Survival of Sclerotia of Rhizoctonia solani AG3PT and Effect of Soil‐Borne Inoculum Density on Disease Development on Potato

Sclerotia produced by a single isolate of Rhizoctonia solani AG3PT were buried in small plot experiments to investigate the effects of sclerotial production method, soil type and burial depth on sclerotial viability in field soil. The factor with the greatest effect on sclerotial viability, defined as the percentage of sclerotia germinating on agar following retrieval, in all experiments was the duration of burial. After 18 months, on average across all experiments, 20% of retrieved sclerotia were viable. A comparison between sclerotia produced in vitro on malt yeast extract agar and in vivo using micropropagated tubers in field soil found no significant differences between the two production methods on sclerotial viability. Burial in field soil at 20‐cm depth was found to significantly reduce sclerotial viability to 50% compared to 60% at 5 cm. In two pot experiments, amending the growing medium and soil with increasing inoculum densities of R. solani was found to increase stem number, stem canker and black scurf severity regardless of whether this soil‐borne inoculum was derived from mycelium or sclerotia. Black scurf incidence and severity were assessed 30–32 days posthaulm destruction and found to be similar for a range of sclerotial soil‐borne inoculum densities (1.0 × 10^?1 g/kg d.w. soil to 6 × 10^?3 g/kg d.w. soil). The significance of these findings in relation to pathogen survival, detection in soil and disease development is discussed.     

Evidences of biological control capacities of Streptomyces spp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet (Beta vulgaris L.)

Ten antibiotic-producing Streptomyces spp. isolated from Moroccan soils were evaluated for their ability to inhibit in vitro Sclerotium rolfsii development. Four isolates having the greatest pathogen inhibitory capabilities were subsequently tested for their ability to inhibit sclerotial germination in sterile soil. This test was carried out by using biomass inoculum, culture filtrate, and spore suspension of the isolates as treatment. Treatment with biomass inoculum and culture filtrate gave the highest inhibition of sclerotia. Biological control tests against Sclerotium rolfsii damping-off of sugar beet seeds showed that the selected Streptomyces isolates reduced significantly the disease severity, the J-2 isolate being the more potent. In addition, treatment with the isolate J-2 resulted in a significant increase (P ≤ 0.05) in seedling development compared to the control. All antagonistic Streptomyces selected here were able to grow in the rhizosphere soil from infected sugar beet culture.     

Extruded Granular Formulation with Biomass of Biocontrol Gliocladium virens and Trichoderma spp. to Reduce Damping-off of Eggplant Caused by Rhizoctonia solani and Saprophytic Growth of the Pathogen in Soil-less Mix

Extruded granular formulations containing rice flour, gluten, Pyrax, vermiculite, canola oil, and fermentor-produced biomass of isolates of Gliocladium virens (Gl-3, Gl-21 and Gl-32), Trichoderma hamatum (TRI-4 and 31-3), T. harzianum (Th-32 and Th-87) and T. viride (Tv-101) were evaluated for their effect on the reduction of eggplant damping-off caused by Rhizoctonia solani, reduction of pathogen inoculum and proliferation of the isolates in a soil-less mix. Granules with all isolates except 31-3 significantly (P < 0.01) reduced damping-off, and granules with Gl-3, Gl-21, Gl-32, TRI-4 and Th-87 yielded stands comparable to that (90%) of the non-infested control. Granules with isolates Gl-21 and TRI-4 were the most effective in the reduction of saprophytic growth of R. solani, and there was a significant inverse correlation (r ² = - 0.82) between eggplant stand and saprophytic growth of the pathogen over all treatments. Isolate propagules proliferated to about 10⁷ colony-forming units (CFU) g^?1 of soil-less mix after a 6-week incubation, but there was no correlation between the number of CFU and eggplant stand or saprophytic growth reduction of the pathogen. Granules with Gl-21 and TRI-4 amended to pathogen-infested soil-less mix at a rate as low as 0.06% significantly (P < 0.05) reduced damping-off and pathogen saprophytic growth, and a rate of 0.25% of Gl-21 granules resulted in an eggplant stand comparable to that of the non-infested control. There was no significant correlation between the rate of granule amendment and the proliferation of Gl-21 and TRI-4. Granules of Gl-21 and TRI-4 also significantly prevented the spread of R. solani in flats of eggplant seedlings when the biocontrol granules were applied to the soil-less mix 1 day before the pathogen inoculum.     

Relationship between the population of viable sclerotia ofSclerotium rolfsii in soil to cropping sequence in the Nigerian savanna

Populations of viable sclerotia ofSclerotium rolfsii were highest in soil in a field in which tomato was planted for three successive years before sampling and in one in which tomato followed groundnut in the 2 years prior to this study. The lowest sclerotial numbers were recorded in fields in which groundnut followed maize or in which maize or sorghum was the last crop before sampling.     

Effect of copper-induced oxidative stress on sclerotial differentiation and antioxidant properties of Penicillium thomii PT95 strain

Penicillium thomii PT95 strain was able to form abundant orange, sand-shaped sclerotia in which carotenoids were accumulated. The aim of this work was to determine the effects of copper-induced oxidative stress on the sclerotial differentiation and antioxidant properties of PT95 strain. The results showed that the time of exudates initiation, sclerotial initiation and sclerotial maturation of PT95 strain were advanced in 1–2 days under the copper-induced oxidative stress growth conditions. The analytical results of sclerotial biomass, carotenoids content in sclerotia showed that copper-induced oxidative stress favored the sclerotial differentiation and biosynthesis of carotenoids. Under the copper-induced oxidative stress growth conditions, the total phenolics content and DPPH free radical scavenging activity of sclerotia of this fungus were decreased as compared with the control. However, the oxidative stress induced by a lower amount of CuSO₄ in media could enhance significantly the reducing power of sclerotia.     

Effects of forest fertilization on nitrogen leaching and soil microbial properties in the Northern Calcareous Alps of Austria

Several boreal and alpine forests are depleted in nutrients due to acidification. Fertilization may be a remedy, but rapidly-soluble salts (N, P, K, Mg) may pose nitrate problems for the groundwater or decrease microbial activity.With the aim to investigate potential nitrogen leaching after fertilization we set up an experiment employing intact soil cores (11 cm diameter, 20–40 cm long) from a mixed forest and a Picea abies stand (soil type Rendsina) in the Northern Calcareous Alps of Austria. The cores were fertilized with a commercial NPK fertilizer or a methylene-urea-apatite-biotite (MuAB) fertilizer at a rate corresponding to 300 kg N ha^-1 and incubated for 28 weeks together with unfertilized controls. Both soil water (retrieved 5 cm below the soil surface) and leachate were analyzed for nitrate and ammonium in regular intervals. After the incubation, soil microbial biomass and basal repiration were determined and a nitrogen mineralization assay was performed.For the control, in the soil water and leachate maximum NH₄ ⁺ and NO₃ ^- concentrations of 5 and 11 mg N L^-1, respectively, were found. Compared to that, MuAB fertilizer resulted in a slow increase of NH₄ ⁺ and NO₃ ^- in the soil water (up to 11 and 35 mg N L^-1 respectively) and in the leachate (4 mg NH₄ ⁺-N L^-1 and 44 mg NO₃ ^--N L^-1). Highest nitrogen loads were found for the fast release NPK fertilizer, with NH₄ ⁺ and NO₃ ^- concentrations up to 170 and 270 mg N L^-1, respectively, in the soil water. NH₄ ⁺-N levels in the leachate remained below 5, while NO₃-N levels were up to 190 mg L^-1. Fast- release NPK caused a significant decrease of microbial biomass and basal respiration. These parameters were not affected by MuAB fertilizer.The results suggest that the MuAB fertilizer may be an ecologically appropriate alternative to fast-release mineral fertilizers for improving forest soils.     

Sclerotial development, melanin production and lipid peroxidation bySclerotium rolfsii

Melanin pigments constituted 13.9% (W/W) of sclerotial walls ofSclerotium rolfsii. The lipid and ash contents in sclerotial walls were twice those in hyphal walls of the fungus. Progress in culture age and maturation of sclerotia were always accompanied by increased levels of lipid peroxidation products and melanin. Lipid peroxidation and melanin formation may thus proceed in parallel during sclerotial biogenesis and maturation. Both these processes are strongly affected by Fe²⁺ and by antioxidant vitamins (ascorbic acid), microelements (selenium) and mercapto compounds (glutathione). Myceliogenic germination and lytic activityvia melanin production can thus be affected by (anti)oxidants that could potentially be used for controlling sclerotia-producing fungi without using traditional toxic fungicides.     

Nitrogen uptake in needles of Scots pine (Pinus sylvestris L.) when exposed to gaseous ammonia and ammonium fertilizer in the soil

Young saplings of Pinus sylvestris L. were exposed to gaseous NH₃ at 53 or 105 g m^–3 for one year in open-top chambers. Saplings received ¹⁵N-labelled (NH₄)₂SO₄ via the soil. To examine the importance of foliar N uptake, changes in the concentration of total and labelled N in the needles were followed. Increase in needle biomass and N concentration were found in trees exposed to NH₃, confirming that atmospheric NH₃ acted as a N fertilizer. NH₃ had a greater and quicker effect than (NH₄)₂SO₄: compared with the growth in ambient air, the N concentration in the needles exposed to NH₃ had increased by 49% in four months, while the increase after highest N-fertilization (200 kg N ha^–1 y^–1) was only 8%. The small contribution of NH₄ ⁺ fertilization to the total N concentration was not due to a deficient N uptake: the ¹⁵N concentration in the needles increased significantly with time. On the other hand, NH₃ uptake in shoots may have a negative effect on the NH₄ ⁺ root uptake. The relation between plant N and atmospheric NH₃ concentration was non-linear and possible reasons for this observation are discussed. Fumigation with NH₃ significantly decreased the ratios of K/N and P/N, showing that fumigation disrupted the nutrient balance.     

Biochemical Investigations of Sclerotial Exudates of Sclerotium rolfsii and their Antifungal Activity

Exudates from sclerotia of two Sclerotium rolfsii isolates (one causing collar rot in Cicer arietinum, isolate VC971, and the other leaf spots in Rauvolfia serpentina, isolate VL016) were assayed for their antifungal activity against 26 fungi consisting of plant parasites as well as saprophytes. Spore germination of all the test fungi was affected by the exudates reaching 100% in some cases. Foliar spray with exudates of isolate VL016 significantly reduced disease incidence of balsam (Impatiens balsamina) powdery mildew caused by Erysiphe cichoracearum and pea (Pisum sativum) powdery mildew caused by Erysiphe pisi, under field conditions. Characterization of exudates from 25 isolates of S. rolfsii revealed pH ranging from 3.8 to 5.3 and colour from light yellow to deep yellow. Among the phenolic acids found in the exudates were tannic, gallic, caffeic, vanillic, ferulic, chlorogenic and cinnamic acids. Oxalic acid was also found in varied amounts. Among the phenolic acids, ferulic acid was found to be present at high concentration in exudates of most isolates (3.9–153.4 μg/ml). The antioxidant properties of phenolics, which generally inhibit fungal morphogenesis including spore germination along with the antifungal nature of some phenolics are chiefly attributed to the inhibitory effect of sclerotial exudates of S. rolfsii. Additionally, both the isolates VC971 and VL016 showed almost similar antifungal activities despite they are of different origin and thereby demonstrate the antifungal nature of sclerotial exudates.     

Isolation of a non-sclerotial mutant ofSclerotium rolfsii and its cellulolytic activity

A mutant strain EMS-1 ofSclerotium rolfsii lacking the ability to develop mature sclerotia was isolated following chemical mutagenesis of macerated sclerotia with ethyl methane-sulfonate. The mutant failed to form sclerotia even in the presence of lactose, threonine or iodo-acetic acid which promoted sclerotial development in the wild strain and the UV-8 mutant. EMS-1 exhibited higher (1.5 – 3.0 times) cellulase and hemicellulase activity compared to the wild strain. Possible correlation between sclerotial morphogenesis and cellulase and/or oxalic acid production is discussed.     

Biological control of Sclerotinia disease by Aspergillus sp. on oilseed rape in the field

Sclerotinia sclerotiorum causes serious yield losses to many crops worldwide. Aspergillus sp. Asp-4, previously shown to inhibit germination of sclerotia of S. sclerotiorum in vitro and in the field, was evaluated in field trials for suppression of this pathogen on oilseed rape. Spray application of Asp-4 to the soil prior to sowing rice in a rice–oilseed rape rotation resulted in a significant reduction in incidence of Sclerotinia stem rot on oilseed rape compared with the non-treated control in two field trials. This application of Asp-4 also resulted in a significant reduction in germination of sclerotia relative to the non-treated control in these field trials, suggesting that this reduction in sclerotial germination led to disease control. Microscopic examination demonstrated that Asp-4 could effectively colonise external and internal portions of sclerotia of S. sclerotiorum in vitro. Incubation of Asp-4 with sterile sclerotial material induced production of β-glucanase and chitinase activities by this isolate; β-glucanase and chitinase being potentially capable of degrading the glucan and chitin polymeric components of sclerotia. Incubation of Asp-4 with sterile sclerotial material also resulted in a significant reduction in dry weight of this sclerotial material relative to the non-treated control in 96?h in vitro experiments. Experiments reported here indicate that Aspergillus sp. Asp-4 has promise as a biological control agent for S. sclerotiorum on oilseed rape. Experiments reported here suggest that disease control results from inhibition of germination of sclerotial resting structures due to mycoparasitic colonisation by Asp-4.     

Potential for biocontrol of sclerotinia rot of carrot with foliar sprays of Contans® WG (Coniothyrium minitans)

A glasshouse and field trial were conducted to evaluate foliar sprays of Contans® WG (Coniothyrium minitans) conidial suspensions for control of sclerotinia rot of carrot and infection of Sclerotinia sclerotiorum sclerotia by C. minitans. In the glasshouse trial, foliar sprays (1×10⁴–10⁸ conidia mL^?1) decreased the viability of sclerotia recovered from diseased plants and increased infection by C. minitans. In the field trial, three successive foliar sprays applied at 14-day intervals failed to reduce foliage disease severity, but significantly reduced viability of sclerotia recovered from diseased plants/crop debris and increased infection by C. minitans. No significant differences in sclerotial viability or infection were observed between two conidial concentrations (2.4 and 4.8×10⁶ conidia mL^?1). Foliar sprays of Contans® WG have potential for reducing viability of sclerotia produced on diseased foliage.     

Fertilization rates and clay fixed ammonium in two Quebec soils

Clay fixed NH₄ ⁺ can provide a significant sink for fertilizer N, as well as a source of N for plant uptake. Knowledge or soil NH₄ ⁺ fixing capacity and release for crops is necessary to develop long-term fertilizer programs. Field experiments with corn (Zea mays L.) were carried out to investigate soil NH₄ ⁺ fixing capacity and subsequent release as influenced by fertilizer rates using ¹⁵N in a Ste. Rosalie clay (fine, mixed, frigid, Typic Humaquept) and a Chicot sandy clay loam (fine-loamy, mixed, frigid, Typic Hapludalf). With high N rates increased NH₄ ⁺ fixation occurred only in the Ste. Rosalie soil. At the end of the first growing season, fertilizer N recovery as clay fixed NH₄ ⁺ for high and normal rates of fertilizer in the Ste. Rosalie soil was 17.8% and 28.7%, respectively and the recovery for the high and normal rates in the Chicot soil was 4.6 and 10.5%, respectively. Significant amounts of clay fixed NH₄ ⁺-N were released in the soil profile in the second year after ¹⁵N application on the Chicot soil. Recently clay fixed fertilizer NH₄ ⁺N was released more rapidly than that of the native fixed NH₄ ⁺, from the surface layer of the Ste. Rosalie soil. The fertilizer fixed NH₄ ⁺ seems to be in a more labile N pool than the native fixed NH₄ ⁺-N in the Chicot soil.     

Trichoderma koningii as a potential parasite of sclerotia of Sclerotium rolfsii

Three different inoculum forms of Trichoderma koningii were tested in vitro for their ability to parasitize the sclerotia of Sclerotium rolfsii. Tests were conducted under two temperature regimes and three incubation periods. Wheat bran was proved to be the most potent inoculum form of the antagonist in reducing 0the viability of the sclerotia. Microscopical observations revealed the presence of hyphae, chlamydospores and conidia of T. koningii in the medullar tissues of the sclerotia. This is the first report of the effect of different inoculum forms of T. koningii on the sclerotia of S. rolfsii and of propagule (chlamydospores and conidia) formation of the antagonist inside the sclerotia of S. rolfsii.     

Comparison of the efficiency of urea,aqueous ammonia and ammonium sulphate as nitrogen fertilizers

Summary Nitrogen-15 labelled urea, aqueous NH₃ and (NH₄)₂SO₄ were applied to soils contained in pots. The fertilizers were injected in 5 cm³ of solution, 3.5 cm below the soil surface, to simulate a fertilizer band in the field. Ryegrass (Lolium perenne) was planted, and several cuttings and roots were harvested. Efficiency was determined as the recovery of fertilizer-N in the plant tissues and soil.Total recovery varied from 94 to 100%. There was no significant difference between the total recovery of the 3 fertilizer forms, although recovery in the soil component was lower for (NH₄)₂SO₄ than for urea or NH₃. There was a significant difference in total recovery between soils due to the soil component. Only small amounts of¹⁵N were not recovered, whereas laboratory experiments reported elsewhere had demonstrated that substantial gaseous losses of N as N₂, N₂O and NO +NO₂ occurred in these soils during nitrification of added NH₃ fertilizer.     

Production, Survival and Evaluation of Liquid Culture-produced Inocula of Coniothyrium minitans Against Sclerotinia sclerotiorum

Growth of Coniothyrium minitans on potato dextrose broth was compared with that on an inexpensive molasses-yeast liquid medium at 18-22°C in static culture. Biomass and conidial production were, in general, similar, although the rate of biomass production was quicker and conidial production was slightly greater per unit volume of medium in the molasses-yeast medium. Air-dried biomass from molasses-yeast liquid culture containing mycelia, pycnidia and conidia of C. minitans was mixed (12%, w/w) with kaolin to give a kaolin-biomass dust. The ability of C. minitans to survive and subsequently infect and reduce the viability of sclerotia of Sclerotinia sclerotiorum from this kaolin-biomass dust was found to be little affected by storage for 48 weeks between 4 and 15°C but was decreased by higher storage temperatures. The kaolin-biomass dust preparation did not differ from a standard maizemeal-perlite inoculum of C. minitans in its ability to infect sclerotia of S. sclerotiorum or reduce their viability or carpogenic germination in glasshouse and field pot bioassays. Further, when either inoculum was applied once to glasshouse soil naturally infested with S. sclerotiorum prior to planting three successive crops of lettuce, the pattern of disease control, reduction of sclerotial numbers/ plot, infection of sclerotia, reduction of sclerotial viability and survival in soil were similar for both inocula. The potential for the commercial development of liquid-culture-produced inocula of C. minitans is discussed.     

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.     

Distribution and recovery of15N after fertilization of Douglas-fir saplings with different nitrogen sources

Summary Four-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) saplings planted in pots with a sand and peat mix (11) were fertilized at the rate of 200 kg N/ha of (¹⁵NH₂)₂CO (U-15),¹⁵NH₄NO₃ (A-15) and NH₄ ¹⁵NO₃(An-15). They were placed in a shadehouse and watered regularly to maintain soil moisture at field capacity over periods of one and two years. Quantity of¹⁵N in foliage generally increased from old to current growth, irrespective of the nitrogen source. Utilization of¹⁵N fertilizers by saplings after the first and second growing seasons following fertilization was greatest with nitrate labelled ammonium nitrate AN-15, and nearly equal for urea U-15 and ammonium labelled ammonium nitrate A-15. The soil immobilized more fertilizer nitrogen-15 from U-15 and A-15 than from AN-15. Data from the present study, in which leaching losses of fertilizer were minimized, demonstrated that in terms of nitrogen uptake by the saplings the nitrate fertilizer was superior to ammonium fertilizer.     

Factors affecting germination, mycoparasitism, and survival of Sporidesmium sclerotivorum.

Macroconidia of Sporidesmium sclerotivorum, a mycoparasite of Sclerotinia spp., germinated after 3 days in soil adjacent to sclerotia of S. minor and on membrane filters placed on soil containing sclerotia. Germination increased with time up to 18 days and with concentration of sclerotia. Conidia as distant as 9 mm from single sclerotia germinated. Germination of conidia was maximum on a sclerotial agar medium in the range of pH 5 to pH 7. Cultivation of S. sclerotivorum parasitically on living sclerotia proceeded optimally in moist, fine quartz sand amended with 1 to 2% (w/w) sclerotia and 0.07% (w/w) CaCO3, at 25 degrees C. Infection of sclerotia in sand reached 100% by 5 weeks. Conidia production paralled infection resulting in logarithmic increase in numbers; a maximum of 3 x 10(5) to 4 x 10(5) conidia/g was reached in 6 to 12 weeks. Viability of air-dried sand-sclerotial cultures of S. sclerotivorum was reduced after 1 and 6 days, but viability was undiminished in air-dried soil. Sporidesmium sclerotivorum survived in moist and air-dried soils stored at room temperature for 15 months.     

Effects of supplemental nitrogen rate and source on biomass production by three weed species in fallow vegetable land

In 1983, a study was conducted to evaluate three weed species for their biomass yield potential on a fallowed tomato land with various nitrogen (N) rates and sources. Dogfennel (Eupatorium capillifolium (Lam.)Small), sida (Sida rhombifolia L.), and pigweed (Amaranthus hybridus L.), were grown with three N rates: residual, 1x and 2x (1x = 56 kg ha⁻¹ N) and with two N sources: NH₄NO₃ and isobutylidene diurea (IBDU). Sida produced the greatest fresh and dry biomass yields and percentage dry matter, 43·3, 23·3 Mg ha⁻¹ and 54·1%, respectively. Yields of the three crops increased with increasing N rates and were higher with NH₄NO₃ than with IBDU. Overall, sida at the 2x N rate with NH₄NO₃ had the highest fresh, 73·8 Mg ha⁻¹, and dry biomass, 37·9 Mg ha⁻¹, yield. Lowest residual soil total soluble salt concentration, 170 ppm, was found after pigweed, in plots which did not receive additional N. Additional N application increased residual soil total soluble salts after each of the three weeds. Thus, additional N, in NH₄NO₃ form, was necessary for high yields in a fallowed vegetable land, but the additional fertilizer also increased soil total soluble salts, regardless of the plant species.