A direct observation technique for studies on rhizoplane and rhizosphere colonization

Summary A slide incubation chamber was described which allowed small plants to be grown from seed and the root systems to be observed microscopically. A fluorescence stain, the ammonium salt of 8-anilino-1-naphthalene sulfonic acid, was applied to the soil in which the roots were growing and the stained microorganisms on the roots and in the rhizosphere were counted. A statistical pattern analysis technique, the two-within-four randomization test, was used to analyze the data obtained from quadrats on the roots. Distinct colonization patterns and colony growth, especially of bacteria, were easily distinguished with the technique.     

Antibiotic-resistantArthrobacter sp. andPseudomonas sp. responses in the rhizosphere of blue grama after herbage removal

Summary Streptomycin-resistant Pseudomonas and Arthrobacter were isolated from semi-arid grassland soil and their relative responses in the rhizosphere of blue grama (Bouteloua gracilis) subjected to herbage removal were evaluated. Using plants grown in normal soil, the two bacteria showed differential responses to herbage removal, which were most marked in the rhizoplane, where the Pseudomonas showed a two-log unit increase over a 60 hour period, while Arthrobacter, in contrast, exhibited a one-log unit decrease in viable counts for at least 48 hours after defoliation, responses which are similar to those observed in root exudate medium experiments by earlier workers. These results suggest that the rhizoplane may be a critical environment for interaction of these two types of microorganisms, and that sequential responses of the root-associated soil microorganisms may occur after herbage removal from this important rangeland plant. These responses are most likely associated with increased exudate release following herbage removal, which has been best documented using blue grama grown under sterile conditions.     

Relationship between survival rates of associative nitrogen-fixers on roots and yield response of plants to inoculation

Abstract: The population dynamics of associative nitrogen-fixers Azospirillum lipoferum 137, Arthrobacter mysorens 7, Flavobacterium sp. L30 and phosphate-solubilizing strain Agrobacterium radiobacter 10 in soil and the rhizoplane of inoculated plants was studied in pot and field experiments. All of the present strains were able to actively colonize the rhizoplane of barley, wheat, oat, tomatoes, rape, and alfalfa. For the most part the population size and dynamics of introduced bacteria depended only slightly on the plant genotype and soil conditions. The overall pictures of survival of the strains in soil and on plant roots were similar. The reliable effect of inoculation on plants was observed only in individual cases. No correlation was established between survival of introduced bacteria and their effect on plant development. It was concluded that the influence of plants on survival of bacteria was not specific. In contrast, the plant response to inoculation was conditioned to a greater extent by the plant genotype.     

Comparison of the community structures of ammonia-oxidizing bacteria and archaea in rhizoplanes of floating aquatic macrophytes

Some common floating aquatic macrophytes could remove nutrients, such as nitrogen, from eutrophic water. However, the relationship between these macrophytes and the ammonia-oxidizing microorganisms on their rhizoplanes is still unknown. In this study, we examined communities of ammonia-oxidizing archaea (AOA) and bacteria (AOB) on the rhizoplanes of common floating aquatic macrophytes (Eichhornia crassipes, Pistia stratiotes and Ipomoea aquatic) in a eutrophic reservoir.The results show that AOB were the predominant ammonia-oxidizer on the three rhizoplanes. The principal AOB were Nitrosomonas europaea and Nitrosomonas ureae clades. The principal group of AOA was most similar to the clone from activated sludge. The ratio of AOB amoA gene copies to AOA varied from 1.36 (on E. crassipes) to 41.90 (on P. stratiotes). Diversity of AOA was much lower than that of AOB in most samples, with the exception of P. stratiotes.     

Rhizosphere micro-organisms in relation to the apple replant problem

Summary One of the factors giving rise to soil sickness in apple orchards is the rhizosphere microflora. The composition of the microbial coenosis in the rhizosphere changes with increasing age of the apple trees. An increase in the counts of micromycetes and actinomycetes and a decrease in bacterial counts was found in agreement with the decreasing pH of the rhizosphere soil. The presence of fluorescent pseudomonads in the rhizosphere of old apple trees was rare, but the planting of apple seedling into sick soil induced their proliferation. The relative proportion of individual genera of micromycetes changed according to the tree age; fungi of the genus Mucor were more often found in the rhizosphere of younger trees than in that of older ones while fungi of the genus Penicillium had an opposite trend. Biological tests showed that Penicillium fungi form the majority of the phytotoxic microflora. The amount of phytotoxic micromycetes was higher in ‘sick’ soil as compared with control soil in which apple trees had not been grown for at least 15 years. Higher numbers of phytotoxic micromycetes were isolated also from the rhizosphere of apple seedlings grown in ‘sick’ soil as compared with those growing in control soil. An increase in the amount of phytotoxic micromycetes in apple tree rhizosphere could be induced by mere addition of 5% (w/w) ‘sick’ soil to the soil in which apple trees were grown for the first time. By adding sterilized ‘sick’ soil, the amount of phytotoxic micromycetes in the apple seedling rhizosphere was not affected. Increased numbers of phytotoxic micromycetes affected negatively the growth of apple trees and the morphology of apple tree roots. This demonstrated the possibility of transfer of a factor participating in the etiology of soil sickness in apple orchards.     

Free amino acids in the seed and root exudates in relation to the nitrogen requirements of rhizosphere soil Fusaria

Summary The amounts of amino acids in seed exudates were generally higher than in root exudates of the same plant. The spectra and relative abundance of amino acids in both plants were similar but they were generally more abundant in cowpea exudates than in sorghum. Glutamic acid and alanine were the most abundant amino acids in the seed and root exudates of both plants. The proportions of the amino acids in the seed exudates were comparable to that stored in the seeds. Many of the major amino acids identified in the exudates were also found to support thein vitro growth ofFusarium spp. isolated from the rhizosphere and rhizoplane. This suggests that the amino acids exuded might contribute signficantly to Fusaria nutrition and its consequent predominance around the root. The significance of this pathogenesis is also discussed.     

Roots of the wetland plants Typha latifolia and Phragmites australis are inhabited by methanotrophic bacteria in biofilms

Wetland plants create partly aerobic conditions in the rhizosphere by releasing oxygen to the waterlogged substrate. The present study was conducted to characterise the arrangement of rhizobacteria, especially those active in methane oxidation, in root-associated biofilms of wetland plants. Root cross sections sampled from Typha latifolia L. (broadleafed cattail) and Phragmites australis (Cav.) Trin. ex Steud. (common reed) were scanned using light and electron microscopy. Methane-oxidising bacteria were identified and quantified by immunological labelling of the α-subunit of the methanol dehydrogenase (α-MDH; encoded in mxaF). On roots of both species there was a diverse subset of bacteria arranged in a microbial biofilm around the roots’ exodermis. Similar bacterial densities in the root-associated biofilm were detected in more basal regions and closer to the root tip. Many microbes carried notable internal membrane systems that are characteristic of methanotrophic bacteria. This morpho-anatomical characterisation was confirmed by immunogold labelling with α-MDH antibodies. Quantification of labelled bacteria revealed that 34–43% of the biofilm bacteria were potentially capable of methane turnover. These findings confirm the presence of methane-oxidising bacteria in the root-associated biofilms of the two common macrophytes T. latifolia and P. australis. This implies that the methanotrophs participate essentially in the microbial processes related to oxygen-releasing roots of wetland plants.     

Nitrogen fixation in the rhizosphere and rhizoplane of barley

Summary Aerobic and anaerobic N₂-fixing bacteria developed in the rhizosphere of barley seedlings and exhibited N₂ase activity when seedlings were grown in sterilized sand-nutrient cultures containing low levels of combined nitrogen. The source of the N₂-fixing bacteria appeared to be the seed. Average daily rates up to 0.9 μmoles C₂H₄ h⁻¹ g⁻¹ dry root tissue were measured, but the intensity of the activity was affected by moisture levels and concentration of combined N in the rhizosphere. Removal and washing of the roots did not remove the activity, and roots remained active even after surface-sterilization. An unidentified aerobic N₂-fixing bacterium was isolated from the rhizoplane of active barley roots. Inoculation of barley seedlings with the aerobic N₂-fixing bacterium enhanced N₂ase activity of excised roots 10-fold, with average rates of 0.9, 1.1 and 1.3 μmoles h⁻¹ g⁻¹ dry root assayed under pO₂ of 0.01, 0.02 and 0.04 atm respectively. The aerobic N₂-fixing bacterium also exhibited N₂ase activity when inoculated into the rhizosphere of oat, rice and wheat seedlings. Microscopic observations of sterilized live and stained barley roots suggest that the aerobic N₂-fixing bacterium is an endophyte which infects root tissue and metamorphoses into vesicle-like structures.     

Synergistic effects of biofilm-producing PGPR strains on wheat plant colonization,growth and soil resilience under drought stress

Drought stress substantially impedes crop productivity throughout the world. Microbial based approaches have been considered a potential possibility and are under study. Based on our prior screening examination, two distinct and novel biofilm-forming PGPR strains namely Bacillus subtilis-FAB1 and Pseudomonas azotoformans-FAP3 are encompassed in this research. Bacterial biofilm development on glass surface, microtiter plate and seedling roots were assessed and characterized quantitatively and qualitatively by light and scanning electron microscopy. Above two isolates were further evaluated for their consistent performance by inoculating on wheat plants in a pot-soil system under water stresses. Bacterial moderate tolerance to ten-day drought was recorded on the application of individual strains with wheat plants; however, the FAB1 + FAP3 consortium expressively improved wheat survival during drought. The strains FAB1 and FAP3 displayed distinct and multifunctional plant growth stimulating attributes as well as effective roots and rhizosphere colonization in combination which could provide sustained wheat growth during drought. FAB1 and FAP3-induced alterations cooperatively conferred improved plant drought tolerance by controlling physiological traits (gs, Ci, E, iWUE and P_N), stress indicators (SOD, CAT, GR, proline and MDA content) and also maintained physico-chemical attributes and hydrolytic enzymes including DHA, urease, ALP, protease, ACP and β glucosidase in the soil. Our findings could support future efforts to enhance plant drought tolerance by engineering the rhizobacterial biofilms and associated attributes which requires in-depth exploration and exploiting potential native strains for local agricultural application.     

Effect of root exudates on the spore germiantion of rhizosphere and rhizoplane mycoflora of chilli (Capsicum annuum L.) cultivars

Summary From root exudates of three cultivars of chilli (Capsicum annuum L.) 12 amino acids and 7 sugars were detected. Methionine, d-1- phenylalanine, citrulline and d-xylose were detected only from the root exudates of resdistant cultivars. The root exudates of resistant variety inhibited spore germination of the pathogen (Fusarium oxysporum f. sp.capsici), but that of susceptible variety enhanced spore germiantion of the same. Spore germiantion of antagonistic fungi (Trichderma viride andAspergillus sydowi) was also influenced by the root exudates of resistant and susceptible varieties, but the influence was different.Spore germiantion of a number of rhizosphere fungi was studied and in general root exudate of susceptible cultivar enhanced spore germiantion of majority of fungi, but spore germination of antagonistic fungi against the pathogen was inhibited. However, root exudate of resistant cultivar stimulated spore germination of antagonistic fungi.