Split-Root Assays Using Trifolium subterraneum Show that Rhizobium Infection Induces a Systemic Response That Can Inhibit Nodulation of Another Invasive Rhizobium Strain

Subterranean clover plants possessing two equally infectible and robust lateral root systems (“split roots”) were used in conjunction with several specific mutant strains (derived from Rhizobium trifolii ANU843) to investigate a systemic plant response induced by infective Rhizobium strains. This plant response controls and inhibits subsequent nodulation on the plant. When strain ANU843 was inoculated onto both root systems simultaneously or 24, 48, 72, or 96 h apart, an inhibitory response occurred which retarded nodulation on the root exposed to the delayed inoculum but only when the delay period between inocula was greater than 24 h. Equal numbers of nodules were generated on both roots when ANU843 was inoculated simultaneously or 24 h apart. The ability to infect subterranean clover plants was required to initiate the plant inhibitory response since preexposure of one root system to non-nodulating strains did not retard the ability of the wild-type strain to nodulate the opposing root system (even when the delay period was 96 h). Moreover, the use of specific Tn5-induced mutants subtly impaired in their ability to nodulate demonstrated that the plant could effectively and rapidly discriminate between infections initiated by either the parent or the mutant strains. When inoculated alone onto clover plants, these mutant strains were able to infect the most susceptible plant cells at the time of inoculation and induce nitrogen-fixing nodules. However, the separate but simultaneous inoculation on opposing root systems of the parent and the mutant strains resulted in the almost complete inhibition of the nodulation ability of the mutant strains. We concluded that the mutants were affected in their competitive ability, and this finding was reflected by poor nodule occupancy when the mutants were coinoculated with the parent strain onto a single root system. Thus the split-root system may form the basis of a simple screening method for the ranking of competitiveness of various rhizobia on small seeded legumes.     

The Flavin Content of Clovers Relative to Symbiosis with a Riboflavin-requiring Mutant of Rhizobium trifoli

A riboflavin-requiring auxotroph of Rhizobium trifolii (T1/D-his(r)-15) formed ineffective root nodules on red clover and on two cultivars of subterranean clover, but produced almost fully effective nodules on several other cultivars of subterranean clover. Fluorescence and bioassay measurements of the flavin content of the roots and shoots of these cultivars revealed no differences between cultivars which could be correlated with the differences in symbiotic response. The concentration of flavin in nodules formed by the auxotroph (in the absence of riboflavin), by the effective parent strain (T1), or by a partly effective mutant (penicillin-resistant) of T1 was roughly proportional to the effectiveness of the nodules. Effective nodules contained 20 times as much flavin, and ineffective nodules 3 to 4 times as much flavin as non-nodulated root tissue. Approximately 20 to 30% of the flavins in both root and nodule tissue was flavin adenine dinucleotide and 70 to 80% was riboflavin + flavin mononucleotide. Most of the flavin adenine dinucleotide in macerated nodules was associated with host cell fragments, and none was detected in a cell-free fraction. Bacteroids accounted for approximately 20% of flavins in effective nodules and also contained more riboflavin + flavin mononucleotide than cultured rhizobial cells. The total flavin content of noninoculated roots increased from about 1.2 nmoles to 1.7 nmoles flavin/g of tissue after 3 days' exposure to 80 mum riboflavin. Exposure of only the upper or lower portion of preinoculated roots indicated negligible translocation, as effective nodulation occurred only on parts of the root in direct contact with riboflavin. Plants grown in a medium containing combined nitrogen (100 or 300 mum nitrogen added as (NH(4))(2)SO(4)), but no added riboflavin showed an increased root flavin content (about 2.1 nmoles flavin/g tissue) and a partly effective response when inoculated with the mutant. Nitrogen also promoted some upward translocation of exogenous riboflavin in the roots.     

Studies on the Characterization of Root Morphology of White Clover Infected by Transconjugant of Rhizobium leguminosarum

White clover plants were inoculated with transconjugant strain' 290 which was obtained from introduction of host specific nodulation genes of wild-type Rhizobium trifolii strain ANU 843 to Rhizobium leguminosarum strain 300. The characterization of root morphology of white clover induced by the transconjugant was observed and compared to the plants induced by the parent strains. White clover started tO form a typical root hair curling inoculated with transconjugant strain 290 24h after inoculation, at 48h a part of cell wall of root hair was degradated, infection thread was observed in the infected root hair cell, cortical cell divisions occurred extensively. All these characterizations were similar to that infected by strain ANU 843. Plant inoculation test indicated that no nodule was formed when inoculated by R. leguminosarum strain 300, while plants nodulated when inoculated with transconjugant strain 290 as well as R. trifolii ANU 843. This suggests that introduction of host specific nodulation genes of R. trifolii results in conferring the nodulation ability of R. leguminosarum on white clover.     

Symbiotic and competitive properties of motility mutants of Rhizobium trifolii TA1

Non-motile mutants of Rhizobium trifolii defective in either flagellar synthesis or function were isolated by transposon Tn5 mutagenesis. they were indistinguishable from motile control strains in growth in both laboratory media and in the rhizosphere of clover roots. When each non-motile mutant was grown together with a motile strain in continuous culture, the numbers of motile and non-motile organisms remained in constant proportion, implying that their growth rates were essentially identical. When inoculated separately onto clover roots, the mutants and wildtype did not differ significantly in the number of nodules produced or in nitrogen fixing activity. However, when mixtures of equal numbers of mutant and wild-type cells were inoculated onto clover roots, the motile strain formed approximately five times more nodules than the flagellate or non-flagellate, non-motile mutants, suggesting that motility is a factor in competition for nodule formation.     

Interaction of Azospirillum and Rhizobium Strains Leading to Inhibition of Nodulation

Rhizobium-Azospirillum interactions during establishment of Rhizobium-clover symbiosis were studied. When mixed cultures of Azospirillum and Rhizobium trifolii strains were simultaneously inoculated onto clover plants, no nodulation by R. trifolii was observed. R. trifolii ANU1030, which nodulated clover plants without attacking root hairs, i.e., does not cause root hair curling (Hac⁻), did not show inhibition of nodulation when inoculated together with Azospirillum strains. Isolation of bacteria from surface-sterilized roots showed that azospirilla could be isolated both from within root segments and from nodules. Inhibition of nodulation could be mimicked by the addition of auxins to the plant growth medium.     

Characterization of the anomalous infection and nodulation of subterranean clover roots by Rhizobium leguminosarum 1020

Anomalous nodulation of Trifolium subterraneum (subterranean clover) roots by Rhizobium leguminosarum 1020 was examined as a model of modified host-specificity in a Rhizobium-legume symbiosis. Consistent with previous reports, these nodules (i) appeared most often at sites of secondary root emergence, (ii) were ineffective in nitrogen fixation and (iii) were as numerous as nodules formed by an effective Rhizobium trifolii strain. R. leguminosarum 1020, grown on agar plates or in the clover root environment, did not bind the white clover lectin, trifoliin A. This strain did not attach in high numbers, and did not induce shepherd's crooks or infection threads, in subterranean clover root hairs. However, R. leguminosarum 1020 did cause branching, moderate curling and other deformations of root hairs. The bacteria probably entered the clover root through breaks in the epidermis at sites of lateral root emergence. The anomalous nodulation was inhibited by nitrate. Only trace amounts of leghaemoglobin were detected in the nodules by Western blot analysis. The nodules were of the meristematic type and initially contained well-developed infection, bacteroid and senescent zones. Infection threads were readily found in the infection zone of the nodule. However, the bacteroid-containing tissue senesced more rapidly than in the effective symbiosis between subterranean clover and R. trifolii 0403. This anomalous nodulation of subterranean clover by R. leguminosarum 1020 suggests a naturally-occurring alternative route of infection that allows Rhizobium to enlarge its host range.     

Tn5 mutagenesis of Rhizobium trifolii host-specific nodulation genes result in mutants with altered host-range ability

Summary A 14 kb DNA fragment from the Sym plasmid of the Rhizobium trifolii strain ANU843, known to carry common nodulation nod and host specific nodulation hsn genes, was extensively mutagenised with transposon Tn5. A correlation between the site of Tn5 insertion and the induced nodulation defect led to the identification of three specific regions (designated I, II, III) which affected nodulation ability. Twenty-three Tn5 insertions into region I (ca. 3.5 kb) affected normal root hair curling ability and abolished infection thread formation. The resulting mutants were unable to nodulate all tested plant species. Tn5 insertions in regions II and III resulted in mutants which showed an exaggerated root hair curling (Hac⁺⁺) response on clover plants. Ten region II mutants which occurred over a 1.1 kb area showed a greatly reduced nodulation ability on clovers and produced aborted, truncated infection threads. Tn5 insertions into region III (ca. 1.5 kb) altered the outcome of crucial early plant recognition and infection steps by R. trifolii. Seven region III mutants displayed host-range properties which differed from the original parent strain. Region III mutants were able to induce marked root hair distortions, infection threads, and nodules on Pisum sativum including the recalcitrant Afghanistan variety. In addition region III mutants showed a poor nodulation ability on Trifolium repens even though the ability to induce infection threads was retained on this host. The altered host-range properties of region III mutants could only be revealed by mutation and the mutant phenotype was shown to be recessive.     

The allelopathic potential of alfalfa root medicagenic acid glycosides and their fate in soil environments

Summary The allelopathic effect of alfalfa (Medicago media Pers.) and red clover (Trifolium pratense L.) root saponins on winter wheat seedling growth and the fate of these chemicals in soil environments were studied. Seed germination, seedling and test fungus growth were suppressed by water and by alcohol extracts of alfalfa roots, and by crude saponins of alfalfa roots, indicating that medicagenic acid glycosides are the inhibitor. Powdered alfalfa roots inhibited wheat seedling growth when added to sand. At concentrations as low as 0.25% (w/w) the root system was completely destroyed whereas seedling shoots suffered little damage. Red clover roots caused some wheat growth inhibition when incorporated to sand, but their effect was much lower than in the alfalfa root treatment. Soil textures had a significant influence on the inhibitory effect of alfalfa roots. The inhibition of seedling growth was more pronounced on light than on heavy soils. This was attribted to the higher sorption of inhibitors by heavy soils. Incubation of alfalfa roots mixed into loose sand, coarse sand, loamy sand and clay loam for a period of 0–8 days resulted in decreased toxicity to bothT. viride and wheat seedlings. This decrease occurred more quickly in heavier soils than in loose sand, due to the hydrolysis of glycosides by soil microorganisms. Soil microbes were capable of detoxifying medicagenic acid glycosides by partial hydrolysis of sugar chain to aglycone. These findings illustrate the importance of medicagenic acid glycosides as an inhibitor of wheat seedling growth, and of their fate in different soil environments.     

A STUDY OF THE BACTERIA ASSOCIATED WITH THE ROOTS OF SUBTERRANEAN CLOVER AND WIMMERA RYE-GRASS

SUMMARY: A study of the bacteria from the surfaces of roots of subterranean clover ( T. subterraneum L.) and Wimmera rye-grass ( L. rigidum Gaud.) revealed that 21 genera were represented among the isolates from clover and 16 genera among those from rye-grass. Bacteria showing branched forms predominated and accounted for 63% of the 151 clover isolates and 78% of the 167 grass isolates. Most of these were identified as Arthrobacter , but from clover a significant proportion were Nocardia -like types. Members of the genera Mycoplana, Micromonospora, Mycobacterium , and Mycococcus were also identified among the branching forms.
Although the soil had been inoculated with effective rhizobia and the clover plants were effectively nodulated only one of the 318 isolates was capable of nodulating subterranean clover. The majority of the isolates were chromogenic and Gram-negative, produced acid from glucose and ammonia from peptone, were catalase-positive and grew best aerobically. Approximately half the isolates liquefied gelatin and produced hydrogen sulphide from peptone.     

Hair Curling Induced in Heterologous Legumes and Monocots by Flavonoid-Treated Rhizobia

The curling of root hairs and the deformation response wereobserved when white clover was infected with homologous strainsof Rhizobium leguminosarum biovar trifolii 4S and 0403. In thecase of Rhizobium meliloti NZ and Rhizobium leguminosarum biovarviciae 128C53, however, curling was only induced when thesebacteria were pretreated with flavonoids: luteolin in the caseof R. meliloti and naringenin for R.I. viciae. The same resultswere obtained with oat, a monocotyledonous non-leguminous plant.The two flavonoids mentioned are secreted from the host plantsand induce the expression of genes for root hair curling (Hac)on Sym plasmid in homologous rhizobia, therefore, the curlingresponse in both white clover and oat appears to be correlatedwith the activation of the Hac genes. These results suggestthat a factor(s) that activates the Hac genes, such as 7,4'-dihydroxyflavonewhich is known as the factor required by R. I. trifolii, issecreted from the oat roots. (Received June 12, 1989; Accepted November 9, 1989)     

Feedback regulation of nodule formation in alfalfa

When high dosages of wild-type Rhizobium meliloti RCR2011 were inoculated at two different times, 24 h apart, onto either the primary roots of alfalfa (Medicago sativa L.) seedlings or onto lateral roots on opposite sides of a split-root system, the number of nodules generated by the second inoculum was much smaller than the number generated by the first inoculum. These results provide evidence that alfalfa has an active, systemic mechanism for feedback control of nodulation. Non-nodulating mutants and delayed, weakly nodulating mutants did not elicit a discernable suppression of nodulation by subsequently inoculated wild-type cells. An appreciable number of Rhizobium infections thus seem required to elicit the suppressive response. Mutants in nodulation regions II_b and II_a nodulated extensively in the initially susceptible region of the root, but nodule initiation by these mutants was 100–1000 times less efficient, respectively, than the parent. Nodules formed by these mutants emerged 1 d later than normal. The II_b mutants elicited a relatively strong suppression of nodulation in younger parts of the root, but region-II_a mutants elicited only a weak response. These results indicate that elicitation of the regulatory response need not be proportional to nodule formation and imply that genes in region II_a play an important role in elicitation. At high dosages, the region-II mutants induced the development of thick, short roots in a considerably higher percentage of plants than the wild-type bacteria. Nodules generated by wild-type isolates and region-II mutants did not emerge in strict acropetal sequence, probably because some infections developed more slowly than others. Prior exposure of the root to non-nodulating mutants resulted in nodulation by the parent in regions of the root otherwise too mature to be susceptible, indicating that exposure to these mutants may affect the sequence of root development.Abbreviations RT root tip - EH smallest emergent root hair - Tsr thick, short roots This is contribution No. 79-88 of the Ohio Agricultural Research and Development Center     

The Isolation and Partial Characterization of the Lipopolysaccharides from Several Rhizobium trifolii Mutants Affected in Root Hair Infection

The lipopolysaccharides (LPSs) from Rhizobium trifolii ANU843 and several transposon (Tn5) symbiotic mutants derived from ANU843 were isolated and partially characterized. The mutant strains are unable to induce normal root hair curling (Hac- phenotype) or nodulation (Nod-phenotype) in clover plants. The LPSs from the parent and mutants are very similar in composition. Analysis by PAGE shows that the LPSs consist of higher and lower molecular weight forms. The higher molecular weight form of the LPSs exists in several aggregation states when PAGE is done in 0.1% SDS but collapses into a single band when PAGE is done in 0.5% SDS. Mild acid hydrolysis of all the LPSs releases two polysaccharides, PS1 and PS2. Immunoblots of the PAGE gels and enzyme linked immunosorbant assay inhibition assays show that the PS1 fractions contain the immunodominant sites of the LPSs and that these sites are present in the higher molecular weight form of the LPSs. All the PS1 fractions contain methylated sugars, 2-amino-2,6-dideoxyhexose, heptose, glucuronic acid, and 2-keto-3-deoxyoctonic acid (KDO). All the PS2 fractions contain galacturonic acid, mannose, galactose, and KDO. The PS2 fractions have a molecular weight of about 700. The KDO is present at the reducing end of both the PS1 and the PS2 fractions. The PS1 and PS2 fractions from the mutants contain more glucose than these fractions from the parent. The LPS from a deletion mutant contains less acyl groups than the other LPSs. Immunoblots of the LPSs show that the parent and nod A mutant LPSs contain an additional antigenic band which is not observed in the other LPSs.     

Exogenous Ethylene Inhibits Nodulation of Pisum sativum L. cv Sparkle

Exogenous ethylene inhibited nodulation on the primary and lateral roots of pea, Pisum sativum L. cv Sparkle. Ethylene was more inhibitory to nodule formation than to root growth; nodule number was reduced by half with only 0.07 μL/L ethylene applied continually to the roots for 3 weeks. The inhibition was overcome by treating roots with 1 μm Ag⁺, an inhibitor of ethylene action. Exogenous ethylene also inhibited nodulation on sweet clover (Melilotus alba) and on pea mutants that are hypernodulating or have ineffective nodules. Exogenous ethylene did not decrease the number of infections per centimeter of lateral pea root, but nearly all of the infections were blocked when the infection thread was in the basal epidermal cell or in the outer cortical cells.     

The Growth and Carbon Allocation Patterns of White Clover (Trifolium repens L.) Plants of Contrasting Branching Structure

The growth, morphology and carbon allocation patterns of F1progeny white clover (Trifolium repens L.) plants selected foreither low (‘LBF’) or high (‘HBF’) frequencyof stolon branching were compared in two controlled-environmentexperiments. Selections from within both a small-leaved (‘GrasslandsTahora’) and a large-leaved (‘Grasslands Kopu’)clover cultivar were compared, and plants were grown under arelatively lenient defoliation treatment (expt 1) or under threelevels of defoliation seventy (expt 2). Carbon allocation patternswere measured by ¹⁴CO₂ pulse-chase labelling using fully unfoldedleaves on the main (parent) stolon. LBF and HBF displayed consistent differences in the selectedcharacter though, within cultivars, the difference between selectionswas most pronounced for Kopu. The selections developed fundamentallydifferent branching structures resulting from differences inbranching frequency, with total branch weight per plant averaging122 mg for LBF and 399 mg for HBF (mean of both experiments).More C moved from parent stolon leaves to branches in HBF thanin LBF (mean 22.6% vs. 15.1% respectively of the ¹⁴C exportedfrom source leaves). More C also moved to stolon tissue in HBF,but, counterbalancing this and the difference in allocationto branches, less moved to developing leaves and roots on theparent stolon itself compared to LBF. However, the total weightof developing leaves and roots per parent stolon was generallygreater in HBF than in LBF, probably reflecting greater C importby these sinks from the higher number of branches present perplant in the former selection. HBF plants were consistentlylarger at harvest than LBF plants. There were no defoliationtreatment x selection interactions in C allocation patternsin expt 2. The implications of the results for plant performancein grazed pastures are discussed. Branching, carbon translocation, defoliation, growth, morphology, Trifolium repens, white clover     

Variation in Binding and Virulence of Agrobacterium tumefaciens Chromosomal Virulence (chv) Mutant Bacteria on Different Plant Species

Chromosomal virulence (chv) mutants of Agrobacterium tumefaciens have been reported to be deficient in binding to cells of zinnia, tobacco, and bamboo. The mutants are nonpathogenic on stems of Kalanchoë, sunflower, tomato, Jerusalem artichoke, and tobacco, but they cause tumors on tubers of Solanum tuberosum. We used a root cap cell binding assay to test ability of cells from individual plants of 13 different plant species to bind parent or chv mutant bacteria. The same plants were then inoculated to test for disease response. Cells from nine of the plant species were grossly deficient in their abilities to bind mutant bacteria, and the plants inoculated with mutant bacteria failed to form tumors. In contrast, root cap cells as well as root hairs and root surfaces of S. tuberosum, S. okadae, and S. hougasii bound chv mutant bacteria as well as wild type. Nevertheless, S. tuberosum roots inoculated with mutant bacteria did not develop tumors. Although S. okadae plants inoculated with mutant bacteria formed a few tumors, and S. hougasii developed as many tumors in response to chv mutants as in response to the parent strain, the tumors induced by mutant bacteria were smaller.     

Effect of Graft and Sexual Hybridization on the Nodulation of Trifolium ambiguum M.B.

Trifolium ambiguum M.B. is a potentially important clover species,but it has not been used widely as a forage legume because itnodulates sparsely or not at all and the nodules formed areusually ineffective. In some compatible grafts of T. hybridumon T. ambiguum effective nodules developed on the roots of thestock. In addition, effective nodulation was obtained on thesexual hybrid between these two species. The investigation dealswith the effectiveness of nodulation of the graft and sexualhybrids and also of the two host species when inoculated withisolates from the graft hybrid, the sexual hybrid, T. hybridum,and from effectively nodulating T. ambiguum.     

Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection.

During analysis of early events in the infection and nodulation of Vicia hirsuta roots inoculated with normal and mutant strains of Rhizobium leguminosarum and strains containing cloned nodulation (nod) genes, a number of novel observations were made. (i) Alternating zones of curled and straight root hairs were seen on roots of V. hirsuta inoculated with the wild-type strain of R. leguminosarum. This phasing of root hair curling was not seen if plants were grown under continuous light or continuous dark conditions. (ii) Reduced nodulation and delayed nodule initiation was observed with a strain carrying a Tn5 mutation in the nodE gene. In addition the phased root hair curling was absent, and root hair curling was observed along the length of the root. (iii) The nodABC genes cloned on a multicopy plasmid in a wild-type strain inhibited nodulation but induced a continuous root hair curling response. Those few nodules that eventually formed were found to contain bacteria which had lost the plasmid carrying the nodABC genes. (iv) With a strain of Rhizobium cured of its indigenous symbiotic plasmid, but containing the cloned nodABCDEF genes, continuous root hair curling on V. hirsuta was observed. However, no infection threads were observed, and surprisingly, it did appear that initial stages of nodule development occurred. Observations of thin sections of these early developing nodules indicated that early nodule meristematic divisions may have occurred but that no bacteria were found within the nodules and no infection threads were observed either within the nodule bumps or within any of the root hairs. It was concluded that for normal infections to occur, precise regulation of the nod genes is required and that overexpression of the root hair curling genes inhibits the normal infection process.     

A non-nodulating alfalfa mutant displays neither root hair curling nor early cell division in response to Rhizobium meliloti.

The early events in the alfalfa-Rhizobium meliloti symbiosis include deformation of epidermal root hairs and the approximately concurrent stimulation of cell dedifferentiation and cell division in the root inner cortex. These early steps have been studied previously by analysis of R. meliloti mutants. Bacterial strains mutated in nodABC, for example, fail to stimulate either root hair curling or cell division events in the plant host, whereas exopolysaccharide (exo) mutants of R. meliloti stimulate host cell division but the resulting nodules are uninfected. As a further approach to understanding early symbiotic interactions, we have investigated the phenotype of a non-nodulating alfalfa mutant, MnNC-1008 (NN) (referred to as MN-1008). Nodulating and non-nodulating plants were inoculated with wild-type R. meliloti and scored for root hair curling and cell divisions. MN-1008 was found to be defective in both responses. Mutant plants inoculated with Exo- bacteria also showed no cell division response. Therefore, the genetic function mutated in MN-1008 is required for both root hair curling and cell division, as is true for the R. meliloti nodABC genes. These observations support the model that the distinct cellular processes of root hair curling and cell division are triggered by related mechanisms or components, or are causally linked.     

Activities and survival of endophytic bacteria in white clover (Trifolium repens L.)

In this study, the genera, abundance, and activities of endophytic bacteria in field-grown white clover (Trifolium repens) and the fate of introduced antibiotic-tolerant bacteria in white clover tissues were investigated. Pseudomonas, Pantoea, and Corynebacterium were the most frequently isolated endophytic bacteria genera, whereas Xanthomonas, Microbacterium, and Cellulomonas occurred less frequently. The average bacterial populations in stolons and roots were approximately 100,000 colony-forming units (CFU) (g wet mass)-1. Of the 28 strains tested for activity, none were chitinolytic or able to inhibit the root pathogen Codinaea fertilis in vitro. However, Fusarium oxysporum and Cylindrocladium scoparium were inhibited by one and five strains, respectively. Four of seven strains tested depressed clover seedling growth. In pot experiments, colonization and recovery of spontaneous rifampicin-tolerant mutants (Rif+) of bacteria were studied in clover plants for periods up to 20 weeks. The strains used, sourced from white clover (endophytic and rhizoplane) and organic compost, had previously shown growth promotion potential of white clover seedlings by increasing plant mass and decreasing nematode numbers. In one experiment in this present study, five Rif+ strains were individually inoculated onto white clover seedlings, all five were re-isolated from shoots after 6 weeks and four strains were re-isolated after 20 weeks (numbers of Rif+ bacteria ranged from 51 to 200 CFU (g wet mass)-1). No Rif+ bacteria were isolated from root tissue at either time. In the second experiment, conducted with two strains of Rif+ bacteria, the population was highest in the shoots (range>500 CFU of Rif+ bacteria (g shoot fresh mass)-1) in weeks 2 and 3, declining to <200 CFU in week 5. Again, no Rif+ bacteria could be detected in roots. No Rif+ bacteria were recovered after 14 weeks for one of the strains. It appears that the main route of bacterial entry into seedlings was through stomata and that bacteria remained in the aerial parts of plants rather than migrating to the roots.     

Exopolysaccharide mutants of Rhizobium loti are fully effective on a determinate nodulating host but are ineffective on an indeterminate nodulating host.

By Tn5 mutagenesis of Rhizobium loti PN184 (NZP2037 str-1) and selection for nonfluorescence of colonies on Calcofluor agar, eight independently generated expolysaccharide (EPS) mutants (three smooth and five rough) were isolated. The parent strain, PN184, was found to produce an acidic EPS. This EPS was produced. with reduced O acetylation, by the smooth EPS mutants but not by the rough EPS mutants. Lipopolysaccharide was isolated from all mutants and was identical to that of PN184 as defined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All mutants were resistant to lysis by R. loti bacteriophage phi 2037/1. Cosmids that complemented the mutations in the rough EPS mutants were isolated from a pLAFR1 gene library of NZP2037 by complementation of the nonfluorescent phenotype. The genes identified were shown to be unlinked and located on the chromosome. All mutants were fully effective when inoculated onto Lotus pedunculatus, a determinate nodulating host, but were ineffective, inducing the formation of very small nodules or tumorlike growths, when inoculated onto Leucaena leucocephala, an indeterminate nodulating host. These results, obtained in an isogenic Rhizobium background, support suggestions that acidic EPS is required for effective nodulation of indeterminate nodulating legumes but is not required for effective nodulation of determinate nodulating legumes.