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

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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

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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.

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ABSTRACT

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

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

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

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

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of

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

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

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

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

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

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

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

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KEYWORDS

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

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

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inhibition

NAA-K+

affected

both,

spores

and

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19

20

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INTRODUCTION

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

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

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

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

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

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higher concentrations repressed germination. The inhibition of growth by exogenous

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

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

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

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

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

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Fusarium graminearum showed reduced mycelial growth, delayed conidial germination

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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The filamentous ascomycete fungi belonging to the genus Fusarium comprises several

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

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

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

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

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

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

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

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

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

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

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

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MATERIALS AND METHODS

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

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

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

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

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

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

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

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

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

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

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

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

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sp. cubense (accession number HQ694500).

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Conidial germination experiments. To analyzed conidial germination, different

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

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



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

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

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

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

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The assay was repeated three times.

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

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

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

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

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

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

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

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

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

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

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

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

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

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measured every day until 8 days.

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Conidial production. The spores produced during mycelial growth inhibition experiments

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

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


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

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distilled water. Spores were counted using a haemocytometer.

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

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

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

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

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

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

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

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

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fluorescence and scanning electron microscopy respectively.

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

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

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

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

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

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

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

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

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

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

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

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


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

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cybernetics EUA).

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

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

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

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

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

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

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

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

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

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

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

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Images were used to determined fractal dimension and shape factors.

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Determination of fractal dimension of texture and shape factor of Fusarium

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

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

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

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

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

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

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


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

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and r is the lateral length of box size.

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

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

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

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

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

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texture of surface.29,30

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

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

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

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

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

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

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using binarized images of Fusarium spores from grey scale images.

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

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

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

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

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

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



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

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kept at -70 °C until RNA extraction.

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

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

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

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

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

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

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

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

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transcripts was determined with ImageJ software.

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

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

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

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

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RESULTS AND DISCUSSION

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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affecting membrane integrity.40

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

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

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

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

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

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

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

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

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

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

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

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

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

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