Suppression of Blue Mold Symptom Development in Satsuma Mandarin Fruits Treated by Low-Intensity Blue LED Irradiation

Abstract

Blue mold (Penicillium italicum) is a major postharvest pathogen of citrus. Here, we investigated the mitigating effect of blue light-emitting diode (LED) irradiation on blue mold in satsuma mandarin (Citrus unshiu Marc.) fruit after harvesting. We examined the growth and development of blue mold subjected to blue LED irradiation of two different intensities in vitro. High-intensity blue LED with an emission peak of 465 nm and fluency of 80 µmol·m⁻²·s⁻¹, and low-intensity blue LED with an emission peak of 465 nm and fluency of 8 µmol·m⁻²·s⁻¹ significantly reduced the growth of P. italicum. In addition, wounded fruits inoculated with P. italicum were irradiated with high- and low-intensity blue LED for 6 days and changes in blue mold symptoms were monitored. The results indicate that not only high-intensity, but also low-intensity blue LED treatment significantly reduced blue mold symptoms in fruit; the most pronounced inhibitory effect was exerted on P. italicum sporulation. These results show that treatment with low-intensity blue LED irradiation is sufficient to reduce blue mold symptom development and is a promising safe approach to control postharvest spoilage in mandarin fruit.

Introduction

Wounded citrus fruits are frequently spoiled by blue mold during postharvest handling. The blue mold Penicillium italicum is a major postharvest pathogen of citrus. Satsuma mandarin (Citrus unshiu Marc.) is mainly harvested from October to December, a portion of which is stored at normal or low temperatures (5 – 8°C) until March. Shizuoka Prefecture in Central Japan is famous for the production of high quality satsuma mandarin ‘Aoshima unshu’. In Shizuoka Prefecture, the economic loss of satsuma mandarin from various postharvest diseases during storage is estimated to be approximately 20% of the total yield. Currently, long-term storage is more complicated because of global warming, and rind puffing of mandarin fruits is now more common. Puffy fruits are frequently damaged during harvest, transport, and storage, and can be easily infected and decayed by fungi.

Measures to control postharvest decay and long-term storage have been extensively investigated (Ikeda et al., 2004; Izumi, 1999; Kitagawa and Tani, 1983; Murata and Yamawaki, 1992), and storage technology has been improved by using high temperatures, chemical fungicides, and the other methods as postharvest treatments.

At present, chemical fungicides are used for controlling blue mold; however, their use is being increasingly limited due to growing public concern regarding health and environmental issues, including the development of fungal resistance. Alternative methods of postharvest disease management are expected to reduce the application of chemical fungicides. Efficient decay control of ripe satsuma mandarins is important for postharvest storage because of the high economic value of the harvested commodity.

Several authors have proposed alternative approaches to postharvest treatment for the effective control of citrus fruit decay. Salt dipping or spraying have been widely investigated in this respect (Cerioni et al., 2012; Cerioni et al., 2013; Youssef et al., 2012). Cerioni et al. (2012) reported the control of lemon green and blue molds by a combination of hydrogen peroxide followed by inorganic salt treatment. Some researchers have reported that ultraviolet-C (UV-C) radiation and heat exposure induce accumulation of phytoalexin scoparone (6,7-dimethoxycoumarin) and improve the resistance of citrus fruit to decay (Kim et al., 1991; Rodov et al., 1992; Kuniga et al., 2006). Rodov et al. (1992) showed that illumination with UV-C (254 nm) of kumquat fruit inoculated with P. digitatum Sacc. prevents decay for 2 days after treatment. However, it has been reported that UV-C irradiation induces significant citrus peel damage (such as browning) when applied at high doses (D'hallewin et al., 1999). On the other hand, Liao et al. (2013) have reported that relative to red, green and white light, irradiation with blue light at a fluency of 40 µmol·m⁻²·s⁻¹ reduces various symptoms in mature tangerine fruits, such as the development of blue and green molds (Penicillium digitatum). Little information is available regarding the antifungal effect of blue light on the decay of satsuma mandarin fruit, a thin peel citrus variety. Specifically, the relationship between symptom development and light intensity has not been elucidated. The objective of this study was to investigate the possibility of controlling blue mold in satsuma mandarin fruit by blue LED (light-emitting diode) irradiation. We examined the changes in symptom development of mold-inoculated fruits irradiated with high-intensity and low-intensity blue LED.

Materials and Methods

In vitro postharvest fungal growth and development under blue LED irradiation with different intensities

Penicillium italicum was obtained from infected citrus fruits at Shizuoka Prefectural Agriculture and Forestry Research Institute Fruit Tree Research Center, and cultured on potato dextrose agar (PDA; 39 g·L⁻¹) at 25°C in the dark. A suspension was prepared by harvesting spores from the sporulation zone of 7-day-old cultures and suspending in sterile distilled water containing 0.1% Triton X-100. Spore concentration was adjusted to 1 × 10³ spores per ml using a hemocytometer, and 30 µL of suspension was applied to PDA and cultured for 48 h at 25°C under darkness. Mycelium (2.25 mm²) was transferred to a new PDA plate and cultured at 25°C under irradiation with blue LED of high-intensity (LED-80; emission peak, 465 nm; fluency, 80 µmol·m⁻²·s⁻¹; Yamato Industrial Co., Ltd., Shizuoka, Japan) and low-intensity (LED-8; emission peak, 465 nm; fluency, 8 µmol·m⁻²·s⁻¹; Tokyo Rikakikai Co., Ltd., Tokyo, Japan) (Fig. 1). The light intensity of this examination was determined in reference to a previous report (Liao et al., 2013). Dark-treated plates (no irradiation) were used as control. Radial growth of the sporulation and mycelial zones was measured with a ruler every day for 3 days after the treatment. Five petri dishes were utilized per treatment. The experimental setup of blue LED irradiation in vitro is shown in Fig. 2 (left).

Fig. 1.

Relative light-emitting diode (LED) power distribution.

Fig. 2.

Experimental setup of blue LED irradiation in vitro (left) and in vivo (right).

Effect of the irradiation with blue LED of different intensities on blue mold symptom development in satsuma mandarin fruits

Satsuma mandarin fruits (C. unshiu Marc. ‘Aoshima unshu’) were harvested from an orchard at Shizuoka Prefectural Agriculture and Forestry Research Institute Fruit Tree Research Center in December 2013. Harvested fruits were maintained at room temperature for 5 days, and then stored for 2 weeks in the dark at 8°C until the experiment. A spore suspension was prepared as described above, and adjusted to 1 × 10⁵ spores per mL using a hemocytometer. A 2-mm deep × 1-mm diameter incision was made between the pedicel and lateral part of the fruit with a needle equipped with a stopper to ensure wound uniformity, and 2 µL of the suspension was applied to the wound with a precision pipette. The inoculated fruits were maintained under LED-80 and LED-8 irradiation for 6 d in a chamber with 85% to 90% relative humidity (RH) and at 25°C; the control treatment was performed under the same conditions in the dark. The experimental setup of blue LED irradiation in vivo is shown in Fig. 2 (right). The diameters of the soft rot area, mycelial area, and sporulation area (Fig. 3) were measured with a ruler daily starting at day 3 until day 6 after treatment. Twenty-five fruits were utilized per treatment. The number of infected fruits was determined to calculate the disease incidence according to the formula:

Fig. 3.

Measurement of the spoilage areas.

Disease incidence = (number of fruits exhibiting symptoms area/total number of examined fruits) × 100.

Statistical analysis    The data are expressed as the mean ± standard error (S.E.). Statistical analysis was performed using statistical functions in Microsoft Office Excel 2007 for Windows (Microsoft Corporation, Redmond, WA, USA). Differences between means were evaluated by Tukey's multiple range test.

Results and Discussion

As shown in Fig. 4, the in vitro mycelial zone for LED-80 and LED-8 treatments was 47% and 81% of that with dark treatment. The sporulation zone for LED-80 and LED-8 treatment was 6% and 68% of that with dark treatment. The average radial growth of the sporulation zone under LED-80, LED-8, and dark exposure was 0.3, 3.0, and 4.4 mm·d⁻¹, respectively.

Fig. 4.

Effect of blue light-emitting diode (LED) irradiation on blue mold symptom development in vitro. Vertical bars indicate standard error (SE) (n = 5). Different letters indicate significant differences between the treatments; P < 0.05 by the Tukey's multiple range test.

Liao et al. (2013) reported that the growth of P. italicum and P. citri under the blue light-dark regime or continuous blue light (fluency, 40 µmol·m⁻²·s⁻¹) exposure resulted in reduced mycelial radial growth, which is consistent with our results. Furthermore, our data indicated that low-intensity blue LED irradiation can control the in vitro growth of P. italicum.

Changes in blue mold symptom development in vivo are illustrated in Fig. 5. The diameter of the soft rot area did not significantly differ between the LED-treated and untreated fruits until 5 days after inoculation. At day 6, LED-80 treatment significantly reduced the diameter of the growth area: the soft rot area was 68% that under dark treatment. However, no significant difference in the soft rot area was found between LED-8 and dark treatments. The mycelial area was not clearly developed at day 3 after inoculation. However, at days 4, 5 and 6 the difference between the treatments was evident: the smallest diameter mycelial area was observed after LED-80 treatment, followed by LED-8, and the dark control. The reduction in the mycelial area for LED-80 and LED-8 was 90% and 47%, respectively, at day 5 and 44% and 30%, respectively, at day 6 compared to control. The significant difference in mycelial area between LED-80 and LED-8 eventually disappeared at day 6. In vitro, the mycelial zone for LED-80 was markedly suppressed, demonstrating less radial growth than the LED-8 treatment, until 2 d after inoculation; however, the mycelial growth from days 2 through 3 did not differ between the LED-80 and LED-8 treatments. The rate of mycelial area increase might differ depending on the elapsed time since inoculation and exposure to blue light. Ortuno et al. (2011) reported that the symptom diameter of mature lemon fruits inoculated with P. digitatum increased at a faster rate from days 4 through 6 than the earlier time points. They suggested that the increase in symptom diameter differs among citrus cultivars. Further studies to analyze the physiological mechanism of blue mold are required. The sporulation area was not obvious until 4 d after inoculation. Five days after inoculation, sporulation was significantly suppressed by LED-80 and LED-8 compared to the control; the suppression was especially evident at day 6. LED-80 showed stronger suppression of fungal growth than LED-8: the reduction in the diameter of the sporulation area at day 6 for LED-80 and LED-8 was 91% and 48%, respectively, compared to dark treatment. At day 6, the disease incidence in the soft rot area for the dark, LED-8, and LED-80 treatments was 92%, 88% and 72%, respectively, whereas in the mycelial area, it was 92%, 76%, and 64%, respectively (Fig. 6). In the sporulation area, LED-8 and LED-80 treatments showed a reduction of the disease incidence (56% and 16%, respectively) compared to the dark treatment (92%).

Fig. 5.

Effect of blue light-emitting diode (LED) irradiation on blue mold symptom development in satsuma mandarin fruits. Vertical bars indicate the standard error (SE) (n = 25). Different letters indicate significant differences between the treatments; P < 0.05 by Tukey's multiple range test.

Fig. 6.

Effect of blue light-emitting diode (LED) irradiation on the incidence of satsuma mandarin fruit disease caused by Penicillium italicum (day 6 after inoculation).

Suppression of the soft rot area by blue light irradiation could be due to a reduction in the activity of polygalacturonase, a fungally produced pectolytic enzyme (Liao et al., 2013). The diameter of the soft rot area did not significantly differ among samples until 5 d after inoculation, and increases in area were markedly suppressed by LED-80 treatment 6 d after inoculation. These results indicate that it may take over 5 d for blue light to induce a defensive response in citrus peel, which is consistent with previous findings (Alferez et al., 2012; Liao et al., 2013). Liao and Burns (2010) reported diurnal fluctuations and light regulation of phospholipase A2 expression and enzymatic activity in citrus leaf and fruit tissues, and have suggested that accompanying diurnal changes in lipophilic second messengers may participate in the regulation of physiological processes associated with phospholipase A2 activity. Their study indicates that blue light in particular is involved in the modulation of phospholipase A2 gene expression and activity in citrus tissues, including fruit flavedo. The increase of phospholipase A2 activity in flavedo in response to blue light exposure was associated with the reduction in citrus postharvest decay (Alferez et al., 2012). Similarly, our results suggest that the effect of low-intensity blue LED may be related to this enzyme activity in satsuma mandarins.

To the best of our knowledge, this is the first report showing that low-intensity blue LED irradiation with the fluency of 8 µmol·m⁻²·s⁻¹ reduces blue mold sporulation in C. unshiu Marc. ‘Aoshima unshu’. However, this study did not clarify in detail the antifungal response mechanism in citrus fruit. Further studies using biochemical and molecular approaches are required to evaluate the relationship between blue light, host response, and suppression of fungal growth. Furthermore, the response to the pathogen in citrus fruit can vary depending on the maturity and storage environment, and it is important to establish conditions to reduce fruit decay by blue light irradiation under all possible storage environments.

Short-term blue light irradiation, e.g., 30 – 60 min, may not be as effective in inhibiting mold growth and inducing antifungal response as UV-C irradiation. However, in this study, blue light caused no visible damage to the peel of satsuma mandarin even during continuous long-term (6 d) irradiation, whereas UV could induce significant damage (such as browning) of citrus peel when used at higher doses (D'hallewin et al., 1999). Vilanova et al. (2013) reported that decay incidence and lesion diameter in Valencia oranges inoculated with P. digitatum at different times after wounding were significantly decreased with the increase in the time between wounding and inoculation. Blue light irradiation can inhibit fungal growth and prevent the infection of wounded fruits before lesion closure, which may lead to a reduction in decay rate during storage.

Prior to the implementation of LED treatment to actual storage conditions, it is important to develop an irradiation regime. The main difficulty appears to be the limited accessibility of mandarins to irradiation, as there are over 100 fruits in a container, which are stacked several containers high in the storage room. Therefore, before practical application of blue LED in actual storage, we must verify the ability of blue LED to control fruit decay due to naturally occurring infections using non-inoculated mandarins. Subsequently, this technology, i.e., visible light decay control, can be combined with other preservation methods such as temperature and moisture conditioning. Further research is required to develop alternative methods of postharvest disease management.

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