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Molecular profiling of microbial communities and its correlation to environmental parameters of grassland ecosystems
Author
Kee, Jennifer Peck Wai
Supervisor
Chia, Tet Fatt
Abstract
Soil microorganisms are essential in healthy grassland development. They are involved in nutrient cycling; provide resistance to diseases and produce phytohormones to aid plant growth; among other known benefits (Badri, et al., 2009; Spaepen, Vanderleyden, & Remans, 2007). The diversity of soil microorganisms associated with thriving grasslands however remains vague. Studies were conducted on Axonopus compressus, Bermuda tifdwarf and Pennisetum purpureum. These are common tropical grasses, which occupy extensive open spaces, and are important agricultural undercover grasses for crops and golf courses.
Naturally growing A. compressus were assayed for plant, soil physiology and mineral content with two contrasting groups identifiable as healthy and unhealthy. Molecular techniques targeting hypervariable regions on marker genes were used in gene sequencing and length heterogeneity to profile the associated microbial community structures.
946 sequences amplified from the 16S rRNA gene, bacterial intergenic spacer (B-ITS) and fungal intergenic spacer (F-ITS) regions revealed discrete microbial compositions associated with healthy A. compressus in comparison to unhealthy A. compressus. Taxonomical identities were assigned for 39-53% of sequences and showed affiliation to mostly N-fixing microbes. The majority of the sequenced 16S rRNA genes (47-61%) was assigned to uncultured bacteria without any taxonomical assignments and suggests current incomplete knowledge of soil bacteria. As a large proportion of soil microbes were unidentifiable, length heterogeneity polymerase chain reaction (LH-PCR) for 16S rRNA gene and automated ribosomal intergenic spacer analysis (ARISA) for B-IGS and F-ITS regions were explored. Richness, Shannon’s diversity and evenness indices were significantly higher in length heterogeneity than gene identification. Richness was detected at 91-117; 449-500 and 43-97 for LH-PCR; B-ARISA and F-ARISA respectively. Both methods revealed higher bacteria diversity in healthy bulk soil while fungal diversity was highest in unhealthy bulk soil. Also, microbes in healthy A. compressus grasslands were more evenly distributed in speciation.
The impact of chemical additive additions on Bermuda tifdwarf (common turfgrass) and soil microbial composition was examined. Commonly used inorganic fertilizers (Floranid); organic fertilizers (C-Humus); Floranid with additions of iron (Lesco); herbicides (Garlon 250); fungicides (Knockdown 500C) and plant growth regulators (Primo Maxx) were assayed over 20 weeks. Overall health of Bermuda tifdwarf was observed to decline in turf condition with major shifts in microbial diversity at week 20. This was expected as chemical additives required for healthy development was provided in isolation. Knockdown with Floranid however, were effective in maintaining turf conditions with increased bacterial richness and diversity.
Lastly, bacterial communities were investigated in P. purpureum during its recovery from a bush fire that stretched over 8 acres of grassland. P. purpureum grew rapidly and re-occupied the grassland within 28 days. Molecular microbial profiling showed distinct changes. Genus-specific amplification using 16S rRNA gene and BARISA profiles revealed highly unstable microbial community structures between days 4 to 21, which stabilized gradually from day 24 onwards. Spore forming Bacillus and plant pathogen Erwinia species were detected at high relative abundances during the initial recovery from the bush fire while nitrogen-fixing bacteria species (Azospirillum, Bradyrhizobium and Rhizobium) became dominant from day 18 onwards. B-ARISA were high in relative abundances of bacteria species 12 months after bush fire similar to unburnt P. purpureum grasslands.
We can conclude that microbial diversity and community structure are correlated to plant and soil physiologies to health states in grasslands. Healthy grasses had higher microbial richness and diversity (H’) compared to unhealthy grasses. ARISA is effective in distinguishing soil microbial communities in different grasslands compared to marker gene sequence identification.
Naturally growing A. compressus were assayed for plant, soil physiology and mineral content with two contrasting groups identifiable as healthy and unhealthy. Molecular techniques targeting hypervariable regions on marker genes were used in gene sequencing and length heterogeneity to profile the associated microbial community structures.
946 sequences amplified from the 16S rRNA gene, bacterial intergenic spacer (B-ITS) and fungal intergenic spacer (F-ITS) regions revealed discrete microbial compositions associated with healthy A. compressus in comparison to unhealthy A. compressus. Taxonomical identities were assigned for 39-53% of sequences and showed affiliation to mostly N-fixing microbes. The majority of the sequenced 16S rRNA genes (47-61%) was assigned to uncultured bacteria without any taxonomical assignments and suggests current incomplete knowledge of soil bacteria. As a large proportion of soil microbes were unidentifiable, length heterogeneity polymerase chain reaction (LH-PCR) for 16S rRNA gene and automated ribosomal intergenic spacer analysis (ARISA) for B-IGS and F-ITS regions were explored. Richness, Shannon’s diversity and evenness indices were significantly higher in length heterogeneity than gene identification. Richness was detected at 91-117; 449-500 and 43-97 for LH-PCR; B-ARISA and F-ARISA respectively. Both methods revealed higher bacteria diversity in healthy bulk soil while fungal diversity was highest in unhealthy bulk soil. Also, microbes in healthy A. compressus grasslands were more evenly distributed in speciation.
The impact of chemical additive additions on Bermuda tifdwarf (common turfgrass) and soil microbial composition was examined. Commonly used inorganic fertilizers (Floranid); organic fertilizers (C-Humus); Floranid with additions of iron (Lesco); herbicides (Garlon 250); fungicides (Knockdown 500C) and plant growth regulators (Primo Maxx) were assayed over 20 weeks. Overall health of Bermuda tifdwarf was observed to decline in turf condition with major shifts in microbial diversity at week 20. This was expected as chemical additives required for healthy development was provided in isolation. Knockdown with Floranid however, were effective in maintaining turf conditions with increased bacterial richness and diversity.
Lastly, bacterial communities were investigated in P. purpureum during its recovery from a bush fire that stretched over 8 acres of grassland. P. purpureum grew rapidly and re-occupied the grassland within 28 days. Molecular microbial profiling showed distinct changes. Genus-specific amplification using 16S rRNA gene and BARISA profiles revealed highly unstable microbial community structures between days 4 to 21, which stabilized gradually from day 24 onwards. Spore forming Bacillus and plant pathogen Erwinia species were detected at high relative abundances during the initial recovery from the bush fire while nitrogen-fixing bacteria species (Azospirillum, Bradyrhizobium and Rhizobium) became dominant from day 18 onwards. B-ARISA were high in relative abundances of bacteria species 12 months after bush fire similar to unburnt P. purpureum grasslands.
We can conclude that microbial diversity and community structure are correlated to plant and soil physiologies to health states in grasslands. Healthy grasses had higher microbial richness and diversity (H’) compared to unhealthy grasses. ARISA is effective in distinguishing soil microbial communities in different grasslands compared to marker gene sequence identification.
Date Issued
2012
Call Number
QR111 Kee
Date Submitted
2012