Biology and mycovirus-assisted biological control of Sclerotinia sclerotiorum infecting vegetable and oilseed crops

Abstract

Plant disease caused by pathogenic fungal infection causes maximum crop damage. Among different fungal diseases, rot caused by Sclerotinia spp.; is a primary concern for vegetables and oilseed industry. Disease management using Chemical fungicides is a potential hazard and leads to the development of many fungicide-resistant strains. Hypovirulence associated mycoviruses is a possible environment-friendly solution, and current studies are aiming to exploit their potential as biocontrol agents. The use of the mycovirus mediated hypovirulent approach has emerged as a new technique to identify successful biocontrol agents. Most mycoviruses are known to have RNA genomes, double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA). A total of six dsRNA mycoviruses and a one ssDNA mycovirus have been reported from Sclerotinia sclerotiorum till date which includes the most recent entry as published by Hamid and his group in 2018. In contrast to dsRNA mycovirus, ssDNA mycovirus reported from Sclerotinia sclerotiorum has significant potential to be used as a biocontrol agent in the fields. Despite several reports on mycoviruses of Sclerotinia, not much could be done to explore its commercial importance. The present review describes the recent developments in the area of mycoviruses of Sclerotinia sclerotiorum and associated biocontrol potential.     

Mycoviruses of filamentous fungi and their relevance to plant pathology

Mycoviruses (fungal viruses) are reviewed with emphasis on plant pathogenic fungi. Based on the presence of virus-like particles and unencapsidated dsRNAs, mycoviruses are common in all major fungal groups. Over 80 mycovirus species have been officially recognized from ten virus families, but a paucity of nucleic acid sequence data makes assignment of many reported mycoviruses difficult. Although most of the particle types recognized to date are isometric, a variety of morphologies have been found and, additionally, many apparently unencapsidated dsRNAs have been reported. Until recently, most characterized mycoviruses have dsRNA genomes, but ssRNA mycoviruses now constitute about one-third of the total. Two hypotheses for the origin of mycoviruses of plant pathogens are discussed: the first that they are of unknown but ancient origin and have coevolved along with their hosts, the second that they have relatively recently moved from a fungal plant host into the fungus. Although mycoviruses are typically readily transmitted through asexual spores, transmission through sexual spores varies with the host fungus. Evidence for natural horizontal transmission has been found. Typically, mycoviruses are apparently symptomless (cryptic) but beneficial effects on the host fungus have been reported. Of more practical interest to plant pathologists are those viruses that confer a hypovirulent phenotype, and the scope for using such viruses as biocontrol agents is reviewed. New tools are being developed based on host genome studies that will help to address the intellectual challenge of understanding the fungal–virus interactions and the practical challenge of manipulating this relationship to develop novel biocontrol agents for important plant pathogens.     

Mycoviruses and virocontrol

Viruses are widespread in all major groups of fungi. The transmission of fungal viruses occurs intracellularly during cell division, sporogenesis, and cell fusion. They apparently lack an extracellular route for infection. Recent searches of the collections of field fungal isolates have detected an increasing number of novel viruses and lead to discoveries of novel genome organizations, expression strategies and virion structures. Those findings enhanced our understanding of virus diversity and evolution. The majority of fungal viruses have dsRNA genomes packaged in spherical particles, while ssRNA mycoviruses, possessing or lacking the ability to form particles, have increasingly been reported. This review article discusses the current status of mycovirus studies and virocontrol (biocontrol) of phytopathogenic fungi using viruses that infect them and reduce their virulence. Selected examples of virocontrol-associated systems include the chestnut/chestnut blight/hypovirus and fruit trees/white root rot fungus/mycoviruses. Natural dissemination and artificial introduction of hypovirulent fungal strains efficiently contributed to virocontrol of chestnut blight in European forests. Attempts to control white root rot with hypovirulence-conferring mycoviruses are now being made in Japan.     

Non-detection of mycoviruses in amphibian chytrid fungus (Batrachochytrium dendrobatidis) from Australia

Mycoviruses may influence the pathogenicity of disease-causing fungi. Although mycoviruses have been found in some chytrid fungi, limited testing has not detected them in Batrachochytrium dendrobatidis (Bd), the cause of the devastating amphibian disease, chytridiomycosis. Here we conducted a survey for mycovirus presence in 38 Bd isolates from Australia (n = 31), Brazil (n = 5) and South Korea (n = 2) with a combination of modern high-throughput sequencing and conventional dsRNA cellulose chromatography. Mycoviruses were not detected in any isolates. This result was unexpected, given the long evolutionary history of Bd, as well as the high prevalence of mycoviruses in related fungal species. Given our widespread sampling in Australia and the limited number of Bd introductions, we suggest that mycoviruses are uncommon or absent from Australian Bd. Testing more isolates from regions where Bd originated, as well as regions with high diversity or low fungal virulence may identify mycoviruses that could aid in disease control.     

The use of mycoviruses in the control of forest diseases

Fifteen families of mycoviruses have been described and 80% of these catalogued. However, their evolutionary relationship with fungi is not clear. The mycovirus genome can be formed by single- or double-stranded RNA or single-stranded DNA. The effects of mycoviruses range from the induction of a cryptic state (asymptomatic) to promotion of hyper- or hypovirulence in the host. Horizontal transmission of mycoviruses is determined by the presence of different vegetative compatibility types and mating types. Biocontrol of chestnut blight (Cryphonectria parasitica) has been found to be a successful mycovirus-based treatment and is considered a model in forest disease management. Development of this type of biological control tool for use in other forest pathologies requires a sound knowledge of viral symptomatology and transmission. The present review focuses on the application of mycoviruses and the prospects for future use in the biological control of forest diseases as well as on advances in mycovirus-applied research in forestry, landscape and culture of woody plants.     

Deep sequencing of mycovirus‐derived small RNAs from Botrytis species

RNA silencing is an ancient regulatory mechanism operating in all eukaryotic cells. In fungi, it was first discovered in Neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in Cryphonectria parasitica and, later, in several fungal species. There is little evidence of the antiviral potential of RNA silencing in the phytopathogenic species of the fungal genus Botrytis. Moreover, little is known about the RNA silencing components in these fungi, although the analysis of public genome databases identified two Dicer‐like genes in B. cinerea, as in most of the ascomycetes sequenced to date. In this work, we used deep sequencing to study the virus‐derived small RNA (vsiRNA) populations from different mycoviruses infecting field isolates of Botrytis spp. The mycoviruses under study belong to different genera and species, and have different types of genome [double‐stranded RNA (dsRNA), (+)single‐stranded RNA (ssRNA) and (–)ssRNA]. In general, vsiRNAs derived from mycoviruses are mostly of 21, 20 and 22 nucleotides in length, possess sense or antisense orientation, either in a similar ratio or with a predominance of sense polarity depending on the virus species, have predominantly U at their 5′ end, and are unevenly distributed along the viral genome, showing conspicuous hotspots of vsiRNA accumulation. These characteristics reveal striking similarities with vsiRNAs produced by plant viruses, suggesting similar pathways of viral targeting in plants and fungi. We have shown that the fungal RNA silencing machinery acts against the mycoviruses used in this work in a similar manner independent of their viral or fungal origin.     

Isolation,molecular characterization and virome analysis of culturable wood fungal endophytes in esca symptomatic and asymptomatic grapevine plants

Europe is the world largest grape producer, but in recent years, the report of diseases due to infection by grapevine trunk pathogens (GTPs) is becoming one of the main constraints for viticulture. Among grapevine trunk diseases (GTDs), esca syndrome is one of the most complex, characterized by simultaneous infection of several fungi, which leads to important reduction in yield and quality. Previous characterization of fungal isolates associated with esca syndrome leads to the recognition of at least three important players: Phaeocremonium minimum, Phaemoniella chlamydospora and Fomitiporia mediterranea. Here we isolated and characterized molecularly fungal endophytes directly from field wood tissues of plants showing or not esca symptoms. In addition, to better characterize such collection, a deep RNA sequencing (100 M reads in paired-end) to screen for mycovirus presence was performed. Thirty-nine viral genomes were detected, 38 of which were putative new viral species; some of these viruses infected GTPs, including P. minimum and F. mediterranea. In this work, we reported for the first time a curated collection of grapevine fungal endophytes identifying the associated mycoviruses some of which could be employed in future biotechnological exploitation as biological control agents for sustainable plant protection.     

Double-Stranded RNA Mycovirus Infection of Aspergillus fumigatus Is Not Dependent on the Genetic Make-Up of the Host

Aspergillus fumigatus is a fungus that causes opportunistic infections in immunocompromised patients, with high morbidity and mortality. In its turn, A. fumigatus can become infected with mycoviruses. Most mycoviruses have a dsRNA genome and can cause fungal hypovirulence. For that reason, mycoviruses could theoretically be used as therapeutic tools to combat fungal infections. We determined if a certain genetic make-up of A. fumigatus was associated with the presence of mycoviruses in 86 clinical A. fumigatus isolates. Mycovirus screening was performed by isolating dsRNA from mycelial cultures using a Trizol/Chloroform method. The genetic relatedness of dsRNA infected A. fumigatus was determined by cell surface protein (CSP) typing and determination of the mating type. Sixteen (18.6%) of the 86 clinical A. fumigatus isolates contained dsRNA. The A. fumigatus collection could be divided into 11 different CSP types. DsRNA infected A. fumigatus isolates had similar CSP types as non-infected isolates. In both cases, the CSP types t01, t02, t03 and t04 were the most prevalent and the distribution comparable to the CSP types observed in other Dutch collections. Mating types MAT1-1 and MAT1-2 were evenly distributed among all A. fumigatus strains, regardless of CSP type. No difference was observed in mycovirus infections between MAT1-1 and MAT1-2 isolates. DsRNA mycovirus infections in A. fumigatus are not related to either CSP or mating type and therefore represent an interesting future therapeutic tool to combat fungal infections.     

Presence of Transposons and Mycoviruses in Botrytis cinerea Isolates Collected from a German Grapevine Growing Region

Transposons and infection of fungal strains with mycoviruses can have significant effects on distinctive phenotypic traits of phytopathogenic fungi such as mycelial growth and sporulation, pathogenicity or fungicide resistance. Two transposable elements (TE), Boty and Flipper, are known to be associated with the ubiquitous fungus Botrytis cinerea. In addition, the presence of two types of ssRNAsRNA viruses, BVX and BVF, has been reported in B. cinerea. In this study, we assessed the genetic diversity of B. cinerea isolates, all sampled within a small‐sized German viticultural area (‘Rheingau’) by examining and classifying them according to the presence of TEs and mycoviruses. A subset of the isolates was further analysed with microsatellite markers to determine the origin of particular isolates with or without one or both mycoviruses. Virtually all isolates (98%) sampled in two different years (2008 and 2010) were screened positive for the presence of a transposon. Presence of one or both B. cinerea mycoviruses was confirmed for 37% of the analysed isolates sampled in 2010, representing the first record of B. cinerea mycoviruses in German isolates. Assignment on individual B. cinerea isolates to different genetic groups was independent of the presence or absence of a mycovirus or a transposable element, respectively. Furthermore, we found no correlation between the presence of either a mycovirus or a transposable element and different viticultural management practices, soil properties or levels of nitrogen fertilization applied to the respective vineyards. However, mycelial growth of B. cinerea strains containing mycovirus BVF was significantly reduced at lower temperatures.     

Aspergillus mycoviruses are targets and suppressors of RNA silencing

RNA silencing can function as a virus defense mechanism in a diverse range of eukaryotes, and many viruses are capable of suppressing the silencing machinery targeting them. However, the extent to which this occurs between fungal RNA silencing and mycoviruses is unclear. Here, three Aspergillus dsRNA mycoviruses were partially characterized, and their relationship to RNA silencing was investigated. Aspergillus virus 1816 is related to Agaricus bisporus white button mushroom virus 1 and suppresses RNA silencing through a mechanism that alters the level of small interfering RNA. Aspergillus virus 178 is related to RNA virus L1 of Gremmeniella abietina and does not appear to affect RNA silencing. The third virus investigated, Aspergillus virus 341, is distantly related to Sphaeropsis sapinea RNA virus 2. Detection of mycovirus-derived siRNA from this mycovirus demonstrates that it is targeted for degradation by the Aspergillus RNA silencing machinery. Thus, our results indicate that Aspergillus mycoviruses are both targets and suppressors of RNA silencing. In addition, they suggest that the morphological and physiological changes associated with some mycoviruses could be a result of their antagonistic relationship with RNA silencing.     

Parasitism and maintenance of diversity in a fungal vegetative incompatibility system: the role of selection by deleterious cytoplasmic elements

In fungi, horizontal transmission of deleterious cytoplasmic elements is reduced by the vegetative incompatibility system. This self/non-self recognition system may select for greater diversity of fungal incompatibility phenotypes in a frequency-dependent manner but the link between the diversity of fungal phenotypes and the virulence of cytoplasmic parasites has been poorly studied. We used an epidemiological model to show that even when transmission between incompatibility types is permitted, parasite pressure can lead to high levels of polymorphism for vegetative incompatibility systems. Moreover, high levels of polymorphism in host populations can select for less virulent cytoplasmic parasites. This feedback mechanism between parasite virulence and vegetative incompatibility system polymorphism of host populations may account for the general avirulence of most known mycoviruses. Furthermore, this mechanism provides a new perspective on the particular ecology and evolution of the host/parasite interactions acting between fungi and their cytoplasmic parasites.     

Characterization of Mycoviruses and Analyses of Chitinase Secretion in the Biocontrol Fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Metarhizium anisopliae is the best-characterized entomopathogen and is used to control insect pests in sugar cane plantations in Brazil on a commercial scale. We have previously reported the infection of some M. anisopliae strains by dsRNA mycoviruses. Here we describe the purification and characterization of the viruses (MaV-A1, MaV-M5, MaV-RJ) in terms of dsRNA content, capsid proteins, electron microscopy, Western blot, and hybridization patterns. One spontaneous mutant lost some of the high molecular weight dsRNA components and showed significant alterations in colony morphology and spore production, suggesting that viral genes interfere with fungal phenotype. A comparison between dsRNA mycovirus-free and infected M. anisopliae isolates showed that virus-free isolates have increased endochitinase secretion. By comparing the following parameters: the buoyant density in CsCl of the presumed virions; the number and estimated molecular weight of the dsRNA components and the molecular mass of the capsid proteins to other mycoviruses previously described, we suggest the inclusion of MaV-A1 and MaV-M5 in the family Totiviridae and MaV-RJ in the family Partitiviridae. Received: 25 September 2001 / Accepted: 1 February 2002     

葡萄孢菌真菌病毒研究进展(综述)

分析寄生于葡萄孢中真菌病毒的种类及其对寄主真菌的影响,描述其中5种病毒的形态及基因组结构,根据葡萄孢真菌病毒的研究现状提出未来研究方向,为葡萄孢真菌病毒的深入研究和利用提供参考。     

Evolutionary genomics of mycovirus-related dsRNA viruses reveals cross-family horizontal gene transfer and evolution of diverse viral lineages

ABSTRACT: BACKGROUND: Double-stranded (ds) RNA fungal viruses are typically isometric single-shelled particles that are classified into three families, Totiviridae, Partitiviridae and Chrysoviridae, the members of which possess monopartite, bipartite and quadripartite genomes, respectively. Recent findings revealed that mycovirus-related dsRNA viruses are more diverse than previously recognized. Although an increasing number of viral complete genomic sequences have become available, the evolution of these diverse dsRNA viruses remains to be clarified. This is particularly so since there is little evidence for horizontal gene transfer (HGT) among dsRNA viruses. RESULTS: In this study, we report the molecular properties of two novel dsRNA mycoviruses that were isolated from a field strain of Sclerotinia sclerotiorum, Sunf-M: one is a large monopartite virus representing a distinct evolutionary lineage of dsRNA viruses; the other is a new member of the family Partitiviridae. Comprehensive phylogenetic analysis and genome comparison revealed that there are at least ten monopartite, three bipartite, one tripartite and three quadripartite lineages in the known dsRNA mycoviruses and that the multipartite lineages have possibly evolved from different monopartite dsRNA viruses. Moreover, we found that homologs of the S7 Domain, characteristic of members of the genus phytoreovirus in family Reoviridae are widely distributed in diverse dsRNA viral lineages, including chrysoviruses, endornaviruses and some unclassified dsRNA mycoviruses. We further provided evidence that multiple HGT events may have occurred among these dsRNA viruses from different families. CONCLUSIONS: Our study provides an insight into the phylogeny and evolution of mycovirus-related dsRNA viruses and reveals that the occurrence of HGT between different virus species and the development of multipartite genomes during evolution are important macroevolutionary mechanisms in dsRNA viruses.     

Mycoviruses of Botrytis cinerea isolates from different hosts

Botrytis cinerea (teleomorph Botryotinia fuckeliana) is a necrotrophic plant pathogenic fungus that causes grey mould and enormous economic losses worldwide in different crops. Control of B. cinerea is difficult due to the appearance of fungicide‐resistant isolates, and the diversity in virulence due to genetic variability and, perhaps, the infection with mycoviruses or fungal viruses. The discovery of mycoviruses and their possible application as biocontrol agents, as well as their use as tools to study the plant–pathogen interaction, has encouraged their study in B. cinerea. Herein, we have analysed the occurrence of mycoviruses in Spanish B. cinerea isolates to approach a better understanding of the interactions among viruses, fungi and plants in this pathosystem. Fifty‐five percent of the B. cinerea isolates analysed contained double‐stranded RNA (dsRNA) elements, and the number of dsRNA elements, their relative concentration and size were variable among isolates. Some of these dsRNAs were related to the presence of virus like rod or isometric particles, and to cellular degeneration and malformed mitochondria. We have also demonstrated that a 3 kb dsRNA present in 55% of the isolates having dsRNA elements was a mycovirus genome. Partial sequence of that mycovirus presented high identity in nucleotide and amino acid sequence with Botrytis cinerea mitovirus 1 (BcMV1). Analysis of the genetic distance within Spanish BcMV1 sequences showed the existence of different isolates of this mitovirus inside the Spanish B. cinerea population analysed. This is the first report of the variability of dsRNA elements and the partial genome sequence of a mitovirus associated with Spanish B. cinerea isolates and the genetic diversity within Spanish isolates of BcMV1.     

抗真菌植物基因工程的策略和进展

所有高等植物都受多种真菌的侵害,水稻的240多种病害中真菌性痫害占90％。,可见真菌病害是世界范围内危害作物产蘑的主要因素之一,是长期以来作物育种学家一直在努力攻克的难题。目前国     

The Ustilaginales as plant pests and model systems

The Ustilaginales are a vast and diverse group of fungi, which includes the plant pathogenic smuts that cause significant losses to crops worldwide. Members of the Ustilaginales are also valuable models for the unraveling of fundamental mechanisms controlling important biological processes. Ustilago maydis is an important fungal model system and has been well studied with regard to mating, morphogenesis, pathogenicity, signal transduction, mycoviruses, DNA recombination, and, recently, genomics. In this review we discuss the life cycles of members of the Ustilaginales and provide background on their economic impact as agricultural pests. We then focus on providing a summary of the literature with special attention to topics not well covered in recent reviews such as the use of U. maydis in mycovirus research and as a model for understanding the molecular mechanisms of fungicide resistance and DNA recombination and repair.     

Tick pathogenicity,thermal tolerance and virus infection in Tolypocladium cylindrosporum

Tolypocladium cylindrosporum is a fungus which has been isolated from soil, from asymptomatic plants as an endophyte and has been shown to be pathogenic to several species of arthropods. The objective of the present work was to study a collection of T. cylindrosporum strains to evaluate the characteristics of this fungus as a bioacaricide. The pathogenicity of five different strains of T. cylindrosporum was tested against two tick species, Ornithodoros erraticus and Ornithodoros moubata. Both tick species were susceptible to all the fungal strains. Mortality was greater for O. erraticus and differed among the five developmental stages of the ticks tested and among the fungal treatments. Mean mortality rates were close to 60% for O. erraticus, similar to those reported for other entomopathogenic fungi used for this purpose. The responses of 11 different strains of the fungus to 22°C and 30°C were also studied. Significant differences in temperature tolerance occurred among the strains and growth inhibition was observed at 30°C. Several mycoviruses were found infecting 5 of the 11 strains. However, no clear relationship was found between the presence of viruses and fungal growth or pathogenicity.     

Nine viruses from eight lineages exhibiting new evolutionary modes that co-infect a hypovirulent phytopathogenic fungus

Mycoviruses are an important component of the virosphere, but our current knowledge of their genome organization diversity and evolution remains rudimentary. In this study, the mycovirus composition in a hypovirulent strain of Sclerotinia sclerotiorum was molecularly characterized. Nine mycoviruses were identified and assigned into eight potential families. Of them, six were close relatives of known mycoviruses, while the other three had unique genome organizations and evolutionary positions. A deltaflexivirus with a tripartite genome has evolved via arrangement and horizontal gene transfer events, which could be an evolutionary connection from unsegmented to segmented RNA viruses. Two mycoviruses had acquired a second helicase gene by two different evolutionary mechanisms. A rhabdovirus representing an independent viral evolutionary branch was the first to be confirmed to occur naturally in fungi. The major hypovirulence-associated factor, an endornavirus, was finally corroborated. Our study expands the diversity of mycoviruses and potential virocontrol agents, and also provides new insights into virus evolutionary modes including virus genome segmentation.