Bacterial and fungal metabolites of rhizospheric microflora of cotton antagonistic to Rhizoctonia solani Kühn

Summary Growth response ofRhizoctonia solani Kühn, the damping-off fungus, to metabolites of selected antagonistic rhizospheric bacteria and fungi of some Egyptian cotton varieties, namely, two strains ofBacillus subtilis Cohn,Aspergillus terreus Thom, andAspergillus flavus Link produced in culture media containing nitrate- or ammonium-nitrogen sources, proved the potency ofB. subtilis metabolites in inhibitingR. solani mycelial growth whether from nitrate- or ammonium-nitrogen culture media. Metabolite filtrates ofB. subtilis (II) are more potent than those ofB. subtilis (I). Increasing concentration of bacterial metabolite filtrates resulted in a decreased mycelial dry weight ofR. solani. The bacterial inhibitory factor forR. solani mycelial growth is partially affected by heat. Metabolite filtrates ofA. terreus from nitrate-nitrogen are slightly more potent than from ammonium-nitrogen culture media while an opposite relation is evident withA. flavus metabolites. Growth responses ofR. solani to different experimental dilutions of metabolite filtrates ofA. terreus andA. flavus proved the intervention of the nutritive factor in witholding growth of the damping-off fungus.     

Identification ofRhizobium strains and evaluation of their competitiveness

Six strains ofRhizobium leguminosarum bv.viciœ, three strains ofBradyrhizobium japonicum and three strains ofRhizobium fredii were evaluated by the polymerase chain reaction (PCR). The possibility of identification of individual rhizobial strains and the way of product analysis were verified. The result of amplifications proved rich spectra along the whole length scale. Numerous identical bands could be found in related strains. Verification of the expected identity of some strains confirmed the applicability of this method for identification of individual bacterial strains of generaRhizobium andBradyrhizobium. Furthermore, competitiveness of two strains ofR. leguminosarum bv.viciœ against the native rhizobial population was evaluated in a pot experiment. When using PCR as the identification method, the presence of the strains in host plant's nodules was ascertained after inoculation by different rates of inoculum strains. With increasing the inoculum rate, the presence of inoculum strains in pea nodules also increased. On the basis of mathematical models by Amarger and Lobreu the competitiveness of the mentioned strains was estimated at certain inoculum rates. Both tested strains displayed a higher competitiveness than native rhizobia in the soil used. As they are also effective N₂ fixators (one strain being HUP⁺), one may expect successful field inoculations with them.     

Extracellular polysaccharides of the genusRhizobium

This paper reports an investigation of the extracellular polysaccharides produced by 26 strains ofRhizobium andAgrobacterium. Strains ofRhizobium leguminosarum andR. phaseoli produced a water-soluble polysaccharide containing glucose, glucuronic acid and 4-0-methylglucuronic acid. These substances were also identified in the polysaccharide of a single strain fromLotus uliginosus. Glucose was the only detectable component in the polysaccharide produced by strains ofAgrobacterium radiobacter andA. tumefaciens. The polysaccharides obtained from slow-growing rhizobia were not freely water-soluble. Glucose, mannose, rhamnose, galactose and 4-0-methylglucuronic acid were identified as components of this extracellular material.These results are related to previous studies on rhizobial taxonomy and to the infection process in legumes.     

Influence of arbuscular-mycorrhizal fungi, Rhizobium meliloti strains and PGPR inoculation on the growth of Medicago arborea used as model legume for re-vegetation and biological reactivation in a semi-arid mediterranean area

Medicago arborea can be used for re-vegetationpurposes under semiarid conditions. These woody legumes have the ability toforman association with arbuscular mycorrhizal (AM) fungi and rhizobial bacteria,which can be maximised by microorganisms producing certain stimulatingmetabolites acting as plant growth promoting rhizobacteria (PGPR). The effectsof single and combined inoculations using microorganisms with different andinteractive metabolic capacities, namely three Glomusspecies, two Rhizobium meliloti strains (a wild type, WTand its genetically modified derivative GM) and a plant growth promotingrhizobacterium, (PGPR), were evaluated. All three inoculated AM fungi affectedMedicago growth in different ways. Differences weremaintained when soil was co-inoculated with each of the rhizobial strains (WTorGM) and the PGPR. Mycorrhizal fungi were effective in all cases, but the PGPRonly affected plant growth specific microbial situations. PGPR increased growthof G. mosseae-colonised plants associated withRhizobium WT strain by 36% and those infected byG. deserticola when associated with the rhizobial GMstrainby 40%. The most efficient microbial treatments involved mycorrhizalinoculation, which was an indication of the AM dependency of this plantspecies.Moreover, PGPR inoculation was only effective when associated with specificmycorrhizal endophytes (G. mosseae plus WT andG.deserticola plus GM rhizobial strain). The reduced root/shoot (R/S)ratio resulting from PGPR inoculation, was an indication of more effective rootfunction in treated plants. AM colonisation and nodule formation wereunaffectedby the type of AM fungus or bacteria (rhizobial strain and/or PGPR). AM fromnatural soil were less infective and effective than those from the collection.The results supported the existence of selective microbial interactionsaffecting plant performance. The indigenous AM fungi appeared to be ineffectiveand M. arborea behaved as though it was highly dependentonAM colonisation, which implied that it must have a mycorrhizal association toreach maximum growth in the stressed conditions tested. Optimum growth ofmycorrhizal M. arborea plants was associated with specificmicrobial groups, accounting for a 355% increase in growth overnodulatedcontrol plants. The beneficial effect of PGPR in increasing the growth of awoody legume, such as M. arborea under stress, was onlyobserved with co-inoculation of specific AM endophytes. As a result of theinteraction, only shoot biomass was enhanced, but not as a consequence ofenhancing of the colonising abilities of the endophytes. The growthstimulation,occurring as a consequence of selected microbial groups, may be critical anddecisive for the successful establishment of plants under Mediterraneanclimaticand soil conditions.     

Colonization ofRetama raetam seeds by fungi and their significance in seed germination

Examination by scanning electron microscopy and incubation on potato-dextrose agar medium showed that dry seeds ofRetama raetam were externally free of fungi. When planted in sandy loam soil, the seeds become colonized with eleven soil-borne fungal species. The fungi were isolated on cellulose agar, pectin agar and lignin agar media.Aspergillus flavus, A. niger, A. fumigatus, Penicillium capsolatum andFusarium oxysporum had broad occurrence and were recovered on all the three media. The production of hydrolytic enzymes by the isolated fungi depends on the substrate and species.Penicillium capsolatum, P. spinulosum andA. niger had wide enzymatic amplitude and they were able to produce cellulolytic, pectolytic and lignolytic activities on corresponding substrates as well as on seed-coat-containing media. The lignolytic activities of the isolated species exceptChaetomium bostrychodes andTrichoderma viride were enhanced by applying the seed-coat materials as C- source rather than lignin. SoakingR. raetam seeds in culture filtrates of most of the fungi grown on seed-coat-supplemented media induced a pronounced and distinct stimulating effect on seed germination. The most effective filtrates were those ofP. capsolatum, P. spinulosum andSporotrichum pulverulentum.     

Interactions between<Emphasis Type="Italic"> Trichoderma pseudokoningii</Emphasis> strains and the arbuscular mycorrhizal fungi<Emphasis Type="Italic"> Glomus mosseae</Emphasis> and<Emphasis Type="Italic"> Gigaspora rosea</Emphasis>

The interaction between Trichoderma pseudokoningii (Rifai) 511, 2212, 741A, 741B and 453 and the arbuscular mycorrhizal fungi Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe BEG12 and Gigaspora rosea Nicolson & Schenck BEG9 were studied in vitro and in greenhouse experiments. All T. pseudokoningii strains inhibited the germination of G. mosseae and Gi. rosea except the strain 453, which did not affect the germination of Gi. rosea. Soluble exudates and volatile substances produced by all T. pseudokoningii strains inhibited the spore germination of G. mosseae. The germination of Gi. rosea spores was inhibited by the soluble exudates produced by T. pseudokoningii 2212 and 511, whereas T. pseudokoningii 714A and 714B inhibited the germination of Gi. rosea spores by the production of volatile substances. The strains of T. pseudokoningii did not affect dry matter and percentage of root length colonization of soybean inoculated with G. mosseae, except T. pseudokoningii 2212, which inhibited both parameters. However, all T. pseudokoningii strains decreased the shoot dry matter and the percentage of AM root length colonization of soybean inoculated with Gi. rosea. The saprotrophic fungi tested seem to affect AM colonization of root by effects on the presymbiotic phase of the AM fungi. No influence of AM fungi on the number of CFUs of T. pseudokoningii was found. The effect of saprotrophic fungi on AM fungal development and function varied with the strain of the saprotrophic species tested.     

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.     

Antigenic affinities of the root-nodule bacteria of legumes

Antisera prepared against 58 strains of root-nodule bacteria and against 16 strains belonging to the genusAgrobacterium were tested against 113 strains ofRhizobium, 20 strains ofAgrobacterium and 20 strains of other, possibly related, bacteria.Three serologically distinct groups of root-nodule bacteria were noted: (1)Rh. trifolii, Rh. leguminosarum andRh. phaseoli; (2)Rh. lupini, Rh. japonicum andRhizobium spp.; (3)Rh. meliloti. Strains ofRh. meliloti showed serological affinities withA. radiobacter andA. tumefaciens. All groups showed wider flagellar than somatic agglutination, and many different serotypes were apparent.The groupings obtained from this investigation are compared with those derived from other taxonomic studies, and the use of serological methods in rhizobial classification is discussed.     

The effect of volatile and gaseous metabolites of swelling seeds on germination of fungal spores

Effects of volatile and gaseous metabolites of swelling seeds of pea, bean, wheat, corn, cucumber, tomato, lentil, carrot, red pepper and lettuce on germination of spores of five genera of fungi were found to depend rather on the fungal than on the plant genus. Germination of spores ofBotrytis cinerea, Mucor racemosus andTrichoderma viride was most severely inhibited. Spores ofVerticillium dahliae were less sensitive and germination of spores ofFusarium oxysporum was inhibited only in two cases. On the other hand, exudates of pea and bean stimulated germination of spores ofFusarium oxysporum. Also spores ofTrichoderma viride germinated better in an atmosphere enriched with exuded metabolites of swelling lettuce seeds. When carbon dioxide produced by the swelling seeds was absorbed in potassium hydroxide, spores ofTrichoderma viride andVerticillium dahliae did not germinate at all, the inhibitory effects of volatile and gaseous exudates on germination of spores ofMucor racemosus were accentuated, and also the percentage of germinated spores ofFusarium oxysporum decreased. Germination of spores ofBotrytis cinerea was not influenced. Absorption of volatile and gaseous metabolites in a solution of potassium permanganate decreased in most cases their inhibitory effects, particularly inBotrytis cinerea.     

Growth of dimorphic human pathogenicfungi on media containing cycloheximide and chloramphenicol

Summary Studies of 24 strains ofBlastomyces dermatitidis confirmed previously published results that the yeast-phase of this fungus is more sensitive than the mycelial-phase to cycloheximide and chloramphenicol.Studies of 5 strains each ofHistoplasma capsulatum, Paracoccidioides brasiliensis andSporotrichum schenckii show that that these species also have a similar yeast-phase mycelial -phase sensitivity differential in regard to these antibiotics.A cycloheximide resistant strain ofB. dermatitidis was developed from a sensitive strain.The experimental results support the general practice of using 0.5 mg/ml cycloheximide and 0.05 mg/ml chloramphenicol in media for the isolation of the four fungi at 25° C. The results indicate, however, that some strains would not be recovered at 37° C with similar concentrations of these antibiotics.It is recommended that a concentration of not more than 0.2 mg/ml chloramphenicol should be used to preserve sputum which is subsequently to be cultured forB. dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis orS. schenckii.     

Pectinolytic enzymes associated with the soft rots ofCitrus sinensis caused byAspergillus aculeatus andBotryodiplodia theobromae

Botryodiplodia theobromae andAspergillus aculeatus causing soft rots ofCitrus fruits were grown on wheat offal medium. The filtered washings of the wheat offal cultures were then assayed and found to contain pectinmethylesterases (PME) and polygalacturonases (PG). The two pectinases were also detected in large quantities from the filtrates of rotted fruits ofCitrus sinensis incited by each of the two test fungi. Two enzyme filtrates, one from rotted fruits ofCitrus sinensis and the other made up of pectinases from fungal cultures were introduced onto sterilised degreened fruits. Each of the two filtrates induced rot symptoms similar to those incited by the test fungi.Aspergillus aculeatus produced less PME and more PG thanBotryodiplodia theobromae.     

Thiamine pyrophosphate,a growth factor forTreponema vincentii andTreponema denticola

Growth ofTreponema vincentii N-9 in complex media was stimulated by culture filtrates ofT. minutum. Thiamine pyrophosphate (TPP) in nanogram quantities could substitute for the stimulatory factor inT. minutum culture filtrates. Characterization of the filtrates indicated that the stimulatory factor was similar to TPP in heat stability and ultrafiltration properties. The oral treponemesT. vincentii andT. denticola required TPP for maximum growth, whereas the genital treponemesT. minutum, T. phagedenis, andT. refringens do not require TPP. The presence of TPP activity in culture filtrates of the genital treponemes was confirmed by using a strain ofNeisseria gonorrhoeae auxotrophic for TPP. TPP activity was not found in culture filtrates of oral treponemes. Culture filtrates of 13 of 14 species representing eight genera found in the microbial flora of the oral cavity also contained TPP activity.     

Penicillin production and its mode of inheritance inAspergillus nidulans

Previous workers have shown that some strains ofAspergillus nidulans produce penicillin-like substances. In the present studies, shake-flask cultures of 101 wild-type strains ofA. nidulans, representatives of 18 different heterokaryon-compatible groups, were examined and filtrates of most found to inhibit the growth of a strain ofBacillus subtilis sensitive to penicillin, although members of two of these groups had no detectable antibiotic activity. Five strains with antibacterial properties were chosen for detailed investigation as well as two genetically labelled derivatives obtained from one of these after ultraviolet light treatments; one derivative had a similar antibiotic yield to its original wild-type parent but the other was selected as having increased antibiotic yield. The antibiotic produced by these seven strains was by all tested criteria, including chromatographic and electrophoretic behaviour, indistinguishable from penicillin. A heterokaryon test between the two mutants indicated that antibiotic productivity was under nuclear control.     

Lysogeny among mycobacteria

Our investigations to detect naturally lysogenic strains of mycobacteria were limited to 1 strain ofMycobacterium smegmatis, 4 strains ofMycobacterium borstelense var.niacinogenes, and to 5 strains ofMycobacterium marinum (Syn:Mycobacterium balnei), all together 10 strains. They were chosen because as a sign of lysis they secrete a large quantity of cytoplasmatic components (nucleic acids proteins, amino acids etc.) into the fluid medium (for instance phosphate buffer), in which they are suspended. In a first series of experiments culture filtrates were tested on 84 strains of slowly and rapidly growingMycobacterium species as indicator strains. Using this method free phage particles were only found in the culture filtrate of 1 strain,Mycobacterium smegmatis SN 46, isolated from a patient with achalasia. Phage particles could not be found in the filtrates of the other 9 probably lysogenic strains. In a second series of experiments more closely related indicator strains were used. The 10 probably lysogenic strains were cultured in bovine serum or antiphage-antiserum containing medium and single selected colony cultures a small part of which showed sensitivity to the filtrates. The released and adapted phages, designated as B₂₄, B₃₀, B₃₂, B₃₃, B₃₄ and B₃₅ have a very narrow host range. The plaques are very small and turbid. On electron micrographs the temperate phages B₂₄, B₃₀ and B₃₅ exhibit the typical head-tail morphology. The head of the temperateborstelense var.niacinogenes phage B₃₀ is 45 nm in diameter, the tength of tail is about, 120nm. The average dimensions of the long head ofsmegmatis phage B₂₄ are 40 × 80 nm, the tail is about 160 nm long. The balnei phage B₃₅ is very similar morphologically to phage B₃₀. The head is about 50 nm in diameter, the length of tail about 160 nm. The phage sensitive variants are not “carrier” strains. Their phage sensitivity is not a stable property. After several culture passages in serum-free medium the variants regain their phage immunity completely and release phages like the lysogenic parent strains. The sensitive variants must therefore be considered to be also lysogenic. TheMycobacterium borstelense var.niacinogenes phages are serologically very related. Dedicated to Academician Ivan Málek on the occasion of his 60th birthday     

Ectomycorrhizal fungal species and strains differ in their ability to produce free and conjugated polyamines

Production of free and conjugated polyamines by one strain of Laccaria proxima (Boud.) Maire, three strains (H, O, K) of Paxillus involutus (Batsch) Fr., and one strain of Pisolithus tinctorius was studied in vitro. Spermidine (Spd) was the main polyamine in the 4-week-old mycelium of all the fungi. It was mainly present in the free form, but it also occurred in conjugated forms. Paxillus involutus strain H released large amounts of free putrescine (Put), and the Pisolithus tinctorius released a compound probably related to cadaverine (Cad). On the other hand, these two fungi contained less conjugated polyamines than the other fungi. In addition to the amounts, the forms (perchloric acid soluble and insoluble) of conjugated polyamines in the mycelium varied between species and strains. L. proxima contained nearly as much insoluble conjugated Spd as free Spd, whereas Paxillus involutus strains O and K contained relatively large amounts of soluble conjugated Spd. The results suggest that ectomycorrhizal fungal species and strains differ in their ability and need to produce conjugated polyamines. The small amounts of soluble conjugated polyamines found in the culture filtrates indicate that some specific conjugated polyamines may be involved in polyamine translocation across the plasma membrane.     

Metabolites of rhizobacteria antagonistic towards fungal plant pathogens

Biological control of insect, plant pathogens and weeds is the only major alternative to the use of pesticides in agriculture and forestry. A double-layer technique was used for isolation of antagonistic bacteria from rhizosphere against plant pathogenic fungi. Four potential rhizobacteria was selected in dual culture plate method based on their antifungal activity against several soil-borne fungal plant pathogens. The selected rhizobacteria, identified based on their morphological, biochemical and molecular traits, belong to the species of fluorescentPseudomonas (SAB8, GM4) andBacillus (A555, GF23). The active antifungal metabolites produced by these strains in culture filtrates were tested for the growth inhibition ofFusarium semitectum used as test fungus. The active fraction of antifungal metabolite/(s) from fluorescentPseudomonas (SAB8, GM4) and their effects on hyphal growth were observed under microscope. Two kinds of alterations were detected: inhibition of hyphal tip elongation and an extensive branching of hyphae with closer septa.     

Production of antifungal metabolites by the ectomycorrhizal fungusPisolithus tinctorius strain SMF

Summary An ectomycorrhizal fungus,Pisolithus tinctorius strain SMF, isolated from a basidiocarp removed from the roots of a recently fallen old growth fir in the Smoky Mountains of Tennessee, was characterized for its in vitro production of antifungal metabolites. On solid mediumP. tinctorius SMF strongly inhibited growth of strains ofFusarium solani, Geotrichum candidum, Phanerochaete chrysosporium, andVerticillium dahliae, all species known to be plant pathogens. Evidence from paired colony growth inhibition studies on agar plates indicated that production of antifungal agents byP. tinctorius SMF may be enhanced by close physical contact with other fungi. The antifungal activity ofP. tinctorius SMF was much greater than that of several culture collection strains ofP. tinctorius. The culture collection strains either showed no or very limited activity. The antifungal activity was associated with an apparently inducible metabolism ofP. tinctorius SMF and with the production of darkly colored water soluble phenolic metabolites. Small scale fermentation studies showed that the phenolics are readily producible by submerged culture fermentation. This is the first report of submerged culture production of antifungal metabolites by an ectomycorrhizal fungus.     

Effect of rhizosphere fungi on seed germination

Summary The effect of culture filtrates of some common rhizosphere fungi on the seed germination ofDichanthium annulatum, Bothriochloa pertusa andSetaria glauca was studied. Surface sterilized seeds were soaked in the fungal culture filtrates for 24 hours and after several washings in distilled water transferred to moist filter paper in Petri dishes and the percentage germination recorded. No significant inhibition or stimulation of germination was noticed in any of the treatments. The only exception wasTrichoderma viride which inhibited the germination ofD. annulatum seeds to some extent.     

Development,persistence and regeneration of foraging ectomycorrhizal mycelial systems in soil microcosms

Development of extraradical mycelia of two strains each of Paxillus involutus and Suillus bovinus in ectomycorrhizal association with Pinus sylvestris seedlings was studied in two dimensions in non-sterile soil microcosms. There were significant inter- and intra-specific differences in extraradical mycelial growth and morphology. The mycelial systems of both strains of P. involutus were diffuse and extended more rapidly than those of S. bovinus. Depending on the strain, P. involutus mycelia were either highly plane filled, with high mass fractal dimension (a measure of space filling) or sparse, low mass fractal dimension systems. Older mycelial systems persisted as linear cords interlinking ectomycorrhizal tips. S. bovinus produced either a mycelium with a mixture of mycelial cords and diffuse fans that rapidly filled explorable area, or a predominately corded mycelium of minimal area cover. In the soil microcosms, mass fractal dimension and mycelial cover tended to increase with time, mycelia encountering litter having significantly greater values. Results are discussed in terms of the ecology of these fungi, their foraging activities and functional importance in forest ecosystems.     

Pectolytic enzymes activity and pathogenicity ofGaeumannomyces graminis var.tritici and related Phialophora-like fungi

Gaeumannmyces graminis var.tritici (Ggt), Phialophora sp. (lobed hyphopodia) andPhialophora graminicola vere grown in a liquid medium with pectin and on autoclaved wheat roots (root media) and the activity of pectolytic enzymes in culture filtrates was measured. Most strains of the fungi exhibited polygalacturonate trans-eliminase activity but no pectin methylesterase activity was detected.Ggt polygalacturonase was found in culture filtrates from all the media used whilePhialophora sp. did not exhibit activity of this enzyme in the unbuffered root media. No polygalacturonase activity was demonstrated forP. graminicola. A correlation was found (r=0.548) betweenin vitro polygalacturonase activity and the pathogenicity ofGgt to wheat seedlings.