Canine olfactory detection of a vectored phytobacterial pathogen, Liberibacter asiaticus, and integration with disease control.

Early detection and rapid response are crucial to avoid severe epidemics of exotic pathogens. However, most detection methods (molecular, serological, chemical) are logistically limited for large-scale survey of outbreaks due to intrinsic sampling issues and laboratory throughput. Evaluation of 10 canines trained for detection of a severe exotic phytobacterial arboreal pathogen, Candidatus Liberibacter asiaticus (CLas), demonstrated 0.9905 accuracy, 0.8579 sensitivity, and 0.9961 specificity. In a longitudinal study, cryptic CLas infections that remained subclinical visually were detected within 2 wk postinfection compared with 1 to 32 mo for qPCR. When allowed to interrogate a diverse range of in vivo pathogens infecting an international citrus pathogen collection, canines only reacted to Liberibacter pathogens of citrus and not to other bacterial, viral, or spiroplasma pathogens. Canines trained to detect CLas-infected citrus also alerted on CLas-infected tobacco and periwinkle, CLas-bearing psyllid insect vectors, and CLas cocultured with other bacteria but at CLas titers below the level of molecular detection. All of these observations suggest that canines can detect CLas directly rather than only host volatiles produced by the infection. Detection in orchards and residential properties was real time, ∼2 s per tree. Spatiotemporal epidemic simulations demonstrated that control of pathogen prevalence was possible and economically sustainable when canine detection was followed by intervention (i.e., culling infected individuals), whereas current methods of molecular (qPCR) and visual detection failed to contribute to the suppression of an exponential trajectory of infection.

First report of Passion fruit green spot virus in yellow Passion fruit (Passiflora edulis f. flavicarpa) in Casanare, Colombia.

Passiflora edulis, commonly known as passion fruit, is a vine species of passionflower native to South America. In Colombia, yellow passion fruit (P. edulis f. flavicarpa) is the most important species in terms of net production and local consumption. Recently two brevipalpus transmitted cileviruses, (i) passion fruit green spot virus (PfGSV) and (ii) hibiscus strain of citrus leprosis virus C2 (CiLV-C2H) were detected in passion fruit in Brazil and Hawaii, respectively (Ramos-González et al., 2020, Olmedo-Velarde et al., 2022). CiLV-C2H infects both citrus and hibiscus in Colombia (Roy et al., 2015, 2018) but there was no report of PfGSV elsewhere apart from Brazil and Paraguay (Costa-Rodrigues et al., 2022). Apart from emerging begomovirus diseases, five major viruses are known to infect passion fruit in Colombia: soybean mosaic virus (SMV), cowpea aphid-borne mosaic virus, passion fruit yellow mosaic virus, cucumber mosaic virus, and a tentative Gulupa bacilliform badnavirus A (Cardona et al., 2022). Current findings of CiLV-C2H in passion fruit and PfGSV in hibiscus motivated us to investigate the possibilities of cilevirus infection in passion fruit in Colombia. During surveys, along with healthy yellow passion fruit leaves, five symptomatic plant samples from Meta and three from Casanare were collected before sent to the Molecular Plant Pathology Laboratory at Beltsville, MD under APHIS permit. Passion fruit samples from Meta showed leaf mottling, rugose mosaic, and leaf distortion, whereas leaf variegation, chlorotic spots, yellowing, green spots in senescent leaves and green vein banding were observed in the Casanare samples (Supp. Fig. 1). Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen, USA). To know the potential cilevirus infection in these samples, three PfGSV specific (Ramos-González et al. 2020) and a CiLV-C2 generic primer pairs (Olmedo-Velarde et al. 2021) were used in the RT-PCR assays. All five passion fruit samples from Meta failed to produce either CiLV-C2 or CiLV-C2H or PfGSV amplicon whereas all three Casanare samples successfully amplified 321, 244 and 299 nts of PfGSV-RNA1 and -RNA2 amplicons using C13F/C13R, C6F/C6R and C8F/C8R primers, respectively. Bi-directional amplicon sequencing followed by BlastN analysis revealed ≥99% nt identity with the PfGSV-RNA1 (MK804173) and -RNA2 (MK804174) genome sequences. An optimized ribo-depleted library preparation protocol was utilized to prepare two cDNA libraries using the RNA extracts of a PfGSV suspected positive (Casanare) and a negative (Meta) samples (Chellappan et al., 2022). HTS libraries of Casanare and Meta samples resulted in 22.7 to 29.5 million raw reads, respectively. After adapter trimming and filtering, clean reads were mapped to the Arabidopsis thaliana reference genome and unmapped reads were de novo assembled (Chellappan et al., 2022). BlastN analysis from the assembled contigs identified 1-3 contigs corresponding to PfGSV-RNA1 and -RNA2, respectively, from Casanare sample whereas 3 contigs of SMV were identified in Meta passion fruit sample. No other virus sequence was obtained from either of the libraries. Assembled contigs covered 99.33% of the RNA1 and 94.42% of the RNA2 genome, with read depths of 64,474 and 119,549, respectively. Meta sample contigs (OP564897) covered >99% of the SMV genome, which shared >99% nt identity with the Colombian SMV isolates (KY249378, MW655827). Both RNA-1 (OP564895) and -2 (OP564896) segments of the Casanare isolate shared 99% nt identity with PfGSV isolate (MK804173-74). Our discovery identified PfGSV in Colombia, for the first-time outside Brazil and Paraguay. The findings of PfGSV in yellow passion fruit increases the potential threat and possibility of PfGSV movement via Brevipalpus sp. from passion fruit to other hosts.

Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico.

The genus Dichorhavirus contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus Clerodendrum, and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit-specific reverse transcription PCR (RT-PCR) assay. During assay validation, OFV-Cit-specific RT-PCR failed to produce an amplicon from some samples with clear symptoms of OFV-Cit. Characterization of this virus revealed that dichorhavirus-like particles were found in the nucleus. High-throughput sequencing of small RNAs from these citrus plants revealed a novel citrus strain of OFV, OFV-Cit2. Sequence comparisons with known orchid and citrus strains of OFV showed variation in the protein products encoded by genome segment 1 (RNA1). Strains of OFV clustered together based on host of origin, whether orchid or citrus, and were clearly separated from other dichorhaviruses described from infected citrus in Brazil. The variation in RNA1 between the original (now OFV-Cit1) and the new (OFV-Cit2) strain was not observed with genome segment 2 (RNA2), but instead, a common RNA2 molecule was shared among strains of OFV-Cit1 and -Cit2, a situation strikingly similar to OFV infecting orchids. We also collected mites at the affected groves, identified them as Brevipalpus californicus sensu stricto, and confirmed that they were infected by OFV-Cit1 or with both OFV-Cit1 and -Cit2. OFV-Cit1 and -Cit2 have coexisted at the same site in Toliman, Queretaro, Mexico since 2012. OFV strain-specific diagnostic tests were developed.

Enhanced Serologically Based Detection of Liberibacters Associated with Citrus Huanglongbing.

'Candidatus Liberibacter spp.' are associated with the most devastating disease of citrus Huanglongbing (HLB). In previous work, we established an in situ tissue print method for the detection of 'Ca. L. asiaticus' (CLas) in sweet orange. We optimized the protocol by preincubation of the anti-Omp antibody with 5% (w/v) extract of healthy rough lemon. This simple process eliminated cross reactions between citrus and the antibody. The optimized protocol enhanced the application of the polyclonal antibody, and we demonstrate detection of CLas from all parts of the world, including isolates from Japan, Thailand, Vietnam, Pakistan, Saudi Arabia, Brazil, the United States, and a selection of strains from China representative of the diversity extant there. The assay also was used to detect four isolates of 'Ca. L. africanus' (CLaf) representative of the diversity present in South Africa. The corresponding outer membrane genes of representative isolates were cloned and sequenced. The coding sequences were highly conserved, and isolates of CLas and CLaf shared 53.8 to 55.9% identity between species at the amino acid level. The optimized protocol is efficient for recognition of both CLas and CLaf in phloem cells of different citrus tissues regardless of geographic origin of the HLB samples. The method is simple and scales well to match the urgent need for accurate, sensitive, and high-throughput screening of HLB bacteria, and may play an important role especially for plant inspection and quarantine programs.

Transcriptomic analyses reveal physiological changes in sweet orange roots affected by citrus blight.

BACKGROUND: Citrus blight is a very important progressive decline disease of commercial citrus. The etiology is unknown, although the disease can be transmitted by root grafts, suggesting a viral etiology. Diagnosis is made by demonstrating physical blockage of xylem cells that prevents the movement of water. This test was used to identify symptomatic trees from four commercial groves in Florida. Total RNA extracts of phloem-enriched scaffold root tissues were prepared from seven trees that failed to take up water and from one healthy tree. These RNA extracts were used for transcriptomic analyses using paired end RNA-Seq from an Illumina 2500 system. The expression of transcripts annotated as polyprotein of citrus endogenous pararetrovirus were estimated by both RT-qPCR and RNA-Seq. RESULTS: Transcripts from seven RNA-Seq libraries from trees affected by citrus blight were compared to a control tree. 129-148 million RNA fragments (two paired-end reads/fragment) were generated per library and were mapped to the sweet orange reference genome. In response to citrus blight stress, genes encoding aquaporins, proteins with water channel activity and several cellulose synthase genes were down-regulated, whereas genes involved in lignin and glucosinolate biosynthesis were up-regulated. Transcripts encoding proteins in pathways of carbohydrate metabolism, nucleotide synthesis, signaling, hormone metabolism, secondary metabolism, transport, and biotic stress pathways were overwhelmingly down regulated in all libraries. CONCLUSION: Reduced water intake and xylem plugging were observed in the trees tested and the changes in their transcriptome were analyzed. Plants adapted to reduced water flow by regulating primary and secondary metabolism, nuclear transport and hormone associated pathways. The patterns of energy generation, transcription, translation and protein degradation were consistent with irreversible decline. The down regulation of cellulose synthase transcripts and up regulation of transcripts related to lignin production likely lead to an imbalance in the pathways leading to wood formation, and may lead to the blockage of the xylem vessels seen as the cardinal symptom of citrus blight. Transcripts of a pararetrovirus were elevated in the transcriptome of roots used in this study.

Synthesis of ABBV-168, a 2'-Bromouridine for the Treatment of Hepatitis C.

ABBV-168 is a dihalogenated nucleotide under investigation for the treatment of hepatitis C virus. Three synthetic routes aimed at achieving the stereoselective installation of the C2' gem-Br,F substitution and subsequent Vorbruggen glycosylation were explored to prepare the penultimate nucleoside intermediate. Development culminated in a route to ABBV-168 featuring a de novo chromatography-free furanose synthesis, protecting group-directed Vorbruggen glycosylation, and highly selective phosphoramidation to furnish the API.

Transcriptional analysis of sweet orange trees co-infected with 'Candidatus Liberibacter asiaticus' and mild or severe strains of Citrus tristeza virus.

BACKGROUND: Citrus worldwide is threatened by huanglongbing (HLB) and tristeza diseases caused by 'Candidatus Liberibacter asiaticus' (CaLas) and Citrus tristeza virus (CTV). Although the pathogens are members of the α-proteobacteria and Closteroviridae, respectively, both are restricted to phloem cells in infected citrus and are transmitted by insect vectors. The response of sweet orange to single infection by either of these two pathogens has been characterized previously by global gene expression analysis. But because of the ubiquity of these pathogens where the diseases occur, co-infection by both pathogens is very common and could lead to increased disease severity based on synergism. We therefore co-inoculated sweet orange trees with CaLas and either a mild or a severe strain of CTV, and measured changes of gene expression in host plants. RESULTS: In plants infected with CaLas-B232, the overall alteration in gene expression was much greater in plants co-inoculated with the severe strain of CTV, B6, than when co-infected with the mild strain of CTV, B2. Plants co-infected with CaLas-B232 and either strain of CTV died but trees co-infected with CTV-B2 survived much longer than those co-infected with CTV-B6. Many important pathways were perturbed by both CTV-B2/CaLas-B232 and/or CTV-B6/CaLas-B232, but always more severely by CTV-B6/CaLas-B232. Genes related to cell wall modification and metal transport responded differently to infection by the pathogens in combination than by the same pathogens singly. The expressions of genes encoding phloem proteins and sucrose loading proteins were also differentially altered in response to CTV-B2 or CTV-B6 in combination with CaLas-B232, leading to different phloem environments in plants co-infected by CaLas and mild or severe CTV. CONCLUSIONS: Many host genes were expressed differently in response to dual infection as compared to single infections with the same pathogens. Interactions of the pathogens within the host may lead to a better or worse result for the host plant. CTV-B6 may exert a synergistic effect with CaLas-B232 in weakening the plant; on the other hand, the responses activated by the mild strain CTV-B2 may provide some beneficial effects against CaLas-B232 by increasing the defense response of the host.

Characterization and purification of proteins suitable for the production of antibodies against 'Ca. Liberibacter asiaticus'.

The citrus disease huanglongbing (HLB), which is caused by 'Candidatus Liberibacter asiaticus' (CaLas), is one of the most devastating pathogens of citrus, and with no effective method of control, poses a serious threat to citrus production throughout the world. In a previous study we described the production of single chain antibodies against several CaLas proteins that provide the basis for efficient and accurate detection of CaLas in citrus tissues. The isolation of a sufficient amount of purified antigen is a key step in the production of functional antibodies. The current report details purification procedures for six protein antigens used to select recombinant and produce polyclonal antibodies. These proteins include a flagellar biosynthesis protein (FlhA), a dinucleoside polyphosphate hydrolase (InvA), a portion of the major outer membrane protein (OmpA), a component of type IV pilus (CapB), the polysialic acid capsule expression protein (KpsA) and the outer membrane efflux protein (TolC). Results of purification under completely native or denatured conditions were not satisfactory. Therefore different hybrid purification conditions were optimized for each of the different proteins. The results of bioinformatic analysis also indicated that the six proteins contained a great diversity of potential antigenic epitopes, which varied in number, and that the antigenic clusters were not uniformly distributed throughout the proteins. The purified proteins are useful for the development of highly specific antibodies capable of differentiating specific strains of Liberibacter.

Transcriptome analysis of sweet orange trees infected with 'Candidatus Liberibacter asiaticus' and two strains of Citrus Tristeza Virus.

BACKGROUND: Huanglongbing (HLB) and tristeza, are diseases of citrus caused by a member of the α-proteobacteria, 'Candidatus Liberibacter asiaticus' (CaLas), and Citrus tristeza virus (CTV) respectively. HLB is a devastating disease, but CTV strains vary from very severe to very mild. Both CaLas and CTV are phloem-restricted. The CaLas-B232 strain and CTV-B6 cause a wide range of severe and similar symptoms. The mild strain CTV-B2 doesn't induce significant symptoms or damage to plants. RESULTS: Transcriptome profiles obtained through RNA-seq revealed 611, 404 and 285 differentially expressed transcripts (DETs) after infection with CaLas-B232, CTV-B6 and CTV-B2. These DETs were components of a wide range of pathways involved in circadian rhythm, cell wall modification and cell organization, as well as transcription factors, transport, hormone response and secondary metabolism, signaling and stress response. The number of transcripts that responded to both CTV-B6 and CaLas-B232 was much larger than the number of transcripts that responded to both strains of CTV or to both CTV-B2 and CaLas-B232. A total of 38 genes were assayed by RT-qPCR and the correlation coefficients between Gfold and RT-qPCR were 0.82, 0.69, 0.81 for sweet orange plants infected with CTV-B2, CTV-B6 and CaLas-B232, respectively. CONCLUSIONS: The number and composition of DETs reflected the complexity of symptoms caused by the pathogens in established infections, although the leaf tissues sampled were asymptomatic. There were greater similarities between the sweet orange in response to CTV-B6 and CaLas-B232 than between the two CTV strains, reflecting the similar physiological changes caused by both CTV-B6 and CaLas-B232. The circadian rhythm system of plants was perturbed by all three pathogens, especially by CTV-B6, and the ion balance was also disrupted by all three pathogens, especially by CaLas-B232. Defense responses related to cell wall modification, transcriptional regulation, hormones, secondary metabolites, kinases and stress were activated by all three pathogens but with different patterns. The transcriptome profiles of Citrus sinensis identified host genes whose expression is affected by the presence of a pathogen in the phloem without producing symptoms (CTV-B2), and host genes whose expression leads to induction of symptoms in the plant (CTV-B6, CaLas-B232).

Direct generation of oxygen-stabilized radicals by H• transfer from transition metal hydrides.

Transition-metal hydrides generate α-alkoxy radicals by H• transfer to enol ethers. We have measured the rate constant for transfer from CpCr(CO)3H to n-butyl vinyl ether and have examined the chemistry of radicals generated by such transfers. Radicals from appropriate substrates undergo 5-exo cyclization, with higher diastereoselectivity than the analogous all-carbon radicals. From such radicals it is straightforward to make substituted tetrahydrofurans.

History and Diversity of Citrus leprosis virus Recorded in Herbarium Specimens.

Leprosis refers to two diseases of citrus that present similar necrotic local lesions, often surrounded by chlorotic haloes on citrus. Two distinct viruses are associated with this disease, one that produces particles primarily in the nucleus of infected plant cells (Citrus leprosis virus nuclear type [CiLV-N]; Dichorhavirus) and another type that produces particles in the cytoplasm of infected plant cells (Citrus leprosis virus cytoplasmic type [CiLV-C]; Cilevirus). Both forms are transmitted by Brevipalpid mites and have bipartite, single-stranded, RNA genomes. CiLV-C and CiLV-N are present in South and Central America and as far north as parts of Mexico. Although leprosis disease was originally described from Florida, it disappeared from there in the 1960s. The United States Department of Agriculture-Agricultural Research Service maintains preserved citrus specimens identified at inspection stations 50 or more years ago with symptoms of citrus leprosis. We isolated RNA from these samples and performed degradome sequencing. We obtained nearly full-length genome sequences of both a typical CiLV-C isolate intercepted from Argentina in 1967 and a distinct CiLV-N isolate obtained in Florida in 1948. The latter is a novel form of CiLV-N, not known to exist anywhere in the world today. We have also documented the previously unreported presence of CiLV-N in Mexico in the mid-20th century.

Identification and Molecular Characterization of Nuclear Citrus leprosis virus, a Member of the Proposed Dichorhavirus Genus Infecting Multiple Citrus Species in Mexico.

Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Querétaro, Mexico. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)-long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5' untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reverse-transcription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants.

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.

Ultrastructural Changes and Putative Phage Particles Observed in Sweet Orange Leaves Infected with 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB), also known as citrus greening, is currently the most destructive citrus disease. Anatomical analyses of HLB-affected sweet orange were carried out by light and electron microscopy. As compared with healthy citrus, the phloem plasmodesmata were plugged with callose, and in some samples the phloem was collapsed. Chloroplast structures were deformed. Prophage sequences occupy a significant portion of the genome of 'Candidatus Liberibacter asiaticus' and have been used to distinguish strains from Yunnan and Guangdong provinces in China and Florida. Interestingly, a large number of possible putative phage particles were observed attached on the surface of 'Ca. L. asiaticus' cells in plants inoculated with strain FJ3 from Fujian Province, China. Phage particles have been observed previously only in periwinkle plants artificially inoculated in Florida with 'Ca. L. asiaticus' that carried the SC1-type prophage. PCR assays verified the presence of the SC1-type prophage sequences previously described from this bacterium in Florida in the FJ3 isolate. This is the first time that suspected phage particles have been observed in sweet orange trees infected with 'Ca. L. asiaticus.'

Enantioselective olefin metathesis with cyclometalated ruthenium complexes.

The success of enantioselective olefin metathesis relies on the design of enantioenriched alkylidene complexes capable of transferring stereochemical information from the catalyst structure to the reactants. Cyclometalation of the NHC ligand has proven to be a successful strategy to incorporate stereogenic atoms into the catalyst structure. Enantioenriched complexes incorporating this design element catalyze highly Z- and enantioselective asymmetric ring opening/cross metathesis (AROCM) of norbornenes and cyclobutenes, and the difference in ring strain between these two substrates leads to different propagating species in the catalytic cycle. Asymmetric ring closing metathesis (ARCM) of a challenging class of prochiral trienes has also been achieved. The extent of reversibility and effect of reaction setup was also explored. Finally, promising levels of enantioselectivity in an unprecedented Z-selective asymmetric cross metathesis (ACM) of a prochiral 1,4-diene was demonstrated.

Studies toward the total synthesis of pluraflavin A.

A synthetic strategy towards the potent cytostatic agent pluraflavin A has been developed. Formation of the enantioenriched anthrapyran core bearing a halogen atom enabled the introduction of the α C-aryl glycoside by Stille cross-coupling and subsequent hydrogenation of the aryl glycal. Chemo- and stereoselective O-glycosylations of α oliose and ß 3-epi vancosamine residues afforded a fully glycosylated aromatic core. Attempts to install the dimethylamino group of the C-disaccharide suggest that introduction of an azide group by displacement and subsequent reduction may pave the way to the total synthesis of pluraflavin A.

Effect of double-bond substituents on the rate of cyclization of α-carbomethoxyhex-5-enyl radicals.

Rate constants have been calculated, and compared with experimental results, for the cyclizations of 1-carbomethoxy-1-methyl-5-hexenyl radicals (2) with various substituents on C6. The calculations have been done by DFT at the B3LYP/6-311++G** level of theory. They show considerable interaction between C5 and the radical centers even in the ground state of all of the radicals 2. Experimentally, the radicals have been generated by H(•) transfer to the corresponding acrylate esters 1 and the yields of cyclized products compared to the calculated rate constants. (The "cyclized products" include those from cyclohydrogenation, 4, and those from cycloisomerization, 9.) Two phenyl substituents on C6 (2i), or a phenyl and a methyl substituent (2g, 2h), increase the rate of cyclization, but a single phenyl substituent on C6 produces a greater increase. The calculations show that the two phenyl substituents are twisted in the transition state for cyclization, while a single phenyl substituent remains flat in that transition state. A methyl substituent on C6 along with a single phenyl causes the phenyl to twist in the transition state and decreases the rate constant for cyclization below that of the H/Ph-substituted 2e, 2f.