Limitations of In Vitro Strain Screening Methods for the Selection of Sclerotinia spp. as Potential Mycoherbicides against the Perennial Weed Ranunculus acris

Nineteen isolates of Sclerotinia sclerotiorum and three isolates of S. minor were inoculated on to excised tissues and intact plants of Ranunculus acris (giant buttercup), to evaluate their pathogenicity. All isolates proved pathogenic, with S. sclerotiorum being more pathogenic than S. minor on both excised tissues and intact plants. Seven of the S. sclerotiorum isolates were more pathogenic than the others on excised tissues, but no significant differences in pathogenicity were found between any of the isolates when they were inoculated on to intact plants. The results of this study indicate that the excised tissue method cannot be used to predict whole plant mortality, nor, therefore, the mycoherbicide potential of strains of S. sclerotiorum for this perennial weed.     

Barley Yellow Dwarf Luteovirus (BYDV) Infectivity of Alate Aphid Vectors in Northeast Spain

Rhopalosiphum padi and Sitobion avenae alates were collected from colonised winter cereals and maize in N.E. Spain and fed on young wheat plants for 7–10 days in the glasshouse. Then, aphids were killed and the plants on which aphids reproduced were kept in the glasshouse for 30–40 days. ELISA of infested plants was made using polyclonal and monoclonal antisera against PAV-, RPV- and MAV-like isolates. In autumn and spring, MAV serotypes were transmitted by S. avenae and R. padi , mainly in mixed infections with PAV serotypes. This possibly explains the high frequency of MAV-like isolates and their previously recorded year-to-year stability in maize, grain and forage winter cereals and cereal volunteers. PAV-like isolates were rarely transmitted by S. avenae and its spread thus depends almost exclusively on R. padi. These results confirm the importance of forage cereals and cereal volunteers as virus sources for winter cereal infection in the autumn, and the latter as a source of BYDV for maize in spring.     

Factors Influencing the Efficacy of the Potential Microbial Herbicide Alternaria alternata (Fr.) Keissler on Amaranthus retroflexus (L.)

Alternaria alternata , applied to run-off at 107 conidia ml -1 and given a 6 h dew period, caused an 88.5% reduction in dry weight of 4 to 5 true-leaf plants of Amaranthus retroflexus . This level of control was achieved whether the inoculated plants were grown in the glasshouse or outside. Formulation of the pathogen in oilseed rape emulsion reduced its dew period requirement from 24 h or more to 8 h or less. For example, with 106 conidia ml -1 , equivalent effects were found with 8 h dew with emulsion formulation compared to 48 h dew with the Tween formulation. Allowing spray deposits to dry on inoculated plants for ca 7 h, before exposing to dew increased the efficacy of the pathogen.     

Development of microbial consortia as a biocontrol agent for effective management of fungal diseases in Glycine max L.

Thirty isolates of bacteria and six isolates of Trichoderma were isolated from fertile agricultural soil and evaluated for their antagonistic activity against phytopathogens like Macrophomina phaseolina and Sclerotinia sclerotiorum, under in vitro conditions. Different isolates showed varying degrees of antagonism. The three most antagonistic bacteria Pseudomonas aeruginosa (MBAA1), Bacillus cereus (MBAA2) and Bacillus amyloliquefaciens (MBAA3) and one fungi Trichoderma citrinoviride (MBAAT) were selected as the most effective isolates as biocontrol agents. The present study was undertaken to develop a plant growth promoting microbial consortium to reduce the disease incidence in Glycine max both under in vitro and in vivo conditions. Biocontrol attributes such as ammonia, siderophore, enzymes like β-1,3 glucanase, chitinase and cellulase were more potential in consortia in comparison to single isolates. Plants treated with consortia?+?pathogen showed lower disease incidence in comparison to single antagonist?+?pathogen and pathogen infested control (p?≤?0.05). Maximum disease control was observed in potted plants treated with S. sclerotiorum?+?MBAA1?+?MBAAT showing only 15.8% disease incidence in comparison to Sclerotinia infested control, in which disease incidence was 97%. Seed bacterised with MBAA1?+?MBAAT exhibited enhanced seed germination of G. max up to 68% along with subsequent increase in other plant growth parameters. Considerable increase in seedling vigour index (1863.2) and chlorophyll content (13.518?mg/g) was observed in seeds treated with MBAA1?+?MBAAT in plants infected with M. phaseolina.     

Origin of Early Attacks of Sclerotinia Stem Rot on Winter Oilseed Rape (Brassica napus sub. sub. sp. oleifera var. biensis) in the UK

Studies in artificially infested field plots of winter oilseed rape showed that mycelia originating from both sclerotia and ascospores of Sclerotinia sclerotiorum can initiate early attacks of sclerotinia stem rot. Disease symptoms appeared in early November in plots previously infested with sclerotia in September. There was a 6–7 week period from artificially inoculating plants with ascospores in October to observing disease symtoms in December. The primary sites of infection for both typesof inocula were the laminae and petioles of senescent and frost-damaged leaves. Following primary infection, mycelia of the pathogen spread by plant to plant contact, causing secondary infections of neighbouring plants. Sclerotia developed on diseased plants and high numbers were returned to the soil. The importance of these findings to the epidemiology of the disease are discussed.     

Involvement of alternative oxidase in the regulation of growth, development, and resistance to oxidative stress of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a cosmopolitan, filamentous, fungal pathogen that can cause serious disease in many kinds of crops. Alternative oxidase is the terminal oxidase of the alternative mitochondrial respiratory pathway in fungi and higher plants. We report the presence of this alternative pathway respiration and demonstrate its expression in two isolates of S. sclerotiorum under unstressed, normal culture conditions. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, severely inhibited the mycelial growth of S. sclerotiorum both on potato dextrose agar plates and in liquid culture media. Inhibition of alternative oxidase could influence the growth pattern of S. sclerotiorum, as salicylhydroxamic acid treatment induced obvious aerial mycelia growing on potato dextrose agar plates. Under the treatment with salicylhydroxamic acid, S. sclerotiorum formed sclerotia much more slowly than the control. Treatment with hydrogen peroxide in millimolar concentrations greatly decreased the growth rate of mycelia and delayed the formation of sclerotia in both tested S. sclerotiorum isolates. As well, this treatment obviously increased their alternative pathway respiration and the levels of both mRNA and protein of the alternative oxidase. These results indicate that alternative oxidase is involved in the regulation of growth, development, and resistance to oxidative stress of S. sclerotiorum.     

Physiological and Molecular Features of the Pathosystem Arabidopsis thaliana L.-Sclerotinia sclerotiorum Libert

The fungal pathogen Sclerotinia sclerotiorum Libert causes rot diseases on many crops worldwide and large economic losses occur frequently because of a lack of resistant varieties. The pathogenesis of S. sclerotiorum and the molecular basis of plant responses to the pathogen are poorly understood. In the present investigation, the process of S. sclerotiorum infection in Arabidopsis thaliana L., a plant that is highly susceptible to this fungus, was analysed. In addition, the defense activation in the host was investigated. A convenient inoculation method using millet grain was developed for S. sclerotiorum in Arabidopsis. The fungus rapidly infected the plants, probably through ball- or cushion-like infection structures. Visible symptoms developed within 24 h and plants were killed 72 h after inoculation. Cellulase, the main enzyme that caused host tissues to rot, was secreted by S. sclerotiorum in a pH-dependent manner. Oxalic acid, another pathogenic factor secreted by the fungus, induced necrotic lesions on the leaves, infection with S. sclerotiorum strongly induced the production of the pathogenesis-related （PR） proteins β-1,3-glucanase and chitinase in Arabidopsis. Furthermore, the PR gene PDF. 1 was induced, but not PR1, indicating that the pathogen activated basal defense of jasmonic acid/ethylene dependence, which is consistent with its necrotrophic characteristics. This pathosystem for Arabidopsis-S. sclerotiorum could provide an approach for the analysis of the interactions between S. sclerotiorum and other crops, thereby facilitating genetic manipulation techniques for controlling this pathogen.     

Evaluation of entomopathogenic fungus Metarhizium anisopliae against soil-dwelling stages of cabbage maggot (Diptera: Anthomyiidae) in glasshouse and field experiments and effect of fungicides on fungal activity

The effect of two isolates of the entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin (389.93 and 392.93) on root-feeding stages of cabbage root fly, Delia radicum (L.), was studied under glasshouse and field conditions. In glasshouse studies, the effect of drenching a suspension of conidia (concentration 1 x 10(8) m(-1), 40 ml per plant, applied on four occasions) onto the base of cabbage plants infested with D. radicum eggs was compared with mixing conidial suspension into compost modules (concentration 1 x 10(8) ml(-1), 25 ml per plant) used to raise seedlings. Drench application reduced the mean number of larvae and pupae recovered per plant by up to 90%, but the compost module treatment had no statistically significant effect. Both application methods reduced the emergence of adult flies from pupae by up to 92%. Most conidia applied as a drench application remained in the top 10-cm layer of compost. Applications of the fungicides iprodione and tebuconazole, which are used routinely on brassica crops, were compatible with using M. anisopliae 389.93 against D. radicum under glasshouse conditions, even though these fungicides were inhibitory to fungal growth on SDA medium. In a field experiment, drench applications of M. anisopliae 389.93 to the base of cauliflower plants at concentrations of 1 x 10(6) to 1 x 10(8) conidia ml(-1) did not control D. radicum populations, although up to 30% of larval cadavers recovered supported sporulating mycelium. Drench applications often exhibited considerable lateral movement on the soil surface before penetrating the ground, which may have reduced the amount of inoculum in contact with D. radicum larvae.     

Biological control of Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of white mold, by Pseudomonas species on canola petals

Sclerotinia sclerotiorum is an important pathogen on canola. Due to the public concern over pesticide use, alternative methods of disease control, such as biological control, should be considered. Several bacterial strains were isolated from canola and soja plants. Inhibition of S. sclerotiorum by bacterial strains in vitro was assayed on PDA medium in dual culture test. Eight Pseudomonas sp. strains (PB-3, PB-4, PB-5, PB-6, PB-7, PB-8, PB-10 and PB-11) caused inhibition zone against 5. sclerotiorum hyphal growth. The biocontrol potential of the bacteria was tested in a plant assay. Disease suppression was investigated using a petal inoculation technique. Canola petals were pretreated with bacteria, and then inoculated with 5. sclerotiorum ascospores 24 h later. Greenhouse experiment showed that application of Pseudomonas sp. strains (1 x 10(8) cfu ml(-1)) effectively suppressed S. sclerotiorum (1 x 10(5) ascospores ml(-1)) on petals and all of them achieved significant (P<0.01) disease suppression. Fourteen days after inoculation, strain PB-3 had 88/7% disease control and strain PB-4 had 69/9% disease control. Result from all studies indicates PB-3 to be effective biocontrol against S. sclerotiorum of canola. PB-3, PB-4, PB-7, PB-8, PB-10 and PB-11 were identified as Pseudomonas fluorescens biovar III. PB-5 and PB-6 was identified as Pseudomonas fluorescens biovar II. Strains PB-3, PB-4, PB-6, PB-10 and PB-11 produced protease and HCN. Strain PB-5 produce protease; no HCN.     

Effect of Application Time on the Efficacy of Sclerotinia sclerotiorum as a Mycoherbicide for Cirsium arvense Control in Pasture

An experiment was conducted in sheep-grazed pasture in three regions of New Zealand over three years to evaluate the effect of application time on the efficacy of a dry granule myceliumon-wheat formulation of Sclerotinia sclerotiorum for the biological control of Cirsium arvense . At each site, the experimental mycoherbicide was applied to a previously untreated plot in each month of the year for three years at a dose of 50 g m -2 . Applications made during the spring and early summer months of October, November and December significantly reduced the ground cover of C. arvense for 67, 67 and 44%, respectively of these applications. Reduced ground covers ranged from 38 to 81% of the cover on untreated plots. Applications in late summer and autumn were less effective. Correlations of ground cover by C. arvense with climate parameters suggested that free moisture promoted treatment efficacy, but that intense rainfall after treatment reduced efficacy through wash off. The importance of leaf wetness for the efficacy of the mycoherbicide was confirmed by comparing disease development and mortality in C. arvense shoots with and without enhanced moisture levels under field conditions. A water miscible formulation applied as a slurry was less dependent upon leaf wetness than the dry granule formulation.     

Diagnosis and management of Sclerotinia stem rot (white mould) of lentils in Greece

The paper describes the field-level symptoms, the identification and management of Sclerotinia stem rot of lentil caused by the soilborne plant pathogen Sclerotinia sclerotiorum in Greece. Regarding symptoms at a field level, initially plants before flowering turn yellow with roots and the base of the plants become brown; then rotten plants exhibit a dry stem and die. On the diseased tissue, at the base of the stem, the typical white mycelium and the resting bodies (sclerotia) were observed. According to our pathogenicity studies in vitro, on the infected plant tissues the fungus first develop a characteristic fluffy white mycelium which will give rise to large black sclerotia, the most obvious evidence of plants infected with S. sclerotiorum. Finally, concerning evaluation of fungicides, isolates of S. sclerotiorum were sensitive to thiophanate-methyl and to triazole fungicides. Thiophanate-methyl and triazole fungicides proved to be most effective in controlling the disease emerged from mycelium or sclerotia.     

Genotypic characteristics in populations of Sclerotinia sclerotiorum from New York State,USA

White mould, caused by the fungus Sclerotinia sclerotiorum, is one of the most destructive diseases of beans globally. In New York State, USA, white mould causes substantial losses in soybean, snap, dry and succulent baby lima beans, which are grown successively in intensive crop rotations. Management strategies for white mould in these crops are reliant upon the prophylactic use of fungicides. No complementary information on the genetic structure of the populations of S. sclerotiorum in New York State, USA is available. Twenty isolates of S. sclerotiorum were collected from symptomatic bean plants within each of 10 fields across New York State, USA in 2014. Eight microsatellite (SSR) markers were used to characterise the genotypic diversity of the hyphal‐tipped isolates. Twenty‐four multilocus genotypes (MLGs) were detected within the population but one MLG was most prevalent. Although STRUCTURE analysis identified two subpopulations, these subpopulations were not associated with geographic location, suggesting no spatial structure to the population. In addition, the pathogen populations were predominantly clonal, with some evidence of infrequent outcrossing. These findings may assist in understanding the durability of management strategies for white mould and support the selection of representative isolates for host resistance screening for pathogen populations in the sampling area.     

Stilbella aciculosa: A Potential Biocontrol Fungus against Rhizoctonia solani

A semi‐solid fermentation product of the potential biocontrol fungus Stilbella aciculosa was formulated on wheat bran: water (1:1, w/w) and incubated 5, 10 and 15 days before addition to soil infested with the pathogen Rhizoctonia solani. Generally, preparations did not reduce survival of the pathogen in infested beet seed but they did prevent saprophytic growth of the pathogen from beet seed into soil. The magnitude of reduction by the 15‐day‐old inoculum was greater than that by the 5‐day‐old inoculum. Ten‐day‐old bran preparations of S. aciculosa at rates of 0.5 and 1.0% (w/w) in soil prevented post‐emergence damping‐off of cotton, radish and sugar beet in the glasshouse and a rate of 1.0% gave stands similar to those in the non‐infested control soil. The antagonist, grown on perlite formulated with molasses, cornmeal, alfalfa tissue or corn stover, prevented damping‐off of cotton in a naturally infested soil. However, the stands were not as great as that in soil planted with pentachloronitrobenzene (PCNB)‐treated seed. Toxic metabolites, produced by S. aciculosa developing on various substrates, slightly inhibited the growth of R. solani in culture and induced cytoplasmic leakage of the pathogen mycelium.     

Oxalic acid production and its role in pathogenesis of Sclerotinia sclerotiorum

Abstract Sunflower plants were inoculated with a virulent isolate of Sclerotinia sclerotiorum and with the same isolate nutritionally conditioned to produce small amounts of oxalic acid. The preconditioned isolate behaved as hypovirulent. Tomato plants were inoculated with four S. sclerotiorum isolates of increasing virulence. A close correlation among disease severity, accumulation of oxalic acid, decrease in pH and inhibition of polyphenoloxidase in both infected host tissues was demonstrated. Oxalic acid production as an important factor of virulence in S. sclerotiorum is emphasized and its effect on the phenolic metabolism of the host via inhibition of polyphenoloxidase is suggested.     

捕食线虫丝孢菌的菌寄生性测定

研究了节丛孢Arthrobotrys、单顶孢Monacrosporium和隔指孢Dactylella三个捕食线虫丝孢菌属16个菌株,对水稻立枯丝核菌RhizoctoniasolaniAG1、大豆核盘菌Sclerotiniasclerotiorum、茄科镰刀菌Fusariumsolani和恶疫霉Phytophthoracactorum四种常见土壤植物病原真菌的菌寄生性。结果表明供试菌可以通过弹簧式菌丝圈缠绕、类附着胞结构吸附、简单的菌丝缠绕或者贴附寄主菌丝生长四种方式寄生病原菌。其中,绝大多数菌株对立枯丝核病菌有寄生作用,一些供试真菌对其它三种病原真菌有寄生现象。利用孢子液浸泡法测定了其中5种捕食线虫真菌对核盘菌菌核的寄生能力,显示有较高寄生率。     

Potato bacterial wilt suppression and plant health improvement after application of different antioxidants

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease that often threatens potato production and exportation. The potential of four antioxidants (seaweed extract (SWE), yeast, chitosan and ascorbic acid (ASA)) in controlling the disease was evaluated in vitro, under glasshouse and field conditions. The field experiment was conducted in two naturally infested locations: Wardan, Giza (sandy soil), and Talia, Minufiya (silty clay soil). Only chitosan showed antibacterial properties against the pathogen in vitro. SWE, yeast and chitosan showed disease suppression under both glasshouse and field conditions. The disease suppression was accompanied by an increase in the ratio of soil copiotrophic to oligotrophic bacteria. The three antioxidants increased plant nitrogen content, decreased soil OM content and decreased C/N ratio. Disease suppression after chitosan application was clearly observed only in Wardan area, which was characterized by a higher soil alkalinity. A high percentage of antagonistic fluorescent strains similar to Pseudomonas putida group were detected for chitosan‐treated plants in Wardan area (sandy soil). ASA drastically decreased the count of the pathogen in soil, but was conducive to the pathogen in plant tissues. A remarkable increase in microbial (bacterial and fungal) soil and rhizosphere diversity as indicated by PCR‐DGGE analysis for bacterial 16S rRNA and fungal 18S rRNA was recorded. In Talia area (silty clay soil), the soil microbial community was more stable and was in general resistant to the disease where the soils were characterized by high electrical conductivity. SWE, yeast and ASA significantly increased crop production in Talia area only.     

Population structure of Sclerotinia sclerotiorum in an Australian canola field at flowering and stem-infection stages of the disease cycle.

Populations of the ascomycete pathogen Sclerotinia sclerotiorum sampled from a canola field were analysed using microsatellite markers. Fifty isolates were collected from ascospore-infested canola petals and, later in the season, another 55 isolates were obtained from stem lesions; these isolates were used to compare inoculum and disease-causing populations. Fifty-five unique haplotypes were identified, with gene diversity ranging from 0.40 to 0.71. Genotypic diversity was higher in the inoculum population than it had been in the previous year, but analysis of molecular variance (AMOVA) showed that less than 10% of the variation was attributable to differences between the 2 years. Genotypic disequilibrium measures were consistent with the occurrence of both clonal reproduction and out-crossing. There was no significant population subdivision between the ascospore and stem-lesion populations, as measured with fixation indices (R(ST) = 0.015, p = 0.90) and AMOVA, suggesting that there are no genetically defined subgroups of isolates more likely to proceed from petal colonization to cause stem infection. This might be because S. sclerotiorum possesses wide-ranging pathogenicity mechanisms that account for the lack of host specificity observed to date.     

Biological control of the wheat root rot caused by Fusarium graminearum using some PGPR strains in Saudi Arabia

The aim of this study was to evaluate the efficacy of selected bacterial strains against the wheat soil‐borne pathogen Fusarium graminearum under greenhouse conditions. The most potent isolates were 3 isolates out of 18 isolates, which have numbers 3, 9 and 10 with in vitro inhibition index 42.5%, 41.3% and 46.3% respectively. Isolates 3 and 10 were selected for the following experiments. Isolates 3 and 10 were identified as Bacillus subtilis MAA03 and Pseudomonas fluorescens MAA10, respectively according to International Identification Keys and, confirmed by using Biolog system and 16S rDNA where the strains exhibited more than 99.5% sequence identity. Their close taxonomic relationship was further documented by phenotypic similarities. The using of B. subtilis and P. fluorescens separately or in mixture as biocontrol agent against F. graminearum on wheat significantly increased the final germination percent, the mean daily germination and germination index of wheat cultivar, while the mean germination time was significantly decreased relative to infested control. The final infection percent, the mean daily infection and infection index were decreased significantly, while the mean infection time was significantly increased relative to infested control. The use of P. fluorescens as biocontrol agent was the most efficient than B. subtilis or in mixture and the best treatment was seed coating. The application of B. subtilis and P. fluorescens separately or in combination significantly affected the growth parameters of wheat cultivar Tabuki, the root length was significantly increased in seed coating and seed soaking treatments, while non‐significantly decreased in case of soil drench treatment relative to infested control. Shoot length was significantly decreased in case of seed coating treatment relative to infested control. The shoot fresh and dry weights were significantly increased in seed coating and seed soaking treatments relative to infested control. The root fresh and dry weights were significantly increased in seed coating and seed soaking treatments relative to infested control. The number of leaves was significantly increased in all treatments relative to infested control.     

Cyclosporine A from a nonpathogenic Fusarium oxysporum suppressing Sclerotinia sclerotiorum

AIMS: To evaluate the antagonistic activity of Fusarium oxysporum nonpathogenic fungal strain S6 against the phytopathogenic fungus Sclerotinia sclerotiorum and to identify the antifungal compounds involved. METHODS AND RESULTS: The antagonistic activity of Fusarium oxysporum strain S6 was determined in vitro by dual cultures. The metabolite responsible for the activity was isolated by chromatographic techniques, purified and identified by spectroscopic methods as cyclosporine A. The antifungal activity against the pathogen was correlated with the presence of this metabolite by a dilution assay and then quantified. Cyclosporine A caused both growth inhibition and suppression of sclerotia formation. In a greenhouse assay, a significant increase in the number of surviving soybean (Glycine max) plants was observed when S. sclerotiorum and F. oxysporum (S6) were inoculated together when compared with plants inoculated with S. sclerotiorum alone. CONCLUSION: Fusarium oxysporum (S6) may be a good fungal biological control agent for S. sclerotiorum and cyclosporine A is the responsible metabolite involved in its antagonistic activity in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Cyclosporine A has not been previously described as an inhibitor of S. sclerotiorum. Its minimum inhibitory concentration (MIC) of 0.1 microg disc(-1) makes it suitable to use as a biofungicide. In vivo experiments showed that F. oxysporum (S6) is a good candidate for the biocontrol of S. sclerotiorum in soybean.     

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.