*3.4. Pot Culture Experiments*

In the pot culture experiments, NEAU-HV9 and actinomycin D effectively suppressed the development of bacterial wilt caused by *R. solanacearum* (Table 4, Figure S10). The negative control treatment had 73.9% relative disease incidence. For strain NEAU-HV9 and actinomycin D, the control efficacies of the formulations were 82% and 100%, respectively.

**Table 4.** Effect of the strain NEAU-HV9 and actinomycin D on the incidence and control of tomato bacterial wilt in pot culture experiments.


Wilt incidence (WI) was calculated as the percentage of leaves that were completely wilted. Control efficacy was calculated using the following formula: control efficacy (%) = 100 × (WI of control − WI of treatment)/WI of control. Data shown are the mean of three replications. Means within the same column followed by the same letter are not significantly different (*p* = 0.05) according to Fisher's least significant difference test.

#### **4. Discussion**

Soil-borne diseases have caused a significant decline in yield in the monoculture tomato field [3]. *Ralstonia solanacearum* is an important soil-borne bacterial plant pathogen which is distributed all over the world [5]. Recently, the biological control of soil-borne diseases has attracted more attention due to its environmental friendliness and high efficiency [54]. Therefore, isolation, screening and application of highly efficient antagonistic microorganisms is a key factor in biological control. With this outlook, a *Streptomyces* sp. strain NEAU-HV9 was isolated and found to exhibit antibacterial activities against *R. solanacearum* in the present study. By using 16S rRNA gene sequence analysis, combined with morphological, cultural and physiological characteristics, the results showed that strain NEAU-HV9 belongs to members of the genus *Streptomyces* and was most closely related to *Streptomyces panaciradicis* 1MR-8T and *Streptomyces sasae* JR-39<sup>T</sup> with gene sequence similarities of 98.90% and 98.89%, respectively.

Actinobacteria, particularly *Streptomyces*, are ubiquitous in the rhizosphere soil and can protect plant from pathogenic fungi/bacteria [55], so they have always been used in agriculture [56]. For instance, several *Streptomyces* species such as strains CAI-24, CAI-121, CAI-127, KAI-32 and KAI-90 have been used as BCAs against *Fusarium* wilt in chickpea plants [57]. The *Streptomyces* sp. CB-75, selected from banana rhizosphere soil, showed antifungal activity against 11 plant pathogenic fungi [54]. In this study, the *Streptomyces* sp. NEAU-HV9 exhibited strong antagonistic activity against *R. solanacearum*. According to the study of Singh et al., susceptibility of early stages of tomato seedlings toward the pathogen was confirmed by root-inoculation of *R. solanacearum* in early stages of tomato seedlings [22]. The antagonistic strains should reach a certain amount to demonstrate a significant biocontrol effect [58,59]. In this study, we inoculated very high numbers of *R. solanacearum* and very high levels of *Streptomyces* sp. NEAU-HV9 (10<sup>9</sup> cfu mL−1) in small tubes in the TR 1 group. After culturing for seven days, all tomato seedlings were as healthy as the CK 1 control group (Table 3). There are only *R. solanacearum* and *Streptomyces* sp. NEAU-HV9 in this artificial system, which can better prove that a single NEAU-HV9 was able to be effective against *R. solanacearum*. On the seventh day, more than 90% of seedlings inoculated with *R. solanacearum* were found to be killed, but water-inoculated control seedlings were not wilted/dried [23]. Freshly grown tomato seedlings are too small to carry out detailed disease assessment, and can only be described as healthy, healthy wilted or dried. In the tests of this study, tomato seedlings inoculated with suspension (107 or 10<sup>8</sup> cfu mL<sup>−</sup>1) of NEAU-HV9 and *R. solanacearum* showed healthy wilted and dried disease phenotypes at different levels, while all tomato seedlings inoculated with suspension (10<sup>9</sup> cfu mL−1) of NEAU-HV9 and *R. solanacearum* were healthy (Figure 4). The results indicated that there are only *R. solanacearum* and *Streptomyces* sp. NEAU-HV9 in this artificial system, which can better prove that a single NEAU-HV9 was able to be effective against *R. solanacearum*. In addition, strain NEAU-HV9 effectively controlled *R. solanacearum* on larger plants in pot culture experiments (Table 4). Thus, the test presented in this study is viable for a preliminary screening of antagonistic actinobacterial strains against *R. solanacearum* and has important aspects with respect to reduced time, space consumption and economics. Meanwhile, the results showed the possibility of using *Streptomyces* sp. NEAU-HV9 as bioinoculant for *R. solanacearum*.

A wide range of bioactive secondary metabolites with anti-inflammatory, antibacterial, antifungal, antialgal, antimalarial and anticancer activities were produced by actinomycetes. Actinomycetes have produced about two-thirds of available antibiotics that have great practical value [60,61]. For example, *Streptomyces* TP-A0595 produced an antagonist that was determined as 6-prenylindole and effective against *Alternaria brassicicola* by inhibiting the formation of infection hyphae [62]. *Streptomyces griseus* H7602 produced a monomer compound that has suppressive effect on infection by *Phytophthora capsici* [63]. Some well-known antibiotics have been isolated from *Streptomyces* and used as fungicides. Many types of antibiotics with high antibacterial activity were produced from *Streptomyces spectabilis*, including streptovaricin [64], desertomycin [65] and spectinomycin [66], and they have high application value in the pharmaceutical industry [67]. Actinomycin D (or Dactinomycin) is a proverbial antitumor-antibiotic drug, which belongs to the actinomycin family and was isolated from *Streptomyces*. Actinomycin D has been demonstrated to have various biological activities. Gram-negative bacteria were largely inhibited by using 10–100 mg per liter concentrations [68]. Actinomycin D produced by the bacterium *Streptomyces hydrogenans* IB310 was effective against both bacterial and fungal phytopathogens [51], and the authors proposed that actinomycin D might be developed as an antibacterial agent used in agriculture. However, there are no reports on antibacterial activities against *R. solanacearum* and it is not currently used in agriculture. In this study, *Streptomyces* sp. NEAU-HV9, which showed strong antibacterial activity against *R. solanacearum*, was isolated and identified. To learn more about the chemical nature of the antibacterial activity of the culture filtrate, the active compound actinomycin D was finally obtained. In this paper, we tested the in vitro antibacterial activity of actinomycin D against *R. solanacearum* and obtained a MIC value of 0.6 mg L−<sup>1</sup> (0.48 μmol L<sup>−</sup>1), which was many fold lower than other reported new natural antibacterial agents [69], synthesized antibacterial agents and those of commercial fungicides including gentamicin and streptomycin [70]. The antibacterial activity of actinomycin D against *R. solanacearum* tomato seedlings treated with 1 × MIC and 2 × MIC were determined. None of the seedlings inoculated with actinomycin D exhibited disease symptoms and the phytotoxic rating of actinomycin D was similar to that of a water control. Thus, actinomycin D was not phytotoxic at a concentration of 0.6 mg L−<sup>1</sup> (0.48 μmol L<sup>−</sup>1). The results suggest that actinomycin D might be useful as a candidate pesticide for the treatment of *Ralstonia solanacearum* in tomato.
