A comparison of cultural characteristics and infectivity ofFrankia isolates from root nodules ofCasuarina species

Summary The isolations of three new strains ofFrankia were made from root nodules ofCasuarina cunninghamiana growing aeroponically. Two strains, HFPCCI1 and HFPCcI2 isolated by Lopez are typicalFrankia strains, producing sporangia among filamentous mats in culture and, in the absence of combined nitrogen, forming vesicles and showing acetylene reduction. They are red-pigmented and, although failing to nodulateCasuarina hosts, effectively nodulatedElaeagnus andHippophae. A third strain HFPCcI3 isolated by Zhang from the same source, also a typicalFrankia, can form sporangia and vesicles in culture and reduce acetylene, is unpigmented, fails to nodulateElaeagnus but effectively nodulatesC. cunninghamiana andC. equisetifolia. Comparisons are made among all of theCasuarina isolates in our collection from around the world (twelve in all) with regard to their cultural characteristics and capacity to infect host plant species. Questions are raised about the specificity of the various isolates and their possible affinities. Opportunities are suggested for inoculation of seedlings for forestry and field application using the infective, effective strains now available.     

Effect of the flavonoid quercetin on culture and isolation ofFrankia fromCasuarina root nodules

The growth of threeFrankia strains (HFPCcl3, ORS021001, ORS020607), isolated from nodules ofCasuarina, was unaffected by 5–15 μmol/L quercetin. Re-isolation ofFrankia strain HFPCc13 fromCasuarina root nodules was carried out with the addition of 15 μmol/L quercetin to three different media. The flavonoid significantly reduced fungal contaminants in the isolation plates and increased the number ofFrankia colonies per plate compared to controls without the flavonoid.     

Frankia inoculation,soil biota,and host tissue amendment influence Casuarina nodulation capacity of a tropical soil

The effects of soil biota, Frankia inoculation and tissue amendment on nodulation capacity of a soil was investigated in a factorial study using bulked soil from beneath a Casuarina cunninghamiana tree and bioassays with C. cunninghamiana seedlings as capture plants. Nodulation capacities were determined from soils incubated in sterile jars at 21 °C for 1, 7, and 28 days, after receiving all combinations of the following treatments: ± steam pasteurization, ± inoculation with Frankia isolate CjI82001, and ± amendment with different concentrations of Casuarina cladode extracts. Soil respiration within sealed containers was determined periodically during the incubation period as a measure of overall microbial activity. Soil respiration, and thus overall microbial activity, was positively correlated with increasing concentrations of Casuarina cladode extracts. The nodulation capacity of soils inoculated with Frankia strain Cj82001 decreased over time, while those of unpasteurized soils without inoculation either increased or remained unaffected. The mean nodulation capacity of unpasteurized soil inoculated with Frankia CjI82001 was two to three times greater than the sum of values for unpasteurized and inoculated pasteurized soils. Our results suggest a positive synergism between soil biota as a whole and Frankia inoculum with respect to host infection.     

Effect of carbon source on growth,nitrogenase and uptake hydrogenase activities of Frankia isolates from Casuarina sp.

The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was studied. In addition, regulation of nitrogenase and uptake hydrogenase activity by carbon sources was investigated. For each of the three isolates, JCT287, KB5 and HFPCcI3, growth was greatest on the carbon sources pyruvate and propionate. In general the carbon sources which gave the greatest growth gave the highest levels of nitrogenase activity, but repressed the activity of uptake hydrogenase. The regulation of growth, uptake hydrogenase activity and nitrogenase activity is discussed.     

Effect of vesicular-arbuscular mycorrhizal fungi and phosphate-solubilizing bacteria on growth and nutrition of soybean in a neutral-calcareous soil amended with32P-45Ca-tricalcium phosphate

Summary The optimum conditions for growth ofFrankia sp. HFPCcI3 isolated fromCasuarina cunninghamiana, were studied in batch culture using defined media. Maximum growth (doubling time was 24 h)_was achieved at 33°C and at pH 6.3 with pyruvate and NH ₄ ⁺ as the sole C and N sources, respectively. Removal of NH ₄ ⁺ from the culture medium resulted in vesicle differentiation which was paralleled by induction of acetylene reduction activity. Growth on atmospheric N₂ was optimal with combined pyruvate and glucose as the carbon sources and displayed a doubling time of about 48 h. Comparisons in growth and N₂-fixing activity ofFrankia strains grown in a variety of cultural conditions demonstrate the range of behavior among the strains.     

Conservation of nif sequences in Frankia

Summary Southern blots of Frankia total DNAs were hybridized with nifHDK probes from Rhizobium meliloti, Klebsiella pneumoniae and Frankia strain Arl3. Differences between strains were noted in the size of the hybridizing restriction fragments. These differences were more pronounced among Elaeagnus-compatible strains than among Alnus- or Casuarina-compatible strains. Gene banks constructed for Frankia strains EUN1f, HRN18a, CeD and ACoN24d were used to isolate nif-hybridizing restriction fragments for subsequent mapping and comparisons. The nifH zone had the highest sequence conservation and the nifH and nifD genes were found to be contiguous. The complete nucleotide sequence of the nifH open reading frame (ORF) from Frankia strain Arl3 is 861 bp in length and encodes a polypeptide of 287 amino acids. Comparisons of these nucleic acid and amino acid sequences with other published nifH sequences suggest that Frankia is most similar to Anabaena and Azotobacter spp. and K. pneunoniae and least similar to the Gram-positive Clostridium pasteurianum and to the archaebacterium Methanococcus voltae.     

The isolation,culture and infectivity of aFrankia strain fromGymnostoma papuanum (Casuarinaceae)

A strain ofFrankia was isolated fromGymnostoma papuanum(Casuarinaceae) nodules harvested from rooted cuttings which had been inoculated with a suspension of crushedCasuarina equisetifolia nodules. Designated HFPGpI1 (catalogue #HFP021801), this strain is pigmented and similar to other pigmentedFrankia strains in cultural characteristics. A previously unknown spiraled hyphal morphology was observed at very low frequency in some cultures of this strain. HFPGpI1 is infective and effective onG. papuanum but not on anyCasuarina species tested. It also infects members of the family Elaeagnaceae andMyrica gale. The host plantG. papuanum can be infected with a wide range ofFrankia isolates and thus can be considered a promiscuous host, unlike its close relatives in the genera Casuarina and Allocasuarina which are very restrictive as to which strains may nodulate them.     

Host-Frankia specificity within the Casuarinaceae

Summary Fifteen species from three genera of the Casuarinaceae were inoculated with suspensions ofFrankia prepared from single nodule-lobes collected from different species and genera within the Casuarinaceae. Host-endophyte specificity was expressed mainly at the generic level. There was marked cross-inoculation within Casuarina and little nodulation ofCasuarina species from Allocasuarina sources with the exception of 3 sources ofFrankia fromA. torulosa which showed a high tendency to nodulateCasuarina species. Few sources from Casuarina nodulated species of Allocasuarina and while cross-inoculation within Allocasuarina was frequent it was less marked than within Casuarina. SomeFrankia inocula had wider host ranges than others, nodulating outside the genus or series of origin. It was not possible to determine if these apparent wider ranges in host spectra reflected genotypic differences betweenFrankia or were associated with the presence of more than oneFrankia strain in some inocula.     

Host plant growth response to inoculation withFrankia

Optimum growth conditions and inoculation regimes were determined for severalFrankia strains isolated from both Alnus and Casuarina host plants. Growth conditions were estabilished that allowed a reduction in generation time to less than 15 hours for certain Alnus derivedFrankia. Differences in plant growth response were observed with differing inoculum levels and soil mixtures. Elite strains of Alnus derivedFrankia were isolated that elicited similar growth reponses in allAlnus species tested; however, differences were observed betweenFrankia strains and plant growth response of variousCasuarina species tested.     

Composition of<Emphasis Type="Italic">Casuarina</Emphasis> leaf litter and its influence on<Emphasis Type="Italic">Frankia-Casuarina</Emphasis> symbiosis in soil

Plant needles ofCasuarina equisetifolia were collected and analyzed in parallel with soil analysis. In three strains ofFrankia—symbionts ofCasuarina—their infectivity and plant performance was determinedin vitro after soil amendment with different leaf litter concentrations. Only one strain was able to nodulate the plant at all litter concentrations (0.5, 3 and 5%) although the nodules were very small. However, all treated plants grew poorly; their growth was reduced by approximately 90% (for 5% litter concentration) compared to plants grown on untreated soil, on the basis of total dry mass. Inhibition of nodulation can be attributed to high concentrations of some elements and compounds that were either found inC. equisetifolia litter or originally found in soil (i.e. chloride, cyanide, copper, manganese and phenols). In general, plant growth decreased as more litter was added. Plant total nitrogen content was also reduced after increasing the litter concentration. The inhibitory effect of high litter concentrations was mainly on plant growth and to a lesser extent on plant nodulation byFrankia strains.     

Improving <Emphasis Type="Italic">Casuarina</Emphasis> growth and symbiosis with <Emphasis Type="Italic">Frankia</Emphasis> under different soil and environmental conditions—review

Casuarinas are very important plants for their various uses and survival in adverse sites or harsh environments. As nitrogen fixation, in symbiosis with Frankia, is an important factor for the survival of these plants under various conditions, the basis for selecting both effective and tolerant Frankia strains and Casuarina spp., are provided. Enhancement of the symbiotic relationship between Frankia and Casuarina, by mycorrhizal infection and other biofertilizing microorganisms such as Bacillus and Azospirillum, is reflected by superior plant growth. Casuarina leaf litter is also a great source for both inorganic and organic nutrients. Therefore, careful management of the top soil layer under Casuarina trees is very important. Litter decomposition ratio is affected by many physical chemical and biological factors including temperature, moisture conditions, lignin, and C-to-N and N-to-P ratios in addition to soil biota. In general, here the above relations are discussed and an alleviation model is presented for important disturbances of natural and human origin made in soil and environment, especially in the dry regions. In conclusion, we suggest how to optimize the nitrogen fixation and plant growth under the prevalent conditions.     

Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes

Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif⁺-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10⁴ cells.     

Production of growth hormones inCasuarina cunninghamiana root nodules induced byFrankia strain HFPCgI4

Summary Measurements of auxin and cytokinin activities in extracts ofCasuarina root nodules were made. The nodules were induced either by pure culture ofFrankia strain CgI4 or by crushed nodule inoculum. Levels of cytokinin activity were significantly higher in root nodules induced by pureFrankia culture than in those induced by crushed nodule inoculum. However, the reasons for this are unknown. Seasonal variation in levels of cytokinin activity inCasuarina nodules has also been detected.Dedicated to the memory of Professor John G. Torrey     

Occurrence of Myrica-nodulating Frankia in Hawaiian volcanic soils

The ability of Hawaiian volcanic soils to nodulate actinorhizal Myrica cerifera, Casuarina equisetifolia, and Alnus glutinosa was determined using a host-plant bioassay. Myrica-nodulating Frankia occurred in five volcanic deposits with depositional ages ranging from 20 to 162 years before present. The oldest deposit had a mean estimated nodulation capacity from 450 to 1200 times greater than those of the younger deposits. Only the oldest deposit had high moisture content, high organic matter content, and increased vegetative cover, including an abundance of actinorhizal M. faya. Casuarina- and Alnus-nodulating Frankia were not detected in any of these volcanic deposits.     

Dinitrogen fixation in a water-stressed Alnus clone is limited by host xerotolerance

The osmotolerance, rather than the halotolerance, of the endosymbiont predicted the xerotolerance of acetylene reduction by Alnus nodulated withFrankia ARgP5 ^AG . Cloned plants ofAlnus glutinosa (L.) Gaertn. AG8022-16 were subjected to water stress under controlled conditions in an environmental growth chamber. Transpiration, stomatal conductance, and leaf water potential had decreased after successive 10 day periods of moderate (75% of water demand) and severe (50% of water demand) water stress. After severe stress had wilted the plants, reducing leaf water potential to –2.10 MPa, nitrogenase activity had fallen to 2.51 M per plant per hour. The reported rapid turnover of nitrogenase implies thatFrankia mycelium was metabolically active at this low water potential, a water potential at which no Alnus-derivedFrankia has been reported active. Although ARgP5 ^AG was similar to other such strains in halotolerance (lower limitca.–1.25 MPa), the low water potential limit for growth with glucose (a non-assimilated osmoticum) wasca.–2.53 MPa. Nitrogenase activity was apparently more limited by host xerotolerance than by endophyte xerotolerance.Journal article J-5400 of the Oklahoma Agriculture Experiment Station, Oklahoma State University, Stillwater, OK 74078, USA.     

Studies of an effective strain ofFrankia fromAllocasuarina lehmanniana of the Casuarinaceae

Summary Seedlings ofCasuarina spp. andAllocasuarina spp. were grown from seed in the greenhouse and inoculated with a nodule suspension fromC. equisetifolia. Plants ofCasuarina spp. nodulated regularly and were effective in nitrogen-fixation. Only one species ofAllocasuariona, A. lehmanniana formed root nodules. Using these plants as source of inoculum, the isolation of a newFrankia sp. HFPA11I1 (HFP022 801) was made and the strain was grown in pure culture.Frankia sp. HFPA11I1 grows well in a defined medium and shows typical morphological characteristics. In media lacking combined nitrogen, the filamentours bacterium forms terminal vesicles in abundance and differentiaties large intrahyphal or terminal sporangia containing numerous spores. This strain, used as inoculum, nodulates effectively seedlings ofC. equisietifolia andC. cunninghamiana, forming nodules with verically-growing nodule roots. Although effective in acetylene reduction, the endophyte within the nodules is filamentous and lacks veiscles. When used to inoculated seedlings ofA llocasuarina lehmanniana, Frankia sp. HFPA11I1 induces root nodules which are coralloid and lacking nodule roots. The nodules are effective in acetylene reduction and the filamentous hyphae ofFrankia within the nodule lobes lack vesicles. Effective nodulation inA. Lehmanniana depends upon environmental conditions of the seedlings and proceeds much more slowly than in Casuariana.     

Survival ofFrankia strains introduced into soil

Factors affecting the establishment of Alnus/Frankia symbioses were studied partly by following the survival ofFrankia strains exposed to different soil conditions, and partly by investigating the effect of pH on nodulation. TwoFrankia strains were used, both of the Sp⁻ type (sporangia not formed in nodules). One of the strains sporulated heavily, while the other formed mainly hyphae. The strains originated fromAlnus incana root nodules growing in soils of pH 3.5 and 5.0. The optimum pH for their growth in pure culture was found to be 6.7 and 6.2, respectively. The strains were introduced into twoFrankia-free soils, peat and fine sand. Their survival, measured as the persistance of nodulation capacity using the plant infection technique, was followed for 14 months. The survival curves of the strains were similar despite the morphological differences between the strains in pure culture. The nodulation capacities declined over time both at 14 and 22°C. Survival was better in soils limed to a pH above 6 than in soils at their original pH (peat 2.9, fine sand 4.2). The effect of pH on nodule formation in Alnus seedlings by theFrankia strains was studied in liquid culture. The number of nodules increased linearly within the pH range studied (3.5–5.8). No nodules were formed at pH 3.5.     

Different genetic groups of Frankia within the root nodules of Casuarina growing in Mexico

This study was carried out to examine the possible genetic diversity that may occur among Frankia strains that nodulate C. equisetifolia in Mexico growing in the site of introduction, the cost of the Golf of Mexico, as well in the highlands. DNA extracted from reference cultures of Casuarina infecting Frankia strains, from field collected nodules (two trees of each of 14 sites and 3–5 analyzed lobes of each nodule) or from young nodules obtained from plants growing at the growth chamber and inoculated with field nodules, were used as the template in PCR reaction with primers targeting two DNA regions, one of the ribosomal operon and the other in the nif operon. PCR products were analyzed by using a set of recommended restriction enzymes. Six PCR-RFLP groups were detected after digestion with combination of enzymes BstUI and CfoI of the nif region, and four PCR-RFLP groups with enzymes NciI and ScrFI in the ribosomal region. Reference strains showed similar patterns and were assigned in group 1 along with an uncultured strain. nifD-K PCR-RFLP groups allowed a better understanding of the diversity among nodular Frankia strains than those observed in the rrs PCR-RFLP groups.     

Glasshouse evaluation of the growth ofAlnus rubra andAlnus glutinosa on peat and acid brown earth soils when inoculated with four sources ofFrankia

The effects of soil type (an acid peat and 2 acid brown earths) andFrankia source (3 spore-positive crushed nodule inocula and spore-negative crushed nodules containing the singleFrankia ArI5) on nodulation, N content and growth ofAlnus glutinosa andA. rubra were determined in a glasshouse pot experiment of two years duration. Plants on all soils required additional P for growth. Growth of both species was very poor on peat withA. glutinosa superior toA. rubra. The former species was also superior toA. rubra on an acid brown earth with low pH and low P content. Some plant-inoculum combinations were of notable effectivity on particular soils but soil type was the major source of variation in plant weight. Inoculation with crushed nodules containingFrankia ArI5 only gave poor infection of the host plant, suggesting that inoculation with locally-collected crushed nodules can be a preferred alternative to inoculation withFrankia isolates of untested effectivity. Evidence of adaptation ofFrankia to particular soils was obtained. Thus, while the growth of all strains was stimulated by mineral soil extracts, inhibitory effects of peat extracts were more apparent with isolates from nodules from mineral soils than from peat, suggesting that survival ofFrankia on peat may be improved by strain selection.