Enhancement of soybean nodulation by Bacillus cereus UW85 in the field and in a growth chamber.

Seed treatments with Bacillus cereus UW85 increased nodulation of soybeans in three field seasons and in three different sterilized soils in the growth chamber. In the field, 28 and 35 days after planting, UW85-treated plants had 31 to 133% more nodules than untreated plants. From 49 days after planting until seed harvest, there were no significant differences between nodulation of UW85-treated plants and untreated control plants. In the growth chamber, in sterilized soil-vermiculite mixtures, at 28 days after planting, UW85 seed treatments enhanced nodulation by 34 to 61%, indicating that the increase in nodulation was not dependent on the soil flora.     

Variable stability of antibiotic-resistance markers in Bacillus cereus UW85 in the soybean rhizosphere in the field

We compared the stability of antibiotic-resistance markers in strains derived from Bacillus cereus UW85 in culture media and in the soybean rhizosphere in a growth chamber and in the field. We studied two independent, spontaneous mutants resistant to neomycin, three independent, spontaneous mutants resistant to streptomycin, and strains carrying plasmid pBC16, which encodes tetracycline resistance. Antibiotic-resistance markers were maintained in populations of all UW85 derivatives in culture and in the rhizosphere of soybeans grown in soil in a growth chamber. In two field experiments, antibiotic resistance was substantially lost in rhizosphere populations of B. cereus as early as 14 or as late as 116 days after planting. To distinguish between death of the inoculated strain and loss of its marker, we tested populations of B. cereus for other phenotypes (orange pigmentation, plasmid-borne resistance to tetracycline, and biocontrol activity) that are typical of UW85-derivatives used as inoculum, but atypical of the indigenous populations of B. cereus , and these phenotypes were maintained in populations from which the marker was lost. In general, neomycin-resistance markers were maintained at a higher frequency than streptomycin-resistance markers, and maintenance of antibiotic-resistance markers varied with position on the root and with the year of the experiment. In a semi-defined medium, the UW85 derivatives grew at the same rate as the wild type at 28°C, but most grew more slowly than the wild type at 16°C, demonstrating that antibiotic resistance can affect fitness under some conditions. The results suggest that the stability of antibiotic-resistance markers should be assessed in the ecosystems in which they will be studied.     

The negative effects of soil microorganisms on plant growth only extend to the first weeks

土壤微生物对植物生长的负面影响只延续到最初几周 土壤微生物群落可以显著影响植物的生长表现。在本文中,我们提出一个问题：土壤微生物群落对植物生长的影响可以持续多久。我们监测了早期、中期和晚期3个阶段的植物生长速率,在无菌土壤或活土壤中对一种菊科植物疆千里光(Jacobaea vulgaris)进行了两次分别为49天和63天的生长实验。在第3个实验中,我们用4种不同的时间处理方法研究了种植前土壤接种时间对该植物相对生长速率的影响。研究结果表明,3个实验中,在无菌土壤和活土壤中生长的植物的生物量差异都增加了。此外,在前2–3周,灭菌土壤中植物的相对生长速率仅显著高于活土壤中植物的相对生长速率。在第3个实验中,植物生物量随着接种和种植之间时间的增加而减少。总体而言,这些结果表明,疆千里光在无菌土壤中的生长优于在活土壤中。土壤接种对植物生物量的负面影响似乎可以延伸到整个生长期,但源于最初几周发生的对相对生长速率的负面影响。     

DIFFERENTIAL GERMINATION OF RAY AND DISC ACHENES IN HEMIZONIA INCRESCENS (ASTERACEAE)

The differential germination responses of ray and disc achenes of Hemizonia increscens (Asteraceae) were compared in field and laboratory investigations in order to gain insight into the ecological and evolutionary significance of heterocarpy. In the field, 200 ray and 200 disc achenes were placed in native, sterilized soil in a series of cleared, randomized, replicated plots. In a nearby plot a similar number of achenes were placed in plastic petri dishes in which high moisture conditions were maintained. Disc achene germination occurred under relatively minimal moisture conditions (<1 cm rainfall for 19 days) and relatively mild temperature regimes (21–7 C). Disc achene germination began three days after planting in the field plot and four days after they were put into the field petri dishes. In contrast, the onset of ray achene germination occurred 21 days after planting in the field plot and 19 days after planting in the field petri dishes. Averages of 2.05 and 2.71 disc achenes/day germinated in the field plot and field petri dishes, respectively. These contrasted with averages of 0.57 and 0.50 ray achenes/day germinated in the field plot and in the field petri dishes, respectively. A total of 67.5% and 69% disc achenes germinated in the field plot and the field petri dishes, but only 18% and 16.5% ray achenes germinated in the field plot and field petri dishes, respectively. Three separate treatments, using 100 ray and 100 disc achenes in each, were performed in laboratory growth chambers: 1) nicking the fruit coat, 2) excising the embryo, and 3) leaving the fruit coat intact. Onset of germination for all disc achene treatments occurred after three days. No significant differences were found among the three disc achene treatments in timing, rate, or germination percentage. All three disc treatments in the laboratory closely paralleled those for disc achenes in the field plots in time and germination percentage, but rates of germination were not as high. Germination of the nicked and excised ray achenes treatments began after four days, while germination of the untreated ray achenes began after 27 days. Untreated ray achenes in the laboratory paralleled the ray achenes in both field experiments in rate and germination percentage, but were delayed in time of germination. The nicked and excised ray achene treatments, however, were similar to the disc achene treatments in time of germination, and were not significantly different from disc achenes in rate of germination. These data suggest 1) that ray and disc achenes are markedly different in germination under identical conditions in field and laboratory experiments, and 2) this difference in germination response may be due to the thicker pericarp of the ray achenes.     

Nodulation and symbiotic nitrogen fixation of cowpea (Vigna unguiculata (L.) Walp)

Summary Rhizobium strains CIAT 301, CIAT 79 and SLM 602 were tested and found effective in the nodulation and nitrogen fixation of cowpea cv. MI-35 (Vigna unguiculata (L.) Walp) plants in growth chamber experiments. Fresh weight of nodules increased with plant age initially and stabilized in 20–30 days from planting, followed by a secondary flush of nodule growth after 30 days. Apparent nitrogen fixation per gram nodule fresh weight reached a maximum in 20–30 days after planting and then decreased, even though a flush of new nodules was produced.     

Response of introduced Bradyrhizobium strains infectinga promiscuous soybean cultivar

Two field experiments were established to assess the competitiveness of foreign bradyrhizobia in infecting the promiscuous soybean cultivar TGX 536-02D. Seeds were inoculated with antibiotic mutants of the bradyrhizobia strains before planting after land preparation. Soybean plants were harvested at pre-determined days after planting for estimating nodule number, nodule dry weight, nodule occupancy, shoot dry weight and seed yield. Results show that nodule number and dry weight significantly increased and showed great variability at 84 days after planting (DAP), probably due to differences in the ability of inoculant bradyrhizobia to form nodules with the soybean cultivar TGX 536-02D. Increased shoot dry weight, %N, total N and seed yield were a result of increased nodulation by the effective and competitive inoculant Bradyrhizobium strains. Strain USDA 110 occupied the highest percentage of nodule sites because it was more competitive than the other Bradyrhizobium strains. These results show that there was high potential for increasing growth and seed yield of the promiscuous soybean cultivar TGX 536-02D by inoculation with foreign Bradyrhizobium strains.     

Influence of tomato genotype on growth of inoculated and indigenous bacteria in the spermosphere

We previously demonstrated a genetic basis in tomato for support of the growth of a biological control agent, Bacillus cereus UW85, in the spermosphere after seed inoculation (K. P. Smith, J. Handelsman, and R. M. Goodman, Proc. Natl. Acad. Sci. USA 96:4786-4790, 1999). Here we report results of studies examining the host effect on the support of growth of Bacillus and Pseudomonas strains, both inoculated on seeds and recruited from soil, using selected inbred tomato lines from the recombinant inbred line (RIL) population used in our previous study. Two tomato lines, one previously found to support high and the other low growth of B. cereus UW85 in the spermosphere, had similar effects on growth of each of a diverse, worldwide collection of 24 B. cereus strains that were inoculated on seeds and planted in sterilized vermiculite. In contrast, among RILs that differed for support of B. cereus UW85 growth in the spermosphere, we found no difference for support of growth of the biocontrol strains Pseudomonas fluorescens 2-79 or Pseudomonas aureofaciens AB254. Thus, while the host effect on growth extended to all strains of B. cereus examined, it was not exerted on other bacterial species tested. When seeds were inoculated with a marked mutant of B. cereus UW85 and planted in soil, RIL-dependent high and low support of bacterial growth was observed that was similar to results from experiments conducted in sterilized vermiculite. When uninoculated seeds from two of these RILs were planted in soil, changes in population levels of indigenous Bacillus and fluorescent Pseudomonas bacteria differed, as measured over time by culturing and direct microscopy, from growth patterns observed in the inoculation experiments. Neither RIL supported detectable levels of growth of indigenous Bacillus soil bacteria, while the line that supported growth of inoculated B. cereus UW85 supported higher growth of indigenous fluorescent pseudomonads and total bacteria. The vermiculite system used in these experiments was predictive for growth of B. cereus UW85 inoculated on seeds and grown in soil, but the patterns of growth of inoculated strains-both Bacillus and Pseudomonas spp.-did not reflect host genotype effects on indigenous microflora recruited from soil to the spermosphere.     

Application of gibberellic acid to the surface of soybean seed (t Glycine max (L.) Merr.) and symbiotic nodulation,plant development,final grain and protein yield under short season conditions

In short-season soybean production areas, low soil temperature is the major factor limiting soybean establishment, nodulation and nitrogen fixation. Gibberellic acid (GA) pretreatment of crop seeds can overcome low soil temperature inhibition of seed germination and seedling development. However, previous studies have found that the application of GAs decreased legume nodulation and nitrogen fixation under optimal growth conditions. A field experiment was conducted under short season conditions in eastern Canada to determine whether the application of GA₃ to soybean seed could accelerate germination, and increase plant nodulation and nitrogen fixation. The results indicated that GA₃ application accelerated seedling emergence but decreased plant nodulation and nitrogen accumulation at early plant growth stages. However, these initial negative effects were overcome as the plants developed. Gibberellic acid applied to soybean seed at the time of planting did not influence final grain and protein yield.     

Action of 2,4-DB and dalapon on the symbiotic properties ofLotus corniculatus (birdsfoot trefoil)

Summary Field trials carried out in 1965 and 1966 showed that 2,4-DB, alone or in combination with dalapon, reduced nodulation and tended to decrease the efficiency of nitrogen fixation in birdsfoot trefoil. Dalapon appeared to enhance the inhibitory action of 2,4-DB on nodulation. No obvious cytological differences could be detected in the nodules or in the isolated bacteroids of field-treated and untreated plants. Under growth chamber conditions, 2,4-DB drastically reduced trefoil growth and nodulation particularly in treatments where the herbicide came directly in contact with the plants. It appears that the reduction in nodulation and nitrogen fixation is a result of plant damage and abnormal root growth caused by 2,4-DB application.Autoradiographs indicated that the translocation of the herbicide was rapid, with detectable concentrations observed in young leaves, leafveins, roots, and nodules 12 hours after leaf-feeding of 2,4-DB-1-C¹⁴. The radio-activity appeared to accumulate with time (up to 5 days) in the growing root tips and nodules. Fractionation of excised nodules from trefoil plants demonstrated the presence of radioactivity in the cell debris, bacteroids, 29,000g pellet, plant ribosomes, and the soluble portion. The greatest accumulation of radioactivity occurred in the soluble fraction.The degradation of 2,4-DB and 2,4-D in trefoil was demonstrated by the evolution of C¹⁴O₂ from non-nodulated and aseptically growing plants leaf-fed with 2,4-DB-1-C¹⁴ or 2,4-D-1-C¹⁴.4-(2,4-dichlorophenoxy) butyric acid.2,2 dichloropropionic acid.     

Compatibility of systemic acquired resistance and microbial biocontrol for suppression of plant disease in a laboratory assay

Systemic acquired resistance (SAR) and microbial biocontrol each hold promise as alternatives to pesticides for control of plant diseases. SAR and Bacillus cereus UW85, a microbial biocontrol agent, separately suppress seedling damping-off diseases caused by oomycete pathogens. The purposes of this study were to investigate how expression of SAR affected the efficacy of biocontrol by UW85 and if UW85 treatment of plants induced SAR. We devised a laboratory assay in which seedling damping-off disease, induction of SAR, and growth of UW85 could be quantified. Seedlings of Nicotiana tabacum Xanthi nc were germinated on moist filter paper and transferred after 7 days to water agar plates (40 seedlings per plate). Zoospores of oomycete pathogens (Pythium torulosum, Pythium aphanidermatum, or Phytophthora parasitica) were applied at concentrations that caused 80% seedling mortality within 10 days. Seedling mortality was dependent on zoospore inoculum concentration. The level of disease suppression caused by treatment with UW85 depended on the UW85 dose applied. SAR was induced with 0.5-mM salicylic acid or 0.1-mM 2,6-dichloroisonicotinic acid. Expression of an SAR-related gene was confirmed by northern analysis with a probe prepared from a tobacco PR-1a cDNA. Induction of SAR suppressed disease caused by each of the oomycete pathogens, but did not alter the growth of UW85 on roots. Treatment of seedlings with UW85 did not induce the expression of PR-1a. The combination of induction of SAR and treatment with UW85 resulted in additive suppression of disease as measured by seedling survival.     

Influence of Tomato Genotype on Growth of Inoculated and Indigenous Bacteria in the Spermosphere

We previously demonstrated a genetic basis in tomato for support of the growth of a biological control agent, Bacillus cereus UW85, in the spermosphere after seed inoculation (K. P. Smith, J. Handelsman, and R. M. Goodman, Proc. Natl. Acad. Sci. USA 96:4786–4790, 1999). Here we report results of studies examining the host effect on the support of growth of Bacillus and Pseudomonas strains, both inoculated on seeds and recruited from soil, using selected inbred tomato lines from the recombinant inbred line (RIL) population used in our previous study. Two tomato lines, one previously found to support high and the other low growth of B. cereus UW85 in the spermosphere, had similar effects on growth of each of a diverse, worldwide collection of 24 B. cereus strains that were inoculated on seeds and planted in sterilized vermiculite. In contrast, among RILs that differed for support of B. cereus UW85 growth in the spermosphere, we found no difference for support of growth of the biocontrol strains Pseudomonas fluorescens 2-79 or Pseudomonas aureofaciens AB254. Thus, while the host effect on growth extended to all strains of B. cereus examined, it was not exerted on other bacterial species tested. When seeds were inoculated with a marked mutant of B. cereus UW85 and planted in soil, RIL-dependent high and low support of bacterial growth was observed that was similar to results from experiments conducted in sterilized vermiculite. When uninoculated seeds from two of these RILs were planted in soil, changes in population levels of indigenous Bacillus and fluorescent Pseudomonas bacteria differed, as measured over time by culturing and direct microscopy, from growth patterns observed in the inoculation experiments. Neither RIL supported detectable levels of growth of indigenous Bacillus soil bacteria, while the line that supported growth of inoculated B. cereus UW85 supported higher growth of indigenous fluorescent pseudomonads and total bacteria. The vermiculite system used in these experiments was predictive for growth of B. cereus UW85 inoculated on seeds and grown in soil, but the patterns of growth of inoculated strains—both Bacillus and Pseudomonas spp.—did not reflect host genotype effects on indigenous microflora recruited from soil to the spermosphere.     

Influence of Meloidogyne incognita on the Water Relations of Cotton Grown in Microplots

The effects of Meloidogyne incognita on the growth and water relations of cotton were evaluated in a 2-year field study. Microplots containing methyl bromide-fumigated fine sandy loam soil were infested with the nematode and planted to cotton (Gossypium hirsutum L.). Treatments included addition of nematodes alone, addition of nematodes plus the insecticide-nematicide aldicarb (1.7 kg/ha), and an untreated control. Meloidogyne incognita population densities reached high levels in both treatments where nematodes were included. Root galling, plant height at harvest, and seed cotton yield were decreased by nematode infection. In older plants (89 days after planting [DAP]), leaf transpiration rates and stomatal conductance were reduced, and leaf temperature was increased by nematode infection. Nematode infection did not affect (P = 0.05) leaf water potential in either young or older plants but lowered the osmotic potential. The maximum rate and cumulative amount of water flowing through intact plants during a 24-hour period were lower, on both a whole-plant and per-unit-leaf-area basis, in infected plants than in control plants. Application of aldicarb moderated some of the nematode effects but did not eliminate them.     

Effect of Canavanine from Alfalfa Seeds on the Population Biology of Bacillus cereus

Bacillus cereus UW85 suppresses diseases of alfalfa seedlings, although alfalfa seed exudate inhibits the growth of UW85 in culture (J. L. Milner, S. J. Raffel, B. J. Lethbridge, and J. Handelsman, Appl. Microbiol. Biotechnol. 43:685–691, 1995). In this study, we determined the chemical basis for and biological role of the inhibitory activity. All of the alfalfa germ plasm tested included seeds that released inhibitory material. We purified the inhibitory material from one alfalfa cultivar and identified it as canavanine, which was present in the cultivar Iroquois seed exudate at a concentration of 2 mg/g of seeds. Multiple lines of evidence suggested that canavanine activity accounted for all of the inhibitory activity. Both canavanine and seed exudate inhibited the growth of UW85 on minimal medium; growth inhibition by either canavanine or seed exudate was prevented by arginine, histidine, or lysine; and canavanine and crude seed exudate had the same spectrum of activity against B. cereus, Bacillus thuringiensis, and Vibrio cholerae. The B. cereus UW85 populations surrounding canavanine-exuding seeds were up to 100-fold smaller than the populations surrounding non-canavanine-exuding seeds, but canavanine did not affect the growth of UW85 on seed surfaces. The spermosphere populations of canavanine-resistant mutants of UW85 were larger than the spermosphere populations of UW85, but the mutants and UW85 were similar in spermoplane colonization. These results indicate that canavanine exuded from alfalfa seeds affects the population biology of B. cereus.     

Plant Growth-Promoting Bacteria Facilitate the Growth of Barley and Oats in Salt-Impacted Soil: Implications for Phytoremediation of Saline Soils

Plant growth-promoting bacteria (PGPB) strains that contain the enzyme 1-amino- cyclopropane-1-carboxylate (ACC) deaminase can lower stress ethylene levels and improve plant growth. In this study, ACC deaminase-producing bacteria were isolated from a salt-impacted (～50 dS/m) farm field, and their ability to promote plant growth of barley and oats in saline soil was investigated in pouch assays (1% NaCl), greenhouse trials (9.4 dS/m), and field trials (6–24 dS/m). A mix of previously isolated PGPB strains UW3 (Pseudomonas sp.) and UW4 (P. sp.) was also tested for comparison. Rhizobacterial isolate CMH3 (P. corrugata) and UW3+UW4 partially alleviated plant salt stress in growth pouch assays. In greenhouse trials, CMH3 enhanced root biomass of barley and oats by 200% and 50%, respectively. UW3+UW4, CMH3 and isolate CMH2 also enhanced barley and oat shoot growth by 100%–150%. In field tests, shoot biomass of oats tripled when treated with UW3+UW4 and doubled with CHM3 compared with that of untreated plants. PGPB treatment did not affect salt uptake on a per mass basis; higher plant biomass led to greater salt uptake, resulting in decreased soil salinity. This study demonstrates a method for improving plant growth in marginal saline soils. Associated implications for salt remediation are discussed.     

Quantitative comparison of the laboratory and field competitiveness of Rhizobium leguminosarum biovar phaseoli.

Rhizobium leguminosarum bv. phaseoli KIM5s outcompeted strain CE3 in bean (Phaseolus vulgaris L.) root nodulation when plants were grown at any of three field sites, each with a different soil type and indigenous population, or in the laboratory in a sterilized sand, a sterilized peat-vermiculite mixture, or a nonsterile field soil. A mathematical model describing nodulation competitiveness was empirically derived to evaluate the relative competitiveness of the two strains under these conditions. This model relates the proportional representation of the two strains in the inoculum to the proportional representation of nodules occupied by each strain or both strains and provides a measure of competitiveness, which is referred to as the competitiveness index. Statistical comparisons of competitiveness indices showed that the relative competitiveness of KIM5s and CE3 remained constant when the two strains were applied in a constant ratio over a range of inoculum concentrations, from 10(3) to 10(7) cells per seed, and when they were applied in various ratios to six P. vulgaris cultivars. Furthermore, the relative competitiveness of KIM5s and CE3 in the laboratory did not differ significantly from their relative competitiveness at the three field sites studied. Thus, a study of the basis for nodulation competitiveness of KIM5s and CE3 in the laboratory has the potential to provide an understanding of competitiveness both in the laboratory and in the field.     

Quantitative comparison of the laboratory and field competitiveness of Rhizobium leguminosarum biovar phaseoli

Rhizobium leguminosarum bv. phaseoli KIM5s outcompeted strain CE3 in bean (Phaseolus vulgaris L.) root nodulation when plants were grown at any of three field sites, each with a different soil type and indigenous population, or in the laboratory in a sterilized sand, a sterilized peat-vermiculite mixture, or a nonsterile field soil. A mathematical model describing nodulation competitiveness was empirically derived to evaluate the relative competitiveness of the two strains under these conditions. This model relates the proportional representation of the two strains in the inoculum to the proportional representation of nodules occupied by each strain or both strains and provides a measure of competitiveness, which is referred to as the competitiveness index. Statistical comparisons of competitiveness indices showed that the relative competitiveness of KIM5s and CE3 remained constant when the two strains were applied in a constant ratio over a range of inoculum concentrations, from 10(3) to 10(7) cells per seed, and when they were applied in various ratios to six P. vulgaris cultivars. Furthermore, the relative competitiveness of KIM5s and CE3 in the laboratory did not differ significantly from their relative competitiveness at the three field sites studied. Thus, a study of the basis for nodulation competitiveness of KIM5s and CE3 in the laboratory has the potential to provide an understanding of competitiveness both in the laboratory and in the field.     

Effect ofLupinus seed diffusates onBradyrhizobium sp. growth and nodulation of lupine

Seeds of three species of lupine (Lupinus termis, L. triticale andL. albus) were tested to determine if the seed contains diffusable substances toxic to bradyrhizobia.L. albus seeds were less toxic to bradyrhizobia, followed byL. triticale. Six strains ofBradyrhizobium were evaluated for their resistance to the toxic substances in lupine seeds. Zones of growth inhibition were determined on yeast-mannitol-agar medium surrounding surface-sterilized seed. The effect of surface sterilization of seeds by different chemical treatments on seed toxicity was assessed. Seeds soaked in water for 1 h before placing on agar surface significantly decreased the inhibition zone. Also, the effect of soaking seeds in water for 4 h before planting and inoculation on nodulation, nitrogen fixation and plant growth were investigated. Addition of seed diffusate to soaked seeds significantly decreased nodulation and plant growth. Autoclaving the seed diffusate had no effect on the toxicity of the seed diffusate. Addition of the absorbent polyvinylpolypyrrolidone (PVPP) to seed diffusates significantly decreased the inhibitory effect of seed diffusate on nodulation and plant growth. Seed diffusate substances were water-soluble, heat-stable and partially bound to PVPP.     

Genetic allelism and linkage tests of a soybean gene,Rfg1, controlling nodulation with Rhizobium fredii strain USDA 205

A soybean gene, Rfg1, controlling nodulation with strain USDA 205, the type strain for the fast-growing species Rhizobium fredii, was tested for allelism with the Rj4 gene. The Rj4 gene conditions ineffective nodulation primarily with certain strains of the slow-growing soybean microsymbiont, Bradyrhizobium elkanii. The F2 seeds of the cross of the cultivars Peking, carrying the alleles rfg1, Rj4, i (controlling inhibition of seed coat color) and W1 (controlling flower color), and Kent, carrying the alleles Rfg1, rj4, i-i and w1, were evaluated for nodulation response with strain USDA 205 by planting surface disinfested seeds in sterilized vermiculite in growth trays and inoculating with a stationary phase broth culture of strain USDA 205 at planting. Plants were classified for nodulation response visually after four weeks growth and transplanted to the field for F3 seed production. Flower color, purple (W1) vs white (w1), was determined in the field. The allele present at the i locus was determined by classification of F3 seed coat color. The F3 seeds were planted in growth trays and inoculated with strain USDA 61 of Bradyrhizobium elkanii to determine the genotype for the Rj4 locus. The Rfg1 and Rj4 genes were determined to be located at separate loci. Chi-square analysis for linkage indicated that Rfg1 segregated independently of the Rj4, I and W1 loci.     

Inoculation of pea (Pisum sativum L.) by Rhizobium leguminosarum bv. viceae preincubated with naringenin and hesperetin or application of naringenin and hesperetin directly into soil increased pea nodulation under short season conditions

In short season areas, low soil temperature is the major limiting factor for symbiotic nitrogen fixation of legume. One greenhouse and four field experiments were conducted in 1999 to determine whether the pre-incubation of Rhizobium leguminosarum bv. viceae with hesperetin and naringenin or application of these compounds onto the seed surface or into the seed furrow at the time of planting can increase pea nodulation and final grain yield. The results from these experiments clearly indicated that application of naringenin and hesperetin by either pre-incubating R. leguminosarum bv. viceae prior to inoculation of plant or directly applying onto the seed surface or into seed furrow at the time of planting can increase pea nodulation, and plant pod numbers. Interactions existed between symbiotic signal compounds and pea cultivars or R. leguminosarum bv. viceae strains. However, there was no impact on the final grain yield by the treatments from the field experiments. The effects of these treatments on the final grain yield have to be farther tested.     

Escape of Steinernema feltiae from Alginate Capsules Containing Tomato Seeds

The entomogenous nematode Steinernema feltiae was encapsulated in an alginate matrix containing a tomato seed. When these capsules were placed on 0.8% agar for 7 days, the seed germinated and ca. 20% of the nematodes escaped from the capsules, whereas only 0.1% escaped from capsules without seeds. When capsules containing nematodes and a seed were planted into sterilized or nonsterilized soil, nematodes escaped to infect Galleria mellonella larvae. When seed in capsules containing ca. 274 nematodes per capsule were planted in nonsterilized soil, Galleria mortality was 90% 1 week later. Galleria mortality declined to 27%, 23%, and 0% in weeks 2, 4, and 8 postplant, respectively. In sterilized soil, Galleria mortality was 96% and did not differ significantly from the nonsterilized soil in week 1, but was significantly higher in sterilized soil over nonsterilized soil for week 2 (81%) and week 4 (51%). When capsules containing nematodes only were used, Galleria mortality was 71% in sterilized soil 1 week after planting and 58%, 33%, and 12% in weeks 2, 4, and 8 postplant, respectively. In nonsterilized soil, Galleria mortality was 8%, 30%, 21%, and 28% after 1, 2, 4, and 8 weeks, respectively, using only encapsulated nematodes. When the number of nematodes per capsule was increased to ca. 817, Galleria mortality was 92 % or higher in sterilized soil from week 1 to week 4.