RFLP analysis for species separation in the generaBipolaris andCurvularia

Restriction fragment length polymorphism (RFLP) of the total DNA ofBipolaris andCurvularia species was analysed using arbitrarily chosen genomic clones of DNA fromCurvularia lunata andBipolaris maydis as probes. Clear differences among species in both genera, resulting in different banding positions, were obtained with some probe-enzyme combinations. Intraspecific polymorphism in banding positions with these probe-enzyme combinations was slight. These analyses allow discrimination between the species. DNA fingerprinting with intrageneric probes is a potentially useful tool for species separation and identification inBipolaris andCurvularia when coupled with another characteristic such as conidial morphology.Curvularia aeria comb. nov. was proposed forCurvularia lunata var.aeria on the basis of differences in RFLP banding patterns and differences in conidial morphology.     

Mycotic keratitis caused by Curvularia lunata var. aeria

A mycotic keratitis case which was caused by Curvularia lunata var. aeria in a patient with an injury in the eye is described. The diagnosis was based on the mycologic analysis of several samples taken from the ulcer of cornea. In vitro tests of the sensitivity of the isolated species to several antifungal drugs were made. The results were related to the response in vivo to the treatment.     

Survival of Curvularia lunata var. aeria in soil

Spore suspensions from 7 day old cultures of Curvularia lunata (Wakker) Boedijn var. aeria (Batista, Lima and Vasconcelos) M.B. Ellis were added to soil samples originally devoid of Curvularia propagules. The test fungus disappeared after six weeks of inoculation irrespective of soil amendments, like the application of green manure, urea, superphosphate, or soil cropped to Sorghum (dura) or cotton seedlings.     

Methanogens in the human intestinal tract and oral cavity

Curvularia lunata var.aeria was grown in YPD (yeast extract, peptone, and dextrose) medium (pH 6.5) at 28°C with varying concentrations (10–40 g/L) of glucose for the production of rifamycin oxidase. Enzyme activity and glucose concentration were found to be indirectly related to the production of black intracellular pigment by the organism. Depletion of glucose level and rise of culture pH initiate the synthesis of pigment. Carboxymethylcellulose (CMC) was used as a carbon source to improve the enzyme yield, but utilization of the substrate in the reactor was much less. Compared with 10 g/L of CMC in the medium, low or high concentrations of CMC did not yield any better result. Addition of glucose in YPC (yeast extract, peptone, and CMC) medium did not increase the enzyme activity, and glucose was rapidly utilized byC. lunata, forming pellets rather than mycelia.     

Variability among strains of Trichoderma harzianum in their ability to reduce take-all and to produce pyrones

Antagonism tests on agar-plates and glasshouse screening indicated that three isolates of Trichoderma harzianum varied in their ability to antagonize the take-all fungus (Gaeumannomyces graminis var. tritici). Isolate 71 which was the most effective in suppressing take-all of wheat, produced two pyrones and other undetermined analogues. Isolates of T. koningii and T. hamatum shown to suppress take-all, produced a simple pyrone compound. Although T. harzianum isolates 70 and 73 did not produce any pyrones, they reduced the disease albeit to a much lesser extent than isolate 71; with isolate 73 showing distinct host growth promotion effects. It is proposed that the success of isolate 71 of T. harzianum was related to the pyrones it produces and that the ability of isolates 70 and 73 to reduce take-all may be related to mechanisms other than those involving antibiotics.     

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.     

Trichoderma species for biocontrol of soil-borne plant pathogens of pasture species

Soil-borne plant pathogens such as Rhizoctonia solani (Kuhn), Pythium ultimum (Trow) and Sclerotinia trifoliorum (Eriks) can reduce grass and forage legume establishment. The potential for biocontrol of these pathogens by Trichoderma fungi was evaluated. Following dual culture assays, nine Trichoderma isolates (five of Trichoderma atroviride and one each of Trichoderma hamatum, Trichoderma koningiopsis, Trichoderma viride and Trichoderma virens) were chosen for assessment in pot experiments. In the presence of R. solani, perennial ryegrass (Lolium perenne L.) emergence was increased by 60–150% by two isolates of T. atroviride and by 35–212% by the isolate of T. virens, with the increase depending on growing medium and amount of pathogen inoculum. Red clover (Trifolium pratense L.) emergence in the presence of S. trifoliorum was significantly increased by two T. atroviride isolates and the T. hamatum isolate. In the presence of P. ultimum, white clover (Trifolium repens L.) emergence was increased by 25–42% by one isolate of T. atroviride and the T. hamatum isolate. However, for all three pasture species, some Trichoderma isolates reduced seedling emergence. Seedling growth (shoot and root fresh weight/plant) of the three pasture species was significantly increased by one or more T. atroviride isolates. On the basis of these results for both disease reduction and growth promotion, four T. atroviride isolates were selected for field assessment as biocontrol agents of soil-borne pathogens of pasture species.     

Cellulose degradation and cellulase activity of five cellulolytic fungi

Biodegradation of pure cellulose powder, bagasse and wheatstraw by five cellulolytic fungi,Aspergillus niger, Chaetomium globosum, Scopulariopsis brevicaulis, Trichoderma koningii andTrichothecium roseum, was studied in solid culture conditions. Minimum degradation was with pure cellulose. Bagasse and wheatstraw were the most suitable for growth and activity of cellulolytic fungi. All fungi contained cellulase activity.

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Résumé On a étudié la biodégradation de la poudre de cellulose pure, de la bagasse et de la paille de froment chez cinq moisissures cellulolytiques,Aspergillus niger, Chaetomium globosum, Scopulariopsis brevicaulis, Trichoderma koningii etTrichothecium roseum, en condition de culture sur milieu solide. La dégradation minimum a lieu avec la cellulose pure. La bagasse et la paille de froment offrent la meilleure croissance et la meilleure activité cellulolytique fungale. Toutes les moisissures exhibent l'activité de la cellulase.

     

拮抗北细辛菌核病木霉菌的分离、鉴定及生防效果

【背景】菌核病是北细辛根部主要病害之一,木霉菌作为目前应用最广泛的生物防治真菌,利用木霉菌防治北细辛菌核病是目前研究的热点。【目的】通过稀释分离法对健康北细辛植株根际土壤进行菌株分离,以期筛选出有效拮抗北细辛菌核病的生防木霉菌。【方法】以北细辛菌核病菌为靶标菌,采用平板对峙培养、挥发性与非挥发性物质抑菌的方法对分离得到的木霉菌进行筛选,采用生长速率法对筛选出的木霉菌的发酵液进行抑菌效果测定,并采用硫代巴比妥酸法测定筛选出的木霉对北细辛菌核病菌的丙二醛(Malondialdehyde,MDA)含量、紫外吸收法测定过氧化氢酶(Catalase,CAT)活性、氮蓝四唑法测定超氧化物歧化酶(Superoxide Dismutase,SOD)活性、愈创木酚法测定过氧化物酶(Peroxidase,POD)活性的影响。【结果】从土壤中分离出木霉菌共14株,通过形态学和ITS-RPB2双基因联合构建系统发育树,鉴定其为哈茨木霉(Trichoderma harzianum)、钩状木霉(Trichoderma hamatum)、拟康氏木霉(Trichoderma koningiopsis)、深绿木霉(Tr...     

Ovine III-thrift in Nova Scotia. 7. Trichoderma hamatum isolated from pasture soil

Trichoderma hamatum accounts for about 1.5% of the fungal propagules isolated from pasture soil at Nappan, Nova Scotia. Greater numbers of propagules of this fungus were isolated in June and September than in July, August and October, with the apparent optimum growth temperature in the field being about 18 ° C. Unlike the fungal flora collected, propagules of T. hamatum were not randomly distributed throughout the experimental plot. Fifty three percent of isolates of T. hamatum (or 1% of the fungal propagules isolated) in laboratory culture produced toxic, water-soluble metabolites. Two of these metabolites are isonitrile acids.     

Biocontrol of Sclerotinia sclerotiorum infection of cabbage by Coniothyrium minitans and Trichoderma spp.

Nine fungal isolates [Clonostachys rosea (1), Coniothyrium minitans (1), Trichoderma crassum (1), T. hamatum (4), T. rossicum (1) and T. virens (1)] were tested in two bioassays for their ability to degrade sclerotia and reduce apothecial production and carpogenic infection of cabbage seedlings. C. minitans LU112 reduced apothecial production in both bioassays, with T. virens LU556 significantly reducing apothecial production in the sclerotial parasitism assay. Both isolates also reduced sclerotial viability in this assay to 5% for C. minitans and 22% for T. virens. C. minitans LU112 and T. virens LU556 reduced the infection of cabbage seedlings in the pot bioassay 126 days after sowing but not after 147 days, partly due to ascospore cross-infection between treatments. C. minitans LU112, T. virens LU556 and T. hamatum LU593 as maizemeal-perlite (MP) soil incorporation and transplant potting mix incorporation were evaluated for their ability to control Sclerotinia sclerotiorum disease of cabbage in field experiments. S. sclerotiorum infection of cabbage was reduced by 46–52% and 31–57% by both C. minitans LU112 and T. hamatum LU593 as MP soil incorporations, respectively, in the two field experiments. T. virens LU556 MP soil incorporation and C. minitans LU112 and T. hamatum LU593 transplant potting mix incorporations reduced S. sclerotiorum disease in the first experiment but not in the second experiment. A commercial C. minitans LU112 formulation, C. Mins LU112 WG, also significantly reduced S. sclerotiorum disease by 59%. Soil incorporation of C. minitans and T. hamatum was shown to have potential to control S. sclerotiorum disease in cabbage.     

Rhizocorallium hamatum (Fischer-Ooster 1858), a Zoophycos-like trace fossil from deep-sea Cretaceous-Neogene sediments

Rhizocorallium hamatum (Fischer-Ooster 1858) is a trace fossil of the Zoophycos group, which is distinguished by its mostly horizontal, branched spreite lobes. It has so far, been ascribed mainly to Zoophycos, but the latter should be limited to forms having helical whorls, which are absent in R. hamatum. It has also been ascribed to Phycosiphon, which, however, shows J-shaped spreite lobes, while the lobes of R. hamatum are U-shaped. R. hamatum is very similar to R. commune var. irregulare, but the latter displays a distinctly wider marginal tunnel with respect to lobe width. R. hamatum occurs from the Turonian to Eocene, possibly from the Hauterivian to Oligocene, but mostly from Maastrichtian to Palaeocene, deep sea, mainly turbiditic sediments rich in marl. The tracemaker, probably a ‘worm’-like invertebrate, ingested an organic-rich mud of the background sediment and relocated it into the middle to deep tiers within the underlying turbiditic marl, mostly in form of faecal pellets packed within the spreite lobes, for further use as a food resource. This way of feeding was a response to food deficiency on the deep-sea floor.     

Inhibitory effects of sodium silicate on the fungal growth and secretion of cell wall‐degrading enzymes by Trichothecium roseum

Trichothecium roseum causes decay in muskmelons, apples, tomatoes and mangoes, which leads to economic losses. In this study, we investigated the effect of sodium silicate on the growth of T. roseum and the cell wall‐degrading enzymes (CWDEs) secreted by the hyphae. The results indicated that sodium silicate significantly inhibited mycelial growth and spore germination of T. roseum. The sodium silicate treatment also retarded the secretion of several CWDEs, including pectate lyase (PL), polygalacturonic acid transeliminase (PGTE), pectin methyltranseliminase (PMTE), pectin methylgalacturonase (PMG), polygalacturonase (PG), cellulase (Cx) and β‐glucosidase. These results suggest that sodium silicate exerts its effects on T. roseum through direct inhibition of its growth and secretion of CWDEs.     

Precise Detection and Tracing of Trichoderma hamatum 382 in Compost-Amended Potting Mixes by Using Molecular Markers

Randomly amplified polymorphic DNA (RAPD) analysis and the PCR assay were used in combination with dilution plating on a semiselective medium to detect and enumerate propagules of Trichoderma hamatum 382, a biocontrol agent utilized in compost-amended mixes. Distinct and reproducible fingerprints were obtained upon amplification of purified genomic DNA of T. hamatum 382 with the random primers OPE-16, OPH-19, and OPH-20. Three amplified DNA fragments of 0.35 (OPE-16_0.35), 0.6 (OPH-19_0.6), and 0.65 (OPH-20_0.65) kb were diagnostic for T. hamatum 382, clearly distinguishing it from 53 isolates of four other Trichoderma spp. tested. Some isolates of T. hamatum shared these low-molecular-weight fragments with T. hamatum 382. However, RAPD analysis of isolates of T. hamatum with all three random primers used in consecutive PCR tests distinguished T. hamatum 382 from other isolates of T. hamatum. These three RAPD amplicons were cloned and sequenced, and pairs of oligonucleotide primers for each cloned fragment were designed. Use of the primers in the PCR assay resulted in the amplification of DNA fragments of the same size as the cloned RAPD fragments from genomic DNA of T. hamatum 382. A combination of dilution plating on a semiselective medium for Trichoderma spp. and PCR, with the RAPD primers OPH-19, OPE-16, and OPH-20 or the three sequence-characterized primers, was used successfully to verify the presence of T. hamatum 382 propagules in nine different soil, compost, and potting mix samples. All 23 Trichoderma isolates recovered on semiselective medium from commercial potting mixes fortified with T. hamatum 382 were identified as T. hamatum 382, whereas 274 Trichoderma isolates recovered from the other nine samples were negative in the PCR assay. Thus, this highly specific combination of techniques allowed detection and enumeration of propagules of T. hamatum 382 in fortified compost-amended potting mixes. Sequence-characterized amplified region markers also facilitated the development of a very simple procedure to amplify DNA of T. hamatum 382 directly from fortified compost-amended potting mixes.     

Biocontrol of Sclerotinia lettuce drop by Coniothyrium minitans and Trichoderma hamatum

Fungal isolates, with known activity against Sclerotinia spp. in laboratory assays, were tested for their ability to control Sclerotinia minor in four field experiments (1998–2000). In the first experiment, eight fungal isolates (Trichoderma hamatum LU595, LU593, LU592, Trichoderma virens LU555 and LU556, Coniothyrium minitans LU112, Clonostachys rosea LU115 and Trichoderma rossicum LU596) were evaluated by incorporating spore suspensions into transplant potting mix and planting lettuce seedlings into a S. minor infested field site. At harvest, Trichoderma hamatum LU595, LU593, T. virens LU555 and C. minitans LU112 reduced disease by 30–50% compared with the untreated control under very high disease pressure (100%). In further field experiments C. minitans LU112 and T. hamatum LU593, applied as maizemeal–perlite soil amendments or incorporated into the potting mix, reduced S. minor disease over a range of disease pressures (29–91%). Disease control was equivalent or greater than that achieved with the standard carbendazim fungicide treatment. Both isolates were shown to effectively colonize the lettuce rhizosphere and surrounding soil and this colonization may have protected the roots from infection by S. minor. Multiple applications of C. minitans LU112 or T. hamatum LU593 formulations gave no added disease control compared with a single application at planting. Commercial formulations of both C. minitans LU112 and T. hamatum LU593 applied as transplant treatments, solid substrate soil amendments or as a spore drench gave consistent disease control and are currently being developed further.     

Interactions among vegetable-infesting aphids,the fungal pathogen Metarhizium anisopliae (Ascomycota: Hypocreales) and the predatory coccinellid Cheilomenes lunata (Coleoptera: Coccinellidae)

Entomopathogenic fungi are among biocontrol agents being considered for the control of aphids on a variety of crops. Predatory coccinellids, although generalist, are also among important natural enemies that must be conserved for aphid management. Laboratory studies were carried out to investigate the interaction between three vegetable-infesting aphids, Metarhizium anisopliae isolate ICIPE 62 and the coccinellid predator Cheilomenes lunata. At a concentration of 1?×?10⁸?conidial?ml^–1, the fungus was found to cause mortality of 7.5% to C. lunata, compared to 2.5% mortality in the control at 10?days post-treatment. Female adult C. lunata to which fungus-infected aphids were offered as prey never accepted them as food source in non-choice bioassays. However, live and dead non-infected aphids were fed upon. In choice bioassay, a total of 1–3 out of 24 infected non-sporulating aphids per species (average of 0.1–0.4 aphids per arena) were consumed by 48?h-starved C. lunata within a period of 60?min, but avoided sporulating cadavers. Foraging adult C. lunata enhanced the spread of conidia of M. anisopliae from infected cadavers to fourth instars Aphis gossypii feeding on okra (0.8–15.0% mortality), Brevicoryne brassicae (3.3–15.0% mortality) and Lipaphis pseudobrassicae (0.8–14.2% mortality) on kale plants. Results of this study demonstrate compatibility between M. anisopliae and C. lunata, and could provide a sustainable strategy for effective management of aphids on crucifers and okra cropping systems.     

Microbial transformation of rifamycin B: A new extracellular oxidase fromCurvularia lunata

Summary A highly active extracellular rifamycin oxidase was isolated fromCurvularia lunata var.aeri. The enzyme has a pH optimum of 6.5 and temperature optimum of 50°C.     

Trichoderma hamatum: Its hyphal interactions withRhizoctonia solani andPythium spp.

The mode of hyphal interaction and parasitism ofPythium spp. andRhizoctonia solani byTrichoderma hamatum was studied by both phase-contrast and Nomarski differential interference-contrast microscopy. Directed growth of the mycoparasite toward its host was observed. In the area of interaction,T. hamatum produced appressorial-like structures attached to the host cell wall. Subsequently, several different types of interactions occurred.T. hamatum either grew parallel to and along the host hypha or coiled around its host. In the contrast regions the parasite formed bulbular or hook-like structures that contained granular cytoplasm. In other cases the parasite penetrated into and grew within the mycelium ofR. solani orP. ultimum. As a consequence of the attack, the host hypha became vacuolated, shrank, collapsed, and finally disintegrated. These observations suggest the involvement of parasitism followed by lysis rather than involvement of antibiotics in this host-mycoparasite relationship.     

Production of docosahexaenoic acid byThraustochytrium roseum

Summary When threeThraustochytrium stains were cultivated in liquid media containing 2.5% starch and 0.2% yeast extract, initial pH 6.0, with shaking under fluorescent light for five days at 25°C, similar biomass yields were observed (9.7–10.3 g L^–1). Contents of docosahexaenoic acid (DHA) in biomass varied: 0.15, 3.55 and 6.40% w/w forT. striatum ATCC 24473,T. aureum ATCC 34304 andT. roseum ATCC 28210, respectively. In further studies,T. roseum produced a maximum titer of 0.85 g of DHA per liter of culture broth. The DHA content of total lipids ranged from 46–49% w/w.     

Production of acetyl esterase during wood biodegradation byCoriolus versicolor

A role of acetyl esterase in wood biodegradation byCoriolus versicolor was examined by the assay of enzyme production and the chemical analysis of decayed wood meal of Japanese beech (Fagus crenata). Enzyme assay demonstrated that the degradation proceeded in two stages and acetyl esterase production was correlated with the cellulolytic and xylanolytic enzyme production in the second stage, not with the production of phenol-oxidizing enzymes. From the results of chemical analysis, acetyl and xylose contents in wood meal were observed to decrease simultaneously in the second stage. In contrast, rapid decrease of lignin was recognized during the initial three wk of incubation, and it was closely related with the production of phenol-oxidizing enzymes in the first stage. These results show that acetyl esterase ofC. versicolor participates in the degradation of acetylxylan and acts with the cellulolytic and xylanolytic systems, not with the ligninolytic system.