Abstract:

Banana bacterial wilt caused by Xanthomonas campestris pv. musacearum has caused significant
decline in livelihoods dependant on the banana/musa enterprise in the great lakes region of Eastern
Africa. First line control methods using cultural packages are not sustainable for long term
application and yet resistant material is yet to be availed to the producers. The pathogen is also
largely unknown and thus the need to understand host pathogen interaction at the molecular level.
Recently, affordable genome sequencing has eased the application of genomic, post-genomic, and
functional genomic approaches to problems of direct relevance to parasitic diseases in plants,
humans and animals. Comparative genomic analysis of xanthomonas species pathogenic on musa
was conducted as a means of unraffling the virulence factors associated with these strains. It refers
to discovering how have Xanthomonas spp. are adapted for host- and tissue-specific pathogenesis,
understanding bacterial plant pathogenesis and host- driven pathogen adaptation and potential to
develop practical applications for crop protection through better disease control and prevention.
Next generation sequencing of Xanthomonas campestris pv. musacearum (Xcm), Xanthomonas
vasicola vasculorum (Xvv) and other musa associated xanthomonas was used to discover
evolutionary relationships and composition of genetic factors in the strains. First the research
sought to identify potential molecular markers of Xcm which information can be used in proposing
a set of genetic material (primers) that are useful for diagnosis between banana infecting Xcm
strains. Previous studies in other bacteria in this genus suggest that advanced draft genome
sequences are valuable resources for molecular studies on their interaction with plants and could
provide valuable tools for diagnostics and detection. Molecular markers are widely used in plant
genetic research and breeding. Single-Nucleotide Polymorphisms (SNPs) are currently the marker
of choice due to their large numbers in virtually all populations of individuals.
Secondly this study demonstrated the use genome-wide sequencing to systematically search
genetic differences among Xvv and Xcm isolates. Comparative genome sequencing of bacteria is
a powerful means of detecting sequence diversity among closely related, but distinct populations.
Comparative whole-genome information about strain specific DNA variation has important
implications for the development of new molecular markers for detection, pathovar classification,
disease epidemiology and understanding evolutionary relationships. In nature, pathogenpopulations with high genetic diversity have high evolutionary potential and thus are more likely
to overcome host genetic resistance than pathogen populations with low genetic diversity. The
resulting changes in population structure or virulence can lead to resistance breakdown. This is
particularly true in agricultural production systems in which mono-culture is a common practice
such as banana, maize and sugar cane production. Under these conditions, the frequency of
pathogen genotypes with increased virulence may increase and ultimately lead to resistance
breakdown and increased disease incidence. Therefore, availability of such genomic information
on coding and noncoding- polymorphic loci will help in linking variability in pathogenicity of
different strains to differences in their genetic backgrounds and monitoring changes in their genetic
diversity. This study was undertaken to identify and characterize the genomes of six Xvv and two
Xcm strains which identified variations are directly applicable for the development of molecular
tools for pathogen detection and strain characterization, in understanding disease epidemiology
and pathogen biology, and the development of novel disease management strategies.
Restriction endonuclease analysis (REA) and Restriction fragment length polymorphism (RFLP)
analysis of genomic DNA are approaches to classify and differentiate strains. Some studies have
developed fast molecular-based genus specific PCR diagnostic kits that are also independent of
bacterial isolation and cultivation. Internal controls however are required by multiplexing with
primers targeting specific genes which can further differentiate strains. Therefore genotypic
characterization based on the analysis of restriction fragment length polymorphism of virulence
factor fragment PCR products was conducted on selected members of the Xvm and Xvv from
varying geographical locations. This study showed that indeed there can be differences in the
effectors across bacterial strains and within strains of the same species and other clusters conserved
in gram-negative bacteria. This variation indicates inherent capacity for host specialization using
inbuilt resistance “R”genes of musa genomes, a response that can be explored to further understand
the interaction of Xvm on susceptible and resistant hosts.
Finally it sought to genetically understand the interaction of selected banana-associated
xanthomonas bacteria that are non-hypersensitive response and pathogenicity (Hrp). Xanthomonas
albilineans is the causal agent of leaf scald, a major disease of sugarcane (Saccharum spp.).
Xanthomonas albilineans closely related to Xanthomonas species isolated from banana in Samoa and Xanthomonas sacchari belong to hyacinthi group together with X. theicola, X. hyacinthi and
X. translucens. Comparative genomic analyses showed that most of the known pathogenicity
factors from other Xanthomonas species are conserved in X. albilineans, with the notable absence
of two major determinants of the “artillery” of other plant pathogenic species of Xanthomonas: the
xanthan gum biosynthesis gene cluster, and the type III secretion system Hrp (hypersensitive
response and pathogenicity). Six major clusters of xanthomanas based on Intergenic spacer
sequences (ITS) as the phylogenetic tree with cluster I comprising X. vasicola and X. campestris
whereas X. albilineans and X. sacchari are in Cluster II-VI. These are known to use a different
infection mechanism from the Type Three Secretion System (TTSS).