Stenotrophomonas indicatrix, the Novel Gammaproteobacteria Isolated from Chlorotic Streaks Symptoms on Maize Leaves and its Seed Transmission

Stenotrophomonas indicatrix, the Novel Gammaproteobacteria Isolated from Chlorotic Streaks Symptoms on Maize Leaves and its Seed Transmission | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Stenotrophomonas indicatrix, the Novel Gammaproteobacteria Isolated from Chlorotic Streaks Symptoms on Maize Leaves and its Seed Transmission Petra Aguirre-Rayo, Hilda Victoria Silva-Rojas, Leopoldo E. Mendoza-Onofre, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9078755/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Stenotrophomonas indicatrix is an aerobic, gram-negative bacterium isolated from dirty plates and ponded water, which was recently described as a novel species. To date, little is known about its presence in other substrates or environments or if it is involved with diseases in humans, plants, or animals. This bacterium is closely related to the S. maltophilia group, which has been reported to cause infections in Mexican maize race plants. Thus, this research attempts to clarify the causal agents of the chlorotic streaks on maize leaves and the twisting of the apical meristem on the Mexican “Chalqueño” race. Eighty plants exhibiting both symptoms were recovered from maize growers' fields in the Central Highland Valley of Mexico. Small portions of symptomatic leaf tissue were disinfested and placed on King's B agar medium. After 5 d, bacterial colonies developed. Overall, 30 isolates were identified by phylogenetic reconstruction of the 16S rRNA and gyrB sequences as S. indicatrix . The measures of biofilm biomass were not related to those of pathogenicity. To monitor transmission through the seed, the red fluorescent protein (RFP) marker was used to transform strains of the bacterium. The expression of the rfp gene in bacteria and seeds was visualized by confocal microscopy. The labeled S. indicatrix showed strong RFP expression. Control seeds did not show red fluorescence. At 12 and 24 h, colonization was visualized in the endosperm, at 48 h in the embryo, and at 72 h in the plumule tissue, evidencing the transmission of S. indicatrix through seeds. Therefore, producers must consider this bacterium when choosing new varieties or seeds due to the risk of transmission to subsequent generations. Bacterial disease biofilm maize pathogen molecular identification seed-transmission Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Maize ( Zea mays L.) is one of the most important cereal crops worldwide, ranking fifth with a production of 1.241 billion t. Almost all regions produce this crop, with the Americas accounting for 51.4% of global production [ 7 ]. Of the top 10 maize-producing countries worldwide, Mexico ranks fifth with a production of 23,402,006 t [ 7 ]. White corn is used for human consumption (52%), livestock feed (19%), and self-consumption (18%), with exports accounting for 6%, losses for 4%, and seed for planting accounting for 1%. Yellow corn is used for livestock feed (76%), starch production (18%), self-consumption (2%), and human consumption (2%), with losses of 2% [ 43 ]. During maize production in the field, a new symptom of chlorotic leaf stripe was recently detected in the Highland Valleys of Mexico. This symptom resembled the chlorotic streaks caused by the bacterium Pantoea agglomerans [ 26 ]. Additionally, Stenotrophomonas maltophilia strains have also been isolated from leaves with chlorotic stripes, and recently, Stenotrophomonas indicatrix was associated with this symptomatology. The genus Stenotrophomonas was first described by Palleroni and Branbury in 1993 as gen. nov. It was reclassified thanks to the confusion caused by the species Stenotrophomonas maltophilia , which was identified for the first time in 1943 with the name Bacterium booker . Later taxonomic studies designated it as Pseudomonas maltophilia in 1961 [ 16 ], Xanthomonas maltophilia in 1983 [ 46 ], and finally, it was placed in a new genus " Stenotrophomonas" in 1993 [ 33 ]. Currently, the genus Stenotrophomonas comprises 27 species considered as child taxa with a validly published and correct name ( https:/lpsn.dsmz.de/genus/stenotrophomonas ). Among those recently described are S. bentonitica [ 40 ], S. pictorum [ 30 ], S. lactitubi [ 48 ], S. indicatrix [ 48 ], S. beteli [ 28 ], S. capsici [ 35 ], S. forensis [ 28 ], S. hibiscicola [ 28 ], S. lacuserhaii [ 3 ], S. mori [ 3 ], S. pennii [ 32 ], S. pigmentata [ 22 ], S. riyadhensis [ 24 ], and S. muris [ 1 ]. The species within this genus exhibit significant adaptability, suggesting that they are engaged in a continual process of evolution and diversification. This is evidenced by the emergence of species complexes among certain Stenotrophomonas [ 13 ]. Stenotrophomonas indicatrix is a new species within the genus Stenotrophomonas , which was recently described [ 48 ] by comparison of its genome with other Stenotrophomonas , and shows obvious differences to be recognized as a new species closely related to S. maltophilia and S. lactitubi [ 8 ]. On the other hand, S. indicatrix and S. maltophilia are genetically similar. Some accessions possess a common formate dehydrogenase locus, and it is likely that one of their ancestors acquired by homologous recombination the loci of the S. maltophilia strain, thus creating a genetic divergence between the two species. So far, S. indicatrix has been isolated from dirty plates [ 48 ] and from a water sample from an oligotrophic pond in Germany [ 9 ]. However, little is known about its presence in other substrates or environments or if it is involved with diseases in humans, plants, or animals. This bacterium has been described as strictly aerobic in the form of motile, gram-negative bacilli. The colonies are bright circular, smooth, and whitish-yellow, growing at 10–37°C, with a pH of 6.0 to 9.0 [ 48 ]. Generally, little is known about the ability of S. indicatrix to form communities known as biofilms. This lifestyle enables them to be more resistant to antibiotics and adverse agents [ 17 ]. Biofilms are formed by a biopolymer matrix that provides stability and protection, which contains polysaccharides, proteins, and microbial cells adherent to one another above a living or non-living surface [ 4 , 15 ]. A significant group of biofilm-forming bacteria has been reported causing disease in monocot and dicotyledonous plants, including maize [ 44 ]. To study seed transmission, fluorescent protein marking is an effective method for investigating promoter activity, gene expression dynamics, cellular and subcellular protein localization, and the growth and development of organisms [ 50 , 52 ]. These markers can be monitored non-destructively in living cells and tissues [ 29 ]. Therefore, this research aimed to determine whether the phytopathogenic maize bacterium Stenotrophomonas indicatrix is ​​seed-transmitted using the RFP marker. The presence of plants with symptoms of chlorotic streaks or stripes has increased in the maize-producing areas of the Central High Valleys region of Mexico. This implies the need to establish management strategies to reduce the incidence of bacterial diseases as a potential agent of chlorotic areas and the risk of being transmitted through the seed. Although S. indicatrix has been associated with maize plants, little is known about its transmission. This research seeks to clarify whether this bacterium is seed-transmitted and to evaluate its biofilm-producing capacity. Materials and methods Bacterial isolation from maize leaves In the spring-summer 2022 season, 117 maize plants were sampled based on the presence of chlorotic leaf streaks and twisting of the apical meristem (Fig. 1 ). These plants were recovered from experimental maize plots and growers' fields from Tlaxcala, Puebla, Morelos, and the State of Mexico, located in the Central Highland Valleys of Mexico at an altitude between 2230 and 2250 m. A small portion of leaves was excised and washed in running water. After that, the leaves were disinfected using 1% sodium hypochlorite (w/v) (NaClO) for 1 min and then rinsed three times with sterile distilled water. Later, they were placed into sterile paper towels to eliminate the excess of water. Six individual leaf portions (3 mm 2 ) per plant were placed on King’s B agar medium with the following composition (g L − 1 ): 1.5 g of K 2 HPO 4 , 1.5 g of MgSO 4 •7H 2 O, 1.0 mL of glycerin, 20 g of peptone, and 16 g of agar [ 42 ]. Petri plates were incubated at 28°C for 5 d. Once bacterial growth was observed from leaf cuttings, a portion of each individual bacterial colony was taken with a loop and cross-streaked on Petri plates with fresh culture medium (King’s B). After 3 d of incubation, the bacterial colony forming units (CFU) were purified for Gram staining, oxidation-fermentation (OF) metabolism, KOH string test, and motility assessment. Molecular identification DNA extraction was performed from purified bacterial cultures based on the 2% CTAB (Tris-HCl 10 mM pH 8.0; EDTA•2 H 2 O 20 mM; CTAB 2%; NaCl 1.4 M) protocol [ 5 ] with the addition of sodium acetate (C 2 H 3 NaO 2 3M). The tubes containing the DNA were placed to dry at room temperature for at least 30 min, and bacterial pellets were resuspended with 50 µL of HPLC water. The DNA was verified by spectrophotometry in a NanoDrop 2000 UV-Vis (Thermo Scientific, USA). Only samples with values of the A260/A280 and A260/A230 ratios of 1.8 to 2.1 were used for PCR amplifications. The 16S rDNA amplification was performed with primers 8F (5´-AGAGTTTGATCCTGGCTCAG-3´) and 1492R (5´-GGTTACCTTGTTACGACTT-3´) that yield a fragment size of approximately 1500 base pairs [ 47 ]. The gyrB amplification was performed with primers XgyrB1F (5´-ACGAGTACAACCCGGACAA-3´) and XgyrB1R (5´-CCCATCARGGTGCTGAAGAT-3´) that yield a fragment size of approximately 850 base pairs [ 49 ]. The PCR reaction mix was prepared in a final volume of 15 µL containing 7.86 µL of HPLC water, 3 µL of 5× PCR buffer, 0.6 µL of dNTPs (20 µM of each), 0.18 µL of each primer (10 µM) (Sigma, USA), 3 µL of genomic DNA (20 ng), and 0.18 µL (2U) of Go Taq DNA polymerase (Promega, USA). The PCR reaction was carried out in a C1000 Touch thermal cycler (Bio-Rad, USA) as follows: in the case of 16S rDNA, an initial denaturation of 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 2 min, alignment at 59°C for 1 min, and extension at 72°C for 1.5 min, with a final extension of one cycle at 72°C for 5 min. In the case of gyrB , an initial denaturation of 95°C for 10 min, followed by 39 cycles of denaturation at 95°C for 30 s, alignment at 50°C for 30 s, and extension at 72°C for 36 s, with a final extension of one cycle at 72°C for 10 min. PCR products were verified by horizontal electrophoresis in 1.5% agarose gels (SeaKem, USA) stained with GelRed (Biotium, USA) and run at 88 volts for 1.5 h. The gels were visualized with the infinity imaging system in the Infinity-3026 WL/C/26MX transilluminator (Vilber Lourmat, Germany). Amplicons were cleaned with the enzyme ExoSAP-IT (Affymetrix, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The sequencing reaction was prepared in a MicroAmp Optical 96-well reaction plate using a Big Dye Terminator v3.1 kit (Applied Biosystems, USA) in a total volume of 20 µL containing 4.0 µL (2.5×) of Ready Reaction Premix, 2.0 µL (5×) of Big Dye Sequencing Buffer, 4 pM aliquots of each internal primer 518F (5’-CCAGCAGCCGCGGTAATACG-3’) and 800R (5’- CTACCAGGGTATCTAAT-3’) [ 10 ] and 2.0 µL of the template DNA. The thermocycler program was set according to the manufacturer instructions. Later, plates were washed with an EDTA/ethanol protocol. The amplicons were sequenced in both directions in a Genetic Analyzer 3130 (Applied Biosystems, Thermo Fisher Scientific, USA) at the Colegio de Postgraduados (COLPOS) facilities in Montecillo, Mexico. To obtain consensus sequences for each isolate, DNA sequences from both strands were assembled with BioEdit Sequence Alignment Editor v7.0.5 [ 14 ] and compared with the BLASTn ( https://blast.ncbi.nlm.nih.gov/ ) in the GenBank of the National Center for Biotechnology Information (NCBI) to find regions of local similarity between sequences with significant alignments. The phylogenetic reconstruction of the isolates obtained here was carried out with the 16S rRNA and gyrB gene sequences, including the 16 representative isolates, along with the 15 Stenotrophomonas type strains downloaded from the GenBank. All sequences were aligned and trimmed using MAFFT v7 [ 19 ] with default parameters and concatenated via Mesquite [ 25 ]. Bayesian inference (BI) was performed using Markov Chain Monte Carlo (MCMC) in Mr. Bayes v3.2.1 [ 37 ]. Analyses with two chains in both runs were considered with randomly chosen trees for 1 million generations and sampled every 1000 generations. The first 25% of trees were discarded as burn-in, and the posterior probabilities were determined for the remaining trees. Xanthomonas vasicola strain LMG 736 T was considered as an outgroup. Sequences derived from this study were deposited in the GenBank database under accession numbers MZ823472–MZ823487 for 16SrRNA and OM479820–OM479849 for gyrB . Biofilm production test This test was performed using the microtiter-plate test according to the modified technique of Stepanovic et al. [ 45 ]. The 30 isolates were seeded in test tubes with 5 mL of King’s B liquid medium (1.5 g of K 2 HPO 4 , 1.5 g of MgSO 4 7H 2 O, 1.0 mL of glycerin, and 20 g of peptone) [ 42 ]. Once all the isolates had been seeded, they were allowed to grow in an incubator at 30°C for 24 h. After bacteria growth, a 1:100 dilution was made in test tubes with King´s B liquid medium. The biofilm determination was conducted three times. The samples were seeded in 96-well ELISA plates in three different plates to be able to differentiate the growth according to the required times. Four wells of a sterile ELISA plate were filled with 200 µL of bacterial suspension each. Negative control wells contained only King’s B liquid medium. The plates were covered and incubated at 30°C for 24, 48, and 72 h. Then the contents of each well were aspirated, and each well was washed three times with 200 µL of sterile phosphate buffer saline (PBS 1×, pH 7.2). The plates were shaken vigorously to remove all non-adherent bacteria. The remaining bound bacteria were fixed with 200 µL of 99% methanol (J. T. Baker, USA) per well, and after 15 min the plates were emptied and allowed to dry. The plates were then stained for 5 min with 200 µL of 2% crystal violet per well. The excess stain was rinsed off by placing the plate under running tap water. After the plates were air-dried, the dye bound to the adherent cells was solubilized with 200 µL of 33% (v/v) glacial acetic acid (J. T. Baker, USA) per well. The optical density (OD) of each well was measured at 562 nm using a microplate reader (Biotek ELx808, USA) with the Kc4 program. The reading was done twice: (i) before the addition of glacial acetic acid, as in the standard microtiter plate assay at 620 nm, and (ii) after adding glacial acetic acid at 562 nm. The test was conducted in triplicate, and the averages were included in the data analysis (Table 1 ). The 33% glacial acetic acid was used as a negative control. The cutoff OD (ODc) was defined as three standard deviations above the mean OD of the negative control. The isolates were classified as biofilm producers as: absent (A) (OD < ODc), weak (W) (ODc < OD<2×ODc), moderate (M) (2×ODc < OD 4×ODc) [ 45 ] Table 1 Percentage of incidence of chlorotic leaf stripe symptoms in the Mexican maize "Chalqueño" race, inoculated with Stenotrophomonas indicatrix and percentage of biofilm producer by the 30 S. indicatrix isolates Treatments Incidence (%) a Biofilm formation b S1 19.87 ± 0.58 AB W S2 0.00 ± 0.00 B M S3 31.10 ± 0.58 AB M S4 23.33 ± 0.58 AB M S5 17.80 ± 0.00 AB W S6 33.33 ± 1.00 AB S S7 37.15 ± 1.53 AB M S8 50.00 ± 1.73 A W S9 33.33 ± 1.00 AB W S10 25.00 ± 1.00 AB W S11 25.43 ± 1.15 AB M S13 50.00 ± 1.15 A S S14 41.93 ± 0.58 AB W S16 39.30 ± 1.53 AB W S17 33.33 ± 1.00 AB W S18 38.90 ± 2.00 AB W S19 27.00 ± 1.15 AB W S20 42.07 ± 0.58 AB W S21 29.40 ± 1.00 AB M S22 26.20 ± 0.58 AB M S24 50.13 ± 1.00 A W S25 45.23 ± 1.00 AB W S26 44.43 ± 0.58 AB M S27 34.30 ± 1.00 AB M S28 32.53 ± 1.00 AB W S29 46.67 ± 1.15 AB M S30 0.00 ± 0.00 B W S31 43.33 ± 1.15 AB W S32 30.83 ± 1.15 AB W S33 51.77 ± 1.00 A W Control 0.00 ± 0.00 B A p-value 0.0004 a Different capital letters in the same column denote significant differences among treatments ( p < 0.05) b Numbers highlighted in red show the highest percentage results obtained, n = 3. Isolates were classified according biofilm producer as: absent (A) (OD < ODc), weak (W) (ODc < OD<2×ODc), moderate (M) (2×ODc < OD 4×ODc) [ 32 ] Use of red fluorescent protein to monitor the transmission of Stenotrophomonas indicatrix in maize seeds Escherichia coli bacteria containing the pBbE8k-RFP plasmid were seeded in solid Luria-Bertani (LB) medium (tryptone, 10 g; yeast extract, 5 g; NaCl, 10 g; agar, 16 g L − 1 ) at 37°C, supplemented with kanamycin at 50 µg mL − 1 . The culture of S. indicatrix , the causal agent of yellow stripe disease in corn, was grown in King's B medium (MgSO 4 .7 H 2 O, 1.5 g; K 2 HPO 4 , 1.5 g; glycerol, 15 mL; peptone, 20 g; agar, 18 g L − 1 ) at 28°C for 4 d. Competent cells were then obtained and transformed with the pBbE8k-RFP plasmid previously obtained from the E. coli strain. Preparation of competent cells of Stenotrophomonas indicatrix To obtain isolated colonies, 100 µL of a dilution of S. indicatrix (108 CFU mL − 1 ) was seeded onto a Petri dish containing solid LB medium and incubated overnight at 28°C. A fresh colony was added to a 50 mL solution of LB medium containing 500 µL of Solution 1 (1M MgCl 2 and 1M MgSO 4 ). This solution was incubated at room temperature and shaken at 250 ×g until the absorbance (550 nm) reached between 0.3 and 0.5. A total of 5 mL of the liquid culture and 2 mL of Solution 1 were added to a flask containing 100 mL of LB medium; it was incubated at room temperature (20°C) and shaken at 250 × g for 4 to 6 h until unsaturated turbidity was obtained. The flask was then placed on ice and shaken to allow rapid cooling. From this stage onwards, all material was kept cold (between 0 and 4°C). Twenty-five milliliters of the cold culture were transferred to pre-cooled, sterile 30-mL Corex centrifuge tubes, which were centrifuged at 4000 × g for 10 min at 4°C, and the supernatant was discarded. The cells were gently resuspended in a final volume of 4 mL of Buffer II (CaC l2 . 2 H 2 O 0.1 M) at 4°C and incubated on ice for 60 min, centrifuged again at 4000 × g for 10 min at 4°C, and the supernatant was discarded. Using a sterile micropipette tip, the cells were resuspended again in a final volume of 400 µL of Buffer III (CaC l2 . 2 H 2 O 0.1 M: glycerol (85:15) v/v) at 4°C and incubated for 10 min, after which the cells were ready for transformation. To preserve the cells, aliquots of 200 to 500 µL were placed in pre-cooled 1.5 mL microcentrifuge tubes and rapidly frozen in an ultra-freezer at -80°C. Transformation procedure This procedure was performed according to Sambrook et al. (1989) [ 39 ]. Aliquots of 100 µL of competent S. indicatrix cells were placed in pre-chilled tubes and kept on ice until use. A tube containing water (5 µL) was included as a positive control. Aliquots of 10 µL of the pBbE8k-RFP plasmid were added per tube, gently mixed cold, and incubated overnight at 4°C. Transformed bacteria were separated from untransformed bacteria based on a growth test in King's B medium supplemented with 50 µg mL − 1 kanamycin. Visualization of fluorescent colonies To confirm the fluorescence of the transformed colonies, a bacterial smear was prepared in sterile distilled water. Individual bacteria were visualized using a multiphoton fluorescence microscope (LSM 880, Axio Imager 2, Carl Zeiss). The Ch2 GaAsP fluorescence channel was used with an excitation wavelength of 543 nm and an emission wavelength of 652 nm. The images were digitized using Carl Zeiss Image 3.13 (ZEN 3.13) software for Windows. Seed inoculation The procedure proposed by Zavaleta-Mancera et al. (2007) [ 51 ] was applied. A transformed colony of S. indicatrix was grown in LB medium supplemented with 50 µg mL − 1 kanamycin for 24 h at 28°C. The culture was then suspended in M9 minimal salt medium (12.8 g of Na₂HPO₄, 3 g of KH₂PO₄, 0.5 g of NaCl, 1 g of NH₄Cl, 20 mL of 1M C₆H₁₂O₆, 2 mL of 1M MgSO₄ in 1 L distilled water) (Davis et al., 1986) for 48 h, from which the inoculum was prepared at a dilution of 10 − 6 CFU mL − 1 . Approximately 100 corn seeds were placed in 800 mL of the 10 − 6 CFU mL − 1 bacterial suspension for 2 h. The inoculated seeds were then placed in Petri dishes containing filter paper previously moistened with sterile distilled water and incubated at 28°C to promote germination. The progress of the infection was monitored at 0, 12, 24, 48, and 72 h after inoculation. Fluorescence microscopy The microscopic study was carried out with five seeds for each time interval. Initially, the seeds were fixed in a buffer solution of phosphates and 8% sucrose for 48 h. After 48 h of seed fixation, the seeds were cut using a LEICA CM 1860 UV cryostat, and the sections obtained were mounted on slides for immediate viewing under a multiphoton fluorescence microscope (LSM 880, Axio Imager 2, Carl Zeiss). Results Bacterial isolates From April to July 2022, maize plants showing chlorotic stripes on the leaves and twisting of the apical tissue were recovered in Tlaxcala, Puebla, Morelos, and the State of Mexico, located in the Central Highland Valleys region. When symptomatic tissue plants were processed, the presence of bacterial colonies rose after 5 d and were then purified to obtain pure cultures. The morphological appearance of colonies in King’s B agar medium was light yellow, smooth, and circular. Physiological tests indicated that they were gram-negative, strictly aerobic, KOH positive, and motile with an unpleasant odor. Overall, five isolates were selected for further analyses. Molecular identification The 2% CTAB protocol used for DNA extraction of the bacterial isolates in this study resulted in good-quality DNA with absorbance values ranging from 1.8 to 2.1. The amplified PCR products with the primers targeting the 16S rRNA gene yielded a band of approximately 1500 base pairs (bp), and 850 bp for gyrB . In addition, the consensus sequences of the 30 isolates that were compared in the GenBank database suggested that they belonged to the genus Stenotrophomonas with a maximum identity of 99 to 100%. The phylogenetic tree showed a clearly differentiated clade conformed by all type strains of Stenotrophomonas species described to date. On the other hand, a second clade in which S. indicatrix , S. lactitubi , S. maltophilia , and S. pavani were grouped (blue shadow). Surprisingly, the 16 representative isolates included in this study clustered with S. indicatrix . In the phylogenetic tree, S. maltophilia and S. pavani were identified as the closest relatives to the cluster containing the isolates from this study (Fig. 2 ). The isolates exhibiting the highest incidence of chlorotic stripe symptoms are highlighted in green text. Additionally, the phylogenetic tree was rooted with Xanthomonas vasicola strain LMG 736 (Fig. 2 ). Biofilm formation The biofilm test showed that S. indicatrix isolates can form biofilm, with some isolates exhibiting higher optical density (ODc) values than others. The results were categorized based on the adherence of the bacteria to the plate as follows: strongly adherent (S), moderately adherent (M), weakly adherent (W), and non-adherent (A). The strongly adherent isolates included S6 (1.98) and S13 (2.18) ODc; the moderately adherent isolates were S2 (0.98), S3 (0.98), S4 (0.68), S7 (0.77), S11 (1.15), S21 (1.20), S22 (1.13), S26 (0.65), S27 (0.62), and S29 (0.77) ODc; the weakly adherent isolates comprised S1 (0.58), S5 (0.57), S8 (0.35), S9 (0.53), S10 (0.52), S14 (0.41), S16 (0.41), S17 (0.50), S18 (0.36), S19 (0.50), S20 (0.53), S24 (0.47), S25 (0.49), S28 (0.57), S30 (0.36), S31 (0.48), S32 (0.23), and S33 (0.47) ODc; the control treatment was classified as non-adherent (A). It is important to note that pathogenic S. indicatrix possesses the ability to form biofilm. However, the observed biofilm values did not correlate with pathogenicity in maize (Table 1 ). Visualization of fluorescent colonies The formation of S. indicatrix biofilm was confirmed using confocal microscopy. Challenges arose when attempting to visualize the bacteria because they produce polysaccharides that obstruct the passage of photons, preventing fluorescence emission. Therefore, it was necessary to wash the sample with phosphate buffer, which enabled the observation of bacteria emitting red fluorescence. Images were obtained showing colonies of Stenotrophomonas indicatrix , with red staining in those incorporating the RFP cassette, clearly distinguishing bacillus-shaped bacterial cells emitting red fluorescence (Fig. 3 ). Seed inoculation The results obtained from maize demonstrated that bacterial labeling of S. indicatrix with the rfp gene allowed the visualization and localization of the bacteria within seed cells. Images were captured from various sections of maize seeds inoculated with S. indicatrix , as well as from uninoculated seeds (controls), to facilitate a comparison of bacterial fluorescence and seed autofluorescence (Fig. 4 ). Transmission of S. indicatrix to seeds Monitoring was achieved within the maize seed at different inoculation times (12, 24, 48, and 72 h), confirming its transmission by seed. During the first 12 hours after inoculation (hai), the presence of the bacterium was noted in the endosperm tissue cells (Fig. 5 ). At 24 hai, images revealed the bacterium in endosperm cells, which exhibited a halo around them; this halo may indicate the activation of the plant's defense mechanisms in response to the pathogen (Fig. 5 ). By 48 hai, evidence of bacterial colonization in the embryo was observed, with bacterial cells visible in the intercellular spaces and within the embryo itself (Fig. 6 ). At 72 hai, bacterial colonization in the plumule tissue was noted; however, no further observations could be made at this time as the samples disintegrated rapidly (Fig. 7 ). Several 3D images of the embryo were also recorded, showing the thickness of the tissue sections and the colonization of bacillus-shaped bacteria between the cells, embedded between the intercellular spaces of the embryo (Fig. 8 ). This monitoring process serves as a tool for investigating the transmission of pathogens through seeds and examining the internal origins of pathogenic bacterial infections in seeds that are significant for agriculture. Additionally, it has potential applications in the study of other phytopathogenic organisms, including fungi and viruses. Discussion Bacterial diseases affecting maize have been reported worldwide. Although some of the diseases are considered of minor importance, the incidence and severity of them depend mainly on the environmental conditions and the resistance or susceptibility of the maize varieties, inbreds, or hybrids. Most of these diseases are associated with leaf blight, particularly bacterial leaf blight (BLB) caused by Paracidovorax avenae , in which no differences in BLB infections were detected among hybrids with yellow, white, or bicolored kernels [ 18 , 34 ]. Infection typically occurs through stomata in leaves within whorls that can harbor large populations of P. avenae and epiphytic bacteria [ 27 ]. Leaf streak symptoms are also described in bacterial leaf streak (BLS) caused by Xanthomonas vasicola , a bacterium previously reported affecting sugarcane [ 41 ] and recently identified in sweet maize plantations in some states of the United States corn belt [ 21 ]. Other bacterial diseases in maize include bacterial streaks and leaf spot caused by Robbsia andropogonis ; Goss’s bacterial wilt and blight caused by Clavibacter nebraskensis ; Holcus spot caused by Pseudomonas syringae ; and Stewart’s bacterial wilt caused by Pantoea stewartii , which induces plant wilting mainly in the United States corn belt [ 6 , 31 ]. Leaf spot disease caused by Pantoea ananatis has been reported in Poland [ 20 ]. In Indonesia, bacterial wilt in maize plants infected by Pantoea sp., as well as leaf blight and vascular wilt caused by Pantoea agglomerans , have been reported [ 36 ]. In Mexico, leaf streak and stalk rot in maize have been associated with the spread of Burkholderia gladioli [ 11 ], which also causes lesions on maize ear leaves in the United States [ 23 ]. In Argentina, maize leaf spot disease has also been attributed to P. ananatis [ 2 ], while in South Africa this bacterium has been identified as the causal agent of brown stem rot [ 12 ]. In 2007, maize plantations in the Highland Valley of Mexico showed symptoms of chlorotic streaks on leaves near the State of Mexico. From these symptoms, a yellow bacterium was consistently isolated and initially identified as belonging to the Stenotrophomonas maltophilia complex [ 44 ]. However, multilocus sequence analysis (MLSA) later showed that these isolates clustered with the recently described Stenotrophomonas indicatrix [ 48 ]. Similar leaf symptoms observed in maize under field conditions were reproduced in this study by inoculating S. indicatrix strains into the same maize race grown under greenhouse conditions. The symptoms closely resembled those associated with Pantoea agglomerans , particularly when chlorotic streaks appear before the onset of leaf waviness in maize [ 26 , 38 ]. These observations indicate that further research is needed to clarify the bacterial relationships involved in chlorotic leaf streak in maize, as several bacterial genera, including S. indicatrix , may be associated with these foliar symptoms. According to GenBank records, genomes of S. indicatrix strains have been obtained from diverse sources, including environmental water samples from an oligotrophic pond in Göttingen, Germany (GCA_004551575.1); soil samples from Denmark (GCA_019285675.1); dirty dishes in Germany (GCA_002750975.1); a rotten pear in France (GCA_013450245.1); a plant sample in Michigan (GCA_019969795.1); sandy loam soil from a Belgian potato storage facility (GCA_002750995.1); soil from a community garden in Michigan (GCA_019969765.1); heavy metal-contaminated soil in France (GCA_001499755.1); the rhizosphere of Astragalus bisculcatus in England (GCA_000613205.1); soil in Lebanon (GCA_003999895.1); and sunflower roots in South Africa (GCA_017565455.1). Overall, S. indicatrix has been associated with chlorotic leaf streak disease in maize plants in the Highland Valley region of Mexico. Significant variation was observed among the five bacterial isolates in their ability to induce chlorotic leaf streak symptoms. All strains caused disease, with incidence ranging from 15 to 52%, significantly affecting plant growth. A major concern with bacterial diseases is their potential transmission through seeds, particularly in maize. S. indicatrix was confirmed to be seed-transmitted by monitoring bacterial presence in maize seeds at different inoculation times (12, 24, 48, and 72 h after inoculation). At 12 h after inoculation (hai), the bacterium was observed in endosperm tissue cells. At 24 hai, bacterial cells were still present in the endosperm and showed a halo around them, possibly indicating activation of plant defense responses during the plant-pathogen interaction. At 48 hai, bacterial colonization of the embryo was detected, with cells located in intercellular spaces and inside embryo tissues. By 72 hai, bacterial colonization was observed in the plumule tissue. Biofilm assays showed that S. indicatrix isolates are capable of forming biofilms, although some isolates exhibited higher optical density (ODc) values than others. Biofilm formation was further verified using confocal microscopy. Initial observations were difficult because the bacteria produce polysaccharides that block photon passage and prevent fluorescence emission. After washing with phosphate buffer to remove these polysaccharides, bacterial cells became visible, emitting red fluorescence under the microscope. For these reasons, the findings of this study on S. indicatrix as a new phytopathogenic bacterium of corn are of great importance, as they reveal typical symptoms of the disease, seed transmission, and the bacterium's ability to form biofilm. These bacteria could be used in the process of selecting maize varieties that are genetically resistant to the phytopathogen that causes the yellow streaks and twisting of the apical meristem. It could likewise be used in the design and development of disease management strategies in order to prevent an epidemic that could potentially affect the production of seeds nationwide. Declarations Acknowledgements Special thanks to the National Council for Humanities, Sciences and Technologies (SECIHTI-Mexico) for financial support to P. Aguirre-Rayo during D.C studies. Compliance with Ethical Standards: Funding: To the seed pathology laboratory of the postgraduate program in genetic resources and seed productivity-production, for funding through projects PS23-4006, PS23-4035, and PS25-4001. Conflict of Interest: Authors declare that they have no conflict of interest. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors. Author Contribution P.A.R. design the research, analysis, wrote the main manuscript text, H.V.S.R. design the research, analysis, wrote the main manuscript text, supervision, and financial support, L.M.O. supervision and financial support, A.A.G. data analysis, A.R.P. supervision, O.J.A.G. supervision, P.A.R. and H.V.S.R. prepared figures 1-8 and table 1.All authors reviewed the manuscript. References Afrizal A, Jennings SAV, Hitch TCA, Riedel T, Basic M, Panyot A, Treichel N, Hager FT, Wong EO, Wolter B, Viehof A, von Strempel A, Eberl C, Buhl EM, Abt B, Bleich A, Tolba R, Blank LM, Navarre WW, Kiessling F, Horz HP, Torow N, Cerovic V, Stecher B, Strowig T, Overmann J, Clavel T (2022) Enhanced cultured diversity of the mouse gut microbiota enables custom-made synthetic communities. 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Phytopathology 105:S4128 Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M (2000) A modified microtiter-plate test for quantification of Staphylococci biofilm formation. J Microbiol Method 40(2):175–179 Swings J, De Vos P, Van Den Mooter M, De Ley J (1983) Transfer of Pseudomonas maltophilia Hugh 1981 to the genus Xanthomonas as Xanthomonas maltophilia (Hugh 1981) comb. nov. Int J Syst Bacteriol 33:409–413 Turner S, Pryer KM, Miao VP, Palmer JD (1999) Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J Eukaryot Microbiol 46(4):327–338 Weber M, Schünemann W, Fuβ J, Kämpfer P, Lipski A (2018) Stenotrophomonas lactitubi sp. nov. and Stenotrophomonas indicatrix sp. nov., isolated from surfaces with food contact. Int J Syst Evol Microbiol 68(6):1830–1838 Young JM, Park DC, Shearman HM, Fargier E (2008) A multilocus sequence analysis of the genus Xanthomonas . 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Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 26 Apr, 2026 Reviews received at journal 19 Apr, 2026 Reviewers agreed at journal 16 Apr, 2026 Reviewers agreed at journal 16 Apr, 2026 Reviewers agreed at journal 16 Apr, 2026 Reviewers invited by journal 16 Apr, 2026 Editor assigned by journal 14 Mar, 2026 Submission checks completed at journal 11 Mar, 2026 First submitted to journal 09 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9078755","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626828725,"identity":"465f96bb-086d-4515-a06a-cd68f315bcd1","order_by":0,"name":"Petra Aguirre-Rayo","email":"","orcid":"","institution":"College of Postgraduate","correspondingAuthor":false,"prefix":"","firstName":"Petra","middleName":"","lastName":"Aguirre-Rayo","suffix":""},{"id":626828726,"identity":"da8ca6c3-f805-427e-970b-3d4acacd49c8","order_by":1,"name":"Hilda Victoria Silva-Rojas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACAwbGxgMMDDYMDOwkaGkAakljYGAmXgsDA1DLYRK0mPMfbjjMU3Nenp+Z9+DDHwzbEhsIabGckQjUcuy24cxmvmRjHobbxoQddoOx4eAMttsJBod5zKQZGG7LEdZy/iBQy79zYC2SPxhu8xDWciCx4cDHtgNgLRI8RNlyA6SlLxnqFwNi/HL++MMHCd/s5PnZe4EhVnGbcIghAZAvDEhQD9UyCkbBKBgFowALAAAzFD4Wa4L71gAAAABJRU5ErkJggg==","orcid":"","institution":"College of Postgraduate","correspondingAuthor":true,"prefix":"","firstName":"Hilda","middleName":"Victoria","lastName":"Silva-Rojas","suffix":""},{"id":626828727,"identity":"aaba0940-570c-4c31-9a93-2d968442db87","order_by":2,"name":"Leopoldo E. Mendoza-Onofre","email":"","orcid":"","institution":"College of Postgraduate","correspondingAuthor":false,"prefix":"","firstName":"Leopoldo","middleName":"E.","lastName":"Mendoza-Onofre","suffix":""},{"id":626828728,"identity":"a8ef1ca8-995b-41e9-81c8-419e8c1e9967","order_by":3,"name":"Andrés Aguilar-Granados","email":"","orcid":"","institution":"Center for Research and Advanced Studies of the National Polytechnic Institute","correspondingAuthor":false,"prefix":"","firstName":"Andrés","middleName":"","lastName":"Aguilar-Granados","suffix":""},{"id":626828729,"identity":"cd91a042-2da1-4cd8-8311-70c9fed635c1","order_by":4,"name":"Alejandrina Robledo-Paz","email":"","orcid":"","institution":"College of Postgraduate","correspondingAuthor":false,"prefix":"","firstName":"Alejandrina","middleName":"","lastName":"Robledo-Paz","suffix":""},{"id":626828730,"identity":"970ed0c1-7232-4872-aa3a-0b925687b5e8","order_by":5,"name":"Oscar Javier Ayala-Garay","email":"","orcid":"","institution":"College of Postgraduate","correspondingAuthor":false,"prefix":"","firstName":"Oscar","middleName":"Javier","lastName":"Ayala-Garay","suffix":""}],"badges":[],"createdAt":"2026-03-10 03:54:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9078755/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9078755/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107631547,"identity":"ec71228a-3247-4a42-8d1d-82ffe4a31b65","added_by":"auto","created_at":"2026-04-23 11:50:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1223661,"visible":true,"origin":"","legend":"\u003cp\u003eChalqueño maize plants (Mexican variety) grown in the field, showing symptoms of chlorotic leaf streak. A. Plant in phenological stage R1 showing symptoms of new shoot strangulation, causing atrophy and failure to flower; B and C. Plant in phenological stage V8 showing symptoms of chlorotic leaf streak\u003c/p\u003e","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9078755/v1/ce1bceef67e2fc9411618c2c.png"},{"id":107707265,"identity":"a128119c-136b-4ce4-9309-a731129df972","added_by":"auto","created_at":"2026-04-24 09:19:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39560,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree constructed with the Bayesian inference method using of 16 representative isolates of \u003cem\u003eStenotrophomonas indicatrix\u003c/em\u003e, and references sequences of the type strains. Yellow line shows the \u003cem\u003eindicatrix\u003c/em\u003e clade that includes the sequences obtained in this study and the strain type. Green text represents those bacterial strains that induced highest percentages of chlorotic leaf stripe incidence. Bar indicates substitution per site. The bootstrap support values are shown in the nodes. \u003cem\u003eXanthomonas vasicola\u003c/em\u003estrain LMG 736 was used as outgroup bacterium\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9078755/v1/3ee80cfb8cf275bfd453ad33.png"},{"id":107631549,"identity":"faaeae73-1dd4-45b1-b5a2-d4234d497fe1","added_by":"auto","created_at":"2026-04-23 11:50:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":276347,"visible":true,"origin":"","legend":"\u003cp\u003eColonies of \u003cem\u003eS. indicatrix\u003c/em\u003e transformed with the pBbE8k-RFP plasmid. A. Bacillus-shaped bacterial cells emitting red fluorescence, on a gray background. B. Bacillus-shaped bacterial cells emitting red fluorescence, on a black background\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9078755/v1/1107c2e5174d3904e2909cd2.png"},{"id":107707555,"identity":"95f87f45-c697-4b2d-ae1e-c5b513af2afa","added_by":"auto","created_at":"2026-04-24 09:20:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1416747,"visible":true,"origin":"","legend":"\u003cp\u003eCross section of maize seed. A. Seed without inoculation, control treatment. B. Seed inoculated with transformed \u003cem\u003eS. indicatrix\u003c/em\u003e\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9078755/v1/9ce0894d495c49230ecf2df8.png"},{"id":107631550,"identity":"a18bb972-2cf2-424c-a00b-f551001bb4ea","added_by":"auto","created_at":"2026-04-23 11:50:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":393096,"visible":true,"origin":"","legend":"\u003cp\u003eEndosperm tissue cells colonized by \u003cem\u003eS. indicatrix.\u003c/em\u003e A. Fluorescent bacterial cells present in endosperm 12 hours after inoculation. B and C. Fluorescent bacterial cells in endosperm 24 hours after inoculation, with a halo present around the bacteria\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-9078755/v1/8021ca222666ef66a7b6de54.png"},{"id":107705938,"identity":"0d2e562d-35d2-40c0-8124-87c3d2248c9a","added_by":"auto","created_at":"2026-04-24 09:15:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":830950,"visible":true,"origin":"","legend":"\u003cp\u003eEmbryo tissue cells colonized by \u003cem\u003eS. indicatrix\u003c/em\u003e. A and B. Fluorescent \u003cem\u003eS. indicatrix\u003c/em\u003e bacteria colonizing the intercellular spaces of the embryo, 48 h after inoculation. C. Fluorescent \u003cem\u003eS. indicatrix\u003c/em\u003e bacteria colonizing inside the embryo cells, 48 h after inoculation. D. Embryo cells from the control sample, without bacterial inoculation\u003c/p\u003e","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-9078755/v1/15b657b90ed0d85ccbcf87ef.png"},{"id":107631552,"identity":"70895988-59bb-46ca-82a3-d82b6445afc4","added_by":"auto","created_at":"2026-04-23 11:50:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1766834,"visible":true,"origin":"","legend":"\u003cp\u003eCross-section of corn plumule tissue showing colonization by fluorescent bacteria 72 hours after inoculation\u003c/p\u003e","description":"","filename":"OnlineFig7.png","url":"https://assets-eu.researchsquare.com/files/rs-9078755/v1/d102b3abba1f25bccb36a27d.png"},{"id":107631553,"identity":"fcf1a561-0cb6-4b9b-8eac-96a444f63a70","added_by":"auto","created_at":"2026-04-23 11:50:47","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":733878,"visible":true,"origin":"","legend":"\u003cp\u003e3D image of embryo cells colonized by \u003cem\u003eS. indicatrix\u003c/em\u003e. A. Bacteria colonizing embryo cells viewed in 3D from the transverse plane. B. Bacteria colonizing embryo cells viewed in 3D from the vertical axis of the section\u003c/p\u003e","description":"","filename":"OnlineFig8.png","url":"https://assets-eu.researchsquare.com/files/rs-9078755/v1/1de24bf6764d3c11311f0a49.png"},{"id":107709291,"identity":"e4a293d1-7bf6-437e-b466-ce538437201f","added_by":"auto","created_at":"2026-04-24 09:35:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4496652,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9078755/v1/0e423f6b-9670-45ae-a1b7-f06131908ccd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stenotrophomonas indicatrix, the Novel Gammaproteobacteria Isolated from Chlorotic Streaks Symptoms on Maize Leaves and its Seed Transmission","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMaize (\u003cem\u003eZea mays\u003c/em\u003e L.) is one of the most important cereal crops worldwide, ranking fifth with a production of 1.241\u0026nbsp;billion t. Almost all regions produce this crop, with the Americas accounting for 51.4% of global production [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Of the top 10 maize-producing countries worldwide, Mexico ranks fifth with a production of 23,402,006 t [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. White corn is used for human consumption (52%), livestock feed (19%), and self-consumption (18%), with exports accounting for 6%, losses for 4%, and seed for planting accounting for 1%. Yellow corn is used for livestock feed (76%), starch production (18%), self-consumption (2%), and human consumption (2%), with losses of 2% [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring maize production in the field, a new symptom of chlorotic leaf stripe was recently detected in the Highland Valleys of Mexico. This symptom resembled the chlorotic streaks caused by the bacterium \u003cem\u003ePantoea agglomerans\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e strains have also been isolated from leaves with chlorotic stripes, and recently, \u003cem\u003eStenotrophomonas indicatrix\u003c/em\u003e was associated with this symptomatology.\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eStenotrophomonas\u003c/em\u003e was first described by Palleroni and Branbury in 1993 as gen. nov. It was reclassified thanks to the confusion caused by the species \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e, which was identified for the first time in 1943 with the name \u003cem\u003eBacterium booker\u003c/em\u003e. Later taxonomic studies designated it as \u003cem\u003ePseudomonas maltophilia\u003c/em\u003e in 1961 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], \u003cem\u003eXanthomonas maltophilia\u003c/em\u003e in 1983 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and finally, it was placed in a new genus \"\u003cem\u003eStenotrophomonas\"\u003c/em\u003e in 1993 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, the genus \u003cem\u003eStenotrophomonas\u003c/em\u003e comprises 27 species considered as child taxa with a validly published and correct name (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps:/lpsn.dsmz.de/genus/stenotrophomonas\u003c/span\u003e\u003cspan address=\"https://lpsn.dsmz.de/genus/stenotrophomonas\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Among those recently described are \u003cem\u003eS. bentonitica\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], \u003cem\u003eS. pictorum\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], \u003cem\u003eS. lactitubi\u003c/em\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], \u003cem\u003eS. indicatrix\u003c/em\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], \u003cem\u003eS. beteli\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], \u003cem\u003eS. capsici\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], \u003cem\u003eS. forensis\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], \u003cem\u003eS. hibiscicola\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], \u003cem\u003eS. lacuserhaii\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], \u003cem\u003eS. mori\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], \u003cem\u003eS. pennii\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], \u003cem\u003eS. pigmentata\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], \u003cem\u003eS. riyadhensis\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and \u003cem\u003eS. muris\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The species within this genus exhibit significant adaptability, suggesting that they are engaged in a continual process of evolution and diversification. This is evidenced by the emergence of species complexes among certain \u003cem\u003eStenotrophomonas\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eStenotrophomonas indicatrix\u003c/em\u003e is a new species within the genus \u003cem\u003eStenotrophomonas\u003c/em\u003e, which was recently described [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] by comparison of its genome with other \u003cem\u003eStenotrophomonas\u003c/em\u003e, and shows obvious differences to be recognized as a new species closely related to \u003cem\u003eS. maltophilia\u003c/em\u003e and \u003cem\u003eS. lactitubi\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. On the other hand, \u003cem\u003eS. indicatrix\u003c/em\u003e and \u003cem\u003eS. maltophilia\u003c/em\u003e are genetically similar. Some accessions possess a common formate dehydrogenase locus, and it is likely that one of their ancestors acquired by homologous recombination the loci of the \u003cem\u003eS. maltophilia\u003c/em\u003e strain, thus creating a genetic divergence between the two species.\u003c/p\u003e \u003cp\u003eSo far, \u003cem\u003eS. indicatrix\u003c/em\u003e has been isolated from dirty plates [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and from a water sample from an oligotrophic pond in Germany [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, little is known about its presence in other substrates or environments or if it is involved with diseases in humans, plants, or animals. This bacterium has been described as strictly aerobic in the form of motile, gram-negative bacilli. The colonies are bright circular, smooth, and whitish-yellow, growing at 10\u0026ndash;37\u0026deg;C, with a pH of 6.0 to 9.0 [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGenerally, little is known about the ability of \u003cem\u003eS. indicatrix\u003c/em\u003e to form communities known as biofilms. This lifestyle enables them to be more resistant to antibiotics and adverse agents [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Biofilms are formed by a biopolymer matrix that provides stability and protection, which contains polysaccharides, proteins, and microbial cells adherent to one another above a living or non-living surface [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A significant group of biofilm-forming bacteria has been reported causing disease in monocot and dicotyledonous plants, including maize [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo study seed transmission, fluorescent protein marking is an effective method for investigating promoter activity, gene expression dynamics, cellular and subcellular protein localization, and the growth and development of organisms [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. These markers can be monitored non-destructively in living cells and tissues [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, this research aimed to determine whether the phytopathogenic maize bacterium \u003cem\u003eStenotrophomonas indicatrix\u003c/em\u003e is ​​seed-transmitted using the RFP marker.\u003c/p\u003e \u003cp\u003eThe presence of plants with symptoms of chlorotic streaks or stripes has increased in the maize-producing areas of the Central High Valleys region of Mexico. This implies the need to establish management strategies to reduce the incidence of bacterial diseases as a potential agent of chlorotic areas and the risk of being transmitted through the seed. Although \u003cem\u003eS. indicatrix\u003c/em\u003e has been associated with maize plants, little is known about its transmission. This research seeks to clarify whether this bacterium is seed-transmitted and to evaluate its biofilm-producing capacity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial isolation from maize leaves\u003c/h2\u003e \u003cp\u003eIn the spring-summer 2022 season, 117 maize plants were sampled based on the presence of chlorotic leaf streaks and twisting of the apical meristem (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These plants were recovered from experimental maize plots and growers' fields from Tlaxcala, Puebla, Morelos, and the State of Mexico, located in the Central Highland Valleys of Mexico at an altitude between 2230 and 2250 m.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA small portion of leaves was excised and washed in running water. After that, the leaves were disinfected using 1% sodium hypochlorite (w/v) (NaClO) for 1 min and then rinsed three times with sterile distilled water. Later, they were placed into sterile paper towels to eliminate the excess of water. Six individual leaf portions (3 mm\u003csup\u003e2\u003c/sup\u003e) per plant were placed on King\u0026rsquo;s B agar medium with the following composition (g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 1.5 g of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1.5 g of MgSO\u003csub\u003e4\u003c/sub\u003e\u0026bull;7H\u003csub\u003e2\u003c/sub\u003eO, 1.0 mL of glycerin, 20 g of peptone, and 16 g of agar [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Petri plates were incubated at 28\u0026deg;C for 5 d.\u003c/p\u003e \u003cp\u003eOnce bacterial growth was observed from leaf cuttings, a portion of each individual bacterial colony was taken with a loop and cross-streaked on Petri plates with fresh culture medium (King\u0026rsquo;s B). After 3 d of incubation, the bacterial colony forming units (CFU) were purified for Gram staining, oxidation-fermentation (OF) metabolism, KOH string test, and motility assessment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular identification\u003c/h3\u003e\n\u003cp\u003eDNA extraction was performed from purified bacterial cultures based on the 2% CTAB (Tris-HCl 10 mM pH 8.0; EDTA\u0026bull;2 H\u003csub\u003e2\u003c/sub\u003eO 20 mM; CTAB 2%; NaCl 1.4 M) protocol [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] with the addition of sodium acetate (C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e3\u003c/sub\u003eNaO\u003csub\u003e2\u003c/sub\u003e 3M). The tubes containing the DNA were placed to dry at room temperature for at least 30 min, and bacterial pellets were resuspended with 50 \u0026micro;L of HPLC water. The DNA was verified by spectrophotometry in a NanoDrop 2000 UV-Vis (Thermo Scientific, USA). Only samples with values of the A260/A280 and A260/A230 ratios of 1.8 to 2.1 were used for PCR amplifications.\u003c/p\u003e \u003cp\u003eThe 16S rDNA amplification was performed with primers 8F (5\u0026acute;-AGAGTTTGATCCTGGCTCAG-3\u0026acute;) and 1492R (5\u0026acute;-GGTTACCTTGTTACGACTT-3\u0026acute;) that yield a fragment size of approximately 1500 base pairs [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The \u003cem\u003egyrB\u003c/em\u003e amplification was performed with primers XgyrB1F (5\u0026acute;-ACGAGTACAACCCGGACAA-3\u0026acute;) and XgyrB1R (5\u0026acute;-CCCATCARGGTGCTGAAGAT-3\u0026acute;) that yield a fragment size of approximately 850 base pairs [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The PCR reaction mix was prepared in a final volume of 15 \u0026micro;L containing 7.86 \u0026micro;L of HPLC water, 3 \u0026micro;L of 5\u0026times; PCR buffer, 0.6 \u0026micro;L of dNTPs (20 \u0026micro;M of each), 0.18 \u0026micro;L of each primer (10 \u0026micro;M) (Sigma, USA), 3 \u0026micro;L of genomic DNA (20 ng), and 0.18 \u0026micro;L (2U) of Go\u003cem\u003eTaq\u003c/em\u003e DNA polymerase (Promega, USA).\u003c/p\u003e \u003cp\u003eThe PCR reaction was carried out in a C1000 Touch thermal cycler (Bio-Rad, USA) as follows: in the case of 16S rDNA, an initial denaturation of 95\u0026deg;C for 2 min, followed by 30 cycles of denaturation at 95\u0026deg;C for 2 min, alignment at 59\u0026deg;C for 1 min, and extension at 72\u0026deg;C for 1.5 min, with a final extension of one cycle at 72\u0026deg;C for 5 min. In the case of \u003cem\u003egyrB\u003c/em\u003e, an initial denaturation of 95\u0026deg;C for 10 min, followed by 39 cycles of denaturation at 95\u0026deg;C for 30 s, alignment at 50\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 36 s, with a final extension of one cycle at 72\u0026deg;C for 10 min. PCR products were verified by horizontal electrophoresis in 1.5% agarose gels (SeaKem, USA) stained with GelRed (Biotium, USA) and run at 88 volts for 1.5 h. The gels were visualized with the infinity imaging system in the Infinity-3026 WL/C/26MX transilluminator (Vilber Lourmat, Germany). Amplicons were cleaned with the enzyme ExoSAP-IT (Affymetrix, Thermo Fisher Scientific, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eThe sequencing reaction was prepared in a MicroAmp Optical 96-well reaction plate using a Big Dye Terminator v3.1 kit (Applied Biosystems, USA) in a total volume of 20 \u0026micro;L containing 4.0 \u0026micro;L (2.5\u0026times;) of Ready Reaction Premix, 2.0 \u0026micro;L (5\u0026times;) of Big Dye Sequencing Buffer, 4 pM aliquots of each internal primer 518F (5\u0026rsquo;-CCAGCAGCCGCGGTAATACG-3\u0026rsquo;) and 800R (5\u0026rsquo;- CTACCAGGGTATCTAAT-3\u0026rsquo;) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and 2.0 \u0026micro;L of the template DNA. The thermocycler program was set according to the manufacturer instructions. Later, plates were washed with an EDTA/ethanol protocol. The amplicons were sequenced in both directions in a Genetic Analyzer 3130 (Applied Biosystems, Thermo Fisher Scientific, USA) at the Colegio de Postgraduados (COLPOS) facilities in Montecillo, Mexico.\u003c/p\u003e \u003cp\u003eTo obtain consensus sequences for each isolate, DNA sequences from both strands were assembled with BioEdit Sequence Alignment Editor v7.0.5 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and compared with the BLASTn (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in the GenBank of the National Center for Biotechnology Information (NCBI) to find regions of local similarity between sequences with significant alignments.\u003c/p\u003e \u003cp\u003eThe phylogenetic reconstruction of the isolates obtained here was carried out with the 16S rRNA and \u003cem\u003egyrB\u003c/em\u003e gene sequences, including the 16 representative isolates, along with the 15 \u003cem\u003eStenotrophomonas\u003c/em\u003e type strains downloaded from the GenBank. All sequences were aligned and trimmed using MAFFT v7 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] with default parameters and concatenated via Mesquite [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Bayesian inference (BI) was performed using Markov Chain Monte Carlo (MCMC) in Mr. Bayes v3.2.1 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Analyses with two chains in both runs were considered with randomly chosen trees for 1\u0026nbsp;million generations and sampled every 1000 generations. The first 25% of trees were discarded as burn-in, and the posterior probabilities were determined for the remaining trees. \u003cem\u003eXanthomonas vasicola\u003c/em\u003e strain LMG 736\u003csup\u003eT\u003c/sup\u003e was considered as an outgroup. Sequences derived from this study were deposited in the GenBank database under accession numbers MZ823472\u0026ndash;MZ823487 for 16SrRNA and OM479820\u0026ndash;OM479849 for \u003cem\u003egyrB\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eBiofilm production test\u003c/h3\u003e\n\u003cp\u003eThis test was performed using the microtiter-plate test according to the modified technique of Stepanovic et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The 30 isolates were seeded in test tubes with 5 mL of King\u0026rsquo;s B liquid medium (1.5 g of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1.5 g of MgSO\u003csub\u003e4\u003c/sub\u003e 7H\u003csub\u003e2\u003c/sub\u003eO, 1.0 mL of glycerin, and 20 g of peptone) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Once all the isolates had been seeded, they were allowed to grow in an incubator at 30\u0026deg;C for 24 h. After bacteria growth, a 1:100 dilution was made in test tubes with King\u0026acute;s B liquid medium.\u003c/p\u003e \u003cp\u003eThe biofilm determination was conducted three times. The samples were seeded in 96-well ELISA plates in three different plates to be able to differentiate the growth according to the required times. Four wells of a sterile ELISA plate were filled with 200 \u0026micro;L of bacterial suspension each. Negative control wells contained only King\u0026rsquo;s B liquid medium. The plates were covered and incubated at 30\u0026deg;C for 24, 48, and 72 h. Then the contents of each well were aspirated, and each well was washed three times with 200 \u0026micro;L of sterile phosphate buffer saline (PBS 1\u0026times;, pH 7.2). The plates were shaken vigorously to remove all non-adherent bacteria. The remaining bound bacteria were fixed with 200 \u0026micro;L of 99% methanol (J. T. Baker, USA) per well, and after 15 min the plates were emptied and allowed to dry. The plates were then stained for 5 min with 200 \u0026micro;L of 2% crystal violet per well. The excess stain was rinsed off by placing the plate under running tap water.\u003c/p\u003e \u003cp\u003eAfter the plates were air-dried, the dye bound to the adherent cells was solubilized with 200 \u0026micro;L of 33% (v/v) glacial acetic acid (J. T. Baker, USA) per well. The optical density (OD) of each well was measured at 562 nm using a microplate reader (Biotek ELx808, USA) with the Kc4 program. The reading was done twice: (i) before the addition of glacial acetic acid, as in the standard microtiter plate assay at 620 nm, and (ii) after adding glacial acetic acid at 562 nm. The test was conducted in triplicate, and the averages were included in the data analysis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The 33% glacial acetic acid was used as a negative control. The cutoff OD (ODc) was defined as three standard deviations above the mean OD of the negative control. The isolates were classified as biofilm producers as: absent (A) (OD\u0026thinsp;\u0026lt;\u0026thinsp;ODc), weak (W) (ODc\u0026thinsp;\u0026lt;\u0026thinsp;OD\u0026lt;2\u0026times;ODc), moderate (M) (2\u0026times;ODc\u0026thinsp;\u0026lt;\u0026thinsp;OD\u0026lt;4\u0026times;ODc), and strong (S) (OD\u0026thinsp;\u0026gt;\u0026thinsp;4\u0026times;ODc) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage of incidence of chlorotic leaf stripe symptoms in the Mexican maize \"Chalque\u0026ntilde;o\" race, inoculated with \u003cem\u003eStenotrophomonas indicatrix\u003c/em\u003e and percentage of biofilm producer by the 30 \u003cem\u003eS. indicatrix\u003c/em\u003e isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncidence (%)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiofilm formation\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e50.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73 A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e50.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e41.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 AB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e42.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 AB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e50.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e45.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 AB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e44.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 AB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e46.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 AB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e43.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 AB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 AB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e51.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ep-value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.0004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003ea\u003c/sup\u003eDifferent capital letters in the same column denote significant differences among treatments (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003eb\u003c/sup\u003eNumbers highlighted in red show the highest percentage results obtained, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3. Isolates were classified according biofilm producer as: absent (A) (OD\u0026thinsp;\u0026lt;\u0026thinsp;ODc), weak (W) (ODc\u0026thinsp;\u0026lt;\u0026thinsp;OD\u0026lt;2\u0026times;ODc), moderate (M) (2\u0026times;ODc\u0026thinsp;\u0026lt;\u0026thinsp;OD\u0026lt;4\u0026times;ODc), strong (S) (OD\u0026thinsp;\u0026gt;\u0026thinsp;4\u0026times;ODc) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eUse of red fluorescent protein to monitor the transmission of\u003c/b\u003e \u003cb\u003eStenotrophomonas indicatrix\u003c/b\u003e \u003cb\u003ein maize seeds\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e bacteria containing the pBbE8k-RFP plasmid were seeded in solid Luria-Bertani (LB) medium (tryptone, 10 g; yeast extract, 5 g; NaCl, 10 g; agar, 16 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 37\u0026deg;C, supplemented with kanamycin at 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The culture of \u003cem\u003eS. indicatrix\u003c/em\u003e, the causal agent of yellow stripe disease in corn, was grown in King's B medium (MgSO\u003csub\u003e4\u003c/sub\u003e .7 H\u003csub\u003e2\u003c/sub\u003eO, 1.5 g; K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1.5 g; glycerol, 15 mL; peptone, 20 g; agar, 18 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 28\u0026deg;C for 4 d. Competent cells were then obtained and transformed with the pBbE8k-RFP plasmid previously obtained from the \u003cem\u003eE. coli\u003c/em\u003e strain.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of competent cells of\u003c/b\u003e \u003cb\u003eStenotrophomonas indicatrix\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo obtain isolated colonies, 100 \u0026micro;L of a dilution of \u003cem\u003eS. indicatrix\u003c/em\u003e (108 CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was seeded onto a Petri dish containing solid LB medium and incubated overnight at 28\u0026deg;C. A fresh colony was added to a 50 mL solution of LB medium containing 500 \u0026micro;L of Solution 1 (1M MgCl\u003csub\u003e2\u003c/sub\u003e and 1M MgSO\u003csub\u003e4\u003c/sub\u003e). This solution was incubated at room temperature and shaken at 250 \u0026times;g until the absorbance (550 nm) reached between 0.3 and 0.5. A total of 5 mL of the liquid culture and 2 mL of Solution 1 were added to a flask containing 100 mL of LB medium; it was incubated at room temperature (20\u0026deg;C) and shaken at 250 \u0026times; g for 4 to 6 h until unsaturated turbidity was obtained.\u003c/p\u003e \u003cp\u003eThe flask was then placed on ice and shaken to allow rapid cooling. From this stage onwards, all material was kept cold (between 0 and 4\u0026deg;C). Twenty-five milliliters of the cold culture were transferred to pre-cooled, sterile 30-mL Corex centrifuge tubes, which were centrifuged at 4000 \u0026times; g for 10 min at 4\u0026deg;C, and the supernatant was discarded. The cells were gently resuspended in a final volume of 4 mL of Buffer II (CaC\u003csub\u003el2\u003c/sub\u003e. \u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003eO 0.1 M) at 4\u0026deg;C and incubated on ice for 60 min, centrifuged again at 4000 \u0026times; g for 10 min at 4\u0026deg;C, and the supernatant was discarded. Using a sterile micropipette tip, the cells were resuspended again in a final volume of 400 \u0026micro;L of Buffer III (CaC\u003csub\u003el2\u003c/sub\u003e.\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003eO 0.1 M: glycerol (85:15) v/v) at 4\u0026deg;C and incubated for 10 min, after which the cells were ready for transformation. To preserve the cells, aliquots of 200 to 500 \u0026micro;L were placed in pre-cooled 1.5 mL microcentrifuge tubes and rapidly frozen in an ultra-freezer at -80\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eTransformation procedure\u003c/h3\u003e\n\u003cp\u003eThis procedure was performed according to Sambrook et al. (1989) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Aliquots of 100 \u0026micro;L of competent \u003cem\u003eS. indicatrix\u003c/em\u003e cells were placed in pre-chilled tubes and kept on ice until use. A tube containing water (5 \u0026micro;L) was included as a positive control. Aliquots of 10 \u0026micro;L of the pBbE8k-RFP plasmid were added per tube, gently mixed cold, and incubated overnight at 4\u0026deg;C. Transformed bacteria were separated from untransformed bacteria based on a growth test in King's B medium supplemented with 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e kanamycin.\u003c/p\u003e\n\u003ch3\u003eVisualization of fluorescent colonies\u003c/h3\u003e\n\u003cp\u003eTo confirm the fluorescence of the transformed colonies, a bacterial smear was prepared in sterile distilled water. Individual bacteria were visualized using a multiphoton fluorescence microscope (LSM 880, Axio Imager 2, Carl Zeiss). The Ch2 GaAsP fluorescence channel was used with an excitation wavelength of 543 nm and an emission wavelength of 652 nm. The images were digitized using Carl Zeiss Image 3.13 (ZEN 3.13) software for Windows.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSeed inoculation\u003c/h2\u003e \u003cp\u003eThe procedure proposed by Zavaleta-Mancera et al. (2007) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] was applied. A transformed colony of \u003cem\u003eS. indicatrix\u003c/em\u003e was grown in LB medium supplemented with 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e kanamycin for 24 h at 28\u0026deg;C. The culture was then suspended in M9 minimal salt medium (12.8 g of Na₂HPO₄, 3 g of KH₂PO₄, 0.5 g of NaCl, 1 g of NH₄Cl, 20 mL of 1M C₆H₁₂O₆, 2 mL of 1M MgSO₄ in 1 L distilled water) (Davis et al., 1986) for 48 h, from which the inoculum was prepared at a dilution of 10\u0026thinsp;\u0026minus;\u0026thinsp;6 CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Approximately 100 corn seeds were placed in 800 mL of the 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bacterial suspension for 2 h. The inoculated seeds were then placed in Petri dishes containing filter paper previously moistened with sterile distilled water and incubated at 28\u0026deg;C to promote germination. The progress of the infection was monitored at 0, 12, 24, 48, and 72 h after inoculation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFluorescence microscopy\u003c/h3\u003e\n\u003cp\u003eThe microscopic study was carried out with five seeds for each time interval. Initially, the seeds were fixed in a buffer solution of phosphates and 8% sucrose for 48 h. After 48 h of seed fixation, the seeds were cut using a LEICA CM 1860 UV cryostat, and the sections obtained were mounted on slides for immediate viewing under a multiphoton fluorescence microscope (LSM 880, Axio Imager 2, Carl Zeiss).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBacterial isolates\u003c/h2\u003e \u003cp\u003eFrom April to July 2022, maize plants showing chlorotic stripes on the leaves and twisting of the apical tissue were recovered in Tlaxcala, Puebla, Morelos, and the State of Mexico, located in the Central Highland Valleys region. When symptomatic tissue plants were processed, the presence of bacterial colonies rose after 5 d and were then purified to obtain pure cultures. The morphological appearance of colonies in King\u0026rsquo;s B agar medium was light yellow, smooth, and circular. Physiological tests indicated that they were gram-negative, strictly aerobic, KOH positive, and motile with an unpleasant odor. Overall, five isolates were selected for further analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMolecular identification\u003c/h2\u003e \u003cp\u003eThe 2% CTAB protocol used for DNA extraction of the bacterial isolates in this study resulted in good-quality DNA with absorbance values ranging from 1.8 to 2.1. The amplified PCR products with the primers targeting the 16S rRNA gene yielded a band of approximately 1500 base pairs (bp), and 850 bp for \u003cem\u003egyrB\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn addition, the consensus sequences of the 30 isolates that were compared in the GenBank database suggested that they belonged to the genus \u003cem\u003eStenotrophomonas\u003c/em\u003e with a maximum identity of 99 to 100%. The phylogenetic tree showed a clearly differentiated clade conformed by all type strains of \u003cem\u003eStenotrophomonas\u003c/em\u003e species described to date. On the other hand, a second clade in which \u003cem\u003eS. indicatrix\u003c/em\u003e, \u003cem\u003eS. lactitubi\u003c/em\u003e, \u003cem\u003eS. maltophilia\u003c/em\u003e, and \u003cem\u003eS. pavani\u003c/em\u003e were grouped (blue shadow). Surprisingly, the 16 representative isolates included in this study clustered with \u003cem\u003eS. indicatrix\u003c/em\u003e. In the phylogenetic tree, \u003cem\u003eS. maltophilia\u003c/em\u003e and \u003cem\u003eS. pavani\u003c/em\u003e were identified as the closest relatives to the cluster containing the isolates from this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The isolates exhibiting the highest incidence of chlorotic stripe symptoms are highlighted in green text. Additionally, the phylogenetic tree was rooted with \u003cem\u003eXanthomonas vasicola\u003c/em\u003e strain LMG 736 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm formation\u003c/h2\u003e \u003cp\u003eThe biofilm test showed that \u003cem\u003eS. indicatrix\u003c/em\u003e isolates can form biofilm, with some isolates exhibiting higher optical density (ODc) values than others. The results were categorized based on the adherence of the bacteria to the plate as follows: strongly adherent (S), moderately adherent (M), weakly adherent (W), and non-adherent (A). The strongly adherent isolates included S6 (1.98) and S13 (2.18) ODc; the moderately adherent isolates were S2 (0.98), S3 (0.98), S4 (0.68), S7 (0.77), S11 (1.15), S21 (1.20), S22 (1.13), S26 (0.65), S27 (0.62), and S29 (0.77) ODc; the weakly adherent isolates comprised S1 (0.58), S5 (0.57), S8 (0.35), S9 (0.53), S10 (0.52), S14 (0.41), S16 (0.41), S17 (0.50), S18 (0.36), S19 (0.50), S20 (0.53), S24 (0.47), S25 (0.49), S28 (0.57), S30 (0.36), S31 (0.48), S32 (0.23), and S33 (0.47) ODc; the control treatment was classified as non-adherent (A). It is important to note that pathogenic S. indicatrix possesses the ability to form biofilm. However, the observed biofilm values did not correlate with pathogenicity in maize (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eVisualization of fluorescent colonies\u003c/h2\u003e \u003cp\u003eThe formation of \u003cem\u003eS. indicatrix\u003c/em\u003e biofilm was confirmed using confocal microscopy. Challenges arose when attempting to visualize the bacteria because they produce polysaccharides that obstruct the passage of photons, preventing fluorescence emission. Therefore, it was necessary to wash the sample with phosphate buffer, which enabled the observation of bacteria emitting red fluorescence. Images were obtained showing colonies of \u003cem\u003eStenotrophomonas indicatrix\u003c/em\u003e, with red staining in those incorporating the RFP cassette, clearly distinguishing bacillus-shaped bacterial cells emitting red fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSeed inoculation\u003c/h2\u003e \u003cp\u003eThe results obtained from maize demonstrated that bacterial labeling of \u003cem\u003eS. indicatrix\u003c/em\u003e with the \u003cem\u003erfp\u003c/em\u003e gene allowed the visualization and localization of the bacteria within seed cells. Images were captured from various sections of maize seeds inoculated with \u003cem\u003eS. indicatrix\u003c/em\u003e, as well as from uninoculated seeds (controls), to facilitate a comparison of bacterial fluorescence and seed autofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTransmission of\u003c/b\u003e \u003cb\u003eS. indicatrix\u003c/b\u003e \u003cb\u003eto seeds\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMonitoring was achieved within the maize seed at different inoculation times (12, 24, 48, and 72 h), confirming its transmission by seed. During the first 12 hours after inoculation (hai), the presence of the bacterium was noted in the endosperm tissue cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). At 24 hai, images revealed the bacterium in endosperm cells, which exhibited a halo around them; this halo may indicate the activation of the plant's defense mechanisms in response to the pathogen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). By 48 hai, evidence of bacterial colonization in the embryo was observed, with bacterial cells visible in the intercellular spaces and within the embryo itself (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). At 72 hai, bacterial colonization in the plumule tissue was noted; however, no further observations could be made at this time as the samples disintegrated rapidly (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral 3D images of the embryo were also recorded, showing the thickness of the tissue sections and the colonization of bacillus-shaped bacteria between the cells, embedded between the intercellular spaces of the embryo (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This monitoring process serves as a tool for investigating the transmission of pathogens through seeds and examining the internal origins of pathogenic bacterial infections in seeds that are significant for agriculture. Additionally, it has potential applications in the study of other phytopathogenic organisms, including fungi and viruses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBacterial diseases affecting maize have been reported worldwide. Although some of the diseases are considered of minor importance, the incidence and severity of them depend mainly on the environmental conditions and the resistance or susceptibility of the maize varieties, inbreds, or hybrids. Most of these diseases are associated with leaf blight, particularly bacterial leaf blight (BLB) caused by \u003cem\u003eParacidovorax avenae\u003c/em\u003e, in which no differences in BLB infections were detected among hybrids with yellow, white, or bicolored kernels [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInfection typically occurs through stomata in leaves within whorls that can harbor large populations of P. \u003cem\u003eavenae\u003c/em\u003e and epiphytic bacteria [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Leaf streak symptoms are also described in bacterial leaf streak (BLS) caused by \u003cem\u003eXanthomonas vasicola\u003c/em\u003e, a bacterium previously reported affecting sugarcane [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and recently identified in sweet maize plantations in some states of the United States corn belt [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Other bacterial diseases in maize include bacterial streaks and leaf spot caused by \u003cem\u003eRobbsia andropogonis\u003c/em\u003e; Goss\u0026rsquo;s bacterial wilt and blight caused by \u003cem\u003eClavibacter nebraskensis\u003c/em\u003e; Holcus spot caused by \u003cem\u003ePseudomonas syringae\u003c/em\u003e; and Stewart\u0026rsquo;s bacterial wilt caused by \u003cem\u003ePantoea stewartii\u003c/em\u003e, which induces plant wilting mainly in the United States corn belt [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLeaf spot disease caused by \u003cem\u003ePantoea ananatis\u003c/em\u003e has been reported in Poland [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In Indonesia, bacterial wilt in maize plants infected by \u003cem\u003ePantoea\u003c/em\u003e sp., as well as leaf blight and vascular wilt caused by \u003cem\u003ePantoea agglomerans\u003c/em\u003e, have been reported [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In Mexico, leaf streak and stalk rot in maize have been associated with the spread of \u003cem\u003eBurkholderia gladioli\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], which also causes lesions on maize ear leaves in the United States [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In Argentina, maize leaf spot disease has also been attributed to \u003cem\u003eP. ananatis\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], while in South Africa this bacterium has been identified as the causal agent of brown stem rot [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn 2007, maize plantations in the Highland Valley of Mexico showed symptoms of chlorotic streaks on leaves near the State of Mexico. From these symptoms, a yellow bacterium was consistently isolated and initially identified as belonging to the \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e complex [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, multilocus sequence analysis (MLSA) later showed that these isolates clustered with the recently described \u003cem\u003eStenotrophomonas indicatrix\u003c/em\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Similar leaf symptoms observed in maize under field conditions were reproduced in this study by inoculating \u003cem\u003eS. indicatrix\u003c/em\u003e strains into the same maize race grown under greenhouse conditions. The symptoms closely resembled those associated with \u003cem\u003ePantoea agglomerans\u003c/em\u003e, particularly when chlorotic streaks appear before the onset of leaf waviness in maize [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. These observations indicate that further research is needed to clarify the bacterial relationships involved in chlorotic leaf streak in maize, as several bacterial genera, including \u003cem\u003eS. indicatrix\u003c/em\u003e, may be associated with these foliar symptoms.\u003c/p\u003e \u003cp\u003eAccording to GenBank records, genomes of \u003cem\u003eS. indicatrix\u003c/em\u003e strains have been obtained from diverse sources, including environmental water samples from an oligotrophic pond in G\u0026ouml;ttingen, Germany (GCA_004551575.1); soil samples from Denmark (GCA_019285675.1); dirty dishes in Germany (GCA_002750975.1); a rotten pear in France (GCA_013450245.1); a plant sample in Michigan (GCA_019969795.1); sandy loam soil from a Belgian potato storage facility (GCA_002750995.1); soil from a community garden in Michigan (GCA_019969765.1); heavy metal-contaminated soil in France (GCA_001499755.1); the rhizosphere of \u003cem\u003eAstragalus bisculcatus\u003c/em\u003e in England (GCA_000613205.1); soil in Lebanon (GCA_003999895.1); and sunflower roots in South Africa (GCA_017565455.1). Overall, \u003cem\u003eS. indicatrix\u003c/em\u003e has been associated with chlorotic leaf streak disease in maize plants in the Highland Valley region of Mexico.\u003c/p\u003e \u003cp\u003eSignificant variation was observed among the five bacterial isolates in their ability to induce chlorotic leaf streak symptoms. All strains caused disease, with incidence ranging from 15 to 52%, significantly affecting plant growth. A major concern with bacterial diseases is their potential transmission through seeds, particularly in maize. \u003cem\u003eS. indicatrix\u003c/em\u003e was confirmed to be seed-transmitted by monitoring bacterial presence in maize seeds at different inoculation times (12, 24, 48, and 72 h after inoculation). At 12 h after inoculation (hai), the bacterium was observed in endosperm tissue cells. At 24 hai, bacterial cells were still present in the endosperm and showed a halo around them, possibly indicating activation of plant defense responses during the plant-pathogen interaction. At 48 hai, bacterial colonization of the embryo was detected, with cells located in intercellular spaces and inside embryo tissues. By 72 hai, bacterial colonization was observed in the plumule tissue.\u003c/p\u003e \u003cp\u003eBiofilm assays showed that \u003cem\u003eS. indicatrix\u003c/em\u003e isolates are capable of forming biofilms, although some isolates exhibited higher optical density (ODc) values than others. Biofilm formation was further verified using confocal microscopy. Initial observations were difficult because the bacteria produce polysaccharides that block photon passage and prevent fluorescence emission. After washing with phosphate buffer to remove these polysaccharides, bacterial cells became visible, emitting red fluorescence under the microscope.\u003c/p\u003e \u003cp\u003eFor these reasons, the findings of this study on \u003cem\u003eS. indicatrix\u003c/em\u003e as a new phytopathogenic bacterium of corn are of great importance, as they reveal typical symptoms of the disease, seed transmission, and the bacterium's ability to form biofilm. These bacteria could be used in the process of selecting maize varieties that are genetically resistant to the phytopathogen that causes the yellow streaks and twisting of the apical meristem. It could likewise be used in the design and development of disease management strategies in order to prevent an epidemic that could potentially affect the production of seeds nationwide.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e Special thanks to the National Council for Humanities, Sciences and Technologies (SECIHTI-Mexico) for financial support to P. Aguirre-Rayo during D.C studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e To the seed pathology laboratory of the postgraduate program in genetic resources and seed productivity-production, for funding through projects PS23-4006, PS23-4035, and PS25-4001.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e Authors declare that they have no conflict of interest. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e This article does not contain any studies with human participants or animals performed by any of the authors.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.A.R. design the research, analysis, wrote the main manuscript text, H.V.S.R. design the research, analysis, wrote the main manuscript text, supervision, and financial support, L.M.O. supervision and financial support, A.A.G. data analysis, A.R.P. supervision, O.J.A.G. supervision, P.A.R. and H.V.S.R. prepared figures 1-8 and table 1.All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAfrizal A, Jennings SAV, Hitch TCA, Riedel T, Basic M, Panyot A, Treichel N, Hager FT, Wong EO, Wolter B, Viehof A, von Strempel A, Eberl C, Buhl EM, Abt B, Bleich A, Tolba R, Blank LM, Navarre WW, Kiessling F, Horz HP, Torow N, Cerovic V, Stecher B, Strowig T, Overmann J, Clavel T (2022) Enhanced cultured diversity of the mouse gut microbiota enables custom-made synthetic communities. 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J Fungi 9(4):493\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZavaleta-Mancera HA, Valencia-Botin AJ, Mendoza-Onofre LE, Silva-Rojas HV, Valadez-Moctezuma E (2007) Use of green fluorescent protein to monitor the colonization of \u003cem\u003ePseudomonas syringae\u003c/em\u003e subsp. \u003cem\u003esyringae\u003c/em\u003e on wheat seeds. Microsc Microanal 13(2):298\u0026ndash;299\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng QH, Zhang XW, Xu KP, Jiang JG (2014) Application of fluorescently labeled tracer technique for detection of natural active macromolecules in Chinese medicine. Drug Metab Rev 46(1):57\u0026ndash;71\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false},"keywords":"Bacterial disease, biofilm, maize pathogen, molecular identification, seed-transmission","lastPublishedDoi":"10.21203/rs.3.rs-9078755/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9078755/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eStenotrophomonas indicatrix\u003c/em\u003e is an aerobic, gram-negative bacterium isolated from dirty plates and ponded water, which was recently described as a novel species. To date, little is known about its presence in other substrates or environments or if it is involved with diseases in humans, plants, or animals. This bacterium is closely related to the \u003cem\u003eS. maltophilia\u003c/em\u003e group, which has been reported to cause infections in Mexican maize race plants. Thus, this research attempts to clarify the causal agents of the chlorotic streaks on maize leaves and the twisting of the apical meristem on the Mexican \u0026ldquo;Chalque\u0026ntilde;o\u0026rdquo; race. Eighty plants exhibiting both symptoms were recovered from maize growers' fields in the Central Highland Valley of Mexico. Small portions of symptomatic leaf tissue were disinfested and placed on King's B agar medium. After 5 d, bacterial colonies developed. Overall, 30 isolates were identified by phylogenetic reconstruction of the 16S rRNA and \u003cem\u003egyrB\u003c/em\u003e sequences as \u003cem\u003eS. indicatrix\u003c/em\u003e. The measures of biofilm biomass were not related to those of pathogenicity. To monitor transmission through the seed, the red fluorescent protein (RFP) marker was used to transform strains of the bacterium. The expression of the \u003cem\u003erfp\u003c/em\u003e gene in bacteria and seeds was visualized by confocal microscopy. The labeled \u003cem\u003eS. indicatrix\u003c/em\u003e showed strong RFP expression. Control seeds did not show red fluorescence. At 12 and 24 h, colonization was visualized in the endosperm, at 48 h in the embryo, and at 72 h in the plumule tissue, evidencing the transmission of \u003cem\u003eS. indicatrix\u003c/em\u003e through seeds. Therefore, producers must consider this bacterium when choosing new varieties or seeds due to the risk of transmission to subsequent generations.\u003c/p\u003e","manuscriptTitle":"Stenotrophomonas indicatrix, the Novel Gammaproteobacteria Isolated from Chlorotic Streaks Symptoms on Maize Leaves and its Seed Transmission","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 11:50:35","doi":"10.21203/rs.3.rs-9078755/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-26T15:05:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-19T15:52:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277047703702690716332905907713376788923","date":"2026-04-16T14:00:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224992914357770701269412413081871113346","date":"2026-04-16T09:01:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50761569490340826139244347108320212835","date":"2026-04-16T07:40:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-16T07:19:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-14T13:55:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-11T15:33:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Current Microbiology","date":"2026-03-10T03:37:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":false,"email":"","identity":"current-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Current Microbiology","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":false,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0fd249f6-c304-42c8-a017-460cf194a58e","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T11:50:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 11:50:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9078755","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9078755","identity":"rs-9078755","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}