| Mangrove has important ecological, economic and social benefits. Because of long-term human damage, the mangrove area reduced, stand quality decreased, and protective effects declined. Study on PGPR of mangrove afforestation species is helpful to utilize mangrove microbial resources, and develop effective bacteria inoculants based on excellent strains. Inoculation of PGPR promotes the growth of mangrove plants and enhances their resistance. This utilization of inoculated seedlings should increase the survival rate of mangrove reforestation and promote the construction of coastal shelter forest system. The present study tries to screen and identify PGPR strains from the rhizosphere of mangrove plants, evaluate their inoculation effects, develop PGPR microbeads inoculants by using advanced experimental techniques and analysis methods, such as conventional bacteria selection and identification methods, acetylene reduction method, molybdenum-antimony anti-colorimetry, Salkowski colourimetry, Biolog automated microbial identification system, 16s rDNA sequence analysis. The results obtained were as follows:1. There were abundant PGPR strains colonized on rhizosphere of mangrove afforestation species. Plant samples were collected for PGPR screen from Zhanjiang, Zhu hai and Dongzhai Harbor. Total 20 nitrogen fixing strains and 35 phosphorus solubilizing strains were isolated from 6 mangrove species, including Bruguiera gymnorrhiza,Kandelia candel,Sonneratia apetala,Rhizophora stylosa,Bruguiera sexangula and Sonneratia caseolaris. According to identification, seven excellent nitrogen fixing strains belonged to Azotobacter (1 strain),Pseudomonas (1strain),Bacillus(4 strains),Ochrobactrum(1 strain). And seven excellent phosphorus solubilizing strains belonged to Pseudomonas (1 strain) and Bacillus (6 strains).2. Using modified OAB medium, 20 nitrogen fixing strains were screened from the the rhizosphere of mangrove plants. Most of these strains showed positive nitrogenase activity ranged from 50.72 to 385.6 nmol C2H4·h-1·mL-1. Only two strains of NGWB4-wy1 and NGHHL2-r14 had their C2H4 production higher than 200 nmol C2H4·h-1mL-1. A batch of (35 strains) phosphorus solubilizing strains with different phosphorus solubilizing abilities, were isolated by using modified SRMS1 medium. There were three strains with marked phosphorus solubilizing intensities, including PGHHL-y7 180.2 mg·L-1 ) , PGWB-y2(158.9 mg·L-1), PGWB-wp5(153.2 mg·L-1). Results of Salkowski colourimetry showed that all tested strains ( i.e. 9 nitrogen fixing strains and 9 phosphorus solubilizing strains ) could secrete IAA, which ranged from 11.40 to 14.10μg·mL-1.3. On the basis of nitrogenase activity and IAA secretion, 15 strains of nitrogen fixing bacteria were selected for single-inoculation pot test. The results showed that inoculation of nitrogen fixing strains significantly increased the height, biomass, nitrogen and phosphorus levels of K. candel and B. gymnorrhiza seedlings. According to different performances on growth indicators, nitrogen and phosphorus levels, 7 strains were screened with marked effects on growth promotion, including NGWB3-w14, NGWB4-wy1, NLQQ2-w14, NGHHL2-r14, NLQQ2-r14, NGWB2-y1, NLQQ3-wy4. As to the K. candel seedlings, height increased 30.09%~65.19%, below-ground biomass increased 61.64%~79.13%, above-ground biomass increased 55.97%~75.21% compared to the control. As to the B. gymnorrhiza seedlings, the corresponding values were 11.98%~23.81%, 37.93%~51.90%, 38.00%~63.78% respectively.4. On the basis of phosphorus solubilizing activity and IAA secretion, 16 strains of phosphorus solubilizing bacteria were selected for single-inoculation pot test. The results showed that inoculation of phosphorus solubilizing strains significantly increased the seedling growth, nitrogen and phosphorus levels of K. candel and B. gymnorrhiza. According to different performances on growth indicators, nitrogen and phosphorus levels, 7 strains were screened with marked effects on plant growth promotion, including PGWB-wp5, PGHHL-y7, PGWB-y8, PLQQ-y12, PGWB-y2, PGML-y1, PGWB-y6. As to the K. candel seedlings, the height increased 31.92%~45.61%, below-ground biomass increased 49.28%~70.56%, above-ground biomass increased 45.80%~67.71% compared to the control. As to the B. gymnorrhiza seedlings, the corresponding values were 20.17%~26.79%, 34.87%~51.20%, 55.92%~67.53%.5. Inoculation effects of PGPR strains on cold resistance of Avicennia germina seedlings were studied through the outdoor pot experiment. Inoculation not only promoted seedling growth but also enhanced the cold resistance of A. germina. The mortality rate of the control group was 60%, while those treatment groups 10% ~ 50%.6. Using the interactive combinations of nitrogen-fixing strain and phosphorus solubilizing strain, mixed inoculation pot experiments were conducted in greenhouse. Results showed that mixed inoculation could promote the growth of K. candel and B. gymnorrhiza seedlings significantly. According to different performances on growth indicators, nitrogen and phosphorus levels, there were 3 strain combinations having marked growth promoting effects, i.e.Group15 ( NGHHL2-r14+ PGWB-y8+ NGWB3-w14+ PGML-y1),Group 6(NGWB2-y1+ PGHHL-y7),Group 9(NLQQ2-w14+ PGWB-wp5. As to the K. candel seedlings, height increased 30.09%~65.19%, basal diameter increased 10.03%~13.27%, below-ground biomass increased 85.90%~99.33%, total biomass increased 116.8%~131.8%, leaf area increased 93.66%~105.2% compared to the control. As to the B. gymnorrhiza seedlings, the corresponding values were 22.60%~24.38%, 9.85%~10.92%, 101.0%~106.6%, 97.17%~116.6%, 74.53%~85.47%.. 7. Mixed inoculation experiments were carried out in mangrove nursery. Results showed that mixed inoculation promoted the seedling growth of K. candel and B. gymnorrhiza significantly. By analysis of variance and multiple comparisons, the growth indicators, nitrogen and phosphorus levels of these inoculated seedlings had significant differences compared to the control. The growth-promoting effect was the best when treated with Group15. As to the K. candel seedlings, height increased 25.76%, below-ground biomass increased 42.22%, total biomass increased 47.70% compared to the control. As to the B. gymnorrhiza seedlings, the corresponding values were 15.15%, 35.50%, 42.20%. 8. Microbead inoculants were explored according to the piercing-solidifying method. A special equipment for the production of microbeads was processed and manufactured, and a complete technological process was formed. Several single-factor tests were conducted to determine suitable conditions for microbead production. The results were as follows, 2.5% (mass fraction) sodium alginate, 2.0% (mass fraction) CaC12, with the volume ratio of bacterium solution and Sodium Alginate at 1:2, with the curing time of 1h. The validity of PGPR microbead inoculants reached at least 6 months after drying process. Microbead inoculants could colonize in the rhizosphere of K.candel, and release bacteria slowly. According to the pot experiments, effects of microbead inoculants on the plant growth promoting were better than those of liquid inoculants.
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