NIRS Footprint of Bio-Fertilizers from Hay Litter-Bags

The biofertilization of crops using microbial biota in the soil (MBS) is a modern practice that is used to sustain fertility. MBS agents can promote the yield and health of crops, by luxuriating in the shoot as well as in the root systems. Farmers devoted to systematic MBS fertilization are creating a “Symbiotic” (S) form of agriculture, which offers a greater advantage of resilience than Conventional (C) or organic farming. Since MBS is involved in organic matter degradation, hay-litter-bag probes can be used to reflect a global functionality of the active soil, in the short-medium term. It is here shown that the NIRS hay-litter-bag technique, intended not as mass decay but as a quality evolution of the hay probes, can be modelled as a valid footprint of S vs. C soils. A patented MBS was used in eight experiments in which litter-bags from an S treated thesis were compared with equivalent litter-bags from a non-inoculated C thesis. The chemical signature of the S vs. C in the litter-bag composition was a percentage decrease of sugars and fibres. A smart NIRS device was used to discriminate the origin of the S vs. C litter-bags and a sensitivity of 71% (P<0.0001) was obtained. External validations on 37 S farms showed that three NIRS models discriminated the true positive S spectra, with a sensitivity of 90% as single and 98% as compound probabilities The NIRS radiation of the hay-litter-bags confirmed the results of the S vs. C agriculture soil footprint. Moreover, the SCIO-NIR devices also made it possible to connect the S farms in a smart network. DOI : 10.14302/issn.2639-3166.jar-18-2084 Corresponding author: Giorgio Masoero, Accademia di Agricoltura di Torino, Via A. Doria 10, 10123, Torino, Italy. E-mail: giorgioxmasoero@gmail.com Running Title: Smart NIRS network for bio-fertilizers


Introduction
Biofertilizer arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) are prominent protagonists in the sustainability search for global agriculture 1 , also concerning the horizons of the BioAg Alliance 2 and Engineering 3 .Their potential can be spread to the different agricultural systems described by Narain 4 , thanks to the properties of resilience inductors 5 .A meta-analysis of field studies on the responses of wheat to AMF 6 has highlighted that field AMF inoculation can be proposed as an effective agronomic practice for wheat production, with aboveground biomass increases of around 20%, as assessed under Indian 7 and in high 8,9 or low 10 Italian input conditions.AMF phenotypes are expressed in accord to the Law of the Minimum 11 .Phosphorus acquisition efficiency is the key feature 12,13 , but Thirkell et al. 14 managed to resolve the paradox of nitrogen: while N-mineral fertilization has been shown to elicit luxuriating and strong mutualism, similar responses have been found to be lacking following the addition of Norganic substances; the Authors, have shown that allowing hyphae access to an organic material can improve the total N and P content, with a simultaneous and substantial increase in the plant biomass (+66% for both the hypogeal and epigeal).The use of fertilizer microbial biotas of the soil (MBS), even at a minimal density of 14 AMF spores per maize seed 8 , has multiple effects: acidification of the roots and stem 15 ; greater resistance to disease 16 ; fortifications of the functional properties, such as the antioxidant potential 17,18 .
Several beneficial effects have been observed along the forage-milk-meat chain 19,20,21 .

The agricultural market crisis in Italy and
Europe has led to a diversification of the supply of products, and also of the methods adopted to obtain different sustainable productions.For some time, several both conventional and organic farmers have engaged in a so called "Symbiotic Agriculture" (S) 22 , in which a systematic use of MBS biofertilizers is adopted.
Considering the chemical parameters of the multifaceted soil fertility that could be rapidly predicted by means of an NIRS examination of the soil bulk sample 23 , lacking of objective rapid measurements able to assess the microbial status of agrarian soils in the present work we aim to demonstrate that a biofertilizing change is real in biochemical functioning mechanisms, and that such a change can easily be testified.
The use of Litter-bags is a technique that has long been adopted in soil studies on microfauna evolution 24 , as well as on mass and / or CHN decay driven by fungi .The idea of coupling a litter-bag to a smart-NIRS technique has sprung from the availability of a new instrument that has been tested successfully with iced milk 26 on live rabbits 27 and for meat discrimination 21 .

Experimental Procedure
The method presented in this study excludes showed a less pronounced downward trend.

Litter-Bag
The easiest and most repeatable substrate for field-scale purposes was identified as being a hay for small animals ("Vita Verde Small Animal Alpine Hay", by  was calculated for each model.In order to formalize an "NIRS biofertilizer footprint", the best three models were then considered for single and for compound probabilities of false negative, by applying a "symbiotic" score predicted from the three independent models to each spectrum: a value of 1 was scored for the S grade and a value of 0 for the C grade.The total symbiotic score of a litter-bag thus varied from 0 (Conventional, nine C=0 from the three models applied to the three spectra) to 3 (fully Symbiotic, when the three models all predicted S=1).The compound probability of the non-S outcome, that is, the false-negative cases, was then fitted from the 318 S spectra.

Eight
The composition of the litter-bags was analysed by means of a mixed one-way model considering the soil type (S vs. C) fixed and the effect of the experiments random 30 .

NIRS Discrimination of the Litter-Bag Origin
The calibration of the SCIO spectra from experiments 1-8 is reported in Tab. 2. The average AKA reclassifications were 71% for S (P<0.0001) and 62% for C (P<0.0001) , with variation coefficients of about 25% between experiments.The results were confirmed from the validation sets in experiments 9 and 10 (Tab. 2), where overall classification ratings of 78±4% for year A and 71±5% for year B were obtained.Among the eight models, numbers 2, 3 and 5 were the best ranking ones for the A and also for the B years : in fact, their average classification ability was 89.9±3.1% and 90.1±3.6%,respectively.Compounding information concerning the algebraic formula of the crop maturity index for growing plants, the regression sign of the percentage on the days from sowing was positive for NDF, ADF and indigestible NDF, while it was negative for ash, crude protein, NDF digestibility and digestible-NDF.After haymaking, in the underground environment the grasses composing the litter-bags started an ontogeny involution, as a result of biotic and abiotic factors, but also because of BMS action.The observed rise in protein may be a sign of increased MBS growth 25,31 , and the net result was that the fibrolytic communities elicited the attacks of the carbohydrates.In terms of crop maturity index, the BMS increased the evolution of the litter-bags towards a more mature type of residue, as can be observed in Fig. 1, where the lines of the S and C trends cross.Our results suggest that, as expected, BMS promotes the mechanisms that are favourable for an early maturation of the residual organic matter in the root horizon, and multi-annual observations are necessary 32 .BMS management is based on the inoculation of aerobic microbes, but, because of a luxuriating rhizosphere, and in spite of respiration-fermentation processes, the net long term result could improve the carbon footprint of the whole plant-soil system, and thus raise its sustainability.supported by EU agricultural policies, while the pro-MBS route has been totally neglected, in spite of the phosphate crisis that is expected from 2030 onwards 33 .
The results of the present work are in agreement with Leolini's 34 results (ibid Table 12) obtained from a litter-bag quality study conducted on six natural sites in Spain, in which four litter types were considered.In fact, as a result of some mild differentiated activities, a significant linear increase was observed in the Spanish study for the ash and ether extract percentages, but the digestible NDF also increased, while no labile constituent was reduced.In The outlooks on the use of NIRS regard both plant tissues and canopies 37,38 as well as soil quality for precision agriculture purposes 39 , all of which require new approaches to acquire soil data on landscapes 40,23 .Direct NIR scanning of the soil horizons has also been proposed as a valid and practical tool to monitor the ontogeny and heterogeneity of detritus in soil, which is useful for the assessment of the carbon and nitrogen budget of the soil 41 , but even for the macro components of soil biota: in fact, Zormoza et al. 42 found very high r-squares for AMF (0.91), Fungi (0.80), Protozoa (0.73), Actinomycetes (0.92) and Bacteria Gram+ (0.91), and also for enzymatic activities, while direct NIR scanning was not so reliable for exchangeable P (0.46).Vol-1 Issue 1 Pg.no. 31

Conclusions
Obtaining knowledge about functional soil biota is expensive, as well as long and hard to achieve.
operation and is based on a footprint of a summary microbial transformation of a standard hay litter-bag evaluated according the percentage variation of the composition on a short-medium term.The work of Santoni 28 has indicated how the more numerous recalcitrant compounds (hemicellulose, ash, ether extract, crude protein and lignin) showed a parabolic upward trend that pointed out an attenuation of the accumulation percentage and a decrease after a maximum at around 60 d.In parallel, the labile substances (cellulose in the NDF and crude fibre) experiments were set up under different conditions for calibration purposes in order to observe the NIR spectra and decomposition of the litter-bags, as well as the S vs. C discrimination ability.The involved crops were : Lolium, Wheat, Coffea, Grapevine, Pear, Quercus and Olive (Tab.1), and the litter-bag experiment concerned 106 C-type litter-bags, which were compared with 143 S-type litter-bags in two complex of 249 FT-Perkin Elmer and of 698 NIRS-SCIO spectra.The common denominator of the trials was the fertilisation of the soil with a patented MB, Micosat F ® (www.micosat.it),a consortium based on: AMF from finely ground cultivated sorghum roots, containing spores and ifae of Funneliformis coronatus GO01 and GU53, F. caledonium GM24, F. intraradices GB67 and GG32, F. mosseae GP11 and GC11, F. viscosum GC41; spp.SB14, Streptomyces spp.SA51, Beauveria spp.BB48, Trichoderma viride, T. harzianum, Trichoderma harzianum TH01, Trichoderma atroviride TA28, Trichoderma spp.; rhizosphere bacteria: Bacillus subtilis BA41, Pseudomonas fluorescens PN53, Pseudomonas spp.PT65 and Pochonia chlamidosporia, in the relative percentage of 40% crude inoculum (AM fungi) and 21.6% bacteria and saprotrophic fungi.In order to validate the litter-bag-NIR-SCIO technique, 37 farms belonging to the "La Granda quality food consortium" (Fossano, It), which started to use a systematic biofertilization of their fields five-six years ago in order to develop a Symbiotic production chain, introduced 89 litter-bags into S-type fields and meadows.The validation experiment lasted two years (A, B) and 318 spectra were obtained.The eight models that predicted the S vs. C type from the calibration experiments were applied to the validation data-set spectra.The classification percentage of the Stype spectra correctly predicted as S-type (sensitivity)

Figure 1 .
Figure 1.Relative deviation of the litter-bag residues from the hay composition after 60 d of landfilling for the Symbiotic and Conventional groups and litter maturity tendency enhanced in the S vs C conditions.

Figure 2 .
Figure 2. Average NIR spectra of the Litter-bags measured by the two instruments.It is possible to consider how short but rich the SCIO range is.
the agrarian and well cultivated soils of the present work, the litter quality appeared to be modified to a great extent, and the MBS attacked the labile components, which arithmetically enhanced the percentages of the counterparts.In the MOLTE long-term organic experiment 26 (ibid Fig. 72), only a mild footprint of the Organic vs. Conventional soil was found by means of a multivariate analysis of the predicted constituents (R 2 0.18±0.04)or, rather, in the direct NIR spectra discrimination (R 2 0.32±0.06).Litter-bag decomposability, intended as mass decay, can be related to NIR spectra 35 , and these correlations were also present in the data from Florence University: in 34 R 2 0.46 for the residual mass; in 28 0.74 for the residual mass and 0.90 for the k decay term (recalculated).In the present work, the quantitative aspect of litter-bags was omitted, because of operational difficulties at a large scale, but also after results from the MOLTE experiment which elicited a more meaningful structural and functional relationship from the variability of the litter-bag quality than from considering the total lost mass or the exponential decay.MBS activities in litter-bag matrices mainly depend on the unexplored vast communities in the foreign soil, and to a lesser extent on the microbiome of the hay.The outcomes of litter-bag modifications are also modulated by abiotic factors, such as the pabulum conditions, i.e. thermic, water and mainly the redox-oxygen availability.According to 36 , an incubation of litter-bags for two months could allow the net N mineralization to be estimated, and in the present BMS framework, an N preservation appeared.The effect of the MBS treatment on the symbiotic fingerprint appeared to be quite consistent and repeatable for the five significantly varied constituents.NIRS Discrimination of the Litter-Bag Origin As far as the discrimination problem is concerned, can these five signs be considered a valid support to obtain an univocal response that could help to testify the use of MBS as biofertilizers?The chemical composition of litter-bags needs a chemometric deconvolution of a broad NIR-IR spectra (714-3333 nm), which could be obtained from high-quality scientific instruments.These devices represent a valid tool to help understand some mechanisms, but are less portable for a large-scale dimension.Thus, thanks to the overtones and combinations of the organic molecules in the electromagnetic spectrum, originating in the IR region, a surprisingly small but rich NIR spectra 740-1070 nm (Fig. 2) can be capitalized on by means of vibrational spectroscopy.For field sampling and analyses operations on a smart-farm basis, the S footprint should be directly searched for in the electromagnetic spectrum.Considering the immensity of the biotas in different farms and crops, a rational choice among local models could protect against gross biases.The between-farm validation adopted in the present work is similar to a local vs.global chemometric procedure, utilized to manage large NIR datasets of soils in a betterway23 .The false negative litter-bags, with an S grade of 1, were mostly concentrated on two farms.This may have been the result of a real inefficacy of the BMS for those particular management conditions.
Moreover, problems in use may arise.Smart sensors that match offline solutions in performance while enabling size reductions, low power consumption, low unit costs, low maintenance costs and data fusion43 are currently being investigated, however far from practical solutions.The proposed rapid comparative method of over 90% success is limited to some Italian farmers organized to monitor their progressive results from fields with probative results of litter-bags over the years.A relevant feature is that it would be possible to testify a future yield, even before harvesting.Above all, this form of indirect certification of the production process, based on the microbial soil footprint instead of a direct NIRS discrimination of the products, would eradicate difficult searches for specific markers of the S footprint in the final product.The natural increase in functional compounds in symbiotic farming products, and mainly in antioxidants, is a scientifically proven fact.However, as we are moving in a context of biological variability, it is unlikely that there will be no overlapping of one or a few characteristic substances between symbiotic and conventional products that are statistically evident at the individual level (and not only of averages detected in experimental trials with several replicates).A diffuse web network could capitalise on this diffusive system of harmonized sampling and smart NIRS analyses, as suggested by Klakegg44 referring to its potential use in future everyday cases.Vol-1 Issue 1 Pg.no.33 studio del processo di decomposizione di diverse specie daovescio nel dispositivo sperimentale Montepaldi Long Term Experiment (MOLTE) per il confronto di sistemi colturali biologici e convenzionali.Tesi Magistrale Università di Firenze Anno Accademico 2013/2014:1-131.29.Tassone S, Masoero G, Peiretti PG, 2014.Vibrational spectroscopy to predict in vitro digestibility and the maturity index of different forage crops during the growing cycle and after freeze-or oven-drying treatment.Animal Feed Sci.Techn.194:12-25.

Table 2 .
Calibration of NIR-SCIO spectra in eight experiments for the Conventional © and Symbiotic (S) footprint of litter-bags and validation on 37 Symbiotic farms of single and the best three chained models.Values in classification percentages (C->C and S->S = Sensitivity).

Table 3 .
Classification probability of the Symbiotic grade 3, 2 and the compound classification (3 or 2) >1, or false negative cases, from the three best equations in the validation of the 318 Symbiotic spectra.Vol-1 Issue 1 Pg.no.28

Table 4 .
Composition of the hay and of the litter-bags in the Conventional (C) and Symbiotic (S) fields.