Factors Affecting the Parasitic Activity of Gliocladium virens on Sclerotia of Sclerotinia sclerotiorum and a Note on its Host Range

Aspects of the biology of Gliocladium virens and parasitism of sclerotia of Sclerotinia sclerotiorum in soil were studied. G. virens parasitized and decayed sclerotia of S. sclerotiorum, S. minor, Botrytis cinerea, Sclerotium, rolfsii and Macrophomina phaseolina on laboratory media and caused a reduction in survival of sclerotia of S. sclerotiorum in soil. It was active over a broad range of soil moisture levels and over the entire agricultural soil pH range. The main factor limiting its use as a biological control agent was its temperature requirements.     

Germination of sclerotia of Botrytis tulipae, the cause of tulip fire

Sclerotia of Botrytis tulipae (Lib) Lind. buried in field soil germinated mainly in winter and early spring to produce conidiophores and conidia and then decayed. Except when buried very late in the year sclerotia germinated and decayed in the season following burial. In the laboratory, sclerotia from pure culture germinated in two ways. On sterile water-agar they formed colourless mycelium but no conidiophores; the rate of germination was greatest at 25 d?C. On unsterile filter paper and on soil they produced mainly conidiophores and conidia; the rate of germination was greatest at 5 d?C and was enhanced by previous cold treatment. It was unaffected by soil-moisture or pH. Primary infections of tulips arose from sclerotia placed in the soil but only when they were close to the shoot tip or developing stem. It is suggested that, because of their limited survival, soil-borne sclerotia are of importance only when tulips are planted in annual succession.     

Inhibition and Stimulation Effects in Communities of Wood Decay Fungi: Exudates from Colonized Wood Influence Growth by Other Species

The effects of exudates from uncolonized and from partly decayed beech wood on the extension rates of 16 later stage decay fungi were investigated. The partly decayed wood had been colonized by the pyrenomycete Eutypa spinosa, or the basidiomycetes Fomes fomentarius, Stereum hirsutum, and Trametes versicolor, all known as common early decay agents in European beech forests. Sterilized wood pieces were placed onto 0.5% malt agar, opposite to small agar plugs containing the test fungi. The latter showed very variable and species-specific growth responses to the various wood types. The presence of uncolonized wood stimulated extension rates in many species, whereas the four previously decayed wood types had variable stimulatory or inhibitory effects. Wood decayed by S. hirsutum resulted in reduced extension rate, delayed growth, or total inhibition in the majority of species, thus it is suggested that this species uses secondary metabolites in a defensive strategy. A single species was, however, stimulated in the presence of S. hirsutum-decayed wood. In contrast, the presence of wood decayed by F. fomentarius was stimulatory to 45% of the species. The other previously decayed wood types generally resulted in more variable responses, depending upon species. The results are discussed in an ecological context and it is suggested that the exudates from the partly decayed wood that are responsible for the reported effects may function as infochemicals, structuring microbial communities in wood.     

The resistance of wood chemically modified with isocyanates: the role of moisture content in decay suppression

This paper discusses the moisture content of sound and decayed Corsican pine (Pinus nigra) after modification with isocyanates {n-butyl isocyanate (BuNCO) and 1,6-diisocyanatehexane (HDI)} at specified intervals of weight percent gain (WPG). The main effects of decay fungi (brown and white rot) and levels of modification (WPG) on moisture contents of modified samples are also examined. Corsican pine reacted with isocyanates enhanced the hydrophobic (restrained water) nature of wood. The equilibrium moisture content of chemically modified wood decreases progressively with increasing WPG. Basidiomycete decay tests demonstrated protection by chemical modification. Wood moisture contents after soil block testing are significantly influenced by decay fungi and by the extent of reaction (WPG). Moisture uptake and susceptibility to decay of modified wood are higher when exposed to Coniophora puteana than other decay fungi. Corsican pine cross-linked by reaction with HDI is less susceptible to decay and is more hydrophobic than samples reacted with the single-site reactant BuNCO at comparable WPG.     

Survival of Sclerotium cepivorum Sclerotia and Fusarium oxysporum Chlamydospores in Soil Amended with Cruciferous Residues

The use of cruciferous plant residues to reduce the amount of sclerotia of Sclerotium cepivorum and chlamydospores of Fusarium oxysporum f. sp. lycopersici in soil was investigated. Air‐dried and crushed mustard (Brassica juncea) added to the soil effectively reduced the viability of fungal propagules. Consequently, the reduction of white rot of onion, caused by S. cepivorum and wild of tomato caused by F. oxysporum was observed. The addition of rapeseed (Brassica. napus cv. Bolko and B. napus cv. Gorczanski) residues to soil also resulted in a significant decrease of number of S. cepivorum sclerotia but the effect on F. oxysporum chlamydospores was variable. Introduction of the plant material increased the total number of bacteria, spore‐forming bacteria, fluorescent pseudomonads, actinomycetes, and fungi in soil. One year after the soil amendment, the amount of sporeforming bacteria in treatments with cruciferous residues was higher as compared to the control soil without plant residues. The possible contribution of the decomposition of plant residues and soil micro‐organisms to the reduction of the pathogens population is discussed.     

Breakdown of fresh and dried <Emphasis Type="Italic">Rhizophora mucronata</Emphasis> leaves in a mangrove of Southwest India

Patterns of fungal colonization, mass loss and biochemical changes during the decomposition of predried and fresh (naturally fallen) leaves of Rhizophora mucronata were studied in a southwest mangrove of India. Dried and fresh leaves in litter bags were introduced at the mid-tide zone and retrieved after 1, 2, 4, 8, 10, 12 and 14 weeks. On incubation in the laboratory, a total of 5 ascomycetes and 18 anamorphic fungi were recorded. The majority of anamorphic taxa were natural inhabitants of the phyllosphere of senescent leaves. Following two weeks of exposure, they were largely replaced by marine fungi (ascomycetes and anamorphic fungi). More taxa were recovered from dried than from fresh leaves, and predrying accelerated the initial rate of mass loss. Ergosterol levels were much lower than those reported from vascular plant detritus exposed in other aquatic habitats. Both ergosterol and nitrogen levels peaked after between 4 weeks and 8 weeks of exposure; ergosterol levels subsequently declined, while nitrogen remained stable in predried leaves and fell in fresh leaves. The dynamics of remaining mass for the first 8 weeks of exposure were best described by a double-exponential decay model. The decay rate then appeared to accelerate, and the second phase was best described by a single exponential decay model. The apparent breakpoint coincided with an increase in the salinity of the mangrove swamp.     

Nitrogen uptake in barley after spring incorporation of 15N-labelled Italian ryegrass into sandy soils

A 5-month laboratory incubation experiment was conducted to study the immobilization-mineralization of N in soil to which dried or composted ¹⁵N labelled ryegrass (Lolium italicum L.) had been added. Cellulose was added to dried ryegrass to give a C/N ratio similar to that of composted ryegrass. Exchangeable NH₄ ⁺ and NO₃ ^–, HCl-hydrolyzable N forms, microbial biomass N, NaOH-soluble and insoluble N were monitored during incubation. Dried ryegrass brought about a significant increase in total and labelled exchangeable NH₄ ⁺, while a rapid immobilization and a subsequent slow release of exchangeable NH₄ ⁺ was observed in soil with composted ryegrass, together with a resistance to degradation of the labelled humic substances. Compounds synthesized during the composting process and resistant to microbial decomposition probably caused an increase in the amino-acid fraction of soil. These findings suggest that composting can reduce the risk of N losses.     

The effect of species of Allium on soil bacteria in relation to germination of sclerotia of Sclerotium cepivorum Berk

Extracts made from Allium species were shown to exhibit marked antibiotic properties in a variety of tests. However, no evidence was obtained to suggest that intact Allium seedlings exude antibiotic compounds in sufficient quantity to cause inhibition of bacteria in the soil or in assay tests in the laboratory. Germination of sclerotia of Sclerotium cepivorum was induced by Allium extracts which were too dilute to cause antibiotic responses in laboratory tests using a number of bacteria. Synthetic methyl-methanethiolsulphinate had little effect on germination of sclerotia. The specific reversal, by Allium species and their extracts, of the mycostatic effect of unsterile soil on sclerotia of S. cepivorum does not appear to be due to antibiotic effects.     

A direct observation technique for evaluating sclerotium germination by Macrophomina phaseolina and effects of biocontrol materials on survival of sclerotia in soil

Germination of sclerotia of Macrophomina phaseolina was quantified by direct microscopic observation following application of experimental treatments in vitro and incubation of sclerotia in soil. To assay germination, pieces of agar containing sclerotia were macerated in dilute, liquid cornmeal agar on glass slides; thinly spread; and incubated in a saturated atmosphere for 18–22 h. Germinated sclerotia then were identified by morphological features of germ hyphae. Frequencies of germination were similar in three dilute agar media. Germination was not affected by air-drying sclerotia for 2 weeks, but it was significantly reduced after 4 weeks and greatly reduced or eliminated after 6 or 8 weeks. Survival of sclerotia for 14 days in soil was greatest at 50, 75, and 100% moisture-holding capacity, less at 0 and 25%, and least at 125% (flooded soil). Incorporation of ground poultry litter into soil at 5% by weight reduced survival of sclerotia after 13 days, and incorporation of litter at 10% nearly eliminated it. These results indicate that the direct-observation technique may be used to evaluate animal wastes and other agricultural byproducts for biocontrol activity against sclerotia of M. phaseolina in soil.     

Compatible solutes of sclerotia of Mycoleptodiscus terrestris under different culture and drying conditions

Mycoleptodiscus terrestris is a plant pathogen which has been shown to be effective in controlling invasive aquatic weeds in inundative biocontrol applications. The preferred propagule for production and application is the sclerotium. In the current study, we evaluated the accumulation of carbohydrates and polyols in the sclerotia of M. terrestris under different culture and drying regimes. The carbohydrates and polyols screened for represent a class of compatible solutes found in fungi. The results show mannitol and trehalose are the major analytes identified in the sclerotia of M. terrestris. We identified higher levels of mannitol and trehalose in liquid-culture produced samples relative to solid-state produced samples. The impact of fermentation time was examined and shown to impact solute levels. In addition, the drying regime was varied to produce samples dried to different moisture contents. The experiments show greater drying led to higher mannitol levels, while trehalose levels remained constant. Rapid drying of the sclerotia in a fluidized-bed dryer also show rapid accumulation of mannitol and trehalose, which suggest the enzyme activities needed for production are readily available. The findings confirm mannitol and trehalose are important metabolites in M. terrestris and their concentrations are responsive to osmotic stress conditions.     

Ultrastructure and germinability of Verticillium dahliae microsclerotia parasitized by Talaromyces flavus on agar medium and in treated soil

The interactions between microsclerotia (ms) of the fungal plant pathogen Verticillium dahliae and the mycoparasite Talaromyces flavus were followed in soil and on agar medium. Germinability of ms, which had been incubated for 14 days in soil treated with 0.5% of a T. flavus ‐ wheat bran preparation, decreased from 84% to 17%, as compared with 81% and 74% in untreated soil and in soil treated with a sterilized biocontrol preparation respectively. Germinability of ms which had been buried in treated soil for 4 days decreased to 70%, all ms being parasitized by T. flavus. Upon transfer of the ms to untreated soil for 10 more days, germinability decreased further to 20%, indicating that T. flavus continued to parasitize sclerotia in the untreated soil. Scanning electron micrographs showed heavy fungal colonization and typical T. flavus conidia on the surface of the ms buried in the treated soil, but not in control soils. Transmission electron micrographs of ms incubated with T. flavus on agar revealed parsitism involving invasion of some host cells by means of small penetration pegs; the host cell walls were mainly lysed at their site of contact with the parasite hyphal tips. Further colonization of the ms cells occurred simultaneously with the degradation of the invaded host cell contents, rather than the cell walls. Mycoparasitism of V. dahliae ms by T. flavus hyphae may be involved in the biological control of verticillium wilt disease.     

Fungal species residing in the sclerotia of <Emphasis Type="Italic">Polyporus umbellatus</Emphasis>

Sclerotia of Polyporus umbellatus is a traditional Chinese herb. The sclerotia can survive in soils for long time and their growth depends upon a symbiotic association with Armillariella mellea. But it is unclear whether other fungi reside or play a role in the sclerotia. In this study, wild sclerotial samples were collected from seven provinces, which span southwest to northeast China. A total of 148 fungal isolates were recovered from the sclerotia of P. umbellatus and classified into 19 morphological taxa. Seventeen belonged to five genera: Fusarium, Eurotium, Penicillium, Geomyces and Mucor. The fungi found within the sclerotia varied depending on the province from which they were collected. The possible role of these fungi is discussed.     

Studies on the survival of Mycosphaerella pinodes and Ascochyta pisi

Laboratory and field experiments on two pea-pod and leaf-spot pathogens, Mycosphaerella pinodes and Ascochyta pisi, have indicated that the former species may survive overwinter in soil whether introduced as pycnidiospores, mycelium, chlamydospores or sclerotia. The survival of pycnidiospores appears to be due at least in part to their transformation into chlamydospores in the soil. A. pisi was much less successful in soil; the mycelium inoculum survived, somewhat sporadically, and spore viability was reduced, while the total number of spores declined markedly. Investigations on the competitive saprophytic ability of these pathogens showed that by the agar plate method M. pinodes could be classed as a moderately successful saprophyte, though the Butler and Park methods indicated only intermediate and poor success respectively. A. pisi was adjudged a poor saprophyte by all three methods. Saprophytic growth and survival in soil of both fungi was generally better at 2 °C than at 15 °C.     

Soil salinity delays germination and limits growth of hyphae from propagules of arbuscular mycorrhizal fungi

Colonisation of plant roots by some arbuscular mycorrhizal (AM) fungi is reduced in the presence of sodium chloride (NaCl), probably due to a direct effect of NaCl on the fungi. However, there appear to be differences between the fungi in their ability to colonise plants in the presence of NaCl. This experiment tested the hypothesis that propagules of different isolates and species of AM fungi from saline and nonsaline soils would differ in their ability to germinate and grow in the presence of NaCl in the soil solution. Spores or pieces of root colonised by a range of AM fungi were incubated between filters buried in soil to which NaCl had been added at concentrations of 0, 150 or 300 mM in the soil solution. At regular intervals, filters were removed from the soil and both the percentage of propagules which had germinated and the length of proliferating hyphae were determined. Germination of spores of AM fungi studied was delayed in the presence of NaCl, but the fungi differed in the extent to which germination was inhibited. Two isolates of Scutellospora calospora reached maximum germination in 300 mM NaCl, but neither of two isolates of Acaulospora laevis germinated in the presence of NaCl. Germination of spores of the other fungi, including some isolated from saline soil, fell between these extremes. For some fungi, the specific rate of hyphal extension was reduced by NaCl. For others, the specific rate of growth was similar in the presence of NaCl to that in the control treatment, but overall production of hyphae was reduced in the NaCl treatments because germination was reduced.     

The effect of soil desiccation on the nutrient status of Eucalyptus regnans F. Muell seedlings

The poor growth of young Eucalyptus regnans seedlings in undried soil from the mature forest of E. regnans can be overcome by previously air-drying the soil or by adding sufficient amounts of complete soluble fertilizer or equivalent concentrations of P (as NaH₂PO₄) and N (as NaNO₃). A factorial pot experiment in which phosphate and nitrate were added to undried soil indicated that P was the primary deficiency for young seedlings and that response to N did not occur until this lack was satisfied. In dried soil, seedlings also responded to additions of complete fertilizer but most of this effect was due to N rather than P. Field trials in the mature forest also indicated greater growth in dried soil than undried soil and confirmed a response of young seedlings to superphosphate. In pot experiments, the concentration of P and N per g plant dry weight after four months was relatively constant irrespective of the final size of the plant. Seedlings in dried soil extracted up to 15 times more P than did those grown in undried soil. In general, chemical analysis of soil indicated more extractable P and N from dried soil although this was not always consistently so. Soil desiccation resulted in an increase in soil surface area due to the fragmentation of larger peds and to an increase in the number of microfractures which remained in the soil crumbs after rewetting. Mycorrhiza are likely to be important since the differentiation of the growth response of seedlings in dried and undried soil, which occurred at 5–6 weeks, corresponded with the establishment of full ectomycorrhizal development (80% root tips). The factors concerned with the increase in fertility after air-drying are discussed.Abbreviations GR Growth Ratio     

Antifungal activity of stilbenes in in vitro bioassays and in transgenic<Emphasis Type="Italic"> Populus</Emphasis> expressing a gene encoding pinosylvin synthase

The effect of two stilbene compounds, pinosylvin and resveratrol, on the growth of several fungi was evaluated in plate tests. Wood decay tests were carried out with birch and aspen samples impregnated with the two stilbenes. In plate experiments, resveratrol had an enhancing effect on growth at concentrations where pinosylvin was already enough to prevent the growth of most fungi studied. Pinosylvin impregnated at 0.2% (w/w) concentration significantly reduced the decay caused by all fungi except Phellinus tremulae. In contrast, a resveratrol content of 0.8%, did not protect the wood from decay. A pinosylvin-synthase-encoding gene from Pinus sylvestris was transferred into aspen (Populus tremula) and two hybrid aspen clones (Populus tremula×tremuloides) by Agrobacterium tumefaciens-mediated transformation. Transgenic plants accumulated pinosylvin synthase-specific mRNA and showed stilbene synthase enzyme activity in vitro. Transgenic aspen line H4 showed increased resistance to Phellinus tremulae, while two hybrid aspen transformants decayed faster than the control trees. However, we were unable to detect the accumulation of stilbenes in the transgenic plantlets.Communicated by P. Debergh     

Population dynamics of Macrophomina phaseolina in relation to disease management: A review

Macrophomina phaseolina (Tassi) Goid. causes seedling blight, charcoal rot, leaf blight, stem and pod rot on over 500 plant species in different parts of the world. The pathogen survives as sclerotia formed in host tissues which are released into the soil as tissue decay. Low soil moisture is considered the more important predisposing factor for M. phaseolina-induced diseases than high temperature. The intensity of the disease on a crop is related to the population of viable sclerotia in the soil and abiotic factors. The influence of various management strategies in reducing the number of viable propagules of the pathogen in the soil has been studied in order to minimize the impact of the disease. Any management approach that reduces inoculum density in the soil may reduce disease incidence on the host. However, to reduce inoculum density, quantitative determination of viable propagules from soil is necessary in order to understand the effect of management strategies on the population dynamics of this pathogen. Considerable work has been done on organic amendments, changing crop sequences with tolerant crops, fumigants, herbicides and tillage in managing M. phaseolina populations in the soil and the resulting disease. Solarization has been used in controlling M. phaseolina in different countries where this pathogen is causing disease on economically valuable crops. However, this method of soil disinfestation was effective in eliminating viable populations at the top soil layer although by combining other approaches its effectiveness was improved at lower soil depth. Use of biological control agents with or without organic amendments or after solarization has emerged to be a practical management approach in the control of M. phaseolina. In this paper, an attempt has been made to review those research findings where the influence of various management approaches on survival of M. phaseolina mainly sclerotia have been investigated.     

Symbiosis of a termite and a sclerotium-forming fungus: Sclerotia mimic termite eggs

Brown balls, of a similar size but different shape to termite eggs, were found frequently in the piles of eggs of the subterranean termite Reticulitermes speratus. rDNA analysis identified the ball as the sclerotia of the fungus, Fibularhizoctonia sp. nov, which is phylogenetically closest to decay fungi, Athelia spp. Laboratory observation showed that the workers gathered the eggs and the sclerotia indiscriminately, even if they were widely scattered in a Petri dish, and piled them up in a specific place for egg care. We compared the morphology of the eggs with that of sclerotia of Fibularhizoctonia spp. and Athelia spp. in relation to egg carrying behaviour, and found that the workers could only carry the Fibularhizoctonia spp. sclerotia whose diameters were similar to the short diameter of the eggs. We also conducted a bioassay using termite eggs and dummy eggs (glass beads and sea sand) of two diameter-classes, coated with or without the egg-derived chemicals. The workers recognized the eggs based on a combination of the size, shape, and chemical cues. All the results suggested that the sclerotia mimic the eggs both morphologically and chemically. Finally, we found that the workers suppressed germination of sclerotia, and termite egg survival increased in the presence of sclerotia only if they were tended by the workers. If the workers were removed experimentally, the sclerotia germinated and grew by exploiting termite eggs. These results suggest that the sclerotia protect termite eggs from putative pathogens.     

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.     

An FT-IR study of the changes in chemical composition of bamboo degraded by brown-rot fungi

The objective of this study was to use FT-IR analysis to investigate the chemical composition of aged and un-aged bamboo specimens, with and without node sections, decayed by brown-rot fungi. Specimens were exposed to two brown-rot fungi, Coniophora puteana and Poria placenta, for 8 weeks after which decay was assessed by weight loss and FT-IR spectra analysis. Depending on the bamboo section examined, the aging process reduced decay resistance of specimens. Weight loss (measured as a percentage) decreased from the top to the bottom portion of bamboo culms. The presence of nodes in the specimens increased weight loss caused by P. placenta attack, and caused only a slight increase in weight loss from C. puteana attack. Significant chemical changes in bamboo were observed after fungal degradation, as revealed by FT-IR analyses. Consistent with the degradation mechanism of brown-rot fungi, lignin was essentially un-degraded or modified. Both brown-rot fungi caused a sharp decrease in the carbonyl absorption area. Surprisingly, cellulose peaks of degraded specimens were nearly similar to the peaks of control specimens. Aging treatments and biodegradation affected the crystalline structure of bamboo specimens. Poria placenta degraded wood components faster and changed the crystallinity more than C. puteana did, in accordance with the weight losses due to decay.