Effect of an AM fungal consortium and Pseudomonas on the growth and nutrient uptake of Eucalyptus hybrid

 Eucalyptus is an important tree species used for afforestation of large tracts of marginal and wastelands. Eucalyptus-arbuscular mycorrhizal fungal (AMF) interactions in seedling establishment and growth promotion have been inadequately dealt with. Efforts were made to assess the role of AMF-pseudomonad (PRS9, plant growth promotory fluorescent Pseudomonas) interactions in growth promotion and nursery establishment of E. hybrid. Seedlings were subjected to six different treatments: (i) uninoculated control, (ii) 400 AM spores, (iii) 800 AMF spores, (iv) PRS9 (v) 400 AMF spores + PRS9, (vi) 800 AMF spores + PRS9, with the different P regimes of 10, 20 and 30 ppm. Root length, shoot length, root and shoot fresh and dry weights were maximal at 400 AMF spores and 20 ppm soil P. Shoot P content was maximal at 800 AMF spores followed by 400 AMF spores and 400 AMF spores + PRS9. In general, plant growth was greater at 20 ppm P. Root P content increased significantly with 400 AMF spores followed by 800 at 20 ppm P. Independent of soil P levels, the quality index of mycorrhizal treatments without PRS9 was significantly higher than the treatments including PRS9 or PRS9 alone. Mycorrhizal inoculation efficiency was superior at 10 ppm P irrespective of the treatment. AM alone (400 spores) significantly improved the inoculation efficiency. PRS9 in association with AM fungi inhibited growth promotion and nutrient uptake Accepted: 8 September 1999     

Biotechnology and bioremediation: successes and limitations

With advances in biotechnology, bioremediation has become one of the most rapidly developing fields of environmental restoration, utilizing microorganisms to reduce the concentration and toxicity of various chemical pollutants, such as petroleum hydrocarbons, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, phthalate esters, nitroaromatic compounds, industrial solvents, pesticides and metals. A number of bioremediation strategies have been developed to treat contaminated wastes and sites. Selecting the most appropriate strategy to treat a specific site can be guided by considering three basic principles: the amenability of the pollutant to biological transformation to less toxic products (biochemistry), the accessibility of the contaminant to microorganisms (bioavailability) and the opportunity for optimization of biological activity (bioactivity). Recent advances in the molecular genetics of biodegradation and studies on enzyme-tailoring and DNA-shuffling are discussed in this paper.     

Biotransformation of sterols: selective cleavage of the side chain

This review elaborates on the recent development of microbial sterol biotransformation systems. Particular emphasis is laid on the new enzymatic approach investigating the cleavage of sterol side chain. New developments in the area of immobilized cell system and use of organic media along with recent reviews on side chain cleavage are discussed.     

A Population-Genetic Lens into the Process of Gene Loss Following Whole-Genome Duplication

Whole-genome duplications (WGDs) have occurred in many eukaryotic lineages. However, the underlying evolutionary forces and molecular mechanisms responsible for the long-term retention of gene duplicates created by WGDs are not well understood. We employ a population-genomic approach to understand the selective forces acting on paralogs and investigate ongoing duplicate-gene loss in multiple species of Paramecium that share an ancient WGD. We show that mutations that abolish protein function are more likely to be segregating in retained WGD paralogs than in single-copy genes, most likely because of ongoing nonfunctionalization post-WGD. This relaxation of purifying selection occurs in only one WGD paralog, accompanied by the gradual fixation of nonsynonymous mutations and reduction in levels of expression, and occurs over a long period of evolutionary time, “marking” one locus for future loss. Concordantly, the fitness effects of new nonsynonymous mutations and frameshift-causing indels are significantly more deleterious in the highly expressed copy compared with their paralogs with lower expression. Our results provide a novel mechanistic model of gene duplicate loss following WGDs, wherein selection acts on the sum of functional activity of both duplicate genes, allowing the two to wander in expression and functional space, until one duplicate locus eventually degenerates enough in functional efficiency or expression that its contribution to total activity is too insignificant to be retained by purifying selection. Retention of duplicates by such mechanisms predicts long times to duplicate-gene loss, which should not be falsely attributed to retention due to gain/change in function.     

Biology and interactions of two distinct monopartite begomoviruses and betasatellites associated with radish leaf curl disease in India

Background

Emerging whitefly transmitted begomoviruses are major pathogens of vegetable and fibre crops throughout the world, particularly in tropical and sub-tropical regions. Mutation, pseudorecombination and recombination are driving forces for the emergence and evolution of new crop-infecting begomoviruses. Leaf curl disease of field grown radish plants was noticed in Varanasi and Pataudi region of northern India. We have identified and characterized two distinct monopartite begomoviruses and associated beta satellite DNA causing leaf curl disease of radish (Raphanus sativus) in India.

Results

We demonstrate that RaLCD is caused by a complex of two Old World begomoviruses and their associated betasatellites. Radish leaf curl virus-Varanasi is identified as a new recombinant species, Radish leaf curl virus (RaLCV) sharing maximum nucleotide identity of 87.7% with Tomato leaf curl Bangladesh virus-[Bangladesh:2] (Accession number AF188481) while the virus causing radish leaf curl disease-Pataudi is an isolate of Croton yellow vein mosaic virus-[India] (CYVMV-IN) (Accession number AJ507777) sharing 95.8% nucleotide identity. Further, RDP analysis revealed that the RaLCV has a hybrid genome, a putative recombinant between Euphorbia leaf curl virus and Papaya leaf curl virus. Cloned DNA of either RaLCV or CYVMV induced mild leaf curl symptoms in radish plants. However, when these clones (RaLCV or CYVMV) were individually co-inoculated with their associated cloned DNA betasatellite, symptom severity and viral DNA levels were increased in radish plants and induced typical RaLCD symptoms. To further extend these studies, we carried out an investigation of the interaction of these radish-infecting begomoviruses and their associated satellite, with two tomato infecting begomoviruses (Tomato leaf curl Gujarat virus and Tomato leaf curl New Delhi virus). Both of the tomato-infecting begomoviruses showed a contrasting and differential interaction with DNA satellites, not only in the capacity to interact with these molecules but also in the modulation of symptom phenotypes by the satellites.

Conclusion

This is the first report and experimental demonstration of Koch's postulate for begomoviruses associated with radish leaf curl disease. Further observations also provide direct evidence of lateral movement of weed infecting begomovirus in the cultivated crops and the present study also suggests that the exchange of betasatellites with other begomoviruses would create a new disease complex posing a serious threat to crop production.     

Ecological Significance of Microdiversity: Coexistence Among Casing Soil Bacterial Strains Through Allocation of Nutritional Resource

A combination of cultivation-based methods with a molecular biological approach was employed to investigate whether bacteria with identical 16S rRNA gene sequences can represent distinct eco- and genotypes. A set of eight bacterial strains wherein three were Pseudomonas putida and rest were Acinetobacter calcoaceticus, were isolated from casing soils community by conventional plating. These strains had identical 16S rRNA gene sequences and represented the dominant phylotype in the plateable fraction. Each strain utilized a specific combination of 154 carbon substrates, and the niche overlap indices were low, suggesting that each strain occupied a different ecological niche. Our results have implications for assessment of the diversity and biogeography of bacteria and increase the perception of natural diversity beyond the level of 16S rRNA gene sequences. It is worthwhile approach to explore prokaryotic diversity in different ecological niches.     

Shelf-life enhancement of bio-inoculant formulation by optimizing the trace metals ions in the culture medium for production of DAPG using fluorescent pseudomonad R62

Statistical experimental design was used to optimize the concentration of trace elements for production of antifungal compound, 2,4-diacetylphloroglucinol (DAPG), from fluorescent pseudomonad R62 in shake-flask cultivation. The selection of the trace metal ions, influencing DAPG production, was done using Plackett-Burman design (PBD). Only Zn(2+), Mn(2+) and MoO(4)(2-) were the most significant components (p<0.05). A quadratic model was used to fit the response. Application of response surface methodology (RSM) revealed that the optimum values of the salts of the trace elements Zn(2+) (ZnSO(4)·7H(2)O), Mn(2+) (MnCl(2)·4H(2)O), and MoO(4)(2-) (Na(2)MoO(4)·2H(2)O) were 83, 42 and 135μM, respectively, to achieve 125 mg/L of DAPG, which was nearly 13-fold more compared to its production in basal synthetic medium in shake flask. The studies in 14L bioreactor resulted in 135 mg/L of DAPG at the end of 36 h of cultivation. The culture broth containing 125 mg/L of DAPG was found to be sufficient for keeping the bio-inoculant viable in non-sterile talcum powder-based formulations (which contained 25μg DAPG/g carrier) when stored at 28°C for 6 months. The structure of the purified DAPG was confirmed using (1)H NMR and mass spectrometry.     

UV radiation reduces epidermal cell expansion in Arabidopsis thaliana leaves without altering cellular microtubule organization

Upon chronic UV treatment pavement cell expansion in Arabidopsis leaves is reduced, implying alterations in symplastic and apoplastic properties of the epidermal cells. In this study, the effect of UV radiation on microtubule patterning is analysed, as microtubules are thought to serve as guiding rails for the cellulose synthase complexes depositing cellulose microfibrils. Together with hemicelluloses, these microfibrils are regarded as the load-bearing components of the cell wall. Leaves of transgenic plants with fluorescently tagged microtubules (GFP-TUA6) were as responsive to UV as wild type plants. Despite the UV-induced reduction in cell elongation, confocal microscopy revealed that cellular microtubule arrangements were seemingly not affected by the UV treatments. This indicates an unaltered deposition of cellulose microfibrils in the presence of UV radiation. Therefore, we surmise that the reduction in cell expansion in UV-treated leaves is most probably due to changes in cell wall loosening and/or turgor pressure.Key words: arabidopsis, cell expansion, GFP-TUA6, leaf development, microtubule cytoskeleton, UV radiationPhotosynthetic functions such as solar light capture and carbon fixation are highly evolved features of plant leaves. To fulfil these functions in an optimal way, leaf development needs to be tuned to environmental conditions. Leaves are continuously exposed and subjected to environmental influences, which serve as co-regulators of leaf and plant development.¹ This ability of plants to adapt, secures the plant''s survival, even under non-optimal conditions. An example of a regulatory environmental parameter is solar light, indispensable for photosynthesis but potentially causing photoinhibition and/or UV-radiation stress. The highly energetic ultraviolet B (UV-B) rays of short wavelengths (280–315 nm) can both cause damage, as well as induce a range of specific metabolic and morphogenic plant responses. It was reported before that exposure to low dose UV radiation reduces Arabidopsis leaf size due to a decreased cell size.² Expansion of leaf epidermal cells of Arabidopsis thaliana is the combined action of promotion and restriction of growth, resulting in the typical irregular sinuous pavement cells. It has been postulated that cellulose microfibrils are responsible for generating a force opposing isotropic expansion by creating neck regions in between outgrowing lobes.³ As the microtubule cytoskeleton is believed to serve as guiding rails for the cellulose synthase complexes (CESAs),⁴ the deposition of the cellulose fibrils is intimately linked to the cortical microtubule arrangement. We have studied the UV-effect on microtubule organisation in leaf epidermal cells whose expansion had decreased upon this UV radiation. Microtubules in the adaxial pavement cells of the fourth leaf were monitored on several successive days in a transgenic line containing GFP fused to tubulin A6.⁵ The chronic UV treatment was started on day 0 when the plants were 2 weeks old, using UV exposure conditions as described in reference ². First the responsiveness of the GFP-TUA6 plants to UV radiation was evaluated. Similar to wild type (WT) plants,² the GFP-TUA6 plants had smaller leaves following 8 days of UV treatment (t-test, p < 0.01) (Fig. 1). This was caused by a significant reduction in the generalized cell area average of all measured cells, irrespective of the location within the leaf (Fig. 1; t-test, p < 0.01). In more detail, the average cell area within the base, middle and top zones of the GFP-TUA6 leaf was systematically lower in UV-treated leaves from 8 days after the treatment started onwards (data not shown).Open in a separate windowFigure 1Effect of UV radiation on leaf and cell area after different days of UV radiation. Open asterisks indicate a statistically significant difference in leaf area between UV-treated and control plants, black asterisks indicate statistically significant difference in cell area (t-test, *p < 0.05, **p < 0.01, ***p < 0.001). Error bars indicate the standard error for five different leaves at all measured time-points and 600, 170 and 180 cells at day 0, 8 and 12 respectively.As GFP-TUA6 leaves were as responsive to UV radiation as wild type leaves, confocal microscopy was used to visualize the organisation of the cortical microtubules facing the outer periclinal wall of the adaxial epidermis. No clear difference in microtubule (re)organization could be detected during the development of pavement cells, and throughout the UV treatment period. As shown in Figure 2 at day 2, pavement cells with comparable areas are similarly shaped in control and UV-irradiated plants and contain similar microtubule arrangements (Fig. 2 and marked cells). This means that microtubule organization is not directly affected by the UV exposure and that shape development proceeds in an analoguous manner as under control conditions. This lack of alteration in the microtubule arrangement can be observed for cells at the leaf tip, which were already in the process of lobe formation at the start of the exposure period, as well as for cells at the leaf base. Under our growth conditions, and in the monitored leaf number 4, cell proliferation still took place in this part of the leaf and lobes only started to appear on the cell surface. As microtubules are linked to the deposition of cellulose microfibrils, it can be assumed that no alterations in cellulose deposition occur upon UV treatment either. We can therefore conclude that the process of lobe formation and microtubule patterning is not impeded and that only the extent of cell expansion is restricted upon UV exposure.Open in a separate windowFigure 2Microtubule pattern in control and UV-exposed leaves visualized using GFP-TUA6 and confocal microscopy. Both images are from cells at the mid zone of the fourth leaf at day 2. Microtubules are similarly arranged in equally shaped and sized cells of control and UV-exposed leaves. The marked cells show a pattern whereby the tubules are centred in the neck regions between two outgrowing lobes.According to the Lockhart equation,⁶ cell (wall) growth is modulated by wall biomechanics and turgor pressure. Concerning turgor pressure, no clear differences in this factor between UV-exposed and control plants of Lactuca sativa L.⁷ and Pisum sativum⁸ could be observed, reinforcing the idea that especially the modulation of cell wall properties is the main factor causing the observed UV-induced reduction in cell expansion. Some reports indicate differential expression of wall loosening enzymes like expansins or xyloglucan endotransglycosylase/hydrolases (XTHs),^9,10 or cell wall strengthening enzymes as particular peroxidases⁷ after UV exposure. Another key event could involve UV-mediated changes in the phenylpropanoid pathway, which may cause changes in the lignin biosynthesis. As shown by the literature^11–14 lignin may well be an important modulator of cell wall architecture in Arabidopsis and therefore alterations in lignin synthesis could form the basis for morphological modifications. Further research on the cell wall properties of UV-treated plants may resolve this uncertainty.As a general conclusion we can state that the patterning of microtubules is not altered, but that alterations in cell wall composition or arrangements are the most plausible candidates for the observed reduction in pavement cell expansion upon chronic UV treatment.