**1. Introduction**

Tomato is one of the world's most important vegetable crops, with a global annual yield of approximately 160 million tons [1,2]. In China, long term continuous cropping is the main planting practice for tomato, which has led to serious soilborne diseases [3]. *Ralstonia solanacearum* [4] is an important soilborne bacterial plant pathogen [5]. Bacterial wilt caused by *R. solanacearum* is a serious and common disease, which reduces the yield of tomato and many other crops in tropical, subtropical, and warm-temperature regions of the world [6]. Because of worldwide distribution and a large host range of more than 200 plant species in 50 families, including pepper, tomato, tobacco, potato, peanut, and banana, this soil bacterium has been recognized as one of the causative agents of bacterial wilt disease and is one of the leading models in pathogenicity [5]. In the absence of host plants, this bacterium can

be free-living as a saprophyte in the soil or in water [7]. Plant breeding, field sanitation, crop rotation, and use of bactericides have met with only limited success for *R. solanacearum* [8]. Furthermore, pathogenic microbial multi-drug resistance is also increasing. Therefore, new natural resources and antibiotics for suppressing this soilborne disease are needed.

Various recent studies have showed that biological control of bacterial wilt disease could be achieved using antagonistic bacteria [8,9]. The suppressive effect of some antagonistic bacteria on *R. solanacearum* was reported by Toyota and Kimura [10]. Moreover, the use of antagonistic bacteria to be effective in control of *R. solanacearum* has been proved by Ciampi-Panno et al. under field conditions [8]. *Streptomycetes* are gaining interest in agriculture as plant growth promoting (PGP) bacteria and/or biological control agents (BCAs) [11,12]. The *Streptomyces* genus comprises Gram-positive bacteria which show a filamentous form; they can grow in various environments. Several *Streptomyces* species such as *S. aureofaciens*, *S. avermitilis*, *S. lividans*, *S. humidus*, *S. hygroscopicus*, *S. lydicus*, *S. plicatus*, *S. olivaceoviridis*, *S. roseoflavus*, *S. scabies* and *S. violaceusniger* have been used to control soilborne diseases due to their greatly antagonistic activities by production of various antimicrobial substances [13–15].

*Actinobacteria* are famous for producing a variety of natural bioactive metabolites. *Streptomyces* is an important source of bioactive compounds among all members of antibiotic production, accounting for two-thirds of commercially available antibiotics [16]. Actinomycins belonging to a family of chromopeptide lactones are produced by various *Streptomyces*. Among several antibiotics produced by this genus, actinomycins are prominent. More than 20 naturally-occurring actinomycins were isolated and observed to have commonality of two pentapeptidolactone moieties with an actnoyl chromophore [17]; however, they differ in functional and/or positional group. Among actinomycins, actinomycin D has been widely studied and used clinically as an anticancer drug, especially in the treatment of childhood rhabdomyosarcoma, infantile kidney tumors and several other malignant tumors [18,19]. However, no reports have been published on actinomycin D against phytopathogen *R. solanacearum*.

In the existing protocol for virulence assays, one-month old tomato plantlets are soil-inoculated with the bacterium and wilting symptoms, if any, are observed and recorded. In usual ground work, tomato seeds are sown to obtain seedlings that take 5–6 days to sprout. Seedlings are then transferred to pots containing soil and grown in a greenhouse for about one month. Following this, plants are shifted to a growth chamber where plants are inoculated with the pathogen by soil drench or the stem inoculation method [20,21]. Using this approach, it usually takes 40 days to perform a single virulence assay. The infection achieved in this way is generally not axenic as the soil conditions used are not devoid of other bacterial communities that can colonize the plant during its growth prior to the infection study. Singh et al. [22] described a simple assay to study the pathogenicity of *R. solanacearum* on freshly grown tomato seedlings instead of fully-grown tomato plants. From seed germination to completion of the infection process, the study takes around 15 to 20 days. Pathogenicity due to *R. solanacearum* was also demonstrated when there is no significant plant growth since no mineral/growth inducing factors have been added into the water [23]. Under this same condition, there are reports of the bacterium's survivability without any growth [24]. The death of tomato seedlings was actually occurring due to the presence of *R. solanacearum* in the water. On the basis of the previous study, we have discussed an approach to study biological assays in tomato seedlings.

In this study, a *Streptomyces* sp., NEAU-HV9, was isolated and showed strong antimicrobial activity against *R. solanacearum*. The taxonomic identity of NEAU-HV9 was determined by a combination of 16S rRNA gene sequence analysis with morphological and physiological characteristics. The potential control of actinomycin D produced by the strain NEAU-HV9 against *R. solanacearum* was also investigated.
