Final Report Summary - ITRIS (Interaction Trichoderma-Stenotrophomonas and its effect on plant growth and health)
Interaction Trichoderma-Stenotrophomonas and its effect on plant growth and health (iTRIS)
Coordinator of the project: Univ-Prof Dipl-Biol Dr.rer.nat. Gabriele Berg (gabriele.berg@tugraz.at)
Name of the Researcher: Dr. Gabriele Alfano (gabrielealfano@tugraz.at)
Aim of the research is to gain novel insights into the molecular ecological interaction between the fungal BCA Trichoderma velutinum G1/8 and one of the most promising candidate for application as a BCA and plant growth promotion, Stenotrophomonas rhizophila DSM14405T. Detailed fundamental studies of the interactions between the two microorganisms, its potential synergistic effect against the fungal soil-borne pathogen Verticillium dahliae on plant growth and health and on the role of secondary metabolites and VOCs are necessary to understand the ecology of this interesting interaction, and to lay the basis for potential biotechnological applications. Experimental objectives, work performed and results achieved in the second year of the project (01/062014-31/05/2015):
1.Interaction Trichoderma velutinum G1/8 - Stenotrophomonas rhizophila DSM14405T (P69) in vitro
Dual cultures trials, and role played by Volatile Organic Compounds (VOCs) in the interaction have been carried out to gain more details into the nature and the mechanisms involved in the interaction.
In dual-cultures on Waksman Agar (WA), Trichoderma spp. were significantly but temporarily inhibited. This might be due to competition for nutrients, however since no inhibition zone developed it would seems that S rhizophila P69 doesn’t have the molecular repertoire to inhibit the fungi before the direct contact. More likely, P69 can partially inhibit or slow the fungi down through the emission of VOCs and only after having directly attached to the fungal cell wall it secretes antifungal metabolites and cell wall degrading enzymes. In several previous reports, species from the genus Stenotrophomonas exhibited strong antifungal activity towards Verticillium dahliae, Rhizoctonia solani, Sclerotinia sclerotiorum on plate tests and Fusarium solani in plant tests on cucumber in salinated soil. However, there are no reports about the direct inhibition effect toward Trichoderma spp. Conversely, under nutrient-poor conditions (on diluted WA) WAd, the growth of Trichoderma spp. was not significantly affected. This different and contrasting P69 behavior, depending on nutrient availability, has never been described before and we could only assume is the result of a switch in cell metabolism which cause the emission of specific molecular signals which in turn mediate the establishment and the nature itself of the interaction. In VOCs mediated interaction trials, volatiles produced by S. rhizophila P69 grown under nutrient rich conditions (NA) caused significant (P<0.05) inhibition of all Trichoderma spp. on both nutrient rich and poor conditions. For all Trichoderma spp. grown on PDAd the inhibition was always higher than PDA. VOCs produced by P69 grown on nutrient-poor medium (NAd) did not significantly (P>0.05) affect the growth of T. velutinum G1/8 and T. viride GB7, whereas they caused significant (P<0.05) growth stimulation of T. atroviride P1. T. atroviride P1 was stimulated up to 31.6% on PDAd after 48 h of co-incubation with P69. Results from transcriptomic analysis show that the biggest changes in P69 gene expression are mainly due to the lack of nutrients. Significant gene expression changes were found comparing the treatments from experiment 1 (samples 1 and 3, nutrient-rich conditions) with treatments from experiment 2 (samples 5 and 7, nutrient-poor conditions) where the nutrient availability conditions were different.
2.Trichoderma velutinum G1/8 - Stenotrophomonas rhizophila DSM14405T (P69) interaction with V. dahliae in vitro
In dual-cultures on WA, V25 was fully stopped and only partially able to grow into, but not to go through P69 colonies. As stated before, this might be due to competition for nutrients and even in this case no inhibition zone was formed. Conversely, on WAd, V25 was significantly inhibited. Trials to assess V25 viability in co-colture with P69 revealed that V25 hyphae were very highly colonized by P69 cells after 3-4 days on WA and 4-7 days on WAd. Colonization seems to start from hyphal tips. Probably, as a consequence of this intense bacterial colonization, V25 hyphae dye. The same behavior was observed for both media, however on WAd it was delayed. Interestingly at 15 and 30 days of co-culture on WA, the P69-colonized fungal mycelium did not show any staining probably because of the loss of internal and external hyphal structure. This phenomenon is hard to explain, so far we can only hypothesize that fungal disruption led to the release of free radicals, or other antibacterial compounds. This different behavior leads us to think that the nature of the interaction S. rhizophila-V. dahliae and S. rhizophila-Trichoderma spp. is different.
3.Plant colonization by Trichoderma velutinum G1/8 - Stenotrophomonas rhizophila DSM14405T (P69)
For these studies we have used tomato plants as a model. Effect of single or combined application of the microorganisms on growth of tomato plants was assessed. Single application of S rhizophila P69 did not cause a significant increase of roots and shoots of tomato plantlets compared to the control. Treatments with single application of T velutinum G1/8 and T atroviride P1 caused the significant increase of tomato roots and shoots. The treatment with the combined application of S rhizophila P69 and T velutinum G1/8 resulted in the significant increase in tomato roots fresh weight compared to treatments with single application of Trichoderma spp, but not in the shoots fresh weight. The treatment with the combined application of S rhizophila P69 and T atroviride P1/8 resulted in the significant increase in tomato roots and shoots fresh weight compared to the control, but actually not statistically different from treatments with single application of Trichoderma spp.
4.Plant-growth promotion and biocontrol of Verticillium wilt of tomato and pepper plant
In a second in vivo experiment, the plant pathogen V dahliae V25 was added to assess the biocontrol activity of P69 and G1/8 in single and combined application. The biocontrol activity was measured in terms of average fresh weight/plant of tomato. Single application of P69 and V25 do not seem to cause significant effect on both root and shoot fresh weight, whereas only G1/8 cause a significant increase of both. Combined application P69+V25 do not show any remarkable difference compared to the control, whereas the application G1/8+P69 caused the significant increase of tomato shoots fresh weight. The best results seems to have been obtained with the combined application of both BCAs (P69+G1/8) to contrast the adverse effect of V25. In this case the significant increase of both root and shoot fresh weight has been obtained. However, the experiment was designed to be carried in a short time (3 weeks), this probably didn’t allow the pathogen to grow properly and to cause evident symptoms on plants. Longer trials may give better results.
Conclusion
The nature of the interactions P69-Trichoderma spp. and P69-V. dahliae V25 was found to be highly dependent on nutrient availability and on the fungal counterpart interacting with P69. Dual-cultures, volatile mediated interactions and viability experiments carried out on nutrient rich media showed a significant inhibition of the tested fungal species. However, under nutrient poor conditions, the interaction observed for the same microorganisms resulted different. P69 behavior was found to be dependent on the fungal counterpart: Trichoderma spp. were not affected or stimulated whereas V. dahliae was significantly inhibited by P69. This leads to the suggestion that under nutrient rich conditions P69 competes very effectively for nutrients causing the significant growth inhibition of fungal counterparts. Conversely, under nutrient poor conditions, it seems that a specific molecular crosstalk can lead to divergent mechanisms ranging from mutual support or neutral interaction for Trichoderma spp. to a competition/parasitism for V. dahliae V25. Trichoderma spp. were always found to emit a higher number of VOCs than V25. This might suggest that Trichoderma spp. have a higher “predisposition” to establish a volatile crosstalk with others microbial members of the community than V25. Probably, S. rhizophila P69 together with other members of the microbial community, could contribute to the establishment and shaping of a stable and beneficial microbial rhizosphere community which globally takes part to plant growth promotion and protection. Our results provide evidence that various abiotic parameters might be critical for the outcome after an encounter, hence additional synergistic interactions with other species are crucial for the particular advantage. In the underlying case Trichoderma spp. were shown to pose the potential to reinforce the antagonistic advantages of a specific S. rhizophila strain. This could be of particular importance to define microbial-based strategies for biocontrol and plant-growth promotion especially under extreme/adverse conditions.
Coordinator of the project: Univ-Prof Dipl-Biol Dr.rer.nat. Gabriele Berg (gabriele.berg@tugraz.at)
Name of the Researcher: Dr. Gabriele Alfano (gabrielealfano@tugraz.at)
Aim of the research is to gain novel insights into the molecular ecological interaction between the fungal BCA Trichoderma velutinum G1/8 and one of the most promising candidate for application as a BCA and plant growth promotion, Stenotrophomonas rhizophila DSM14405T. Detailed fundamental studies of the interactions between the two microorganisms, its potential synergistic effect against the fungal soil-borne pathogen Verticillium dahliae on plant growth and health and on the role of secondary metabolites and VOCs are necessary to understand the ecology of this interesting interaction, and to lay the basis for potential biotechnological applications. Experimental objectives, work performed and results achieved in the second year of the project (01/062014-31/05/2015):
1.Interaction Trichoderma velutinum G1/8 - Stenotrophomonas rhizophila DSM14405T (P69) in vitro
Dual cultures trials, and role played by Volatile Organic Compounds (VOCs) in the interaction have been carried out to gain more details into the nature and the mechanisms involved in the interaction.
In dual-cultures on Waksman Agar (WA), Trichoderma spp. were significantly but temporarily inhibited. This might be due to competition for nutrients, however since no inhibition zone developed it would seems that S rhizophila P69 doesn’t have the molecular repertoire to inhibit the fungi before the direct contact. More likely, P69 can partially inhibit or slow the fungi down through the emission of VOCs and only after having directly attached to the fungal cell wall it secretes antifungal metabolites and cell wall degrading enzymes. In several previous reports, species from the genus Stenotrophomonas exhibited strong antifungal activity towards Verticillium dahliae, Rhizoctonia solani, Sclerotinia sclerotiorum on plate tests and Fusarium solani in plant tests on cucumber in salinated soil. However, there are no reports about the direct inhibition effect toward Trichoderma spp. Conversely, under nutrient-poor conditions (on diluted WA) WAd, the growth of Trichoderma spp. was not significantly affected. This different and contrasting P69 behavior, depending on nutrient availability, has never been described before and we could only assume is the result of a switch in cell metabolism which cause the emission of specific molecular signals which in turn mediate the establishment and the nature itself of the interaction. In VOCs mediated interaction trials, volatiles produced by S. rhizophila P69 grown under nutrient rich conditions (NA) caused significant (P<0.05) inhibition of all Trichoderma spp. on both nutrient rich and poor conditions. For all Trichoderma spp. grown on PDAd the inhibition was always higher than PDA. VOCs produced by P69 grown on nutrient-poor medium (NAd) did not significantly (P>0.05) affect the growth of T. velutinum G1/8 and T. viride GB7, whereas they caused significant (P<0.05) growth stimulation of T. atroviride P1. T. atroviride P1 was stimulated up to 31.6% on PDAd after 48 h of co-incubation with P69. Results from transcriptomic analysis show that the biggest changes in P69 gene expression are mainly due to the lack of nutrients. Significant gene expression changes were found comparing the treatments from experiment 1 (samples 1 and 3, nutrient-rich conditions) with treatments from experiment 2 (samples 5 and 7, nutrient-poor conditions) where the nutrient availability conditions were different.
2.Trichoderma velutinum G1/8 - Stenotrophomonas rhizophila DSM14405T (P69) interaction with V. dahliae in vitro
In dual-cultures on WA, V25 was fully stopped and only partially able to grow into, but not to go through P69 colonies. As stated before, this might be due to competition for nutrients and even in this case no inhibition zone was formed. Conversely, on WAd, V25 was significantly inhibited. Trials to assess V25 viability in co-colture with P69 revealed that V25 hyphae were very highly colonized by P69 cells after 3-4 days on WA and 4-7 days on WAd. Colonization seems to start from hyphal tips. Probably, as a consequence of this intense bacterial colonization, V25 hyphae dye. The same behavior was observed for both media, however on WAd it was delayed. Interestingly at 15 and 30 days of co-culture on WA, the P69-colonized fungal mycelium did not show any staining probably because of the loss of internal and external hyphal structure. This phenomenon is hard to explain, so far we can only hypothesize that fungal disruption led to the release of free radicals, or other antibacterial compounds. This different behavior leads us to think that the nature of the interaction S. rhizophila-V. dahliae and S. rhizophila-Trichoderma spp. is different.
3.Plant colonization by Trichoderma velutinum G1/8 - Stenotrophomonas rhizophila DSM14405T (P69)
For these studies we have used tomato plants as a model. Effect of single or combined application of the microorganisms on growth of tomato plants was assessed. Single application of S rhizophila P69 did not cause a significant increase of roots and shoots of tomato plantlets compared to the control. Treatments with single application of T velutinum G1/8 and T atroviride P1 caused the significant increase of tomato roots and shoots. The treatment with the combined application of S rhizophila P69 and T velutinum G1/8 resulted in the significant increase in tomato roots fresh weight compared to treatments with single application of Trichoderma spp, but not in the shoots fresh weight. The treatment with the combined application of S rhizophila P69 and T atroviride P1/8 resulted in the significant increase in tomato roots and shoots fresh weight compared to the control, but actually not statistically different from treatments with single application of Trichoderma spp.
4.Plant-growth promotion and biocontrol of Verticillium wilt of tomato and pepper plant
In a second in vivo experiment, the plant pathogen V dahliae V25 was added to assess the biocontrol activity of P69 and G1/8 in single and combined application. The biocontrol activity was measured in terms of average fresh weight/plant of tomato. Single application of P69 and V25 do not seem to cause significant effect on both root and shoot fresh weight, whereas only G1/8 cause a significant increase of both. Combined application P69+V25 do not show any remarkable difference compared to the control, whereas the application G1/8+P69 caused the significant increase of tomato shoots fresh weight. The best results seems to have been obtained with the combined application of both BCAs (P69+G1/8) to contrast the adverse effect of V25. In this case the significant increase of both root and shoot fresh weight has been obtained. However, the experiment was designed to be carried in a short time (3 weeks), this probably didn’t allow the pathogen to grow properly and to cause evident symptoms on plants. Longer trials may give better results.
Conclusion
The nature of the interactions P69-Trichoderma spp. and P69-V. dahliae V25 was found to be highly dependent on nutrient availability and on the fungal counterpart interacting with P69. Dual-cultures, volatile mediated interactions and viability experiments carried out on nutrient rich media showed a significant inhibition of the tested fungal species. However, under nutrient poor conditions, the interaction observed for the same microorganisms resulted different. P69 behavior was found to be dependent on the fungal counterpart: Trichoderma spp. were not affected or stimulated whereas V. dahliae was significantly inhibited by P69. This leads to the suggestion that under nutrient rich conditions P69 competes very effectively for nutrients causing the significant growth inhibition of fungal counterparts. Conversely, under nutrient poor conditions, it seems that a specific molecular crosstalk can lead to divergent mechanisms ranging from mutual support or neutral interaction for Trichoderma spp. to a competition/parasitism for V. dahliae V25. Trichoderma spp. were always found to emit a higher number of VOCs than V25. This might suggest that Trichoderma spp. have a higher “predisposition” to establish a volatile crosstalk with others microbial members of the community than V25. Probably, S. rhizophila P69 together with other members of the microbial community, could contribute to the establishment and shaping of a stable and beneficial microbial rhizosphere community which globally takes part to plant growth promotion and protection. Our results provide evidence that various abiotic parameters might be critical for the outcome after an encounter, hence additional synergistic interactions with other species are crucial for the particular advantage. In the underlying case Trichoderma spp. were shown to pose the potential to reinforce the antagonistic advantages of a specific S. rhizophila strain. This could be of particular importance to define microbial-based strategies for biocontrol and plant-growth promotion especially under extreme/adverse conditions.