Periodic Report Summary - ECOMYCORRHIZA (The effects of agronomic practices conducive to organic agriculture on the diversity and function of arbuscular mycorrhizal fungi)
Arbuscular mycorrhizal fungi (AMF) extensively form symbiotic relations with most plants which in most cases result in benefits to the plant including improved nutrition, amelioration of drought stress, improved soil structure, and protection from soil pathogens. AMF are considered key microbial players in low-input agricultural systems like organic farms. Selected efficient AMF could be used as soil inoculum in organic agriculture to benefit plant growth. Crop protection in such organic farms relies on pesticides of biological origin like azadirachtin, spinosad and other natural products. The application of by-products of local agricultural industry like olive mill wastewaters (OMWs) might suppress soil plant pathogens and/or improve soil fertility. Although all the above practices are conducive to organic farms there are no data regarding their effects on AMF and fungal community. We aimed to investigate:
1) the diversity and function of AMF in vegetable organic farms in Greece
2) to isolate, characterise and propagate pure cultures of AMF from organic farms in Greece and assess their efficacy to promote plant growth at the laboratory as well as at the field scale
3) to assess the effects of agronomic practices including crop protection and fertilisation via application of wastewaters enriched in organic C on the structure and function of the AMF community in plant roots.
During the first 18 months of the project the objectives which had been set were achieved. Therefore, an initial survey was conducted in vegetable organic farms, identified mainly by the accreditation organisms DIO (see http://www.dionet.gr online), and Biohellas (see http://www.bio-hellas.gr/gr online). In total, thirty one organic vegetable farm fields were sampled from 20 farms, and root samples were used as inoculum in order to establish trap-cultures and assess the diversity of AMF in organic farms in Greece. An initial microscopic grouping of AMF spores was performed. However, a more thorough assessment of the biodiversity of AMF fungi in these samples collected from organic farms is in progress using molecular biological techniques.
However, the initial groups of spores prepared were utilized as inoculum for the gradual development of pure AMF cultures through a series of sequential trap-cultures in maize. This led to the establishment of five potentially pure AMF cultures. Molecular characterisation of these pure cultures showed that four out of the five were pure and were identified as different species of the genus Glomus, G. intraradices (MC3), G. etunicatum (MC4) and G. mosseae (MC10, MC22). The mixed AMF culture was a mixture of spores of G. mosseae and G. etunicatum. These results were verified by cloning and sequencing of the most variable region of the 18S rRNA gene of fungi using primers AML1 - AML2. The results were also verified by DGGE analysis.
Subsequently the pure AMF cultures isolated were assessed in a pot experiment with pepper and tomato plants for their efficiency to colonize plants and promote plant growth and P uptake. These pot experiments showed that the AMF cultures colonised successfully the roots of pepper compared to a lower level of colonisation observed in the tomato plants by all fungi. The AMF cultures did not significantly promote plant growth (shoot and root biomass) during the duration of the experiment but the mixed culture 27 which comprised spores of G. mosseae and G. etunicatum drastically and significantly increased P concentration in plant roots in peppers. The absence of growth promotion by our isolates could be attributed to the limitations of time and space for the growth of plants in the pots. Thus, the full potential of the AMF cultures isolated will be tested in the field in the second period of the project as has planned from the beginning.
Finally, the effects of plant protection products (PPP) used in organic farming and of wastewaters which are commonly used as organic fertilisers in organic farming was assessed in pot experiments. The short-term effects of OMW on the soil-plant system, regarding the diversity, structure and root colonisation capacity AMF and the respective growth response of Vicia faba L, commonly used as green manure, were evaluated. A compartmentalised pot system that allowed the establishment of an AMF community in one compartment (feeder) and the application of three OMW dose levels in an adjacent second compartment (receiver) was used. At 0, 10, and 30 days after OMW treatment (DAT), V. faba pre-germinated seeds were seeded in the receiver compartment. At harvest, shoot and root dry weights, soil hyphal length and AMF root colonisation were recorded in the receiver plants.
In addition, OMW effects on AMF diversity in plant roots were studied by DGGE. A transient effect of OMW application was observed; plant growth and AMF colonisation were initially inhibited whereas total soil hyphal length was stimulated, but in cases differences were alleviated when seeding was performed 30 DAT. Similarly, changes induced in the structure of the root AMF community were of transient nature. Cloning and sequencing of all the major DGGE bands showed that roots were colonised mainly by uncultured Glomus spp. The transient effects of OMW on the structure and function of AM fungi could be attributed to a direct toxicity of OMW-phenolics or to an indirect effect via alteration of soil nutritional status. Our results indicate that soil application of OMW would cause transient changes in the AM fungal colonisation of V. faba plants, which, reciprocally, would not, impair their long-term plant growth promoting ability.
In a second pot experiment, we assessed the effects of pesticides of botanical origin including azadirachtin, spinosad, pyrethrum, a terpenoids mixture and the fungicide carbendazim (negative control) on a mixed AMF inoculum isolated as previously describe. A non-pesticide treated AMF-inoculated control and a non-AMF inoculated control were also included in the study. Pesticides were soil applied two days prior to inoculation with the AMF mixture. Pepper plants were transplanted at the day of inoculation (0 days) and harvested at 20 and 40 DAT. The percentage of AMF root length colonisation, root and shoot biomass, and plant height were determined in order to assess the effects of pesticide treatments on plant growth. In addition, the effects of pesticides on the structure of the AMF community in plant roots were also determined at 20 and 40 days using a PCR-DGGE as described before.
We found that terpens promoted both shoot and root growth, although this was not linked with significantly higher root colonisation by AMF compared to AMF-inoculated control samples. On the other hand, pyrethrum significantly suppressed root colonization by AM fungi, but this was not linked with any inhibition in plant growth. Regarding the effects of pesticides on the structure of AMF community in plant roots, azadirachtin directly or indirectly suppressed certain members of the AMF community. Thus, azadirachtin had a negative effect on the diversity of AMF in plant roots whereas it increased root colonization by AMF compared to non-pesticide treated control. These results are the first to investigate the effects of botanical pesticides on the structure of the AMF community in plant roots. The website of the project has been launched, and can be found at http://ecomycorrhiza.bio.uth.gr.
In the second period of the project a series of field experiments will be conducted in order to verify the findings of the pot experiments. It is expected that the outcome of the field experiments will provide realistic and robust evidence for the function of the isolated AMF cultures and will further reinforce are original findings that certain PPP will have adverse effects on the biodiversity of AMF. This will have implications for the use of these chemicals in organic farming.
1) the diversity and function of AMF in vegetable organic farms in Greece
2) to isolate, characterise and propagate pure cultures of AMF from organic farms in Greece and assess their efficacy to promote plant growth at the laboratory as well as at the field scale
3) to assess the effects of agronomic practices including crop protection and fertilisation via application of wastewaters enriched in organic C on the structure and function of the AMF community in plant roots.
During the first 18 months of the project the objectives which had been set were achieved. Therefore, an initial survey was conducted in vegetable organic farms, identified mainly by the accreditation organisms DIO (see http://www.dionet.gr online), and Biohellas (see http://www.bio-hellas.gr/gr online). In total, thirty one organic vegetable farm fields were sampled from 20 farms, and root samples were used as inoculum in order to establish trap-cultures and assess the diversity of AMF in organic farms in Greece. An initial microscopic grouping of AMF spores was performed. However, a more thorough assessment of the biodiversity of AMF fungi in these samples collected from organic farms is in progress using molecular biological techniques.
However, the initial groups of spores prepared were utilized as inoculum for the gradual development of pure AMF cultures through a series of sequential trap-cultures in maize. This led to the establishment of five potentially pure AMF cultures. Molecular characterisation of these pure cultures showed that four out of the five were pure and were identified as different species of the genus Glomus, G. intraradices (MC3), G. etunicatum (MC4) and G. mosseae (MC10, MC22). The mixed AMF culture was a mixture of spores of G. mosseae and G. etunicatum. These results were verified by cloning and sequencing of the most variable region of the 18S rRNA gene of fungi using primers AML1 - AML2. The results were also verified by DGGE analysis.
Subsequently the pure AMF cultures isolated were assessed in a pot experiment with pepper and tomato plants for their efficiency to colonize plants and promote plant growth and P uptake. These pot experiments showed that the AMF cultures colonised successfully the roots of pepper compared to a lower level of colonisation observed in the tomato plants by all fungi. The AMF cultures did not significantly promote plant growth (shoot and root biomass) during the duration of the experiment but the mixed culture 27 which comprised spores of G. mosseae and G. etunicatum drastically and significantly increased P concentration in plant roots in peppers. The absence of growth promotion by our isolates could be attributed to the limitations of time and space for the growth of plants in the pots. Thus, the full potential of the AMF cultures isolated will be tested in the field in the second period of the project as has planned from the beginning.
Finally, the effects of plant protection products (PPP) used in organic farming and of wastewaters which are commonly used as organic fertilisers in organic farming was assessed in pot experiments. The short-term effects of OMW on the soil-plant system, regarding the diversity, structure and root colonisation capacity AMF and the respective growth response of Vicia faba L, commonly used as green manure, were evaluated. A compartmentalised pot system that allowed the establishment of an AMF community in one compartment (feeder) and the application of three OMW dose levels in an adjacent second compartment (receiver) was used. At 0, 10, and 30 days after OMW treatment (DAT), V. faba pre-germinated seeds were seeded in the receiver compartment. At harvest, shoot and root dry weights, soil hyphal length and AMF root colonisation were recorded in the receiver plants.
In addition, OMW effects on AMF diversity in plant roots were studied by DGGE. A transient effect of OMW application was observed; plant growth and AMF colonisation were initially inhibited whereas total soil hyphal length was stimulated, but in cases differences were alleviated when seeding was performed 30 DAT. Similarly, changes induced in the structure of the root AMF community were of transient nature. Cloning and sequencing of all the major DGGE bands showed that roots were colonised mainly by uncultured Glomus spp. The transient effects of OMW on the structure and function of AM fungi could be attributed to a direct toxicity of OMW-phenolics or to an indirect effect via alteration of soil nutritional status. Our results indicate that soil application of OMW would cause transient changes in the AM fungal colonisation of V. faba plants, which, reciprocally, would not, impair their long-term plant growth promoting ability.
In a second pot experiment, we assessed the effects of pesticides of botanical origin including azadirachtin, spinosad, pyrethrum, a terpenoids mixture and the fungicide carbendazim (negative control) on a mixed AMF inoculum isolated as previously describe. A non-pesticide treated AMF-inoculated control and a non-AMF inoculated control were also included in the study. Pesticides were soil applied two days prior to inoculation with the AMF mixture. Pepper plants were transplanted at the day of inoculation (0 days) and harvested at 20 and 40 DAT. The percentage of AMF root length colonisation, root and shoot biomass, and plant height were determined in order to assess the effects of pesticide treatments on plant growth. In addition, the effects of pesticides on the structure of the AMF community in plant roots were also determined at 20 and 40 days using a PCR-DGGE as described before.
We found that terpens promoted both shoot and root growth, although this was not linked with significantly higher root colonisation by AMF compared to AMF-inoculated control samples. On the other hand, pyrethrum significantly suppressed root colonization by AM fungi, but this was not linked with any inhibition in plant growth. Regarding the effects of pesticides on the structure of AMF community in plant roots, azadirachtin directly or indirectly suppressed certain members of the AMF community. Thus, azadirachtin had a negative effect on the diversity of AMF in plant roots whereas it increased root colonization by AMF compared to non-pesticide treated control. These results are the first to investigate the effects of botanical pesticides on the structure of the AMF community in plant roots. The website of the project has been launched, and can be found at http://ecomycorrhiza.bio.uth.gr.
In the second period of the project a series of field experiments will be conducted in order to verify the findings of the pot experiments. It is expected that the outcome of the field experiments will provide realistic and robust evidence for the function of the isolated AMF cultures and will further reinforce are original findings that certain PPP will have adverse effects on the biodiversity of AMF. This will have implications for the use of these chemicals in organic farming.