Induction of defense responses in longkong fruit (Aglaia dookkoo Griff.) against fruit rot fungi by Metarhizium guizhouense

Accepted Manuscript Induction of defense responses in longkong fruit (Aglaia dookkoo Griff.) against fruit rot fungi by Metarhizium guizhouense Thanunchanok Chairin, Vasun Petcharat PII: DOI: Reference:

S1049-9644(17)30114-7 http://dx.doi.org/10.1016/j.biocontrol.2017.05.012 YBCON 3596

To appear in:

Biological Control

Received Date: Revised Date: Accepted Date:

10 January 2017 20 May 2017 26 May 2017

Please cite this article as: Chairin, T., Petcharat, V., Induction of defense responses in longkong fruit (Aglaia dookkoo Griff.) against fruit rot fungi by Metarhizium guizhouense, Biological Control (2017), doi: http://dx.doi.org/ 10.1016/j.biocontrol.2017.05.012

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

1

Induction of defense responses in longkong fruit (Aglaia dookkoo Griff.) against

2

fruit rot fungi by Metarhizium guizhouense

3

Thanunchanok Chairin1,2*, Vasun Petcharat1

4

1

5

University 15 Kanjanavanich Rd. Hat Yai, Songkhla, 90110 Thailand

6

2

7

University 15 Kanjanavanich Rd. Hat Yai, Songkhla, 90112 Thailand

8

*

9 10 11

12

13

14

15

16

17

18

19

20

Department of Pest Management, Faculty of Natural Resources, Prince of Songkla

Pest Management Biotechnology and Plant Physiology Laboratory, Prince of Songkla

Corresponding author: [email protected]

2

21

Abstract

22

The defense responses of longkong fruit were induced by an entomopathogenic

23

fungus, Metarhizium guizhouense. The longkong fruits were dipped in a spore

24

suspension of M. guizhouense PSUM04 at concentration of 104 spore mL -1 for 5 sec,

25

then an enzyme assay, a protein assay and Polyacrylamide Gel Electrophoresis (PAGE)

26

were done. It was found that the longkong fruit presented small necrotic spots on their

27

peels within 24 hours. β-1,3-glucanase and chitinase were detected in peel extracts at

28

1.33 ± 0.09 and 0.12 ± 0.004 U mL -1, respectively. The sodium dodecyl sulphate

29

(SDS)-PAGE showed protein bands that emerged after the fungal treatment when

30

compared to untreated control. These proteins were a 25-27 kDa group of chitinase

31

isoforms, and β-1,3-glucanase at 43 kDa. From gel diffusion method, crude extracted

32

from the fruit peel inhibited the mycelial growth of Botrytis sp. and Fusarium sp. as

33

34.9 ± 3.1 and 29.3 ± 5.0%, respectively. Moreover, the observation from Scanning

34

electron microscope (SEM) showed abnormal shapes, twisting and cracking in both

35

fungal mycelia and less dense mycelial growth for Fusarium sp. Thus, M. guizhouense

36

PSUM04 was not only a bio insecticide but it was also be the biotic inducer of plant

37

defense responses against pathogenic fungi.

38 39

Key words:

40

Metarhizium guizhouense

41

Plant defense response

42

Antifungal

43

β-1,3-glucanase

44

Chitinase

3

45 46

1. Introduction

47

Longkong (Aglaia dookkoo Griff.) is an economically important tropical fruit in

48

southern Thailand. There is a high demand of longkong because this fruit is juicy and

49

has a pleasant taste (Sapii et al., 2000). Longkong contains a variety of nutrients,

50

including high levels of antioxidants, carbohydrates, vitamins and minerals, while the

51

amounts of protein and fat are low (Ketsa and Paull, 2008). However, postharvest

52

diseases negatively impacts longkong fruit quantity and quality. Fruit rot is one of the

53

most serious postharvest diseases of many fruits, with pathogen infections occurring

54

throughout postharvest processing until consumption. Most fruit rots are caused by

55

fungi, follow by bacteria, and occasionally viruses. The fruit which drop or without

56

stem cause fruit rot rapidly during storage. Light-brown to dark-brown lesion develop

57

on fruit after that white or grayish fungal mycelia cover the fruit rapidly. As the fungi

58

spread, a semisoft decay occur, resulting in large rotted areas over time. The fungal

59

pathogens of fruit rot such as in the genera Aspergillus, Botrytis, Colletotrichum,

60

Fusarium, Lasiodiplodia and Phomopsis (Coates et al., 2003; Lim and Sangchote,

61

2003).

62

The plant defense responses include three main pathways: (a) hypersensitive

63

response (HR), (b) systemic acquired resistance (SAR) that includes induction of

64

pathogenesis-related (PR) proteins and (c) inducted systemic resistance (ISR) (Shoresh

65

et al., 2010). Chitinase and β-1,3-glucanases are known PR proteins that hydrolyze the

66

major components, chitin and β-1,3-glucan, of fungal cell walls and inhibit fungal

67

growth (Droby, 2011). It has been suggested that chitinase and β-1,3-glucanases may

68

function in defense against fungal pathogens such as Botrytis cinerea (Daulagala, 2014),

4

69

Colletotrichum gloeosporioides (Ali et al., 2014), Penicillium expansum (Chan and

70

Tian, 2006), P. digitatum (Inkha and Boonyakiat, 2010) and Phomopsis asparagi (Lu et

71

al., 2008).

72

The entomopathogenic fungus Metarhizium guizhouense has potential to control

73

various insect pests of plants and livestock (Boucias and Pendland, 1998). Not only it is

74

an insect pathogen but some species of Metarhizium are also associated with plants that

75

the fungus colonize the plant rhizosphere, endophytes, and possibly even plant-growth-

76

promoting agents (Hu and St. Leger, 2002). Some studies have shown that M.

77

guizhouense can produce chitinase (Thaochan and Chandrapatya, 2016). However, there

78

is no prior report on using M. guizhouense as a biotic agent to induce the defense

79

responses in plants.

80

The biochemical defensed response in plants have been widely studied in leaves and

81

roots, but only in a few cases in ripening fruits (Prusky et al., 2013). Thus, the purpose

82

of this research was to investigate the induction of plant defense responses in

83

postharvest longkong fruit by the entomopathogenic fungus, M. guizhouense PSUM04

84

and their antifungal activity against postharvest fruit rot fungi.

85

2. Materials and methods

86

2.1. Fungal cultivation

87

M. guizhouense PSUM04 from National Biological Control Research Center,

88

Southern Region, Faculty of Natural Resources, Prince of Songkla University, Hat Yai,

89

Thailand (Thaochan and Chandrapatya, 2016), was selected for use as the biotic inducer

90

in this study. M. guizhouense PSUM04 was cultured by solid state cultivation, modified

91

from the method of Thongkeawyuan and Chairin (2016). Three mycelial plugs of 7-day-

92

old M. guizhouense PSUM04 on potato dextrose agar (PDA) were transferred to

5

93

colloidal chitin media and incubated at 30oC, under static conditions for 10 days. Spores

94

of the fungus were suspended in 20 mL of 50 mM potassium phosphate buffer (KPB) at

95

pH 7.0, and this suspension was used as the spore suspension.

96

Fruit rot pathogens, Botrytis sp. and Fusarium sp., isolated from postharvest rotting

97

longkong fruit collected from local orchards in Phatthalung, Nakhon Si Thammarat and

98

Songkhla provinces, Thailand. Pure cultures of fungi were obtained as stock cultures

99

and established on PDA (incubated at 30oC). The 5-day-old Botrytis sp. and Fusarium

100

sp. were used as the fungal pathogens.

101

2.2. Fruit preparation

102

Longkong fruit were harvested at their mature stage from local orchards in

103

Phatthalung, Nakhon Si Thammarat and Songkhla provinces. Healthy longkong fruit

104

were surfaced disinfected with 2% of NaOCl for 3 min and rinsed with distilled water.

105

The fruit were dipped for 15 sec in the spore suspension of M. guizhouense PSUM04, at

106

a concentration of 1  104 spores mL -1, containing 0.05% of tween 80, then washed

107

with distilled water and air-dried at room temperature. The enzyme activities and SDS -

108

PAGE banding of fungal-treated longkong were compared with untreated longkong

109

fruit (control).

110

2.3. Enzyme assays

111

Chitinase and β-1,3-glucanase activity were measured using colloidal chitin and

112

laminarin as the substrates, respectively following the 3,5-dinitrosalicylic acid (DNS)

113

method (Miller, 1959). The reaction mixture contained 250 µL of crude sample and 1%

114

(w/v) substrate in 250 µL of 50 mM KPB pH 7.0 for colloidal chitin and 50 mM acetate

115

buffer pH 5.5 for laminarin and incubated for 30 min in 50oC water bath. Reducing

116

sugar released in mixtures were determined by recording the absorbance at 575 nm for

6

117

chitinase and 550 nm for β-1,3-glucanase. One unit (U) of chitinase activity was defined

118

as releasing 1 µmol of N-acetly-D-glucosamine from the substrate per min and one unit

119

of β-1,3-glucanase activity was defined as releasing 1 µmol of glucose from the

120

laminarin per min.

121

2.4. Protein extraction

122

The extraction of total proteins was performed according to the method of Zheng et

123

al. (2007) with slight modifications. The longkong peels were ground in a small mortar

124

and the sample was suspended in 12 mL of SDS extraction buffer, containing 0.5%

125

(w/v) SDS, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA and 464 mL of water. The

126

suspended sample was incubated at 90-94oC for 8 min and centrifuged at 8000 g at 4oC

127

for 15 min. After that the resulting supernatant was mixed with 25 mL of 13% TCA in

128

acetone solution, containing 10% (w/v) TCA and 20 mM DTT in 100% ice-cold

129

acetone and 28 mM mercaptoethanol in a ratio of 1/10 (w/v). The solution was

130

incubated at -20 oC for 2 hours and the proteins were precipitated by centrifugation at

131

14,000 g, at 4oC for 15 min. After 2 washes with 100% ice-cold acetone, the pellet was

132

air-dried and re-suspended in SDS-PAGE sample buffer.

133

2.5. Protein assay

134

The total protein concentration was determined according to the procedure of

135

Bradford (1976) using bovine serum albumin (BSA) as standard. The reaction mixture

136

containing 0.1 mL of sample and 5 mL of protein reagent (Coomassie Brilliant Blue G-

137

250 dissolved in 95% ethanol and 85% (w/v) of phosphoric acid added to this). The

138

developed color was measured at 595 nm after 5 min incubation.

139

2.6. Polyacrylamide gel electrophoresis (PAGE)

7

140

The apparent molecular masses of proteins were determined by SDS – PAGE

141

analysis, both for healthy fruit and treated fruit that were compared. The SDS-PAGE

142

was carried out on mini-gels (12% separating gel and 4% stacking gel) according to the

143

procedure of Laemmli (1970), with marker ladder of proteins from 250 to 10 kDa

144

(BioLabs Inc.). The samples were loaded at either end of the gel, and electrophoresis

145

was performed at 100 V for the stacking gel and at 120 V for the separating gel.

146

2.7. Antifungal activity assay

147

Crude enzyme was extracted from longkong fruit peel and performed to assess the

148

potential control in laboratory scale against fruit rot pathogens, Botrytis sp. and

149

Fusarium sp. Gel diffusion method were used to investigate the mycelial growth

150

inhibition on PDA by crude enzyme. There were 3 treatments comprised distilled water

151

(control), extraction buffer and crude enzyme extracted from fruit peel. Tested plates

152

were incubated at 30oC for 4 days. The experimental design used was completely

153

randomized design (CRD) with 3 replicates and the experiment was repeated 3 times.

154

Growth inhibition was calculated by measuring the clearing zone between fungal

155

colonies and crude enzyme well by using the formula:

156

Percentage inhibition =

 100

157

Where C is the radial growth of fungus from the center to the control well and T is the

158

radial growth of fungus from the center to the crude enzyme well.

159

2.8. Scanning electron microscope (SEM) observations

160

Mycelial plug samples were evaluated the effect of antifungal activity on mycelial

161

morphological change. These plugs were after 1 hour incubation at 37 oC with crude

162

extracted from treated longkong fruit peel and control plugs were incubated with

163

autoclaved distilled water. The mycelial plugs were fixed in 0.5 ml of 3%

8

164

glutaraldehyde for 24 hour at 4 oC and then dehydrated sequentially in 30, 50, 60, 70,

165

80, 90 and finally in 100% ethanol (three times) for 15 min at each concentration. The

166

dehydrated samples were dried with critical point drying (CPD) method, coated with

167

gold and observed with a SEM (JSM-5800 LV, JEOL, USA) at Scientific Equipment

168

Center, Prince of Songkla University, Hat Yai, Thailand.

169

2.9. Statistical analysis

170

The results were analyzed by one-way analysis of variance (ANOVA) and Duncan’s

171

multiple range test (p < 0.05) using SPSS version 11.5. All tests were carried out in

172

triplicate.

173

3. Results and discussion

174

The plant defense responses include three main pathways: HR, SAR and ISR

175

(Shoresh et al., 2010). The mechanism of entomopathogenic fungus, M. guizhouense

176

PSUM04 to trigger plant defense responses was not completely understood. However,

177

in this study presented some of the defense response of longkong fruit. The fruit

178

presented small HR or necrotic spots on the peel surfaces within 24 h after treatment

179

with a spore suspension of M. guizhouense PSUM04. The necrotic spots did not invade

180

through the peel into the fruit. The necrotic tissue had associated PR proteins

181

production: the chitinase activity was 0.12 ± 0.004 U mL -1 at 24 h and slightly increased

182

to 0.17 ±0.02 U mL -1 at 48 h after inoculation. The activities of β-1,3-glucanase were

183

1.33 ±0.09 and 1.51 ±0.12 U mL

184

observations of a similar kind by Brown and Swinburne (1980), the banana fruit

185

responded to Colletotrichum musae by producing antifungal compounds that were

186

isolated from the necrotic tissues within the peels. The β-1,3-glucanase and chitinase

187

were detectable on the first day after fungal treatment. Ali et al. (2014) found that the

-1

at 24 and 48 h, respectively (Table 1). In

9

188

dragon fruit plant could produce β-1,3-glucanase and chitinase on the first day after C.

189

gloeosprioides inoculation, with activities 0.6 and 0.1 U g-1 (fresh weight), respectively,

190

and the enzyme activities increased to maximum on day 18.

191

The proteins in the samples were separated by one-dimensional polyacrylamide gel

192

electrophoresis and stained with Coomassie brilliant blue R-250, to observe protein

193

bands. The SDS-PAGE analysis presented clear differences in the expression of major

194

proteins between the fungal-treated longkong and the untreated control. The protein

195

bands were assigned molecular weights in the 25-50 kDa range, based on a molecular

196

weight standard (Fig. 1). It has been reported that the 25-27 kDa proteins are a group of

197

chitinase isoforms (Punja and Zhang, 1993). In similar observations by Daulagala

198

(2014), chitinase had apparent 26 kDa molecular mass when detected in banana pulp

199

extracts. The 43 kDa protein was tentatively identified as β-1,3-glucanase isoform, in

200

agreement with Porat et al. (1999) that identified the protein band at 43 kDa as β-1,3-

201

endoglucanase, from grapefruit peel tissue subjected to SDS-PAGE.

202

The PR proteins, β-1,3-glucanase and chitinase have been found in some fruits, such

203

as banana (Daulagala, 2014), grapefruit (Porat et al., 1999), orange (Inkha and

204

Boonyakiat, 2010) and papaya (Chen et al., 2007). Although, this study is the first to

205

report PR protein induction in longkong fruit. The induction of PR proteins helps

206

control plant diseases (Ali et al., 2014; Inkha and Boonyakiat, 2010). Chan and Tian

207

(2006) reported that the protein content of sweet cherry fruit plays a role in its resistance

208

against postharvest blue mold. In this study, PR protein, β-1,3-glucanase and chitinase

209

extracted from longkong fruit peel presented the antifungal potential, exhibited the

210

inhibition zone against Botrytis sp. and Fusarium sp. on PDA plate. While, the fungal

211

mycelia growth normally through the wells filled with distilled water and extraction

10

212

buffer (50 mM KPB) control (Fig. 2). The percentage inhibition were 34.9 ± 3.1 and

213

29.3 ± 5.0% for Botrytis sp. and Fusarium sp., respectively. The inhibition rate were

214

significantly different (p<0.05) between extraction buffer control and crude which

215

contained PR proteins (Fig. 3).

216

Cheng et al. (2009) presented that most pathogenic fungi have cell walls composed

217

of complex polymer of glucans, where chitin acts as a structural backbone arranged in

218

regularly ordered layers. Cell walls of filamentous fungi consist of up to 20% or more

219

of chitin, which can be found throughout the whole cell wall of hyphae (Ruiz-Herrera,

220

1991). Thus, breakdown of the fungal cell wall plays a role to inhibit the pathogenic

221

fungal growth. The observation from SEM in this study showed that crude extracted

222

from the peel of Metarhizium-treated longkong contained the PR protein, β-1,3-

223

glucanase and chitinase, caused morphological change of Botrytis sp. and Fusarium sp.

224

mycelia. These observation showed abnormal shapes, twisting and cracking in both

225

fungal mycelia (Fig. 4 and 5) and less dense mycelial growth for Fusarium sp. (Fig. 5b

226

and d). Wang et al. (2013) presented the major damage of the mycelia and the spores of

227

Aspergillus parasiticus caused by cultured with cell-free culture filtrate of Serratia

228

marcescens which contained chitinase. In plant defense responses, the hyphal

229

degradation or debris may related to the non- fungal hyphal recognition, which, in

230

observations of a similar kind by Cortes et al. (1998), as suggested of the non-

231

Trichoderma hyphal recognition would lead to the strong release of lytic enzymes such

232

as β-1,3-glucanases and chitinases, resulting in the digestion of the major cell wall

233

components.

234

From our preliminary test, spore suspension of Metarhizium and enzyme produced

235

from Metarhizium under solid state cultivation could delayed rotting and browning of

11

236

longkong fruit for 24 and 48 hour, respectively (data not shown). Thus, M. guizhouense

237

PSUM04 was not only a bio insecticide but it was also appear potential for biological

238

control of postharvest disease in laboratory experiment and be the biotic inducer of

239

plant defense responses against pathogenic fungi. Furthermore, the potential of M.

240

guizhouense PSUM04 to control postharvest disease in the field need to be conducted.

241

4. Conclusions

242

Hypersensitive response and PR proteins production were induced by the

243

entomopathogenic fungus, M. guizhouense PSUM04 in longkong fruit. Crude extracted

244

from the peel of Metarhizium-treated longkong exhibited the antifungal activity against

245

postharvest fruit rot fungi, Botrytis sp. and Fusarium sp.

246

Acknowledgement

247

This work was supported by the Research and Development Office, Prince of Songla

248

University, Songkhla, Thailand [grant number NAT570507S]. Moreover, we are

249

grateful to Associate Professor Dr. Seppo Karrila, Faculty of Science and Industrial

250

Technology, Prince of Songkla University, Surat Thani Campus for helping to improve

251

the draft manuscript.

252

References

253

Ali, A., Zahid, N., Manickam, S., Siddiqui, Y., Alderson, P.G., Maqbool, M., 2014.

254

Induction of lignin and pathogenesis related proteins in dragon fruit plants in

255

response to submicron chitosan dispersions. Crop Protection 63

256 257

88.

Boucias, D.G., Pendland, J.C., 1998. Principle of Insect Pathology. Kluwer Academic Publishers, Boston, USA.

12

258

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram

259

quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.

260

72, 248 254.

261

Brown, A.E., Swinburne, T.R., 1980. The resistance of immature banana fruits to

262

anthracnose (Colletotrichum musae (Berk. and Curt.) Arx.). J. Phytopathol. 99, 70

263

80.

264

Chan, Z., Tian, S., 2005. Induction of H2O2-metabolizing enzymes and total protein

265

synthesis by antagonistic yeast and salicylic acid in harvested sweet cherry fruit.

266

Postharvest Biol. Tec. 39, 314 320.

267

Chen, Y., Hsu, L., Huang, I., Tsai, T., Lee, G., Shaw, J., 2007. Gene cloning and

268

characterization of a novel recombinant antifungal chitinase from papaya (Carica

269

papaya). J. Agr. Food Chem. 55, 714 722.

270

Coates, L.M. Sangchote, S., Johnson, G.L., Sittigul, C., 2003. Diseases of longan,

271

lychee and rambutan. p. 307-325. In: Ploetz, R.C., ed. Diseases of Tropical Fruit

272

Crops. CAB International, New York, USA.

273

Cheng, Y., Hong, T., Liu, C., Meng, M., 2009. Cloning and functional characterization

274

of a complex endo-β-1,3-glucanase from Paenibacillus sp. Appl. Microbiol.

275

Biotechnol. 81, 1051–1061.

276

Cortes, C., Gutierrez, A., Olmedo, V., Inbar, J., Chet, I., Herrera-Estrella, A., 1998. The

277

expression of genes involved in parasitism by Trichoderma harzianum is triggered

278

by a diffusible factor. Mol. Gen. Genet. 260, 218–225.

279 280

Daulagala, P.W.H.K.P., 2014. Expression of chitinase with antifungal activities in ripening banana fruit. Trop. Plant Res. 1, 72–79.

13

281

Hu, G., St. Leger, R., 2002. Field studies using a recombinant mycoinsecticide

282

(Metarhizium anisopliae) reveal that it is rhizosphere competent. Appl. Environ.

283

Microb. 68, 6383–6387.

284

Inkha, S., Boonyakiat, D., 2010. Induction of resistance to Penicillium digitatum in

285

tangerine fruit cv. Sai Num Phung flavedo by hot water treatment. Songklanakarin

286

J. Sci. Technol. 32, 445–451.

287 288 289 290 291 292

Ketsa, S., Paull, E.R., 2008. Meliaceae. p. 468-472. In: Janick, J.; Paull, E.R., eds. The Encyclopedia of Fruits and Nuts. CAB International, New York, USA. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lim, T.-K., Sangchote, S., 2003. Diseases of durian. p. 241-251. In: Ploetz, R.C., ed. Diseases of Tropical Fruit Crops. CAB International, New York, USA.

293

Lu, G., Jian, W., Zhang, J., Zhou, Y., Cao, J., 2008. Suppressive effect of silicon

294

nutrient on Phomopsis stem blight development in Asparagus. Horticulture Sci. 43,

295

811–817.

296 297

Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Biochem. 31, 426–428.

298

Porat, R., Lers, A., Dori, S., Cohen, L., Weiss, B., Daus A., Wilson, C.L., Droby, S.,

299

1999. Induction of chitinase and β-1,3-endoglucanase proteins by UV irradiation

300

and wounding in grapefruit peel tissue. Phytoparasitica 27, 233–238.

301

Prusky, D., Alkan, N., Mengiste, T., Fluhr, R., 2013. Quiescent and necrotrophic

302

lifestyle choise during postharvest disease development. Annu. Rev. Phytopathol.

303

51, 155–176.

14

304 305 306 307

Punja, Z.K., Zhang Y-.Y., 1993. Plant chitinases and their roles in resistance to fungal diseases. J. Nematol. 25, 526–540. Ruiz-Herrera J., 1991. Fungal cell wall: structure, synthesis and assembly. Boca Raton: CRC Press.

308

Sapii, A.T., Norlia, Y., Muda, P., Lin, T.S., 2000. Postharvest quality changes in

309

dokong (Lansium domesticum Corr.) harvested at different stages of ripeness. p.

310

201-205. In: Johnson, G.I.; To, L.V.; Duc, N.D.; Webb, M.C., eds. Quality

311

Assurance in Agricultural Produce. ACIAR Proceeding, Australia.

312 313 314 315

Shoresh, M., Harman, G.E., Mastouri, F., 2010. Induced systemic resistance and plant responses to fungal biocontrol agents. Annu. Rev. Phytopathol. 48, 21–43. Thaochan, N., Chandrapatya, A., 2016. The phenotypic and metabolic properties of Metarhizium guizhouense on Corcyra cephalonica. Mycosphere 7, 214–225.

316

Thongkeawyuan, A., Chairin, T., 2016. Selection of carbon and nitrogen sources for

317

protease production by Metarhizium anisopliae PSUM02 under solid state

318

cultivation. Khon Kaen Agri. J. 44, (Suppl.) 1, 924–929.

319

Wang, K., Yan, P-S., Cao, L-X., Ding, Q-L., Shao, C., Zhao, T-f., 2013. Potential of

320

chitinolytic Serratia marcescens strain JPP1 for biological control of Aspergillus

321

parasiticus and aflatoxin. Biomed Res. Inter., 2013, 1–7.

322

Zheng, Q., Song, J., Doncaster, K., Rowland, E., Byers, D., 2007. Qualitative and

323

quantitative evaluation of protocols for apple and strawberry fruit suitable for two-

324

dimentional electrophoresis and mass spectrometry analysis. J. Agr. Food Chem.

325

55, 1663–1673.

326 327

15

328 329 330 331 332

Fig. 1. Protein bands of SDS-PAGE from longkong peel extracts after Metarhizium

333

guizhouensePSUM04 treatment at 24 h (lane 2) and at 48 h (lane 3), along with

334

untreated control (lane 1). Lane M provides a calibration “ladder” formed by standard

335

proteins of known molecular weights.

336 337

Fig. 2. Inhibition zone of crude enzyme from longkong fruit peel against mycelial

338

growth of Botrytis sp. (right) and Fusarium sp. (left), where b means the well filled with

339

50 mM potassium phosphate buffer pH 7.0, c means the well filled with distilled water

340

(control), and e means the well filled with crude enzyme. Bar = 1 cm.

341 342

Fig. 3. Mycelial growth inhibition of Fusarium sp. and Botrytis sp. by crude extracted

343

from longkong fruit peel (gray bar) and extraction buffer, 50 mM potassium phosphate

344

buffer pH 7.0 (black bar). Bar represent standard deviation of three replications and the

345

same letter are not significantly different (p>0.05) according to Duncan’s multiple range

346

test.

347 348

Fig. 4. Scanning electron microscope micrographs showing the mycelia of Botrytis sp.;

349

(a), (c) treated with distilled water (control) and (b), (d) treated with longkong fruit peel

350

crude extract.

351

16

352

Fig. 5. Scanning electron microscope micrographs showing the mycelia of Fusarium

353

sp.; (a), (c) treated with distilled water (control) and (b), (d) treated with longkong fruit

354

peel crude extract.

355

17

356 357

18

358 359

19

360 361

20

362 363

21

364 365

22

366

Table 1

367

Enzyme activities of longkong fruit peel extracts at 24 and 48 h post inoculation. Sample

368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384

*

Chitinase (U mL-1)*

β-1,3-glucanase (U mL-1)*

24 h

48 h

24 h

48 h

Control

0.00 ± 0.00

0.02 ± 0.001

0.00 ± 0.00

0.00 ± 0.00

Inoculated

0.12 ± 0.004

0.17 ±0.02

1.33 ±0.09

1.51 ±0.12

Mean ± Standard deviation (3 replications)

23

385

Highlights

386

 Metarhizium elicited a defense responses in longkong fruit within 24 hours.

387

 Treated longkong fruit produced PR proteins in necrotic peel areas.

388

 Crude extracted from the peel showed antifungal activity against fruit rot fungi.

389