Plant Growth Promotion Assessment in Rice Plant Enhanced by Inoculation of Rhizobacteria

In the search of efficient biofertilizer, nine efficient strains of PGPR were evaluated by inoculation in two different rice varieties, Saryu-52 and Malviya Dhan-36 in the gnotobiotic conditions using two different media FCN-medium and soil extract medium. Agrobacterium sp. strain BN-2A showed better result in respect to other eight isolates in the total length, fresh weight, number of roots and chlorophyll-a content. By the inoculation Agrobacterium sp. strain BN-2A, total length increased in Saryu-52 (22.6%) and in Malviya Dhan-36 (52.1%), fresh weight increased in Saryu-52 (42.4%) and in Malviya Dhan-36 (68.8%) and chlorophyll-a increased in Saryu-52 (76.6%) and in Malviya Dhan-36 (37.1%). Similarly in soil extract medium, inoculation of Agrobacterium sp. strain BN-2A alone showed better result in comparison to mixture of nine strains. To prove that colonization indeed occurs, gusA reporter gene was tagged with the most efficient isolate Agrobacterium sp. strain BN-2A and colonization in the rice root was confirmed by gusA staining and histochemical analysis of gusA staining. Therefore, BN-2A has best potential to be used as biofertilizer.DOI: http://dx.doi.org/10.3126/av.v4i0.12362Academic Voices Vol.4 2014: 73-84

THE USE OF gusA REPORTER GENE TO MONITOR THE SURVIVAL OF INTRODUCED BACTERIA IN THE SOIL

An effective marker to monitor the survival of introduced bacteria in the soil is required for further evaluation of their beneficial effects on plant growth. This study tested the use of gusA gene as a marker to trace the fate of three Gram negative bacteria in the root, rhizosphere, and soil. The study was conducted at the laboratory and greenhouse of the National Institute of Molecular Biology and Biotechnology, Philippines from January to December 2001. Isolates TCaR 61 and TCeRe 60, and Azotobacter vinelandii Mac 259 were selected as test bacteria based on their ability to produce indole-3acetic acid and solubilize precipitated phosphate, which may promote plant growth in the field. These bacteria were marked with gusA reporter gene from Escherichia coli strain S17-1(λ-pir) containing mTn5SSgusA21. The gusA (β-glucuronidase) gene from the donor (E. coli) was transferred to each bacterium (recipient) through bacterial conjugation in mating procedures using tryptone-yeast agar followed by the selection of the transconjugants (bacteria receiving gusA) in tryptone-yeast agar supplemented with double antibiotics and X-GlcA (5bromo-4chloro- 3indoxyl-β-D-glucuronic acid). The antibiotics used were rifampicin and either streptomycin or spectinomycin based on antibiotic profiles of the donor and recipients. The results showed that the insertion of gusA gene into bacterial genomes of the recipient did not impair its phenotypic traits; the growth rates of the transconjugants as well as their ability to produce indole-3acetic acid and solubilize precipitated phosphate in pure culture were similar to their wild types. All transconjugants colonized the roots of hot pepper (Capsicum annuum L.) and survived in the rhizosphere and soil until the late of vegetative growth stage. The distinct blue staining of transconjugants as the expression of gusA gene in media containing X-GlcA coupled with their resistance to rifampicin and streptomycin or spectinomycin made them easier to be recognized and evaluated.<br /><br />

THE USE OF gusA REPORTER GENE TO MONITOR THE SURVIVAL OF INTRODUCED BACTERIA IN THE SOIL

An effective marker to monitor the survival of introduced bacteria in the soil is required for further evaluation of their beneficial effects on plant growth. This study tested the use of gusA gene as a marker to trace the fate of three Gram negative bacteria in the root, rhizosphere, and soil. The study was conducted at the laboratory and greenhouse of the National Institute of Molecular Biology and Biotechnology, Philippines from January to December 2001. Isolates TCaR 61 and TCeRe 60, and Azotobacter vinelandii Mac 259 were selected as test bacteria based on their ability to produce indole-3acetic acid and solubilize precipitated phosphate, which may promote plant growth in the field. These bacteria were marked with gusA reporter gene from Escherichia coli strain S17-1(λ-pir) containing mTn5SSgusA21. The gusA (β-glucuronidase) gene from the donor (E. coli) was transferred to each bacterium (recipient) through bacterial conjugation in mating procedures using tryptone-yeast agar followed by the selection of the transconjugants (bacteria receiving gusA) in tryptone-yeast agar supplemented with double antibiotics and X-GlcA (5bromo-4chloro- 3indoxyl-β-D-glucuronic acid). The antibiotics used were rifampicin and either streptomycin or spectinomycin based on antibiotic profiles of the donor and recipients. The results showed that the insertion of gusA gene into bacterial genomes of the recipient did not impair its phenotypic traits; the growth rates of the transconjugants as well as their ability to produce indole-3acetic acid and solubilize precipitated phosphate in pure culture were similar to their wild types. All transconjugants colonized the roots of hot pepper (Capsicum annuum L.) and survived in the rhizosphere and soil until the late of vegetative growth stage. The distinct blue staining of transconjugants as the expression of gusA gene in media containing X-GlcA coupled with their resistance to rifampicin and streptomycin or spectinomycin made them easier to be recognized and evaluated.<br /><br />

Patterns of Expression of a Lolitrem Biosynthetic Gene in the Epichloë festucae–Perennial Ryegrass Symbiosis

Lolitrem B is synthesized by Epichloë festucae in associations with Pooid grasses. A complex cluster of at least 10 genes (ltm genes) is required for its synthesis. An early step in this pathway is catalyzed by ltmM, a symbiosis-expressed gene. PltmM-gusA reporter gene analysis was used to monitor ltmM gene expression patterns in planta. The minimum promoter length required for high-level gusA expression in infected seedlings is in the range of 480 to 782 bp. gusA was expressed by the endophyte in all infected vegetative plant tissues and in epiphyllous hyphae. Spikelets from reproductive tillers were analyzed at different developmental stages. During pre-anthesis, gusA expression was observed in all infected floral organs except the immature gynoecium. In post-anthesis florets, gene expression occurred almost exclusively in the gynoecium. Expression of gusA by the endophyte was observed in germinating seeds 24 h postimbibition and seedlings older than 6 days postimbibition in hyphae from the mesocotyl to the tip of the emerging first leaf. This work provides a detailed analysis of the spatial and temporal expression patterns of a symbiosis-expressed gene in planta.

Auxin induction is a trigger for root gall formation caused by root-knot nematodes in white clover and is associated with the activation of the flavonoid pathway

We studied the expression of the auxin responsive promoter (GH3) fused to the gusA reporter gene in white clover (Trifolium repens cv. Haifa) during the initiation of root galls by root-knot nematodes (Meloidogyne javanica) to investigate whether nematode infection affects auxin distribution in developing galls. In search for a plant signal that would mediate changes in auxin location we studied the induction of the flavonoid pathway because flavonoids can act as auxin transport regulators. Three chalcone synthase (CHS1, CHS2 and CHS3) promoter:gusA fusions were examined in transgenic plants and flavonoids were detected using fluorescence microscopy. Within 24 h post inoculation CHS:gusA expression occurred around the invading nematode. At 48 h post inoculation CHS:gusA expression and flavonoids were detected throughout the infection site, followed by high GH3:gusA expression in the gall 48–72 h post inoculation. Initially (48–72 h post inoculation) high GH3:gusA expression in giant cell precursors was followed by low expression in the enlarging giant cells (96–120 h post inoculation), suggesting that auxin is needed as a trigger for giant cell initiation but not for later enlargement. We suggest that nematodes control auxin distribution in the root and that flavonoids could be responsible for controlling auxin accumulation.