Efficiency of various selective media in determining Pythium population in soil

Of 15 selective media recommended for isolation and enumeration ofPythium spp. directly from soil, corn meal agar (CMA) supplemented with agar, sucrose, minor elements, thiamine, rose bengal, pimaricin, pentachloronitrobenzene and vancomycin (MPVM) was the most efficient. Streptomycin (30–50 ppm) and rose bengal (33–60 ppm) as used in certain tested media effectively suppressed development of bacteria and actinomycetes. However, these chemicals adversely affected germination of spores and mycelial growth and thereby the recovery ofPythium spp. from soil. Media containing pimaricin (5 to 100 ppm) were more effective than those with nystatin (40 ppm) in suppressing development of nonphycomycetous fungi on isolation plate. MPVM with pimaricin at 5 ppm was more efficient than that with 10 ppm of the antibiotic in recoveryingPythium from soil. However, there was no difference in recovery ofPythium by this medium containing rose bengal at 5 ppm or at 10 ppm, butPythium colonies were more dense and better delineated when the medium contained 10 ppm of rose bengal. CMA containing pimaricin (5–100 ppm) and vancomycin (200 ppm) permitted occasionally development of a large number ofMortierella and bacterial colonies from certain soils, that interfered with accurate determination of colonies of certainPythium spp. on the plates. Vancomycin at 300 ppm, as used in MPVM, substantially reduced development of bacterial colonies compared to 200 ppm of the antibiotic. Surface-soil dilution-plate was more effective than the soil-dilution-plate method in reducing bacteria andMortierella colonies on isolation plates without affecting recovery ofPythium. The importance of basal medium, complement of antimicrobial agents, and isolation methods for efficiency of selective medium in recovery ofPythium spp. directly from soil is discussed.     

Actinophages and restriction enzymes fromMicromonospora species (Actinomycetales)

Summary To develop a screening procedure for the detection of restriction endonucleases in micromonosporae and catellatosporae based on efficiency of plating, eight different actinophages were isolated from soils enriched withMicromonospora species and one fromCatellatospora-enriched soil. The lytic actinophages all contained double-stranded DNA and the majority appeared, when examined by electron microscopy, to belong to Ackermann's type B1 since they had isometric heads and noncontractile tails. One actinophage was classified as type C1 because of its isometric head and very short noncontractile tail. The host ranges of the actinophages were determined on strains ofMicromonospora and selected species from other actinomycete genera of cell wall chemotype II. Type II restriction enzymes were isolated fromM. echinospora ssp.echinospora (ATCC 15837),M. purpurea (ATCC 15835) andM. zionensis (LL-100-125) and were designatedMecI,MpuI andMziI, respectively. Restriction enzymesMecI andMpuI are isoschizomers ofXhoI, whileMziI is an isoschizomer ofPvuII.     

Studies on the distribution of alkalophilic and alkali-tolerant soil fungi I

Soil fungi were isolated from two different soil types using alkaline and slightly acidic media (alkaline cornmeal agar (AC-MA), pH 9.7; cornmeal agar (CMA), pH 6.0) to study their distribution. Different species were obtained on each isolation medium. The number of species ofAcremonium andFusarium increased on ACMA, though many species growing well in acidic conditions were not detected on ACMA. Most of the fungi isolated on ACMA, especially from the alkaline soils, were alkalophiles or alkali-tolerants that can grow at pH 10.Acremonium alternatum, A. furcatum, Acremonium sp. 6,Gliocladium cibotii (YBLF 575),Phialophora geniculata, Stachylidium bicolor andStilbella annulata were alkalophilic, of whichAcremonium sp. 6 was the most pronounced alkalophile. Ability to grow under alkaline conditions, as well as under acidic conditions, was common in manyAcremonium species. The use of alkaline medium facilitates the isolation of alkalophilic soil fungi.     

Soil salinity and pH in Japanese mangrove forests and growth of cultivated mangrove plants in different soil conditions

Soil conditions of mangrove forests in southern Japan were found to correlate largely with zonal distributions of the species.Kandelia candel grew in soils with low salinity and low pH,Avicennia marina, Rhizophora stylosa andSonneratia alba in soils with high salinity and high pH, andBruguiera gymnorrhiza in soil with a wide range of pH but limited range of salinity.Lumnitzera racemosa colonized soil with a wide range of pH and medium salinity. Seedlings ofKandelia candel, Bruguiera gymnorrhiza andRhizophora stylosa were planted in soils with differing salinity and pH. Optimum seedling growth ofKandelia, Bruguiera andRhizophora occurred when plants were cultivated in soils similar to those of their natural habitats, suggesting that growth of mangrove species and their zonal distributions were regulated by salinity and soil pH.     

The mycoflora of groundnut rhizosphere,soil and geocarposphere on light,medium and heavy soils and its relations to aspergillus flavus

Summary A comparative study was carried out in groundnut fields in Israel of the mycoflora of rhizosphere (R) and soil adhering to geocarps (G) and of the soil (S).Of a total of 157 fungal species isolated and listed, 133 occurred in R, 96 in S, and 86 in G. Ten species considered rare or worthy of special note are briefly discussed.Species ofAspergillus were most numerous in heavy soil, species ofPenicillium in light soil, and species ofFusarium in medium soil.Penicillium funiculosum andP. rubrum were the species occurring with greatest frequency, and these were equally common in R, S, and G of light and medium soil. Soil inoculation withA. flavus depressed the number of species in the mycoflora of R and S.Quantitative comparison of R, S, and G on the three soils showed that numbers for R exceeded those for S and G, forP. funiculosum andP. rubrum on all soils, and for several Aspergilli on heavy soil. Among fungi more prevalent in S than in R and G wasA. flavus on medium and heavy soil. No special affinity of any one of the 3 major groups (Aspergillus, Penicillium, Fusarium) for the mycoflorae of R or S or G has been found. The relative number of colonies of all fungi in R and S (R/S ratio) was in almost all cases in excess of 1 on all soil types.This research is supported by grant number FG-161 of the United States Department of Agriculture, to whom the author is indebted.     

Nitrate production in the rhizosphere of Plantago species

Summary The production of nitrate in an old established dune grassland soil and its uptake by plants was studied by comparing amounts of mineral nitrogen and numbers of nitrifying bacteria in the rhizosphere on the one hand, and on the other accumulated nitrate and levels of nitrate reductase (NaR) of individual plants of three Plantago species,i. e., P. major, P. lanceolata andP. coronopus. For these three Plantago species andP. media basal levels of NaR in the absence of nitrate were determined in plants grown in culture solutions. The basal NaR levels ofP. major andP. media (species occurring on nutrient-rich soils) were significantly higher than those ofP. lanceolata andP. coronopus (species found on nutrient-poor soils). NaR activity increased in the presence of nitrate and was suppressed by ammonium.From the numbers of nitrifying bacteria in the rhizosphere and NaR activity in the leaves it was concluded that nitrate was produced in the root environments of the three Plantago species and that the compound was taken up by the plants. NaR activities and numbers of nitrifying bacteria were higher for individuals ofP. major than for those ofP. lanceolata andP. coronopus. No correlation was found between the ammonium levels and the numbers of nitrifying bacteria in the soil, and no indications of inhibition of nitrifying bacteria in the rhizosphere were obtained. For individuals ofP. lanceolata a correlation was found between the numbers of nitrifying bacteria in the soil and NaR activity in the leaves. The results are discussed in relation to the ecological habitats of the three species.Grassland Species Research Group Publication No.38.     

Glasshouse evaluation of the growth ofAlnus rubra andAlnus glutinosa on peat and acid brown earth soils when inoculated with four sources ofFrankia

The effects of soil type (an acid peat and 2 acid brown earths) andFrankia source (3 spore-positive crushed nodule inocula and spore-negative crushed nodules containing the singleFrankia ArI5) on nodulation, N content and growth ofAlnus glutinosa andA. rubra were determined in a glasshouse pot experiment of two years duration. Plants on all soils required additional P for growth. Growth of both species was very poor on peat withA. glutinosa superior toA. rubra. The former species was also superior toA. rubra on an acid brown earth with low pH and low P content. Some plant-inoculum combinations were of notable effectivity on particular soils but soil type was the major source of variation in plant weight. Inoculation with crushed nodules containingFrankia ArI5 only gave poor infection of the host plant, suggesting that inoculation with locally-collected crushed nodules can be a preferred alternative to inoculation withFrankia isolates of untested effectivity. Evidence of adaptation ofFrankia to particular soils was obtained. Thus, while the growth of all strains was stimulated by mineral soil extracts, inhibitory effects of peat extracts were more apparent with isolates from nodules from mineral soils than from peat, suggesting that survival ofFrankia on peat may be improved by strain selection.     

Long-term changes in frequencies ofPythium iwayamai andP. paddicum in upland- and paddy-field soils

The long-term changes in the frequencies ofPythium iwayamai andP. paddicum in upland- and flooded paddy-field soils were compared. The frequencies of both species fell in both soils within the first 6 months after they had been buried. After 36 and 48 months,P. iwayamai showed higher frequency thanP. Paddicum in upland-field soil, whileP. paddicum showed higher frequency thanP. iwayamai in paddy-field soil. These findings indicate respectively higher colonization ability to organic matters ofP. iwayamai in upland-field soil andP. paddicum in paddy-field soil.This study was partly supported by a Grant-in-Aid (no. 63560048) from the Ministry of Education, Science and Culture, Japan.     

Methanogenium,a new genus of marine methanogenic bacteria,and characterization ofMethanogenium cariaci sp. nov. andMethanogenium marisnigri sp. nov.

A new genus of marine methanogenic bacteria and two species within this genus are described.Methanogenium is the proposed genus andMethanogenium cariaci the type species. Cells of the type species are Gram-negative, peritrichously flagellated, irregular cocci with a periodic wall surface pattern. Colonies formed by these bacteria are yellow, circular and umbonate with entire edges. The DNA base composition is 52 mol% guanine plus cytosine. Formate or hydrogen and carbon dioxide serve as substrates for growth. Cells ofMethanogenium marisnigri are of similar shape but smaller diameter thanM. cariaci. The colonies ofM. marisnigri are convex, and the DNA base composition is 61 mol % G+C. Formate or hydrogen and carbon dioxide are growth substrates. Sodium chloride is required for growth of both methanogens.Abbreviations SDS sodium dodecylsulfate - PIPES piperazine-N,N-bis (2 ethanesulfonic acid) - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid     

Comparison of four media for the isolation ofAspergillus flavus group fungi

Four agar media used to isolate aflatoxin producing fungi were compared for utility in isolating fungi in theAspergillus flavus group from agricultural soils collected in 15 fields and four states in the southern United States. The four media wereAspergillus flavus andparasiticus Agar (AFPA, 14), the rose bengal agar described by Bell and Crawford (BCRB; 3), a modified rose bengal agar (M-RB), and Czapek's-Dox Agar supplemented with the antibiotics in BC-RB (CZ-RB). M-RB was the most useful for studying the population biology of this group because it permitted both identification of the greatest number ofA. flavus group strains and growth of the fewest competing fungi. M-RB supported an average of 12% moreA. flavus group colonies than the original rose bengal medium while reducing the number of mucorales colonies and the number of total fungi by 99% and 70%, respectively. M-RB was successfully employed to isolate all three aflatoxin producing species,A. flavus, A. parasiticus andA. nomius, and both the S and L strains ofA. flavus. M-RB is a defined medium without complex nitrogen and carbon sources (e.g. peptone and yeast extract) present in BC-RB. M-RB should be useful for studies on the population biology of theA. flavus group.Abbreviations M-RB Modified Rose Bengal Agar - CZ-RB Czapeks Rose Bengal Agar - BC-RB Bell and Crawford's Rose Bengal Agar - AFPA Aspergillus flavus andparasiticus agar     

Persistence ofBacillus thuringiensis andBacillus cereus in soil supplemented with grass or manure

Summary Persistance of inocula ofBacillus thuringiensis spores, parasporal crystals, andBacillus cereus spores in soil supplemented with dried-grass or partly composted, dried-chicken manure (100 mg supplement per 900 mg soil,^–0.01 MPa water availability, 25°C) were monitored over a period of up to 64 days by dilution plating and bioassay with larvae ofPieris brassicae. The inoculantB. thuringiensis population increased 22 x in level in grass-supplemented soil, but declined in manure-supplemented soil to 0.22 x the original level. TheB. cereus inocula declined in both soil treatments to approximately 0.1 x the original level. Insecticidal activity of theB. thuringiensis parasporal crystal decreased exponentially in grass and manuresupplemented soils, with half-lives of approximately 9.5 and 8.5 days respectively.     

Absence of a role for lytic microorganisms in the decline of bacteria andSaccharomyces introduced into soil

The populations ofKlebsieila pneumoniae, Escherichia coli, Enterobacter aerogenes, andPseudomonas sp. fell following their addition to soil, but species lysing these gram-negative bacteria were not detected. The numbers ofStaphylococcus aureus andMicrococcus flavus fell by more than four orders of magnitude and ofSaccharomyces cerevisiae by more than two orders after their addition to soil. Organisms lysing these gram-positive bacteria were present in soil, but their numbers did not increase as a result of the additions. Lytic activity againstS. aureus was detected in soil filtrates, but this activity was not enhanced by inoculation of soil with the bacterium. Addition of cycloheximide to soil suspensions delayed the fall in abundance ofM. flavus but did not suppress the lytic populations. We conclude that lysis is not responsible for the decline of bacteria orS. cerevisiae added to soil.     

Bacteriolytic activities of the free-living soil amoebae,Acanthamoeba castellanii,Acanthamoeba polyphaga andHartmannella vermiformis

Bacteriolytic activities of axenically grown free-living soil amoebaeAcanthamoeba castellanii, Acanthamoeba polyphaga andHartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined. A spectrophotometric assay revealed that the specific bacteriolytic activities of bothAcanthamoeba species were higher as those of the threeHartmannella strains.Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus andPseudomonas fluorescens were more easily lysed than the other bacteria tested.Agrobacterium tumefaciens, Klebsiella aerogenes andSerratia marcescens were hardly affected at all by the amoebal bacteriolytic activities. Among the Gram-negative bacteria we observed differences in lysis sensitivity while the Gram-positive bacteria tested were sensitive to lysis. Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3–10 was performed to separate the bacteriolytic isoenzymes of amoebae. Bacteriolytic patterns were shown by using an activity assay in which lysis bands were formed in the agar/bacteria gel-overlay. The activity assay revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae. Distinct differences between typical pI points of bacteriolytic activities inAcanthamoeba andHartmannella were shown. Bacteriolytic activities ofHartmannella were more pronounced and observed in the isoelectric points (pI) range of 4.0–9.3 while forAcanthamoeba the range was pI 4.5–8.9.Abbreviations IEF isoelectric focusing - PAA-IEF polyacrylamide-isoelectric focusing - CCAP culture collection of algae and protozoa - AS amoeba saline medium - pI isoelectric points     

Inhibition of fungi,actinomycetes and bacteria by Stachybotrys atra

The effect ofStachybotrys atra on fungi, actinomycetes and bacteria was studied in agar culture. Of the 27 genera and 52 species of fungi, 6 species of actinomycetes and 5 genera and 10 species of bacteria tested,S. atra was found to inhibit 95.7 % fungi, all the actinomycetes and 83.3 % of bacteria.Aspergillus ustus, 2 unidentified species ofFusarium, Rhizobium trifolii andPseudomonas spp. were however not inhibited. In experiments in soil,S. atra inhibited the growth ofTrichoderma viride. S atra was ineffective whereT. viride andS. atra were inoculated simultaneously or whereT. viride was added 7 days earlier. Besides,S. atra was not found to controlMacrophomina phaseoli infection on cotton. Soil culture ofS. atra showed phytotoxic effect on cotton seedlings.S. atra produced a dark brown to black thermostable toxic metabolite in the medium. The culture filtrate retarded the growth ofM. phaseoli in agar culture. Despite the importance ofS. atra as a fungistatic agent with wide antimicrobial spectrum, the use ofS. atra as a protectant against plant pathogens seems to be limited because of its phytotoxicity on cotton.     

Differentiation ofMelampsora rust species on willows in Japan using PCR-RFLP analysis of ITS regions of ribosomal DNA

To develop a reliable method for identifyingMelampsora species parasitic on willows in Japan, we differentiated 10Melampsora species by PCR-RFLP analysis. Internal transcribed, spacer (ITS) regions, including 5.8S ribosomal DNA, of 63 collections of 10Melampsora species and 4 collections of unidentified species were amplified by PCR. The fragments from the 67 collections varied in size (approximately 880 bp, 860 bp and 840 bp). The restriction sites in the amplified DNA fragments were mapped after the RFLP analysis using four restriction enzymes,Dra I,EcoRI,SspI andTaqI. All the collections were divided into 11 RFLP types. In the 6 species,M. caprearum, M. epiphylla, M. kamikotica, M. larici-urbaniana, M. microsora andM. yezoensis, the RFLP type was species-specific. The RFLP type ofM. chelidonii-pierotii andM. coleosporioides was identical. The collections ofM. epitea were separated into three RFLP types. One of these three types was identical with the type ofM. humilis. It is suggested that the PCR-RFLP analysis of ITS regions is a useful and reliable method for species identification ofMelampsora. Contribution No. 131, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Crossfostered drones ofApis cerana (Fabricius, 1793) andApis koschevnikovi (Buttel-Reepen, 1906) fly at their species specific mating times

Summary Reciprocal transfer of sealed drone brood between colonies ofApis cerana andApis koschevnikovi was successful and resulted in four colonies (two of each species) with a mixed drone population. Flights ofApis cerana drones occurred between 14.00 and 16.15 regardless whether they were in a conspecific or alien colony.Apis koschevnikovi drones also flew at their species specific time from 16.45 to 18.30. A variance estimation revealed that 99.4% of the total variance depended on the species of the drone. In contrast to theApis drone's general biological dependence upon the colony, crossfostered drones ofApis cerana andApis koschevnikovi showed an unexpected autonomy in chosing their mating flight time.     

Occurrence and survival ofAzospirillum spp. in temperate regions

In order to evaluate the suitability ofAzospirillum spp. as a crop inoculant in temperate regions, the natural occurrence, distribution and survival ofAzospirillum after seed inoculation in Belgian agricultural soils was studied.Azospirillum was present in most of the fields examined, but concentrations never exceeded 1000 cfu per g soil or per g roots. Under field conditions none of the known species was found to be localized inside the roots of barley, wheat, rye, maize or grasses. Also, the distribution ofA. brasilense SpBr 14 within the root system of hydroponic-grown wheat was studied by immunofluorescence. From the rhizosphere samples of the field crops investigated, a number of microaërophilic, diazotrophic bacteria were isolated and identified asA. lipoferum, found only on maize and grass roots, andA. brasilense, present under all crops. In contrast toA. brasilense, A. lipoferum was able to use different amino-acids and some derivatives as sole carbon and nitrogen sources. Use of a peat-based seed inoculant resulted in the establishment of theAzospirillum spp. in the rhizosphere of field-grown winter barley and winter wheat. The established population survived during winter without appreciable change in numbers, but there was no indication of active growth during spring or summer.     

Accumulation of nutrients by eucalyptus seedlings from acidic and calcareous soils of the cerrado region of Central Brazil

Summary The accumulation of nutrients by seedlings ofEucalyptus grandis andE. saligna from a latosol and two other fertile soils was compared. Both species performed well in a calcareous soil and an acidic but fertile gallery forest soil but not in the dystrophic latosol.E. grandis accumulated more nutrients from the calcareous soil thanE. saligna. In the other two soils these two species showed similar growth and nutrient accumulation. In spite of the high acidity, manganese toxicity did not seem to be a limiting factor in the latosol or the gallerty forest soil. Correlations among the shoot concentrations were not always the same as those among the total contents of the nutrients in the aerial biomass.     

Nest site selection by fire ant queens

Summary We examined nest site selection by foundresses of the polygyne form ofSolenopsis invicta. In the laboratory, foundresses were allowed to nest in control soil vs. soil inoculated with refuse from a colony ofPheidole dentata, a natural enemy. In a second experiment foundresses chose between control andS. invicta soil. More foundresses (100%) avoided the soil of a natural enemy than the soil of a conspecific colony (79%) that may accept them into the nest. Foundresses from this polygyne ant appear to avoid other colonies in general, but avoid heterospecific colonies more.     

Competition between soil bacteria and fusarium

Summary The phenomenon of competition has been characterized in liquid medium and sterile soil systems using a variety of soil bacteria andFusarium oxysporum f.cubense as test organisms. For most of the bacteria, suppression of the fungus was the result of a biologically induced nitrogen deficiency, this effect being reversed by the addition of excess inorganic nitrogen. High populations of competitors were found in two soils of neutral pH, but no isolates competed in the acid San Alejo loam.Agrobacterium radiobacter was able to compete when San Alejo loam was limed to about pH 6.6. Inhibition of the fungus by a number of gram-positive, spore-forming rods could not be accounted for in terms of competition for nutrients or by antibiotic production in artificial media.The competitive ability ofA. radiobacter when tested in twelve Central American soils was found to be related to pH in acid and neutral environments but was correlated with texture, organic-matter content and total nitrogen in soils of intermediate pH. In all soils where inhibition occurred, the competitive effect was overcome by additions of inorganic nitrogen. Excluding the group ofBacillus spp., the competitive ability of soil bacteria was related to the ability to develop in the absence of amino acids and growth factors but could not be correlated with growth rates of the bacteria in soil or liquid medium.It is suggested that competition for nutrients is a significant means of ecological control among members of the soil microflora and, together with competitive interactions for space and oxygen, may be the major factors governing the biological control of soil-borne fungi.The investigation was supported in part by a grant from the United Fruit Company. Agronomy Paper No. 471.