Rania Aydi Ben Abdallah

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.

Variation in Chitosan and Salicylic Acid Efficacy Towards Soil-borne and Air-borne Fungi and Their Suppressive Effect of Tomato Wilt Severity

Two resistance inducers (RIs), chitosan and salicylic acid (SA), were assessed in vitro for their antifungal activity against ten tomato phytopathogenic fungi i.e. Fusarium oxysporum f. sp. lycopersici, F. oxysporum f. sp. radicis-lycopersici, F. solani, Verticillium dahliae, Rhizoctonia solani, Colletotrichum coccodes, Pythium aphanidermatum, Sclerotinia sclerotiorum, Botrytis cinerea, and Alternaria solani. The impact of these RIs, applied as soil drench, on Verticillium wilt, Fusarium wilt, and Fusarium Crown and Root Rot severity and on growth parameters of tomato cv. Rio Grande plants were also investigated. Chitosan (0.5-4 mg/ml) and SA (1-25 mM) inhibited mycelial growth of all pathogens in Potato Dextrose Agar (PDA) medium in a concentration-dependent manner, with the greatest inhibition achieved using the highest chitosan and SA concentrations. Inter specific variations in sensitivity to chitosan and SA were detected. P. aphanidermatum and S. Sclerotiorum were the most sensitive to both RIs. Single treatments with chitosan (4 mg/ml) and SA (10 mM) resulted in varied degree of protection against wilt diseases. Chitosan-and SA-based treatments resulted in 42.1-73.68, 60.86-78.26 and 45- 50% reductions in wilt severity, as compared to VD-, FOL- and FORL-inoculated and untreated controls, respectively. All growth parameters noted were enhanced using RIs compared to pathogen-inoculated controls. In fact, SA-based treatment had significantly increased plant height, root and aerial part fresh weights by 17.94, 52.17 and 33.33%, by 23.01, 55.40 and 29.72%, and by 17.72, 50 and 46.84%,respectively, while compared to VD-, FOL- and FORL-inoculated and untreated plants. Chitosan-treated plants showed increment in their height, root and aerial part fresh weights by 13.81, 62.16 and 38.97%, respectively, compared to FORL-inoculated and untreated control. Results from this investigation showed that SA and chitosan may be used as potential inducers of systemic acquired resistance for successfully controlling fungal tomato diseases in Tunisia.

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.

Comparative Efficacy of Potassium Salts Against Soil-borne and Air-borneFungi and Their Ability to Suppress Tomato Wilt and Fruit Rots

Potassium sorbate (PS), potassium bicarbonate (PB) and dipotassium hydrogenphosphate (DPHP) were assessed for their antifungal activity against Fusarium oxysporum f. sp. lycopersici (FOL), F. oxysporum f. sp. radicis-lycopersici (FORL), F. solani, Verticillium dahliae (VD), Rhizoctonia solani, Colletotrichum coccodes, Pythium aphanidermatum, Sclerotinia sclerotiorum, Botrytis cinerea and Alternaria solani. They were screened for their ability to suppress Verticillium and Fusarium wilts, and Fusarium Crown and Root Rot (FCRR), for their effects on tomato growth, and for their potential control of Botrytis, Alternaria, Rhizoctonia and Anthracnose fruit rots. PS (0.25-1.5%), DPHP (0.1-0.6 M) and PB (0.1-0.6 M) inhibited fungal growth in a concentration-dependent manner, with the greatest inhibition achieved using the highest concentrations. Inter specific variations in sensitivity were detected with P. aphanidermatum, S. Sclerotiorum and B. cinerea being the most sensitive to all salts. Single treatments using PS (0.25%), PB (50 mM) and DPHP (50 mM) resulted in varied degree of protection against wilts. PS led to 50, 78.26 and 65% lower wilt severity as respectively compared to VD-, FOL- and FORL-inoculated controls. PS had significantly increased plant height, root and aerial part fresh weights by 20.61, 30.76 and 33.02%, respectively, compared to FORL-inoculated plants and had improved root fresh weight by 42.18 and 32.87% compared to FOL-and VDinoculated plants, respectively. PB-based treatment led to 60.86 and 30% lower Fusarium wilt and FCRR severity but did not suppress Verticillium wilt. DPHP suppressed only Fusarium wilt by 65.21%. Used as fruit treatment, DPHP and PS significantly decreased Botrytis, Rhizoctonia, Alternaria and Anthracnose fruit rots by 46.68 and 30.81%, 14.04 and 15.74%, 20 and 31.67%, and 19.17 and 25.24%, respectively, compared to inoculated and untreated controls. PB-based treatment resulted in 12.83% significantly lower Rhizoctonia fruit rot. These results showed that PS may be used as potential abiotic agent for successfully controlling fungal tomato diseases.

Effect of Fodder Radish (Raphanus sativus L.) Green Manure on PotatoWilt, Growth and Yield Parameters

Potato is threatened by several soil-borne fungi causing wilt and root rots. In this study, two fodder radish (Raphanus sativus L.) (FR) cultivars (cvs. Boss and Defender), used as green manure preceding a potato crop, were evaluated for their suppressive effects against wilt incidence and severity, potato growth and yield as compared to animal manure. The essay was carried out in a completely randomised design with three types of organic amendment and two potato cultivars (cvs. Spunta and Royal). Incidence of potato wilting noted 100 days post planting (DPP) was high, exceeding 70%, for all soil amendments tested. The extent of vascular discoloration varied depending on amendments used where cv. Defender behaved as the control while the highest extent was noted on potato plants grown in cv. Boss amended plots. As compared to animal manure, the application of cvs. Boss and Defender had increased by 48.43 and 41.28% the incidence of vascular discoloration on cv. Spunta, respectively, while on cv. Royal, only cv. Defender had reduced this parameter by 16.32%. Fungal isolations performed from roots and stems revealed the involvement of several soil-borne pathogens in the recorded plant wilting. Soil manuring using cvs. Boss and Defender FR resulted in significant increment in average stem number per plant and aerial part fresh weight by 22.79 and 21.32% and by 34.62 and 27.03%, respectively, as compared to animal manure. At 100 DPP, potato root fresh weight increase by 8.7 and 33.49% was noted on cv. Spunta compared to 30.34 and 23.48% recorded on cv. Royal. Potato tuber yield was improved by 38.28 and 10.7% and by 28.44 and 27.62% in cvs. Spunta and Royal, respectively, relative to animal manure. The use of FR as green manure may be implemented in the integrated management of soil-borne diseases for the enhancement of potato yield.

Soil-borne and Compost-borne Aspergillus Species for BiologicallyControlling Post-harvest Diseases of Potatoes Incited by Fusariumsambucinum and Phytophthora erythroseptica

Nine isolates of Aspergillus spp., isolated from soil and compost were tested in vitro and in vivo for their antifungal activity against Fusarium sambucinum and Phytophthora erythroseptica, the causal agents of the Fusarium dry rot and pink rot of potato tubers. Tested using the dual culture method, the pathogen growth of F. sambucinum and P. erythroseptica was inhibited by 27 to 68% and 16 to 25% by all Aspergillus species, respectively. The highest inhibitory activity against both pathogens was induced by the isolate CH12 of A. niger. A significant reduction of the mycelial growth of both pathogens tested using the inverse double culture method involves the presence of volatile antifungal metabolites. Their effectiveness was also evaluated as tuber treatment prior to inoculation with the pathogens. The highest effectiveness in reducing Fusarium dry rot severity was recorded on tubers treated with the isolate CH12 of A. niger. This study also revealed that the efficacy of Aspergillus spp. as biocontrol agents may be enhanced by varying the timing of their application. In fact, the lesion diameter of dry and pink rots was reduced by 54-70 and 52% with preventive application, respectively. However, this parameter decreased by 21-48 and 47% when the Aspergillus spp. were applied simultaneously with pathogens, respectively. Similarly, diseases’ severity, estimated based on average penetration of F. sambucinum and P. erythroseptica, was reduced by 57-77 and 55% with preventive treatments and by 29-68 and 44% with simultaneous application, respectively. This study reveals that Aspergillus spp., isolated from compost and soil, exhibits an interesting antifungal activity toward F. sambucinum and P. erythroseptica and may represent a potential source of biopesticide. Testing of their culture filtrates, their organic extracts and their toxicity may give additional information on their safe use as biocontrol agents.

Coniella granati (Saccardo) a New Potential Threat to Pomegranate (Punica granatum L.) in Tunisia Causing Twig Dieback and Fruit Rot

The disease caused by Coniella granati (Saccardo) (syn. Pilidiella granati, Saccardo) is an expanding threat to pomegranate (Punica granatum L.) cultivation and industry and is rapidly emerging in almost all pomegranateproducing regions of the world. The current study presents the first attempt to characterize C. granati associated with pomegranate dieback, shoot blight and fruit rot in Tunisia. Pathogenic isolates were identified based on their cultural and morphological characteristics and molecular data. Collected C. granati isolates were shown able to grow between 10°C and 30°C with an optimal mycelial growth at 20-25°C but they did not grow at 35°C. The fungus was able to grow 4-10 pH with an optimum growth at pH 4-5 for Cg1 and Cg2 isolates. Potato Dextrose Agar (PDA), Carrot Agar (CA) and Oatmeal Agar (OA) followed by pomegranate juice agar (PJA) and Malt Extract Agar (MEA) favored its mycelial growth. Pathogen growth was reduced under continuous light in comparison with the 12 h light/12 h dark regime and was significantly slowest under 24 h dark regime. Inoculated to pomegranate cv. Gabsi fruits, C. granati isolates induced soft rot within 9 days following incubation at 25°C and a complete fruit rot after 15 days. Leaves were highly susceptible to C. granati infection and completely degenerated 5 days post-inoculation. C. granati isolates were found to be pathogenic on pomegranate cv. Gabsi attached shoots and detached branches, giving rise to brown necrotic lesions. Keeping in view the importance of the pomegranate crop and the destructive nature of Coniella induced disease, further studies are needed to verify the pathogen host range, its aggressiveness towards the mostly grown Tunisian pomegranate varieties and to search for suitable control methods.

Effect of Temperatures and Culture Media on Sclerotium rolfsii Mycelial Growth, Sclerotial Formation and Germination

Sclerotium rolfsii is a serious soilborne phytopathogenic fungus causing serious yield loss in crops of high economic importance. In this study, the effects of temperatures and culture media were evaluated on S. rolfsii mycelial growth, sclerotial production and germination. The three isolates tested grew over a range of temperatures from 10°C to 35°C but not at 5°C and 40°C. Radial growth and dry mycelium production were highest at 30°C. Sclerotial initiation started on the 3rd day after incubation at 30°C and 35°C, and mature sclerotia were observed after 15 and 6 days of incubation, respectively. Optimal sclerotial production and the dry weight of 100 sclerotia varied depending on isolates and temperatures and occurred at 25°C-35°C. No mature sclerotia were produced at 5°C, 10°C, 15°C and 40°C. Sclerotial germination, noted after 24 h of incubation, was 96%-100% at 25°C-35°C, declined at 15°C-20°C, and was totally inhibited at 5°C, 10°C and 40°C even after 72 h of incubation. For all S. rolfsii isolates, optimal radial growth occurred on OAT medium followed by CZA, MYEA and PDA. When grown on PDA and OAT media, S. rolfsii developed cottony colonies with abundant mycelium whereas on WA and CZA pathogen growth was thin and scanty. Colonies formed on the other media developed sparse and flat mycelia. For sclerotial formation, WA and CZA were the most suitable culture media but no dark brown sclerotia were observed on NA and YDA. The highest sclerotial formation was observed on PDA and CZA. The highest sclerotium weight was produced by Sr3 isolate on PDA whereas for Sr1 and Sr2 isolates, sclerotial weights were significantly comparable on PDA, MYEA and CZA. Sclerotial germination was optimal on all tested media except NA. The significance of these findings on S. rolfsii biology is discussed.