Studying Sclerotinia sclerotiorum-infecting viruses collected from Saskatchewan for their potential role in disease control
Term: 4 years, beginning 2024
Status: Ongoing
Researcher(s): Sean Prager, U of S
SaskCanola Investment: $313,375
Total Project Cost: $626,750
Funding Partners: WGRF
Background
1. Identification and detection of mycoviruses present in Sclerotinia sclerotiorum (SS) collected from Saskatchewan.
2. Characterization of viruses for their potential biocontrol activities against SS.
3. Developing an efficient and effective mass production protocol for mycoviruses exhibiting biocontrol activity.
Project Description
Efficient control methods are necessary to protect the Prairies crops from Sclerotinia sclerotiorum.
SS causes significant damage to sunflower, flax, alfalfa, and other pulses. Alfalfa seed production fields in the Canadian Prairies have been experiencing epidemics of Blossom blight caused by SS since 1993, with occurrences being common in most years. SS is also common in dry bean fields and disease surveys conducted in Alberta showed incidents of white mould disease caused by SS in up to 100% of surveyed fields in some years. In dry beans, under non-irrigated conditions, every single unit percentage increase in the disease incidence is estimated to reduce yield by 7 to 26 kg/ha. Some canola and pulses do show some level of resistance or tolerance, but effective disease control still needs fungicide application.
Biological control is appealing for the control of plant diseases as it is acceptable for conventional/organic cultivation and can have a minimal negative impact on producers, consumers, and the environment. Currently, two biological control agents (Contans and Serenade Opti) are available for sclerotinia stem rot control in Canada. Serenade Opti contains the bacteria Bacillus subtilis which stimulates plant defence mechanisms and suppresses disease. Contans contains an obligate mycoparasite (Coniothyrium minitans) that infects the SS fungus and controls the disease.
Mycoviruses (fungi-infecting viruses) can affect fungal growth, reproduction, infection and fungal establishment in their host and can be used as biocontrol agents against plant-pathogenic fungi. Nearly 20% of the mycoviruses are estimated to cause deleterious effects on fungi and reduce fungal virulence (causes hypovirulence) and therefore these viruses have the potential to be used as biocontrol agents to control plant pathogenic fungi. For example, in early 1950, a fungal pathogen Cryphonectria parasitica, a causative agent for chestnut blight, devastated the European Chestnut and Oak species. However, an epidemic of chestnut blight disease slowed in Europe due to the emergence of a hypovirulent strain of C. parasitica. Further studies showed that a mycovirus Cryphonectria hypovirus 1 (CHV1) is responsible for the hypovirulence of C. parasitica. However, the application of the hypovirulent C. parasitica for the control of chestnut blight in North America provided little success which highlights the need for a better understanding of complex virusfungus- plant-environment interactions for the development of successful control strategies.
The potential of mycoviruses in controlling SS disease was studied as early as 1990 in Canada.
Dr. Boland's group from the University of Guelph reported a hypovirulent SS isolate from soil samples collected in the USA that exhibited the presence of double-stranded RNA (dsRNA), a hallmark of the RNA virus. The group showed the transmission of the hypovirulence to wildtype isolates and reported the presence of double-membrane bodies in the infected cells during microscopy suggesting the presence of unencapsidated mycovirus or mycovirus-like agents. Similarly, another hypovirulent isolate of SS collected from sunflowers in Manitoba exhibited the presence of dsRNA. Although the hypovirulence trait is transmissible to wildtype isolates, the dsRNA, in this case, is not shown to be associated with hypovirulence as the dsRNA-free progeny also showed hypovirulence. Unfortunately, previous studies on hypovirulence and associated dsRNA were not further examined possibly due to the technical challenges associated with the extraction, purification, and identification of mycoviruses in the past.
The SS isolate characterized by Dr. Boland's group showed slower growth and atypical colony morphology that differed from the other wildtype isolates. In comparison to the wildtype SS, the hypovirulent isolate produced 64% smaller lesions. The hypovirulence trait can be transmitted into a virulent isolate during hyphal anastomosis and the new hypovirulent isolate produces an 80% smaller lesion. Nevertheless, the group deemed this hypovirulent isolate unsuitable for its use in biocontrol activity against SS due to the higher number of mycelial compatibility groups that existed in SS. However, Dr. Boland's group showed suppression of dollar spot on turfgrass caused by Sclerotinia homeocarpa (Sh), another species of Sclerotinia, in both lab and field conditions. Suppression of the disease upon application of the hypovirulent isolate of the Sh was observed within 10 days post inoculation and the disease suppression was still significant after a year suggesting their rapid as well as prolonged efficacy.
Until recently, all known mycoviruses lacked an extracellular phase capable of infecting fungal cells by passing through the rigid cell wall and they were considered endogenous viruses. Mycoviruses including the CHV1, lack an extracellular phase and strictly depend on transmission through sexual spores, asexual structures, or via hyphal fusion with a compatible fungal strain. The heterokaryon incompatibility (fungal non-self-recognition system) reduces the transmission of mycoviruses between incompatible fungal strains. SS is known to have diverse incompatibility groups that can significantly reduce virus transmission via hyphal anastomosis which possibly explains the declining interest in the study of mycoviruses for biocontrol agents. However, the identification of a new virus called Sclerotinia sclerotiorum hypovirulence associated DNA virus 1 (SsHADV-1), which is shown to have an extracellular route of transmission, has revived the research interest towards the identification and utilization of potential mycovirus against SS.
SsHAVD-1 shows many interesting characteristics suitable for a biocontrol agent.
The SsHADV-1 is a small (2.4 kb) circular single-stranded DNA virus with coding regions for a coat protein and a replication protein. It is the first and founding member of the family Genomoviridae, which now represents more than 230 species of viruses. SsHADV-1 was first isolated and characterized by a group working on a hypovirulence isolate of SS in Wuhan, China. Compared with the virus-free SS strain, the virus-infected SS strain produces smaller spores and takes a longer time to make sclerotinia. Under laboratory conditions, spraying of virus particles on plant leaves reduces the SS-induced lesions and protects the plant from the disease. A recent study showed the virus’s remarkable ability to downregulate SS pathogenicity factor genes and its ability to convert necrotrophic SS into a beneficial fungus. The SsHADV-1 infected SS induces genes involved in plant growth and enhances the expression of Brassica napus defence-related genes. Furthermore, wheat plants treated with the SsHADV-1 infected hypovirulent SS isolate showed an increased abundance of plant growth- promoting microorganisms in the soil rhizosphere and protected against Fusarium head blight and stripe rust disease. This provides additional benefits to the farmers who follow pulses/canola-wheat in their crop rotation schedule where remnants of hypovirulent SS from previous seasons’ application on SS-susceptible crops (pulses/canola) can increase the production of next year's wheat crop.
SsHADV-1 can infect Sclerotinia minora and S. nivalis in addition to SS but its host range is narrower and it can not infect other fungi from the same family including Coniothyrium minitants, an active ingredient of the biocontrol agent Contans. The virus cannot infect Botrytis cinerea, but B. napus plants inoculated with SsHADV-1 showed resistance against Botrytis cinerea under laboratory conditions. Particles of the SsHADV-1 virus remain stable on the leaf for more than two weeks. While the virus does not infect plants, it can infect the mushroom sciarid fly (Lycoriella ingenue) and this fly serves as a transmission vector for the virus. In addition, the virus can transmit between vegetatively incompatible strains of the fungus which allows the virus to spread between multiple SS strains in the field. The narrow host range of the virus, its stability in the environment, its ability to infect the host extracellularly and the use of a mycophagous insect as a vector for transmission are some of the beneficial attributes making SsHADV-1 suitable and well suited as a biocontrol agent.
Identification of known and novel viruses via Next generation sequencing (NGS)
Previously, studies from Canada showed dsRNA from hypervirulence isolates of SS but they were unable to characterize the viruses possibly due to the technical difficulties of extraction, purification, and characterization of these viruses. However, recent advancement in sequencing techniques together with the development of bioinformatics tools/techniques and the availability of public databases with a large number of virus sequences has significantly improved our ability to detect and characterize these viruses. Next-generation sequencing (NGS) provides an opportunity to sequence indiscriminately from the samples including the genome of intracellular pathogens such as mycoviruses. Metagenomic studies for the identification/detection of viruses can be carried out using total RNA/DNA, dsRNA or siRNA extracted from the infected samples or from purified virion-associated nucleic acids. Each of these methods has advantages and disadvantages; for example, the total RNA/DNA extracted from infected samples overwhelmingly has DNA/RNA from the host. This method can detect viruses that are present in a higher titer but can be difficult to use for viruses that remain at a lower concentration. Increasing sequencing depth can help but it increases the cost and amount of data for analysis. The other methods such as the sequencing of dsRNA or siRNA can target viral genomic sequences but require additional time, skills, and reagents for nucleic acid extraction and purification. Similarly, sequencing of dsRNA may not detect DNA viruses and negative-stranded RNA viruses which usually have low dsRNA accumulation.
The sequence information obtained from NGS can be further used to detect the presence of known viruses or these sequences can be further analyzed using bioinformatic tools to discover novel viruses. A number of such tools have been developed and they are publicly available. NGS of DNA/RNA extracted from fungi to identify the presence of viruses has been adapted for many plants' pathogenic/symbiotic fungi including SS.
Summary
Currently, there is no report of characterized SS-infecting mycoviruses from Canada, but previous reports showed the presence of dsRNA of unknown origin. The presence of hypovirulent strains in Canadian isolates of SS has been described in the literature but the details on the underlying mechanism are lacking. Diverse mycoviruses including the isolates of SsHADV-1 have been reported from China, Egypt, Australia, New Zealand, and the USA. Studies show the possibility of SsHADV-1 as a biocontrol agent but the presence of this virus and its effectiveness to control Saskatchewan isolates of SS is not known. In addition, the potential of other mycoviruses in controlling the pathogenicity of the SS or the ability of other mycoviruses to induce plant defence responses is lacking in the literature. Therefore, this study will explore the possibility of controlling one of the most destructive fungal pathogens of Saskatchewan, Sclerotinia sclerotiorum using mycoviruses isolated from Canada.