Disease Management in the Genomics Era-Summaries of Focus Issue Papers.

The genomics revolution has contributed enormously to research and disease management applications in plant pathology. This development has rapidly increased our understanding of the molecular mechanisms underpinning pathogenesis and resistance, contributed novel markers for rapid pathogen detection and diagnosis, and offered further insights into the genetics of pathogen populations on a larger scale. The availability of whole genome resources coupled with next-generation sequencing (NGS) technologies has helped fuel genomics-based approaches to improve disease resistance in crops. NGS technologies have accelerated the pace at which whole plant and pathogen genomes have become available, and made possible the metagenomic analysis of plant-associated microbial communities. Furthermore, NGS technologies can now be applied routinely and cost effectively to rapidly generate plant and/or pathogen genome or transcriptome marker sequences associated with virulence phenotypes in the pathogen or resistance phenotypes in the plant, potentially leading to improvements in plant disease management. In some systems, investments in plant and pathogen genomics have led to immediate, tangible benefits. This focus issue covers some of the systems. The articles in this focus issue range from overall perspective articles to research articles describing specific genomics applications for detection and control of diseases caused by nematode, viral, bacterial, fungal, and oomycete pathogens. The following are representative short summaries of the articles that appear in this Focus Issue .

Preparation of Mealybugs (Hemiptera: Pseudococcidae) for Genetic Characterization and Morphological Examination.

Mealybugs (Hemiptera: Pseudococcidae) are economically significant agricultural pests on many different crops. Because of their small size and lack of easily visible characters for identification, determination of their taxonomic status is difficult and requires technical competency to prepare a slide-mounted specimen. The standard mounting technique does not allow for analysis of the genome of the specimen. Conversely, preparatory techniques for genetic analysis of mealybugs cause either loss of the entire individual or physical damage that can make morphology-based identification difficult. This study describes a simple protocol that does not impact physical integrity of the specimen for fixation and microscopic examination yet enables simultaneous DNA extraction for DNA-based identification of four mealybug species. All species prepared yielded high quality slide mounts, identified as Planococcus citri Risso, Pseudococcus viburni Signoret, Rhizoecus kondonis Kuwana, or Rhizoecus californicus Ferris. DNA extracted in this manner had higher purity and yield in the final eluate than in samples extracted using standard methods. DNA extracted was successfully amplified by polymerase chain reaction using primers for the cytochrome oxidase I gene and subsequently sequenced for all specimens. This protocol is likely to be applicable to other Hemiptera taxa that are preserved by slide mounting, allowing for both the preparation of a high-quality voucher specimen for morphological identification and simultaneous analysis of DNA for the same specimen. The methods used are technically less challenging than current standard procedures.

Genetically engineered virus-resistant plants in developing countries: current status and future prospects.

Plant viruses cause severe crop losses worldwide. Conventional control strategies, such as cultural methods and biocide applications against arthropod, nematode, and plasmodiophorid vectors, have limited success at mitigating the impact of plant viruses. Planting resistant cultivars is the most effective and economical way to control plant virus diseases. Natural sources of resistance have been exploited extensively to develop virus-resistant plants by conventional breeding. Non-conventional methods have also been used successfully to confer virus resistance by transferring primarily virus-derived genes, including viral coat protein, replicase, movement protein, defective interfering RNA, non-coding RNA sequences, and protease, into susceptible plants. Non-viral genes (R genes, microRNAs, ribosome-inactivating proteins, protease inhibitors, dsRNAse, RNA modifying enzymes, and scFvs) have also been used successfully to engineer resistance to viruses in plants. Very few genetically engineered (GE) virus resistant (VR) crops have been released for cultivation and none is available yet in developing countries. However, a number of economically important GEVR crops, transformed with viral genes are of great interest in developing countries. The major issues confronting the production and deregulation of GEVR crops in developing countries are primarily socio-economic and related to intellectual property rights, biosafety regulatory frameworks, expenditure to generate GE crops and opposition by non-governmental activists. Suggestions for satisfactory resolution of these factors, presumably leading to field tests and deregulation of GEVR crops in developing countries, are given.

Biology and Molecular Characterization of Cucurbit leaf crumple virus, an Emergent Cucurbit-Infecting Begomovirus in the Imperial Valley of California.

Cucurbit leaf crumple virus (CuLCrV) is an emergent and potentially economically important bipartite begomovirus first identified in volunteer watermelon plants in the Imperial Valley of southern California in 1998. Field surveys indicated that CuLCrV has become established in the Imperial Valley; and field plot studies revealed that CuLCrV primarily infects cucurbits, including cantaloupe, squash, and watermelon. Full-length DNA-A and DNA-B clones of an Imperial Valley isolate of CuLCrV were obtained by polymerase chain reaction (PCR) with overlapping primers. These clones were infectious in various cucurbits and common bean (cv. Topcrop); symptoms included stunted growth and leaf crumple, curl, and chlorosis. CuLCrV was not sap-transmissible, and immunolocalization and DNA in situ hybridization studies revealed that it is phloem-limited. A CuLCrV agroinoculation system was generated, and host range studies revealed differential susceptibility in cucurbits, with squash, watermelon, cantaloupe, and honeydew melon being most to least susceptible, respectively. Germplasm screening studies identified a number of resistant cantaloupe and honeydew melon cultivars. The genome organization of this CuLCrV isolate (CuLCrV-CA) is similar to other bipartite begomoviruses, and phylogenetic analysis placed CuLCrV in the Squash leaf curl virus (SLCV) cluster of New World bipartite begomoviruses. A CuLCrV-specific PCR test was developed which allows for differentiation from other begomoviruses, including SLCV.

The N-terminus of the Begomovirus nuclear shuttle protein (BV1) determines virulence or avirulence in Phaseolus vulgaris.

The BV1 gene of the bipartite Begomovirus genome encodes a nuclear shuttle protein (NSP) that is also an avirulence determinant in common bean. The function of the NSP of two common bean-infecting bipartite begomoviruses, Bean dwarf mosaic virus (BDMV) and Bean golden yellow mosaic virus (BGYMV), was investigated using a series of hybrid DNA-B components expressing chimeric BDMV and BGYMV NSP, and genotypes of the two major common bean gene pools: Andean (cv. Topcrop) and Middle American (cvs. Alpine and UI 114). BDMV DNA-A coinoculated with HBDBG4 (BDMV DNA-B expressing the BGYMV NSP) and HBDBG9 (BDMV DNA-B expressing a chimeric NSP with the N-terminal 1 to 42 amino acids from BGYMV) overcame the BDMV resistance of UI 114. This established that the BDMV NSP is an avirulence determinant in UI 114, and mapped the domain involved in this response to the N-terminus, which is a variable surface-exposed region. BDMV DNA-A coinoculated with HBDBG10, expressing a chimeric NSP with amino acids 43 to 92 from BGYMV, was not infectious, revealing an essential virus-specific domain. In the BGYMV background, the BDMV NSP was a virulence factor in the Andean cv. Topcrop, whereas it was an avirulence factor in the Middle American cultivars, particularly in the absence of the BGYMV NSP. The capsid protein (CP) also played a gene pool-specific role in viral infectivity; it was dispensable for infectivity in the Andean cv. Topcrop, but was required for infectivity of BDMV, BGYMV, and certain hybrid viruses in the Middle American cultivars. Redundancy of the CP and NSP, which are nuclear proteins involved directly or indirectly in viral movement, provides a masking effect that may allow the virus to avoid host defense responses.

The Lettuce infectious yellows virus (LIYV)-encoded P26 is associated with plasmalemma deposits within LIYV-infected cells.

Cytological, immunological, and mutagenesis approaches were used to identify the viral factors associated with the formation of plasmalemma deposits (PLDs) in whole plants and protoplasts infected by Lettuce infectious yellows virus (LIYV). Transmission electron microscopy and immunogold labeling using polyclonal antibodies to four of the five LIYV RNA 2-encoded large proteins, capsid protein (CP), minor capsid protein (CPm), HSP70 homolog (HSP70h), and P59, showed specific labeling of LIYV virions or virion aggregates around the vesiculated membranous inclusions, but not PLDs in LIYV-infected Nicotiana benthamiana, Nicotiana clevelandii, Lactuca sativa, and Chenopodium murale plants, and Nicotiana tabacum protoplasts. In contrast, antibodies to the RNA 2-encoded P26 showed specific labeling of PLDs but not virions in both LIYV-infected plants and protoplasts. Virion-like particles (VLPs) were seen in protoplasts infected by all LIYV RNA 2 mutants except for the CP (major capsid protein) mutant. PLDs were more difficult to find in protoplasts, but were seen in protoplasts infected by the CP and CPm mutants, but not in protoplasts infected by the P26, HSP70h, or P59 mutants. Interestingly, although the CPm mutant showed VLPs and PLDs, the PLDs did not show associated virions/virion-like particles as was always observed for PLDs seen in protoplasts infected by wild-type LIYV. Immunoblot analyses performed on purified LIYV virions showed that P26 was not detected with purified virions, but was detected in the cell wall, 1000 g and 30,000 g pellet fractions of LIYV-infected plants. These data suggest that P26 is associated with the LIYV-induced PLDs, and in contrast to the other RNA 2-encoded large proteins, P26 is not a virion protein.

Functional analysis of proteins involved in movement of the monopartite begomovirus, Tomato yellow leaf curl virus.

The functional properties of proteins [capsid protein (CP), V1, and C4] potentially involved with movement of the monopartite begomovirus, Tomato yellow leaf curl virus (TYLCV), were investigated using microinjection of Escherichia coli expressed proteins and transient expression of GFP fusion proteins. The TYLCV CP localized to the nucleus and nucleolus and acted as a nuclear shuttle, facilitating import and export of DNA. Thus, the CP serves as the functional homolog of the bipartite begomovirus BV1. The TYLCV V1 localized around the nucleus and at the cell periphery and colocalized with the endoplasmic reticulum, whereas C4 was localized to the cell periphery. Together, these patterns of localization were similar to that of the bipartite begomovirus BC1, known to mediate cell-to-cell movement. However, in contrast to BC1, V1 and C4, alone or in combination, had a limited capacity to move and mediate macromolecular trafficking through mesophyll or epidermal plasmodesmata. Immunolocalization and in situ PCR experiments, conducted with tomato plants at three stages of development, established that TYLCV infection was limited to phloem cells of shoot apical, leaf, stem, and floral tissues. Thus, the V1 and/or C4 may be analogs of the bipartite begomovirus BC1 that have evolved to mediate TYLCV movement within phloem tissue.

Bean dwarf mosaic virus BV1 protein is a determinant of the hypersensitive response and avirulence in Phaseolus vulgaris.

The capacities of the begomoviruses Bean dwarf mosaic virus (BDMV) and Bean golden yellow mosaic virus (BGYMV) to differeBean dwarf mosaic viru certain common bean (Phaseolus vulgaris) cultivars were used to identify viral determinants of the hypersensitive response (HR) and avirulence (avr) in BDMV. A series of hybrid DNA-B components, containing BDMV and BGYMV sequences, was constructed and coinoculated with BDMV DNA-A (BDMV-A) or BDMVA-green florescent protein into seedlings of cv. Topcrop (susceptible to BDMV and BGYMV) and the BDMV-resistant cvs. Othello and Black Turtle Soup T-39 (BTS). The BDMV avr determinant, in bean hypocotyl tissue, was mapped to the BDMV BV1 open reading frame and, most likely, to the BV1 protein. The BV1 also was identified as the determinant of the HR in Othello. However, the HR was not required for resistance in Othello nor was it associated with BDMV resistance in BTS. BDMV BV1, a nuclear shuttle protein that mediates viral DNA export from the nucleus, represents a new class of viral avr determinant. These results are discussed in terms of the relationship between the HR and resistance.

A New Bipartite Geminivirus (Begomovirus) Causing Leaf Curl and Crumpling in Cucurbits in the Imperial Valley of California.

During fall 1998, volunteer watermelons (Citrullus lunatus L. (Thunb.) Matsum. & Nakai) showing leaf curl, crumpling, and yellowing symptoms were found in a commercial honeydew melon (Cucumis melo L. subsp. melo Inodorus group) field in the Imperial Valley of California. The plants were infected with a begomovirus (family Geminiviridae, genus Begomovirus) based on (i) a positive response in squash blots probed with a general begomovirus DNA probe (1) and (ii) amplification of DNA-A (≈1.2 kb) and DNA-B (≈1.4 kb) fragments by polymerase chain reaction (PCR) with degenerate DNA-A (PAL1v1978/PAR1c496) and DNA-B (PBL1v2040/PBR1c970) primers, respectively (3). The DNA-A and -B fragments were cloned and sequenced (GenBank accession nos. AF224760 [DNA-A] and AF224761 [DNA-B]). The DNA-A and -B fragments had a nearly identical (99.5%) common region (CR) of 186 (DNA-A) and 187 (DNA-B) nucleotides, indicating they were from the same begomovirus. Database searches conducted with these sequences revealed no high degree of sequence identity (i.e., >90%) with other begomoviruses, including Squash leaf curl virus (SqLCV [2]) from southern California. The partial AC1 sequence (669 nt) was most identical to Tomato severe leaf curl virus (ToSLCV) from Guatemala (83%) and SqLCV (81%), the partial AV1 sequence (135 nt) was most identical to Tomato golden mosaic virus from Brazil (84%) and SqLCV (81%), and the CR was most identical to Squash yellow mottle virus from Costa Rica (81%), ToSLCV (81%), and SqLCV (77%). The partial BV1 sequence (465 nt) was most identical to Bean calico mosaic virus and SqLCV (72%), and the partial BC1 sequence (158 nt) was most identical to SqLCV (75%). Watermelon seedlings bombarded with a DNA extract from infected watermelon volunteers developed crumpling and distortion symptoms, whereas seedlings bombarded with gold particles alone developed no symptoms. Geminivirus infection in symptomatic seedlings was confirmed by PCR. These results suggest a new begomovirus caused the disease symptoms in the watermelon volunteers. Leaf crumpling and curling symptoms were not observed in spring melons in the Imperial Valley in 1999, but on 2 July and 17 August 1999, cantaloupe (C. melo L. subsp. melo Cantalupensis group), muskmelon (C. melo L. subsp. melo Cantalupensis group), and watermelon plants with leaf crumpling and yellowing were found. These plants were infected with the new begomovirus based on sequence analysis of PCR-amplified DNA-A fragments (97 to 98% identity for CR and partial AC1 sequence). A survey of fall melons, conducted 23 to 24 September 1999, revealed widespread symptoms of leaf curl and crumpling on new growth of muskmelon plants in all seven commercial fields examined (estimated incidence 25 to 50%) and on watermelon volunteers. No such symptoms were observed on leaves of honeydew melons. Symptomatic muskmelon and watermelon leaves, collected from eight locations throughout the Imperial Valley, were infected with the new begomovirus based on sequence analysis of PCR-amplified DNA-A fragments. Thus, a new begomovirus has emerged in the Imperial Valley; the name Cucurbit leaf crumple virus (CuLCrV) is proposed. References: (1) R. L. Gilbertson et al. Plant Dis. 75: 336, 1991. (2) S. G. Lazarowitz and I. B. Lazdins. Virology 180:58, 1991. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Bean golden yellow mosaic virus from Chiapas, Mexico: Characterization, Pseudorecombination with Other Bean-Infecting Geminiviruses and Germ Plasm Screening.

ABSTRACT The complete nucleotide (nt) sequences of the cloned DNA-A (2644 nts) and DNA-B (2609 nts) components of Bean golden yellow mosaic virus (BGYMV-MX) from Chiapas, Mexico were determined. The genome organization of BGYMV-MX is similar to that of other Western Hemisphere bipartite geminiviruses (genus Begomovirus). Infectivity of the cloned BGYMV-MX DNA components in common bean (Phaseolus vulgaris) plants was demonstrated by particle bombardment and agroinoculation. BGYMV-MX was identified as a BGYMV (previously type II BGMV) isolate based on sequence analyses, sap-transmissibility, and pseudorecombination experiments with other bean-infecting begomoviruses. On the basis of differences in the DNA-B hypervariable region, symptom phenotype, and properties of infectious pseudorecombinants, BGYMV-MX may represent a distinct strain of BGYMV. Pseudorecombination experiments further established that BGYMV symptom determinants mapped to DNA-B, and that BGYMV-MX was most closely related to BGYMV from Guatemala. A Tomato leaf crumple virus (TLCrV) DNA-A/BGYMV-MX DNA-B pseudorecombinant was infectious in bean, establishing that a viable reassortant can be formed between begomovirus species from different phylogenetic clusters. Bean germ plasm representing the two major gene pools (Andean and Mesoamerican) was screened for response to BGYMV-MX with three methods of inoculation: sap-inoculation, particle bombardment, and agroinoculation. Andean germ plasm was very susceptible and similar results were obtained with all three methods, whereas Mesoamerican germ plasm showed resistance to BGYMV-MX, particularly with agroinoculation.

Nucleotide sequence of both genomic RNAs of a North American tobacco rattle virus isolate.

The complete sequence of a North American tobacco rattle virus (TRV) isolate, 'Oregon yellow' (ORY), was determined from cDNA and RT-PCR clones derived from the two genomic RNAs of this isolate. The RNA-1 is 6790 bases and RNA-2 is 3261 bases. The sequence of TRV-ORY RNA-1 was similar to RNA-1 to TRV isolate SYM, and differs in 48 nucleotides. TRV-ORY RNA-1 was one base shorter than--SYM, and had 47 base substitutions resulting in 12 amino acid substitutions of which 4 were conservative. The RNA-2 of TRV-ORY was distinct from RNA-2 of other characterized TRV isolates and contained three open reading frames (ORFs) that could potentially code for proteins of MW 22.4 kDa, 37.6 kDa and 17.9 kDa. Based on the homology of the predicted amino acid sequence with those of other tobraviruses. ORF1 of RNA-2 encodes the coat protein (CP). The protein sequence of ORF2 had regions of limited similarity with those of ORF2 of two other TRV isolates and pea early browning tobravirus. The ORF3 was unique to TRV-ORY. Phylogenetic analysis of tobravirus CPs indicated that TRV-ORY was most closely related to pepper ringspot tobravirus and TRV-TCM. The relationship of tobravirus CPs to other rod-shaped tubular plant viruses is also discussed.

Penicillinase-based enzyme-linked immunosorbent assay for the detection of plant viruses.

A penicillinase (PNC)-based, enzyme-linked immunosorbent assay (ELISA) was standardized to detect maize mosaic virus (MMV) in sorghum leaf extracts, peanut mottle virus (PMV) in pea leaf extracts, and tomato spotted wilt virus (TSWV) in peanut leaf extracts. Rabbit Fc-specific antibodies were conjugated with PNC by a single step glutaraldehyde bridge. Among several indicators tested, bromothymol blue (BTB) was found suitable for measuring PNC activity under simulated conditions. Two reagents, starch-iodine complex (SIC) and a mixed pH indicator, containing bromocresol purple and BTB (2:1) used earlier for the PNC-based ELISA, were compared with BTB for utilization in the PNC-based ELISA. SIC gave a slightly higher virus titre than BTB or the mixed pH indicator, but it often gave nonspecific reactions. Sodium or potassium salts of penicillin-G at 0.5-1.0 mg/ml and BTB at 0.2 mg/ml were found to be suitable as substrate-indicator mixture for PNC-based ELISA. The sensitivity of the PNC system was comparable to those of the alkaline phosphatase (ALP) and horseradish peroxidase (HRP) systems in detecting MMV, PMV, and TSWV. The PNC conjugate could be used at a greater dilution than those of the ALP and HRP conjugates and the BTB substrate mixture was stable for at least 3 weeks at 4 degrees C. Penicillin is readily available in developing countries, and at a substantially lower cost than p-nitrophenyl phosphate, the commonly used substrate for ALP in the plate ELISA. Thus the PNC-based ELISA provides a less expensive means for assaying plant viruses by ELISA.