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Stimulation of plant growth and biocontrol by Bacillus amyloliquefaciens subsp. plantarum FZB42 engineered for improved action
Chemical and Biological Technologies in Agriculturevolume 1, Article number: 12 (2014)
During the last decade, the use of plant-root colonizing bacteria with plant growth-promoting activity has been proven as an efficient and environmental-friendly alternative to chemical pesticides and fertilizers. Biofertilizer and biocontrol formulations prepared from endospore-forming Bacillus strains are increasingly applied due to their long shelf life, which is comparable with that of agrochemicals. Today, spore suspensions from natural representatives of mainly Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus pumilus are available. However, these biofertilizers, directly prepared from environmental strains, are sometimes hampered in their action and do not fulfill in each case the expectations of the appliers (Borriss R, Bacteria in agrobiology: plant growth responses, Springer, 2011, pp. 41-76). This review will focus on several ways to improve the action of B. amyloliquefaciens subsp. plantarum FZB42T, the type strain for the group of plant-associated B. amyloliquefaciens strains. We are focusing here on genomics and genetic engineering techniques as helpful tools for developing more powerful biofertilizer and biocontrol agents.
In recent years, use of biologicals in plant protection is steadily increasing and begins to replace, in part, chemical pesticides. An increasing number of farmers are recognizing the need for other avenues for pest control that are not as damaging to the environment and the land. Henceforth, they are turning to biopesticides to prevent pest damage in a more ecological-friendly manner that includes targeted applications, lower residues, and fewer applications. According to a comprehensive study of BCC Research, global markets for biopesticides will grow from US$54.8 billion in 2013 to US$61.8 billion in 2014. This is estimated to reach US$83.7 billion by 2019, with a 5-year compound annual growth rate (CAGR) of 6.3% from 2014 through 2019 (http://www.bccresearch.com/market-research/chemicals/biopesticides-chm029e.html). Thereby, biological preparations from spore-forming Bacillus spp. are preferred, because their long-term viability facilitates the development of commercial products. Unfortunately, their success in agricultural application is still hampered by insufficient knowledge about basic mechanisms of interactions between bacilli and plants, although some progress has been made in last decade .
Plant-associated Bacillus amyloliquefaciens strains belonging to subsp. plantarum are distinguished from other representatives of endospore-forming B. amyloliquefaciens by their ability to colonize plant rhizosphere, to stimulate plant growth, and to suppress competing phytopathogenic bacteria and fungi. Due to their biofertilizer and biocontrol properties, they are becoming increasingly important as a natural alternative to chemical pesticides and other agrochemicals. We have focused our research on B. amyloliquefaciens FZB42T, the type strain for B. amyloliquefaciens subsp. plantarum. Comparative genome analysis, transposon mutagenesis, and transcriptome and proteome analysis of this model organism are valuable means to evaluate its plant growth-promoting activity. A network of research activities was established in frame of national and international programs to elucidate the interaction of the beneficial bacterium with plants, plant pathogens, and the microbial community living on plant roots. The outcome of this research will contribute to the development of an efficient and environmental-friendly plant protection agent. In order to reveal the specific genomic features linked with the properties beneficial for plant growth and biocontrol, we have sequenced the whole genome of FZB42 as the first example of gram-positive plant beneficial bacteria .
Land plants and bacteria have shared the same environment for approximately 360 to 480 million years . The contact between them has developed into various dependencies on both sides. Plants and certain rhizobacteria form mutually beneficial associations mediated through an exchange of chemical metabolites .
Plant growth-promoting Bacilli, like FZB42, are able to propagate in the rhizosphere, the soil environment influenced by plant roots. This environment is highly competitive due to the nutrient-rich rhizodeposits consisting of a wide variety of compounds derived from sloughed off root cells and tissues, mucilages, and soluble exudates originating from intact roots . However, roots respond to signals that stimulate defense responses (salicylic and jasmonic acids) by exuding a range of secondary metabolites, such as saponins, glucosinolates, hydroxamic acids, and naphthoquinones, which are inhibiting the growth of many ‘ordinary’ (not adapted to plant colonization) bacteria or fungi in this area.
The ability of FZB42 to colonize the rhizoplane is a precondition for plant growth promotion . Using a GFP-tagged derivative , the fate of bacterial root colonization was recently studied. It ruled out that the bacterium behaves distinctly in colonizing root surfaces of different plants. In contrast to maize, FZB42 colonized preferentially root tips when colonizing Arabidopsis thaliana. On duckweed, Lemna minor, FZB42 accumulated preferably along the grooves between epidermal cells of roots and in the concave spaces on ventral sides of fronds. In vitro studies performed with maize seedlings revealed that the segment within 2 to 8 cm distance from the basal site of the primary root was the most colonized region by FZB42. On the contrary, few bacterial cells could be observed within the range of 2 cm of root tip. In general, the green fluorescent FZB42 were decreasingly observed from the upper part of a root down to the root tip. Scanning electron microscopy confirmed the presence of FZB42 on root hairs, where the bacterial cells were usually associated with a wealth of presumed root exudates . In lettuce (Lactuca sativa) seedlings, bacterial colonization occurred mainly on the primary roots and root hairs, as well as on root tips and adjacent border cells. Occurrence of labelled bacteria decreased towards the root tips of the lateral roots, and no colonization of the finer roots was observed .
Genomics and other -omics techniques are useful for dissecting interactions between FZB42 and plants
The whole genome sequence of the type strain of plant-associated B. amyloliquefaciens subsp. plantarum, FZB42T, has been determined in 2007, as the first representative of gram-positive, plant growth-promoting bacteria. Its 3,918-kb genome, containing an estimated 3,695 protein coding sequences (CDS), lacks extended phage insertions, which occur ubiquitously in the related Bacillus subtilis 168 genome. The B. amyloliquefaciens genome reveals a huge potential to produce secondary metabolites, including the polyketides bacillaene, macrolactin, and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes . A comparison of its genomic sequence with that of the B. amyloliquefaciens-type strain DSM7T revealed significant differences in the genomic sequences of both strains . The strains have in common 3,345 CDS residing in their core genomes; while 547 and 344 CDS were found to be unique in FZB42T and DSM7T, respectively. Notably, the gene clusters encoding non-ribosomal synthesis of antibacterial polyketides difficidin and macrolactin , are absent in DSM7T. For comparison, B. subtilis 168T has a similar number of CDS in common with B. amyloliquefaciens strains DSM7T and FZB42T (3,222 and 3,182 CDS, respectively). Meanwhile, besides FZB42T, the genomes of other B. amyloliquefaciens plantarum strains have become available . The core genome formed by 15 B. amyloliquefaciens plantarum genomes includes 3,151 genes, the pan-genome more than 6,000 genes, suggesting a high degree of flexibility in the genomes of such plant-associated B. amyloliquefaciens strains. Nevertheless, as has been shown in a previous study, the genomes of the plantarum subsp. are well distinguished from the non-plant-associated amyloliquefaciens subsp. . In addition, except DSM7T, the genomes of three other representatives of the subsp. amyloliquefaciens have been published, enabling a comparative genome analysis of plant root-associated and free-living soil B. amyloliquefaciens strains. Fifty-four genes were identified as being unique for subspecies plantarum and did not occur in the free-living soil bacterium B. amyloliquefaciens subsp. amyloliquefaciens, e.g., gene clusters involved in the synthesis of difficidin and macrolactin and in carbon metabolism (Table 1). Genes involved in ribosomal synthesis of several bacteriocins, such as mersacidin , plantazolicin , and amylocyclicin , were detected in several representatives of B. amyloliquefaciens subsp. plantarum but are not part of the plantarum core genome. We conclude that most of the genes unique in subsp. plantarum are involved in plant-bacteria interactions. In order to support this idea, we have performed transposon mutagenesis and transcriptome and proteome analysis of FZB42 exposed directly to plants or plant root exudates ,,. Adding of root exudates up to a final concentration of 250 mg dry weight per liter of culture medium was found sufficient to cause a significant response of the FZB42 transcriptome and proteome during transient growth stage. Among the 302 genes with significantly altered expression by root exudates, 189 were annotated with known functions. The transcription of 46 genes involved in carbon and nitrogen utilization was altered in response to root exudates, with 43 of them being upregulated.
A total of 12 genes encoding enzymes involved in the Embden-Meyerhof-Parnas (EMP) pathway (including pgi encoding for glucose-6-phosphate isomerase) and the TCA cycle were significantly upregulated. Nearly a quarter of the genes with altered transcription (46 out of 189) were involved in uptake or utilization of nutrients. This observation corroborated that root exudates serve as energy sources in the interaction between roots and rhizobacteria. A representative selection of genes involved in plant-bacteria interaction is compiled in Table 2.
Plant growth promoting bacilli engineered for enhanced efficiency
An important feature of plant growth promoting rhizobacteria (PGPRs) is their root colonization activity . After identifying genes involved in root colonization and other plant-bacteria interactions, markerless gene targeting techniques (strains without linked antibiotic resistance marker) are useful techniques in order to generate strains with enhanced rhizosphere competence. Enhanced root colonization and biocontrol activity was gained in B. amyloliquefaciens SQR9 by disruption of the global regulator abrB gene . Other genes, involved in the expression of antimicrobial compounds can also be targeted. The global regulator gene degU was shown to control non-ribosomal synthesis of bacillomycin D  and bacilysin  in FZB42, for example.
Alternatively, reisolating of improved plant growth-promoting strains after being exposed to the natural environment for a distinct time interval, e.g., one vegetation period, is a promising approach . Sequences of the unique restriction modification systems (RM) can serve as a kind of molecular ‘barcode’, facilitating specific strain identification in the environment. In contrast to Pseudomonas fluorescens and some other gram-negative bacteria, bacilli are known as comparable ‘weak’ colonizers of plant root surfaces, and plant growth-promoting bacilli are hardly detected later than 3 months after their application .
We have developed a specific method to detect FZB42 in environmental samples, previously treated with B. amyloliquefaciens (Rhizovital®, ABiTEP GmbH, Berlin, Germany), by combining specific methods of enrichment and molecular detection. Five months after its application in field trials, we took soil samples for reisolating FZB42 derivatives. We obtained colonies with the typical morphology of FZB42, after a complex enrichment procedure consisting of the following steps: (1) resuspending of 2.5-g sample material in 25 ml distilled water under shaking for 2 h, (2) boiling for 1 h, (3) 10 ml of the suspension was added to 40 ml liquid minimal medium with lactose as single carbon source for 1 to 2 days until rod-like bacteria became visible in the microscopic sample, and (4) plating of the 10-1 to 10-5 diluted samples onto lactose (0.1%) minimal agar plates with 0.1% azure dye-stained hydroxyl ethyl cellulose (AZCL HE). Colonies hydrolyzing AZCL and displaying the typical morphology of FZB42 (rough, flat, dendritic, translucent, white) were analyzed after 3 days for presence of an unique 785-bp DNA fragment by PCR using primers PRBrm5215 5′…TGATGGAGTAAATAATAAGGCTGG and PRBrm6000 5′… AATACATCTAAAGTTGCATCCACC. Amplification with two other primer pairs (Table 3) indicating presence of the nrs and pzn gene clusters was also found useful for examining the obtained colonies by either colony PCR or using isolated chromosomal DNA as template. The isolated colonies were examined for their ability to colonize plant roots using microbial standard techniques , and the genomes of the selected clones were sequenced to detect mutations possibly responsible for their improved capability to colonize plant roots. We propagated the most promising clones to obtain samples used in greenhouse and field trials. By using that approach, it was possible to obtain isolates with improved ability to colonize plant roots without engineering their genomes. Field trials to demonstrate enhanced positive response by the plant are underway.
In the following, we describe in more detail, a possibility to obtain more efficient strains by applying genetic engineering techniques in the plant growth-promoting strain FZB42. This work has been performed in the laboratory of Xuewen Gao, Nanjing Agriculture University, China. We have to acknowledge that at present, use of such engineered PGPR strains under field conditions is refused by the public, at least in Europe. However, in light of a steadily increasing world's population growing from 7 billion now to 8.3 billion in 2025 , innovative approaches for getting higher harvest yields without using increasing amounts of agrochemicals should not longer be excluded, given that their use is safe and without harmful consequences for human beings and nature. Careful environmental studies are a precondition before releasing genetic engineered bacteria into the environment.
Case study: expression of the Harpin gene enhances biocontrol activity of FZB42
The plant immune system has gained recognition as a major factor in the growth and development of plants and the resistance to disease, predation, and environmental stress. The hrp (‘harp’) genes encode type III secretory proteins enabling many phytopathogenic bacteria to elicit a hypersensitive response (HR) on non-host or resistant host plants and induce pathogenesis on susceptible hosts. The HR is a rapid localized death of the host cells that occurs upon pathogen infection and, together with the expression of a complex array of defense-related genes, is a component of plant resistance. The plant genes create a cascade of effects which promote a systemic acquired resistance (SAR) throughout the plant. Beneficial effects on plant growth and health have been reported .
The hrp genes were first identified in Pseudomonas syringae pv. phaseolicola, a bean pathogen , and then in the plant pathogen Erwinia amylovora by the group of Steven Beer at Cornell University . An optimized technology for producing the E. amylovora Harpin in a recombinant Escherichia coli strain was subsequently developed . Today, Plant Health Care (PHC) promotes Harpinaß as foliar applicant and seed treatment on the global crop market.
Xanthomonas oryzae pv. oryzicola, the cause of bacterial leaf streak in rice, possesses clusters of hrp genes that determine its ability to elicit a HR in non-host tobacco and pathogenicity in host rice ,. The hpa1 gene of Xanthomonas oryzae pv. oryzae encodes a 13-kDa glycine-rich protein with a composition similar to those of the harpins in Xanthomonas spp. and PopA in Rhizoctonia solanaceum. The hpa1 XooC gene was cloned and expressed in E. coli BL21 . It is a member of the Harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease and insect resistance in plants, and enhancing plant growth. Despite completely different sequence, its function was found very similar to that of the E. amylovora Harpin protein . Transgenic tobacco plants expressing the hpa1 XooC gene were constructed but were found unable to induce hypersensitive cell death (HCD) .
The hpaG XooC gene had been cloned on an expression plasmid in B. subtilis OKB105, a derivative of B. subtilis 168 which is able to produce surfactin  and to colonize plant roots. Application experiments in tomato plants demonstrated that OKB105 expressing HpaGXooC was improved in its biocontrol activity . However, after 100 generations, the HpaGXooC expression plasmid pM43HF is unstable in B. subtilis, which does not allow the use of this system under large-scale conditions in practice . In order to overcome this difficulty, the groups from Nanjing Agricultural University and Humboldt University decided to use the plant growth-promoting model strain FZB42 as a host for establishing a durable and efficient HpaGXooC expression system . In order to avoid proteolytic destruction of the recombinant harpin gene product, we removed the two main extracellular proteases Apr and Npr from FZB42. Chromosomal integration of two hpa1 genes cloned from X. oryzae under the control of the strong P43 promoter allowed stability and constitutive expression of the hpa1 gene product in FZB42. The experiment was described extensively in , but unfortunately in Chinese. Here, we present a short outline.
Briefly, two recombinant Harpin protein integration vectors, pGAprEHS (Figure 1) and pUNprEKHS (Figure 2), were constructed. The two vectors contained the powerful P43 promoter and the nprB signal peptide, which were fused with the gene encoding HpaGXooC. In addition, the vectors, which were unable to replicate freely in Bacillus cells, contained parts of the aprE and nprE sequences of FZB42, allowing their target-specific integration into the FZB42 chromosome. Transformation of pGAprEHS into competent cells of FZB42 resulted in the removal of the two main proteases, AprE and NprE, and yielding FZB42AN (Figure 3A). Subsequently, two copies of the hpa1 gene were inserted into the former protease gene sites in FZB42AN using plasmid pUNprEKHS. The resulting engineered strain FZBHarpin (Table 4) contained two copies of the hpa1 gene and the antibiotic marker fragments (lox-Km, lox-Spec, Cre-lox).
The tobacco (Nicotiana tabacum cv. NC89) seeds were first soaked in FZB-derived strain suspensions with a final concentration of 1 × 108 CFU for 12 h and then disinfested in a 15% (w/v) solution of sodium hypochlorite for 15 min and washed three times with sterilized distilled water. These seeds were sown onto square Petri dishes (10 cm2) containing solidified Murashige medium. Each treatment included five plates with ten seeds each, and the experiment was replicated three times. The petri dishes were incubated in an illuminated incubator (200 μE m−2 s−1 at 25°C) with a 16-h day and 8-h night cycle. The root length was measured after 4 to 5 weeks.
FZB42, FZBAN, and FZBHarpin were cultivated in Landy medium at 30° and 200 rpm. Bacterial cultures were taken after 24, 48, and 72 h, respectively, and were infiltrated into the intercellular space of tobacco leaves (N. tabacum L. ‘xanthi’). The Landy medium and the Harpin protein purified from E. coli (50 ug · ml−1) served as the negative and positive controls, respectively. The development of the HR was registered after incubation for 24 to 36 h at room temperature.
The rice cultivar ‘Fengyou 22’ was used in this study. FZB-derived strains were shaken at 200 rpm at 30°C for 72 h. The culture was adjusted to about 1 × 108 CFU ml−1 with sterile distilled water for use. The rice seeds were surface-sterilized using sodium hypochlorite (15%, v/v) for 15 min, washed three times in sterile water, and allowed to germinate for 2 to 3 days at 25°C. Then, the seeds were soaked in the Bacillus spore suspension at 25°C for 2 h. The seeds were sown into sterile soil pots, containing a mixture of vermiculite and organic manure (1:1, w/w). Five plants are placed in each pot, and each treatment includes ten pots. These pots were cultivated in a greenhouse at 18°C to 30°C. After 45 days, the FZB suspension was diluted to 108 CFU ml−1 and used for spray treatment. After 1 day, the treated pots were inoculated with the bacterial leaf blight pathogen X. oryzae pv. oryzae POX99 (ca. 109 CFU ml−1). Bacterial leaf blight symptoms were assessed 21 days after inoculation. The height of the plants was measured.
Results and discussion
Quantitative real time PCR (qPCR) revealed constitutive expression of the two Harpin genes in the transgenic FZBHarpin strain (Figure 4). Although the harpin gene products were not detected in the supernatant of FZBHarpin by SDS-PA gel electrophoresis, their effect on tobacco plants were clearly visible: HR on tobacco was induced by supernatants taken from FZBHarpin cultures, demonstrating that biological active Harpin protein was secreted into the medium (Figure 5). Moreover, the plant growth-promoting effect of FZB42 was found to be increased in the FZB42Harpin derivative, as demonstrated by enhanced root growth. The average root length in FZB42Harpin was increased by 30% compared to the untreated control (Figure 6).
Greenhouse experiments demonstrated efficacy of FZBHarpin in biocontrolling rice bacterial blight. The control efficacy of FZB42Harpin was 51.9%. In addition, a plant growth-promoting effect by FZB42Harpin exceeding that of FZB42 was also detected (Table 5). Before applying the recombinant FZB42Harpin strain in field trials, removal of the two resistance markers flanked by the Cre-lox recombinase recognition sites via site-directed recombination has to be performed.
Outlook: how to improve acceptance for use of genetic engineered bacteria for enhancing crop yield?
Today, application and release of genetic engineered bacteria directly in the environment is not accepted by the public, and governmental regulations are contradictory for use of such microorganisms in enhancing crop yield. One reason is the presence of resistance genes in transgenic strains, which have been introduced in the bacteria during the allelic replacement process, and methods avoiding use of such marker genes are therefore highly desirable.
Today there are several methods for marker removal available. Wang et al.  developed a simple and efficient B. subtilis genome editing method in which targeted gene(s) could be inactivated by single-stranded PCR product(s) flanked by short homology regions, and in-frame deletion could be achieved by incubating the transformants at 42°C. In this process, homologous recombination was promoted by the lambda beta protein synthesized under the control of promoter PRM in the lambda cI857 PRM-PR promoter system on a temperature-sensitive plasmid pWY121.
Alternatively, site-specific recombination systems are capable of eliminating antibiotic resistance markers, if they are flanked by recombinase recognition sites as it is the case in FZB42Harpin. In a previous study , a Cre-lox setting was established that allowed the efficient removal of resistance genes from the genomes of Staphylococcus carnosus and Staphylococcus aureus. Two cassettes conferring resistance to erythromycin or kanamycin were flanked with wild-type or mutant lox sites, respectively, and used to delete single genes and an entire operon. After transformation of the cells with a newly constructed cre expression plasmid, genomic eviction of the resistance genes was observed in approximately one out of ten candidates analyzed and subsequently verified by PCR. Due to its thermo-sensitive origin of replication, the plasmid can be eliminated at non-permissive temperatures, and markerless deletion mutants can be obtained. Before applying the engineered FZB42Harpin under non-containment conditions, we have to perform marker removal by one of the methods described above.
Of course, marker removal is not the only precondition for improved acceptance of genetically engineered strains when released into the environment. As stated above, careful case studies demonstrating that no harmful effects caused by genetic engineered strains are urgently needed. In applying genetic engineered plant growth-promoting bacteria, we have to distinguish two different levels:
Engineered strains without foreign genes but containing useful mutations in genes affecting the beneficial effect of the bacterium in terms of plant growth promotion and biocontrol of pathogens. Given that no resistance marker has been introduced, it might be unimportant whether the useful mutation has been introduced by a targeted allele exchange or has been evolved after applying a natural selection procedure. We believe that such strains will be accepted in the future when their improved action has been convincingly demonstrated.
Engineered strains containing genes from bacteria. Such bacteria will be considered as ‘recombinant’ , also when the donor bacteria occur in the same natural environment. This was the case in the example described here. Ironically, the harpin gene isolated from a pathogen bacterium was shown to act beneficial when cloned and expressed in FZB42. However, long-term environmental studies are necessary to demonstrate that such recombinant bacteria do not harm the environment by novel recombination events with other microorganisms occurring in the same environment.
Biologicals prepared from beneficial microbes are useful and environmental-friendly tools for developing a sustainable and efficient agriculture. In this context, genomic analysis and genetic engineering of promising beneficial microbes are helpful for obtaining improved bioformulations. This strategy should enable us to save considerable amounts of agrochemicals, especially chemical fertilizers, and chemical pesticides.
JQQ is scientific coworker of Jiangsu Academy of Agricultural Sciences, Nanjing. HJW and RH are graduate students in the laboratory of XWG, Nanjing Agriculture University, Nanjing. RB is Prof. em. of Humboldt University Berlin and Director of Research at ABiTEP GmbH Berlin.
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The work of X. Gao was supported by grants from the National High-Tech R&D Program of China (2012AA101504), the Special Fund for Agro-Scientific Research in the Public Interest (20130315), the National Natural Science Foundation of China (31471811), and the Doctoral Fund of Ministry of Education of China (20100097120011). R.B. wishes to thank for the support given by the European's Seventh Framework Programme (FP/2007-2013) under Grant Agreement no. 312117.
The authors declare that they have no competing interests.
JQQ carried out the molecular genetic studies and application experiments with FZB42Harpin and helped to draft the manuscript. HJW and RH carried out the molecular genetic studies and application experiments with FZB42Harpin. XWG conceived of the study, and participated in its design and coordination and helped to draft the manuscript. RB designed the experiments and wrote the manunuscript. All authors read and approved the final manuscript.