Non-nodulating Species Research Articles

NIN-at the heart of NItrogen-fixing Nodule symbiosis.

Legumes and actinorhizal plants establish symbiotic relationships with nitrogen-fixing bacteria, resulting in the formation of nodules. Nodules create an ideal environment for nitrogenase to convert atmospheric nitrogen into biological available ammonia. NODULE INCEPTION (NIN) is an indispensable transcription factor for all aspects of nodule symbiosis. Moreover, NIN is consistently lost in non-nodulating species over evolutions. Here we focus on recent advances in the signaling mechanisms of NIN during nodulation and discuss the role of NIN in the evolution of nitrogen-fixing nodule symbiosis.

Frankia nepalensis sp. nov., a non-infective non-nitrogen-fixing isolate from root nodules of Coriaria nepalensis Wall

Strains CN4T, CN6, CN7 and CNm7 were isolated from root nodules of Coriaria nepalensis from Murree in Pakistan. They do not form root nodules on C. nepalensis nor on Alnus glutinosa although they deformed root hairs of Alnus. The colonies are bright red-pigmented, the strains form hyphae and sporangia but no N2-fixing vesicles and do not fix nitrogen in vitro. The peptidoglycan of strain CN4T contains meso-diaminopimelic acid; whole cell sugars consist of ribose, mannose, glucose, galactose and rhamnose. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown lipids represent the major polar lipids; MK-9(H4) and MK-9(H6) are the predominant menaquinones (>15 %), and iso-C16 : 0 and C17 : 1ω8c are the major fatty acids (>15 %). The results of comparative 16S rRNA gene sequence analyses indicated that strain CN4T is most closely related to Frankia saprophytica CN 3T. An MLSA phylogeny using amino acids sequences of AtpD, DnaA, FtsZ, Pgk and RpoB, assigned the strain to cluster 4 non-nodulating species, close to F. saprophytica CN 3T , Frankia asymbiotica M16386T and Frankia inefficax EuI1cT with 0.04 substitutions per site, while that value was 0.075 with other strains. Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between CN4T and all species of the genus Frankia with validly published names were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 27.8 and 83.7 %, respectively. The four strains CN4T, CN6, CN7 and CNm7 had dDDH (98.6–99.6 %) and ANI values that grouped them as representing a single species. CN4T has a 10.76 Mb genome. CN4T was different from its close phylogenetic neighbours with validly published names in being red-pigmented, in having several lantibiotic-coding clusters, a carbon monoxide dehydrogenase cluster and a clustered regularly interspaced short palindromic repeats (CRISPR) cluster. The results of phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain CN4T (=DSM 114740T = LMG 32595T) to a novel species, with CN4T as type strain, for which the name Frankia nepalensis sp. nov. is proposed.

Comparison of the seasonal and successional variation of asymbiotic and symbiotic nitrogen fixation along a glacial retreat chronosequence

Climate change is resulting in accelerated retreat of glaciers worldwide and much nitrogen-poor debris is left after glacier retreats. Asymbiotic dinitrogen (N2) fixation (ANF) can be considered a ‘hidden’ source of nitrogen (N) for non-nodulating plants in N limited environments; however, seasonal variation and its relative importance in ecosystem N budgets, especially when compared with nodulating symbiotic N2-fixation (SNF), is not well-understood. In this study, seasonal and successional variations in nodulating SNF and non-nodulating ANF rates (nitrogenase activity) were compared along a glacial retreat chronosequence on the eastern edge of the Tibetan Plateau. Key factors regulating the N2-fixation rates as well as the contribution of ANF and SNF to ecosystem N budget were also examined. Significantly greater nitrogenase activity was observed in nodulating species (0.4–17,820.8 nmol C2H4 g−1 d−1) compared to non-nodulating species (0.0–9.9 nmol C2H4 g−1 d−1) and both peaked in June or July. Seasonal variation in acetylene reduction activity (ARA) rate in plant nodules (nodulating species) and roots (non-nodulating species) was correlated with soil temperature and moisture while ARA in non-nodulating leaves and twigs was correlated with air temperature and humidity. Stand age was not found to be a significant determinant of ARA rates in nodulating or non-nodulating plants. ANF and SNF contributed 0.3–51.5 % and 10.1–77.8 %, respectively, of total ecosystem N input in the successional chronosequence. In this instance, ANF exhibited an increasing trend with successional age while SNF increased only at stages younger than 29 yr and then decreased as succession proceeded. These findings help improve our understanding of ANF activity in non-nodulating plants and N budgets in post glacial primary succession.

Contributions of Leguminosae to young and old stands of neotropical forests under different environmental conditions

Potentially nodulating Leguminosae species had high density and biomass in young stands of forests in Northeast Brazil, except in a dry forest on a low-fertility soil. Leguminosae species, especially potentially nodulating ones, appear to be important components of tropical secondary forests, although information is still scarce concerning their contributions to richness and biomass under different environmental conditions. To evaluate the contribution of Leguminosae to the aboveground biomass and density of young and old forest stands in six sites, with different climate and soil conditions, in northeastern Brazil. The biomass of plants was estimated using allometric equations in ten plots in one young and one old forest stand in each site. Leguminosae were separated into nodulating and non-nodulating species based on literature. Leguminosae, mainly nodulating species, displayed the largest number of species among all families and the largest biomass and density in young stands. There was more similarity in Leguminosae species composition between young and old stands in dry than in humid forests. Nodulating species make considerable contributions to the biomass of the initial successional stages of forests in Northeast Brazil — confirming the importance of the family for natural forest regeneration, especially in dry environments.

A Homeotic Mutation Changes Legume Nodule Ontogeny into Actinorhizal-Type Ontogeny.

Some plants fix atmospheric nitrogen by hosting symbiotic diazotrophic rhizobia or Frankia bacteria in root organs known as nodules. Such nodule symbiosis occurs in 10 plant lineages in four taxonomic orders: Fabales, Fagales, Cucurbitales, and Rosales, which are collectively known as the nitrogen-fixing clade. Nodules are divided into two types based on differences in ontogeny and histology: legume-type and actinorhizal-type nodules. The evolutionary relationship between these nodule types has been a long-standing enigma for molecular and evolutionary biologists. Recent phylogenomic studies on nodulating and nonnodulating species in the nitrogen-fixing clade indicated that the nodulation trait has a shared evolutionary origin in all 10 lineages. However, this hypothesis faces a conundrum in that legume-type and actinorhizal-type nodules have been regarded as fundamentally different. Here, we analyzed the actinorhizal-type nodules formed by Parasponia andersonii (Rosales) and Alnus glutinosa (Fagales) and found that their ontogeny is more similar to that of legume-type nodules (Fabales) than generally assumed. We also show that in Medicago truncatula, a homeotic mutation in the co-transcriptional regulator gene NODULE ROOT1 (MtNOOT1) converts legume-type nodules into actinorhizal-type nodules. These experimental findings suggest that the two nodule types have a shared evolutionary origin.

The Evolutionary Aspects of Legume Nitrogen-Fixing Nodule Symbiosis.

Nitrogen-fixing root nodule symbiosis can sustain the development of the host plants under nitrogen-limiting conditions. Such symbiosis occurs only in a clade of angiosperms known as the nitrogen-fixing clade (NFC). It has long been proposed that root nodule symbiosis evolved several times (in parallel) in the NFC. Two recent phylogenomic studies compared the genomes of nodulating and related non-nodulating species across the four orders of the NFC and found that genes essential for nodule formation are lost or pseudogenized in the non-nodulating species. As these symbiosis genes are specifically involved in the symbiotic interaction, it means that the presence of pseudogenes and the loss of symbiosis genes strongly suggest that their ancestor, which still had functional genes, most likely had a symbiosis with nitrogen-fixing bacteria. These findings agree with the hypothesis that nodulation evolved once at the common ancestor of the NFC, and challenge the hypothesis of parallel evolution. In this chapter, we will cover the current understandings on actinorhizal-type and legume nodule development, and discuss the evolution of the legume nodule type.

Assessment of Polygala paniculata (Polygalaceae) characteristics for evolutionary studies of legume\u2013rhizobia symbiosis

Root nodule (RN) symbiosis is a mutualistic interaction observed between nitrogen-fixing soil bacteria and nodulating plants, which are scattered in only four orders of angiosperms called nitrogen-fixing clade. Most of legumes engage in RN symbiosis with rhizobia. Molecular genetic analyses with legumes and non-leguminous nodulating plants revealed that RN symbiosis utilizes early signalling components that are required for symbiosis with arbuscular mycorrhizal (AM) fungi. However detailed evolutionary processes are still largely unknown. Comparative analyses with non-nodulating species phylogenetically related to legumes could be better strategies to study the evolution of RN symbiosis in legumes. Polygala paniculata is a non-leguminous species that belongs to a family different from legumes but that is classified into the same order, Fabales. It has appropriate characteristics for cultivation in laboratories: small body size, high fertility and short lifecycles. Therefore, we further assessed whether this species is suitable as a model species for comparative studies with legumes. We first validated that the plant we obtained in Palau was truly P. paniculata by molecular phylogenetic analysis using rbcL sequences. The estimated genome size of this species was less than those of two model legumes, Lotus japonicus and Medicago truncatula. We determined conditions for cultivation in vitro and for hairy root formation from P. paniculata seedlings. It would facilitate to investigate gene functions in this species. The ability of P. paniculata to interact with AM fungi was confirmed by inoculation with Rhizophagus irregularis, suggesting the presence of early signalling factors that might be involved in RN symbiosis. Unexpectedly, branching of root hairs was observed when inoculated with Mesorhizobium loti broad host range strain NZP2037, indicating that P. paniculata has the biological potential to respond to rhizobia. We propose that P. paniculata is used as a model plant for the evolutionary study of RN symbiosis.

Dryas as a Model for Studying the Root Symbioses of the Rosaceae.

The nitrogen-fixing root nodule symbiosis is restricted to four plant orders: Fabales (legumes), Fagales, Cucurbitales and Rosales (Elaeagnaceae, Rhamnaceae, and Rosaceae). Interestingly all of the Rosaceae genera confirmed to contain nodulating species (i.e., Cercocarpus, Chamaebatia, Dryas, and Purshia) belong to a single subfamily, the Dryadoideae. The Dryas genus is particularly interesting from an evolutionary perspective because it contains closely related nodulating (Dryas drummondii) and non-nodulating species (Dryas octopetala). The close phylogenetic relationship between these two species makes Dryas an ideal model genus to study the genetic basis of nodulation by whole genome comparison and classical genetics. Therefore, we established methods for plant cultivation, transformation and DNA extraction for these species. We optimized seed surface sterilization and germination methods and tested growth protocols ranging from pots and Petri dishes to a hydroponic system. Transgenic hairy roots were obtained by adapting Agrobacterium rhizogenes-based transformation protocols for Dryas species. We compared several DNA extraction protocols for their suitability for subsequent molecular biological analysis. Using CTAB extraction, reproducible PCRs could be performed, but CsCl gradient purification was essential to obtain DNA in sufficient purity for high quality de novo genome sequencing of both Dryas species. Altogether, we established a basic toolkit for the culture, transient transformation and genetic analysis of Dryas sp.

Diversification of cytokinin phosphotransfer signaling genes in Medicago truncatula and other legume genomes

BackgroundLegumes can establish on nitrogen-deprived soils a symbiotic interaction with Rhizobia bacteria, leading to the formation of nitrogen-fixing root nodules. Cytokinin phytohormones are critical for triggering root cortical cell divisions at the onset of nodule initiation. Cytokinin signaling is based on a Two-Component System (TCS) phosphorelay cascade, involving successively Cytokinin-binding Histidine Kinase receptors, phosphorelay proteins shuttling between the cytoplasm and the nucleus, and Type-B Response Regulator (RRB) transcription factors activating the expression of cytokinin primary response genes. Among those, Type-A Response Regulators (RRA) exert a negative feedback on the TCS signaling. To determine whether the legume plant nodulation capacity is linked to specific features of TCS proteins, a genome-wide identification was performed in six legume genomes (Cajanus cajan, pigeonpea; Cicer arietinum, chickpea; Glycine max, soybean; Phaseolus vulgaris, common bean; Lotus japonicus; Medicago truncatula). The diversity of legume TCS proteins was compared to the one found in two non-nodulating species, Arabidopsis thaliana and Vitis vinifera, which are references for functional analyses of TCS components and phylogenetic analyses, respectively.ResultsA striking expansion of non-canonical RRBs was identified, notably leading to the emergence of proteins where the conserved phosphor-accepting aspartate residue is replaced by a glutamate or an asparagine. M. truncatula genome-wide expression datasets additionally revealed that only a limited subset of cytokinin-related TCS genes is highly expressed in different organs, namely MtCHK1/MtCRE1, MtHPT1, and MtRRB3, suggesting that this “core” module potentially acts in most plant organs including nodules.ConclusionsFurther functional analyses are required to determine the relevance of these numerous non-canonical TCS RRBs in symbiotic nodulation, as well as of canonical MtHPT1 and MtRRB3 core signaling elements.

Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis.

The root nodule symbiosis of plants with nitrogen-fixing bacteria affects global nitrogen cycles and food production but is restricted to a subset of genera within a single clade of flowering plants. To explore the genetic basis for this scattered occurrence, we sequenced the genomes of 10 plant species covering the diversity of nodule morphotypes, bacterial symbionts, and infection strategies. In a genome-wide comparative analysis of a total of 37 plant species, we discovered signatures of multiple independent loss-of-function events in the indispensable symbiotic regulator NODULE INCEPTION in 10 of 13 genomes of nonnodulating species within this clade. The discovery that multiple independent losses shaped the present-day distribution of nitrogen-fixing root nodule symbiosis in plants reveals a phylogenetically wider distribution in evolutionary history and a so-far-underestimated selection pressure against this symbiosis.

Size, structure and nitrogen content of seeds of Fabaceae in relation to nodulation

Seed weight in Fabaceae ranges over seven orders of magnitude from 0.1 mg to 334 g. The frequency distribution is strongly right skewed with a single modal peak at 18 mg. The two sets of morphological characters, the presence or absence of endosperm and the anatomical form of the cotyledons, interact to define eight structurally and functionally distinct seed types. The predominant seed type is endospermic with foliar cotyledons. The other seed types have evidently arisen from it by transfer of storage function from endosperm to cotyledons, culminating in exendospermic seeds with storage cotyledons. This has probably happened repeatedly in disparate tribes and genera throughout the family. There is a highly significant allometric relationship between the logarithms of the weight of nitrogen in the seed and the total seed weight, with the datasets for nodulating and non-nodulating species yielding significantly different regression equations. The rate of increase in weight of nitrogen is lower than the rate of increase in total seed weight, and this discrepancy is greater in non-nodulating than in nodulating species. A consequence of the discrepancy is an overall inverse relationship between seed weight and nitrogen concentration. Seeds of the same type are significantly larger and have a higher absolute nitrogen content, but a lower nitrogen concentration in non-nodulating than in nodulating species.

Phenotypic and genotypic characterization of rhizobia associated with Acacia saligna (Labill.) Wendl. in nurseries from Algeria

Twenty seven rhizobial strains associated with Acacia saligna grown in northern and southern Algeria were characterized, including generation time, host-range, the 16S rRNA gene and 16S–23S rRNA intergenic spacer restriction patterns, 16S rRNA gene sequence analysis and tolerance to salinity and drought. Cross inoculation tests indicated that 11 slow-growing isolates from northern nurseries were able to nodulate introduced Australian acacias exclusively, whereas 16 fast-growing isolates, mainly from southern nurseries, were capable of also nodulating native acacias. Restriction patterns and sequence analysis of the 16S rRNA gene showed that strains of the first group belonged to Bradyrhizobium while strains of the second group were related to Sinorhizobium meliloti and Rhizobium gallicum. Interestingly, five strains of the first group formed a distinct cluster phylogenetically close to Bradyrhizobium betae, a non-nodulating species causing tumour-like deformations in sugar beet roots. Bradyrhizobium strains were in general more sensitive to NaCl and PEG than the S. meliloti and R. gallicum representatives. Among the latter, strains S. meliloti BEC1 and R. gallicum DJA2 were able to tolerate up to 1 M NaCl and 20% PEG. This, together with their wide host-range among Acacia species, make them good candidates for developing inoculants for A. saligna and other acacia trees growing in arid areas.

Novel expression pattern of cytosolic Gln synthetase in nitrogen-fixing root nodules of the actinorhizal host, Datisca glomerata.

Gln synthetase (GS) is the key enzyme of primary ammonia assimilation in nitrogen-fixing root nodules of legumes and actinorhizal (Frankia-nodulated) plants. In root nodules of Datisca glomerata (Datiscaceae), transcripts hybridizing to a conserved coding region of the abundant nodule isoform, DgGS1-1, are abundant in uninfected nodule cortical tissue, but expression was not detectable in the infected zone or in the nodule meristem. Similarly, the GS holoprotein is immunolocalized exclusively to the uninfected nodule tissue. Phylogenetic analysis of the full-length cDNA of DgGS1-1 indicates affinities with cytosolic GS genes from legumes, the actinorhizal species Alnus glutinosa, and nonnodulating species, Vitis vinifera and Hevea brasilensis. The D. glomerata nodule GS expression pattern is a new variant among reported root nodule symbioses and may reflect an unusual nitrogen transfer pathway from the Frankia nodule microsymbiont to the plant infected tissue, coupled to a distinctive nitrogen cycle in the uninfected cortical tissue. Arg, Gln, and Glu are the major amino acids present in D. glomerata nodules, but Arg was not detected at high levels in leaves or roots. Arg as a major nodule nitrogen storage form is not found in other root nodule types except in the phylogenetically related Coriaria. Catabolism of Arg through the urea cycle could generate free ammonium in the uninfected tissue where GS is expressed.

Software dependent phenetic relationships of non-nodulating African species ofAcacia

The phenetic analysis of non-nodulatingAcacia species by Harrier et al. (1997) was repeated to illustrate how different computer programs may generate alternative UPGMA trees for the very same data, even in the absence of data input order effects (ties). For example, all Harrier et al.'s UPGMA dendrograms produced by software from the Scottish Agricultural Statistics Service differed from those obtained by the packages NTSYS and MVSP87. Particularly, the positions ofA. albida, A. rovumae, andA. pentagona, as well as the relationships betweenDiacanthae andTriacanthae were affected by this phenomenon. Hence, whenever clustering techniques are used, care should be taken to consider possible software-dependent caveats and artefacts. Nevertheless, all programs provided clusterings that largely coincided with the subgeneric and sectional groupings proposed by Vassal (1972) although the positions of some species varied depending on whether morphological or molecular data were considered (e.g.A. albida andA. rovumae).

Rhizobiology and nitrogen fixation of some tree legumes native to Sri Lanka

Rhizobiology of four tree legumes, Abarema bigemina (endemic), Adenanthera bicolor (endemic), Humboldtia laurifolia (indigenous) and Pericopsis mooniana (indigenous), was studied by: (1) observations of their in situ root nodulation, (2) characterization and authentication of rhizobia isolated from them, and (3) an examination of the infectivity and effectivity of the rhizobial isolates using the respective hosts and the broad-spectrum host, siratro (Macroptillium atropurpureum). Investigations were also conducted to evaluate the effect of rhizobial inoculation on growth and N fixation of the valuable timber species, P. mooniana, during its early stages of growth. All the hosts revealed in situ nodulation, except A. bicolor which had nodule-like swellings on its roots. While four rhizobial isolates were obtained from A. bigemina, four from H. laurifolia and 18 from P. mooniana, no authentic rhizobia were isolated from A. bicolor. The nodule-like structures did not show acetylene reduction activity, and the plants failed to nodulate upon inoculation with rhizosphere soil. A. bicolor was therefore tentatively assigned as a non-nodulating species. This report includes the first description of root nodules in the endemic species A. bigemina and the indigenous species H. laurifolia and the isolation and characterization of rhizobia from them. Significant differences were observed among the isolates with regard to their infectivity and effectivity. One isolate from P. mooniana was outstanding in its infectivity and effectivity on the original host, as well as on siratro. Inoculation of P. mooniana with this isolate together with a reference strain significantly improved nodulation and N fixation and gave a 50% increase in plant dry matter under a low level of N fertilizer, during a 12-month growth period. It is suggested that the nursery culture of P. mooniana could be improved by inoculation under low N-input conditions. The ability of these rhizobia to persist in the soil for a 12-month period was observed by the use of strains marked with antibiotics.

Pre-infection events in non-nodulating species of African Acacia

Non-nodulation occurs sporadically throughout the family Leguminosae, the genus Acacia is unusual as there are a range of species which are consistently non-nodulating, but which are closely related to species able to form nodules. A comparative study of these and coexisting related nodulating species has been made to establish whether the non-nodulating species exhibited any pre-infection characteristics which are typical of their nodulating counterparts. The non-nodulating species did not form any nodule-like structures, but exhibited pre-infection characteristics such as rhizobial attachment similar to that of their nodulating counterparts. The root exudate of nodulating A. polyacantha contained stimulatory compounds which aided binding of rhizobia to the roots of the non-nodulating species and nodulation was completely inhibited when A. polyacantha was co-cultured with a non-nodulating species.

Early Nodulin Genes are Not Markers of the Capacity of Woody Legumes to Nodulate

Knowing whether trees can develop nodules in which nitrogen is fixed is important for managing inputs during culture. Early nodulin genes, such as ENOD2 and ENOD12, could function as molecular markers for the capacity to nodulate if they are conserved only in species that nodulate. Several nodulating species are known to possess these genes; but, their occurrence has not been studied among non-nodulating taxa, including Cercis canadensis L. (redbud), Gleditsia triacanthos L. var. inermis Willd. (honey locust), and Gymnocladus dioica (L.) C. Koch (Kentucky coffee tree). Our objective was to determine the relationship between the capacity to nodulate and the occurrence of putative ENOD2 or ENOD12 genes by probing the genomes of these non-nodulating species and the genomes of two legumes that nodulate, Albizia julibrissin Durazz. (silk tree) and Laburnum alpinum (Mill.) Bercht. & J. Presl (Scotch laburnum). ENOD2 and ENOD12 cDNA clones from Glycine max (L.) Merill (soybean) and Pisum sativum L. (pea), respectively, and cloned ENOD2 PCR fragments from Maackia amurensis Rupr. & Maxim. (Amur maackia) and Styphnolobium japonicum (L.) Schott (Japanese pagodatree) were used as probes for Southern hybridizations. Sequences from genomes of silk tree, Scotch laburnum, honey locust, and Kentucky coffee tree hybridized to ENOD2 probes on Southern blots. Putative ENOD12 sequences were detected in the genomes of Scotch laburnum, redbud, and honey locust. Thus, we conclude that ENOD2 and ENOD12 can not be used as markers for the capacity to nodulate.

Nodulação e crescimento de 49 leguminosas arbóreas nativas da Amazônia em viveiro

Os ecossistemas periodicamente inundados, embora representem 25% da área total da Amazônia, contribuem com 75% da produção madeireira. Tanto esses ecossistemas como os de terra firme apresentam grande diversidade de espécies da família Leguminosae. Algumas dessas espécies já são exploradas economicamente de forma extrativista, mas o potencial econômico da maioria permanece inexplorado. Conhecer o comportamento dessas espécies em condições de viveiro é o primeiro passo para compreender sua biologia e estabelecer plantios nas áreas já desmatadas. Neste trabalho, desenvolvido no Campus do INPA, em Manaus, durante 1985 e 1986, são apresentadas informações sobre o crescimento inicial e nodulação de 49 espécies arbóreas nativas, de ecossistemas de terra firme (16) ou periodicamente inundados, (33) da Amazônia. Plântulas recém-germinadas foram transplantadas, em condições de viveiro, para sacos plásticos que continham cinco diferentes substratos que consistiram de areia adubada, de uma mistura de argila com areia 3:2 (v/v), de material do horizonte A de um glei húmico, e de material do horizonte A de um podzólico vermelho-amarelo, adubado ou não. A maioria das espécies desenvolveu-se bem nos cinco substratos. Algumas espécies cresceram melhor nos substratos, de maior fertilidade. Espécies oriundas de ecossistemas inundados cresceram melhor que as oriundas de terra firme. Foi observada nodulação em 30 espécies. Entre as que apresentaram maiores taxas de crescimento, havia tanto espécies nodulíferas como não-nodulíferas, indicando que a fixação biológica de N2 não é o único mecanismo eficiente de absorção de nitrogênio em leguminosas tropicais. As espécies que apresentaram as maiores taxas de crescimento foram: Enterolobium maximum, Swartzia polyphylla e Vatairea guianensis.

A study of the symbiotic importance and location of nod gene inducing compounds in two widely nodulating and two non-nodulating tropical tree species

Previous work led us to consider the role of nod gene-inducing compounds in related nodulating and non-nodulating Acacia species. Could the nodulation status of the non-nodulating plants be determined by an inability to produce and exude compounds that the related nodulating species were capable of delivering to the rhizobial symbionts? Also, it seemed to have been assumed previously that results gathered for extracts of seedlings or root tissue would reflect the situation for compounds exuded freely into the rhizosphere. The growth medium used in this investigation gave an opportunity to test this theory with the added bonus of allowing root exudates to be collected from a limited number of plants. The two non-nodulating species were found to lack neither the ability to produce potentially inducing compounds nor the means to exude them into the rhizosphere. However, it was demonstrated, through the distribution of potentially inducing compounds within the plants and their exudates, that the components of tissue extractions did not necessarily reflect the components exuded into the rhizosphere.

Phenetic investigation of non-nodulating African species ofAcacia (Leguminosae) using morphological and molecular markers

Morphological and RAPD markers were used to assess the relationships among nodulating and non-nodulating species of AfricanAcacia. Non-nodulating species of AfricanAcacia are only found within subg.Aculeiferum sect.Monacanthea. African species of sect.Monacanthea examined were found to form a group distinct from the other African species examined on a morphological and molecular basis. All lack the ability to nodulate, suggesting that non-nodulation may be used as a taxonomic tool. The species of sect.Aculeiferum were separated by RAPD and morphological analysis into two groups depending on whether they were armed with prickles in pairs and/or prickles in threes, or solitary. A third group of species was identified within sect.Acacia: further subdivision of this group was achieved into subsectt.Pluriseriae andUniseriae. The position ofA. albida relative to other AfricanAcacia species was found to be distinct but not totally independent of the genus. The partitioning and distribution of the genetic variability within the genus is further elucidated by the RAPD analysis of populations ofAcacia species. A population analysis ofA. polyacantha demonstrated geographical and site-specific variation.