An Improved Growth Medium to Assess Mycelial Compatibility Groups in Sclerotium rolfsii

Mycelial compatibility is assayed mainly by pairing mycelial plugs of field isolates on Petri dishes with agar media. Although methodologically simple, mycelial compatibility testing requires an artificial growth medium that permits the identification of compatible and incompatible interactions. In this work, several growth media were studied to assess consistently mycelial interactions between Sclerotium rolfsii isolates. A modification of Patterson’s medium with an increment of 25% glucose from the original concentration at a rate of 23.4 g/l and amended with 180 μl/l of red food colouring was the most effective combination for enhancing the size, density and distinctiveness of the aversion zone between incompatible isolates. This medium allowed the unequivocal identification of compatible and incompatible reactions of a set of five S. rolfsii isolates, which could be determined quickly after 5 days of incubation in the dark at 25°C. This new formulation improved significantly and consistently the assessment of the aversion zone reaction that was visible as a red line on the colony reverse as compared to that assessed using previous media formulations, for which the visualization of aversion zones was scarcely discernible. The utility of the improved growth medium was validated by microscopic observations of the contact area of hyphal pairings between isolates of S. rolfsii in microscope slide cultures.     

Analysis of Mycelial Growth Rates and RAPD-PCR Profiles in a Population of Gaeumannomyces graminis var. tritici Originating from Wheat Plants Grown from Fungicide-treated Seed

Linear mycelial growth rates of 70 isolates of Gaeumannomyces graminis var. tritici on agar medium amended or unamended with the fungicide silthiofam were not correlated. Mycelial growth rate was not influenced by the fungicide applied to the seed of the plants from, which the isolates originated. DNA polymorphism determined by randomly amplified polymorphic DNA (RAPD) polymerase chain reaction was used to assess genetic variation among isolates. Thirty RAPD markers generated with five arbitrary 10‐mer primers revealed DNA polymorphism suitable for assessing variability in this fungal population. Cluster analysis of RAPD data identified two groups at the 54% similarity level. There was a significant relationship between the presence of 11 markers and sensitivity to silthiofam.     

Partial Characterization of the Antimicrobial Activity of Streptomyces antimycoticus FZB53

The paper describes experiments aimed at evaluating the sensitivity of different fungi, most of them plant pathogens and bacteria towards Streptomyces antimycoticus FZB53, a biocontrol agent that, when applied as a seed treatment, in previous studies has shown good activity against different seed‐borne fungal diseases. When incorporated into agar media, the filtrate from shake cultures of S. antimycoticus FZB53 inhibited the mycelial growth or spore germination, respectively, of a broad spectrum of fungi. The most sensitive of the fungi tested was Fusarium culmorum. The inhibitory activity could be removed from the culture filtrate by extraction with ethyl acetate. When ethyl acetate extracts of the pellet and supernatant obtained by centrifugation of the shake culture were added to the agar medium, inhibition of mycelial growth of F. culmorum was restored, especially with the extracts of the pelleted biomass. Autoclaving of the culture filtrate reduced the inhibition of F. culmorum but completely eliminated the inhibitory activity against Fusarium graminearum. Among the bunt fungi tested, spore germination of Tilletia tritici was more sensitive to the culture filtrate of S. antimycoticus FZB53 than spore germination of Ustilago avenae and U. tritici. Separation by thin layer chromatography (tlc) and spraying with different reagents showed that ethyl acetate extracts from shake cultures or biomass scraped from agar media contained several hydrophobic metabolites. When eluted from the tlc‐plates, the material from one of the spots had strong antifungal activity against spore germination of T. tritici and mycelial growth of F. culmorum, respectively. Ethyl acetate extracts from biomass of S. antimycoticus FZB53 prevented the growth of the tested Gram‐positive bacteria, namely Clavibacter michiganensis and different species of Bacillus. The results indicated that these bacteria were at least as sensitive towards the metabolites of S. antimycoticus FZB53 as F. culmorum. The tested Gram‐negative bacteria were not affected.     

A preliminary study on the effects of salicylic and jasmonic acids on Ganoderma boninense growth,mycelial hydrophobicity,and media pH under in vitro assays

Potato dextrose agar amended with salicylic acid (SA) suppressed growth of all Ganoderma boninense isolates. However, mycelial growth inhibitory effects varied with times, SA concentrations and G. boninense isolates. At 150 ppm of SA or higher, growth suppression diminished with time and mycelial growth recovery was observed. In contrast, jasmonic acid (JA) unexpectedly improved growth of G. boninense. At 21 day-after-inoculation, pH changes and hydrophobicity for the melanised mycelia were lower compared to non-melanised mycelia. The degree of inhibitory or promotional effects on the growth of G. boninense and the level of G. boninense mycelial hydrophobicity by SA and JA were isolate-dependent.     

Physiological characterization of the dandelion bioherbicide,Sclerotinia minor IMI 344141

The fungus Sclerotinia minor (IMI 344141) is being developed as a biological control for dandelion and other broadleaf weeds in turfgrass environments. Being a microbial pest control agent (MPCA), the S. minor strain must be characterized to show relatedness to like organisms and to distinguish the MPCA from related microorganisms. Phenotypic variation among 30 isolates of S. minor, collected from different regions and hosts, was studied on potato dextrose agar (PDA) and oatmeal agar (OMA). Isolates varied significantly in sclerotia shape (length/width ratio) and number, but did not vary in colony morphology or growth rates. There was high diversity (0.6) among the mycelial compatibility groups (MCG) as seven multi-member and 11 single member groups were recognized. Isolates were categorized into highly virulent, virulent, moderately virulent, and hypo virulent based on 48 h post mycelial growth on detached dandelion leaves. When assessed on dandelion plants in the greenhouse, isolate IMI 344141 ranked the highest in biocontrol efficacy, reduction of above- and below-ground biomass, and reduction in dandelion survival. Oxalic acid production was not correlated with isolate aggressiveness or growth rate and did not vary among isolates of the same MCG. IMI 344141 can be phenotypically distinguished from the other tested S. minor isolates by performing vegetative compatibility testing and counting sclerotia produced on standard 9-cm diameter PDA plates. IMI 344141 produces <100 sclerotia/plate.     

Effects of cultural conditions on the mycelial growth of healthy and virus-infected cultivated mushroom, Agaricus bisporus

There was a significant inverse correlation (P= 0=001) between concentrations of mushroom viruses 1 and 2 in sporophores and amounts of mycelial growth on malt agar of isolates taken from them. Increasing virus concentrations decreased linear growth of one mushroom strain from 76 mm (healthy) to 35 mm (mildly infected) and 7 mm (severely infected) when incubated at 25°C for 3 wk. Mycelial growth rates of isolates from healthy and from virus-infected mushrooms were compared on eleven agar media. All media clearly differentiated between healthy and severely infected isolates, but fewer separated healthy from mildly infected isolates. Those that did contained maltose, sucrose or starch as carbon source. Media containing peptone usually gave better differentiation than those with other sources of nitrogen, but the best differentiation was obtained with malt agar. Growing healthy and infected isolates on a range of media affected their subsequent growth on malt agar, the growth of some isolates apparently being changed permanently after 2 months on some of the different media. Whereas none of the infected isolates grew less rapidly after this treatment, the growth of some of the mildly infected isolates improved to such an extent that they could no longer be distinguished from healthy isolates. After heat-treatment (1–6 wk at 33°C), mycelial growth rates of infected isolates were increased, but viruses 1 and 2 were not always eliminated unless the heat-treatment was begun immediately after subculture. Mycelial growth rate and colony characters are not infallible criteria of the presence or absence of virus, a feature of particular significance when checking the health of mushroom spawn.     

In vitro effect of fermented maize slurry and ethanol extracts of five plants against Sclerotium rolfsii Sacc. of tomato (Lycopersicon esculentum Mill.)

Sclerotium rolfsii Sacc. is an important fungal pathogen affecting the production of tomato (Lycopersicon esculentum Mill.). Conventional control methods using chemicals are expensive, which constitute environmental hazards. This has necessitated the search for alternatives in botanicals. Ethanol and supernatant solution of fermented maize slurry extracts from Ocimum gratissimum, Cymbopogon citratus, Xylopia aethiopica, Aframomum melegueta and Allium sativum were evaluated. In vitro test was carried out at 1, 2, 3, 4 and 5% concentrations against the pathogen. Mycelial growth was significantly reduced in the following descending order: O. gratissimum?>?A. melegueta?>?A. sativum?>?X. aethiopica?>?C. citratus. Supernatant solution extracts of O. gratissimum and A. melegueta at 5% concentration gave highest mycelial reduction (0.00?mm), respectively, while O. gratissimum ethanol extract recorded highest mycelial reduction (0.23?mm) among the ethanol extracts. Supernatant solution of fermented maize slurry extracts of O. gratissimum and A. melegueta at 5% concentration was more effective in reducing mycelial growth of S. rolfsii.     

Antagonism between Gliodadium roseum,Trichoderma harzianum,or Trichothecium roseum and Phytophthora megasperma f. sp. glydnea

The antagonism between Gliodadium roseum, Trichoderma harzianum, or Trichothecium roseum and Phytophthora megasperam f. sp. glycinea (Pmg), cause of Phytophthora rot of soybeans (Glycine max), was studied. G. roseum, T. harzianum, and 17 isolates of T. roseum were grown separately on modified Czapek-Dox medium (MCD) in the dark for 25 days at 25 °C. Culture filtrates of T. roseum at 0.5% and 20.0% concentrations inhibited mycelial growth of Pmg at 3.1% and 90.4%; and those of G. roseum at –0.7% and 44.0%; and of T. harzianum at 0.7% and 46.0%, respectively. Culture filtrates of T. roseum at 0.5% concentration inhibited zoosporangenesis of Pmg at 98.0% and at 1.0% concentration prevented it. Culture filtrates of G. roseum and T. harzianum at 5% concentration significantly (P=0.05) inhibited zoosporangenesis of Pmg, but differences were not significant from that on MCD. Culture filtrates of 17 isolates of T. roseum inhibited mycelial growth and zoosporangenesis of Pmg at different percentages with different concentrations with those of isolate 9 showing the greatest inhibition of both. Mycelial growth of most of 16 races of Pmg was prevented at 10% concentration of the culture filtrates of isolate SS-2 of T. roseum, and all 16 races, except race 6, was prevented at 20% concentration. Zoosporangenesis of all races of Pmg was prevented at 2% the culture filtrate of SS-2. Culture filtrates of SS-2 inhibited zoosporangenesis of Pmg in soil.     

Lauric Acid Exhibits Antifungal Activity Against Plant Pathogenic Fungi

This study aimed at examining the effects of the saturated fatty acid lauric acid on mycelial growth of Rhizoctonia solani and Pythium ultimum and on infection of barley seedlings with Blumeria graminis f. sp. hordei. Mycelial growth of R. solani and P. ultimum in agar culture was significantly reduced by lauric acid at concentrations of 100 μm and above, while no fungal growth occurred in liquid culture at concentrations above 50 μm . Application of lauric acid at concentrations ranging from 250 to 1000 μm to barley leaves before or after inoculation with B. graminis f. sp. hordei led to significant reductions in infection. This study provides the first report of the activity of lauric acid against plant pathogenic fungi and indicates the need for investigation of its mechanism of action.     

First Report of Sclerotium rolfsii in Dandelion in Parana,Brazil

Taraxacum officinale (dandelion) is a medicinal plant that occurs in various countries and is also reported as an invasive plant in some parts of the world. Recently, a severe case of southern blight was observed in dandelion in the Medicinal Plant House at the State University of Maringa, Umuarama, State of Parana, Brazil. A dense, cottony mycelial growth and the formation of sclerotia were observed on the plants. The fungus was isolated, inoculated into healthy plants and re‐isolated. Samples were sequenced for rDNA regions ITS4 and ITS5. The inoculated plants presented symptoms of southern blight, beginning at the base of the plant and eventually killing the plant. DNA analysis revealed a 99% species identity index for Athelia rolfsii (anamorph: Sclerotium rolfsii).     

Changes in soil microflora associated with control of Sclerotium Rolfsii by Furfuraldehyde

The efficacy of 2‐furfuraldehyde for control of Sclerotium rolfsii was studied in laboratory and greenhouse experiments. Mycelial growth of the fungus was reduced proportionally with concentrations of 0.1–0.5 ml furfuraldehyde l^‐1 agar medium, and viability of sclerotia diminished on exposure to 2‐furfuraldehyde vapours. Detectable populations of bacteria and fungi, including Trichoderma spp., were reduced significantly (9=0.05) when furfuraldehyde was added to the agar used for soil dilution plates of untreated soil. Repeated treatments of natural soil with the fumigant significantly increased populations of Trichoderma spp. and bacteria, but diminished numbers of actinomycetes. Increasing dosages applied to soil artificially infested with S. rolfsii caused a reduction of disease on lentil, Lens culinaris. Results indicate that the compound, when applied to field soil, changes the composition of soil microflora and has potential for integrated control of S. rolfsii.     

Die Beurteilung des Einflusses qualitativer Prüffaktoren (Infektionstermine,Sorten) auf den Befall mit Hilfe der Kontingenztafelanalyse

Mycelial growth of some wood‐rotting fungi was studied on a solid modified medium MS (Murashige and Skoog, 1962) with indole‐3‐acetic acid at concentrations of 10^‐6 to 10^‐3 M. The IAA concentrations of 10^‐6 M and 10^‐5 M inhibited mycelial growth of the fungus Phaeolus schweinitzii, Laetiporus sulphureus and Pleurotus ostreatus while the same concentrations stimulated mycelial growth of the fungus Stereum rugosum. The IAA concentrations of 10^‐6 M stimulated mycelial growth in Piptoporus betulinus and temporarily stimulated mycelial growth in Heterobasidion annosum. The IAA concentration of 10^‐4 M appeared critical for wood‐rotting fungi. The IAA concentration of 10^‐3 M inhibited mycelial growth in all the fungi under study.     

Suppression by Zygorrhynchus moelleri of Growth and Sclerotium Production by Rhizoctonia solani and Sclerotinia sclerotiorum

As indicated by reduced cellulolysis, Zygorrhynchus moelleri suppressed mycelial growth in Rhizoctonia solani and Sclerotinia sclerotiorum. Sclerotium production by both pathogenic fungi was also reduced by Z. moelleri in dual sand-oatmeal cultures. The viability of sclerotia produced by S. sclerotiorum, but not those produced by R. solani, was greatly reduced. Sclerotium production by S. sclerotiorum on celery and tomato segments was reduced to a much greater extent when Z. moelleri was applied to the plant tissue 24 h before the pathogen than when applied at the same time or 24 h after the pathogen.     

Extracellular Chitinases of Fluorescent Pseudomonads Antifungal to Fusarium oxysporum f. sp. dianthi Causing Carnation Wilt

Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.     

In vitro evaluation of combination of Trichoderma harzianum and chitosan for the control of sapstain fungi

In vitro assays were undertaken to evaluate the control of two sapstain fungi, Leptographium procerum and Sphaeropsis sapinea by a combination of chitosan or chitosan oligomer and an albino strain of Trichoderma harzianum. Spore germination and hyphal growth of the test fungi were assessed on media amended with chitosan or chitosan oligomer with and without T. harzianum using either simultaneous inoculation with test fungus or inoculation 1, 2, or 3 days after pre-infection with test fungus.There was no mycelial growth of the test fungi regardless of chitosan concentrations used when either L. procerum or S. sapinea was simultaneously inoculated with T. harzianum. However, the dose–response of chitosan or chitosan oligomer on the test fungi was apparent when T. harzianum was not simultaneously inoculated with test fungus but introduced later. There was a greater growth reduction at higher concentrations (0.075–0.1% v/v) of chitosan, and overall chitosan oligomer was more effective than chitosan aqueous solution.Chitosan alone was able to restrict or delay the germination of spores but the combination of chitosan and T. harzianum inhibited spore germination and hence colony formation of test fungi regardless of time delay.     

Trichoderma harzianum and its Metabolite 6-Pentyl-alpha-pyrone Suppress Fusaric Acid Produced by Fusarium moniliforme

The interaction of the pathogen Fusarium moniliforme and two antagonistic Trichoderma harzianum isolates was studied especially with respect to their secondary metabolites fusaric acid (FA) and 6‐pentyl‐alpha‐pyrone (6PAP). Among 10 isolates of F. moniliforme screened for FA production on maize kernels, the isolate 8 accumulated the highest amount of FA (678 μg/g). Mycelial growth and production of FA by isolate 8, determined in different liquid media revealed that the highest biomass and FA were produced in Czapek Dox Broth (CDB) followed by Richard’s solution. The amount of FA per gram mycelial dry weight reached its maximum in CDB and Richard’s solution after 14 days of incubation. Mycelial growth and conidia production of both Trichoderma isolates (T16 and T23) were retarded by increasing concentrations of FA in agar medium. At FA concentration of 300 mg/ml the radial mycelial growth of the isolates T16 and T23 were retarded by 32.5% and 45%, respectively. Conidia production was diminished in a similar extent as mycelial growth. Both T. harzianum isolates were capable to degrade FA in potato dextrose broth medium, particularly when lower doses of FA were present. In the presence of 50 mg/ml FA in the culture medium, the isolates T23 and T16 reduced FA by 51.4% and 88.4%, respectively, 9 days post‐inoculation. The antifungal metabolite 6PAP, isolated from T. harzianum T23 cultures, was introduced at different concentrations into 2‐day‐old cultures of F. moniliforme. After further 5 days of incubation of F. moniliforme in the presence of 6PAP, the FA contents per gram mycelial dry weight were significantly decreased compared to control cultures where 6PAP was absent. Dosages of 300 and 400 mg/l of 6PAP in the cultures retarded FA accumulations by 62.5% and 77.2%, respectively. The current results, however, provided the first evidence for activity of 6PAP, as a Trichoderma secondary metabolite, on degrading/synthesis suppression of the Fusarium toxin FA.     

Induced tolerance of Sclerotium cepivorum to antibiotics in the presence of onion exudates

The onion white rot pathogen Sclerotium cepivorum was cultured on agar media containing 2% malt extract and one of the antifungal antibiotics, endomycin, griseofulvin, venturicidin and cycloheximide at concentrations that reduced but did not prevent growth of mycelium. When onion seeds or agar discs impregnated with diffusates from onion bulbs were placed on the antibiotic media, radial growth of the fungus was greatly increased, and there was a profuse development of aerial mycelium. Gaseous diffusates from onion tissue and from impregnated agar discs were also effective. On the antibiotic media, tomato, cabbage and radish seeds did not stimulate the growth of S. cepivorum and the onion exudates did not stimulate the growth of four other fungi. This and other evidence is considered to show that the stimulation of growth of S. cepivorum was not caused by any direct effect on the antibiotics but by a tolerance of the fungus to them, which was specifically induced by an exudate from its host plant. The phenomenon may be related to the reported reversal by onion extracts of the inhibitory effects of soil mycostasis on germination of sclerotia of the fungus.     

Effects of sodium chloride on growth of ectomycorrhizal fungal isolates in culture

We studied the tolerance of ectomycorrhizal (ECM) fungi to sodium chloride (NaCl) to find the best fungus to aid growth of Pinus thunbergii. Four ECM fungi, Cenococcum geophilum, Pisolithus tinctorius, Rhizopogon rubescens, and Suillus luteus, were grown in liquid MMN media with five different concentrations of NaCl for 30 days, and their mycelial weights were determined. Mycelial weights of P. tinctorius and R. rubescens were not significantly different between 0 mM and 200 mM, whereas those of C. geophilum and S. luteus decreased with increasing NaCl concentration, indicating that the former two species were more tolerant to higher NaCl concentrations than the latter species. We further studied the intraspecific differences in NaCl tolerance of nine P. tinctorius isolates. They were grown on MMN agar media with six different concentrations of NaCl for 21 days, and their radial growth was measured. In total, the hyphal growth at 25 mM NaCl was significantly higher than those at the other NaCl concentrations, and EC₅₀ values were confirmed at between 50 mM and 200 mM. Among the isolates, Pt03 and Pt21 showed measurable growth at 200 mM; the growth of Pt03 was not significantly different between 0 mM and 200 mM. The results indicate that there are intraspecific variations in NaCl tolerance of Pisolithus species.     

Selection of antagonistic soil microbes against a few fungal pathogens of piper betle L

Summary Against the fungal pathogens,Phytophthora parasitica, Sclerotium rolfsii, Colletotrichum capsici andGlomerella cingulata responsible for leaf and foot rot of betel vines, 3 fungus, 9 actinomycetes and 4 bacterial antagonists were screened out from 61 fungus, 28 actinomycetes and 4 bacterial organisms isolated from 3 sources of soils. One each from the 3 groups of antagonists when further tested, was found quite effective against the pathogens towards neutral side of H-ion concentration. The antagonists (P. citrinum, Streptomyces sp. and bacterial organism B-7) were also found effective against 18 among 32 isolated fungus organisms from potted soil (collected from betel vine soil) and except one which happened to beP. citrinum, the rest had no adverse effect upon their growth.This work is a part of a scheme supported by Food and Agriculture Council of Pakistan and conducted in Jute Research Institute.     

Effect of some antibiotics on Sclerotium cepivorum Berk., the cause of white rot of onion

The effect of some antibiotics onSclerotium cepivorum, the cause of white rot of onion was studied in agar culture and soil. The growth ofS. cepivorum was inhibited in Czapek Dox yeast agar containing 50µg of gliotoxin, viridin, actidione and 100µg of patulin per ml of the medium. Lower concentrations of the antibiotics retarded the growth of the fungus. In soil, patulin had no effect in the control ofS. cepivorum infection of onion seedlings. Concentration of actidione of 5µg/g of soil completely controlled white rot infection but severely stunted the growth of onion seedlings; 40µg/g of actidione killed the seedlings. Despite the importance of actidione as a fungistatic agent its use on onion is limited by its phytotoxicity.