Studies On Biocontrol Bacteria Of Pine Seedlings Damping-off And Patterns Of Their Colonization

Slash pine(Pinus elliottii) is an important afforesting tree species in Hubei Province and in the southern China. The turpentine is also the important raw chemical material. During slash pine seedling production, damping-off can often cause severe damages to seedlings production and afforesting quality because of its endangering. Studies from China and other countries indicated that pine seedling damping-off is caused by a number of fungal pathogens including genera Rhizoctonia, Pythium, Fusarium, Alternaria, Botrytis, and Pestalotia etc. The kinas of fungal pathogens vary in different areas and localities. The objectives of this study were to study dominant pathogens of pine seedling damping-off; to determine the distribution and variation of rhizobacteria; using Pseudomonas fluorescens and Burkholderia cepacia and other introduced and native biocontrol agents, to elucidate colonization of biocontrol bacteria in rhizosphere, and to evaluate the effectiveness of disease suppression of the biocontrol bacteria with molecular technology. Slash pine seedlings were used as host plants in this study. Results and conclusions of the research are as follows:(1) Distribution and population dynamics of microorganisms in pine seedling rhizospherc.The large quantities of microorganisms were present in the soil of slash pine rhizosphere. Populations of fungi were 1×10~5～3.5×10~6 CFU(colony forming units)/g of soil, but populations of bacteria were 10~7～10~8CFU/g of soil. The peak of fungal population was in September, but that of bacteria is in April after transplantation. Proliferation of bacteria is faster than fungi, and the time of bacterial colonization on rhizoplane was also earlier than fungi.(2) Dominating pathogenic fungi of slash pine seedling damping-off and their pathogenicities.The predominant pathogens in slash pine nurseries of Hubei Province were Rhizoctonia solani Kühn, Fusarium oxysporum(Fr.) Keissl. and Alternaria alternata Schl.. R. solani was the most virulent, and its infection rate reached 92.6% at 7th day. F. oxysporum is the second and the highest infection rate is 89.5% at 7th day. The pathogenicity of A. alternata was 48% at 7th day and weaker than other pathogens, but able to reach to 80% at 10th day.(3) Screening and identifying biocontrol bacteria. We selected 6 strains of the strongest virulent pathogens in genus Fusarium as target. Antagonistic test was conducted on plates, 320 bacteria isolates originated from slash pine rhizosphere were used to screen antagonists. We gained 27 antagonistic bacteria strains by selective media, wherein 12 strains were weak antagonist, 5 strains, intermediate antagonists and 10 strains, strong antagonists. Then 10 strong antagonistic strains were identified by 16S rDNA(ribosomal DNA) sequence analysis. The results were 7 strains of Burkholderia cepacia Yabuuchi et al., 2 strains of Bacillus subtilis(Ehrenberg) Cohn and one unknown strain.(4)Colonization patterns of bioeontrol bacteria in the pine seedling rhizosphere.We examined the antagonism on plates using 8 strains of Pseudomonas fluorescens Q2-87, 1M1-96, HT5-1, Pf-5, MVP1-4, FFL1R8, 2-79, Q37-87 introduced from the USA and one indigenous Burkholderia cepacia strain C23 from pine nurseries. The target pathogenic fungi are Rhizoctonia solani A, B, Fusarium oxysporum and Alternaria alternata. Results confirmed that all biocontrol bacteria suppressed Alternaria and Rhizoctonia well, but showed weak antagonism against Fusarium. C23 and Q2-87 were assured to be better antagonist against Rhizoctonia and Alternaria. After inoculation on root in the greenhouse and field, the results applying molecular assay(terminal dilution assay) showed that both Pseudomonas fluorescens Q2-87 and Burkholderia cepacia C23 colonized well.This study had screened and obtained the indigenous biocontrol bacteria from pine seedling rhizosphere, achieved the assured antagonistic effects in the greenhouse and field, primarily eluciated the colonizing patterns of biocontrol bacteria. It is an important progress about biocontrol researches of pine seedling damping-off in China. The results of this study established the foundation of exploiting biological control and solving the difficult problem of controlling pine seedling damping-off. Furthermore, biocontrol research of pine damping-off will be helpful to promote biocontrol of forest root disease into a higher level.