Real-time monitoring of Ralstonia solanacearum infection progress in tomato and Arabidopsis using bioluminescence imaging technology

Background Ralstonia solanacearum, one of the most devastating bacterial plant pathogens, is the causal agent of bacterial wilt. Recently, several studies on resistance to bacterial wilt have been conducted using the Arabidopsis-R. solanacearum system. However, the progress of R. solanacearum infection in Arabidopsis is still unclear. Results We generated a bioluminescent R. solanacearum by expressing plasmid-based luxCDABE. Expression of luxCDABE did not alter the bacterial growth and pathogenicity. The light intensity of bioluminescent R. solanacearum was linearly related to bacterial concentrations from 104 to 108 CFU·mL−1. After root inoculation with bioluminescent R. solanacearum strain, light signals in tomato and Arabidopsis were found to be transported from roots to stems via the vasculature. Quantification of light intensity from the bioluminescent strain accurately reported the difference in disease resistance between Arabidopsis wild type and resistant mutants. Conclusions Bioluminescent R. solanacearum strain spatially and quantitatively measured bacterial growth in tomato and Arabidopsis, and offered a tool for the high-throughput study of R. solanacearum-Arabidopsis interaction in the future.

Effectiveness of bioactive compounds in amending the soil infested with soil-borne Ralstonia solanacearum of potato

Three natural bioactive compounds, viz. cow dung, bee propolis and turmeric powder were applied to amend the Ralstonia solanacearum infested soil of potato. Sun dried cow dung @ 30 t/ha, aqueous extraction of turmeric powder @ 30 kg/ha and propolis @ 5 lit/ha were applied to the inoculated soil by mixing with field soil in SAU (Dhaka) and BARI (Gazipur). Data on pH, organic matter, total cfu/ml and avirulent cfu/ml of the treatment applied soil and per cent disease index (PDI) of bacterial wilt of potato were recorded. Significant difference in soil pH and per cent organic matter (OM) occurred in all the amended soil as compared to control. A decreased total cfu/ml of R. solanacearum occurred in all the treated soil, but propolis (8.2 X 107) and cow dung (1.1 X 108) showed the lowest count. However, in increasing the avirulent count of R. solanacearum, turmeric powder (1.8 X 109) showed the highest count compared to other amended soil. Significant disease reduction over control was also observed in all the treatments, but propolis (45.65%) and turmeric powder (43.48%) showed the best results in wilt disease reduction. J. Biodivers. Conserv. Bioresour. Manag. 2021, 7(1): 43-50

Metabolomic Evaluation of Ralstonia solanacearum Cold Shock Protein Peptide (csp22)-Induced Responses in Solanum lycopersicum

Ralstonia solanacearum, the causal agent of bacterial wilt, is one of the most destructive bacterial plant pathogens. This is linked to its evolutionary adaptation to evade host surveillance during the infection process since many of the pathogen’s associated molecular patterns escape recognition. However, a 22-amino acid sequence of R. solanacearum-derived cold shock protein (csp22) was discovered to elicit an immune response in the Solanaceae. Using untargeted metabolomics, the effects of csp22-elicitation on the metabolome of Solanum lycopersicum leaves were investigated. Additionally, the study set out to discover trends that may suggest that csp22 inoculation bestows enhanced resistance on tomato against bacterial wilt. Results revealed the redirection of metabolism toward the phenylpropanoid pathway and sub-branches thereof. Compared to the host response with live bacteria, csp22 induced a subset of the discriminant metabolites, but also metabolites not induced in response to R. solanacearum. Here, a spectrum of hydroxycinnamic acids (especially ferulic acid), their conjugates and derivatives predominated as signatory biomarkers. From a metabolomics perspective, the results support claims that csp22 pre-treatment of tomato plants elicits increased resistance to R. solanacearum infection and contribute to knowledge on plant immune systems operation at an integrative level. The functional significance of these specialized compounds may thus support a heightened state of defense that can be applied to ward off attacking pathogens or toward priming of defense against future infections.

Saponins accumulation and antimicrobial activities on shallot (Allium cepa L.) from marginal land

Saponins are one of the secondary metabolites found in Shallot (Allium cepa L.), particularly in the roots. Microclimate differences in the cultivation area are thought to have a significant impact on the production of secondary metabolites, such as saponins. This research aimed to observe the saponins content in the root of shallot plants cultivated in marginal agricultural land and their antimicrobial activity against bacteria (Ralstonia solanacearum) and fungus (Fusarium oxysporum). This research was observational research with a random sampling method. The samples were collected from the shallot plantation with two different cultivation conditions. Two varieties of ‘Bima' and 'Tiron' cultivated by farmers in sandy coastal land Samas, Bantul were used. The plants were harvested at 1, 1.5, and 2 months after planting, respectively. The crude saponins extract was used to test antimicrobial activity. Shallot plants cultivated in marginal coastal sandy land produced higher saponins accumulated in their roots. The saponins production increased along with the maturity of shallot plants, both cultivated in marginal coastal sandy land and regular paddy field. The saponins extracted from the roots of shallots cultivated in both marginal and regular land showed higher antimicrobial activity than antifungal activity. Saponin merupakan salah satu metabolit sekunder yang terdapat pada bawang merah (Allium cepa L.), terutama pada bagian akar. Perbedaan iklim mikro pada lahan budidaya diduga akan berpengaruh terhadap produksi metabolit sekunder termasuk saponin. Tujuan penelitian ini adalah untuk mengetahui kandungan saponin pada bagian akar tanaman bawang merah yang dibudidayakan di lahan pertanian marginal serta aktivitas antimikrobanya terhadap bakteri (Ralstonia solanacearum) dan jamur (Fusarium oxysporum). Penelitian ini merupakan penelitian observasi dengan metode pengambilan sampel secara acak. Sampel berasal dari perkebunan bawang merah dengan dua lahan budidaya yang berbeda. Digunakan dua varietas yaitu 'Bima' dan 'Tiron' yang dibudidayakan oleh petani di daerah pantai Samas,kabupaten Bantul. Bahan tanaman dipanen pada tiga waktu berbeda, yaitu 1 bulan, 1,5 bulan dan 2 bulan berturut-turut setelah tanam. Ekstrak kasar saponin digunakan untuk menguji aktivitas antimikrobanya. Tanaman bawang merah yang dibudidayakan di lahan marginal pasir pantai menghasilkan saponin yang lebih tinggi yang terakumulasi pada akarnya. Produksi saponin semakin meningkat seiring dengan umur tanaman bawang merah, baik yang dibudidayakan di lahan marginal pasir pantai maupun di lahan sawah biasa. Saponin yang diekstraksi dari akar bawang merah yang dibudidayakan di lahan marginal dan lahan biasa menunjukkan aktivitas antimikroba yang lebih tinggi daripada aktivitas antijamurnya.

Biosynthesis of Silver Chloride Nanoparticles by Rhizospheric Bacteria and Their Antibacterial Activity against Phytopathogenic Bacterium Ralstonia solanacearum

Ralstonia solanacearum is the most destructive pathogen, causing bacterial wilt disease of eggplant. The present study aimed to develop green synthesis and characterization of silver chloride nanoparticles (AgCl-NPs) by using a native bacterial strain and subsequent evaluation of their antibacterial activity against R. solanacearum. Here, a total of 10 bacterial strains were selected for the biosynthesis of AgCl-NPs. Among them, the highest yield occurred in the synthesis of AgCl-NPs using a cell-free aqueous filtrate of strain IMA13. Ultrastructural observation revealed that the AgCl-NPs were spherical and oval with smooth surfaces and 5–35 nm sizes. XRD analysis studies revealed that these particles contained face-centered cubic crystallites of metallic Ag and AgCl. Moreover, FTIR analysis showed the presence of capping proteins, carbohydrates, lipids, and lipopeptide compounds and crystalline structure of AgCl-NPs. On the basis of phylogenetic analysis using a combination of six gene sequences (16S, gyrA, rpoB, purH, polC, and groEL), we identified strain IMA13 as Bacillus mojavensis. Three kinds of lipopeptide compounds, namely, bacillomycin D, iturin, and fengycin, forming cell-free supernatant produced by strain IAM13, were identified by MALDI-TOF mass spectrometry. Biogenic AgCl-NPs showed substantial antibacterial activity against R. solanacearum at a concentration of 20 µg/mL−1. Motility assays showed that the AgCl-NPs significantly inhibited the swarming and swimming motility (61.4 and 55.8%) against R. solanacearum. Moreover, SEM and TEM analysis showed that direct interaction of AgCl-NPs with bacterial cells caused rupture of cell wall and cytoplasmic membranes, as well as leakage of nucleic acid materials, which ultimately resulted in the death of R. solanacearum. Overall, these findings will help in developing a promising nanopesticide against phytopathogen plant disease management.

Phylogenetic distribution of Ralstonia solanacearum species complex populations in potato in Kenya

Ralstonia solanacearum is a pathogen causing bacterial wilt disease of potato, resulting in 70% potato production losses in Kenya. A study was conducted to determine the diversity of Ralstonia solanacearum species complex strains within the main potato-growing regions of Kenya. Potato tubers were collected from different potato-growing regions of Kenya from visibly wilted potato plants, including tomato and irrigation water and cultured for pathogen isolation. Genomic DNA was isolated from 135 purified cultures of RSSC isolates and PCR amplified using multiplex and sequevar primers targeting the endoglucanase partial gene sequences. Pathogenicity test using R. solanacearum strain (phylotype II sequevar I) was done on Kenya Karibu, Shangi, Chulu, Wanjiku and Money Maker cultivars. Phylogenetic analysis of the partial endoglucanase gene identified two genospecies, R. pseudosolanacearum sp. nov (1.5%) and R. solanacearum (98.5%). All R. solanacearum strains clustered in sequevar I and were distributed in all the potato-growing regions surveyed. The cultivars were grown in a greenhouse for two cycles in a randomized complete block design and inoculated with R. solanacearum strain. The severity scores were assessed and the area under disease progress curve (AUDPC) was determined. All the cultivars tested for pathogenicity exhibited wilting symptoms at varying intervals after infection, with none showing complete resistance to R. solanacearum. Cultivar Shangi exhibited minimum disease severity and progression of 41.14% and AUDPC of 1041.7, respectively while Kenya Karibu was the most susceptible with a high progression rate of 68.24% and AUDPC of 1897.5, respectively. Money Maker, Chulu and Wanjiku showed no significant difference in disease severity depicting a simultaneous rate of infection among them. These findings provide valuable information to better understand the pathogen genetic diversity in Kenya and how it spreads.