The importance of water potential range tolerance as a limiting factor on Trichoderma spp. biocontrol of Sclerotinia sclerotiorum

Biological control of fungal phytopathogens is often more variable in efficacy compared with disease suppression achieved by conventional pesticide use. Matching the environmental range of a potential biocontrol agent with that of the target phytopathogen is necessary if consistent disease suppression is to be achieved under field conditions. Strains of Trichoderma that could parasitise sclerotia of Sclerotinia sclerotiorum had their spore germination and mycelial growth (five strains) and ability to parasitise sclerotia (two strains) tested under a range of water potentials under laboratory conditions. Relative mycelial growth and germination of all strains decreased with decreasing osmotic and matric potentials, with matric potential having a greater impact on growth and germination over the range examined. Trichoderma harzianum LU698 mycelial growth was the least affected by decreasing osmotic potential than the other isolates, and Trichoderma atroviride LU141 growth least affected by decreasing matric potential. The germination of LU698 and LU144 was also generally less affected by decreasing osmotic potential, although generally decreasing matric potential had the greatest affect on the germination of LU698 along with T. atroviride LU132. Soil treatments of LU698 and Trichoderma asperellum LU697 reduced sclerotial viability in all but the lowest soil water potential tested, with LU698 being most effective at ?0.1 and ?0.3 MPa after 28 days and LU697 most effective at ?0.01 and ?1.5 MPa after 28 days. We conclude that differences in the tolerance of potential biocontrol agents to changing water potential is an important experimental factor to consider when assaying biocontrol or making predictions of biocontrol efficacy in the field.     

Effects of water potential on mycelial growth,sclerotial production,and germination of Rhizoctonia solani from potato

The effects of osmotic and matric potential on mycelial growth, sclerotial production and germination of isolates of Rhizoctonia solani [anastomosis groups (AGs) 2-1 and 3] from potato were studied on potato dextrose agar (PDA) adjusted osmotically with sodium chloride, potassium chloride, glycerol, and matrically with polyethylene glycol (PEG) 6000. All isolates from AGs 2-1 and AG-3 exhibited fastest mycelial growth on unamended PDA (−0.4 MPa), and growth generally declined with decreasing osmotic and matric potentials. Growth ceased between −3.5 and −4.0 MPa on osmotically adjusted media, and at −2.0 MPa on matrically adjusted media, with slight differences between isolates and osmotica. Sclerotium yield declined with decreasing osmotic potential, and formation by AG 2-1 and AG-3 isolates ceased between −1.5 and −3.0 MPa and −2.5 and −3.5 MPa, respectively. On matrically adjusted media, sclerotial formation by AG 2-1 isolates ceased at −0.8 MPa, whereas formation by AG-3 isolates ceased at the lower matric potential of −1.5 MPa. Sclerotial germination also declined with decreasing osmotic and matric potential, with total inhibition occurring over the range −3.0 to −4.0 MPa on osmotically adjusted media, and at −2.0 MPa on matrically adjusted media. In soil, mycelial growth and sclerotial germination of AG-3 isolates declined with decreasing total water potential, with a minimum potential of −6.3 MPa permitting both growth and germination. The relevance of these results to the behaviour of R. solani AGs in soil and their pathogenicity on potato is discussed.     

Impact of osmotic and matric water stress on germination, growth, mycelial water potentials and endogenous accumulation of sugars and sugar alcohols in Fusarium graminearum

Studies were conducted to determine the effect of osmotic (NaCl, glycerol) and matric (PEG 8000) water stress on temporal germination and growth of two F. graminearum strains over the water potential range of -0.7 to -14.0 MPa at 15 and 25 C. The effect on endogenous water potentials and accumulation of sugars and sugar alcohols also were measured. For both strains, germination occurred rapidly over the same range of osmotic or matric potential of -0.7 to -5.6 MPa after 4-6 h incubation. At lower osmotic and matric potentials (-7.0 to -8.4 MPa), there was a lag of up to 24 h before germination. Optimum germ-tube extension occurred between -0.7 and -1.4 MPa for both strains but varied with the solute used. Growth was optimal at -1.4 MPa and 25 C in response to matric stress, with the minimum being about -8.0 and -11.2 MPa at 15 and 25 C, respectively. In contrast, F. graminearum grew fastest at -0.7 MPa and was more tolerant of solute stress modified with either glycerol or NaCl with a minimum of about -14.0 MPa at 15 and 25 C. A decrease in the osmotic/matric water potential of the media caused a large decrease in the mycelial water potential (Ψ(c)) as measured by thermocouple psychrometry. In general, the concentration of total sugar alcohols in mycelia increased as osmotic and matric potential were reduced to -1.2 MPa. However, this increase was more evident in mycelia from glycerol-amended media. The quality of the major sugar alcohol accumulated depended on the solute used to generate the water stress. The major compounds accumulated were glycerol and arabitol on osmotically modified media and arabitol on matrically modified media. In response to matric stress, the concentration of trehalose in colonies generally was higher in the case of osmotic stress. In each water-stress treatment there was a good correlation between Ψ(c) and total sugar alcohol content.     

Fungi isolated from Sclerotia ofSclerotium cepivorum and from soil and their effects upon the pathogen

Summary Data are presented on the antagonistic effects of the fungi isolated from sclerotia ofSclerotium cepivorum and from nonrhizosphere soil taken from around the roots of infected onions upon mycelial growth and sclerotial germination ofS. cepivorum. Most of the isolated fungi especiallyPenicillium species were antagonistic to mycelial growth. Sclerotial germination was slightly inhibited by diffusates of these fungal isolates. Testing the antifungal effect of someAllium extracts against the fungal isolates by the inhibition zone method showed that garlic extract has the greatest antifungal effects and onion extract is the least potent. However, spore germination tests indicated that onion extract completely inhibits the spore germination of all test fungi. The role of host-plant extracts in stimulating sclerotial germination is discussed.     

Effects of temperature,pH and water potential on growth of four fungi with disease biocontrol potential

Effects of temperature, pH and water potential on blomass production or hyphal extension of Gliocladium virens (G20) and three Trichoderma isolates were determined in vitro. Optimum blomass production occurred between 20 and 30°C and at pH ranges between 4.6 and 6.8. Two isolates of T. viride grew at 5°C and G. virens grew at 35°C but no isolates grew at 40°C. Hyphal extension rates and conidial germination of all fungi declined with decreasing water potential over the range -0.7 to -14.0 MPa. In general, growth rates for each isolate were lower on potato/dextrose agar with water potential adjusted with polyethylene glycol than when adjusted with NaCl or glycerol. No mycelial growth or spore germination occurred on agar at-14.0 MPa.The authors are with the Microbiology and Crop Protection Department, Horticulture Research International, Littlehampton, West Sussex BN17 6LP, UK. J.M. Whipps is the corresponding author     

Germination and seedling growth of carrots under salinity and moisture stress

Poor crop stand is a common problem in saline areas. Germination and seedling emergence may be depressed as a result of impeded aeration, saline or dry conditions. In this study, we examined the effects of salinity and moisture stress and their interactions on seed germination and seedling growth of carrots. Variable soil matric and osmotic potentials were either obtained by equilibrating soil salinized to different degrees on a 0.5 MPa ceramic plate soil moisture extractor or by adding different amounts of salt solutions to the same mass of air-dried soil, based on a previously determined soil moisture release curve, and allowing to equilibrate for 1 week. Germination decreased significantly in the investigated silty soil (Aquic Ustifluvent) at soil moisture potentials higher than −0.01 MPa, whereas osmotic potentials as low as −0.5 MPa did not influence germination. Matric potentials of −0.3 and −0.4 MPa, respectively, resulted in a strong decrease (35–95%) of germination and delayed germination by 2 to 5 days in the silty soil to which different amounts (18 and 36%, respectively) and sizes (0.8–1.2 mm and 1.5–2.2 mm, respectively) of sand particles had been added. No effect of sand and grain diameter was detected. Germination was not affected by comparable osmotic potentials. Seedling growth showed a much higher sensitivity than germination to decreasing matric potentials, but was not affected by osmotic potentials ranging from −0.05 to −0.5 MPa. Optimum shoot growth occurred at matric potentials between −0.025 and −0.1 MPa. Shoot and root growth decreased markedly at matric potentials higher than −0.01 MPa. Fresh weight of shoots decreased gradually at matric potentials lower than −0.2 MPa. Root growth was significantly increased at matric potentials of −0.1 to −0.3 MPa, whereas comparable osmotic potentials did not have equivalent effects. It is concluded that germination and seedling growth are differently affected by comparable matric and osmotic stresses and that water stress exerts a more negative effect than salt stress.     

Factors Affecting Biological Control of Sclerotinia sclerotiorum by Fungal Antagonists

Studies were conducted to determine the effects of soil moisture (9, 16 or 24% w/w) and temperature (5, 15, 20 or 25°C) on the control of sclerotia of Sclerotinia sclerotiorum by five fungal agents in sterile and natural field soil. All five biocontrol agents were effective in reducing the survival of sclerotia of S. sclerotiorum in sterile soil under dry (9% moisture) or wet (24% moisture) conditions at 20°C, but only Coniothyrium minitans was effective in natural soil. Coniothyrium minitans was the most effective in reducing sclerotial viability at the temperature range of 15–25°C. Trichoderma virens was effective against sclerotia of S. sclerotiorum to a lesser extent than C. minitans , and in non-autoclaved soil, it performed best at 25°C. Although Epicoccum purpurascens , Talaromyces flavus and Trichothecium roseum were effective against sclerotia of S. sclerotiorum in some instances, they were less effective than C. minitans and T. virens . Sclerotia of S. sclerotiorum conditioned for myceliogenic germination were more vulnerable to attack by the biocontrol agents than dormant sclerotia. The implications are discussed with respect to enhancement of biological control of crop diseases caused by S. sclerotiorum in different geographic regions.     

Individual and Combined Effects of Certain Pesticides on Rhizoctonia solani, Sheath Blight Pathogen of Rice

The effect of different pesticides (fungicide, herbicide and insecticide) on the mycelial growth and formation and germination of sclerotia of sheath blight pathogen of rice, Rhizoctonia solani Kühn was studied in nutrient medium and in natural soil. Among the herbicides and insecticides, fenitrothion (insecticide) and thiobencarb (herbicide) were the most effective in inhibiting the mycelial growth and sclerotial formation. These pesticides effected significant inhibition of both mycelialgrowth and sclerotial formation at 7.5 to 10μg/ml concentration while fungicides carbendazim and tolclofosmethyl effected almost complete inhibition at 2.5 μg/ml. Interestingly, a synergistic increase in the toxicity to mycelial growth, sclerotial formation and sclerotial germination was noticed when the fungicide, mancozeb and the herbicide, thiobencarb were applied at subtoxic concentration in combination. Such synergistic interactions between pesticides in a combination leading to increased toxicity suggest that under the current practice of applying fungicides, herbicides and insecticides either simultaneously or in rotation, certain combinations may help in reducing the dose of a fungicide in disease control.     

Molasses supplementation promotes conidiation but suppresses aflatoxin production by small sclerotial Aspergillus flavus

AIMS: To find a supplemental ingredient that can be added to routinely used growth media to increase conidial production and decrease aflatoxin biosynthesis in small sclerotial (S strain) isolates of Aspergillus flavus. METHODS AND RESULTS: Molasses was added to three commonly used culture media: coconut agar (CAM), potato dextrose agar (PDA), and vegetable juice agar (V8) and production of conidia, sclerotia, and aflatoxins by A. flavus isolate CA43 was determined. The effect of nitrogen sources in molasses medium (MM) on production of conidia, sclerotia and aflatoxins was examined. Water activity and medium pH were also measured. Conidia harvested from agar plates were counted using a haemocytometer. Sclerotia were weighed after drying at 45 degrees C for 5 days. Aflatoxins B(1) and B(2) were quantified by high-performance liquid chromatography. Addition of molasses to the media did not change water activity or the pH significantly. Supplementing CAM and PDA with molasses increased conidial production and decreased aflatoxins. Two-fold increased yield of conidia was found on MM, which, like V8, did not support aflatoxin production. Adding ammonium to MM significantly increased the production of sclerotia and aflatoxins, but slightly decreased conidial production. Adding urea to MM significantly increased the production of conidia, sclerotia and aflatoxins. CONCLUSIONS: Molasses stimulated conidial production and inhibited aflatoxin production. Its effect on sclerotial production was medium-dependent. Water activity and medium pH were not related to changes in conidial, sclerotial or aflatoxin production. Medium containing molasses alone or molasses plus V8 juice were ideal for conidial production by S strain A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into molecular events associated with the utilization of molasses may help to elucidate the mechanism(s) that decreases aflatoxin biosynthesis. Targeting genetic parameters in S strain A. flavus isolates may reduce aflatoxin contamination of crops by reducing the survival and toxigenicity of these strains.     

In vitro inhibitory effects of rosemary and sage extracts on mycelial growth and sclerotial formation and germination of Sclerotinia sclerotiorum

The anti-fungal efficacy for two Labiate plants, rosemary (Rosmarinus officinalis L.) and Greek sage (Salvia fructicosa Mill.), against Sclerotinia sclerotiorum fungus (Lib.) de Bary has been investigated. The inhibitory effect of these plants as crude leaf ethanolic extract on the radial mycelial growth as well as on sclerotial production and germination was measured in vitro at various concentrations (stock?=?0.5?g dry leaf powder/ml ddH₂O) in the growth medium. In general, rosemary extract revealed a remarkable anti-fungal effect against the fungus, being more inhibitory than Greek sage in this respect. This was evident as total inhibition of radial mycelial growth by rosemary occurred at 10% extract concentration, while sage was half as potent producing such an effect at double the concentration (20%). Both rosemary and sage extracts were more inhibitory to sclerotial formation than to mycelial growth as the fungus ceased to produce any sclerotia at the lower concentrations of 5 and 5–10%, respectively. In addition, rosemary was highly effective in inhibiting sclerotia germination as total inhibition of germination occurred at 20% extract concentration at three?days and onward after incubation. Moreover, at this level, the survival of sclerotia was totally lost when examined after 12?days of incubation. For sage, inhibition of sclerotial germination/death was only 20% at 12th day of incubation. The results of this study indicate that the extracts of rosemary and Greek sage leaves could become natural alternatives to synthetic fungicides to manage diseases of S. sclerotiorum.     

In vitro water stress bioassays with the nematophagous fungus Pochonia chlamydosporia: Effects on growth and parasitism

The biocontrol potential of Pochonia chlamydosporia, a fungus with parasitic activity against economically important plant-parasitic nematodes, can be influenced by abiotic factors such as water availability. The objective of this study was to evaluate the effects of different water stress regimes on in vitro growth, sporulation, germination and parasitism of P. chlamydosporia isolates. The osmotic water potential of 1.7% corn meal agar (CMA) was modified by addition of potassium chloride (KCl) or glycerol, and the matric water potential was modified using polyethylene glycol (PEG 8000). The fungus was able to grow over a range of potentials but radial growth rates decreased with the increase of osmotic and matric stress. No growth was observed at −10 MPa on 1.7% CMA amended with glycerol and at −7.1 MPa on medium with PEG 8000 but all isolates were able to resume growth when transferred onto unmodified 1.7% CMA. The production of chlamydospores was repressed in both osmotic and matric modified media. Although the production of conidia increased in medium modified with KCl, the germination rate was lower. Spores/hyphal fragments remained viable in all isolates that were previously inoculated onto media with growth-limiting water potential (−10 MPa on 1.7% CMA amended with glycerol and −10 MPa on medium with PEG 8000). The percentage of viable conidia produced on 1.7% CMA, after inoculation under osmotic or matric stress conditions for 25 days, was over 74.5% in all isolates (osmotic stress) and ranged from 1% (Pc1) to 65.8% (Pc280) (matric stress). The in vitro infection of potato cyst nematodes, Globodera rostochiensis eggs by P. chlamydosporia isolates, grown under these limiting conditions, was studied using a standard bioassay. The percentage of parasitized eggs was significantly higher under osmotic stress except for isolates Pc2 and Pc3. P. chlamydosporia spores/hyphal fragments can remain viable at water potentials limiting for growth, for prolonged periods of time, suggesting that the osmoregulation mechanisms, used to compensate water stress, affect in vitro sporulation and increased pathogenicity. Knowledge on water requirements of P. chlamydosporia enables a better understanding of its survival and growth strategies in the soil environment and could aid the development of effective strategies to increase the production and quality of inoculum, thus contributing to the implementation of biosafe, sustainable management strategies against plant-parasitic nematodes.     

Variability in Indian isolates of Sclerotium rolfsii

Variability among 26 isolates of Sclerotium rolfsii collected from various hosts/soil samples and localities in India is reported. The isolates varied in colony morphology, mycelial growth rate, sclerotium formation, teleomorph production and sclerotial size and color. Out of 26 isolates, only 4 produced the teleomorph stage on Cyperus rotundus rhizome meal agar medium. Mycelial incompatibility among the isolates was also seen, and out of 325 combinations, only 29 combinations (8.9%) showed compatible reactions. Based on mycelial compatibility, 13 vegetative incompatibility groups (VCG) were identified among the isolates. HPLC analysis of the ethyl acetate fraction of culture filtrates of the isolates revealed 10-22 peaks. Six peaks were identified as gallic, oxalic, ferulic, indole-3-acetic acid (IAA), chlorogenic, and cinnamic acids. Oxalic, IAA, and cinnamic acids were present in the culture filtrates of all the isolates in varying amounts. The other three phenolic acids were not detected in some of the isolates. A comparative HPLC analysis of sclerotial exudate, sclerotia, mycelia, and culture filtrates of two S. rolfsii isolates (leaf spot- and collar rot-causing) producing different symptoms on their respective hosts revealed variation in the content of phenolic acids, IAA, and oxalic acid.     

Antifungal effects of essential oils from oregano and fennel on Sclerotinia sclerotiorum

AIMS: The antifungal effects of essential oils of oregano (Origanum syriacum var. bevanii) and fennel (Foeniculum vulgare) were evaluated against Sclerotinia sclerotiorum. Effects of the essential oils on morphological structures of hyphae and sclerotia were studied under light and scanning electron microscopes (SEM). METHODS AND RESULTS: Inhibitory effects of volatile and contact phases of the essential oils used were determined on hyphae and sclerotia. Both essential oils have a marked antifungal effect against S. sclerotiorum. Soil amendment with essential oils has significant effect on reducing sclerotial viability. Both essential oils significantly inhibited the fungal growth in soil, thereby increasing the number of surviving tomato seedling by 69.8% and 53.3%, respectively. Light and SEM observations on pathogen hyphae and sclerotia revealed considerable morphological alterations in hyphae and sclerotia. CONCLUSIONS: The significant reduction in the mycelial growth and germination of sclerotia would greatly reduce the pathogen inoculum source. This may influence the rate of disease development in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the reduction in the number of diseased plants in infested soil amended with essential oils, we concluded that oregano and fennel essential oils could be used as possible bio fungicides alternative to synthetic fungicides against phytopathogenic fungi.     

Osmotic and matric potential effects on growth, sugar alcohol and sugar accumulation by Aspergillus section Flavi strains from Argentina

AIMS: The effect of osmotic and matric potential stress on growth and sugar alcohols (polyols: glycerol, erythritol, arabitol and mannitol) and sugars (trehalose and glucose) accumulation in toxigenic and nontoxigenic colonies of Aspergillus flavus and A. parasiticus was evaluated. METHODS AND RESULTS: Growth of Aspergillus section Flavi with significant reductions at 20 and 30 degrees C was more sensitive to changes in matric potential, between 60 and 100% in the range of -7 to -14 MPa. No significant differences were found between toxigenic and nontoxigenic strains for both species. Total polyol accumulation in unamended maize meal agar medium (-0.75 MPa water potential) was higher at 30 than 20 degrees C. The major change in concentrations of endogenous sugars and total polyols was in matrically amended medium (with PEG 8000) at -7 and -10 MPa. Accumulation of glucose, arabitol, mannitol and erythritol content of A. flavus and A. parasiticus mycelial colonies was greater in normal unstressed maize meal agar medium (-0.75 Mpa) at 20 degrees C. This was modified by solute and matric stress. CONCLUSIONS: The data showed relative sensitivity to osmotic and matric potential, and temperature, and the impact on growth rates, polyol and sugar accumulation in mycelia of A. flavus and A. parasiticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The matric potential effects on growth may be of particular importance for growth and survival in environments with low-matric potential stress. The tolerance of spoilage fungi such as Aspergillus section Flavi to such modifications could increase the potential for spoilage and mycotoxin production in such substrates. This knowledge is important for understanding the relative ecological fitness of these aflatoxigenic species and in the development of prevention strategies for their control.     

Polyamine metabolism during sclerotial development of Sclerotinia sclerotiorum

A study on polyamine metabolism and the consequences of polyamine biosynthesis inhibition on the development of Sclerotinia sclerotiorum sclerotia was conducted. Concentrations of the triamine spermidine and the tetramine spermine, as well as ornithine decarboxylase and S-adenosyl-methionine decarboxylase activities, decreased during sclerotia maturation. In turn, the concentration of the diamine putrescine was reduced at early stages of sclerotial development but it increased later on. This increment was not related to de novo biosynthesis, as demonstrated by the continuous decrease in ornithine decarboxylase activity. Alternatively, it could be explained by the release of putrescine from the conjugated polyamine pool. α-Difluoro-methylornithine and cyclohexylamine, which inhibit putrescine and spermidine biosynthesis, respectively, decreased mycelial growth, but did not reduce the number of sclerotia produced in vitro even though they disrupted polyamine metabolism during sclerotial development. It can be concluded that sclerotial development is less dependent on polyamine biosynthesis than mycelial growth, and that the increase of free putrescine is a typical feature of sclerotial development. The relationship between polyamine metabolism and sclerotial development, as well as the potential of polyamine biosynthesis inhibition as a strategy for the control of plant diseases caused by sclerotial fungi are discussed.     

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.     

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.     

Biological control of Botrytis gray mould and Sclerotinia drop in lettuce

Research was carried out to evaluate the effectiveness of the biological control of two most important fungal diseases of lettuce (Lactuca sativa L.): 1) Botrytis Gray Mould caused by Botrytis cinerea Pers. ex Fr.; 2) Sclerotinia Drop caused by two pathogenic fungi, Sclerotinia sclerotiorum (Lib.) De Bary and/or Sclerotinia minorJagger. Biological control in lettuce was carried out: 1) using Coniothyrium minitans Campbell, an antagonist fungus that attacks and destroys sclerotia within the soil; 2) identifying lettuce genotypes showing less susceptibility or tolerance. The object of this research was to find control strategies to reduce chemical treatments. The use of resistant varieties is one of the most economical ways to control vegeTable diseases. The lettuce genotypes showing in preliminary trials the best behaviour to the sclerotial diseases were compared in a randomized complete block experiment design and replicated four times. Observations were carried out from February up to April registering the number of diseased plants and yield. The pathogens were isolated on PDA medium and identified. The isolates grown onto PDA plates, after incubation for 6 weeks, allowed obtaining sclerotia that were the target of C. minitans in biological control trials. In laboratory, in controlled conditions, 27 small plots (30 cm in diameter each) with disinfected soil were performed. In 18 plots 9 sclerotia were inoculated (per plot, three of each fungus) and in 9 plots of them a suspension of the antagonist fungus was added. Subsequently, three lettuce varieties were transplanted. For each variety were compared: 1) untreated plots; 2) treated plots with sclerotia only; 3) treated plots with sclerotia and C. minitans suspension. The number of diseased plants was recorded. According to symptom evaluation scale, ranged from 0 (no disease) up to 10 (100% necrotic leaves or dead plants) the plants were grouped into infection classes, calculating the McKinney index. In greenhouse trials, "LM 1307" genotype showed less significant susceptibility to Botrytis Gray Mould (0-2% of affected plants), while "Ninja" and "Charmy" showed 4-11% and 16-26% of diseased plants, respectively. The yields were 69.7, 62.7, 55.3 t/Ha, respectively. In laboratory tests, the McKinney index gave the following results: no disease in all untreated plants; 38.3, 54.2 and 89.2% in "LM 1307", "Ninja" and "Charmy" treated with sclerotia only, respectively; 2.5, 7.5 and 20.8% in "LM 1307", "Ninja" and "Charmy" treated with sclerotia and C. minitans, respectively. In conclusion, the less susceptibility of the genotypes to sclerotial diseases and the use of hyperparasites of sclerotia of phytopathogenic fungi exhibited best results.     

Aviglycine and propargylglycine inhibit conidial germination and mycelial growth of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>f. sp. <Emphasis Type="Italic">luffae</Emphasis>

Two inhibitors, aviglycine and propargylglycine, were tested for their ability to suppress methionine synthesis thus inhibit conidial germination and mycelial growth of Czapek-Dox liquid medium grown Fusarium oxysporum f. sp. luffae μM. The linear inhibition range for mycelial growth was about 7.6–762.9 μM. Although aviglycine did not completely inhibit both conidial germination and mycelial growth, it showed significant inhibitory effect at 1.5 μM. The inhibition range for propargylglycine against conidial germination and mycelial growth were from 0.08 to 8841 μM and from 0.8 to 884.1 μM, respectively. Propargylglycine inhibited conidial germination and mycelial growth at a concentration of 8841 μM. The EC₅₀ values of aviglycine were 1 μM for conidial growth and 122 μM for mycelial growth, and the EC₅₀ values of propargylglycine were 47.7 μM for conidial growth and 55.6 μM for mycelial growth. Supplement of methionine released inhibition of aviglycine or propargylglycine to conidial germination. In addition, a mixture of aviglycine (1.5 μM) and propargylglycine (8841 μM) showed additive inhibitive effect than applied alone on 10 isolates. From these results, both aviglycine and propargylglycine exhibited inhibitory activity, and suggest that they can provide potential tools to design novel fungicide against fungal pathogens.     

Response of microbial activity and community structure to decreasing soil osmotic and matric potential

Low soil water content (low matric potential) and salinity (low osmotic potential) occur frequently in soils, particularly in arid and semi-arid regions. Although the effect of low matric or low osmotic potential on soil microorganisms have been studied before, this is the first report which compares the effect of the two stresses on microbial activity and community structure. A sand and a sandy loam, differing in pore size distribution, nutrient content and microbial biomass and community structure, were used. For the osmotic stress experiment, salt (NaCl) was added to achieve osmotic potentials from ?0.99 to ?13.13 MPa (sand) and from ?0.21 to 3.41 MPa (sandy loam) after which the soils were pre-incubated at optimal water content for 10d. For the matric stress experiment, soils were also pre-incubated at optimal water content for 10d, after which the water content was adjusted to give matric potentials from ?0.03 and ?1.68 MPa (sand) and from ?0.10 to 1.46 MPa (sandy loam). After amendment with 2% (w/w) pea straw (C/N 26), soil respiration was measured over 14d. Osmotic potential decreased with decreasing soil water content, particularly in the sand. Soil respiration decreased with decreasing water potential (osmotic?+?matric). At a given water potential, respiration decreased to a greater extent in the matric stress experiment than in the osmotic stress experiment. Decreasing osmotic and matric potential reduced microbial biomass (sum of phospholipid fatty acids measured after 14 days) and changed microbial community structure: fungi were less tolerant to decreasing osmotic potential than bacteria, but more tolerant to decreasing water content. It is concluded that low matric potential may be more detrimental than a corresponding low osmotic potential at optimal soil water content. This is likely to be a consequence of the restricted diffusion of substrates and thus a reduced ability of the microbes to synthesise osmolytes to help maintain cell water content. The study also highlighted that it needs to be considered that decreasing soil water content concentrates the salts, hence microorganisms in dry soils are exposed to two stressors.