Trophic preferences of the pathogen Ralstonia solanacearum and consequences on its growth in xylem sap.

Ralstonia solanacearum is one of the most destructive pathogens worldwide. In the last 30 years, the molecular mechanisms at the origin of R. solanacearum pathogenicity have been studied in depth. However, the nutrition status of the pathogen once inside the plant has been poorly investigated. Yet, the pathogen needs substrates to sustain a fast-enough growth, maintain its virulence and subvert the host immunity. This study aimed to explore in-depth the xylem environment where the pathogen is abundant, and its trophic preferences. First, we determined the composition of tomato xylem sap, where fast multiplication of the pathogen occurs. Then, kinetic growth on single and mixtures of carbon sources in relation to this environment was performed to fully quantify growth. Finally, we calculated the concentration of available metabolites in the xylem sap flux to assess how much it can support bacterial growth in planta. Overall, the study underlines the adaptation of R. solanacearum to the xylem environment and the fact that the pathogen assimilates several substrates at the same time in media composed of several carbon sources. It also provides metrics on key physiological parameters governing the growth of this major pathogen, which will be instrumental in the future to better understand its metabolic behavior during infection.

The regulation of BbLaeA on the production of beauvericin and bassiatin in Beauveria bassiana.

Beauvericin and bassiatin are two valuable compounds with various bioactivities biosynthesized by the supposedly same nonribosomal peptide synthetase BbBEAS in entomopathogenic fungus Beauveria bassiana. To evaluate the regulatory effect of global regulator LaeA on their production, we constructed BbLaeA gene deletion and overexpression mutants, respectively. Deletion of BbLaeA resulted in a decrease of the beauvericin titer, while overexpression of BbLaeA increased its production by 1-2.26 times. No bassiatin could be detected in ΔBbLaeA and wild type strain of B. bassiana, but 4.26-5.10 µg/mL bassiatin was produced in OE::BbLaeA. Furthermore, additional metabolites with increased production in OE::BbLaeA were isolated and identified as primary metabolites. Among them, 4-hydroxyphenylacetic acid showed antibacterial bioactivity against Ralstonia solanacearum. These results indicated that BbLaeA positively regulates the production of beauvericin, bassiatin and various bioactive primary metabolites.

A high-throughput virulence screening method for the Ralstonia solanacearum species complex.

Ralstonia solanacearum species complex strains are the causative agents for wilting diseases of many plants, including the economically important brown rot of potato. We developed a high-throughput virulence screen that is implemented in 96-well microtiter plates using seedlings grown in soft water agar to save space, effort, and resources. Nicotiana glutinosa was determined to be the most effective host for this assay, and we confirmed bacterial growth and systemic spread in inoculated seedlings. In our assay, N. glutinosa seeds were sown quickly and easily on top of individual water agar wells of a 96-well plate by pipetting out desired number of seeds in an aqueous suspension. They were inoculated on the same day by first touching a bacterial colony with an autoclaved toothpick and then stabbing the toothpick into the center of the water agar well. Such inoculation method resulted in inocula above a threshold of 2 × 104 CFU per well achieving consistent virulence results and enabling reduction of inoculum preparation efforts to facilitate high-throughput screening. Our assay is suitable for forward genetic screening of a large number of strains, isolates or mutants for disease symptoms under both cool (20 °C) and warm (28 °C) temperature conditions before detailed studies can be narrowed down to a manageable number of desired candidates. Our virulence screen method provides a valuable tool for future work in understanding genetics of virulence of Rssc, especially cool virulence of the highly regulated race 3 biovar 2 group of R. solanacearum, leading toward development of effective control strategies.

Induction of spontaneous phenotype conversion in Ralstonia solanacearum by addition of iron compounds in liquid medium.

Ralstonia solanacearum is a soil-borne pathogen that causes bacterial wilt in plants. The wild-type strain of R. solanacearum undergoes spontaneous phenotype conversion (PC), from a fluidal to non-fluidal colony morphology. PC mutants are non-pathogenic due to reduced virulence factors, and can control wilt diseases as biological control agents. The induction factors of PC in R. solanacearum are currently unclear. Here, we investigated the effect of iron treatment on bacterial growth of wild-type strain and PC mutant, and PC of the wild-type strain in liquid medium. Interestingly, PC was frequently induced in the single cultured wild-type strain by iron treatment; however, PC was not induced in the co-culture. In a co-culture of both strains, the PC mutant showed increased growth compared to the wild-type strain by iron treatment. Furthermore, we investigated the effects of iron treatment on the bacterial growth and PC of the wild-type strain under different culture conditions of medium type (MM broth, BG broth, and water medium), iron compounds, and pH. In BG broth, PC occurred frequently regardless of iron treatment. In MM broth, the optimal conditions for high frequency induction of PC by iron treatments were treatment of iron (III) EDTA, and under pH 7-8. Conversely, PC was not induced by iron treatment in water medium and in MM broth under pH 5 conditions. Common to the culture conditions wherein PC was not induced by iron treatment, the bacterial density of the wild-type strain was as low as 106 CFU mL-1 or less. Finally, we investigated the effects on bacterial growth and PC of the wild-type strain by the iron treatment and addition of culture filtrate after cultivation of the wild-type strain at high concentration. In medium containing only the culture filtrate, PC did not occur. However, in medium containing the culture filtrate and iron, PC occurred frequently. Our results thus suggest that high-density growth of the wild-type strain as well as the presence of iron are involved in inducing PC in R. solanacearum.

A bacterial effector protein uncovers a plant metabolic pathway involved in tolerance to bacterial wilt disease.

Bacterial wilt caused by the soil-borne plant pathogen Ralstonia solanacearum is a devastating disease worldwide. Upon plant colonization, R. solanacearum replicates massively, causing plant wilting and death; collapsed infected tissues then serve as a source of inoculum. In this work, we show that the plant metabolic pathway mediated by pyruvate decarboxylases (PDCs) contributes to plant tolerance to bacterial wilt disease. Arabidopsis and tomato plants respond to R. solanacearum infection by increasing PDC activity, and plants with deficient PDC activity are more susceptible to bacterial wilt. Treatment with either pyruvic acid or acetic acid (substrate and product of the PDC pathway, respectively) enhances plant tolerance to bacterial wilt disease. An effector protein secreted by R. solanacearum, RipAK, interacts with PDCs and inhibits their oligomerization and enzymatic activity. Collectively, our work reveals a metabolic pathway involved in plant resistance to biotic and abiotic stresses, and a bacterial virulence strategy to promote disease and the completion of the pathogenic life cycle.

Effects of integrated biocontrol on bacterial wilt and rhizosphere bacterial community of tobacco.

Bacterial wilt as a soil-borne disease was caused by Ralstonia solanacearum, and seriously damages the growth of tobacco. Integrated biocontrol method was explored to control bacterial wilt. Nevertheless, the long-term effects of the integrated biocontrol method on soil bacterial community, soil physicochemical properties and the incidence of bacterial wilt are not well understood. In this study, B. amyoliquefaciens ZM9, calcium cyanamide and rice bran were applied to tobacco fields in different ways. The disease index and incidence of tobacco bacterial wilt (TBW), soil physicochemical properties, colonization ability of B. amyoliquefaciens ZM9, and rhizopshere bacterial community were investigated. The results showed that the integrated application of B. amyoliquefaciens ZM9, rice bran and calcium cyanamide had the highest control efficiency of TBW and bacteria community diversity. Additionally, the integrated biocontrol method could improve the colonization ability of B. amyoliquefaciens ZM9. Furthermore, the integrated biocontrol method could effectively suppress TBW by regulating soil physicochemical properties, promoting beneficial bacteria and antagonistic bacteria of rhizopshere soil. This strategy has prospect of overcoming the defects in application of a single antagonistic bacteria and provides new insights to understand how to improve the colonization capacity of antagonistic bacteria and control efficacy for TBW.

Design, synthesis, crystal structure and in vitro antimicrobial activity of novel 1,2,4-triazolo[1,5-a]pyrimidine-containing quinazolinone derivatives.

A series of novel 1,2,4-triazolo[1,5-a]pyrimidine-containing quinazolin-4(3H)-one derivatives (8a-8o) were designed, synthesized and assessed for their in vitro antibacterial and antifungal activities in agriculture. All the title compounds were completely characterized via 1H NMR, 13C NMR, HRMS and IR spectroscopic data. In particular, the molecular structure of compound 8f was further corroborated through a single-crystal X-ray diffraction measurement. The turbidimetric method revealed that some of the compounds displayed noticeable bactericidal potencies against the tested plant pathogenic bacteria. For example, compounds 8m, 8n and 8o possessed higher antibacterial efficacies in vitro against Xanthomonas oryzae pv. oryzae with EC50 values of 69.0, 53.3 and 58.9 µg/mL, respectively, as compared with commercialized agrobactericide bismerthiazol (EC50 = 91.4 µg/mL). Additionally, compound 8m displayed an EC50 value of 71.5 µg/mL toward Xanthomonas axonopodis pv. citri, comparable to control bismerthiazol (EC50 = 60.5 µg/mL). A preliminary structure-activity relationship (SAR) analysis was also conducted, based on the antibacterial results. Finally, some compounds were also found to have a certain antifungal efficacy in vitro at the concentration of 50 µg/mL.

Identification of Type III Secretion Inhibitors for Plant Disease Management.

Bacterial plant pathogens are among the most devastating threats to agriculture. To date, there are no effective means to control bacterial plant diseases due to the restrictions in the use of antibiotics in agriculture. A novel strategy under study is the use of chemical compounds that inhibit the expression of key bacterial virulence determinants. The type III secretion system is essential for virulence of many Gram-negative bacteria because it injects into the plant host cells bacterial proteins that interfere with their immune system. Here, we describe the methodology to identify bacterial type III secretion inhibitors, including a series of protocols that combine in planta and in vitro experiments. We use Ralstonia solanacearum as a model because of the number of genetic tools available in this organism and because it causes bacterial wilt, one of the most threatening plant diseases worldwide. The procedures presented can be used to evaluate the effect of different chemical compounds on bacterial growth and virulence.

Antimicrobial Meroterpenoids and Erythritol Derivatives Isolated from the Marine-Algal-Derived Endophytic Fungus Penicillium chrysogenum XNM-12.

One new meroterpenoid-type alkaloid, oxalicine C (1), and two new erythritol derivatives, penicierythritols A (6) and B (7), together with four known meroterpenoids (2-5), were isolated from the marine algal-derived endophytic fungus Penicillium chrysogenum XNM-12. Their planar structures were determined by means of spectroscopic analyses, including UV, 1D and 2D NMR, and HRESIMS spectra. Their stereochemical configurations were established by comparing the experimental and calculated electronic circular dichroism (ECD) spectra for compound 1, as well as by comparison of the optical rotations with literature data for compounds 6 and 7. Notably, oxalicine C (1) represents the first example of an oxalicine alkaloid with a cleaved α-pyrone ring, whereas penicierythritols A (6) and B (7) are the first reported from the Penicillium species. The antimicrobial activities of compounds 1-7 were evaluated. Compounds 1 and 6 exhibited moderate antibacterial effects against the plant pathogen Ralstonia solanacearum with minimum inhibitory concentration (MIC) values of 8 and 4 µg/mL, respectively. Compound 6 also possesses moderate antifungal properties against the plant pathogen Alternaria alternata with a MIC value of 8 µg/mL.

A Bacterial Effector Protein Hijacks Plant Metabolism to Support Pathogen Nutrition.

Many bacterial plant pathogens employ a type III secretion system to inject effector proteins within plant cells to suppress plant immunity. Whether and how effector proteins also co-opt plant metabolism to support extensive bacterial replication remains an open question. Here, we show that Ralstonia solanacearum, the causal agent of bacterial wilt disease, secretes the effector protein RipI, which interacts with plant glutamate decarboxylases (GADs) to alter plant metabolism and support bacterial growth. GADs are activated by calmodulin and catalyze the biosynthesis of gamma-aminobutyric acid (GABA), an important signaling molecule in plants and animals. RipI promotes the interaction of GADs with calmodulin, enhancing the production of GABA. R. solanacearum is able to replicate efficiently using GABA as a nutrient, and both RipI and plant GABA contribute to a successful infection. This work reveals a pathogenic strategy to hijack plant metabolism for the biosynthesis of nutrients that support microbial growth during plant colonization.

The essential genome of Ralstonia solanacearum.

Ralstonia solanacearum is a scientifically/economically important plant pathogenic bacterium. The plant disease caused by R. solanacearum causes huge economic losses, and efficient control measures for the disease remain limited. To gain a better system-level understanding of R. solanacearum, we generated a near-saturated transposon insertion library of R. solanacearum GMI1000 with approximately 240,000 individual insertion mutants. Transposon sequencing (Tn-seq) allowed the mapping of 70.44%-80.96% of all potential insertion sites of the mariner C9 transposase in the genome of R. solanacearum and the identification of 465 genes essential for the growth of R. solanacearum in rich medium. Functional and comparative analyses of essential genes revealed that many basic physiological and biochemical processes such as transcription differ between R. solanacearum and other bacteria. A comparative analysis of essential genes also suggested that 34 genes might be essential only for Ralstonia group bacteria, whereas another 16 essential genes are unique to Ralstonia, providing high-priority candidate targets for developing R. solanacearum-specific drugs.

Antibacterial Activities of Novel Dithiocarbamate-Containing 4H-Chromen-4-one Derivatives.

To aid the development of novel antibacterial agents that possess a innovative mechanism of action, we built a series of novel dithiocarbamate-containing 4H-chromen-4-one derivatives. We evaluated the activities of the derivatives against three plant pathogens Xanthomonas oryzae pv oryzae (X. oryzae pv o.), Ralstonia solanacearum (R. solanacearum), and Xanthomonas axonopodis pv citri (X. axonopodis pv c.). The results of the antibacterial bioassay showed that most of the target compounds displayed good inhibitory effects against X. oryzae pv o. and X. axonopodis pv c. Remarkably, compound E6 showed the best in vitro antibacterial activity against X. axonopodis pv c., with an EC50 value of 0.11 µg/mL, which was better than those of thiodiazole copper (59.97 µg/mL) and bismerthiazol (48.93 µg/mL). Compound E14 exhibited the best in vitro antibacterial activity against X. oryzae pv o., with an EC50 value of 1.58 µg/mL, which was better than those of thiodiazole copper (83.04 µg/mL) and bismerthiazol (56.05 µg/mL). Scanning electron microscopy analysis demonstrated that compounds E6 and E14 caused the rupture or deformation of the cell membranes for X. axonopodis pv c. and X. oryzae pv o., respectively. In vivo antibacterial activity test and the defensive enzymes activity test results indicated that the compound E14 could reduce X. oryzae pv o. more effectively than thiodiazole-copper or bismerthiazol.

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease.

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

Sustainable management strategies for bacterial wilt of sweet peppers (Capsicum annuum) and other Solanaceous crops.

Pepper bacterial wilt is caused by the bacterial pathogen, Ralstonia solanacearum. It is the most destructive disease of many Solanaceous crops such as potatoes, tobacco, pepper, tomatoes and eggplant and is a significant source of crop loss worldwide. Physical, cultural and chemical controls have been employed to combat this destructive disease. However, none of these strategies has been able to control the disease completely due to the broad host range and genetic diversity of the pathogen, its prolonged survival in the soil and survival on vegetation as a latent infection. Owing to co-management strategies, biological control is the best approach for human health and environmental friendly motivations. It makes use of various antagonistic rhizobacteria and epiphytic species such as Bacillus cereus, Pseudomonas putida, Bacillus subtilis, Paenibacillus macerans, Serratia marcescens, Bacillus pumilus and Pseudomonas fluorescens, which compete with and ultimately inhibit the growth of the pathogen. The possible mechanisms of biocontrol by these species involve multifaceted interactions between the host, pathogen and the antagonists. These can involve competition for nutrients and space, plant-mediated systemic resistance, siderophore production and production of extracellular cell wall degrading enzymes to inhibit or suppress the growth of the bacterial wilt agent.

Chemical Composition, Algicidal, Antimicrobial, and Antioxidant Activities of the Essential Oils of Taiwania flousiana Gaussen.

Taiwania flousiana (T. flousiana) Gaussen is a precious wood in the family Taxodiaceae. This study investigated the chemical components of the essential oil from the stem bark of T. flousiana and its algicidal, antifungal, and antioxidant properties. Sixty-nine compounds representing 89.70% of the stem bark essential oil were identified by GC-MS. The essential oil showed strong anti-algae, anti-bacteria, and anti-fungus activities against the tested species, and antioxidant activities. The IC50 values of the essential oil against chlorophyll a, chlorophyll b, and the total chlorophyll of Spirogyra communis (a species of algae), 24-96 h after the treatment, ranged from 31.77 to 84.92 µg/mL, while the IC50 values of butachlor ranged from 40.24 to 58.09 µg/mL. Ultrastructure changes revealed by the transmission electron microscopy indicated that the main algicidal action sites were the chloroplast and cell wall. The essential oil showed antifungal activities on Rhizoctonia solani (EC50 = 287.94 µg/mL) and Colletotrichum gloeosporioiles (EC50 = 378.90 µg/mL). It also showed bactericidal activities on Ralstonia solanacearum and Staphylococcus aureus, with zones of inhibition (ZOIs) being 18.66 and 16.75 mm, respectively at 40 µg/disk. Additionally, the essential oil possessed antioxidant activity estimated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method (IC50 = 33.51 µg/mL; IC50 value of the positive control ascorbic acid was 7.98 µg/mL). Thus, the essential oil of this plant might be used as a possible source of natural bioactive molecules in agrochemical industry as well as in food and cosmetic industries.

Biochar amendment controlled bacterial wilt through changing soil chemical properties and microbial community.

Long-term continuous cropping has led to epidemic of bacterial wilt disease in Southern China. Bacterial wilt disease is caused by Ralstonia solanacearum and difficult to control. In order to control bacterial wilt, rice hull biochar was applied to soil with different doses (0, 7.5, 15, 30 and 45 t ha-1) in a field trial. After three years, the influence of biochar on soil properties, incidence of bacterial wilt and microbial community were characterized. Biochar amendment significantly suppressed bacterial wilt through changing soil chemical properties and microbial composition. Compared with control, disease incidence and index of biochar amendments (7.5, 15, 30, and 45 t ha-1) significantly decreased. Disease incidence and index of biochar amendment (15 t ha-1) were the lowest. Compared to the unamended control, contents of soil organic matter in biochar amendments (15, 30 t ha-1), available nitrogen in biochar amendment (15 t ha-1), and urease activity in biochar amendments (7.5, 15 t ha-1) significantly increased. Biochar amendments (15, 30, and 45 t ha-1) increased the relative abundances of potential beneficial bacteria (Aeromicrobium, Bacillus, Bradyrhizobium, Burkholderia, Chlorochromatium, Chthoniobacter, Corynebacterium, Geobacillus, Leptospirillum, Marisediminicola, Microvirga, Pseudoxanthomonas, Telmatobacter). Biochar amendments (7.5, 30, and 45 t ha-1) reduced the relative abundances of denitrifying bacteria (Noviherbaspirillum, Reyranella, Thermus). Biochar amendments (7.5, 15, and 45 t ha-1) significantly decreased pathogen Ralstonia abundance. Overall, application of biochar effectively controlled bacterial wilt through sequestering more carbon and nitrogen, enriching specific beneficial bacteria and decreasing pathogen abundance. This study revealed the potential of biochar in control of bacterial wilt.

Involvement of a PadR regulator PrhP on virulence of Ralstonia solanacearum by controlling detoxification of phenolic acids and type III secretion system.

Ralstonia solanacearum can metabolize ferulic acid (FA) and salicylic acid (SA), two representative phenolic acids, to protect it from toxicity of phenolic acids. Here, we genetically demonstrated a novel phenolic acid decarboxylase regulator (PadR)-like regulator PrhP as a positive regulator on detoxification of SA and FA in R. solanacearum. Although the ability to degrade SA and FA enhances the infection process of R. solanacearum toward host plants, PrhP greatly contributes to the infection process besides degradation of SA and FA. Our results from the growth assay, promoter activity assay, RNA-seq and qRT-PCR revealed that PrhP plays multiple roles in the virulence of R. solanacearum: (1) positively regulates expression of genes for degradation of SA and FA; (2) positively regulates expression of genes encoding type III secretion system (T3SS) and type III effectors both in vitro and in planta; (3) positively regulates expression of many virulence-related genes, such as the flagella, type IV pili and cell wall degradation enzymes; and (4) is important for the extensive proliferation in planta. The T3SS is one of the essential pathogenicity determinants in many pathogenic bacteria, and PrhP positively regulates its expression mediated with the key regulator HrpB but through some novel pathway to HrpB in R. solanacearum. This is the first report on PadR regulators to regulate the T3SS and it could improve our understanding of the various biological functions of PadR regulators and the complex regulatory pathway on T3SS in R. solanacearum.

The antibiotic activity and mechanisms of active metabolites (Streptomyces alboflavus TD-1) against Ralstonia solanacearum.

OBJECTIVES: In order to elucidate the antibacterial activity and mechanism of S. alboflavus TD-1 active metabolites, the minimal inhibitory concentration of R. solanacearum and other effects on cell wall, cell membrane, nucleic acid, protein and cell morphology were studied. Besides, based on LCMS-IT-TOF, the active metabolites of S. alboflavus TD-1 were preliminarily analyzed. RESULTS: In this study, We found that the active metabolites had obvious inhibitory effect on R. solanacearum, and the minimal inhibitory concentration (MIC) of R. solanacearum was 3.125 mg/mL. And the treatment of 10 mg/mL active metabolites can increase the permeability of R. solanacearum membranes, destroy the cell wall integrity, inhibit the synthesis of bacterial nucleic acids and proteins, and cause leakage of bacterial nucleic acids and proteins, obstruct the normal expression of proteins and destroy their bacterial morphology. At the same time, We speculated the molecular weights corresponding to the six compounds were 618, 615, 615, 615, 646, 646, respectively among the active metabolites, and it was found that were highly unstable. CONCLUSIONS: The active metabolites produced by S. alboflavus TD-1 liquid fermentation contain components that can significant inhibitory effects on R. solanacearum. It had the potential to develop biocontrol agents against bacterial wilt and be a kind potential sources for the preparation of functional anti-pathogenic microbial agents.

Antibacterial activity of three essential oils and some monoterpenes against Ralstonia solanacearum phylotype II isolated from potato.

The present study describes the possibility of using some essential oils and monoterpens as bioagents against the growth of Ralstonia solanacearum, a causal bacterium of potato brown rot disease. Eight isolates of the bacterium were recovered from infected potato tubers, showing typical symptoms of the disease, Isolates were identified as R.solanacearum phylotype II, based on biochemical and physiological characteristics, as well as, at the molecular level through PCR analysis. Three essential oils extracted from Corymbia citriodora (leaves), Cupressus sempervirens (aerial parts), and Lantana camara (aerial parts) were evaluated for their antibacterial activity against eight isolates of R. solanacearum phylotype II. Results demonstrated that L. camara essential oil (concentration 5000 µg/mL) had the highest effects against the RsMo2, RsSc1 and Rs48, with inhibition zone (IZ) values of 17.33, 16.33, and 17.50 mm, respectively, also against Rs2 (IZ 14.33 mm), and RsIs2 (IZ 16 mm). C. citriodora oil showed the highest activity against RsBe2 (IZ 14 mm), RsFr4 (IZ 13.66 mm) and RsNe1 (IZ 13.66 mm). Gas Chromatography-Mass Spectrometry (GC-MS-FID) analyzed the chemical composition of these essential oils. It was proved that L. camara leaves contains mainly trans-caryophyllene (16.24%) and α-humulene (9.55%), in C. citriodora oil were α-citronellal (56.55%), α-citronellol (14.89%), and citronellol acetate (13.04%), and in Cup. sempervirens aerial parts were cedrol (22.17%), and Δ3-carene (18.59%). Five monoterpenes were evaluated against the most resistance Ralstonia isolate RsFr5 to the three studied essential oils and found that limonene had the highest effect against it compared with the lowest thymol. The results proved the strong bio effects of the essential oil from L. camara leaves as a natural product contained monoterpenes that can inhibit the growth of tested R. solanacearum phylotype II isolates.

Optimization of Ralstonia solanacearum cell growth using a central composite rotational design for the P(3HB) production: Effect of agitation and aeration.

The intracellular accumulation of polyhydroxyalkanoates (PHAs) normally occurs after cell growth, during the second fermentation stage and under nutrient-limited conditions in the presence of a carbon excess. However, some microorganisms are able to accumulate PHAs as poly(3-hydroxybutyrate) [P(3HB)] during the first fermentation stage, the cell growth phase, without nutrient limitation, once they have been reported to utilize type II metabolism during the polymer accumulation phase. This study evaluated the effect of aeration and agitation on cell growth and P(3HB) accumulation in Ralstonia solanacearum RS, performed in a bioreactor for 24h at 32°C. A 22 central composite rotational design (CCRD) was used, with agitation (150 to 250 rpm) and aeration (0.3 to 1 vvm) as independent variables and optical density (OD600nm), dry cell weight (DCW), and P(3HB) yield as dependent variables. A significant polymer accumulation, until 70% of P(3HB), was observed, proving that R. solanacearum RS exhibited metabolism type II, regardless of the aeration process. The best results were obtained for 1 vvm and 250 rpm (+1, +1), with values of OD600nm (18.04) and DCW (4.82 g.L-1).