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.     

Pathogenicity of Sclerotinia sclerotiorum on Ranunculus acris in Dairy Pasture

Fifty-four isolates of Sclerotinia sclerotiorum from Ranunculus acris and other natural hosts were applied as mycelial infested kibbled wheat onto 6 month-old R. acris plants in two glasshouse screening experiments. Most isolates (90%) did not differ in their pathogenicity towards R. acris. One isolate, S. sclerotiorum G45, was selected based on its ability to cause severe disease and suppress regeneration of R. acris. A field experiment was conducted to determine the efficacy of S. sclerotiorum (G45) against R. acris in infested dairy pastures in the Takaka Valley, Golden Bay, New Zealand. Isolate G45 was formulated as a wettable powder and was applied as a slurry at 20 and 40 ml/plant in December 1995. After 10 weeks, regeneration from the crown of treated plants was apparent and a second application of S. sclerotiorum was made in February 1996. Best control of R. acris was obtained when the plants were inoculated in full flower in December. At the first time of treatment, the 40 ml application of S. sclerotiorum slurry reduced the total dry weight of R. acris by an average of 57%. The second application had no effect on total dry weight, possibly because moisture levels were not sufficient for S. sclerotiorum infection. This study confirmed S. sclerotiorum to be an aggressive pathogen of R. acris under both glasshouse and field conditions. As a result, this pathogen has potential as a mycoherbicide for R. acris. Further experiments are required to explore ways of enhancing the efficacy of S. sclerotiorum against R. acris by manipulation of the host, pathogen and environment.     

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.     

Pathogenicity Determinants of Sclerotinia sclerotiorum and Their Association to Its Aggressiveness on Brassica juncea

White rot or stem rot caused by Sclerotinia sclerotiorum is one of the most destructive fungal diseases that have become a serious threat to the successful cultivation of oilseed Brassicas. The study was designed with an aim to investigate the association between the pathogenic aggressiveness and pathogenicity determinants of this pathogen specifically in Brassica for the first time. For this, a total of 58 isolates of S. sclerotiorum from different geographical regions were collected and purified. These isolates were inoculated on a Brassica juncea cv. RL-1359 and they exhibited high level of variation in their disease progression. The isolates were grouped and then 24 isolates were selected for the biochemical analysis of pathogenicity determinants. The isolates varied significantly with respect to their total organic acids, oxalic acid production and pectin methyl esterase and polygalacturonase activity. The oxalic acid production corresponded to the disease progression of the isolates; the isolates with higher oxalic acid production were the more aggressive ones and vice-versa. This is, in our knowledge, the first study to establish a correlation between oxalic acid production and pathogenic aggressiveness of S. sclerotiorum on B. juncea. However, the pectinases’ enzyme activity did not follow the trend as of disease progression. These suggest an indispensable role of oxalic acid in pathogenicity of the fungus and the potential to be used as biochemical marker for preliminary assessment of pathogenic aggressiveness of various isolates before incorporating them in a breeding program.     

Identification and characterization of Sclerotinia sclerotiorum NADPH oxidases

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. ΔSsnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum.     

Pathogenic characterization and study of the mycelial groups of compatibility in Sclerotium cepivorum Berk

We have studied the pathogenicity and the formation of mycelial compatibility groups of (MCG) in 12 isolates of the fungus Sclerotium cepivorum Berk from the states of Táchira, Mérida, Trujillo in Venezuela and Pamplona in Colombia. The pathogenicity was evaluated according to severity and virulence. In vitro confrontation tests were used to check the presence of compatible and incompatible interactions expressed by MCG when different isolates of the fungus were put together in a petri dish. When pathogenicity was evaluated, we found high levels of disease with isolates T-9, C-1 and TR-10. The isolate T-9 was the most virulent and agressive, as the symptoms began at day 12 and at day 19 and 100% of the nine inoculated plants died. Three responses were observed when confronting S. cepivorum isolates: a reaction of compatibility or anastomosis and two reactions of incompatibility. Two MCG were found, represented by isolates T-8 (Táchira) and TR-3 (Trujillo), which showed incompatibility when faced with other isolates. These results provide a basis upon which the genetic characterization of S. cepivorum can be established.     

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.     

An endophyte of the tropical forage grass Brachiaria brizantha: isolating, identifying, and characterizing the fungus, and determining its antimycotic properties

Brachiaria, predominantly an African genus, contains species, such as B. brizantha, an apomictic C4 grass, that are commercially important forage grasses in tropical America, where they now cover about 55 million hectares. From B. brizantha accession CIAT 6780, we isolated an endophytic fungus that may be economically significant. The fungus was identified as Acremonium implicatum (J. Gilman & E.V. Abott). 18S rDNA and ITS rDNA sequences were used to characterize isolates of the endophyte, and showed that they belonged to the Acremonium genus, being close to A. strictum and A. kiliense. Using the random amplified polymorphic DNA (RAPD) technique, involving arbitrary primers of 10 bases, we showed that the isolates were highly similar to each other. Antiserum produced from a monoconidial culture of A. implicatum isolated from B. brizantha 6780, differentiated the isolates consistently in line with the DNA data. When we compared endophyte-free with endophyte-infected B. brizantha CIAT 6780 plants, both artificially inoculated with the pathogenic Drechslera fungus, we found that the endophyte-infected plants had fewer and smaller lesions than did the endophyte-free plants. Sporulation of Drechslera sp. on artificially inoculated leaf sheath tissues was also much less on tissue infected with the endophyte.     

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.     

A Comparison of Auxotrophic and Wild Strains of Sclerotinia sclerotiorum Used as a Mycoherbicide Against Californian Thistle ( Cirsium arvense )

Two auxotrophic mutant strains of Sclerotinia sclerotiorum were tested in the greenhouse for pathogenicity on Cirsium arvense (Californian thistle) with and without amino acid amendments. An arginine auxotrophic mutant, with an amendment of the amino acid, followed an identical disease progress curve to that of the wild strain of the pathogen from which it was derived. However, when deprived of the amino acid amendment it was still highly pathogenic. A leucine auxotrophic mutant demonstrated poor pathogenicity without a leucine amendment, but improved pathogenicity with the addition of the amino acid. However, both of these treatments were inferior to the two wild strains tested and the arginine auxotroph with and without amendments. A field experiment was conducted on C. arvense stems in permanent pasture to compare the pathogenicity of amended auxotrophic strains and wild strains of S. sclerotiorum applied as a granule in a wheat-based carrier. The two wild strains gave significant reductions in thistle cover within 3 months of treatment, and subsequent reductions in thistle stem height and density during the following season. There was no evidence that the auxotrophic strains reduced thistle cover in the season the treatments were applied, but they did reduce subsequent stem density in the following spring. To determine disease carry-over associated with the wild and auxotrophic strains of the pathogen, rape was planted into subplots over the next three consecutive seasons. Despite substantial populations of sclerotia being present in the soil, especially in the first season after treatment of the thistles, no disease of rape caused by S. sclerotiorum was detected over the three seasons in any of the plots. Sclerotium populations of S. sclerotiorum in the soil declined by over 50% between 20 and 32 months after treatment, but there was no decline over the subsequent 12 months. The trial demonstrated that the auxotrophic strains were less field fit compared with the wild strains and that the presence of inoculum and a susceptible host to S. sclerotiorum were not the only prerequisites for disease development. It was concluded that use of a trap crop following treatment is not a suitable method for determining the risk of using this pathogen as a mycoherbicide in pasture.     

Development of an insect model for the in vivo pathogenicity testing of yeasts

Conventional in vivo assays to determine the relative pathogenicity of yeast isolates rely upon the use of a range of mammalian species. The purpose of the work presented here was to investigate the possibility of using an insect (Galleria mellonella) as a model system for in vivo pathogenicity testing. The haemolymph of G. mellonella larvae was inoculated with PBS containing different concentrations of stationary phase yeasts of the genus Candida by injection at the last pro-leg. Larvae were incubated at 30 degrees C and monitored over 72 hours. Results indicate that G. mellonella can be killed by the pathogenic yeast Candida albicans and by a range of other Candida species but not to a significant extent by the yeast Saccharomyces cerevisiae. The kill kinetics for larvae inoculated with clinical and laboratory isolates of C. albicans indicate the former class of isolates to be more pathogenic. Differences in the relative pathogenicity of a range of Candida species may be distinguished using G. mellonella as a model. This work indicates that G. mellonella may be employed to give results consistent with data previously obtained using mammals in conventional in vivo pathogenicity testing. Larvae of G. mellonella are inexpensive to culture, easy to manipulate and their use may reduce the need to employ mammals for routine in vivo pathogenicity testing with a concomitant reduction in mammalian suffering.     

Electrophoretic Karyotypes of Sclerotinia sclerotiorum

Electrophoretic karyotypes (EKs) of 83 isolates were variable within agricultural and natural populations of Sclerotinia sclerotiorum, as well as among S. sclerotiorum, Sclerotinia minor, and Sclerotinia trifoliorum. Variation in EKs was not observed within six mitotic or three meiotic lineages of isolates. EKs of 8 to 10 chromosome-sized DNAs were observed. Homologous and heterologous probes hybridized to four linkage groups.     

Colonization of root tissues and protection against Fusarium wilt of rye (Secale cereale) by nonpathogenic rhizosphere strains of Fusarium culmorum

Colonization of rye (Secale cereale) tissues by nonpathogenic rhizosphere Fusarium culmorum isolates DEMFc2 and DEMFc5 and a pathogenic strain DEMFc37, and their effect on plant fresh weight were studied in pot experiments. Both rhizosphere isolates colonized the epidermis and the cortex but were not found in vessels, while the pathogen colonized all three layers of root cells. The numbers of pathogen CFU isolated from plant tissues were much higher than those of the rhizosphere isolates in spite of the same number of macroconidia used as inoculum (1 × 10⁵ g⁻¹ of soil). Inoculation of seedlings with DEMFc2 resulted in a 20% increase, with DEMFc5 in more than a 20% reduction, and with DEMFc37 in a 38% reduction of shoot fresh weight of 14-day-old plants. Pre-colonization of plants with (either of) the rhizosphere isolates and subsequent inoculation with the pathogen resulted in plant weights the same as those observed in plants inoculated with the rhizosphere strain alone. The disease severity index for shoots of plants pre-colonized with DEMFc2 was reduced from class 4 (86% diseased plants) observed for plants inoculated with the pathogen alone to class 2 (average of 8% diseased plants) when pre-treated with the rhizosphere strain. The CFU number of the pathogen isolated from the interior of roots of plants pre-colonized with the rhizosphere isolates was as low as 10% of the number isolated from plants inoculated with the pathogen alone. A study of in vitro interactions between the rhizosphere isolates and the pathogen suggests that changes in plant colonization by the pathogen and its effect on fresh weight of plants pre-colonized with the rhizosphere isolates were not connected with inhibition of its growth by a direct action of the rhizosphere isolates. The results suggest that strain DEMFc2 can be considered as a potential biocontrol agent.     

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.     

Pathogenicity of Fusarium graminearum Schwabe isolates from Poland towards seedlings of cereal species

Ten isolates ofFusarium graminearum Schwabe originating from diseased cereal plants and kernels were tested for pathogenicity to various cultivars of wheat, rye, triticale and oats. The isolates varied greatly in their pathogenicity to the seedlings of the species, and were most pathogenic to rye and triticale, less pathogenic to barley and wheat and least pathogenic to oats.     

两株致病力不同的核盘菌形态变化研究

比较了强弱致病型不同的核盘菌Ep-1PNA5和Ep-1PN的菌落、菌丝、细胞的形态变化。结果发现强致病型的Ep-1PNA5在平板上的菌落呈圆形均匀扩展,气生菌丝少,形成的菌核多,细胞的原生质均匀浓密,内部结构完整。弱致病型的Ep-1PN菌落呈扇形扩展,不均匀,气生菌丝发达,形成的菌核少,菌丝顶端分枝异常,有原生质外渗,且原生质不均匀,内部结构不完整。Ep-1PN致病力减弱和生长抑制可能与其菌丝结构和细胞形态变异有关。     

Endophytic and pathogenic isolates of the cacao fungal pathogen Moniliophthora perniciosa (Tricholomataceae) are indistinguishable based on genetic and physiological analysis

We evaluated the genetic and physiological variability of Moniliophthora perniciosa obtained from healthy and diseased branches of cacao (Theobroma cacao) plants. The diversity of the isolates was evaluated by RAPD technique and by studies of virulence and exoenzyme production. The genetic variability of endophytic and pathogenic M. perniciosa was evaluated in association with pathogenicity assays. RAPD analysis showed eight genetic groups, which were not related to plant disease status (healthy versus diseased branches). Isolates from cacao were included in three groups, excluding isolates from other host plants. Pathogenicity and enzyme analysis showed that the virulence of the isolates is not related to exoenzyme production. This is the first evidence that M. perniciosa colonizes healthy parenchymatic tissues, showing that endophytic behavior may occur in this species.