PATHOLOGICAL, BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF THE SEED-BORNE BACTERIA "PANTOEA SPP., XANTHOMONAS SPP. AND PSEUDOMONAS SPP." FROM SOLANACEOUS PLANTS IN EGYPT

The seed-borne bacterial diseases of family solanaceae cause significant economic losses worldwide. In the present study, fourteen bacterial isolates were recovered from seeds of different varieties of tomato, eggplant, black nightshade and tobacco.  The seed samples were collected from different locations in Egypt. Isolated bacteria were identified based on morphological, physiological and biochemical tests as well as colonial morphology aspects on the differential medium, yeast extract dextrose calcium carbonate (YDC) besides the selective media (PA 20, King's B and Tween B). Pathogenicity of the isolated bacteria was assessed for causing graywall, bacterial speck and bacterial spot symptoms on tomato seedlings. Disease symptoms were recognized on leaves and stems of the inoculated seedlings. Re-isolation from collected seeds of the inoculated tomato plants was performed. Results indicated that Pantoea spp. was an endophytic bacteria, Pseudomonas spp. was an epiphytic bacteria, while Xanthomonas spp. was both epiphytic and endophytic bacteria. Furthermore, cellular fatty acids composition were identified and quantified to give a profile for tested bacterial isolates. Moreover, the bacterial isolates were identified at the molecular level via PCR reactions utilizing the 16S rRNA gene. Partial DNA sequences were analyzed using BLAST tool revealed that the inferred 16S rRNA partial sequences of the 7 isolates  showed similarity to Pantoea ananatis (3 isolates), Pseudomonas syringae pv. tomato (2 isolates) and Xanthomonas vesicatoria (2 isolates). To the best of our knowledge the bacterium associated with the graywall seems to be first report of P. ananatis isolated from tomato fruits and black nightshade seeds in Egypt.


INTRODUCTION
Seed production has been moved to semi-arid regions to escape seed-borne pathogens. Seed-borne bacterial diseases continue to be problematic and cause significant economic losses worldwide. Infested seeds are responsible for thereemergence of diseases of the past, movement of pathogens across international borders, or the introduction of diseases into new areas (Gitaitis andWalcott, 2007). Family Solanaceae, included important economical, medicinal crops and weeds in the world. One of them is Tomato (Lycopersicon esculentum Mill.) which cultivated for its fruits, having economic importance for domestic consumption, export and food industries. Egypt ranks fifth in the world for tomato production, 6.62 million metric tons (FAO, 2018). Tomato seeds are subjected to infection by bacterial seed-borne diseases such as graywall disease caused by Pantoea ananatis [formerly, Erwinia ananas] (Stall et al., 1970 andBoyle, 1994). The genus Pantoea is a diverse group of yellowpigmented, rod shaped Gram-negative bacteria in the family Enterobacteriaceae. Some of the first members were recognized as plant pathogens causing wilting, soft rot and necrosis in different host plants ( Walterson and Stavrinides, 2015). P. ananatis causes disease symptoms in a wide range of economically important agricultural crops and forest tree species worldwide, which associated with plants as an epiphyte and endophyte bacteria (Coutinho and Venter, 2009 ; Gi Yoon Shin et al., 2019). Moreover, bacterial speck disease of tomato which caused by Pseudomonas syringae pv. tomato can also reduce yield when it severely affects leaves early in the growing season. The diseases is developed due to high humidity and low night temperatures (Kolomiets et al., 2017) .The bacterium can be moved from plant to plant via splashing water or on hands and gardening tools causes a serious outbreak on tomato plants grown in commercial fields (Sahin, 2001 ;Shenge et al., 2010). Bacterial spot of tomato, caused by Xanthomonas vesicatoria, is a potentially devastating disease that can lead to unmarketable fruit and even plant death. Bacterial spots can occur wherever tomatoes are grown, but are found most frequently in warm, wet climates, as well as in greenhouses (Shenge et al., 2010;Kolomiets et al., 2017). The pathogens enter plants through natural openings (e.g., stomates), as well as through wounds. Disease development is favored by warm, wet weather. Wind-driven rain can contribute to more severe disease as the pathogens are splashed and spread to healthy leaves and fruit. The species-specific PCR assays, biochemical and serological analyses were used to identify the Xanthomonas species associated with bacterial leaf spot of tomato as diagnostic protocols for this disease (Manjula et al., 2017;Roach et al., 2018;Vancheva et al., 2018).Therefore, the present study was conducted to achieve the following objectives: to isolate the seed-borne bacterial pathogens from solanaceous plants collected from different locations in Egypt, to identify and characterize the isolated bacteria through cultural, biochemical tests and fatty acid profiling ,as well as, molecular techniques through 16S rRNA gene sequences.

Sampling collection
Seeds of different varieties of some selected vegetable crops and weeds which belonging to family Solanaceae were collected as following: 6 varieties of tomato seeds (Solanum esculentum) namely Castle Rock, Super strain B, Peto 86, Super Jakal, Gs 12 and local variety were purchased from local markets at Alexandria Governorate. Moreover, one tomato seed sample of Super Jakal variety was obtained from naturally infected fruits collected from Assiut Governorate. Two varieties of eggplant seeds (S. melongena) namely local variety and Black Beauty, as well as, one local variety of pepper seeds (Capsicum annuum) were purchased from local markets at Alexandria Governorate. Seeds of datura (Datura stramonium and D. metel), tobacco (Nicotiana tabacum) and black nightshade (S. nigrum) were obtained from Plant Pathology Institute, Agricultural The seed-borne bacterial diseases of family solanaceae cause significant economic losses worldwide. In the present study, fourteen bacterial isolates were recovered from seeds of different varieties of tomato, eggplant, black nightshade and tobacco. The seed samples were collected from different locations in Egypt. Isolated bacteria were identified based on morphological, physiological and biochemical tests as well as colonial morphology aspects on the differential medium, yeast extract dextrose calcium carbonate (YDC) besides the selective media (PA 20,King's B and Tween B). Pathogenicity of the isolated bacteria was assessed for causing graywall, bacterial speck and bacterial spot symptoms on tomato seedlings. Disease symptoms were recognized on leaves and stems of the inoculated seedlings. Re-isolation from collected seeds of the inoculated tomato plants was performed. Results indicated that Pantoea spp. was an endophytic bacteria, Pseudomonas spp. was an epiphytic bacteria, while Xanthomonas spp. was both epiphytic and endophytic bacteria. Furthermore, cellular fatty acids composition were identified and quantified to give a profile for tested bacterial isolates. Moreover, the bacterial isolates were identified at the molecular level via PCR reactions utilizing the 16S rRNA gene. Partial DNA sequences were analyzed using BLAST tool revealed that the inferred 16S rRNA partial sequences of the 7 isolates showed similarity to Pantoea ananatis (3 isolates), Pseudomonas syringae pv. tomato (2 isolates) and Xanthomonas vesicatoria (2 isolates). To the best of our knowledge the bacterium associated with the graywall seems to be first report of P. ananatis isolated from tomato fruits and black nightshade seeds in Egypt.

ARTICLE INFO
Research Center (ARC), Giza, Egypt. The above mentioned varieties were used for isolation trials.

Isolation procedures
Isolation and detection of presumptive bacterial isolates from the above mentioned seed varieties were carried out using either dilution plating of seed extracts on semi-selective media or direct seed plating methods, as well as, liquid assay (LA) method. Symptomless and morphologically infected seed samples were divided into 2 groups (I, II) for isolation trials. In group I, seeds were non surface sterilized to detect epiphytic. In group II seeds were surface sterilized to detect only the endophytic bacteria. Then seeds were macerated in the phosphate buffer saline (PBS-Tween 20). All seed varieties in each group were incubated overnight for 14 hrs at 4ºC in the same buffer at a ratio of 3 mL of buffer to 1 gram of seeds (v:w). (www.wordseed.org/--/Tomato_Xanthomonas_spp_010). Ten-fold serial dilutions of either seed suspension or its extracts in PBS buffer were prepared. The resultant dilutions (0.1 mL) was spread onto two semi selective media, PA 20 medium for isolation of Pantoea spp.

Direct plating on semi-selective media and liquid assay methods
These methods were used for detection of Pseudomonas spp. from non-surface sterilized seeds (Group I). In direct plating method, 100 seed of each variety were planted on King's B medium (King et al. 1954). Seeds were distributed in ten plates, 10 seeds in each plate then incubated at 28ºC for 4-5 days. In liquid assay method, 0.1 gram of non-surface sterilized seeds of each variety was soaked in 10 mL sterile saline solution (0.85% NaCl) for 24 hrs at room temperature 25±2 ºC then, 0.1 mL of the suspension aliquots were spread on King's B medium. Isolated colonies were transferred on YDC medium for further identification.

Inoculation of tomato seedlings
Pure cultures of (P) and (X) bacterial isolates were cultivated on NA medium for 48 hrs. at 28ºC,whereas, (Ps) isolates was cultivated on King's B (KB) medium for 48 hrs at 28ºC. Bacterial cells were collected and suspended in sterile distilled water (SDW). In case of (P) isolates, bacterial suspension was adjusted to ca. 10 7 CFU/mL. Four weeks-old tomato seedlings (Alissa F1 variety) were inoculated by injecting bacterial suspension into the stem. Then, stem-inoculated plants were placed in plastic bags to increase the relative humidity (RH) and maintained in a greenhouse. The development of symptoms was recorded after 15 days. In case of (Ps) and (X) , bacterial suspensions (ca. 10 8 CFU/mL) were atomized with a hand-held sprayer until run-off. After inoculation, plants were placed in plastic bags and maintained in a greenhouse. Development of symptoms was observed daily (Milijasevic et al., 2009). Control plants were treated in a similar way using sterile water. All inoculation tests were replicated three times.

Inoculation of tomato fruits
Immature tomato fruits (Alissa F1 variety) were inoculated with the suspension of (P) isolates by injection the bacterial suspension (ca. 10 7 CFU/mL) into the fruits using sterile syringe. Disease symptoms were recorded after 14 days. Healthy fruits injected by sterile water were served as a control.

Re-isolation from artificially infected tomato Tomato Seedlings and fruits
Re-isolation from tomato seedlings was performed after 6 weeks from artificial infection. Leaves and stems of infected tomato seedlings (Alissa F1variety) were gently washed with tap water, cut into small pieces and soaked into a few drops of SDW for 30 min. The resultant suspension was streaked on nutrient agar (NA) medium. Bacterial spots and speck lesions were excised and surface sterilized by dipping in 70% ethanol for 2 seconds followed by two successive rinses in SDW. Lesions were crushed in 0.5 mL SDW, and the resultant suspension was streaked on NA medium. All inoculated plates were incubated for 3 days at 28ºC and a single colony of the predominant colonies was selected and purified by repeated streaking on the same medium, then examined for further studies. Moreover, reisolation was performed from fruits showing disease symptoms as previously mentioned.

Tomato seeds
Re-isolation from seeds which collected from tomato fruits was performed after 9 weeks from artificial infection of tomato seedlings as previously described (EL-Meneisy, 2005).

Cultural, morphological, physiological and biochemical Tests
Bacterial isolates were identified using cultural, morphological, physiological and biochemical characteristics, which were conducted by performing the standard tests The above mentioned tests were applied on the obtained bacterial isolates which included: cell shape, sporulation, motility, Gram staining, catalase, oxidase, starch hydrolysis, gelatin liquefaction, arginine dihydrolase, nitrate reduction, levan production and acid production from mannose, cellobiose, mannitol and sorbitol. Cultural characteristics of the bacterial isolates were studied on different culture media such as PA 20 medium for Pantoea spp., King's B medium for Pseudomonas spp. and Tween B medium for Xanthomonas spp., glycerol agar, NA and YDC media were used, also bacterial growth of Pseudomonas spp. and Xanthomonas spp. isolates was tested on PA 20 medium (Goszczynska et al., 2006a).

Extraction of total cellular lipids
Total lipids were extracted from seven bactrerial isolates grown on NB medium for 48 hrs as described by Kates (1972). Bacterial cells were harvested after growth for 24 hrs at room temperature with occasional shaking by centrifugation at 3000 g for 10min. Bacterial pellet (0.5 g) was transferred to screw cape tube capacity 20 mL and extracted with 5 mL methanol-chloroform mixture (2:1 v/w). The organic layer was transferred to another tube, and 5 ml of methanolchloroform mixture was added to re-extract the pellet for 2 hrs. The organic solvent was decanted and collected to the first extract 2.5 mL chloroform and 2.5 mL of distilled water were add to each tube, mixed well, and left for phases separation. The chloroform lower layer containing total lipids was withdrawn into a screw cape tube 20 mL capacity. Chloroform was evaporated at 40ºC under stream of nitrogen and the traces of water were removed by drops of benzene. Total lipids extract was kept refrigerated under nitrogen (N2) gas.

Preparation of methyl esters of fatty acids
The methyl esters of fatty acids (MEsFA) were prepared from total lipids as described by Radwan (1978). The total lipids extract in the screw cape tube was dissolved in 2.0 mL benzene and 10 mL of the esterification reagent (1% sulphuric acid in absolute methanol) were added. The tubes were caped under nitrogen and heated at 90ºC for 90 minutes. Ten mL of distilled water were added to the cooled tubes and the MEsFA were extracted with 5 mL benzene and the extracts were dried using anhydrous sodium sulfate (5 g), received in 10 mL capacity vials, kept under N2 and used for gas liquid chromatography (GLC) analysis.

Gas liquid chromatography of MEsFA
The MEsFA were analyzed using Shimadzu-8 A, GLC equipped with flam ionization detector and ordinary glass column (ID 3 mm X 2.5 m.) of 5 % diethylene glycol succinate on chromosorb Q 80/100 mesh. The following conditions were used for GLC analysis:column temperature 160ºC, detector temperature 270ºC, flow rates of nitrogen 20 mL/ min, H2 75 mL/ min, air 0.5 mL/ min, chart speed 2.5 mm/ min. Standard MEsFA and their retention times were used for identification. The area under each peak was measured by the triangulation methods and expressed as percentage of each fatty acid with regard to the total area.

Extraction of genomic DNA
Bacterial genomic DNA was extracted by boiling one mL of a suspension containing ca. 1x10 10 CFU/mL for 10 min. DNA was separated by centrifugation for 5 min at 11.000 xg (Yahiaoui-Zaidiet al.,2003).

PCR product of electrophoresis and visualization
Two µL of loading dye was added prior to loading of 10 µl per gel slot. Electrophoresis was performed at 100 volt with 0.5 x Tris-EDTA-borate (TBE) (Tris base, 108 g/L boric acid, 55 g/L and 0.5 M EDTA with a pH of 8, 40 ml for 10 x) as running buffer in 1.5% agarose gel cast in 0.5 x TBE gel and then the gel was stained in 0.5 µg/mL (w/v) ethidium bromide solution and distained in deionized water. Finally, the gel was visualized with a UV transilluminator at 254 nm.

Purification of PCR products
QIAquick PCR purification kit (Qiagen, Germany) was used to purify the amplified products of 16S rRNA gene.

Sequencing of 16S rRNA gene
The amplified product (1550 bp) of 16S rRNA was sequenced by Big Dye terminator cycle sequencing kit. Sequencing products were purified using Centri-Sep spin columns and were resolved on an ABIPRISM model 310 automated DNA sequencer at the Sigma Scientific Services Company.

Alignment and phylogenetic analysis
Pair-wise and multiple DNA sequence alignment were carried out using CLUSTALW (1.82) http://www.ebi.ac.uk/clustalw . Bootstrap neighbor-joining tree generated using MEGA version 7.0 (Kumar et al., 2016) from CLUSTALW alignment. Comparisons with sequences in the GenBank database were achieved in BLASTN searches at the National Center for Biotechnology Information site (http://www.ncbi.nlm.nih.gov). The obtained sequences in the current study were deposited in European Nucleotide Archive (ENA) under accession numbers from LN880270 to LN880276.

Isolation trials from collected seeds
Seed samples of different varieties of tomato, eggplant, tobacco, black nightshade, pepper and datura were collected in this study. Only one seed sample was obtained from naturally infected tomato fruits (Super jakal variety) showed typical symptoms of gray wall disease. Different bacterial colonies were observed on glycerol agar, nutrient agar, yeast extract dextrose-CaCo3 (YDC), as well as, semi selective media, PA20, King's B and Tween B (Table 1). According to their colonies characteristics on the previous semi selective media, three types of bacterial isolates were detected: Type I [typical colonies of Pantoea spp. (P1 , P2 , P3 , P4 and P5)] on PA20 medium, colonies were yellow, 3-4 mm in diameter, shiny, drop shaped with small, granular, darker inclusions inside and yellow lighter zones around colonies, Type II [Typical colonies of Pseudomonas spp. (Ps1, Ps2, Ps3, Ps4, Ps5 and P6)] on King's B medium, colonies were white, circular and mucoid and Type III [typical colonies of Xanthomonas spp. (X1, X2 and X3)] on Tween B medium, colonies were raised, circular and yellow surrounded by zones of white crystals giving a fried-egg appearance (Fig.1).

Tomato seedlings
All the bacterial isolates Type I (P1,P2,P3,P4 and P5), Type II (Ps1, Ps2, Ps3, Ps4, Ps5 and P6) and Type III ( X1, X2 and X3) were tested for their pathogenicity on tomato seedlings Alissa F1 and Gs Nada varieties. Inoculation with isolates of type I, stems appeared shrivel and wither, moreover, discolored water-conducting tissue and chlorosis was appeared on leaves (Figs. 2) which were suspected to belong to Pantoea spp. Inoculation with isolates of type II showed necrotic spots surrounded by a chlorotic halo appearing on leaves (Fig. 3) which were suspected to belong to Pseudomonas spp. In case of inoculation with type III bacterial isolates, leaves appeared water soaked lesions and became brown color (Fig. 4) which was suspected to belong to Xanthomonas spp.

Figure 4
Artificially infected tomato seedlings "Alissa F1" with Type III isolates (X1 and X2) of Xanthomonas spp. showing water soaked lesions on leaves which became brown colour (arrows).

Tomato fruits
All isolates of Pantoea spp. (P) were tested for their pathogenicity on tomato fruits (Alissa F1) and showed grey areas around the inoculation site (Fig. 5).

Re-isolation from artificially infected seedlings Tomato seedlings and fruits
Re-isolation from infected seedlings with isolated bacteria appeared identical to those colonies of Pantoea spp., Pseudomonas spp. and Xanthomonas spp. Re-isolation from inoculated fruits on selective media produced yellow colonies typical to Pantoea spp.

Tomato seeds
Re-isolation from non-surface sterilized seeds from infected seedlings with P1 isolate appeared typical graywall disease symptoms didn't revealed colonies identical to Panteoa spp. Otherwise non surface-sterilized seeds from infected seedlings with Ps2 isolate and X3 isolate showed typical bacterial speck and spot diseases symptoms and produced colonies identical to Pseudomonas spp. and Xanthomonas spp. respectively. Re-isolation from macerated surface-sterilized seeds revealed colonies identical to Pantoea spp. (P1 isolate) and Xanthomonas spp. (X1 isolate), otherwise didn't revealed colonies identical to Pseudomonas spp. (Ps2 isolate).

Molecular identification through 16S rRNA gene
The following partial sequences were obtained for Pantoea spp. isolates P1, P2 and P3,Psendomonas spp. isolates Ps1, Ps2 and Xanthomonas spp. isolates X1 and X2. Search in databases to identify the bacteria was achieved in BLAST search at the NCBI. The search revealed that the sequence corresponding to each individual isolate of P1, P2 and P3 was almost identical (99% homology) to that of Pantoea ananatis whereas, the sequence of each of the Ps1 and Ps2 was almost similar to that of Psendomonas syringae pv. tomato. The Genbank accession numbers of the bacterial isolates were listed in Table (5). While the sequence of each of the X1 and X2 was almost similar (97% homology) to that of Xanthomonas vesicatoria. Pseudomonas syringae pv. tomato LN880273 Ps2 Pseudomonas syringae pv. tomato LN880274 X1 Xanthomonas vesicatoria LN880275 X2 Xanthomonas vesicatoria LN880276

Alignment and phylogenetic analysis
The phylogenetic tree generated in this study for the P.ananatis isolates P1, P2 and P3 revealed that two main clusters do exist. Cluster I included the two out-group isolates Pectobacterium carotovorum subsp. carotovorum "Pcc" (AB680280) and Erwinia tracheiphila "E.trach" (NR044924). Cluster II divided into two Sub-clusters: Sub-cluster 1 divided further into two Groups: Group 1 contained the two isolates of P.ananatis isolated from tomato P1(LN880270) and P2 (LN880271) besides the P.ananatis isolates collected from Genbank. However, Group 2 included only one isolate P3 (LN880272) which isolated from Black nightshade. Sub-cluster 2 included only one isolate KJ670108 (Fig.10). While, the phylogenetic tree generated for the Pseudomonas syringae pv.tomato isolates revealed that two main clusters do exist. Cluster I contained our two isolates Ps1 (LN880273) and Ps2 (LN880274). Whereas Cluster II included the other isolatesof Ps. Syringae pv. tomato collected from Genbank (Fig.11).
In case of phylogenetic tree generated in this study for the Xanthomonas vesicatoria isolates revealed that two main clusters do exist. Cluster I included only one isolate KP84443. Cluster II divided into two Sub-clusters: Sub-cluster 1 divided further into two Groups: Group 1 contained the two isolates contained isolates collected from Genbank KU301883, HF585549, AF123088 and NR026388. Group II included the two isolates X1 (LN880275) and X2 (LN880276) (Fig.12) Figure 10 phylogenetic tree of Pantoea ananatis (P1, P2 and P3 isolates) obtained in this study and validly related bacteria from the alignment of 16S rRNA sequences. The scale appearing at the bottom indicates linkage distance.

Figure 11
Phylogenetic tree of Pseudomonas syringae pv. tomato (Ps1 and Ps2 isolates) obtained in this study and validly related bacteria from the alignment of 16S rRNA sequences.The scale appearing at the bottom indicates linkage distance.

Figure 12
Phylogenetic tree of Xanthomonas vesicatoria (X1 and X2 isolates) obtained in this study and validly related bacteria from the alignment of 16S rRNA sequences. The scale appearing at the bottom indicates linkage distance.

DISCUSSION
Characterization of the population structure, diversity, and evolution are the main factors for understanding the pathogen biology and providing information necessary for the development of effective means for disease control (Vancheva et al., 2018). Results of the pathological behaviors of the isolated tomato seedborne bacteria, their cultural, morphological and physiological characters, as well as molecular techniques (16S rRNA gene sequence) were indicated that Pantoea ananatis was associated with graywall symptoms (Stall et al., 1970), Pseudomonas syringae pv. tomato was the causal agent of bacterial speck disease and Xanthomonas vesicatoria was associated with bacterial spot disease The use of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy has been by far the most common housekeeping genetic marker (Ashmawy et al., 2020). 16S rRNAgene was used to identify the tested isolates and study the genetic variability among 3 isolates of Pantoea spp., 2 isolates of Pseudomonas spp. and 2 isolates of Xanthomonas spp. Results obtained of all tested isolates gave one band in the right expected molecular length. DNA sequences of tested isolates revealed that the sequences belong to P.ananatis, P. syringae pv. tomato and X. campestris pv. vesicatoria. Such findings agreed with data obtained with Krawczyket al., (2010) and Mbegaet al., (2012). The bacterium associated with the tomato (Solanum esculentum) and the alternative host black nightshade (S. nigrum) seeds appeared to be P. ananatis on the basis of 16S rRNA gene sequence. This apparently is the first report of P. ananatis as a bacterial pathogen isolated from tomato and black nightshade seeds in Egypt. Alternative hosts such as several crop and weed species have been suspected to potentially play a role in the spread or survival of bacterial diseases (Ocimati et al., 2018). The current study determined the potential risk posed by black nightshade (S. nigrum) as alternative hosts to P. ananatis.