Studies on Possible Modes of Action and Tolerance to Environmental Stress Conditions of Different Biocontrol Agents of Foliar Diseases in Maize

The present study evaluates the possible modes of action of antagonistic bacteria and their tolerance to UV radiation, temperature and osmotic stress. The partial 16S-23S rRNA gene sequencing of eight antagonistic bacteria had a high match with three bacterial genera: Curtobacterium, Pantoea and Bacillus. In this study, the three Bacillus isolates showed the most relevant production of enzymes, volatile organic compounds and antibiosis against Exserohilum turcicum. Respect to UV radiation and temperature Pantoea and Bacillus isolates were more tolerant; whereas the eight isolates were tolerant to osmotic stress in varying degree. The three Bacillus isolates have the greatest potential as biocontrol agents for foliar diseases in maize. The antagonistic action could be explained through different modes of action such as enzymes, volatile organic compounds and/or direct antibiosis by other secondary metabolites. Bacillus isolates tolerance to environmental stresses including UV radiation, temperature and osmotic stress is relevant for survival and persistence on the leaf surface. This work provides new information about the mode of action of antagonistic bacteria with proven efficacy against maize leaf pathogens. In addition, it provides information about the tolerance of antagonistic bacteria against different stress conditions. The data of the present study could contribute to the development of a successful foliar biofungicide.


Introduction
Maize (Zea mays L.) is one of the most important crops in Argentina. During the 2017/2018 growing season the sowing area was increased by 35% reaching 5.4 million hectares [1]. Among the factors that limit yield, two foliar diseases have become important in recent years. Common rust is an endemic disease in maize growing area caused by Puccinia sorghi, occurring all seasons with varying degrees of intensity according to hybrid susceptibility, pathogen biotypes and environmental conditions during the crop cycle [2]. On the other hand, Northern Corn Leaf Blight (NCLB) caused by Exserohilum turcicum, is a foliar disease in which the fungus destroys foliage causing a decrease in yield that varies from 28% to 91% [3].
Fungicides based on strobilurin + triazoles are the most effective method to control foliar diseases [4]. However, the dependence on chemical control has caused undesirable side effects such as food contamination, environmental dispersal and higher production costs [5]. Consequently, biological control is presented as a viable alternative to achieve the management of pests in various crops.
Biological control is defined as an "environmentally-friendly" strategy using microorganisms or their derivatives to reduce a targeted pathogen. Furthermore, the narrow spectrum of biological control agents (BCAs) should provide an optimum efficacy without affecting non-targeted organisms [6]. The action of the microbial antagonists can result from direct (parasitism, antibiosis or competitions) or indirect ecological interactions (induction of resistance). The different mechanisms of action are probably never mutually exclusive. It is known that a successful BCA has several mechanisms of action capable of working synergistically [7].
The development of new BCAs is a stepwise approach which begins with the screening of a large number of potential antagonists. Reservoirs for such microorganisms are broad although the screening of antagonists directly isolated from the host plant and surrounding environment seems more appropriate [8]. Taking into account that both E. turcicum and P. sorghi are foliar pathogens, previous work in our laboratory was carried out to evaluate the epiphytic bacterial community that can share the niche with pathogens in similar stages. In the pre-selection, antagonistic interactions bacteria/pathogens were evaluated over competition for nutrients and reduction effect on growth parameters [9]. Later, the effectiveness of bacterial antagonists in reducing the disease severity was evaluated in greenhouse assays [10] and field trials [11].
The phylloplane is a source of bacterial antagonists much less used than the rhizosphere. In this environment, the bacteria population may be exposed to one or more stresses such as fluctuating temperature and UV radiation [12]. On the other side, changes in water potential may present the most difficult challenge to bacterial survival. Reductions in osmotic potential can eventually lead to bacterial desiccation [13]. Our working hypothesis is that a better knowledge of the

Biological Control Agents (BCAs)
Eight potential BCAs isolated from maize leaves from fields located in Córdoba province, Argentina, were selected based on antagonistic ability evaluated in a previous study [9]. The cultures are deposited in the culture collection of the

Detection of Protease
To determine the protease production, 100 µl of suspensions of the antagonist bacteria were plates on LB agar (10 g tryptone, 5 g yeast extract, 10 g NaCL, 12 g agar and 950 ml distilled water) containing 3% skim milk. Plates were incubated at 25˚C for 4 days. The formation of a transparent halo around the bacterial colonies was classified as positive [17].

Detection of β-1,3-Glucanase
The quantification of enzymatic activity was evaluated following the microplate method described by Zheng and Wozniak [18] with some modification. Starting ter bath, preheated to approximately 99˚C for 10 min. Finally, it was cooled on ice and the absorbance at 450 nm was read. The standard glucose curve was prepared using concentrations between 2 -800 μM in duplicate, placing 30 μl in each well, following the same procedure described above.
From this standard curve, the net OD 450nm of an assay sample was converted to the amount of reducing sugar released by β-1,3-glucanase from the substrate laminarin. The net OD 450nm was calculated by subtracting the average OD 450nm of the duplicate substrate-control wells.

Production of Volatile Compounds
The antagonistic bacterial strains were streaked on trypticase soy agar plates

Characterization of Bacterial Volatiles Compounds by Gas Chromatography-Mass Spectrometry
Solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) were performed using the methodology developed by Khabbaz et al. [17].

Detection of Antibiotic Compounds Production
The technique developed by Bautista-Rosales et al. [19] for yeasts was adapted to evaluate mechanisms of antibiosis in the bacterial strains. The selected antago-

Heat Shock Survival of Bacterial Cells
The bacterial cells were obtained from 150 ml of TSB medium inoculated and incubated on a rotary shaker (140 rpm) at 25˚C. In order to choose a suitable temperature treatment for heat shock induction of tolerance, when cultures reached the stationary phase of growth, the cells were harvested by centrifugation at 7.000 g for 10 min at 10˚C. Then the cells were re-suspended in 2 mL of phosphate-buffered solution (PBS), and cell suspensions incubated in a water bath at 45˚C for 30 min. To determine the survival rates after heat shock, the cell paste was serially diluted in PBS. An inoculum of 100 µl of each bacterial suspension was spread onto TSA medium. A new count of viable cells was done after 24 h of incubation at 25˚C. Three replicates per treatment were used and repeated twice [21]. Survival at 45˚C was expressed as logarithmic values of N and N 0 , where N refers to the bacteria count following exposure to heat shock, and

Sensitivity to Osmotic Stress
The bacterial cells were grown on TSA medium osmotically modified by adding different amount of ionic solute (NaCl) and non-ionic solutes (glycerol and glucose) [22]. The water potential (Ψ) of the unmodified medium was −1.38 MPa, and it was selected as control treatment. The Ψ of TSA medium was adjusted to

Statistical Analysis
Data analyses were performed by analysis of variance (ANOVA) using InfoStat version 2013 [23]. Mean separation and comparisons for survival to UV and sensitivity to osmotic stress were made by Duncan test at a probability level of p < 0.05 and p < 0.01, respectively. Mean separation and comparison for heat shock survival were made using a DGC Test (p < 0.01).

Identification of BCAs
Analysis of 16S-23S rRNA sequences of antagonistic bacteria had a high match with three bacterial genera: Curtobacterium, Pantoea and Bacillus (Table 1). In the isolates identified molecularly as belonging to the genus Bacillus, the identification was complemented with a biochemical characterization. The API system showed that two Bacillus isolates EM-A7 and EM-A8 have a high similarity index with B. subtilis while the Bacillus EM-A6 showed a test with weak discrimination among Bacillus species. Given the complexity in the taxonomy of this genus, it is necessary in the future to carry out a phylogenomic analysis that allows us to confirm the identification of these BCAs.

Detection of Hydrolytic Enzymes
All antagonist bacteria showed no chitinolytic capacity in the chitin-based medium. When determining the proteolytic enzymes, the three Bacillus isolates demonstrated a high protease activity on LB agar supplemented with 3% skim milk. Curtobacteruim isolates showed small haloes around bacterial colonies indicating a slight capacity protease, while the Pantoea isolates did not show proteolytic activity ( Table 2). On the other side, all bacteria showed ability to digest glucan. Table 2 shows the glucose released using laminarin as a substrate.

Determination of Antibiosis
The production of volatile compounds with antibiosis capacity on E. turcicum growth in sealed double plate assays determined that all isolates were producers of volatile compounds. Pantoea EM-A4 showed the highest production of volatile compounds, followed by Pantoea EM-A5 and Bacillus EM-A6 and EM-A8 (Table 2). Gas chromatography-mass spectrometry analysis of extracted volatile compounds identified 16 volatile organic compounds. As shown in Table 3, Pantoea EM-A4 produced the highest amount of volatile compounds (8 compounds), including the production of indole and tetrhahydrofuran-2-propil. The Bacillus EM-A7 and EM-A8 release high levels of silanediol, dimethyl, and cyclopentasiloxane, decamethyl, respetively. On the other hand, during evaluating the production of antibiotic compounds, it was determined that all antagonists were not producers of non-peptidic antibiotics (filtrated and sterilized extracts). However, when the inoculation was performed using viable cells, the three Bacillus isolates showed a total inhibition of E. turcicum growth. The two Pantoea isolates allowed a slight growth of the pathogen and the three Curtobacterium isolates did not produce antibiotic compounds (Table 2). Bacterial growth of BCAs before and after heat shock treatment is shown in

Discussion
Phyllosphere microbioma could contribute to the health of plant species through surface protection against pathogens [12]. In the present study, eight epiphytic bacteria previously evaluated in greenhouse and field trials experiments against foliar fungal pathogens [10] [11], were characterized for hydrolytic enzymes production, volatile compounds and antibiosis and tolerance to UV radiation, temperature and osmotic stress.
The three Curtobacterium isolates showed the lowest production of metabolites and enzymes, with a slight production of volatile compounds with antibiosis action. These isolates were selected in previous studies because they showed to decrease the growth rate of E. turcicum and one of them had shown inhibition at distance in dominance tests [9]. Regarding Pantoea EM-A4 and EM-A5, both isolates showed significant inhibition of E. turcicum in sealed double plate assays suggesting the action of volatile compounds that could play a vital role in pathogen inhibition [24]. The GC-MS profile indicated that Pantoea EM-A4 produced eight compounds while Pantoea spp. EM-A5 produced five detectable compounds, being in both isolates the indole the most relevant compound. Indole controls diverse aspects of bacterial physiology, such as spore formation, plasmid stability, drug resistance, biofilm formation and virulence in indole-producing bacteria [25] [26]. By the other hand, Pantoea isolates did not produce proteolytic and chitinolytic enzymes, and a low production of β-1,3-glucanase was detected. Also antibiotics production was slight. These results suggest that the possible mode of Bacillus isolates showed a relevant production of enzymes, volatile organic compounds and antibiosis. The three Bacillus isolates showed a relevant protease production, standing out Bacillus EM-A6 and EM-A8 and β-1,3-glucanase activity, one of the enzymes responsible for lysis and degradation of cell wall and sclerotium wall in fungi [30]. Production of extracellular lytic enzymes such proteases and β-1,3-glucanase by bacteria might contribute to their antagonistic mechanism, which in many situations may partially explain the biological control of plant disease [31]. Also a significant production of volatile compounds such as silanediol, dimethyl cyclopentaxiloxane and decamethyl was determined in Bacillus isolates. Diverse authors have also reported the volatiles compound production by different Bacillus species and their effect on mycelial growth, spore germination and tube elongation in different fungal pathogens [28] [32].
In addition the three Bacillus isolates also showed in vitro antibiosis against E. turcicum, generating in many cases a total growth inhibition. Members of this genus produce several biologically active molecules with antimicrobial activity such as polyketides and cyclic lipopeptides [33] [34]. Further studies are necessary to analyse the gene clusters encoding for the biosynthesis of secondary metabolites with antifungal activity in these BCAs. This is relevant in the Bacillus EM-A8 that caused the greatest effect on reducing the severity of northern leaf blight in previous field studies [10] [11].
The leaf surface is a continuously fluctuating physical environment and their resident microbes are subjected to changes in the microclimate. Adaptations conferring tolerance to stress may be critical to survival of epiphytic bacteria used as BCA on phyllosphere conditions. A tolerance strategy requires the ability to tolerate direct exposure to environmental stresses on leaf surfaces, including UV radiation and low water availability [13]. In our study, Curtobacterium isolates with a strong pigmentation were found to be the most sensitive to UV radiation. This is in contrast with previous studies which showed that organisms resistant to radiation are usually intensively pigmented; containing UV-absorbing compounds [20]. By the other side, Pantoea and Bacillus isolates showed higher resistance to UV radiation than Curtobacterium isolates in spite of differences in pigmentation. In the present study epiphytic bacteria were exposed to heat shock under temperature condition that can occur on phyllosphere in summer, during the period that maize is susceptible to be infected by the pathogen. Once again Pantoea and Bacillus isolates were more tolerant to heat shock than Curtobacterium isolates. In a previous study we demonstrated that osmotic tolerance of B. amyloliquefaciens helps to maintain survival population after heat shock [21]. The reduction in free water on leaf surface may cause increases in the concentration of solutes, resulting in osmolarities that may be sufficient high to damage bacte- with glucose and NaCl generated flat and small colonies. In this sense, the acclimatization of BCAs using glycerol could be help to improve the protection on leaf surface because it stimulates the production of extracellular polysaccharides (EPS).

Conclusion
The present work complements previous studies performed in the greenhouse and field trials and shows that the three Bacillus isolates have the greatest potential as BCAs for foliar diseases in maize. Bacillus isolates showed a good enzymatic production, synthesis of volatile organic compounds and direct antibiosis towards E. turcicum. Further studies are necessary to analyse the gene clusters encoding for the biosynthesis of secondary metabolites associated with the antifungal activity. In addition, these isolates showed tolerance to environmental stresses including UV radiation, temperature and osmotic stress which are important factors for survival on the leaf surface and their persistence in future foliar applications. Furthermore, the information obtained about tolerance to different stress conditions will be useful to select the type and formulation process in order to obtain an effective product for biocontrol of foliar disease.

Conflicts of Interest
The authors declare that they have no conflict of interest.