Preparation And Evaluation Of An Engineered Pseudomonas As Microbial Inoculation In Field Trials

Garlic and ginger are important flavoring agents used for culinary purposes with considerable economic benefits,which have been widely planted in China for more than two thousand years.Although garlic and ginger have a long history of tillage in China,there has been a lack of scientific and systematic cultivation management and practices.Excessive synthetic chemical fertilizers and pesticides usage is a common practice among farmers for achieving short-term economic benefits in recent years,which not only affected the quality of garlic and ginger,but also weaken the disease resistance of plants.Moreover,various environmental issues,such as soil degradation and agricultural diffuse pollution,resulted from long-term continuous cropping have became a threat to food scarcity and agricultural sustainable development.Microbial inoculants commonly used as biofertilizers or biocontrol agents are made up of one or more specific and cultivable beneficiary microbes,which can promote plant growth or disease prevention by both direct and indirect mechanisms.Microbial inoculation is less harmful to the environment and less expensive than synthetic chemical fertilizers,which offers a reliable alternative to the use of agrochemicals in agricultural practices.Pseudomonas protegens is a well-characterized plant growth-promoting strain and an efficient biocontrol bacterium.P.protegens CHAO can colonize the rhizosphere of a wide range of hosts,however,it does not contain the nitrogenase enzyme system.Our previous studies have obtained an genetically engineered strain CHA0-ΔretS-nif with an introduced 49 kb nif gene island isolated from Pseudomonas stutzeri DSM 4166 and the retS gene deletion in chromosome.The recombinant derivative had displayed high levels of nitrogenase activity and improved bactericidal ability.This study aims to ascertain whether the CHA0-ΔretS-nif inoculation is effective to enhance garlic and ginger production using mass-produced liquid microbial agents.Experiments for garlic were performed from October 8,2017 to May 23,2018 in Yutai County,Jining City,Shandong Province,China.CHA0-ΔretS-nif inoculation caused an evident increase in plant growth when compared to the uninoculated control group.The growth-promoting traits had a significant impact on garlic production（P<0.05）.The yields of garlic bulbs and sprouts in the CHA0-ΔretS-nif-inoculated group had an increase of 10.93%and 17.99%,respectively,when compared to the control.No significant differences were noted in the contents of vitamin C,nitrate and soluble solids in garlic cloves between the groups,however,an accumulation of allicin was improved by CHA0-ΔretS-nif inoculation.When compared to the control,the content of allicin in the CHA0-ΔretS-nif-inoculated group significantly increased by 17.86%（P<0.05）.Root rot is a common disease that has significant effects on garlic yields and economic income.Disease index in the CHA0-ΔretS-nif inoculated group（2.65%）was significantly lower than the control（3.31%）（P<0.05）.Overall,CHA0-ΔretS-nif inoculation significantly improved the growth of garlic plants,increased the yields of garlic bulb and sprout,displayed inhibition of root rot and enhanced allicin content in garlic cloves.When the chemical N application rate decreased by 25%,disease index in the CHA0-ΔretS-nif-inoculated group decreased to 2.72%,and the yields of garlic bulbs and sprouts and the content of allicin in garlic cloves were increased by 10.55%,11.30%and 14.29%,respectively,when compared to the control.Results indicated that CHA0-ΔretS-nif inoculation can partially substitute chemical N-fertilization,and contribute to plant growth and N production efficiency,and there was no loss in the quality of garlic bulbs.Moreover,the application of CHA0-ΔretS-nif with a decreased level of chemical N-fertilizer significantly increased the total microbial numbers and the bacterial population size in the garlic rhizosphere,when compared to the control（P<0.05）,indicating that CHA0-ΔretS-nif inoculation can improve the rhizosphere microbial community structure and bacterial population size.Ginger trials were performed from April 2,2018 to October 15,201 8 in Beimeng County,Changyi City,Shandong Province,China.CHA0-ΔretS-nif inoculation led to an evident increase in plant growth when compared to the uninoculated control group.The total yields of ginger in the CHA0-ΔretS-nif-inoculated group had an significant increase of 12.93%compared to the control（P<0.05）.CHA0-ΔretS-nif inoculation also led to significant effects on whole plant N and K acquisition.After harvest,whole plant N and K acquisition significantly increased by 29.05%and 23.19%,respectively,when compared to the control（P<0.05）.However,significant effects were not found between the groups on plant P content.The total protein content in ginger rhizomes of CHA0-ΔretS-nif-inoculated group significantly increased by 34.65%compared to the control（P<0.05）.No significant impacts were found between the groups on the contents of polyphenol,flavonoid,starch,and soluble sugars in ginger rhizomes.The yield of ginger,whole plant N and K accumulation,and the total protein content in ginger rhizomes in the CHA0-ΔretS-nif inoculated group increased by 3.33%,18.17%,28.04%and 31.26%,respectively,compared to the control even when the chemical N application rate decreased by 15%.However,the beneficial increment in ginger yield was not found when the chemical N inputs decreased by 30%;which suggested that CHA0-ΔretS-nif inoculation can partially substitute chemical N-fertilization and plant growth would be suppressed due to excessive reduction of chemical N-fertilizer inputs.Furthermore,the application of CHA0-ΔretS-nif with a decreased level of N-fertilizer significantly increased the maximal numbers of earthworms,microbes and bacteria at the rhizosphere soil of ginger plant compared to the control（P<0.05）.These results indicated that CHA0-ΔretS-nif inoculated plants can achieve more N that provided by biological nitrogen fixation,and genetically modified CHA0-ΔretS-nif can be used as microbial inoculants for vegetable farming,which is less harmful to the environment,to mitigate the inorganic-N pollution from traditional farming practices.Agrobacterium-mediated plant transformation is a crucial way for crop genetic manipulation,which has been established can increase yield and quality of crop plants.A.rhizogenes within the family Rhizobiaceae,now renamed as Rhizobium rhizogenes,also has the ability to infect wounded higher plant cells,which is widely used for the construction of transgenic plants with inheritable features.However,the fundamental biological mechanisms of various functions of R.rhizogenes involved in plant genetic transformation are still unknown,and genetic engineering in R.rhizogenes is difficult due to the absence of an efficient recombineering technology.Red/ET recombineering can catalyze accuracy and fluency homologous recombination in Escherichia coli by use of linear DNA substrates,e.g.PCR products,double-stranded or single-stranded synthetic oligonucleotides,flanking short homology arms.Red/ET recombination has a promising application to manipulate E.coli chromosomes and high copy plasmids.However,adapting the phage lambda Red system to different bacteria is problematic.Host-specific phage-derived recombineering systems have been established in some other species extending this DNA engineering technology to interesting new hosts and promoting the development of bacterial genome editing.This study aims to establish a powerful Red-like recombineering system in R.rhizogenes type strain NBRC 13257 for bacterial genetic manipulation and functional analysis based on the expression of a pair of RecET-like recombinases derived from its closely related species,and provide efficient and versatile genetic engineering tools for Agrobacterium species.Discovery of a new and efficient genetic engineering technology for R.rhizogenes will broaden the capacity for fundamental research on this genus and for its usage as a transgenic vehicle.Since the lambda Red system can not perform efficiently in R.rhizogenes NBRC 13257 and orthologs to Redβ/β or RecE/T were not identified in genomes of NBRC 13257,genomes and phage genomes of Agrobacterium and Rhizobium were searched for potential recombinases with PSI-BLAST program in the nonredundant database of NCBI using the coding sequences of Redβ,RecT,and Pluβ as queries.RecE-like 5’-3’ exonuclease and RecT-like annealing protein involved in recombineering are usually located within an operon.Four recombinase operons were chosen for establishing a robust and efficient recombineering system for R.rhizogenes.The four operons also contain genes encoding some proteins with unknown functions,which are collectively referred to as hypothetical proteins.All recombinase operons were constructed based on broad-host-range expression vectors with the recombinase genes under the control of the tetracycline-regulated promoter.Transformation efficiency determines optimal recombineering and thus the electroporation method of NBRC 13257 was optimized.The highest transformation efficiency was obtained using culture entered log phase（OD600 reaching around 0.4-0.6）and electrocompetent cells were prepared using SH buffer（10%sucrose+2μM HEPES）at room temperature.The optimal electric field strength was 1900 V mm-1.When the dosage of foreign transferred DNA was 1500 ng,the transformation efficiency of NBRC 13257 was 2.6×105 transformants per μg of DNA by using the optimized transformation conditions.The recombinase expression plasmids used to assess homologous recombination efficiencies were transformed into NBRC 13257,respectively.Only the RecEThRHI483 system exhibited recombination activity between dsDNA substrates flanked by 80 bp homology arms in NBRC 13257.Addition of Redy or Pluy only slightly increased the recombineering efficiency of the RecEThRHI483 system.Deletion of the hypothetical protein of the RecEThRHI83 operon resulted in a decrease of the recombination efficiency by about half in NBRC 13257.Finally,genome modifications of NBRC 13257 and two gene deletion mutants were successfully achieved by using the newly developed P1uγEThRHI483 system and PCR substrates flanked by 80 bp homology arms.The most suitable induction temperature and time were 28℃ and 0.5 h for the recombineering system.