Fenton Reaction Facilitates The Fungal Infection On The Unicellular Alga Haematococcus Pluvialis and Discovery of a Biosafe and Environment-Friendly Chemical That Blocks The Infection

Background The green microalga Haematococcus pluvialis is used as cell factories for producing astaxanthin, the high-value natural compound with multiple biological functions. However, H. pluvialis is prone to the infection by a parasitic fungus Paraphysoderma sedebokerense, which is the most devastating threat to the mass culture of H. pluvialis all over the world. Little is known for the mechanisms underlying the infection process, though it is of great essential for developing effective measures to mitigate the pathogen threatening for the natural astaxanthin industry. Results We observed that there were heat-stable substances with small molecular weight produced during the infection process, which significantly stimulated the parasitism process and enhanced the susceptibility of H. pluvialis cells to the pathogen. Systematic approaches including multi-omics, biochemical and imaging analysis were deployed to uncover the identity of the metabolites and the underlying mechanisms. Two metabolites, 3-hydroxyanthranilic acid and hordenine were identified and proved to stimulate the infection via driving fenton reaction mediated oxidative stress to H. pluvialis. The reaction generated hydroxyl radicals to disrupt the subcellular components of the algal cells and to make the algal cells more susceptible to the infection. Based on these findings, a biosafe and environment-friendly antioxidant butylated hydroxyanisole was selected to inhibit the fungal infection, which completely abolished the infection at 12 ppm. Conclusions This study provide for the first time, a framework to dissect the functions of secondary metabolites in the interaction between the unicellular algal H. pluvialis and its fungi parasite, indicating that oxidative degradation is a strategy used for the fungal infest. Eliminating the oxidative burst through adding antioxidant butylated hydroxyanisole could be an effective measure to reduce parasitic infection in H. pluvialis mass culture.

Medicinal Mushrooms Ophiocordyceps Sinensis and Paecilomyces Hepiali

The fungi of the genus Ophiocordyceps sinensis (Berk.) belong to entomopathogenic fungi. Ophiocordyceps sinensis has been used as a tonic and medicinal product in China for more than 2,000 years. A number of scientific papers have described the clinical use of this parasitic fungus with subsequent biological and pharmacological effects. The various chemical compounds identified in these fungi are responsible for a wide range of biological activity: cordycepin, cordycepic acid, D-mannitol, polysaccharides, nucleotides, proteins, amino acids, and unsaturated fatty acids. Our research focused on the determinations of the biologically active chemical compounds in extracts from cultivated Ophiocordyceps fungi using UV/VIS (Ultraviolet/Visible) spectrophotometry and NMR (Nuclear Magnetic Resonance) spectroscopy. The testing of antimicrobial activity of extracts against the collection strains of Escherichia coli and Staphylococcus aureus demonstrated that the percentage of RIZD (relative inhibition zone diameter) ranged from 83 % to 166 %. The increased antimicrobial activity against E. coli was observed in comparison with that against S. aureus.

A Valsa mali Effector Protein 1 Targets Apple (Malus domestica) Pathogenesis-Related 10 Protein to Promote Virulence

To successfully colonize the plants, the pathogenic microbes secrete a mass of effector proteins which manipulate host immunity. Apple valsa canker is a destructive disease caused by the weakly parasitic fungus Valsa mali. A previous study indicated that the V. mali effector protein 1 (VmEP1) is an essential virulence factor. However, the pathogenic mechanism of VmEP1 in V. mali remains poorly understood. In this study, we found that the apple (Malus domestica) pathogenesis-related 10 proteins (MdPR10) are the virulence target of VmEP1 using a yeast two-hybrid screening. By bimolecular fluorescence (BiFC) and coimmunoprecipitation (Co-IP), we confirmed that the VmEP1 interacts with MdPR10 in vivo. Silencing of MdPR10 notably enhanced the V. mali infection, and overexpression of MdPR10 markedly reduced its infection, which corroborates its positive role in plant immunity against V. mali. Furthermore, we showed that the co-expression of VmEP1 with MdPR10 compromised the MdPR10-mediated resistance to V. mali. Taken together, our results revealed a mechanism by which a V. mali effector protein suppresses the host immune responses by interfering with the MdPR10-mediated resistance to V. mali during the infection.

Effects of ergotamine on the central nervous system using untargeted metabolomics analysis in a mouse model

The ergot alkaloid ergotamine is produced by Claviceps purpurea, a parasitic fungus that commonly infects crops and pastures of high agricultural and economic importance. In humans and livestock, symptoms of ergotism include necrosis and gangrene, high blood pressure, heart rate, thermoregulatory dysfunction and hallucinations. However, ergotamine is also used in pharmaceutical applications to treat migraines and stop post-partum hemorrhage. To define its effects, metabolomic profiling of the brain was undertaken to determine pathways perturbed by ergotamine treatment. Metabolomic profiling identified the brainstem and cerebral cortex as regions with greatest variation. In the brainstem, dysregulation of the neurotransmitter epinephrine, and the psychoactive compound 2-arachidonylglycerol was identified. In the cerebral cortex, energy related metabolites isobutyryl-L-carnitine and S-3-oxodecanoyl cysteamine were affected and concentrations of adenylosuccinate, a metabolite associated with mental retardation, were higher. This study demonstrates, for the first time, key metabolomic pathways involved in the behavioural and physiological dysfunction of ergot alkaloid intoxicated animals.

Powdery mildew resistance of barley accessions from Dagestan

Powdery mildew caused by the parasitic fungus Blumeria graminis (DC.) Golovin ex Speer f. sp. hordei Marchal is one of the most common diseases of barley. Growing resistant varieties can significantly minimize harmful effects of the pathogen. The specificity in the interaction between the fungus and its host plant requires a continuous search for new donors of the resistance trait. The powdery mildew resistance of 264 barley accessions from Dagestan and genetic control of the trait in resistant forms were studied under field and laboratory conditions. Forty-seven barley lines carrying previously identified powdery mildew resistance genes were also examined. During three years, the experimental material was evaluated under severe infection pressure at the Dagestan Experiment Station of VIR (North Caucasus, Derbent). Juvenile resistance against the Northwest (St. Petersburg, Pushkin) pathogen population was evaluated in a climatic chamber. The genetic control of B. graminis resistance in the selected accessions was studied with the application of hybridological and molecular analyses. The level of genetic diversity of Dagestan barley for effective resistance to powdery mildew is very low. Only two accessions, VIR-23787 and VIR-28212, are resistant against B. graminis at both seedling and adult plant stages. The high-level resistance of breeding line VIR-28212 originating from barley landrace VIR-17554 (Ep-80 Abyssinien) from Ethiopia is controlled by the recessive gene mlo11. Accession VIR-17554 is heterogeneous for the studied trait, with the powdery mildew resistant genotypes belonging to two varieties, dupliatrum (an awnless phenotype) and nigrinudum (an awned phenotype). In accession VIR-23787, a recessive resistance gene distinct from the mlo11 allele was identified. This accession is supposed to be protected by a new, effective pathogen resistance gene.

First report of coffee leaf rust caused by Hemileia vastatrix on coffee (Coffea arabica) in Hawaii

Coffee is one of the most economically valuable specialty crops for which Hawaii is famous. It is produced commercially on >6,900 acres across six islands by more than 1,470 growers. It has a raw crop value of $55.9 million, while the value-added benefits of coffee-related industries exceed $148.5 million (USDA, 2021). In addition to high product quality, Hawaii also has the distinction of being the last major coffee growing region that is free of coffee leaf rust (CLR), a highly damaging disease caused by the obligate parasitic fungus Hemileia vastatrix Berk. & Broome. On October 21, 2020, a coffee grower in Maui County, Hawaii reported the presence of coffee leaf rust-like symptoms on coffee plants (var. ‘Typica’) at their farm with many trees heavily defoliated. Foliar symptoms consisted of yellowish-orange, circular lesions that often coalesced. On abaxial surfaces, these lesions appeared powdery. Urediniospores were mostly reniform, (25)27-34(36) × (17)18-28(29) µm, strongly echinulated on the upper (convex) surface and smooth on the lower (concave) surface, with hyaline to pale yellow-orange walls. Urediniospores incubated in molecular grade water for 10 min at 95 oC served as template for PCR targeting the internal transcribed spacer region of fungi using primers ITS1/ITS2, ITS3/ITS4, and ITS1/ITS4 (White et al., 1990). Amplification products underwent direct Sanger-based sequencing. Following primer sequence trimming, the sequence reads were assembled using CAP3 (Huang and Madan, 1999) and deposited in GenBank (Accession MW228837). Blastn analysis revealed > 99% nucleotide identity with isolates of H. vastatrix from Mexico (eg. KX260251) and Brazil (eg. MF627828). A voucher material from which both the morphological and molecular assays were performed was deposited in the National Fungus Collection (BPI 924818). Subsequent surveys on the islands of Hawaii, Lanai, and Oahu revealed the presence of coffee plants with symptoms of coffee leaf rust. To confirm pathogenicity, urediniospores from a symptomatic plant growing in Holualoa, Hawaii, were collected in gelatin capsules using a G-R Electric Manufacturing Portable Vacuum Pump with a mini cyclone spore adapter. The concentration of spores was adjusted to 1 x 105 spores/ml in sterile water using a Brightline Hemocytometer. The spore suspension was brushed onto the abaxial side of leaves from two C. arabica var. ‘Typica’ plants using a camel hair paintbrush. Two control plants were mock-inoculated with sterile water. Plants were placed in a dark humid chamber set at 22 oC for 48 hours, after which it was adjusted to 12 hours light under cool white fluorescent lighting. After 20 days small, chlorotic spots were visible on the adaxial leaf surface and a few spots contained orange urediniospores on the abaxial surface. Lesions expanded by day 34, followed by necrosis of the center area of several leafspots and leaf abscission was observed at day 70. The experiment was conducted twice. H. vastatrix was confirmed by both morphological and molecular examination of urediniospores recovered from the observed lesions. All mock-inoculated control plants remained asymptomatic. To our knowledge, this is the first report of CLR on coffee in Hawaii. This discovery is of great concern since CLR threatens not only the yield and quality of Hawaii-grown coffee, but also the economic viability of this historic and culturally important industry. Statewide monitoring for CLR continues and further work is needed to mitigate the impact of this discovery through the development of short- and long-term management strategies.

The Density and Viability of Metarhizium anisopliae Conidia on Several Growth Media

Metarhizium anisopliae is a parasitic fungus on insects, and thus called entomopathogenic fungus. This fungus is used as a biological control agent for insect pests. Fungal propagation can be done using a variety of media. The purpose of this study was to analyze the growth of M. anisopliae on four types of media, with conidial density and viability as the growth parameters. This research was conducted at the Laboratory of Microbiology, Universitas Negeri Semarang. This study was an experimental research used a Completely Randomized Design (CRD) with one factor and four treatments: Control (PDA/Potato Dextrose Agar medium), Treatment I (ELSA/Extract Larvae Sucrose Agar medium), Treatment II (CWSA/Coconut Water Sucrose Agar medium), Treatment III (CWELSA/Coconut Water and Extract Larvae Sucrose Agar medium). The and results showed that there was an effect of growth media on the density and viability of M. anisopliae conidia. CWELSA media had the highest conidial density (2.91 x 108 cfu/mL) and viability (97.17%). CWSA media had 2.82 x 108 cfu/mL and 95.33%. PDA media had 2.25 x 108 cfu/mL and 92.83%. ELSA media had 1.64 x 108 cfu/mL and 90.83%. The high conidial density and viability of M. anisopliae is CWELSA medium. This study is as an alternative growth medium to improve the quality of M. anisopliae propagation.

The role of Dicer-dependent RNA interference in regulating cross-species communication during fungus-fungus interactions

Dicer-like (DCL) proteins play a vital role in transcriptional and post-transcriptional gene silencing, also known as RNA interference (RNAi), by cleaving double-stranded RNAs or single-stranded RNAs with stem-loop structures into small RNAs . Although DCL-mediated RNAi can regulate interspecific communication between pathogenic/mutualistic organisms and their hosts, its role in parasitic fungus-fungus interactions is yet to be investigated . In this study, we deleted dcl genes in the mycoparasitic fungus Clonostachys rosea and analyzed the transcriptome and secondary metabolome to characterize the regulatory functions of DCL-dependent RNAi in mycoparasitism. Deletion of dcl2 resulted in a mutant with reduced growth rate, pigment production and antagonism towards the plant pathogenic fungus Botrytis cinerea . Moreover, the Δ dcl2 mutant displayed a reduced ability to control fusarium foot rot disease on wheat, caused by Fusarium graminearum , and reduced production of 62 secondary metabolites (SM) including yellow‐coloured sorbicillinoids. Transcriptome sequencing of the in vitro interaction between the C. rosea Δ dcl2 strain and B. cinerea or F. graminearum identified downregulation of genes coding for transcription factors, membrane transporters, hydrolytic enzymes and SM biosynthesis enzymes putatively involved in antagonistic interactions, in comparison with the C. rosea wild type interaction. Sixty-one putative novel microRNA-like RNAs (milRNAs) were identified in C. rosea , and 11 was upregulated in the Δ dcl2 mutant. In addition to putative endogenous gene targets, these DCL2-dependent milRNAs were predicted to target B . cinerea and F. graminearum virulence factor genes, which showed an increased expression during interaction with the Δ dcl2 mutant incapable of producing the targeting milRNAs. This paper constitutes the first step in elucidating the role of RNAi in mycoparasitism, with important implications for biological control of plant diseases. This study further indicates a possible cross-species regulatory activity of fungal milRNAs, emphasizing a novel role of RNAi in fungal interactions and ecology.

Integrated management of Meloidogyne incognita on tomato using combinations of abamectin, Purpureocillium lilacinum, rhizobacteria, and botanicals compared with nematicide

Background Acceptable alternative eco-friendly tools in the present study were tested to control the root-knot nematode, Meloidogyne incognita, on greenhouse-cultivated vegetables. The nematicidal effect of rhizobacteria (Pseudomonas and Serratia), egg parasitic fungus (Purpureocillium lilacinum), abamectin (Streptomyces avermitilis), and 3 botanicals (colocynth, Citrullus colocynthis; moringa, Moringa oleifera; marigold, Tagetes erecta L.) singly or in combination was tested against M. incognita, in comparison with emamectin benzoate. Results In vitro treatments revealed that egg hatching and juvenile mortality were influenced by the type of bioagents, plant species of botanicals, and exposure time. All the tested bioagents and botanicals displayed nematicidal potential via their ovicidal and larvicidal action on egg hatching and J2 mortality of M. incognita. Three and 5 days post-treatment, abamectin and emamectin benzoate were more effective than P. lilacinum, Serratia and Pseudomonas, and C. colocynthis in inhibiting egg hatching: 96.31 and 94.88%; 95.79 and 94.05%; 94.11 and 94.46%; 85.54 and 87.28%; 88.87 and 84.30%, respectively. On the other hand, after 10 days, P. lilacinum gave the highest inhibition percentage (99.00%), followed by abamectin (89.25%). However, the difference was insignificant compared with the inhibition percentage of rhizobacteria, Serratia and Pseudomonas (88.69%; p ≤ 0.05). Moreover, juvenile mortality was 100.0, 96.80, and 91.60% after 10 days of treatment, respectively. However, botanicals showed a lower effect on egg hatching and juvenile mortality. Under greenhouse conditions, potential antagonism towards M. incognita by application the mixture of biocontrol agents and botanicals was more effective in controlling M. incognita than single treatments. Conclusions The combination of abamectin and/or emamectin benzoate with P. lilacinum and rhizobacteria was the most effective against M. incognita, followed by rhizobacteria and P. lilacinum, not only in decreasing galls and reproduction of M. incognita but also in increasing plant growth of tomato parameters than the control. The application of various bioagents including abamectin might be a potential antagonism strategy against phytonematodes in protected agricultural areas.

Integrated Strategies to Managing Meloidogyne Incognita and Soil Borne Fungus Infesting Cucumber in Two Consecutive Crop Seasons Under Protected Cultivation

Relative efficacy of various approaches to managing Meloidogyne incognita and soil borne fungus, Fusarium oxysporum f. sp. cucumerinum in cucumber for two seasons was tested under protected cultivation. Several management practices adopted such as chemicals (fumigant, non-fumigant, and fungicide), organic amendments (Neem cake, leaves and oil, soil and seed treatment), and biocontrol agents (the egg-parasitic fungus, Purpureocillium lilacinum), all practices combine for the management of disease. Eleven treatments with untreated control in a randomized block design. Two significant parameters were measured: plant growth parameter (plant height, dry root weight and yield) and disease parameters (galls/plant, final nematode population, egg masses/plant and fungal incidence). All treatments significantly improved plant growth parameters and reduced nematode reproduction as compared to untreated check. The integration of formalin and neem oil seed treatment favor the low root galling index compared to all other treatments in both the seasons. Integration of formalin and neem oil seed treatment reduced nematode population and fungus incidence and increased the yield of cucumber during both the seasons.