Decay severity

4. IN VITRO AND IN VIVO DISINFECTION AND BIOCONTROL TREATMENTS

5.6 Results and Discussion

5.6.1 Decay severity

Table 5.1 presents the decay severity on the surface of kumquat fruit due to P. digitatum.

The treatment and storage period had a highly significant (P≤0.001) influence on the decay severity of kumquat fruit. No visible mould growth was observed between Days 0 and 7 for all treatments. On Day 14, a notable increase in the mould formation was measured at 4.48% of the surface area of samples treated with chlorinated water only.

Table 5.1 Changes in the decay severity (%) due to Penicillium digitatum encountered in kumquat fruit over a 28-day storage period subjected to different integrated pre-packaging treatments

Treatment Storage Period (Days)

0 7 14 21 28

Chlorinated water 0.00^a 0.00^a 4.48^ab 8.41^b 13.62^c

Anolyte water 0.00^a 0.00^a 0.00^a 1.57^a 4.08^ab

Hot water 0.00^a 0.00^a 0.00^a 0.79^a 1.31^a

Biocontrol (B13) 0.00^a 0.00^a 1.05^a 1.05^a 3.01^ab

Chlorinated water + HWT 0.00^a 0.00^a 0.00^a 4.71^ab 4.71^ab

Chlorinated water + B13 0.00^a 0.00^a 0.00^a 1.36^a 1.36^a

Chlorinated water + HWT + B13 0.00^a 0.00^a 0.00^a 0.52^a 2.62^a

Anolyte water + HWT 0.00^a 0.00^a 1.81^a 1.81^a 1.81^a

Anolyte water + B13 0.00^a 0.00^a 0.22^a 3.50^ab 4.28^ab

Anolyte water + HWT + B13 0.00^a 0.00^a 0.00^a 0.00^a 0.00^a

HWT + B13 0.00^a 0.00^a 0.00^a 0.00^a 0.00^a

Control 0.00^a 0.00^a 0.00^a 0.00^a 0.00^a

Significance

Treatment (A) **

Storage Period (B) **

AB *

CV (%) 20.6

NS, *, ** Non-significant or significant at P≤0.05 or P≤0.001, respectively. Means within a column followed by the same letter(s) are not significantly different from each other according to Duncan’s Multiple Range Test (P≤0.05), (n=3). CV, Coefficient of variation; HWT, hot water treatment; +,

‘combined with’.

On Day 28 the mould formation had grown substantially, amounting to 13.62% (Figure 5.1).

Figure 5.1 Penicillium digitatum-infected kumquat fruit from Day 0 to Day 28 treated with chlorinated water only

Chlorinated water alone resulted in the greatest decay severity, in which a total of 66%

of the fruit in this batch displayed visible signs of mould development, as indicated in Table 5.2. This was followed by the biocontrol treatment alone with 44% of fruit displaying visible decay. This corresponds with the findings by Abraham et al. (2012) in which the use of the yeast biocontrol is better suited as a preventative treatment rather than as a curative treatment. The combination treatment of anolyte water + hot water + B13 did not develop any mould throughout the 28-day storage period. A similar trend was observed in the combination treatment of biocontrol with hot water as well as in the control fruit. Obagwu and Korsten (2003) also found a significant reduction in the blue and green mould of oranges due to the combination of hot water (45°C for 120 seconds) and biocontrol (Bacillus F1). The anolyte water and hot water treatment resulted in a mould formation of 1.81% on Day 14, which remained constant for the remaining storage period. Similarly, chlorinated water and hot water had a constant decay severity of 4.71%.

Hot water only and the combination of chlorinated water and B13 resulted in low decay severity of 1.31% and 1.36%, respectively, by Day 28.

Day 0 Day 7 Day 14 Day 21 Day 28 Tissue breakdown and softening

Table 5.2 Percentage of decayed fruit due to Penicillium digitatum

Treatment Storage Period (Days) *Total % of

decayed fruit

0 7 14 21 28

Chlorinated water 0 0 22 11 33 66

Anolyte water 0 0 0 11 22 33

Hot water 0 0 0 11 11 22

Biocontrol (B13) 0 0 22 11 11 44

Chlorinated water + HWT 0 0 0 11 11 22

Chlorinated water + B13 0 0 0 11 11 22

Chlorinated water + HWT + B13 0 0 0 11 11 22

Anolyte water + HWT 0 0 11 11 11 33

Anolyte water + B13 0 0 11 11 11 33

Anolyte water + HWT + B13 0 0 0 0 0 0

HWT + B13 0 0 0 0 0 0

Control 0 0 0 0 0 0

*Total percentage of decayed fruit at the end of the 28-day storage period. HWT, hot water treatment;

+, ‘combined with’.

The two-way interaction between the treatment and storage period had a significant influence on the decay severity as a result of P. digitatum at P≤0.05. As time progressed the decay caused by P. digitatum increased (Hong et al., 2007; Schirra et al., 2011). Hong et al. (2007) and Sen et al. (2007) attributed the reduction in decay in hot water treated citrus fruit to the melting and redistribution of natural epicuticular wax to seal cracks on the fruit surface. This creates a barrier for pathogen penetration. The reduction in decay could also be due to the host-pathogen interaction, where the combined effect of the pathogen and the hot water treatment induced resistance in the fruit peel. Hot water treatments also resulted in a reduction in the epiphytic microorganism population, which may prove to be beneficial (Hong et al., 2007). Biocontrol treatments have been found to be more effective in reducing decay when combined with other treatments such as hot water (Hong et al., 2014). The presence of B13 on the fruit surface colonises wounds by using up the nutrients produced by the wound (Abraham et al., 2012). Therefore, Penicillium spp. spores are unable to sporulate due to the lack of available nutrients.

However, B13 is most effective when applied as a preventative treatment (Abraham et al., 2012).

The additional initial use of either chlorinated water or anolyte water as a disinfectant to remove some of the previously existing surface pathogens resulted in a lower decay severity. Furthermore, with the action of the hot water treatment to induce fruit resistance, as in the case of combined anolyte water, hot water and B13, no incidence of decay was observed due to the disinfecting action of the anolyte water, the curative action of the hot water and the preventative action of the B13. As mentioned in Section 4.5.2.1, anolyte water is described as having disinfecting properties and the B13 as having preventative properties. The addition of hot water further reduced the onset of decay due to the curative property that it is believed to possess (Ben-Yehoshua et al., 2005). Kim et al. (1991) observed an increase in scoparone in citrus fruit after inoculation with P. digitatum, which further increased after a heat treatment at 36°C for 3 days. The induced concentration of scoparone was sufficient to reduce fungal growth in lemon fruit. This also demonstrated the presence of pathogens to elicit fruit resistance. Lesar (2002) found that a dilution of anolyte water of 1:5 and 1:10 resulted in 100% spore eradication on citrus fruit with an exposure time ranging from 30 to 300 seconds. No visible decay of blue mould was observed on kumquat fruit for all treatments. This could be due to blue mould being more prevalent at cooler temperatures (10C) whereas at room temperatures (25C) green mould develops at a faster rate (Brown, 1994). Schirra et al. (2011) also observed green mould to be the main decay agent in kumquat fruit. Therefore, the results obtained for the development of blue mould on the surface of the kumquat fruit has subsequently been omitted from this section. The results demonstrated that combined pre-packaging treatments proved to be more beneficial in inhibiting decay caused by green mould, compared to individual treatments. In particular the treatments of (1) anolyte water + hot water + B13 and (2) hot water + B13 were most beneficial in preventing decay caused by P. digitatum in kumquat fruit.