Identification of an ice-nucleating bacterium and its ice nucleation properties

An ice-nucleating bacterium, KUIN-2, was isolated from carrot leaves. The ice-nucleating bacterium was found in the white colony group. KUIN-2 was identified as Pseudomonas viridiflava from its taxonomic characteristics. When KUIN-2 was cultured aerobically in a medium consisting of Trypticase soy broth (pH 6.0) for 24 h at 18°C, the ice-nucleating activity of KUIN-2 cells was obtained. Ice nucleation was detected at −2.8°C in cell suspensions (1.4 × 10⁸ cells/ml) of KUIN-2. The nucleation frequency of KUIN-2 was greatly inhibited by the addition of urea or N-ethylmaleimide.     

Isolation and Characterization of a Novel Ice-nucleating Bacterium,Pseudomonas sp. KUIN-4, Which Has Stable Activity in Acidic Solution

A novel ice-nucleating bacterium, KUIN-4 was isolated from a cherry leaf, which was unsusceptible to frost injury. Strain KUIN-4 was identified as Pseudomonas sp. from its characteristics and taxonomies; the optimum temperature and pH for its growth were 18°C and 4.5, respectively. When strain KUIN-4 was cultured aerobically in CYE medium (pH 4.5) for 48 h at 18°C, the ice-nucleating activities of strain KUIN-4 cells and culture broth (extracellular ice-nucleating matter) after removal of the cell were obtained. The ice-nucleating temperature, T₅₀ (°C), was indicated to be –2.8°C in cell suspensions (4.25 x 10⁷ cell/ml) of strain KUIN-4. Also, it had become apparent that the ice-nucleating activity involved class A, B, and C structures as judged by its freezing difference spectrum in D₂O versus H₂O. The ice-nucleating activity of this strain as well as other ice-nucleating bacteria was significantly decreased by heat treatment (40°C, 30 min). The ice-nucleating activity from this strain had unique features, which was stable under acidic conditions (pH 3.5–5.0) and was weakly inhibited by denaturants and protein-modifying reagents.     

Identification of a novel ice-nucleating bacterium of Antarctic origin and its ice nucleation properties

A novel ice-nucleating bacterium (INB) was isolated from Ross Island, Antarctica. INBs could be isolated more frequently than was generally thought. INB strain IN-74 was found in the white colony group. Strain IN-74 was identified from its taxonomic characteristics as a novel INB, Pseudomonas antarctica IN-74. When strain IN-74 was cultured aerobically in a medium consisting of the ice-nucleating broth (pH 7.0) for 6 days at 4 degrees C, the ice-nucleating activity of strain IN-74 cells was obtained. Strain IN-74 cells produced ice nuclei only at extremely low growth temperatures. The nuclei appeared to be less thermolabile than those of INB Pseudomonas fluorescens KUIN-1. The freezing difference spectra in D2O and H2O at ice-nucleating temperature for strain IN-74 cells and conventional INBs (Pseudomonas fluorescens KUIN-1, Pseudomonas viridiflava KUIN-2, and Pseudomonas syringae C-9) exhibited different curves.     

Determination of the Chemical Form of Fluorine in Tea Infusions by 19F-NMR

A novel marine ice-nucleating bacterium, KUIN-5, was isolated from a marine algae, Monostroma latissum. Strain KUIN-5 was identified as a Pseudomonas sp. from its characteristics and taxonomies; the optimum temperature and pH for its growth were 25°C and 6.0, respectively. When strain KUIN-5 was aerobically cultured in Carlucci-Pramer medium (pH 6.0) for 50 h at 25°C, the highest ice-nucleating activity of the cells among the media for marine bacteria was obtained, and the ice-nucleating temperature, T₅₀, was indicated to be ? 3.2°C. Also, the optimum concentration of NaCl for the growth in this medium, which was prepared with distilled water instead of seawater, was 2.0% (w/v) and then the ice-nucleating activity was inversely proportional to the NaCl concentration. Moreover, when strain KUIN-5 was cultured in Davis medium under optimum conditions, it produced insoluble polysaccharide (IPS) in the culture. The maximum amount of IPS production by strain KUIN-5 was 84.5 mg/ml of medium under optimum conditions. Therefore, this IPS was isolated and could be identified as cellulose, based on TLC or HPLC of the acid hydrolysate, and GC-MS of the acetylated polyalcohol prepared by periodate oxidation and Smith degradation of this polysaccharide. This is the first report of cellulose production by a marine ice-nucleating bacterium.     

Identification of a Compound in Chamaecyparis taiwanensis Inhibiting the Ice-nucleating Activity of Pseudomonas fluorescens KUIN-1

Inactivation of the ice-nucleating activity of Pseudomonas fluorescens KUIN-1 by compounds in the leaves from coniferous trees were investigated, and the inactivated material was identified. Intact cells of the strain KUIN-1 and the acetone or methanol extracts of leaves of various coniferous trees were allowed to react for 30 min at 18°C. Antinucleation compounds were obtained from Chamaecyparis taiwanensis. When the acetone extract from the leaves of coniferous trees was added to the cell suspension (about 10⁶ cells/ml) in 50 mM potassium phosphate buffer (pH 7.0), the ice nucleating temperature, T₅₀, was significantly decreased (T₅₀<-5°C). This inhibitor was isolated by using TLC, then identified as hinokitiol based on UV-VIS, IR, and mass spectral data. When intact cells of the strain KUIN-1 were incubated with hinokitiol, limonene, and α-pinene of the principal constituent of the leaves of coniferous trees in 50 mM potassium phosphate buffer (pH 7.0), the ice-nucleating activity decreased, but not in α-terpinene. Furthermore, the ice-nucleating activities from other ice-nucleating bacteria also decreased in the presence of hinokitiol. This inhibition was proportional to the concentration of hinokitinol. The pH and thermal stabilities of the ice-nucleating activity of the cells were changed by the addition of hinokitiol (10 mM).     

Ice-nucleating activity of Pseudomonas fluorescens

Pseudomonas fluorescens KUIN-1 can cause the freezing of water at relatively warm termperatures (−2.9 to −30°C). Glucose, glycerol, and citric acid were good substrates yielding cells with high ice-nucleating activity. Ammonium salts were good nitrogen sourcfes yielding cells with high ice-nucleating activity. The ice-nucleating activity of KUIN-1 was not affected by pH between pH 5.5 to pH 8.0. The ice-nucleating activity of cells was greatly influenced by heat treatment.     

Cinnamtannin A2, a Tetrameric Procyanidin,Increases GLP-1 and Insulin Secretion in Mice

The ice-nucleating bacterium, Pantoea agglomerans IFO12686, induces the cryoptotective protein (CRP) by cold acclimation at 12°C. The CRP was purified to apparent homogeneity by various chromatographies. We found that the purified CRP was a monomer of approximately 29,000 according to gel filtration chromatography and SDS-PAGE, and was a heat-stable protein. The CRP could protect freeze-labile enzymes, lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH) and isocitrate dehydrogenase (iCDH), against freezing-thawing denaturation. The activity of the CRP was about 3.5×10⁴ times more effective than bovine serum albumin (BSA) and 2×10⁶ times than COR26 from the ice-nucleating bacterium Pseudomonas fluorescens KUIN-1. We confirmed that the CRP was a novel protein, as judged by the a different molecule mass from the already-known cryoprotectants, and has an extremely high cryoprotective activity.     

A Cold Acclimation Protein with Refolding Activity on Frozen Denatured Enzymes

We found that a cold acclimation protein from an ice-nucleating bacterium, Patoea ananas KUIN-3, has refolding activity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the cold shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refolding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH₄)₂SO₄ precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,000 each with a molecular mass of 200,000 and had refolding activity against denatured enzymes, which were denatured by heat-treatment at 100°C, cryopreservation at -20°C, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was Met-Arg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high level of activity at low temperature (12°C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82×10^-10, 4.35×10^-9, 8.98×10^-12, and 3.05×10^-11, respectively. The affinity of Hsc25 for frozen danatured enzymes was higher than the affinity for heat denatured enzymes when compared with the affinity of GroEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation.     

Identification of a compound in Chamaecyparis taiwanensis inhibiting the ice-nucleating activity of Pseudomonas fluorescens KUIN-1

Inactivation of the ice-nucleating activity of Pseudomonas fluorescens KUIN-1 by compounds in the leaves from coniferous trees were investigated, and the inactivated material was identified. Intact cells of the strain KUIN-1 and the acetone or methanol extracts of leaves of various coniferous trees were allowed to react for 30 min at 18 degrees C. Antinucleation compounds were obtained from Chamaecyparis taiwanensis. When the acetone extract from the leaves of coniferous trees was added to the cell suspension (about 10(6) cells/ml) in 50 mM potassium phosphate buffer (pH 7.0), the ice nucleating temperature, T50, was significantly decreased (T50 < -5 degrees C). This inhibitor was isolated by using TLC, then identified as hinokitiol based on UV-VIS, IR, and mass spectral data. When intact cells of the strain KUIN-1 were incubated with hinokitiol, limonene, and alpha-pinene of the principal constituent of the leaves of coniferous trees in 50 mM potassium phosphate buffer (pH 7.0), the ice-nucleating activity decreased, but not in alpha-terpinene. Furthermore, the ice-nucleating activities from other ice-nucleating bacteria also decreased in the presence of hinokitiol. This inhibition was proportional to the concentration of hinokitinol. The pH and thermal stabilities of the ice-nucleating activity of the cells were changed by the addition of hinokitiol (10 mM).     

The ice Nucleation Activity of Pseudomonas fluorescens Migula and its Inhibition by Various Chemicals

The ice nucleation activity (INA) of three strains of Pseudomonas fluorecens, nos 553, 554 and 606, isolated by the Institute for Pathogen Diagnostics in Ascherleben, Germany, was determined. Under equal growth conditions and at given test temperatures the ice nucleation frequency spectra of the isolates differed slightly. The fraction of cells which acted as ice nuclei increased with falling temperatures. Below ?5°C the nucleation frequency rose from 10^-8 to 10^-3. Between, 0 and ?10°C only a fraction of approximately 2 to 5 × 10^-3 cells performed ice nucleation activity. Fifteen newly synthesized chemicals showed no or only a very slight intrinsic INA at ?5°C and ?7°C. The compounds were used as antinucleators against INA-exhibiting bacteria. In INA-exhibiting suspensions of isolate 553 bacterial ice nuclei were reduced after treatment with the 15 compounds. Dependent on the compounds, a nucleation frequency of ?8.32 to ?5.10 was detected at ?5°C. At ?7°C, the frequency amounted to ?7.89 to ?5.05. As the temperature was lowered to ?10°C in bacterial suspensions which were treated with 9 (of the 15) compounds, a remainder of 1.79 to 5.91 × 10^-6 cells retained ice nucleation activity. The most pronounced inhibitory effect was noted for the compounds 1989/6255, 1989/6436 and 1990/6158. In a 10-fold dilution of isolate 553 the compound 1989/6153 inhibited ice nucleation between 0 and 10°C so strongly that it was about 100 times below the control. The ‘tube-freezing’ method showed that on excised corn leaves treated with 1989/6259 and 1990/6155, the bacterial INA decreased while the super-cooling was more pronounced. ‘Frostgard’, 1986/6205, 1986/6199 and 1989/6259 inhibited most INA-exhibiting bacteria on corn seedlings. Compared to inoculated plants, a significantly higher percentage of treated plants survived at ?2 and ?3°C.     

Transfer of ampicillin resistance from Salmonella Typhimurium DT104 to Escherichia coli K12 in food

Aims: To investigate the transfer of antibiotic resistance from a donor Salmonella Typhimurium DT104 strain to a recipient Escherichia coli K12 strain. Methods and Results: Mating experiments were conducted in broth, milk and ground meat (beef) at incubation temperatures of 4, 15, 25 and 37°C for 18 and 36 h. Ampicillin‐resistance transfer was observed at similar frequencies in all transfer media at 25 and 37°C (10^?4 to 10^?5 log₁₀ CFU ml g^?1, transconjugants per recipient) for 18 h. At 15°C, transfer was observed in ground meat in the recipient strain (10^?6, log₁₀ CFU g^?1, transconjugants per recipient), but not in broth or milk. At 4°C, transfer did not occur in any of the examined mediums. Further analysis of the E. coli K12 nal^R transconjugant strain revealed the presence of a newly acquired plasmid (21 kbp) bearing the β‐lactamase gene bla_TEM. Transconjugants isolated on the basis of resistance to ampicillin did not acquire any other resistant markers. Conclusion: This study demonstrates the transfer of antibiotic resistance in food matrices at mid‐range temperatures. Significance and Impact of the Study: It highlights the involvement of food matrices in the dissemination of antibiotic‐resistant genes and the evolution of antibiotic‐resistant bacteria.     

Methanohalophilus profundi sp. nov., a methylotrophic halophilic piezophilic methanogen isolated from a deep hypersaline anoxic basin

A novel anaerobic methylotrophic halophilic methanogen strain SLHTYRO^T was isolated from a deep hypersaline anoxic basin called “Tyro” located in the Eastern Mediterranean Sea. Cells of SLHTYRO^T were motile cocci. The strain SLHTYRO^T grew between 12 and 37 °C (optimum 30 °C), at pH between 6.5 and 8.2 (optimum pH 7.5) and salinity from 45 to 240 g L⁻¹ NaCl (optimum 135 g L⁻¹). Strain SLHTYRO^T was methylotrophic methanogen able to use methylated compounds (trimethylamine, dimethylamine, monomethylamine and methanol). Strain SLHTYRO^T was able to grow at in situ hydrostatic pressure and temperature conditions (35 MPa, 14 °C). Phylogenetic analysis based on 16S rRNA gene and mcrA gene sequences indicated that strain SLHTYRO^T was affiliated to genus Methanohalophilus within the order Methanosarcinales. It shared >99.16% of the 16S rRNA gene sequence similarity with strains of other Methanohalophilus species. Based on ANIb, AAI and dDDH measurements, and the physiological properties of the novel isolate, we propose that strain SLHTYRO^T should be classified as a representative of a novel species, for which the name Methanohalophilus profundi sp. nov. is proposed; the type strain is SLHTYRO^T (=DSM 108854 = JCM 32768 = UBOCC-M-3308).     

Production and Properties of Alkaline Lipase from Alcaligenes sp. Strain No. 679

For the production of extracellular lipase by Alcaligenes species No. 679, NaNO₃, polyoxyethylene alkyl ether, Fe⁺⁺, sodium citrate and fructose were found to be effective. The enzyme was prepared by acetone precipitation from the filtrate of the culture broth of this strain. The enzyme was most active at pH 9.0 and 50°C, while 35% of its activity was lost on heat treatment at 60°C for 10 min. Sodium salts of bile acids stimulated the enzyme activity. This lipase could hydrolyse natural fats and oils as well as olive oil. During the hydrolysis of olive oil, monoglyceride was found to accumulate up to 70 mol percent. This lipase possesses special properties similar to those of pancreatic lipase as shown in the comparative experiments.     

Biodegradation and metabolic pathway of sulfamethoxazole by Pseudomonas psychrophila HA-4, a newly isolated cold-adapted sulfamethoxazole-degrading bacterium

Sulfamethoxazole is a common antibiotic that is frequently detected in wastewater and surface water. This study investigated the biodegradation and metabolic pathway of sulfamethoxazole by Pseudomonas psychrophila HA-4, a cold-adapted bacterium. Strain HA-4, which uses sulfamethoxazole as its sole source of carbon and energy, was isolated at a low temperature (10 °C) and identified as P. psychrophila by physico-biochemical characterization and 16S rRNA gene sequence analysis. Strain HA-4 removed sulfamethoxazole at temperatures ranging from 5.0 °C to 30 °C, with the maximal removal rate at 10 °C. The maximal removal rate of sulfamethoxazole by strain HA-4 was 34.30 % after 192 h at 10 °C. The highest percentage of unsaturated fatty acid was determined to be 23.03 % at 10 °C, which adheres to the characteristic for cold-adapted psychrophiles and psychrotrophs. At low concentrations of sulfamethoxazole, the growth kinetics correlated well with the Haldane model. The single-substrate parameter values of sulfamethoxazole on cell growth were determined to be μ _max?=?0.01 h^?1, K _s?=?20.91 mg/l and K _i?=?170.60 mg/l. Additionally, the major intermediates from sulfamethoxazole biodegradation by strain HA-4, including aniline, 3-amino-5-methylisoxazole, 4-aminothiophenol and sulfanilamide, were identified by GC-MS and high-resolution mass spectrometry (HR-MS) analysis. The results demonstrate that strain HA-4 has the potential to degrade sulfamethoxazole at low temperatures.     

Isolation and general characterization of a heat-stable proteinase from Pseudomonas fluorescens AFT 36

An extracellular proteinase from Pseudomonas fluorescens, strain AFT 36, was isolated to homogeneity by chromatography on DEAE-cellulose and Sephadex G-150; a 230-fold increase in specific activity with a recovery of 53% was obtained. The enzyme was optimally active at pH 6.5 and 45°C; activity declined rapidly at higher temperatures but significant activity persisted down to 4°C. Activity was strongly inhibited by 10^?3 M EDTA and was partially restored by addition of Zn²⁺, Ca²⁺ or Co²⁺. The K_m values on methylated casein and sodium caseinate were 18.2 and 7.1 mg/ml, respectively. The enzyme was very labile in phosphate buffer and in a milk salts buffer at 55°C but was very stable in the latter at more than 80°C.     

The occurrence of ice nucleation activity in pathogenic bacterial species

We tested the nucleation activity (INA) of 122 strains of plant pathogenic bacteria (12 varieties and 15 subspecies) stored in collection of the University of Göttingen (GSPB). The strains are isolates from diverse host plants and different geographic regions. One-hundred and seven isolates belong to the Pseudomonads, nine to the genus Erwinia and six to the Xanthomonas. The INA was analysed by ?3°, ?5°, ?7° and ?9°C. The observed value of INA cells ranged from non-detectable to a maximum concentration of ice nuclei in a range from ?7.85 at ?5°C to ?2.63 at ?3°C in 1.82 × 10³ cfu to 3.3 × 10³ cfu per ml. The data indicated that 71 (58.2%) of the 122 strains had INA cells, and 51 (41.8%) were inactive. The highest amount of strains with INA cells we found in Pseudomonads (69). In comparison only one strain was active at Erwinia and at Xanthomonas, 46 strains were isolated from the genus Phaseolus vulgaris and 6 from the genus Beta vulgaris. The other isolates with ice active cells belonged to the 13 other plant species. The 51 inactive pathovars were isolated from 21 different culture plants. The pathogens under test were isolated in 16 different countries, mainly in Germany and USA.     

Psychrotrophic Bacteria Isolated from a Constantly Warm Tropical Environment

Psychrotrophic bacteria are known to occur in temperate, constantly cold, and artificially cooled environments. This is the first report of their occurrence in a constantly warm (ca. 24°–35°C) tropical environment. Soil samples taken from two sites along the southeastern coastal zone of Jamaica yielded growth of psychotrophic bacteria after 3–4 weeks of enrichment culture in 1/30 strength tryptic soy broth, 20 mg L⁻¹ cycloheximide at 2°C. Growth of individual isolates at 2°C was confirmed. Isolates include aerobic and fermentative Gram-negative rods and sporeforming (Bacillus sp.) and non-sporeforming (Aureobacterium sp.) Gram-positive rods. We determined the effect of temperature on growth rate in four isolates. Strain Y1 has an unusually wide temperature range for growth, 2°–44°C, resembling that of Listeria monocytogenes. In strain R1 the optimum temperature for growth occurred unusually near the maximum temperature for growth. Strains R2 and Y2 displayed cardinal temperatures typical of known psychotrophs but appear to have evolved enhanced growth potential near the optimum temperature in response to a constantly warm environment. Received: 30 April 1997 / Accepted: 9 August 1997     

Utilization of oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater by Flavobacterium johnsoniae

Aims: To obtain a bacterial strain that can be used to quantify biodegradable polysaccharides at concentrations of a few micrograms per litre in freshwater. Methods and Results: Flavobacterium johnsoniae strain A3 was isolated from tap water supplemented with laminarin, pectin or amylopectin at 100 μg C l^?1 and river Rhine water. The organism utilized 14 of 23 oligo‐ and polysaccharides, and 1 of 9 monosaccharides, but none of the sugar acids, sugar alcohols, carboxylic acids or aromatic acids tested at 10 μg C l^?1. Amino acids promoted growth of strain A3, but not in coculture with assimilable organic carbon (AOC) test strain Pseudomonas fluorescens P17, which utilized these compounds more rapidly than strain A3. Compounds released by strain P17 and AOC test strain Spirillum sp. NOX grown on acetate promoted the growth of strain A3 at N_max values of ≥ 2 × 10⁵ CFU ml^?1 of strain P17 and ≥ 5 × 10⁵ CFU ml^?1 of strain NOX. Significant growth of strain A3 was observed in surface water and in tap water in the presence of strain P17 (N_max P17 < 2 × 10⁵ CFU ml^?1). Conclusions: Strain A3 utilizes oligo‐ and polysaccharides at microgram‐per‐litre levels. In surface water and in tap water, the organism was able to utilize compounds that were not utilized by strain P17. These compounds may include oligo‐ and/or polysaccharides. Significance and Impact of the Study: Phytoplanktonic and bacterial polysaccharides can constitute an important biodegradable fraction of natural organic matter in water and may promote growth of heterotrophic bacteria during water treatment and drinking water distribution. Strain A3 can be used to quantify a group of compounds that includes oligo‐ and polysaccharides at microgram‐per‐litre levels in freshwater.     

The effect of bacterial strain and temperature changes on the nitrogenase activity of Lotus pedunculatus root nodules

After 40 days of growth at 25°C, Lotus pedunculatus cav., cv. Maku plants infected with Rhizobium loti strain NZP2037 displayed similar relative growth rates but had twice the nodule mass and only one third the whole plant dry weight of plants infected with Bradyrhizobium sp. (Lotus) strain CC814s. In the NZP2037 symbiosis, the rate of CO₂ evolution (per g dry weight of nodulated root) was 1.6 times as high as that in the CC814s symbiosis while the rate of C₂H₂ reduction (per g dry weight of nodule) was only 48% of that in the CC814s symbiosis. Studies of the effect of short term temperature changes on the gas exchange characteristics (CO₂ and H₂ evolution, C₂H₂ reduction) of these symbioses revealed wide differences in the optima for C₂H₂ reduction. Nodules infected with NZP2037 displayed maximal C₂H₂ reduction rates [157 μmol (g dry weight nodule)^?1 h^?1] at 12°C, whereas nodules infected with CC814s were optimal at 30°C [208 μmol (g dry weight nodule)^?1 h^?1]. These short term studies suggested that differences in temperature optima for N₂ may have partially accounted for the poorer effectivity, at 25°C, of strain NZP2037 when compared with strain CC-814s. The relative efficiency [RE = 1 – (H₂ evolution/C₂H₂ reduction)] of N₂ fixation varied widely with temperature in the two symbioses, but there was a general trend toward higher RE with lower temperatures. The ratio of CO₂ evolution: C₂H₂ reduction (mol/mol) in nodulated roots infected with CC814s was constant (ca 10 CO₂/C₂H₂) between 5°C and 30°C, whereas in plants infected with NZP2037 it reached a minimal value of 3.3 CO₂/C₂H₂ at 10°C and was 19 CO₂/C₂H₂ at the growing temperature (25°C).     

Evaluation of Pochonia chlamydosporia var. chlamydosporia as a control agent of Meloidogyne javanica on pistachio

The potential of isolates of Pochonia chlamydosporia var. chlamydosporia as biocontrol agents for root-knot nematodes was investigated in vitro and on pistachio plants. On potato dextrose agar, growth of all isolates started at temperatures above 10°C, reached maximum between 25 and 28°C and slowed down at 33°C. On water agar, all isolates parasitized more than 85% of the eggs of Meloidogyne javanica at 18°C after 3 weeks. Filtrates of isolates grown on malt extract broth did not cause more than 5% mortality on second-stage juveniles of M. javanica after 48 h of incubation. A single application of 10×10³ chlamydospores (produced on sand–barley medium) g^–1 soil, was applied to unsterilised soil planted with pistachio cv. Kalehghochi, and plants were inoculated with 3000 nematode eggs. After 120 days in the glasshouse, nematode multiplication and damage were measured. Ability of fungus isolates to survive in the soil and to grow on roots were estimated by counting colony forming units (cfu) on semi-selective medium. Fungal abundance in soil increased nearly 3-fold and 10×10³ and 20×10³ cfu g^–1 root of pistachio were estimated in pots treated with isolates 40 and 50, respectively. Strain 50 was more abundant in soil and on the roots, infected more eggs (40%) on the roots and controlled 56% of total population of M. javanica on pistachio roots, whereas isolate 40 parasitized 15% of the eggs on the roots and controlled ca. 36% of the final nematode population.