Post on 07-Mar-2023
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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