In vitro antagonistic activity of native bacteria isolated from soils of the argentine Pampas against Fusarium tucumaniae and Fusarium virguliforme

1 Cátedra de Fitopatología, Facultad de Agronomía, Universidad de Buenos Aires, Argentina. 2 Cátedra de Microbiología Agrícola, Facultad de Agronomía, Universidad de Buenos Aires, Argentina. 3 Instituto de Investigaciones en Biociencias Agrícolas y Ambientales (INBA-CONICET/UBA), Facultad de Agronomía, Universidad de Buenos Aires, Argentina. 4 Centro de Referencia de Micología (CEREMIC), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.


INTRODUCTION
Soybean is the main crop in Argentina.In the last seasons, the area planted to soybean in Argentina was about 20 million hectares per year (Carmona et al., 2015).Sudden Death Syndrome (SDS) is a soybean *Corresponding author.E-mail: sautua@agro.uba.ar.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License disease caused by at least four Fusarium species, but in Argentina F. tucumaniae and F. virguliforme are the predominant.These are soil-borne pathogens commonly found in Argentina's pampas region and are important causes of crop losses (Scandiani et al., 2010).The fungus infects soybean roots and, under appropriate conditions, toxin-dependent symptoms develop in the aerial tissues after flowering and during pod fill, leading to rapid necrosis (Hartman et al., 2015).Under monoculture and no-till conditions in the argentine Pampas region the presence of SDS has intensified (Scandiani et al., 2010).Because of the difficulty in obtaining resistant soybeans varieties, the impossibility of fungicides to move towards the roots basipetally, wide host range of the pathogen and its ability to survive in the soil with resistance structures (chlamydospores), common management strategies such as genetic resistance, seed treatment with fungicides and crop rotation do not provide adequate control of SDS (Scandiani et al., 2010).In this context, biological control appears as an alternative and interesting tool.
Plant growth promoting rhizobacteria (PGPR) have been widely reported and recognized to have the potential for PGP and for their ability to antagonize the growth of fungal pathogens in crops such as maize, rice, potato, wheat and canola (Siddiqui et al., 2006).In soybean, PGPR were successfully tested against Macrophomina phaseolina (Simonetti et al., 2015) and Pythium ultimum (León et al., 2009).
There are few reports on the antagonistic effect of bacterial isolates on soybean Fusarium species causing SDS (Xing and Westphal, 2007;Agaras et al., 2012).It is reported that the chance of finding bacterial strains effective for biocontrol increases if the isolates are obtained from pathogen suppressive soils and from the same environment in which they will be used (Cook and Baker, 1983).The aim of the present preliminary study was to evaluate indigenous PGPR previously isolated from soy fields in Argentina's pampas region for their in vitro antagonistic effects on the control of F. tucumaniae and F. virguliforme.

MATERIALS AND METHODS
Ten PGPR strains that were previously isolated and identified by Simonetti et al. (2015) for their in vitro antagonistic capacity against M. phaseolina, were tested for their in vitro inhibitory capacity against the fungal pathogen F. tucumaniae (Table 1).
The fungal strains used in this study were originally isolated from infected soybean plants showing SDS root rot and provided by Centro de Referencia de Micología (CEREMIC), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (Table 1).

Assay 1
All bacterial strains were tested for their ability to inhibit the mycelial growth of F. tucumaniae149-12.Each bacterial isolate was streaked as a band on the edge of a PDA 90-mm diameter plate and incubated for 24 h at 28 ± 2°C.Then, a 6 mm diameter mycelial disc of F. tucumaniae 149-12 was taken from the margin of a growing colony and placed onto the centre of previously inoculated potato-dextrose-agar (PDA) plates.The Petri dishes were sealed by parafilm and incubated at room temperature in the dark.Plates containing only the fungal mycelial plug were maintained as control.

Assay 2
The bacterial strains that did not show genetic relationship with potentially hazardous bacteria in the assay 1 were tested for their ability to inhibit the growth of F. virguliforme 101-03 and F. tucumaniae 149-12 using in vitro dual-culture assay (Simonetti et al., 2012).Each bacterial isolate was prepared in nutrient broth (NB) and incubated for 48 h at 28 ± 2°C in order to use them in stationary phase.Fungi were maintained on PDA at 24 ± 2°C for one week.A 6 mm diameter mycelial plug was taken from the margin of a growing colony and placed centrally in a Petri dish containing PDA medium.Two drops (2 µL) of each bacterial culture previously prepared were placed in a straight line 3 cm away from the center of the plate and drops of sterile water served as control.
All these experiments were performed in triplicate.After incubation period of 11 days at 24 ± 2°C, mycelium growth inhibition was calculated as I=[(C-T)/C]×100, where C is the mycelium diameter in control, and T is the mycelium diameter in bacteria-inoculated plates.
Data were analyzed using analysis of variance and differences between means were tested using Tukey test with an overall risk level of 5%.

DISCUSSION
Altogether, these results suggest that strain 54 (B.subtilis) displays antifungal features mainly towards F. tucumaniae 149-12, one of the causing agents of soybean SDS.The original contribution of this study is the isolation and testing of bacteria originating from the Pampas region.
These findings are in accordance with those of Xing and Westphal (2007), who found antagonism of B. subtilis against 12 isolates of F. virguliforme.On the other hand, our results differ with those found by Agaras et al. (2012) where Pseudomonas strain SMMP3 antagonized the growth of several pathogenic fungi, including the F. tucumaniae isolate CCC 132-11.
Because good results obtained in vitro cannot always be dependably reproduced under field conditions, these in vitro results should be confirmed by in planta experiments.In this way, Agaras et al. (2012) carried out both greenhouse and field trials using soybean seeds inoculated with Pseudomonas strain SMMP3.This inoculation treatment caused a reduction of SDS ratings (incidence, severity) and a decrease in the AUDPC (area under disease progress curves) values.Nevertheless, these effects were not statistically meaningful (P>0.05).This may be due to the many factors that affect the effectiveness of the bacteria in natural conditions (Badri et al., 2009).Isolated bacterial strains should be rhizospheric competent, able to survive and colonize in the rhizospheric soil (de Souza et al., 2015).
Further studies on the effects of this strain on the growth of soybean plants and on the SDS control will uncover the mechanisms and potential of this bacterial isolate.However, it has been previously described that most cases of naturally occurring biological control result from mixtures of antagonists, rather than from high populations of a single antagonist (Myresiotis et al., 2012).Moreover, root-infecting Fusarium species attack soybean seedlings in the first developmental stages, thus additional tests as for example seed treatment with multiple strain inoculation might be required to improve the degree of SDS control.20020130100604BA Universidad de Buenos Aires, Argentina.

Figure 1 .
Figure 1.Photographs (A) and figure (B) of the bacteria strains tested for their ability to inhibit the mycelial growth of F. tucumaniae 149-12, in assay 1 (columns with different letters are significantly different P<0.05); Photographs of B. subtilis strain 54 and its control, tested for their ability to inhibit the mycelial growth of F. tucumaniae 149-12 (C) in assay 2; and C. vietnamense strain 110 and its control, tested for their ability to inhibit the mycelial growth of F. virguliforme 101-03 (D) in assay 2.

Table 1 .
Identification of PGPR and fungal strains used in the antagonistic trials.

Table 2 .
F. tucumaniae and F. virguliforme mycelial growth inhibition caused by four selected strains of native bacteria isolated from soils of the argentine Pampas, in assay 1 and 2.Means with different letters within a column are significantly different (P<0.05).