Ahlem Nefzi

Endophytic Bacteria from Datura stramonium for Fusarium WiltSuppression and Tomato Growth Promotion

Ten nonpathogenic bacterial isolates, recovered from Datura stramonium organs, and successfully colonizing the internal stem tissues of tomato cv. Rio Grande were screened for their ability to suppress tomato Fusarium wilt, caused by Fusaium oxysporum f. sp. lycopersici (FOL), and to enhance tomato growth. S37 and S40 isolates were found to be the most effective in decreasing leaf yellowing by 94-88% and the vascular browning extent by 96-95%, respectively, as compared to FOL-inoculated and untreated control. A significant enhancement of growth parameters was recorded on tomato plants inoculated or not with the pathogen. The two bioactive isolates were morphologically and biochemically characterized and identified using 16S rDNA sequencing genes as Stenotrophomonas maltophilia str. S37 and Bacillus mojavensis str. S40. Screened in vitro for their antifungal activity toward FOL, these strains led to 43.8 and 39% decrease in pathogen radial growth and to the formation of an inhibition zone of about 11.37 and 12.12 mm in diameter, respectively. S. maltophilia str. S37 and B. mojavensis str. S40 were found to be chitinase-, protease- and pectinase-producing strains but only S. maltophilia str. S37 was able to produce the volatile metabolite hydrogen cyanide. Indole-3-acetic acid production, phosphate solubilizing ability and pectinase activity were investigated for elucidating their plant growth promoting traits and their endophytic colonization ability. To our knowledge, this is the first report on endophytic bacteria from D. stramonium exhibiting Fusarium wilt suppression potential and plant growth-promoting ability on tomato.

Endophytic bacteria from Datura metel for plant growth promotion and bioprotection against Fusarium wilt in tomato

ABSTRACT Nine non-pathogenic bacterial isolates, recovered from Datura metel organs and able to colonise the internal stem tissues of tomato cultivar Rio Grande, were screened for their ability to suppress tomato Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici (FOL), and to enhance plant growth. S33 and S85 isolates tested were found to be the most effective in decreasing Fusarium wilt severity by 94–95% compared to FOL-inoculated and untreated control. A significant enhancement of growth parameters was recorded on tomato plants inoculated or not with FOL. Both isolates were characterised and identified using 16S rDNA sequencing genes as Stenotrophomonas sp. str. S33 (KR818084) and Pseudomonas sp. str. S85 (KR818087). Screened in vitro for their antifungal activity towards FOL, these isolates led to 38.7% and 22.5% decrease in pathogen radial growth and to the formation of an inhibition zone of 12.75 and 8.37 mm respectively. Stenotrophomonas sp. str. S33 and Pseudomonas sp. str. S85 were found to be chitinase-, protease- and pectinase-producing strains but unable to produce hydrogen cyanide. Production of indole-3-acetic acid-like compounds, phosphate solubilising ability and pectinase activity were investigated for elucidating their plant growth-promoting traits and their endophytic colonisation ability.

Isolation of Endophytic Bacteria from Withania Somnifera and Assessment of their Ability to Suppress Fusarium Wilt Disease in Tomato and to Promote Plant Growth

Four nonpathogenic and putative endophytic bacterial isolates, recovered from Withania somnifera fruits (S7, S8 and S9) and stems (S15), were evaluated for their in vivo and in vitro antifungal activity against Fusarium oxysporum f. sp. lycopersici (FOL), and their plant-growth promoting ability. Tomato plants challenged and/or not with FOL and treated using these bacterial isolates exhibited a significant increment in their growth parameters (plant height, aerial part fresh weight, maximum root length, and root fresh weight). The strong suppressive effect against Fusarium wilt was achieved using two isolates (namely S15 and S8) leading to 92-96% lower disease severity compared to pathogen-inoculated and untreated control. Both isolates were characterized and only the isolate S8 was identified as Alcaligenes faecalis subsp. faecalis str. S8 (KR818077) using 16S rDNA gene sequencing. The unidentified bacterial isolate S15 had improved germination of bacterized tomato seeds relative to the untreated ones. Tested using streak and sealed plates methods, diffusible and volatile compounds from S15 and S8 isolates inhibited FOL by 10.7-16.8% and 53.8-20.7%, respectively. Moreover, an inhibition zone (8.5-8.25 mm) was formed around FOL colonies using the disc diffusion method. Alcaligenes faecalis subsp. faecalis str. S8 and the unidentified bacterium str. S15 were shown able to produce chitinolytic, proteolytic and pectinolytic enzymes and hydrogen cyanide. Production of indole-3-acetic acid and phosphate solubilizing ability were also investigated for elucidation of their plant growth-promoting traits.

Antifungal activity of aqueous and organic extracts from Withania somnifera L. against Fusarium oxysporum f. sp. radicis-lycopersici

The aim of this study was to evaluate the in vitro antifungal activity of aqueous and organic extracts from native Withania somnifera L. leaves, stems, and fruits against Fusarium oxysporum f. sp. radicis-lycopersici (FORL), the causal agent of Fusarium Crown and Root Rot disease in tomato. Aqueous and organic extracts (used at 1, 2, 3 and 4%) were added to molten Potato Dextrose Agar (PDA) medium. After pathogen challenge, cultures were incubated at 25°C for 5 days. All extracts tested, whatever the concentrations used, showed a strong antifungal activity toward targeted pathogen. FORL response to the different extracts assessed using the poisoned food technique, varied depending on plant organs, concentrations tested and organic solvent used for extraction. For aqueous extracts, fruit extract used at 2% exhibited the highest antifungal potential where FORL growth was decreased by 56.27%, relative to the untreated control, compared to 52 and 45.34% achieved using stem and leaf extracts at 3%, respectively. The highest antifungal activity of organic extracts was registred at the highest concentration used (4%). FORL was found to be more sensitive to fruit extracts than those from leaves and stems. Among the three organic extracts tested, butanolic fractions were the most active against FORL growth. The highest antifungal potential expressed by 62.03% decrease in pathogen radial growth was displayed by butanolic stem extracts applied at 4%. These results indicate that native W. somnifera plants may be exploited as potential source of allelochemicals biologically active against FORL.

Endophytic Bacillus spp. from Wild Solanaceae and Their Antifungal Potential against Fusarium oxysporum f. sp. lycopersici Elucidated Using Whole Cells, Filtrate Cultures and Organic Extracts

Six isolates of culturable bacteria, isolated from stems of wild Solanaceae species (Datura metel, Solanum nigrum, S. elaeagnifolium, and Nicotiana glauca), were assessed for their antifungal activity against F. oxysporum f. sp. lycopersici (FOL), the causal agent of the tomato Fusarium wilt. Blast analysis of 16S rDNA sequencing genes homology showed that the isolates belonged to the genus Bacillus (Bacillus cereus str. S42, B. tequilensis str. SV39, B. subtilis str. SV41, B. methylotrophicus str. SV44, B. amyloliquefaciens subsp. plantarum str. SV65, and B. mojavensis str. SV104). The mycelium growth of FOL was significantly reduced by 36 to 46% by diffusible metabolites and by 18 to 21% by volatile compounds. Cell-free cultures were found to be mostly active when issued from 4 days-old cultures where FOL growth inhibition significantly varied from 31.1 to 59.5%. Active metabolites present in the cell-free cultures were extracted with n-butanol and chloroform. Both organic extracts exhibited antifungal potential towards FOL higher than that induced by the two commercial products i.e. Bavistin® (50%, chemical fungicide) and Bactospeine® (16000UI/mg, biopesticide). This study clearly indicates that endophytic Bacillus spp. from wild Solanaceae species can be used as natural sources of bioactive metabolites towards FOL. Wild Solanaceae are frequent in Tunisia and were not explored as potent sources of candidate antagonistic bacteria. In view of the endogenous progress of the pathogen via the vascular tissues, the use of endophytic bacteria can suppress tomato Fusarium wilt disease.

Evaluation of the Growth-Promoting Potential of Endophytic Bacteria Recovered from Healthy Tomato Plants

The study examined the plant growth-promoting potential of 38 endophytic bacterial isolates recovered from healthy tomato plants. Eight isolates were selected as the most efficient in improving the plant height by 30.5-35.9%, the aerial part fresh weight by 47.4 -56.9%, the maximum root length by 17.3- 28.3%, and the root fresh weight by 44.7- 58.8% compared to control. They were morphologically and biochemically characterized and identified using 16S rDNA sequencing genes as Stenotrophomonas maltophilia CT12, S. maltophilia CT13, S. maltophilia CT16, Pseudomonas geniculata CT19, B. amyloliquefaciens CT32, B. subtilis subsp. inaquosorum CT43, B. licheniformis SV4, and B. subtilis SV5. All isolates were shown able to produce indole-3-acetic acid and displayed pectinolytic activity. Phosphate solubilization ability was recorded in S. maltophilia CT13, S. maltophilia CT16, B. subtilis subsp. inaqosorum CT43 and B. licheniformis SV4. This study demonstrates that healthy tomato plants may be naturally colonized by beneficial endophytic bacteria with growth-promoting potential useful for the improvement of tomato growth.

A putative endophytic Bacillus cereus str. S42 from Nicotiana glauca for biocontrol of Fusarium wilt disease in tomato and gas chromatography-mass spectrometry analysis of its chloroform extract

Abstract A putative endophytic Bacillus cereus str. S42 (KP993206), recovered from surface-sterilised stems of Nicotiana glauca was assessed in vitro and in vivo for its antifungal potential towards Fusarium oxysporum f. sp. lycopersici (FOL). Pathogen sporulation was significantly inhibited by B. cereus str. S42. FOL mycelial growth was reduced using its whole-cell suspensions, cell-free culture supernatant and chloroform extract. Its extracellular metabolites remained effective after heating at 50–100 °C with a decline in their activity was observed beyond 100 °C, when added with proteinase K and/or after pH adjustment to 2 and 12. Chitinase gene was detected using PCR amplification. Gas chromatography–mass spectrometry analysis of its chloroform extract matched phthalic acid, dibutyl ester with high level of similarity. B. cereus str. S42 cell-free culture supernatant and whole-cell suspensions had significantly suppressed Fusarium wilt severity by 87–96% and enhanced tomato growth by 39–79% compared to FOL-inoculated and untreated control.