cellulase and diacetylfluorescein

Degradation of soil properties following deforestation and long-term soil cultivation may lead to decreases in soil microbial diversity and functional stability. In this study, we investigated the differences in the stability (resistance and resilience) of microbial community composition and enzyme activities in adjacent soils under either native tropical forest (FST) or in agricultural cropping use for 14 years (AGR). Mineral soil samples (0 to 5 cm) from both areas were incubated at 40 degrees C, 50 degrees C, 60 degrees C, or 70 degrees C for 15 min in order to successively reduce the microbial biomass. Three and 30 days after the heat shocks, fluorescein diacetate (FDA) hydrolysis, cellulase and laccase activities, and phospholipid-derived fatty acids-based microbial community composition were measured. Microbial biomass was reduced up to 25% in both soils 3 days after the heat shocks. The higher initial values of microbial biomass, enzyme activity, total and particulate soil organic carbon, and aggregate stability in the FST soil coincided with higher enzymatic stability after heat shocks. FDA hydrolysis activity was less affected (more resistance) and cellulase and laccase activities recovered more rapidly (more resilience) in the FST soil relative to the AGR counterpart. In the AGR soil, laccase activity did not show resilience to any heat shock level up to 30 days after the disturbance. Within each soil type, the microbial community composition did not differ between heat shock and control samples at day 3. However, at day 30, FST soil samples treated at 60 degrees C and 70 degrees C contained a microbial community significantly different from the control and with lower biomass regardless of high enzyme resilience. Results of this study show that deforestation followed by long-term cultivation changed microbial community composition and had differential effects on microbial functional stability. Both soils displayed similar resilience to FDA hydrolysis, a composite measure of a broad range of hydrolases, supporting the concept of high functional redundancy in soil microbial communities. In contrast, the resilience of the substrate-specific activities of laccase and cellulase were lower in AGR soils, indicating a less diverse community of microorganisms capable of producing these enzymes and confirming that specific microbial functions are more sensitive measurements for evaluating change in the ecological stability of soils.

Topics: Agriculture; Biomass; Brazil; Cellulase; Ecosystem; Enzyme Stability; Fluoresceins; Hot Temperature; Laccase; Soil; Soil Microbiology; Trees

The development of novel strategies against plant viral diseases relies on a better understanding of molecular virus-host interactions. Here, we report an easy, efficient and reproducible protocol for Arabidopsis protoplast isolation and transfection to study the infection and replication of a potyvirus, Plum pox virus (PPV). Macerozyme and cellulose were used to release protoplasts from Arabidopsis leaf tissues, and polyethylene glycol-mediated DNA uptake was employed for transfection of a PPV infectious clone. Protoplast viability was monitored by fluorescein diacetate staining, and transfection efficiency was estimated by transient expression of the green fluorescent protein. The protocol allowed production of 95% viable mesophyll protoplasts and a successful transfection rate of 35%. The system was used further in a time-course experiment to investigate PPV viral RNA accumulation. It was found that 3 h post-transfection (hpt) in the transfected protoplasts viral RNA increased by about 150-fold and progressively accumulated to reach the maximum at 12 hpt. Viral RNA then decreased dramatically at 24 hpt reaching 40% of its peak level. Considering the availability of the whole genome microarrays, and other genomic resources of Arabidopsis, the synchronized single-cell (protoplast) infection system will be useful for elucidating early molecular events associated with PPV infection.

Topics: Arabidopsis; Cellulase; DNA, Viral; Fluoresceins; Fluorescent Dyes; Genes, Reporter; Green Fluorescent Proteins; Kinetics; Microscopy, Confocal; Plant Diseases; Plant Leaves; Plum Pox Virus; Protoplasts; RNA, Viral; Staining and Labeling; Transfection