A bacterial formula with native strains as alternative to chemical fertiliser for tomato crop

doi:10.21203/rs.3.rs-2258949/v1

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A bacterial formula with native strains as alternative to chemical fertiliser for tomato crop

https://doi.org/10.21203/rs.3.rs-2258949/v1

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published 02 Apr, 2023

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Worldwide productivity of tomato is threatened by biotic and abiotic stress factors. To sustain and guarantee an adequate yield of tomato crops, agricultural practices have been based on the intensive use of fertilisers with negative impacts on the environment. An eco-friendly and sustainable alternative to the traditional cultivation methods is the bioaugmentation approach, using tailor-made microbial consortia. Eight indigenous strains, isolated from the soil of “Terra-Sole” farm in the coastal plain of Pula (Sardinia - Italy), were selected in the laboratory for their plant growth promoting (PGP) functions. The beneficial effects of the bacterial formula, including genera Delftia, Pseudomonas, Paenarthrobacter, Phyllobacterium, Bacillus, and Acinetobacter, were tested in three subsequent field trials carried out at the company greenhouse, with different tomato varieties (Camone, Oblungo, Cherry). The results indicate that the inoculation of the indigenous bacterial formula repeated at the different stages of plant growth, regardless of the tomato variety, represents an effective strategy to obtain a fruit yield comparable to that obtained with chemical fertilisers. The application of proper biofertilisation could thus substitute the use of expensive and polluting chemicals without compromising the tomato yield.

plant growth-promoting bacteria

tomato crop

bioaugmentation

sustainable agriculture

bacterial inoculum

biofertiliser

Tomato (Solanum lycopersicum L.) represents one of the most important crops because of its great economic and nutritional value (Cordero et al. 2018; Čechura et al. 2021). Global annual tomato production, increasing by 300% over the last four decades (Giuliani et al. 2019), is currently around 180 million tons (Wu et al. 2022). European tomato production, according to FAOSTAT data (Statistics Division of the Food and Agriculture Organisation of the United Nations 2022), represents around 13% of global production and Italy is the main producer in the European Union with almost 75% of its production oriented towards processing and the rest for fresh consumption (Čechura et al. 2021). In 2021, on a surface of over 71 thousand hectares, Italy produced over 6 million tonnes of processing tomatoes, up by 17% compared to 2020, placing itself as the second producing country in the world after the United States and clearly ahead of China (ANICAV Italy’s National Industrial Association of Vegetable Food Preserves 2021). Italian tomato production is spatially concentrated in the northern area of Emilia-Romagna region and in the south regions of Puglia and Campania. Nevertheless, this cultivation represents one of the most important crops from an economic point of view, also for other local communities such as Sardinia region. Despite the last Italian tomato harvesting season being very successful, the world scenario is characterised by a production that in the last year has remained stable, while the tomato consumption around the globe is constantly increasing (François-Xavier Branthôme 2022a).

With demand for tomato products at a historical high, there is even more pressure on growers to cope with water deficiency, alternative crop competition, increased input costs, and other economic factors. To continue to be competitive, Italian tomato growers need to improve their productivity and efficiency, reducing average costs per unit of output. Until now, the strategy to get high yield enough to allow farmers to gain a profit margin, taking in account the low price of tomatoes, was a very intensive agroecosystem, based on a large exploitation of chemical fertilisers, pesticides, herbicides, and water (Giuliani et al. 2019; Morra et al. 2021). Anyway, with rising costs of chemicals, this intensive production system has become very expensive and, in the long run, it could have high costs also in terms of environmental impact and reduction of agricultural yields. In fact, intensive agriculture contributes to degrade soil quality, changes its structure and its water holding capacity and increases surface runoff and loss of nutrients, causing water sources eutrophication and leading to food safety decline and public health hazards (Manfredi et al. 2019; Ye et al. 2020; Kumar et al. 2022). Moreover, the traditional tomato growing practices are not suited to cope with environmental problems connected to the effects of the ongoing climatic changes, which are expected to be even larger in the upcoming years (Giuliani et al. 2019). We are going to have a more tropical and drier Europe because of the water deficit; annual precipitation is going to decrease and there will be the need for more water to produce one ton of tomatoes because of the increase in the evaporation rate (François-Xavier Branthôme 2022b). The greenhouse farming, frequently used to meet demand for tomato growth particularly during the colder seasons, should also shift its focus from maximising total production to decreased use of irrigation, the main source of water for crop growth in the enclosed space (Wu et al. 2021). Furthermore, the intensive greenhouse production involves chemical fertiliser application that may affect soil chemical and biological quality (Wu et al. 2022). In light of this and taking into account that too much water and fertiliser applied can jeopardise fruit quality, compromise profit and enhance soil pollution (Wu et al. 2022), the greenhouse cultivations also need more efficient and low-cost management practices.

Within the new agricultural technologies, the crop inoculation with plant growth-promoting bacteria (PGPB) as biofertilisers has emerged as a sustainable and environment-friendly method, for improving soil fertility and plant growth, simultaneously reducing the application of synthetic fertilisers, and maximising the efficiency in the use of resources (Flores-Félix et al. 2021; Kumar et al. 2022). Moreover, the cost of biological fertilisers can be competitive in relation to the production cost of chemical fertilisers (Lobo et al. 2019). Many studies report the application of beneficial microbiomes as a valid tool to combat the deterioration of cultivable topsoil caused by traditional agricultural practices, like overgrazing and tilling, and by the over utilisation of chemical fertilisers throughout the years (Cordero et al. 2018; Sah et al. 2021).

Soil quality and microbial rich diversity are crucial for supporting high quality plants growth. Approximately, one gram of soil contains 4000 distinct bacterial genomes (Sah et al. 2021). Among these microbes, PGPB exert their growth-promoting effect through different mechanisms that may be direct, such as the improvement of nutrient availability and the phytohormone production, or indirect, as the competition with harmful soil microorganisms, the enhancement of symbiotic relations, and the contribution towards mitigation of abiotic and biotic stresses (Cordero et al. 2018; Oleńska et al. 2020; Kumar et al. 2022).

The beneficial effects of PGPB have been demonstrated in many studies on species of agronomic interest including cucumber (Gamalero et al. 2008), pepper (del Amor and Cuadra-Crespo 2012), pumpkin, corn, broccoli, lettuce (Angulo et al. 2020). However, tomato is one of the plants most widely used in PGPB application, not only because of its high economic value but also because, being a fast-growing plant species, allows evaluating a high number of strains to select those best performing (Cordero et al. 2018). Nonetheless, a small number of studies compared the effects of the biofertilisation and chemical fertilisers, and even fewer included a stress factor in the comparison (Cordero et al. 2018; Angulo et al. 2020). Moreover, previous research on different crops concluded that PGPB inoculation must be combined with chemical treatment to have the best results on plant growth (Bona et al. 2018; Cordero et al. 2018; Angulo et al. 2020). The same assertion was reported for the tomato species: several authors wrote about the positive effect of microbial inoculation under environmental or biological stresses such as drought, salinity (Mannino et al. 2020; Riva et al. 2021), nutrient deficiency in soils, and pathogens; anyway, the few studies that compared bio- and chemical fertilisation, described a lower yield in fruit production when chemicals have been completely replaced by PGPB inoculations (Adesemoye et al. 2009; Ye et al. 2020). The results of these mentioned works do not encourage the tomato growers to apply the biofertilisation.

There is a great debate on the development of microbial inoculants for agriculture (Kaminsky et al. 2019). Anyway, the greater or lesser success of the biofertilisation could depend on different experimental factors such as the application of a single strain or consortium (Compant et al. 2019), the choice of introducing autochthonous or allochthonous microorganisms (Ortiz et al. 2015), the composition of the chosen inoculum (Adesemoye et al. 2009), the frequency and period of application (He et al. 2019; Riva et al. 2021). Based on a careful literature review about different methods of biofertilisation, the present study aims to propose and test an effective and advantageous customised strategy of bioaugmentation that could be attractive for the farmers. The experimentation described in this paper was carried out in the commercial greenhouse of the farm “Cooperativa Santa Margherita Terra e Sole'' (Pula, Sardinia - Italy) with the aim to improve the sustainability of the agroecosystem by using endemic bacteria as biofertilisers to replace the chemical fertilisation. Three tomato varieties (Camone, Oblungo, Cherry) were tested in three consecutive growing seasons, to assess the more suitable inoculum composition and frequency.

Soil Sampling and Physiological profile at community-level

Samples from the bulk soil of 10 tomato plants (about 100 g from each plant) were randomly collected in the greenhouse of the farm “Cooperativa Santa Margherita Terra e Sole” (Pula, Sardinia - Italy) in September 2018. The soil samples were stored in sterile tubes at RT (room temperature) and transported to the laboratory where they were mixed to create a composite sample for microbiological and chemical analyses. From the composite sample, 50 g were taken in triplicate and placed each in 500 mL of 0.1% sodium pyrophosphate in sterile bottles and agitated in an orbital shaker at 180 rpm for 90 minutes at RT.

The soil suspension (containing the bacterial community) was inoculated into Biolog ECO Plates (Biolog Inc., Hayward, CA, USA), according to Sprocati et al. (2014) to analyse the Community-Level Physiological Profile (CLPP). Briefly, soil suspension was diluted 1:10 and dispensed into the 96 wells of the microplates (100 µL per well) containing 32 different substrates in triplicate. The plates were incubated at 28°C in the dark and were read every 24 hours in the microplate reader using a double wavelength (OD₅₉₀ - OD₇₅₀). Kinetic analysis was performed using average well colour development (AWCD) as a parameter that captures an integral fingerprinting of carbon sources utilisation. AWCD was calculated as the arithmetic mean of the OD values of all the wells in the plate per reading time (Garland 1996). Patterns of substrates utilisation for each soil were recorded and analysed with the Microlog software (version 5.0). Heatmap was generated with Excel (Windows Office 13). Data are the mean of six individual OD values.

Chemical and mineralogical analysis of soil

Mineralogy of the agricultural soil was investigated by powder X-rays diffraction (PXRD) at the University of Cagliari laboratories. Samples were dried at RT, then were manually ground using an agate mortar, and the analysis were carried out using laboratory θ–2θ equipment (Panalytical, Almelo, Netherlands) with Ni-filtered Cu Kα1 radiation (λ = 1.54060 Å), operating at 40 kV and 40 mA, and an X′celerator detector. Chemistry and trace chemistry of the sample were investigated by X-rays fluorescence (XRF) (Potts and Webb 1992). Soil samples were grinded and pressed into solid pellets. Soil analyses were performed with a Rigaku ZSX Primus II WDXRF spectrometer at the Structural Crystallography Centre (CRIST), Florence, Italy. The contents of C, H and N in soil samples were determined by Perkin Elmer 2400 CHN analyser (Hitachi, Tokyo, Japan).

Bacteria isolation and identification

The total heterotrophic bacterial community was enumerated by plating serial dilutions (up to 10^− 5) of the same soil suspension, on different agar media: Tryptic Soy Agar (Laboratorios Conda, Madrid, Spain) as general medium, Mineral Medium (Schmidt and Schlegel 1989) for oligotrophic bacteria selection, and Nitrogen free (NF) Agar (Dobereiner J et al. 1976) to select the nitrogen fixing bacteria (N-fix). The plates were incubated at RT for several weeks until the microbial growth was observed and the heterotrophic bacterial strains were isolated. NF plates were incubated in a microaerophilic atmosphere at RT up to 14 days before proceeding with the isolation of the N-fix isolates. Chemicals for media preparation and for analytical assays were purchased at Carlo Erba (Milano, Italy) unless otherwise specified.

For strains identification, single colony 16S r-DNA amplification was performed by polymerase chain reaction (PCR) with the Euroclone Gradient One thermocycler (Euroclone, Milano, Italy) using the universal Eubacteria primers 9bmf (5’- GAG TTT GAT YHT GGC TCA G -3’) and 1512r (5’- ACG GHT ACC TTG TTA CGA CTT − 3’) to amplify the 16S rRNA gene (ca. 1,500 bp), according to the procedure described by Mühling et al. (2008). Sequence similarity searches were conducted using the BLAST network service of the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/) to identify the nearest relatives of the partially sequenced 16S rRNA genes. The phylogenetic analysis was performed using MEGA X (Kumar et al. 2018). The phylogenetic tree of the aligned sequences was constructed using the neighbour-joining method (Tamura et al. 2004). The sequences generated in this study have been deposited in the GenBank database under accession numbers OP159876 to OP159914. All strains were stored in 20% glycerol at – 80°C for long-term storage.

PGP assays and bacterial formula assembling

The isolated strains were tested for specific plant growth promoting activities such as nitrogen fixation, phosphate and potassium solubilisation (P-sol and K-sol), indo-acetic acid (IAA) and siderophore production.

Nitrogen fixers were selected or tested on a NF solid medium (described in the previous paragraph) on plates incubated at RT and in a microaerophilic environment (Mirza and Rodrigues 2012). The Pikovskaya’s medium (PVK) was used to select strains able to solubilise phosphates (Gupta et al. 1994). Potassium solubilisation was assessed according to the protocol suggested by Zhang (Zhang and Kong 2014), by using solid Aleksandrov medium(Hu et al. 2006) and adding 0.2% of potassium and aluminium silicate in different form: ignimbrite, trachytic lava, microcline (Setiawati and Mutmainnah 2016). The capacity of the strains to produce indole was evaluated with the protocol suggested by Patten and Glick (2002). To detect siderophores production, the isolated strains were tested by chrome azurol S (O-CAS) assay, prepared according toSchwyn and Neilands (1987) followingPérez-Miranda et al. (2007) modification.

The tailor-made microbial formula T-S was assembled on the basis of the best-performing PGP traits. On this consortium the tests described above were repeated over the time in order to verify the maintenance of PGP properties.

Field experiments

Three field experiments were carried out, using three varieties of tomato with different growing seasons: Camone DRW7723 (Bayer,Leverkusen, Germany); Oblungo Artù (Sementiera Medhermes, Ragusa, Italy); and Cherry Dantesco (Vilmorin Italia, Bologna, Italy). The plants were grown in a portion of a commercial greenhouse (“Santa Margherita Terra e Sole”) at Santa Margherita di Pula (Sardinia, Italy). The greenhouse has a surface of 3000 m², with natural light and no cooling/heating supplies, provided with drip irrigation. The distance between the rows was 1 m, while the distance between adjacent plants within a row was 50 − 30 cm depending on the tomato variety. In each experimentation, two entire rows (about 200 plants) were treated with conventional chemical fertiliser (F) and were separated by two rows of plants inoculated with the bacterial Formula T-S (B). According to previous analysis (data not shown) the greenhouse soil was classified as sandy-loam (silt, 22%; clay, 13%; and sand, 65%) with pH 7.25.

The home-made chemical fertiliser was composed by ammonium nitrate 25 g/L, potassium nitrate 25 g/L, calcium nitrate 75 g/L (YaraTera, Italia), magnesium sulphate 25 g/L (DRT, Turkey), potassium sulphate 25 g/L (Haifa Hiberia, Spain), iron chelates -EDDHA 3,8% (Valagro, Italy), phosphoric acid and microelements (Jisa, Spain). The fertiliser mixture was provided by fertirrigation, according to the variety needs. The bacterial Formula T-S was composed of 8 PGP strains selected as described above and inoculated into the soil by watering each plant with 400 mL of bacterial suspension (density about 10⁷ CFU/mL).

The three experimental campaigns were carried out with tomato varieties having shifted growing season and different requirements on water supply and temperature. In order to establish the optimal number of microbial applications (bio fertilisation), the output of the first campaign was used to adjust the following field trials. The first experimentation was performed in the period October 2019-June 2020, on Camone tomato plants. Microbial inoculation on the two selected plant rows was repeated two times (the former in October 2019 and the latter in February 2020), while the chemical fertiliser, on the other two rows, was provided every day by fertigation. The second experimental campaign was performed between October 2020 and February 2021, on the Oblungo variety. The microbial inoculations were provided monthly, while the chemical fertiliser was applied every day. The latest experimental campaign, which began in May 2021 and ended in October 2021, involved the study of Cherry tomatoes, the application of monthly microbial inoculations and daily chemical fertilisation.

Tomato growth parameters and productivity analysis

The principal parameters of plant growth and productivity (plant height, fruit weight and number) were monitored according to the physiological growth of the plant varieties, in order to compare the effectiveness of the two treatments: chemical fertilisation and microbial inoculation. The parameters were expressed as an average of 30 plants per treatment.

The evaluation of the weight and number of tomatoes collected per plant was carried out: i) for the first campaign from May to June 2020; ii) for the second campaign from November 2020 to February 2021; iii) for the third campaign from August to September 2021.

Statistical analyses

The experimental data (average plant height, average weight of the harvest of tomatoes, average number of collected tomatoes) were subjected to statistical analyses, one-way analysis of variance (ANOVA) and Tukey HSD-test, through Xlstat software, in order to evaluate any statistically significant difference between the groups of microbial inoculation and chemical fertilisation, at a significance level α = 0.05.

Results of the tests (K-sol, P-sol, N-fix, IAA, O-CAS) for the evaluation of PGP properties of the microbial formula, were subjected to principal component analysis (PCA), in order to evaluate any difference among bacterial strains with regard to metabolic characteristics. Results were obtained by considering for each bacterial strain the relative percentage of the expressed variables.

Soil characterization

The chemical composition of the farm soil (Table 1) shows high amounts of silica (73.82%) and aluminium oxides (14.29%), the presence of abundant K (K₂O, 6.82%), and P in traces around 600 ppm. The mineralogical association, determined by XRD analyses, consists of quartz (SiO₂)k-feldspar (orthoclase/microcline, KAlSi₃O₈), plagioclase (NaAlSi₃O₈) and phyllosilicates such as phlogopite (KMg₃(Si₃Al)O₁₀(OH)₂), muscovite (KAl₂(AlSi₃O₁₀)(OH)₂), illite (K_0.65Al_2.0[Al_0.65Si_3.35O₁₀](OH)₂) and kaolinite (Al₂Si₂O₅(OH)₄). These results are in agreement with the geological setting of the area, characterised by the widespread occurrence of granitic rocks (Barca et al. 2009). Quartz, feldspars and muscovite are primary components of granitoids, whereas kaolinite and illite are weathering products of feldspars and muscovite, respectively. CHN analysis results indicate C values in the low range. The technique detects total C from carbonate minerals and organic materials; as the soil is poor in carbonate this amount has to be referred to organic carbon. Moreover, the soil is poor in hydrogen, while it contains appreciable amounts of N.

Table 1

Chemical composition of the composite soil sample collected before the experimental campaigns, Wt oxides (%), CHN (%) and Trace Elements (ppm).Standard error is ± 2%
Wt oxides (%)									CHN (%)
SiO2	TiO2	Al₂O₃	MgO	Fe₂O₃	MnO	CaO	Na₂O	K₂O	C	H	N
73.82	0.27	14.29	0.26	2.00	0.06	0.49	1.76	6.82	0.67	nd	2.34
Trace elements (ppm)
P	S	Cl	Cu	Zn	Cr	Rb	Sr	As	Zr	Y
655	391	394	51	39	255	394	53	27	100	82

Functional diversity analysis of microbial soil communities

Average Well Colour Development (AWCD) was used as an indicator of microbial activity in Biolog analysis. The AWCD showed a lag phase of 24 h and increased rapidly after 48 h of incubation, reaching the plateau after 7 days of incubation in ECOPlates, indicating a high metabolic activity (Fig. 1).

All the groups of substrates (carbohydrates, P-sugars, carboxylic acids, aminoacids, amines and polymers) contained in the ECOplates were efficiently used by the microbial community (Fig. 1), showing a high functional diversity (96%). Among the substrates, only D,L a-glycerol-phosphate was below the threshold value (OD 0.2). The carbon sources readily metabolised, with OD values higher than 1.0 after 48 h of incubation, were b-Methyl-D-Glucoside, D-Mannitol, D-Cellobiose, N-Acetyl-D-Glucosamine, Itaconic Acid, D-Malic Acid, 4-Hydroxybenzoic Acid, L-Asparagine, L-Serine, and Glycogen. After 72h of incubation α-D-Lactose, D-Galactonic Acid Lactone, L-Arginine, L-Phenylalanine, Phenylethylamine, Tween 40, Tween 80, and α-Cyclodextrin were metabolised. The slowest kinetic reaction was with D-Xylose, i-Erythritol, α-D-Glucose-1-Phosphate, D-Galacturonic Acid, Pyruvic Acid Methyl Ester, D-Glucosaminic Acid, α-Ketobutyric Acid, Glycyl-L-Glutamic Acid, 2-Hydroxybenzoic Acid, and L-Threonine. The substrates γ-Hydroxybutyric Acid and Putrescine were poorly metabolised and reached OD values lower than 1.0.

Isolation and identification of bacteria

The microbial load of the heterotrophic population was 3×10⁷ CFU g^− 1 of soil. Based on differential colony morphologies, a total of 40 bacterial strains were isolated and identified using 16S rRNA gene sequencing to species level (Table 2 and Table S1 in Supplementary Material).

Table 2

Plant growth promoting (PGP) traits of the isolated bacteria. Positive reaction in increasing scale (+), (++), (+++); negative reaction (–), weak reaction (+/-), ng: no growth. Strains in bold are those composing the Formula T-S.
			PGP Trait
	Isolate	Closest relative in GenBank	N-Fix	O-CAS	P-sol	K-sol	IAA	N°traits
Alpha-proteobacteria	IN3	Phyllobacterium phragmitis	+	++	+/-	+	++	5
	IN8	Sphingobium mellinum	+	-	-	++	+	3
	IN9	Ensifer sesbaniae	+	-	-	-	++	2
	IN10	Ensifer sesbaniae	+	-	+/-	++	+	4
Actinobacteria	ITA5	Paenarthrobacter nitroguajacolicus	+	+	-	-	++	3
	ITA9	Paenarthrobacter nicotinovorans	+	+	+/-	++	-	4
	ITA12	Microbacterium phyllosphaerae	+	-	ng	-	-	1
	IN5	Microbacterium hydrocarbonoxydans	+	-	-	++	+	3
	IN7	Arthrobacter celericrescens	+	-	+	-	-	2
	ITA16	Streptomyces cyaneus	+	-	+/-	-	++	3
Bacilli	ITA15	Bacillus subtilis	+	+++	+/-	+	+	5
	ITA7	Bacillus subtilis	+	-	+/-	-	+	3
	ITA20	Bacillus subtilis	+	-	-	-	++	2
	ITA17	Bacillus tequilensis	+	++	+/-	++	-	4
	ITA10	Bacillus haynesii	+/-	-	+/-	+	-	3
	ITA23	Bacillus haynesii	+	-	+/-	++	-	3
	ITA26	Bacillus amiloliquefaciens	+	++	+/-	++	-	4
	ITA4	Mesobacillus subterraneus	-	-	ng	ng	-	0
	ITA24	Fredinandcohnia onubensis	+	-	ng	++	-	2
	ITA6	Cytobacillus depressus	-	-	ng	-	-	0
	ITA3	Paenibacillus xylanilyticus	+	-	-	-	+	2
	ITA11	Paenibacillus lautus	+	-	ng	-	+/-	2
	ITA18	Peribacillus asahii	-	-	ng	++	+++	2
	ITA19	Peribacillus asahii	+	-	ng	-	-	1
	ITA21	Rossellomorea vietnamensis	+/-	-	ng	ng	-
Beta-proteobacteria	IN1	Delftia lacustris	+	+/-	+/-	-	+	4
Beta-proteobacteria	ITA2	Delftia lacustris	+	-	-	ng	-	1
Gamma-proteobacteria	ITA14	Pseudomonas plecoglossicida	+	++	+	++	+/-	5
	IN11	Pseudomonas plecoglossicida	+	+/-	++	+	+++	5
	IN14	Pseudomonas plecoglossicida	+	-	+	+++	-	3
	ITA13	Pseudomonas mosselii	+	++	+	++	-	4
	ITA22	Pseudomonas cuatrocienegasensis	+/-	-	-	++	++	3
	ITA25	Pseudomonas mediterranea	+	-	+	++	++	4
	IN13	Pseudomonas monteilii	+	-	+	+	-	3
	IN15	Pseudomonas resinovorans	+	++	-	+++	+	4
	IN16	Pseudomonas alcaligenes	+	+	ng	ng	++	3
	IN4	Acinetobacter venetianus	+	-	+/-	+	-	3
	ITA1	Acinetobacter venetianus	+	-	-	ng	++	2
	IN2	Enterobacter cloacae	+	-	+/-	++	+++	4
	IN12	Enterobacter cloacae	+	+/-	+/-	++	+	5

The 40 strains isolated from the cultivable fraction of the heterotrophic population were examined together with the reference strains of related taxa and the phylogenetic status is shown in the cladogram compiled by Neighbour-Joining methods (Fig. 2). The most represented class was Bacilli, with seven genera and 11 species; 13 strains belong to 3 genera of the class γ-Proteobacteria (Pseudomonas, Enterobacter, Acinetobacter); 6 strains belong to 4 genera of the class Actinobacteria (Streptomyces, Microbacterium, Paenarthrobacter and Arthrobacter); 2 strains of Delftia lacustris belong to the class β-Proteobacteria; 4 strains belong to 3 genera of the class α-Proteobacteria (Ensifer, Sphingobium, and Phyllobacterium).

Strains Characterization for PGP traits

After isolation and identification, all strains were characterised for PGP traits (Table 2). Within the 40 isolated, about 93% were positive for nitrogen-fixation; 53% for phosphate solubilisation, 58% for potassium solubilisation, 58% were able to produce IAA and 33% siderophores. In particular, eight isolates from the cultivable fraction (IN11, ITA5, IN3, ITA14, ITA15, ITA17, IN1, and IN4) were selected to be part of the bacterial Formula T-S, due to their multiple PGP activities. According to the ecology-based approach, the Formula was assembled reflecting as much as possible the original composition of the native community, and included 8 species belonging to the genera Delftia, Pseudomonas, Paenarthrobacter, Phyllobacterium, Bacillus, and Acinetobacter (indicated by red dots in Fig. 2).

The Formula T-S was prepared considering the capacity of these eight microbial strains to hold the largest number of PGP properties and reproducing as much as possible the original composition of the cultivable fraction of native community. In the Formula T-S all the strains are nitrogen-fixers. Most of the eight strains were able to produce different amounts of IAA. Ps. plecoglossicida IN11 was the best IAA producer and the most efficient phosphate solubilizer, while Ps. plecoglossicida ITA14 and Bacillus tequilensis ITA17 were excellent potassium-solubilizing bacteria. Except for Acinetobacter venetianus (IN4), all the selected bacteria were able to produce siderophores. This common feature was particularly marked in Bacillus subtilis (ITA15).

The PGP traits [i.e. the ability to fix atmospheric nitrogen (N-fix), to solubilise phospates (P-sol), to solubilise potassium (K-sol), to produce IAA and siderophores (O-CAS)] of the isolated microbial community were subjected to principal components analysis (PCA). Figure 3a shows the biplot of PCA obtained for all the isolated microbial strains, where the first two components (PC1 and PC2) account for the 42.68% and 33.86% of the variance respectively. The parameter N-fix resulted to be not decisive for the discrimination of bacteria, since almost all strains showed nitrogen fixing properties (Table 2). On the contrary, O-CAS property was found to be limited to only few bacteria, those selected for the microbial formula. The main separation between the strains selected to compose the Formula T-S and the rest of the microbial community is observed along the PC1 (Fig. 3a). The selected strains (black dots) included those with higher capacity to express the overall plant growth promoting traits within the community. In fact the selected bacterial strains are distributed mainly in the right side of the biplot (positive values of PC1), influenced by the contribution of most of the variables considered. The strains selected for the Formula T-S showed a wide distribution in five different clusters as result of their multiple PGP traits, and they are grouped on the basis of their metabolic similarities, as observed in the biplot (Fig. 3b) in which the first two components (PC1 and PC2) account for the 55.76% and 30.90% of the variance respectively. Among the eight selected bacteria, ITA15 strain showed the higher capacity to produce siderophores than the others, and clusters separately (Fig. 3b).

Field experiments

Figure 4 shows the plant average height, measured at different stages of plant growth, during the three field experiments in two different soil treatments: biofertilisation (B) and chemical fertilisation (F). In the first and in the second field experiment, the average trend of plant growth was similar for both bio- and chemical fertilisation (Fig. 4a and 4b).In the same month, no significant differences were observed between treatment with bacteria and chemical fertilisation, either for the first (p = 0.952 in March; p = 0.801 in April; p = 0.904 in May; p = 0.997 in June) or for the second experiment (p = 0.999 in October; p = 0.999 in 2nd November; p = 0.996 in 24th November; p = 0.998 in December), as shown in Fig. 4a and 4b. In the third field experiment, the average trend of plant growth was similar in the first period, with no significant difference in June (p = 1.00) and slight difference in 7th July (p = 0.037). The last period (from 23rd to 30th July) was an exception: an average higher plant height was observed in the case of chemical fertiliser, with a significant difference (p < 0.0001).

The average weight of tomatoes per plant in the three field experiments is shown in Fig. 5. The first field experiment revealed a significant difference of the average weight (p < 0.0001) between biofertilisation (B) and chemical fertilisation (F), with much higher values in the case of chemical fertiliser application. On the contrary, in the other two experiments, the average weight was very similar between B and F (second experiment p = 1.00; third experiment p = 0.989). Likewise, in the first experiment a higher number of tomatoes was collected in the group of chemical fertilisation (p = 0.0003), while similar results were observed during the second and third experiments for both groups (p = 0.9999).

This was the first work in which the efficiency of the complete substitution of chemical fertilisation with a bioaugmentation strategy was evaluated in a tomato farm in Sardinia. When the research began, it was known that the goal was very ambitious, especially for Sardinia Island that is among the hotspots for climate change (Marras et al. 2021), because of its central location in the Mediterranean Sea. Indeed, the consequent impoverishment of soil fertility, also due to increasing temperature and changes in precipitation (Mondal 2021; Caloiero and Guagliardi 2021), led to a greater demand for chemical fertilisers to cope with the decrease in agricultural yields. Therefore, considering the unsustainability of the traditional intensive production system, based only on chemical fertilisation, the far-sighted farmers involved in this study recognized the importance of testing alternative methods to produce tomatoes, reducing environmental impacts and facing the effects of climate change.

The choice to apply the emerging bioaugmentation approach based on a combination of microorganisms with different PGP traits was due to the results reported in various studies showing that microbial consortia have the potential to increase plant growth much more than inoculants with a single bacterial species (He et al. 2019; Compant et al. 2019; Mitter et al. 2021). However, a smart and knowledge-driven selection of consortia and strains is required (Tosi et al. 2020). Indeed, as reported in literature, no microbial inoculant can be universal for all systems, and the effectiveness may be affected by many factors such as the ability of inoculated microorganisms to persist in soil, depending on their compatibility with the environmental characteristics and the degree of spatial competition with other organisms in the target niche (Mannino et al. 2020) or the interactions between a specific plant type and the selected PGP strains (Adesemoye et al. 2009; Mitter et al. 2021). In order to increase the success of the inoculum establishment, but also the probability of finding bacteria that exhibit the desired PGP effects, we investigated the culturable indigenous microbial community, functionally linked with chemical characteristics of native soil and with the needs of the target tomato species, to select and to locally isolate pre-adapted bacteria to be used as bioaugmentation strategy (Sprocati et al. 2014).

The forty isolated bacteria and, among them, the eight PGPB selected to be combined in the Formula T-S for the field experiments, belonged to the cultivable fraction of the tomato soil bacterial community. Although this soil was subject to agricultural management, its bacterial community seemed to preserve a peculiar taxonomic (Fig. 2 and Table S1) and functional biodiversity (Fig. 1). Moreover, the observed bacterial density (3 × 10⁷ CFU g − 1) was about an order of magnitude higher than what normally found in other agricultural soils sampled in summer (Bevivino et al. 2014; Bhowmik et al. 2019). While taking in account that microorganism load may vary within and between different soil types and conditions (Vieira and Nahas 2005), the observed bacterial load could indicate an acceptable quality of soil microbiome.

In vitro tests were carried out to assess the PGP potential of all the isolates and to choose the best strains to perform field bioaugmentation. The rationale of the choice for the bacterial formula composition was the combination of microorganisms with the highest number of PGP traits, (Fig. 3a) but also with different and complementary capacities potentially inducing positive effects on plant physiology and fruit yield (Fig. 3b). Moreover, according to the ecology-based approach suggested by (Hu et al. (2016), in order to improve the survival and functioning of bacteria in the tomato soil microbiome, the formula was assembled including different species reflecting as much as possible the original microbial community biodiversity (Fig. 2).

All the eight bacteria composing Formula T-S were diazotrophic microbes, able to convert atmospheric nitrogen into ammonia (Table 2). The biologically fixed nitrogen, more sustainable than chemical fertilisers and less available for leaching and volatilization, allows the replenishment of soil total nitrogen content and regulates the crop growth and yield. An increase of the root system development and a more efficient nutrient uptake by the plant are also due to the production of IAA (Kumar et al. 2020). This biostimulant capacity was very high in Pseudomonas plecoglossicida (IN11), Paenarthrobacter nitroguajacolicus (ITA5) and Phyllobacterium phragmitis (IN3) belonging to genera already tested as efficient IAA producers (Menéndez et al. 2020; Pérez-Rodriguez et al. 2020; Riva et al. 2021) and, to a lesser extent, in all the other isolates, except Bacillus tequilensis (ITA17) and Acinetobacter venetianus (IN4). These last two bacteria, and in particular Bacillus tequilensis (ITA17) were chosen, together with Pseudomonas Plecoglossicida (ITA14), for the excellent ability to solubilise potassium (K-sol) in laboratory tests, as already reported in literature for the respective genera (Ahmad et al. 2016; Etesami et al. 2017; Saxena et al. 2020; Ashfaq et al. 2020). Soil at the tomato commercial greenhouse in this study, originating from weathering processes of granite minerals (Table 1), showed the presence of microcline, muscovite and phlogopite, which are an important source of insoluble potassium. The availability of K to tomato plants could be enhanced by the potassium-solubilizing bacteria in Formula T-S, able to fulfil the potassium requirement of the crops in alternative to chemical fertilisers. In addition, phosphorus (P) is an essential nutrient required for diverse plant metabolic processes such as respiration, biosynthesis, photosynthesis, energy transfer, and signal transduction but, in many agricultural lands, it is slightly bioavailable (Kumar et al. 2022). Bacteria belonging to different genera such as Arthrobacter, Bacillus, Beijerinckia, Burkholderia, Enterobacter, Pseudomonas, Erwinia, Mesorhizobium, Flavobacterium, Rhodococcus and many others, are able to solubilize phosphates converting them into a form that plants can absorb for their growth (Lobo et al. 2019; Shilev 2020). For this PGP trait, two strains of P. plecoglossicida (IN11 and ITA14), were chosen as the most efficient phosphate-solubilizing microbes among all the tested isolates. Another important nutrient for plant cells is Fe but, despite its abundance on Earth, it is not widely accessible in soils, when it is present in complexes of hydroxides and oxyhydroxides. Different PGPB, such as Pseudomonas, Bacillus, and Phyllobacterium found in this study, possess the ability to synthesise siderophores, Fe-chelating compounds having a high affinity to Fe³⁺ and forming complexes that lead to the mobilisation of Fe (reduced to Fe²⁺), making it bioavailable and absorbable by the plant roots (Shilev 2020; Pérez-Rodriguez et al. 2020; Flores-Félix et al. 2021). Moreover, it is known that siderophore-producing bacteria play a crucial role not only in growth promotion but also in biocontrol activity, by competing for Fe³⁺ with the pathogens in the rhizosphere (Kumar et al. 2022). For instance, several strains of Bacillus subtilis have been reported to suppress fungal pathogens in plants using siderophores (Manasa et al. 2021; Kumar et al. 2022). In agreement with these literature data, the highest siderophores production observed in this study was performed by two strains of Bacillus (ITA15, ITA17).

Thus, all the eight strains tested as possible biofertiliser are phylogenetically affiliated to bacterial species that, according to the authors mentioned above, present a PGP potential. Moreover, in terms of biosafety, all the selected isolates belong to the risk group 1, as stated in the reference document provided by the German Committee on Biological Agents – ABAS, TRBA 466 (2020), and their use in field does not imply particular concern on human health. A further advantage of using native microorganisms is to avoid the risk of introducing foreign strains, which could prove dangerous once in contact with the indigenous community, as also reported by Mahmud et al. (2021).

After the positive results obtained by in vitro screening for the selected potential PGP bacteria, in agreement with the bottom-up approach suggested by Riva et al. (2021), we performed field experiments to test the real effect of these bacterial inoculants on plant production of three different tomato varieties. For this purpose, we carried out the experiments in a commercial greenhouse, with the aim to generate meaningful information for researchers and, at the same time, to minimise eventual loss of profit for growers. In fact, to avoid any reduction in tomato production, the experimental design did not include negative controls, represented by plants without fertilisation. Anyway, the greenhouse experimentations were essential because it is known that lab screening and small-scale experiments may provide only limited information and success in the open field is often variable. In other studies, some consortia with multiple PGP-activities showed low efficiency when applied in field experiments (Cardinale et al. 2015; Compant et al. 2019; Riva et al. 2021), while other bacteria, that in typical assays did not display a promising set of PGP activities, proved to be the best growth promoters when tested directly on plants (Cardinale et al. 2015). Therefore, if field experiments are fundamental to select the most efficient bioinoculant, it is equally important to perform long-term experiments of biofertilisation and to evaluate the PGP effect exerted throughout the plant life cycle and especially in fruit production (Riva et al. 2021). As highlighted in a recent work, plants require distinct types of microbial activities at different stages of growth (He et al. 2019) so, not only the co-inoculation of bacterial strains with different properties, but also the frequency of PGPB application, could influence the plant performances. In the present work, when the number of microbial inoculations was optimised (from two initial biofertilisation to monthly applications per growing season), an improvement on tomato yield, comparable with the results obtained with chemical fertilization, was observed (Fig. 5). These results confirmed that the inoculation of PGPB at different stages of plant growth, regardless of the tomato variety, represents an efficient strategy to improve fruit yield. The importance of the present study, hence, lies in the fact that, for the first time, it was possible to demonstrate the complete replacement of chemicals by biofertiliser to sustain and guarantee an adequate tomato yield, contrary to what was claimed by Adesemoye et al. (2009) or, more recently, by Ye et al. (2020), that indicated microbial inoculation as a promising complement of synthetic fertilisers but not as their valid substitute.

In conclusion, the successful strategy described in this paper depends on the application of different suggestions, coming from our previous research and from literature, that were combined in a bioaugmentation based on the co-inoculation of autochthonous bacteria, selected for their different and complementary PGP abilities and for their different taxonomic affiliations, applied at different stages of tomato plant growth. Still, some important aspects remain to be investigated: 1) in-depth investigation of the microbial ecology of the soil target to enrich the bioaugmentation formula with microbial inoculants not considered in this work, i.e. cyanobacteria and mycorrhiza, 2) the effect of the biofertiliser on tomatoes quality in terms of organoleptic properties, the content of Vitamin C, and the nitrate accumulation as suggested by Ye et al. (2020); 3) the effect of Formula T-S on the chemical and biological properties of the soil in a continuous cropping system and 4) the potential of this approach in the scenario of increasing drought and salinity caused by the climate change.

The results obtained in this work imply that the application of biofertilisers could substitute the use of expensive and polluting chemicals without compromising the tomato yield, encouraging to continue on this strategic line, which responds to the need to move towards the replacement of chemical-based agricultural practices with sustainable practices.

PGP

Plant growth promoting

PGPB

Plant growth promoting bacteria

Room temperature

CLPP

Community-level physiological profile

AWCD

Average well colour development

PXRD

Powder X-rays diffraction

XRF

X-rays fluorescence

CRIST

Structural crystallography centre

CFUs

Colony-forming units

Nitrogen free

N-fix

Nitrogen fixing

PCR

Polymerase chain reaction

P-sol

phosphate solubilisation

K-sol

potassium solubilisation

IAA

Indo-acetic acid

PVK

Pikovskaya’s medium

O-CAS

Overlaid chrome azurol S

Chemical fertiliser

Bacterial formula T-S (biological fertiliser)

ANOVA

Analysis of variance

- Ethical Approval: Not applicable

-Consent to Participate: Not applicable

-Consent to Publish: Not applicable

-Authors Contributions: GDG coordinated the project. ARS, CA coordinated the research activity. GDG, ARS, GM, PP, CA, and FT contributed to the conception and design of the study. GDG, DM, PAM, CA and PP collected soil samples. DM ED, and PAM performed mineralogical and chemical analyses and data elaboration. TC, PP, CA, GM and FT performed bacteria isolation and characterization. PP and FT performed sequence analysis. TC, CI, PP, CA, GM and FT prepared the inocula. TC, ARS, GDG, ED, DM and PAM set up the greenhouse experiment and data collection.CI performed statistical analysis and plant data interpretation. PP and CI wrote the first draft of the manuscript. CA, ED, and GDG wrote sections of the manuscript. All authors read and approved the final manuscript.

-Funding: This work was supported by “SUPREME: developing tools for SUstainable food PRoduction in mEditerranean area using MicrobEs”-id: ERANETMED2 - 72 – 094.

-Competing Interests: The authors declare no competing interests. The authors have no relevant financial interests to disclose

-Availability of data and materials: Supplementary data are provided in a file

Acknowledgements: The authors are grateful to Giulia Siclari and the Siclari family for their contribution in running the field experiments.

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A bacterial formula with native strains as alternative to chemical fertiliser for tomato crop

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Journal Publication

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Abstract

Figures

Introduction

Material And Methods

Soil Sampling and Physiological profile at community-level

Chemical and mineralogical analysis of soil

Bacteria isolation and identification

PGP assays and bacterial formula assembling

Field experiments

Tomato growth parameters and productivity analysis

Statistical analyses

Results

Soil characterization

Functional diversity analysis of microbial soil communities

Isolation and identification of bacteria

Strains Characterization for PGP traits

Field experiments

Discussion And Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1