Assessing the Carboxymethylcellulose Copper-Montmorillonite Nanocomposite for Controlling the Infection of Erwinia carotovora in Potato (Solanum tuberosum L.)

A novel antimicrobial formulation based on carboxymethylcellulose (CMC) spray-coated Cu2+ intercalated montmorillonite (MMT) nanocomposite material was prepared and its morphology, internal structure, and bonding interactions were studied. Meanwhile, the antibacterial efficacy and release behavior of Cu2+ was also determined. PXRD patterns indicated the intercalation of Cu2+, while FTIR spectra and TGA traces confirmed the association of Cu−MMT with CMC. SEM study revealed the improvement of nanocomposites by CMC, without disturbing the clay structure. TEM and EDAX studies indicated the distribution of Cu (copper) throughout the composite. In vitro antibacterial assays performed with Erwinia carotovora revealed effective bacterial growth suppression, indicating the potential of this material in controlling soft rot of potatoes (Solanum tuberosum); also observed was a connection between growth inhibition and concentration of CMC spray coats indicating a positive relationship between Cu2+ release and concentration of the CMC coatings. The activity pattern of the nanocomposite displayed a significant degree of sustained-release behavior.


Introduction
Nanoscience-based products and applications have fetched a greater attention in a range of fields including the biocide industry. Cu is a major active ingredient of chemicals with cidal effects because it is effective against numerous plant and mammalian diseases [1,2]. The use of Cu in agricultural applications goes back to the 19th century. Inorganic Cu compounds with fungicidal and bactericidal properties, and as fertilizer additives are very popular due to their low cost [3]. Another advantage possessed by copper is that bacteria and fungi cannot build up resistance against it as they do with antibiotics and synthetic fungicides that are organic in origin. Among the Cu based biocides, Cu(NO 3 ) 2 , CuSO 4 .5H 2 O, Cu(OCl) 2 , Cu(OH) 2 , and Cu 2 O are more common, with the metallic copper encapsulated formulations are more effective in terms of extended activity than nonencapsulated forms as encapsulation reduces the losses caused by unwanted release [39] whilst the clay component in the composite improves the structural properties of the nanocomposite [40].

Preparation of Cu 2+ Exchanged MMT (Cu-MMT)
A portion of 9 g of Na−MMT (Sigma Aldrich, St. Louis, MO, USA) were weighed and mixed with 200 mL of 0.05 M CuSO 4 .5H 2 O (Sigma-Aldrich) solution at room temperature (25 • C) and after stirring the mixture was kept overnight. The resulting solution was centrifuged for 20 min at 5000 rpm. Then it was decanted, and the remaining solid material was washed three times with distilled water. Following this, the material was oven-dried for 6 h at 80 • C. After drying it, a sample of the Cu-MMT was analyzed for the percentage of copper by weight using a Thermo Scientific ICE 3500 atomic absorption spectrophotometer (AAS).

Preparation of CMC Spray-Coated Cu-MMT Nanocomposites
CMC (CDH laboratories, New Delhi, India: Viscosity 1% at 25 • C, 1200-2400 cps) at different concentrations, specifically 2.5, 5 and 7.5 g/L were prepared using distilled water heated up to 80 • C. After adding CMC, the solution was continuously stirred for 2 h and then cooled down to room temperature. The three solutions at room temperature were poured into a spray gun in turn. Prepared Cu−MMT from each composite was spread on Petri dishes (2.5 g each) uniformly. Each Petri dish with Cu−MMT was sprayed with different strengths of the CMC solutions while slightly shaking the dishes. After spraying, the composites were kept in a drying oven at 60 • C for 6 h to remove moisture.

Structural Properties
PXRD patterns of all synthesized samples were recorded using a Bruker D8 Focus Xray powder diffractometer using Cu K radiation (=0.154 nm) over a 2θ range of 3-65 • with a step size of 0.02 • and a step time of 1s. The nature of chemical bonding of the synthesized samples was determined using a Bruker Vertex 80 FTIR spectrometer, by spanning the range from 600 to 4000 cm −1 using the attenuated total reflectance technique. The thermal behavior of the synthesized samples was studied by performing TGA (TA Instruments SDTQ600). The samples (10-15 mg) were heated from ambient temperature to 100 • C (ramp 10 • C/min) in a nitrogen environment (100 cm 3 /min N 2 flow rate). The particle size and the morphology of the synthesized samples were studied using a HITACHI SU6600 SEM and Philips CM30 TEM and energy dispersive X-ray analysis (EDAX).

Antibacterial Properties Experiment 1: Bacterial Growth Inhibition Test
In-vitro antibacterial activity was assessed using gram-negative bacterium Erwinia carotovora, which was isolated from potatoes showing soft rot symptoms which were collected from a field in Nuwara-Eliya, Sri Lanka. A single bacterial colony of E. carotovora obtained through sub-culturing was inoculated into 50 mL of nutrient broth (Sigma-Aldrich) and shake-incubated at 30 • C for 72 h. Then the bacterial cell concentration of the culture was determined by the dilution plate technique. At the same time, Petri dishes with nutrient agar were prepared and wells were bored in the middle of the solidified agar plates. Each well was incorporated with composites of 20, 40, or 60 mg and the wells were again filled with nutrient agar. The composites were exposed to UV light for a period of 6 h before adding them into the petri dishes. Then 100 µL of the E. carotovora culture was spread on the surface of the material in the Petri dishes and incubated at 29 ± 1 • C for 24 h. After the incubation period, the diameter of the inhibition zones corresponding to each weight Nanomaterials 2021, 11, 802 4 of 16 level was measured using a Vernier caliper. The experiment was replicated thrice and the average values of the diameters of the inhibition zones were obtained.

Experiment 2: Potato Tuber Inoculation Test
Certified, disease-free potatoes (variety Granola) were used for inoculation. First, the tubers were thoroughly washed with water and then surface sterilized with NaOCl 1% solution. After that, the outer peels were removed. Following that, they were sliced into pieces measuring approximately 2.5 × 2 × 0.5 cm 3 , which were considered as replicates. Each treatment was comprised of 10 replicates of potato pieces weighing approximately 60 g each (optimum economic size of cut potato that can be used as planting material, according to Mayakaduwa et al. [15]. The following treatments were done using all three CMC spray-coated Cu−MMT nanocomposites (2.5, 5, and 7.5 g/L CMC). The potato pieces in each replicate were treated with either 20 mg, 40 mg, or 60 mg of each composite (approximately 2 mg, 4 mg, or 6 mg/replicate). As control one group was treated with only Erwinia. In each of the above treatments, every replicate was inoculated with 10 µl of 1.2 × 10 5 CFU/mL Erwinia cell suspension. All inoculations were done under aseptic conditions. After three days of inoculation, observations were made and the areas of infection were measured, from which the percentages of infection were calculated using the formula, [(area infected/ total area of the tissue piece) × 100] and the values were averaged.
A factor-factorial analysis was performed to determine the effect of concentration of CMC spray coating and weight of nanocomposites on the percentage of infection in the above two experiments.

Soil Release Study
A soil release study was conducted as in soil medium, an interaction takes place between the tubers treated with composites and soil. Accordingly, One Cu−MMT−CMC composite (Cu−MMT−CMC 5.0 g/L) out of three was tested for its release behavior and the release of Cu was compared with pure CuSO 4 .5H 2 O and uncoated Cu-MMT. Cu−MMT−CMC 5.0 g/L was selected as it was stable in the presence of atmospheric water. The test was conducted using two potato growing soil types (i.e., sandy soil with low organic matter content and loamy soil with high organic matter content) packed in two leaching columns for seven days. The leachates were analyzed for the quantification of Cu by means of atomic absorption spectrophotometry (AAS) using a Thermo Scientific ICE 3500 spectrophotometer. The data were plotted as the cumulative percent release of Cu over time (days). A schematic representation of all experimental procedures is provided in Figure 1.

PXRD Characterization
The PXRD results provide information about the purity of materials and the crystalline phases present in the prepared nanocomposite. PXRD patterns for MMT, CMC, and Cu−MMT−CMC nanocomposites are shown in Figure 2. Basal dspacing values were determined using X-ray diffraction. Accordingly, Na-MMT showed a diffraction peak at 2θ = 7.40°: 1.19 nm (corresponding interlayer d001 value according to the Bragg equation). Na + ions present in the interlayer space of MMT exchange with Cu 2+ ions, causing the d001 value to shift to 1.23 nm as hydrated Cu 2+ ions cause the interlayer distance to increase [41]. Many researchers have reported dspacing changes in Cu-MMT composites with values ranging from about 1.21 to 1.27 [22,28,31,[42][43][44][45][46][47][48]. Small new reflections at 2θ = 13 in the diffraction patterns of Cu-MMT could be due to amorphous Cu(OH)2. H2O [44] while dspacing of clays vary with the level of hydration. After spray coating CMC on Cu−MMT particles, interlayer space remains unchanged as it is a surface coating on the solid Cu-MMT particles. It is important to mention that the spray coating of CMC was preferred over the direct addition of Cu−MMT into the solution as the acidity of Cu 2+ ions might lead to coagulation of CMC.

PXRD Characterization
The PXRD results provide information about the purity of materials and the crystalline phases present in the prepared nanocomposite. PXRD patterns for MMT, CMC, and Cu−MMT−CMC nanocomposites are shown in Figure 2. Basal d spacing values were determined using X-ray diffraction. Accordingly, Na-MMT showed a diffraction peak at 2θ = 7.40 • : 1.19 nm (corresponding interlayer d 001 value according to the Bragg equation). Na + ions present in the interlayer space of MMT exchange with Cu 2+ ions, causing the d 001 value to shift to 1.23 nm as hydrated Cu 2+ ions cause the interlayer distance to increase [41]. Many researchers have reported d spacing changes in Cu-MMT composites with values ranging from about 1.21 to 1.27 [22,28,31,[42][43][44][45][46][47][48]. Small new reflections at 2θ = 13 in the diffraction patterns of Cu-MMT could be due to amorphous Cu(OH) 2 . H 2 O [44] while d spacing of clays vary with the level of hydration. After spray coating CMC on Cu−MMT particles, interlayer space remains unchanged as it is a surface coating on the solid Cu-MMT particles. It is important to mention that the spray coating of CMC was preferred over the direct addition of Cu−MMT into the solution as the acidity of Cu 2+ ions might lead to coagulation of CMC.

FTIR Analysis
FTIR technique is used to study the bonding interactions involve within the nanocomposite as intermolecular bonds play an important role in the release kinetics of active ingredients present in the prepared nanocomposite. FTIR spectra for MMT, CMC, and Cu−MMT−CMC are shown in Figure 3. CMC shows a band at 2908 cm −1 due to C-H stretching of the -CH2 groups and the band due to ring stretching of -COOappears at 1600 cm −1 . In addition, the bands in the region 1350-1450 cm −1 are due to symmetrical deformations of -CH2 and -COH groups. The bands due to -CH2OH stretching mode and -CH2 vibrations appear at 1070 and 1020 cm −1 , respectively [49]. MMT shows a characteristic absorption band at 3400 cm −1 due to the O-H stretching of adsorbed water and the shoulder at 3628 cm −1 due to structural -OH groups of MMT. Peaks are seen at 995 cm −1 and 1125 cm −1 are due to Si-O stretching vibrations of MMT layers [47]. In the FTIR spectrum of Cu−MMT−CMC, O-H stretching of adsorbed water of MMT shows two lobes at 3350 and 3170 cm −1 . In addition, the shoulder at 3628 cm −1 due to structural OH of MMT has shifted slightly to 3622 cm −1 .

FTIR Analysis
FTIR technique is used to study the bonding interactions involve within the nanocomposite as intermolecular bonds play an important role in the release kinetics of active ingredients present in the prepared nanocomposite. FTIR spectra for MMT, CMC, and Cu−MMT−CMC are shown in Figure 3. CMC shows a band at 2908 cm −1 due to C-H stretching of the -CH 2 groups and the band due to ring stretching of -COO − appears at 1600 cm −1 . In addition, the bands in the region 1350-1450 cm −1 are due to symmetrical deformations of -CH 2 and -COH groups. The bands due to -CH 2 OH stretching mode and -CH 2 vibrations appear at 1070 and 1020 cm −1 , respectively [49]. MMT shows a characteristic absorption band at 3400 cm −1 due to the O-H stretching of adsorbed water and the shoulder at 3628 cm −1 due to structural -OH groups of MMT. Peaks are seen at 995 cm −1 and 1125 cm −1 are due to Si-O stretching vibrations of MMT layers [47]. In the FTIR spectrum of Cu−MMT−CMC, O-H stretching of adsorbed water of MMT shows two lobes at 3350 and 3170 cm −1 . In addition, the shoulder at 3628 cm −1 due to structural OH of MMT has shifted slightly to 3622 cm −1 .

Thermal Analysis
The thermal analysis provides the thermal stability of the materials prepared. Here the weight change of the material is determined by heating the sample at a constant rate. TGA and differential thermal analysis (DTA) traces for Cu−MMT−CMC nanocomposites are shown in Figure 4. All three Cu−MMT−CMC nanocomposites show similar thermal events. Up to 200 °C , a total weight loss of approximately 15% occurs due to dehydration of surface absorbed water in CMC and MMT in the nanocomposite. Furthermore, at around 250 °C , a 2% weight loss occurs due to the removal of water of the crystallization of CuSO4. The thermal event around 300 °C is due to the degradation of CMC [47] in the composites and the losses are in keeping with the concentration of CMC sprayed onto the composites. For instance, the 2.5, 5, and 7.5 g L −1 concentration levels show 2%, 3%, and 4% of weight loss, respectively. The thermal peak at 500 °C is due to further degradation of CMC residues. Those losses are approximately 3%, 5%, and 6% and correspond to the concentrations of 2.5, 5, and 7.5 g/L, respectively. The prominent thermal event at around 600-700 °C is due to the collapse of the layered structure of MMT [47]. At 800 °C another thermal event could be observed due to the removal of SO2 and O2 from CuSO4 leaving behind CuO. Finally, at 1050 °C another thermal event occurs due to the removal of O2 from CuO leaving a residue of Cu2O ( Figures S1 and S2).

Thermal Analysis
The thermal analysis provides the thermal stability of the materials prepared. Here the weight change of the material is determined by heating the sample at a constant rate. TGA and differential thermal analysis (DTA) traces for Cu−MMT−CMC nanocomposites are shown in Figure 4. All three Cu−MMT−CMC nanocomposites show similar thermal events. Up to 200 • C, a total weight loss of approximately 15% occurs due to dehydration of surface absorbed water in CMC and MMT in the nanocomposite. Furthermore, at around 250 • C, a 2% weight loss occurs due to the removal of water of the crystallization of CuSO 4 . The thermal event around 300 • C is due to the degradation of CMC [47] in the composites and the losses are in keeping with the concentration of CMC sprayed onto the composites. For instance, the 2.5, 5, and 7.5 g L −1 concentration levels show 2%, 3%, and 4% of weight loss, respectively. The thermal peak at 500 • C is due to further degradation of CMC residues. Those losses are approximately 3%, 5%, and 6% and correspond to the concentrations of 2.5, 5, and 7.5 g/L, respectively. The prominent thermal event at around 600-700 • C is due to the collapse of the layered structure of MMT [47]. At 800 • C another thermal event could be observed due to the removal of SO 2 and O 2 from CuSO 4 leaving behind CuO. Finally, at 1050 • C another thermal event occurs due to the removal of O 2 from CuO leaving a residue of Cu 2 O (Figures S1 and S2).  SEM technique is used to obtain information on the surface topography of prepared nanocomposites. As seen in Figure 5a,b SEM images show that the plate-like layered structure of MMT continues to remain even after the formation of the composite which facilitates the controlled release behavior of active ingredients present in the nanocomposite. Furthermore, no accumulations of CMC could be seen, confirming the relative uniformity of the spray coats.

Microscopy Scanning Electron Microscopy (SEM)
SEM technique is used to obtain information on the surface topography of prepared nanocomposites. As seen in Figure 5a,b SEM images show that the plate-like layered structure of MMT continues to remain even after the formation of the composite which facilitates the controlled release behavior of active ingredients present in the nanocomposite. Furthermore, no accumulations of CMC could be seen, confirming the relative uniformity of the spray coats.  SEM technique is used to obtain information on the surface topography of prepared nanocomposites. As seen in Figure 5a,b SEM images show that the plate-like layered structure of MMT continues to remain even after the formation of the composite which facilitates the controlled release behavior of active ingredients present in the nanocomposite. Furthermore, no accumulations of CMC could be seen, confirming the relative uniformity of the spray coats. intercalated layers. Figure 6b shows the Cu particles intercalated in MMT layers along with the CMC polymer in the form of a network in a dark field TEM image.
of clay layers and a region of small intercalated tactoids together with a few intercalated layers. Figure 6b shows the Cu particles intercalated in MMT layers along with the CMC polymer in the form of a network in a dark field TEM image.
Besides, Figure 6c shows a nanoscale TEM−EDAX elemental mapping in which MMT layers consisting of O, Al, and Si were detected as the primary component of the MMT while CMC polymer consisting of C was detected along with the C which emanates from the sample substrate. S was observed in varying proportions throughout the nanocomposite which arises from sulphate residues present in the nanocomposite. As seen in Figure  6c TEM−EDAX image of Cu shows that Cu is distributed more evenly over the MMT layer compared to other elements.

Experiment 1: Bacterial Growth Inhibition Test
The cell concentration of the bacterial culture was determined as 3 × 10 9 CFU/ mL through the dilution plate technique. Figure 7 shows the results of a bacterial inhibition test for all three Cu−MMT−CMC nanocomposites whereas Table 1 shows the diameters of the inhibition zones observed. The results clearly indicate that the diameter of the inhibition zone increased with the number of composites tested, indicating the antibacterial activity of all types of nanocomposites used in the study against E. carotovora.
The output of factor-factorial analysis can be interpreted in terms of a particular weight level if a slight reduction of the diameter of the inhibition zone can be observed when the concentration of the CMC spray coat increases. An interaction effect between factors was not significant when the p-value was 0.102 (p > 0.05). However, the main effects, the specifical concentration of spray coat and weight were highly significant correspondingly with p = 0.006 and p = 0.000 values (p < 0.05) to the diameter of the inhibition zone. Besides, Figure 6c shows a nanoscale TEM−EDAX elemental mapping in which MMT layers consisting of O, Al, and Si were detected as the primary component of the MMT while CMC polymer consisting of C was detected along with the C which emanates from the sample substrate. S was observed in varying proportions throughout the nanocomposite which arises from sulphate residues present in the nanocomposite. As seen in Figure 6c TEM−EDAX image of Cu shows that Cu is distributed more evenly over the MMT layer compared to other elements.

In Vitro Antibacterial Activity Against Plant Pathogenic Erwinia carotovora 3.2.1. Experiment 1: Bacterial Growth Inhibition Test
The cell concentration of the bacterial culture was determined as 3 × 10 9 CFU/ mL through the dilution plate technique. Figure 7 shows the results of a bacterial inhibition test for all three Cu−MMT−CMC nanocomposites whereas Table 1 shows the diameters of the inhibition zones observed. The results clearly indicate that the diameter of the inhibition zone increased with the number of composites tested, indicating the antibacterial activity of all types of nanocomposites used in the study against E. carotovora. This indicates Cu 2+ ions can be released into the medium by penetrating through the polymer network of the medium. Therefore, it can be inferred that the mobility of Cu 2+ is more or less in step with the current range of concentration. Compared to the polymercoated nanocomposites, the negative controls, i.e., CMC, MMT (Na + ), or MMT−CMC did not show any antibacterial activity (data not shown) against E. carotovora.  Prior to the antibacterial test, the average Cu content of the composites in triplicate was determined through AAS and found to be 9.2%, 8.6% and 8.2% of Cu by weight respectively, which corresponded to spray coats of 2.5, 5.0 and 7.   Prior to the antibacterial test, the average Cu content of the composites in triplicate was determined through AAS and found to be 9.2%, 8.6% and 8.2% of Cu by weight respectively, which corresponded to spray coats of 2.5, 5.0 and 7. The output of factor-factorial analysis can be interpreted in terms of a particular weight level if a slight reduction of the diameter of the inhibition zone can be observed when the concentration of the CMC spray coat increases. An interaction effect between factors was not significant when the p-value was 0.102 (p > 0.05). However, the main effects, the specifical concentration of spray coat and weight were highly significant correspondingly with p = 0.006 and p = 0.000 values (p < 0.05) to the diameter of the inhibition zone.
This indicates Cu 2+ ions can be released into the medium by penetrating through the polymer network of the medium. Therefore, it can be inferred that the mobility of Cu 2+ is more or less in step with the current range of concentration. Compared to the polymercoated nanocomposites, the negative controls, i.e., CMC, MMT (Na + ), or MMT−CMC did not show any antibacterial activity (data not shown) against E. carotovora.

Experiment 2: Potato Tuber Inoculation Test
The percent infection was decreased with the increased concentration of the CMC spray coats and increased weight of nanocomposites applied to potato tuber pieces while the control pieces showed higher infection percentages compared to other treatments (Table 2; Figure S3). Here the Cu−MMT−CMC 2.5 g/L (60 mg) showed the lowest percentage of infection. Through the factor-factorial analysis, it was revealed that the concentration of the spray coat has a significant effect on percentage infection with the p-value of 0.0016 (p < 0.05). Similarly, the weight of the nanocomposite also showed a significant effect on the percentage of infection (p < 0.001). Moreover, the interaction effect of concentration of the spray coat and weight of the nanocomposite was also significant as p-value was 0.0057 (p < 0.05).  Cu based compounds have been proven to be efficacious against many plant pathogenic bacteria including Erwinia spp. [50]. Cu compounds are widely used against many Erwinia spp. including E. amylovora in apple and pear, E. mangiferae in mango and E. trachiphila in cucurbits [51]. Among the Cu based compounds, CuSO 4 .5H 2 O is in common usage for controlling plant pathogenic bacteria [23]. In-vitro experimental results have proven its ability to control plant pathogenic bacteria species belonging to the genus Pectobacterium (formerly in the genus Erwinia) [52].
Interestingly, with respect to the potato crop, Abo-Elyousr et al. [5] have demonstrated that spraying CuSO 4 had the highest controlling effect on soft rot caused by E. carotovora. In addition to that, Zhang et al. [53] reported that potato soft rot caused by E. carotovora can be effectively controlled by using CuSO 4 . Furthermore, Gracia-Garza et al. [54] demonstrated that treatment of greenhouse-grown calla lilies (Zantedeschia sp.) with sub-irrigation laced with Cu and Cu (OH) 2 reduced the soft rot incidence of E. carotovora without compromising the plant growth.
Several other types of bacteria besides the one tested in the present study were studied. For instance, Cu−MMT composites were prepared and tested with E. coli and it was found they suppressed the growth of this organism [27,28,[31][32][33]; this showed that the efficaciousness and ability of Cu-MMT were superior to Na-MMT [55]. Ag-MMT was also tested against mesophilic bacteria and bacteria present in lactic acid by Costa et al. [34] and against E. faecium by Magaña et al. [30], which showed it to be efficacious in all these cases. MMT exchanged with Cu, Ag, and Zn, when tested against E. coli, Pseudomonas ostreatus and P. cinnabarinus had shown promising results in respect of suppression of growth of those bacteria [29]; in the same study, it was reported that Cu−MMT is efficacious over Cu 2+ . Furthermore, certain studies had reported that Cu 2+ intercalated MMT can bind with polymers to enhance the sustained release nature of Cu 2+ into the medium and act against E. coli and S. aureus effectively. For example, this led to the development of a Cu−MMT-epoxy resin [35]. Furthermore, epoxy matrices with CuO nanoparticles embedded in MMT [37] and Cu 2 O embedded in octadecyl amine-modified MMT [21] have also been studied and found to be very effective at suppressing the growth of E. coli. Therefore, the present study provides new insights into employing Cu−MMT-polymer composites for controlling plant diseases.
In general, positively charged biomaterial-i.e., polymer surfaces show an antimicrobial effect on adhering Gram-negative bacteria [56]. Replacement of Na + in Na−MMT by Cu 2+ increases the presence of positive charges on the surface and in the interlayer space of MMT where the Gram-negative bacteria tend to get attached. E. carotovora is a Gram-negative bacterium that is highly vulnerable to Cu 2+ [29,31]. The addition of a biopolymer like CMC on Cu−MMT particles further enhances the release of Cu 2+ ions by absorbing moisture from the environment. Moisture absorption by CMC due to its highly hydrophilic carboxylic group causes the CMC composite to swell to a marked extent, depending mainly upon the pH of the medium [57,58]. The MMT also increases the swelling capacity of certain composites, i.e., CMC-g-poly (acrylamide)-MMT [59].
In Sri Lanka, losses caused by E. carotovora are significant in certain economically important crops [60,61]. As observed, farmers in Sri Lanka tend to cut the seed potato instead of the whole tuber to minimize the costs incurred. Under such situations, infestations could be higher as the cut surfaces facilitate the penetration process. Furthermore, Cu 2+ is not only efficacious against bacteria, but it also affects soil-borne fungi such as Phytopthora spp. Its broad range of action against organisms that destroy crops has favored higher usage of Cu 2+ throughout the world.

Soil Release Study
The cumulative amounts of Cu released plotted against time from CuSO 4 .5H 2 O, Cu−MMT, and Cu−MMT−CMC were measured using a sandy soil sample. The results are shown in Figure 8. Overall, the Cu releasing pattern from pure Cu (from CuSO 4 ) and Cu−MMT nanocomposite are similar when compared to Cu−MMT−CMC nanocomposite, which nearly leveled off after the third day. Therefore, the present study provides new insights into employing Cu−MMT-polymer composites for controlling plant diseases. In general, positively charged biomaterial-i.e., polymer surfaces show an antimicrobial effect on adhering Gram-negative bacteria [56]. Replacement of Na + in Na−MMT by Cu 2+ increases the presence of positive charges on the surface and in the interlayer space of MMT where the Gram-negative bacteria tend to get attached. E. carotovora is a Gramnegative bacterium that is highly vulnerable to Cu 2+ [29,31]. The addition of a biopolymer like CMC on Cu−MMT particles further enhances the release of Cu 2+ ions by absorbing moisture from the environment. Moisture absorption by CMC due to its highly hydrophilic carboxylic group causes the CMC composite to swell to a marked extent, depending mainly upon the pH of the medium [57,58]. The MMT also increases the swelling capacity of certain composites, i.e., CMC-g-poly (acrylamide)-MMT [59].
In Sri Lanka, losses caused by E. carotovora are significant in certain economically important crops [60,61]. As observed, farmers in Sri Lanka tend to cut the seed potato instead of the whole tuber to minimize the costs incurred. Under such situations, infestations could be higher as the cut surfaces facilitate the penetration process. Furthermore, Cu 2+ is not only efficacious against bacteria, but it also affects soil-borne fungi such as Phytopthora spp. Its broad range of action against organisms that destroy crops has favored higher usage of Cu 2+ throughout the world.

Soil Release Study
The cumulative amounts of Cu released plotted against time from CuSO4.5H2O, Cu−MMT, and Cu−MMT−CMC were measured using a sandy soil sample. The results are shown in Figure 8. Overall, the Cu releasing pattern from pure Cu (from CuSO4) and Cu−MMT nanocomposite are similar when compared to Cu−MMT−CMC nanocomposite, which nearly leveled off after the third day.  As shown in the results, at the end of the first 12 h, Cu−MMT−CMC cumulative release percentage was around 2.2. The release percentage gets gradually increased to five times of initial quantity until day 5 and then gets leveled off just over 10.5%. Initially, there is a 1% of Cu release from Cu-MMT and then slightly increased release behavior up to 4.5% of Cu at 120 h which is similar to the Cu−MMT−CMC nanocomposite. Nearly two times lower release of Cu from Cu-MMT than Cu−MMT−CMC may be due to the non-covalent intercalation of Cu inside the MMT layers that induces the slow release even though they had similar kinetics. The burst release of Cu−MMT−CMC nanocomposite (coated) compared to Cu−MMT can be due to CMC coating. CMC absorbs and retains water which swells to facilitate dissolution of more trapped Cu within MMT and after that release through diffusion to the surrounding.
Based on the results obtained for pure Cu, the instantaneous Cu release was observed on the first 12 h which is around 7%. Despite this, the rest release pattern of pure Cu showed moderately increasing behavior reaching nearly 9% around day 7. However, the comparative analysis indicated that the nanocomposite had a slow-release behavior followed by instantaneous release due to the absence of modifications compared to the other two formulations. Thus, it reveals that the addition of CMC to the Cu−MMT (Cu−MMT−CMC) nanocomposite might concentrate much of the copper inside nanocomposite giving the ability to release Cu in a gradually increasing manner and the same time Cu ions which have been loaded to the intercalated MMT(Cu−MMT) layers enabling them to be released in a slow and controlled manner. In general, the release pattern of Cu from Cu−MMT−CMC gives a better and controlled release behavior in comparison to the other two forms.
In contrast, the leachate of potato soil with higher organic matter content did not contain Cu. Thus, releasing around 10% of Cu from the composite incorporated into the sandy soil is considerable. The reasons behind on not containing Cu in the leachate might be the complexation and coagulation of Cu with humic acids [62], which might be an underlying cause that prevents releasing Cu in this case.

Conclusions
Through the current work, Cu intercalated montmorillonite nanocomposites coated with three different concentration of CMC spray were formulated and characterized for their essential properties. FTIR spectra and TGA traces confirmed the association of Cu−MMT with CMC. The plate-like appearance of MMT was maintained even after nanocomposite formation which is vital for controlled release behavior. All three Cu−MMT−CMC nanocomposites showed antibacterial activity against E. carotovora making them suitable for tuber treatments. Furthermore, in the agar plate experiment, antimicrobial activity was slightly reduced when the concentration of the CMC spray coats increased; this implied that the Cu 2+ ions released into the medium by penetrating through the polymer network were slightly affected by the CMC coating on the Cu−MMT particles. When the composite was tested for the release of Cu 2+ , it was found that it could release Cu 2+ instantaneously, followed by a controlled release pattern. Furthermore, it carries a great commercial potential as clays and biodegradable CMC are environmentally friendly, biocompatible, and economically viable.