Detection Of ACC Deaminase Genes And The Application Of Bacteria Containing ACC Deaminase

Plants produce high levels of ethylene under abiotic and biotic stresses and inhibit plant growth. Bacteria containing1-aminocyclopropane-l-carboxylate (ACC) deaminase can catalyze the degradation of ACC, the immediate precursor of ethylene, and thus reduce ethylene levels and alleviate the inhibition of plant growth under stress. Bacteria containing ACC deaminase have great application potentials in agriculture, forestry and environmental protection. Therefore, it is important to establish efficient methods to screen and identify bacteria containing ACC deaminase. Our lab has established two methods to screen and identify bacteria containing ACC deaminase. One is a colorimetric method based on ninhydrin reaction with ACC to measure bacterial consumption of ACC. The other one is using the broad-spectrum and specific CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) primers to amplify the ACC deaminase structural gene (acdS). Based on the two methods, a number of bacteria containing ACC deaminase were screened out. In this study, we firstly revised the acdS amplification method and previous results. Secondly, acdS genes were detected from a number of rhizobia isolated from black locust (Robinia pseudoacaciu). Thirdly, some bacteria containing ACC deaminase were identified as pathogens. Fourthly, I made an attempt to detect acdS and ACC deaminase activity from the plant growth-promoting endophytic fungus Pirifurmospora indica. Finally,I tried to combine bacteria containing ACC deaminase with P. indica to protect oil rape from fungal pathogen Sclerotinia sclerotiorum.The new primer CODEHOPacdSr4was designed using the CODEHOP strategy based on the conserved region including the essential Leu322residue of ACC deaminases. Using the CODEHOPacdSrf3/acdSr4primer pair, the amplified fragment covers the conserved region including the essential Glu295residue and thus can tell the true acdS from other homologues. Amplification using CODEHOPacdSf3/acdSr4, sequencing and phylogenetic analysis of acdS from ACC deaminase-containing bacteria previously isolated from rice roots showed that Herbaspirillum and Pseudomonas strains may obtained acdS via past horizontal gene transfer but intergeneric horizontal transfer of acdS did not occur among the bacteria isolated from the same habitat.Ethylene suppresses the root infection and nodulation of rhizobia on legumes. Rhizobia containing ACC deaminase can lower the ethylene level and thus have high infection and nodulation ability. Amplification of acdS from82CCBAU rhizobial strains isolated from black locust was performed using CODEHOPacdSf3/acdSr3and CODEHOPacdSf3/acdSr4primers. Twenty-nine strains containing acdS were screened out. Phylogene analysis of their16S rDNA sequences showed that some of them were novel rhizobia strains, and6strains might acquire their acdS gene through horizontal transfer. A Mesorhizohium strain2-4containing acdS was screened out from25isolates obtained from nodules on black locust growing in Wuhan, Hubei Province. Inoculation experiments showed that the strain2-4nodulated efficiently and greatly increased the dry weight of black locust seedlings. This indicated that acdS-PCR is an effective method in screening efficient rhizobia from a large number of isolates.We made an attempt to screen bacteria promoting the growth of rice plants from the43strains containing ACC deaminase but found that some Burkholderia and Herbaspirillum strains are deleterious to rice seedlings. Further inoculation of the43strains on tobacco leaves, onion bulbs, apicot and kumquat fruits indicated that the Burkholderia and Herbaspirillum strains were pathogenic. These results alert us to detect the pathogenicity of bacteria containing ACC deaminase before application.ACC deaminase activity and accIS were not detected from the P. indica fungus. Therefore, the P. indica-mediated plant growth promotion under stress is not related to ACC deaminase.The Variovorax sp. SaNR1strain contains ACC deaminase and promotes growth of the host Sedum alfredii plants. Variovorax sp. SaNR1and P. indica showed no antagonism towards S. sclerotiorum. Inoculation experiments showed that Variovorax sp. SaNR1promoted oil rape root elongation and may protect the oil rape from S. sclerotiorum. Although P. indica did not protect the oil rape from S. sclerotiorum alone but may control S. sclerotiorum desease together with Variovorax sp. SaNR1.