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Functional genomics analysis of a phyllospheric Pseudomonas spp with potential for biological control against coffee rust

Background Pseudomonas spp. promotes plant growth and colonizes a wide range of environments. During the annotation of a Coffea arabica ESTs database, we detected a considerable number of contaminant Pseudomonas sequences, specially associated with leaves. The genome of a Pseudomonas isolated from coffee leaves was sequenced to investigate in silico information that could offer insights about bacterial adaptation to coffee phyllosphere. In parallel, several experiments were performed to confirm certain physiological characteristics that could be associated with phyllospheric behavior. Finally, in vivo and in vitro experiments were carried out to verify whether this isolate could serve as a biocontrol agent against coffee rust and how the isolate could act against the infection. Results The isolate showed several genes that are associated with resistance to environmental stresses, such as genes encoding heat/cold shock proteins, antioxidant enzymes, carbon starvation proteins, proteins that control osmotic balance and biofilm formation. There was an increase of exopolysaccharides synthesis in response to osmotic stress, which may protect cells from dessication on phyllosphere. Metabolic pathways for degradation and incorporation into citrate cycle of phenolic compounds present in coffee were found, and experimentally confirmed. In addition, MN1F was found to be highly tolerant to caffeine. The experiments of biocontrol against coffee leaf rust showed that the isolate can control the progress of the disease, most likely through competition for resources. Conclusion Genomic analysis and experimental data suggest that there are adaptations of this Pseudomonas to live in association with coffee leaves and to act as a biocontrol agent. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-022-02637-4.


Background
Coffee (Coffea arabica L) is one of the most consumed brewed drink in the world, being one of the most valued commodities on the market [1]. The coffee production chain has a great socio-economic impact, moving around US$ 90 billion a year and involving around 500 million people worldwide, including producers, rural workers, traders and coffee producers [2]. Although more than 100 species of the genus Coffea have been described, only Coffea arabica and Coffea canephora are effectively cultivated and commercialized. C. arabica is an autogamous allotetraploid plant, the result of Open Access *Correspondence: jorge.mondego@sp.gov.br; jmcmondego@gmail.com de Sousa et al. BMC Microbiology (2022) 22:222 a recent cross between C. canephora, or related species, and C. eugenioides [3]. Such autogamy leads to a narrow genetic basis, aggravated by the fact that most of the C. arabica planted today have only two varieties as base populations, Typica and Bourbon [4]. Because of this narrow genetic basis, C. arabica plantations are quite susceptible to diseases, especially coffee rust.,caused by the biotrophic fungus Hemileia vastatrix [5]. Traditionally, disease control is based on copper-based pesticides, but this practice has been restricted by the new environmental guidelines [6]. Thus, biological control has been a good alternative for the control of coffee rust, especially in organic cultivation [7]. Bacteria of the genus Pseudomonas comprise a diverse group of microorganisms that can be isolated from different environments including fresh water, salt water, soil, internal tissues of plants, and food [8]. Investigation about Pseudomonas spp. associated with plants leads to great interest because of their benefits on plant host by producing auxins and siderophores, which improve plant growth and development by increasing yield, mediating resistance against other pathogens and providing competitive advantages [9]. As a result, these bacteria have been recognized as potential tools for the development of biofertilizers and biocontrol products for agriculture [10].
Pseudomonas have been detected in coffee tissues [11]. Moreover, during annotation of the coffee expressed sequence tag (EST) database for the Brazilian Coffee Genome Project (BCGP; [4]), Coffea arabica was found to contain a large amount of 'contaminant' bacterial sequences on the surface of leaves, most of which belonged to Pseudomonas spp.. In this study, we report a new biocontrol strain of Pseudomonas (Pseudomonas sp. MN1F), which was isolated from coffee leaves, and analyzed experimental and genomic data through two perspectives: 1) the adaptive mechanisms that allow survival on the leaf surface; 2) the possible effect as a biocontrol agent against coffee rust.

Identification and genomic information
The isolate named Pseudomonas sp. MN1F was confirmed as belonging to Pseudomonas genus with proximity to Pseudomonas putida and Pseudomonas monteilii ( Figure S1). Bacterial sequencing was performed using Illumina technology. After trimming and assembly, genome length was estimated at 6 Mbp, consistent with other Pseudomonas sequenced genomes [12,13]. C + G content, number of coding sequences and number of tRNAs were 62,9%, 5.575 and 45, respectively. Global genomes statisitics are depicted in Table 1. Forty-eight insertion sequences (IS) were found, 34 of which were similar to Pseudomonas syringae sequences (Table S1).
In order to evaluate whether MN1F uses coffee molecules as energy sources, genes that are involved in the degradation of phytocompounds were searched. MN1F possess the genetic machinery required for the degradation of benzoate, quinate, cinnamate, and salicylate; phenolics commonly found in coffee plants [15][16][17]. The interconnection between these degradation systems culminates with the formation of acetyl-CoA, a precursor for citrate synthesis (Fig. 1). The degradation pathways of benzoate and salicylate converge on the conversion into catechol by BenD and SH enzymes, respectively. The quinate pathway converges on the benzoate pathway through the formation of 3-oxoadipate-enol-lactone, but the reverse may also occur when  (Fig. 2).

EPS production and caffeine resistance
Exopolysaccharides (EPS) are involved in regulating mechanisms that promote tolerance to desiccation in bacteria and fungi [18]. Based on the fact that leaf surface is an environment that is poor in water [19,20], EPS production at increasing osmotic pressure was measured during MN1F growth at minimal media plus PEG. MN1F responded to induced drought stress by producing large amounts of EPS ( Fig. 3; dark grey, > 10 g/L at high PEG concentrations). After extensive genome annotation, no genes were found for caffeine degradation. To evaluate whether MN1F could incorporate caffeine by a non-canonical pathway, we grew bacteria in media with glucose (0-2 g) and NH 4 Cl (0-8 g) as carbon and nitrogen primary sources, respectively. The intention was to check whether MN1F induce cells to "replace" glucose and NH 4 Cl with caffeine. MN1F was not able to grow in media containing caffeine in low concentrations of caffeine (Fig. S2).
We evaluated MN1F resistance to high concentrations of caffeine (Fig. 4). Different caffeine concentrations were used (0.8, 2, 3, 6, 10, 15 g/mL) and measured bacterial growth after 8 h of incubation. MN1F was more resistant to caffeine than E. coli control (Fig. 4), indicating some degree of resistance to caffeine.

Biocontrol against coffee leaf rust
The presence of Type I and VI secretion systems in MN1F gave us a hint that this bacteria could be used as   In vivo antagonism was verified by calculating the H. vastatrix incidence in response to MN1F application (Fig. 6a). The treatments with the fungicide azoxistrobin + cyproconazole (Fungi), the resistance inducer acibenzolar-S-methyl (ASM) and MN1F inoculant showed a positive effect in delaying the progress of infection, since all had a lower incidence than control plants. MN1F treatments ANTP (antagonism), INDP (induction) and INDP/ANTP (antagonism and induction) showed a final effect similar to the fungicide and ASM. However, after the final measurement, the application of MN1F few minutes before infection (ANTP) showed the best result of all treatment with an incidence of 12%, a better result than chemical treatments with 18% of incidence. Treatment with application two days and few minutes before infection (INDP/ANTP) proved to be inferior (around 18% of incidence) to ANTP alone. In addition. treatment with MN1F also reduced the average number of lesions per plant by about fourfold (Fig. 6b) compared to the untreated control, but there was no significant difference (p > 0.05) between treatment with MN1F and the other treatments (with BION and fungicide).
The MN1F strain showed ability to inhibit 40% of uredospores germination, when applied as a solution in the same concentration of in vitro conditions (Fig. 7). Therefore, this effect over the germination, which is essential for the infection process, may be related with the control of the disease in the coffee seedlings in the treatment ANTP. However, because the coffee seedlings treated with MN1F showed lower incidences of coffee rust until the last evaluation (54 days after the inoculation), probably other mechanisms of control, such as induction of resistance, may also be acting in the biocontrol of the disease.

Discussion
The phyllosphere is reported to be a harsh environment to colonize because it imposes numerous abiotic stresses on the microbial community: variations in temperature, low availability of nutrients and water, the effect of winds and rain and UV radiation [19,20]. Taking it into account, we searched for genes in the genome of MN1F that would help to explain its leaf surface colonizer behavior, especially with respect to tolerance to abiotic stresses.
For instance, genome analysis indicated that MN1F has several heat-shock and cold-shock proteins, which are consistent with an environment where the temperature can abruptly change [20]. Genes related to osmotic/ drought stress were also found, either by controlling the proline/betaine balance [21] or by controlling the accumulation of glycerol that helps with environmental osmo-adjustments [22]. In addition, the presence of Aquaporin Z, which also acts in freezing tolerance [23], suggests that MN1F had several means of dealing with desiccation. This perception was confirmed in PEG-induced drought stress experiment (Fig. 3), where even at very high concentrations of PEG6000, there was still bacterial presence. EPS synthesis would also assist the biofilm production [24], as well as fimbriae synthesis [25]. The bacterial extracellular matrix built in certain regions of the leaf is crucial to establishment of microbial communities, where the biofilm would serve not only for protection, but also to maximize the search for resources [19,20,26].
Another characteristic of MN1F is the degradation network of phenolic compounds that leads to the citrate cycle ( Figs. 1 and 2). It is noteworth that some of these compounds have antimicrobial activity, especially salicylate, which mediates the immune response of plants [27]. Therefore, the degradation and incorporation pathways of these phenolics would not only serve as carbon sources in an environment naturally poor in resources [19,20], but also as protection against the harmful effects of the compounds. In fact, phenolic compounds produced by leaf tissue help to regulate microbial community [27].
Caffeine is a compound abundant in coffee leaves. Although no genes were found for degradation of caffeine, and growth experiments verified no use of caffeine as carbon and nitrogen sources ( Figure S2), MN1F has a high tolerance to caffeine (Fig. 4). This data suggests that MN1F has another tolerance mechanism instead of caffeine degradation. It is possible that MN1F has a potential caffeine extrusion mechanism. This high tolerance may have been selected by the leaf environment rich in caffeine, which would give MN1F an advantage to colonize leaves of the coffee tree.
Pseudomonas was previously reported as a biocontrol agent for coffee rust [6,28]. In this work, we showed that MN1F decreased the incidence and severity of coffee rust under laboratory and greenhouse conditions. In fact, in the greenhouse experiments, inoculation with MN1F proved even better than the fungicide in terms of incidence when the bacteria inoculation was done before the inoculation of the pathogen. The loss of effect of the fungicide is a phenomenon observed in coffee crops due to the selection of resistant H. vastatrix strains [29]; however, in our experiment we observed that on the 40 th and 47 th days after the inoculation, the fungicide managed to significantly reduce the damage by the pathogen. From day 47 to day 54 there was a decrease in effectiveness, something that also happened with resistant inductor ASM; although the mechanisms of action of the two products are different. It is suggested that over time, chemical compounds lose their effectiveness, requiring a new application; one of the problems of chemical pest control [30]. On the other hand, in all treatments with MN1F there was a delay in the evolution of the symptoms. The application of the biocontrol agent before the fungus infection (ANTP) was the treatment that gave the most positive results, perhaps because there is an immediate antagonistic effect on the germination of uredospores, hindering the subsequent establishment of the pathogen [6,28]. This antagonism may be due to the production of some anti-fungal compounds or the rapid occupation of leaf sites for the establishment of bacteria, making it difficult for the fungus to compete for resources [20]. However, the experiment with the application of the filtered supernatant showed that there was no significant effect on H. vastratrix infection, which corroborates with the analysis of the genome that predicts that MN1F does not produce anti-fungal compounds as seen in other biocontrol agents. Curiosly, this data is in contrast to Haddad et al., [31], which found that P. putida P286 supernatant was equally effective than the bacterial cells at reducing the H. vastatrix uredospores germination, most likely through the action of antifungal compounds. Nevertheless, our results suggest that MN1F biocontrol mechanisms are dependent on the direct bacteria/fungus interaction.
We suggest that competition for resources may be one of these mechanisms as well seen for rhizosphere bacteria [32,33]. Another possibility to be considered concerns the type VI secretion system (T6SS). It is known that this system is linked not only to the virulence of pathogens, but also in the colonization of commensal and beneficial bacteria [34]. It has been discovered that bacteria can use T6SS to deliver effectors with antifungal activity [35]. MN1F has T6SS and also five copies for RHS proteins that can have exotoxin activity in both eukaryotic and prokaryotic targets using T6SS as delivery system [36]. We suggest that, given the need for direct interaction between bacteria and fungi for biocontrol in this model, MN1F could use T6SS as a delivery system for RHS toxins as seen in others Pseudomonas with biocontrol activity [37].
Pre-applications of MN1F with two days before H. vastatrix infection (INDP) showed good results, but less effective than ANTP. Interestingly when applied before and after infection (ANTP + INDP) the effect is worse when compared to ANTP. We suggest that there may be a competition mechanism between the first population (coming from the first application) and the second population (coming from the second application). In fact, in the rhizosphere the establishment of several different populations of bacteria can spoil the intended biocontrol action [38]. This behavior, added to the fact that the time of application influences the use of MN1F, must be taken into account when planning the application of the bacteria to improve its biocontrol ability.

Conclusions
Genome analysis and physiological tests showed that Pseudomonas sp. MN1F has characteristics consistent with a phyllosphere bacteria, being probably a constant and adapted resident on the coffee leaf. MN1F also showed antagonism against H. vastatrix, what indicates this bacterial isolate as a potential biocontrol tool for coffee rust disease.

Isolation of Pseudomonas spp. MN1F
Leaves of a six-month-old C.  [39]. The plates were then incubated at 28ºC and the growth of individual fluorescent colonies characteristic of Pseudomonas was monitored within 48 h after plating. A colony was isolated and named as MN1F. Bacterial identification was performed using TYGS program (https:// tygs. dsmz. de/), which digitally performs a DNA-DNA hybridization and calculates/compares the G + C contents between the studied genome and those deposited in the NCBI [40].

Genome sequencing, annotation and functional analysis
DNA from MN1F was extracted with Trizol ® reagent (Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer's specifications. DNA was sequenced using the Illumina MiSeq platform at the Carolina Center for Genomic Sciences (CCGS) at the University of North Carolina (UNC), Chapel Hill. Following quality check, 300-bp PE libraries were produced to determine the orientation and relative position of the contigs produced by sequencing. The libraries were constructed using the TruSeq sample preparation kit (Illumina). The assembly of the reads was carried out with Velvet assembler [41]. Phylogenomics was performed using the Type Strain Genome Server-TYGS program (https:// tygs. dsmz. de/) [39]. Gene prediction was completed using GeneMark and Glimmer [42,43]. The functional and comparative annotation of bacterial genomes was performed using the RAST program according to the developers' specifications [44]. Metabolic maps were drawn based on the KEGG tool [45]. Mobile elements were identified using ISFinder (https:// www-is. bioto ul. fr/) according to Siguier et al. (2006) [46]. The potential production of secondary metabolites was examined using antiSMASH 2.0 software according to Blin et al. (2013) [14].

EPS production and caffeine tolerance
To examine EPS (exopolysaccharide) production in response to water stress, bacteria (MN1F and E. coli as a control) were grown in liquid King

Degradation and incorporation of phenolics
Tests for degradation of aromatic phytochemicals, which are commonly found in coffee such as benzoate, quinate, cinnamate and salicylate were carried out. The first round of growth was carried out in solid medium with the following composition: 0.2 g/L K 2 HPO 4 ; 1 g/L NaCl; 0.15 g/L CaCl 2 2H 2 O,; 0.4 g/L MgCl 2 6H 2 O,; 0.5 g/L NH 4 Cl,; 0.5 g/L KCl,; 0.5 g/L cysteine-HCl,; 0.5 g/L yeast extract,; 5 mM cinnamate or benzoate or salicylate or quinate as the primary source of carbon and agar. A second round of cultivation, now in a liquid medium, was carried out under the same conditions described previously with 5 mM cinnamate or benzoate or salicylate or quinate, which would be degraded as the acetate concentration increased. Phytocompounds were measured with the HPLC analytical column (Agillent) Waters µBondapak C18 (3.9 × 300 mm, 10 µm) and detected in an API-5000 Triple Quadruple mass analyzer. The ESI (-)-MS was performed at adjusted voltages of -4000 V and 70 V.

Preparation of the microorganisms for biocontrol essays
MN1F was grown in 250 mL of LB medium at 28 °C during 12 h. The inoculum was slightly centrifuged to allow the pellet to be collected without destroying the cells and resuspended in saline (0.9% NaCl) until it reached a concentration of 10 8 cells per ml. This suspension was used for spray on the leaves. The H. vastarix uredospores were collected by scraping the abaxial region of the leaves one day before the experiment. The uredospores were packed in closed eppendorfs and placed in the refrigerator until the experiment. The final concentration of the inoculant was 0.02 g of uredospores per liter of saline (0.02 g/L).

In laboratory verification of antagonistic potential and effect of extracelular metabolites on infection progress
For Twenty-five uL of MN1F, uredospores, saline and supernatant were applied to the abaxial part of the discs that were incubated in the dark for 24 h. After this period, the plates were left incubated in 12 h of photoperiod, 500-1000 lx, 22 ± 2 °C, and approximately 100% relative humidity. Thirty days later, the severity of the lesions (measured by the percentage of injured area) was evaluated using images of leaves digitized by a digital camera and estimated using Téllez et al., approach [48]. The experimental design was randomized blocks (n = 3), each replicate consisting of three leaves.

Greenhouse antagonism evaluation
After checking the antagonism in detached leaves, plants of C. arabica IAC 62 of 6 months old, were used for greenhouse experiments. Six treatments were carried out: Control-Infected plants without treatment. ASM-Acibenzolar-S-methyl (plant defense inducer analogous to salicylic acid) formulated as BION 500 WG ® containing 50 g/kg of active ingredient (Syngenta). Plants were sprayed two days before H. vastatrix inoculation with a 0.01 g/L solution of ASM.. Fungi-Fungicide Priori Xtra (containing 200 g/L of azoxystrobin and 80 g/L of cyproconazol). Plants were sprayed two days before H. vastatrix inoculation,with a 1.25 ml of commercial product per liter of water. INDP-Plants inoculated with MN1F two days before H. vastatrix inoculation. ANTP-Plants inoculated with MN1F a few minutes before H. vastatrix inoculation. ANTP/INDP-Plants were inoculated two days before and a few minutes before the inoculation with MN1F.
Uredospores were inoculated by high pressure spray. Plants were placed in a dark humid chamber (near 100% humidity) for 48 h. Each treatment contained 16 plants and the first three pairs of leaves from bottom to top were evaluated at each evaluation. Three evaluations were performed after the appearance of the first symptoms at 40, 47 and 54 days after inoculation. The incidence was estimated using the formula (Number of leaves with lesions / Total number of leaves) × 100. The average number of injuries per plant was also evaluated. The trial was set up in a randomized blocks with 6 treatments, each one consisting of sixteen plants.

Effect of MN1F on uredospore germination
The suspension of bacteria and fungi used previously was used for the uredospore germination inhibition test. The substrate used was potato-dextrose-agar. The effect of the suspension of bacteria was evaluated after 8 h, in the dark and at a temperature of 24 °C. Saline solution 0.9% NaCl was used as control. Microphotographs were taken from the uredospores with a digital camera (AxioCam ICc1) mounted on a compound microscope (Carl Zeiss, Primo Star). For each treatment, 3000 uredospores were selected, and the number of germinated spores was recorded. The experiment was performed in triplicate and an analysis of variance was performed to verify if there was a difference between treatments. The percentage of inhibition of uredospore germination was calculated with the formula: I = [(C-D)/T] × 100, where I = percentage of non-germinated uredospores; C = number of germinated uredospores in the control treatment; T = number of germinated uredospores in the experimental treatment.