The Diversity And Pathogenicity Of Burkholderia Cepacia Complex Strains In The Rhizospheres Of Main Cereal Crops

Burkholderia cepacia is a gram-negative bacterium commonly found in soil, Water, plant rhizosphere, patients and hospital environment. It was first described as a phytobacterium. Subsequently, it emerged as an opportunistic human pathogen in hospital, and caused "cepacia sydrome" resulting in death of the patients in China and other countries. However, B. cepacia strains have attracted considerable interest as plant pest antagonists, plant growth-promoting rhizobacteria and bioremediation agents of toxic substances. Recently, B. cepacia has been classified into ten genotypically distinct but phenotypically similar species (genomovars) referring to the B. cepacia complex (Bcc). The current findings showed that the natural environmental may serve as a "reservoir" for pathogenic strains, and the rhizospheres of crop plants were regarded as abundant sources of Bcc strains. However, little information is available for Bcc populations from natural environment, especially in the rhizosphere of major cereal crops in China.The distribution and diversity of Bcc population were surveyed in the rhizospheres of rice, maize and barley with monoculture for several years for understanding Bcc genomovars in different crop rhizosphere and providing key information in risk analysis of Bcc. Based on Bcc isolates from the rhizospheres and clinical sources, the virulence study of the Bcc strains in alfalfa model was performed to detect the virulence degree of different genomovar strains. The pathogenicity of different Bcc genomovar strains was tested on onion bulbls, and antagonistic screening of Bcc isolates against phytopathogenic fungi was also conducted in this study for more promising and safe strains in biocontrol.The RFLP assay with enzyme HaeIII was performed to detect genetic variability among 139 Bcc isolates from the rhizosphere of rice and maize. The results showed that four different RFLP patterns (A, B, H and K) were found among 78 Bcc isolates from rice rhizosphere, and four other different patterns (A, E, I and M) were also detected among 61 Bcc isolates from maize rhizosphere, revealing considerable variability among the Bcc isolates.The identification of Bcc genomovars was performed among 139 Bcc isolates from the rhizosphere of rice and maize, and 14 Bcc isolates from clinical samples by a combination of recA-HaeIII restriction fragment length polymorphism assays, species-specific PCR tests and recA gene sequence analysis. Three Bcc species including B. cepacia, B. cenocepacia recA lineage IIIB and B. vietnamiensis; were recovered from rice rhizosphere. The B. vietnamiensis isolates showed highest numbers with76.9% of the total. To our knowledge, this is the first report of B. vietnamiensis dominating in the rice rhizosphere. B. cenocepacia dominated the maize rhizosphere, followed by B. cepacia, B. pyrrocinia and B. vietnamiensis. It indicated that predominant Bcc species vary dramatically in the rhizosphere of maize and rice. B. cepacia and B. cenocepacia recA lineage IIIA were found among the Bcc isolates from clinical sources and the later was higher with 64.3% of the total.Twenty Bcc isolates sharing the recA RFLP pattern were PCR positive with B. cenocepacia IIIA-specific primers. Two representatives of these isolates, M229 and M279, clustered with B. cenocepacia recA lineage IIID strains with the least robust group within the phylogenetic analysis (BCL≤28%). The existence of a low bootstrap value suggests that further species diversity may be present. These Bcc isolates have therefore not been assigned to a genomovar. One isolate (R456) that gave a unique RFLP profile could not be assigned to a genomovar by recA gene sequence analysis. The recA gene sequence of this isolate had 97% identity to those from several genomovars including B. cenocepacia recA lineage IIIB, B. stabilis and B. pyrrocinia, and it formed a single discrete cluster within the phylogenetic tree. This indicates that the isolate is equally divergent from several of the other genomovars. However, its recA sequence had 99% identity to that of the previously described strain BC14 with which they probably belong to a novel Bcc group.Sixty-seven Bcc isolates from natural environment and clinical samples were tested for virulence in the alfalfa model, and PCR tests were also performed to detect the genes with BCESM and cblA. The results showed that the strains of B. cepacia and B. cenocepacia caused symptoms of yellow leaves, stunted roots, and brown necrotic lesions on alfalfa seedlings with mean percentages of the infected seedlings by B. cepacia and B. cenocepacia from clinical sources being 69% and 68%, respectively. Among the Bcc strains from natural environment, B. cenocepacia recA lineage IIIB strains caused symptoms in 55% of the alfalfa seedlings; Some strains of B. cepacia did not cause symptoms of seedlings; Most strains of B. vietnamiensis and B. pyrronicia did not cause symptoms of seedlings. It indicated that B. cenocepacia recA lineage IIIB strains from natural environment were similar to B. cenocepacia recA lineage IIIA and B. cepacia strains from clinical sources in symptoms of the seedlings, and they may be potential human opportunistic pathogen. The genes with BCESM and cblA did not been detected among these Bcc strains.Twenty representative Bcc strains from'natural environment and clinical sources were tested for hypersensitive reaction (HR) on tobacco and pathogenicity tests on onion. All strains including B. cepacia, B. cenocepacia and B. vietnamiensis, except for one B. pyrrocinia strain, had HR on tobacco, revealing that most Bcc strains may be pathogenic. B. cepacia and B. cenocepacia strains from clinical sources induced symptoms on onion which were similar to those caused by strain LMG1222. It indicated these strains were both human opportunistic pathogen and onion phytopathogen. Among the Bcc strains from natural environment, B. cepacia and B. cenocepacia strains caused symptoms on onion; B. vietnamiensis produced small dot lesions on onion without extending after inoculation for 14 d; Strains of 5. pyrrocinia did not cause symptoms on onion.Seventy-seven Bcc strains were selected to screen for their antagonism against pathogenic fungi of crops in vitro. Thirty-four Bcc strains, 54.5% of the taotal showed antagonistic effect on Fusarium oxysporum f. sp. cucumerinum Owen and Botrytis cinerea. The percentage of antagonistic bacteria was highest in B. vietnamiensis, and lowest in B. cenocepacia; 23 B. vietnamiensis strains significantly inhibited growth of the two pathogens, showing 15-20 mm of inhibition zone. And they also had higher antagonistic activity against Rhizoctonia solania, Phytophthara capsici Leonian and other Fusarium oxysporum. Four antagonistic bacteria were selected to investigate for antagonistic activities of their culture filtrates. It showed that the culture filtrates of the bacteria had certain inhibitory effect on Rhizoctonia solania and Botrytis cinerea, and no effect on Fusarium oxysporium. The four antagonistic bacteria could be considered as safe strains since they were almost aviruience on alfalfa seedlings, not pathogenic on onion and the virulence genes with BCESM and cblA were not detected.