Identification of bldA as a Major Regulator of Virulence in the Sweetpotato Soil Rot Pathogen Streptomyces ipomoeae and Characterization of Distinct Clostridium Sweetpotato Soft Rot Isolates

Abstract

Streptomyces ipomoeae is the causative agent of Streptomyces soil rot on sweetpotato, a disease characterized by extensive necrosis of both adventitious and storage roots. While Streptomyces potato scab pathogens produce a phytotoxin (thaxtomin A), which is induced in the presence of cellobiose and suberin, S. ipomoeae produces a less-modified, phytotoxin (thaxtomin C), whose inducer has not been identified. To investigate transcriptional regulation of thaxtomin C production, we inserted a promoter involved in thaxtomin C synthesis upstream of the gusA reporter in an S. ipomoeae strain. Reporter gene expression was significantly upregulated in the presence of size-fractionated sweetpotato extract (SPE). The result indicated that the inducer molecule(s) of S. ipomoeae thaxtomin gene expression present in SPE is likely to be low MW. We also found that suberin purified from either sweetpotato or potato promoted cell differentiation in S. ipomoeae, which raises the possibility that secondary metabolite production, including thaxtomin C, may also be regulated by suberin in this species. The thaxtomin gene cluster of S. ipomoeae contains an unusually large number of TTA codons.The bldA regulatory gene encodes the only tRNA that recognizes this rare Streptomyces codon. The bldA gene was studied to elucidate the translational regulation of thaxtomin C production and virulence in S. ipomoeae. Our results showed that a S. ipomoeae ΔbldAmutant was avirulent on both sweetpotato adventitious and storage roots, in part because it is likely unable to produce thaxtomin C. Overall, the results suggest that bldA gene may be a global regulator of virulence in S. ipomoeae. Flooding of sweetpotatoes in the field leads to development of soft rot on the storage roots while they remain submerged or on subsequent harvest and storage. Two distinct bacterial strains isolated from soft-rotted sweetpotato storage roots retrieved from an intentionally flooded field were confirmed to cause soft rot disease on sweetpotato and other vegetable crops ina previous study. These strains were further characterized here by several phenotypic and biochemical properties, as well as by phylogenetic analysis. Overall, our results demonstrate that the strains likely represent multiple previously unknown plant-pathogenic Clostridium species that can cause soft rot disease on sweetpotato.