Benzothiazole (BTH) Induced Resistance of Navel Orange Fruit and Maintained Fruit Quality during Storage

Abstract

Current research aimed at studying the effect of benzothiazole (BTH) on the fruit quality and resistance against Penicillium italicum (P. italicum). Recently, a synthetically prepared novel BTH was introduced that elicits the induction of resistance against various diseases of fruits. However, little was reported on the effect of BTH on the disease resistance and fruit quality of postharvest navel orange fruit. In this study, 50 mg·L⁻¹ BTH significantly reduced the decay rate of fruits during 36 days of storage at 20 ± 0.5°C ($P<0.05$). BTH markedly inhibited the weight loss rate in fruits ($P<0.05$) and effectively maintained higher soluble solid content (SSC), titratable acid (TA), and vitamin C (VC) content compared with control navel orange fruits. Further, BTH significantly suppressed the increase of disease incidence and lesion area of orange fruits challenged with P. italicum ($P<0.05$). BTH treatment significantly enhanced antioxidant capacity (DPPH, ABTS radical scavenging activity, and reducing power), and superoxide dismutase (SOD) and peroxidase (POD) activities were significantly increased, while the activity of catalase (CAT) was opposite to the former ($P<0.05$). The activities of β-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), and chalcone isomerase (CHT) were significantly higher in BTH-treated navel orange fruits ($P<0.05$). Our results suggested that BTH treatment may be a promising treatment for maintaining the quality and inhibiting blue mold of postharvest navel orange in the future.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Navel orange (Citrus sinensis L., Osbeck) is pretty popular among the consumers worldwide and is a favorite fruit due to its unique taste and high nutritional value. However, navel orange is susceptible to pathogenic fungi. Penicillium italicum and P. digitatum, which cause blue mold and green mold, are the major fungi [1, 2] and account for up to 60–80% of the total fungal decay during citrus fruit storage [3, 4]. Postharvest diseases are being controlled through multiple approaches specifically by applying synthetic fungicides. However, the excessive and misuse of fungicides caused resistance of pathogens against synthetic fungicides and caused environmental degradation and human health issues which led to a worldwide trend for exploring novel natural alternatives with reduced or no side effects. The induction of the resistance using various biological, chemical, or physical means may become potential strategies for controlling postharvest decaying of fruits [5]. Some exogenous compounds such as chitosan [6], salicylic acid [7], terpene limonene [4], and nitric oxide [8] have been used to induce disease resistance of postharvest orange fruits. BTH is a newly reported analogue of naturally occurring salicylic acid (SA) reported in plants and is highly effective for induction of systemic-acquired resistance (SAR) in plants to protect from various microbial diseases. Moreover, it has been reported as nontoxic to plants without any unpleasant environmental impacts [9]. BTH exposure can induce resistance for diseases and even wound-mediated suberization in fruits and vegetables such as muskmelon [10], banana [11], strawberry [12], tomato [13], and potato [14, 15] and effectively reduce the occurrence of disease. However, according to the authors’ knowledge, no study explained the efficacy of BTH for the control of blue mold in navel orange.

The current study aimed to examine how BTH affects the growth of postharvest green mold caused by P. italicum and quality of navel orange fruits under storage. Decay rate and fruits’ quality for 36 days of storage at 20 ± 0.5°C were investigated, and antioxidant capacity, antioxidant enzymes activities, and disease resistance related enzymes activities in navel orange fruits following inoculation with P. italicum were also determined.

2. Materials and Methods

2.1. Navel Fruits and BTH Treatment

Navel orange fruits (Citrus sinensis L. Osbeck cv. Newhall) were harvested from the orchard in Ganzhou City, Jiangxi Province, China, at a commercially mature period with a mean soluble solid content (SSC) of 12 °Brix. Fruits of uniform size and free of wound were picked out for the experiments. The navel orange fruits were randomly classified in two groups (each group comprised of over 300 fruits) and exposed with 50 mg·L⁻¹ of BTH (containing 0.05% Tween-80) and deionized H₂O (control, containing 0.05% Tween-80) for 10 min. After that, the fruits were air-dried for further 2 h at room temperature, and all navel oranges were packed in PE bags (1 orange per bag) and were kept at 20 ± 0.5°C, 85% relative humidity (RH) for 36 days.

Certain quality parameters were measured at 6, 12, 18, 24, 30, and 36 days after storage at 20 ± 0.5°C using 10 fruits of each replicate. This measurement of newly picked navel orange fruits prior to experimental studies was named as 0 day, and each treatment was replicated three times.

2.2. Decay Rate and Fruit Quality Parameters Assay

The fruits decay process under room temperature storage (20 ± 0.5°C, RH 85%–95%) was visually examined for 60 fruits in each treatment group. If there were visible decay symptoms, fruits were considered to be decayed. The percentage decaying of the fruits was counted on every 6th day of storage, and the decay rate of fruits was tabulated by taking the percentage of the total number of fruits with decayed fruits accounting the total number investigated.

The weight loss was defined as the percentage of the reduced weight to the initial weight of navel orange fruit during storage. SSC of fruit juice was assayed by a hand-held refractometer (ATAGO PAL-1, Tokyo, Japan) and expressed as °Brix. TA in fruit juice was assayed using the titration method and expressed as a percentage of citric acid. Vitamin C content was determined by spectrophotometric method [16] and expressed as mg 100 g⁻¹ FW.

2.3. Pathogen Preparation and Inoculation

A second experiment was then conducted to determine the lesion area and inoculation infection rate. For this reason, fruits treated with BTH or deionized water (as control) were inoculated with P. italicum. P. italicum was isolated and purified from infected navel orange fruits. The P. italicum was cultivated in PDA medium at 25°C for a week, and then, spore suspension (1 × 10⁵ spores per milliliter) was prepared with sterile water. Navel orange fruits were pierced at two opposite points (3 mm deep × 3 mm wide) in the middle, and then, 10 μL of spore suspension was injected in each wounded area. During storage, the number of fruits with obvious disease symptoms was recorded. Three replicates of 20 fruits in each group were used for inoculation infection rate and lesion area determination. The pericarp tissue around the lesion of 10 fruits per replicate was sampled, dipped into liquid nitrogen, and stored at −80°C for further analysis. There were three replicates in each treatment.

2.4. The Decay Rate, Disease Incidence, and Lesion Area

The disease incidence of navel orange challenged with P. italicum was done by the percentage of the number of infected wounds with decay accounting the total wounds per replicate. The lesion diameter of the inoculated fruits was measured by vernier caliper. The lesion area was calculated as follows: lesion area = (mm²) = π × (d/2)².

2.5. Determination of Antioxidant Capacity

Navel orange samples (5 g) were added to 30 ml methanol solution and extracted by ultrasonic assist for 40 min and then centrifuged at 13,000xg for 15 min. The supernatant was collected to determine antioxidant capacity of navel orange fruit. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity, 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging capacity, and reducing power were determined by Sun et al. [17]. DPPH radical scavenging capacity was measured at 517 nm. ABTS radical scavenging capacity was measured at 734 nm. For determination of reducing power, the absorbance was measured at 700 nm.

2.6. Assays of Enzyme Activities

1.0 g of navel orange was homogenized in 8 mL of 50 mM pH 7.8 PBS containing 0.8 g·L⁻¹ polyvinylpyrrolidone (PVP) and 1 mM ethylenediaminetetraacetic acid (EDTA) and then centrifuged at 12,000xg for 15 min at 4°C. The supernatants were used for the SOD, POD, and PPO activity assays. Frozen pericarp tissue (1 g) was ground with 8 mL of 50 mM PBS, containing 2% PVP and 5 mM (dithiothreitol) DTT for CAT extraction. SOD activity was assayed following the steps of Prochazkova et al. [18]. CAT and POD activities were assayed according to previous report [17]. PPO activity was assessed following Liu et al. [19]. SOD, CAT, POD, and PPO activity was expressed as U·mg⁻¹ protein min⁻¹.

Frozen pericarp tissues were ground to finally powder form in 8 mL of 50 mM precooled boric acid buffer (pH 8.8) containing 0.5 g PVP, 5 mM β-mercaptoethanol, and 2 mM EDTA for PAL extraction. PAL activity was assayed following the steps of Lu et al. [20]. Frozen pericarp tissue was ground in 8 mL of 100 mM acetic acid buffer (pH 5.0) containing 1 mM EDTA and 5 mM β-mercaptoethanol for GLU and CHT extraction. GLU activity and CHT activity were assayed as described by Chen et al. [2]. PAL activity was expressed as U·mg⁻¹ protein h⁻¹. GLU and CHT are expressed as U·mg⁻¹ protein. Soluble protein content in all enzyme extracts was determined following Bradford method [21], using bovine serum albumin as standard.

2.7. Statistical Analysis

Data were analyzed with SPSS (18.0 version). The significance of difference between the data was determined by Duncan’s multiple range test.

3. Results

3.1. Effect of BTH on Decay Rate of Navel Orange

The decay rate of navel orange fruit increased with the extension of storage time at room temperature storage (Figure 1(a)). BTH treatment significantly inhibited the decay rate of navel oranges stored at 20°C ($P<0.05$). At the 36th day of storage, the decay rate of BTH treated orange fruit was only 6.67%, which was a quarter of the control group.

[figures omitted; refer to PDF]

3.2. Effect of BTH on Disease Incidence and Lesion Area

The lesion diameter had a gradual enlargement starting at the third day after exposure to P. italicum. BTH significantly reduced the disease incidence (Figure 1(b)) and lesion diameters (Figure 1(c)) in navel orange fruit inoculated with P. italicum compared with its respective controls ($P<0.05$).

3.3. Effect of BTH on Fruit Quality of Navel Orange

During storage, weight loss increased in all groups, and weight loss in BTH treated navel orange fruits was significantly lower than its control group after 12 days of storage ($P<0.05$) (Figure 2(a)). The SSC in all samples was initially increased and then declined. Compared with the control, the navel orange fruit treated with BTH showed significantly higher SSC after 18 days of storage ($P<0.05$) (Figure 2(b)). TA content increased slightly at first and then decreased. The TA content in the BTH-treated navel orange fruits was significantly higher than that in the control group ($P<0.05$) at the early stage, and there was no significant difference after 24 days of storage (Figure 2(c)). VC content of the navel orange fruits in control gradually decreased. There was an increase in BTH treated navel orange fruits on day 18 and VC content of the BTH treatment maintained significantly higher VC content after 18 days of storage (Figure 2(d)).

[figures omitted; refer to PDF]

3.4. Effect of BTH on Antioxidant Capacity

DPPH, ABTS radical scavenging activity, and reducing power exhibited a similar trend after inoculation and showed a sharp increase initially and followed by a decrease (Figures 3(a)–3(c)). The fruits exposed to BTH depicted significantly higher DPPH radical scavenging potential, ABTS radical scavenging activity, and reducing power during storage ($P<0.05$).

[figures omitted; refer to PDF]

3.5. Effect of BTH on Antioxidant Enzymes Activities

The changes of the activity of SOD, CAT, and POD activities in navel orange fruits challenge with P. italicum are shown in Figure 4. SOD, CAT, and POD activities in navel orange initially arose in the storage period and then gradually declined at later storage periods (Figure 4). The SOD and POD activities reached the peak on day 4 after storage, but BTH treated navel fruits reached the peak on day 2 and were significantly higher than those of control navel orange fruits ($P<0.05$). The activity of CAT in BTH treated navel orange fruits was significantly lower than that of control fruits ($P<0.05$).

[figures omitted; refer to PDF]

3.6. Effect of BTH on Defense-Related Enzymes Activities

The activities of PPO, PAL, GLU, and CHT in the navel orange fruits challenge with P. italicum were initially increased and later were decreased (Figure 5). PPO activity in BTH treated fruits was significantly lower than its control on the sixth day in storage ($P<0.05$). The activity of PAL in the BTH treatment significantly higher than that of control navel orange after four days of storage ($P<0.05$), while GLU and CHT activities were significantly higher compared with respective control after 48 hours of storage ($P<0.05$).

[figures omitted; refer to PDF]

4. Discussion and Conclusion

BTH was one of the inducers that were known to have potential for application to induce SAR production in fruits and vegetables [9]. In this current study, 50 mg·L⁻¹ BTH significantly reduced the decaying rate of navel fruits during 20°C storage ($P<0.05$). BTH significantly inhibited weight loss of navel fruits ($P<0.05$) and effectively maintained high SSC, VC, and TA contents at the later storage. So, BTH can reduce the decay rate and maintain quality of navel orange during storage. Further, we used P. italicum-inoculated navel orange fruits to investigate how BTH reduced the decay rate of navel orange fruits.

ROS is a signaling molecule regulating plant disease resistance against pathogens, and ROS accumulation can inhibit pathogen infection by inducing hypersensitive responses (HR) [22]. However, high level of ROS can cause oxidative damage, which might make tissues more susceptible to pathogens. Therefore, the existence of antioxidant enzymes and nonenzymatic antioxidants plays an important role in maintaining ROS at a nontoxic level. SOD, CAT, and POD are crucial antioxidant enzymes that efficiently scavenge ROS [23]. BTH remarkably raised the SOD and POD activities but had inhibitory effects on CAT level (Figure 4). Similar phenomena were also observed in chitosan treated navel oranges [24]. Increasing of SOD and POD activities by BTH may protect navel orange fruits cells against oxidative damage. Herein, BTH depicted significantly higher DPPH, ABTS scavenging activity, and reducing power (Figure 3), which is being considered as an additional mechanism involved in inhibiting the increase of disease incidence (Figure 1). Therefore, increasing of SOD and POD activities and the higher antioxidant capacity in BTH treated navel orange fruits help improve disease resistance capability.

The metabolic pathway of phenylpropanoid is quite essential and provides multiple substances especially phenolics, lignin, and hundreds of flavonoids directly with disease resistance. POD, PAL, and PPO are crucial enzymes taking part in phenylpropanoid pathway that leads to the biological synthesis of lignin [25]. Anam et al. [26] had found that an increase in POD and PPO activities in SA treated citrus fruits, and there was a clear relationship between increased POD and PPO activities and disease resistance against blue mould in citrus species. BTH significantly enhanced PAL activity and POD activity (Figures 3(a) and 2(d)), and it indicated that BTH induces navel orange fruits resistance to diseases via improving the phenylpropanoid metabolism pathway in this study. β -1, 3-glucanase (GLU) and chitinase (CHT) are two important plant pathogenesis-related proteins [27] which can hydrolyze alone or synergistically to destroy fungal cell wall structures and thus have direct antibacterial effects. In this study, BTH treatment significantly increased the GLU activity and CHT activity in the navel orange fruits challenge with P. italicum, and it indicated that enhancing disease resistance related enzyme activities was also one of the important mechanisms of BTH induced disease resistance in navel orange fruits.

In summary, our results demonstrated that BTH had promising effects on improving resistance against postharvest blue mold disease in navel orange. The elevated disease resistance in BTH treated orange fruits may be attributed to enhanced antioxidant capacity and antioxidant enzyme activities to the ROS homeostasis, and BTH induced the key enzymes activities in defense response. BTH may be a promising treatment for maintaining the quality and inhibiting blue mold of postharvest navel orange in the future.

Acknowledgments

This study was financed by the Natural Science Foundation of China (no. 31560219).

References

[1] J. Chen, Y. Shen, C. Chen, C. Wan, "Inhibition of key citrus postharvest fungal strains by plant extracts in vitro and in vivo: a review," Plants, vol. 8 no. 2,DOI: 10.3390/plants8020026, 2019.

[2] C. Chen, X. Peng, R. Zeng, M. Chen, C. Wan, J. Chen, "Ficus hirta fruits extract incorporated into an alginate-based edible coating for Nanfeng Mandarin preservation," Scientia Horticulturae, vol. 202, pp. 41-48, DOI: 10.1016/j.scienta.2015.12.046, 2016.

[3] S. Zheng, G. Jing, X. Wang, Q. OuYang, L. Jia, N. Tao, "Citral exerts its antifungal activity against Penicillium digitatum by affecting the mitochondrial morphology and function," Food Chemistry, vol. 178, pp. 76-81, DOI: 10.1016/j.foodchem.2015.01.077, 2015.

[4] N. Tao, Y. Chen, Y. Wu, X. Wang, L. Li, A. Zhu, "The terpene limonene induced the green mold of citrus fruit through regulation of reactive oxygen species (ROS) homeostasis in Penicillium digitatum spores," Food Chemistry, vol. 277, pp. 414-422, DOI: 10.1016/j.foodchem.2018.10.142, 2019.

[5] G. Romanazzi, S. M. Sanzani, Y. Bi, S. Tian, P. Gutiérrez Martínez, N. Alkan, "Induced resistance to control postharvest decay of fruit and vegetables," Postharvest Biology and Technology, vol. 122, pp. 82-94, DOI: 10.1016/j.postharvbio.2016.08.003, 2016.

[6] G. Romanazzi, E. Feliziani, M. Santini, L. Landi, "Effectiveness of postharvest treatment with chitosan and other resistance inducers in the control of storage decay of strawberry," Postharvest Biology and Technology, vol. 75, pp. 24-27, DOI: 10.1016/j.postharvbio.2012.07.007, 2013.

[7] M. H. Aminifard, S. Mohammadi, H. Fatemi, "Inhibition of green mould in blood orange ( Citrus sinensisvar . Moro) with salicylic acid treatment," Archives of Phytopathology and Plant Protection, vol. 46 no. 6, pp. 695-703, DOI: 10.1080/03235408.2012.749740, 2013.

[8] L. Zhu, R. Yang, Y. Sun, "Nitric oxide maintains postharvest quality of navel orange fruit by reducing postharvest rotting during cold storage and enhancing antioxidant activity," Physiological and Molecular Plant Pathology, vol. 113,DOI: 10.1016/j.pmpp.2020.101589, 2021.

[9] N. Benhamou, R. R. Bélanger, "Benzothiadiazole-mediated induced resistance to Fusarium oxysporum f. sp. Radicis-lycopersici in tomato," Plant Physiology, vol. 118 no. 4, pp. 1203-1212, DOI: 10.1104/pp.118.4.1203, 1998.

[10] X. Li, Y. Bi, J. Wang, "BTH treatment caused physiological, biochemical and proteomic changes of muskmelon ( Cucumis melo L.) fruit during ripening," Journal of Proteomics, vol. 120, pp. 179-193, DOI: 10.1016/j.jprot.2015.03.006, 2015.

[11] S. Zhu, B. Ma, "Benzothiadiazole- or methyl jasmonate-induced resistance to Colletotrichum musae in harvested banana fruit is related to elevated defense enzyme activities," The Journal of Horticultural Science and Biotechnology, vol. 82 no. 4, pp. 500-506, DOI: 10.1080/14620316.2007.11512265, 2007.

[12] X. Zhang, Y. Sun, Q. Yang, L. Chen, W. Li, H. Zhang, "Control of postharvest black rot caused by Alternaria alternata in strawberries by the combination of Cryptococcus laurentii and Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester," Biological Control, vol. 90, pp. 96-101, DOI: 10.1016/j.biocontrol.2015.05.018, 2015.

[13] R. M. S. Tariq, K. P. Akhtar, A. Hameed, N. Ullah, M. Y. Saleem, I. U. Haq, "Determination of the role of salicylic acid and Benzothiadiazole on physico-chemical alterations caused by Cucumber mosaic virus in tomato," European Journal of Plant Pathology, vol. 150 no. 4, pp. 911-922, DOI: 10.1007/s10658-017-1332-4, 2018.

[14] H. Jiang, Y. Wang, C. Li, "The effect of benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) treatment on regulation of reactive oxygen species metabolism involved in wound healing of potato tubers during postharvest," Food Chemistry, vol. 309,DOI: 10.1016/j.foodchem.2019.125608, 2020.

[15] C. Wang, L. Chen, C. Peng, "Postharvest benzothiazole treatment enhances healing in mechanically damaged sweet potato by activating the phenylpropanoid metabolism," Journal of the Science of Food and Agriculture, vol. 100 no. 8, pp. 3394-3400, DOI: 10.1002/jsfa.10373, 2020.

[16] L. B. Pfendt, V. L. Vukašinović, N. Z. Blagojević, M. P. Radojević, "Second order derivative spectrophotometric method for determination of vitamin C content in fruits, vegetables and fruit juices," European Food Research and Technology, vol. 217 no. 3, pp. 269-272, DOI: 10.1007/s00217-003-0746-8, 2003.

[17] Y. Sun, W. Zhang, T. Zeng, Q. Nie, F. Zhang, L. Zhu, "Hydrogen sulfide inhibits enzymatic browning of fresh-cut lotus root slices by regulating phenolic metabolism," Food Chemistry, vol. 177, pp. 376-381, DOI: 10.1016/j.foodchem.2015.01.065, 2015.

[18] D. Prochazkova, R. K. Sairam, G. C. Srivastava, D. V. Singh, "Oxidative stress and antioxidant activity as the basis of senescence in maize leaves," Plant Science, vol. 161 no. 4, pp. 765-771, DOI: 10.1016/s0168-9452(01)00462-9, 2001.

[19] H. Liu, W. Jiang, Y. Bi, Y. Luo, "Postharvest BTH treatment induces resistance of peach ( Prunus persica L. cv. Jiubao) fruit to infection by Penicillium expansum and enhances activity of fruit defense mechanisms," Postharvest Biology and Technology, vol. 35 no. 3, pp. 263-269, DOI: 10.1016/j.postharvbio.2004.08.006, 2005.

[20] L. Lu, H. Lu, C. Wu, "Rhodosporidium paludigenum induces resistance and defense-related responses against Penicillium digitatum in citrus fruit," Postharvest Biology and Technology, vol. 85, pp. 196-202, DOI: 10.1016/j.postharvbio.2013.06.014, 2013.

[21] M. M. Bradford, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding," Analytical Biochemistry, vol. 72 no. 12, pp. 248-254, DOI: 10.1016/0003-2697(76)90527-3, 1976.

[22] M. S. Aghdam, J. R. Fard, "Melatonin treatment attenuates postharvest decay and maintains nutritional quality of strawberry fruits (Fragaria × anannasa cv. Selva) by enhancing GABA shunt activity," Food Chemistry, vol. 221, pp. 1650-1657, DOI: 10.1016/j.foodchem.2016.10.123, 2017.

[23] D. Lacan, J.-C. Baccou, "High levels of antioxidant enzymes correlate with delayed senescence in nonnetted muskmelon fruits," Planta, vol. 204 no. 3, pp. 377-382, DOI: 10.1007/s004250050269, 1998.

[24] K. Zeng, Y. Deng, J. Ming, L. Deng, "Induction of disease resistance and ROS metabolism in navel oranges by chitosan," Scientia Horticulturae, vol. 126 no. 2, pp. 223-228, DOI: 10.1016/j.scienta.2010.07.017, 2010.

[25] A.-R. Ballester, M. T. Lafuente, "LED Blue Light-induced changes in phenolics and ethylene in citrus fruit: implication in elicited resistance against Penicillium digitatum infection," Food Chemistry, vol. 218, pp. 575-583, DOI: 10.1016/j.foodchem.2016.09.089, 2017.

[26] M. Anam, T. S. Shahbaz, A. K. Sajid, U. M. Aman, "Salicylic acid and jasmonic acid can suppress green and blue moulds of citrus fruit and induce the activity of polyphenol oxidase and peroxidase," Folia Horticulture, vol. 31 no. 1, pp. 195-204, DOI: 10.2478/fhort-2019-0014, 2019.

[27] M. Heil, "Induced systemic resistance (ISR) against pathogens - a promising field for ecological research," Perspectives in Plant Ecology, Evolution and Systematics, vol. 4 no. 2, pp. 65-79, DOI: 10.1078/1433-8319-00015, 2001.