Attempts to eradicate graft-transmissible infections through somatic embryogenesis in <Emphasis Type="Italic">Citrus</Emphasis> ssp. and analysis of genetic stability of regenerated plants

Ability to detect Pseudocercospora macadamiae infection in macadamia husk at least four months before symptoms become visible will aid the development of disease control measures. This study examined the distinctness of P. macadamiae within the phylogenetic lineages of the genus Pseudocercospora. In addition, we developed two quantitative PCR (qPCR) assays, as rapid diagnostic tools, for early detection and quantification of P. macadamiae in planta. Phylogenetic analysis of concatenated sequences of four gene loci (large subunits, internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and actin of 47 P. macadamiae isolates showed that P. macadamiae is a distinct species in the genus Pseudocercospora. P. macadamiae isolates were partitioned into subunits in the cluster but the grouping of the isolates was regardless of location. Nucleotide diversity (0.02) and the coefficient of genetic differentiation (0.07) were low in the P. macadamiae population. Two qPCR primer sets, based on ITS (PMI) and TEF-1α (PME) were designed that consistently amplified P. macadamiae in fungal cultures (Ct = 16.93 ± 0.11 and Ct = 21.20 ± 0.11, respectively) and in planta (Ct = 32.36 ± 0.28 and Ct = 38.07 ± 1.20, respectively). The PMI primers also detected species in the genus Pseudocercospora, while PME was more specific and robust for quantification of P. macadamiae. Both primer sets detected P. macadamiae in asymptomatic tissue samples and strongly differentiated various stages of disease progression, which revealed approximately 10-fold increase in fungal biomass between each consecutive stage of symptom development.     

Genetic analysis of Tunisian fig (Ficus carica L.) cultivars using amplified fragment length polymorphism (AFLP) markers

Genetic diversity of forty fig cultivars collected from five regions in Tunisia was investigated using amplified fragment length polymorphism (AFLP). A total of 342 reproducible bands amplified with six AFLP primer combinations were obtained. The high percentage of polymorphic bands (%PB) of 97.5 and the resolving power (Rp) collective rate value of 143 were scored. In addition, the polymorphism information content (PIC) values varied from 0.61 to 0.87 with an average of 0.77. Although cluster (UPGMA) and principal components analyses indicate that the cultivars’ clustering made independently both from the geographical origin, horticultural classifications and/or from the sex of trees. In addition, the observed variation suggests considerable differentiation among fig cultivars. The present data supports the common origin of the fig cultivars. Analysis of molecular variance (AMOVA) revealed that average Φ_ST value overall loci was 0.026, and the overall distribution pattern of molecular variation indicated that about 97.43% of the total variance was accounted by the within-region variance component. The remaining 2.5% (P < 0.001) of the variation was founded among cultivars of the prospected regions. Our results proved that AFLP markers are useful for germplasm discrimination as well as for investigation of fig patterns variation. The information may be useful to define conservation management program.