Micro-nano bubble water pre-treatments: antifungal action against Colletotrichum gloeosporioides sensu lato, curative control of anthracnose and impact on guava fruit quality

Abstract Anthracnose in guava fruit ( Psidium guajava L.) is one of the major problems in the fruit industry. This study investigated the antifungal effects of air and ozone (O 3 ) micro-nano bubble (MNB) water against the identified fungal pathogen in- vitro and in- vivo (on ‘Fan Retief’ guava fruit). The internally transcribed spacer (ITS) universal primers (ITS1 and ITS4 pair) were used to conduct a polymerase chain reaction (PCR) on extracted fungal DNA. C olletotrichum gloeosporioides (C. gloeosporioides) sensu lato was accurately identified as the isolate from diseased guava tissues. For the in- vitro study, 1 mL of spore suspension (1 × 10 6 spores/mL) of C. gloeosporioides sensu lato was exposed to 9 mL of 200 mg/L sodium hypochlorite (NaOCl) for 5 min, and air-MNB or O 3 -MNB for 30 min each, while the untreated suspension was used as the control. The microstructural and ultrastructural changes of the spores were analysed using confocal laser scanning microscope (CLSM) and scanning-transmission electron microscope (STEM). Fresh guava fruits were inoculated with 1×10 6 spores/mL of C. gloeosporioides sensu lato and washed with distilled water (control), 200 mg/L of NaOCl for 5 min, air- or O 3 -MNB for 30 min, and assessed for disease severity. A separate batch of non-inoculated fruits were pre-treated and packaged in non-hermetic polyethylene clamshells and stored for 21 days at 13 °C, 90 ± 2% relative humidity. Peel colour, weight loss, respiration rates (RR), total soluble solids (TSS), titratable acidity (TA) and TSS/TA ratio were monitored. Spore viability tests via CLSM revealed that NaOCl led to ≈76.45% dead spores, followed by O 3 -MNB (≈54.44%), air- (≈39.27%), and control (≈8%). STEM micrographs showed vacuolization under both MNB and NaOCl treatments. While NaOCl showed significant ( P < 0.005) inhibitory effects against anthracnose, O 3 -MNB water was comparable to NaOCl at day 6 of storage. Treatment with O 3 -MNB better maintained fruit quality and slowed down rapid ripening as observed by the low weight loss, RR and TSS/TA. This study provides alternatives to chlorine-based washing for fruits.