Medium molecular weight chitosan (deacetylation degree 75% - 85%; Sigma-Aldrich) was used for the preparation of chitosan nanoparticles. Glacial acetic acid and methanol were purchased from Fermont Chemicals Inc. Ethanol was bought from J.T. Baker.

Colletotrichum gloeosporioides were isolated from Carica papaya L. and Annona muricata L., Alternaria alternata from Ficuscarica and Rosmarinus officinalisat Morelos, México and the isolates were maintained on Potato Dextrose-Agar (PDA) in petri plates at temperature 28˚C. To maintain pathogenicity of the fungus, periodic inoculations and reisolations from infected fruitsand leaves were carried out.

Nanoparticles synthesized were added to PDA after sterilization media and poured into Petri plates (60 × 15 mm). A five-mm agar disc of a 9 days old colony of the pathogen was placed at the center of each plate and incubated at 28˚C for 8 to 14 days in the dark. The colony diameter was recorded for each treatment until fungal colonies in the control treatment reached the edge of the plate. The percentage of inhibition was% I = [(C - T)/C] × 100 where C represents the growth in the non-amended control and T in the treatment. For in vitro studies sporulation and germination were measured as previously described [18] . There were six replicates for each treatment. Control Petri plates contained only PDA and Sportak as commercial fungicide was used for comparison.

A completely randomized design was used for statistical analysis. One-way analysis of variance (ANOVA) with a significance level of P < 0.05 was applied. Similarly, when significant differences were found, a comparison of means was performed using Tukey’s multiple comparison test. A confidence interval of 95% was employed. The analysis was performed using a SigmaStat 3.5 program.

Figure 1 shows the size distribution profiles for chitosan nanoparticles and the nanoparticles added with the botanical extracts. In Figure 1(a), the size distribution for CSNPs is observed. Z-average particle size obtained was 406.6 ± 25.5 nm and Zeta potential value was −12.3 to −12.9. Figure 1(b) and Figure 1(c) show the profiles for chitosan nanoparticles added with the botanical extracts having a Z-average particle size of 521.8 ± 85.9 nm and 304.2 ± 31.7 nm and Zeta potential values of −4.85 to −7.86 mV and −40.0 to −43.8 mV for EEA-CSNPs and EMN-CSNPs, respectively.

Comparing the size distribution of CSNPs, EEA-CSNPs and EMN-CSNPs it can be seen, that the narrower size distribution was for EEA-CSNPs, followed by EMN-CSNPs and finally the CSNPs. On the other hand, the smallest value of Z-average particle size was for the EMN-CSNPs (304.2 ± 31.7 nm), followed by CSNPs (406.6 ± 25.5 nm) and finally EEA-CSNPs (521.8 ± 85.9 nm).

Zeta potential value for CSNPs was −12.3 to −12.9 mV. When EEA-CSNPs is added to CSNPs Zeta potential value decreased (−4.85 to −7.86 mV). However, when EMN-CSNPs are incorporated to CSNPs, the value is increased (−40.0 to −43.8 mV). Addition of bioactive components to chitosan solutions, affects the

surface positive charges of chitosan changing the stability of the nanoparticles. It has been reported in the literature, that a stable suspension must have a Zeta potential minimum value of ± 30 mV [19] . From the results, it can be seen that the most stable suspension was EMN-CSNPs. Also, the lowest particle size (304.2 ± 31.7 nm), was obtained for EMN-CSNPs.

In Figure 2, the FTIR spectra of the CSNPs, botanical extracts and CSNPs loaded with the different botanical extracts are observed. In Figure 2(a) results for ethanolic extract from Vaccinium corymbosum are shown, and in Figure 2(b) results for methanolic extract from Byrsonima crassifolia are seen.

The characteristic absorption bands for CSNPs are: between 3750 and 2750 cm⁻¹ related to -CH, -OH and -NH stretching, at 2921 cm⁻¹ corresponding to -CH, at 1635 cm⁻¹ related to amide I (-NH₂), at 1375 cm⁻¹ for -CH deformation and at 1027 cm⁻¹ corresponding to stretching vibrations of C-O-C for the aromatic ring. The band between 878 and 676 cm⁻¹ belongs to pyranoside ring (Figure 2(a) and Figure 2(b)).

On the other hand, in Figure 2(a) for the EEA, the characteristic absorption

bands are almost similar, only varying in intensity. They main peaks are: between 3750 - 3100 cm⁻¹ for-CH and -OH, at 2900 cm⁻¹ corresponding to -CH, at 1717 and 1594 cm⁻¹ for -C=O and at 1079 cm⁻¹ for C-O-C.

After Vaccinium corymbosum incorporation to the CSNPs (Figure 2(a)). The bands at around 2900 cm⁻¹ corresponding to -CH, the bands at 1717 and at 1594 cm⁻¹ for -C=O and the band at 1079 cm⁻¹ for C-O-C were broadened (green circle), indicating EEA incorporation into the CSNPs. However, after incorporation of the EMN to CSNPs, for Byrsonima crassifolia small changes (green circle)were observed in characteristic absorption bands in the FTIR spectra (Figure 2(b)).

Table 1 shows the results in vitro evaluations of chitosan C (2%) and the CSNPs and EEA-CSNPs (0.05%) on A. alternata development after 8 - 14-day incubation at 28˚C. The results obtained were significantly (P < 0.05) different among treatments, the CSNPs and EEA-CSNPs (250 and 500 μl) shown high micelial inhibition and germination inhibition on A. alternata from Fig and Rosemary. The highest sporulation was obtained in CS treatment (9.3 × 10⁷ spores/ml) followed by the EEA-CSNPs (250 to 500 μl) treatments with few spore formation (0.33 a 1.57 × 10⁷ spores/ml) and CSNPs (500 μl) treatments shown 0.00 spores/ml in A. alternata from Rosemary. Similar results were published by [20] which tested the antimicrobial activity of chitosannanoparticles with and without thyme essential oil against C. gloeosporioides. The results showed that oil addition caused the total inhibition of this fungus. In this work, the results showed that CSNPs (500 μl) without EEA caused the total germination inhibition of A. alternata from Rosemary.

C = chitosan; CSNPs = chitosan nanoparticles; EEA = ethanolic blueberry extract. Mean in the same column followed by different letters are statistically different according to the Tukey test (P < 0.05).

Table 2 shows the results in vitro evaluations of chitosan CS (2%) and the CSNPs and EMN-CSNPs (0.05%) on C. gloeosporioides from papaya and soursop development after 8 - 14-day incubation at 28˚C. The results obtained were significantly (P < 0.05) different among treatments. The PDA and CS control showed the lowest mycelial inhibition, EMN-NPQ with 500 and 750 μl and the commercial fungicide showed the highest mycelial inhibition. A dose response effect was observed in the treatments of CSNPs and EMN-CSNPs as the volume increase, increased the mycelial inhibition in both isolates of C. gloeosporioides. Inhibition of germination was 0% to 11% in the treatments with CS and CSNPs, with the treatment of EMN-CSNPs and the fungicide of 47% to 100% inhibitionon C. gloeosporioides from papaya, similar values were obtained over C. gloeosporioides from soursop. Chookhongkga, Sopondilok, and Photchanachai [21] evaluated the effect of chitosan nanoparticles on mycelial growth of Rhizopus sp., C. capsici, C. gloeosporioides and A. niger, finding lower mycelial growth of 2.8, 2.2, 2.4 and 5.5 mm, respectively, at 0.6% concentration. Other studies showed that applying chitosan nanoparticles at 0.1% inhibited mycelial growth of A. alternata, Macrophomina phaseolina and Rhizoctonia solani [22] . Zahid, Alderson, Ali, Maqbool, y Manickam [23] reported that low molecular weight chitosan nanoparticles at 1% concentration had the best inhibitory effect on conidial germination.