Interactions between Onychiurus armatus and Trichoderma harzianum in take-all disease suppression in a simple experimental system

In a simple experimental system with wheat plants, interactions between the collembolan Onychiurus armatus and Trichoderma harzianum, a soil-borne fungus parasite of several plant pathogenic fungi, were studied in the presence of Gaeumannomyces graminis var. tritici, one of the most important foot and root fungal pathogens of cereals world-wide. Trichoderma harzianum was used according to two different modalities: fungal inoculum applied to seed or mixed with substrate. The isolate of T. harzianum proved to be ineffective against the pathogenic fungus, independent of the application modality, as shown in the experiments where this fungus was used alone, whereas Collembola used alone significantly reduced disease severity. However, the mode by which T. harzianum was applied significantly influenced the disease control ability of Collembola. In fact, only when T. harzianum was coated to seed were springtails able to reduce the disease caused by G. graminis var. tritici.     

Fungal communities in fallow soil before and after amending with pine sawdust

Pine sawdust (9 kg m⁻²) was ploughed into soils, intended for future forestry plantations, that had been left for 3 or 6 years after previously being used for agriculture. Two years after the amendment, soil pH had changed in both sites and the C:N ratio had increased. Total fungal populations decreased after the treatment. A small decrease in the number of species was not statistically significant. Trichoderma harzianum, an important biological control fungus, increased considerably after sawdust application whilst some fungi, such as Penicillium spp., Pseudogymnascus roseus and partially Mucorales decreased. The practical implications of the increase in T. harzianum density for the health of the young forests are discussed.     

Population dynamics of soil microorganisms associated with fungicide-dusted caladium seedpieces

The effects of three fungicidal dusts (captan ; a mixture of benomyl, thiram and streptomycin sulfate; and a mixture of chloroneb, thiram and streptomycin sulfate) on the short-term populations dynamics of fungi and bacteria associated with caladium seedpieces planted in raw muck soil were investigated. Both germinating seedpieces and seedpieces which were decomposing after the removal of eyes were used. To determine microbial populations, dilutions of a comminuted suspension of a seed-piece and surrounding soil were plated on selective media at 0,2,4, 8 and 12 weeks after seedpieces were planted. Addition of seedpieces to soil resulted in increases in populations in the following approximate chronological order: fluorescent Pseudomonas spp, Pythium spp, total bacteria, Fusarium spp and other genera of fungi. Most organisms assayed reached higher populations on decomposing than on germinating seedpieces. The effects of the fungicidal dusts on populations of microorganisms associated with seedpieces were not related to changes in populations that followed incorporation of the dusts into soil without seedpieces. The fungicidal dusts reduced total fungal population increases on germinating, but not on decomposing, seedpieces. Bacterial population increases were similar for all treatments of germinating seedpieces, but were prolonged on decomposing, fungicide-dusted seedpieces, as compared to controls.     

Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum

The filamentous fungi Trichoderma spp. is currently developed as biocontrol agents against many plant pathogens. Recent studies have shown that these fungi are able to infect nematode eggs and juveniles. In this research, biological control of root-knot nematode (Meloidogyne javanica) by Trichoderma harzianum BI was investigated in greenhouse and laboratory experiments. Results showed that different concentrations (10²–10⁸ spores/ml) of T. harzianum BI decreased nematode infection and other parameters significantly, compared to control. T. harzianum BI was able to penetrate nematode egg mass matrix and significantly decreased nematode egg hatching level. Specific activities of resistance-related enzymes, namely peroxidase (POX), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) increased significantly in T. harzianum BI inoculated plants. Maximum activities of POX, PPO and PAL were observed at the 5, 5 and 6 days after inoculation, respectively. Chitinase activity was also increased in culture filtrates of T. harzianum BI grown on wheat bran moistened with salt solution supplemented with colloidal chitin or nematode eggs. Maximum activity of chitinase was recorded at the 4 days after inoculation, in media supplemented with colloidal chitin (1.15 U/min per ml) and nematode eggs (0.85 U/min per ml). Results suggested that direct parasitism of eggs through the increase in extracellular chitinase activity, which would be indicator of eggs infection capability, and inducing plant defense mechanisms leading to systemic resistance are two main suppression mechanisms used by T. harzianum BI against nematode.     

APPLICATION OF TRICHODERMA HARZIANUM T22 AS A BIOFERTILIZER POTENTIAL IN MAIZE GROWTH

This study was conducted to investigate the effect of Trichoderma harzianum (T22) on the growth and development of maize (Zea mays) plants. T. harzianum was applied to the grains in two different treatments, either by inoculating the soil with air-dried mycelia of T. harzianum or by treating the seeds with different concentrations of the metabolic solution (as the culture supernatant of T. harzianum) before sowing. Comparisons were made between the two treatments to determine if any beneficial effects for improving the growth of maize plants occurred. Results revealed that application of the highest concentration of air-dried mycelia and all concentrations of the metabolic solution of T. harzianum caused increases in all measured parameters which include growth parameters, chlorophyll content, starch content, nucleic acids content, total protein content and phytohormone content of maize plants but the magnitude of these increases was much more pronounced in case of treating the seeds with the metabolic solution of T. harzianum.     

Interactions between the arbuscular mycorrhizal fungus Glomus mosseae and plant growth-promoting fungi and their significance for enhancing plant growth and suppressing damping-off of cucumber (Cucumis sativus L.)

This study investigated the interactions between the arbuscular mycorrhizal fungus (AMF) Glomus mosseae and the plant growth-promoting fungi (PGPF) Penicillium simplicissimum GP17-2 and Trichoderma harzianum GT3-2 in relation to their colonization of roots and the rhizosphere of cucumber (Cucumis sativus L.), and their effect on plant growth and suppression of the damping-off pathogen Rhizoctonia solani. Combined inoculation of T. harzianum with G. mosseae increased the percentage of AMF root colonization, but the presence of P. simplicissimum had no significant effect on mycorrhizal formation. The existence of G. mosseae decreased the population development of T. harzianum in and/or around the roots, however, growth of P. simplicissimum was not affected. Both PGPF isolates were effective in increasing the plant shoot and root dry weight, G. mosseae, however, was not. Nevertheless, co-inoculation of G. mosseae with T. harzianum synergistically enhanced plant growth. A comparable additive effect on plant growth was not observed when P. simplicissimum was combined with G. mosseae. Treating plants with the PGPF either simultaneously with the pathogen or prior to pathogen infection suppressed damping-off disease. G. mosseae was significantly effective in reducing disease severity when inoculated prior to the pathogen but not when inoculated simultaneously with the pathogen. Interestingly, the levels of disease protection achieved by the single inoculation of P. simplicissimum, T. harzianum or G. mosseae were significantly increased by combined inoculation of each PGPF with G. mosseae.     

Isotopic fractionation by saprotrophic microfungi: Effects of species, temperature and the age of colonies

Saprotrophic fungi represent an important resource for a number of fungivorous and omnivorous soil animals, but little is known about the patterns of isotopic fractionation by soil fungi. We grew five common species of saprotrophic microfungi in laboratory cultures on simple artificial substrate based on carbohydrates derived either from C3 or C4 plants. Fungal cultures were kept at 15, 20 or 25 °C. Isotopic composition of carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) in bulk fungal tissue was determined after 11, 21 and 32 days. The fractionation of carbon and nitrogen stable isotopes was species-specific, but generally did not differ in C3- and C4-based growth media. The Zygomycete Mucor plumbeus did not differ in δ¹³C from the carbon source used, though Ascomycetes (Alternaria alternata, Cladosporium cladosporioides, Trichoderma harzianum and Ulocladium botrytis) were depleted in heavy carbon relative to the carbon source by 0.5-0.9‰. Three species were significantly depleted in ¹⁵N relative to the sodium nitrate that was used as a single source of nitrogen. In all species, δ¹⁵N but not δ¹³C tended to increase with the age of fungal colonies. The effect of temperature on δ¹⁵N was weak and inconsistent in different species. In contrast, all fungi except T. harzianum accumulated more ¹³С at 25 °C than at 15 °C. The overall variation in the isotopic signatures of saprotrophic fungi growing in identical conditions reached 8‰ for δ¹⁵N and 2.5‰ for δ¹³C due to species-specific differences in the isotopic fractionation and the age of individual fungal colonies. This variation should be incorporated into the interpretation of the isotopic composition of fungivorous soil animals.     

Effect of some rare earth elements on the growth and lanthanide accumulation in different Trichoderma strains

Accumulation of rare earth elements (REE) in the soil may be due to the use of REE enriched fertilizers and to contamination by REE containing wastes. Although widely used in China for soil and foliar dressing of crops, little is known about the effect of REE applications on the soil microbial community. The effect of REE on the growth of biological control strains of Trichoderma atroviride and Trichoderma harzianum was investigated in vitro using either a mix of different REE containing different amounts of lanthanum, cerium, praseodymium, neodymium, gadolinium nitrate and lanthanum nitrate alone in comparison to treatments with potassium nitrate and water. In plate tests applied concentrations ranged from 0.1 mM to 300 mM for lanthanum and REE mix and from 0.1 mM to 900 mM for the potassium solution. In liquid culture tests applied concentrations ranged from 0.001 mM to 100 mM for lanthanum and REE mix and from 0.003 mM to 900 mM for the potassium solution. ICP-MS, TEM and TEM X-ray microanalysis were used to study the accumulation of REE in fungal biomass. All the Trichoderma strains showed a good tolerance to the presence of REE in the culture media. Some growth enhancing effects were observed in liquid cultures of T. harzianum strains but not in T. atroviride. Accumulation of REE in fungal biomass, both at intracellular level and in the extracellular matrix, was observed.     

Isotope ratio mass spectrometry identifies soil microbial biocontrol agents having trophic relations with the plant pathogen Armillaria mellea

An understanding of the types of interactions that take place between plant pathogens and other microorganisms in the natural environment is crucial in order to identify new potential biocontrol agents. The use of microorganisms labelled with stable isotopes is a potentially useful method for studying direct parasitisation of a given pathogen or assimilation of the pathogen's metabolites by microorganisms. A microorganism labelled with a stable isotope can be monitored in the environment and isotope ratio mass spectrometry can detect whether it is directly parasitised or its metabolites are used by other microorganisms. In this study, we isolated 158 different species of fungi and bacteria from soil and assayed their biocontrol potential against a plant pathogen (Armillaria mellea) by coupling a dual-culture test with mass spectrometry analysis of the ¹³C isotope in the microorganisms in presence of ¹³C-labelled A. mellea. The microorganisms affected the pathogen by means of antibiosis phenomena (total or partial inhibition of pathogen growth, alteration of its morphology) and by antagonism, probably resulting from competition for space and nutrients or from mycoparasitism. Isotope ratio mass spectrometry was used to identify direct trophic interactions between microorganisms and the pathogen as in dual cultures as in soil microcosms. Six fungi and one bacterium were found to display the best active trophic behaviour against the pathogenin dual cultures; three microorganisms were discarded due to their plant pathogen potential. Trichoderma harzianum, Pseudomonas fluorescens and Rhodosporidium babjevae were selected to carry out the experiments. T. harzianum inhibited pathogen development (rate of inhibition 80 ± 0.19%) and its δ ¹³C values increased (244.03 ± 36.70‰) in contact with ¹³C-labelled A. mellea. Lower levels of antagonism and correspondingly lower assimilation of ¹³C were detected in P. fluorescens and R. babjevae. Only T. harzianum maintained mycoparasitic activity in the soil microcosm, showing a δ ¹³C value of 1.97 ± 2.24‰ after one month in co-presence with the labelled pathogen. This study provides support for the use of isotope ratio mass spectrometry as an additional tool in screening for potential biocontrol agents.     

Wheat straw and cellulolytic fungi application increases nodulation, nodule efficiency and growth of fenugreek (Trigonella foenum-graceum L.) grown in saline soil

This study was performed to evaluate the ability of cellulolytic fungi and wheat straw incorporation to improve the nodulation, growth and nitrogen status of fenugreek grown in saline soils. NaCl addition to the growth medium at rates of 0.5 and 1% strongly decreased the enzymatic activity of the ten tested moulds. Three of these fungi, Aspergillus niger, Chaetomium globosum and Trichoderma harzianum, showed the highest enzymatic activity. The three moulds have the ability to degrade straw in the presence of NaCl and T. harzianum was the best straw degrader. Inoculating the plants with Rhizobium meliloti strain TAL1373 and cellulolytic fungi slightly promoted nodulation, growth and nitrogen accumulation when plants were grown with the addition of 0.5% NaCl when compared to plants inoculated with R. meliloti alone. However, application of wheat straw with cellulolytic fungi significantly enhanced growth, nodulation and nodule efficiency at 0.5 and 1.0% salinity. The greatest values of nodulation and growth parameters were obtained with a straw-Trichoderma harzianum combination. Cellulolytic fungi and wheat straw increased the concentration of Ca, Mg and K in the shoots and roots of plants. The increase in dry matter production and N content was mainly due to improved N₂ fixation reflected by enhanced formation and growth of nodules as well as nitrogenase activity. Received: 20 January 1997     

Mycorrhizae, biocides, and biocontrol 3. Effects of three different fungicides on developmental stages of three AM fungi

The effects of biocide use on nontarget organisms, such as arbuscular mycorrhizal (AM) fungi, are of interest to agriculture, since inhibition of beneficial organisms may counteract benefits derived from pest and disease control. Benomyl, pentachloronitrobenzene (PCNB) and captan were tested for their effects on the germination and early hyphal growth of the AM fungiGlomus etunicatum (Becker & Gerd.),Glomus mosseae (Nicol. & Gerd.). Gerd. and Trappe andGigaspora rosea (Nicol & Schenck) in a silty-clay loam soil placed in petri plates. Application of fungicides at 20 mg active ingredient (a.i) kg^?1 soil inhibited spore germination by all three AM-fungal isolates incubated on unsterilized soil for 2 weeks. However, fungicides applied at 10 mg a.i. kg^?1 soil had variable effects on AM-fungal isolates. Fungicide effects on germination and hyphal growth of G.etunicatum were modified by soil pasteurization and CO₂ concentration in petri plates and also by placing spores below the soil surface followed by fungicide drenches. Effects of fungicides on mycorrhiza formation and sporulation of AM fungi, and the resulting host-plant response, were evaluated in the same soil in associated pea (Pisum sativum L.) plants. Fungicides applied at 20 mg a.i. kg^?1 soil did not affect the root length colonized byG. etunicatum, but both benomyl and PCNB reduced sporulation by this fungus. Benomyl and PCNB reduced the root length colonized byG. rosea at 48 and 82 days after transplanting. PCNB also reducedG. mosseae-colonized root length at 48 and 82 days, but benomyl only affected root length colonized byG. mosseae at the earlier time point. Only PCNB reduced sporulation byG. mosseae, consistent with its effect on root length colonized by this fungus. captan reduced the root length colonized by G. rosea at 48 days, but not at 82 days, and reduced colonization byG. mosseae at 82 days, but not at 48 days. Captan did not affect sporulation by any of the fungi.G. rosea spore production was highly variable, but benomyl appeared to reduce sporulation by this fungus. Overall,G. etunicatum was the most tolerant to fungicides in association with pea plants in this soil, andG. rosea the most sensitive. Benomyl and PCNB were overall more toxic to these fungi than captan. Interactions of AM fungi and fungicides were highly variable and biological responses depended on fungus-fungicide combinations and on environmental conditions.     

Parasitism of sclerotia of Sclerotium rolfsii by trichoderma harzianum

The ability of Trichoderma harzianum isolate 203 to attack the soil-borne plant pathogen Sclerotium rolfsii is apparently connected with the production by the isolates of chitinase and β-(1,3)-glucanase inside the attacked sclerotia during parasitism.SEM and TEM micrographs show that the mycoparasite degraded walls of sclerotial cells and the attacked cells lost their cytoplasmic contents. It is assumed that T. harzianum utilizes sclerotial cell contents thus enabling it to sporulate intensively on the sclerotial surface and inside the digested cells.     

Interactions between Sclerotium rolfsii and Trichoderma spp: Relationship between antagonism and disease control

Ten isolates of Trichoderma spp were examined for their ability to antagonize growth and to parasitize mycelium of Sclerotium rolfsii (Sr-1) on agar media, to inhibit germination of sclerotia of S. rolfsii on natural soil plates and to sporulate on the sclerotia, and to protect bean seedlings against the pathogen in the greenhouse. A high negative correlation (r = ?0.844) was observed between plant stand in the greenhouse and sclerotial germination on soil plates but not with antagonism on agar plates. Three isolates of T. harzianum (Th-7, Th-20, WT-6) and one of T. hamatum (TRI-4) were especially effective in reducing sclerotial germination and controlling disease in the greenhouse. Three isolates of Trichoderma spp (WT-6, TMP, and TRI-4), effective in reducing sclerotial germination of isolate Sr-1, also prevented sclerotial germination in four out of five additional S. rolfsii isolates studied.     

Diversity of potential microbial parasites colonizing sclerotia of Macrophomina phaseolina in soil

The colonization of Macrophomina phaseolina sclerotia by microbial parasites was evaluated in unsterilized field soil at different levels of soil moisture (0,-5, and-10 kPa) and temperature (20, 30, and 40°C). The maximum colonization of sclerotia was recorded in soil held at-5 or-10 kPa at 30–40°C. Trichoderma harzianum isolate 25–92 and Pseudomonas fluorescens isolate 4–92 were recorded as potential sclerotial parasites, and they significantly (P=0.05) reduced the germination of sclerotia by 60–63%. Cells of P. fluorescens and buffer-washed conidia of T. harzianum were completely agglutinated at 28°C with crude agglutinin of M. phaseolina. The ability of different antagonists to parasitize the sclerotia were correlated with the agglutination ability of the antagonists.     

Effect of microbial culture filtrates on the growth of sal (Shorea robusta gaertn.) leaf litter fungi

Experiments were made on the nature of fungal competition on sal leaf-litter with emphasis on the antibiotic action of culture filtrates. The culture filtrates of Mortierella subtilissima, Aspergillus candidus, A. flavus, A. niger, Penicillium rubrum, Papulaspora sp., Staphylococcus aureus and Bacillus subtilis were found most effective in suppressing the growth of various leaf-litter fungi. Dominant fungi which displayed best competitive tolerance to the staling products of culture filtrates were Aspergillus flavus, A. niger, A. sclerotiorum, A. terreus and Trichoderma harzianum.     

Influence of a pyrethroid insecticide on soil fungi

The selective effect of the pyrethroid insecticide (Polytrin) on soil fungi was studied at three doses when incorporated in soil or in agar or liquid medium. Polytrin induced an inhibitory effect on soil fungi after 2, 5 and 40 days when applied at certain levels and a stimulatory effect after 20 days of treatment with the high dose. When incorporated in the agar medium, Polytrin decreased the total counts of fungi, particularly, Aspergillus and Penicillium at the high dose (10.0 ppm). In liquid medium, it showed no significant effect except in case of A. terreus which was significantly enhanced by the three doses.     

Effects of Single and Combined Inoculations with Azospirillum brasilense and Trichoderma harzianum on Seedling Growth or Yield Parameters of Wheat (Triticum vulgaris L., Giza 168) and Corn (Zea mays L., Hybrid 310)

Inoculation of wheat and corn grains with formulations of Azospirillum brasilense significantly increased seedling growth parameters of wheat and corn compared to untreated controls. Inoculation with Azospirillum and supplemental Trichoderma harzianum free or coimmobilized in calcium alginate resulted in significant increase in all plant growth parameters in addition to improving plant nutrient-content [phosphorus (P), potassium (K), and calcium (Ca)]. Grain treatments with T. harzianum alone or in a combination with A. brasilense were protected from invasion by Fusarium in a pot experiment. Nitrogen (N) fixation was investigated by A. brasilense free or double inoculated with T. harzianum in soil amended with different C-sources; also, phosphate solubilization was tested by these two organisms. Single and double inoculation with A. brasilense and/or T. harzianum improved wheat yield growth parameters in addition to seed protein; therefore, immobilized and coimmobilized formulations could be used as biofertilizer and biopesticide, and might be recommended to avoid the extensive use of the agrochemicals.     

施用生物有机肥抑制香蕉镰刀菌萎蔫病的研究

Fusarium wilt is one of the most serious diseases of banana plants caused by soil-borne pathogen Fusarium oxysporum f.sp. cubense(FOC). In this study a pot experiment was conducted to evaluate the effects of different bio-organic fertilizers(BIOs) on Fusarium wilt of banana, including the investigations of disease incidence, chitinase and β-1,3-glucanase activities of banana plants, and FOC populations as well as soil rhizosphere microbial community. Five fertilization treatments were considered, including chemical fertilizer containing the same N, P and K concentrations as the BIO(control), and matured compost mixed with antagonists Paenibacillus polymyxa SQR-21 and Trichoderma harzianum T37(BIO1), Bacillus amyloliquefaciens N6(BIO2), Bacillus subtilis N11(BIO3), and the combination of N6 and N11(BIO4). The results indicated that the application of BIOs significantly decreased the incidence rate of Fusarium wilt by up to 80% compared with the control. BIOs also significantly promoted plant growth, and increased chitinase andβ-1,3-glucanase activities by 55%–65% and 17.3%–120.1%, respectively, in the banana roots. The population of FOC in the rhizosphere soil was decreased significantly to about 104 colony forming units g-1with treatment of BIOs. Serial dilution plating and denaturing gradient gel electrophoresis analysis revealed that the application of BIOs increased the densities of bacteria and actinomycetes but decreased the number of fungi in the rhizosphere soil. In general, the application of BIOs revealed a great potential for the control of Fusarium wilt disease of banana plants.     

Limitations to using benomyl in evaluating mycorrhizal functioning

Arbuscular mycorrhizal (AM) grasses compete for nutrients with ectomycorrhizal (EM) pine in the southeastern United States. Our objective was to determine if benomyl could be used to selectively inhibit the function of AM and thereby reduce grass competition in the field. The effects of Benlate (active ingredient: benomyl) in the greenhouse and field were evaluated. No effect was observed on pine inoculated with Pisolithus tinctorius in the greenhouse. Colonized root length of benomyl-treated Zea mays L. plants inoculated with Glomus sp. in the greenhouse remained static over time and the response was not dose dependent at concentrations of 0, 20, 60 and 150kg benomyl ha^–1 equivalent. In contrast, colonization of nontreated plants increased over time. In the field, a minimal reduction of grass colonization was observed following four applications of benomyl ranging from 5 to 20kgha^–1. We conclude that benomyl can successfully inhibit development of AM fungi under controlled conditions in the greenhouse with no inhibitory effects on the EM fungus P. tinctorius; however, in the field several factors may interfere with the effect of benomyl on AM fungi. These factors include: (a) the presence of ground cover which obstructs penetration of the fungicide to the soil, (b) timing of application in relation to mycorrhizal development, and (c) the application method of benomyl, a soil drench being preferable to a foliar spray. Received: 30 September 1996     

Effect of mixing soil saprophytic fungi with organic residues on the response of Solanum lycopersicum to arbuscular mycorrhizal fungi

The effect of the dual inoculation with arbuscular mycorrhizal (AM) and saprophytic fungi and a combination of wheat straw and sewage sludge residues were studied by determining their effect on dry weight of tomato and on chemical and biochemical properties of soil. Incubation of organic residue (sewage sludge combined with wheat straw) with saprophytic fungi and plant inoculation with mycorrhizal fungi was essential to study plant growth promotion. Soil application of organic residues increased the dry weight of tomato inoculated with Rhizophagus irregularis. The greatest shoot dry mass was obtained when the organic residues were incubated with Trichoderma harzianum and applied to AM plants. However, the greatest percentage of root length colonized with AM in the presence of the organic residues was obtained with inoculation with Coriolopsis rigida. The relative chlorophyll was greatest in mycorrhizal plants regardless of the presence of either saprophytic fungus. The presence of the saprophytic fungi increased soil pH as the incubation time increased. Soil nitrogen and phosphorus contents and acid phosphatase were stimulated by the addition of organic residues, and contents of N and P. Total N and P content in soil increased when the organic residue was incubated with saprobe fungi, but this effect decreased as the incubation period of the residue with saprobe fungi increased. The same trend was observed for soil β‐glucosidase and fluorescein diacetate activities. The application of organic residues in the presence of AM and saprophytic fungi seems to be an interesting option as a biofertilizer to improve plant growth and biochemical parameters of soils.