Hypovirulent Isolates Research Articles

Characterisation of Cryphonectria hypovirus 1 strains in Turkey and their transmission to various vegetative compatibility types of Cryphonectria parasitica

Characterization of the Cryphonectria parasitica population was initially done by a phenotypical assessment of 40 in vitro grown isolates obtained from 52 healing cankers collected from eight important chestnut-growing provinces of Turkey. The results of Bavendamm test, often correlated indirectly to hypovirulence, suggested 31 possibly hypovirulent and 9 virulent isolates. PCR tests amplified two regions of ORFs A and B of Cryphonectria hypovirus 1 (CHV-1) from 36 of 40 isolates. The PCR test confirmation was more sensitive than the Bavendamm test. Partial ORFA sequencing revealed 36 CHV-1 haplotypes belonging to Italian subtype (I), with all hypovirulent isolates being of EU-1 vc type. The CHV-1 from 10 native EU-1 isolates were first transferred to six European vc type testers, EU-2, EU-3, EU-5, EU-7, EU-26, and EU-44, having heteroallelism at one vic locus. The presence of the vic locus difference generally reduces virus transmission. The easiest and highest frequency virus transfer was obtained by vic4 and vic6 allelic differences, while the differences vic2 and vic7 made the transfer more challenging. Finally, in this study we successfully transferred CHV-1 to an EU-1 isolate obtained from the Bursa province to an EU-12 European tester isolate.

Binucleate Rhizoctonia induced tomato resistance against Rhizoctonia solani via affecting antioxidants and cell wall reinforcement

Isolates of Rhizoctonia solani (AG-3 PT, AG-4 HG-I, AG-4 HG-II) and one binucleate Rhizoctonia sp. (BNR) belonging to AG-Bb were investigated for pathogenicity on tomato cultivar Mobil. The BNR isolate revealed the lowest virulence and it was used as biocontrol agent against R. solani AG-4 HG-II, which showed the highest virulence on tomato. Inoculation of tomato plants with the hypovirulent BNR isolate reduced the disease symptoms of R. solani and induced resistance. Resistance induction was observed not only on the plants simultaneously inoculated with BNR and R. solani, but also when the plants were inoculated by the BNR and R. solani with time intervals. The peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT) activities and expression levels of the corresponding genes in tomato plants increased after R. solani or BNR inoculation. The highest level of antioxidant activities and expression of their genes, lignin and callose formation were observed in the plants inoculated with the BNR and R. solani, simultaneously. The BNR inoculation reduced H2O2 accumulation. The highest level of priming was observed for the POX among other antioxidants tested via application of the BNR. Treatment with potassium cyanide (as a POX inhibitor) reduced basal resistance and BNR-induced resistance (BNR-IR) via reduction of lignification and callose deposition in tomato plants. These findings demonstrated the role of antioxidant enzymes, mainly the POX, in both basal resistance and BNR-IR. Therefore, redox state and antioxidants are involved in cell wall strengthening via lignin and callose formation, as important defense components which decrease the pathogen progress in plant tissues.

Kinetic and proteomic studies in milk show distinct patterns among major Listeria monocytogenes clones

Listeria monocytogenes, a contaminant of raw milk, includes hypervirulent clonal complexes (CC) like CC1, CC4, and CC6, highly overrepresented in dairy products when compared to other food types. Whether their higher prevalence in dairy products is the consequence of a growth advantage in this food remains unknown. We examined growth kinetics of five L. monocytogenes isolates (CC1, CC4, CC6, CC9, and CC121) at 37 and 4 °C in ultra-high temperature (UHT) milk and raw milk. At 4 °C, hypovirulent CC9 and CC121 isolates exhibit better growth parameters in UHT milk compared to the hypervirulent CC1, CC4, and CC6 isolates. CC9 isolate in raw milk at 4 °C exhibited the fastest growth and the highest final concentrations. In contrast, hypervirulent isolates (CC1, CC4, and CC6) displayed better growth rates in UHT milk at 37 °C, the mammalian host temperature. Proteomic analysis of representative hyper- (CC1) and hypovirulent (CC9) isolates showed that they respond to milk cues differently with CC-specific traits. Proteins related to metabolism (such as LysA or different phosphotransferase systems), and stress response were upregulated in both isolates during growth in UHT milk. Our results show that there is a Listeria CC-specific and a Listeria CC-common response to the milk environment. These findings shed light on the overrepresentation of hypervirulent L. monocytogenes isolates in dairy products, suggesting that CC1 and CC4 overrepresentation in dairy products made of raw milk may arise from contamination during or after milking at the farm and discard an advantage of hypervirulent isolates in milk products when stored at refrigeration temperatures.

Molecular genetic variability of Cryphonectria hypovirus 1 associated with Cryphonectria parasitica in South Tyrol (northern Italy).

Cryphonectria hypovirus 1 (CHV-1) has been widely studied and used as a biocontrol agent because of its ability to infect the chestnut blight fungus, Cryphonectria parasitica, and to reduce its virulence. Knowledge about the hypovirus, its presence, and diversity is completely lacking in South Tyrol (northern Italy), which may obstruct biocontrol measures for chestnut blight based on CHV-1. This work aimed to study the occurrence of CHV-1 infecting C. parasitica in South Tyrol and to perform a genetic characterization of the hypovirus. In South Tyrol, CHV-1 was found to occur in 29.2% of the fungal isolates investigated, varying in frequency between different regions and chestnut stands. Twenty-three haplotypes based on partial cDNA (complementary DNA) sequences of open reading frame (ORF)-A and 30 haplotypes based on partial cDNA sequences of ORF-B were identified among 47 and 56 hypovirulent fungal isolates, respectively. Phylogenetic analysis showed that all the haplotypes belonged to the Italian subtype of CHV-1 and that they were closely related to the populations of Italy, Switzerland, Croatia and Slovenia. Evidence of recombination was not found in the sequences and point mutations were the main source of diversity. Overall, this study indicated that the prevalence of CHV-1 in South Tyrol is low compared to many other central and western European populations and determined a need to actively impose biocontrol measures. Using sequence analysis, we identified some variants of interest of CHV-1 that should be studied in detail for their potential use in biocontrol.

Various Population Structures of Cryphonectria parasitica in Cáceres (Spain) Determine the Feasibility of the Biological Control of Chestnut Blight with Hypovirulent Strains

Chestnut blight, caused by Cryphonectria parasitica, is a severe disease that may be biologically controlled by the use of hypovirulent strains, but the diversity of the pathogen population affects biocontrol feasibility. Villuercas–Ibores–Jara, the Jerte Valley and La Vera are the main chestnut production districts in Cáceres (central-western Spain). The purpose of this study is to determine the Vegetative Compatibility Groups (VCGs) and mating types in these districts and to identify hypovirulent isolates to be used in biological control. The VCGs were determined by the merging/barrage response; PCR tests were used for the mating type determination and CHV-1 hypovirus detection. In total, 40 sites were surveyed and 269 isolates were obtained, most of them (227) from the Villuercas–Ibores–Jara district, where EU11 was the predominant VCG (88.1%) with EU1 (6.6%) and EU12 (4.4%) being also present. In the Jerte Valley and La Vera, EU1 (61.9%), EU11 (11.9%) and EU12 (11.9%) were the predominant VCGs. Both mating types were detected (48% MAT-1; 50% MAT-2) and in general, only one mating type was found in each site. The presence of Cryphonectria hypovirus 1 (CHV-1), subtype I, was identified in only one isolate (VCG EU11) from Villuercas–Ibores–Jara. The characteristics of the C. parasitica population in this district and the occurrence of CHV-1 hypovirus support the potential of successful biological control in Villuercas–Ibores–Jara using hypovirulent strains, while in the Jerte Valley and La Vera only preventive measures are recommended.

Genetic diversity of Cryphonectria parasitica causing chestnut blight in South Tyrol (northern Italy)

European chestnut (Castanea sativa) is threatened by the invasive fungus Cryphonectria parasitica, which causes chestnut blight. The virulence of the fungus can be reduced by a group of mycoviruses that can spread among vegetatively compatible strains through hyphal anastomosis. Hypovirulent isolates are used as biocontrol agents, but their efficiency can be diminished by restricted hyphal anastomosis if the variability of vegetative compatibility (vc) types in a population is high. Sexual reproduction could increase the vc type diversity and further complicate biocontrol in a region. Therefore, knowledge of genetic diversity of C. parasitica is important to assess the effectiveness of a biological control program. The present study was performed in the Autonomous Province of Bozen-Bolzano (South Tyrol) in northern Italy, where chestnut cultivation provides an additional income to farmers. The genetic characterization of C. parasitica isolates from 35 chestnut stands and one forest population in different districts of South Tyrol was performed based on the analysis of vegetative incompatibility loci, the mating type locus, and the internal transcribed spacer (ITS) region. In this study, a total of 23 different vc types were found all over South Tyrol with a Shannon diversity index of 1.86. EU-2, EU-1, and EU-13 were the most widespread vc types comprising 51%, 13%, and 9% of the fungal isolates, respectively. Both mating types were present in the region with a ratio close to 1:1. Three different haplotypes were identified based on ITS sequence analysis, which pointed to two introduction events of the fungus to the region and allowed placing C. parasitica from South Tyrol into a larger phylogeographic context.

Amphibian resistance to chytridiomycosis increases following low‐virulence chytrid fungal infection or drug‐mediated clearance

Abstract Amphibian biodiversity is experiencing ongoing declines due in part to the infectious disease, chytridiomycosis. Efforts to mitigate the effects of the causal agent of chytridiomycosis, Batrachochytrium dendrobatidis (Bd), in the wild have not been wholly effective. Translocations are an important management tool for amphibians, and immunizations represent a possible strategy for preparing amphibians for release across a landscape where Bd exists. We evaluated the utility of using an isolate of Bd that was shown to be hypovirulent to the relict leopard frog Rana onca as a transmissible inoculum for promoting chytridiomycosis resistance. We conducted a co‐housing experiment to determine if the isolate we used could be passed between R. onca without increasing in virulence. We then followed with an experiment where frogs that were exposed to the hypovirulent isolate were then challenged with a virulent Bd isolate. In other experiments, we evaluated whether Bd infections followed by clearance with itraconazole (an antifungal) could increase resistance to chytridiomycosis in R. onca and Rana pipiens (northern leopard frog). We found that our hypovirulent Bd inoculation was transmissible between hosts, did not cause chytridiomycosis and was effective at imparting long‐term chytridiomycosis resistance. Rana onca inoculated with the hypovirulent Bd isolate had lower pathogen burdens and were 55 times more likely to survive infections by a virulent Bd isolate than non‐inoculated frogs. For both species, prior exposure to Bd followed by infection clearance with itraconazole resulted in significantly increased survivorship and lower pathogen burdens as compared to controls that had no prior Bd exposure. Rana onca that were previously exposed to Bd were more than 15 times more likely to survive infections. Previously exposed R. pipiens survived in higher proportions than controls, but with weaker statistical support. Synthesis and Applications. Translocations are an important management tool for many amphibian conservation programs. For amphibian species that have experienced declines due to the disease chytridiomycosis [caused by the pathogen Batrachochytrium dendrobatidis (Bd)], the success of translocations may be limited due to the persistent threat of the disease in the wild. We completed a series of experiments aimed at increasing amphibian resistance to chytridiomycosis using low‐virulence, transmissible Bd inoculations and Bd infection followed by clearance with an antifungal (itraconazole). Our low‐virulence Bd inoculations were transmissible, did not cause disease and provided long‐term resistance against chytridiomycosis. Similarly, infection with Bd and clearance with itraconazole led to increased resistance to chytridiomycosis in subsequent exposures. The use of a low‐virulence Bd inoculation could be further developed into a transmissible vaccine, propagating the protective benefits beyond individuals that can be directly inoculated. At this stage, we advise that using the alternative approach of infecting frogs with Bd from translocation sites followed by clearance of infections with itraconazole may be sensible for increasing host resistance to chytridiomycosis.

Divergent RNA viruses in Macrophomina phaseolina exhibit potential as virocontrol agents

Macrophomina phaseolina is an important necrotrophic phytopathogenic fungus and cause extensive damage in many oilseed crops. Twelve M.phaseolina isolates with diverse biological phenotypes were selected for a high-throughput sequencing-based metatranscriptomic and bioinformatics analysis to identify viruses infecting M.phaseolina. The analysis identified 40 partial or nearly complete viral genome segments, 31 of which were novel viruses. Among these viral sequences, 43% of the viral genomes were double-stranded RNA (dsRNA), 47% were positive single-stranded RNA (ssRNA+), and the remaining 10% were negative sense-stranded RNA (ssRNA−). The 40 viruses showed affinity to 13 distinct viral lineages, including Bunyavirales (four viruses), Totiviridae (three viruses), Chrysoviridae (five viruses), Partitiviridae (four viruses), Hypoviridae (one virus), Endornaviridae (two viruses), Tombusviridae (three viruses), Narnaviridae (one virus), Potyviridae (one virus), Bromoviridae (one virus), Virgaviridae (six viruses), ‘Fusagraviridae’ (five viruses), and Ourmiavirus (four viruses). Two viruses are closely related to two families, Potyviridae and Bromoviridae, which previously contained no mycovirus species. Moreover, nine novel viruses associated with M.phaseolina were identified in the family Totiviridae, Endornaviridae, and Partitiviridae. Coinfection with multiple viruses is prevalent in M.phaseolina, with each isolate harboring different numbers of viruses, ranging from three to eighteen. Furthermore, the effects of the viruses on the fungal host were analyzed according to the biological characteristics of each isolate. The results suggested that M.phaseolina hypovirus 2, M.phaseolina fusagravirus virus 1-5 (MpFV1-5), M.phaseolina endornavirus 1-2 (MpEV1-2), M.phaseolina ourmia-like virus 1-3 (MpOLV1-3), M.phaseolina mitovirus 4 (MpMV4), and M.phaseolina mycobunyavirus 1-4 (MpMBV1-4) were only detected in hypovirulent isolates. Those viruses associated with hypovirulence might be used as biological control agents as an environmentally friendly alternative to chemical fungicides. These findings considerably expand our understanding of mycoviruses in M.phaseolina and unvailed the presence of a huge difference among viruses in isolates from different hosts in distant geographical regions. Together, the present study provides new knowledge about viral evolution and fungus-virus coevolution.

Competitive saprophytic ability of the hypovirulent isolate QT5-19 of Botrytis cinerea and its importance in biocontrol of necrotrophic fungal pathogens

This study determined the competitive saprophytic ability (CSA) of the hypovirulent isolate QT5-19 of Botrytis cinerea on potato dextrose agar (PDA) at 20 °C. Meanwhile, importance of the QT5-19 CSA in biocontrol against virulent isolates of B. cinerea and Sclerotinia sclerotiorum (a close relative of B. cinerea), and the mechanisms for the QT5-19 CSA were elucidated. The results showed that QT5-19 had higher CSA than the virulent isolates 08168 (B. cinerea) and EP-1PNA367 (S. sclerotiorum) on potato dextrose agar. The hyphal fragments of QT5-19 exhibited an effective suppression against infection of leaves of oilseed rape by the hyphae of the two virulent fungal pathogens through competition. QT5-19 was detected to be able to produce volatile organic compounds (VOCs) with antifungal activity against B. cinerea and S. sclerotiorum, whereas the VOCs from the virulent isolate RoseBc-3 of B. cinerea had no detectable antifungal activity. The QT5-19 VOCs effectively reduced disease severity of strawberry fruit rot caused by two virulent fungal pathogens. These results suggest that QT5-19 may achieve success of competition through extensive mycelial growth and production of antifungal volatiles. This study provided an example of using hypovirulent isolates to control virulent isolates of B. cinerea and S. sclerotiorum.

Hypovirulent effect of the Cryphonectria hypovirus 1 in British isolates of Cryphonectria parasitica.

Chestnut blight, caused by Cryphonectria parasitica, is controlled in many European countries by the naturally occurring mycovirus Cryphonectria hypovirus 1 (CHV-1). During surveys of recently identified chestnut blight outbreak in England, CHV-1 was detected in several individuals of the pathogen isolated from affected trees. We investigated two of these CHV-1-infected isolates (L-6 and Db-1) as potential biocontrol agents for deployment in the UK comparing their virulence against virus-free (M1275) and hypovirulent (M784) European isolates by inoculating sweet chestnut seedlings. Both the European CHV-1 M784 hypovirulent isolate and UK L-6 isolate formed significantly smaller lesions in sweet chestnut seedling bark than the other three isolates (Db-1, and virulent isolates FTC121 and M1275). The highest virus concentration was detected in isolate M784, followed by L-6, with the lowest concentration in isolate Db-1. White colony colouration indicative of hypovirulence was common in colonies re-isolated from smaller lesions, and the same isolates also tended to be slower growing in culture, have a higher virus concentration, and caused less epicormic growth and fewer stromata to be present in plants. L-6 and Db-1 virus sequences, respectively, matched the virus haplotype E-5 detected previously in Switzerland and a mutation of the same subtype I haplotype. Isolate L-6 could potentially act as biocontrol for chestnut blight outbreaks in the UK but further laboratory and field experiments are needed. © 2019 Crown copyright. Pest Management Science © 2019 Society of Chemical Industry.

Isolation, Identification and the Abilities of Fungi Associated with Agarwood from Bangka Belitung Island to Induce Agarwood Compounds

Agarwood is one of the non-timber forest products that have high economic value. Agarwood is widely used to make incense, perfume and other products. Sapwood on agarwood is a group of secondary metabolites of agarwood plants that form a lump and have a certain color and aroma. The fragrant aroma of sapwood on agarwood is formed due to pathogenic infection of the agarwood tree. Until now, most studies of fungi forming sapwood are only oriented to virulent pathogenic fungi in nature and have never been reported to form hypovirulent agarwood. This study aimed to evaluate the potential of fungi originating from sapwood on agarwood especially those that are hypovirulent in inducing sesquiterpene compounds. This study included exploration, isolation, identification, and induction of fungi associated with sapwood on agarwood from four districts in the Bangka Belitung Islands Province. Hypovirulence test in cucumber sprouts and sesquiterpene induction test on agarwood plantlets was conducted in the laboratory. Based on the isolation results, 48 fungal isolates associated with sapwood on agarwood were found: fungi of genus Fusarium, Trichoderma, Aspergillus, Penicillium, Curvularia, Peniophora, and six isolates were unidentified. Based on the hypovirulence test on cucumber sprouts, 46 isolates of the fungus 46 isolates were virulent and 2 isolates were hypovirulent (isolates 4A and 17A). The induction of sesquiterpene compounds on the agarwood plant was employed using 5 sample isolates consisting of 4 virulent isolates (2A, 7A, 18A, and 25A) and 1 hypovirulent isolate (Isolate 4A). The results showed that hypovirulent isolates were able to produce sesquiterpenes even in small amounts compared with virulent isolates. Isolates produced many sesquiterpene compounds were isolates 18A (Fusarium sp.). Sesquiterpene compounds formed were pinene, terpineol, patchouli alcohol, trimethyl-naphthalene, beta-caryophyllene, camphor, eugenol, trimethyl- benzene, phenanthrene, citronella, eucalyptol, 4-hydroxy-4-methyl-2-pentanone. In this study also found fungi associated with sapwood on agarwood which had never been reported by previous researchers, Peniophora sp. (isolate 25A).

Genetic Diversity of Cryphonectria hypovirus 1, a Biocontrol Agent of Chestnut Blight, in Croatia and Slovenia.

Transmissible hypovirulence associated with Cryphonectria hypovirus 1 (CHV1) has been used for biological control of chestnut blight, devastating disease of chestnut caused by the fungus Cryphonectria parasitica. The main aims of this study were to provide molecular characterization of CHV1 from Croatia and Slovenia and to reveal its genetic variability, phylogeny, and diversification of populations. Fifty-one CHV1 haplotypes were detected among 54 partially sequenced CHV1 isolates, all belonging to Italian subtype (I). Diversity was mainly generated by point mutations while evidence of recombination was not found. The level of conservation over analyzed parts of ORF-A proteins p29 and p40 varied, but functional sites were highly conserved. Phylogenetic analysis revealed close relatedness and intermixing of Croatian and Slovenian CHV1 populations. Our CHV1 isolates were also related to Swiss and Bosnian hypoviruses supporting previously suggested course of CHV1 invasion in Europe. Overall, this study indicates that phylogeny of CHV1 subtype I in Europe is complex and characterized with frequent point mutations resulting in many closely related variants of the virus. Possible association between variations within CHV1 ORF-A and growth of the hypovirulent fungal isolates is tested and presented.

Enhanced hypovirus transmission by engineered super donor strains of the chestnut blight fungus, Cryphonectria parasitica, into a natural population of strains exhibiting diverse vegetative compatibility genotypes

Horizontal transmission of virulence attenuating hypoviruses of Cryphonectria parasitica is restricted by an allorecognition system termed vegetative incompatibility (vic). A super donor formulation of two engineered C. parasitica strains (SD328/SD82) with gene disruptions at four of six vic loci transmitted hypovirus to strains in the laboratory independent of vic genotype. We now report the transmission of hypovirus by the SD328/82 formulation to a diverse, natural C. parasitica population infecting American chestnut in a forest setting. Hypovirulent (HV) isolates were recovered from 94% of cankers treated with the hypovirus-infected SD328/82 formulation compared to 51% of cankers treated with a hypovirus-infected EU5/6 formulation (strains having the same vic genotypes as SD strains but lacking vic gene disruptions). Overall, the SD328/82 formulation transmitted hypovirus into more divergent vic genotypes compared to the EU5/6 formulation. These results demonstrate the SD328/82 formulation can serve as an enhanced hypovirus vector for highly divergent C. parasitica populations.

Changes in Cryphonectria parasitica Populations Affect Natural Biological Control of Chestnut Blight.

Invasive species, especially plant pathogens, have a potential to completely eradicate native plant species and remodel landscapes. Tripartite interactions among sweet chestnut (Castanea sativa), chestnut blight-causing invasive fungus Cryphonectria parasitica, and hyperparasitic virus Cryphonectria hypovirus 1 (CHV1) were studied in two populations. The number of different vegetative compatibility (vc) types of C. parasitica more than doubled over the 10 years, while the hypovirulence incidence dropped in one population and slightly increased in the other one. Over the course of our 3-year monitoring experiment, the prevalence of hypovirulent isolates obtained from monitored cankers increased slowly (i.e., more hypovirulent isolates were being obtained from the same cankers over time). Within studied cankers, considerable changes in vc type and CHV1 presence were observed, indicating a highly dynamic system in which virulent and hypovirulent mycelia, sometimes of discordant vc types, often appeared together. The increase in hypovirulence prevalence did not have any observable curative effect on the cankers and, occasionally, reactivation of healed cankers by new, virulent C. parasitica isolates was observed. Both short- and long-term observations and revalidation of the infected plant populations are necessary to accurately estimate disease progress and formulate an adequate disease management strategy.

Phenotypic and Molecular Investigations on Hypovirulent Cryphonectria parasitica in Italy.

Chestnut blight is caused by the fungus Cryphonectria parasitica. As one of the most ecologically important diseases of Castanea spp., C. parasitica can rapidly kill trees. In Europe, mitigation of disease severity took place spontaneously through colonization of C. parasitica by mycoviruses, which reduced the virulence of the fungus. In the framework of a survey, 138 C. parasitica isolates were identified, and virulent/hypovirulent phenotypes were determined through morphological properties and pathogenicity tests. For a pool of four hypovirulent isolates, dsRNA was extracted, cDNA synthesized, and a library subjected to next-generation sequencing. The bioinformatics analysis allowed detecting and reconstructing the complete genome of Cryphonectria hypovirus 1 (CHV-1), denoted as CHV-1 Marche. When compared with the available genomes of other hypoviruses that affected the virulence of C. parasitica, available in databases, CHV-1 Marche showed some nucleotide diversity. The approach used in this study was effective to explore the virome inside a pool of hypovirulent C. parasitica isolates. Next-generation sequencing allowed us to exclude the presence of any other ssRNA and dsRNA viruses infecting the fungus and determine CHV-1 as the only responsible of hypovirulence of C. parasitica in the analyzed samples.

BIOLOGICAL AND MOLECULAR STUDIES ON FUSARIUM WILT DISEASE OF TOMATO

Tomato (Solanum lypersicum Mill.) is ranked number 1 among fruits and vegetables with 14% of the total production worldwide (Food & Agriculture Organization of the United Nations, FAO, 2003). Fusarium wilt disease is only responsible for 10 to 50% of these losses (Lukyanenko 1991). In Egypt, the losses due to tomato wilt disease reached up to 67% (Lasheen 2009). Different Fusarium oxysporum isolates were tested under greenhouse conditions to determine their virulence potential toward tomato plants. Severity and development of Fusarium wilt disease was recorded 4, 6 and 8 weeks after pathogen inoculation. All the tested isolates were pathogenic and varied significantly with regard to their virulence potential on tomato under the test conditions. The ability of a Fusarium isolate to infect particular plant species dependent on specific genes encoding host determining ‘virulence factors’ that distinguish virulent from avirulent strains. Eight fungal proteins were identified from xylem sap of infected plants, encompassing the small secreted proteins called SIX1, Six2, Six3, Six4, Six5, Six6, Six7 and SIX 8. The results showed that the tested isolates were varied regarding to their pathogenicity potential toward tomato plants under greenhouse conditions. On the other hand, the amplicons of SIX1, SIX5 and SIX 7 were detected with most hyper virulent isolates while no amplicons for any tested SIX genes were observed with the nonpathogenic isolate (Fo162) as well as with the most hypo virulent isolates. These results, suggested that SIX1, SIX5 and SIX6 may play a distinct role in virulence potential of these Fusarium oxysporum isolates toward tomato plants under Egyptian conditions .

Occurrence of hypovirus-infected Cryphonectria parasitica isolates in northern Spain: an encouraging situation for biological control of chestnut blight in Asturian forests

The presence of hypovirulent (hypovirus-infected) isolates of the chestnut blight fungus Cryphonectria parasitica in Asturias, northern Spain, is first reported by using both morphological and molecular assessments. Hypovirulence was detected in 16 of the 69 councils where the fungus was previously observed. In total 30 of the 863 C. parasitica isolates analyzed were found to be hypovirus-infected. The isolates belonged to the two main vegetative compatibility (vc) types (EU-1 and EU-13) of the region. Asturian hypoviruses were genetically characterized by sequencing of ORFA and ORFB specific regions of the hypoviral RNA. The results show that 70% of the investigated hypovirulent isolates contained the Spanish subtype (subtype E) while 30% isolates the German subtype (D) of Cryphonectria hypovirus 1 (CHV-1). The presence of hypovirulent isolates compatible with the dominant vc types and the low vc diversity in this region favours the application of biological control. An initial disease management effort could be applied by using the hypovirus of subtype D, whereas further research related to the biocontrol properties of the subtype E hypovirus should be conducted.

Natural dissemination of hypovirulent Cryphonectria parasiticastrain used for biological control of chestnut blight

Chestnut blight, caused by Cryphonectria parasitica Murr. Barr, is the most serious disease of chestnut trees in Turkey on which several studies related to biological control were conducted. The aim of this study was to determine the cause of the occurrence of healing cankers on untreated chestnut trees and the role of carriers in the spread of hypovirulence by laboratory tests, and to discover the effect of some environmental factors (temperature and relative humidity) on the natural spreading of hypovirulence. The study was carried out in a chestnut grove where biological control studies of the disease had been performed in previous years. Bark samples were collected from healed cankers and 388 possibly hypovirulent isolates were obtained. Two of them that were white converted virulent isolates to hypovirulent ones, but others developed yellowish cream-colored mycelia and did not convert virulent isolates. dsRNA analysis was performed on randomly selected possible hypovirulent isolates collected from healed cankers. After the virus concentration had increased, dsRNA-positive reactions were obtained for 56.73% of the isolates. Thirty-one possible hypovirulent isolates from Formicidae and Gastropoda did not convert virulent isolates to hypovirulent ones. dsRNA analysis was performed on these isolates and 61.29% of them containing dsRNA were observed in the invertebrates collected from cankers that had healed from hypovirulence. It was also observed that there was no significant difference among the date of average temperatures and date of relative humidity from 3 different altitudes. It was determined that the reason for the occurrence of the healed cankers on untreated trees was the natural dissemination of hypovirulence, ants and snails might have played a role in the natural dissemination of hypovirulence, and healed cankers mostly occurred on the western sides of shaded slopes.

Characterization of the Mycelial Compatibility Groups and Mating Type Alleles in Populations of Sclerotinia minor in Central China.

Ninety-five single-sclerotium isolates were obtained from lettuce and weeds in three counties in central China. They were identified belonging to Sclerotinia minor based on colony morphology and the S. minor-specific DNA marker. Mycelial compatibility groups (MCGs) and the mating type (MAT) alleles in these isolates were determined using the methods of paired cultures and specific PCR, respectively, and the MCG data were used to calculate Shannon's H index (H) and Simpson index (S), thereby evaluating diversity of S. minor. Eight MCGs (MCG1 to MCG8) and two MAT alleles (Inv+, Inv-) were identified in these isolates. Low diversity was detected for the total 95 isolates (H = 1.748, S = 0.786). Isolates of different MCGs or with different MAT alleles did not significantly differ (P > 0.05) in mycelial growth rate on potato dextrose agar (PDA, 20°C) or lesion diameter on lettuce leaves (20°C), but slightly differed in the number of sclerotia produced on PDA (20°C). Furthermore, this study reported five new host plants of S. minor in China, including Capsella bursa-pastoris, Oenanthe javanica, Fragaria gracilis, Ranunculus ternatus, and Salvia plebeia, and identified three hypovirulent isolates. These results broaden our understanding about the population biology of S. minor.

Co-infection of a hypovirulent isolate of Sclerotinia sclerotiorum with a new botybirnavirus and a strain of a mitovirus.

BackgroundSclerotinia sclerotiorum, a notorious plant fungal pathogen, causes yield loss of many crops and vegetables, and is a natural host of a diverse viruses with positive-sense RNA (+ssRNA), negative-sense RNA (−ssRNA), double-stranded RNA (dsRNA), or DNA genomes. Mixed-infection with multiple related or unrelated mycoviruses is a common phenomenon in S. sclerotiorum. However, a single strain co-infected with dsRNA and + ssRNA viruses has not been reported in S. sclerotiorum.ResultsWe report two unrelated viruses, Sclerotinia sclerotiorum botybirnavirus 2 (SsBRV2) with a bipartite dsRNA genome and Sclerotinia sclerotiorum mitovirus 4 (SsMV4/AH16) with a + ssRNA genome, which were originally detected in a single hypovirulent strain AH16 of S. sclerotiorum. SsMV4/AH16 has a typical genome of mitovirus and is a strain of mitovirus SsMV4. The genome of SsBRV2 consists of two separated dsRNA segments. The large dsRNA segment is 6159 bp in length and only has a single open reading frame (ORF) encoding a putative 1868-aa polyprotein with a conserved RNA dependent RNA polymerase (RdRp) domain. The small dsRNA segment is 5872 bp in length and encodes a putative 1778-aa protein. Phylogenetic analysis using RdRp conserved domain sequences revealed that SsBRV2 is phylogenetically related to the previously reported three bipartite viruses SsBRV1, Botrytis porri RNA virus 1 (BpRV1), and soybean leaf-associated botybirnavirus 1 (SlaBRV1). Electron microscopy demonstrated that SsBRV2 forms rigid spherical virions with a diameter of approximately 40 nm in infected mycelia. The virion of SsBRV2 was successfully introduced into a virus-free strain, which provides conclusive evidence that SsBRV2 confers hypovirulence on phytopathogenic fungus S. sclerotiorum.ConclusionsA bisegmented dsRNA virus (SsBRV2/AH16) and a nonsegmented + ssRNA virus (SsMV4/AH16) were characterized in a hypovirulent strain AH16 of S. sclerotiorum. SsMV4/AH16 is a strain of a reported mitovirus, whereas SsBRV2 is a new botybirnavirus. SsBRV2 is the causal agent of hypovirulence on S. sclerotiorum. Our findings supplied a first evidence that a single S. sclerotiorum strain is co-infected by dsRNA and + ssRNA mycoviruses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0550-2) contains supplementary material, which is available to authorized users.