Relative Contribution of Windbreak, Copper Sprays, and Leafminer Control for Citrus Canker Management and Prevention of Crop Loss in Sweet Orange Trees.

The management of citrus canker, caused by Xanthomonas citri subsp. citri, has been widely studied in endemic areas because of the importance of the disease in several citrus-producing countries. A set of control measures is well established, but no study has investigated the efficiency of each measure individually and their combination for disease suppression. This study comprised a 3-year field study to assess the relative contribution of three measures for the control of citrus canker and reduction of crop losses. Windbreak (Wb), copper sprays (Cu), and leafminer control (Lc) were assessed in eight different combinations in a split-split plot design. The orchard was composed of 'Valencia' sweet orange trees grafted onto 'Rangpur' lime. Casuarina cunninghamiana trees were used as Wb. Cu and Lc sprays were performed every 21 days throughout the year. Individually, Cu showed the highest contribution for canker control, followed by Wb. Lc had no effect on reducing citrus canker. Wb+Cu showed the highest efficiency for control of the disease. This combination reduced the incidence of diseased trees by approximately 60%, and the incidence of diseased leaves and fruit by ≥90% and increased the yield in 2.0- to 2.6-fold in comparison with the unmanaged plots. Cu sprays were important for reducing disease incidence and crop losses, whereas Wb had an additional contribution in minimizing the incidence of cankered, non-marketable fruit. The results indicated that the adoption of these measures of control may depend on the characteristics of the orchard and destination of the production.

Little evidence that farmers should consider abundance or diversity of arbuscular mycorrhizal fungi when managing crops.

Contents Summary 1092 I. Introduction 1093 II. Investigating activity of AMF in agroecosystems 1093 III. Crop benefit from AMF: agronomic and mycorrhizal literature differ 1094 IV. Flawed methodology leads to benefits of mycorrhizas being overstated 1094 V. Rigorous methodology suggests low colonisation by AMF can sometimes reduce crop yield 1095 VI. Predicting when mycorrhizas matter for crop yield 1096 VII. Crop genotype 1099 VIII. Fungal genotype 1100 IX. Complex interactions between the mycorrhizal fungal and soil microbial communities 1102 X. Phosphorus-efficient agroecosystems 1102 XI. Conclusions 1103 Acknowledgements 1104 References 1104 SUMMARY: Arbuscular mycorrhizal fungi (AMF) are ubiquitous in agroecosystems and often stated to be critical for crop yield and agroecosystem sustainability. However, should farmers modify management to enhance the abundance and diversity of AMF? We address this question with a focus on field experiments that manipulated colonisation by indigenous AMF and report crop yield, or investigated community structure and diversity of AMF. We find that the literature presents an overly optimistic view of the importance of AMF in crop yield due, in part, to flawed methodology in field experiments. A small body of rigorous research only sometimes reports a positive impact of high colonisation on crop yield, even under phosphorus limitation. We suggest that studies vary due to the interaction of environment and genotype (crop and mycorrhizal fungal). We also find that the literature can be overly pessimistic about the impact of some common agricultural practices on mycorrhizal fungal communities and that interactions between AMF and soil microbes are complex and poorly understood. We provide a template for future field experiments and a list of research priorities, including phosphorus-efficient agroecosystems. However, we conclude that management of AMF by farmers will not be warranted until benefits are demonstrated at the field scale under prescribed agronomic management.

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice.

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. 'W. Murcott' mandarin (a hybrid of 'Murcott' and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of 'W. Murcott' mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of 'W. Murcott' mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3-5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic 'W. Murcott' mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.

Host-specific effects of soil microbial filtrates prevail over those of arbuscular mycorrhizae in a fragmented landscape.

Plant-soil interactions have been shown to determine plant community composition in a wide range of environments. However, how plants distinctly interact with beneficial and detrimental organisms across mosaic landscapes containing fragmented habitats is still poorly understood. We experimentally tested feedback responses between plants and soil microbial communities from adjacent habitats across a disturbance gradient within a human-modified tropical montane landscape. In a greenhouse experiment, two components of soil microbial communities were amplified; arbuscular mycorrhizal fungi (AMF) and a filtrate excluding AMF spores from the soils of pastures (high disturbance), coffee plantations (intermediate disturbance), and forest fragments (low disturbance), using potted seedlings of 11 plant species common in these habitats (pasture grass, coffee, and nine native species). We then examined their effects on growth of these same 11 host species with reciprocal habitat inoculation. Most plant species received a similar benefit from AMF, but differed in their response to the filtrates from the three habitats. Soil filtrate from pastures had a net negative effect on plant growth, while filtrates from coffee plantations and forests had a net positive effect on plant growth. Pasture grass, coffee, and five pioneer tree species performed better with the filtrate from "away" (where these species rarely occur) compared to "home" (where these species typically occur) habitat soils, while four shade-tolerant tree species grew similarly with filtrates from different habitats. These results suggest that pastures accumulate species-specific soil enemies, while coffee plantations and forests accumulate beneficial soil microbes that benefit pioneer native plants and coffee, respectively. Thus, compared to AMF, soil filtrates exerted stronger habitat and host-specific effects on plants, being more important mediators of plant-soil feedbacks across contrasting habitats.

Tale of the Huanglongbing Disease Pyramid in the Context of the Citrus Microbiome.

The Huanglongbing (HLB) disease pyramid is composed of Liberibacters, psyllid vectors, citrus hosts, and the environment. The epidemiological outcomes for Liberibacter-associated plant diseases are collectively determined by the inherent relationships among plant-Liberibacters-psyllids, and how various environmental factors affect plant-Liberibacter-psyllid interactions. Citrus-Liberibacter-psyllid interactions occur in a complex microbiome system. In this review, we focus on the progress in understanding the HLB disease pyramid, and how the microbiome affects the HLB disease pyramid including the interaction between HLB and the citrus microbiome; the interaction between Liberibacters and psyllids; the interaction between Liberibacters and gut microbiota in psyllids; and the effect of HLB on selected above- and belowground citrus pathogens. Their implications for HLB management are also discussed.

Management of Huanglongbing of Citrus: Lessons from São Paulo and Florida.

São Paulo, Brazil, and Florida, USA, were the two major orange production areas in the world until Huanglongbing (HLB) was discovered in São Paulo in 2004 and Florida in 2005. In the absence of resistant citrus varieties, HLB is the most destructive citrus disease known because of the lack of effective tools to reduce spread of the vector, Diaphorina citri (Asian citrus psyllid), and transmission of the associated pathogen, Candidatus Liberibacter asiaticus. In both countries, a three-pronged management approach was recommended and begun: planting only disease-free nursery trees, effective psyllid control, and removal of all symptomatic trees. In Brazil, these management procedures were continued and improved and resulted in relatively little overall loss of production. In contrast, in Florida the citrus industry has been devastated with annual production reduced by approximately 80%. This review compares and contrasts various cultural and pest management strategies that have been used to reduce infection by the pathogen and increase tolerance of HLB in the main orange-growing regions in the world.

Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mechanisms of bacterial virulence and host range.

BACKGROUND: Citrus bacterial canker is a disease that has severe economic impact on citrus industries worldwide and is caused by a few species and pathotypes of Xanthomonas. X. citri subsp. citri strain 306 (XccA306) is a type A (Asiatic) strain with a wide host range, whereas its variant X. citri subsp. citri strain A(w)12879 (Xcaw12879, Wellington strain) is restricted to Mexican lime. RESULTS: To characterize the mechanism for the differences in host range of XccA and Xcaw, the genome of Xcaw12879 that was completed recently was compared with XccA306 genome. Effectors xopAF and avrGf1 are present in Xcaw12879, but were absent in XccA306. AvrGf1 was shown previously for Xcaw to cause hypersensitive response in Duncan grapefruit. Mutation analysis of xopAF indicates that the gene contributes to Xcaw growth in Mexican lime but does not contribute to the limited host range of Xcaw. RNA-Seq analysis was conducted to compare the expression profiles of Xcaw12879 and XccA306 in Nutrient Broth (NB) medium and XVM2 medium, which induces hrp gene expression. Two hundred ninety two and 281 genes showed differential expression in XVM2 compared to in NB for XccA306 and Xcaw12879, respectively. Twenty-five type 3 secretion system genes were up-regulated in XVM2 for both XccA and Xcaw. Among the 4,370 common genes of Xcaw12879 compared to XccA306, 603 genes in NB and 450 genes in XVM2 conditions were differentially regulated. Xcaw12879 showed higher protease activity than XccA306 whereas Xcaw12879 showed lower pectate lyase activity in comparison to XccA306. CONCLUSIONS: Comparative genomic analysis of XccA306 and Xcaw12879 identified strain specific genes. Our study indicated that AvrGf1 contributes to the host range limitation of Xcaw12879 whereas XopAF contributes to virulence. Transcriptome analyses of XccA306 and Xcaw12879 presented insights into the expression of the two closely related strains of X. citri subsp. citri. Virulence genes including genes encoding T3SS components and effectors are induced in XVM2 medium. Numerous genes with differential expression in Xcaw12879 and XccA306 were identified. This study provided the foundation to further characterize the mechanisms for virulence and host range of pathotypes of X. citri subsp. citri.

Evidence for acquisition of copper resistance genes from different sources in citrus-associated xanthomonads.

ABSTRACT We determined that multiple and independent introductions of copper resistance genes have taken place for strains of Xanthomonas citri subsp. citri from Argentina and strains of X. alfalfae subsp. citrumelonis from Florida. This study compared the partial nucleotide sequences of principal copper resistance genes copL, copA, and copB from X. citri subsp. citri and X. alfalfae subsp. citrumelonis to strains of other Xanthomonas spp. resistant to copper that were isolated from 12 different countries or territories. The survey confirmed that the copLAB gene cluster is present in many species of Xanthomonas from different parts of the world. Alignment of partial nucleotide sequences of copper resistance genes among the copper-resistant (Cu(R)) strains of Xanthomonas detected homology of ≥92, ≥96, and ≥91% for copL, copA, and copB, respectively. Grouping of strains based on branching patterns of phylogenetic trees was similar for copL and copA but differed for copB. When the three genes were concatenated and analyzed using various phylogenetic methods, it appeared that the plasmid had been horizontally transferred and various populations were mutating based on selection pressure unique to geographic regions. Although high homology of the genes among the strains indicated that the copper resistance in xanthomonads has a common origin, the slight differences in nucleotide sequences within groups of strains indicated that Cu(R) genes have been independently exchanged among species of Xanthomonas throughout the world.

Plant mitochondrial introns as genetic markers - conservation and variation.

Plant genomes are comprised of nuclear, plastid and mitochondrial components characterized by different patterns of inheritance and evolution. Genetic markers from the three genomes provide complementary tools for investigations of inheritance, genetic relationships and phenotypic contributions. Plant mitochondrial genomes are challenging for universal marker development because they are highly variable in terms of size, gene order and intergenic sequences and highly conserved with respect to protein-coding sequences. PCR amplification of introns with primers that anneal to conserved, flanking exons is effective for the development of polymorphic nuclear genome markers. The potential for plant mitochondrial intron polymorphisms to distinguish between congeneric species or intraspecific varieties has not been systematically investigated and is possibly constrained by requirements for intron secondary structure and interactions with co-evolved organelle intron splicing factors. To explore the potential for broadly applicable plant mitochondrial intron markers, PCR primer sets based upon conserved sequences flanking 11 introns common to seven angiosperm species were tested across a range of plant orders. PCR-amplified introns were screened for indel polymorphisms among a group of cross-compatible Citrus species and relatives; two Raphanus sativus mitotypes; representatives of the two Phaseolus vulgaris gene pools; and congeneric pairs of Cynodon, Cenchrus, Solanum, and Vaccinium species. All introns were successfully amplified from each plant entry. Length polymorphisms distinguishable by gel electrophoresis were common among genera but infrequent within genera. Sequencing of three introns amplified from 16 entries identified additional short indel polymorphisms and nucleotide substitutions that separated Citrus, Cynodon, Cenchrus and Vaccinium congeners, but failed to distinguish Solanum congeners or representatives of the Phaseolus vulgaris major gene pools. The ability of primer sets to amplify a wider range of plant species' introns and the presence of intron polymorphisms that distinguish congeners was confirmed by in silico analysis. While mitochondrial intron variation is limited in comparison to nuclear introns, these exon-based primer sets provide robust tools for the amplification of mitochondrial introns across a wide range of plant species wherein useful polymorphisms can be identified.

Chemotactic Responses of Xanthomonas with Different Host Ranges.

Xanthomonas citri pv. citri (Xcc) (X. citri subsp. citri) type A is the causal agent of citrus bacterial canker (CBC) on most Citrus spp. and close relatives. Two narrow-host-range strains of Xcc, Aw and A*, from Florida and Southwest Asia, respectively, infect only Mexican lime (Citrus aurantifolia) and alemow (C. macrophylla). In the initial stage of infection, these xanthomonads enter via stomata to reach the apoplast. Herein, we investigated the differences in chemotactic responses for wide and narrow-host-range strains of Xcc A, X. euvesicatoria pv. citrumelonis (X. alfalfae subsp. citrumelonis), the causal agent of citrus bacterial spot, and X. campestris pv. campestris, the crucifer black rot pathogen. These strains of Xanthomonas were compared for carbon source use, the chemotactic responses toward carbon compounds, chemotaxis sensor content, and responses to apoplastic fluids from Citrus spp. and Chinese cabbage (Brassica pekinensis). Different chemotactic responses occurred for carbon sources and apoplastic fluids, depending on the Xanthomonas strain and the host plant from which the apoplastic fluid was derived. Differential chemotactic responses to carbon sources and citrus apoplasts suggest that these Xanthomonas strains sense host-specific signals that facilitate their location and entry of stomatal openings or wounds.

Comparative genomic analysis of Xanthomonas axonopodis pv. citrumelo F1, which causes citrus bacterial spot disease, and related strains provides insights into virulence and host specificity.

Xanthomonas axonopodis pv. citrumelo is a citrus pathogen causing citrus bacterial spot disease that is geographically restricted within the state of Florida. Illumina, 454 sequencing, and optical mapping were used to obtain a complete genome sequence of X. axonopodis pv. citrumelo strain F1, 4.9 Mb in size. The strain lacks plasmids, in contrast to other citrus Xanthomonas pathogens. Phylogenetic analysis revealed that this pathogen is very close to the tomato bacterial spot pathogen X. campestris pv. vesicatoria 85-10, with a completely different host range. We also compared X. axonopodis pv. citrumelo to the genome of citrus canker pathogen X. axonopodis pv. citri 306. Comparative genomic analysis showed differences in several gene clusters, like those for type III effectors, the type IV secretion system, lipopolysaccharide synthesis, and others. In addition to pthA, effectors such as xopE3, xopAI, and hrpW were absent from X. axonopodis pv. citrumelo while present in X. axonopodis pv. citri. These effectors might be responsible for survival and the low virulence of this pathogen on citrus compared to that of X. axonopodis pv. citri. We also identified unique effectors in X. axonopodis pv. citrumelo that may be related to the different host range as compared to that of X. axonopodis pv. citri. X. axonopodis pv. citrumelo also lacks various genes, such as syrE1, syrE2, and RTX toxin family genes, which were present in X. axonopodis pv. citri. These may be associated with the distinct virulences of X. axonopodis pv. citrumelo and X. axonopodis pv. citri. Comparison of the complete genome sequence of X. axonopodis pv. citrumelo to those of X. axonopodis pv. citri and X. campestris pv. vesicatoria provides valuable insights into the mechanism of bacterial virulence and host specificity.

Molecular characterization of copper resistance genes from Xanthomonas citri subsp. citri and Xanthomonas alfalfae subsp. citrumelonis.

Copper sprays have been widely used for control of endemic citrus canker caused by Xanthomonas citri subsp. citri in citrus-growing areas for more than 2 decades. Xanthomonas alfalfae subsp. citrumelonis populations were also exposed to frequent sprays of copper for several years as a protective measure against citrus bacterial spot (CBS) in Florida citrus nurseries. Long-term use of these bactericides has led to the development of copper-resistant (Cu(r)) strains in both X. citri subsp. citri and X. alfalfae subsp. citrumelonis, resulting in a reduction of disease control. The objectives of this study were to characterize for the first time the genetics of copper resistance in X. citri subsp. citri and X. alfalfae subsp. citrumelonis and to compare these organisms to other Cu(r) bacteria. Copper resistance determinants from X. citri subsp. citri strain A44(pXccCu2) from Argentina and X. alfalfae subsp. citrumelonis strain 1381(pXacCu2) from Florida were cloned and sequenced. Open reading frames (ORFs) related to the genes copL, copA, copB, copM, copG, copC, copD, and copF were identified in X. citri subsp. citri A44. The same ORFs, except copC and copD, were also present in X. alfalfae subsp. citrumelonis 1381. Transposon mutagenesis of the cloned copper resistance determinants in pXccCu2 revealed that copper resistance in X. citri subsp. citri strain A44 is mostly due to copL, copA, and copB, which are the genes in the cloned cluster with the highest nucleotide homology (≥ 92%) among different Cu(r) bacteria.

Agrobacterium-mediated transient expression in citrus leaves: a rapid tool for gene expression and functional gene assay.

In this study, we present a method for transient expression of the type III effector AvrGf1 from Xanthomonas citri subsp. citri strain A(w) in grapefruit leaves (Citrus paradisi) via Agrobacterium tumefaciens. The coding sequence of avrGf1 was placed under the control of the constitutive CaMV 35S promoter in the binary vectors pGWB2 and pGWB5. Infiltration of grapefruit leaves with A. tumefaciens carrying these constructs triggered a hypersensitive response (HR) in grapefruit 4 days after inoculation. When transiently expressed in grapefruit leaves, two mutants, AvrGf1ΔN116 and AvrGf1ΔC83, failed to induce an HR. Moreover, using bioinformatics tools, a chloroplast transit signal was predicted at the N terminus of AvrGf1. We demonstrated chloroplast localization by using an AvrGf1::GFP fusion protein, where confocal images revealed that GFP fluorescence was accumulating in the stomatal cells that are abundant in chloroplasts. Transient expression in citrus has the potential for aiding in the development of new disease defense strategies in citrus.

Entomopathogenic nematodes, phoretic Paenibacillus spp., and the use of real time quantitative PCR to explore soil food webs in Florida citrus groves.

Quantitative real-time PCR (qPCR) is a powerful tool to detect and quantify species of cryptic organisms such as bacteria, fungi and nematodes from soil samples. As such, qPCR offers new opportunities to study the ecology of soil habitats by providing a single method to characterize communities of diverse organisms from a sample of DNA. Here we describe molecular tools to detect and quantify two bacteria (Paenibacillus nematophilus and Paenibacillus sp.) phoretically associated with entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematodae. We also extend the repertoire of species specific primers and TaqMan® probes for EPNs to include Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema feltiae and Steinernema scapterisci, all widely distributed species used commercially for biological control. Primers and probes were designed from the ITS rDNA region for the EPNs and the 16S rDNA region for the bacteria. Standard curves were established using DNA from pure cultures of EPNs and plasmid DNA from the bacteria. The use of TaqMan probes in qPCR resolved the non-specificity of EPN and some bacterial primer amplifications whereas those for Paenibacillus sp. also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two species that are not phoretically associated with nematodes. The primer-probe sets for EPNs were able to accurately detect three infective juvenile EPNs added to nematodes recovered from soil samples. The molecular set for Paenibacillus sp. detected the bacterium attached to Steinernema diaprepesi suspended in water or added to nematodes recovered from soil samples but its detection decreased markedly in the soil samples, even when a nested PCR protocol was employed. Using qPCR we detected S. scapterisci at low levels in a citrus grove, which suggested natural long-distance spread of this exotic species, which is applied to pastures and golf courses to manage mole crickets (Scapteriscus spp.). Paenibacillus sp. (but not P. nematophilus) was detected in low quantities in the same survey but was unrelated to the spatial pattern of S. diaprepesi. The results of this research validate several new tools for studying the ecology of EPNs and their phoretic bacteria.

Soil Application of SAR Inducers Imidacloprid, Thiamethoxam, and Acibenzolar-S-Methyl for Citrus Canker Control in Young Grapefruit Trees.

Soil applications of inducers of systemic acquired resistance (SAR) imidacloprid, thiamethoxam, or acibenzolar-S-methyl, at various rates and application frequencies, were evaluated for control of citrus canker caused by Xanthomonas citri subsp. citri in a field trial of 3- and 4-year-old 'Ray Ruby' grapefruit trees in southeastern Florida. Reduction of foliar incidence of canker produced by one, two, or four soil applications of imidacloprid, thiamethoxam, and acibenzolar-S-methyl was compared with 11 foliar sprays of copper hydroxide and streptomycin applied at 21-day intervals. In the 2008 and 2009 crop seasons, canker incidence on each set of vegetative flushes was assessed as the percentage of the total leaves with lesions. By the end of the 2008 season, despite above-average rainfall and a tropical storm event, all treatments significantly reduced foliar incidence of citrus canker on the combined spring-summer-fall flushes. Sprays of copper hydroxide and streptomycin were effective for reducing canker incidence on shoot flushes produced throughout the season compared with the untreated control, whereas soil-applied SAR inducers reduced foliar disease depending on rate, frequency, and timing of application. Except for the treatment of four applications of acibenzolar-S-methyl at 0.2 g a.i. per tree or two applications of imidacloprid, SAR inducers were ineffective for reducing foliar disease on the flushes that were present during the tropical storm. In 2009, all treatments significantly reduced the incidence of foliar canker on the combined spring-summer-fall flushes but not all treatments of spring-summer flushes with SAR inducers were effective compared with the untreated control. Hence, depending on rate, frequency, and timing of application, soil-applied SAR inducers reduced incidence of canker on foliar flushes of young grapefruit trees under epidemic conditions.

Negative plant-soil feedbacks are stronger in agricultural habitats than in forest fragments in the tropical Andes.

There is now strong evidence suggesting that interactions between plants and their species-specific antagonistic microbes can maintain native plant community diversity. In contrast, the decay in diversity in plant communities invaded by nonnative plant species might be caused by weakening negative feedback strengths, perhaps because of the increased relative importance of plant mutualists such as arbuscular mycorrhizal fungi (AMF). Although the vast majority of studies examining plant-soil feedbacks have been conducted in a single habitat type, there are fewer studies that have tested how the strength and direction of these feedbacks change across habitats with differing dominating plants. In a fragmented montane agricultural system in Colombia, we experimentally teased apart the relative importance of AMF and non-AMF microbes (a microbial filtrate) to the strength and direction of feedbacks in both native and nonnative plant species. We hypothesized that native tree species of forest fragments would exhibit stronger negative feedbacks with a microbial filtrate that likely contained pathogens than with AMF alone, whereas nonnative plant species, especially a highly invasive dominant grass, would exhibit overall weaker negative feedbacks or even positive feedbacks regardless of the microbial type. We reciprocally inoculated each of 10 plant species separately with either the AMF community or the microbial filtrate originating from their own conspecifics, or with the AMF or microbial filtrate originating from each of the other nine heterospecific plant species. Overall, we found that the strength of negative feedback mediated by the filtrate was much stronger than feedbacks mediated by AMF. Surprisingly, we found that the two nonnative species, Urochloa brizantha and Coffea arabica, experienced stronger negative feedbacks with microbial filtrate than did the native forest tree species, suggesting that species-specific antagonistic microbes accumulate when a single host species dominates, as is the case in agricultural habitats. However, negative feedback between forest trees and agricultural species suggests that soil community dynamics may contribute to the re-establishment of native species into abandoned agricultural lands. Furthermore, our finding of no negative feedbacks among trees in forest fragments may be due to a loss in diversity of those microbes that drive diversity-maintaining processes in intact tropical forests.

Control of Citrus Canker in Greenhouse and Field with a Zinc, Urea, and Peroxide Ternary Solution.

Accumulation of toxic copper in soil and development of copper-resistant pests are emerging challenges currently faced by the agricultural community worldwide. As an alternative, we have developed a ternary zinc chelate solution (TSOL) pesticide where zinc ions are the primary active ingredient. The material is composed of zinc, urea, and hydrogen peroxide. Urea was chosen as it is widely used as a plant fertilizer and can also bind to both zinc and hydrogen peroxide. No phytotoxicity was observed with TSOL on Meyer lemon (Citrus × meyeri) seedlings at a field spray rate of 800 µg/mL Zn metal concentration. Antimicrobial studies showed that TSOL exhibited improved killing efficacy against Escherichia coli and Xanthomonas alfalfae compared to Zn ions alone. Citrus canker field trials in a grapefruit (Chrysopelea paradisi) grove over three years showed that TSOL provided comparable disease protection to copper products at an equivalent or lower metal content.

Small-Group Technology-Assisted Instruction: Virtual Teacher and Robot Peer for Individuals with Autism Spectrum Disorder.

The authors combined virtual reality technology and social robotics to develop a tutoring system that resembled a small-group arrangement. This tutoring system featured a virtual teacher instructing sight words, and included a humanoid robot emulating a peer. The authors used a multiple-probe design across word sets to evaluate the effects of the instructional package on the explicit acquisition and vicarious learning of sight words instructed to three children with autism spectrum disorder (ASD) and the robot peer. Results indicated that participants acquired, maintained, and generalized 100% of the words explicitly instructed to them, made fewer errors while learning the words common between them and the robot peer, and vicariously learned 94% of the words solely instructed to the robot.

Multimodal Generally Recognized as Safe ZnO/Nanocopper Composite: A Novel Antimicrobial Material for the Management of Citrus Phytopathogens.

Copper (Cu) bactericides/fungicides are used extensively for crop protection in agriculture. Concerns for Cu accumulation in soil, Cu leaching into the surrounding ecosystem, and development of Cu resistance in phytopathogenic bacteria are evident. While there is no suitable alternative to Cu available to date for agricultural uses, it is possible to reduce Cu per application by supplementing with Zn and improving Cu bioavailability using nanotechnology. We have prepared a non-phytotoxic composite material consisting of generally recognized as safe ZnO 800 particles and nanocopper-loaded silica gel (ZnO-nCuSi). The morphology of the ZnO-nCuSi material was characterized using scanning electron microscopy, showing ZnO particles dispersed in the silica gel matrix. ZnO-nCuSi demonstrated strong in vitro antimicrobial properties against several model plant bacterial species. Two consecutive year field efficacy results showed that agri-grade ZnO-nCuSi was effective in controlling citrus canker disease at less than half the metallic rate of the commercial cuprous oxide/zinc oxide pesticide.