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The Metabolic Elicitors of the Pseudomonas fluorescens N 21.4 Strain as Effective Biotechnological Inoculants for the Cultivation of Blackberry
* 1 , 2 , 2 , 3 , 3
1  Universidad San Pablo-CEU Universities
2  Plant Physiology, Pharmaceutical and Health Sciences Department, Faculty of Pharmacy, Universidad San Pablo-CEU Universities, 28668 Boadilla del Monte (Madrid), Spain
3  Plant Physiology Pharmaceutical and Health Sciences Department, Faculty of Pharmacy, Universidad San Pablo-CEU Universities, 28668 Boadilla del Monte (Madrid), Spain


The current need to provide sufficient and quality food to a growing population is linked to the development of new agricultural techniques based on improving the resistance of crops against biotic and abiotic stress. Since the consumption of blackberry is becoming very popular for its benefits on human health and it is a crop with a lack of specific inoculants able to boost plant resistance, we looked for an efficient, affordable, and sustainable biotechnological inoculant made of metabolic elicitor molecules of the beneficial rhizobacterium Pseudomonas fluorescens N 21.4 to get a more stress-resilient crop. For this, we inoculated the live rhizobacterium as positive control, since its capacity to elicit blackberry secondary defensive metabolism has been previously demonstrated (Garcia-Seco et al. 2015), and its metabolic elicitors to commercial cultivars of blackberry (Rubus cv. Loch Ness). We measured stress markers such as photosynthetic parameters, oxidative stress enzymatic activity, pathogenesis-related proteins and polyphenolic composition of the leaves, and the bioactive content of the fruit in two stages of ripening.

Our results showed that the metabolic elicitor-inoculated plants were more prepared to cope with stress since higher values of photosynthesis were observed, as well as lower enzymatic activity and an activation of pathogenesis-related proteins. Furthermore, the leaves and the fruits of the inoculated plants showed an enhancement in the concentration of beneficial polyphenols, highlighting the increase in epicatechin (Martin-Rivilla et al. 2020). This would mean having better quality fruits and would revalorize the leaves pruning as a potential source of polyphenols, providing an added value to the crop while following the premises of the circular economy.

The elicitation of the blackberry secondary defensive metabolism therefore demonstrates metabolic elicitors’ capacity to reinforce plant immune system and to increase the synthesis of beneficial bioactives, and this work suggest them as effective and ecofriendly plant inoculants.

Keywords: Blackberry; Pseudomonas fluorescens N 21.4; Metabolic elicitors; Secondary defensive metabolism; Circular economy; Epicatechin.
Comments on this paper
Paula Garcia Fraile
ME inoculum question

I wanted to know more about the relation between the analysis of photosynthetic parameters and the plant stress resistance.

On the other hand, I wanted to know your opinion about the safety of the strain applied as bioinoculant to the crops, considering that some studies have found strains of the P. fluorescens species associated to some human diseases (see

Thank you.
Helena Martin-Rivilla
Hello Paula!
Thank you for your comments!
About the relation between the analysis of photosynthetic parameters and the plant stress resistance:

A fundamental parameter that reflects the health status and the stress levels of a plant is photosynthesis because the photosynthetic apparatus is the first physiological function to be affected by stress. In our work, plants treated with the strain N 21.4 and with its ME had a photosynthetic apparatus working optimally, were less stressed, and therefore were in better health conditions. The proper photosynthesis performance in treated plants was deduced due to their lower values of F0 (minimal fluorescence yield), since high values of this parameter indicate malfunctions of the photosystem II (PSII) and general photosynthesis damage. On the other hand, Fv/Fm parameter is an estimator of the maximal photochemical efficiency of PSII (PSII maximum capacity to transfer electrons to the electron transport chain) and it is used to identify loss of function of PSII reaction centers. A decrease in Fv/Fm ratio, especially under stress conditions means photoinhibition. However, in our treated plants, higher values of Fv/Fm were observed compared to control plants. Therefore, the results of F0 and Fv/Fm were consistent between them and both reflected that bacterial treatments with strain N 21.4 and with its ME supposed a better functioning of the entire photosynthetic apparatus with greater potential to channel electrons to the electron transport chain and thus to generate more energy. Our treatments decreased photosynthetic damage caused by stress and improved photosynthetic performance and efficiency, increasing energy production for the plant. The plant could use this extra energy (from a more efficient photosynthesis) to synthetize target secondary metabolites related to defense and to face to stress, therefore plant resistance to stress could be greater.
In summary, it is in this greater energy from photosynthesis and in the consequent greater capacity to synthesize molecules that fight stress where the relationship between the analysis of photosynthetic parameters and the resistance of plants to stress is found.

About your second comment:

As you say, some strains of Pseudomonas fluorescens have been shown to cause problems in humans, especially in immunosuppressed people. The inoculation of live microorganisms in agricultural systems can also suppose biosecurity problems and niche competition with other rhizospheric microorganisms. For all this, the final objective of our work is to identify the secondary metabolites produced by the microorganisms that are the triggers of the beneficial effects in the lants. Thus, living microorganisms could be replaced by inert and innocuous molecules in the production of plant inoculants, these products maintaining the same effectiveness.

I hope my explanations have helped you.

Beatriz Ramos
Pseudomonas and human disease
Thanks for your comment.
I would like to add more information about our experience with bacterial inoculations in greenhouse and field experiments in which we have studied modification of microbial communities.
We have shown that delivering bacterial strains to plant roots causes a disruption of the existing microbial communities in the first hours post inoculation that lasts up to 2 weeks and this perturbation fades along time, showing that the inoculum has integrated in the rhizosphere communities and colonized the roots; positive effects on plant fitness are associated to low perturbation of the rhizosphere communities. When the perturbation lasts, there are no positive effects on plant growth. With these experiments we demonstrated that bacteria are integrated in the system and we believe they remain attached to the roots.
Furthermore, we have also experienced the need to inoculate every 2 weeks as when inoculations are preformed every 4 weeks, effects disappear. So this suggests that soil "invasion" by bacteria is very unlikely to occur.
So, in answer to your question, a correct management and handling of the bio fertilizer should effectively prevent a hypothetical infection in healthy humans, and duration of inoculum in the system is a good indicator of the low risk associated to these products.
I will be pleased to have further discussion about this matter.