Brassica carinata Seed Meal as Soil Amendment and Potential Biofumigant

Abstract

Brassicaceae products have been extensively studied for their biofumigant activity; despite this, few investigate their effect on soil proprieties. This paper aims to describe the effect on soil fertility by adding the seed meal of Brassica carinata at three different doses corresponding to field dose (3 tons/ha), 10 and 100 fold this dose in organic soil. The organic carbon balance was evaluated by analysing the oxidisable, humified and mineralised carbon fractions. Microbial activity was measured by enzymes linked to the carbon, phosphorous and sulphur cycles—β-glucosidase, phosphatase and arylsulphatase, respectively. The amount corresponding to 30 t/ha was the best solution for the mineralised carbon and humic carbon ratio. In contrast, there was a substantial increase in the organic substance’s mineralisation level at the maximum dose, not offset by a relative rise in humification. No repression was observed in the metabolic activity of the microorganisms, their abundance or the main enzymatic activities and, in the conditions tested, a release of only a volatile isothiocyanate occurred, limiting the biofumigant effect. Therefore, these combined factors allow us to assert that the amendment with this type of material at the intermediate dose could positively affect the phosphorus cycle, the soil structure, and fertility.

1. Introduction

The International Federation of Organic Agriculture Movements (IFOAM) defines the overarching goal of organic farming as follows: “Organic agriculture is a production system that sustains the health of soils, ecosystems and people. It relies on ecological processes, biodiversity and cycles adapted to local conditions rather than the use of inputs with adverse effects. Organic agriculture combines tradition, innovation and science to benefit the shared environment and promote fair relationships and a good quality of life for all involved” [1]. In this agricultural system, the use of synthetic molecules such as pesticides and mineral fertiliser is strictly prohibited [2,3], and the use of organic amendments is recommended for a positive balance of organic matter [4,5], which is the centre of physical, chemical and biological soil fertility [6,7].

Brassica carinata seed meal can be considered among the products obtained from the energy supply chain and for industrial use, particularly for producing biodiesel from vegetable oil [8]. The importance of renewable energy sources from crops is evident from the policy of the European Community that, in the last ten years, has promoted a policy of programming and incentives in this sector [9]. Some authors have pointed out that although production in vegetable oil is lower when compared with rapeseed, on the contrary, biomass production resulted in very interesting prospecting important uses such as direct energetic use [10] and high-value biochemicals extraction [11,12].

Moreover, in comparison with rapeseed, Brassica carinata has some favourable aspects: the greater hardiness, the ability to optimise the poor spring rainfall making it more resistant to water stress conditions [8,13,14], the less need for nutrients that results in a substantial saving of nitrogen to supply, the greater tolerance to some parasites [15,16] and the non-existent phenomenon of dehiscence of the pods [12,17]. Soil incorporation of selected Brassica sp. plants as catch crop green manures or derived materials as biofumigant pellets represents a sustainable technique for soil disinfection [18,19,20] and fertility management that is an alternative to the chemical soil fumigants [21].

The 100% vegetable composition of these materials makes it possible to incorporate a significant amount of organic matter (OM) in soil [22], which is a fundamental benefit for soil fertility [23]. Indeed, it is well known that OM plays a positive role in physical, chemical and biological soil properties [24,25]. In addition to these benefits, typical of organic fertiliser and green manure crop management, biofumigant plants [26] and materials add a clear allelopathic effect on several soil pests and pathogens [27]. In fact, Brassica cells contain the defensive system glucosinolate-myrosinase (GLs-Myr) [28,29], which is able to release, by enzymatic hydrolysis, a number of biologically active compounds, including main isothiocyanates and, at lower concentrations, nitriles and thiocyanates [30,31,32]. These compounds are recognised for their well-known biocidal activity [33] against fungi [34,35], insects [36,37,38] and wireworms [39]. There are no univocal results in the literature regarding the potential adverse effect of Brassica spp. on the positive soil microorganisms. Some studies report fungistatic effects at high doses [40] and a reduction in fungal richness [41]. As for the beneficial bacterial communities, Cohen et al. [42] argue that Brassica favours them, while other authors note a decrease in an abundance which is of a transitory type [41,43]. The effects of classic organic amendments on nutrient availability and soil enzymatic activities are widely discussed in the literature [44]. In this context, soil enzymes are a fundamental indicator of the quality of this natural matrix, considering their crucial involvement in the main geochemical processes of plant nutrients [45]. Although there are many studies on the effects of soil management and regular amendments on respiration and enzymatic activities of the soil [46,47,48,49,50,51], few papers refer to the effect of biofuel production by-products, in particular to the impact of seed meals [22,52,53].

The aim of the present paper was to evaluate the amendment potential of seed meal of Brassica carinata in a laboratory experiment with a soil cultivated in an organic farming system with Alfa alfa for 13 years and added at three different doses of seed meal corresponding to field dose (3 ton ha⁻¹), 10 fold and 100 fold the field dose. The effect on soil fertility was evaluated by studying the cycle of organic matter in terms of humic and mineralised carbon and the analysis of the enzymatic activities linked to the carbon, phosphorus, and sulphur cycles.

2. Materials and Methods

2.1. Soil and Seed Meal

Soil samples were collected from the top 30 cm layer of an Alfa alfa field cultivated in the last four years under organic management at the Experimental Farm of Marche Polytechnic University, Agugliano, Ancona, Italy (43°57′ N, 13°43′ E). This soil was cleaned of plant residues, air dried, and sieved at 2 mm, before the start of the test. It was a Vertic Eutrudept [54] named “Hypocalcic Calcisols” [55], with a clay loam texture (37% clay, 40% silt and 23% sand), a bulk density of 1.41 g cm⁻³, a soil pH of 8.01 and an initial organic carbon content of 8.2 g kg⁻¹ [56].

Brassica carinata seed meal, used as the tested organic amendment in the present study, was obtained by grinding pellets from the mechanical squeezing to get oil from Brassica c. seeds. The oil extraction, which led to pellets as a waste product of the process, was carried out with a continuous press in the laboratory of the Agricultural Engineering Area of Marche Polytechnic University. During the pressing phase, the temperature remained below 60 °C to avoid the denaturation of myrosinase, an endogenous enzyme of plant tissues, essential for the degradation of GLS and the subsequent release of isothiocyanates. The chemical characterisation of the organic amendment used was: pH 5.28, organic carbon (48%), total nitrogen (5.7%), P₂O₅ (2%), K₂O (2.4%), total sulphur (1.5%), and total magnesium (0.7%).

2.2. Experimental Set Up

Brassica c. seed meal was incorporated into the soil at a rate of 0.1 g (Trial “D”), 1 g (Trial “10D”) and 10 g (Trial “100D”) per 100 g of soil. The first dose corresponds to 3 tons, a quantity normally distributed per hectare as a soil amendment [57]; the other two doses were tested only for experimental purposes considering that they are not applicable in agricultural practice, but their detection could provide important general information on the transformation of organic matter in the soil.

Treated and non-treated (control) soils were placed in a 1 L glass container, in three replicates per treatment and for the control, and maintained at 20 ± 3 °C in the laboratory with soil moisture of 60%. The lids had a membrane to allow the withdrawal with a syringe to sample the air with the possible products of the glucosinolates hydrolysis; this analysis was performed immediately after the set up of the treated containers.

After one week of the start of the trial, parameters such as oxidisable organic carbon, pH (H₂O) and total fungal and bacterial count were evaluated in each replicate.

The enzymatic activities of ß-glucosidase, alkaline phosphatase and arylsulphatase were evaluated at 0, 3, 6, 9, 12, 18, 20, 23, 30, 45, and 60 days of the incubation period, while after eight months from the start of the trial, the humic (HA) and fulvic acids (FA) of total extractable carbon (TEC), were measured in each replicate.

A parallel trial with 8 soil samples (3 treatments plus one control × 3 replicates) was set up to monitor the Soil Basal Respiration (SBR).

2.3. Soil Organic Carbon Analysis

The oxidisable total organic carbon (TOC) was determined in each replicate by oxidising soil organic carbon with acidic dichromate and titrating excess dichromate with ferrous sulphate following the Walkley-Black method [58] with some modification; to not underestimate organic carbon, the protocol was set up without external heating as previously described by Vischetti et al. [59]. From the calculation of TOC, the estimation of Organic Matter (OM) was obtained using the standard correction factor of 1.724.

Organic carbon fractions were performed by extracting total organic carbon in an alkaline solution following the Schnitzer protocol [60], with slight modifications as reported in Vischetti et al. [59].

2.4. Soil Basal Respiration

The monitoring of basal respiration was evaluated with 30 g of soil (of control and the treated soils) placed in a sealed jar together with a container with NaOH following the method described by Isermeyer [61] with the modification proposed by Bloem et al. [62].

The content of mineralised C, expressed as CO₂, is calculated with the formula [63]:

$$\mathrm{mg}\mathrm{C}-{\mathrm{CO}}_2{\mathrm{g}}_{\mathrm{s}.\mathrm{s}}^{-1}{\mathrm{h}}^{-1}=\frac{\left({\mathrm{V}}_0-\mathrm{V}\right)\times \mathrm{M}\times \mathrm{E}}{20\times {\mathrm{h}}_{\mathrm{inc}}}$$

where V₀ and V are the volumes of HCl used to titrate the blank and the sample, M is the molarity of titrant, E = 6 is the equivalent weight of carbon in CO₂, and h_inc are the hours of incubation.

2.5. Enzymatic Activities

The ß-glucosidase, alkaline phosphatase and arylsulphatase enzymatic activities were determined according to Tabatabai [64], Juma and Tabatabai [65] and Klose and Tabatabai [66], respectively.

2.6. Volatiles Compounds Analysis

Glucosinolates and their derived forms are detected and identified by GC–MS (Clarus 600 S, Perkin Elmer, Waltham, MA, USA). The GC–MS was equipped with a Rxi 5 Sil-MS capillary column (30 m 0.32 mm ID 0.25 mm film thickness, RESTEK, Milan, Italy). An aliquot of the headspace of the container containing the soil/seed meal mix was taken by piercing the silicone septum and injected into the analyser using a 50 µL gas-tight syringe. The chromatographic conditions for the analysis were the same as described by Toscano et al. [67]. The mass selective detector was operated in Full Ion mode (50–550 m/z). Analytes were identified by comparing total mass spectra with the National Institute of Standards and Technology (NIST) library.

2.7. Bacterial and Fungal Populations Investigation

In order to assess the total fungal and bacterial count, soil samples from each experimental thesis were weighed as much as 5 g and then suspended in 50 mL of distilled water and shaken for 30 min. After which, 1 mL of suspension was mixed with 9 mL of distilled water. Ten serial dilutions 1:10 were obtained. From each dilution, 50 µL was transferred on culture plates of Potato Dextrose Agar (PDA; Oxoid, Basingstoke, UK) supplemented with 0.2% of cycloheximide fungicide for bacteria count determination, or streptomycin + ampicillin (100 mg/L each) for fungal specie investigation, and incubated at 37 °C for 48 h. Then, the culture plates were investigated for 5 days to determine the colony forming units (CFU)/g soil. Each sample dilution was repeated in four replicas.

2.8. Statistical Analysis

Soil chemical and biochemical parameters were analysed by a two-way ANOVA and Tukey’s Honest Significant Differences (HSD) test (significance 95%). Assumption of normality was tested beforehand through the Shapiro–Wilk test. All statistical analyses were performed using JMP.10 (SAS Institute Inc., Cary, NC, USA).

3. Results and Discussions

3.1. Soil Structure

The more evident physical effect of adding the seed meal of Brassica carinata to the soil was the change in the soil structure (Figure S1), which was positively influenced by the addition of the seed meal through the formation of macro and micro aggregates and a progressive reduction in soil roughness. The soil treated at the highest dose of seed meal was fine and well structured with respect to the control soil in the same experimental conditions, and a positive effect on the soil structure could be expected when this amendment is added [68,69].

3.2. Soil pH

pH variation in the soil samples was from 8.01 in the control to 7.82, 7.77 and 7.21 in the samples added with D, 10D and 100D, respectively. The acidity of the seed meals (pH = 5.28) could be the factor that contributed to the pH decrease, and this could be useful when the meal is applied to alkaline soils. Despite this, other authors argued that pH changes by approximately 0.5 after adding B. carinata pellets were considered negligible [57].

3.3. Soil Organic Carbon

The amendment effect of adding B. carinata seed meal on the soil organic matter balance was studied by the measurement of the total organic carbon, the humic carbon, the fulvic and humic components, and the balance between mineralised and humified organic carbon.

The total organic carbon (TOC) measured as organic carbon oxidised present in the soil samples untreated and treated with seed meal at different doses is reported in Figure 1.

In the control soil, the TOC value of 8.18 g kg⁻¹ was not significantly different from the TOC of the soil added at the field-dose D of seed meal, while the addition of the meal at doses of 10D and 100D resulted in TOC significantly higher than the control soil with a maximum value of 24.83 g kg⁻¹ at 100D dose.

Conversion of the TOC values in percent organic matter content (OM%), which is the usual way to display analytical results, is obtained by the OM% = TOC% × 1.724 [70].

The resulted values were 1.41, 1.51, 1.75 and 4.28% for control soil, D, 10D and 100D soils, respectively; being the soil under investigation a clay loam soil, where OM% results in a low content in the range of 1.2–2.2% and in high content in the range >3.0%, it can be concluded that only the addition of seed meal at 100D really improves the OM content of the soil under experiment, supplying a very high amount of fresh organic matter. In contrast, the addition of the meal at doses D and 10D let stand the soil in the same class of OM content as the control soil, even if a slight increase is recorded, similarly to what was observed by Serrano-Pérez et al. [57] at the dose of 3 ton/ha.

Humification and mineralisation processes of organic carbon added with the amendment should be adequate and balanced to supply the right amount of organic carbon, which, in turn, should provide the proper amount of humus for sustainable management of organically farmed soils.

Nine months after the treatment, the humified organic carbon was determined in all samples, and the results are shown in Figure 2.

The total humic carbon as a sum of fulvic and humic acids at 10D and 100D doses was significantly different from the control, with increases of 16.8 and 139.4%, respectively. Similarly, in the study by Galvez et al. [52], a significant increase in extractable carbon is measured compared to the control in the test treated with 20 tons/ha (therefore, values similar to the 30 tons/ha of the 10D test in the present study) of rapeseed meal from biodiesel production.

FA fraction resulted significantly different from the control at 10D and 100D with increases of 34 and 105%, respectively. HA fraction resulted significantly different from the control only at 100D dose with an increase of 155%.

During the experiment, a balance between humified and mineralised organic carbon was made to understand the fate of the organic matter added at the different doses. The mineralising rate has been measured as SBR during two months, and the results are reported in Figure 3, together with the calculation of organic carbon mineralised during the same period.

Between 20 and 30 days from the start of the test, all those with brassica seed meals show a breathing flow (Figure 3a) that seems to reach certain stability, although this plateau seems less evident at the higher dose (100D). This pattern of baseline respiration was also observed in other studies [71].

The trend shows significant mineralisation (Figure 3b) at 10D and 100D doses and, at the end of the two months, very high soil respiration was observed in the 100D sample, indicating that the increased addition of organic carbon in this sample produced high mineralisation until 4 fold the control sample. Dose-dependent mineralisation was also observed by Wang et al. [71] with Brassica juncea seed meal.

Organic amendments should positively affect the right balance of organic carbon in organic farming, i.e., the humification rate should be sustained by an adequate mineralisation rate. The ratio between mineralised and humified carbon in each treatment with respect to the control gives the following results: 0.11, 0.53, 2.07, respectively, for trials D, 10D and 100D. In the present experiment, a very high humification rate was observed in the 100D samples, but, at this dose, also the mineralisation rate was very high. The consequence that in this situation, it is possible to observe a “priming effect” [72,73], which consists of an increase in the decomposition rate of OM caused by the addition of fresh organic material at high doses that should determine a global growth of microbial activity and, in turn, a decreasing of the total organic carbon in the soil [49], in contrast with the primary purpose of the amendment.

Considering the increase in OM observed at all doses tested, using B. carinata seed meal as an alternative fertiliser to chemical ones was confirmed, as stated by Mazzoncini et al. [74].

3.4. Soil Enzymatic Activities

Enzymatic activities are sensible biomarkers to verify the influence of adding an organic amendment to soils [75]. Some enzymes are involved in the nutrient cycle in soil, such as β-glucosidases (C cycle), phosphatases (P cycle) and arylsulphatases (S cycle) and are active in the mineralisation of natural organic compounds [76,77].

β-glucosidases are involved in the hydrolysis of cellobiose in glucose and supply information about the decomposition of organic matter of cellulosic origin. β-glucosidase activity is related to soil management, suggesting that organic amendments to soils furnish substrates for these enzymes and positively affect microbial growth [78,79,80]. Figure 4 shows the trend of β-glucosidases activity in the soil control and added Brassica seed meals at different doses.

The first observation that can be made is that no inhibition of the β-glucosidase activity was found following the addition of seed meal, indicating that the presence of glucosinolate compounds in the soil does not affect the enzyme functionality and was not toxic against the microbial community able to degrade cellulose.

A significant increase in the activity was observed only at 100D dose for the first 30 days after application, which returned to the initial values at day 60. The trend indicated that thanks to high levels of organic material, there was an initial attack on cellulosic residues followed by a return to values in the same range as the control soil when the organic residues have been hydrolysed.

The arylsulphatase activity (Figure 5) is a good indicator of sulphur availability in soils, being the sulphate in an available form for plant nutrition only after the hydrolysis of the sulphuric esters made by these enzymes [81].

Additionally, significant differences were observed only at 100D (increases of 48.5% with respect to the control) for this enzyme, but this time the effect persisted for the entire experiment period. The presence of organic sulphur in the Brassica seeds could explain this behaviour, a good source of sulphur availability in soil.

Phosphatases are vital enzymes in the phosphorous cycle in soils [82], and they promote the release of inorganic phosphorous from organic forms [83]. The phosphatase activity is mainly extracellular and located in the humus/enzyme complexes, and its study allows the control of the effect of the organic residues on soils. The alkaline phosphatase activity is reported in Figure 6.

Significant differences in phosphatase with respect to the control have been observed for 10D and 100D doses. In 10D treatment, an increase in phosphatase activity was measured from 7 to 30 days from the application, while 100D treatment showed higher gains but starting from day 14, and the effect also remained at the end of the experiment (60 days). The Brassica seeds contain a high natural concentration of phosphorous (phospholipids, phosphine), which allowed an increase in alkaline phosphatase in the 10D and 100D samples, more promptly evident at 10D dose but more stable during the time for the 100D dose due to the high content of organic phosphorous in this sample which can sustain the phosphatase activity for a long time, allowing a noticeable release of phosphorous for plant nutrition.

In general, a limited increase in enzymatic activities was observed after the application of seed meal at the highest doses. The limited extent of the increase in the aforementioned enzymatic activities in the present study could be due to a depressing effect on the part of the only GLS hydrolysis product measured (allyl-isothiocyanate) reported in the following chapter. However, this depressing effect is very limited; in other studies, it seems that allyl-isothiocyanate does not produce a significant bio-fumigant effect on enzymatic activities [84].

Other studies conducted with B. carinata pellets at similar doses to 10D β-glucosidase and phosphatase activities were not significantly different from the control [57,85]. On the contrary, for Zaccardelli et al. [22], the β-glucosidase and phosphatase are significantly increased compared to control after one month, and the arylsulphatase seems to be influenced in the short term with a slight increase compared to the control followed by a stabilisation as observed in the present study at the same dose tested (10D).

3.5. Biofumigant Effect of Brassica Seed Meal

In the present work, no observation about the possible biocidal activity of the B. carinata seed meal against nematode and wireworms was performed, but the microbial population was checked to understand if, under the conditions tested, control of microbial pathogens takes place.

The analysis of air trapped immediately after treatment revealed in the treated soil samples (Figure 7) only the presence of allyl-isothiocyanate (AITC), which is the major GLS in this specie [40,86]. Thus, in the experimental conditions tested, the release of hydrolysis products of glucosinolates was somewhat difficult, and the biofumigant action could be very poor when seed meals are used as biofumigation products.

At the end of the experiment, all the samples undergo the microbial count for the microorganisms present. Total fungal count (at 7 days after inoculation in PDA media) showed significant differences only between 100D and the control: Rhizopus spp. and Penicillium spp at 1.1 × 10³ and only Rhizopus spp. at 1.1 × 10⁶ for fungi count. While 2 × 10³ and 9 × 10⁴ for bacteria count, respectively, for control and 100D treated soil were found, a similar trend of increases in the bacterial population was observed in other studies after the addition of Brassicacea seed meal [41,42].

At D dose (3 ton ha⁻¹), no significant differences were found between control and treated samples, demonstrating that, at the field dose, no control of any microorganisms was performed in all samples, either for fungi or bacteria populations, as revealed by Zaccardelli et al. [87].

The increase in the microbial population is certainly due to the addition of a high amount of organic material of seed meal; the preponderance of the saprophytic fungi of the Rhizopus strain had a mask effect versus other microbial dynamics and did not allow determination of where there was effective control of pathogen species.

4. Conclusions

Soil amendment with Brassica carinata seed meal to improve its fertility has shown positive effects in terms of increasing the total organic carbon content and humified carbon.

Furthermore, no repression of microorganisms’ metabolic activity was observed for respiration, abundance, and enzymatic activities related to the carbon, sulphur and phosphorus cycles.

Among the tested doses, the intermediate 10D (30 t/ha) was the best solution, as together with a significant percentage of mineralised carbon, a rather substantial portion of humic fractions will increase the reserves of organic substance. This is a desirable condition in cases where synthetic mineral fertilisation is not granted or is not economically advantageous (organic farming and extensive agriculture).

On the other hand, the maximum dose of 100D had a ratio of mineralised/humified carbon higher than 1; therefore, these doses could lead, in the long term, to an impoverishment of the OM contained in the soil, thus creating the opposite effect compared to that desired by the amendment.

Based on the results obtained, it is proposed to use B. carinata seed flour as an organic amendment. The usefulness of this practice could manifest itself as crucial for the enrichment of the soil in phosphorus and in cultural alternations that involve the inclusion of plants of the Leguminosae family in nitrogen thanks to their nitrogen-fixing capacity.