Plant growth-promoting rhizobacteria (PGPR) in Cannabis sativa ‘Finola’ cultivation: An alternative fertilization strategy to improve plant growth and quality characteristics
Graphical abstract
Introduction
In order to increase crop yields and quality, the application of chemical fertilizers in agriculture has increased over time with implications in terms of higher production costs as well as of environmental threats regarding water pollution (ground water, lakes, rivers), air (GHG emission) and soil (reduction of pH and exchangeable bases, low organic matter content) (Savci, 2012). A promising alternative to chemical fertilizers is represented by plant growth-promoting rhizobacteria (PGPR) which, since they firstly definition (Kloepper and Schroth (1978), have been efficiently applied in agriculture due to their positive effect on crop productivity and ecosystem functioning (Vejan et al., 2016). These bacteria are able to promote plant growth and development through several mechanisms, such as: atmospheric nitrogen fixation, siderophores production (siderophores chelate iron to be available to the plant root), minerals solubilization (particularly phosphorus), production of plant-growth promoting substances (such as auxins, cytokinins, gibberellins), and the synthesis of several other growth-promoting compounds (e.g. enzymes) (Glick and Bashan, 1997; Pérez-Montaño et al., 2014). These microorganisms, which associate with almost all plant species (Rosenblueth and Martínez-Romero, 2006), belong to the genera Acinetobacter, Alcaligenes, Arthrobacter, Azospirillium, Azotobacter, Bacillus, Beijerinckia, Burkholderia, Enterobacter, Erwinia, Flavobacterium, Gluconacetobacter, Herbaspirillum, Rhizobium, Serratia, and others still unknown (James et al., 2001; Roncato-Maccari et al., 2003; Sturz and Nowak, 2000). In particular Azospirillum sp., by stimulating elongation of root systems (Dobbelaere et al., 1999), formation of lateral and adventitious roots (Molina-Favero et al., 2008), root hairs (Fulchieri et al., 1993), and root hairs branching (Jain and Patriquin, 1985), plays a major role in the improvement of crop growth and yield under several environmental and soil conditions (Bashan and de-Bashan, 2010; Pereg et al., 2016). Azospirillum species have demonstrated their potentialities as biofertilizers applied both alone (Mehnaz, 2015), or in consortium with other microbial species (Raja et al., 2006; Rajasekar and Elango, 2011; Sarma et al., 2015; Shahzad et al., 2017): Azospirillum brasilense with Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, and Burkholderia ambifaria, demonstrated its beneficial effects on Solanum lycopersicum L. plant growth and development (Botta et al., 2013). Similar benefits have been recorded with the employment of Gluconacetobacter diazotrophicus on Saccharum officinarum L. and Sorghum bicolor L. (Muthukumarasamy et al., 2002; Yoon et al., 2016), of Herbaspirillum seropedicae on Oryza sativa L., Sorghum bicolor L., and Zea mays L. (James et al., 2001; Gyaneshwar et al., 2002; Roncato-Maccari et al., 2003; Canellas et al., 2013) and of Burkholderia ambifaria on Sorghum bicolor L. and Zea mays L. (Di Cello et al., 1997; Chiarini et al., 1998, 2006; Compant et al., 2008;).
It is known PGPR positive influence on secondary plant metabolism (Jain et al., 2014; Khalid et al., 2017; Kilam et al., 2015; Kiprovski et al., 2016) although the mechanisms behind this effect has not been completely clarified.
In Italy the cultivation of industrial hemp, (Cannabis sativa L.) in the past disappeared both for economic and administrative reasons (Cappelletto et al., 2001), has been recently recovered in several regions, i.e. Abruzzo principally with cv. ‘Finola’ due to its short growth cycle and height. Nowadays the plant offers several potential utilizations in biological, alimentary, and industrial fields (Fike, 2016), mostly thanks to its secondary metabolites with different biological properties, including cannabinoids - mainly Δ9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD) - as well as terpenoid-like and phenolic compounds (Flores-Sanchez and Verpoorte, 2008; Matteucci et al., 2016). In particular, in the past two decades, hemp female inflorescences and their cannabinoids have played an increasing role in medicine for their therapeutic effects towards the treatment of a large number of disorders (Grotenhermen and Müller-Vahl, 2016). At the same time, the antioxidant activity associated to polyphenolic compounds is usually exploited in the pharmaceutical and food industry (Abootalebian et al., 2016; Pellegrini et al., 2018). In this context, a key role is recovered by the analytical techniques that allows the identification and quantification of these bioactive compounds (e.g. liquid chromatography tandem-mass spectometry) as well as the widely adopted in vitro assays for evaluating the antioxidant capacity with easy and low-cost procedures (e.g. radical scavenging and total phenolic content assays) (Sun et al., 2018).
To date, there are not available data on the potential benefits of root inoculation with PGPR on biomass yield, growth, as well as on secondary metabolites production/accumulation in industrial hemp. Besides, mineral nutrition represents a critical issue, in particular N supply which, although essential, seems to exert positive effects only at limited range of application doses on biomass growth and development (Finnan and Burke, 2013; Amaducci et al., 2015; Tang et al., 2017): Campiglia et al. (2017) recently assessed that rates from 50 to 100 Kg N ha−1 are adequate under several farm conditions. Hence, the present work represents a first attempt aimed at investigating the effects of a consortium of PGPR (Azospirillum brasilense, Gluconacetobacter diazotrophicus, Burkholderia ambifaria, and Herbaspirillum seropedicae) on female plants of an industrial dioecious hemp variety (cv. ‘Finola’), on morphological and physiological traits, and especially on precise quality characteristics. To estimate the effects of such consortium, a comparison with a nitrogen mineral nutrition was imposed. To test our hypothesis, a two-phase experiment, under controlled conditions, was carried out: one preliminary in vitro, to study seed inoculum growth and hemp root colonization, and one under greenhouse to test PGPR biofertilization potential, on plant growth parameters, cannabinoid content, total phenolic accumulation, and antioxidant capacity.
Section snippets
Finola
Cannabis sativa ‘Finola’ (previously “FIN-314”) is a dioecious cultivar characterized by early sowing and flowering, low branching and short length, with smaller seeds and lower levels of biomass recovery than other hemp varieties; Finola was first accepted in Finland in the list of official plant cultivars, in 2003 and was published in the EU Common Catalogue (Callaway, 2004). Finola, as well as the other hemp cultivars, is a low-maintenance crop, which needs no herbicides or other pesticides;
PGPR colonization of Hemp seedling roots
The PGPR consortium ability to colonize root tissues was investigated in 8 days-old seedlings of hemp by scanning electron microscopy (SEM). The obtained SEM images are reported in Fig. 2. From the images is clearly visible that: (i) the bacteria actively stimulate root hair proliferation (Fig. 2A), (ii) adhere to root surface (Fig. 2B), and (iii) penetrate inside the inner root tissues (Fig. 2C). A comparison between treated and control root surface is shown in Fig. 3. In this Figure the
Summary/conclusions
This preliminary study allowed obtaining information on the utilization of PGPR as biofertilizer for C. sativa ‘Finola’. In vitro study revealed an excellent ability of the bacteria to adhere to the roots surface, and to colonize root vascular tissues of hemp seedling. From our in vivo results it emerges that the application of PGPR could be very interesting since it improves not only growth and plant physiological status, but also plant secondary metabolites accumulation and antioxidant
Authors contribution
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Declaration of interest
None.
Acknowledgments
The Authors are greatly thankful to Alessandro Palumbo of “Hemp Farm Italia” for providing plant seeds material and to Maria Di Giammatteo and Lorenzo Arrizza of “Centro di Microscopie – Università degli dell’Aquila” for their support with the Scanning Electron Microscopy.
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These authors contributed equally to this article.