An evaluation in vitro and in vivo of rhizobacteria in the biocontrol of Radopholus similis in banana

Rhizobacteria were isolated from the rhizosphere of banana cultivars ‘Prata Comum’ and ‘Prata Anã’ and from crotalaria and marigold in Tryptic Soy Agar and King B-agar media. Bacteria were used for valuations of the in vitro and in vivo. The promising isolates were submitted to identification by polymerase chain reaction. Among the one hundred and fifty two evaluated, twenty three showed toxicity to R. similis after twenty-four and thirty six hours. Five presented deleterious effect on the population of R. similis in the soil under in vivo conditions. Of the ten submitted to identification, three corresponded to the Bacillus genus and two to the Pseudomonas genus. The bacteria evaluated have antagonistic potential against R. similis.


INTRODUCTION
Bananas are affected by Radopholus similis, considered the most important nematode in this crop (Araya, 2004).The strategies applied to R. similis control include chemical control, use of resistant varieties and crop rotation.Nematicides are costly and lead to environmental risks and fruit contamination (Athman et al., 2006).The use of resistant varieties presents obstacles in obtaining the resistance associated to desirable agronomic characteristics.On the other hand, crop rotation alone is not considered an appropriate method since it does not provide satisfactory economic return to farmers.
Rhizobacteria have been shown to be an important component to promote biological control of plant diseases (Mondino and Vero, 2006).The use of these microorganisms is an alternative strategy to disease control, which is intensely investigated aiming the reduction of pesticide use.A great number of reports are found in the literature regarding bacteria of the Pseudomonas and Bacillus genera acting as biocontrol agents of plant pathogens.The biocontrol of nematodes by bacteria happens by direct production of toxic compounds, modification of root exudates or induction of resistance in the host plant (Kloepper and Ryu, 2006;Lian et al., 2007).Recent surveys report that the bacteria Pseudomonas synxantha, Pseudomonas fluorescens, Bacillus subtilis, and Stenotrophomonas maltophilia showed toxic effect on eggs and juveniles under laboratory conditions indicating the occurrence of the production of antimicrobial metabolites (Ludwig et al., 2013).Chaves et al. (2009) selecting microorganisms for the biocontrol of R. similis, reported the occurrence of biocontrol percentage of 92% provided by Bacillus and significant increases in biomass production in banana plantlets of the 'Grand Naine' cultivar.
This study tries to isolate rhizobacteria from different plant species.It also evaluates them as to the biocontrol potential of R. similis through in vitro and in vivo tests and identifies the best isolates at genus level.

MATERIALS AND METHODS
For the isolation of rhizobacteria, samples of roots from two banana cultivars (Prata Comum' and 'Prata Anã') and from two species of plants known for their antagonistic affect to nematodes (Crotalaria -Crotalarea junceae and Marigold -Taegetes patula) were used.The serial dilution technique and Tryptic Soy Agar and King Bagar medium were adopted (Mariano and Kloepper, 2000).The isolates were preserved in Nutrient Broth Yeast extract (NBY) (Schaad, 1988) medium containing 15% of glycerol and subsequently stored at -80°C in ultrafreezer.
After obtainment, the rhizobacteria were submitted to the extraction of metabolites by subculturing the isolates in Tryptic Soy Broth liquid medium.After 15 days the medium with the culture was centrifuged at 10000 rpm (7504 g) for 15 min at 25°C and the supernatant containing the metabolites placed in sterilized glass bottles sterilized stored in freezer at -4°C (Sousa et al., 2006 modified) Preliminary in vitro tests were performed with a smaller number of isolates before starting the tests with all the isolates.For these tests, 150 ml of the extract containing bacterial metabolites and 50 ml of an aqueous suspension with 10 nematodes were used; both placed in Eppendorf tubes, and incubated in (BOD) chamber at 25°C.The number of dead nematodes was quantified after 12, 18, 24, 36 and 48 h with the aid of a microscope.For the biocontrol tests, conducted after the preliminary tests, the number of dead nematodes was quantified after 24 and 36 h.
For the in vivo tests, plantlets of the 'Grand Naine' cultivar and the 10 most promising isolates obtained in the in vitro evaluation were used.This experiment consisted of 22 treatments: 10 rhizobacteria alone, 10 rhizobacteria inoculated with nematodes, inoculation with nematodes alone and an absolute control, adopting the block randomized design with six replicates per treatment.For the preparation of the bacterial suspensions to be used in the treatment of the plantlets, 50 ml of sterilized distilled water were added in each Petri dish containing the rhizobacteria grown for 48 h.Afterwards, the concentrations of these suspensions were adjusted to OD540 = 0.5 equivalent to 10 9 cfu ml -1 , in the spectrophotometer.The plantlets were treated by immersion of the roots in the suspensions for 10 min.Plantlets were transplanted to plastic tubes containing autoclaved substrate composed of 50% vermiculite and 50% organic compound (prepared by composting of 50% grass, 20% orange peel, 20% cocoa cake, 10% castor bean cake and 30 L cassava pressed juice diluted in water, 1:1) (Nascimento, 2010).
After 45 days, the plants were transplanted to pots containing 2.0 kg of soil and 10 days later inoculation was performed with 1000 specimens of Radopholus similis per seedling using a 5 ml syringe.

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After 60 days of cultivation, plant height, stem diameter, dry weight of shoot and rhizome, were evaluated and these data used to calculate the growth rate of plants.The level of root damage and the number of nematodes in the roots and soil were also calculated.
The 10 isolates evaluated in the in vivo experiment were submitted to PCR technique for identification at genus level.The bacterial DNA extraction was performed by means of subcultivation of the isolates in microtubes containing 200 ml of extraction buffer (0.05 M NaOH, 0.25% SDS), followed by heat treatment (20 min 100°C), centrifugation (1 min at 10000 rpm) and collection of the supernatant containing the DNA.PCR reactions were then carried out with specific primers for the genus Pseudomonas (PSMG and 9-27) (Johnsen et al., 1999) and Bacillus (B-K1 / F and B-K1 / R) (Wu et al., 2006).The amplification products were subjected to electrophoresis in 1% agarose gel and photographed in UV transilluminator and photodocumented.The analysis was performed by the presence of specific bands for each bacterial genus; Pseudomonas (445 bp) and Bacillus (1114 bp).Data from the in vitro tests were transformed to arcsin √x/100 whereas the data of the in vivo test were subjected to logarithmic transformation.Data from the preliminary in vitro test were submitted to the Tukey test at 5% and Scott and Knott test at 5% was applied to the other in vitro tests and in vivo tests, using the program SAS.

RESULTS
A total of 152 isolates were obtained, 77 in King B-agar medium and 75 in Tryptic Soy Agar (Table 1).In the preliminary test, differences between treatments began to occur at 24 h of exposure of the nematodes to the bacterial metabolites, and again observed after 36 h applying the Tukey test (Figure 1).After 42 h, 100% of the specimens were dead, however maintaining the proportional difference in relation to the control treatment.
According to these data, the effect of bacterial metabolites on the nematodes after 24, 36 and 42 h using the Tukey test was observed.The first two periods were considered most appropriate to evaluate the antagonistic potential of isolates against R. similis.Among the bacterial isolates evaluated, 64 showed nematicidal effect and of these, 23 showed toxic effects to the nematodes after 24 and 36 h.Four treatments differed among themselves in the evaluation period.The isolate 17KB-CR, obtained from the rhizosphere of Crotalaria, provided 100% of mortality after 24 h (Table 2).
In the in vivo experiment no differences were detected for plant growth rate.There were also no significant differences in the damage level of roots and in the number of nematodes in the root system.However, there was a reduction in the final population of R. similis in the soil for half of the treatments (19TSA-PA, 8KB-PA, 12KB-PA, 9KB-PA e 17KB-PA) (Figure 2).
Among the 10 isolates with the highest suppressive effects after 24 and 36 h, five were identified by PCR.Three belonged to the Bacillus (2TSA-PA, 3KB-PC, PC-36TSA), and two to the Pseudomonas genus (8KB-PA-PA 9KB).These two isolates from Pseudomonas genus promoted reduction of the population of nematodes in the soil (Figure 2).

DISCUSSION
The period of exposure of nematodes to the metabolites under in vitro conditions, directly influenced the mortality rate of juveniles and adults of R. similis.In the work performed by Ludwig et al. (2013), mortality increase of second stage juveniles (J2) of Meloidogyne javanica in all bacterial suspensions tested when the exposure time increased from 24 to 48 h was detected.Similar results were observed in this study when the expansion of the exposure time of juveniles and adults of R. similis to bacterial metabolites caused an increase in the percentage of dead nematodes.
According to some authors, soil that contain native bacteria were able to produce toxic compounds against nematodes, which are compounds resulting from microbial metabolism (Rodrígez-Kábana, 1991;Tian et al., 2007;Machado et al., 2012).In this study, rhizobacteria probably synthesized some antimicrobial compound that caused the death of R. similis under in vitro conditions, since only in the presence of the culture medium the mortality rate was much lower.Most rhizobacteria (9 isolates) that stood out in evaluations were obtained from the banana cultivar 'Prata Anã'.Therefore, there is the possibility of rhizospheric microorganisms to be related with the characteristic of moderate natural resistance displayed by the cultivar 'Prata Anã' to R. similis (Embrapa, 2009).This fact could explain the results found in this study.None of the isolates were able to reduce the penetration of R. similis in the roots of the plants under in vivo conditions.Probably, the rhizobacteria used in this experiment did not promote mechanisms of interference in the pathogenhost relationship.According to Siddiqui and Mahmood (1999), two mechanisms of action may contribute for the reduction of the infection root rate: (i) production of metabolites that reduce the communication between host plant and nematode and (ii) degradation of root exudates that control the behavior of nematodes.The effect of nonparasitic rhizobacteria in the impediment of the nematodes penetration possibly occurs due to a link between the bacteria and lectins on the root surface.In this case, the recognition process is controlled by interactions between lectins on the root surface and carbohydrates in the nematode cuticle.
Five out of the ten rhizobacteria selected in the in vitro test reduced the final population of nematodes in the soil under in vivo conditions.Among these, two belong to the  Pseudomonas genus.This decrease was probably due to the multiplication of these bacteria, since the species of this genus is capable of adapting well in the rhizosphere, promoting mechanisms of action characteristic of the genus.The direct antagonism derived by the production of antibiotics or by competition for essential nutrients such as iron is one of the mechanisms used by species of Pseudomonas for the biocontrol of nematodes in the soil (Melo, 2015).The species P. fluorescens is characterized as being able to use different carbon sources.Furthermore, these species compete with native microflora showing great aggressiveness in colonizing the rhizosphere (Stirling, 1991;Mazzola et al., 1992;Rizvi et al., 2012).
In this study, it was possible to identify bacteria belonging to the Bacillus and Pseudomonas genera with biocontrol potential, showed under in vitro conditions.According to Romeiro and Garcia (2009), certain beneficial bacteria such as rhizobacteria, when dispensed in plants, are able to promote the biological control of diseases by direct antagonism, by induction of resistance or by both mechanisms.Bacteria of the Bacillus and Pseudomonas are among the most common and that also act effectively.Isolates of B. subtilis and Pseudomonas spp.obtained from soil, as well being antagonistic to pathogenic bacteria and fungi, also have potential to act as biological control of nematode agents (Kerry, 2000;Carrión, 2012).
Therefore rhizobacteria of the Bacillus and Pseudomonas genera have potential to be used in the biocontrol of R. simillis in banana not only under in vitro conditions but also under in vivo conditions.

Figure 1 .
Figure 1.Inactivation curve of R. similis according to the exposure time in suspensions of bacterial metabolites.

Figure 2 .
Figure 2. Number of nematodes in the soil after the growth of banana plantlets inoculated with different bacterial isolates.Means followed by same letter do not differ statistically by Scott-Knott test at 5%.

Table 1 .
Number of rhizobacteria found in the culture medium and original plant.

Table 2 .
Percentage of dead nematodes in suspension of metabolites produced by rhizobacteria after 24 and 36 h of exposure.
1Lowercase letters compare treatments in each period and uppercase letters compare the same treatments between periods. 2 Means followed by same letter do not differ by Scott-Knott test at 5%.