Abstract
Virulent strains of Rhodococcus fascians cause a range of disease symptoms, many of which can be mimicked by application of cytokinin. Both virulent and avirulent strains produce a complex of cytokinins, most of which can be derived from tRNA degradation. To test the three current hypotheses regarding the involvement of cytokinins as virulence determinants, we used PCR to detect specific genes, previously associated with a linear virulence plasmid, including two methyl transferase genes (mt1 and mt2) and fas4 (dimethyl transferase), of multiple strains of R. fascians. We inoculated Pisum sativum (pea) seeds with virulent and avirulent strains of R. fascians, monitored the plants over time and compared these to mock-inoculated controls. We used RT-qPCR to monitor the expression of mt1, mt2, and fas4 in inoculated tissues and LC-MS/MS to obtain a comprehensive picture of the cytokinin complement of inoculated cotyledons, roots and shoots over time. The presence and expression of mt1 and mt2 was associated with those strains of R. fascians classed as virulent, and not those classed as avirulent. Expression of mt1, mt2, and fas4 peaked at 9 days post-inoculation (dpi) in cotyledons and at 15 dpi in shoots and roots developed from seeds inoculated with virulent strain 602. Pea plants inoculated with virulent and avirulent strains of R. fascians both contained cytokinins likely to have been derived from tRNA turnover including the 2-methylthio cytokinins and cis-zeatin-derivatives. Along with the isopentenyladenine-type cytokinins, the levels of these compounds did not correlate with virulence. Only the novel 1- and 2-methylated isopentenyladenine cytokinins were uniquely associated with infection by the virulent strains and are, therefore, the likely causative factors of the disease symptoms.
Highlights
IntroductionRhodococcus fascians (Tilford, 1936; Goodfellow, 1984) is a soil-borne, Gram positive Actinobacterium, some strains of which are reported to be growth-promoting epiphytes (Francis et al, 2016; Savory et al, 2017), whereas other strains have the ability to exist as epiphytes and can invade a wide variety of host plants and exist as endophytes causing fasciation symptoms (Lacey, 1939; Miller et al, 1980; Savory et al, 2017)
TZ, tZR, tZRMP, tZOG, tZROG, iP, iPR, iPRMP were reported in pea cotyledons, shoots and roots inoculated with virulent strain 602 and avirulent strain 589 as well as in mockinoculated controls
Mock controls and avirulent strains clustered randomly between virulent strains. It is well-known that both virulent and avirulent strains of R. fascians secrete a similar array of cytokinins into culture medium (e.g., Scarbrough et al, 1973; Armstrong et al, 1976; Murai et al, 1980; Eason et al, 1996; Pertry et al, 2009)
Summary
Rhodococcus fascians (Tilford, 1936; Goodfellow, 1984) is a soil-borne, Gram positive Actinobacterium, some strains of which are reported to be growth-promoting epiphytes (Francis et al, 2016; Savory et al, 2017), whereas other strains have the ability to exist as epiphytes and can invade a wide variety of host plants and exist as endophytes causing fasciation symptoms (Lacey, 1939; Miller et al, 1980; Savory et al, 2017). Virulent bacteria often feature different life styles and may be asymptomatic leaf surface colonizers, epiphytes, before they invade the apoplast and feature an endophytic lifestyle. This movement toward the endophytic compartment may be coordinated by quorum sensing decisions (Dulla and Lindow, 2008) which may apply to R. fascians (Stes et al, 2013). Fasciation symptoms of sweet peas caused by virulent strains are characterized by a release from apical dominance, the outgrowth of swollen fleshy lateral stems (Lacey, 1936; Tilford, 1936), reduced shoot and leaf growth (Lacey, 1936) and reduced root growth (Oduro and Munnecke, 1975). The symptoms displayed can depend on the method of inoculation (Goethals et al, 2001; Dolzblasz et al, 2018)
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