Capsid structure of a fungal dsRNA megabirnavirus reveals its previously unidentified surface architecture.

Rosellinia necatrix megabirnavirus 1-W779 (RnMBV1) is a non-enveloped icosahedral double-stranded (ds)RNA virus that infects the ascomycete fungus Rosellinia necatrix, a causative agent that induces a lethal plant disease white root rot. Herein, we have first resolved the atomic structure of the RnMBV1 capsid at 3.2 Å resolution using cryo-electron microscopy (cryo-EM) single-particle analysis. Compared with other non-enveloped icosahedral dsRNA viruses, the RnMBV1 capsid protein structure exhibits an extra-long C-terminal arm and a surface protrusion domain. In addition, the previously unrecognized crown proteins are identified in a symmetry-expanded cryo-EM model and are present over the 3-fold axes. These exclusive structural features of the RnMBV1 capsid could have been acquired for playing essential roles in transmission and/or particle assembly of the megabirnaviruses. Our findings, therefore, will reinforce the understanding of how the structural and molecular machineries of the megabirnaviruses influence the virulence of the disease-related ascomycete fungus.

Physicochemical characteristics and flavor profiles of fermented fish sauce (budu) during fermentation in commercial manufacturing plant.

This study was done by dynamically monitoring the changes in bacterial composition, physicochemical characteristics, and volatile substances during fermentation of fermented fish sauce (budu). The degree of hydrolysis, TCA-soluble peptides, and nitrogen contents increased as the fermentation time progressed. A continuous decrease in peroxide value and thiobarbituric acid reactive substances was noted over 60 days of fermentation. A total of 44 volatile compounds were detected, and increases in volatile compounds, such as 2-methylbutanal, 3-methylbutanal, benzaldehyde, and 2-ethyl furan, with low odor thresholds values, might contribute to budu's flavor of the final product. Additionally, the relationship between evolving microbiota and the formation of flavor compounds was analyzed, and halophilic lactic acid bacteria was identified to be the most important bacterial contributing to flavor and aroma development. This finding will provide important information for improving the quality of budu in terms of flavor characteristics.

Phytopathogenic fungus hosts a plant virus: A naturally occurring cross-kingdom viral infection.

The transmission of viral infections between plant and fungal hosts has been suspected to occur, based on phylogenetic and other findings, but has not been directly observed in nature. Here, we report the discovery of a natural infection of the phytopathogenic fungus Rhizoctonia solani by a plant virus, cucumber mosaic virus (CMV). The CMV-infected R. solani strain was obtained from a potato plant growing in Inner Mongolia Province of China, and CMV infection was stable when this fungal strain was cultured in the laboratory. CMV was horizontally transmitted through hyphal anastomosis but not vertically through basidiospores. By inoculation via protoplast transfection with virions, a reference isolate of CMV replicated in R. solani and another phytopathogenic fungus, suggesting that some fungi can serve as alternative hosts to CMV. Importantly, in fungal inoculation experiments under laboratory conditions, R. solani could acquire CMV from an infected plant, as well as transmit the virus to an uninfected plant. This study presents evidence of the transfer of a virus between plant and fungus, and it further expands our understanding of plant-fungus interactions and the spread of plant viruses.

Identification of Viruses and Viroids Infecting Tomato and Pepper Plants in Vietnam by Metatranscriptomics.

Tomato (Lycopersicum esculentum L.) and pepper (Capsicum annuum L.) plants belonging to the family Solanaceae are cultivated worldwide. The rapid development of next-generation sequencing (NGS) technology facilitates the identification of viruses and viroids infecting plants. In this study, we carried out metatranscriptomics using RNA sequencing followed by bioinformatics analyses to identify viruses and viroids infecting tomato and pepper plants in Vietnam. We prepared a total of 16 libraries, including eight tomato and eight pepper libraries derived from different geographical regions in Vietnam. We identified a total of 602 virus-associated contigs, which were assigned to 18 different virus species belonging to nine different viral genera. We identified 13 different viruses and two viroids infecting tomato plants and 12 viruses and two viroids infecting pepper plants with viruses as dominantly observed pathogens. Our results showed that multiple infection of different viral pathogens was common in both plants. Moreover, geographical region and host plant were two major factors to determine viral populations. Taken together, our results provide the comprehensive overview of viral pathogens infecting two important plants in the family Solanaceae grown in Vietnam.

Megabirnavirus structure reveals a putative 120-subunit capsid formed by asymmetrical dimers with distinctive large protrusions.

Rosellinia necatrix megabirnavirus 1 (RnMBV1) W779 is a bi-segmented dsRNA virus and a strain of the type species Rosellinia necatrix megabirnavirus 1 of the family Megabirnaviridae. RnMBV1 causes severe reduction of both mycelial growth of Rosellinia necatrix in synthetic medium and fungal virulence to plant hosts, and thus has strong potential for virocontrol (biological control using viruses) of white rot. The structure of RnMBV1 was examined by cryo-electron microscopy and three-dimensional reconstruction at 15.7 Å resolution. The diameter of the RnMBV1 capsid was 520 Å, and the capsid was composed of 60 asymmetrical dimers in the T = 1 (so-called T = 2) lattice that is well conserved among dsRNA viruses. However, RnMBV1 has putatively 120 large protrusions with a width of ∼ 45 Å and a height of ∼ 50 Å on the virus surface, making it distinguishable from the other dsRNA viruses.

Biological properties and expression strategy of rosellinia necatrix megabirnavirus 1 analysed in an experimental host, Cryphonectria parasitica.

Rosellinia necatrix megabirnavirus 1 (RnMBV1) with a bipartite dsRNA genome (dsRNA1 and dsRNA2) confers hypovirulence to its natural host, the white root rot fungus, and is thus regarded as a potential virocontrol (biocontrol) agent. Each segment has two large ORFs: ORF1 and partially overlapping ORF2 on dsRNA1 encode the major capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), whilst ORF3 and ORF4 on dsRNA2 encode polypeptides with unknown functions. Here, we report the biological and molecular characterization of this virus in the chestnut blight fungus, Cryphonectria parasitica, a filamentous fungus that has been used as a model for mycovirus research. Transfection with purified RnMBV1 particles into an RNA-silencing-defective strain (Δdcl-2) of C. parasitica and subsequent anastomosis with the WT strain (EP155) resulted in stable persistent infection in both host strains. However, accumulation levels in the two strains were different, being ~20-fold higher in Δdcl-2 than in EP155. Intriguingly, whilst RnMBV1 reduced both virulence and growth rate in Δdcl-2, it attenuated virulence without affecting significantly other traits in EP155. Western blot analysis using antiserum against recombinant proteins encoded by either ORF1 or ORF2 demonstrated the presence of a 250 kDa protein in purified virion preparations, suggesting that RdRp is expressed as a CP fusion product via a -1 frameshift. Antiserum against the ORF3-encoded protein allowed the detection of 150, 30 and 23 kDa polypeptides specifically in RnMBV1-infected mycelia. Some properties of an RnMBV1 mutant with genome rearrangements, which occurred after transfection of Δdcl-2 and EP155, were also presented. This study provides an additional example of C. parasitica serving as a versatile, heterologous fungus for exploring virus-host interactions and virus gene expression strategies.

Unveiling the microbial dynamics in vermicomposting with coir pith as earthworm substrate.

This study explored the impact of incorporating coir pith, a byproduct of the coconut industry, into the vermicomposting substrate of Eudrilus eugeniae earthworms. The groups were compared based on their diets: cow manure only or cow manure mixed with varying amounts of coir pith. The aim was to assess the effects of coir pith on earthworm growth, mortality and the microbial community involved in vermicomposting. Earthworms fed with higher proportions of coir pith (70 % w/w) experienced reduced growth (0.81 g/worm) and increased mortality (24.67 %) after 5 weeks of vermicomposting. These effects were attributed to the high level of total phenolic content in the system. Coir pith required specific bacteria for digestion and detoxification, and excessive intake disrupted the earthworms' digestion, thus hindering nutrient absorption. The study also examined the microbial composition of the vermicast samples and identified variations based on the diet. Bacterial taxa involved in lignocellulose degradation, such as Bacteriodota, Azospirillum, Chitinophagaceae, Marinomonas and Pantoea, exhibited decreased abundances in treatments with coir pith. Conversely, the abundances of potentially harmful bacteria, such as Aeromonas, increased with higher coir pith inclusion levels. This pioneering investigation sheds light on the feasibility of coir pith use in vermicomposting and emphasises the importance of optimising earthworm diets to enhance microbial ecological functions and improve vermicompost quality.

TOR Signaling Tightly Regulated Vegetative Growth, Conidiation, Oxidative Stress Tolerance and Entomopathogenicity in the Fungus Beauveria bassiana.

Beauveria bassiana degenerates after repeated subcultures, demonstrating declined conidiation and insect virulence. The target of rapamycin (TOR) kinase conserved among eukaryotes is the master regulator of cellular physiology and is likely involved in culture degeneration. Indeed, the levels of TOR-associated proteins increase over successive subcultures. Here, CRISPR/Cas9 locus engineering introduced the inducible Tet-On promoter upstream of the TOR kinase 2 gene tor2 in B. bassiana. The mutant PTet-Ontor2 'T41' was verified for the Tet-On integration via PCR analyses and provided a model for evaluating the fungal phenotypes according to the tor2 expression levels, induced by doxycycline (Dox) concentrations. At 0 µg·mL-1 of Dox, T41 had 68% of the wild type's (WT) tor2 expression level, hampered radial growth and relatively lower levels of oxidative stress tolerance, conidiation and virulence against Spodoptera exigua, compared to those under the presence of Dox. A low dose of Dox at 0.1-1 µg·mL-1 induced tor2 upregulation in T41 by up to 91% compared to 0 µg·mL-1 of Dox, resulting in significant increases in radial growth by 8-10% and conidiation by 8-27%. At 20 µg·mL-1 of Dox, which is 132% higher than T41's tor2 expression level at 0 µg·mL-1 of Dox, T41 showed an increased oxidative stress tolerance and a decrease in growth inhibition under iron replete by 62%, but its conidiation significantly dropped by 47% compared to 0 µg·mL-1 of Dox. T41 at 20 µg·mL-1 of Dox had a strikingly increased virulence (1.2 day lower LT50) against S. exigua. The results reflect the crucial roles of TOR kinase in the vegetative growth, conidiation, pathogenicity and oxidative stress tolerance in B. bassiana. Since TOR upregulation is correlated with culture degeneration in multiple subcultures, our data suggest that TOR signaling at relatively low levels plays an important role in growth and development, but at moderate to high levels could contribute to some degenerated phenotypes, e.g., those found in successive subcultures.

Lactic Acid Bacteria from Gamecock and Goat Originating from Phitsanulok, Thailand: Isolation, Identification, Technological Properties and Probiotic Potential.

From independent swab samples of the cloaca of indigenous gamecocks (CIG), anus of healthy baby goats (AHG), and vagina of goats (VG) originating from Phitsanulok, Thailand, a total of 263 isolates of lactic acid bacteria (LAB) were collected. Only three isolates, designated C707, G502, and V202, isolated from CIG, AHG, and VG, respectively, exhibited an excellent inhibitory zone diameter against foodborne pathogenic bacteria when evaluated by agar spot test. Isolates C707 and G502 were identified as Enterococcus faecium, whereas V202 was identified as Pediococcus acidilactici, based on 16S rRNA sequence analysis. When foodborne pathogenic bacteria were co-cultured with chosen LAB in mixed BHI-MRS broth at 39°C, their growth was suppressed. These LAB were found to be capable of surviving in simulated stomach conditions. Only the isolate G502 was able to survive in the conditions of simulated intestinal juice. This research suggests that selected LAB could be used as a food/feed supplement to reduce foodborne pathogenic bacteria and improve the safety of animal-based food or feed.

Hypovirulence of Colletotrichum gloesporioides Associated with dsRNA Mycovirus Isolated from a Mango Orchard in Thailand.

The pathogenic fungus Colletotrichum gloeosporioides causes anthracnose disease, which is an important fungal disease affecting the production of numerous crops around the world. The presence of mycoviruses, however, may have an impact on the pathogenicity of the fungal host. Here, we describe a double-stranded RNA (dsRNA) mycovirus, which was isolated from a field strain of C. gloeosporioides, Ssa-44.1. The 2939 bp genome sequence comprises two open reading frames (ORFs) that encode for a putative protein and RNA-dependent RNA polymerase (RdRp). The Ssa-44.1 mycovirus is a member of the unclassified mycovirus family named Colletotrichum gloeosporioides RNA virus 1 strain Ssa-44.1 (CgRV1-Ssa-44.1), which has a phylogenetic similarity to Colletotrichum gleosporioides RNA virus 1 (CgRV1), which was isolated from citrus leaves in China. In C. gloeosporioides, CgRV1-Ssa-44.1 was shown to be linked to hypovirulence. CgRV1-Ssa-44.1 has a low spore transfer efficiency but can successfully spread horizontally to isogenic virus-free isolates. Furthermore, CgRV1-Ssa-44.1 had a strong biological control impact on C. gloeosporioides on mango plants. This study is the first to describe a hypovirulence-associated mycovirus infecting C. gloeosporioides, which has the potential to assist with anthracnose disease biological management.

Infection of Two Heterologous Mycoviruses Reduces the Virulence of Valsa mali, a Fungal Agent of Apple Valsa Canker Disease.

Mycovirus infection has been widely shown to attenuate the virulence of phytopathogenic fungi. Valsa mali is an agriculturally important fungus that causes Valsa canker disease in apple trees. In this study, two unrelated mycoviruses [Cryphonectria hypovirus 1 (CHV1, genus Hypovirus, and single-stranded RNA) and Mycoreovirus 1 (MyRV1, genus Mycoreovirus, double-stranded RNA)] that originated from Cryphonectria parasitica (chestnut blight fungus) were singly or doubly introduced into V. mali via protoplast fusion. CHV1 and MyRV1 stably infected V. mali and caused a reduction in fungal vegetative growth and virulence. Co-infection of both viruses further reduced the virulence of V. mali but compromised the stability of CHV1 infection and horizontal transmission through hyphal anastomosis. Infections of MyRV1 and, to a lesser extent, CHV1 up-regulated the transcript expression of RNA silencing-related genes in V. mali. The accumulation of CHV1 (but not MyRV1) was elevated by the knockdown of dcl2, a key gene of the RNA silencing pathway. Similarly, the accumulation of CHV1 and the efficiency of the horizontal transmission of CHV1 during co-infection was restored by the knockdown of dcl2. Thus, CHV1 and MyRV1 are potential biological control agents for apple Valsa canker disease, but co-infection of both viruses has a negative effect on CHV1 infection in V. mali due to the activation of antiviral RNA silencing by MyRV1 infection.

Interplays of enzyme, substrate, and surfactant on hydrolysis of native lignocellulosic biomass.

Tracking enzyme, substrate, and surfactant interactions to reach maximum reducing sugar production during enzymatic hydrolysis of plant biomass may provide a better understanding of factors that limit the lignocellulosic material degradation in native rice straw. In this study, enzymes (Cellic Ctec2 cellulase and Cellic Htec2 xylanase) and Triton X-100 (surfactant) were used as biocatalysts for cellulose and xylan degradation and as a lignin blocking agent, respectively. The response surface model (R2 = 0.99 and R2-adj = 0.97) indicated that Cellic Ctec2 cellulase (p < 0.0001) had significant impacts on reducing sugar production, whereas Cellic Htec2 xylanase and Triton X-100 had insignificant impacts on sugar yield. Although FTIR analysis suggested binding of Triton X-100 to lignin surfaces, the morphological observation by SEM revealed similar surface features (i.e., smooth surfaces with some pores) of rice straw irrespective of Triton X-100. The reducing sugar yields from substrate hydrolysis with or without the surfactant were comparable, suggesting similar exposure of polysaccharides accessible to the enzymes. The model analysis and chemical and structural evidence suggest that there would be no positive effects on enzymatic hydrolysis by blocking lignins with Triton X-100 if high lignin coverage exists in the substrate due to the limited availability of hydrolyzable polysaccharides.

Omnipresence of Partitiviruses in Rice Aggregate Sheath Spot Symptom-Associated Fungal Isolates from Paddies in Thailand.

Partitiviruses are one of the most prevalent double-stranded RNA viruses that have been identified mostly in filamentous fungi and plants. Partitiviruses generally infect host fungi asymptomatically but infrequently exert significant effect(s) on morphology and virulence, thus being considered a potential source of biological control agents against pathogenic fungi. In this study, we performed a screening for mycoviruses of a collection of Thai isolates of rice fungal pathogen Rhizoctonia oryzae-sativae, a causal agent of rice aggregated sheath spot disease. As a result, 36% of tested isolates carried potentially viral double-stranded RNAs with sizes ranging from 2 to 3 kbp. By conventional cDNA library construction and RNA-seq, we determined six new alphapartitiviruses that infected three isolates: tentatively named Rhizoctonia oryzae-sativae partitivirus 1 to 6 (RosPV1-6). Furthermore, RT-PCR detection of each virus revealed their omnipresent nature in different R. oryzae-sativae isolates. Although virus-curing of basidiomycetous fungi is generally difficult, our repeated attempts successfully obtained virus-free (for RosPV1, RosPV2, and uncharacterized partitiviruses), isogenic strain of R. oryzae-sativae TSS190442. The virus-cured strain showed slightly faster colony growth on the synthetic media and severe symptom development on the rice sheath compared to its virus-infected counterpart. Overall, this study shed light on the distribution of partitiviruses in R. oryzae-sativae in a paddy environment and exemplified a virus-curing protocol that may be applicable for other basidiomycetous fungi.

A novel bipartite double-stranded RNA Mycovirus from the white root rot Fungus Rosellinia necatrix: molecular and biological characterization, taxonomic considerations, and potential for biological control.

White root rot, caused by the ascomycete Rosellinia necatrix, is a devastating disease worldwide, particularly in fruit trees in Japan. Here we report on the biological and molecular properties of a novel bipartite double-stranded RNA (dsRNA) virus encompassing dsRNA-1 (8,931 bp) and dsRNA-2 (7,180 bp), which was isolated from a field strain of R. necatrix, W779. Besides the strictly conserved 5' (24 nt) and 3' (8 nt) terminal sequences, both segments show high levels of sequence similarity in the long 5' untranslated region of approximately 1.6 kbp. dsRNA-1 and -2 each possess two open reading frames (ORFs) named ORF1 to -4. Although the protein encoded by 3'-proximal ORF2 on dsRNA-1 shows sequence identities of 22 to 32% with RNA-dependent RNA polymerases from members of the families Totiviridae and Chrysoviridae, the remaining three virus-encoded proteins lack sequence similarities with any reported mycovirus proteins. Phylogenetic analysis showed that the W779 virus belongs to a separate clade distinct from those of other known mycoviruses. Purified virions approximately 50 nm in diameter consisted of dsRNA-1 and -2 and a single major capsid protein of 135 kDa, which was shown by peptide mass fingerprinting to be encoded by dsRNA-1 ORF1. We developed a transfection protocol using purified virions to show that the virus was responsible for reduction of virulence and mycelial growth in several host strains. These combined results indicate that the W779 virus is a novel bipartite dsRNA virus with potential for biological control (virocontrol), named Rosellinia necatrix megabirnavirus 1 (RnMBV1), that possibly belongs to a new virus family.

Identification of a Novel Hypovirulence-Inducing Hypovirus From Alternaria alternata.

Mycoviruses are wide spread throughout almost all groups of fungi but only a small number of mycoviruses can attenuate the growth and virulence of their fungal hosts. Alternaria alternata is an ascomycete fungus that causes leaf spot diseases on various crop plants. In this study, we identified a novel ssRNA mycovirus infecting an A. alternata f. sp. mali strain isolated from an apple orchard in China. Sequence analyses revealed that this virus is related to hypoviruses, in particular to Wuhan insect virus 14, an unclassified hypovirus identified from insect meta-transcriptomics, as well as other hypoviruses belonging to the genus Hypovirus, and therefore this virus is designed as Alternaria alternata hypovirus 1 (AaHV1). The genome of AaHV1 contains a single large open-reading frame encoding a putative polyprotein (∼479 kDa) with a cysteine proteinase-like and replication-associated domains. Curing AaHV1 from the fungal host strain indicated that the virus is responsible for the slow growth and reduced virulence of the host. AaHV1 defective RNA (D-RNA) with internal deletions emerging during fungal subcultures but the presence of D-RNA does not affect AaHV1 accumulation and pathogenicities. Moreover, AaHV1 could replicate and confer hypovirulence in Botryosphaeria dothidea, a fungal pathogen of apple white rot disease. This finding could facilitate better understanding of A. alternata pathogenicity and is relevant for development of biocontrol methods of fungal diseases.

Genome rearrangement of a mycovirus Rosellinia necatrix megabirnavirus 1 affecting its ability to attenuate virulence of the host fungus.

Rosellinia necatrix megabirnavirus 1 (RnMBV1) is a bi-segmented double-stranded RNA mycovirus that reduces the virulence of the fungal plant pathogen R. necatrix. We isolated strains of RnMBV1 with genome rearrangements (RnMBV1-RS1) that retained dsRNA1, encoding capsid protein (ORF1) and RNA-dependent RNA polymerase (ORF2), and had a newly emerged segment named dsRNAS1, but with loss of dsRNA2, which contains two ORFs of unknown function. Analyses of two variants of dsRNAS1 revealed that they both originated from dsRNA1 by deletion of ORF1 and partial tandem duplication of ORF2, retaining a much shorter 5' untranslated region (UTR). R. necatrix transfected with RnMBV-RS1 virions showed maintenance of virulence on host plants compared with infection with RnMBV1. This suggests that dsRNAS1 is able to be transcribed and packaged, as well as suggesting that dsRNA2, while dispensable for virus replication, is required to reduce the virulence of R. necatrix.