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Plant growth-promoting bacteria (PGPB) colonize plants and enhance their growth by different mechanisms. Some of these microorganisms may represent a potential threat to human, animal or plant health; however, their use might be approved in parts of Europe if they have been recommended as plant growth enhancers. The current regulatory framework has resulted in a fragmented, contradictory system, and there is an urgent need to establish harmonized protocols for the predictability, efficiency, consistency and especially the safety of PGPB for human and animal health and for the environment. In response to current efforts to update biosafety policies and provide alternative methods to replace the use of vertebrate animals, we propose a panel of tests and an evaluation system to reliably determine the biosafety of bacterial strains used as PGPB. Based on the results of different tests, we propose a scoring system to evaluate the safety of candidates for PGPB within the limitations of the assays used.
Bacteria that are beneficial for plant growth have been used since the 1970s to increase crop production (
Plant growth-promoting bacteria can be found associated to plant roots, shoots, and leaves, or in the fruits or seeds (
The European Parliament and the Council of the European Union are working to produce new regulations for 2017. Currently, Regulation (EC) No 2003/2003 aims to regulate the correct use of materials intended mainly to provide nutrients to plants, regardless of whether microorganisms or other types of products are involved. Regulation (EC) No 1107/2009 recognizes explicitly that “plant protection products may involve risks and hazards for humans, animals and the environment, especially if placed on the market without having been officially tested and authorized and if incorrectly used.” This regulation further establishes that “in the interest of predictability, efficiency and consistency, criteria procedures and conditions for the authorization of plant protection products should be harmonized, account being taken of general principles of protection of human and animal health and the environment.” Regulatory harmonization, however, cannot rely on animal tests as noted in “the development of non-animal test methods should be promoted in order to produce safety data relevant to human and to replace animal studies currently in use.” Moreover, Directive 2010/63/EU specifies that “minimized animal testing and tests on vertebrates should be undertaken as a last resort.”
In light of the need to comply with current regulations aimed to ensure human and environmental safety, we developed a set of biosafety tests for PGPB that assess the potential impacts of the products released by these microorganisms on microbial metabolism (Microtox® testing in
In the tests reported here we also considered whether, according to EC Regulation No 1107/2009 the microorganisms “present a clear benefit for plant production.”
The results of all tests were translated into a scoring system to generate what we termed the
In this work we piloted the EHSI to test the safety of a minimum of 108 cells of
The bacterial strains we used were
Sensitivity assays in
Light emission by
Bacterial killing of
These bioassays were carried out according to
Earthworm reproduction tests (
The toxicity of bacterial extracts to
Pathogenicity in an animal model was tested with a modification of the method of
Growth promotion of pepper plants inoculated with PGPB candidates was tested according to
All tests were performed independently tree times. For statistical testing, analysis of variance (ANOVA) was used for each test with a significance level of
The potential effect of PGPB candidates on microbial communities was assessed in two different areas: microbial viability (sensitivity assay) and microbial metabolism (bioluminescence assay). The premise is that secondary metabolites produced and released to the environment can be collected from the growth medium, most likely during the idiophase. Therefore we studied the effect on
Apart from its potential effect on the nematode community in the environment, the addition of a PGPB candidate to soil or plants can potentially alter the abundance of non-pest herbivores, predators and parasitoids of pest species, or pollinators, and this in turn will affect the environmental equilibrium. Green lacewings (
Although published reports are available on the impact of chemicals (
The addition of PGPB candidates to enhance plant growth should not have any detrimental effect on other plant species. To address this concern, we tested both bacterial strains in pepper plants (
The addition of
The laboratory mouse (
Although models of pathogenesis based on the interaction between bacterial pathogens and higher organisms such as
Environmental and human safety index (EHSI) tests included and mouse pathogenicity tests with results of
Maximum Possible Value (MPV) | Score for test strain |
Modeling for EHSI Categories | |||||
---|---|---|---|---|---|---|---|
Sensitivity test with |
CFUs/mL | A reduction of 50% indicates possible influence on soil microbiota | Soil microbiota | 10 | 10 | 5 | Mortality |
Microtox® Test ( |
EC50 | Genus |
Freshwater organisms | 5 | 5 | 1.25 | Development |
Effects extrapolated to microbiota | Soil microbiota | ||||||
Bioassay with |
No. Adults | Nematofauna are good markers of the status of soil microbiota | Soil microbiota | 6 | 6 | 1.5 | Reproduction |
No. Juveniles | Nematofauna are good markers of the status of soil organisms | Soil surface/Underground organisms | 10,5 | 10,5 | 2.625 | Reproduction | |
No. Eggs | Human health | 6 | 6 | 1.5 | Reproduction | ||
No. Deaths | 25 | 25 | 6.25 | Mortality | |||
Bioassay with |
Length | Good markers of transfer in the food chain and sensitive biological control organisms | Soil surface organisms | 1 | 1 | 1 | Development |
Weight | Beneficial organisms | 2 | 2 | 2 | Development | ||
No. Deaths | 3.75 | 3.75 | 3.75 | Mortality | |||
Bioassay with |
Length | Good markers of transfer in the food chain and sensitive biological control organisms | Soil surface organisms | 1 | 1 | 1 | Development |
Weight | Beneficial organisms | 2 | 2 | 2 | Development | ||
No. Deaths | 3,75 | 3.75 | 3.75 | Mortality | |||
Bioassay/Ecotoxicity test with |
Length | Earthworms are good markers of the status of soil organisms since they are involved in the health of soil microbiota and soil ecosystems: nutrients, structure | Soil surface/Underground organisms | 2 | 2 | 2 | Development |
Weight | 3 | 3 | 3 | Development | |||
No. Juveniles | Soil microbiota | 4,5 | 4.5 | 4.5 | Reproduction | ||
No. Ootheca | Beneficial organisms | 3 | 3 | 3 | Reproduction | ||
DaphtoxKit Test ( |
EC50 | Organisms greatly affected by changes in their environment | Freshwater organisms | 7,5 | 5.625 | 1.875 | Mortality |
Test of bacterial effects on plants (based on pepper, |
Shoot length | The plant-microorganism balance | Soil microbiota | 1 | 1 | 1 | Development |
Dry weight (DW) | Agricultural species can carry pathogens that affect farm animals and humans (as habitat and food) | Human mealth | 2 | 2 | 2 | Development | |
Relative water content (RWC) | Surface organisms | 1 | 1 | 1 | Development | ||
100 | 98.125 | 50 | Maximum Score = 100 | ||||
Bioassay with CD1 laboratory mice ( |
Final weight | Mice are good markers of transfer in the food chain | Surface organisms | 15 | 15 | 15 | Development |
Weight gain | 35 | 35 | 8.75 | Development | |||
No. Deaths | Model of pathogenicity in humans | Human health | 50 | 50 | 25 | Mortality | |
100 | 100 | 48.75 | Maximum Score = 100 |
We used the Delphi method to integrate the results from our panel of tests into a single value (
We termed the scale of values the
Maximum scores for different assays were weighted depending on the importance of each test and its relevance for human and environmental safety. For example, the highest possible score for
The score for the development parameter (D) was based on length and weight results obtained in
To calculate the score for the reproduction parameter (R), we used the number of eggs, juveniles and adults from
A PGPB candidate that does not alter any of the prespecified values used as safety indicators would obtain the maximum score of 100 and therefore would be considered safe to use under the assayed conditions. Intermediate scores vary depending on the magnitudes of the effects in different assays. If the difference compared to the negative control scores (i.e., the maximum score) is very small or negligible, and falls within the first quartile Q1 (0–24%), the final score is calculated by multiplying by 1.0. For example, if
To validate the potential applicability of this scoring system we calculated the EHSI for
Environmental and human safety index (EHSI) tests included and mouse pathogenicity tests.
Environmental and human safety index |
Score for test strain. Modeling for EHSI Categories | ||||||||
---|---|---|---|---|---|---|---|---|---|
Sensitivityy test with |
CFUs/mL | 5 | 5 | 5 | 2.5 | 10 | 10 | 10 | 10 |
Microtox® Test ( |
EC50 | 1.25 | 1.25 | 1.25 | 1.25 | 2.5 | 2.5 | 2.5 | 2.5 |
Bioassay with |
No. Adults | 1.5 | 1.5 | 1.5 | 1.5 | 3 | 3 | 3 | 3 |
No. Juveniles | 2.625 | 2.625 | 2.625 | 2.625 | 5.25 | 5.25 | 7.875 | 5.25 | |
No. Eggs | 1.5 | 1.5 | 1.5 | 1.5 | 3 | 3 | 4.5 | 3 | |
No. Deaths | 0 | 0 | 0 | 0 | 6.25 | 6.25 | 12.25 | 6.25 | |
Bioassay with |
Length | 0.75 | 1 | 1 | 1 | 1 | 0.75 | 1 | 1 |
Weight | 1.5 | 1.5 | 1 | 1 | 2 | 1.5 | 2 | 2 | |
No. Deaths | 0 | 0 | 0 | 0 | 2.8125 | 2.8125 | 3.75 | 2.8125 | |
Bioassay with |
Length | 0.5 | 0.5 | 0.5 | 0.5 | 0.75 | 0.75 | 0.75 | 0.75 |
Weight | 0.25 | 0.25 | 0.25 | 0.25 | 1 | 1.5 | 2 | 1.5 | |
No. Deaths | 0 | 0 | 0 | 0 | 1.875 | 2.8125 | 3.75 | 2.8125 | |
Bioassay/Ecotoxicity test with |
Length | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
Weight | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | |
No. Juveniles | 3.375 | 3.375 | 2.25 | 2.25 | 2.25 | 3.375 | 4.5 | 3.375 | |
No. Ootheca | 2.25 | 2.25 | 2.25 | 1.125 | 3 | 2.25 | 2.25 | 2.25 | |
DaphtoxKit® Test ( |
EC50 | 1.875 | 1.875 | 1.875 | 1.875 | 3.75 | 3.75 | 3.75 | 3.75 |
Test of bacterial effects on plants (based on pepper, |
Shoot length | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Dry weight | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | |
RWC | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | |
31.375 | 31.625 | 30 | 26.375 | 57.4375 | 58.5 | 72.875 | 59.25 |
Bacteria that will be released into the environment to promote plant growth should be safe for humans, animals and the environment. At present, however, there are no internationally harmonized, reliable protocols to evaluate the safety of these bacterial strains. We propose a panel of tests and an evaluation system to accurately determine the safety of bacterial strains. Our set of assays holds the potential to reduce the number of vertebrate animals needed for biosafety testing. We hope that these tests will help policy makers in their efforts to develop new regulations. The EHSI is a new instrument that holds the potential to facilitate the prediction of potential harms to human health and the environmental caused by organisms that are under investigation for use as PGPB. The combination of tests in microorganisms and pathogenicity assays in laboratory mice can help reduce the need to use vertebrates in experimental research – one of the aims of current 3Rs policies (reduce, replace, refine) regulating the use of animals for research purposes. The modular nature of the EHSI makes it easy to interchange target organisms depending on the local environmental. For example, if a reduction is observed in the local bee population and no river or fresh water habitats are located near target crops for a particular PGPB, assays in
JV and SA have performed the experimental assays. JV, JG-L, and AN have performed the statistical analysis. JV and MM have designed and written the article.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Dr. Estefanía Hinarejos (IAB S.L., Spain) and Dr. Gloria Andrea Silva-Castro (University of Granada) for their useful discussions, and K. Shashok for improving the use of English in the manuscript.