Correlation of Soil Physiochemical Properties, Microorganism Numbers, and Bacterial Communities Following Unburned and Burned Sugarcane Harvest

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Introduction
Te burning of agricultural residues is a universal phenomenon and can cause global air quality decline [1,2] and contribute to greenhouse gas emissions [3].Air pollution and greenhouse gases are caused by carbon dioxide, methane, nitrous oxide, and fuorinated gases [4].Furthermore, the particulate matter generated from biomass burning has negative efects on both the environment and human health [5,6].Burning agricultural residues are a serious threat to soil security and food chain sustainability.For example, the efect of burning wheat residues on soil quality has caused nutrient losses in both developing and developed countries [7].Sugarcane burning practices negatively afect soil quality, human health, and harvested sugarcane moisture loss [8].During the agricultural harvesting season in Tailand (November to February), burning on cultivated land is a major problem, especially the preharvest burning of sugarcane.
Sugarcane is a crucial source of sugar and ethanol production in Tailand, with 1.74 million ha planted with sugarcane producing 2.00 million in the production year 2020/21 [9].Annually, sugar factories purchase sugarcane from farmers as either fresh sugarcane (73.6%; 4.90 million t) or burned sugarcane (26.4%; 1.76 million t).Although government measures have become more stringent to limit the practice of burning during harvesting, farmers still burn sugarcane.Substantial losses of carbon, nitrogen, and organic matter (OM) due to sugarcane residue burning have been reported and have resulted in the deterioration of soil quality [8,10].Increases in soil temperature during preharvest burning afect soil microorganisms and water content [11,12].Rachid et al. [13] reported that under diferent management regimes (preharvest burn and mechanical, unburnt harvest, or green sugarcane), there were signifcant changes in the total structure, ammoniaoxidizing, and denitrifying bacterial communities.In addition, soil microorganisms play critical roles in soil OM decomposition, nutrient availability, and cycling [14,15].Tus, the soil microbiome must be managed to ensure reliable agricultural production and efective control of plant diseases [16].It is known that burning afects soil bacteria and soil properties; therefore, this work extends the investigation of the relationship between soil bacterial communities and soil chemical properties as a changing relationship or not and how it afects soil properties.Te purpose of the current research was to compare and study correlations in the efects of unburned and burned sugarcane harvesting on the soil properties and the population structure of soil microorganisms to better understand the ecological impact of burning sugarcane.

Soil Sample Collection and Physicochemical
Analysis before Planting and after Harvesting.Composite samples in a zig-zag pattern were collected from a homogeneous plot of land (8-10 ha), consisting of a mixture of 10-20 subsamples [17].Samples were collected at a depth of 0-30 cm in a Kamphaeng Saen soil series in Bo Suphan subdistrict, Song Phi Nong district, Suphan Buri province (14.165747 °N, 99.800659 °E), Tailand.Some of the soil properties before planting sugarcane were analyzed.Te potential of hydrogen (pH) was measured using a pH meter.Soil samples were processed using H 2 SO 4 -Na 2 SO 4 -Se mixture digestion as specifed to analyze the total nitrogen content based on the Kjeldahl method for analyzing soil OM [18].Te phosphorus content was analyzed according to Bray and Kurtz [19], potassium was analyzed using atomic absorption [20], and the cation exchange capacity (CEC) was determined using the method of 1 N ammonium acetate, pH 7.0 [21].Te soil moisture content was analyzed based on oven-drying at 105 °C for 24-48 h [22] and was kept in a desiccator until the weight was stable.Te percentage of soil moisture is calculated according to the formula: (weight of moist soilweight of dry soil)/weight of dry soil.

Sugarcane Planting and
Harvesting.Khon Kaen 3, a sugarcane cultivar in the frst ratoon cane, was used in the current research.Two diferent treatments were carried out for sugarcane harvest (unburned and burned sugarcane felds) with 3 replications.Te plot size was 5 rows planted × 12 m (1.4-1.8 m between planted rows).Fertilizer was applied following recommendations according to the soil analysis for sugarcane by the Department of Agriculture, Tailand (N:P 2 O 5 :K 2 O at the rate of 2.88 : 0.96 : 2.88 kg•ha −1 , respectively).Te composite soil samples were collected from 10-20 points in each plot at a depth of 0-30 cm.Te soil from felds growing sugarcane aged 12 months was collected following unburned and burned sugarcane harvesting for soil analysis, microbial counts, and deoxyribonucleic acid (DNA) extraction.Te samples were kept on ice before transporting to a laboratory for microbial analysis.

Microbial Counts.
A soil solution dilution method was used in this experiment.Te soil solution was diluted to a range of 10 −1 -10 −6 with 3 replications.Ten, 0.1 ml of the appropriate dilution solution was pipetted into a Petri dish.Diferent types of microorganism-specifc media were used: nutrient agar (NA) for bacteria, actinomycetes isolation agar (AA) for actinomycetes, potato dextrose agar (PDA) for fungi (HIMEDIA), Pikovskaya (PVK) medium for phosphate-soluble bacteria [23], and N-free medium (NF) for nitrogen-fxing bacteria [24].Microorganisms were counted after incubation at 30 °C for 24 h for bacteria and for 7 days for actinomycetes and fungi.Te numbers of microorganisms were compared using the logarithm of colonyforming units (log CFU) per 1 g of dry soil weight (Dw).

DNA Preparation and Metagenomics
. DNA was extracted from 0.25 g of soil using a NucleoSpin soil DNA Purifcation Kit (Macherey-Nagel, Germany) following the protocol supplied by the manufacturer.DNA samples were quality-checked based on 1% agarose gel electrophoresis and quantifed using a Nanodrop spectrophotometer (Maestro, Taiwan).A sample of DNA (20-30 ng) was used to generate amplicons.Te library preparation and sequencing were used for the V3 and V4 hypervariable regions of the prokaryotic 16S ribosomal RNA (rRNA) gene.Te library was quantifed to 10 nM; PE250/FE300 paired-end sequencing was performed according to the Illumina MiSeq/NovaSeq (Illumina, USA) instrument manual.Processing of the 16S rRNA libraries was performed using the quantitative insights into microbial ecology (QIIME) software, version 1.9.1 [25].Te resulting sequence for the operational taxonomic unit (OTU) clustering used the VSEARCH clustering software, version 1.9.6 [26].Nonmetric multidimensional scaling (NMDS) was used to display beta diversity visualization, based on the distance between using the Bray-Curtis metric.Te OTU analysis was used with a random sampling of the sample sequences to calculate the indices Ace, Chao1, Shannon, Simpson alpha diversity and Good's coverage, and community species sequence (sequence similarity was set to 97%).Te diversity indexes were calculated using the following equation: Ace index [27]: R � S 0 + a 0 where S 0 : the number of taxa observed at least once in a sample and a 0 : the unknown number of species present in the community but not observed.Chao's index [28,29]: where S max : maximum no. of species, S obs : number of species observed in diferent samples, a: singletons (number of species represented by one individual each), and b: doubletons (number of species represented by two individuals each).Shannon's index [30]: H ′ �  R i�1 P i ln P i and Simpson's index [31]: where Pi: fraction of the entire population made up of 2 Applied and Environmental Soil Science species i, and R: numbers of species encountered.Good's coverage index [32]: 1 − F1/N where F1: the number of singleton OTUs, and N: the total number of individuals or the sum of abundances for all OTUs.

Statistical Analysis and Correlation.
Analysis of variance was carried out using the R software, version 2.15.3 [33] with diference comparisons between mean values using Duncan's new multiple range test.Te diference between treatments was tested as either signifcant or highly signifcant (P < 0.05 and P < 0.01, respectively).Te correlation coefcient was displayed in a heat map of the relationships between quantitative variables using the Minitab, version 16.2.0[34] software.

Changes in Soil Physiochemical Properties after Harvesting
Unburned and Burned Sugarcane.Te soil physiochemical properties at a soil depth of 0-30 cm were analyzed after unburned and burned sugarcane harvesting (Table 1).Tere were no changes in the OM, available phosphorus, and exchangeable potassium contents or in the CEC of the soil.Te most obvious changes in soil properties for the burned sugarcane harvesting compared to the unburned were in the reduced levels for the pH (reduced from 5.54 to 4.60) and the soil moisture (1.42%) and total nitrogen (0.01%) contents.With burned sugarcane harvest, the CEC and phosphate-solubilizing bacteria were positively correlated with Gaiella, Conexibacter, and Nitrospira.Te OM, available phosphorus, and exchangeable potassium were positively correlated with Streptomyces, C. Solibacter, Byobacter, and Bradyrhizobium.Fungi and nitrogen-fxing bacteria were positively correlated with Paenibacillus.Conversely, the OM, available phosphorus, and exchangeable potassium were negatively correlated with soil moisture content, fungi, nitrogen-fxing bacteria, Paenibacillus, and Paracoccus.Te pH was negatively correlated with actinomyces.Te CEC was negatively correlated with bacteria, nitrogen-fxing bacteria, and Paracoccus.

Discussion
Te results clearly showed that burning the sugarcane affected the measured soil properties.Te pH was more acidic, the moisture loss was about 20%, and the total nitrogen was reduced compared to not burning (Table 1).Tese results were consistent with those of Flores-Jiménez et al. [36], who reported volatilized nitrogen nutrients and water loss from the soil during sugarcane burning.Arocena and Opio [37] reported that a pH decrease was generally caused by the loss of base cations during burning.Te opposite efect was reported by the authors of reference [38], who compared to the control (unburned) sites for pine forest and oak forest, the soil pH levels of burnt pine and oak forests were higher by 0.41 and 0.78 units, respectively.In the current study, the numbers of bacteria, actinomycetes, and fungi decreased.In addition, phosphate-solubilizing and nitrogen-fxing bacteria numbers were reduced (Figure 1).Te results were consistent with the results of Mills and Alley [39] who reported heterotrophic bacteria decreasing to 76% at a depth of 0-3 cm and up to 90% at a depth of 5-10 cm after burning of stubble, with Azotobacter (nitrogen-fxing bacteria) and nitrifying bacteria destroyed in the topsoil.Signifcant reductions in nitrogen-related bacteria in the rhizosphere soil and sulfur cycle functions accounted for the continuous efective reduction of the total nitrogen and sulfur contents in the rhizosphere soil of sugarcane along with the decrease in the sugarcane yield and sugar content [40].However, the authors of reference [38] found that the bacterial population tended to increase after the fre because of more available carbon sources.In the current study, the bacterial structure in the soil changed after burning during sugarcane harvest (Table 2 and Figure 3).Notably, a large proportion of Paenibacillus was found in the soil after burned sugarcane harvest (Figure 3).Tese endospore-forming bacteria are heat-tolerant [41] and can survive in a burned crop.Several known species of Paenibacillus sp. are known to colonize the plant rhizosphere, promoting the growth and productivity of crops, including rice, maize, pumpkin, poplar, and switchgrass [42].Candidatus Koribacter, Gaiella, Pseudomonas, and Sphingomonas in the soil after unburned sugarcane harvesting had greater relative abundance than in soil after burned sugarcane harvesting.Tese bacteria are benefcial for sugarcane growth, with C. Koribacter in the Acidobacteria related to nutrient mineralization [43], while Sphingomonas and Gaiella decompose lignocellulose and contribute to nutrient cycling [44].S. paucimobilis ZJSH1 promoted plant growth through nitrogen fxation and various phytohormones, including salicylic acid, indole-3acetic acid, zeatin, and abscisic acid [45].Te diversity of Pseudomonas spp. was associated with nitrogen fxation and indole-3-acetic acid production in sugarcane in Guangxi, China [46], and the efect of Pseudomonas fuorescens with other inocula reduced the dose of phosphate fertilizer and phosphorus accumulation in sugarcanes at the end of the cycle [47].Tese results were related to the work of Kirkby   6 Applied and Environmental Soil Science and Fattore [48], who reported that the stubble retained in soils had higher microbial biomass and microbial activity than in stubble-burned soils.Turning the harvest from burning to green (unburned) reportedly positively afected many soil properties, such as carbon stock, microbial biomass, soil enzymatic activity, and soil aggregation [49,50].Crop residues from sugarcane harvesting provide ecosystem services, nutrient recycling, soil biodiversity, water storage, carbon accumulation, and soil erosion control and restrict weed infestation [51].Terefore, soil management using unburned sugarcane harvest and allowing the waste products to decompose in the feld should be a better option than burned sugarcane harvest.With burned sugarcane harvest, the available phosphorus and exchangeable potassium had a positive correlation with the dominant bacterial community (Figure 4).Te reason for the increase in nutrients was the carbon dioxide released from burning [52], combined with water to produce carbonic acid that afected the soil pH; thus, the pH controls the soil biology and soluble nutrients [53].Another reason for the dominance of the Bacillus genus in the current research was its reported efectiveness in dissolving phosphorus and potassium [54,55].However, the moisture, actinomyces, bacteria, and fungi had a high negative correlation with the dominant bacterial community in the current research; with burning, some groups of bacteria decreased, while others increased.Te cellulolytic and amylolytic populations slightly decreased, but the ammonium oxidizers are positively afected by fre [56].A similar situation was evident in the current research, where the phyla Acidobacteria and the genera C. solibacter and C. koribacter, Streptomyces spp., and Bradyrhizobium, which produce cellulolytic or amylolytic enzymes decreased, while an ammonia-oxidizing bacteria Paenibacillus increased.Te moisture also afects bacterial numbers in the soil [57].However, further research is required because diferent soil series and the soil management methods associated with postharvest burning may change the bacterial community structure and its relationship to soil properties.

. Conclusion
Burning during sugarcane harvest resulted in reductions in soil properties (pH and the total moisture and nitrogen contents) and the number of microorganisms.Te correlation between soil properties and soil bacteria population structure changed patterns in unburned and burned sugarcane harvest, with the bacterial structure in the burned  sugarcane harvest decreasing.Tese bacteria are important for soil fertility and quality that afect plant growth.However, spore-forming bacteria survived the sugarcane burning, of which Bacillus was correlated with phosphorus and potassium solubilization.

Figure 1 :Figure 2 :
Figure 1: (a) Numbers of soil microorganisms and (b) numbers of nitrogen-fxing and phosphate-solubilizing bacteria in unburned and burned sugarcane plots.Te bar indicates ± standard error and * � P <0.05.

Figure 3 :
Figure 3: Relative abundance of major bacteria by (a) phylum and (b) genus from soil samples of unburned and burned sugarcane plots.

Figure 4 :
Figure 4: Correlation coefcients of physiochemical properties, microbial count, and bacterial structure of (a) unburned and (b) burned soils.Te heat map correlation levels are colored in green (positive) or red (negative) hues, while no correlations are shown in yellow.

Table 2 .
Te numbers of bacteria, actinomycetes, and fungi were signifcantly diferent between unburned and burned sugarcane harvest (Figure1(a)).Te numbers of bacteria, actinomycetes, and fungi were 5.68, 4.76, and 4.73 log CFU g Dw −1 , respectively, in unburned sugarcane harvest compared to 4.40, 3.34, and 3.65 log CFU g Dw −1 , respectively, in burned sugarcane.Te soil samples after unburned and burned sugarcane harvest were used to generate V3-V4 16SrRNA gene profles.Bacterial diversity and abundance were evaluated.In total, 1,295,634 reads were achieved with an average count of 215,939 per batch of samples, and in total, 367 OTUs were generated, of which 318 were shared by unburned and burned sugarcane harvesting.Te NMDS ordination (Figure2) showed the structural diferences and separation of soil bacteria communities of the unburned and burned sugarcane harvest.Te values for Ace, Chao 1, Shannon, and Simpson indices were high for both abundance and evenness of the species present in a community for unburned sugarcane harvest compared with burned sugarcane harvest.Good coverage was found at 1.0 for unburned and burned sugarcane harvest.

Table 1 :
Soil physiochemical properties after unburned and burned sugarcane harvesting at soil depth 0-30 cm.
Dongia.Bacteria had a positive correlation with actinomyces, Burkholderia, Sphingomonas, Tumebacillus, and Bradyrhizobium, while fungi had a positive correlation with Paenibacillus, Bacillus, Streptomyces, Pseudomonas, and Jatrophihabitans.Te CEC, total nitrogen, and nitrogenfxing bacteria were negatively correlated with Ammoniphilus.Te pH had a negative correlation with the fungi, Paenibacillus, Bacillus, Streptomyces, while the available phosphorus had a negative correlation with the CEC, Nitrospira, Candidatus Solibacter, Gemmatimonas, Byobacter, and Occallatibacter.

Table 2 :
Soil bacterial diversity indices and richness estimates of unburned and burned sugarcane harvest determined using Illumina MiSeq sequencing analysis.