Evaluation of in vitro inhibition of mycelial growth of Fusarium solani f. sp. piperis by different products in Brazil

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, CML2353, 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.

pathogens are responsible for 13.3% of such damages.
The black pepper (Piper nigrum L.), which accounts for ~57,800 tons of national agricultural produce (IBGE, 2016), has been affected by root rot, also known as fusariosis (Kimati et al., 1997).This severe disease can cause a lot of damage to the crop, with an annual reduction of 3% in the cultivated area and production.A healthy pepper crop has a productive cycle potential of 12 years on average.This disease reduces the cycle by 5 to 6 years (Tremacoldi, 2014).The causal agent is Fusarium solani (Mart.)Appel & Wr.emend.Snyd.& Hans.f.sp.piperis, Albuquerque (Teleomorphs Nectria haematococca Berk.& Br.f. sp.piperis Albuq.).The Fusarium species are widely distributed in the soil, and in an adverse environment, form a resistant structure called chlamydospore, which can remain viable for more than 20 years (Pfenning and Lima, 2007).After infection, the fungi settlesdown in the vascular system of the plant, hindering the absorption of water and nutrients (Bedendo, 1995).Once inside the root system, the fungi are initially limited to the root or the plant base and, at some point, begin to spread to the vascular system.The damage is due to colonization of xylem vessels by hyphae and microconidia; hypertrophy and hyperplasia of the cambium, xylem, and phloem; destruction of xylem fibers and amyloplasts in parenchymatous cells; and production of gels by the plant (Ortiz et al., 2014).Eventually, the combination of the fungal growth in the vascular system, fungal toxins such as naphthoquinones and fusaric acid (Rocha et al., 2016), and defense structures produced by the plant hamper the absorption and transport of water, causing wilt and death of the plant (Wheeler and Rush, 2001).
Strategies for F. solani sp.piperis control are limited because there is still no information on resistant cultivars and an effective fungicide does not exist (or is not officially approved in Brazil).In vitro, studies are needed to identify the products with the possible ability to control the fungus.To this end, various compounds are being tested.Benzimidazole fungicides are used extensively in agriculture due to strong systemic activity against a great number of fungal species (Reis et al., 2001).Carbendazim, a systemic fungicide with a benzimidazole chemical group, exerts both preventive and curative action (Kus and Altanlar, 2003).Among the products with great potential antifungal stand out chitosan, silicon and phosphite.Such products have not been tested effectively in black pepper as an alternative method of controlling fusarium or were used on a small scale without scientific evidence.Thus, various control methods are used to minimize the severity of the disease.Chitosan, a high-molecular-weight polysaccharide, has many physicochemical and biological properties (El-Ghaouth et al., 1994), e.g., antimicrobial activity against some fungi (yeasts) and bacteria (Allan and Hadwiger, 1979;Roller and Covill, 1999).Among mineral nutrients used in pest management, silicon (Si) stands out as an element that reduces severity of major diseases in several crops (Epstein, 1999).Silicon can act on the constitution of the physical barrier to prevent penetration of fungi and affects the signals between the host and the pathogen, resulting in more rapid and extensive activation of pre-and post-formed defense mechanisms in the plant (Chérif et al., 1994;Epstein, 1999), e.g., by increasing the synthesis of phenolic compounds and peroxidase, polyphenoloxidase, chitinase, and glucosidase (Fauteux et al., 2005).Phosphites are characterized by their effectiveness in controlling downy mildew and various diseases caused by genus Phytophthora (Ouimette and Coffey, 1989), exert acropetal and basipetal systemic action and suppress foliar and root diseases (Guest and Grant, 1991).Furthermore, they have high stability in plants and may remain active for substantial periods (Smillie et al., 1989).Regarding the mechanism of action of phosphites, some authors discuss direct action on the pathogen (Fenn and Coffey, 1984;Fenn and Coffey, 1985;McGrath, 2004).Others suggest that the mechanism is indirect, via activation of plant defense mechanisms (Nemestothy and Guest, 1990;Saindrenan et al., 1990) or a combination of direct and indirect effects (Smillie et al., 1989;Jackson et al., 2000).
The objective of the present study was to evaluate the effects of alternative antifungal agents such as chitosan, silicon, and phosphite as well as the known fungicide carbendazim on mycelial growth of F. solani f. sp.piperis in vitro.

F. solani f. sp. piperis isolates
The isolates that w e tested w ere CML-2466 and CML-2353 (Coleção Micológica de Lavras, Universidade Federal de Lavras -Minas Gerais State) and E-637 and E-596 (Incaper-Instituto Capixaba de Pesquisa, Assistência Técnica e Extensão Rural -Espírito Santo State).The isolates w ere maintained in Petri dishes containing potato dextrose agar (PDA) in refrigerator at 4°C.Every month, an agar disk (5mm) from a pure culture of F. solan iw as placed in the center of a PDA plate containing the same medium.The plates w ere incubated at 25°C in biochemical oxygen demand (B.O.D.), w ith the photoperiod of 12 h.

Preparation of chitosan, silicon, phosphite, and carbendazim concentrations
Chitosan w as added to the PDA medium at concentrations of 0.5, 1.0, 1.5, 2.0%, 2.5, and 3.0%.Chitosan (Fagron) w as extracted in acetic acid and diluted in w ater to a concentration of 2% at pH 4.4.This substance has high viscosity and w as diluted w ith sterilized distilled w ater to obtain the desired concentrations.The other products added to the culture medium at the follow ing 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 ; and carbendazim (Carbomax 500) at 0.83, 1.67, 2.50, 3.34, 4.16, or 5.0 ml L -1 .As controls, w e used Petri dishes containing PDA medium supplemented w ith 2% of acetic acid for the treatment w ith 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 0.25 and 0.5 gL -1 , 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 and E-596 were completely inhibited at the various concentrations of carbendazim.Carbendazim exerted a fungistatic effect on the isolates CML-2466 and E-637,    and the effect was not dosedependent.The inhibition of growth of these isolates was 60 and 80%, respectively (Figure 1D, Table 1D).

DISCUSSION
The absence of inhibition of mycelial growth by chitosan suggested that the F .solani f. sp.piperis isolates can use this substance as anadditional carbon source.This is possibly because chitosan is a polysaccharide, and probably, the fungus uses it as a source of nutrients for its growth.Nascimento et al. ( 2007), studying fungi causing grapevine trunk diseases, found that chitosan inhibited the growth of all fungi tested except Neonectria liriodendri, which grew at all the concentrations analyzed.
According to Baños et al. (2004) and Bhaskara-Reddy et al. ( 1998), mycelial growth and sporulation of Penicillium digitatum and Alternaria alternata, respectively, were stimulated by the presence of chitosan.These authors believed that such behavior may be a response to stress caused by the chitosan.Several studies have shown that the biological activity of chitosan is significantly dependent upon its molecular weight, acetylation degree (Alfredsen et al., 2004;Wu et al., 2004;Torr et al., 2005), pH of the medium (Devlieghere et al., 2004), and the microorganism membrane characteristics (Qi et al., 2004).In general, the lower the molecular weight and degree of acetylation of chitosan, the greater the efficacy atreducing the growth and multiplication of microorganisms (Goy et al., 2009).The other possibility is the unusual pH of the culture medium, which remained acidic (about 4.0).The ability of fungi to grow in wider pH ranges is associated with the presence of pH-regulatory systems.These regulatory systems are mediated by differential production of extracellular enzymes and metabolites as a function of pH of the medium (Denison, 2000).It is likely that this pH adjustment mechanism also exists in F. solani.This phenomenon may be associated with fungal survivability for long periods in the soil, even under adverse conditions.In control plates where we added acetic acid, there was no growth for any of the isolates tested.Sholberg et al. (2000) reported that the inhibitory effect of acetic acid on microorganisms is due to the reduction in pH as well as the ability of the coupled molecules of acetic acid to pass easily through the membrane of conidia, exerting its toxic effect by reduc ing the cellular protoplasm.This mechanism may explain the inhibition of mycelial growth of F. solani in control plates containing only PDA with added acetic acid.Chitosan's effects on growth of microorganisms are well known, but the mechanisms underlying its antifungal action have not been fully elucidated.The response to this possible antifungal agent may vary depending on the pathogen (El-Ghaouth et al. 1992).
Our results suggest that silicon does not have direct action on the F. solani isolates tested because it induced mycelial growth at all concentrations.Silicon probably acts as a resistance inducer in the plant.Similar results were reported by Carré-Missio et al. (2010), who studied the effect of silicon on Pestalotia leaf spotin cultivated strawberry.In vitro results showed that silicon at the dose of 8 g L -1 does not inhibit mycelial growth ofPestalotia longisetula.In another study, the growth of Fusarium spp.and Verticillium spp.were enhanced at silicon concentrations of 5 and 10 ml L -1 , respectively (Kaiser et al. 2005).Generally, silicates do not act directly on microorganisms that cause diseases in plants, but have alternative mechanisms of action, which in some casesbecause of their beneficial effect on the plant-may reduce abiotic and biotic types of stress (Zambolim et al. 2012).In the literature, there are reports of reduced and increased intensity of diseases in plants after treatment with silicon (Zambolim and Ventura, 1996).Silicon can act locally by inducing defensive reactions in cells and can also contribute to systemic resistance by increasing the production of stress hormones.Nonetheless, the exact mechanism by which silicon modulates signaling in plants remains unclear.Evidence suggests that silicon can act as an enhancer of plant defense responses or as a strategic signaling proteins.Silicon can therefore interact with several key components of the plant stress response-related signaling pathways, leading to effective resistance to pathogenic fungi.In agreement with the results of our study, Araújo et al. (2008), while studying Colletotrichum gloeosporioides, showed that potassium phosphite (Fitofós K®) has a direct effect on this fungus, almost completely inhibiting the mycelial growth in vitro.Potassium phosphite was tested against Penicillium expansum, which causes postharvest blue mold infections on apple fruits; this compound completely inhibited the mycelial growth (Amiri and Bompeix, 2011).In a study made by Lobato et al. (2010), phosphate exerted a fungicidal effect on pathogens of potatoes: F. solani, Rhizoctonia solani, and Streptomyces scabies.According to Guest and Grant (1991), phosphites inhibit the growth of pathogens in plants via a complex mechanism of action.The first stage is a direct fungistatic effect, which is dependent on the concentration of phosphite that accumulates in the fungus.This, in turn, is influenced by the concentration of phosphate, and the effectiveness of the phosphite oxidation system.The second step is a change in the metabolism of the pathogen, such that a faster and more effective defensive response by the plant can develop.These alterations imply a reduction in the amount of suppressor molecules on the pathogen's surface or an increase in the number of receptors exposed to agonists in host cells, or both, suggesting that phosphites may have multiple modes of action.As for the direct action on the pathogen, it is known that phosphorous acid and its derivatives act by inhibiting the process of oxidative phosphorylation in Oomycetes (McGrath, 2004).In general, the effects of phosphites on the phytopathogens are mediated by the formation of membrane pores due to damage to the plasma membrane and cell wall of the hyphae, probably because of transcription changes in genes that encode proteins involved in the biosynthesis of their components and other parts of the overall cellular metabolism.
These changes compromise the morphology, physiology, and sporulation of the fungus, interfering with the parasitism (Smillie et al., 1989;King et al., 2010).The indirect action of phosphate involves activation of plant defense mechanisms such as stimulation of the production of phytoalexins (Guest and Grant, 1991;Daniel and Guest, 2006) or lignification and production of phenols (Nojosa et al., 2005).
The biological activity of benzimidazoles (such as carbendazim) is mediated by interference with the formation and functioning of microtubules in eukaryotic cells.The affinity of benzimidazole for tubulin is the main factor determining its fungicidal activity.The stronger the binding affinity of the compound for tubulin, the more sensitive is the organism to the fungicide.Also, resistance to carbendazim is described as a change in the -tubulin protein, which reduces the binding of the fungicide to this protein (Osmani and Oakley, 1991).In our study, carbendazim showed different effects when administered to the fungus.In two F. solani isolates, CML-2466 and E-637, this compound had a fungistatic effect at all the doses analyzed, whereas for CML-2353 and E-596 isolates, this compound showed a fungitoxic effect.According to Sultana and Ghaffar (2013), carbendazim completely inhibits colony growth of F. oxysporum.When tested on mycelial growth of Rhizoctonia solani, carbendazim reduced it by 86% in vitro (Schurt et al., 2013).In addition to in vitro results, chitosan (El-Ghaouth et al., 1994), silicon (Epstein, 1999) and phosphites (Ouimette and Coffey, 1989) due to resistance induction characteristics are being field tested by us in Black pepper plants, to evaluate the behavior of such plants inoculated with F. solani.
In summary, chitosan and silicon did not inhibit the growth of F. solani and instead promoted the growth of most isolates.Carbendazim exerted growth control in 50% of the isolates and in the other 50%, had a fungistatic effect, and these effects were not dosedependent.Among the products tested for possible inhibition of the mycelial growth of F. solani f. sp.piperis in vitro, the action of phosphite stands out: 100% inhibition in isolates CML-2466, CML-2353, E-637, andE-596.