In vitro antifungal / fungistatic activity of manganese phosphite against soybean soil-borne pathogens

Resumen. Las pudriciones de raíz y tallo (PRYT) en el cultivo de soja causadas por patógenos habitantes del suelo son enfermedades comúnmente encontradas en campos de soja, y son una de las causas más importantes de pérdidas económicas. La sensibilidad micelial de Fusarium virguliforme, Fusarium tucumaniae, Sclerotinia sclerotiorum y Macrophomina phaseolina fue evaluada en medio de cultivo agar papa glucosa (25 mL) suplementado con diferentes concentraciones (μg/mL) de fosfito de manganeso (PhiMn) diluido en agua (0; 25; 37,5; 50; 100; 200; 300; 400; 500; 800 y 1000). La sensibilidad del crecimiento micelial fue analizada usando análisis de regresión lineal logarítmico. Se calculó la concentración de PhiMn necesaria para inhibir el 50% del crecimiento micelial (CI50). Los valores de CI50 fueron desde 105 μg/mL (Fusarium spp.) hasta 409 μg/mL (M. phaseolina). La formación de esclerocios fue completamente inhibida a 500 μg/mL. Los resultados del presente estudio representan el primer reporte de la acción fungicida/fungistática in vitro del Phi contra los hongos que causan las PRYT en el cultivo de soja.


INTRODUCTION
Soybean is the main crop in Argentina, with a planted area of 19.78 million hectares in the 2013/2014 growing season.Root and stem rots caused by soil-borne pathogens are diseases commonly found in soybean fields and one of the most important causes of crop losses (Gupta et al., 2012;Leandro et al., 2012;Walters et al., 2013).In addition to killing plants, root and stem rots can slow down or stop plant growth, causing premature defoliation and decreasing the number and weight of seeds.Rhizoctonia solani Kühn, Fusarium tucumaniae Aoki, O´Donnell, Homma & Lattanzi, F. virguliforme O'Donnell & T. Aoki, Macrophomina phaseolina (Tassi) Goidanish, Pythium spp., Phytophthora sojae (Kaufman & Gerdman) and Sclerotinia sclerotiorum (Lib.)De Bary, are among the most important soil-borne pathogens causing root and stem rots, and yield-reducing diseases (Hartman et al., 1999;Scandiani et al., 2010;Ploper et al., 2011;Carmona et al., 2015;Grijalba & Gally, 2015).All these soybean soil-borne pathogens attack many species of cultivated plants, and develop different survival structures such as chlamydospores, microsclerotia, sclerotia and oospores.This is why the common management strategies, such as genetic resistance, seed treatment with fungicides and crop rotation, generally fail to provide adequate control of soil-borne diseases (Dorrance et al., 2009;Gupta et al., 2012;Leandro et al., 2012).
The objective of the present study was to evaluate the in vitro mycelial sensitivity of root and stem rot pathogens in soybean to manganese phosphite (MnPhi), at different concentrations.
Colony diameters were measured with a precision ruler in two perpendicular directions, when the mycelial growth in the control (0 MnPhi µg/mL) reached the dish edge three days after the inoculation with S. sclerotiorum, and six days after the inoculation with M. phaseolina.For slow-growing pathogens, such as Fusarium species, the diameter of the fungal colony was measured 26 days post-inoculation, even though the mycelium did not reach the dish edge.
The influence of pH on growth inhibition due to MnPhi addition was evaluated for each treatment.The pH of the PDA either supplemented or not with each concentration of MnPhi was measured with a pH meter before the addition of mycelial discs of each pathogen isolate.An additional control treatment was carried out by seeding mycelial discs of all pathogens in PDA acidified up to pH 3 with tartaric acid 10% v/v.
A completely randomized experimental design was used for the in vitro tests with three repetitions within each assay.Experimental units were represented by a Petri dish.The assays were repeated three times.The mycelial growth sensitivity bioassays were analyzed using logarithmic linear regression analysis.The MnPhi concentrations needed to inhibit 50% of the mycelial growth (IC 50 ) were calculated from the generated equations.Data from the pH tests were analyzed using ANOVA, and differences between means were tested using Fisher's LSD test.Statistical analyses were carried out using INFOSTAT software (professional version 1.1).

RESULTS
The results from the in vitro bioassay showed that while the concentration of MnPhi increased, the mycelial growth of all pathogens decreased (Fig. 1).The coefficients of determination for the IC 50 equations ranged from 0.79 to 0.98 (Table 1).The highest IC 50 was determined for M. phaseolina followed by S. sclerotiorum.The Fusarium spp.isolates were the most sensitive to MnPhi, with an IC 50 ranging from 105 to 114 µg/mL (Fig. 1).

Pathogens
Linear observed for M. phaseolina, since a concentration of 800 µg/ mL completely inhibited the mycelial growth and a concentration of 500 µg/mL inhibited microsclerotia formation (Fig. 1).For the three isolates of Fusarium spp., the mycelial growth was very slow, and even in the control it failed to cover the entire plate at 26 days post-seeding (Fig. 1).Manganese phosphite concentrations of 500 µg/mL or above completely inhibited the mycelial growth of the three isolates of Fusarium spp.(Fig. 1).The pH ranged from 5.5 (0 µg/mL MnPhi) to 3 (1000 µg/ mL MnPhi).The mycelia of M. phaseolina and S. sclerotiorum growing on media acidified with tartaric acid showed no dif-FYTON ISSN 0031 9457 (2017) 86: 265-269 ferences (P<0.0001) with the control.By contrast, Fusarium spp.were partially affected (up to 30%) (P<0.0001) by acidification, showing total inhibition of mycelial growth at pH 4.

DISCUSSION
The results obtained are consistent with those reported by Lobato et al. (2010) and Araújo et al. (2010) in relation to in vitro inhibition of true fungi such as Fusarium solani, Rhizoctonia solani, Streptomyces scabies and Colletotrichum gloeosporioides by Phi.Lobato et al. (2010) found higher IC 50 for F. solani, being 1280 µg/mL for CaPhi, >3560 µg/mL for KPhi and 680 µg/mL for CuPhi.In the case of oomycetes such as P. infestans and P. plurivora, IC 50 were lower.Dalio et al. (2014) found an IC 50 of 34 µg/mL for mycelial growth inhibition and of 2.9 µg/mL for zoospore inhibition of P. plurivora by Phi.
The media supplemented with 25 and 1000 µg/mL of Mn-Phi had a pH of 5 and 3, respectively.At pH values between 3 and 5, M. phaseolina and S. sclerotiorum were not significantly inhibited (Csöndes et al., 2011).Therefore, the inhibition of mycelial growth observed in the present study is mainly attributed to the effect of MnPhi and not to the acidification of the medium.In contrast, the effect of acidification by MnPhi could partially contribute to the mycelial growth inhibition of Fusarium species causing sudden death syndrome (Sanogo & Yang, 2001).According to Lobato et al. (2010), the activity of Phi cannot be attributed to a single reason, and probably would result from a combination of several factors such as the concentration of phosphite anion, nature of cation, acidification of the medium and type of pathogen.In this way and according to Lobato et al. (2010), when acidification is not important, the concentration of phosphite anion plays the main role in inhibiting the growth of pathogens.Most fungi tolerate a wide pH range, but the optimum range of growth is between 5.0 and 6.5.Thus, microorganisms that are in media at pHs above or below the optimum range will have impaired development (Araújo et al., 2010).However, the type of pathogen or fungal species would also be an important influential factor.In this regard, Lobato et al. (2010) determined that the pH could partially contribute to mycelial growth inhibition in other fungal species (up to 30%), including those of the Fusarium solani species complex.
There is still no conclusive explanation regarding the biochemical mode of action of Phi against pathogens (Dalio et al., 2014).It might be possible that in vivo interactions of Phi act in a direct way (Smilie et al., 1989), reducing fungal growth or generating hyphal disruption (King et al., 2010).We found no studies on the effects of Phi on the formation of sclerotia produced by pathogens.
The results of the present study represent the first report of direct in vitro fungicidal/fungistatic action of Phi against soybean fungi, which are causal agents of soil-borne diseases.Further in vivo research is needed to assess the impact of Phi applied on the soil or plant to analyze whether the control achieved in this work in vitro is also attainable in the plant.In this regard, Simonetti et al. (2015) tested MnPhi alone or in combination with PGPR bacteria to control M. phaseolina in soybean under greenhouse conditions; it was the first work to report effective control of M. phaseolina (up to 37%) using MnPhi as seed treatment.
In the same way, some preliminary results related to the effect of MnPhi seed treatment on field development of soybean sudden death were reported (Carmona et al., 2013).