Table_1_A biotroph sets the stage for a necrotroph to play: ‘Candidatus Phytoplasma solani’ infection of sugar beet facilitated Macrophomina phaseolina root rot.DOCX

‘Candidatus Phytoplasma solani’ (stolbur phytoplasma) is associated with rubbery taproot disease (RTD) of sugar beet (Beta vulgaris L.), while Macrophomina phaseolina is considered the most important root rot pathogen of this plant in Serbia. The high prevalence of M. phaseolina root rot reported on sugar beet in Serbia, unmatched elsewhere in the world, coupled with the notorious tendency of RTD-affected sugar beet to rot, has prompted research into the relationship between the two diseases. This study investigates the correlation between the occurrence of sugar beet RTD and the presence of root rot fungal pathogens in a semi-field ‘Ca. P. solani’ transmission experiment with the cixiid vector Reptalus quinquecostatus (Dufour), in addition to naturally infected sugar beet in the open field. Our results showed that: (i) Reptalus quinquecostatus transmitted ‘Ca. P. solani’ to sugar beet which induced typical RTD root symptoms; (ii) Macrophomina phaseolina root rot was exclusively present in ‘Ca. P. solani’-infected sugar beet in both the semi-field experiment and naturally infected sugar beet; and that (iii) even under environmental conditions favorable to the pathogen, M. phaseolina did not infect sugar beet, unless the plants had been previously infected with phytoplasma.</p

COG categories assigned to expressed gene products identified by RNA-Seq and/or proteome analysis (orange) versus the deduced protein content of '<i>Ca</i>. P. mali' strain AT (green).

<p>Values indicate total number reached in each category.</p

Primer pairs used in (q)RT-PCR reactions.

<p>Primer pairs used in (q)RT-PCR reactions.</p

Phylogenetic tree constructed by parsimony analyses of the deduced peptide sequences of acetate kinase of available <i>Mollicutes</i> in HAMAP database, <i>Spiroplasma citri</i>, <i>Clostridium botulinum</i>, <i>Erysipelothrix rhusiopathiae</i>, <i>Bacillus subtilis</i> subsp.

<p><i>subtilis</i>, <i>Lactobacillus plantarum</i><b>, </b><i>Enterococcus faecalis</i><b>, </b><i>Streptococcus pneumonia</i><b>, </b><b>and </b><i>Lactococcus lactis</i><b> subsp. </b><i>lactis</i><b> employing </b><i>Escherichia coli</i><b> and </b><i>Salmonella typhimurium</i><b> as outgroup.</b> One of the two most parsimonious trees is shown. Accession numbers are given in parentheses. Numbers on the branches are bootstrap values obtained for 1,000 replicates (only values above 60% are shown). The tree is drawn to scale, with branch lengths calculated using the average pathway method, and are in the units of the number of changes over the whole sequence. The scale bar represents 50 amino acid substitutions.</p

Experimental hosts and genes used in qRT-PCR experiments with their correlating average (Ø) C_T and ΔC_T values after normalization (short amplicons).

<p>Experimental hosts and genes used in qRT-PCR experiments with their correlating average (Ø) C_T and ΔC_T values after normalization (short amplicons).</p

Phylogenetic tree constructed by parsimony analyses of deduced malate dehydrogenase peptide sequences of available phytoplasmas, <i>Clostridium botulinum</i> strains ATCC3502 and ATCC19397, <i>Bacillus subtilis</i> subsp. <i>subtilis</i>, and <i>Bacillus cereus</i> employing <i>Escherichia coli</i> strain K12 as outgroup.

<p>Accession numbers are given in parentheses. Numbers on the branches are bootstrap values obtained for 1,000 replicates (only values above 60% are shown). The tree is drawn to scale, with branch lengths calculated using the average pathway method, and are in the units of the number of changes over the whole sequence. The scale bar represents 20 amino acid substitutions.</p

Genome circle of ‘<i>Ca</i>. P. mali’ strain AT highlights gene expression.

<p>Circular patterns (from outside to inside): 1 (outer circle), scale in base pairs of the chromosome; 2 (black), predicted protein coding sequences; 3, tRNAs (grey) & rRNAs (green); 4 (dark blue), identified proteins of ‘<i>Ca</i>. P. mali’; 5 (red), expressed genes of ‘<i>Ca</i>. P. mali’ identified by RNA-Seq; 6 (dark green), expressed genes (proteome and transcriptome) without similarity to NRPROT entries excluding the <i>Acholeplasmataceae</i> entries; 7 (magenta), assigned proteins of the mulberry dwarf phytoplasma; and 8 (olive and pink), G + C skew. Expressed genes located in the terminal ends (identical in sequence) were marked twice.</p

Experimental hosts and genes used in qRT-PCR experiments with their correlating average (Ø) C_T and ΔC_T values after normalization (long amplicons).

<p>Experimental hosts and genes used in qRT-PCR experiments with their correlating average (Ø) C_T and ΔC_T values after normalization (long amplicons).</p

RT-PCR confirming the expression of <i>pgi</i>, <i>pfkA</i>, <i>fba</i>, <i>tpiA</i>, <i>pduL</i>, <i>ackA</i>, <i>degV</i> and SAP11-like gene.

<p>RNA was obtained from <i>Nicotiana occidentalis</i>, <i>Malus domestica</i>, <i>Catharanthus roseus</i> infected by ‘<i>Ca</i>. P. mali’ strain AT. The RT-PCR products were separated on a 1.4% TAE agarose gel. Lane number nine was used for negative control applying water as template (example SAP11-like gene). The product size of around 200 bp was estimated by the 50 bp DNA ladder (Lifetechnologies) loaded on first and last lane of each gel.</p