Page 12 - Volume 5, Issue 2, July 2012

© Benaki Phytopathological Institute
Skandalis et al. 40
HrpZ protein fused to a plant signal pep-
tide enhanced “rhizomania” resistance both
in a model plant ( Nicotiana benthamiana)
and sugar beet, the natural host of BNYVV
(Pavli et al., 2011). However, the exact action
site of harpins remains unknown. Tampak-
aki and Panopoulos (2000) suggested that
the harpin receptor could be extracellular
in transgenic tobacco expressing HrpZ of P.
syringae pv. phaseolicola, while other groups
proposed the plasma membrane as the lo-
cation of harpin receptor using immune-cy-
tological experiments (Lee et al., 2001).
Harpin’s potential culminated from basic
and applied research that led to the regis-
tration and placing in the market plant pro-
tection products with various harpins as the
active ingredients, by companies such as
EDEN Bioscience, which launched Messen-
gerTM and ProActTM as biopesticides for yield
enhancement and disease management.
This novel crop management technolo-
gy offers the advantages of being environ-
mentally friendly as it does not leach, bio-
acccumulate, or persist in the environment,
and leaves no detectable residues on treat-
ed crops. Nevertheless, its small share in the
global market indicates that there are limita-
tions to this technology and probably space
for improvement.
Hrp-based Diagnostics
Conventional methods for detection
of bacterial plant pathogens relies on cul-
ture-based methods, biochemical and se-
rological typing (immunofluorescence,
enzyme-linked immunosorbent assay -
ELISA), protein profiling (SDS-PAGE), fat-
ty acid methyl-ester (FAME) profiling and
pathogenicity confirmation testing. How-
ever, detection is increasingly depending
on polymerase chain reaction (PCR) - based
techniques, which mainly focus on amplifi-
cation of the 16S rDNA gene and the 16S-23S
internal transcribed spacer by genera/spe-
cies specific primers, combined occasional-
ly with simple restriction fragment length
polymorphisms (RFLPs) and repetitive-se-
quence-based PCR (REP-PCR) analysis. A list
of internationally agreed diagnostic proto-
cols for regulated pests is published by the
European and Mediterranean Plant Protec-
tion Organization (EPPO, 2012). Other primer
target genes usually include genes involved
in basic cellular functions, as for example the
Rhs gene family (Park et al., 2006; Albuquer-
que et al., 2012) and gyrase B (gyrB) (Weller
et al. , 2007). Moreover, several specific prim-
ers have been designed on genera and spe-
cies specific genes, such as the genes con-
trolling production of bacterial phytotoxins,
such as phaseolotoxin or coronatine for the
detection of the bean halo blight (Schaad et
al. , 1995) and the bacterial speck pathogen
of tomato (Cuppels, 2006). The main draw-
backs so far of PCR-based methods are sen-
sitivity, cross reaction with other bacteria,
and false negatives/positives, usually due
to the DNA extraction method and/or the
plant tissue. Real time, multiplex and com-
petitive PCR protocols have been developed
to overcome such limitations (Pastrik, 2000;
Berg et al., 2006; Chen et al. , 2010).
The use of primers targeting functional
systems that only pathogenic bacteria pos-
sess, such as the T3SS, could offer an alterna-
tive tool to overcome the aforementioned
limitations. In this direction, there have
been reports for the detection of Gram-
negative pathogens by PCR amplification
of hrc genes by means of genus, species or
pathovar specific primers. In their pioneer-
ing work, at a time when full annotations of
the hrp cluster were not available, the Stall
and Bonas group (Leite et al., 1994) had fore-
seen the potential of hrp genes for diagnos-
tic purposes. They have used genomic DNA
of a X. campestris pv. vesicatoria strain and
DNA from plasmids that contained cloned
parts of the hrp cluster to develop oligonu-
cleotide primers specific for different hrp re-
gions, which amplified DNA from X. fragar-
iae and 28 pathovars of X. campestris. More
recently, other groups have also used the
same set of primers for Xanthomonas species
either per se or as a basis for designing new
primer sets against additional Xanthomonas
species and pathovars, based on the load of
available sequences. Indeed, Obradovich et
al. (2004) differentiated the X. campestris pv.