Naphthalene Acetic Acid Potassium Salt (NAA-K+) Affects Conidial

Oct 18, 2016 - The response to exogenous addition of naphthalene acetic acid potassium salt (NAA-K+) to Fusarium oxysporum f. sp radici-lycopersici AT...
0 downloads 11 Views 2MB Size
Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Naphthalene Acetic Acid potassium salt (NAA-K+) Affects Conidial Germination, Sporulation, Mycelial Growth, Cell Surface Morphology and Viability of Fusarium oxysporum f. sp radici-lycopersici and F. oxysporum f. sp cubense in vitro María Karina Manzo-Valencia, Laura Valdés-Santiago, Lino Sanchez-Segura, and Doralinda Guzman-de-Pena J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03105 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 22, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

Journal of Agricultural and Food Chemistry

Naphthalene Acetic Acid potassium salt (NAA-K+) Affects Conidial Germination, Sporulation, Mycelial Growth, Cell Surface Morphology and Viability of Fusarium oxysporum f. sp radici-lycopersici and F. oxysporum f. sp cubense in vitro María Karina Manzo-Valenciaa, Laura Valdés-Santiagoa, Lino Sánchez-Segurab, Dora Linda Guzmán-de-Peñaa* a

Departamento de Biotecnología y Bioquímica, Unidad Irapuato Centro de Investigación y

Estudios Avanzados-IPN, Km 9.6 Libramiento Norte Irapuato-León, 36821, Irapuato, Guanajuato México. b

Departamento de Ingeniería Genética, Unidad Irapuato Centro de Investigación y Estudios

Avanzados-IPN, Km 9.6 Libramiento Norte Irapuato-León, 36821, Irapuato, Guanajuato México. Corresponding author *

Phone: +52 462 6239648; E-mail: [email protected]

Notes The authors declare no conflict of interest.

1 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 36

1

ABSTRACT

2

The response to exogenous addition of Naphthalene Acetic Acid potassium salt (NAA-K+ )

3

to Fusarium oxysporum f. sp radici-lycopersici ATCC 60095 and F. oxysporum f. sp.

4

cubense isolated from Michoacan Mexico soil is reported. The in vitro study showed that

5

NAA-K+ might be effective in the control of Fusarium oxysporum. Exogenous application

6

of

7

mycelium stages of the fungi. Viability testing using acridine orange and propidium iodide

8

showed that NAA-K+ possess fungal killing properties, doing it effective in the destruction

9

of conidia of this phytopathogenic fungi. Analysis of treated spores by Scanning Electron

10

Microscopy showed changes in shape factor and fractal dimension. Moreover, NAA-K+

11

repressed the expression of brlA and fluG genes. The results disclosed here, give evidences

12

of the use of this synthetic growth factor as substances of bio-control that present

13

advantages and, the methods of application in situ should be explored.

14

KEYWORDS

15

Fusarium oxysporum f. sp radici-lycopersici, F. oxysporum f. sp. cubense, naphthalene

16

acetic acid potassium salt, brlA, fluG, mycelial growth inhibition, spore germination

17

inhibition

NAA-K+

affected

both,

spores

and

18

19

20

21

2 ACS Paragon Plus Environment

Page 3 of 36

Journal of Agricultural and Food Chemistry

22

23

INTRODUCTION

24

The presence of the natural growth regulator indole-3-acetic acid (IAA) in fungi has been

25

long-standing supported, even though its role and mode of action in not well understood.

26

However, it has been suggested that IAA is involved in the physiology and regulation of

27

gene expression in fungi.1-5 It has been observed that at lower concentrations IAA promoted

28

conidial germination as well as elongation of germ tubes in Neurospora crassa, while

29

higher concentrations repressed germination. The inhibition of growth by exogenous

30

addition of IAA has been also reported in Ustilago maydis, Fusarium oxysporum and

31

Fusarium delphinoides.6-8 Another study found that exogenous addition of IAA inhibited

32

mycelial growth in Fusarium oxysporum f. sp. cubense, and the presence of IAA in this

33

fungus was found later.9,10 A similar effect was reported in Fusarium culmorum were IAA

34

repressed the mycelium growth rate as well as sporulation and germination.11 Lately,

35

Fusarium graminearum showed reduced mycelial growth, delayed conidial germination

36

and anormal hyphae branching by the addition of IAA.12 In recent research studies Sun et

37

al. tested different concentration of IAA and the authors conclude that IAA behaves either

38

as stimulant or inhibitor, it depends on the fungal-strain and the IAA levels.5,13 A synthetic

39

derivative of IAA is the growth-regulator naphthalene acetic acid (NAA); NAA is

40

chemically similar to IAA (Figure 1). Interestingly, NAA exhibits an antifungal effect

41

resembling the effects on fungi observed by the addition of IAA. Sclerotinia sclerotiorum,

42

Fusarium culmorum and Colletotrichum spp. treated with NAA presented less mycelial

43

growth compared with the control.11,14,15 Exogenous addition of NAA also, reduced spore

44

germination, germ tube growth in Alternaria solani.16 In fact, it has been suggested the 3 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 36

45

application of NAA in susceptible plant cultivars as resistance inductor.16 On the other

46

hand, NAA has been widely used to improve multiple features in several plants, and its

47

efficiency on the stimulation of plant growth is well documented.17-19 The use of

48

naphthalene acetates (1-NAA, its salts, ester, and acetamide) as pesticide and as a plant

49

growth regulator has been endorsed by the U.S. Environmental Protection Agency (U.S.

50

EPA), in fact, it is estimated that 20, 000 lbs/year of naphthalene acetates are applied in the

51

U.S.20 Furthermore, naphthalene acetates are used on food/feed crops as spray solution in a

52

1-2% or even as a dust, powder or other kind of formulation.20 Accordingly, with the

53

agency´s human health and environmental risk, naphthalene acetates are metabolized by

54

human body after 48 h of exposure, as final products were found glycine and glucuronic

55

acid conjugates, substances that are not of toxicological concern.20 EPA determined that

56

“the naphthalene acetates show low acute toxicity, are not mutagenic, and are not expected

57

to be carcinogenic”.20 An innovative use to NAA-K+ has been given as fungicide to

58

prevent and control mycotoxin contamination of cereal grains (Mexican patent number

59

307901). Sporulation, mycelial growth and sterigmatocystin biosynthesis in Aspergillus

60

nidulans was reduced with the addition of NAA-K+ (0.5 mM). Moreover, in Aspergillus

61

parasiticus, the addition of 5 mM NAA-K+ inhibited aflatoxin biosynthesis and growth.21

62

The filamentous ascomycete fungi belonging to the genus Fusarium comprises several

63

agronomically serious phytopathogens.22,23 Moreover, F. oxysporum pathogens present

64

forma specialis (f. sp.) showing host specificity, F. oxysporum f. sp. radici-lycopersici is a

65

radish and tomato wilt pathogen, whereas F. oxysporum f. sp. cubense is the causal agent of

66

banana wilt diseases.22,24 Fusarium oxysporum is one of the more versatile genus,

67

comparative genomic analysis has revealed sequences (lineage-specific regions) containing

68

transposable elements responsible for host specificity and pathogenicity.25 The response of 4 ACS Paragon Plus Environment

Page 5 of 36

Journal of Agricultural and Food Chemistry

69

Fusarium oxysporum f. sp. radici-lycopersici, F. oxysporum f. sp. cubense to NAA-K+

70

exposure has not been studied and, there is no information about the effect of NAA-K+ at

71

level of microscopic cell morphology and transcriptional expression. The goal of this

72

research reported here was to determine the effect of NAA-K+ on two pathogen special

73

forms of Fusarium oxysporum in terms of morphological, physiological and genetic level.

74

MATERIALS AND METHODS

75

Reagents and solutions. 1-Naphthaleneacetic acid potassium salt was purchased from

76

Sigma (St. Louis, MO). Acridine orange and propidium iodide were obtained from Sigma

77

Chemical Co. (St. Louis, MO). Acridine orange (0.1% dissolve in sterile distilled water)

78

and propidium iodide (2% dissolve in phosphate buffered saline pH 7.4) stock solutions

79

were prepared separately and store in the dark at 4°C.

80

Fungal material. F. oxysporum f. sp. radici-lycopersici 60095 was derived from the

81

American Type Culture Collection (ATCC). Isolates of Fusarium were collected from

82

banana soil from Michoacan, Mexico. The soilborne fungal isolates were analyzed

83

morphologically and those with colony features that resembled Fusarium were selected to

84

DNA isolation and, the amplification and sequencing of the 5.8S rRNA region using the

85

primers ITS1/ITS4.26 A BLAST analysis of the amplicon sequences confirmed the identity

86

of the isolated selected to this study SFM4 with 100% of homology with F. oxysporum f.

87

sp. cubense (accession number HQ694500).

88

Conidial germination experiments. To analyzed conidial germination, different

89

concentrations of NAA-K+ were tested (0, 0.5, 1, 2, 3, 4, 5, 8, 10 mM); spore suspension of

90

1x106 conidias/well were inoculated into microtiter plates (24 well) containing 500 µl of

5 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 36

91

Potato Dextrose Broth (PDB) medium and incubated for 15 h. Five replicates for each

92

concentration were performed. Microscopic observations (x20) of samples (15 µl/well)

93

were done and, germinated and not germinated spores were counted on 5 fields per slide

94

(≈300 spores) with five replicates n=5 for reporting the percentage of spore germination.

95

The assay was repeated three times.

96

Mycelial inhibition experiments. The examination of the effect of NAA-K+ on the

97

mycelial growth was performed using PDA plates containing different concentrations of the

98

compound (0,1, 5, and 10 mM). Each plate with 20 ml of medium was inoculated at the

99

centre with mycelium of each strain. The hyphal plugs (consisting of fragments of

100

mycelium) of 4-mm diameter were taken from actively growing cultures of 15-days-old

101

inoculated with 1x106spores/plate and incubated at 28°C in the dark. Each treatment

102

including the control plates (with no NAA-K+ addition) was performed three times with

103

five replicate plates per treatment. Fungal growth (colony diameter) was measured in two

104

directions. Mean of these data was used to calculated mycelial percentage inhibition

105

(percentage growth inhibition = (C-T)/C x 100, where C = colony growth (mm) in the

106

control and T= colony growth in the tested control plate.27 Growth rates of the colonies

107

were calculated by plotting the colony diameter against time and, the slope of the linear

108

regression of the curve represented the growth rate (mm/day).28 The colony diameter was

109

measured every day until 8 days.

110

Conidial production. The spores produced during mycelial growth inhibition experiments

111

were quantified. Fungal spore suspension was recovered from de colony surface adding 10

112

ml of Triton (0.01%) to the plates at the end of the treatment (8 days). The spore suspension

6 ACS Paragon Plus Environment

Page 7 of 36

Journal of Agricultural and Food Chemistry

113

was centrifuged 10 min /10000 rpm and the pellet was resuspended in 3 ml of sterile

114

distilled water. Spores were counted using a haemocytometer.

115

Sample preparation for microscopic analysis. Spore suspension (1x107) of Fusarium

116

oxysporum f. sp. radici-lycopersici and F. oxysporum f. sp. cubense were inoculated

117

separately into flasks (25 ml volume) containing 10 ml of PDB medium with the three

118

following treatments: control PDB; PDB + 5 mM NAA-K+; PDB + 10 mm NAA-K+ (3

119

replicates for each treatment with three repetitions). All treatments were incubated without

120

agitation at 28 °C for 72h. After incubation time samples were centrifuged at 10, 000 rpm

121

for 5 min, the pellet was washed twice with 1ml of sterile distilled water. The pellet was

122

resuspended in 200 µl of sterile distilled water and, 50 and 2 µl of this sample was taken for

123

fluorescence and scanning electron microscopy respectively.

124

Staining of conidia with acridine orange and propidium iodide. Spore suspension was

125

obtained as described in 2.5 section, 50 µl of this sample was taken and diluted with 50 µl

126

of sterile distilled water. Subsequently, 20 µl of propidium iodide 2% plus 40 µl of acridine

127

orange 0.1% were added to the sample and it was incubated at room temperature for 5

128

minutes under dark conditions. Spores treated with the dyes were recovered by

129

centrifugation at 10 000 rpm for 5 minutes, the pellet was resuspended in 100 µl of sterile

130

distilled water. Microscopic observations of samples (10 µl) were done. Fungal spores (≈

131

300 with three repetitions n=3) either green-fluorescent or red-fluorescent were counted.

132

Fluorescence microscopy. The viability of fungal spores was observed in fluorescence

133

microscope (BX-50, Olympus, Japan) at 20X/0.50, UPlan-FL (α-0.17) coupled to a digital

134

camera Lumenera-Infinity 3 (Lumenera, Canada) with UV lamp for illumination.

135

Excitation filters 450-550 nm for acridine orange and 550-600 nm for propidium iodide 7 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 36

136

were used. Processing of the images was done with software Image Pro premier 9.1 (Media

137

cybernetics EUA).

138

Scanning Electron Microscopy (SEM) sample preparation. The morphology of

139

Fusarium oxysporum f. sp. radici-lycopersici and F. oxysporum f. sp. cubense spores was

140

examined by SEM (EVO LS40, Zeiss, Germany), coupled to coolstage -25°C to +50°C at

141

50Pa with ambient at 17ºC (Deßen EVO® XVP® Coolstage, UK). Samples were treated as

142

described above (section 2.5), 2 µl of this spore suspension were fixed on the holder using a

143

double-sided carbon tape mounted on stub and, incubated for 5 minutes at 37°C in a closed

144

petri dish for drying. Afterward, colloidal gold was applied using sputter coater (Fullam

145

EMS-76V) for 2 min. The conditions of operation in all experiments were 25 kV High

146

Voltage (EHT), 2.508 Fil I Target, 20 mm working distance and 600 ± 5Spot Size. SEM

147

micrographs were captured at 10.5kx, magnification and size of 1024x768 pixels captured

148

in grey scale. In this format, one assigns a grey scale with 0 for black and 255 for white.

149

Images were used to determined fractal dimension and shape factors.

150

Determination of fractal dimension of texture and shape factor of Fusarium

151

oxysporum f. sp. radici-lycopersici and F. oxysporum f. sp. cubense. The roughness and

152

complexity of the cell wall in Fusarium oxysporum f. sp. radici-lycopersici and Fusarium

153

oxysporum f. sp. cubense were measured by fractal dimension theory, using Sliding

154

Differential Box Counting (SDBC) method.29 Fractal dimension was calculated using the

155

“Frac-Lac” plug-in in ImageJ software (National Institutes of Health, Bethesda, MD, USA).

156

The fractal dimension was the quantitative parameter directly related to surface roughness

157

(texture) of the cell walls. Fractal dimension was obtained from a plot of the log(box count)

8 ACS Paragon Plus Environment

Page 9 of 36

Journal of Agricultural and Food Chemistry

158

versus log(box size) and the calculation of slope (Eq. A.1), where N is the number of boxes

159

and r is the lateral length of box size.

160

In the SEM images of Fusarium oxysporum f. sp radici-lycopersici and F. oxysporum f. sp.

161

cubense, the cells were cropped and transferred into a new image of 450x450 pixels at 8

162

bits with white background and storage in tagged image file format (.tiff). The fractal

163

dimension measures in 2-D grayscale images are limited to the range of values from 2

164

(smooth image) to 3 (image high roughness), which increases according to changes in the

165

texture of surface.29,30

166

On the other hand, the shape of the Fusarium spores can be described by shape factor

167

parameter. Shape factor or circularity is based on the projected area of the cell (A) and the

168

overall perimeter (P) of the projection (Eq. A.2) according to Bouwman et al (2004).31

169

Shape factor values of circularity are limited to the range of values from 0 to 1, where unity

170

represents a perfect sphere and values near zero represent longer or rougher shapes.31 This

171

morphometric parameter was measured by Sigma Scan Pro software (V5.0, SPSS, USA)

172

using binarized images of Fusarium spores from grey scale images.

173

Semi-quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

174

Liquid medium (25 mL) of PDB was inoculated with a spore suspension (1x107) of F.

175

oxysporum f. sp radici-lycopersici and F. oxysporum f. sp. cubense separately and

176

incubated in darkness at 28°C for 12 h to obtained primordial germ tubes, then NAA-K+ (5

177

and 10 mM) was added, as control treatments samples without NAA-K+ were used. After

178

48 h of additional incubation RNA was obtained, at the end of the incubation period (60 h),

9 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 36

179

samples were centrifuged at 6000 rpm for 10 min to remove the medium, and the pellet was

180

kept at -70 °C until RNA extraction.

181

Total RNA was extracted with TRIZOL reagent (Invitrogen) from F. oxysporum f. sp

182

radici-lycopersici and F. oxysporum f. sp. cubense. RNA was reverse transcribed with 1 µg

183

of total RNA using First Strand synthesis kit (Invitrogen) according to the manufacter´s

184

instructions. The cDNA obtained from F. oxysporum under different conditions was used as

185

template to the amplification of genes fluG and brlA together with the reference gene β-

186

actin as internal control. The conditions selected (Tm 60°C and 25-35 cycles) corresponded

187

to the exponential phase of the PCR, allowing the comparison of cDNA from identical

188

reactions. Primers used for genes amplification are shown in Table 1. Band intensity of

189

transcripts was determined with ImageJ software.

190

Statistical analysis. Data analyses of experimental data were performed with R software

191

version 3.3.0 (The R foundation for statistical computing).32 The results were statistically

192

significant when the p-value of one-way analysis of variance (ANOVA) test was < 0.05.

193

Differences between values were evaluated by Tukey´s test (α = 0.05).

194

RESULTS AND DISCUSSION

195

NAA-K+ inhibits spore germination of F. oxysporum f. sp. radici-lycopersici and F.

196

oxysporum f. sp. cubense. Conidial germination of F. oxysporum f. sp. radici-lycopersici

197

and F. oxysporum f. sp. cubense was analyzed under optimal conditions (nutrient-rich

198

medium). After 12 h of incubation, almost 20% of spores presented germ tube length twice

199

longer that spore, and at 24 h 100% of spores have been germinated and formed mycelium.

200

On a first attempt, different concentration of NAA-K+ was added to the medium and, after

10 ACS Paragon Plus Environment

Page 11 of 36

Journal of Agricultural and Food Chemistry

201

24 h of incubation, complete inhibition of microconidia and macroconidia germination was

202

observed at all NAA-K+ concentrations tested (10, 25, 50 and 100 mM), while the control

203

without NAA-K+ addition presented 100 % of conidial germination (data not shown). Since

204

treatment with 10 mM inhibited completely conidial germination, lower NAA-K+

205

concentrations were proved: 0, 0.5, 1, 2, 3, 4, 5 and 10 mM (Figure 2A). For conducting

206

this second attempt, 15h of incubation was selected because at that time spores had been

207

germinated and mycelium did not interfere with the counting. Complete spore germination

208

inhibition was observed with the addition of 2-10 mM. However, at 0.5 and 1 mM the

209

percentage of spore germination was 27 and 5 % (73 and 95 % of spore inhibition)

210

respectively (Figure 2A). Our data are consistent with earlier reports indicating that NAA

211

inhibits spore germination in Sclerotinia sclerotiorum and Alternaria solani.16,33 However,

212

in Fusarium mangiferae lower concentrations of NAA enhanced conidia germination.3

213

Compounds capable of inhibit spore germination are needed since this is a key process in

214

the infection on any fungal pathogen as well as food borne diseases.35,36 In order to explain

215

how NAA-K+ could inhibit spore germination, it was necessary to review the fate of NAA

216

in other organisms. In the ectomycorrhizal fungus Pisolithus arhizus NAA is metabolized

217

generating 1,2-dihydroxyl-1,2-dihydronaphthalene-1-acetic acid γ-lactone and 4-hydroxy-

218

naphthalene acetic acid as major products.37 On the other hand, some bacteria such as

219

Pseudomonas are able to use NAA as sole carbon source, in this case the degradation of

220

NAA starts with the decarboxylation of the acid and its, replacement by a hydroxyl group

221

yielding a dihydroxy naphthalene which is unstable and susceptible to reduction giving as a

222

result catechol or substituted catechol, compound able to enter the tricarboxylic acid

223

cycle.38,39 In this line, Bhajbhuje attributes the effect of NAA on spore germination

11 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 12 of 36

224

inhibition to the hydrolytic by-products generating during the degradation of NAA that

225

could induce cell wall damage,16 in fact, some agents inhibit spore germination through

226

affecting membrane integrity.40

227

To find out if spore germination had been delaying, we carried out the same assay, with the

228

next concentrations of NAA-K+ : 1, 5 and 10 mM and longer incubation time (1x107 spores

229

were inoculated in 25 ml of PDB and incubated for 15 days at 28°C). At the end of the

230

experiment, growth was quantified by mycelial dry weight. We observed no growth

231

measured at 10 mM in both fungi (Figure 2B), compared with 93 mg obtained in the

232

control. Further, when we probed 1 mM, the effect was slightly attenuated with 68 and 48

233

mg of mycelial dry weight in F. oxysporum f. sp. radici-lycopersici and F. oxysporum f. sp.

234

cubense respectively (Figure 2B). These results suggested that spores treated with high

235

concentration of NAA-K+ are unable to recover their capacity to growth.

236

NAA-K+ is able to inhibit mycelial growth of F. oxysporum f. sp. radici-lycopersici and

237

F. oxysporum f. sp. cubense. High concentrations of NAA-K+ completely suppressed

238

active mycelial growth of both strains of F. oxysporum on PDA significantly (P=