Efficient expressions of reporter genes in the industrial filamentous fungus Sclerotium rolfsii mediated by Agrobacterium tumefaciens

Sclerotium rolfsii (teleomorph Athelia rolfsii) is one of the plant pathogenic basidiomycetes, which causes severe stem-rot disease in hundreds of plants and produces important metabolites, such as scleroglucan and TF-specific lectin. However, further molecular biological research on this filamentous fungus is severely plateaued out due to the lack of genetic methods. In this study, the A. tumefaciens strain LBA4404 harboring a binary vector containing the basta resistance gene fused with three reporters (DsRed, tdTomato, and GUSPlus) respectively, driven by the SrGPD promoter, was used for genetic transformation of S. rolfsii. The results showed that the three reporter genes were all effectively expressed in S. rolfsii. This study also showed that the intron of the SrGPD promoter is not necessary for transgene expression in this fungus. Besides, we showed that these reporters’ signals could be observed easily but in a short time window. The efficient Agrobacterium-mediated transformation system and the three reporter gene plasmids for S. rolfsii developed in this study are of significance in overcoming current limitations of no available transformation and genetic manipulation techniques in S. rolfsii, facilitating further genetic manipulations and gene function exploration.     

Impact of salinity stress on soil-borne fungi of sugarbeet

The sugarbeet cultivar Kaumera was found to be highly susceptible to infection by the root-rot pathogens Rhizoctonia solani and Sclerotium rolfsii in the absence of salinity stress. Under this environmental condition, R. solani was more efficient than S. rolfsii in producing cell wall-degrading enzymes in infected hypocotyls. Xylanase and galactanase were most effective. The rate of cell wall degradation by R. solani was nearly 2.5 times that of S. rolfsii when cells walls of healthy hypocotyls were used as sole carbon substrate for the in vitro produced crude enzymes.Under salinity stress the pathogenicity and the performance of cell wall-degrading enzymes of R. solani and S. rolfsii varied profoundly. Pathogenicity studies showed that R. solani appeared to be more tolerant than S. rolfsii of the salinity stresses applied, and relatively more virulent to cv Kaumera. The activities of cell wall enzymes of R. solani decreased and those of S. rolfsii increased with increased salt concentration when cell wall material was used as a sole carbon source. The metabolic products produced under salinity stress by R. solani and R. solani in the cell wall amended culture media shifted the initial pH towards neutrality or slight alkalinity for R. solani and to high acidity for S. rolfsii.When model substrates were used, xyland and galactan were the most responsive substrates for degradation by the cell wall enzymes of the two fungi studied. The rate of degradation was higher for S. rolfsii than for R. solani. The excessive acidity in salt stressed S. rolfsii culture media suggested reduced activities of the enzymes involved in cell wall degradation in vivo. This may explain the decreased virulence potentialities.     

Short chain regioselectively hydrolyzed scleroglucans induce maturation of porcine dendritic cells

Branched β-1,3/1,6-glucans (scleroglucan) were produced by cultivation of Sclerotium rolfsii ATCC 15205. Regioselective hydrolysis at the β-1,3-linkage of the cell-free and purified polysaccharide was performed in borosilicate glass bottles at pH 5, 121°C, and 1 bar for 72 h. The mixture was divided into four molar mass fractions by stepwise cross-flow filtration using different cutoffs. In vitro studies revealed that scleroglucan hydrolysates with a low molar mass of less than 5 kDa significantly stimulated the activation and maturation of porcine monocyte derived dendritic cells (MoDC) by upregulation of CD40 and CD80/86 as well as by reduction of antigen uptake. MoDC treated with low molar mass scleroglucan showed a considerable increase in the amounts of secreted proinflammatory cytokine tumor necrosis factor alpha and stimulated the proliferation of lymphocytes. Therefore, scleroglucan molecules of low molecular weight are able to induce activation and maturation of porcine DC, which are key initiators of inflammatory and adaptive immune responses, and could provide improved protection against infectious diseases.     

Integrated Control of Sclerotium rolfsii on Groundnut in South Africa

Sclerotium rolfsii Sacc. causes disease of numerous crop plants worldwide, including groundnuts. Control of this pathogen is difficult as it produces sclerotia which overwinter in the soil to emerge as inoculum and cause disease the following season. Various chemical, biological and cultural control strategies have been suggested and implemented, some of which have reduced disease incidence in the field. No studies have yet been undertaken in South Africa to control this disease on groundnut, either chemically, biologically or by cultural practices. In this study, several strategies were investigated for the control of S. rolfsii on groundnuts. Difenoconazole was identified as a fungicide that could possibly be applied in combination with Trichoderma harzianum, a biological antagonist of S. rolfsii, above carbendazim and flusilazole, and chlorothalonil. Difenoconazole significantly reduced the growth rate of S. rolfsii but not of T. harzianum. The cultivation of infected fields with an inversion plough significantly reduced infection of groundnuts by S. rolfsii and also improved the quality of the produce, while yield was not increased. Lower plant density increased the incidence of disease in an infected field, and is therefore not considered to be a viable form of cultural control.     

Degradation of oxalic acid (OA) producing Sclerotium rolfsii (Sacc.) by organic biocides

The aim of the study for the importance of oxalic acid produced by Sclerotium rolfsii during the invasion of host tissue during pathogenesis acts synergistically with endopolygalacturonase, lowering the pH of the infected tissues to a level optimal for the activity of this enzyme. Oxalic acid was the principal toxic agent produced in the culture filtrates of S. rolfsii and it was responsible for the death of host cells. The calcium present in structural pectates can be strongly chelated by oxalic acid. As a consequence, plant tissues are rendered more susceptible to invasion by S. rolfsii. Oxalic acid produced by S. rolfsii was very much reduced by Trichoderma viride (TVB1) (0.79 mg/ml culture filtrate), Pseudomonas fluorescens (SBHRPF2) 0.93 mg, neem cake (10%) (0.87 mg/ml), Lippia nodiflora (10%) and Lantana camera (10%) which were recorded as 2.12 and 2.25 mg/ml. The organic biocides that interfered with S. rolfsii led to reduction of oxalic acid production, which specifically reduced the disease incidence, and the oxalic acid degradation was a useful approach as a disease control strategy.     

Mechanisms of biocontrol of soil-borne plant pathogens by Rhizobacteria

Bacterial antagonism, responsible for biological control, may operate by antiobiosis, competition or parasitism. Parasitism relies on lytic enzymes for the degradation of cell walls of pathogenic fungi. Serratia marcescens was found to be an efficient biocontrol agent of Sclerotium rolfsii and Rhizoctonia solani under greenhouse conditions. Populations of 10⁵ or 10⁶ colony forming units g^-1 soil were the most effective. Drench and drip application of S. marcescens suspension were more effective in controlling S. rolfsii than spraying, mixing in soil or seed coating. The highest population density of the bacteria in the rhizosphere was found on the proximal portion of the root, decreasing significantly until the tips, where it increased again. The isolated Serratia, found to possess chitinolytic activity, was able to release N-acetyl D-glucosamine from cell walls of S. rolfsii. The gene coding for chitinase was cloned into Escherichia coli and the enzyme was uniquely excreted from the bacterium into its growth medium. When S. rolfsii was sprayed by partially purified chitinase produced by the cloned gene, rapid and extensive bursting of the hyphal tips was observed. This chitinase preparation was effective in reducing disease incidence caused by S. rolfsii in beans and R. solani in cotton, under greenhouse conditions. A similar effect was obtained when a viable E. coli cell, containing the plasmid with the chitinase gene (pLCHIA), was applied. It appears that genetic engineering of the lytic enzymes, such as chitinase which play an important role in plant disease control, may improve the efficacy of biocontrol agents.     

Increased accumulation of phenolic metabolites in groundnut (Arachis hypogaea L.) genotypes contribute to defense against Sclerotium rolfsii infection

Abstract

Fungal infections cause several metabolic changes to the plants, which can affect its physiology and survival in various ways. In the present study, we have analysed various phenolic compounds and activity of oxidative enzymes in healthy and Sclerotium rolfsii-infected groundnut genotypes. Increased phenolics content and higher activity of oxidative enzymes was observed in the tolerant genotype (CS 19, GG 16) followed by susceptible genotype (GG 20, TG 37A). Among the phenolic compounds tested, chlorogenic acid content has increased greatly in leaf, stem and root of infected tolerant genotypes compared to the respective controls. In vitro growth of S. rolfsii showed significant inhibition at concentrations 500 and 1000 µg/mL of phenolic compounds in the radial growth inhibition assay. These results have strongly suggested that, higher accumulation of chlorogenic acid could be an important factor in imparting resistance and protecting groundnut against S. rolfsii infection in tolerant genotypes.     

Biological control of the invasive alien weed Solidago canadensis: combining an indigenous fungal isolate of Sclerotium rolfsii SC64 with mechanical control

Solidago canadensis L. is a major invasive weed that is highly tolerant to disturbances and difficult to control in China. In order to develop a rapid non-chemical control strategy for this weed in heterogeneous environments, we investigated different treatments including mechanical control (cutting and hoeing) and inoculation with an indigenous pathogen, Sclerotium rolfsii SC64, which was isolated from S. canadensis and applied by means of a solid formulation. Greenhouse and field trials were conducted to test how the control regimes (i.e. individual treatment methods, combination of these methods and different treatment timing) influence control efficiency. The fungal isolate S. rolfsii SC64 caused 70% plant mortality and fresh weight reduction of S. canadensis under 150 cm growth stage, and efficacy increased to 80% when the above-ground material was removed. However, the use of cutting, hoeing or treating with S. rolfsii SC64, on its own, did not provide sufficient control of S. canadensis. Cutting treatments performed in July and September only eliminated sexual reproduction of S. canadensis. Combination of cutting, hoeing and treating with isolate SC64 during the growing season in May, July and September was able to kill more than 90% of the ramets. This combination of methods not only eliminated sexual reproduction of S. canadensis, but also destroyed its underground stems and prevented its regrowth. Therefore, this integrated approach may provide an optima control strategy for S. canadensis.     

The occurrence of sclerotium rot on Momordica charantia caused by Sclerotium rolfsii in Korea

Sclerotium rolfsii is one of the most destructive pathogens and thought to affect a broad range of plant hosts. In July 2014, an occurrence of sclerotium rot was observed on bitter melon (Momordica charantia L.) in Jinju, South Korea. The rot symptoms were most developed on stems and fruit near the soil line, and infected bitter melon plants withered and eventually died. White mycelial mats with numerous sclerotia were produced on diseased stems and fruit near the soil surface. Based on the morphological characteristics, pathogenicity tests, and DNA sequencing and phylogenetic analysis of the internal transcribed spacer (ITS) ribosomal RNA (rRNA) gene region, the causal fungus was identified as S. rolfsii Saccardo. This is the first report of sclerotium rot caused by S. rolfsii on bitter melon in Korea. The recent occurrence of sclerotium rot on bitter melon poses a potential threat to its production in Korea.     

Involvement of phenazines and lipopeptides in interactions between Pseudomonas species and Sclerotium rolfsii, causal agent of stem rot disease on groundnut

Aims: To determine the role of phenazines (PHZ) and lipopeptide surfactants (LPs) produced by Pseudomonas in suppression of stem rot disease of groundnut, caused by the fungal pathogen Sclerotium rolfsii. Methods and Results: In vitro assays showed that PHZ‐producing Pseudomonas chlororaphis strain Phz24 significantly inhibited hyphal growth of S. rolfsii and suppressed stem rot disease of groundnut under field conditions. Biosynthesis and regulatory mutants of Phz24 deficient in PHZ production were less effective in pathogen suppression. Pseudomonas strains SS101, SBW25 and 267, producing viscosin or putisolvin‐like LPs, only marginally inhibited hyphal growth of S. rolfsii and did not suppress stem rot disease. In contrast, Pseudomonas strain SH‐C52, producing the chlorinated LP thanamycin, inhibited hyphal growth of S. rolfsii and significantly reduced stem rot disease of groundnut in nethouse and field experiments, whereas its thanamycin‐deficient mutant was less effective. Conclusions: Phenazines and specific lipopeptides play an important role in suppression of stem rot disease of groundnut by root‐colonizing Pseudomonas strains. Significance and Impact of the Study: Pseudomonas strains Phz24 and SH‐C52 showed significant control of stem rot disease. Treatment of seeds or soil with these strains provides a promising supplementary strategy to control stem rot disease of groundnut.     

The effect of mode of application of Bacillus subtilis CA32r on control of Sclerotium rolfsii on Capsicum annuum

Abstract

Sclerotium rolfsii Sacc. the causal agent of collar and root rot of chili (Capsicum annuum L.), is one of the most important soil-borne pathogens in Sri Lanka. Bacillus subtilis CA32r, a stable spontaneous kanamycin resistant isolate, showing antagonism in a Petri plate assay, was selected for greenhouse pot experiments to control S. rolfsii. An ethyl acetate extract of the culture filtrate of B. subtilis CA32r inhibited radial colony growth as well as germination of sclerotia of the pathogen in vitro, indicating the presence of antifungal compound(s) in the culture extract. B. subtilis CA32r was investigated for its effectiveness as a biological control agent against S. rolfsii infecting transplanted chilli seedlings in greenhouse pot experiments. CA32r significantly decreased the disease incidence in terms of lesion development on stem base and roots depending on the mode of the bacterial application. Seed bacterization and soil application alone did not protect chilli plants, but root bacterization prior to the transplanting of seedlings significantly decreased the disease incidence caused by S. rolfsii. However, even in the combined treatment, seed bacterization and soil application, did not protect chilli plants. The best protection was achieved by combination of root bacterization prior to transplant and soil application of CA32r. Root bacterization resulted in maintaining higher numbers of bacteria at the collar region of chilli plants and may have shielded the most vulnerable area from the pathogen, resulting enhanced protection. Since the application of CA32r resulted in a significant reduction of the number of viable S. rolfsii propagules in the soil indicates that B. subtilis CA32r possesses not only protective but also eradicative potential.     

Determining biomass in viscous culture broths of Sclerotium rolfsii during β-glucan production: hexosamine assay as an indirect alternative technique

Micro-Kjeldahl determination of protein content in the cells was not suitable for the determination of biomass because of the changing composition of the cells. The significant correlation was found during cultivation between the values of the hexosamine assay and those corresponding to the dry weight method (r = 0.98, slope = 1.04) which showed the validity of both hexosamine and dry weight methods, with the former being a simple, reliable and not time consuming alternative for the biomass estimation of Sclerotium rolfsii during scleroglucan production.     

Cross‐Species Alleviation of Biotic and Abiotic Stresses by the Endophyte Pseudomonas aeruginosa PW09

A wheat endophytic bacterium (Pseudomonas aeruginosa PW09) was evaluated for its ability to trigger an induced systemic resistance response in cucumber against biotic and abiotic stresses. PW09 was applied to cucumber seeds, and the seedlings were subjected to Sclerotium rolfsii infection and NaCl (150 mm ). The role of PW09 was evaluated in alleviating the stresses by assessing plant mortality due to S. rolfsii infection and biomass accumulation under NaCl stress as well as at the physiological level through phenylpropanoid metabolism, antioxidant activities and proline accumulation. The endophyte reduced seedling mortality by 60% and increased biomass accumulation significantly under S. rolfsii (7%) and NaCl (18%) stresses, respectively, compared with endophyte‐untreated seedlings. Application of PW09 also induced higher accumulation of proline (1.3‐ and 1.4‐fold) and total phenolics (1.2‐ and 1.1‐fold) and activities of polyphenol oxidase (4.3‐ and 1.5‐fold), phenylalanine ammonia lyase (1.29‐ and 1.27‐fold) and superoxide dismutase (2.5‐ and 1.39‐fold) under S. rolfsii and NaCl stresses, indicating the ability of the wheat endophyte PW09 in alleviating both biotic and abiotic stresses in cucumber.     

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.     

Acquired resistance of Alternaria solani and Sclerotium rolfsii to Polyoxin-D

Acquired resistance to the antibiotic polyoxin-D was studied in two phytopathogenic fungi, Alternaria solani Sorauer, and Sclerotium rolfsii Sacc. The ED₅₀ value of the antibiotic for A. solani was 100 μg/ml and S., rolfsii 200 μg/ml. A. solani and S. rolfsii could be trained to tolerate concentrations upto 1.000 μg/ml and 2.200 μg/ml respectively. The acquired resistance in both cases was not lost on continued subculturing in fungicide-free, media. On transfer to fungicide-free media, Polyoxinresistant strains of both the fungi showed faster growth rate and appreciable reduction in sporulation compared to the original strains. The adapted strain of A. solani showed cross-resistance to Cycloheximide and Difolatan but not to Hinosan and Bayleton.     

A high-affinity Zn2+ uptake system controls growth and biosynthesis of an extracellular,branched β-1,3-β-1,6-glucan inSclerotium rolfsii ATCC 15205

The plant pathogenic fungus imperfectusSclerotium rolfsii ATCC 15025 requires Zn²⁺ for both growth and biosynthesis of an extracellular, branched β-1,3-β-1,6-glucan in a completely defined mineral medium. Upon rising the external zinc concentration an increased yield of glucan inversely proportional to the yield of biomass was found in the cultivations with the bioreactor, but not in those with shake flasks. The complete carbon balance presented includes oxalic acid as an additional metabolite, secreted in rather high amounts due to the reduced oxygen supply in the viscous culture suspension. Only low amounts of zinc were accumulated. The successful development of an assay for the uptake of⁶⁵Zn²⁺ by homogeneous suspensions of zinc-depleted cultures ofS. rolfsii is reported. An energy-dependent highly specific Zn²⁺ uptake system, sufficient for growth and glucan synthesis, but no efflux system was demonstrated inS. rolfsii.     

Reactor kinetics for submerged aerobic biofilms

Sclerotium rolfsii ATCC 15205 was cultivated in continuous culture (48 l reactor volume) for scleroglucan production. The maximum volumetric productivity (Q_Pvmax) amounted to 7.2 g/ld and was more than twice as high as in comparable batchwise cultivations. In addition, the relation between (a) volumetric productivity (Q_P [g/ld]) and product yield (Y_{Glucan/Glucose} [1]), (b) volumetric productivity and product quality (M_W [g/mol]), and (c) product quality and impeller tip speed (Nd [m/s]) was studied in continuous culture. It was found, that an increase in volumetric productivity led to a decline in product yield and product quality. Furthermore, an impeller tip speed of >0.7 m/s decreased the attainable product quality considerably. Based on these results, the impact of the operational setpoint of the process in terms of oxygen supply and reactor scale on the economics of scleroglucan production was discussed. In contrast to batchwise cultivation, scleroglucan production in continuous culture proved to be not feasible under non-aseptic conditions.     

<Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis> strain 1P (MTCC 8707) a novel bacterial antagonist from the North Western Indian Himalayas

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a rhizospheric, Gram positive, rod shaped, yellow pigmented bacterium isolated from an apple orchard rhizospheric soil, on nutrient agar plates incubated at 4°C. The species level identification was arrived on the basis of 16S rRNA gene sequencing. The sequence showed 98% similarity with sequences of E. acetylicum available in the public domain. The strain was positive for siderophore and HCN production. In separate invitro assays it was found to inhibit the growth and development of Rhizoctonia solani, Sclerotium rolfsii, Pythium and Fusarium oxysporum. The volatile compound produced by the bacterium was found to be the most potent in inhibiting the hyphal development of R. solani, S. rolfsii, Pythium and F. oxysporum by 45.55, 41.38, 28.92 and 39.74% respectively. Commonly observed deformities caused by the diffusible and volatile compounds produced by the bacterium included hyphal inhibition, constriction and deformation. Under pot culture conditions the bacterium improved the germination and early growth parameters of pea (Pisum sativum) in the presence of R. solani and S. rolfsii.     

Comparative Antagonistic Properties of Gliocladium virens and Trichoderma harzianum on Sclerotium rolfsii and Rhizoctonia solani—its Relevance to Understanding the Mechanisms of Biocontrol

An isolate of Trichoderma harzianum which is less effective than G. virens in suppressing S. rolfsii and R. solani was compared with G. virens for various mechanisms of antagonism in vitro, viz., antagonism in dual culture/hyphal parasitism, parasitism of sclerotia and antibiosis. G. virens and T. harzianum were equally effective in parasitizing the hyphae of R. solani. Only T. harzianum parasitized the hyphae of S. rolfsii, and the two antagonists were comparable with respect to antibiosis on the test pathogens. However, G. virens readily parasitized the sclerotia of the test pathogens and was found to be more effective than T. harzianum in destroying the sclerotia. Under SEM, G. virens was found to colonize, penetrate, and sporulate inside the sclerotia of the test pathogens.Parasitism of sclerotia is suggested as the principal mechanism of biological control of S. rolfsii and R. solani by G. virens.     

Low-cost method for the preservation of Sclerotium rolfsii Proimi F-6656: Inoculum standardization and its use in scleroglucan production

Summary The Castellani's Method for the preservation of Sclerotium rolfsii in sterile distilled water was tested. Culturing on Potato Dextrose Yeast extract (PDY) slants, the current system used, was also evaluated. Preservation of sclerotia according to the Castellani's method allowed the strain survival for more than two years. Comparing with the strain periodically activated, a critical decrease (about 80%) in -glucan synthesizing capacity was detected for mycelium preserved either on PDY slants or in water. Activation of stored sclerotia followed by subculturing in liquid Production Medium (PM) allowed preparation of homogeneous suspensions for batch fermentations, and scleroglucan concentrations achieved were similar to those with the strain periodically activated.