Comparison Of The Genomovars Of Burkholderia Cepacia Complex From The Farmland And Hospital

The Burkholderia cepacia, is a gram-negative bacterium widely distributed in agricultural and clinical environment. It has been divided into 9 genomovars called Burkholderia cepacia complex (Bcc). The strains of Bcc had been mainly used as biological control agent in China, though some of them were the onion pathogens or opportunistic pathogens of humans causing "cepacia sydrome" resulted in many deaths of the patients with cystic fibrosis in several countries. It has been known that the strains of plant pathogens, human opportunistic pathogens and biological control agent are undistinguishable. Little is known about the genomovars of Bcc from farmland and hospital in China up-to-now. It is extremely necessary to know the genomovars of Bcc existed in Chinese farmland and determine whether they had the same risk as the dangerous clinical strains.Forty-three samples of soil and irrigation water were collected from the farmland of Guangxi, Zhejiang, Hainan, and Hebei province. Twenty-six isolates from the farmland were identified as Bcc strains after growing on the Bcc selective medium and the phenotypic tests.And 41 clinical isolates from hospital of Liaoning and Zhejiang province were identified as Bcc strains. The phenotypic tests including Biolog and FAMEs only can identify the strains at Bcc level but not the genomovarsThe sequencing analysis of 16S rRNA gene commonly used for idetification of bacteria can not distinguish the genomovars of Bcc due to their high homoeology. Comparison of the identification by PCR with Bcc-specific and. genomovar-specific . primers and sequencing analysis based on recA gene and by Biolog and FAMEs were conducted at the present stduy. The results showed that 59 strains(25 from the farmland and 34 from hospital) out of the 67 strains identified by Biolog and FAMEs were confirmed as Bcc by the molecule biological methods. The misidentification percentage of Biolog and FAMEs were 11.94 %. It has been found that the Bcc strains from the farmland were genomovar I, II, IV and V, and the genomovar I was the most predominant one followed by the genomovar V The Bcc strains from thehospital were genomovar I, II, IIIA, HID, and V, and the genomovarlllA was the most predominant while the genomovar I also had higher isolation frequency in the hospital. PCR with Bcc-specific and genomovar-specific primers based on recA gene were the major methods for primarily identification of Bcc genomovars due to their simple procedure and high reliability. However, the sequencing analysis based on recA still was a important supplementary me"ans for low sensitivity of PCR with some recA gene genomovar-specific primers for Bcc strains from the farmland (eg. genomovar II) and lack of genomovar DC-specific primers. The results also showed that some genomovars had their unique colony characteristics, the colonies of genomovar I were olivaceous, colorless of genomovar IV colonies, and opal of the most genomovarlllcolonies..The results for distinguishing the genomovar I strains of the predominant Bcc from the farmland and the hospital showed that all the methods of RFLP and sequencing analysis of recA gene, and hypersensitive reaction in tobacco could not distinguish them, however, the toxicity to mammal tested by alfalfa plant model, the replacement of mammal model, could distinguish the virulent and avirulent ones, which laid the firm foundation for us to safely use the Bcc strains in biocontrol and bioremediation. It was the first report of distinguishing the genomovar I strains of Bcc from the two different sources by using alfalfa plant model to test the toxicity to mammal and the further study on its mechanism was necessary. The toxicity to mammal of the different Bcc genomavars from the farmland was firstly studied in China by alfalfa model, the replacement of mammal model. It showed that the toxicity of the Bcc strains from the farmland was distinctly weaker than the clinical strains. The disease infection percentage of the all Bcc strains from the farmland tested including the genomovar I, H and genomovar IVon the alfalfa seedlings did not show significant difference compared with the check at P=l% level. The percentage of the all Bcc clinical strains of genomovar I and genomovar III on the alfalfa seedlings showed significant difference compared with the check at P=l% level. The infection perentage of the Bcc strains of genomovar V from the farmland on the alfalfa seedlings was variable, 4 of them showed significant difference compared with thecheck at P=l% level and 3 of them were not. However, all the Bcc strains of genomovar V from the farmland showed significant difference in the disesaese infection compared with the Bcc strains of same genomovar from the clinical at P=l% level. Fifty-nine Bcc strains were tested for their antagonism against the 3 phytobacterial and 1 fungal pathogens in vitro. It revealed that the antagonism of the Bcc strains against the phytobacteria was low and higher against the fungal pathogen. The strain 91 from the farmland showed safety to mammal by test on the plant model and high effectivity in suppressing the Botrytis cinerea, causing vegetable gray mold had high potential to develop a biological control agent.