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).

REFERENCES

1. Alt-Mo$ rbe J, Ku$ hlmann H, Schro$ der J. 1989. Di�erences in induction of Ti plasmid virulence genesäirG and äirD, and continued control of äirD expression by four external factors. MolecularP lant–Microbe Interactions 2 : 301–308.

2. Alt-Mo$ rbe J, Neddermann P, von Lintig J, Weiler EW, Schro$ der J. 1988. Temperature-sensitivestep in Ti plasmid äir-region induction and correlation with cytokinin secretion by Agrobacteria.Molecular and General Genetics 213 : 1–8.

3. Anderson AR, Moore LW. 1979. Host specificity in the genus Agrobacterium. Phytopathology 69 : 320–323.4. Ankenbauer RG, Nester EW. 1990. Sugar-mediated induction of Agrobacterium tumefaciens virulence

genes : structural specificity and activities of monosaccharides. Journal of Bacteriology 172 : 6442–6446.5. Bean SJ, Gooding PS, Mullineaux PM, Davies DR. 1997. A simple system for pea transformation.

Plant Cell Reports 16 : 513–519.6. Byrne MC, McDonnell, RE, Wright MS, Carnes MG. 1987. Strain and cultivar specificity in the

Agrobacterium-soybean interaction. Plant Cell Tissue and Organ Culture 8 : 3–15.7. Cangelosi GA, Ankenbauer RG, Nester EW. 1990. Sugars induce the Agrobacterium virulence genes

through a periplasmic binding protein and a transmembrane signal protein. Proceedings of the NationalAcademy of Sciences, USA 87 : 6708–6712.

8. Confalonieri M, Balestrazzi A, Biso� S. 1994. Genetic transformation of Populus nigra byAgrobacterium tumefaciens. Plant Cell Reports 13 : 256–261.

9. De Bondt A, Eggermont K, Penninckx I, Goderis I, Broekaert WF. 1996. Agrobacterium-mediatedtransformation of apple (Malus¨domestica Borkh.) : an assessment of factors a�ecting regeneration oftransgenic plants. Plant Cell Reports 15 : 549–556.

10. DeCleene M, DeLey J. 1976. The host range of crown gall. Botanical Reäiews 42 : 389–466.11. Deng W, Pu X, Goodman RN, Gordon MP, Nester EW. 1995. T-DNA genes responsible for

inducing a necrotic response on grapevines. Molecular Plant–Microbe Interactions 8 : 538–548.

Agrobacterium tumefaciens virulence and supervirulence 77

12. Firoozabady E, Moy Y, Courtney-Gutterson N, Robinson K. 1994. Regeneration of transgenic rose(Rosa hybrida) plants from embryogenic tissue. Bio}Technology 12 : 609–613.

13. Hobbs SLA, Jackson JA, Mahon JD. 1989. Specificity of strain and genotype in the susceptibility ofpea to Agrobacterium tumefaciens. Plant Cell Reports 8 : 274–277.

14. Hood EE, Clapham DH, Ekberg I, Johannson T. 1990. T-DNA presence and opine production intumors of Picea abies (L.) Karst induced by Agrobacterium tumefaciens A281. Plant Molecular Biology 14 :111–117.

15. Hood EE, Fraley RT, Chilton M-D. 1987. Virulence of Agrobacterium tumefaciens strain A281 onlegumes. Plant Physiology 83 : 529–534.

16. Hood EE, Gelvin SB, Melchers LS, Hoekema A. 1993. New Agrobacterium helper plasmids for genetransfer to plants. Transgenic Research 2 : 208–218.

17. Hood EE, Helmer GL, Fraley RT, Chilton M-D. 1986. The hypervirulence of Agrobacteriumtumefaciens A281 is encoded in a region of pTi Bo542 outside of T-DNA. Journal of Bacteriology 168 :1283–1290.

18. Jin S, Komari T, Gordon MP, Nester EW. 1987. Genes responsible for the supervirulence phenotypesof Agrobacterium tumefaciens A281. Journal of Bacteriology 169 : 4417–4425.

19. Knauf VC, Panagopoulos CG, Nester EW. 1982. Genetic factors controlling the host range ofAgrobacterium tumefaciens. Phytopathology 72 : 1545–1549.

20. Lin B-C, Kado CI. 1977. Studies on Agrobacterium tumefaciens. VII. Avirulence induced by temperatureand ethidium bromide. Candian Journal of Microbiology 23 : 1554–1561.

21. Lowe BA, Krul WR. 1991. Physical, chemical, developmental, and genetic factors that modulate theAgrobacterium-Vitis interaction. Plant Physiology 96 : 121–129.

22. Maheswaran G, Welander M, Hutchinson JF, Graham MW, Richards D. 1992. Transformationof apple rootstock M26 with Agrobacterium tumefaciens. Journal of Plant Physiology 139 : 560–568.

23. Martin GC, Miller AN, Castle LA, Morris JW, Morris RO, Dandekar AM. 1990. Feasibilitystudies using b-glucuronidase as a gene fusion marker in apple, peach, and radish. Journal of theAmerican Society for Horticultural Science 115 : 686–691.

24. Mathews H, Wagoner W, Cohen C, Kellogg J, Bestwick R. 1995. E�cient genetic transformationof red raspberry, Rubus ideaus L. Plant Cell Reports 14 : 471–476.

25. McKently AH, Moore GA, Doostdar H, Niedz RP. 1995. Agrobacterium-mediated transformation ofpeanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants. Plant Cell Reports14 : 699–703.

26. Morris JW, Castle LA, Morris RO. 1989. E�cacy of di�erent Agrobacterium tumefaciens strains intransformation of pinaceous gymnosperms. Physiological and Molecular Plant Pathology 34 : 451–461.

27. Morris JW, Morris RO. 1990. Identification of an Agrobacterium tumefaciens virulence gene inducerfrom the pinaceous gymnosperm Pseudotsuga menziesii. Proceedings of the National Academy of Sciences USA87 : 3614–3618.

28. Mourgues F, Chevreau E, Lambert C, De Bondt A. 1996. E�cient Agrobacterium-mediatedtransformation and recovery of transgenic plants from pear (Pyrus communis L.). Plant Cell Reports 16 :245–249.

29. Nam J, Matthysse AG, Gelvin SB. 1997. Di�erences in susceptibility of Arabidopsis ecotypes to crowngall disease may result from a deficiency in T-DNA integration. The Plant Cell 9 : 317–333.

30. Nester EW, Gordon MP, Amasino RM, Yanofsky M. 1984. Crown gall : a molecular andphysiological analysis. Annual Reäiew of Plant Physiology 35 : 387–413.

31. Owens y de Novoa C, Conner AJ. 1991. Responses of Rubus genotypes to strains of Agrobacterium.Journal of Genetics and Breeding 45 : 359–368.

32. Pen4 a L, Cervera M, Jua! rez J, Navarro A, Pina JA, Dura! n-Vila N, Navarro L. 1995. Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants. Plant Cell Reports 14 :616–619.

33. Pen4 a L, Cervera M, Jua! rez J, Ortega C, Pina JA, Dura! n-Vila N, Navarro L. 1995. High e�ciencyAgrobacterium-mediated transformation and regeneration of citrus. Plant Science 104 : 183–191.

34. Pen4 a L, Cervera M, Jua! rez J, Navarro A, Pina JA, Navarro L. 1997. Genetic transformation oflime (Citrus aurantifolia Swing.) : factors a�ecting transformation and regeneration. Plant Cell Reports,in press.

35. Pu X-A, Goodman RN. 1992. Induction of necrosis by Agrobacterium tumefaciens on grape explants.Physiological and Molecular Plant Pathology 41 : 245–254.

36. Pythoud F, Sinkar VP, Nester EW, Gordon MP. 1987. Increased virulence of Agrobacterium rhizogenesconferred by the vir region of pTiBo542: application to genetic engineering of poplar. Bio}Technology5 : 1323–1327.

37. Raharjo SHT, Herna! ndez MO, Zhang YY, Punja ZK. 1996. Transformation of pickling cucumberwith chitinase-encoding genes using Agrobacterium tumefaciens. Plant Cell Reports 15 : 591–596.

78 M. Cervera et al.

38. Samac DA. 1995. Strain specificity in transformation of alfalfa by Agrobacterium tumefaciens. Plant CellTissue and Organ Culture 43 : 271–277.

39. Sarria R, Caldero! n A, Thro AM, Torres E, Mayer JE, Roca WM. 1994. Agrobacterium-mediatedtransformation of Stylosanthes guinensis and production of transgenic plants. Plant Science 96 : 119–127.

40. Sciaky D, Montoya AL, Chilton M-D. 1978. Fingerprints of Agrobacterium Ti plasmids. Plasmid 1 :238–253.

41. Scott Grayburn W, Vick BA. 1995. Transformation of sunflower (Helianthus annuus L.) followingwounding with glass beads. Plant Cell Reports 14 : 285–289.

42. Sheng J, Citovsky V. 1996. Agrobacterium-plant cell DNA transport : have virulence proteins, willtravel. The Plant Cell 8 : 1699–1710.

43. Shimoda N, Toyoda-Yamamoto A, Nagamine J, Usami S, Karayama M, Sakagami Y,Machida Y. 1990. Control of expression of Agrobacterium äir genes by synergistic actions of phenolicsignal molecules and monosaccharides. Proceedings of the National Academy of Sciences USA 87 :6684–6688.

44. Stachel SE, Messens E, Van Montagu M, Zambryski P. 1985. Identification of the signal moleculesproduced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318 :624–629.

45. Turk SCHJ, Melchers LS, den Dulk-Ras H, Regensburg-Tuı$nk AJG, Hooykaas PJJ. 1991.Environmental conditions di�erentially a�ect äir gene induction in di�erent Agrobacterium strains. Roleof the VirA sensor protein. Plant Molecular Biology 16 : 1051–1059.

46. Veluthambi K, Krishnan M, Gould JH, Smith RH, Gelvin SB. 1989. Opines stimulate inductionof the äir genes of Agrobacterium tumefaciens Ti plasmid. Journal of Bacteriology 171 : 3696–3703.

47. Vernade D, Herrera-Estrella A, Wang K, Van Montagu M. 1988. Glycine betaine allows enhancedinduction of the Agrobacterium tumefaciens äir genes by acetosyringone at low pH. Journal of Bacteriology170 : 5822–5829.

48. Warkentin TD, McHughen A. 1991. Crown gall transformation of lentil (Lens culinaris Medik.) withvirulent strains of Agrobacterium tumefaciens. Plant Cell Reports 10 : 489–493.

49. Yanofsky M, Lowe B, Montoya A, Rubin R, Krul W, Gordon MP, Nester E. 1985. Molecular andgenetic analysis of factors controlling host range in Agrobacterium tumefaciens. Molecular and GeneralGenetics 201 : 237–246.

50. Yanofsky M, Montoya A, Knauf V, Lowe B, Gordon M, Nester E. 1985. Limited-host rangeplasmid of Agrobacterium tumefaciens : molecular and genetic analysis of transferred DNA. Journal ofBacteriology 163 : 341–348.

51. Yanofsky MF, Nester EW. 1986. Molecular characterization of a host range determining locus fromAgrobacterium tumefaciens. Journal of Bacteriology 166 : 244–250.