Development of Primers and Probes for Genus and Species Specific Detection of 'Candidatus Liberibacter Species' by Real-Time PCR.

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease impacting citrus production. The disease is associated with three different 'Candidatus Liberibacter species', 'Ca. Liberibacter asiaticus', 'Ca. Liberibacter americanus', and 'Ca. Liberibacter africanus', which induce similar and overlapping symptoms. When HLB-symptomatic trees are tested, one of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. The 16S rRNA-based assays to detect the three species are species-specific and must be performed sequentially. We describe a single assay that detected all species of 'Ca. Liberibacter' at the genus level, providing increased convenience. Recent molecular analyses of 'Ca. Liberibacter species' and other bacteria suggest that the rpoB gene (encoding the ß-subunit of RNA polymerase) provides an alternative target for bacterial identification. We report here the design of a single pair of degenerate primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three citrus 'Ca. Liberibacter species', enabling detection of 'Ca. Liberibacter' at the genus level. In addition, species-specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus- and species-specific assays were validated in both SYBR Green I and TaqMan formats, and with both plant and insect extracts that contained the pathogen. These one-step qPCR diagnostic methods are useful for the detection of all species of Liberibacter infecting citrus. In addition, the degenerate genus-specific primers and probe successfully detected 'Ca. Liberibacter solanacearum', a psyllid-transmitted pathogen associated with disease in tomato, carrot, and potato.

Stem-Pitting Citrus tristeza virus Predominantly Transmitted by the Brown Citrus Aphid from Mixed Infections Containing Non-Stem-Pitting and Stem-Pitting Isolates.

Citrus tristeza virus (CTV) is a phloem-limited Closterovirus that produces a variety of symptoms in various Citrus spp. One of these symptoms is stem pitting (SP). SP does not occur in all Citrus spp. but when it does it may cause low tree vigor, decline, and an economic reduction in fruit size and yield. Historically, the first appearance of CTV-SP in a citrus area often occurs after the introduction of the most efficient CTV vector, the brown citrus aphid (BCA), Toxoptera citricida. Hypotheses for this association range from the introduction of these strains in new planting materials to the increased ability of BCA to transmit SP strains from existing CTV sources. It is known that CTV often exists as a complex of isolates or subisolates. Single and multiple BCA transmissions have been used to separate different genotypes or strains of CTV from mixed CTV infected plants. This study was initiated to determine what the BCA transmits when an exotic severe SP CTV isolate B12 from Brazil or B408 from Dominican Republic are mixed with a non-SP (NSP) isolate, FS627 from Florida. Biological and molecular data was generated from grafted mixtures of these isolates and their aphid-transmitted subisolates. Single-strand conformation polymorphism patterns of the 5' terminal region of open reading frame (ORF) 1a, the overlapping region of ORF1b and ORF2, and the major coat protein gene region of NSP and SP CTV-grafted plants remained unchanged but the patterns of doubly inoculated plants varied. The haplotype diversity within SP isolates B12, B408, and mixtures of NSP and SP isolates (FS627/B12 and FS627/B408) and aphid-transmitted subisolates from doubly inoculated plants was determined by analysis of the haplotype nucleotide sequences. Aphid transmission experiments, symptoms, and molecular analyses showed that SP-CTV was more frequently transmitted with or without NSP-CTV from mixed infections.

Acquisition and Transmissibility of U.S. Soybean dwarf virus Isolates by the Soybean Aphid, Aphis glycines.

Soybean dwarf virus (SbDV) exists as several distinct strains based on symptomatology, vector specificity, and host range. Originally characterized Japanese isolates of SbDV were specifically transmitted by Aulacorthum solani. More recently, additional Japanese isolates and endemic U.S. isolates have been shown to be transmitted by several different aphid species. The soybean aphid, Aphis glycines, the only aphid that colonizes soybean, has been shown to be a very inefficient vector of some SbDV isolates from Japan and the United States. Transmission experiments have shown that the soybean aphid can transmit certain isolates of SbDV from soybean to soybean and clover species and from clover to clover and soybean with long acquisition and inoculation access periods. Although transmission of SbDV by the soybean aphid is very inefficient, the large soybean aphid populations that develop on soybean may have epidemiological potential to produce serious SbDV-induced yield losses.

Murraya paniculata and Related Species as Potential Hosts and Inoculum Reservoirs of 'Candidatus Liberibacter asiaticus', Causal Agent of Huanglongbing.

Huanglongbing (HLB), considered to be the most serious insect-vectored bacterial disease of citrus, is transmitted in nature by the Asian citrus psyllid Diaphorina citri and the African citrus psyllid Trioza erytreae. D. citri was discovered in southern Florida in 1998 and the HLB disease in 2005. Both have become established throughout citrus-producing areas of Florida. Murraya species are widely grown in southern Florida as ornamental hedges and are readily colonized by D. citri vectors. Colonies of D. citri, isolates of 'Candidatus Liberibacter asiaticus' from Taiwan and Florida, and the Murraya species were established in the BSL-3 biosecurity facility at Fort Detrick. In controlled inoculation experiments, D. citri transmitted 'Ca. L. asiaticus' into M. paniculata (34/36 plants) and M. exotica (22/23 plants), but not into Bergera (Murraya) koenigii. Disease symptoms rarely developed in Murraya plants; however, positive infections were determined by conventional and real-time polymerase chain reaction (PCR). Back-inoculations of 'Ca. L. asiaticus' from M. paniculata to Madam Vinous sweet orange resulted in disease development in 25% of the inoculated plants. Considerable variability was observed in infection rates, titer, and persistence of 'Ca. L. asiaticus' in infected Murraya.

Cultivation of 'Candidatus Liberibacter asiaticus', 'Ca. L. africanus', and 'Ca. L. americanus' associated with huanglongbing.

A new medium designated Liber A has been designed and used to successfully cultivate all three 'Candidatus Liberibacter spp.,' the suspect causative agents of huanglongbing (HLB) in citrus. The medium containing citrus vein extract and a growth factor sustained growth of 'Ca. Liberibacter spp.' for four or five single-colony transfers before viability declined. Colonies, positive for 'Ca. L. asiaticus' by a 16s-based rDNA real-time polymerase chain reaction (RT-PCR) assay and sequencing, were irregular-shaped, convex, and 0.1 to 0.3 mm after 3 to 4 days. Suspect 'Ca. L. asiaticus' and 'Ca. L. americanus' cells were observed in infected tissue and on agar culture by scanning electron microscopy. The cells were ovoid to rod shaped, 0.3 to 0.4 by 0.5 to 2.0 microm, often with fimbriae-like appendages. Two strains of 'Ca. L. asiaticus' and one of 'Ca. L. americanus' grown on Liber A medium were pathogenic on citrus and could be isolated from noninoculated tissues of inoculated trees and seedlings 9 and 2 months later, respectively. The identity was confirmed by RT-PCR and 16s rDNA sequencing. This is the first report of the cultivation and pathogenicity of 'Ca. L. asiaticus' and 'Ca. L. americanus' associated with symptoms of HLB.

Prunus Host Range of Plum pox virus (PPV) in the United States by Aphid and Graft Inoculation.

Plum pox (Sharka) is a serious virus disease of stone fruits caused by the Plum pox virus (PPV). To determine which species could function as potential hosts and virus reservoirs, we used aphid transmission and bud or chip grafting to evaluate the susceptibility of commercial, ornamental, and wild Prunus species to isolates of PPV found in Pennsylvania, USA. Following inoculation, test trees were observed for symptoms, analyzed by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), back-assayed to healthy peach, and followed through at least four cold-induced dormancy (CID) cycles over 4 years. Thirty-one of 33 Prunus species and cultivars were systemically infected following aphid transmission. Systemic infection could not be detected in P. cerasus (sour cherry) and P. × 'Snofozam' (Snow Fountains) despite repeated aphid inoculation attempts. Following grafting of PPV-infected budwood, all 40 species and varieties became infected, although species differed in their susceptibility. Within most species, some individual plants remained PPV negative throughout the study despite repeated inoculations. Infection in some species could be detected only through quantitative reverse transcription (RT)-PCR. Most species displayed clear symptoms, were highly positive by ELISA and RT-PCR, and could be back-inoculated into peach seedlings following CID. Our results indicate that a wide range of native and ornamental Prunus species are susceptible to U.S. isolates of PPV-D.

An assessment model for rating high-threat crop pathogens.

ABSTRACT Natural, accidental, and deliberate introductions of nonindigenous crop pathogens have become increasingly recognized as threats to the U.S. economy. Given the large number of pathogens that could be introduced, development of rapid detection methods and control strategies for every potential agent would be extremely difficult and costly. Thus, to ensure the most effective direction of resources a list of high-threat pathogens is needed. We address development of a pathogen threat assessment model based on the analytic hierarchy process (AHP) that can be applied world-wide, using the United States as an illustrative example. Previously, the AHP has been shown to work well for strategic planning and risk assessment. Using the collective knowledge of subject matter expert panels incorporated into commercial decision-making software, 17 biological and economic criteria were determined and given weights for assessing the threat of accidental or deliberately introduced pathogens. The rating model can be applied by experts on particular crops to develop threat lists, especially those of high priority, based on the current knowledge of individual diseases.

Transmission of Xylella fastidiosa, Causal Agent of Citrus Variegated Chlorosis, by the Glassy-Winged Sharpshooter, Homalodisca coagulata.

Citrus variegated chlorosis (CVC) is an economically important disease of citrus in Brazil and Argentina. The causal pathogen is a strain of Xylella fastidiosa transmitted by several sharpshooter species. The glassy-winged sharpshooter (GWSS), Homalodisca coagulata, has become an important new pest of citrus and grapevines in California, where it transmits X. fastidiosa strains to several crops including grapes, oleander, and almonds. Transmission studies over a 3-year period at the USDA BSL3-P containment facility at Fort Detrick, MD, utilizing California field-collected GWSS, a Brazilian strain of CVC, and Madam Vinous sweet orange seedlings, have shown a consistent although low level of transmission of CVC. Test plants were observed for CVC symptoms, analyzed by polymerase chain reaction using species-specific primers for X. fastidiosa, membrane entrapment immunofluorescence, and scanning electron microscopy. X. fastidiosa was not detected in field-collected GWSS but was detected in GWSS following feeding on CVC-infected source plants. Transmission of the CVC strain of X. fastidiosa by GWSS increases the risk of establishment of CVC in the United States if it were introduced.

Molecular Analyses of Citrus tristeza virus Subisolates Separated by Aphid Transmission.

Citrus tristeza virus (CTV) exists in field isolates as a complex of virus isolates. This complex may contain both mild and severe CTV. Using single and multiple aphid transmissions, subiso-lates of the various field isolates were separated. Some CTV isolates that tested negative with the monoclonal antibody MCA13 consisted of MCA13-positive subisolates. Using primers to specific and variable regions of the CTV genome, molecular profiles of the isolates and subisolates were generated and compared. The profiles of the subisolates sometimes were very different from the parent field isolates from which they were transmitted.

Transmission of the Citrus Variegated Chlorosis Bacterium Xylella fastidiosa with the Sharpshooter Oncometopia nigricans.

Citrus variegated chlorosis (CVC) is an economically important, destructive disease in Brazil and is caused by the bacterium Xylella fastidiosa Wells. The bacterium has been found to be transmitted in Brazil by sharpshooter leafhoppers (Cicadellidae). Sharpshooters are present in most citrus growing areas of the United States. The sharpshooter leafhopper, Oncometopia nigricans Walker, frequently is found feeding on citrus in Florida. This sharpshooter transmits the X. fastidiosa strains that cause Pierce's disease of grape and ragweed stunt. Research was initiated to determine if O. nigricans was capable of vectoring the X. fastidiosa that causes CVC. In 59 different transmission tests, using 1 to 57 insects per test, transmission of the bacterium was observed 12 times (20.3%). Symptom development in the greenhouse was not a reliable indicator of transmission. Transmission was verified by specific polymerase chain reaction-based assays. Individual insects were able to transmit the bacterium. This information on sharpshooter transmission of CVC is needed to assess the threat posed by the CVC disease to the citrus industries in the United States.

Histology of Sweet Orange Stem Pitting Caused by an Australian Isolate of Citrus tristeza virus.

Some strains of the Citrus tristeza virus (CTV) cause stem pitting in sweet orange (Citrus sinensis (L.) Osbeck). This abnormality causes tree decline and reduction in fruit size and yield of affected citrus trees. Stem-pitting symptoms can occur on trunks, on all sizes of limbs, and on the twigs where fruit are produced. Variously sized pits or grooves in the wood often contain a yellow gum. Irregular growth of the phloem occurs in the area of these xylem pits. The histology of stem pitting caused by an Australian CTV isolate was studied in sweet orange using light and electron microscopy. Using scanning electron microscopy, details of the wood pits containing the gumming material were revealed. In thin sections of bark tissue, outgrowths of the phloem tissue were found at various intervals that corresponded to the pits in the wood. Higher numbers of viral inclusions were detected in the phloem outgrowths than were present in the other sieve elements.

Identification, characterization, and relatedness of luteovirus isolates from forage legumes.

ABSTRACT Virus isolates from forage legumes collected from eight different states were identified as luteoviruses closely related to soybean dwarf luteovirus dwarfing (SbDV-D) and yellowing (SbDV-Y) described in Japan. All isolates produced reddened leaf margins in subterranean clover and were transmitted in a persistent manner by Acrythosiphon pisum, but not by Aulacorthum solani. Specific monoclonal antibodies raised against SbDV-Y were differentially reactive with endemic isolates. Immunoblots probed with a SbDV-D polyclonal antiserum showed single 26-kDa coat protein bands, confirming close serological relatedness to SbDV. Analyses of genomic and subgenomic double-stranded RNAs and northern blot analyses confirmed genomic relatedness to SbDV. Based on our results, we conclude that the U.S. luteovirus isolates studied comprise a strain or strains of the soybean dwarf virus that have clovers as common hosts and the pea aphid as a common vector.

Sequence and expression in Escherichia coli of the coat protein gene of the dwarfing strain of soybean dwarf luteovirus.

The nucleotide sequence of the coat protein gene of the dwarfing (D) strain of soybean dwarf luteovirus (SbDV) was determined from cloned cDNA. The gene contains 600 nucleotides and encodes a protein of 200 amino acids with a calculated molecular mass of 22.2 kDa. A major portion of the coat protein open reading frame (ORF) was expressed in Escherichia coli as a pET fusion protein and the product was detected by western blot analysis using SbDV-D polyclonal antibodies. Comparison of the deduced coat protein amino acid sequence to that from the yellowing (Y) strain of SbDV demonstrated 88% identity.

Occurrence and Partial Characterization of a New Mechanically Transmissible Virus in Mashua from the Ecuadorian Highlands.

Mashua (Tropaeolum tuberosum) is a tuber crop indigenous to the Andean highlands and of economic value to its native people as a food and medicinal crop. Field plants often exhibit symptoms typical of virus infection, including interveinal chlorosis, leaf cupping, distortion, and stunting. Using an herbaceous host range, at least 1 mechanically transmissible virus was detected in each of 10 accessions. All virus isolates tested reacted to potyvirus group antiserum in indirect enzyme-linked immunosorbent assays (ELISA). Results of monoclonal antibody testing using antigen coated plate (ACP) ELISA, where virions are disrupted to expose cryptotopes, indicated that isolate Tt 4 has epitopes in common with maize dwarf mosaic potyvirus A (MDMV-A). Polyclonal antiserum produced to purified preparations of the Tt 4 isolate reacted in indirect ELISA to Tt 4, the common strain of potato potyvirus Y (PVY-C), less strongly to tobacco etch potyvirus (TEV) and potato potyvirus A (PVA), but not to 11 other potyviruses, including MDMV-A. Conversely, the Tt 4 isolate reacted strongly in indirect ELISA to antisera to bean common mosaic potyvirus (BCMV) and watermelon mosaic potyvirus 2 (WMV-2), but not antisera to 12 other potyviruses. Our data suggest that isolate Tt 4 is a distinct potyvirus closely related to and sharing external epitopes with PVY-C, TEV, PVA, WMV-2, and BCMV, and buried epitopes with MDMV-A. The virus has been tentatively named Tropaeolum mosaic potyvirus (TropMV). A survey of T. tuberosum in Ecuador revealed that TropMV was widespread, being detected by ACP-ELISA in 34 of 46 accessions at the National Institute for Agricultural Research's Santa Catalina Research Station, Quito, and all eight production fields surveyed.

Prunus tomentosa as a Diagnostic Host for Detection of Plum Pox Virus and Other Prunus Viruses.

The efficacy of seedlings of Prunus persica cv. GF 305, P. persica cv. Siberian C, and P. tomentosa (Nanking Cherry) as diagnostic indicators of plum pox infection, and of P. tomentosa for other Prunus viruses was evaluated by graft-inoculation with eight different strains or isolates of plum pox virus (PPV) representative of the Marcus (M) and Dideron (D) serogroups; and one isolate each of Prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), and sour cherry green ring mottle virus (GRMV). The initial PPV symptoms that developed in P. tomentosa within 30 days after inoculation were chlorotic banding along the midrib spreading to lateral veins from the leaf base upward, giving the appearance of a chlorotic oak-leaf pattern. Symptoms caused by PPV-M could be distinguished from those caused by PPV-D. Virus titers in infected P. tomentosa and GF 305 were higher than those in Siberian C when measured by triple antibody sandwich enzyme-linked immunosorbent assay. Infections by PNRSV, PDV, and GRMV were evident with the first flush of vegetative growth.

Nucleotide sequence and E. coli expression of the coat protein gene of the yellowing strain of soybean dwarf luteovirus.

The nucleotide sequence of the coat protein gene of the yellowing (Y) strain of soybean dwarf virus (SbDV) was determined from cloned cDNA. The gene contains 600 nucleotides and encodes a protein of 200 amino acids with a calculated M(r) of 22,200. The identity of the open reading frame (ORF) encoding the coat protein was confirmed by expression of an Escherichia coli beta-galactosidase fusion protein and detection by a dot blot immunoassay. Sequence comparisons of the deduced coat protein amino acids to several luteoviruses demonstrated that the SbDV-Y coat protein ORF shares greatest similarity with bean leaf roll virus (BLRV) (65%).

Maize streak virus genes essential for systemic spread and symptom development.

The entire genome of single component geminiviruses such as maize streak virus (MSV) consists of a single-stranded circular DNA of ~2.7 kb. Although this size is sufficient to encode only three average sized proteins, the virus is capable of causing severe disease of many monocots with symptoms of chlorosis and stunting. We have identified viral gene functions essential for systemic spread and symptom development during MSV infection. Deletions and gene replacement mutants were created by site-directed mutagenesis and insertion between flanking MSV or reporter gene sequences contained in Agrobacterium T-DNA derived vectors. Following Agrobacterium-mediated inoculation of maize seedlings, the mutated MSV DNAs were excised from these binary vectors by homologous recombination within the flanking sequences. Our analyses show that the capsid gene of MSV, while not required for replication, is essential for systemic spread and subsequent disease development. The ;+' strand open reading frame (ORF) located immediately upstream from the capsid ORF and predicted to encode a 10.9 kd protein was also found to be dispensable for replication but essential for systemic spread. By this analysis, MSV sequences that support autonomous replication were localized to a 1.7 kb segment containing the two viral intergenic regions and two overlapping complementary ;-' strand ORFs. Despite the inability of the gene replacement mutants to spread systemically, both inoculated and newly developed leaves displayed chlorotic patterns similar to the phenotype observed in certain developmental mutants of maize. The similarity of the MSV mutant phenotype to these developmental mutants is discussed.