1,721,036 research outputs found
BIOLOGICAL AND MOLECULAR PROPERTIES OF POTATO VIRUS S (PVS) AND THE EFFECT OF PVS ON LATE BLIGHT RESISTANT POTATO GENOTYPES
Full text linkThesis (Ph.D.), Department of Plant Pathology, Washington State UniversityPotato (Solanum tuberosum) is one of the most important crops grown in Washington State. In 2011, Washington growers raised 160,000 acres of potatoes with an average yield of 615 CWT per acre, 98 CWT total, with a farm gate value of over $734 million. Late blight, caused by Phytophthora infestans, is an extremely devastating disease of potato worldwide. Defender is the only cultivar with foliar and tuber resistance to this disease in the U.S. Under field conditions, however, this cultivar exhibits high susceptibility to infection by Potato virus S (PVS, family Betaflexiviridae, genus Carlavirus ). My research reproduced this phenotype under controlled conditions and demonstrated the tuber/seed transmission and proved modified Koch's postulates.Department of Plant Pathology, Washington State Universit
Transcriptome analysis of capsicum chlorosis virus-induced hypersensitive resistance response in Bell capsicum
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Weed-infecting viruses in a tropical agroecosystem present different threats to crops and evolutionary histories.
Full text linkIn the Caribbean Basin, malvaceous weeds commonly show striking golden/yellow mosaic symptoms. Leaf samples from Malachra sp. and Abutilon sp. plants with these symptoms were collected in Hispaniola from 2014 to 2020. PCR tests with degenerate primers revealed that all samples were infected with a bipartite begomovirus, and sequence analyses showed that Malachra sp. plants were infected with tobacco leaf curl Cuba virus (TbLCuCV), whereas the Abutilon sp. plants were infected with a new bipartite begomovirus, tentatively named Abutilon golden yellow mosaic virus (AbGYMV). Phylogenetic analyses showed that TbLCuCV and AbGYMV are distinct but closely related species, which are most closely related to bipartite begomoviruses infecting weeds in the Caribbean Basin. Infectious cloned DNA-A and DNA-B components were used to fulfilled Koch's postulates for these diseases of Malachra sp. and Abutilon sp. In host range studies, TbLCuCV also induced severe symptoms in Nicotiana benthamiana, tobacco and common bean plants; whereas AbGYMV induced few or no symptoms in plants of these species. Pseudorecombinants generated with the infectious clones of these viruses were highly infectious and induced severe symptoms in N. benthamiana and Malachra sp., and both viruses coinfected Malachra sp., and possibly facilitating virus evolution via recombination and pseudorecombination. Together, our results suggest that TbLCuCV primarily infects Malachra sp. in the Caribbean Basin, and occasionally spills over to infect and cause disease in crops; whereas AbGYMV is well-adapted to an Abutilon sp. in the Dominican Republic and has not been reported infecting crops
Development of a universal RT-PCR assay for grapevine vitiviruses.
Full text linkThe genus Vitivirus in the family Betaflexiviridae includes eleven viruses known to infect grapevine: grapevine vitiviruses A, B, D, E, F, G, H, I, J, L and M (GVA-GVM). Three of these viruses, GVA, GVB and GVD, have been associated with the etiology of rugose wood disease in grapevine and cause agronomically significant losses. The other vitiviruses were more recently discovered and their effects on grapevine are undetermined. To certify grape material for propagation as virus tested, an updated reverse transcription PCR (RT-PCR) assay to detect all known vitiviruses is desirable. To accomplish this, multiple grapevine vitivirus sequences were aligned at the amino acid level to search for conserved motifs. Two highly conserved motifs were found at an ideal distance for RT-PCR detection in the RNA-dependent RNA polymerase region of the replicase protein. The amino acid motifs were back translated to create degenerate primers and used to successfully amplify all eleven grapevine vitiviruses. The RT-PCR primers were used to test a panel of vitivirus-infected vines for inclusivity as well as vines infected with closely related viruses in the Betaflexiviridae family (i.e. grapevine pinot gris virus and grapevine rupestris stem pitting-associated virus) for exclusivity. Broader use of these primers to detect vitiviruses in other plant hosts was investigated. In summary, an end-point RT-PCR assay that detects all the known grapevine vitiviruses and potentially other members of the genus Vitivirus has been developed. The universal assay represents an alternative to individual assays to reduce the work associated with the diagnosis of vitiviruses, including for regulatory purposes
BIOLOGICAL, EPIDEMIOLOGICAL AND MOLECULAR INSIGHTS INTO THRIPS-IRIS YELLOW SPOT TOSPOVIRUS PEST COMPLEX
Full text linkThesis (Ph.D.), Department of Plant Pathology, Washington State UniversityIris yellow spot tospovirus (IYSV) (genus Tospovirus, family Bunyaviridae), transmitted by Thrips tabaci L. causes an economically important disease in both onion bulb and seed crop in the USA and other onion-growing regions of the world. Onion thrips as a pest alone can cause up to >60 % crop loss. Besides Allium spp, several weeds were found to be hosts of IYSV.
IYSV isolates collected from different states in the USA were evaluated to determine the existence of biologically distinct strains. On the basis of the ability to cause systemic infection, disease severity, senescence and death of the inoculated plants, isolates were delineated as mild or severe isolates.
Since the genome structure of only the small (S) RNA of IYSV was known, the large (L) and medium (M) RNAs of the virus were sequenced. The L RNA was 8,880 nucleotides in length, coding the 331.17 kDa RNA-dependent RNA polymerase in the viral complementary (vc) strand. The M RNA was 4,817 nucleotides long coding the movement protein (34.7kDa) in the viral sense and the glycoprotein precursor (128.4 kDa) in the vc strand.
An ELISA protocol was developed for detecting IYSV in single adult thrips using a polyclonal antiserum produced against the nonstructural protein (NSs) coded by the small (S) RNA. The approach enabled estimating the proportion of viruliferous thrips among the field-collected thrips. This will help better understand the epidemiology of IYSV.
To understand the molecular basis of the emergence of new tospoviruses, a system was developed to study virus-virus interactions. It was found that two distinct and economically important tospoviruses, IYSV and Tomato spotted wilt virus (TSWV) complement each other to overcome host defense. The small RNA expression profiles of IYSV and TSWV in single-and dually-infected datura plants showed that systemic leaves of dually-infected plants had reduced levels of TSWV N gene-specific small interfering RNAs (siRNAs). This identifies a new role for the viral gene silencing suppressor in potentially modulating the biology and host range of viruses and underscores the important role of virally-coded suppressors of gene silencing in virus infection of plants.Department of Plant Patholog
GENOMIC CHARACTERIZATION AND MOLECULAR INVESTIGATIONS INTO NEGATIVE-STRANDED RNA VIRUSES OF PLANTS
Full text linkThesis (Ph.D.), Department of Plant Pathology, Washington State UniversityTospoviruses are economically important viruses affecting a wide range of field and horticultural crops worldwide. Tospoviruses contain large (L) RNA, medium (M) RNA and small (S) RNA. Sequence analysis of the NSs gene in S RNA and the deduced protein sequences revealed two amino acid motifs that are conserved. Using Tomato spotted wilt virus (TSWV) as a model, the role of these motifs in suppressor activity of NSs was investigated. Using site-directed point mutations in two conserved motifs, glycine, lysine and valine/threonine (GKV/T) and tyrosine and leucine (YL), and an assay to measure the reversal of gene silencing in Nicotiana benthamiana line 16c, I showed that substitutions in these motifs abolished suppressor activity of the NSs protein, indicating that these two motifs are essential for the suppressor function of tospoviruses. RNA silencing-based approaches are successful for resistance using short regions of the viral genome. A new artificial micro RNA approach to confer resistance to was developed by Prof Mitter in collaboration with Prof Pappu. The candidate TSWV sequences (21nt in length) were introduced into an Arabidopsis amiRNA backbone. The effect of amiRNA constructs to impart resistance to TSWV was evaluated using transient assays in N. benthamiana as well as in transformed N. tabacum plants. In general, amiRNA constructs targeting the N gene were more effective against TSWV infection, while those specific to the NSs gene were not. This approach should be broadly applicable to other tospoviruses as well as other viruses that defy control due to lack of host plant resistance. EMDV is a rhabdovirus and consists of a large single-stranded RNA in negative sense. To obtain the complete RNA genome sequence from infected Agapanthus plants, deep-sequencing was carried out and the resulting sequences were assembled. De novo assembly of contigs, along with RACE to obtain the terminal sequences, showed that the viral genome is of 13,100-nt length and had 85.6% identity with the known EMDV genome from Greece. Sequence analysis showed five conserved motifs in the L gene, which may be useful in developing virus-specific as well as genus-specific detection tools for virus diagnosis and management.Department of Plant Pathology, Washington State Universit
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Characterizing Douglas-fir tissue colonization by the "sudden oak death" pathogen, Phytophthora ramorum
Full text linkIn 2001, Koch's Postulates confirmed Douglas-fir as a host for P. ramorum. Naturallyinfected saplings have been observed in California forests, and studies on artificially inoculated Douglas-fir stems and shoots have established susceptibility parameters. In 2009, P. ramorum stem cankers were recently observed on 8-year old plantation grown Douglas-fir in Great Britain. These findings substantiate the importance of research to characterize the behavior of P. ramorum in Douglas-fir and assess the potential risk that this pathogen poses to Douglas-fir ecosystems. Goals of this research included determining which tissues are colonized by P. ramorum, whether woody tissues can support sporulation, the likelihood of stem infections occurring through intact bark, and pathogen viability in foliage. Methods included isolation, ELISA, and histology of infected stem tissues; RT-PCR and isolation to determine colonization of foliage, and baiting studies to determine the ability of bark to reduce colonization of Rhododendron leaves by the pathogen. ELISA showed that pathogen proteins were detectable in the phloem, cambium, and superficial xylem, with infrequent detection in asymptomatic tissues. The pathogen was not able to be isolated from non-discolored tissues. ELISA and isolation results were highly positively correlated (r2 =0.62, p=0.78), and histological observations supported results from these techniques. Bark reduced infection on leaf baits by up to 83%. Pathogen DNA was detectable in foliage using qPCR methods, but the pathogen could not be isolated. Evidence of sporulation in stem tissues was not observed. The pathogen may infect into the shallow xylem tissues of wounded Douglas-fir stems, but spore formation is not apparent. The lack of isolation from non-discolored tissues suggests that proteins detected by ELISA may be elicitins secreted in advance of hyphae. The inability to isolate from needles may indicate chemical inhibitors that render the pathogen non-viable subsequent to initial infection. The ability of Douglas-fir bark to suppress infection of leaf baits suggests that bark is inhibitory to P. ramorum and that stem infection through intact bark may be limited. Characterizing P. ramorum colonization of Douglas-fir is important for understanding host susceptibility and pathogen behavior to enhance our ability to assess risk and thwart the spread of this exotic pathogen
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Investigating virus-host and virus-virus interactions using Iris yellow spot virus and Tomato spotted wilt virus (Tospovirus ; Bunyaviridae) as models
Full text linkTospoviruses (Tospovirus; Bunyaviridae) account for major economic losses in terms of both yield quantity and quality in numerous field and horticultural crops worldwide. Sensitive molecular assays were developed to investigate virus-host and virus-virus interactions in planta using Iris yellow spot virus (IYSV), which causes localized infections, and Tomato spotted wilt virus (TSWV), which causes systemic infections, as model tospoviruses, with Datura stramonium as the model host. For virushost interactions, the distribution and accumulation of the nucleocapsid (N) gene of each virus were determined in individually inoculated D. stramonium. To investigate the interactions between the two viruses in the same host, the viruses were simultaneously inoculated and the distribution and levels of each virus determined. Quantitative real-time PCR showed that TSWV was capable of replicating in inoculated and spreading to all tested non-inoculated leaves. In contrast, the host restricted IYSV only to inoculated leaves; no IYSV N gene was detected in the noninoculated leaves. There was no change in terms of distribution or titer levels of TSWV in dually infected plants. However, in the presence of TSWV, IYSV overcame the host defense system and spread to some non-inoculated leaves. In addition, IYSV titers increased more than 1000-fold in inoculated leaves in the presence of TSWV. To further investigate if IYSV was exclusively dependent on TSWV gene products for movement, qPCR protocols were developed for analysis of IYSV movement protein (NSm) and silencing suppressor (NSs) gene expression. The IYSV NSs gene was detected in all the samples while NSm was only expressed in 8% of the samples. Overall, these results suggest that IYSV is only capable of overcoming D. stramonium defense mechanism in the presence of TSWV. A unilateral type of synergistic interaction is evident between IYSV and TSWV, and only the former benefits. However, at this stage it is not clear whether it is exclusively the TSWV NSm gene, or both NSs and NSm, that is required for IYSV movement. This interaction could have economic and epidemiological impact by significantly reducing yield and increasing vector transmission efficiency. The assays are useful in studying virus-host interactions at the molecular level
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TITER AND DISTRIBUTION OF HOP STUNT VIROID AND HOP LATENT VIROID INFECTING HOPS
Full text linkHop stunt viroid and Hop latent viroid have proven recalcitrant to attempts to eliminatethem. This project was undertaken to understand the localization of the two viroids within thehop cultivars, ‘Comet,’ ‘Chinook,’ ‘Canadian Red,’ and ‘Wuerttemberger’ under cold and darkstress with the goal of improving viroid elimination therapies. With micro-shoot tip culture,HSVd elimination was successful in 4% to 9% of regenerated meristems and HLVd eliminationwas successful 19% to 54% in ‘Comet’ and ‘Chinook,’ respectively. HLVd was not eliminated in‘Canadian Red’ and ‘Wuerttemberger.’ In situ hybridization showed that HSVd was present inthe ground meristem, but not the tunica, while HLVd was not present in the first ~500 μM in‘Comet’ and ‘Chinook.’ HLVd was present in the ground meristem of ‘Wuerttemberger.’ Thislocalization may explain the difficulty of eradicating the viroids from infected hops. Darktherapy increased viroid titers and is not advisable. Though cold therapy decreased viroid titers,meristems failed to regenerate. Unexpectedly, sourcing shoots from plants during spring thatexperienced a natural dormancy period showed an increase in HLVd elimination (ranging from50% to 100%) in cvs. ‘Cascade,’ ‘Centennial,’ ‘Triumph,’ and ‘Wuerttemberger.’ This increase inHLVd elimination, accompanied by a 13-fold increase in HSVd elimination, was observed in‘Comet’ plants that experienced an artificially induced spring
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THE EFFECT OF HEAT STRESS AND BACTERIA-DELIVERED PLANT ELICITORS ON PLANT IMMUNITY AGAINST ROOT-KNOT NEMATODES
Full text linkPlant-parasitic nematodes are one of the most economically important plant pathogens, especially for food crops in agriculture. Root-knot nematodes (RKNs) are especially destructive and are a prime focus of research for management strategies. The most common strategies for combating RKNs included nematicides and resistant crop cultivars. However, resistant cultivars may lose effectiveness at high temperatures and some commercial nematicides are being phased out due to health and environmental concerns. Our goal has been to look for novel means of nematode control and determining the effects of excessive heat on potato responses to RKNs. In terms of novel nematode control, it has been shown that bacteria can deliver plant elicitor peptides to the roots to prime plant immunity before nematode infection. Here we show that that Bacillus subtilis can be engineered to produce and secrete the tomato elicitor peptide, SlPep6. Application of this bacteria on tomato plant roots successfully reduced the galling caused by the RKN, Meloidogyne hapla. This bacteria delivery system of an elicitor activates defenses present in the host plant. Crop resistance is an established and effective management strategy for RKNs. However, there have been accounts of crop resistance breaking at high temperatures. This is concerning for growers who depend on resistant varieties to protect their crop plants, especially in light of rising temperatures due to climate change. The cultivated potato breeding line PA99N82-4 is normally resistant to the RKN, Meloidogyne chitwoodi race 1. PA99N82-4 potato plants exposed to excessive heat (36˚C) for 24 hours had significantly more M. chitwoodi galls on their roots than plants grown at 25˚C. This was also the case for plants exposed to three hours of heat (36˚C) for five consecutive days, mimicking realistic mid-day heat spikes that crop plants might undergo. This indicates that root resistance conferred in PA99N82-4 may not remain effective as temperatures rise. Priming plant immunity with transgenic bacteria as well as incorporating heat-stable resistance into crop cultivars are effective ways to diminish the damage caused by RKNs. These strategies should be considered for RKN management in order to better protect crop plants and improve global food security
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