Isolation and characterization of Bacillus endophytic strains producers for non ribosomal lipopeptides NRLPs from tomato

Received on: 21.08.2017 Revised on: 15.12.2017 Accepted on: 03.02.2018 Tomato (Solanum lycopersicum) are considered one of the most important vegetable crops and infected by numbers of different diseases. Studying the use of biological alternatives, instead of chemical substances against plant diseases became necessary for the treatment by beneficial microorganisms endophytes, which can excrete natural products benefits to plant in reducing disease severity, promoting growth and inducing plant defence mechanisms. In this work, three endophytes strains were isolated from tomato stems and their 16srDNA have been found to belong to Bacillus species. The first strain was named BMG100, the second BMG101 and the third BMG102. Two Bacillus strains BMG100 and BMG101 have been found to harbour synthetases genes from three lipopeptides families; surfactin, plipastatin, and iturin (mycosubtilin) which can be detected by degenerated primers designed to detect the presence of synthetases genes encoding lipopeptides. The lipopeptides production was proved by their quantification using High Performance Liquid Chromatography (HPLC), whereas BMG100 produced 105, 178 and 293 mg/L of plipastatin, mycosubtilin and surfactin, respectively, BMG101 produced 385 mg/L of surfactin and 236 mg/L of mycosubtilin, while BMG102 showed no lipopeptides production.


INTRODUCTION
In Egypt, tomato (Solanum lycopersicum) are considered one of the most important vegetable crops and infected by numbers of different diseases.Studying the use of biological alternatives instead of chemical substances against plant diseases became necessary (Ongena et al., 2004).Beneficial microorganisms' endophytes can enhance plant resistance system to a wide range of pathogens, as bacteria, fungi, and viruses (Ongena et al., 2007).Endophytes are considered groups of beneficial microorganisms which reside inside tissues of healthy and normal plant without any infectious symptoms (Hallmann et al., 1997;Robert et al., 2007).These endophytes can promote plant growth and plant disease management by producing metabolite substances.These substances are considered novel natural products for agricultural, medicinal, and industrial applications (Bacon and Hinton., 1999;Misaghi and Donndelinger., 1990;Pleban et al., 1995;Sturz and Kinpinski., 2004).Several studies have reported that some endophytes have the ability to excrete antimicrobial agents; surfactin consider one of these compounds, which is cyclic lipoheptapeptide biosurfactant produced by Bacillus subtilis various strains.It has shown inhibitory activities against some phyto-pathogenic bacteria, fungi, viruses, and mycoplasma (Nissen-Meyer and Nes., 1997;Phae et al., 1990).
The aim of this work is to select endophytic bacteria isolated from tomato stems and to investigate the excretion of lipopeptides as natural products, which are important for the biocontrol of tomato bacterial diseases.

Endophytic bacteria isolation from Tomato plants
Tomato plants were collected from Faculty of Agriculture farm, Ain-Shams University.The surface of stems was sterilized according to (Maurice et al., 2009;Wen et al., 2011).The attached dust and soil were removed from samples by washing with tap water, then, 70% ethanol for one minute and 5% sodium hypochlorite for five minutes were used to sterilize samples tissues.After that, samples were rinsed with sterilized distilled water three times, outer tissues were removed, and then were cut into fragments of 0.5-cm-long.The fragments were sterilized one more time with 70% ethanol for 10 seconds and rinsed with sterilized distilled water three times.The fragments were incubated on YPDA medium (pH 7.2) at 28 o C until the appearance of colonies.The colonies were purified and cultured on YPDA slants at 28 o C.
At the same time, a negative control was performed without surface sterilization and a positive control of 100 µl of the last rinsed water were inoculated on YPDA medium.

Endophytic bacteria identification from tomato plant by amplifying the 16S rDNA
Twelve colonies were grown on YPDA medium from five different tomato plants, their total bacterial DNA were extracted by Wizard Genomic DNA Purification Kit [Promega].Bacterial 16S rDNA universal primers were used as described by (Teng et al., 2006) 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACCTTGTTACGACTT-3'.PCR conditions started with initial denaturation step at 94°C for two minutes and followed by 35 cycles of 45 sec at 94°C, annealing step at 50°C for 45 s sec and elongation step at 72°C for 1.5 min and a final extension at 72°C for 10 min.

Non-Ribosomal Lipopeptides genes detection by degenerated primers in tomato endophytic isolates
Degenerated primers for surfactin, fengycin, mycosubtilin, and kurstakin families were used to detect the presence or absence of Non-Ribosomal Lipopeptides genes.The PCR conditions were performed as described (Tapi et al., 2006;Hussein and Fahim., 2017).

PCR amplification and cloning of endophytic bacterial 16S rDNA
The obtained fragments amplified by PCR with bacterial 16S rDNA universal primers were extracted and purified using Zymoclean Tm Gel DNA Recovery Kit (Epigenetics Company).pGEM ® -TEasy Vector was used to ligate purified PCR products and then, transformed into E. coli DH5α.Transformants were grown on LB plates supplemented with 50 mg/L ampicillin.White single colonies were chosen and their plasmid DNA were extracted using Plasmid miniPREP Kit (GeneDireX).Plasmids were double digested with EcoRI to verify the insertion of interested fragments then, amplified fragments were migrated on 1.2% agarose gel electrophoresis.Cloned products were sequenced and sequences were aligned with the GenBank databases (BLAST) online software of NCBI.

Detection of Non Ribosomal Lipopeptides production from isolated endophytic strains by HPLC.
Microbial fermentation was lanced in Landy Modified medium in 100 mL flasks with filling volume of 10% at 30 °C for 48 h (Hussein and Fahim., 2016).The culture was centrifuged (stationary phase) at 15,000 rpm at 4°C for 20 min to remove the bacterial cells.The total yield was collected before analysis by HPLC in C18 column, the mobile phase was acetonitrile -water -trifluoroacetic acid (80:20:0.5[vol/vol/vol]) (Fahim et al., 2012).

Phylogenetic tree based on 16S rRNA partial gene sequence
The phylogenetic tree based on alignment of 16S rRNA gene sequence of tomato endophytic Bacillus strains with other Bacillus strains indicated that all Bacillus strains could be divided into two main clusters from the same node.The first cluster includes the endophytic Bacillus strain named BMG102, while the second cluster divided into subclusters.The first subcluster includes B. subtilis strain EBs4 and the second divided into two subgroups.The first subgroup includes B. subtilis strain EBs5 and the second subgroup divided into two subgroups.The first subgroup includes; B. subtilis strain FR033, B. subtilis strain N391, while the second subgroup includes several subgroups.One of these subgroups divided into two subgroups; the first includes B. subtilis strain MZS1, while the second subgroup divided into two main subgroups; the first includes endophytic strain Bacillus BMG100 and the second includes the endophytic strain Bacillus BMG101 (Figure 2).

Non-Ribosomal Lipopeptides genes detection by degenerated primers in tomato endophytic isolates.
Three fragments were amplified with Srf (surfactin), Pps (plipastatin), and Myc (mycosubtilin) primers from Bacillus sp.BMG100 endophytic strain which were 425, 893 and 419 bp of length, respectively.From Bacillus sp.BMG101 endophytic strain two fragments were amplified with Srf and Myc primers which were 431 and 416 bp of length, while no fragments were amplified with the five degenerated primers for the endophytic strain BMG102 strain as mentioned in table 5.No fragments were amplified with Krs (kurstakin) primers.
The use of Fen primers was amplified fragment about 450 bp of length from Bacillus sp.BMG100 belongs to plipastatin synthetases genes, which was confirmed before by (Hussein and Fahim., 2017), because Fen primers are lower specificity than the other primers due to their high degeneracy.Also, at the absence of these genes, other genes harbour adenylation and thiolation domains are detected.In general, degenerated primers approach led to detect the presence of NRPS genes in two tomato endophytic isolates, this was confirmed before by (Tapi et al., 2010) who reported that the use of degenerated primers are helpful in screening the non-ribosomal synthetases genes in Bacillus spp.which lead to detect new NRPS molecules and it facilitate the study and genetic potential knowledge of these molecules biosynthesis.Lipopeptides quantification of production by HPLC revealed the presence of plipastatin, mycosubtilin, and surfactin from Bacillus sp.BMG100 with, 105, 178 and 293 mg/L, respectively, while 385 mg/L of surfactin and 236 mg/L of mycosubtilin were produced by Bacillus sp.BMG101 (Figure 3).BMG102 showed no lipopeptides production.These results proved that all the isolates of tomato stems had the ability to produce lipopeptides except isolate number 3 named BMG102 which showed no lipopeptides production by HPLC.It was also found that endophytic isolates are co-producers of three families of lipopeptides; surfactin, fengycin and mycosubtilin (iturin) which known as inducers of systemic resistance system in plant (Hussein et al., 2016).Also, the ability of B. subtilis strain M4 a co-producer strain of surfactin, iturin and fengycin lipopeptides families mixture to reduce disease incidence caused by Pythium aphanidermatum and Colletotrichum lagenarium on tomato was proven by (Ongena et al., 2004).

CONCLUSION
Three families of lipopeptides; surfactin, and or plipastatin and iturin (mycosubtilin) have been identified of two endophytic strains isolated from tomato.

Figure 2 :Figure 3 :
Figure 2: Phylogenetic tree based on alignment of 16S rRNA gene sequence of tomato endophytic Bacillus strains with other Bacillus strains