Download For example, Gall diseases on the roots of tobacco plants were first

BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
The crown is the point at the soil line where the main root joins the stem where is
galls typically form at. But the galls may also develop on secondary or lateral roots
and on the main stem and branches above the soil line
The study of the development of crown gall disease in plants is important, not only
because the disease affects a wide range of dicotyledonous plants (especially those
in the rose family, including fruit trees and raspberries as well as roses), but also
because of the nature of the developmental changes that occur. Understanding
tumorigenesis and crown gall development could provide important insights into
plant hormone signaling pathways, carbon and nitrogen metabolism, and sourcesink relationships. (Eckardt-2007)
Furthermore, The virulence mechanism of the pathogene turns out to be unique
among interactions between prokaryotic pathogens and eukaryotic hosts
For example, Gall diseases on the roots of tobacco plants were first found in
a field of Kawai-mura, Iwate Prefecture, Japan in 1995. Since then, the disease has
continued to occur in the same field. A. tumefaciens was isolated from the galls of
the tobacco plants, and the Agrobacterium tobacco strains were fully characterized
by pathogenicity tests, reinoculation of healthy tobacco plants, growth on
selective medium, and biochemical properties in this study. This report is the first
of crown gall of tobacco caused by A. tumefaciens in the field.
Several changes have been proposed related to the no-menclature and taxonomy of
Agrobacterium
Symptoms and signs
Crown gall is identified by overgrowths appearing as galls on roots and at the base
or "crown" of woody plants such as pome (e.g., apple, pear) and stone (e.g.,
cherry, apricot) fruit and nut (e.g., almond, walnut) trees (Figure 1). Crown galls
are also formed on ornamental woody crops such as roses, Marguerite daisies, and
Chrysanthemum spp. as well as on vines and canes such as grapevines (Figure 2)
and raspberries. Marguerite daisies, chrysanthemums and grapevines can become
infected systemically. Occasionally, galls have been observed on field crops such
as cotton, sugar beets, tomatoes, beans (Figure 3) and alfalfa (Figure 4), but the
Agrobacterium tumefaciens 2
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
disease does not impact such crops economically. Crown gall is caused by
Agrobacterium tumefaciens, a Gram-negative, bacilliform bacterium that is
normally associated with the roots of many different plants in the field. This
bacterium can survive in the free-living state in many soils with good aeration
such as sandy loams where crown gall diseased plants have grown. The bacterium
can also survive on the surface of roots (rhizoplane) of many orchard weeds.
Plants representing over 93 plant families are susceptible to crown gall as judged
by experimental inoculations. Owing to their high susceptibility to crown gall,
plants such as Jimson weed (Datura stramonium) and sunflower (Helianthus
annuus) are used as assay hosts for testing the degree of virulence of A.
tumefaciens. Also, Kalanchoë daigmontiana (also known as Bryophyllum) is used
for assaying A. tumefaciens, but the plant is less sensitive than Datura.
Pathogen Biology
Agrobacterium tumefaciens is a Gram-negative rod-shaped bacterium that is
commonly found in the rhizosphere of many plants, where it survives on root
exudates. It will infect a plant only through a wound site (which often occurs in
nursery stock through transplanting and grafting and in vineyards through
pruning).
Agrobaterium is widely recognized for its ability to transfer foreign DNA into
plant cells, whereby T-DNA becomes integrated into the plant genome. Certain
phenolic compounds produced by the plant (including acetosyringone) cause the
induction of agrobacterial virulence genes encoding, among other proteins, an
endonuclease that excises T-DNA from the bacterial tumor-inducing plasmid. The
T-DNA then becomes integrated into the plant genome, and T-DNA genes are
expressed via the plants normal transcriptional and translational machinery. Some
of the salient features of crown gall disease were reviewed by Nester et al. (1984),
and a review concerning T-DNA transfer was presented by Gelvin
(2003(.(Eckardt-2006)
So Agrobacterium tumefacience is one of the transgenic organism witch contains
genes foreign to its own genome (Griffiths 1999). Such organisms can be used for
research or for specialized commercial applications.
Agrobacterium tumefaciens 3
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
There are multiple ways to construct transgenic organisms. Expression plasmids
are widely used tools, especially in the genetic modification of plants. The
expression plasmid is a circular piece of DNA that contains an origin of
replication, the appropriate regulatory regions located 5’ to the site in which the
gene of interest is inserted, and a criterion for selection. The origin of replication
is important to assure the replication of the plasmid within the host bacterium
(Darnell 1990). Containing the correct regulatory regions, such as promoters,
allows for the transcription and translation of the inserted gene, ultimately
resulting in the expression of the gene. Having a mechanism for selection enables
for the discrimination between self-ligated (which do not contain the gene of
interest) and recombinant plasmids (which do contain the gene of interest).
Selectable bacterial genes, such as antibiotic resistance, are often used for the
selection process. Expression is finally achieved when the plasmid is reinserted
into bacteria, which have replication, transcription, and translation machinery
(Griffiths 1999).
The Taxonomy of the genus Agrobacterium:
The genus is divided into species largely based on pathogenic properties, although
other physiological characteristics correlate with pathogenic
properties. The major species are A. radiobacter (nonpathogenic), A. tumefaciens
(the causative agent of crown gall tumors), A. rhizogenes (the causative agent of
hairy root disease), and A.vitis (the causative agent of tumors and necrotic disease
on grapevines). There are also less well studied proposed species such as A. rubi
isolated from cane galls on Rubrus species. Agrobacteria also have been divided
into biotypes (biovars) based on physiological properties. Biovar 1, which includes
most strains of A. tumefaciens, has no growth factor requirements and will grow in
the presence of 2% NaCl.
Most strains produce 3-ketolactose. All biovars produce acid from mannitol and
adonitol. Biovar 1 bacteria also produce acid from dulcitol, melizitose, ethanol,
and arabitol. Some biovar 1 strains are able to grow at 37°C. However, they may
lose the Ti plasmid, which is required for virulence, when grown at this
temperature. Biovar 2 includes most strains of A. rhizogenes. These bacteria
require biotin for growth. They fail to grow in the presence of 0.5% NaCl or at
37°C. Some biovar 2 strains can grow on tartrate producing alkali. Biovar 3 strains
Agrobacterium tumefaciens 4
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
include most A. vitis strains. Some authors also include some A. tumefaciens
strains in this group. Like biovar 1 strains, these bacteria will grow in the presence
of 2% NaCl but generally do not grow at 37°C.
Both biovar 2 and 3 strains fail to produce 3 ketolactose. Biovar 3 strains can
produce alkali from tartrate. Some biovar 3 strains require biotin for growth (Table
2). Selective growth media for various biovars have been reported and are
described in the section on isolation of agrobacteria (Table 1). Biovars 1 and 3
contain both strains with wide and others with narrow host ranges (Kerr et al.,
1977b).( Matthysse-2005)
Agrobacterium tumifaciens fall into 3 biovars which differ in their host range,
metabolic characteristics. All pathogenic strains harbor a tumor-inducing (Ti)
plasmid that encodes the classified by the types of opines induced and
metabolized. Some strains also harbor additional plasmids, but previous studies
focused heavily on Ti plasmid, inducing the recently completed sequences of
nonpaline-agrocinopine-type plasmid.( Goodner -2001)
Agrobacterium tumefaciens genom and plasmid:
Most of the work on Agrobacterium tumefaciens, since its identification as the
causal agent in crown gall disease of dicotyledonous plants at the turn of the
century, has rightfully focused on the mechanism of tumor induction. Since most
of the virulence genes lie on the Ti plasmid, the chromosomal complement of A.
tumefaciens has been relatively understudied
Initial chromosomal maps for A. tumefaciens, based on chromosome mobilization
and recombination of genetic markers, suggested a single circular chromosome .
However, recent physical mapping data strongly suggests that A. tumefaciens has
two chromosomes, one circular chromosome of ~3 Mbp and one linear
chromosome of ~2.1 Mbp This chromosome organization appears to be a
conserved trait throughout the genus. While multiple chromosomes have been
found in some other eubacteria (Schrammeijer-2003)
Analysis of the entire Agrobacterium tumefaciens C58 genome by pulsed-field gel
electrophoresis (PFGE) reveals four replicons: two large molecules of 3,000 and
2,100 kb, the 450-kb cryptic plasmid, and the 200-kb Ti plasmid. Digestion by
PacI or SwaI generated 12 or 14 fragments, respectively. The two megabase-sized
replicons, used as probes, hybridize with different restriction fragments, showing
Agrobacterium tumefaciens 5
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
that these replicons are two independent genetic entities. A 16S rRNA probe and
genes encoding functions essential to the metabolism of the organism were found
to hybridize with both replicons, suggesting their chromosomal nature. In PFGE,
megabase-sized circular DNA does not enter the gel. The 2.1-Mb chromosome
always generated an intense band, while the 3-Mb band was barely visible. After
linearization of the DNA by X-irradiation, the intensity of the 3-Mb band
increased while that of the 2.1-Mb remained constant. This suggests that the 3-Mb
chromosome is circular and that the 2.1-Mb chromosome is linear. To confirm this
hypothesis, genomic DNA, trapped in an agarose plug, was first submitted to
PFGE to remove any linear DNA present. The plug was then recovered, and the
remaining DNA was digested with either PacI or SwaI and then separated by
PFGE. The fragments corresponding to the small chromosome were found to be
absent, while those corresponding to the circular replicon remained, further proof
of the linear nature of the 2.1-Mb chromosome.(Goodner 2001)
The plant pathogen Agrobacterium tumefaciens has a tumour-inducing (Ti)
plasmid of which part, the transfer (T)-region, is transferred to plant cells during
Agrobacterium tumefaciens 6
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
the infection process. As a result, the infected plant cells are triggered to divide,
leading to the formation of crown gall tumours. The tumorous growth of the
infected plant cells is caused by expression of the oncogenes located on the TDNA. The vir region, also present on the Ti plasmid, encodes the Vir proteins,
which mediate the processing of the T-region and the transfer of a single-stranded
(ss) DNA copy of this region, the T-strand, into the recipient cells. One of the Vir
proteins, the nicking enzyme VirD2, remains covalently attached to the 5' end of
the T-strand. VirD2 pilots the T-strand to the plant cell nucleus by virtue of its
nuclear localisation signal (NLS). Recently, we showed that, in addition to the
nucleoprotein complex, A.tumefaciens translocates the Vir proteins VirE2 and VirF
directly into plant cells. Transport occurs via the VirB/VirD4 type IV secretion
channel. The ssDNA-binding protein VirE2 and the F-box protein VirF play a role
in the process of plant transformation. The VirE2 protein protects the T-strand and
is involved in its transport into the nucleus. VirF can interact through its F-box
with plant homologues of the yeast Skp1 protein. However, the target of VirF
remains unknown so far. The virF operon contains a single gene, but the adjacent
virE operon embraces three genes, namely virE1, virE2 and virE3. The VirE1
protein plays an important role in the transport of VirE2 by acting as a chaperone
of this protein and thus preventing at the same time premature binding to the Tstrand and VirE2 aggregate formation. The function of the VirE3 protein has not
been established, but virFvirE3 double mutants are more strongly attenuated in
virulence than virF mutants (B.Schrammeijer, P.Zuiderwijk and P.J.J.Hooykaas,
unpublished results). (Schrammeijer-2003)
Over gall genome structure for Agrobacterium tumefaciens C58:
Agrobacterium tumifaciens C58 genome consists of a circular chromosome, and 2
plasmids: the tumor inducing plasmid pTiC58 and a second plasmid, p AtC58 the
genome was sequenced and assembled with standard methods the sequence of all
four DNA molecules is available on GenBank.
The two chromosomes contain all of the genes stable RNAs and housekeeping
proteins involved in assential cellular functions and prototrophic growth . the
circular chromosome contains a putative origin of replication (Cori) of
Caulobacter crescentus. The liner chromosome in the otherhand, has a plasmidtype replication system of the same type found on pTiC58 and pAtC58. This
Agrobacterium tumefaciens 7
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
system, encoded by the repABC genes, expresses a pair of segregation
proteins(RepA and RepB) and an origin binding replication initiation protein
(RepC). Thus hepothesized that the liner chromosome is evolutionarily derived
from a plasmid. The plasmid origin of an extra chromosome had been predicted
for multichromosome genomes of the α-proteobacteria and has been found in more
distantly related origansims such as Vibrio cholerae
Gene density is very similar between the two chromosomes. However,genes
involved in most essential processes are significantly overrepresented on the
circular chromosome. The dinucleotide signatures of the two plasmids are quite
similar to each other and to related plasmids from the other members of the
Rhizobiaceae family.
More than 6000 base pairs of near-perfect sequence identity extend across the two
ribosomal RNA (rRNA) gene clusters on each of the two chromosomes. The
chromosomes also share some shorter regions of greater than 90% sequence
identity with pAtC58 . Transcription of all rRNA gene clusters is oriented away
from the DNA replication origins, with those on the linear chromosome in the
same orientation. A number of housekeeping genes are located between the linear
chromosome's rRNA operons, and one might expect frequent recombination
resulting in lethal events.
The linear chromosome is covalently closed linear molecule so the telomeres of
the linear chromosome are covantly closed. Apparently possessing hairpin loops of
tge telomers the sequence which made by Brad Goodner et al 2001 came within
several hundred bases of the telomersand additional sequence is presented by
wood et al. However, the sequence of the putative hairpin loop is not yet available.
The proximal regions of both telomers are similar in overall architecture but very
different in sequence. These regions contain portions of several insertion
sequences (IS) elements with intervening DNA of additional repeated and unique
sequence. They are rich in potential secondary structure and contain numerous
short sequence repeats.(goodner 2001)
Agrobacterium tumefaciens biotypes and biovars
Based on some distinct phenotypic differences, A. tumefaciens isolates were
originally classified into three biotypes or biovars (biotype I, II and III; biovar 1, 2,
and 3). Biotype I or biovar 1 strains produce 3-ketosugars and usually have wide
host ranges. Biotype II or biovar 2 strains mainly classify as the hairy root-forming
Agrobacterium tumefaciens 8
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
organism, A. rhizogenes. Biotype III or biovar 3 isolates are mainly confined to
grapevines, prefer L-tartaric acid over glucose and produce polygalacturonase.
Because grapevine isolates formed a distinct group verified by DNA homology
studies and were frequently limited in host-range to grapevines, biovar 3 strains
have been reclassified into one species, A. vitis. Agrobacterium rubi strains infect
canes of the genus Rubus, representing blackberry and raspberry
DNA replication and the cell cycle:
DNA replication is synchronized with the A.tumifaciens cell cycle (***),
A feat requiring coordination of four DNA molecules and two different classes of
replication origin: Cori and repABC. The precise signal that initiates DNA
replication is not yet clear, although in the related α-proteobacterium Coulobacter,
many proteins are subject to cell cycle synchronized transcriptional control and
proteolysis. Because the initiation signal must be interpretable by both types of
replication origin, it is unlikely to be transduced by a single origin-specific binding
protein.
Prossesive DNA replication is performed by DNA polymerase III (pol III), and
A.tumefaciens carries for paralogs of the dnaE gene incoding the polIII α
(polymerase) subunit. These dnaE genes fall into distinct sequence families,
designated as categories A and B. The category A gene of the circular
chromosome is conserved in all sequenced α-proteobacteria and probably encodes
the primary replication enzyme. Each of the A,tumefaciens repABC replicons
(linear chromosome,pTiC58, and pAtC58) encodes a category B dnaE gene within
an operon containing two conserved gene of unknown function. The operon is
present in all fully sequenced α-proteobacteria except the Rickettsia species and
may encode a novel DNA polymerase complex.(Goodner 2001)
Agrobacterium tumefaciens 9
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
Plant transformation and tumorigenesis:
(Aloni et al 2005) Tumor growth is initiated by the integration and expression of
the T-DNA of the bacterial Ti plasmid within the plant nuclear DNA. The T-DNA
encodes enzymes catalyzing the synthesis of high levels of auxin, cytokinin and
opines (Zambryski et al. 1989). Such tumors are characterized by high percentage
of transformed cells, which may reach up to 100% transformation (Rezmer et al.
1999).
For many years, plant tumors induced by A. tumefaciens were considered
unorganized, or only partly organized masses (Sachs 1991). However, an analysis
of the three-dimensional pattern of phloem and xylem in Agrobacterium-induced
crown galls unveiled a sophisticated vascular network of continuous vascular
bundles extending from the host plant up to the tumor margin (Aloni et al. 1995).
The development of these bundles indicates release of free auxin by the A.
tumefaciens-transformed plant cells up to the surface of the fast-growing tumor.
(Aloni et al 2005)
Agrobacterium tumefaciens 10
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
Agrobacterium vitis-induced crown galls cause poor xylem development in
grapevine, which impairs water flow into the young parts of the shoot above the
gall (Agrios 1988). Furthermore, crown galls do not regenerate the disrupted
epidermis and cuticle to protect against water evaporation (Aloni et al. 1995;
Schurr et al. 1996), and the enlarged and unorganized callus shape of the tumor
surface increases transpiration substantially at the tumor surface, about 15 times
higher compared with host leaves, and 7.5 times higher compared with leaves of
noninfected castor bean plants (Schurr et al. 1996; Wächter et al. 2003). In the
centripetal direction the crown gall causes the development of pathologic xylem
within the host stem characterized by narrow vessels, giant vascular rays and by
the absence of fibers (Aloni et al. 1995). These anatomical features have triggered
the propounding of the gall-constriction hypothesis (Aloni et al. 1995) to explain
the mechanism that gives priority in water supply to the growing gall over the host
shoot. This hypothesis proposes that a growing gall retards the development of its
host shoot by decreasing vessel diameter in the shoot tissues close to the tumor,
which substantially reduces water supply to the upper parts of the shoot. It was
further postulated that the controlling signal that induces the narrow vessels in the
host is the phytohormone ethylene (Aloni et al. 1995), which is known to reduce
the diameter of vessels (Yamamoto et al. 1987). It was also suggested that the high
auxin levels induced by the T-DNA-encoded oncogenes stimulate this ethylene
production (Aloni et al. 1995). These ideas were experimentally confirmed by
showing that tumor-induced ethylene is a limiting and controlling factor of crown
gall morphogenesis; very high ethylene levels are produced continuously by
growing crown galls during several weeks (Figure 1); up to 140 times more
ethylene than in wounded, but not infected control stems, reaching a maximum at
five weeks after infection (Aloni et al. 1998; Wächter et al. 1999). The vigorous
ethylene synthesis in galls is enhanced by high levels of auxin and cytokinin
(Wächter et al. 1999, 2003). Furthermore, this ethylene emission induces the
synthesis of considerable concentrations of abscisic acid in the tumor and host
leaves; as a consequence, transpiration in the leaves slows down to 10% of that of
uninfected plants.
(Terakora et al 2006) Agrobacterium tumefaciens and A. vitis strains that harbor Ti
plasmids induce crown gall tumors upon infection of dicotyledonous plants. TDNAs from most Ti plasmids contain the three well-characterized genes ipt (tmr),
Agrobacterium tumefaciens 11
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
iaaM (tms1) and iaaH (tms2), which are involved in biosynthesis of cytokinin and
auxins, respectively, and responsible for the formation of the crown gall tumors.
This region also encodes a gene called 6b, which exhibits an oncogenic effect on
certain plant species (Hooykaas et al. 1988, Tinland et al. 1989). The 6b genes
from various Ti plasmids stimulate ipt- and iaaM/iaaH-induced division of cells
(Tinland et al. 1989, Wabiko and Minemura 1996) and induce the formation of
shooty calli when discs from leaves that express the 6b gene from pTiAKE10
(AK-6b) are incubated in the absence of exogenous phytohormones in the culture
medium (Wabiko and Minemura 1996). Therefore, the AK-6b gene appears to
play a role in the proliferation of plant cells, which might be related to the action
of the plant growth regulators auxin and cytokinin (Kitakura et al. 2002).
It has been reported that transgenic tobacco plants that express 6b genes from
various sources show abnormal leaf morphology. Transgenic plants of Nicotiana
rustica in which the T-6b gene (from pTiTm4) is driven by the heat-shock
promoter generate tubular leaves upon heat shock treatment (Tinland et al. 1992),
and the transgenic tobacco plants that express AK-6b develop small leaf-like
structures from veins of the abaxial leaf surface, some of which are extremely
asymmetric along the midvein (Wabiko and Minemura 1996).
Recently, Helfer et al. (2003) reported that AB-6b (from pTiAB4) transgenic
tobacco plants formed extra cell layers in the abaxial side of leaves and displayed
alterations in flower morphology, and that AB-6b transgenic Arabidopsis plants
generated radial symmetrical tubes on the abaxial side of the leaves. Northern blot
analysis of cell cycle genes in AB-6b-transformed leaves, however, showed no
significant difference in levels of transcripts of these genes compared with those in
untransformed leaves (Helfer et al. 2003). However, the relationship between
severity of phenotypes generated by the 6b gene and levels of transcripts of genes
related to cell division and organ development has yet to be extensively
investigated.
It also remains to be examined how the phenotypes are directly related to cell
division. In addition, expression of AK-6b affects levels of accumulation of
various metabolites including phenolics in plants (Galis et al. 2004, Kakiuchi et al.
2006),though the genetic basis for such effects is unclear. The 6b protein has been
shown to be localized to plant nuclei and associated with a nuclear protein of
tobacco named NtSIP1 (Kitakura et al. 2002). NtSIP1 has an amino acid sequence
Agrobacterium tumefaciens 12
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
that is similar to a tri-helix motif, which is known to be a DNA-binding sequence
in the rice transcription factor GT-2 (Dehesh et al. 1992), and promotes nuclear
localization of the 6b protein. A chimeric 6b protein that is fused to the DNAbinding domain of yeast GAL4 protein activates transcription of a reporter gene in
tobacco cells. We have recently identified other binding proteins, which were also
predicted to be nuclear proteins (our unpublished data). Based on these
observations, it is proposed that 6b might function as a transcriptional co-activator/mediator by interacting with NtSIP1 (Kitakura et al.2002). However, it has
not been examined whether nuclear localization of 6b protein is essential for the
generation of 6b-related phenotypes. Recently, it has been reported the T-6b protein moves through leaf cells (Grémillon et al. 2004).
(Goodner et al 2001) Genes involved in plant transformation and tumorigenesis
are located on all four genetic elements. The circular chromosome harbors the
well-studied chvAB genes required for synthesis of the extracellular β-1,2-glucan
involved in binding to plant cells; the chvGI, chvE, and ros genes involved in
regulation of Ti plasmid vir genes; and the chvD ,chvH,and acvB genes. The linear
chromosome harbors the exoC(pgm) gene required for synthesis of the
extracellular β-1,2-glucan and succinoglucan polysaccharides, and the cellulose
synthesis (cel) genes involved in binding to plant cells. Plasmid pAtC58 contains
the attachment(att) genes involved initial specific attachment of bacterium to plant
cells ,as well as a second, partial att locus. pAtC58 is reportedly dispensable for
virulence, raising the question of whether there is a virulence requirement for att.
Ti plasmid fall into several opine groups, and the three plasmid sequences now
available permit detailed analysis of their relationships. The order of genes on the
nopaline-agrocinopine-type plasmids pTiC58 and pTi-SAKURA are virtually
identical. Major exceptions include one large insertion on pTiC58 and four smaller
insertions on pTi-SAKURA. In contrast, the consensus octopine-type plasmid
shares only five major gene clusters with the nopaline-type plasmids. Many
regional differences among these plasmids can be circumstantially linked to
mobile DNA elements. Most of the pTiC58-specific genes are involved in
metabolism and transport and probably allow the bacterium to scavenge additional
nutrients. However, for a few genes unique to a given Ti plasmid, there is direct or
circumstantial evidence that they play a role in tumorigenesis on particular hosts.
Agrobacterium tumefaciens 13
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
Both pTiC58 and pTi-SAKURA encode a probable NUDIX hydrolase, which may
degrade altered nucleotides or other toxic compounds present in the plant wound
environment.
(Kawagoshi et al 2005) A. radiobacter biovar 3 strain F2/5 is the most studied
agent for biological control of grape crown gall and is effective against A.
tumefaciens biovar 3 strains (Burr and Reid 1993). However, biological control by
F2/5 is specific to grapevine; F2/5 is not effective on nongrapevine host plants
such as tomato, sunflower, or Kalanchoe daigremontiana. Other strains of A.
radiobacter biovar 3 should be tested for their ability to inhibit gall formation on
stems of tomato seedlings because other strains that inhibit galls on grapevine has
also been found to inhibit galls on tomato. Antibiotic production by strains
identified as A.radiobacter biovar 3 was indicated by an inhibitory zone in the
bacterial lawn around the test strain (Table 6, Fig. 3). This inhibitory substance
can be considered a type of bacteriocin because it was produced by biovar 3
strains of A.radiobacter and inhibited A. tumefaciens biovar 3. VAR03-1 and
VAR03-21 caused an inhibitory zone against all tested indicator strains of A.
tumefaciens biovar 3.
As far as we know, there is no effective method for controlling crown gall of
grapevine in the field anywhere in the world.
(Aloni et al 2005) discussed some ideas were experimentally confirmed by
showing that tumor-induced ethylene is a limiting and controlling factor of crown
gall morphogenesis; very high ethylene levels are produced continuously by
growing crown galls during several weeks curve-1; up to 140 times more ethylene
than in wounded, but not infected control stems, reaching a maximum at five
weeks after infection (Aloni et al. 1998; Wächter et al. 1999). The vigorous
ethylene synthesis in galls is enhanced by high levels of auxin and cytokinin
(Wächter et al. 1999, 2003). Furthermore, this ethylene emission induces the
synthesis of considerable concentrations of abscisic acid in the tumor and host
leaves; as a consequence, transpiration in the leaves slows down to 10% of that of
uninfected plants (Veselov et al. 2003).
Agrobacterium tumefaciens 14
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
(Reader et al 2005) Infection of plants by pathogenic strains of Agrobacterium
tumefaciens causes crown gall tumors with devastating economic consequences.
The most successful bacterial biocontrol agent, nonpathogenic A. radiobacter
strain K84, prevents disease by production of the "Trojan horse" toxin agrocin 84.
Biocontrol of crown gall tumors by agrocin 84 thus targets a tRNA synthetase in
the pathogen.
(Goff et al 1985) The growth of crown-gall cells cultured in vitro (Nicotiana
tabacum L. cv. White Burley and Parthenocissus tricuspidata cv. Veitchii) is
inhibited by alstonine (BG-8), a plant alkaloid, the anti-cancer effect of which has
previously been demonstrated on animals and plants. The growth of normal cells is
only slightly affected. The inhibitory effect of BG-8 on crown-gall cells is
antagonized by indole-3-acetic acid (IAA) added to the culture medium. Kinetin
associated with IAA does not prevent this inhibitory effect. BG-8 present in the
culture medium containing the two types of hormones seems to modify the later
hormonal requirement of Parthenocissus crown-gall tissues.
BG-8 exhibits high binding affinity for crown-gall DNA and, therefore, strongly
inhibits its in vitro synthesis. The alkaloid has practically no effect on DNA from
healthy cells. The inhibition by BG-8 is dependent on the level of DNA strand
separation and on the origin and nature of the tissues (crown-gall DNA is more
destabilized than healthy DNA; DNA from habituated tissues is intermediate).
IAA and kinetin have opposite effects on the in vitro strand separation of the
DNAs from crown-gall cells and, consequently, antagonistic effects on DNA
replication (IAA stimulates and kinetin inhibits). It is possible to establish a close
relationship between in situ development of crown-gall tissues of the two species
studied (in the presence or absence of BG-8 or cell-growth factors), in vitro DNA
synthesis and DNA strand separation.
Agrobacterium tumefaciens 15
BACTERIAL GALLS AND CANKERS
GALLS – CROWN GALL
(Hodgson et al 1951) In addition to its role as an essential nutrient for
microorganism, tryptophan, under certain conditions, also may be inhibitor.
Agrobacterium tumefaciens 16