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Discovery of new and efficient genetic engineering technologies for Agrobacterium will broaden the capacity for fundamental research on this genus and for its utilization as a transgenic vehicle. In this study, we aim to develop an efficient recombineering system for Agrobacterium species. We examined isolates of Agrobacterium and the closely related genus Rhizobium to identify pairs of ET-like recombinases that would aid in the recombineering of Agrobacterium species. Four pairs of ET-like recombinases, named RecETh1h2h3h4 AGROB6 , RecETh1h2P3 RHI597 , RecET RHI145 , and RecETh RHI483 , were identified in Agrobacterium tumefaciens str. B6, Rhizobium leguminosarum bv. trifolii WSM597, Rhizobium sp. LC145, and Rhizobium sp. Root483D2, respectively. Eight more candidate recombineering systems were generated by combining the new ET-like recombinases with Redγ or Pluγ. The PluγET RHI145 system, RecETh1h2h3h4 AGROB6 system, and PluγETh RHI483 system were determined to be the most efficient recombineering system for the type strains A. tumefaciens C58, A. tumefaciens EHA105, and R. rhizogenes NBCR13257, respectively. The utility of these systems was demonstrated by knocking out the istB and istA fusion gene in C58, the celI gene in EHA105, and the 3’−5’ exonuclease gene and endoglucanase gene in NBCR13257. Our work provides an effective genetic manipulation strategy for Agrobacterium species. IMPORTANCE Agrobacterium is a powerful transgenic vehicle for the genetic manipulation of numerous plant and fungal species and even animal cells. In addition to improving the utility of Agrobacterium as a transgenic vehicle, genetic engineering tools are important for revealing crucial components that are functionally involved in T-DNA translocation events. This work developed an efficient and versatile recombineering system for Agrobacterium . Successful genome modification of Agrobacterium strains revealed that this new recombineering system could be used for the genetic engineering of Agrobacterium .

BACKGROUND Excessive nitrogen (N) fertilization in glasshouse fields greatly increases N loss and fossil‐fuel energy consumption resulting in serious environmental risks. Microbial inoculants are strongly emerging as potential alternatives to agrochemicals and offer an eco‐friendly fertilization strategy to reduce our dependence on synthetic chemical fertilizers. Effects of a N‐fixing strain Pseudomonas protegens CHA0‐ΔretS‐nif on ginger plant growth, yield, and nutrient uptake, and on earthworm biomass and the microbial community were investigated in glasshouse fields in Shandong Province, northern China. RESULTS Application of CHA0‐ΔretS‐nif could promote ginger plant development, and significantly increased rhizome yields, by 12.93% and 7.09%, respectively, when compared to uninoculated plants and plants treated with the wild‐type bacterial strain. Inoculation of CHA0‐ΔretS‐nif had little impact on plant phosphorus (P) acquisition, whereas it was associated with enhanced N and potassium (K) acquisition by ginger plants. Moreover, inoculation of CHA0‐ΔretS‐nif had positive effects on the bacteria population size and the number of earthworms in the rhizosphere. Similar enhanced performances were also found in CHA0‐ΔretS‐nif‐inoculated ginger plants even when the N‐fertilizer application rate was reduced by 15%. A chemical N input of 573.8 kg ha−1 with a ginger rhizome yield of 1.31 × 105 kg ha−1 was feasible. CONCLUSIONS The combined application of CHA0‐ΔretS‐nif and a reduced level of N‐fertilizers can be employed in glasshouse ginger production for the purpose of achieving high yields while at the same time reducing the inorganic‐N pollution from traditional farming practices. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.