Isolation and structural characterization of a non-diketopiperazine phytotoxin from a potato pathogenic Streptomyces strain

Abstract Two Streptomyces spp. strains responsible for potato common scab infections in Uruguay which do not produce diketopiperazines were identified through whole-genome sequencing, and the virulence factor produced by one of them was isolated and characterized. Phylogenetic analysis showed that both pathogenic strains can be identified as S. niveiscabiei, and the structure of the phytotoxin was elucidated as that of the polyketide desmethylmensacarcin using MS and NMR methods. The metabolite is produced in yields of ∼200 mg/L of culture media, induces deep necrotic lesions on potato tubers, stuns root and shoot growth in radish seedlings, and is comparatively more aggressive than thaxtomin A. This is the first time that desmethylmensacarcin, a member of a class of compounds known for their antitumor and antibiotic activity, is associated with phytotoxicity. More importantly, it represents the discovery of a new virulence factor related to potato common scab, an economically-important disease affecting potato production worldwide. Graphical Abstract


Introduction
Streptomyces species are gram-positive, filamentous, saprophytic bacteria known for their capacity to produce useful and biologically-important secondary metabolites. Although several hundred species are known to date, only a few cause plant diseases (Loria et al. 2006). Potato common scab, characterized by the presence of necrotic lesions with a corky texture on the surface of Solanum tuberosum tubers (Loria et al. 1997), is the most economically-important plant disease caused by Streptomyces spp. The best-studied pathogens responsible for common scab are S. scabiei (Lambert and Loria 1989a), S. acidiscabies (Lambert and Loria 1989b), S. turgidiscabies (Miyajima et al. 1998), and S. europaeiscabiei (Bouchek-Mechiche et al. 2000). Other pathogenic Streptomyces strains that cause the disease include S. stelliscabiei, S. reticuliscabiei (Bouchek-Mechiche et al. 2000), S. luridiscabiei, S. puniscabiei, and S. niveiscabiei (Park et al. 2003). Recently, some pathogenic strains of S. bottropensis were also reported (Leiminger et al. 2012;Wanner 2009;Zhou et al. 2017). Plant pathogenicity in Streptomyces is normally associated with the presence of thaxtomins (King and Calhoun 2009), a group of 2,5-diketopiperazine phytotoxic secondary metabolites which are essential for the induction of the characteristic symptoms of common scab on potato tubers (King et al. 1991). The non-ribosomal peptide synthase modules txtA and txtB are among the genes responsible for the biosynthesis of the thaxtomins. They can be part of a mobile pathogenicity island (PAI) that includes nec1 and tomA (Kers et al. 2005;Huguet-Tapia et al. 2014), two genes that encode for a necrogenic protein and a homologous of tomatinase, respectively (Seipke and Loria 2008). Pathogenic Streptomyces strains that do not produce thaxtomins have also been reported (Cao et al. 2012;Fyans et al. 2016;Natsume et al. 2005), and some virulent strains lack the txtA and txtB genes required for the biosynthesis of these diketopiperazines (Jordaan and van der Waals 2016;Lapaz et al. 2017;P ankov a et al. 2012;Wanner 2004). Indeed, other phytotoxic metabolites that may contribute to common scab symptoms have been described (Cao et al. 2012;Natsume et al. 2005Natsume et al. , 1996. In a previous study we described the isolation and characterization of a collection of 70 pathogenic Streptomyces strains from necrotic lesions on potato tubers and soil samples in Uruguay (Lapaz et al. 2017). Our search for the txtA, txtB, nec1, and tomA genes revealed that 17% of the isolates did not contain any of them. In this report we present results from whole-genome sequencing of representative strains from the collection, as well as the isolation and structural characterization of a non-diketopiperazine metabolite responsible for the pathogenicity on potato of isolates lacking all four genes from the PAI. As shown below, these pathogenic strains can be identified as S. niveiscabiei and produce desmethylmensacarcin (1, Figure 2), a polyketide that causes deep necrotic lesions on potato tubers and which is comparatively more aggressive than thaxtomin A.

Results and discussion
Our search for novel pathogenicity factors in Streptomyces spp. causing potato common scab in Uruguay involved a group of five representative strains from a collection of 70 isolates (Lapaz et al. 2017). In addition to the unidentified pathogenic strains ST1015 and ST1020, in which the genes of the aforementioned PAI were not detected and were thus the main interest of the study, three strains which had the nec1, tomA, txtA, and txtB genes and were identified as S. scabiei (ST129), S. europaeiscabiei (ST1229), and S. acidiscabies (ST105) were included. Initial efforts involved the identification of the strains lacking the PAI genes, and for that purpose a phylogenetic tree incorporating the strains under scrutiny together with 40 pathogenic and non-pathogenic Streptomyces strains was constructed. As shown in Figure 1, strains ST105, ST129, and ST229 clustered with those of their respective species. On the other hand, strains ST1015 and ST1020 clustered with S. niveiscabiei, a species responsible for potato common scab which was originally isolated and characterized in Korea (Park et al. 2003). To our knowledge, this is the first report of this pathogenic Streptomyces specie in Uruguay.
The next step involved the isolation and characterization of the phytotoxins produced by S. niveiscabiei strains ST1015 and ST1020. The culture supernatants of ST1015, the most aggressive of the two strains, induced deep necrosis in potato tuber slices and inhibited root and shoot growth in radish seedlings ( Figure S1), confirming the existence of these phytotoxic compounds. The supernatants of all strains in the group were then analyzed by HPLC following reported protocols (Johnson et al., 2007). As expected, the three strains that bear the txtA and txtB genes, as well as the S. acidiscabies reference strain, produced thaxtomin A, whereas the diketopiperazine was not detected in strains ST1020 nor ST1015 ( Figure S2). However, a preparative TLC performed on lyophilized culture supernatants of strain ST1015 allowed us to isolate a metabolite in relatively good purity and yields of $200 mg/L of culture media. The compound was unrelated to thaxtomin A, and its activity on the potato tuber and radish seedlings phytotoxicity assays was comparable to that of the original supernatants.
The phytotoxin presented protonated and sodiated molecular ion peaks at m/z 407. 1324 Figure S5). In addition, and in agreement with the molecular formula deduced from the HR-ESIMS data and the ionization pattern observed in the ESIMS 2 spectrum, the remaining 1 H and 13 C signals are at relatively low fields and hint to a highly oxygenated carbon skeleton. The absorption at 3447 cm À1 in the IR spectrum also suggests the presence of hydroxyl groups. Indeed, the 1 H resonances at d H 4. 86, 4.68, 3.75, and 3.64 have no correlations to 13 C atoms in the HSQC spectrum and disappear when the experiments are recorded in CD 3 OD (Table S2, Figure S7). This reveals four alcohol functionalities, and analysis of the COSY and HMBC spectra shows that one of them is quaternary and the remaining three are tertiary. The 13 C resonances at d C 208.2 and 192.8 and IR bands at 1723 and 1688 cm À1 are consistent with aliphatic and aromatic ketones, respectively. The other four unsaturations are due exclusively to rings, and based on chemical shift and coupling constant analysis two of these correspond to oxiranes. Homo and heteronuclear correlations indicate that one of these forms a bridged ring system with 13 C signals at d C 65.3 and 63.4, and the other one is part of a butenonyl oxide fragment with 13 C resonances at d C 58.0, 56.8 and 17.8, and their corresponding 1 H signals at d H 3.96, 3.10 and 1.49. Further examination of the data, and in particular of the HMBC spectrum, confirmed the assignments summarized above and allowed us to elucidate the structure of the phytotoxin as that of desmethylmensacarcin (1, Figure 2). This was confirmed by comparison with optical rotation, IR, and NMR data reported for this polyketide by Arnold (2002), which, together with mensacarcin (2, Figure 2), was isolated by this author from a S. bottropensis strain found in a soil sample in Germany.
Once identified, the phytotoxicity of polyketide 1 was compared to that of thaxtomin A in a potato tuber slice assay. As shown in Figure S3, both compounds are phytotoxic in the 0.1-10.0 mg/mL concentration range. However, it is clear that desmethylmensacarcin is more aggressive and produces deeper necrosis in the tuber tissue compared to the superficial lesions caused by the 2,5-diketopiperazine, making this compound a likely virulence factor of strain ST1015. The biological activity of this type of polyketides has been extensively studied. Indeed, the antitumor activity and mode of action of its methylated derivative (2) is well documented (Arnold 2002;Maier et al. 2014;Plitzko et al. 2017). Similarly, there are a number of reports regarding the antitumor and antibacterial activity of cervicarcin (3), a desmethylated analog of compound 1 produced by S. ogaensis (Ohkuma et al. 1962;Marumo et al. 1964). However, this is the first time that a compound from this class is associated with phytotoxic activity. It is also worth noting that desmethylmensacarcin was originally isolated from S. bottropensis (Arnold 2002), and that some strains of this species are associated with potato common scab. While all the reported S. bottropensis pathogenic strains carry the txtA and txtB genes, thaxtomin production was not confirmed for any of them (Leiminger et al. 2012;Wanner 2009;Zhou et al. 2017). Therefore, the phytotoxic activity on potato of these pathogenic strains could be due to thaxtomins, polyketides such as 1, or a combination of the two classes of metabolites.

Conclusions
In summary, we described the identification of a Streptomyces niveiscabiei strain responsible for potato common scab infections in Uruguay, and showed that the phytotoxin associated with its pathogenicity is the polyketide desmethylemensacarcin. This is a significant finding, as it represents the discovery of a new virulence factor related to an economically-important disease that affects potato production worldwide. It is also the first time that a compound of this class, which are known for their antitumor and antibiotic activity, is associated with phytotoxicity.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Funding from the Comisi on Sectorial de Investigaci on Cient ıfica (awards IIM2 81/2015 and I þ D 166/2016), the Comisi on Acad emica de Posgrado, and the Programa de Desarrollo de las Ciencias B asicas is acknowledged. We also wish to thank Marcos Colazzo for recording the ESIMS 2 spectra, and Horacio Heinzen and Guillermo Valdomir for their assistance in procuring the HRESIMS data.