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Postharvest Biology and Technology 67 (2012) 138–143

Contents lists available at SciVerse ScienceDirect

Postharvest Biology and Technology

journa l h o me pa g e: www.elsev ier .com/ locate /postharvbio

echanism of antifungal action of borate against Colletotrichum gloeosporioideselated to mitochondrial degradation in spores

uequn Shi1,2, Boqiang Li2, Guozheng Qin, Shiping Tian ∗

ey Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Xiangshan Nancincun 20, Haidian District, Beijing 100093, China

r t i c l e i n f o

rticle history:eceived 22 November 2011ccepted 3 January 2012

eywords:

a b s t r a c t

Anthracnose caused by Colletotrichum gloeosporioides Penz. is one of the most important diseases in har-vested mango fruit. In this study, we investigated the effect of borate on spore germination of the fungalpathogen in vitro and anthracnose control in harvested mango fruit, and observed mitochondrial damagein the spores under borate exposure, in order to evaluate the mechanism of its antifungal action. Borate

orateeactive oxygen speciesolletotrichum gloeosporioidesangoitochondria

treatment significantly inhibited spore germination of the fungal pathogen and effectively controlledanthracnose in harvested mango fruit. Borate treatment at 20 mM furthermore seems to induce reactiveoxygen species (ROS) generation in the fungal spores. Mitochondrial degradation after 6 h treatment of thespores with borate was observed with both laser scanning confocal and transmission electron microscopy.These results suggest that mitochondrial degradation is involved in the mechanism of antifungal activityof borate against C. gloeosporioides.

© 2012 Elsevier B.V. All rights reserved.

. Introduction

Mango (Mangifera indica Linn.) is an important tropical fruitecause of its favorable flavour and high marketing value. How-ver, diseases caused by various pathogens usually result in greatconomic losses in harvested mango fruit (Tian et al., 2010).nthracnose caused by Colletotrichum gloeosporioides (Penz.) is onef the most serious diseases in mango fruit, as the pathogen canttack various parts of mango and cause latent infection in smallruit (Pitkethley and Conde, 2007; Kefialew and Ayalew, 2008).pplication of synthetic fungicides is the major approach for con-

rol of fruit diseases; for example, hot benomyl dips have beeneported to effectively control anthracnose of harvested mangoruit (Kim et al., 2007). However, using fungicides at high con-entrations over a long time can result in pathogen resistanceTian, 2006). Additionally, public concern about fungicide residuess urging researchers to find new technologies for fruit disease

ontrol.

Recently, a number of reports have demonstrated that somentifungal compounds, such as oxalic acid (Zheng et al., 2007),

∗ Corresponding author. Tel.: +86 10 62836559; fax: +86 10 82594675.E-mail address: tsp@ibcas.ac.cn (S. Tian).

1 The author now works in College of Environment and Plant Protection, Keyaboratory of Protection and Development Utilization of Tropical Crop Germplasmesources, Hainan University, Hainan Province, China.2 These authors contributed equally to this work.

925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.postharvbio.2012.01.003

salicylic acid (Tian et al., 2007), calcium (Zhu et al., 2010) and plantextracts (Regnier et al., 2008; Linde et al., 2010), can effectivelycontrol postharvest diseases in mango fruit. In previous studies,we found that borate could significantly limit the pathogenicityof Penicillium expansum (Qin et al., 2007), and effectively controlpostharvest diseases in fruit, such as gray mould rot caused byBotrytis cinerea in table grapes (Qin et al., 2010) and anthracnosecaused by C. gloeosporioides in mango fruit (Shi et al., 2011). Inaddition, we showed that the antifungal action of borate is associ-ated with antioxidant enzymes and oxidative damage of the fungalpathogen.

It is well known that mitochondria are the main source of cellu-lar adenosine triphosphate (ATP) and play a central role in a varietyof cellular processes (Chan, 2006; Mucha et al., 2009). Moreover,mitochondria are also the major endogenous sources of reactiveoxygen species (ROS), whose accumulation can lead to oxidation ofmacromolecules, resulting in mtDNA mutations, aging and cell apo-ptosis (Osiewacz, 2002; Kowaltowski et al., 2009). However, littleinformation is available about whether borate can cause mitochon-drial damage of fungal spore cells, and whether the mitochondrialdamage is specifically related to inhibiting fungal growth.

The main objective of this study was to comprehensively explorethe antifungal mode of borate against the fungal pathogen, withparticular attention to the changes of mitochondria in C. gloeospo-

rioides spores treated with or without borate. Moreover, effects ofborate on spore germination of C. gloeosporioides in vitro and onanthracnose disease control of harvested mango fruit were inves-tigated.

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. Materials and methods

.1. Fungal isolates

The fungus C. gloeosporioides was isolated from diseased tissuesf symptomatic mango fruit. The virulence of the isolates was ver-fied by inoculating mango fruit with 5 �L of a spore suspension1 × 104 spores mL−1). Then, the pathogen was purified via singlepore isolation on potato dextrose agar (PDA) and maintained inDA slants at 10 ◦C according to the method of Cloh (1999).

.2. Effect of borate on spore germination of C. gloeosporioides

The effect of borate on spore germination of C. gloeosporioidesas assayed in potato dextrose broth (PDB) medium following theethod described by Qin et al. (2003). Spores were collected by

dding sterile distilled water to the surface of the culture and gen-ly scrubbing with a sterile spatula. Then, spores were incubatedn PDB medium with different concentrations (10, 20 and 30 mM)f potassium tetraborate with or without pH adjustment. The PDBedium without borate served as the control. The inhibition effi-

acy of borate at different concentrations was evaluated on sporeermination after 8 h of incubation at 25 ◦C. At least 100 spores inach treatment were investigated. Each treatment contained threeeplicates and the entire experiment was repeated twice.

.3. Effect of borate on reactive oxygen species generation

The oxidant-sensitive probe 2′, 7′-dichlorodihydrofluoresceiniacetate (DCHF-DA) was used to assess the intracellular ROS levels

n C. gloeosporioides according to the methods of Chen and Dickman2005). Spores of C. gloeosporioides were cultured in PDB mediumupplemented with 0 or 20 mM potassium tetraborate (withoutdjusting the pH) and incubated for 2, 4, 6 and 8 h. Then, the sporesere washed with 10 mM potassium phosphate buffer (pH 7.0), and

ncubated for 5 min in the same buffer containing 10 �M DCHF-DAdissolved in dimethyl sulfoxide). After being washed twice withotassium phosphate buffer, spores were examined under a Zeissxioskop microscope (Carl Zeiss, Oberkochen, Germany) and theercentage of spores stained by DCHF-DA in each treatment calcu-

ated. DCHF-DA is commonly used to determine intracellular ROS,articularly H2O2 (Chen and Dickman, 2004).

.4. Effect of borate on spore mitochondria by laser scanningonfocal microscopy and transmission electron microscopy (TEM)

The spores were incubated in liquid PDB medium containing0 mM potassium tetraborate as described above or suspended inDB medium without borate as a control for 2, 4, 6 and 8 h at 25 ◦C.itoTracker® Orange CMTMRos probes (Invitrogen) were added to

he fungal inoculation at final concentrations of 500 nM for 5 min.luorescence of MitoTracker® Orange CMTMRos stained sporesas detected using a Zeiss LSM 510 META laser scanning confo-

al microscope (LSCM). At least 20 spores were examined for eachreatment with three replications. MitoTracker® Orange CMTM-os is a mitochondrion-selective stain, and has been widely used

or studying mitochondrial distribution and functionality (Chidat al., 2004; Czarna et al., 2010). Accumulation of the stain in cellss dependent upon membrane potential which is one of the mostmportant parameters indicating mitochondrial functionality.

For TEM analysis, the spores treated with 0 and 20 mM potas-ium tetraborate for 2, 4, 6 and 8 h as described above were

xed overnight in 2.5% formaldehyde and 2% paraformaldehyde in.1 M sodium cacodylate buffer (SCB) at a pH 7.2, and centrifuged16,000 × g for 5 min at 4 ◦C). Gels of 1–2 mm3 were prepared bydding 3% low gelling temperature agarose in SCB to the pellet.

echnology 67 (2012) 138–143 139

After thorough rinsing with 0.1 M SCB, the gels were post-fixedwith 1% osmium tetroxide in 0.1 M SCB for 4 h at room temperature,and dehydrated with 15 min stages in an ascending acetone series.The samples were embedded in spurr’s resin. Ultrathin sectionswere obtained using a diamond knife and stained by soaking in2% uranyl acetate for 15 min, post-stained in lead citrate for 1 min.Mitochondrial alteration in the spores was analysed using a JEOL1230 transmission electron microscope at 80 kV.

2.5. Effect of borate on anthracnose disease on mango fruit

Fresh, almost ripe mango fruit (Mangifera indica L. cv. Tai Nong1) were harvested from commercial orchards in the southern partof Hainan province, Lingshui county. The surface of the mangowas sterilized by immersion in 70% ethanol for 1 min and pre-pared for inoculation by inflicting a single 1-mm deep wound inthe middle of each fruit with a sterile needle. Each wound was theninoculated with the pathogen C. gloeosporioides by placing 5 �L ofspore suspension (1×104 spores mL−1) on the wound. The inocu-lated fruit were incubated overnight in a sterile box at 28 ◦C beforedipping it in the potassium tetraborate at concentration of 20 mMfor 30 min. Fruit dipped in sterile distilled water served as the con-trol. In addition, the effect of borate treatments was also comparedwith 100 �g a.i. mL−1 benomyl. The treated fruit were incubated ina moist plastic box at 28 ◦C for 6 d and disease development wasassessed by measuring the diameter of the anthracnose lesion onmango fruit. Each treatment consisted of 50 fruit with 3 replicationsand the experiment was repeated twice.

2.6. Statistical analysis

All statistical analyses were performed with SPSS version 11.5(SPSS Inc., Chicago, IL, USA) and analysed by one-way analysis ofvariance (ANOVA). Mean separations were performed by Duncan’smultiple range tests. Differences at P = 0.05 were considered signif-icant.

3. Results

3.1. Effect of borate on spore germination of C. gloeosporioides

As shown in Table 1, borate was effective in inhibiting sporegermination and germ tube elongation of C. gloeosporioides follow-ing a concentration-dependent trend (P < 0.05). After incubation for8 h at 25 ◦C, germination of C. gloeosporioides spores was stronglyinhibited by borate at the concentration of 20 mM. The inhibitoryefficacy of borate solutions with normal pH was better than that ofsolutions with an adjusted pH of 7.0.

3.2. Measurement of reactive oxygen species (ROS)

ROS generation was monitored when C. gloeosporioides wasexposed to 20 mM borate for 2, 4, 6 and 8 h at 25 ◦C (Fig. 1). After2 h of incubation, only 5.4% spores in the control treatment werestained by DCHF-DA, implying poor ROS production at the time.However, in the borate treatment about 58.2% of spores werestained. With an increased incubation period, even higher levels ofROS were detected in borate-treated C. gloeosporioides spores com-pared with the control. The highest percentage of stained sporeswas observed following 6 h of incubation with 20 mM borate.

3.3. Effect of borate on mitochondria distribution by laser

scanning confocal microscopy

As shown in Fig. 2A, mitochondria in untreated spores werepresent throughout the intracellular space during incubation. In

140 X. Shi et al. / Postharvest Biology and Technology 67 (2012) 138–143

Table 1Effects of borate on spore germination of C. gloeosporioides after 8 h incubation at 25 ◦C.

CK (PDB) Borate (mM, pH 7.0) Borate (mM, pH not adjusted)

10 20 30 10 20 30

Spore germination (%) 97.5 ± 7.4a 53.5 ± 6.4b 27.6 ± 6.5c 15.5 ± 5.4c 45.6 ± 5.2b 14.5 ± 3.0c 9.5 ± 4.4c

Germ tube length (�m) 144 ± 24.6a 60.5 ± 8.6b 8.5 ± 4.2c

Mean values followed by the different letters differ significantly within a row according t

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ig. 1. Effect of borate on the generation of reactive oxygen species in spores of C.loeosporioides. Spores were cultured at 25 ◦C in PDB medium supplemented with0 mM potassium tetraborate or not. Vertical bars indicate standard deviations.

ontrast, abnormal distribution of mitochondria was observed inorate-treated spores. After exposure to borate for 4 and 6 h, sporeells had many spaces void of mitochondria, while these organellesould only be found around the cell periphery after 8 h exposureFig. 2B).

.4. Effect of borate on mitochondria analysed by TEM

For spores incubated for 2 h in PDB medium without borate,itochondria were obvious, and appeared normal in morphology

Fig. 3A). With prolonged incubation in PDB, numerous mitochon-ria were observed within the spores (Fig. 3B–D). Within the first

h, no significant difference in number and morphology of mito-hondria was found between borate-treated spores and the control.

ig. 2. Effect of borate on the distribution of mitochondria in spores of C. gloeosporioides. Setraborate (B) or not (A). After incubated at the indicated times, spores were stained wit

5.5 ± 2.4c 38.5 ± 8.3b 6.0 ± 2.5c 5.0 ± 2.4c

o Duncan’s multiple range tests (P < 0.05), respectively.

However, after 4 h borate incubation, the number of mitochondriain the spore was markedly fewer and many small vacuoles werefound (Fig. 3F). After 6 h of incubation in borate, distortion of mito-chondrial cristae appeared. After 8 h, mitochondria were almostcompletely degraded (Fig. 3G and H).

3.5. Effect of borate on anthracnose disease on mango fruit

Borate was effective in inhibiting the lesion diameter of anthrac-nose (P < 0.05). The application of borate reduced the mean lesiondiameter from 2.6 cm in the untreated control fruit to 1.4 cm inborate-treated fruit after 6 d storage at 28 ◦C. This was as effectiveas the regular fungicide used for anthracnose control in mango,Benomyl (at a concentration of 100 �g a.i. mL−1) (Fig. 4A and B).

4. Discussion

Boron is an essential microelement and has important physio-logical functions for plant growth (O’Neill et al., 2001; Xuan et al.,2001; Simoglou and Dordas, 2006) and quality improvement in fruit(Strong et al., 2001; Plich and Wójcik, 2002; Hafez and Haggag,2007). In recent years, borate has been used as an antifungal com-pound to control postharvest diseases in various fruit (Rolshausenand Gubler, 2005; Qin et al., 2010; Thomidis and Exadaktylou, 2010;Shi et al., 2011). In previous studies, we found that germinationand germ tube elongation of C. gloeosporioides were significantlyinhibited by the addition of 15 mM borate to the PDB. Mango inflo-rescences sprayed with borate significantly increased the numberof fruit that set per branch compared to the water-sprayed con-trol, and lower disease incidence of anthracnose during postharvestperiods was observed (Shi et al., 2011). In addition, we found that

borate at 1% concentration could inhibit blue mould rot caused byP. expansum in harvested apple fruit, and were able to demonstratethat the mechanisms involved changing expression of antioxidantproteins and hydrolytic enzymes, based on comparative analysis

pores were cultured at 25 ◦C in PDB medium supplemented with 20 mM potassiumh the staining MitoTracker® . Bars represent 20 �m.

X. Shi et al. / Postharvest Biology and Technology 67 (2012) 138–143 141

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ig. 3. Effect of borate on the mitochondria in spores of C. gloeosporioides by TEM

or 2 h (A), 4 h (B), 6 h (C) and 8 h (D); spores treated with 20 mM potassium tetrabolack arrows indicate mitochondria in spores. L: lipid bodies, N: nucleus, S: septum

f cellular and extracellular proteome (Qin et al., 2007). Thomidisnd Exadaktylou (2010) reported that borate treatment was effec-ive in controlling brown mould rot by Monilinia laxa in peach.in et al. (2010) proved that borate at 1% concentration could

copy. Untreated spores served as control were incubated in PDB medium at 25 ◦Cere incubated in the same culturing condition for 2 h (E), 4 h (F), 6 h (G) and 8 h (H).cuoles. Bars represent 0.5 �m.

inhibit gray mould rot by Botrytis cinerea in table grapes storedat room temperature or at 0 ◦C. Furthermore, it provided evidencethat boron-reduced gray mould decay of table grapes was directlyrelated to the disruptive effect of boron on the cell envelope of the

142 X. Shi et al. / Postharvest Biology and

Fig. 4. Effect of borate treatment on disease severity in mango fruit (cv. Tai Nong 1)after inoculated with 1 × 104 mL−1 spores of C. gloeosporioides. (A) The mean diseaselesion diameter was measured after the fruit were inoculated and stored in 28 ◦Cfor 6 d. (B) Symptoms in inoculated mango fruit stored in 28 ◦C for 6 d. Mean valuespt

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resent in each bar followed by different letters are significantly different accordingo Duncan’s multiple range tests (P < 0.05). Vertical bars indicate standard deviations.

athogen, resulting in the breakdown of the cell membrane andoss of cytoplasmic materials from the hyphae (Qin et al., 2010).n the current study, we found that borate significantly inhibitedpore germination of C. gloeosporioides (Table 1) and reduced theesion diameter of anthracnose disease in harvested mango fruitfter artificially inoculating pathogen spores (Fig. 4). These resultsndicate that borate has a broadly inhibiting effect on postharvestungal pathogens. Moreover, borate solutions with adjusted pHpH 7.0) also exhibited a lower inhibitory effect, suggesting thatH only partially influenced the antifungal action of boron against. gloeosporioides. The same result has been reported in B. cinereaausing gray mould rot of table grapes (Qin et al., 2010).

ROS may cause oxidative damage to cellular compounds andead to cellular dysfunction or cell death (Angelova et al., 2005;ircu and Aw, 2010). In this study, we used an oxidant-sensitiverobe DCHF-DA to investigate the ROS accumulation in C. gloeospo-ioides spores, and observed that borate-treated spores showed aigher ratio of stained cells than that in control (Fig. 1), indicating

hat borate induced ROS accumulation in C. gloeosporioides spores.OS accumulation can cause oxidative damage of the fungal sporend result in lower spore germination. It is well known that theitochondrial respiratory chain is the major endogenous source of

Technology 67 (2012) 138–143

ROS, particularly when the mitochondria are damaged (Osiewacz,2002; Hipkiss, 2006; Shi et al., 2011). Based on confocal microscopyand TEM, we found abnormal distribution and serious degradationof mitochondria in borate-treated spores (Figs. 2 and 3), indicat-ing that borate treatment can lead to mitochondrial damage. Aviset al. (2009) reported that abnormal mitochondria appeared inconidia of Fusarium sambucinum after incubation for 10 min with100 mM AlCl3 which was also considered as a potential antifungalagent. Therefore, it suggests that mitochondrial degradation of fun-gal spores may be one of the important modes by which antifungalcompounds inhibit fungal growth.

In summary, we conclude that borate treatment can stimu-late ROS accumulation in fungal spores, resulting in mitochondrialdamage, which may act as the antifungal mechanism of borateinhibiting spore germination of C. gloeosporioides and controllinganthracnose in mango fruit. Additionally, application of borate ispromising as an alternative to synthetic fungicides for postharvestdisease control in mango fruit.

Acknowledgments

The study was supported by the National Natural ScienceFoundation of China (31030051), by the Knowledge Innovation Pro-gram of the Chinese Academy of Sciences (KSCX2-EW-G-6) andNational High-Technology Development Program of China (863,SS2012AA101010).

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