Goodman (1982)

and Zubrzycki and Zubrzycki (1987). However, in our

experiments, concentration of 101 cfu ml1 resulted in 63%

infection of the plants with CLM (third instar stage) and

87% of the plants with CLM (pupal stage), demonstrating

that infection is enhanced by injuries caused by CLM (third

instar and pupal stages) compared to other treatments.

Belasque et al. (2001) also observed that higher infection

rates were obtained when citrus leaves were infested by

larvae or pupae of CLM.

Infection occurring subsequent to mechanical wounding

and CLM infestation (third instar and pupal stages)

presented lower MIPs than infection of intact leaves or

CLM infestation (egg and first instar stages,). The lowest

MIPs (6 d) were observed in the CLM treatment (third

instar and pupal stage) inoculated with 106 cfu ml1, and

the highest MIPs were observed in intact leaves (up 30 d) at

101 and 102 cfu ml1. Low MIP and high disease incidence

resulted in higher values for the AUDPC* for CLM injury

(pupal stage), differing from other treatments (po0:05),

followed by CLM (third instar stage) and mechanical

wounding treatments. AUDPC retains the maximum

information relative to an epidemic. Verniere et al. (2003)

observed high correlations between AUDPC and all otherepidemic variables of citrus canker on Tahiti lime.

For all leaf treatments tested, disease severity increased

with increasing inoculum concentration. The relationship

was exponential with the logarithm of the concentration.

The greatest disease severity developed with the CLM

injury (third instar and pupal stages, po0:01). The

difference in disease severity between CLM egg stage

injury and CLM pupal stage injury, which was most

noticeable at 106 cfu ml1, indicates a strong influence of

CLM developmental stage on the subsequent infection

with Xac. The increase in disease severity with CLM (egg to

pupa stages) is related to the area injured. Feeding

activities of CLM directly expose mesophyll cells to

infection with Xac. Therefore, the larger the CLM growth

stage, the bigger the feeding galleries formed, and the

greater opportunity for Xac to infect. At the egg stage,

CLM injury is virtually zero and at the first instar stage

there is only a small area of injury parallel to the main vein.

However, at the third instar stage, the CLM gallery can

occupy about two-thirds of the leaf area, and at pupa stage

the gallery can cover the whole leaf area.

Infection of leaves by Xac in the presence of the egg and

first instar stage of CLM was probably via stomata. There

was no evidence for dissemination of Xac by CLM in the

interior of the galleries during feeding activities, because at

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Fig. 2. Effect of leaf treatment and inoculum concentration on the standardized area under the disease progress curve (AUDPC*) for the incidence (%

leaves) of Tahiti lime infected with Asiatic citrus canker. Leaf treatments are intact leaves (control), mechanically wounded, and infested with citrus leaf

miner at the egg stage, first instar, third instar or pupal stages. Inoculum concentrations are 10 1 (1), 102 (2), 104 (4) and 106 (6)cfu ml1. At each inoculum

concentration, columns with the same letter do not differ (po0:05).



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all concentrations tested the disease severity in leaves with

either the egg or first instar stages was similar to intact

leaves.

Both intact and mechanically wounded leaves showed

similar disease severity, which probably derives from the

method used to quantify disease. All canker lesions were

assessed on intact leaves, but only lesions associated with

the injured area were assessed for the mechanical wounding

treatment, and Xac infection was restricted to the area of

mechanical wounding (six sites punctured with a histolo-

gical needle). The greater leaf injury resulting from

CLM infestation at the third instar and pupal stages

favors Xac infection and subsequent citrus canker in-

cidence and severity, even at low concentrations of the

pathogen (101 cfu ml1). The high severity values observed

from CLM injury for the third instar and pupal stages

(five times greater than in intact leaves) is probably related

to the amount of damage that exposes mesophyll cells to

direct Xac infection. Appropriate pest management

approaches are needed to reduce the effect that CLM

has in increasing the incidence and severity of citrus

canker.

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0.0

0.5

1.0

1.5

2.0

2.5

3.0

0

2

4

6

8

10

12

0

1 2 3 4 6

2

4

6

8

10

12

Disease

severity(%)

Log (cfu/ml)

IL MW

ES 1I

3I PS

Y= 0.12exp(0.33X)

R2=0.98

Y= 0.08exp(0.56X)

R2=0.99

Y= 0.33exp(0.55X)

R2=0.99

Y= 0.40exp(0.49X)

R2=0.98

Y= 0.02exp(0.73X) *1

R2=0.99 *2

Y= 0.06exp(0.73X)

R2=0.99

5 1 2 3 4 6

Log (cfu/ml)

5

Fig. 3. The effect of leaf treatment and inoculum concentration on the severity (% leaf area infected) of Asiatic citrus canker in leaves of Tahiti lime. Leaf

treatments are: intact leaf (IL), mechanically wounded (MW) and infested with various stages of citrus leaf miner: egg stage (ES), first instar (1I), third

instar (3I) and pupal stages (PS). Each point represents the mean of 2 repetitions. Vertical bars represent standard errors. ( *1) Exponential model:

Y b0 exp (b1X), where b0 and b1 are equation parameters; Y is disease severity (%); and X is inoculum concentration (log(cfu ml1)). (*2) Coefficient of

determination of the exponential model.



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References

Belasque, J., Parra, A.L., Chagas, M.C.M., Behe, C., Ayres, A.J., Parra,

J.R.P., Hartung, J.S., 2001. Interaction of citrus bacterial canker,

citrus leaf miner and pest management. Phytopathology 91, S7.

Bergamin Filho, A., Amorim, L., Gottwald, T.R., Laranjeira, F.F.,

2001. Spatial distribution of citrus canker in Sao Paulo, Brazil. In:

International Workshop on Plant Disease Epidemiology, Proceedings

of the International Society of Plant Pathology, Ouro Preto, Brazil,

vol. 8, pp. 2829.

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0.0

0.3

0.6

0.9

1.2

1.5

1.8

0

2

4

6

8

10

12

3rdinstar

1stinstar

Eggstage

4thinstar

Pupastage

2ndinstar

3rdinstar

1stinstar

Eggstage

4thinstar

Pupastage

2ndinstar

CLM stages development

Diseaseseverity(%)

Y= 0.09exp(0.36X) *1

R2=0.90 *2

Y= 0.26exp(0.24X)

R2=0.78

Y= 2.66exp(0.25X)

R2=0.87

Y= 1.06exp(0.24X)

R2=0.72

(A) (B)

(C) (D)

Fig. 4. The effect of citrus leaf miner (CLM) injury at different stages on severity (% leaf area infected) of Asiatic citrus canker in leaves of Tahiti lime at

inoculum concentrations of 101 (A), 102 (B), 104 (C) and 106 (D) cfu ml1. Each point represents the mean of 2 repetitions. Vertical bars represent standard

errors. (*1) Exponential model: Y b0 exp (b1X), where b0 and b1 are equation parameters; Yis disease severity (%); and X is CLM stage: egg stage, first

instar, second instar, third instar and pupa stage. (*2) Coefficient of determination of the exponential model.

Table 1Contrast analysis of severity of citrus canker for each leaf treatment and each inoculum concentration

Contrasted treatments Inoculum concentration (cfu ml1)

101 102 104 106

Intact leafmechanical wound 0.07a 0.15 0.17 0.62

Intact leafCLMb (egg stage) 0.02 0.05 0.40 0.72

Intact leafCLM (first instar) 0.04 0.12 0.50 0.90

Intact leafCLM (31 instar) 0.31 0.65 2.88 5.89

Intact leafCLM (pupal stage) 0.47 0.69 2.78 7.29

Mechanically woundedCLM (egg stage) 0.09 0.10 0.23 1.34

Mechanically woundedCLM (first instar) 0.03 0.03 0.33 1.52

Mechanically woundedCLM (third instar) 0.24 0.5 2.71 6.51

Mechanically woundedCLM (pupal stage) 0.4 0.54 2.61 7.91

CLM (egg stage)CLM (first instar) 0.06 0.07 0.10 0.18CLM (egg stage)CLM (third instar) 0.33 0.60 2.48 5.17

CLM (egg stage)CLM (pupa stage) 0.49 0.64 2.38 6.57

CLM (first instar)CLM (third instar) 0.27 0.53 2.38 4.99

CLM (first instar)CLM (pupal stage) 0.43 0.57 2.28 6.39

CLM (third instar)CLM (pupal stage) 0.16 0.04 0.1 1.4

Contrasted treatments are significantly different at po0.01 by contrast analyses.aValues of difference between contrasted treatments means.bCLM citrus leaf miner.



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