Experimental fungi

In the present study seven fungi of importance in postharvest were evaluated:Botrytis cinerea,Monilia fructicola,Penicillium digitatum,Penicilliumsp.,Colletotrichum gloeosporioides,Fusarium solaniandRhizopus stolonifer.In the Table 1 details the information for each isolate.

Testing biological effectiveness of the SES on spore germination and germ tube development

An inoculation increment was made for the selected strains in PDA culture medium. The fungi were incubated at 27 °C for 7-10 days in a BINDER-BD® incubator. From each of the genera, a conidial solution of 1 × 10⁵spores mL^-1in sterile distilled water was prepared. An electrolyzed super oxidation solution with neutral pH (SES) was used, which was provided by Esteripharma, SA. of CV. The value of pH (pH 7.03 ±0.02), concentration of active chlorine (54 ±1 mg^-1 L) and potential oxidation-reduction was observed after the electrolysis and until the end of the experiment. (862 ±3.9 mV) were completely stable.

Dilutions were made with the SES and the conidial solution in sterile distilled water at concentrations of 3, 5, 6, 8, 18, 24, 27, 29, 36 and 43 mg L^-1of active chlorine. The spores remained 5 min at each concentration. From each dilution (treatment) eight samples (replicates) of 100 μLwere taken and placed at four equidistant points on a Petri dish with PDA culture medium; the beads were covered with a coverslip, then the boxes were capped and incubated at 27 °C. For each treatment, an absolute control was included. In the absolute control, the spores were immersed in sterile distilled water replacing the SES.

Percent inhibition of spore germination was determined. The estimation was performed by counting 100 spores at eight randomly selected points, one spot per coverslip, in the clear field at 10x of a Leica® model DM1000 optical microscope. The number of spores germinated on the PDA culture medium was recorded at 24 and 48 h after sowing.

Also, photographs were taken of the spores, and the software Leica LAS® length germ tube (µm) spores 24 and 48 h was measured. A completely randomized design with eight replicates per treatment was used for the statistical analysis, where the experimental unit was the spore concentration covered with the coverslip. An analysis of variance (Anova) was performed and comparison of means (Tukeyp≤ 0.05) using SAS (^{SAS Institute, Inc., 2010}).

Effectiveness of SES on Botrytis cinerea

InBotrytisisolated blackberry in the state of Colima, SES had effect in reducing spore germination at all concentrations at 24 h at concentration intervals of 2-43 ppm active chlorine germination was reduced by 100%. At 48 h 100% reduction was observed at 4 and 18-43 ppm (Table 2). The development of the germ tube was inhibited at 24 h at concentrations of 2 to 43 ppm, while at 48 h the total inhibition was reached from 4 to 43 ppm (Table 3). For the case ofBotrytisisolated from blackberry in Jalisco, similar results were observed, where concentrations of 2 to 43 ppm were not statistically different at 24 h and 100% reduction in germination was presented at concentrations of 3, 4 and 18-43 ppm at 48 h, 100% reduction was observed at 1, 3, 4, and 18-43 ppm (Table 2).

In the length of the germ tube, at 24 h the inhibition was reached at 3, 4 and 18-43 ppm, while at 48 h it was present at concentrations of 1, 3, 4 and 18-43 ppm (Table 3). Several studies have reported that electrolyzed water has asporicidal effect onBotrytis(^{Huanget al., 2008}; ^{Craveroet al., 2016} and ^{Felizianiet al., 2016}). ^{Guentzelet al. (2010} and ²⁰¹¹) reported that electrolyzed water close to neutrality (pH= 6.3- 6.5) inhibitedB. cinereaisolated from grape and strawberry at concentrations of 25 to 100 ppm of active chlorine.

Effectiveness of SES onColletotrichum gloeosporioides

InColletotrichummango isolated in Nayarit, SES inhibited 100% germination of spores at 24 h in concentrations of 3, 6-24 and 29-43 ppm, whereas in 48 h, all concentrations showed total inhibition of spores (Table 4). The SES inhibited germ tube development at all concentrations at 24 h. At 48 h, concentrations of 2-43 ppm inhibited 100% growth, except at 27 ppm, where the length was 6 μ (Table 5).

ForColletotrichumin mango fruits from Sinaloa, inhibition in germination was 100% at concentration intervals of 6-43 ppm at 24 h, and from 5-43 ppm at 48 h (Table 4). Similar effects were observed in the length of the germ tube (Table 5). InColletotrichumguava fruits, SES inhibited 100% spore germination and germination development at all concentrations at 24 and 48 h (Table 4 and 5). For the case ofColletotrichumin lychee fruits, at 24 h concentrations of 3 to 18 ppm and 27 to 43 ppm inhibited 100% germination, while at 48 h, all concentrations with SES inhibited 100% germination of spores (Table 4). In the length of the germ tube, at 24 h their development was inhibited at concentration intervals of 3-18 ppm and 27-43 ppm. At 48 h, all concentrations with SES inhibited the development of the germ tube (Table 5).

Based on the results obtained, it can be determined that SES has a control effect onColletotrichumspores, which causes anthracnose in mango, lychee and guava, at concentrations equal to or greater than 6 ppm. ^{Huanget al. (2008)} and ^{Hatiet al. (2012)} mentioned that electrolyzed water has shown sporicidal effect onColletotrichum. In a strawberry study (Fragaria × ananassa), ^{Hirayamaet al. (2016)}, reported that neutral electrolyzed water (pH= 6.5-7.5) completely eliminated the conidia ofC. fruticolaat 10 ppm. According to ^{Bucket al.(2002)},Colletotrichumis a fungus that has a thin wall, which makes it susceptible to damage with electrolyzed water. This is because it induces cell disruption, which causes alterations in cellular processes and consequently cell death (^{Hatiet al., 2012}).

Effectiveness of SES onFusarium solani

The results onFusariumisolated from chile showed that SES at concentrations of 3 to 43 ppm reduced spore germination by 100% at 24 h; at 48 h only the concentration at 5 ppm reduced germination 81.45%, the rest of the concentrations reduced it by 100% (Table 6). In the germ tube length concentrations of 3 to 43 ppm completely inhibited the development of the tube at 24 h, and at 48 hatconcentrations of 3 to 4 and from 6 to 43 ppm there was no tube growth (Table 7). On the other hand,Fusariumisolated from stevia presented results similar toFusariumisolated from chili, both in the percentage of inhibition of spore germination and in the length of the germinative tube (Table 6 and 7). ^{Bucket al. (2002)} reported that electrolyzed water reduces spore germination inFusarium.

Similar results were reported in F.oxysporumf.sp.lycopersiciwith acid electrolyzed water (^{Abbasi and Lazarovits, 2006}). For its part ^{Audenaertet al. (2012)} determined that the electrolyzed neutral water had a sporicidal effect onF. graminearum. The data obtained in this research suggest that the SES has sporicidal effect and in the development of theFusariumgerminative tube at concentrations up to 3 ppm.

Effectiveness of SES onMonilinia fructicola

In the percentage reduction in spore germination all SES concentrations were not statistically different (p≤ 0.05) in the two evaluations (24 and 48 h); in the first evaluation the percentage of reduction was 77 to 100%, where the concentrationsof6to43 ppm inhibitedthegermination 100%, for the last evaluation the reduction percentage was between 66 and 100%, and the concentrations that totally inhibited germination were 3, 8, and 24 to 43 ppm (Table 8). For the germ tube length, concentrations of 6 to 43 ppm inhibited the development of the tube at 24 h, whereas at 48 h the inhibition was present at 3, 8 and from 24 to 43 ppm (Table 9 ).

In general, it can be inferred that concentrations of SES equal to or greater than 24 ppm significantly affect the germination and development of theMoniliniagerm tube. Similar results were reported by^{Al-Haqet al. (2001)}; ^{Huanget al. (2008)}; ^{Hatiet al. (2012}); ^{Rahmanet al. (2016)}: ^{Felizianiet al. (2016)}, who mentioned that electrolyzed water has control effects onMonilinia. For its part ^{Guentzelet al. (2010)} reported that electrolyzed water near neutrality (pH= 6.3-6.5) inhibitedM. fructicolaat concentrations of 25 to 100 ppm of active chlorine.

Effectiveness of SES onPenicillium digitatumand Penicilliumsp .

ForPenicilliumcase isolated from Persian lemon from Nayarit, SES concentrations of 18 to 43 ppm reduced germination of 100% spores at 24 and 48 h (Table 10). On the other hand, total inhibition of the germ tube at 24 and 48 h was observed in the concentration range of 18 to 43 ppm (Table 11). For the case ofPenicilliumisolated from Mexican lemon from Colima, the 100% reduction at 24 h was obtained at concentrations of 4 to 43 ppm, while at 48 h it was from 8 to 43 ppm (Table 10).

On the other hand, in the germ tube length, total inhibition at 24 h was presented in the concentration range of 4 to 43 ppm, whereas at 48 h it was in the range of 8 to 43 ppm (Table 11). InPenicilliumisolated from papaya, similar effects were also observed; in the reduction of spore germination, at 24 h the concentration range of 3 to 43 ppm reduced germination by 100% and at 48 h at concentrations of 5 and 8-43 ppm (Table 10). The length of the germ tube was inhibited at the same concentrations as in spore germination (Table 11). Whangchaiet al. (2009) reported that acid electrolyzed water (pH= 4.84) at a concentration of 215 ppm of active chlorine completely inhibited the development ofP. digitatumspores in mandarin (Citrusxtangerina) after two minutes of immersion.

One year later ^{Whangchaiet al.(2010)} reported that acid electrolyzed water (pH = 3.9) at 102 ppm of active chlorine had the same effect on the same fungus from one minute of immersion. In contrast to the above, the results obtained in this research suggest that concentrations with SES higher than 8 ppm have sporicidal effects onPenicillium.

Effectiveness of SES onRhizopus stolonifer

The total reduction ofRhizopusspore germination in soursop was presented at concentration intervals of 5-24 and 29-43 ppm at 24 h, while at 48 h concentrations of 5,

6, 18, 24, 29 and 43 ppm also inhibited 100% (Table 12). On the other hand, in the length of the germinative tube the concentration intervals 5-24 and 29-43 ppm totally inhibited their development in 24 and 48 h, the rest of the concentrations had similar or greater growth to the control (Table 13).

For the case ofRhizopusisolated from yaca, concentrations with SES of 18 to 43 ppm were observed to reduce spore germination by 100% in 24 and 48 h; On the other hand, it is important to note that the concentration of 8 ppm was not statistically different from the above concentrations, and that the reduction in spore germination was slightly lower in 24 and 48 h (Table 12). In the length of the germ tube the concentrations of 18 to 43 ppm completely inhibited the development of the tube in both evaluations, 24 and 48 h. However, it should be noted that the concentration of 8 ppm was not statistically different in the concentration range of 18 to 43 ppm at 48 h (Table 13).

Although the genusRhizopusand specificallyR. stoloniferis one of the most important post-harvest fungi (^{Spadaro and Droby, 2006}), there is little information on the effects of electrolyzed water onRhizopusspores; however, the results indicate that concentrations equal to or greater than 18 ppm of SES have significant effects on the germination and development ofRhizopusspores.