Antifungal activity of Stenotrophomonas maltophilia in Stomoxys calcitrans larvae Atividade antifúngica de Stenotrophomonas maltophilia em larvas de Stomoxys calcitrans

The microbiota present in Stomoxys calcitrans larvae may assist their survival in contaminated environments through production of inhibitory substances. Bacteriological identification methods, the polymerase chain reaction (PCR) and scanning electron microscopy (SEM) were used to detect a bacterium naturally present in mucus and macerated S. calcitrans larvae. The antifungal activity was determined based on the results from disk diffusion tests on an artificial solid medium. The bacterium was identified as Stenotrophomonas maltophilia and presented antifungal activity against Beauveria bassiana sensu lato isolates CG 138, CG 228 and ESALQ 986. This result suggests that the larval microbiota is a factor that can compromise the use of B. bassiana s.l. fungus for biological control of S. calcitrans larvae.


Introduction
Stomoxys calcitrans (Linnaeus, 1758) (Diptera: Muscidae), known as the stable fly, parasitizes different animal species for its blood meal.This parasitism supports transmission of pathogenic microorganisms that cause losses in livestock.The fly's immature stages can be found in decomposing plant material (cane sugar, coffee straw or grass), in association with urine, feces and uneaten animal feed (BITTENCOURT; CASTRO, 2004;BITTENCOURT, 2012).Accumulation of sugarcane by-products contributes towards stable fly outbreaks and consequently towards population imbalance (LEITE et al., 2013).Oda and Arantes (2010) conducted a study with the aim of identifying the causes of high S. calcitrans infestation in the municipality of Planalto, SP, Brazil.They observed that larvae were found in the cane fields with the vinasse, and that inadequate vinasse management in the sugar cane processing plant and poor management of organic matter by farmers favored expansion of the S. calcitrans population.
In an ecological context, microorganisms should be considered to be important components of insect biology (DILLON; DILLON, 2004).According to Romero et al. (2006), S. calcitrans larvae are dependent on the presence of natural symbiotic microbiota.Lysyk et al. (1999) evaluated S. calcitrans larval survival in rearing medium supplemented with pure or mixed bacteria cultures.They reported that addition of Acinetobacter sp., Empedobacter breve, Flavobacterium odoratum or Escherichia coli favored larval development.In another study, microbiota with pathogenic potential for humans and animals and bacteria used for biological control of arthropods were isolated from adult flies; 33 bacterial species from the families Bacillaceae, Enterobacteriaceae and Micrococcaceae were identified (CASTRO et al., 2008).
This study aimed to identify and report for the first time the S. maltophilia bacteria present in mucus and macerate of S. calcitrans larvae, using bacteriological methods, the polymerase chain reaction (PCR) technique and scanning electron microscopy (SEM), and to evaluate the bacterial antifungal activity by means of the disk diffusion test.

Mucus and macerate of Stomoxys calcitrans larvae
Groups of nine 9-day-old larvae were used for collecting mucus and obtaining larval macerate.The larvae were washed in sterile distilled water and placed in sterile cryogenic tubes (1.2 ml) for mucus production (1 hour).After removing the larvae, 900 µl of cold sterile phosphate buffer solution (PBS) was added (0.1 M; NaCl 1.5 M; pH 7.4).Larval macerate was obtained by placing larvae in sterile microcentrifuge tubes (1.5 ml) with 900 µl of buffer solution (PBS) followed by maceration using a cone stick and storage in sterile cryogenic tubes (1.2 ml).The entire procedure was performed under a laminar flow hood.The samples were manipulated on ice and stored in an ultrafreezer (-80 °C).

Bacterial identification
The bacterial isolate from the mucus and macerate of S. calcitrans larvae was transferred to Petri dishes (9 cm) containing brain and heart infused agar (BHI) (52.0 g/l, MERCK  ) and was incubated at 27 °C for 48 hours.After bacterial growth, the plates were kept under refrigeration (4 °C) (KONEMMAN et al., 2001).
Macroscopic characteristics (colony aspects) and microscopic characteristics (optical microcopy after Gram staining) were evaluated and the catalase test (KONEMMAN et al., 2001) and molecular biology techniques were applied, in order to identify the bacterium species.
The 16S rRNA gene sequence was automatically aligned using ClustalW, and then corrected manually.The sequences were subjected to BLAST analysis (ALTSCHUL et al., 1997) with the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov), and the closest relatives were included in the phylogenetic analysis.Phylogenetic analysis of 16S rRNA was performed using the neighbor-joining method and Kimura two-parameter (K2P) with the MEGA5 software package (TAMURA et al., 2011).A total of 1000 bootstrap replicates were performed.
To prepare the fungal suspensions, the isolates were removed from the Petri dishes (9 cm) with a scalpel, and then suspended and agitated (vortex, 2 min.) in a sterile solution (distilled water and 0.01% Tween).A Neubauer chamber was used to obtain a concentration of 1 × 10 8 con/ml.The viability of the conidia was assessed by inoculating the suspensions in Petri dishes containing PDA (39 g/l, DIFCO  ) and YE (1%, MERC  ) and counting the numbers of germinated and ungerminated conidia (ALVES, 1998;FERNANDES et al., 2009).

Disk diffusion test in artificial solid medium
Ten microliters of fungal suspension at a concentration of 1 × 10 8 con/ml, from the three isolates (CG 138, CG 228 and ESALQ 986) were spread evenly with a sterile swab in Petri dishes containing PDA (39 g/l) and YE (1%).Similarly to the method used by Hunt (1986) and Urbanek et al. (2012), three filter paper disks (6 mm in diameter) were autoclaved (120 °C for 20 min) and dried in a Pasteur oven (100 °C), and then were immersed in the macerate of the larvae.After the procedure, the disks were placed in dishes cultured with the fungal suspension.The same procedure was carried out with the mucus samples.These dishes (three dishes/isolate) were incubated for three days at 25 ± 1 °C and 70-80% RH.The fungal inhibition zone was verified on the second and third days after the start of the experiment.The assays were carried out twice to obtain more reliable results.

Results
Similarity analyses based on the 16S rRNA sequence (~1,450 bp), on a representative strain, revealed that these isolates belonging to the genus Stenotrophomonas were more closely related to the S. maltophilia strain K279a (accession number AM743169.1).Phylogenetic analysis based on the 16S rRNA gene of a representative isolate was compared with the most closely related bacterial strains retrieved from the database, and these confirmed the result (Figure 1).
The macroscopic characteristics of the bacterial colony were: regular borders, brightness, light brown color, smoothness and convex and mucoid appearance (Figure 2a).According to the microscopic characteristics, the bacterium was Gram-negative, with a bacillary straight or slightly curved form.The catalase test was positive.Bacillary bacteria and bacterial ultrastructures such as a long polar flagellum and microfibers were also viewed when the larval surface was observed using SEM (Figure 2b).
The disk diffusion test (Figure 2c) showed bacterial growth on all the filter paper disks.The bacteria grown on the disks had the same macro characteristics as observed in the BHI Petri dish colonies (Figure 2a).An inhibition zone was formed around the disk in the dishes cultured with the three fungal isolates (Figure 2c).Disks immersed in larval mucus or immersed in macerated larvae all presented the inhibition zone.The conidial suspensions used in the present study had 100% viability.

Discussion
Molecular biology-based techniques provide rapid and precise bacterial identification, while traditional techniques based on morphological and physiological characteristics are inconclusive (VIDEIRA et al., 2004).For this reason, PCR was used to identify the bacterium.Furthermore, SEM revealed bacterial ultrastructures (Figure 1) similar to those described by others in identifying S. maltophilia (OLIVEIRA-GARCIA et al., 2002).
According to Konemman et al. (2001) and Almeida et al. (2005), S. maltophilia can be obtained from different sources.Oliveira et al. (2000) identified it in the microbiota from the digestive tract of female Lutzomyia longipalpis flies and Chansang et al. (2010) isolated the same species on the surface of Siphunculina funicola flies.However, there are no reports of this species as part of the S. calcitrans larval microbiota, and therefore the present report can be considered to be its first description in this host.
The metabolic versatility of S. maltophilia (RYAN et al., 2009) allows it to colonize human urine (KONEMMAN et al., 2001), canine urine (KRALOVA-KOVARIKOVA et al., 2012), crocodile kidney (septicemia) (HARRIS; ROGERS, 2001) and buffalo liver (PETRIDOU et al., 2010).However, there is no description of its presence in the urine or feces of cattle, horses or other production animals.Accordingly, the presence of S. maltophilia in plants, soil and water (ALMEIDA et al., 2005), and in urine and fecal materials, may be one of the reasons that makes it part of the microbiota of S. calcitrans larvae, since the immature stages of this fly develop in moist contaminated environments comprised of a mixture of urine, feces, soil and decaying plant (BITTENCOURT; CASTRO, 2004;BITTENCOURT, 2012).
In the literature, these microorganisms' ability to produce substances that inhibit growth and affect competing agents has been reported (DILLON; DILLON, 2004).Some metabolites synthesized by S. maltophilia, such as pyrrolnitrin and maltophilia, allow this bacterium to survive in polymicrobial niches (KERR, 1996;ALMEIDA et al., 2005).Kerr (1996) 2009) reported that S. maltophilia can be used for biological control of phytopathogenic fungi such as Fusarium graminearum and Verticilium dahliae.The disk diffusion test (Figure 1c) aimed to demonstrate that the bacterium naturally obtained from the mucus and macerate of S. calcitrans larvae has antifungal activity against three B. bassiana s.l.isolates.This result suggests that the larval microbiota/microenvironment is a factor that can affect the use of B. bassiana s.l.fungus for biological control of S. calcitrans larvae.
S. maltophilia was identified in S. calcitrans larvae and larval mucus.This bacterium has antifungal activity against B. bassiana s.l.CG 138, CG 228 and ESALQ 986 isolates.Knowledge of the microbiota of S. calcitrans larvae is of considerable importance for integrated pest management in which entomopathogenic fungi are applied, since the substances produced by these bacteria can disrupt them.Accordingly, studies are required in order to select tolerant and effective fungal isolates for biological control of the immature stages of S. calcitrans.

Figure 1 .
Figure 1.Neighbor-joining phylogenetic tree based on partial 16S rRNA gene sequences from the Brazilian isolate and reference strains of Stenotrophomonas and other related bacteria.The database accession numbers are indicated after the bacterial names.Bootstrap analyses were performed with 1000 cycles.The dendrogram was constructed using the MEGA4 program, and the clustering was carried out using the neighbor-joining method.Bootstrap values of 450% are indicated at nodes.Scale bar = 0.002 substitutions per nucleotide position.