Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations

Many of the microbial inoculants all over the world are based on solid peat formulations. This has been mostly true for well developed legume inoculants based on selected rhizobial strains, due to peat bacterial protection properties. Six carriers (bagasse, cork compost, attapulgite, sepiolite, perlite and amorphous silica) were evaluated as alternatives to peat. Compost from the cork industry and perlite were superior to peat in maintaining survival of different rhizospheric bacteria. Other tested materials were discarded as potential carriers for soybean rhizobia. Also, different liquid culture media have been assayed employing mannitol or glycerol as C sources. Some media maintained more than 10⁹ cfu ml^?1 of Sinorhizobium (Ensifer) fredii SMH12 or Bradyrhizobium japonicum USDA110 after 3 months of storage. Rhizobial survival on pre-inoculated seeds with both solid and liquid formulations previously cured for 15 days led to a higher bacterial numbers in comparison with recently made inoculants. An additional curing time of solid inoculants up to 120 days had a beneficial effect on rhizobial survival on seeds. The performance of different formulations of two highly effective soybean rhizobia strains was assayed under field conditions. Soybean inoculated with cork compost, perlite and liquid formulations produced seed yields that were not significantly different to those produced by peat-based inoculants.     

Effect of some fungicide seed treatments on the survival of Bradyrhizobium japonicum and on the nodulation and yield of soybean [Glycine max. (L) Merr.]

Several commercial fungicide seed treatments were evaluated for their possible effect on the survival of Bradyrhizobium japonicum on seeds and on the nodulation and yield of soybeans in a greenhouse and a field experiment. quinolate Pro (carbendazim and oxine copper), Vitavax 200FF (carboxin and thiram), and Monceren (pencycuron) had a small effect or no effect on the survival of B. japonicum and on the nodulation and yield of soybeans. They can thus be considered compatible with soybean seed inoculation. Germipro UFB (carbendazim and iprodione), Apron 35J (metalaxyl), and Tachigaren (hymexazol) decreased B. japonicum survival and the nodulation and yield of soybeans and thus cannot be considered compatible with soybean seed inoculation.     

EFFECT OF FOLIAR BORON APPLICATION ON GROWTH,REPRODUCTION, AND OIL QUALITY OF OLIVE TREES CONDUCTED UNDER A HIGH DENSITY PLANTING SYSTEM

Boron (B) foliar treatments (300 mg L^?1 as Solubor DF) were applied at two different dates in 2006 and 2007, prior to flowering and just after fruit set, on olive (Olea europaea L.) trees with no visual symptoms of B deficiency. Leaf B level increased after the first application as compared to control (?B). After July treatment, leaf B levels in ?B and +B treated trees increased when compared to the first sampling date. Foliar B application did not significantly affect vegetative growth in either year. During the first year of study (considered as an “on year”), B application had no significant effect on several phenological characteristics including fruit set, yield oil contents and oil quality. In the second year (“off year”), B sprays improved blooming rate, which increased from 20% in ?B to 30% in + B treated trees, and olive yield, which increased by 27% in response to B.     

Water-facilitated dispersal of inoculant Bradyrhizobium japonicum in soils

Summary Studies were performed to assess the influence of percolating water and an advancing wetting front on the transport of Bradyrhizobium japonicum in sand and silt loam soils, and to assess the influence of clay content on water-facilitated dispersal of these bacteria in a sand amended with various amounts of kaolin. The data obtained showed that movement of B. japonicum in soil was dependent upon water movement and that both percolating water and an advancing wetting front readily transported bacteria in coarse-textured soils. Percolation with the equivalent of 10 cm of rainfall dispersed B. japonicum throughout 40-cm columns containing sand and silt loam soils. Percolation with 5 cm of water was sufficient to disperse B. japonicum throughout 20-cm columns of these soils but did not transport these bacteria below the surface 4 cm of a sand amended with 12% kaolin. Our finding that cells of B. japonicum are readily transported by an advancing wetting front indicates that non-saturated flow of soil water contributes to dispersal of inoculum in soils.     

Rhizobiophage effects on nodulation,nitrogen fixation,and yield of field-grown soybeans (Glycine max L. Merr.)

Summary Previous laboratory and greenhouse studies have shown that phages significantly reduce soil populations of homologous rhizobia. Reductions in nodulation and N₂ fixation have also been observed. The purpose of the current study was to examine the effect of a phage specific ofBradyrhizobium japonicum USDA 117 on nodulation, nodule occupancy, N₂ fixation and soybean growth and yield under field conditions. The phage was inoculated in combination withB. japonicum USDA 117 and/orB. japonicum USDA 110 (resistant strain) into a rhizobia-free sandy loam soil and planted toGlycine max (L.) Merr. Williams. When the phage was applied to soil inoculated withB. japonicum USDA 117 alone, significant reductions in nodule weight and number, shoot weight, foliar N, nitrogenase activity, and seed index were observed. When, however, the soil also contained the non-homologous strain,B. japonicum USDA 110, no significant effects on any of these parameters were found. Nodule occupancy by competing strains ofB. japonicum USDA 110 and USDA 117 was also affected by the phage. In soil which did not contain the phage, 46% and 44% of the identified nodules were occupied by USDA 110 and 117, respectively. When the phage was present in the soil, nodule occupancy byB. japonicum USDA 117 was reduced to 23%, while occupancy byB. japonicum USDA 110 was increased to 71%. These results suggest that nodulation by selected strains of rhizobia can be restricted and nodulation by more effective, inoculated strains can be increased through the introduction of a homologous phage to soils.     

Repeated sequence RSα is diagnostic for Bradyhizobium japonicum and Bradyrhizobium elkanii

The genome of Bradyrhizobium japonicum and B. elkanii contains multiple copies of the repeated DNA sequence RSα. A collection of 18 B. japonicum, 4 B. elkanii and 72 other bacterial strains was screened by polymerase chain reaction (PCR) using a pair of primers specific for RSα. Only strains of B. japonicum and B. elkanii gave the predicted amplification product. Restriction analysis of PCR products obtained from different strains of B. japonicum showed that the RSα sequence was generally conserved. The usefulness of RSα as a specific probe for Bradyrhizobium strains capable of nodulating soybean was also demonstrated. Received: 11 May 1995     

Effects of some Nigerian Bradyrhizobium inoculants on the performance of three cowpea cultivars (Vigna unguiculata) in two field plots

Summary We assessed the effectiveness of three locally made lignite, subbituminous coal and cowmanure-based cowpea Bradyrhizobium inoculants in comparison with a peat-based imported Bradyrhizobium incoculant in a two-field plot investigation. The local inoculants were prepared by incorporating three rhizobia strains (Ife CR9, Ife CR15 and Bradyrhizobium japonicum) into each of the above carrier materials and were used to inoculate three cowpea seed varieties: TVU 1190, IT 82E-60 and Ife brown. With lignite-based He CR9 inoculated into TVU 1190 seeds, total N content of the plants was 178.6 mg/plant compared with only 64.3 mg/plant for the uninoculated nitrate-free control plants. With Nigerian lignite, sub-bituminous coal and cow manure as carriers for cowpea rhizobia, the cowpea yield of the inoculated plants increased by 72%, 54% and 10%, respectively, compared with uninoculated plants, while the peat-based inoculant gave a 25% increase in cowpea yield. With lignite-based Ife CR9 inoculated into Ife brown seeds, total N content of the plants was 149.1 mg/plant, but with inoculation by lignite-based B. japonicum, total N content of the treated Ife brown plant was 132.4 mg/plant. Thus, the native Ife CR9 strain seems to be slightly better adapted to tropical conditions than the imported B. japonicum.     

Genetic diversity and phylogeny of indigenous rhizobia from cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.]

16S rRNA RFLP, 16S rRNA sequencing, 16S-23S rRNA Intergenetic Spacer (IGS) RFLP and G-C rich random amplified polymorphic DNA (RAPD) assays were conducted to genetically characterise indigenous cowpea [Vigna unguiculata (L.) Walp.] rhizobia from different geographic regions of China. Isolated cowpea rhizobia comprised six 16S rRNA genospecies. Genotype I was composed of 14 isolated strains and the reference strains of B. japonicum and B. liaoningense. This group was divided into two sub-groups respectively related to B. japonicum and B. liaoningense by 16S rRNA sequencing, IGS restriction fragment length polymorphism and RAPD assays. Genotype II composed of 27 isolates from a variety of geographic regions. Four different assays confirmed this group was genetically distinct from B. japonicum and B. liaoningense and probably represent an uncharacterised species. Strains isolated from Hongan, Central China and B. elkanii were grouped to genotype III. Strain DdE4 was solely clustered into genotype IV and related to Rhizobium leguminosarum. Genotypes V and VI consisted of six fast-growing isolates and clustered with reference strain of Sinorhizobium fredii. Comparing with the miscellaneous slow-growing isolates, fast-growing isolates mainly isolated from cowpea cultivar Egang I exhibited strict microbe–host specificity except SjzZ4. Nucleotide sequences reported were deposited in the GenBank with the accession numbers DQ786795–DQ786804.     

Long-Term Spreading of Olive Mill Wastewater on Olive Orchard: Effects on Olive Production,Oil Quality,and Soil Properties

In Italy, the law no 574 of 1996 permits and regulates the disposal of olive mill wastewater (OMW), the liquid by-product obtained in oil mill when olive fruits are processed to extract virgin olive oil, by its controlled spreading on cultivated soil. With the aim to verify the long-term effects of the practice on olive production, oil quality, and physical–chemical and microbiological characteristics of soil, different amounts of OMW were spread in February, for 9 years consecutively, on soil cultivated with olive trees. The results obtained confirmed that the controlled spreading of OMW is not harmful for the plant production. On the contrary, OMW spread on olive grove is useful for the fertility of soil and the growth of microflora for the important supply of nitrogen (N), phosphorus (P), potassium (K), and, above all, of organic matter. The evaluated biological properties indicated that OMW spreading stimulates the growth of soil fungal and fungus-like communities and aerobic N₂ fix micro-organisms. With regard to the oil quality, the data indicate that no significant differences were ascertained on the analyzed parameters.     

Dual inoculation with strains of Bradyrhizobium japonicum and Azospirillum brasilense to improve growth and biological nitrogen fixation of soybean (Glycine max L.)

The effects of inoculation with Bradyrhizobium japonicum and Azospirillum brasilense strains on the growth of soybean were evaluated with regard to the estimation of N₂ fixation using the ¹⁵N isotope dilution technique. Inoculation, in general, increased the dry mass of soybean as well as nitrogen content. Dual inoculation with a mixture of B. japonicum and A. brasilense strains was superior over single inoculation with B. japonicum. Nitrogen fixed (Ndfa) varied according to inoculant and soil conditions. Percentages of nitrogen derived from air (% Ndfa) using a non-nodulating isoline were 72% and 76% for B. japonicum and B. japonicum plus A. brasilense, respectively, in non-sterile soil. A similar but higher trend was recorded in sterilized soil, in which the percentages of N₂ fixed were 81% and 86% for single and dual inoculation, respectively. The correlation coefficient between N₂ fixed and N uptake (r=0.94) and dry mass (r=0.89) was significant. Application of special bacterial inoculants in agricultural systems of Egypt seems to be a promising technology and could be used for improving soybean growth as well as soil fertility, thus minimizing environmental pollution. Received: 10 January 1996     

CO-INOCULATION OF SOYBEAN (GLYCINE MAX) WITH ACTINOMYCETES AND BRADYRHIZOBIUM JAPONICUM ENHANCES PLANT GROWTH,NITROGENASE ACTIVITY AND PLANT NUTRITION

The aim of this study was to determine the potential of the endophytic actinomycetes that produce plant growth promoters used as co-inoculants with Bradyrhizobium japonicum to promote the growth of soybean. These endophytes exhibited the potential to enhance plant growth, nitrogenase activity of root nodules and plant nutrient uptake. Co-inoculum of B. japonicum with Nocardia alba conferred the maximum yield of root and shoot dry weight. All single-inoculated actinomycetes strains had the ability to enhance plant growth. Noc. alba and Nonomuraea rubra increased total plant dry weight up to 2.14-fold and 2.11-fold, respectively, when compared to the uninoculated controls. Co-inoculations of B. japonicum with each of Noc. alba, Non. Rubra, and Actinomadura glauciflava increased acetylene reduction activity up to 1.7 to 2.7-fold. For plant mineral composition, all of co-inoculation treatments significantly increased the nutrient levels of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe) and zinc (Zn) within a soybean plant.     

Response of soybean cultivars toward inoculation with three arbuscular mycorrhizal fungi and Bradyrhizobium japonicum in the alluvial soil

The aim of this study was to assess the comparative efficacy of three arbuscular mycorrhizal fungi (AMF) combined with cultivar specific Bradyrhizobium japonicum (CSBJ) in soybean under greenhouse conditions. Soybean seeds of four cultivars namely JS 335, JS 71-05, NRC 2 and NRC 7 were inoculated with three AM fungi (Glomus intraradices, Acaulospora tuberculata and Gigaspora gigantea) and CSBJ isolates, individually or in combination, and were grown in pots using autoclaved alluvial soil of a non-legume cultivated field of Ajmer (Rajasthan). Assessment of the data on nodulation, plant growth and seed yield revealed that amongst the single inoculations of three AMF, G. intraradices produced the largest increases in the parameters studied followed by A. tuberculata and G. gigantea indicating that plant acted selectively on AMF symbiosis. The dual inoculation with AMF + CSBJ further improved these parameters demonstrating synergism between the two microsymbionts. Among all the dual treatments, G. intraradices + B. japonicum brought about the largest increases in the studied characteristics particularly in seed weight per plant that increased up to 115.19%, which suggested that a strong selective synergistic relationship existed between AMF and B. japonicum. The cv. JS 335 exhibited maximum positive response towards inoculation. The variations in efficacy of different treatments with different soybean cultivars indicate the specificity of the inoculation response. These results provide a basis for selection of an appropriate combination of specific AMF and Bradyrhizobium which could further be utilized for verifying the symbiotic effectiveness and competitive ability of microsymbionts under field conditions of Ajmer region.     

Cloning and characterization of a NADP+-malic enzyme gene from Bradyrhizobium japonicum USDA110

Malic enzymes have been considered to play a key role in energy metabolism for nitrogenase reaction in bacteroids. To elucidate the physiological role of the malic enzymes in Bradyrhizobium japonicum bacteroids, a putative malic enzyme gene Bjtme1 was cloned by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes and draft sequence data of the Bradyrhizobium japonicum USDA110 genome sequence project. To confirm the characteristics of the Bjtme1 gene, the protein encoded by this gene was over-expressed using a pET32a(+) system and it exhibited a NADP⁺-malic enzyme (EC 1.1.1.40) activity, indicating that Bjtme1 was the gene of the NADP⁺-malic enzyme. This is the first report on the cloning and characterization of the NADP⁺-malic enzyme gene from B. japonicum, and the gene structure was compared with that of NADP⁺-malic enzyme genes of other rhizobia.     

Oleaster (var. sylvestris) and subsp. cuspidata are suitable genetic resources for improvement of the olive (Olea europaea subsp. europaea var. europaea)

The olive belongs to Olea, a complex genus and to a species with six subspecies. Subsp. europaea includes both the cultivated olive and the oleaster, the wild ancestor of the olive. Little is known on the phenotype of fruits from subsp. cuspidata. We aimed to compare europaea and cuspidata trees for oil content and composition and to verify whether natural hybrids may exist between the two subspecies. Specimens were from Kenya and putative natural hybrids between cuspidata and europaea were from Stellenbosch (South Africa). Cultivar and oleaster trees were sampled in France (continental and Corsica), Italy (Continental and Sardinia) and Tunisia. We have examined the fruit (drupe) structure between subsp. cuspidata and europaea and extracted oil from the pulp, seed and total drupe. Comparison for oil content was made between the cultivated olive and the oleaster and some cuspidata trees from Kenya. A few of cuspidata individuals from Botanical gardens do not enable phenotyping for oil content and composition. Oil composition analyses were performed for the main fatty acids to compare the three taxa. We used microsatellite markers at 11 loci to compute genetic distances between cuspidata, oleaster and cultivar trees and to reveal eventual hybrids. The SSR polymorphisms were huge between the two subspecies and they revealed that putative hybrids were true hybrids sampled around olive orchards. The whole comparison of oil content shows that olive cultivars display higher oil content than the subsp. cuspidata and that the oleaster trees are intermediate, whereas for oil composition of the drupe, cuspidata from Kenya shows less oleic acid than europaea. However, the cuspidata trees show seed oil composition similarities with the other two taxa. The discussion deals with possible reasons to explain the differences and of the possible uses of these taxa for breeding both the olive and brown olive. H. Hannachi and H. Sommerlatte have equal contribution in the article.     

Field response of legumes to inoculation with plant growth-promoting rhizobacteria

Carrier-based (soil/FYM, 1:1) plant growth-promoting rhizobacteria (PGPR) isolates (Bacillus subtilis, Klebsiella planticola and Proteus vulgaris) were tested individually and in combination with Bradyrhizobium japonicum and Rhizobium leguminosarum biovar viciae under field conditions on soybean and lentil crops, respectively, under field conditions. Inoculation of soybean (Glycine max) cv. Pusa 22 with B. subtilis produced maximum nodule number, mass and nitrogenase activity (acetylene reduction activity, ARA) followed by B. japonicum (SB 271). Maximum soybean yield was registered with the coinoculation of B. japonicum and B. subtilis over an uninoculated control. Maximum nodulation in the lentil (Lens culinaris) cv. L 4147 was obtained with a combination of R. leguminosarum (L-12-87) and P. vulgaris inoculation followed by a single inoculation with Rhizobium and B. subtilis. None of the PGPR isolates either singly or in coinoculation with R. leguminosarum could significantly influence the yield of the lentil crop.     

Survival and potential denitrifying activity of Azospirillum lipoferum and Bradyrhizobium japonicum inoculated into sterilized soil

Summary Potential denitrifying activity and population dynamics of Azospirillum lipoferum (137C) and Bradyrhizobium japonicum (G2sp) inoculated into a -sterilized soil were studied for a period of 3 weeks. The denitrifying enzyme potential of soil inoculated independently with each bacterial species was strongly stimulated by the presence of a plant (Zea mays L.). Simultaneous inoculation of both bacteria also produced a higher denitrifying enzyme potential than simple inoculation. Even with double inoculation, the presence of a plant did not modify the evolution of the activity. The response of the population dynamics to these treatments followed a different pattern. The population dynamics of A. lipoferum was not affected by the presence of the plant or by the presence of B. japonicum. In contrast, the presence of both a plant and of A. lipoferum seemed to promote the growth of B. japonicum.     

Nodulation competition among Bradyrhizobium japonicum strains as influenced by rhizosphere bacteria and iron availability

Summary Bacteria isolated from the root zones of field-grown soybean plants [Glycine max (L.) Merr.] were examined in a series of glasshouse experiments for an ability to affect nodulation competition among three strains of Bradyrhizobium japonicum (USDA 31, USDA 110, and USDA 123). Inocula applied at planting contained competing strains of B. japonicum with or without one of eleven isolates of rhizosphere bacteria. Tap-root nodules were harvested 28 days after planting, and nodule occupancies were determined for the bradyrhizobia strains originally applied. Under conditions of low iron availability, five isolates (four Pseudomonas spp. plus one Serratia sp.) caused significant changes in nodule occupancy relative to the corresponding control which was not inoculated with rhizosphere bacteria. During subsequent glasshouse experiments designed to verify and further characterize these effects, three fluorescent Pseudomonas spp. consistently altered nodulation competition among certain combinations of bradyrhizobia strains when the rooting medium did not contain added iron. This alteration typically reflected enhanced nodulation by USDA 110. Two of these isolates produced similar, although less pronounced, effects when ferric hydroxide was added to the rooting medium. The results suggest that certain rhizosphere bacteria, particularly fluorescent Pseudomonas spp., can affect nodulation competition among strains of R. japonicum. An additional implication is that iron availability may be an important factor modifying interactions involving the soybean plant, B. japonicum, and associated microorganisms in the host rhizosphere.Paper No. 10648 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA     

Soil microbial diversity and activity in a Mediterranean olive orchard using sustainable agricultural practices

Sustainable soil management of orchards can have positive effects on both soils and crop yields due to increases in microbial biomass, activity and complexity. The aim of this study was to investigate medium‐term effects (12 yr) of two different management practices termed ‘sustainable’ (ST) and ‘conventional’ (CT) on soil microbial composition and metabolic diversity of a rainfed mature olive orchard in Southern Italy. ST included no‐till, self‐seeding weeds (mainly graminaceous and leguminosae), and mulch derived from olive tree prunings, whilst CT was managed by frequent tillage and included severe pruning with residues removed from the orchard. Microbial analyses were carried out by culture‐dependent methods (microbial cultures and Biolog^®). Molecular methods were used to confirm the identification by light microscopy of the isolates of fungi and Streptomyces. Significantly more culturable fungi and bacteria were found in ST than in CT. The number of fungal groups in ST was also significantly greater than in CT. Overall and substrate‐specific Biolog^® metabolic diversity indices of microbial communities and soil enzyme activities were greater in ST. The results demonstrate that soil micro‐organisms respond positively to sustainable orchard management characterized by periodic applications of locally derived organic matter. This study confirms the need to encourage farmers with orchards in the Mediterranean basin to practise soil management based on organic matter inputs associated with zero tillage to improve soil functionality.     

Characterization of local rhizobia in Thailand and distribution of malic enzymes

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD⁺-dependent (DME: EC 1.1.1.39) and NADP⁺-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was performed to survey the distribution of the malic enzyme genes among the local rhizobia, which are similar to those of B. japonicum. DNA probes (ME1 for DME and ME2 for TME) were prepared by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes. Southern blot analysis with ME1 as a probe showed a single band in about half of the isolates, especially in B. japonicum and R. leguminosarum strains, suggesting the wide distribution of such DME genes among local rhizobia. In contrast, Southern blot analysis with ME2 as a probe detected a single band only in five B. japonicum strains, suggesting that the TME genes, which are similar to those of B. japonicum, would be unique in a group of B. japonicum.     

Serological properties and intrinsic antibiotic resistance of soybean bradyrhizobia isolated from Thailand

A group of Bradyrhizobium strains isolated from soybean plants in Thailand did not correspond to any known DNA homology groups of Bradyrhizobium japonicum and Bradyrhizobium elkanii reported by Hollis et al. (J. Gen. Microbiol., 123, 215–222, 1981). To clarify the phenotypic characteristics of the group, serological properties and intrinsic antibiotic resistance (IAR) profile of 94 Thai strains were compared with those of USDA and Japanese strains. Indirect ELISA tests for each Thai strain were performed agaiIl.st polyclonal antisera prepared against 15 USDA standard serotype strains of B. japonicum and B. elkanii. Among the 94 Thai strains tested, 36 which were previously identified as B. elkanii, with the exception of one strain, were strongly responsive to an antiserum prepared against USDA 31. The remaining 58 strains, with the exception of two strains, showed multiple cross reactions which were peculiar to the Thai strains. These serological reaction patterns did not correspond to any known serogroups labeled as B. japonicum and B. elkanii. In the IAR test, the taxonomically unknown Thai soybean bradyrhizobia exhibited a high level of resistance to neomycin (50 µg/mL), polymyxin (50 µg/mL), nalidixic acid (15 µg/mL), and kanamycin (15 µg/mL). Kanamycin could thus be useful in combination with neomycine and nalidixic acid for distinguishing between the unknown Thai strains and strains of B. japonicum and B. elkanii. Our results demonstrated that the unknown Thai strains were serologically and IAR-phenotypically remote from both B. japonicum and B. elkanii.