Production of a Fungistatic Substance by <Emphasis Type="Italic">Pseudallescheria boydii</Emphasis> Isolated from Soil Amended with Vegetable Tissues and Its Significance

Four fungal isolates that were able to use vegetable tissues for multiplication in soil were isolated and identified as Pseudallescheria boydii based on morphological characteristics and ITS sequence similarity. When grown in broth prepared from the same vegetable tissues used in soil amendment, all these isolates of P. boydii produced a substance capable of reducing the disease incidence of black leaf spot of spoon cabbage caused by Alternaria brassicicola and inhibiting the germination of A. brassicicola conidia. The substance, which was fungistatic, was very stable under high temperature and high or low pH value. It was soluble in polar solvents and insoluble in non-polar solvents. Molecular weight estimation and ion exchange ability tests suggest that the fungistatic compound has a molecular weight between 500 and 1,000 and has no charge on its molecule. Results from this study suggest the possession of a strong competitive saprophytic ability by P. boydii, which in turn may explain the widespread occurrence of this human pathogen in soil. Production of a fungistatic substance when P. boydii was grown in broth prepared from vegetable tissues suggests the importance of antibiotic production in its competitive saprophytic colonization of organic matters in soil.     

Antifungal activity of venenatine, an indole alkaloid isolated fromAlstonia venenata

The indole alkaloid venenatine exhibited antifungal activity against some plant pathogenic and saprophytic fungi. Venenatine in an aqueous acetic acid solution inhibited spore germination of all the 10 tested fungi, Fusarium udum, Alternaria brassicicola, Ustilago cynodontis and Aspergillus flavus showed an especially high sensitivity towards this compound, exhibiting germination levels below 10%. The spore germination and colony development of the parasitic fungus Erysiphe pisi, which causes powdery mildew in pea (Pisum sativum), on excised leaves of pea was also significantly affected. Pre-inoculation rather than post inoculation treatment of the leaves was more inhibitory against spore germination and colony development.     

Soil fungistasis: elevation of the exogenous carbon and nitrogen requirements for spore germination by fungistatic volatiles in soils.

Axenic, washed conidia of Fusarium solani f. sp. phaseoli, Aspergillus flavus, and Verticillium albo-atrum were placed on washed Difco purified agar discs along with an inorganic salt solution containing various levels of carbon and nitrogen substrates. These discs were exposed to volatiles from six soils (pH 5.1-8.6). Fusarium solani macroconidial germination was inhibited mostly by volatiles from soils of pH 5.1, 6.1, 7.0, and 7.5, but high levels of glucose and NH4Cl reversed this inhibition, raising germination to that of no-soil, no-carbon or nitrogen controls. Conidial germination of A. flavus was inhibited mainly by volatiles from high pH (7.0, 7.8, and 8.6) soils, and increased levels of glucose plus an amino acid mixture nullified this inhibition. Volatiles from soils of pH 5.1, 6.1, and 7.5 stimulated A. flavus conidial germination. Assays after the removal of CO2 from the air above soil of pH 5.1 demonstrated that volatiles inhibitory to A. flavus were produced by this soil. Assays indicated that a KOH-soluble compound was a fungistatic soil volatile to F. solani macroconidial germination. The nullification by carbon and nitrogen substrates of F. solani and A. flavus inhibition caused by soil volatiles parallels that for soil fungistasis. Conidial germination of V. albo-atrum was markedly stimulated by volatiles in all soils tested, and was not affected by removal of CO2. Inhibitory soil volatiles may increase the nutritional requirements for spore germination of certain fungi.     

Effect of a saprophytic fungus on the growth and the lead uptake, translocation and immobilization in Dodonaea viscosa

Phytoremediation is a feasible alternative to remediate soils polluted with toxic elements, which can be enhanced by manipulating plant-microbe interactions. Regarding this, free-living saprophytic fungi that interact beneficially with roots have been scarcely studied. Thus, the aim of this study was to assess the effect of a saprophytic fungus, Lewia sp., on the plant growth and the ability of Dodonaea viscosa to phytoaccumulate or phytostabilize soluble and insoluble sources of lead in a solid support. The growth of D. viscosa was influenced by both Pb and Lewia sp. While seedlings exposed to Pb showed a decrease in biomass production, in seedlings grown without Pb the biomass was stimulated by Lewia sp. The fungus strongly stimulated the weight-to-length ratio in roots. Regardless of the treatment, D. viscosa accumulated 4.4-6.5 times more Pb in roots than in shoots, conducting to low translocation factors (< 0.2). The presence of Lewia sp. significantly improved Pb accumulation, achieving high bioconcentration factors (> 22), which was attributed to an increased bioavailability and uptake of Pb due to the fungus. This study demonstrated that Lewia sp. could improve Pb-phytostabilization by D. viscosa in soils polluted with soluble and insoluble forms of Pb.     

Colonization of rye green manure and peanut fruit debris by Aspergillus falvus and Aspergillus niger group in field soils.

Aspergillus flavus and Aspergillus niger group colonization of deep-plowed, decomposing rye green manure cover crops in peanut field soils was studied in four fields during 1972 and 1973; colonization of decomposing peanut fruits was studied in 1972 in two fields. A. flavus colonization of rye and peanut fruits was greater in soils of heavy texture, and an A. flavus population as high as 165 propagules per g of soil was observed in soil adjacent to rye, whereas A. flavus populations in soils not associated with rye were 18 propagules per g of soil or lower. Highest A. flavus populations in soil adjacent to decomposing peanut fruits were usually comparable to populations associated with rye. Little decomposing rye or peanut fruit colonization was generally observed by the A. flavus competitor, A. niger group. A. flavus may maintain or increase its inoculum potential by colonization of these and other moribund plant tissues.     

The Saprophytic Status of Fusarium oxysporum Schl: Causing Vascular Wilt of Oil Palm

The droplet plating method described, especially when used inconjunction with a baiting technique, facilitates the demonstrationof Fusarium oxysporum in soil. This fungus was present in soilfrom wilt-free areas of oil palm plantations as well as in soilabout wilt-diseased palms. Hyphal fusions between soil isolatesand isolates from wilted palms could be demonstrated. The funguspersisted for periods of at least ¹ year in naturally infestedsoils under a variety of moisture conditions; it also survivedin inoculated alien soil forms of Ieast ¹year. Soil forms ofthe fungus had a high competitive saprophytic ability, competitivelycolonizing sterilized soils and sand, and organic materialsadded to soils and mixed cultures. The pathogenic isolate, aswell as the soil isolates, exhibited characters belonging tosoil-inhabiting fungi, namely continued persistance in soil:tolerance, in respect of growth and reproduction, to antagonism:and the ability competitively to colonize dead organic materialin soils. The pathogenic form is considered to be a soil inhabitant.     

Occurrence and activity of phosphate-solubilizing fungi from coconut plantation soils

Summary The occurrence of phosphate-solubilizing fungi in coconut plantation soil types was investigated. The laterite, alluvial and clayey soils harboured more of the P-solubilizing fungi than the sandy soils. The isolated P-solubilizing fungi solubilized 26 to 74 per cent of the tricalcium phosphate in 5 to 15 days. The competitive saprophytic ability of the active P-solubilizing fungi in soil varied between the isolates. Eight fungi with high P-solubilizing capacity and high competitive saprophytic ability were recognised. They have better capacity to survive in soil and express their role in P-solubilization.Contribution No. 291 of the Central Plantation Crops Research Institute, Kasaragod, India     

A COMPARATIVE STUDY OF OPHIOBOLUS GRAMINIS AND FUSARIUM CULMORUM IN SAPROPHYTIC COLONIZATION OF WHEAT STRAW

The competitive saprophytic ability of Ophiobolus graminis has been compared with that of Fusarium cultnorum. Test pieces of wheat straw were buried in maize-meal-sand inoculum mixed in various proportions with unsterilized soil, according to the technique already described by Butler (1953). Some possible sources of variation in the results obtained by this technique have been investigated. Saprophytic colonization of the straw tissues by Ophiobolus graminis appeared to decline as invasion progressed from the outer to the inner tissues, presumably owing to continued competition with other saprophytes. An experiment using as inoculum the stubble of plants naturally infected by O. graminis suggests that saprophytic colonization of wheat straw by the take-all fungus can occur only to a negligible extent in the field.     

Effects of inoculum density and substrate type on saprophytic survival of Phytophthora drechsleri, the causal agent of gummosis (crown and root rot) on pistachio in Rafsanjan, Iran

In this study, sampling was carrid out on soils around pistachio trees in various regions of Rafsanjan, Iran. Following isolation and identification of Phytophthora isolates, the predominant species was found to be P. drechsleri and used for further investigation. For studying saprophytic survival of the fungus, soils collected from different areas were combined and autoclaved. Sterile soil was divided into 10 parts and mixed with fungal inoculum at various concentrations of 0-9% (w/w) separately. Each soil part (100g) was placed in 15cm diameter plastic pot. Some soils in pots were supplemented with sterile wheat straw whereas others were mixed with pistachio leaves surface sterilized with 5% (v/v) sodium hypochlorite. After 3, 6 and 9 weeks of incubation, five leaves or straws samples were taken from each replicate and cultured on CMA-PARPH medium and the fungal colony formation was monitored. The experiment was performed using completely randomized design with factorial experiments including three factors (substrate type, inoculum density and time), 10 treatments (0-9 g inoculum levels) and nine replicates. The results showed that the type of substrate (wheat straw and pistachio leaf) was very important for the fungal saprophytic survival in that this was significantly greater for the pistachio leaves. Time was also considered another critical factor for the fungal survival. With passing incubation time, saprophytic survival of the fungus declined. Further, it was demonstrated that increasing inoculum density would result in longer survivability of P. drechsleri and maximum fungal survival on substrate was obtained when inoculum density was at 9% (w/w).     

Saprophytic behaviour of Cephalosporium maydis and C. acremonium

The competitive saprophytic ability (CSA) of Cephalasporium maydis was smaller at 30 C when measured by the agar-plate modification than by the original Cambridge method. The agar-plate method suggested that C. maydis was a less competitive saprophyte than C. acremonium although both were low in CSA.
C. acremonium grows and sporulates well on organic and synthetic media. C. maydis grows faster but is more exacting nutritionally and is less able to decompose cellulose or maize straw than C. acremonium . Neither fungus produced pectolytic culture filtrates and both were susceptible to antibiotics produced by soil micro-organisms.
C. maydis survived on maize straw much longer than C. acremonium . In field soils C. maydis colonized and survived in supplemented wheat bran poorly and not below the top 20 cm of soil.     

Interaction between Streptococcus lactis and Aspergillus flavus on production of aflatoxin

The inoculation of Aspergillus flavus spores into a culture of Streptococcus lactis in Lablemco tryptone broth medium resulted in little or no aflatoxin accumulation even though the growth of the fungus was not hindered. The drop in pH and reduced nutrient levels in the medium as a result of the S. lactis growth were not the cause of the observed inhibition. The inhibition was not eliminated by the addition of carbohydrate equal to the amount used by the bacterium before the inoculation with the fungus. Aflatoxin levels were also markedly reduced when S. lactis was inoculated into a growing A. flavus culture. In addition to inhibiting the synthesis of aflatoxin, S. lactis also degraded preformed toxin. A. flavus, on the other hand, not only reduced the growth of S. lactis but also affected the morphology of the bacterial cell; the cells became elongated and formed long chains. S. lactis produced and excreted the inhibitor into the medium late in its growth phase. The inhibitor was a heat-stable low-molecular-weight compound. Chloroform extracts of A. flavus grown in the presence of S. lactis were toxic to Bacillus megaterium but did not exhibit mutagenic or carcinogenic activity in the Salmonella/mammalian microsome mutagenicity test.     

Interaction between Streptococcus lactis and Aspergillus flavus on production of aflatoxin.

The inoculation of Aspergillus flavus spores into a culture of Streptococcus lactis in Lablemco tryptone broth medium resulted in little or no aflatoxin accumulation even though the growth of the fungus was not hindered. The drop in pH and reduced nutrient levels in the medium as a result of the S. lactis growth were not the cause of the observed inhibition. The inhibition was not eliminated by the addition of carbohydrate equal to the amount used by the bacterium before the inoculation with the fungus. Aflatoxin levels were also markedly reduced when S. lactis was inoculated into a growing A. flavus culture. In addition to inhibiting the synthesis of aflatoxin, S. lactis also degraded preformed toxin. A. flavus, on the other hand, not only reduced the growth of S. lactis but also affected the morphology of the bacterial cell; the cells became elongated and formed long chains. S. lactis produced and excreted the inhibitor into the medium late in its growth phase. The inhibitor was a heat-stable low-molecular-weight compound. Chloroform extracts of A. flavus grown in the presence of S. lactis were toxic to Bacillus megaterium but did not exhibit mutagenic or carcinogenic activity in the Salmonella/mammalian microsome mutagenicity test.     

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.     

Quantification in soil and the rhizosphere of the nematophagous fungus Verticillium chlamydosporium by competitive PCR and comparison with selective plating

A competitive PCR (cPCR) assay was developed to quantify the nematophagous fungus Verticillium chlamydosporium in soil. A gamma-irradiated soil was seeded with different numbers of chlamydospores from V. chlamydosporium isolate 10, and samples were obtained at time intervals of up to 8 weeks. Samples were analyzed by cPCR and by plating onto a semiselective medium. The results suggested that saprophytic V. chlamydosporium growth did occur in soil and that the two methods detected different phases of growth. The first stage of growth, DNA replication, was demonstrated by the rapid increase in cPCR estimates, and the presumed carrying capacity (PCC) of the soil was reached after only 1 week of incubation. The second stage, an increase in fungal propagules presumably due to cell division, sporulation, and hyphal fragmentation, was indicated by a less rapid increase in CFU, and 3 weeks was required to reach the PCC. Experiments with field soil revealed that saprophytic fungal growth was limited, presumably due to competition from the indigenous soil microflora, and that the PCR results were less variable than the equivalent plate count results. In addition, the limit of detection of V. chlamydosporium in field soil was lower than that in gamma-irradiated soil, suggesting that there was a background population of the fungus in the field, although the level was below the limit of detection. Tomatoes were infected with the root knot nematode (RKN) or the potato cyst nematode (PCN) along with a PCN-derived isolate of the fungus (V. chlamydosporium isolate Jersey). Increases in fungal growth were observed in the rhizosphere of PCN-infested plants but not in the rhizosphere of RKN-infested plants after 14 weeks using cPCR. In this paper we describe for the first time PCR-based quantification of a fungal biological control agent for nematodes in soil and the rhizosphere, and we provide evidence for nematode host specificity that is highly relevant to the biological control efficacy of this fungus.     

Effect of liming on spore germination,germ tube growth and root colonization by vesicular-arbuscular mycorrhizal fungi

Summary The effect of soil acidity on spore germination, germ tube growth and root colonization of vesicular-arbuscular mycorrhizal (VAM) fungi was examined using a Florida Ultisol. Soil samples were treated with 0, 4, 8 and 12 meq Ca/MgCO₃/100 g soil and each lime level received 0, 240, and 720 ppm P as superphosphate. Corn (Zea mays L.) was planted in the soil treatments, inoculated with eitherGlomus mosseae orGigaspora margarita spores and grown for 31 days. Acid soil inhibits mycorrhizal formation byG. mosseae through its strong fungistatic effect against the spores. The dolomitic lime increased mycorrhizal formation by both fungal species.G. margarita is much less sensitive to acidic conditions thanG. mosseae. Al ions are a very important component of the fungistatic property against the VAM symbiosis. VAM fungus adaptation may be important for plants growing on infertile acid soils if soil inoculation with these fungi is to contribute significantly to low-input technology for tropical agricultural systems.     

Quantification in Soil and the Rhizosphere of the Nematophagous Fungus Verticillium chlamydosporium by Competitive PCR and Comparison with Selective Plating

A competitive PCR (cPCR) assay was developed to quantify the nematophagous fungus Verticillium chlamydosporium in soil. A γ-irradiated soil was seeded with different numbers of chlamydospores from V. chlamydosporium isolate 10, and samples were obtained at time intervals of up to 8 weeks. Samples were analyzed by cPCR and by plating onto a semiselective medium. The results suggested that saprophytic V. chlamydosporium growth did occur in soil and that the two methods detected different phases of growth. The first stage of growth, DNA replication, was demonstrated by the rapid increase in cPCR estimates, and the presumed carrying capacity (PCC) of the soil was reached after only 1 week of incubation. The second stage, an increase in fungal propagules presumably due to cell division, sporulation, and hyphal fragmentation, was indicated by a less rapid increase in CFU, and 3 weeks was required to reach the PCC. Experiments with field soil revealed that saprophytic fungal growth was limited, presumably due to competition from the indigenous soil microflora, and that the PCR results were less variable than the equivalent plate count results. In addition, the limit of detection of V. chlamydosporium in field soil was lower than that in γ-irradiated soil, suggesting that there was a background population of the fungus in the field, although the level was below the limit of detection. Tomatoes were infected with the root knot nematode (RKN) or the potato cyst nematode (PCN) along with a PCN-derived isolate of the fungus (V. chlamydosporium isolate Jersey). Increases in fungal growth were observed in the rhizosphere of PCN-infested plants but not in the rhizosphere of RKN-infested plants after 14 weeks using cPCR. In this paper we describe for the first time PCR-based quantification of a fungal biological control agent for nematodes in soil and the rhizosphere, and we provide evidence for nematode host specificity that is highly relevant to the biological control efficacy of this fungus.     

Lignin-modifying enzymes of flavodon flavus, a basidiomycete isolated from a coastal marine environment

A basidiomycetous fungus Flavodon flavus (Klotzsch) Ryvarden (strain 312), isolated from decaying sea grass from a coral lagoon off the west coast of India, mineralized nearly 24% of 14C-labeled synthetic lignin to 14CO2 in 24 days. When grown in low-nitrogen medium (2.4 mM N) this fungus produced three major classes of extracellular lignin-modifying enzymes (LMEs): manganese-dependent peroxidase (MNP), lignin peroxidase (LIP), and laccase. Low MNP and laccase activities were seen in high-nitrogen medium (24 mM N), but no LIP activity was seen. In media containing lignocellulosic substrates such as pine, poplar, or sugarcane bagasse as the sole source of carbon and nitrogen, relatively high MNP and moderate levels of laccases were seen, but LIP production either was not seen or was minimal. LME production was also seen in media prepared with artificial seawater. Fast protein liquid chromatography and isoelectric focusing resolved LMEs into four isozymes each of MNP and LIP, while laccase isozymes were resolved into two groups, one group containing seven isozymes (pIs 4 to 6) and the other group containing at least three isozymes (pIs < 3). The molecular masses of the different isozymes were 43 to 99 kDa for MNP, 40 and 41.5 kDa for LIP, and 43 and 99 kDa for laccase. F. flavus showed effective degradation of various dye pollutants in media prepared with or without artificial seawater. This is the first report on the production of all three major classes of LMEs by F. flavus and points to the bioremediation potential of this organism in terrestrial as well as marine environments.     

Differential protein expression in response to the phytoalexin brassinin allows the identification of molecular targets in the phytopathogenic fungus Alternaria brassicicola

The effects of the cruciferous phytoalexin brassinin on the protein expression patterns of the phytopathogenic fungus Alternaria brassicicola were investigated. Cell-free protein extracts of mycelia of A. brassicicola induced with brassinin at 0.50 and 0.10 mm were fractionated, and the proteins in soluble fractions were separated by two-dimensional electrophoresis. Spots corresponding to differentially expressed proteins were digested and analysed by liquid chromatography-electrospray ionization-mass spectrometry. The number of differentially expressed proteins was significantly higher in mycelia treated with brassinin at 0.50 mm (96 protein spots) than in mycelia treated with brassinin at 0.10 mm (18 protein spots). The majority of differentially expressed proteins included proteins involved in metabolism, processing, synthesis and several heat shock proteins (HSPs). Brassinin concentrations below 0.30 mm induced HSP90, a protein involved in the regulation of morphogenetic signalling in fungi, suggesting that 0.30 mm is a minimal concentration of brassinin necessary for the protection of brassicas against A. brassicicola. These results reveal that HSP90 is a potential target for inhibition in stressed A. brassicicola and confirm that brassinin has strong detrimental effects on A. brassicicola, suggesting that its detoxification by the fungus suppresses an important defence layer of the plant.     

Bdellovibrio bacteriovorus Parasitizing Rhizobium in Western Australia

S ummary . Bdellovibrio bacteriovorus strains which parasitize Rhizobium meliloti, R. trifolii, Agrobacterium tumefaciens and A. radiobacter have been found in Western Australian soils. Rhizobium lupini was not lysed by any strain. Some Bdellovibrio cells pass through 0·22 μm membrane filters, whilst the 0·45 μm filters allow several saprophytic soil bacteria to pass. The strength of the medium is shown to be of great importance for the detection or counting of Bdellovibrio cells. The Bdellovibrio strains are capable of destroying large numbers of rhizobia in the laboratory.     

Survival of Alternaria cheiranthi in soil

Summary Experiments were conducted to study the survival of Alternaria cheiranthi in wet and dry soil. As free spores, survival was demonstrated only in air-dry soil. In wet soil, lysis of the germ tubes commenced after the second day. On wallflower seeds, the pathogen was viable until the seeds were decomposed. It was concluded that the fungus showed a low competitive saprophytic ability in the soil