Regulation of nitrogen metabolism in salt tolerant and salt sensitive Frankia strains.

Effect of salinity (0, 50, 100, 250, 500 and 750 mM NaCl) was observed on some important physiological parameters of nitrogen metabolism such as nitrate uptake, intracellular and extracellular ammonium status and activities of nitrogenase, nitrate reductase, nitrite reductase and glutamine synthetase among Frankia strains differing in their salt tolerance capacity. Nitrogenase activity closely followed the growth pattern with regular decline on NaCl supplementation. All the other enzymes showed optimum activity at 100 mM and declined further. Co-regulation of the nitrate uptake system and sequential enzyme activities plays a crucial role in governing the nitrogen status of strains during salt stress. HsIi10 experiencing minimum decline in enzyme activities and best possible nitrogen regulation under NaCl replete condition showed adequate nutritional management. Among all the strains, HsIi10 proved to be salt tolerant on account of above features while the salt sensitive strain HsIi8 lacked the ability to regulate various steps of nitrogen metabolism during salinity, and thus Frankia strain HsIi10 can potentially serve as a potential biofertilizer in the saline soil.

In vitro synthesis of Frankia and mycorrhiza with Casuarina equisetifolia and ultrastructure of root system.

Casuarina equisetifolia is one of the ecologically and economically important tropical coastal trees nodulated by nitrogen-fixing actinomycete Frankia and forming symbiotic associations with both ecto- and endomycorrhizal fungi. The present study aims at the ultrastructural study of interactions between C. equisetifolia, Frankia, and mycorrhiza. C. equisetifolia seeds were sterilised and germinated under in vitro condition. The seedlings were transferred to conical flasks containing vermiculite and saw dust with Hoagland's solution. After 30 days, the inoculum of AM fungus--Glomusfasciculatum (A), ectomycorrhizal fungus-Pisolithus tinctorius (E) and actinorhizal Frankia (F) were inoculated individually and in various combinations, (A+E), (A+F), E+F) and (A+E+F). After 90 days, the experimental plant roots and nodules were harvested for assessment of growth characters of mycorrhizal and actinorhizal association by light and scanning electron microscope methods. C. equisetifolia roots were infected with arbuscles and vesicles of G. fasciculatum; P. tinctorius formed fungal sheath but no Hartig net. Large number of cortical cells were seen infected with Frankia, hyphae of Frankia were frequently seen penetrating from cell to cell directly through cell walls and Frankia occupied majority of the cell volume.

Caracterização morfológica, fisiológica e infectividade em planta de estirpes de Frankia isoladas de nódulos de Casuarina / Morphological, physiological and plant infectivity characterization of Frankia strains isolated from Casuarina’s nodules

Frankia são microrganismos capazes de fixar N2 quando em simbiose com espécies arbóreas, denominadas plantas actinorrízicas. Este estudo foi conduzido para caracterizar morfologicamente, fisiologicamente e avaliar a nodulação de quatro estirpes referência de Frankia (HFPCcI3, JCT287, KB5 e F59) e 12 estirpes(IPRF) isoladas de nódulos de Casuarina. Todas as estirpes (Referência e IPRF) foram Gram-positivas e 50% foram “acid-fast”. As estirpes de Frankia alcalinizaram o meio de cultura, exceto IPRF006, IPRF008e IPRF010. As colônias das estirpes F59, IPRF002, IPRF004, IPRF005 e IPRF011, produziram melanina. Entre as estirpes referência, apenas a JCT287 cresceu no meio de cultura com pH 5,5, enquanto no meio com pH 6,0 as duas, JCT287 e KB5, apresentaram crescimento. A análise de regressão mostrou uma relação linear (Y = 67,56+ 3,88X e R2=0,5862, p<0,05) e coeficiente de correlação de Pearson (r=0,766,p<0,05) entre número de esporos e hifas (120 a 480 UFC mL-1) e proteína total (18 a 145 µg mL-1). Foi observado que a estirpe F59 apresentou uma maior proporção de proteína total (50%) do que a JCT287(7,0%) e, que as estirpes IPRF mostraram valores entre 17,5 e 29,3%. Todas as estirpes produziram compostos indólicos com valores variando de 5,9 a 98,8 µM.

Frankia are soil microorganisms that form symbiosis with roots of tree species called actinorhizal plants and are capable of fixing atmospheric N2. This study was carried out to characterize morphologically, physiologically and to assess the nodulation of four Frankia reference strains (HFPCcI3, JCT287, KB5and F59) and 12 (IPRF) isolated from root nodules of Casuarina plants. All strains (Reference and IPRF)were characterized as Gram-positive and 50% as acid-fast. The Frankia strains produced alkali in the culture medium, except the IPRF006, IPRF008 and IPRF010. The colonies of strains F59, IPRF002, IPRF004,IPRF005, and IPRF011 produced melanin. Among reference strains, only JCT287 grew in culture mediawith pH 5.5, while with pH 6.0 both strains JCT287 and KB5 presented growth. The regression analysis showed a linear relationship (Y = 67.56+ 3.88X and R2=0.5862, p<0.05) and Pearson’s correlation coefficient(r=0.766, p<0.05) between number of spores and hyphae (120 to 480 UFC mL-1) and total protein (18 to 145µg mL-1). It was observed that the strains F59 had a higher proportion of total protein 50.0% than JCT287with 7.0% and that the IPRF strains showed values between 17.5 and 29.3%. All strains presented ability to produce indolic compounds in growth media with values ranging from 5.9 to 98.8 µM.

Morphological variation, histology, histochemistry and nutrient contents of Frankia root nodules in Casuarina equisetifolia.

Root nodules were collected from the existing plantation of Casuarina equisetifolia from three different agroclimatic zones of Tamil Nadu, India. Morphological variation, histology, histochemistry and nutrient contents of the nodules were studied. The nodules were found to be simple, dichotomously branched and coralloid. The stele in simple and branched nodules reached up to the tip of the nodules. Structure of Frankia nodules was described using histochemical methods to elucidate the nature of micro-symbiont within the nodules. The nodule consisted of a distinct periderm enclosing the cortex. The cortex showed patches of infected cells interspersed among the uninfected ones. Inside the cortex was a stele bound by an endodermis, which contained high amount of tannin. The micro-symbiont was observed in the nodule tisues in hyphal form and spore packed sporocorp was also seen in the cortex. The histochemical tests revealed the presence of proteins, insoluble polysaccharides and polyphenols.

El efecto de la interacción Frankia - micorrizas - micronutrientes en el establecimiento de árboles Aliso (Alnus acuminata) en sistemas silvopastoriles / The effects of the interaction Frankia - mycorrhizal fungi - microelements in the establishment of Alder trees (Alnus acuminata) in tree grass systems

Frankia es un género de microorganismos llamados actinomicetos capaces de inducir la formación de nódulos radiculares fijadores de nitrógeno atmosférico en algunas angiospermas no leguminosas, denominadas plantas actinorrízicas. El desarrollo de fijación de Nitrógeno (N) simbiótico envuelve múltiples procesos que se llevan a cabo en compartimentos subcelulares endosimbióticos donde Francia produce los nódulos de la raíz que convierten el N2 en forma combinada. La simbiosis micorriza y nódulo es generalmente sinérgica, promueve el crecimiento vegetal, la multiplicación de las micorrizaspropicia un ambiente de mayor competencia, no favorable para los agentes patógenos. A lo anterior se debe sumar la participación de microelementos como el molibdeno(Mo), el cual es constituyente dela nitrogenasa y cuya deficiencia en el medio causa un efecto directo y negativo en la fijación del N; con el boro(B,) donde su deficiencia produce una desestabilización de la cubierta protectora contra el oxígeno, afectando considerablemente la nodulación de la planta y con el cobalto(Co), forman un componente estructural de la coenzima de la vitamina B12 en el proceso de fijación de N. Sin embargo, muchos aspectos relacionados de estos microelementos en la interrelación y de la simbiosis micorriza y Frankia se desconocen. Por lo anterior, es importante determinar sus efectos sobre los microorganismos del suelo y el desarrollo de los nódulos; para intepretar una óptima manifestación, sin embargo su complejidad hace que su efecto no sea predecible bajo todas las condiciones ni para todas las especies. Por lo tanto, es importante incentivar la investigación al respecto, así como profundizar en el conocimiento de sus principios de funcionamiento, interacciones y mostrar los resultados encontradosen su uso en árboles promisiorios para sistemas silvopastoriles trópico alto como el aliso (Agnus acuminata), especie forestal útil en la solución de problemas de degradación de suelos y deforestación.

Polymorphism in Alnus based Frankia of Darjeeling.

DNA samples extracted from the root nodules of Alnus nepalensis, collected from 10 different locations of Darjeeling hills, were used to assess the genetic diversity of Frankia. The DNA samples from the nodules of naturally growing plants were used as templates in PCR, targeting different genomic regions of Frankia, namely distal, middle and proximal parts of 16S rRNA gene and nifH-D IGS region with locus specific primers. The PCR products were digested with a number of frequent (4-base) cutter restriction endonucleases. Bands were scored as present (1) or absent (0) and the clustering was done using NTSYSpc. Distinct polymorphism was found among the nodules collected from different parts of the region and those of same geographic area. These results demonstrate that genetic diversity is indeed present among the naturally occurring Frankia of Darjeeling, India.

Symbiosis between Frankia and actinorhizal plants: root nodules of non-legumes.

In actinorhizal symbioses, filamentous nitrogen-fixing soil bacteria of the genus Frankia induce the formation of nodules on the roots of a diverse group of dicotyledonous plants representing trees or woody shrubs, with one exception, Datisca glomerata. In the nodules, Frankia fixes nitrogen and exports the products to the plant cytoplasm, while being supplied with carbon sources by the host. Possibly due to the diversity of the host plants, actinorhizal nodules show considerable variability with regard to structure, oxygen protection mechanisms and physiology. Actinorhizal and legume-rhizobia symbioses are evolutionary related and share several features.

Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts.

While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region of rrn operon of Frankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS1 region of the nuclear rrn operon of Alnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.