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Physiological and Molecular Plant Pathology (1998) 52, 67–78
Virulence and supervirulence of Agrobacterium
tumefaciens in woody fruit plants
M. Cervera, M. M. Lo
!pez, L. Navarro and L. Pen4 a*
Dpto. ProteccioU n Vegetal y BiotecnologıUa, Instituto Valenciano de Investigaciones Agrarias (IVIA), Apartado Oficial,46113-Moncada, Valencia, Spain
(Accepted for publication November 1997)
The ability of two wild-type (C58 and Ach5) and one transconjugant (A281) Agrobacteriumtumefaciens strains to incite tumours on some woody fruit species of the Rosaceae, Vitaceae andRutaceae was determined in comparison to tobacco and tomato plants. Agrobacterium strainA281 has been reported to be supervirulent on tobacco and tomato. Here, the supervirulentphenotype of A281 was clearly shown on tobacco plants but not on two di�erent tomato cultivars.On woody fruit hosts, relative virulence induced by each Agrobacterium strain was dependent onthe infecting strain and the host species. On Rutaceae, A281 showed a supervirulent phenotype,while on Rosaceae and on grapevine the responses to all Agrobacterium strains were highly variableand supervirulence was never observed. It is suggested that di�erent responses observed ondi�erent host species are probably due to the specificity of the Agrobacterium strain–hostinteraction. Implications of these results for the concept of supervirulence associated with strainA281 and its disarmed derivatives are discussed. # 1998 Academic Press Limited
INTRODUCTION
Agrobacterium tumefaciens is a well-known soil pathogen. It causes crown gall disease
in most dicotyledonous plants [10]. During infection, Agrobacterium transfers a
specific portion of its tumour inducing (Ti) plasmid, the T-DNA fragment, to
the plant genome. The subsequent expression of genes included in the T-DNA leads
to tumour formation. T-DNA carries genes involved in the synthesis of plant growth
hormones and the production of opines which are tumour-specific compounds.
Agrobacteria are usually classified on the basis of the type of opines specified by the
bacterial T-DNA. The virulence (äir) region, also in the Ti plasmid, is responsible for
almost all the actions involved in T-DNA transfer. To regulate infection of plants,
Agrobacterium has evolved a two component signal transduction system composed of
the virulence proteins encoded by äirA and äirG genes. These proteins sense signal
molecules secreted by wounded plant cells and activate the expression of other äir
genes, thereby initiating the process of T-DNA transport. Another component, located
on the Agrobacterium chromosome, is involved in bacterial chemotaxis towards and
attachment to the wounded plant cell [42].
*To whom correspondence should be addressed.
0885–5765}98}020067≠12 $25.00}0}pp970135 # 1998 Academic Press Limited
68 M. Cervera et al.
The ability of Agrobacterium to transfer a fragment of DNA to plants has been
exploited as a mechanism for introducing foreign DNA into plant genomes. The simple
replacement of the wild-type T-DNA coding region with any DNA sequence of interest
has made Agrobacterium the most useful transformation tool. Big e�orts have been
made to understand the factors that modulate the virulence of Agrobacterium in order
to establish e�cient plant transformation systems for di�erent hosts. Bacterial host
range and virulence determinants have been attributed to structural and organizational
di�erences either in T-DNA or in äir loci [30, 49–51] and to di�erences in chromosomal
loci [31]. The role of the host genome in Agrobacterium–host compatibility has not
received as much consideration as that of the pathogen [29].
The e�ect of äir gene expression on transformation e�ciency and host range has been
exemplified by the results of studies using A. tumefaciens strain A281. This strain is
capable of eliciting larger, earlier-appearing tumours on some plant species, compared
to other strains [17, 18]. Furthermore, the determinants of hypervirulence or
supervirulence of A281 were used to genetically transform recalcitrant tree species that
were only weakly susceptible to common strains [14, 36]. The supervirulent phenotype
induced by A281 was attributed to the äirG region of its Ti plasmid, pTiBo542, since,
when transferred to a non-supervirulent strain it conferred traits of increased virulence
and extended the host range [12]. The importance of äirG from pTiBo542 as an enhancer
for plant genetic transformation has been clearly established. New helper Ti plasmids
that assemble the optimal virulence characteristics of pTiBo542, as strains EHA101 or
EHA105 [16] have been constructed and are being extensively used to improve the
ability of Agrobacterium to introduce foreign DNA into a wide range of herbaceous
and woody plants [5, 9, 12, 24, 25, 28, 32–34, 39, 41].
To analyse the role of the host genome in Agrobacterium-host interactions, we have
investigated the virulence of A. tumefaciens strain A281, compared to that of the
common strains Ach5 and C58, on a wide range of agronomically important woody
fruit crops of the families Rosaceae, Rutaceae and grapevine, using tobacco and tomato
as control hosts in which strain A281 was considered to be supervirulent [17, 18]. The
results could provide new information on the relationships between A. tumefaciens
strains and their hosts and aid in the choice of proper Agrobacterium vectors for
transformation of these woody fruit species.
MATERIALS AND METHODS
Bacterial strains
Agrobacterium tumefaciens C58 and Ach5 are wild-type strains, harbouring respectively
pTiC58 and pTiAch5 plasmids. Strain C58 was originally isolated from Prunus aäium
L. [40] and was obtained from the IVIA Collection of Plant Pathogenic Bacteria.
Strain Ach5 was originally isolated from Prunus domestica L. [20] and was kindly
supplied by Dr. X. Nesme (Univ. of Lyon, France). Strain A281 contains a Ti plasmid
from the wild-type strain Bo542, originally isolated from dahlia (Dahlia rosea Cav.),
that has been transconjugated in planta into the C58 chromosomal background [40].
Strain A281 was kindly supplied by Dr. M.-D. Chilton (Ciba-Geigy, U.S.A.) and by
Dr. E. Nester (Univ. of Washington, Seattle, U.S.A.). Each strain belongs to a di�erent
opine group, as defined by their Ti plasmids : C58 is a nopaline strain, Ach5 is an
Agrobacterium tumefaciens virulence and supervirulence 69
Table 1Tobacco (Nicotiana tabacum L. cä Xanthi) and tomato (Lycopersicon esculentum L.) responses toAgrobacterium infection: number of deäeloping tumours, final tumour formation frequency, tumour weight
and plant height
Number oftumours
(number ofinoculations)*
Final tumourformationfrequency
Tumourweight (g) Plant height
Species Strain 4 weeks 8 weeks (%) ≥SE† (cm) ≥SE†
First Tobacco A281 21 (21) 21 (21) 100 2±32≥0±48 a 31±4≥4±6 aexperiment Ach5 15 (24) 24 (24) 100 0±92≥0±11 b 62±0≥8±0 b
C58 18 (18) 18 (18) 100 0±70≥0±07 b 54±8≥7±2 bControl 0 (9) 0 (9) 0 78±3≥3±0 b
First Tomato A281 58 (60) 58 (60) 96±7 3±51≥0±31 a 133±4≥4±4 aexperiment cv. Roma Ach5 55 (60) 58 (60) 96±7 6±27≥0±51 b 142±6≥2±9 ab
C58 57 (60) 60 (60) 100 4±35≥0±29 a 146±6≥4±0 bControl 0 (9) 0 (9) 0 138±7≥2±7 ab
Second Tobacco A281 21 (21) 21 (21) 100 1±11≥0±13 a 103±1≥3±4 aexperiment Ach5 12 (12) 12 (12) 100 0±81≥0±04 b 119±8≥4±6 b
C58 9 (21) 21 (21) 100 0±59≥0±04 b 114±2≥4±9 bControl 0 (12) 0 (12) 0 140±3≥3±2 c
Second Tomato A281 23 (24) 24 (24) 100 2±55≥0±55 a 139±5≥3±7 aexperiment cv. Roma Ach5 19 (21) 21 (21) 100 2±64≥0±16 a 155±9≥2±6 b
C58 15 (21) 21 (21) 100 2±76≥0±38 a 153±3≥3±9 bcControl 0 (6) 0 (6) 0 139±5≥0±5 ac
Second Tomato A281 23 (24) 24 (24) 100 4±60≥0±31 a 116±8≥5±7 aexperiment cv. Rutgers Ach5 24 (24) 24 (24) 100 4±29≥0±39 a 129±3≥5±0 a
C58 23 (24) 24 (24) 100 2±94≥0±18 b 124±8≥5±4 aControl 0 (12) 0 (12) 0 114±8≥15±9 a
*The number of developing tumours, scored visually 4 and 8 weeks after inoculation. Thetotal number of inoculations per plant species and strain is indicated in brackets.
†Di�erent letters indicate statistically significant di�erences in an F-test (LSD, confidencelevel 95%).
octopine strain and A281 is an L,L-succinamopine strain. The three strains were kept
as glycerinated stocks at Æ70 ∞C and, when required, cultured on YPGA medium
(3 g l�" yeast extract, 5 g l�" bactopeptone, 10 ml l�" glycerol and 20 g l�" agar) for
2 days at 25 ∞C.
Plant species and growing conditions
Apricot (Prunus armeniaca L. cv. Canino), peach (P. persica (L.) Batsch cv. GF 305), plum
(P. domestica L. cv. Mirabolano), almond (P. dulcis Mill. cv. Ferragnes), apple (Malus
domestica Borkh. cv. Ozark Gold), pear (Pyrus communis L. cv. Castell), grapevine (Vitis
äinifera L. cv. Monastrell), loquat (Eriobotrya japonica (Thunb.) Lindl. cv. Algerie)
and a rose rootstock (Rosa indica L.) were obtained from a commercial nursery
(Orero Nursery, Spain). Sweet orange (Citrus sinensis (L.) Osb. cv. Pineapple), lime (C.
aurantifolia (Chritsm.) Swing. cv. Mexican), clementine (C. clementina Hort. ex Tan. cv.
Clemenules) and citrange (C. sinensis (L.) Osb.¨Poncirus trifoliata (L.) Raf cv. Carrizo)
70 M. Cervera et al.
Table 2Woody fruit plant responses to Agrobacterium infection: number of deäeloping tumours, final tumour
formation frequency and final tumour weight
Number of tumours(number of inoculations)*,†
Final tumourformationfrequency Tumour weight
Family}Species Strain 4 weeks 8 weeks 20 weeks 28 weeks (%)‡ (g) ≥SE§
RosaceaeApricot A281 21 (21) 21 (21) 100 a 0±85≥0±15 a(Prunus armeniaca L. Ach5 21 (21) 21 (21) 100 a 1±01≥0±16 acv. Canino) C58 21 (21) 21 (21) 100 a 1±01≥0±08 a
RosaceaePeach A281 21 (21) 21 (21) 100 a 0±71≥0±09 a(Prunus persica (L.) Ach5 20 (21) 20 (21) 95±2 a 1±02≥0±17 aBatsch. cv. GF305) C58 21 (21) 21 (21) 100 a 0±91≥0±12 a
RosaceaePlum A281 0 (18) 0 (18) 2 (18) 2 (18) 11±1 a 0±03(Prunus domestica L. Ach5 0 (18) 0 (18) 3 (18) 6 (18) 33±3 a 0±02cv. Mirabolano) C58 8 (21) 13 (21) 16 (21) 19 (21) 90±5 b 9±03≥3±34
RosaceaeAlmond A281 21 (21) 21 (21) 100 a 0±67≥0±16 a(Prunus dulcis Mill. Ach5 10 (21) 11 (21) 52±4 b 0±08≥0±04 bcv. Ferragnes) C58 21 (21) 21 (21) 100 a 1±34≥0±16 c
RosaceaeApple A281 0 (18) 0 (18) 0 (18) 1 (18) 5±6 a 0±01(Malus domestica Ach5 0 (18) 0 (18) 0 (18) 0 (18) 0 aBorkh. cv. OzarkGold)
C58 0 (21) 0 (21) 0 (21) 4 (21) 19 a 0±02
RosaceaePear A281 0 (21) 0 (21) 2 (21) 2 (21) 9±5 a 0±06(Pyrus communis L. Ach5 0 (21) 0 (21) 1 (21) 1 (21) 4±8 a 0±33cv. Castell) C58 4 (21) 14 (21) 15 (21) 15 (21) 71±4 b 6±63≥2±42
RosaceaeLoquat A281 0 (21) 0 (21) 0 (21) 0 (21) 0(Eriobotrya japonica Ach5 0 (21) 0 (21) 0 (21) 0 (21) 0(Thumb.) Lindl.cv. Algerie)
C58 0 (21) 0 (21) 0 (21) 0 (21) 0
RosaceaRose A281 10 (18) 10 (18) 12 (18) 66±7 a 1±20≥0±34 a(Rosa indica L.) Ach5 1 (18) 4 (18) 4 (18) 22±2 b 0±47≥0±30 a
C58 10 (18) 15 (18) 15 (18) 83±3 a 3±19≥0±69 bVitaceae
Grapevine A281 1 (24) 1 (24) 3 (24) 4±2 a(Vitis äinifera L. Ach5 0 (6) 2 (6) 3 (6) 50 b 0±01cv. Monastrell) C58 7 (21) 21 (21) 21 (21) 100 c 0±55≥0±17
RutaceaeSweet orange A281 2 (30) 20 (30) 24 (30) 24 (30) 80 a 0±17≥0±04 a(Citrus sinensis (L.) Ach5 0 (30) 0 (30) 0 (30) 0 (30) 0 bOsb. cv. Pineapple) C58 0 (30) 2 (30) 7 (30) 7 (30) 23±3 c 0±24≥0±10 a
RutaceaeLime A281 13 (30) 30 (30) 30 (30) 30 (30) 100 a 0±61≥0±07 a(C. aurantifolia Ach5 0 (30) 2 (30) 6 (30) 7 (30) 23±3 b 0±07≥0±06 b(Christm.) Swing.cv. Mexican)
C58 1 (30) 21 (30) 25 (30) 26 (30) 86±7 a 0±52≥0±10 a
Agrobacterium tumefaciens virulence and supervirulence 71
Table 2 (cont.)
Number of tumours(number of inoculations)*,†
Final tumourformationfrequency Tumour weight
Family}Species Strain 4 weeks 8 weeks 20 weeks 28 weeks (%)‡ (g) ≥SE§
RutaceaeClementine A281 3 (30) 10 (30) 12 (30) 12 (30) 40 a 0±41≥0±18(C. clementina Hort. ex Ach5 0 (30) 0 (30) 0 (30) 0 (30) 0 bTan. cv. Clemenules) C58 0 (30) 0 (30) 0 (30) 0 (30) 0 b
RutaceaeCitrange A281 6 (30) 21 (30) 28 (30) 28 (30) 93±3 a 0±41≥0±09 a(C. sinensis (L.) Ach5 0 (30) 2 (30) 7 (30) 7 (30) 23±3 b 0±08≥0±05 bOsb.¨Poncirustrifoliata (L.) Raf.cv. Carrizo)
C58 0 (30) 23 (30) 27 (30) 27 (30) 90 a 0±94≥0±11 c
*The number of developing tumours, scored visually 4, 8, 20 and 28 weeks after inoculation.The total number of inoculations per plant species and strain is indicated in brackets.
†Three or four plants of each species (six for citrus) served as control plants. No tumourswere shown on them.
‡Data of tumour frequency were compared in a chi-square test (confidence level 95%).Di�erent letters indicate statistically significative di�erences.
§Di�erent letters indicate statistically significant di�erences in an F-test (LSD, confidencelevel 95%).
were obtained from the Citrus Germplasm Collection of the IVIA. Tobacco (Nicotiana
tabacum L. cv. Xanthi) was obtained from a seedstock of the IVIA. Tomato (Lycopersicon
esculentum L.) cv. Roma seeds were obtained from Batlle Seeds, while cv. Rutgers
seeds were provided by Dr. F. Nuez (Universidad Polite! cnica of Valencia, Spain) from
the Tomato Germplasm Bank of the UPV (Valencia, Spain).
Plants were grown in individual 2±5 l pots containing a mixture of 55% sphagnum
peat and 45% siliceous sand, and were fertilized weekly. They were kept inside
temperature-controlled greenhouses at 24–26}15–16 ∞C day}night temperatures, with
relative humidities between 60–80%.
Inoculations
For each Agrobacterium strain and host pair, three inoculations per plant were
performed at the same time. Plant stems were wounded three times with a sterile scalpel,
at 5–7 cm spacings with the first wound 5 cm from the soil. Five microlitres of
A. tumefaciens suspension in water (OD&*&nm
E 0±3), from 48 hour-old cultures on
YPGA medium, were added to the wounds with a micropipette. The wounds were then
covered with plastic wraps for 2 weeks to prevent desiccation. Control plants
were also wounded and 5 µl of sterile water were applied per wound. All the
inoculations were performed in April, when the woody plants were actively growing
and tumour development was allowed to progress for 8–28 weeks, depending on the
species (Tables 1 and 2). Experiments with tobacco and tomato were repeated in
the same season for 2 consecutive years. Tobacco plants were 2- to 3-months old
(30–40 cm high in the first experiment and 70–80 cm high in the second
experiment), tomato plants were 2-months old (50–60 cm high) and woody plants were
72 M. Cervera et al.
4- to 6-month -old when inoculated. In the first experiment, strain A281,
supplied by Dr. Nester, was inoculated into tobacco, tomato cv. Roma and woody
fruit plants, and in the second experiment, A281, supplied by Dr. Chilton, was
inoculated into tobacco, tomato cvs Roma and Rutgers.
Scoring of Agrobacterium–host interactions
Tumour formation was monitored visually and, once formed, diameter of tumours was
measured every 2 weeks. After 2–7 months, when tumour growth stopped, tumours
were excised and weighed, after recording their position on the plant (upper, inter-
mediate or lower). In the case of tobacco and tomato, plant height was also measured.
Photographic records of tumours were made.
The relative virulence of Agrobacterium strains was evaluated per strain and host
species according to the following factors : (i) frequency of tumour formation,
calculated as the number of tumours per total number of inoculations¨100, (ii) final
weight of tumours, and (iii) time of appearance of tumours. According to Jin et al. [17],
the supervirulence of A281 is related to its ability to induce large, early-appearing
tumours on a wider range of plants than do other A. tumefaciens strains.
Statistical analysis
To compare mean tumour weight among upper, intermediate and lower inoculation
positions, and mean tumour weight per Agrobacterium strain-host plant interaction, the
F-test from a one-factor analysis of variance was used. Data of tumour formation
frequency were also statistically compared in a chi-squared test.
RESULTS AND DISCUSSION
Virulence of Agrobacterium strains on tobacco and tomato plants
Tobacco and tomato were primarily used as positive controls and as a reference of the
supervirulent phenotype induced by strain A281. Responses of tobacco and tomato
plants to the three Agrobacterium strains are given in Table 1. Both species are natural
hosts for Agrobacterium tumefaciens and a high tumour formation frequency (practically
100%) appeared in all cases as expected. No significant di�erences in tumour
formation frequency and weight were observed among upper, intermediate and lower
inoculation sites, so all tumours were considered independent replicates.
Figure 1 illustrates typical tumours observed in tobacco and tomato plants 2 months
after infection. Whereas tumours induced by strains Ach5 and C58 showed the
hypertrophy characteristic of crown gall in these host species, exhibiting light green to
light brown colours, those induced by strain A281 became progressively dark brown
6 weeks after inoculation. Although necrosis incited by A281 could have slightly
a�ected the final weight of tumours on both tobacco and tomato plants, these tumours
continued proliferating as well as those produced by strains Ach5 and C58 until 8
weeks postinoculation (results not shown).
Strain A281 was the most consistent in inducing tumours on tobacco. These tumours
were significantly larger in two di�erent experiments than those induced by strains
Ach5 and C58 (Table 1), in agreement with the results of supervirulence of A281 on
tobacco previously reported by Hood et al. [17] and Jin et al. [18]. Furthermore,
tumour necrosis seemed to be related to the supervirulent phenotype of A281.
Agrobacterium tumefaciens virulence and supervirulence 73
(a) (b)
(c) (d)
Fig. 1. Tumours incited by A. tumefaciens strains A281 (A), Ach5 (B) and C58 (C) on: (a)tobacco, (b) Rutgers tomato (both corresponding to the second experiment, see text), (c) plum,and (d) lime. In each photograph the stem labelled T corresponded to a control plant.Photographs were taken before excision and weighing the tumours, 8 weeks after inoculationfor tobacco and tomato, and 28 weeks for plum and lime.
(a) (b)
Fig. 2. (a) Roma tomato plants infected by A281 (left) and C58 (right). The necrotic phenotypeof tumours induced by A281 is evident in the photograph. (b) Several tumours excised fromthe stem of lime plants, induced by A281 (left column), Ach5 (middle column), and C58 (rightcolumn). The necrotic response is evident in tumours incited by A281 strain.
74 M. Cervera et al.
Formation of such large tumours caused serious damage to the growth of tobacco
plants, as it is shown in Table 1. Tobacco plants inoculated with A281 did not reach
the average height of plants inoculated with the other Agrobacterium strains. Strains
Ach5 and C58 were equivalent in tumour formation frequency and weight on tobacco
(Table 1).
On tomato, we expected a supervirulent response to strain A281. Rutgers was the
tomato cultivar in which Hood et al. [17] described the hypervirulent phenotype of this
strain. However, when considering factors such as tumour formation frequency, speed
of appearance of tumours, tumour size and final plant height, a hypervirulent
phenotype for A281 was not evident in any of the experiments (Table 1), although
the necrotic response of tumours was observed on both tomato cultivars (Fig. 1b and
2a). On Roma tomato, significant di�erences in virulence among strains were
observed in the first experiment (Ach5 induced the largest tumours) but not in the
second. On Rutgers tomato, A281 and Ach5 produced tumours of similar weight and
larger than those induced by C58.
Di�erent responses on cvs Rutgers and Roma could be explained by the
Agrobacterium strain-host cultivar specificity which has been extensively reported for
a number of plant species, including grapevine [19], soybean [7], pea [13], Pseudotsuga
menziesii [26, 27], Rubus [31] and alfalfa [38]. Absence of supervirulence on tomato
plants infected with strain A281 and di�erences in virulence of Ach5 on Roma in the
two experiments could be attributed to environmental and}or physiological factors.
In fact, levels of äir gene expression are strongly influenced by physical conditions
exerting their e�ects through the äirA-äirG regulatory system. In addition to the
requirement for particular inducer molecules, such as acetosyringone and several
phenolic compounds [44], monosaccharides [4, 7, 43], opines [46] and a range of
metabolites [26, 47], it has been reported that temperature and pH conditions
modulate äir gene activation [1, 2, 45, 47].
Virulence of Agrobacterium strains in woody fruit plants
Table 2 shows the responses of woody fruit plants to Agrobacterium infection. Again,
no significant di�erences were observed among upper, intermediate and lower
inoculation sites. Therefore, each tumour was considered to be an independent
replicate (Table 2).
Within the Rosaceae morphology was comparable in tumours produced by the
three strains on each host species. Tumours exhibited single lobes and light green
to brown colours in all cases (Fig. 1c). However, Agrobacterium strains produced
di�erent virulence responses in each host species (Table 2).
For Prunus, whereas the three strains were equivalent in frequency, weight and
appearance of tumours on apricot and peach, C58 incited the largest tumours and
Ach5 was the least tumorigenic in almond, and C58 was consistently the most virulent
in plum (Table 2). All the four Prunus species are natural hosts of A. tumefaciens and this
could explain the high susceptibility to the bacteria of these species compared to
other rosaceous hosts. Furthermore, strain C58 was originally isolated from plum,
possibly explaining the greater responsiveness of this species to C58.
Di�erent responses to Agrobacterium were also found among the pear and apple
(Table 2). Strain C58 was clearly the most virulent on pear, producing larger tumours
Agrobacterium tumefaciens virulence and supervirulence 75
earlier and at much higher frequencies than strain A281 or Ach5. Inoculations
on apple were not so responsive. Di�culties in inducing tumours on apple stems have
been reported previously [3, 23, 23]. Tumours occurred much later at 28 weeks
postinoculation and in low frequency and of small size. C58 was as virulent as A281,
but both of them were more virulent than Ach5 (Table 2).
On rose, tumours of strains C58 and A281 were of equal frequency and appearance
but C58 incited larger tumours than A281. Ach5 was the less e�cient (Table 2).
Loquat was not susceptible to any of the three strains tested. These results clearly
showed that strain A281 did not incite a supervirulent phenotype on any of the
Rosaceae hosts tested.
On grapevine, strain C58 was the most virulent in terms of rate of appearance, larger
size and higher frequency of tumours (Table 2). The other two strains produced very
small tumours on this host species (Table 2). Strain A281 has never been reported to
be supervirulent on grapevine. Curiously, Lowe and Krul [21] found a necrotic
response in nodal stem segments of Vitis äinifera L. cv Steuben inoculated with A281
and other Agrobacterium strains. This reaction occurs in certain Agrobacterium strain-
Vitis cultivar interactions [35, 49]. This progressive necrosis, rather than tumour
formation, has been attributed to the sensitivity of grapevine cells to elevated levels of
auxins encoded by at least gene tms 6b from the T-DNA of Agrobacterium tumefaciens
strain A281 [11]. We did not observe such reactions in response to any of the
inoculations of the three Agrobacterium strains on Monastrell grapevine.
Although the Rutaceae are not considered natural hosts for Agrobacterium tumefaciens,
the four species tested were responsive to one or more strains. Strain Ach5 was not
e�ective on sweet orange and clementine, and only produced small tumours at low
frequencies on lime and citrange. Strain A281 was the most virulent in all the Citrus
species, since it induced tumours rapidly and in high frequency. On sweet orange
and lime, tumour weight was comparable in plants inoculated with strains A281
and C58. However, strain C58 was incapable of inciting tumours in clementine. On
citrange, A281 induced earlier-appearing tumours but C58 was as e�cient as A281
in tumour formation and incited larger tumours than A281 (Table 2). Interestingly,
tumours induced by strain A281 on the four Rutaceae hosts showed the same
morphology as those produced by this strain on tobacco and tomato (Fig. 1d and 2b).
In fact, a progressive blackening of tumours was observed after 3 months from
inoculation. When tumours incited by A281 were excised, 7 months postinoculation,
necrosis was severely a�ecting their morphology and final weight, thus explaining
the larger size of tumours produced by strain C58 on citrange, and equivalent sizes of
tumours produced by A281 and C58 on sweet orange and lime. Since tumour necrosis
seems to be strongly correlated with the supervirulent phenotype A281, and this
strain incited early-appearing tumours at higher frequencies than the other
Agrobacterium strains, A281 can be considered supervirulent on the four Rutaceae
hosts tested. Consequently, we have been using a disarmed derivative of this strain,
EHA105 [16], to develop e�cient transformation systems for citrange, sweet orange
and lime [32–34]. The hypervirulence of A281 on woody hosts has been found
in subsequent experiments on citrange and other citrus hosts, where it caused
similar necrosis, earlier appearance of tumours and higher tumour formation frequency
(Ghorbel et al., unpublished work).
76 M. Cervera et al.
In summary, strain A281 expressed a supervirulent phenotype on some hosts
but not in others. In our research on woody fruit species, supervirulence was an
exception rather than a rule. Moreover, other reports have shown that A281 did not
induce a supervirulent response on a number of plant species including herbaceous and
woody hosts [6, 8, 13, 15, 31, 38, 48]. The generic term supervirulent has been widely
used to define the Agrobacterium strain A281 and its disarmed derivatives,
independently of the host species [9, 12, 24, 28, 37, 39, 41]. However, supervirulence
data about this strain in the literature are equivocal and it seems that the definition
is being used too loosely. As a recent example, Nam et al. [29] have found that
‘‘ somewhat surprisingly, inoculation of Arabidopsis root segments with Agrobacterium
strain A281 (harbouring the supervirulent Ti plasmid pTiBo542) did not result in
e�cient tumorigenesis ’’. We have shown here that A. tumefaciens-host species
compatibility is unpredictable probably due to the highly specific interactions between
genotypes of both organisms. This can play a role in epidemiological, control of crown
gall, and transformation experiments and suggests that preliminary inoculation tests,
prior to such assays, would be always advisable.
We thank A. Navarro for excellent technical assistance, J. A. Pina for the acquisition
of plants, J. Jua! rez for his help with the photographic material, J. Cubero for his
critical advice, and Dr. F. Nuez (Univ. Polite! cnica of Valencia, Spain) for
providing Rutgers tomato seeds. We also thank Dr. M.-D. Chilton (Ciba-Geigy,
U.S.A.) and Dr. E. Nester (Univ. of Washington, Seattle, U.S.A.) for supplying strain
A281, and Dr. X. Nesme (Univ. of Lyon, France) for supplying strain Ach5. This work
was supported by a grant (No. SC97-102) from the Instituto Nacional de
Investigaciones Agrarias (INIA).
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