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.     

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.     

Effective nodulation ofCoriaria myrtifolia L. induced byFrankia strains fromAlnus glutinosa L. (Gaertn.)

The present contribution covers the cross-inoculation between two actinorhizae belonging to different genera and families, mainlyAlnus glutinosa andCoriaria myrtifolia. Frankia strains isolated fromA. glutinosa received from the Netherlands (LDAgp₁r₁, LDAgn₁) and from Scotland (UGL010708), induced a fully effective nodulation onC. myrtifolia. The same effect was caused by a nodule extract fromA. glutinosa. The reverse, a crushed-nodule inoculum fromC. myrtifolia nodulated all theA. glutinosa seedlings, though nodules formed were less effective than those induced by the other inocula. Re-isolation of thoseFrankia strains from the nodules formed onA. glutinosa was readily obtained, whereas attempts to re-isolate them from the nodules formed onC. myrtifolia failed, suggesting that isolation procedures different to those employed should be tried.     

Nodulation patterns of actinorhizal plants in the family rosaceae

Patterns of nodulation, growth, andFrankia — host specificity have not been well characterized for the actinorhizal genera in the family Rosaceae because of the scarcity ofFrankia isolates from these taxa. Furthermore, the few isolates available from actinorhizal Rosaceae have consistently failed to nodulate plants from the host genus. In a series of experiments, species of rosaceousDryas, Cowania, Cercocarpus, Fallugia, andPurshia were inoculated withFrankia isolates, crushedDryas actinorhizae, and neoglacial soils to ascertain whether any of these inocula would effectively induce nodulation. Neoglacial soils from Alaska and Canada nodulated not only the localDryas drummondii, but alsoCercocarpus betuloides, Cowania mexicana andPurshia tridentata from distant and ecologically diverse locales as well as nonrosaceous, actinorhizal species ofAlnus, Elaeagnus, Myrica, andShepherdia. But of eightFrankia isolates, including two fromPurshia tridentata and one fromCowania mexicana, none were able to induce nodulation onPurshia orCowania species. Globular, actinorhizae-like nodules incapable of acetylene reduction were produced onC. betuloides inoculated withFrankia isolates. Crushed nodule suspensions fromDryas drummondii nodulated rosaceousCowania, Dryas andPurshia, as well as non-rosaceousElaeagnus, Myrica, andShepherdia species. Nodules produced by inoculation ofCowania mexicana andPurshia tridentata with crushed, dried nodule suspensions fromDryas drummondii reduced acetylene to ethylene, indicating nitrogenase activity for these nodulated plants. These data suggest that a similar microsymbiont infects the actinorhizal genera in the family Rosaceae.     

Devising an <Emphasis Type="Italic">in vitro</Emphasis> nodulation system for two actinorhizal plants: <Emphasis Type="Italic">Allocasuarina verticillata</Emphasis> (Lam.) L. Johnson and <Emphasis Type="Italic">Casuarina glauca</Emphasis> sieb. ex spreng (Casuarinaceae)

Summary The purpose of this study was to establish an efficient in vitro nodulation device for producing actinorhizal root nodules on Allocasuarina verticillata and Casuarina glauca. Seeds from the two species were germinated aseptically and seedlings with at least two photosynthetic branchlets and a 3–5 cm long root system were transferred into Petri dishes containing a biphasic (solid/liquid) medium. To assess the nodulation capacity, four different culture media were tested. As soon as the root system developed and spread adequately on the surface of the medium, plants were deprived of nitrogen for at least 1 wk and inoculated with the Frankia strain. The time course nodulation for A. verticillata showed that the basal Hoagland medium supplemented with CaCO₃ and KNO₃ was most efficient, with 83% of plantlets forming nodules, while the medium supplemented with CaCO₃ reached 100% nodulation for C. glauca. This procedure can provide a valuable tool for the study of early events of actinorhizal nodulation and spatio-temporal expression of symbiotic genes in transgenic Casuarinaceae.     

Root nodulation studies inAeschynomene aspera

Summary Fifteen isolates of nodule bacteria were isolated from root and stem nodules ofAeschynomene aspera and they were characterized as Rhizobium by well known laboratory tests. All these isolates together with other efficient strains of known rhizobia belonging to different cross-inoculation groups were evaluated for their nodulation abilities onAeschynomene aspera, Cajanus cajan (pigeon pea),Cicer arietinum (chickpea),Pisum sativum (pea),Trifolium repens (clover),Medicago sativa (lucerne),Lens culinaris (lentil),Glycine max (soybean),Vigna sinensis (cowpea),Vigna radiata (mung bean),Vigna mungo (urd bean) andArachis hypogea (peanut). The results demonstrated that Rhizobium fromAeschynomene could form nodules only on its homologous host (Aeschynomene) but not on other legumes tested. Secondly, none of the rhizobia of other cross-inoculation groups could nodulateA. aspera.     

Nematodes (Longidorus sp. andTylenchorhynchus microphasmis loof) in growth and nodulation of sea buckthorn (Hippophaë rhamnoides L.)

Summary Hippophaë rhamnoides seedlings were grown in sterilized and unsterilized soil from a decliningH. rhamnoides scrub, to which different numbers ofLongidorus sp. andTylenchorhynchus microphasmis were added. In sterilized and unsterilized soil, retardation of growth, content of dry matter in the shoots, and incidence of deformed short lateral roots of test plants were positively correlated with counts of both nematode species. Nitrogen content in the shoots, nodulation on the roots of test plants and increase increase in nematodes were negatively correlated with the initial number of both nematode species in sterilized soil. In unsterilized soil, an unknown biotic factor was present that reduces growth ofH. rhamnoides, nodulation and multiplication of the nematodes. This factor seems to interact with the nematodes in reducing growth ofH. rhamnoides.Deceased.     

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.     

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.     

Feedback regulation of nodule formation in Hippophaë rhamnoides

Seedlings of Hippophaë rhamnoides possessing two equally infectible root systems (split roots) were used in conjunction with specific Frankia strains to investigate plant control over nodulation. When a wild-type Frankia strain was inoculated onto both root systems simultaneously or 1, 2, 4, or 8 weeks apart, an inhibitory response occurred which retarded nodulation on the root exposed to the delayed inoculum. Similar suppressive responses were also observed when two different wild-type Frankia inocula were applied onto opposite sides of a split-root system at different times. The depressed response shown by the delayed inoculum was more pronounced as the delay period was increased. The roots exposed to the delayed inoculum displayed a complete lack of nodulation when the delay was 4 or 8 weeks. The nodulation response on the root inoculated first depended on subsequent inoculation of the second root system of the plant, so that maximum nodulation of the first root was observed when the second root was unnodulated. These results provide evidence that sea buckthorn has an active, systemic mechanism for feedback control of nodulation that suppresses further nodule formation and prevents excessive nodulation. The significance of these results to the understanding of nodule ontogeny is discussed.     

Variable compatibility of clonedAlnus glutinosa ecotypes against ineffectiveFrankia strains

Nodulation tests onin-vitro propagated clones ofAlnus glutinosa ecotypes (forest ecotype, pioneer ecotype) withFrankia strains originating from both ecotypes indicated differences in host-plant compatibility. Inoculated plants of the pioneer ecotype clone were not infected by strains, that were unable to fix nitrogen in pure culture. Nodulation could only be induced on the clone of the forest ecotype, but no nitrogen-fixing activity could be detected. Ultra-structural observations of the nodules by SEM and TEM indicated that ineffectivity of these strains was correlated with the lack of vesicles in the infected cells. Cells were only filled with hyphae: neither sporangia nor vesicles could be detected. In contrast, effective nodules could be obtained on both alder clones after inoculation with an effective strain, showing normal development of vesicle clusters in infected cells. In pure culture the ineffective strains produced no vesicles; sporangia were found only during early stage of growth. The results demonstrate the existence ofFrankia strains which were either non-infective or ineffective on different clones ofAlnus glutinosa.     

Enhanced nodulation of soybean by Bradyrhizobium in the presence ofPseudomonas fluorescens

Experiments were undertaken to determine the effect ofPseudomonas fluorescens on nodulation of soybean by two strains ofBradyrhizobium japonicum, USDA I-110 and 61A76.Pseudomonas fluorescens can enhance the nodulation ability ofB. japonicum. Preincubation ofB. japonicum withP. fluorescens before inoculation further increased the level of nodulation.     

Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.     

Phenotypic and genetic diversity among rhizobia isolated from three Hedysarum species: H. spinosissimum,H. coronarium and H. flexuosum

Cultural, physiological and biochemical properties of 18 strains of rhizobia isolated from root nodules of the forage legume H. spinosissimum were compared with those of rhizobia from the related species H. coronarium (15 strains) and H. flexuosum (four strains). On the basis of 43 characteristics the 37 strains of Hedysarum rhizobia could be divided into two groups by numerical analysis. The H. spinosissimum rhizobia formed the first group and the second group comprised the strains from H. coronarium and H. flexuosum. The reference Rhizobium leguminosarum bv. viceae strain 250A was clustered with the rhizobia from H. coronarium and H. flexuosum. By contrast Bradyrhizobium sp. (Arachis) reference strain 280A was not clustered with any of the strains tested, indicating that the H. spinosissimum rhizobia differ from both Rhizobium and Bradyrhizobium. Serological data also discriminate between H. spinosissimum and H. coronariumrhizobia but not between the latter and H. flexuosum strains. The strains tested exhibit a high degree of specificity for nodulation and nitrogen fixation. We also determined the16SrRNA gene sequence of H. spinosissimum rhizobia (four strains), H. coronarium (two strains) and H. flexuosum (two strains) and found that the four H. spinosissimum isolates share a 98% identity among each other in this region but they showed less than 92% identity to the H. coronarium and H. flexuosum isolates. The H. spinosissimum isolates were closely related to both Mesorhizobium loti and M. ciceri, sharing 97% identity with each species.     

Variation with soil depth,topographic position and host species in the capacity of soils from an Australian locale to nodulateCasuarina andAllocasuarina seedlings

Sandy alluvial soils in a floodplain supporting a native stand ofCasuarina cunninghamiana Miq. produced about three times as many nodulated seedlings and more than twice as many nodules per nodulated seedling on roots of baitedCasuarina spp. than did clay loam red earth soils from the adjacent valley slope. Moist and well-aerated subsurficial alluvial sands had the greatest nodulation capacity of all the soils sampled. For all topographic positions, soil samples from depths greater than 20 cm promoted 76% more nodulated Casuarina seedlings than samples from the surficial 20 cm.Seedlings of three provenances ofC. cunninghamiana, together with seedlings ofC. glauca Sieb. ex Spreng.,C. cristata F. Muell ex Miq. andC. obesa Miq. developed significantly more nodules per pot and nodules per nodulated seedling in soils from this locale than seedlings of twoCasuarina equisetifolia Forst. provenances. Seedlings of two provenances ofAllocasuarina torulosa (Ait.) L. Johnson had fewer than 1% nodulated seedlings, a significantly lower level by far than that ofCasuarina seedlings.A. torulosa provenances also had significantly fewer nodulated seedlings per pot and nodules per nodulated seedling than all Casuarina hosts excepting one poorly-nodulated provenance ofC. equisetifolia.Nodulated seedlings of allCasuarina species had the capacity to fix atmospheric N₂, as indicated by acetylene-reduction capability. The presence of yellow cladodes and low rates of acetylene reduction per plant forC. cristata Miq. suggest that this association was poorly effective.     

Occurrence and infective potential of the endophyte ofHippophaë rhamnoides L. ssp.rhamnoides in coastal sand-dune areas

Summary The infective potential (IP) of nodule homogenates from field-grownHippophaë rhamnoides L. ssp.rhamnoides was determined by counting the number of nodules formed on test plants after inoculation with various dilutions of the homogenates. The IP was almost constant,i.e. 10⁵ to 10⁶ per gram of fresh nodule material. Methods to store nodule material without loss of IP were tested. The IP of air-dried nodule powders stored at 6°C hardly decreased during a period of more than a year.Data are presented on the IP of soil samples from sites representing various stages of dune formation. BeforeH. rhamnoides appeared, the IP was low: 1 to 36 nodules were formed on test plants per kg of soil. This low IP was due to low numbers of infective endophyte particles in these soils. During the succession of theH. rhamnoides scrub, the IP of the soil increased, due to the increase in the number of endophyte particles in the soil. Gradually, however, nodulation was limited by other environmental factors. The nature of these factors is discussed.     

Speed of nodulation and competitive ability among strains of Rhizobium leguminosarum bv phaseoli

This study examines the speed of nodulation of 20 strains of Rhizobium leguminosarum bv phaseoli, and relates this trait to the competitive performance of these strains with Phaseolus vulgaris L. At 25/20°C day/night temperature, and with 10⁷ cells applied per growth pouch, there was a strong positive correlation between the speed of nodulation and the competitiveness of strains with the nod ⁺ fix^– reference strain UMR 1116. Strains UMR 1084, 1125, 1165, 1173 and 1384 combined good competitive performance with extensive nodulation in the uppermost root regions. When inoculant levels in the RTM studies were reduced to 10³ cells per pouch no correlation between the apparent competitiveness of strains and their speed in nodulation was evident, presumably because cells had to undergo multiplication before infection. Nodulation was also delayed when growth temperatures were raised to 31/26°C, but a correlation was still evident between competitive performance and nodulation in the region 0.1 to 5.0 mm below the RTM at the time of inoculation. From these results speed of nodulation can be used to estimate the competitive potential of Rhizobium strains, but only under carefully regulated conditions. The effects of inoculation level and temperature on the relationship between speed of nodulation and strain competitiveness could explain the inconsistent results obtained in earlier studies on this topic.Journal paper No. 16962, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

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.     

Diversity ofFrankia strains isolated from a single alder stand

One hundredFrankia strains isolated from variousAlnus species in a single alder stand were tested for plasmid presence. Plasmid DNA was observed in five of the frankiae strains and was analyzed. We found that plasmids with a similar molecular weight exhibited in fact minor divergences in restriction patterns. The genetic diversity among the five isolates which contained plasmids and seven isolates which contained no plasmid DNA were examined by using restriction endonucleas digestions, Southern hybridization ofnifHDK,nifAB genes, andFrankia cryptic DNA fragments determined at random. Results indicate that genomic DNA digestion patterns and Southern hybridizations to anifHDK probe were not able to discriminate between closely related frankiae. On the other hand, plasmid presence, Southern hybridization to anifAB proble or to a crypticFrankia probe allowed us to delineate groupings of these isolates.     

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.