Evaluation of citrus somatic hybrids for tolerance to

Phytophthora nicotianae and citrus tristeza virus

Francisco de Assis Alves Mourao Filho a,*, Rafael Pio a, Beatriz Madalena Januzzi Mendes b,Fernando Alves de Azevedo a, Evandro Henrique Schinor a, Fabio Albuquerque Entelmann a,

Andre Siqueira Rodrigues Alves a, Tatiana Eugenia Cantuarias-Aviles a

a Universidade de Sao Paulo, Escola Superior de Agricultura ‘‘Luiz de Queiroz’’, Departamento de Producao Vegetal,

Caixa Postal 9, 13418-900 Piracicaba, SP, Brazilb Universidade de Sao Paulo, Centro de Energia Nuclear na Agricultura, 13400-970 Piracicaba, SP, Brazil

Received 7 February 2007; received in revised form 10 September 2007; accepted 1 October 2007

Abstract

Somatic hybridization is a biotechnology tool that can be used in citrus breeding programs to produce somatic hybrids with the complete genetic

combination of both parents. The goal of this work was to test the reaction of citrus somatic hybrids that may be useful as rootstocks to trunk and root

infections caused by Phytophthora nicotianae van Breda de Haan (P. parasitica Dastur) and to citrus tristeza virus (CTV). The somatic hybrids

evaluated were ‘Caipira’ sweet orange (Citrus sinensis L. Osbeck) + ‘Rangpur’ lime (C. limonia Osbeck), ‘Caipira’ sweet orange + ‘Cleopatra’

mandarin (C. reshni hort. ex Tanaka), ‘Caipira’ sweet orange + ‘Volkamer’ lemon (C. volkameriana V. Ten. & Pasq.), ‘Caipira’ sweet orange + rough

lemon (C. jambhiri Lush.), ‘Cleopatra’ mandarin + ‘Volkamer’ lemon, ‘Cleopatra’ mandarin + sour orange (C. aurantium L.), ‘Rangpur’ lime +

‘Sunki’ mandarin (C. sunki (Hayata) hort. ex Tanaka), ‘Ruby Blood’ sweet orange (C. sinensis L. Osbeck) + ‘Volkamer’ lemon, ‘Rohde Red’ sweet

orange (C. sinensis L. Osbeck) + ‘Volkamer’ lemon, and ‘Valencia’ sweet orange + Fortunella obovata hort. ex Tanaka. For P. nicotianae trunk and

root infection assays, plants of the somatic hybrids, obtained from 9-month semi-hardwood cuttings, were evaluated and compared with diploid citrus

rootstock cultivars after mycelia inoculation in the trunk or spore infestation in the substrate, respectively. ‘Cleopatra’ mandarin + sour orange,

‘Rangpur’ lime + ‘Sunki’ mandarin, ‘Cleopatra’ mandarin + ‘Volkamer’ lemon, ‘Ruby Blood’sweet orange + ‘Volkamer’ lemon, ‘Rohde Red’sweet

orange + ‘Volkamer’ lemon, and ‘Caipira’ sweet orange + ‘Volkamer’ lemon had less trunk rot occurrence, whereas the somatic hybrids ‘Cleopatra’

mandarin + ‘Volkamer’ lemon, ‘Cleopatra’ mandarin + sour orange, ‘Caipira’ sweet orange + ‘Volkamer’ lemon, and ‘Caipira’ sweet orange +

‘Rangpur’ lime were tolerant to root rot. For CTVassays, plants of the somatic hybrids along with tolerant and intolerant rootstocks were budded with a

mild strain CTV-infected or healthy ‘Valencia’sweet orange budwood. Differences in average scion shoot length indicated that the hybrids ‘Cleopatra’

mandarin + sour orange and ‘Valencia’ sweet orange + Fortunella obovata were intolerant to CTV.

# 2007 Elsevier B.V. All rights reserved.

Keywords: CTV; Disease resistance; Protoplast fusion; Rootstock; Trunk rot

www.elsevier.com/locate/scihorti

Available online at www.sciencedirect.com

Scientia Horticulturae 115 (2008) 301–308

1. Introduction

Although citrus (Citrus spp.) is affected by many pathogens,

Phytophthora species are considered one of the most important

soilborne problems leading to considerable losses worldwide,

especially in the economically important production regions

(Erwin and Ribeiro, 1996; Graham and Menge, 2000). Among

several diseases caused by this pathogen, trunk rot and root rot are

* Corresponding author. Tel.: +55 19 34294190; fax: +55 19 34294385.

E-mail address: famourao@esalq.usp.br (F.A.A. Mourao Filho).

0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2007.10.004

considered the most relevant. The symptoms of foot rot are

characterizedby injuries to the bark or the roots nearground level.

Symptoms include necrosis and gum exudation, and the disease is

called gummosis. Pytophthora species may also cause extensive

root rot leading to scion dieback and possible tree death.

Phytophthora nicotianae van Breda de Haan (P. parasitica

Dastur) and P. citrophthora (Smith & Smith) Leonian are

considered the most common Phytophthora species affecting

citrus orchards in Brazil. However, P. nicotianae has been most

frequently associated with the disease, detected over 95% of

nurseries and orchards in Sao Paulo State (Medina Filho et al.,

2004).

F.A.A. Mourao Filho et al. / Scientia Horticulturae 115 (2008) 301–308302

Citrus tristeza virus (CTV) (Closterovirus) is considered the

most important virus pathogen affecting citrus production

worldwide. Several isolates have been identified that have been

associated with quick-decline disease of trees budded on sour

orange (Citrus aurantium L.) and other intolerant rootstocks. In

addition, other isolates have caused severe stem-pitting disease

in susceptible scion cultivars even when budded onto tolerant

rootstocks (Garnsey and Lee, 1993). CTV is spread by several

species of aphids, especially the citrus brown aphid (Toxoptera

citricidus Kirkaldy), the most efficient vector. This virus

disease is now endemic in several economically important

production regions around the world (Garnsey, 1999). The

development and identification of tolerant rootstocks is one of

the main strategies for the control of quick-decline disease

caused by CTV (Bar-Joseph et al., 1989).

The incorporation of biotechnology tools into conventional

citrus breeding programs may facilitate the development of

cultivars with superior characteristics. Among these techni-

ques, protoplast fusion, which produces somatic hybrids

(allotetraploids), has high potential. These hybrids maintain

the entire nuclear genetic combination of both parents since no

meiotic segregation occurs in somatic hybridization. Applica-

tions of somatic hybridization in citrus include the development

of seedless scions for the fresh market and improved rootstocks

with potential for tree size control (Mourao Filho et al., 1996;

Grosser and Gmitter, 2005).

In scion improvement, the production of new cultivars may

be achieved by a number of strategies. Somatic hybrids may be

directly used as tetraploid scions (Guo et al., 2004) or involved

in interploid crosses to produce seedless triploid cultivars

(Viloria and Grosser, 2005). Initial evaluation of some of this

material has already been carried out, such as triploid hybrids of

‘Lakeland’ limequat screened for citrus canker resistance

(Viloria et al., 2004).

On the other hand, citrus somatic hybridization may also be

applied to rootstock improvement in an effort to combine all of

the necessary biotic and abiotic resistances with superior

adaptation and productivity. Besides the desirable character-

istics combined in the somatic hybrids, plants budded on

tetraploid rootstocks are generally smaller, which could lead to

reduced harvest costs and greater production efficiency. In

Florida, more than 70 somatic hybrids that can potentially be

used as rootstocks have already entered into commercial field

trials. Preliminary results from these trials have shown that

somatic hybrid rootstocks can produce adequate yields of high-

quality sweet oranges (Citrus sinensis L. Osbeck) on small trees

(Grosser and Gmitter, 2005). However, prior to establishing

trials in the field, somatic hybrids can be screened for resistance

against primary diseases such as Phytophthora sp. and CTV.

During the last decade, research on somatic hybridization of

citrus has also been conducted in Brazil. The experiments

carried out by Mendes-da-Gloria et al. (2000a) and by Benedito

et al. (2000) on the induction of embryogenic calli and on

protoplast isolation of several rootstocks resulted in somatic

hybridization and post-fusion plant regeneration, with the

confirmation of somatic hybrids from 13 different parental

combinations. Many of these hybrids may be tolerant to CTV

and to citrus blight (Mendes-da-Gloria et al., 2000b; Mendes

et al., 2001; Costa et al., 2003), and some may be tolerant to

Phytophthora sp. (Calixto et al., 2004).

The aim of this work was to verify possible tolerance to

trunk and root infections caused by P. nicotianae and to CTV in

citrus somatic hybrids that may potentially be used as

rootstocks.

2. Materials and methods

2.1. Plant material

Somatic hybrid plants of ‘Caipira’ sweet orange (Citrus

sinensis L. Osbeck) + ‘Rangpur’ lime (C. limonia Osbeck),

‘Caipira’ sweet orange + ‘Cleopatra’ mandarin (C. reshni hort.

ex Tanaka), ‘Caipira’ sweet orange + ‘Volkamer’ lemon (C.

volkameriana V. Ten. & Pasq.), ‘Caipira’ sweet orange + rough

lemon (C. jambhiri Lush.), ‘Cleopatra’ mandarin + ‘Volkamer’

lemon, ‘Cleopatra’ mandarin + sour orange (C. aurantium L.),

‘Rangpur’ lime + ‘Sunki’ mandarin (C. sunki (Hayata) hort. ex

Tanaka), ‘Ruby Blood’ sweet orange (C. sinensis L.

Osbeck) + ‘Volkamer’ lemon, ‘Rohde Red’ sweet orange (C.

sinensis L. Osbeck) + ‘Volkamer’ lemon, and ‘Valencia’ sweet

orange + Fortunella obovata hort. ex Tanaka (Costa et al.,

2003; Mendes et al., 2001; Mendes-da-Gloria et al., 2000a)

were obtained from 15-cm semi-hardwood cuttings removed

from 6- to 8-year-old mother plants, treated in an indole-3-

butyric acid solution (1000 mg L�1) for 10 min, and rooted in

an intermittent mist chamber for 100 days. Plants were grown in

expanded polystyrene trays filled with Plantmax CitrusTM

potting mix.

Seeds of ‘Sunki’ mandarin, ‘Cleopatra’ mandarin, ‘Rang-

pur’ lime, ‘Volkamer’ lemon, sour orange, ‘Caipira’ sweet

orange, ‘Davis A’ trifoliate orange (Poncirus trifoliata L. Raf.)

(negative control) and ‘Sicilian’ lemon (C. limon (L.) Burm. f.)

(positive control) were sown in tubelets (12 cm � 2.7 cm, filled

with 125 cm3 Plantmax CitrusTM potting mix) and transplanted

after 90 days to be used in the screening trials for Phytophthora

sp. tolerance. In the screening trial for CTV tolerance, plants of

‘Rangpur’ lime, ‘Davis A’ trifoliate orange (negative control),

and sour orange (positive control) were also produced from

seeds.

All the plants were grown in plastic bags (32 cm � 24 cm,

5 L) containing Rendmax CitrusTM potting mix supplemented

with OsmocoteTM 22-04-08 (250 g/25 kg potting mix), were

fertilized weekly (Kristalon HydroTM: 0.84 g L�1 and Tenso

Iron HydroTM: 0.08 g L�1), systematically irrigated, and

maintained over elevated benches in a greenhouse covered

with aphid-proof netting.

2.2. Reaction of somatic hybrids to trunk infection with

P. nicotianae

A completely randomized block design was used, with 18

treatments (ten somatic hybrids and eight cultivars), with six

replications and four inoculations per plot, with 432 inocula-

tions in total.

F.A.A. Mourao Filho et al. / Scientia Horticulturae 115 (2008) 301–308 303

Leaves growing at 30-cm height were removed and the

petiole surface was disinfected with 70% alcohol. Two

inoculations per plant, at 10 and 20 cm height, were done on

opposite sides, using the needle method (Siviero, 2001). An

incision to a 3-mm depth was done with an iron needle,

previously soaked in 96% alcohol and flamed. The needle’s

surface was then scraped on the border of P. nicotianae colonies

growing in Petri dishes with a diluted carrot liquid medium

(CA) to collect fungus mycelia, and then placed in the incision

area of the stem. A 2-cm width cotton strip imbibed in distilled

water was wrapped around the stem area containing the

inoculum, and covered with a double surgical bandage. Cotton

strips were wetted daily with distilled water in the morning

hours.

Plants were evaluated after 35 days of inoculation by

measuring the length and the average wounded area under the

bark, as observed directly on the wood surface, over the

cambium zone. Wounded areas were plotted on transparent

paper, based on the color alteration produced by the incision,

and they were then measured with a LICORTM LI-3000 area

meter.

2.3. Reaction of somatic hybrids to root infection by P.

nicotianae

A completely randomized design was used following a split-

plot spatial arrangement, with four replications. Plots were

constituted by infected (I) and control (C) plants, placed on

separated benches, representing the main treatment. The 18

genotypes (ten somatic hybrids and eight cultivars) comprised

the sub-plots, considered the secondary treatment, which were

randomly allocated within each plot. Each sub-plot consisted of

three plants, with a total of 12 infected plants and 12 non-

infected plants, comprising 432 evaluated plants in total.

To produce the inoculum for infecting the potting mix,

pathogenic sporangia were obtained from a spore suspension

following the method described by Ribeiro and Baumer (1977)

and modified by Feichtenberger et al. (1984). Mycelium discs

of 0.25 cm2 area were removed from the borders of 4-day-old

isolated colonies grown on plastic Petri dishes of

90 mm � 15 mm with 25 mL of CA liquid medium, and were

then transferred to plastic Petri dishes with diluted CA medium

(2 mL carrot juice and 8 mL distilled water, autoclaved at

121 8C for 29 min). Petri dishes with 10 discs each were kept at

25 8C, under continuous fluorescence light, for 24 h. The CA

medium was aseptically removed and replaced by 10 mL of

autoclaved distilled water. The inoculated Petri dishes were

then incubated at 25 8C for 48 h. Additional Petri dishes filled

with 25 mL of CA medium and containing 7-day-old mycelia

of the same isolate, which grew in the dark at 25 8C under

B.O.D. conditions, were also obtained for further propagule

preparation. Petri dishes containing the diluted CA liquid

medium with the sporangia were refrigerated at 10–12 8C for

15 min before preparing the propagule suspension to induce

zoospore release. The liquid of each Petri dish (�10 mL

zoospore suspension) was then removed and transferred to a

500-mL Erlenmeyer flask. The remaining 10 discs and the

content of 7-day-old pathogenic mycelium of one Petri dish

were added to the Erlenmeyer flask, and the total volume was

adjusted to 300 mL, by refilling with cold distilled water. The

spore suspension was divided in two parts, and 150 mL of the

suspension was applied in each plastic bag containing one

single plant. Control plants received the same volume to

distilled water only.

On the day of infection, eight holes, 10-cm deep, were

placed in each plastic bag using a glass stick to facilitate the

diffusion of spore suspension in the substrate and to cause some

wounds to the roots. Plants were regularly irrigated during the

trial, avoiding water running out from the bottom of the plastic

bag. Daily minimum and maximum air temperatures for the

period of the experiment were 19.5 and 32.1 8C, respectively.

After 60 days of substrate inoculation, infected plants (I) and

control plants (C) were removed from the plastic bags and were

carefully washed with running tap water. Disease severity was

evaluated by three people using a 5-point scale (1: no growth

reduction; 5: strong growth reduction) based on visual

observation of the root system, and qualitative features of

the aerial plant organs. The mean value of these three

measurements was then calculated by the method of Broadbent

and Gollnow (1992). Averages of leaf number, dry mass of

aerial organs, and dry root mass were also collected. These data

was used to compute the growth difference between infected

and control plants (losses) and the reduction index, calculated

by the following expression: (infected plants/control

plants) � 1 � 100.

2.4. Reaction of somatic hybrids to CTV

The tolerance of somatic hybrids to CTV was evaluated by

the test of transmissibility in greenhouses, i.e., through tissue

union (grafting) with budwood from mild strain CTV-infected

plants of ‘Valencia’ sweet orange, which were used at the same

time as scion and CTV inoculum. The CTV complex used as

CTV source in the experiment was selected by Muller and

Costa (1977) from a ‘Pera’ sweet orange tree (C. sinensis L.

Osbeck), and used for cross-protection in the same cultivar.

When indexed in ‘Mexican’ lime (C. aurantifolia, Christm.

Swingle), it produces mild CTV symptoms, without symptoms

of quick decline and stem pitting. The stability and high degree

of protection of this CTV strain has led for the use of this clone

in the cross protection of the certified budwood program in Sao

Paulo State, Brazil (Costa and Muller, 1980). The mother plants

were grown in pots, under aphid-proof net. Before budwood

harvest, an ELISA test was performed on the mother plants

infected with CTV to confirm the presence of the virus (ELISA

PTA, using the polyclonal anti-serum).

A completely randomized experimental design was used

following a split-plot spatial arrangement, with four replica-

tions. The plots consisted of plants infected with CTV (I) and

healthy plants (H), placed on separated benches, comprising the

main treatment. The 13 genotypes (ten somatic hybrids and

three cultivars) formed the sub-plots, which was considered the

secondary treatment, and were randomly distributed within

each plot. Each sub-plot consisted of three plants, with a total of

F.A.A. Mourao Filho et al. / Scientia Horticulturae 115 (2008) 301–308304

12 healthy plants and 12 infected plants, comprising 312

evaluated plants.

Budding was done by the ‘‘inverted T-bud’’ method, with

one single bud per plant. After budding, the budwood zone was

wrapped with a plastic strip. Sprouts of the rootstock plants

were periodically removed below and above the inserted bud.

After 25 days, the plastic strips were removed and the aerial

part was cut at 5 cm above the inserted bud to promote bud

break.

Trial evaluations started 30 days after removal of the plastic

strip or 55 days after inoculation (budding). The mean shoot

length was measured during the first vegetative flush of the

scion (‘Valencia’ sweet orange), as well as visual observations

were carried out to detect CTV symptoms. These evaluations

were repeated 85 and 105 days after inoculation. Additional

data collection included the determination of the scion growth

difference between healthy and infected plants, and the

reduction index calculated by the following expression:

(infected plants/control plants) � 1 � 100, in each evaluation.

At the end of the trial, Valencia sweet orange plants budded

onto every genotype were grouped, and a 2 mm2 leaf piece was

removed from the scion of both healthy and CTV infected

plants for ELISA testing. The ELISA PTA method was

employed, using the polyclonal anti-serum provided by Centro

APTA Citros Sylvio Moreira, Limeira, Brazil.

Data analyses of the three experiments included analysis of

variance and mean comparison by the Scott–Knott’s test

(P � 0.05).

3. Results and discussion

3.1. Reaction of somatic hybrids to trunk infection with

P. nicotianae

Smaller wounded areas were observed in ‘Davis A’ trifoliate

orange than all of the other rootstocks. Sour orange and

Table 1

Length and area of wounds of citrus rootstocks and somatic hybrids 35 days after

Genotype

‘Davis A’ trifoliate orange

Sour orange

‘Cleopatra’ mandarin + sour orange

‘Cleopatra’ mandarin

‘Rangpur’ lime

‘Sunki’ mandarin

‘Volkamer’ lemon

‘Rangpur’ lime + ‘Sunki’ mandarin

‘Cleopatra’ mandarin + ‘Volkamer’ lemon

‘Ruby Blood’ sweet orange + ‘Volkamer’ lemon

‘Rohde Red’ sweet orange + ‘Volkamer’ lemon

‘Caipira’ sweet orange + ‘Volkamer’ lemon

‘Caipira’ sweet orange + ‘Cleopatra’ mandarin

‘Caipira’ sweet orange + ‘Rangpur’ lime

‘Caipira’ sweet orange

‘Caipira’ sweet orange + rough lemon

‘Valencia’ sweet orange + Fortunella obovata

‘Sicilian’ lemon

Any two means within a column not followed by the same letter are significantly

‘Cleopatra’ mandarin + sour orange can be classified as more

resistant to P. nicotianae than the rootstocks ‘Cleopatra’

mandarin, ‘Rangpur’ lime, ‘Sunki’ mandarin and ‘Volkamer’

lemon and the somatic hybrids ‘Rangpur’ lime + ‘Sunki’

mandarin, ‘Cleopatra’ mandarin + ‘Volkamer’ lemon, ‘Ruby

Blood’ sweet orange + ‘Volkamer’ lemon, ‘Rohde Red’ sweet

orange + ‘Volkamer’ lemon and ‘Caipira’ sweet orange +

‘Volkamer’ lemon, which can be classified as moderately

susceptible. ‘Caipira’ sweet orange and the somatic hybrids

‘Caipira’ sweet orange + ‘Cleopatra’ mandarin, ‘Caipira’ sweet

orange + ‘Rangpur’ lime, ‘Caipira’ sweet orange + rough

lemon and ‘Valencia’ sweet orange + Fortunella obovata

(classified as susceptible), and ‘Sicilian’ lemon can be

classified as extremely susceptible to P. nicotianae (Table 1).

The results observed in the cultivated rootstocks are

consistent with those found in previous studies, in which

‘Davis A’ trifoliate orange was classified as highly resistant,

‘Rangpur’ lime was classified as moderately susceptible and

‘Sicilian’ lemon was classified as extremely susceptible to P.

nicotianae, as determined by plant inoculation with the needle

method (Siviero et al., 2002). Field evaluation results of

rootstock resistance/susceptibility cited in previous investiga-

tions support the reactions observed with the inoculations

performed in this work (Feichtenberger, 2001).

Regarding the somatic hybrids, ‘Cleopatra’ mandarin + sour

orange showed resistance as did one of its parents, sour orange,

which is different from the other parent, ‘Cleopatra’ mandarin,

which is considered moderately susceptible. The same situation

occurred with the hybrids ‘Ruby Blood’ sweet orange +

‘Volkamer’ lemon, ‘Rohde Red’ sweet orange + ‘Volkamer’

lemon, and ‘Caipira’ sweet orange + ‘Volkamer’ lemon, which

behaved similarly to their moderately susceptible parent,

‘Volkamer’ lemon, whereas its other parent, sweet orange, is

susceptible to trunk rot. On the other hand, ‘Caipira’ sweet

orange + ‘Cleopatra’ mandarin and ‘Caipira’ sweet orange +

‘Rangpur’ lime somatic hybrids were susceptible to trunk rot,

inoculation with Phytophthora nicotianae in the trunk

Wound length (mm) Wound area (mm2)

3.5 a 10.0 a

6.9 b 21.4 b

7.5 b 21.8 b

9.6 c 32.8 c

9.4 c 30.8 c

10.1 c 33.5 c

11.1 c 32.6 c

9.1 c 32.0 c

9.6 c 32.5 c

9.2 c 30.7 c

10.7 c 31.7 c

11.1 c 33.9 c

12.5 d 38.5 d

12.3 d 38.5 d

12.9 d 41.2 d

17.1 f 49.8 f

14.9 e 46.0 e

45.7 g 151.1 g

different at P � 0.05 (Scott–Knott’s test).

F.A.A. Mourao Filho et al. / Scientia Horticulturae 115 (2008) 301–308 305

similar to the ‘Caipira’ sweet orange parent, since the other

parents are classified as moderately susceptible.

Sweet orange has raised great interest as a rootstock because

it confers resistance to blight, high production and good fruit

quality, but its use is very limited due to its high susceptibility to

Phytophthora spp. (Matheron et al., 1998). Therefore, ‘Ruby

Blood’ sweet orange + ‘Volkamer’ lemon, ‘Rohde Red’ sweet

orange + ‘Volkamer’ lemon and ‘Caipira’ sweet orange +

‘Volkamer’ lemon somatic hybrids have good prospects in

evaluations under field conditions.

Evaluation of several genotypes regarding Phytophthora

spp. inoculation using the disc method in adult citrus plants

grown in the field showed that the wounded area under the bark

of the hybrid ‘Rangpur’ lime � ‘Sunki’ mandarin did not differ

from that of the progenitors (Medina Filho et al., 2003).

3.2. Reaction of somatic hybrids to root infection by

P. nicotianae

Less leaf drop was observed in root-infected plants of ‘Davis

A’ trifoliate orange and ‘Cleopatra’ mandarin + sour orange

somatic hybrids than the other rootstocks tested. On the other

hand, a significant leaf drop was recorded for ‘Sunki’ mandarin,

‘Caipira’ sweet orange and ‘Sicilian’ lemon rootstocks, as well

as in ‘Caipira’ sweet orange + ‘Cleopatra’ mandarin, ‘Caipira’

sweet orange + rough lemon and ‘Valencia’ sweet orange +

Fortunella obovata somatic hybrids. Plants of ‘Volkamer’

lemon and ‘Cleopatra’ mandarin had leaf drop reduction

indexes of 6.4% and 8.5%, respectively (Table 2).

Regarding the dry mass of aerial organs, lower losses were

recorded in ‘Davis A’ trifoliate orange, ‘Cleopatra’ mandarin,

Table 2

Leaf number loss, dry mass of aerial organs, root dry mass, reduction index, and roo

with P. nicotianae in the substrate

Genotype Leaf number loss

C � Ib (number) RIc (

‘Davis A’ trifoliate orange 0.3 a 0

Sour orange 6.8 c 12.6

‘Volkamer’ lemon 4.4 c 6.4

‘Rangpur’ lime 5.6 c 10.2

‘Cleopatra’ mandarin 4.4 c 8.5

‘Sunki’ mandarin 8.7 d 12.2

‘Caipira’ sweet orange 9.1 d 14.5

‘Sicilian’ lemon 10.4 d 12.0

‘Cleopatra’ mandarin + sour orange 2.0 b 12.9

‘Cleopatra’ mandarin + ‘Volkamer’ lemon 4.1 c 12.0

‘Rangpur’ lime + ‘Sunki’ mandarin 4.1 c 11.2

‘Caipira’ sweet orange + ‘Cleopatra’ mandarin 9.1 d 23.1

‘Caipira’ sweet orange + rough lemon 8.0 d 19.7

‘Caipira’ sweet orange + ‘Volkamer’ lemon 5.6 c 13.2

‘Caipira’ sweet orange + ‘Rangpur’ lime 4.2 c 11.8

‘Rohde Red’ sweet orange + ‘Volkamer’ lemon 5.9 c 16.2

‘Ruby Blood’ sweet orange + ‘Volkamer’ lemon 6.1 c 18.3

‘Valencia’ sweet orange + Fortunella obovata 12.5 d 43.0

Any two means within a column not followed by the same letter are significantlya Five-point severity scale (1 = no visual growth reduction a 5 = strong visual grb Difference between control (C) and infected (I) plants of citrus rootstocks andc Reduction index (RI) = (infected plants/control plants) � 1 � 100.

‘Cleopatra’ mandarin + sour orange, ‘Cleopatra’ mandarin +

‘Volkamer’ lemon, and ‘Caipira’ sweet orange + ‘Rangpur’

lime. On the other hand, high dry mass losses were observed for

‘Sunki’ mandarin, ‘Caipira’ sweet orange, ‘Sicilian’ lemon, and

‘Valencia’ sweet orange + Fortunella obovata (Table 2). Yet,

‘Davis A’ trifoliate orange showed lower root dry mass losses,

followed by ‘Cleopatra’ mandarin, ‘Cleopatra’ mandarin +

sour orange, ‘Caipira’ sweet orange + rough lemon, and

‘Caipira’ sweet orange + ‘Rangpur’ lime (Table 2). The

reduction index (RI) is consistent for genotype classification

concerning the losses produced by P. nicotianae, both in the

roots and in the aerial parts (Medina Filho et al., 2004).

Visual observations of the root system for the severity of

feeder root rot and qualitative aspects of the aerial parts

indicated that there was no difference between ‘Davis A’

trifoliate orange infected and non-infected plants, whereas

differences were observed with ‘Volkamer’ lemon, ‘Cleopatra’

mandarin, ‘Cleopatra’ mandarin + sour orange, ‘Cleopatra’

mandarin + ‘Volkamer’ lemon, ‘Caipira’ sweet orange +

‘Volkamer’ lemon and ‘Caipira’ sweet orange + ‘Rangpur’

lime. More evident differences were observed in the plants of

‘Sunki’ mandarin, ‘Caipira’ sweet orange, ‘Sicilian’ lemon, and

‘Valencia’ sweet orange + Fortunella obovata (Table 2).

‘Cleopatra’ mandarin + ‘Volkamer’ lemon somatic hybrid

infected plants showed adequate growth as expected, since both

progenitors involved in hybridization are considered tolerant to

P. nicotianae. The tolerance of ‘Cleopatra’ mandarin + sour

orange and ‘Caipira’ sweet orange + ‘Volkamer’ lemon somatic

hybrids to root and feeder-root rots is due the progenitors

‘Cleopatra’ mandarin and ‘Volkamer’ lemon, which are also

tolerant to the pathogen’s infestation. An additional feature was

t rot severity of citrus rootstocks and somatic hybrids 60 days after inoculation

Dry mass of aerial organs Root dry mass Root rot

severitya

%) C � I (g) RI (%) C � I (g) RI (%)

0.1 a 2.4 0.1 a 3.4 1.0 a

1.4 c 9.5 0.7 c 11.6 3.6 c

1.3 c 2.9 1.1 c 6.8 2.3 b

1.2 c 11.5 1.0 c 15.1 3.6 c

0.6 b 6.2 0.5 b 13.1 2.1 b

3.0 e 22.2 1.1 c 27.2 4.0 d

3.6 e 26.7 2.2 d 32.8 4.1 d

3.9 e 14.7 3.1 e 26.5 3.7 d

0.4 b 10.8 0.3 b 12.8 2.0 b

0.7 b 6.9 0.7 c 14.7 2.3 b

0.9 c 13.8 0.9 c 25.9 3.2 c

1.6 c 17.4 1.0 c 25.8 3.6 c

0.9 c 11.6 0.6 b 20.5 3.2 c

1.3 c 8.4 1.1 c 14.5 2.5 b

0.7 b 7.7 0.6 b 16.9 2.6 b

1.2 c 11.7 1.1 c 21.3 2.9 c

1.4 c 14.8 1.1 c 23.7 3.1 c

2.0 d 34.4 1.1 c 59.9 4.9 e

different at P � 0.05 (Scott–Knott’s test).

owth reduction).

somatic hybrids.

Table 3

Shoot length and reduction index of ‘Valencia’ sweet orange budded on citrus rootstocks and somatic hybrids after inoculation with CTV

Genotype Shoot lengtha (cm) RIb (%) Shoot length (cm) RI (%) Shoot length (cm) RI (%)

Days after inoculation

55 85 105

Sour orange 3.2 Ca 18.9 20.8 Bc 54.8 31.7 Ac 64.6

‘Davis A’ trifoliate orange 0.2 Aa 0 0.4 Aa 0 1.0 Aa 0

‘Rangpur’ lime 2.1 Aa 8.9 1.5 Aa 3.7 6.1 Aa 10.3

‘Caipira’ sweet orange + ‘Rangpur’ lime 0.5 Ba 0 0.4 Ba 0 5.0 Aa 7.5

‘Caipira’ sweet orange + ‘Cleopatra’ mandarin 2.9 Aa 4.9 2.3 Aa 11.9 5.5 Aa 9.3

‘Caipira’ sweet orange + ‘Volkamer’ lemon 1.4 Aa 6.7 3.1 Aa 10.9 5.0 Aa 9.7

‘Caipira’ sweet orange + rough lemon 0.7 Aa 0.1 4.8 Aa 14.4 5.2 Aa 9.9

‘Cleopatra’ mandarin + ‘Volkamer’ lemon 2.7 Aa 10.2 2.0 Aa 2.8 2.5 Aa 3.7

‘Cleopatra’ mandarin + sour orange 5.7 Ca 17.7 13.9 Bb 38.4 31.2 Ac 59.9

‘Rangpur’ lime + ‘Sunki’ mandarin 1.1 Aa 0 1.2 Aa 0 0.6 Aa 10.0

‘Ruby Blood’ sweet orange + ‘Volkamer’ lemon 1.4 Aa 3.5 1.1 Aa 0 3.4 Aa 4.7

‘Rohde Red’ sweet orange + ‘Volkamer’ lemon 2.7 Aa 17.5 1.4 Aa 2.9 5.1 Aa 9.1

‘Valencia’ sweet orange + Fortunella obovata 0.9 Ca 0 6.6 Ba 42.0 11.5 Ab 52.7

Any two means within a column not followed by the same lower-case letter or within a row not followed by the same upper-case letter are significantly different at

P � 0.05 (Scott–Knott’s test).a Difference between control (C) and infected (I) plants of citrus rootstocks and somatic hybrids.b Reduction index (RI) = (infected plants/control plants) � 1 � 100.

F.A.A. Mourao Filho et al. / Scientia Horticulturae 115 (2008) 301–308306

observed in the ‘Caipira’ sweet orange + ‘Rangpur’ lime

somatic hybrid, which had increased tolerance compared to

both parents (Feichtenberger, 2001).

The overall analysis of the reaction of the rootstocks and

somatic hybrids regarding trunk rot and root and feeder-root

rots caused by P. nicotianae indicated that ‘Cleopatra’

mandarin + sour orange was resistant to trunk rot and tolerant

to feeder-root rot, whereas ‘Cleopatra’ mandarin, ‘Volkamer’

lemon, ‘Cleopatra’ mandarin + ‘Volkamer’ lemon and ‘Cai-

pira’ sweet orange + ‘Volkamer’ lemon, classified as tolerant to

feeder-root rot, behaved as moderately susceptible to trunk rot.

‘Caipira’ sweet orange + ‘Rangpur’ lime somatic hybrid

behaved as tolerant to feeder-root rot, but susceptible to trunk

rot. This finding indicates that the relationship between

resistance to trunk infection and tolerance to root rot caused

by Phytophthora spp. is not always adequate. Therefore, the

analysis of new citrus rootstocks should include both trunk

infection and root and feeder-root rot responses in order to

collect information on the genotype behavior in the presence of

these two important diseases caused by Phytophthora spp. Sour

orange is an example of this fact, which is considered highly

tolerant to trunk rot, despite its low tolerance to root rot

(Graham, 1990).

3.3. Reaction of somatic hybrids to CTV

‘Valencia’ sweet orange trees budded onto sour orange

rootstock, and ‘Cleopatra’ mandarin + sour orange and

‘Valencia’ sweet orange + Fortunella obovata somatic hybrids

showed significant differences in average scion shoot length 55,

85, and 105 days after inoculation (or budding), with a

progressive reduction of shoot height, i.e., for these genotypes

the difference between healthy and infected plants increased

over the evaluation period. It is important to point out that

‘Valencia’ sweet orange healthy and infected plants budded

onto ‘Caipira’ sweet orange + ‘Rangpur’ lime somatic hybrid

showed differences only 105 days after inoculation (Table 3).

No differences were observed between plants on somatic

hybrids and control plants after 55 days, whereas after 85 days,

plants on ‘Cleopatra’ mandarin + sour orange and sour orange

were significantly different from the others (13.9 and 20.8 cm

height, respectively). Yet, after 105 days, the difference

increased to 31.2 and 31.7 cm, respectively, and at this date,

the hybrid ‘Valencia’ sweet orange + Fortunella obovata was

also affected, with a 11.5-cm loss in scion shoot length due to

the presence of CTV. Plants budded on other somatic hybrids

and ‘Rangpur’ lime and ‘Davis A’ trifoliate orange controls did

not suffer significant losses, thus demonstrating their tolerance

to CTV.

‘Valencia’ sweet orange plants budded onto sour orange and

‘Cleopatra’ mandarin + sour orange somatic hybrid had higher

reduction indexes 55 days after inoculation, whose values

increased 85 days after inoculation. The last evaluation, 105

days after inoculation, confirmed the intolerance to CTV of

these genotypes. Shoot growth completely stopped in

‘Valencia’ sweet oranges infected with CTV and budded onto

sour orange and the somatic hybrids ‘Cleopatra’ mandarin +

sour orange and ‘Valencia’ sweet orange + Fortunella obovata,

with reduction indexes of 64.6%, 60.0% and 52.6%,

respectively. On the other hand, plants on other genotypes

had lower reduction indexes than those on ‘Rangpur’ lime,

which is considered to be tolerant to CTV (Carlos et al., 1997)

(Table 3).

A strong yellowing of young leaves and the presence of

small leaves, which generally remained twisted up to 3 months

after budding, were observed. In more severe infections, growth

is almost completely stopped and the plant decays (Costa et al.,

1954).

‘Valencia’ sweet orange + Fortunella obovata is intolerant

to CTV probably due to the Fortunella obovata progenitor,

F.A.A. Mourao Filho et al. / Scientia Horticulturae 115 (2008) 301–308 307

since the hybrids of this species showed intolerance to CTV

(Bennett and Costa, 1949). The hybrid ‘Cleopatra’ mandar-

in + sour orange intolerance to CTV probably came from the

sour orange progenitor, which is classified as intolerant. It

should be noted that not all the somatic hybrids which have the

sour orange as one of their progenitors are necessarily

intolerant to CTV, just as, for instance, the somatic hybrids

sour orange + Carrizo citrange, sour orange + ‘Palestine’ sweet

lime lemon and sour orange + ‘Rangpur’ lime, which were

tolerant to CTV in a greenhouse study, whereas the hybrid sour

orange + ‘Flying Dragon’ trifoliate orange was susceptible,

despite the fact that ‘Flying Dragon’ trifoliate orange is tolerant

to the virus (Grosser et al., 1996, 1998, 2000).

The results reported herein indicate that most of the somatic

hybrids evaluated had adequate tolerance to trunk and root rot

caused by P. nicotianae. Somatic hybrid combinations

involving sour orange or Fortunella obovata as one of the

progenitors were intolerant to CTV. All other somatic hybrid

combinations with good performance herein, especially those

with tolerance to CTV, should be evaluated in the field in future

investigations.

Acknowledgements

The authors thank Fundacao de Amparo a Pesquisa do Estado

de Sao Paulo (FAPESP) and Fundo Paulista de Defesa da

Citricultura (Fundecitrus) for financial support. The authors are

also grateful to Eduardo Feichtenberger (UPD-Sorocaba, APTA

Regional, SAA) for kindly providing the Phytophthora isolate;

Dr. Jorgino Pompeu Junior (Centro APTACitros Sylvio Moreira)

for kindly providing the healthy and CTV-infected budwood of

‘Valencia’ sweet orange, and Dr. Jay Lee Schell for critical

comments. Francisco de Assis Alves Mourao Filho and Beatriz

Madalena Januzzi Mendes acknowledge Conselho Nacional de

Desenvolvimento Cientıfico e Tecnologico (CNPq) for fellow-

ship. Rafael Pio acknowledges Coordenacao de Aper-

feicoamento de Pessoal de Nıvel Superior (CAPES) for

fellowship.

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