Use of Coniothyrium minitans and other microorganisms for reducing Sclerotinia sclerotiorum

Biological control agents (BCAs) Bacillus subtilis QST 713, Coniothyrium minitans CON/M/91-08, Streptomyces lydicus WYEC 108, and Trichoderma harzianum T-22 were evaluated for their efficacy in the reduction of survival of sclerotia and production of apothecia of Sclerotinia sclerotiorum under controlled environments. A growth chamber assay was conducted where 25 sclerotia were buried in pots containing potting soil, and BCAs were drenched into the soil at various concentrations, and five soybean seeds were planted in each pot. The presence and number of S. sclerotiorum apothecia were recorded daily. Sclerotinia sclerotiorum sclerotia were retrieved six weeks after seeding and viability was assessed on water agar plates. All BCAs were effective in reducing S. sclerotiorum inoculum at various efficacies. In general, efficacy was positively correlated with the rate of application. At the rate of application when the efficacy did not change significantly by increasing the rate, the BCAs had various reductions of apothecia and sclerotia. B. subtilis reduced apothecia and sclerotia by 91.2 and 29.6%, respectively; C. minitans reduced apothecia and sclerotia by 81.2 and 50%, respectively; Streptomyces lydicus reduced apothecia and sclerotia by 100 and 29.6%, respectively; Trichoderma harzianum reduced apothecia and sclerotia by 80.5 and 31.7%, respectively. In addition, the commercial strain of C. minitans CON/M/91-08, and a wild Michigan strain of C. minitans W09 were compared for their growth and sclerotial reduction. W09 had faster growth rate than the commercial strain, indicating potential diversities of biological control strains to be studied.     

Studies on the survival of Mycosphaerella pinodes and Ascochyta pisi

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

Field management of Sclerotinia stem rot of soybean using biological control agents

Biological control agents (BCAs) were evaluated for their efficacy on reducing the number of sclerotia of Sclerotinia sclerotiorum (Lib.) de Bary in the soil and on Sclerotinia stem rot in soybean production systems in Michigan. BCAs included Coniothyrium minitans CON/M/91–08 (Product name: Contans®WG), Streptomyces lydicus WYEC 108 (Actinovate®AG), Trichoderma harzianum T-22 (PlantShield®HC), and Bacillus subtilis QST 713 (Serenade®MAX). At two field locations, soil artificially infested with S. sclerotiorum sclerotia, was treated by incorporating the above BCAs in the topsoil before planting and boscalid was applied as a foliar fungicide at growth stage R1 as a positive control. C. minitans was the most effective BCA and reduced the disease severity index (DSI) by 68.5% and the number of sclerotia of S. sclerotiorum in the soil by 95.3%. S. lydicus and T. harzianum reduced DSI by 43.1% and 38.5% and sclerotia in soil by 90.6% and 70.8%, respectively. B. subtilis only had a marginal effect on S. sclerotiorum. Populations of Bacillus, Streptomyces, Trichoderma spp., and C. minitans collected from soil samples and at 3, 28, 71, and 169 days after BCA application indicated that the population of Streptomyces, Trichoderma spp., and C. minitans did not change significantly throughout the season, which may be the reason for their effectiveness.     

Decay of Sclerotia of Botrytis cinerea by Trichoderma spp. at Low Temperatures

In vitro, tests were conducted at 10°C and 5°C against sclerotia of Botrytis cinerea with 58 isolates of Trichoderma spp., highly antagonistic at 24°C but differing in their cold tolerance. Some isolates macerated and colonized sclerotia even at 5 °C. With 19 isolates of Trichoderma spp. less than 10 % of the sclerotia remained viable after 42 d at 5 °C. Conidia ol some Trichoderma spp. germinated at 5 °C within a few days and reached germination rates higher than 80 %. It seems to be feasible to use selected isolates of Trichoderma spp. for biological control of sclerotia of ß. cinerea also during the colder season.     

Effect of Moisture and Temperature on the Survival of Sclerotia of Sclerotium rolfsii in Soil

The sclerotia of Sclerotium rolfsii Sacc. survived in natural soil for 225 days under controlled moisture at 50% water holding capacity (WHC) after which there was a progressive reduction in the population of viable sclerotia. At 390 days only 48% were recovered. Sclerotia survived well at moisture contents upto 75% WHC but at 100% the population declined rapidly and none were recovered after 60 days. The contents of the sclerotia were found to lyse without germination leaving hollow rinds. Such lysis was found to be favoured between 25 and 40°C. At and below 20°C no such lysis was recovered and more than 80% sclerotia were recovered even after 60 days.     

Isosclerone,a New Metabolite of Sclerotinia sclerotiorum (Lib.) De Bary

Isosclerone, mp 74~76°C, , a new metabolite of Sclerotinia sclerotiorum, was isolated and its absolute structure was determined as (4S)-3,4-dihydro-4,8-dihydroxy-1(2H)-naphthalenone. It stimulated the root elongation of rice seedlings by ca. 30% at concentrations of 1~10 ppm, and inhibited the growth of shoots and roots at high concentrations above 50 ppm.     

Survival of Rhizoctonia solani AG‐1 IA,the Causal Agent of Rice Sheath Blight,under Different Environmental Conditions

The ability of Rhizoctonia solani AG‐1 IA, the causal agent of rice sheath blight, to survive in diseased rice straw and as sclerotia and mycelia was investigated. After storage for 10 months at 4°C, 25°C and non‐air‐conditioned natural room temperature (NRT, temperature range from 6°C to 35°C), sclerotia placed inside a desiccator, soaked in sterile water or immersed in wet paddy soil were viable. In contrast, only 15% of sclerotia in dry paddy soil survived. Survival of mycelia was severely affected by temperature and humidity. After 10 months in a desiccator at 4°C, 55% of mycelia samples could survive, whereas at 25°C and NRT, mycelial samples survived for only 7 and 5 months, respectively. However, mycelia stored in sterile water at constant temperatures (4°C or 25°C) survived for 10 months. A certain amount of UV radiation had no obvious effect on the survival of sclerotia or mycelia. The survival rate of the fungus in diseased rice straw stored for 16 months could reach 100% at 4°C, 50% at 25°C and 35% at NRT. The survival rates of the pathogen in diseased rice straw buried in dry, wet and flooded paddy soils after 10‐month storage at NRT were 75, 100 and 100%, respectively, indicating that soil humidity is a crucial factor for the survival of this fungus.     

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.     

Sporenkeimung von Phoma lingam (Tode ex. Fr.) Desm. und Resistenzprüfung bei Raps im Gewächshaus

Spore germination of Phoma lingam (Tode ex. Fr.) Desm. and methods to determine resistance of oil seed rape in the greenhouse It was the aim of this investigation to obtain more insight into the epidemiology of Phoma lingam (Tode ex. Fr.) Desm. (stat. gen. Leptosphaeria maculans (Desm.) Ces. et de Not.) and to improve the existing methods for resistance testing. In laboratory experiments the differing demands on temperature of both, the sexual as well as the non sexual phase were observed. Many ascospores developed germ tubes after eight hours at 4-8°C whilst pycnidiospores needed 24 hours at 16°C to have similar development. In greenhouse experiments young plants were infected by spraying or by placing a droplet of spore suspension onto cotyledons or leaves. Generally, ascospores were more virulent than pycnidiospores. The ascospores were obtained from old rape stalks which could be stored at -18°C without losing virulence. The most severe attack was observed after incorporating infested oat kernels (3 % w/ w) into soil, but the difference between cultivars vanished which was already low with the other methods, and which did not always correspond with results obtained in the field at stage 85, so that all these methods are not as suitable as those in the field. The distribution of pycnidiospores is also possible by adhering to the seed after threshing. The infection of the seedlings from this source was more pronounced in steamed than in unsteamed soil. The re-isolation of P. lingam increased as well from plants grown in steamed soil. Furthermore, pycnidiospores are distributed by wind during combining to neighbouring fields, already prepared at that time for rape sowing.     

Responses of skilletfish Gobiesox strumosus to high temperature and low oxygen stress

This study quantified physiological responses of skilletfish Gobiesox strumosus exposed to thermal and oxic stress. Fish acclimated at 12, 22 and 32° C had low oxygen tolerance values (mean ±s.d .) of 0·40 ± 0·09, 0·40 ± 0·08 and 0·35 ± 0·03, and critical thermal maxima (mean ±s.d .) of 33·2 ± 0·5, 38·1 ± 0·0 and 39·5 ± 0·3° C, respectively. Furthermore, G. strumosus were oxygen conformers at all acclimation temperatures, i.e. the fish allowed oxygen consumption rates to decrease with ambient oxygen concentration. High temperature tolerance, low oxygen tolerance and decreasing metabolic rates during hypoxic events allow the fish to survive harsh environmental conditions encountered in their natural environment.     

Fattening Pelibuey lambs with sugar cane tops and corn complemented with or without slow intake urea supplement

One hundred and eighty Pelibuey lambs were fattened for 120 days with three treatments evaluating in situ DM disappearance, voluntary DM or OM intake, rumen degradation, rate of passage, NH₃ and VFA concentration, apparent digestibility, ruminal pH, total fermentable carbohydrates and weight gains. The first diet (T1), 60 lambs (25.7 ± 0.7 kg BW) plus two cannulated sheep were fed 100% sugar cane tops (SCT) per day; treatment 2 (T2), 60 lambs (25.4 ± 0.4 kg BW) plus two cannulated sheep diet was SCT supplemented with 200 g DM of slow intake urea supplement (SIUS) (a 4% urea mixture) per day. Treatment 3 (T3), 60 sheep (25.5 + 0.5 kg BW) plus two cannulated animals, fed SCT and corn crop chopped (C) mixture (40:60%) were supplemented with 200 g SIUS per day. Ruminal kinetics were determined thereafter in four adult ruminal cannulated sheep in metabolic cages. A 100% corn diet was added to the second trial (T4). At all times, fibrous forages were available and exceeded VDMI. Daily BW gain by SCT animals was 70 g/d compared to 135 g/d for SCT/SIUS and 218 g/d for SCT/C/SIUS diets (P < 0.05). Total forage intake was similar for T1 and T2 treatments and higher for T3 (P < 0.05). Kinetics observation showed a total DMI variation increasing from 474 g/d for T1; 597 g/d for T4 to 797 g/d for T2, and 917 g/d for T3, being different for the last two treatments compared with the first two (P < 0.001) and similar among SIUS diets. Rumen pH diminished from 6.8 to 6.7 at 2 h, when SIUS was offered, and stayed above 6.5 during the 12 h of sampling when SIUS was the only supplement and 6.3 when corn was added, while in SCT lambs, ruminal pH decreased to 6.2 by 6 h and rose again to 6.4 after 12 h favoring bacterial multiplication. Ammonia concentration and digestion of potential digestible and indigestible fractions were significantly augmented (P < 0.05) by SIUS. In vivo nitrogen digestibility was different (P < 0.05) among diets: T1, T2, T3, and T4 (36.5%, 74.1%, 76.5%, and 39.4%, respectively). In vivo digestibility of DM, OM, cellulose and hemicellulose was similar among T2 and T3 groups but different when T1 or T4 diets were offered (P < 0.05). NDF digestibility was higher for T3 (77.1%) and T2 (75.1%) but different from T1 (57.1%) and T4 (59.1%) (P < 0.01). In situ DM disappearance did not show differences among T2 and T3 diets at 9, 12, 24 and 48 h of incubation but differed in percentage for T1 and T4. Digestion rate constant (k_d) was similar among SIUS diets 0.038 and 0.039, but different from T1 and T4 diets 0.023; 0.021 (P < 0.05). True digestibility of NDF was higher (P < 0.05) in SCT/SIUS/C (32.5%) and SCT/SIUS (32.7%) compared with SCT (26.2%) and C (27.4%). Passage of NDF was different (P < 0.05) between T1 (0.059 h⁻¹) and T4 (0.061 h⁻¹) from 0.079 (T2) and 0.081 k_p/h (T3). The half-time (t_1/2) disappearance for hemicellulose was higher (P < 0.05) for SCT/SIUS (22.1 h) and SCT/SIUS/C (29.2 h) as compared to SCT (18.1 h). Kinetic performance among SCT and C diet did not have a statistical difference. Utilization of fibrous forages by lambs was higher (P < 0.05) for SIUS diets. VDMI and apparent digestibility showed better performance for DM and OM, lower acetate molar proportions and greater feed intake by both SIUS diets which was reflected in body weight gain.     

Synthesis of Pogostone-Inspired Dehydroacetic Acid Derivatives and Their Antifungal Activity against Sclerotinia sclerotiorum (Lib.) de Bary

In an effort to exploit the natural antifungal pogostone, its simplified scaffold dehydroacetic acid (DHA) was used as a lead compound to semi-synthesize 56 DHA derivatives ( I1 – 48 , II , III , and IV1 – 6 ). Among them, compound IV4 exhibited the most potent antifungal activity with 11.0 μM EC₅₀ against mycelial growth of Sclerotinia sclerotiorum (Lib.) de Bary whose sclerotia production was also completely suppressed at this concentration. Furthermore, IV4 could completely inhibit infection cushion formation of S. sclerotiorum on rape leaves and achieved a preventive efficacy of 90.2 % at 500 μM, which was on the same level as that of commercial boscalid at 30 μM (88.7 %). The results of physiological and ultrastructural studies indicated that IV4 might disrupt the cell membrane permeability or induce the imbalance of mitochondrial membrane potential homeostasis to exert the antifungal mode of action. Besides, the robust and predicative three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed and discussed herein.     

Some environmental factors affect growth and antibiotic production by the mycoparasite Coniothyrium minitans

The effects of temperature and pH on growth and antibiotic production by three isolates of Coniothyrium minitans (Conio, Contans and IVT1), known to produce the macrolide antibiotic macrosphelide A, were examined in modified Czapek Dox broth (MCD). Antibiotic production was determined by incorporating heated (60°C for 5 min) C. minitans spent culture filtrates of MCD (10%, v/v) into potato dextrose broth and assessing the ability of the filtrates to inhibit growth of S. sclerotiorum. All isolates grew over the temperature range of 10–30°C, with the optimum at approximately 15–20°C. Antibiotics were produced by all isolates at 10–30°C. Culture filtrates of MCD from all isolates incorporated into PDB inhibited growth of S. sclerotiorum by >50%, whereas there was a reduction in inhibition at 30°C for Conio and IVT1 but not Contans. All three isolates grew over the pH range of 3–7, with greater biomass production in buffered pH 3–5 than the unbuffered control (pH 4.8) media. Antibiotics were produced by all isolates at pH 3–5. Culture filtrates of MCD from all three isolates grown at pH 3–5 inhibited growth of S. sclerotiorum, with the greatest effect on inhibition observed at pH 3. There were no differences in growth inhibition between isolates at pH 3 and 4, but culture filtrates from Conio grown at pH 5 inhibited S. sclerotiorum more than those of IVT1 grown at the same pH. The significance of these results for biocontrol and optimizing antibiotic production by C. minitans is discussed.     

Colonisation of sclerotia of Sclerotinia sclerotiorum by a fungivorous nematode

Aphelenchoides saprophilus nematodes fed on sclerotia, mycelium, and alginate-formulated pellets of Sclerotinia sclerotiorum, mycelium of Trichoderma harzianum, and mixed fungal cultures. As many as 500 nematodes were found inside individual sclerotia. Results suggest potential impacts of fungivory on S. sclerotiorum and its ecological interactions with plant hosts and biocontrol fungi.     

Seed germination ecology of Viola calaminaria,an endangered metallophyte with a narrow distribution

Viola calaminaria is an endangered metallophyte endemic to a small area close to the border between Belgium, Germany and the Netherlands, where it grows on rock outcrops rich in heavy metals (zinc, lead and cadmium). Because V. calaminaria reproduces mainly by seeds, it is of crucial importance to understand its germination requirements. Germination percentage and speed at constant (11–25°C) and alternating (23/09°C) temperatures were investigated in five large populations. Germination percentage was positively correlated to seed weight. Germination was low (<25%) at 11 and 16°C, intermediate (around 65%) between 20 and 25°C and the highest (93%) at the alternating temperature regime (23/09°C). V. calaminaria is a slow germinator requiring 41 days on average to germinate at 23/09°C and considerably more at 20 to 25°C (105 days on average). Our results also highlighted that the species is desiccation tolerant and can therefore be safely conserved under standard seed bank conditions.     

Symbiont transmission and reproductive mode influence responses of three Hawaiian coral larvae to elevated temperature and nutrients

Elevated temperatures and nutrients are degrading coral reef ecosystems, but the understanding of how early life stages of reef corals respond to these stressors remains limited. Here, we test the impact of temperature (mean ~ 27 °C vs. ~ 29 °C) and nitrate and phosphate enrichment (ambient, + 5 µM nitrate, + 1 µM phosphate and combined + 5 µM nitrate with 1 µM phosphate) on coral larvae using three Hawaiian coral species with different modes of symbiont transmission and reproduction: Lobactis scutaria (horizontal, gonochoric broadcast spawner), Pocillopora acuta (vertical, hermaphroditic brooder) and Montipora capitata (vertical, hermaphroditic broadcast spawner). Temperature and nutrient effects were species specific and appear antagonistic for L. scutaria and M. capitata, but not for P. acuta. Larvae survivorship in all species was lowest under nitrate enrichment at 27 °C. M. capitata and L. scutaria survivorship increased at 29 °C. However, positive effects of warming on survivorship were lost under high nitrate, but phosphate attenuated nitrate effects when N/P ratios were balanced. P. acuta larvae exhibited high survivorship (> 91%) in all treatments and showed little change in larval size, but lower respiration rates at 29 °C. Elevated nutrients (+N+P) led to the greatest loss in larvae size for aposymbiotic L. scutaria, while positive growth in symbiotic M. capitata larvae was reduced under warming and highest in +N+P treatments. Overall, we report a greater sensitivity of broadcast spawners to warming and nutrient changes compared to a brooding coral species. These results suggest variability in biological responses to warming and nutrient enrichment is influenced by life-history traits, including the presence of symbionts (vertical transmission), in addition to nutrient type and nutrient stoichiometry.

     

Influence of Temperature and Relative Humidity on Germinability of Mucor piriformis Spores

Studies were conducted to determine the influence of temperature and relative humidity (RH) on germinability and viability of Mucor piriformis spores. Spores did not survive when stored at 35 °C and their survival rate decreased rapidly at 30 °C; however, spores remained viable for more than 1 year at 0 °C. RH also significantly affected spore viability. Spores held at 26 °C and 100% RH no longer germinated after 35 days, while those held at 75 or 90% RH germinated for 65 days. At 20 °C, RH had little effect on spore germinability. The effect of temperature and RH on percentage spore germination also varied. At all temperatures studied, spore viability decreased more rapidly with time at 100% RH than at 75 or 90% RH. The least favorable, temperature-humidity combination, 30 °C and 100% RH, decreased spore germination from 100% to less than 1% in 14 days.     

Pre-germinated conidia of Coniothyrium minitans enhances the foliar biological control of Sclerotinia sclerotiorum

The relatively slow germination rate of Coniothyrium minitans limits its control efficiency against Sclerotinia sclerotiorum. Pre-germinated conidia of C. minitans enhanced its efficiency significantly: in foliar experiments with oilseed rape, hyphal extension of S. sclerotiorum was inhibited by 68%, while formation of sclerotia was completely inhibited when pre-germinated conidia were applied.Revisions requested 27 July 2004; Revisions received 7 September     

Colonization of Sclerotinia sclerotiorum sclerotia by a biocontrol isolate of Trichoderma harzianum,and effects on myceliogenic germination

Sclerotia of Sclerotinia sclerotiorum were incubated on cultures of Trichoderma harzianum. Myceliogenic germination decreased by 50% within 1 day and continued to decrease over time. Quantitative PCR showed a decrease in Sclerotinia DNA for older sclerotia, but not fresh sclerotia. Trichoderma DNA increased and persisted inside older sclerotia but not fresh sclerotia.     

Secondary metabolites produced by Propionicimonas sp. (ENT-18) induce histological abnormalities in the sclerotia of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a highly aggressive pathogen that causes great economic losses, especially in temperate climates. Several biological control agents are available, but actinobacteria have seldom been used to control this fungus. Our objective was to evaluate the efficiency and ultrastructural effects of the secondary metabolites produced by the ant-associated actinobacterium Propionicimonas sp. ENT-18 in controlling the sclerotia of S. sclerotiorum. We demonstrated total inhibition of sclerotia treated with 62.5 μg/10 μl of an ethyl acetate extract of compounds produced by ENT-18, and calculated an LC₅₀ of 1.69 μg/sclerotia. Histological and ultrastructural analysis indicated that the cells of the treated sclerotia were severely damaged, suggesting direct action of the biomolecule(s) produced by the actinobacterium ENT-18 on the cell structure of the medullae and rind cell wall. This is the first report demonstrating a novel property of Propionicimonas sp.—antifungal activity against S. sclerotiorum.