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Forest litter decomposition is an essential component of global carbon and nutrient turnover. Invertebrates play important roles in litter decomposition, but the regional pattern of their effects is poorly understood. We examined 476 case studies across 93 sites and performed a meta-analysis to estimate regional effects of invertebrates on forest litter decomposition. We then assessed how invertebrate diversity, climate and soil pH drive regional variations in invertebrate-mediated decomposition. We found that (1) invertebrate contributions to litter decomposition are 1.4 times higher in tropical and subtropical forests than in forests elsewhere, with an overall contribution of 31% to global forest litter decomposition; and (2) termite diversity, together with warm, humid and acidic environments in the tropics and subtropics are positively associated with forest litter decomposition by invertebrates. Our results demonstrate the significant difference in invertebrate effects on mediating forest litter decomposition among regions. We demonstrate, also, the significance of termites in driving litter mass loss in the tropics and subtropics. These results are particularly pertinent in the tropics and subtropics where climate change and human disturbance threaten invertebrate biodiversity and the ecosystem services it provides., (© 2024 The Authors. Ecology Letters published by John Wiley & Sons Ltd.)

Chronic pain is becoming increasingly prevalent and burdensome both worldwide and in the United Kingdom. Due to the complexity of chronic pain and the therapeutic challenge associated, management is often difficult and requires multidisciplinary care encompassing a combination of pharmacological and non-pharmacological strategies. Conventional analgesic treatments, such as opioids and anticonvulsants, are effective in less than half of chronic pain sufferers and are typically limited to short-term use to prevent complications associated with long-term use such as tolerance and dependence. Consequently, research and clinical interest in alternative management options for chronic pain have increased in recent years, with ketamine being one example under investigation. However, since ketamine has been licensed as an anaesthetic for decades, it has bypassed the traditional scrutinous drug development sequence that is typically seen for therapeutics marketed for pain. As such, data supporting the unlicensed administration of ketamine for chronic pain management is lacking and is being outpaced by the rates of off-label use in pain clinics. Recent limited evidence suggests that ketamine, when given as an intravenous infusion in subanaesthetic doses for refractory pain patients, may provide modest analgesic effects in nearly all aetiologies of chronic pain, with side effects common but typically mild. However, there are concerns over the safety of this practice due to the paucity of robust supportive evidence and the accompanying lack of clinical guidelines or standardised protocols. This review shall summarise the literature examining the use of subanaesthetic-dose ketamine infusions for chronic pain to comment on the current level of evidence, with limitations of existing research and future recommendations discussed., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2023.)

Deadwood is a large global carbon store with its store size partially determined by biotic decay. Microbial wood decay rates are known to respond to changing temperature and precipitation. Termites are also important decomposers in the tropics but are less well studied. An understanding of their climate sensitivities is needed to estimate climate change effects on wood carbon pools. Using data from 133 sites spanning six continents, we found that termite wood discovery and consumption were highly sensitive to temperature (with decay increasing >6.8 times per 10°C increase in temperature)-even more so than microbes. Termite decay effects were greatest in tropical seasonal forests, tropical savannas, and subtropical deserts. With tropicalization (i.e., warming shifts to tropical climates), termite wood decay will likely increase as termites access more of Earth's surface.

Soil invertebrates make significant contributions to the recycling of dead plant material across the globe. However, studies focussed on the consequences of decomposition for plant communities largely ignore soil fauna across all ecosystems, because microbes are often considered the primary agents of decay. Here, we explore the role of invertebrates as not simply facilitators of microbial decomposition, but as true decomposers, able to break down dead organic matter with their own endogenic enzymes, with direct and indirect impacts on the soil environment and plants. We recommend a holistic view of decomposition, highlighting how invertebrates and microbes act in synergy to degrade organic matter, providing ecological services that underpin plant growth and survival., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

Tree mortality rates are increasing within tropical rainforests as a result of global environmental change. When trees die, gaps are created in forest canopies and carbon is transferred from the living to deadwood pools. However, little is known about the effect of tree-fall canopy gaps on the activity of decomposer communities and the rate of deadwood decay in forests. This means that the accuracy of regional and global carbon budgets is uncertain, especially given ongoing changes to the structure of rainforest ecosystems. Therefore, to determine the effect of canopy openings on wood decay rates and regional carbon flux, we carried out the first assessment of deadwood mass loss within canopy gaps in old-growth rainforest. We used replicated canopy gaps paired with closed canopy sites in combination with macroinvertebrate accessible and inaccessible woodblocks to experimentally partition the relative contribution of microbes vs. termites to decomposition within contrasting understorey conditions. We show that over a 12 month period, wood mass loss increased by 63% in canopy gaps compared with closed canopy sites and that this increase was driven by termites. Using LiDAR data to quantify the proportion of canopy openings in the study region, we modelled the effect of observed changes in decomposition within gaps on regional carbon flux. Overall, we estimate that this accelerated decomposition increases regional wood decay rate by up to 18.2%, corresponding to a flux increase of 0.27 Mg C ha -1  year -1 that is not currently accounted for in regional carbon budgets. These results provide the first insights into how small-scale disturbances in rainforests can generate hotspots for decomposer activity and carbon fluxes. In doing so, we show that including canopy gap dynamics and their impacts on wood decomposition in forest ecosystems can help improve the predictive accuracy of the carbon cycle in land surface models., (© 2021 John Wiley & Sons Ltd.)

Termites perform key ecological functions in tropical ecosystems, are strongly affected by variation in rainfall, and respond negatively to habitat disturbance. However, it is not known how the projected increase in frequency and severity of droughts in tropical rainforests will alter termite communities and the maintenance of ecosystem processes. Using a large-scale termite suppression experiment, we found that termite activity and abundance increased during drought in a Bornean forest. This increase resulted in accelerated litter decomposition, elevated soil moisture, greater soil nutrient heterogeneity, and higher seedling survival rates during the extreme El Niño drought of 2015-2016. Our work shows how an invertebrate group enhances ecosystem resistance to drought, providing evidence that the dual stressors of climate change and anthropogenic shifts in biotic communities will have various negative consequences for the maintenance of rainforest ecosystems., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Ants are diverse and abundant, especially in tropical ecosystems. They are often cited as the agents of key ecological processes, but their precise contributions compared with other organisms have rarely been quantified. Through the removal of food resources from the forest floor and subsequent transport to nests, ants play an important role in the redistribution of nutrients in rainforests. This is an essential ecosystem process and a key energetic link between higher trophic levels, decomposers and primary producers. We used the removal of carbohydrate, protein and seed baits as a proxy to quantify the contribution that ants, other invertebrates and vertebrates make to the redistribution of nutrients around the forest floor, and determined to what extent there is functional redundancy across ants, other invertebrate and vertebrate groups. Using a large-scale, field-based manipulation experiment, we suppressed ants from c. 1 ha plots in a lowland tropical rainforest in Sabah, Malaysia. Using a combination of treatment and control plots, and cages to exclude vertebrates, we made food resources available to: (i) the whole foraging community, (ii) only invertebrates and (iii) only non-ant invertebrates. This allowed us to partition bait removal into that taken by vertebrates, non-ant invertebrates and ants. Additionally, we examined how the non-ant invertebrate community responded to ant exclusion. When the whole foraging community had access to food resources, we found that ants were responsible for 52% of total bait removal whilst vertebrates and non-ant invertebrates removed the remaining 48%. Where vertebrates were excluded, ants carried out 61% of invertebrate-mediated bait removal, with all other invertebrates removing the remaining 39%. Vertebrates were responsible for just 24% of bait removal and invertebrates (including ants) collectively removed the remaining 76%. There was no compensation in bait removal rate when ants and vertebrates were excluded, indicating low functional redundancy between these groups. This study is the first to quantify the contribution of ants to the removal of food resources from rainforest floors and thus nutrient redistribution. We demonstrate that ants are functionally unique in this role because no other organisms compensated to maintain bait removal rate in their absence. As such, we strengthen a growing body of evidence establishing ants as ecosystem engineers, and provide new insights into the role of ants in maintaining key ecosystem processes. In this way, we further our basic understanding of the functioning of tropical rainforest ecosystems., (© 2017 The Authors. Journal of Animal Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society.)

Anthropogenic activities are causing species extinctions, raising concerns about the consequences of changing biological communities for ecosystem functioning. To address this, we investigated how dung beetle communities influence seed burial and seedling recruitment in the Brazilian Amazon. First, we conducted a burial and retrieval experiment using seed mimics. We found that dung beetle biomass had a stronger positive effect on the burial of large than small beads, suggesting that anthropogenic reductions in large-bodied beetles will have the greatest effect on the secondary dispersal of large-seeded plant species. Second, we established mesocosm experiments in which dung beetle communities buried Myrciaria dubia seeds to examine plant emergence and survival. Contrary to expectations, we found that beetle diversity and biomass negatively influenced seedling emergence, but positively affected the survival of seedlings that emerged. Finally, we conducted germination trials to establish the optimum burial depth of experimental seeds, revealing a negative relationship between burial depth and seedling emergence success. Our results provide novel evidence that seed burial by dung beetles may be detrimental for the emergence of some seed species. However, we also detected positive impacts of beetle activity on seedling recruitment, which are probably because of their influence on soil properties. Overall, this study provides new evidence that anthropogenic impacts on dung beetle communities could influence the structure of tropical forests; in particular, their capacity to regenerate and continue to provide valuable functions and services., (© 2016 The Author(s).)

Functional diversity indices are used to facilitate a mechanistic understanding of many theoretical and applied questions in current ecological research. The use of mean trait values in functional indices assumes that traits are robust, in that greater variability exists between than within species. While the assertion of robust traits has been explored in plants, there exists little information on the source and extent of variability in the functional traits of higher trophic level organisms. Here we investigated variability in two functionally relevant dung beetle traits, measured from individuals collected from three primary forest sites containing distinct beetle communities: body mass and back leg length. In doing so we too addressed the following questions: (i) what is the contribution of intra vs. interspecific differences in trait values; (ii) what sample size is needed to provide representative species mean trait values; and (iii) what impact does omission of intraspecific trait information have on the calculation of functional diversity (FD) indices from naturally assembled communities? At the population level, interspecific differences explained the majority of variability in measured traits (between 94% and 96%). In accordance with this, the error associated with calculating FD without inclusion of intraspecific variability was low, less than 20% in all cases. This suggests that complete sampling to capture intraspecific variance in traits is not necessary even when investigating the FD of small and/or naturally formed communities. To gain an accurate estimation of species mean trait values we encourage the measurement of 30-60 individuals and, where possible, these should be taken from specimens collected from the site of study.

Background: Invasive predators may change the structure of invaded communities through predation and competition with native species. In Europe, the invasive signal crayfish Pacifastacus leniusculus is excluding the native white clawed crayfish Austropotamobius pallipes., Methodology and Principal Findings: This study compared the predatory functional responses and prey choice of native and invasive crayfish and measured impacts of parasitism on the predatory strength of the native species. Invasive crayfish showed a higher (>10%) prey (Gammarus pulex) intake rate than (size matched) natives, reflecting a shorter (16%) prey handling time. The native crayfish also showed greater selection for crustacean prey over molluscs and bloodworm, whereas the invasive species was a more generalist predator. A. pallipes parasitised by the microsporidian parasite Thelohania contejeani showed a 30% reduction in prey intake. We suggest that this results from parasite-induced muscle damage, and this is supported by a reduced (38%) attack rate and increased (30%) prey handling time., Conclusions and Significance: Our results indicate that the per capita (i.e., functional response) difference between the species may contribute to success of the invader and extinction of the native species, as well as decreased biodiversity and biomass in invaded rivers. In addition, the reduced predatory strength of parasitized natives may impair their competitive abilities, facilitating exclusion by the invader.

Klebsiella spp. are a common cause of mastitis, milk loss, and culling on dairy farms. Control of Klebsiella mastitis is largely based on prevention of exposure of the udder to the pathogen. To identify critical control points for mastitis prevention, potential Klebsiella sources and transmission cycles in the farm environment were investigated, including oro-fecal transmission, transmission via the indoor environment, and transmission via the outdoor environment. A total of 305 samples was collected from 3 dairy farms in upstate New York in the summer of 2007, and included soil, feed crops, feed, water, rumen content, feces, bedding, and manure from alleyways and holding pens. Klebsiella spp. were detected in 100% of rumen samples, 89% of water samples, and approximately 64% of soil, feces, bedding, alleyway, and holding pen samples. Detection of Klebsiella spp. in feed crops and feed was less common. Genotypic identification of species using rpoB sequence data showed that Klebsiella pneumoniae was the most common species in rumen content, feces, and alleyways, whereas Klebsiella oxytoca, Klebsiella variicola, and Raoultella planticola were the most frequent species among isolates from soil and feed crops. Random amplified polymorphic DNA-based strain typing showed heterogeneity of Klebsiella spp. in rumen content and feces, with a median of 4 strains per 5 isolates. Observational and bacteriological data support the existence of an oro-fecal transmission cycle, which is primarily maintained through direct contact with fecal contamination or through ingestion of contaminated drinking water. Fecal shedding of Klebsiella spp. contributes to pathogen loads in the environment, including bedding, alleyways, and holding pens. Hygiene of alleyways and holding pens is an important component of Klebsiella control on dairy farms., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

During recent climate warming, many insect species have shifted their ranges to higher latitudes and altitudes. These expansions mirror those that occurred after the Last Glacial Maximum when species expanded from their ice age refugia. Postglacial range expansions have resulted in clines in genetic diversity across present-day distributions, with a reduction in genetic diversity observed in a wide range of insect taxa as one moves from the historical distribution core to the current range margin. Evolutionary increases in dispersal at expanding range boundaries are commonly observed in virtually all insects that have been studied, suggesting a positive feedback between range expansion and the evolution of traits that accelerate range expansion. The ubiquity of this phenomenon suggests that it is likely to be an important determinant of range changes. A better understanding of the extent and speed of adaptation will be crucial to the responses of biodiversity and ecosystems to climate change.

A dominant allele at the D locus (also known as I in diploid potato) is required for the synthesis of red and purple anthocyanin pigments in tuber skin. It has previously been reported that D maps to a region of chromosome 10 that harbors one or more homologs of Petunia an2, an R2R3 MYB transcription factor that coordinately regulates the expression of multiple anthocyanin biosynthetic genes in the floral limb. To test whether D acts similarly in tuber skin, RT-PCR was used to evaluate the expression of flavanone 3-hydroxylase (f3h), dihydroflavonol 4-reductase (dfr) and flavonoid 3',5'-hydroxylase (f3'5'h). All three genes were expressed in the periderm of red- and purple-skinned clones, while dfr and f3'5'h were not expressed, and f3h was only weakly expressed, in white-skinned clones. A potato cDNA clone with similarity to an2 was isolated from an expression library prepared from red tuber skin, and an assay developed to distinguish the two alleles of this gene in a diploid potato clone known to be heterozygous Dd. One allele was observed to cosegregate with pigmented skin in an F(1) population of 136 individuals. This allele was expressed in tuber skin of red- and purple-colored progeny, but not in white tubers, while other parental alleles were not expressed in white or colored tubers. The allele was placed under the control of a doubled 35S promoter and transformed into the light red-colored cultivar Désirée, the white-skinned cultivar Bintje, and two white diploid clones known to lack the functional allele of D. Transformants accumulated pigment in tuber skin, as well as in other tissues, including young foliage, flower petals, and tuber flesh.

This study was designed to explore the relationship between cow and udder cleanliness scores and the risk of isolation of Klebsiella spp. from lower hind legs and teat ends, respectively. The distribution of Klebsiella species was compared among isolates from teat ends, legs, and cases of clinical mastitis obtained from 2 dairy farms in New York State, with 850 and 1,000 cows, respectively. Farms were visited twice approximately 4 wk apart in August and September 2007 to obtain cleanliness scores and swabs from legs and teats. Isolates of Klebsiella clinical mastitis from each farm were collected from July through October 2007. Two studies were conducted. In the first study, whole-cow cleanliness of a purposive sample of 200 lactating cows was scored using a 4-point scale, and swabs were taken from their lower hind legs. In the second study, udder cleanliness of a separate convenience sample of 199 lactating cows was scored in the milking parlor, and swabs were taken from their teat ends before and after premilking udder preparation. Prevalence of Klebsiella spp. on legs and teat ends before udder preparation was 59 and 60%, respectively. Logistic regression was used to explore the association between isolation of Klebsiella spp. and cleanliness scores. Cow cleanliness scores and udder cleanliness scores were not associated with detection of Klebsiella on legs and on teats before udder preparation, respectively. After udder preparation, 43% of previously Klebsiella positive teat end samples remained positive, with significant differences between farms and months. Teats from dirty udders were significantly more likely to test positive for Klebsiella after udder preparation than teats from clean udders. The proportion of Klebsiella pneumoniae and Klebsiella oxytoca isolates was similar for isolates from teat end swabs and clinical mastitis cases, supporting the notion that the presence of Klebsiella on teat ends may lead to opportunistic intramammary infections. Udder cleanliness scores could be used as a management tool to monitor the risk of exposure to Klebsiella spp. on teat ends.

The potato P locus is required for the production of blue/purple anthocyanin pigments in any tissue of the potato plant such as tubers, flowers, or stems. We have previously reported, based on RFLP mapping in tomato, that the gene coding for the anthocyanin biosynthetic enzyme flavonoid 3',5'-hydroxylase (f3'5'h) maps to the same region of the tomato genome as P maps in potato. To further evaluate this association a Petunia f3'5'h gene was used to screen a potato cDNA library prepared from purple-colored flowers and stems. Six positively hybridizing cDNA clones were sequenced and all appeared to be derived from a single gene that shares 85% sequence identity at the amino acid level with Petunia f3'5'h. The potato gene cosegregated with purple tuber color in a diploid F1 sub-population of 37 purple and 25 red individuals and was found to be expressed in tuber skin only in the presence of the anthocyanin regulatory locus I. A potato f3'5'h cDNA clone was placed under the control of a doubled CaMV 35S promoter and introduced into the red-skinned cultivar 'Desiree'. Tuber and stem tissues that are colored red in Desiree were purple in nine of 17 independently transformed lines.

A disease with symptoms similar to elm yellows (EY) was noticed in the early 1990s in suburban Chicago, IL. More than 1,000 mature American elms (Ulmus americana) have since died. Infected trees varied in the incidence and severity of canopy yellowing, leaf epinasty, butterscotch discoloration, and wintergreen odor of the phloem, but all developed a sparse and clumpy crown, uniformly necrotic phloem, and died within 2 years of showing canopy symptoms. Because symptoms were expressed irregularly and phytoplasma detection results by a commercial diagnostic company were inconsistent, a study was initiated to determine if EY phytoplasma was the causal agent. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods using universal or EY phytoplasma specific primers were employed to detect putative phytoplasma(s) associated with 10 trees of varied disease severity within the outbreak region and 10 asymptomatic trees from an uninfected area (controls). Nested PCR using universal primers revealed that 90% of trees from the outbreak region were positive for phytoplasma while asymptomatic elms from another location (controls) tested negative. Phytoplasma-positive trees ranged in disease severity from 1 (asymptomatic) to 5 (near death). Inner bark samples chiseled from the lower trunk had higher phytoplasma detection rates than foliage or drill shavings. RFLP analyses and DNA sequencing of 16S rDNA indicated that the phytoplasma recovered from dying elms in Arlington Heights is not related to the reference EY phytoplasma (group16SrV). It is most closely related to clover proliferation (CP) phy-toplasma (group 16SrVI), and we have designated it Illinois Elm Yellows (ILEY) phytoplasma, and assigned it to a new taxonomic subgroup (16SrVI-C). EY phytoplasma was not detected in any samples, but two ILEY phytoplasma positive trees also were positive for aster yellows (AY) phytoplasma. ILEY phytoplasma was not detected in local leafhopper populations trapped in elm trees between May and September 2000. This is the first report of a phytoplasma related to CP phytoplasma causing elm yellows disease symptoms.

The cultivated potato (Solanum tuberosum) shares similar biology with other members of the Solanaceae, yet has features unique within the family, such as modified stems (stolons) that develop into edible tubers. To better understand potato biology, we have undertaken a survey of the potato transcriptome using expressed sequence tags (ESTs) from diverse tissues. A total of 61,940 ESTs were generated from aerial tissues, below-ground tissues, and tissues challenged with the late-blight pathogen (Phytophthora infestans). Clustering and assembly of these ESTs resulted in a total of 19,892 unique sequences with 8,741 tentative consensus sequences and 11,151 singleton ESTs. We were able to identify a putative function for 43.7% of these sequences. A number of sequences (48) were expressed throughout the libraries sampled, representing constitutively expressed sequences. Other sequences (13,068, 21%) were uniquely expressed and were detected only in a single library. Using hierarchal and k means clustering of the EST sequences, we were able to correlate changes in gene expression with major physiological events in potato biology. Using pair-wise comparisons of tuber-related tissues, we were able to associate genes with tuber initiation, dormancy, and sprouting. We also were able to identify a number of characterized as well as novel sequences that were unique to the incompatible interaction of late-blight pathogen, thereby providing a foundation for further understanding the mechanism of resistance.

The polymerase chain reaction (PCR) employing phytoplasma-specific ribosomal RNA primer pair P1/P7 consistently amplified a product of expected size (1.8 kb) from 29 of 36 symptom-less Virginia creeper (Parthenocissus quinquefolia) plants growing in southern Florida. Restriction fragment length polymorphism analysis of P1/P7-primed PCR products indicated that most phytoplasmas detected in Virginia creeper were similar to phytoplasmas composing the elm yellows (16SrV) group. This relationship was verified by reamplification of P1/P7 products using an elm yellows (EY) group-specific rRNA primer pair fB1/rULWS1. rDNA products (1,571 bp) were generated by group-specific PCR from 28 phytoplasma-positive plants and 1 negatively testing plant identified by earlier P1/P7-primed PCR. Analysis of 16S rDNA sequences determined the Virginia creeper (VC) phytoplasma to be phylogenetically closest to the European alder yellows (ALY) agent, an established 16SrV-C subgroup strain. However, presence or absence of restriction sites for endonucleases AluI, BfaI, MspI, RsaI, and TaqI in the 16S rRNA and 16-23S rRNA intergenic spacer region of the VC phytoplasma collectively differentiated this strain from ALY and other 16SrV group phytoplasmas. Failure to detect the VC phytoplasma by PCR employing nonribosomal primer pair FD9f/FD9r suggests that this newly characterized agent varies from known European grapevine yellows (flavescence dorée) phyto-plasmas previously classified as 16SrV subgroup C or D strains.

Elms (genus Ulmus) of six clonal cultivars representing Eurasian species and hybrids were grafted when 2 to 3 years old with bark patches from U. rubra infected with an elm yellows phytoplasma or were left untreated as controls. The cultivars were U. glabra × minor 'Pioneer', U. minor × parvifolia 'Frontier', U. parvifolia 'Pathfinder', U. wilsoniana 'Prospector', and the complex hybrids 'Homestead' and 'Patriot'. Trees were evaluated for infection and symptoms 1 or 2 years after inoculation. Infection was detected via the 4',6-diamidino-2-phenylindol e·2HCl (DAPI) fluorescence test in 26 of 86 grafted trees representing five cultivars. Infection of selected trees was confirmed by polymerase chain reaction (PCR) amplification of a fragment of phytoplasmal rDNA, and the phytoplasma was identified by restriction fragment length polymorphism (RFLP) analysis of the amplified DNA using restriction enzymes AluI, RsaI, and TaqI. Elm yellows phytoplasma was also identified by nested PCR and RFLP analysis in two of seven inoculated, healthy-appearing, DAPI-negative trees and one noninoculated control tree. All RFLP profiles were identical to that of reference strain EY1. Phytoplasma-associated symptoms, observed in five cultivars, included suppressed growth, progressive size reduction of apical shoots and leaves, chlorosis, foliar reddening, witches'-brooms, and dieback. Phyto-plasma was not detected in cv. Homestead. Possible resistance of this cultivar to elm yellows phytoplasma was indicated by localized phloem necrosis in stems below inoculum patches.

Five cultivars of Fraxinus americana (white ash) and five of F. pennsylvanica (green ash) were graft-inoculated with three strains of ash yellows phytoplasmas at Ames, IA, and with thrsee other strains at Ithaca, NY. A sixth green ash cultivar was tested only in New York. Trees were allowed to grow in field plots for 3 years. Infection was detected via the DAPI (4', 6-diamidino-2-phenylindole 2HCl) fluorescence test. Incidence of witches'-brooms on infected trees was greater on white ash than green ash and varied significantly among phytoplasma strain treatments at both locations. Volume growth of infected ash, averaged across cultivars over 2 years in Iowa and 3 years in New York, was 49 and 59%, respectively, as great as that of noninfected trees. Foliar greenness was reduced significantly by infection at both locations, and this reduction was positively correlated with growth reduction. Cultivars at each location varied significantly in growth of noninfected trees and in growth of diseased trees relative to that of nonin-fected trees (a measure of phytoplasma tolerance), but cultivar means for these variables in Iowa were not significantly correlated with those in New York. Green ash cvs. Bergeson, Dakota Centennial, and Patmore and white ash cv. Autumn Applause were above average in tolerance at both locations. Phytoplasma strains at each location varied significantly in aggressiveness as indicated by host growth suppression.

Twelve strains of phytoplasmas belonging to the ash yellows (AshY) group, from across the known range of AshY and representing six host species, were assessed for differences in ability to suppress growth and cause chlorosis in graft-inoculated Fraxinus pennsylvanica (green ash) and Catharanthus roseus (periwinkle). In each of two experiments with ash and one with periwinkle, different strains caused significantly different degrees of growth suppression and loss of foliar greenness. These growth and color impacts were positively and significantly correlated among experiments and between ash and periwinkle, indicating strain variation in aggressiveness. After two strains that differed in aggressiveness were coinoculated to periwinkle plants, polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) assays of DNA from leaves remote from the inoculation sites revealed the presence of the aggressive strain sooner and more frequently than that of the less aggressive strain. Thus, aggressiveness was associated with more rapid multiplication and/or movement than was achieved by the less aggressive strain. When either strain was inoculated 11 weeks before the other into the same plant, only the initial strain could be detected after a further 12 weeks of incubation. Thus, the initial strain or its effect on the host may have interfered with multiplication and/or long-distance movement of the second strain. A concept of preemptive dominance is proposed to account for detection by primary PCR of only single phytoplasma strains in plants that may harbor two or more strains.

Restriction fragment length polymorphism (RFLP) analyses were performed on polymerase chain reaction (PCR) amplimers of phytoplasmal DNA from eight samples obtained from Ulmus spp. (elms) affected by elm yellows (EY) in Italy and the United States, from Catharanthus roseus infected with strain EY1, and from five other plant species infected with phytoplasmas of the EY group sensu lato (group 16SrV). RFLP profiles obtained with restriction enzyme TaqI from ribosomal DNA amplified with primer pair P1/P7 differentiated elm-associated phytoplasmas from strains originally detected in Apocynum cannabinum, Prunus spp., Rubus fruticosus, Vitis vinifera, and Ziziphus jujuba. RFLP profiles obtained similarly with BfaI differentiated strains from A. cannabinum and V. vinifera from other phytoplasmas of group 16SrV. Elm-associated strains from within the United States had two RFLP patterns in ribosomal DNA based on presence or absence of an RsaI site in the 16S-23S spacer. Elm-associated phytoplasma strains from Italy were distinguished from those of American origin by RFLPs obtained with MseI in the same fragment of non-ribosomal DNA. Strain HD1, which was discovered in A. cannabinum associated with EY-diseased elms in New York State, was unique among the strains studied.

Phytoplasmas associated with the plant diseases ash yellows (AshY, occurring in Fraxinus) and lilac witches'-broom (LWB, occurring in Syringa) represent a putative species-level taxon. Phytoplasmal DNA from 19 ash or lilac sources across the known geographic range of AshY (71-113 degrees W) was examined to determine if AshY and LWB phytoplasmas are a coherent group, if variability exists in both conserved and anonymous DNA, and if variability in 16S rDNA is related to host or geographic origin. The 16S rRNA gene and the 16S-23S spacer were amplified using primer pair P1/P7 and analysed using 15 restriction enzymes. RFLPs were detected in digests obtained with Alul, Hhal or Taql, for a total of four RFLP profile types. Sequencing of the amplimers from strains AshY1T, AshY3, AshY5 and LWB3 (which represent the four 16S rDNA RFLP profile types) revealed only three positions in the 16S rRNA gene and one position in the 16S-23S spacer at which differences occurred; these were single nucleotide substitutions. Sequence homology between any two strains was > 99.8%. A portion of a ribosomal protein operon, amplified with primer pair rpF1/R1 from each of the four strains noted above, was analysed with six restriction enzymes, resulting in the detection of two RFLP profiles with Msel. Southern analysis, utilizing two non-specific probes from other phytoplasma groups, revealed three RFLP profile types in anonymous chromosomal DNA of strains representing the four 16S rDNA genotypes. Two strains, AshY3 and LWB3, had unique combinations of characters in the various assays. On the basis of RFLP profiles, the strains from the other plants sampled comprised two groups. The grouping was not clearly related to host or geographic origin. The genome size of strain AshY3 was estimated from PFGE data to be 645 kbp. Phylogenetic analysis of a 1423 bp 16S rDNA sequence from strains AshY1T, AshY3, AshY5 and LWB3, together with sequences from 14 other mollicutes archived in GenBank, produced a tree on which the AshY and LWB strains clustered as a discrete group, consistent with previous analyses utilizing only type strain AshY1T. Thus, the AshY phytoplasma group is coherent but heterogeneous. The name 'Candidatus Phytoplasma fraxini' is proposed for this group.

Growth responses of different ash (Fraxinus) species and rootstock-scion combinations to ash yellows (AshY) phytoplasmas were compared in greenhouse experiments by expressing each measurement as a proportion of the final average value of the variable in noninoculated, own-rooted control trees. Phytoplasmal infection suppressed shoot growth of white ash (F. americana) and green ash (F. pennsylvanica) beginning when buds opened, but did not suppress velvet ash (F. velutina) until after 60 days of growth. AshY-associated growth losses in height, stem diameter, and root volume, averaged across two experiments, were 80, 93, and 98%, respectively, in white ash; 60, 57, and 79% in green ash; and 23, 0, and 12% in velvet ash. Growth in height, but not in stem diameter or root volume, of diseased white ash on velvet ash rootstock was significantly greater than growth of diseased own-rooted white ash. White ash witches'-brooms grafted onto healthy velvet ash continued to grow but did not produce vigorous, dominant shoots. Growth of diseased velvet ash on white ash roots was severely suppressed in comparison with that of diseased own-rooted velvet ash. Management of AshY through the use of tolerant genotypes may require tolerance in both scions and rootstocks.