Michael J. Melzer

Diversity and mealybug transmissibility of ampeloviruses in Pineapple

Mealybug wilt of pineapple (MWP) is one of the most destructive diseases of pineapple (Ananas comosus) worldwide. At least one Ampelovirus species, Pineapple mealybug wilt associated virus-2 (PMWaV-2), and mealybug feeding are involved in the etiology of MWP. A previously undescribed Ampelovirus sharing highest homology with PMWaV-1 and a putative deletion mutant sharing highest homology with PMWaV-2 were detected with reverse transcription-polymerase chain reaction (RT-PCR) assays using degenerate primers. Results were verified with additional sequence information and by immunosorbent electron microscopy. Sequence homology between the virus tentatively designated PMWaV-3, and PMWaV-1 and PMWaV-2, decreases toward the N-terminal across the HSP70 homolog, small hydrophobic protein, and RNA-dependent RNA polymerase open reading frames (ORF). Putative PMWaV-3 could not be detected with four different monoclonal antibodies specific for PMWaV-1 and PMWaV-2. The potential deletion mutant spanning the N-terminal of the HSP70 region was obtained from a pineapple accession from Zaire maintained at the USDA-ARS National Clonal Germplasm Repository in Hawaii. Putative PMWaV-3, like PMWaV-1 and PMWaV-2, is transmissible separately or in combination with other PMWaVs by Dysmicoccus brevipes and D. neobrevipes mealybugs. Plants infected with PMWaV-3 that were continuously exposed to mealybugs did not develop symptoms of MWP in the absence of PMWaV-2. Specific RT-PCR assays were developed for detection of putative PMWaV-3 and the deletion mutant.

Characterization of a Virus Infecting Citrus volkameriana with Citrus Leprosis-Like Symptoms

A Citrus volkameriana tree displaying symptoms similar to citrus leprosis on its leaves and bark was found in Hawaii. Citrus leprosis virus C (CiLV-C)-specific detection assays, however, were negative for all tissues tested. Short, bacilliform virus-like particles were observed by transmission electron microscopy in the cytoplasm of symptomatic leaves but not in healthy controls. Double-stranded (ds) RNAs ≈8 and 3 kbp in size were present in symptomatic leaf tissue but not in healthy controls. Excluding poly(A) tails, the largest molecule, RNA1, was 8,354 bp in length. The ≈3 kbp dsRNA band was found to be composed of two distinct molecules, RNA2 and RNA3, which were 3,169 and 3,113 bp, respectively. Phylogenetic analyses indicated that the RNA-dependent RNA polymerase (RdRp) domain located in RNA1 was most closely related to the RdRp domain of CiLV-C. A reverse-transcription polymerase chain reaction assay developed for the detection of this virus was used to screen nearby citrus trees as well as Hibiscus arnottianus plants with symptoms of hibiscus green spot, a disease associated with infection by Hibiscus green spot virus (HGSV). All nearby citrus trees tested negative with the assay; however, symptomatic H. arnottianus plants were positive. All three RNAs were present in symptomatic H. arnottianus and were >98% identical to the RNAs isolated from C. volkameriana. We contend that the virus described in this study is HGSV, and propose that it be the type member of a new virus genus, Higrevirus.

Diversity of double-stranded RNA viruses within populations of entomopathogenic fungi and potential implications for fungal growth and virulence

Naturally occurring isolates as well as sev- eral culture collection isolates of the entomopatho- genic fungi, Metarhizium anisopliae, Beauveria bassi- ana, and Metarhizium flavoviride were assessed for the presence of double-stranded RNA (dsRNA). DsRNA was present in the mycelium of 28 of 73 iso- lates (38.4%) of M. anisopliae and 2 of 12 isolates (16.7%) of B. bassiana collected from southern On- tario soil samples. DsRNA was also present in myce- lium of 2 of 3 isolates of M. anisopliae and 5 of 6 isolates of M. flavoviride obtained from culture col- lections. DsRNA banding patterns were variable and, in total, eighteen dsRNA banding patterns were iden- tified, containing from one to eight bands ranging from 5.2 to 0.5 kbp in size. Partially purified virion preparations from selected dsRNA-containing isolates provided similar banding patterns to dsRNAs extract- ed from mycelium. Comparison of 12 dsRNA-con- taining isolates with 10 dsRNA-free isolates indicated dsRNAs were not responsible for the large variation in growth rate, conidiospore production, and insect virulence observed in M. anisopliae. However, a com- parison between two isogenic strains, one with ds- RNA and one without, showed a significantly in- creased growth rate and conidiospore production on potato dextrose agar (PDA) amended with dodine, as well as significantly increased virulence, in the dsRNA-free isolate. These results indicate that dsRNA

Role Bending: Complex Relationships Between Viruses, Hosts, and Vectors Related to Citrus Leprosis, an Emerging Disease

Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.

Genetic algorithm approach for the closest string problem

A fundamental aspect of post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) is the requirement of sequence homology between the transgene and viral or messenger RNAs being targeted. For example, virus-resistant transgenic plants are resistant only to viruses that are closely related (i.e. high sequence homology) to the virus from which the transgene was derived. One idea for broadening this resistance is to devise an artificial sequence that incorporates the sequence variation found in a viral population. This requires an algorithm which can determine an artificial sequence with an optimal (or at least a 90-95% ) homology to all of the viral sequences in a population. The genetic algorithm (GA) presented in this paper serves this purpose. It should be of great value to all researchers who utilize PTGS or RNAi. In the context of coding theory, the task is to find the radius of a code S /spl sub/ {A, C, G, T} /sup n/. In computational biology this problem is commonly referred to as the closest string problem. Experimental results suggest that this NP-complete optimization problem can be approached well with a custom-built GA.

Genetic polymorphisms in three subtilisin-like protease isoforms (Pr1A, Pr1B, and Pr1C) from Metarhizium strains.

Restriction fragment length polymorphisms (RFLP) were examined in three isoforms of a gene family encoding subtilisin-like proteases (Pr1A, Pr1B, and Pr1C) in several isolates of the entomopathogenic fungus Metarhizium anisopliae. RFLP variation was not observed in any of the Pr1 genes from isolates within the same genetically related group. Between genetically related groups and between isolates from disparate geographical areas, the greatest variation in RFLP patterns was observed for Pr1A. When variation does occur at Pr1B and Pr1C, it was generally observed at an EcoRI site. Metarhizium anisopliae var. majus strain 473 and a M. flavoviride isolate were most dissimilar in RFLP patterns at all Pr1 genes when compared to the M. anisopliae strains. We suggest that Pr1 genes represent a gene family of subtilisin-like proteases and that the Pr1A gene encodes for the ancestral subtilisin-like protease which has subsequently duplicated and rearranged within the genome.

An assemblage of closteroviruses infects Hawaiian ti (Cordyline fruticosa L.)

The ti plant (Cordyline fruticosa L.) is culturally important throughout most of Polynesia and has considerable economic importance in Hawai’i where the foliage is commonly used in cultural ceremonies as well as food and ornamental industries. In Hawai’i, ringspot symptoms were recently observed on leaves of the common green variety of ti growing in Kahalu’u on the island of O’ahu, and Wailuku and Hana on the island of Maui. High molecular weight double-stranded (ds)RNAs were isolated from the leaves of symptomatic plants as well as plants without symptoms. A cDNA library derived from the dsRNAs present in symptomatic plants was generated and sequenced. These sequences indicated at least four distinct clostero-like viruses were present in the plants, and phylogenetic analyses suggested they were most closely related to Little cherry virus 1, an unassigned member of the family Closteroviridae. The 16,883 nucleotide genome of one of these viruses was determined and predicted to contain ten open reading frames with an organization typical of closteroviruses. Reverse-transcription PCR revealed this virus was present in both symptomatic and asymptomatic ti plants, making it unlikely to be responsible for the observed ringspot symptoms. We propose the name Cordyline virus 1 (CoV-1) for this virus and include it as a new, unassigned member of the family Closteroviridae.

Genetically related isolates of the entomopathogenic fungus Metarhizium anisopliae harbour homologous dsRNA viruses

Several isolates of the entomopathogenic Metarhizium anisopliae that harboured dsRNA viruses of similar electrophoretic band sizes (1.8 and 2.0 kbp) were assessed for homologies of the dsRNA by Northern analysis. The isolates were also characterised genetically by RAPD and VCG. Similarly sized dsRNA, as visualised by electrophoresis, were not always homologous, suggesting that the comparison of dsRNA based solely on electrophoretic banding patterns is an unreliable method of dsRNA characterisation. Several isolates, but not all, harbouring multiple dsRNA patterns, including a 1.8 and 2.0 kbp doublet, also showed homologies to strains harbouring only the dsRNA banding doublet. This suggests that mixed infections of different dsRNA elements are found in M. anisopliae. Genetically similar fungi, based on RAPD banding patterns and vegetative compatibility, were more likely to harbour genetically related dsRNA. The findings suggested that dsRNA elements in M. anisopliae are horizontally transferred to genetically related isolates or are maintained through clonal lineages.

Pineapple bacilliform CO virus: Diversity, Detection, Distribution, and Transmission

Members of the genus Badnavirus (family Caulimovirdae) have been identified in dicots and monocots worldwide. The genome of a pineapple badnavirus, designated Pineapple bacilliform CO virus-HI1 (PBCOV-HI1), and nine genomic variants (A through H) were isolated and sequenced from pineapple, Ananas comosus, in Hawaii. The 7,451-nucleotide genome of PBCOV-HI1 possesses three open reading frames (ORFs) encoding putative proteins of 20 (ORF1), 15 (ORF2), and 211 (ORF3) kDa. ORF3 encodes a polyprotein that includes a putative movement protein and viral aspartyl proteinase, reverse transcriptase, and RNase H regions. Three distinct groups of putative endogenous pineapple pararetroviral sequences and Metaviridae-like retrotransposons encoding long terminal repeat, reverse-transcriptase, RNase H, and integrase regions were also identified from the pineapple genome. Detection assays were developed to distinguish PBCOV-HI1 and genomic variants, putative endogenous pararetrovirus sequences, and Ananas Metaviridae sequences also identified in pineapple. PBCOV-HI1 incidences in two commercially grown pineapple hybrids, PRI 73-114 and PRI 73-50, was 34 to 68%. PBCOV-HI1 was transmitted by gray pineapple mealybugs, Dysmicoccus neobrevipes, to pineapple.

First report of Tomato yellow leaf curl virus in Hawaii.

Tomato yellow leaf curl disease, caused by the begomovirus Tomato yellow leaf curl virus (TYLCV; family Geminiviridae), is an economically important disease of tomato (Solanum lycopersicum L.) that can be very destructive in tropical and subtropical regions (1). In October 2009, tomato plants showing stunted new growth, interveinal chlorosis, and upward curling of leaf margins were reported by a residential gardener in Wailuku, on the island of Maui. Similar symptoms were observed in approximately 200 tomato plants at a University of Hawaii research farm in Poamoho, on the island of Oahu in November 2009. The similarity between these symptoms and those of tomato yellow leaf curl disease and the presence of whiteflies (Bemisia spp.), the vector of TYLCV, suggested the causal agent was a geminivirus such as TYLCV. Total nucleic acids were extracted from a tomato plant sample from Wailuku and Poamoho and used in a PCR assay with degenerate primers PAR1c715 and PAL1v1978 for geminivirus detection (4). The ~1.5-kbp amplicon expected to be produced from a geminivirus template was generated from the symptomatic tomato plant samples but not from a greenhouse-grown control tomato plant. The amplicons were cloned by the pGEM-T Easy vector (Promega, Madison, WI). Three clones from each sample were sequenced, revealing 97 to 99% nucleotide identity to TYLCV sequences in GenBank and a 98.9% nucleotide identity between the Wailuku (Accession No. GU322424) and Poamoho (Accession No. GU322423) isolates. A multiplex PCR assay for the detection and discrimination between the IL and Mld clades of TYLCV was also performed on these isolates (2). A ~0.8-kbp amplicon was generated from both isolates confirming the presence of TYLCV and their inclusion into the TYLCV-IL clade (2). Seven symptomatic and three asymptomatic tomato plant samples from Poamoho were tested for TYLCV using a squash-blot hybridization assay (3) utilizing a digoxigenin-labeled probe derived from the ~1.5-kbp PCR amplicon. All symptomatic tomato plants and one asymptomatic tomato plant were found to be infected with TYLCV. How the virus entered Hawaii and how long it has been present is unknown. The most plausible route is through infected plant material such as an asymptomatic alternative host rather than viruliferous whiteflies. It appears TYLCV is not a recent introduction into Hawaii since the Wailuku gardener observed similar disease symptoms for a few years before submitting samples for testing. In January 2010, TYLCV was also detected in two commercial tomato farms on Oahu, posing a serious threat to the states