Vol. 10(47), pp.

1992-1998, 21 December, 2016

DOI: 10.5897/AJMR2016.8292
Article Number: B8E6F4162128
ISSN 1996-0808
Copyright © 2016
African Journal of Microbiology Research
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJMR

Full Length Research Paper

Evaluation of in vitro inhibition of mycelial growth of

Fusarium solani f. sp. piperis by different products in
Brazil
Verônica D’Addazio 1*, Renata Aparecida Ahnert dos Santos 2, Arthur Salomão Bastos Leitão 2,
Marcelo Barreto da Silva2, Adriano Alves Fernandes 2 and Antelmo Ralph Falqueto 2
1
Universidade Federal do Espírito Santo (UFES) - Av. Fernando Ferrari, 514 – Goiabeiras, 29075-910 - Vitória, ES,
Brazil.
2
Universidade Federal do Espírito Santo (UFES) - Centro Universitário Norte do Espírito Santo – CEUNES, Rodovia BR
101 Norte, Km 60 - Bairro Litorâneo, 29932-540 - São Mateus, ES, Brazil.
Received 3 September, 2016; Accepted 28 October, 2016

The black pepper (Piper nigrum L.) is one of the most popular peppers in the world. Nonetheless, there
are several limitations on cultivation, resulting in reduced production or a complete loss of the crop.
The major disease affecting this crop is fusarium root rot caused by Fusarium solani f. sp. piperis,
which is responsible for decimating whole crops in Brazil, with losses reaching millions of dollars per
year. So far, there is no effective control measure against this fungus and no cultivars resistant to it. In
this study, in vitro effects of different products on colony growth was evaluated. Carbendazim,
chitosan, silicon, and phosphate were tested against F.solani f. sp. piperis isolates CML-2466, CML-
2353, E-637, and E-596. Chitosan and silicon did not inhibit mycelial growth of any of the isolates, while
phosphite inhibited mycelial growth by 100%. Carbendazim was found to be fungitoxic for isolates
CML-2353 and E-596 and fungistatic for CML-2466 and E-637, inhibiting the mycelial growth of these
isolates by 60 and 80%, respectively. There were no dose effects of the products tested.

Key words: Fusarium solani, Black pepper, chitosan, silicon, phosphite, carbendazim

INTRODUCTION

Among the major factors limiting agricultural production According to Nojosa et al. (2015), the losses for the
are infections with fungi, bacteria, viruses, and Brazilian agribusiness can be as high as $55 billion
nematodes, insects, mites, and weeds (Kreyci and annually due to diseases of crops, which is equivalent to
Menten, 2013). It is estimated that, agricultural losses the average annual loss of 7.7% or 25 million tons of
due to pest attacks reach US $1.4 trillion, or almost 5% of agricultural produce. According to the Food and
global gross domestic product (GDP) worldwide. Agriculture Organization(FAO) of the United Nations (UN)

*Corresponding author. E-mail: v [email protected] Tel: +55 (27) 3312-1548.

Author(s) agree that this article remains permanently open access under the terms of the Creativ e Commons Attribution
License 4.0 International License
 D’Addazio et al. 1993

pathogens are responsible for 13.3% of such damages. element that reduces severity of major diseases in
The black pepper (Piper nigrum L.), which accounts for several crops (Epstein, 1999). Silicon can act on the
~57,800 tons of national agricultural produce (IBGE, constitution of the physical barrier to prevent penetration
2016), has been affected by root rot, also known as of fungi and affects the signals between the host and the
fusariosis (Kimati et al., 1997). This severe disease can pathogen, resulting in more rapid and extensive
cause a lot of damage to the crop, with an annual activation of pre-and post-formed defense mechanisms in
reduction of 3% in the cultivated area and production. A the plant (Chérif et al., 1994; Epstein, 1999), e.g., by
healthy pepper crop has a productive cycle potential of increasing the synthesis of phenolic compounds and
12 years on average. This disease reduces the cycle by 5 peroxidase, polyphenoloxidase, chitinase, and -
to 6 years (Tremacoldi, 2014). The causal agent is glucosidase (Fauteux et al., 2005). Phosphites are
Fusarium solani (Mart.) Appel & Wr. emend. Snyd. & characterized by their effectiveness in controlling downy
Hans.f. sp. piperis, Albuquerque (Teleomorphs Nectria mildew and various diseases caused by genus
haematococca Berk. & Br. f. sp. piperis Albuq.). The Phytophthora (Ouimette and Coffey, 1989), exert
Fusarium species are widely distributed in the soil, and in acropetal and basipetal systemic action and suppress
an adverse environment, form a resistant structure called foliar and root diseases (Guest and Grant, 1991).
chlamydospore, which can remain viable for more than Furthermore, they have high stability in plants and may
20 years (Pfenning and Lima, 2007). After infection, the remain active for substantial periods (Smillie et al., 1989).
fungi settlesdown in the vascular system of the plant, Regarding the mechanism of action of phosphites, some
hindering the absorption of water and nutrients (Bedendo, authors discuss direct action on the pathogen (Fenn and
1995). Once inside the root system, the fungi are initially Coffey, 1984; Fenn and Coffey, 1985; McGrath, 2004).
limited to the root or the plant base and, at some point, Others suggest that the mechanism is indirect, via
begin to spread to the vascular system. The damage is activation of plant defense mechanisms (Nemestothy and
due to colonization of xylem vessels by hyphae and Guest, 1990; Saindrenan et al., 1990) or a combination of
microconidia; hypertrophy and hyperplasia of the direct and indirect effects (Smillie et al., 1989; Jackson et
cambium, xylem, and phloem; destruction of xylem fibers al., 2000).
and amyloplasts in parenchymatous cells; and production The objective of the present study was to evaluate the
of gels by the plant (Ortiz et al., 2014). Eventually, the effects of alternative antifungal agents such as chitosan,
combination of the fungal growth in the vascular system, silicon, and phosphite as well as the known fungicide
fungal toxins such as naphthoquinones and fusaric acid carbendazim on mycelial growth of F. solani f. sp. piperis
(Rocha et al., 2016), and defense structures produced by in vitro.
the plant hamper the absorption and transport of water,
causing wilt and death of the plant (Wheeler and Rush,
MATERIALS AND METHODS
2001).
Strategies for F. solani sp. piperis control are limited F. solani f. sp. piperis isolates
because there is still no information on resistant cultivars
and an effective fungicide does not exist (or is not The isolates that w e tested w ere CML-2466 and CML-2353
officially approved in Brazil). In vitro, studies are needed (Coleção Micológica de Lavras, Universidade Federal de Lavras -
Minas Gerais State) and E-637 and E-596 (Incaper– Instituto
to identify the products with the possible ability to control
Capixaba de Pesquisa, Assistência Técnica e Extensão Rural -
the fungus. To this end, various compounds are being Espírito Santo State). The isolates w ere maintained in Petri dishes
tested. Benzimidazole fungicides are used extensively in containing potato dextrose agar (PDA) in refrigerator at 4°C. Every
agriculture due to strong systemic activity against a great month, an agar disk (5mm) from a pure culture of F. solan iw as
number of fungal species (Reis et al., 2001). placed in the center of a PDA plate containing the same medium.
Carbendazim, a systemic fungicide with a benzimidazole The plates w ere incubated at 25°C in biochemical oxygen demand
(B.O.D.), w ith the photoperiod of 12 h.
chemical group, exerts both preventive and curative
action (Kus and Altanlar, 2003). Among the products with
great potential antifungal stand out chitosan, silicon and Preparation of chitosan, silicon, phosphite, and carbendazim
phosphite. Such products have not been tested concentrations
effectively in black pepper as an alternative method of
Chitosan w as added to the PDA medium at concentrations of 0.5,
controlling fusarium or were used on a small scale 1.0, 1.5, 2.0%, 2.5, and 3.0%. Chitosan (Fagron) w as extracted in
without scientific evidence. Thus, various control methods acetic acid and diluted in w ater to a concentration of 2% at pH 4.4.
are used to minimize the severity of the disease. This substance has high viscosity and w as diluted w ith sterilized
Chitosan, a high-molecular-weight polysaccharide, has distilled w ater to obtain the desired concentrations. The other
many physicochemical and biological properties (El- products added to the culture medium at the follow ing
Ghaouth et al., 1994), e.g., antimicrobial activity against concentrations w ere: silicon (SiO2) at 0.25, 0.50, 1.0, 1.5, 2.0, or 3.0
g L-1; phosphite (Phosethyl Al) at 1.0, 2.0, 3.0, 4.0, 5.0, or 6.0 g L-1;
some fungi (yeasts) and bacteria (Allan and Hadwiger, and carbendazim (Carbomax 500) at 0.83, 1.67, 2.50, 3.34, 4.16,
1979; Roller and Covill, 1999). Among mineral nutrients or 5.0 ml L-1. As controls, w e used Petri dishes containing PDA
used in pest management, silicon (Si) stands out as an medium supplemented w ith 2% of acetic acid for the treatment w ith
1994 Afr. J. Microbiol. Res.

chitosan (Control 1) and/or only the PDA medium (Control 2). After
solidification, a fungal mycelial disc 5mm in diameter, 15 days old,
w as transferred to the center of each Petri dish (68-mm diameter).
This procedure w as performed for each F.solani isolate. The plates
w ere sealed w ith parafilm and maintained in B.O.D at 25°C, w ith a
photoperiod of 12 h.

Effect of different products on m ycelial grow th of F. solani

isolates

The mycelial grow th of F. solani isolates w as assessed daily by

measuring the diameter of the colonies in orthogonal directions by
means of a pachymeter, until the colonies in control treatments
reached the edge of the board. The percent grow th inhibition w as
calculated according to Guo et al. (2006), using the follow ing
formula: antifungal index (%) = (1 – Da/Db)  100, w here Da w as
the diameter of the zone of grow th in the test plates, and Db w as
the diameter of grow th zone in the control plate.

Statistical analysis

The experiment w as performed using randomized block design

(RBD) w ith 28 treatments and five repetitions for each isolate of F.
solani. Each repetition consisted of a Petri dish. Each experiment
w as repeated three times. The significance of treatment effects on
radial grow th among isolates w as tested w ith analysis of variance
(ANOVA). Where significant F values w ere obtained, Tukey’s all
pairw ise comparison test, w hich includes a correction for multiple
comparisons, w as used to assess the significance of differences
betw een meansin the statistical softw are ASSISTAT 7.1 beta (Silva
and Azevedo, 2009).

RESULTS

The mycelial growth of F. solani was not inhibited by any

chitosan concentrations tested except for the test plate
PDA with added acetic acid. The presence of chitosan
favored the growth of the four fungal isolates: CML-2466
and E-637 reaching the edge of the plate after 9 days of
incubation, and CML-2353 and E-596 showed maximal
growth after 11 days (Figure 1A). The fungi were also
seeded on agar-agar with added chitosan (same
concentrations) or agar-agar only and all reached the
edge of the plate after 9 days of growth (data not shown).
Silicon, at the concentrations tested, did not inhibit fungal
growth (Figure 1B). For CML-2353, at concentrations of
-1
0.25 and 0.5 gL , the inhibition rate was 25.28 and
22.07%, respectively. At other concentrations, the
inhibition rate was below 11%. The other strains grew
normally at all concentrations of silicon tested (Table 1B).
Phosphite proved to be effective in inhibiting the mycelial
growth of fungi under all our experimental conditions
(Figure 1C). None of the plates showed mycelial growth
at the tested doses of phosphite.
Carbendazim was 100% effective against two of the
four isolates tested. The mycelial growth of CML-2353
Figure 1. Effect of chitosan (A), silicon (B), phosphite and E-596 were completely inhibited at the various
(C) and carbendazim (D) on the mycelial grow th of F.
solani isolates. Control 1: PDA medium supplemented concentrations of carbendazim. Carbendazim exerted a
w ith 2% of acetic acid; Control 2: only PDA medium. fungistatic effect on the isolates CML-2466 and E-637,
 D’Addazio et al. 1995

Table 1. Colony diameter (C.D.) and percent grow th inhibition (P.I.) of F. solani isolates in chitosan (A ), silicon (B), phosphite (C) and
carbendazim (D).

A
CML 2466** CML 2353** E-637** E-596**
Chitosan
(%) C.D. C.D. P.I. C.D. P.I. C.D. P.I.
P.I. (%)
(mm) (mm) (%) (mm) (%) (mm) (%)
0c c
Control 1 100 0 100 0b 100 0b 100
b b a a
Control 2 60.95 0 51.58 0 63.16 0 62.03 0
0.5 68.00 a -11.57 65.56 a -27.10 68.00 a -7.66 65.04 a -4.86
1.0 68.00 a -11.57 65.53 a -27.05 68.00 a -7.66 63.76 a -2.79
1.5 68.00 a -11.57 62.16 a -20.51 68.00 a -7.66 64.64 a -4.21
2.0 68.00 a -11.57 62.49 a -21.15 68.00 a -7.66 52.95 a 14.63
2.5 68.00 a -11.57 62.21 a -20.61 67.85 a -7.43 61.96 a 0.12
3.0 68.00 a -11.57 64.17 a -24.41 68.00 a -7.66 64.49 a 4.30
V.C. (%) = 4.72 7.96 4.12 12.71
B
CML 2466 n.s. CML 2353* E-637** E-596 n.s.
-1
Silicon (g L ) C.D. P.I. C.D. P.I. C.D. P.I. C.D. P.I.
(mm) (%) (mm) (%) (mm) (%) (mm) (%)
Control 2 60.95 a 0 51.58 a 0 63.16 ab 0 62.03 a 0
a b b a
0.25 62.48 -2.51 38.54 25.28 58.00 8.17 64.63 -24.22
62.50 a 40.20 ab ab
0.5 -2.54 22.07 64.57 -2.24 63.43 a -21.92
a ab a a
1.0 64.33 -5.54 46.15 10.53 68.00 -7.66 64.90 -24.73
1.5 64.43 a -5.72 47.82 ab 7.29 68.00 a -7.66 63.38 a -21.81
a ab
2.0 67.78 -11.20 50.78 1.56 68.00 a -7.66 65.13 a -25.18
3.0 68.00 a -11.57 50.66 ab 1.78 68.00 a -7.66 59.33 a -14.03
V.C. (%) = 9.82 12.47 5.98 12.05
C
CML 2466** CML 2353** E-637** E-596**
Phosphite
C.D. P.I. C.D. P.I. C.D. C.D. P.I.
(g L-1) P.I. (%)
(mm) (%) (mm) (%) (mm) (mm) (%)
a a a a
Control 60.95 0 51.58 0 63.16 0 62.03 0
0b 0b b b
1.0 100 100 0 100 0 100
b b b b
2.0 0 100 0 100 0 100 0 100
b b
3.0 0 100 0 100 0b 100 0b 100
b b b b
4.0 0 100 0 100 0 100 0 100
5.0 0b 100 0b 100 0b 100 0b 100
6.0 0b 100 0b 100 0b 100 0b 100
V.C. (%) = 34.0 13.35 28.70 10.34
D
CML 2466** CML 2353** E-637** E-596**
Carbendazim
-1 C.D. P.I. C.D. P.I. C.D. P.I. C.D. P.I.
(ml L )
(mm) (%) (mm) (%) (mm) (%) (mm) (%)
a a
Control 2 60.95 0 51.58 0 63.16 a 0 62.03a 0
0.83 23.45 b 61.52 0b 100 14.18 b 77.55 0b 100
1.67 19.77 b 67.57 0b 100 11.81 b 81.30 0b 100
b b b b
2.50 20.61 66.19 0 100 11.65 81.55 0 100
3.34 23.67 b 61.16 0b 100 13.50 b 78.63 0b 100
b b b b
4.16 21.89 64.09 0 100 12.09 80.86 0 100
5.0 23.49 b 61.46 0b 100 11.40 b 81.96 0b 100
V.C. (%) = 16.56 13.35 14.73 10.34
Averages follow ed by the same letter are not statistically different among themselves, by Tukey test. V.C. = Variation coefic ient; **
significant at 1% probability (p < 0.01); * significant at 5% probability (0.01 = < p < 0.05); ns not significant (p > = 0.05).
1996 Afr. J. Microbiol. Res.

and the effect was not dosedependent. The inhibition of acts as a resistance inducer in the plant. Similar results
growth of these isolates was 60 and 80%, respectively were reported by Carré-Missio et al. (2010), who studied
(Figure 1D, Table 1D). the effect of silicon on Pestalotia leaf spotin cultivated
strawberry. In vitro results showed that silicon at the dose
-1
DISCUSSION of 8 g L does not inhibit mycelial growth ofPestalotia
longisetula. In another study, the growth of Fusarium spp.
The absence of inhibition of mycelial growth by chitosan and Verticillium spp. were enhanced at silicon
-1
suggested that the F .solani f. sp. piperis isolates can use concentrations of 5 and 10 ml L , respectively (Kaiser et
this substance as anadditional carbon source. This is al. 2005). Generally, silicates do not act directly on
possibly because chitosan is a polysaccharide, and microorganisms that cause diseases in plants, but have
probably, the fungus uses it as a source of nutrients for alternative mechanisms of action, which in some cases—
its growth. Nascimento et al. (2007), studying fungi because of their beneficial effect on the plant—may
causing grapevine trunk diseases, found that chitosan reduce abiotic and biotic types of stress (Zambolim et al.
inhibited the growth of all fungi tested except Neonectria 2012). In the literature, there are reports of reduced and
liriodendri, which grew at all the concentrations analyzed. increased intensity of diseases in plants after treatment
According to Baños et al. (2004) and Bhaskara-Reddy et with silicon (Zambolim and Ventura, 1996). Silicon can
al. (1998), mycelial growth and sporulation of Penicillium act locally by inducing defensive reactions in cells and
digitatum and Alternaria alternata, respectively, were can also contribute to systemic resistance by increasing
stimulated by the presence of chitosan. These authors the production of stress hormones. Nonetheless, the
believed that such behavior may be a response to stress exact mechanism by which silicon modulates signaling in
caused by the chitosan. Several studies have shown that plants remains unclear. Evidence suggests that silicon
the biological activity of chitosan is significantly can act as an enhancer of plant defense responses or as
dependent upon its molecular weight, acetylation degree a strategic signaling proteins. Silicon can therefore
(Alfredsen et al., 2004; Wu et al., 2004; Torr et al., 2005), interact with several key components of the plant stress
pH of the medium (Devlieghere et al., 2004), and the response-related signaling pathways, leading to effective
microorganism membrane characteristics (Qi et al., resistance to pathogenic fungi.
2004). In general, the lower the molecular weight and In agreement with the results of our study, Araújo et al.
degree of acetylation of chitosan, the greater the efficacy (2008), while studying Colletotrichum gloeosporioides,
atreducing the growth and multiplication of showed that potassium phosphite (Fitofós K®) has a
microorganisms (Goy et al., 2009). The other possibility is direct effect on this fungus, almost completely inhibiting
the unusual pH of the culture medium, which remained the mycelial growth in vitro. Potassium phosphite was
acidic (about 4.0). The ability of fungi to grow in wider pH tested against Penicillium expansum, which causes
ranges is associated with the presence of pH-regulatory postharvest blue mold infections on apple fruits; this
systems. These regulatory systems are mediated by compound completely inhibited the mycelial growth (Amiri
differential production of extracellular enzymes and and Bompeix, 2011). In a study made by Lobato et al.
metabolites as a function of pH of the medium (Denison, (2010), phosphate exerted a fungicidal effect on
2000). It is likely that this pH adjustment mechanism also pathogens of potatoes: F. solani, Rhizoctonia solani, and
exists in F. solani. This phenomenon may be associated Streptomyces scabies. According to Guest and Grant
with fungal survivability for long periods in the soil, even (1991), phosphites inhibit the growth of pathogens in
under adverse conditions. In control plates where we plants via a complex mechanism of action. The first stage
added acetic acid, there was no growth for any of the is a direct fungistatic effect, which is dependent on the
isolates tested. Sholberg et al. (2000) reported that the concentration of phosphite that accumulates in the
inhibitory effect of acetic acid on microorganisms is due fungus. This, in turn, is influenced by the concentration of
to the reduction in pH as well as the ability of the coupled phosphate, and the effectiveness of the phosphite
molecules of acetic acid to pass easily through the oxidation system. The second step is a change in the
membrane of conidia, exerting its toxic effect by reduc ing metabolism of the pathogen, such that a faster and more
the cellular protoplasm.This mechanism may explain the effective defensive response by the plant can develop.
inhibition of mycelial growth of F. solani in control plates These alterations imply a reduction in the amount of
containing only PDA with added acetic acid. Chitosan’s suppressor molecules on the pathogen’s surface or an
effects on growth of microorganisms are well known, but increase in the number of receptors exposed to agonists
the mechanisms underlying its antifungal action have not in host cells, or both, suggesting that phosphites may
been fully elucidated. The response to this possible have multiple modes of action. As for the direct action on
antifungal agent may vary depending on the pathogen the pathogen, it is known that phosphorous acid and its
(El-Ghaouth et al. 1992). derivatives act by inhibiting the process of oxidative
Our results suggest that silicon does not have direct phosphorylation in Oomycetes (McGrath, 2004). In
action on the F. solani isolates tested because it induced general, the effects of phosphites on the phytopathogens
mycelial growth at all concentrations. Silicon probably are mediated by the formation of membrane pores due to
 D’Addazio et al. 1997

damage to the plasma membrane and cell wall of the ACKNOWLEDGEMENTS

hyphae, probably because of transcription changes in
genes that encode proteins involved in the biosynthesis This study is a part of a Doctor of Science thesis of V.
of their components and other parts of the overall cellular D’Addazio in the Programa de Pós-graduaçãoem
metabolism. These changes compromise the Biologia Vegetal, Universidade Federal do Espírito Santo.
morphology, physiology, and sporulation of the fungus, V. D’Addazio is supported by CAPES. The authors thank
interfering with the parasitism (Smillie et al., 1989; King et Ludwig Pfenning, Ph.D., (Coleção Micológica de Lavras -
al., 2010). The indirect action of phosphate involves UFLA) and José Aires Ventura (Incaper – Instituto
activation of plant defense mechanisms such as Capixaba de Pesquisa, Assistência Técnica e Extensão
stimulation of the production of phytoalexins (Guest and Rural) for kindly providing the F. solani isolates. This
Grant, 1991; Daniel and Guest, 2006) or lignification and study was supported by FAPES- Fundação de Amparo à
production of phenols (Nojosa et al., 2005). Pesquisa e Inovação do Espírito Santo (Grant TO
The biological activity of benzimidazoles (such as 986/2014) to A. R. Falqueto.
carbendazim) is mediated by interference with the
formation and functioning of microtubules in eukaryotic
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