Activity of natural occurring entomopathogenic fungi on nymphal and adult stages of Philaenus spumarius.

The spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) is the predominant vector of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) in Apulia, Italy and the rest of Europe. Current control strategies of the insect vector rely on mechanical management of nymphal stages and insecticide application against adult populations. Entomopathogenic fungi (EPF) are biological control agents naturally attacking spittlebugs and may effectively reduce population levels of host species. Different experimental trials in controlled conditions have been performed to i) identify naturally occurring EPF on P, spumarius in Northwestern Italy, and ii) evaluate the potential for biocontrol of the isolated strains on both nymphal and adult stages of the spittlebug. Four EPF species were isolated from dead P. spumarius collected in semi-field conditions: Beauveria bassiana, Conidiobolus coronatus, Fusarium equiseti and Lecanicillium aphanocladii. All the fungal isolates showed entomopathogenic potential against nymphal stages of P. spumarius (≈ 45 % mortality), except for F. equiseti, in preliminary trials. No induced mortality was observed on adult stage. Lecanicillium aphanocladii was the most promising fungus and its pathogenicity against spittlebug nymphs was further tested in different formulations (conidia vs blastospores) and with natural adjuvants. Blastospore formulation was the most effective in killing nymphal instars and reducing the emergence rate of P, spumarius adults, reaching mortality levels (90%) similar to those of the commercial product Naturalis®, while no or adverse effect of natural adjuvants was recorded. The encouraging results of this study pave way for testing EPF isolates against P, spumarius in field conditions and find new environmentally friendly control strategies against insect vectors of X. fastidiosa.

Rice false smut virulence protein subverts host chitin perception and signaling at lemma and palea for floral infection.

The flower-infecting fungus Ustilaginoidea virens causes rice false smut, which is a severe emerging disease threatening rice (Oryza sativa) production worldwide. False smut not only reduces yield, but more importantly produces toxins on grains, posing a great threat to food safety. U. virens invades spikelets via the gap between the 2 bracts (lemma and palea) enclosing the floret and specifically infects the stamen and pistil. Molecular mechanisms for the U. virens-rice interaction are largely unknown. Here, we demonstrate that rice flowers predominantly employ chitin-triggered immunity against U. virens in the lemma and palea, rather than in the stamen and pistil. We identify a crucial U. virens virulence factor, named UvGH18.1, which carries glycoside hydrolase activity. Mechanistically, UvGH18.1 functions by binding to and hydrolyzing immune elicitor chitin and interacting with the chitin receptor CHITIN ELICITOR BINDING PROTEIN (OsCEBiP) and co-receptor CHITIN ELICITOR RECEPTOR KINASE1 (OsCERK1) to impair their chitin-induced dimerization, suppressing host immunity exerted at the lemma and palea for gaining access to the stamen and pistil. Conversely, pretreatment on spikelets with chitin induces a defense response in the lemma and palea, promoting resistance against U. virens. Collectively, our data uncover a mechanism for a U. virens virulence factor and the critical location of the host-pathogen interaction in flowers and provide a potential strategy to control rice false smut disease.

Metagenomic sequencing for detection and identification of the boxwood blight pathogen Calonectria pseudonaviculata.

Pathogen detection and identification are key elements in outbreak control of human, animal, and plant diseases. Since many fungal plant pathogens cause similar symptoms, are difficult to distinguish morphologically, and grow slowly in culture, culture-independent, sequence-based diagnostic methods are desirable. Whole genome metagenomic sequencing has emerged as a promising technique because it can potentially detect any pathogen without culturing and without the need for pathogen-specific probes. However, efficient DNA extraction protocols, computational tools, and sequence databases are required. Here we applied metagenomic sequencing with the Oxford Nanopore Technologies MinION to the detection of the fungus Calonectria pseudonaviculata, the causal agent of boxwood (Buxus spp.) blight disease. Two DNA extraction protocols, several DNA purification kits, and various computational tools were tested. All DNA extraction methods and purification kits provided sufficient quantity and quality of DNA. Several bioinformatics tools for taxonomic identification were found suitable to assign sequencing reads to the pathogen with an extremely low false positive rate. Over 9% of total reads were identified as C. pseudonaviculata in a severely diseased sample and identification at strain-level resolution was approached as the number of sequencing reads was increased. We discuss how metagenomic sequencing could be implemented in routine plant disease diagnostics.

Cyclophilin acts as a ribosome biogenesis factor by chaperoning the ribosomal protein (PlRPS15) in filamentous fungi.

The rapid transport of ribosomal proteins (RPs) into the nucleus and their efficient assembly into pre-ribosomal particles are prerequisites for ribosome biogenesis. Proteins that act as dedicated chaperones for RPs to maintain their stability and facilitate their assembly have not been identified in filamentous fungi. PlCYP5 is a nuclear cyclophilin in the nematophagous fungus Purpureocillium lilacinum, whose expression is up-regulated during abiotic stress and nematode egg-parasitism. Here, we found that PlCYP5 co-translationally interacted with the unassembled small ribosomal subunit protein, PlRPS15 (uS19). PlRPS15 contained an eukaryote-specific N-terminal extension that mediated the interaction with PlCYP5. PlCYP5 increased the solubility of PlRPS15 independent of its catalytic peptide-prolyl isomerase function and supported the integration of PlRPS15 into pre-ribosomes. Consistently, the phenotypes of the PlCYP5 loss-of-function mutant were similar to those of the PlRPS15 knockdown mutant (e.g. growth and ribosome biogenesis defects). PlCYP5 homologs in Arabidopsis thaliana, Homo sapiens, Schizosaccharomyces pombe, Sclerotinia sclerotiorum, Botrytis cinerea and Metarhizium anisopliae were identified. Notably, PlCYP5-PlRPS15 homologs from three filamentous fungi interacted with each other but not those from other species. In summary, our data disclosed a unique dedicated chaperone system for RPs by cyclophilin in filamentous fungi.

Advances in the biological control of phytoparasitic nematodes via the use of nematophagous fungi.

Agricultural production is one of most important activities for food supply and demand, that provides a source of raw materials, and generates commercial opportunities for other industries around the world. It may be both positively and negatively affected by climatic and biological factors. Negative biological factors are those caused by viruses, bacteria, or parasites. Given the serious problems posed by phytoparasitic nematodes for farmers, causing crop losses globally every year, the agrochemical industry has developed compounds with the capacity to inhibit their development; however, they can cause the death of other beneficial organisms and their lixiviation can contaminate the water table. On the other hand, the positive biological factors are found in biotechnology, the scientific discipline that develops products, such as nematophagous fungi (of which Purpureocillium lilacinum and Pochonia chlamydosporia have the greatest potential), for the control of pests and/or diseases. The present review focuses on the importance of nematophagous fungi, particularly sedentary endoparasitic nematodes, their research on the development of biological control agents, the mass production of fungi Purpureocillium lilacinum and Pochonia chlamydosporia, and their limited commercialization due to the lack of rigorous methods that enable the anticipation of complex interactions between plant and phytopathogenic agents.

Isolation and Purification of a New Bacillus Subtilis Strain from Deer Dung with Anti-microbial and Anti-cancer Activities.

OBJECTIVE: Bacillus strains are well known for their natural bioactive products that have antimicrobial and/or anti-cancer activities. Many of Bacillus' structurally unique metabolites can combat human diseases, including cancers. However, because Bacillus' metabolites are so abundant, few have been studied extensively enough to fully characterize their chemical constitutions and biological functions. METHODS: In this study, we focused on the isolation and purification of a new Bacillus strain, and determined the effects of its metabolites on bacteria and cancer cells. Our study focused on a new strain of Bacillus isolated from deer dung. Based on BLAST results, this isolate belongs to Bacillus subtilis, and therefore we named the strain Bacillus subtilis NC16. Congo red assay was used to test the cellulase activity. The inhibition zone was measured to test the antimicrobial activity. CCK-8, wound healing and flow cytometry were used to test the anti-cancer activity. RESULTS: Metabolites from Bacillus subtilis NC16 have both antimicrobial and anti-cancer activities. They can both suppress the growth of Trichoderma vride and Staphylococcus aureus, and inhibit the proliferation and promote the apoptosis of non-small cell lung cancer cell lines. CONCLUSION: Our results suggest that Bacillus subtilis NC16 can not only degrade cellulose, but its metabolites may be sources of antibiotics and anti-cancer drugs.

A novel transcription factor UvCGBP1 regulates development and virulence of rice false smut fungus Ustilaginoidea virens.

Ustilaginoidea virens, causing rice false smut (RFS) is an economically important ascomycetous fungal pathogen distributed in rice-growing regions worldwide. Here, we identified a novel transcription factor UvCGBP1 (Cutinase G-box binding protein) from this fungus, which is unique to ascomycetes. Deletion of UvCGBP1 affected development and virulence of U. virens. A total of 865 downstream target genes of UvCGBP1 was identified using ChIP-seq and the most significant KEGG enriched functional pathway was the MAPK signaling pathway. Approximately 36% of target genes contain the AGGGG (G-box) motif in their promoter. Among the targets, deletion of UvCGBP1 affected transcriptional and translational levels of UvPmk1 and UvSlt2, both of which were important in virulence. ChIP-qPCR, yeast one-hybrid and EMSA confirmed that UvCGBP1 can bind the promoter of UvPmk1 or UvSlt2. Overexpression of UvPmk1 in the ∆UvCGBP1-33 mutant restored partially its virulence and hyphae growth, indicating that UvCGBP1 could function via the MAPK pathway to regulate fungal virulence. Taken together, this study uncovered a novel regulatory mechanism of fungal virulence linking the MAPK pathway mediated by a G-box binding transcription factor, UvCGBP1.

Activation of defense response in common bean against stem rot disease triggered by Trichoderma erinaceum and Trichoderma viride.

White mold and stem rot is a common disease of Phaseolus vulgaris caused by Sclerotinia sclerotiorum. Biological control is a promising alternative for the control of this disease. In the present study, two Trichoderma spp., T. erinaceum and T. viride, and the consortium of both were evaluated as biocontrol agents against sclerotinia stem rot disease. The results revealed that T. erinaceum (NAIMCC-F-02171) and T. viride (NAIMCC-F-02500) when applied alone, significantly suppressed the infection rate of S. sclerotiorum and increased the rate of survival of plants by 74.5%. On the contrary, the combination of both the Trichoderma spp. was found to be more effective in reducing stem rot by 57.2% and increasing the survival of plants by 87.5% when compared to the individual Trichoderma applications. Further, the exogenous supplementation of Trichoderma activated antioxidative machineries, such as peroxidase, polyphenol oxidase, superoxide dismutase, catalase, and ascorbic acid in the plant. Besides, hydrogen peroxide and superoxide-free radical accumulation were also found to be reduced when T. erinaceum and T. viride were used either individually or in combination under the pathogen-challenged condition. Additionally, the photopigments in the bioprimed plants were markedly increased. Moreover, the combined inoculation of the two isolates yielded the highest records of growth parameters (root weight, shoot length, and leaf weight) compared with individual inoculation. Therefore, based on the above results, it was concluded that the combination of T. erinaceum and T. viride can be effectively used as an alternative to control white mold and stem rot caused by S. sclerotiorum.

Effects of Trichoderma harzianum on Photosynthetic Characteristics and Fruit Quality of Tomato Plants.

The beneficial role of fungi from the Trichoderma genus and its secondary metabolites in promoting plant growth, uptake and use efficiency of macronutrients and oligo/micro-nutrients, activation of plant secondary metabolism and plant protection from diseases makes it interesting for application in environmentally friendly agriculture. However, the literature data on the effect of Trichoderma inoculation on tomato fruit quality is scarce. Commercially used tomato cultivars were chosen in combination with indigenous Trichodrema species previously characterized on molecular and biochemical level, to investigate the effect of Trichoderma on photosynthetic characteristics and fruit quality of plants grown in organic system of production. Examined cultivars differed in the majority of examined parameters. Response of cultivar Gruzanski zlatni to Trichoderma application was more significant. As a consequence of increased epidermal flavonols and decreased chlorophyll, the nitrogen balance index in leaves has decreased, indicating a shift from primary to secondary metabolism. The quality of its fruit was altered in the sense of increased total flavonoids content, decreased starch, increased Bioaccumulation Index (BI) for Fe and Cr, and decreased BI for heavy metals Ni and Pb. Higher expression of swolenin gene in tomato roots of more responsive tomato cultivar indicates better root colonization, which correlates with observed positive effects of Trichodrema.

Antagonistic fungal enterotoxins intersect at multiple levels with host innate immune defences.

Animals and plants need to defend themselves from pathogen attack. Their defences drive innovation in virulence mechanisms, leading to never-ending cycles of co-evolution in both hosts and pathogens. A full understanding of host immunity therefore requires examination of pathogen virulence strategies. Here, we take advantage of the well-studied innate immune system of Caenorhabditis elegans to dissect the action of two virulence factors from its natural fungal pathogen Drechmeria coniospora. We show that these two enterotoxins have strikingly different effects when expressed individually in the nematode epidermis. One is able to interfere with diverse aspects of host cell biology, altering vesicle trafficking and preventing the key STAT-like transcription factor STA-2 from activating defensive antimicrobial peptide gene expression. The second increases STA-2 levels in the nucleus, modifies the nucleolus, and, potentially as a consequence of a host surveillance mechanism, causes increased defence gene expression. Our results highlight the remarkably complex and potentially antagonistic mechanisms that come into play in the interaction between co-evolved hosts and pathogens.

Morphological and molecular identification of four isolates of the entomopathogenic fungal genus Akanthomyces and their effects against Bemisia tabaci on cucumber.

The cotton whitefly, Bemisia tabaci Gen. (Hem., Aleyrodidae), is a key pest of many vegetables. Entomopathogenic fungi are promising microbial control agents against B. tabaci, but limited information is available concerning indigenous Iranian isolates. In this study, three isolates of Akanthomyces lecanii (PAL6, PAL7, and PAL8) and one isolate of A. muscarius (AGM5) were obtained from citrus hemipteran pests, Pulvinaria aurantii Cock. and Aphis gossypii Glover, in Mazandaran province, northern Iran. The isolates were then morphologically and molecularly identified. The efficacies of five different agar media for vegetative growth and conidiation of each isolate were determined. Potato dextrose agar was the medium on which the fungal mycelia developed at a relatively high rate. However, the highest rate of conidiation was found on Sabouraud dextrose agar. To determine the effects of the isolates on B. tabaci, a dose-response bioassay was carried out to estimate lethal concentration (LC50) and lethal time (LT50) values of each fungal isolate to second instar nymphs. The mean LC50 values of A. lecanii isolates ranged from 4.22 × 106 to 7.35 × 1013 conidia ml-1 at 5 to 7 days after the treatment. For A. muscarius, the values varied from 9.2 × 104 to 8.7 × 1010 conidia ml-1 at 5 to 7 days after the treatment. The lowest and the highest mean LC50 values were observed for A. mucarius (AGM5) and A. lecanii (isolate PAL6), respectively. The mean LT50 values of A. lecanii and A. muscarius isolates were 7.1-9.0 and 4.9-7.2 days, respectively. The LT50 values of A. muscarius were significantly lower than the other isolates. Overall, all isolates, especially A. muscarius (AGM5), exhibited appropriate potential as a biological control agent against B. tabaci.

Evaluation of Seven Essential Oils as Seed Treatments against Seedborne Fungal Pathogens of Cucurbita maxima.

Essential oils are gaining interest as environmentally friendly alternatives to synthetic fungicides for management of seedborne pathogens. Here, seven essential oils were initially tested in vivo for disinfection of squash seeds (Cucurbita maxima) naturally contaminated by Stagonosporopsis cucurbitacearum, Alternaria alternata, Fusarium fujikuro, Fusarium solani, Paramyrothecium roridum, Albifimbria verrucaria, Curvularia spicifera, and Rhizopus stolonifer. The seeds were treated with essential oils from Cymbopogon citratus, Lavandula dentata, Lavandula hybrida, Melaleuca alternifolia, Laurus nobilis, and Origanum majorana (#1 and #2). Incidence of S. cucurbitacearum was reduced, representing a range between 67.0% in L. nobilis to 84.4% in O. majorana #2. Treatments at 0.5 mg/mL essential oils did not affect seed germination, although radicles were shorter than controls, except with C. citratus and O. majorana #1 essential oils. Four days after seeding, seedling emergence was 20%, 30%, and 10% for control seeds and seeds treated with C. citratus essential oil (0.5 mg/mL) and fungicides (25 g/L difenoconazole plus 25 g/L fludioxonil). S. cucurbitacearum incidence was reduced by ~40% for plantlets from seeds treated with C. citratus essential oil. These data show the effectiveness of this essential oil to control the transmission of S. cucurbitacearum from seeds to plantlets, and thus define their potential use for seed decontamination in integrated pest management and organic agriculture.

Quantitative Proteomics Analysis Reveals the Function of the Putative Ester Cyclase UvEC1 in the Pathogenicity of the Rice False Smut Fungus Ustilaginoidea virens.

Rice false smut is a fungal disease distributed worldwide and caused by Ustilaginoidea virens. In this study, we identified a putative ester cyclase (named as UvEC1) as being significantly upregulated during U. virens infection. UvEC1 contained a SnoaL-like polyketide cyclase domain, but the functions of ketone cyclases such as SnoaL in plant fungal pathogens remain unclear. Deletion of UvEC1 caused defects in vegetative growth and conidiation. UvEC1 was also required for response to hyperosmotic and oxidative stresses and for maintenance of cell wall integrity. Importantly, ΔUvEC1 mutants exhibited reduced virulence. We performed a tandem mass tag (TMT)-based quantitative proteomic analysis to identify differentially accumulating proteins (DAPs) between the ΔUvEC1-1 mutant and the wild-type isolate HWD-2. Proteomics data revealed that UvEC1 has a variety of effects on metabolism, protein localization, catalytic activity, binding, toxin biosynthesis and the spliceosome. Taken together, our findings suggest that UvEC1 is critical for the development and virulence of U. virens.

Comparative Performance of Sanitizers in Managing Plant-to-Plant Transfer and Postharvest Infection of Calonectria pseudonaviculata and Pseudonectria foliicola on Boxwood.

Calonectria pseudonaviculata and Pseudonectria foliicola causing the infamous "boxwood blight" and "Volutella blight," respectively, are a constant threat to the boxwood production and cut boxwood greenery market. Both pathogens cause significant economic loss to all parties (growers, retailer, and customers) in the horticultural chain. The objective of this study was to evaluate efficacy of disinfesting chemicals (quaternary ammonium compound [QAC], peroxy, acid, alcohol, chlorine, and cleaner) in preventing plant-to-plant transfer of C. pseudonaviculata and P. foliicola via cutting tools, as well as reduction of postharvest boxwood blight and Volutella blight disease severity in harvested boxwood greenery. First, an in vitro study was conducted to select products and doses that completely or near-completely inhibited conidial germination of C. pseudonaviculata and P. foliicola. The selected treatments were also tested for their ability to reduce plant-to-plant transfer of C. pseudonaviculata and P. foliicola and manage postharvest boxwood blight and Volutella blight in boxwood cuttings. For the plant-to-plant transfer study, Felco 19 shears were used as a tool for mechanical transfer of fungal conidia. The blades of Felco 19 shears were exposed to a conidial suspension of C. pseudonaviculata or P. foliicola by cutting a 1-cm-diameter cotton roll that had been dipped into a fungal suspension. Disease-free boxwood rooted cuttings (10-cm height) were pruned with the contaminated shears. The Felco 19 shears were equipped with a mounted miniature sprayer connected to a pressurized reservoir of treatment solution that automatically sprayed the blade and plant surface while cutting. The influence of accumulated sap on the shear blade was studied through 1- or 10-cut pruning variable on test plants and screened for the efficacy of treatments. Then, the boxwood rooted cuttings were transplanted and incubated in room conditions (21°C, 60% RH) with 12 h of fluorescent light; data evaluation on disease severity was done weekly for a month. Disease progress (area under disease progress curve [AUDPC]) was calculated. In another study, postharvest dip application treatments were used for the management of postharvest boxwood blight or Volutella blight on boxwood cuttings. The harvested boxwood cuttings were inoculated with a conidial suspension of C. pseudonaviculata or P. foliicola and then dipped into treatment solution 3 days afterward. The treated boxwood cuttings were kept in room conditions, and boxwood blight or Volutella blight disease severity as well as marketability (postharvest shelf life) was assessed every 2 days for 1 week. A significant difference between treatments was observed for reduction of boxwood blight or Volutella blight severity and AUDPC. The treatments [Octyl decyl dimethyl (ODD) + dioctyl dimethyl (DoD) + didecyl dimethyl (DdD) + dimethyl benzyl (DB)] ammonium chloride (AC) (Simple Green D Pro 5), 2-propanol + didecyl dimethyl ammonium chloride (DDAC) (0.12%; KleenGrow), and dimethyl benzyl ammonium chloride (DBAC) + dimethyl ethylbenzyl ammonium chloride (DEAC) (GreenShield) were the most effective in reducing the plant-to-plant transfer of boxwood blight and Volutella blight when pruned with contaminated Felco 19 shears. In addition to the three effective treatments above, acetic acid (2.5%; vinegar), 2-propanol + DDAC (0.06%), sodium hypochlorite (Clorox), and potassium peroxymonosulfate + NaCl (2%; Virkon) were effective in reducing postharvest boxwood blight, whereas DBAC + DBAC (Lysol all-purpose cleaner), ethanol (70% [ethyl alcohol]), and DDAC + DBAC (Simple Green D Pro 3 plus) were effective in reducing Volutella blight disease severity and AUDPC, and they also maintained better quality and longer postharvest shelf life of boxwood cuttings when applied as a dip treatment. The longer postharvest shelf life of boxwood cuttings noted may be attributed to reduced disease severity and AUDPC resulting in healthy boxwood cuttings.

The N-terminus of an Ustilaginoidea virens Ser-Thr-rich glycosylphosphatidylinositol-anchored protein elicits plant immunity as a MAMP.

Many pathogens infect hosts through specific organs, such as Ustilaginoidea virens, which infects rice panicles. Here, we show that a microbe-associated molecular pattern (MAMP), Ser-Thr-rich Glycosyl-phosphatidyl-inositol-anchored protein (SGP1) from U. virens, induces immune responses in rice leaves but not panicles. SGP1 is widely distributed among fungi and acts as a proteinaceous, thermostable elicitor of BAK1-dependent defense responses in N. benthamiana. Plants specifically recognize a 22 amino acid peptide (SGP1 N terminus peptide 22, SNP22) in its N-terminus that induces cell death, oxidative burst, and defense-related gene expression. Exposure to SNP22 enhances rice immunity signaling and resistance to infection by multiple fungal and bacterial pathogens. Interestingly, while SGP1 can activate immune responses in leaves, SGP1 is required for U. virens infection of rice panicles in vivo, showing it contributes to the virulence of a panicle adapted pathogen.

The efficacy of 8-hydroxyquinoline derivatives in controlling the fungus Ilyonectria liriodendri, the causative agent of black foot disease in grapevines.

AIM: The purpose of this study was to evaluate the in vitro and in vivo efficiency of derivatives of 8-Hydroxyquinoline (8HQ) in controlling the fungus Ilyonectria liriodendri. METHODS AND RESULTS: The in vitro tests consisted of assessing its susceptibility to the minimal inhibitory concentration (MIC) and the inhibition of mycelial growth. While the in vivo tests consisted of applying and assessing the most effective products for the protection of wounds, in both preventive + curative and curative forms. The MIC values for PH 151 (6·25 µg ml-1 ) showed better results when compared to the fungicides tebuconazole (>50 µg ml-1 ) and mancozeb (12·5 µg ml-1 for strain 176 and 25 µg ml-1 for strain 1117). PH 151 significantly inhibited mycelial growth, while mancozeb did not differ from the control. In in vivo tests, PH 151 again demonstrated excellent results in vitro, especially when applied preventively. CONCLUSIONS: The derivative of 8HQ PH 151 was effective in controlling the fungus I. liriodendri in vitro and proved to be a promising option for protecting wounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study points to the prospect of an effective and safe preventive antifungal product, which would enable the use of pesticides in vine culture to be reduced.

Arabidopsis Defense against the Pathogenic Fungus Drechslera gigantea Is Dependent on the Integrity of the Unfolded Protein Response.

Drechslera gigantea Heald & Wolf is a worldwide-spread necrotrophic fungus closely related to the Bipolaris genus, well-known because many member species provoke severe diseases in cereal crops and studied because they produce sesterpenoid phytoxins named ophiobolins which possess interesting biological properties. The unfolded protein response (UPR) is a conserved mechanism protecting eukaryotic cells from the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER). In plants, consolidated evidence supports the role of UPR in the tolerance to abiotic stress, whereas much less information is available concerning the induction of ER stress by pathogen infection and consequent UPR elicitation as part of the defense response. In this study, the infection process of D. gigantea in Arabidopsis thaliana wild type and UPR-defective bzip28 bzip60 double mutant plants was comparatively investigated, with the aim to address the role of UPR in the expression of resistance to the fungal pathogen. The results of confocal microscopy, as well as of qRT-PCR transcript level analysis of UPR genes, proteomics, microRNAs expression profile and HPLC-based hormone analyses demonstrated that ophiobolin produced by the fungus during infection compromised ER integrity and that impairment of the IRE1/bZIP60 pathway of UPR hampered the full expression of resistance, thereby enhancing plant susceptibility to the pathogen.

Cyclopeptides from the Mushroom Pathogen Fungus Cladobotryum varium.

Three new cyclopeptides with serial Phe residues were identified with the aid of HPLC-DAD analysis, from the culture broth of Cladobotryum varium, a fungal pathogen causing mushroom cobweb disease. Cladoamides A (1) and B (2) have two consecutive N-methylphenylalanine units in the destruxin class cyclic depsipentapeptide framework, while cladoamide C (3) has a three consecutive Phe motif in a cyclopentapeptide structure. Of these three cyclopeptides, 1 showed potent autophagy-inducing activity at 10 µg/mL, comparable to a positive control, rapamycin. For the determination of the absolute configurations of the Ile residues in 1 and 3, new conditions for separating Ile and allo-Ile, using a pentafluorophenyl-bonded solid phase and methanolic solvent, were established within the analytical scheme of the advanced Marfey's method, thus offering a convenient alternative to the C3 Marfey's method, which requires elution with a three-solvent mixture. The sequence of two d-Phe and one l-Phe in 3 was determined through NMR chemical shift prediction by DFT-based calculations and chemical synthesis, which demonstrated the significance of noncovalent interactions in the accurate calculation of stable conformers for peptides with multiple aromatic rings.

Principal component analysis of the biological characteristics of entomopathogenic fungi in nutrient-limited and cuticle-based media.

Media formulated with insect cuticle (0.5% and 1%; Sphequit Sph®), with a reduction in nutrients (» Sabouraud dextrose agar + yeast [SDAY]) and commercial media (potato dextrose agar, Sabouraud dextrose agar) were evaluated for the cultivation of Beauveria bassiana, Cordyceps javanica (Isaria javanica [Bally] Samson & Hywel-Jones), and Metarhizium robertsii. By using principal component analysis, it was determined that the » SDAY and Sph formulations have greater advantages than commercial media for the development of fungi. The » SDAY and Sph (0.5% and 1%) improved hydrophobicity, radial growth rate, germination, conidia yield, and virulence in B. bassiana; in M. robertsii, they favored conidia yield, germination, and virulence, and in C. javanica, the » SDAY and Sph 0.5% media enhanced conidia yield, germination, radial growth rate, and virulence. We suggest that these formulations are an alternative to commercial culture media as they are cheaper and appropriate to improve the growth characteristics and virulence of the three strains evaluated. Some applications of culture media are suggested, and the importance of multivariate analysis as an exploratory tool to carry out the choice of culture media in a suitable way for the development of mycoinsecticides is also discussed.

Comprehensive identification of lysine 2-hydroxyisobutyrylated proteins in Ustilaginoidea virens reveals the involvement of lysine 2-hydroxyisobutyrylation in fungal virulence.

Lysine 2-hydroxyisobutyrylation (Khib ) is a newly identified post-translational modification (PTM) that plays important roles in transcription and cell proliferation in eukaryotes. However, its function remains unknown in phytopathogenic fungi. Here, we performed a comprehensive assessment of Khib in the rice false smut fungus Ustilaginoidea virens, using Tandem Mass Tag (TMT)-based quantitative proteomics approach. A total of 3 426 Khib sites were identified in 977 proteins, suggesting that Khib is a common and complex PTM in U. virens. Our data demonstrated that the 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. Network analysis of the modified proteins revealed a highly interconnected protein network that included many well-studied virulence factors. We confirmed that the Zn-binding reduced potassium dependency3-type histone deacetylase (UvRpd3) is a major enzyme that removes 2-hydroxyisobutyrylation and acetylation in U. virens. Notably, mutations of Khib sites in the mitogen-activated protein kinase (MAPK) UvSlt2 significantly reduced fungal virulence and decreased the enzymatic activity of UvSlt2. Molecular dynamics simulations demonstrated that 2-hydroxyisobutyrylation in UvSlt2 increased the hydrophobic solvent-accessible surface area and thereby affected binding between the UvSlt2 enzyme and its substrates. Our findings thus establish Khib as a major post-translational modification in U. virens and point to an important role for Khib in the virulence of this phytopathogenic fungus.