Glutamate wastewater as a culture medium for <Emphasis Type="Italic">Azospirillum rugosum</Emphasis> production and its impact on plant growth

Monosodium glutamate wastewater (MSGW) was used as a culture medium for Azospirillum rugosum strain IMMIB AFH-6 inoculant (In) by optimizing the dilution at 2.5% and pH 7.0 ± 0.1. A. rugosum growth studies were carried out in shake flasks and subsequently tested for plant growth promotion by seed germination bioassay and greenhouse pot experiments. The highest colony-forming units (cfu) 8.58 and 8.87 log cfu mL⁻¹, respectively, at 48 h of growth were recorded in MSGW and MSGW with yeast extract 0.04%. Seed bioassay results revealed that A. rugosum grown on MSGW stimulated the root and shoot elongation of maize (Zea mays L.) and pai tsai (Brassica chinensis L.). Pai tsai recorded significantly higher dry matter production when treated with A. rugosum and chemical fertilizers than control. The MSGW is a suitable culture medium for A. rugosum growth, and it is comparable to commercial media when tested for agricultural production.     

Pre-inoculation with arbuscular mycorrhizal fungi suppresses root knot nematode (<Emphasis Type="Italic">Meloidogyne incognita</Emphasis>) on cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

A pot experiment was conducted to evaluate the influence of pre-inoculation of cucumber plants with each of the three arbuscular mycorrhizal (AM) fungi Glomus intraradices, Glomus mosseae, and Glomus versiforme on reproduction of the root knot nematode Meloidogyne incognita. All three AM fungi tested significantly reduced the root galling index, which is the percentage of total roots forming galls. Numbers of galls per root system were significantly reduced only in the G. intraradices + M. incognita treatment. The number of eggs per root system was significantly decreased by AM fungus inoculation, no significant difference among the three AM fungal isolates. AM inoculation substantially decreased the number of females, the number of eggs g⁻¹ root and of the number of eggs per egg mass. The number of egg masses g⁻¹ root was greatly reduced by inoculation with G. mosseae or G. versiforme. By considering plant growth, nutrient uptake, and the suppression of M. incognita together, G. mosseae and G. versiforme were more effective than G. intraradices.     

Effects of cow manure and sewage sludge on the activity and kinetics of <Emphasis Type="SmallCaps">l</Emphasis>-glutaminase in soil

A study was conducted to investigate the effects of cow manure and sewage sludge application on the activity and kinetics of soil l-glutaminase. Soil samples were collected from a farm experiment in which 0, 25, and 100 Mg ha⁻¹ of either cow manure or sewage sludge had been applied annually for 4 consecutive years to a clay loam soil (Typic Haplargid). A chemical fertilizer treatment had also been applied. Results indicated that the effects of chemical fertilizer and the solid waste application on pH in the 18 surface soil (0–15 cm) samples were not significant. The organic C content, however, was affected significantly by the different treatments, being the greatest in soils treated with 100 Mg ha⁻¹ cow manure, and the least in the control treatment. l-Glutaminase activity was generally greater in solid-waste applied soils and was significantly correlated (r = 0.939, P < 0.001) with organic C content of soils. The values of l-glutaminase maximum velocity (Vmax) ranged from 331 to 1,389 mg NH₄ ⁺–N kg⁻¹ 2 h⁻¹. Values of the Michaelis constant (K _m) ranged from 35.1 to 71.7 mM. Organic C content of the soils were significantly correlated with V _max (r = 0.919, P < 0.001) and K _m (r = 0.763, P < 0.001) values. These results demonstrate the considerable influence that solid waste application has on this enzymatic reaction involved in N mineralization in soil.     

Salt tolerance in <Emphasis Type="Italic">Zea mays</Emphasis> (L). following inoculation with <Emphasis Type="Italic">Rhizobium</Emphasis> and <Emphasis Type="Italic">Pseudomonas</Emphasis>

This study aimed to investigate the effect of inoculation with plant growth-promoting Rhizobium and Pseudomonas species on NaCl-affected maize. Two cultivars of maize (cv. Agaiti 2002 and cv. Av 4001) selected on the basis of their yield potential were grown in pots outdoors under natural conditions during July. Microorganisms were applied at seedling stage and salt stress was induced 21 days after sowing and maintained up to 50% flowering after 120 days of stress. The salt treatment caused a detrimental effect on growth and development of plants. Co-inoculation resulted in some positive adaptative responses of maize plants under salinity. The salt tolerance from inoculation was generally mediated by decreases in electrolyte leakage and in osmotic potential, an increase in osmoregulant (proline) production, maintenance of relative water content of leaves, and selective uptake of K ions. Generally, the microbial strain acted synergistically. However, under unstressed conditions, Rhizobium was more effective than Pseudomonas but under salt stress the favorable effect was observed even if some exceptions were also observed. The maize cv. Agaiti 2002 appeared to be more responsive to inoculation and was relatively less tolerant to salt compared to that of cv. Av 4001.     

Benefits of inoculation of the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) crop with efficient and competitive<Emphasis Type="Italic"> Rhizobium tropici</Emphasis> strains

Cropping in low fertility soils, especially those poor in N, contributes greatly to the low common bean (Phaseolus vulgaris L.) yield, and therefore the benefits of biological nitrogen fixation must be intensively explored to increase yields at a low cost. Six field experiments were performed in oxisols of Paraná State, southern Brazil, with a high population of indigenous common bean rhizobia, estimated at a minimum of 10³ cells g^–1 soil. Despite the high population, inoculation allowed an increase in rhizobial population and in nodule occupancy, and further increases were obtained with reinoculation in the following seasons. Thus, considering the treatments inoculated with the most effective strains (H 12, H 20, PRF 81 and CIAT 899), nodule occupancy increased from an average of 28% in the first experiment to 56% after four inoculation procedures. The establishment of the selected strains increased nodulation, N₂ fixation rates (evaluated by total N and N-ureide) and on average for the six experiments the strains H 12 and H 20 showed increases of 437 and 465 kg ha^–1, respectively,in relation to the indigenous rhizobial population. A synergistic effect between low levels of N fertilizer and inoculation with superior strains was also observed, resulting in yield increases in two other experiments. The soil rhizobial population decreased 1 year after the last cropping, but remained high in the plots that had been inoculated. DGGE analysis of soil extracts showed that the massive inoculation apparently did not affect the composition of the bacterial community.     

Effects of long-term mineral fertilization on microbial biomass,microbial activity,and the presence of <Emphasis Type="Italic">r</Emphasis>- and <Emphasis Type="Italic">K</Emphasis>-strategists in soil

Long-term effects of mineral fertilization on microbial biomass C (MBC), basal respiration (R _B), substrate-induced respiration (R _S), β-glucosidase activity, and the r–K-growth strategy of soil microflora were investigated using a field trial on grassland established in 1969. The experimental plots were fertilized at three rates of mineral N (0, 80, and 160 kg ha⁻¹ year⁻¹) with 32 kg P ha⁻¹ year⁻¹ and 100 kg K ha⁻¹ year⁻¹. No fertilizer was applied on the control plots (C). The application of a mineral fertilizer led to lower values of the MBC and R _B, probably as a result of fast mineralization of available substrate after an input of the mineral fertilizer. The application of mineral N decreased the content of C extracted by 0.5 M K₂SO₄ (C _ex). A positive correlation was found between pH and the proportion of active microflora (R _S/MBC). The specific growth rate (μ) of soil heterotrophs was higher in the fertilized than in unfertilized soils, suggesting the stimulation of r-strategists, probably as the result of the presence of available P and rhizodepositions. The cessation of fertilization with 320 kg N ha⁻¹ year⁻¹ (NF) in 1989 also stimulated r-strategists compared to C soil, probably as the result of the higher content of available P in the NF soil than in the C soil.     

Promotion of mycorrhiza formation and growth of willows by the bacterial strain <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. 23L on fly ash

Re-vegetation of fly ash, the principal by-product of coal fired power stations, is hampered by its unfavourable chemical and physical properties for plant growth. In the present study, we evaluated the use of inoculation with a mycorrhiza-associated bacterial strain (Sphingomonas sp. 23L) to promote mycorrhiza formation and plant growth of three willow clones (Salix spp.) on fly ash from an over-burdened dump in a pot experiment. The high pH_H2O (8.7) and low nitrogen content (N_t = 0.1 g kg⁻¹) in combination with hydrophobicity of the particle surfaces caused low plant growth. Inoculation of the willows with Sphingomonas sp. 23L improved the nitrogen uptake by plants, increased plant growth and stimulated formation of ectomycorrhizae with an autochthonous Geopora sp. strain on all three willow clones. The ectomycorrhiza formed by the Geopora sp. was morphologically and anatomically described. The inoculation significantly increased the shoot growth of two Salix viminalis clones and the root growth of a S. viminalis x caprea hybrid clone. We conclude that inoculation with mycorrhiza promoting bacterial strains might be a suitable approach to support mycorrhiza formation with autochtonous site-adapted ectomycorrhizal fungi in fly ash and thereby to improve re-vegetation of fly ash landfills with willows.     

<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.     

Responses of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">niveum</Emphasis> to exogenously added sinapic acid in vitro

To assess the influence of phenolic acids from plant root exudates on soil pathogens, we studied the effect of sinapic acid added to chemically defined media on the growth and virulence factors of Fusarium oxysporum f. sp. niveum. Sinapic acid inhibited the growth and conidial formation and germination of F. oxysporum f. sp. niveum by 6.7–8.8% and 11.2–37.3%, respectively. Mycotoxin production by F. oxysporum f. sp. niveum was stimulated by 81.6–230.7%. Pectinase, proteinase, cellulase, and amylase activities were stimulated at a lower concentration of sinapic acid, while they were inhibited at a higher concentration. It is concluded that sinapic acid inhibited the growth and conidial germination of F. oxysporum f. sp. niveum and decreased the pathogenic enzymes’ activity at higher doses.     

<Emphasis Type="Italic">Erigeron annuus</Emphasis> (L.) Pers., as a green manure for ameliorating soil exposed to acid rain in Southern China

Background, aims, and scope  Increasing soil acidification is a growing concern in southern China. The traditional green manures applied in the fields mostly comprise legumes that tend to accelerate soil acidification. Moreover, acid deposition can act as a source of nitrogen. Hence, we looked for new plant species that would enhance nutrient concentrations when used as green manure and would reduce soil acidity or at least not worsen it. Materials and methods  We studied the use of Erigeron annuus (L.) Pers. for ameliorating acid soil in a pot experiment with simulated acid rain (SAR) treatments (pH 5.8 to 3.0) in an open area in Guangzhou City. The pots were divided into two groups named A and B groups. On day 0, pots of A group were filled with soil and planted with Erigeron annuus seedlings. Pots of B group were only filled with soil as the control. On day 40, seedlings of E. annuus were harvested and buried in the corresponding pots. On day 54, two seeds of Phaseolus vulgaris L. were sown in each pot in both groups. The growth and bean yield of P. vulgaris seedlings were then used to evaluate the effects of E. annuus on acid soil. Plant and/or soil samples were collected on days 0, 40, 54, and 150; corresponding parameters were measured. Results  Results showed that E. annuus could maintain a good growth even on very acid soil. On day 40, the pH decreased significantly (P < 0.0001) in the B group pots without E. annuus compared with the A group. On day 54, after E. annuus was buried as a manure, the soil pH of all A group treatments except the pH 4.0 treatment showed a significant increase compared to day 40 (P < 0.01). At the same time, the application of E. annuus as a manure produced a significant increase of soil K and P (P < 0.001), Ca (P < 0.05), and Mg (P < 0.001) concentrations of all A group SAR treatments compared to their B group counterparts (except control pots for Ca). The soil exchangeable K and available P concentration doubled, and Ca and Mg increased by around 25% in the presence of the E. annuus manure application. Discussion  The higher soil pH in the A group than B group on the day 40 was due to a great absorption of NO₃ ⁻ by the roots of E. annuus. The soil pH increase after E. annuus was applied to the soil of A group was attributed to the release of high amount of K, the mineralization of organic N, and the oxidation of organic acid anions. Nutrient increase in the A group after E. annuus application was mostly the result of the nutrient release during the residue decomposition. The amelioration of the soil was effective as demonstrated by the enhanced growth and bean yield of P. vulgaris seedlings on the manured soil compared to the seedlings grown on a control that was not manured. Conclusions   E. annuus could maintain a good growth in the acid lateritic field soil. Cultivating this plant and applying it to the soil with a rate of 1.6 ton ha⁻¹ doubled the soil K and P concentrations and increased soil exchangeable Ca and Mg concentrations by around 25%. This species would be a good green manure candidate for growing in the acid soils of southern China. Application of E. annuus also has beneficial effects on crop growth through reduced Al toxicity and cation leaching. Recommendations and perspectives  Since E. annuus would improve soil pH and nutrient concentrations with minimum care, it is recommended for treating acid soils with poor yield whenever a low-cost solution is required.     

Transfer of leaf rust and stripe rust resistance from <Emphasis Type="Italic">Aegilops umbellulata</Emphasis> Zhuk. to bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Aegilops umbellulata acc. 3732, an excellent source of resistance to major wheat diseases, was used for transferring leaf rust and stripe rust resistance to cultivated wheat. An amphiploid between Ae. umbellulata acc. 3732 and Triticum durum cv. WH890 was crossed with cv. Chinese Spring Ph ^I to induce homoeologous pairing between Ae. umbellulata and wheat chromosomes. The F₁ was crossed to the susceptible Triticum aestivum cv. ‘WL711’ and leaf rust and stripe rust resistant plants were selected among the backcross progenies. Homozygous lines were selected and screened against six Puccinia triticina and four Puccinia striiformis f. sp. tritici pathotypes at the seedling stage and a mixture of prevalent pathotypes of both rust pathogens at the adult plant stage. Genomic in situ hybridization in some of the selected introgression lines detected two lines with complete Ae. umbellulata chromosomes. Depending on the rust reactions and allelism tests, the introgression lines could be classified into two groups, comprising of lines with seedling leaf rust resistance gene Lr9 and with new seedling leaf rust and stripe rust resistance genes. Inheritance studies detected an additional adult plant leaf rust resistance gene in one of the introgression lines. A minimum of three putatively new genes—two for leaf rust resistance (LrU1 and LrU2) and one for stripe rust resistance (YrU1) have been introgressed into wheat from Ae. umbellulata. Two lines with no apparent linkage drag have been identified. These lines could serve as sources of resistance to leaf rust and stripe rust in breeding programs.     

Mitigation of salt stress in wheat seedlings by a <Emphasis Type="Italic">gfp</Emphasis>-tagged <Emphasis Type="Italic">Azospirillum lipoferum</Emphasis>

Root colonization and mitigation of NaCl stress on wheat seedlings were studied by inoculating seeds with Azospirillum lipoferum JA4ngfp15 tagged with the green fluorescent protein gene (gfp). Colonization of wheat roots under 80 and 160 mM NaCl stress was similar to root colonization with this bacterial species under non-saline conditions, that is, single cells and small aggregates were mainly located in the root hair zone. These salt concentrations had significant inhibitory effects on development of seedlings, but not on growth in culture of gfp-A. lipoferum JA4ngfp15. Reduced plant growth (height and dry weight of leaves and roots) under continuous irrigation with 160 mM NaCl was ameliorated by bacterial inoculation with gfp-A. lipoferum JA4ngfp15. Inoculation of plants subjected to continuous irrigation with 80 mM NaCl or to a single application of either NaCl concentration (80 or 160 mM NaCl) did not mitigate salt stress. This study indicates that, under high NaCl concentration, inoculation with modified A. lipoferum reduced the deleterious effects of NaCl; colonization patterns on roots were unaffected and the genetic marker did not induce undesirable effects on the interaction between the bacterium and the plants.     

Evaluation of 3,4-dimethylpyrazole phosphate as a nitrification inhibitor in a <Emphasis Type="Italic">Citrus</Emphasis>-cultivated soil

 The objectives of this work were to evaluate the inhibitory action on nitrification of 3,4-dimethylpyrazole phosphate (DMPP) added to ammonium sulphate nitrate [(NH₄)₂SO₄ plus NH₄NO₃; ASN] in a Citrus-cultivated soil, and to study its effect on N uptake. In a greenhouse experiment, 2 g N as ASN either with or without 0.015 g DMPP (1% DMPP relative to NH₄ ⁺-N) was applied 6 times at 20-day intervals to plants grown in 14-l pots filled with soil. Addition of DMPP to ASN resulted in higher levels of NH₄ ⁺-N and lower levels of NO₃ ^–-N in the soil during the whole experimental period. The NO₃ ^–-N concentration in drainage water was lower in the ASN plus DMPP (ASN+DMPP)-treated pots. Also, DMPP supplementation resulted in greater uptake of the fertilizer-N by citrus plants. In another experiment, 100 g N as ASN, either with or without 0.75 g DMPP (1% DMPP relative to NH₄ ⁺-N) was applied to 6-year-old citrus plants grown individually outdoors in containers. Concentrations of NH₄ ⁺-N and NO₃ ^–-N at different soil depths and N distribution in the soil profile after consecutive flood irrigations were monitored. In the ASN-amended soil, nitrification was faster, whereas the addition of the inhibitor led to the maintenance of relatively high levels of NH₄ ⁺-N and NO₃ ^–-N in soil for longer than when ASN was added alone. At the end of the experiment (120 days) 68.5% and 53.1% of the applied N was leached below 0.60 m in the ASN and ASN+DMPP treatments, respectively. Also, leaf N levels were higher in plants fertilized with ASN+DMPP. Collectively, these results indicate that the DMPP nitrification inhibitor improved N fertilizer efficiency and reduced NO₃ ^– leaching losses by retaining the applied N in the ammoniacal form. Received: 31 May 1999     

Solubilization of inorganic phosphates and plant growth promotion by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Two of 187 fungal isolates (Aspergillus niger 1B and 6A) displaying superior phosphate (P) solubilization and hydrolytic enzyme secretion were studied using P forms of calcium (Ca-P), iron (Fe-P), and aluminum (Al-P). Phosphate solubilization in a sucrose-basal salt (SB) broth was increased and pH decreased by both isolates. In Ca-P medium, solubilization for 6A was approximately 322 μg P mL⁻¹ and pH decreased by 4.2 units to 2.3 in 72 h. However, when pH value of the SB broth was lowered to 2.5 using HCl, 65.3  ±  0.4 μg mL⁻¹ of P was released from Ca-P, whereas trace amounts of P were released from Fe-P and Al-P. Both isolates displayed enhanced Al-P solubilization using NH₄Cl rather than KNO₃ as the N source; final pH values were not significantly different. With Ca-P, gluconic acid was predominantly produced by 1B and 6A, whereas oxalic acid predominated with Fe-P and Al-P. Addition of gluconic acid (final concentration of 8.5 μmol mL⁻¹) to Ca-P-supplemented SB lowered pH (2.9) and solubilized phosphate (146.0 ± 1.0 μg mL⁻¹). Similarly, addition of oxalic acid (final concentration 6.6 μmol mL⁻¹) to Ca-P- and Fe-P-amended media solubilized P (60.2 ± 0.9 and 21.6 ± 2.1 μg mL⁻¹, respectively), although these quantities were significantly lower than those detected in unamended SB. The presence of unidentified P solubilized compound(s) in the dialyzed (MW>500) supernatant warrants further study. In pot experiments, significant increases in plant (Brassica chinensis Linn.) dry weight and N and P contents were observed with the addition of isolate 6A, when a small amount of organic fertilizer together with either rock phosphate (South African apatite) or Ca-P served as the main P sources.     

Evaluation and utilization of <Emphasis Type="Italic">Aegilops</Emphasis> and wild <Emphasis Type="Italic">Triticum</Emphasis> species for enhancing iron and zinc content in wheat

Grains of 80 accessions of nine species of wild Triticum and Aegilops along with 15 semi-dwarf cultivars of bread and durum wheat grown over 2 years at Indian Institute of Technology, Roorkee, were analyzed for grain iron and zinc content. The bread and durum cultivars had very low content and little variability for both of these micronutrients. The related non-progenitor wild species with S, U and M genomes showed up to 3–4 folds higher iron and zinc content in their grains as compared to bread and durum wheat. For confirmation, two Ae. kotschyi Boiss. accessions were analyzed after ashing and were found to have more than 30% higher grain ash content than the wheat cultivars containing more than 75% higher iron and 60% higher zinc than that of wheat. There were highly significant differences for iron and zinc contents among various cultivars and wild relatives over both the years with very high broad sense heritability. There was a significantly high positive correlation between flag leaf iron and grain iron (r = 0.82) and flag leaf zinc and grain zinc (r = 0.92) content of the selected donors suggesting that the leaf analysis could be used for early selection for high iron and zinc content. ‘Chinese Spring’ (Ph ^I ) was used for inducing homoeologous chromosome pairing between Aegilops and wheat genomes and transferring these useful traits from the wild species to the elite wheat cultivars. A majority of the interspecific hybrids had higher leaf iron and zinc content than their wheat parents and equivalent or higher content than their Aegilops parents suggesting that the parental Aegilops donors possess a more efficient system for uptake and translocation of the micronutrients which could ultimately be utilized for wheat grain biofortification. Partially fertile to sterile BC₁ derivatives with variable chromosomes of Aegilops species had also higher leaf iron and zinc content confirming the possibility of transfer of required variability. Some of the fertile BC₁F₃ and BC₂F₂ derivatives had as high grain ash and grain ash iron and zinc content as that of the donor Aegilops parent. Further work on backcrossing, selfing, selection of fertile derivatives, leaf and grain analyses for iron and zinc for developing biofortified bread and durum wheat cultivars is in progress. Nidhi Rawat, Vijay K. Tiwari, and Neelam Singh have contributed equally to the work.     

Genetic Characterization of Brazilian Annual <Emphasis Type="Italic">Arachis</Emphasis> Species from Sections <Emphasis Type="Italic">Arachis</Emphasis> and <Emphasis Type="Italic">Heteranthae</Emphasis> using RAPD Markers

The genus Arachis is divided into nine taxonomic sections. Section Arachis is composed of annual and perennial species, while section Heteranthae has only annual species. The objective of this study was to investigate the genetic relationships among 15 Brazilian annual accessions from Arachis and Heteranthae using RAPD markers. Twenty-seven primers were tested, of which nine produced unique fingerprintings for all the accessions studied. A total of 88 polymorphic fragments were scored and the number of fragments per primer varied from 6 to 17 with a mean of 9.8. Two specific markers were identified for species with 2n = 18 chromosomes. The phenogram derived from the RAPD data corroborated the morphological classification. The bootstrap analysis divided the genotypes into two significant clusters. The first cluster contained all the section Arachis species, and the accessions within it were grouped based upon the presence or absence of the ‘A’ pair and the number of chromosomes. The second cluster grouped all accessions belonging to section Heteranthae.     

A new mycorrhizal helper bacterium, <Emphasis Type="Italic">Ralstonia</Emphasis> species,in the ectomycorrhizal symbiosis between <Emphasis Type="Italic">Pinus thunbergii</Emphasis> and <Emphasis Type="Italic">Suillus granulatus</Emphasis>

In a Robinia-pseudoacacia-dominated coastal forest in Tottori prefecture Japan, the growth and survival of Pinus thunbergii seedlings and the natural regeneration of P. thunbergii was disturbed by R. pseudoacacia. In order to improve the growth of P. thunbergii seedling in the Tottori sand dune, we tried to find a mycorrhiza helper bacteria (MHB) from P. thunbergii mycorrhizosphere in a Tottori sand dune. Two MHB, Ralstonia sp. and Bacillus subtilis, were selected from the nine bacterial species isolated from the mycorrhizosphere of P. thunbergii. The bacterial effect on the ectomycorrhizal fungus Suillus granulatus was investigated by confrontation assay and a microcosm experiment. The confrontation assay showed that Ralstonia sp. promoted the hyphal growth of S. granulatus. Moreover, the S. granulatus–P. thunbergii symbiosis was significantly stimulated by Ralstonia sp. and B. subtilis. Ralstonia sp. and B. subtilis were regarded as MHB associated with P. thunbergii. This is the first report of Ralstonia sp. as an MHB.     

Characterization of fertile amphidiploid between <Emphasis Type="Italic">Raphanus sativus</Emphasis> and <Emphasis Type="Italic">Brassica alboglabra</Emphasis> and the crossability with <Emphasis Type="Italic">Brassica</Emphasis> species

A fertile amphidiploid × Brassicoraphanus (RRCC, 2n = 36) between Raphanus sativus cv. HQ-04 (2n = 18, RR) and Brassica alboglabra Bailey (2n = 18, CC) was synthesized and successive selections for seed fertility were made from F₄ to F₁₀. F₁₀ plants exhibited good fertility with 14.9 seeds per siliqua and 32.3 g seeds per plant. Cytological observation revealed that frequent secondary pairing occurred among 3 chromosome pairs in pollen mother cells of plants (F₄) with lower fertility, but not of plants with high fertility (F₁₀). GISH analysis indicated that these F₁₀ plants included the expected 18 chromosomes from R. sativus and B. alboglabra, respectively, but they lost approximately 27.6% R. sativus and 35.6% B. alboglabra AFLP (amplified fragment length polymorphism) bands. The crossability of the Raphanobrassica with R. sativus and 5 Brassica species (13 cultivars) were investigated. Seeds or F₁ seedlings were easy to be produced from crosses × Brassicoraphanus × R. sativus, and B. napus, B. juncea and B. carinata × Brassicoraphanus. Fewer seeds or seedlings were obtained from crosses × Brassicoraphanus × B. napus, B. juncea and B. carinata. However, few seeds were harvested in the reciprocals of × Brassicoraphanus with B. rapa and B. oleracea. The possible cause of fertility improvements and the potential of the present × Brassicoraphanus for breeding were discussed.     

Leaf litter ingestion and assimilation by two endemic pill millipedes (<Emphasis Type="Italic">Arthrosphaera</Emphasis>)

This study aims to determine leaf litter preference, consumption rate, growth rate, food conversion efficiency, and quality of fecal pellets of two endemic pill millipedes (Arthrosphaera dalyi and Arthrosphaera davisoni) of the Western Ghats of India by laboratory microcosm experiments. Among seven combinations of three plantation leaf litters offered in 4-day trial, top three preferred combinations were selected for 4-week trial. In 4-week trial, preference of mixed litter diet was higher than single litter diet, which resulted in enhanced growth as well as food conversion efficiency of millipedes. Among Hopea , Pongamia , and Areca litters, A. dalyi preferred Hopea + Pongamia, and its consumption was significantly correlated with contents of organic carbon (P < 0.05; r = –0.97) and nitrogen (P < 0.01; r = 0.99), while growth rate with phosphorus content (P < 0.05; r = 0.97) and food conversion efficiency with contents of organic carbon (P < 0.05; r = 0.98) and calcium (P < 0.01; r = –0.99). Among Areca , Elettaria , and Coffea litters, Areca + Elettaria  + Coffea was most preferred by A. davisoni, which was significantly correlated with organic carbon content (P < 0.05; r = 0.98) and food conversion efficiency with calcium content (P < 0.0001; r = 0.99). The food conversion efficiency, however, was the highest in millipedes fed with Areca  +  Elettaria. The present study demonstrated increased nitrogen and phosphorus contents and decreased phenolic content and C/N ratio in fecal pellets of pill millipedes fed with plantation litter, and thus, these millipedes play an important role in leaf litter mineralization and soil enrichment in plantations Western Ghats.     

Karyotypic studies in wild germplasm of <Emphasis Type="Italic">Arachis</Emphasis> (Leguminosae)

The karyomorphology for eight diploid species of Arachis belonging to three sections has been described for the first time, Sect. Extranervosae: A. macedoi (2n = 20m) and A. retusa (2n = 14m + 6sm); Sect. Heteranthae: A. sylvestris (2n = 16m + 4sm); Sect. Procumbentes: A. chiquitana (2n = 18m + 2sm); Sect. Arachis: A. cruziana (2n = 18m + 2sm), A. herzogii (2n = 18m + 2sm), A. simpsonii (2n = 20m) and A. williamsii (2n = 20m). A pair of satellited chromosomes was observed in all species. A chromosomes were found in A. chiquitana, A. herzogii and A. simpsonii. Karyotypic differences between sections were observed, but not enough to establish a characteristic karyotype pattern for each section. However, the species may be differentiated by the presence of A chromosomes, the type and position of satellites, and the karyotype formulae. These results are discussed with regard to karyotype evolution in Arachis to contribute to understanding the role of chromosome changes in the evolution of the genus.